THE BEHAVIOR OF SOME HYDANTOIN DERIVATIVES IN METABOLISM. II.

Z-THIOHYDANTOINS.

BY HOWARD B. LEWIS.

(From the Shefield Laboratory of Physiological chemistry, Yale UniveTeity, New Haven, Conn.)

(Received for publication, February 11, 1913.)

In a former paper,l it has been shown that the hydantoin nuc- leus is excreted unchanged in the urine, when introduced into the organism of the cat, rabbit or dog. The present study deals with the behavior of some thiohydantoins in which sulphur replaces the oxygen in the a-position of the hydantoin nucleus.

NH-CO I c=o I

NH-CH2

NH-CO I c=s I

NH-CH2 Hydantoin 2-Thiohydantoin

The behavior of compounds containing this type of sulphur linkage is especially interesting in view of the recent studies of Johnson and his co-workers.2 In these the probable existence of sulphur in the protein molecule in forms other than as cystine or cysteine groupings is discussed, and the possible occurrence of thio- amide sulphur, -NHCS-, corresponding to the acid amide form, -NHCO-, present in polypeptides is suggested. This type of re- placement of oxygen by bivalent sulphur is well represented in the thiohydantoins. It must be pointed out, however, that the -CS - group attached to two nitrogen atoms as in thiohydantoins is much

1 This Journal, xiii, pp. 347-56, 1912. * Johnson: this Journal, ix, pp. 331-2,439-48,449-63,1911; xii, pp. 175-96,

1912. 245

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246 Behavior of Hydantoin Derivatives

more stable than the -CS- group placed between a nitrogen and a carbon atom as in a thioamide, R-CS-NH2, or as in dithiopiper- azine, the anhydride of the thiopolypeptide, recently prepared by Johnson and Burnham. In the latter compound the sulphur may be readily split off as hydrogen sulphide by boiling with hydrochloric acid.

Since thiohydantoins contain the thioamide type of sulphur grouping, a study of the ability of the organism to oxidize and ex- crete the sulphur of these compounds should throw some light on the possible behavior of similar sulphur groupings, whose presence in the protein molecule has been suggested.

Tt has recently been shown that a compound which closely resembles the thiohydantoins may be obtained from the ergot of rye. Barger and Ewins4 have found that the base ergothioneine isolated from ergot by Tanret? is probably the betaine of a-amino- /3-2-thioglyoxaline-4 (or 5)-propionic acid.

NH-CH I

c=s II

AH--!.cH~.cH~.N(~H~)~ I I

co-o Ergothioneine

The simplest compound which contains this type of sulphur combination is thiourea, NH,-CS-NH,, the behavior of which has repeatedly been the subject of study.” Thiourea is non-toxic, is excreted unchanged in the urine, and does not increase the oxi- dized sulphur content of the urine. After the administration of thiourea to rabbits, Pohl’ reports the elimination of an alkyl

8 Johnson and Burnham: this Journal, ix, pp. 449-63,1911. ’ Barger and Ewins: Journ. of Chem. Sot. (London), xcix, pp. 233641,

1911. b Tanret: Journ. de pharm. et de chim., xxx, pp. 145-53, 1909; Compt.

rend. de Z’Acad. des Sci., cxlix, pp. 222-24, 1909. *Cf. Lange: Inaugural Dissertation, Restock, 1892; Jahresber. u. d.

Fortsch. d. Thierchem., xxii, p. 67, 1892; Sato: Zeitschr. f. physiol. &em., lxiii, pp. 378-96, 1909; Maauda: ibid., lxvii, p. 28, 1910.

7 Pohl: Arch. f. exp. Path. u. Pharm., Ii, pp. 34145, 1904.

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Howard B.~ -Lewis 247

sulphide, probably ethyl sulphide, in the breath. No sulphide could be detected in the urine. The greater part of the thiourea was excreted unchanged and only a few milligrams of alkyl sul- phide were obtained from the expired air.

In the present work, two preliminary experiments in which 2 grams of thiourea were injected subcutaneously into rabbits were made. In confirmation of the work of Pohl, a peculiar garlic-like odor was observed in the breath five hours after the injection, but no alkyl sulphide could be obtained from the urines by the method of Abel.8 The total sulphate-sulphur content of the urine was unchanged, while the “neutral sulphur” was increased in pro- portion to the amount of sulphur injected as thiourea. The urines of the experimental days gave strong reactions with potassium ferrocyanide and acetic acid, as described for thiourea by Sato.9 No toxic effects were apparent.

In the experiments to be recorded with the thiohydantoins the animals used were, with one exception, rabbits, which were main- tained on a uniform diet of carrots and oats. The urine was col- lected from the bladder by gentle pressure at the same hour daily. The substances, when fed, were dissolved in water and intro- duced through a stomach tube. The routine analytical proce- dures included the Kjeldahl-Gunning method for total nitrogen, Folin’s method for total sulphate-sulphur, and Benedict’s methods10 for urea and total sulphur. “Neutral sulphur” was obtained by difference. It is of interest to note that all of the compounds studied give the color reaction with phosphotungstic acid and sodium carbonate described by Folin for uric acid.

%Thiohydantoin.

NH-CO

Ls I I I

NH-CH,

8 Abel: Zeitschr. f. physiol. Chem., xx, pp. 253-78, 1895. Q Sato: Biochem. Zeitschr., xxiii, pp. 45-6, 1910. 10 S. R. Benedict: this Journal, vi, pp. 363-71, 1909 (total sulphur);

ibid., viii, pp. 405-22, 1910 (urea).

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248 Behavior of Hydantoin Derivatives

This compound was prepared from hippuric acid and ammonium thiocyanate as described by Johnson and Nicolet.ll Its purity was established by its melting point (226-7’) and a Kjeldahl nitrogen determination (24.12 per cent N). The compound had a slight red-yellow color and when dissolved in water gave a yellowish solution. With picric acid and an alkali a color resem- bling that given by creatinine in Jaff&s test was obtained.

The substance proved to be toxic for rabbits, doses of 0.5 gram causing death within twelve hours. The most noticeable symp- toms were loss of muscular control and power of co-ordination, dyspnoea and convulsions. The degree of toxicity is shown by the following illustrative protocol and the accompanying table (I) which summarizes all the results obtained.

Rabbit 14. Weight 1.4 kgms. 8.SO a.m. Received 0.5 gram of Z-thio- hydantoin subcutaneously. Resumed eating on return to cage.

9.00 Has stopped eating. 10.00 Apparently normal. 11.00 Apparently normal except for slightly increased respiration. 12.00 Restless. Poor control of hind legs. Tremors. Rapid respira-

tion. 1.00 Lack of coordination. Control of hind legs lost. Dyspnoea. 2.00 An occasional convulsion. 6.66 More quiet. Convulsions less frequent. Dyspnoea. 8.10 Urinates. Urine deep orange red. 3.46 Violent convulsions, dyspnoea. Pupils very greatly dilated. 4.00 Dead. Autopsy. Muscles stained yellow red at point of injection. Fluid all

absorbed. Viscera appear normal. Bladder empty. Lungs and heart congested.

Examination of Urine. Color deep orange red. Jaffe’s picric acid test very brilliant. Albumin test negative. Reduction test with Benedict’s solution gives a black precipitate of copper sulphide.

11 Johnson and Nicolet: Journ. Amer. Chem. Sot., xxxiii, p. 1973, 1911; Johnson: Amer. Chem. Journ., xlix, pp. 68-9, 1913.

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Howard B. Lewis

TABLE I.

Toxicity of d-Thiohydantoin.

249

lNI?.fdL td6ETlmD OF ADM6INIITB*TmN z%3ULrn

______~~ kgms. grams

Rabbit 12.. . . . 1.70 1.17 per 0s Death in 3+ hours. Rabbit 13.... 1.67 0.59 per 0s Death in 4J hours. Rabbit 14. . . 1.40 0.31 subcutaneously Death in 7+ hours. Rabbit 15.....i 1.58 0.125 subcutaneously Death in 24 hours. Rabbit 16. . . / 1.48 1 0.066 subcutaneously Refused food for 2 days.

Restless. Recovered. Rabbit 19.. . . 1 .SO ; 0.277 subcutaneously Preparation 2.* Death

in 7f hours. Rabbit 22.... 1.92 0.260 subcutaneously Preparation 3.* Death

93 hours. Rabbit 27. . . 1.78 0.056 subcutaneously Recovery. Refuses food

*After the earlier experImenta hsd shown the marked toxicity. a new preparation 8 was pm- ed and especially purified.

r Prqmration S was a prepsratIon used In experiments with rab-

its 19, 22. 27 and 29, and cat C, especially purffied by Mr. Ben H. Nicolet to whom I am indebted for thin and other asslatanae.

The toxicity of this comppund must be attributed to the sulphur which replaces the oxygen in the hydantoin nucleus. While approximately 0.125 gram per kilo body weight is the lethal dose for the thiohydantoins, amounts of over 1.5 grams of hydantoin per kilo have been fed to rabbits without any toxic effects.

It was first planned to study the distribution of sulphur in the urine after the administration of the thiohydantoin in order to note any oxidation of the sulphur. But the relatively small size of the dose which is necessary to prevent a fatal outcome of the experiment made such a study unpromising. An attempt was made in the case of rabbits 22,27, 28,29, and cat C to identify the unchanged thiohydantoin in the urine.

Many attempts to isolate the thiohydantoin as such were un- successful. At length a method of procedure was adopted which, while it gave positive evidence of the presence of unchanged thiohydantoin in the urine, did not furnish absolute proof. Thio-

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250 Behavior of Hydantoin Derivatives

hydantoins when boiled with chloroacetic acid are desulphurizedlz with the formation of hydantoins and thioglycollic acid. The latter acid may be detected by a delicate color reaction described by Heffter.ls A very dilute solution of thioglycollic acid gives, on the addition of sodium nitroprusside and an alkali, a purpie- red color, changing quickly to brown-red and soon disappearing. This reaction is also given by the alkyl sulphides, ethyl and benzyl mercaptans, cysteine, cy- and 8-thiolactic acids and thiophenol.

The urines were first tested for this reaction before treatment with chloroacetic acid to rule out the presence of the alkali sul- phides and the other above-mentioned compounds which give this reaction. In no case was any purple color obtained from the dilute urine. The urine was then concentrated on the water bath to small volume, 2-3 grams of chloroacetic acid added, and the mixture boiled with a return condenser from four to six hours. The contents of the flask were cooled, treated with animal charcoal to decolorize, and the color reaction carried out. In all the experi- mental urines the reaction was positive. Normal urines which were treated in the same manner never gave positive reactions. To avoid the color of the urine, which interfered with a delicate test, the liquid after boiling with chloroacetic acid was in some cases evaporated to dryness and extracted with ether, in which thio- glycollic acid is soluble. The ether was then removed by evapora- tion, the residue taken up in water, and the color reaction carried out. The reactions were more brilliant when the test was per- formed in this way.

While the possibility that there may be other compounds formed in the body, which yield thioglycollic acid on boiling with chloro- acetic acid, is not entirely excluded, the demonstration of the pres- ence of thioglycollic acid after the chloroaeetic acid treatment in the experimental urines gives a strong indication of the presence of un- changed thiohydantoin. 2-Thiohydantoin, like hydantoin, is elim- inated probably unchanged by the rabbit. Unlike hydantoin it is very toxic for rabbits, 0.125 gram per kilo being a lethal dose.

1* Johnson, Pfau and Hodge: Journ. Amer. Chem. Sot., xxxiv, pp. 1041-48, 1912.

18 Heffter: Me&in.-naturwiss. Archiv, i, p. 81, 1908.

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Howard B. Lewis 251

d-Thio+nethylhydantoin.

NH-CO I c=s I I

INH-cH.cH~

2-Thio4methylhydantoin was prepared by the action of ammonium thiocyanate on alanine as described by Johnson.” Its purity was shown by its melting point (161’) and a Kjeldahl nitrogen determination (21.53 per cent N).

2-Thio4methylhydantoin was toxic for rabbits although far less so than the 2-thiohydantoin. The lethal dose was 0.6 gram per kilo body weight, or more than three times that of Z-thio- hydantoin. The symptoms were very similar to those produced by the 2-thiohydantoin, although the convulsions were less severe than with the latter. Most marked was the intense albuminuria in all those cases in which the fatal dose was given. A study of the typical protocol given below, together with the tabular sum- mary (table. II), will best serve to illustrate the toxicity.

Rabbit 84. fiVeight 1.7 kilos. Dec. 18, 8.80 a.m. Received 1 gram of 2-thio-4-methylhydantoin subcutaneously.

11.00 Drowsy. Sits with eyes closed. 18.00 Tremors. Runs excitedly around cage when roused. 8.00 Very excitable. Runs around cage. Occasional convulsions. 6.00 Quiet.

December 18. 8.00 a.m. Drowsy. Urine deep yellow. Protein tests positive (Heller’s, picric acid, heat coagulation). No casts.

Animal quiet all day. Ate no food. Dec. 14. 8.00 a.m. Animal dead in cage. Body still warm. Urine,

protein test strongly positive. Autopsy revealed nothing abnormal.

r4 Johnson: this Journal, xi, pp. 97-101, 1912; Amer. Chem. Journ., xlix, pp. 6%9,1913.

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252 Behavior of Hydantoin Derivatives

TABLE II.

Toxicity of .%ThioJ-Methylhydantoin.

*Nm.M.

-

.-

Rabbit 20.. . Rabbit 21. . . Rabbit 20.. . :

WEXGHT

kpns.

2.1 1.9 2.1

Rabbit,31.... 1.6

Rabbit 33.. . . 1.2 Rabbit 34. . . 1.7

Rabbit 35. . . 1.8

Rabbit 36.. 1.7

_- ------y

~--

grams

0.25 0.25 0.50

0.93

0.83 0.58

0.69

0.58

1 per OS No symptoms. isubcutaneously No symptoms. ! per OS Restless. Refuses food

for two days. subcutaneously Death in 31 hours. Ai-

bumin test positive. subcutaneously Death in 1) hours. subcutaneously Death in 2 days. Al-

bumin test positive. ! per OS Death in 5) hours. Al-

bumin test positive. per OS Death in 3 days. Al-

bumin test negative.

No attempt was made to identify the unchanged thiohydantoin in the urine.

The chief interest in the above experiments lies in the lowered toxicity of the sulphur hydantoms due to the substitution of a methyl group for one hydrogen in the 4-position.

%Thiohydantoin-&acetic acid.

NH-CO I c-s I

NH-CH-CHs-COOH

This hydantoin was prepared from ammonium thiocyanate and asparagine as described by Johnson and GuestI A determination of its melting point (222’) and nitrogen content (16.09 per cent N) showed its purity. This compound is rather difficultly soluble in water; hence in the experiments in which it was used sodium car- bonate was added to form the sodium salt which is more soluble.

I5 Johnson and Guest: Amer. C%em. Journ., xlviii, pp- 10%9, 1912; John- son: ibid., xlix, pp. 68-9, 1913.

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Howard B. Lewis 253

After several preliminary experiments had shown that Zthio- hydantoin4acetic acid was non-toxic for rabbits and tbat as large a dose as 2 grams had no obvious effects on the animal, a study of the sulphur elimination and distribution in the urine was made.

Rabbit SO. Daily diet, 250 grams of carrots and 25 grams of oats. This was completely consumed. On the day of the injec- tion the animal showed no symptoms, except a refusal to eat for several hours. On the fourth day, a subcutaneous injection of 2 grams of 2-thiohydantoin4acetic acid was made (N content = 0.321 gram, S content = 0.368 gram). The protocol follows.

Rabbit SO; Weight, 1.69 kgms. ispmwc’ TOT*L g YOLDYE oaav- N d I ’ i LmE.4 TJRlO.4

ITY +NHrN N+N;~~F wy “$jy NEU- /

+NH:--N c S TR.tL s ‘;r / NEU~

’ s I _~____.~, cc. gram *mm I percent gram gram gsam pwm per cent per cent

1 85 1.040 0.675 0.555 82.2 0.120 0.06670.05750.0092 86.2 13.8 2 125 1.020 0.720 0.615 85.4 0.105 0.06420.05360.0106 81.9 18.1 3 125 1.030 0.570 0.465 81.7 0.105 0.06290.04970.0132 79.0 21.0 4 loo 1.040 1.088 0,735 5 125 1.0231 0.705 0.570

67.6 0.353 0.34780.03060.3172 8.8 91.2 80.8 0.135 0.05670.03710.01~~ 65.4 34.6

Rabbit 40. Diet, 25 grams of oats and 250 grams of carrots. This diet was consumed as usual on the day of the administration of the 2-thiohydantoin4acetic acid. On the fourth day 1.75 grams of 2-thiohydantoin4acetic acid were given per OS (S con- tent = 0.322 gm.) No toxic symptoms were noted. The proto- col follows.

Rabbit 40; Weight, 1.88 kgms.

-I- CC.

100 1.015 180 1.014 150 1.020 160 1.023 so 1.036

160 1.015 160 1.019

-7 I

-

-

TOTAL S

gran

0.0718 0.0767 0.1167 0.3824 0.1596 0.0980 0.0946

,.~_.~ gram gram per cent 1 per cent

0.0561 0.0157 78.1 21.9 0.0566 0.0201 74.8 26.2 0.0809 0.0358 69.3 30.7 0.0531 0.2244 20.6 79.6 0.0790 0.0806 49.5 I 50.5 0.0691 0.0289 70.5 / 29.5 0.0728 i 0.0218 77.0 23.0

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254 Behavior of Hydantoin Derivatives

In a third experiment in which 1.75 grams of 2-thiohydantoin- 4-acetic acid were given per OS to a rabbit of 1.5 kilos, similar results were obtained, the “neutral sulphur” rising from a preliminary average of 23.2 per cent to 88.8 per cent on the day of the injection.

In these three experiments on rabbits there was no evidence of an oxidation of the sulphur by the organism, a reaction which should result in an increased total sulphate-sulphur elimination. In no case was the sulphate-sulphur increased on the day of the injection. In each instance, however, the “neutral sulphur” elimination was increased in proportion to the amount of sulphur given as 2-thiohydantoin+Lacetic acid. No attempt was made to recover the unchanged hydantoin from the urine.

It is interesting to note that these results, which show that the sulphur in this type of combination is not oxidized by the organism, are in agreement with the results obtained by SteudeP and Mendel and Myers.” The former working with 2-thio+Lmethyluracil found that it was excreted unchanged by the organism of the dog. The latter studied the distribution of sulphur in, the urine after the administration of 2-thiouracil to rabbits and found no increase in the oxidized sulphur, but a marked increase in the “neutral sulphur” of the urine. These findings all agree in demonstrating the stability of thioamide sulphur in the organism.

Experiments on rabbits in which doses of 0.44 and 0.79 gram per kilo of 2-thiohydantoin+Gacetamide were given per OS to rabbits demonstrated that in such amounts this substance is non-toxic for rabbits.

The lack of toxicity of 2-thiohydantoi&-acetic acid for the rabbit would seem to indicate that the substitution of alkyl groups or their oxidation products for a hydrogen in the 4-position dimin- ishes the toxicity shown by the sulphur in 2-thiohydantoin. The increase in the molecular weight of the substituted alkyl group, in the substances studied at least, gradually decreases the toxicity. Thus the lethal dose of the unsubstituted thiohydantoin was found to be about 0.125 gram, the substitution of a methyl group raised the fatal dose to 0.6 gram, while substitution of an acetic acid or acetamide group caused a loss of toxicity. It is of interest to know whether this theory of decreased toxicity will be confirmed by a

I6 Steudel: .k??eilschr. f. physiol. Chem., xxxix, pp. 13642, 1903. “Mendel and Myers: Amer. Journ. of Physiol., xxvi, pp. 77-105, 1910.

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Howard B. Lewis 255

study of the effect of other radicals, substituted both in the 4-posi- tion as in the compounds studied and in other positions in the ring. An investigation of the effects of such substitution is proposed.

SUMMARY.

2-Thiohydantoin is toxic for rabbits. The substitution of an alkyl group in the 4-position decreases the toxicity. 2-Thio- Pmethylhydantoin is less toxic than 2-thiohydantoin, Pvhile 2- thiohydantoin&acetic acid is not toxic in doses of 2 grams. 2-Thio+methylhydantoin in fatal doses causes an albuminuria in rabbits. The sulphur contained in 2-thiohydantoins is not oxidized in the organism of the rabbit, but is excreted probably unchanged.

I wish to acknowledge my indebtedness to Professor Lafayette IX Mendel under whose direction this work has been carried out and to Professor Treat B. Johnson who has aided in the questions of organic chemistry involved.

ADDENDUM.

Through the courtesy of Professor Treat B. Johnson, a study of one of the thiopolypeptides already referred to was made possible. The compound with which the experiments were carried out was dithiodimethylpiperazine, the preparation and properties of which will be described in a later paper from Professor Johnson’s labora- tory. The new compoundis very insoluble in water and was admin- istered as the sodium salt. It was first suspended in water, sodium hydroxide added in the cold until solution took place, and the whole immediately neutralized. These precautions were made necessary because of the ease with which dithiodimethylpiperazine splits off sulphur in the presence of free alkali. As has already been pointed out, this thioamide type of sulphur combination is very unstable.

The relation of this thiopolypeptide derivative to the thio- hydantoins which have been shown to be toxic may be seen from a comparison of the structural formulae.

NH -CH*CHs NH-CO I I

c=s c=s c=s I I I I I

CH3. CH-NH NH -CH .CHs Dithiodimethylpiperazine 2-Thio+methylhydantoin

TBE JOURriA& OF BIOLOBICAL mEm8TRY, “cm. XIV, i-30. 3.

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256 Behavior of Hydantoin Derivatives

Two -CS.NH.CH.CHa groupings make up the thiopolypeptide derivative. This same grouping occurs once in the thiohydantoin. Hence from a theoretical standpoint, the toxicity of the thiopip- erazine derivative should be greater than that of the thiohydan- toin. This was found to be the case. Two typical protocols follow.

Rabbit 49. Weight 1.70 kgms. 10.07 a.m. Received 0.3 gram (= 0.176 gram per kilogram body weight) dithiodimethylpiperazine, prepared as described above, subcutaneously.

10.96 Tremors. Very restless. Dyspnoea. 10.90 Slight convulsions. 10.36 Dyspnoea very marked. Animal gasps, gnashes teeth. 10.40 Tremors more marked. Pupils widely dilated. 10.46 Animal has a very violent convulsion in the course of which it

forces open the cover of the cage. Immediately following tetanic symptoms, not clonic, appear.

10.66 Convulsions very frequent. As animal was apparently dying ether was given to neutralize the effects of convulsions. Animal became quiet immediately and on being allowed to recover from the anesthesia showed convulsions again.

19.06 The animal had been etherized for more than an hour, but the increasing violence and frequency of the convulsions made deeper anesthe- sia constantly necessary. The animal was now allowed to recover from anesthesia. Immediately, extreme dyspnoea, labored breathing and con- vulsions began.

1636 Very weak. 1.16 Dead.

Autopsy. Nothing abnormal. Urine: trace of albumin. Rabbit 46. Weight 1.8 kgms. 9.00 a.m. Received 0.3 gram dithiodi-

methylpiperazine prepared as described above per OS. 9.07 Tremors. 9.10 Convulsions. 9.16 Lies on side in convulsions. 9.99 Extreme dyspnoea. 9.SS Very weak.

10.00 Death. Autopsy. Nothing abnormal except congested heart and lungs.

The close parallelism between the symptoms above described and those produced by alkali sulphides or hydrogen sulphide immediately suggests that an explanation of the toxicity may be found in the liberation of sulphides in the organism from the un- stable sulphur linkage. Further investigations on this substance are in progress.

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Howard B. Lewis2-THIOHYDANTOINS

METABOLISM. II:HYDANTOIN DERIVATIVES IN

THE BEHAVIOR OF SOME

1913, 14:245-256.J. Biol. Chem. 

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