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The diastereoselective synthesis and biological activity of piperidine-3,4-diol and piperidine-3-ol-derived pyrrolotriazine inhibitors of anaplastic lymphoma kinase (ALK) are described. Although piperidine-3,4-diol and piperidine-3-ol derivatives showed comparable in vitro ALK activity, the latter subset of inhibitors demonstrated improved physiochemical and pharmacokinetic properties. Furthermore, the stereochemistry of the C3 and C4 centers had a marked impact on the in vivo inhibition of ALK autophosphorylation. Thus, trans-4-aryl-piperidine-3-ols (22) were more potent than the cis diastereomers (20)., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The spread of intra-abdominal cancers is a vexing clinical problem for which there is no widely effective treatment. We discovered previously that (2E)-3-[(4-tert-butylphenyl)sulfonyl]acrylonitrile (1) induced cancer cell apoptosis during adhesion to normal mesothelial cells which line the peritoneum. We recently demonstrated that the sulfonylacrylonitrile portion of 1 and hydrophobic aryl substitution were essential for pro-apoptotic activity in cancer cells. Here we synthesized a diverse series of analogues of 1 in order to improve the efficacy and pharmaceutical properties. Analogues and 1 were compared in their ability to cause cancer cell death during adhesion to normal mesothelial cell monolayers. Potent analogues identified in the in vitro assay were validated and found to exhibit improved inhibition of intra-abdominal cancer in two clinically relevant murine models of ovarian and pancreatic cancer spread and metastasis, highlighting their potential clinical use as an adjunct to surgical resection of cancers.

Introduction: Anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase from the insulin receptor superfamily, is implicated in the oncogenesis of numerous cancers including anaplastic large-cell lymphoma, non-small-cell lung cancer, diffuse large B-cell lymphoma, inflammatory myofibroblastic tumors, glioblastoma, as well as neuroblastoma. The root cause for these specific cancers has been identified as aberrant ALK kinase activity, which has been shown to be associated with specific gene translocations, single-point mutations, gene amplification and/or overexpression. The direct inhibition of ALK with small-molecule inhibitors represents a viable therapeutic intervention that has achieved clinical proof of concept., Areas Covered: Small-molecule ALK inhibitors covered in the patent literature from 2010 to September 2013 are described. Relevant peer-reviewed journal articles that describe discovery and development of the above-identified ALK inhibitors are also discussed. Keyword-based (e.g., ALK, anaplastic lymphoma kinase) literature searches were conducted in Scifinder®., Expert Opinion: Novel ALK inhibitors continued to be discovered at a fast pace over the covered period, with many distinct chemotypes emerging. Crizotinib received FDA approval in 2011, and six additional ALK inhibitors have entered clinical trials. The focus of ALK research appears to have shifted toward inhibitors that display activity against resistant mutants unearthed in clinical studies with crizotinib.

Members of the JAK family of nonreceptor tyrosine kinases play a critical role in the growth and progression of many cancers and in inflammatory diseases. JAK2 has emerged as a leading therapeutic target for oncology, providing a rationale for the development of a selective JAK2 inhibitor. A program to optimize selective JAK2 inhibitors to combat cancer while reducing the risk of immune suppression associated with JAK3 inhibition was undertaken. The structure-activity relationships and biological evaluation of a novel series of compounds based on a 1,2,4-triazolo[1,5-a]pyridine scaffold are reported. Para substitution on the aryl at the C8 position of the core was optimum for JAK2 potency (17). Substitution at the C2 nitrogen position was required for cell potency (21). Interestingly, meta substitution of C2-NH-aryl moiety provided exceptional selectivity for JAK2 over JAK3 (23). These efforts led to the discovery of CEP-33779 (29), a novel, selective, and orally bioavailable inhibitor of JAK2.

Anaplastic lymphoma kinase (ALK) is a promising therapeutic target for the treatment of cancer, supported by considerable favorable preclinical and clinical activities over the past several years and culminating in the recent FDA approval of the ALK inhibitor crizotinib. Through a series of targeted modifications on an ALK inhibitor diaminopyrimidine scaffold, our research group has driven improvements in ALK potency, kinase selectivity, and overall pharmaceutical properties. Optimization of this scaffold has led to the identification of a potent and efficacious inhibitor of ALK, 25b. A striking feature of 25b over previously described ALK inhibitors is its >600-fold selectivity over insulin receptor (IR), a closely related kinase family member. Most importantly, 25b exhibited dose proportional escalation in rat compared to compound 3 which suffered dose limiting absorption preventing further advancement. Compound 25b exhibited significant in vivo antitumor efficacy when dosed orally in an ALK-positive ALCL tumor xenograft model in SCID mice, warranting further assessment in advanced preclinical models.

Mutations in the BRAF gene have been identified in approximately 7% of cancers, including 60% to 70% of melanomas, 29% to 83% of papillary thyroid carcinomas, 4% to 16% colorectal cancers, and a lesser extent in serous ovarian and non-small cell lung cancers. The V600E mutation is found in the vast majority of cases and is an activating mutation, conferring transforming and immortalization potential to cells. CEP-32496 is a potent BRAF inhibitor in an in vitro binding assay for mutated BRAF(V600E) (K(d) BRAF(V600E) = 14 nmol/L) and in a mitogen-activated protein (MAP)/extracellular signal-regulated (ER) kinase (MEK) phosphorylation (pMEK) inhibition assay in human melanoma (A375) and colorectal cancer (Colo-205) cell lines (IC(50) = 78 and 60 nmol/L). In vitro, CEP-32496 has multikinase binding activity at other cancer targets of interest; however, it exhibits selective cellular cytotoxicity for BRAF(V600E) versus wild-type cells. CEP-32496 is orally bioavailable in multiple preclinical species (>95% in rats, dogs, and monkeys) and has single oral dose pharmacodynamic inhibition (10-55 mg/kg) of both pMEK and pERK in BRAF(V600E) colon carcinoma xenografts in nude mice. Sustained tumor stasis and regressions are observed with oral administration (30-100 mg/kg twice daily) against BRAF(V600E) melanoma and colon carcinoma xenografts, with no adverse effects. Little or no epithelial hyperplasia was observed in rodents and primates with prolonged oral administration and sustained exposure. CEP-32496 benchmarks favorably with respect to other kinase inhibitors, including RAF-265 (phase I), sorafenib, (approved), and vemurafenib (PLX4032/RG7204, approved). CEP-32496 represents a novel and pharmacologically active BRAF inhibitor with a favorable side effect profile currently in clinical development., (©2012 AACR.)

Anaplastic lymphoma kinase (ALK) is constitutively activated in a number of human cancer types due to chromosomal translocations, point mutations, and gene amplification and has emerged as an excellent molecular target for cancer therapy. Here we report the identification and preclinical characterization of CEP-28122, a highly potent and selective orally active ALK inhibitor. CEP-28122 is a potent inhibitor of recombinant ALK activity and cellular ALK tyrosine phosphorylation. It induced concentration-dependent growth inhibition/cytotoxicity of ALK-positive anaplastic large-cell lymphoma (ALCL), non-small cell lung cancer (NSCLC), and neuroblastoma cells, and displayed dose-dependent inhibition of ALK tyrosine phosphorylation in tumor xenografts in mice, with substantial target inhibition (>90%) for more than 12 hours following single oral dosing at 30 mg/kg. Dose-dependent antitumor activity was observed in ALK-positive ALCL, NSCLC, and neuroblastoma tumor xenografts in mice administered CEP-28122 orally, with complete/near complete tumor regressions observed following treatment at doses of 30 mg/kg twice daily or higher. Treatment of mice bearing Sup-M2 tumor xenografts for 4 weeks and primary human ALCL tumor grafts for 2 weeks at 55 or 100 mg/kg twice daily led to sustained tumor regression in all mice, with no tumor reemergence for more than 60 days postcessation of treatment. Conversely, CEP-28122 displayed marginal antitumor activity against ALK-negative human tumor xenografts under the same dosing regimens. Administration of CEP-28122 was well tolerated in mice and rats. In summary, CEP-28122 is a highly potent and selective orally active ALK inhibitor with a favorable pharmaceutical and pharmacokinetic profile and robust and selective pharmacologic efficacy against ALK-positive human cancer cells and tumor xenograft models in mice.

The vast majority of cancer patients die from metastasis, the process by which cancer cells spread to secondary tissues through body fluids. Peritoneal carcinomatosis is a type of metastasis in which cancer cells gain access to the intra-abdominal cavity and then implant in the peritoneum, the thin tissue that lines the abdominal wall and internal organs. Unfortunately, peritoneal carcinomatosis can occur following surgical resection of intra-abdominal malignancies. We previously reported proapoptotic activity of (2E)-3-[[4-(1,1-dimethylethyl)phenyl]sulfonyl]-2-propenenitrile (BAY 11-7085, 1) on colon and pancreatic cancer cells during adhesion and demonstrated that this compound could significantly inhibit peritoneal carcinomatosis in mice.(1,2) In order to determine the chemical basis of the anti-metastatic properties of BAY 11-7085, a series of analogs were synthesized and evaluated for their ability to induce apoptosis in pancreatic and ovarian cancer cells during adhesion to mesothelial cells, which line the surface of the peritoneum. The co-culture assay results were validated using a murine peritoneal carcinomatosis model. These analogs may greatly benefit patients undergoing surgical resections of colorectal, pancreatic, and ovarian cancers depending on their tolerability., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Chemical strategies to mitigate cytochrome P450-mediated bioactivation of novel 2,7-disubstituted pyrrolo[2,1-f][1,2,4]triazine ALK inhibitors are described along with synthesis and biological activity. Piperidine-derived analogues showing minimal microsomal reactive metabolite formation were discovered. Potent, selective, and metabolically stable ALK inhibitors from this class were identified, and an orally bioavailable compound (32) with antitumor efficacy in ALK-driven xenografts in mouse models was extensively characterized.

A novel set of 2,4,8,22-tetraazatetracyclo[14.3.1.1(3,7).1(9,13)]docosa-1(20),3(22),4,6,9(21),10,12,16,18-nonaene macrocycles were prepared as potential anaplastic lymphoma kinase (ALK) inhibitors, designed to rigidly lock an energy-minimized bioactive conformation of the diaminopyrimidine (DAP) scaffold, a well-documented kinase platform. From 13 analogues prepared, macrocycle 2m showed the most promising in vitro ALK enzymatic (IC(50) = 0.5 nM) and cellular (IC(50) = 10 nM) activities. In addition, macrocycle 2m exhibited a favorable kinase selectivity preference for inhibition of ALK relative to the highly homologous insulin receptor (IR) kinase (IR/ALK ratio of 173). The inclusive in vitro biological results for this set of macrocycles validate this scaffold as a viable kinase template and further corroborate recent DAP/ALK solid state studies indicating that the inverted "U" shaped conformation of the acyclic DAPs is a preferred bioactive conformation.

A novel 2,7-disubstituted-pyrrolo[2,1-f][1,2,4]triazine scaffold has been designed as a new kinase inhibitor platform mimicking the bioactive conformation of the well-known diaminopyrimidine motif. The design, synthesis, and validation of this new pyrrolo[2,1-f][1,2,4]triazine scaffold will be described for inhibitors of anaplastic lymphoma kinase (ALK). Importantly, incorporation of appropriate potency and selectivity determinants has led to the discovery of several advanced leads that were orally efficacious in animal models of anaplastic large cell lymphoma (ALCL). A lead inhibitor (30) displaying superior efficacy was identified and in depth in vitro/in vivo characterization will be presented.

The incorporation of R,R-1,2-diaminocyclohexane at C4 in a series of 2,4-diaminopyrimidines led to a number of ALK inhibitors in which optimized activity was achieved by conversion of the 2-amino group into a methanesulfonamide. Tumor growth inhibition was observed when an orally bioavailable analog was evaluated in a Karpas-299 tumor xenograft mouse model., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Elaboration of the SAR around a series of 2,4-diaminopyrimidines led to a number of c-Met inhibitors in which kinase selectivity was modulated by substituents appended on the C4-aminobenzamide ring and the nature of the C2-aminoaryl ring. Further lead optimization of the C2-aminoaryl group led to benzoxazepine analogs whose pharmaceutical properties were modulated by the nature of the substituent on the benzoxazepine nitrogen. Tumor stasis (with partial regressions) were observed when an orally bioavailable analog was evaluated in a GTL-16 tumor xenograft mouse model. Subsequent PK/PD studies suggested that a metabolite contributed to the overall in vivo response., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Dysregulation of the anaplastic lymphoma kinase (ALK) is implicated in a variety of cancers. A series of tetrahydropyrido[2,3-b]pyrazines was constructed as ring-constrained analogs of a known aminopyridine kinase scaffold. Chemistry was developed to rapidly elaborate the SAR, structural elements impacting ALK inhibitory activity were exploited, and kinase selective analogs were identified that inhibit ALK with IC(50) values approximately 10 nM (enzyme) and approximately 150 nM (cell)., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The ubiquitin-proteasome pathway plays a central role in regulation of the production and destruction of cellular proteins. These pathways mediate proliferation and cell survival, particularly in malignant cells. The successful development of the 20S human proteasome inhibitor bortezomib for the treatment of relapsed and refractory multiple myeloma has established this targeted intervention as an effective therapeutic strategy. Herein, the potent, selective, and orally bioavailable threonine-derived 20S human proteasome inhibitor that has been advanced to preclinical development, [(1R)-1-[[(2 S,3 R)-3-hydroxy-2-[(6-phenylpyridine-2-carbonyl)amino]-1-oxobutyl]amino]-3-methylbutyl]boronic acid 20 (CEP-18770), is disclosed.