We have addressed the key deficiency of noncovalent pyridinone acetamide thrombin inhibitor L-374,087 (1), namely, its modest half-lives in animals, by making a chemically stable 3-alkylaminopyrazinone bioisostere for its 3-sulfonylaminopyridinone core. Compound 3 (L-375,378), the closest aminopyrazinone analogue of 1, has comparable selectivity and slightly decreased efficacy but significantly improved pharmacokinetics in rats, dogs, and monkeys to 1. We have developed an efficient and versatile synthesis of 3, and this compound has been chosen for further preclinical and clinical development.

No abstract

No abstract

The interaction of thrombin with several potent and selective ␣-ketoamide transition state analogs was characterized. L-370,518 (H-N-Me-D-Phe-Pro-t-4-aminocyclohexylglycyl N-methylcarboxamide) a potent (K i ‫؍‬ 90 pM) and selective (>10 4 -fold versus trypsin) ketoamide thrombin inhibitor was shown to bind thrombin via a two-step reaction wherein the initially formed thrombin-inhibitor complex (EI 1 ) rearranges to a more stable, final complex (EI 2 ). A novel sequential stopped-flow analysis showed that k ؊1 , the rate constant for dissociation of EI 1 , was comparable to k 2 , the rate constant for conversion of EI 1 to EI 2 (0.049 and 0.035 s ؊1, respectively) indicating that formation of the initial complex EI 1 is partially rate controlling. Replacement of the Nterminal methylamino group in L-370,518 with a hydrogen (L-372,051) resulted in a 44-fold loss in potency (K i ‫؍‬ 4 nM) largely due to an increase in k ؊1 . Consequently in the reaction of L-372,051 with thrombin formation of EI 1 was not rate controlling. Replacement of the P1 N-methylcarboxamide group of L-370,518 with an azetidylcarboxamido (L-372,228) produced a 58-fold increase in the value of the equilibrium constant (K ؊1 ) for dissociation of EI 1 . Nevertheless, L-372,228 was a 2-fold more potent thrombin inhibitor (K i ‫؍‬ 40 pM) than L-370,518 due to its 16-fold higher k 2 and 10-fold lower k ؊2 values. The desketoamide analogs of L-370,518 and L-372,051, namely L-371,912 and L-372,011, inhibited thrombin via a one-step process. The K i value for L-371,912 and the K ؊1 value for its ␣-ketoamide analog, L-370,518, were similar (5 and 14 nM, respectively). Likewise, the K i value for L-372,011 and the K ؊1 value for its ␣-ketoamide analog, L-372,051, were similar (330 and 285 nM, respectively). These observations are consistent with the view that the ␣-ketoamides L-370,518 and L-372,051 form initial complexes with thrombin that are similar to the complexes formed by their desketoamide analogs, and in a second step the ␣-ketoamides react with the active site serine residue of thrombin to form a more stable hemiketal adduct.Our pursuit of novel antithrombotic agents has focused on direct inhibitors of thrombin, a trypsin-like serine proteinase that plays a key role in thrombosis. One strategy for the design of potent thrombin inhibitors is to replace the substrate P1 1 carboxamido group with an electrophilic keto or aldehyde group (1). Cyclotheonamide A is a naturally occurring proteinase inhibitor from a marine sponge (Theonella) that contains such a potency-enhancing keto group (2-4). Cyclotheonamide A is a potent (K i ϭ 1 nM) reversible inhibitor of thrombin (5, 6) and several other trypsin-like proteinases (5, 7). Various ␣-ketoamide derivatives of tripeptide substrates of thrombin have been prepared in an attempt to identify selective, potent, reversible inhibitors of thrombin (8, 9). Placement of a t-4-AChxGly 2 residue at the P1 position in ␣-ketoamide thrombin inhibitors provided excellent selectivity for thrombin, relative to tryspin...

No abstract