Mitigation of reactive metabolite formation for a series of 3-amino-2-pyridone inhibitors of Bruton's tyrosine kinase (BTK).

Reactive metabolites have been putatively linked to many adverse drug reactions including idiosyncratic toxicities for a number of drugs with black box warnings or withdrawn from the market. Therefore, it is desirable to minimize the risk of reactive metabolite formation for lead molecules in optimization, in particular for non-life threatening chronic disease, to maximize benefit to risk ratio. This article describes our effort in addressing reactive metabolite issues for a series of 3-amino-2-pyridone inhibitors of BTK, e.g. compound 1 has a value of 459pmol/mg protein in the microsomal covalent binding assay. Parallel approaches were taken to successfully resolve the issues: establishment of a predictive screening assay with correlation association of covalent binding assay, identification of the origin of reactive metabolite formation using MS/MS analysis of HLM as well as isolation and characterization of GSH adducts. This ultimately led to the discovery of compound 7 (RN941) with significantly reduced covalent binding of 26pmol/mg protein.

Pyrido[2,3-d]pyrimidines: discovery and preliminary SAR of a novel series of DYRK1B and DYRK1A inhibitors.

DYRK1B is a kinase over-expressed in certain cancer cells (including colon, ovarian, pancreatic, etc.). Recent publications have demonstrated inhibition of DYRK1B could be an attractive target for cancer therapy. From a data-mining effort, the team has discovered analogues of pyrido[2,3-d]pyrimidines as potent enantio-selective inhibitors of DYRK1B. Cells treated with a tool compound from this series showed the same cellular effects as down regulation of DYRK1B with siRNA. Such effects are consistent with the proposed mechanism of action. Progress of the SAR study is presented.

Identification of novel, potent and selective inhibitors of Polo-like kinase 1.

A series of pyrimidodiazepines was identified as potent Polo-like kinase 1 (PLK1) inhibitors. The synthesis and SAR are discussed. The lead compound 7 (RO3280) has potent inhibitory activity against PLK1, good selectivity against other kinases, and excellent in vitro cellular potency. It showed strong antitumor activity in xenograft mouse models.

Pyrazolobenzodiazepines: part I. Synthesis and SAR of a potent class of kinase inhibitors.

A novel series of pyrazolobenzodiazepines 3 has been identified as potent inhibitors of cyclin-dependent kinase 2 (CDK2). Their synthesis and structure-activity relationships (SAR) are described. Representative compounds from this class reversibly inhibit CDK2 activity in vitro, and block cell cycle progression in human tumor cell lines. Further exploration has revealed that this class of compounds inhibits several kinases that play critical roles in cancer cell growth and division as well as tumor angiogenesis. Together, these properties suggest a compelling basis for their use as antitumor agents.

Discovery of a highly potent, orally active mitosis/angiogenesis inhibitor r1530 for the treatment of solid tumors.

A new series of 7,8-disubstituted pyrazolobenzodiazepines based on the lead compound 1 have been synthesized and evaluated for their effects on mitosis and angiogenesis. Described herein is the design, synthesis, SAR, and antitumor activity of these compounds leading to the identification of R1530, which was selected for clinical evaluation.

Discovery of RG7112: A Small-Molecule MDM2 Inhibitor in Clinical Development.

The p53 tumor suppressor is a potent transcription factor that plays a key role in the regulation of cellular responses to stress. It is controlled by its negative regulator MDM2, which binds directly to p53 and inhibits its transcriptional activity. MDM2 also targets p53 for degradation by the proteasome. Many tumors produce high levels of MDM2, thereby impairing p53 function. Restoration of p53 activity by inhibiting the p53-MDM2 interaction may represent a novel approach to cancer treatment. RG7112 (2g) is the first clinical small-molecule MDM2 inhibitor designed to occupy the p53-binding pocket of MDM2. In cancer cells expressing wild-type p53, RG7112 stabilizes p53 and activates the p53 pathway, leading to cell cycle arrest, apoptosis, and inhibition or regression of human tumor xenografts.

MDM2 small-molecule antagonist RG7112 activates p53 signaling and regresses human tumors in preclinical cancer models.

MDM2 negatively regulates p53 stability and many human tumors overproduce MDM2 as a mechanism to restrict p53 function. Thus, inhibitors of p53-MDM2 binding that can reactivate p53 in cancer cells may offer an effective approach for cancer therapy. RG7112 is a potent and selective member of the nutlin family of MDM2 antagonists currently in phase I clinical studies. RG7112 binds MDM2 with high affinity (K(D) ~ 11 nmol/L), blocking its interactions with p53 in vitro. A crystal structure of the RG7112-MDM2 complex revealed that the small molecule binds in the p53 pocket of MDM2, mimicking the interactions of critical p53 amino acid residues. Treatment of cancer cells expressing wild-type p53 with RG7112 activated the p53 pathway, leading to cell-cycle arrest and apoptosis. RG7112 showed potent antitumor activity against a panel of solid tumor cell lines. However, its apoptotic activity varied widely with the best response observed in osteosarcoma cells with MDM2 gene amplification. Interestingly, inhibition of caspase activity did not change the kinetics of p53-induced cell death. Oral administration of RG7112 to human xenograft-bearing mice at nontoxic concentrations caused dose-dependent changes in proliferation/apoptosis biomarkers as well as tumor inhibition and regression. Notably, RG7112 was highly synergistic with androgen deprivation in LNCaP xenograft tumors. Our findings offer a preclinical proof-of-concept that RG7112 is effective in treatment of solid tumors expressing wild-type p53.

Preclinical evaluation of the novel multi-targeted agent R1530.

PURPOSE: This study describes the antiproliferative activity of the multikinase inhibitor R1530 in vitro and its antitumor and anti-angiogenic activity, pharmacokinetics, and tolerability in vivo. METHODS: The antiproliferative activity of R1530 was investigated in a range of human tumor, endothelial and fibroblast cell lines. Tolerability and antitumor activity were assessed in mice bearing a range of human tumor xenografts, and anti-angiogenic properties were established in the murine corneal pocket assay. R1530 pharmacokinetics in mice were established. RESULTS: R1530 strongly inhibited human tumor cell proliferation. Growth factor-driven proliferation of endothelial and fibroblast cells was also inhibited. Significant tumor growth inhibition was demonstrated in a lung cancer xenograft model with a range of once daily, weekly and twice-weekly doses of R1530 (3.125-50 mg/kg qd, 100 mg/kg qw, 100 mg/kg biw). Daily doses were most effective in the lung cancer model and also had significant growth inhibitory effects in models of colorectal, prostate, and breast tumors. Tumor regression occurred in all models treated with the maximum tolerated daily dose (50 mg/kg). The doses of 25 and 50 mg/kg qd resulted in biologically significant increased survival in all tested models. After oral administration in nude mice, R1530 showed good tissue penetration. Exposure was dose dependent up to 100 mg/kg with oral administration. CONCLUSIONS: R1530 has demonstrated activity against a range of tumor models in vitro and in vivo and is an effective inhibitor of angiogenesis. These findings support the approach of targeting multiple pathways in the search for potential agents with improved anticancer properties.