Proof of Principle for Local Delivery of a c-Met Inhibitor.

The reported proof of principle study demonstrated the feasibility of local delivery of a c-Met inhibitor (VXc-140) in a subcutaneous xenograft tumor model. VXc-140 was formulated in a wafer delivery system for direct implantation into the tumor. Systemic and local tumor exposure of VXc-140 was analyzed. High tumor exposures coupled with fast release of compound were associated with significant tumor regression and reduction in tumor levels of phosphorylated c-Met. High VXc-140 tumor-to-plasma ratios (∼42 at the tumor periphery) were achieved. The tumor response achieved (7/11 partial response) with VXc-140 with the local delivery in the wafer (4 mg over 15 days) was comparable to the regression observed (11/15 partial response) for VXc-140 in the oral delivery (∼8 mg total administered once a day for 2 weeks). Notably, the plasma levels in animals implanted with VXc-140 wafers ranged from 2 to 4 µM, which, although higher than trough levels achieved with oral administration, were well below oral Cmax levels (∼42 µM) suggesting that toxicities associated with Cmax exposure may be reduced or eliminated by local delivery. The high tumor to plasma exposure of VXc-140 and the efficacy observed with local wafer delivery warrants further exploration into the utility of local delivery.

Novel Ranking System for Identifying Efficacious Anti-Influenza Virus PB2 Inhibitors.

Through antigenic drift and shifts, influenza virus infections continue to be an annual cause of morbidity in healthy populations and of death among elderly and at-risk patients. The emergence of highly pathogenic avian influenza viruses such as H5N1 and H7N9 and the rapid spread of the swine-origin H1N1 influenza virus in 2009 demonstrate the continued need for effective therapeutic agents for influenza. While several neuraminidase inhibitors have been developed for the treatment of influenza virus infections, these have shown a limited window for treatment initiation, and resistant variants have been noted in the population. In addition, an older class of antiviral drugs for influenza, the adamantanes, are no longer recommended for treatment due to widespread resistance. There remains a need for new influenza therapeutic agents with improved efficacy as well as an expanded window for the initiation of treatment. Azaindole compounds targeting the influenza A virus PB2 protein and demonstrating excellent in vitro and in vivo properties have been identified. To evaluate the in vivo efficacy of these PB2 inhibitors, we utilized a mouse influenza A virus infection model. In addition to traditional endpoints, i.e., death, morbidity, and body weight loss, we measured lung function using whole-body plethysmography, and we used these data to develop a composite efficacy score that takes compound exposure into account. This model allowed the rapid identification and ranking of molecules relative to each other and to oseltamivir. The ability to identify compounds with enhanced preclinical properties provides an opportunity to develop more-effective treatments for influenza in patients.

Discovery of thienoimidazole-based HCV NS5A inhibitors. Part 2: non-symmetric inhibitors with potent activity against genotype 1a and 1b.

The discovery of non-symmetric thienoimidazole-containing HCV NS5A inhibitors is described. The inhibitors herein reported display high potencies against both genotype 1a and 1b. In this follow-up manuscript, we discuss the importance of the linker aromaticity to achieve high potency, particularly against genotype 1a.

Discovery of thienoimidazole-based HCV NS5A inhibitors. Part 1: C2-symmetric inhibitors with diyne and biphenyl linkers.

The discovery of C2-symmetric bis-thienoimidazoles HCV NS5A inhibitors is herein reported. Two straightforward approaches to access the requisite diyne and biphenyl linker moieties are described. This study revealed the paramount importance of the aromatic character of the linker to achieve high genotype 1a potency.

Synthesis and evaluation of NS5A inhibitors containing diverse heteroaromatic cores.

Inhibitors of the HCV NS5A nonstructural protein are showing promising clinical potential in the treatment of hepatitis C when used in combination with other direct-acting antiviral agents. Current NS5A clinical candidates such as daclatasvir, ledipasvir, and ombitasvir share a common pharmacophore that features a pair of (S)-methoxycarbonylvaline capped pyrrolidines linked to various cores by amides, imidazoles and/or benzimidazoles. In this Letter, we describe the evaluation of NS5A inhibitors which contain alternative heteroaromatic replacements for these amide mimetics. The SAR knowledge gleaned in the optimization of scaffolds containing benzoxazoles was parlayed toward the identification of potent NS5A inhibitors containing other heteroaromatic replacements such as indoles and imidazopyridines.

Benzimidazole-containing HCV NS5A inhibitors: effect of 4-substituted pyrrolidines in balancing genotype 1a and 1b potency.

The treatment of HCV with highly efficacious, well-tolerated, interferon-free regimens is a compelling clinical goal. Trials employing combinations of direct-acting antivirals that include NS5A inhibitors have shown significant promise in meeting this challenge. Herein, we describe our efforts to identify inhibitors of NS5A and report on the discovery of benzimidazole-containing analogs with subnanomolar potency against genotype 1a and 1b replicons. Our SAR exploration of 4-substituted pyrrolidines revealed that the subtle inclusion of a 4-methyl group could profoundly increase genotype 1a potency in multiple scaffold classes.

Discovery of Thienoimidazole-Based HCV NS5A Genotype 1a and 1b Inhibitors.

The discovery of potent thienoimidazole-based HCV NS5A inhibitors is herein reported. A novel method to access the thienoimidazole [5,5]-bicyclic system is disclosed. This method gave access to a common key intermediate (6) that was engaged in Suzuki or Sonogashira reactions with coupling partners bearing different linkers. A detailed study of the structure-activity relationship (SAR) of the linkers revealed that aromatic linkers with linear topologies are required to achieve high potency for both 1a and 1b HCV genotypes. Compound 20, with a para-phenyl linker, was identified as a potential lead displaying potencies of 17 and 8 pM against genotype 1a and 1b replicons, respectively.

Expression, purification and characterization of the human membrane transporter protein OATP2B1 from Sf9 insect cells.

OATP2B1 is an important member of the organic anion transporting polypeptides (OATP) family and is implicated in the intestinal and hepatic disposition of endo- and xenobiotics. The purpose of this work was to produce a highly purified protein for use as a reference standard for quantification of OATP2B1 in human tissue and in vitro assay systems. Here, we report the successful expression, purification and characterization of OATP2B1 in a heterologous expression system. Protein expressed by the Sf9-baculovirus expression system is functionally active as demonstrated by saturable uptake kinetics with a K(m) of 5.9+/-0.76 microM for estrone-3-sulfate. OATP2B1 was extracted from Sf9-membranes with ABS-14-4 detergent and purified using a one-step FLAG-tag purification method. Yield of OATP2B1 from Sf9 cells was 1.1mg per liter of culture, for a final recovery of 1.8%. SDS-PAGE resolution and Western blot of purified protein displayed multiple banding of OATP2B1-specific protein, which was thoroughly investigated to confirm homogeneity of the sample. C-terminal FLAG-tag purification and immunoblot detection, together with N-terminal sequencing, confirmed the presence of only full-length protein. Treatment with endoglycosidases had little effect on the migration pattern in SDS-PAGE, suggesting that multiple banding was not due to different glycosylation states of the protein. Amino acid analysis further confirmed the homogeneity of the protein with a calculated extinction coefficient of 80,387 cm(-1) M(-1). Physical, biochemical and functional characterization show that purified human OATP2B1 is pure, homogeneous and appropriate for use as a standard to quantitate expression of OATP2B1 in in vitro systems and tissue samples.

Discovery of thiadiazoles as a novel structural class of potent and selective PDE7 inhibitors. Part 2: metabolism-directed optimization studies towards orally bioavailable derivatives.

The synthesis and optimization of pharmacokinetic parameters of structurally novel small PDE7 inhibitors is discussed.

Spiroquinazolinones as novel, potent, and selective PDE7 inhibitors. Part 2: Optimization of 5,8-disubstituted derivatives.

The optimization of 5,8-disubstituted spirocyclohexane-quinazolinones into potent, selective, soluble PDE7 inhibitors with acceptable in vivo pharmacokinetic parameters is presented.