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.     

Studies Designed to Increase the Stability and Antiviral Activity (HCMV) of the Active Benzimidazole Nucleoside,TCRB

Abstract

The potent activity of 2,5,6-trichloro-1-(ß-D-ribofuranosyl)benzimidazole (TCRB) against Human Cytomegalovirus with the concomitant low cellular toxicity at concentrations that inhibit viral growth prompted considerable interest in this research area. This interest was moderated by the pharmacokinetic studies of TCRB in rats and monkeys that revealed the instability of TCRB in vivo. These studies suggested that the instability was due to a cleavage of the glycosidic bond in vivo which released the heterocycle (2,5,6-trichlorobenzimidazole) into the bloodstream. This prompted us to initiate synthetic studies designed to increase the stability of the glycosidic bond of TCRB and BDCRB. Several synthetic approaches to address this and other problems are presented.     

Design,Synthesis, and Antiviral Evaluation of 2-Deoxy-D-Ribosides of Substituted Benzimidazoles as Potential Agents for Human Cytomegalovirus Infections

Abstract

Stereoselective glycosylation of 2,5,6-trichlorobenzimidazole (1b), 2-bromo-5,6-dichlorobenzimidazole (1c), 5,6-dichlorobenzimidazole (1d), 5,6-dichlorobenzimidazole-2-thione (1e), 5,6-dichloro-2-(methylthio)benzimidazole (1f), 2-(benzylthio)-5,6-dichlorobenzimidazole (1g), and 2-chloro-5,6-dimethylbenzimidazole (1h) with 2-deoxy-3,5-di-O-p-toluoyl-α-D-erythro-pentofuranosyl chloride was achieved to give the desired β nucleosides 2b-h. Subsequent deprotection afforded the corresponding free β-D-2-deoxyribosides 3b-h. The 2-methoxy derivative 3i was synthesized by the treatment of 2b with methanolic sodium methoxide. Displacement of the 2-chloro group of 2b with lithium azide followed by a removal of the protective groups gave the 2-azido-5,6-dichlorobenzimidazole derivative (5). The 2-amino derivative (6) was obtained by hydrogenolysis of 5 over Raney nickel. 5,6-Dichloro-2-isopropylamino-1-(2-deoxy-β-D-erythro-pentofuranosyl)benzimidazole (10) was prepared using 2′-deoxyuridine (7), N-deoxyribofuranosyl transferase and 1d followed by functionalization of the C2 position. Antiviral evaluation of target compounds established that compounds 3b and 3c were active against human cytomegalovirus (HCMV) at non-cytotoxic concentrations. The activity of these 2-deoxy ribosides, however, was less than the activity of the parent riboside, 2,5,6-trichloro-1-β-D-ribofuranosylbenzimidazole (TCRB). Compared to TCRB, 3b and 3c were somewhat more cytotoxic and active against herpes simplex virus type 1. Compounds 3d-i with other substituents in the 2-position were inactive against both viruses and non-cytotoxic. In contrast, compounds with amine substituents in the 2-position (5, 6, 10) were active against HCMV albeit less so than TCRB. These results establish that 2-deoxy-D-ribosyl benzimidazoles are less active against the DNA virus HCMV than are the corresponding D-ribosides.     

Design, synthesis, and antiviral evaluation of 2-deoxy-D-ribosides of substituted benzimidazoles as potential agents for human cytomegalovirus infections

Stereoselective glycosylation of 2,5,6-trichlorobenzimidazole (1b), 2-bromo-5,6-dichlorobenzimidazole (1c), 5,6-dichlorobenzimidazole (1d), 5,6-dichlorobenzimidazole-2-thione (1e), 5,6-dichloro-2-(methylthio)benzimidazole (1f), 2-(benzylthio)-5,6-dichlorobenzimidazole (1g), and 2-chloro-5,6-dimethylbenzimidazole (1h) with 2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranosyl chloride was achieved to give the desired beta nucleosides 2b-h. Subsequent deprotection afforded the corresponding free beta-D-2-deoxyribosides 3b-h. The 2-methoxy derivative 3i was synthesized by the treatment of 2b with methanolic sodium methoxide. Displacement of the 2-chloro group of 2b with lithium azide followed by a removal of the protective groups gave the 2-azido-5,6-dichlorobenzimidazole derivative (5). The 2-amino derivative (6) was obtained by hydrogenolysis of 5 over Raney nickel. 5,6-Dichloro-2-isopropylamino-1-(2-deoxy-beta-D-erythro- pentofuranosyl)benzimidazole (10) was prepared using 2'-deoxyuridine (7), N-deoxyribofuranosyl transferase and 1d followed by functionalization of the C2 position. Antiviral evaluation of target compounds established that compounds 3b and 3c were active against human cytomegalovirus (HCMV) at non-cytotoxic concentrations. The activity of these 2-deoxy ribosides, however, was less than the activity of the parent riboside, 2,5,6-trichloro-1-beta-D-ribofuranosylbenzimidazole (TCRB). Compared to TCRB, 3b and 3c were somewhat more cytotoxic and active against herpes simplex virus type 1. Compounds 3d-i with other substituents in the 2-position were inactive against both viruses and non-cytotoxic. In contrast, compounds with amine substituents in the 2-position (5, 6, 10) were active against HCMV albeit less so than TCRB. These results establish that 2-deoxy-D-ribosyl benzimidazoles are less active against the DNA virus HCMV than are the corresponding D-ribosides.     

Benzimidazole 2′-Isonucleosides: Design,Synthesis, and Antiviral Activity of 2-Substituted-5,6-Dichlorobenzimidazole 2′-Isonucleosides

Abstract

2,5,6-Trihalogenated benzimidazole-β-D-ribofuranosyl nucleosides and 2-substituted amino-5,6-dichlorobenzimidazole-β-L-ribofuranosyl nucleosides are potent and selective inhibitors of human cytomegalovirus (HCMV). The D-ribofuranosyl analogs are metabolized rapidly in vivo rendering them unsuitable as drug candidates. The primary source of instability is thought to be the anomeric bond. The synthesis of a series of chemically stable benzimidazole-2′-isonucleosides is presented. The synthetic schemes employed are based on nucleophilic displacements of a 2′-tosylate from carbohydrate intermediates with 2-bromo-5,6-dichlorobenzidazole. 2-Bromo and 2-isopropyl amino analogs with 3′- and 5′-oxo and deoxy substitutions were prepared. The benzimidazole-2′-isonucleosides presented here demonstrated reduced activity against HCMV when compared to other D-ribofuranosyl benzimidazole analogs. In addition, they were not found to be inhibitors of HIV.     

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-(β-D-ribofuranosyl)indole (FTCRI) and 3-cyano-2,5,6-trichloro-1-(β-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 FTCRI and CTCRI directly. None of the synthesized analogs demonstrated potent antiviral activity without some corresponding cytotoxicity.     

Studies designed to increase the stability and antiviral activity (HCMV) of the active benzimidazole nucleoside, TCRB.

The potent activity of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) against Human Cytomegalovirus with the concomitant low cellular toxicity at concentrations that inhibit viral growth prompted considerable interest in this research area. This interest was moderated by the pharmacokinetic studies of TCRB in rats and monkeys that revealed the instability of TCRB in vivo. These studies suggested that the instability was due to a cleavage of the glycosidic bond in vivo which released the heterocycle (2,5,6-trichlorobenzimidazole) into the bloodstream. This prompted us to initiate synthetic studies designed to increase the stability of the glycosidic bond of TCRB and BDCRB. Several synthetic approaches to address this and other problems are presented.     

Dramatic effects of 2-bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole riboside on the genome structure, packaging, and egress of guinea pig cytomegalovirus

The halogenated benzimidazoles BDCRB (2-bromo-5,6-dichloro-1-beta-D-riborfuranosyl benzimidazole riboside) and TCRB (2,5,6-trichloro-1-beta-D-riborfuranosyl benzimidazole riboside) were the first compounds shown to inhibit cleavage and packaging of herpesvirus genomes. Both inhibit the formation of unit length human cytomegalovirus (HCMV) genomes by a poorly understood mechanism (M. R. Underwood et al., J. Virol. 72:717-715, 1998; P. M. Krosky et al., J. Virol. 72:4721-4728, 1998). Because the simple genome structure of guinea pig cytomegalovirus (GPCMV) provides a useful model for the study of herpesvirus DNA packaging, we investigated the effects of BDCRB on GPCMV. GPCMV proved to be sensitive to BDCRB (50% inhibitory concentration = 4.7 microM), although somewhat less so than HCMV. In striking contrast to HCMV, however, a dose of BDCRB sufficient to reduce GPCMV titers by 3 logs (50 microM) had no effect on the quantity of GPCMV genomic DNA that was formed in infected cells. Electron microscopy revealed that this DNA was in fact packaged within intranuclear capsids, but these capsids failed to egress from the nucleus and failed to protect the DNA from nuclease digestion. The terminal structure of genomes formed in the presence of BDCRB was also altered. Genomes with ends lacking a terminal repeat at the right end, which normally exist in an equimolar ratio with those having one copy of the repeat at the right end, were selectively eliminated by BDCRB treatment. At the left end, BDCRB treatment appeared to induce heterogeneous truncations such that 2.7 to 4.9 kb of left-end-terminal sequences were missing. These findings suggest that BDCRB induces premature cleavage events that result in truncated genomes packaged within capsids that are permeable to nuclease. Based on these and other observations, we propose a model for duplication of herpesvirus terminal repeats during the cleavage and packaging process that is similar to one proposed for bacteriophage T7 (Y. B. Chung, C. Nardone, and D. C. Hinkle, J. Mol. Biol. 216:939-948, 1990).     

Synthesis of Pyrrolo[2,3-d]pyrimidines that are Structurally Related to Methylated Guanosines from tRNA and the Nucleoside Q Analogs,PreQ0 and PreQ1

Abstract

The N-3- and N-2-methylated analogs of several 5-substituted 2 amino-7-(β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidin-4-ones were synthesized from 5-cyano-2,4-dichloro-7-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (10). These compounds are analogs of nucleoside Q that are methylated in a manner similar to some of the naturally occurring methylated guanosines.

     

Nucleosides and Nucleotides. 98. Synthesis and Antitumor Activities of 5-Ethynylimidazole-4-carboxamide and -carbonitrile Derivatives

Abstract

5-Ethynyl-1-(2-deoxy-β-D-ribofuranosyl)imidazole-4-carbonitrile (4) and -carboxamide (5) and 5-ethynyl-1-(5-deoxy-β-D-ribofuranosyl)imidazole-4-carbonitrile (11) and -carboxamide (12) have been synthesized from the corresponding 5-iodo derivatives 2 and 7 by a palladium-catalyzed cross-coupling reaction with (tri-methylsilyl)acetylene. The aglycons, 5-ethynylimidazole derivatives 14 and 15 were synthesized by the hydrolytic cleavage of the corresponding nucleosides. The antileukemic activity of these nucleosides and base analogues are also described.     

New Pyrimidine Cyclonucleosides with Hydrogenated Aglycones: Synthesis and X-Ray Structures

Abstract

Acid catalysed transformations of (6S)-6,5′-anhydro-6-hydroxy-1-(2′,3′-O-isopropylidene-β-D-ribofuranosyl)hexahydropyrimidine-2-thione are studied. (6R)-6,2′-anhydro-6-hydroxy-1-(α-D-ribofuranosyl)hexahydropyrimidine-2-thione was formed as a thermodynamically stable product. Two intermediates, (6S)-6,5′-anhydro-6-hydroxy-1-(β-D-ribofuranosyl)hexahydropyrimidine-2-thione and 6-hydroxy-1-(D-ribosyl)hexahydropyrimidine-2-thione and products of cleavage of glycosidic bond were identified in the reaction mixtures. Results of X-ray structural determination of the synthesised nucleosides are presented.     

Resistance of human cytomegalovirus to the benzimidazole L-ribonucleoside maribavir maps to UL27

1-(beta-D-Ribofuranosyl)-2,5,6-trichlorobenzimidazole (TCRB) and its 2-bromo analog, BDCRB, are potent and selective inhibitors of human cytomegalovirus (HCMV) DNA processing and packaging. Since they are readily metabolized in vivo, analogs were synthesized to improve biostability. One of these, 1-(beta-L-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94; maribavir), inhibits viral DNA synthesis and nuclear egress. Resistance to maribavir was mapped to UL97, and this viral kinase was shown to be a direct target of maribavir. In the present study, an HCMV strain resistant to TCRB and BDCRB was passaged in increasing concentrations of maribavir, and resistant virus was isolated. This strain (G2) grew at the same rate as the wild-type virus and was resistant to both BDCRB and maribavir. Resistance to BDCRB was expected, because the parent strain from which G2 was isolated was resistant due to known mutations in UL56 and UL89. However, no mutations were found in UL97 or other relevant open reading frames that could explain resistance to maribavir. Because sequencing of selected HCMV genes did not identify the resistance mutation, a cosmid library was made from G2, and a series of recombinant G2 wild-type viruses were constructed. Testing the recombinants for sensitivity to maribavir narrowed the locus of resistance to genes UL26 to UL32. Sequencing identified a single coding mutation in ORF UL27 (Leu335Pro) as the one responsible for resistance to maribavir. These results establish that UL27 is either directly or indirectly involved in the mechanism of action of maribavir. They also suggest that UL27 could play a role in HCMV DNA synthesis or egress of HCMV particles from the nucleus.     

4′- C -Methyl-β-D-ribofuranosyl Purine and Pyrimidine Nucleosides Revisited

Abstract

In order to evaluate their antiviral properties, a series of 4′-C-methyl-β-D-ribofuranosyl purine and pyrimidine nucleosides has been prepared. Unfortunately, none of these 4′-branched nucleosides showed any antiviral activity or cytotoxcity when tested against HIV, HBV, and Yellow Fever virus.     

Introduction of Fluorine Into the C8 Position of Purine Nucleosides

Abstract

We have synthesized 2-amino-6,8-difluoro-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine (3) from 2-amino-6,8-dichloro-9-(2,3,5-tri-O-acetyl-ß-D-ribofuranosyl)purine (1) in a two-step procedure. The reaction of 3 with anhydrous ammonia in dry 1,2-dimethoxyethane gave 2,8-diamino-6-fluoro-9-(2,3,5-tri-O-acetyl-ß-D-ribofuranosyl)purine (4) in 64.1% yield. Compound 4 was deaminated with t-butylnitrite in tetrahydrofuran to give 2-amino-6-fluoro-9-(2,3,5-tri-O-acetyl-ß-D-ribofuranosyl)purine (6). The ¹H, ¹⁹F, and ¹³C NMR spectral data were determined and evaluated for each of the compounds.     

Role of a mutation in human cytomegalovirus gene UL104 in resistance to benzimidazole ribonucleosides

The benzimidazole D-ribonucleosides TCRB and BDCRB are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. Two HCMV strains resistant to these compounds were selected and had resistance mutations in genes UL89 and UL56. Proteins encoded by these two genes are the two subunits of the HCMV "terminase" and are necessary for cleavage and packaging of viral genomic DNA, a process inhibited by TCRB and BDCRB. We now report that both strains also have a previously unidentified mutation in UL104, the HCMV portal protein. This mutation, which results in L21F substitution, was introduced into the genome of wild-type HCMV by utilizing a recently cloned genome of HCMV as a bacterial artificial chromosome. The virus with this mutation alone was not resistant to BDCRB, suggesting that this site is not involved in binding benzimidazole nucleosides. As in previous proposals for mutations in UL104 of murine cytomegalovirus and HCMV strains resistant to BAY 38-4766, we hypothesize that this mutation could compensate for conformational changes in mutant UL89 and UL56 proteins, since the HCMV terminase is likely to interact with the portal protein during cleavage and packaging of genomic DNA.     

Nucleosides and Nucleotides. 140. Synthesis and Antileukemic Activity of 5-Carbon-Substituted 1-β-d-Ribofuflranosylimidazole-4-Carboxamides

Abstract

Synthesis of 5-carbon-substituted 1-β-d-ribofuranosylimidazole-4-carboxamides are described. Treatment of 5-iodo derivative 8 with methyl acrylate in the presence of palladium catalyst gave (E)-5-(2-carbomethoxyvinyl)-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)imidazole-4-carboxamide (9), followed by appropriate manipulations to afford various 5-carbon-substituted imidazole derivatives 1–7. The antileukemic activities of these imidazole nucleosides are also described.

     

Synthesis of 1,3-Dl-O-Acetyl-5-O-Benzoyl-2-O -(O-Carboran-1-Ylmethyl)- D-Ribofuranose. A General Precursor for the Preparation of Carborane-Containing Nucleosides for Boron Neutron Capture Therapy

Abstract

An eight-step synthesis of 1,3-di-O-acetyl-5-O-benzoyl-2-O-(o-carboran-1-ylmethyl)-D-ribofuranose 9 was carried out from 1,2:5,6-O-isopropylidene-α-D-allofuanose 1. Condensation of 9 with trimethylsilyl protected uracil in the presence of trimethylsilyl trifluoro-methanesulfonate, and subsequent deblocking of the resulting 1-[3-O-acetyl-5-O-benzoyl-2-O-(o-carboran-1-ylmethyl)-D-ribofuranosyl]uracil 10 (>95& β-configuration) by alkaline hydrolysis, yielded 1-[2-O-(o-carboran-1-ylmethyl)-β-D-ribofuranosyl]uracil 11.     

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.     

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-(β-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-butyldimethylsilyl-2,3-O-isopropylidene-β-D-ribofuranosyl-4-(1,2,4-oxadiazol-3-yl)imidaz-oles (6a-d), with different substituents at the 5-position of the 1,2,4-oxadiazole, were synthesized from 5-amino-1-(β-D-ribofuranosyl)imidazole-4-carboxamide (AICA Ribose, 3). It was found that 5-amino-1-(5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-β-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-(β-D-ribofuranosyl)imidazo[4,5-c]pyrazole (2) as a 5:5 fused analog of adenosine (1).