Imidazotriazines: Spleen Tyrosine Kinase (Syk) Inhibitors Identified by Free-Energy Perturbation (FEP).

There has been significant interest in spleen tyrosine kinase (Syk) owing to its role in a number of disease states, including autoimmunity, inflammation, and cancer. Ongoing therapeutic programs have resulted in several compounds that are now in clinical use. Herein we report our optimization of the imidazopyrazine core scaffold of Syk inhibitors through the use of empirical and computational approaches. Free-energy perturbation (FEP) methods with MCPRO+ were undertaken to calculate the relative binding free energies for several alternate scaffolds. FEP was first applied retrospectively to determine if there is any predictive value; this resulted in 12 of 13 transformations being predicted in a directionally correct manner. FEP was then applied in a prospective manner to evaluate 17 potential targets, resulting in the realization of imidazotriazine 17 (3-(4-(3,4-dimethoxyphenylamino)imidazo[1,2-f][1,2,4]triazin-2-yl)benzamide), which shows a tenfold improvement in activity relative to the parent compound and no increase in atom count. Optimization of 17 led to compounds with nanomolar cellular activity.

Discovery of isoquinolinone indole acetic acids as antagonists of chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2) for the treatment of allergic inflammatory diseases.

Previously we reported the discovery of CRA-898 (1), a diazine indole acetic acid containing CRTH2 antagonist. This compound had good in vitro and in vivo potency, low rates of metabolism, moderate permeability, and good oral bioavailability in rodents. However, it showed low oral exposure in nonrodent safety species (dogs and monkeys). In the current paper, we wish to report our efforts to understand and improve the poor PK in nonrodents and development of a new isoquinolinone subseries that led to identification of a new development candidate, CRA-680 (44). This compound was efficacious in both a house dust mouse model of allergic lung inflammation (40 mg/kg qd) as well as a guinea pig allergen challenge model of lung inflammation (20 mg/kg bid).

Diazine indole acetic acids as potent, selective, and orally bioavailable antagonists of chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2) for the treatment of allergic inflammatory diseases.

New classes of CRTH2 antagonists, the pyridazine linker containing indole acetic acids, are described. The initial hit 1 had good potency but poor permeability, metabolic stability, and PK. Initial optimization led to compounds of type 2 with low oxidative metabolism but poor oral bioavailability. Poor permeability was identified as a liability for these compounds. Addition of a linker between the indole and diazine moieties afforded a series with good potency, low rates of metabolism, moderate permeability, and good oral bioavailability in rodents. 32 was identified as the development track candidate. It was potent in cell based, binding, and whole blood assays and exhibited good PK profile. It was efficacious in mouse models of contact hypersensitivity (1 mg/kg b.i.d.) and house dust (20 mg/kg q.d.) when dosed orally. In sheep asthma, administration at 1 mg/kg iv completely blocked the LAR and AHR and attenuated the EAR phase.

Discovery of 2-[1-(4-chlorophenyl)cyclopropyl]-3-hydroxy-8-(trifluoromethyl)quinoline-4-carboxylic acid (PSI-421), a P-selectin inhibitor with improved pharmacokinetic properties and oral efficacy in models of vascular injury.

Previously, we reported the discovery of PSI-697 (1a), a C-2 benzyl substituted quinoline salicylic acid-based P-selectin inhibitor. It is active in a variety of animal models of cardiovascular disease. Compound 1a has also been shown to be well tolerated and safe in healthy volunteers at doses of up to 1200 mg in a phase 1 single ascending dose study. However, its oral bioavailability was low. Our goal was to identify a back up compound with equal potency, increased solubility, and increased exposure. We expanded our structure-activity studies in this series by branching at the alpha position of the C-2 benzyl side chain and through modification of substituents on the carboxylic A-ring of the quinoline. This resulted in discovery of PSI-421 with marked improvement in aqueous solubility and pharmacokinetic properties. This compound has shown oral efficacy in animal models of arterial and venous injury and was selected as a preclinical development compound for potential treatment of such diseases as atherosclerosis and deep vein thrombosis.

Bromodecarboxylation of quinoline salicylic acids: increasing the diversity of accessible substituted quinolines.

Quinoline salicylic acids underwent bromodecarboxylation at room temperature upon treatment with N-bromosuccinimide. A wide variety of functional groups was tolerated. Several one-pot transformations were also carried out, allowing the preparation of diverse 4-substituted quinolines.

Characterization of the novel P-selectin inhibitor PSI-697 [2-(4-chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[h] quinoline-4-carboxylic acid] in vitro and in rodent models of vascular inflammation and thrombosis.

P-selectin plays a significant and well documented role in vascular disease by mediating leukocyte and platelet rolling and adhesion. This study characterizes the in vitro activity, pharmacokinetic properties, and the anti-inflammatory and antithrombotic efficacy of the orally active P-selectin small-molecule antagonist PSI-697 [2-(4-chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[h] quinoline-4-carboxylic acid; molecular mass, 367.83]. Biacore and cell-based assays were used to demonstrate the ability of PSI-697 to dose dependently inhibit the binding of human P-selectin to human P-selectin glycoprotein ligand-1, inhibiting 50% of binding at 50 to 125 microM. The pharmacokinetics of PSI-697 in rats were characterized by low clearance, short half-life, low volume of distribution, and moderate apparent oral bioavailability. A surgical inflammation model, using exteriorized rat cremaster venules, demonstrated that PSI-697 (50 mg/kg p.o.) significantly reduced the number of rolling leukocytes by 39% (P < 0.05) versus vehicle control. In a rat venous thrombosis model, PSI-697 (100 mg/kg p.o.) reduced thrombus weight by 18% (P < 0.05) relative to vehicle, without prolonging bleeding time. Finally, in a rat carotid injury model, PSI-697 (30 or 15 mg/kg p.o.) administered 1 h before arterial injury and once daily thereafter for 13 days resulted in dose-dependent decreases in intima/media ratios of 40.2% (P = 0.025) and 25.7% (P = 0.002) compared with vehicle controls. These data demonstrate the activity of PSI-697 in vitro and after oral administration in animal models of both arterial and venous injury and support the clinical evaluation of this novel antagonist of P-selectin in atherothrombotic and venous thrombotic indications.

Identification of a novel class of selective Tpl2 kinase inhibitors: 4-Alkylamino-[1,7]naphthyridine-3-carbonitriles.

We have previously reported the discovery and initial SAR of the [1,7]naphthyridine-3-carbonitriles and quinoline-3-carbonitriles as Tumor Progression Loci-2 (Tpl2) kinase inhibitors. In this paper, we report new SAR efforts which have led to the identification of 4-alkylamino-[1,7]naphthyridine-3-carbonitriles. These compounds show good in vitro and in vivo activity against Tpl2 and improved pharmacokinetic properties. In addition they are highly selective for Tpl2 kinase over other kinases, for example, EGFR, MEK, MK2, and p38. Lead compound 4-cycloheptylamino-6-[(pyridin-3-ylmethyl)-amino]-[1,7]naphthyridine-3-carbonitrile (30) was efficacious in a rat model of LPS-induced TNF-alpha production.

Inhibitors of tumor progression loci-2 (Tpl2) kinase and tumor necrosis factor alpha (TNF-alpha) production: selectivity and in vivo antiinflammatory activity of novel 8-substituted-4-anilino-6-aminoquinoline-3-carbonitriles.

Tumor progression loci-2 (Tpl2) (Cot/MAP3K8) is a serine/threonine kinase in the MAP3K family directly upstream of MEK. Recent studies using Tpl2 knockout mice have indicated an important role for Tpl2 in the lipopolysaccharide (LPS) induced production of tumor necrosis factor alpha (TNF-alpha) and other proinflammatory cytokines involved in diseases such as rheumatoid arthritis. Initial 4-anilino-6-aminoquinoline-3-carbonitrile leads showed poor selectivity for Tpl2 over epidermal growth factor receptor (EGFR) kinase. Using molecular modeling and crystallographic data of the EGFR kinase domain with and without an EGFR kinase-specific 4-anilinoquinazoline inhibitor (erlotinib, Tarceva), we hypothesized that we could diminish the inhibition of EGFR kinase by substitution at the C-8 position of our 4-anilino-6-aminoquinoline-3-carbonitrile leads. The 8-substituted-4-anilino-6-aminoquinoline-3-carbonitriles were prepared from the appropriate 2-substituted 4-nitroanilines. Modifications to the C-6 and C-8 positions led to the identification of compounds with increased inhibition of TNF-alpha release from LPS-stimulated rat and human blood, and these analogues were also highly selective for Tpl2 kinase over EGFR kinase. Further structure-activity based modifications led to the identification of 8-bromo-4-(3-chloro-4-fluorophenylamino)-6-[(1-methyl-1H-imidazol-4-yl)methylamino]quinoline-3-carbonitrile, which demonstrated in vitro as well as in vivo efficacy in inhibition of LPS-induced TNF-alpha production.

Synthesis and biological evaluation of quinoline salicylic acids as P-selectin antagonists.

Leukocyte recruitment of sites of inflammation and tissue injury involves leukocyte rolling along the endothelial wall, followed by firm adherence of the leukocyte, and finally transmigration of the leukocyte across cell junctions into the underlying tissue. The initial rolling step is mediated by the interaction of leukocyte glycoproteins containing active moieties such as sialyl Lewisx (sLex) with P-selectin expressed on endothelial cells. Consequently, inhibition of this interaction by means of a small molecule P-selectin antagonist is an attractive strategy for the treatment of inflammatory diseases such as arthritis. High-throughput screening of the Wyeth chemical library identified the quinoline salicylic acid class of compounds (1) as antagonists of P-selectin, with potency in in vitro and cell-based assays far superior to that of sLex. Through iterative medicinal chemistry, we identified analogues with improved P-selectin activity, decreased inhibition of dihydrooratate dehydrogenase, and acceptable CYP profiles. Lead compound 36 was efficacious in the rat AIA model of rheumatoid arthritis.

2-(4-Chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[H]quinoline-4-carboxylic acid (PSI-697): identification of a clinical candidate from the quinoline salicylic acid series of P-selectin antagonists.

P-selectin-PSGL-1 interaction causes rolling of leukocytes on the endothelial cell surface, which subsequently leads to firm adherence and leukocyte transmigration through the vessel wall into the surrounding tissues. P-selectin is upregulated on the surface of both platelets and endothelium in a variety of atherosclerosis-associated conditions. Consequently, inhibition of this interaction by means of a small molecule P-selectin antagonist is an attractive strategy for the treatment of atherosclerosis. High-throughput screening and subsequent analoging had led to the identification of compound 1 as the lead candidate. Herein, we report the continuation of this work and the discovery of a second-generation series, the tetrahydrobenzoquinoline salicylic acids. These compounds have improved pharmacokinetic properties, and a number of them have shown oral efficacy in mouse and rat models of atherogenesis and vascular injury. The lead 31 (PSI-697), is currently in clinical development for the treatment of atherothrombotic vascular events.

Inhibition of Tpl2 kinase and TNFalpha production with quinoline-3-carbonitriles for the treatment of rheumatoid arthritis.

The synthesis and structure-activity studies of a series of quinoline-3-carbonitriles as inhibitors of Tpl2 kinase are described. Potent inhibitors of Tpl2 kinase with selectivity against a panel of selected kinases in enzymatic assays and specificity in cell-based phosphorylation assays in LPS-treated human monocytes were identified. Selected inhibitors with moderate activity in human whole blood assay effectively inhibited LPS/D-Gal induced TNFalpha release when administered intraperitoneally in mice.

Inhibition of Tpl2 kinase and TNF-alpha production with 1,7-naphthyridine-3-carbonitriles: synthesis and structure-activity relationships.

The synthesis and structure-activity studies of a series of 6-substituted-4-anilino-[1,7]-naphthyridine-3-carbonitriles as inhibitors of Tpl2 kinase are described. The early exploratory work described here may lead to the discovery of compounds with significant therapeutic potential for treating rheumatoid arthritis and other inflammatory diseases.

Quinic acid derivatives as sialyl Lewis(x)-mimicking selectin inhibitors: design, synthesis, and crystal structure in complex with E-selectin.

A search for noncarbohydrate sLe(x) mimics led to the development of quinic acid derivatives as selectin inhibitors. At Wyeth we solved the first cocrystal structure of a small molecule, quinic acid, with E-selectin. In the cocomplex two hydroxyls of quinic acid mimic the calcium-bound fucose of the tetrasaccharide sLe(x). The X-ray structure, together with structure based computational methods, was used to design quinic acid based libraries that were synthesized and evaluated for their ability to block the interaction of sLex with P-selectin. A large number of analogues were prepared using solution-phase parallel synthesis. Selected compounds showed decrease in leukocyte rolling in the IVM mouse model. Compound 2 inhibited neutrophil influx in the murine TIP model and demonstrated good plasma exposure.