The four members of the Janus family of nonreceptor tyrosine kinases play a significant role in immune function. The JAK family kinase inhibitor, tofacitinib 1, has been approved in the United States for use in rheumatoid arthritis (RA) patients. A number of JAK inhibitors with a variety of JAK family selectivity profiles are currently in clinical trials. Our goal was to identify inhibitors that were functionally selective for JAK1 and JAK3. Compound 22 was prepared with the desired functional selectivity profile, but it suffered from poor absorption related to physical properties. Use of the phosphate prodrug 32 enabled progression to a murine collagen induced arthritis (CIA) model. The demonstration of a robust efficacy in the CIA model suggests that use of phosphate prodrugs may resolve issues with progressing this chemotype for the treatment of autoimmune diseases such as RA.

PI3Kδ plays an important role controlling immune cell function and has therefore been identified as a potential target for the treatment of immunological disorders. This article highlights our work toward the identification of a potent, selective, and efficacious PI3Kδ inhibitor. Through careful SAR, the successful replacement of a polar pyrazole group by a simple chloro or trifluoromethyl group led to improved Caco-2 permeability, reduced Caco-2 efflux, reduced hERG PC activity, and increased selectivity profile while maintaining potency in the CD69 hWB assay. The optimization of the aryl substitution then identified a 4'-CN group that improved the human/rodent correlation in microsomal metabolic stability. Our lead molecule is very potent in PK/PD assays and highly efficacious in a mouse collagen-induced arthritis model.

A series of potent dual JAK1/3 inhibitors have been developed from a moderately selective JAK3 inhibitor. Substitution at the C6 position of the pyrrolopyridazine core with aryl groups provided exceptional biochemical potency against JAK1 and JAK3 while maintaining good selectivity against JAK2 and Tyk2. Translation to in vivo efficacy was observed in a murine model of chronic inflammation. X-ray co-crystal structure determination confirmed the presumed inhibitor binding orientation in JAK3. Efforts to reduce hERG channel inhibition will be described.

Cytosolic Phospholipase A2α (cPLA2α) selectively cleaves the sn-2 position of arachidonyl-glycerophospholipids to generate free arachidonic acid. This arachidonic acid is in turn metabolized to a variety of inflammatory mediators including leukotrienes, prostaglandins and thromboxanes. The lysophospholipid remaining after arachidonic acid cleavage can be acetylated to form yet another inflammatory mediator, platelet activating factor, or PAF. Selective inhibition of cPLA2α would provide a novel therapeutic with applications in many disease states including osteoarthritis, rheumatoid arthritis, and asthma. The development of a class of novel and selective indole based inhibitors of cPLA2α is presented. This class of cPLA2α inhibitors is unique in that SAR defined a specific pharmacophore that is completely unrelated to lipophilicity. These inhibitors represent a breakthrough in that they show excellent correlation between activity in isolated enzyme assays and that seen in whole blood assays. These are also the first class of inhibitors that show a predictable correlation between in vitro potency, exposure and in vivo efficacy. Examples that delineate the required pharmacophore, demonstrate good correlations in the assays and show in vivo efficacy will be presented.

A new class of Janus kinase (JAK) inhibitors was discovered using a rationally designed pyrrolo[1,2-b]pyridazine-3-carboxamide scaffold. Preliminary studies identified (R)-(2,2-dimethylcyclopentyl)amine as a preferred C4 substituent on the pyrrolopyridazine core (3b). Incorporation of amino group to 3-position of the cyclopentane ring resulted in a series of JAK3 inhibitors (4g-4j) that potently inhibited IFNγ production in an IL2-induced whole blood assay and displayed high functional selectivity for JAK3-JAK1 pathway relative to JAK2. Further modifications led to the discovery of an orally bioavailable (2-fluoro-2-methylcyclopentyl)amino analogue 5g which is a nanomolar inhibitor of both JAK3 and TYK2, functionally selective for the JAK3-JAK1 pathway versus JAK2, and active in a human whole blood assay.

A novel series of p38 MAP kinase inhibitors with high selectivity for the p38α isoform over the other family members including the highly homologous p38β isoform has been identified. X-ray co-crystallographic studies have revealed an unprecedented kinase binding mode in p38α for representative analogs, 5c and 9d, in which a Leu108/Met109 peptide flip occurs within the p38α hinge region. Based on these findings, a general strategy for the rational design of additional promising p38α isoform selective inhibitors by targeting this novel binding mode is proposed.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Pyrrolo[2,1-f][1,2,4]triazine based inhibitors of p38α have been prepared exploring functional group modifications at the C6 position. Incorporation of aryl and heteroaryl ketones at this position led to potent inhibitors with efficacy in in vivo models of acute and chronic inflammation.

The discovery and characterization of 7k (BMS-582949), a highly selective p38α MAP kinase inhibitor that is currently in phase II clinical trials for the treatment of rheumatoid arthritis, is described. A key to the discovery was the rational substitution of N-cyclopropyl for N-methoxy in 1a, a previously reported clinical candidate p38α inhibitor. Unlike alkyl and other cycloalkyls, the sp(2) character of the cyclopropyl group can confer improved H-bonding characteristics to the directly substituted amide NH. Inhibitor 7k is slightly less active than 1a in the p38α enzymatic assay but displays a superior pharmacokinetic profile and, as such, was more effective in both the acute murine model of inflammation and pseudoestablished rat AA model. The binding mode of 7k with p38α was confirmed by X-ray crystallographic analysis.

The synthesis and structure-activity relationships (SAR) of p38α MAP kinase inhibitors based on a 5-amino-pyrazole scaffold are described. These studies led to the identification of compound 2j as a potent and selective inhibitor of p38α MAP kinase with excellent cellular potency toward the inhibition of TNFα production. Compound 2j was highly efficacious in vivo in inhibiting TNFα production in an acute murine model of TNFα production. X-ray co-crystallography of a 5-amino-pyrazole analog 2f bound to unphosphorylated p38α is also disclosed.

The design, synthesis, and structure-activity relationships (SAR) of a series of 2-aminothiazol-5-yl-pyrimidines as novel p38α MAP kinase inhibitors are described. These efforts led to the identification of 41 as a potent p38α inhibitor that utilizes a unique nitrogen-sulfur intramolecular nonbonding interaction to stabilize the conformation required for binding to the p38α active site. X-ray crystallographic studies that confirm the proposed binding mode of this class of inhibitors in p38 α and provide evidence for the proposed intramolecular nitrogen-sulfur interaction are discussed.

Abstract 2198 Poster Board II-175 Dasatinib (SPRYCEL®), a small molecule tyrosine kinase inhibitor is 325-fold more potent against BCR-ABL than imatinib. Targeting BCR-ABL in chronic myeloid leukemia (CML), dasatinib offers the most favorable benefit-risk ratio with the dose regimen of 100-mg once daily (in comparison with 3 other treatment arms: 50 mg BID, 140 mg QD and 70 mg BID. Duration of cytogenetic response and progression-free survival were similar across all 4 arms, but there was significantly less frequent grade 3-4 neutropenia, thrombocytopenia, anemia and pleural effusion in the 100-mg QD arm compared to the other 3 arms combined (Shah et al, J Clin Oncol 26:3204, 2008). The undiminished efficacy of once daily dosing occurs despite the fact that orally administered dasatinib has a pronounced peak-to-trough plasma pharmacokinetics profile and a relatively short half-life (∼3-5 h), which allows for complete recovery of BCR-ABL kinase activity within 8-12 hrs after a daily dose. The clinical efficacy of once daily dasatinib supports the notion that continuous target (BCR-ABL) inhibition is not required for anti-leukemic activity although the truncated exposure may help to ameliorate other side-effects of “on” or “off” targets inhibition. In addition to BCR-ABL, dasatinib potently inhibits SRC-family kinases (SFKs) with an IC50 in the low single digit nM range; SFKs play key roles in T cell activation. Based on continuous exposure studies, it had been suggested that dasatinib may act as an immunomodulator in vivo. We investigated the effects of variation in exposure duration in vivo and in vitro on the anti-leukemic and T cell activation inhibition activities of dasatinib in preclinical models. Methods. Anti-leukemic activity was determined in vitro in K562 and KU812 CML cell lines, and in vivo as xenografts in K562. BCR-ABL kinase activity was monitored with a phospho-specific CRKL antibody. Human T cells were isolated from PBMC by rosetting with sheep red blood cells and stimulated with anti-CD3/CD28 antibodies for 48 h. Cytokines production were measured by ELISA. T cell proliferation was determined at 3 days by 3 H-thymidine incorporation. Immunocompetency of dasatinib treated mice were determined using the mouse cardiac allograft model and the in vivo MLR T cell proliferation model. Results. Transient (1-6 h) dasatinib exposure of CML cells that caused >80% inhibition of phospho-CRKL is highly cytotoxic. Degree of cytotoxicity directly correlates with the magnitude of BCR-ABL kinase inhibition. In vivo single dose of 30 mg/kg dasatinib administered IV was highly cytotoxic to K562 xenografts as determined by in vivo-in vitro colony formation assay. Intermittent IV dosing regimens of dasatinib (Q4D or Q7D) were effective against K562 xenografts. Dosing regimen in mice (5 mg/kg, PO) that closely mimic the pharmacokinetics of 100 mg oral dose in human was equally efficacious as administering the same dose in 2 split doses (BID, 2.5 mg/kg, PO). In terms of effects on T cell activation, a linear relationship was observed between serum concentration and in vitro T cell proliferation IC50 values. Modeling the human PK profile, delayed dasatinib treatment of T cells after T cell stimulation in vitro led to a time dependent decrease in potency as measured by both IC50 and Emax values. Comparison of serum adjusted IC50 values from these studies to the human PK profile suggests that dasatinib at the approved 100-mg once-daily dose would permit T cell activation on a daily basis. A similar pattern was observed in preclinical in vivo models. Dasatinib was found to be completely protective in a mouse model of cardiac allograft rejection at a dose of 25 mg BID, whereas a dose of 15 mg BID was not protective. In the MLR model, dasatinib inhibits T cell proliferation at 50 mg/kg but at the clinically relevant dose of 5 mg/kg was completely devoid of T cell inhibitory effects. Taken together, these results suggest that dasatinib may be able to provide anti-leukemic activity while avoiding suppression of T cell activation at clinically relevant doses. Disclosures: Schieven: Bristol-Myers Squibb Co: Employment. Zhang: Bristol-Myers Squibb Co: Employment. Pitt: Bristol-Myers Squibb Co: Employment. McGlinchey: Bristol-Myers Squibb Co: Employment. Menard: Bristol-Myers Squibb Co: Employment. Smykla: Bristol-Myers Squibb Co: Employment. Susulic: Bristol-Myers Squibb Co: Employment. Wen: Bristol-Myers Squibb Co: Employment. Wiebesiek: Bristol-Myers Squibb Co: Employment. Townsend: Bristol-Myers Squibb Co: Employment. Lee: Bristol-Myers Squibb Co: Employment.

Dasatinib potently inhibits Src-family kinases in addition to BCR-Abl and blocks T cell responses in vitro, suggesting potential utility in the treatment of autoimmune disease. A linear relationship was observed between serum concentration and in vitro T cell proliferation IC50 values. Modeling the human PK profile, delayed dasatinib treatment of T cells after T cell stimulation in vitro led to a time dependent decrease in potency as measured by both IC50 and Emax values. Comparison of serum adjusted IC50 values from these studies to the human PK profile suggests that dasatinib at the approved 100 mg QD dose would not be able to achieve a blockade of T cell activation on a daily basis. A similar pattern was observed in preclinical in vivo models. Dasatinib was found to be completely protective in a mouse model of cardiac allograft rejection at a dose of 25 mg BID, whereas a dose of 15 mg BID was not protective. By contrast, dasatinib at a dose of 3 mg/kg BID was highly efficacious in the mouse collagen induced arthritis model. Taken together, these results suggest that dasatinib may be able to provide anti-inflammatory effects while avoiding suppression of T cell activation at clinically relevant doses.

A novel structural class of p38 mitogen-activated protein (MAP) kinase inhibitors consisting of substituted 4-(phenylamino)-pyrrolo[2,1- f][1,2,4]triazines has been discovered. An initial subdeck screen revealed that the oxindole-pyrrolo[2,1- f][1,2,4]triazine lead 2a displayed potent enzyme inhibition (IC 50 60 nM) and was active in a cell-based TNFalpha biosynthesis inhibition assay (IC 50 210 nM). Replacement of the C4 oxindole with 2-methyl-5- N-methoxybenzamide aniline 9 gave a compound with superior p38 kinase inhibition (IC 50 10 nM) and moderately improved functional inhibition in THP-1 cells. Further replacement of the C6 ester of the pyrrolo[2,1- f][1,2,4]triazine with amides afforded compounds with increased potency, excellent oral bioavailability, and robust efficacy in a murine model of acute inflammation (murine LPS-TNFalpha). In rodent disease models of chronic inflammation, multiple compounds demonstrated significant inhibition of disease progression leading to the advancement of 2 compounds 11b and 11j into further preclinical and toxicological studies.

Rational design, synthesis, and SAR studies of a novel class of benzothiazole based inhibitors of p38 alpha MAP kinase are described. The issue of metabolic instability associated with vicinal phenyl, benzo[d] thiazol-6-yl oxazoles/imidazoles was addressed by the replacement of the central oxazole or imidazole ring with an aminopyrazole system. The proposed binding mode of this new class of p38 alpha inhibitors was confirmed by X-ray crystallographic studies of a representative inhibitor (6a) bound to the p38 alpha enzyme. (C) 2008 Published by Elsevier Ltd.