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  • Signaling through Axl can lead

    2024-06-11

    Signaling through Axl can lead to activation of the PI3K/Akt and Ras/MAPK pathways which promote cellular phenotypes (e.g., transformation, migration) consistent with pro-survival and pro-metastatic effects., , Inhibition of Axl activity by a variety of approaches (including pharmacologic, dominant negative, and siRNA) has been demonstrated to decrease cell proliferation and migratory activity, in addition to reducing cell viability and metastasis of Axl-expressing xenograft tumors in mice., , , , Accordingly, there has been interest in developing potent small-molecule inhibitors of Axl as anti-cancer therapies. Recently, the first Axl specific small molecule inhibitor, BGB324 (originally discovered by Rigel as R-428), from BergenBio entered the clinic and Phase 2 trials are planned. ()., , The 1-imidazole-2-carboxamide series was initially discovered from a screen of in-house kinase inhibitors. Elaboration of the series quickly led to GSK 2837808A sale (), a potent Axl inhibitor with enzymatic potency of 33nM and cellular potency of 3.6μM in a phospho-Axl ELISA assay (). Besides the 100-fold difference between the enzyme and cell potency, compound () lacked selectivity against other kinases (data not shown). Compound () also had low microsomal stability and significant hERG inhibition. prediction of metabolism for compound () predicted oxidation of the imidazole A-ring and phenyl B-ring as the major metabolites (data not shown). The prediction was confirmed by experimental metabolite identification. In addition, compound () showed poor exposure in a rat PK study GSK 2837808A sale (F=13%, CL=20mL/kg/min) which is likely due to poor absorption. These were the main issues targeted in improving compound () as an Axl inhibitor. To aid the design of molecules with improved kinase potency and selectivity, we modeled compound () into a previously obtained crystal structure of Mer(I650M). The active site of TAM subfamily member Mer is lined with the same residues as Axl with the exception of Ile650. The I650M mutation renders the Mer active site 100% sequence identical to the Axl active site and provides an adequate surrogate crystallographic platform (Note: because Mer was used in crystallographic and modeling studies, Mer residue numbers are used in the following discussion). Modeling studies of compound () in Mer(I650M) predicted a binding mode in which the imidazole N-H and amide carbonyl oxygen bind to the hinge (Pro672 and Met674, respectively) while the sulfonamide oxygen interacts with Lys619 of the salt bridge (). The model also suggests the methyl sulfonamide binds with the methyl oriented into a pocket formed by Met650, Met730 and Ala740. With regards to the ligand conformation, modeling revealed an intramolecular H-bond network created by the amide N-H interacting with 1) the imidazole nitrogen and 2) the thiazole nitrogen. Based on the MetID results and modeling data, A-ring modifications were pursued to improve metabolic stability which was also thought to modulate kinase selectivity via interaction with the gatekeeper, Leu671. In the model, B-ring modifications were expected to affect the oxidation of the phenyl ring system based on the prediction of metabolism while providing the appropriate vectors to probe the region immediately outside of the active site pocket. The synthetic route to compound () is shown in . Conversion of Cbz-protected amino acid () to the corresponding α-bromo ketone () was accomplished in a high yielding three-step sequence (>95%). Thiazole ring formation proceeds cleanly by heating () with 2-nitrobenzothioamide in high yield and carried forward as the hydrobromide salt () without purification. Cbz deprotection was performed with hydrobromic acid in acetic acid to afford (). Hexane washings of the Cbz deprotection before neutralization eliminated any -benzyl byproduct. Reaction of the resultant amine () with methanesulfonyl chloride proceeded in high yields to give the corresponding sulfonamide (). Reduction of the nitro group of () was achieved in a few hours using platinum on carbon/ammonium vanadate/triphenylphosphite system. Upon completion, the reaction required simple filtration to afford () which was used in the final step without purification. The final amide formation was performed by coupling 2-imidazole-carboxylic acid with EDC/HOBt at elevated temperature to give ().