Discovery of a novel class of potent HCV NS4B inhibitors: SAR studies on piperazinone derivatives.

HTS screening identified compound 2a (piperazinone derivative) as a low micromolar HCV genotype 1 (GT-1) inhibitor. Resistance mapping studies suggested that this piperazinone chemotype targets the HCV nonstructural protein NS4B. Extensive SAR studies were performed around 2a and the amide function and the C-3/C-6 cis stereochemistry of the piperazinone core were essential for HCV activity. A 10-fold increase in GT-1 potency was observed when the chiral phenylcyclopropyl amide side chain of 2a was replaced with p-fluorophenylisoxazole-carbonyl moiety (67). Replacing the C-6 nonpolar hydrophobic moiety of 67 with a phenyl moiety (95) did not diminish the GT-1 potency. A heterocyclic thiophene moiety (103) and an isoxazole moiety (108) were incorporated as isosteric replacements for the C-6 phenyl moiety (95), resulting in significant improvement in GT-1b and 1a potency. However, the piperazonone class of compounds lacks GT-2 activity and, consequently, were not pursued further into development.

Synthesis of stable isotope labeled analogs of the anti-hepatitis C virus nucleotide prodrugs PSI-7977 and PSI-352938.

In order to support bioanalytical LC/MS method development and plasma sample analysis in preclinical and clinical studies of the anti-hepatitis C-virus nucleotides, PSI-7977 and PSI-352938, the corresponding stable isotope labeled forms were prepared. These labeled compounds were prepared by addition reaction of the freshly prepared Grignard reagent (13)CD(3)MgI to the corresponding 2 '-ketone nucleosides followed by fluorination of the resulting carbinol with DAST. As expected, these 2 '-C-(trideuterated-(13)C-methyl) nucleotide prodrugs showed similar anti-HCV activity to that of the corresponding unlabeled ones.

Discovery of a ß-d-2'-deoxy-2'-α-fluoro-2'-ß-C-methyluridine nucleotide prodrug (PSI-7977) for the treatment of hepatitis C virus.

Hepatitis C virus (HCV) is a global health problem requiring novel approaches for effective treatment of this disease. The HCV NS5B polymerase has been demonstrated to be a viable target for the development of HCV therapies. ß-d-2'-Deoxy-2'-α-fluoro-2'-ß-C-methyl nucleosides are selective inhibitors of the HCV NS5B polymerase and have demonstrated potent activity in the clinic. Phosphoramidate prodrugs of the 5'-phosphate derivative of the ß-d-2'-deoxy-2'-α-fluoro-2'-ß-C-methyluridine nucleoside were prepared and showed significant potency in the HCV subgenomic replicon assay (<1 µM) and produced high levels of triphosphate 6 in primary hepatocytes and in the livers of rats, dogs, and monkeys when administered in vivo. The single diastereomer 51 of diastereomeric mixture 14 was crystallized, and an X-ray structure was determined establishing the phosphoramidate stereochemistry as Sp, thus correlating for the first time the stereochemistry of a phosphoramidate prodrug with biological activity. 51 (PSI-7977) was selected as a clinical development candidate.

Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977.

A phosphoramidate prodrug of 2'-deoxy-2'-α-fluoro-ß-C-methyluridine-5'-monophosphate, PSI-7851, demonstrates potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. PSI-7851 is a mixture of two diastereoisomers, PSI-7976 and PSI-7977, with PSI-7977 being the more active inhibitor of HCV RNA replication in the HCV replicon assay. To inhibit the HCV NS5B RNA-dependent RNA polymerase, PSI-7851 must be metabolized to the active triphosphate form. The first step, hydrolysis of the carboxyl ester by human cathepsin A (CatA) and/or carboxylesterase 1 (CES1), is a stereospecific reaction. Western blot analysis showed that CatA and CES1 are both expressed in primary human hepatocytes. However, expression of CES1 is undetectable in clone A replicon cells. Studies with inhibitors of CatA and/or CES1 indicated that CatA is primarily responsible for hydrolysis of the carboxyl ester in clone A cells, although in primary human hepatocytes, both CatA and CES1 contribute to the hydrolysis. Hydrolysis of the ester is followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the spontaneous elimination of phenol and the production of an alaninyl phosphate metabolite, PSI-352707, which is common to both isomers. The removal of the amino acid moiety of PSI-352707 is catalyzed by histidine triad nucleotide-binding protein 1 (Hint1) to give the 5'-monophosphate form, PSI-7411. siRNA-mediated Hint1 knockdown studies further indicate that Hint1 is, at least in part, responsible for converting PSI-352707 to PSI-7411. PSI-7411 is then consecutively phosphorylated to the diphosphate, PSI-7410, and to the active triphosphate metabolite, PSI-7409, by UMP-CMP kinase and nucleoside diphosphate kinase, respectively.

PSI-7851, a pronucleotide of beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine monophosphate, is a potent and pan-genotype inhibitor of hepatitis C virus replication.

The hepatitis C virus (HCV) NS5B RNA polymerase facilitates the RNA synthesis step during the HCV replication cycle. Nucleoside analogs targeting the NS5B provide an attractive approach to treating HCV infections because of their high barrier to resistance and pan-genotype activity. PSI-7851, a pronucleotide of beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine-5'-monophosphate, is a highly active nucleotide analog inhibitor of HCV for which a phase 1b multiple ascending dose study of genotype 1-infected individuals was recently completed (M. Rodriguez-Torres, E. Lawitz, S. Flach, J. M. Denning, E. Albanis, W. T. Symonds, and M. M. Berry, Abstr. 60th Annu. Meet. Am. Assoc. Study Liver Dis., abstr. LB17, 2009). The studies described here characterize the in vitro antiviral activity and cytotoxicity profile of PSI-7851. The 50% effective concentration for PSI-7851 against the genotype 1b replicon was determined to be 0.075+/-0.050 microM (mean+/-standard deviation). PSI-7851 was similarly effective against replicons derived from genotypes 1a, 1b, and 2a and the genotype 1a and 2a infectious virus systems. The active triphosphate, PSI-7409, inhibited recombinant NS5B polymerases from genotypes 1 to 4 with comparable 50% inhibitory concentrations. PSI-7851 is a specific HCV inhibitor, as it lacks antiviral activity against other closely related and unrelated viruses. PSI-7409 also lacked any significant activity against cellular DNA and RNA polymerases. No cytotoxicity, mitochondrial toxicity, or bone marrow toxicity was associated with PSI-7851 at the highest concentration tested (100 microM). Cross-resistance studies using replicon mutants conferring resistance to modified nucleoside analogs showed that PSI-7851 was less active against the S282T replicon mutant, whereas cells expressing a replicon containing the S96T/N142T mutation remained fully susceptible to PSI-7851. Clearance studies using replicon cells demonstrated that PSI-7851 was able to clear cells of HCV replicon RNA and prevent viral rebound.

Mechanism of action of (-)-(2R,4R)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl) thymine as an anti-HIV agent.

(-)-(2R,4R)-1-(2-Hydroxymethyl-1,3-dioxolan-4yl)thymine (DOT) is a thymidine analogue that has potent in vitro activity against wild-type and nucleoside reverse transcriptase inhibitor (NRTI)-resistant HIV. For nucleoside analogues to inhibit viral replication, they must be metabolized to the active triphosphate, which inhibits the viral reverse transcriptase (RT). Using purified enzymes, the kinetics of DOT phosphorylation, inhibition of wild-type and drug-resistant HIV-1 reverse transcriptase activity, and excision of DOT-5'-monophosphate (DOT-MP) from a chain-terminated primer were examined. DOT was phosphorylated by human thymidine kinase-1 (TK-1) but not by other pyrimidine nucleoside kinases, including the mitochondrial thymidine kinase (TK-2). Resistance to NRTIs involves decreased binding/incorporation and/or increased excision of the chain-terminating NRTI. RTs containing the D67N/K70R/T215Y/K219Q or T695-SS/T215Y mutations show enhanced removal of DOT-MP from terminated primer as well as approximately four-fold decreased binding/incorporation. The Q151M and K65R mutations appear to cause decreased inhibition by DOT-TP. However, both the K65R and Q151M mutations show decreased excision, which would confer greater stability on the terminated primer. These opposing mechanisms could offset the overall resistance profile and susceptibility. Little or no resistance was observed with the enzymes harbouring mutations resistant to lamivudine (M184V) and non-nucleoside RT inhibitors (K103N).