Analogues structurally related to anaplastic lymphoma kinase (ALK) inhibitor 1 were optimized for metabolic stability. The results from this endeavor not only led to improved metabolic stability, pharmacokinetic parameters, and in vitro activity against clinically derived resistance mutations but also led to the incorporation of activity for focal adhesion kinase (FAK). FAK activation, via amplification and/or overexpression, is characteristic of multiple invasive solid tumors and metastasis. The discovery of the clinical stage, dual FAK/ALK inhibitor 27b, including details surrounding SAR, in vitro/in vivo pharmacology, and pharmacokinetics, is reported herein.

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).

A substantial body of evidence supports the utility of antiangiogenesis inhibitors as a strategy to block or attenuate tumor-induced angiogenesis and inhibition of primary and metastatic tumor growth in a variety of solid and hematopoietic tumors. Given the requirement of tumors for different cytokine and growth factors at distinct stages of their growth and dissemination, optimal antiangiogenic therapy necessitates inhibition of multiple, complementary, and nonredundant angiogenic targets. 11-(2-Methylpropyl)-12,13-dihydro-2-methyl-8-(pyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (11b, CEP-11981) is a potent orally active inhibitor of multiple targets (TIE-2, VEGF-R1, 2, and 3, and FGF-R1) having essential and nonredundant roles in tumor angiogenesis and vascular maintenance. Outlined in this article are the design strategy, synthesis, and biochemical and pharmacological profile for 11b, which completed Phase I clinical assessing safety and pharmacokinetics allowing for the initiation of proof of concept studies.

Abstract 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. Mol Cancer Ther; 11(3); 670–9. ©2011 AACR.

The elaboration of a novel scaffold for the inhibition of JAK2 and FAK kinases was targeted in order to provide a dual inhibitor that could target divergent pathways for tumor cell progression.

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.

Homogeneous cellular assays can streamline product detection in the drug discovery process. One commercially available assay employing time-resolved fluorescence resonance energy transfer (TR-FRET) that detects phosphorylated products was used to evaluate inhibitors of the receptor tyrosine kinase AXL in a cell line expressing an AXL-green fluorescent protein fusion protein. This TR-FRET assay was modified to evaluate the phosphorylation state of the AXL family member MER in a cell line expressing MER with a V5 tag by adding a fluorescein-labeled anti-V5 antibody. This homogeneous cellular assay was further modified to evaluate the nonreceptor tyrosine kinase focal adhesion kinase (FAK) in cell lines that expressed an untagged kinase by the inclusion of a commercially available anti-FAK antibody conjugated with an acceptor dye. The methods described here can be further adapted for TR-FRET detection of other cellular kinase activities.

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.

An understanding of the dynamics of drug-target interactions is important in the drug discovery process. Information related to the binding kinetics of a drug toward its target or off-target aids in determining the efficacy or toxicity of a drug. Biophysical techniques such as surface plasmon resonance (SPR) have been available for over 20 years, but have been predominantly utilized to characterize protein-protein interactions. With improvements in instrument sensitivity and data analysis software, interactions between proteins (such as kinases) and small molecules have been successfully evaluated. More recently, the LanthaScreen Eu kinase binding assay for characterizing kinase inhibitors has been described. This assay monitors displacement of an Alexa Fluor 647-labeled tracer from the ATP-binding site of an epitope-tagged kinase by a test compound. Such behavior results in a decrease in time-resolved fluorescence energy transfer signal. In this report, a side-by-side comparison of the LanthaScreen Eu kinase binding assay and the SPR method was performed using inhibitors of focal adhesion kinase. The two methods yielded comparable results and identified compounds with time-dependent inhibition and relatively slow dissociation.

A series of potent anaplastic lymphoma kinase (ALK) inhibitors based on a 7-amino-6-chloro-3H-imidazo[4,5-b]pyridine scaffold were identified through rational design from a 5-chloro-2,4-diaminopyrimidine pharmacophore, maintaining key binding elements, favourable lipophilic interactions and orienting the side chains into favoured trajectories. Importantly, potency and selectivity determinants from the parent series were directly applicable to the new scaffold. This highly focused strategy led to the identification of several lead inhibitors that displayed potent activity in enzyme and cellular assays as well as pronounced oral bioavailability.

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.

A substantial body of evidence supports the utility of antiangiogenesis inhibitors as a strategy to block or attenuate tumor-induced angiogenesis and inhibition of primary and metastatic tumor growth in a variety of solid and hematopoietic tumors. Given the requirement of tumors for different cytokine and growth factors at distinct stages of their growth and dissemination, optimal antiangiogenic therapy necessitates inhibition of multiple, complementary, and nonredundant angiogenic targets. 11-(2-Methylpropyl)-12,13-dihydro-2-methyl-8-(pyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (11b, CEP-11981) is a potent orally active inhibitor of multiple targets (TIE-2, VEGF-R1, 2, and 3, and FGF-R1) having essential and nonredundant roles in tumor angiogenesis and vascular maintenance. Outlined in this article are the design strategy, synthesis, and biochemical and pharmacological profile for 11b, which completed Phase I clinical assessing safety and pharmacokinetics allowing for the initiation of proof of concept studies.

A novel set of 2,4,8,22-tetraazatetracyclo[14.3.1.13,7.19,13]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 (IC50 = 0.5 nM) and cellular (IC50 = 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.

Anaplastic lymphoma kinase (ALK) was originally identified as the oncogenic NPM (nucleophosmin)-ALK fusion protein due to a t (2;5) chromosomal translocation in anaplastic large cell lymphomas (ALCL). Many other chromosomal rearrangements or gene mutations/amplification leading to enhanced ALK activity have subsequently been identified and characterized in a number of human cancer types. The recent reports of EML4 (echinoderm microtubule-associated protein-like 4)-ALK oncogenic proteins in non-small cell lung cancer (NSCLC) and the identification of ALK activating point mutations and gene amplification in neuroblastoma have indicated ALK as a potential major therapeutic target for human cancers. 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 (IC50 of 3 nM) and NPM-ALK tyrosine phosphorylation in ALCL cells (IC50 of 20-30 nM). It is selective over a broad panel of protein kinases and a panel of receptors and ion channels with greater than 300-fold selectivity for insulin receptor. CEP-28122 induced concentration-dependent growth inhibition and cytotoxicity of ALK-positive ALCL and NSCLC cells with minimal activity against ALK-negative lymphoma and leukemia cells as well as ALK-negative NSCLC cells at concentrations up to 3 μM. CEP-28122 exhibited favorable oral bioavailability (F = 37-71% across species) with adequate tissue distribution in rodents. It displayed dose-dependent inhibition of NPM-ALK tyrosine phosphorylation in human ALCL tumor xenografts in mice with complete target inhibition (> 90%) for more than 12 h following single oral dosing at 30 mg/kg. Dose-dependent anti-tumor activity was observed in NPM-ALK-positive Sup-M2 and Karpas-299 ALCL tumor xenografts and EML4-ALK-positive NCI-H2228 and NCI-H3122 tumor xenografts in mice dosed with CEP-28122, bid, po, with complete or near complete tumor regressions observed following 2 weeks of treatment with CEP-28122 at 30 mg/kg or higher. Treatment of mice bearing Sup-M2 tumor xenografts or primary human ALCL tumorgrafts with CEP-28122 at 55 or 100 mg/kg bid, po, for 4 weeks led to sustained tumor regression in all mice, with no tumor re-emergence in any mouse up to 60 days post cessation of CEP-28122 treatment. On the contrary, CEP-28122 displayed marginal anti-tumor activity against ALK-negative lymphoma and NSCLC tumor xenografts under the same dosing regimens. It was well tolerated with all dosing regimens in mice and rats with no overt toxicity and no compound-related body weight loss. CEP-28122 advanced into preclinical development based on its potency, selectivity and overall favorable pharmacological, pharmaceutical and safety profiles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3574. doi:10.1158/1538-7445.AM2011-3574