Isoxazolines as potent antagonists of the integrin alpha(v)beta(3).

Starting with lead compound 2, we sought to increase the selectivity for alpha(v)beta(3)-mediated cell adhesion by examining the effects of structural changes in both the guanidine mimetic and the substituent alpha to the carboxylate. To prepare some of the desired aminoimidazoles, a novel reductive amination utilizing a trityl-protected aminoimidazole was developed. It was found that guanidine mimetics with a wide range of pK(a)'s were potent antagonists of alpha(v)beta(3). In general, it appeared that an acylated 2-aminoimidazole guanidine mimetic imparted excellent selectivity for alpha(v)beta(3)-mediated adhesion versus alpha(IIb)beta(3)-mediated platelet aggregation, with selectivity of approximately 3 orders of magnitude observed for compounds 3g and 3h. It was also found in this series that the alpha-substituent was required for potent activity and that 2,6-disubstituted arylsulfonamides were optimal. In addition, the selective alpha(v)beta(3) antagonist 3h was found to be a potent inhibitor of alpha(v)beta(3)-mediated cell migration.

Disubstituted indazoles as potent antagonists of the integrin alpha(v)beta(3).

A new series of indazole-containing alpha(v)beta(3) integrin antagonists is described. Starting with lead compound 18a, variations in a number of structural features were explored with respect to inhibition of the binding of beta(3)-transfected 293 cells to fibrinogen and to selectivity for alpha(v)beta(3) over GPIIbIIIa, another RGD-binding integrin. Indazoles attached to a 2-aminopyridine or 2-aminoimidazole by a propylene linker at the indazole 1-position and to a diaminopropionate derivative via a 5-carboxylate amide provided the best potency with moderate selectivity. Several differences in the SAR of the diaminopropionate moiety were observed between this series and a series of isoxazoline-based selective GPIIbIIIa antagonists. Compound 34a (SM256) was a potent antagonist of alpha(v)beta(3) (IC(50) 2.3 nM) with 9-fold selectivity over GPIIbIIIa.

The characterization of potent novel warfarin analogs.

Racemic sodium warfarin, Coumadin, is widely used in the prevention of thromboembolic disease. The present study was undertaken to characterize three novel classes of warfarin analogs, and to compare them with the warfarin enantiomers. All three classes of compounds inhibit vitamin K epoxide reductase, the enzyme inhibited by racemic warfarin. The alcohol and the ester analogs have reduced protein binding compared with R-(+)-warfarin. The ester and the fluoro-derivatives have similar in vivo anticoagulant activity in the rat to that of S-(-)-warfarin. Thus, it is possible to synthesize novel warfarin analogs that differ from racemic warfarin or its enantiomers in certain selected properties.

Warfarin analog inhibition of human CYP2C9-catalyzed S-warfarin 7-hydroxylation.

Human metabolism of the S-warfarin enantiomer is catalyzed primarily by cytochrome P4502C9 (CYP2C9), which, because of the enzyme's broad drug substrate specificity, leads to drug-S-warfarin interactions. Several warfarin analogs have been synthesized and used to determine whether they exhibit diminished interactions with CYP2C9. The kinetics of the warfarin analogs' inhibition of human liver microsomal CYP2C9 catalyzed metabolism of S-warfarin to S-7-hydroxywarfarin have been investigated. R- and S-7-fluorowarfarin were both predominantly competitive inhibitors, whereas racemic 6-fluorowarfarin and racemic 6,7,8-trifluorowarfarin were predominantly mixed inhibitors with some competitive inhibition. For the alcohols produced by reductive methylation of the side chain of R- and S-warfarin, the R-enantiomer did not inhibit S-warfarin metabolism, whereas the S-enantiomer was primarily a competitive inhibitor. The fluorine substituted warfarins and the S-warfarin alcohol apparently bind with high affinity to CYP2C9. Thus their use clinically (if efficacious) would not prevent CYP2C9 associated warfarin-drug interactions. The R-warfarin alcohol did not inhibit CYP2C9 catalyzed metabolism of S-warfarin and is less likely than warfarin to participate in CYP2C9 associated warfarin-drug interactions.

Human cytomegalovirus US3 and UL36-38 immediate-early proteins regulate gene expression.

We have established the ability of the human cytomegalovirus (HCMV) UL36-38 and US3 immediate-early (IE) gene products to alter gene expression in human cells by using transient transfection assays. The cellular heat shock protein 70 (hsp70) promoter was transactivated following cotransfection with the HCMV IE regions in nonpermissive HeLa cells by UL36-38, US3, or IE1 and in permissive human diploid fibroblasts (HFF) by IE1 or IE2. Moreover, hsp70 expression was synergistically increased in HeLa cells cotransfected with US3 and UL36, with US3 and UL37, or with US3 and UL37x1. The synergistic transactivation of hsp70 expression by US3 and UL36-38 was not observed in HFF cells. Synergy was also not observed in HeLa cells between US3 and UL38, an early gene product encoded by the UL36-38 IE locus. Synergistic transactivation of hsp70 expression in HeLa cells required the syntheses of UL36-38 and US3 IE proteins, since nonsense mutants were not functional. hsp70 expression increased with increasing amounts of transfected US3 and UL37 DNA and occurred at the level of stable hsp70-promoted RNA. In contrast to the broad hsp70 response, promoters from the HCMV UL112 early gene and another cellular gene, brain creatine kinase, both responded strongly only to singly transfected IE2 in HeLa cells. Nevertheless, IE2 transactivation of the UL112 promoter was further stimulated by cotransfection of IE1 or of UL36-38 in both HeLa and HFF cells. Thus, different patterns of promoter transactivation and interactions between HCMV IE gene products in transactivation were found in HFF cells and in HeLa cells. These results establish the ability of the HCMV US3 and UL36-38 proteins to alter cellular and viral gene expression and are consistent with involvement of cellular transcription factors in HCMV IE regulation of gene expression.