POLYMERIZATION OF &HYDROXYINDOLES AND ITS RELATION TO THE FORMATION OF MELANIN

BY AVERY A. MORTON AND WILSON R. SLAUNWHITE, JR.

(From the Department of Chemistry, Massachusetts Institute of Technology, Cambridge)

(Received for publication, January 11, 1949)

Melanins are probably the most widely distributed pigments in the ani- mal kingdom. In the laboratory one can be formed by oxidative processes from 3,4-dihydroxyphenylalanine. On excellent grounds (1) an inter- mediate product (2) is judged to be an o-quinone indoline, I, or one of its tautomers, for example II or II’. Recent spectroscopic measurements (3) clearly favor I.

0

\ \ 3=

[$HCO~H OJ~>HO~~H ~~@JJco~H

///N

0 H

(1) (II) (II?

Subsequent oxidation of this intermediate yields a melanin. The course of this change is not understood, although it seems clear (l-3) that tauto- merit changes occur to I so that structures of the type of II or II’ form.

Some light might be thrown on the subject if the behavior of monohy- droxyindoles was known. 5-Hydroxyindole (4) is stable unless it happens to be formed in acid solution (5), where it can suffer the same type of vinyl polymerization which indole and skatole (6) undergo. 6-Hydroxy- indole has not yet been prepared. Good reasons exist for supposing that such a compound would have unusual polymerizing activity. For instance, the 3 position of indole is normally very active as seen in the Mannich re- action, certain condensation reactions, and the behavior with Grignard reagents. Mild oxidation, such as is used for changing hydroquinones to quinones, should convert a 6-hydroxyindole to a quinomethane, III. Qui- nomethanes contain an unsymmetrically substituted vinyl system and as a class are highly susceptible to polymerization. The simplest quino- methane, IV, has never been prepared, but if two phenyl groups are pres- ent, as in the well known fuchsone, V, there is sufficient stability to permit isolation. The as-diphenylquinodimethane, (CsH&C=CsH4=CH2, can- not be isolated (7) because of its great tendency to polymerize. While higher molecular weight analogues, such as dinaphthylquinodimethane,

259

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260 MELANIN FORMATION

(CNHT)&=CBH~=CHZ, can be isolated, they polymerize readily. In gen- eral a vinyl group unsymmetrically substituted and with 1 carbon part of the ring seems highly disposed toward polymerization.

These facts led us to study compounds (a) in which an o-quinone might not, or could not, form and (6) in which a single phenyl group is at the 3 position in order to facilitate the isolation of what might otherwise prove to be an exceedingly unstable monomer. Such a phenyl group is often used’ in organic problems because of its stabilizing nature and, in the present instance, was expected to impart desirable characteristics. The two com- pounds selected were 2-carboxy-6-hydroxy-3-phenylindole (VI) and its 5-methyl homologue (VII), which, apart from the features noted, are very similar to the indole, II, from 3,4-dihydroxyphenylalanine.

Ho)$)TOzH ;f$OzH

H H

(VI) (VII)

Compound VI or its methyl ester could be isolated only in impure form although the 6-benzyloxy-3-phenylindole-2-carboxylate that immediately preceded VI was quite stable. The product obtained was always low in carbon, probably because of oxygen uptake at position 5. Causse’s (8) test with antimony trifluoride for o-diphenols was positive. The 5 posi- tion would be active because of the combined influence of the 6-hydroxy and the amino group. When the 5 position was blocked, as was done later in the preparation of VII, no oxygen was taken up. Hence the desired product, VI, was probably contaminated with the 5,6-dihydroxy com- pound (VIII).

Isolation of pure VII proved feasible. Its oxidation with potassium chlorate-vanadium pentoxide or with ferric chloride, both mild reagents

1 For example, phenyl groups stabilize carbonium ions so that the isolation of the stable carbonium triarylcarbonium perchlorate is possible. It also slows down the tautomeric process of nitromethane so that the change can be observed. It is responsible for the unreactivity of chlorine in chiorobenaene.

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A. A. MORTON AND W. R. SLAUNWRITE, JR. 261

CeHs

COzH

H

(VIII)

suitable for converting hydroquinones to quinones, yielded dark amorphous polymers, insoluble in common organic solvents and in dilute alkali. It is therefore clear that the 5 position is unnecessary for conversion to a melanin-like product. Polymerization takes place with great ease, even when the course of oxidation is restricted to the quinomethane path and a phenyl group is present to impart stability.

( C-C-),

;c ‘I I HC

( /c\N//cco2H C-CH

/ 0

(IX)

While a vinyl function appears to be present in the hypothetical pri- mary oxidation product from VII and can cause polymerization, the actual course is obscured by one or more secondary reactions. A vinyl type of polymerization might give a structure, IX, for which the empirical com- position would be 2 hydrogen atoms less than for the monomer. But the products from polymerization showed higher percentages of carbon with loss in acidity. Carbon dioxide could not have been lost, because that would require a very large increase in percentage of carbon. Furthermore the ratio of carbon to nitrogen remained approximately constant at 16:1, rather than dropping to 15: 1. The general change in composition indi- cates a condensation and the simplest one would be loss of water, the OH coming from the carboxyl group and the H from the NH of another un- oxidized molecule. The over-all oxidation-condensation process would then be expressed by the equation

~C~~HIIOJN-~H-H~~EC~~H,~O~N~

The composition of the polymers produced in acetic acid was not far from this value. See Table I.

The argument that the carboxyl group participates in an ordinary con-

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262 MELANIN FORMATION

densation as a secondary reaction during the polymerization seems to be sustained from the results in alcohol in which esterification could occur. In this case the ratio of atoms of carbon to nitrogen would approach 18 the more successfully esterification competes with the alternative conden- sations. The data in Table I are consistent with this view.

TABLE I Comparison of Atoms in Polymers from 6-Hydroxy-5-methyl-S-phenylindole-d-

carboxylic Acid -

I Ratio of atoms in polymer

C

Monomer.................................. 16 Polymer (theory).......................... 16

“ (found in HOAc with KClOa*V20J. 16.05 WithFeCl~................................ 16.4 Polymer less H20; theory for oxidation-

condensation............................ 16 Polymer (found in EtOH with KC103.Vz06). 17.20 WithFeC13................................ 17.3 Polymer; theory for oxidation-esterification 18

N II

1 13 1 11 1 11.3 1 12.9

1 9 1 12.55 1 14.7 1 15

-

--

-

0

3 3 2.46 2.1

2.5 2.69 2.74 3

These results do not of course prove that the polymerization of a com- pound, such as II’ or VIII, which has the hydroxyl group at Cs, must take place through the quinomethane path, but it is conceivable that polymer- ization does occur that way and that the hydroxyl group at Cs activates, but does not directly participate in, the process. Attention should be drawn to the fact that, if oxidation had taken place to give a quinoimine X, as the initial step, the product would still be equivalent, in degree 0;

COPH +--+ H CO,H

(a (XI)

oxidation, to a quinomethane, XI, since the two structures can be pic- tured as contributing to resonance in a hybrid, as indicated by the formulas. The second form could then be the one that would yield the polymer.

The methods of syntheses of the two compounds are outlined in Dia- grams 1 and 2. In general, each preparation involved the application of a Japp-Klingemann condensation to an appropriate intermediate in which the phenolic hydroxyl was protected by a benzyl group. The latter was then removed by aluminum chloride.

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A. A. MORTON AND W. R. SLAUNWHITE, JR. 263

An attempt was made to prepare l-methyl-3-phenyl-6-hydroxyindole in which the tendency for the 5 position to oxidize might be lessened. The method chosen necessitated the formation of the unsymmetrical hydra- zine, WL-C~H&H~OC~H~N(CH~)NH~, which was to be used for the prepara- tion of a hydrazone and a Fischer indole synthesis. The scheme pro- ceeded satisfactorily through the preparation of the nitroso compound, m-CeH&HzOCsH~N(CH3)NO, but met failure in the reduction to the hydrazine.

Diagram 1. Method of Preparation of Compound VI

(XII to xv> (XVI and VI) XII,R=H,R’=H XV, R = CaH&H2, R’ = H

XI& ‘I = “ “ = COCH3 XVI, “ = “ “ = C2Hs

XIV, “ = C&H&H,, R’ = COCH, VI, “ = H, R’ = H

Diagram 9. Method for Preparation of Compound VII

::(-)NO, - :@R; - :@$:’

H Cmul (XVIII to XX) (XXI and VII)

XVIII, R = H, R’ = 0 XXI, R = C&H&Hz, R’ = CzHs XIX, “ = C&H&H,, R’ = 0 VII, “ = H, R’ = H xx “ = “ I<

, =H

An attempt was made also to prepare VI by way of the O-methyl in- stead of the 0-benzyl ether. The experiments were successful until the last stage of cleavage, in which extensive polymerization took place to give a black solid.

The authors are greatly indebted to Professor Charles H. Blake, of the Biology Department, for calling our attention to this problem of melanins and for many profitable discussions on their occurrence and properties. One of us, W. R. S., is also indebted to the American Cancer Society for a predoctoral fellowship during this study.

EXPERIMENTAL

S-Benzyloxyacetanilide, XIV--r+Hydroxyacetanilide (9) (220 gm., 1.46 moles) was dissolved in 600 ml, of dry methanol containing 81.5 gm. (1.46

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264 MELANIN FORMATION

moles) of potassium hydroxide and methanol and water were removed under reduced pressure. Benzyl chloride (204 gm. or 1.6 moles), pre- viously dried over calcium chloride, and 315 ml. of anhydrous ethanol were then added to the solid potassium phenoxide and the mixture was re- fluxed on a steam bath for 2 hours. The mixture, when cooled and filtered, yielded a brown solid, and the mother liquor yielded more after being diluted with 100 ml. of water. The combined precipitates were washed with 500 ml. of cold 1 N sodium hydroxide and then with ice water. The yield was 259 gm. or 74 per cent. A sample was alternately crystallized from benzene, ethyl acetate, and alcohol until a white product, which melted at 122.5-123.5” (corrected), was obtained. This material was very soluble in chloroform and acetone, was soluble in nitromethane, etha- nol, and ethyl acetate, and was insoluble in petroleum ether, ether, cy- clohexane, benzene, and carbon tetrachloride.

AnaZysis-C&HI~O~N. Calculated. C 74.68, H 6.27, N 5.81 Found. “ 74.35, “ 6.42, “ 6.01

The above reaction was even better when the m-hydroxyacetanilide was refluxed with benzyl chloride in acetone that contained potassium car- bonate. The yield was 85 per cent.

m-Benxyloxyaniline, XV-This compound was prepared by refluxing 145 gm. of the anilide, XIV, with 336 gm. (6 moles) of potassium hy- droxide in 2 liters of 90 per cent alcohol for 6 hours. The alcohol was then removed by distillation and the residue was extracted with ether. The extract was washed, dried over Drierite, and distilled (171’, 2 mm.). When redistilled, this oil solidified on standing. The pure product melted at 61-62.5’. It was very soluble in ether, ethanol, acetone, chloroform, carbon tetrachloride, and 1 N hydrochloric acid, and was insoluble in benzene, cyclohexane, and petroleum ether.

Analysis-CIsHltON. Calculated. C 78.36, H 6.58, N 7.03 Found. “ 78.24, “ 6.53, “ 6.84

The p-toluenesulfonyl derivative melted at 112-112.5” (corrected) and was soluble in acetone and chloroform, insoluble in ether, ethyl acetate, alcohol, benzene, cyclohexane, and alkali.

Analysis-CZ~HI~O~NS. Calculated. C 67.95, H 5.43, N 3.96 Found. “ 67.95, “ 5.33, “ 4.08

Ethyl S-Phenyl-6-benxyloxyindole-W-carboxylate, XVI--m-Benzyloxyani- line, 24.9 gm. or 0.125 mole, was diazotized in a solution of 8.7 gm. (0.126 mole) of sodium nitrite in 20 ml. of water, 150 gm. of ice, and 35 ml. of concentrated hydrochloric acid. This solution was then added at once to a solution of 27.5 gm. (0.125 mole) of ethyl ar-benzylacetoacetate (10)

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A. A. MORTON AND W. R. SLAUNWHITE, JR. 265

in 190 ml. of alcohol, 40 ml. of water, and 15 gm. (0.375 mole) of sodium hydroxide. After 5 minutes, 300 ml. of water were added. The black oily precipitate was washed by decantation and then dried. This oil was dissolved in 50 ml. of dry alcohol and the solution saturated with dry hy- drogen chloride. Refluxing half an hour caused the separation of ammo- nium chloride. Addition of water precipitated a dark resinous mass after a few hours. Extraction of this crude material with petroleum ether gave a light colored solid which could be crystallized from alcohol, ethyl acetate, carbon tetrachloride, or nitromethane. Eventually a 17 per cent yield of tiny white square plates that melted at 165-165.5” (corrected) was ob- tained .

Analysis-CZ~HLXO~N. Calculated. C 77.60, H 5.70, N 3.76 Found. “ 77.37, “ 5.86, “ 3.59

3-Phenyl-6-hydroxyindole-%carboxylic acid, VI-In a special creased flask (11) with inverted bottom, equipped with a mercury-sealed stirrer and reflux condenser, were placed 3.80 gm. (0.01 mole) of the ester, XVI, 4.1 gm. (0.03 mole) of anhydrous aluminum chloride, and 200 ml. of dry ben- zene. The mixture was refluxed with stirring for 1 hour. The contents were then poured on ice and acidified with hydrochloric acid. The ice- cold solution was immediately extracted with ether and the ether in turn with sodium hydroxide. Ice was added to the alkaline layer and the red alkaline solution titrated with concentrated hydrochloric acid until a copi- ous white precipitate appeared. The precipitate was filtered, washed until neutral to litmus, and dried at reduced pressure. A light brown solid that melted at 170-175” was obtained. It was soluble in sodium carbonate but the neutralization equivalent was high, being 264 instead of the calcu- lated 253. Furthermore, the amount of solid obtained was 2.62 gm., or 20 mg. more than the theoretical amount.

The above results indicated that some of the product had taken up oxygen. The 5 position was suspected and a test with antimony trifluo- ride for adjacent phenolic groups was made. The antimony trifluoride was dissolved in a saturated sodium chloride solution and the solution was neutralized with sodium carbonate solution until the precipitate which formed no longer dissolved. The solution was then filtered. The crude acid, VI, was dissolved in a volume of methanol equal to that of the in- organic solution. The two solutions were then warmed and mixed. A precipitate immediately formed, indicative of the diphenolic compound. The precipitate was filtered, washed with water to remove antimony and sodium salts, and then with methanol to remove organic acid. A small amount of black solid remained when the residue was dried.

That all of the compound had not been oxidized to a diphenolic product

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266 MELANlN FORMATION

was indicated by the results of an attempt to esterify the crude acid, VI, (2.52 gm.) with dry hydrochloric acid in 200 ml. of alcohol. The tempera- ture was maintained below 60” for 50 minutes. When the phenolic prod- uct was recovered by extraction and sublimation, the analysis suggested loss of nearly all of the carboxyl group. Accordingly the trace of ester was removed by saponification with 10 per cent sodium hydroxide, under nitro- gen atmosphere, a few mg. of sodium bisulfite also being added in order to prevent oxidation. The neutral material, 6-hydroxy-3-phenylindole, re- covered from these steps consisted of 200 mg. of a white solid that melted at 175-177” (corrected).

Analysis-CMHIION. Calculated. C 80.33, H 5.30, N 6.69 Found. “ 79.97, “ 5.36, “ 6.63

Two further attempts were made to obtain a pure ester from the crude acid, VI; one was with the silver salt-methyl iodide method and the other was with diazomethane. In general, neither of these processes was success- ful. The product was usually a brown solid. Sublimation at 2 P and 150” yielded lighter colored material, some even white, but none that cor- responded exactly with the value for the pure methyl ester. Each frac- tion was low in carbon and high in oxygen, indicative of contamination with the oxidized product.

4-N&o-W-hydroxytoluene, XVIII-The synthesis of VII began with tech- nical nitrotoluidine, XVII, and followed the method of Ullmann and Fitz- enkam (12). The yellow crude product melted at 113-116’, and was used in the next step. The recorded value is 118’.

4-N&o-Menzyloxytoluene, XIX-The benzyl ether was made by re- fluxing overnight 8.5 gm. (0.056 mole) of XVIII with 5.8 gm. (0.042 mole) of anhydrous potassium carbonate and 7.6 gm. (0.06 mole) of dry benzyl chloride in 150 ml. of acetone. The mixture was stirred gently to prevent bumping. The product was washed with 1 per cent sodium hydroxide and then with water. The yield of crude ether was 74 per cent. Re- crystallization successively from alcohol, petroleum ether, and methanol (decolorizing carbon used in the last) gave white microscopic needles which melted at 75.5-76” (corrected). The product was very soluble in ethyl acetate, benzene, and acetone, soluble in alcohol and methanol, and in- soluble in water.

AnaEysis-CMHI~OSN. Calculated. C 69.12, H 5.39, N 5.76 Found. “ 68.84, “ 5.46, “ 5.75

4-Amino-HwnxyZoxytoZuene, XX-On a steam bath 6.7 gm. (0.03 mole) of stannous chloride dihydrate, 8 ml. (0.1 mole) of concentrated hydro- chloric acid, and 20 ml. of alcohol were heated to boiling in a beaker. Ad- dition of 2.43 gm. (0.01 mole) of crude XIX caused a rapid exothermic re-

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A. A. MORTON AND W. R. SLAUNWHITE, JR. 267

action. In 10 minutes a copious white precipitate appeared. After 5 more minutes the beaker was placed on ice, and the pasty solid recovered by filtration. Addition of 10 per cent sodium hydroxide produced an oil which was extracted with ether. Addition of a small amount of concen- trated hydrochloric acid then yielded 1.7 gm. (70 per cent) of a yellow solid. Sublimation at 1 mm. in a large test-tube gave first a yellow solid and then a pure white hygroscopic solid. The latter melted at 216.5-217” with de- composition. This hydrochloride of XX was soluble in hot alcohol and insoluble in alcohol, ether, benzene, acetone, and water.

Analysis-CI~HI~ONCI. Calculated. C 67.33, H 6.46, N 5.61 Found. “ 67.21, “ 6.38, “ 5.84

Addition of alkali to the hydrochloride yielded an oil, XX, which dis- tilled at 157” and 1 mm. and had a refractive index, nE5 = 1.6022.

Analysis-CI~HISON. Calculated. C 78.84, H 7.09, N 6.57 Found. “ 78.70, “ 7.07, “ 6.71

Ethyl S-Phenyl-5-methyl-6-benxyloxyindole-S-car~oxylate, XXI-This com- pound was made by the procedure used for XVI from 3 gm. (0.012 mole) of the crude hydrochloride of XX, 0.9 gm. of sodium nitrite, 24 ml. (0.024 mole) of 1 N hydrochloric acid, 1.45 gm. (0.036 mole) of sodium hydroxide in 5 ml. of water, and 2.65 gm. (0.012 mole) of ethyl benzylacetoacetate in 20 ml. of alcohol. The crude brown product amounted to 3.2 gm., 69 per cent yield. A cold methanol mash left a light brown solid which was crystallized from cyclohexane and then decolorized with carbon and re- crystallized from the same solvent. The final yield was 1.8 gm. (39 per cent) of white microscopic needles that melted at 173.5-174.5” (corrected). It was very soluble in ethyl acetate, benzene, ether, and acetone, soluble in hot methanol, ethanol, or cyclohexane, and insoluble in petroleum ether.

AnaZ;ysis-C&,HSSOZN. Calculated. C 77.91, H 6.00, N 3.64 Found. “ 77.97, “ 6.13, “ 3.74

S-Phenyl-6-methyl-6-hydroxyindole-d-carboxylic Acid, VII-This com- pound was made by the method used for VI from 1.0 gm. (0.0026 mole) of the ester, XXI, 1.05 gm. (0.0078 mole) of aluminum chloride, and 100 ml. of dry benzene. The yield was 585 mg. (84 per cent) of nearly white solid that melted at about 195”, and was very soluble in methanol, ethanol, ether, and acetone and insoluble in benzene and chloroform. Repeated crystallization from hot benzene and the use of decolorizing carbon would not remove all the color. The light tan needles melted at 205-205.5” (corrected) with decomposition. Analysis showed a small trace of ash still present.

Analysis-CI~H~~O~N. Calculated. C 71.90, H 4.90, N 5.24 Found. “ 71.34, “ 4.92, “ 5.19, ash 0.61

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268 MELANIN FORMATION

Oxidative Polymerization of VII-O.5 gm. (0.00188 mole) of VII was dissolved in 40 ml. of glacial acetic acid. A small trace of insoluble residue was removed by filtration. A solution (5 ml.) of vanadium pentoxide (1 to 2 mg.) in 1 N hydrochloric acid and 2 ml. (0.001 mole) of 0.5 N potas- sium chlorate was added. The solution immediately became jet-black. The next day the reaction mixture was filtered and the precipitate washed thoroughly with water. When dried, the purple-black solid, Fraction a, weighed 0.1 gm.

Analysis-Found, C 74.87, H 4.42, N 5.44, ash, trace

The filtrate was allowed to stand 5 more days and more polymer was collected. The dark brown solid, Fraction b, was washed and dried as before. This fraction had a neutralization equivalent of the order of 10,000. When allowed to stand overnight in 10 per cent sodium hydroxide, only a little of Fraction b dissolved. None of Fraction a dissolved. Sub- sequently, after being filtered and washed, part of Fraction b dissolved to give a dark green solution. The residue was red. Carbon dioxide re- precipitated the polymer. Fraction b, C 73.11, H 4.95.

Ferric chloride (4.0 ml. of 1 N solution) was used as the oxidizing agent in place of the chlorate. The black solid isolated by the end of 1 day was 0.137 gm. C 76.39, H 5.04, N 5.43, ash, trace.

In alcohol solution the quantities of reagents were the same as in acetic acid. Potassium chlorate yielded 0.054 gm. of a red solid after standing overnight. C 74.77, H 4.59, N 5.06, ash, trace. Ferric chloride, by the end of 1 day had produced 0.073 gm. of a black solid. C 74.09, H 5.28, N 4.98.

When oxidizing agents were not used, no precipitation occurred. N-Methyl-m-benxyloxyaniline-Sodium sand (4.6 gm. or 0.2 mole) was

made by vigorous agitation in hot xylene in the high speed stirring ap- paratus (11) under an a.tmosphere of nitrogen. The xylene was cooled to 60” and pure dry m-benzyloxyacetanilide, XIV, (48.2 gm. or 0.2 mole) was added with rapid stirring to insure thorough mixing. The temperature was raised to 100” and held at 100-110” for 1 hour. A rapid evolution of hydrogen occurred and the solution gradually became homogeneous. The heater was then removed and 18.9 gm. (0.15 mole) of dry methyl sulfate were added in small portions. When this highly exothermic re- action was completed, the solution was stirred an additional 15 minutes and then cooled and filtered. Xylene was removed from the filtrate by distillation at reduced pressure to give an oil. Meanwhile the jelly-like product on the filter was dissolved in water and more of the oil recovered. To the combined oil fraction 55 gm. of potassium hydroxide in 500 ml. of 90 per cent alcohol were added. The mixture was refluxed for 48 hours.

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A. A. MORTON AND W. R. SLAUNWFiITE, JR. 269

The alcohol was removed by distillation and the residue extracted with ether. The ether extract was washed, dried with sodium sulfate, and distilled. At 156-165” and 1 mm., 40 gm. or 94 per cent of a straw-colored liquid were obtained. This product was dissolved in acetone, cooled to -72”, and filtered through a bed of solid carbon dioxide. The filtrate was then distilled twice and the N-methyl-m-benzyloxyaniline collected at 144-145’, 1 mm., as a pale yellow liquid; n”,” = 1.5960.

Analysis-ClaHlsON. Calculated. C 78.84, H 7.09, N 6.57 Found. “ 78.90, “ 7.19, “ 6.82

The acetyl derivative melted at 70-72”. It was soluble in all common solvents except cyclohexane and petroleum ether.

Analysis--CI~HITO~N. Calculated. C 75.27, H 6.71, N 5.49 Found. “ 75.26, “ 6.80, “ 5.59

The picrate was obtained when a boiling dilute hydrochloric acid solu- tion was mixed with a boiling saturated aqueous solution of picric acid. The product was crystallized from alcohol and then from ethyl acetate to give large yellow-brown, four-sided prisms that melted at 138-138.5’ (corrected).

AnaZysis-C~oH~~0~N~. Calculated. C 54.30, H 4.10, N 12.67 Found. “ 54.40, ‘I 4.19, “ 12.80

N-Nitroso-N-methyl-m-benzylmyaniline-The above compound (1.0 gm. or 0.0047 mole) was partially dissolved in 82 ml. of 0.1 N hydrochloric acid. The mixture was cooled to 15“ and an aqueous solution of 0.4 gm. (0.006 mole) of sodium nitrite was added rapidly with stirring. After 10 minutes the orange-colored solution was extracted with ether. From the extract 0.9 gm. (88 per cent) of a brown-red solid was recovered. Crystal- lization from 1: 1 ether-petroleum ether and then from methanol yielded orange-colored crystals that melted at 62-62.5”. It was soluble in chloro- form, ether, benzene, ethyl acetate, and hot cyclohexane, slightly soluble in methanol and alcohol, and insoluble in cyclohexane and petroleum ether.

Analysis-CUHMO~N~. Calculated. C 69.40, H 5.82, N 11.57 Found. I‘ 69.70, “ 6.01, “ 11.58

Reduction of this compound with zinc dust and acetic acid failed to give the hydrazine.

Ethyl S-Phenyl-6-methoxyindole-b-car~oxylate~-~isidine, 6.1 gm. or 0.05 mole, was subjected to a Japp-Klingemann condensation with 11 gm. (0.05 mole) of ethyl benzylacetoacetate, sodium nitrite, hydrochloric acid, and sodium hydroxide in the same manner and the same proportions as were used in the preparation of XVI. The resinous product was

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270 MELANIN FORMATION

crystallized alternately from alcohol and ethyl acetate until white needles, m.p. 176-176.5”, were obtained.

Analysis-CrsHr~0aN. Calculated. C 73.22, H 5.79, N 4.74 Found. ” 73.24, “ 6.08, ‘I 4.64

When the ether was refluxed with aluminum chloride in chlorobenzene, a black solid was obtained.

SUMMARY

6-Hydroxy-3-phenylindole-2-carboxylic acid, similar in structure to a possible intermediate in polymerization of a hydroxyphenylalanine to mela- nin except for the stabilizing phenyl group, has been prepared and found to undergo easy oxidation at the 5 position to give probably the 5,6- dihydroxy compound.

The 5 position has been blocked by a methyl group and the correspond- ing 5-methyl-6-hydroxy-3-phenylindole-2-carboxylic acid can be isolated. It undergoes polymerization in the presence of mild oxidizing agents to give a melanin-like product.

The results are interpreted as showing that the 5 position is not neces- sarily involved in the polymerization of a 5,6-dihydroxyindole to melanin, since a quinomethane structure that involves the 6 position would be sufficient to give the polymer.

BIBLIOGRAPHY

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2. Raper, H. S., Biochem. J., 21, 189 (1927); J. Chem. Sot., 125 (1938). Verr, W. L. C., Rec. trav. chim. Pays-Bas, 68,949 (1939). Harley-Mason, J., J. Chem. Sot., 1244 (1948) .

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Jr.Avery A. Morton and Wilson R. Slaunwhite,

MELANINRELATION TO THE FORMATION OF

6-HYDROXYINDOLES AND ITS POLYMERIZATION OF

1949, 179:259-270.J. Biol. Chem. 

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