1Si4
E.
c. ’I‘AYLOR, JR., C. E;. C A I N AND HARVEY M. L O U X
I COXlRIBUTION FROM
Pteridines. XI.
THE
NOYES CHEMICAL LABORATORY, UNIVERSITY O F
Vol. i(i ILLINOIS]
The Structure of Wieland’s “Bis-alloxazine”l
BY E. C. TAYLOR, JR., C. K. CAINAND HARVEY M. Loux RECEIVEDDECEMBER 28, 1953 The product of the reaction between 5,6-diamino-2,4-dihydroxypyrimidine sulfate arid alloxan has been shoivn to he 2,4,5, 7-tetrahydroxypyrimido(5,4-g)pteridine (IV), in agreement with the original postulate of W i e l a r ~ d . ~The reaction between alloxan and 5,6-diaminopyrimidines in dilute acid solution appears t o be general and leads by way of a colored intermediate (corresponding in structure to 111) t o derivatives of pyrimido( 5,4-g)pteridine. This conclusion is supported by several independent syntheses of derivatives of this ring system. The formation of the colored intermediate serves as a sensitive qualitative test either for alloxan or for the 5,6-diaminopyrimidine.
The reaction of alloxan with aromatic 1,2-dia- loxan with 5,G-diarnino-!?,-l-dihydroxypyrimidine mines has been widely used to prepare a host of het- gives 2,4,5,7-tetrahydroxypyrimido(3,4-g)pteridine erocyclic compounds.2 The first reported use of a (IV),12in support of the original postulate of IVie 5 ,G-diaminopyrimidine in this reaction was by H Sachs and Meyerhein~,~ who described the condenO,(S\,O trls-l,s\l -01-1 sation of alloxan with 1,3-diniethyl-c5,6-dianiino-2,4(lH,3H)pyrimidinedione in neutral solution to give 1,3-dimethyl-2,4(1H,3H)diketo-7-hydroxy-ti/I pteridinecarbonylurea. Subsequently a number of Oli 9 condensations of alloxan with 5,G-diaminopyrimiI1 I dines have been carried ~ u t , ~ - lprincipally O in acidic Route I Route I1 solution”; it has been assumed that tricyclic structures similar to IV or VI have been formed. ’Iliieland and co-workers’ showed that the reacOH tion between alloxan and 5,6-diamino-2,4-dihydroxypyrirnidine involved a purple-bronze intermediate I11 which was isolated and characterized and which was readily transformed by heating in basic solution into a final product for which structure IV 1 was proposed, and for which the name “bis-alloxaOH OH OH zine” was suggested. It was recognized that the reI11 \. action might have proceeded by way of the isomeric intermediate V to VI, but structure IV was preferred 0 1i for the final product because of the known greater I reactivity of the 5-amino group in the 5,G-diaminopyrimidine and the 5-carbonyl group in alloxan. No direct proof was offered, however, to support this assignment of structure. Other workers either have not indicated which isomer was p r o d u ~ e d , ~ ~ ~ ~ ’ ~ OH OH OH or have assigned the formulation IV7-9 or VIfito the \.I IT products without experimental verification. It has now been found that the reaction of al- land that the condensation reaction proceeds by route 1. Thus, IV was cleaved with strong aque(1) This investigation was supported in part b y a research grant (C-2031 E T ) from t h e National Cancer Institute of t h e Kational Inous sodium hydroxide by the method of Weijlard, stitutes of IIealth, Public Health Service. Tishler and Erickson13 to give a diaminopyrazine(2) For example, see 0. Kuliling, B e y . , 24, Z t i 3 (1891); 27, 21 lfi
I
.i
(1894); 28, 1968 (1895); K . Kuhn and F.Weygand, ibid., 67, 140!3, 1459 (1934); 68, 1282 (1935); I I . Rudy and K . E. Kramer, i b i d . . 71B, 1234 (1938); H. R u d y and 0 . h k j e r , ibid., 71B, 1213, 1323 (1938); 72B, 933 (1939); A. JIcCoubrey and W,Webster, J . Chein. Soc., 1719 (1948). (3) F. Sachs and G . hteyerheim, Rei.., 41, 3957 (1908). (4) R. Robinson and 31. I,. Tomlinson, J . Chetn. Sac., 467 (1933). ( 2 ) R. K u h n and A . H . Cook, Be?., 70B,761 (1837). (6) B. Hepner, I. Kelner, A . Simonberg and H . Kaltman, Co16g. ~ h i n z i,i z d . C o m p f .v e n d . , 17, I, 228 11937). (7) H. Wieland, A. Tartter and R . Purrmann, .1?1u.,646, 200 (1940). (8) H . S . Forrest, R . Hull, 11. J . Rodda and A . R . Todd, J . Chem. S O L . , 3 (1951). (9) E. 11. Gal, Ergevieitfia, 7 , 2G1 (1951). (10) H. Brederick, I. Hennig, W. Pfleiderer a n d 0. Deschler, Ber., 86, 845 (1953). (11) Experiments in this Laboratory have shown t h a t t h e condensation products of alloxan and 5,6-diaminopgrimidines in basic solution rliffer from the products obtained i n acidic solution. Similar resillti Iidve IJeen demonstrated recently in t h e condensation of alloxan and o phenylenediamine (R. B . Darlolv, 11. R . Ing and I. h l . Lewis. J . C h i n . Soc.. 3212 (19.313: 17. 1’. Kinp :iud 1. W. Clark-Lewis, i b i d . , 3379 (1951)). T h i s phasc 01 t h e [irobleni i i being investigatcd further.
H?K--fKj
N
----,
VI12
HOOC-\Sp-COOI
I
OH
II‘
OH
H.S
I
-’\ ‘I-“c hT, 11
I
TI1 (12) Various names have been suggested for these compounds. Robinson (ref. 4) referred t o his product as a “bis-p),rimidazine”; Wieland (ref, 7) proposed t h e name “bis-alloxazine”; Todd (ref. 8) employed t h e systematic name 1,3,6,8,9,1O-hexa-aza-anthracene and also suggested “diazaisoalloxazine” for a 9-alkyl derivative; and Gal (ref. 9), who apparently believed he had prepared members of this series for the first time, proposed the name “7,9-diaza-alloxazine” and “pyrimido(4,5-b)pteridine.” We believe t h e shortened systematic name “pyrimido(5,4-g)pteridine” t o be advantageous. (13) J. Weijlard, M . Tishler and A. E. Erickson, T H I S J O U R N A L , 6 1 , 802 (1945).
STRUCTURE OF WIELAND’S “BIs-ALLOXAZINE”
April 5 , 1954
1873
dicarboxylic acid which was not characterized but method, was identical with the “bis-alloxazine” which was decarboxylated directly to 2,B-diamino- prepared by Wieland’s method and that XI1 was identical with the product of the condensation of pyrazine (VII), Further evidence in favor of a pyrimido(5,4-g)- 2,4,5,6-tetraminopyrimidineand alloxan. It thus pteridine structure for the reaction product of a 5,G- seems a valid generalization that the tricyclic strucdiaminopyrimidine and alloxan was found in the ob- tures obtained from 5,G-diaminopyrimidines and alservations that (1) the condensation of 5,5-dibromo- loxan are derivatives of pyrimido(5,4-g)pteridine. 16d Methylation of IV with methyl iodide and potasor 5,5-dichlorobarbituric acid with 5,G-diamino-2,4dihydroxypyrimidine gave a product identical with sium carbonate in acetone gave 1,3,G&tetrarnethylthe alloxan condensation product IV, and ( 2 ) the 2,d,3,7 ( 1H,3H,BH,8H)pyrimido ( 5,4 - g) pteridine tetrone (XIII), m.p. 403-404”, apparently identical condensation of 2-amino-4-hydroxy-6-keto-5,5-dichlorodihydropyrimidine (VIII) with 5,G-diam- with the product, m.p. 403O, obtained by Timmis16 ino-2,4-dihydroxypyrimidine (11) yielded a product from the condensation of 1,3-dimethyl-6-amino-3X identical with that obtained from 2,5,6-triamino- nitroso-2,4(1Hj3H)pyrimidinedioneand dimethyl4-hydroxypyrimidine (IX) and alloxan. These barbituric acid. Bredereck’O recently has reported results are in agreement with the recent confirma- the preparation of a “tetramethyl-bis-alloxazine,” m.p. 390°, by the treatment of 1,3-dimethyl-Gamino- 5 - acetylamino - 2,4(1Hi3H)pyrimidinedione with sulfuric acid, but no decision was made as to whether the product was a derivative of IV or V I . In all probability the product was identical with ~111.17 CH1
jl CH,1-
o=(S)4X\/A-\-o
CHr-NyN/Y-CHs 0
0 XIII
.Irecent patentla claims the synthesis of XI11 by the condensation of 1,3-dimethyl-5,6-diamino-2,4(lH,3H)pyrimidinedione with dimethylalloxan, followed by heating the resulting reaction mixture H with 0.5 N sodium hydroxide for two hours. However, the product was reported to melt a t 20G-207’ and is obviously not identical with XIII. We have found that when XI11 is heated a t reflux with 1 N sodium hydroxide for three hours, 2,G-his0 OH (methylamino) - N - methyl - X’ - methylpyrazineIX 3,5-dicarboxamide (XIV), m.p. 231-2325’, is tion by \.l.7ilson14 that the reaction between a 5,(j- formed in S1% yield. The lability of XI11 to basic diaminopyrimidine and an acid chloride, ester or hydrolysis also has been noted by Bredereck, lo who acid occurs exclusively with the 5-amino group. stated that mild alkaline hydrolysis of XI11 gave a Furthermore, T i m m i ~ recently ~ ~ , ~ ~has reported pteridine derivative which was not identified. the synthesis of a number of pyrimido(5,4-g)pteri- Hence, the above reaction product, m.p. 206-20i0, dines by an independent and unequivocal method is probably either N, 1,3-trimethyl-7-methylaminoinvolving the condensation of barbituric acid with 2,4-( lH,3H)-pteridinedione-6-carboxamide or a 6-amino-5nitrosopyrimidines (XI). Using his mixture of hydrolysis products. method, we have prepared 2,4,5,7-tetrahydroxyThe reaction between 5,tbdiaminopyriniidines and alloxan to give colored intermediate condensaH tion products (corresponding in structure to 111) R--/S?-XHI O/,s\O , has been observed by a number of worker^,^,^,^ and il + +s=o serves as a sensitive qualitative test for the presence of e i t h s reactant. This reaction has been of I R-,,;s\ds, ,A-& 1I:, 0 I /--OH Ri particular value in our work with the pteridines bej XI ,9S/$ cause of the intense color given by a trace of most 3,G-diaminopyrimidines and alloxan in 0.5 N hydroI R’ OH (16a) NOTEADDEDIN P R O O F . - D r . G. H. Hitchings a n d Dr. E. A . x
!
I
-
-
-
+
I‘
pyrimido(5,4-g)pteridine (IV) and 2,4-diamino-5,7dihydroxypyrimido(5,4-g)pteridine (XII). By comparison of infrared spectra (Nujol mull), i t was shown that a sample of IV, prepared by Timmis’s (14) W. Wilson, J . C‘heiiz. .SOL.,1157 (1948). (15) G. M. Timmis, X a l w e , 164, 139 (1949). ( l e ) G. M . Timmis, U. S. Patent 2,581,889 (Jail. 8, 1952); C . A , , 46, 7594 (1052).
Falco of t h e Wellcome Research Laboratories have communicated the information t h a t they also have prepared IV by t h e methods of Wieland a n d Timmis and found t h e two products t o be identical by analysis and comparison of ultraviolet absorption spectra. (17) We will report later on a n unequivocal Fynthesis of t h e isomeric “bis-alloxazine” VI. T h e tetramethyl derivative of VI melts a t 358300° and thus provides final confirmation of t h e above structural assignments (18) 0. DeGarmo, U. S. Patent 2,5ti1,324 (July 24, 1U31); C. A 46, 1505 (1952).
187.6
E. C. TAYLOR, JR., C. K. CAINAND HARVEY M. Loux
1701.
76
ment with Sorit, the filtrate was acidified t o p H 6 with hydrochloric acid and the precipitated solid collected by filtration and washed with water followed by acetone to give 0.1 g. (quantitative). The product was purified by dissolving in dilute sodium hydroxide, treating with Norit and filtering into boiling dilute hydrochloric acid and was obtained as a light yellow microcrystalline solid which darkened slightly without decomposition upon heating to 360'. Experimentalz0 Anal. Calcd. for CsH&703: C, 38.9; H, 2.0; K, 30.7. The 2,4,5,7-Tetrahydroxypyrimido(5,4-g)pteridine(IV). Found: C, 38.6; H, 2.2; N,39.8. Method A.-This compound was prepared by the method of Method B.-A mixture of 1.0 g. (0.0018 mole) of 5,6\I'ieland7 from 5,6-diamino-2,4-dihydrox).pyrimidine sulfate diamino-2,4-dihydroxypyrimidine sulfate, 1.15 g. (0.0058 and alloxan. mole) of 2-amino-1-hydroxy-6-keto-5,5-dichlorodihydropyMethod B.-This compound was also prepued by the rimidineZ2and 50 ml. of glacial acetic acid was heated under method of T i m m i ~ ' ~from J ~ 6-amino-2,4-dihydroxy-5-nitroreflux for three hours. The usual deep red color charactersopyrimidine and barbituric acid. Method C .-A mixture of 2.0 g. of 5,6-diamino-2,1-dihp- istic of the intermediate half-condensation product appeared, but on further heating i t faded rapidly to yellow with the droxypyrimidine sulfate, 2.5 g. of 5,5-dichlorobarbituric simultaneous separation of a yellow solid. The rewtion acid and 73 ml. of glacial acetic acid was heated under reflux for two hours. A deep red color appeared at the beginning mixture was cooled, filtered and the solid washed well with of the reaction which gradually lightenel to orange as heat- water followed bv acetone. The solid was then suspended in 100 ml. of boiling 0.1 N hydrochloric acid, the pH ading prdceeded. The cooled reaction mixture was filtered justed to 10 with ammonium hydroxide, the solution heated to give a red-orange solid which was washed thoroughly for a few minute4 and then reacidified to p H 5 with hydrowith acetone and then suspended in 100 ml. of boiling water chloric acid; yield 1.01g. (S7.5%). The product was idenmade alkaline to p H 10 with ammonium hydroxide. The tical with the product obtained by method A as shown b: a color of the suspended solid rapidly changed from orange to yellow. After heating the alkaline mixture for several comparison of infrared spectra (Nujol mull). 2,4-Diamino-5,7-dihydroxypyrimido(5,4-g)pteridine (XII) . minutes, the p H was adjusted to 5 with acetic acid and the yellow solid collected by filtration; yield 1.5 g. (5870). Method A.-To a solution of 3.0 g. (0.0135 mole) of 2,4,5,6tetraminopyrimidine bisulfite in 50 ml. of 0.5 N hydrochloComparable results were obtained using 5,5-dibromobarbiric acid was added a solution of 2.5 g. (0.0156 mole) of :Aturic acid. loxan monohydrate in 10 ml. of water. The deep purple The products from methods A, B and C were identical color of the half-condensation product appeared immedias shown by a comparison of their infrared spectra (Sujol ately upon mixing, but after a few minutes lightened to mull). orange with the simultaneous separation of an orange solid. 2,6-Diaminopyrazine (VIII .-Ten grams of 2,4,5,7-tetrnAfter adjusting the p H to 9 with dilute sodium hydroxide, hydroxypyrimido(5,4-g)pteridine mas added t o 65 ml. of the reaction mixture was boiled for ten minutes, reacidified sodium hydroxide solution and the mixture wag heated to p H 6 with hydrochloric acid, cooled t o 0' and filtered. in a steel autoclave a t 170" for three hours. Addition of The orange solid was thoroughly washed with water followed 18% hydrochloric acid to the cooled bomb contents caused by acetone; yield 2.2 g. (66%). The product was purified copious evolution of carbon dioxide and the separation of by suspending in boiling 0.1 iV hydrochloric acid, filtering 2,6-diamino-3,5-pyrazinedicarboxylic acid as a dark red and then resuspending in boiling water and filtering. An solid; yield 7.1 g. (89.5%). Two grams of this m a t e r i J orange microcrystalline solid was obtained which did not was added to 30 ml. of 80% sulfuric acid and the solution darken upon heating t o 360". heated for 30 minutes at 195-200". The black reaction Annl. Calcd. for CeH6Ss02: C, 39.0; H, 2.5; S , 43.5. mixture was cooled, added to 75 g. of ice, and the resulting Found: C, 39.0; H , 2 . 5 ; N,45.6. solution made strongly alkaline with sodium hydroxide. It was then extracted for 20 hours with peroxide-free ether, Method B.-This compound was also prepared by the the ether extract dried over sodium sulfate, and the ether method of Timmis'6,16 from 2,6-diamino-4-hydrosy-Lnitroremoved by evaporation. Recrystallization of the residue sopyrimidine and barbituric acid. I t was identical with the from methylene chloridepetroleum ether gave 0.23 g. of product obtained by method A as shown by a comparison yellow needles, m.p. 133-136" dec. The reported decom- of infrared spectra (Nujol mull). position point of 2,G-diaminopyrazine is 136°.21 The low 1,3,6,8-Tetramethyl-2,4,5,7( lH,3H,6H,SH)pyrimido( 5,4yield of diaminopyrazine obtained bv this degradation is not ehteridmetetrone (XIII).-A mixture of 5 2 . of 2.4.5.7-tetrasurprising in view of its known instability. Gy&oxypyrimido(5;4-g)pteridine (IV), 45 ml. of methyl 2-Amino-4,5,7-trihydroxypyrimido(5,4-g)pteridine ( X ) . iodide, 50 g of potassium carbonate and 150 ml. of acetone Method A.-A solution of 5.0 g. of freshly recrystallized was heated under reflux for 24 hours. Addition of 100 ml. of 2,5,6-triamino-4-hydroxypyrimidinebisulfite (0.024 mole) water followed bv filtration gave a green-yellow solid which in 12 ml. of boiling water was added t o a solution of 4.0 g. mas placed in a Soxhlet cup and extracted with chloroform (0.025 mole) of alloxan monohydrate in 6 ml. of water. for 48 hours. A light yellow solid separated from the Immediately upon mixing a deep purple color appeared with chloroform extract which was collected by filtration, dried the simultaneous separation of a purple solid. After cool- and sublimed at 280" (0.1 mm.) t o give 1.2 g. of a white ing to O " , the reaction mixture was filtered and the solid crystalline solid, m.p. 403-404'. An additional 0.5 g. of washed with a small amount of ice-cold water followed by pure product was obtained by sublimation of the chloroformacetone; yield 4.5 g. (71%). The microcrystalline halfinsoluble residue; total yield 1.7 g. ( 2 8 % ) . condensation product (corresponding in structure to 111) A n d . Calcd. for ClzH12S604: C, 47.4; H, 4.0; N, 27.6. turned orange upon heating above 300°, indicating ring clo- Found: C, 47.5; H , 3.9; X, 27.9. sure to 2-amino-4,~,7-trihydroxyp~~rimido(5,1-g)pteridine; 2,6-Bis-( methylamine )-N-methyl-N'-methylpyrazine-3,5it was dried for analysis a t 140" in VQCUO over phosphorus dicarboxamide (XIV).-A mixture of 1 .0 g. of XI11 and 30 pentoside for ten hours. ml. of 1 N sodium hydroxide was hekited under reflux for 2.,5 Anal. Calcd. for C ~ H ~ ~ : I C, O I30.2; : H , 2.7; N, 37.0. hours, 13 ml. of ethanol added and heating continued for an Found: C , 36.2; H, 2.8; N, 37.0. additional 30 minutes. Cooling caused the separation of King closure to 2-amino-4,6,7-trihydroxypyritnido(5,4- yellow crystds which were collected by filtration and washed g)pteridine was effected by boiling 0.4 g. of the above halfwith cold ethanol; yield O.F7 g. (817,), m.p. 230-232'. condensation product in 10 ml. of 0.1 N sodium hydroxide Sublimation a t 180' (0.6 mm.) gave dense yellow crystals, for a period of ten minutes. During this time, the color r11.p. -031-232 .so. lightened from a deep cherry-red t o yellow. After tre,iti l ? t ~ l . C d c d . for C10Hi6N602: c, 47.6; H,6.4; N , 33.3. Fouud: C, 47.3; €1, 6.1; N, 33.3. (19) W. Trauhe, B e r . , 33, 1371 (1900).
chloric acid. Although the coior is much less intense with pyrimidines lacking an enolizable group in either the 2- or 4-positions, this test has been found to be more convenient and reliable than the nitric acid method employed by Traube.19
(20) T h e microanalyses were performed by hIrs. I,ucy Cliang, iilrs. Esther Fett a n d hlr. Joseph Nemeth. T h e infrared spectra were determined by hlr. James Rrader. (21) K. H. Schaaf and P. E. Spoerri, THISJ O U R N A L , 71, 2043 ( 1919).
U R R A N A , 11.I.INOIS
(22) R. B. Angier, J . 13. hlowat, J. H. Roothe, C. W. Waller. J. Semh, n. I,. Hutchings, E. L. R. Stokstad and Y .SubhaRow, i h i d . , 7 0 , 1274 (1048).
