Studies on Aziactones and Rhodanines Derived From Thiophene Aldehydes

Citation preview

r

FORDHAM UNIVERSITY GRADUATE SCHOOL

19LP.

This dissertation prepared under my direction by

Bernard F. Cro^e

entitled .............-

• U.-.

- ./rC.-i.... :e.__ PiLheDirUPiF.:

Deiil M't u DDG.u THiOPHeMe DLDaHXDDS

has been accepted in partial fulfilment of the requirements for the

Degree of

1)0c tOP

Dr. Friedrich Nord (Faculty A d vise r)

r

Studies on Azlactones and Rhodanines Derived from Thiophene Aldehydes

By Bernard F . Crowe B.S., Fordham College, 1943 M.S., Fordham University, 1948

DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF CHEMISTRY AT FORDHAM UNIVERSITY

NEW YORK 1950

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ProQuest Number: 10992988

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is d e p e n d e n t upon the quality of the copy subm itted. In the unlikely e v e n t that the a u thor did not send a c o m p le te m anuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a n o te will ind ica te the deletion.

uest ProQuest 10992988 Published by ProQuest LLC(2018). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

i H

r TABLE OP CONTENTS page I. List of Tables andFigures........ ii Acknowledgements.................... iii

II. III.

General Introduction.............

1

Instrumental Techniques..........

3

Materials................

7

IV. Azlactones A. Introduction.

8

B. Experimental..............

15

C. Discussion....................

30

V. Rhodanine Synthesis A. Introduction.........

35

B. Experimental................... 39 G. Discussion............ VI.

61

ft -2-Thienylalanines

A. Introduction..................

65

B. Experimental........

68

C. Discussion...... .............. 81 VII. Summary........... ...............85 Bibliography........................

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ii

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LIST OF TABLES AND FIGURES PAGE

TABLE 1.

Azlactones Prepared from Thiophene Aldehydes and Hippuric Acid.

II.

III. IV.

Rhodanine Condensation Products of Thiophene Aldehydes.

47

Rhodanine Cleavage Products.

51

3(2-Thienyl)-2-oximinopropionic acids derived through the Rhodanine Syntheses.

V.

Ultra-violet absorption spectrum of potassium chromate.

2.

.

23

Ultra-violet absorption spectrum of 2-methyl-4(2-thenal)5-oxazolone.

6

22

Ultra-violet absorption spectra of di- and trisubstituted azlactones.

5.

__ 6 _

Ultra-violet absorption spectra of monosubstituted azlactones.

4.

__5__

Ultra-violet absorption spectrum of azobenzene.

3.

75

PAGE

FIGURE .

56

-2-Thienylalanines prepared by the Rhodanine Synthesis.

1

54

24

Ultra-violet absorption spectra of Rhodanine s.

45

ill

A cknowledgement s

The author wishes to express his gratitude to his parents and sister for their guidance and assistance• This investigation was aided, in part, by a grant from the Office of Naval Research. This study was carried out under the direction of Dr. P. F. Nord.

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Studies on Azlactones and Rhodanines Derived from Thiophene Aldehydes.

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Introduction Aromatic aldehydes have been used as starting materials in a variety of syntheses.

As a particular example, conden­

sations between aldehydes and compounds containing an active methylene group have proved to be of considerable interest and importance.

For the preparation of amino acids, such

substances as hydantoin, thiohydantoin, diketopiperazine, hippuric acid, acetylglycine and rhodanine have been employed. The condensation products formed by the latter three reagents have been shown to serve as versatile intermediates for the synthesis of many other products as well.

The unsaturated

azlactones resulting from aldehyde condensations with hippuric acid and acetylglycine have been utilized to obtain c^-keto acids, arylacetic acids, styrylamides and isoquinoline derivatives.

Rhodanine intermediates have been

converted to arylthionopropionic acids, arylacetonitriles, arylacetic acids and

arylethylamines.

While differences in reactivity exist between benzene and its analog, thiophene, due to the influence of the hetero sulfur atom, their aldehydes and other derivatives in many instances show some similarity in behavior.

The study of the

reactions of benzaldehyde has been covered extensively in the literature but that of 2- and 3-thenaldehyde has been neg­ lected until comparatively recent times.

As late as 1941

Steinkopf wrote "Thiophenaldehyde sind nicht viele bekannt.1’ This situation may be attributed in part to the lack of suitable methods for the preparation of thiophene aldehydes. L

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The application of the Sommelet reaction (36) and N-methylformanilide synthesis (71) to the thiophene series has ameliorated this condition.

The discovery that diverse

thiophene products possess important physiological prop­ erties has captured the attention of numerous scientific workers and other interests which have oriented the efforts of their researches to this field.

Topics which have been

investigated include amines displaying pressor action (15) and antihistaminic properties (17, 27, 28, 29, 74, 101), narcotics (18, 19, 26, 30, 31, 47, 57, 98, 100) and amino acid antagonists (10, 12, 25, 32, 33, 34, 35, 37, 48, 54, 68 ). Inasmuch as azlactones and rhodanines have been demon­ strated to be flexible tools for organic syntheses, their study in the thiophene series was undertaken.

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II.

Instrumental Techniques

1

A Beckman, Model DU, quartz spectrophotometer was used for the ultra-violet absorption measurements presented here. The solvent used for the azlactones was chloroform while absolute alcohol was employed for the rhodanines.

The con­

centrations used for these measurements varied between 0.06 g. and 0.0006 g. per liter of solution.

The absorption

data of the azlactones and rhodanines was plotted as (log£ + N) versus wavelength expressed in millimicrons, where IT is equal to an arbitrary figure whose value for each particular curve is indicated as part of the legend. The experimental points were taken at intervals of 2 millimicrons in the spectral region from 250 to 300 millimicrons and at intervals of 5 millimicrons in the spectral region from 300 to 430 millimicrons.

The absorption

peaks of each compound recorded were measured at intervals of 1 millimicron.

Three methods were used to check the

calibration of the wave length scale.

The absorption curve of an

alkaline potassium chromate solution was found to coincide with that published by the National Bureau of Standards (83). The comparison of these curves is presented in Figure 1. The absorption curve of an alcoholic solution of azobenzene was found to be identical with that previously obtained by Mull (82) for the same instrument and that recorded by Erode (22). These data are presented in Figure line at 656.3

mj±

2

.

Lastly, the °(-hydrogen

was used, as described by the National

Technical Laboratories (1 1 ). L

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The optical rotations reported were obtained with a standard half-shade type polarimeter (C.P. G-oerz, Berlin) using a 4 decimeter, water-jacketed polarimeter tube. The refractive indices recorded were obtained with a Spencer Abbe-type refractometer. A water pump and constant temperature bath permitted optical rotations and refractive indices to be measured at specified temperatures.

FIG ORE 1 u l t r a v io l e t

5 “

a b s o r p t io n

spectrum

K^ChO«f *BuREflU o r S T A N D A R D S A B S O R B A N C Y D A T A P L U S 0.1

d— ©FORDH/i/i BE C KH AN A B S O R B A N C Y D A T A

1.0 /

©

0. 9 . /

/

OB

*7

©

it

w

I \

©

/

©

/ /

0

\

0

0.6

//

I

/

H Q

0

.£

/ /

* ©

/ 1

©

0.+

* •

/

0 I

t

/

\

&

\1

/

7

\

V

n V ' \

/

\

0

w

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©

© ‘ \'

u

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w

Q

.

I

! I

0.3

0.2

I

ww ©

/

/

0.1

5>

\

/

.0

0.0 zso

300

A (w/t)

A B S ORBANC Y

0 .7.

350

400

6

FIGURE

o

2

f

AZOBENZE N E

AZOBEtfZENE -0.3300 7.///tep - O.OUlf./atCr IN ABSOLUT? ET H A N O L - K£CoRD£D PR. M O L L -

(* 7

I3r

43.

/4 Z 0 B ^ \ J Z £ / v £ ~ O . O G C , f f . / l i t e r - O . D K ° ( p f . / j r t e h

IN A B S O L U T E E T H A A / O k - / ^ O .

700

9oo

900

1000

HOO

/zoo

7.

Ill. Materials 1* Thiophene aldehydes were synthesized as described in References (71, 72). 2. Hippuric acid, m.p. 186-187°C., was obtained from Eimer and Amend, N.Y., N.Y. 3. Acetylglycine, m.p. 207-208°C., was prepared as outlined in Organic Syntheses, Coll. Vol. II, page 11 (1943). 4. Lithium aluminum hydride was obtained from Metal Hydrides Incorporated, Beverly, Massachusetts. 5. 3,4-Dimethylthiophene, b.p. 143-144°C. , 71^ = 1.5212, was provided by the Socony-Vacuum Oil Company, Paulsboro, New Jersey. 6

. Fresh-frozen beef pancreas was supplied by the United Dressed Beef Company, N.Y., N.Y.

7. Chloroacetyl chloride was procured from Eimer and Amend, N.Y., N.Y. 8

. Phenyl isothiocyanate and phenyl isocyanate were obtained from Eimer and Amend, N.Y., N.Y.

9. Triketohydrindene hydrate was supplied by Eimer and Amend, N.Y., N.Y.

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IV. Azlactones Azlactones may be divided into two general groups, saturated and unsaturated, as indicated by I and II respectively:

I

c=o

n

Three systems of nomenclature have been used for their description so that they may be considered as de­ rivatives of amino acids, of oxazolone and of dihydrooxazole.

Chemical Abstracts and Beilsteinfs Handbueh favor

the second system. The saturated azlactones arise through the treatment of ©(-acylamino acids with acetic anhydride.

In 1908-1910

Mohr and co-workers (78, 79, 80) prepared several compounds of this type according to the following scheme:

R-c h -c oxh NH i

R-c/y------- c=o

C=0 i R

i

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9

It was found later that treatment of

el -( oi

-haloacyl)

-amino acids with acetic anhydride and pyridine results in dehydrohalogenation and dehydration and affords unsaturated azlactones (13, 14).

The following mechanism was proposed

by Bergmann and Stern (13):

r - ch x - c h - c o x h t

C—O

kchx - c

C=0

RCH=C

PYRIDINE

HM ~

ACETIC

AMHYDR)J>E

Ci -0

N

0

c

X-CHR

II

cm

The Erlenmeyer azlactone synthesis consists in the condensation of an aldehyde with an acylglycine in the presence of acetic anhydride and sodium acetate.

The

azlactones so produced are of the unsaturated type and the reaction may be represented thus:

RCHO +

R - C H = C -- C = 0

CHx- C 0 z H N H

SOVtUH

ACETATE

r

ACETIC

fl/vJHYDRl D£*

«

R

y

a[i

v C (

R

L

yL

10

r -i Plochl (91) was the first to use this method, in 1883, with benzaldehyde and hippuric acid but neglected the proof of structure of the product*

Ten years later Erlenmeyer began

a long series of papers in which he elucidated the struc­ ture (20, 42), widened the scope of the reaction to include other aldehydes, both aromatic and aliphatic, (44,45,46), and established the utility of unsaturated azlactones as intermediates in the synthesis of o(-keto (39,41) and c(

-amino acids (43,44).

Consequently, the designation of

this reaction bears his name* For practical purposes this reaction was generally considered as being limited to aromatic aldehydes ando(, /3 -unsaturated aldehydes.

However, recent work (51) has

demonstrated that substitution of lead acetate for sodium acetate has made the extension to simple aliphatic alde­ hydes feasible. Of the many acylglycines which have been used, hip­ puric acid and acetylglycine have received the most atten­ tion.

The condensations of aldehydes of the furan, pyrrole,

thiophene, indole, naphthalene, pyrene, chromone, coumarane and thiazole series have been found to follow that of benz­ aldehyde in behavior (2 ). In the thiophene series azlactones have been utilized both as derivatives and as intermediates for further syn­ theses.

Campaigne and LeSuer (26) prepared 2-phenyl-4

(3-thenal) 5-oxazolone for the identification of 3thenaldehyde. L

Barger and Easson (10) applied the Erlen.j

r meyer azlactone synthesis to

2 -thenaldehyde

preparation of /^-2 -thienylalanine.

for the

"

It is interesting to

note that in this study an example of the difference between reactions in the benzene and thiophene series may be found.

Whereas,

ft-phenylalanine

may be prepared from

2-phenyl-4-benzal-5-oxazalone by the action of red phos­ phorous and hydriodic acid (56), 2-phenyl-4(2-thenal) 5-oxazolone did not yield the corresponding amino acid when subjected to the same conditions (10). ft -2 -thienylalanine

However,

was obtained in stepwise fashion by

an opening of the azlactone ring to the cl -benzamido-/3 2 -thienylacrylic

acid with dilute sodium amalgam and

hydrolysis of the benzamido group to the free amino acid with 10% HC1 (10). The observations of Yuan and Li (102) have shown that 2 -thenaldehyde

diethylacetal may be condensed with hippuric

acid in yields approximately the same as those realized from the free aldehyde. As a further contribution to the knowledge extant concerning the properties of thiophene compounds, a number of substituted thiophene aldehydes were subjected to acylglycine condensations.

In addition to the usual

physical constants, the ultra violet absorption spectra of the thienyl substituted azlactones so obtained were recorded in figures 3,4,5 and their relative data on pages 21 and 25. No attempt was made to analyze these results in the light of theoretical considerations other than to point out major

12

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shifts of absorption peaks and changes of curve shape with variations of the position of substituents on the thiophene nucleus.

Asahina (7,8) has made a study of the ultra-violet

absorption spectra of the azlactones, diketopiperazines and hydantoins derived from benzaldehyde and furfural.

Stuckey

(99) has investigated the effect of alkyl substitutions of the hydantoin ring on the ultra-violet absorption spectrum of 5-benzylidenehydantoin.

Abe (1) has found that the max­

imum absorption bands of

2 -acetylthiophene

lie at 266 itijjl and 267

respectively.

my.

and

2 -acetylfuran

Such examples from

the literature indicate that when sufficient observations have been collected and correlated a greater insight may be obtained as to the nature of organic compounds.

Ferguson

(50) in his review article entitled "Relationship between Absorption Spectra and Chemical Constitution of Organic Molecules", while pointing out the inability, at present, of theoretical organic chemists to predict absorption spectra for compounds other than very simple ones and the limitations of the deductions which can be made from absorption data, has this to say:

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"There are several reasons, however, why organic chemists should, in the meantime, continue their search for correlations between molecular structure and spectra until, through a fusion of empirical and theoretical methods, it will be possible to define all the laws for the absorption of light by organic molecules. For instance, qualitative relationships between the color and the chemical constitution of organic molecules serve as a wieldy tool to elucidate the structure of and to characterize new compounds. Many cases are found in the literature where the structure of a compound was selected from among several possibilities on the basis of its absorption spectra. In this respect, j

H. N. Jones has found that systems of condensed aromatic rings have associated with them a char­ acteristic spectrum by means of which a particular ring system may be recognized. Further, an accurate compilation of quantitative data on the spectra of pure compounds will provide material for the quantum theorists.” All but one of the thiophene azlactones described in the experimental section were prepared by using hippuric acid as the acylglycine. with yields in Table 1. one aldehyde,

These products have been listed Acetylglycine was utilized with

2 -thenaldehyde,

to demonstrate its ability to

condense with thiophene aldehydes.

The 2-methyl-4(2-thenal)

5-oxazolone so obtained was shown to be susceptible to ring opening resulting in the formation of cK -acetamido-/3 -2 thienylacrylic acid.

The scheme of these reactions is as

follows: Sodiuai ACETIC

C=0

acetate aw w vdride

H

CHzC0zH i i

CH} /tCETOWE

H ^O

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Two new thiophene aldehydes, 3 ,4-dimethyl-2-thenalde­ hyde and 3,4,5-trimethyl-2-thenaldehyde were obtained by applying the N-methylformanilide (71) and modified WolffKishner (72) reactions to 3 ,4-dimethylthiophene. The susceptibility of thiophene azlactones to alcoholysis was found to parallel that of the benzene series.

This was demonstrated by applying Nieoletfs

method (87) to 2-phenyl-4(2-thenal)5-oxazolone.

With a

methanol solution of sodium methylate this azlactone was opened rapidly to methyl e(-benzamido-/3-( 2 -thienyl)acrylate.

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Experimental Preparation of Azlactones The modified procedure (24) of Kropp and Decker (73) was employed for the synthesis of all the azlactones des­ cribed here, except for that of 2-methyl-4(2-thenal)5oxazolone for which the method of Herbst and Shemin (65) was used.

2-Phenyl-4(2-thenal)5-oxazolone.

2-Thenaldehyde (10.14 g. ,

0.09 mole), 7.42 g. (0.09 mole) of freshly fused sodium acetate, 16.2 g. (0.09 mole) of hippuric acid and 27.7 g. (0.27 mole) of acetic anhydride were placed in a 500-cc. round-bottom flask and heated on the steam bath for a few minutes with manual stirring.

The yellow paste which

formed was left on the steam bath for two hours.

Then

30 cc. of ethanol was added and the product placed in the ice-box overnight.

The crude azlactone was filtered and

washed with hot water.

After recrystallization from

aqueous ethanol, 15.85 g. (6 8 $) of 2-phenyl-4(2-thenal) 5-oxazolone were obtained in the form of yellow needles, m.p. 174.5-175.5°C.1 Analysis. Calculated for C^HgOgNS: C,65.88; H,3.53; N,5.49 Found:

C,66.10; H,3.66; N,5.69

^Ref. (10) gives m.p. 175°C.

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16 r 2-Phenyl-4(5-methyl-2-thenal)5-oxazolone. 5-Methyl-2-thenal­ dehyde (10 g., 0.079 mole), 6.5 g. (0.079 mole) of freshly fused sodium acetate, 14.2 g. (0.079 mole) of hippuric acid and 24.3 g. (0.25 mole) of acetic anhydride were reacted as above.

There was obtained 12.4 g. (58%) of 2-phenyl-4 o (5-methyl-2-thenal)5-oxazolone, m.p. 152-153 C.

Analysis. Calculated for C-^gH-^OgNS: C,66.91: H,4.09; N,5.20 Found

C,67.00; H,3.94; N,5.40

2-Phenyl-4(3-methyl-2-thenal)5-oxazolone. 3-Methyl-2thenaldehyde (1 2 . 6 g.,

0.1

mole),

8.2

g. (0 . 1 mole) of

freshly fused sodium acetate, 17.9 g. (0.1 mole) of hip­ puric acid and 30.6 g. (0.3 mole) of acetic anhydride after reaction as above yielded 16.2 g. (60%) of

2 -phenyl-

4(3-methyl-2-thenal)5-oxazolone, m.p. 151-152°C. Analysis. Calculated for C1 5 H 1 1 0gNS: C,66.91; H,4.09; N,5.20 Found:

C,66.87; H,4.07; N,5.51

2-Phenyl-4(5-ethyl-2-thenal)5-oxalolone.

5-Ethyl-2-thenal-

dehyde (14.0 g., 0.1 mole), 8.2 g. (0.1 mole) of freshly fused sodium acetate, 17.9 g. (0.1 mole) of hippuric acid and 30.6 g. (0.3 mole) of acetic anhydride when treated as above gave 17 g. (60%) of 2-phenyl-4(5-ethyl-2-thenal) 5-oxazolone, m.p. 107.5-109°C. Analysis. Calculated for C^gH^^OgNS: C,67.84; H,4.59; N,4.94 Found L

C ,68.02; H,4.48; N,4.94 -I

2-Phenyl-4(5-propyl-2-thenal)5-oxalolone. 5-Propyl-2thenaldehyde (15.4 g., 0.1 mole), was condensed with 17.9 g. (0 . 1 mole) of hippuric acid in the presence of

8.2

g.

(0 . 1 mole) of freshly fused sodium acetate and 30.6 g. (0.3 mole) of acetic anhydride.

The crude material

obtained was washed with cold rather than hot water because of its lower melting point.

Recrystallization

from aqueous ethanol yielded 17.8 g. (60%) of 2-phenyl-4 (5-propyl-2-thenal)5-oxazolone, m.p. 97-98.5°C. Analysis. Calculated for C^^H^^OgNS: C,68.69; H,5.05; N,4.71 Pound:

C,68.80; H,5.28; N,4.90

2-Phenyl-4(5-chloro-2-thenal)5-oxazolone. 5-Chloro-2thenalaehyde (14.7 g. (0.1 mole), 8.2 g. (0.1 mole) of freshly fused sodium acetate, 17.9 g. (0.1 mole) of hippuric acid and 30.6 g. (0.3 mole) of acetic anhydride when treated as above produced 17.8 g. (61%) of

2 -phenyl-

4(5-chioro-2-thenal)5-oxazolone, m.p. 182.5-183.5°C. Analysis. Calculated for C-^HQClOgNS:C,58.03;H,2.76;N,4.83 Found:

C,58.40;H,2.74;N,5.19

2-Phenyl-4(5-bromo-2-thenal)5-oxazolone. 5-Bromo-2-thenaldehyde (19.1 g. (0.1 mole), 8.2 g. (0.1 mole) of freshly fused sodium acetate, 17.9 g. (0.1 mole) of hippuric acid and 30.6 g. (0.3 mole) of acetic anhydride were treated as above and yielded 21.4 g. (64%) of 2-phenyl-4(5-bromo2-thenal)5-oxazolone, m.p. 186-187°C.

18 r Analysis. Calculated for C^^HgBr02NS:C,50.29;H,2.39;N,4.19 Pound

C,50.10;H,2.25;N,4.33

2-Phenyl-4(3,4-dimethyl-2-thenal) 5-oxazolone. 3,4-Dimethyl2-thenaldehyde (4.2 g., 0.03 mole), 2.46 g. (0.03 mole) of freshly fused sodium acetate, 5.37 g. (0.05 mole) of hippuric acid and 9.2 g. (0.09 mole) of acetic anhydride were reacted as described above and produced 4.6 g. (54%) of 2-pbenyl-4(3,4-dimethyl-2-thenal)5-oxazolone, m.p. 195-196°C. Analysis. Calculated for C1 6 H2 3 O 2 NS: C,67.84; H,4.59 Found

C,67.78; H,4.38

2-Phenyl-4(3,4,5-trimethyl-2-thenal)5-oxazolone.

3,4,5-

Trimethyl -2-thenaldehyde (3.08 g., 0.02 mole), 1.64 g. (0.02 mole) of freshly fused sodium acetate, 3.58 g. (0.02 mole) of hippuric acid and 6.13 g. (0.06 mole) of acetic anhydride when reacted as above yielded 3.61 g. (61%) of

2 -phenyl-4(3,4,5 -trimethyl-2 -thenal)5-oxazolone,

m.p. 193.5-194.5°C. Analysis. Calculated for C ^ H ^ O g N S : C,6 8 .6 8 ; H,5.05 Pound:

C ,68.47; H,4.79

2-Phenyl-4(4,5-dime thyl-2-thenal)5-oxazolone.

4,5-

Dimethyl-2-thenaldehyde (0.59 g., 0.004 mole), 0.35 g. (0.004 mole) of freshly fused sodium acetate, 0.77 g. (0.004 mole) of hippuric acid and 1.32 g. (0.012 mole) of acetic anhydride were reacted as above and yielded 0.9 g. L

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p (75%) of 2-phenyl-4(4,5 -dimethyl-2-thenal)5-oxazolone, m.p. 205.2-206°C. Analysis.

Calculated for C^gH^OgNS: C,67.84: Pound:

C,67.94;

H,4.59 H,4.36

2-Phenyl-4(2,5-dimethyl-5-thenal)5-oxazolone. 2,5-Dimethyl3 -thenaldehyde

(0.5 g. , 0.004 mole), 0.3 g. (0.004 mole) of

freshly fused sodium acetate, 0.65 g. (0.004 mole) of hippuric acid and

1.1

g. (0 . 0 1 1 mole) of acetic anhydride

when treated as above produced 0.8 g. (79%) of 2-phenyl-4 (2,5-dimethyl-3-thenal)5-oxazolone, m.p. 142.5-143.5°C. Analysis.

Calculated for C1 5 H 1 3 O 2 NS: C,67.85; Found:

0,68.18;

H,4.59 H,4.77

2-Phenyl-4(2,3,5-trimethyl-4-thenal)5-oxazolone. 2,3,5Trimethyl-4-thenaldehyde (0.84 g . , 0.005 mole), 0.45 g. (0.005 mole) of freshly fused sodium acetate, 0.98 g. (0.005 mole) of hippuric acid and 1.68 g. (0.015 mole) of acetic anhydride when reacted as above yielded 0.3 g. (18%) of 2-phenyl-4(2,3,5-trimethyl-4-thenal)5-oxazolone, m.p. 101-102.5°C. Analysis.

Calculated for C1 7 H 1 5 O2 NS: C,6 8 .6 8 ; H,5.05 Found:

C,68.87;

H,4.95

2-Phenyl-4(3-thianaphthal)5-oxazolone. 3-Thianaphthaldehyde (4.05 g., 0.025 mole), 2.05 g. (0.025 mole) of freshly fused sodium acetate, 4.47 g. (0.025 mole) of hippuric acid

20 r and 7.65 g. (0.075 mole) of acetic anhydride when reacted

i

as above yielded 5.26 g. (69%) of 2-phenyl-4(3 -thianaphthal) 5-oxazolone, m.p. 223-223.5°C. Analysis. Calculated for C1 QH 1 1 0 2 NS: C,70.81; H,3.61 Pound:

C,70.70; H,3.81

2-Methyl-4(2-thenal)5-oxazolone. A mixture of 2-thenalde­ hyde (42 g., 0.37 mole), acetylglycine (29.3 g., 0.25 mole), sodium acetate (15 g., 0.19 mole) and acetic anhydride (67 g., 0.63 mole) was heated on the steam bath in a 500-ml. Erlenmeyer flask, loosely corked, with occasional stirring for 20 minutes.

At the end of this time complete solution

had been achieved and the flask was fitted with a reflux condenser and its contents refluxed for one hour over a low flame. night.

The flask was then left in the ice-box over­

The resultant solid which had formed was broken up

with 75 cc. of water, filtered on a Buchner funnel and washed with cold water.

The crude yield after drying in

vacuo over PgOg and KOH amounted to 40.65 g. (56%). A sample recrystallized from absolute alcohol had a m.p. of 131.5-132.5°C. Analysis. Calculated for CgH^OgNS: N,7.26 Found:

L

N,7.27

21

Absorption Maxima of Compounds Prepared by the Erlenmeyer Azlactone Synthesis__________ Aldehyde

Maxima of Resultant Azlactone 'm jll

£ x | 0 3

Lo 273 429

L

37.6

4.03 4.57

10.8

4,5-dimethyl-2-thenaldehyde

272 421-2

11.9 39.1

4.07 4.59

2 ,5-dimethyl-3-thenaldehyde

272 390-1

10.0

34.1

4.00 4.53

2,3,5-trimethyl-2-thenaldehydei 273 387-8

10.7 17.3

4.03 4.24

-J

22

FIGURE 3

8.0

_1

6.0

S.O

ABSORPTION OF

SPECTRA

23

F I G U R E 48

Dl-AA/D

ABSORPTION SPECTRA O F TRISUBSTITUTED A2LACTONES

H-

300

A (-nyi)

400

FIGURE

5

A B S O R P T I O N S P E C T 'RUM

OF 2 - M E T H Y L - 4 ( 2 - THE N f l L ) F - O X f l Z O l O N E

4.5

3.5

2.5

250

30 0

35"0 A

(w/l)

400

25

Legend for Figure 3 2-phenyl-4(2-thenal)5-oxazolone

--------------N = 0.0

2-phenyl-4( 3-methyl-2 -thenal) 5-oxazolone

N = 1.0

2-phenyl-4(5-methyl-2-thenal)5-oxazolone

.N = 2.0

2-phenyl-4( 5-chloro-2-thenal )5-oxazolone---------N = 3.0

Legend for Figure 4 2-phenyl-4( 3-thianaphthal )5-oxazolone— : --------- N = 0.0 2-phenyl-4(2,5-dimethyl-3-thenal)5-oxazolone---- N = 1.0 2-phenyl-4(2,3,5-trimethyl-4-thenal)5-oxazolone— ^*N= 1.5 2-phenyl-4( 3,4-dime thyl-2-thenal )5-oxazolone.... N — 2.0 2-phenyl-4(4,5-dimethyl-2-thenal)5-oxazolone---- N = 2.5 2-phenyl-4 (3,4,5-trimethyl-2-the nal)5-oxazolone— N =3.0

Legend for Figure 5 2-me thyl -4 (2- thenal)5-oxazolone----------------- -----

26 r

i

o(-Acetamido-/3-2-thlenylacrylic acid To a mixture of 328 cc. of acetone and 127 cc. of water, contained in a one-liter round bottom-flask fitted with a reflux condenser, was added 35.2 g. (0.18 mole) of 2-methyl-4(2-thenal)5-oxazolone.

The mixture was refluxed

for 4 hours and the acetone removed on the steam bath at normal pressure.

After addition of 292 cc. of water, the

solution was boiled for five minutes and filtered.

The

residue was boiled with 250 cc. of water, filtered and the combined filtrates left in the ice-box overnight, whereupon the acid precipitated.

The product was

filtered with suction and dried in a vacuum dessicator over £*2^5 an2 and co-workers (35) obtained /3-2-thienylalanine from

2-

chloromethylthiophene by the application of the phthalimidomalonic and acetamidomalonic ester syntheses in yields of 51% and 67% respectively. Since Granacher (61) obtained ft

ft -phenylalanine

and

-2 -furylalanine by the reduction of their corresponding

oximinopropionic acids, this procedure was applied to five thiophene oximinopropionic acids which had previously been prepared through the rhodanine method.

These researches

were made with the dual purpose of examining another reaction as a test of the extent to which the benzenethiophene analogy holds, and also of determining the use­ fulness of the rhodanine synthesis for the preparation of ft

-2-thienylalanine and some of its 5-thienyl substituted

products. The method of Henze and Speer (64) is adaptable to the formation of hydantoins from a variety of aldehydes by refluxing them with potassium cyanide and ammonium carbonate.

This scheme has the advantage of not requiring

expensive hydantoin as a starting material. therefore tried with

2 -thenaldehyde

Its use was

but the results were

negative. du Vigneaud and Ferger amplified the study of the nanti phenyl alanineeffect of/3-2 -thienyl alanine by util­ izing its D and L optical isomers in growth inhibition experiments both on microrganisms (48) and on young rats (49).

These investigations revealed that f t -2-thienyl-L-

alanine and^-2-thienyl-DL-alanine exert inhibitory activ­ ity toward Saccharomyces cerevisiae, Escherichia coli and Lactobacillus delbrueckii, LD5, in the ratio of 2:1 on a weight basis, whereas/3 -2 -thienyl-D-alanine had no activity. All three forms of the amino acid inhibited the growth of young rats.

The enantiomorphs used in these experiments

were obtained by the chemical resolution of the racemic acid with brucine.

This method of resolution is quite

tedious in comparison with some enzymatic ones.

Greenstein

and co-workers (55) resolved racemic phenylalanine, tyro­ sine and tryptophan into their optically pure isomers by subjecting their N-chloroacetylated derivatives to asym­ metric hydrolysis by a purified beef pancreas carboxypeptidase preparation. In view of the significance of the optical isomers of ( 3 - 2 -thienylalanine in in vivo studies, it was deemed ad­

visable to attempt the enzymatic resolution (8 6 ) of this unnatural amino acid according to the method described for natural amino acids (55,62,93).

Preparation of the ^-2-Thienylalanines The reduction procedure used for these syntheses is essentially the same as that reported in the literature (61).

This method, exemplified by the detailed descrip­

tion of the preparation of/^-2 -thienylalanine, was used for all the amino acids obtained with the exception of / 3 -(5-chloro-2 -thienyl)-alanine which required an adjust­ ment of the pH of the reaction mixture to induce precip­ itation.

/ 3 - s -Thienylalanlne.

3 -(2-Thienyl)-2-oximinopropionic

acid

(3.8 g. , 0.021 mole) was dissolved in 77 cc. of absolute alcohol and 150 g. of

2%

sodium amalgam was added in small

portions with heating on the steam-hath.

The solution was

kept acidic by the addition of lactic acid at irregular intervals.

After all the amalgam had been added, the

alcoholic solution was decanted and left in the refriger­ ator overnight. was obtained.

Upon filtration 2.29 g. of the amino acid The filtrate was evaporated to a brown

syrup which was dissolved in a minimum amount of absolute alcohol and chilled.

This gave an additional 0.1 g. of

amino acid and a total yield of 2.39 g. (6 8 %) lization from

68%

•

Recrystal­

ethanol in the presence of Norit produced

white crystals which exhibited a positive ninhydrin re­ action, m.p. 273-275°C. when the oil bath was preheated to 270 C.

Reference (10) gives a m.p. of 274-275°C. under

these conditions.

Reference (37) gives a m.p. of 243-245°C.

69 when the oil bath is heated at a rate of

8 °/minute.

Analysis. Calculated for C^HgOgNS: C,49.12; E,5.26; N,8.18 Pound:

C,49.20; H,5.14; N,8.27

The phenylurea derivative was prepared by dissolving 1 g. of the amino acid in 10 cc. of

NaOH with cooling

and shaking this solution with 0.9 g. of phenyl isocyanate. The small amount of diphenylurea which formed was filtered off and the thienylureide precipitated with dilute HC1. White crystals were obtained from dilute ethanol, m.p. 175-176°C. when the oil bath was preheated to 165°C.

With

no preheating the m.p. 164-164.5°C. was obtained. Analysis. Calculated for ^^H^O^EgS: C,57.93; H,4.82 Found:

C,57.70; H,4.82

/ 3 - ( 5-Methyl-2 -thienyl)-alanine. 3-(5-Methyl-2-thienyl)-2 oximinopropionic acid (4.98 g. , 0.025 mole) was dissolved in 75 cc. of absolute alcohol, reduced with 200 g. of 2% sodium amalgam and treated as above.

In this manner there

was obtained a yield of 5.42 g.

o f f i - ( 5-methyl-2-

(74%)

thienyl)-alanine which exhibited a positive ninhydrin reaction.

Recrystallization from hot water produced white

crystals, m.p. 253-255°C. when the oil bath was heated at a rate of

6 °/minute.

Analysis. Calculated for C8 H 1 1 02 NS: C,51.89; H,5.94; N,7.56 Pound:

^Ref.

(66)

C,52.10; H,6.14; N,7.70

gives a m.p. of 165-166°C.(capillary) and 182°

(Dennis melting point bar).

The phenylurea derivative was prepared as described above and gave white crystals, when re crystallized from dilute ethanol, which had a m.p. of 162.5-163°C. Analysis. Calculated for

C,59.21; H,5.26

Pound:

C,59.49; H,4.99

/ 3 -(5-Sthyl-2-thienyl)-alanine.

3-(5-Ethyl-2-thienyl)-2-

oximinopropionic acid (5.5 g., 0.026 mole) was dissolved in 80 cc. of absolute alcohol, reduced with 225 g. of 2% sodium amalgam and treated as above. tained a yield of 3.7 g.

(71%)

There was thus ob­

of/^-(5-ethyl-2-thienyl)

-alanine which showed a positive ninhydrin reaction.

Re­

crystallization from hot water gave white crystals, m.p. 235-238°C. when the oil bath was heated at a rate of

6 °/

minute. Analysis. Calculated for CgH^OgNS: C,54.27; H,6.53; N,7.03 Pound:

C,54.56; H,6.24; N,7.19

The phenylurea derivative, prepared as above, when recrystallized from dilute ethanol gave white crystals, m.p. 165.5-166.5°C. Analysis. Calculated for Pound: /3

C,60.37; H,5.66 C,60.48; H,5.41

-(5-Propyl-2-thienyl)-alanine. 3-(5-Propyl-2-thienyl)-2-

oximinopropionic acid (9.08 g., 0.04 mole) was dissolved in 135 cc. of absolute alcohol, reduced with 400 g. of 2% sodium amalgam and treated as above.

There was obtained

71 ra yield of 5,7 g. (67%) of/3-(5-propyl-2-thienyl)-alanine which exhibited a positive ninhydrin reaction.

n

Recrystal­

lization from hot water gave white crystals, m.p. 217-220°C. when the oil bath was heated at a rate of

6 °/minute.

Analysis. Calculated for CpoH-^OgNS: C,56.34; H,7.04; N,6.57 Found:

C,56.74; H,6.93; N,6.72

The phenylure-a. derivative was prepared as above.

Re­

crystallization from dilute ethanol gave white crystals, m.p. 158-159°C. Analysis. Calculated for

: C,61.44; H,6.02

Found:

C,61.76; H,6.08

-(5-Chloro-2-thienyl)-alanine.

3-(5-chloro-2-thienyl)-2-

oximinopropionic acid (14 g., 0.064 mole) was dissolved in 215 cc. of absolute alcohol and reduced with 600 g. of sodium amalgam.

2%

The reaction mixture was decanted diluted

with water to twice the original volume and allowed to stand for 24 hours in the refrigerator.

The f t -(5-chloro-2-

thienyl)-alanine which had precipitated was filtered with suction, washed with absolute alcohol and after drying over KOH in vacuo amounted to 7.2 g. (55%).

This product too

exhibited a positive ninhydrin reaction.

Recrystallization

from hot water yielded white crystals, m.p. 226-228°C., when the oil bath was heated at a rate of

6 °/

minute.

Analysis. Calculated for C^HqCIC^NS: C,40.87; H,3.89; N,6.81 Found:

L

C,40.97; H,3.85; N,6.82

-I

72

r

The phenylurea derivative was prepared as above and

after recrystallization from dilute ethanol the white crystals so obtained had a m.p. of 163.5-164°C. Analysis. Calculated for C^H-j^ClOgNgS: C,51.77; H,4.00 Pound:

C,51.58; H,4.12

Attempted Preparation of/ 3 - ( 5-bromo-2-thienyl)-alanine. 3-(5-Bromo-2-thienyl)2-oximinopropionic acid (5 g., 0.019 mole) was dissolved in 85 cc. of absolute alcohol, reduced with

200

g. of

2%

sodium amalgam and treated as above.

The product which precipitated out of solution gave a positive ninhydrin reaction but analyzed for ^ - 2 -thienylalanine.

The yield amounted to

2

g. (61:1) and after

recrystallization from hot water had a m.p. of 273-275°C. when the

oil bath was preheated to 270°C.

Analysis. Calculated for C^HgOgNS: Found: The

C,49.12; H,5.26; N,8.18 C,-49.00; H,5.27; N,8.22

phenylurea derivative was prepared as above and

after recrystallization from aqueous ethanol had a m.p. of 175-176°C. when the oil bath was preheated to 165°C.

A

mixed melting point with an authentic sample of this ureide showed no depression.

L

n

TABLE V

/3-2-Thienylalanines Prepared by the Rhodanine Synthesis

Starting Material

Product

%

Yield

2 -Thenaldehyde

ft -2 -1 hienylalanine

56

5-Methyl-2-thenaldehyde

f t - ( 5-methyl-2-thienyl)

65

-alanine 5-Ethyl-2-thenaldehyde

f t -(5-ethyl-2-thienyl)

62

-alanine 5-Propyl-2-thenaldehyde

f t - { 5-propyl-2-thienyl)

55

-alanine 5-Chloro-2-thenaldehyde

f t - [ 5-chloro-2-thienyl)

47

-alanine 5-Bromo-2-thenaldehyde

ft-

2-thienylalanine

50

74 r Attempted Preparation of /?-2 -thienylalanine from

n

2-Phenyl-4( 2-thenal)5-oxazolone ._____ __ 2-Phenyl-4(2-thenal)5-oxazolone (9.7 g. , 0.038 mole), 7.45 g. of red phosphorous and 51 g. of acetic anhydride were placed in

1

-liter three-necked, round-bottomed flask

fitted with a reflux condenser, a mechanical stirrer and a dropping funnel.

Over a period of one half-hour, 38.2 cc.

of 50% hydriodic acid were added dropwise with stirring. The mixture was then refluxed for 3 hours and filtered free of unreacted phosphorous.

The filtrate was evaporated to

dryness in vacuo with heating on a water bath.

Forty cc.

of water were added to the dry residue and the evaporation repeated.

Seventy cc. of water and 70 cc. of ether were

added to the dry residue with shaking to effect solution. The aqueous layer was removed and extracted three times with 30 cc. portions of ether.

The aqueous layer was heated

to boiling and neutralized to Congo red with 15% ammonia. An amorphous gray-white solid settled out after cooling which was only slightly soluble in boiling water and which gave a negative ninhydrin test.

Attempted Preparation of 5-(2-thenal)hydantoin. 2-Thenalde­ hyde (2.24 g., 0.02 mole) was dissolved in 50 cc. of 50% aqueous ethanol contained in a 500 cc. round-bottomed flask fitted with a reflux condenser. of ammonium carbonate and

2.6

To this solution 9.12 g.

g. of potassium cyanide were

added and the mixture heated at 58-60°C. for 2 hours. L

rThe solution was then evaporated to 2/3 of the original volume and chilled.

A small amount of white solid pre­

cipitated which was insoluble in all common organic sol­ vents and water.

Most hydantoins can be recrystallized

easily from aq. alcohol (64).

The hydantoin from benzalde-

hyde was prepared concurrently with the above attempt and was found to possess the solubility properties described in the literature (64).

Enzymatic Resolution of /3~2-Thienyl-DL-alanine. Beef Pancreas Carboxypeptidase The enzyme was obtained by the method of Anson (6 ) with some modifications. Ten kilograms of fresh-frozen beef pancreas, which had been stored at -14°C., were allowed to thaw at room temper­ ature for* six hours.

The exudate which appeared during

this process was collected for use later.

The thawed pan­

creas was cut into slices, ground with a meat grinder and finally homogenized with a

2%

solution in a Waring blender.

aqueous sodium chloride The macerated mass plus the

exudate was placed in a 35 liter glass vat with a total of 50 liters of 2% sodium chloride solution and 2.3 liters of toluene, stirred mechanically for an hour and then left overnight.

The next morning the fat which had formed was

skimmed off and discarded.

The rest of the mixture was

filtered through cheese cloth.

5N acetic acid was added

to the filtrate until it was green to bromcresol green.

This acidified filtrate was filtered in two stages in order to obtain a clear filtrate.

The first filtration was ac­

complished by adding Hy-Flo Super-Cel (Johns-Manville) to the solution and filtering on a Buchner funnel through coarse filter paper.

This filtrate was then passed through

a Buchner funnel fitted with No. 1 Whatman filter paper on the bottom, a layer of Hy-Flo Super-Cel and coarse filter paper on top.

There were thus obtained 22.7 liters of clear

yellow filtrate.

To this 8876 g. of solid ammonium sulfate

were added with stirring, and the resultant solution left to stand overnight.

The next morning the white precipitate

which had formed and floated to the surface was filtered through a fluted filter funnel.

The yellow filtrate was

discarded and the precipitate dialyzed against running distilled water until a negative sulfate test was obtained with calcium chloride.

This took approximately 24 hours.

The dialyzed material was centrifuged at 1700 r.p.m. for 20 minutes.

The supernatant liquid was discarded and the

crude enzyme preparation which had settled out was suspended in 480 cc. of distilled water and stored at

Chloroacetyl-/3

-14°C.

-2-thienyl-DL-alanine

Applying the method described for the chloroacetylation of L-phenylalanine (52), 9.5 g. (0.055 mole) of^-2-thienylDL-alanine was dissolved in 56 cc. of IN NaOH and placed in an ice-bath. To this solution were added, with shaking and in small portions, 83 cc. of IN NaOH and a solution of

77

r 8.8

i g. of chloroacetyl chloride in ether.

Dry HG1 gas was

then passed into the alkaline mixture for 30 minutes which precipitated the chloroacetylated compound as an oil. After standing overnight in the cold, the oil solidified giving a yield of 11.35 g. (82%) of chloroacetyl-/3-2th i e nyl-DL-alanine.

Recrystallization from hot water in

the presence of Norit gave 9.75 g. (71%) of white

crystals,

m.p. 127-128°C•

Analysis. Calculated for CgH^lNO^S: N,5.65 Found:

N,5.85

To verify the identity of the chloroacetylated com­ pound, it was converted to the unsaturated azlactone,

2-

methyl-4(2-thenal)-5-oxazolone, by treatment with acetic anhydride and pyridine (14).

Recrystallization from abso­

lute alcohol afforded yellow needles, m.p. 131-132.5°C.

A

sample prepared by the Erlenmeyer azlactone synthesis had a m.p. of 131.5-132.5°C.

A mixed melting point of the two

samples showed no depression. Analysis. Calculated for CgHyOgNS: N,7.26 Found:

N,7.34

78 r Resolution Procedure ft

-2-Thienyl-L-alanine. Chloroacetyl~/3-2-thienyl-DL-

alanine (9.5 g., 0.058 mole) was suspended in water and dissolved by the cautious addition of stirring.

6N

LiOH with vigorous

Sixty cc. of the enzyme suspension was thawed at

room temperature and the enzyme brought into solution by adjusting the pH to 7.6 with 0.2M LiOH.

After the insoluble

globulins were removed by filtration, 75 cc. of Macllvaine buffer (pH 7.6) were added and this solution mixed with the substrate forming a total volume of about 300 cc. digestion was carried out at 37°C. for 48 hours.

The The mix­

ture was then acidified with glacial acetic acid to pH 5.0 and evaporated in vacuo to half of its original volume. The/3-2-thienyl-L-alanine which was filtered off at this point amounted to

1.2

g. (36%) after washing with cold

water and absolute alcohol and recrystallization from hot water in the presence of Norit.

White crystals were ob­

tained, m.p. 238-244°C. when the oil bath was heated at a 21° rate of 8 /minute;[^J^ - -31.4°(1.0834 g. in 50 cc. of water).

Reference (48) gives a m.p. of 239-244°C. and

21°

= ~31.7° ( 1 % aqueous solution). Analysis. Calculated for C^HgNOgS: Found:

11,8.18 N,8.25

Chloroacetyl-/3-2-thienyl -D-ala n i n e .

The filtrate was concentrated further in vacuo, layered ^with ethyl acetate, acidified to pH 2.0 with cone. HC1 and

79

r

extracted five times with ethyl acetate.

The extract was

"i

dried over anhydrous sodium sulfate, evaporated in vacuo to an oil which crystallized after being washed with pet­ roleum ether.

Upon filtration there was obtained 3.2 g.

(67f o ) of chloroacetyl-/^-2 -thienyl-D-alanine. This product after recrystallization from hot water had a m.p. of 119.5120°C. and

— Zl°

=

-47.2°(0.9487 g. in 50 cc. of absolute

alcohol). Analysis. Calculated for CgH^oClNOgS: N,5.65 Found:

N,5.90

Chloroacetyl-f t -2 -thienyl-L-alanine In order to check the specific rotation of the chloracetylated D-isomer, chloroacetyl-/3-S-thienyl-L-alanine was prepared as above from/^-2 -thienyl-L-alanine and gave

21

°

a m.p. of 120.5-121.5°C. and

- +46.5° (0.8468 g. in

50 cc. of absolute alcohol). Analysis. Calculated for CgH^oClNOgS: N,5.65 Found:

N,5.65

/^-2-Thienyl-D-alanine. Chloroacetyl-f t -2 -thienyl-D-alanine (1.35 g., 0.055 mole) was refluxed for 70 minutes with 17 cc. of IN HBr and evaporated to dryness in vacuo on a tepid water bath.

The white residue was dissolved in absolute

alcohol and the pH adjusted to 5.2 with 4N LiOH whereupon 0.65 g. (69f o ) of f t -2 -thienyl-D-alanine precipitated. After recrystallization from hot water the m.p. was 239-246°C.

80 r

when the oil bath was heated at a rate of L°

ZI ° 0

d

=

n

/minute;

+ 31.4° (0.2624 g. in 25 cc. of water).

Reference (48) gives

Zl°

- 4-31.6 (1% aqueous solution).

Analysis. Calculated for CyEgNOgS: Found:

L

8

N,8.18 N,8.38

n

/3-2 -Thienylalanines Discussion The sodium amalgam reductions of 3-{2 -thienyl)-2 oximinopropionic acid and its 5-thienyl substituted methyl, ethyl and propyl homologs proceeded readily to give the desired amino acids in substantial yields.

However, when

this same procedure was applied to 3(5-chloro-2-thienyl)-2oximinopropionic and 3(5-bromo-2-thienyl)-2-oximinopropionic acids some unexpected results were obtained.

Under normal

conditions the free amino acid precipitated from the re­ duction solution when chilled.

In the case of the chloro

compound no solid material was deposited even when the re­ action mixture was allowed to remain in the refrigerator for one week.

It was then found that by diluting the re­

action mixture to twice the original volume with water t h e ^ -(5-chloro-2-thienyl)-alanine appeared in a few minutes.

It was noted that since small additions of lactic

acid were used to keep the reduction media acidic, the pH of all the solutions, after reaction was complete, was about 5*7.

The effect of dilution with water to twice the orig­

inal volume was to lower the pH to 4,9.

This dilution

failed to improve the yield of any of the amino acids except the chloro substituted one* The reduction of 3-(5-bromo-B-thienyl)-oximinopropionic acid appeared to behave in the same manner as did the un­ substituted and alkylated acids.

A white product crystal­

lized from the reaction mixture and exhibited a positive

ninhydrin reaction.

Analyses for carbon, hydrogen and

nitrogen, however, coincided with those required for /3 -2 -thienylalanine. The melting point of the phenylurea derivative was identical with that prepared from a known sample of/^-2 -thienylalanine and when mixed with it showed no depression of melting point.

This amino acid gave neg­

ative qualitative tests for halogen by the Beilstein and magnesium and potassium carbonate fusion methods.

The

identity of 3-(5-bromo-2-thienyl)-2 -oximinopropionic acid had been proved by analyses and the method of its preparation. In addition, it gave a positive qualitative test for halogen. It was therefore concluded that the bromine atom had under­ gone hydrogenolysis in the attempted reduction of 3-(5-bromo2 -thienyl)-2 -oximinopropionic

to/3-(5-bromo-2-thienyl)-

alanine and /3 -2 -thienylalanine was obtained instead.

The

literature contains two other examples of the removal of bromine from a thiophene ring with hydrogen but these took place in the presence of palladium deposited on charcoal (32,81). The yield of /3 -2 -thienylalanine prepared through the rhodanine synthesis is 39% when computed from thiophene. On this basis, this figure compares favorably with du Vigneaud's method (37f o ) and Dittmerfs method (32f o

) .

It is within the realm of possibility that the alkyl and chloro thienylalanines which were also procured in good yields may prove to be useful In biological investigations. The application of the beef pancreas carboxypeptidase

83 preparation resulted in the resolution of /3-2-thienyl-DLalanine but only when the original procedure was modified. G-reenstein reported bringing the enzyme suspension into solution by the addition of a minimum amount of lithium chloride.

When this method was tried, a considerable

amount of insoluble material remained.

Anson (6 ) in pre­

paring crystalline carboxypeptidase described the enzyme suspension under discussion as being completely soluble in sodium hydroxide and partially soluble in barium hydroxide. This description was found to be accurate.

The solvent

power of lithium hydroxide was investigated, too, and found to lie between sodium hydroxide and barium hydroxide. Since the chloroacetylated amino acid is dissolved in lithium hydroxide prior to digestion, the

enzyme

prep­

aration was also dissolved this way and left only a slight residue. In the actual resolution, two major differences be­ tween phenylalanine and (3 -2 -thienylalanine became apparent. Chloroacetyl-L-phenylalanine is completely hydrolyzed by beef pancreas carboxypeptida.se in the absence of buffer. Under these circumstances while some optically pure ft

-2 -thienyl-L-ala.nine was obtained from ch l o r o a c e t y l - / 3

-8 -thienyl-DL-alanine, the chloroacetylated D-compound was evidently contaminated with unhydrolyzed chloro­ acetylated L-isomer because its specific rotation was The chloroacetylated L-isomer when hydrolyzed with L

2N

HC1 yielded a free amino acid with no

optical activity.

However, w h e n buffer was added to the

digestion mixture optically pure/3-2-thienyl-L-alanine and chloroacetyl-/3-2-thienyl-D-alanine were obtained.

In the isolation of D-phenylalanine, chloroacetyl-Dphenylalanine was hydrolyzed by refluxing with 2N HC1 and then evaporated to dryness in vacuo.

The residue was

evaporated to dryness twice more with water to remove excess HG 1 . When this procedure was applied to chloroaeetyl-/3-2 -thienyl-D-alanine racemization occurred. However, substitution of IN HBr in the hydrolysis step followed by only one evaporation to dryness obviated this difficulty.

The excess HBr was removed with LiOH thus

forming LiBr which was readily soluble in absolute alcohol at pH 5.2 whereas /^-2 -thienyl-D-alanine precipitated from solution.

VII. Summary 1

. The Erlenmeyer azlactone synthesis, using hippurie acid

as the acylglycine component, has been applied to a series of alkyl and halogen substituted thiophene aldehydes. 2

. The Erlenmeyer azlactone synthesis, using acetylglycine

as the acylglycine component, has been applied to

2 -then-

aldehyde. 3. Rhodanine was shown to condense with a number of alkyl and halogen substituted thiophene aldehydes. 4. The utilization of rhodanine condensation products for syntheses in the thiophene series was demonstrated by the preparation of thienylthiopyruvic acids, thienyloximinopropionic acids and /3-2-thienylalanines from

2 -thenalrhodanine

and a variety of alkyl and halogen substituted thenalrhodanines.

2 -Thenalrhodanine

acetonitrile,

was also used to prepare

2 -thienylacetamide, 2 -thienylacetic

2 -thienyl-

acid and

/3-2 -thienylethylamine. 5. Racemic

2 _t hie nyl alanine

was resolved into its optical

isomers through the asymmetric hydrolysis of its chloro­ acetyl derivative using purified beef pancreas carboxypeptidase preparation. 6

. The following compounds which were synthesized have not

been reported previously in the literature: 2-phenyl-4(5-methyl-2-thenal)5-oxazolone 2 -phenyl-4(3-methyl-2 -thenal)5-oxazolone 2 -phenyl-4(5-ethyl-2-thenal)5-oxazolone

2-phenyl-4(5-propyl-2-thenal)5-oxazolone

86 2-phenyl-4( 5-chloro-2-thenal)5-oxazolone

n

2-phenyl-4( 5-bromo-2-thenal) 5-oxazolone 2-phenyl-4(3 ,4-dimethyl-2-thenal)5-oxazolone 2-phenyl-4(3,4,5-tririiethyl-2-thenal)5-oxazolone 2-phenyl-4 (4,5-dimethyl-2-thenal)5-oxazolone 2-phenyl-4(2,5-dimethyl-3-thenal)5-oxazolone 2-phenyl-4( 2 ,3,5-trimethyl-4-thenal) 5-oxazolone 2-phenyl-4 (3-thianaphtlial)5-oxazolone 2-phenyl-4(2 -thenal)5-oxazolone 5-methyl-2 -thenalrhodanine 3-methyl-2-thenalrhodanine 5-ethyl-2-thenalrhodanine 5-propyl-2 -thenalrho danine 5-chloro-2-thenalrhodanine 5-bromo-2-thenalrhodanine 3-thianaphthalrhodanine 3.4-dimethyl-2-thenalrhodanine 3.4.5-trimethyl-2 -thenalrhodanine 3.4-diiiiethyl-2-thenaldehyde 3.4.5-trimethyl-2-thenaldehyde 2 -thienylthiopyruvic

acid

3-methyl-2-thienylthiopyruvic acid 5-methyl-2-thienylthiopyruvic ac id 5-et hyl-2 -thienylthiopyruvic ac id 5-propyl-2-thienylthiopyruvic ac id 5-chioro-2 -1 h ienylt hiopyruvic ac id 5-bromo-2-thienylthiopyruvic acid

87 3-(2-thienyl)-2-oximinopropionic acid 3-(5-methyl-2-thienyl)-2-oximinopropionic acid 3-(5-ethyl-2-thienyl)-2-oximinopropionic acid 3-(5-propyl-2-thienyl)-2-oximinopropionic acid 3-(5-chloro-2-thienyl)-2 -oximinopropionic ac id 3-(5-bromo-2-thienyl)-2 -oximinopropionic acid /^-( 5-methyl-2-thienyl)-alanine f t - (5-ethyl-2 -thienyl)-alanine f t -(5-propyl-2 -thienyl)-alanine ft

-(5-chloro-2-thienyl)-alanine

chloroacetyl-f t -2 -thienyl-DL-alanine chloroacetyl-f t -2 -thienyl-L-alanine chloroacetyl-ft -2 -thienyl-D-alanine

L

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L

VITA NAME

Bernard F. Crowe

DATE OE BIRTH

Nov. 2, 1921

M M T A H I

Incarnation Grammar School, N.Y., N.Y. 1935

SCHOOL

Graduated HIGH SCHOOL Graduated

De Witt Clinton H.S. 1939

BACCALAUREATE DEGREE

B.S. Fordham College 1943

Date OTHER DEGREE

M.S. Fordham University 1948

MILITARY- SERVICE

U. S. Army Air Force Mar. 1943 - July 1946

PUBLICATIONS (co-author)

Syntheses of New Azlactones from Thiophene Aldehydes, Nature, 163, 876 (1949). Azlactones and Rhodanines Prepared from 2-Thenaldehyde and some Substituted 2-Thenaldehydes, J. Org. Chem., 15, 81 (1950). Enzym­ atic Resolution of Raeemic ^-2-Thienylalanine, Arch. Biochem., 25, 460 (1950). Enzymatic Res­ olution of Racemic/3-2Thienylalanine and Prep­ aration of some Sub­ stituted /3-2-Thienylalanines, J. Org. Chem. (in press).