Michael S. South

Structure-based drug design of pyrazinone antithrombotics as selective inhibitors of the tissue factor VIIa complex

Structure-based drug design coupled with polymer-assisted solution-phase library synthesis was utilized to develop a series of pyrazinone inhibitors of the tissue factor/Factor VIIa complex. The crystal structure of a tri-peptide ketothiazole complexed with TF/VIIa was utilized in a docking experiment that identified a benzyl-substituted pyrazinone as a P(2) surrogate for the tri-peptide. A 5-step PASP library synthesis of these aryl-substituted pyrazinones was developed. The sequence allows for attachment of a variety of P(1) and P(3) moieties, which led to synthesis pyrazinone 23. Compound 23 exhibited 16 nM IC(50) against TF/VIIa with >6250x selectivity versus Factor Xa and thrombin. This potent and highly selective inhibitor of TF/VIIa was chosen for pre-clinical intravenous proof-of-concept studies to demonstrate the separation between antithrombotic efficacy and bleeding side effects in a primate model of thrombosis.

Synthesis of 2-pyridones as tissue factor VIIa inhibitors

2-Pyridones were prepared from 2,6-dibromopyridine via a multi-step synthesis. A variety of chemical transformations, including regioselective nucleophilic addition and selective nitrogen alkylation, afforded the penultimate intermediate 9. A combination of two-dimensional NMR techniques to unequivocally assign the structure of 9 is described. Compound 9 was then used in a Suzuki coupling and further derivatized to afford the targeted tissue Factor VIIa inhibitors. These compounds were tested in several serine protease enzyme assays with biological activity reported.

Polymer-Assisted solution-Phase (PASP) parallel synthesis of an α-Ketothiazole library as tissue factor VIIa inhibitors

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.

Multi-step polymer-assisted solution-phase (PASP) library synthesis of functionalized diaminobenzamides.

A parallel solution-phase library synthesis of functionalized diaminobenzamides is described. The four-step library synthesis is accomplished using polymer-assisted solution-phase (PASP) synthesis techniques. This high-yielding, multi-step sequence utilizes sequestering resins for the removal of reactants, reactant by-products, and employs a resin capture/release strategy as a key library synthesis step. Step one of the sequence relies on the displacement of an activated fluoro-group from the aromatic ring of 1a, b with a variety of primary amines to introduce the first diversity position. Step two is hydrolysis of the benzoate ester to a benzoic acid which is subsequently captured on a polyamine resin, washed, and released to give 4a, b in pure form. Step three utilizes PASP resins to mediate the amide coupling of a benzoic acid with a variety of primary amines to give the aminonitrobenzamides 5a, b and introduces the second diversity position. Step four is the parallel reduction of the aminonitrobenzamides 5a, b to the functionalized diaminobenzamides 6a, b. This library synthesis proceeds with high overall purities which average 80 % over the 4-step sequence.