Identification of N-Methyl Nicotinamide and N-Methyl Pyridazine-3-Carboxamide Pseudokinase Domain Ligands as Highly Selective Allosteric Inhibitors of Tyrosine Kinase 2 (TYK2).

As a member of the Janus (JAK) family of nonreceptor tyrosine kinases, TYK2 plays an important role in mediating the signaling of pro-inflammatory cytokines including IL-12, IL-23, and type 1 interferons. The nicotinamide 4, identified by a SPA-based high-throughput screen targeting the TYK2 pseudokinase domain, potently inhibits IL-23 and IFNα signaling in cellular assays. The described work details the optimization of this poorly selective hit (4) to potent and selective molecules such as 47 and 48. The discoveries described herein were critical to the eventual identification of the clinical TYK2 JH2 inhibitor (see following report in this issue). Compound 48 provided robust inhibition in a mouse IL-12-induced IFNγ pharmacodynamic model as well as efficacy in an IL-23 and IL-12-dependent mouse colitis model. These results demonstrate the ability of TYK2 JH2 domain binders to provide a highly selective alternative to conventional TYK2 orthosteric inhibitors.

Highly Selective Inhibition of Tyrosine Kinase 2 (TYK2) for the Treatment of Autoimmune Diseases: Discovery of the Allosteric Inhibitor BMS-986165.

Small molecule JAK inhibitors have emerged as a major therapeutic advancement in treating autoimmune diseases. The discovery of isoform selective JAK inhibitors that traditionally target the catalytically active site of this kinase family has been a formidable challenge. Our strategy to achieve high selectivity for TYK2 relies on targeting the TYK2 pseudokinase (JH2) domain. Herein we report the late stage optimization efforts including a structure-guided design and water displacement strategy that led to the discovery of BMS-986165 (11) as a high affinity JH2 ligand and potent allosteric inhibitor of TYK2. In addition to unprecedented JAK isoform and kinome selectivity, 11 shows excellent pharmacokinetic properties with minimal profiling liabilities and is efficacious in several murine models of autoimmune disease. On the basis of these findings, 11 appears differentiated from all other reported JAK inhibitors and has been advanced as the first pseudokinase-directed therapeutic in clinical development as an oral treatment for autoimmune diseases.

Discovery and structure-based design of 4,6-diaminonicotinamides as potent and selective IRAK4 inhibitors.

The identification of small molecule inhibitors of IRAK4 for the treatment of autoimmune diseases has been an area of intense research. We discovered novel 4,6-diaminonicotinamides which potently inhibit IRAK4. Optimization efforts were aided by X-ray crystal structures of inhibitors bound to IRAK4. Structure activity relationship (SAR) studies led to the identification of compound 29 which exhibited sub-micromolar potency in a LTA stimulated cellular assay.

Discovery of highly potent, selective, covalent inhibitors of JAK3.

A useful and novel set of tool molecules have been identified which bind irreversibly to the JAK3 active site cysteine residue. The design was based on crystal structure information and a comparative study of several electrophilic warheads.

Discovery of ((4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-methylpyrrolo[1,2-f][1,2,4]triazine-6-carbonyl)(propyl)carbamoyloxy)methyl-2-(4-(phosphonooxy)phenyl)acetate (BMS-751324), a Clinical Prodrug of p38α MAP Kinase Inhibitor.

In search for prodrugs to address the issue of pH-dependent solubility and exposure associated with 1 (BMS-582949), a previously disclosed phase II clinical p38α MAP kinase inhibitor, a structurally novel clinical prodrug, 2 (BMS-751324), featuring a carbamoylmethylene linked promoiety containing hydroxyphenyl acetic acid (HPA) derived ester and phosphate functionalities, was identified. Prodrug 2 was not only stable but also water-soluble under both acidic and neutral conditions. It was effectively bioconverted into parent drug 1 in vivo by alkaline phosphatase and esterase in a stepwise manner, providing higher exposure of 1 compared to its direct administration, especially within higher dose ranges. In a rat LPS-induced TNFα pharmacodynamic model and a rat adjuvant arthritis model, 2 demonstrated similar efficacy to 1. Most importantly, it was shown in clinical studies that prodrug 2 was indeed effective in addressing the pH-dependent absorption issue associated with 1.

Discovery of pyrrolo[1,2-b]pyridazine-3-carboxamides as Janus kinase (JAK) inhibitors.

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.

The identification of novel p38α isoform selective kinase inhibitors having an unprecedented p38α binding mode.

A novel series of p38 MAP kinase inhibitors with high selectivity for the p38α isoform over the other family members including the highly homologous p38ß 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.

Discovery of 4-(5-(cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-N-propylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (BMS-582949), a clinical p38α MAP kinase inhibitor for the treatment of inflammatory diseases.

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.

Utilization of a nitrogen-sulfur nonbonding interaction in the design of new 2-aminothiazol-5-yl-pyrimidines as p38α MAP kinase inhibitors.

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.

Synthesis and SAR of new pyrrolo[2,1-f][1,2,4]triazines as potent p38 alpha MAP kinase inhibitors.

A novel series of compounds based on the pyrrolo[2,1-f][1,2,4]triazine ring system have been identified as potent p38 alpha MAP kinase inhibitors. The synthesis, structure-activity relationships (SAR), and in vivo activity of selected analogs from this class of inhibitors are reported. Additional studies based on X-ray co-crystallography have revealed that one of the potent inhibitors from this series binds to the DFG-out conformation of the p38 alpha enzyme.

Design, synthesis, and anti-inflammatory properties of orally active 4-(phenylamino)-pyrrolo[2,1-f][1,2,4]triazine p38alpha mitogen-activated protein kinase inhibitors.

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.

Synthesis and SAR of p38alpha MAP kinase inhibitors based on heterobicyclic scaffolds.

The synthesis and structure-activity relationships (SAR) of p38alpha MAP kinase inhibitors based on heterobicyclic scaffolds are described. This effort led to the identification of compound (21) as a potent inhibitor of p38alpha MAP kinase with good cellular potency toward the inhibition of TNF-alpha production. X-ray co-crystallography of an oxalamide analog (24) bound to unphosphorylated p38alpha is also disclosed.

Synthesis and activity of N-acyl azacyclic urea HIV-1 protease inhibitors with high potency against multiple drug resistant viral strains.

As part of our efforts to identify potent HIV-1 protease inhibitors that are active against resistant viral strains, structural modification of the azacyclic urea (I) was undertaken by incorporating acyl groups as P(1)' ligands. The extensive SAR study has yielded a series of N-acyl azacyclic ureas (II), which are highly potent against both wild-type and multiple PI-resistant viral strains.

The discovery of orally active triaminotriazine aniline amides as inhibitors of p38 MAP kinase.

A new structural class of triaminotriazine aniline amides possessing potent p38 enzyme activity has been discovered. The initial hit (compound 1a) was identified through screening the Pharmacopeia ECLiPS compound collection. SAR modification led to the identification of a short acting triaminotriazine aniline methoxyamide (compound 1m) possessing in vitro and in vivo oral activity in animal models of acute and chronic inflammatory disease. An X-ray crystal structure of compound 1m in this class, cocrystallized with unactivated p38 alpha protein, indicates that these compounds bind to the ATP binding pocket and possess key H-bonding interactions within a deeper cleft. Hydrogen bonding between one of the triazine nitrogens and the backbone NH of the Met109 residue occurs through a water molecule. The methoxyamide NH and carbonyl oxygen are within H-bonding distance of Glu71 and Asp168.

Rational design and synthesis of an orally active indolopyridone as a novel conformationally constrained cannabinoid ligand possessing antiinflammatory properties.

A series of unique indazoles and pyridoindolones have been rationally designed and synthesized as novel classes of cannabinoid ligands based on a proposed bioactive amide conformation. This has led to the discovery of the novel indolopyridone 3a as a conformationally constrained cannabinoid ligand that displays high affinity for the CB2 receptor (K(i)(CB2) = 1.0 nM) and possesses antiinflammatory properties when administered orally in an in vivo murine inflammation model.