Optimization of a Novel Quinazolinone-Based Series of Transient Receptor Potential A1 (TRPA1) Antagonists Demonstrating Potent in Vivo Activity.

There has been significant interest in developing a transient receptor potential A1 (TRPA1) antagonist for the treatment of pain due to a wealth of data implicating its role in pain pathways. Despite this, identification of a potent small molecule tool possessing pharmacokinetic properties allowing for robust in vivo target coverage has been challenging. Here we describe the optimization of a potent, selective series of quinazolinone-based TRPA1 antagonists. High-throughput screening identified 4, which possessed promising potency and selectivity. A strategy focused on optimizing potency while increasing polarity in order to improve intrinsic clearance culminated with the discovery of purinone 27 (AM-0902), which is a potent, selective antagonist of TRPA1 with pharmacokinetic properties allowing for >30-fold coverage of the rat TRPA1 IC50 in vivo. Compound 27 demonstrated dose-dependent inhibition of AITC-induced flinching in rats, validating its utility as a tool for interrogating the role of TRPA1 in in vivo pain models.

Discovery of N-(4-(3-(2-aminopyrimidin-4-yl)pyridin-2-yloxy)phenyl)-4-(4-methylthiophen-2-yl)phthalazin-1-amine (AMG 900), a highly selective, orally bioavailable inhibitor of aurora kinases with activity against multidrug-resistant cancer cell lines.

Efforts to improve upon the physical properties and metabolic stability of Aurora kinase inhibitor 14a revealed that potency against multidrug-resistant cell lines was compromised by increased polarity. Despite its high in vitro metabolic intrinsic clearance, 23r (AMG 900) showed acceptable pharmacokinetic properties and robust pharmacodynamic activity. Projecting from in vitro data to in vivo target coverage was not practical due to disjunctions between enzyme and cell data, complex and apparently contradictory indicators of binding kinetics, and unmeasurable free fraction in plasma. In contrast, it was straightforward to relate pharmacokinetics to pharmacodynamics and efficacy by following the time above a threshold concentration. On the basis of its oral route of administration, a selectivity profile that favors Aurora-driven pharmacology and its activity against multidrug-resistant cell lines, 23r was identified as a potential best-in-class Aurora kinase inhibitor. In phase 1 dose expansion studies with G-CSF support, 23r has shown promising single agent activity.

Development of novel azabenzofuran TRPA1 antagonists as in vivo tools.

The transient receptor potential ankyrin 1 (TRPA1) channel is activated by noxious stimuli including chemical irritants and endogenous inflammatory mediators. Antagonists of this channel are currently being investigated for use as therapeutic agents for treating pain, airway disorders, and itch. A novel azabenzofuran series was developed that demonstrated in vitro inhibition of allyl isothiocyanate (AITC)-induced (45)Ca(2+) uptake with nanomolar potencies against both human and rat TRPA1. From this series, compound 10 demonstrated in vivo target coverage in an AITC-induced flinching model in rats while providing unbound plasma concentrations up to 16-fold higher than the TRPA1 rat IC50.

Synthesis of 4-substituted chlorophthalazines, dihydrobenzoazepinediones, 2-pyrazolylbenzoic acid, and 2-pyrazolylbenzohydrazide via 3-substituted 3-hydroxyisoindolin-1-ones.

Herein we describe a general three-step synthesis of 4-substituted chlorophthalazines in good overall yields. In the key step, N,N-dimethylaminophthalimide (8a) directs the selective monoaddition of alkyl, aryl, and heteroaryl organometallic reagents to afford 3-substituted 3-hydroxyisoindolinones 9b, 9i-9am. Many of these hydroxyisoindolinones are converted to chlorophthalazines 1b-1v via reaction with hydrazine, followed by chlorination with POCl(3). We have also discovered two novel transformations of 3-vinyl- and 3-alkynyl-3-hydroxyisoindolinones. Addition of vinyl organometallic reagents to N,N-dimethylaminophthalimide (8a) provided dihydrobenzoazepinediones 15a-15c via the proposed ring expansion of 3-vinyl-3-hydroxyisoindolinone intermediates. 3-Alkynyl-3-hydroxyisoindolinones react with hydrazine and substituted hydrazines to afford 2-pyrazolyl benzoic acids 16a-16d and 2-pyrazolyl benzohydrazides 17a-17g rather than the expected alkynyl phthalazinones.

Discovery of potent and highly selective thienopyridine Janus kinase 2 inhibitors.

Developing Janus kinase 2 (Jak2) inhibitors has become a significant focus for small molecule drug discovery programs in recent years due to the identification of a Jak2 gain-of-function mutation in the majority of patients with myeloproliferative disorders (MPD). Here, we describe the discovery of a thienopyridine series of Jak2 inhibitors that culminates with compounds showing 100- to >500-fold selectivity over the related Jak family kinases in enzyme assays. Selectivity for Jak2 was also observed in TEL-Jak cellular assays, as well as in cytokine-stimulated peripheral blood mononuclear cell (PBMC) and whole blood assays. X-ray cocrystal structures of 8 and 19 bound to the Jak2 kinase domain aided structure-activity relationship efforts and, along with a previously reported small molecule X-ray cocrystal structure of the Jak1 kinase domain, provided structural rationale for the observed high levels of Jak2 selectivity.

Discovery of triazine-benzimidazoles as selective inhibitors of mTOR.

mTOR is part of the PI3K/AKT pathway and is a central regulator of cell growth and survival. Since many cancers display mutations linked to the mTOR signaling pathway, mTOR has emerged as an important target for oncology therapy. Herein, we report the discovery of triazine benzimidazole inhibitors that inhibit mTOR kinase activity with up to 200-fold selectivity over the structurally homologous kinase PI3Kα. When tested in a panel of cancer cell lines displaying various mutations, a selective inhibitor from this series inhibited cellular proliferation with a mean IC(50) of 0.41 µM. Lead compound 42 demonstrated up to 83% inhibition of mTOR substrate phosphorylation in a murine pharmacodynamic model.

Preclinical evaluation of AMG 900, a novel potent and highly selective pan-aurora kinase inhibitor with activity in taxane-resistant tumor cell lines.

In mammalian cells, the aurora kinases (aurora-A, -B, and -C) play essential roles in regulating cell division. The expression of aurora-A and -B is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis, making them attractive targets for anticancer therapy. AMG 900 is an orally bioavailable, potent, and highly selective pan-aurora kinase inhibitor that is active in taxane-resistant tumor cell lines. In tumor cells, AMG 900 inhibited autophosphorylation of aurora-A and -B as well as phosphorylation of histone H3 on Ser(10), a proximal substrate of aurora-B. The predominant cellular response of tumor cells to AMG 900 treatment was aborted cell division without a prolonged mitotic arrest, which ultimately resulted in cell death. AMG 900 inhibited the proliferation of 26 tumor cell lines, including cell lines resistant to the antimitotic drug paclitaxel and to other aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358), at low nanomolar concentrations. Furthermore, AMG 900 was active in an AZD1152-resistant HCT116 variant cell line that harbors an aurora-B mutation (W221L). Oral administration of AMG 900 blocked the phosphorylation of histone H3 in a dose-dependent manner and significantly inhibited the growth of HCT116 tumor xenografts. Importantly, AMG 900 was broadly active in multiple xenograft models, including 3 multidrug-resistant xenograft models, representing 5 tumor types. AMG 900 has entered clinical evaluation in adult patients with advanced cancers and has the potential to treat tumors refractory to anticancer drugs such as the taxanes.

Discovery of a potent, selective, and orally bioavailable pyridinyl-pyrimidine phthalazine aurora kinase inhibitor.

The discovery of aurora kinases as essential regulators of cell division has led to intense interest in identifying small molecule aurora kinase inhibitors for the potential treatment of cancer. A high-throughput screening effort identified pyridinyl-pyrimidine 6a as a moderately potent dual inhibitor of aurora kinases -A and -B. Optimization of this hit resulted in an anthranilamide lead (6j) that possessed improved enzyme and cellular activity and exhibited a high level of kinase selectivity. However, this anthranilamide and subsequent analogues suffered from a lack of oral bioavailability. Converting the internally hydrogen-bonded six-membered pseudo-ring of the anthranilamide to a phthalazine (8a-b) led to a dramatic improvement in oral bioavailability (38-61%F) while maintaining the potency and selectivity characteristics of the anthranilamide series. In a COLO 205 tumor pharmacodynamic assay measuring phosphorylation of the aurora-B substrate histone H3 at serine 10 (p-histone H3), oral administration of 8b at 50 mg/kg demonstrated significant reduction in tumor p-histone H3 for at least 6 h.

Pyridyl-pyrimidine benzimidazole derivatives as potent, selective, and orally bioavailable inhibitors of Tie-2 kinase.

Selective small molecule inhibitors of Tie-2 kinase are important tools for the validation of Tie-2 signaling in pathological angiogenesis. Reported herein is the optimization of a nonselective scaffold into a potent and highly selective inhibitor of Tie-2 kinase.

Synthesis, structural analysis, and SAR studies of triazine derivatives as potent, selective Tie-2 inhibitors.

A novel class of selective Tie-2 inhibitors was derived from a multi-kinase inhibitor 1. By reversing the amide connectivity and incorporating aminotriazine or aminopyridine hinge-binding moieties, excellent Tie-2 potency and KDR selectivity could be achieved with 3-substituted terminal aryl rings. X-ray co-crystal structure analysis aided inhibitor design. This series was evaluated on the basis of potency, selectivity, and rat pharmacokinetic parameters.

Evolution of a highly selective and potent 2-(pyridin-2-yl)-1,3,5-triazine Tie-2 kinase inhibitor.

Inhibition of angiogenesis is a promising and clinically validated approach for limiting tumor growth and survival. The receptor tyrosine kinase Tie-2 is expressed almost exclusively in the vascular endothelium and is required for developmental angiogenesis and vessel maturation. However, the significance of Tie-2 signaling in tumor angiogenesis is not well understood. In order to evaluate the therapeutic utility of inhibiting Tie-2 signaling, we developed a series of potent and orally bioavailable small molecule Tie-2 kinase inhibitors with selectivity over other kinases, especially those that are believed to be important for tumor angiogenesis. Our earlier work provided pyridinyl pyrimidine 6 as a potent, nonselective Tie-2 inhibitor that was designed on the basis of X-ray cocrystal structures of KDR inhibitors 34 (triazine) and 35 (nicotinamide). Lead optimization resulted in pyridinyl triazine 63, which exhibited >30-fold selectivity over a panel of kinases, good oral exposure, and in vivo inhibition of Tie-2 phosphorylation.

Alkynylpyrimidine amide derivatives as potent, selective, and orally active inhibitors of Tie-2 kinase.

The recognition that aberrant angiogenesis contributes to the pathology of inflammatory diseases, cancer, and myocardial ischemia has generated considerable interest in the molecular mechanisms that regulate blood vessel growth. The receptor tyrosine kinase Tie-2 is expressed primarily by vascular endothelial cells and is critical for embryonic vasculogenesis. Interference with the Tie-2 pathway by diverse blocking agents such as soluble Tie-2 receptors, anti-Tie-2 intrabodies, anti-Ang-2 antibodies, and peptide-Fc conjugates has been shown to suppress tumor growth in xenograft studies. An alternative strategy for interfering with the Tie-2 signaling pathway involves direct inhibition of the kinase functions of the Tie-2 receptor. Herein we describe the development of alkynylpyrimidine amide derivatives as potent, selective, and orally available ATP-competitive inhibitors of Tie-2 autophosphorylation.