Identification, synthesis and SAR of amino substituted pyrido[3,2b]pyrazinones as potent and selective PDE5 inhibitors.

A new class of potent and selective PDE5 inhibitors is disclosed. Guided by X-ray crystallographic data, optimization of an HTS lead led to the discovery of a series of 2-aryl, (N8)-alkyl substituted-6-aminosubstituted pyrido[3,2b]pyrazinones which show potent inhibition of the PDE5 enzyme. Synthetic details and some structure-activity relationships are also presented.

Investigation of aminopyridiopyrazinones as PDE5 inhibitors: Evaluation of modifications to the central ring system.

Efforts to improve the potency and physical properties of the aminopyridiopyrazinone class of PDE5 inhibitors through modification of the core ring system are described. Five new ring systems are evaluated and features that impart improved potency and improved solubility are delineated.

Synthesis, crystal structure, and activity of pyrazole-based inhibitors of p38 kinase.

A series of pyrazole inhibitors of p38 mitogen-activated protein (MAP) kinase were designed using a binding model based on the crystal structure of 1 (SC-102) bound to p38 enzyme. New chemistry using dithietanes was developed to assemble nitrogen-linked substituents at the 5-position of pyrazoles. Calculated log D was used in tandem with structure-based design to guide medicinal chemistry strategy and improve the in vivo activity of a series of molecules. The crystal structure of an optimized inhibitor, 4 (SC-806), in complex with p38 enzyme was obtained to confirm the hypothesis that the addition of a basic nitrogen to the molecule induces an interaction with Asp112 of p38 alpha. A compound identified from this series was efficacious in an animal model of rheumatic disease.

Structure-based design and synthesis of pyrazinones containing novel P1 'side pocket' moieties as inhibitors of TF/VIIa.

We describe the structure-based design, synthesis, and enzymatic activity of a series of substituted pyrazinones as inhibitors of the TF/VIIa complex. These inhibitors contain substituents meta to the P(1) amidine designed to explore additional interactions with the VIIa residues in the so-called 'S(1) side pocket'. A crystal structure of the designed inhibitors demonstrates the ability of the P(1) side pocket moiety to engage Lys192 and main chain of Gly216 via hydrogen bond interactions, thus, providing additional possibility for chemical modification to improve selectivity and/or physical properties of inhibitors.

Synthesis and X-ray crystal structures of substituted fluorobenzene and benzoquinone inhibitors of the tissue factor VIIa complex.

Multistep syntheses of substituted benzenes and benzoquinone inhibitors of tissue Factor VIIa are reported. The benzene analogues were designed such that their substitution pattern would occupy and interact with the S(1), S(2), and S(3) pockets of the tissue Factor VIIa (TF/VIIa) enzyme. The compounds exhibited modest potency on TF/VIIa with selectivity over Factor Xa and thrombin. The X-ray crystal structures of the targeted fluorobenzene 12a and benzoquinone 14 inhibitors bound to TF/VIIa were obtained and will be described.

Design, synthesis, and crystal structure of selective 2-pyridone tissue factor VIIa inhibitors.

Targeted 2-pyridones were selected as tissue Factor VIIa inhibitors and prepared from 2,6-dibromopyridine via a multistep synthesis. A variety of chemical transformations, including regioselective nucleophilic addition, selective nitrogen alkylation, and a Suzuki coupling, afforded the targeted tissue Factor VIIa inhibitors. The pyridone core was selected as a replacement for the pyrazinone core of noncovalent tissue Factor VIIa inhibitors and designed such that their substitution pattern would occupy and interact with the S(1), S(2), and S(3) pockets of the tissue Factor VIIa enzyme. These compounds were tested in several serine protease enzyme assays involved in the coagulation cascade exhibiting modest activity on tissue Factor VIIa with excellent selectivity over thrombin and Factor Xa. Finally, an X-ray crystal structure of inhibitor 14a bound to tissue Factor VIIa was obtained and will be described.

Synthesis and crystal structures of substituted benzenes and benzoquinones as tissue factor VIIa inhibitors.

Several multistep syntheses of substituted benzenes are reported. The benzene analogues were designed such that their substitution pattern would occupy and interact with the S(1), S(2), and S(3) pockets of the tissue Factor VIIa enzyme. A variety of chemical transformations including nucleophilic additions, reductive aminations, Stille couplings, and polymer-assisted solution-phase (PASP) techniques were used to prepare key intermediates and final products. The initial analogues identified some weakly active compounds which ultimately led to a 340 nM (IC(50)) tissue Factor VIIa inhibitor with selectivity over other related enzymes. The structure-activity relationship of these inhibitors and the synthetic progression from the discovery of the lead compound to the development of potent analogues will be discussed. The X-ray crystal structures of fluorobenzene 50c and benzoquinone 54 inhibitors complexed with the TF/VIIa enzyme will also be described.

Polymer-assisted solution-phase library synthesis and crystal structure of alpha-ketothiazoles as tissue factor VIIa inhibitors.

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.

Design, parallel synthesis, and crystal structures of pyrazinone antithrombotics as selective inhibitors of the tissue factor VIIa complex.

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.

A novel mechanism of cyclooxygenase-2 inhibition involving interactions with Ser-530 and Tyr-385.

A variety of drugs inhibit the conversion of arachidonic acid to prostaglandin G2 by the cyclooxygenase (COX) activity of prostaglandin endoperoxide synthases. Several modes of inhibitor binding in the COX active site have been described including ion pairing of carboxylic acid containing inhibitors with Arg-120 of COX-1 and COX-2 and insertion of arylsulfonamides and sulfones into the COX-2 side pocket. Recent crystallographic evidence suggests that Tyr-385 and Ser-530 chelate polar or negatively charged groups in arachidonic acid and aspirin. We tested the generality of this binding mode by analyzing the action of a series of COX inhibitors against site-directed mutants of COX-2 bearing changes in Arg-120, Tyr-355, Tyr-348, and Ser-530. Interestingly, diclofenac inhibition was unaffected by the mutation of Arg-120 to alanine but was dramatically attenuated by the S530A mutation. Determination of the crystal structure of a complex of diclofenac with murine COX-2 demonstrates that diclofenac binds to COX-2 in an inverted conformation with its carboxylate group hydrogen-bonded to Tyr-385 and Ser-530. This finding represents the first experimental demonstration that the carboxylate group of an acidic non-steroidal anti-inflammatory drug can bind to a COX enzyme in an orientation that precludes the formation of a salt bridge with Arg-120. Mutagenesis experiments suggest Ser-530 is also important in time-dependent inhibition by nimesulide and piroxicam.

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.

Polymer-assisted solution-phase (PASP) parallel synthesis of an alpha-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.

The crystal structure, mutagenesis, and activity studies reveal that patatin is a lipid acyl hydrolase with a Ser-Asp catalytic dyad.

Patatin is a nonspecific lipid acyl hydrolase that accounts for approximately 40% of the total soluble protein in mature potato tubers, and it has potent insecticidal activity against the corn rootworm. We determined the X-ray crystal structure of a His-tagged variant of an isozyme of patatin, Pat17, to 2.2 A resolution, employing SeMet multiwavelength anomalous dispersion (MAD) phasing methods. The patatin crystal structure has three molecules in the asymmetric unit, an R-factor of 22.0%, and an R(free) of 27.2% (for 10% of the data not included in the refinement) and includes 498 water molecules. The structure notably revealed that patatin has a Ser-Asp catalytic dyad and an active site like that of human cytosolic phospholipase A(2) (cPLA(2)) [Dessen, A., et al. (1999) Cell 97, 349-360]. In addition, patatin has a folding topology related to that of the catalytic domain of cPLA(2) and unlike the canonical alpha/beta-hydrolase fold. The structure confirms our site-directed mutagenesis and bioactivity data that initially suggested patatin possessed a Ser-Asp catalytic dyad. Alanine-scanning mutagenesis revealed that Ser77 and Asp215 were critical for both esterase and bioactivity, consistent with prior work implicating a Ser residue [Strickland, J. H., et al. (1995) Plant Physiol. 109, 667-674] and a Ser-Asp dyad [Hirschberg, H. J. H. B., et al. (2001) Eur. J. Biochem. 268, 5037-5044] in patatin's catalytic activity. The crystal structure aids the understanding of other structure-function relationships in patatin. Patatin does not display interfacial activation, a hallmark feature of lipases, and this is likely due to the fact that it lacks a flexible lid that can shield the active site.

Solution structure and backbone dynamics of the catalytic domain of matrix metalloproteinase-2 complexed with a hydroxamic acid inhibitor.

MMP-2 is a member of the matrix metalloproteinase family that has been implicated in tumor cell metastasis and angiogenesis. Here, we describe the solution structure of a catalytic domain of MMP-2 complexed with a hydroxamic acid inhibitor (SC-74020), determined by three-dimensional heteronuclear NMR spectroscopy. The catalytic domain, designated MMP-2C, has a short peptide linker replacing the internal fibronectin-domain insertion and is enzymatically active. Distance geometry-simulated annealing calculations yielded 14 converged structures with atomic root-mean-square deviations (r.m.s.d.) of 1.02 and 1.62 A from the mean coordinate positions for the backbone and for all heavy atoms, respectively, when 11 residues at the N-terminus are excluded. The structure has the same global fold as observed for other MMP catalytic domains and is similar to previously solved crystal structures of MMP-2. Differences observed between the solution and the crystal structures, near the bottom of the S1' specificity loop, appear to be induced by the large inhibitor present in the solution structure. The MMP-2C solution structure is compared with MMP-8 crystal structure bound to the same inhibitor to highlight the differences especially in the S1' specificity loop. The finding provides a structural explanation for the selectivity between MMP-2 and MMP-8 that is achieved by large inhibitors.

Molecular docking and high-throughput screening for novel inhibitors of protein tyrosine phosphatase-1B.

High-throughput screening (HTS) of compound libraries is used to discover novel leads for drug development. When a structure is available for the target, computer-based screening using molecular docking may also be considered. The two techniques have rarely been used together on the same target. The opportunity to do so presented itself in a project to discover novel inhibitors for the enzyme protein tyrosine phosphatase-1B (PTP1B), a tyrosine phosphatase that has been implicated as a key target for type II diabetes. A corporate library of approximately 400 000 compounds was screened using high-throughput experimental techniques for compounds that inhibited PTP1B. Concurrently, molecular docking was used to screen approximately 235 000 commercially available compounds against the X-ray crystallographic structure of PTP1B, and 365 high-scoring molecules were tested as inhibitors of the enzyme. Of approximately 400 000 molecules tested in the high-throughput experimental assay, 85 (0.021%) inhibited the enzyme with IC50 values less than 100 microM; the most active had an IC50 value of 4.2 microM. Of the 365 molecules suggested by molecular docking, 127 (34.8%) inhibited PTP1B with IC50 values less than 100 microM; the most active of these had an IC50 of 1.7 microM. Structure-based docking therefore enriched the hit rate by 1700-fold over random screening. The hits from both the high-throughput and docking screens were dissimilar from phosphotyrosine, the canonical substrate group for PTP1B; the two hit lists were also very different from each other. Surprisingly, the docking hits were judged to be more druglike than the HTS hits. The diversity of both hit lists and their dissimilarity from each other suggest that docking and HTS may be complementary techniques for lead discovery.