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Chronic hepatitis C (CHC) is a major liver disease caused by the hepatitis C virus. The current standard of care for CHC can achieve cure rates above 95%; however, the drugs in current use are administered for a period of 8-16 weeks. A combination of safe and effective drugs with a shorter treatment period is highly desirable. We report synthesis and biological evaluation of a series of 2',3'- and 2',4'-substituted guanosine nucleotide analogues. Their triphosphates exhibited potent inhibition of the HCV NS5B polymerase with IC

Recent cases of severe toxicity during clinical trials have been associated with antiviral ribonucleoside analogs (e.g. INX-08189 and balapiravir). Some have hypothesized that the active metabolites of toxic ribonucleoside analogs, the triphosphate forms, inadvertently target human mitochondrial RNA polymerase (POLRMT), thus inhibiting mitochondrial RNA transcription and protein synthesis. Others have proposed that the prodrug moiety released from the ribonucleoside analogs might instead cause toxicity. Here, we report the mitochondrial effects of several clinically relevant and structurally diverse ribonucleoside analogs including NITD-008, T-705 (favipiravir), R1479 (parent nucleoside of balapiravir), PSI-7851 (sofosbuvir), and INX-08189 (BMS-986094). We found that efficient substrates and chain terminators of POLRMT, such as the nucleoside triphosphate forms of R1479, NITD-008, and INX-08189, are likely to cause mitochondrial toxicity in cells, while weaker chain terminators and inhibitors of POLRMT such as T-705 ribonucleoside triphosphate do not elicit strong in vitro mitochondrial effects. Within a fixed 3’-deoxy or 2′-C-methyl ribose scaffold, changing the base moiety of nucleotides did not strongly affect their inhibition constant (Ki) against POLRMT. By swapping the nucleoside and prodrug moieties of PSI-7851 and INX-08189, we demonstrated that the cell-based toxicity of INX-08189 is mainly caused by the nucleoside component of the molecule. Taken together, these results show that diverse 2′ or 4′ mono-substituted ribonucleoside scaffolds cause mitochondrial toxicity. Given the unpredictable structure-activity relationship of this ribonucleoside liability, we propose a rapid and systematic in vitro screen combining cell-based and biochemical assays to identify the early potential for mitochondrial toxicity.

Influenza viruses are responsible for seasonal epidemics and occasional pandemics which cause significant morbidity and mortality. Despite available vaccines, only partial protection is achieved. Currently, there are two classes of widely approved anti-influenza drugs: M2 ion channel blockers and neuraminidase inhibitors. However, the worldwide spread of drug-resistant influenza strains poses an urgent need for novel antiviral drugs, particularly with a different mechanism of action. Favipiravir (T-705), a broad-spectrum antiviral agent, has shown potent anti-influenza activity in cell-based assays, and its riboside (2) triphosphate inhibited influenza polymerase. In one of our approaches to treat influenza infection, we designed, prepared, and tested a series of C-nucleoside analogues, which have an analogy to 2 and were expected to act by a similar antiviral mechanism as favipiravir. Compound 3c of this report exhibited potent inhibition of influenza virus replication in MDCK cells, and its triphosphate was a substrate of and demonstrated inhibitory activity against influenza A polymerase. Metabolites of 3c are also presented.

Norovirus (NoV) is a positive-sense single-stranded RNA virus that causes acute gastroenteritis and is responsible for 200,000 deaths per year worldwide. No effective vaccine or treatment is available. Recent studies have shown that the nucleoside analogs favipiravir (T-705) and 2′- C -methyl-cytidine (2CM-C) inhibit NoV replication in vitro and in animal models, but their precise mechanism of action is unknown. We evaluated the molecular interactions between nucleoside triphosphates and NoV RNA-dependent RNA polymerase (NoVpol), the enzyme responsible for replication and transcription of NoV genomic RNA. We found that T-705 ribonucleoside triphosphate (RTP) and 2CM-C triphosphate (2CM-CTP) equally inhibited human and mouse NoVpol activities at concentrations resulting in 50% of maximum inhibition (IC 50 s) in the low micromolar range. 2CM-CTP inhibited the viral polymerases by competing directly with natural CTP during primer elongation, whereas T-705 RTP competed mostly with ATP and GTP at the initiation and elongation steps. Incorporation of 2CM-CTP into viral RNA blocked subsequent RNA synthesis, whereas T-705 RTP did not cause immediate chain termination of NoVpol. 2CM-CTP and T-705 RTP displayed low levels of enzyme selectivity, as they were both recognized as substrates by human mitochondrial RNA polymerase. The level of discrimination by the human enzyme was increased with a novel analog of T-705 RTP containing a 2′- C -methyl substitution. Collectively, our data suggest that 2CM-C inhibits replication of NoV by acting as a classic chain terminator, while T-705 may inhibit the virus by multiple mechanisms of action. Understanding the precise mechanism of action of anti-NoV compounds could provide a rational basis for optimizing their inhibition potencies and selectivities.

T-705 (favipiravir) is a new antiviral agent in advanced clinical development for influenza therapy. It is supposed to act as an alternative substrate for the viral polymerase, causing inhibition of viral RNA synthesis or virus mutagenesis. These mechanisms were also proposed for ribavirin, an established and broad antiviral drug that shares structural similarity with T-705. We here performed a comparative analysis of the effects of T-705 and ribavirin on influenza virus and host cell functions. Influenza virus-infected cell cultures were exposed to T-705 or ribavirin during single or serial virus passaging. The effects on viral RNA synthesis and infectious virus yield were determined and mutations appearing in the viral genome were detected by whole-genome virus sequencing. Besides, the cellular nucleotide pools were quantified as well as direct inhibition of the viral polymerase enzyme. We demonstrate that the anti-influenza effect of ribavirin is based on IMP dehydrogenase inhibition, which results in fast and profound GTP depletion and an imbalance in the nucleotide pools. In contrast, T-705 acts as a potent and GTP-competitive inhibitor of the viral polymerase. In infected cells, viral RNA synthesis is completely inhibited by T-705 or ribavirin at ≥50 μM, whereas exposure to lower drug concentrations induces formation of non-infectious particles and accumulation of random point mutations in the viral genome. This mutagenic effect is two-fold higher for T-705 than for ribavirin. Hence, T-705 and ribavirin both act as purine pseudobases, but profoundly differ with regard to the mechanism behind their antiviral and mutagenic effects on influenza virus. ispartof: Antimicrobial Agents and Chemotherapy vol:60 issue:11 pages:6679-6691 ispartof: location:United States status: published

The HIV-1 trans-activation responsive element (TAR) RNA 59-residue stem-loop interacts with the HIV trans-activator protein Tat and other cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibition of these interactions blocks full-length HIV transcription and hence replication. We have found that three types of 12-residue oligonucleotide analogues, namely, a 2'-O-methyl oligoribonucleotide (OMe), a chimeric oligonucleotide containing 7xOMe and 5x5-methyl C locked nucleic acid (LNA) residues, and a peptide nucleic acid (PNA), inhibit Tat-dependent in vitro transcription in HeLa cell nuclear extract equally efficiently (50% inhibition at 100-200 nM) and sequence specifically. The results are correlated with surprisingly similar binding strengths to a model 39-residue TAR under transcription conditions. A 12-mer containing 11 contiguous LNA residues was less effective in both Tat-dependent transcription inhibition and TAR 39 binding. Anti-TAR 3'-carboxyfluorescein- (FAM-) labeled OMe and OMe/LNA chimeric 12-mers were also efficient Tat-dependent in vitro transcription inhibitors as were 3'-FAM-labeled OMe oligonucleotides containing some phosphorothioate (PS) linkages. By use of a HeLa cell line containing stably integrated plasmids expressing firefly luciferase under HIV-LTR/Tat dependence as well as a Renilla luciferase constitutive control, we showed submicromolar, selective, dose-dependent, and sequence-dependent intracellular inhibition of Tat-TAR trans activation by the anti-TAR 3'-FAM 12-residue 7xOMe/5xLNA oligonucleotide when delivered by cationic lipid. No intracellular activity was observed for the corresponding anti-TAR 3'-FAM OMe 12-mer. An alternating PS-containing 3'-FAM OMe 12-mer oligonucleotide exhibited partial inhibition of trans-activation activity, but this was correlated with a similar effect on control gene expression, suggesting nonspecific inhibition.

Invasiveness, the ability of certain tumour cells to migrate beyond their natural tissue boundaries, often leads to metastasis, and usually determines the fatal outcome of cancer. The need for anti-invasive agents has led us to search for possibly active compounds among alkaloids and polyphenolics. One hundred compounds were screened in an assay based on the confrontation of invasive human MCF-7/6 mammary carcinoma cells with fragments of normal embryonic chick heart in vitro. Anti-invasive activity was frequently found among chalcones having a prenyl group. Six compounds were found to inhibit invasion when added to the culture medium at concentrations as low as 1 μM. For at least three of them the anti-invasive effect could be associated with a cytotoxic effect on the MCF-7/6 cells, but not on the heart tissue. This selective cytotoxicity was substantiated by different methods, such as histology and growth assays (volume measurements, cell counts, MTT and sulforhodamine B assays). The anti-invasive effects of the compounds could neither be ascribed to induction of apoptosis nor to the promotion of cell-cell adhesion. Our data indicate that among the alkaloids and polyphenolics a number of molecules can inhibit growth and invasion of human mammary cancer cells via selective cytotoxicity.

Sonogashira coupling strategies were employed to synthesize new furo[2,3-d]pyrimidin-2(3H)-one (FuPyrm) 2'-deoxynucleoside analogues. Partial or complete reduction of ethyne-linked compounds afforded ethenyl- and ethyl-linked derivatives. Levels of inhibition of varicella-zoster virus (VZV), human cytomegalovirus (HCMV), a broad range of other DNA and RNA viruses, and several cancer cell lines were evaluated in cell cultures. The anti-VZV potency decreased with increasing rigidity of the side chain at C6 of the FuPyrm ring in the order dec-1-yn-1-yldec-1-en-1-yldecan-1-yl. In contrast, compounds with a rigid ethynyl spacer between C6 of the FuPyrm ring and a 4-alkylphenyl moiety were more potent inhibitors of VZV than the corresponding derivatives with an ethyl spacer. Replacement of the phenyl moiety in 6-(4-alkylphenyl) derivatives with a pyridine ring (in either regioisomeric orientation) gave analogues with increased solubility in methanol but reduced anti-VZV potency, and replacement with a pyrimidine ring reduced the anti-VZV activity even further. The pyridine-ring-containing analogues were approximately 20-fold more potent inhibitors of VZV than acyclovir but were approximately 6-fold less potent than BVDU and approximately 60-fold weaker than the most active 6-(4-pentylphenyl)-substituted prototype.

Derivatives of the 2'-deoxynucleoside of furo[2,3-d]pyrimidin-2(3H)-one with long-chain alkyl (or 4-alkylphenyl) substituents at C6 exhibit remarkable anti-VZV (varicella-zoster virus) potency and selectivity, and analogous 2',3'-dideoxynucleoside derivatives show anti-HCMV (human cytomegalovirus) activity. We now report a synthetic approach that enables the preparation of long-chain 6-(alkyn-1-yl)furo[2,3-d]pyrimidin-2(3H)-ones in which the rodlike acetylene spacer replaces the 4-substituted-phenyl ring at C6. Analogues with methyl, beta-d-ribofuranosyl, beta-d-arabinofuranosyl, and 2-deoxy-beta-d-erythro-pentofuranosyl substituents at N3 have been prepared. Long-chain derivatives at C6 in the 2'-deoxynucleoside series showed virus-encoded nucleoside kinase-sensitive anti-VZV activity. Surprisingly, 3-methyl-6-(octyn-1-yl)furo[2,3-d]pyrimidin-2(3H)-one (prepared as a negative anti-VZV test control) exhibited anti-HCMV activity, which supports the possibility of development of non-nucleoside anti-HCMV agents originating from uncomplicated derivatives of such bicyclic ring systems.

In search of active nucleoside 5'-triphosphate mimics, we have synthesized a series of AZT triphosphate mimics (AZT P3Ms) and evaluated their inhibitory effects on HIV-1 reverse transcriptase as well as their stability in fetal calf serum and in CEM cell extracts. Reaction of AZT with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, followed by treatment of the phosphite intermediate 2 with pyrophosphate analogues, yielded the cyclic triphosphate intermediates 4b-4f, which were subjected to boronation and subsequent hydrolysis to give AZT 5'-alpha-borano-beta,gamma-bridge-modified triphosphates 6b-6f in moderate to good yields. Reaction of the cyclic intermediate 4d with iodine, followed by treatment with a series of nucleophiles, afforded the AZT 5'-beta,gamma-difluoromethylene-gamma-substituted triphosphates (7b-7i). Several different types of AZT P3Ms containing alpha-P-thio (or dithio) and beta,gamma-difluoromethylene (13,14), alpha,beta-difluoromethylene and gamma-P-methyl(or phenyl) (15,16), and alpha-borano-beta,gamma-difluoromethylene and gamma-O-methyl/phenyl (11,12) were also synthesized. The effectiveness of the compounds as inhibitors of HIV-1 reverse transcriptase was determined using a fluorometric assay and a poly(A) homopolymer as a template. A number of AZT P3Ms exhibited very potent inhibition of HIV-1 reverse transcriptase. Modifications at the beta,gamma-bridge of triphosphate rendered the AZT P3Ms 6b-6f with varied activities (K(i) from 9.5 to >>500 nM) while modification at the alpha,beta-bridge of triphosphate led to weak AZT P3M inhibitors. The results imply that the AZT P3Ms were substrate inhibitors, as is AZT triphosphate. The most active compound, AZT 5'-alpha-R(p)()-borano-beta,gamma-(difluoromethylene)triphosphate (AZT 5'-alphaB-betagammaCF(2)TP) (6d-I), is as potent as AZT triphosphate with a K(i)() value of 9.5 nM and at least 20-fold more stable than AZT triphosphate in the serum and cell extracts. Therefore, for the first time, a highly active and stable nucleoside triphosphate mimic has been identified, which is potentially useful as a new type of antiviral drug. The promising triphosphate mimic, 5'-alpha-borano-beta,gamma-(difluoromethylene)triphosphate, is expected to be valuable to the discovery of nucleotide mimic antiviral drugs.

The HIV-1 Tat protein interaction with its RNA recognition sequence TAR is an important drug target and model system for the development of specific RNA-protein inhibitors. 2′-O-methyl oligoribonucleotides complementary to the TAR apical stem-loop effectively block Tat binding in vitro. Substitution by 5-propynylC or 5-methylC LNA monomeric units into a 12-mer 2′-O-methyl oligoribonucleotide leads to stronger inhibition, as does a 12-mer PNA. 10–16 mer 2′-O-methyl oligoribonucleotides give sequence- and dose-dependent inhibition of Tat-dependent transcription of an HIV DNA template in HeLa cell nuclear extract. Inhibition is maintained for the substituted 12-mer analogues but is poorer for PNA and is not correlated with TAR binding strength.

T-705 (Favipiravir) is a broad-spectrum antiviral molecule currently in late stage clinical development for the treatment of influenza virus infection. Although it is believed that T-705 potency is mediated by its ribofuranosyl triphosphate (T-705 RTP) metabolite that could be mutagenic, the exact molecular interaction with the polymerase of influenza A virus (IAVpol) has not been elucidated. Here, we developed a biochemical assay to measure the kinetics of nucleotide incorporation by IAVpol in the elongation mode. In this assay, T-705 RTP was recognized by IAVpol as an efficient substrate for incorporation to the RNA both as a guanosine and an adenosine analog. Compared to natural GTP and ATP, the discrimination of T-705 RTP was about 19- and 30-fold, respectively. Although the single incorporation of the ribonucleotide monophosphate form of T-705 did not efficiently block RNA synthesis, two consecutive incorporation events prevented further primer extension. In comparison, 3'-deoxy GTP caused immediate chain termination but was incorporated less efficiently by the enzyme, with a discrimination of 4,900-fold relative to natural GTP. Collectively, these results provide the first detailed biochemical characterization to evaluate the substrate efficiency and the inhibition potency of nucleotide analogs against influenza virus polymerase. The combination of ambiguous base-pairing with low discrimination of T-705 RTP provides a mechanistic basis for the in vitro mutagenic effect of T-705 towards influenza virus.