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 ClRB and BTCRB against RCMV was quantified by measurement of plaque formation.

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.

Benzimidazole analogs inhibit human herpesvirus 6.

Several benzimidazole nucleoside analogs, including 1H-β-D-ribofuranosyl-2-bromo-5,6-dichlorobenzimidazole (BDCRB) and 1H-β-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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

Inhibition of human cytomegalovirus replication by benzimidazole nucleosides involves three distinct mechanisms.

The benzimidazole nucleosides 2-bromo-5,6-dichloro-1-(beta-d-ribofuranosyl)benzimidazole (BDCRB) and 2-isopropylamino-5,6-dichloro-1-(beta-l-ribofuranosyl)benzimidazole (1263W94, or maribavir) are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. These inhibitors act by two different mechanisms: BDCRB blocks the processing and maturation of viral DNA, whereas maribavir prevents viral DNA synthesis and capsid nuclear egress. In order to determine by which of these two mechanisms other benzimidazole nucleosides acted, we performed time-of-addition studies and other experiments with selected new analogs.

Facile synthesis of 1-substituted 2-amino-3-cyanopyrroles: new synthetic precursors for 5,6-unsubstituted pyrrolo[2,3-d]pyrimidines.

1-Benzyl-3-cyanopyrrole-2-carbonyl azide (5) underwent a Curtius rearrangement followed by quenching with alcohols to form the corresponding carbamates (6a-c). The carbamates 6a,b were unblocked to give the desired 2-amino-1-benzyl-3-cyanopyrrole (1a). A more facile procedure was subsequently developed for the synthesis of 1-substituted 2-amino-3-cyanopyrroles.

Synthesis and antiviral evaluation of some novel tricyclic pyrazolo[3,4-b]indole nucleosides.

Novel pyrazolo[3,4-b]indole nucleoside analogs were synthesized from the corresponding 3-formyl-2-chloroindole and 3-cyano-2-chloroindole nucleosides by treatment with hydrazine. Very few examples of pyrazolo[3,4-b]indole heterocycles have been published in the literature and this is the first synthesis of nucleoside analogs containing this heterocycle. These new pyrazolo[3,4-b]indole nucleosides were active against human cytomegalovirus and herpes simplex virus type 1, but this activity was not well separated from cytotoxicity.

Rhesus cytomegalovirus is similar to human cytomegalovirus in susceptibility to benzimidazole nucleosides.

Rhesus and human cytomegalovirus (RhCMV and HCMV, respectively) exhibit comparable inhibition by benzimidazole nucleosides, including 2,5,6-trichloro-(1-beta-d-ribofuranosyl)benzimidazole (TCRB), and pyrrolo[2,3-d]pyrimidines. The two HCMV protein targets of TCRB, UL89 and UL56, are highly conserved with their RhCMV homologues. These data indicate that infection of rhesus macaques with RhCMV represents a useful model to test novel anti-HCMV drugs.

Activities of benzimidazole D- and L-ribonucleosides in animal models of cytomegalovirus infections.

Since human cytomegalovirus (HCMV) does not infect or replicate in nonhuman cells and tissues, there are few animal models currently available for evaluation of antiviral therapies for these infections. In the present studies, we utilized two different models in which severe combined immunodeficient (SCID) mice were implanted with human fetal tissue that was subsequently infected with HCMV. In one model, human fetal retinal tissue was implanted into the anterior chamber of the SCID mouse eye, and in the second, human fetal thymus and liver (thy/liv) tissues were implanted under the kidney capsule.