ABSTRACT
A solution-phase synthesis of an alpha-ketothiazole library of the general form D-Phe-L-AA-Arg-alpha-ketothiazole is described. The five-step synthesis is accomplished using a combination of polymeric reagents and polymer-assisted solution-phase purification concepts, including reactant-sequestering resins, reagent-sequestering resins, and tagged reagents. The multistep synthesis affords desired alpha-ketothiazole products in excellent purities and yields. A variety of L-amino acid inputs were used to probe the S2 pocket of tissue Factor VIIa enzyme to influence both potency and selectivity. An X-ray crystal structure of compound 10k bound to the TF/VIIa complex was obtained that explains the observed selectivity. The alpha-ketothiazoles were found to be potent, reversible-covalent inhibitors of tissue Factor VIIa, with some analogues demonstrating selectivity over thrombin.
Subject(s)
Combinatorial Chemistry Techniques/methods , Factor VIIa/antagonists & inhibitors , Ketones/chemistry , Thiazoles/chemical synthesis , Thiazoles/pharmacology , Thromboplastin/antagonists & inhibitors , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Factor VIIa/genetics , Factor VIIa/metabolism , Humans , Inhibitory Concentration 50 , Models, Molecular , Polymers/chemistry , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/metabolism , Thrombin/antagonists & inhibitors , Thrombin/metabolism , Thromboplastin/genetics , Thromboplastin/metabolismABSTRACT
Structure-based drug design (SBDD) and polymer-assisted solution-phase (PASP) library synthesis were used to develop a series of pyrazinone inhibitors of the Tissue Factor/Factor VIIa (TF/VIIa) complex. The crystal structure of a tripeptide-alpha-ketothiazole complexed with TF/VIIa was utilized in a docking experiment to identify the pyrazinone core as a starting scaffold. The pyrazinone core could orient the substituents in the correct spatial arrangement to probe the S1, S2, and S3 pockets of the enzyme. A multistep PASP library synthesis was designed to prepare the substituted pyrazinones varying the P1, P2, and P3 moieties. Hundreds of pyrazinone TF/VIIa inhibitors were prepared and tested in several serine protease enzyme assays involved in the coagulation cascade. The inhibitors exhibited modest activity on TF/VIIa with excellent selectivity over thrombin (IIa) and Factor Xa. The structure-activity relationship of the pyrazinone inhibitors will be discussed and X-ray crystal structures of selected compounds complexed with the TF/VIIa enzyme will be described. This study ultimately led to the synthesis of compound 34, which exhibited 16 nM (IC50) activity on TF/VIIa with >6250 x selectivity vs Factor Xa and thrombin. This potent and highly selective inhibitor of TF/VIIa was chosen for preclinical, intravenous proof-of-concept studies to demonstrate the separation between antithrombotic efficacy and bleeding side effects in a nonhuman primate model of electrolytic-induced arterial thrombosis.
Subject(s)
Factor VIIa/antagonists & inhibitors , Fibrinolytic Agents/chemical synthesis , Fibrinolytic Agents/pharmacology , Pyrazines/chemical synthesis , Pyrazines/pharmacology , Thromboplastin/antagonists & inhibitors , Antithrombin III/pharmacology , Binding Sites , Combinatorial Chemistry Techniques/methods , Crystallography, X-Ray , Drug Design , Factor VIIa/chemistry , Factor VIIa/genetics , Fibrinolytic Agents/chemistry , Humans , Inhibitory Concentration 50 , Models, Molecular , Pyrazines/chemistry , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Serine Proteinase Inhibitors/pharmacology , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/pharmacology , Thrombin/antagonists & inhibitors , Thromboplastin/chemistryABSTRACT
A solution-phase synthesis of an alpha-ketothiazole library of the general form D-Phe-L-AA-L-Arg-alpha-ketothiazole is described. The five-step synthesis is accomplished using a combination of polymeric reagents and polymer-assisted solution-phase purification protocols, including reactant-sequestering resins, reagent-sequestering resins, and tagged reagents. The multi-step synthesis affords the desired alpha-ketothiazole products in excellent purities and yields. A variety of L-amino acid inputs were used to probe the S2 pocket of the tissue factor (TF) VIIa enzyme to influence both potency and selectivity. An X-ray crystal structure of compound 10e bound to the TF/VIIa complex was obtained that explains the observed selectivity. The alpha-ketothiazoles were found to be potent, reversible-covalent inhibitors of tissue factor VIIa, with some analogues demonstrating selectivity versus thrombin.