Melissa Vasbinder

Discovery of Checkpoint Kinase Inhibitor (S)-5-(3-Fluorophenyl)-N-(piperidin-3-yl)-3-ureidothiophene-2-carboxamide (AZD7762) by Structure-Based Design and Optimization of Thiophenecarboxamide Ureas.

Checkpoint kinases CHK1 and CHK2 are activated in response to DNA damage that results in cell cycle arrest, allowing sufficient time for DNA repair. Agents that lead to abrogation of such checkpoints have potential to increase the efficacy of such compounds as chemo- and radiotherapies. Thiophenecarboxamide ureas (TCUs) were identified as inhibitors of CHK1 by high throughput screening. A structure-based approach is described using crystal structures of JNK1 and CHK1 in complex with 1 and 2 and of the CHK1-3b complex. The ribose binding pocket of CHK1 was targeted to generate inhibitors with excellent cellular potency and selectivity over CDK1and IKKβ, key features lacking from the initial compounds. Optimization of 3b resulted in the identification of a regioisomeric 3-TCU lead 12a. Optimization of 12a led to the discovery of the clinical candidate 4 (AZD7762), which strongly potentiates the efficacy of a variety of DNA-damaging agents in preclinical models.

Discovery of a novel class of 2-ureido thiophene carboxamide checkpoint kinase inhibitors.

Checkpoint kinase-1 (Chk1, CHEK1) is a Ser/Thr protein kinase that mediates the cellular response to DNA-damage. A novel class of 2-ureido thiophene carboxamide urea (TCU) Chk1 inhibitors is described. Inhibitors in this chemotype were optimized for cellular potency and selectivity over Cdk1.

Discovery of a Novel Class of Dimeric Smac Mimetics as Potent IAP Antagonists Resulting in a Clinical Candidate for the Treatment of Cancer (AZD5582)

A series of dimeric compounds based on the AVPI motif of Smac were designed and prepared as antagonists of the inhibitor of apoptosis proteins (IAPs). Optimization of cellular potency, physical properties, and pharmacokinetic parameters led to the identification of compound 14 (AZD5582), which binds potently to the BIR3 domains of cIAP1, cIAP2, and XIAP (IC50 = 15, 21, and 15 nM, respectively). This compound causes cIAP1 degradation and induces apoptosis in the MDA-MB-231 breast cancer cell line at subnanomolar concentrations in vitro. When administered intravenously to MDA-MB-231 xenograft-bearing mice, 14 results in cIAP1 degradation and caspase-3 cleavage within tumor cells and causes substantial tumor regressions following two weekly doses of 3.0 mg/kg. Antiproliferative effects are observed with 14 in only a small subset of the over 200 cancer cell lines examined, consistent with other published IAP inhibitors. As a result of its in vitro and in vivo profile, 14 was nominated as a candidate for clinical development.

Discovery and Optimization of a Novel Series of Potent Mutant B-Raf V600E Selective Kinase Inhibitors.

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.

Small molecule inhibitor of apoptosis proteins antagonists: a patent review

Introduction: The family of inhibitor of apoptosis proteins (IAPs) plays a key role in the suppression of proapoptotic signaling; hence, a small molecule that disrupts the binding of IAPs with their functional partner should restore apoptotic response to proapoptotic stimuli in cells. The continued publication of new patent applications of IAP antagonists over the past 4 years is a testament to the continued interest surrounding the IAP family of proteins. Areas covered: This review summarizes the IAP antagonist patent literature from 2010 to 2014. Monovalent and bivalent Smac mimetics will be covered as well as two new developments in the field: IAP antagonists coupled to or merged with other targeted agents and new BIR2 selective IAP antagonists. Expert opinion: In addition to the well-explored scaffolds for monovalent and bivalent Smac-mimetics, some companies have taken more drastic approaches to explore new chemical space – for example, fragment-based approaches and macrocyclic inhibitors. Furthermore, other companies have designed compounds with alternative biological profiles – tethering to known kinase binding structures, trying to target to the mitochondria or introducing selective binding to the BIR2 domain. An overview of the status for the four small molecule IAP antagonists being evaluated in active human clinical trials is also provided.

Identification of azabenzimidazoles as potent JAK1 selective inhibitors.

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.

Adventures in Scaffold Morphing: Discovery of Fused Ring Heterocyclic Checkpoint Kinase 1 (CHK1) Inhibitors

Checkpoint kinase 1 (CHK1) inhibitors are potential cancer therapeutics that can be utilized for enhancing the efficacy of DNA damaging agents. Multiple small molecule CHK1 inhibitors from different chemical scaffolds have been developed and evaluated in clinical trials in combination with chemotherapeutics and radiation treatment. Scaffold morphing of thiophene carboxamide ureas (TCUs), such as AZD7762 (1) and a related series of triazoloquinolines (TZQs), led to the identification of fused-ring bicyclic CHK1 inhibitors, 7-carboxamide thienopyridines (7-CTPs), and 7-carboxamide indoles. X-ray crystal structures reveal a key intramolecular noncovalent sulfur-oxygen interaction in aligning the hinge-binding carboxamide group to the thienopyridine core in a coplanar fashion. An intramolecular hydrogen bond to an indole NH was also effective in locking the carboxamide in the preferred bound conformation to CHK1. Optimization on the 7-CTP series resulted in the identification of lead compound 44, which displayed respectable drug-like properties and good in vitro and in vivo potency.

Discovery and Optimization of a Novel Series of Highly Selective JAK1 Kinase Inhibitors

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 ∼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).

Abstract 979: Discovery of the JAK1 selective kinase inhibitor AZD4205

Janus kinases are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling, with constitutive activation of JAK/STAT pathways associated with a wide variety of malignancies. Elevated JAK/STAT signalling leading to increased activation of STAT3 is reported in a wide variety of cancers, including breast, liver, prostate, colorectal, head and neck, oesophageal, pancreatic, bladder, and non-small cell lung, and is implicated in the pathogenesis of diffuse large B-cell lymphoma and nasopharyngeal carcinomas. Overall, up to 70% of human tumours are linked to persistent elevated STAT3 activity which can be associated with poorer prognosis in many of these settings. In addition, elevated pSTAT3 is observed in response to chemotherapy treatment, and also in response to treatment with inhibitors of oncogenic signalling pathways such as EGFR, MAPK and AKT, and is associated with resistance or poorer response to agents targeting these pathways. In many of these cases, JAK1 is believed to be a primary driver of STAT3 phosphorylation and signalling, suggesting inhibition of JAKs as a therapeutic approach to treat these potential resistance mechanisms. The mixed JAK1/2 kinase inhibitor ruxolitinib is approved for the treatment of myeloproliferative neoplasms including intermediate or high risk myelofibrosis and polycythemia vera and has been tested in a variety of tumor settings. Since JAK2 is essential for the signal transduction downstream of erythropoietin, thrombopoietin and related receptors that control erythrocyte and megakaryocyte expansion, dosing of inhibitors that target JAK2 can be limited by toxicities such as thrombocytopenia and anaemia. Starting from a non-kinome selective screening hit, structure-based design was used to optimise a series of aminopyrimidines that led to JAK1-selective candidate drug AZD4205. This compound demonstrates ATP competitive binding with IC50’s in a high ATP concentration enzyme assay against JAK1 of 73 nM (Ki = 2.8 nM), with high selectivity against JAK2 and JAK3 with IC50’s of 13,233 nM and >30,000 nM respectively. In addition it showed potent inhibition of p-STAT3 in a cell based assay of JAK1 activity with an IC50 of 128 nM and excellent selectivity across the kinome. In summary, AZD4205 is a highly potent JAK1-selective kinase inhibitor with excellent preclinical pharmacokinetics with potential for further clinical development. The optimization from screening hit to first disclosure of this candidate drug will be presented. Citation Format: Jason G. Kettle, Qibin Su, Neil Grimster, Sameer Kawatkar, Scott Throner, Richard Woessner, Huawei Chen, Geraldine Bebernitz, Kristen Bell, Erica Anderson, Linette Ruston, Jon Winter-Holt, Paul Lyne, Melissa Vasbinder, Claudio Chuaqui. Discovery of the JAK1 selective kinase inhibitor AZD4205 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 979. doi:10.1158/1538-7445.AM2017-979

Abstract B100: Structure-based design of AZ7732 a novel in vivo active beta-alanine-derived pan-IAP inhibitor.

Both monomeric and dimeric SMAC (Second Mitochondria-derived Activator of Caspaces) mimetics acting as IAP (Inhibitor of apoptosis proteins) anatgonists have been reported in the clinic as well as extensively in the literature (1). The first four amino acids in the N-terminal of SMAC (AVPI) are critical for binding to IAP proteins. Reported medicinal chemistry exploration of the AVPI template has primarily consisted of variation to the VPI position in the amino-terminal of the SMAC peptide. Structural illucidation of XIAP bound to IAP inhibitors has revealed a critical role for the alanine with dense hydrogen bonding, electrostatic and hydrophibic complementarity with the protein. To our knowledge, exploration of alanine modifications has been limited and generally led to significant reduction in potency. Using the molecular modeling software SuperStar(2), we investigated the publically available co-crystal structures of Smac-mimetics with cIAP1 and hypothesized that homologating the basic amine might be tolerated. Applying this strategy, we report on the successful transfer of a beta-alanine warhead to a number of monomeric scaffolds. The resulting novel monomers maintained cIAP1/2 potency albeit with a reduction in xIAP potency. We report here the first co-crystal structure of xIAP baculoviral IAP repeat 3 domain (BIR3) with a beta-alanine derived monomer. Examination of the binding site contacts in the co-crystal structure provided further insight into the optimization of the warhead. Herein we describe the synthesis, SAR and SPR of this novel warhead and the discovery of beta-alanine derived pan-IAP inhibitors. We show that the SAR can be transferred to dimers and is invariant to the position of dimerization. We report our efforts to optimize the series and mitigate Cyp3A4 inhibition. This work led to the discovery of AZ7732, a novel dimeric SMAC-mimetic; a pan inhibitor of IAPs (cIAP BIR3 IC50 = 12 nM, XIAP BIR3 IC50 = 13 nM, and XIAP BIR2 IC50 = 30 nM); potent in cells as a single agent (MDA-MB231 cIAP degradation IC50 = 0.2 nM, GI50 = 0.4 nM) and is synergistic in vitro in combination with gemcitabine. AZ7732 has favorable in vivo PK with physical properties suitable for IV dosing. AZ7732 is active in vivo as a single agent. Once weekly dosing in MDA-MB231 led to dose-dependent tumor growth inhibition with stasis achieved at 2.5 mpk, ¼ MTD. In conclusion, Structure-based design and medicinal chemistry efforts have successfully identified novel monomeric and dimeric SMAC mimetics leading to the discovery of a novel in vivo active dimeric pan-IAP inhibitor. (1) Fulda et al, Nat. Rev. Drug Disc., 11, 109-123, 2012. (2) M. L. Verdonk, et al, J. Mol. Biol., 289, 1093-1108, 1999 Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B100. Citation Format: Jamal C. Saeh, Brian Aquila, Daniel Russell, Edward Hennessy, Alex Hird, Melissa Vasbinder, Andrew Ferguson, Bin Yang, Maureen Hattersley, Naomi Laing, Terry MacIntyre, Troy Patterson, Galina Repik, Michael Rooney, Haiyun Wang, Dave Witson, Li Sha, Donald Cook, Paula Lewis, John Lee, Danyang Li, Victor Kamhi, Vibha Oza, Charles Omer. Structure-based design of AZ7732 a novel in vivo active beta-alanine-derived pan-IAP inhibitor. [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 B100.