[CONTRIBUTION FROM TEE DEPARTMENT OF CHEMISTRY, BROOKLYN COLLEQE]

THE MUREXIDE QUESTION DAVID DAVIDSON AND ELIAS EPSTEIN

Received August 17, 1956

INTRODUCTION

Thie current structural formula for murexide (I) represents it as the ammonium salt of an as yet unisolated acid, purpuric acid (11). It was proposed practically simultaneously by Piloty,' Mohlau,2 and Slimmer and Stiegli t~.~

NB-CO CO-NH NH-CO CO-NH

CO (2-N-CH CO CO C-N-C CO I I I I I 1 I I I 1 II 1 I 1 I I

NH-CO CO-NH NH-CO [NH4]OC-NH I I1

This structure serves to explain the formation of murexide by (a) the oxida- tion of uramil (III),4s8 (a) the direct condensation of uramil with alloxan (IV),2J,4 (e) the action of ammonium salts on alloxantine or a mixture of alloxan and ammonium dialurate,lJ,6 and (d ) the action of alloxan on 7-alkyl~ramils.'~~ I t also accounts for the production of other purpurates by ( e ) metathesis of murexide with salts,1s3 (f) the condensation of alloxan with alkali salts of ~ r a m i l , ~ (9) the oxidation of alkali salts of uramil,'%* and (h) the action of amines or aminoacids on alloxan or alloxantine,1~2*6*7 although auxiliary hypotheses are necessary to account for (d) and ( h ) .

The most conspicuous chemical property of murexide is its hydrolysis by acids, in which uramil (111) and alloxan (IV) are obtained, accompanied by more or less alloxantine (V).l , 2 , 3 , 4 , 8

1 PILOTY, A m . , 333, 22 (1904). 2 MYOHLAU, Ber., 37, 2686 (1904); MOHLAU AND LITTER, J . prakt. Chem., 121, 7 3 ,

449 (1906). SILIMMER AND STIEQLITZ, Am. Chem. J . , 31, 661 (1904).

4 WOHLER AND LIEBIQ, Ann., 26, 319 (1838). 5 H ~ ~ ~ ~ ~ ~ , J . Chem. SOC., 87, 1791 (1905).

S'PRECKER, Ann., 123, 363 (1862). TRAUBE, Ber., 44, 3145 (1911).

8 BEILSTEIN, Ann., 107, 176 (1858). 305

306 DAVID

NH-CO NH-CO

CO CHNHz CO CO

NH-CO NH-CO

I I I 1 I 1 I I

I11 IV

DAVIDSON AND ELIAS EPSTEIN

NH-COOH CO-NH NH-CO CO-NH

co c- co co C=N--c co I I / I 1 I / I / II 1

ONH, \

V T’I

This occurrence of alloxantine has not been accounted for satisfactorily. Piloty watt even inclined to consider alloxantine a primary hydrolytic product although uramil and alloxan are more consistent with the azo- methine structure of purpuric acid (11). He favored an alternative for- mula (VI) for murexide, and ascribed the production of alloxantine and ammonia on the one hand and of uramil and alloxan on the other to con- current reactions, although the latter formula makes this no more plausi- ble than the former. The unconvincing character of this explanation is reflected in the following statement of Johnson and Hahn:9

“It might be assumed further that the resolution of this complex [purpuric acid] on hydrolysis would serve to define this relation but unfortunately the experi- mental evidence is confusing.” [p. 2791

It thus appears desirable either (1) to suggest a new formula for murex- ide, which accounts for its close relationship to alloxantine, or (2) to offer a plausible explanation for the formation of alloxantine from the present structure.

ALLOXANTINE

Among the proponents of the azomethine structure for murexide, Piloty, alone, emphasized the close relationship between alloxantine and murexide. Thus, aa mentioned above, he considered alloxantine a pri- mary hydrolytic product of murexide. He also implied that the formation of murexide from alloxantine and ammonium salts did not proceed through the intermediary uramil, as was suggested by other writer^.*^^^^ It is well, therefore, to consider the structure of alloxantine before proceeding further.

In aqueous solution it appears to be largely dissociated10 into alloxan (IV) and dialuric acid (VII). Attempts to form derivatives of it have led to derivatives of its dissociation products.ll Nevertheless, oBe may seriously consider the

Alloxantine is characterized by its instability.

9 JOHNBON AND HAHN, Chem. Rev., 13, 193 (1933). 10 BIILMANN AND BENTZON, Ber., 61, 522 (1918). 11 BILTZ AND PAETZOLD, Ann., 453, 64 (1923).

THE MUREXIDE QUESTION 307

molecular structure of alloxantine, since it yields unique insoluble salts among which may be mentioned the beautiful purple barium salt which is used to detect alloxantine. This unusual salt led Hantzsch and Retinger" to revive the old pinacol formula (V) in preference to the hemiacetal for- mula of Piloty' and of Slimmer and St iegl i t~.~ Hantzsch and Retinger compared the barium salt of alloxantine with the metal ketyls." The dissociation of alloxantine into alloxan and dialuric acid may then be con- sidered a disproportionation of the ketyl radicals." Another fact in favor of this view is the formation of analogs of alloxantine from alloxan and acyl derivatives of dialuric acid.ls In these acylated dialuric acids the hydroxyl group necessary for the formation of a hemiacetal is presumably converted to an ester group. The formation of alloxantine from dialuric acid (VII) and alloxan (IV) may, therefore, be represented as follows:

CO-NH NH-CO OH CO-NH

co I I I I 1 r-- --. 1

' I I I Rh.-- ..____..- -e-'

_----___ NH-CO

co co NH-GO CO-NH

HOCH co -+ co c NH-CO

IV VI1 V

MUREXIDE AN ANALOG OF ALLOXANTINE (?)

The foregoing considerations led to the suggestion of the following formula (VIII) for the structure of murexide.

NH-CO CO-NH NH-CO OH CO-NH

co co NH-CO H2N CO-NH

co c I I

NH-CO H2N CO-NH

I I I I I co b/ I 1,"z I I

VI11 IX

All the methods of preparing murexide are readily explained by it. Pur- puric acid is then represented as an intermediate between alloxantine and murexide (IX). Formula IX differs from formula I1 by a mole of water. It is significant that the metallic purpurates described by Piloty' and by Slimmer and Stieglita2 contain a mole of water (based on 11). The forma- tion (of murexide from alloxantine would thus be represented by the re- placement of two acidic hydroxyls by two amino groups (amide formation). The production of alloxantine on hydrolysis would be represented by the

l2 E[ANTZSCH, Ber., 54,1271 (1921); RETINQER, J . Am. Chem. SOC., 39, 1059 (1917). SCHLENK AND THAL, Ber., 46, 2840 (1913).

I* MICHAELIS, Chem. Rev., 16, 243 (1935). I5 E ~ E H R E N D AND FRIEDERICHS, Ann., 344, 1 (1906).

308 DAVID DAVIDSON AND ELIAS EPSTEIN

reverse reaction, the replacement of amide groups by acidic hydroxyls. The production of uramil and alloxan could be accounted for by the disproportionation of the intermediate purpuric acid (IX). This formula would have great advantage in explaining the behavior of the alkyl am- monium purpurates of Mohlau and Litter? It may be noted that the foregoing hypothesis relates murexide to the highly-colored meriquinones.14

LEUCOMUREXIDE

An experimenturn crucis for testing this formula was found in reduction. While Piloty‘ has claimed that reducing agents cause a scission of the murexide molecule, it has recently been found that murexide is readily reduced by sodium hydrosulfite to a colorless leuco compound without disrupting the molecule.16 Now formula VI11 requires the formation of two moles of uramil on reduction, while formula I permits the absorption of two atoms of hydrogen by the azomethine bond. The colorless product obtained from murexide was readily distinguished from uramil by the ease with which it was reconverted to murexide by air or potassium ferri- cyanide.

THE HYDROLYSIS OF URAMIL

The suggestion of a murexide structure based on analogy with alloxantine having been disposed of, attention was next directed toward explaining the occurrence of alloxantine in the hydrolysis of murexide on the basis of the azomethine structure. Since alloxantine may arise from alloxan and dialuric acid, and alloxan is a recognized hydrolytic product of murex- ide, the problem reduces itself to tracing the origin of the dialuric acid. Uramil is a t once suspected.

In 1904, Piloty wrote relative to the hydrolysis of murexide:‘

“Bei diesem Versuche ist eine Einwirkung der geringen Menge verdunnter Saure auf das Uramil and auch auf das Alloxan etwa unter Bildung von Alloxantin- ausgeschlossen. Reines Alloxan unter den gleichen Verhaltnissen mit verdiinnter Salzsaure behandelt, zeigt selbst nach langerer Zeit keine Veranderung, vor allem werden keine isolierbaren Mengen Alloxantin gebildet.” [p. 331

The conviction that uramil was stable under these conditions was so strong that Piloty does not appear to have troubled to test it. The in- fluence of his dictum may be traced in Mohlau’s remark:2

“. . . und da es ausgeschlossen erscheint, dass Uramil unter den Bedingungen dieser Spaltung in Ammoniak und Dialursaure zerfallt. . . . ” [p. 26871

and in Johnson and Hahn’s splendid review?‘

Is (a) KUHN AND LYMAN, Ber., 69B, 1547 (1936), and (b) DAVIDSON, J . Am. Chem. Soc., 68, 1821 (1936).

THE MUREXIDE QUESTION 309

“Moreover there is again no instance recorded in the literature of an amino group in the 5-position being easily split off from a pyrimidine by hydrolysis.” [p. 27111

On the other hand, it appears that Liebig and Wohler4 found that uramil was decomposed by dilute sulfuric acid yielding “uramilic acid.” Piloty and Finckh” found “uramilic acid” to be ammonium hydurilate and, concerning the decomposition of ammonium thionurate, a progenitor of uramil, they state:

i‘ Neben diesen Krystallen t r i t t stets unverandertes thionursaures Salz und Uramil, zuweilen auch Alloxantin auf.”

Furthermore, Techowl* reported that 1,3-dimethyluramil was completely hydrolyzed by boiling hydrochloric acid to 1,3-dimethyldialuric acid, which, on aerial oxidation, yielded crystalline tetramethylalloxantine. Behrendl9 also claims that 5aminouracil is converted to isobarbituric acid by hydrochloric acid.

I t is now found that the alleged stability of uramil toward hydrolysis is illusory. Actually, treatment for one or two minutes with boiling 6 N hydrochloric acid produces sufficient dialuric acid to yield a purple precipi- tate of barium alloxantinate with barium hydroxide or a colorless precipi- tate of sodium dialurate with sodium acetate. The behavior of uramil toward barium hydroxide before and after treatment with hydrochloric acid may, in fact, be used as a convenient test for uramil.

Quantitative experiments indicated erratic rates of hydrolysis with dif- ferent samples of uramil. The hypothesis was proposed that oxidizing agents accelerate the hydrolysis, while reducing agents hinder it. Since uramil may be oxidized to alloxan (in acid medium), the following mecha- nism seems a plausible explanation for the catalyzed hydrolysis of uramil. Uramil (111) and alloxan (IV) first undergo an oxidation-reduction reac- tion, yielding alloxan-imine (X) and dialuric acid (VI). The alloxan- imine hydrolyzes readily (as is characteristic of imines) regenerating alloxan, which may go through the cycle again, converting another mole of uramil to dialuric acid and ammonia, and so on.2o

NH-CO NH-CO NH-CO NH-CO

(20 CHNHz + CO CO == CO C:NH + CO CHOH

IQH-CO

I I I I I I I I I I I 1

NH-CO I I

NH-CO I I

NH-CO I11 IV X VI1

l’ I’ILOTY AND FINCKH, Ann., 359, 71 (1904). 18 (a) TECHOW, Ber., 27,3083 (1894). Compare (b) FISHER AND JOHNSON, J. Am.

Chem. Soc., 64, 2040 (1932). l9 BEHREND, Ann., as, 38 (1885). 20 I’INCK AND HILBERT, J. Am. Chem. soc. , 64, 710 (1932), report a closely related

dehydrogenation reaction involving fluorenylamine.

310 DAVID DAVIDSON AND ELIAS EPSTEIN

Alloxan thus appears to be a true catalyst for the hydrolysis of uramil. Table I represents the time required for the dissolution of 0.5 g. of uramil by 25 cc. of 2.5 N hydrochloric acid at the boiling point in the presence of various proportions of alloxan. Thus, for example, 0.1 g. of alloxan tetra- hydrate causes 0.5 g. of uramil to dissolve (ie., hydrolyze) in seven minutes. Ferric chloride is equally effective in promoting the hydrolysis of uramil.

TABLE I TIME REQUIRED FOR HYDROLYSIS OF 0.5 g. URAMIL BY 25 cc. OF 2.5 N HCl IN THE

PRESENCE OF ALLOXAN

TIME I ALLOXAN

0.015 g. 0.025 0.050 0.075 0.100

>180 min. 87 16 10 7

~

TABLE I1 TIME REQUIRED FOR HYDROLYSIS OF 0.5 g. URAMIL IN THE PRESENCE OF 0.050 g.

ALLOXAN BY HCl OF DIFFERENT STRENQTHS

TIME HCl I 1.0 N 2.5 6 . 0

35 min. 16 5

TABLE I11 RECOVERY OF URAMIL FROM 1 g. AFTER REFLUXINQ FOR 2 HOURS WITH 50 cc. 6 iV

HC1 IN THE PRESENCE OF STANNOUS CHLORIDE

SnClr2HzO

0.16 g. 0.32 0.50 1.08

On the other hand, as little as one I

RECOVERY

51 % 89 92 98

illigram of &annous chloride greatly retards the reaction. This effect of small amounts of tin or of alloxan explains the variable behavior of different samples of uramil. Larger amounts of stannous chloride protect the uramil almost completely. Thus, 98 per cent. of the uramil employed was recovered after boiling a gram of uramil for two hours with 50 cc. of 6 N hydrochloric acid containing one

THE MUREXIDE QUESTION 311

gram of stannous chloride. This result accounts for the success of Hartman and Sheppard's method of preparing uramil.*l' (Table 111.)

When equivalent quantities of uramil and alloxan are treated with boil- ing 6 N hydrochloric acid, they are immediately converted into alloxantine and ammonia, the intermediate dialuric acid combining with the regener- ated alloxan to form alloxantine. It is now possible to explain the results obtained in the hydrolysis of murexide. Assuming that alloxan and uramil are the primary products of the reaction, it becomes clear that more or less alloxantine will be formed from these, according to the mechanism suggested above, depending on the strength of the acid employed, the temperature, and the time of contact. (Tables I, 11.)

THE HYDROLYSIS OF DIALURIC ACID

In the hydrolysis experiments just discussed tests for the hydrolytic products (dialuric acid or alloxantine) were obtained except when the hydrolysis was prolonged for several hours. It thus seemed that the dialuric acid appearing in the hydrolysis of uramil was itself undergoing hydrolysis involving the scission of the pyrimidine ring. Accordingly dialuric acid was subjected to boiling with 6 N hydrochloric acid. While a test for dialuiic acid could still be obtained after 30 minutes, at the end of two hours the test was negative. Tartronic acid was isolated in the form of its baxium salt.

THE MECHANISM OF FORMATION OF MUREXIDE

The formation of murexide from alloxantine and ammonium salts has been explained2** as follows. Alloxantine dissociates into alloxan and dialuiic acid. Dialuric acid then reacts with the ammonium salt to form uramil which condenses with alloxan to form purpuric acid, the ammonium salt of which is murexide. While dialuric acid is known to be converted to uramil by ammonium this reaction appears to be much slower than the formation of murexide under discussion. This suggests an in- direct, formation of uramil, which may proceed according to the following suggested ucheme. Alloxan condenses with ammonia to form alloxan- imine (hypothetical) which is reduced by the dialuric acid present to uramil. This condenses with alloxan or with alloxan-imine to form pur- puric acid. The postulated oxidation-reduction is the reverse of that given above in the mechanism of the hydrolysis of uramil.

In the preparation of murexide by the action of mercuric oxide on uramil, alloxan-imine is undoubtedly the primary product of oxidation. The production of murexide by the action of 7-alkyluramils on alloxan or

*l HARTMAN AND SHBPPARD, Org. Syntheses, 12, 84 (1932). 2% BILTZ AND DAMM, Ber., 48, 3668 (1913).

3 12 DAVID DAVIDSON AND ELIAS EPSTEIN

alloxantine in the presence of ammonium carbonate’” requires further consideration. It may be supposed that oxidation-reduction plays a r81e here too. For example, with 7-ethyluramil (XI) alloxan may react as follows, being converted to alloxan-ethylimine (XII).

NH-CO NH-CO

CO CO + CO CHNHCH2CH3 ;=f

NH-CO NH-CO

I I I 1 I 1 I I

IV XI

NH-CO NH-CO NH-CO

CO CHOH + CO C:NCH&H, -+ CO CHN:CHCHB I I I 1 I 1

I I I I NH-CO

I I NH-CO N H - C O

VI XI1 XI11

From this point, two alternatives are possible: (1) since, as a result of the redox reaction, both alloxan and dialuric acid are present, murexide may be formed as outlined above for alloxantine; or (2) alloxan-ethylimine (XII) may isomerize to ethylidene-uramil (XIII)23 which then reacts with alloxan, eliminating acetaldehyde. Mohlau2 claims to have detected ethyl alcohol (iodoform test) in this reaction. This test would, of course, apply equally well to acetaldehyde.

Condensa- tion yields alloxan-alkylimines (like XII) which may then be converted to purpuric acid and an aldehyde according to the second mechanism given just above. If ammonia is absent, the amine combines with the purpuric acid to form an alkylammonium purpurate. Traube’ obtained a good yield of benzaldehyde from the action of benzylamine on alloxan. In the reaction between aminoacids and alloxan, the same mechanism may be applied, if , in addition, decarboxylation is postulated to occur, following the isomerization step.6*7s24

The action of amines on alloxan may be explained similarly.

23 INGOLD AND WILSON, J . Chem. SOC., 1933, 1500. 24 (a ) HURTLEY AND WOOTON, ibid., 99, 288 (1911), ( b ) HARDING AND WARNEFORD,

J . Biol. Chem., 26, 319 (1916), and (c) FRANKE, Biochem. Z., 268, 297 (19331, offer other mechanisms. Support for the present mechanism will be presented in a forth- coming paper from this laboratory.

THE MUREXIDE QUESTION 313

I -

l l NH--CO

I I NH-CO

NH-CO NH-CO I I I I

I I I I CO CHN:CRCOOH ---+ CO CHN:CHR + Cot

NH-CO NH-CO 1 (alloxan)

purpuric acid + RCHO

EXPERIMENTAL

Uric acid was oxidized with potassium chlorate according to Biltz and Damm.22 From the resulting alloxan solution, alloxan tetrahydrate was obtained by chilling, alloxantine by reduction with stannous chloride in the and dialuric acid by reduction with stannous chloride in the hot.

Uramil was prepared from dialuric acid by the method of Biltz and Damm22 and from barbituric acid by the method of Hartman and Sheppard,21 as well as by the following improved method. A mixture of 12.8 g. (0.10 mole) of barbituric acid, 7.5 g. (0.11 mole) of sodium nitrite, and 500 cc. of water is heated with stirring until the acid dissolves and a deep purple solution of sodium violurate forms. The hot solution is filtered and then heated to boiling in a 3-1. flask. In the meantime a solution of sodium hydrosulfite is prepared by shaking 60 g. of the salt with 400 cc. of water and 100 cc. of concentrated ammonia in a well-stoppered flask. This solu- tion iei quickly filtered by suction and added at once to the boiling violurate solution. Vigorous boiling is continued for about 30 minutes to remove the ammonia and complete the precipitation of the uramil which appears as snow-white, silky needles. The mixture is then cooled to room temperature and filtered by suction. The product is washed by removing it from the filter, stirring it up to a paste with water and rlafiltering. This is repeated and the resulting cake washed with water and finally with methanol. After drying a t loo", a yield of 13.6 g. (95% of theory) of uramil is obtained.

Anlzl. Murexide was prepared from alloxantine by the method of Davidson.16b Hydrolysis of Murexide to Alloxantine.-While previous writers have reported

varying amounts of alloxantine to be formed in the hydrolysis of murexide, i t is possible to obtain principally alloxantine as follows. To 1.00 g. of murexide was added 50 cc. of dilute hydrochloric acid (1:4), The mixture was boiled for one to two minutes, filtered hot from a small residue, and then cooled rapidly. A nearly colorless, granular precipitate was obtained, which was recognized as alloxantine by its solubility, its purple barium salt, and its conversion to murexide. Yield, 0.68 g. (60% of theory).

Action of Alloxan on Uramil i n Acid Solution.-While the action of alloxan on uramil in alkaline solution yields purpurates (see Introduction), their interaction

Calc'd for C4HsNaOa: N, 29.4; Found: N, 28.9.

26 E ~ L T Z , Ber., 46, 3673 (1912).

3 14 DAVID DAVIDSON AND ELIAS EPSTEIN

in acid solution has not been reported. A mixture of 1.43 g. of uramil, 2.14 g. of alloxan tetrahydrate, and 25 cc. of dilute hydrochloric acid (1:l) was boiled for two minutes and then diluted with 50 cc. of boiling water. This resulted in complete solution of the reagents. On cooling, 2.32 g. (72% theoretical) of coarse, granular crystals characteristic of alloxantine separated, which was identified as described above.

Hydrolysis of Uramil to Dialuric Acid (Catalyzed by Alloxan).-One g. of uramil and 0.1 g. of alloxan tetrahydrate were refluxed in 50 cc. of 6N hydrochloric acid. The uramil dissolved completely in five minutes. The solution was then evaporated t o dryness under diminished pressure. The residue waa treated with 100 cc. of warm water, and the mixture was filtered from a small insoluble residue (0.02 g.). The clear filtrate was treated with 15 g. of sodium acetate, which precipitated 0.78 g. of sodium dialurate.16 This was identified by its solubility in dilute hydrochloric acid, the precipitation of a purple barium salt from the acid solution by barium hydroxide, and its blue ferric salt (with ferric chloride and ammonia).

The experiments represented in Tables I, 11, and I11 were performed by refluxing the materials in a 200-cc. flask attached to a condenser to which was fitted a down- comer which dipped into mercury. This served t o prevent access of air to the reaction mixture.

Hydrolysis of Dialuric Acid.-One gram of dialuric acid was refluxed with 50 cc. of 6 N hydrochloric acid for two hours. The solution was then evaporated under diminished pressure and the residue taken up in water. Precipitation with barium acetate solution in the hot yielded 0.3 g. of a dense, white salt which was filtered off, washed with dilute acetic acid and with water, and dried at 120'. It did not melt below 350" and contained no nitrogen.

Calc'd for CaHzBaOs: Ba, 53.9; Found: Ba, 54.0. Anal.

SUMMARY

1. The hydrolysis of murexide by acid may yield alloxantine, or a mix- ture of uramil and alloxan, or all three products, depending on conditions.

2. Alloxan and uramil interact in acid solution to form alloxantine. 3. Uramil is susceptible of hydrolysis to dialuric acid in the presence of

alloxan or other oxidizing agents, but resists hydrolysis in the presence of reducing agents.

4. Dialuric acid is readily hydrolyzed to tartronic acid by boiling 6N hydrochloric acid.

5. The formation of murexide, the hydrolysis of murexide, and the catalyzed hydrolysis of uramil may be explained by assuming the follow- ing reversible redox reaction :

Alloxan + Uramil Dialuric Acid + Alloxan-imine. 6. The action of primary amines and of alpha-aminoacids on alloxan

may be plausibly explained by a mechanism involving (a) condensation, (b) isomerization of the azomethine formed in (a), (c) interaction of the resulting Schiff base with alloxan.