The work in this paper describes the optimization of the 3-(3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine chemical series as potent, selective allosteric inhibitors of AKT kinases, leading to the discovery of ARQ 092 (21a). The cocrystal structure of compound 21a bound to full-length AKT1 confirmed the allosteric mode of inhibition of this chemical class and the role of the cyclobutylamine moiety. Compound 21a demonstrated high enzymatic potency against AKT1, AKT2, and AKT3, as well as potent cellular inhibition of AKT activation and the phosphorylation of the downstream target PRAS40. Compound 21a also served as a potent inhibitor of the AKT1-E17K mutant protein and inhibited tumor growth in a human xenograft mouse model of endometrial adenocarcinoma.

Abstract Bruton's tyrosine kinase (BTK) is a non receptor tyrosine kinase and belongs to the SRC-related TEC subfamily. The role of BTK in the B cell receptor (BCR) signaling pathway is well defined and is critical for full activation of phospholipase-C γ and MAPK as well as calcium mobilization. More than 95% of human lymphomas are B cell lymphoma and among them over 70% expresses BCR. Studies in lymphoma cell lines in vitro have demonstrated the role of BTK in BCR signaling. We have performed in vitro kinase profiling to determine percent inhibition and IC50 for kinase families of interest. An extensive cellular characterization including anti-proliferative assays, Western blot analysis for evaluation of phospho BTK (pY223) and cell cycle analysis was performed in DLBCL TMD8 cells. An in vivo tumor xenograft model was employed to further evaluate the target knockdown and efficacy against tumor growth. Compounds 1 and 2 were found to exhibit potent inhibition of BTK (IC50 0.6 nM), Src family (IC50 0.6-10 nM) and Trk isoforms (IC50 2-4 nM). Inhibition of cell proliferation was accompanied by reduction of phosphorylation of BTK. In addition to the anti-proliferative activity, following a single oral dose, inhibition of BTK phosphorylation (pY223) was observed in vivo in a TMD8 tumor xenograft model. The growth of TMD8 tumor xenografts was markedly suppressed after daily oral administration for 12 days of these two compounds. A series of potent multi non-receptor tyrosine kinase inhibitors targeting the Tec, Src and Trk family of kinases have been discovered and characterized. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B270. Citation Format: Nivedita D. Namdev, Susan Cornell-Kennon, Yi Yu, Jason Hill, Cathy Bull, Laurie Volak, Jianqiang Wang, Deirdre Lowe, Xuibin Gu, Steffi Koerner, Magdi Moussa, David Vensel, Yanbin Liu, Hui Wu, Chang-Rung Chen, Daniel T. Dransfield, Mark A. Ashwell. Discovery and preliminary characterization of novel tyrosine kinase inhibitors with selectivity to BTK. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B270.

This paper describes the implementation of a biochemical and biophysical screening strategy to identify and optimize small molecule Akt1 inhibitors that act through a mechanism distinct from that observed for kinase domain ATP-competitive inhibitors. With the aid of an unphosphorylated Akt1 cocrystal structure of 12j solved at 2.25 Å, it was possible to confirm that as a consequence of binding these novel inhibitors, the ATP binding cleft contained a number of hydrophobic residues that occlude ATP binding as expected. These Akt inhibitors potently inhibit intracellular Akt activation and its downstream target (PRAS40) in vitro. In vivo pharmacodynamic and pharmacokinetic studies with two examples, 12e and 12j, showed the series to be similarly effective at inhibiting the activation of Akt and an additional downstream effector (p70S6) following oral dosing in mice.

Abstract Herein we describe the implementation of a biochemical and biophysical screening strategy to discover small molecules that inhibit Akt through a mechanism distinct from ATP-competitive inhibitors. A series of novel derivatives of the core scaffold 3H-imidazo[4,5-b]pyridine were identified and optimized. These Akt inhibitors demonstrated potent inhibition of intracellular Akt and downstream targets including PRAS40 activation in vitro. Pharmacodynamic and pharmacokinetic studies in vivo demonstrated the effectiveness of the series at inhibiting the activation of Akt and an additional downstream effector (p70S6) following oral dosing in mice. Co-crystallization studies with un-phosphorylated Akt1 revealed that as a consequence of binding these novel, potent and selective, ATP-independent inhibitors the ATP binding cleft is occupied by non-polar residues which are associated as tight clusters. The cleft is closed with a ‘hydrophobic lock’ which may function to sterically exclude the binding of both ATP and ATP-competitive inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-1. doi:1538-7445.AM2012-LB-1

Abstract Utilization of hydrophobic motifs present in auto-inhibited protein kinases has resulted in the identification of a series of 5,6-dihydrobenzo [h]quinazolin-2-amines with activity as fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors. Herein we describe the combination of a proprietary in silico design process, a new screening paradigm using an array of biochemical and biophysical technologies in conjunction with an established parallel chemistry process for the identification and optimization of a series of novel FGFR inhibitors. These potent FGFR inhibitors exhibit a preference for the inactive form of the kinase, are non-ATP competitive, and exhibit robust cellular pharmacodynamic inhibition as well as in vitro anti-proliferative effects in cells dependent on FGFR and significant anti-tumor activity in appropriate xenograft models in vivo. The design strategy, synthesis, structure activity relationships and in vitro and in vivo biology of selected inhibitors will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3905. doi:1538-7445.AM2012-3905

Abstract Dysregulation of members of the FGFR tyrosine kinase family has been increasingly implicated in a number of human cancers, including gastric, breast, endometrial, and bladder carcinomas. A proprietary structure-based design paradigm was employed to identify inhibitors which favor a mode of binding that is distinct from the commonly described ATP competitive inhibitors. A molecular template was identified (ARQ 523) which, upon further modification, provided molecules that were shown to inhibit FGFR kinases in the low micromolar range, to bind to FGFR2 in an enantiomeric-specific fashion, to bind to unphosphorylated FGFR2 with a KD of 5 µM; and, after pre-incubation with inactive FGFR2, to prevent a fluorescent analogue of ATP from binding to the enzyme. We have designated this novel type of kinase inhibitor as “ATP-exclusionary” or “Type IV” to differentiate these inhibitors from extant molecules. Following a lead optimization campaign, biochemical potencies of 1 nM or less against FGFR kinases with corresponding increases in binding affinities to FGFR2 in biophysical assays were documented in several compounds in two distinct series. These compounds showed sub-micromolar activity in both FGFR2-dependent pharmacodynamic and cytotoxicity assays, and demonstrated a moderately high degree of selectivity across the human kinome. A representative compound showed marked FGFR2 pharmacodynamic suppression and corresponding growth inhibition in KATO III and SNU-16 human gastric carcinoma cells. In addition, growth of SNU-16 tumor xenografts in athymic mice was markedly suppressed (58% regression as compared to vehicle-treated controls) after daily intraperitoneal administration for 9 days. A paired Ba/F3 xenograft model was also employed, using both Ba/F3 cells transfected with either FGFR2 or the unrelated insulin receptor. Significant tumor growth inhibition (77% inhibition as compared to vehicle-treated controls) was observed in treated mice bearing FGFR2-transfected Ba/F3 tumors but not in the insulin receptor transfectants. In summary, we have achieved proof-of-principle of a structure-based kinase inhibitor design paradigm for the identification of a series of FGFR kinase inhibitors that exhibits a preference for the inactive form of the kinase and excludes ATP upon binding. One advanced lead molecule demonstrated the ability to exert profound anti-tumor effects against cancer cell lines in which FGFR2 is the dominant oncogenic driver. The opportunity now exists to significantly expand the number of new chemotypes available for drug discovery against disease-relevant kinases in oncology and in other therapeutic areas, since it is predicted that approximately half of the human kinome is amenable to this novel mode of inhibition. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A139.

3,4-Dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione (ARQ 501; beta-lapachone) showed promising anticancer activity in phase I clinical trials as monotherapy and in combination with cytotoxic drugs. ARQ 501 is currently in multiple phase II clinical trials. In vitro incubation in fresh whole blood at 37 degrees C revealed that ARQ 501 is stable in plasma but disappears rapidly in whole blood. Our data showed that extensive metabolism in red blood cells (RBCs) was mainly responsible for the rapid disappearance of ARQ 501 in whole blood. By comparison, covalent binding of ARQ 501 and/or its metabolites to whole blood components was a minor contributor to the disappearance of this compound. Sequestration of intact ARQ 501 in RBCs was not observed. Cross-species metabolite profiles from incubating [(14)C]ARQ 501 in freshly drawn blood were characterized using a liquid chromatography-mass spec-trometry-accurate radioactivity counter. The results show that ARQ 501 was metabolized more rapidly in mouse and rat blood than in dog, monkey, and human blood, with qualitatively similar metabolite profiles. Six metabolites were identified in human blood using ultra-high performance liquid chromatography/time-of-flight mass spectrometry, and the postulated structure of five metabolites was confirmed using synthetic standards. We conclude that the primary metabolic pathway of ARQ 501 in human blood involved oxidation of the two adjacent carbonyl groups to produce dicarboxylic and monocarboxylic metabolites, elimination of a carbonyl group to form a ring-contracted metabolite, and lactonization to produce two metabolites with a pyrone ring to form a ring-contracted metabolite. Metabolism by RBCs may play a role in clearance of ARQ 501 from the blood compartment in cancer patients.

ARQ 501 (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b] pyran-5,6-dione), a synthetic version of beta-Lapachone, is a promising anti-cancer agent currently in multiple Phase II clinical trials. Promising anti-cancer activity was observed in Phase I and Phase II trials. Metabolism by red blood cells of drugs is an understudied area of research and the metabolites arising from oxidative ring opening (M2 and M3), decarbonylation/ring contraction (M5), and decarbonylation/oxidation (M4 and M6) of ARQ 501 offer a unique opportunity to provide insight into these metabolic processes. Since these metabolites were not detected in in vitro incubations of ARQ 501 with liver microsomes and were structurally diverse, confirmation by chemical synthesis was considered essential. In this report, we disclose the synthetic routes employed and the characterization of the reference standards for these blood metabolites as well as additional postulated structures, which were not confirmed as metabolites.

ARQ 501 (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b] β -lapachone) showed promising anticancer activity in Phase I clinical trials as monotherapy and in combination with cytotoxic drugs. ARQ 501 is currently in multiple Phase II clinical trials. I n vitro incubation in fresh whole blood at 37 ºC revealed that ARQ 501 is stable in plasma, but disappears rapidly in whole blood. Our data showed that extensive metabolism in red blood cells (RBCs) was mainly responsible for the rapid disappearance of ARQ 501 in whole blood. By comparison, covalent binding of ARQ 501 and/or its metabolites to whole blood components was a minor contributor to the disappearance of this compound. Sequestration of intact ARQ 501 in RBCs was not observed. Cross-species metabolite profiles from incubating [ 14 C]-ARQ 501 in freshly drawn blood were characterized using liquid chromatography-mass spectrometry-accurate radioactivity counter. The results show that ARQ 501 was metabolized more rapidly in mouse and rat blood than in dog, monkey and human blood, with qualitatively similar metabolite profiles. Six metabolites were identified in human blood using ultra-high performance liquid chromatography/time-of-flight mass spectrometry, and the postulated structure of five metabolites was confirmed using synthetic standards. We conclude that the primary metabolic pathway of ARQ 501 in human blood involved oxidation of the two adjacent carbonyl groups to produce dicarboxylic and monocarboxylic metabolites, elimination of a carbonyl group to form a ring-contracted metabolite, and lactonization to produce two metabolites with a pyrone ring to form a ring-contracted metabolite. Metabolism by RBCs may play a role in clearance of ARQ 501 from the blood compartment in cancer patients.