Discovery of Non-Nucleotide Small-Molecule STING Agonists via Chemotype Hybridization.

The identification of agonists of the stimulator of interferon genes (STING) pathway has been an area of intense research due to their potential to enhance innate immune response and tumor immunogenicity in the context of immuno-oncology therapy. Initial efforts to identify STING agonists focused on the modification of 2',3'-cGAMP (1) (an endogenous STING activator ligand) and other closely related cyclic dinucleotides (CDNs). While these efforts have successfully identified novel CDNs that have progressed into the clinic, their utility is currently limited to patients with solid tumors that STING agonists can be delivered to intratumorally. Herein, we report the discovery of a unique class of non-nucleotide small-molecule STING agonists that demonstrate antitumor activity when dosed intratumorally in a syngeneic mouse model.

Synthesis and Evaluation of Novel Tetrahydronaphthyridine CXCR4 Antagonists with Improved Drug-like Profiles.

Our first-generation CXCR4 antagonist TIQ15 was rationally modified to improve drug-like properties. Introducing a nitrogen atom into the aromatic portion of the tetrahydroisoquinoline ring led to several heterocyclic variants including the 5,6,7,8-tetrahydro-1,6-naphthyridine series, greatly reducing the inhibition of the CYP 2D6 enzyme. Compound 12a demonstrated the best overall properties after profiling a series of isomeric tetrahydronaphthyridine analogues in a battery of biochemical assays including CXCR4 antagonism, CYP 2D6 inhibition, metabolic stability, and permeability. The butyl amine side chain of 12a was substituted with various lipophilic groups to improve the permeability. These efforts culminated in the discovery of compound 30 as a potent CXCR4 antagonist (IC50 = 24 nM) with diminished CYP 2D6 activity, improved PAMPA permeability (309 nm/s), potent inhibition of human immunodeficiency virus entry (IC50 = 7 nM), a cleaner off-target in vitro safety profile, lower human ether a-go-go-related gene channel activity, and higher oral bioavailability in mice (% FPO = 27) compared to AMD11070 and TIQ15.

Amino-Heterocycle Tetrahydroisoquinoline CXCR4 Antagonists with Improved ADME Profiles via Late-Stage Buchwald Couplings.

This work surveys a variety of diamino-heterocycles as an isosteric replacement for the piperazine substructure of our previously disclosed piperarinyl-tetrahydroisoquinoline containing CXCR4 antagonists. A late-stage Buchwald coupling route was developed for rapid access to final compounds from commercial building blocks. Among 13 analogs in this study, compound 31 embodying an aza-piperazine linkage was found to have the best overall profile with potent CXCR4 inhibitory activity and favorable in vitro absorption, distribution, metabolism, and excretion (ADME) properties. An analysis of the calculated physiochemical parameters (ROF, cLogD) and the experimental ADME attributes of the analogs lead to the selection of 31 for pharmacokinetic studies in mice. Compared with the clinical compound AMD11070, compound 31 has no CYP450 3A4 or 2D6 inhibition, higher metabolic stability and PAMPA permeability, greatly improved physiochemical parameters, and superior oral bioavailability (%F = 24). A binding rationale for 31 within CXCR4 was elucidated from docking and molecular simulation studies.

Tetrahydroisoquinoline CXCR4 Antagonists Adopt a Hybrid Binding Mode within the Peptide Subpocket of the CXCR4 Receptor.

The rationale for the structural and mechanistic basis of a tetrahydroisoquinoline (THIQ) based series of CXCR4 antagonists is presented. Using the previously reported crystal structures which reveal two distinct binding sites of CXCR4 defined as the small molecule (IT1t or minor) binding pocket and peptide (CVX15 or major) binding pocket, we hypothesized our THIQ small molecule series could bind like either molecule in these respective receptor configurations (IT1t versus CVX15 based poses). To this end, a thorough investigation was performed through a combination of receptor mutation studies, medicinal chemistry, biological testing, conformational analysis, and flexible docking. Our findings showed that the CVX15 peptide-based CXCR4 receptor complexes (red pose) were consistently favored over the small molecule IT1t based CXCR4 receptor configurations (blue pose) to correctly explain the computational and mutational studies as well as key structural components of activity for these small molecules.

Design, Synthesis, and Pharmacological Evaluation of Second-Generation Tetrahydroisoquinoline-Based CXCR4 Antagonists with Favorable ADME Properties.

CXCR4 is a G-protein-coupled receptor that interacts with its cognate ligand, CXCL12, to synchronize many physiological responses and pathological processes. Disruption of the CXCL12-CXCR4 circuitry by small-molecule antagonists has emerged as a promising strategy for cancer intervention. We previously disclosed a hit-to-lead effort that led to the discovery of a series of tetrahydroisoquinoline-based CXCR4 antagonists exemplified by the lead compound TIQ15. Herein, we describe our medicinal-chemistry efforts toward the redesign of TIQ15 as a result of high mouse-microsomal clearance, potent CYP2D6 inhibition, and poor membrane permeability. Guided by the in vitro ADME data of TIQ15, structural modifications were executed to provide compound 12a, which demonstrated a reduced potential for first-pass metabolism while maintaining CXCR4 potency. Subsequent SAR studies and multiparameter optimization of 12a resulted in the identification of compound 25o, a highly potent, selective, and metabolically stable CXCR4 antagonist possessing good intestinal permeability and low risk of CYP-mediated drug-drug interactions.

Discovery of N-Alkyl Piperazine Side Chain Based CXCR4 Antagonists with Improved Drug-like Properties.

A novel series of CXCR4 antagonists with piperidinyl and piperazinyl alkylamine side chains designed as butyl amine replacements are described. Several of these compounds showed similar activity to the parent compound TIQ-15 (5) in a SDF-1 induced calcium flux assay. Preliminary structure-activity relationship investigations led us to identify a series containing N-propyl piperazine side chain analogs exemplified by 16 with improved off-target effects as measured in a muscarinic acetylcholine receptor (mAChR) calcium flux assay and in a limited drug safety panel screen. Further efforts to explore SAR and optimize drug properties led to the identification of the N'-ethyl-N-propyl-piperazine tetrahydroisoquinoline derivative 44 and the N-propyl-piperazine benzimidazole compound 37, which gave the best overall profiles with no mAChR or CYP450 inhibition, good permeability in PAMPA assays, and metabolic stability in human liver microsomes.

Synthesis of Novel Tetrahydroisoquinoline CXCR4 Antagonists with Rigidified Side-Chains.

A structure-activity relationship study of potent TIQ15-derived CXCR4 antagonists is reported. In this investigation, the TIQ15 side-chain was constrained to improve its drug properties. The cyclohexylamino congener 15a was found to be a potent CXCR4 inhibitor (IC50 = 33 nM in CXCL12-mediated Ca2+ flux) with enhanced stability in liver microsomes and reduced inhibition of CYP450 (2D6). The improved CXCR4 antagonist 15a has potential therapeutic application as a single agent or combinatory anticancer therapy.

Synthesis and SAR of 1,2,3,4-Tetrahydroisoquinoline-Based CXCR4 Antagonists.

CXCR4 is the most common chemokine receptor expressed on the surface of many cancer cell types. In comparison to normal cells, cancer cells overexpress CXCR4, which correlates with cancer cell metastasis, angiogenesis, and tumor growth. CXCR4 antagonists can potentially diminish the viability of cancer cells by interfering with CXCL12-mediated pro-survival signaling and by inhibiting chemotaxis. Herein, we describe a series of CXCR4 antagonists that are derived from (S)-5,6,7,8-tetrahydroquinolin-8-amine that has prevailed in the literature. This series removes the rigidity and chirality of the tetrahydroquinoline providing 2-(aminomethyl)pyridine analogs, which are more readily accessible and exhibit improved liver microsomal stability. The medicinal chemistry strategy and biological properties are described.

Discovery of Tetrahydroisoquinoline-Containing CXCR4 Antagonists with Improved in Vitro ADMET Properties.

CXCR4 is a seven-transmembrane receptor expressed by hematopoietic stem cells and progeny, as well as by ≥48 different cancers types. CXCL12, the only chemokine ligand of CXCR4, is secreted within the tumor microenvironment, providing sanctuary for CXCR4+ tumor cells from immune surveillance and chemotherapeutic elimination by (1) stimulating prosurvival signaling and (2) recruiting CXCR4+ immunosuppressive leukocytes. Additionally, distant CXCL12-rich niches attract and support CXCR4+ metastatic growths. Accordingly, CXCR4 antagonists can potentially obstruct CXCR4-mediated prosurvival signaling, recondition the CXCR4+ leukocyte infiltrate from immunosuppressive to immunoreactive, and inhibit CXCR4+ cancer cell metastasis. Current small molecule CXCR4 antagonists suffer from poor oral bioavailability and off-target liabilities. Herein, we report a series of novel tetrahydroisoquinoline-containing CXCR4 antagonists designed to improve intestinal absorption and off-target profiles. Structure-activity relationships regarding CXCR4 potency, intestinal permeability, metabolic stability, and cytochrome P450 inhibition are presented.

Macrocyclic pyrrolobenzodiazepine dimers as antibody-drug conjugate payloads.

Macrocyclic pyrrolobenzodiazepine dimers were designed and evaluated for use as antibody-drug conjugate payloads. Initial structure-activity exploration established that macrocyclization could increase the potency of PBD dimers compared with non-macrocyclic analogs. Further optimization overcame activity-limiting solubility issues, leading to compounds with highly potent (picomolar) activity against several cancer cell lines. High levels of in vitro potency and specificity were demonstrated with an anti-mesothelin conjugate.

Structure-Based Design of Selective Janus Kinase 2 Imidazo[4,5-d]pyrrolo[2,3-b]pyridine Inhibitors.

Early hit to lead work on a pyrrolopyridine chemotype provided access to compounds with biochemical and cellular potency against Janus kinase 2 (JAK2). Structure-based drug design along the extended hinge region of JAK2 led to the identification of an important H-bond interaction with the side chain of Tyr 931, which improved JAK family selectivity. The 4,5-dimethyl thiazole analogue 18 demonstrated high levels of JAK family selectivity and was identified as a promising lead for the program.

Discovery of a Highly Selective JAK2 Inhibitor, BMS-911543, for the Treatment of Myeloproliferative Neoplasms.

JAK2 kinase inhibitors are a promising new class of agents for the treatment of myeloproliferative neoplasms and have potential for the treatment of other diseases possessing a deregulated JAK2-STAT pathway. X-ray structure and ADME guided refinement of C-4 heterocycles to address metabolic liability present in dialkylthiazole 1 led to the discovery of a clinical candidate, BMS-911543 (11), with excellent kinome selectivity, in vivo PD activity, and safety profile.

Antibody-drug conjugates: current status and future directions.

Antibody-drug conjugates (ADCs) aim to take advantage of the specificity of monoclonal antibodies (mAbs) to deliver potent cytotoxic drugs selectively to antigen-expressing tumor cells. Despite the simple concept, various parameters must be considered when designing optimal ADCs, such as selection of the appropriate antigen target and conjugation method. Each component of the ADC (the antibody, linker and drug) must also be optimized to fully realize the goal of a targeted therapy with improved efficacy and tolerability. Advancements over the past several decades have led to a new generation of ADCs comprising non-immunogenic mAbs, linkers with balanced stability and highly potent cytotoxic agents. Although challenges remain, recent clinical success has generated intense interest in this therapeutic class.

Pyrazole and pyrimidine phenylacylsulfonamides as dual Bcl-2/Bcl-xL antagonists.

5-Butyl-1,4-diphenyl pyrazole and 2-amino-5-chloro pyrimidine acylsulfonamides were developed as potent dual antagonists of Bcl-2 and Bcl-xL. Compounds were optimized for binding to the I88, L92, I95, and F99 pockets normally occupied by pro-apoptotic protein Bim. An X-ray crystal structure confirmed the proposed binding mode. Observation of cytochrome c release from isolated mitochondria in MV-411 cells provides further evidence of target inhibition. Compounds demonstrated submicromolar antiproliferative activity in Bcl-2/Bcl-xL dependent cell lines.

Discovery of N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-4-ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (BMS-777607), a selective and orally efficacious inhibitor of the Met kinase superfamily.

Substituted N-(4-(2-aminopyridin-4-yloxy)-3-fluoro-phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamides were identified as potent and selective Met kinase inhibitors. Substitution of the pyridine 3-position gave improved enzyme potency, while substitution of the pyridone 4-position led to improved aqueous solubility and kinase selectivity. Analogue 10 demonstrated complete tumor stasis in a Met-dependent GTL-16 human gastric carcinoma xenograft model following oral administration. Because of its excellent in vivo efficacy and favorable pharmacokinetic and preclinical safety profiles, 10 has been advanced into phase I clinical trials.

Discovery of orally active pyrrolopyridine- and aminopyridine-based Met kinase inhibitors.

A series of acylurea analogs derived from pyrrolopyridine and aminopyridine scaffolds were identified as potent inhibitors of Met kinase activity. The SAR at various positions of the two kinase scaffolds was investigated. These studies led to the discovery of compounds 3b and 20b, which demonstrated favorable pharmacokinetic properties in mice and significant antitumor activity in a human gastric carcinoma xenograft model.

Identification of pyrrolo[2,1-f][1,2,4]triazine-based inhibitors of Met kinase.

An amide library derived from the pyrrolo[2,1-f][1,2,4]triazine scaffold led to the identification of modest inhibitors of Met kinase activity. Introduction of polar side chains at C-6 of the pyrrolotriazine core provided significant improvements in in vitro potency. The amide moiety could be replaced with acylurea and malonamide substituents to give compounds with improved potency in the Met-driven GTL-16 human gastric carcinoma cell line. Acylurea pyrrolotriazines with substitution at C-5 demonstrated single digit nanomolar kinase activity. X-ray crystallography revealed that the C-5 substituted pyrrolotriazines bind to the Met kinase domain in an ATP-competitive manner.

Synthesis and SAR of pyrrolotriazine-4-one based Eg5 inhibitors.

Synthesis and SAR of substituted pyrrolotriazine-4-one analogues as Eg5 inhibitors are described. Many of these analogues displayed potent inhibitory activities in the Eg5 ATPase and A2780 cell proliferation assays. In addition, pyrrolotriazine-4-one analogue 26 demonstrated in vivo efficacy in an iv P388 murine leukemia model. Both NMR and X-ray crystallographic studies revealed that these analogues bind to an allosteric site on the Eg5 protein.

Exploiting the Pd- and Ru-catalyzed cycloisomerizations: enantioselective total synthesis of (+)-allocyathin B2.

Pd- and Ru-catalyzed cycloisomerizations of 1,6-enynes are compared and contrasted. Such considerations led to the enantioselective synthesis of a cyathin terpenoid, (+)-allocyathin B(2) (1). The synthesis features a Pd-catalyzed asymmetric allylic alkylation (AAA) to install the initial quaternary center, a Ru-catalyzed diastereoselective cycloisomerization to construct the six-membered ring, and a diastereoselective hydroxylative Knoevenagel reaction to introduce the final hydroxyl group. We demonstrate for the first time a mechanism-based stereochemical divergence in Pd- and Ru-catalyzed cycloisomerization reactions as well as in creation of alkene geometry with alkynes bearing a carboalkoxy group. Mechanistic rationalization is proposed for these observations.

Total synthesis of (+)-allocyathin B2.

The first enantioselective synthesis of (+)-allocyathin was achieved. The synthesis features a Pd-catalyzed asymmetric allylic alkylation to install the first quaternary center, a Ru-catalyzed diastereoselective cycloisomerization to construct the six-membered ring, and a diastereoselective hydroxylative Knoevenagel reaction to introduce the final hydroxyl group. The unusual olefin isomerization of the Ru-catalyzed cycloisomerization was discussed and exploited for the synthesis.