Identification of two novel and potent competitive inhibitors of the glucose-6-phosphatase catalytic protein.

AIM: In this study, we show that inhibitors of the glucose-6-phosphatase (G-6-Pase) catalytic protein could be an alternative approach to the recent G-6-Pase T1-translocase inhibitors to target this enzyme for the treatment of type 2 diabetes. METHOD: The active enantiomers of 4-methoxyphenyl-[4-(4-methoxyphenyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-5-yl]methanone (Compound A-1) and 4-methoxyphenyl-[4-(4-trifluoromethoxyphenyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-5-yl]methanone (Compound B-1) were characterized as inhibitors of the G-6-Pase catalytic protein using pig and rat liver microsomes and cultured rat hepatocytes. RESULTS: Both compounds were found to be potent competitive inhibitors of the G-6-Pase catalytic protein obtained from pig and rat liver microsomes. The K(i) values (microM) were calculated to be 0.61 +/- 0.02 and 0.63 +/- 0.08 for compound A-1 and B-1 on intact pig microsomes, and 0.27 +/- 0.02 and 0.29 +/- 0.06 on disrupted pig microsomes. The corresponding values for rat liver microsomes were found to be 3.3 +/- 0.6 and 4.0 +/- 1.2 for compound A-1 and B-1 on intact microsomes, and 1.54 +/- 0.1 and 1.21 +/- 0.1 on disrupted microsomes. Compound A-1 was also able to inhibit pyrophosphatase activities from both intact and disrupted microsomes with equal potency (IC50; 0.43-0.55 microm). Using cultured rat hepatocytes and glycerol as the substrate, these compounds were able to prevent glucose production up to 60% with a concomitant increase in the G-6-P content (2.3-fold) using compound A-1. No increase in glycogen levels was seen. CONCLUSION: These data demonstrated that these compounds were more potent inhibitors on G-6-Pase obtained from pig microsomes and were able to penetrate the microsomal membrane. The hepatocyte data further support the kinetic data, and are also consistent with the evoked mechanism of action.

Glucose-6-phosphatase catalytic enzyme inhibitors: synthesis and in vitro evaluation of novel 4,5,6,7-tetrahydrothieno[3,2-c]- and -[2,3-c]pyridines.

The discovery of the first class of potent glucose-6-phosphatase catalytic site inhibitors, substituted 4,5,6,7-tetrahydrothieno[3,2-c]- and -[2,3-c]pyridines, is described. Optimisation of this series involved solution phase combinatorial synthesis and very potent compounds were prepared with IC50 values down to 140 nM. The structure activity relationship (SAR) of these compounds indicates that: a tetrahydrothieno[3,2-c]pyridine core ring system and the isomeric [2,3-c] system are equipotent and much better than the corresponding benzo analogue, 1,2,3,4-tetrahydro-isoquinoline. The 4-substituent of the tetrahydrothieno[3,2-c]pyridine ring has to be a phenyl group, optionally substituted with a lipophilic 4-substituent, such as trifluoromethoxy or chloro. The 5-substituent of the tetrahydrothieno[3,2-c]pyridine ring has to be a substituted benzoyl; anisoyl and (E)-3-furan-3-ylacryloyl are the best of the investigated groups. Substitution in the benzoyl ortho position seems to be forbidden, whereas substitution in the meta position is tolerated only if a methoxy para substituent is present. These SAR findings were parallel to those obtained in the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine system. Enantioselectivity in enzyme recognition was observed and the activity resided in all cases only in one of the enantiomers.

ZG-1494 alpha, a novel platelet-activating factor acetyltransferase inhibitor from Penicillium rubrum, isolation, structure elucidation andbiological activity.

A novel inhibitor of platelet-activating factor (PAF) acetyltransferase, an essential enzyme in the remodeling pathway of platelet-activating factor synthesis, was identified by a high throughout screen of natural product extracts of microbial origin. The compound, ZG-1494 alpha, was isolated from an ethyl acetate extract of a culture broth of Penicillium rubrum through bioassay guided fractionation. The structure of ZG-1494 alpha was determined by spectroscopic methods. A key feature of the structure, which is relatively rare among natural products, is the 5-hydroxy-3-pyrrolin-2-one moiety. A 13C-13C INADEQUATE was utilized to unambiguously determine the regiochemistry of this molecule.