Synthesis and properties of the quaternary strychnine salts

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SYNTHESIS AND PROPERTIES OF THE QUATERNARY STRYCHNINE SALTS

A Thesis Presented to the Faculty of the College of Pharmacy The University of Southern California

In Partial Fulfillment of the Requirements for the Degree Master of Science

by Thomas John Haley February 1942

UMI Number: EP63455

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T h is thesis, w ritte n by

t$so£-

THOMAS JOHN HALEY und er the direction o f

ALa.. F a c u lt y

C o m m itte e ,

a n d a p p r o v e d by a l l its m e m b e r s , has been presented to a n d accepted by the C o u n c il on G ra d u a te S tu d y an d Research in p a r t ia l f u l f i l l ­ m e n t o f th e r e q u i r e m e n t s f o r the d e g re e o f

MASTER

OIENCE

D ean

Secretary D ate.... EeJir.uar.^^...i9.42..

Faculty Committee

}

Lihatrman

9

.

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TABLE OF CONTENTS SECTION

PACE INTRODUCTION..................................

I.

THE CHEMISTRY OF DRUGS WITH CURARIFORM ACTIVITY

1

Organic and inorganic bases

................

2

....................

8

THE PHYSIOLOGICAL AND PHARMACOLOGICAL EFFECTS

IV.

10 10

Organic and inorganic bases

................

11

. . ................

12

THE THERAPEUTIC USE OF CURARIFORM DRUGS

....

14

Spastic and dyxtonic muscle conditions . . . .

14

Treatment of t e t anus........................

is

Shock treatment of mental conditions........

18

EXPERIMENTAL.................................. Synthesis of strychnine alkyl halides

V.

.

Curare and its alkaloids....................

Alkaloidal double salts III.

1

Curare and its alkaloids....................

Alkaloidal double salts II.

vi

• • • •

19 19

Synthesis of methyl-strychnine ..............

20

Physical properties of synthesized compounds .

21

Pharmacological and physiological effects

27

. .

C O N C L U S I O N S ..................................

B I B L I O G R A P H Y .........................................

37 40

LIST OF CHARTS CHART 1.

PAGE Inorganic Salts and Alphatic Ammonium Bases Name* Formula, Molecular weight, and Effect . . .

£•

Aromatic Ammonium Bases Name, Formula, Molecular weight, and Effect . . .

3.

9

Comparison of Reaction Time Strychnine methochloride and Curarine chloride

5.

6

Alkaloids and Alkaloidal Double Salts Name, Formula, Molecular weight, and Effect . . .

4.

3

Effects of Compounds on Intact Animals

. . . . . .

.

16 31

LIST OF GRAPHS PAGE

GRAPH 1*

Response of Asphyxiated Nerve to Action Current after Poisoning with Curariform Drugs . . . . .

17

LIST OF PLATES PLATE

PAGE

1*

Microphotographs..................................

2.

Kymograph Records

...................

26

INTRODUCTION The history of curare is an old one, it has long been known to the civilized world*

In the sixteenth century Sir

Walter Raleigh'*' brought the first samples to England, but nothing was done with it until Pelouse and Bernard^ investi­ gated the action of the drug in 1850.

In 1864 the alkaloid r*

curarine was isolated from curare by Preyer.

After spending

two years learning the manufacture of curare from the natives, G-ill4 returned to this country with sufficient quantity of the drug to enable a more comprehensive study of its properties. Boehme^ prepared a sample of the alkaloid curarine in 1898; this material has subsequently been used for the deter­ mination of its action on muscle and nerve preparations and also for comparison with the various synthetics and other alkaloids used as substitutes for curare.

In the group of

materials prepared as substitutes for curare we find many of the onium salts (Ing,^ 1936), including the quaternary ammonium salts, simple phosphonium, arsonium, stibonium, sulfonium and iodonium salts as well as the metho-halides of n

several alkaloids (Crum-Brown and Fraser, 1927).

O

1869, and Frankel,

It is on the metho-halide compounds that much of the

recent work has been done in an effort to obtain a suitable source of supply of a drug having a curariform activity equal to curare itself, as the curare is difficult to obtain and the different samples vary in strength and action.

This paper

vi will discuss, first the standing of the various compounds having a curariform activity as well as curare itself in re­ spect to their chemical and physical nature, physiological action and their application to modern therapeutics and secondly the physical and physiological properties of strych­ nine methyl iodide, struchnine ethyl iodide, strychnine isopropyl iodide and methyl strychnine with respect to their methods of synthesis, purification, melting point solubility, crystaline structure and physiological action in the animal body. The purpose of this investigation is to determine whether the compounds studied actually have curariform activity which would be applicable to human therapeutics, also the factors which might limit its use to lower animals.

These factors in­

clude solubility in water, length of time of activity in the animal body and toxicity to the animal body.

SECTION I TEE CHEMISTRY OF DRUGS WITH CURARIFORM ACTIVITY The actual chemical composition of curare and its alka­ loids curine and curarine is not known but the source of this potent drug is from the leaves, stems, bark, and roots of a variety of Strychnos plants, notably Strychnos toxifera and Strychnos Castelaeni and also from Cocculus toxifera.

Curare

is a brownish-black, shiny, resinoid mass soluble in cold water and diluted alcohol.

The difference in the varieties

of curare is usually designated by the containers in which they are supplied to the importers, namely, tubocurare (bamboo tubes), calabash curare (gourds), and pot curare (earthen­ ware pots). Some investigators believe the action of curare is in­ dependent of its chemical structure while others

(Frankel,^

1927) believe it is due to the quaternary nitrogen atom in the molecule,

although the exact chemical nature of the sub­

stance is still a mystery*

One of the alkaloids, curine,

which has no curariform activity, can be readily converted into curarine, which has marked curariform activity, by the addition of a metho-halide, showing that the ternary nitrogen atom in the molecule has been converted into a quaternary nitrogen atom* Some inorganic salts have shown curariform activity,

notably ammonium iodide and magnesium sulfate; the action in the former is very slight but the latter has enough activity to be used in the convulsive shock treatment of mental dis­ eases as a nerve cnd-plate paralysant.

This curariform activ­

ity of magnesium sulfate was discovered by Jolyet and Cahours in 1869 and later confirmed by Meltzer9 in 1907. The alphatic ammonium bases have been investigated by Frankel8 and Ings and the effect of the different groupings may be seen in Chart 1.

Although there seems to be no definite

point at which these bases lose their curariform activity due to the increase in molecular weight and to the increase in the length of the side chain, it would be expected that the com­ pounds as well as those of the aromatic series, in Chart 2, would become less effective as the above weights become greater and the chain length increases.

This would correspond to the

principles upon which Schmiedebergs R u l e s ^ of the action of alphatic compounds are based. 1. The physiological activity of substances (espec­ ially alphatic) depends chiefly on physical properties. 2. The readiness of absorption is very important, as with no absorption there can be no physiological action. 3. Volatility and solubility in water are of great importance as the higher non-volatile members of the paraffin series exert no action. A study of Chart 1 shows that the first series of com­ pounds were substituted into the ammonium group starting with ammonium hydroxide to give a compound with a quaternary nitrogen

3 CHART 1 INORGANIC SALTS AND ALPHATIC AMMONIUM BASES

Name

Formula

Molecular weight

Effect

Ammonium Iodide

NH4I

144.96

C ____

Magnesium Sulfate

MgS04

120.38

C

Ammonium Cyanide

NE4CN

44.06

C

Ethyl Ammonium Chloride

C2H5NH3C1

81.52

C

Amyl Ammonium Chloride

C5E11NH3C1

123.57

C

Amyl Ammonium Iodide

C5H11NH3I

202.88

C

Amyl Ammonium Sulfate dimethyl Ammonium Chloride• Dimethyl Ammonium Iodide Diethyl Ammonium Chloride Diethyl Ammonium Iodide Diethyl Ammonium Sulfate Trimethyl Ammonium Iodide Triethyl Ammonium Chloride Triethyl Ammonium Iodide Triethyl Ammonium Sulfate Tetramethyl Ammonium Iodide Tetraethyl Ammonium Iodide ‘Tetraamyl Ammonium Iodide Tetramethyl Ammonium Formate Tetraethyl Arsonium Cadminuin Iodide

C 5H11NH3NH4S 04

185.19

C

81.52

C

(CH3)2NH2I

160.83

C

(C2H5)2NH2C1

109.56

C

(C2H5)2NH2I

188.86

C

(C2H5)2(NH2)2S04

170.16

C

(CH3)3NHI

174.84

C

(C2H5)3NHC1

137.59

c

(C2H5)3NHI

216.89

c

(C2H5)3NHNH4SC4

216.24

c

(CH3)4NI

188.86

c

(C2H5)4NI

244.92

C M N

(C5H11)4NI

413.12

c

(CH3)4N00CH

119.11

D

(C2H5)4AsCdI

418.24

C

(CH3)2NH2C1

4 CHART 1 (continued) INORGANIC SALTS AND ALPHATIC AMMONIUM BASES

Name

Formula

Methyl Triethyl Stibonium Iodide Methyl Triethyl Stibonium Hydrate Tetraethyl Phosphonium Iodide tetraethyl Arsonium Zinc Iodide tetraethyl Arsonium Iodide Trimethyl Sulfonium Hydrate Trimethyl Sulfonium Iodide Tetramethyl Arsonium Iodide tetramethyl Phosphonium Iodide tetramethyl Stibonium Iodide triethyl Sulfonium Bromide tetraethyl Ammonium Chloride C c S M N P

-

Molecular weight

Effect

CH3(C2H5)SbI

338.66

C

CH3(C2H5)3SbOH

240.91

c

(C2H5) 4PI

261.90

c

(C2H5)4AsZnI

371.21

c

(C2H5)4AsI

305.83

S c

(CH3)3S0H

94.14

C

(CH3)3SI

191.89

C M N

(CH3)4AsI

205.83

M N

(CH3)4PI

249.76

C M N

(CH3)4SbI

296.61

M N

(C2H5)3SBr

199.10

N

(C2H5)4NC1

165.89

N

great curariform activity (paralysis of motor end-plates). lesser curariform activity (paralysis of motor end-plates), strychnine activity (stimulation to spinal cord). muscarine activity (stimulation of parasynpathetic nerves). nicotine activity (paralysis of autonomic ganglia). picrotoxin activity (stimulation to central nervous system).

5 atom, in each case the hydrogen content of the ammonium group was decreased and the alphatic groups were increased until a tetra alphatic compound was formed. The second series of compounds were substituted arsonium, stibonium, phosphonium, or sulfonium bases always following the above sequence of substitution to form a quaternary base.

All

the above bases exist as ternary or quaternary compounds and it is only when they are in the quaternary form that they show curariform activity.

These compounds are listed in Chart 2.

As is the case with many compounds showing curariform activity there are secondary reactions occuring at the same time which in many cases are undesirable, particularly those side reactions similar to muscarine and nicotine, which are in some instances great enough to mask the desired effect of curariform activity.

Reference to the charts will show those

compounds having such properties.

Also in some instances the

compounds showed a lesser margin of safety in that the compound became lethal in very low concentrations, this was particularly evident with ethyl atropine and methyl atropine which were among the alkaloids which Frankel studied. With the aromatic ammonium bases there was even greater variation and the curariform activity was often accompanied by an action on the central nervous system. aromatic bases studied are shown on Chart 2.

The numerous It is seen on

this chart that the original alphatic bases were used as a

6 CEART 2 AROMATIC AMMONIUM BASES

Name Phenyl dimethyl ethyl ammonium iodide Phenyl dimethyl amyl ammonium iodide Phenyl dimethyl amyl ammonium hydrate Phenyl triethyl ammonium iodide Toluyl diethyl emyl ammonium iodide Ditoluyl diethyl ammonium iodide Yoluyl diethyl amyl ammonium iodide Toluyl triethyl ammonium hydrate Thimethyl menthy1 ammonium hydrate Trimethyl ammonium hydroxide valeric acid Eexa-methyl-tetra methylene di-ammonium hydroxide Hexa-methyl-penta methylene di~ammonium hydroxide Trimethyl beta imidazolyl ethyl ammonium hydroxide Phenyl ethyl pyrazole ammonium tr. ln-methy1-3.3-dimethyl indolium exyhydrate

Formula

Molecular weight

EfTec'

C6H5(CH3)2G2H5NI

264.92

C

C6H5(CH3)2C5H11NI

306.96

C

C6H5(CH3)2C5H11N0H

209.21

C

C6H5(C2H5)3NI

292.94

C

CH3C6H5(C2H5)2C5H11NI

348.99

C

(CH3C6H5)2(C2H5)2NI

368.96

C

CH3C6H55 C2H513G5H11NI

324.99

c

CH3C6H5{C2H5)3HI

209.19

c

(CH3)3CH3C6H9CE(CH3)2N0H

215.24

c

(CH3)3NOOCC4E9

160.15

c

(CK3)6(CH2)4N2

174.22

c

((CH3)3(HH)(CH2)2)2

188.24

c

(CH3)3(CHNCHNCH)C2H5N

155.16

c

C6H5C2H5N(CH2)3NH(NE2)

193.17

c

CH3(CH3)2(C8H7N)OH

179.14

C P

Methyl aniline

C6E5NHCH3

107.08

G

Ethyl aniline

C6E5NHC2H5

121.09

C

Amyl aniline

C6H5NHC5H11

163.14

c

Methyl quinoline

CH3G9H6N

143.08

NC

Ethyl quinoline

C2E5C9H6N

157.09

NC

Amyl quinoline Oxvp.+.hvi Quinoline ammonium chloride

C5E11C9H6N

199.14

NC

C2E50C9E6NNE4C1

236.59

NC

7 CHART 2 (continued) AROMATIC AMMONIUM BASES

Name

Formula

Molecular - weight

Effect

Diquinoline dimethyl sulfate

(C9H6)2S04(CH3)2

384.63

NC

Methyl violet

(C6H5)3CH

244.IS

D

Methyl iodide

CH3I

129.78

C D

Quinoline

C9H7N

129.06

NC

NC - no curariform activity* D - Digitalis effect (stimulating to the cardiac muscles).

8 starting point and the phenyl and tolyl groups were substituted into the alphatic compounds in place of one of the original alphatic groups of the ammonium base, also it is shown that for the most part ammonium iodide was used as the starting compound as was the case with the alphatic series of compounds.

In

the case of the indol base studied the primary action was cur­ ariform even to its paralysis of the respiratory tract but the secondary action resembled a typical picrotoxin convulsion. In the alkaloids investigated we find alphatic groups being substituted into the molecule in a manner quite different from that employed in the other series of compounds, namely the methyl, ethyl, or amyl group was not attached to the ternary nitrogen atom alone but it was attached to both the ternary nitrogen atom and to a carbonyl group which had been formed when the alkaloid was converted into the alkaloidal acid.

The

other method, employed for alkaloids, converted the alkaloid into a double salt by the addition of an alkyl halide to the ternary nitrogen atom forming a quaternary nitrogen derivative. This bond is very unstable and subject to hydrolysis giving the free alkaloid.

This may be observed by examination of the

formula of any of the alkaloidal compounds listed in the charts. In the aforementioned charts it may be seen that the increase in the molecular weight of the compounds by the addition of other radicles while it may affect the solubility and diffusibiiity of the compound apparently does not restrict its action, as the action depends upon the formation of the quaternary salt.

9 CHART 3 ALKALOIDS AND ALKALOIDAL DOUBLE SALTS Name

Formula

Molecular .

Effect

______

Berberine

C20H1905N

353*16

Methyl piperidine

C5H10NCH3

99.11

Methyl atropine

C17H2203NCH3

303.20

NC A

Methyl strychnine

C21H2102N2CH3

348.20

S

Ethyl strychnine

C21H2102N2C2H5

362.22

S

Methyl brucine

C22H2504N2CH3

396.24

S

Ethyl brucine

C22H2504N2C2H5

410.25

S

Methyl cinchonine

C19H210N2CH3

308.20

NC

Amyl cinchonine

C19H210H2C5H11

364.27

NC

Methyl quinine

C20H2302N2CH3

338.22

NC

Methyl quinidine

C20H2302N2CH3

338.22

NC

Methyl cocaine

C18H204NCH3

329.19

NC

Methyl codoine

C18H203NCH3

313.19

C s

Methyl morphine

C17H1803NCH3

299.17

c

Dimethyl conine

C3H7C5H10N(CH3)2

173.19

NC

Curarine

Unknown

317.00

C

Curare

Unknown

Unknown

C

Ethyl nicotine

C10H13N2C2H5

290.16

NC N

Methyl thebaine

C19H2003NCH3

301.19

C

Methyl Veratrine

C37H52011NCH3

701.44

NC

Amyl veratrine Cinchonine iodic acid methyl ester

C37H52011NC5H11

757.51

NC

C19H22N20(1CH2)(CO)(0)CH3

481.99

NC

Conine

C3H7C5H10N

127.24

NC

Spartine Benzyl bromide C15H2 6N2(C6H5Br)

391.18

C D

Spartine

234.22

C D

C15H26N2

S NC

SECTION II THE PHYSIOLOGICAL AND PHARMACOLOGICAL EFFECTS In a discussion of the effects of curare and other drugs with a similar physiological and pharmacological action it would he well to review the physiology of that part of the nervous system which is affected by those drugs, namely the motor end-plates of the peripherial nervous system, as well as the central nervous system which is affected by the undesirable side reactions which often accompany the paralysis that develops The nerve impulse passes through a reflex arc from the receptor to the effector and it is not affected by any curari­ form drugs except at the effector junction of the motor endplates with the muscle.

The ganglionic cells of the sympathe­

tic nervous system still secrete acetylcholine but this chemical upon which the effector response is believed to be dependent does not cause its customary response (Brown, Dale, and Feldberg, 1936).

It is believed that the sympathetic nervous system is

not affected by curariform drugs. The effects on the central nervous system are due to stimulation and all parts of the cerebro-spinal axis are af­ fected, particularly the spinal cord in which instead of a simple reflex are mechanism exciting a discrete motor response the minimal sensory impulse spread diffusely up and down the cord causing a tetanic convulsion.

11 The absorption, fate of the drug in the body, and its excretion should also claim our attention as this will in most cases decide the mode of administration.

Curare and curarine

are absorbed in the gastro-intestinal tract to a very limited extent and this prevents any toxic accumulation of these drugs in the tissues, there is also the possibility that the drug may be destroyed in the gastro-intestinal tract before it can exert its action.

Any of the drug which is absorbed is partially

destroyed in the liver and the remainder is excreted unchanged by the kidneys.

With impaired renal function there is the

possibility of increased concentration in this organ, with the subsequent toxic reactions.

These drugs must be given hypo­

dermically to exert their action. Of the other drugs investigated there is no record

of

any work having been done on their fate in the body or their excretion, with however the exception of strychnine methochloride and strychnine methoiodide as investigated by Cowan and Ing^*2 in 1934, these drugs were absorbed similarly to curare and curarine if given hypodermically and their fate in the body and their excretion were similar to curare and curarine. The oral administration of strychnine metho-halides is contra­ indicated due to the possibility of hydrolysis to the free alkaloid by the secretions in the gastro-intestinal tract with subsequent strychnine poisoning.

This would be

true no matter

which alkaloidal base was used and the poisoning would be that

12 characteristic of the alkaloid used to form the base. Y/ith the other aforementioned synthetics on which there is no data concerning their absorption, fate

in the body or

excretion, it is probable that they are detoxified in the liver and excreted in the urine in a similar

manner to curare.

Recently it has been reported that an alkaloid obtained from the seeds of Erthrina corralloides is effective both orally and hypodermically (Burman,13 1939), also that its margin of safety was greater than that of curare. Upon the introduction of curare or other drugs with curariform activity into the body it is of prime importance that the symptoms of curare poisoning be recognized.

The first

reaction is a paralysis of the muscles of the fingers, toes, eyes, and ears; gradually those of the limbs, neck and trunk are affected, then lastly those of the diaphragm.

This results

in asphyxia due to the inability of muscular contraction which also prevents asphyxial convulsions.

There is no direct toxic

action on the heart but cardiac failure results from anoxia due to the respiratory arrest.

Artificial respiration and the

injection of either 1:2000 prostigmin solution or 1:1000 epinepherine solution aids in recovery which takes place in the reverse order of the paralysis. The use of magnesium sulfate which also has a curariform activity is limited to intravascular injection, as orally the only effect obtained is that of catharticls.

The absorption

of magnesium sulfate in the body is limited and its excretion when given orally is handled almost entirely by the gastro­ intestinal tract although a small portion is excreted by the kidneys; with hypodermic injection it is excreted entirely by the kidneys although retention for two or three days may occur (Soloman).^

Poisoning by this drug shows a different

action on the heart than that shown by curare, in that there is a slowing of the heart rate with moderate dilation, then incoordination and finally sudden dilation and standstill. All other reactions on the animal body resemble curare.

SECTION III THE THERAPEUTIC USE OF CURARIFORM DRUGS The use of curare by early investigators with resultant toxic manifestations and death, caused the abandonment of its use in therapeutics until recent times.

The recent knowledge

that curare is of varying potency is probably the contributing factor which lead to its early abandonment by the physician although the aqueous or alcoholic extracts as now prepared and standardized appear to be reliable in cases where a relaxation of the musculature is desired (Burraan,^ 1937). In his recent application of these extracts Burman was able to produce beneficial results in cases of spastic and dystonic muscle states in humans.

In the cases treated the

muscular relaxation enabled the patients to use muscles which they had been unable to use for years.

It is shown that they

were able to walk without too much discomfort and were able to write their names more legibly than before treatment with curare.

Although both intravenous and intramuscular injec­

tions were employed the former required only half the amount necessary as when the latter was used, and even with repeated injections (every two or three days) no harmful effects were observed.

It should be emphasized however, that a complete

knowledge of the toxic manifestations of the drug and their antidotes be known before its application.

Either 1:2000

15 solution of prostigmin or 1:1000 epinephrine solution as well as artificial respiration should he used should the occasion arise.

It was also found the certain groups of muscles showed

more sensitivity to curare action than others. In the same publication Burman stated that the effects of erythrodine were different than curare in their action on the nervous system in that the cranial nerves were affected before the motor nerves. The use of the synthetic ammonium bases in human thera­ peutics has not been attempted as yet but Cowan and Ing^2 tested the effects on the excised nerve from the leg of the walking crab (Maia squinado) and on sartorius nerve-muscle preparations of the frog (Rana Pipiens).

With the frog nerve-

muscle preparation which was asphyxiated with hydrogen at 19° C. the nerve failed to respond after five hours and two hours later when oxygen was administered a response of about 50 per cent above the initial response was observed v/hen the nerve had been treated with strychnine methoiodide of a strength of 1 millimole per liter and v/hen treated with tetraammonium iodide of a strength of 2 millimoles per liter there was no response.

Work was also done on intact animals and this is

shown on Chart 4, the other work is shown on Graph 1.

Quinine

methochloride has been employed by Harvey^e using decerebrated cats.

Both of these investigations showed that these prepara­

tions while giving promise of ultimate usage in human therapeutics

16 CHART 4 COMPARISON OF REACTION TIME

Molecular weight

Material

Concentration in mm/L 1.0 0.1

Strychnine methoiodide

476

3 minutes

6.3 minutes

Kings Curarune Chloride

317

3.7 minutes

7.0 minutes

Boehm*s Curarine Chloride

317

3.8 minutes

7.0 minutes

Mean time in minutes required for complete paralysis of sartorius nerve muscle preparations to indirect stimulation. Temperature 17.0° C.

Material Curarine Chloride (no reflex excitability) Strychnine Methoiodide

Concentration 0.1 mm/L (0.016-0.032 Mg)

Frog weight

Paralysis time

Dose

80 Gm.

12 hours complete in 10-15 minutes

0.1 mm/L

80 Gm.

0.5 hour 1.0 cc incomplete under skin paralysis reflex excitability enhanced

0.1 mm/L

80 Gm.

5 minutes partial paralysis 10 minutes further advanced 15 minutes complete

0.5-1.0cc (dorsal or ventral lymph sac)

2-3 cc (dorsal lymph sac)

Twitellings and other central symptoms modified by altering method of administration or temperature at which experiment is carried out. In some experiments at 23.0° C. central symptoms less marked.

17 GRAPH 1

f init ial 1- 1-4G

Hs 1 0 5 4 ■’j■- ITim© in h0urs Tigure 0”* “Cowan ~L. “_ ! ..T*.L:7~L iThe action current in frogs .asphyxiation in hydrogen at

,

L~

a T* Physiol. ”■ "7ZZiV_II"”.7..! . .._i____ !_. nerve during and after 19° C.

Ai-:M7t©lLjj?>0i^^ mm/l* of B - after;poisoning with; 1 mm/L t f strychnine metlfioiod^Lde• ,C - control after soaking for one and one-half hoars in huffared Ringers aoiution [

18 were still in the experimental stages#

Quinine methochloride

was similar to erythtodine in one respect in that it was effective orally# The treatment of tetanus was another conditions in which curare and curarine were employed but as yet none of the other substitutes have been used#

Success in this type of

medication has been reported by C o l e ^ and by W e st^® in 1936, and M i t c h e l l ^

in 1934 using curare, 1 9 3 5 using

curarine#

The principal usage of curare or drugs with a curariform activity is as a preliminary medication in the shock treatment of mental diseases; here complete relaxation of the musculature of the patient is a prerequisite to success, otherwise traumatic fracture may occur, due to the violent contraction of the muscles.

The complete relaxation of the

patient also has some psychological benefit in that the patient shows no fear or apprehension of the impending shock treatment. Using either aqueous or alcoholic extracts of curare by hypodermic injection, Bennett20 reported that the action of curare on the motor nerve end-plates of all striated mus­ culature prevented traumatic fracture in the properly curarized patient unless there was a serious pathological condition in the bone.

The dosage of curare used as preliminary medication

was 0.1 of the lethal dose per kilogram body weight for humans.

SECTION IV EXPERIMENTAL A thorough investigation of the literature revealed that there was no information on the synthesis, or physical properties of the strychnine alkyl halides or methyl strych­ nine although it was stated that strychnine methoiodide could he purified by recrystalization from water. In the synthesis of the struchnine alkyl halides car­ ried out in this laboratory two methods were employed which gave splendid results. 1. In the first method, the strychnine alkaloid and the alkyl halide were placed in a covered vessel and left for the period of one week after which the product was filtered by suction and washed successively with benzene, ethyl alcohol and ether and then dried.

The resulting crude product was

then dissolved in hot water and concentrated by partial vacuum distillation, after which it was crystalized out by lowering the temperature of the concentrated solution with an ice bath. This purified salt is in the form of small crystals of definite shape depending on the particular alkyl halide used to form the quaternary salt. 2. In the second method, the strychnine and the alkyl halide are refluxed for a period of sixteen hours and then the product is purified as in the first method. The particular alkyl halides employed form the quaternary

20 strychnine salts were methyl iodide, ethyl iodide and isopropyl iodide.

The mode of introduction of the alkyl halide into the

strychnine molecule depends on the ternary nitrogen atom and the mechanism of the addition is similar in all instances as shown by the following formula.

Strychnine

Methyl iodide

Strychnine methyl iodide

As there is only one ternary nitrogen atom in the strychnine molecule this reaction should take place in the manner described, as the other nitrogen atom is in the form of an imide grouping and is not reactive.

(Henry,^1 1939.)

With methyl strychnine, however, the method of production differs in that after the strychnine methyl iodide has been purified it is reduced with freshly prepared silver hydroxide to form the methyl strychnine and remove the iodine atom from the compound forming silver iodide which precipitates and is removed from the solution by suction filtration.

The

resulting solution is red colored and is evaporated to a small volume to effect separation of the salt which crystalizes out in the form of feather-like crystals.

The salt is purified

by washing with benzene, ethyl alcohol and ether.

The removal

of the iodine and the different crystal structure shows that this salt is not the same as strychnine alkaloid or strychnine methyl iodide.

21 The purification processes of the strychnine salts as well as the methyl strychnine must be repeated to remove all traces of iodides and free iodine which is shown by their brown color as well as the brown color of their solutions; this color is effectively removed in the washing with organic solvents, particularly with the alcohol and ether.

Care must

also be exercised in the partial vacuum distillation of their solutions as too great heat causes a breakdown of the product. This step cannot be used in the case of methyl strychnine, as the constant flow of air through the solution causes an oxida­ tion of the product. The following diagram shows the apparatus employed in the partial vacuum distillation. F

) A - water line 3 - condensing flask C - vacuum line D - 1 L Claisen flask E - constant level water bath F - capillary tube

DETERMINATION CF MELTING POINTS In the determination of the melting points using an emergent stem thermometer and a cottonseed oil bath it was

22 found that the compounds decomposed before melting.

The follow­

ing table shows the decomposition points of the compounds as they were determined in groups of five. Melting Points in Degrees Centigrade

Compound

Strychnine methyl iodide 287° Strychnine ethyl iodide 299° Strychnine isopropyl iodide 240° Methyl strychnine 275°

290° 291° 248° 199°

285° 291° 247° 275°

287° 275° 250° 279°

286° 293° 248° 263°

When compared with strychnine alkaloid which melts at 268° C. and boils at 270° it is seen that the compounds are not strych­ nine but are salts of the nature of strychnine nitrate which also decomposes before melting.

This property of the above

compounds is one that can be used to differentiate between not only strychnine alkaloid and any one of the compounds, but could be used to differentiate between each of the compounds themselves. DETERMINATION OF SOLUBILITY In the purification process the insolubility of the com­ pounds was used as a method of precipitating them out in crys­ tal ine form.

In hot water these compounds were very soluble

but as the temperature was lowered to room temperature they crystalized out rapidly.

By using small quantities and trying

to dissolve them in varying amounts of water it was found that the compounds were soluble as shown in the following table.

23 Gm. Soluble in I q q CCt Water""

Compound Strychnine methyl iodide Strychnine ethyl iodide Strychnine isopropyl iodide Methyl sti'ychnine

0*1 gm* 0.1 gm. 0.06 gm. 0.025gm.

_ Temperature 23° 23° 23° 23°

C. C. C. C.

The above solubilities were determined with saturated solutions and they were checked by adding additional quantities of the compounds to the solutions, filtering and weighing the filtrate after drying.

The solubility of the above compounds is very

low but it is higher than that of strychnine alkaloid which is only soluble 1 gm. in 6420 cc. of water. DETERMINATION OF OPTICAL ACTIVITY The specific rotation of the compounds was also investi­ gated by making solutions of the compounds containing 0.05 gm. per 200 cc. of water and observing the optical rotation of the compounds in a polariscope.

The polariscope tube had a length

of 20 centimeters and the source of light was a sodium lamp. The following table gives the results of this experiment. Compound

Reading

Strychnine methyl iodide Strychnine ethyl iodide Strychnine isopropyl iodide Methyl strychnine

No rotation No rotation No rotation - 5° laevorotcry

Temperature 20° 20° 20° 20°

C. C. C. C.

Although methyl strychnine was the only compound to show optical activity it might be expected that the other compounds would also have this property as the parent substance, strychnine alkaloid, is laevorotcry.

This test could be used to distinguish

24 between strychnine and any of the above compounds,even methyl strychnine as the amount of optical activity would determine whether the material was strychnine or methyl strychnine.

The

loss of optical activity by the other three compounds may p e r ­ haps be due to structural changes in the molecule during the formation of the quaternary salt.

DETERMINATION OF REFRACTIVE INDEX The refractive index of the solutions of the compounds was checked and the compounds showed no refraction of light, which may have been due to the strength of the solution used. The solutions were saturated but even then contained very little active ingredient, due to the low solubility of the compounds.

The determinations were carried out using an

Abbe Refractometer.

This physical property will be determined

again as soon as it is possible to build a crystal large enough to enable it to be prepared for this type of operation. DETERMINATION OF CRYSTALLINE STRUCTURE As crystaline structure is often used as a rapid method of preliminary identification of a compound, microphotographs were taken with a 116 Eastman Box Brownie Camera, using the 4mm lens of a Bausch-Lomb Microscope for magnification. exposure time was thirty seconds.

The

By comparison with the crys­

taline microphotograph of strychnine alkaloid it is seen that

25 the strychnine alkyl halides as well as methyl strychnine have definite crystaline structure which is different for each com­ pound.

These differences in crystaline structure could be

used with the other physical properties as a method of iden­ tification of these compounds.

The complete set of microphoto­

graphs is shown on Plate 1. REACTION TO OFFICIAL STRYCHNINE TESTS In the National Formulary VT are found the official identification tests for strychnine alkaloid.

These tests

will determine qualitatively the presence of strychnine in any combination so that it is possible to determine whether the strychnine has been changed by the synthesis procedure which it had undergone to form the quaternary salts.

The results of

these tests are as follows: 1. Reaction of a saturated solution to litmus paper. Compound

Degree of Acidity

Strychnine methyl iodide Strychnine ethyl iodide Strychnine isopropyl iodide Methyl strychnine

Strongly acid Weakly acid Weakly acid Weakly acid

In this test the solutions of the last three but

slight change in the color of the

compoundscaused

litmus paper,while

the

first compound caused a rapid change of the blue litmus paper to red on contact with the solution. 2. Reaction with sulfuric acid containing 1 per cent ammonium vanadate.

26

PLATE 1 MICROPHOTOGRAPHS

S T R Y C H N I N E M E T H Y L IO D I D E

STRYCHNINE ISOPROPYL IODIDE

ST R Y C H N I N E E T H Y L I O D I D E

METHYL STRYCHNINE

27 Strychnine methyl iodide changed the color of the solution from purple to orange red, to cherry red. Strychnine ethyl iodide changed the color of the solution from purple to orange red to cherry red. Strychnine isopropyl iodide changed the color of the solution from blue to cherry red.

Methyl strychnine changed the color of the solution from blue to cherry red. In all cases the reaction was positive for strychnine, thus showing that the compound actually contained strychnine as a part of the molecule, otherwise if any change had taken place during the synthesis the test would have been negative in so far as the strychnine was concerned. 3.

Reaction when a small fragment of potassium dichromat

is added to a solution of about 0.05 gm of the compound in lcc. of sulfuric acid. Strychnine methyl iodide, strychnine ethyl iodide, strychnine isopropyl iodide and methyl struchnine all gave the same color change of from deep blue to deep violet to purplish-red to cherry red to orange. In all cases the test for strychnine was positive, thus con­ firming the first test for strychnine, also in this test the purple color seen at the start of the test showed the splitting of the compound and the liberation of free iodine due to hydrolysis of the compound in the presence of acid and confirms the presence of iodine in the compound. DETERMINATION OF PHYSIOLOGICAL ACTIVITY ON INTACT ANIMALS As a specific pharmacological action had been described

28 in the literature in regard to this type of compound, the pharmacological properties of the compounds were determined. At first a concentration of 1 millimole per liter was used but this was later modified to a concentration of 1 milligram per cubic centimeter, as the latter strength is the one used in human curarization and also as the former acted only as a stimulant to the animal and not as a paralyzing agent. The animal used was the common frog, Rana Pipens, and animals of all weights were used from 135.9 gm. to 30.1 gm. although it was found that animals weighing about 40.0 gm. gave the best results. The solutions of the compounds were made up in quanti­ ties of 50 cc. and placed in rubber capped vials.

These

solutions showed no deterioration after standing for two weeks at room temperature and were just as effective paralyzing agents as when first prepared. These compounds show a definite curariform activity except for strychnine isopropyl iodide and the paralysis in­ duced is typical of curare in that it progresses in the same manner.

First the toes and extremities of the limbs, then the

eyelids and finally the respiratory tract, at which time the skin respiratory mechanism takes over the function of respira­ tion; with the onset of the respiratory paralysis the muscula­ ture of the animal becomes completely flacid.

The stimulus

used to determine the degree of paralysis is a direct pricking

29 of the muscles with a pithing needle*

Recovery proceeded in

the same manner as curare with the respiratory mechanism recovering first and the rest of the muscles recovering in the opposite manner of the paralysis. Complete paralysis of the animal takes place in from three to seven minutes and complete recovery takes from four to fifteen hours depending on the body weight of the animal and the dosage administered.

The effective dosage has been

determined to be 1 mg. of cative ingredient per each 20 gm. of body weight.

The dosage is of course determined by the

solubility of the compound also.

Of all the compounds tested

the strychnine methyl iodide is the best paralyzing agent followed by strychnine ethyl iodide and methyl strychnine in that order.

There is no typical strychnine convulsion with

the above compounds but the recovery period shows a strych­ nine effect similar to that produced during the curare recovery period. With strychnine isopropyl iodide the effect observed is a typical strychnine convulsion which begins in seven minutes and progresses very rapidly to the acute stage,at which time the animal is stimulated to violent tetanic convul­ sions by the slightest tapping on the laboratory table and finally even the blowing of a stream of air on the animal’s back produces the convulsion. in about thirty minutes.

This condition leads to death

30 The heart rate of the animals was taken "before injection and at intervals of five minutes after injection.

With strych­

nine isopropyl iodide the heart rate of the animal increased to a point where it was impossible to get an accurate reading of the beats per minute.

With the other three compounds the

heart rate is not increased but decreases gradually, finally reaching the stage where a rate of twenty beats a minute may be observed.

In one animal of small size (31.1 gm.) there was

a period of about six hours in which it was impossible to determine the heart rate at all although even with this type of apnea the animal recovered and was given further injections without accumulative effects due to the previous dosage. The respiration of the animals used was comparatively steady with the initial rate varying from sixty to sixty-four beats per minute and gradually decreasing as the paralysis progressed and finally ceasing, at which time it was observed that the skin respiratory mechanism took over the function of supplying the system with oxygen. All the experiments were conducted on intact animals after it was discovered that pithing disrupted the details of the paralysis causing side reactions which covered the recov­ ery stages of the experiment.

The results on four of the

twelve animals are shown in Chart 5. DETERMINATION OF EFFECT ON NERVE-MUSCLE PREPARATION When it was discovered that the compounds with the exception

31 CHART 5 EFFECTS OF COMPOUNDS ON INTACT ANIMALS

Age ~ Young adult frog

Young adult frog

Young frog

"weight 68/min.

54.S gm.

Respiration 68/min.

Dropped to 64, then to 52

68/min.

Strychnine ethyl iodide lcc. lmgm/cc Respiratory paralysis starts with gasping in six minutes, labored breathing in 12 min­ utes. Animal unable to turn over, muscular twitchings in lower extremities 18 minutes. Complete paralysis of eye lids and partial paralysis of the limbs in 30 minutes. Paralysis wears out in 3 hours and recovery sets in with muscular twitching, re­ covery complete in 4 hours.

48*5 gm.

64/min.

Dropped to 48/min. in 30 min.

40/min.

Sub!!“ “

Strychnine methyl iodide 2cc lmgm/cc. Respiratory failure began with gasping in 3 minutes, paralysis complete in 7 min­ utes, slight twitching of muscles in 13 minutes. Re­ flex action restored in limbs in 75 minutes, greater re­ sponse to stimuli in 1 1/2 hours. Recovery complete in 3 hours.

33/gm.

60/min.

Strychnine isopropyl iodide 2 cc. 0/5mgm/cc Typical strychnine convulsion in 7 minutes. Response to tapping on table or stream of air was very violent in 12 minutes. Death in 1 hour.

33 CHART 5 (continued) EFFECTS OF COMPOUNDS ON INTACT ANIMALS

Age Young frog

Heart rate

Weight

Respiration

52/min.

33 gm.

68/min.

Substance injected and effect Methyl strychnine 2ce. 0.025 mgm/cc. Sluggish and gasping for breath in 3 minutes, all reflexes present in 30 minutes. Complete recovery in 75 minutes, at which time strychnine-like ef­ fects were observed.

33 of strychnine isopropyl iodide produced a curariform action in the intact animal, a further check was made to determine where this action took place and if it took place at the motor end-plates in the muscle as it does with curare. A nerve muscle preparation from the leg of the frog, Rana Pipens, was prepared, using the gastronemius muscle and the sciatic nerve, the kymograph and its mountings were assembled, the electrical connections and key, signal and inductorium in the primary and platinum electrodes in the secondary circuit were connected, the muscle mounted and the nerve was drawn across a watch glass containing salt solution was placed on the moist chamber base, the recording points adjusted and the records were taken on a fast drum.

Electrical

stimulus was applied by touching the nerve with the platinum electrode and pressing the key which applied the make and break shocks to the nerve.

The first records were to establish

a normal control then saturate solutions containing the active compounds were dropped on the watch glass and the nerve stimu­ lated until there was no further response*

The response was

abolished as follows. Strychnine methyl iodide in thirty minutes. Strychnine ethyl iodide in forty-three minutes. Methyl strychnine in forty-eight minutes. The muscle was then stimulated and a very definite re­ sponse was exhibited which showed that the nerve end-plates were not conducting the stimulus to the muscle as the muscle

34 still responded to a direct stimulus.

The kymograph records

of this experiment are shown in Plate 2.

mjtss K g m m j s n

sm / s m m

\Ia i"

lrlTfint

I

hh‘

?•=>h s