Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of the T cell receptor signaling pathway and is therefore a target of interest for immunooncology. Nonselective HPK1 inhibitors may affect other kinase components of T cell activation, blunting the beneficial impact of enhanced T cell activity that results from HPK1 inhibition itself. Here, we report the discovery of pyrazine carboxamide HPK1 inhibitors and their optimization through structure-based drug design to afford a highly selective HPK1 inhibitor, compound 24 (AZ3246). This compound induces IL-2 secretion in T cells with an EC50 of 90 nM without inhibiting antagonistic kinases, exhibits pharmacokinetic properties consistent with oral dosing, and demonstrates antitumor activity in the EMT6 syngeneic mouse model.

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T-cell signaling. Inhibition of HPK1 with small molecules has been shown to reinvigorate the immune system toward fighting tumours in preclinical models, thus it holds promise as a therapeutic strategy in cancer immunotherapy. Herein we report a series of pyrazine carboxamide pyrazoles as selective inhibitors of HPK1. Key to our approach was the development of late-stage functionalisation chemistry which allowed for rapid SAR generation. Through these efforts, we discovered difluoroethyl pyrazole 16a, an in vivo tool which elicited the desired pharmacodynamic response in mice. Further, we describe the optimization of synthetic chemistry which could support preclinical studies of a member of this series of substituted pyrazoles.

JAK-STAT cytokines are critical in regulating immunity. Persistent activation of JAK-STAT signaling pathways by cytokines drives chronic inflammatory diseases such as asthma. Herein, we report on the discovery of a highly JAK1-selective, ATP-competitive series of inhibitors having a 1000-fold selectivity over other JAK family members and the approach used to identify compounds suitable for inhaled administration. Ultimately, compound 16 was selected as the clinical candidate, and upon dry powder inhalation, we could demonstrate a high local concentration in the lung as well as low plasma concentrations, suggesting no systemic JAK1 target engagement. Compound 16 has progressed into clinical trials. Using 16, we found JAK1 inhibition to be more efficacious than JAK3 inhibition in IL-4-driven Th2 asthma.

Spleen tyrosine kinase (SYK) is a non-receptor cytoplasmic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signalling, inhibition of SYK has been a target of interest in a variety of diseases. Herein, we report the use of structure-based drug design to discover a series of potent macrocyclic inhibitors of SYK, with excellent kinome selectivity and in vitro metabolic stability. We were able to remove hERG inhibition through the optimization of physical properties, and utilized a pro-drug strategy to address permeability challenges.

ROS1 rearrangements account for 1-2% of non-small cell lung cancer patients, yet there are no specifically designed, selective ROS1 therapies in the clinic. Previous knowledge of potent ROS1 inhibitors with selectivity over TrkA, a selected antitarget, enabled virtual screening as a hit finding approach in this project. The ligand-based virtual screening was focused on identifying molecules with a similar 3D shape and pharmacophore to the known actives. To that end, we turned to the AstraZeneca virtual library, estimated to cover 1015 synthesizable make-on-demand molecules. We used cloud computing-enabled FastROCS technology to search the enumerated 1010 subset of the full virtual space. A small number of specific libraries were prioritized based on the compound properties and a medicinal chemistry assessment and further enumerated with available building blocks. Following the docking evaluation to the ROS1 structure, the most promising hits were synthesized and tested, resulting in the identification of several potent and selective series. The best among them gave a nanomolar ROS1 inhibitor with over 1000-fold selectivity over TrkA and, from the preliminary established SAR, these have the potential to be further optimized. Our prospective study describes how conceptually simple shape-matching approaches can identify potent and selective compounds by searching ultralarge virtual libraries, demonstrating the applicability of such workflows and their importance in early drug discovery.

JAK1, JAK2, JAK3, and TYK2 belong to the JAK (Janus kinase) family. They play critical roles in cytokine signaling. Constitutive activation of JAK/STAT pathways is associated with a wide variety of diseases. Particularly, pSTAT3 is observed in response to the treatment with inhibitors of oncogenic signaling pathways such as EGFR, MAPK, and AKT and is associated with resistance or poorer response to agents targeting these pathways. Among the JAK family kinases, JAK1 has been shown to be the primary driver of STAT3 phosphorylation and signaling; therefore, selective JAK1 inhibition can be a viable means to overcome such treatment resistances. Herein, an account of the medicinal chemistry optimization from the promiscuous kinase screening hit 3 to the candidate drug 21 (AZD4205), a highly selective JAK1 kinase inhibitor, is reported. Compound 21 has good preclinical pharmacokinetics. Compound 21 displayed an enhanced antitumor activity in combination with an approved EGFR inhibitor, osimertinib, in a preclinical non-small-cell lung cancer (NSCLC) xenograft NCI-H1975 model.

The design and synthesis of a novel series of 2,6-disubstituted pyrazine derivatives as CK2 kinase inhibitors is described. Structure-guided optimization of a 5-substituted-3-thiophene carboxylic acid screening hit (3a) led to the development of a lead compound (12b), which shows inhibition in both enzymatic and cellular assays. Subsequent design and hybridization efforts also led to the unexpected identification of analogs with potent PIM kinase activity (14f).

Cyclin‐dependent kinase (CDK) 12 knockdown via siRNA decreases the transcription of DNA‐damage‐response genes and sensitizes BRCA wild‐type cells to poly(ADP‐ribose) polymerase (PARP) inhibition. To recapitulate this effect with a small molecule, we sought a potent, selective CDK12 inhibitor. Crystal structures and modeling informed hybridization between dinaciclib and SR‐3029, resulting in lead compound 5 [(S)‐2‐(1‐(6‐(((6,7‐difluoro‐1H‐benzo[d]imidazol‐2‐yl)methyl)amino)‐9‐ethyl‐9H‐purin‐2‐yl)piperidin‐2‐yl)ethan‐1‐ol]. Further structure‐guided optimization delivered a series of selective CDK12 inhibitors, including compound 7 [(S)‐2‐(1‐(6‐(((6,7‐difluoro‐1H‐benzo[d]imidazol‐2‐yl)methyl)amino)‐9‐isopropyl‐9H‐purin‐2‐yl)piperidin‐2‐yl)ethan‐1‐ol]. Profiling of this compound across CDK9, 7, 2, and 1 at high ATP concentration, single‐point kinase panel screening against 352 targets at 0.1 μm, and proteomics via kinase affinity matrix technology demonstrated the selectivity. This series of compounds inhibits phosphorylation of Ser2 on the C‐terminal repeat domain of RNA polymerase II, consistent with CDK12 inhibition. These selective compounds were also acutely toxic to OV90 as well as THP1 cells.

Janus kinases (JAKs) have been demonstrated to be critical in cytokine signaling and have thus been implicated in both cancer and inflammatory diseases. The JAK family consists of four highly homologous members: JAK1-3 and TYK2. The development of small-molecule inhibitors that are selective for a specific family member would represent highly desirable tools for deconvoluting the intricacies of JAK family biology. Herein, we report the discovery of a potent JAK1 inhibitor, 24, which displays, similar to 1000-fold selectivity over the other highly homologous JAK family members (determined by biochemical assays), while also possessing good selectivity over other kinases (determined by panel screening). Moreover, this compound was demonstrated to be orally bioavailable and possesses acceptable pharmacokinetic parameters. In an in vivo study, the compound was observed to dose dependently modulate the phosphorylation of STAT3 (a downstream marker of JAK1 inhibition).

We have identified a class of azabenzimidazoles as potent and selective JAK1 inhibitors. Investigations into the SAR are presented along with the structural features required to achieve selectivity for JAK1 versus other JAK family members. An example from the series demonstrated highly selective inhibition of JAK1 versus JAK2 and JAK3, along with inhibition of pSTAT3 in vivo, enabling it to serve as a JAK1 selective tool compound to further probe the biology of JAK1 selective inhibitors.

expression and STAT5 activation. AZD1208 causes cell cycle arrest and apoptosis in MOLM-16 cells, accompanied by a dose-dependent reduction in phosphorylation of Bcl-2antagonistofcelldeath,4EBP1,p70S6K,andS6,aswellasincreasesincleavedcaspase3 and p27. Inhibition of p4EBP1 and p-p70S6K and suppression of translation are the most representative effects of Pim inhibition in sensitive AML cell lines. AZD1208 inhibits the growth of MOLM-16 and KG-1a xenograft tumors in vivo with a clear pharmacodynamic-pharmacokinetic relationship. AZD1208 also potently inhibits colony growth and Pim signaling substrates in primary AML cells from bone marrow that are Flt3 wild-type or Flt3 internal tandem duplication mutant. These results underscore the therapeutic potential of Pim kinase inhibition for the treatment of AML. ( Blood . 2014;123(6):905-913)

Abstract The PIM serine/threonine kinase family, composed of three highly homologous members, PIM-1, PIM-2 and PIM-3, are upregulated in leukemias and lymphomas, including AML, NHL and CLL, highlighting the potential of these kinases as therapeutic targets in these indications. Over-expression of PIM-1 or PIM-3 has also been observed several solid tumors, in particular prostate, pancreatic, gastric, bladder and hepatocellular cancers. PIM kinases are downstream effectors of many cytokine and growth factor signaling pathways and are direct transcriptional targets of STAT transcription factors activated by these pathways, thereby mediating cell proliferation and survival. We have identified novel, potent and highly selective inhibitors of the PIM family kinases. The synthesis, X-ray crystallographic binding mode, and SAR of this benzylidene-1,3-thiazolidine-2,4-dione series are described. Examples from this series exhibit single digit nanomolar potency against all three PIMs, and have been shown to be selective across a panel of more than 440 kinases, with inhibition found for only approximately 3% of the panel, and at least 10-fold selectivity over kinases outside the PIM family. The compounds additionally are stable in rat microsomes, have high aqueous solubilities, and are not potent against the hERG ion channel. The series has antiproliferative activity in a panel of AML cells, has excellent pre-clinical pharmacokinetic properties, and lead compounds have shown strong tumor growth inhibition in vivo in mouse AML xenograft models. From this series, AZD1208 has recently entered Phase I clinical trials for AML and solid tumors. Citation Format: Michelle L. Lamb, Les A. Dakin, Michael H. Block, Huawei Chen, Erin Code, James E. Dowling, Xiaomei Feng, Andrew D. Ferguson, Isabelle Green, Alexander W. Hird, Tina Howard, Dennis Huszar, Erika K. Keeton, Paul D. Lyne, Hannah Pollard, Michael Rooney, Jon Read, Allan J. Wu, Tao Zhang, Xiaolan Zheng. Novel inhibitors of PIM-1, PIM-2, and PIM-3 protein kinases: medicinal chemistry leading to AZD1208. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2353. doi:10.1158/1538-7445.AM2013-2353

B-Raf represents an attractive target for anticancer therapy and the development of small molecule B-Raf inhibitors has delivered new therapies for metastatic melanoma patients. We have discovered a novel class of small molecules that inhibit mutant B-Raf(V600E) kinase activity both in vitro and in vivo. Investigations into the structure-activity relationships of the series are presented along with efforts to improve upon the cellular potency, solubility, and pharmacokinetic profile. Compounds selectively inhibited B-Raf(V600E) in vitro and showed preferential antiproliferative activity in mutant B-Raf(V600E) cell lines and exhibited selectivity in a kinase panel against other kinases. Examples from this series inhibit growth of a B-Raf(V600E) A375 xenograft in vivo at a well-tolerated dose. In addition, aminoquinazolines described herein were shown to display pERK elevation in nonmutant B-Raf cell lines in vitro.

Upregulation of Pim kinases is observed in several types of leukemias and lymphomas. Pim-1, -2, and -3 promote cell proliferation and survival downstream of cytokine and growth factor signaling pathways. AZD1208 is a potent, highly selective, and orally available Pim kinase inhibitor that effectively inhibits all three isoforms at <5 nM or <150 nM in enzyme and cell assays, respectively. AZD1208 inhibited the growth of 5 of 14 acute myeloid leukemia (AML) cell lines tested, and sensitivity correlates with Pim-1 expression and STAT5 activation. AZD1208 causes cell cycle arrest and apoptosis in MOLM-16 cells, accompanied by a dose-dependent reduction in phosphorylation of Bcl-2 antagonist of cell death, 4EBP1, p70S6K, and S6, as well as increases in cleaved caspase 3 and p27. Inhibition of p4EBP1 and p-p70S6K and suppression of translation are the most representative effects of Pim inhibition in sensitive AML cell lines. AZD1208 inhibits the growth of MOLM-16 and KG-1a xenograft tumors in vivo with a clear pharmacodynamic-pharmacokinetic relationship. AZD1208 also potently inhibits colony growth and Pim signaling substrates in primary AML cells from bone marrow that are Flt3 wild-type or Flt3 internal tandem duplication mutant. These results underscore the therapeutic potential of Pim kinase inhibition for the treatment of AML.

Abstract Genomic rearrangement of Anaplastic Lymphoma Kinase (ALK) has been observed in several tumor types including 60-80% anaplastic large cell lymphoma (ALCL) and 3-6% of non small cell lung cancer (NSCLC). Although the ALK inhibitor crizotinib has clinical efficacy in selected ALK positive NSCLC patients, the majority of patients who show initial responses eventually relapse. Various mechanisms leading to resistance have been proposed and include ALK amplification and resistance mutations, as well as alternative pathway drivers including EGFR, cKIT and, more recently, IGF1R. We have discovered a novel and potent inhibitor of ALK, AZD3463 with a Ki value of 0.75nM which also inhibits additional receptor tyrosine kinases including insulin growth factor receptor (IGF1R) with equivalent potency. AZD3463 inhibits ALK in cells as demonstrated by its ability to decrease ALK autophosphorylation in tumor cell lines containing ALK fusions including DEL (ALCL NPM-ALK), H3122 (NSCLC EML4-ALK) and H2228 (NSCLC EML4-ALK). Inhibition of ALK is associated with perturbations in downstream signaling including ERK, AKT and STAT3 pathways leading to preferential inhibition of proliferation in the ALK fusion containing cell lines in vitro. AZD3463 also demonstrates the ability to dose dependently inhibit pALK in xenograft tumors in vivo resulting in stasis (H3122) or regression (DEL, H2228). AZD3463 retains good activity against a number of clinically relevant crizotinib resistant mutations including the gatekeeper mutant L1196M where equivalent potency to wild type ALK is observed in vitro and in vivo in EML4-ALK containing BAF3 cell lines. To further assess the potential ability of AZD3463 to overcome additional resistance mechanisms, antiproliferative activity was assessed in multiple crizotinib resistant cell lines independently derived in vitro from H3122 cells as well as a patient derived crizotinib relapsed model. These resistant cell lines contain multiple resistance mechanisms including the L1196M gatekeeper and T115Ins mutations, ALK amplification and/or secondary drivers including EGFR and IGF1R. AZD3463 retains antiproliferative potency within 4 fold of parental H3122 cells for 10 out of 12 of these acquired resistance models in vitro. In summary, AZD3463 is a potent ALK inhibitor which inhibits additional kinases including IGF1R and has activity in a number of crizotinib resistant models driven by multiple resistance mechanisms. Citation Format: Lisa Drew, Jane Cheng, Jeffrey Engelman, Douglas Ferguson, Ryohei Katayama, Brenda McDermott, Jamal Saeh, Alice Shaw, Minhui Shen, Dan Widzowski, Allan Wu, Graeme Smith. AZD3463, a novel ALK/IGF1R inhibitor, overcomes multiple mechanisms of acquired resistance to crizotinib. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 919. doi:10.1158/1538-7445.AM2013-919

Abstract Translocations linking anaplastic lymphoma kinase (ALK) to multiple fusion partners were identified in anaplastic large cell lymphoma (ALCL), inflammatory myofibroblastic tumors, esophageal squamous cell carcinomas, neuroblastoma, and in non small cell lung cancer (NSCLC). This fusion leads to constitutive activation of ALK and has potent transforming activity. The recent approval of Crizotinib validated clinically the usefulness of treating EML4-ALK +ve patients with an Alk inhibitor. We report two novel series of compounds that potently inhibit ALK both in vitro and in vivo. Two series of pyrazolimidazolpyridine compounds, with good physical properties, were identified as part of our screening effort as potent ALK inhibitors in ALK enzyme assays. Subsequent testing of the mode of action of these compounds in Del cell lines containing NPM-ALK fusions demonstrated strong inhibition of phospho-ALK. In vivo, oral administration of 10 mg/kg of a pyrazolimidazopyridine compound in a Del xenograft model in SCID mice resulted in greater than 90% inhibition of phospho-ALK over 6 hours. In conclusion, we have identified two series of pyrazolimidazopyridine compounds as potent ALK inhibitors both in vitro and in vivo. 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 2916. doi:1538-7445.AM2012-2916

Abstract Despite the success of Crizotinib in treating NSCLC patients with EML4-ALK fusions, cancers eventually develop resistance via a variety of mechanisms including mutations in the ATP binding site of the kinase domain. A series of 5-aminopyrazol-imidazopyridine compounds were identified to potently inhibit anaplastic lymphoma kinase and were found to be active against a number of mutations in vitro but suffered from strong Cyp inhibition and were found to be metabolically unstable by incubation in human hepatocytes and microsomes. Installation of a pyrimary alcohol group on the heteroaryl ethyl group of the scaffold consistently improved Cyp3A4 liability, and led to improvement in physical and DMPK properties as well as improvement of their metabolic stability in human Heps. The lead compound from this series was orally administrated to SCID mice in a Del xenograft model and achieved greater than 90% phospho-ALK inhibition over 6 hours post dose at 10 mg/kg dose. The lead compound was subsequently tested against Crizotinib-resistant ALK mutations in enzyme assays, and was shown to inhibit the clinically relevant ALK mutations, including L1196M. In enzyme assays, it was inactive against G1269S mutation, consistent with modeling studies. Subsequent profiling in an FDCP cell line over expressing ALK G1269S mutation showed potent anti-proliferative activity, suggesting off-target activity. Further characterization of this series identified AurB inhibition to be a key cell cycle kinase responsible for the off-target activity in the engineered cell line. The off target activity hinders it from assessing the primary pharmacology responsible for its preclinical efficacy in disease relevant models. In conclusion, we have identified a novel orally bioavailable compound that is a potent dual active ALK and AurB inhibitor that is active against clinically-relevant gatekeeper mutant. 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 3910. doi:1538-7445.AM2012-3910

In this paper we describe a series of 3-cyano-5-aryl-7-aminopyrazolo[1,5-a]pyrimidine hits identified by kinase-focused subset screening as starting points for the structure-based design of conformationally constrained 6-acetamido-indole inhibitors of CK2. The synthesis, SAR, and effects of this novel series on Akt signaling and cell proliferation in vitro are described.

Translocations linking anaplastic lymphoma kinase (ALK) to multiple fusion partners were identified in anaplastic large cell lymphoma (ALCL), inflammatory myofibroblastic tumors, esophageal squamous cell carcinomas, neuroblastoma, and in non small cell lung cancer (NSCLC). This fusion leads to constitutive activation of ALK and has potent transforming activity. The recent approval of Crizotinib validated clinically the usefulness of treating EML4-ALK +ve patients with an Alk inhibitor. We report two novel series of compounds that potently inhibit ALK both in vitro and in vivo. Two series of pyrazolimidazolpyridine compounds, with good physical properties, were identified as part of our screening effort as potent ALK inhibitors in ALK enzyme assays. Subsequent testing of the mode of action of these compounds in Del cell lines containing NPM-ALK fusions demonstrated strong inhibition of phospho-ALK. In vivo, oral administration of 10 mg/kg of a pyrazolimidazopyridine compound in a Del xenograft model in SCID mice resulted in greater than 90% inhibition of phospho-ALK over 6 hours. In conclusion, we have identified two series of pyrazolimidazopyridine compounds as potent ALK inhibitors both in vitro and in vivo. 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 2916. doi:1538-7445.AM2012-2916