Purine analog inhibitors of xanthine oxidase: structure activity relationships and proposed binding of the molybdenum cofactor

A number of new hypoxanthine analogs have been prepared as substrate inhibitors of xanthine oxidase. Most noteworthy inhibitory new hypoxanthine analogs are 3-(m-tolyl)pyrazolo[1,5-a]pyrimidin-7-one (47), ID50 0.06 μM and 3-phenylpyrazolo[1,5-a]pyrimidin-7-one (46), ID50 0.40 μM. 5-(p-Chlorophenyl)pyrazolo[1,5-a]pyrimidin-7-one (63) and the corresponding 5-nitrophenyl derivative 64 exhibited an ID50 of 0.21 and 0.23 μM, respectively. 7-Phenylpyrazolo[1,5-a]-s-triazin-4-one (40) is shown to exhibit an ID50 of 0.047 μM. The structure-activity relationships of these new phenyl substituted hypoxanthine analogs are discussed and compared with the xanthine analogs 3-m-tolyl- and 3-phenyl-7-hydroxypyrazolo[1,5-a]pyrimidin-5-ones (90) and (91), previously reported from our laboratory to have ID50 of 0.025 and 0.038 μM, respectively. The presence of the phenyl and substitutedphenyl groups contribute directly to the substrate binding of these potent inhibitors. This work presents an updated study of structure-activity relationships and binding to xanthine oxidase. In view of the recent elucidation of the pterin cofactor and the proposed binding of this factor to the molybdenum ion in xanthine oxidase, a detailed mechanism of xanthine oxidase oxidation of hypoxanthine and xanthine is proposed. Three types of substrate binding are viewed for xanthine oxidase. The binding of xanthine to xanthine oxidase is termed Type I binding. The binding of hypoxanthine is termed Type II binding and the specific binding of alloxanthine is assigned as Type III binding. These three types of substrate binding are analyzed relative to the most potent compounds known to inhibit xanthine oxidase and these inhibitors have been classified as to the type of inhibitor binding most likely to be associated with specific enzyme inhibition. The structural requirements for each type of binding can be clearly seen to correlate with the inhibitory activity observed. The chemical syntheses of the new 3-phenyl- and 3-substituted phenylpyrazolo[1,5-a]pyrimidines with various substituents are reported. The syntheses of various 8-phenyl-2-substituted pyrazolo-[1,5-a]-s-triazines, certain s-triazolo[1,5-a]-s-triazines and s-triazolo[1,5-a]pyrimidine derivatives prepared in connection with the present study are also described.

[1]  M. Stevens,et al.  Pyrazolo-as-triazines. Part I , 1966 .

[2]  Y. Makisumi.  Studies on the Azaindolizine Compounds. X. Synthesis of 5, 7-Disubstituted Pyrazolo [1, 5- a ] pyrimidines , 1962 .

[3]  S. Plescia,et al.  An unequivocal synthesis of 7-oxopyrazolo[1,5-a] pyrimidines , 1972 .

[4]  K. Imai.  STUDIES ON NUCLEIC ACID ANTAGONISTS. VII. SYNTHESIS AND CHARACTERIZATION OF 1,4,6-TRIAZAINDENES(5H-PYRROLO(3,2-D)PYRIMIDINES). , 1964, Chemical & pharmaceutical bulletin.

[5]  R. K. Robins,et al.  6-aminopyrazolo[4,3-c]pyridin-4(5H)-one, a novel analogue of guanine. , 1977, Journal of medicinal chemistry.

[6]  J. Baldwin,et al.  3,5-disubstituted 1,2,3,4-triazoles, 1 new class of xanthine oxidase inhibitor. , 1975, Journal of Medicinal Chemistry.

[7]  R. Bray,et al.  The mechanism of action of xanthine oxidase. The relationship between the rapid and very rapid molybdenum electron-paramagnetic-resonance signals. , 1979, The Biochemical journal.

[8]  Robins Rk,et al.  Synthesis and antimicrobial activity of certain imidazo[1,2-a]pyrimidines. , 1975 .

[9]  H. R. Beilfuss,et al.  The Structure of Certain Polyazaindenes. III. 1,2,3a,7- and 1,3,3a,7-Tetrazaindenes , 1959 .

[10]  K. Rajagopalan,et al.  Characterization of the molybdenum cofactor of sulfite oxidase, xanthine, oxidase, and nitrate reductase. Identification of a pteridine as a structural component. , 1980, The Journal of biological chemistry.

[11]  Thomas Novinson,et al.  The chemistry of 4-hydrazino-7-phenylpyrazolo[1,5-a] -1,3,5-triazines , 1974 .

[12]  K. Rajagopalan,et al.  In vitro reconstitution of demolybdosulfite oxidase by a molybdenum cofactor from rat liver and other sources. , 1977, The Journal of biological chemistry.

[13]  R. K. Robins,et al.  Novel heterocyclic nitrofurfural hydrazones. In vivo antirypanosomal activity. , 1976, Journal of medicinal chemistry.

[14]  H. Gray,et al.  The molybdenum site of sulfite oxidase. Structural information from x-ray absorption spectroscopy , 1979 .

[15]  B. R. Baker,et al.  Irreversible enzyme inhibitors XCII. Inhibition of xanthine oxidase by some purines and pyr midines , 1967 .

[16]  R. K. Robins,et al.  Aromaticity in heterocyclic systems. II. the application of N.M.R. in a study of the synthesis and structure of certain lmidazo[1,2-c]pyrimidines and related pyrrolo[2,3-d]pyrimidines , 1964 .

[17]  S. Bel,et al.  The Synthesis and Reactions of Some Imidazo[1,2-a]pyrimidines1 , 1960 .

[18]  R. K. Robins,et al.  Purines. II. The Synthesis of Certain Purines and the Cyclization of Several Substituted 4,5-Diaminopyrimidines1 , 1953 .

[19]  R. K. Robins.  Potential Purine Antagonists. I. Synthesis of Some 4,6-Substituted Pyrazolo [3,4-d] pyrimidines1 , 1956 .

[20]  W. F. Wood,et al.  Irreversible enzyme inhibitors. CXXVI. Hydrocarbon interaction with xanthine oxidase by phenyl substituents on purines and pyrazolo[3,4-d]pyrimidines. , 1968, Journal of medicinal chemistry.

[21]  R. K. Robins,et al.  Potential Purine Antagonists XXI. Preparation of Some 9-Phenyl-6-substituted Purines1 , 1959 .

[22]  Y. Makisumi.  Synthesis of Potential Anticancer Agents. IV. 5, 7-Disubstituted s-Triazolo [2, 3-a] pyrimidines. , 1961 .

[23]  R. K. Robins,et al.  Studies on the mode of oxidation of pyrazolo(3,4-d)pyrimidine by aldehyde oxidase and xanthine oxidase. , 1969, Biochemical pharmacology.

[24]  牧角 徳夫,et al.  Synthesis of Potential Anticancer Agents. III. 7-Substituted s-Triazolo [2, 3-a]-pyrimidines and their Halogenated Compounds , 1959 .

[25]  K. Rajagopalan,et al.  Structural and metabolic relationship between the molybdenum cofactor and urothione. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[26]  J. Olson,et al.  The mechanism of action of xanthine oxidase. , 1974, The Journal of biological chemistry.

[27]  R. K. Robins,et al.  Potential Purine Antagonists. XII. Synthesis of 1-Alkyl(aryl)-4,6-disubstituted Pyrazolo[3,4-d]pyrimidines1 , 1958 .

[28]  S. Goldfinger.  Treatment of gout. , 1971, The New England journal of medicine.

[29]  V. Massey,et al.  On the mechanism of inactivation of xanthine oxidase by allopurinol and other pyrazolo[3,4-d]pyrimidines. , 1970, The Journal of biological chemistry.

[30]  B. R. Baker.  Irreversible enzyme inhibitors. 93. Hydrophobic bonding to xanthine oxidase by some phenylpurines. , 1967, Journal of pharmaceutical sciences.

[31]  T. Hawkes,et al.  The structure of the inhibitory complex of alloxanthine (1H-pyrazolo[3,4-d]pyrimidine-4,6-diol) with the molybdenum centre of xanthine oxidase from electron-paramagnetic-resonance spectroscopy. , 1984, The Biochemical journal.

[32]  J. Bordas,et al.  X-ray absorption spectroscopy of xanthine oxidase. The molybdenum centres of the functional and the desulpho forms. , 1980, The Biochemical journal.

[33]  R. K. Robins,et al.  Aromaticity in Heterocyclic Systems. I. The Synthesis and Structure of Certain 4,6-Dihydroxyimidazo[4,5-c]pyridines1 , 1963 .

[34]  K. Hodgson,et al.  The molybdenum site of xanthine oxidase. Structural evidence from x-ray absorption spectroscopy , 1979 .

[35]  R. K. Robins,et al.  Synthesis and enzymic activity of some novel xanthine oxidase inhibitors. 3-Substituted 5,7-dihydroxypyrazolo(1,5-alpha)pyrimidines. , 1976, Journal of medicinal chemistry.

[36]  P. D. Cook,et al.  Synthesis of 3-deazaguanine, 3-deazaguanosine, and 3-deazaguanylic acid by a novel ring closure of imidazole precursors. , 1976, Journal of the American Chemical Society.

[37]  Roland K. Robins,et al.  Potential Purine Antagonists. VI. Synthesis of 1-Alkyl- and 1-Aryl-4-substituted Pyrazolo[3,4-d]pyrimidines , 1956 .

[38]  P. D. Cook,et al.  Synthesis of 6‐amino‐1,2,3‐triazolo[4,5‐c] pyridin‐4(5H)one (8‐aza‐3‐deazaguanine) and 6‐amino‐1‐(β‐D‐ribofuranosyl)‐1,2,3‐triazolo[4,5‐c] pyridin‐4(5H)one (8‐Aza‐3‐deazaguanosine) via novel ring closure procedures , 1980 .

[39]  R. K. Robins,et al.  Clarification of the ring-closure of certain 4,5-diaminopyrimidines to 6-substituted 8-alkylpurines. , 1973, Journal of the Chemical Society. Perkin transactions 1.

[40]  G. Hitchings,et al.  Ribonucleosides of allopurinol and oxoallopurinol. Isolation from human urine, enzymatic synthesis, and characterization. , 1967, The Journal of biological chemistry.

[41]  W. N. Kelley,et al.  Allopurinol: Alteration in Pyrimidine Metabolism in Man , 1970, Science.

[42]  H. Simmonds,et al.  The conversion of 4-hydroxypyrazolo[3,4-d]pyrimidine (allopurinol) into 4,6-dihroxypyrazolo[3,4-d]pyrimidine (Oxipurinol) in vivo in the absence of xanthine-oxen oxidoreductase. , 1969, The Biochemical journal.

[43]  R. K. Robins,et al.  Synthesis of unsymmetrical 2,4-dialkylpyrazolo[1,5-a]-1,3,5-triazines , 1974 .

[44]  R. K. Robins,et al.  Synthesis and antischistosomal activity of certain pyrazolo[1,5-a]pyrimidines. , 1975, Journal of medicinal chemistry.

[45]  J. Berg,et al.  Structural results relevant to the molybdenum sites in xanthine oxidase and sulfite oxidase. Crystal structures of MoO2L, L = (SCH2CH2)2NCH2CH2X with X = SCH3, N(CH3)2 , 1979 .

[46]  G. Banchelli,et al.  2-Phenylpyrazolo[1,5-a]pyrimidin-7-ones. A new class of nonsteroidal antiinflammatory drugs devoid of ulcerogenic activity. , 1983, Journal of medicinal chemistry.

[47]  J. Sprague,et al.  4-Trifluoromethylimidazoles and 5-(4-pyridyl)-1,2,4-triazoles, new classes of xanthine oxidase inhibitors. , 1975, Journal of medicinal chemistry.

[48]  R. K. Robins,et al.  Pyrazolopyrimidines as inhibitors and substrates of xanthine oxidase. , 1957, The Journal of biological chemistry.

[49]  E. Taylor,et al.  STUDIES IN PURINE CHEMISTRY. 13. SYNTHESIS OF THE 5-AZA ANALOGS OF ADENINE AND HYPOXANTHINE. , 1965, Journal of the American Chemical Society.

[50]  Stephen F Martin,et al.  The synthesis of lmidazo[4,5-d] pyridazines. VI. v-Triazolo[4,5-d] pyridazines, pyrazino[ 2,3-d ] pyridazines and 7H-Imidazo [4,5-d] tetrazolo[ 1,5-b] pyridazine , 1969 .

[51]  J. Lafferty,et al.  SYNTHESIS AND MUSCLE RELAXANT PROPERTIES OF 3-AMINO-4-ARYLPYRAZOLES. , 1964, Journal of medicinal chemistry.

[52]  K. Rajagopalan,et al.  Molybdenum sites of sulfite oxidase and xanthine dehydrogenase. A comparison by EXAFS , 1981 .

[53]  K. Tanaka,et al.  STUDIES ON NUCLEIC ACID ANTAGONISTS. VI. SYNTHESIS OF 1,4,6-TRIAZAINDENES(5H-PYRROLO(3,2-D)PYRIMIDINES). , 1964, Chemical & pharmaceutical bulletin.

[54]  R. K. Robins,et al.  SYNTHESIS AND BIOLOGICAL ACTIVITY OF SOME NUCLEOSIDES RESEMBLING GUANOSINE: IMIDAZO[1, 2‐a] PYRIMIDINE NUCLEOSIDES , 1975 .

[55]  R. K. Robins,et al.  A study of electrophilic substitution in the pyrrolo[2,3-d]pyrimidine ring† , 1969 .

[56]  C. Walsh.  Suicide substrates: mechanism-based enzyme inactivators with therapeutic potential , 1983 .

[57]  R. K. Robins,et al.  Synthesis and enzymic activity of various substituted pyrazolo[1,5-a]-1,3,5-triazines as adenosine cyclic 3',5'-phosphate phosphodiesterase inhibitors. , 1982, Journal of medicinal chemistry.

[58]  J. Davoll.  26. Pyrrolo[2,3-d]pyrimidines , 1960 .

[59]  B. Lynch,et al.  Synthesis and biological evaluation of xanthine oxidase inhibitors. Pyrazolo(3,4-d)pyrimidines and pyrazolo(3,4-b)pyridines. , 1975, Journal of medicinal chemistry.

[60]  R. K. Robins,et al.  Pyrazoles. I. Synthesis of 4-hydroxypyrazolo[3,4-d]-v-triazine. A new analog of hypoxanthine. , 1968, Journal of pharmaceutical sciences.

[61]  J. Druey,et al.  Heilmittelchemische Untersuchungen in der heterocyclischen Reihe. Mitteilung. Pyrazolo‐(3,4‐d)‐pyrimidine , 1956 .

[62]  A. Goldin,et al.  5‐Azapurines and the structures of sym‐triazole intermediates , 1972 .

[63]  J. Wyngaarden,et al.  Drugs and uric acid. , 1969, Annual review of pharmacology.

[64]  R. Merényi,et al.  Synthesen mit heterocyclischen Aminen, I. Reaktionen des 3(5)‐Amino‐pyrazols mit α,β‐ungesättigten Estern. Darstellung und Charakterisierung isomerer Oxo‐dihydro‐pyrazolo‐pyrimidine , 1970 .

[65]  B. R. Baker,et al.  Irreversible enzyme inhibitors. CXXV. Active-site-directed irreversible inhibitors of xanthine oxidase derived from arylpurines and pyrazolo[3,4-d]pyrimidines bearing a terminal sulfonyl fluoride. , 1968, Journal of medicinal chemistry.

[66]  S. Hamilton,et al.  Ischemic injury in the cat small intestine: Role of superoxide radicals , 1982 .

[67]  R. K. Robins,et al.  Potential Purine Antagonists. XIX. Synthesis of Some 9-Alkyl(aryl)-2-amino-6-substituted Purines and Related V-Triazolo [d]pyrimidines1 , 1959 .

[68]  V. Massey,et al.  The existence of nonfunctional active sites in milk xanthine oxidase: reaction with functional active site inhibitors. , 1970, Vitamins and hormones.

[69]  R. K. Robins,et al.  The synthesis and chemical reactions of certain pyrazolo[1,5-a]-1,3,5-triazines† , 1974 .

[70]  R. Miller,et al.  Kinetic properties and inhibition of orotidine 5'-phosphate decarboxylase. Effects of some allopurinol metabolites on the enzyme. , 1973, The Journal of biological chemistry.

[71]  R. K. Robins,et al.  The synthesis of certain 8-cyano-2,4-disubstituted-imidazo[1,5-a] pyrimidines , 1974 .

[72]  R. K. Robins,et al.  Imidazo[1,2-a]-s-triazine nucleosides. Synthesis and antiviral activity of the N-bridgehead guanine, guanosine, and guanosine monophosphate analogues of imidazo[1,2-a]-s-triazine. , 1978, Journal of medicinal chemistry.

[73]  R. Klein,et al.  New synthesis of 5H-pyrrolo[3,2-d]pyrimidines via pyrimido[5,4-c]pyridazines , 1978 .

[74]  R. K. Robins,et al.  Synthesis of 6-amino-1,2,4-triazolo[1,5-a]pyrazin-4(5H)one, a bridgehead analog of guanine , 1977 .

[75]  Lilly Capuano,et al.  Heterocyclisierungen, IX Darstellung von Pyrazolo-, Triazolo-, Oxazolo- und Thiazolo-s-triazinen mit Brückenkopf-Stickstoff sowie eines N-Äthoxycarbonyl-isopurins , 1971 .

[76]  R. Roblin,et al.  Studies in Chemotherapy. VIII. Methionine and Purine Antagonists and their Relation to the Sulfonamides1 , 1945 .

[77]  V Massey,et al.  Tight binding inhibitors of xanthine oxidase. , 1981, Pharmacology & therapeutics.