Fructose-1,6-bisphosphatase inhibitors. 1. Purine phosphonic acids as novel AMP mimics.

Inhibition of FBPase is considered a promising way to reduce hepatic gluconeogenesis and therefore could be a potential approach to treat type 2 diabetes. Herein we report the discovery of a series of purine phosphonic acids as AMP mimics targeting the AMP site of FBPase, which was achieved using a structure-guided drug design approach. These non-nucleotide purine analogues inhibit FBPase in a similar manner and with similar potency as AMP. More importantly, several purine analogues exhibited potent cellular and in vivo glucose-lowering activities, thus achieving proof-of-concept for inhibiting FBPase as a drug discovery target. For example, compounds 4.11 and 4.13 are as equipotent as AMP with regard to FBPase inhibition. Furthermore, compound 4.11 inhibited glucose production in primary rat hepatocytes and significantly lowered blood glucose levels in fasted rats.

Bis[(para-methoxy)benzyl] phosphonate prodrugs with improved stability and enhanced cell penetration.

A series of substituted bis[(para-methoxy)benzyl] (bisPMB) esters of 1-naphthalenemethylphosphonate (NMPA) were synthesized and evaluated as phosphonate prodrugs. BisPMB NMPA esters (4b and 4c) with significantly improved aqueous stability were identified that also resulted in increased intracellular levels of NMPA following prodrug incubation with primary rat hepatocytes.

Optimization of subsite binding to the beta5 subunit of the human 20S proteasome using vinyl sulfones and 2-keto-1,3,4-oxadiazoles: syntheses and cellular properties of potent, selective proteasome inhibitors.

Beginning with the peptide sequence Cbz-Ile-Glu(OtBu)-Ala-Leu found in PSI (3), a series of vinyl sulfones (VS) were synthesized for evaluation as inhibitors of the chymotrypsin-like activity of the 20S proteasome. Variations at the key P3 position confirmed the importance of a long side chain capped with a hydrophobic group for optimal potency, consistent with a model of binding to the S3 subsite. The tert-butyl glutamic ester initially used at P3 gave plasma unstable, insoluble compounds and was replaced with the better isostere, N-beta-neopentyl asparagine. The inhibitors were shortened by replacing the N-terminal Cbz-isoleucine with a p-tosyl group without loss of potency. Small l-amino acids were used at P2, where d-substitution was not tolerated. The resulting optimized P4-P3-P2 sequence was grafted onto a novel proteasome inhibitor warhead, 2-keto-1,3,4-oxadiazoles (KOD), to produce reversible, subnanomolar proteasome inhibitors that were 1000-fold selective versus cathepsin B (CatB), cathepsin S (CatS), and trypsin-like as well as PGPH-like proteasome activity. A number of compounds in both the VS and the KOD series exhibited growth inhibitory effects against the human prostate cancer cell line PC3 at submicromolar concentrations.

Design and synthesis of selective keto-1,2,4-oxadiazole-based tryptase inhibitors.

Using a scaleable, directed library approach based on orthogonally protected advanced intermediates, we have prepared a series of potent keto-1,2,4-oxadiazoles designed to explore the P(2) binding pocket of human mast cell tryptase, while building in a high degree of selectivity over human trypsin and other serine proteases.

Design and synthesis of tri-ring P3 benzamide-containing aminonitriles as potent, selective, orally effective inhibitors of cathepsin K.

We have prepared a series of achiral aminoacetonitriles, bearing tri-ring benzamide moieties and an aminocyclohexanecarboxylate residue at P2. This combination of binding elements resulted in sub-250 pM, reversible, selective, and orally bioavailable cathepsin K inhibitors. Lead compounds displayed single digit nanomolar inhibition in vitro (of rabbit osteoclast-mediated degradation of bovine bone). The best compound in this series, 39n (CRA-013783/L-006235), was orally bioavailable in rats, with a terminal half-life of over 3 h. 39n was dosed orally in ovariectomized rhesus monkeys once per day for 7 days. Collagen breakdown products were reduced by up to 76% dose-dependently. Plasma concentrations of 39n above the bone resorption IC50 after 24 h indicated a correlation between functional cellular and in vivo assays. Inhibition of collagen breakdown by cathepsin K inhibitors suggests this mechanism of action may be useful in osteoporosis and other indications involving bone resorption.

pH Dependence of inhibitors targeting the occluding loop of cathepsin B.

Potent and selective cathepsin B inhibitors have previously been synthesized based upon the natural product cysteine protease inhibitor E-64. X-ray crystal data indicates that these compounds interact through their free carboxylate with the positively charged histidine residues located on the prime-side of the active site within the occluding loop of cathepsin B. Herein, we examine the pH dependence of two prime-side-binding compounds. In each case there is a dramatic decrease in k(inact)/K(I) as the pH is raised from 4 to 7.8 corresponding to a single ionization of pK(a) 4.4. These results suggest that targeting of the occluding loop of cathepsin B may be a poor inhibitor design strategy if the enzyme environment has a pH greater than 5.5. However, this type of inhibitor may be a useful tool to help elucidate the role and the environment of cathepsin B in invading tumors.

Peptidic 1-cyanopyrrolidines: synthesis and SAR of a series of potent, selective cathepsin inhibitors.

1-Cyanopyrrolidines have previously been reported to inhibit cysteinyl cathepsins (Falgueyret, J.-P. et al., J. Med. Chem. 2001, 44, 94). In order to optimize binding interactions for a given cathepsin and simultaneously reduce interactions with the other closely related enzymes, small peptidic substituents were introduced to the 1-cyanopyrrolidine scaffold, either at the 2-position starting with proline or at the 3-position of aminopyrrolidines. The resulting novel compounds proved to be micromolar inhibitors of cathepsin B (Cat B) but nanomolar to picomolar inhibitors of cathepsins K, L, and S (Cat K, Cat L, Cat S). Several of the compounds were >20-fold selective versus the other three cathepsins. SAR trends were observed, most notably the remarkable potency of Cat L inhibitors based on the 1-cyano-D-proline scaffold. The selectivity of one such compound, the 94 picomolar Cat L inhibitor 12, was demonstrated at higher concentrations in DLD-1 cells. Although none of the compounds in the proline series that was tested proved to be submicromolar in the in vitro bone resorption assay, two Cat K inhibitors in the 3-substituted pyrrolidine series, 24 and 25 were relatively potent in that assay.