Phthalazinone-based lactams and cyclic ureas as ROCK2 selective inhibitors.

Derivatives of lactam, cyclic urea and carbamate were explored as aniline amide replacements in a series of phthalazinone-based ROCK inhibitors. Potent ROCK2 inhibitors such as 22 were identified with excellent overall kinase selectivity as well as good isoform selectivity over ROCK1.

Discovery of a phenylpyrazole amide ROCK inhibitor as a tool molecule for in vivo studies.

Structure-activity relationship optimization on a series of phenylpyrazole amides led to the identification of a dual ROCK1 and ROCK2 inhibitor (25) which demonstrated good potency, kinome selectivity and favorable pharmacokinetic profiles. Compound 25 was selected as a tool molecule for in vivo studies including evaluating hemodynamic effects in telemeterized mice, from which moderate decreases in blood pressure were observed.

Recent encounters with atropisomerism in drug discovery.

Atropisomerism is stereochemistry arising from restricted bond rotation that creates a chiral axis. Atropisomers are subject to time-dependent inversion of chirality via bond rotation, a property which in drug molecules introduces complexity and challenges for drug discovery and development processes. Greater recognition of the occurrence of atropisomerism and improved characterization techniques have helped medicinal chemists successfully advance atropisomeric drug molecules. This review provides recent examples of atropisomerism encountered in medicinal chemistry efforts and the strategies used to address the accompanying challenges.

Neutral macrocyclic factor VIIa inhibitors.

Factor VIIa (FVIIa) inhibitors have shown strong antithrombotic efficacy in preclinical thrombosis models with limited bleeding liabilities. Discovery of potent, orally active FVIIa inhibitors has been largely unsuccessful due to the requirement of a basic P1 group to interact with Asp189 in the S1 binding pocket, limiting their membrane permeability. We have combined recently reported neutral P1 binding substituents with a highly optimized macrocyclic chemotype to produce FVIIa inhibitors with low nanomolar potency and enhanced permeability.

Synthesis and P1' SAR exploration of potent macrocyclic tissue factor-factor VIIa inhibitors.

Selective tissue factor-factor VIIa complex (TF-FVIIa) inhibitors are viewed as promising compounds for treating thrombotic disease. In this contribution, we describe multifaceted exploratory SAR studies of S1'-binding moieties within a macrocyclic chemotype aimed at replacing cyclopropyl sulfone P1' group. Over the course of the optimization efforts, the 1-(1H-tetrazol-5-yl)cyclopropane P1' substituent emerged as an improved alternative, offering increased metabolic stability and lower clearance, while maintaining excellent potency and selectivity.

Design and synthesis of potent, selective phenylimidazole-based FVIIa inhibitors.

Heterocyclic amide isosteres were incorporated into a phenylglycine-based tissue factor/factor VIIa (TF-FVIIa) inhibitor chemotype, providing potent inhibitors. An X-ray co-crystal structure of phenylimidazole 19 suggested that an imidazole nitrogen atom effectively mimics an amide carbonyl, while the phenyl ring forms key hydrophobic interactions with the S1' pocket. Exploration of phenylimidazole substitution led to the discovery of potent, selective and efficacious inhibitors of TF-FVIIa.

Design and synthesis of bicyclic pyrazinone and pyrimidinone amides as potent TF-FVIIa inhibitors.

Bicyclic pyrazinone and pyrimidinone amides were designed and synthesized as potent TF-FVIIa inhibitors. SAR demonstrated that the S2 and S3 pockets of FVIIa prefer to bind small, lipophilic groups. An X-ray crystal structure of optimized compound 9b bound in the active site of FVIIa showed that the bicyclic scaffold provides 5 hydrogen bonding interactions in addition to projecting groups for interactions within the S1, S2 and S3 pockets. Compound 9b showed excellent FVIIa potency, good selectivity against FIXa, Xa, XIa and chymotrypsin, and good clotting activity.

Nonbenzamidine acylsulfonamide tissue factor-factor VIIa inhibitors.

Aminoisoquinoline and isoquinoline groups have successfully replaced the more basic P1 benzamidine group of an acylsulfonamide factor VIIa inhibitor. Inhibitory activity was optimized by the identification of additional hydrophobic and hydrophilic P' binding interactions. The molecular details of these interactions were elucidated by X-ray crystallography and molecular modeling. We also show that decreasing the basicity of the P1 group results in improved oral bioavailability in this chemotype.

Atropisomer Control in Macrocyclic Factor VIIa Inhibitors.

Incorporation of a methyl group onto a macrocyclic FVIIa inhibitor improves potency 10-fold but is accompanied by atropisomerism due to restricted bond rotation in the macrocyclic structure, as demonstrated by NMR studies. We designed a conformational constraint favoring the desired atropisomer in which this methyl group interacts with the S2 pocket of FVIIa. A macrocyclic inhibitor incorporating this constraint was prepared and demonstrated by NMR to reside predominantly in the desired conformation. This modification improved potency 180-fold relative to the unsubstituted, racemic macrocycle and improved selectivity. An X-ray crystal structure of a closely related analogue in the FVIIa active site was obtained and matches the NMR and modeled conformations, confirming that this conformational constraint does indeed direct the methyl group into the S2 pocket as designed. The resulting rationally designed, conformationally stable template enables further optimization of these macrocyclic inhibitors.

Discovery of a Highly Potent, Selective, and Orally Bioavailable Macrocyclic Inhibitor of Blood Coagulation Factor VIIa-Tissue Factor Complex.

Inhibitors of the tissue factor (TF)/factor VIIa complex (TF-FVIIa) are promising novel anticoagulants which show excellent efficacy and minimal bleeding in preclinical models. Starting with an aminoisoquinoline P1-based macrocyclic inhibitor, optimization of the P' groups led to a series of highly potent and selective TF-FVIIa inhibitors which displayed poor permeability. Fluorination of the aminoisoquinoline reduced the basicity of the P1 group and significantly improved permeability. The resulting lead compound was highly potent, selective, and achieved good pharmacokinetics in dogs with oral dosing. Moreover, it demonstrated robust antithrombotic activity in a rabbit model of arterial thrombosis.

P2Y6 receptor potentiates pro-inflammatory responses in macrophages and exhibits differential roles in atherosclerotic lesion development.

BACKGROUND: P2Y(6), a purinergic receptor for UDP, is enriched in atherosclerotic lesions and is implicated in pro-inflammatory responses of key vascular cell types and macrophages. Evidence for its involvement in atherogenesis, however, has been lacking. Here we use cell-based studies and three murine models of atherogenesis to evaluate the impact of P2Y(6) deficiency on atherosclerosis. METHODOLOGY/PRINCIPAL FINDINGS: Cell-based studies in 1321N1 astrocytoma cells, which lack functional P2Y(6) receptors, showed that exogenous expression of P2Y(6) induces a robust, receptor- and agonist-dependent secretion of inflammatory mediators IL-8, IL-6, MCP-1 and GRO1. P2Y(6)-mediated inflammatory responses were also observed, albeit to a lesser extent, in macrophages endogenously expressing P2Y(6) and in acute peritonitis models of inflammation. To evaluate the role of P2Y(6) in atherosclerotic lesion development, we used P2Y(6)-deficient mice in three mouse models of atherosclerosis. A 43% reduction in aortic arch plaque was observed in high fat-fed LDLR knockout mice lacking P2Y(6) receptors in bone marrow-derived cells. In contrast, no effect on lesion development was observed in fat-fed whole body P2Y(6)xLDLR double knockout mice. Interestingly, in a model of enhanced vascular inflammation using angiotensin II, P2Y(6) deficiency enhanced formation of aneurysms and exhibited a trend towards increased atherosclerosis in the aorta of LDLR knockout mice. CONCLUSIONS: P2Y(6) receptor augments pro-inflammatory responses in macrophages and exhibits a pro-atherogenic role in hematopoietic cells. However, the overall impact of whole body P2Y(6) deficiency on atherosclerosis appears to be modest and could reflect additional roles of P2Y(6) in vascular disease pathophysiologies, such as aneurysm formation.

Design and Synthesis of Phenylpyrrolidine Phenylglycinamides As Highly Potent and Selective TF-FVIIa Inhibitors.

Inhibitors of the Tissue Factor/Factor VIIa (TF-FVIIa) complex are promising novel anticoagulants that show excellent efficacy and minimal bleeding in preclinical models. On the basis of a zwitterionic phenylglycine acylsulfonamide 1, a phenylglycine benzylamide 2 was shown to possess improved permeability and oral bioavailability. Optimization of the benzylamide, guided by X-ray crystallography, led to a potent TF-FVIIa inhibitor 18i with promising oral bioavailability, but promiscuous activity in an in vitro safety panel of receptors and enzymes. Introducing an acid on the pyrrolidine ring, guided by molecular modeling, resulted in highly potent, selective, and efficacious TF-FVIIa inhibitors with clean in vitro safety profile. The pyrrolidine acid 20 showed a moderate clearance, low volume of distribution, and a short t 1/2 in dog PK studies.

Design and synthesis of bicyclic pyrimidinone-based HCV NS3 protease inhibitors.

A series of bicyclic pyrimidinone-based HCV NS3 protease inhibitors was synthesized via selective C8 position functionalization. Substituted phenylamides and phenylureas were preferred in the S2 binding pocket.

On the power of chemical synthesis: immunological evaluation of models for multiantigenic carbohydrate-based cancer vaccines.

Synthetic carbohydrate cancer vaccines have been shown to stimulate antibody-based immune responses in both preclinical and clinical settings. The antibodies have been observed to react in vitro with the corresponding natural carbohydrate antigens expressed on the surface of tumor cells, and are able to mediate complement-dependent and/or antibody-dependent cell-mediated cytotoxicity. Furthermore, these vaccines have proven to be safe when administered to cancer patients. Until recently, only monovalent antigen constructs had been prepared and evaluated. Advances in total synthesis have now enabled the preparation of multivalent vaccine constructs, which contain several different tumor-associated carbohydrate antigens. Such constructs could, in principle, serve as superior mimics of cell surface antigens and, hence, as potent cancer vaccines. Here we report preclinical ELISA-based evaluation of a TF-Le(y)-Tn bearing construct (compound 3) with native mucin glycopeptide architecture and a Globo-H-Le(y)-Tn glycopeptide (compound 4) with a nonnative structure. Mice were immunized with one or the other of these constructs as free glycopeptides or as keyhole lymphet hemocyanin conjugates. Either QS-21 or the related GPI-0100 were coadministered as adjuvants. Both keyhole lymphet hemocyanin conjugates induced IgM and IgG antibodies against each carbohydrate antigen, however, the mucin-based TF-Le(y)-Tn construct was shown to be less antigenic than the unnatural Globo-H-Le(y)-Tn construct. The adjuvants, although related, proved significantly different, in that GPI-0100 consistently induced higher titers of antibodies than QS-21. The presence of multiple glycans in these constructs did not appear to suppress the response against any of the constituent antigens. Compound 4, the more antigenic of the two constructs, was also examined by fluorescence activated cell sorter analysis. Significantly, from these studies it was shown that antibodies stimulated in response to compound 4 reacted with tumor cells known to selectively express the individual antigens. The results demonstrate that single vaccine constructs bearing several different carbohydrate antigens have the potential to stimulate a multifaceted immune response.

Principles of mucin architecture: structural studies on synthetic glycopeptides bearing clustered mono-, di-, tri-, and hexasaccharide glycodomains.

The structural characteristics of a mucin glycopeptide motif derived from the N-terminal fragment STTAV of the cell surface glycoprotein CD43 have been investigated by NMR. In this study, a series of molecules prepared by total synthesis were examined, consisting of the peptide itself, three glycopeptides having clustered sites of alpha-O-glycosylation on the serine and threonine side chains with the Tn, TF, and STF carbohydrate antigens, respectively, and one with the beta-O-linked TF antigen. Additionally, a glycopeptide having the sequence SSSAVAV, triglycosylated with the Le(y) epitope, was investigated. NMR data for the tri-STF-STTAV glycopeptide were used to solve the structure of this construct through restrained molecular dynamics calculations. The calculations revealed a defined conformation for the glycopeptide core rooted in the interaction of the peptide and the first N-acetylgalactosamine residue. The similarity of the NMR data for each of the alpha-O-linked glycopeptides demonstrates that this structure persists for each construct and that the mode of attachment of the first sugar and the peptide is paramount in establishing the organization of the core. The core provides a common framework on which a variety of glycans may be displayed. Remarkably, while there is a profound organizational effect on the peptide backbone with the alpha-linked glycans, attachment via a beta-linkage has little apparent consequence.