Purine nucleosides HxR or GdR (2.5 micrograms/mL blood) were added to EDTA-treated cynomolgus monkey whole blood in vitro, alone or with the PNP inhibitor CI-1000 (1 microgram/mL), mixed, and the concentration of nucleosides remaining in plasma followed as a function of time. The half-lives of GdR and HxR in control blood were 1.2 and < 1 min, respectively, and were extended to 17.8 and 39.8 min, respectively, by coaddition of CI-1000. In contrast, a structural analog of CI-1000, CI-972, when tested in parallel at 1 microgram/mL, had markedly less effect on the breakdown of either nucleoside. The ability of CI-1000 to retard nucleoside breakdown in blood in vitro may be a predictor of in vivo activity, and can be viewed as an early and essential biochemical consequence of PNP inhibition culminating in immunosuppression.

An in-parallel comparison is presented of the in vitro and in vivo properties of two 9-deazaguanine analog inhibitors of purine nucleoside phosphorylase (PNP), CI-972 [8-amino-9-deaza-9-(3-thienylmethyl)guanine] and PD 141955 [9-deaza-9-(3-thienylmethyl)guanine] (published Ki values of 0.83–8.0 and 0.08 μM, respectively). Despite structural similarities, PD 141955 was considerably more potent and active in all systems studied. The respective ic50 values for inhibition of MOLT-4 cell growth in the absence and presence of 10 μM 2'-deoxyguanosine (GdR) were > 50 and 5.06 μM for CI-972 and 15.4 and 0.061 μM for PD 141955. PD 141955 induced accumulation of dGTP in GdR-treated MOLT-4 and CEM cells at log-lower concentrations than were required of CI-972, and the magnitude of dGTP accumulation in PD 141955-treated T cell cultures was markedly greater (e.g. 366 vs 100 pmol106 CEM cells at 10 μM). PD 141955 administered orally produced a dose-dependent elevation of plasma inosine and guanosine in rats over a broad concentration range. Mean plasma inosine concentrations following a 150 mgkg p.o. dose peaked at 6.21 and 13.2 μM in CI-972 and PD 141955-treated rats, respectively. Low levels of inosine were detectable at 50 μgkg following oral administration of PD 141955.

Purine nucleoside phosphorylase (PNP) is a purine-metabolizing enzyme in the purine cascade and has been a target for drug design for sometime. A series of potent human PNP inhibitors, pyrrolo[3,2-d]pyrimidines (9-deazaguanines), has been synthesized and evaluated in the enzyme assay and in the cell line assay using MOLT-4 (T-cell) and MGL-8 (B-cell) lymphoblasts for selectivity. One of the compounds, 2,6-diamino-3,5-dihydro-7-(3-thienylmethyl)-4H-pyrrolo [3,2-d]pyrimidin-4-one (11c; CI-972), was found to be moderately potent, competitive, and reversible inhibitor of PNP with K(i) = 0.83-mu-M. It was also found to be selectively cytotoxic to MOLT-4 lymphoblasts (IC50 = 3.0-mu-M) but not to MGL-8 lymphoblasts and was evaluated further. Compound 11c (CI-972) is under development in the clinic.

PD 116124 (8-amino-2'-nordeoxyguanosine; 2,8-diamino-1,9-dihydro-9- ([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)-6H-purin-6-one) is a competitive, reversible inhibitor of human purine nucleoside phosphorylase with an apparent inhibition constant of 0.41 microM. In a cell line system using human MOLT-4 and CEM T lymphoblasts and human MGL-8 and NC-37 B lymphoblasts, PD 116124 failed to inhibit [3H]thymidine uptake at concentrations up to 500 microM. However, in the presence of 10 microM 2'-deoxyguanosine (dGuo), a noninhibitory dGuo concentration by itself, PD 116124 produced potent inhibition of growth of both T cell lines but not of either B cell line. Significant elevation of intracellular 2'-deoxyguanosine triphosphate was observed in both inhibited T cell lines but not in either B cell line. Greater and more sustained accumulation of 2'-deoxyguanosine triphosphate was observed in T lymphoblasts cultured with PD 116124 plus dGuo than with dGuo only. PD 116124 was only weakly inhibitory in human mixed lymphocyte cultures (IC50 approximately equal to 1420 microM), but in the presence of 10 microM dGuo, the IC50 for PD 116124 was reduced to 108.7 microM. Administration of PD 116124 p.o. to normal male Wistar rats caused dose-dependent elevation of plasma inosine up through 500 mg/kg. Maximal inosine elevation occurred at 30 min after dosing, and elevation was significant even 24 hr after dosing. Guanosine was also elevated, although not in a dose-dependent manner. Administration of PD 116124 i.v. produced marked and statistically significant elevation of both inosine and guanosine.(ABSTRACT TRUNCATED AT 250 WORDS)

A series of 3-substituted hypoxanthines (6-10, 14-17) and related analogues (22, 23) have been synthesized as inhibitors of purine nucleoside phosphorylase (PNP), which may conceivably act as T-cell-selective immunosuppressive agents with potential utility in autoimmune disorders such as rheumatoid arthritis, in organ transplantations, and in T-cell leukemias. The compounds were evaluated for their PNP activity by a radiochemical assay and also for their cytotoxic effects on a T-lymphoblastoid cell line (MOLT-4). Appropriate substitutions on 3-benzylhypoxanthine (7a) (IC50 in PNP assay, 112 microM; IC50 in MOLT-4 assay, 204.2 microM) increase potency: 8-amino (17a; 42.6, 65.2), 2-hydroxy (9a; 13.4, 28.6), 2-amino (10a; 11.4, 29.1), and 2,8-diamino (16a; 5.0, 11.9). Variation of the 3-aryl substituents of 16a as in 16b-d has thus far failed to further increase potency. Replacement of the 6-oxygen function in 7a with the analoguous nitrogen or sulfur functions, as in 22a and 23a, resulted in little change in activity. Other variations including the increase of the 3-aliphatic chain length as in 6h and 7h (n = 2), the substitution of the phenyl ring with electron-withdrawing groups as in 7e-g, and replacement of the 2-hydrogen with methylthio as in 8a and 14a resulted in decrease of activity. The values for 16a-d represent moderate but significant activities, as compared to the most active inhibitor presently known, 8-amino-9-thienylguanine (1c; 0.17, 0.82). 2,8-Diamino-3-substituted hypoxanthines (16a-d) represent a novel structural type hitherto unreported in the literature, and efficient methodologies for their synthesis were developed in the present studies. The formation of the aminoimidazole moiety occurred through a base-catalyzed 1,5-(O----N)-carbamimidoyl rearrangement (13 to 14, 20 to 16).

CI-972 (2,6-diamino-3,5-dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3,2- d]pyrimidin-4-one monohydrochloride, monohydrate) is a competitive inhibitor of PNPase (E.C. 2.4.2.1., Ki = 0.83 microM) entering clinical trials as a T cell-selective immunosuppressive agent. Neither CI-972 (less than or equal to 50 microM) nor dGuo (less than or equal to 10 microM) inhibited [3H]Thd uptake by human MOLT-4 (T cell) or MGL-8 (B cell) lymphoblasts, but in the presence of 10 microM dGuo, the IC50 for CI-972 decreased to 3.0 microM for MOLT-4 but remained at greater than 50 microM for MGL-8. Inhibition of MOLT-4 growth was associated with an increase in dGTP that was dependent on CI-972 concentration and inhibited by 2'-deoxycytidine. Growth could not be restored by hypoxanthine or adenine. No alterations in GTP pools were noted in MOLT-4, and neither GTP nor dGTP were altered in MGL-8.

CI-959 (5-methoxy-3-(1-methylethoxy)-N-1H-tetrazol-5-yl-benzo[b]-thiophene-2-carboxamide), an antiallergy compound, blocked release of IL-2 from Con A stimulated rat splenocytes and human lymphocytes with respective IC50s of 19.1 and 23.1 μM. Inhibition of IL-2 production required the presence of CI-959 in culture medium for the first 9 hr. CI-959 also inhibited Con A-stimulated rat and human lymphocyte proliferation with IC50s of 4.7 and 5.4 μM, respectively. Inhibition of the Con A proliferative response could not be overcome by exogenous recombinant human IL-2 (300 units/ml) in either the rat or human systems. Although potent in the human mixed lymphocyte reaction (IC50=3.5 μM), CI-959 was less effective in blocking the PHA response (IC50=43.9 μM), and had minimal effect on the release of IL-1 and TNFα from LPS-stimulated human monocytes. These findings suggest that CI-959 selectively inhibits some lymphocyte functions, as opposed to monocyte functions, and that among these is the production of IL-2.

CI-972 (2,6-diamino-3,5-dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3, 2-d]pyrimidin-4-one monohydrochloride, monohydrate) is a novel inhibitor of PNP (Ki = 0.83 microM) under development as a T cell-selective immunosuppressive agent. CI-972 inhibited proliferation (3H-thymidine uptake) of human MOLT-4 (T cell) but not MGL-8 (B cell) lymphoblasts with respective IC50s of 3.0 and greater than 50 microM when tested with 10 microM 2'-deoxyguanosine. Without addition of exogenous 2'-deoxyguanosine, CI-972 was not inhibitory to any human T or B lymphoblastoid cell line tested. 2'-Deoxycytidine (10 microM), but not hypoxanthine or adenine, restored MOLT-4 cell growth. Inhibition of 3H-thymidine uptake in MOLT-4 cells correlated with accumulation of dGTP, while alterations in guanine nucleotides were not observed. 2'-Deoxycytidine (10 microM) also blocked dGTP accumulation in MOLT-4 cells. CI-972 showed activity in vivo over a broad dose range: At 5-150 mg/kg p.o., CI-972 produced dose-dependent elevation of plasma inosine one hr after administration to rats (mean maximum of 2.62 vs. 0.06 microM in controls). Guanosine was also significantly elevated in a concentration-dependent manner, although the effect was not as impressive. Plasma nucleosides remained statistically-significantly elevated for up to four hr following a single oral dose of CI-972.

Previously, we have described the synthesis and biological activity of 2,8-diamino-1,9-dihydro-9-(2-thienylmethyl)-6H-purin-6-one (PD 119229; Cl-950) as a potent and competitive PNP inhibitor. As a part of our continuing efforts to develop a PNP inhibitor for autoimmune diseases, we have synthesized a series of pyrrolo[3,2-d]pyrimidines as PNP inhibitors. In this series, 2,6-diamino-3,5-dihydro-7-(3- thienylmethyl)-4H-pyrrolo-[3,2-d]pyrimidin-4-one (Cl-972) was found to be a potent, competitive inhibitor of PNP with Ki of 0.83 microM. It was also found to be selectively cytotoxic to human MOLT-4 (T cell) (IC50 = 3.0 microM) but non-toxic to MGL-8 (B cell) lymphoblasts. Cl-972 is under development as a potential T-cell selective immunosuppressive agent. Synthesis and biological activities of the series are discussed.

CI-949 (5-methoxy-3-(1-methylethoxy)-1-phenyl-N-1H-tetrazol-5-yl-1H-indole-2-carboxamide,l-arginine salt), an antiallergy compound was found to be a weak inhibitor of IL-1 release from LPS-stimulated murine peritoneal exudate cells and human peripheral blood leukocytes, with IC50s of 186.2 and 267.9 μM, respectively. CI-949 was also a poor inhibitor of release of IL-2 from Con A-stimulated rat spelenocytes (37% inhibition at 100 μM). CI-949 did produce concentration-related inhibition of the response of human lymphocytes to PHA and Con A (IC50s=44.7 and 21.5 μM, respectively) as well as in the mixed lymphocyte reaction (MLR) (IC50=16.8 μM). The clinical significance of these latter findings is unknown at present.

A series of 8-amino-9-substituted guanines was synthesized and their activity evaluated against human purine nucleoside phosphorylase (PNP). All compounds were found to be potent inhibitors of human PNP (IC50s: 0.17–126 μM). They were also selectively cytotoxic to MOLT-4 lymphoblasts in the presence of a nontoxic amount (10 μM) of the PNP substrate, 2′-deoxyguanosine (GdR). The most potent of these analogs, 2,8-diamino-1,9-dihydro-9-(2-thienylmethyl)-6H-purin-6-one (8-amino-9-(2-thienyl-methyl)guanine; PD 119,229) has an IC50 of 0.17 μM (Ki=0.067 μM), significantly more potent than the known standard, 8-aminoguanosine (IC50=1.40 μM). Thus it represents the most potent PNP inhibitor known to date when tested without limiting the concentration of inorganic phosphate.

PD 119,229 [8-amino-9-(2-thienylmethyl)guanine] is a novel and potent inhibitor of human erythrocyte purine nucleoside phosphorylase (PNP) with a Ki of 0.067 μM. In a cell line assay using human MOLT-4 (T cell) and MGL-8 (B cell) lymphoblasts, PD 119,229 alone had no effect on the growth of either cell line at the highest concentration tested (100 μM). However, in the presence of a nontoxic concentration of 2′-deoxyguanosine (10 μM), the IC50 values of PD 119,229 for MOLT-4 and MGL-8 were 0.9 and >100 μM, respectively. The inhibition of growth of MOLT-4 was accompanied by a 40-fold increase in dGTP and a two-fold reduction in GTP, while no alteration in nucleotide prolife was noted in MGL-8. Both the inhibition of growth of MOLT-4 and the accumulation of dGTP were substantially prevented by coaddition of 2′-deoxycytidine.

8-Amino-9-(2-thienylmethyl)guanine (PD 119,229; 2,8-diamino-1,9-dihydro-9-(2-thienylmethyl)-6H-purine-6-one monohydrochloride) is a potent inhibitor of human purine nucleoside phosphorylase (PNP). The effects of orally administered PD 119,229 on the plasma concentration of the PNP substrates, inosine and guanosine, were determined using normal rats. In time course studies following administration of a single 3 mg/kg dose of PD 119,229, both inosine and guanosine were statistically significantly elevated as soon as one hr postdose. Plasma inosine elevation was maximal ten hr after dosing, reaching a mean of 2.13 μM (14.2-fold vehicle). Guanosine was maximally elevated at three hr following a single 3 mg/kg oral dose, reaching a mean of 0.77 μM (4.5-fold vehicle). In dose-response studies in which blood specimens were obtained one hr following oral administration of PD 119,229 at doses of 1.5 to 50 mg/kg, maximal mean inosine elevation (1.71 μM or 57-fold vehicle) occurred at 15 mg/kg, with a plateauing or decline in inosine concentration noted at higher doses. The maximal mean plasma guanosine concentration was achieved at 50 mg/kg (mean of 0.2 μM, or 6.7-fold vehicle). Substantially greater nucleoside elevation was not observed following multiple oral 15 mg/kg doses, nor when nucleoside levels were assessed at two to four hr following a large oral dose. However, inosine levels reaching 15 μM, and guanosine concentrations approaching 2 μM, were occasionally noted in individual rats. It is concluded that oral administration of PD 119,229 can simulate, in normal rats, one of the more readily detectable biochemical abnormalities of the PNP deficient state in humans.

The sodium salt of (I) reacts with cyanogen bromide, yielding the thiazolopyrimidine (II).

8-Aminoguanine is a potent inhibitor of purine nucleoside phosphorylase (PNP) and also a substrate of PNP. Two thio isosteres of 8-aminoguanine, 2,5-diaminothiazolo[5,4-d]pyrimidin-7(6H)-one (2) and 2,4-diaminothiazolo[4,5-d]pyrimidin-7(6H)-one (3), which cannot be substrates of PNP, were synthesized and evaluated for their inhibitory activity against PNP. They were found to be weak inhibitors of PNP and to be noncytotoxic for MOLT-4 T-cells in culture.

Annals of the New York Academy of SciencesVolume 451, Issue 1 p. 313-314 Effects of 8-Aminoguanosine, an Inhibitor of Purine Nucleoside Phosphorylase, on Plasma Nucleosides in Wistar Rats RICHARD B. GILBERTSEN, RICHARD B. GILBERTSEN Warner-Lambert/Parke-Davis Pharmaceutical Research Ann Arbor, Michigan 48105Search for more papers by this authorMI K. DONG, MI K. DONG Warner-Lambert/Parke-Davis Pharmaceutical Research Ann Arbor, Michigan 48105Search for more papers by this author RICHARD B. GILBERTSEN, RICHARD B. GILBERTSEN Warner-Lambert/Parke-Davis Pharmaceutical Research Ann Arbor, Michigan 48105Search for more papers by this authorMI K. DONG, MI K. DONG Warner-Lambert/Parke-Davis Pharmaceutical Research Ann Arbor, Michigan 48105Search for more papers by this author First published: October 1985 https://doi.org/10.1111/j.1749-6632.1985.tb27125.xCitations: 8AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References 1 Cohen, A., D. Doyle, D. W. Martin, Jr & A. J. Ammann. 1976. N. Engl. J. Med. 295: 1449. 2 Stoop, J. W., B. J. M. Zegers, G. F. M. Hendrickx, L. H. S. van Heukelom, G. E. J. Staal, P. K. de Bree, S. K. Wadman & R. E. Ballieux. 1977. N. Engl. J. Med. 296: 651. 3 Edwards, N. L., E. W. Gelfand, D. Biggar & I. H. Fox. 1978. J. Lab. Clin. Med. 91: 736. 4 Hartwick, R. A. & P. R. Brown. 1977. J. Chromatogr. 143: 383. Citing Literature Volume451, Issue1Adenosine Deaminase in Disorders of Purine Metabolism and in Immune DeficiencyOctober 1985Pages 313-314 ReferencesRelatedInformation