1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.

S-Substituted glutathiones were prepared and investigated as inhibitors of the enzyme, glyoxalase I. A nonpolar region exists on the enzyme and plays an important role in the formation of an enzyme-inhibitor complex. The amount of inhibition of glyoxalase increases as the alkyl chain is lengthened from methyl to n-octyl. Several S-arylglutathiones were also prepared in order to take advantage of this nonpolar region on the enzyme. For example, S-(p-bromobenzyl)glutathione ( 14) gave about 920-fold better binding than the previously reported S-methylglutathione (1).

The anabolism of 1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, a selective inhibitor of human immunodeficiency virus (HIV), was characterized in human T-lymphoblastoid CD4+ CEM cells. 1592U89 was ultimately anabolized to the triphosphate (TP) of the guanine analog (-)-carbovir (CBV), a potent inhibitor of HIV reverse transcriptase. However, less than 2% of intracellular 1592U89 was converted to CBV, an amount insufficient to account for the CBV-TP levels observed. 1592U89 was anabolized to its 5'-monophosphate (MP) by the recently characterized enzyme adenosine phosphotransferase, but neither its diphosphate (DP) nor its TP was detected. The MP, DP, and TP of CBV were found in cells incubated with either 1592U89 or CBV, with CBV-TP being the major phosphorylated species. We confirmed that CBV is phosphorylated by 5'-nucleotidase and that mycophenolic acid increased the formation of CBV-TP from CBV 75-fold. However, mycophenolic acid did not stimulate 1592U89 anabolism to CBV-TP. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not inhibit CBV-TP formation from CBV or 1592U89, whereas the adenylate deaminase inhibitor 2'-deoxycoformycin selectively inhibited 1592U89 anabolism to CBV-TP and reversed the antiviral activity of 1592U89. 1592U89-MP was not a substrate for adenylate deaminase but was a substrate for a distinct cytosolic deaminase that was inhibited by 2'-deoxycoformycin-5'-MP. Thus, 1592U89 is phosphorylated by adenosine phosphotransferase to 1592U89-MP, which is converted by a novel cytosolic enzyme to CBV-MP. CBV-MP is then further phosphorylated to CBV-TP by cellular kinases. This unique activation pathway enables 1592U89 to overcome the pharmacokinetic and toxicological deficiencies of CBV while maintaining potent and selective anti-HIV activity.

We report the first application of pronucleotide (ProTide) technology to the antiviral agent abacavir (Ziagen), used for the treatment of HIV infection. The phenylmethoxyalaninyl phosphoramidate of abacavir was prepared in good yield in one step. Also prepared was the corresponding phosphoramidate of the guanine nucleoside analogue "carbovir". The antiviral profile of each of the parent nucleosides was compared to that of the phosphoramidate ProTides. A significant (28- to 60-fold) increase in anti-HIV potency was noted for the ProTide of abacavir but not for that of carbovir. These findings were in agreement with the markedly higher (ca. 37-fold) levels of carbovir triphosphate that are formed in CEM cells upon response to the abacavir ProTide compared with the parent abacavir compound. In contrast the anti-HBV potency of both abacavir and carbovir were improved (10- and 20-fold, respectively) by ProTide formation. As in CEM cells, the abacavir ProTide provided significantly enhanced carbovir triphosphate levels in HepG2 2.2.15 cells over that of the parent nucleoside. On the basis of these data, a series of phosphoramidate analogues with structural variation in the ester and amino acid regions were prepared and their antiviral profiles described. In addition, the pharmacokinetic disposition of the abacavir phenylethoxyalaninyl phosphoramidate was evaluated in Cynomolgus monkeys.

The selectivity of benzylpyrimidines for bacterial dihydrofolate reductases was studied by using equilibrium and kinetic techniques. Trimethoprim [2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine] and a series of close structural analogues with different methoxy group substitutions on the benzyl showed in vitro Escherichia coli antibacterial activities that varied according to their degree of substitution. Trimethoprim, the most potent analogue tested, was 400-fold more active than benzylpyrimidine, and the monomethoxy and dimethoxy analogues were of intermediate antibacterial activity. The relative antibacterial potencies of all the compounds were directly proportional to their E. coli form 1 dihydrofolate reductase Ki values, as determined by classical enzyme kinetics.

We report the application of phosphoramidate pronucleotide (ProTide) technology to the antiviral agent carbocyclic L-d4A (L-Cd4A). The phenyl methyl alaninyl parent ProTide of L-Cd4A was prepared by Grignard-mediated phosphorochloridate reaction and resulted in a compound with significantly improved anti-HIV (2600-fold) and HBV activity. We describe modifications of the aryl, ester, and amino acid regions of the ProTide and how these changes affect antiviral activity and metabolic stability. Separate and distinct SARs were noted for HIV and HBV. Additionally, ProTides were prepared from the D-nucleoside D-Cd4A and the dideoxy analogues L-CddA and D-CddA. These compounds showed more modest potency improvements over the parent drug. In conclusion, the ProTide approach is highly successful when applied to L-Cd4A with potency improvements in vitro as high as 9000-fold against HIV. With a view to preclinical candidate selection we carried out metabolic stability studies using cynomolgus monkey liver and intestinal S9 fractions.

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