We have designed and developed novel and selective TLR7 agonists that exhibited potent receptor activity in a cell-based reporter assay. In vitro, these agonists significantly induced secretion of cytokines IL-6, IL-1 beta, IL-10, TNFa, IFNa, and IP-10 in human and mouse whole blood. Pharmacokinetic and pharmacodynamic studies in mice showed a significant secretion of IFN alpha and TNF alpha cytokines. When combined with aPD1 in a CT-26 tumor model, the lead compound showed strong synergistic antitumor activity with complete tumor regression in 8/10 mice dosed using the intravenous route. Structure-activity relationship studies enabled by structure-based designs of TLR7 agonists are disclosed.

Dual activation of the TLR7 and TLR8 pathways leads to the production of type I interferon and proinflammatory cytokines, resulting in efficient antigen presentation by dendritic cells to promote T-cell priming and antitumor immunity. We developed a novel series of TLR7/8 dual agonists with varying ratios of TLR7 and TLR8 activity for use as payloads for an antibody-drug conjugate approach. The agonist-induced production of several cytokines in human whole blood confirmed their functional activity. Structure-activity relationship studies guided by structure-based drug design are described.

We describe the design, synthesis, and structure-activity relationship (SAR) of heterobifunctional RET ligand-directed degraders (LDDs) derived from three different second-generation RET inhibitors. These LDDs are composed of a target binding motif (TBM) that binds to the RET protein, a linker, and a cereblon binding motif (CBM) as the E3 ligase recognition unit. This led to the identification of a series of pyrazolopyridine-based heterobifunctional LDDs, as exemplified by compound 39. LDD 39 demonstrated high in vitro inhibitory and degradation potency against both RET wild-type and the two representative mutants, V804M and G810R. Importantly, in PK/PD studies, 39 exhibited a differentiated and favorable in vivo profile compared to the corresponding tyrosine kinase inhibitor (TKI), compound 3. Robust and sustained degradation of total-RET (tRET) protein and inhibition of phospho-RET (pRET) signaling were observed in TPC-1 xenograft tumors driven by RET and the RET/G810R mutant following a single dose of LDD 39 at 15 and 75 mg/kg, respectively.

Small molecule toll-like receptor (TLR) 7 agonists have gathered considerable interest as promising therapeutic agents for applications in cancer immunotherapy. Herein, we describe the development and optimization of a series of novel TLR7 agonists through systematic structure-activity relationship studies focusing on modification of the phenylpiperidine side chain. Additional refinement of ADME properties culminated in the discovery of compound 14, which displayed nanomolar reporter assay activity and favorable drug-like properties. Compound 14 demonstrated excellent in vivo pharmacokinetic/pharmacodynamic profiles and synergistic antitumor activity when administered in combination with aPD1 antibody, suggesting opportunities of employing 14 in immuno-oncology therapies with immune checkpoint blockade agents.

Supplementary Figures 1-6 from Antitumor and Antiangiogenic Activities of BMS-690514, an Inhibitor of Human EGF and VEGF Receptor Kinase Families

Supplementary Tables 1-3, Figure Legends 1-6 from Antitumor and Antiangiogenic Activities of BMS-690514, an Inhibitor of Human EGF and VEGF Receptor Kinase Families

Abstract The chromosome 9p21 (chr9p21) locus is deleted in almost 10% of all cancer types. This locus includes the CDKN2A gene that encodes the critical tumor suppressors p19-ARF and p16-INK4a. Methylthioadenosine phosphorylase (MTAP), a gene proximal to CDKN2A, is co-deleted in 80%-90% of tumors with CDKN2A deletion. MTAP plays a critical role in the methionine salvage pathway, and the deletion of MTAP results in the accumulation of its substrate methythioadenosine (MTA). Accumulation of MTA partially inhibits the activity of the arginine methyltransferase PRMT5, causing MTAP deficient cancer cells to be more sensitive to the genetic knockdown of PRMT5. In contrast to genetic knockdown, sensitivity to pharmacological inhibition of PRMT5 does not appear to stratify with MTAP status. However, currently known PRMT5 inhibitors all possess SAM competitive or uncompetitive MOIs, which generally require displacement of MTA from the active site for binding. We hypothesize that leveraging the high MTA state induced by MTAP deficiency will require an inhibitor that can bind PRMT5 without disrupting bound MTA. Here we report the discovery of a PRMT5 inhibitor with a novel binding mode that is compatible with MTA binding. While this compound possesses the desired MOI, it shows only modestly increased potency toward MTAP-null cells. Mathematical simulations of different inhibitor mechanisms indicate that the degree of selectivity that can be achieved depends on the difference in MTA levels between MTAP-null and WT cells. Our in vitro data suggest that the elevation in intracellular MTA concentrations that occurs with MTAP deletion is not sufficient to confer significantly increased sensitivity to PRMT5 inhibition. We anticipate that the therapeutic index that can be achieved between MTAP-null tumor cells and PRMT5-sensitive normal tissues will be similarly limited in vivo. Citation Format: Rohit Malik, Peter K. Park, Christopher M. Barbieri, Yuval Blat, Steven Sheriff, Carolyn A. Weigelt, Lisa M. Kopcho, Muge Celiktas, Max Ruzanov, Joseph G. Naglich, Jennifer L. Price, Mary Harner, Kevin M. Omalley, JIngjing Deng, William Schmitz, Guo Li, Zheming Ruan, Lan-ying Qin, Gerald J. Duke, Iyoncy Rodrigo, Mark R. Witmer, David G. Harden, Shilpa Demes, Brian J. Arey, Matt Soars, Brian E. Fink, Ashvinikumar V. Gavai, Gregory D. Vite, Charles F. Voliva. A novel MTA non-competitive PRMT5 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1140.

We describe our efforts to introduce structural diversity to a previously described triazole-containing N1-carboline series of bromodomain and extra-terminal (BET) inhibitors. N9 carbolines were designed to retain favorable binding interactions that the N1-carbolines possess. A convergent synthetic route enabled modifications to reduce clearance, enhance physicochemical properties, and improve the overall in vitro profile. This work led to the identification of a potent BET inhibitor, (S)-2-{8-fluoro-5-[(3-fluoropyridin-2-yl)(oxan-4-yl)methyl]-7-[4-(2H3)methyl-1-methyl-1H-1,2,3-triazol-5-yl]-5H-pyrido[3,2-b]indol-3-yl}propan-2-ol (10), a compound with enhanced oral exposure in mice. Subsequent evaluation in a mouse triple-negative breast cancer tumor model revealed efficacy at 4 mg/kg of N9-carboline 10.

Inhibition of the bromodomain and extra-terminal (BET) family of adaptor proteins is an attractive strategy for targeting transcriptional regulation of key oncogenes, such as c-MYC. Starting with the screening hit 1, a combination of structure-activity relationship and protein structure-guided drug design led to the discovery of a differently oriented carbazole 9 with favorable binding to the tryptophan, proline, and phenylalanine (WPF) shelf conserved in the BET family. Identification of an additional lipophilic pocket and functional group optimization to optimize pharmacokinetic (PK) properties culminated in the discovery of 18 (BMS-986158) with excellent potency in binding and functional assays. On the basis of its favorable PK profile and robust in vivo activity in a panel of hematologic and solid tumor models, BMS-986158 was selected as a candidate for clinical evaluation.

We describe our efforts to identify structurally diverse leads in the triazole-containing N1-carboline series of bromodomain and extra-terminal inhibitors. Replacement of the N5 "cap" phenyl moiety with various heteroaryls, coupled with additional modifications to the carboline core, provided analogs with similar potency, improved pharmacokinetic properties, and increased solubility compared to our backup lead, BMS-986225 (2). Rapid SAR exploration was enabled by a convergent, synthetic route. These efforts provided a potent BET inhibitor, 3-fluoropyridyl 12, that demonstrated robust efficacy in a multiple myeloma mouse tumor model at 1 mg/kg.

Abstract Bromodomain and extraterminal (BET) proteins recognize acetylated lysine residues for the purpose of transcriptional regulation of genes, including those involved in stem cell renewal and oncogenes such as MYC. Inhibition of BET proteins BRD2, BRD3, BRD4, and BRDT represents a promising treatment option for patients with cancer. However, little is known about the consequence of BET inhibition on normal stem cell renewal processes, such as hematopoiesis. This study explored the mechanistic effects of BET inhibition on bone marrow (BM) hematopoiesis. Rats treated with BET inhibitors JQ1, BMS-X, or BMS-986158 for 4 days in vivo showed dose-dependent pan-cellular BM atrophy and reduction of hematopoietic progenitors of myeloid and erythroid lineage (combination of anti-rat CD45, CD11b, anti-granulocyte, CD71, anti-erythroid, and CD90 via flow cytometry) and consequential reductions in circulating platelet and reticulocyte counts, with complete reversibility within 10 days of stopping treatment with BET inhibitors. Primary rat BM stem and progenitor cells treated with BET inhibitors in vitro were evaluated with the colony-forming unit assay and resulted in dose-dependent reduction of multiple lineage progenitor colonies, especially the erythroid and megakaryocyte lineages. To further elucidate pathways involved in BET-related BM atrophy, erythropoiesis and thrombopoiesis genes regulated by GATA1, a BRD-associated transcription factor, from rat BM, as well as rat and human whole-blood samples exposed to BET inhibitor(s) were evaluated via RNAseq and RT-PCR. Dose-dependent responses in genes involved in erythropoiesis (Alas2, ABCme, PBG-D, HMBS) and thrombopoiesis (NFE2, PF4, GP1Bb, MPL) were observed after 4 days of treatment with BMS-986158. In a clinical trial (NCT02419417), patients with solid tumors treated with BMS-986158 demonstrated reversible thrombocytopenia and downregulation of NFE2, PF4, and HMBS expression, similar to that observed in rats. GATA1 was also downregulated in rat BM, with target engagement in rat and human demonstrated by the induction of HEXIM1 transcription, a pharmacodynamic (PD) biomarker of growth inhibition and apoptosis induced by BRD4 inhibition. Overall, our results suggest inhibition of BET signaling causes target-related, dose-dependent repression of hematopoietic progenitors through alterations of GATA1-associated erythropoiesis and thrombopoiesis regulation in rat BM and human blood samples, and these effects are reversible on cessation of BET inhibitor treatment. This is the first in vivo study demonstrating the mechanism of BET inhibition resulting in GATA1-associated repression of hematopoietic progenitors that is correlated to clinical pharmacokinetics and PD (Chen X, et al. AACR 2020) and is translatable from preclinical evaluation to clinical experience. Citation Format: Cindy Zhang, Ke Xu, Julie Panzica-Kelly, Jennifer Price, Denise Bounous, Shodeinde Coker, Kezi Unsal-Kacmaz, Danielle Greenawalt, Ashvinikumar Gavai, Ronald Fleming, Karen Augustine-Rauch, Richard Westhouse. Inhibition of BET signaling leads to reversible GATA1-associated repression of hematopoietic progenitors: translation from preclinical assessment to clinical development [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1742.

C-terminal Src kinase (CSK) functions as a negative regulator of T cell activation through inhibitory phosphorylation of LCK, so inhibitors of CSK are of interest as potential immuno-oncology agents. Screening of an internal kinase inhibitor collection identified pyridazinone lead 1, and a series of modifications led to optimized compound 13. Compound 13 showed potent activity in biochemical and cellular assays in vitro and demonstrated the ability to increase T cell proliferation induced by T cell receptor signaling. Compound 13 gave extended exposure in mice upon oral dosing and produced a functional response (decrease in LCK phosphorylation) in mouse spleens at 6 h post dose.

Abstract Background: Regulation and recognition of covalent chromatin modifications by cellular proteins are key determinants of gene expression. Bromodomain and extra-terminal (BET) proteins, including BRD2, BRD3, BRD4, and BRDT, bind directly to acetylated lysine on histone tails to promote gene transcription. Oncogenes such as c-MYC, BCL2, and ASCL1, are directly regulated by BET proteins. Here, we report the preclinical evaluation of BMS-986158, an orally bioavailable, potent, and selective BET inhibitor. Methods: BMS-986158 activity was assessed in tumor growth assays across solid and hematologic cancer cell lines in vitro and in vivo in patient-derived xenografts (PDX). Dose-response gene expression profiling of ex vivo blood from healthy donors was used to identify transcripts exhibiting a pharmacodynamic effect with BMS-986158 treatment. Results: Tumor growth inhibition (TGI) with BMS-986158 was observed across different solid and hematologic cancer cell lines. Mutations in chromatin regulators were identified among BMS-986158–sensitive cell lines. BMS-986158 also demonstrated antitumor activity (TGI ≥ 70%) in 24 of 82 PDX models tested (Table). Genetic alterations that correlate with BMS-986158 responsiveness will be presented. Finally, an ex vivo dose-response profiling study in healthy human blood identified 124 genes responsive to BMS-986158, including chemokines and chemokine receptors. Conclusions: BMS-986158, a potent and selective BET inhibitor, demonstrated robust anti-proliferative activity in vitro across a broad range of cancer cell lines. Consistent with the in vitro data, preclinical antitumor activity was observed in various PDX tumor models. Furthermore, gene expression profiling studies in human blood samples identified select target genes responsive to BMS-986158 treatment that may be useful as clinical pharmacodynamic markers. TGI assay in PDX modelsTumor TypeTotal sample, nResponders, naAdenocarcinoma319Lung squamous cell carcinoma217Breast cancer82Brain cancer10Colorectal cancer63Esophageal cancer31Gallbladder cancer21Head and neck cancer31Mixedb70Total8224a Responders were defined as having > 70% TGIb Mixed tumor types include large cell neuroendocrine carcinoma of the lung, liver, liver metastases from rectum, mixed adenocarcinoma, neuroendocrine tumor, and non-small cell lung cancer Citation Format: Susan Wee, Donald Jackson, Heshani Desilva, Maya Dajee, Julie Carman, Petra Ross-MacDonald, Jinping Gan, Richard A. Westhouse, Christine Huang, Zheng Yang, Michael Poss, John T. Hunt, Gregory D. Vite, Ashvinikumar V. Gavai. Preclinical antitumor activity of BMS-986158, an oral BET inhibitor, for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5792.

Abstract Background: The bromodomains and extra-terminal domain (BET) proteins are a family of 4 adapter proteins, BRD2, BRD3, BRD4, and BRDT, that bind to specific acetylated lysine residues on the histone tails of chromatin and recruit additional proteins to regulate gene transcription. The c-MYC oncogene, which is amplified and deregulated in 40% to 70% of all cancers, is directly regulated by BET proteins. Preclinical studies provide a strong rationale for pursuing transcriptional regulation via BET inhibition in cancer treatment (Lenhart, et al. Mol Cancer Ther. 2015;14:2167-2174; Filippakopoulos, et al. Nature. 2010;468:1067-1073). Here, we present results of crystal structure-guided structure-activity relationship (SAR) studies that resulted in the identification of BMS-986158, a highly potent BET inhibitor. Methods: Using fluorescence resonance energy transfer (FRET), we screened a library of compounds and identified a carbazole series of BET inhibitors. Alkylation of the carbazole nitrogen resulted in a 10-fold boost in potency against BET. We then created a differently oriented carbazole series and, subsequently, a carboline series of compounds to improve potency and pharmaceutical properties. A thermal shift assay was used to evaluate selectivity for binding to the BET family of bromodomains. Results: Crystal structure and subsequent SAR studies demonstrated that the isoxazole moiety formed critical interactions with the BET bromodomains. Lead compounds demonstrated potent binding to BRD4 and reduction in c-MYC expression and proliferation in cell lines such as KMS-11. Accessing a second lipophilic pocket in the BRD4 binding site increased potency significantly. Modification of the lead series from a carbazole carboxamide to a carboline resulted in significant improvement in pharmaceutical properties and led to the identification of BMS-986158, which demonstrated in vitro and in vivo potency against a variety of tumor types. In c-MYC-driven cancer cell lines, BMS-986158 caused dose-dependent downregulation of c-MYC expression and induced cancer cell death. BMS-986158 demonstrated > 70% tumor growth inhibition at tolerated doses in patient-derived xenograft models (lung, colorectal, and triple-negative breast cancers). Antitumor activity in mice and pharmacokinetic properties in animal studies support oral dosing in humans. Conclusions: Structure-based drug design led to the discovery of BMS-986158, a highly potent BET inhibitor. With promising antitumor activity in preclinical studies, BMS-986158 is currently being evaluated in a phase 1/2a clinical trial in patients with advanced cancers. Citation Format: Ashvinikumar V. Gavai, Derek Norris, David Tortolani, Daniel O'Malley, Yufen Zhao, Claude Quesnelle, Patrice Gill, Wayne Vaccaro, Tram Huynh, Vijay Ahuja, Dharmpal Dodd, Christopher Mussari, Lalgudi Harikrishnan, Muthoni Kamau, John S. Tokarski, Steven Sheriff, Richard Rampulla, Dauh-Rurng Wu, Jianqing Li, Huiping Zhang, Peng Li, Dawn Sun, Henry Yip, Yingru Zhang, Arvind Mathur, Haiying Zhang, Christine Huang, Zheng Yang, Asoka Ranasinghe, Celia D'Arienzo, Ching Su, Gerry Everlof, Lisa Zhang, Nirmala Raghavan, John T. Hunt, Michael Poss, Gregory D. Vite, Richard A. Westhouse, Susan Wee. Discovery of clinical candidate BMS-986158, an oral BET inhibitor, for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5789.

An enantioselective synthesis of (S)-7-amino-5H,7H-dibenzo[b,d]azepin-6-one (S-1) is described. The key step in the sequence involved crystallization-induced dynamic resolution (CIDR) of compound 7 using Boc-D-phenylalanine as a chiral resolving agent and 3,5-dichlorosalicylaldehyde as a racemization catalyst to afford S-1 in 81% overall yield with 98.5% enantiomeric excess.

An oxidative intermolecular enolate heterocoupling reaction was employed for the synthesis of anti-2,3-disubstituted succinic acid mono- and differentially protected diesters. Tactical approaches to access all the diastereomers are discussed. The method was applied to the synthesis of a potent anticancer agent, BMS-906024.