Chemistry of Penicillin 9781400874910

Table of contents :
Preface
Contents
I. Brief History of the Chemical Study of Penicillin
II. The Earlier Investigations relating to 2-Pentenylpenicillin
III. The Chemistry of n-Amylpenicillin up to December 1943
IV. Status of the Research on the Structure of Benzylpenicillin in December 1943
V. Isolation and Characterization of the Various Penicillins
VI. Penillic Acids and Penillamines
VII. Review of Certain Investigations on the Structure of Benzylpenicillin during 1944-1945
VIII. Some Inactivation and Degradation Reactions not included in Chapters IV and VII
IX. Desthiobenzylpenicillin and Other Hydrogenolysis Products of Benzylpenicillin
X. The Thiocyanate Derivative of Benzylpenicillin Methyl Ester
XI. The X-Ray Crystallographic Investigation of the Structure of Penicillin
XII. Identification and Crystallography of Penicillins and Related Compounds by X-Ray Diffraction Methods
XIII. Infrared Spectroscopic Studies on the Structure of Penicillin
XIV. Other Physical Methods
XV. The Constitution of Penicillins
XVI. Penicillamine, Its Analogs and Homologs
XVII. Penilloaldehydes and Penaldic Acids
XVIII. The Penilloic and Penicilloic Acids and Their Derivatives and Analogs
XIX. Biosynthesis of Penicillins
XX. Chemical Modifications of Natural Penicillins
XXI. Oxazoles and Oxazolones
XXII. Attempted Syntheses of Penicillins
XXIII. The Condensation of Oxazolones and D-Penicillamine and the Resultant Antibiotic Activity
XXIV. Methyl Benzylpseudopenicillinate
XXV. Thiazolidines
XXVI. The Chemistry of β-Lactams
XXVII. The γ-Lactam of Benzylhomopenicilloic Acid and Related Compounds
XXVIII. Synthetic Benzylpenicillin
XXIX. Assay of Penicillins
XXX. Appendix
SUBJECT INDEX

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THE CHEMISTRY OF PENICILLIN

THE CHEMISTRY OF PENICILLIN REPORT ON A COLLABORATIVE INVESTIGATION BY AMERICAN AND BRITISH CHEMISTS UNDER THE JOINT SPONSORSHIP OF THE OFFICE OF SCIENTIFIC RESEARCH AND DEVELOPMENT, WASHINGTON, DC., AND THE MEDICAL RESEARCH COUNCIL, LONDON. COMPILED UNDER THE AUSPICES OF THE NATIONAL ACADEMY OF SCIENCES, WASHINGTON, DC., PURSUANT TO A CONTRACT WITH THE OFFICE OF SCIENTIFIC RESEARCH AND DEVELOPMENT ·$· φ φ

E D I T O R I A L B O A B D I HANS T . CLARKE JOHN R. JOHNSON SIR ROBERT ROBINSON

PRINCETON UNIVERSITY PRESS · 1949 PRINCETON, NEW JERSEY

Copyright, 1949, by Princeton University Press London: Geoffrey Cumberlege, Oxford University,Press

Under the terms of the contract for the publication of The Chemistry of Penicillin the publisher has agreed to waive its rights under the copyright after five years from the date of publication. Thereafter this volume will be in the public domain and dedicated to the public.

Printed in the United States of America By the Maple Press, York, Pennsylvania

PREFACE THIS monograph represents an attempt to record in detail results of experimental and theoretical studies carried out in a unique, internationally collabora­ tive effort to ascertain the chemical constitution of penicillin and to devise methods for its synthesis. The earliest work on the production of penicillin was carried out in Sir Howard Florey's laboratory at Oxford, England, and the first chemical experi­ ments on the nature of the antibiotic were also initi­ ated in Oxford before there was any question of industrial cooperation. When that stage was reached the Therapeutic Research Corporation of Great Britain set up a Penicillin Sub-Committee of their Research Panel to deal with the production and chemistry of penicillin. The progress reports, rendered to this committee' as occasion arose, were known as the "PEN" reports; those which have a direct bearing on the structure of penicillin are included as source material for this monograph. In general, the chemical information contained in these reports was not published in the scientific press, but was privately communicated to recog­ nized workers in the field. In October 1942, the-Ministry of Supply set up a General Penicillin Committee. It was then decided that the existing Penicillin Sub-Committee should be enlarged to include other interested workers, among whom were Imperial Chemical (Pharma­ ceuticals) Ltd., and should continue its task of coordinating the work on production and purifica­ tion of penicillin to the stage fit for clinical use, reporting to the General Penicillin Committee. The name of the Sub-Committee was changed at this time to the Penicillin Producer's Conference or, as it was briefly called, the Penicillin Conference. At the same time it was arranged that the unofficial Conference of Chemists, which had been set up under the chairmanship of the late Dr. F. L. Pyman, should continue its function of handling information . on the chemistry and structure of penicillin; the reports of this Chemists' Conference were made available to the Chairman of the General Penicillin Committee of the Ministry of Supply. By this time, experiments relating to the produc­ tion of penicillin had been instituted in the labors tories of certain American pharmaceutical manu­ facturers (Merck & Co., Inc., E. R. Squibb and Sons, and subsequently Chas. Pfizer & Co., Inc.). The information secured by these firms was communicated to the Committee on Medical Research and transmitted by it, via the Medical

Research Council, to the Therapeutic Research Corporation, Imperial Chemical Industries, and various academic groups in Britain. In October 1943, when many of the structural features of the penicillin molecule were becoming clear, the problem of the synthesis became a press­ ing one, and to handle this aspect the Medical Research Council set up a Committee for PenicilliQ Synthesis "to initiate, coordinate and make investi­ gations on the synthesis of penicillin and analogues." The confidential reports which were issued and exchanged were known as the "CPS" reports. At the same time, the Committee on Medical Research of the Office of Scientific Research and Development in Washington, which had already undertaken the coordination of chemical work on penicillin in the United States, came to an agree­ ment with the Medical Research Council for an exchange of information on anything which had a bearing on the problem of the synthesis of peni­ cillin. A limited group (see below) of industrial and academic research organizations engaged, under contract with the Office of Scientific Research and Development, to collaborate in studies "in con­ nection with (i) the chemical structure of penicillin and (ii) the synthesis of penicillin or a therapeutic equivalent." In general, the American partici­ pants filed progress reports at monthly intervals. These reports bear index letters showing the groups in which they originated, followed by a serial number. During the progress of the collaboration, copies of all reports were filed with American and British coordinators for distribution to the various partici­ pants in both countries. However, during ' the first half of 1944 a considerable delay was incurred by the lapse of time necessary to complete the formal agreements at the Government level, and> subsequently, in spite of every endeavor to secure prompt transmission, the time lag involved in the transatlantic exchange occasionally resulted in unwitting duplication of experimental effort. . The dates on which reports were received are indicated in the Appendix. When the confidential reports were written, it was never intended that they should be made avail­ able in their original form to a wider circle of readers. They were essentially interim reports, jottings from laboratory note-books, hastily produced and circulated so that all engaged in· the urgent co­ operative effort could take immediate advantage ν

PREFACE TABLE I

Participating Group

Abbreviation

•Boots Pure Drug Company, Ltd. •British Drug Houses, Ltd. Cambridge University, Department of Chemistry ) Cambridge University, Department of Colloid Science J •Glaxo Laboratories, Ltd. Imperial Qhomical Industries, Ltd. (Alkali Division) \ Imperial Chemical (Pharmaceuticals) Ltd. J Imperial College of Science, London, Department of Organic Chemistry The London Hospital Medical Unit Manchester University, Department of Chemistry •May & Baker, Ltd. National Institute for Medical Research, Hampstead, London 1I Oxford University, Department of Crystallography Oxford University, Dyson Perrins Laboratory I Oxford University, Sir William Dunn School of Pathology | Oxford University, Department of Physical Chemistry ) •Wellcome Foundation, Ltd. Abbott Laboratories U. S. Department of Agriculture, Northern Regional Research Laboratory Cornell University Medical College, Department of Biochemistry Cornell University Medical College, Russell Sage Institute Cutter Laboratories Federal Security Agency, Food and Drug Administration Harvard University, Department of Chemistry Heyden Chemical Corporation University of Illinois, Department of Chemistry Eli Lilly and Company Merck & Co., Inc. University of Michigan, Department of Chemistry University of Michigan, Department of Physics National Bureau of Standards Parke, Davis and Company Chas. Pfizer & Co., Inc. The Rockefeller Institute for Medical Research Shell Development Company Squibb Institute for Medical Research Stanford University, Department of Biology The Upjohn Company Winthrop Chemical Company, Inc. 4cMembere

Boots B.D.H. Cambridge Glaxo I.C.I. I.C.S. London Hosp. Manchester May & Baker N.I.M.R. Oxford Wellcome Abbott N.R.R.L. Cornell Bioch. Cornell R.S. Cutter F.D.A. Harvard Heyden Illinois Lilly Merck Mich. Chem. Mich. Phys. Bur. Stand. Parke-Davis Pfizer Rockefeller Shell Squibb Stanford Upjohn Winthrop

of the Therapeudc Research Corporation of Great Britain, Ltd.

of any recorded gain in knowledge. The same consideration applies to speculations which were advanced as quickly as possible in the common interest. In Table I are listed the names of the individual participant groups, the abbreviations employed to designate these in the text of the monograph, and, in the case of the American participants, the prefix indicating group of origin. In addition to those listed in Table I, the following grQups agreed to participate, but were not called upon for scientific contributions: Naval Medical Research Institute, Bethesda, Maryland Dr. B. E. Warren, Massachusetts Institute of Technology Dr. Frank C. Whitmore, Pennsylvania State College

Shortly after the cessation of hostilities it waa decided by the Office of Scientific Research and De­ velopment and the Medical Research Council that the findings should be published in a monograph, rather than in the scientific press. This decision was reached on the following grounds: (a) The simultaneous release of all the data would throw an insupportable burden on the journals, (b) The ir­ reversibility of the interdigitation of the researches of the collaborating groups made it impracticable to assign individual achievements to individual in­ vestigators. In view of the probability that the monograph could not be completed prior to the dissolution of the OflSce of Scientific Research and Development, it was necessary to secure for the work the sponsorship of a permanent organization. This function was kindly undertaken by the Na­ tional Academy of Sciences, under contract with the OflBce of Scientific Research and Development.

PREFACE At a conference held early in 1946, attended by scientific representatives of the majority of the. American participant groups and by two members of the British Commonwealth Scientific Office representing British participants, the plan for publication in monograph form was unanimously approved. The distribution of subject matter into monograph chapters was determined, and authors for these chapters were selected. These selections were made on the basis of special familiarity with the field and it was agreed that authors should regard themselves as impartial reporters of perti­ nent information, irrespective of source. In gen­ eral, the chapter topics were distributed according to organic chemical classification and physicochemical techniques. Owing to the extreme importance of penicillin as a military weapon, all of the information secured during the period of active collaboration bore a high security classification. By January 1, 1946, all participants had been released from their obliga­ tion to hold the information in secrecy, but they agreed to refrain from any independent publication that might, in the opinion of the Editorial Board, interfere with the plans for the monograph. How­ ever, with the sanction of all participants a brief account of the principal findings had been published

vii

in Science and in Nature in December 1945. This was followed by similar publications in Science in November 1940, June 1947, and November 1947. Further steps were taken to make the declassified information available to persons especially inter­ ested. A few sets of the progress reports were filed by the Medical Research Council in April 1947 in selected scientific libraries in Britain, and in the United States similar sets were filed with the Patent Office in March 1946, and in the Office of Technical Services of the Department of Commerce in July 1947. The Editorial Board owes much to the helpful advice and encouragement of Dr. A. N. Richards, Chairman of the Committee on Medical Research of the Office of Scientific Research and Develop­ ment, and Dr. Harold King, Secretary of the Penicillin Synthesis Committee. Its labors were lightened in no small measure by the assiduous co­ operation of Mrs. Helen C. Moreland and Dr. A. H. Cook, who carried out the distribution of reports and other material necessary to the authors in their arduous task of preparing the various chapters. Inestimable aid in the revision of manuscript and proof was rendered by Mr. Herbert S. Bailey, Jr. of Princeton University Press. To all of these the Board expresses its sincere appreciation. .

CONTENTS Preface I. Brief History of the Chemical Study of Penicillin Hans T. Clarke, John R. Johnson, and Robert Robinson II. The Earlier Investigations relating to 2-Pentenylpenicillin E. P. Abraham, W. Baker, W. R. Boon, C. T. Calam, H. C. Carrington, E. Chain, H. W. Florey, G. G. Freeman, R. Robinson, and A. G. Sanders

ν 3 10

III. The Chemistry of n-Amylpenicillin up to December 1943 A. H. Cook, and I. M. Heilbron

38

IV. Status of the Research on the Structure of Benzylpenicillin in December 1943 Robert L. Peck and Karl Folkers

52

V. Isolation and Characterization of the Various Penicillins / 76 0. Wintersteiner, W. R. Boon, H. C. Carrington, D. W. MacCorquodale, F. H. Stodola, J. L. Wachtel, R. D. CoghilJ, W. C. Risser, J. E. Philip, and 0. Touster VI. PenillicAcidsandPenillamines !...... 106 A. H. Cook VII. Review of Certain Investigations on the Structure of Benzylpenicillin during 1944-1945 . 144 Robert L. Peck and Karl Folkers VIII. Some Inactivation and Degradation Reactions not included in Chapters IV and VII . . 207 0. Wintersteiner, Η. E. Stavely, J. D. Dutcher, and M. S. Spielman IX. Desthiobenzylpenicillin and Other Hydrogenolysis Products of Benzylpenicillin Edward Kaczka and Karl Folkers X. The Thiocyanate Derivative of Benzylpenicillin Methyl Ester Vincent du Vigneaud and Donald B. Melville

243 269

XI. The X-Ray Crystallographic Investigation of the Structure of Penicillin 310 D. Crowfoot, C. W. Bunn, B. W. Rogers-Low, and' A. Turner-Jones XII. Identification and Crystallography of Penicillins and Related Compounds by X-Ray Dif­ fraction Methods . ......... 367 G. L. Clark, W. I. Kaye, K. J. Pipenberg, and N. C. Schieltz XIII. Infrared Spectroscopic Studies on the Structure of Penicillin H. W. Thompson, R. R. Brattain, Η. M. Randall and R. S. Rasmussen XIV. OtherPhysicalMethods R. B. Woodward, A. Neuberger, and N. R. Trenner XV. The Constitution of Penicillins John R. Johnson, Robert B. Woodward and Robert Robinson XVI. Penicillamine, Its Analogs and Homologs Harry M. Crooks

382 415 440, 455

XVII. Penilloaldehydes and Penaldic Acids Ellis V. Brown

473

KVIIL The Penilloic and Penicilloic Acids and Their Derivatives and Analogs Ralph Mozingo and Karl Folkers

535

XIX. Biosynthesis of Penicillins Otto K. Behrens XX. Chemical Modifications of Natural Penicillins R. D. Coghill, F. H. Stodola, and J. L. Wachtel ix

657 680

χ

CONTENTS

XXI. Oxazoles and Oxazolones J. W. Cornforth XXII. Attempted Syntheses of Penicillins W. E. Bachmann and M. W. Cronyn XXIII. The Condensation of Oxazolones and D-Penicillamine and the Resultant Antibiotic Activity V. du Vigneaud, J. L. Wood, and Μ. E. Wright XXIV. MethylBenzylpseudopenicillinate '. . J. H. Hunter, J. W. Hinman, and Η. E. Carter XXV. Thiazolidines A. H. Cook and I. M. Heilbron XXVI. The Chemistry of /3-Lactams · s. A. Ballard, D. S. Melstrom, and C. W. Smith XXVII. The γ-Lactam of Benzylhomopenicilloic Acid and Related Compounds Vincent du Vigneaud and Frederick H. Carpenter XXVIII. Synthetic Benzylpenicillin. Vincent du Vigneaud, Frederick H. Carpenter, Robert W. Holley, Arthur H. Livermore, and Julian R. Rachele XXIX. Assay of Penicillins John V. * Scudi and Η. B. Woodruff XXX. Appendix

688 849 892 909 921 973 1004 1018 1025 1043

THE CHEMISTRY OF PENICILLIN

BRIEF HISTORY OF THE CHEMICAL STUDY OF PENICILLIN HANS T. CLARKE,1 JOHN R. JOHNSON,2 AND SIR ROBERT ROBINSON»

During the decade following the discovery of penicillin (Fleming, Brit. J. Exp. Path., 10; 226 (1929)) relatively little information was secured as to its chemical nature. Fleming had reported it to be soluble in water and in alcohol, but insoluble in ether or chloroform, and that its thermal stability in solution was maximal at neutrality. Three years later Clutterbuck, Lovell, and Raistrick (Biochem. J., 26, 1907 (1932)) found that penicillin could be produced in a synthetic medium. They also showed that when solutions of penicillin at pH 7.2 were extracted with ether, some of the anti­ bacterial activity was transferred to the ether, but that when the process was carried out at pH 2, the extraction was almost complete. They likewise noted that the antibiotic extract was sensitive to oxidants and was readily inactivated by evaporation in acid and alkaline solution, but moderately stable at pH 5-6. After a further interval of three years Raistrick's findings were confirmed, in general, by Reid («7. Bact., 29, 215 (1935)), who found in addition that the activity was lost on dialysis and that penicillin was adsorbed on charcoal. In 1940 Chain, Florey, Gardner, Heatley, Jen­ nings, Orr-Ewing, and Sanders (Lancet, 289, 226 (1940)) prepared penicillin in solid, though inhomogeneous, form and reported on its effectiveness in vino against various pathogenic organisms. In the same year Abraham and Chain (Nature, IJfi, 837 (1940)) described the preparation from B. coli of an enzyme, penicillinase, which inactivates penicillin. In 1941 Abraham, Chain, Fletcher, Gardner, Heatley, Jennings, and Florey (Lancet, 241, 177 .(1941)) published details of a procedure for the concentration of penicillin from culture fluid ob­ tained with the use of Raistrick's synthetic medium. This involved extraction, from acid solution with organic liquids and further purification by chroma­ tographic procedures. In this way there was secured (Chain, Nature, 148, 758 (1941); Biochem. J., 86, 4 (1943)) the barium salt of an acid which proved to be stable when absolutely dry or in organic solvents. In 1942 Abraham and Chain (Nature, 149, 328 1 Columbia

University. * Cornell University. * Oxford University.

(1942)), by further refinements of the process, succeeded in producing a barium salt with an activity of 450-500 U./mg. This product was later found to contain nitrogen (Abraham, Baker, Chain, Florey, Holiday, and Robinson, Nature, 149, 356 (1942)), but the suggested provisional formula, C24H32Oi0N2Ba, was soon found to be incorrect. One impurity detected was barium furoate. The significance of the nitrogen content was indicated by its linear relation to antibiotic potency. On hydrolysis this substance yielded carbon dioxide, a volatile acid, and the crystalline salt of a base. The authors recognized that their material was not homogeneous, so that these data could not be inter­ preted with certainty. It was also observed (Abraham and Chain, Brit. J. Exp. Path., 28, 103 (1942)) that penicillin is inactivated by primary alcohols, by organic bases, and by various metallic salts, for example those of copper and zinc. The basic compound referred to above received the name penicillamine; it was shown (Abraham, Chain, Baker, and Robinson, Nature, 151, 107 (1943)) to be a primary amine and to contain one strongly arid one weakly acidic group. The hydrochloride was at first assigned the formula CeHnO4N-HCl, and later C6HuO2SN-HCl. In so far as the Oxford work was concerned, the recogni­ tion of sulfur in penicillin (see below) followed directly from the study of an oxidation product of penicillamine. The analytical results indicated the uptake of three oxygen atoms and thus suggested conversion of a thiol to a sulfonic acid. The signifi­ cance of penicillamine as an integral part of penicil­ lin became obvious when it was obtained from penicillin having an activity of 1,000 U./mg. on the Oxford scale of that date. In the meantime the study of penicillin was taken up in other laboratories. A product showing 750 U./mg. was obtained at the Imperial College of Science, London (Catch, Cook, and Heilbron, Nature, 150, 633 (1942)), where it was observed that penicillin on degradation yielded a product which appeared to be an amino acid. The con­ version of penicillin in acid solution into a crystal­ line product termed penillic acid was recorded early in 1943 by chemists in the Wellcome Research Laboratories (Duffin and Smith, Nature, 151, 251 (1943)). In the United States penicillin was

4

THE HISTORY OF THE CHEMICAL STUDY OF PENICILLIN

obtained in the form of a crude ammonium salt (Meyei, Hobby, Dawson, Schwenk, and Fleischer, Science, 96, 20 (1942)), which had an activity of 240 U./ing. (Hobby, Meyer, Chaffee, and Dawson, J. Bact., JtS, 65 (1942)), but no evidence was presented as to its chemical nature. On treatment with diazoalkanes penicillin con­ centrates were found to yield esters which proved to be notably more stable than the salts. They showed little activity in vitro but were antibiotically active in vivo. The benzhydryl ester was split by catalytic hydrogenation with regeneration of in vitro activity (Meyer, Hobby, and Chaffee, Science, 97, 205 (1943)). It was later shown that the methyl and ethyl esters could be hydrolyzed to ac­ tive penicillins by treatment with sodium hydroxide or sodium bicarbonate solution (Merck, M.12b; Hickey, Science, 101, 462 (1945)). By 1943 recognition of the potential military importance of penicillin had led to restriction of chemical information on the subject. Investiga­ tion was continued with increasing intensity in both academic and industrial research laboratories, but, in general, the results were privately communicated to other recognized workers in the field rather than to the scientific press. This exchange of informa­ tion on the chemistry of penicillin was effected in Britain at first through unofficial conferences of interested workers and later by those sponsored by the Ministry of Supply and the Medical Research Council. There were also special agreements be­ tween certain pharmaceutical manufacturers in England and in the United States. Information secured by the American firms was subsequently communicated to the Committee on- Medical Re­ search and disseminated through it. A similar procedure was followed in England, where the par­ ticipating groups reported to the Medical Research Council. These two government agencies arranged for the international exchange of information bear­ ing on the chemistry of penicillin. During the first half of 1943 progress in the chemical studies was made principally in Britain, where in addition to penicillamine, 2-pentenylpenillic acid and 2-pentenylpenillamine were isolated as conversion products of the impure preparations then available. Experimental work in the United States was at first primarily directed towards problems of production and purification. It was found in the Northern Regional Research Laboratory and in the Merck and Squibb labora­ tories that chromatographic procedures, the efficacy of which had been demonstrated in Britain for the concentration of penicillins in the form of their free acids, could advantageously be applied to the more stable sodium sa,lts. The partition chromatography of Martin and Synge was adapted for the purifica­ tion and separation of the penicillins by chemists of Imperial Chemical Industries, Ltd. In the surjimer of 1943 MacPhillamy, Wintersteiner, and Ali-

cino, of the Squibb group, succeeded (S.S) in crystallizing the sodium salt-of benzylpenicillin. This important achievement, which made possible the accurate chemical study of the pure compound, immediately led to the recognition by the same investigators of sulfur as a constituent ,of the mole­ cule. Coincidentally, the presence of sulfur in (impure) barium penicillin, as well as in penicil­ lamine, penicillaminic acid, penillic acid and other well-defined derivatives of penicillin, was inde­ pendently discovered by the chemists in Oxford (Abraham, Baker, Chain, and Robinson, PEN.88). Soon afterward the Oxford workers reported the crystallization of alkali metal salts of their penicillin. At about the same time it became clear that the penicillin which had been obtained in crystalline form in the United States was not identical with the penicillin with which the British investigators had been working. Among other differences be­ tween the favo was the far greater reluctance of the latter to crystallize. The chemical distinction between them was clearly brought to light during the middle of 1943 by observations in several quarters. One was the demonstration by Stodola, Wachtel, and Coghill, of the Northern Regional Research Laboratory, that the two varieties of penicillin give different, though analogous, crys­ talline derivatives when the free acids of the respective penicillins are treated with benzylamine. The second observation was the demonstration in the Merck laboratories that the analytical data on crystalline penillic acids showed that there were at least two penicillins.4 The American preparations had been found (Merck, Report for April through July 1943) to yield phenylacetic acid on hydrolysis, whereas the antibiotic studied in Britain yielded 2-hexenoic acid under similar conditions (Abraham, Chain, Baker, and Robinson, Report dated Novem­ ber 8, 1943). Very convincing evidence was also obtained by the chemists of Imperial Chemical Industries, Ltd., at an early stage of the develop­ ment. The main constituents of their own penicillin and of Merck penicillin were chromatographically separated (Imperial Chemical Industries, Report dated August 4, 1943). This was later confirmed by direct comparison of the derived penillic acids(I.C.I., Report dated November 18, 1943). It appears that these reports had a limited circulation. On the other hand, penicillamine having the same configuration was obtained from both types. After some uncertainty as to its constitution (Abraham, Chain, Baker, and Robinson, PEN.88), penicil­ lamine was recognized by the Oxford workers to be /3,/3-dimethylcysteine. This was demonstrated by chemical (Abraham, Chain, Baker, Cornforth, 4 Systems of nomenclature using letters (in the United States) and Roman numerals (in Britain) were initiated late in 1943. However, in the interests of clarity and uniformity» worker? in the field agreed early in 1946 on the nomenclature now in use, which involves a designatory prefix such as "benzyl." ,

THE HISTORY OF THE CHEMICAL STUDY OF PENICILLIN Cornforth, and Robinson, PEN.100) and crystallographic comparison with a synthetic sample (Crowfoot and Low, PEN.lOl). Penicillamine had in the meantime been shown to yield thiazolidines on condensation with carbonyl compounds, and it was suspected that the same ring system was present in the penicillin molecule (Abraham, Chain, Baker, and Robinson, PEN.97). Grounds for this suspi­ cion had been furnished by the observation (Abra­ ham, Baker, Chain, and Robinson, PEN.91) that when the Oxford penicillin was decomposed by mercuric chloride it yielded a carbonyl compound, probably an aldehyde, as well as penicillamine. This aldehyde was characterized in the form of a crystalline 2,4-dinitrophenylhydrazone and a con­ densation product with dimedone, and was shown to have the composition C8Hi3O2N (Abraham, Chain, Baker, and Robinson, PEN.97). It was recognized as hexenoylaminoacetaldehyde and as the source of the glyoxal-osazones which were early obtained from mother liquors resulting after hydrolysis and separation of penicillamine. Mean­ while the Imperial College of Science group (Bentley, Catch, Cook, Elvidge, Hall, and Heilbron, PEN.99; 102; 105) had shown that the antibiotic studied in Britain could be reduced to a dihydro derivative which was biologically active and was recognized as a "natural" penicillin. It afforded n-caproic acid on hydrolysis. The position of the double bond in the hexenoic acid obtained from the unreduced antibiotic was determined at Oxford by permanganate oxidation to propionaldehyde. The Imperial College workers found that the penilloaldehyde from their dihydropenicillin afforded a dinitrophenylhydrazone which could be further changed to the glyoxal osazone. They therefore suggested that the aldehyde was n-caproylaminoacetaldehyde. In both series the identity of -the penilloaldehydes was confirmed by synthesis. Analogous conclusions and hypotheses were attained independently and simultaneously by American workers. The chemists in the Squibb Institute (Dutcher, MacPhillamy, Stavely, and Wintersteiner, S.Sa) and in the Merck labora­ tories (M.l) produced evidence that the carbonyl compound secured by treatment of the American crystalline penicillin with mercuric chloride was phenylacetylaminoacetaldehyde. It was, for exam­ ple, found to be oxidized to phenaceturic acid. At the same time the Merck group demonstrated that when the product formed by the action of benzylamine upon benzylpenicillin was treated with mer­ curic chloride and penicillamine was liberated, the benzylamine group remained in amide linkage in the residual portion of the molecule. A strictly analo­ gous result was obtained with the methyl ester which resulted from the action of methanol upon benzylpenicillin. It was also shown by Boon, Carrington, and Freeman (CPS.28) that the methyl ester of pentenylpenicillin, on treatment with mercuric

6

chloride, yielded the methyl ester cf penicillamine. The identity of the acid group in penicillin with the carboxyl group of penicillamine was thus definitely established by two independent methods. In the recognition of degradation products of penicillin invaluable aid was rendered by X-ray crystallographic measurements. For instance, the 2,4-dinitrophenylhydrazone of the penillic aldehyde secured in Oxford was shown by this method (Crow­ foot and Low, PEN.112) to be identical with the hydrazone prepared from synthetic 2 hexenoyl­ aminoacetaldehyde (Abraham, Chain, Baker, and Robinson, PEN.111). On the basis of these and many other findings the Oxford workers proposed (Abraham, Chain, Baker, and Robinson, PEN.108) the thiazolidineoxazolone S C5H9C=N CH-CH ^C(CH3)2 I l l l OCO NH CHCOOH formula as the simplest expression for their penicil­ lin. An exactly corresponding formula was inde­ pendently proposed by the Merck, Squibb, and Abbott groups for benzylpenicillin. However, the chemists in both the Oxford and the Merck labora­ tories drew attention to the fact that the presence of a basic group, indicated by this structure, could not be detected in penicillin, and both proposed, as a possible alternative, the /3-lactam structure S C6H9CONH CH-CH I I CO-N-

nvC(CH ) 8 s

I CHCOOH

At this point it was hoped that, as the penicillins were relatively simple compounds, synthetic meth­ ods for their production could be developed without much difficulty. The urgency of the need for large quantities of penicillin by the military forces made imperative the intensive exploration of this field on a wider front and on a basis of international col­ laboration. At the instance of the Director of the Office of Scientific Research and Development and the Chairman of the Committee on Medical Re­ search in Washington, and the Secretary of the Medical Research Council in London, the necessary diplomatic agreements were reached. The Ameri­ can group of collaborators was enlarged by the inclusion of eight more industrial research labora­ tories and ten academic laboratories. Contracts between these organizations and the governmental agencies were entered into, according to the terms of which each contractor undertook to conduct experimental investigations in connection with the chemical structure of penicillin and the synthesis of penicillin or a therapeutic equivalent. All eon-

6

THE HISTORY OF THE CHEMICAL STUDY OF PENICILLIN

tractors undertook to report to their government all pertinent information available to them at that time and thereafter to furnish monthly progress reports/ All of this information was transmitted as expeditiously as possible to each contractor in Britain and in the United States. In the United States the contracts, which began during December and January 1943-1944, remained in force until November 1, 1945, in the case of the industrial organizations' and December 31, 1945, in the case of the academic institutions. In Britain, as already stated, the early collaboration was an informal by­ product of a Penicillin Production Committee sponsored by the Ministry of Supply. On January 1,1944, the Medical Research Council set up a Com­ mittee on Penicillin Synthesis (CPS) under the chairmanship of Sir Robert Robinson, and this included representatives of the industrial' groups, academic centers, and also of the Ministry of .Supply. The regular exchange of British and American reports then began but there was delay in the first months so that much work was dupli­ cated. Attention is drawn elsewhere to the more important consequences of this situation. The unrestricted exchange of current information at frequent intervals resulted in as close a collabora­ tion as is possible among a widely distributed group of laboratory teams, but no attempt was made to avoid duplication of effort in the various labora­ tories.- In consequence it is difficult or impossible, except in a relatively few specialized phases of the joint effort, to assign sole scientific credit for indi­ vidual findings secured during the period covered by the contracts.7 In the outline that follows no attempt is made to do more than touch upon some of the more significant results. In the winter of 1943-1944 an important problem was the confirmation and clarification of the struc­ ture assigned to penicilloic acids, the primary product of the hydrolysis of penicillins. The major part of the light thrown on this problem was supplied by the Merck laboratories. The D (or "unnatural") configuration of penicillamine was es­ tablished by successive treatment with phenyl isocyanate and with Raney nickel catalyst, whereby * A complete file of these reports has been deposited with the U. S. Department of Commeroef Office of' Tebhniosl Servioes, from whom reproductions of desired portions can be obtained on request. Copies of these reports are also filed in scientific libraries in Britain. * The industrial participants in the chemical projects performed the subject work of their contracts without financial aid from their govern­ ments. In both countries, however, grants in support of their work were made to the academic groups. * Shortly after the eontrabts between OSED and the industrial partici­ pants had expired, a brief summary announcement of the principal findings was published in Soience (,IOMt 627 (1945)) and in Nature (15β, 766 (1945)). At a conference, attended by scientific representa­ tives of all the cooperating groups, held on January 9, 1946, it was dedded that detailed reports of the resultssecured under the collaborative program should, in general, be published in a monograph rather than in individual papers in the scientific press. However, provision was made for the publication, in advance of the monograph, of papers which, in the opinion of the Editorial Board, would not cotiflict with the plan. Several such articles have appeared. When the monograph was planned, it was hoped that its' writing could be completed in six months; unfortunately this estimate proved unduly optimistic.

the sulfur atom was replaced by hydrogen; the product was identical with the phenylureido deriva­ tive of D-valine (Merck, M.l). As stated in Science (102, β%7 (1945)), the consti­ tution of the penaldic acid, with which penicillamine is combined in benzylpenicilloic acid; was estab­ lished (Merck, M.l) by its conversion on cata­ lytic hydrogenation into cyclohexylacetylalanine. Under less drastic conditions of hydrogenation corresponding derivatives of serine were obtained. As these amino acid derivatives were optically inactive, they yielded no information as to the configuration of that part of the penicilloic acid molecule. This information was secured only later when it was shown in the Merck laboratories (M.62) that under suitable conditions of hydrogena­ tion derivatives of ^alaniner which-hast the "nat­ ural" configuration possessed by the ammo acids of proteins, were formed. Derivatives of penaldic acid were synthesized in several laboratories by the condensation of ethyl formate or orthoformate with esters of phenylacetyl glycine, and when the products were condensed with synthetic D-penicillamine, esters of penicilloic acids were obtained. In a very complete Study in the Merck laboratories (M.28), methyl esters of three of the four theoretically possible diastereoisomers of the penicilloic acids derived frotn D-peni­ cillamine were synthesized and it was shown that at least two of these were formed by the action of methanol upon benzylpenicillin.. Somewhat later, esters of the fourth isomeric form of penicilloic acid were synthesized in the Squibb laboratories (S.33). It has already been mentioned that theconversion of penicillins into penillic acids contributed largely to the recognition of the existence of more than one variety of penicillin. The constitution of benzylpenillic acid was confirmed by synthesis in· two laboratories during 1944. An optically inactive monomethyl ester was prepared by Cook, Elvidge, and Heilbron (CPS.199) by condensing the methyl ester of phenylthioacetyldiethokyalanine with DLpenicillamine. The dimethyl ester of optically , active penillic acid, identical with the product from benzylpenicillin, was synthesized by the Merck group (M.49) by condensing the Schiff-base from N-formyl-e-formylglycine methyl ester and benzylamine with the methyl ester of D-penicillamine. Attempts, made in many laboratories, to produce penicillins by anhydrization of penicilloates, met with almost universal failure. It is worthy of note, however, that they did provide the first syn­ thetic material possessing antibiotic activity even ' though the potencies were small and not proved to be of penicillin type (Imperial College of Science, CPS.5). In the early stages of the research, the oxazolone, or azlactone, structure indicated above was that to which most attention was paid, but as the work progressed increasing difficulty was experienced in applying it to the penicillins. This

THE HISTORY OF THE CHEMICAL STUDY OF PENICILLIN was especially the case in connection with, the physico-chemical investigations such as the electrometric studies by Neuberger (CPS.54/ 266) and many others. A number of structures which had been proposed at the outset were excluded, during the first half of 1944, by the observation that

7
Ci6H20N2O3S + CO2 XV IX

C6H6CH2CONH-CH2-C /I HO2C NH XVH

C(CH3)2 I CH-CO2H

malonaldehydic acid moiety; in structure XVII, the I C3 fragment, —NHCH2C— is an aminopyruvic I CO2H acid moiety. Because of the great ease of the decarboxylation of benzylpenicilloic acid, and be­ cause an α-amino acid C3— fragment seemed more probable biogenetically, structure XVI was preferred for benzylpenicilloic acid. Since benzylpenilloic acid (X) had been cleaved successfully into peni­ cillamine (I) and phenylacetylaminoacetaldehyde (V), it appeared that benzylpenicilloic acid (XVI) or (XVII) should theoretically cleave into the cor­ responding carbonyl degradation products XVIII or XIX in addition to penicillamine. From the CeH6CH2CONH-CH-CHO

Ao2H XVIII CeH6CH2CONHCH2C-O CO2H XIX extreme lability of this carboxy group of unknown position, it was evident that some acid derivative of benzylpenicilloic acid would have to be used if the degradation with mercuric chloride were to be suc­ cessful (Merck, M.l, 7). Benzylpenaldic Acid. During studies on the purification of penicillin concentrates, an ethereal solution of benzylpenicillin obtained from the "1249-fermentation" was treated with benzylamine. A crystalline benzylamine derivative of the penicillin was obtained which melted at 130131°. This product was a hydrate, and it was shown that one molecule of benzylamine was present as a salt of the acidification of the penicillin and a benzylamine residue was present in the molecule. The composition of this product, C2g-

THE STRUCTURE OF BENZYLPENICILLIN TO DECEMBER 1943 H38N4O5S, corresponded with the formula Ci4H20N2O4S for the penicillin (A2-pentenylpenicillin). A corresponding benzylamine derivative was made from a penicillin concentrate obtained from the "832-fermentation" and was found to melt at 136-137°. The composition of this second benzyl­ amine derivative, C30H3SN4O6S (monohydrate), corresponded with the formula Ci6Hi8N2O4S for the penicillin (benzylpenicillin). It was assumed that the second molecule of benzylamine entered the penicillin molecule without the loss of water. These products aided the characterization of the two penicillins, the existence of which had already been shown. These products also offered promise in the determination of the structure of penicillin (Stodola, NRRL, Monthly Progress Report, 14, p. 5; 16, pp. 4, 5). On the basis of structures XVI and XVII for benzylpenicilloic acid, it appeared that structures XX and XXI would represent the benzylamine derivative of benzylpenicillin, and that degradation of the derivative with mercuric chloride should give the benzylamide of either acid XVIII or XIX and permit differentiation of the formulas (Merck, M.l, 8).

59

with 2,4-dinitrophenylhydrazine, and semicarbazide. A crystalline hydrazone which melted at 239-240°, and a crystalline semicarbazone which melted at 216-217° were obtained. The analyses of the 2,4-dinitrophenylhydrazone and the semi­ carbazone were in agreement with the formulas C24H22N6O6 and Ci9H2IN5O3, respectively. These formulas are those which were anticipated for either carbonyl compound XVIII or XIX. Another portion of the filtrate from the mercuric mercaptide was freed of mercuric ions and concentrated to dryness. The residue was recrystallized from methanol and gave an optically inactive compound which melted at 163-164° and had the composition C20H24N2O4. This compound was found to contain two methoxyl groups. Since the experimental conditions for the isolation of this substance were those for acetal and not ketal formation, the forma­ tion of this compound strengthened the belief that the aminomalonaldehydic structure (XVIII) was correct. Another sample of the benzylamine derivative was treated with mercuric chloride, diluted with dioxane, treated with Raney nickel catalyst, and then hydrogenated with a platinum catalyst. A crystalline compound was obtained S

CeH5CH2CONHCH-CH C(CH3)2 I I I C6H5CH2NHCO NH- -CHCO2H-NH2CH2C6H5 XX

S

/

C6H5CH2CONH-CH2-C C(CH3)2 /I C6H5CH2NH-CO NH- -CH-CO2H-NH2CH2C6H6 XXI

It was evident that the benzylamine derivative contained no labile carboxy group since a solution •of it ^n a mixture of ethanol and dilute sulfuric acid yielded no carbon dioxide when refluxed. The mild conditions for the formation of the benzylamine derivative seemed to preclude the possibility of the attachment of the labile carbonyl group between the two nitrogen atoms to form a ureide (Stodola, Wachtel, and Coghill, NRRL, unreported details). The addition of an ethereal solution of benzyl­ amine to an ethereal solution of benzylpenicillin caused partial separation of the crystalline benzyl­ amine salt of benzylpenicillin which melted at 100°. An ethereal or alcoholic solution of this -salt reacts with more benzylamine to form the characteristic derivative melting at 136-137° (Merck, Report for October 19JiS, p. 12). A solution of the benzylamine derivative of benzylpenicillin in aqueous methanol was mixed with a solution of mercuric chloride. The precip­ itated mercuric mercaptide of penicillamine was removed. Portions of the filtrate were treated

from the hydrogenation of the carbonyl degradation product which melted at 192-194° and had the composition Ci8H32N2O3. It was identical with a synthetic specimen of the cyclohexylmethylamide of cyclohexylacetyl-DL-serine (XXII). Therefore, C6HuCH2CONH-CH-CH2OH I CONHCH2C6HU XXII

structure XXIII is established for the carbonyl degradation product of the benzylamine derivative of benzylpenicillin, and structure XVIII is estab­ lished for the corresponding free acid. •*, This free acid (XVIII) was designated benzylpenaldic acid. It is now evident that the 2,4-dinitrophenylhydra­ zone and the semicarbazone have structures XXIV C6H5CH2CONH-CH-CHO I CONHCH2C6H5 XXIII

60

T H E STRUCTURE OF BENZYLPENICILLIN TO D E C E M B E R 1943

and XXV, and that the acetal has structure XXVI.

The racemic nature of the benzylamide of benzylpenaldic acid dimethyl acetal (XXVI) and the serine derivative (XXII) is apparently due to enolization of the carbonyl derivative (XXIII) as follows (Merck, M.l, 7-9).

The authentic specimen of the cyclohexylmethylamide of cyclohexylacetyl-DL-serine was synthesized

as follows. DL-Serine (XXVII) was converted into the phenylacetyl derivative (XXVIII) which was

methylated with diazomethane to give an oily ester (XXIX). The benzylamide of phenylacetyl-DLserine (XXX) was prepared from the ester by reaction with benzylamine and hydrogenated with a platinum catalyst to give the perhydro derivative

(XXII). The dimethoxy derivative (XXVI) was compared later and found to be identical with a synthetic specimen of the benzylamide of benzylpenaldic acid dimethyl acetal (XXVI) which waa prepared by the reaction between benzylamine and the methyl ester of benzylpenaldic acid dimethyl acetal (XXXI) (Merck, M.l, 8, 9).

A parallel degradation, which was carried out simultaneously with the degradation of the benzylamine derivative, was the degradation of the "methanol-inactivation product" of benzylpenicillin. When a solution of sodium benzylpenicillin in methanol was refluxed, the specific rotation of the solution decreased rapidly and became constant at about [a]o +140° for a 0.4% solution. The methanol-reaction product was biologically inactive. Distillation of the solvent left an amorphous product. However, treatment of the amorphous methanol-inactivation product with benzylamine in ethereal solution gave a crystalline salt which

melted at 136-138°. The composition, C24H31N30=,S, of this salt was in agreement with structure XXXII, which could be formulated similarly to the benzylamine derivative (XX). A solution of this methanol-inactivation product in water was treated stepwise with mercuric chloride and 2,4-dinitrophenylhydrazine. A crystalline dinitrophenylhydrazone, m.p. 183-181°, of the composition C18H17N5O7 was obtained. When another portion of the solution was treated with dimedone, a crystalline methone derivative, m.p. 157-158°, was obtained. An oily precipitate of a phenylhydrazone was also obtained which gave a pyrazolone, Ci 7 Iii S N 3 02, when it was heated at 100° in vacuo. A solution of the aldehydic degradation -product was freed of the mercuric mercaptide and then of mercuric ions and evaporated to dryness. Treatment of the residue with methanolic hydrogen chloride gave a crystalline acetal of m.p. about 94°. An aqueous solution of the aldehydic degradation product was diluted with dioxane and hydrogenated with a platinum catalyst. A crystalline product, m.p. 155-159°, which had the composition

THE STRUCTURE OF BENZYLPENICILLIN TO DECEMBER 1943 C11H19NO3 was obtained. This hydrogenated derivative of the aldehydic degradation product was identical with a synthetic specimen of cyclohexylacetyl-DL-alanine (XXXIII) and was not identical with a synthetic specimen of the cyclohexylacetylDL-serine (XXIV). It is evident that the ^-hydroxy group which was produced by hydrogenation of the formyl group underwent hydrogenolysis to the alanine derivative (XXXIII).

These data established structure XXXV for the aldehydic degradation product, i.e., methyl benzylpenaldate, and were in agreement with structure

61

product of sodium benzylpenicillin was prepared and characterized as a crystalline benzylamine salt in a manner similar to that described above (Squibb, S.l, 7; 5, 1). Isolation of the 2,4-dinitrophenylhydrazone derivative of free benzylpenaldic acid

(XXXIX) appears to be impossible because of the ease of decarboxylation of benzylpenaldic acid in acidic solutions (Squibb, S.12, 6). When the free acid of the benzylamine derivative of benzylpenicillin was dissolved in methanol, treated appropriately with mercuric chloride for the formation and precipitation of penicillamine, and then treated with 2,4-dinitrophenylhydrazine, a hydrazone of the expected composition, C24H22N 6 0 6 , was obtained (Stodola, Wachtel, and Coghill, NRRL, unreported details). a-Benzylamide of D-a-Benzylpenicilloic Acid, a-Methyl D-a-Benzylpenicilloate. After the establishment of structure XVIII for benzylpenaldic acid, structure XL for the free acid of the benzylamine derivative of benzylpenicillin was accepted immediately by many chemists on the penicillin

XXXII for the methanol-inactivation product of benzylpenicillin. The dinitrophenylhydrazone and the acetal of the aldehydic degradation product have structures XXXVI and XXXVII, respectively.

The dinitrophenylhydrazone was identical with a synthetic specimen of the hydrazone prepared from the sodium salt of methyl a-formyl-a-phenylacetamidoacetate XXXVIII. The latter compound was prepared from phenylacetylglycine methyl ester (XXXIX) and methyl formate (Merck, M.l, 9, 10, 12;6, 4).

The establishment of structure XXIII for the benzylamide of benzylpenaldic acid and structure XXXV for methyl benzylpenaldate was confirmed by the preparation and characterization of the 2,4dinitrophenylhydrazones of these two aldehydic degradation products. The methanol-inactivation

program. Further evidence in confirmation of this structure, which resulted from a comparison of "natural" and synthetic desthio derivatives, was obtained in 1944 and is described in Chapter XVIII. In accord with this later evidence and stereochemical considerations, this benzylamine derivative of benzylpenicillin became known as the a-benzylamide of D-a-benzylpenicilloic acid. Similarly, structure XLI was generally accepted

for the methanol-inactivation product of benzylpenicillin on the basis of structure XXXV for methyl benzylpenaldate. Evidence on the confirmation of structure XLI was also obtained in 1914 when three pairs of "natural" and synthetic a-alkyl N-benzoylD-a-benzylpenicilloates were found to be identical (Chapter X V I I I ) . The methanol-inactivation product was designated a-methyl D-a-benzylpenicilloate as a result of these later stereochemical studies.

62

THE STRUCTURE OF BENZYLPENICILLIN TO DECEMBER 1943

Quantitative acetylation of the benzylamine derivative of benzylpenicillin (XX) showed that two acetyl groups were bound, one by the benzyl­ amine of the salt group and the other by the >NH group of the thiazolidine ring. The acetylated product, which was known later to have structure XLII, did not give a precipitate with mercuric S CeH6CH2CONH-CH

C6H6CH2NHio

/\

CH

C(CH8)2

N

CH-CO2H

COCH3 XLII chloride in solution and 3-acetyl-4-thiazolidinecarboxylic acid gave no precipitate with this reagent. The acetylated product and 3-acetyl-4thiazolidinecarboxylic acid did not bind hydrogen chloride, and 4-thiazolidinecarboxylic acid did bind hydrogen chloride. This behavior of the acetylated product was recognized immediately to be compati­ ble with the possible presence of a weakly basic thiazolidine nucleus in the compound. (Stodola, Wachtel and Coghill, NRRL, unreported details.) Benzylpenillic Acid. When an aqueous solution of crystalline sodium benzylpenicillin was treated with one equivalent of hydrochloric acid, the ob­ served rotation of the selution began to increase immediately and became constant after standing overnight. Crystals appeared in the solution after about four hours. This crystalline product was identical in melting point and composition, Ci6Hi8N2O4S, with the benzylpenillic acid which had been previously prepared from amorphous benzylpenicil­ lin concentrates (Merck, Reportfor September IdJ t S, pp. 5-7). The potentiometric titration of benzylpenillic acid with alkali showed that the compound was dibasic and the midpoints of the titration occurred at pH 3.1-3.3 and pH 7.6-7.7. Polarographic analysis of benzylpenillic acid indicated the absence of a sulfhydryl group (Merck, M.S, 5,6). It was found that benzylpenillic acid yielded somewhat more than one mole of carbon dioxide when it was heated at 190-200° for about thirty minutes (Merck, Reportfor October 1943). A comparison was made of the action of mercuric chloride on benzylpenillic acid and benzylpenicillin. Cleavage of both of these compounds in methanol solution with mercuric chloride took place very rapidly at 25°. Cleavage of dimethyl penillate under comparable conditions took place more slowly. The mercuric mercaptide of penicillamine partially precipitated in the experiment with benzyl­ penicillin, but the solution remained clear in the experiment with benzylpenillic acid. It was found that the reaction of benzylpenillic acid with mer­ curic chloride in water solution was quite different

from the corresponding reaction with benzyl­ penicillin. Benzylpenillic acid was converted into benzylpenillamine (cf. following section) and carbon dioxide, whereas benzylpenicillin was converted into penicillamine, carbon dioxide, and phenylacetylaminoacetaldehyde (Merck, Reportfor October 1943, pp. 10-12). When it was recognized that benzylpenicilloic acid probably had structure XV, it was immediately realized that the known facts about benzylpenillic acid were in agreement with structure XLIII CO2H I CH S / \ / \ N CH C(CH3)2

Il

C6H6CH2C

I

N XLIII

I

CH-CO2H

(Merck, M.l, 12). The correctness of this struc­ ture was substantiated by later structural degrada­ tions and total synthesis as described in Chapter VI. Benzylpenillamine. The reaction of A2-pentenylpenillic acid with mercuric chloride in aqueous solution to form a new compound of the formula Ci3H20N2O2S was first described by British investi­ gators (Abraham, Chain, Baker, and Robinson, Pen.79). This product was named A2-pentenylpenillamine. When a solution of mercuric chloride was added to an aqueous solution of benzylpenillic acid, a mercuric mercaptide precipitated. Aeration of the suspension with nitrogen for several hours yielded 0.81 mole of carbon dioxide per mole of benzylpenillic acid. The precipitate was removed, suspended in water, treated with one equivalent of hydrochloric acid, and decomposed with hydrogen sulfide. The filtrate from the mercuric sulfide yielded a crystalline hydrochloride, m.p. 174°, [α]ο —70.7°, which had the composition Ci6Hi8N2O2S-HCl. A picrate of the base was also obtained which had the composition Ci6Hi8N2O2S-C6H3N8O7. This product, benzylpenillamine, was stable in 0.1 N sulfuric acid at the reflux tempera­ ture for two and one-half hours. Benzylpenillamine appeared to possess a free mercapto group since it gave a strong blue color with ferric chloride solution and an intense nitroprusside reaction. It also coupled in alkaline solution with diazotized sulfanilic acid to give an orange-red colored solution. The ultraviolet absorption spectrum showed end absorption with possibly a maximum at 2200 A, E m about 10,000 (Squibb, Report for September to October 1943, p. 8; S.l, 8). When benzylpenillic acid was dissolved in metha­ nol and treated with mercuric chloride, the rotation of the solution decreased markedly. No precipitate formed when the reaction was carried out in metha­ nol instead of water. After the mercuric ions were removed from the solution, crystalline benzyl-

THE STRUCTURE OF BENZYLPENICILLIN TO DECEMBER 1943 penillamine, Ci5Hi8N2O2S-HCl, was obtained by evaporation. In another experiment one equiva­ lent of carbon dioxide was collected quantitatively. Potentiometric titration of benzylpenillamine hydro­ chloride gave a neutral equivalent of 328; calc., 326 (Merck, Report for October 1943, pp. 7-8). The chemical and physical properties of benzyl­ penillamine were considered to be in agreement with structure XLIV for this degradation product (Merck, M.l, 13). Later studies on the structure and synthesis of benzylpenillamine substantiated CH HS / \ \ N CH C(CH3)2 C6H6CH2C-

NXLIV

-CH-CO2H

structure XLIV (Chapter VI). Benzylpenicillin. Structure XVI was generally accepted for benzylpenicilloic acid. The outstand­ ing supporting evidence for this structure was provided by the characterization of the benzylamide (XXIII) and the methyl ester (XXXV) of benzylpenaldic acid, which were obtained from the benzylamine derivative of benzylpenicillin and the methanol-inactivation product of benzylpenicillin. The formulation of structure XL for the benzylS C6H6CH2CONH-CH-CH (a)

XVI XL XLI

—CO HO— C6H6CH2HNCH3O-

NH

oxazolone structure (XLV) appeared to be a possible N

/\

C6H6CH2C

CH-CH

C(CH3)2

O

CO NH XLV

CH-CO2H

structure for benzylpenicillin. This structure was considered favorably, because it was known that oxazolones reacted with amines and alcohols as did benzylpenicillin. For example, 4-methyl-2phenyl-5 (4)-oxazolone (Mohr and Stroschein, Ber., 42, 2521 (1909); J. Prakt. Chem., (2) 81, 478 (1910)) reacted readily with aniline in ethereal solution as with ethanol to give the corresponding anilide or ethyl ester. Other formulas were considered and it was stated that "the four-membered lactam fused upon a five-membered heterocyclic ring (the /3-lactam structure, XLVI) appears unlikely unless the strain involved could account for the reactivity to methanol and benzylamine" (Merck, M.l, 10, 11). It was impossible to demonstrate by titration the presence of a weakly basic group in benzyl­ penicillin. A substance of structure XLV would appear to have a weakly basic group (Merck, M.8, 7). S

C(CH3)2

CH-CO2H

S

/\

CeH5CH2CONH-CH-CH

/\

C(CH3)2

CO-NXLVI

(β)

amine derivative and structure XLI for the methanol inactivation product also established the carboxy group (/3- in later nomenclature) of the penicillamine moiety as the free carboxy group of benzylpenicillin. The characterization of the sulfinic acid cor­ responding to the methyl ester of penicillamine from a reaction of mercuric chloride with methyl benzyl­ penicillin also showed that the carboxy group of the penicillamine moiety was free in benzylpenicillin (Squibb, S.5, 5). Benzylpenicilloic acid (XVI) itself was obtained as a solid disodium salt by adding one equivalent of sodium hydroxide to an aqueous solution of sodium benzylpenicillin, and evaporating the water after hydrolysis was complete (Merck, M.6, 4). It was also evident that it was the carboxyl group (a- in later nomenclature) of the penaldic acid moiety of benzylpenicilloic acid (XVI) which con­ stituted the "labile group" of benzylpenicillin. The structure of benzylpenicillin could be formu­ lated from the structure of benzylpenicilloic acid by the elimination of a molecule of water which involved the α-carboxy group. The thiazolidine-

63

-CH-CO2H

The thiazolidine-oxazolone structure (XLV) ap­ peared at the time to explain additional data which were being accumulated on the reactions of sodium benzylpenicillin with various reagents. However, it was recognized that "the possibility that there exist in penicillin unusual strained ring systems capable of undergoing facile rearrangements should not be entirely dismissed." Oxidation of sodium benzylpenicillin with ammoniacal silver oxide gave a crystalline sulfur-free product (Chapter VIII). A malonimide structure (XLVII) was tentatively con­ sidered for this oxidation product on the basis that it might be derived from groupings such as XLVIII S—

/ -CH-CH

—CH-CH or CO—N— XLVIII

I -C

S-

I N—

OH XLIX

/ C6H5CH2CONH-CH

CO \

CO XLVII

NH

64

THE STRUCTURE OF BENZYLPENICILLIN TO DECEMBER 1943

or XLIX in benzylpenicillin. However, structure XLVII was dismissed as improbable when the lack of data on malonimides in the literature was con­ sidered (Squibb, S.l, 8, 9,11, 13). The thiazolidine-oxazolone structure (XLV) was also considered "to be compatible with the majority of the data" by the cooperating laboratories in the midwestern area (Abbott, Lilly, Parke-Davis, and Upjohn) as given in a summary report (A.I, 4). Ai-PentenylpenicilIin and n-Amylpenicillin. In connection with studies on "penicillin C," "penillic acid G" and "penillamine G," and tjie differentia­ tion of these three compounds from "penicillin F," "penillic acid F," and "penillamine F," it was con­ cluded that "penicillin F" (A2-pentenylpenicillin) had the composition CHH2UN2O4S. It was also concluded that the sole difference between "peni­ cillin G" and "penicillin F" was that between phenylacetic acid moiety and an unknown aliphatic acid moiety of the composition C 5 H 9 CO 2 H. Deductions from the melting points of some new 2,4-dinitrophenylhydrazones of aliphatic acylaminoacetaldehydes when compared with data reported by British investigators (Catch, Cook, Hall, and Heilbron, Pen.83·, Abraham, Baker, Chain, and Robinson, Pen.91; Catch, Cook, Elvidge, Hall, and Heilbron, Pen.99) made it appear very probable that "dihydropenicillin F" had a CHs(CH2)4gro'up in place of the C6H5CH2- group in "penicillin G." Similar deductions made it probable that "penicillin F" had either a CH3CH=CHCH2CH.r group or a CH3CH2CH=CHCH2-group (Merck, M.2). The chemistry of A2-pentenyl- and n-amylpenicillin is reviewed fully in Chapters II and III. E X P E R I M E N T A L

2

Experiments on Benzylpenicillin Concentrates. ACID HYDROLYSIS

OP

CONCENTRATES

OF

BENZYLPENICILLIN

(Merck, Report for November 194% to February 1943, pp. 5, 6; and unreported details). A solution of 424 mg. of a con­ centrate of benzylpenicillin, 1,050 units/mg., in 20 cc. of 4 N sulfuric acid and 20 co. of ethanol was heated at the reflux temperature while a stream of nitrogen was passed through the solution. The evolved gases were passed through absorption tubes containing standard barium hydroxide solution. Evolution of carbon dioxide was com­ plete within about thirty minutes. The heating was con­ tinued for two hours. The titration showed that 31 mg. of carbon dioxide was evolved. The solution was steam dis­ tilled and yielded volatile acid equivalent to 2.30 cc. of 0.1019 N sodium hydroxide. The aqueous solution was cooled and extracted with ether. Evaporation of the solvent yielded 95 mg. of a crystalline residue. Sublimation of the residue at 30-40° in vacuo gave 55 mg. of sublimate and 27 mg. of residue. Crystallization of the residue from benzene-petroleum ether yielded a product which melted at 126-128°. The melting point was unchanged when this product was mixed with authentic α-furoic acid. The sublimate was recrystallized from petroleum ether and the * During 1942-1943 it was the custom of the Merck and Squibb Laboratories to submit summary reports rather than reports with full experimental details. The inclusion of experimental details in reports began in 1944 during the overall collaboration under the auspices of the Office of Scientific Research and Development. Consequently, it has been necessary to include in this chapter certain unreported details for work summarized during the 1942-1943 period.

product melted at 75-76.5°. No depression of the melting point was observed when this product was mixed with authentic phenylacetic acid. The ultra-violet absorption spectrum of the crude mixture of acids indicated that approximately equimolar amounts of a-furoic acid and phenylacetic acid were present. After ether extraction the aqueous hydrolysis solution was made alkaline with barium hydroxide, freed of barium sulfate, and distilled. A small amount of ammonia was obtained and converted to the chloroplatinate for identifi­ cation. The aqueous solution was then quantitatively freed of barium ions with sulfuric acid. The solution gave a positive ninhydrin reaction. An amino-nitrogen determi­ nation showed the presence of 16.6 mg. of amino-nitrogen. Evaporation to dryness left a partially crystalline residue which was soluble in ethanol. This residue gave a blue color with ferric chloride solution, a positive ninhydrin test, and a precipitate with phosphotungstic acid solution. Benzoylation of 38 mg. of the residue by the SchottenBaumann procedure gave 31 mg. of a crystalline product. Recrystallization from water yielded crystals which melted at 198-200°. BENZYLPENILLIC ACID FROM BENZYLPENICILLIN CONCEN­ TRATES (Merck, Reportfor April to July 1943, pp. 4, 5; and

unreported data). A solution of 2.083 g. of a concentrate of barium benzylpenicillin, activity 472 units/mg., in 200 cc. of water was acidified to pH 2.05 with dilute sulfuric acid and kept at 37° for two hours. The barium sulfate, 880 mg., was removed by filtration and the filtrate was extracted several times with ether in a separatory funnel. The ether extract was evaporated to dryness in vacuo and left an amorphous residue; weight, 893 mg. The aqueous solution was frozen and evaporated from the frozen state to a volume of about 5 cc. On allowing this solution to stand for a short time, crystals were deposited. The crystals weighed 131 mg. and melted at 179-181°. A sample of such material weighing 113 mg. was dissolved in methanol and recrystal­ lized by the addition of acetone. After four recrystallizations there was obtained 38 mg. of material which melted constantly at 186-187° (dec.); [CHSO2C (CH3)2— group. The Zerewitinoff determination on benzylpenicillin methyl ester itself was not entirely satisfactory, because of apparent decomposition of the com­ pound in anisole at 95° (Merck, M.12b, 2; 62, 7). The exchange of hydrogen for deuterium, when sodium benzylpenicillin was equilibrated with heavy water, showed the presence of one active hydrogen atom and was a satisfactory determina­ tion (Cornell Bioch., D.ll, 1). The methyl ester of benzylpenicillin sulfone (ΧΧΠ) reacted rapidly with benzylamine in chloro­ form solution at 25° and the reaction was almost completed in two hours as evidenced by the decrease in the optical rotation. A crystalline product was obtained from this reaction which was identified as the benzylamide of a-phenylacetamidoj8-benzylaminoacrylic acid (XXX). This benzylamide (XXX) formed solvates which show consider­ able variation in the melting point depending upon the method which is used. After recrystallization from methanol or ethanol, the benzylamide2 (XXX) melted at 66-67° or 118-119°, respectively, and -C==CHOC2H6 HBrNC6H6CH2C

C 6 H 5 CH 2 NH 2

CO

ester with benzylamine to give the a-benzylamide of D-a-benzylpenicilloic acid (XXXIII) had been established (Chapter XVIII), it was not to be S / V C(CHs)2 CeH6CH2CONHCH-CH IC( C6H6CH2NHCO

O

XXXII

hco2H

O

O \ /

S

/\

C6H6CH2CONHCH-CH C6H6CH2NHCO

C(CH3)2

NH

CH-CO2CH3

XXXIV

expected that the reaction of benzylamine with the sulfone of benzylpenicillin methyl ester (ΧΧΠ) would produce the corresponding penicilloate sul­ fone (XXXIV) as a stable compound. The in­ herent instability of a compound of structure XXXTV had been realized (Merck, M.56, 1; cf. α-aminosulfone derivatives, Meyer, J. prakt. Chem., (2) 68, 167 (1901); KohIer and Reimer, Am. Chem. J., 31, 163 (1904)). If the penicilloate sulfone (XXXIV) were the first unstable intermediate formed, it would be ex­ pected to rearrange into a penicillamine derivative XXXV or (less probably) the sulfone XXXVI. Further reaction with benzylamine would then proC6H5CH2NH3+

SO2-

CeH6CH2CONHC=CH I I C6H6CH2NHCO NH-

\/

-A

NH-

XXXIII

C(CH3)2 -in-CO2CH3

XXXV

CeH6CH2CONHC=CH CeH6CH2NHCO

Brady's

XXX

CeH6CH2CONHCH-CH CeH6CH2NHCO

O

NHCH2CeH6 Reagent

N-NHC6H3(NO2)2

O \ / S

/\C(CH )

CeH6CH2CONHC=CH I I C6H6CH2NHCO NH2

3 2

I CH-CO2CH3

XXXI

XXXVI

contained alcohol of crystallization correspond­ ing to the solvent used. Treatment of this benzyl­ amide (XXX) with Brady's reagent yielded the 2,4-dinitrophenylhydrazone of the benzylamide of benzylpenaldic acid (XXXI). The benzylamide (XXX) was also obtained by the reaction of ben­ zylamine with 2-benzyl-4-ethoxymethylene-5(4)oxazolone hydrobromide (XXXII) (Merck, M.56, 7; 59, 14-15; 12a, 8). Although the reaction of benzylpenicillin methyl

duce the insoluble benzylamide of a-phenylacetamido-/3-benzylaminoacrylic acid (XXX) (Merck, M.59, 2-3). A solution of benzylpenicillin methyl ester sulfone (XXII) in 1:1 methanol-chloroform solution or in dioxane containing 1 % N-ethylpiperidine was unchanged after sixteen hours and the sulfone was recovered. However, when N-ethylpiperidine was added to the solution of the sulfone in methanolchloroform, reaction took place readily. It ap­ peared that the unstable penicilloate sulfone (XXXVII) was formed which rearranged into the

* The anhydrous form of this compound melted at 132-134° (Upjohn,

u.e, 5).

INVESTIGATIONS ON BENZYLPENICILLIN, 1944-1945 penicillaminic acid derivative XXXVIII. The latter compound apparently was responsible for the ultraXXII

155

in glacial acetic acid solution with hydrogen peroxide (Merck, M.64, 8-9). The reaction of oxalyl chloride with methyl phenaceturate, N-methylphenylacetamide, and N-benzylphenylacetamide in ether solution gave oxazolidine-4,5-diones which have structures XLI, XLIa and XLIb (Abbott, A.18, 1; Chapter VIII). It was also found that xylene could be employed as

the solvent for the preparation of the oxazolidinedione (XLI), and that oxalyl chloride reacted with methyl /3-benzoxy-a-phenylacetamidoacrylate, methyl benzylpenaldate dimethyl acetal, methyl (3-methoxy-a-pheny lacetamidoacrylate, and dimethyl N-benzoyl-D-7-benzylpenicilloate to give the new oxazolidine-diones XLII-XLV, respectively. Xylene was used as the solvent for the preparation of these

violet absorption band at 2,775 A of the product in absolute ethanol solution. The product was readily hydrolyzed by water, and when treated with Brady's reagent in methanol, yielded the 2,4dinitrophenylhydrazone (XL) of methyl benzylpenaldate (XXXIX) (Merck, M.59, 11-12). When toluene or pyridine solutions of the methyl ester of benzylpenicillin sulfone were heated at 100° for thirty to sixty minutes, an acidic substance was formed which showed an ultraviolet absorption band at 3,180-3,190 A, characteristic of a benzylpenicillenate derivative (Merck, M.59, 13). The methyl ester of benzylpenicillin sulfone was much more stable than the methyl ester of benzylpenicillin in a mixture of 0.2 N hydrochloric acid and dioxane or in glacial acetic acid. The sulfone was recovered from the hydrochloric acid-dioxane solution after sixty hours at 25° and from glacial acetic acid solution after nineteen hours at 25° (Merck, M.59, 13-14). A treatment of the methyl ester of benzylpenicillin sulfone in glacial acetic acid solution with ozone or 30% hydrogen peroxide resulted in an 86-95% recovery of the starting material (Merck, M.68, 7). It was not found possible to obtain a crystalline product corresponding to /3-methvl N-acetyl-D-abenzylpenicilloate sulfone when benzylpenicillin methyl ester was inactivated in glacial acetic acid and then the product was oxidized with hydrogen peroxide. Neither was it possible to prepare the methyl ester of benzylpenicillin sulfone by oxidation

new oxazolidine-diones and the yields were 60-80 per cent. The dione (XLV) from the D-7-benzyl-

penicilloate was a pale yellow compound which melted at 203-204°. These oxazolidine-diones showed ultraviolet absorption maxima of E M 9,600-12,800 at 3,475-3,525 A in tetrachloroethane solution (Merck, M.60, 14; 65, 5, 6).

156

INVESTIGATIONS ON BENZYLPENICILLIN, 1944-1945

The methyl ester of benzylpenicillin sulfone (XXII) was allowed to react with oxalyl chloride in xylene solution at 25° for forty minutes. The product showed an ultraviolet absorption band at 3,250 A, which is nat characteristic of the oxazolidine-diones, but which could be due to the presence of the benzylpenicillenate XLVL Upon the as-

lethal doses of Diplococcus pneumoniae Type I, #37. One hundred per cent survival was observed when a single dose of 0.3 mg. of the sulfone or 300' units of sodium benzylpenicillin was injected subcutaneously. Complete survival was observed on oral administration of 5 mg. of the sulfone or 1,000 units of sodium benzylpenicillin (Merck, M.59, 15-20). Methyl Ester of Benzylpenicillin Sulfoxide. The HO2S oxidation of various thiazolidines with sodium \C(CH ) metaperiodate showed that rupture of the thiazoliN -C==CH 3 2 dine nucleus occurred for those compounds having Il I I I an unsubstituted >NH group, whereas N-acylC6H5CH2C CO NH CHCO2CH3 thiazolidines readily yielded sulfoxides (Sykes and Todd, CPS.526). This behavior of thiazolidines to oxidation by sodium metaperiodate paralleled XLVI the behavior of thiazolidines to oxidation by hydro­ sumption that the absorption is accounted for by gen peroxide. The latter studies originated with the benzylpenicillenate XLVI which has a sulfinic Ratner and Clarke (J. Am. Chem. Soc., 59, 200 acid group and which would be quantitatively (1937)) who prepared the sulfoxide and the sulfone similar to methyl benzylpenicillenate in absorption of 3-acetyl-4-thiazolidinecarboxylic acid and the intensity, the extinction indicated that about 50% sulfone of acetylthiazolidine by oxidation under of the sulfone was transformed into this product. various conditions. These studies were extended Since about 50 % of the sulfone was recovered from to benzylpenicilloic acid derivatives with similar the reaction, not more than a few per cent of the results, sulfones being obtained by oxidation with sulfone could have reacted with oxalyl chloride to potassium permanganate or other oxidizing agents give the oxazolidine-dione derivative (Merck, M.65, (Merck, M.S9, 4). 6). It was found (CPS.526) that 3-acetyl-5,5-diThe methyl ester of benzylpenicillin sulfone methyl-2-phenyl-4-thiazolidinecarboxylic acid was reacts at a faster rate with benzylamine and with oxidized by sodium metaperiodate to give the cor­ aqueous alkali than does the methyl ester of benzyl­ responding sulfoxide. The same reaction took penicillin (Merck, M.61, 6, 7). place with hydrogen peroxide. Numerous model The ultraviolet and'infrared absorption spectra of compounds including substances with a free —SH the methyl ester of benzylpenicillin and its sulfone group and substances with an N-unsubstituted were found to be similar. The infrared absorption thiazolidine nucleus were subjected to oxidation by spectrum of N-methylphenylacetamide was com­ periodate, iodate, or iodine, and it appeared that pared with that of N-methylcyclohexylacetamide, the end product was a thiolsulfonate or a sulfonic and the infrared absorption spectrum of benzyl­ acid. Oxidation of benzylpenicillin by sodium penicillin sulfone methyl ester was compared with metaperiodate was carried out in the hope of secur­ that of hexahydrobenzylpenicillin sulfone methyl ing clarification of the structural problems. How­ ester. Both pairs of compounds showed strikingly ever, attempts to isolate the oxidation product from similar absorption spectra which clearly indicate benzylpenicillin were not successful. Application that the contribution of the phenyl group in these of the conditions (Merck, M.56, 7) used to make compounds is not significant. The presence of benzylpenicillin sulfone methyl ester to oxidation strong bands at 6.55 μ in the spectra of both amides experiments with sodium metaperiodate readily and of the strong bands at 6.66 μ in the spectra of yielded a crystalline sulfoxide of the methyl ester of both sulfones is significant. These results provided benzylpenicillin (XLVII) (CPS.526, 5). This sulf­ evidence that the band at 6.66 μ in the spectrum oxide melted at 123° and had a composition which of benzylpenicillin does not arise from the substi­ tuted phenyl group in benzylpenicillin. The re­ O sults also support the assumption (Shell, Sh.2, 5) that the band at 6.66 μ arises from a monosubstisT tuted amide group (Merck, M.61, 3; 64, 5, 6). /\ CeHsCH2CONHCH-CH · C(CH3)2 The extremely low solubility of the methyl ester I I I of benzylpenicillin sulfone in water did not permit CO-N CHCO2CH3 a satisfactory test of its activity in vitro. When the XLVII sulfone ester was treated first with a 0.1 N sodium hydroxide-pyridine mixture at about zero for one minute, diluted with buffer and tested, the solution corresponded to the benzylpenicillin sulfoxide showed 40 U/mg. The sulfone was tested for methyl ester hemihydrate. It was biologically in­ efficacy in vivo in mice which had been injected with active when tested under conditions where benzyl-

INVESTIGATIONS ON BENZYLPENICILLIN, 1944-1945 penicillin methyl ester showed an activity of 70 U/mg. The formation of a sulfoxide from benzylpenicillin methyl ester in this way is an additional piece of evidence showing the closer resemblance of benzylpenicillin to N-acylthiazolidines than to simple thiazolidines containing a free > NH group. The reactions of the sulfoxides were then studied (Sykes and Todd, CPS.677, 1). 3-Acetyl-5,5dimethyl-2-phenyl-4-thiazolidinecarboxylic acid (XLVIII) is stable to alkali but is readily decom­ posed by mineral acid to give benzaldehyde and S C6H5CH I CH3CON

which had previously been obtained from the benzylamine degradation of benzylpenicillin sulfone methyl ester (Merck, M.56, 7; 59, 2, 14). With Brady's reagent this product gave the 2,4-dinitrophenylhydrazone of the benzylamide of benzylpenaldic acid (XXXI) (CPS.677, 2; M.59, 14). C6H6CH2CONH-C==CH I I C6H6CH2NHCO NHCH2C6H6 XXX

C6H6CH2CONH-CH

CH

C6H6CH2NHCO

/\

C(CH3)2 I CHCO2H

XLVIII

penicillamine. The corresponding sulfoxide, on the other hand, is stable to acid, but treatment with warm alkali yields benzaldehyde and a-acetamido-/3,/3-dimethylacrylic acid, with elimination of hydrogen sulfide. The corresponding sulfone re­ sembles the sulfoxide in its instability to alkali, hydrogen sulfide being eliminated, but it also breaks up readily with acid to give benzaldehyde and a resinous acid. Benzylpenicillin sulfoxide methyl ester shows remarkable stability towards acids (Merck, M.59, 21). This sulfoxide is stable in methanol, but when treated with methanolic hydro­ gen chloride it undergoes slow decomposition yield­ ing phenylacetamidoacetaldehyde. The sulfoxide is less stable to acids than simple N-acylthiazoli­ dines, but is more stable than benzylpenicillin methyl ester. Benzylpenicillin sulfoxide methyl ester was ap­ parently hydrolyzed to benzylpenicillin sulfoxide (XLIX) when allowed to stand with sodium hydroxO T

s

/\

CeH6CH2CONHCH-CH I I CO-N

C(CH3)2 I CH-CO2H

XLIX

ide in aqueous methanol or dioxane, but underwent more extensive change on refluxing with alkali. The product in the latter case was a pale yellow resin which could not be crystallized, but which when treated with benzylamine yielded a product which appeared to be the benzylamine salt of benzylpenicilloic acid sulfoxide (CPS.677, 2). The reaction of benzylamine with benzylpenicillin sulfoxide methyl ester at room temperature yielded a sulfur-free product which had the properties of the benzylamide of /3-benzylamino-a-phenylacetamidoacrylic acid (XXX) (CPS.677), a substance

157

N-NC6H3(NO2)2

XXXI

The γ-lactam of a-benzamido-4-carboxy-5,5-dimethyl-2-thiazolidinepropionic acid (L) (Cornell Bioch., D.26; CPS.677, 5; Chapter XXVII) was readily oxidized with sodium metaperiodate to the CH2

S

/ \ /\

C6H6CONH-CH I CO

CH I N

C(CH3)2 I CH-CO2H

L

corresponding sulfoxide. This sulfoxide was fairly stable to cold alkali but decomposed on heating the alkaline solution to give the aldehydo-acid LI which was isolated as a 2,4-dinitrophenylhydrazone, and a-amino-/3,/3-dimethylacrylic acid which was CH2CHO I C6H6CONH-CH I COOH LI

isolated in the form of its acetyl derivative. Broadly speaking, the alkaline degradation of the sulfoxide of the γ-lactam of a-benzamido-4-carboxy-5,5-dimethyl-2-thiazolidinepropionic acid proceeded in a manner analogous to that of N-acylthiazolidine sulfoxides, except that the large acyl residue was removed from the nitrogen of the a-amino-β,βdimethylacrylic acid, presumably by a secondary reaction; it differed markedly from the correspond­ ing degradation of benzylpenicillin sulfoxide methyl ester. Although alkaline treatment of benzylpenicillin sulfoxide methyl ester appeared to yield benzylpeni­ cilloic acid sulfoxide, the latter compound could not be prepared by oxidation of benzylpenicilloic acid with periodate (Sykes and Todd, CPS.526). The degradation results are compatible with a 0-lactam structure if it is assumed that the course of alka­ line fission is determined by the stability of the >N—CO— linkage. If attack on the sulfinyl group, with consequent ring fission, occurs before hydrolytic removal of the N-acyl group, then

158

INVESTIGATIONS ON BENZYLPENICILLIN, 1944-1945

degradation to a derivative of «-amino-/?,/3-dimethylacrylic acid will occur; if the contrary is the case, then the product is simply the alkali-stable thiazolidine sulfoxide containing a free >NH group (Sykes and Todd, CPS.677, 3). Benzylpenillonic Acid and Methyl Benzylpenillonate. During a study of the stability of benzylpenicillin methyl ester (LII) it was discovered that heating a solution of the ester in p-cymene at 170° caused complete inactivation and gave rise to a new isomeric compound. Phenaceturic acid (LIII) was also formed as a result of degradation (Merck, M M , 1). S CeH5CH2CONH CH-CH

J i CO-N

sxC(CH

3)2

i CH-CO CH 2

3

LII C6H6CH2CONHCH2COOH + C17H20N2O4S LIII The new isomeric compound was designated methyl benzylpenillonate. It is neutral, melts at 152-154°, and shows [a]D22 +318° (in methanol) and +298° (in chloroform). Benzylpenicillin methyl ester melts at 97-98° and shows [a]D22 +276° (in methanol) and +175° (in chloroform). A molecular weight determination on methyl benzylpenillonate gave a value of 313 (calc., 348) (Merck, M.S6, 1). It was apparent that this new compound is a molec­ ular rearrangement product of benzylpenicillin. It was subsequently found that methyl benzyl­ penillonate was formed from benzylpenicillin methyl ester when a solution of the latter in xylene was heated at 140° for thirty minutes. The rearrange­ ment was also effected by refluxing for three hours a toluene solution of benzylpenicillin methyl ester to which had been added a crystal or two of iodine. The yield of once-recrystallized methyl benzylpenil­ lonate from this latter procedure was about 33% (Merck, M.86, 1). The formation of phenaceturic acid was negligible when toluene was used as solvent for the rearrangement reaction. Methyl benzylpenillonate showed the presence of one active hydrogen atom by the Zerewitinoff deter­ mination. Under the same conditions, N-methylphenylacetamide showed one, methyl phenaceturate showed one, and phenylacetyl-DL-alanyl-D-valine methyl ester showed two active hydrogen atoms (Merck, M.86, 1; 52, 7). In the ultraviolet, methyl benzylpenillonate showed maxima characteristic of phenyl-group absorption with Em 470 at 2,575 A (benzylpenicillin methyl ester showed Em 250 at 2,575 A) and end absorption about the same as for benzylpenicillin methyl ester (Merck, M.52, 7). In the infrared, methyl benzylpenillonate showed bands at 2.65 μ (weak), 5.81 μ, 6.08 μ and 6.66 μ (weak) (Shell,

Sh.4, Fig. 30; Merck, MSJ i ., 4). It was reported later (Shell, Sh.7, 79) that a 20% solution of methyl

benzylpenillonate in chloroform showed a weak band at 2.87 μ. Saponification of methyl benzylpenillonate with methanolic sodium hydroxide gave benzylpenillonic acid, m.p. 185-187°, [a]D +342°. Potentiometric titration of the acid showed it was monobasic and gave a neutral equivalent of 342 (calc. 342) with ρKa 3.0. The ultraviolet absorption spectrum of the acid was about the same as that of the methyl ester (Merck, M.86, 3). The sodium salt of benzylpenillonic acid was prepared by neutralizing a sample of the free acid with the calculated amount of standard sodium hydroxide. By freeze-drying the solution, the salt was' isolated as an amorphous white powder, [a]D +306° (Merck, M.57, 9). The formation of phenaceturic acid (LIII) during the rearrangement reaction, when p-cymene or xylene was used as the solvent, was evidently due to thermal decomposition of methyl benzylpenillonate. When methyl benzylpenillonate was heated in p-cymene solution at 150-170°, phenaceturic acid was produced. No phenaceturic acid was formed if a little pyridine was present in the solution, and the methyl benzylpenillonate was recovered (Merck, M 46, 19). Alkaline hydrolysis of methyl benzylpenillonate yielded phenylacetic acid. The water-soluble por­ tion of the hydrolysis mixture gave a blue color with ferric chloride and a precipitate with mercuric chloride. Decomposition of the mercuric chloride precipitate by means of hydrogen sulfide, and evaporation of the aqueous filtrate to dryness, yielded a crystalline hydrochloride. This crystal­ line product gave, a blue color with ferric chloride solution and was identified as penicillamine (Merck, M.39, 2). Acid hydrolysis of methyl benzylpenillonate with hot methanolic hydrochloric acid for four and onehalf hours caused nearly complete loss of the optical rotation. A positive ninhydrin test and a blue color with ferric chloride were given by the hydrolysis solution which indicated the presence of penicill­ amine. Ether extraction of the solution removed methyl phenylacetate which was saponified to phenylacetic acid. Methyl benzylpenillonate was not hydrolyzed by 0.02 N hydrochloric acid in 50% methanol at 75° for two and one-half hours (Merck, M.39, 3; Ifi, 18). Hydrolysis of methyl benzyl­ penillonate was then carried out by dissolving it in a mixture of equal parts of methanol and concentrated hydrochloric acid and refluxing the solution for two hours. Evaporation of the solution left a crystal­ line residue from which ether extracted crystalline methyl phenaceturate, m.p. 87-89° (Merck, M.Jfi, 18). Hydrolysis of methyl benzylpenillonate with hot 0.1 N hydrochloric acid in 60% dioxane for four

INVESTIGATIONS ON BENZYLPENICILLIN, 1944-1945 and one-half hours caused a decrease in optical rotation. The solution was continuously extracted with petroleum ether which removed phenylacetic acid and some starting material. The aqueous residual solution was evaporated to a glassy residue which gave a blue color with ferric chloride solution, a positive Folin-Denis test for the sulfhydryl-group, and a positive ninhydrin test. A solution of this residue in aqueous sodium bicarbonate slowly deposited crystalline material. This product melted above 200° and was not identified (Merck, M.50, 9; 52, 6). Methyl benzylpenillonate did not react with benzylamine in xylene solution at the reflux tem­ perature. However, after methyl benzylpenillonate was heated with benzylamine at 150-170° for seventy-five minutes, there was obtained phenaceturic acid benzylamide, m.p. 179-180° (Merck, M.4S, 4). Comparison of the potentiometric titration data of benzylpenillonic acid and N-acylpenicillamine derivatives showed that benzylpenillonic acid has the partial structure LIV. The hydrolytic deS C10H10NO < -CO-NLIV

C(CH3)2 I -CHCO2H

gradations and the reactions of benzylpenillonic acid with methanol and with benzylamine showed that phenaceturic acid or its derivatives and penicillamine are key degradation products. Fur­ ther structural information was gained from the results of degradations of the hydrogenolysis products of methyl benzylpenillonate and benzyl­ penillonic acid. Hydrogenolysis of methyl benzylpenillonate with Raney nickel catalyst in methanol gave crystalline methyl desthiobenzylpenillonate, m.p. 104-106°. The analytical data on this substance were in agreement with the formula C1JH22N2Oi, and showed that the hydrogenolysis had resulted in the removal of the sulfur atom from methyl benzyl­ penillonate and its replacement with two hydrogen atoms (Merck, M.4-6, 20; 50, 7-8). In the infrared, methyl desthiobenzylpenillonate showed bands at 5.84 and 6.04 μ, but did not show any band in the 2.5-3.3 μ region (Merck, M.64, 3; 68, compound 53; 70, 3). Thus methyl desthio­ benzylpenillonate does not have an NH group. Desthiobenzylpenillonic acid was obtained by saponification of methyl desthiobenzylpenillonate with methanolic sodium hydroxide. The acid crystallized from acetone, melted at 211-213° and showed [ct]D +22° (Merck, M.50, 9). Desthiobenzylpenillonic acid was also prepared by hydro­ genolysis of an aqueous solution of sodium benzyl­ penillonate with Raney nickel catalyst. When the

159

acid was recrystallized by careful acidification of an aqueous solution of its sodium salt, there was ob­ tained desthiobenzylpenillonic acid hemi-hydrate, m.p. 194-195°. Potentiometric titration of the hemi-hydrate gave a neutral equivalent of 314 (calc., 313). Recrystallization of the hemi-hydrate from acetone gave the anhydrous acid, m.p.· 211-213° (Merck, M.50, 8, 9). Acid hydrolysis of desthiobenzylpenillonic acid with hydrochloric acid in aqueous dioxane for seventeen hours "yielded phenylacetic acid (Merck, M.52, 6). Methyl desthiobenzylpenillonate was heated at 150-190° with benzylamine, and phenyl­ acetic acid benzylamide, m.p. 121-122°, was ob­ tained (Merck, M.52, 6). Synthetic phenylacetic acid benzylamide was prepared for comparison by the reaction of phenylacetyl chloride with benzyl­ amine in pyridine solution. The synthetic product melted at 122-123° (Merck, M.52, 7). Hydrolysis of desthiobenzylpenillonic acid was then carried out with a 3:2 mixture of concentrated hydrochloric acid and water in a sealed tube at 120° for six hours. The hydrolyzate was diluted with water and freed of phenylacetic acid by ether extraction. The residual aqueous solution was then evaporated to a partially crystalline residue. Extraction of the residue with acetone left white crystals. The acetone extract on evaporation yielded a partially crystalline residue. From a portion of the acetone-insoluble material, there was obtained by treatment with y>(p-hydroxyphenylazo)-benzenesulfonic acid, a crystalline salt which melted at 235-240° (dec.). This salt was shown to be identical with the glycine salt of p-(p-hydroxyphenylazo)-benzenesulfonic acid (LV). N=

=N

/\ OH .

SO3H-NH2CH2COOH LV

C6H5CONHCH2CO2H LVI

Benzoylation of another portion of the acetoneinsoluble material by the Schotten-Baumann pro­ cedure yielded hippuric acid (LVI). Benzoylation of the acetone-soluble material by the SchottenBaumann method gave a good yield of N-benzoylDL-valine (LVII) (Merck, M.6Jh 4). CH(CH3)2 C6H5CONHCH-CO2H LVII Desthiobenzylpenillonic acid was hydrolyzed at 120° with hydrochloric acid, as described above,

160

INVESTIGATIONS ON BENZYLPENICILLIN, 1944-1945

and then the hydrolyzate was diluted with water, buffered to a pH of 4.5 and treated with dimedone for reaction with formaldehyde (Yoe and.Reid, Ind. Eng. Chem., Anal. Ed., 13, 238 (1941)). There was obtained a yield of the formaldehyde-dimedone condensation product LVIII corresponding to about 90% of .the amount calculated for one molar H2 H2 (CH3). /\=0 O= (CH3)2

-CH2

H. O

Y

H2

O

LVIII

equivalent per mole of desthiobenzylpenillonic acid (Merck, M.68, 5). The identification of phenylacetic acid, glycine, valine, and formaldehyde as hydrolysis products of desthiobenzylpenillonic acid and the other abovementioned data on benzylpenillonic acid are in agreement with structure LIX for desthiobenzylC6H6CH2CO ϊί

/\

CH2 I CO

CH2 CH(CH3)2 ι ι -N CHCO2H LIX

penillonic acid. Since benzylpenillonic acid differs from desthiobenzylpenillonic acid only in the re­ placement of the sulfur atom by two hydrogen atoms, structure LX corresponds to benzylpenilC6H6CH2CO N

S

CH2

CH

C(CH3)2

CO-

-NLX

-CHCO2H

Ionic acid (Merck, M.68, 2). During the course of the elucidation of the struc­ ture of benzylpenillonic acid, the synthesis of methyl benzylpenillonate was achieved. The syn­ thesis was effected by heating at the reflux tem­ perature a solution of methyl benzylpenicillenate (LXI) in toluene with addition of a few crystals of HS

-C=

N-

C6H6CH2C

CO

=CH NH-

O LXI iodine (Merck, M.lfi, 18).

\

C(CH3)2

-CHCO2CH3

Both "natural" and synthetic methyl benzyl­ penicillenate were dissolved in toluene containing a very small amount of iodine. The solutions were refluxed for three hours, then evaporated to dryness. Solutions of the residues were seeded with "natural" methyl benzylpenillonate, and methyl benzylpenil­ lonate crystallized. The synthetic product, m.p. 151-152°, [a]D +322 to +326°, showed no depres­ sion of the melting point when mixed with methyl benzylpenillonate obtained from benzylpenicillin methyl ester (Merck, M.lfi, 18, 20; 50, 6).3 The use of a minute amount of sulfuric acid for the synthesis of methyl benzylpenillonate was found to give approximately the same yields as when iodine was employed. No significant formation of methyl benzylpenillonate was observed when a toluene solution of methyl benzylpenicillenate with no iodine present was heated for three and one-half hours. The rotation was unchanged (Merck, M.50, 6). Saponification of synthetic methyl benzylpenil­ lonate with methanolic sodium hydroxide gave excellent yields of benzylpenillonic acid, m.p. 185186° (Merck, M.50, 7). A study of the mechanism of the rearrangement of benzylpenicillin methyl ester to methyl benzylpenil­ lonate was carried out by following the changes in the optical rotation, ultraviolet absorption, and biological activity of a refluxing solution of benzyl­ penicillin methyl ester in toluene containing a minute amount of iodine. The rotation decreased to a minimum value of about [«]D25 +137° after three minutes, then rose to a value of [a]D25 +193° after six minutes, and became constant at [a]D26 +210° after about twelve minutes. In the ultra­ violet, a maximum appeared at once at 3,200 A which rose in intensity to a peak value of EM 1,507 after six minutes of the heating period. The EM at 3,200 A decreased to about 674 after twelve minutes, and then slowly decreased. The biological activity dropped rapidly; no detectable activity re­ mained after about nine minutes. The immediate drop in rotation and loss of biological activity coin­ ciding with the rise in intensity of the band at 3,200 A indicate intermediate formation of methyl benzylpenicillenate. Methyl benzylpenilloi^ate could be isolated from aliquots removed after about twelve minutes of the heating period (Merck, M.lfi, 20). Benzylpenicillin methyl ester was heated for six hours in a sublimation apparatus at 130-135° at a pressure of 1-3 microns. A light yellow gum was slowly deposited on the condensing surface which 8 Jansen and Robinson (private communication) have effected a direct synthesis of methyl benzylpenillonate (analysis, undepressed mixed m.p., and comparison of properties) by reaction of 2-benzyl-5(4)oxazolone and methyl 5,5-dimethylthiazoline-4-carboxylate in cold ethereal solution. This indicates a possible mechanism of the rearrange· ment of methyl benzylpenicillenate, that is, formation of the oxazolonethiazolidine, scission to the above-mentioned components, and resynthesis. It is of interest that this view was foreshadowed by R. B. Woodward (letter to K. F. of January 2, 1945) from theoretical considerations.

INVESTIGATIONS ON BENZYLPENICILLIN, 1944-1945 amounted to about half of the total weight of original material. Treatment of the sublimate with a drop of acetone caused the whole mass to crystallize, m.p. 148-150°; [a]D25 +325°. The sublimate was nearly pure methyl benzylpenillonate. The sublimation residue showed an ultra­ violet absorption maximum of Em 3,280 at 3,250 A after fifteen minutes of the heating period, of E M 4,690 at 3,300 A after one hour, and of Em 4,400 after another hour. Some methyl benzylpenillonate could be isolated with difficulty from the final sublimation residue (Merck, M.62, 4). How­ ever, when synthetic methyl benzylpenicillenate was similarly heated at 120-125° at 1-3 microns pressure, a sublimate was obtained which also crystallized on treatment with a drop of acetone, but this crystalline product melted at 185-187°. Recrystallization gave a compound which melted at 192-194° and was identical with authentic a-phenylacetamido-j3-methoxyacrylic acid (LXII). CH3OCH

LXIII, and benzylpenicillenic acid has structure LXIV. Benzylpenicillenic acid (LXIV) has been HS N Il RC

C==CH I I CO NH-

C(CH3)2 I -CH-CO2H

\/ O

LXIII S

/\

C6H5CH2CONHCH—CH I I CO-N LXV

C(CH3)2 I CH-CO2H

HS N Il C6H6CH2C

\C(CH )

C==CH I I CO NH

3 2

I CH-CO2H

\/

C6H6CH2CONH-C-CO2H LXII This compound must have been present as an impurity in the crude methyl benzylpenicillenate. No methyl benzylpenillonate could be isolated in this experiment on the thermal treatment of crude methyl benzylpenicillenate. It appeared possible that in these sublimation experiments, methyl benzylpenicillin was being transformed directly (i.e., not via methyl penicillenate, although this substance is present) into methyl benzylpenillonate through ring expansion for diminution of ring tension (Merck, M.68, 3; 76, 2). An analogous rearrangement of methyl D-n-amylpenicillenate to methyl D-n-amylpenillonate has been shown to occur (Merck, M.50, 10; 76, 4). Crystalline synthetic methyl D-n-amylpenicillenate was dissolved in toluene containing a trace of iodine, and the solution was heated at the reflux temperature for three hours. The solution was evaporated to dryness to give a pale brown gum, [a]D +207°. The starting material showed an ultraviolet absorption band of Em 28,000 at 3,175 A, but the rearrangement product showed only a shoulder of Em 1,030 at 3,175 A. A study of the change in optical rotation showed that refluxing of the solution for one hundred minutes caused an increase in rotation of the solute to [a]D25 +236°. The product was isolated as a glassy solid. Chro­ matographic purification yielded a colorless amor­ phous product, [a]D25 +314°. The substance on analysis gave results which were in agreement with the formula Ci6H24N2OIS; and infrared bands at 5.80 and 6.05 μ were observed (Merck, M.76, 4). Benzylpenicillenic Acid and Related Compounds. The penicillenic acids have the general structure

161

O

LXIV characterized as a molecular rearrangement product of benzylpenicillin (LXV). Benzylpenicillenic acid is an oxazolone, and bears a close structural relation­ ship to the oxazolone-thiazolidine structure (LXVI) which was considered at one time as a possible structure of benzylpenicillin. The knowledge of the chemistry of benzylpenicillenic acid is partly S NIl C6H6CH2C

-CH-CH CO

NH

C(CH3)2 CH-CO2H

/

O LXVI S / \ -CH-CH C(CH3)2 I I I CO NH CH-CO2H

NIl RC

/

O LXVII due to the results of experiments on the degradation of benzylpenicillin, and partly to the results of experiments on syntheses which were designed to yield compounds of general structure LXVII. It was soon realized that the characterizable products of the syntheses had the penicillenic acid structure (LXIII) rather than the oxazolone-thiazolidine structure (LXVII). However, the reaction of

162

INVESTIGATIONS ON BENZYLPENICILLIN, 1944-1945

penicillenic acids (LXIII) with an amine (RNH2) or an alcohol (ROH) gives penicilloates (LXVIII); and

grade benzylpenicillin with mercuric chloride to theoxazolone (LXIX). Theseexperimentsinvolved

S

/\

R'CONHCH—CH

C(CH3)2

N C6H5CH2C

(RNH—, RO-)—CO NH-CH-CO2H LXVIII in this sense they give the same reaction products that would be expected from compounds of ox­ azolone-thiazolidine structure (LXVII). There are possible stereochemical differences, however, in the penicilloate(s) (LXVIII) which may be produced from the penicillenic acids (LXIII) or the possible oxazolone-thiazolidines (LXVII). It was concluded that compounds of the oxazolone-thiazolidine structure (LXVII) are very unstable and readily rearrange into the penicillenic acids (LXIII). Certain investigations led to the conclusion that the oxazolone-thiazolidine (LXVI) can be formed from benzylpenicillin, but that it is so unstable that it is capable of only transitory existence. The unstable oxazolone-thiazolidine (LXVI) has been designated pseudobenzylpenicillin and is reviewed in Chapter XXIV. Most of the degradative and synthetic work on the chemistry of benzylpenicillenic acid (LXIV) and related penicillenates. (LXIII) emerged some­ what intermittently between December 1943 and October 1944. Certain aspects of these develop­ ments on benzylpenicillenic acid and related com­ pounds are reviewed in the following paragraphs. Probably the most characteristic physical prop­ erty of the benzylpenicillenic acids is their pro­ nounced ultraviolet absorption band at about 3,200 A. Benzylpenicillenic acid was first detected in solution and as a contaminent in solid materials by the observation of the appearance of this band at 3,200 A. A solution of benzylpenicillin in chlo­ roform, which had been manipulated at room tem­ perature or below, was found to yield material which showed an ultraviolet absorption band at 3,200 A. This band at 3,200 A also appeared when an aqueous solution of sodium benzylpenicillin was allowed to stand one day or longer. Furthermore, this band was observed in the spectra of some prepa­ rations of methyl and ethyl esters of benzylpenicil­ lin. In the case of the esters, the substance responsible for the band at 3,200 A was apparently extracted with the benzylpenicillin prior to the reac­ tion with diazomethane or diazoethane. Thus, this substance which is responsible for the band 3,200 A and was later designated penicillenic acid can be formed from benzylpenicillin under very mild conditions (Merck, M.3, 9; 12c, 19; Squibb,S.l, 12). Because of the early interest in the oxazolonethiazolidine structure (LXVI) for benzylpenicillin, experiments were initiated in an attempt to de­

CH-CHO

CO \ κ O

N

C=CHOH

Il

I

C6H5CH2C

CO

\/ O

LXIX

the treatment of benzylpenicillin with mercuric chloride in ethyl acetate and acetone solutions, but satisfactory results were not obtained (Merck, M.6, 1; 8, 4, 5). Subsequently, the treatment of benzylpenicillin with mercuric chloride in an organic solvent was extended to the methyl ester. It was found that the treatment of the methyl ester of benzylpenicil­ lin in ethyl acetate solution with mercuric chloride yielded a product which showed an ultraviolet absorption band of Em 11,000 at 3,200 A. The specific rotation of this product was much lower, i.e. +61°. When benzylpenicillin methyl ester was treated with acetic anhydride at the reflux temperature for two hours, the resulting material showed an absorption band of Em 20,000 at 3,300 A (Merck, MM, 7, 8). Synthetic studies were described in which the condensation of DL-penicillamine methyl ester (LXX) with 4-ethoxymethylene-2-phenyl-5(4)-oxazolone (LXXI) yielded a crystalline product, methy phenylpenicillenate (LXXII). Structure LXXII was assigned to the condensation product after a rather extensive study of its properties and reactions. N

C=CHOC2H5

C6H5C

+

CO

HSC(CH3)2 I H2NCHCO2CH3

\/ O

LXXI

LXX HS

NIl

C6H5C

CO

=CH \

\C(CH )

3 2

NH

I CHCO2CHs

O LXXII Similar reactions between DL- and D-penicillamine methyl ester (LXX) and 2-benzyl-4-methoxymethylene-5(4)-oxazolone (LXXIII) gave the methyl DLand D-benzylpenicillenates (LXXIV) which were obtained in non-crystalline form. The latter product showed an ultraviolet absorption band

INVESTIGATIONS ON BENZYLPENICILLIN, 1944-1945

163 HS

N-

-C==CHOCH3 CO

C6H6CH2C

HSC(CH3)2 +

H2N-CH-CO2CH3

O LXXIII

C6H6CH2C

-C=CH CO

NH-

C(CH3)2 I -CH-CO2R

\/

O LXXVI R = H, m.p. 147° Em 28,000; 3,225 A LXXVII R = CH3, m.p. 54-55° EM 28,000; 3,200 A

-C=CH I \ CO NH-

C6H6CH2C

O LXXIV LXXV

LXX

of E m 16,000 at 3,200 A. The condensation of D-penicillamine hydrochloride with 2-benzyl4-methoxymethylene-5(4)-oxazolone gave crude non-crystalline D-benzylpenicillenic acid (LXXV) (Merck, M.10, 13; 12a, 13, 17; 15c, 21-23; 23, 19, 21; 37, 16, 23; 1&, 2; 47, 3; 76, 1; Upjohn, U.16, 23). It is this crude D-benzylpenicillenic acid which con­ tains the small amount of synthetic benzylpenicillin which has been discussed separately in Chapter XXIII. It appears that the purest specimen of methyl D-benzylpenicillenate was one which showed an ultraviolet absorption band of Em 20,600-21,600 at 3,175 A. This specimen was prepared by the reaction of both freshly prepared D-penicillamine methyl ester and 2-benzyl-4-methoxymethylene5(4)-oxazolone in benzene solution at room tem­ perature. A solution of the light yellow oily product in chloroform was washed with phosphate buffer, and after recovery showed a band of E M 20,600 at 3,175 A. After passing a solution of this product in benzene over acid-washed alumina, the product showed a band of Em 21,600 at 3,175 A (Merck, M.76, 1). It was clear that the compound formed from benzylpenicillin or its methyl ester which shows a band at 3,200 A is probably identical with benzylpenicillenic acid or its methyl ester. However, further evidence on this relationship was needed, since the oily nature of the products made satis­ factory purification and comparison of the products difficult (Merck, M.Jfi, 3). This further evidence, which proved the identity of the "natural" and synthetic benzylpenicillenic acids resulted from several subsequent lines of investigation. The properties of a few synthetic compounds, which were crystalline and pure, were known which supported the penicillenic acid structure (LXIII). The condensation of D-S-benzylpenicillamine and its methyl ester (Chapter XVI) with 2-benzyl-4C6H6CH2S N-

N-

C(CH3)2 I -CHCO2R

R = CH3 R =H

methoxymethylene-5(4)-oxazolone gave S-benzylbenzylpenicillenic acid (LXXVI) and its methyl ester (LXXVII). Both of these crystalline penicillenates showed the characteristic ultraviolet absorp­ tion band at 3,200-3,225 A. The condensation product (LXXVIII) of DL-alanine and 2-benzyl-4methoxymethylene-5(4)-oxazolone showed a similar absorption band. The ultraviolet absorption band CH3 C=CH I \ CO NH-CH-CO2H

N I! C6H6CH2C

\/

O LXXVIII m.p. 147-148° Em 26,000; 3,200 A at 3,200 A is shifted to about 3,000 A in compounds such as 2-benzyl-4-methoxymethylene-5(4)-oxazolone (LXXIII) which do not have the penicillamine moiety attached to the conjugated chromophoric moiety (LXXIX). These comparisons of spectra substantiated the interpretation that the penicill­ amine and benzyloxazolone moieties were present in the benzylpenicillenic acid. The ultraviolet absorption band at 3,200 A for benzylpenicillenic N —C

C=CH·

C=O

\/

O LXXIX

acid is shifted to 3,500 A for phenylpenicillenic acids (Merck, M.12c, 4; 15b, 18; 23, 18; 30, 8, 10; lft, 16; 46, 3). An ethereal solution of benzylpenicillin methyl ester was treated with mercuric chloride to give an insoluble mercuric complex. Decomposition of the complex with hydrogen sulfide in a buffer solution yielded a "neutral" gummy product in which it was believed that "the labile carboxyl group re­ mains intact." This "neutral" gummy product had a low specific rotation ([a]D +20.8°) and reacted with benzylamine to give crystalline products melting at 115-125° and 154-157°. Structure LXXX was considered possible for the benzylamine reaction product (m.p. 154-157°) (Abbott, A.6, 3-5). Later work on this gummy product, which

164

INVESTIGATIONS ON BENZYLPENICILLIN, 1944-1945

was described as a "neutral oil," showed that it exhibited an ultraviolet absorption band with EM 11,700 at 3,150 A. This "neutral oil" reacted with benzylamine to give a low yield of a benzylamide which melted at 116-118°. The ultraviolet absorption spectrum of the benzylamide, m.p. 116-118°, was in agreement with that expected for structure LXXXI and not for structure LXXX (Abbott, A.8, 2-5). The composition of the insoluble mercuric complex obtained from mercuric chloride and benzylpenicillin methyl ester seemed to have the composition CivHuN^SHgCl-HgCl* (Abbott, A.15, 3). When pinene was added to the ethereal reaction solution to remove the hydrogen chloride, there was no significant loss of the benzylpenicillin methyl ester (Abbott, A.12, 2-3). The azlactonization of synthetic /3-methyl D-7benzylpenicilloate (LXXXII) with benzoyl chloride and pyridine in chloroform solution gave a neutral gum which had an ultraviolet absorption band of E m 18,000 at 3,200 A and was relatively pure methyl benzylpenicillenate (LXXIV) (Merck, M.37, 20). This formation of the methyl benzylpenicil-

lenate by azlactonization of the penicilloate LXXXII was substantiated by the azlactonization of /3-methyl D-rc-amylpenicilloate (LXXXIII) to a pure crystalline compound which corresponded in chemical and physical properties to methyl D-namylpenicillenate (LXXXIV) (Merck, M.47, 2-3). The condensation of D-penicillamine methyl ester (LXX) with 2-amyl-4-methoxymethylene-5(4)-oxazolone (LXXXV) also gave the crystalline methyl D-n-amylpenicillenate (LXXXIV) (Merck, M.50, 11).

The purity of the methyl D-benzylpenicillenate (LXXIV) which was prepared by the azlactonization reaction was comparable (spectra comparison EM 18,000, 3,200 A) to the average product (calc. and found analytical differences, C, —0.52%; H, +0.08%; N, - 0 . 2 % ) which was prepared by the condensation reaction. As noted above, the purest available preparation of methyl D-benzylpenicillenate showed E M 21,600, 3,200 A. The preparation of methyl D-benzylpenicillenate is reproducible. The product has been found to contain impuitiesrelated to the oxazolone, such as a-phenylacetarmido /3-methoxyacrylic acid (LXII). Many attempts to obtain methyl D-benzylpenicillenate in pure crystalline form failed. When methyl D-benzylpenicillenate, prepared by either the azlactonization reaction or the condensation reaction, was allowed to react with benzylamine, a benzylamide melting sharply at 116-118° was obtained. (There was no depression of the melting point of a mixture of the benzylamides derived from the two sources.) Thus, it was highly probable that this benzylamide was identical with the one melting at 116-118° which had been reported from the Abbott Laboratories (Merck, M.37, 20, 23). However, it is diastereoisomeric with the a-benzylamide of /3-methyl D-a-benzylpenicilloate (LXXXI), m.p. 68-70°, which is derived directly

from benzylpenicillin without any known change of configuration. ' Methyl D-benzylpenicillenate (LXXIV) was also

INVESTIGATIONS ON BENZYLPENICILLIN, 1944-1945 obtained

by

the

azlactonization

of

/3-methyl

165

synthetic crystalline specimen of the sodium salt

D-7-benzylpenicilloate (LXXXII) when phosphorus , of 2-benzyl-4-hydrox.ymethylene-5 (4)-oxazolone

tribromide and pyridine were used as reagents. In this experiment, the crude penicillenate reacted with benzylamine to give a mixture of diastereoisomeric α-benzylamides of /3-methyl D-benzylpenicilloates (LXXXI). One fraction of crystals of the benzylamides melted at 154-160° and ap­ parently corresponded to the benzylamide fraction from the Abbott Laboratories described as melting at 154-157° (Merck, M.37, 18). The Abbott procedure for the preparation of methyl benzylpenicillenate from benzylpenicillin methyl ester in ether solution with mercuric chloride was repeated. The ultraviolet absorption of the penicillenate was compared with that of a freshly prepared synthetic sample which was ob­ tained by the condensation reaction; the two prepa­ rations had substantially identical bands at 3,200 A. The "natural" penicillenate reacted with benzyl­ amine and a benzylamide (LXXXI) melting at 116.5-118.5° was readily obtained. A mixture of this "natural" benzylamide and the synthetic ben­ zylamide (m.p. 116-118°, obtained from the con­ densation product) melted without any depression. The specific rotations of the "natural" and syn­ thetic benzylamides were also identical. Thus, the identity of the benzylamine reaction products of "natural" and synthetic methyl benzylpenicillenates was established. Furthermore, the dis­ tinctive character of methyl benzylpenicillenate which is obtained from benzylpenicillin methyl ester by mercuric chloride in an inert solvent was apparently established. On the basis of this structural evidence, and because of the existence of numerous synthetic compounds of related struc­ ture, the trivial name penicillenic acid was intro­ duced (Merck, M.lfi, 6, 10, 11). The transformation of (3-methyl D-7-benzylpenicilloate via methyl D-benzylpenicillenate into the α-benzylamide of (3-methyl D-benzylpenicilloate has been confirmed (Upjohn, U.16, 23, 24). When "natural" methyl benzylpenicillenate (LXXIV) was treated with sodium hydroxide solu­ tion, the molecule was cleaved and a crystalline sodium salt was obtained which was identical with a HS =CH

NIl C6H5CH2C

CO

NH-

(LXXXVI). By these stepwise reactions, a crystal­ line degradation product having a known oxazolone structure has been obtained from benzylpenicillin (Merck, M.lfi, 11-12). The possibility that the crystalline sodium salts do not have structure LXXXVI but have struc­ ture LXXXVII instead was eliminated by the nature -C-CO2R

NC6H5CH2C

\ / 0

R = Na

LXXXVIII

R = H

of the properties of 2-benzyl-4-carboxyoxazole (LXXXVIII). The oxazole (LXXXVIII) does not give an enol color test with ferric chloride as does the oxazolone (LXXXVI) and has a very different ultraviolet absorption spectrum (Merck, M.lfi, 24-25). A precedent for the cleavage reaction of the methyl benzylpenicillenate with sodium hydroxide was the conversion of the crystalline methyl phenylpenicillenate (LXXII) into the crystalline sodium salt of 4-hydroxymethylene-2-phenyl-5(4)-oxazolone (LXXXIX) by the same experimental condi­ tions (Merck, M.12b, 15-16). HS -C=

NC6H6C

=CH

CO

NH-

C(CH3)2 I -CH-CO2CH3

\/ O

LXXII N Il C6H5C

C=CHONa I CO

\/

O LXXXIX

"Natural" methyl benzylpenicillenate was sub­ jected to hydrogenolysis treatment with Raney nickel catalyst to produce methyl desthiobenzylpenicillenate (XC) which was treated with sodium N

C(CH3)2 I -CHCO2CH3

LXXXVII CH

C—CH

C6H5CH2C

CO

CH(CH3)2

NH-CHCO2CH3

O XC

O LXXIV N C6H5CH2C

S C=CHONa CO

\/

O LXXXVI

=CH -C^I CO H2N

NIl C6H5CH2C O

XCI

C(CH3)2 I CH-CO2CH3

166

INVESTIGATIONS ON BENZYLPENICILLIN, 1944-1945

hydroxide solution to give the same crystalline sodium salt of the oxazolone (LXXXVI). This intermediate hydrogenolysis step precludes the possibility that methyl benzylpenicillenate has the alternative structure XCI. The hydrogenolysis reaction in this case would have produced valine methyl ester (XCII) and 2-benzyl-4-methylene5(4)-oxazolone (XCIII) (or a further hydrogenation product), and the alkaline hydrolysis reaction to

give the oxazolone (LXXXVI) would be impossible (Merck, M.Jfi, 12-14). Methyl DL-desthiobenzylpenicillenate (XC) was synthesized by condensing DL-valine with 2-benzyl4-methoxymethylene-5(4)-oxazolone to give the crystalline penicillenate which was then treated with diazomethane. Methyl D-desthiobenzylpenicillenate (XC) was synthesized by condensing directly D-valine methyl ester with the oxazolone. In each case, the products were oils which showed ultraviolet absorption bands of E M 24,000 -26,000 at 3,200 A (Merck, M.50, 10; 47, 2). It is apparent from the available evidence that benzylpenicillin (LXV) (and also the methyl ester of benzylpenicillin) rearranges under the influence of certain experimental conditions into an unstable isomer, probably pseudobenzylpenicillin (LXVI), and thence into the stable benzylpenicillenic acid (LXIV). There is no evidence which would support a postulated intermediary formation of the unsaturated thio-/3-lactam (XCIV).

(9-Methyl D-phenylpenicilloate (XCV) was converted into the methyl D-phenylpenicillenate (LXXII) by azlactonization with benzoyl chloride and pyridine, or with phosphorus tribromide and pyridine, or with acetic anhydride. The penicillenates thus obtained reacted with benzylamine to give the corresponding a-benzylamide of /3-methyl D-phenylpenicilloate (XCVI) (Merck, M.37, 17, 19, 20).

The condensation of L-cysteine methyl ester (XCVII) with 4-ethoxymethylene-2-phenyl-5(4)oxazolone (LXXI) gave a crystalline product which melted at 124-125° and was solvated with benzene. The product showed an ultraviolet absorption band at 3,500 A corresponding to methyl desdimethylphenylpenicilloate (XCVIII) (Merck, M.ISc, 23). Treatment of /3-methyl DL-desdimethylbenzylpenicilloate (XCIX) with benzoyl, chloride and pyridine for azlactonization gave an amorphous product corresponding in ultraviolet absorption to methyl DL-desdimethylbenzylpenicillenate (C) (Merck, M.S3, 10).

INVESTIGATIONS ON BENZYLPENICILLIN, 1944-1945 N-

-C=CHOC2H5

CeH6C

CO \ / O

HSCH2

+

H2N-CH-CO2CH3

·

LXXI

XCVII

CH2 CO

CfiHr1C

CH-CO2CH3

NH XCVIII

C6H6CH2CONHCH-CH HO2C

CH2

NH- -CHCO2CH3 XCIX HS

-C=

NIl C6H6CH2C

=CH

CO

CH2 I -CHCO2CH3

NH-

O

Condensation of D- and DL-penicillamine methyl esters with 4-ethoxymethylene-2-styryl-5(4)-oxazolone gave crystalline methyl D- and DL-styrylpenicillenates (Cl) (Merck, M.23, 23-24; see also HS -C=

NIl

C6H6CH==CH-C

CO

=C NH-

C(CH3)2 I -CH-CO2CH3

O

CI Robinson, Abraham, Baker, Chain and Robinson, CPS.198, 1).

The reactions of synthetic methyl benzylpenicillenate with mercuric acetate (Squibb, S.58, 8) and thiocyanic acid (Squibb, S.S8, 1, 4, 8) are described in Chapters VIII and X. E X P E R I M E N T A L Saponification of the Methyl Ester of Benzylpenicillin in Ethanol-Water Solution (Merck, M.41, 5). Using potentiometric saponification technique, the hydrolysis of the methyl ester of benzylpenicillin was studied in 66 % ethanol-water solution. Sufficient 1.03 N sodium hydroxide was added to raise the apparent pH of the solution to 12.9. Alkali was then added portion-wise to hold the ρH constant as hy­ drolysis progressed, until one equivalent had been used. This required eighty-nine minutes at 24°. The system was then titrated with 1.03 N hydrochloric acid and was found to have a binding span of pHji = 5.1, and a span length equal to 92 % of one theoretical equivalent. The titration system was allowed to stand at a pH of 2.5 and 24° for twenty hours, following which it was back titrated with 1.03 N sodium hydroxide. Back titration showed that only

167

25% of the acidic group corresponding to the original Hi^ = 5.1 span remained. The decarboxylation of benzyl-' penicilloic acid under these conditions was known to involve the α-carboxyl group. The results therefore indicated that at least 75 % of the original methyl ester of benzylpenicillin must have been hydrolyzed to yield /3-methyl benzylpenicilloate under the conditions used. To account for the 25% of binding in the ρH 5.1 region following the ρH 2.5 treatment, two explanations appeared possible. Twentyfive per cent of the benzylpenicillin methyl ester may have been saponified to free benzylpenicillin which was then rapidly converted to free benzylpenicilloic acid at pH 12.9. The latter would be decarboxylated at pH 2.5 to benzylpenilloic acid which would be manifested on subsequent back titration. This appeared to be unlikely since under such circumstances the residual binding region should have moved to a higher pH. Benzylpenilloic acid is known to be a weaker acid than β-methyl benzylpenicilloic acid. Actually the forward and reverse binding spans lay in identical pH regions. A more Ukely explanation is that the decarboxylation of the /3-methyl benzylpenicilloate was merely incomplete under the arbitrary conditions of twenty hours at 24° and ρΉ 2.5. No trace of any binding in the region of pTT above 7 was observed at any time; hence no benzylpenillic acid was formed at pH 2.5. Benzylpenicillin Methyl Ester from the Triethylamine Salt of Benzylpenicillin (Pfizer, P.B4, 12). A solution of 5 g. of the triethylamine salt of benzylpenicillin in 100 cc. of dioxane was treated with a slight excess of an ethereal solu­ tion of diazomethane. After standing for one-half hour, the mixture was evaporated in vacuo and the residue crystal­ lized from ethyl acetate-petroleum ether. In this way there was obtained 3.4 g. of the methyl ester of benzylpenicillin, m.p. 93-94°, in the first two crops. This is 85% of the theoretical amount. When this product was mixed with an authentic specimen of benzylpenicillin methyl ester, there was no depression of the melting point. Action of Methanol on Sodium Benzylpenicillin and Benzylpenicillin Methyl Ester (Pfizer, P.Uh 10). A 1% solution of the methyl ester of benzylpenicillin in absolute methanol had MD +289°. After twenty-four hours, it still had [23

0.33 1.83 6.17 24

+255 121 66 49

1 2 3

St. C1

4960 3880 5370 7675

1.5 hrs.2 3 hrs.2

5900 5350 6430 7750

6170 5950 6940 7800

Standard conditions. Time allowed for color development before final dilution. Calculated on thiolamine added.

Per cent SH3 (3 hr. value)

75 72 84 95

219

The remainder of the solution was worked up after 29 hours. Assay of a sample withdrawn at that time showed that practically all antibiotic activity had been destroyed. The solution was acidified to pH 2 with sulfuric acid and extracted with a total of 500 cc. of ethyl acetate in 5 portions. The combined extracts on evaporation in vacuo yielded 513 mg. of a slightly yellow solid. The crude product, analyzed after drying at 25° in vacuo, gave values in agreement with benzylpenicilloic acid. Found: N, 7.45; S, 7.70 Calc. for Ci6H20O6N2S: N, 7.95; S, 9.05 The crude product was purified by removing some material insoluble in chloroform, and reprecipitating the chloroform soluble portion twice from chloroform-toluene. The result­ ing product had a [ = —48° in metha­ nol). The product was dissolved in a small volume of ethyl acetate. The solution on standing deposited 38 mg. of a crystalline product melting at 184-189°. Recrystallization from the same solvent afforded 28 mg. of clear-cut elongated plates melting at 190-192°. HD24 = +4.0° (0.70% in methanol). The ultraviolet absorption curve (95% ethanol) showed maxima at 236 πιμ ( EM = 9,200) and 274 πιμ ( EM = 15,800) and minima at 225 ηιμ (EM = 8,200) and 250 πιμ (Em = 5,800). Calc. C19H23O4N3S2: C 54.16, H 5.50, N 9.98, OC2H610.70 Found: C 53.94, H 5.72, N 10.08, OC2H610.41 The original aqueous mother liquor was thoroughly ex­ tracted with ether. The combined, dried ether extracts on evaporation yielded 1.18 gm. of a white solid melting at 90-100°, [α]υ = —42° in methanol. The absorption spec­ trum of this product was qualitatively similar to that of thiocyanate derivative B in that it displayed the main band at 277 ηιμ (EM = 233). It was dissolved in ethyl acetate, from which it deposited 312 mg. of crystalline material melting at 93-95°. Three recrystallizations from the same solvent yielded 84 mg. of feathery rosettes melting at 155-165° after softening at 145° (sample dried in vacuo at 110°, since the desiccator-dry product tenaciously retained ethyl acetate). The material was soluble in one equivalent of sodium bicarbonate solution. The ultraviolet absorption spectrum (95 % ethanol) showed maxima at 234 m/i ( EM = 10,000) and 278 m/i (EM = 15,000), and minima at 225 ηιμ (Em = 9,000) and 252 (Em = 6,000). Calc. C17H19O4N3S2: C 51.90, H 4.87, Neut. Equiv. 393 Found: C 51.46, H 4.82, Neut. Equiv. 402 Reaction of α-Methyl D-a-Benzylpenicilloate with Am­ monium Thiocyanate in Acetic Acid (SMO, 5). From a boiling solution of crystalline α-methyl D-a-benzylpenicilloate (28.6 mg., prepared according to S.86, 25) and am­ monium thiocyanate (12.1 mg., 2 moles) in 1.0 cc. of acetic acid, 0.1 cc. aliquots were withdrawn (after brief cooling to room temperature) at 0.5, 1 and 3 hour intervals. EM for the maximum at 277 Difi was 5,700, 8,300 and 10,300, respec­ tively, for these periods. In the preparative experiment 936 mg. of the ester were heated with 392 mg. of ammonium thiocyanate in 7 cc. of acetic acid for 3 hours. Addition of ice water to the chilled solution precipitated an oil, which was extracted from the mixture with ethyl acetate. The extract was washed with several portions of half-saturated sodium bicarbonate solu­ tion (total 75 cc.), then with water, and dried. Removal of the solvent gave 191 mg. of residue which partially crystal­ lized on treatment with methanol-water (28 mg., m.p. 198218°). Two recrystallizations from the same solvent yielded rods melting at 225-230° (soft. 212°). A mixture with the higher-melting (220-227°) of the two methanol-alkali isomerization products from thiocyanate derivative B (S.49, 4) melted at 221-229°. [a]D = -1.8° (0.446% in methanol). Calc. C18H21O4N3S2: C 53.05, H 5.20 Found: C 53.01, H 5.10 The ultraviolet absorption spectrum in 95 per cent ethanol showed maxima at 237 ιημ ( EM = 9,200) and 275 ηιμ ( EM = 15,800). The bicarbonate solution was acidified to pH 2.5 and extracted with ethyl acetate. The dried extract after evaporation in vacuo yielded 656. mg. of an oil which crystal­ lized on seeding with the corresponding acidic product obtained from α-ethyl D-7-benzylpenicilloate (S.49, 4). The crystalline portion (229 mg., m.p. 92-105°) was recrys­ tallized three times from ethyl acetate and then melted at 139-152° (soft. 132°). [a]D23 = -96.2° (0.670% in metha­ nol). The product, which was markedly hygroscopic, was

THIOCYANATE DERIVATIVE OF METHYL BENZYLPENICILLINATE

305

dried in vacuo at 110° for the analysis and determination of the physical constants.

tion spectrum (95 % ethanol) was essentially identical with that of the higher-melting, dextrorotatory isomer.

Calc. CnH19O4N3S2: C 51.90, H 4.87 Found: C 50.90, H 4.99

Calc. CnHi6O3N2S2: C 45.83, H 5.60 Found: C 45.71, H 5.46

The ultraviolet absorption spectrum showed maxima at 277 ταμ (EM = 14,500) and at 230 ταμ (EM = 9,300), the latter with a shoulder around 234 πιμ (EM = 8,900). Alkali Isomerization of the Thiohydrouracil-Thiazolidine Carboxylic Acid from α-Ethyl D-7-Benzylpenicilloate (Squibb, S.50, 7; 53, 7). A solution of the crystalline acidic product (S.49, 4) (157 mg., 0.40 millimole) in ethanol (5 cc.) was alkalinized with 0.80 cc. of 1 N NaOH (2 equiva­ lents) and the volume made up with water to 10.0 cc. The following rotation changes were observed:

The original aqueous filtrate was repeatedly extracted with ether. The residue of the ether extract (0.6 gm.) was triturated with 6 % sodium bicarbonate solution. Acidifica­ tion and extraction of the latter with ether yielded 0.29 gm. of a partly crystalline residue, which was triturated with ether. The ether-insoluble portion (25 mg.) melted at about 260° and had an [ CH3-C CH-CN

SCH2C6H5 XXVII

RNH2

-• VI

CH3 CH3-C I S

CH-COOH I NH

CH3 NHR I l CH3-C CH-CN

CH3 NHR I l ' CH3-C CH-COOH

SCH2C6H6 R1

R2 XXXI

SCH2C6H5

XXVIII

XXIX CH3

CH3

CH3-C-CH-COOH I I HS NHR(HX) XXX

CH3-C-CH-COOH I I HS NH-CHR1R2 XXXII Figure 4.

adsorption) if the aluminum was precipitated as the hydroxide and the penicillamine isolated from the filtrate. A somewhat different route to 4-carboxy-5,5-dimethyl-2-thio-thiazolidone (XXI) was afforded by the condensation of N-dithio-carbethoxyglycine (XXII) with acetone in the presence of acetic anhydride and sodium acetate to give 2-ethylmercapto-4-isopropylidene-thiazolone (XXIII), which with hydrogen sulfide and triethylamine gave the thiol acid (XXIV) as its triethylamine salt. Warming the latter with mineral acid gave the carboxy-thio-thiazolidine (XXI). Synthesis by the Strecker Reaction. Figure 4. (Catch, Cook, Harris, and Heilbron, CPS.678.) Although the utilization of the Strecker reaction in the synthesis of penicillamine was one of the early synthetic methods tried, it was abandoned because of the failure of a-bromoisobutyraldehyde diethylacetal to react with sodium benzyl mercaptide in the desired manner (Squibb, S.8; 9). The Imperial College group (CPS.678), however, found that the bromo-aldehyde prepared from the bromoacetal gave an almost quantitative yield of a-benzylmercaptoisobutyraldehyde (XXV), which in turn yielded the cyanhydrin (XXVI) and aminonitrile

duced; thus, substitution of methylamine for ammonia in the conversion of the cyanohydrin (XXVI) gave the N-methyl homolog (XXVIII) in essentially quantitative yield. Alternative Resolution and Racemization Meth­ ods. Although the original resolution of S-benzylN-formyl-DL-penicillamine has already been de­ scribed as well as the racemization procedure for the less desirable L isomer, new procedures were devised for use with DL-penicillamine. The Merck group (M.26, 5, 6) found that N-formyl-DL-penicillamine gave a crystalline salt with brucine and that the salt of N-formyl-D-penicillamine crystallized directly in satisfactory yield and purity. A racemization procedure for the L form was not reported. The second resolution procedure was that developed by the Wellcome group (Copp, Duffin, Smith, and Wilkinson, CPS.108) in which isopropylidene-DL-penicillamine (VIII) was formylated to IX and this formyl derivative resolved through the brucine salt. Here again it was the salt of the D-penicillamine derivative which first separated. The same workers later (CPS.S19) reported the racemization of the l derivative by treatment with acetic anhydride and subsequently found (CPS.B85) that the brucine salt itself could

460

PENICILLAMINE

be racemized by acetic anhydride in benzene. The resolved N-formyl-isopropylidene-D-penicillamine was readily hydrolysed by mineral acid to D-penicillamine hydrochloride (VI). Cook, Elvidge, and Heilbron (CPS.37S), using this procedure, found that a considerable saving in time and effort could be effected, by converting the incompletely resolved brucine salts to the thiazolidine acids, which then gave the isomers of high purity with a single crys­ tallization from benzene-petroleum ether or, better, water. The optical component present in excess separated in pure form in the first fractions. Syntheses of Penicillamine Analogs and Homologs. The synthesis of an intermediate which would lead to N-methyl penicillamine has been described (p. 459). However, a versatile synthesis of N-substituted /S-thiol amino acids was discovered by the Upjohn group (U.18, 2-6; 19, 1) and N-methyl- and N-isopropyl-cysteine as well as D and L N-methyl-penicillamine were prepared. This procedure was based on the discovery that the simple thiazolidines (XXXI) cleave with sodium in liquid ammonia with subsequent hydrolysis to give the corresponding N-substituted amino acid (ΧΧΧΠ). The Cornell group (D.29) in a study of this reaction found that under strictly anhydrous conditions the major product was dimeric in nature (e.g. thiazolidine-4-carboxylic acid gave, apparently, Ν,Ν'-ethylene-bis-cysteine) but with one molecular proportion of water present the simple cysteine derivative was isolated. In the course of the problem a number of amino acids analogous to penicillamine were prepared in which the basic carbon skeleton was varied. In their initial "present knowledge" report the Lilly group reported having prepared a-amino-/3-mercaptovaleric acid at a time when the structure of penicillamine had not been clarified. The details of this synthesis (L.5) showed that it was prepared in an entirely similar manner to the Oxford peni­ cillamine synthesis, propionaldehyde being sub­ stituted for acetone in the preparation of the initial oxazolone. The same results were later reported by the Wellcome group (Copp, DufBn, Smith, and Wilkinson, CPS.672) who also prepared the oxazol­ one by heating N-benzoyl-/3-hydroxy-norvaline with acetic anhydride. The homologous /3-mercapto-leucine was pre­ pared by the Cornell group (D.l, 8-10), through the azlactone prepared from N-chloroacetyl-leucine, to check the method of synthesis after difficulties were encountered in the addition of benzyl mercaptan to 4-isopropylidene-2-methyl-5(4)-oxazolone. A further homolog, /3-mercapto-isoleucine, "homopenicillamine," was prepared by the Abbott group (A.IS, 2-5) following the procedure of Merck for penicillamine. Although not a homolog, but of interest because of its possible use in preparing penicillin without a free carboxyl group, a-amino-j8-thiol-isobutane was

prepared by the Imperial College group (Arnstein, Cook, and Heilbron, CPS.6Jfi) by addition of benzyl mercaptan to α-nitro-isobutene with subse­ quent reduction of the nitro- and benzylthio- groups. Reactions of Penicillamine. Penicillamine was found to react in most respects very similarly to cysteine. Indeed, it was to a considerable extent upon this similarity that the structure was deduced (Abraham, Baker, Chain, and Robinson, PEN.97). The reaction of penicillamine with carbonyl com­ pounds to form thiazolidines (cf. Schubert, J. Biol. Chem., Ill, 671 (1935); 114, 341 (1936); Ratner and Clarke, J. Am. Chem. Soc., 59, 200 (1937)) is treated extensively in Chapter XXV. The intro­ duction of the mercapto group in the penicillamine synthesis is more difficult than in the less substi­ tuted analogs. Penicillamine was degraded to isobutyraldehyde by standing overnight with aqueous mercuric chloride while cysteine was stable for at least five days under the same conditions (Squibb, S.19, 8). In contrast it is more stable than cysteine to the action of hot sodium plumbite solution (Squibb, S.21, 7). The oxidation of penicillamine to /8,/3'-dithiobis-valine, analogous to the formation of cystine from cysteine, was found to proceed less readily than with the lower homolog (Merck, M.15, 13; 69,18; Cornell Bioch., D.17); and the product, once formed, proved to be considerably more stable than the simpler compound. Thus, although it behaves polarographically and toward the action of iodine as does cystine, it gives no color as cystine does when tested with alkaline sodium nitroprusside in the presence of sodium cyanide, and none when tested by the Folin-Merenzi reaction; the disulfide bond is cleaved by sodium in liquid ammonia, as in cystine (Cornell Bioch., D.17). The oxidation to penicillaminic acid (dimethylcysteic acid) was mentioned earlier in this chapter. The oxidizing agent usually used was bromine water. Hydrogen peroxide in acetic acid also effects this conversion as shown by the Squibb workers (S.5, 2), who thus obtained the benzylammonium salt of penicillaminic acid from the benzylammonium salt of benzylpenicilloic a-benzylamide and the methyl ester from the methyl ester of benzylpenicillin (S.14, 5). An intermediate oxidation stage, the sulfinic acid, was obtained by the Squibb workers (S.6, 5), the methyl ester of the sulfinic acid being isolated from the reaction of mercuric chloride on methyl benzylpenicillinate in ether. The formation of an insoluble precipitate on treatment with mercuric chloride was of particular value in the original isolation of penicillamine from penicillin (Abraham, Baker, Chain, and Robin­ son, PEN.91, et ante). Esters were prepared by the action of the appropriate alcohol in the presence of anhydrous mineral acid. A very useful procedure for obtaining penicillamine esters directly from

N-formyl (or isopropylidene) penicillamine involved treatment of these derivatives with hydrogen chloride in the appropriate alcohol (Boon, Carrington, Davies, Jones, and Ramage, CPS. 479). N-Acyl derivatives were prepared, for the most part, by action of the acyl halide or anhydride under

Details of preparation are given only for typical examples; physical constants for the various derivatives are given in the concluding table. Unsuccessful Attempted Syntheses of Penicillamine. A wide variety of reactions attempted durintr the course of the Droblem did not lead to

Figure 5.

Schotten-Baumann conditions, although a number were also prepared by the indirect method of acylation of a thiazolidine and treatment of the acyl thiazolidine with mercuric chloride, the carbonyl compound being released and the acyl penicillamine regenerated from the mercuric chloride precipitate by the action of hydrogen sulfide (Imperial College, CPS.5, 4; Cook, Elvidge, Hall, Heilbron, and Shaw, CPS.270, 2). N-Formyl penicillamine was also prepared by heating penicillamine hydrochloride with formamide (Allen, Boon, Carrington, Gaubert, and Levi, CPS.B25, 2, 3). Acylation through the use of the acid azides was used in the case of some of the more highly substituted derivatives (Mich. Chem., B.8, 3). More stringent conditions for direct acylation of penicillamine led to formation of the corresponding 2-thiazoline-4-carboxylic acids. Sodium-liquid ammonia reduction of N-acyl-S-benzyl-penicillamines also yielded the N-acyl penicillamines.

successful penicillamine syntheses. One of the most promising of these was the preparation of /3-benzylmercapto-dimethylpyruvic acid (XVII) from the /3-bromo compound (XVI). Although the mercapto compound formed an oxime (Squibb, S.7, 2; 11, 3) and a phenylhydrazone (Hems, Holland, and Robinson, CPS.224, 3, 4; Pfizer, P.8, 9; 9, 10; 11, 2; 12, 9), neither of these derivatives could be reduced to penicillamine nor would the benzylmercapto-dimethylpyruvic acid react in the Strecker reaction (CPS.224) although evidence was obtained for a good yield of the cyanhydrin. Diethyl /3-1-benzylmercaptoisopropylmalonate (XXXIV), prepared from isopropylidene-malonic ester (XXXIII) (Abbott, A.6, 8; 7, 1; Heyden, H.S, 1, Pfizer, P.8, 11; 13, 1; Winthrop, W.S,1; 4,1; 6, 4), did not give the expected reaction products with butyl or amyl nitrite (W.S, 4, 17; H.S, 1), with benzene diazonium chloride or potassium phenyl diazotate (P.8, 11), with bromine (A.5, 4; 6, 8) or

462

PENICILLAMINE

with bromine followed by ammonia (W.5, 4), nor did the rtionoethyl ester react with hydrazine to give the hydrazide (W.5, 4). Diethyl α,β-dibromoisopropylmalonate (XXXV), formed readily from XXXIII (Pfizer, P.7, 5; 11, 3; Squibb, S.8, 1), did not give satisfactory products with Na2S2 or thiourea (S.8, 1, 2), nitrosyl chloride, or amyl nitrite (S.9, 7). The use of iodine monochloride in place of bromine on dimethylacrylic acid (XXXVII) (.P.9, 12) was unsuccessful. The use of ethyl a-cyano-0,jS-dimethylacrylate (XXXVI) in place of the malonic ester was successful as to the addition of benzyl mercaptan (P.8,11; W.6,1, 4) but no farther. The reaction of /3,/3-dimethylacrylic acid (XXXVII) with benzyl mercaptan did not proceed (Squibb, S.7, 1), nor did the action of nitrosyl chloride or amyl nitrite lead to the expected products (S.9, 7) either with the acid or the ethyl ester. Attempts to replace the halogen of a-chloro-/3-hydroxy-isovaleric acid (XXXVIII) by the use of ammonia or potassium phthalimide were not successful (Pfizer, PJ, 22; 7, 5; Squibb, S.8, 2). A sustained attempt to derive a penicillamine synthesis from a-bromo (or hydroxy)-/3-sulfo-isovaleric acid by the Pfizer group also failed (P.7, 8; 8, 12; 9, 12, 13; 10, 5; 14, 2). Acetone was found to condense with rhodanine to give the isopropylidene compound (XL) by the Oxford (Abraham, Baker, Chain, and Robinson, CPS.75, 2, 3) and the Merck (M.35, 3) groups. Hydrolysis of the compound was unsatisfactory under most conditions, although with mild treat­ ment a small amount of what appeared to be a dimeric form of'the sulfur analog of /3,/3-dimethylpyruvic acid was isolated. 5-Isopropylidene-hydantoin (XLI) was prepared by the Imperial Chemical (Boon, Carrington and Jones, CPS.20) and the Merck groups (M.26, 2; 85, 3), and the benzyl mercaptan addition product (XLII) was prepared by the former. This product could not be hydrolysed to S-benzyl-penicillamine. The 2-thiohydantoin corresponding to (XLI) was prepared by the Glaxo group (Dupre, Hems, and Robinson, CPS.88) and converted to the S-benzylisopropyl-thiohydantoin, but all attempts to con­ vert this to S-benzyl-penicillamine failed. The thiohydantoin (XLI) was utilized, however, as a starting material for the preparation of valine by reduction and hydrolysis (Beer, King, Waley; Abraham, Baker, Chain, and Robinson, CPS.244) • E X P E R I M E N T A L S-Benzyl-DL-penicillamine (V) and N-Benzoyl-S-benzylDL-penicillamine (Abraham, Baker, Chain, Cornforth, Cornforth, and Robinson, PEN.100). A yield of 86 g. of the azlactone (I) was obtained from 240 g. of hippuric acid by the method of Ramage and Simonsen (J. Chem. Soc., 1935, 534). Using an excess, 50 moles, of acetone the Northern Regional Research Laboratory (C.2, 1) secured 62.5 % of the theoretical yield. The azlactone (91 g.) in dry benzene (450 cc.) was slowly added to a solution of benzyl mercaptan (51 cc.) in methan-

olic sodium methoxide (2.25 g. of sodium in 450 cc. dry methanol) at 5-10°. After twenty-four hours the solution was made acid to Congo Red and evaporated in vacuo. The oily residue was dissolved in a hot mixture of acetic acid (1,125 cc.), cone, hydrochloric acid (325 cc.), and water (625 cc.). The mixture was boiled under reflux until a clear solution resulted (% hour) and then concentrated hydrochloric acid (600 cc.) was added gradually to the boiling solution, so that not much oil separated, about three hours. After a further two hours refluxing3 the solution was evaporated in vacuo and the residue treated with ether and N hydrochloric acid. The whole was filtered. An insoluble crystalline residue (4 gm.) upon recrystallization from benzene gave leaflets, m.p. 156-159° of derivative IV. Calc. CiiHsiOjNS: C, 66.5; H, 6.1 Found: C, 67.2; H, 6.2 The aqueous filtrate was separated from the ether, concen­ trated in vacuo to 600 cc. and neutralized to litmus with ammonia. The total crystalline precipitate (V) amounted to 26 g. and was crystallized from water for analysis, m.p. 197-198° (dec.). Calc. Ci2H17O2NS: C, 60.3; H, 7.1 Found: C, 60.7; H, 7.1 N-Formyl-S-benzyl-DL -penicillamine (IV, R = H) (PEN.100, 3). The above acid (25 g.) was dissolved in absolute formic acid (200 cc.), and stirred at 60°, a tem­ perature maintained by the gradual addition of acetic anhydride (70 cc.). The solution was cooled, water (70 cc.) was added, and after twenty minutes the whole was evapo­ rated in vacuo from a bath at 50°. The residue was recrystallized from acetone-benzene. The yield of N-formyl derivative was 23.6 g., m.p. 155-156°; a portion after recrystallization from alcohol had m.p. 158-159°. Calc. CisH17O8NS: C, 58.4; H, 6.4; N, 5.2 Found: C, 58.2; H, 6.3; N, 5.2 Resolution and Hydrolysis of S-Benzyl-N-formyl-DLpenicillamine (PEN.100, 3). The formyl derivative (21 g.) was dissolved in η-butyl alcohol (160 cc.) and anhydrous brucine (31 g.) was added. The solution was warmed, treated with charcoal, and filtered. An extra 20-25 cc. of butanol was used in these operations. Water (10 cc.) was then added (no brucine salt separated until this was done). After twelve hours at room temperature and twenty-four hours at 0° the crystals were collected, washed with a little butanol and dried in the air (27.4 g.). The colorless prisms (called fraction A) melted at 99-100° and had Md20 —13.5° (c = 1 % in 50 % ethyl alcohol). The filtrate from A was concentrated in vacuo to 113 cc., and 10 cc. water was then added. After six hours at 0° the crystals (called fraction B) were collected as before (16.0 g.); [ck]D21 —16.0°.

The filtrate from B was concentrated to 50 cc. in vacuo, and water (10 cc.) was added. After twenty-four hours at 0° fraction C (1 g.) was collected, [25 +150°) was converted to the dimethyl ester with ethereal diazomethane and isolated as a glassy solid. This was dissolved in ether and treated with ethereal p-toluenesulfonic acid. Fine needles ap­ peared. After two recrystallizations from ethanol-ether, the product weighed 15 mg.; m.p. 135-138° (micro-block). A few crystals did not melt until 143°. When mixed with a sample of synthetic dimethyl D-a-benzylpenicilloate p-toluenesulfonate, m.p. 134-138° (micro-block), there was no depression of the melting point. In all cases there appeared to be a few crystals which did not melt until 143°. The rotation was [α]η +41.7° (c = 0.984 in ethanol). Calc. for C26H82N2O8S2: C, 54.31; H, 5.84; N, 5.07 Found: C, 54.02; H, 5.55; N, 5.48

PENILLOIC AND PENICILLOIC ACIDS p-Toluenesulfonic Acid Salt of Synthetic Dimethyl (M.43, 5; 44, H)· Purified per­ chloric acid salt of dimethyl D-a-benzylpenicilloate (0.5 g.) was decomposed by suspending it in sodium bicarbonate solution and extracting the free base into chloroform. The chloroform solution was dried over sodium sulfate and con­ centrated under reduced pressure to leave a colorless gum. The gum was dissolved in ether and treated with an ethereal solution containing an excess of p-toluenesulfonic acid. A gummy precipitate, which formed immediately, quickly solidified when scratched. The product was crys­ tallized from absolute ethanol-ether to yield felted needles, m.p. 145-147° (capillary, placed in bath at 135°); m.p. 134138° (micro-block); [a]d22 +53.7° (c = 0.475 in ethanol). D-a-Benzylpenicilloate

Calc. for C26H82O8N2S2: C, 54.31; H, 5.84; N, 5.07 Found: C, 54.57; H, 5.67; N, 5.34 Dimethyl N4-Nitroso-D-a-benzylpenicilloate (M.73 , 6). α-Methyl D-a-benzylpenicilloate (500 mg.) was treated with a slight excess of diazomethane in 30 ml. of ethyl ether. The solid dissolved completely and nitrogen was liberated. The solution was concentrated to remove unused diazo­ methane, then diluted with ethyl ether to 50-ml. volume. The ether solution was washed twice with 20-ml. portions of 5% sodium bicarbonate solution and twice with 20-ml. por­ tions of water. After filtering, the ether was removed in vacuo leaving a crystalline residue of dimethyl D-a-benzyl­ penicilloate. The diester was dissolved in 25 ml. of 15% hydrochloric acid and the solution was cooled to 5°. A solu­ tion containing 100 mg. (10% excess) of sodium nitrite in 5 ml. of water was cooled to 5° and poured into the acid solution. An immediate precipitation of the N-nitroso derivative resulted. The precipitate was separated by centrifuging, washed with 25 ml. of ice water and centrifuged again. The N-nitroso derivative was fairly stable be­ low room temperature but rapidly turned to a gum and decomposed at room temperature. Dimethyl D-|S-Benzylpenicilloate (M.S7, 8). Condensa­ tion of D-penicillamine methyl ester and methyl penaldate in boiling toluene gave a reaction product which was con­ verted to the hydrochloride, then crystallized from ether, yielding approximately 30% of dimethyl n-7-benzylpenicilloate hydrochloride. The ethereal mother liquor was diluted to 100 ml. volume, then washed with four 50-ml. portions of 5% sodium bicarbonate. The ether layer was then filtered and evaporated to dryness, giving the free ester, which was taken up in 25 ml. of ether. From the solution, after five days, 3.1 g. of partly gel-like, partly crystalline product was obtained. Recrystallization yielded 260 mg. of material as soft needles melting at 113-114°. This product gave no depression in melting point on mixing with the dimethyl u-/3-benzylpenicilloate secured by heating di­ methyl D-7-benzylpenicilloate in xylene. Conversion of Dimethyl D-7- and D-a-Benzylpenicilloate into Dimethyl D-/3-Benzylpenicilloate (M.S7, 8; 50, 19). A solution of 1.00 g. of dimethyl D-7-benzylpenicilloate in 10 ml. xylene was heated under reflux for three hours. The xylene was then removed in vacuo and the residue crystallized from ether. After two successive recrystallizations, 120 mg. of the D-/S-isomer was obtained as long silky needles melting at 113-114°. The compound sharply de­ pressed the melting point of dimethyl n-7-benzyIpeniciHoate. It showed typical penicilloate ultraviolet absorption; [«Id26 +24.5° (c = 0.79 in methanol). Calc. for C18H24O6N2S: C, 56.82; H, 6.32; N, 7.37 Found: C, 56.93, 56.94; H, 6.83, 6.84; N, 7.67 One batch of dimethyl D-7-benzylpenicilloate was un­ affected by this treatment. A solution made from 100 mg. of dimethyl D-7-benzylpenicilloate from this "stable" batch, dissolved in 10 ml. of toluene was treated with ten micro­ grams of iodine and refluxed for two hours. There was obtained 11 mg. of recrystallized dimethyl D-^-benzylpenicil-

615

loate melting at 113-114° which showed no melting point depression with an authentic sample. Approximately the same result was secured on heating dimethyl D-a-benzyl­ penicilloate in toluene containing iodine, 16% of the diffi­ cultly crystallized dimethyl D-/3-benzylpenicilloate, m.p. 112-114°, being secured. The [a]D25 values of the crude, treated a- and 7-benzylpenicilloates were +25° and +26° (c = 1 in methanol). In refluxing xylene solution containing iodine, the di­ methyl D-α- and D-7-benzylpenicilloates reacted as in toluene. Conversion of α-Methyl D-7-Benzylpenicilloate to Di­ methyl i)-|3-Benzylpenicilloate (M.40, 13). A solution of 10.0 g. of α-methyl D-r-benzylpenicilloate in 150 ml. of methanol was refluxed for thirty-six hours. The solution was then concentrated in vacuo to a thick syrup which was taken up in 200 ml. of ether. After standing for forty-eight hours, 2.407 g. of α-methyl D-7-benzylpenicilloate melting at 164-165° was recovered. The mother liquor material weighed 7.412 g. This was treated in chloroform-ether solution with excess diazomethane. The unused diazo­ methane and ether were removed and the chloroform solution was washed with dilute bicarbonate solution and evaporated to dryness, giving 7.501 g. of mixed dimethyl D-benzylpenicilloates. The product was dissolved in 100 ml. of ether and chilled to 0° for one hour. A colorless gel precipi­ tated weighing 4.108 g. This fraction, when crystallized from ether containing a trace of methanol gave 1.2 g. of the dimethyl D-^-benzylpenicilloate melting at 113-114°. Conversion of Dimethyl D-7-Benzylpenicilloate to Di­ methyl D-/3-Benzylpenicilloate (M.40, 12). A solution of 10.0 g. of dimethyl D-7-benzylpenicilloate in 120 ml. of methanol containing 0.2 ml. of concentrated hydrochloric acid was refluxed for five hours. The solution was evapo­ rated to dryness in vacuo and the residue was dissolved in 50 ml. of methanol containing 1.0 g. of hydrogen chloride. The solution was concentrated to approximately 30-ml. volume, then 200 ml. of ether was added. After three days the crystalline hydrochloride which deposited was removed by filtration. The product melted at 91-93° with preliminary softening and weighed 7.518 g. Decomposition of this hydrochloride by shaking with chloroform and dilute bi­ carbonate and subsequent crystallization of the free base from ether gave 6.380 g. of crude dimethyl D-7-benzylpenicilloate melting at 100-102°. The mother liquor from the hydrochloride crystallization was shaken with three 50-ml! portions of 5% sodium bicarbonate solution. The ether layer, evaporated to dryness, gave 2.412 g. of amorphous base. By crystallization from ether, 780 mg. of dimethyl 1 D-/3-benzylpenicilloate, m.p. 113-114°, was secured. Stability of Dimethyl D-7-Benzylpenicilloate in Pyridine {M.40, 12). A 5% solution of dimethyl D-7-benzy lpenicilloate in pyridine showed od26 +5.70° (1 dcm.) on mixture and ο®'5 +,5.79° after heating one hour at 100°. After twenty hours heating at 100°, starting material was re­ covered in excellent yield. A solution of 500 mg. of dimethyl D-7-benzylpenicilloate in a mixture of 10 ml. of pyridine and 10 ml. of methanol was refluxed for twenty hours. The starting material was reT covered in excellent yield. Conversion of the α-Ethyl /3-Methyl i)-/3-Benzylpenicilloate to the Dimethyl D -/3-Benzylpenicilloate (M.44, 17; 47, 15). Exactly 0.236 g. of α-ethyl /3-methyl D-/3-benzylpenicilloate, m.p.101-102°, was dissolved in 5 ml. of methanol and 2 ml. of water and a drop of phenolphthalein solution added. The solution was treated with one equivalent (6.37 ml. of 0.094 N solution) of sodium hydroxide. Oa working up in the usual manner, the acidic fraction was obtained as a white fluffy solid. This was taken up in ether and esterified with diazomethane to give 170 mg. of neutraj oil. ,' The product appeared to be chromatographically homo,geneous; it was eluted from activated alumina by ether con­ taining 2% of methanol. From dilute methanol was depos^

616

PENILLOIC AND PENICILLOIC ACIDS

ited poorly crystalline material of m.p. 107-111°; Wd26 +18° (c = 2.12 in absolute ethanol). Slow concentration at room temperature of an absolute ethanol solution of the material gave crystals melting between 110 and 114°. These observa­ tions indicate that α-ethyl /3-methyl D-/j-benzylpcnicilloate, m.p. 101-102°, is configurationally related to dimethyl D-(3benzylpenicilloate, m.p. 113-114° and [α]η23 +24.5° (in methanol). In confirmation of this, a mixture of the latter compound and the material of m.p. 110-114° described above melted at 112.5-114°. Hydrogenolysis of Dimethyl D-/3-Benzylpenicilloate (M.50,

25). A solution of 250 mg. of dimethyl D-/3-benzylpenicilloate in 5 ml. of dioxane and 15 ml. of water was treated with 3 g. of freshly prepared Raney nickel catalyst. The mixture was stirred for 1.5 hours at room temperature and then 0.5 hour at 65°. The nickel was removed by filtration and washed with 15 ml. of methanol. The combined filtrate and washings were evaporated to a few milliliters in vacuo up to 50°. The oil was extracted from the water with ether and the extract was then washed well with water. The ether layer gave 98 mg. of colorless oil upon evaporation. This oil could not be crystallized. No crystalline salt was found. The oil gave a negative azide sulfhydryl test; [α]ο26 +22° (c = 1 in methyl alcohol). Calc. for C18H2EOSN2: C, 61.70; H, 7.48; N, 8.00 Found: C, 61.99; H, 7.47; N, 8.01 Dimethyl N4-Benzoyl-D-/3-benzylpenicilloate (M.44, 15).

α-Methyl N4-benzoyl-D-/3-benzylpenicilloate, m.p. 227-229°, was methylated in a methanol-ether solution with diazomethane. After removal of the solvents, the residue was crystallized from ether by adding petroleum ether. The product crystallized in needles, m.p. 83-84°. Calc. for C25H28NsOeS: C, 61.96; H, 5.82; N, 5.78 Found: C, 61.28; H, 5.91; N, 5.81 Dimethyl N4-Benzoyl-D-(3-benzylpenicilloate and the Con­ figuration of the Dimethyl D-Benzylpenicilloate Melting at 113-114° (Μ.ψ, 18). A solution of 525 mg. of dimethyl D-0-benzylpenicilloate

melting at 113-114° in 3.34 g. of dimethylaniline was treated at 0° with 1.93 g. of benzoyl chloride. After one-half hour, the solution was removed from the ice bath and let stand at room temperature over­ night. The reaction mixture was diluted with 25 ml. of ether, then washed with six equal volumes of N hydro­ chloric acid and three equal volumes of 5.0% sodium bi­ carbonate solution. The ether layer was evaporated to dry­ ness and the residue extracted with three 10-ml. volumes of petroleum ether to remove benzoyl chloride. Efforts to crystallize the dark colored residue were unsuccessful. The product, dissolved in 20 ml. of 2:1 ether-petroleum ether mixture, was passed over a Brockmann alumina column (20 cm. long, 1.5 cm. diameter). Elution was continued with pure ether. The first 50-ml. eluate gave 325 mg. of almost colorless product from which was secured by crystal­ lization from ether-petroleum ether, then thrice from ether, 41 mg. of constant melting rod-like crystals melting at 79-80° (micro-block), [α]ϋ22 +12.7° (c = 1 in ethanol). This N-benzoyl derivative did not depress the melting point of authentic dimethyl N4-benzoyl-D-/3-benzylpenicilloate prepared by methylation of α-methyl N4-benzoyl-D-/3benzylpenicilloate. The observed constants on the latter material were: m.p. 80-81° (micro-block); [a]n22 +14.5° (c = 1 in ethanol). Calc. for C25H28OeN2S: C, 61.97; H, 5.82; N1 5.78 Found: C, 61.38, 61.50; H, 5.58, 6.05; N, 6.05 Preparation of Synthetic Dimsthyl ο-γ-Benzylpenicilloate (M.28 , 7; cf. Shell, Sh.3, 22). To a solution of 2 g. of syn­

thetic α-methyl D-7-benzylpenicilIoate in chloroform, ethereal diazomethane was added until the yellow color persisted. After a few minutes the solvent was removed under reduced pressure. Ether was added to the residue. On scratching,

the material crystallized; yield, 1.47 g. The substance was recrystallized from acetone-ether; m.p. 109-110°. Calc.for C18H24O5N2S: C, 56.81;H, 6.32; N, 7.36; OCH3,16.3 Found: C, 56.44; H, 6.41; N, 7.27; OCH3, 14.7 Dimethyl D-7-Benzylpenicilloate (Upjohn, U .11, 16; 17, 3). To a suspension of 435.3 mg. of finely divided α-methyl D-7-benzylpenicilloate in 25 ml. of anhydrous ether was added an excess of freshly prepared, cold, dry ethereal diazomethane followed by approximately 0.5 ml. of methanol. The mixture was swirled at room temperature occasionally during the lively evolution of gas. When essentially all of the solid had dissolved, the reaction mixture was warmed gently to complete the reaction and remove excess diazomethane. The solution was filtered and the filtrate washed once with 5.0% sodium bicarbonate, then twice with water, and dried over anhydrous magnesium sulfate. The dried solution was filtered and the filtrate allowed to evaporate spontaneously whereupon the diester crystallized in burrs of slender white needles; yield, 408.4 mg. (90.6%); m.p. 107-109°; Md28 +100° (c = 0.13 in absolute ethanol). Calc. for C18H24N2O5S: C, 56.81; Ή, 6.32; N, 7.36 Found: C, 57.04; H, 6.45; N, 7.42 To a solution of 0.361 mg. of the dimethyl D-7-benzylpenicilloate in absolute ethanol was added 0.257 mg. of mercuric chloride in absolute ethanol. The volume was made up to 10 ml. After seventy minutes, the reaction mix­ ture showed an absorption maximum at 2,800-2,810 A; Eloin.1% 258.3. Regeneration of Dimethyl D-7-Benzylpenicilloate from /3-Methyl D-T-Benzylpenicilloate (Merck, M.40, 9; 41, 14).

To a chloroform solution of 2.15 g. of /3-methyl D-7-benzylpenicilloate (prepared by saponification of dimethyl D-7benzylpenicilloate), ethereal diazomethane was added until the yellow color persisted. Evaporation of the solvent under reduced pressure gave an oil which crystallized on rubbing with ether; yield, 0.83 g.; m.p. 106-108°. Authentic di­ methyl D-7-benzylpenicilloate melted at 108-109°. A mixed melting point was 108-109°. An ethanol solution containing 0.524 g. per 100 ml. of dimethyl D-benzylpenicilloate was treated with one equiva­ lent of mercuric chloride per mole of substrate. The rota­ tion changed as follows: Time after HgCU addition

[«]D24

+112° + 35° + 30° + 26° + 24.2° + 24.2° + 24.2° After 160 minutes it showed a 2,800 A band,

EM

7,460.

When the above experiment was repeated with one equiva­ lent of benzylamine added before addition of the mercuric chloride, the following change took place: Time

Md24 -18.6° -19.6° -21.6° -21.6°

After 86 minutes it showed a band of 2,800 A of EM 17,300.

PENILLOIC AND PENICILLOIC ACIDS Dimethyl D-7 -Benzylpenicilloate from α-Methyl v-yBenzylpenicilloate (Squibb, S.29, 2). A solution of 3.0 g. of α-methyl D-7-benzylpenieilloate in chloroform was treated with ethereal diazomethane until the yellow color persisted. The solution was then concentrated under reduced pressure and the residue dissolved in a small volume of chloroform. The addition of ether to this solu­ tion caused the precipitation of 2.23 g. of solid, m.p. 1ΙΟ­ Ι10.5°; [a]D22 +122° (c = 1 in methanol). Merck reported a melting point of 109-110°. A second crop of 0.5 g. was obtained by the addition of hexane to the mother liquor. This second crop of material melted at 108-109°. Preparation of Dimethyl D-7 -Benzylpenicilloate from D-7 -Benzylpenicilloic Acid (S.S1, 2). To an ethereal sus­ pension of 100 mg. of benzylpenicilloic acid, prepared by the hydrolysis of α-ethyl D-7-benzylpenicilloate and isolated via the lead salt, ethereal diazomethane was added in small portions until the yellow color persisted. After thirty minutes, the solution was concentrated in a stream of nitro­ gen. The crystalline residue was washed with a small amount of absolute ether. The product weighed 60 mg. and melted at 100-105°. The product was dissolved in chloroform, filtered from a small insoluble fraction and concentrated to a syrup. Crystallization was started by the addition of a small amount of ether. The crystals were filtered off and washed with ether. They weighed 40 mg. and melted at 106-108°. A mixed melting point with a sample of dimethyl ester (m.p. 110-110.6°) was 108-110°. The rotation was [α]η25 +111° (c = 1 in ethanol) as compared with [α]ο25 +122° (c = 1 in ethanol) for the dimethyl ester prepared from α-methyl D-7 -benzylpenicilloate. Heats of Combustion and Formation of Dimethyl D-7Benzylpenicilloate (Rossini, Prosen and Johnson, BS.2, 1). Measurements of the heats of combustion were made with the apparatus and procedure described in J. Res. Nat. Bur. Standards, 27, 289 (1941) and 33, 255 (1944). The procedure was modified in several details since this compound con­ tained sulfur. These modifications were used to obtain a more clear-cut reaction in the bomb and to improve the analysis of the products of cpmbustion. Before combustion, the samples were dried for eighteen hours in vacuo over phosphorus pentoxide. The material was formed into pellets for combustion, with about 0.75 g. being used in each experiment. A pellet of about 0.75 g. of NBS Standard Sample benzoic acid was burned along with each sample. The amount of reaction in each calorimetric experiment was determined from the mass of carbon dioxide produced from the sample. There was calculated the Heats of Combustion and Formation of Dimethyl D-7 -Benzylpenicilloate

Formula

C18H24O6N2S

Heat of formation, b + AHf 0 at 25°C.

Heat of No. of Mass of C O 2 formed combustion, 6 -AHc 0 expts. Mass of CO 2 calcd. at 25°C.

2

0.99698 + 0.00040

kcal/mole kcal/mole 2279.3 ±2.8 -233.5 ±2.8

AHc 0 represents the heat evolved in the combustion of the given sample in the solid state in gaseous oxygen to form gaseous carbon dioxide and liquid water and gaseous nitrogen and crystalline sulfur (rhombic) at 25° and constant pressure, with all the reactants and products in their appropriate standard reference states, according to the following reaction:

617

ratio of the mass of carbon dioxide formed in the combustion to the mass" of carbon dioxide calculated stoichiometrically from the mass of sample placed in the bomb for combustion. From the measured heat of combustion per mole of carbon dioxide produced there was calculated the heat of combustion per gram-formula-weight. Combination with the appro­ priate values for the heats of formation of carbon dioxide and water yielded values for the heats of formation of the compound from the elements. The results are summarized in the preceding table. The uncertainties given are esti­ mated overall uncertainties. Epimerization of Dimethyl D-7 -Benzylpenicilloate in Methanol with Sodium Methoxide (Merck, M.Jft, 13). Dimethyl D-7-benzylpenicilloate (5 g.) was dissolved in 50 ml. of absolute methanol in which 82 mg. of sodium had been dissolved. This solution showed a specific rotation of MD28 +41.0° but after refluxing for three hours, the rota­ tion could not be observed because of the dark color. The solution was cooled, diluted with 100 ml. of chloroform and washed twice with 50-ml. portions of water. The filtered chloroform solution was concentrated to dryness in vacuo to yield 3.39 g. of an amber colored gum. Attempts to obtain a crystalline hydrochloride of this dimethyl D-benzylpenicilloate werg unsuccessful. Perchloric Acid Salt of Dimethyl D-7 -Benzylpenicilloate CM.47, 10). To a solution of 760 mg. of dimethyl D-7-benzylpenicilloate in glacial acetic acid, 0.3 g. of 70% perchloric acid was added. When the solution was diluted with ether, a gel was formed. This material was removed by filtration, washed with ether and dissolved in acetone. Addition of ether to the acetone solution caused the separation of clusters of slender needles. (The attempted use of acetic acid-ether and ethanol-ether for crystallization had previously been found to result in gel formation.) The product was crystal­ lized a second time from acetone-ether and dried at 56° (1 mm.). It melted at 126-127° (capillary, placed in the bath at 110°) with some preliminary sintering at 120°. The rotation was [α]η22 +93.5° (c = 0.984 in ethanol). Calc. for Ci8H24O6N2S-HClO4: C, 44.93; H, 5.25; N, 5.83 Found: C, 44.88; H, 5.48; N , 5.92 Dimethyl Desthio-D -7 -benzylpenicilloate (M.50, 19). A solution of 1 g. of dimethyl D-7-benzylpenicilloate in 15 ml. of pure dioxane was diluted with 15 ml. of water and 20 g. of Raney nickel catalyst was added. The suspension was stirred at room temperature for 1.5 hours, then at 70° for fifteen minutes. The catalyst was removed by filtration. The filtrate was concentrated in vacuo to a volume of approximately 5 ml. The crystals that formed during the concentration were separated by filtration and dried. The yield of crystals was 240 mg., m.p. 77-79°. Extraction of the aqueous concentrate with chloroform and removal of the solvent in vacuo, etc., yielded 230 mg. of gum that did not crystallize even when seeded with the crystals isolated in the preceding step. Two recrystallizations of the crystals from ether-petroleum ether gave a product with the constant melting point of 79° and [a]D25 +28.0° (c = 1.0 in methyl alcohol). Calc. for Ci8H26N2O6: C, 61.69; H, 7.47; N, 7.99 Found: C, 61.90; H, 7.13; N, 8.33

a

OisH 24 OsN2S(e) 4~

02(g) = 18 C0 2 (g) -(- 12 H 2 Od)

N 2 (K) -I" S(c.rh)

b

AHf 0 represents the increment in the heat content of the process of forming the given compound in the solid state, from its elements, at 25°, with all the reactants and products in their appropriate standard refer­ ence states, according to the following reaction: 18 C(c, graphite) + 12 H2(g) + % 02(g) + Na(«) + S(c,rh)

= Cl8H2406N2S(c)

Dimethyl Desthio-D -7 -benzylpenicilloate Hydrochloride (M.56, 12). An ether solution (3 ml.) of 37 mg. of dimethyl desthio-D-7-benzylpenicilloate (M.50, 19) was treated with an excess of anhydrous hydrogen chloride. The resulting oily precipitate readily crystallized after standing for a few minutes; m.p. 144-148°. After two recrystallizations from methanol-ether, the slender needles melted at 145-148° (micro-block); [α]ο25 +30.6° (c = 3.92 in methanol). Calc. for Ci8H27N2OiiCl: C, 55.83; H, 7.04; N, 7.25 Found: C, 55.79; H, 7.00; N, 7.45

618

PF,NTTT,OTC, AND PENICILLOIC ACIDS

, Dimethyl N4-Formyl-D-7-benzylpenicilloate (M.29, 6; 8§, 6). To a suspension of α-methyl N4-formyl-rr-7-benzylpenicilloate in chloroform, an excess of diazomethane in ether was added. Reaction took place readily. Evapora­ tion of the solvent rapidly under reduced pressure gave a solid powder. The material could not be recrystallized, however, since treatment with solvents gave an oil. The oil was purified by distillation in a molecular still and a fraction which collected at a bath temperature of 125-130° at 0.5 micron was-analyzed. Calc. for Ci8H24OeN2S: C, 55.86; H, 5.92; N, 6.86 Found: C, 56.04; H, 6.03; N, 6.91 1 Treatment of dimethyl D-7-benzylpenicilloate with formic acid and acetic anhydride gave a similar product. • Dimethyl N4-Formyl-D-7-benzylpenicilloate (Upjohn, U.12, 3). Using the procedure of the University of Michigan group (B.S, 5), 175 mg. of crystalline dimethyl ϋ-7-benzylpenicilloate was treated with a mixture of 2 ml. of formic afeid and 1 ml. of acetic anhydride. The solution was all6wfid to stand at room temperature for twenty hours and the excess reagents were removed in vacuo. The glassy residue was highly soluble in ether and sparingly soluble in petroleum ether. All attempts to crystallize the glass failed. Without further characterization, the crude product was used for treatment with phosphorus pentasulfide (below). Dimethyl N4-Formyl-D-7-thionbenzylpenicilloate (U.12, 4). The glass presumed to be the N-formyl compound (121 mg.) was heated under reflux with 55 mg. of phos­ phorus pentasulfide in 25 ml. of benzene (procedure based on that of Gatewood and Johnson, J. Am. Chem. Soc., 48, 2900 (1926)). After heating for 1.5 hours, the solvent was removed in vacuo and the residue digested with several portions of petroleum ether. In this way 20 mg. of resinous solid was obtained. Dimethyl N4-Acetyl-D-7-benzylpenicilloate (Merck, M.47, 13). An ether solution of diazomethane was added to a solution of 6.10 g. of α-methyl N4-acetyl-D-7-benzylpenicilloate (14.9 millimoles) in 100 ml. of chloroform until the yellow color persisted. Evaporation of the solvent gave an oil "which partially crystallized on standing overnight. Ether was added and the product filtered; yield, 5.45 g., m.p. 103-105°; second crop, 0.64 g., m.p. 102-103°; total yield, 6.09 g. (97%). The material was recrystallized from abetone-ether. The analytical sample melted at 102-103°.

The equivalent weight was found to be 455 (theory 450) by adding an excess of lithium hydroxide solution and back-titrating with hydrochloric acid. It appears that one ester group was readily hydrolyzed. Dimethyl N4-Benzoyl-D-7-benzylpenicilloate (M.16, 1; 33, 7). To 7 g. of α-methyl N 4-benzoyl-D-7-benzy lpenicilloate, suspended in 150 ml. of chloroform, ethereal diazometh­ ane was added with swirling until the yellow color persisted. The solvent was evaporated under reduced pressure. A colorless oil remained, which became crystalline on stirring with ether. The dimethyl N4-benzoyl-D-7-benzylpenicilloate, m.p. 137-138°, weighed 6.7 g. It was recrystallized from acetone-ether; m.p. 137-138°. Calc. for C26H28O6N2S: C, 61.96; H, 5.82; N, 5.78 Found: C, 61.90; H, 6.04; N, 5.98 Dimethyl N4-Benzoyl-D-7-benzylpenicilloate Sulfone (M.39, 4). Dimethyl N4-benzoyl-D-7-benzylpenicilloate (32.0 mg.) was dissolved in 1.0 ml. of glacial acetic acid. To the solution were added small portions of an aqueous solution of potassium permanganate (104.6 mg. in 50 ml. of water) until a permanent permanganate color persisted, in all 6.0 ml. being required. This corresponds to approxi­ mately two atoms of oxygen per mole of penicilloate. Then after addition of 1.0 ml. of glacial acetic acid, sulfur dioxide gas was passed into the solution, decolorizing it almost instantly. Concentration to 0.5 ml. caused the separation of a colorless oil which was extracted with two 10-ml. por­ tions of ether. Evaporation of the ether left 19.1 mg. of needles melting at 169-170° (micro-block). Recrystalliza­ tion from ether did not raise the melting point of the di­ methyl N4-benzoyl-D-7-benzylpenicilloate sulfone. Calc. for C26H28N2O8S: C, 58.13; H, 5.46; N, 5.42 Found: C, 58.31; H, 5.51; N, 5.80

Attempts were made to cause the α-methyl ester group to form a benzylamide by treatment with benzylamine. Only starting material was recovered even after refluxing in xylene solution. Preparation of Dimethyl N4-Methoxalyl-D-7-benzylpenicilloate (M.60, 17; 63, 22). An excess of ethereal diazo­ methane was added to a suspension in chloroform of α-methyl N4-methoxalyl-D-7-benzylpenicilloate. The sus­ pended material dissolved as it reacted. Removal of the solvents at reduced pressure left a gum which crystallized from a mixture of acetone, ether and petroleum ether. Recrystallization from the same mixture of solvents gave elongated prisms, m.p. 150-151°; [«]D23 +183° (c = 1.18 in ethanol).

Oxidation of dimethyl N4-benzoyl-D-7-benzylpenicilloate was also carried out on 26.6 mg. using 0.02 ml. of 30% hydrogen peroxide in 0.1 ml. of glacial acetic acid and allow­ ing it to stand at room temperature for four days. tThe solu­ tion was treated with solid manganese dioxide, filtered and the filtrate was evaporated to dryness in vacuo and extracted with ether. Evaporation of the ether left a gum which was crystallized by adding petroleum ether to its benzene solution. The product melted at 163-168° (micro-block). Recrystallization from ether gave 14.3mg. of crystals melting at 167—168° (micro-block). With perbenzoic acid in chloroform, dimethyl NMjenzoyl-D-7-benzylpenicilloate used up an amount of the reagent corresponding to two atoms of oxygen per mole of penicilloate. Reaction of Dimethyl N4-Benzoyl-D-7-benzylpenicilloate Sulfone with Oxalyl Chloride (M.65, 6). Finely powdered dimethyl N^benzoyl-D^-benzylpenicilloate sulfone (300 mg.) was treated with 10 ml. of oxalyl chloride-xylene mixture (1:5). Solution was completed in approximately ten minutes. After forty minutes, the mixture was evaporated to dryness in vacuo and the residue was crystallized from benzene-petroleum ether, giving 199 mg. of product; m.p. 180-185°. After two recrystallizations from benzenepetroleum ether, the oxazolidinedione, pale yellow needles, melted at 203-204°; [ajo25 —68° (c = 0.37 in benzene); Em 9,600 at 3,525 A (in tetrachloroethane).

Gale, for C21H26O8N2S: C, 54.04; H, 5.63; N, 6.01 tfound: C, 54.29; H, 5.82; N, 6.05

Calc. for C 2T H 26 Oi0SN 2 : N, 4.91 Found: N, 4.63

Calc. for C20H26OiN2S: C, 56.85; H, 6.20; N, 6.63 Found: C, 57.12; H, 6.03; N, 6.23

Dimethyl N4-Isobutyryl-D-7-benzylpenicilloate (M.66, 1.6). To a suspension of 45 g. of α-methyl N4-isobutyryl-D7-benzylpenicilloate in ether, diazomethane was added until the reaction was complete. The ester crystallized readily; yield 16.5 g. After recrystallization froin ether the melting point was constant at 123—124°. Calc. for C22Ha0N2OeS: C, 58.65; H, 6.71; N, 6.22 Found: C, 58.87; H, 6.76; N, 6.21

Attempted Reaction of Dimethyl N4-Benzoyl-D-7-benzylpenicilloate with Nitrous Fumes (M.54, 6). A solution of 1 g. of dimethyl N4-benzoyl-D-7-benzylpenicilloate (m.p. 137-138°) in 5 ml. of acetic acid was cooled to 8°. Nitrous fumes were passed in until the solution was green (fifteen minutes). The mixture was allowed to stand at 8° for an additional hour, and then at 25° for fifteen minutes. The solution was poured into water and the resulting precipitate

PENILLOIC AND PENICILLOIC ACIDS filtered, washed with water, and dried in vacuo. The product weighed 0.8 g. and melted at 124-132°. It gave a negative Liebermann (nitrosamine) test. The material on recrystallization from acetone-ether-petroleum ether yielded 0.5 g. of unchanged starting material, m.p. 136.5-138°. Attempted Reactions of Dimethyl N1-Benzoyl-D-Ybenzylpenicilloate with Acetic Anhydride (M.56, 13). A solution of 2.00 g. (4.17 millimoles) of dimethyl N4-benzoylD-7-benzylpenicilloate, m.p. 137-138°, in 3.94 ml. (41.7 milli­ moles) of acetic anhydride was heated for twenty-five minutes at 100°. The colorless solution was cooled and 5 ml. of water and 0.65 ml. of pyridine were added. Crystals began to form immediately. After ten minutes 4.13 ml. of 1.95 N sulfuric acid and 20 ml. of water were added. The crystals weighed 1.95 mg. and melted at 136-137.5° after recrystallization from acetone-ether-petroleum ether. A solution of 1 g. of dimethyl N4-benzoyl-D-7-benzylpenicilloate in a mixture of 3 ml. of pyridine, 3 ml. of acetic anhydride and 9 ml. of xylene was refluxed for one hour. Water was then added to decompose the acetic anhydride. Sulfuric acid equivalent to the pyridine present was added and the solution was extracted with ether. The ethereal solution was washed with bicarbonate, the ether was re­ moved by distillation, and the residue was crystallized from acetone-ether-petroleum ether, yielding 0.80 g. of starting material, m.p. 136.5-137.5°, in the first crop. The bicar­ bonate washings (acid fraction) were examined for phenylacetic acid but none could be detected. In a final experiment, 1.00 g. of dimethyl N4-benzoyl-D-7benzylpenicilloate was refluxed for two hours with 4 ml. of acetic anhydride. The solution was cooled, and water and a small amount (0.65 ml.) of pyridine were added. Un­ changed starting material (0.94 g.) of m.p. 136-137° crys­ tallized from the aqueous solution. Cleavage of Dimethyl N4-Benzoyl-D-7-benzylpenicilloate with Potassium Hydroxide in Methanol (M.3S, 8). To 1 g. of dimethyl N4-benzoyl-D--Y-benzylpenicilloate (2.07 milli­ moles), dissolved in 10 ml. of methanol, 1.97 ml. of 1.05 N potassium hydroxide in methanol (2.07 millimoles) was added. After standing overnight, the methanol was evaporated under reduced pressure. Ether and water were added to the residue. The ether layer yielded 0.17 g. of material, m.p. 119-125°. After recrystallization from acet­ one-ether, it melted at 137-138°. An authentic sample of methyl /3,/S-dimethyl-a-benzoylaminoacrylate melted at 137-138°. A mixed melting point showed no depression. Dimethyl N4-Phenylaeetyl-D-7-benzylpenieilloate (M.29, 13). To 1 g. of synthetic dimethyl D-7-benzylpenicilloate (6.23 millimoles), dissolved in 10 ml. of benzene, 0.63 g. (5.2 millimoles) of dimethylaniline and 0.41 g. (2.63 milli­ moles) of phenylacetyl chloride were added. After standing overnight, the benzene solution was extracted thoroughly with dilute hydrochloric acid, washed with water, dried and evaporated. Theresiduewasan oil which did not crystallize. Dimethyl N4-Carbophenoxy-D-7-benzylpenicilloate (M.5S, 14). A solution of 0.5 g. of α-methyl N4-carbophenoxy-D-7benzylpenicilloate (M.33, 3) in chloroform was treated with an excess of diazomethane in ether. Removal of the solvents at reduced pressure left an oil which crystallized when ether was added. Recrystallization from acetone-ether-petro­ leum ether gave prismatic needles (0.4 g.), m.p. 133-134° (capillary). Calc. for C26H28O7SN2: C, 59.96; H, 5.64; N, 5.60 Found: C, 60.01; H, 5.62; N, 5.46 Preparation of Dimethyl D-S-Benzylpenicilloate from D-S-Benzylpenicilloic Acid (Squibb, S.33, 1; 36, 23). A suspension of 0.3 g. of D-S-benzylpenicilloic acid (obtained by the decomposition of its copper salt) in anhydrous ether was treated with an ethereal solution of diazomethane until the evolution of nitrogen had ceased and the yellow color persisted. The ethereal solution was then concentrated in a current of nitrogen. The residue was dissolved in

619

chloroform and the solution centrifuged to remove a small amount of insoluble material. Concentration of the chloro­ form solution gave a viscous syrup. The addition of anhy­ drous ether to the syrup caused crystallization of a solid, m.p. 80-103°. After two recrystallizations from a chloro­ form-ether mixture, 145 mg. of solid, m.p. 111-114°, was obtained; [ajn23 —40° (c = 1 in methanol). Calc. for C18H21O6N2S: C, 56.84; H, 6.32; N, 7.39; OCH3, 16.32 Found: C, 56.55; H, 6.21; N, 7.48; OCH3, 16.47 A mixed melting point of this ester with dimethyl D-7-benzylpenicilloate (m.p. 110-110.5°) showed a marked depres­ sion, being 90-102°. The ultraviolet absorption of the dimethyl ester is almost identical with that of α-ethyl D-7-benzylpenicilloate and D-benzylpenicilloic acid. On addition of one equivalent of mercuric chloride to the alcoholic solution the characteristic "penicilloate band" at 2800 A developed to high intensity

EM 10,000.

A second esterification was carried out on 1.3 g. of D-Sbenzylpenicilloic acid (obtained through the copper salt). In this run, the chloroform solution of the product was washed with saturated sodium bicarbonate solution to remove any unreacted acid. The diester, isolated in the same manner as indicated previously, weighed 0.56 g. and melted at 115-117°; [«]D2S —43° (c = 1 in methanol). Levorotatory Dimethyl D-S-Benzylpenicilloate (S.36, 24). A suspension of 100 mg. of cupric salt of u-S-benzylpenicilloic acid, prepared from the copper salt from benzylpenicillin (S.33, 11), in 50 ml. of dry ether was treated with ethereal diazomethane until solution resulted and a slight excess of the reagent was present. The solution was evaporated and the residue taken up in a little chloroform. On addition of ether and hexane the diester was obtained in form of large prisms. It melted at 11&-117° after softening at 115° and showed no depression when mixed with the di­ methyl D-S-benzylpenicilloate, m.p. 114-116°, obtained via the cupric salt from D-7-benzylpenicilloic acid. [α]η23 —35° (c = 1.0 in methanol). Calc. for Ci8H24O6N2S: C, 57.05; H, 6.45 Found: C, 56.82; H, 6.35 Regeneration of Dimethyl D-S-Benzylpenicilloate from /3-Methyl D-S-Benzylpenicilloate (S.33, 2). A suspension of 100 mg. of β-methyl D-S-benzylpenicilloate in anhydrous ether was treated with ethereal diazomethane until the yellow color persisted. Upon removal of the ether in a cur­ rent of nitrogen an oil was obtained. This was dissolved in a small amount of ether and allowed to stand at 0° for fortyeight hours. The crystalline material was centrifuged off, washed with ether and recrystallized from a mixture of chloroform and ether. The ester, which weighed 7.2 mg., melted at 108-111°; [Q:]D2S —30.5° (c = 0.7 in methanol). A mixed melting point of this compound with a sample of the dimethyl ester, m.p. 115-117°, [«ID25 —43°, was 113-115°. Dimethyl N4-Benzoyl-D-S-benzylpenicilloate from Di­ methyl D-S-Benzylpenicilloate (S.33, 3; 35, 1; 37, 1). The benzoylation was carried out by the procedure for α-ethyl /3-methyl N4-benzoyl-D-7-benzylpenicilloate (Merck, M.JiIt, 15). To 200 mg. of dimethyl D-S-benzylpenicilloate, MD23 —43° (c = 1 in methanol), dissolved in 5 ml. of an­ hydrous benzene and 1.28 g. of dimethylaniline, a solution of 0.67 g. of benzoyl chloride in 2 ml. of benzene was added dropwise. The solution was allowed to stand at room tem­ perature for forty-eight hours, diluted with benzene and washed with saturated sodium bicarbonate solution, 5% hydrochloric acid, saturated sodium bicarbonate solution and finally with saturated sodium chloride solution. The benzene solution was dried over sodium sulfate and then concentrated at room temperature to an oil. The excess benzoyl chloride was decomposed by heating with aqueous pyridine on the steam bath for five minutes. The product

620

PENILLOIC AND PENICILLOIC ACIDS

was extracted with benzene which was then washed with dilute hydrochloric acid, saturated sodium bicarbonate solution and saturated sodium chloride solution. The benzene solution was dried over sodium sulfate and con­ centrated under reduced pressure to an oil, which did hot crystallize. The oil was dissolved in benzene and hexane was added until the solution became cloudy. The solution was then chromatographically adsorbed on an alumina column. The column was then eluted with hexane, hexane-ether solution, ether, ether-chloroform solution, chloroform and finally methanol. The methanol eluates upon concentration yielded an oil which was granulated by trituration with hexane. The solid, after crystallization from a mixture of ether and hexane, weighed 135 mg.; m.p. 109-110°. Recrystallization from ether raised the melting point to 110-110.5°; MD25 +26.6° (c = 0.94 in methanol).

worked Up in the usual manner to yield a yellow oil, presum­ ably a mixture of dimethyl D-benzylpenicilloates.

In a typical isolation, 12.8 g. of methylated mother liquors from the preparation of α-methyl D-7-benzylpenicilloate was dissolved in 675 ml. of benzene and 1,020 ml. of petroleum ether added. The turbid solution was put on a column of 282 g. of alumina which had been acid-washed, but not especially heat-activated. The column was then washed with 800 ml. of a 40% benzene-60% petroleum ether mixture. The fractionation which followed is indicated below. Frac­ tion num­ ber

Calc. for C26H28O6N2S: C, 61.98; H, 5.78; N, 5.78 Found: C, 61.60; H, 5.79; N, 5.93 In another preparation, a solution of 95 mg. of dimethyl D-5-benzylpenicilloate and 65 mg. of dimethylaniline in 4 ml. of benzene was treated with a solution of 38 mg. of benzoyl chloride in 2 ml. of benzene. The reaction mixture was allowed to stand at room temperature for four days. Benz­ ene (20 ml.) was added and the solution washed with dilute hydrochloric acid, saturated sodium bicarbonate solution and saturated sodium chloride solution. The benzene solution was dried over magnesium sulfate and freeze-dried. The addition of a small amount of ether caused the solid to dissolve and then immediately crystallize out. Hexane was added to complete the crystallization. The solid was collected and washed with a small amount of ether. The product weighed 75 mg. and melted at 110-111°; [«ID25 +26.0° (c = 1 in methanol). A mixed melting point of this product with a sample of the previously prepared N-benzoyl derivative showed no depression. Hydrolysis of Dimethyl D-a-, D-/3-,. and D-7-Benzylpenicilloates to Methyl Benzylpenaldate (Merck, Μ.Β0, 25). Solutions of 18 mg. of dimethyl ο-α-, τ>-β-, and D-7-benzylpenicilloates in 1 ml. of methyl alcohol were prepared. To each were added 1 ml. of a saturated solution of 2,4-dinitrophenylhydrazine in 2 JV hydrochloric acid and then 5 ml. of water. In two hours at room temperature crystalliza­ tion began. In fifteen hours approximately 5 mg. quanti­ ties of methyl benzylpenaldate 2,4-dinitrophenylhydrazone were secured. The products melted at 180-182° and showed no depression on admixture with authentic 2,4-dinitrophenylhydrazone of methyl benzylpenaldate. Preparation of Dimethyl D-Benzylpenicilloate in Aqueous Solution (M.S7, 21). A solution of 2 g. of 2-benzyl-4hydroxymethylene-5(4)-oxazolone in 15 ml. of methanol was refluxed for fifteen minutes, cooled to room temperature and added to a solution of 2.1 g. of D-penicillamine methyl ester hydrochloride and 1.05 g. of potassium acetate in 15 ml. of water. On standing overnight at room temperature, an oil separated. The methanol was removed in vacuo and the water was decanted. The oil was then dissolved in 150 ml. of ethyl ether and washed with 50 ml. of water. The filtered ether solution was concentrated to dryness in vacuo to yield 2.4 g. of clear yellow gummy dimethyl D-benzylpenicilloate. A portion of the gum was converted to a crystalline hydro­ chloride, m.p. 102°. The melting point did not change on recrystallization. The specific rotation was [«]D28 +63.8°. A sample of the dimethyl D-benzylpenicilloate prepared by the toluene procedure yielded a hydrochloride, m.p. 110° and [«]D28 +91°. There was no depression in mixed melting point. Dimethyl N4-Methyl-D-benzylpenicilloate (M.47, 17; 50, 26; 63, 12). The mother liquors from the preparation of α-methyl D-7-benzylpenicilloate, in methanol-ether solution were treated with a small excess of diazomethane and then

1 2 •

3 4 5 6 7 8

9

10 11 12 13 14 15 16 17

Eluents

40% Benzene-60% Ether (2500 ml.) 50% Benzene-50% Ether (600 ml.) 60% Benzene-40% Ether (600 ml.) 70% ijenzene-30 % Ether (600 ml.) 80% Benzene-20% Ether (275 ml.) 80% Benzene-20% Ether (275 ml.) 80% Benzene-20% Ether (275 ml.) 90% Benzene-10 % Ether (275 ml.)

Residue

Remarks

Pet. 0 Pet. 0 Pet. 0 Pet.

0 Pet. 0.005 g. Noncrystallizaable oil. Pet. 0.024 g. Noncrystallizable oil. Pet. 0.037 g. Noncrystallizable oil. Pet. 0.050 g. Ether-pet. ether gave mixture of crystals and oil. 90% Benzene-10 % Pet. 0.098 g. Ether-pet. Ether (275 ml.) ether gave crystals: 29.8 mg. 90% Benzene-10 % Pet. 0.106 g. Crystals: 47.9 Ether (275 ml.) mg. 95% Benzene-5% Pet. 0.107 g. Crystals: 43.6 Ether (275 ml.) mg. 95% Benzene-5% Pet. 0.112 g. Crystals: 47.9 Ether (275 ml.) mg. 100% Benzene (275 ml.) 0.109 g. Crystals: 29.9 mg. 100% Benzene (275 ml.) 0. 111g. Poorly crystal­ line material: 25.1 mg. 100% Benzene (275 ml.) 0.116g. Noncrystallizable oil. 100% Ether (600 ml.) 1.040 g. Oil. 100% Methanol (600 ml.) 10.061 g. Yellow oil.

As indicated in the table, fractions 9-14 gave on crystalliza­ tion from ether-petroleum ether a total of 0.224 g. of a product, m.p. 77-80°. The mother liquors from these crystallizations were combined and allowed to evaporate slowly at room temperature. The residue crystallized on standing for several days. The material was taken up in ether, decolorized with Norit, filtered, concentrated, and petroleum ether added. Long, slender needles (150 mg.), m.p. 77-80°, were obtained. The total yield of material, m.p. 77-80°, was 0.374 g. (2.9%). Recrystallization from ether-petroleum ether afforded 0.324 g. (2.5%) of crystal» melting at 83-84° (micro-block), [O]D!' +27° (c = 0.785 in absolute ethanol). Further recrystallizations from aqueous methanol gave material which melted at 83-84.5° (micro-

PENILLOIC AND PENICILLOIC ACIDS block). It has been characterized as a dimethyl N1-HiethylD-benzylpenicilloate. Calc. for Ci9H26 N 2O5S: C, 57.85; H, 6.64; N, 7.10; OCH 3 , 15.7; N-CH 3 , 3.8 Found: C, 57.76, 57.88; H, 6.41, 6.46; N, 7.37, 7.09; OCH3, 16.0, 13.4; N-CH 3 , 3.0 In ethanol solution this material showed an ultraviolet absorption qualitatively similar to, although lower than, that of the known dimethyl D-benzylpenicilloates. E m (M. Wt. = 380) A

2520 2580 2645

Compound of m.p. 84-86°

Dimethyl D-/3-Benzylpenicilloate

340 (Peak) 280 (Peak) 195 (Peak)

665 (Peak) 665 (Peak) 512 (Inflection Point)

In absolute ethanol solution containing one equivalent of mercuric chloride a band formed slowly at 2,825 A, reaching a final value of Em 1930 after twenty-three hours. Under these conditions the known dimethyl D-benzylpenicilloates form an absorption band at the same wave-length which grows rapidly to a much higher value (EM about 16,000). A potentiometric titration indicated that this compound reacts rapidly with approximately one mole of alkali to give the salt of a monobasic acid of pK 3.5. The methyl benzylpenaldate moiety was isolated after treatment with mercuric chloride in aqueous methanol. Partial Saponification and Remethylation of Dimethyl N4-Methyl-D-benzylpenicilloate (M.50, 29). An aqueous methanolic solution of 119 mg. of the pure material was treated with one equivalent of sodium hydroxide solution and the free acid isolated in the usual manner. The product (110 mg.) was dissolved in ether containing a trace of methanol and an ethereal solution of diazomethane added. On working up, 86 mg. (78%) of crystals melting at 82.5-84° was readily obtained. The product did not depress the melt­ ing point of a sample of starting material. Dimethyl 6-Methyl-D-benzylpenicilloate and Its Saponifi­ cation to the /3-Methyl 6-Methyl-D-benzylpenicilloate (Squibb, S.41, 1). A mixture of 1.24 g. of D-penicillamine methyl ester hydrochloride and 2.0 g. of methyl a-phenylacetamido-a-methyl-/3,/3-diethoxypropionate was fused for fifteen minutes at 115°. The cooled melt was dissolved in 50 ml. of methanol and 57.2 ml. of 0.2174 N sodium hydroxide was added with cooling. The solution was allowed to stand for twenty-four hours at room temperature. The methanol was then distilled off under reduced pressure and the faintly alkaline aqueous solution decanted from the slight gummy deposit which had formed. The aqueous layer was neutralized with dilute acetic acid and 8 ml. of a 10% lead acetate solution was added. The precipitate which formed immediately was collected after the solution had been allowed to stand at 0° for fifteen minutes. The lead salt was washed with cold water and weighed. 1.45 g. after drying. A suspension of the lead salt in 70 ml. of water was treated with hydrogen sulfide to remove the lead as lead sulfide. The solution was filtered and the precipi­ tate washed with water. The filtrate and washings were combined and freeze-dried to give 0.95 g. of /3-methyl 6-methyl-D-benzylpenicilloate. Calc. for Ci8H24N2O5S: C, 56.80; H, 6.32; neut. equiv., 380 Calc. for Ci8H24N2O5S HH 2 O: C, 55.50; H, 6.43; neut. equiv., 389 Found: C, 55.76; H, 5.97; neut. equiv., 376

621

The dimethyl 6-methyl-D-benzylpenicilloate was also prepared by treating an ethereal solution of α-methyl 6-methyl-D-benzylpenicilloate (S.S9, 2) with ethereal diazomethane. The neutral fraction from this reaction, dimethyl 6-methyl-D-benzylpenicilloate, was saponified with one equivalent of alkali under conditions similar to those described above. The reaction mixture was acidified and the /3-methyl 6-methyl-D-benzylpenicilloate extracted. The crude acid had a neutral equivalent of 503 (theory, 380). Dimethyl N7-Benzyl-D-benzylpenicilloate (Merck, M.76, 5). An amorphous sample of α-methyl N'-benzyl-D-benzylpenicilloate (p. 587) was esterified with diazomethane and yielded an oil which partially crystallized in the form of slender needles after standing for several days. The amount of crystalline material obtained was insufficient to permit purification. Hydrogenolysis of Dimethyl N7-Benzyl-D-benzylpenicilloate (M.76, 6; 78, 3). Hydrogenolysis of dimethyl N7-benzyl-D-benzylpenicilloate was carried out in an effort to obtain dimethyl N7-benzyl-desthio-D-benzylpenicilloate. A solution of 1 g. of the oily dimethyl ester, presumably a mixture of stereoisomers, in 50 ml. of methanol was heated and stirred on a steam cone for ninety minutes with 10 g. of Raney nickel catalyst. The catalyst was removed by centrifuging, and was extracted with additional methanol. Concentration of the combined methanol solutions under reduced pressure left a light yellow gummy residue which did not yield any crystals. The sulfur-free oil was extracted repeatedly with 2.5 N hydrochloric acid. When the acid extract was made alkaline with sodium bicarbonate a small amount of oil was precipitated. This material was extracted into chloro­ form, and the chloroform extract was concentrated under reduced pressure to leave an oil. The oil was dissolved in ether, and the addition of an ethereal solution of p-toluenesulfonic acid caused the precipitation of an oil which failed to crystallize. The bulk of the hydrogenolysis product consisted of a neutral oil which cannot be extracted into either hydro­ chloric acid or sodium bicarbonate solution. No crystals were obtained from this neutral fraction. The crude hydrogenolysis product seems to contain little or no acidic material. Dimethyl α-Thiol-D-benzylpenicilloate (Reaction of MethylBenzylpenicillinwithMethylMercaptan) (M.64, 5). To a solution of 224 mg. of methyl benzylpenicillin in 2 ml, of benzene in a small ampoule was added 2 ml. of methyl mercaptan and a small drop of N-ethylpiperidine. The ampoule was sealed and allowed to stand at 25° for four days. The solution was then evaporated in vacuo to a glassy residue which was dissolved in benzene, washed with dilute acid and with water, dried over magnesium sulfate and evaporated in vacuo to a glassy solid. The substance showed an activity of 0.2 U./mg. calculated as methyl benzylpenicillin (less than 4 % of the original activity) when assayed in vitro. It had [a]D +20° (c = 0.765 in methanol). Calc. for Ci8H24 N 2O4S2: C, 54.52; H, 6.10 Found: C, 55.50; H, 6.00 Preparation of α-Ethyl /3-Methyl D-a-Benzylpenicilloate (Squibb, S.2%, 2). A suspension of 500 mg. of α-ethyl D-abenzylpenicilloate in 5 ml. of anhydrous ether was treated with an excess of diazomethane in ether solution. The solid went into solution and nitrogen was evolved. The reaction mixture was allowed to stand for eighteen hours. The slow evaporation of the ether during this time caused the formation of crystals on the side of the container. These were removed for subsequent use as seed crystals. The volume of the solution was made up to 50 ml. and the solution washed with dilute acetic acid, water, then sodium bicarbonate solution. After drying over magnesium sulfate, the ether solution was concentrated to 2 ml. and seeded. The crystals, after filtering and washing with hexane,

622

PENILLOIC AND PENICILLOIC ACIDS

weighed 390 mg. and melted at 83-86°. After recrystallization from ether the product melted at 85-86°. Calc. for Ci9H26O6N2S: C, 57.58; H, 6.60 Found: -C, 57.93; H, 6.55 a;-Ethyl /3-Methyl D-a-Benzylpenicilloate Perchlorate from α-Ethyl /3-Methyl D-/3-Benzylpenicilloate (Merck, M-47, 15). To a solution of 633 mg. of α-ethyl /8-methyl D-/3-benzylpenicilloate in 4 ml. of glacial acetic acid was added a solution of 1.0 equivalent of 70% perchloric acid in acetic acid. Immediately after mixing, the solution was diluted with ether until it became turbid. On standing overnight at room temperature, 622 mg. (78%) of nicely crystalline material was obtained. The product melted at 121-136° (micro-block), [24 —19.4°; this was unchanged after thirty-six minutes, at which time the solution had E m 17,100 at 2,800 A. In the presence of pyridine the rotation was [a] D 24 +20.2° after eight minutes and changed to [a] D 24 +17.1° after thirty-six minutes. The ultraviolet absorption spectrum of the final solution showed E m 11,500 at 2,800 A. α-Ethyl ^-Methyl D-Benzylpenicilloate (Upjohn, U.ll, 14). α-Ethyl D-benzylpenicilloate (1.122 g.) was finely pow­ dered and suspended in 50 ml. of dry ether. To the suspen­ sion was added an excess of an anhydrous ethereal solution of diazomethane and approximately 1 mi. of anhydrous methanol. When the brisk reaction had subsided and the solid dissolved, excess diazomethane and solvent were removed by warming gently on the steam bath and finally in a current of nitrogen. The clear viscous residue was taken up in ether, filtered and washed once with 5% sodium bi­ carbonate, then twice with water, and dried over anhydrous magnesium sulfate. The solution was allowed to evaporate spontaneously and then dried in a vacuum desiccator over phosphorus pentoxide. The water-clear oil crystallized slowly, crystallization being complete after about four days; yield, 1.07 g. A small sample of the crude crystalline ma­ terial was triturated with ice-cold 50 % ethanol and the crys­ talline material separated by centrifugation. After repeating this procedure and drying in a stream of nitrogen, the crys­ talline product melted at 84-85.5°. Calc. for Ci9H26N2O5S: C, 57.85; H, 6.64; N, 7.10 Found: C, 57.61; H, 6.52; N, 7.04

PENILLOIC AND PENICILLOIC ACIDS

624

Reaction of α-Ethyl /3-Methyl D-Benzylpenicilloate with Phosphorus Tribromide (Parke-Davis, PD.19, 3). To a solution of 1.00 g. (2.53 millimoles) of α-ethyl /3-methyl Dbenzylpenicilloate in 125 ml. of absolute ether was added 1.37 g. (0.48 ml., 5.06 millimoles) of phosphorus tribromide. A flocculent white precipitate formed immediately. The reaction mixture was kept in the refrigerator for seventeen hours. The white solid was rapidly filtered in an atmosphere of dry nitrogen and washed on the filter with a total of 100 ml. of dry ether. After being desiccated for one hour in vacuo over phosphorus pentoxide, the white, fluffy, hygroscopic salt was suspended in 50 ml. of dry ether, cooled in ice and treated with an excess of ethereal diazomethane. The lat­ ter reacted with the evolution of nitrogen. Concentration of the solution in a stream of dry nitrogen caused the separa­ tion of some sticky white solid. The product was dissolved in a minimum of dry ether, filtered and placed in the refrigerator. Crystals separated which were collected, washed with a little cold ether and desiccated over phosphorus pentoxide; m.p. 84-85°; weight 0.3 g. A mixed melting point deter­ mination with starting material gave a value of 85-86°. Removal of ether left a pale yellow viscous oil. The results suggested that the phosphorus tribromide forms an addition compound which tends to epimerize the diester, m.p. 85-86°, to a mixture of diastereoisomers. α-Ethyl /3-Methyl N4-Methyl-D-benzylpenicilloate (Merck, M.44, 16; 5S, 12). A previous section reported the chroma­ tography of material obtained by epimerizing α-ethyl x>-7-benzylpenicilloate, removing unchanged α-ethyl D-γbenzy lpenicilloate by crystallization from ether, and esterifying the residue with diazorilethane. The isolation of α-ethyl /3-methyl D-jS-benzylpenicilloate from some middle fraction^ was described. The forefractions of this chromatograph were combined and rechromatographed. On long standing in ether-petro­ leum ether solution, material from the first elutions yielded a small quantity of crystals, m.p. 90-92°. After two recrystallizations from benzene-petroleum ether the compound melted constantly at 92-93° (micro-block); [α]ι>23 +30° (ethanol). In an effort to obtain more material for characterization 4,98 g. of epimerized, esterified material from α-ethyl D-γbenzylpenicilloate was chromatographed over alumina. The first two fractions (eluted by 50% benzene-50% petro­ leum ether and by 60% benzene-40% petroleum ether) gave 15 mg. (0.3%) of crystalline material. After one recrystallization from ether-petroleum ether the product (5 mg.) melted at 91-93°. The ultraviolet absorption of this compound in ethanol solution resembles closely that of the dimethyl N4-HiethylD-benzylpenicilloate. Compound of Wave­ Dimethyl N4-Methyl-D-benzylpeni- m.p. 92-93° length cilloate EM (Assumed A Em (M. Wt. 394) M. Wt. 409)

2,520 2,580 2,645

352 (peak) 290 (peak) 202 (peak)

307 (peak) 286 (peak) 192 (peak)

Upon addition of one equivalent of mercuric chloride to the ethanol solution a band formed slowly at 2,830 A, reaching a value of Em 1,720 after nineteen hours. This behavior is almost identical with that of the dimethyl N4-Hiethyl-Dbenzylpenicilloate. To a solution of 5.5 mg. of the compound of m.p. 92-93° in 0.13 ml. of methanol was added 0.8 ml. of saturated aque­ ous mercuric chloride solution. The white precipitate was

extracted with water and the aqueous extract treated with 1 ml. of a saturated solution of 2,4-dinitrophenylhydrazine in 2 N hydrochloric acid. The resulting precipitate was washed repeatedly with water and then dried. The ma­ terial (0.4 mg.) melted at 186-190° (micro-block). Recrystallization from methanol gave yellow needles, m.p. 189-191°. The melting point of benzylpenaldate ethyl ester 2,4-dinitrophenylhydrazone (m.p. 191-192.5°) was not depressed by admixture. The final identification of the compound, m.p. 92—93°, as an α-ethyl /3-methyl N 4-methy1-D-benzylpenicilloati follows from its independent preparation from dimethyl N4-HiethylD-benzylpenicilloate. α-Ethyl /3-Methyl N4-Methyl-D-benzylpenicilloate from Dimethyl N4-Methyl-D-benzylpenicilloate (M.S3, 14). A solution of 82.5 mg. of pure dimethyl N4-methyl-D-benzylpenicilloate, m.p. 83-84°, in 2.1 ml. of methanol and 0.8 ml. of water was treated over a period of four hours with 2.02 ml. of 0.1076 N sodium hydroxide. The resulting solution was allowed to stand overnight and was then worked up in the usual manner, yielding 80 mg. of acidic product. This was dissolved in ether and treated with a small excess of diazoethane. The solvent was removed and the residue crystal­ lized readily from ether-petroleum ether, giving 43 mg. of crystals, m.p. 90.5-92.5°. It was recrystallized twice from benzene-petroleum ether, giving fine needles, m.p. 92-93°. Calc. for C20H28N2O6S: C, 58.66; H, 6.89; N, 6.84 Found: C, 58.61; H, 6.72; N, 6.29 A mixture of this product and the material of m.p. 92-93° described in the preceding section showed no depression of melting-point. The stereochemical configuration of the two N-methylpenicilloates is undetermined. However, the fact that the two are· configurationally identical follows rigorously from the conversion of one to the other, since it has been demon­ strated (M.50, 29) that the saponification of the a-carbomethoxy group in the dimethyl N 4-methyl-D-benzylpenicil­ loate proceeds with retention of configuration. Attempted Preparation of α-Phenyl /3-Methyl D-Benzylpenicilloate {M.29, 10). To 9 g. of 2-benzyl-4-hydroxymethylene-5(4)-oxazolone in 200 ml. of dry toluene was added 4.1 g. of phenol in 10 ml. of dry toluene; the mixture was heated on a steam bath for twenty-five minutes. One-half of the resultant clear solution was treated with 2.45 g. of D-penicillamine methyl ester in 10 ml. of dry toluene and the solution refluxed with a water trap for nine­ teen minutes. In the first four minutes, 0.15 ml. of water had distilled; a total of 0.18 ml. was obtained compared to a theory of 0.22 ml. The toluene was removed by concentrat­ ing in vacuo and the residue was dissolved in 400 ml. of ethyl ether. An oil separated on concentrating, but it redissolved readily on slight warming. The solution slowly darkened on standing but crystals did not separate. α-Benzyl /3-Methyl D-Benzylpenicilloate (Upjohn, JJ.11, 24). A suspension of 239.7 mg. of finely divided amorphous α-benzyl D-benzylpenicilloate in 15 ml. of dry ether was treated with an excess of cold, dry ethereal diazomethane. Evolution of gas began immediately; the reaction was accelerated by the addition of fifteen drops of methanol. The reaction was completed within about ten minutes and excess diazomethane was removed by warming very gently on the steam bath. The slightly colored residual ethereal solution was treated with Darco at room tempera­ ture for a few minutes and filtered into a separatory funnel. The solution was washed once with 5% sodium bicarbonate, twice with water and dried over anhydrous magnesium sulfate. After filtering from the drying agent the solution' was allowed to evaporate spontaneously; yield, 146.6 mg. (58.2 %) of a cloudy, viscous gum. For analysis the material was dried in high vacuum at room temperature. Calc. for C24H28N2O6S: C, 63.13; H, 6.18; N, 6.14 Found: C, 63.70; H, 6.57; N, 5.76

PENILLOIC AND PENICILLOIC ACIDS α-Benzyl /J-Methyl D-Benzylpenicilloate (U.lSa, 6; 16, 18). Two grams (0.01 mole) of D-penicillamine methyl ester hydrochloride and 4.0 g. (0.01 mole) of the benzylamine derivative of benzyl benzylpenaldate (second crop material not recrystallized) were warmed in 43 ml. of ethanol until all of the solid dissolved and for ten minutes thereafter. The yellow solution was filtered and carefully diluted with water and seeded. Water (room temperature) was added until the solution became quite turbid and droplets of oil became visible. The oil was dissolved by warming, and the solution allowed to cool to room temperature. Within a few minutes crystallization started, and after one hour the nearly solid mass of crystals was placed in the refrigerator overnight. The crystals were filtered, washed with cold 50% ethanol, pressed as dry as possible, then thoroughly dried in a vacuum oven at 40°. The yield of diester melting at 96-98° was 3.3 g. or 71.5%. The compound was analyzed without further purification. Recrystallization can, how­ ever, be effected readily from a variety of solvents including dilute alcohol, ether, light petroleum, and ether-heptane mixtures. Calc. for C2IH28N2O6S: C, 63.13; H, 6.18; N, 6.14 Found: C, 62.97; H, 6.34; N, 6.30 The crude mixture of diesters was prepared as before in 90% yield; m.p. 91-96° (turbid melt and previous soften­ ing); [5 +55.0°. On standing overnight at room temperature the solution showed [afo25 +33.3°. Hydrazine Salt of the α-Hydrazide of D-a-Benzylpeni­ cilloic Acid (M.60, 16). To a solution of 230 mg. of α-methyl D-a-benzylpenicilloate in 3 ml. of absolute methanol was added 0.2 g. of 85 % hydrazine hydrate. After refluxing for ten minutes, the solution was concentrated to dryness in vacuo to yield a transparent gum which was triturated with 20 ml. of absolute ethyl ether until all of it was transformed into a flaky white solid. It was dried at room temperature.

631

Calc. for Ci6H26N6O4S-H2O: C, 46.14; H, 6.77 Found: C, 46.21; H, 6.80 α-Hydrazide of D-Y-Benzylpenicilloic Acid (Squibb, S.S9, 3). A solution of 600 mg. of the hydrazine salt in 5 ml. of water was cooled to 5° in an ice bath and dilute hydrochloric acid added dropwise until precipitation of the white solid was complete. The mixture was then filtered and the solid washed twice with cold water. After two recrystallizationa from aqueous alcohol, it melted at 154°, resolidified, and remelted at 188° (dec.). Calc. for Ci6H22N4O4S: C, 52.46; H, 6.01; N, 15.30 Found: C, 52.27; H, 5.72; N, 15.07 α-Hydrazide of D -7-Benzylpenicilloic Acid (Merck, Af.47, 4, 20; 57, 12; 60, 15). To a solution of 2 g. of α-methyl D-7-benzylpenicilloate in 20 ml. of absolute methanol, 1 ml. of 85% hydrazine hydrate was added. After refluxing for ten minutes, the solution was concentrated to dryness in vacuo and the colorless gum was crystallized from methanolether to yield 1.53 g. of the hydrazine salt, m.p. 144-146" (dec.). Recrystallization from methanol-ether yielded 0.9 g.; m.p. 146-146.5° (dec.). The 0.9 g. of the hydrazine salt of the α-hydrazide was dissolved in 2 ml. of water at 10° and the solution was acidified to ρII 4.5 with 1 N hydrochloric acid when precipi­ tation seemed to be complete. The precipitate was filtered, washed with 5 ml. of cold water, dried in vacuo to yield 0.80 g. of a white solid, m.p. 141-143°, followed by solidifica­ tion and remelting at 187-189° (dec.). The 0.8 g. of the acid was recrystallized from methanol to yield 0.4 g. of crystals melting at 148°, followed by solidification and remelting at 191-192° (dec.).

Calc. for Ci6H22N4O4S: N, 15.26 Found: N, 15.8 A solution of 1 g. of the hydrazine salt of α-hydrazide of acid, m.p. 145-146°, in 2 ml. of water was cooled to freezing with a methanol-dry ice bath. The slushy mixture was acidified to pH 4.5 by the dropwise addi­ tion of 1 N hydrochloric acid, care being taken to keep the mixture at 0°. The precipitate was centrifuged, supernatant liquid was decanted, and the precipitate was washed with 4 ml. of cold water (5°), then centrifuged again. The solid was dried in vacuo overnight to yield 0.84 g. of material melting at 125-126° followed by solidification and remelting at 183-187°. D-7-benzylpenicilloic

Calc. for Ci6H22N4O4S-H2O: C, 50.04; H, 6.30 Found: C, 50.38; H, 6.28 When the α-hydrazide of D-7-benzy lpenicilloic acid was recrystallized several times from methanol or aqueous methanol (warm) the melting point rose steadily until a product melting constantly at 221-222° was obtained. This was found to be identical by mixed melting point determina­ tion with 4-phenylacetamido-5-pyrazolone. Calc. for CiiHi1N3O2: C, 60.82; H, 5.07 Found: C, 60.86; H, 5.21 Hydrazine Salt of the α-Hydrazide of D-7-Benzylpenicilloic Acid {M.44-, 15; 4-7, 20; 50, 21). A solution of 1 g. of α-methyl D-7-benzylpenicilloate in 10 ml. of absolute ethanol was treated with 1 ml. of 85 % hydrazine hydrate, refluxed for ten minutes, and concentrated to dryness in vacuo. The residue was recrystallized from absolute methan­ ol-ether to yield 0.83 g. of crystals melting at 145-146° (dec.). The analyses indicated that one molecule of hydrazine was bound to two molecules of the acid hydrazide. When this salt was recrystallized from methyl alcohol con­ taining a few added drops of hydrazine hydrate (3 drops per 5 ml.) a salt was obtained where the ratio of hydrazine to the

632 acid hydrazide is one to one. (dec.).

PENILLOIC AND PENICILLOIC ACIDS This salt melted at 145-146°

The analysis indicated that this substance is /3-methyl D-benzylpenicilloate α-hydrazide.

Calc. for CieH2eNeO1S: C, 48.28; H, 6.58; N, 21.11 Found: C, 48.19; H, 6.68; N, 21.13

Calc. for Ci7H21N4O1S: C, 53.6; H, 6.4; N, 14.7 Found: C, 53.1; H, 6.8; N, 14.3

Hydrazine Salt of the α-Hydrazide of D-7-Benzylpenicilloic Acid (Squibb, S.35, 3; 37, 2). A suspension of 4.0 g. of α-ethyl D-7-benzylpenicilloate in 5 ml. of absolute alcohol was cooled in an ice bath and 2 ml. of 85 %hydrazine hydrate added. The ester went into Solution immediately. The re­ action mixture was then allowed to stand at room tempera­ ture for two days. After the first day, some solid had precipitated in the reaction mixture. Anhydrous ether (20 ml.) was added and the suspension filtered. The solid which weighed 3.8 g. melted at 120-135°. After two recrystallizations from methanol containing a small amount of hydrazine hydrate, it melted at 145-146° (dec.) (reported melting point 145-146° (dec.), Merck, M.60, 21). A suspension of 1.9 g. of α-ethyl D-7-benzylpenicilloate in 15 ml. of absolute ethanol was cooled in an ice bath and 2 ml. of anhydrous hydrazine added. The clear solution was allowed to stand for a week at room temperature and was then poured into anhydrous ether. A red oil precipi­ tated out which granulated upon the addition of a second portion of anhydrous ether. The ether solution deposited long needles upon slow evaporation. These were recrystallized from anhydrous ether and melted at 115-116°, and are apparently the hydraz­ ide of phenylacetic acid; m.p. 116°.

Addition of mercuric chloride to an aqueous solution of the compound yielded a heavy precipitate. The super­ natant failed to give the usual aldehyde tests. After removal of the mercury with hydrogen sulfide and concentra­ tion of the filtrate a crystalline product was obtained which after recrystallization from water melted at 210-213°.

Calc. for C8Hi0N2O: C, 64.00; H, 6.67; N, 18.67 Found: C, 63.91; H, 6.83; N, 18.77

α-Hydrazide of ^-Methyl D-Y-Benzylpenicilloate (Merck, To 0.5 g. of finely powdered hydrazine salt of the α-hydrazide of D-7-benzylpenicilloic acid (m.p. 146°) a solution of 1 g. of diazomethane in 200 ml. of ether was added. An instantaneous reaction occurred with the vigorous evolution of nitrogen, solution of the solid, and the appearance of a small amount of gummy material that slowly went into solution with the liberation of more nitro­ gen. In approximately twenty minutes the gum had dissolved completely. The excess diazomethane and ether were removed in vacuo to yield a colorless oil. The gum was dissolved in 25 ml. of ether, in which it is very soluble, and the ether solution was washed once with 20 ml. of 5 % sodium bicarbonate solution, followed by a wash with 20 ml. of water. Evaporation of the ether solution to dryness in vacuo yielded 0.44 g. of a colorless gum. Attempts to crystallize this ester were not successful.

α-Hydrazide of DL-Benzylpenicilloic Acid (Mich. Chem., B.1S, 2). A simple way to obtain the α-hydrazide of DL-benzylpenicilloic acid is to add hydrochloric acid to a solution of the hydrazine salt. To a solution of 1 g. of the hydrazine salt in 3 ml. of water was added 0.5 N hydro­ chloric acid until the solution was just acid to methyl orange. The α-hydrazide which precipitated was filtered and washed with water; weight, 0.75 g.; m.p. 150°, followed by solidification and remelting at 194°. A second crop was obtained by concentration of the filtrate. The α-hydraz­ ide is not very soluble in water or alcohol but dissolves readily in a warm mixture of the two, from which it can be crystallized. Calc. for CieH22NiO4S: C, 52.44; H, 6.05 Found: C, 52.08; H, 5.98 Hydrazine Salt of the α-Hydrazide of DL-Benzylpenicilloic Acid (B.12, 6; 13, 2). This compound was best pre­ pared by adding 0.83 ml. of aqueous 85% hydrazine hydrate to a cold solution of 2.1 g. of ethyl DL-benzylpenicilloate in 5.5 ml. of absolute ethanol and allowing the solution to stand overnight in a stoppered flask at room temperature. The yield of crystals which precipitated (first crop) was 1.06 g.; m.p. 149-151° with frothing. More of the product can be obtained in the form of the hydrazide acid by the procedure described below. Calc. for Ci6H2eNeO1S: C, 48.22; H, 6.58 Found: C, 47.82; H, 6.74 The sodium salt of α-ethyl DL-benzylpenicilloate was found to react rapidly with hydrazine. Reaction of Benzylpenicillin Methyl Ester with Hydrazine (Squibb, S.34-, 15). To a solution of benzylpenicillin methyl ester (165 mg.) in 15 ml. of ether, 0.7 ml. of 85 % hydrazine hydrate was added. After shaking a few minutes a gelatinous precipitate formed, which was collected and washed with ether (167 mg., m.p. 140-142°). Attempted crystallization from several solvents failed to change the gelatinous consistency of the product, Md +78° (in ethanol).

Calc. for CuH„N302: C, 60.82; H, 5.11; N, 19.34 Found: C, 60.62; H, 5.42; N, 19.18 The melting point and composition indicate that the compound is 4-phenylacetamido-5-pyrazolone, obtained previously from the hydrazide of benzylpenaldic acid acetal with acetic acid (Mich. Chem., B.9, 2) and from a-phenylacetamido-/3,/3-dimethoxypropionic acid and from crude α-methyl D-benzylpenicilloate with hydrazine (Merck, M.47, 4, 20). The ultraviolet spectrum of the ethanolic solution agrees with that given(M.47, 20; solvent not stated). In the spectrum of the aqueous solution the maximum at 2,470 A is not in evidence. A . . . 2,200 2,250 2,300 2,400 2,450 2,500 2,600 2,700 EM . · 8,500 8,100 8,200 8,900 9,100 9,100 7,700 4,500 (ethanol)

M.60, 21).

Calc. for Ci7H24N4O1S: N, 14.72; OCH3, 8.15 Found: N, 14.69; OCH3, 8.78 Dihydrazide of D-7-Benzylpenicilloic Acid (M.47, 21; Squibb, S.39, 2). A solution containing 1 g. of dimethyl D-7-benzylpenicilloate and 1.5 ml. of 85 % hydrazine hydrate in 10 ml. of absolute ethanol was refiuxed for ten minutes. The solution was cooled to room temperature and petroleum ether was added to the point of incipient turbidity. After· refrigeration overnight, 0.998 g. of crystals was obtained; m.p. 172-173° (dec.). After 4 recrystallizations from methanol-petroleum ether the melting point was constant at 189° (dec.). Calc. for Ci8H24N6OaS: C, 50.51; H, 6.35; N, 22.09 Found: C, 50.40; H, 6.64; N, 22.49 One gram of dimethyl D-7-benzylpenicilloate was dissolved in 10 ml. of absolute ethanol and 1.5 ml. of 85% hydrazine hydrate added. The solution was allowed to stand at room temperature overnight and the crystalline solid which had then formed was filtered off. After two crystallizations from 95 % alcohol, the dihydrazide melted at 196-197° with decomposition. Calc. for Ci6H21N6O3S: C, 50.53; H, 6.32; N, 22.11 Found: C, 50.48; H, 6.53; N, 22.11

PENILLOIC AND PENICILLOIC ACIDS Dihydrazide of D -S-Benzylpenicilloic Acid (Squibb, S.39, 3). To a solution of 200 mg. of dimethyl D-ii-benzylpenicilloate in 2 ml. of absolute ethanol, 0.3 ml. of anhydrous hydraz­ ine was added. The solution was allowed to stand over­ night at room temperature, and then diluted with 3 ml. of water. Crystals formed upon scratching; these were filtered off and after two recrystallizations from aqueous ethanol melted at 187-189° with decomposition. Calc. for Ci6H24N6O3S: C, 50.53; H, 6.32; N, 22.11 Found: C, 50.48; H, 6.44; N, 21.91

633

133-134°. After drying at 100° over phosphorus pentoxide, oil pump vacuum for two hours, the melting point was around 120° (not sharp), but the product seemed to have retained its crystalline structure. Calc. for C23H27N3O1S: C, 62.5; H, 6.12; N, 9.5 Found: C, 62.87; H, 6.26; N, 9.55 The experiment was repeated, but the preparation (m.p. 132-134°) was analyzed air-dry: Calc. for C30H38N4O6S: C, 63.6; H, 6.7; N, 9.9 Found: C, 63.88; H, 6.52; N, 10.3

A mixed melting point of the dihydrazide of π-γ-benzylpenicilloic acid and the dihydrazide of D-i-benzylpenicilloic acid was 177-185°. Benzylammonium Salt of α-Methylamide of D -Benzylpenicilloic Acid (Merck, M.15b, 3). The methylamide of benzylpenicilloic acid was prepared by treating sodium benzylpenicillin with an aqueous solution of methylamine at room temperature. The crystalline benzylamine salt was obtained, melting at 131-137° (micro-block).

When 100 mg. of amorphous sodium salt (1100 U./mg:; flat culture; 3 strains derived from original Squibb strain 144) was treated as above, the first precipitate from the reaction in ether solution consisted of a yellow oil; crystals were obtained from supernatant on standing at room tem­ perature after no more oily material deposited. The yield of twice recrystallized product was 15 mg.; m.p. 128-129°. It was air-dried.

Calc. for C24H32NiOiS-J^H2O: C, 59.85; H, 6.91; N, 11.63 Found: C, 59.91, 59.54; H, 6.81, 7.01; N, 11.81,11.83

Calc. for C28H38N4O5S: C, 62.0; H, 7.0; N, 10.3 Found: ' C, 62.2; H, 6.96; N, 10.29

Benzylammonium Salt of α-Benzylamide of D -Benzylpenicilloic Acid and D-2-Pentenylpenicilloic Acid (Coghill, Stodola and Wachtel, NRRL-CMR Report No. 16, 4). To an undried ether solution of free penicillin containing about 3,000 U./ml. was added one-fifth of its volume of a 10 % solution of benzylamine in ether. The solution became cloudy at once and soon oily droplets appeared on the sides of the test tube. In about ten minutes fine blades appeared and in a few hours all the oil had been converted to crystals. After standing overnight the crystals were rapidly filtered and washed several times with ether. For recrystallization, the product was dissolved in a minimum amount of 85 % alcohol at room temperature and ether added to cloudi­ ness. On standing, long, thin blades appeared. These were collected and dried in air. The weight of the derivative usually equaled the weight of the penicillin used. The benzylamine derivative obtained from the 1249 fermentation melted after three recrystallizations at 130-131°. Elementary analysis showed the composition C28Hi8NiO5S. Drying to constant weight at 50° and IO-5 mm. showed the product to be a monohydrate. It could also be demonstrated that a molecule of benzylamine is present as a salt by shaking a chloroform solution of the derivative with dilute hydrochloric acid. That the product remaining in the chloroform still had another benzylamine residue in the molecule was proved by absorption spectrum. Alkaline hydrolysis also liberated benzylamine from this free acid, which was not obtained in crystalline form. It can be deduced that the acid would have the composi­ tion C28H38NiOsS - (H2O + C7H9N) = C2iH27N304S. If it be further assumed that the second molecule of benzyl­ amine enters the molecule without the loss of water, the formula of this penicillin would be Ci4Hi8N2O4S. The benzylamine derivative prepared from the submerged 832 penicillin was found to resemble the compound just described, although there were some differences. The 832 derivative melted at 136-137° when pure. It was also a monohydrate and analyzes for Cs0H38N406S. Making deductions for one molecule of water and two molecules of benzylamine, a formula of Ci6Hi8N2O4S is indicated for this penicillin. It is also significant that the X-ray diffraction patterns of the two benzylamine derivatives were distinctly different. Benzylamine Derivative from Barium Benzylpenicillin (Squibb, S.Sa, 3; cf. Pfizer, P.l, 20). An amorphous barium salt (50 mg. of 1200-1500 U./mg. material from deep culture strain 832, made from acetone insoluble material giving good yield of sodium salt crystals) was treated with benzyl­ amine as above (NRRL-CMR Report No. 16, 4). The yield of twice recrystallized derivative was 15 mg.; m.p.

The analytical results support the idea that flat culture 144 penicillin differs from deep culture 832 penicillin by 2 C-atoms. Attempts to prepare the crystalline sodium salt from the former material were unsuccessful except in one experiment in which a small amount of crystals with [a]r> +340° was obtained. The amount was insufficient for analysis. Benzylammonium Salt of α-Benzylamide of D -Benzylpenicilloic Acid (Merck, Report for October, 1943, p. 12; M.S, 4; IS, 1). A sample of benzylpenicillin free acid (from the crystalline sodium salt) in wet ether solution was treated with ethereal benzylamine. A precipitate of white needle crystals, m.p. 100°, immediately appeared and was filtered off after standing at room temperature for thirty minutes. The filtrate after standing overnight deposited a further crop of needle crystals, m.p. 133-134°. Recrystallization from 85 % ethanol by addition of ether yielded needles melting at 135-136°. The corresponding product from submerged 832 penicillin melted at 135-136° (Coghill, Stodola and Wachtel, NRRL-CMR Report No. 16, 4, and previous private communication). Potentiometric titration with alkali indicated that the substance was a benzylamine salt of a monobasic acid, with an equivalent weight of 551 (calc. 566) and ρ11½ 9.55. Potentiometric titration with acid indicated that the substance was a salt of a monobasic acid (pK 3.96) with an equivalent weight of 555. The ultraviolet absorption in water was:

Wave-length A

E (mol.) M.W. = 548

2,800 2,575 2,450 2,350

25 650 500 850

This absorption resembles that observed for benzylpeni­ cilloic acid if allowance is made for the presence of the two additional phenyl groups. Benzylammonium Salt of the α-Benzylamide of D -Benzylpenicilloic Acid from Benzylpenicillin (NRRL, C.B, 1 and unreported details). To a solution of 1.12 g. of the sodium salt of benzylpenicillin (1300 U./mg.) in 180 ml. of ice-cold water was added 13 ml. of ice-cold 1:1 phosphoric acid. The solution was at once shaken vigorously with 230 ml. of ice-cold ether. The aqueous solution was drawn off and

634

PENILLOIC AND PENICILLOIC ACIDS

extracted with two 155-mI. portions of cold ether. The combined ether extracts were carefully decanted into a flask which was cooled in ice for ten minutes. (Failure to allow the water to separate completely leads to a low-melting product difficult to purify.) The ether solution was then filtered and to the filtrate was added gradually from a separatory funnel a solution of 10 ml. of benzylamine in 100 ml. of ether. In a few minutes oil and crystals appeared. The tightly stoppered flask was allowed to stand at room tem­ perature overnight, during which time the oil was converted completely to crystals. Filtration gave 1.42 g. (80%) of white product, m.p. 136-137° (capillary). ^ The sodium salt of benzylpenicillin (100 mg.) was dis­ solved in 15 ml. of water and the solution cooled to 0°. After acidification to pH 2 with phosphoric acid, the cloudy solution was extracted rapidly with three 15-ml. portions of ether. The combined ether extracts were allowed to stand for ten minutes in a cylinder cooled in ice. The ether solu­ tion was decanted, warmed to room temperature and treated with 1 ml. of benzylamine in 9 ml. of ether, added dropwise. The oily droplets soon crystallized in the form of long blades. After standing overnight at room temperature, the colorless crystals were removed by filtration and dried in air; weight, 143 mg. (90%); m.p. 137-138° (capillary); [a]DJ3 +109°. Calc. for C3OH38N4O6S: C, 63.58; H, 6.76; N, 9.89; S, 5.66 Found: C, 63.38, 63.23; H, 6.58, 6.58; N, 9.83; S, 5.64 The derivative was crystallized without change in melting point by solution in the minimum amount of 85 % alcohol at room temperature and the addition of ether to cloudiness. To show that the derivative contained no labile carbon atom removable as carbon dioxide, 100 mg. was refluxed in a stream of nitrogen for one hour with 3 ml. of 95 % alcohol and 3 ml. of 0.2 N sulfuric acid. Passage of the nitrogen through barium hydroxide solution gave only a trace of 1 barium carbonate. Quantitative acetylation of the derivative with acetic anhydride gave a value of 2.70% NH (calc. 2.65% for one NH).

A simple and rapid method for the preparation of this compound was developed. The sodium salt of benzylpenicillin (103 mg.) was dissolved in the minimum amount of water necessary for solution at room temperature. To this solution was added 0.15 ml. of benzylamine. After one hour the reaction mixture was diluted with 2 ml. of water and 3 ml. of 1 AT hydrochloric acid added dropwise. The white precipitate was filtered and washed with a little water. After air-drying for a few minutes it weighed 96 mg. (75 %). Ten milligrams of this product was dissolved in two drops of 85 % alcohol and one drop of benzylamine. Careful addition of ether to avoid gel formation gave 7.5 mg. of needles melting at 137-138° (44 %). The overall yield was 33%. «-Benzylamide of D-Benzylpenicilloic Acid ("Natural") (Merck, M.12b, 2; 15b, 4). The benzylamine salt of the α-benzylamide of D-benzylpenicilloic acid ("natural") was dissolved in water, acidified to ρH 2 and extracted with ether. The ether solution was filtered and evaporated to a small volume whereupon crystals separated. The product was filtered and dried. It melted at 110-113° (micro-block); [«]D +88° (in methanol). Calc. for C2sH27N3O4S: C, 62.56; H, 6.16; N, 9.52 Found: C, 62.68; H, 6.60; N, 9.41 From the mother liquors on evaporation a second crop of needles was obtained melting at 118-122° (micro-block). α-Benzylamide of D-Benzylpenicilloic Acid (Coghill, Stodola, Wachtel, Unreported experiments). The sodium salt of benzylpenicillin (580 mg.) was dissolved in 2 ml. of dimethylformamide and 0.50 ml. of benzylamine added. After several weeks at room temperature, dry ether was added to the reaction mixture. The precipitated sodium

salt was filtered off and found to weigh 755 mg. It was dissolved in a minimum amount of hot water, cooled to room temperature and. the clear solution acidified with 1.78 ml. (1 equivalent) of 0.97 N hydrochloric acid. The white precipitate of free acid was separated. It weighed 650 mg. and melted at 100-105°. Crystallization from aqueous acet­ one gave 201 mg. of needles which started to sinter at 113° and melted at 119-121°. For analysis, the compound was dried at 56° (0.1 mm.); any higher temperature was found to produce some alteration. Calc. for C23H27N3O4S: C, 62.56; H, 6.17; N, 9.52 Found: C, 62.5; H, 6.10; N, 9.37 The benzylamine derivative was prepared from the free acid. Ether was added to a solution of 21 mg. of the free acid in the minimum amount of 85 % alcohol containing two drops of benzylamine; the development of turbidity was followed by the' deposition of fine needles; yield, 15 mg. Crystallization from alcohol-ether gave 5 mg. of needles melting at 137-138°. This product was shown by mixed melting point and X-ray powder patterns.to be identical with the benzylamine derivative of benzylpenicillin (benzylammonium salt of α-benzylamide of D-benzylpenicilloic acid). α-Benzylamide of D-Benzylpenicilloic Aeid from Triethylamine Salt of Benzylpenicillin (Pfizer, P.26, 7). A solution of 1 g. of the triethylamine salt of benzylpenicillin in 50 ml. of chloroform was treated with 1 ml. of benzylamine and allowed to stand overnight at room temperature. At the end of this time the bioassay showed no activity. The solution was evaporated to dryness in vacuo and triturated with benzene to give a crude product melting at 135-136°. This was recrystallized from ieri-butanol to give 0.86 g. of what appeared to be the benzylamine salt of a-benzylamide of D-benzylpenicilloic acid melting at 137-138°; [«'d +119°. Calc. for C30H36N4O4S-H2O: C, 64.51; H, 6.45; N, 10.03 Found: C, 63.47; H, 6.84; N, 9.95, 9.84 The salt was dissolved in water containing a little sodium hydroxide, acidified and extracted with chloroform. The chloroform solution was evaporated to dryness in vacuo and the residue crystallized from ieri-butanol. The a-benzyl­ amide of D-benzylpenicilloic acid thus obtained melted at 140-145° and had [afo +140°. Calc. for C23H27N3O4S: C, 62.58; H, 6.12, N, 9.52 Found: C, 62.10; H, 7.21; N, 9.70, 9.60 α-Benzylamide of D-Benzylpenicilloic Acid (Upjohn, {7.7, 18). To a solution of 43.2 mg. of α-phenylacetylamino/3-benzylaminocrylic acid benzylamide in 2.0 ml. of slightly warm 95 % ethanol was added 19.2 mg. of D-penicillamine hydrochloride in 1.0 ml. of water. One drop of concentrated hydrochloric acid was added and the solution allowed to stand at room temperature overnight. A small amount of material which crystallized was redissolved by very gentle warming. At the end of the next twenty-four hours the few crystals which had separated were again brought into solution by gently warming the reaction mixture, and after a total of sixty hours the reaction mixture was evaporated to dry­ ness in a stream of nitrogen. The white, crystalline residue was stirred with 3.0 ml. of 5 % sodium bicarbonate solution whereupon essentially all the material dissolved. After filtering and washing with water, the combined filtrate (pH 8) was acidified to ρH 3 with normal hydrochloric acid. The white, fluffy precipitate was collected, washed re­ peatedly with water and dried at 63° in a stream of nitrogen. The white product sintered at 89-96.5°, softened at 97-101°, and melted at 101-106°; yield, 7.5 mg. Calc. for C23H27N3O4S: C, 62.56; H, 6.17; N, 9.52 Found: C, 62.60; H, 6.29; N, 9.39

PENILLOIC AND PENICILLOIC ACIDS

635

The combined filtrate and washings from the above com­ pound was brought to pH 5 with 5 % sodium bicarbonate, and evaporated in a nitrogen jet to a small volume thus yielding an additional 10.2 mg. of slightly colored, somewhat impure product softening at 90° and melting at 92-100°. Benzylammonium Salt of the a-Benzylamide of D-Benzylpenicilloic Acid (Coghill, Stodola and Wachtel, CJh 1). The sodium salt of benzylpenicillin (10 mg.) dissolved in 1.0 ml. of acetic anhydride was heated at 60° for thirty minutes. The almost colorless solution was lyophilized and the residue dissolved in 1 ml. of water. Conversion to the benzylamine derivative in the manner previously de­ scribed (NRRL-CMR Report No. 16, 4) gave 13.5mg. (85% of theory) of needles melting at 134-135°. Admixture with the benzylamine derivative of m.p. 136-137° gave a melting point of 135-136°. Benzylammonium Salt of the α-Benzylamide of D-Benzylpenicilloic Acid (Merck, M.15a, 4; 15c, 4; 23, 7; cf. Upjohn, U.7, 15). The benzylamide of benzylpenaldic acid was allowed to condense with D-penicillamine hydrochloride at room temperature in aqueous alcohol solution. After fifteen hours the product was isolated by evaporating the alcohol with a current of air (pH such that addition of neither dilute sodium hydroxide nor hydrochloric acid produced an oil) and separating the oil. This solidified by trituration with water. An analytical sample, m.p. 95-110°, was obtained by two precipitations from an alkaline solution by hydrochloric acid; m.p. 95-110°.

α-Benzylamide of D-Benzylpenicilloic Acid Hydrochloride (NRRL, C.4, 3). The free acid from the benzylamine derivative of benzylpenicillin (shown to be free of benzyl­ amine by ninhydrin test) was dissolved in alcohol and 0.5 ml. of 1 ΛΓ hydrochloric acid. The solution was concentrated in vacuo to a glass, dissolved in tertiary butyl alcohol, and lyophilized. The fluffy product was left at about 0.1 mm. pressure for 1.5 hours in order to eliminate mechanical retention of hydrochloric acid. A Carius chlorine analysis showed that 0.65 mole of hydrogen chloride had been re­ tained as a salt. These experiments were repeated with the following results: Under identical conditions, 4-thiazolidinecarboxylic acid yielded a product containing 0.9 mole of hydrogen chloride; in another experiment 0.81 mole. N-Acetyl-4thiazolidinecarboxylic acid showed no retention of hydrogen chloride, as proved by negative silver nitrate test and no depression in melting point on mixing with the starting material. The free acid from the benzylamine derivative of benzylpenicillin was able to retain 0.59 mole of hydrogen chloride. Hydrogenolysis of α-Benzylamide of D-Benzylpenicilloic Acid (Merck, M.15b, 8; 22, 10). "Natural" D-benzylpenicilloic acid α-benzylamide when dissolved in 0.5% aque­ ous sodium carbonate and treated with excess nickel catalyst at 70° for thirty minutes gave a crystalline desthiobenzylamide, m.p. 201-202° (dec.) (micro-block). The analysis agreed with that expected for the desthiobenzylamide.

Calc. for C23H27N3O4S: C, 62.56; H, 6.17; N, 9.52 Found: C, 62.39, 62.97; H, 6.76, 6.61; N, 9.38

Calc. for C23H29N3O4: C, 67.13; H, 7.10; N, 10.21 Found: C, 67.42; H, 7.40; N, 10.08

A crystalline benzylamine salt was obtained by dissolving the free acid in 85 % ethanol, adding one equivalent of benzylamine, then adding ether to the point of saturation and cooling; m.p. 180-181° (corr.); EM 640 at 2,580 Α; [α]η23 +78° (c = 0.528 in ethanol).

Synthetic benzylpenicilloic acid α-benzyIaniidc in aqueous sodium carbonate solution (0.5%) failed to react with Raney nickel catalyst at room temperature, but at 75° the reaction was apparently complete in thirty minutes. The product obtained by acidifying the alkaline solution and extracting with chloroform was amorphous. A crystalline product was obtained from an alkaline solution of the acid when acidified and allowed to stand or from an aqueous methanol solution. It melted over a wide range. By fractional crystallization two products were isolated. Com­ pound A melted at 202° (dec.) (micro-block) and gave no depression of the mixed melting point with the product above obtained from the "natural" benzylpenicilloic «-benz­ ylamide ;[α]β — 24.6° (c = 0.298 in 0.1 N sodium hydroxide).

Calc. for C30H36N4O4S: C, 65.67; H, 6.61; N, 10.21 Found: C, 65.97; H, 6.73; N, 10.66 This product titrated potentiometrically in 25 % alcohol with sodium hydroxide as a benzylammonium salt of a mono­ basic acid of equivalent weight 539 (calc. 549) and 9.29. With hydrochloric acid, the pH^ of the acid function was 4.01 and the equivalent weight 540. The pHj^ of the acid function for the "natural" analog was observed to be 3.93 {M.3, 4). With one equivalent of mercuric chloride in ethanol the rotation fell from Md23 + 78° to a final value of [α]ι>23 —22° in 10 minutes. The solution then had Em 17,700 at 2,825 A. Benzylammonium Salt of α-Benzylamide of D-Benzylpenicilloic Acid (Upjohn, U.7, 23). To 34.6 mg. of ethyl D -benzylpenicilloate was added 0.1 ml. of redistilled benzyl­ amine; the mixture was slowly swirled for five minutes, when practically all of the solid had dissolved yielding a some­ what viscous, colorless, slightly turbid solution. After eighty-four hours at room temperature the mixture was diluted with dry ether. Upon rubbing and scratching, the viscous gum which precipitated at first soon solidified. The solid was washed repeatedly with dry ether, with centrifuging after each washing, and was finally dried at 65° in a cur­ rent of nitrogen. The white powder thus obtained weighed 34.7 mg. and had an indistinct melting point. When placed in the cold bath and the temperature raised slowly the product began to sinter at 102°, softened and foamed at 112°, and gradually melted to a clear yellow melt at 122140°. When placed in the bath previously heated to 97°, the product sintered immediately, began foaming at 102° and was completely melted (clear yellow melt) at 152.5°. Repetition of the aminolysis using 54.0 mg. of ethyl D-benzylpenicilloate gave 60 mg. (77 %) of pure white solid, m .p. 144-146° (micro-block). Calc. for C30H36N4O4S: C, 65.67; H, 6.61; N, 10.21 Found: C, 64.99; H, 7.01; N, 10.00

Calc. for C23H29N3O4: C, 67.13; H, 7.10; N, 10.21 Found: C, 67.44; H, 7.51; N, 10.54, 10.31 The other product (apparently not pure) melted at 153-157° and gave the following analysis. Calc. for C23H29N3O4: C, 67.13; H, 7.10; N, 10.21 Found: C, 69.68; H, 6.51; N, 9.71 The compound may be the second possible diastereoisomer in an impure condition. α-Benzylamide of Desthio-D-benzylpenicilloic Acid from Desthiobenzylpenicillin (M.52, 8). A solution of 65 mg. of desthiobenzvlpenicillin in 3 ml. of dioxane containing 0.25 ml. of benzylamine was refluxed for three hours. The solution was then cooled and evaporated to a viscous oil under reduced pressure at 25°. This residue was dissolved in 4 ml. of water and on acidification with dilute hydro­ chloric acid gave a slightly yellow-colored crystalline precipi­ tate which was washed with water and dried; yield 52 mg., m.p. 187-191°. After two recrystallizations from 75% methyl alcohol, the melting point was constant at 204-205° (dec.) (micro-block). When mixed with a sample of the α -benzylamide of desthio -D -benzylpenicilloic acid derived from benzylpenicillin (M.22, 10) there was no depression of the melting point. Calc. for C23H29N3O4: C, 67.13; H, 7.10; N, 10.21 Found: C, 66.80; H, 7.18; N, 9.97

636

PENILLOIC AND PENICILLOIC ACIDS

α-Benzylamide of N4-Acetyl-D-benzylpenicilloic Acid {M.23, 7; 25, 4). Ketene was passed into a cooled solution of 2 g. of the α-benzylamide of D-benzylpenicilloic acid in 18 ml. of ethanol for forty-five minutes. After standing overnight, the ethanol was removed in vacuo and most of the residue dissolved in dilute sodium hydroxide. The product was then precipitated by pouring the alkaline solu­ tion into an excess of dilute hydrochloric acid. Two more reprecipitations yielded a product, m.p. 95-105°; MD23 0° (c = 1.13 in ethanol); [«!n23 +12° (c = 1.10 in methyl ethyl ketone); EM 4,500 at 2,580 A. Calc. for C26H29N8O6S: C, 62.09; H, 6.05; N, 8.69 Found: C, 62.16; H, 6.03; N, 8.40 Benzylammonium Salt of α-Benzylamide of "Natural" N4-Benzoyl-D-benzylpenicilloic Acid (M.S6, 5; 39, 11). Benzylpenicillin free acid was converted to the a-benzylamide of D-benzylpenicilloic acid benzylamine salt with excess benzylamine in the usual manner. From the salt, m.p. 132-136° (micro-block), the free acid was obtained as a glass. A solution of 50 mg. of the free acid in 0.3 ml. of dry pyridine was treated with 0.016 ml. of benzoyl chloride. The mixture was allowed to stand at room temperature for one hour and then evaporated in vacuo. The residue was dissolved in chloroform and extracted with 0.1 N hydro­ chloric acid and then with water. The chloroform was next extracted with N sodium bicarbonate solution. The bicarbonate solution was acidified and extracted with chloroform to remove the acid fraction which weighed 53 mg. This crude oil was extracted with hot petroleum ether to remove a small amount of benzoic acid. The residue failed to crystallize as the free acid but yielded a crystalline salt with ethereal benzylamine. Recrystallization from methanol and ether gave needles, m.p. 111-112° (micro-block); [C*]D +31° (c = 0.9 in methanol). In the ultraviolet the compound had an end absorption about 2,300 A of EM 7,700 which is typical of N4-benzoyl deriva­ tives of benzylpenicilloates. The compound appears to be a hydrate. Calc. for C37H40N4O8S-H2O: C, 66.24; H, 6.31; N, 8.35 Found: C, 66.48; H, 5.81; N, 8.81 Benzoylation of α-Benzylamide of Benzylpenicilloic Acid (Abbott, A.10, 6). To 25 mg. of α-benzylamide of benzylpenicilloic acid in 1 ml. of dry pyridine was added 1.4 equiva­ lents of benzoyl chloride in pyridine. After one-half hour at room temperature, the pyridine and benzoyl chloride were evaporated in vacuo and the residue was dissolved in 2 ml. of chloroform. After washing once with 0.5 ml. of 0.01 N hydrochloric acid and four times with 0.5-ml. portions of water, the chloroform solution was dried over sodium sulfate and evaporated to give a glass which was then triturated with ether. Since the residue was somewhat soluble in ,ether, the latter was evaporated and the residue washed three times with hexane. The product was then precipitated from an ether solution by means of hexane. The melting point of 101-105° was raised to 107-110° by precipitation from ether-hexane. The yield was approximately 75%.

concentrated solution of sodium chloride. It was recrystallizedfromwarm water; m.p. 178°. Calc. for C80HaiN4OeSNa: C, 61.84; H, 5.36 Found: C, 61.47; H, 5.65 Ultraviolet absorption in ethanol shows a single sharp band at 2,400 A of Em 14,500. This is in accord with the assigned structure; [a]D23 —38° (c = 0.431 in ethanol). Optical rotation ninety minutes after treatment with one equivalent of mercuric chloride per mole in ethanol was HD23 —41° (c = 0.687). Obviously no reaction had oc­ curred with the mercuric chloride. Potentiometric titration of this compound in 50% alcohol gave results indicating its ρHH to be 3.86 and its equivalent weight 570 (calc. value 570.6). In view of the presence of alcohol, the true pHy2 value in water is at least 0.5 to 1 pH unit lower, i.e., 3.4 to 2.9. α-Benzylamide of (3-Methyl D-a-Benzylpenicilloate (Ab­ bott, A.8, 5). The benzylamine degradation product (400 mg.) of crystalline sodium benzylpenicillin was con­ verted to the free acid with dilute hydrochloric acid in the presence of ether at ice temperature. Evaporation of the ethereal solution to a small volume and then to dryness yielded two crops of needles melting at 104-107° and 102.5105°, respectively. The weight of combined product was 270 mg. (87%). A solution of 220 mg. of the acid in ether containing a small amount of methanol was treated with diazomethane. Removal of the solvents and unchanged diazomethane gave a quantitative yield of the α-benzylamide of j3-methyl D-benzylpenicilloate. Calc. for C24H29N8O4S: N, 9.23 Found: N, 8.99 The material was not obtained in crystalline form. It was obtained also by direct treatment of the benzylamine degradation product of benzylpenicillin with a solution of diazomethane in ether. On treating this substance dissolved in ethanol with mercuric chloride an absorption peak was formed at 2,780 A with EM 8,080 without pyridine, EM 8,910 in the presence of pyridine. α-Benzylamide of /3-Methyl D-Benzylpenicilloate (Up­ john, U.7, 16). A cold ethereal solution of diazomethane was added portionwise with swirling to 50 mg. of crude D-benzylpenicilloic acid α-benzylamide suspended in 10 ml. of anhydrous ether. A brisk evolution of gas occurred and the majority of the acid dissolved. Excess diazomethane and ether were removed by gentle warming on a steam bath, the last traces of ether being removed in a jet of nitrogen. The residue of crude material, a colorless glass, weighed 53.7 mg. The crude product was redissolved in ether, filtered and washed, first with 5% sodium bicarbonate solution and then with water. The organic layer was dried over an­ hydrous magnesium sulfate, filtered and the solvent allowed to evaporate spontaneously; yield, 36.9 mg.; m.p. 118-122° (capillary).

Calc. for CsoH81N8O6S: C, 66.05; H, 5.68; N, 7.70 Found: C, 65.63; H, 5.90; N, 7.69

Calc. for C24H29N3O4S: C, 63.27; H, 6.42; N, 9.22 Found: C, 63.21; H, 6.54; N, 9.02

Sodium Salt of α-Benzylamide of N4-PhenylcarbamylAcid (Merck, M.ISc, 4; 25, 6). An aqueous solution of the sodium salt of the α-benzylamide of D-benzylpenicilloic acid was shaken with phenyl isocyanate until no more reaction occurred. The diphenylurea was then removed by filtration and the α-benzylamide of N4-phenylcarbamyl-D-benzylpenicilloic acid precipitated by adding an excess (to keep any starting material in solution) of hydrochloric acid. This was dissolved in dilute sodium hydroxide and the sodium salt of the α-benzylamide of N4-carbamyl-D-benzylpenicilloic acid was salted out with a

α-Benzylamide of β-Methyl D-Benzylpenicilloate from Benzylpenicillin Methyl Ester (Merck, M.39, 7). To a solu­ tion of 190 mg. of benzylpenicillin methyl ester in 10 ml. of methanol, a slight excess of benzylamine was added. After three hours at room temperature, the rotation fell to HD26 +90°. The residue, in chloroform solution, was extracted with dilute hydrochloric acid, bicarbonate and water; then dried. Evaporation of the chloroform solution to dryness under reduced pressure at 25° gave a gum-like residue which was crystallized from ethanol and water; m.p. 68-70° (micro-block).

D-benzylpenicilloic

PENILLOIC AND PENICILLOIC ACIDS Calc. for C24H29NsO4S: C, 63.27; H, 6.42 Found: C, 63.01; H, 6.52 α-Benzylamide of /S-Methyl D-Benzylpenicilloate from Benzylpenicillin Methyl Ester under Attempted Anhydrous Conditions (M.39, 7). To a solution of 100 mg. of benzylpenicillin methyl ester in 4 ml. of dioxane (purified and distilled over sodium), 0.06 ml. of benzylamine was added. The mixture was allowed to stand for three hours. (All operations were performed in a dry-cabinet, where the reac­ tion mixture was kept during the standing period.) The solvent was removed under reduced pressure at 25°; the residue was washed with petroleum ether (30-60°) and then crystallized from ethanol and water. The crude crystalline product melted at 68-71° (micro-block). α-Benzylamide of /3-Methyl D-Benzylpenicilloate from the Benzylamine Salt of the α-Benzylamide of Benzylpenicilloic Acid (M.39, 7). The "natural" benzylamine salt of the α-benzylamide of D-benzylpenicilloic acid (45 mg.) was first converted to the free acid and then, with diazomethane, to the /3-methyl ester. The gum-like residue obtained from ether solution was purified as in the above preparation and then crystallized from ethanol and water; m.p. 68-70° (micro-block); Hd25 +62.2° (c = 0.45 in methanol). Calc. for C24H29N3O4S: C, 63.27; H, 6.42; N, 9.23 Found: C, 63.24; H, 6.38; N, 9.40 α-Benzylamide of /3-Methyl D-Benzylpenicilloate from Benzylpenicillin Methyl Ester (Pfizer, P.l7, 7; 19, 15). A 1 % solution of benzylpenicillin methyl ester in benzene was treated with two equivalents of benzylamine. The solution at the outset had [a]D +281°; at the end of four hours it had I«]D +115°; after twenty-four hours [a]D +63°; after ninety-six hours [α]ϋ +62°. Thesolutionwasthenwashed with dilute hydrochloric acid and water, dried over sodium sulfate and lyophilized. The α-benzylamide of /3-methyl D-benzylpenicilloate was obtained in amorphous form. Calc. for C24H29NsO4S: N, 9.23 Found: N, 9.34, 9.37 The α-benzylamide of /3-methyl D-benzylpenicilloate was prepared by treating 500 mg. of benzylpenicillin methyl ester in 20 ml. of dioxane with 0.5 ml. of benzylamine. Atthe end of six hours the solvent was removed and the benzylamide was crystallized from aqueous methanol. There was ob­ tained 540 mg. of product melting at 81-83°; [α]ϋ23 +68° (c = 0.5 in methanol). The constants have been reported (Merck, M.39, 7) to be m.p. 68-70° and [a]D23 +62.2° (c = 0.45 in methanol). Identification of the Benzylamides from "Natural" and Synthetic Methyl Benzylpenicillenate (Merck, M.46, 10). To a solution of 31 mg. of "natural" methyl D-benzylpenicillenate in 1 ml. of ether was added 0.02 ml. of benzylamine. After standing overnight at room temperature, 17 mg. of crystals melting at 107-114° (micro-block) was obtained. Recrystallization from methanol-ether gave 11 mg. of product, m.p. 116.5-118.5°; [afo22 +7.4° (c = 0.539 in benzene). A sample was recrystallized from benzene-ether. Calc. for C24H29NsO4S: C, 63.27; H, 6.42; N, 9.22 Found: C, 63.04; H, 6.15; N, 9.18 The melting point of a mixture of this benzylamide and the benzylamide of melting point 116° obtained from synthetic methyl D-benzylpenicillenate (M.37, 23) was not depressed. α-Benzylamide of /3-Methyl D-Benzylpenicilloate (M.37, 19). To 1.8 g. of /3-methyl D--y-benzylpenicilloate (3.22 millimoles) in 15 ml. of anhydrous pyridine, cooled in an icesalt bath, 0.48 g. of phosphorus tribromide (1.8 millimoles) was added. An orange color was produced, and a white precipitate (pyridine hydrobromide) deposited. After standing for ten minutes in the cooling bath and one hour at

637

room temperature, the mixture was filtered. The pyridine solution was evaporated and the residue dissolved in chloro­ form. After washing with dilute hydrochloric acid and then with aqueous sodium bicarbonate, the chloroform solution was dried and evaporated. The residue was treated with ether. A few milligrams of a semi-solid material remained undissolved. Benzylamine was added to a portion of the ether solution. After standing for about thirty-six hours at room temperature, ball-shaped crystal masses had deposited; m.p. 114-116°. Crystals reformed at 130°and finally melted at 154-158°. On recrystallization from methanol-ether the melting point became 115-117° (capillary); after a second recrystallization, m.p. 154-160°; third recrystallization, m.p. 154-160° with previous softening. Calc. for C24H29NsO4S: C, 63.27; H, 6.42; N, 9.22 Found: C, 63.17; H, 6.33; N, 9.30 α-Benzylamide of /3-Methyl D-Benzylpenicilloate (M.37, 23; l f i , 10; cf. Upjohn, U.16, 24). A solution of 1.00 g. of D-penicillamine methyl ester and 1.33 g. of 2-benzyl-4methoxymethylene-5(4)-oxazolone in 25 ml. of toluene was concentrated to dryness at room temperature immediately after preparation. The concentration required approxi­ mately fifteen minutes. The residual gum was dissolved in 50 ml. of chloroform and washed with three 30 ml. portions of 0.2 M phosphate buffer. The chloroform layer was filtered and evaporated to dryness, giving 1.964 g. of product as a straw-colored gum which became yellow on standing; [α]ϋ 25 +40.0° (c = 0.5 in benzene solution). In alcohol solu­ tion the product shows ultraviolet absorption at 3,175 A, EM 18,000. Calc. for Ci7H20N2O4S: C, 58.60; H, 5.79; N, 8.04 Found: C, 58.08; H, 5.87; N, 8.24 A solution of 115 mg. of the condensation product in 2.5 ml. of ether was treated with 0.7 ml. of benzylamine. After standing overnight at room temperature, 55 mg. of crystalline benzylamide melting at 116° had deposited. The product was recrystallized from ether containing a trace of methanol without change in melting point. The compound did not depress the melting point of the benzylamide, melt­ ing at 116-118°, secured (M.37, 20; see under a-(|8'-phenylethylamide) below) from the benzoyl chloride azlactonization of /3-methyl D-benzylpenicilloate; [α]ο25 +83°*(E = 0.542 in benzene). α-Benzylamide of /3-Methyl D-Benzylpenicilloate from Pseudobenzylpenicillin Methyl Ester Hydrochloride (Ab­ bott, A.15, 4; cf. Upjohn, XJ.16, 8). Pseudobenzylpenicillin methyl ester hydrochloride (108.6 mg.) was suspended in 2 ml. of dry ether and 100 mg. of benzylamine dissolved in 1 ml. of ether was added. The suspended material im­ mediately became gummy. It was triturated with 0.5 ml. of ieri-butyl alcohol until it became friable. After stand­ ing, the ether solution was washed with water, with dilute hydrochloric acid, with pH 7 buffer and finally with water. After drying with sodium sulfate the solution was evaporated, leaving 45 mg. of product. Crystallization from methanolether gave 20 mg. (tight balls of needles) melting at 104112°. Recrystallization of this from methanol-ether gave 7.3 mg. of product melting at 163-165° and which gave no depression of melting point when mixed with a previously prepared sample of the high melting form of α-benzylamide of /3-methyl D-benzylpenicilloate. Calc. for C24H29N3O4S: N, 9.23 Found: N, 9.54 α-Benzylamide of 3-Methyl N4-Acetyl-D-benzylpenicilloate (Merck, MJtJl, 13). To an ether solution of 1.00 g. of the acetic anhydride-pyridine reaction product of /3-methyl D-7-benzylpenicilloate, which had been washed with sodium bicarbonate solution, 0.31 ml. of benzylamine was added. In a few minutes an oil separated. After standing over-

PENILLOIC AND PENICILLOIC ACIDS

638

night, crystals formed; yield, 0.43 g.; m.p. 128-144° (microblock). The material was purified by two recrystallizations from acetone-ether. After drying at 56° in vacuo it melted at 147-149° (micro-block). Calc. for C26HaiN3O6S: C, 62.75; H, 6.28; N, 8.44; acetyl, 8.65 Found: C, 62.82; H, 6.23; N, 8.65; acetyl, 14.2 Apparently, in the acetyl determination about 65% of the theoretical amount of phenylacetic acid distilled over and was titrated along with the acetic acid. This behavior was observed with other compounds containing both the acetyl and phenylacetyl groups. The benzylamide described above has also been obtained from the reaction product of /3-methyl D-7-benzylpenicilloate and an excess of acetic anhydride at 50°. o-Benzylamide of D-Benzylpenicilloic Acid (Upjohn, U.7,21; 10, 3). A solution of 100 mg. of crude benzylpenaldic acid benzylamide and 60 mg. of L-penicillamine in 35 ml. of 60% ethanol was allowed to stand at room temperature overnight. A solution of 27 mg. of sodium bicarbonate in 1 ml. of water was added and after seven hours the resulting clear solution was concentrated on a steam bath, using an air jet, until a gummy precipitate formed. The precipitate was washed with water by decantation and then extracted with 1% sodium bicarbonate. On acidification of this extract, a white solid was obtained. After washing with water and drying at 60° in vacuo for one hour the product melted at 100-125° (dec.). Calc. for C21H27NJO4S: C, 62.56; H, 6.17; N, 9.52 Found: C, 62.49; H, 5.87; N, 9.29 α-Benzylamide of /3-Methyl DL-Benzylpenicilloate (Merck, M.12a, 18; 12c, 16). The crude condensation product between DL-penicillamine methyl ester and the 2-benzyl-4methoxymethylene-5(4)-oxazolone reacted with benzylamine to give a low yield of crystals melting at 182° and analyzing satisfactorily for an α-bcnzylamide of /3-methyl DL-benzylpenicilloate. The ultraviolet absorption spectrum in ethanol indicated that the thiazolidine ring was closed. Wave-length (A) •

EM

2,700 2,580 2,450 2,350

170 420 340 820

-nitrophenyl)-4-benzylthiomethylene-5-oxazolone with either aniline or benzylamine gave the corresponding aminomethyleneoxazolones, the ring being evidently so much stabilized by the p-nitrophenyl group that the side-chain became the more reactive centre (CPS.641). The oxazolone ring in 2-benzyl-4-ethylthiomethylene-5-oxazolone was opened by benzylamine to the benzylamide of 0-ethylthio-a-phenyIacetamidoacrylic acid (LXXII; R = C 6 H 6 CH 2 ; R' = C 2 H 6 ) (CPS.888). The action of ethyl mercaptan containing a trace of sodium mercaptide on 2-phenyl-4-ethylthiomethylene-5-oxazolone gave ethyl phenylthiolpenaldate diethylthioacetal (LXXIII; R = C2H6) ; this reaction is analogous to that of 2-phenyl-4-ethoxymethylene5-oxazolone with ethanol. With benzyl mercaptan the product was benzyl phenylthiolpenaldate dibenzylthioacetd, exchange having occurred as well as addition (CPS.542). RiSCH=C-CO2H I NHCOR LXXII

(RS)2CH-CH-COSR I NHCOC6H6 LXXIII

756

OXAZOLONES

OC4H8NCH=CCONC4H8O I NHCOCH2C6H6 LXXIV (iv) 4-Aminomethylene-5-oxazoIone$. The only acid hydrolyses of this type of substance have been carried out in the presence of 2,4-dinitrophenylhydrazine; phenylacetamidoacetaldehyde 2,4-dinitrophenylhydrazone was thus produced from 2-benzyl-4-aminomethylene-5-oxazolone (Merck. M.12a), and from 2-benzyl-4-benzylaminomethylene-5-oxazolone (Upjohn, U.ll). A few cases of alkaline hydrolysis have been recorded and these have led to hydroxymethyleneoxazolones. Alcoholysis of 2-phenyl-4-benzylaminomethylene-5-oxazolone has been carried out in the presence of 2,4-dinitrophenylhydrazine and acid (Merck, M.33; Barber, Gregory, Slack, Stickings and Woolman, CPS.£34); the product was the dinitrophenylhydrazone of ethyl phenylpenaldate. 2-Benzyl~4~ morpholinomethylene-5-oxazolone on heating with morpholine gave the morpholide (LXXIV) of βmorpholino-a-phenylacetamidoacrylic acid. On the other hand, it is reported (Barber, Gregory, Slack, Stickings and Woolman, CPS.66) that the ring in 2-phenyl-4-anilinomethylene-5-oxazolone could not be opened "even by the most drastic treatment with aniline," though benzyl mercaptan in acetic acid appeared to open the ring. (c) REACTIONS INVOLVING BOTH FUNCTIONAL GROUPS. The reaction of 2-benzyl-4-ethoxymethylene-5-oxazolone with benzylamine in ether under mild conditions led to 2-benzyl-4-benzylaminomethylene-5-oxazolone in the normal way (Up­ john, U.ll; NRRL, C.10; Merck, M.47); this sub­ stance was found to exhibit dimorphism. With an excess of benzylamine a product was formed, either from 2-benzyl-4-ethoxymethylene-5-oxazolone or its "hydrobromide" (Merck, M.12a; Upjohn, U.7; 6), which gave rise to a good deal of confusion owing to its variable properties. It crystallized from ethanol (or methanol) with one molecular propor­ tion of the solvent, which it retained rather tena­ ciously. This fact was not at first suspected and led to such formulations as LXXV and LXXVI, for it had been found that the substance gave the benzylamide of benzylpenaldic acid on treatment with alcoholic hydrochloric acid (or the 2,4-dinitrophenylhydrazone of this benzylamide when dinitrophenylhydrazine was also present) (Merck, M.16c; Upjohn, U.7), and with cysteine the thiazolidine (LXXVII) appeared to be formed (Merck, M.12a). Later (Merck, M.60; 61) the presence of alcohol in the crystals was recognized, and it was shown that the hydrolysis with acid led to equimolecular amounts of benzylamine and benzyl­ penaldic benzylamide. The substance, then, was clearly the benzylamide (LXX) of β-benzylaminoα-phenylacetamidoacrylic acid. This had already been recognized as a product of the action of benzyl­

amine on the oxazolone "hydrobromide" (Upjohn, U.6); apparently the isolation procedure had avoided the use of alcohol (or the crystals had been effectively freed from solvent by drying) so that accurate analytical data could be obtained. With morpholine and 2-benzyl-4-ethoxymethylene-5-oxazolone "hydrobromide," both 2-benzyl-4-morpholinomethylene-5-oxazolone and β-morpholino-aphenylacetamidoacrylic acid morpholide (LXXIV) were isolated; the conversion of the former sub­ stance to the latter has been mentioned above (Merck, M.23; 28). N—-CCHO Il Il C6H6CH2C-O-CNHCH2CeH6 LXXV N Il C6H6CH2C

CHCHO I CO

\ / N \

CH2C6H6

LXXVI CH2-S—CH-CH-CONHCH2C6H6 HO2C CH

NH NH-COCH2C6H6 LXXVII CH2-COX NH-COCH2C6H6 LXXVIII

A curious fission reaction must be mentioned here. The action of piperidine on the "hydrobromide" of 2-benzyl-4-ethoxymethylene-5-oxazolone gave not only 4-piperidinomethylene-2-benzyl-5-oxazolone but some of the piperidide (LXXVIII; X = NC6Hi0) of phenaceturic acid; an analogous prod­ uct (LXXVIII; X = NC4H8O) was obtained with morpholine (Merck, M.15c). This removal of the 4-methine group, which in these examples is per­ haps split off as a Ν,Ν'-dialkylformamidine, may proceed via the aminomethyleneoxazolone, for it was found that 2-phenyl-4-benzylaminomethylene5-oxazolone on brief boiling with benzylamine gave hippuric acid benzylamide (Barber, Gregory, Slack, Stickings and Woolman, CPS.234). An analogous and even more surprising case was the formation of, hippuric acid by the action of cold acetic acid on methyl D-phenylpenicillenate (LXXIX). In this instance the other fission product was perhaps the thiazoline (LXXX). (CH3)2C-SH N CC6H6 I . 1 1 CH3O2C-CH-NH-CH=C-CO-O LXXIX

OXAZOLONES

757

captan to give 2-phenyl-4-cyanamidomethylene-5oxazolone (King, King; Abraham, Baker, Chain and Robinson, CPS.632). 4-Benzamidopyrazolone The behavior of 2-phenyl-4-ethoxymethylene-5oxazolone with hydrazine and certain other reagents constitutes a special case. The oxazolone with hydrazine in alcohol gave a bright red substance, evidently 2-phenyl-4-hydrazinomethylene-5-oxazolone (LXXXI; R = H), for it gave an azide with nitrous acid. This substance melted when placed in a bath at 120°, but on slow heating melted much higher and with loss of the red color (Barber, Gregory, Langford, Slack, Stickings and Woolman, CPS.385). Later (Slack, CPS.4I6; Barltrop; Abraham, Baker, Chain and Robinson, CPS.463) it was shown that this behavior was due to rearrangement, the product of heating being 4-benzamidopyrazolone (LXXXII; R = H), identical with material made from hydrazine and ethyl /3-ethoxy-a-benzamidoacrylate.

A similar rearrangement may be suspected in the product from phenylhydrazine and 2-phenyl-4ethoxymethylene-5-oxazolone (Mich. Chem., B.2). This substance, placed in a bath at 170-180°, melted, resolidified, and then melted at a higher temperature; on slow heating or after melting once only the higher melting-point could be found. The product obtained after recrystallization of the melt was identical with the substance, evidently l-phenyl-4benzamido-5-pyrazolone (LXXXII; R = C 6 H 6 ), obtained from ethyl phenylpenaldate and phenylhydrazine. It seems probable that the initial substance was 2-phenyl-4-phenylhydrazinomethylene-5-oxazolone (LXXXI; R = C 6 Hs), and rearranged to the pyrazolone on melting. The reaction of acetamidine with 2-phenyl-4ethoxymethylene-5-oxazolone gave the acetamidinomethylene derivative (LVI; R = C 6 H5; X = — N H — C ( C H 3 ) = N H ) , which in the presence of sodium ethoxide was rearranged to 2-methyl-4-hydroxy-5benzamido-pyrimidine (LXXXIII; R = CH 3 ). Similarly 2-phenyl-4-guanidinomethylene-5-oxazolone and 2-phenyl-4-ureidomethylene-5-oxazolone gave the pyrimidines (LXXXIII; R = NH 2 — and H O — ) ; in the interesting case of 2-phenyl-4-(o:-pyridylamino)-methylene-5-oxazolone the p r o d u c t was a divinylene pyrimidone (LXXXIV). 2-Phenyl4-(S-methylthioureido)methylene-5-oxazolone (LXXXV) on similar treatment lost methyl mer-

(LXXXII; R = H) was cyclized by phosphoryl chloride to the oxazolopyrazole (LXXXVI) (Barltrop; Abraham, Baker, Chain and Robinson, CPS.463), and this reaction was also applied {CPS. 632) to prepare the oxazolopyrimidines (LXXXVII; R = C H 3 a n d N H 2 ) from the pyrimidones (LXXXIII; R = CH 3 and NH 2 ). These ring systems are new.

Chromoisomerism in Aminomethyleneoxazolones. The bright red 2-phenyl-4-hydrazinomethylene-5oxazolone (LXXXI; R = H) has already been

OXAZOLONES

758

mentioned. It has been found (Robinson, Wilson; Abraham, Baker and Chain, CPS.681) that this is a labile form, and that a more stable orange form was obtainable. The latter showed a melting-point below that of the pyrazolone (LXXXII; R = H); rearrangement, however, took place rapidly on melting. The structure was proved by prepara­ tion of a benzylidene derivative and a disubstituted hydrazine (LXXXVIII) which resulted from further condensation with 2-phenyl-4-ethoxymethylene-5oxazolone. 2-Phenyl - 4 - aminomethylene - 5 - oxazolone (Barber and Slack, CPS.Jfi), and the cor­ responding acetamidino and guanidino compounds (King, King; Abraham, Baker, Chain and R. Robin­ son, CPS.682) have also been shown to exist in two differently-colored forms, the deeper-colored being the more labile in each case. It has been suggested (Barltrop; Abraham, Baker, Chain and Robinson, CPS.4-68) Robinson, Wilson; Abraham, Baker, and Chain, CPS.681) that the more deeplycolored forms are chelated cts forms, e.g., LXXXIX (the electronic surplus is to be regarded as shared by the oxazolone ring as a whole), and that the more stable varieties are the trans forms. In support of this hypothesis it may be mentioned that the scarlet 2-phenyl-4-hydrazinomethylene-5-oxazolone is insoluble, unlike the orange form, in dilute acid, and also seems to be more readily rearranged to the pyrazolone, to judge from the behavior on heating. H2N

δO-CO

\

NH

\ / C=CH

/

CeH6C=N

LXXXIX

HOCR'=C—CO—O I

N=

CR

XC 4 - (α-Hydroxyalkylidene) -5-oxazolones. Some members of the class XC have been prepared; as might be expected they closely resemble the 4-hydroxymethylene-5-oxazolones but with some interesting discrepancies. 2-Styryl-4-(a-hydroxyethylidene)-5-oxazolone (XC; R = C6H6CH=CH; R' = CH3) was prepared from cinnamoylglycine, acetic anhydride and potassium acetate (Bentley, Robinson; Abraham, Baker, Chain and Robinson, CPS.441); hippuric acid similarly gave 2-phenyl4-(a-hydroxyethylidene)-5-oxazolone (XC; R = C6H6; R' = CH3) (Elliott, Hems and F. A. Robinson, CPS.490), but it was found better to prepare this from sodium hippurate and acetic anhydride; 2-phenyl-4-(oc-hydroxypropylidene)-5-oxazolone (XC;

R = C6H6; R' = C2H6) could also be prepared in this way {CPS.490). A few reactions were studied.

The styryloxazolone with methyl sulphate gave a high-melting methyl derivative which contained the N-methyl group (Herzig-Meyer) and was apparently the enol-betaine (XCI). With diazomethane this substance was also produced, along with a lower-melting isomer which was evidently the O-ether, for the hydroxyethylideneoxazolone was regenerated readily on hydrolysis and the ultraviolet absorption also resembled the parent substance (and 2-styryl-4-hydroxymethylene-5-oxazolone) (CPS.441). An O-methyl ether was simi­ larly obtained from 2-phenyl-4-hydroxyethylidene5-oxazolone and diazomethane (CPS.490). -OC (CH3)=C-CO-O

CH3N= +

=C-CH=CHC6H6 XCI

CH3COCHCO2C2H6 NHCOCH=CHC6H6 XCII

So far as they were studied the ring-opening reactions of these oxazolones resembled those of the 4-hydroxymethylene-5-oxazolones. 2-Phenyl4-(a-hydroxyethylidene)-5-oxazolone was found to be stable to hot 2 N alkali under conditions which destroyed the hydroxymethylene analogue ( CPS. 490); the 2-styryl compound was also reported to be stable to alkali (CPS.441)· The sodium deriva­ tive of XC (R = C6H6; R' = CH3) was also stable to alcoholic sodium ethoxide. Hydrolysis of XC (R = C6H6; R' = CH3) with 2 N hydrochloric acid gave aminoacetone (CPS.490). With alcohol followed by 2,4-dinitrophenylhydrazine, the styryl oxazolone (XC; R = C6H6CH=CH; R' = CH3) gave the 2,4-dinitrophenylhydrazone of ethyl a-cinnamoylaminoacetoacetate (XCII) (CPS.441)· Ultraviolet Absorption of Oxazolones. Some general remarks are necessary on the ultraviolet absorption behavior of oxazolones, as this property has frequently helped in their identification. Sim­ ple oxazolones of type I show only end absorption unless the 2-substituent is an aryl or similar group. However, the type II oxazolones show character­ istic absorption bands of high intensity in the region 2,600-3,600 A, the longest wave-lengths being found when R is aryl and when R' is attached through an oxygen or particularly a nitrogen or sulphur atom. An illustrative table is given below (Table VI); the absorption spectra of other com­ pounds are given with their preparation in the experimental section. PART III

Reaction of 4-Heteromethylene-5-oxazolones with Penicillamine and Similar Substances. One of the principal objects in making 4-heteromethyl-

OXAZOLONES

759

TABLE VI Solvent

5-Oxazolone

^mfti (A)

EM

End absorpti on only

Reference

M,12c

2-Benzyl-4,4-dimethyl-

Cyclohexane

2-Phenyl-4-benzyl-

Ether

2,440

17,300

SM

2-Phenyl-4-phenyl-

Ether

2,450

20,000

S.53

2-Phenyl-4-benzylidene-

?

2,500 2,600 3,620

12,200 12,200 43,400

W.10

2-Benzyl-4-hydroxymethylene-

Aq. alkali

2,400 3,000

6,000 16,700

MSO

2-Benzyl-4-ethoxymethylene-

Ether

2,800

14,650

C.10

2-Benzyl-4-aminom ethylene-

95 % alcohol

2,350 (plateau) 3,100

4,290 23,100

M.l 2c

Methanol

2,400 (plateau) 3,225

7,500 28,000

M.12c

Methyl benzylpenicillenate

Ethanol

3,175

20,600

MM

Methyl phenylpenicillenate

Ethanol-Chloroform

2,400 2,800 3,500

12,000 6,000 34,000

M.12c

2-Benzyl-4-ethylthiomethylene-

Ethanol

3,350

20,250

CPS.388

2-Phenyl-4-ethylthiomethylene-

Methanol

3,540

29,800

CPS.S88

(CH3)2C-CH-NH-CH=C-CO-O CeH6CHi

C^O2H

A

(W C H 2

6

6

ene-5-oxazolones was, naturally, to condense them with penicillamine, and the possibility that such a condensation might lead to penicillin was enter­ tained at a very early stage. As work proceeded, it became evident that 4-hydroxymethylene-5oxazolones and their derivatives showed little tendency to smooth formation of thiazolidines in the manner of normal aldehydes; and owing to the frequently complex and intractable nature of the products, most of the experiments led to no definite result. These indecisive experiments are sum­ marized in Table VII. Some of them, as indicated, led to products having a trace of antibiotic activity. Under certain conditions, and chiefly by the use of penicillamine methyl ester, it was found possible to obtain definite products, and collect some evi­ dence of their constitution. These will be men­ tioned in greater detail. (i) Reactions with 4-Hydroxymethylene-5-oxazolones. The condensation of 2-phenyl-4-hydroxymethylene-5-oxazolone with L-cysteine in aque­ ous solution at pH 2 gave carbon dioxide and 2-benzoylaminomethylthiazolidine-4-carboxylic acid

(XCIII); another experiment with ρII initially 4-5 gave some indication that coupling with cysteine preceded decarboxylation (Mich. Chem., B.2). A condensation of 2-amyl-4-hydroxymethylene-5-oxazolone with DL-penicillamine at pH 5 gave DLamylpenilloic acid (XCIV) and carbon dioxide; these

were accompanied by other products which could not be isolated (Attenburrow, Elliott, Hems, and Robinson, CPSl CH2-S

\

CH-CH2NHCOC6H6

HO2C-CH-NH XCIII (CH3)2C

S

\

CHCH2NHCOCBHu

HO2C-CH-NH XCIV (CH3)2C-CH-NH-CO HS

SH

CO- -NH- -CH-C(CH3)2 XCV

The condensation of 2-benzyl-4-hydroxymethylene-5-oxazolone with DL-penicillamine methyl ester (Mich. Chem., B.8) in dioxan gave a neutral product, m.p. 210-212°, which was recrystallized from ethyl acetate. It then had m.p. 198-199° and appeared to consist of the diketopiperazine (XCV) of DL -penicillamine. Another group (Merck, M.28) obtained the product m.p. 211-212° in the same way;

OXAZOLONES

736 Condensation of Oxazolones

Group R

Group X

Co-cyclohexyl-5,5dimethylthiazolidine hydrochloride.. N-Acetyl derivative α -Ethyl w-amy lpenicilloate N-Acetyl derivative 4-Carboxy-2,2,5,5-tetramethylthiazolidine Hydrochloride 4-Carboxy-2,5,5-trimethylthiazolidine hydrochloride 4-Carboxy-2-acetyl-2,5,5-trimethylthiazolidine Thiazolidine-4-carboxylic acid 2-Ethylthiazolidine-4-carboxylic acid... 4-Carboxy-2-ethyl-5,5-dimethylthiazolidine hydrochloride 2-Phenylthiazolidine 4-Carboxy-2-(2'-hydroxyphenyl)-5,5dimethylthiazolidine 0,N-Diacetyl derivative 2-(3'-Methoxy-4'-hydroxyphenyl)-5,5dimethylthiazolidine-4-carboxylic acid Ο,Ν-Diacetyl derivative 2- (a,0-Dihydroxyethyl)-5,5-dimethylthiazolidine-4-carboxylic acid 2-Carbethoxymethyl-2,5,5-trimethylthiazolidine-4-carboxylic acid 2-(2'-Hydroxynaphthyl)-5,5-dimethylthiazolidine-4-carboxylic acid Benzylpenicillin (triethylamine salt) Benzylpenillic acid DL -Penicillamine hydrochloride DL -N-Acetylpenicillamine DL -S-Benzylpenicillamine DL -Cysteine hydrochloride L -Cystine |3-Mercapto-ethylamine Benzyl thiol Thioacetic acid 2- (p-Chlorobenzeneazo-carbethoxymethyl)-5,5-dimethylthiazolidine-4carboxylic acid Penicillaminic acid

XaIOi

Iodine produc­ tion

1.05 1.1

+

0.99 1.08 2.98 0.96

+

con­ sumed

—

—

+ —

1.0 0.91

+ +

0.95

+

0.92 0.91 0.91

+ + +

1.1 1.17

+ +

1

2.2 1.1

-

2.1 1.03

—

2.2

+

2.1

-

2.0 1.2 3.2 0.9 1.0 Nil 0.97 0.97 1.1 0.6 0.45 3.0 Nil

2

1 — —

+ +

3

+ —

+ + + +

1 Yellow 2 3

color. Brown color. Uptake at 60°, 2 moles (Sykes and Todd, CPS.677).

sulphoxide, which is stable both to periodate and iodate. Up to this time several undescribed thiazolidines are described in the experimental portion, among them 2-(a,P-dihydroxyethyl)-5,5-dimethylthiazolidine4-carboxylic acid. This compound was originally synthesized in the hope of preparing from it 2-aldehydo-5,5-dimethylthiazolidine-4-carboxylic acid; attempts to do so broke down as a result of accompanying oxidation at the sulphur atom (Sykes and Todd, CPS.526; 677).

929

N-Acylated thiazolidines differed markedly from their sulphoxides in their stability to acid and alkali, although both were stable to methanol and to mercuric chloride. Thus, 3-acetyl-2-phenyl5,5-dimethylthiazolidine-4-carboxylic acid was sta­ ble to alkali but was readily decomposed by mineral acid giving benzaldehyde and penicillamine. The corresponding sulphoxide, on the other hand, was stable to acid, but treatment with warm alkali yielded benzaldehyde together with a-acetamido-/3/3-dimethylacrylic acid, sulphur being eliminated. 4-Carboxy-3-acetyl-2-phenyl-5,5-dimethylthiazolidine sulphone resembled the sulphoxide in its instability to alkali, sulphur being eliminated, but it also broke up readily with acid giving benzalde­ hyde and a resinous acid (Sykes and Todd, CPS. 677). Methyl benzylpenicillin sulphone showed remarkable stability towards acids (Merck, M.59). Thiazolidine-4-carboxylic acids unsubstituted on the ring nitrogen were readily oxidized by iodine, the extent depending on the 2-substituents. It seemed probable that the oxidation was concerned with a thiol fission product rather than with the thiazolidine sulphur atom as such. N-Substituted thiazolidine-4-carboxylic acids (such as N-acetyl and N-carbomethoxy derivatives) were not oxidized by iodine; this reflects the greater stability of the ring system in such compounds, since N-acetylation of amino acids does not prevent their oxidation by iodine (Lilly, L.19; Upjohn, U.lSa, 14). In oxida­ tions with bromine the distinction is not so marked. Various thiazolidines, including one containing a N-carbethoxy group (4-carboxy-3-carbethoxy-2-i'sopropyl thiazolidine), reduced about 6 equivalents of bromine in 1 N solution in acetic acid but 4-carboxy3-acetyl-thiazolidine consumed almost 3 equivalents of bromine under similar conditions (Upjohn, U.J.)· N-Unsubstituted thiazolidines also appear to be oxidized (presumably as their thiol cleavage products) by ammoniacal silver solution and by Tollens reagent (Squibb, SAJt.; 12a) though ir­ regularities were apparent (Lilly, L.J). 4-Carboxy3-acetyl-2-phenylthiazolidine gave with Tollens reagent the corresponding thiazolidine sulphoxide (Lilly, L.4), and oxidation of several N-acyl thi­ azolidines with potassium permanganate gave the sulphones (Merck, M.6S). Reaction of Some Thiazolidines with Carbon Disulphide or Phosgene. For some time attempts were made to prepare XV (R = H or Me; R' = H) CO2R-CH-N-

-CS

Me2C · S · CHCHR'NH XV which it was hoped might have reacted, in the form of its ester, with lithium benzyl to give compounds with the ring structure of penillic acid; earlier a similar intermediate containing a "penicilloate" carboxyl group was of interest as possibly affording

TfflAZOLIDINES

930

Me2C=CCO I I NHCSN-R XXII

a route to "tricyclic penicillin" derivatives. Ex­ periments to condense XVI (R = H or Me) as hydrochloride or free base with carbon disulphide CO2RCH-NH Me2A-S-CH-CH2NH2 XVI were at first unpromising and a means of facilitat­ ing formation of the required bicyclic thiazolidines seemed to be offered by the hypothetical compound XVII. CS2EtNHCH2CH(OEt)2 XVII Reaction of ethyl dithiochloroformate and aminoacetaldehyde diethyl acetal in cold aqueous sodium bicarbonate only afforded XVII as an intermediate, as on distillation of the product ethyl mercaptan was lost to give a compound of. composition cor­ responding to the β,β-diethoxyethyl isothiocyanate (XVIII). That the product was so constituted was confirmed by the fact that it reacted at room tem­ perature with ethereal aniline to give the phenylthiourea XIX (R = Ph) and with benzylamine to give the benzylthiourea XIX (R = CH2Ph). With aqueous 2,4-dinitrophenylhydrazine, XVIII gave a crystalline derivative of composition in fair agreeCH(OEt)2-CH2NCS XVIII

was obtained by reacting penicillamine with the iso­ thiocyanate XVIII in alkaline solution. Acyl derivatives of penicillamine often pass readily into derivatives of β,/3-dimethylacrylic acid (Bentley, Catch, Cook, Elvidge, Hall and Heilbron, PEN.114; Cook, Elvidge, Hall, Heilbron and Shaw, CPS.270) and cyclization of the postulated kind is paralleled by a similar reaction (Bentley, Catch, Cook, Heil­ bron and Shaw, CPS.267), the nature of which was completely proved (Cook, Heilbron and Shaw, CPS.S11). The formation of thiohydantoins, how­ ever, cannot always proceed so easily for reaction of penicillamine ethyl ester with phenyl isothio­ cyanate gave a crystalline product with composition indicating it to be the thiourea XXI (R = Ph; R' = Et). On the other hand penicillamine and phenyl isothiocyanate led to 3-phenyl-5-isopropylidene-2thiohydantoin (CPS.S11) and another cyclization of this kind may be mentioned. Ethyl formylacetate diethylacetal condensed with penicillamine to give 4-carboxy-5,5-dimethyl-2-carbethoxymethyl-thiazolidine hydrochloride (XXIII), which also reacted readily with phenyl isothiocyanate to give the neutral fused-ring thiazolido-thiohydantoin XXIV.

CO 2 HCH~NHHCI

CH(OEt)2-CH2-NH-CSNHR XIX ment with structure XX. It therefore seemed that XVIII might have been a suitable intermediate for N-NH-C6H3(NO2)2 Il CH-CH2-NH-CS-NHNH-C6Hs(NO2)2 XX the preparation of XV and it was accordingly treated with penicillamine methyl ester. The crystalline product, Ci2H20OsN2S, contained no thiol grouping nor did it develop thiol properties on hydrolysis; it was devoid of basic properties and was indeed a pseudo acid; finally, although the thiocyanate grouping was no longer evident, carbonyl functions were still present and the compound gave a dinitrophenylhydrazone C14Hi4O5N6S. It can hardly be doubted, therefore, that the primary reaction is the C02R'CH NH I I Me2C-SH CS-NHR XXI formation of the thiourea XXI (R' = Me; R = CH2CH(OEt)2) which cyclizes in an undesired direction losing hydrogen sulphide to give 3-(/3,/3diethoxyethyl)-5-isopropylidene-2-thiohydantoin (XXII; R = CH2CH(OEt)2). The same compound

Me2C · S · CH-CH2CO2Et XXIII Me2C

CH-CO

S-CH(CH2CO2Et)-N-CS-NPh XXIV Under rather different conditions an acid Ci6Hie 03N2S2 was formed, presumably by the alternative cyclization to give the thiazolido-thiouracil XXV. Me2C-CH(CO2H)-N-CS-NPh I I I g__ CH-CH2CO XXV \ / C

\ / C

(!!H2NH CS-NHCH2 XXVI The dihydrochloride of thiazolidine XVI (R = H) readily gave the ester XVI (R = Me) with diazomethane. Many experiments were made to convert this ester into the bicyclic compound of type XV; the most successful involved long refluxing with carbon disulphide, hydrogen sulphide being evolved. Two compounds were eventually obtained though in rather small yield; both had compositions

THIAZOLIDINES corresponding to C9Hi4O2N2S2 but one had m.p. 187-188° (Compound A) and was almost insoluble in ether, while the other had m.p. 117-118° (Com­ pound B) and was more soluble in ether. Both were soluble in cold aqueous sodium hydroxide though that of m.p. 187-188° was perhaps less ready to dissolve. These products were formed in different relative quantities in different experiments; for example, before their isomerism was appreciated it was thought that one might have been the thio­ urea XXVI and an attempt to prevent its formation by carrying out the condensation in very dilute solution with respect to the initial thiazolidine led only to the lower melting isomeride. It was at one time thought that they might have been stereoisomeric but the difference in physical properties hardly supported this belief. Nor could one be the uncyclized isothiocyanate XXVII for neither had any notable reaction with organic bases. Neither contained a polarographically detectable thiol CO2Me-CH-NH I I Me2C · S • CH-CH2NCS XXVII CO2MeCH-N

C-SH

Me2C-S-CH-CH(R)-N XXVIII group: the low melting compound formed a complex with mercuric chloride, insoluble in methanol and water, from which it was recovered quantitatively on treatment with hydrogen sulphide. Neither ester was soluble in cold 2 N hydrochloric acid, and attempts to convert the low melting isomer into the second compound by heating alone, or with various reagents, were unsuccessful. On mild alkaline or acid hydrolysis both esters yielded the same acid, m.p. 197-198°, C8Hj2O2N2S2. It is considered that the low-melting isomer, Compound B, is to be represented by XV (R = Me; R' = H), and Compound A as the pseudothiourea XXIX (R = Me; R' = H). The acid was regarded as XXIX (R = R' = H), isomerization CO2R-CH-N C=NH I I I Me2C · S • CH-CH(R')-S XXIX occurring during hydrolysis in the case of Compound B. On reaction of Compound B with mercuric chlo­ ride the mercury derivative of the isomeric XXVIII (R = H) is formed. Similarly, Compound B formed a white precipitate with ethereal benzyl magnesium chloride, presumably a magnesium complex of XXVIII (R = H), from which it was recovered unchanged on decomposition with acid. In order to obtain more evidence about the isom­ erization, 4-carboxy-5,5-dimethy'i-2-methylamino-

931

methyl-thiazolidine (XXXI; R = H) was prepared as its dihydrochloride, and converted into its ester (XXXI; R = Me) by reaction with diazomethane: CO2Me-CH-N CO I I I Me2C-S-CH-CH2-NH XXX CO2RCH-NH Me2C • S · CH-CH2NHMe XXXI analysis however, indicated that two methyl groups had been introduced, and no crystalline product was obtained on subsequent reaction with carbon disulphide. In contrast to these results, after interaction of XVI (R = Me) with phosgene in the presence of sodium bicarbonate only the single ester (XXX) or an isomeride was isolated, but as the product was only obtained in small yield, the possibility of other products being formed is not excluded. As intermediates in further penillic acid syntheses it was necessary to prepare XV (R = H or Me; R' = CO2Me). 4-Carbomethoxy-5,5-dimethyl-2carbomethoxyaminomethyl-thiazolidine was ob­ tained from its dihydrochloride (Abraham, Baker, Chain and Robinson, CPS.342) by treating with potassium bicarbonate and extracting with benzene. On reaction with carbon disulphide in a methanolether solution, two isomeric compounds CnHi 6 O4N2S2, were again obtained. One compound (designated A) had m.p. 219°, and was insoluble in ether and only sparingly soluble in methanol: Compound B, m.p. 162°, was soluble in ether, and more soluble in methanol. By analogy with the previously described compounds, these compounds are formulated as XXIX (R = Me; R' = CO2Me) and XXVIII (R = CO2Me) respectively (Bentley, Cook, Elvidge, Heilbron and Shaw, CPS.S87; Bentley, Cook and Heilbron, CPS.430). N-/3-Hydroxyethyl-thiazolidines. When at­ tempts were made to synthesize thiazolidineoxazolones from penicillamine, it was clear that the NH2 group, and in thiazolidines the NH group, offered possibilities which precluded the attainment of the final objective. N-Methylpenicillamine was therefore synthesized with a view to preparing "protected" N-methylthiazolidines, and was con­ densed with formaldehyde (Upjohn, U.18, 2; see p. 944). The use of the /3-hydroxyethyl group was found to be more convenient, and was investi­ gated in detail. When i'sopropylidene penicillamine was heated with a chloroform solution of ethylene oxide, preferably in presence of boron trifluoride, it was converted into a nonacidic crystalline material of composition corresponding to CioHnO2NS. This material was unaffected by acetic anhydride but on

932

THIAZOLIDINES

shaking with cold baryta afforded an acid which reverted to the original material on mild dehydration. The primary product must therefore be represented as the lactone XXXII.

The thiazolidine ring (XXXII) was ruptured, though not very satisfactorily by aqueous mercuric chloride; with moderately strong hydrochloric acid, the isopropylidene residue was removed and ~N-$-hydroxyethylpenicillamine hydrochloride (XXXIII) was obtained. This general route to XXXII seemed the least troublesome, for the direct interaction of ethylene oxide and penicillamine, penicillamine ester or S-benzylpenicillamine gave mixtures doubtless due to the formation of tertiary amines and other obvious complications. On the other hand other thiazolidines reacted as cleanly as the tsopropylidene compound. Thus isopropylidene penicillamine methyl ester afforded 4;~carbomethoxy-2,2,h,b-tetramethyl-Z-(f}-hydroxyethyl)-thiazolidine (XXXIV) with ethylene oxide in the presence of a little boron trifluoride. On warming XXXIV with acetic anhydride it did not undergo acetylation, but instead lost the elements

cillamine to react with acetone. For this reason, it seemed advantageous (for reactions more allied to synthesis of penicillin analogues) to insert the hydroxyethyl grouping or lactone ring at a later stage. Accordingly, a-ethyl n-amylpenicilloate was treated with ethylene oxide in cold chloroform, using a little boron trifluoride as catalyst. Again, the crystalline product was an acid to which constitution XXXVII (R = n-C 5 Hii) was ascribed. When XXXVII was dehydrated with acetic anhydride the lactone XXXVIII (R = n-C s Hn) was formed, which also arose when the initial reaction was carried out at a higher temperature.

XXXVII

(R = n-CsHu)

was treated

with

2

equivalents of alkali overnight, and the lyophically dried sodium salt (regarded as the disodium penicilloate corresponding to XXXVII) was treated with acetic anhydride at 60° for thirty minutes, or 90° for one hour.

After removal of acetic anhydride, and

solution of the residue in neutral buffer, solution, small activities were observed (slightly more than 1 U./mg.), the starting material being inactive. Clearly, the most reasonable manner of accounting for this activity was to postulate formation of the lactone-oxazolone XXXIX (R = 71-C5H11).

No re-

action was observed between methyl benzylpenicillin and ethylene oxide, the ester being recovered of methanol to yield the lactone XXXII. 4-Carboxy-5,5-dimethyl-2-phenylthiazolidine reacted with ethylene oxide in chloroform solution containing boron trifluoride, and at low temperature yielded a crystalline acid of composition corresponding to the N-p-hydroxyethyl derivative (XXXV); this compound was also produced in the absence of a catalyst. Treatment of XXXV with acetic anhydride gave the lactone (XXXVI) also obtained by the direct reaction with ethylene oxide at a higher temperature.

Whereas XXXII was more stable than the parent thiazolidine, XXXIII was less ready than peni-

unchanged (Bentley, Cook and Heilbron, CPS.527). a-Ethyl benzylpenicilloate reacted similarly with ethylene oxide, without the aid of an additional catalyst, to give a-ethyl ~N-fi-hydroxyethyl-benzylpenicilloate (XXXVII; R = C6H 6 CH 2 ), which passed into the corresponding lactone (XXXVIII; R = C6H5CH2) on treating, either with acetic anhydride, or warm dilute mineral acid. The relationship between these compounds was confirmed in that XXXVII gave with hydrazine a water-soluble hydrazide hydrazinium salt (XL) whereas XXXVIII gave the dihydrazide (XLI). When XXXVII was treated with cold aqueous baryta a mixture resulted which was eventually resolved into three pure compounds. The first was N-fi-hydroxyethyl-benzylpenicilloic acid (XLII) obtained as a hydrate and also as a butanolate. XLII gave a dibenzylammonium salt. It was somewhat surprising to find that XLII was much more stable

XXXVIII above). propionate was also prepared and condensed with The third product from XXXVII was devoid of D-penicillamine to give O-i-carboxy-5,5-dimethyl-2acidity and appeared analytically to be the lactone methylamino-carbomethoxymethyl-thiazolidine hydro­ XLIV. In attempts to convert XLII into an oxazol- chloride (Lilly, L.20). Similarly starting from one it was treated with acetic anhydride in pyridine. phenylacetyl-sarcosine methyl ester the preparation There was clearly some degree of degradation, for of methyl N-methyl-benzylpenaldate was at­ when the gummy product was treated with benzyl- tempted, but the product obtained was of doubtful amine, phenylacetylbenzylamide was formed; the nature (Upjohn, U.19). Other work achieved fell into two sections, (a) main product, however, was not a benzylamide but the benzylammonium salt (XLV). It seems there­ the preparation of N-substituted penaldates, their fore that the sole action of acetic anhydride under acetals and the model reactions of such compounds, these conditions was to close the lactone ring and and (b) the preparation and properties of the there was no evidence of the formation of an oxazol- N-substituted penicilloates derived therefrom. (a) An attempt to prepare the ethyl ester of one ring. Meanwhile the dihydrazide (XLI) reacted with nitrous acid, the azide giving with benzylamine N-benzyl-/3,/3-diethoxyalanine (L) by direct con­ the lactone-benzylamide (XLVI). The closure of the densation of benzylamine and ethyl a-chloro-^,/3lactone ring under these conditions is noteworthy. diethoxypropionate was unsuccessful but the parent Small biological activities were observed in this acid (L) was successfully synthesized as follows: series only irregularly and were not regarded as Ph-CH2N(CHO)-CH2-COOEt -> significant (Bentley, Cook and Heilbron, continua­ XLVHI tion of CPS.527). Miscellaneous Phenyl-penilloic and -penicilloic Ph-CH2-N (CHO) -C(=CHOR) -COOEt Derivatives, and Some N-Alkyl and N-Aralkyl XLIX Compounds. Formylation of ethyl hippurate af­ forded ethyl formylhippurate and ethyl hippuroylI hippurate (Erlenmeyer, Annalen, 887, 251 (1904)). CH(OEt)2-CH(NH-CH2Ph)-COOH The diethyl acetal of the former was more satis­ L factory for condensation with penicillamine, giving N-Benzylglycine ethyl ester (Mason and Widner, α-ethyl phenylpenicilloate, hydrolyzed to phenylpenicilloic acid. The latter was however readily J. Chem. Soc., 65, 188 (1894)) was converted into decarboxylated to phenylpenilloic acid (Barber, its N-formyl derivative (XLVIII) which reacted Gregory, Slack, Stickings and Woolman, CPS.66; smoothly with potassium ethoxide and ethyl see also Imperial College, CPS.5) and on treatment formate to give a-(N-benzylformamido)-fi-hydroxywith acetic anhydride gave no evidence of dehydra­ acrylic ethyl ester (XLIX; R = H). This was tion but rather of acetylation, and no significant further characterized as its enol acetate (XLIX; biological activity was produced (Newbery and R = Ac) and enol benzoate (XLIX; R = COPh). Treatment of XLIX (R = H) with alcoholic hydro­ Raphael, CPS.206). In the hope of synthesizing an N-methyl or gen chloride and subsequent hydrolysis with N-benzyl analogue of "tricyclic penicillin" (XLVII), caustic soda (cf. Lilly, L.18; 20) gave a rather poor yield of L (Copp, Duffin, Smith and Wilkinson, CO2H-CH-N-CRO CPS.861). Attempts to convert L into N-benzylbenzylpenaldic acid diethyl acetal (Li; R = H; NH CH(OEt)2-CH-CO2R Me2C · S · CH-CH CO XLVII R'-NCOR" LI which might be-achieved more readily than that of penicillin itself, the following syntheses were CH(OH)=C-CO2Et initiated and continued since some of the reactions I R-N-COR' originally pro posed for the preparation of analogues of XLVII could equally well have given the cor­ LII

935

TfflAZOLIDINES R' = R" = CH2Ph) gave rise to a gum (Copp, f)uffin, Smith and Wilkinson, CPS.!/>4) and an alternative route to this compound via LII (R = R' = CH2Ph) by the formylation of N-benzylphenaceturate also failed (Winthrop, W.10, 8; 11, 1). However, other workers were successful in phenylacetylating L to N-benzyl-benzylpenaldic acid diethyl acetal (Merck, M.60, 13). Although the preparation of these phenylacetyl derivatives was difficult, the corresponding benzoyl derivative, ethyl N-benzyl-phenylpenaldate (Li; R = Et; R' = CH2Ph; R" = Ph) was readily pre­ pared by direct formylation of ethyl N-benzylhippurate, and formylation of ethyl N -methylhippurate gave ethyl N-methyl-phenylpenaldate (LII; R = Me; R' = Ph). The ethyl N-benzylhippurate and ethyl N-methylhippurate were prepared by the benzoylation of N-benzylglycine ethyl ester and sarcosine ethyl ester respectively; these esters were further characterized by hydrolysis to N-benzylhippuric acid and N-methylhippuric acid respectively (see Cocker and Lapworth, J. Chem. Soc., 1931, 1894). Both LII (R = Me; R' = Ph) and LII (R = CH2Ph; R' = Ph) reacted readily with aniline and benzylamine to give the corresponding anilino and benzylamino derivatives. Reaction with benzylthiol was very sluggish and after a prolonged period the greater part of the starting material was recovered. With glycine, LII (R = CH2Ph; R' = Ph) gave ethyl a-(N-benzylbenzamido)^-carboxymethylaminoacrylate (LIII), but LII (R = Me; CO2H-CH2NHCH=C (CO2Et)-N(CH2Ph) -COCH2Ph LIII R' = Ph) gave an unidentified product. Attempts to prepare the acetals (LI; R = H; R' = Me or CH2Ph; R" = CH2Ph) by treatment of the penaldates with ethyl orthoformate by the Claisen proc­ ess were unsatisfactory (Copp, Duffin, Smith and Wilkinson, CPS.IfiJ+)· Formylation of N-benzoylsarcosine methyl ester gave methyl N -methyl-phenylpenaldate, further characterized by means of the anilino derivative (Merck, M.63). In order to obtain compounds containing an acyl group more closely allied to natural 2-pentenylpenicillin the formylation of N-benzy Z-N -caproylglycine ethyl ester, prepared from caproyl chloride and N-benzylglycine ethyl ester, was examined. Formylation seemed to proceed as usual and the product, presumably ethyl N-benzyl-amylpenaldate (LII; R = CH2Ph; R' = CoH11), although rather impure, was successfully characterized and identi­ fied as its crystalline benzylamino derivative. Simi­ larly the formylation of ethyl N -methylaccturate gave a-(N-methylacetamido)-P-hydroxyacrylic ethyl ester (LII; R = R' = Me) which readily underwent model reactions with aniline and benzylamine to give crystalline products (Copp, Duffin, Smith and Wilkinson, CPS.IfiJt). The attempted formylation of N-benzyl-'N-2-

hexenoylglycine ethyl ester proceeded abnormally with the formation of what was probably N -benzyl3-(l-butenyl)-2,4.-pyrrolidione (LIV) the same prod-

EtCH=CH-CH-CO-CH2 I ! CO N-CH2Ph LIV net being obtained in the absence of ethyl formate. Presumably it was formed by migration of the double bond in the presence of potassium ethoxide to give N-benzyl-N-2-hexenoylglycine ethyl ester which would then undergo intramolecular condensa­ tion in a manner analogous to that observed for the conversion of methyl N-methvl-benzylpenaldate into N-methyl-3-phenyl-2,4-pyrrolidione (see Merck, M.60, 11). That wandering of the double bond was involved was shown by preparing N-benzyi-N-3hexenoyIglycine ethyl ester from 2-hexenoyl chloride and N-benzylglycine ethyl ester and treating it with potassium ethoxide under the same conditions as before when the same compound N-benzyl-3-(lbutenyl)-2,4-pyrrolidione, was obtained, only in rather better yield. This facile wandering of the double bond was somewhat unexpected but it has been shown (Hugh, Kon and Linstead, J. Chem. Soc., 1927, 2585) that a very small amount of sodium ethoxide (actually V-5 ο mole) converts isopulegone to pulegone extremely quickly, and thus potassium ethoxide might catalyze the three carbon isomerization here recorded (Copp, Duffin, Smith and Wilkinson, CPS.642). (b) As a preliminary experiment, XLIX (R = H) was condensed with DL-penicillamine but the prod­ uct, possibly a mixture of stereoisomers of 4-carboxy- 5,5-dimethyl -2-N-benzylf ormamido-carbethoxymethyl-thiazolidine (LV; R = CH2PhjR' = H) was not obtained crystalline (Copp, Duffin, Smith CO2HCH-NH Me2C • S · CH-CH(CO2Et)-NR-COR' LV and Wilkinson, CPS.361). Ethyl N-benzyl-phenylpenaldate (LII; R = CH2Ph; R' = Ph) also con­ densed with DL-penicillamine to give an amorphous product, analysis of which was in good agreement with that required for Oij-i-carboxy-5,5-dimethyl-2N-benzyl-benzamido-carbethoxymethyl-thiazolidine

(LV; R = CH2Ph; R' = Ph) (Copp, Duffin, Smith and Wilkinson, CPS.Ifi4)· It formed an amorphous hydrochloride which nevertheless analyzed cor­ rectly and it seemed likely that the product was a mixture of stereoisomers, a view supported by the ultraviolet absorption spectrum (see later) (Copp, Duffin, Smith and Wilkinson, CPS.642). Condensation of the methyl ester of N-benzylbenzylpenaldic acid diethyl acetal with D-penicillamine likewise gave an amorphous product, namely D -4-carboxy-5,5-dimethyl-2-N-benzyl-phenylacet-

936

THIAZOLIDINES

amido-carbomethoxymethyl-thiazolidine which was characterized by means of its crystalline benzylamine salt (Merck, M.63, 5). With D-penicillamine methyl N-methyl-phenylpenaldate condensed readily to give crystalline D4-carboxy-5,5-dimethyl-2-N-methyl-benzamido-carbomethoxy methyl-thiazolidine. Esterification with diazomethane afforded the dimethyl ester and hydrolysis with sodium hydroxide gave D-4-carbomethoxy-b,5-dimethyl-2-'N-methyl-benzamido-carboxymethyl-thiazolidine (Merck, M.63). Similarly, ethylN-methyl-phenylpenaldate (LII; R = Et; R' = Ph) condensed with D-penicillamineto give a pure crys­ talline product believed to be a single stereoisomer of T>-4-carboxy-5,5-dimethyl-2-'N-methyl-benzamidocarbethoxymethyl-thiazolidine (LV; R = Me; R' = Ph). The condensation product from DL-penicillamine and crude ethyl N-benzyl-amylpenaldate (LII; R = CH2PI1,· R' = C5H11) on the other hand was unsatisfactory; it formed a resinous hydro­ chloride, the composition of which, however, ap­ proximated to that required for the hydrochloride of LV (R = CH2Ph; R' = CsHn). Attempts to use the pure benzylamine derivative in place of the crude hydroxymethylene derivative led only to recovery of starting material. The condensation product from D-penicillamine and LII (R = R' = Me) could not be isolated (Copp, Duffin, Smith and Wilkinson, CPS.64%)• Attempts to convert LV (R = CH2Ph or Me; R' = Ph) into analogues of XLVII were unsuccess­ ful, due mainly to the lack of reactivity shown by the «-ethyl ester grouping. Attempts to isolate other isomers by benzoylation of the residues also failed since no crystalline compounds were isolated; LV (R = Me; R' = Ph) itself readily yielded a crys­ talline N-benzoyl derivative. CO2H-CH-NH CONHNH2

s

Me2A · · AH —AHNR-COPII LVI CO2H-CH-N

CO

I - S - A H — A H - NR-COPh Me2CLVII It seemed likely that a hydrazide such as LVI (R = Me or CH2Ph) would be converted into LVII (R = Me or CH2Ph) via the corresponding azide but attempts to prepare such hydrazides were unsuccessful. Treatment of LV (R = CH2Ph; R' = Ph) under the conditions successfully used for the preparation of phenylpenicilloic a-hydrazide (Chapter XXI) led only to the recovery of un­ changed material, while more vigorous conditions led only to reduced recovery without the formation of the desired hydrazide. Presumably this lack of reactivity is due to steric hindrance; the model conversion of ethyl N-benzylhippurate to the cor­

responding hydrazide (LVIH) (isolated as its hemihydrate) proceeded only in the presence οϊ considerable excess of hydrazine hydrate, showing that even here steric hindrance was probably present. PhCON(CH2Ph) CH2CONHNH2 LVIII PhCONMe-CH-

-CO

CH-NH-NH LIX Treatment of O-A-carboxy-5,5-dirnethyl-2-N-methylbenzamido-carbethoxymethyl-thiazolidine (LV; R = Me; R' = Ph) with hydrazine hydrate was equally fruitless. Under mild conditions there was no reaction but under more vigorous conditions two compounds were formed: (a) 4-(N-methyl-benzamido)-3-pyrazolone (LIX), the identity of which was demonstrated by synthesis from ethyl Nmethyl-phenylpenaldate (LII; R = Me; R' = Ph) and (b) an unidentified crystalline derivative, certainly not LVI (R = Me). Treatment of LV (R = Me; R' = Ph) with diazomethane gave a gum, presumably D-4-carbomethoxy - 5,5 - dimethyl - 2 - N-methyl - benzamidocarbethoxymethyl-thiazolidine (LX; R = Me; R' = Et). Hydrolysis with one equivalent of sodium hydroxide proceeded very slowly to give an amor­ phous acid and a crystalline neutral product which CO2R-CH-NH CO2R' Me2A ·S · AH-AH-NMe-COPh LX CO2MeC-NH CO2Et I' ι I Me2C CH=C-NMe-COPh LXI appeared to be isopropylidene-carbomethoxymethylaminomethylene-(N-methyl-benzamido)-glycine ethyl ester (LXI). Decomposition of the amorphous acid with mercuric chloride did not appear to liberate any carbon dioxide; treatment of the liber­ ated aldehyde with dinitrophenylhydrazine reagent gave a mixture from which the pure crystalline 2,4dinitrophenylhydrazone of ethyl N-methyl-phenylpenaldate (LII; R = Me; R' = Ph) was isolated. Thus the hydrolysis appeared to have resulted largely in the regeneration of LV (R = Me; R' = Ph) rather than in the production of D-4-carbomethoxy - 5,5 - dimethyl - 2 - N -methyl-benzamidocarboxymethyl-thiazolidine (LX; R = H;R' = Me), a product which was expected by analogy with the hydrolysis of the penicilloate esters. Prior to demonstrating the structure of the acid its de­ hydration to LVII (R = Me) or the corresponding analogue of XLVII was investigated; the chief

TfflAZOLIDINES product was recovered acid together with a little neutral gum which had no antibiotic activity before or after hydrolysis with sodium hydroxide at 0°. CO2R-CH-NCOR' I I Me2C-S- CH-CH(C02R")-NMe-COPh LXII Later work (Shell, Sh.9, 113) suggested the possi­ bility of synthesizing a "penicillin" by pyrolysis of an acid such as LXII (R = R' = alkyl; R" == H). Acetylation of LX (R = H; R' = Et) gave D-4carboxy-3-acetyl-5,5-dimethyl-2-N-methyi-benzamidocarbethoxymethyl-thiazolidine (LXII; R = H; R' = Me;R" = Et.) Esterification gave a gum, presum­ ably D-4-carbomethoxy-3-acetyl-5,5-dimethyl-2-Nmethyl-benzamido-carbethoxymethyl-thiazolidine (LXII; R = R' = Me; R" = Et). Hydrolysis with one equivalent of sodium hydroxide proceeded rather more quickly than for LX (R = Me; R' = Et) to give an amorphous acid of Unknown struc­ ture. When it was treated with mercuric chloride there was no evolution of carbon dioxide and only a very small amount of aldehyde was isolated as an amorphous dinitrophenylhydrazone. This stability to mercuric chloride was confirmed polarimetrically, the rotation (in ethanol) being unchanged after twenty-four hours standing with a slight excess of iTiercuric chloride. This behavior is reminiscent of that of DL-4-carbomethoxy-3-benzoyl-2,2,5,5tetramethyl-thiazolidine but the original acid (LXII; R = H; R' = Me; R" = Et) showed pre­ cisely the same behavior and the amorphous acid may therefore be LXII (R = H; R' = Me; R" = Et) or LXII (R = R' = Me; R" = H) or a mixture of these substances. Pyrolysis of the amorphous acid gave a product with no antibiotic activity but pyrolysis of the resinous ester LXII (R = H; R' = Me; R" = Et) gave a product which had slight though incompletely reproducible antibiotic activ­ ity. The starting material (LXII; R = H; R' = Me; R" = Et) and the amorphous acid obtained from its hydrolysis were both biologically inactive so the active principle must have been formed dur­ ing the pyrolysis. The activities were too low to warrant further work. x>-i-Carboxy-Z-isobutyryl-2-'N-methyl-benzamidocarbethoxymethyl-thiazolidine (LXII; R = H; R' = CHMe2; R" = Et) was prepared and converted into the crystalline T>-4.-carbomethoxy-3-isobutyryl-2N-methyl-benzamido-carbethoxymethyl -thiazolidine (LXII; R = Me; R' = CHMe2; R" = Et). In view of the results already recorded for the acetyl derivative, hydrolysis was not examined. Pyroly­ sis of this diester did not lead to biologically active products. The absorption spectra in ethanol of LII (R = CH2Ph; R' = Ph) and LII (R = Me; R' = Ph), of their derivatives and of LV (R = Me and CH2Ph; R' = Ph) and LVI were of considerable interest.

937

As shown below, LII (R = CH2Ph; R = Ph), LII (R = Me; R' = Ph) and their derivatives had well-defined maxima in the region of 2,500-2,800 A. On the other hand, LV (R = CH2Ph; R' = C5H11), LVI and LXII (R = H; R' = Me; R" = Et) all showed end absorption only with no sign of the maxima observed for the other compounds. Evi­ dently these penicilloates have the structures assigned to them above, being true thiazolidine derivatives and not of the type LXIII. This is further verified by the fact that the 3-acetyl and 3-i'sobutyryl derivatives did not decolorize the sodium azide-iodine reagent, proving the absence of a thiol group. CO2HCH Me2A-SH

Compound

Ethyl N-benzyl-phenylpenaldate (LII; R = CH2Ph; R' = Ph)... Benzylamine derivative Glycine derivative (LIII) Ethyl N-methyl-phenylpenaldate (Ln; R = Me; R' = Ph) Benzylamine derivative LV (R = CH2Ph; R' = CeHn). LV (R = Me and CH2 Ph; R' = Ph). LXII (R = H; R' = Me; R" = Et).

NH

CO2Et

(I)H=C-NR-COPh LXIII

T

EM (min.)

^max. EM A (max.)

2,490 2,650

8,000 5,800

2,680 9,100 2,850 10,400

2,590

3,300

2,850 7,400

2,510 2,560

5,900 6,500 End absorp­ tion only End absorp­ tion only End absorp­ tion only

2,680 7,209 2,850 13,000

—

—

—

Recently (Glickman and Cope, J. Am. Chem. Soc., 67, 1017 (1946)) the ultraviolet absorption spectra of ethyl /3-aminocrotonate and ethyl β-dimethylaminocrotonate (in ethanol) were also shown to have a maximum at 2,740 and 2,840 A (though the intensity of absorption is somewhat greater). The similar location of the maxima shown by the absorption spectra of the above compounds is good evidence that, like ethyl /3-aminocrotonate and ethyl /3-dimethylaminocrotonate, these phenylpenaldates are hydroxymethylene derivatives and that their benzylamine and aniline derivatives are not anils but enamines (Copp, Duffin, Smith and Wilkinson, CPS.642). E X P E R I M E N T A L RELATED TO TEXT OF DISCUSSION Preparation of Thiazolidines: Miscellaneous Syntheses 4-CarbethoxythiazoIidine Hydrochloride (Pfizer, P.9, 2). Cysteine ethyl ester hydrochloride was refluxed in absolute ethanol, containing a trace of hydrogen chloride, with a slight excess of paraformaldehyde. 4-CarbethoxytMaz'ol-

THIAZOLIDINES

938

idine hydrochloride was produced in good yield and crystal­ lized on addition of ether to a warm ethanolic solution; it had m.p. 144-145°. Calc. for C6Hi2O2NSCl: N, 7.1 Found: N, 7.1 D-2-Phenyl-4-carboxy-5,5-dimethylthiazolidine (Merck, MM, 1). D-Penicillamine (1.385 g.) and benzaldehyde (1.145 cc.) in anhydrous dioxan (100 cc.) containing hydro­ gen fluoride (5 g.) was stirred in a copper flask for three hours. After filtration, concentration, and trituration with light petroleum the residue was taken up in ethanol (15 cc.) and water (15 cc.) was added, followed by aqueous sodium acetate to ρII 4. The precipitate was recrystallized from 50% ethanol, giving D-2-phenyl-4-carboxy-5,5-dimethylthiazolidine (1.7 g.; 77%), m.p. 151.5-152° (dec.). α-Methyl D-7-Benzylpenicilloate (MM, 2). D-Penicillamine (2.43 g.) and methyl benzylpenaldate (3.1 g.) were allowed to stand for four hours in anhydrous dioxan (31 cc.) containing hydrogen fluoride (1.55 g.). The mixture was poured into a solution of sodium acetate (6.1 g.) in water (300 cc.) and extracted thrice with chloroform. Evapora­ tion left an oil (4.6 g.). A portion (2.6 g.) of this was dis­ solved in methanol (25 cc.), diluted with water (150 cc.) and extracted into chloroform. Evaporation in vacuo left a light yellow brittle glass of α-methyl D-7-benzylpenicilloate, [a]D +31° (25% in methanol). 2S

Calc. for C17H22O6N2S: C, 55.8; H, 6.0; N, 7.7 Found: C, 56.1; H, 5.6; N, 6.7 Upon standing in methanol-ether, a small quantity of Crystalline product was obtained, m.p. 164°. 2-Keto-5-carboxy-6,6-dimethylthiazan (?) (Elks, Hems and Robinson, CPS.201, 6; Elks, Hems, Robinson and Ryman, CPS.510, 1). A mixture of dihydroxytartaric acid (1.4 g.), penicillamine (1.0 g.) and ethanol (20 cc.) was shaken mechanically for four hours. The precipitate (I) (0.32 g.) was crystallized from ethanol when it had m.p. 182-184° (dec.). Calc. for C7H11OsNS: C, 44.4; H, 5.9; N, 7.4; S, 16.9 Found: C, 44.4; H, 5.9; N, 7.5; S, 16.9 The compound showed maximum light absorption at 2,360 A (EM 4,159). A solution of the compound in water gave a transient blue with ferric chloride, but polarographic exami­ nation gave no indication of a thiol group even after five minutes boiling with N hydrochloric acid. 3-Keto-5-carboxy-6,6-dimethylthiazan (II) (Elks, Hems, Robinson and Ryman, CPS.510, 6). Penicillamine (1 g.) was dissolved in water (10 cc.), magnesium oxide (0.7 g.) added, and the mixture cooled in ice and stirred while chloroacetyl chloride (0.51 cc.) was added slowly. The mixture was stirred for one hour, without cooling, then acidified to Congo Red and extracted thoroughly with chloroform. The solvent was removed in vacuo, and the residue triturated with dry ether. 3-Keto-5-carboxy-6,6-dimethylthiazan crystal­ lized from chloroform-light petroleum in prisms (0.26 g.), m.p. 171-174°. Calc. for C7H11O3NS: C, 44.4; H, 5.9; N, 7.4; S, 16.9 Found: C, 44.5; H, 5.8; N, 7.35; S, 16.6 Maximum light absorption was at 2,850 A. The compound gave no color with ferric chloride and a negative ninhydrin test. Chloroacetyl chloride (0.51 cc.) was added gradually to a cooled, stirred solution of penicillamine (1 g.) and sodium bicarbonate (1.5 g.) in water (20 cc.). The resulting solu­ tion was allowed to stand in ice for thirty minutes and then at room temperature for fifteen minutes. It was then acidified to Congo Red and worked up as in the previous preparation (yield, 0.25 g.).

N-Chloroacetylpenicillamine (100 mg.), prepared as de­ scribed by Barber, Gregory, Slack, Stickings and Woolman ([CPS.234) was dissolved in a solution of sodium bicarbonate (150 mg.) in water (5 cc.). The solution was left at room temperature for one hour, acidified to Congo Red and worked up as above. Colorless prisms (10 mg.) were obtained, m.p. 171-173°, alone or mixed with a specimen of the compound described above. 4-Carbethoxy-2,5,5-trimethyl-Δ2-thiazoline Hydrochloride (Bentley, Catch, Cook, Heilbron and Shaw, CPS.267). DL-Penicillamine ethyl ester hydrochloride (1.5 g.) and thioacetamide (0.6 g.) were finely ground and heated first at 100° (thirty minutes), then at 120° (2.5 hours), hydrogen sulphide being evolved and ammonium chloride separating. The melt was extracted with ether (3 X 15 cc.) and the dried extract concentrated and treated with dry ethereal hydrogen chloride, when 4-carbethoxy-2,5,5-trimethyl-A2thiazoline hydrochloride separated; it crystallized from chloroform-ether in small rods and sublimed at 80-90°/14 mm. in long white deliquescent needles, m.p. 140-141°. Calc. for C9HleO2NSCl: C, 45.5; H, 6.3; N, 5.9; S, 13.5 Found: C, 45.7; H, 6.5; N, 6.0; S, 13.8 4-Carbethoxy-2,5,5-trimethylthiazolidine Hydrochloride (CPS.267). The free base of the above thiazoline (350 mg.), b.p. 60-63°/0.1 mm.,.in ether (80 cc.) was treated with amalgamated aluminium (2.5 g.). Water (8 cc.) was added in portions over three days, the ether evaporated, and the oil dried over phosphorus pentoxide. It was dissolved in dry ether and treated with dry hydrogen chloride. 4-Carbethoxy-2,5,5-trimethylthiazolidine hydrochloride was pre­ cipitated (250.mg.) and crystallized from chloroform-ether, m.p. 113-114°. Calc. for C9H18O2NSCl: C, 45.2; H, 7.5 Found: C, 45.0; H, 7.8 The thiazolidine hydrochloride (120 mg.) was treated with excess aqueous mercuric chloride overnight. The odor of acetaldehyde was soon detected, and from the filtrate, acetaldehyde 2,4-dinitrophenylhydrazone, m.p. 160-161°, (51 mg.) was obtained. 2-Phenylacetamidomethyl-4-carbomethoxy-5,6-dimethylAMhiazoline (Bar ltrop; Abraham, Baker, Chain and Robin­ son, CPS.463). Penicillamine methyl ester hydrochloride (2.0 g.) in water (2.0 cc.) and phenylacetamidoacetimino ethyl ether (2.0 g.) in chloroform (2.0 cc.) were shaken for two hours at room temperature. The product obtained from the chloroform was distilled in bulbs to yield a very viscous oil (1.8 g.), b.p. 180-190°/0.1 mm. Calc. for C16H20O3N2S: C, 60.0; H, 6.3; N, 8.8 Found: C, 60.3; H, 6.3; N, 8.6 The above thiazoline (0.8 g.) in ether (100 cc.) was refluxed with aluminium amalgam (40 g.), water being added over five hours. A colorless oil (0.46 g.) was obtained from the ether, forming a crystalline hydrochloride, m.p. 85-95°. This was treated with mercuric chloride, and addition of 2,4-dinitrophenylhydrazine in 2 N hydrochloric acid gave the dinitrophenylhydrazone of benzylpenilloaldehyde (0.46 g.), m.p. 202-205° after crystallization from ethanol. No depression in m.p. was observed on admixture with an authentic sample. 2-(a-Carboxyisopropyl) -3-isobutyryl-thiazolidine (Shell, (Sh.14, 214). A solution of dimethylketene (15.4 g.) and A2-thiazoline (8.7 g.) in ethyl acetate (150 cc.) was stoppered under nitrogen and left at room temperature for four days. After removal of the solvent, the residue was taken up in ether and extracted first with aqueous sodium bicarbonate and then with 2 N hydrochloric acid. Neutralization of these extracts gave only small quantities of products. However, after a second extraction with aqueous sodium bicarbonate, followed by acidification, a crystalline acid

TfflAZOLIDINES separated. Recrystallization from methanol-water gave 8.87 g. (28%), m.p. 122°. Calo. for C 1 IH 19 O 3 NS: C, 53.9; H, 7.8; N, 5.7; S, 13.1; neut. equiv., 245 Found: C, 53.8; H, 7.8; N, 5.5; S, 13.0; neut. equiv., 253 2-Methyl-2-(a-carboxy-isopropyl)-3-isobutyryl-thiazolidine ( Sh.IZ f , 211). A cold solution of dimethylketene (10.5 g.) and 2-methyl-A 2 -thiazoline (7.6 g.) in ethyl acetate (100 cc.) was stoppered under nitrogen and left for three days. The solution was extracted with aqueous sodium bicarbonate, washed, dried and evaporated. The residue was dissolved in light petroleum (50 cc.), filtered and concentrated to a light yellow oil (23 g.). The infrared spectrum of this compound was compatible with a fused thiazolidine-piperidindione ring system. The oil was refluxed for eighteen hours in a mixture of ethyl acetate (50 cc.), water (15 cc.) and acetic acid (2 cc.). Acidic materials were extracted into aqueous sodium bicarbonate, and the extract was washed and acidified. An oil separated, -Vyhich crystal­ lized on cooling and scratching. Recrystallization from aqueous methanol gave 5.3 g., m.p. 130.5°. Calc. for C 12 H 21 O 3 NS: C, 55.6; H, 8.2; N, 5.4; S, 12.4; neut. equiv., 259 Found: C, 55.4; H, 8.2; N, 5.0; S, 12.5; neut. equiv., 260 2 - Phenyl -2 - (a -carboxy-isopropyl) -3-isobutyryl-thiazolidine ( Sh.8 , 105). The solution of dimethylketene in ethyl acetate derived from α-bromo-isobutyryl bromide (100 g.) was added to 2-phenyl-A 2 -thiazoline (8.2 g.). After stand­ ing overnight, the solution was warmed for thirty minutes at 60°, and the solvent removed in vacuo. Extraction with 10% sodium carbonate and acidification yielded a crystal­ line acid (3.5 g.), which was recrystallized from benzenelight petroleum to give 2-phenyl-2-(a-carboxyisopropyl)-3isobutyryl-thiazolidine, m.p. 157.5-158°. Calc. for C 17 H 23 O 3 NS: C, 63.5; H, 7.2; N, 4.4; neut. equiv., 322 Found: C, 63.8; H, 7.2; N, 4.4; neut. equiv., 324 The residue was purified by repeated crystallization from benzene-light petroleum, with charcoal. 6,1-2',3'Thiazolidine-6-pheny1-3,3,5,5-tetramethyl-2,4-piperidindione (0.8 g.), m.p. 136-137°, was ultimately obtained. Calc. for C 17 H 21 O 2 NS: C, 67.3; H, 7.0 Found: C, 67.3; H, 7.1 A solution of the piperidindione (6 g.) in methyl alcohol (60 cc.) was refluxed for ten hours with N sodium methoxide (0.2 cc.). The solution was concentrated to 30 cc. and the warm solution diluted with water until fcrystals formed. Cooling in ice yielded 2-phenyl-2-(a-carbomethoxy-isopropyl)-3-isobutyryl-thiazolidine, m.p. 127-130°. Recrystallization from benzene-light petroleum yielded the pure ester (4.2 g.), m.p. 137-138°. Calc. for C 18 H 25 O 3 NS: N, 4.2; S, 9.6 Found: N, 4.2; S, 9.7 2- Phenyl-2-(a-carboxy-isopropyl)-3-isobutyryl-thiazolidine was treated with excess ethereal diazomethane. Recrystallization of the residue from benzene-light petroleum yielded 2-phenyl-2- (α-carbomethoxy)-isopropyl-3-isobutyryl-thiazolidine, m.p. 138.5°. D-2-Carboxy isopropyl-3-isobutyryl-4-carbomethoxy-5,5dimethylthiazolidine (Merck, M.66, 2). Dimethylketene (780 mg.) in ethyl acetate (20 cc.) was added to 4-carbomethoxy-5,5-dimethylthiazoline (970 mg.) in a sealed tube, and the solution allowed to stand for two days. The ethyl acetate was washed with sodium carbonate, dried and evap­ orated under reduced pressure, finally at 0.2 mm. pressure to remove dimethylketene dimer. The residue was crystal­ lized from light petroleum. The crude solid (0.15 g. from

939

a total of 0.29 g.) was dissolved in ethyl acetate (5 cc.) and refluxed with water (1.5 cc.) and isobutyric acid (1 drop) for twelve hours. The mixture was shaken with 10% aqueous sodium carbonate and the aqueous solution acidified with hydrochloric acid giving a crystalline product (80 mg.), m.p. 147-149°, unchanged on recrystallization from benzenelight petroleum. Calc. for C 15 H 25 O 6 NS: C, 54.4; H, 7.6; N, 4.2 Found: C, 54.8; H, 7.9; N, 4.3 A further quantity of the acid (0.34 g.) was obtained by hydrolvzing the oily residue from the first light petroleum filtrate. 6,l,2',3'-(4'-Carbomethoxy-5',5'-dimethylthiazolidino)-6phenyl-3,3,5,5-tetramethyl-2,4-piperidindione (Shell, Sh.8, 109). Dimethylketene (4.1 g.) in ethyl acetate (40 cc.) was added to 2-phenyl-4-carbomethoxy-5,5-dimethylthiazoline (14.4 g.) under nitrogen. The mixture was allowed to stand overnight and then refluxed for two-hours. Concen­ tration to 25 cc. and dilution with light petroleum (1 vol.) and cooling and scratching induced crystallization (1 g.). Recrystallization from benzene-light petroleum yielded 0.6 g., m.p. 146°, which was subsequently raised to 154°. Calc. for C 21 H 27 O 4 NS: C, 64.8; H, 7.0 Found: C, 64.5; H, 7.2 L-6,l,2',3'-(4'-Carbomethoxy-5',5'-dimethyl-thiazolidino)2.4-piperidindione (Pfizer, P.28, 11). L-4-Carbomethoxy5.5-dimethylthiazoline hydrochloride (1.07 g.) in dry benzene (10 cc.) suspension was treated with a small excess of diazo­ methane in benzene, and volatile products removed in vacuo at room temperature. Excess ketene (3 moles) was added and the solution allowed to stand overnight at 0°. The solution (20 cc.) was diluted to 50 cc. with benzene, and then run down ac olumn of Brockmann alumina (275 mm. X 12 mm.). The column was washed with benzene (40 cc.), and the benzene rejected. A mixture of dry ether (20 cc.) and benzene (20 cc.), followed by dry ether (40 cc.) was passed through the column. The combined fractions yielded an oil (50 mg.), which crystallized on cooling and scratching; m.p. 99-102°.

Calc. for C 11 H 15 O 4 NS: C, 51.4; H, 5.9; N, 5.5; S, 12.5 Found: C, 51.8; H, 6.7; N, 5.5; S, 12.3 3-Acetyl-4-carbomethoxy-6,5-dimethylthiazolidme (Up­ john, U.21, 7; 22, 2; 23, 1; 24, 1). 4-Carbomethoxy-5,5dimethyl-A 2 -thiazoline (152 mg.) in heptane (6 cc.) was maintained at 50° for fifteen minutes while a rapid stream of ketene was passed in. The solution was kept at 0° for eighteen hours and evaporated in vacuo. A clear yellow oil (170 mg.) was obtained. Calc. for C 9 H 15 O 3 NS: C, 49.7; H, 7.0; N, 6.5; S, 14.7 Found: C, 49.9; H, 6.3; N, 6.4; S, 15.1 Under strictly anhydrous conditions, a pale yellow, amor­ phous solid was obtained. Found: C, 51.2; H, 6.9; N, 6.8; S, 14.8 Condensations of Thiazolidines with Oxazolones 2 -Ph enylacetamido -carbomethoxy-methyl-4-carbomethoxy-thiazolidine (Merck, M.ISc, 21). Cysteine methyl ester hydrochloride (1.71 g.) and 2-benzyl-4-methoxymethylene5(4)-oxazolone (2.1 g.) were heated in pyridine (50 cc.) on the steam bath for ten minutes. The pyridine was evaporated and the residue dissolved in chloroform and washed with water. After evaporation of the chloroform the product crystallized from ethyl acetate; m.p. 188-189°. Calc. for C 16 H 20 O 5 N 2 S: C, 54.5; H, 5.7; N, 8.0 Found: C, 54.9; H, 6.1; N, 8.1 Methyl Phenylpenilloate (Copp, Duffin, Smith and Wil­ kinson, CPS.S88). Penicillamine hydrochloride (4.6 g.)

940

TfflAZOLIDINES

and 2-phenyl-4-ethoxymethylene-5(4)-oxazolone (5.4 g.) in 50% aqueous dioxan (50 cc.) were allowed to stand over­ night, the solution was filtered and the filtrate kept at 60-70° for three hours. Phenylpenilloic acid hydrochloride (3.5 g.) separated on cooling and crystallized from aqueous dioxan in prisms, m.p. 220°. Calc. for Ci4Hi9O3N2SCl: C, 50.8; H, 5.7; N, 8.5; S, 9.7; neut. equiv., 165 Found: C, 50.9; H, 5.9; N, 8.6; S, 9.8; neut. equiv., 165 The methyl ester was obtained from the acid and diazomethane as a liquid, b.p. 120°/0.05 mm. Calc. for Ci5Hs0OjN2S: S, 10.4 Found: S, 10.1 DL-2-Benzamido-carboxybenzylamide-methyl-4-carbo-

methoxy-5,6-dimethylthiazolidine (Mich. Chem., B.3, 3; cf. Merck, MMa, 15). DL-Penicillamine methyl ester (25 mg.) and 2-phenyl-4-ethoxymethylene-5(4)-oxazolone (35 mg.) were allowed to stand overnight in dry ether (1 cc.). Benzylamine (2 drops) was added and the precipitate triturated with ether to give 8 mg., m.p. 205° with softening from 190°. Calc. for C28H27O4NsS: C, 62.6; H, 6.2 Found: C, 62.8; H, 6.5 Condensation also occurred in pyridine. DL-2-Phenylacetamido-carboxy-(a-carbomethoxy-/S-mer-

capto-/9,/3-dimethyl)-ethylamido-methyl-4-carbomethoxy-5, 5-dimethylthiazolidine (Merck, M.23, 22). DL-Penicillamine methyl ester (417 mg.) and 2-benzyl-4-hydroxymethyIene5(4)-oxazolone (514 mg.) were held in dioxan (2 oc.) for twenty-four hours. The product (11 mg.) was crystallized from methanol-ether; m.p. 212-213°. Calc. for C2SHaaOeNsS2: C, 54.1; H, 6.5; N, 8.2; S, 12.5; OCH3, 12.1 Found: C, 54.5; H, 6.9; N, 7.9; S, 13.0; OCH3, 12.0 D-2-Phenylacetamido-carboxy-/3-phenylethylamidomethyl-4-carbomethoxy-6,5-dimethylthiazolidine (M.37,10). D-Penicillamine methyl ester and 2-benzyl-4-methoxymethylene-5(4)-oxazolone were condensed in toluene for sixteen hours at room temperature. The residue was dis­ solved in ether and treated with 2 equivalents of 0-phenylethylamine for sixteen hours and crystallized from ethyl acetate-methanol-light petroleum; m.p. 175-176°. Calc. for C26HsiO4N3S: C, 63.9; H, 6.7; N, 9.0 Found: C, 63.9; H, 6.5; N, 9.3 D -2 - Phenylacetamido - carboxybenzyla midomethyl-4carboxybenzylamide-5,5-dimethylthazolidine (M.37, 10). D-Penicillamine methyl ester and 2-benzyl-4-methoxymethylene-5(4)-oxazolone were condensed in pyridine in the usual manner, and the residue treated with excess benzylamine and refluxed overnight in ethyl acetate. Treatment of a fraction of the oily residue with methanol and clarification with charcoal gave white rods, m.p. 186-188°.

The L-form was prepared similarly (M.23, 11). m.p. 160-161° from aqueous methanol.

It had

Calc. for CuHisO5N2S: C, 53.2; H, 5.4 Found: C, 53.3; H, 5.5 DL-2-Phenylacetamido-carbethoxymethyl-4-carboxy-thiazolidine (M$9, 7). A solution of 2-benzyl-4-hydroxymethylene-5(4)-oxazolone (1.948 g.) in absolute ethanol (15 cc.) was refluxed for eight minutes and treated with DL-cysteine hydrochloride (1.023 g.) in water (15 cc.), followed by potassium acetate (0.5 g.). After 2 days the crystalline product (1.742 g.) was recrystallized from ethan­ ol; m.p. 188°.

Calc. for Ci6H20O6N2S: C, 54.6; H, 5.7; N, 8.0 Found: C, 54.3; H, 5.7; N, 8.1 L-2-Phenylacetamido-carbobenzoxymethyl-4-carboxythiazolidine (Upjohn, 17.7, 32). A solution of 2-benzyl-4hydroxymethylene-5(4)-oxazolone (203 mg.) in dioxan (2 cc.) containing benzyl alcohol (108 mg.) was heated on the steam bath for thirty minutes and added to L-cysteine hydro­ chloride (157 mg.) and potassium acetate (100 mg.) in water (1 cc.) and left overnight. The solid product recrystallized from aqueous dioxan as a white powder, m.p. 160-162°.

Calc. for C2iH2206N2S: N, 6.8 Found: N, 7.1 D-W-Heptylpenilloic Acid Hydrochloride (Pfizer, P.SS, 7). A mixture of 2-n-heptyl-4-hydroxymethylene-5(4)-oxazolone (1.2 g.) and ethanol (18 cc.) was heated on the steam bath for five minutes, cooled and added to D-penicillamine (0.85 g.) in water (8 cc.). The mixture was treated with charcoal and left to stand. Extraction of the product with ether, followed by treatment with anhydrous hydrogen chloride and concentration gave a gum which solidified under fresh ether. RecrystaUization from ethanol-ether gave a product (0.1 g.), m.p. 190-191°; [afc" + 81° (in methanol).

Calc. for Ci6H29O3N2SCl: C, 51.1; H, 8.2; N, 7.9; S, 9.1; Cl, 10.1 Found: C, 51.1; H, 8.4; N, 7.9; S, 9.5; Cl, 10.2 D-2-Cyclohexylacetamido-carbethoxymethyl-4-carboxy-5, 5-dimethylthiazolidine Hydrochloride (P.SS, 7). A mixture of 2-cyclohexylmethyl-4-hydroxymethylene-5(4)-oxazolone (1.3 g.) and ethanol (8 cc.) was warmed on the steam bath until a clear solution was obtained, and then added to D-penicillamine (0.93 g.) in water (8 cc.). An oil was deposited and was extracted with chloroform. An amorph­ ous solid was obtained on removing chloroform and was dissolved in ether and treated with dry hydrogen chloride. The solid product (1.4 g.) was recrystallized from ethanolether; m.p. 182-183° (dec.); [α]η25 + 61° (in methanol).

Calc. for Ci8HsiO6N2SCI: C, 51.1; Η, 7.3; N, 6.6; S, 7.6; Cl, 8.4 Found: C, 51.3; H, 7.5; N, 6.7fS, 7.1; Cl, 8.4

DL-2-Benzylamido-carbomethoxy-methyl-4-carboxy-thiazolidine (M.29, 8). A solution of 2-phenyl-4-hydroxymethylene-5(4)-oxazolone (2.1 g.) in ethanol (15 cc.) was warmed on the steam bath for thirty minutes, treated with DL-cysteine hydrochloride (1.8 g.) and potassium acetate (1.0 g.) in water (15 cc.) and left overnight. The crys­ talline product (2.5 g.; 70%), m.p. 155.5-156°, was a monohydrate.

D-2-Benzamido-carbomethoxymethyl-4-carbomethoxy-5, 5-dimethylthiazolidine Hydrochloride (Merck, M.SS, 9). 2-Phenyl-4-hydroxymethylene-5(4)-oxazolone (1.4 g.) was refluxed in methanol (25 cc.) for twenty-five minutes and the solution concentrated to dryness in vacuo. The result­ ing gum was refluxed for twenty minutes in toluene (50 cc.) with D-penicillamine methyl ester (1.43 g.) and the solution evaporated in vacuo. The resulting amber oil was dissolved in methanol (5' cc.) and acidified with methanolic hydrogen chloride to give, after addition of ether, a crystalline product (1.8 g.), m.p. 182-184°; recrystallized from methanol and ether, m.p. 190°.

Calc. for Ci5Hi8O6N2S-H2O: C, 50.6; H, 5.6; N, 7.9 Found: C, 50.8; H, 5.7; N, 7.7

Calc. for Ci7H23O6N2SCl: C, 50.8; H, 5.8; N, 7.0 Found: C, 50.4; H, 5.9; N, 7.1

Calc. for CsoH34O3N4S: C, 67.9; H, 6.5 Found: C, 67.6; H, 6.5

THIAZOLIDINES

941

D l-2 -Phenylacetamido-carbomethexymethyl-3-formyl-4carboxy-6,5-dimethylthiazolidine (Μ.29, 9). A solution of 2-benzyl-4-hydroxymethylene-5(4)-oxazolone (2 g.) in meth­ anol (15 ce.) was refluxed for ten minutes, added to DL-penicillamine hydrochloride (1.8 g.) and potassium acetate (1 g.) in water (15 cc.) and left overnight. The heavy oil which separated was taken up in sodium bicarbonate solution. After acidification and extraction into chloroform, a brittle residue was obtained (1.28 g.). A portion (300 mg.) was allowed to stand at 0° for twenty-four hours with a mixture of 85% formic acid (2 cc.) and acetic anhydride (1 cc.). Concentration and trituration with ether yielded a white solid, m.p. 175°.

L -2-p -Methoxyphenylacetamido-carbomethoxymethyl-4carboxy-thiazolidine (P.19, 14). 2-p-Methoxybenzy1-4-hydroxymethylene-5(4)-oxazolone (1.9 g.) was refluxed with methanol (25 cc.) for fifteen minutes, and treated with [.,-cysteine hydrochloride (1.58 g.) and potassium bicarbonate (1 g.) in water (25 cc.). After standing overnight, the product (2.84 g.) was collected and crystallized from methanol; m.p. 165-166°.

Calc. for Ci8H22O6N2S-H2O: C, 52.4; H, 5.9 Found: C, 52.9; H, 5.9

Calc. for CieH20O6N2S: C, 52.2; H, 5.4; N, 7.6 Found: C, 51.9; H, 5.4; N, 8.0

D-2-Phenylacetamido-carbomethoxy-methyl-4-carboxy-5, 6-dimethylthiazolidine (Cornell Bioch., D.12, 5). This com­ pound, similarly prepared from DL-penicillamine hydro­ chloride, crystallized from methanol and had m.p. 159-160°.

D -2-p -Methoxyphenylacetamido-carbomethoxymethyl-4carboxy-5,5-dimethylthiazolidine (P.20, 9). D-Penicillamine hydrobromide (from D-4-carboxy-2,2,5,5-tetramethyI-thiazolidine hydrobromide, 7.5 g.) in water (50 cc.) was treated with potassium bicarbonate (2.8 g.) and a solution obtained by refluxing 2-p-methoxybenzyl-4-hydroxymethylene-5(4)oxazolone (6.1 g.) in methanol (50 cc.) for fifteen minutes. The mixture was held overnight, shaken with chloroform, and the chloroform shaken with aqueous potassium bicar­ bonate. Acidification with acetic acid, extraction with benzene, and Iyophilization gave a white powder (0.45 g.).

Calc. for C17H22O6N2S: C, 55.7; H, 6.1; N, 7.6 Found: C1 55.9; H, 5.9; N, 7.1 α -Ethyl D -Benzylpenicilloate (Merck, Μ Λ 6 ) . 2-Benzyl4-hydroxymethylene-5(4)-oxazolone (4.2 g.) and ethanol (25 cc.) were heated on the steam bath for eight minutes, D-penicillamine hydrochloride (3.85 g.) and potassium acet­ ate (2 g.) in water (25 cc.) were added, and after eighteen hours the solution was seeded. Crystal crops totalling 5.5 g. (70%), m.p. 145° (dec.), were collected at intervals of two, five and ten days. The product was recrystallized from hot ethanol (10 cc./g.) by diluting with an equal vol­ ume of water, to form felted needles, m.p. 150° (dec.); Md + 128° (c = 0.391 in ethanol).

Calc. for Ci8H24OsN2S: C, 56.8; H, 6.4; N, 7.4; neut. equiv., 380 Found: C, 57.0; H,'6.5; N, 7.5; neut. equiv., 380 Benzylamine Salt of α-Benzyl D-Benzylpenicilloate (M.29, 9). 2-Benzyl-4-hydroxymethylene-5(4)-oxazolone (6 g.) was refluxed with benzyl alcohol (3.2 g.) in dry benzene (50 cc.) for forty-five minutes. A portion of this solution yielded almost quantitatively a 2,4-dinitrophenylhydrazone, m.p. 182°. Calc. for C21H21O7N6: C, 58.7; H, 4.3; N, 14.3 Found: C, 58.7; H, 4.7; N, 14.4 The remaining benzyl ester in dioxan was added to D-penicillamine hydrochloride (2.86 g.) and potassium acetate (1.5 g.) in water (20 cc.) and left overnight. The solution was concentrated in vacuo, and water and chloro­ form were added. The chloroform extract yielded a brittle solid (6 g.). A portion (1.6 g.) of this in a mixture of ethanol (5 cc.) and ether (2 cc.) was treated with redistilled benzylamine (0.406 g.) in 30% alcoholic ether, and light petroleum was added to incipient turbidity. After one week the crystalline product was recrystallized from ethanollight petroleum, giving the benzylamine salt of a-benzyl-Dbenzylpenicilloate, m.p. 163-164°. The melting point was depressed to 158-160° on admixture with the corresponding natural product, m.p. 162-163°. Calc. for C30H36O6N3S: C, 65.6; H, 6.4; N, 7.6 Found: C, 65.6; H, 6.5; N, 8.0 L-2-p-Acetoxyphenylacetamido-carbethoxymethyl-4-carbethoxy-thiazolidine (Pfizer, P.21, 8). Cysteine ethyl ester hydrochloride (2 g.) and 2-p-acetoxybenzyl-4-ethoxymethylene-5(4)-oxazolone (2.9 g.) were refluxed in pyridine (40 cc.) for ten minutes. The pyridine was removed in vacuo and the residue allowed to stand with ethanol (50 cc.) for two hours. After evaporation of the ethanol the residue was dissolved in chloroform and washed with water. The

residue from the chloroform was lyophilized from benzene; m.p. 55-60°. Calc. for C20H26O7N2S: C, 54.8; H, 5.9; N, 6.4 Found: C, 54.5; H, 5.8; N, 6.3

Calc. for Ci8H24O6N2S: C, 54.5; H, 6.7; N, 7.1 Found: C, 56.4; H, 6.0; N, 6.2 Azothiazolidines 2-p-Chlorobenzeneazo-carbethoxymethyl-4-carboxy-5,5dimethylthiazolidine (Baddiley, Openshaw, Sykes and Todd, CPS.65). Ethyl sodioformylacetate (20 g.) in water (300 cc.) was made acid (Brilliant Yellow) with acetic acid (turbidity) and sodium acetate (20 g.) added, followed, with stirring, by diazotized p-chloroaniline (12.7 g.) in solution (300 cc.) neutralized (Congo Red) with sodium bicarbonate. After five minutes the azo-compounds were extracted with chloroform, chloroform removed, and the residue chromatographed on alumina from ethyl acetate. A lower deep red band was washed out with ethyl acetate and yielded ethyl di-(p-chlorobenzeneazo)-acetate, which separated from benzene-light petroleum in deep red needles, m.p. 138° (about 0.5 g.). Calc. for Ci6Hi4O2N4Cl2: C, 52.7; H, 3.8; N, 15.3 Found: C, 53.2; H, 3.9; N, 14.9 The upper yellow band was washed out with ethariol and yielded ethyl p-chlorohenzeneazo-formylacetate as pale yellow prisms, m.p. 82° (5 g.), from xylene-light petroleum. Calc. for Found:

CiiHiiO3N2CI:

C, 52.0; H, 4.3; N, 11.1 C, 51.8; H, 4.6; Ν, 11.2

The p-chlorophenylhydrazone, obtained in the usual manner, crystallized from acetic acid in yellow needles, m.p. 228°.

Calc. for C17Hi6O2N4Cl2: N, 14.8 Found: N, 15.0 The azoformyl ester (0.5 g.) in warm methanol (20 cc.) was added to DL-penicillamine hydrochloride (0.4 g.) in water (5 cc.) and the mixture left for eight hours at room tempera­ ture and overnight at 0°. 2-p-Chlorobenzeneazo-carbethoxymethyl-4-earboxy-5,5-dimethylthiazolidine separated from dilute ethanol as yellow needles (0.25 g.), m.p. 152°. Calc. for CieH20O4N3SCl: C, 49.8; H, 5.2; N, 10.9 Found: C, 49.9; H, 5.9; N, 10.9 The azoformyl ester (1 g.) in warm 95% ethanol (40 cc.) was added to DL-cysteine (1 g.) in water (10 cc.). The mix-

942

THIAZOLIDINES

ture began to deposit crystals within one hour and after standing overnight the product was recrystallized from methanol. 2-p-Chlorobenzeneazo-carbethoxymethyl-4-carboxy-thiazolidine hydrochloride formed yellow needles (1.4 g.), m.p. 148°. Calc. for Ci4HieO4N3SCl: C, 46.9; H, 4.6; N, 11.7 Found: C, 47.0; H, 4.7; N, 11.2 Methyl Ethoxymethylene-nitroacetate (CPS.65). A mix­ ture of methyl nitroacetate (8.7 g.) (Steinkopf, Annalen, 434, 21 (1923)), ethyl orthoformate (15 g.), and acetic anhydride (19 cc.) was heated under reflux for two hours in an oil bath at 135-140°. Material boiling below 100° was then removed by distillation at atmospheric pressure during a further forty-five minutes and the residue fractionated under reduced pressure. The main fraction (7.46 g.; 58%) distilled at 155-158°/12 mm. A redistilled sample of methyl ethoxymethylene-nitroacetate, b.p. 156-157°/ 12 mm., was a yellow, slightly viscous oil. Calc. for CsHeOsN: C, 41.1; H, 5.1; N, 8.0 Found: C, 42.1; H, 5.5; N, 9.1 The above ethoxymethylene ester reacted exothermically with aniline in methanol solution and a yellow solid rapidly separated. When recrystallized from methanol, methyl anilinomethylene-nitroacetate had m.p. 109-109.5°. Calc. for Ci0H10O4N2: C, 54.0; H, 4.5; N, 12.6 Found: C, 53.7; H, 4.6; N, 13.0 p-Chlorobenzeneazo-formylacetic Acid (Baddiley, Openshaw, Sykes, Todd and Wardleworth, CPS.351). Ethyl p-chlorobenzeneazo-formylacetate (1 g.) was dissolved in methanol (110 cc.) and potassium hydroxide (0.9 g.) in water (5 cc.) added. The solution was kept at 0° for twenty-four hours and made acid to Congo Red with hydrochloric acid. Addition of water (350 cc.) precipitated an oil, which crystal­ lized on standing at 0°; yellow needles (0.7 g.) from ethanol; m.p. 104-105°. Calc. for C0HjO3N2Cl: N, 12.3; neut. equiv., 227 Found: N, 12.1; neut. equiv., 224 2-p-Chlorobenzeneazo-carboxymethyl -4-carboxy-5,5-dimethylthiazolidine (VII; R = H) (CPS.351). p-Chlorobenzeneazo-formylacetic acid (4 g.) was dissolved in boiling methanol (40 cc.) and D!.-penicillamine hydrochloride (4 g.) in water (5 cc.) was added. The mixture was set aside; after one hour crystallization of the product set in, and after twenty-four hours it was collected and recrystallized from methanol. The azo acid VII (R = H) formed yellow needles (4.4 g.), m.p. 165-166°. Calc. for C14H10O4N3SCl: C, 46.9; H, 4.5; N, 11.7; neut. equiv., 179 Found: C, 47.4; H, 4.8; N, 11.3; neut. equiv., 182 When the acid was dissolved in a little pyridine and light petroleum (60-80°) was added, the pyridine salt separated in small yellow rods, m.p. 149° (dec.). Calc. for C14H10O4N3SCl C6H6N: C, 52.3; H, 4.8; N, 12.8 Found: C, 52.4; H, 5.1; N, 12.3 The azo ester VII (R = Et) (0.88 g.) was dissolved in methanol (30 cc.) and a solution of potassium hydroxide (0.32 g.) in water (4 cc.) was added. After forty-eight hours at room temperature the solution was acidified and water (about 100 cc.) added. An oil separated, which quickly solidified. Recrystallization from methanol gave yellow needles, m.p. 165° (dec.), undepressed on admixture with material obtained by the above method. Reduction of the acid with sodium hyposulphite at 65-70° gave p-chloroaniline and a resinous material giving no ninhydrin reaction.

Ethyl 0,0-Diethoxy-a-(p-chlorobenzeneazo)-propionate (CPS.351). Ethyl jo-chlorobenzeneazo-formyl acetate (2.5 g.), ethyl orthoformate (3 g.), ammonium chloride (0.05 g.) and absolute ethanol (6 cc.) were heated together under reflux for four hours. Alcohol and ethyl orthoformate were removed in vacuo and the residual red oil dissolved in ethyl acetate was chromatographed on activated alumina. The acetal passed rapidly through the column as an orangeyellow band and on evaporating the eluate it was obtained as an orange oil (93%). Slow distillation at 140°/10'' mm. gave an oil, which set to a mass of pale yellow needles, in.p. 54-55°. Calc. for C15H2IO4N2Cl: C, 54.7; H, 6.4; N, 8.5 Found: C, 54.4; H, 6.1; N, 9.1 The acetal could not be condensed with penicillamine or its hydrochloride to give a thiazolidine. DL -2-a-03-Hydroxynaphthyl)-4-carboxy-5,5-dimethyl-thiazolidine (CPS.351). DL-Penicillamine (0.85 g.) was added to a solution of 0-hydroxy-a-naphthaldehyde (1 g.) in methanol (10 cc.) and the mixture refluxed till all the peni­ cillamine had dissolved (fifteen minutes). The solution was left overnight and the crystalline precipitate collected and recrystallized from methanol to yield colorless needles (1.1 g.), m.p. 129-130°. Calc. for C16H17O3NS: C, 63.0; H, 5.6; N, 4.6 Found: C, 62.6; H, 5.7; N, 4.5 DL -2-(3'-Methoxy-4'-hydroxyphenyl)-4-carboxy-5,5-di-

methyl-thiazolidine (CPS.351). Vanillin (1 g.) and DL-penicillamine (2 g.) were refluxed in a mixture of n-butanol (20 cc.), ethanol (10 cc.), and water (5 cc.) during thirty minutes, by which time all had gone into solution. The thiazolidine separated on standing overnight, and crystal­ lized from ethanol in needles (1.4 g.), m.p. 184° (dec.). Calc. for C13HnO4NS: C, 55.1; H, 6.1; N, 4.9 Found: C, 55.3; H, 6.2; N, 5.0 DL -2-(2'-Hydroxyphenyl)-4-carboxy-5,5-dimethyl-thiazolidine (CPS.351). Salicylaldehyde (1 g.) and DL-penicillamine were condensed similarly. The product crystallized in needles (1.1 g.), m.p. 180-181° (dec.), from n-butanol, containing 5% ethanol.

Calc. for C12H15O3NS: C, 56.8; H, 5.9; N, 5.5 Found: C, 56.8; H, 6.2; N, 5.2 DL -2-(p -Chlorobenzeneazo-carboxymethyl)-4-carboxy-3, δ,δ-trimethylthiazolidine (CPS.351). The azo-thiazolidine ester VII (R = Et) (0.75 g.) was methylated in methanolic solution with dimethyl sulphate (0.4 cc.) and 3 N sodium hydroxide (2 cc.) at room temperature during twelve hours. The reaction mixture was acidified to Congo Red with hydrochloric acid and diluted with water (200 cc.). The product crystallized from ethanol in yellow needles, m.p.

182°.

Calc. for C16H18O4N3SCl: C, 48.3; H, 4.8 Found: C, 47.8; H, 4.8 Attempts to carry out methylation at higher temperatures led to the production of resinous materials. 2-(p-Chlorobenzeneazo-carbethoxymethyl)-l,3-dibenzyliminazolidine (VIII; R = H) (Baddiley, Kilby and Todd, CPS.3S9). Benzaldehyde (212 g.) was added to ethylenediamine (90 g., 70% aqueous solution). Heat was evolved and on cooling the dibenzylidene derivative crystallized. Ethanol (300 cc.) was added to dissolve solid, and the solution agitated for four hours at 100° with hydrogen (100 atm.) in presence of Raney nickel catalyst. Ethanol was removed, the residue dissolved in hydrochloric acid, and benzaldehyde and neutral material extracted with ether. The acid aqueous layer was made alkaline, extracted with ether, and the product distilled. The bis-benzylamino-

943

TfflAZOLIDINES ethane had b.p. 222°/14 mm. (van Alphen, Rec. Trav. Chim., 64, 93 (1935), gives b.p. 222°/18 mm.) and was accompanied by a considerable amount of the mono-benzylethylenediamine, b.p. 140°/14 mm. (Bleier, Ber.32, 1829 (1899), gives b.p. 162-165°/20 mm.). α,/3-Bis-benzylaminoethane (2.42 g.) and ethyl j»-chlorobenzeneazo-formylacetate (2.55 g.) were warmed together until fusion and reaction took place with separation of water. The mixture was cooled and the red gum dissolved in warm ethanol and left overnight at 0°. The solid which separated was recrystallized from ethanol; yellow needles (3.4 g.), m.p. 88-89°.

and ethyl p-chlorobenzeneazo-formvlacetate was isolated from the mixture as its 2,4-dinitrophenylhydrazone, m.p. 258° (dec.). 2-(p-Chlorobenzeneazo-carbethoxymethyl)-l,3-dibenzyliminazolidine-4-carboxylic Acid (VIII; R = COOH) (CPS. 389). α,β-Bis-benzylaminopropionic acid (2.8 g.) and ethyl p-chlorobenzeneazo-formylacetate (2.5 g.) were refluxed in ethanol (20 cc.) for one hour, cooled and left overnight in the refrigerator. The crystals (2.0 g.) which separated were recrystallized from ethanol to give pale yellow needles, m.p. 137°.

Calc. for C 27 H 29 O 2 N 4 Cl: C, 68.0; H, 6.1; N, 11.8 Found: C, 67.9; H, 5.9; N, 11.3

Calc. for C 28 H 29 O 2 N 4 Cl: C, 64.6; H, 5.6; N, 10.8 Found: C, 64.8; H, 5.8; N, 10.6

Treatment of the iminazolidine with warm dilute acid rapidly opened the ring to yield the original components. Hydrogenation of Azo-iminazolidine Derivative (CPS. S89). The preceding ester VIII (R = H) was recovered unchanged after treatment in ethyl acetate solution with hydrogen and Raney nickel catalyst under a pressure of 90 atm. at 25° during 5.5 hours. Hydrogenation under atmospheric pressure with Adams platinum catalyst led to uptake of 2 moles of hydrogen (presumably debenzylation) but the azo linkage was unaffected. Partial hydrogenation occurred in ethanol solution with hydrogen under 120 atm. pressure at 70-80° for 5.5 hours using Raney nickel catalyst. Evaporation of the filtered solution left a resinous material, which evidently contained free p-chloroaniline since diazotization of a sample followed by coupling with alkaline /3-naphthol gave a red dye. During attempts to work up the product it was treated with cold 5% hydrochloric acid and the mixture extracted with ether. From the aqueous acid extract p-chloroaniline was isolated, and identified by acetylation to p-chloroacetanilide, as well as the hydrochloride of a,8-bis-benzylaminoethane (m.p. and mixed m.p. 305-307°). From the ethereal extract ethyl p-chlorobenzeneazo-formylacetate was isolated as its 2,4-dinitrophenylhydrazone (m.p. and mixed m.p. 260°). No other identifiable product was obtained. It would appear from the nature of the products isolated that some of the original iminazolidine was unaffected by the hydrogenation and subsequently underwent acid fission during working up. 2-(p-Chlorobenzeneazo-carbethoxymethyl)-4-carboxy-5, 6-dimethylthiazolidine [CPS.389). The iminazolidine VIII (R = H) (4.77 g.) in methanol (150 cc.) was mixed with penicillamine hydrochloride (1.85 g.) in water (15 cc.) and the mixture refluxed for forty minutes. On standing over­ night at 0°, a yellow solid (3.9 g.) separated. On recrystallization from ethanol it formed yellow needles, m.p. 149-151° (dec.) according to rate of heating.

The" above product (100 mg.) was heated to boiling for one to two minutes with N sulphuric acid (2 cc.), the mixture cooled, and extracted with ether. From the ether layer ethyl p-chlorobenzeneazo-formylacetate was isolated as its 2,4-dinitrophenylhydrazone (50 mg., m.p. 258°, undepressed by authentic material m.p. 260°), and from the neutralized aqueous layer 1 CO— N— 2-azetidinone

Extent of hydrolysis •

Conditions of hydrolysis

Reference

1,3,3,4-Tetraphenyl-

0.5 N KOH in MeOH reflux 25 hours

Very slight

Staudinger*

l,4-Diphenyl-3,3-dimethyl-

0.5 N KOH in MeOH reflux 25 hours

34%

Staudinger*

1,4-Diphenyl-

5% KOH in MeOH reflux 1 hour

At least 85%

P.8, 1

l-Benzyl-3-methyl-4-phenyl-

1 M NaOBu in BuOH 76°—2 hours

Considerable hydrolysis, from M.64, 9 infra-red data

0.12 N NaOH in EtOH 76°—29 hours

63% hydrolysis, pot. titration

M.64, 11

1 M NaOBu in BuOH 76°—4 hours

No change, from infra-red data

M.64, 9

0.12 N NaOH in EtOH 76°—29 hours

No change, pot. titration

M.64, 11

NaOBu in BuOH 100° max.—1 min.

Rapid hydrolysis, from infra-red M.64, 9 data

1 N NaOH 100°—90 min.

Extensive hydrolysis

l-Cyclohexanemethyl-3-methyl-4cyclohexyl-.

l-Phenyl-3-phenylacetamido-

M.B5, 2

Rapid hydrolysis, from infra-red M.64, 9 data

l-Cyclohexyl-3-cyclohexaneacetamido-. NaOBu in BuOH 100° max.—1 min. 1 N NaOH in EtOH 100°—15 min.

Extensive hydrolysis

Desthiobenzylpenicillin (3-Phenylacetamido-2-azetidinone-l-o-Disovaleric acid)

NaOBu in BuOH 100° max.—1 min.

Rapid hydrolysis, from infra-red M.64, 9 data

Sodium benzylpenicillinate

MeOH at 25°—2 hours

Complete hydrolysis, from infra- Sh.S, 16 red data

M.6S,

8

* Staudinger, Die Ketone, Enke, Stuttgart (1912),'p. 75.

phenyl ring to the corresponding cyclohexyl derivative or the removal of sulfur by hydrogenolysis of a fused thiazolidine-/3-lactam to give the monocyclic /3-Iactam. The first example of the latter reaction was the hydrogenolysis of penicillin to desthiobenzylpenicillin (assuming the /3-lactam structure) carried out by the Merck group and described in Chapter I X . Another example of this reaction which will be described in more detail later is as follows:

Reactions. As mentioned in the introduction to this chapter one of the main objects of the synthesis of model /3-lactams was to study their chemical reactions in relation to those of penicillin. Of particular interest were the rates of acid and alkaline hydrolysis as well as reactions with amines, hydrochloric acid and thiocyanic acid. It was, in general, the consensus of opinion during the early stages of the penicillin synthetic program that the rate of hydrolysis of penicillin was much too rapid to be explained by the /3-lactam structure. Hydrolysis. Both alkaline and acid cleavage of /3-lactams have been studied to a considerable extent. In Table II a number of experiments on various alkaline treatments are presented. The first three /3-lactar s in the table differ only by the type of substituent in the 3-position of the 2-azetidi-

979

THE CHEMISTRY OF /3-LACTAMS TABLE III Acid Hydrolysis of Monocyclic ^-Lactams Substituents on the nucleus I I -C3—4C— I2 1I CO—N— 2-azetidinone

Conditions of hydrolysis

Extent of hydrolysis

Reference

. 2 equivs. HCl in EtOH reflux—1 hr.

No reaction

P.8, 1

l-Benzyl-3-methyl-4-phenyI-

0.12 N HCl in EtOH 76°—86 hr.

No reaction

M.64, 11

l-Cyclohexanemethyl-3-methyl-4-

0.12 N HCl in EtOH 76°—86 hr.

No reaction

M.64, 11

1 N HCl in EtOH 100°—3 hr.

Little, if any, reaction

M.55, 2

Cone. HCl 100°—5 min.

Extensive, to give hydrochloride of amino acid

M.61, 3

1 N HCl in EtOH 23°—52 hr.

52%, pot. titration

M.67, 7

1 N HCl 100°—1 min.

Immediate hydrolysis

M.6S, 8

1 N HCl in EtOH 23"—96 hr.

No hydrolysis, pot. titration

M.67, 7

1,4-Diphenyl-

cyclohexyl-.

l-Phenyl-3-phenylacetamido-

l-Cyclohexyl-3-cyclohexaneacetamido- .

Desthiobenzylpenicillin (3-Phenylacetamido-2-azetidinone-l-a-Disovaleric acid).

none nucleus and, as can be readily seen, the rates of hydrolysis were considerably different. It is possible that these differences can be explained in part by the steric effect of different substituents on the attack of the carbonyl group during hydrolysis. For example, it was shown by Levenson and Smith, J. Am. Chem. Soc., 62, 2324 (1940), that substitu­ tion of phenyl groups in the α-position of ethyl esters of aliphatic acids greatly lowers the rate of saponification. These authors postulated that the phenomenon is caused by a powerful steric effect which more than overcomes any polarization effect of the phenyl groups (except in the case of esters of phenylacetic acid). The fourth and fifth com­ pounds in the table differ only in the degree of saturation of the carbocyclic rings. It is apparent that in this case some factor other than steric effect is responsible for differences in the rates of hydroly­ sis. Along these lines it has been shown by Calvet (J. chim. phys., 80, 140 (1933); Compt. rend., 192, 1569 (1931)), that rates of hydrolysis of amides increase with increasing strength of the parent acid. The fact that phenylacetic acid is a stronger acid (K0 = 5.56 X IO-5) than cyclohexaneacetic acid (Ka = 2.36 X IO-5) would place the /3-lactams under consideration in the expected order of rate of hydrolysis. The amine strengths also probably differ to some extent and this factor should be taken into consideration. The last three /3-lactams (exclusive of penicillin) which contain acylamino

groups a to the /3-Iactam carbonyl hydrolyzed much more rapidly than the preceding ones and ap­ proached the hydrolysis rate of penicillin. This is not surprising inasmuch as it is known that a-acylamino acids are stronger than the corresponding aliphatic acids (Zief and Edsall, J. Am. Chem. Soc., 59, 2047 (1937)). Therefore, as noted above, the amides derived from these acids should hydrolyze more rapidly than the corresponding amides of purely aliphatic acids. The acid hydrolysis (see Table III) of the model /3-lactams proceeds more slowly than the alkaline hydrolysis. Inasmuch as the data are not com­ parable it is not possible to say whether the acyl­ amino substituted /3-lactams hydrolyzed faster than the simple /3-lactams, although it was possible to hydrolyze some of the former but not" the latter under the conditions used. Reaction with Nitrogen Bases. A mixture of hydrazine with l,4-diphenyl-3-methyl-2-azetidinone when heated on the steam cone for ten to fifteen minutes gave a-methyl-/3-phenyl-/3-anilinopropionohydrazide (Mich. Chem., B.16, 4). Four ^-lactams (Compounds 22, 26, 28, and 30 in Table I) have given the N-benzylamide of the corresponding acid on heating with benzylamine at 160°. This reaction was attempted with certain other /3-lactams, specifically l-cyclohexyl-3-cyclohexaneacetamido-2-azetidinone at 100°, without success.

Small amount phenylacet- PD.n, 3 amide; unidentified neutral M.B7, 5 dark oil.

lMgCH(C0 2 C 2 H 6 ) 2 in ether C2H&O2C—CH—CHCEH6 at room temperature io—N—C6H6 Heated 150° ten minutes

( C H , C 0 ) 2 0 2 hr. on steam CH2—CHCaHs cone: Reflux forty-five min. io—N—C6H6

180° for one hour

C6H6N=CHC6H6

CeH 6 NHCH (C,H.) C H 2 C 0 0 H

C,H 6 NHCH(C6H5)CH 2 COOH

C,H 6 CH 2 NHCH 2 —CH—CO 2 C 2 H 6

C,H6N (CHO) CH2CH2CN

HC1 and

CH 2 —CH S I I or derivative HN=C N—C«H 6

At 150-160°

C.H 6 NHCH 2 CH 2 CN

C 2 H 6 OH

C 6 H 6 CH 2 CONHCH—CH 2 1 1 CO—N—C,H 6

H N 0 2 in aqueous medium

C6H6NHCH2CH—CON2H,« 1 CeHsCHaCONH

HC1 in dioxane

C.H 6 CH 2 CONHCH—CH 2 1 1 CO—N—CH 2 C 6 H 6

H N 0 2 in aqueous medium

|

C.H 6 CH 2 CONH

IO

in CH2

N—CeHs

CHj

IO—N—C6H6

C6H 6 CH 2 NHCH 2 CHCON 2 H 3 e

CH2—CHCeHe

CH 3 CH 2 CH 2 CH 2 ONO HC1 in CHC1,

I O — N — C H 2C EH 6

C 6 H 6 CH 2 CONHCH—CH 2

i'O—N—C6H6

CH2—CHCbHS

CeH 6 NHCH(C6H 6 )CH 2 CON 2 Ha°

C 6 HsCH 2 CONH

Product* m.p. 140-141° prob- P.U, 5 ably 4-phenyl-4-(N-acetylanilino)-2-butanone._ Derivative11 with hydrox'ylamine m.p. 150-151°.

BrCH(COOC 2 H 6 ) 2 with Zn C2H6O2CCH—CHCEHG (and Mg) in refluxing toluene 40 minutes io—A—CeHs

PM,

PD.M, 2

6

5

C«H 6 NHCH 2 CH 2 CN product obtained.

only M.60, 1

Gummy product, no evidence M.60, 1 of desired product.

No evidence of azide or /3-lac- M.B3, 8 tam.

Product m.p. 144-147°'

m.p. 227-228°. PM, May be 1,5-diphenyItetrahydroimidazolone.

Product11

Cinnamic acid obtained.

Product m.p. 97.5-98.5°; %N, H.14, 1 3.9. Probably diethyl a-anilinobenzylmalonate.

Reactants consumed; only H.14, 1 tarry products obtained.

W.14, 1

C 6 H 6 N=CHC 6 H6

No reaction.

CH 2 =C(OC 2 H 6 ) 2 CH2—CH—CeH( Mixture heated 180-200° for 90 minutes. (C 2 H 6 0) 2 C - A CeHs or derivatives

Reference

It was hoped to generate H.1S, 2 methylketene in situ from the diazoacetone. N-Benzylideneaniline recovered.

Remarks

C,H6N=CHC,H,

io—A—C.H6

CHjCII—CH—CgQi

Expected product

Ag 2 0, N 2 C H C 0 C H , Heated in dioxane

Other reagents and/or reaction conditions

C e H s N=CHCeH 6

Reactant

TABLE IV Attempted Syntheses of Monocyclic /3-Lactams

GO

s

0

Q

O •=1

O w H g i—( CO H W K!

H W H

to oo ©

In refluxing methyl isobutyl C 6 H 6 CH 2 CONHCH—CH 2 I I ketone, one hour.

C,H6COCH2CO2C2H6

Booi

Cf. Kfitw and Merkel, J. prakt. Chem., 187, 102 (1909).

II2—CH2—D/HCOJCJHI

IO—N—C6H5

C H = C — C EHJ

Heated, various conditions

C 6 H 5 A=CHC0NHC«H S

HN—C,H 6

C,H 6 C=CHCONH 2

CH=C—C$H( 1 1 CO—NH

i o — N — R (R = H or C 6 H 6 )

CH=C—CBUCsHs

IO—N—CEHS

CH=C—C{HT

Expected product

Heated, various conditions

Heated in NH 3 or C«H 6 NH 2

C,H 6 CH 2 COCH 2 CO 2 C 2 H 6

NH2

In refluxing aniline

Other reagents and/or reaction conditions

C6H5COCH2CO2C2H5

Reactant

TABLE IV—(Continued)

compound

P..29, 1

P.29, 1

Reference

HH 02 H »

§

O w

i-3 W M

No crystalline product iso- PD.S7,1 lated.

02

o H >

Kj 30% recovery of starting ma- PD.27, 1 o terial. Gas evolved, 21% ? recovery of starting material.

N,N' = Diphenylurea; un- P.29, 1 identified product m.p. 187189°.

Only product identified m.p. P.29, 1 > 250°: analyzed approx. for C 17 H U ON 2 .

None of desired obtained.

C 6 H 6 I = C H C O N H C 6 H 6 only product identified.

HN—C,H 6

Remarks

to

co

CO

THE CHEMISTRY OF /3-LACTAMS Reaction with Anhydrous Hydrogen Chloride. It was found (Merck, M.57, 1) that when 1-phenyl3-phenylacetamido-2-azetidinone was treated with hydrogen chloride in dioxane for forty-five seconds and then warmed one minute on the steam bath, aniline hydrochloride precipitated when the solu­ tion was cooled. Apparently a-phenylacetamido/3-anilinopropionic acid was not an intermediate in this transformation since it is stable in dioxane containing hydrogen chloride, and even in hot con­ centrated hydrochloric acid. One possible explana­ tion for this formation of aniline hydrochloride might be indicated according to the following reactions: C6H5CH2CONHCH-CH2

HC1

CO-NC6H6 CeH6CH2CONHCH-CH2NHC6H6 I COCl HCl I, or ClH-N-

-CHCH2NHC6H6

-A, CeH6CH2C-O-CO Π CeH6CH2CONHC=CH2 COOH

HCl

+ C6H6NH2-HCl

983

penicillin were converted to 2-benzyIidene-4-methyl-5(2)-oxazolone by heating in anisole (Merck, M.72, 8).

Reaction with Thiocyanic Acid. l-Cyclohexyl-3cyclohexaneacetamido-2-azetidinone with thiocyanic acid gave a thiodihydrouracil (Merck, M.62, 3; 63, 9) by a reaction which resembles that of methyl benzylpenicillinate with thiocyanic acid (Cornell Bioch., D.20, 4). Pyrolysis. Staudinger studied the thermal cleav­ age of several /3-lactams and found that decomposi­ tion occurred in two directions. Reversion of the /3-lactam to the original ketene and imine as well as the formation of an olefin and an isocyanate were observed. In the case of the /3-lactam derived from dimethyl-ketene and benzophenoneanil the reac­ tions were as follows: (CH3)2C=CO + (C6H6)2C=NC6H6 t (CH3)2C-CO (C6H6)2C-NC6H6 1 (CH3)2C=C(C6H6)2 + C6H6N=CO The decomposition rates and the proportion which went in each direction were dependent on the struc­ ture of the /3-lactams. Attempted Syntheses of Monocyclic ^-Lactams. In Table IV a number of unsuccessful attempts to carry out novel preparations of /3-lactams or to synthesize ^-lactams of special interest are men­ tioned. Compounds of Uncertain Structure Postulated as /S-Lactams. There are in the literature several compounds of questionable structure which have been assigned the j3-lactam structure. Kipping and Perkin (J. Chem. Soc., 55, 330 (1889)) proposed an unsaturated /3-lactam structure as shown for the reaction product of ethyl α,ω-diacetylcaproate with anhydrous ammonia.

These reactions are of interest in connection with one of the several mechanisms possible for the benzylpenicillin-benzylpenillic acid rearrangement. Another indication of an intermediate like I or II above in the reaction of dry hydrogen chloride with /3-lactams was obtained with l-cyclohexyl-3-cyclohexaneacetamido-2-azetidinone (Merck, M.69, 5). When this /3-lactam was treated with hydrogen chloride in dry chloroform at room temperature for one minute followed by treatment with benzylamine there was obtained the benzylamide of CH3CO(CH2)4C=CCH3 a-cyclohexaneacetamido-/3-cyclohexylaminopropionic acid. This amide must have arisen from CO—NH some intermediate other than the β-Iactam for the /S-Iactam did not give a benzylamide when heated Bruylants (JBull. acad. Toy. Belg., (5) 7, 252 (1921)) with benzylamine on the steam cone for three hours. proposed the following structures for the reaction Hydrogenolysis. When l,4-diphenyl-2-azetidi- product of glutaronitrile with ethylmagnesium none was refluxed for five minutes in 50 % aqueous bromide: dioxane with Raney nickel /3-phenylpropionanilide NC(CH2)2CH-C(CH2)3CN was obtained in good yield (Pfizer, P.22, 8). or A similar behavior was noted with l-benzyl-3,3I Il CO-N dimethyl-4-phenyl-2-azetidinone (Shell, Sh.9, 123). This reaction was tried with l-phenyl-2-azetidiNC(CH2)2CH-C=CH(CH2)2CN none, l-phenyl-3-phenylacetamido-2-azetidinone (Pfizer, P.23, 10), and desthiobenzylpenicillin CO—NH (Merck, M.52, 10) without evidence of any such From 2-carbethoxy-5-methylcyclohexanone and cleavage. Conversion to 5(2)-Oxazolones. l-Phenyl-3- ammonia K6tz and Merkel (J. prakt. Chem., 187, phenylacetamido-2-azetidinone and desthiobenzyl­ 102 (1909)) obtained ethyl 5-methyl-2-tetrahydro-

984

THE CHEMISTRY OF /S-LACTAMS

anthranilate. This compound lost ethanol on heating at 2Q0° in aniline. Kotz and Merkel sug­ gested the following structure for the product: CO-C-CH2-CH2 I Il I NH-C-CH2-CHCH3 BICYCLIC ^-LACTAMS (THIAZ0LIDINE-/3-LACTAMS) The first attempts to prepare compounds con­ taining the bicyclic thiazolidine-^-lactam ring system were carried out by workers at the labora­ tories of Pfizer, Merck, and Parke-Davis. These unsuccessful experiments involved the reaction of an α-bromoester with a 2-thiazoline in the pres­ ence of zinc or the reaction of a Grignard reagent with an ester of a 2-thiazolidineacetic acid. The first synthesis of a thiazolidine-/3-lactam was accomplished at the Shell laboratories (Sh.7, 70) by the reaction of diphenylketene with 2-phenyl2-thiazoline. The resulting ^-lactam (I) of 2,a,a-triphenyl-2-thiazolidineacetic acid did not resemble (CeH5)2C

C(CeH5)-S

CO-N-CH2-AH2 penicillin in its chemical reactions. However, the position of the infra-red absorption of the carbonyl group in the synthetic thiazolidine-/3-lactam was in good agreement with that of the labile group in penicillin. This discovery provided a potent stimulus for further work in the field of thiazolidineβ-lactams and for attempts to synthesize the ^-lac­ tam structure corresponding to penicillin. In spite of numerous attempts, no other thiazolidine-/3-Iactams have been prepared directly by the reaction of 2-thiazolines with diphenylketene, dimethylketene, or ketene itself. In general, the only definite products isolated have been thiazolidine-2,4-piperidinediones: CO-CR2-CR'-

-S

CR2-CO-N-CH2-CH2 These compounds are formed from one mole of the thiazoline and two moles of the ketene by a reac­ tion analogous to the formation of monocyclic 2,4-piperidinediones from some open-chain imines with ketenes (see p. 976). Two of these piperidinediones, those from di­ methylketene with 2-phenyl-2-thiazoline and with 2-methyl-2-thiazoline, were converted to thiazolidine-j8-lactams by a procedure involving hydrolysis to N-isobutyryl-2-thiazolidine-a-isobutyric acids which on thermal treatment lost isobutyric acid with closure of the β-lactam ring. The two /S-Iactams thus prepared, the lactams of 2-phenyl- and 2-methyl-2-thiazolidine-a-isobutyric acid, proved to be much more resistant to hydrolysis than peni-

(CH3)2C

C(C6H5)-S

Ao-N-CH2-CH2 Π (CH3)2C

C(CH3)-S

CO-N-CH2-AH2 in cillin. However, compound II resembled penicillin in some significant chemical reactions (see p. 987). Furthermore, in both II and ΠΙ the position of the characteristic infra-red absorption of the carbonyl group was in exact agreement with that of the labile group in penicillin. The structure of compound II was definitely established by means of degradative studies. None of the three synthetic thiazolidine-/3lactams had any significant antibiotic activity. /3-Lactam of 2,a,α-Triphenyl-2-thiazolidineacetic Acid. PREPARATION. Diphenylketene reacted smoothly with 2-phenyl-2-thiazoline with moderate evolution of heat (Shell, Sh.7, 81). The thiazolidine-j3-lactam structure (I) has been assigned to the prpduct by analogy with the structure of the prod(C6H5)2C=CO + C6H5C-

N--CH2-A:H

2

(C6H5)2C

C(C6H5)-S

AO—N—CH2—AH2 I uct formed by the reaction of diphenylketene and Ν-benzylideneaniline (Staudinger, Ann., 356, 61, 95 (1907)). (C6H5)2C=CO + CeH6CH=NC6H5 —» (C6H5)2C CHC6H5 I I CO-NC6H6 This thiazolidine-/3-lactam was a crystalline solid for which the analytical data were in good agreement with structure I or with the isomeric ketotrimethylenimine (3-azetidinone) structure IV, which would be formed by reverse addition of diphenylketene to the anil. The known ketoCO-C(C6H6)-S (C6H5)2C

N-CH2- -CH2 IV

trimethylenimine (see p. 975) formed from ethylcarbethoxyketene and N-benzylideneaniline is a very unstable compound. Further confirmation for the /3-lactam structure (I) for the reaction product of diphenylketene and 2-phenyl-2-thiazoline was provided by the agree­ ment of its infra-red absorption with that of the

985

THE CHEMISTRY OF /3-LACTAMS subsequently prepared /ϊ-lactam of 2-phenyl-2thiazolidine-a-isobutyric acid, the structure of which was conclusively proved by degradative studies (see below, p. 985). The infra-red absorption of the /3-lactam of 2,a,a-triphenyl-2-thiazolidineacetic acid (I) showed the following significant bands: a strong and sharp band at 5.655 μ (carbonyl) and a rather weak band at 6.235 μ (phenyl). The x-ray diffraction powder pattern of a sample of the crystalline /3-lactam has been determined (Illinois, Cl.6, 5). REACTIONS. The /3-lactam of 2,a,a-triphenyl-2thiazolidineacetic acid could not be hydrolyzed to the amino acid nor methanolyzed to the correspond­ ing methyl ester. With dilute base under mild conditions there was no change in the /3-lactam; under more drastic conditions the products were diphenylacetic acid and 2-phenyl-2-thiazoline. It seems probable that these products were formed by decomposition of the intermediately formed triphenylthiazolidineacetic acid. This decomposi(C6H5)2C

C(C6H6)-S

H2Q

CO-N-CH2-CH2

/3-Lactam of 2-Phenyl-2-thiazolidine-a-isobutyric Acid. PREPARATION AND STRUCTURE PROOF. The reaction of dimethylketene with 2-phenyl-2-thiazoIine led to the formation of a thiazolidine-piperidinedione, from two molecules of the ketene with one of the thiazoline, rather than a thiazolidine-/3-lactam. The piperidinedione was hydrolyzed to N-isobutyryl-2-phenyl-2-thiazolidine-a-isobutyric acid, which upon heating lost isobutyric acid ("deisobutyrylation") with the formation of the /3-lactam of 2-phenyl-2-thiazolidine-a-isobutyric acid (II) (Shell, Sh.8, 107). 2(CH3)2C=CO + C6H6C-S N-CH2-CH2 CO-C(CH3)2-C(C6H6)-S (CH3)2C

CO

N-CH2-CH2

H2O HOOCC(CH3)2C(C6H6)-S

Δ

(CH3)2CHCON-CH2-CH2 (CH3)2C

C(C6H6)-S

CO-N-CH2-CH2

+ (CH3)2CHCOOH

II (C6H6)2C-

-C(C6H6)

S

COOH NH-CH2-CH2 I (C6H6)2CHCOOH + C6H6CN-CH2-CH2 tion is a reaction analogous to the decomposition of the /3-lactam of /3-phenyl-/3-anilinoisovaleric acid to isobutyric acid and N-benzylideneaniline upon hydrolysis with hydrochloric acid, and to the decom­ position of /3-anilinobenzylmalonic acid to malonic acid and N-benzylideneaniline (Staudinger, Die Ketene, Enke, Stuttgart (1912), pp. 73-74). The products of methanolysis of the /3-lactam (I) were not isolated. The infra-red absorption of the crude oily product indicated that the oil probably consisted of a mixture of methyl diphenylacetate and 2-phenyl-2-thiazoline. The 0-lactam reacted only slowly and incom­ pletely with benzylamine. It did not react with dilute potassium permanganate, but underwent extensive decomposition on treatment with hydro­ gen peroxide in acetic anhydride. Hydrogenolysis of the /3-lactam with Raney nickel at room tem­ perature took place readily, but the products were mixtures which could not be purified. The sulfur was removed by this treatment, but apparently cleavage of a carbon-nitrogen bond also occurred. Two unidentified crystalline products were formed when the /3-lactam was treated with hydrazine.

The thiazolidine-/3-lactam structure (II) for the deisobutyrylation product was established as follows: 1. The presence of the /3-lactam ring was demon­ strated by hydrogenolysis of the deisobutyrylation product to an oil the infra-red spectrum of which was identical with that of an authentic sample of the /3-lactam of /3-ethylamino-/3-phenyl-a,a:-dimethylpropionic acid (V). The authentic sample of V, also obtained as an oil, was prepared by thermal deisobutyrylation of N-isobutyryl-/3-ethylamino-/3phenyl-a,a-dimethylpropionic acid. (CH3)2C

C(C6H5)-S

CO-N-CH2-AH2 H H 2 INi

(CH3)2C CHC6H6 • I I CO-NCH2CH3 V -(CH 3 ) 2 CHCOOHt

HOOC-C(CH3)2-CHC6H6 (CH3)2CHCON-CH2CH3 2. The presence of a thiazolidine ring in the methanolysis product (VI) of the /3-lactam was demonstrated by the formation of a ketone (methyl α-benzoylisobutyrate) upon treatment of the meth-

986

THE CHEMISTRY OF ^-LACTAMS

anolysis product with mercuric chloride. The methyl α-benzoylisobutyrate "was shown to be identical with a sample prepared independently from methyl benzoate and methyl a-bromoisobutyrate in the presence of zinc. (CH3)2C

C(C6H6)-S

CO—N-CH II (CH3)8C-

CHs0h

CH2

-C(CEH6)

" S

HgCl2

COOCH3 NH-CH2-CH2 VI (CH3)2C-COC6H6 COOCH3 "f Zn ' (CH3)2CBrCOOCH3 + CH3OOCC6H6 ' An attempt to prepare methyl 2-phenyl-2thiazolidine-a-isobutyrate (VI) by condensation of methyl α-benzoylisobutyrate with 2-aminoethanethiol was unsuccessful. The infra-red absorption of the thiazolidine-/Slactam (II) showed a strong and sharp absorption band at 5.625 μ (carbonyl) and very weak bands at 5.95 μ and 6.075 μ. The x-ray diffraction powder pattern of a crystal­ line sample of II has been determined (Illinois, Cl.e, 6). REACTIONS. The /3-lactam of 2-phenyl-2-thiazolidine-a-isobutyric acid (II) was resistant to alkaline hydrolysis. It was recovered unchanged after five hours of refluxing with on® equivalent of alkali in dioxane. Under more drastic conditions the mole­ cule was cleaved to fragments which have not been identified. The /3-lactam was slowly but smoothly converted to methyl 2-phenyl-2-thiazolidine-a-isobutyrate by treatment with absolute methanol containing a trace of sodium methoxide. The /3-lactam did not react under moderate condi­ tions with benzylamine or with thiocyanic acid. Treatment with these reagents under more vigorous conditions led to the formation of products which have not been identified. Treatment of the /3-lactam (II) with mercuric chloride gave a mercaptan (VII) which was oxidized to a disulfide (VIII). Both the mercaptan and the disulfide gave Che same hydrogenolysis product, believed to be the. N-ethylamide of a,a-dimethyl-/3phenyl-|8-hydroxypropionic acid (IX). A sample of the latter was prepared independently in less pure form from methyl-α,a-dimethyl-/3-phenyl-/3hydroxypropionate and ethylamine. The /3-lactam reacted slowly with hydrogen chlor­ ide in dry ether to form the same disulfide (VIII) as had been obtained by oxidation of the mercaptan

(VII). Apparently the water and oxygen required for this transformation were taken up from the air. (CH3)2C

C(C6H6)-S

CO-N-CH2-CH2 II

HgCl2 • H's

C6H6COC(CH3)2CONHCH2CH2SH JHL VII T Ni Γ CeH6COC(CH3)2CONHCH2CH2S—"|_3i L VIII J2Ni C6H6CHOHC(CH3)2CONHC2H6 IX The /3-lactam did not react with iodine in glacial acetic acid. However, oxidation with hydrogen peroxide in acetic anhydride gave a compound believed to be the a-disulfoxide [C6H6COC(CH3)2CONHCH2CH2S—]2, I O which was hydrogenolyzed to the N-ethylamide (IX). By means of titration studies with perchloric acid in glacial acetic acid it was shown that the nitrogen atom of the thiazolidine-/3-lactam (H) was neutral even under these conditions of titration. /3-Lactam of 2-Methyl-2-thiazolidine-a-isobutyric Acid. The /3-lactam of 2-methyl-2-thiazolidine-aisobutyric acid (III) was prepared by a procedure (Shell, Sh.14, 212) analogous to that previously described for the 2-phenyl compound (II). The (CH3)2C

C(CH3)—S

CO-N-CH2-CH2 IH thiazolidine-piperidinedione from two moles of dimethylketene and one mole of 2-methyl-2thiazoline could not be isolated in pure form because of its ready hydrolysis to N-isobutyryl-2-methyl-2thiazolidine-a-isobutyric acid This acid upon heating gave up isobutyric acid with the formation of the /3-lactam (III) in 74% yield This compound is the only synthetic thiazolidine/3-lactam containing exclusively aliphatic substituents. Like the 2-phenyl compound it reacted only slowly with methanol containing a trace of sodium methoxide. No other studies of the reactiv­ ity of this /3-lactam have been made. In the infra-red it showed a strong and sharp band at 5.63 μ, and no other band in the region of double-bond absorption. Comparison of Thiazolidine-/3-lactams with Peni­ cillin. Of the three thiazolidine-/3-lactams which have been prepared, the /3-lactam of 2-phenyl-2-

987

THE CHEMISTRY OF /3-LACTAMS thiazolidine-a-isobutyric acid (II) has been the most thoroughly investigated. This thiazolidine/3-lactam in its chemical behavior resembles the penicillins in the following respects: (1) The nitro­ gen atom of the t h ia ζ οIi d i ηe-β-1a c ta m is completely neutral; (2) the sulfur atom of the thiazolidine-/3Iactam is inert to iodine in glacial acetic acid; (3) the thiazolidine-/3-lactam is hydrogenolyzed by Raney nickel to a monocyclic /3-lactam. In the case of the hydrogenolysis reaction, a striking parallel between the behavior of the thiazolidine-/3-lactam (II) and of methyl and sodium benzylpenicillinate is shown by the changes in the positions of their double-bond absorptions in the infra-red upon the removal of the sulfur atoms. The position of absorption (5.62 μ) of the labile group in penicillin is strongly shifted by hydro­ genolysis and it is shifted to the range of absorption of the carbonyl group of monocyclic ^-lactams (about 5.74 μ). As far as ease of hydrolysis and of reaction with benzylamine are concerned, the thiazolidine-/3lactams, I and II, proved to be much less reactive than the penicillins. The (3-lactam (III) was not studied in this connection. All the synthetic (C6H6)2C-C(C6H6)-S I I I CO-N-CH2-CH2 I (CH3)2C C(C6H6)-S I I I CO-N-CH2-CH2 II (CH3)2C

C(CH3)-S

CO-N-CH2-CH2 III thiazolidine-/3-lactams, including the purely ali­ phatic compound (III), were much more slowly methanolyzed than the penicillins. Assuming that the penicillins have the ,S-Iactam formula, then their greater reactivity may be due RCONHCH—CH-S-C (CH3) 2 CO-N

CHCOOH

to the presence of the acylamino group in the «-posi­ tion to the /3-lactam carbonyl group and possibly to the presence of the carboxyl group in the 4-position of the thiazolidine ring. The low reactivity of the /3-lactam carbonyl group in the synthetic thiazolidine-jS-lactams (I, II and III) may be due, at least in part, to the presence of two aryl or alkyl groups in the α-position. In support of the hypothesis that the lability of the ^-lactam ring in the penicillins is in part due to the presence of the acylamino group in the «-posi­ tion, one may cite the work of Calvet showing that

the rates of hydrolysis of amides increase with increasing strength of the parent acid, and that of Zief and Edsall showing that substitution of an acylamino group in the α-position of aliphatic acids increases the acid strength (see discussion on p. 979). The labilizing effect of the a-phenylacetamido group in a monocyclic /3-lactam is shown in Table II. In the synthetic thiazolidine-/3-lactams, the presence of two methyl or two phenyl groups in the α-position to the /3-lactam carbonyl group might well be expected to exert a stabilizing effect on the cyclic amide linkage. No study of the effect of α-methyl or α-phenyl substitution in amides on their rates of hydrolysis is available for comparison. However, Davies and Evans (J. Chem. Soc., 339 (1940) have shown that a great increase in resistance to hydrolysis of ethyl esters of aliphatic acids is produced by substitution on the α-carbon atom of alkyl groups in general and of methyl groups in particular. Similarly, Levenson and Smith (J. Am. Chem. Soc., 62, 2324 (1940)) have shown that the substitution of phenyl groups in the α-position of esters of aliphatic acids greatly lowers the rate of saponification (see p. 978). According to Ingold ( J . C h e m . S o c . , 119, 305 (1921)) and Ingold and Thorpe {J. Chem. Soc., 1318 (1928)), the very presence of a geminal dialkyl or other geminal grouping on a ring exerts a stabiliz­ ing influence on the ring. Attempted Syntheses of Thiazolidine-j3-lactams. REACTION OF KETENE WITH THIAZOLINES. Only one monocyclic ^-lactam, the /3-lactam of /3-anilino/3-phenyl-propionic acid, has been prepared by the reaction of ketene with an imine. The reaction of ketene with 2-thiazoline gave an orange gum which has not been identified. There was no reaction between ketene and 2-phenyl2-thiazoline or benzothiazole, even under drastic conditions. A considerable amount of work has been done on the reaction of ketene with methyl 5,5-dimethyl-2thiazoline-4-carboxylate,' and its 2-methyl and CH-S-C(CH3)2

Il

N-

I

CHCOOCH3

2-phenyl derivatives. Although some crystalline products have been obtained, none of these appears to include a thiazolidine-/3-lactam. Products ob­ tained from two of the thiazoline esters are believed to be thiazolidine-piperidinediones. No crystalline products were obtained from the reaction of ketene with methyl 2-phenylacetamidomethyl-2-thiazoline-4-carboxylate or its 5,5-dimethyl derivative. These reactions are summarized in Table V, which includes brief experimental data and references. REACTION OF DIMETHYLKETENE WITH THIAZO­ LINES. Thiazolidine-piperidinediones have been

988

THE CHEMISTRY OF /3-LACTAMS TABLE V Reaction of Ketene with 2-Thiazolines Thiazoline

Product

Conditions

Orange gum (amide ?)

Analysis of product

Infra-red of product

Reference

Strong band at Sh.14, 216 6.04/.

2-Thiazoline

25° in benzene

2-Phenyl-2-thiazoline

25° and 90-100° No reaction

— .

—

Sh.14, 217

Benzothiazole

25°, 50° and 100° No reaction

—

—

Sh.14, 217

Methyl 5,5-Dimethyl2-thiazoline-4carboxylate

0° in ether

No reaction

— •

—

U.21, 2

Methyl 5,5-Dimethyl2-thiazoline-4carboxylate.

50° in heptane

Yellow oil; may be Good anal, for methyl N-acetyl-5,5- C 9 HuN0 3 S dimethyl-4-thiazolidinecarboxylate

Methyl 5,5-Dimethyl2-thiazoline-4carboxylate.

50° in heptane, Noncrystalline solid, Fair anal, for anhyd. condi- possibly an isoxazo- C9H13NO3S tions line"

Methyl 5,5-Dimethyl2-thiazoline-4carboxylate.

50° in benzene, Light yellow gum, be- Fair anal, for anhyd. condi- lieved to be N-acetyl- C 8 H 16 N0 S S tions penicillamine methyl ester

Methyl 5,5-Dimethyl2-thiazoline-4carboxylate.

(Not described)

Methyl 5,5-Dimethyl2-thiazoIine-4carboxylate

(Not described) Oil

—

5.73, 5.95 n

U.M-22

5.73, 5.99 n

v.n-u

U.S4,

Crystals, m.p. 99- Calc. for C11H15NO4S: 102°; may be the C, 51.40; H, 5.84; N, 5.45; S, 12.45. piperidinedioneb Found: C, 51.84; H, 6.71; N, 5.45; S, 12.32 —

P.28,

—

Methyl 2,5,5-Trimethyl- 30° in benzene, Crystalline solid, m.p. Calc. for CI 2 H 1 TN0 4 S: 2-thiazoline-4C, 53.12; H, 6.32. 150-151°; may be or 180° in carboxylate. Found: C, 53.25, the piperidinedione0 p-cymene 53.12; H, 6.18, 6.18 Methyl 2,5,5-Trimethyl- 0° in benzene 2-thiazoline-4carboxylate.

Product, m.p. 81-83°

Methyl 2-Phenyl-5,5dimethyl-2-thiazoline-4-carboxylate

(Not described)

Crystalline material

Methyl 2-Phenyl-5,5dimethyl-2-thiazoline-4-carboxylate.

0° in benzene

Oil (contained unchanged starting material)

2-Phenyl-5,5-dimethyl2-thiazoline-4carboxylic acid

(Not described)

No crystalline product

2

11

B.17, 1 - 2

B.16, 3; 17-21

Calc. for C, 0 HuNO 3 S: Not indicative of P.29) 82-3$ C, 52.40; H, 6.55; thiazolidine-j3N, 6.12; S, 13.98. lactam Found: C, 52.28; H, 6.61; N, 6.01, 6.04; S, 13.80, 14.06 —

—

B.16,

3

—

—

P.29,

10

M.68,

18

989 THE CHEMISTRY OF /3-LACTAMS TABLE V— {Continued) Thiazoline

Methyl 2-Phenylacetamido-5,5dimethyl-2-thiazoline-4-carboxylate

c

Conditions

Analysis of product

Product

(Not described) No crystalline product

—

—

On hydrolysis with alcoholic NaOH, the supposed piperidinedione,

which may be

Infra-red of product

Reference

B.17, 5; 18, 5

gave an acid melting at 218-220 ,

The methyl ester of the acic^ melts at 73°-75°.

prepared by the reaction of dimethylketene (two moles) with the following thiazolines: 2-methyl2-thiazoline, 2-phenyl-2-thiazoline, methyl 5,5dimethyl-2-thiazoline-4-carboxylate, and methyl 2-phenyl-5,5-dimethyl-2-thiazoline-4-carboxylate. The reaction of dimethylketene with benzothiazole gave a benzothiazoline-piperidinedione. Each of these piperidinediones has been converted to the corresponding N-isobutyryl-2-thiazolidine-a-isobutyric acid, but only the acids derived from 2-mcthyland 2-phenyl-2-thiazoline could be converted to thiazolidine-/3-lactams (see pp. 986 and 985). The reaction of dimethylketene with 2-thiazoline apparently gave the thiazolidine-piperidinedione which, however, hydrolyzed so readily that only N-isobutyryl-2-thiazolidine-a-isobutyric acid was isolated. Heating of this acid did not effect removal of isobutyric acid with formation of a /3-lactam ring. The experimental data for the preparation of the thiazolidine-piperidinediones and the N-isobutyryl2-thiazolidine-a-isobutyric acids are given in Table VI. R E A C T I O N OF D I P H E N Y L K E T E N E W I T H T H I A Z O L -

INES. In only one case has a thiazolidine-/3lactam been prepared directly from a ketene and a 2-thiazoline. This case, the reaction of diphenylketene and 2-phenyl-2-thiazoline, has been discussed on p. 984. The reaction of diphenylketene with some other 2-thiazolines has given products derived from two moles of the ketene and one mole of the thiazolines. The products have been assigned the thiazolidinepiperidinedione structure (X). The compounds

formed by the reaction of diphenylketene with 2-methyl-2-thiazoline, 2-styryl-2-thiazoline, and methyl 2,5,5-trimethyl-2-thiazoline-4-carboxylate, are believed to be piperidinediones of structure X. The crystalline product from benzothiazole and diphenylketene has been assigned the benzothiazoline-piperidinedione structure (XI). The analysis and infra-red absorption of its hydrogenolysis product are in good agreement with those expected for compound (XII), 1,3,3,5,5-pentaphenyl-2,4-piperidinedione. Methanolysis of XI

gave a methyl ester having an analysis in poor agreement with that calculated for the expected methyl N-diphenylacetyl-a,a-diphenyl-2,3-dihydro2-benzothiazoleacetate. However, methanolysis of the hydrogenolysis product (XII) gave a product of which the analysis and the infra-red absorption were in agreement with that expected for methyl N-diphenylacetyl-/3-anilino-a, a-diphenylpropionate (XIII).

REACTION

OF

MISCELLANEOUS

KETENES

WITH

Attempts by the Shell group to prepare a thiazolidine-/3-lactam containing a carbTHIAZOLINES.

990

THE CHEMISTRY OF ^-LACTAMS TABLE VI Reaction of Dimethylketene with 2-Thiazolines Thiazoline

2-Thiazoline

Conditions

Piperidinedione

4 days at 25° in ethyl Not isolated acetate

2-Methyl-2-thiazolineb.... 3 days at 25° in ethyl Oil (see p. 1000) acetate

Acylamino acid

Reference

N-Isobutyryl-2-thiazol- ShAJ ht 214-215 idine-a-isobutyric acid," m.p. 120-122°; recryst. from CHCl3 m.p. 130-131° (see p. Sh.14, 211-212 1000)

2-Phenyl-2-thiazolinec.... 2 days at 25° in ethyl m.p. 136-137° (see p. m.p. 157.5-158° (see p. Sh.8, 105-6 997) 997) acetate Methyl 5,5-Dimethyl-22 days at 25° in ethyl Gummy solid, m.p. 75- m.p. 147.5-148.5°; re­ M.66, 2-3 95° thiazoline-4-carboxylate. acetate cryst. from CeH6-pet. \ ether4 Methyl 2-Phenyl-5,5Overnight at 25°, then m.p. 154°, recryst. from m.p. 191-192°; recryst. Sh.8, 109; 10, 142 dimethyl-2-thiazoline-4- 2 hrs. at reflux in ethyl C6He-pet. ether* from aq. CH3OHf carboxylate. acetate Benzothiazole

4 days at 25° in ethyl m.p. 81-83°, recryst. m.p. 157-158°; recryst. Sh.14, 213-21 4 acetate from CH 3 OH g from isooctaneb

• Anal. Calo. ior CuHnNOiS: C, 53.85; H1 7.81; N, 5.71; S, 13.07; neut. equiv., 245. Found: C, 53.8, 53.9; H, 7.8, 8.0; N, 5.5, 5.5; S, 13.0, 13.3; neut. equiv., 253, 256. b This reaction and the conversion to acylamino acid and thiazolidine-/3-lactam are described in detail on p. 997. 0 This reaction and the conversion to acylamino acid and thiazolidine-/3-lactam are described in detail on p. 995. d D-N-Isobutyryl-4-carbomethoxy-5,5-dimethyl-2-thiazolidine-«-isobutyric acid. Anal. Calc. for CuIiUeChNS: C, 54.35; H, 7.60; N, 4.23. Found: C, 54.81; H, 7.86; N, 4.25. ® The sample which was analyzed melted at 146°. Anal. Calc. for C21H27O4NS: C, 64.75; H, 6.99. Found: C, 64.45, 64.52; H, 7.15, 7.15. f N-Isobutyryl-4-carbomethoxy-5,5-dimethyl-2-phenyl-2-thiazolidine-a-isobutyric acid. Anal. Calc. for C21H28NO6S: neut. equiv., 406. Found: neut. equiv., 390, 392. (Prepared by hydrolysis of the piperidinedione with methanolic NaOH.) β Anal. Calc. for CieHi7NOjS: C, 65.42; H, 6.22; N, 5.09; S, 11.64. Found: C, 65.52; H, 6.34; Nf 5.2, 5.4; S, 11.8, 11.9. h N-Isobutyryl-2,3-dihydro-2-benzothiazole-o!-isobutyric acid. AnaU Calc. for CuHieNOaS: C, 61.41; H1 6.53; N, 4.78; neut. equiv., 293. Found: C, 61.1; H, 6.7; N, 4.4; neut. equiv., 309.

ethoxy group by the reaction of ethylcarbethoxyketene with 2-methyl- and 2-phenyl-2-thiazoline, and of ethylcarbethoxyketene dimer with 2-phenyl2-thiazoline, were unsuccessful (Sh.9, 126). Two unsuccessful attempts to prepare thiazolidine-/3-lactams by the reaction of diazoketones (diazoacetophenone and diazomethyl ethyl ketone) and silver oxide (presumably forming a ketene) with 2-phenyl-2-thiazoline were carried out (ShAJ+, 217). The preparation of some N-alkylbenzamidoketenes was attempted in the Michigan Chemical laboratory (B.21, 6) by the following series of reactions: [CeH5C0NR]Na + COCl2 CH2N

[C6H6CONRCOCl]

' [CeH5CONRCOCHN2]

[C6H5CONRCH=C=O]. (R = methyl, ethyl, or benzyl) The intermediates and the products were not isolated. The crude products, presumably con­ taining the ketenes, were treated with methyl 2,5,5-trimethyl-2-thiazoline-4-carboxylate and gave reaction mixtures which had no bioactivity. An attempted synthesis of methyl benzylpenicillinate involves the reaction of methyl 5,5-dimethyl-

2-thiazoline-4-carboxylate with phenylacetamidoketene, supposedly formed by the following series of reactions in which neither the diazoketone nor the ketene was isolated: phenylacetylcarbamyl chloride + diazomethane —• phenylacetylcarbamyldiazomethane (C6H5CH2CONHCOCHN2) + silver ox­ ide —> phenylacetamidoketene. No products were isolated from the reaction of this supposed ketene with the thiazoline ester, and the crude product had little or no bioactivity (B.16,3). ATTEMPTED CYCLIZATIOX OF N-ACYL-2-THIAZOLIDINEACETIC ACIDS. The successful preparation of two thiazolidine-/3-lactams by thermal removal of isobutyric acid (deisobutyrylation) from two N-isobutyryl-2-thiazolidine-a-isobutyric acids (see pp. 985 and 986) provided the incentive for a great number of attempts to prepare by a similar proce­ dure other thiazolidine-0-lactams, especially some containing acylamino groups in the α-position to the /3-lactam carbonyl group and carbomethoxy groups in the 4-position of the thiazolidine ring. The deacylation experiments were unsuccessful except in the two cases already described in detail. Particularly disappointing was the failure to obtain thiazolidine-/3-lactams which would have been lower homologues of esters of the penicillins from α-acylamino-N-acyl-4-carbalkoxy-2-thiazolidineacetic acids and the failure to obtain methyl

THE CHEMISTRY OF 0-LACTAMS

991

TABLE VII Reaction of DiphenyIketene with 2-Thiazolines Thiazoline

Conditions

Product

M.P. of prod.

2-Phenyl-2-thiazoline 25°, no solvent

Thiazolidine-/3Iactama

156.5-158°

2-Methyl-2-thiazoline 25°, no solvent

Piperidinedione, C32H27N O2S

160.5-161°

Infra-red of prod, (μ)

Anal, of prod.

5.655, 6.235

—

5.72, 6.03, 6.25

25°, then 70°- Piperidinedione,cd 163-164.5° 80° for 5 min., C36H26NO2S no solvent

Piperidinedione

2- Phenylacetamido- (Not described) methyl-5,5-dimethyl-2-thiazoline-4-carboxylic acid.

No product re­ ported

Sh.7-8

Calc. for C32H2;- 5.655, 6.09, 6.24 O2NS: C, 78.5; H, 5.5; N, 2.86. Found: C, 78.2, 78.2; H, 5.6, 5.7; N, 2.5, 2.5

2-Styryl-2-thiazolineb 25°, then 85°, no A dark-red glass, solvent believed to be the piperidine­ dione

Methyl 2,5,5-Tri- (Not described) methyl-2-thiazoline-4-carboxylate.

Reference

Calc. for C36H26- 5.655, 6.085, 6.24 O2NS: C, 80.31; H, 4.78; N, 2.68; mol. wt., 523. Found: C, 81.66, 81.77; H, 5.25, 5.21; N, 2.37, 2.35; niol. wt., 565

215°

(Ultraviolet max. at 2950 A.)

—

Sh.10,

145

Sh.14,

216

Sh.14,

144

M.66,

1

B.17,

5

a Described in detail on p. 996. b Preparation of 2-styryl-2-thiazoline: N-Cinnamoylethanol (b.p. 190-195° (2 mm.)) was prepared in 68% yield by heating cinnamic acid and ethanolamine, and was treated with phosphorus pentasulfide (procedure adapted from Wenker, J. Am. Chem. Soc., 57, 1079 (1935)), to give an 8.4 % yield of crude 2-styryl-2-thiazoline, boiling at 95-120° (0.3 mm.). With picric acid this gave a picrate, m.p. 158-159°, which had the correct analysis for 2-styryl-2-thiazoline picrate. Anal. Calc. for C17H14N4O7S: C f 48.80; H, 3.37; N, 13.39. Found: C, 48.6, 48.8; H, 3.7, 3.7; N, 12.2, 12.2. c Methanolysis of this piperidinedione gave a product having an analysis in rather poor agreement with that calculated for the expected methyl N-diphenylacetyl-a,a:-diphenyl-2,3-dihydro-2-benzothiazoleacetate. Anal. Calc. for CieHsiNOsS: C, 77.84; H, 5.23; N, 2.52. Found; C, 76.1, 75.8; H, 6.0 5.6; N, 2.1. d This piperidinedione on treatment with Raney nickel gave a sulfur-free product believed to be the piperidinedione (XII, p. 989), m.p. 167-169°; infra-red: 5.655, 6.085, 6.25μ. Anal. Calc. for C 35 H 27 NO 2 ; C, 85.19; H, 5.48; N, 2.84. Found: C, 84.0, 84.0; H, 5.7, 5.5; N, 2.8. Methanolysis of this sulfur-free piperidinedione gave a product melting at 145°-146°, which gave analytical results in good agreement with those calculated for methyl N -diphenylacetyl -0 -anilino -a ,A -diphenylpropionate. Anal. Calc. for C BS H JI NO S : C, 82.29, H, 5.90; N, 2.67. Found: C, 82.2; H, 6.2; N, 2.5, 2.6.

benzylpenicillinate from /3-methyl-4-isobutyryl-9benzylpenicilloate. In general, upon heating the various N-acylthiazolidineacetic acids there was no reaction except decarboxylation which took place in some cases. Upon vigorous heating extensive decomposition occurred. These unsuccessful deacylation experiments, ex­ clusive of those designed to give one of the penicil­ lins or a penicillin ester (these are discussed in Chapter XXII) are summarized on pp. 1001-1002, with brief experimental details. Miscellaneous Attempted Syntheses. The un­ successful attempts to prepare thiazolidine-|3lactams by Reformatsky type reactions, by Grignard and organolithium cyclizations, and by other procedures and some of the proposed but untried

syntheses of thiazolidine-/3-lactams are tabulated at the end of the experimental section. E

X

P

E

R

I

M

E

N

T

A

L

PREPARATION OF MONOCYCLIC /3-LACTAMS Cyclization of a β-Am'mo Acid: l,4-Diphenyl-2-azetidinone (Pfizer, P.24, 6). A mixture of 1.2 g. of 3-anilino-(3-phenylpropionic acid and 2.4 ml. of phosphorus trichloride was refluxed for one-half hour. As much of the reagent as possible was then removed under reduced pressure. The gummy residue was washed by decantation twice with 15 ml. portions of water and dissolved in methanol without drying. Crystals soon appeared and, after chilling overnight, were collected. The material weighed 0.6 g. and melted at 154155°. When mixed with an authentic specimen of the ^-lactam (p. 992), there was no [depression of melting point.

992

THE CHEMISTRY OF /3-LACTAMS

Cyclization of 0-Acylamino Acids. SYNTHESIS OP 3,3-DIMETHYIZ-I-ETHYL^-WENYL^-AZETIDINONE (Shell, Sh.9123): 1:ETHYL-6-PHENYL-3,3,5,5-TETRAMETHYL-2, 4-PIPERIDINEDIONE. To 5.6 g. (0.041 mole) of N-benzylidene-ethylamine

(prepared from benzaldehyde and ethylamine) in a cylinder flushed with nitrogen was added a solution of δ.9 g. (0.084 mole) of dimethylketene (p. 997) in 60 ml. of ethyl acetate. The solution became colorless after about six hours but was allowed to stand for twenty hours. The ethyl acetate was re­ moved under reduced pressure to leave a crystalline residue which weighed 8.08 g. (73% yield). One gram of this material was recrystallized from benzene-petroleum ether and gave 0.6 g. of l-ethyl-6-phenyl-3,3,5,5-tetramethyl-2,4piperidinedione as white crystals which melted at 89-90°.

added- dropwise. After about one-half of the ester had been added rapid reaction took place. Following reflux for one-half hour the toluene solution was cooled, washed with concentrated ammonium hydroxide, with water, with 10% hydrochloric acid, and again with water, and then con­ centrated in vacuo to a thick syrup. Finally the syrupy residue was distilled, the fraction boiling at 141-146° (0.10 mm.) (nDa, 1.5698) being retained. Calc. for Ci8Hi7ON: C, 81.2; H, 6.8; N, 5.58. Found: C, 80.9; H, 6.9; N, 5.58. In a second preparation the product boiled at 135° (0.02 mm.), and had np26, 1.5702.

Found: C, 81.0; H, 6.7; N, 5.28. The hydrolysis of this material is discussed on pp. 978-979. Cyclization of /S-Amino Acid Esters with Organometallic Compounds. SYNTHESIS OF 1,4-DIPHENYL-2-AZETIDINONE (Pfizer, P.10, 1). The Grignard reagent prepared from These values indicate that a considerable portion of the 3.27 g. (0.03 mole) of ethylbromide and 0.73 g. of magnesium piperidinedione had been converted to the acylamino acid, in 25 cc. of dry ether was diluted with an equal volume of discussed in the following section. When a sample was ether; to this solution was added 8.1 g. (0.03 mole) of ethyl allowed to stand in a stoppered bottle for two days it was /S-phenyl-/S-anilinopropionate (m.p. 76-76.5°. Calc. for converted to a crystalline material, which was shown by Ci Hi O N: N, 5.20. Found: N, 5.26, 5.19) partially dis­ 7 9 2 melting point, 111.5-112.5°, and mixed melting point, 113- solved and suspended in 50 cc. of dry ether. Reaction was 114.5°, to be identical with the acylamino acid obtained in instantaneous and a tan-colored, partly solid gum separated. the following experiment. The reaction mixture was stirred and refluxed for one-half N-ISOBUTYRYL-/S-ETHYLAMINO-|8-PHENYL-A, FF-DIMETHYLhour after all the ester was added. After cooling to room PROPIONIC ACID. A suspension of 7 g. of the piperidinedione temperature, the ether layer was decanted and the gum was from the previous experiment was refluxed for thirty minutes washed with some fresh ether. The combined ether layer with 25 ml. of 10% sodium carbonate solution. All of the and washings were poured into water containing excess piperidinedione had gone into solution in less than ten ammonium chloride. The ether layer was separated and minutes. The cooled solution was extracted with ether and dried. Subsequent evaporation of the solvent gave 1.8 g. of acidified. The N-isobutyryl-/3-ethylamino-/3-phenyl-»,a-di- the original ester (m.p. 68-70°). The gum was covered with methylpropionic acid which separated was collected on a 100 cc. of toluene and treated with 125-150 cc. of water filter and dried in air; it weighed 7.0 g. (93.5% yield). This containing excess ammonium chloride. Thorough stirring acid was recrystallized from aqueous methanol and melted and crushing was needed to break up the lumps. The tolu­ at 114r-114.5°. ene layer was separated and filtered through a dry paper. The solvent was removed as completely as possible under Calc. for Ci7H25NOs: neut. equiv., 291.38. reduced pressure. The residue was taken up in 100-125 cc. Found: neut. equiv., 290, 292. of boiling methanol and upon cooling 3.5 g. of the β-lactam • 3,3- DIMETHYL- 1-ETHYL-4-PHENYL-2-AZETIDINONE. separated. This material melted at 154.5-155.5° and a A 6.3 g. (0.0216 mole) sample of the above acid was heated in mixture with a sample prepared by the procedure of Gilman a small Claisen flask to 160-170° (20 mm.) for about an hour and Speeter (J. Am. Chem. Soc., 65, 2255 (1943)) showed no (until bubbling stopped). During this time 1.9 g. of iso- depression of melting point. Concentration of the methanol butyric acid was collected. The pressure was then reduced mother liquor gave an additional 1.4 g. of slightly less pure and the product was distilled at 92-100° (2 mm.), yielding material. Thus the yield based on ester consumed was 93.9%. 3.8 g. (86.7%) of the azetidinone. Calc. for C17H23NO2: C, 74.70; H, 8.48. Calc. for Ci7H26NO8: C, 70.07; H, 8.65. Found: C, 73.56, 73.18; H, 8.51, 8.51.

Calc. for Ci3H17NO: C, 76.80; ΐί, 8.43; Ν, 6.89. Found: C, 76.55, 76.12; Η, 8.42, 8.45; Ν, 6.68. VAKIABLES IN 0-LACTAM FORMATION FROM /3-ACYLAMINO ACIDS (Sh.9, 129). A limited study was made of solvents

which might favor the elimination of isobutyric acid from 0-isobutyrylamino acids. With N-isobutyryl-/3-benzylamino-j3-phenyl-a,a-dimethylpropionic acid (Staudinger, Klever and Kober, Ann., 374, 1 (1910)) a 50% conversion of 0-lactam was obtained when the reaction was carried out in refluxing diisobutyl ketone (169°) for thirty minutes. In order to test the possibility that isobutyric acid might catalyze the reaction, the acylamino acid was heated for thirty minutes in refluxing diisobutyl ketone containing isobutyric acid. No improvement in conversion to |8-lactam (50%) was observed. Complete recovery of starting ma­ terial was obtained when the acylamino acid was refluxed in pyridine (116°) or quinoline (160°) for thirty minutes. Reformatsky Reaction with an Imine: I-BENZYL3-METHYL-4-PHENYL-2-AZETIDINONE (Merck, M.68, 4). To 20 g. of N-benzylidenebenzylamine, dissolved in 100 ml. of dry toluene, 8 g. of 20-40 mesh zinc metal (washed with conc. sulfuric acid containing a few drops of nitric acid) was added and the whole brought to boiling. After addition of a crystal of iodine, 14 ml. of ethyl α-bromopropionate was

SYNTHESIS OP 3-BENZAMIDO-1-PHENYL-2-AZETIDIN0NE. ETHYL IS-ANILINO-O-BENZAMIDOACRYLATE (Merck, M.81, 7). Five grams of the sodium salt of ethyl formylhippurate (Erlenmeyer, Ann., 337, 251 (1904)) was added to a mixture of 10 ml. of cold water and 12 ml. of 2.5 N hydrochloric

acid. The oil that formed was extracted with chloroform. The chloroform solution was dried over sodium sulfate and evaporated under reduced pressure. The residual oil was dissolved in 5 ml. of ethanol and 1.8 g. of aniline was added. Heat was evolved. After the solution had stood and cooled a jelly-like precipitate separated. It was collected on a filter and dried; weight, 3.1 g. The material was dissolved in hot ethanol and water was added to incipient cloudiness. After the solution had been cooled, the precipitate was collected on a filter and dried in a vacuum desiccator; weight, 2.7 g., m.p. 144-147°. After two further «crystallizations and drying at 100° (0.1 mm.) the melting point was 135-137°. Calc. for Ci8H18O3N2: C, 69.67; H, 5.85; N, 9.03. Found: C, 69.76; H, 5.68; N, 8.85. ETHYL /S-ANILINO-O-BENZAMIDOPROPIONATE {M.81, 7). Two grams of /3-anilino-a-benzamidoacrylic acid ethyl ester was dissolved in 125 ml. of ethanol and reduced at 70° for one hour under 135 atmospheres of hydrogen with Raney nickel catalyst. The catalyst was removed by filtration and the solvent evaporated under reduced pressure. The residue

THE CHEMISTRY OF /S-LACTAMS was dissolved in ether, petroleum ether was added to incipient cloudiness, and the mixture was allowed to stand in the ice box. Crystals formed; weight, 0.3 g., m.p. 66-69°. After three recrystallizations from ether-petroleum ether, the melting point was 72-73°. Calc. for C18H20O3N2: b, 69.22; H, 6.45; N, 8.97. Found: C, 69.41; H, 6.65; N, 8.87. 3-BENZAMIDO-1-PHENYL-2-AZETIDINONE (M.S1 , 7). To 1.00 g. of /S-anilino-cc-benzamidopropionie acid ethyl ester dissolved in 40 ml. of benzene was added an ether solution of methylmagnesium iodide prepared from 0.16 g. of mag­ nesium, 0.5 ml. of methyl iodide and 10 ml. of ether. The flask was shaken for five minutes and the precipitate col­ lected by eentrifugation. The precipitate was stirred with 10 ml. of water containing 1 g. of ammonium chloride. The insoluble material was collected on a filter; weight, 0.2 g., m.p. 175-180°. After two recrystallizations from methanol the melting point was 205-206°.

The following procedure was used, with minor variations, for the reaction of all the organometallie compounds with the methyl /3-anilino-a-phenylacetamidopropionate. Nine hundred and thirty-six milligrams (0.003 mole) of methyl ^-anilino-a-phenylacetamidopropionate was dis­ solved in 40 ml. of warm benzene. The solution was cooled to room temperature and 5-10 ml. of an ether solution con­ taining 0.006 mole of ethylmagnesium bromide was added with shaking. A precipitate was formpd and, after the mixture was shaken for five minutes, the solid was separated by eentrifugation. A solution containing 1 g. of ammonium chloride in 10 ml. of water was added to the precipitate and the mixture was stirred vigorously. The magnesium com­ pound decomposed and the ^-lactam of jS-anilino-a-phenylacetamidopropionic acid remained as a solid. It was removed by filtration and was crystallized from 35 ml. of methyl alcohol to give 145 mg. (17% yield) of |8-lactam melting at 217-218°. Cyclization of Methyl /3-Anilinoa-phenylacetamidopropionate

Calc. for CI6Hi4O2N2: C, 72.16; H, 5.30; N, 10.52. Found: C, 72.68; H, 5.36; N, 10.85. SYNTHESIS OF 1-PHENYL-3-PHENYLACETAMIDO-2-AZETIDINONB (M.56, 2). The anil of ethyl penaldate was hydrogenated under 2000 lb. pressure at 65-70° for one hour in the

presence of Raney nickel in order to saturatfe the imine grouping. The alcohol was removed in vacuo and the oily residue dissolved in ether and precipitated as the hydro­ chloride by the addition of hydrogen chloride. A sample of the product, ethyl a-phenylaeetamido-/3-anilinopropionate hydrochloride (M.5S, 8) was dissolved in alcohol and an equal volume of ether was added to give crystals, m.p.

993

Reagent

Molar ratio Yield of rea­ { % ) gent to amino ester

Methylmagnesium iodide.

2

35

132-133°.

Ethylmagnesium bromide

1

6

Calc. for C19H22N2O3JICl: C, 62.90; H, 6.39; N, 7.72. Found: C, 62.75; H, 7.29; N, 7.29, 7.70, 8.15.

Ethylmagnesium bromide

2

17

Ethylmagnesium bromide

3

14

Ethylmagnesium iodide. .

2

21

Phenyllithium"

2

9

Terl.- Butylmagnesium, io­

2

0

dide. 7' O C

0.40

σ

ο

0.30

CL

O

0.20 O.IO

2800

2200

3Q00

3200

3400

Wave Length, A Figure 4.

Ultraviolet absorption spectra of the triethylammonium salts of benzylpenicillin: ·, natural; Q, synthetic.

aureus H as the test organism.

The value found was 1,340 U/mg. The calculated value for the triethylammonium salt of benzylpenicillin (based on a value of 1,667 U/mg. for the sodium salt) is 1,363 U/mg. The ratio of the activity found on B. subtilis to that found on Staph, aureus was 0.96 (standard value, 1.00). ULTRAVIOLET ABSORPTION SPECTRUM. The ultraviolet absorption spectra of 0.01% solutions of the triethyl­ ammonium salts of the natural and synthetic benzylpeni­ cillin in 1 % phosphate buffer at pH 6.0 are shown in Figure 4. At this concentration both compounds exhibited only strong end absorption with no characteristic absorption peak. INFRARED ABSORPTION SPECTRUM. The absorption spec­ tra of the triethylammonium salts of the natural and syn­ thetic benzylpenicillin in the infrared region from 770 to 3,750 cm.-1 are shown in Figure 5. The absorption spectra were determined on samples of the crystalline compounds (about 0.5 mg.) mulled in mineral oil between two sodium chloride plates.® Since no attempt was made to obtain • We are indebted to Dr. R. Bowling Barnes and Dr. Robert C. Gore of the Stamford Research Laboratories, American Cyanamid Company, for the preparation of the mineral oil mulls on the sodium chloride plates and for helpful suggestions in the infrared work. The infrared absorption spectra of the triethylammonium salts of both the natural

exactly the same thickness of layers between the salt plates for the two samples, attention must be focused on the loca­ tion of the transmission minima rather than on the relative per cent transmission at any one wave number. The infra­ red absorption spectra shown in Figure 5 were determined in this Laboratory on a Perkin-Elmer Recording Infrared Spectrometer with gain control to compensate for the energy distribution of the Globar source. It should be pointed out that the transmission minima at 1,384, 1,460 and 2,910 (doublet) cm.-1 are due to absorption by the mineral oil.

SUMMARY A detailed description of the isolation of syn­ thetic benzylpenicillin from the condensation of D-penicillamine hydrochloride and 2-benzyl-4-methoxymethylene-5(4)-oxazolone has been presented. and synthetic benzylpenicillin were also determined in the above Labora­ tories as well as in our own on a Perkin-Elmer instrument without the use of a gain control. The curves obtained on the two samples were identical within the limits of the determination.

1024

SYNTHETIC BENZYLPENICILLIN

Figure 5.

Infrared absorption spectra of the triethylammonium salts of synthetic and natural benzylpenicillin.

A comparison has been made between the triethylammonium salts of natural and synthetic benzylpenicillin as to melting point, specific rotation, antibiotic activity, ultraviolet and infrared absorption spectra. It has been found that these properties are identical within the limits of experimental error. From studies on the role of hydrogen chloride in the synthesis of benzylpenicillin, a modified procedure has been devised for the synthesis. This procedure involves the separation of the reaction into two steps. In the first step the D-penicillamine hydrochloride and 2-benzyl-4-methoxy-methylene-5(4)-oxazolone were condensed in

pyridine containing triethylamine to form an intermediate. In the second step, this intermediate product was converted to benzylpenicillin in small yield by the action of hydrogen chloride in hot pyridine. Acknowledgment. The authors wish to express their appreciation to Miss Rachel Jewett and Miss Helen E. Heath' for performing the penicillin assays, and to Miss Josephine E. Tietzman for technical assistance. They would also like to express their appreciation to Dr. Mary Elizabeth Wright for aid in preparation of the manuscript and for many helpful suggestions throughout the course of the investigation.

ASSAY OF PENICILLINS JOHN V. SCUDI 1 AND Η. B. WOODRUFF 2

CONTENTS Physical and Chemical Methods 1025 Biochemical Methods 1025 Titrimetric Methods 1025 Spectrophotometric Method 1027 Polarographic Methods 1028 Colorimetric Methods 1029 Differentiation of the Penicillins. . . . 1031 Microbiological Methods 1031 Requirements of a Penicillin Assay. . . 1031 Designations of Activity 1032 Standards 1032 Serial Dilution Assays 1035 Turbidimetric Assays 1036 Diffusion Assay 1037 Metabolic Activity Assays1 1040 Differential Assays 1041 PHYSICAL AND CHEMICAL METHODS3 This review is concerned with physical and chemi­ cal methods for the assay of penicillin. Only brief abstracts with working directions are given for those procedures already published, but methods taken from unpublished progress reports are presented in detail. The methods are considered, arbitrarily, in the following order: biochemical, titrimetric, spectrographic, polarographic, and colorimetric methods of assay. Finally, the differ­ entiation of the various penicillins is considered. Biochemical Methods of Assay. A. Goth and Μ. T. Bush (J. Ind,. Eng. Chem., Anal. Ed., 16, 451 (1944)) reported a rapid method for the estimation of penicillin which is based upon the ability of penicillin to inhibit the production of nitrite from nitrate by actively growing Staphylococcus aureus cultures. The decreased nitrite formation, as com­ pared to a control, is determined colorimetrically following diazotization and coupling. The test can be completed within 60 to 90 minutes, and it possesses greater accuracy than that of the standard dilution method. The principle upon which the method is based can be used for the estimation of other anti­ biotics which inhibit the growth of Staphylococcus aureus. W orking details of the method areas follows: 1 Merck & Co., Inc. Present address: Pyridium Corporation, Yonkers 2, Ν. Y. ι Merck & Company, Inc. 1 By John V. Scudi.

The test strain of Staphylococcus aureus is grown for 24 hours on a medium containing 2% Difco, and 0.6% sodium chloride. The 24 hour culture is diluted 1 to 4 with an ice-cold medium containing 2% Difco, 0.6% sodium chloride, 0.02% sodium nitrate and 0.05% p-aminobenzoic acid. The suspension is left on ice for 10 to 15 minutes while a standard solution of penicillin and the unknown are diluted with 0.05 M sodium phosphate .to concentra­ tions of 0.5 to 1.5 units per cc. One cc. of each solution is pipetted into 50 cc. Erlenmeyer flasks in duplicate, and 1 cc. of buffer (prepared by mixing equal volumes of 0.05 M mono- and dibasic sodium phosphate) is pipetted into each of 3 control flasks. Five cc. of the ice-cold Staphylococcus suspension are added to each flask and the mixture is incubated at 37° for 60 to 90 minutes. The concentration of the nitrite is then determined as follows: 0.5 cc. is pipetted into 6 cc. of water which contains 1 cc. of 15% trichloroacetic acid. After 3 minutes, 0.5 cc. of a 0.1% solution of N-(l-naphthyl)ethylenediamine dihydrochloride is added. After 3 minutes the color intensity is measured and com­ pared with a standard curve to obtain the nitrite concentration. Knowing the nitrite content, com­ parison with the solutions containing 0.5, 1.0, and 1.5 units of penicillin gives the penicillin content of the unknown. Turner et al. (Turner, J. C., Heath, F. K., and Magasanik, B., Nature, 152, 326 (1943)) observed that urease was inhibited by the crude penicillin preparations available to them at the time when they performed their experiments. They sug­ gested the in vitro inhibition of the enzyme as a basis for the assay of penicillin, but subsequent investi­ gation (Scudi, J. V., and Jelinek, V. C., Science, 100, 312 (1944)) demonstrated that the inhibition is not produced by pure penicillin. Consequently the inhibition of urease is not a measure of penicillin content. Methods involving the use of penicillinase are considered in subsequent sections. Titrimetric Methods of Assay. The early obser­ vation of Abraham and Chain (Abraham, E. P., and Chain, E., Brit. J. Exp. Path., 23, 103 (1942)) that alkaline inactivation of penicillin produces a new acid group, affords a basis for the titrimetric determination of penicillin. Two such methods

1026

ASSAY OF PENICILLINS

were developed in the Pfizer research laboratories (P.30). The methods were not offered to replace the Oxford cup method, but because of their facility and the short time required to perform an analysis, they were believed to be useful for ccintrol purposes. PROCEDTJBB A: 10 cc. of a penicillin solution con­ taining from 60,000 to 150,000 units are pipetted into a 50 cc. beaker equipped with a motor driven stirrer and with glass and calomel electrodes con­ nected to a pH meter. Using 0.1 N sodium hydrox­ ide, sufficient solution is added dropwise to bring the ρH to 8.0. Exactly 10.00 cc. of the standard alkali is added to bring the pH to approximately 12. The solution is allowed to stir at room temperature for 10 minutes and is then titrated back to pH 8.0 with standard 0.1 N hydrochloric acid, noting the volume of acid consumed. The number of penicil­ lin units = (cc. N NaOH—cc. N HCl) X 594,000. The factor 594,000 is based on a molecular weight of 356 for sodium benzylpenicillinate and an estab­ lished potency of 1667 units per milligram. PEOCEDXJRK B: 10 cc. of a penicillin solution con­ taining from 60,000 to 150,000 units are pipetted into a 50 cc. beaker equipped with glass and calomel electrodes as described under Procedure A. The pH is adjusted to 8.0 with 0.1 N sodium hydrox­ ide and 3 cc. of 3% hydrogen peroxide solution (previously adjusted to pH 8.0) are added for each 100,000 units of penicillin. The pH of the solution drops very quickly. The pH is maintained at 8.0 by the dropwise addition of 0.1 N sodium hydroxide. When the pH remains constant at 8.0 or the drop is less than 0.1 unit in 40-60 seconds, the amount of sodium hydroxide used subsequent to the addition of the hydrogen peroxide is recorded. The number of penicillin units = cc. N NaOH X 594,000. The above procedures were carried out with many of the commercial penicillins available on the market as well as on several types of solutions which occur in the course of penicillin production, and satisfactory results were obtained. The results were more reproducible than those obtained by means of the Oxford cup method. With pure crystalline sodium benzylpenicillinate the assay varied from 100% to 102%. The method did not give satis­ factory results in the analysis of highly buffered solutions, low potency samples, or fermentation broths. The application of these procedures to liquors low in penicillin content is under investiga­ tion. Attention is called to the fact that if the bioassay values of the material being tested differ substantially from that of sodium benzylpenicil­ linate (as in the case of those containing sodium p-hydroxybenzylpenicillinate) an appropriate cor­ rection is necessary since the calculation of the factor 594,000 is based solely on the values for sodium benzylpenicillinate. J. W. Foster (Science, 101, 205 (1945)) observed that when penicillin is inactivated by penicillinase a new acid group is formed. Although the reaction

was studied manometrically by means of the carbon dioxide liberated from a carbonate buffer, the reac­ tion was not adapted to the assay of penicillin. Later, J. J. Murtaugh and G. B. Levy (J. Am. Chem. Soc., 67, 1042 (1945)) standardized the reaction for the alkalimetric titration of penicillin. Their method consists of adjusting separately, to pH 8, a 10 cc. aliquot of penicillin (5,000 to 20,000 units) and a 1 to 2 cc. solution containing 1000 to 2000 units of penicillinase. The enzyme is added to the penicillin and the mixture is maintained at pH 6.8 by the gradual addition of 0.02 N sodium hydroxide. After a few minutes the penicillin is inactivated and the pH becomes constant. The titration is then completed by a rapid adjustment to pH 8.0. The method is applicable to solutions of potency over 200-500 units per cc., which do not contain an excessive amount of buffer. An iodometric method for the assay of penicillin preparations was developed at the Squibb Institute for Medical Research (S.51) (Alicino, Ind. and Eng. Chem., Anal. Ed., 18, 619 (1946)). The assay method, which is based on earlier observations (Lilly, L.19) of the behavior of penicillin and certain of its inactivation products toward iodine, involves the differential titration of the penicillin prepara­ tion before and after inactivation by alkali. Analyses are performed as follows: A weighed sample (3-5 mg.) of crystalline sodium benzylpenicillinate, placed in a glass-stoppered flask, is dissolved in 5-10 cc. of water, and treated with 0.5 cc. of 1 N NaOH. The alkaline solution is allowed to stand for 15 minutes and then neutral­ ized with 0.5 cc. of 1 N HCI. A measured excess of 0.01 N iodine solution (about 10 cc.) is added. After 30 minutes the unconsumed iodine is titrated with 0.01 N thiosulfate. It was found that one mg. of sodium benzylpenicillinate will consume under these conditions 2.52 cc. of 0.01 N iodine solution, corresponding to 8.97 atoms of iodine per mole, which is ill good agreement with the range reported from the Lilly Laboratories for penicilloates (8.58.9). This value is reproducible and within fairly wide limits is independent of the penicillin con­ centrations and the excess of iodine used. Sodium 2-pentenylpenicillinate (crystalline, anhydrous) con­ sumed 2.64 cc. of 0.01 N iodine solution per milli­ gram, or 8.8 atoms per mole, showing that the double bond in this compound is inert to iodine under these conditions. This was confirmed by the blank titration (without prior alkali treatment) which showed an uptake of only 0.07 cc. per milli­ gram. However, it is probable that sodium p-hydroxybenzylpenicillinate might give abnor­ mally high values in this procedure. In the assay of unknowns an amount of material corresponding to approximately 1,000-5,000 units is inactivated with alkali, and the neutralized solution is treated with iodine and titrated after 30 minutes as described. A blank titration in

ASSAY OF PENICILLINS which the treatment with alkali is omitted is carried out on a separate sample of the same magnitude. The difference between both titra­ tions gives the amount of penicillin present in terms of mg. of sodium benzylpenicillinate, which figure is then converted to international units by multiplying by 1667. When relatively pure prepa­ rations (800-1,000 U./mg.) were assayed in this manner, the figures agreed well with the bioassay values regardless of the time allowed for contact of the blank sample with excess iodine, the small amounts of iodine-consuming impurities present in such preparations having little influence on the final result. However, with preparations of lower potency containing proportionately greater amounts of iodine-consuming impurities, the iodometric results were considerably lower than the bioassay figures. It was found empirically that the dis­ crepancy between the iodometric and bioassay values was minimized when the blank value arrived at by immediate back-titration was used in com­ puting the results. In the final procedure adopted for the assay of high- as well as low-grade prepara­ tions, the blank sample is therefore back-titrated immediately after the addition of the iodine solu­ tion, whereas with the alkali-inactivated sample the 30-minute interval is maintained. With the use of this procedure good correlation between the iodometric and bioassay values was obtained on a variety of carefully bioassayed solid sodium salt preparations of widely varying potency. Comparison of the values obtained by the iodo­ metric, the alkalimetric (Pfizer) and the two stand­ ard bioassay methods was conducted in the Squibb Biological Laboratories on solutions of commercial sodium salt preparations ranging in potency from 900 to 1,150 U./mg. The data showed that the agreement is in most cases satisfactory enough for practical purposes. It remains to be seen whether the iodometric method can be used other than for the quick assay of high-grade final products. An iodometric method for the assay of penicillin has been devised in the laboratories of the Food and Drug Administration in Washington, by Mundell, Fischbach, and Eble ( Science, 104, 84 (1946); J. Am. Pharm. Assoc., 35, 373 (1946)). Five cc. aliquots of a solution containing 2.5 to 3.5 mg. of sodium benzylpenicillinate are placed in each of two iodine-containing flasks. To one, an equal quantity of 1 N sodium hydroxide is added, and the reaction mixture is permitted to stand at room temperature for 15 min. Five cc. of 1.1 N hydrochloric acid and 15 cc. of 0.01 N iodine are added and after 15 min. the excess of iodine is titrated with 0.01 N thiosulfate. As the end point is approached an occasional drop of 1% starch solution is added. About 5 cc. ©f carbon tetra­ chloride is added and the titration is completed with frequent shaking until all of the iodine is removed from the carbon tetrachloride. To the second

1027

flask, 15 cc. of the 0.01 N iodine solution is added and the titration is completed immediately. The difference between titers divided by 2.52 equals the number of mg. of penicillin in the original aliquot. Spectrophotometric Method of Assay. A spectrophotometric method for the estimation of the penicillins was described by R. M. Herriott ( J . Biol. Chem., 164, 725 (1946)). The method con­ sists of measurements of the absorption at 322 ηΐμ before and after heating for 15 minutes at 100° in 0.3 M acetate buffer pH 4.6, and comparison of the change in absorption with a similarly treated standard penicillin preparation. Two related pro­ cedures were used. The first one, A, when ample quantities of more concentrated solutions were available. The second, B, when the concentration or quantity was low. A. A 2 cc. aliquot of a penicillin solution contain­ ing 30-300 Oxford units per cc. was added to 6 cc. of 0.4 M acetate, pH 4.6. 4 cc. of this solution was pipetted into a 30-50 cc. test tube placed in boiling water for 15 minutes, after which the solution was cooled rapidly to room temperature. During the heating a small funnel in the test tube served as a reflux condenser. The absorption of the heated and unheated aliquots was determined at 322 πΐμ in a Beckman quartz photoelectric spectrophotom­ eter (Gary, H. H., and Beckman, A. O., J. Opt. Soc. Am., 81, 682 (1941)). The change in absorp­ tion was converted to units of penicillin with the aid of a standard curve. B. To 4 cc. of a penicillin solution containing 8-80 Oxford units per cc. was added 0.25 cc. of a 5 M acetate buffer solution, ρH 4.6. The final concentration and pH were the same as in Ai This solution was read in the spectrophotometer at 322 ηΐμ before and after being heated as described in A. Depending on the number of samples to be analyzed at one time the source of heat or volume of water should be such that when the test tubes are introduced the water temperature drops no more than a few degrees. Solutions of penicillin in 0.3 M acetate pll 4.6 do not change absorption values for at least a half hour and once they are heated and cooled there is no appreciable change for several hours. The increase in absorption caused by heating varies almost linearly with increasing penicillin concentrations. If it is assumed that the above treatment produces a complete conversion of penicillin into the absorbing material, then the extinction, Eicm.l% for this substance is about 240. Crystalline ammonium salts of 2-pentenylpencillinic, p-hydroxybenzylpenicillinic and n-heptylpenicillinic acids had nearly the same absorption spectrum as crystalline sodium benzylpenicillinate. This was true after heating as well as before. Thus spectrophotometric analyses are independent of the nature or proportion of these various penicillins. The results of two spectrophotometric analyses

1028

ASSAY OF PENICILLINS

of five different commercial preparations carried out on two different days, using procedure A were com­ pared with bioassays and Scudi colorimetric analyses of the samples. The spectrophotometric results were in substantial agreement with those obtained by the two accepted methods. The data also show that the spectrophotometric results are readily reproducible. A precision of +5% has been obtained with solutions containing 10 to 100 Oxford units per cc. No impurity was found whose absorption at 322 ηιμ is changed on heating under the conditions of the method, but a great many substances either absorb or scatter light of 322 πΐμ and when present in sufficient concentration prevent a precise estima­ tion of the penicillin. Blood, urine and certain microbiological suspensions contain such inter­ fering substances in high concentrations. It may be possible to separate quantitatively the penicillin from these substances and thus render the spectro­ photometric method applicable to such systems. Polarographic Methods of Assay. Polarographic studies of penicillin and related compounds have been conducted in a number of different laboratories (Cornell Bioch., D.6, 13; Merck, M.S; Cook, Hall, Heilbron, and Roberts, CPS.203; Hems, Page, and Robinson, CPS.225; 290; 884). The use of the polarograph for the assay of penicillin was sug­ gested by Cook et al. (CPS.203). These workers found that if the penicillin preparations were inactivated with alkali (30 mins. at pH 11) and the solutions then kept for 60 mins. at 90-95° with JV/10 acid ("penilloic acid"—penicillamine deg­ radation) there was a very marked —SH effect, and the height of the —SH wave was a measure of the activity employed. By taking a known solu­ tion as standard, the biological activity of others could be predicted by means of purely physical measurement. Clearly this was a measure of the derived thiol grouping and could not take account of different penicillins which, it is known, have somewhat different potencies against any one selected organism. However, in view of the fact that the known penicillins are not of widely dif­ ferent molecular weights and mainly occur as mixtures of which only the resultant potency is measured biologically, the present method may, it was suggested, provide a useful subsidiary method, and, from the chemical viewpoint, may have advantages. Hems et al. (Hems, Page, and Robinson, CPS.225) of the Glaxo Laboratories also studied the polaro­ graphic method of assay. They reported that many degradation products of penicillin contain free sulfhydryl groups which yield catalytic steps when examined in an ammoniacal buffer solution containing divalent cobalt. They believed that this step might be used for the estimation of the penicillins and their derivatives. Brdicka (Coll. Czech. Chem. Comm., 5, 112, 148, 328 (1933)) had

found that cysteine and cystine in an ammoniacal buffer solution containing divalent cobalt or nickel (but not trivalent cobalt) give a single catalytic step. This catalytic step occurs at a more nega­ tive potential than corresponds to the reduction of cystine and must, therefore, be caused by the reduced form, cysteine. The step formed by cystine is identical with that obtained with a cysteine solution of double the concentration. The catalytic nature of the step is indicated by the fact that the step obtained with cystine is 500 times as great as that for the reduction of cystine to cysteine. The Cornell group (D.6, 2) reported that penicillamine in ammoniacal buffer solution con­ taining cobaltous chloride showed a polarographic wave like that produced by cysteine. Hems et al. found that this process yields an excellent catalytic step which is about 500 times as high as the anodic step. They suggested that this step should be invaluable for the determination of small quantities of penicillamine, since quantities of the order of 1 X IO-6 molar can be detected. Calibration curves were prepared for cysteine, penicillamine, several thiazolidines and penicillin, under the same polarographic conditions. It was found (Hems, Page, and Robinson, CPS.290) that when the con­ centrations of these substances were expressed in molar quantities (assuming that the potency of the pure penicillin is 1,650 Oxford units per mg.), the calibration curves could, within experimental error, be superposed on each other. This estab­ lished the fact that alkaline inactivation followed by acid hydrolysis of penicillin and simple hy­ drolysis of the thiazolidines liberates the sulfur from these substances quantitatively as free sulf­ hydryl groups. It was originally suggested that the method might be of value for assaying routine commercial samples of penicillin (containing about 200 Oxford units of penicillin per mg.), but it was found later (CPS.S84) that the correlation between the height of the step and the biological activity was not consistently satisfactory for these grades of penicillin. Nevertheless, an accuracy better than 10% was obtained when solutions of purified penicil­ lin were used. The method is more rapid than either the biological or Scudi's method, the standard procedure requiring only about 2.5 hours. The procedure was described as follows: A Cambridge recording polarograph was used, a micro cell (capacity 3 cc.) being used to hold the po­ larograph solutions. The instrument was calibrated to read directly in microamp. by the procedure rec­ ommended by Kolthoff and Lingane (Polarography, New York, 1941). The capillary used had the following characteristics: At a pressure of 29.0 cm. of mercury, the droptime (t) on open circuit in 0.1 N potassium chloride was 2.10 sec., weight of mercury dropping per second (m) = 2.52 mg. and m^tw = 2.09. The symbols are those adopted by Kolthoff and Lingane.

ASSAY OF PENICILLINS The following modified procedure for the routine examination of clinical samples of penicillin was recommended: One cc. of a solution containing between 30 and 150 Oxford units per cc. of penicillin is mixed in a pyrex test-tube with 1 cc. of 0.1 JV caustic soda and 2 cc. of water. The mixture is allowed to stand at room temperature for 30 minutes and then treated with 1 cc. of N hydrochloric acid followed by 5 cc. of water. The mixture is heated in a boil­ ing water bath for 1 hour, cooled, and then added to a freshly prepared mixture of 1 cc. of N am­ monium chloride, 1 cc. of 0.02 M cobalt chloride and 2 cc. of N ammonia in a 15-cc. graduated flask. The solution is made up to 15 cc. with 1 cc. of 0.1% gelatin solution. Three cc. of the solution are transferred to the polarograph cell and examined at a sensitivity of 3^oo over the voltage range of —1.2 to —2.0 volts. The examination is repeated at a second sensitivity depending on the height of the catalytic step observed during the first measure­ ment. When 1 cc. of a solution containing 120 Oxford units of penicillin was treated by the above procedure, a diffusion current of about 11 microamp. was obtained. Colorimetric Methods of Assay. Abraham et al. (Abraham, E. P., Chain, E., Baker, W., and Robin­ son, R., Nature, 151, 107 (1943)) noted that acid hydrolysates of penicillin gave a blue-violet colora­ tion with the ninhydrin reagent, and that the color intensity of the reaction ran parallel to the anti­ bacterial activity of their preparations. More recently, Allinson (Allinson, M. J. C., Proc. Soc. Exp. Biol. Med., 60, 293 (1945)) in a preliminary pa­ per reported on the use of the ninhydrin reagent in the analysis of solutions containing 3 to 9 7 of pure penicillin. In the course of study of the inactivation of benzylpenicillin by hydroxylamines, the Merck Laboratories reported (M.38) that following the hydroxylamine, /3-methylhydroxylamine and βbenzylhydroxylamine reactions with sodium benzylpenicillinate, a characteristic red color is produced on addition of ferric chloride. Methylhydroxamic acid gives an identical color reaction with ferric chloride. This color reaction might serve as the basis of an assay of penicillin. Schematically, the hydroxylamine reaction would be: RCONH-CH—CH-S-C(CH3)2 I I I + N H 2 O H- • CO-N CH-COOH RCONH-CH—CH-S-C(CH3)2

1029

hydrochloride, and 2 cc. of N potassium hydroxide are added. The solutions are mixed and allowed to stand at room temperature for three minutes. One cc. of 2 N hydrochloric acid is added. The flask is placed in an ice bath and cooled. Then one cc. of 2% ferric chloride solution is added and the contents of the flask diluted to the mark with ice cold distilled water. The contents are mixed and the depth of color is immediately determined in a " Cenco" photelometer containing a Wratten H filter (450 ταμ). (The color examined in a Coleman spectrophotometer was found to exhibit maximum absorption at 440-470 ηιμ.) This method is subject to considerable improve­ ment. 1. The color produced is somewhat unstable. It has been found, however, that fading is less rapid in a cold solution than at room temperature. It would appear that the instability of the color is not a function of the penicillin molecule, since the color formed under similar conditions with butyrolactone is also unstable. 2. The sensitivity of the test as described is not as great as may be desired. A difference in penicillin concentration of 150 units results in only 5-6 points difference in photelometer reading. Since the instrument cannot be read closer than +0.5 points, an error of +15 penicillin units is possible. Preliminary tests using free benzylpenicillinic acid in ether solution indicate that a greater sensitivity might thus be attainable. In addition, a considerable increase in sensitivity should be possible by a decrease of the total volume of the solution. Although the specificity of the reagent described above has not been determined completely, sodium benzylpenicilloate (natural and synthetic) and sodium benzylpenillate (natural) give no color. Methyl and ethyl benzylpenicilloate (synthetic) and the methyl and ethyl alcohol inactivation prod­ ucts of benzylpenicillin do give the color. A colorimetric method for the determination of the penicillins, which is based upon the following reaction, was reported (Scudi, J. V. J. Biol. Chem., 164, 183 (1946)). RiNH-CH2CH2NH2 +

I RCONH-CH—CH-S-C(CH3)2 CO—N

CH-COOH

RCONH-CH—CH-S-C(CH3)2 RiNHCH2CH2NHCO

NH-CH-COOH

II

HCNH-CO

NH-CH-COOH

This method was developed in the Lilly Research Laboratories (L.12, 11) and reported as follows: A solution of sodium benzylpenicillinate is placed in a 10 cc. volumetric flask. One cc. of molar borate buffer, pH 9.0, 1 cc. of N hydroxylamine

During the development of the test, methodologic variations were introduced from time to time. The procedure ultimately adopted involves the follow­ ing considerations. The reagent I (N-(l-naphthyl4-azobenzene)ethylenediamine) is a strong base, and the primary amine is the stronger of the two

1030

ASSAY OF PENICILLINS

basic groups. In the presence of acetic acid, which accelerates the reaction, the reagent condenses with the free acid of the penicillins in a water-immiscible solvent. Extraction of the organic solution with sodium hydroxide removes the condensation prod­ uct as the sodium salt, leaving the excess reagent in the organic phase. The weakly basic condensa­ tion product II is then extracted from the acidic solution. Any of the strongly basic reagent I that may have passed into alkaline solution remains in the acidic, aqueous phase. The penicillin con­ tent is then determined colorimetrically in terms of the highly tinctorial dye incorporated into the condensation product II. The reader is referred to the original article for details of the method. The method, which is applicable to solutions containing 10 to 120 micrograms of penicillin, possesses certain specificity requirements. For a substance to be measured as a penicillin, it must be water-soluble at pH 7.0; it must be extractable by chloroform from aqueous solutions at pH 2.0; it must condense with the reagent within a restricted period of time, the product must possess a carboxyl group; and finally, the condensation product must not possess strongly basic properties. That some potential interfering substances do not meet this final requirement is evidenced by the fact that variable amounts of colored material are left in the acid phase in the course of analysis of commercial samples of penicillin. In this connection, it may also be noted that a variety of degradation products of penicillin have been tested with no significant evidence of interference. Despite these specificity requirements, inter­ ferences by other types of substances may be anticipated; for example, ether-soluble keto acids may react with the colored amine to give alkalisoluble products of the Schiff-base or aldehydeammonia type, and lactones, azlactones and esters may react to give amidic compounds. Neverthe­ less, when applied to a large number of commercial samples of penicillin, the method gave results which were satisfactory in comparison with microbiological data (correlation coefficient = 0.964). Further, comparison of the stability characteristics of pure sodium benzylpenicillinate and of the active princi­ ple of commercial preparations also indicated that penicillin was measured specifically. When applied to low potency broths, however, interfering sub­ stances may be detected. If significant amounts are present (which is seldom the case), these can be eliminated readily. The marked lability of the penicillins affords a ready means of differentiating the active principle from inactive interfering substances. A given sam­ ple may be analyzed as usual to give a "total" concentration. An aliquot, exposed to the action of some inactivating agent, may be analyzed to give a "non-penicillin" value. The "total" minus the "non-penicillin " value would be expected to give a

close approximation of the true penicillin concentra­ tion. Such a differential procedure was described: This colorimetric method was studied in the laboratories of the Food and Drug Administration (F.2, 4, 5, 6, 7) and a modification aimed at the elimination of the first step of the assay was evolved (F.7). The procedure worked out is as follows: About 8 mg. of penicillin were dissolved in 2.5 cc. of water and acetone added to make 50 cc. Aliquots of this standard were pipetted into 25 cc. glassstoppered graduated cylinders containing 10 cc. of an ether solution of the dye (10 mg. dye). (For the aliquots smaller than 1.0 cc. an amount of 95% acetone was added to make the total volume of each sample 1.0 cc.) Then 0.1 cc. of glacial acetic acid was added, the solution made to 20 cc. with ether, the mixture thoroughly shaken and allowed to stand for three hours. A 10 cc. aliquot was then pipetted into a separator containing 25 cc. of 0.1 N NaOH and 20 cc. of petroleum ether. The mixture was shaken for one minute; and after the layers had separated, about 80% of the top layer was aspirated off. Three successive washings with 25 cc. each of petroleum ether were then made, shaking each for one minute and aspirating off about 80% each time. Finally a washing with 25 cc. of carbon tetrachloride was made, shaking only about 10 seconds. The CCl4 was drawn off and the assay completed by adding 1 cc. of HCl followed by butanol, etc., as in the original method. A rapid micro-method for the fluorometric determination of penicillin has been reported (Scudi, J. V., and Jelinek, Y. C., J. Biol. Chem., 164, 195 (1946)). The original colorimetric method, which was used extensively in the analysis of com­ mercial penicillin preparations and broths, is more precise and appears to be more specific than existing biological methods. It is, however, limited in sensitivity. In order to extend the range of appli­ cability of the method by increasing its sensitivity, a strongly fluorescent amino acridine (2-methoxy6-chloro-9-(N1-ethylenediamine)-acridine) was synthesized, and the amino-acridine was used in place of the highly tinctorial azo dye originally used. Quantitative measurements of small amounts of pen­ icillin were thus achieved. The amino-acridine con­ denses with penicillin at a more rapid rate than the azo dye. Consequently, it was possible to reduce the condensation time to one hour. Working details of the method are available in the article cited. The method, as presented, is directly applicable to solutions containing 0.0625 to 0.625 micrograms of penicillin per cc. For more dilute solutions, larger volumes may be concentrated as described in the original report. The method is directly ap­ plicable to the analysis of urine or protein-free filtrates of whole blood or plasma. All of the specificity requirements incorporated in the colorimetric method for the determination of penicillin are inherent in the fluorescence method.

ASSAY OF PENICILLINS The method is precise to ± 10 %. It is a compara­ tively simple method which is carried out almost entirely in a separatory funnel, and it is a relatively rapid method which may be used by a trained operator to complete 8 analyses in about 2 hours. Differentiation of the Penicillins. Chemical pro­ cedures, like those described above, which are based upon a key functional group common to all penicillins, yield molar values for all varieties of penicillin without differentiation between the vari­ eties. Clearly, such a differentiation is desirable. Recently H. Fischbach, M. Mundell, and T. E. Eble {Science, 104, 84 (1946)) reported on the successful separation and quantitative determination of so­ dium n-heptylpenicillinate in the presence of the other penicillins. Their procedure, which employs a partition chromatography technique, was reported as follows: Twenty-five grams of silica gel prepared accord­ ing to Gordon et al. (Gordon, A. H., Martin, A. J. P., and Synge, R. L. M., Biochem. J., 87, 79 (1943)) were thoroughly moistened with 12.5 to 16.5 cc. (depending upon the absorbability of the gel) of 20% potassium phosphate, pH 6.4. Washed chloroform was added to the mixture, and the slurry was poured into a glass cylinder of 22 mm. inside diameter, using slight pressure to facilitate the uniform settling of the silica gel. The penicil­ lins (25 to 50 mg.) were extracted at 0° from pH 2.0 buffer into chloroform, and the extracts were passed through the column at 10°. Cold chloro­ form was used to furnish 100 to 200 cc. of eluate. The rate of elution varied between 10 and 35 min­ utes per 25 cc. without causing discernible dif­ ferences. Of the n-heptylpenicillinic acid, 70-75% was removed in the first 25 cc. of eluate and the remainder was removed within the first 3 or 4 fractions. Three to four additional 25 cc. frac­ tions exhibited no activity. From this point on, anesthesia ether saturated with water was used as the eluent until all remaining activity was recovered. A gravimetric method for the determination of sodium benzylpenicillinate in the presence of other penicillins was reported by Sheehan et al. (Sheehan, J. C., Mader, W. J., and Cram, D. J., J. Am. Chem. Soc., 68, 2407 (1946)). The method takes advan­ tage of the sparing solubility of the N-ethylpiperidine salt of benzylpenicillinic acid in amyl acetateacetone mixtures. In actual operation samples of the mixed penicillins are acidified, the acids are extracted with amyl acetate, and, after drying, the N-ethylpiperidine benzylpenicillinate is selectively precipitated. The recovery appears to be quanti­ tative when sodium benzylpenicillinate comprises over 50% of the sample. MICROBIOLOGICAL METHODS4 The method of assay for penicillin most suited to the clinician is the determination in vivo of the * By Η. B. Woodruff.

1031

efficacy of penicillin for the treatment of a specific disease agent. The chemist requires an exact evaluation of the molar concentration of penicillin in solution. Between these extremes, attempts have been made to develop satisfactory methods of assay for penicillin. Biological methods are limited to the effect of penicillin on growth of a microorganism or to its effect on some measurable metabolic activity of a microorganism. During the preliminary studies on penicillin, biological methods of assay were the method of necessity, since no others were available. At the present time, biological methods are the methods of choice, since the modifications in antibacterial activity brought about by mixtures of the various types of penicillinates cannot be foretold by chemical procedures. Requirements of a Penicillin Assay. The re­ quirements of a penicillin assay may vary according to the particular interest and need for accuracy of the individual worker. The main objective is to determine, as accurately as required, the antibac­ terial activity of a particular sample of penicillin. When a high degree of accuracy is not necessary, many factors conducive to precision may be ignored. On the other hand, detection of relatively small differences in potencies is often required and pre­ cautions must be taken to insure this order of sensitivity. The results obtained on any given sample of peni­ cillin must be reproducible in the same and different laboratories. A common language for expressing the activity of penicillin preparations should be used. Preferably, samples should not require pretreatment to make them suitable for assay. Pretreatment becomes a great handicap when applied to a routine procedure. Any method of more than limited value should be amenable to the running of large numbers of tests in routine and yield results in a reasonably short period of time. Entirely fallacious results may result from the assay for penicillin of any substance unless it is shown that penicillin is the only antibiotic present. In specific instances, it may be possible to design assay methods to be specific for penicillin in the assay of a mixture of two known antibiotics. For example, the action of notatin may be eliminated by the choice of an assay medium lacking sugar, with an assay organism of sufficient sensitivity to allow dilution of the sample to insignificant sugar concentration. With the above precautions, peni­ cillin may be assayed in a mixture of penicillin and notatin because the latter requires certain sugars for expression of its antibacterial activity. Except in assays especially designed to eliminate the action of contaminating antibiotics, the penicillin assay methods are applicable to preparations containing penicillin alone. For the determination of the molar concentration of penicillin in a given preparation, it is necessary

1032

ASSAY OF PENICILLINS

that the penicillin be a single type, sodium n-heptylpenicillinate, or sodium benzylpenicillinate, etc., for which the potency of the pure preparation is known. Organisms often respond in a different manner to the various penicillins. Thus, a stand­ ard test organism must be selected. When mix­ tures of penicillins ar.e presented for assay, the microbiological assay indicates the biological activ­ ity of the preparation, but yields no exact figure for the actual quantity by weight of penicillin present. Attempts to determine the latter quantity, will be discussed below under the heading "differential assays." No competitive phenomenon analogous to paminobenzoic acid reversal of sulfonamide bacteriostasis is known for penicillin. Thus, the growth inhibition of bacteria in a medium is a function of penicillin concentration. Introduction of small quantities of impurities contained in the penicillin samples is of little significance. This cannot be interpreted to allow inclusion of high concentra­ tions of solvents or amino acids with the sample for assay. Solvents act synergistically with penicillin in concentrations not bacteriostatic for the test organism and certain amino acids act likewise with specific test organisms. Designations of Activityi Numerous terms have been employed to express penicillin activity. These include the inhibitory dilution, the number of micrograms, and arbitrary units. The dilution unit, or the greatest dilution at which inhibition of a given test organism is obtained, is the traditional method of expression. Rigid standardi­ zation of medium, test organism, and physical factors are required. Standardization on this basis has been found impractical where highly accurate results are required. Utilization of a microgram basis of terminology requires a pure reference standard. This was not available at the time of development of most assay methods. Therefore, arbitrary units were devised for the interim period until a pure reference stand­ ard became available. As mentioned previously, the different antibacterial action of the various penicillinates makes the microgram terminology impractical even at present. Standards. Because of the difficulty in standard­ izing all variables of an assay, considerable discrep­ ancies are obtained in day to day determinations. A standard, of similar composition to the unknown sample, serves to minimize daily fluctuations. Changes in response of the test organism to penicil­ lin are compensated for in calculations of results by the similar change which takes place with the penicillin standard. The concept of a penicillin unit to define the antibacterial potency of the antibiotic substance has been proposed (Chain, Fletcher, Gardner, Jen­ nings, and Florey, Lancet, 241, 177 (1941)). In honor of the research team pioneering in the work,

the term Oxford unit was accepted as the designa­ tion for the unit until it became possible to define an International unit. A standard of partially puri­ fied solution of purely arbitrary strength was established initially as an internal reference stand­ ard for the use by the Oxford team; this unit was defined as "that amount of penicillin which when dissolved in 1 cc. of water gives the same inhibition as this standard." The standard produced an inhibitory zone of 24 mm. by the cup assay pro­ cedure (see below) and was maintained in phosphate buffer solution, saturated with ether, in the ice-chest. Later a quantity of dried solid was prepared at Oxford and adopted by them as a reference stand­ ard, the material being found by them to contain 4.4 units per mg. (Therapeutic Research Corporation, Pen.Cl., 1). Samples of this material were dis­ tributed by the Oxford workers to manufacturers and investigators to assist in establishing a uniform basis of comparison. Secondary standards were established by comparison with the Oxford material. Difficulties soon arose from the necessity of using an unstable material for a standard. Additional problems were presented by the periodic need of establishing new substandards. To meet these problems, an assay panel was organized as a part of the Therapeutic Research Corporation in January of 1943 (Pen.Cl., 1). Problems presented to the panel included indications of non-homogeneity of the Oxford standard (Pen.C2S, 1) and evidence of difference in assay values arising from comparisons of Merck penicillin against the Oxford standard with several microorganisms (Pen.C 18, 1). To eliminate difficulties thought to be due to the exist­ ence of more than one type of penicillin in material being assayed, the panel decided to use a standard strain of organism for its determinations. The panel also assigned values, in terms of the Oxford standard, to two reference standards, samples of which later proved of assistance in defining the International unit of penicillin. (Therapeutic Re­ search Corporation, Pen.121, 6; League of Nations Bulletin 12, No. 2, 194). The T.R.C. suggested large units, as an aid to clinicians, to remain in force on a temporary basis until official standards were established at an International Conference. The new units were the kilo-unit = Ku = 1,000 Oxford units and the mega-unit = Mu = 1,000,000 Oxford units (Pen.C IS, 1). In the fall of 1943 the U. S. Food and Drug Administration was called upon to start a regular program of penicillin assays. The extensive varia­ tion obtained between manufacturer's assay and the F.D.A. assay dictated an extensive cooperative assay for the establishment of master and secondary standards. (A. C. Huntet and W. A. Randall, J. Assoc. Official Agr. Chem., 22, 430 (1944)). Chemically pure sodium salt of benzylpenicillin

ASSAY OF PENICILLINS

served as the master standard. Comparison bydifferent manufacturers of the master standard with standards derived from the Oxford prepara­ tion yielded an average value of 1,600 Oxford units per mg. of master standard. In January 1944, the standard was accepted as official for assays made in the F.D.A. laboratory. Later, to facilitate con­ version of potency designation to a weight basis, the master standard was assigned the value of 1,650 Oxford units per mg. As an alternative expression, ampules could be labeled to contain the antibiotic activity of 60 mg. of pure penicillin sodium, in place of 100,000 Oxford units. A working standard of impure calcium penicillin, distributed to those having a legitimate need for it, and of a potency established by cooperative assay, was maintained by the F.D.A. This preparation contained 370 Oxford units per mg. and was similar in purity to the bulk of commercial penicillin produced at that time (early 1944). At a meeting held at the Royal Society, London, from October 16-19, 1944, an International Con­ ference on Penicillin met for establishment of standardized procedures for the assay of penicillin (Science, 101, 42 (1945)). The following draft of resolution was adopted: (1) That, notwithstanding the existence of more than one penicillin, it is desirable and possible to select and adopt an International Penicillin Standard consisting of a specimen of the pure crystalline sodium salt of Peni­ cillin II or G; and an International Penicillin Working Standard, the specific activity of which has been deter­ mined in relation to that of the International Standard. (2) That the use of these Standard Preparations, for the time being, would meet the needs of practical standard­ ization and render quantitative results obtained in different countries sufficiently comparable. (3) That the offer of the representatives of the United States of America to prepare the material for the International Standard, from contributions generously supplied for the purpose by manufacturers in the United States and the United Kingdom be gratefully accepted, and that the individual contributions be brought into one solution and finally crystallized as one uniform preparation. (4) That, with a view to the eventual replacement of this International Standard by a preparation of identical properties, the physical and chemical constants of the preparation now being adopted shall be accurately determined. (5) That approximately 8 grams of the International Standard shall be prepared and a quantity regarded as adequate to satisfy international requirements shall be deposited with the Department of Biological Standards, the National Institute for Medical Research, Hampstead, London, N. W. 3, on behalf of the Health Organization of the League of Nations. (6) That, on receipt at Hampstead, the International Standard shall be dispensed in suitable quantities into separate containers and after complete desiccation shall be sealed in these containers in pure dry nitrogen gas, by the method and technique hitherto adopted for other International Biological Standards and shall thereafter be maintained in cold storage pending supply to national control centers. (7) That the International Penicillin Working Standard for general distribution shall, for the present, consist of a

(8)

(9)

(10)

(11)

1033

calcium salt of penicillin and that the offer of the Food and Drug Administration of the United States of America to supply such a preparation be gratefully accepted. As in the case of the International Stand­ ard, the International Penicillin Working Standard shall be deposited with the Department of Biological Standards, the National Institute for Medical Re­ search, Hampstead, London, N. W. 3, on behalf of the Health Organization of the League of Nations. It shall be dispensed in suitable quantities, in the manner described above, and stored and distributed under the same conditions as other International Biological Standards. That the International Unit of Penicillin be defined as "the specific penicillin activity contained in 0.6 micro­ gram of the International Penicillin Standard." The International Unit so defined is approximately equivalent to the unit originally adopted by Heatley and other collaborators of Florey (1941) and com­ monly known as the "Oxford" unit. That 2.7 micrograms of the present International Penicillin Working Standard (see paragraph 7 above) be accepted as containing 1 International Unit of Penicillin. That for the determination, by a suitable method of comparative assay, of the specific activity of an unknown preparation of penicillin in terms of the International Standard it is necessary to use a suita­ ble strain of Staphylococcus aureus, and that this strain must have practically equal sensitiveness to the inhibitory actions of Penicillin I, or F, and Penicillin II, or G. The two strains of Staphylococcus aureus which at present can be certified, by the experience of many workers, to fulfill these conditions are those known variously as: (a) No. 6571 of the National Collection of Type Cul­ tures (England). No. NRRL B-314 (Northern Regional Research Laboratory, Peoria, Illinois, U. S. A.). No. 9144 of the American Type Culture Collection (Washington, D. C., U. S. A.). and (b) No. 209-P, the Food and Drug Administration (Washington, D. C., U. S. A.). No. NRRL B-313 (Northern Regional Research Laboratory, Peoria, Illinois, U. S. A.). No. 6538 of the American Type Culture Collection, Washington, D. C., U. S. A.). Both of these cultures may be obtained by application to the National Collection of Type Cultures, The Lister Institute, London, or to the American Type Culture Collection, Georgetown University, Washington, D. C. That the Conference recognizes that it may eventually become necessary and practicable to establish further standards made from other varieties of Penicillin; and recommends that, with a view to such further develop­ ment, efforts should be made to make pure sample of other penicillins available for international exchange among research workers in this field.

Supplemental data concerning the preparation and properties of the International Penicillin Standard, which could not be published due to the prevailing secrecy regulations, were presented in a report from R. D. CoghiIl of NRRL (C.21, 1). Because of secrecy regulations, the report covering the preparation of the International Penicillin Standard was, of necessity, incomplete. This supplemental report describes the method used in the recrystallization of the Standard and supplies further data relative to its composition and properties.

ASSAY OF PENICILLINS

1034

A. Method of Crystallization. The crystallization of the pooled samples of sodium benzylpenicillin was carried out as follows: The 19.0 g. of sodium salt was placed in a 3 X 16 cm. test tube, cooled in an ice bath, and 9.50 ml. of water added. The mixture was worked with a stirring rod at room temperature until all the solid was in solution. Eleven ml. of η-butyl alcohol was then gradually added at room temperature with stirring. The resulting clear solution was filtered under nitrogen pressure through a cotton-asbestos pad, η-butyl alcohol being used for washing. To the clear filtrate was added more η-butyl alcohol, so that the total amount used in the crystallization was 238 ml. (25 times the volume of water used). This solution was allowed to stand at room temperature for two hours, during which time a heavy deposit of crystals (blades) appeared. The material was then treated as de­ scribed in the report previously submitted. B. Elementary Analysis. The crystals, after drying, were analyzed for carbon, hydrogen, nitrogen, sulfur, and sodium. The results, as well as those required by theory, are given below: Per Cent C H N S Na Calc. for CieHl7O4N2SNa: 53.92 4.81 7.86 9.00 6.45 Found: 54.15 4.94 7.90 9.22 6.50 54.14 4.95 7.83 9.01 6.46 8.80

C. Ultraviolet Absorption Spectrum. The ultraviolet absorption of the penicillin was measured in duplicate in 95% alcohol (in which the absorption shows no change for TABLE I Absorption Data of International Penicillin Standard in 95% Ethyl Alcohol (Concentration in g. per 100 ml. of Solution) Wave length (Ang­ stroms)

3450 3400 3350 3300 3250 3200 3150 3100 3050 3000 2950 2900 2875 2850 2825 2800 2775 2750 2725 2700 2690 2685 2680 2675 2670 2665 2660 2655

Absorp­ tion (Ang­ coeffi­ stroms) cient E1(!m.i *

.103 .135 .163 .183 .191 .188 .175 .163 .144 .128 .126 .155 .201 .228 .245 .265 .320 .396 .669 1.39 2.22 2.71 2.91 2.93 3.05 3.28 3.70 4.24

2650 2645 2640 2635 2630 2625 2620 2615 2610 2605 2600 2595 2590 2587 2585 2580 2575 2570 ?565 2560 2555 2550 2545 2540 2535 2530 2525 2520

Elom.1 %

(Ang­ stroms)

Eiom.1 %

4.63 4.78 4.57 4.30 4.14 4.26 4.52 4.87 5.21 5.55 6.00 6.45 6.87 7.12 7.11 7.04 6.84 6.56 6.39 6.41 6.55 6.79 7.17 7.54 7.95 8.36 8.67 8.86

2515 2510 2505 2500 2490 2480 2470 2460 2450 2440 2420 2400 2380 2360 2340 2320 2300 2280 2260 2240 2220 2200 2180 2160 2140 2120 2100

9.01 9.11 9.27 9.53 10.3 11.2 12.2 13.2 14.3 16.0 20.4 24.4 29.4 35.4 43.1 52.0 61.9 80.0 99.8 119 143 175 219 263 304 362 401

TABLE II Ring Diameters, Spacings, and Relative Intensities of Lines in X-Ray Powder Pattern of the International Penicillin Standard Ring diameter cm.

Spacing "d" in A

Intensity

.99 1.83 2.02 2.68 3.15 3.32 3.75 3.91 4.08 4.53 4.73 4.92 5.13 5.40 5.75 5.97 6.28 6.56 6.88 7.14 7.60 7.80 8.34 8.60 9.38

15.60 8.52 7.72 5.84 5.02 4.78 4.31 4.14 4.00 3.65 3.51 3.40 3.27 3.13 2.98 2.89 2.78 2.69 2.59 2.52 2.40 2.35 2.25 2.21 2.08

VS VW VW VS VS VS W S W M M M W S W M M VW M M W W VW VW W

several days). The measurements were made with a Beckman photoelectric spectrophotometer and the peaks and valleys are just fully resolved by using the narrowest nominal band width, which is 10 A. (0.39 mm. slit width) as defined by T. R. Hogness, et al. (J. Phys. Chern., 41, 379 (1937)). The absorption data are given in Table I below. One of these covers the range from 2,100 to 3,400 A., the other covering 2,500 to 2,825 A. in order better to show the peaked absorption. Two portions of the absorption curves of "pure" penicillin preparations vary with different sources of sample and method of purification. The first portion of the spectrum of the International PenicilUn Standard which does not represent pure penicillin arises from the material giving the peak at 3,240 A. This substance forms from penicillin in aqueous solution and represents, in this prepa­ ration, only 0.3% of impurity if the absorption coefficient found by Merck and Company and reported by Shell Development Company (Sh.S, 21) is used. The second portion is the shoulder at 2,850 A., which some of the components making up the original sample did not show at all. This absorption is at the peak position for peni­ cillin X (III), and assuming about half the absorption is due to this impurity, it would represent 0.25%. D. X-ray Powder Pattern. An X-ray powder pattern was taken of the penicillin. The conditions were: selfrectified tube, voltage 37 K.V.P., 15 ma. current, 5.0 cm. sample to film distance, exposure seven hours, pin-hole 0.013 inches, and Ni filtered Cu radiation. The sample was not ground but was packed with pressure into a hole, . . . some of the crystals [were] too large for a smooth powder picture. Table II gives the measured ring diameters, the calculated spacings, and estimated relative intensities. Other specimens of "pure" penicillin treated similarly yield prints showing variations in relative intensities, especially in the outer rings. E. Specific Rotation. The specific rotation of a 2% aqueous solution was found to be [24·8 = +301°.

ASSAY OF PENICILLINS Methods of Assay. The methods of assay may be grouped under several distinct headings. These are the serial dilution, turbidimetric, diffusion, and special methods based on metabolic activity of the test organism. A fifth type, the differential assay, makes use of one of the foregoing methods with several test organisms, for the differentiation of types of penicillin. An attempt will be made to abstract the first report of each method of assay, including the procedure, and to mention significant modifications. For full details the reference should be consulted. Serial Dilution Assays. A serial dilution assay for penicillin was proposed by Fleming (Brit. J. Exper. Path., 10, 226 (1929)) for the accurate estimation of penicillin. The inhibitory power can be accurately titrated by making serial dilutions of penicillin in fresh nutrient broth, and then implanting all the tubes with the same volume of a bacterial suspension and incubating them. The inhibition can then readily be seen by noting the opacity of the broth. For the estimation of the antibacterial power of a mold culture, it is unnecessary to filter as the mold grows only slowly at 37°C., and in 24 hours, when the results are read, no growth of the mold is perceptible. Staphylococcus is a very suitable microbe on which to test the broth as it is hardy, lives well in culture, grows rapidly, and is very sensitive to penicillin.

Later, in a more detailed description (Fleming, Lancet, 242, 732 (1942)), Fleming proposed a broth consisting of peptone (1 gm.), sodium chloride (0.5 gm.), glucose (1 gm.), Andrades' indicator (1 cc.) and water (q.s. to 100 cc.). A 3 mm. loop of a 24 hour broth culture of S. aureus was inoculated into 5 cc. of the medium and serial dilutions of penicillin added. After incubation for 24 hours, the endpoint was determined from the appearance of red color in those dilutions allowing bacterial growth. Clutterbuck, Lovell and Raistrick (Biochem. J., 26, 1907 (1932)) extended the serial dilution assay to include tests with the pneumococcus and H. influ­ enzae. Opacity readings established the endpoint. A standard strain of hemolytic streptococcus (C. 203 M.) was proposed by Hobby, Meyer and Chaffee (Proc. Soc. Exper. Biol. Med., 50, 277 (1942)). Rammelkamp's method (Proc. Soc. Exp. Biol. Med., 51, 95 (1942)) was designed to meet the clinical problem of measuring very low levels of penicillin in blood or other body fluid. Addition of erythrocytes improved the serial dilution method greatly by yielding a sharply defined endpoint, the point of hemolysis. The unknown samples of penicillin are stored at 5°C. until the time of testing. If the samples are known to be contaminated, sterilization is effected by passing them through a Seitz filter. To the first 2 tubes of a series of small culture tubes 0.2 cc. of the unknown sample is added. The tubes, with the exception of the first one in the series, contain 0.2 cc. of veal infusion broth. From the second tube, then, 0.2 cc. of the broth-penicillin sample is removed and serial dilutions

1035

are made. In addition, if the solution to be tested is known to contain a very small quantity of penicillin, a further tube containing 0.5 cc. of the unknown is added to the test. A control run with each determination is made up from a standard of penicillin which is stored at 5°C. in a solution of 0.85% sodium chloride in a concentration of 20 Florey units per cc. This standard is then treated in a manner similar to the unknown samples. The test organism is a Group A strain of hemolytic streptococcus obtained from the blood stream of a patient with erysipelas. The appropriate dilution of a 12-hour broth culture is made in veal infusion broth containing 1 % erythrocytes so that the final number of organisms varies between 1,000 and 10,000 per cc. The inoculum consists of 0.5 cc. of this dilution and is added to each tube as well as to the control series containing dilutions of a known amount of penicillin. The cultures are then incu­ bated for 18 hours, following which the tubes are examined for hemolysis. A 3 mm. loop of the cultures near the endpoint is streaked on blood agar plates as a check of sterility.

Further improvements or modifications include reduction of the assay time 3 to 3½ hours by use of actively multiplying cultures (Wilson, Nature, 152, 475 (1943); Rake and Jones, Proc. Soc. Exper. Biol. Med., 54-, 189 (1943)) and choice of reduction of hemoglobin by Staphylacoccus aureus NRRL 313 grown under anaerobic conditions as the indicator for bacterial growth (Personal Communication, J. D. Thayer, U. S. Public Health Service, Marine Hospital, Staten Island, Ν. Y.) Heilman and Herrell, (An. J. Clin. Path., 15, 7 (1945)) working in the laboratories of the Mayo Clinic, have found an adaptation of the Wright slide-cell technique to be the most satisfactory in their experience for the determination of the amounts of penicillin in the various body fluids. Fleming's modification of the technique, (Fleming, Lancet, 247, 620 (1944); Am. J. Clin. Path., 15, 1 (1945)), as used by the above authors, effects a considerable saving of time and equipment. The materials used are sterile whole human blood defibrinated by shaking with glass beads, a 24-hour broth culture of Streptococcus pyogenes, and penicillin of known strength. Various dilutions of standard penicillin and of serum . . . are mixed with the defibrinated blood, in which the leuko­ cytes have been killed, and are incubated on glass slides divided into sections by strips of paper dipped into hot petrolatum. After incubation, the dilution that completely prevents hemolysis due to S. pyogenes is determined and then multiplied by the concentration of the standard in the same test that completely prevents hemolysis. For ex­ ample, if a 1:4 dilution of the same test substance caused complete inhibition and the endpoint with the standard were 0.03 units per cc., the tested fluid contained 0.12 units of penicillin per cc.

A method of penicillin assay in which variation in repeated runs is insignificant, thus eliminating the need for a standard, has been claimed by Heilman (Am. J. Med. Sci., 207, 477 (1944)). While the method is essentially a serial dilution determination, a semisolid tissue culture serves as the medium. A standard strain of pneumococcus is the test organ­ ism. The method is not recommended for deter­ mining the amount of penicillin in blood or other

1036

ASSAY OF PENICILLINS

tissues of patients, but is strictly, a method for standardizing penicillin. Serial dilution in an agar medium was employed by Clutterbuck, Lovell, and Raistrick (Biochem. J., 26, 1907 (1932)) as a check against their broth assays. Waksman and Reilly (Ind. Eng. Chem. Anal. Ed., 17, 556 (1945)) presented the advantages and limitations of this method for the testing of antibiotics. The advantage of including a number of test organisms on each dilution plate should be emphasized. The characteristic spectrum obtained serves-to verify the identity of the antibiotic under test. Modification to a micro scale was reported by Jackson (J. Bact., 51, 407 (1946)). Turbidimetric Assays. Turbidimetric assays are a natural extension of the serial dilution assay. To obtain a sharp endpoint in turbidity, hemolysis, or any indicator employed in the serial dilution method, increments of 50-100% are usually required in the penicillin concentration. Intermediate levels yield stages of partial inhibition. In many media, when a measurement of degree of inhibition is made at intermediate penicillin levels, a logarithmic relation­ ship can be shown to exist between degree of growth and penicillin concentration. Under favor­ able conditions this type of assay is capable of high precision. Impurities affect the slope of the growth curve. Assays of broths and low-potency products are often questionable when determined by the turbidimetric method, but the method is the most exact biological procedure known for purified preparations. In the first report of a turbidimetric assay applied to penicillin, Foster (J. Biol. Chem., 144, 285 (1942)) described the method as follows:

levels are run on each unknown, depending on how many can be predicted to fall on the central (three-fourths) region of the curve. . . . In practice the log curve may be used as reference; only 3 or 4 points are required to define the log curve.

Pope independently developed a turbidimetric assay, (Therapeutic Research Corporation, Pen. 89(F), 2), which was accurate for purified prepara­ tions. A linear relationship did not exist between penicillin and a logarithmic function of the density of growth when samples containing destruction products of penicillin were assayed. A layer of paraffin over the surface of the culture medium minimized fluctuations in assay results (Therapeutic Research Corporation, Pen.50, 1). Foster and Woodruff ( J . B a c t . , 4 6 , 187 (1943)) showed that the turbidimetric assay could be modified to yield maximum growth of controls and normal standard curves within a 4 hour incubation period by increasing the inoculum of S. aureus, to overcome the log phase. A similar assay with B. adherans as the test organism and incubation under conditions of agitation to promote the growth rate was reported later (Foster and Wilker, J. Baet., 46, 377 (1943)). Standard curve: Five points in duplicate (0, 0.1, 0.2, 0.3 and 0.4 Florey units/10 cc.) define the curve although it is probable that three would suffice. Two hundred cc. of sterile nutrient broth are inoculated with 2.0 cc. of an overnight aerated broth culture of B. adherans (or iS. aureus) and then distributed in 10 cc. amounts into 50 cc. Erlenmeyer flasks. The curve tends to flatten out near the end if the inoculum contains too few or slow growing cells. The standard penicillin solution containing 1.0 Florey unit per cc. is stored in the refrigerator or, preferably, in dry ice. Quantities of 0, 0.1, 0.2, 0.3 and 0.4 cc. of the standard solution are added to the respective flasks in duplicate. Samples: Unknown samples are diluted to contain 1.0 Florey unit per cc. on the basis of the expected potency and 0.1, 0.2 and 0.3 cc. added in duplicate to flasks as above. All dilutions are made in advance and the penicillin added to the medium all at one time. Sterile glassware is not re­ quired; the flasks do not need to be plugged. The flasks are placed on a shaking machine at 37°C. for 3 to 5 hoars, after which growth is stopped immediately by cooling in ice water or by adding a drop of disinfectant solution (5% phenol or formalin). Turbidities are read in the Evelyn photoelectric colorimeter. The standard curve is obtained by plotting per cent transmissible light against penicillin concentration. Potencies of the unknowns are computed from the standard curve, the values obtained for the three levels being averaged. . . . While not furnishing as high an order of accuracy as desirable, this method has found valu­ able application in the production of penicillin.

In this work the Oxford strain of Staphylococcus aureus has been used. To tubes containing 5 ml. of various dilu­ tions of penicillin samples are added 5 ml. of sterile, double strength nutrient broth, inoculated just prior to apportion­ ment. For inoculum, 0.4 ml. of a 20-hour nutrient broth culture of the test organism is added to 100 ml. of the double strength broth. Dilutions are made as follows: The original samples of penicillin are diluted with ice-cold sterile 0.02 M phosphate buffer at ρH 7.2 so as to contain approximately 0.02 unit per ml. These, together with the standard, are kept in an ice bath until all the samples have been treated similarly. Different amounts, namely 0.5, 1.0, 2.0, 3.0, and 5.0 ml., are then added aseptically to tubes previously sterilized with 4.5, 4.0, 3.0, 2.0, and 0 ml. of buffer, respectively. Aseptic precautions should be observed throughout. In assaying dry preparations of penicillin, contaminants are not Joslyn (Science, 99, 21 (1944)) proposed reading a serious factor owing to the ultimate high dilution and short incubation. With penicillium filtrates or penicillin solutions light transmission in the same test tubes in which contaminations may, however, be serious. Such liquids, the culture is grown. Reduction of the assay time when possible, should be obtained aseptically and main­ in a turbidimetric test was accomplished by Lee et al. tained sterile. Otherwise they should be kept cold (0°-5°), or saturated with ether or chloroform to minimize con­ (J. Biol. Chem., 152, 485 (1944)) with a culture of tamination. These solvents are without effect on the test group B streptococcus used for inoculum in the organism under these conditions. active growth phase. Holmes and Lockwood After 16 hours (overnight) incubation at 37°, the tubes (Am. J. Med. Sci., 207, 267 (1944)) successfully are shaken, the contents poured into calibrated Evelyn tubes, and the turbidimetric readings obtained. Galva­ detected amounts of penicillin as low as 0.01 unit per nometer differences (per cent transmissible light) are ml. but were not successful in developing a satis­ factory turbidimetric assay for blood serum. plotted against penicillin concentration. Three to five

ASSAY OF PENICILLINS However, Kirby and Rantz (J. Bact. 4-8, 603 (1944)) proposed a modified method which permitted the determination of penicillin in body fluids. Laborsaving devices were introduced by McMahan (J. Biol. Chem., 153, 249 (1944)) in an assay that proved successful in the routine determination of large numbers of samples, yielding assays more precise than were obtained by diffusion methods. Thermostable factors in the broth were found to cause drift in assay values at the University of Wisconsin (OPRD Report No. 9, 13). The lowest value obtained was found to correspond most closely to the cup assay. The average of a large number of samples indicated that the turbidimetric values were 18% higher than cup assay values. By adding heated broth, in which the penicillin was destroyed, to the standard, valid assays could be obtained turbidimetrically. Improved results with an alternate method of measurement were claimed by Osgood (J. Clin. Invest. 23, 948 (1944)). A relation was found to exist between the growth rates of staphylococci and the penicillin concentration in the medium. Lysis, which often occurred after maximum growth with staphylococci, did not affect growth rate determina­ tions, whereas it introduced errors in the normal method of measurement. Enterococci have been employed in turbidimetric tests (Park, 1944, M.S. Thesis, University of Wisconsin). Streptococcus lactis 30-5 was par­ ticularly adaptable to turbidimetric procedures. Assay of broth activities often yielded anomalous results due to the sensitivity of the test organism to various chemicals added to submerged fermenta­ tions for antifoam properties. A concentration of 0.008 mg./cc. Iinoleic acid or 0.04 mg./cc. saponified corn oil completely prevented growth of the test organism. The systematic errors introduced by these substances different from penicillin are greater than the reduction in random errors of assay with the enterococci, as compared with S. aureus. Therefore, no advantage could be claimed for this method of assay. Diffusion Assay. The detailed reports of Abra­ ham et al. (Lancet, 241, 177 (1941)), which served as a basis for most studies on penicillin, contained the description of a diffusion method of assay. Fleming (Brit. J. Exper. Path., 10, 226 (1929)) had first proposed use of diffusion of penicillin as a rough means of measurement, by incorporating penicillin and agar in a gutter cut in an agar plate. Modifica­ tion of this procedure to a quantitative method was proposed by the Oxford workers. Ordinary nutrient agar plates are seeded with the test organism—Staphylococcus aureus has been used in this work—by allowing a broth culture of the organism to flow over the surface of the agar and draining off the excess broth. The plates are then dried for an hour in the incu­ bator at 37°C. in a special rack which supports the lid of the Petri dish half an inch above the lower part. When dry the seeded plates are removed from the incubator and

1037

can be kept in the refrigerator for one or two days. Cylin­ ders made from short lengths of glass tubing, the dimensions of which will be seen from the inset of Fig. 1, are placed on the agar. The lower edge of the cylinder is carefully ground level and has an internal bevel so that the thin edge tends to sink into the agar and make a water- and bacteria-tight seal. Vitreous porcelain cylinders of the same size and shape, coloured at the non-bevelled end to facilitate their orientation, have also proved satisfactory. The cylinders are filled with the fluid to be tested, and the plates, resting on a block of wood, are incubated for 12-16 hours at 37°C. (If placed directly on the warm incubator shelves moisture may condense on the lids of the Petri dishes and drop on the agar, thus obscuring the results.) By the end of incuba­ tion most of the fluid in the cylinders has disappeared and each cylinder is surrounded by a circular zone where no bacterial growth has occurred. The diameter of the zone depends on the concentration of the penicillin, the type of relation being shown in Fig. 1. [Here reference is made to a standard curve.] The possibilities and limitations of this method of assay have not yet been fully worked out, but the following points may be noted. 1. The diameter in mm. of the zone of inhibition (which we have called the "assay value") is only slightly smaller (1-2 mm.) when the cylinder is half filled then when it is fully filled. 2. The assay value is unaffected by the pH of the fluid being tested, provided it is not strongly buffered and lies within the range ρH 5-8.5. 3. No inhibition is provided by a saturated aqueous solu­ tion of ether or by water containing free droplets of chloroform. 4. Diffusion of penicillin seems to be practically complete in 2-3 hours, and assay values after 14 hours of incu­ bation are only very slightly smaller (0.5-1 mm.) than after a further 8 hours at 37°C. 5. Provided the plate is not jarred the fluid cannot escape from the cylinders, and even if the fluid is not sterile the contaminating bacteria are confined to the inside of the cylinder. 6. The assay value is not affected by the thickness of the agar provided it is between the limits of 3 to 5 mm. 7. The assay value varies slightly with different batches of plates and with the density of bacterial population at the beginning of incubation. For this reason, uneven seeding of the plates must be avoided. 8. Sometimes the clear zone of inhibition is surrounded by a halo of partial inhibition, which varies from a faint ghost to almost complete inhibition. So far no explana­ tion for, or means of controlling, this phenomenon has been discovered. 9. When the antibacterial activity of blood is to be assayed plasma or serum must be used, since red cells tend to form a layer immediately on top of the agar which seri­ ously impedes the diffusion of penicillin and leads to low values. High accuracy cannot be claimed for this method of assay, but if it is done in triplicate (preferably on three different plates), and if the unknown solution is diluted so as to give an assay of not more than 25 mm. (before the curve has flattened out), the error is probably not greater than ±25% and may be considerably less. We have no evidence that under suitable conditions this method is inferior in accuracy to the serial-dilution method and it is certainly many times quicker. In addition, less than 0.25 cc. of fluid is required for each test.

Full practical details were subsequently pub­ lished by Heatley (Biochem. J., 38, 61 (1944)), including quantitative information on limits of accuracy. The F.D.A. 209 strain of S. aureus was found to

1038

ASSAY OF PENICILLINS

form sharp boundaries in the cup assay. Larger circles were obtained with agar of pH 6.0-6.5 than of 7.0-8.0. (NRRL, CMR Report No. 2, 3.) In­ organic salts present in the sample were found to give increased zone size. Crystalline benzylpenicillin, dissolved in M/5 pH 7.0 phosphate buffer, showed an activity of 5,133 units per mg. when assayed against a crystalline standard in M/50 buffer with an assumed potency of 1,650 units per mg. (Merck, M.49, 17). Fleming (Lancet, 242, 732 (1942)) concluded that a porcelain cup to contain penicillin was unneces­ sary. Removal of agar from a plate with a cork borer provided a suitable receptacle to hold the antibiotic. While keeping the basic technique of the Oxford workers, Foster and Woodruff (J. Biol. Chem., 148, 723 (1943)) proposed a modifica­ tion to improve reproducibility of the assay. A spore suspension of a sensitive strain of B. subtilis was substituted for S. aureus. Spore suspensions provide a stable source of inoculum for periods of at least a year when maintained at temperatures below 4°C. Detailed procedures for the cup assay have been reported, including discussions of various influential factors. (Foster and Woodruff, J. Bact., 47, 43 (1944); Schmidt and Moyer, J. Bact., 47, 199 (1944); Univ. of Wisconsin, OPRD Report No. 4, 5). An increase in size of the agar container has been proposed to reduce variation between plates (Stanford University, OPRD Report No. 4, Ij Epstein, Foley, Perrine and Lee, J. Lab. Clin. Med., 29, 319 (1944)). Replacement of the cup as a container for penicillin has proven successful. Filter paper or blotting paper discs of a constant size liberate penicillin into the agar medium, (Vincent, J. G., and Vincent, H. W., Proc. Soc. Exper. Biol. Med., 55, 162 (1944); Sherwood, Falco and de Beer, Science, 99, 247 (1944)). However, the absolute volume of solution added to a disc affects the zone size markedly, while such variations have little effect in the conventional cup assays. Both the size of the zone and slope of the relation­ ship between zone and penicillin were found to vary with increase in diffusion times of penicillin (Univ. of Wisconsin, OPRD Report No. 4, 5). Statistical methods of evaluating the assay have been proposed by Bliss (Science, 100, 577 (1944)) and by Knudsen. (Science, 101, 46 (1945.)) Various types of cylinder guides for placing the assay cups have considerably aided the adaption of the method to a routine procedure (Stanford Univ., OPRD Report No. 4, 1! Oswald and Randall, Science, 101, 99 (1945); Reeves and Schmidt, J. Bact., 49> 395 (1945)). Elimination of cups in favor of Fleming's agar hole method has been proposed (Cholden, J. Bact., ^7, 402 (1944); Therapeutic Research Corporation, Pen.139, 1). For the control of penicillin produced in the United States, the Food and Drug Administration

of the United States Department of Agriculture has proposed an official method of assay, based on the cup procedure, with acceptable modifications. The method has undergone revision at )4, yearly intervals. Portions of the revision of July 1945 (Tests and Methods of Assays to Determine Compliance with Standards for Penicillin Products. Revised July 1945. Food and Drug Administra­ tion, U.S.D.A.) with modifications effective Septem­ ber 8, 1945, (Section 141.1, part 141, Chapter 1, title 21, Federal Register, September 8, 1945),are reported below: I. Sodium Penicillin and Calcium Penicillin A. POTENCY

1 Cylinders (Cups): Make from standard wall pyrex tubing or from glazed porcelain, stainless steel, or aluminum tubing of the same wall thickness (±0.1 mm.), having an outside diameter of 8 mm. (±0.1 mm.), by cutting into 1.0 cm. lengths. Unless made from stainless steel, the cylinders are beveled inside on one end at an angle of 30° to 40°. The beveled surface is ground to a smooth edge. 2. Culture Media: (i) Make nutrient agar, for the assay and for carrying the test organism, as follows: Peptone Pancreatic Digest of Casein YeastExtract Beef Extract Glucose Agar Distilled H2O q.s. PH 6.5-6.6 after sterilization

6.0 gms. 4.0 gms. 3.0 gms. 1.5 gms. l.o gms. 15.0 gms. 1,000 ml.

(ii) Make nutrient broth, for preparing an inoculum of the test organism, as follows: Peptone Yeast Extract Beef Extract NaCl Glucose Dipotassium phosphate Potassium dihydrogen phosphate.. Distilled H2O q.s pH 7.0 after sterilization

5.00 gms. 1.50 gms. 1.50 gms. 3.50 gms. 1.00 gms. 3.68 gms. 1.32 gms. 1,000 ml.

(iii) Media ingredients: (a) The yeast extract used in (i) and (ii) is a peptone-like substance which represents the soluble products of yeast cells, (S. cerevisiae) autolyzed under optimum conditions, clarified and desiccated to a powder. One gram of the extract represents not less than 7.5 gm. of the original yeast. It is a reddish-yellow to brown powder with a character­ istic but not putrescent odor. It is soluble in water, forming a yellowish to brown solution having a slight acid reaction. Its nitrogen content, after drying to constant weight at 100°C., as determined by the Kjeldahl method, is not less than 7.2% and not more than 9.5%. Its residue on ignition, as determined by weighing accu­ rately about 0.5 gm. and heating slowly until it is thoroughly charred, cooling, adding 1.0 ml. of sulfuric acid, and igniting to constant weight, ISvIiot more than 15%. Its loss at IOO0C., as determined by weighing accurately about 1.0 gm. and drying to constant weight at IOO0C., is not more than 5%. It contains no coagulable protein, as determined by the absence of precipitate when a filtered aqueous solution (1 in 20) is heated to boiling. Its chloride content, calculated as sodium chloride, is not more than 5%.

ASSAY OF PENICILLINS It contains no carbohydrate other than that naturally present. (b) The pancreatic digest of casein (bacteriological peptone) used in (i) and (ii) is a grayish-yellow powder, with a characteristic but not putrescent odor. It is freely soluble in water, a 2 % solution having a light yellow color, being free from turbidity or sediment, and having a reaction of pH 6.5 to 7.0; it is insoluble in alcohol or ether. The casein used in the preparation of the digest is good commercial grade or better of acid precipitated casein which meets the following specifications: Ash Moisture Fat Free acid (as lactic acid) Reducing sugars Fineness

not more than 2.5% not more than 8.0 % not more than 0.5% not more than 0.25% trace 100 % through a 20mesh sieve

The sodium chloride content of the pancreatic digest of casein is not more than 1 %. Its loss at IOO0C., as determined by weighing accurately about 2 gm. and drying to constant weight at IOO0C., is not more than 7 %. Its nitrogen content, after drying to constant weight at IOO0C., as determined by the Kjeldahl method, is not less than 10%. Its residue on ignition, as determined by weighing ac­ curately about 0.5 gm. (previously dried to constant weight at IOO0C.) and heating it slowly until it is thoroughly charred, cooling, adding 1.0 ml. of sulfuric acid, and igniting to constant weight, is not more than 15 %. If the peptone meets the above requirements it need not be dried to constant weight for the following tests: It meets the following tests for degree of digestion: Dis­ solve 1 gm. in 10 ml. of distilled water, (a) Stratify a few drops of 10 % acetic acid in 50 % alcohol on about 1 ml. of the solution. No ring or precipitate forms at the junction of the two fluids and, when shaken, no turbidity results, (b) Mix 1 ml. with 4 ml. of a saturated solution of zinc sulfate. A moderate amount of precipitated proteoses is formed. (c) To 1 ml. of filtrate from (b) add 3 ml. of distilled water and 4 drops of saturated bromine water; a distinct reaction for tryptophane is given. It is free from nitrites as determined by the following test: To about 5 ml. of a 2 % solution add (a) a few drops of sulfanilic acid reagent (sulfanilic acid, 0.8 gm; sulfuric acid of sp. gr. 1.84, 5 ml; distilled water, 100 ml.) and (b) a few drops of dimethyl-a-naphthylamine reagent (dimethyl-a-naphthylamine, 0.6 ml; glacial acetic acid, 30 ml; distilled water, 70 ml.). Mix and allow to stand for 15 minutes. No pink or red color develops. It meets the following tests for bacteria-nutrient properties: Prepare media by adding to distilled water—(a) 2 % of the bacteriological peptone, 0.5 % of sodium chloride; (b) 1 % of the bacteriological peptone, 0.5% of sodium chloride; (c) 0.1 % of the bacteriological peptone, 0.5 % of sodium chloride; (d) 1 % of the bacteriological peptone, 0.5 % sodium chloride, 0.5% dextrose; (e) 2% of the bacteriological peptone, 0.5% sodium chloride, 1.5% agar. Adjust the reaction of all media to ρH 7.2-7.4. To medium (a) add sufficient phenol red indicator to give a readable color, tube in Durham fermentation tubes, and autoclave. Inoculate with a loop of 24-hour culture of Escherichia colt. Neither acid nor gas is produced during incubation for 48 hours at 37°C. Inoculate 5 ml. of medium (b) with Eberthella lyphosa. Suspend between the cotton plug and the mouth of the test tube a strip or loop of lead acetate paper so that it hangs about 2 inches above the medium. After incubation at 37°C. for 24 hours the lower tip of the lead acetate paper shows little, if any, darkening; after 48 hours it shows an appreciable amount of brownish blackening (lead sulfide).

1039

Incubate 5 ml. of medium (c) inoculated with E. coli for 24 hours at 37°C.; add about 0.5 ml. of indol reagent (p-dimethylaminobenzaldehyde, 1 gm.; ethyl alcohol, 95 ml.; hydrochloric acid of sp. gr. 1.18, 20 ml.); a distinct pink or red color forms which is soluble in chloroform. Inoculate 5 ml. of medium (d) with Aerobacter aerogenes, and incubate for 24 hours at 37°C. Test by adding to the culture an equal volume of 10% solution of sodium or potassium hydroxide; shake and allow to stand at room temperature for several hours. The presence of acetylmethyl-carbinol is shown by the appearance of a pink color. In lieu of preparing the medium from the individual ingredients specified in paragraphs (2) (i) and (ii) of this section, they may be made from a dehydrated mixture which, when reconstituted with distilled water, has the same composition as such media. 3. Working Standard: Keep the working standard (ob­ tained from the Food and Drug Administration) in tightly stoppered vials, which in turn are kept in larger stoppered tubes containing anhydrous calcium sulfate, constantly at freezing temperature. Weigh out carefully between 4 and 5 mg. of the working standard and dilute with sterile 1 % phosphate buffer (pH 6.0) to make a stock solution of any convenient concentration. Keep this solution at a tempera­ ture of about 10°C.; do not use it later than 3 days after it is made. From this stock solution make daily a working dilution containing 1.0 unit per ml. and another containing 0.25 unit per ml. 4. Preparation of Sample: Dissolve aseptically, in pyrogenfree sterile distilled water, the sample to be tested to make a stock solution containing 5,000 units (estimated) per ml. In the assay for potency, place 1.0 ml. of this solution in a 100 ml. volumetric flask and make up to volume by the addition of sterile distilled water. Transfer 1.0 ml. of this 50 unit (est.) per ml. solution to a flask containing 49 ml. of 1 % phosphate buffer (pH 6.0). Transfer 1.0 ml. of this 1.0 unit (est.) per ml. solution to 3 ml. of buffer to make a solution containing 0.25 unit (est.) per ml. Use these last two dilutions in the assay for potency. 5. Preparation of Plates: Add 21 ml. of agar to each Petri dish (20 X 100 mm.). After the agar has been distributed evenly in the plates and has hardened, store in the refriger­ ator until the following day. (They may be kept several days before use). The test organism is Straphylococcus aureus (F.D.A. 209-P). Maintain the test organism on agar slants and transfer to a fresh agar slant about once a week. Prepare an inoculum for the plates by transferring the cul­ ture from the agar slant into broth and incubate at 37°C. From 16 to 24 hours thereafter add 2.0 ml. of this broth culture to each 100 ml. of agar which has been melted and cooled to 48°C. Mix the culture and agar thoroughly and add 4 ml. to each of the plates containing the 21 ml. of the uninoculated agar. Tilt the plates back and forth to spread the inoculated agar evenly over the surface. Replace the glass covers of these inoculated plates with porcelain covers glazed on the outside. Place four cylinders on the agar surface (beveled end down) so that they are at approximately 90° intervals on a 2.9 cm. radius. In so placing the cylinders drop them from a height of 1A inch, using a mechanical guide or device. 6. Assay: Use four plates for each sample. Fill one cylinder on each plate with the 1.0 unit per ml. dilution, and one with the 0.25 unit per ml. dilution, of the working standard. Add the estimated dilutions of 1.0 unit per ml. and 0.25 unit per ml. of the sample under test to the remain­ ing 2 cylinders on each plate. Carefully place the plates in racks and incubate 16 to 18 hours at 37°C. After incubation measure the diameter of each circle of inhibition to the nearest 0.5 mm. using a colony counter with a mm. scale etched into the supporting glass over the light source. Other measuring devices of equal accuracy may be used. 7. Estimation of Potency and Error: For this purpose reference is made to the charts and nomograph published by Knudsen (Science, 101, 46 (1945); cf. Welch, Randall, and

ASSAY OF PENICILLINS

1040

Knudsen,/. Am.Pharm. Assoc., 86,102 (1946)). Tousethe chart for estimating potency, two values, namely, V and W, are required. For each plate calculate two values: ν = (UL + UH) — (SL + SH) and w = (UH + SH) — (UL + SL), where SH and SL are the diameters of the zones of inhibition in mm. of the 1.0 unit and 0.25 unit dilutions of the standard respect­ ively and UH and UL refer similarly to the corresponding dilu­ tions of the sample under test. The value V is the sum of the ν values for all plates and W is the sum of the w values for all plates. To estimate the potency locate the point on the chart corresponding to the values V and W, and the potency can be read from the radial lines on the chart. The error of the assay is estimated by using the nomo­ graph which requires five values, namely, the potency, V, W, Rt, and Rw. Rv (the range of the v's) is the highest value of ν minus, the lowest value of ν obtained from the individ­ ual plates. Similarly, Rw is the difference between the highest and lowest w values. 'After obtaining these five val­ ues, connect with a straight edge the points corresponding to V and W on the respective scales on the right side of the nom­ ograph. Mark with a pin or sharp pointed-pencil the inter­ section of the straight edge and the diagonal line of the nomograph. Move the straight edge so that it connects the value of Rw on its scale and the diagonal line at the point of the pin. The value for Q is thus determined by the scale value where the straight edge crosses the line labeled "Q." T is obtained by adding the squares of Q and Rv. On the left side of the chart connect the values of T and W with the straight edge and read the value of the ratio (error of assay/potehcy) where the straight-edge intersects the scale of values for the ratio. This value multiplied by the potency equals the percentage error of the assay. The error of the assay calculated here estimates only how closely one assayist can check himself on any given set of dilutions of unknown and standard. It does not include any errors of weighing or errors due to variations in materials or sub­ divisions of a lot of penicillin. Table III gives an example of a typical assay with the five values inserted. The chart for determining potency should not be used for determinations of potency lower than 50 % or higher than 150% of the standard. If the potency lies outside these limits, the assay should be repeated using a higher or lower dilution. The radical lines on the chart beyond these limits permit a rough estimation of potency from a low as 5 % to as high as 1,000% when low values of W are found. If the value of V or W falls outside the limits of the chart, divide

both V and W by the same proper number to bring them into the range of the chart and read the potency from the radical lines as before. If 11.4 Rw is greater than W, the slope of the assay does not differ significantly from zero and the assay is invalid. (The figure 11.4 was obtained by use of Student's "t" test for determining'the significance of a slope.) In certain laboratories it has been noted that with the 4 to 1 ratio, involving concentrations of 0.25 unit for the low dose, the zone of inhibition given by this dose may either be too small for accurate reading or have edges which are poorly defined. In order to permit the use of a higher con­ centration of penicillin for the low dose the third of the attached charts may be used in assays in which the ratio of doses is 2 to 1, i.e., the high dose (SH) is twice the low dose (SL). AS in the previous charts, if the potency lies outside the limits of 50 % to 150 % the assay should be repeated, using a lower or higher dilution. The potencies on this chart have been extended beyond these limits for rough estimation purposes when low values of W are found, namely, for 1, 2, and 3 plate assays. These extensions can also be used for four (or more) plate assays .if both V and W are divided by the same proper number to bring them into the range of the chart. 8. The potency of sodium penicillin and calcium penicillin is satisfactory when assayed by the above method if the immediate containers are represented to contain: 200,000 units or less and contain 85% or more of the number of units so represented. More than 200,000 units and contain 90 % or more of the units so represented.

Metabolic Activity Assays. Until the mode of action of penicillin is elucidated, assays cannot be made on the metabolic function directly affected by penicillin. Those methods based on microbial metabolism, which have been proposed, are merely alternative procedures.for measuring the growth of the test organism. More exact measurements may be made of small quantities of many metabolic products of microorganisms than of increase in cell numbers, therefore such assays may be completed with relatively short incubation periods. Goth and Bush (Ind. Eng. Chem., Anal, Ed., 16, 451 (1944)) investigated nitrite production from nitrate

TABLE III Penicillin Plate-assay Standard

Unknown

SL

SH

Plate No.

0.25 u./ml. mm.

1.0 u./ml. mm.

1 2 3 4

18.0 18.0 18.0 18.0

24.0 24.5 25.5 24.0

72.0

98.0

Sum Range

—

uL estimated 0.25 u./ml. mm.

uH estimated 1.0 u./ml. mm.

18.5 18.0 18.0

24.0 24.0 24.5 24.0

+0.5 -0.5 -1.0 0

11.5 12.5 14.0 12.0

72.5

96.5

—

—

-1.0 = V 1.5 = Rv

50.0 = W 2.5 = Rw

18.0

(UL

ν or + uH) - (SL + sH)

Potency = 97 % of standard Q = 0.1 T = 2.26

Error of Assay = + 3.93 % of standard

w or (SH

+

UH) - (SL

+

UL)

1041

ASSAY OF PENICILLINS by actively growing S. aureus cultures and found that this property would lend itself to the deter­ mination of penicillin. This method is discussed in detail in the first section of this Chapter. A method proposed by Hirsch (Turk Fiziki vi Tabii Ilimler Sosyetesi Yillik Bildirigleri ve Arsivi., 12, 38 (1943-1944)) is based on inhibition of oxygen utilization of actively multiplying S. aureus cul­ tures by penicillin. Manometric procedures are utilized for measurement and a relationship deter­ mined for the percentage of inhibition of O2 uptake compared with the quantity of penicillin added. Four to five hours are required for a single deter­ mination, with 0.01 to 0.05 Oxford units per ml. the smallest quantity that can be detected. The prime advantage of the procedure is the removal of inter­ ference by other products of P. notatum having bacteriostatic activity (notatin). A somewhat different method of measurement has been proposed (Levitov, M. M., Vyshepan, E. D., and Nenasheva, A. M., Biochimia., 10, 491 (1945)), in which a vessel containing the smallest amount of penicillin in which there is no increase in respiration with time is taken as the endpoint. Differential Assays. In January of 1944 indi­ cations of a biological difference in the penicillins isolated from P. notatum 832 and P. notatum 1,249 were reported by W. H. Schmidt (NRRL, CMR Report No. 17, 3). Credit was given to Dr. W. R. Boon, Imperial Chemical Industries, for first noting the existence of more than one compound having the antibiotic activity associated with penicillin. Evi­ dence had been obtained that the Merck penicillin, when assayed against the British standard, yielded divergent assay values depending on the micro­ organisms used in the assay. For one preparation, values of 146 were obtained with S. aureus, 217 with B. subtilis, and 185 with C. bulgaricus (Therapeutic Research Corporation, Pen.ClS, 1; 95, 5). Crystalline sodium benzylpenicillinate from P. notatum 832 showed 1,500 Oxford units with S. aureus NRRL 313 and 1,540 with B. subtilis NRRL 518. The purest material obtained from P. notatum 1,249, mostly of the sodium 2-pentenylpenicillinate type, showed 900 units per mg. with S. aureus and 612 with B. subtilis (NRRL, CMR Report No. 17, 3). As soon as pure crystalline sodium 2-pentenylpenicillinate became available, Schmidt and Ward of NRRL (CMR Report No. 18, 6) were able to propose the use of differential assays for the determination of the proportion of the two types of penicillin present in a mixture. Pure sodium 2-pentenyl- and benzylpenicillinates were equally active for S. aureus, within experi­ mental error, but sodium 2-pentenylpenicillinate was only 66% as effective as sodium benzylpenicil­ linate, for the inhibition of B. subtilis. The relationship of B. subtilis activity for mixtures was linear between the extremes of 100% and 66%. Therefore, the error in determining relative amounts

was three times the standard assay error. Mixtures of the pure penicillins, added to penicillin broth inactivated with acid, yielded true values. Crystalline sodium p-hydroxybenzylpenicillinate was found to contain 900 Oxford units per mg. when measured against a sodium benzylpenicillinate standard with S. aureus and 1,200 units with B. subtilis. It was noted that the presence of the p-hydroxy type in a mixture vitiated the proposed differential assay method of Stodola, Wachtel, Benedict, and Schmidt (NRRL, CMRReportNo. 21, 4). The ratio, B. subtilis assay/5, aureus assay, served as a measure of the response initiated by various penicillins. The ratio for sodium benzyl­ penicillinate is, by definition, 1.00. Marked varia­ tion in the ratio occurred in the determination of sodium p-hydroxybenzylpenicillinate at various times. Schmidt (NRRL, CMR Report No. 22, 3) reported this to be due in large measure to the phase of B. subtilis used for inoculum. Ratios, as determined with the rough phase, were 2.0, with the smooth phase 0.88-1.00, and for an ordinary stock culture about 1.3. No marked phase difference was noted in determinations of ratios for the 2-pentenylpenicillinate. Frequent transfer, high glucose concentration in the medium and a short incubation period (12 hrs. at 30°C.) maintained the smooth phase of B. subtilis, while a sugar content of less than 0.1% and extended incubation period (24 hrs. at 30-37°C.) favored the change to a rough phase. Based on the marked difference in response of S. aureus NRRL 313, B. subtilis 558 (smooth), and B. subtilis 558 (rough), a method was proposed ' TABLE IV P -Hy2-FendroxyBenzyl- tenylpenicillin penicillin benzylpenicillin

NRRL B-313 NRRL B-314 NRRI J B-558 (smooth phase) NRRL B-558 (rough phase)..

Activity vs. B. subtilis (smooth) Activity vs. S. aureus (313) Activity vs. B. subtilis (rough) Activity vs. S. aureus (313) * By definition.

1667* 1667* 1667* 1667*

units per mg 845 1490 935 1440 800 970 970 1200-1700

2-Pentenylpenicillin

p-Hydroxvbenzvlpenicillin

0.65

0.95

0.65

1.42-2.00

1042

ASSAY OF PENICILLINS

whereby the content of any of the above three penicillins in a sample may be approximated. (Schmidt, W. H., Ward, G. E., and Coghill, R. D., J. Bact., 49, 411 (1945)). With this salt as a standard [sodium benzylpenicillinate] the pure sodium salts of penicillins F and X [sodium 2-pentenylpenicillinate and sodium p-hydroxybenzylpenicillinate] have been assayed against Staphylococcus aureus NRRL B-313 (FDA strain 209P), S. aureus NRRL B-314 (Heatley strain), and Bacillus subtilis NRRL B-558 (both smooth and rough phases) by means of the cylinderplate assay method. Typical results are shown in Table IV. The data can be used to calculate the ratios of the activities of the various penicillins against the organisms in question. In view of these different ratios, when a given sample of penicillin is assayed against these three organisms, it is possible to draw tentative conclusions as to the proportions of the various penicillins present. Such conclusions involve the assumption that there are present no other substances possessing antibacterial activity. If the rough phase of B. subtilis is used, each day's inoculum must be standardized against a known pure penicillin X [sodium p-hydroxybenzylpenicillinate], since some variation in the sensitivity of this dissociated strain has been encountered, presumably because of variation in the degree of dissociation. In pre­ senting these data, no claim is made that this method affords a precise means of analysis; it is believed, however, that results thus obtained are, in most cases indicative of composition.

The presence of sodium n-heptylpenicillinate, sodium n-amylpenicillinate or sodium 3-pentenylpenicillinate in the mixture invalidates results obtained by the above method. The differential method may be extended to be specific for each new type of penicillin by choosing test organisms excep­ tionally sensitive or exceptionally resistant to the new penicillin. However, with each additional penicillin the error of the determination increases. With the large number of natural and biologically synthesized penicillins now available, any of which may be present in a given sample, biological meth­ ods may be expected to show only a very general indication of the types present. Thus, two methods are available for expressing the quantity of penicillin in a mixture. (1) By appropriate chemical isolation and analysis or very roughly by the differential microbiological assay procedure, the weight of each type of penicillin may be determined. (2) With a standard test bacterium, an expression of the antibacterial activ­ ity of the preparation may be measured. While most present methods propose a standard strain of S. aureus, modification to an in vivo evaluation against a recognized pathogen may be proposed for future consideration.

APPENDIX

This appendix contains information as to the origin, date of issue and date of receipt of the indi­ vidual progress reports which form the basis of the foregoing chapters. Whenever possible, the names of the investigators who contributed the technical data contained in each report are stated; in some partici­ pant groups, however, the scientific personnel constituted integrated teams, all of the members of which are to be regarded as contributing to every report issued by their group. BRITISH MINISTRY OF SUPPLY PENICILLIN PRODUCTION COMMITTEE

Report No.1

Date of report

Date received by O.S.R.D. (Ν. Y.)

PEN.69 79

15 Feb?1943 19 May 1943

5 Oct. 1946 5 Oct. 1946

81 85 86 87

9 Jun.1943 23 Jun. 1943 10 Jul. 1943

5 Oct. 1946 5 Oct. 1946 5 Oct. 1946 5 Oct. 1946

88

30 Jul. 1943

24 Jan. 1944

89 90 91

4 Aug. 1943 16 Aug. 1943 14 Aug. 1943

24 Jan. 1944 24 Jan. 1944 24 Jan. 1944

93 η

27 Aug. 1943 2 Sep. 1943

24 Jan. 1944 24 Jan. 1944

96 96 97

6 Sep. 1943 16 Sep. 1943

24 Jan. 1944 24 Jan. 1944 24 Jan. 1944

98 99

29 Sep. 1943

24 Jan. 1944 24 Jan. 1944

100

4 Oct. 1943

24 Jan. 1944

101 IOS

4 Oct. 1943 19 Oct. 1943

24 Jan. 1944 24 Jan. 1944

IOS

22 Oct. 1943

24 Jan. 1944

104 105

23 Oct. 1943 25 Oct. 1943

24 Jan. 1944 24 Jan. 1944

106

26 Oct. 1943

24 Jan. 1944

1 The

109

8 Nov. 1943

24 Jan. 1944

110

8 Nov. 1943

24 Jan. 1944

111

17 Nov. 1943

24 Jan. 1944

Group

Investigators

DuiBn1 W. M., and Smith, S. Abraham, E. P., Baker, W., Chain, E., and Robinson, R. DufEn, W. M., and Smith, S. Wellcome Crowfoot, D., and Low, B. Oxford Duffin, W. M., and Smith, S. Wellcome Abraham, E. P., Baker, W., Chain, E., and Rob­ Oxford inson, It. Abraham, E. P., Baker, W., Chain, E., and Rob­ Oxford inson, R. London Hospital Holiday, E. R. Duffin, W. M., and Smith, S. Wellcome Abraham, E. P., Baker, W., Chain, E., and Rob­ Oxford inson, R. Hems, B. A. Glaxo Abraham, E. P., Baker, W., Chain, E., and Rob­ Oxford inson, R. Pope, C. G., and Stevens, M. F. Wellcome Crowfoot, D., and Low, B. Oxford Abraham, E. P., Baker, W., Chain, E., and Rob­ Oxford inson, R. Oxford Crowfoot, D., and Low, B. I.C.S. Catch, J. R., Cook, A. H., Elvidge, J. A., Hall, R. H., and Heilbron, I. M. Oxford Abraham, E. P., Baker, W., Chain, E., Cornforth, J. W., Cornforth, R. H., and Robinson, R. Oxford Crowfoot, D., and Low, B. I.C.S. Bentley, R., Catch, J. R., Cook, A. H., Heilbron, I. M., Hall, R. H., and Elvidge, J. A. Oxford Abraham, E. P., Baker, W., Chain, E., and Rob­ inson, R. Oxford Crowfoot, D., and Low, B. I.C.S. Bentley, R., Catch, J. R., Cook, A. H., Elvidge, J. A., Hall, R. H., and Heilbron, I. M. I.C.S. Bentley, R., Catch, J. R., Cook, A. H., Elvidge, J. A., Hall, R. H., and Heilbron, I. M. Oxford Abraham, E. P., Baker, W., Chain, E., and Rob­ inson, R. I.C.S. Bentley, R., Catch, J. R., Cook, A. H., Elvidge, J. A., Hall, R. H., and Heilbron, I. M. Oxford Abraham, E. P., Baker, W., Chain, E., and Rob­ inson, R. Wellcome Oxford

reports in this series not here listed contained no information relating to the chemical constitution of penicillin.

1043

1044

APPENDIX

Report No.

Date of report

Date received by O.S.R.D. (N. Y.)

Group

Investigators

PEN.112 11S

18 Nov. 1943 25 Nov. 1943

24 Jan. 1944 16 Dec. 1946

Oxford I.C.S.

114

11 Dec. 1943

16 Dec. 1946

I.C.S.

115 116 117 118 119

11 22 2 21 30

1943 1943 1944 1944 1943

16 16 16 16 11

Dec. Dec. Dec. Dec. Nov.

1946 1946 1946 1946 1944

Glaxo Wellcome Oxford Glaxo Glaxo

Crowfoot, D., and Low, B. Bentley, R., Catch, J. R., Cook, A. H., Elvidge, J. A., Hall, R. H., and Heilbron, I. M. Bentley, R., Catch, J. R., Cook, A. H., Elvidge, J. A., Hall, R. H., and Heilbron, I. M. Smith, E. L. Trevan, J. W. Crowfoot, D.,'Low, B., and Schmidt, G. Smith, E. L., and Anderson, F. W. Hems, B. A., Holland, D. 0., and Mathews,

121 122 124 126 127 129 ISO 1SS 138 139 140 141 143 144

24 Feb. 1944 4 Mar. 1944 30 Mar. 1944 Apr. 1944 5 May 1944 31 May 1944 5 Jun. 1944 4 Jul. 1944 3 Oct. 1944 9 Oct. 1944 26 Oct. 1944 15 Nov. 1944 14 Dec. 1944 13 Dec. 1944

11 11 11 11 11 11 11 11 28 8 8 12 12 19

Nov. 1944 Nov. 1944 Nov. 1944 Nov. 1944 Nov. 1944 Nov. 1944 Nov. 1944 Nov. 1944 Dec. 1944 Jan. 1945 Jan. 1945 Mar. 1945 Mar. 1945 Mar. 1945

Wellcome B.D.H. Glaxo Glaxo B.D.H. Wellcome Glaxo B.D.H. B.D.H. Glaxo Glaxo Glaxo Glaxo Wellcome

Pope, C. G., and Stevens, M. F. Stack, M. V., Stewart, D. D., and Mead, Smith, E. L. Goddard, H. W., and Smith, E. L. Stack, M. V., Stewart, D. D., and Mead, Pope, C. G. Ungar, J., and Hunwicke, R. F. Stack, M. V., Stewart, D. D., and Mead, Mead, T. H., Stack, M. V., and Stewart, Campbell, A. H., and Folkes, B. F. Campbell, A. H. Campbell, A. H. Campbell, A. H. Pope, C. G., and Jones, T- S. G.

Dec. Dec. Jan. Jan. Dec.

MEDICAL

RESEARCH

T*. 1V1. AT U

T. H. T. H. T. H. D. D.

COUNCIL

COMMITTEE ON PENICILLIN SYNTHESIS

Date of report

Date received by O.S.R.D. (N. Y.)

Group

Investigators

20 Dec. 1943

5 Jun.1944

Oxford

2 3 4 5 6 7 8 • 9 10 11

8 Jan. 1944 12 Jan. 1944 21 Jan. 1944 26 Jan. 1944 27 Jan. 1944 27 Jan. 1944 1 Feb. 1944 1 Feb. 1944 4 Feb. 1944 7 Apr. 1944

5 5 5 5 5 5 5 5 5 5

12 19

8 Feb. 1944 16 Feb. 1944

5 Jun. 1944 5 Jun. 1944

Boots Wellcome

20

21 Feb. 1944

5 Jun. 1944

I.C.I.

21

23 Feb. 1944

5 Jun. 1944

Oxford

22

26 Feb. 1944

5 Jun. 1944

Oxford

28 24

29 Feb. 1944 1 Mar. 1944

5 Jun. 1944 5 Jun. 1944

, I.C.S. I.C.S.

25

14 Jan. 1944

5 Jun. 1944

Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Not designated Not designated Boon, W. R., Carrington, H. C. and Sexton, W. A. Not designated Not designated Not designated Not designated Not designated Not designated Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Peak, D. A. Copp, F. C., Duffin, W. M., Smith, S., and Wilkinson, S. Boon, W. R., Carrington, H. C., and Jones, W. G. M. Abraham, E. P., Chain, E., Duthie, E. S., Baker, W., and Robinson, R. Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Catch, J. R., Cook, A. H., and Heilbron, I. M. Cook, A. H., Elvidge, J. A., Hall, R. H., Heilbron, I. M., and Shaw, G. Carrington, H. C.

Report No. !

CPS.l

1

Jun. Jun. Jun. Jun. Jun. Jun. Jun. Jun. Jun. Jun.

1944 1944 1944 1944 1944 1944 1944 1944 • 1944 1944

Oxford Wellcome I.C.I I.C.S. Glaxo May and Baker Oxford Manchester B.D.H. Oxford

The numbera not here listed were assigned to American reports.

I.C.I.

APPENDIX Report No.

Date received Date of report by O.S.R.D. (Ν. Y.)

CPS.S6

14 Mar. 1944

5 Jun. 1944

Z7

14 Mar. 1944

5 Jun.1944

28

14 Mar. 1944

5 Jun. 1944

29

18 Mar. 1944

5 Jun. 1944

SO

20 Mar. 1944

5 Jun. 1944

81 82

20 Mar. 1944 20 Mar. 1944

5 Jun. 1944 5 Jun. 1944

88 34 86

22 Mar. 1944 25 Mar. 1944 27 Mar. 1944

5 Jun. 1944 5 Jun. 1944 5 Jun. 1944

86

28 Mar. 1944

5 Jun. 1944

87 88 89 φ U

30 Mar. 1944 30 Mar. 1944 30 Mar. 1944 3 Apr. 1944

5 Jun. 1944 5 Jun. 1944 5 Jun. 1944 5 Jun. 1944 5 Jun.1944

4%

3 Apr. 1944

5 Jun. 1944

43

3 Apr. 1944

15 Jun. 1944

44 46 46 47 48

6 Apr. 1944 5 Apr. 1944 4 Apr. 1944 4 Apr. 1944 7 Apr. 1944

15 Jun. 1944 15 Jun. 1944 15 Jun. 1944 15 Jun. 1944 15 Jun. 1944

49

12 Apr. 1944

15 Jun. 1944

50 51 52 58

17 Apr. 1944 18 Apr. 1944 17 Apr. 1944 19 Apr. 1944

15 Jun. 1944 15 Jun. 1944 15 Jun.1944 15 Jun. 1944

54 55 56

3 Apr. 1944 25 Apr. 1944 26 Apr. 1944

15 Jun. 1944 15 Jun. 1944 15 Jun. 1944

57

26 Apr. 1944

15 Jun. 1944

58

1 May 1944

15 Jun. 1944

59

2 May 1944

15 Jun. 1944

60 61 62 68

5 May 1944 4 May 1944 10 May 1944

15 Jun. 1944 15 Jun. 1944 15 Jun. 1944 15 Jun. 1944

64 66

10 May 1944 24 May 1944

15 Jun. 1944 15 Jun. 1944

66

24 May 1944

15 Jun. 1944

67

26 May 1944

15 Jun. 1944

Group

Oxford

1045

Investigators

Wilson, J. P., Jepson, J. B., Robinson, G. M.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. I.C.I. Boon, W. R., Carrington, H. C., and Freeman, G. G. I.C.I. Boon, W. R., Carrington, H. C., and Freeman, G. G. Oxford Abraham, E. P., Baker, W., Chain, E., and Robin­ son, R. Oxford Jepson, J. B.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. I.C.I. Boon, W. R., Calam, C. T., and Carrington, H. C. Oxford McOmie, J. F. W., Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Glaxo Gibbs, E. M., Hems, Β. A., and Robinson, F. A. Robinson, R. Oxford Oxford Robinson, G. M.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. I.C.S. Cook, A. H., Elvidge, J. A., Heilbron, I. M., and Shaw, G. Topham, A. Manchester Glaxo Dupre, D. J., Hems, Β. A., and Robinson, F. A. May and Baker Not designated May and Baker Barber, H. J., and Slack, R. I.C.I. Boon, W. R., Carrington, H. C., Davies, J. S. H., and Jones, W. G. M. I.C.I. Boon, W. R., Calam, C. T., Carrington, H. C., and Freeman, G. G. Oxford Abraham, E. P., Chain, E., Baker, W., and Robin­ son, R. I.C.S. Not designated May and Baker Newbery, G., and Raphael, R. London Hospital Holiday, E. R. London Hospital Holiday, E. R. Barltrop, J. A., Robinson, G. M.; Abraham, E. P., Oxford Baker, W., Chain, E., and Robinson, R. Boon, W. R., Calam, C. T., Carrington, H. C., and I.C.I. Levi, A. A. Bradley, W., and Evans, A. A. B.D.H. Bradley, W., and Davis, M. B.D.H. Elliott, D. F., Hems, Β. A., and Robinson, F. A. Glaxo Crowfoot, D. M., Low, B. Vf., and Schmidt, G. Oxford M. J. Neuberger, A. N.I.M.R. May and Baker Barber, H. J., Slack, R., and Stickings, C. E. Bradley,'W., MacLean, J. A. R., Pryce, N. A. C., B.D.H. and Skrimshire, G. E. Cornforth, J. W., Cornforth, R. H.; Abraham, E. Oxford P., Baker, W., Chain, E., and Robinson, R. Cornforth, J. W., Cornforth, R. H.; Abraham, E. Oxford P., Baker, W., Chain, E., and Robinson, R. Cornforth, J. W., Cornforth, R. H.; Abraham, E. Oxford P., Baker, W. Chain, E., and Robinson, R. Peak, D. A. Boots Boon, W. R., Carrington, H. C., and Levi, A. A. I.C.I. May and Baker Newbery, G., and Raphael, R. Bentley, R., Catch, J. R., Cook, A. H., Elvidge, I.C.S. J. A., Heilbron, I. M., and Shaw, G. Bradley, W. B.D.H. Baddiley, J., Openshaw, H. T., Sykes, P., and Manchester Todd, A. R. May and Baker Barber, H. J., Gregory, P. Z., Slacki R., Stickings, C. E., and Woolman, A. M. Cook, A. H., Elvidge, J. A., and Heilbron, I. M. I.C.S.

APPENDIX

1046 Date received Date of report by O.S.R.D. (Ν. Y.)

Group

Investigators

30 May 1944

15 Jun. 1944

Oxford

30 May 1944

15 Jun. 1944

Oxford

1 Jun. 1944 1 Jun. 1944

21 Jul. 1944 21 Jul. 1944

I.C.I. Oxford

2 Jun. 1944

21 Jul. 1944

Wellcome

6 Jun. 1944

21 Jul. 1944

Glaxo

12 Jun. 1944

21 Jul. 1944

Oxford

13 Jun. 1944

21 Jul. 1944

Oxford

14 Jun. 1944

21 Jul. 1944

Oxford

17 Jun. 1944 14 Jun. 1944 16 Jun. 1944 16 Jun. 1944

21 Jul. 1944 21 Jul. 1944 21 Jul. 1944 21 Jul. 1944

I.C.S. Boots I.C.I. I.C.I.

16 Jun. 1944 24 Jun. 1944

21 Jul. 1944 31 Aug. 1944

May and Baker Oxford

29 Jun. 1944 29 Jun. 1944 4 Jul. 1944 7 Jul. 1944 7 Jul. 1944

31 Aug. 1944 31 Aug. 1944 26 Aug. 1944 31 Aug. 1944 26 Aug. 1944

Boots Manchester I.C.S. I.C.S. I.C.I.

7 Jul. 1944

31 Aug. 1944

14 Jul. 1944 17 Jul. 1944

31 Aug. 1944 31 Aug. 1944

I.C.S. Oxford

19 Jul. 1944 20 Jul. 1944 20 Jul. 1944

31 Aug. 1944 31 Aug. 1944 31 Aug. 1944

I.C.S. Glaxo Glaxo

31 Jul. 1944 31 Jul. 1944

18 Sept. 1944 18 Sept. 1944

B.D.H. I.C.S.

4 Aug. 1944 31 Jul. 1944 31 Jul. 1944 14 Aug. 1944

18 Sept. 1944 18 Sept. 1944 18 Sept. 1944 18 Sept. 1944

I.C.S. May and Baker May and Baker Oxford

11 Aug. 1944 11 Aug. 1944 11 Aug. 1944 14 Aug. 1944

18 Sept. 1944 18 Sept. 1944 18 Sept. 1944 18 Sept. 1944 18 Sept. 1944

Glaxo Glaxo Glaxo Glaxo I.C.I.

23 Aug. 1944

18 Sept. 1944

May and Baker

26 Aug. 1944

18 Sept. 1944

Oxford

5 Sept. 1944

18 Sept. 1944

Oxford

28 Sept. 1944 15 Sept. 1944

24 Oct. 1944 24 Oct. 1944

N.I.M.R. I.C.S.

King, P. E., Waley, S. G.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Cornforth, J. W., Cornforth, R. H., Dewar, M. J. S.; Abraham, E. P., Baker, W., Ch^in, E., and Robinson, R. Boon, W. R., Carrington, H. C., and Sexton, W. A. Abraham, E. P., Baker, W., Chain, E., and Robin­ son, R. Copp, F. C., DuiBn, W. M., Smith, S., and Wilkin­ son, S. Attenburrow, J., Elliott, D. F., Hems, Β. A., and Robinson, F. A. Abraham, E. P., Baker, W., Chain, E., and Robin­ son, R. Abraham, E. P., Baker, W., Chain, E., and Robin­ son, R. Wilson, J. P.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Cook, A. H., Elvidge, J. A., and Heilbron, I. M. Grew, E. L. Flett, M. St.C., and Horrobin, S. Boon, W. R., Carrington, H. C., and Jones, W. G. M. Gregory, P. Z., and Slack, R. Wilson, J. P.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Brodrick, C. I., and Peak, D. A. Todd, A. R., and Topham1 A. Catch, J. R., Cook, A. H., and Heilbron, I. M. Cook, A. H., Heilbron, I. M., and Shaw, G. Boon, W. R., Carrington, H. C., and Freeman, G. G. Copp, F. C., Duffin, W. M., Smith, S., and Wilkin­ son, S. Bentley, R., Cook, A. H., and Heilbron, I. M. Robinson, G. M.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Cook, A. H., Elvidge, J. A., and Heilbron, I. M. Hems, Β. A., and Robinson, F. A. Clayton, J. C., Elks, J., Hems, Β. A., and Robin­ son, F. A. Not designated Cook, A. H., Hall, R. H., Heilbron, I. M., and Roberts, E. R. Cook, A. H., Heilbron, I. M., and Shaw, G. Harrison, J., and Newbery, G. Newbery, G., and Raphael, R. King, F. E., Muir, I. H. M.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Elks, J., Hems, Β. A., and Robinson, F. A. Hems, Β. A., Holland, D. C., and Robinson, F. A. Hems, B- A., Page, J. E., and Robinson, F. A. Smith, E. L., and Bide, A. E. Boon, W. R., Calam, C. T., Carrington, H. C., and Freeman, G. G. Barber, H. J., Gregory, P. Z., Slack, R., Stickings, C. E., and Woolman, A. M. Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Beer, R. J.S., King, F. E., Waley, S. G.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Neuberger, A. Bentley, R., Catch, J. R., Cook, A. H., Heilbron, I. M., and Shaw, G.

Wellcome »

APPENDIX

, No.

Date received by O.S.R.D. (Ν. Y.)

268

24 Oct. 1944

269 270

24 Oct. 1944 24 Oct. 1944

271

24 Oct. 1944

271j

3 Nov. 1944

272

3 Nov. 1944

273 274

3 Nov. 1944 3 Nov. 1944

276 290 291 298

3 Nov. 1944 3 Nov. 1944 3 Nov. 1944 3 Nov. 1944

305

13 Dec. 1944

311 312

13 Dec. 1944 13 Dec. 1944

313 314 319

13 Dec. 1944 13 Dec. 1944 13 Dec. 1944

325

13 Dec. 1944

327 328 329 340

13 Dec. 1944 2 Jan. 1945 13 Dec. 1944 18 Jan. 1945

341

18 Jan. 1945

342

18 Jan. 1945

346

24 Jan.1945

360

1 Feb. 1945

S61

24 Jan. 1945

369 360 361

1 Feb. 1945 1 Feb. 1945 26 Feb. 1945

362 377

1 Feb. 1945 26 Feb. 1945

378 379

26 Feb. 1945 26 Feb. 1945 26 Feb. 1945 13 Mar. 1945 2 Apr. 1945 13 Mar. 1945 26 Feb. 1945

1047

Investigators

Beer, R. J. S., King, F. E.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Robinson, R. Oxford I.C.S. Cook, A. H., Elvidge, J. A., Hall, R. H., Heilbron, I. M., and Shaw, G. Oxford Abraham, E. P., Baker, W., Chain, E., and Robin­ son, R. Abraham, E. P., Baker, W., Chain, E., and Robin­ Oxford son, R. King. F. E., Waley, S. G.; Abraham, E. P., Baker, Oxford W., Chain, E., and Robinson, R. I.C.S. Cook, A. H., Elvidge, J. A., and Heilbron, I. M. I.C.I. Boon, W. R., Carrington, H. C., Davies, J. S. H., Jones, W. G. M., and Ramage, G. R. B.D.H. Mead, T. H., Stack, Μ. V., and Stewart, D. D. Glaxo Hems, Β. A., Page, J. E., and Robinson, F. A. Catch, J. R., Cook, A. H., and Heilbron, I. M. I.C.S. Smith, E. L., Bide, A. E., Duckworth, A., and Glaxo Graham, W. Boots Brodrick, C. I., Peak, D. A., Short, W. F., Whitmont, F. F., and Wilson, W. I.C.S. Cook, A. H., Heilbron, I. M., and Shaw, G. Bailey, J. L., Bradley, W., Davis, M., Evans, A. B.D.H. A., MacLean, J. A. R., Pryce, N. A. C., and Sayer, F. G. I.C.S. Cook, A. H., Heilbron, I. M., and Levy, A. L. I.C.S. Cook, A. H., Heilbron, I. M., and Graham, A. R. Wellcome Copp, F. C., Duffin, W. M., Smith, S., and Wilkin­ son, S. Oxford King, F. E., Waley, S. G.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Neuberger, A. N.I.M.R. I.C.S. Cook, A. H., Elvidge, J. A., and Heilbron, I. M. Robinson, R. Oxford Copp, F. C., Duffin, W. M., Smith, S., and Wilkin­ Wellcome son, S. Copp, F. C., Duffiii, W. M., Smith, S., and Wilkin­ Wellcome son, S. Abraham, E. P., Baker, W., Chain, E., and Oxford Robinson, R. I.C.I. Boon, W. R., Carrington, H. C., Davies, J. S. H., Jones, W. G. M., Ramage, G. R., and Waring, W.S. May and Baker Campbell, A., Harris, J. 0., Harrison, J., and Newbery, G. Manchester Wardleworth, J., Todd, A. R., Sykes, P., Baddiley, J., and Openshaw, Η. T. Oxford Crowfoot, D., and Rogers-Low, B. W. Oxford Crowfoot, D., and Rogers-Low, B. W. Wellcome Copp, F. C., Duffin, W. M., Smith, S., and Wilkin­ son, S. I.C.I. Bunn, C. W., and Turner-Jones, A. I.C.S. Arnstein, Η. Α. V., Catch, J. R., Cook, A. H., and Heilbron, I. M. May and Baker Newbery, G., and Raphael, R. . Oxford Crowfoot, D., and Rogers-Low, B. W. I.C.S. Cook, A. H., and Heilbron, I. M. Glaxo Smith, E. L., and Goddard, H. W. Glaxo Attenburrow, J., Elliott, D. F., Hems, Β. A., and Robinson, F. A. Glaxo Hems, Β. A., Page, J. E., and Robinson, F. A. May and Baker Barber, H. J., Gregory, P. Z., Langford, K. N., Slack, R., Stickings, C. E., and Woolman, A. M. Oxford

APPENDIX •

t No

Date received Date of report by O.S.R.D. (Ν. Y.)

Group

Investigators

.386

22 Jan. 1945

26 Feb. 1945

I.C.S.

Bentley, R., Cook, A. H., Harris, G., and Heilbron,

387

29 Jan. 1945

13 Mar. 1945

388

5 Feb. 1945

13 Mar. 1945

389 390 409 416 417 418

5 Feb. 1945 5 Feb. 1945 7 Feb. 1945 19 Feb. 1945 28 Feb. 1945 31 Oct. 1944

4η

22 Feb. 1945

I. M.

Bentley, R., Cook, A. H., Elvidge, J. A., Heilbron, I. M., and Shaw, G. Copp, F. C., Duffin, W. M., Smith, S., and Wilk­ Wellcome inson, S. Baddiley, J., Kilby, Β. A., and Todd, A. R. Cambridge Crowfoot, D., and Rogers-Low, B. W. Oxford Boon, W. R., and Leigh, T. I.C.I. May and Baker Slack, R. Brodrick, C. I., Peak, D. A., and Wilson, W. Boots Dewar, M. J. S.; Abraham, E. P., Baker, W., Oxford Chain, E., and Robinson, R. Boon, W. R., Carrington, H. C.', Davies, J. S. H., I.C.I. Jones, W. G. M., Ramage, G. R., and Waring, W. S. Cornforth, J. W., Cornforth, R. H.; Abraham, E. Oxford P., Baker, W., Chain, E., and Robinson, R. Copp, F. C., Duffin, W. M., Smith, S., and Wilk­ Wellcome inson, S. Bentley, R., Cook, A. H., and Heilbron, I. M. I.C.S. Dewar, M. J. S.; Abraham, E, P., Baker, W., Oxford Chain, E., and Robinson, R. Robinson, R. Oxford Cook, A. H., Harris, G., Heilbron, I. M., and I.C.S. Shaw, G. Bentley, H. R., Robinson, G. M.; Abraham, E. P., Oxford Baker, W. Chain, E., and Robinson, R. Richards, R. E., and Thompson, H. W. Oxford Cook, A. H., Elvidge, J. A., Heilbron, I. M., and I.C.S. Levy, A. L. Bailey, J. L., Baker, B., and Bradley, W. B.D.H. Oxford / Wilson, J. P.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Brodrick, C. I., Peak, D. A., and Whitmont, F. F. Boots Bailey, J. L., Bradley, W., Davis, M., Evans, A. B.D.H. A., and MacLean, J. A. R. Cook, A. H., Elvidge, J. A., Heilbron, I. M., and I.C.S. Levy, A. L. Cook, A. H., Graham, A. R., Harris, G., and Heil­ I.C.S. bron, I. M. Barltrop, J. A.; Abraham, E. P., Baker, W., Chain, Oxford E., and Robinson, R. Copp, F. C., Duffin, W. M., Smith, S., and Wilkin­ WeDcome son, S. Bunn, C. W., and Turner-Jones, A. I.C.I. May and Baker Newbery, G., and Raphael, R. Boon, W. R., Carrington, H. C., Davies, J. S. H., I.C.I. Jones, W. G. M., Ramage, G. R., and Waring, W. S. Richards, R. E., and Thompson, H. W. Oxford Elliott, D. F., Hems, Β. A., and Robinson, F. A. Glaxo King, F. E., Waley, S. G.; Abraham, E. P., Baker, Oxford W., Chain, E., and Robinson, R. Cornforth, J. W., Cornforth, R. H.; Abraham, Oxford E. P., Baker, W., Chain, E., and Robinson, R. Cook, A. H., Harris, G., Heilbron, I. M., and I.C.S. Shaw, G. Oxford Thompson, H. W., and Richards, R. E. Oxford Crowfoot, D., and Rogers-Low, B. W. Robinson, R. j Oxford Elks, J., Hems, B.; A., Robinson, F. A., and RyGlaxo man, B. E. j Thompson, H. W., and Richards, R. E. Oxford I.C.S.

13 Mar. 1945 13 Mar. 1945 13 Mar. 1945 2 Apr. 1945 . 2 Apr. 1945 2 Apr. 1945 2 Apr. 1945

423

8 Mar. 1945

4 May 1945

m

13 Mar. 1945

4 May 1945

430 438

6 Mar. 1945 19 Mar. 1945

4 May 1945 4 May 1945

439 440

15 Mar. 1945 19 Mar. 1945

4 May 1945 4 May 1945

441

20 Mar. 1945

4 May 1945

44^

443

22 Mar. 1945 22 Mar. 1945

4 May 1945 4 May 1945

444 445

22 Mar. 1945 23 Mar. 1945

4 May 1945 4 May 1945

446 468

29 Mar. 1945 16 Mar. 1945

4 May 1945 4 May 1945

461

10 Apr. 1945

4 May 1945

462

26 Mar. 1945

2 Jun. 1945

463

10 Apr. 1945

2 Jun. 1945

464

24 Apr. 1945

2 Jun. 1945

465 478 479

9 Apr. 1945 30 Apr. 1945 30 Apr. 1945

2 Jun. 1945 19 Jun. 1945 2 Jun. 1945

480

490 491

5 May 1945 1 May 1945 15 May 1945

19 Jun. 1945 19 Jun. 1945 19 Jun. 1945

492

11 May 1945

19 Jun. 1945

SOO

14 May 1945

19 Jun. 1945

602 608

609 610

21 May 1945 29 May 1945 28 May 1945 29 May 1945

26 Jun. 1945 ; 26 Jun. 1945 26 Jun. 1945 i 19 Jul. 1945

611

30 May 1945

19 Jul. 1945

;

APPENDIX

report

Date received by O.S.R.D. (Ν. Y.)

1049

Group

Investigators

Dewar, M. J. S.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Dewar, M. J. S.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Allan, D., Boon, W. R., Carrington, H. C., Gaubert, P., and Levi, A. A. Sykes, P., and Todd, A. R. Bentley, R., Cook, A. H., and Heilbron, I. M. Bradley, W., Davis, M., and MacLean, J. A. R. Cook, A. H., Elvidge, J. A., and Heilbron, I. M. Thompson, H. W., and Richards, R. E. Copp, F. C., Duffin, W. M., Smith, S., and Wilkin­ son, S. Baddiley, J., Kilby, Β. A., and Todd, A. R. Bunn, C. W., and Turner-Jones, A. Copp, F. C., Duffin, W. M., Smith, S., and Wilk­ inson, S. Dewar, M. J. S., and Robinson, R. Barltrop, J. A., Waley, S. G., King, F. E.; Abra­ ham, E. P., Baker, W., Chain, E., and Robinson, R. Bentley, H. R., and Robinson, R. Sutherland, G. B. B. M., and Darmon, S. E. Cook, A. H., Graham, A. R., and Heilbron, I. M. King, H. Abraham, E. P., Baker, W., and Chain, E. Bradley, W., Davis, M., and Pryce, N. A. C. Cook, A. H., Harris, G., and Heilbron, I. M. Cook, A. H., Harris, G., Heilbron, I. M., and Shaw, G. Copp, F. C., Duffin, W. M., Smith, S., and Wilkin­ son, S. Barber, H. J., Gregory, P. Z., Slack, R., and Stickings, C. E. Brodrick, C. I., Peak, D. A., Whitmont, F. F., and Wilson, W. Elliott, D. F., Hems, Β. A., and Robinson, F. A. Gell, P. G. H , and Harington, C. R. Clayton, J. C., Hems, Β. A., and Robinson, F. A. Cook, A. H., Elvidge, J. A., and Heilbron, I. M. Bradley, W., and Gayler, M. Robinson, R., Wilson, J. P.; Abraham, E. P., Baker, W., and Chain, E. King, T. J., King, F. E.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Barber, H. J., and Slack, R. Boon, W. R., Carrington, H. C., Davies, J. S. H., Gaubert, P., Jones, W. G. M., Ramage, G. R., and Waring, W. S. Catch, J. R., Cook, A. H., and Heilbron, I. M. Arnstein, H. R. V., Cook, A. H., and Heilbron,

τ

1945

19 Jul. 1945

Oxford

τ

1945

19 Jul. 1945

Oxford

1945

19 Jul. 1945

I.C.I.

1945 1945 1945 1945 1945 1945

19 Jul. 1945 19 Jul. 1945 19 Jul. 1945 19 Jul. 1945 19 Jul. 1945 11 Aug. 1945

Cambridge I.C.S. B.D.H. I.C.S. Oxford Wellcome

1945 1945 1945

20 Jul. 1945 20 Jul. 1945 11 Aug. 1945

Cambridge I.C.I. Wellcome

1945 1945

11 Aug. 1945 11 Aug. 1945

Oxford Oxford

1945 1945 1945 1945

Oxford Cambridge I.C.S.

. 1945 1945 1945

11 Aug. 1945 11 Aug. 1945 11 Aug. 1945 17 Sep. 1945 17 Sep. 1945 17 Sep. 1945 17 Sep. 1945 17 Sep. 1945

. 1945

22 Sep. 1945

Wellcome

. 1945

22 Sep. 1945

May and Bakei

. 1945

22 Sep. 1945

Boots

. 1945 1945 . 1945 1945 1945

22 Sep. 1945 22 Sep. 1945 3 Nov. 1945 3 Nov. 1945 21 Nov. 1945 7 Dec. 1945

Glaxo N.I.M.R. Glaxo I.C.S. B.D.H. Oxford

7 Dec. 1945

Oxford

. 1945 1945

10 Dec. 1945 7 Dec. 1945

May and Baker I.C.I.

1945 1945

7 Dec. 1945 7 Dec. 1945

I.C.S. I.C.S.

. 1945

9 Jan. 1946

Wellcome

. 1945

9 Jan. 1946

Wellcome

. 1945 . 1945

7 Dec. 1945 10 Dec. 1945

I.C.I. Oxford

. 1945

9 Jan. 1946

Oxford

. 1945

9 Jan. 1946

Oxford

Oxford B.D.H. I.C.S. I.C.S.

I. M.

Copp, F. C., Duffin, W. M., Smith, S., and Wilk­ inson, S. Copp, F. C., Duffin, W. M., Smith, S., and Wilkin­ son, S. Boon, W. R., Carrington, H. C., and Snow, G. A. Cornforth, J. W., Cornforth, R. H.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Robinson, G. M.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R.

1050

APPENDIX Date received Date of report by O.S.R.D. (Ν. Y.)

Group

Investigators

Goldsworthy, L. J., Robinson, R.; Abraham, E. P., Baker, W., and Chain, E. Jones, T. S. G., Copp, F. C., Duffin, W. M., Smith, S., and Wilkinson, S. Copp, F. C., Duffin, W. M., Smith, S., and Wilkinson, S. Copp, F. C., Duffin, W. M., Smith, S., and Wilkin­ son, S. Newbery, G., Nineham, A. W., and Raphael, R. Sykes, P., and Todd, A. R. Catch, J. R., Cook, A. H., Harris, G., and Heilbron, I. M. Cook, A. H., Elvidge, J. A., and Heilbron, I. M. Robinson, R., Rogers, M. A. T.; Abraham, E. P., Baker, W., and Chain, E. Cornforth, J. W., Fawaz, E.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Felton, D. G. I., King, F. E.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Smith, E. L., Bide, A. E., and Graham, W. Smith, E. L., Bide, A. E., and Graham, W, Copp, F. C., Duffin, W. M., Smith, S., and Wilkin­ son, S. Cornforth, J. W., Cornforth, R. H.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Poole, J. B.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Beer, C. T., Cornforth, J. W.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Jansen, Α. Β. A.; Abraham, E. P., Baker, W., Chain, E., and Robinson, R. Boon, W. R., Carrington, H. C., Jones, W. G. M., Ramage, G. R., Tyler, J., and Waring, W. S. Bunn, C. W., Crowfoot, D., Rogers-Low, B. W., and Turner-Jones, A. Goldsworthy, L. J., and Robinson, R. Sutherland, G. B. B. M., and Darmon, S. E. Sutherland, G. B. B. M., and Darmon, S. E. Thompson, H. W., and Richards, R. E.

26 Nov. 1945

9 Jan. 1946

Oxford

17 Dec. 1945

1 Feb. 1946

Wellcome

10 Dec. 1945

1 Feb. 1946

Wellcome

10 Dec. 1945

1 Feb. 1946

Wellcome

12 Dec. 1945 9 Jan. 1946 14 Jan. 1946

1 Feb. 1946 15 Mar. 1946 15 Mar. 1946

May and Baker Cambridge I.C.S.

16 Jan. 1946 31 Jan. 1946

15 Mar. 1946 26 Mar. 1946

I.C.S. Oxford

31 Jan. 1946

26 Mar. 1946

Oxford

31 Jan. 1946

26 Mar. 1946

Oxford

6 Feb. 1946 12 Feb. 1946 22 Feb. 1946

18 Apr. 1946 18 Apr. 1946 18 Apr. 1946

Glaxo Glaxo Wellcome

22 Feb. 1946

23 Dec. 1946

Oxford

8 Mar. 1946

23 Dec. 1946

Oxford

8 Mar. 1946

23 Dec. 1946

Oxford

12 Mar. 1946

23 Dec. 1946

Oxford

15 Apr. 1946

23 Dec. 1946

I.C.I.

1 May 1946

23 Dec. .1946

Oxford

1 May 1946 27 Apr. 1946 24 Jun. 1946 Mar. 1946

23 Dec. 1946 17 Jun.1946 15 Jul. 1946 7 Feb. 1947

Oxford Cambridge Cambridge Oxford

Abbott Laboratories Carter, Herbert Ε.» Larson, L. W. MacCorquodale, D. W.

Milne, James W. Philip, Julian E. Risser, W. C.

Schenck, J. R. Spielman, M. A. Sylvester, J. C.

Touster, Oscar Weston, A. W. Zaugg, Η. E. Date received

Report No.

Λ.ί 2* S 4 B 1

Consultant. * Circulated in abstract form.

Date of report

30 Dec. 1943

I I

14 Jan. 1944 14 Feb. 1944 ; 14 Mar. 1944 15 Apr. 1944

By 0;S.R.D. (Ν. Y.)

By Medical Research Council

3 Jan. 1944

pp. 1-5, 7 Jul. 1944 pp. 6-11,15 Mar. 1945 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 15 Sep. 1944

•

17 Jan. 1944 17 Feb. 1944 17 Mar. 1944 17 Apr. 1944

APPENDIX

1051 Date received

Report No.

A.6 6a 7 8 9 10 11 12 ISa IS U 16 16 17 18 19 20 Sl SS SS U SB S6

Date of report

15 May 1944 10 Jul. 1944 15 Jun. 1944 30 Jun.1944 15 Jul. 1944 15 Aug. 1944 15 Sep. 1944 15 Oct. 1944 7 Nov. 1944 15 Nov. 1944 15 Dec. 1944 15 Jan. 1945 15 Feb. 1945 15 Mar. 1945 15 Apr. 1945 15 May 1945 15 Jun. 1945 15 Jul. 1945 15 Aug. 1945 15 Sep. 1945 31 Oct. 1945

By O.S.R.D. (Ν. Y.)

By Medical Research Council

17 May 1944 13 Jul. 1944 20 May 1944 17 Jun. 1944 8 Jul. 1944 17 Jul. 1944 17 Aug. 1944 18 Sep. 1944 18 Oct. 1944 18 Oct. 1944 11 Nov. 1944 27 Nov. 1944 22 Dec. 1944 19 Jan. 1945 26 Feb. 1945 23 Mar. 1945 23 Apr. 1945 21 May 1945 22 Jun. 1945 18 Jul. 1945 24 Aug. 1945 22 Sep. 1945 10 Nov. 1945

7 Jul. 1944 8 Sep. 1944 7 Jul. 1944 29 Jun. 1944 26 Jul. 1944 26 Jul. 1944 8 Sep. 1944 3 Oct. 1944 6 Nov. 1944 6 Nov. 1944 23 Nov. 1944 8 Dec. 1944 11 Jan. 1945 7 Feb. 1945 15 Mar. 1945 6 Apr. 1945 5 May 1945 31 May 1945 26 Jul. 1945 1 Aug. 1945 5 Sep. 1945 4 Oct. 1945 20 Nov. 1945

U. S. DEPARTMENT OF AGRICULTURE NORTHERN REGIONAL RESEARCH LABORATORY

Auernheimer, Arthur Η. Benedict, Robert G. Coghill, Robert D. Cowan, John C. Dvonch, William Falkenburg, Lee B. Friedkin, Morris Hilbert, G. E.

Lathrop, Elbert C. MacMillan, Duncan Mehltretter, Charles L. Mellies, Russell L. Melvin, Eugene H. Milner, Reid T. Morell, Samuel A. Olds, David W.

Paschke, Raymond F. Radlove, Sol B. Rist, Carl E. Schieltz, N. C. Schmidt, William H. Schniepp, Lester E. Scholfield, Charles R. Schwab, Arthur W.

Sehnert, Eldon S. Stodola, Frank H. Teeter, Howard M. Van Etten, Cecil H. Von Korff, Richard W. Wachtel, Jacques L. Wolff, Ivan A.

Date received Report No.

C.l 2 S 4 ε 6a 6 7 8 9

Date of report

7 Feb. 1944 12 Feb. 1944 4 Mar. 1944 9 Mar. 1944 10 Apr. 1944 21 Apr. 1944 21 Apr. 1944 25 May 1944 12 Jul. 1944 1 Aug. 1944

By O.S.R.D. (Ν. Y.)

By Medical Research Council

10 Feb. 1944 16 Feb. 1944 8 Mar. 1944 13 Mar. 1944 13 Apr. 1944 27 Apr. 1944 27 Apr. 1944 5 Jun. 1944 21 Jul. 1944 7 Aug. 1944

7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 15 Sep. 1944 7 Jul. 1944 7 Jul. 1944 20 Jun. 1944 26 Jul. 1944 30 Aug. 1944

APPENDIX

1052

Date received Report No.

C.10 115 12» IS 14 W ie> 17 18 19 SO 21» 22 28 24"

Date of report

;

i :

By O.S.R.D. (Ν. Y.)

By Medical Research Council

14 Sep. 1944 14 Sep. 1944 25 Sep. 1944 9 Oct. 1944 8 Nov. 1944 26 Dec. 1944 26 Dec. 1944 19 Feb. 1945 2 Apr. 1945 7 May 1945 8 Jun. 1945 18 Jul. 1945 8 Aug. 1945 24 Sep. 1945 8 Nov. 1945

27 Sep. 1944 27 Sep. 1944 19 Oct. 1944 19 Oct. 1944 21 Nov. 1944 11 Jan. 1945 7 Feb. 1945 3 Mar. 1945 15 Apr. 1945 24 May 1945 29 Jun. 1945 1 Aug. 1945 23 Aug. 1945 4 Oct. 1945 20 Nov. 1945

1 Sep. 1944 1 Sep. 1944 21 Feb. 1944 5 Oct. 1944 6 Nov. 1944 18 Dec. 1944 15 Dec. 1944 15 Feb. 1945 1 Apr. 1945 1 May 1945 1 Jun. 1945 1 Aug. 1945 24 Sep. 1945 1 Nov. 1945

:

Cornell University Medical College DEPARTMENT OP BIOCHEMISTBT

Brown, George B. Carpenter, Frederick H. Dittmer, Karl du Vigneaud, Vincent Funk, Roscoe C., Jr.

Holley, Robert W. Livermore, Arthur H. McKennis, Herbert, Jr. Melville, Donald B.

Partridge, Chester W. H. Ploeser, James Rachele, Julian R. Stevens, Carl M.

Todd, David Wilson, John E. Wood, John L. Wright, Mary Elizabeth

Date received Report No.

D.l 2 S 4 6 6 7 8 9 10 11 IS IS 14 16 16 17 18 19

Date of report By O.S.R.D. (Ν. Y.)

By Medical Research Council

17 Feb. 1944 18 Feb. 1944 18 Feb. 1944 23 Mar. 1944 23 Mar. 1944 7 Apr. 1944 3 May 1944 3 May 1944 9 May 1944 20 May 1944 15 Jun. 1944 16 Jun. 1944 8 Jul. 1944 21 Jul. 1944 27 Jul. 1944 10 Aug. 1944 17 Aug. 1944 25 Aug. 1944 29 Aug. 1944

7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 29 Jun. 1944 29 Jun. 1944 26 Jul. 1944 26 Jul. 1944 30 Aug. 1944 30 Aug. 1944 8 Sep. 1944 8 Sep. 1944 8 Sep. 1944

3 Feb. 1944 3 Feb. 1944 3 Feb. 1944 15 Mar. 1944 15 Mar. 1944 31 Mar. 1944 1 May 1944 1 May 1944 5 May 1944 15 May 1944 12 Jun. 1944 13 Jun. 1944 29 Jun. 1944 18 Jul. 1944 20 Jul. 1944 3 Aug. 1944 14 Aug. 1944 23 Aug. 1944 23 Aug. 1944

• Signed by F. H. Stodola. • Signed by William H. Schmidt, George E. Ward and Robert D. Coghill. Signed by E. H. Melvin and N. C. 8chieltz. 1 Signed by F. H. Stodola, J. L. Wachtel, and R. D. Coghill. • Signed by Robert D. Coghill. 10 Signed by N. C. Schieltz J. L. Wachtel, and M. Friedkin. t 1

APPENDIX

1053 Date received

Report No.

D.20 21 22 23 U 25 26 27 28 29 30 Sl 32 33 34 SS 86 37 38 39

Date of report

20 Oct. 1944 25 Oct. 1944 1 Dec. 1944 18 Dec. 1944 1 Jan. 1945 15 Feb. 1945 20 Feb. 1945 1 Mar. 1945 15 Mar. 1945 30 Mar. 1945 29 May 1945 12 Jun. 1945 2 Jul. 1945 1 Aug. 1945 23 Aug. 1945 15 Sep. 1945 1 Nov. 1945 1 Dec. 1945 15 Dec. 1945 31 Dec. 1945

By O.S.R.D. (Ν. Y.)

By Medical Research Council

26 Oct. 1944' 2 Nov. 1944 4 Dec. 1944 27 Dec. 1944 12 Jan. 1945 27 Feb. 1945 9 Mar. 1945 20 Mar. 1945 3 Apr. 1945 4 Apr. 1945 1 Jun. 1945 15 Jun. 1945 18 Jul. 1945 7 Aug. 1945 31 Aug. 1945 15 Sep. 1945 13 Nov. 1945 17 Jan. 1946 17 Jan. 1946 24 Jan. 1946

8 Nov. 1944 14 Nov. 1944 20 Dec. 1944 11 Jan. 1945 7 Feb. 1945 10 Mar. 1945 28 Mar. 1945 7 Apr. 1945 15 Apr. 1945 15 Apr. 1945 12 Jun. 1945 26 Jul. 1945 1 Aug. 1945 31 Aug. 1945 11 Sep. 1945 19 Sep. 1945 28 Nov. 1945

RUSSELL SAGE INSTITUTE

Furchgott, Robert F.

Rosenkrantz, Harris

Shorr, Ephraim Date received

Report No.

RS.1

Date of report

15 Feb. 1945 .

By O.S.R.D. (Ν. Y.)

By Medical Research Council

9 Mar. 1945

28 Mar. 1945

Cutter Laboratories Beniams, Herman N. Deromedi, Frank D. Dufrenoy, Jean

Hatch, Alden B. Johnson, Frederick F.

Pilcher, Karl S. Pratt, Robertson

Seeberg, Victor P. Winegarden, Howard M. Wolfred, Morris Date received

Report No.

Cu.l 2 5

4

B 6 7 8 9 10

Date of report

15 Jun. 1944 1 Nov. 1944 1 Dec. 1944 1 Feb. 1945 1 Apr. 1945 1 Jun.1945 1 Jul. 1945 1 Aug. 1945 1 Oct. 1945 1 Nov. 1945

By O.S.R.D. (Ν. Y.)

By Medical Research Council

26 Feb. 1945 6 Apr. 1945 8 May 1945 28 May 1945 11 Jun. 1945 15 Jun. 1945 18 Jul. 1945 10 Aug. 1945 10 Oct. 1945 26 Dec. 1945

11 May 1945 11 May 1945 24 May 1945 12 Jun. 1945 20 Jun. 1945 26 Jul. 1945 1 Aug. 1945 31 Aug. 1945 16 Oct. 1945 12 Jan.1946

1054

APPENDIX Federal Security Agency FOOD AND DHUG ADMINISTRATION

Eble, Thomas E. Fischbachl Henry

Grove, Donald C. Herwick, R. P.

Report No.

Date of report

Wiley, Frank

Mundell, Merlin Welsh, Llewellyn H.

Date received

F.l* S 5 4 6 6 7

24 Feb. 1944 10 Oct. 1944 10 Nov. 1944 10 Dec. 1944 10 Jan. 1945 14 Feb. 1945 -15 May 1945

By O.S.R.D. (Ν. Y.)

By Medical Research Council

16 Mar. 1944 13 Oct. 1944 14 Nov. 1944 18 Dec. 1944 15 Jan. 1945 17 Feb. 1945 4 Jun. 1945

7 Jul. 1944 25 Oct. 1944 21 Nov. 1944 11 Jan. 1945 7 Feb. 1945 3 Mar. 1945 15 Jun. 1945

* Signed by George L. Keenan and William V. Eisenberg, Microanalytical Division.

Harvard University DEPARTMENT OP CHEMISTRY

Brutschy, Frederick Georgian, Vlasios

Ross, Sydney D. Wasserman, Harry H.

Kirk, Marjorie-Jane Kornfeld, Edmund C.

Wendler, Norman Woodward, Robert B.

Date received Report No.

Wo.l 2 8 4 ε 6 7 8 9 10

Date of report

19 Sep. 1944 1 Jun. 1945 27 Jul. 1945

By O.S.R.D. (Ν. Y.)

By Medical R^earch Council

21 Sep. 1944 18 Jan. 1945 8 Jun. 1945 30 Jul. 1945 20 Dec. 1945 17 May 1946 17 May 1946 17 May 1946 17 May 1946 17 May 1946

19 Oct. 1944 7 Feb. 1945 20 Jun. 1945 22 Aug. 1945 27 Dec. 1945

Heyden Chemical Corporation Date received Report No. Date of report

Investigators By Medical Research By O.S.R.D. (Ν. Y.) Council

H.l S 5 4 δ

9 May 1944 9 May 1944 9 May 1944 15 Jun. 1944 15 Feb. 1945

22 May 1944 22 May 1944 22 May 1944 15 Jun. 1944 17 Feb. 1945

20 Jun. 1944 20 Jun. 1944 • 20 Jun. 1944 29 Jun. 1944 3 Mar. 1945

6

15 Mar. 1945

17 Mar. 1945

7 Apr. 1945

7

15 Apr. 1945

18 Apr. 1945

5 May 1945

Rigler, Ν. E. Rigler, Ν. E. Curtis, G. W., and Rapoport, H. Rigler, Ν. E. Darken, Μ. A., Rigler, N. E., and Sjolander, N. 0. Darken, Μ. A., Rapoport, H., Rigler, N. E., and Sjolander, N. O. Darken, Μ. A., Rigler, N. E., and Sjolander, N. O.

APPENDIX

1055

Date received Investigators

Report No. Date of report By O.S.R.D. (Ν. Y.)

By Medical Research Council

H.8

15 May 1945

19 May 1945

31 May 1945

9 10

15 Jun. 1945 15 Jul. 1945

21 Jun. 1945 18 Jul. 1945

26 Jul. 1945 1 Aug. 1945

11

15 Aug. 1945

21 Aug. 1945

5 Sep. 1945

η

15 Sep. 1945

17 Sep. 1945

19 Sep. 1945

13

15 Oct. 1945

18 Oct. 1945

20 Oct. 1945

U

31 Oct. 1945

10 Nov. 1945

28 Nov. 1945

Darken, Μ. A., Gorin, G., Rapoport, H., and Sjolander, N. 0. Rapoport, H., and Rigler, Ν. E. Darken, Μ. A., Rapoport, H., Rigler, N. E., and Sjolander, N. 0. Busch, E., Curtis, G. W., Port, W. S., and Rapoport, H. Darken, Μ. A., Gorin, G., Rapoport, H., Rigler, N. E., and Sjolander, N. 0. Darken, Μ. A., Gorin, G., Rapoport, H., Rigler, N. E., and Sjolander, N. 0. Port, W. S., Rigler, N. E., and Ziegler, W. M.

University of Illinois DEPARTMENT OF CHEMISTRY

Date received Date of report

Report No.

Investigators By O.S.R.D. (Ν. Y.) 3 Jul. 1944 12 Aug., 1944 11 Sep. 1944 11 Dec. 1944 11 Apr. 1945 13 Jun. 1945 20 Sep. 1945 25 Jan. 1946

1 Jul. 1944

CLl la 2 5

1 Sep. 1944 1 Dec. 1944 1 Apr. 1945 1 Jun.1945 15 Sep. 1945 '31 Dec. 1945

4

6 6 7

By Medical Research Council 5 Aug. 1944 8 Sep. 1944 27 Sep. 1944 11 Jan.1945 5 May 1945 26 Jul. 1945 4 Oct. 1945

Clark, G. L., and Kaye, W. I. Clark, G. L., and Kaye, W. I. Clark, G. L., and Kaye, W. I. Clark, G. L., and Kaye, W. I. Clark, G. L. Clark, G. L., and Pipenberg, K. J. Clark, G. L., and Pipenberg, K. J. Clark, G. L., and Pipenberg, K. J.

Eli Lilly and Company Behrens, Ο. K. Brown, William L. Carter, Herbert E.11 Clowes, G. H. A. Corse, J. W. Davis, W. W. Dickman, S. R.

Glanz, Alberta R. Hager, G. P. Hunter, Howard L. Jones, Reuben G. Kleiderer, E. C. Krahl: Μ. E.

Leighty, John A. McGuire, James M. Miller, Marjorie J. Noe, Reta E. O'Brien, John H. Parke, Thomas V., Jr.

Rohrmann, E. Schabacker, Dorothea E Shonle, H. A. Van Abeele, Frederick R. Walden, George B. Weber, Dorothy E.

Date received Report No.

L.l S 4 11 Consultant. " Circulated in abstract form only.

Date of report

27 Dec. 1943 13 Jan. 1944 12 Feb. 1944 15 Mar. 1944

By O.S.R.D. (Ν. Y.)

By Medical Research Council

3 Jan. 1944 17 Jan. 1944 16 Feb. 1944 16 Mar. 1944

15 Mar. 1945 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944

APPENDIX

1056

Date received Report No.

L.5 6 7 8 9 10 11 IZ IZa ISb IS U 16 16 17 18 19 ZO BOa Zl ZZ ZS U ZS Z6 27 Z8 Z9 SO Sl

Date of report

28 Mar. 1944 15 Apr. 1944 28 Apr. 1944 15 May 1944 15 Jun. 1944 30 Jun. 1944 15 Jul. 1944 15 Aug. 1944 9 Sep. 1944 15 Oct. 1944 7 Nov. 1944 15 Nov. 1944 18 Nov. 1944 13 Dec. 1944 16 Dec. 1944 15 Dec. 1944 11 Jan. 1945 15 Jan. 1945 15 Feb. 1945 15 Mar. 1945 15 Apr. 1945 15 May 1945 15 Jun. 1945 15 Jul. 1945 15 Aug. 1945 31 Oct. 1945

By O.S.R.D. (Ν. Y.)

By Medical Research Council

10 Apr. 1944 19 Apr. 1944 1 May 1944 17 May 1944 21 Jun. 1944 8 Jul. 1944 20 Jul. 1944 21 Aug. 1944 26 Aug. 1944 8 Sep. 1944 14 Sep. 1944 26 Oct. 1944 11 Nov. 1944 24 Nov. 1944 1 Dec. 1944 16 Dec. 1944 21 Dec. 1944 23 Dec. 1944 1 Feb. 1945 15 Jan. 1945 1 Feb. 1945 21 Feb. 1945 30 Mar. 1945 26 Apr. 1945 21 May 1945 25 Jun. 1945 23 Jul. 1945 1 Sep. 1945 13 Nov. 1945 8 Dec. 1945

7 Jul. 1944 15 Sep. 1944 7 Jul. 1944 7 Jul. 1944 26 Jul. 1944 26 Jul. 1944 26 Jul. 1944 8 Sep. 1944 8 Sep. 1944 21 Sep. 1944 27 Sep. 1944 6 Nov. 1944 23 Nov. 1944 8 Dec. 1944 20 Dec. 1944 11 Jan. 1945 11 Jan. 1945 11 Jan. 1945 15 Feb. 1945 7 Feb. 1945 15 Feb. 1945 15 Mar. 1945 17 Apr. 1945 11 May 1945 31 May 1945 26 Jul. 1945 10 Aug. 1945 9 Oct. 1945 27 Nov. 1945

Merck & Co., Inc. Date received Report No.

M.l Z S 4u 6" 6 7 8 9 10 10a Il 1 3 ISa IZb 18

Date of report By O.S.R.D. (Ν. Y.)

By Medical Research Council

3 Jul. 1942 30 Oct. 1942 31 Mar. 1943 31 Jul. 1943 Sep. 1943 Oct. 1943 Nov. 1943 Nov. 1943 14 Dec. 1943

27 Sep. 1946 18 Jan. 1944 18 Jan. 1944 •18 Jan. 1944 30 Dec. 1943 30 Dec. 1943 28 Dec. 1943 28 Dec. 1943 28 Dec. 1943

14 Feb. 1944 14 Feb. 1944 14 Feb. 1944 14 Feb. 1944

Dec. 1943 Dec. 1943 Jan. 1944 Jan. 1944 31 Jan. 1944 22 Oct. 1945

27 Jan. 1944 27 Jan. 1944 14 Feb. 1944 14 Feb. 1944 14 Feb. 1944 27 Oct. 1945

7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 20 Nov. 1945

29 Feb. 1944 29 Feb. 1944

6 Mar. 1944 6 Mar. 1944

Various British reports in the PEN. series, copies of which were supplied to O.S.R.D. by Merck and Co., Inc.

7 Jul. 1944 7 Jul. 1944

APPENDIX

1057 Date received

Report No.

Date of report By O.S.R.D. (Ν. Y.)

M.12c 12d IS U 16a 16b 16c 16 17 18 19 20 21 21a Slb 21c 21d 22 2S 24 25 26 27 28 29 SO Sl 82 SS 84 86 S6 87 88 89 40 41 42 4S 44 46 46 47 48 49 50 61 52 63 64 65 66 57 68 59 60 61 62 63 64 65

Feb. 1944 18 Feb. 1944 10 Mar. 1944 17 Mar. 1944 Mar. 1944 31 Mar. 1944 31 Mar. 1944 14 Apr. 1944 17 Apr. 1944 18 Apr. 1944 24 Apr. 1944 24 Apr. 1944 28 Jul. 1944

29 Apr. 1944 29 Apr. 1944 29 Apr. 1944 Apr. 1944 Apr. 1944 12 May 1944 31 May 1944 31 May 1944 May 1944 8 Jun. 1944 30 Jun. 1944 30 Jun. 1944 Jun. 1944 27 Jul. 1944 31 Jul. 1944 31 Jul. 1944 Jul. 1944 31 Aug. 1944 31 Aug. 1944 Aug. 1944 29 Aug. 1944 30 Sep. 1944 30 Sep. 1944 Sep. 1944 31 Oct. 1944 31 Oct. 1944 Oct. 1944 30 Nov. 1944 30 Nov. 1944 30 Nov. 1944 30 Dec. 1944 30 Dec. 1944 31 Jan. 1945 31 Jan. 1945 28 Feb. 1945 28 Feb. 1945 28 Feb. 1945 31 Mar. 1945 31 Mar. 1945 31 Mar. 1945 30 Apr. 1945 30 Apr. 1945 30 Apr. 1945 31 May 1945

6 Mar. 1944 6 Mar. 1944 14 Mar. 1944 25 Mar. 1944 6 Apr. 1944 6 Apr. 1944 6 Apr. 1944 18 Apr. 1944 25 Apr. 1944 25 Apr. 1944 4 May 1944 4 May 1944 4 May 1944 12 Aug. 1944 22 Sep. 1944 23 Jan. 1945 23 Jan. 1945 5 May 1944 5 May 1944 5 May 1944 5 May 1944 5 May 1944 20 May 1944 5 Jun. 1944 5 Jun. 1944 5 Jun. 1944 12 Jun. 1944 17 Jul. 1944 17 Jul. 1944 17 Jul. 1944 4 Aug. 1944 4 Aug. 1944 4 Aug. 1944 • 4 Aug. 1944 11 Sep. 1944 11 Sep. 1944 11 Sep. 1944 11 Sep. 1944 11 Oct. 1944 11 Oct. 1944 11 Oct. 1944 8 Nov. 1944 8 Nov. 1944 8 Nov. 1944 16 Dec. 1944 16 Dec. 1944 16 Dec. 1944 12 Jan. 1945 12 Jan. 1945 8 Feb. 1945 8 Feb. 1945 9 Mar. 1945 9 Mar. 1945 9 Mar. 1945 13 Apr. 1945 13 Apr. 1945 13 Apr. 1945 17 May 1945 17 May 1945 17 May 1945 11 Jun. 1945

By Medical Research Council

7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 15 Sep. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 8 Sep. 1944 3 Oct. 1944 7 Feb. 1945 7 Feb. 1945 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 20 Jun. 1944 20 Jun. 1944 20 Jun. 1944 29 Jun. 1944 26 Jul. 1944 26 Jul. 1944 26 Jul. 1944 30 Aug. 1944 30 Aug. 1944 30 Aug. 1944 30 Aug. 1944 27 Sep. 1944 27 Sep. 1944 27 Sep. 1944 27 Sep. 1944 25 Oct. 1944 25 Oct. 1944 25 Oct. 1944 23 Nov. 1944 23 Nov. 1944 21 Nov. 1944 11 Jan. 1945 11 Jan. 1945 11 Jan. 1945 7 Feb. 1945 7 Feb. 1945 27 Feb. 1945 27 Feb. 1945 28 Mar. 1945 28 Mar. 1945 28 Mar. 1945 5 May 1945 5 May 1945 5 May 1945 31 May 1945 31 May 1945 31 May 1945 21 Jun. 1945

APPENDIX

1058

Date received Report No.

Date of report By O.S.R.D. (N. Y.)

M.66 67 68

69 70 71 72 73

74

76 76 77 78 79 80 81

Investigator

Aliminosa, L. M. Anderson, R. C. Arth, Glen E. Babson, R. D. Bacher, F. A. Bacher, M. H. Berg, C. J. Bittenbender, W. A. Bolhofer, W. A. Boos, R. N. Braunmuller, A. R. Brink, N. G. Buhs, R. P. Clark, R. L. Conn, J. B. Cram, D. J. Easton, N. R. Field, Lamar Folkers, K. A. Goldsmith, D. P. J. Graber, R. P. Harris, S. A. Hetrick, J. C.

31 31 30 30 30

May May Jun. Jun. Jun.

1945 1945 1945 1945 1945

31 31 31 31 31 28 28 30 31 31

Jul. 1945 Jul. 1945 Jul. 1945 Aug. 1945 Aug. 1945 Sep. 1945 Sep. 1945 Sep. 1945 Oct. 1945 Oct. 1945

11 Jun. 1945 11 Jun. 1945 27 Jul. 1945 27 Jul. 1945 27 Jul. 1945 1 Aug. 1945 13 Aug. 1945 13 Aug. 1945 13 Aug. 1945 13 Sep. 1945 13 Sep. 1945 17 Oct. 1945 17 Oct. 1945 17 Oct. 1945 16 Nov. 1945 16 Nov. 1945

By Medical Research Council

21 21 10 10 10 22 31 31 31 25 25 29 29 29 30 30

Jun. 1945 Jun. 1945 Aug. 1945 Aug. 1945 Aug. 1945 Aug. 1945 Aug. 1945 Aug. 1945 Aug. 1945 Sep. 1945 Sep. 1945 Oct. 1945 Oct. 1945 Oct. 1945 Nov. 1945 Nov. 1945

Report

M.26 ' Sept. 1943; Oct. 1943; M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 15b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; 40; 42~44; 46; 47; 49; 50; 62-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81 M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 16b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; 40; 42-44; 46; 47; 49; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81 M.l 2d; 14; 24; 26; 29; 33; 35; 37; 40; 42; 44; 62; 63; 66; 66; 68; 69; 72; 73; 75-78; 80; 81 July 1942-Sept. 1943 (Phys. Chem.); Sept. 1943 (Phys. Chem.); M.3; 7; 9; 12c; 16a; 25; 30; 34; 38; 41; 46; 48; 51; 68; 61; 64; 67; 70; 74; 79 Sept. 1943 {Phys. Chem.); M.3; 7; 9; 12c; 15a; 25; 30; 34; 38; 41; 45; 48; 51; 68; 61; 64; 67; 70; 74; 79 M.lSb, c; 16-20; 22-24; 26-29; 31-33; 36-37; 39; 40; 42~44; 46; 47; 49; 60; 62-57; 69; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81 M.62; 63; 66; 66; 68; 69; 72; 73; 75-78; 80; 81 M.24; 26; 29; 33; 35; 37; 39; 40; 42-44; 46; 47; 49; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 76-78; 80; 81 Microanalyses M.12d; 14; 24; 26; 29; 33; 35; 37; 40; 42; 44 M.39; 40; 42-44; 46; 47; 49; SO; 62-67; 69; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81 July 1942-Sept. 1943 (Phys. Chem.); Sept. 1943 {Phys. Chem.); M.3; 7; 9; 12c; 15a; 26; 30; 34; 38; 41; 46; 48; 61; 68; 61; 64; 67; 70; 74; 79 M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 15b, c; 16-20; 22-24; 26-29; 31-33; 36-37; 39; 40; 42-44; 46; 47; 49; 60; 52-67; 69; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81 July 1942-Sept. 1943 (Phys. Chem.); Sept. 1943 (Phys. Chem.); M.3; 7; 9; 12c; 16a; 26; 30; 34; 38; 41; 46; 48; 61; 68; 61; 64; 67; 70; 74; 79 M.12d; 14; 24; 26; 29; 33; 35; 37; 40; 42; 44; 62; 63; 66; 66; 68; 69; 72; 73; 76-78; 80; 81 Sept. 1943 M.24; 26; 29; 33; 36; 37; 39; 40; 42-44; 46; 47; 49; 60; 62-67; 69; 60; 62; 63; 66; 66; 68; 69; 72; 73; 76-78; 80; 81 July-Oct. 1942; Nov. 1942-Feb. 194S; Apr.-July 1943; Sept. 1943; Oct. 1943; M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 15b, c; 16-20; 22-24; 26-29; 31-33; 36-37; 39; Jfl; 42-44; 46; 47; 49; 50; 52-57; 69; 60, 62; 63; 66; 66; 68; 69; 72; 73; 76-78; 80; 81 M.12d; 14; 24; 26; 29; 33; 35; 37; 40; 42; 44 Nov. 1942-Feb. 1943; Apr.-July 1943; Sept. 1943; Oct. 1943; M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 16b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; Jfi; 42-44; 46; 47; 49; 50; 62-57; 69; 60; 62; 63; 66; 66; 68; 69; 72; 73; 76-78; 80; 81 Oct. 1943; M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 16b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; Jfi; 42-44; 46; 47; 49; 60; 62-67; 59; 60; 62; 63; 66; 66; 68; 69; 72; 73; 76-78; 80; 81 July-Oct. 1942 (Phys. Chem.)

APPENDIX Report

Investigator Hoffhine, C. E., Jr. Hoffman, C. H. Humphrey, W. R. Jelinek, V. C. Johnson, Ο. H. Kaczka, E. A. Koniuszy, F. R. Kuna, Martin Ladenburg, Kurt McGregor, John H. McPherson, J. F. Meiss, Ε. H. Mozingo, Ralph Peck, R. L. Phillips, R. F. Pierson, Ε. H. Reiss, Wilhelm Rogers, E. F. Rosalsky, Leonard Scudi, J. V. Sheehan, J. C. Shunk, C. H. Southwick, P. L. Stiller, Ε. T. Thornton, E. J., Jr. Tishler, Max Trenner, N. R. Webb, T. J. Wilson, A. N. Wolf, D. E.

1059

Sept. 1943; Oct. 1943; M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 15b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; 40; 42-44; 46; 47; 49; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81 M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 15b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; 40; 42-44; 46; 47; 49; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81

Microanalyses

M.21; 21a, b, c, d; 71 M.l; 2; 6; 8; 10; 12a, 6, d; 13; 14; 15b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; 40; 42-44; 46; 47; 49; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81 M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 15b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; 40; 42-44; 46; 47; 49; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69;72; 73; 75-78; 80; 81 M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 15b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; 40; 42-44; 46; 47; 49; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81 Nov. 1942-Feb. 1943; Apr-July 1943; Sept. 1943; Oct. 1943; M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 15a; 25; 30; 34; 38; 41; 45; 48; 51; 58; 61; 64; 67; 70; 74; 79 M.l 2d; 14; 24; 26; 29; 33; 35; 37; 40; 42; 44; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81

Microanalyses

Sept. 1943; Oct. 1943; M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 15b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; 40; 42-44; 46; 47; 49; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81

Microanalyses

Sept. 1943; Oct. 1943; M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 15b, c; 16-20; 22-24; 26-29; 31-S3; 35-37; 39; 40; 42-44; 46; 47; 49; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81 July-Oct. 1942; Nov. 1942-Feb. 1943; Apr-July 1943; Sept. 1943; Oct. 1943; M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 15b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; 40; 42-44; 46; 47; 49; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81 M.6; 8; 10; 12a, b, d; 13; 14; 15b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; 40; 42-44; 46; 47; 49; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81 M.l 2d; 14; 24; 26; 29; 33; 35; 37; 40; 42; 44 July 1942-Sept. 1943 (Phys. Chem.); Sept. 1943 (Phys. Chem.); M.3; 7; 9; 12c; 15a; 25; 30; 34; 38; 41; 45; 48; 51; 58; 61; 64; 67; 70; 74; 79 M.l; 2; 6; 8; 10; 12a, b, d, 13; 14; 15b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; 40; 42-44; 46; 47; 49; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81

Microanalyses

M.21; 21a, b, c, d; 71 M.l 2d; 14; 24; 26; 29; S3; 35; 37; 39; 40; 42-44; 46; 47; 43; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81 M.6; 8; 10; 12a, b, d; 13; 14; 15b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; 40; 42-44; 46; 47; 49; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81 M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 15b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; 40; 42-44; 46; 47; 49; 50; 52-57; 59; 60; 62: 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81 July-Oct. 1942; Nov. 1942-Feb. I943

Microanalyses

M.12d; 14; 24; 26; 29; 33; 35; 37; 39; 40; 42-44; 46; 47; 49; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81 July 1942-Sept. 1943 (Phys. Chem.); Sept. 1943 (Phys. Chem.); M.3; 7; 9; 12c; 15a; 25; 30; 34; 38; 41; 45; 48; 51; 58; 61; 64; 67; 70; 74; 79 July 1942-Sept. 1943 (Phys. Chem.); Sept. 1943 (Phys. Chem.); M.3; 7; 9; 12c; 15a; 25; 30; 34; 38; 41; 45; 48; 51; 58; 61; 64; 67; 70; 74; 79 Oct. 1943; M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 15b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; 40; 4244; 46; 47; 49; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81 Sept. 1943; Oct. 1943; M.l; 2; 6; 8; 10; 12a, b, d; 13; 14; 16b, c; 16-20; 22-24; 26-29; 31-33; 35-37; 39; 40; 42-44; 46; 47; 49; 50; 52-57; 59; 60; 62; 63; 65; 66; 68; 69; 72; 73; 75-78; 80; 81

APPENDIX

1060

University of Michigan DEPARTMENT OP CHEMISTRY

Bachmann, Werner E. Chemerda, John M. Cronyn, Marshall W.

Deno, Norman C. Horning, Evan C.

Jenner, Edward L. Johnson, G. Dana

Scott, Lawrence B. Smith, Peter A. S. Warzynski, Raymond J. Date received

Date of report

Report No.

B.l S S

4

6 6 7 8 9 10 11 IS IS U 16 16 17 18 19 SO ν Sl

,

16 Feb. 1944 1 Mar. 1944 1 Apr. 1944 1 May 1944 1 Jun. 1944 1 Jul. 1944 1 Aug. 1944 1 Sep. 1944 1 Oct. 1944 1 Nov. 1944 1 Dec. 1944 1 Jan. 1945 1 Feb. 1945 1 Mar. 1945 1 Apr. 1945 1 May 1945 1 Jun. 1945 1 Jul. 1945 1 Aug. 1945 1 Sep. 1945 21 Dec. 1945

By O.S.R.D. (Ν. Y.)

By Medical Research Council

21 Feb. 1944 4 Mar. 1944 17 Apr. 1944 6 May 1944 5 Jun. 1944 10 Jul. 1944 14 Aug. 1944 11 Sep. 1944 11 Oct. 1944 13 Nov. 1944 11 Dec. 1944 15 Jan. 1945 3 Feb. 1945. 14 Mar. 1945 18 Apr. 1945 21 May 1945 15 Jun. 1945 20 Jul. 1945 20 Aug. 1945 20 Sep. 1945 29 Dec, 1945

7 Jul. 1944 7 Jul. 1944 15 Sep. 1944 7 Jul. 1944 20 Jun. 1944 26 Jul. 1944 8 Sep. 1944 21 Sep. 1944 25 Oct. 1944 23 Nov. 1944 11 Jan. 1945 7 Feb. 1945 15 Feb. 1945 28 Mar. 1945 5 May 1945 31 May 1945 26 Jul. 1945 1 Aug. 1945 5 Sep. 1945 4 Oct. 1945

DEPARTMENT OF PHYSICS

Ashbyl Leonard J.

Dangl, Robert

Fowler, R. G.

Fuson, Nelson

Randall, Η. M.

Date received Report No.

R.l S S 4 6 6a 6 7 8 9 10 11 ISa, b

Date of report

17 Oct. 1944 31 Mar. 1945 30 Apr. 1945 31 May 1945 30 Jun. 1945 1 Aug. 1945 31 Jul. 1945 31 Aug. 1945 30 Sep. 1945 31 Oct. 1945 13 Nov. 1945 30 Nov. 1945 31 Dec. 1945

By O.S.R.D. (Ν. Y.)

By Medical Research Council

19 Oct. 1944 5 Apr. 1945 5 May 1945 10 Jun. 1945 9 Jul. 1945 6 Sep. 1945 31 Jul. 1945 6 Sep. 1945 6 Oct. 1945 15 Nov. 1945 19 Nov. 1945 6 Dec. 1945 3 Jan. 1946

8 Nov. 1944 20 Apr. 1945 11 May 1945 20 Jun. 1945 26 Jul. 1945 11 Sep. 1945 22 Aug. 1945 11 Sep. 1945 10 Oct. 1945 20 Nov. 1945 28 Nov. 1945 15 Jan. 1946

United States Department of Commerce NATIONAL BUREAU OP STANDARDS

Johnson, Walter H.

Prosen, Edward J.

Rossini, Frederick D. Date received

Report No.

BS.l S

Date of report

18 Apr. 1945 20 Sep. 1945

By O.S.R.D. (Ν. Y.)

By Medical Research Council

23 Apr. 1945 24 Sep. 1945

20 Jun. 1945 27 Dec. 1945

APPENDIX

1061

Parke, Davis & Company Alberi, Joseph Τ. Bartz, Quentin R. Binkley, Stephen Β. Brown, John M. Carter, Herbert E.14

Cheney, Lee C. Controulis, John Crooks, Harry M. Hooper, Irving R.

Hummel, Ralph D. Jones, Eldon M. Long, Loren M. Miller, James R.

Piening, John R.; Sweet, Leon A. Vandenbelt, John M. Van House, Russell W.

Date received Report No.

PD.l git S1« 4U 6" 6"

71«

8 9 10 11 IB IS

U 16 16 17 18 19 SO Sl SS SS

U SB S6 S7

Date of report By O.S.R.D. (Ν. Y.)

By Medical Research Council

29 Dec. 1943

3 Jan. 1944

15 Mar. 1945

15 Jan. 1944

17 Jan. 1944 24 Jan. 1944 31 Jan. 1944 31 Jan. 1944 31 Jan. 1944 18 Feb. 1944 17 Mar. 1944 17 Apr. 1944 18 May 1944 16 Jun. 1944 8 Jul. 1944 17 Jul. 1944 17 Aug. 1944 18 Sep. 1944 23 Oct. 1944 11 Nov. 1944 18 Nov. 1944 21 Dec. 1944 22 Jan. 1945 19 Feb. 1945 20 Mar. 1945 21 Apr. 1945 24 May 1945 24 Jun. 1945 11 Jan. 1946

7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 15 Sep. 1944 7 Jul. 1944 29 Jun. 1944 26 Jul. 1944 26 Jul. 1944 8 Sep. 1944 3 Oct. 1^44 8 Nov. 1944 23 Nov. 1944 2 Dec. 1944 11 Jan. 1945 7 Feb. 1945 3 Mar. 1945 7 Apr. 1945 5 May 1945 7 Jun. 1945 26 Jul. 1945 16 Jan. 1946

27 Jan. 1944 27 Jan. 1944 15 Feb. 1944 15 Mar. 1944 14 Apr. 1944 15 May 1944 15 Jun. 1944 30 Jun. 1944 13 Jul. 1944 14 Aug. 1944 15 Sep. 1944 17 Oct. 1944 7 Nov. 1944 15 Nov. 1944 15 Dec. 1944 15 Jan. 1945 15 Feb. 1945 15 Mar. 1945 15 Apr. 1945 15 May 1945 15 Jun.1945 31 Oct. 1945

Chas. Pfizer & Co., Inc. Date received Report No.

P.l S 5 4 δ" 6

Date of report By O.S.R.D. (Ν. Y.)

By Medical Research Council

24 Dec. 1943

6 Jan. 1944

7 Jul. 19441'

2 Jan. 1944 3 Jan. 1944 2 Jan. 1944 6 Jan. 1944 15 Nov. 1943

6 Jan. 1944 6 Jan. 1944 6 Jan. 1944 8 Jan. 1944 18 Jan. 1944

7 Jul. 1944 7 Jul. 19441' 7 Jul. 1944" 7 Jul. 1944" 7 Jul. 1944"

Consultant. Identical with A./. Circulated in abstract form only. ** Transmitted in abstract form only. 11 Published in the J. Biol. Ckem., 1SS, 249 (1944). u

16

11

17

Investigators

Murphy, F. X., Bogert, V. V.,iand Brown, Ε. V. ι Regna, P. P. Smith, W. J. Maxwell, C. E. McMahan, J. R. Brown, Ε. V., and Pasternack, R.

APPENDIX Date received Date of report

Investigators By O.S.R.D. (Ν. Y.) By Medical Research Council

.7

5 Feb. 1944

9 Feb. 1944

7 Jul. 1944

7ι

30 Mar. 1944

31 Mar. 1944

7 Jul. 1944

8

9 Mar. 1944

11 Mar. 1944

7 Jul. 1944

9

4 Apr. 1944

8 Apr. 1944

7 Jul. 1944

10

3 May 1944

6 May 1944

7 Jul. 1944

11

3 May 1944

6 May 1944

7 Jul. 1944

IS

7 Jun. 1944

8 Jun. 1944

29 Jun. 1944

U

IS

7 Jun. 1944 12 Jul. 1944

8 Jun. 1944 14 Jul. 1944

29 Jun. 1944 26 Jul. 1944

IB

.8 Aug. 1944

11 Aug. 1944

30 Aug. 1944

16 17

30 Aug. 1944 8 Sep. 1944

11 Sep. 1944 11 Sep. 1944

21 Sep. 1944 21 Sep. 1944

18

6 Oct. 1944

7 Oct. 1944

19 Oct. 1944

19

8 Nov. 1944

10 Nov. 1944

23 Nov. 1944

20

7 Dec. 1944

8 Dec. 1944

20 Dec. 1944

21

18 Jan. 1945

21 Jan. 1945

7 Feb. 1945

22

8 Feb. 1945

9 Feb. 1945

27 Feb. 1945

28

9 Mar. 1945

12 Mar. 1945

28 Mar. 1945

U

9 Apr. 1945

10 Apr. 1945

5 May 1945

26

8 May 1945

9 May 1945

18 May 1945

26

7 Jun. 1945

10 Jun. 1945

20 Jun. 1945

27

28

28 Jun. 1945 5 Jul. 1945

29 Jun. 1945 9 Jul. 1945

26 Jul. 1945 26 Jul. 1945

29

7 Aug. 1945

10 Aug. 1945

31 Aug. 1945

SO Sl 82

13 Aug. 1945 11 Sep. 1945 9 Oct. 1945-

18 Aug. 1945 14 Sep. 1945 11 Oct. 1945

4 Sep. 1945 18 Sep. 1945 16 Oct. 1945

SS

8 Nov. 1945

9 Nov. 1945

28 Nov. 1945

3

Bogert, V. V., Brown, Ε. V., Maxwell, C. E., Murphy, F. X., and Smith, W. J. Bogert, V. V., Brown, Ε. V., Maxwell, C. E., Murphy, F. X., and Smith, W. J. Bogert, V. V., Brown; Ε. V., Maxwell, C. E., Murphy, F. X., and Smith, W. J. Bogert, V. V., Brown, Ε. V., Maxwell, C. E., Murphy, F. X., and Smith, W. J. Bogert, V. V., Brown, Ε. V., Maxwell, C. E., Murphy, F. X., and Smith, W. J. Bogert, V. V., Brown, Ε. V., and Murphy, F. X. Bogert, V. V., Brown, Ε. V., Maxwell, C. E., and Murphy, F. X. Brown, Ε. V., and Murphy, F. X. Bogert, V. V., Brown, Ε. V., Maxwell, C. E., Murphy, F. X., and Smith, W. J. Bogert, V. V., Brown, Ε. V., and Maxwell, C. E. Brown, Ε. V., and Maxwell, C. E. Bogert, V. V., Brown, Ε. V., Maxwell, C. E., and Murphy, F. X. Bogert, V. V., Brown, Ε. V., Maxwell, C. E., Minieri, P. P., and Murphy, F. X. Bogert, V. V., Brown, Ε. V., Maxwell, C. E., and Murphy, F. X. Bogert, V. V., Brown, Ε. V., Maxwell, C. E., and Murphy, F. X. Bogert, V. V., Brown, Ε. V., and Murphy, F. X. Brown, Ε. V., Maxwell, C. E., and Murphy, F. X. Bogert, V. V., Brown, Ε. V., Maxwell, C. E., and Murphy, F. X. Bogert, V. V., Brown, Ε. V., Maxwell, C. E., and Murphy, F;-X. Bogert, V. V., Brown, Ε. V., and Maxwell, C. E. Bogert, V. V., Brown, Ε. V., and Maxwell, C. E. Regna, P. P. Bogert, V. V., Brown, Ε. V., and Maxwell, C. E. Bogert, V. V., Brown, Ε. V., Maxwell, C. E., and Murphy, F. X. Grenfell, T. C., and Hedger, F. Howard Bogert, V. Y., and Brown, Ε. V. Bogertr V. V., Brown, Ε. V., and Maxwell, C. E. Bogert, V. V., Brown, Ε. V., Maxwell, C. E., and Smith, W. J.

THe Rockefeller Institute for Medical Research Herriott, Roger M.

Northrop, John H. Date received

. No.

Date of report

19 Aug. 1945

By O.S.R.D. (Ν. Y.)

By Medical Research Council

22 Aug. 1945

4 Sep. 1945

APPENDIX

1063

Shell Development Company Ballard, Seaver A. Brattain, R. Robert Harp, William R., Jr.

Holm, Roy T. Magruder, Gordon H. Melstrom, Donald S.

Mitchell, LIoyd J. Norton, Douglas G. Rasmussen, Robert S.

Smith, Curtis W. Tunnicliff, Donald D.

Date received Report No.

Sh.l 2 Za S 4 6 6 7 7a 8 9 10 11 12 IS ISa U 16

Date of report

31 Aug. 1944 30 Sep. 1944 31 Oct. 1944 30 Nov. 1944 31 Dec. 1944 31 Jan. 1945 28 Feb. 1945 31 Mar. 1945 30 Apr. 1945 31 May 1945 30 Jun. 1945 26 Jul. 1945 31 Jul. 1945 31 Aug. 1945 31 Oct. 1945

By O.S.R.D. (Ν. Y.)

By Medical Research Council

19 Sep. 1944 25 Oct. 1944 27 Nov. 1944 14 Nov. 1944 26 Dec. 1944 22 Jan.1945 24 Feb. 1945 21 Mar. 1945 23 Apr. 1945 23 Apr. 1945 21 May 1945 25 Jun.1945 26 Jul. 1945 6 Aug. 1945 27 Aug. 1945 13 Nov. 1945 4 Oct. 1945 13 Nov. 1945

3 Oct. 1944 6 Nov. 1944 8 Dec. 1944 2 Dec. 1944 11 Jan. 1945 7 Feb. 1945 15 Mar. 1945 6 Apr. 1945 11 May 1945 11 May 1945 31 May 1945 26 Jul. 1945 10 Aug. 1945 22 Aug. 1945 5 Sep. 1945 27 Nov. 1945 11 Oct. 1945 28 Nov. 1945

Squibb Institute for Medical Research Date received Report No.

Date of report By O.S.R.D. (Ν. Y.)

By Medical Research Council

S.l

20 Dec. 1943

27 Dec. 1943

14 Feb. 1944

2 S

13 Jan. 1944 1 Aug. 1943

15 Jan. 1944 18 Jan. 1944

7 Jul. 1944

1 Nov. 1943

Apr. 1944

Sa 4" 5

11 ,e

14 Feb. 1944

3 Feb. 1944

7 Jul. 1944

6 7

31 Jan. 1944 1 Feb. 1944

3 Feb. 1944 4 Feb. 1944

7 Jul. 1944 7 Jul. 1944

8

7 Feb. 1944

10 Feb. 1944

7 Jul. 1944

9

1 Mar. 1944

6 Mar. 1944

7 Jul. 1944

9a

3 Mar. 1944

6 Mar. 1944

7 Jul. 1944

1Q20

7 Mar. 1944

14 Mar. 1944

7 Jul. 1944

Investigators

Dutcher, J. D., MacPhillamy, H. B., Stavely, H. E., and Wintersteiner, 0. Wintersteiner, 0., Moore, M. MacPhillamy, H. B., Wintersteiner, 0., and Alicino, J. F. Dutcher, J. D., MacPhillamy, H. B., Stavely, H. E., and Wintersteiner, 0. Adler, M., MacPhillamy, H. B., Dutcher, J. D., Stavely, H. E., Chow, B. F., Menzel, A. E. 0., and Wintersteiner, 0. Chow, B. F., and McKee, C. M. Menzel, A. E. 0., Dutcher, J. D., Stavely, H. E., MacPhillamy, H. B., Anchel, M., and Wintersteiner, 0. Lott, W. A., Bernstein, J., Losee, Κ. A., Dexter, M., Shaw, E., Bergeim, F. H., and Stearns, B. MacPhillamy, H. B., Dutcher, J. D., Stavely, H. E., Menzel, A. E. 0., Chow, B. F., Anchel, M., Adler, M., and Wintersteiner, 0. Bergeim, F. H., Lott, W. A., Bernstein, J., Losee, Κ. A., Dexter, M., Shaw, E., and Stearns, B.

Various British reports in the PEN. series, copies of which were supplied to O.S.R.D. by the Squibb Institute. Interpretation of infrared data from the Department of Physics, University of Michigan.

1064

APPENDIX Date received

Report No.

By O.S.R.D. (Ν. Y.)

By Medical Research Council

1 Mar. 1944

5 Apr. 1944

15 Sep. 1944

12a

1 Apr. 1944

5 Apr. 1944

7 Jul. 1944

12b

1 Apr. Apr. 1944

5 Apr. 1944

IS

25 Apr. 1944

29 Apr. 1944

14

1 May 1944

3 May 1944

7 Jul. 1944

IS

1 May 1944

3 May 1944

7 Jul. 1944

16 80 l 7 iι 18 19

1 May 1944 4 May 1944 8 May 1944 1 Jun. 1944

4 May 1944 10 May 1944 8 Jun. 1944

7 Jul. 1944 7 Jul. 1944 7 Jul. 1944 29 Jun. 1944

20

1 Jun. 1944

8 Jun. 1944

29 Jun. 1944

21

1 Jul. 1944

6 Jul. 1944

22 Aug. 1944

22

1 Jul. 1944

6 Jul. 1944

22 Aug. 1944

28™ U

3 Jul. 1944 1 Aug. 1944

13 Jul. 1944 2 Aug. 1944

30 Aug. 1944 22 Aug. 1944

25

1 Aug. Aug. 1944

2 Aug. Aug. 1944 1944

22 Aug. 1944

22 May 1944 1 Sep. 1944

24 May 1944 5 Sep. 1944

8 Sep. 1944 20 Sep. 1944

28

1 Sep. 1944

5 Sep. 1944

20 Sep. 1944

29

1 Oct. 1944

4 Oct. 1944

19 Oct. 1944

SO

1 Oct. 1944

4 Oct. 1944

19 Oct. 1944

Sl

1 Nov. 1944

3 Nov. 1944

18 Nov. 1944

32

1 Nov. 1944

3 Nov. 1944

18 Nov. 1944

SS

1 Dec. 1944

6 Dec. 1944

11 Jan. 1945

S.ll

26 20 27

11

Investigators

Date of report

An abstract of information contained in

S.S, S.Sa, and S.1S.

7 Jul. 1944

Menzel, A. E. O., Anchel, M., Dutcher, J. D., Wintersteiner, O.; Lott, W. A., Bernstein, J., Losee, Κ. A., Dexter, M., Shaw, E., Biergeim, F. H., and Stearns, B. Dutcher, J. D., MacPhillamy, H. B., Stavely, H. E., Menzel, A. E. O., Anchel, M., and Wintersteiner, 0. Losee, Κ. A., Lott, W. A., Bernstein, J., Dexter, M., Shaw, E., and Stearns, B. Adler, M., MacPhillamy, H. B., and Winter­ steiner, 0. Stavely, H. E., Fried, J., Koerber, W. L., Dutcher, J. D., Anchel, M., Menzel, A. E. 0., and Wintersteiner, O. Bernstein, J., Lott, W. A., Losee, Κ. A., Dexter, M., Shaw, E., and Stearns, B. Wintersteiner, 0., and Coy, N. Wintersteiner, 0., and Moore, M. MacPhillamy, H. B., Dutcher, J. D., Stavely, H. E., Menzel, A. E. 0., Fried, J., Koerber, W. L., Adler, M., and Wintersteiner, 0. Shaw, E., Lott, W. A., Bernstein, J., Losee, Κ. A., Dexter, M., Stearns, B., and McCasland, G. E. Dutcher, J. D., Stavely, H. E., Anchel, M., Fried, J., Menzel, A. E. 0., Alicino, J. F., and Wintersteiner, 0. McDowell, W. B., Lott, W. A., Bernstein, J., Losee, Κ. A., Dexter, M., Shaw, E., Stearns, B., and McCasland, G. E. Wintersteiner, 0., and Coy, N. Stavely, H. E., Dutcher, J. D., Anchel, M., Menzel, A. E. 0., and Wintersteiner, 0. Stearns, B., Lott, W. A., Bernstein, J., Dexter, M., Shaw, E., McDowell, W. B., Nyman, Μ. Μ. A., and McCasland, G. E. Wintersteiner, 0., and Coy, N. Bernstein, J., Lott, W. A., Dexter, M., Shaw, E., Stearns, B., Nyman, Μ. A., McCasland, G. E., DiCarlo, F. J., and Bergeim, F. H. Dutcher, J. D., Stavely, H. E., Menzel, A. E. 0., Anchel, M., and Wintersteiner, 0. Nyman, Μ. A., Lott, W. A., Bernstein, J., Dexter, M., Shaw, E., McCasland, G. E., and DiCarlo, F. J. Stavely, H. E., Dutcher, J. D., Menzel, A. E. 0., Anchel, M., Adler, M., and Winter­ steiner, 0. Dexter, M., Lott, W. A., Bernstein, J., Shaw, E., Stearns, B., McDowell, W. B., Nyman, Μ. A., and DiCarlo, F. J. Anchel, M., Wintersteiner, 0., Menzel, A. E. 0., Dutcher, J. D., Stavely, H. E., and Moore, M. Bernstein, J., Lott, W. A., Dexter, M., Shaw, E., Stearns, B., McDowell, W. B., MeCasland, G. E., and Nyman, M. A.

APPENDIX

1065

Date received Report No. Date of report

Investigators By O.S.R.D. (Ν. Y.)

By Medical Research Council

S.S4

1 Dec. 1944

6 Dec. 1944

11 Jan. 1945

85

2 Jan. 1945

5 Jan. 1945

7 Feb. 1945

36

1 Jan. 1945

5 Jan. 1945

7 Feb. 1945

37

1 Feb. 1945

7 Feb. 1945

17 Feb. 1945

38

1 Feb. 1945

7 Feb. 1945

17 Feb. 1945

39

1 Mar. 1945

12 Mar. 1945

28 Mar. 1945

40

1 Mar. 1945

12 Mar. 1945

28 Mar. 1945

41

2 Apr. 1945

11 Apr. 1945

20 Apr. 1945

4S

1 Apr. 1945

11 Apr. 1945

20 Apr. 1945

48

1 May 1945

14 May 1945

24 May 1945

U

1 May 1945

14 May 1945

24 May 1945

45

1 Jun.1945

11 Jun. 1945

20 Jun. 1945

46

1 Jul. 1945

24 Jul. 1945

10 Aug. 1945

47

1 Aug. 1945

20 Aug. 1945

5 Sep. 1945

48 49

1 Sep. 1945 1 Oct. 1945

13 Sep. 1945 11 Oct. 1945

15 Sep. 1945 16 Oct. 1945

60

1 Nov. 1945

8 Nov. 1945

20 Nov. 1945

61 62

1 Nov. 1945 1 Nov. 1945

8 Nov. 1945 8 Nov. 1945

20 Nov. 1945 20 Nov. 1945

53

22 Jan. 1946

24 Jan. 1946

Anchel, M., Wintersteiner, 0., Menzel, A. E. 0., Stavely, H. E., Dutcher1 J. D., and Moore, M. Shaw, E., Lott, W. A., Bernstein, J., Dexter, M., Stearns, B., and McDowell, W. B. Wintersteiner, 0., Anchel, M., Menzel, A. E. 0., Dutcher, J. D., Stavely, H. E., and Moore, M. Bernstein, J., Lott, W. A., Dexter, M., Shaw, E., McDowell, W. B., and McCasland, G. E. Dutcher, J. D., Stavely, H. E., Anchel, M., Menzel, A. E. 0., Moore, M., and Winter­ steiner, 0. Lott, W. A., Bernstein, J., Dexter, M., Shaw, E., McDowell, W. B., Nyman, Μ. A., and DiCarlo, F. J. Stavely, H. E., Wintersteiner, 0., Anchel, M., Dutcher, J. D., Adler, M., Moore, M., and Menzel, A. E. 0. Bernstein, J., Lott, W. A., "Shaw, E., and Nyman, M. A. Wintersteiner, 0., Stavely, H. E., Adler, M., Menzel, A. E. 0., Moore, M., and Dutcher, J. D. Shaw, E., Lott, W. A., Bernstein, J., and McDowell, W. B. Wintersteiner, 0., Anchel, M.,-Stavely, H. E., Boyack, G. A., Menzel, A. E. 0., Adler, M., and Moore, M. Stavely, H. E., Boyack, G. A., Winter­ steiner, 0., Anchel, M., Dutcher, J. D., Menzel, A. E. 0., Adler, M., and Moore, M. Anchel, M., Stavely, H. E., Boyack, G. A., Moore, M., and Wintersteiner, 0. Wintersteiner, O., Stavely, H. E., Anchel, M., Adler, M., Boyack, G. A., and Moore, M. Anchel, M., Moore, M., and Wintersteiner, 0. Stavely, H. E., Boyack, G. A., Anchel, M., Moore, M., and Wintersteiner, 0. Wintersteiner, 0., Anchel, M., Stavely, H. E., Moore, M., and Boyack, G. A. Alicino, J. P. Shaw, E., Lott, W. A., Bernstein, J., and DiCarlo, F. J. Wintersteiner, 0., Anchel, M., Stavely, H. E., and Moore, M.

Stanford University DEPARTMENT or BIOLOGY

Date received Report No.

Investigators

Date of report By O.S.R.D. (Ν. Y.) By Medical Research Council

Be.l

13 Aug. 1945

30 Aug. 1945

Z

28 Dec. 1945

7 Jan. 1945

11 Sep. 1945

Beadle, George W., Bonner, David M., Horo­ witz, Norman H., and Mitchell, Herschel K. Beadle, George W., Bonner, David M., Horo­ witz, Norman H., and Mitchell, Herschel K.

1066

APPENDIX The Upjohn Company

Bohonos, Nestor Carter, Herbert E.22 Cartland, G. F. Cohen, Harry Emerson, C. H.

Ford, Jared H. Haines, W. J. Hinman, Jack W. • Hunter, J. H. Kolloff, Harold G.

Leach, Byron E. Levin, Robert H. Nathan, Alan H. Rolfson, Stanley T. Tenenbaum, Leon E.

Thomas, Donald G. Wallace, Stuart M. Wesner, Mildred M. Wick, A. N.

Date received Report No.

.V.l Z S 4 6 6 7 8 9 10 11 IH . ISa ISb U IB 16 17 18 19' ZO Zl ZZ ZS U Z4a ZS Z6 Z7 Z8 Z9 SO Sl SS SS 84 M

Consultant.

Date of report

30 Dec. 1943 14 Jan. 1944 8 Feb. 1944 15 Mar. 1944 13 Apr. 1944 9 May 1944 8 Jun. 1944 δ Jul. 1944 30 Jun. 1944 10 Jul. 1944 10 Aug. 1944 10 Sep. 1944 16 Oct. 1944 16 Oct. 1944 7 Nov. 1944 16 Nov. 1944 16 Dec. 1944 15 Jan. 1945 19 Feb. 1945 15 Mar. 1945 15 Apr. 1945 15 May 1945 15 Jun. 1945 15 Jul. 1945 22 Aug. 1945 24 Sep. 1945 15 Sep. 1945 1 Nov. 1945 30 Nov. 1945

10 Dec. 1945 3 Jan. 1946 5 Jan. 1946

By O.S.R.D. (Ν. Y.)

By Medical Research Council

3 Jan. 1944 17 Jan. 1944 17 Feb. 1944 17 Mar. 1944 17 Apr. 1944 15 May 1944 17 Jun. 1944 8 Jul. 1944 8 Jul. 1944 17 Jul. 1944 17 Aug. 1944 18 Sep. 1944 20 Oct. 1944 20 Oct. 1944 11 Nov. 1944 22 Nov. 1944 30 Dec. 1944 22 Jan. 1945 1 Mar. 1945 24 Mar. 1945 19 Apr. 1945 24 May 1945 25 Jun. 1945 23 Jul. 1945 31 Aug. 1945 15 Nov. 1945 24 Sep. 1945 15 Nov. 1945 3 Dec. 1945 14 Dec. 1945 14 Dec. 1945 14 Dec. 1945 14 Dec. 1945 14 Dec. 1945 14 Jan. 1946 14 Jan.1946

7 Jul. 1944 (not circulated) 7 Jul. 1944 7 Jul. 1944 15 Sep. 1944 7 Jul. 1944 29 Jun. 1944 26 Jul. 1944 26 Jul. 1944 26 Jul. 1944 8 Sep. 1944 3 Oct. 1944 8 Nov. 1944 8 Nov. 1944 23 Nov. 1944 2 Dec. 1944 17 Jan. 1945 7 Feb. 1945 25 Mar. 1945 7 Apr. 1945 11 May 1945 7 Jun. 1945 26 Jul. 1945 10 Aug. 1945 11 Sep. 1945 20 Nov. 1945 4 Oct. 1945 28 Nov. 1945

APPENDIX

1067

Winthrop Chemical Company, Inc. Albertson, Noel Γ. Archer, Sydney Buck, J. S. Cavallito, C. J.

Ewing1 G. W. Haskell, Theodore King, John A.

Report No.

Date of report

Kirchner, Frederick Lawson, E. J. McCormack, Jerry R. D.

McMillan, Freeman H. Pratt, Margaret G. Suter, C. M.

Date received By O.S.R.D. (Ν. Y.) W.l 2 S 4 6 6 7 8 9 10 11 12 IS 14 15 16 17

29 Mar. 1944 15 Jan. 1944 15 Apr. 1944 15 May 1944 19 Jun. 1944 19 Jul. 1944 20 Aug. 1944 25 Sep. 1944 18 Oct. 1944 15 Nov. 1944 15 Dec. 1944 15 Jan.1945 15 Feb. 1945 15 Mar. 1945 15 May 1945 15 Jun. 1945 6 Nov. 1945

4 Apr. 1944 4 Apr. 1944 21 Apr. 1944 25 May 1944 3 Jul. 1944 25 Jul. 1944 5 Sep. 1944 29 Sep. 1944 27 Oct. 1944 7 Dec. 1944 27 Dec. 1944 25 Jan. 1945 1 Mar. 1945 24 Mar. 1945 23 May 1945 27 Jun. 1945 17 Nov. 1945

By Medical Research Council 15 Sep. 1944 . (not circulated) 7 Jul. 1944 20 Jun. 1944 26 Jul. 1944 5 Aug. 1944 20 Sep. 1944 13 Oct. 1944 8 Nov. 1944 20 Dec. 1944 11 Jan. 1945 15 Feb. 1945 25 Mar. 1945 7 Apr. 1945 2 Jun. 1945 26 Jul. 1945 28 Nov. 1945

SUBJECT INDEX

INTRODUCTION AND KEY

index, without regard to the names used in different places in the text by authors of individual chapters. Often the complexity of the compounds is such that one has difficulty in recognizing that two or more different names pertain to the same compound and some instances of this will have escaped attention in the preparation of the index. An unfortunate result of the indexing system is that, not infre­ quently, names used in the text will not be found in their original form in the index; conversely, the names appearing in the index may not occur in precisely the same form in the text. It is hoped that the advantage of having all references under one entry will outweigh the attendant disadvantages.

The listing of organic compounds in this index has been rendered difficult by the inherent com­ plexity of the subject matter and by the circum­ stance that the authors of the various chapters have not used a uniform system of nomenclature. To aid in locating specific subjects and compounds each chapter has been provided with a detailed table of contents and numerous cross references have been furnished. In assigning systematic names the general principles and' methods used in Chemical Abstracts have been adopted, with a few exceptions, but the entries have been made without inverting the names of the substituents. The principal func­ Trivial Names of Penicillin Derivatives tional group is included in the name of the parent compound (see Chem. Abs., 39, 5875 (1945)) and Desdimethyl-: prefix used to indicate cysteine prefixes designating substituent groups are placed analogs of penicillin derivatives, in which the two in alphabetical order before the name of the parent methyl groups of the penicillamine residue are compound; thus, a-Caproylamino-4-carboxy-5,5lacking. dimethyl-3-ethyl-2-thiazolidineacetic acid, 4-Carboxy-a - phenylacetamido-2-thiazolinepropionic Desdimethylpenicilloic acids: example, Desdiacid, etc. methylbenzylpenicilloic acid; systematic name, 4Generally accepted trivial or common names Carboxy - a - phenylacetamido - 2 - thiazolidineacetic (e.g., glycine, penicillamine, phenaceturic acid, acid. etc.) are used in preference to systematic names for Desdimethylpenillic acids: example, Desdispecific compounds, and simple derivatives thereof, methylbenzylpenilloic acid, systematic name, 5-benzbut homologs and complex substitution products yl-2,3,7,7a-tetrahydroimidazo[5,l-&]thiazole-3,7are usually entered under systematic names. New dicarboxylic acid. and convenient trivial names devised in the course of the chemical studies of penicillin are listed Desdimethylpenilloic acids: example, Desdibelow, together with type formulas and systematic methylbenzylpenilloic acid, systematic name, names for the more important ones. 2-phenylacetamidomethyl-4-thiazolidinecarboxylic Functional derivatives of organic acids, alde­ acid. hydes, etc., acyl and alkyl derivatives of amines, Desthiopenicillins: example, Desthiobenzylpeniand salts of organic acids and bases are treated in various ways. Where the importance of a specific cillin; systematic name, a-Isopropyl-2-oxo-3derivative or simplification of indexing appeared to phenylacetamido-l-azetidineacetic acid. warrant it, a derivative has been entered separately CH2-N-CH-CH(CH3)2 by its own specific name; e.g., α-Methyl benzylpenicilloate, Benzylpenicilloic α-benzylamide, etc. I I I C6H6CH2CONH-CH—CO CO2H In other instances the functional derivative may be listed as a sub-entry under the name of the parent Desthiopenicillenic acids: example, Desthiobenzcompound; e.g., 4-Thiazolidinecarboxylic acid, methyl ester. Where there is a small number of ylpenicillenic acid; systematic name, iV-(2-benzylreferences to the parent compound and/or its func­ 5-oxo-4(5)-oxazolyIidenemethyl) valine. tional derivatives, all of the references are given Desthiopenicilloic acids: example, Desthiobenzylunder the name of the parent compound without penicilloic acid; iV-(2-Carboxy-2-phenylacetamidoindication of the particular derivative involved; ethyl)valine. thus, references to various derivatives are included Desthiopenillamines: example, Desthiobenzylpenunder the single entry, Heptylpenilloaldehyde. An attempt has been made to list all of the refer­ illamine; systematic name, 2-benzyl-a-isopropyl-lences to a particular compound in one place in the imidazoleacetic acid.

Desthiopenillonic acids: example, Desthiobenzylpenillomc acid; systematic name, a-Isopropyl-5oxo-3-phenylacetyl-l-imidazoleacetic acid.

derivatives of a parent open-chain skeleton of the ' 'N- (penaldo)penicillamine'' type:

Homopenicilloic acids: example, Benzylhomopenicilloic acid; systematic name, 4-Carboxy-5,5dimethyl - a - phenylacetamido - 2 -thiazolidinepropionic acid.

Isopenillic acids: example, 2-Pentenylisopenillic acid; systematic name, 4-Carboxy-a-(l-mercaptoisopropyl)-l-imidazoleacetic acid.

Norpenaldic acid: specific name for the a-formyl derivative of N-formylglycine; systematic name, a-Formamidomalonaldehydic acid.

Penillamines: example, A mylpenillamine; systematic name, 2-Amyl-a-(l-mercaptoisopropyl)-limidazoleacetic acid. Penaldic acids: example, Phenylpenaldic acid; systematic name, Benzamidomalonaldehydic acid. Acetals of the unacylated amino-acid may be named as /3,/3-dialkoxy derivatives of alanine; e.g., j8,|8-Diethoxyalanine. Penicillamine: specific name for /3,/S-dimethylcysteine; systematic name, a-amino-j3-mercaptoisovaleric acid.

The individual penicillins differ from one another merely by having different organic groups as the substituent radicals of the acylamino group (Rof the general formula shown above). The generalized open-chain form of the penicillin skeleton affords a possible basis for a universal numbering system that may be used in connection with any of the trivial names. Penicillinates: examples, Rubidium benzylpenicillinate, Methyl 2-pentenylpenicillinctte. This name is used to designate salts and esters of the penicillins. The terms benzylpenicillin, 2-pentenylpenicillin, etc., as used in the index refer generally to the sodium salt of the penicillin, which is the form in which the penicillins are available normally from the culture media. Penicilloic acids: example, Benzylpenicilloic acid; systematic name, 4 - C a r b o x y - 5 , 5 - d i m e t h y l - a phenylacetamido-2-thiazolidineacetic acid. The derivatives of the carboxyl functions of the penicilloic acids are indicated by using the designation a - for the carboxyl of the penaldic moiety and (3for that of the penicillamine residue. Penillic acids: example, Amylpenillic acid; systematic name, 5-Amyl-2,3,7,7a-tetrahydro-2,2-dimethylimidazo[5, l-6]thiazole-3,7-dicarboxylic acid. Penilloaldehydes: example, 2-Pentenylpenilloaldehyde; systematic name, 3-hexenoylaminoacetaldehyde or N-(formylmethyl)-3-hexenoic amide. Penilloic Acids: example, Benzylpenilloic acid; systematic name, 5,5-Dimethyl-a-phenylacetamidomethyl-4-thiazolidinecarboxylic acid. Penillonic acids: example, Benzylpenillonic acid; systematic name, 2,3,5,6,7,7a-Tetrahydro-2,2-dimethyl-5-oxoimidazo[2,l-6]thiazole-3-carboxylic acid.

Penicillaminic acid: specific name for /3,/3-dimethylcysteic acid; systematic name, a-amino-^sulfoisovaleric acid. Penicillenic acids: example, Benzylpenicillenic acid; systematic name, iV-(2-Benzyl-5-oxo-4(5)-oxazolylidenemethyl)penicillamine. Penicillins: example, 2-Pentenylpenicillin. The individual penicillins are named by adding prefixes designating the radical attached to the carbonyl function of the acylamino group present in the penaldic moiety of the molecule. All of the penicillins may be regarded essentially as cyclized

Pseudopenicillinates: example, Methyl pseudopenicillinate. This name has been assigned to the antibiotically inactive compound obtained by treating methyl benzylpenicillinate with hydrogen chloride, followed by dehydrohalogenation with diazomethane or silver oxide (see Chapter X X I V ) .

SUBJECT INDEX A Absorption spectra, see infrared spec­ troscopy and Ultraviolet spectroscopy, and also sub-entries spectrum, infrared, and spectrum, ultraviolet, under names of individual com­ pounds. 4-Acetamidinomethylene-2-phenyl5-oxazolone, 829 3-Acetamido-2,4-diketopyrrolidine, 847 3-Acetamido-2,4-diketo-l-acetylpyrrolidine, 847 a-Acetamido-jj,/3-dimethylacrylic acid, azlactonization of, 457 conversion to an oxazolone, 465 from penicillamine, 53 from thiazolidine sulfoxides, 157 prepn. of, 465, 733 x-ray crystallography of, 318 N-Acetamidoethyl valine, 135 «-Acetamido-/i-mercaptoisovaleric acid, 842 4-Acetamidomethylene-2-phenyl-5oxazolone, 817 4-Acethy drazinomethylene-2 -phenyl 6-oxazolone, 829 2-Acetonyl-2-methyl-4-thiazoIidinecarboxylic acid, 961 2-Acetonyl-4-thiazolidinecarboxylic acid, 959 2-(l'-Acetoxyamyl)-4-styryl-5-ethoxyoxazole, 698 2-p-Acetoxybenzyl-4-ethoxymethylene-5-oxazolone, 523, 812 2-p-Acetoxybenzyl-4-hydroxymethylene-6-oxazolone, 941 p-Acetoxybenzylpenaldic acid, 515, 812 4-Acetoxymethylene-2-benzyl-5oxazolone, reaction with penicill­ amine, 898 4-Acetoxymethylene-2-phenyl-6oxazolone, 822 p-Acetoxyphenylacetamidoacetal, 121, 139 a-p-Acetoxyphenylacetamido-4-carboxy -2-thiazolidineacetic acid, 941 a-p-Acetoxyphenylacetamido-/3-ethoxyacrylic acid, 812 2 -p -Acetoxyphenylacetamidomethyl 6,6-dimethyl-4-thiazolidine-carboxylic acid, 140 p-Acetoxyphenylacetic acid, 139, 812 p-Acetoxyphenylacetonitrile, 812 p-Acetoxyphenylacetyl chloride, 139, 491, 812 Acetylamino-, see Acetamido-. 4-(N-Acetylanilinomethylene) -2 phenyl-5-oxazolone, 898 N-Acetyl-N-benzylalanine, 944 acid, N-Acetyl-