Benzimidazole nucleosides have been shown to be potent inhibitors of human cytomegalovirus (HCMV) replication in vitro. As part of the exploration of structure-activity relationships within this series, we synthesized the 2-isopropylamino derivative (3322W93) of 1H-␤-D-ribofuranoside-2-bromo-5,6-dichlorobenzimidazole (BDCRB) and the biologically unnatural L-sugars corresponding to both compounds. One of the L derivatives, 1H-␤-L-ribofuranoside-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94), showed significant antiviral potency in vitro against both laboratory HCMV strains and clinical HCMV isolates, including those resistant to ganciclovir (GCV), foscarnet, and BDCRB. 1263W94 inhibited viral replication in a dose-dependent manner, with a mean 50% inhibitory concentration (IC 50 ) of 0.12 ؎ 0.01 M compared to a mean IC 50 for GCV of 0.53 ؎ 0.04 M, as measured by a multicycle DNA hybridization assay. In a single replication cycle, 1263W94 treatment reduced viral DNA synthesis, as well as overall virus yield. HCMV mutants resistant to 1263W94 were isolated, establishing that the target of 1263W94 was a viral gene product. The resistance mutation was mapped to the UL97 open reading frame. The pUL97 protein kinase was strongly inhibited by 1263W94, with 50% inhibition occurring at 3 nM. Although HCMV DNA synthesis was inhibited by 1263W94, the inhibition was not mediated by the inhibition of viral DNA polymerase. The parent benzimidazole D-riboside BDCRB inhibits viral DNA maturation and processing, whereas 1263W94 does not. The mechanism of the antiviral effect of L-riboside 1263W94 is thus distinct from those of GCV and of BDCRB. In summary, 1263W94 inhibits viral replication by a novel mechanism that is not yet completely understood.Human cytomegalovirus (HCMV) is a herpesvirus that causes a benign infection in an estimated 40 to 100% of populations in the United States (reviewed by Sia and Patel [23]). In most cases, HCMV infection is not associated with disease; however, in patients with an immature or compromised immune system, HCMV infection can be a serious or even lifethreatening disease. Four drugs-ganciclovir (GCV), its prodrug valganciclovir, cidofovir, and foscarnet-are currently used for the treatment of systemic HCMV infection; however, there are a number of disadvantages associated with each of these therapies. Cidofovir and foscarnet are available only as intravenous formulations, whereas GCV is given intravenously for initial treatment of systemic disease. With all anti-HCMV drugs currently available, there can be serious side effects associated with prolonged treatment. In addition, the drugs have similar mechanisms of action, all ultimately targeting the HCMV polymerase; therefore, selection of crossresistant HCMV mutants can occur. Thus, there is a need for other therapeutic agents that are safe, potent, and orally bioavailable, with a novel mechanism of action.As part of an ongoing program to develop novel anti-HCMV compounds that could potentially yield new therapeutic agents for treatmen...

A new series of 2-substituted 5,6-dichlorobenzimidazole ribonucleosides has been synthesized and tested for activity against two human herpes viruses and for cytotoxicity. 2,5,6-Trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) was prepared by ribosylation of the heterocycle 2,5,6-trichlorobenzimidazole followed by a removal of the protecting groups. The 2-bromo derivative (BDCRB) was made in a similar fashion from 2-bromo-5,6-dichlorobenzimidazole. In contrast, the 2-iodo derivative presented a more difficult problem since the appropriate heterocycle was unavailable. This prompted us to prepare the 2-amino derivative followed by nonaqueous diazotization and removal of the blocking groups. Biological evaluation revealed marked differences in the activities of these compounds and the closely related known compound 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB). DRB was weakly active against both human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1), (IC50's = 42 and 30 microM, respectively) but was cytotoxic to uninfected human foreskin fibroblasts and KB cells in the same dose range. Similar results were obtained with the heterocycle 2,5,6-trichlorobenzimidazole. In marked contrast, the ribonucleoside of 2,5,6-trichlorobenzimidazole (TCRB) was active against HCMV (IC50 = 2.9 microM, plaque assay; IC90 = 1.4 microM, yield assay) but only weakly active against HSV-1 (IC50 = 102 microM, plaque assay). Little to no cytotoxicity was observed in HFF and KB cells at concentrations up to 100 microM. By changing the substituent at the 2-position from chlorine to bromine (BDCRB), a 4-fold increase in activity against HCMV was observed without any significant increase in cytotoxicity. In contrast, the 2-I and 2-NH2 derivatives were only weakly active against HCMV and HSV-1 with activity not well-separated from cytotoxicity. These data establish that for maximum activity against HCMV with separation from cytotoxicity, ribose is preferred at the 1-position and that Cl or Br is apparently preferred at the 2-position. The activity and selectivity of both TCRB and BDCRB were better than that observed with either ganciclovir or foscarnet.

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Seven arabinosyl, 2'-deoxyribosyl, and ribosyl pyrrolo[2,3-dlpyrimidines were evaluated in vitro for activity against human cytomegalovirus and for cytotoxicity in primary and established cell lines of human origin. The parent ribosyl analogs exhibited little antiviral selectivity owing to high cytotoxicity. In contrast, aratubercidin, ara-toyocamycin, ara-sangivamycin, and deoxysangivamycin exhibited selectivity between antiviral effect (measured by plaque or titer reduction or both) and cytotoxicity (measured microscopically and by incorporation of radioactive precursors into DNA, RNA, and protein). The selectivity (in vitro therapeutic indexes) for these four compounds ranged from 2 to 40. The two sangivamycin analogs were the most potent and selective. Ara-sangivamycin, for example, inhibited virus replication 105-fold at a concentration (10 ,uM) which produced only partial inhibition of cell growth and labeled precursor incorporation. The four arabinosyl and deoxyribosyl nucleosides appeared to act by inhibition of viral DNA synthesis as quantitated by DNA-DNA dot blot hybridization. These four analogs also were tested for activity against two strains of type 1 herpes simplex virus by a plaque reduction assay. Unexpectedly, all compounds inhibited herpes simplex virus to a lesser extent than human cytomegalovirus.

Several 2-substituted alpha-D- and alpha-L-lyxofuranosyl and 5-deoxylyxofuranosyl derivatives of 5,6-dicholro-2-(isopropylamino)-1-(beta-L-ribofuranosyl) benzimidazole (1263W94) and 2,5,6-trichloro-1(beta-D-ribofuranosyl)benzimidazole (TCRB) were synthesized and evaluated for activity against two herpesviruses (HSV-1 and HCMV) and for their cytotoxicity against HFF and KB cells. Condensation of 1,2,3,5-tetra-O-acetyl-L-lyxofuranose (2a) with 2,5,6-trichlorobenzimidazole (1) yielded the alpha-nucleoside 3a. The 2-bromo derivative and 2-methylamino derivative were prepared by treatment of 3a with HBr followed by deprotection or from methylamine, respectively. Compound 3a was deprotected and the resultant nucleoside used to prepare the 2-cyclopropylamino and 2-isopropylamino derivatives. The 2-alkylthio nucleosides were prepared by condensing 2a with 5,6-dichlorobenzimidazole-2-thione followed by deprotection. Alkylation of this adduct gave the 2-methylthio and 2-benzylthio derivatives. Condensation of 5-deoxy-1,2,3-tri-O-acetyl-L-lyxofuranosyl, prepared from L-lyxose, with 1 or 2-bromo-5,6-dichlorobenzimidazole (15), followed by deprotection, gave the 2-chloro or 2-bromo-5'-deoxylyxo-furanosyl derivative, respectively. The cyclopropylamino derivative was prepared from the 2-chloro derivative. All D-isomers were prepared in an analogous fashion from D-lyxose. Either compounds were inactive against HSV-1 or weak activity was poorly separated from cytotoxicity. In contrast, the 2-halogen derivatives in both the alpha-lyxose and 5-deoxy-alpha-lyxose series were active against the Towne strain of HCMV. The 5-deoxy alpha-L analogues were the most active, IC50's = 0.2-0.4 microM, plaque assay; IC90's = 0.2-2 microM, yield reduction assay. All of the 2-isopropylamino or 2-cyclopropylamino derivatives were less active (IC50's = 60-100 microM, plaque assay; IC90's = 17-100 microM, yield reduction assay) and were not cytotoxic. The methylamino, thio, and methylthio derivatives were neither active nor cytotoxic. The benzylthio derivatives were weakly active, but this activity was poorly separated from cytotoxicity. The alpha-lyxose L-isomers were more active in a plaque assay against the AD169 strain of HCMV compared to the Towne strain, thereby providing additional evidence of antiviral specificity.