Tetrahalogenated benzimidazole D-ribonucleosides are active against rat cytomegalovirus.

BACKGROUND: Benzimidazole D-ribonucleosides are potent and selective inhibitors of CMV infection that have been shown to target the viral terminase, the enzyme complex responsible for viral DNA cleavage into single unit-length genomes and subsequent DNA packaging into procapsids. Here, we evaluated the viral inhibition by benzimidazole D-ribonucleosides against rat cytomegalovirus (RCMV). METHODS: Antiviral activity of compounds Cl4RB and BTCRB against RCMV was quantified by measurement of plaque formation. Yield assays and electron microscopy of thin sections was performed using RCMV-infected cells in the presence or absence of the compounds. The effects of Cl4RB and BTCRB on cleavage of concatemers was analyzed by pulsed-field gel electrophoresis. To characterize the behaviour of the antiviral compounds in a more physiological environment, a 3D cell culture model was employed where cells are embedded in an extracellular matrix using rat-tail collagen I. RESULTS: Both compounds had an inhibitory effect against RCMV-E. Electron microscopy revealed that only few virions were formed in RCMV-E infected cells in the presence of the compounds. Pulsed-field gel electrophoresis showed that DNA concatemers failed to be processed in the presence of the compounds. Yield Assays showed a comparable viral growth in the 3D vs. 2D cell culture as well as inhibition in the presence of Cl4RB or BTCRB for RCMV-E/GFP. CONCLUSIONS: These results demonstrate that the tetrahalogenated benzimidazole D-ribonucleosides are effective against RCMV-E by preventing cleavage of concatemeric DNA and nuclear egress of mature capsids.

Human cytomegalovirus resistance to deoxyribosylindole nucleosides maps to a transversion mutation in the terminase subunit-encoding gene UL89.

Human cytomegalovirus (HCMV) infection can cause severe illnesses, including encephalopathy and mental retardation, in immunocompromised and immunologically immature patients. Current pharmacotherapies for treating systemic HCMV infections include ganciclovir, cidofovir, and foscarnet. However, long-term administration of these agents can result in serious adverse effects (myelosuppression and/or nephrotoxicity) and the development of viral strains with reduced susceptibility to drugs. The deoxyribosylindole (indole) nucleosides demonstrate a 20-fold greater activity in vitro (the drug concentration at which 50% of the number of plaques was reduced with the presence of drug compared to the number in the absence of drug [EC50] = 0.34 µM) than ganciclovir (EC50 = 7.4 µM) without any observed increase in cytotoxicity. Based on structural similarity to the benzimidazole nucleosides, we hypothesize that the indole nucleosides target the HCMV terminase, an enzyme responsible for packaging viral DNA into capsids and cleaving the DNA into genome-length units. To test this hypothesis, an indole nucleoside-resistant HCMV strain was isolated, the open reading frames of the genes that encode the viral terminase were sequenced, and a G766C mutation in exon 1 of UL89 was identified; this mutation resulted in an E256Q change in the amino acid sequence of the corresponding protein. An HCMV wild-type strain, engineered with this mutation to confirm resistance, demonstrated an 18-fold decrease in susceptibility to the indole nucleosides (EC50 = 3.1 ± 0.7 µM) compared to that of wild-type virus (EC50 = 0.17 ± 0.04 µM). Interestingly, this mutation did not confer resistance to the benzimidazole nucleosides (EC50 for wild-type HCMV = 0.25 ± 0.04 µM, EC50 for HCMV pUL89 E256Q = 0.23 ± 0.04 µM). We conclude, therefore, that the G766C mutation that results in the E256Q substitution is unique for indole nucleoside resistance and distinct from previously discovered substitutions that confer both indole and benzimidazole nucleoside resistance (D344E and A355T).

Benzimidazole analogs inhibit human herpesvirus 6.

Several benzimidazole nucleoside analogs, including 1H-ß-D-ribofuranosyl-2-bromo-5,6-dichlorobenzimidazole (BDCRB) and 1H-ß-L-ribofuranosyl-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir [MBV]), inhibit the replication of human cytomegalovirus. Neither analog inhibited the related betaherpesvirus human herpesvirus 6 (HHV-6). Additional analogs of these compounds were evaluated against both variants of HHV-6, and two L-analogs of BDCRB had good antiviral activity against HHV-6A, as well as more modest inhibition of HHV-6B replication.

Inhibition of human immunodeficiency virus type 1 by triciribine involves the accessory protein nef.

Triciribine (TCN) is a tricyclic nucleoside that inhibits human immunodeficiency virus type 1 (HIV-1) replication by a unique mechanism not involving the inhibition of enzymes directly involved in viral replication. This activity requires the phosphorylation of TCN to its 5' monophosphate by intracellular adenosine kinase. New testing with a panel of HIV and simian immunodeficiency virus isolates, including low-passage-number clinical isolates and selected subgroups of HIV-1, multidrug resistant HIV-1, and HIV-2, has demonstrated that TCN has broad antiretroviral activity. It was active in cell lines chronically infected with HIV-1 in which the provirus was integrated into chromosomal DNA, thereby indicating that TCN inhibits a late process in virus replication. The selection of TCN-resistant HIV-1 isolates resulted in up to a 750-fold increase in the level of resistance to the drug. DNA sequence analysis of highly resistant isolate HIV-1(H10) found five point mutations in the HIV-1 gene nef, resulting in five different amino acid changes. DNA sequencing of the other TCN-resistant isolates identified at least one and up to three of the same mutations observed in isolate HIV-1(H10). Transfer of the mutations from TCN-resistant isolate HIV-1(H10) to wild-type virus and subsequent viral growth experiments with increasing concentrations of TCN demonstrated resistance to the drug. We conclude that TCN is a late-phase inhibitor of HIV-1 replication and that mutations in nef are necessary and sufficient for TCN resistance.

Susceptibilities of human cytomegalovirus clinical isolates and other herpesviruses to new acetylated, tetrahalogenated benzimidazole D-ribonucleosides.

Recently we characterized two inhibitors targeting the human cytomegalovirus (HCMV) terminase, 2-bromo-4,5,6-trichloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) benzimidazole (BTCRB) and 2,4,5,6-tetrachloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) benzimidazole (Cl(4)RB). The terminase consists of the ATP-hydrolyzing subunit pUL56 and the subunit pUL89 required for duplex nicking. Because mammalian cell DNA replication does not involve cleavage of concatemeric DNA by a terminase, these compounds represent attractive alternative HCMV antivirals. We now have tested these previously identified benzimidazole ribonucleosides in order to determine if they are active against HCMV clinical isolates as well as those of herpes simplex virus type 1, mouse cytomegalovirus, rat cytomegalovirus (RCMV), and varicella-zoster virus (VZV). Antiviral activity was quantified by measurement of viral plaque formation (plaque reduction) and by viral growth kinetics. Interestingly, both BTCRB and Cl(4)RB had an inhibitory effect in ganciclovir (GCV)-sensitive and GCV-resistant clinical isolates, with the best effect produced by Cl(4)RB. Electron microscopy revealed that in cells infected with GCV-sensitive or GCV-resistant isolates, B capsids and dense bodies were formed mainly. Furthermore, pulsed-field gel electrophoresis showed that cleavage of concatenated DNA was inhibited in clinical isolates. In addition, the antiviral effect on other herpesviruses was determined. Interestingly, in plaque reduction assays, BTCRB was active against all tested herpesviruses. The best effects were observed on VZV- and RCMV-infected cells. These results demonstrate that the new compounds are highly active against GCV-resistant and GCV-sensitive clinical isolates and slightly active against other herpesviruses.

Toyocamycin specifically inhibits auxin signaling mediated by SCFTIR1 pathway.

The auxins, plant hormones, play a crucial role in many aspects of plant development by regulating cell division, elongation and differentiation. Toyocamycin, a nucleoside-type antibiotic, was identified as auxin signaling inhibitor in a screen of microbial extracts for inhibition of the auxin-inducible reporter gene assay. Toyocamycin specifically inhibited auxin-responsive gene expression, but did not affect other hormone-inducible gene expression. Toyocamycin also blocked auxin-enhanced degradation of the Aux/IAA repressor modulated by the SCF(TIR1) ubiquitin-proteasome pathway without inhibiting proteolytic activity of proteasome. Furthermore, toyocamycin inhibited auxin-induced lateral root formation and epinastic growth of cotyledon in the Arabidopsis thaliana plant. This evidence suggested that toyocamycin would act on the ubiquitination process regulated by SCF(TIR1) machineries. To address the structural requirements for the specific activity of toyocamycin on auxin signaling, the structure-activity relationships of nine toyocamycin-related compounds, including sangivamycin and tubercidin, were investigated.

Design and synthesis of 1-(beta-D-ribofuranosyl)imidazo[4,5-c]pyrazoles as 5:5 bicyclic analogs of purine nucleosides.

5-Alkylaminopyrazole nucleosides underwent nitrosation to give the corresponding N1-ribosylated 5-alkyl-amino-4-nitrosopyrazoles. The intramolecular cyclo-dehydration reactions of these 5-alkylamino-4-nitrosopyrazoles were carried out in pyridine at reflux temperature to afford the ring-closure N-1 ribosylated imidazo[4,5-c]pyrazoles in good yields.

Identification of acetylated, tetrahalogenated benzimidazole D-ribonucleosides with enhanced activity against human cytomegalovirus.

DNA packaging is the key step in viral maturation and involves binding and cleavage of viral DNA containing specific DNA-packaging motifs. This process is mediated by a group of specific enzymes called terminases. We previously demonstrated that the human cytomegalovirus (HCMV) terminase is composed of the large subunit pUL56 and the small subunit pUL89. While the large subunit mediates sequence-specific DNA binding and ATP hydrolysis, pUL89 is required only for duplex nicking. An excellent inhibitor targeting HCMV terminase is 2-bromo-5,6-dichloro-1-(beta-d-ribofuranosyl)benzimidazole (BDCRB), but it was not developed as an antiviral drug due to its metabolic cleavage in experimental animals. We now have tested several new benzimidazole d-ribonucleosides in order to determine whether these compounds represent new, potent inhibitors. Analysis by bioluminometric ATPase activity assays identified two of the new compounds with a high inhibitory effect, 2-bromo-4,5,6-trichloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) benzimidazole (BTCRB) and 2,4,5,6-tetrachloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl benzimidazole (Cl(4)RB). By using viral plaque formation, viral yield, and viral growth kinetics, we demonstrated that the two compounds BTCRB and Cl(4)RB had antiviral activities similar to that of BDCRB. Interestingly, BTCRB retained its inhibitory activity after preincubation with HFF cells. By use of electron microscopy, we observed an increase of B capsids and a lack of cytoplasmic capsids in the presence of the compounds that correlated with the virus yield. Furthermore, cleavage of concatenated DNA was inhibited by both compounds, and inhibition by BTCRB was shown to be dose dependent. These results demonstrate that the new compounds are highly active against HCMV and act by mechanisms similar but not identical to those of BDCRB.

N-methylpurine DNA glycosylase and 8-oxoguanine dna glycosylase metabolize the antiviral nucleoside 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole.

The rapid in vivo degradation of the potent human cytomegalovirus inhibitor 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BDCRB) compared with a structural L-analog, maribavir (5,6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole), has been attributed to selective glycosidic bond cleavage. An enzyme responsible for this selective BDCRB degradation, however, has not been identified. Here, we report the identification of two enzymes, 8-oxoguanine DNA glycosylase (OGG1) and N-methylpurine DNA glycosylase (MPG), that catalyze N-glycosidic bond cleavage of BDCRB and its 2-chloro homolog, 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole, but not maribavir. To our knowledge, this is the first demonstration that free nucleosides are substrates of OGG1 and MPG. To understand how these enzymes might process BDCRB, docking and molecular dynamics simulations were performed with the native human OGG1 crystal coordinates. These studies showed that OGG1 was not able to bind a negative control, guanosine, yet BDCRB and maribavir were stabilized through interactions with various binding site residues, including Phe319, His270, Ser320, and Asn149. Only BDCRB, however, achieved orientations whereby its anomeric carbon, C1', could undergo nucleophilic attack by the putative catalytic residue, Lys249. Thus, in silico observations were in perfect agreement with experimental observations. These findings implicate DNA glycosylases in drug metabolism.

Interaction of the putative human cytomegalovirus portal protein pUL104 with the large terminase subunit pUL56 and its inhibition by benzimidazole-D-ribonucleosides.

Herpesvirus DNA replication leads to unit length genomes that are translocated into preformed procapsids through a unique portal vertex. The translocation is performed by the terminase that cleaves the DNA and powers the insertion by its ATPase activity. Recently, we demonstrated that the putative human cytomegalovirus (HCMV) portal protein, pUL104, also forms high-molecular-weight complexes. Analyses now have been performed to determine the intracellular localization and identification of interaction partners of pUL104. In infected cells, HCMV pUL104 was found to be predominantly localized throughout the nucleus as well as in cytoplasmic clusters at late times of infection. The latter localization was abolished by phosphonoacetic acid, an inhibitor of viral DNA replication. Immunofluorescence revealed that pUL104 colocalized with pUL56, the large subunit of the HCMV terminase. Specific association of in vitro translated pUL104 with the carboxy-terminal half of GST-UL56C was detected. By using coimmunoprecipitations a direct interaction with pUL56 was confirmed. In addition, this interaction was no longer detected when the benzimidazole-D-nucleosides BDCRB or Cl4RB were added, thus indicating that these HCMV inhibitors block the insertion of the DNA into the capsid by preventing a necessary interaction of pUL56 with the portal. Electron microscopy revealed that in the presence of Cl4RB DNA is not packaged into capsids and these capsids failed to egress from the nucleus. Furthermore, pulsed-field gel electrophoresis showed that DNA concatemers synthesized in the presence of the compound failed to be processed.

Design, synthesis and antiviral activity of novel 4,5-disubstituted 7-(beta-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazines and the novel 3-amino-5-methyl-1-(beta-D-ribofuranosyl)- and 3-amino-5-methyl-1-(2-deoxy-beta-D-ribofuranosyl)-1,5-dihydro-1,4,5,6,7,8-hexaazaacenaphthylene as analogues of triciribine.

The synthesis of several heterocyclic analogues of the biologically important nucleoside antibiotic toyocamycin and the tricyclic nucleoside triciribine (TCN) were prepared along with their 2'-deoxy counterparts. Coupling of 2-nitropyrrole-3,4-dicarboxamide (15) under a variety of conditions with alpha-chloro-2-deoxy-3,4-di-O-toluoyl-D-ribofuranose (16a) gave mixtures of the alpha and beta anomers. A coupling of 15 with 1-chloro-2,3,5-tri-O-benzoyl-D-ribofuranose (18) gave exclusively the beta anomer. Individually, the two pyrrole nucleosides were treated with Pd/C, H2 to reduce the nitro groups and cyclized with nitrous acid, and the corresponding 4-position was functionalized as a triazoyl derivative. Nucleophillic displacement was carried out with ammonia to give a mixture of 4-amino-1-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazine-5-carbonitrile (26) and 2-amino-1-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)pyrrole-3,4-dicarbonitrile (27), the latter being formed via a retro-Diels-Alder reaction. The subsequent addition of hydrogen sulfide, water, methanol, hydroxylamine, cyanamide, hydrazine and methylhydrazine to the 5-cyano group was carried out to give the corresponding analogues. In the case of methyl hydrazine, subsequent treatment with NaOMe in methanol gave the title hexaazaacenaphthylenes. Biological evaluation of the compounds established that the pyrrole (17beta, 19-21) and most of the pyrrolotriazine (22, 24, 28, 32-34) nucleosides were inactive or weakly active against human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1). In contrast 29 and 31 were active against one or both of these viruses but activity was poorly separated from cytotoxicity. In contrast, the 2-aza analogue of sangivamycin (30) was active against HCMV and HSV-1 but this apparent activity was most likely due to its high cytotoxicity. The tricyclic nucleoside 12, was active against its target virus, human immunodeficiency virus type 1 (HIV-1), but this activity was not well separated from cytotoxicity.

Amino acid ester prodrugs of 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole enhance metabolic stability in vitro and in vivo.

2-Bromo-5,6-dichloro-1-(beta-d-ribofuranosyl)benzimidazole (BDCRB) is a potent and selective inhibitor of human cytomegalovirus (HCMV), but it lacks clinical utility due to rapid in vivo metabolism. We hypothesized that amino acid ester prodrugs of BDCRB may enhance both in vitro potency and systemic exposure of BDCRB through evasion of BDCRB-metabolizing enzymes. To this end, eight different amino acid prodrugs of BDCRB were tested for N-glycosidic bond stability, ester bond stability, Caco-2 cell uptake, antiviral activity, and cytotoxicity. The prodrugs were resistant to metabolism by BDCRB-metabolizing enzymes, and ester bond cleavage was rate-limiting in metabolite formation from prodrug. Thus, BDCRB metabolism could be controlled by the selection of promoiety. In HCMV plaque-formation assays, l-Asp-BDCRB exhibited 3-fold greater selectivity than BDCRB for inhibition of HCMV replication. This potent and selective antiviral activity in addition to favorable stability profile made l-Asp-BDCRB an excellent candidate for in vivo assessment and pharmacokinetic comparison with BDCRB. In addition to rapid absorption and sufficient prodrug activation after oral administration to mice, l-Asp-BDCRB exhibited a 5-fold greater half-life than BDCRB. Furthermore, the sum of area under the concentration-time profile (AUC)(BDCRB) and AUC(prodrug) after l-Asp-BDCRB administration was roughly 3-fold greater than AUC(BDCRB) after BDCRB administration, suggesting that a reservoir of prodrug was delivered in addition to parent drug. Overall, these findings demonstrate that amino acid prodrugs of BDCRB exhibit evasion of metabolizing enzymes (i.e., bioevasion) in vitro and provide a modular approach for translating this in vitro stability into enhanced in vivo delivery of BDCRB.

Amino acid ester prodrugs of the antiviral agent 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole as potential substrates of hPEPT1 transporter.

Amino acid ester prodrugs of 2-bromo-5,6-dichloro-1-(beta-d-ribofuranosyl)benzimidazole (BDCRB) were synthesized and evaluated for their affinity for hPEPT1, an intestinal oligopeptide transporter. Assays of competitive inhibition of [(3)H]glycylsarcosine (Gly-Sar) uptake in HeLa/hPEPT1 cells by the amino acid ester prodrugs of BDCRB suggested their 2- to 4-fold higher affinity for hPEPT1 compared to BDCRB. Further, promoieties with hydrophobic side chains and l-configuration were preferred by the hPEPT1 transporter.

Design, synthesis, and antiviral evaluation of some polyhalogenated indole C-nucleosides.

2,5, 6-Trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB), 2-bromo-5, 6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BDCRB) and 2-benzylthio-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BTDCRB) are benzimidazole nucleosides that exhibit strong and selective anti-HCMV activity. Polyhalogenated indole C-nucleosides were prepared as 1-deaza analogs of the benzimidazole nucleosides TCRB and BDCRB. A mild Knoevenagel coupling reaction between an indol-2-thione and a ribofuranose derivative was developed for the synthesis of 2-benzylthio-5, 6-dichloro-3-(beta-D-ribofuranosyl)indole (12). 3-(beta-D-ribofuranosyl)-2,5,6-trichloroindole (16) was prepared from 12 in 4 steps. A Lewis acid-mediated glycosylation method was then developed to prepare the targeted 2-haloindole C-nucleoside 16 stereoselectively in four steps from the corresponding 2-haloindole aglycons. Only 12 was active against HCMV but it also was somewhat cytotoxic.

Synthesis and antiviral activity of some 2-substituted 3-formyl- and 3-cyano-5,6-dichloroindole nucleosides.

A series of dichlorinated indole nucleosides has been synthesized and tested for activity against human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1) and for cytotoxicity. The isopropylidene-protected analogs of the previously reported 3-formyl-2,5,6-trichloro-1-(beta-Dribofuranosyl)indole (FTCRI) and 3-cyano-2,5, 6-trichloro-1-(beta-D-ribofuranosyl)indole (CTCRI) were modified by nucleophilic displacement of the 2-chloro substituent using secondary amines. Deprotection of the intermediates provided 2-substituted analogs of FTCRI and CTCRI in good yield. There was a significant difference in reactivity between the isopropylidene-protected and the fully deprotected FTCRI and CTCRI with respect to nucleophilic displacement of the 2-chloro substituent using dialkylamines. This difference in reactivity was not observed with monoalkylamines or with alkoxides, and the corresponding 2-alkylamino- and 2-methoxy substituted analogs were synthesized from FITCRI and CTCRI directly. None of the synthesized analogs demonstrated potent antiviral activity without some corresponding cytotoxicity.

Synthesis of 3-aminoimidazo[4,5-c]pyrazole nucleoside via the N-N bond formation strategy as a [5:5] fused analog of adenosine.

3-Amino-6-(beta-D-ribofuranosyl)imidazo[4,5-c]pyrazole (2) was synthesized via an N-N bond formation strategy by a mononuclear heterocyclic rearrangement (MHR). A series of 5-amino-1-(5-O-tert-butyldimethylsilysilyl-2,3-O-isopropylidene-beta-D-ribofuranosyl)-4-(1,2,4-oxadiazol-3-yl)imidazoles (6a-d), with different substituents at the 5-position of the 1,2,4-oxadiazole, were synthesized from 5-amino-1-(beta-D-ribofuranosyl)imidazole-4-carboxamide (AICA Ribose, 3). It was found that 5-amino-1-(5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-beta-D-ribofuranosyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)imidazole (6a) underwent the MHR with sodium hydride in DMF or DMSO to afford the corresponding 3-acetamidoimidazo[4,5-c]pyrazole nucleoside(s) (7b and/or 7a) in good yields. A direct removal of the acetyl group from 3-acetamidoimidazo[4,5-c]pyrazoles under numerous conditions was unsuccessful. Subsequent protecting group manipulations afforded the desired 3-amino-6-(beta-D-ribofuranosyl)imidazo[4,5-c]pyrazole (2) as a 5:5 fused analog of adenosine (1).

Synthesis and antiviral evaluation of polyhalogenated imidazole nucleosides: dimensional analogues of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole.

A series of polyhalogenated imidazole nucleosides were designed and synthesized as ring-contracted analogues of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) and its 2-bromo analogue (BDCRB) in an effort to explore the spatial limitation of the active pocket(s) in the target protein(s). 2,4,5-Trichloro-, 2-bromo-4,5-dichloro-, and 2,4,5-tribromoimidazole nucleosides were prepared by a condensation of the preformed heterocycles with the appropriate sugar precursors. The ribofuranosyl and xylofuranosyl analogues were prepared by a direct glycosylation using the Vorbruggen's silylation method and provided exclusively the beta-anomers. The arabinofuranosyl analogues were prepared by the sodium salt method to give both the alpha- and beta-anomers. The absolute configurations were established by 1H NMR spectroscopy. Alkylation of the polyhalogenated imidazoles with the appropriate bromomethyl ethers gave the acyclic acyclovir and ganciclovir analogues. In general, the parent polyhalogenated imidazoles showed some activity against human cytomegalovirus (HCMV) (IC50 approximately 35 microM). However, with the exception of two tribromo analogues (7c, 13c-beta), most of their nucleoside derivatives were inactive against both HCMV and herpes simplex virus type-1 (HSV-1) and were not cytotoxic. The results suggest that the ring-contracted nucleoside analogues of TCRB and BDCRB interacted weakly or not at all with viral and cellular targets.

Design, synthesis, and antiviral activity of certain 3-substituted 2,5,6-trichloroindole nucleosides.

A series of trichlorinated indole nucleosides has been synthesized and tested for activity against human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1) and for cytotoxicity. Modifications of the previously reported 2,5,6-trichloro-1-(beta-d-ribofuranosyl)indole at the 3-position of the heterocycle were designed in part to test our hypothesis that hydrogen bonding is required at that position for antiviral activity. Analogues were synthesized using electrophilic addition at the 3-position or by synthesis of modified indole heterocycles followed by glycosylation and modification of the sugar. Among the modifications at the 3-position, only those analogues with hydrogen-bond-accepting character were active against HCMV (e.g., 3-formyl-2,5,6-trichloro-1-(beta-D-ribofuranosyl)indole, FTCRI, IC50 = 0.23 microM). Conversely, analogues with non-hydrogen-bonding substituents at the 3-position (e.g., 3-methyl-2,5,6-trichloro-1-(beta-D-ribofuranosyl)indole) were much less active (IC50 = 32 microM) than those with the requisite hydrogen-bonding capacity. The 5'-O-acyl analogue of FTCRI was obtained as an intermediate and also found to be a potent inhibitor of HCMV (IC50 < 0.1 microM). The synthesis of some additional 5'-O-acylated analogues did not provide a compound with increased antiviral activity. None of the indole nucleosides had significant activity against HSV-1, and none were cytotoxic to uninfected cells in their antiviral dose range. Results obtained from the antiviral evaluations have validated our hypothesis that hydrogen bonding at the 3-position is required for antiviral activity in this series of chlorinated indole nucleosides.

Synthesis and antiviral activity of 3-formyl- and 3-cyano-2,5,6-trichloroindole nucleoside derivatives.

A series of trichlorinated indole nucleosides has been synthesized and tested for activity against human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1) and for cytotoxicity. The previously reported 3-formyl-2,5,6-trichloro-1-(beta-D-ribofuranosyl)indole (FTCRI) and its 3-cyano homologue (CTCRI) were chemically modified at the 3-position. The formation of hydrazones and oximes of FTCRI was accomplished by a dehydrative addition of the appropriate hydrazine or hydroxylamine derivatives, respectively. A carboxamide oxime and imidate were synthesized from CTCRI by the addition of hydroxylamine or methanol, respectively, to the 3-nitrile substituent. Analogues synthesized from FTCRI generally had less antiviral activity than either FTCRI or CTCRI. However, the derivatives of CTCRI were potent and selective inhibitors of HCMV in vitro. The analogue 2,5,6-trichloro-1-(beta-D-ribofuranosyl)indole-3-carboxamide oxime was especially selective (HCMV IC50 = 0.30 microM, CC50 > 100 microM). None of the analogues had significant activity against HSV-1.

Synthesis, antiviral activity, and mode of action of some 3-substituted 2,5,6-trichloroindole 2'- and 5'-deoxyribonucleosides.

A series of chlorinated indole nucleosides has been synthesized and tested for activity against human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1) and for cytotoxicity. The 2'- and 5'-deoxy derivatives of the reported 3-formyl-2,5,6-trichloro-1-(beta-D-ribofuranosyl)indole (FTCRI) and 3-cyano-2,5,6-trichloro-1-(beta-D-ribofuranosyl)indole (CTCRI) were synthesized by either a modification of the appropriate 3-unsubstituted sugar-modified nucleoside analogues or by a glycosylation of 3-substituted heterocycles with a protected alpha-chlorosugar. The modifications were guided in part by structural similarity to the corresponding series of chlorinated benzimidazole ribonucleosides and the fact that 5'-deoxy analogues of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) are very active against HCMV. The 5'-deoxy analogues of FTCRI and CTCRI were nearly as active as FTCRI and CTCRI, suggesting that the chlorinated benzimidazole nucleosides and the chlorinated indole nucleosides act in a similar manner. This hypothesis was supported by time-of-addition studies using FTCRI and by the resistance of TCRB-resistant strains of HCMV to four different 3-substituted indole ribonucleosides. The 2'-deoxy analogues of the trichlorinated indole nucleosides also had potent antiviral activity, in contrast to decreased activity and selectivity observed for 2'-deoxy TCRB compared to TCRB. In addition, 3-acetyl-2,5,6-trichloro-1-(2-deoxy-beta-d-ribofuranosyl)indole was also active and much less cytotoxic (HCMV IC50 = 0.30 microM, HFF CC50 >100 microM) than previous analogues. None of the analogues had significant activity against HSV-1.