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. β-d-2'-Deoxy-2'-α-fluoro-2'-β-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 β-d-2'-deoxy-2'-α-fluoro-2'-β-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.

A phosphoramidate prodrug of 2-deoxy-2-␣-fluoro-␤-Cmethyluridine-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.Nucleoside analogs have long been the backbone therapy for the treatment of viral diseases such as HIV, HBV, and HSV infections (1-5). Recent studies have suggested that nucleoside analogs may be useful for treating hepatitis C virus (HCV) 3 infection (4, 6 -8). The most advanced anti-HCV nucleoside, RG7128, is a diisobutyrate nucleoside prodrug of ␤-D-2Ј-deoxy-2Ј-␣-fluoro-2Ј-␤-C-methylcytidine (PSI-6130) and is currently in phase IIb clinical studies. PSI-6130 demonstrated potent activity in the subgenomic HCV replicon assay (9); the incubation of radiolabeled PSI-6130 with either replicon cells or primary human hepatocytes resulted in the formation of the 5Ј-mono-, di-, and triphosphate metabolites of . The triphosphate metabolite (PSI-6130-TP) was shown to be a potent inhibitor of HCV NS5B RNA-directed RNA polymerase (RdRp) (11). However, incubation of replicon cells with the uridine analog, PSI-6206, resulted in no inhibition of HCV RNA production due to the inability of PSI-6206 to be phosphorylated by cellular nucleoside kinases to its monophosphate, 12). Biochemical studies showed that PSI-7411 was consecutively phosphorylated to its diphosphate, PSI-7410, by UMP-CMP kinase and its triphosphate, PSI-7409, by nucleoside diphosphate kinase (12). Inhibition studies using the replic...

1H NMR chemical shift assignments were established for Nδ1H (16.9 ppm) and Nε2H (16.1 ppm) of the active-center His57 for the complex of MeOSuc-Ala-Ala-Pro-boroPhe (BoroPhe) with chymotrypsin and for the Cε1H proton (9.2 ppm at low pH and 8.5 ppm at high pH) of His57 in uncomplexed chymotrypsin. The assignment for Cε1H corrects previous assignments and reveals an unusual environment of this carbon-bound proton. The relative NH assignments are reversed from the order of NH assignments previously found for α-lytic protease complexes with boronate inhibitors. Isotopic fractionation factors (H/D) were determined using 1H NMR for hydrogen bonds to the active site histidine in BoroPhe complexes with chymotrypsin and subtilisin E, and for uncomplexed chymotrypsin. Measured fractionation factors accurate to about ±0.1 were 0.82 (pH 10) and 0.64 (pH 3) for the Nδ1H proton of uncomplexed chymotrypsin. In the presence of BoroPhe at pH 6.5, the Nδ1H fractionation factors were 0.65 for the chymotrypsin−inhibitor complex, and 0.53 for the subtilisin−inhibitor complex. Measurements for the Nε2H fractionation factor were 1.05 (uncomplexed chymotrypsin at pH 10), 0.93 (BoroPhe−chymotrypsin at pH 6.5), and 0.76 (BoroPhe−subtilisin at pH 6.5). Both model calculations of isotopic fractionation factors and experimentally determined inhibition constants were used in the analysis of the fractionation-factor results.

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Hepatitis C virus afflicts approximately 180 million people worldwide, and the development of direct acting antivirals may offer substantial benefit compared to the current standard of care. Accordingly, prodrugs of 2'-deoxy-2'-fluoro-2'-C-methylguanosine monophosphate analogues were prepared and evaluated for their anti-HCV efficacy and tolerability. These prodrugs demonstrated >1000 fold greater potency than the parent nucleoside in a cell-based replicon assay as a result of higher intracellular triphosphate levels. Further optimization led to the discovery of the clinical candidate PSI-353661, which has demonstrated strong in vitro inhibition against HCV without cytotoxicity and equipotent activity against both the wild type and the known S282T nucleoside/tide resistant replicon. PSI-353661 is currently in preclinical development for the treatment of HCV.

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.

A combination of 1H and 15N nuclear magnetic resonance experiments have been carried out to assign the two high-frequency 1H resonances that result from the complexation of subtilisin E and MeoSuc-Ala-Ala-Pro-boroPhe, a potent peptideboronic acid inhibitor of both subtilisins from a variety of sources and chymotrypsin. First, it was demonstrated unequivocally using two auxotrophs of Bacillus subtilis that the proton resonances at 16 and 17 ppm pertain to a histidine residue. Next it was shown by both 1D and 2D methods that the two proton resonances pertain to the same histidine. Finally, in the subtilisin E-peptideboronate complex, all of the imidazole proton and nitrogen resonances pertinent to this His64 were assigned as follows:  Nε2 at 183 and Nδ1 at 189 ppm; Nε2H at 16 and Nδ1H at 17.4 ppm; Cε1H at 9.20 and Cδ2H at 7.09 ppm. Using the 1D NOE method demonstrated on the subtilisin−peptideboronate complex, the resonances due to complexation were also assigned in the chymotrypsin−peptideboronate complex. The assignments of the two high-frequency resonances are reversed from those assumed in a previous paper from the current authors (Zhong, S.; Haghjoo, K.; Kettner, C.; Jordan, F. J. Am. Chem. Soc. 1995, 117, 7047−7055), in which the assignments were adopted from the relative chemical shifts assigned on α-lytic protease [Bachovchin, W. W.; Wong, W. Y. L.; Farr-Jones, S.; Shenvi, A. B.; Kettner, C. A. Biochemistry 1988, 27, 7689−7697].

In this letter we report first nonpeptide inhibitors of hepatocyte growth factor (HGF) activation. These compounds inhibit the three proteases (matriptase, hepsin, and HGF activator) required for HGF maturation. We show that 6, 8a, 8b, and 8d block activation of fibroblast-derived pro-HGF, thus preventing fibroblast-induced scattering of DU145 prostate cancer cells. Compound 6 (SRI 31215) is very soluble (91 μM) and has excellent microsome stability (human t 1/2 = 162 min; mouse t 1/2 = 296 min). In mouse 6 has an in vivo t 1/2 = 5.8 h following IV administration. The high solubility of 6 and IV t 1/2 make this compound a suitable prototype "triplex inhibitor" for the study of the inhibition of HGF activation in vivo.