Synthesis and antiviral evaluation of halogenated beta-D- and -L-erythrofuranosylbenzimidazoles.

A series of 2-substituted benzimidazole D- and L-erythrofuranosyl nucleosides were synthesized and tested for activity against herpesviruses and for cytotoxicity. The D-nucleosides 2,5, 6-trichloro-1-(beta-D-erythrofuranosyl)benzimidazole (8a) and 2-bromo-5,6-dichloro-1-(beta-D-erythrofuranosyl)benzimidazole (8b) were prepared by coupling 1,2,3-tri-O-acetyl-beta-D-erythrofuranose (D-6) with the appropriate benzimidazole, followed by removal of the acetyl protecting groups. The 2-isopropylamino (9), 2-cyclopropylamino (10), and 2-mercaptobenzyl (11) derivatives were synthesized by nucleophilic displacements of the C-2 chlorine in the benzimidazole moiety of 8a. The D-nucleoside 4-bromo-5, 6-dichloro-2-isopropylamino-1-(beta-D-erythrofuranosyl)benzimid azo le (17) was prepared by coupling D-6 with the appropriate benzimidazole. The L-erythrofuranosyl derivatives, 5, 6-dichloro-2-isopropylamino-1-(beta-L-erythrofuranosyl)benzimid azo le (21a), its 2-cyclopropylamino analogue (21b), and the 2-isopropylamino analogue (25), were prepared by coupling L-6 with the appropriate benzimidazole. Several of these new derivatives had very good activity against HCMV in plaque and yield reduction assays (IC(50) = 0.05-19 microM against the Towne strain of HCMV) and DNA hybridization assays. Very little activity was observed against other herpesviruses. This pattern is similar to the antiviral activity profile observed for the corresponding ribofuranosides 2,5, 6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (4a), its 2-bromo analogue (4b), and the 2-cyclopropylamino analogue (4c). In comparison, 8a was 15-fold more active against HCMV than 4a, and 8b was 4-fold more active against HCMV than 4b. The 5, 6-dichloro-2-isopropylamino-1-(beta-L-erythrofuranosyl)benzimid azo le (21a) was less active than 4c, which is now in clinical trials for HCMV infection. Both 8a,b had comparable HCMV activity to 4c. Mode of action studies with the D-erythrose analogues established that 8b acted by inhibition of viral DNA processing whereas 9 and 10 may act via a different mechanism. The lack of a 5'-hydroxymethyl group in all members of this series established that antiviral activity occurred without 5'-phosphorylation, a feature required for the activity of most nucleoside analogues.

Synthesis and antiviral activity of 2'-deoxy-4'-thio purine nucleosides.

A series of 2'-deoxy-4'-thioribo purine nucleosides was prepared by trans-N-deoxyribosylase-catalyzed reaction of 2'-deoxy-4'-thiouridine with a variety of purine bases. This synthetic procedure is an improvement over methods previously used to prepare purine 4'-thio nucleosides. The compounds were tested against hepatitis B virus (HBV), human cytomegalovirus (HCMV), herpes simplex virus (HSV-1 and HSV-2), varicella zoster virus (VZV), and human immunodeficiency virus (HIV-1). Cytotoxicity was determined in a number of cell lines. Several compounds were extremely potent against HBV and HCMV and had moderate to severe cytotoxicity in vitro. The lead compound from the series, 2-amino-6-(cyclopropylamino)purine 2'-deoxy-4'-thioriboside, was the most potent and selective agent against HCMV and HBV replication in vitro; however, this analogue was nephrotoxic when tested in vivo.

Influence of stereochemistry on antiviral activities and resistance profiles of dideoxycytidine nucleosides.

beta-L-2',3'-Dideoxycytidine (beta-L-ddC) and beta-L-5-fluoro-2',3'-dideoxycytidine (5-F-beta-L-ddC) were prepared and shown to have potent activity against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). These compounds were compared with beta-D-2',3'-dideoxycytidine (beta-D-ddC) and two beta-L-oxathiolane nucleosides (beta-L-3'-thio-2',3'-dideoxycytidine and beta-L-5-fluoro-3'-thio-2',3'-dideoxycytidine) in terms of anti-HIV and anti-HBV activity, cytotoxicity, and development of HIV-1 resistance. Compared with beta-D-ddC, the beta-L-dideoxycytidine nucleosides had similar anti-HIV-1 activities, significantly greater anti-HBV activities, and decreased toxicities to a B-cell line, T-cell lines, and human bone marrow progenitor cells. HIV-1 strains resistant to beta-D-ddC were susceptible to the beta-L-ddC analogs. Compared with the beta-L-oxathiolane nucleosides, beta-L-ddC and 5-F-beta-L-ddC had similar anti-HIV-1 activities, decreased anti-HBV activities, and greater toxicities to B- and T-cell lines and bone marrow progenitor cells. There were similarities between the beta-L-ddC and beta-L-oxathiolane nucleosides in the rate of development and pattern of resistant HIV-1 selection. While the in vitro activity and cytotoxicity profiles of the beta-L-ddC nucleosides differed from those of the beta-D-ddC and beta-L-oxathiolane nucleosides, the data presented herein suggest that the sugar configuration of a dideoxynucleoside analog may play a major role in the rate of development and the pattern of HIV-1 resistance.

Beta-L-thymidine 5'-triphosphate analogs as DNA polymerase substrates.

beta-L-3'-Deoxythymidine 5'-triphosphate (L-ddTTP) and beta-L-3'-deoxy-2',3'-didehydrothymidine 5'-triphosphate (L-d4TTP) were substrates for human immunodeficiency virus reverse transcriptase, Escherichia coli DNA polymerase I (Klenow), and Sequenase (modified T7 DNA polymerase). The beta-D- and beta-L-enantiomers of 5-methyluridine 5'-triphosphate (rTTP) were inhibitors but not substrates of reverse transcriptase. The steady-state Km values for L-ddTTP and L-d4TTP, with all three enzymes, were 12-70-fold larger than the Km values for the corresponding D-enantiomers. The Km value of reverse transcriptase for L-ddTTP was 50-fold larger than that for D-ddTTP because the Kd for L-ddTTP was 5-fold larger than that for D-ddTTP, and the first-order rate constant for incorporation of L-ddTMP into the template-primer was 10% that of the D-enantiomer. The D- and L-enantiomers had kcat values with reverse transcriptase and Sequenase that were similar to kcat for the natural substrate, thymidine 5'-triphosphate (dTTP). Thus, the rate determining step appeared to be dissociation of the enzyme-chain-terminated template-primer complex. In contrast, kcat values for the L-enantiomers with Klenow were only 0.1% that of dTTP, and the kcat values for the D-enantiomers were 15% the kcat for dTTP. The reduced kcat values were due to a change in rate determining step from dissociation of the Klenow-chain-terminated template-primer complex to an earlier step in the reaction mechanism, presumably catalysis. Thus, these DNA polymerases did not stereospecifically recognize D-nucleoside 5'-triphosphate analogs as substrates.