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Deoxyguanosine Cytotoxicity by a Novel Inhibitor of Furine Nucleoside Phosphorylase, 8-Amino-9-Benzylguanine1

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Deoxyguanosine Cytotoxicity by a Novel Inhibitor of Furine Nucleoside Phosphorylase, 8-Amino-9-Benzylguanine1 [CANCER RESEARCH 46, 519-523, February 1986] Potentiation of 2'-Deoxyguanosine Cytotoxicity by a Novel Inhibitor of Furine Nucleoside Phosphorylase, 8-Amino-9-benzylguanine1 Donna S. Shewach,2 Ji-Wang Chern, Katherine E. Pillóte,Leroy B. Townsend, and Peter E. Daddona3 Departments of Internal Medicine [D.S.S., P.E.D.], Biological Chemistry [P.E.D.], and Medicinal Chemistry [J-W.C., K.E.P., L.B.T.], University ol Michigan, Ann Arbor, Michigan 48109 ABSTRACT to the ADA-deficient disease state (2). PNP is an essential enzyme of the purine salvage pathway, We have synthesized and evaluated a series of 9-substituted catalyzing the phosphorolysis of guanosine, inosine, and their analogues of 8-aminoguanine, a known inhibitor of human purine 2'-deoxyribonucleoside derivatives to the respective purine nucleoside phosphorylase (PNP) activity. The ability of these bases. To date, several inhibitors of PNP have been identified, agents to inhibit PNP has been investigated. All compounds were and most of these compounds resemble purine bases or nucleo found to act as competitive (with inosine) inhibitors of PNP, with sides. The most potent inhibitors exhibit apparent K¡values in K¡values ranging from 0.2 to 290 /¿M.Themost potent of these the range of 10~6to 10~7 M (9-12). Using partially purified human analogues, 8-amino-9-benzylguanine, exhibited a K, value that erythrocyte PNP, the diphosphate derivative of acyclovir dis was 4-fold lower than that determined for the parent base, 8- played K¡values of 5.1 x 10~7 to 8.7 x 10~9 M, depending on aminoguanine. As a metabolically stable compound in human the concentration of inorganic phosphate (13). However, it is not blood, 8-amino-9-benzylguanine was more effective than 8-ami known whether acyclovir diphosphate can attain an intracellular noguanine at potentiating the toxicity of 2'-deoxyguanosine to concentration sufficient to inhibit PNP during i.v. administration MOLT-4 T-lymphoblasts in culture. 8-Amino-9-benzylguanine is of the parent drug to patients. the most potent base or nucleoside inhibitor of human PNP This laboratory has previously reported the PNP inhibitory reported to date, and it is a promising lead compound in the activity of 8-AGR, an analogue of guanosine, in cultured human development of more effective PNP inhibitors. T-lymphoblasts (9). In addition to its activity as a competitive inhibitor of PNP, 8-AGR is a substrate for this enzyme. In fact, its phosphorolytic cleavage product, 8-AG, has been reported to INTRODUCTION be a more potent inhibitor of PNP (10). On the basis of these The discovery of T-cell-related immunodeficiency diseases findings, 8-AGR is assumed to be a more soluble pro-drug form associated with a lack of ADA4 or PNP in patient erythrocytes of 8-AG. 8-AGR can potentiate the cytotoxic effects of dGuo and permit increased accumulation of the 5'-triphosphate of dGuo in has prompted considerable interest in developing inhibitors of these enzymes as a selective approach to cancer chemotherapy mammalian cells in culture (9), thereby mimicking the PNP- (1,2). In the ADA- and PNP-related immunodeficiency states, 2'- deficiency state. While it has been shown that 8-AG can decrease deoxyadenosine or dGuo accumulates, respectively, and these the rate of dGuo phosphorolysis in human red cells (10), its nucleosides are phosphorylated preferentially by immature T- effects on dGuo cytotoxicity have not been reported. From the cells to their toxic 5'-triphosphate derivatives (3-6). Thus, inhi available data it appears that 8-AGR and 8-AG are not sufficiently bition of ADA or PNP in patients with T-cell lymphoproliferative potent to inhibit the high levels of PNP in human blood (10). diseases may result in the selective destruction of T-cells. The Hence, it would be useful to develop PNP inhibitors which could potent ADA inhibitor 2'-deoxycoformycin was developed with bind tightly to the active site of the enzyme without exhibiting this rationale as a selective antileukemic agent and resulted in substrate activity with PNP. We now report the development of T-lymphoblast lysis in vivo (7); however, use of this drug was a new PNP inhibitor, 8-ABG, which is an example of a novel also associated with severe toxicity (8). Administration of an class of PNP-resistant inhibitors. inhibitor of PNP in vivo may result in less toxicity since the effects on the immune system are more selective in the PNP- compared MATERIALS AND METHODS Received 8/15/85; revised 10/23/85; accepted 10/25/85. Synthesis of 9-Substituted Purine Analogues. Proton ' H-NMR spec The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in tra were obtained using a Brucker Wm 360 spectrometer (360 MHz) or accordance with 18 U.S.C. Section 1734 solely to indicate this fact. IBM WP-270SY NMR spectrometer (270 MHz) and are reported in &- 1This investigation was supported by Public Health Service grants CA-26284 units with tetramethylsilane (Me4Si) as the internal standard (Fig. 1). (P. E. D.) and CA-28381 (L. B. T.) awarded by the National Cancer Institute, Melting points are unconnected and were determined on a Thomas- Department of Health and Human Services, and aided by grant CH-299 (L. B. T.) from the American Cancer Society. Hoover melting point apparatus. IR spectra were recorded on a Hewlett- 2Arthritis Foundation Fellow. Packard UV8450 spectrometer, and elemental analyses were obtained 3 To whom requests for reprints should be addressed, at the Department of from M-H-W Laboratories, P. O. Box 15853, Phoenix, AZ 85018. Thin- Biological Chemistry, University of Michigan, Ann Arbor, Ml 48109. 4 The abbreviations used are: ADA, adenosine deaminase (EC 3.5.4.4); 8-ABG, layer chromatography was performed with Analtech silica gel GHLF thin- 8-amino-9-benzylguanine; 8-ABH, 8-amino-9-benzylhypoxanthine; 8-APEG, 8- layer chromatography plates (250 urn). amino-9-phenethylguanine; 8-APG, 8-amino-9-phenylguanine; 8-AG, 8-aminogua 2-Amino-4-phenylammo-5-[1-(3-methoxycarbonyl)thioureido]py- nine; 8-AGR, 8-aminoguanosine; dGuo, 2'-deoxyguanosine; HPLC, high-pressure rimidin-6-one (2a). A solution of sodium nitrite (6.5 g, 0.09 mol) in water liquid chromatography; ICsi, concentration of drug necessary to inhibit cell growth (20 ml) was added to a mixture of 2-amino-4-phenylaminopyrimidin-6- by 50%; PNP, purine nucleoside phosphorylase (EC 2.4.2.1); s, singlet; br s, broad singlet; d, doublet; t, triplet; q, quartet; m, multiplet; NMR, nuclear magnetic one (1a, 6.3 g, 0.03 mol) (14), glacial acetic acid (5 ml), and water (100 resonance; DMSO, dimethyl sulfoxide; DMF, dimethylformamide. ml) with stirring at room temperature. After 5 h, the yellow solid was CANCER RESEARCH VOL. 46 FEBRUARY 1986 519 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1986 American Association for Cancer Research. 8-ABG INHIBITION OF PNP so tallized from a mixture of methanol and water (v:v, 1:1) to afford 3a (1.13 g, 90%). M.p. 230°C; white color changed to brown, >300°C; 'H-NMR (270 MHz, DMSO-de): ¿11.09(s, 1H, NH, D2O exchangeable); 9.40 (s, 1H, NH, D2O exchangeable); 7.88 (d, 2H, Ar—H); 7.26 (t, 2H, Ar—H); H,N 6.98 (t, 1H, Ar—H); 6.46 (s, 2H, NH2); and 3.71 (s, 3H, CH3). C,3H12N603 (300.278) Calculated: C 52.00, H 4.03, N 27.99 Found: C 52.18, H 4.18, N 28.09 8-Amino-9-phenylguanine (5a). A mixture of methyl 6-amino-4-phen- O ylamino-oxazolo[5,4-d]pyrimidine-2-carbamate (3a, 0.8 g, 2.66 mmol) II NHCOCH, and potassium carbonate (0.74 g, 5.36 mmol) in anhydrous methanol (30 ml) was heated at reflux in an oil bath for 5 h. The mixture was evaporated to dryness in vacuo (oil pump) at 60°C,and the residual solid HN was dissolved in water (30 ml). The resulting solution was heated at reflux for 24 h and cooled to room temperature, and the pH was adjusted to 5 with glacial acetic acid. The solid was collected by filtration and washed with water (50 ml). The crude product was dissolved in 0.1 N sodium hydroxide solution and precipitated with glacial acetic acid to give pure 5a (0.51 g, 79%). M.p. >300°C; UV Xm«nm (t x 104): MeOH:DMF, v:v, 9:1:262 (1.3), 298 (0.7); pH 1:253 (1.4), 288 (1.0); and pH 11:259 (1.3); and 'H-NMR (270 MHz, DMSO-d6): ¿10.39(s, 1H, NH, D20 exchangeable); 7.54-7.36 (m, 5H, Ar—H); 6.14 (s, 2H, NH2, D20 exchangeable); and 5.57 (s, 2H, NH2, D2O exchangeable). C,,H10N6O (242.242) Calculated: C 54.54, H 4.16, N 34.69 Found: C 54.59, H 4.24, N 34.56 8-Amino-9-benzylguanine (5b). A solution of methyl 9-benzylgua- nine-8-carbamate (4b, 0.3 g, 0.95 mmol) (16) and potassium hydroxide -CH2CH,- (0.145 g, 2.5 mmol) in water (15 ml) was heated at reflux for 20 h. The mixture was cooled to room temperature, and the pH was adjusted to 5 with glacial acetic acid. The solid was collected by filtration and washed with water (100 ml). The crude product was reprecipitated with 0.1 N sodium hydroxide and glacial acetic acid to afford pure 5b (0.21 g, 86%).
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