Joseph Fargnoli

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 Brivanib Alaninate ((S)-((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate), A Novel Prodrug of Dual Vascular Endothelial Growth Factor Receptor-2 and Fibroblast Growth Factor Receptor-1 Kinase Inhibitor (BMS-540215)

A series of amino acid ester prodrugs of the dual VEGFR-2/FGFR-1 kinase inhibitor 1 (BMS-540215) was prepared in an effort to improve the aqueous solubility and oral bioavailability of the parent compound. These prodrugs were evaluated for their ability to liberate parent drug 1 in in vitro and in vivo systems. The l-alanine prodrug 8 (also known as brivanib alaninate/BMS-582664) was selected as a development candidate and is presently in phase II clinical trials.

Discovery of Pyrrolopyridine-Pyridone Based Inhibitors of Met Kinase : Synthesis, X-ray Crystallographic Analysis, and Biological Activities

Conformationally constrained 2-pyridone analogue 2 is a potent Met kinase inhibitor with an IC50 value of 1.8 nM. Further SAR of the 2-pyridone based inhibitors of Met kinase led to potent 4-pyridone and pyridine N-oxide inhibitors such as 3 and 4. The X-ray crystallographic data of the inhibitor 2 bound to the ATP binding site of Met kinase protein provided insight into the binding modes of these inhibitors, and the SAR of this series of analogues was rationalized. Many of these analogues showed potent antiproliferative activities against the Met dependent GTL-16 gastric carcinoma cell line. Compound 2 also inhibited Flt-3 and VEGFR-2 kinases with IC50 values of 4 and 27 nM, respectively. It possesses a favorable pharmacokinetic profile in mice and demonstrates significant in vivo antitumor activity in the GTL-16 human gastric carcinoma xenograft model.

Differential Mechanisms of Acquired Resistance to Insulin-like Growth Factor-I Receptor Antibody Therapy or to a Small-Molecule Inhibitor, BMS-754807, in a Human Rhabdomyosarcoma Model

Agents targeting insulin-like growth factor-I receptor (IGF-IR), including antibodies and small-molecule inhibitors, are currently in clinical development for the treatment of cancers including sarcoma. However, development of resistance is a common phenomenon resulting in failures of anticancer therapies. In light of this problem, we developed two resistant models from the rhabdomyosarcoma cell line Rh41: Rh41-807R, with acquired resistance to BMS-754807, a small-molecule dual-kinase inhibitor targeting IGF-IR and insulin receptor (IR), and Rh41-MAB391R, with resistance to MAB391, an IGF-IR-blocking antibody. In addition, tumor xenograft models were established from Rh41 and Rh41-807R cell lines. Gene expression and DNA copy number analyses of these models revealed shared as well as unique acquired resistance mechanisms for the two types of IGF-IR inhibitors. Each resistant model used different signaling pathways as a mechanism for proliferation. Platelet-derived growth factor receptor α (PDGFRα) was amplified, overexpressed, and constitutively activated in Rh41-807R cells and tumors. Knockdown of PDGFRα by small interfering RNA in Rh41-807R resensitized the cells to BMS-754807. Synergistic activities were observed when BMS-754807 was combined with PDGFRα inhibitors in the Rh41-807R model in vitro. In contrast, AXL expression was highly elevated in Rh41-MAB391R but downregulated in Rh41-807R. Notably, BMS-754807 was active in Rh41-MAB391R cells and able to overcome resistance to MAB391, but MAB391 was not active in Rh41-807R cells, suggesting potentially broader clinical activity of BMS-754807. This is the first study to define and compare acquired resistance mechanisms for IGF-IR-targeted therapies. It provides insights into the differential acquired resistance mechanisms for IGF-IR/IR small-molecule inhibitor versus anti-IGF-IR antibody.

The Antiangiogenic Activity in Xenograft Models of Brivanib, a Dual Inhibitor of Vascular Endothelial Growth Factor Receptor-2 and Fibroblast Growth Factor Receptor-1 Kinases

Tumor angiogenesis is a complex and tightly regulated network mediated by various proangiogenic factors. The fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) family of growth factors, and associated tyrosine kinase receptors have a major influence in tumor growth and dissemination and may work synergistically to promote angiogenesis. Brivanib alaninate is the orally active prodrug of brivanib, a selective dual inhibitor of FGF and VEGF signaling. Here, we show that brivanib demonstrates antitumor activity in a broad range of xenograft models over multiple dose levels and that brivanib alaninate shows dose-dependent efficacy equivalent to brivanib in L2987 human tumor xenografts. Brivanib alaninate (107 mg/kg) reduced tumor cell proliferation as determined by a 76% reduction in Ki-67 staining and reduced tumor vascular density as determined by a 76% reduction in anti-CD34 endothelial cell staining. Furthermore, Matrigel plug assays in athymic mice showed that brivanib alaninate inhibited angiogenesis driven by VEGF or basic FGF alone, or combined. Dynamic contrast-enhanced magnetic resonance imaging, used to assess the effects of brivanib alaninate on tumor microcirculation, showed a marked decrease in gadopentetate dimeglumine contrast agent uptake at 107 mg/kg dose, with a reduction in area under the plasma concentration-time curve from time 0 to 60 minutes at 24 and 48 hours of 54% and 64%, respectively. These results show that brivanib alaninate is an effective antitumor agent in preclinical models across a range of doses, and that efficacy is accompanied by changes in cellular and vascular activities. Mol Cancer Ther; 9(2); 369–78

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.

Discovery and Validation of Biomarkers that Respond to Treatment with Brivanib Alaninate, a Small-Molecule VEGFR-2/FGFR-1 Antagonist

The process of neovascularization from preexisting blood vessels, referred to as angiogenesis, plays a critical role in both tumor growth and dissemination in multiple cancer types. Currently, there exists a need to identify biomarkers that can both indicate biological activity and predict efficacy at the molecular level for antiangiogenesis drugs which are anticipated to result in tumor stasis rather than regression. To identify such biomarkers, athymic mice bearing L2987 human tumor xenografts were treated with the antiangiogenic agent brivanib alaninate, which is currently under clinical evaluation. This is an orally available and selective tyrosine kinase inhibitor that targets the key angiogenesis receptors vascular endothelial growth factor receptor 2 (VEGFR-2) and fibroblast growth factor receptor 1. In the described studies, tumor samples were collected from these xenografts and RNA was extracted for gene expression profiling on Affymetrix 430A mouse GeneChips. Statistical analysis was done using a defined set of genes identified to be coexpressed with VEGFR-2 from a clinical tumor gene expression profiling database and between tumor samples isolated from brivanib alaninate-treated and untreated mice. Tyrosine kinase receptor 1 (Tie-1), collagen type IV alpha1 (Col4a1), complement component 1, q subcomponent receptor 1 (C1qr1), angiotensin receptor-like 1 (Agtrl1), and vascular endothelial-cadherin (Cdh5) were all identified to be significantly modulated by treatment with brivanib alaninate. These genes, which may be potentially useful as markers of brivanib alaninate activity, were further studied at the protein level in two separate in vivo human colon tumor xenograft models, HCT116 and GEO, using immunohistochemistry-based approaches.

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.

Synergistic Antitumor Activity of Ixabepilone (BMS-247550) Plus Bevacizumab in Multiple In vivo Tumor Models

Purpose: Angiogenesis is a critical step in the establishment, growth, and metastasis of solid tumors, and combination of antiangiogenic agents with chemotherapy is an attractive therapeutic option. We investigated the potential of ixabepilone, the first in a new class of antineoplastic agents known as epothilones, to synergize with antiangiogenic agents to inhibit tumor growth. Experimental Design:In vitro and in vivo cytotoxicity of ixabepilone as single agent and in combination with two targeted antiangiogenic agents, bevacizumab or sunitinib, were examined in preclinical tumor models. Direct effects of the agents against endothelial cells was also examined and compared with the effects of paclitaxel as single agent and in combination with bevacizumab. Results: Ixabepilone showed robust synergistic antitumor activity in combination with bevacizumab and sunitinib in preclinical in vivo models derived from breast, colon, lung, and kidney cancers. The synergistic antitumor effect was greater with ixabepilone compared with paclitaxel. Furthermore, ixabepilone was more effective than paclitaxel at killing endothelial cells expressing P-glycoprotein in vitro and inhibiting endothelial cell proliferation and tumor angiogenesis in vivo. Conclusions: Ixabepilone may enhance the antitumor effects of antiangiogenic therapy by direct cytotoxicity and also indirectly via the killing of tumor-associated endothelial cells. Given that ixabepilone has reduced susceptibility to drug efflux pumps compared with taxanes, these data may explain the increased antiangiogenic and antitumor activity of ixabepilone in combination with antiangiogenic agents. Phase II studies to assess the efficacy and safety of ixabepilone plus bevacizumab in locally recurrent or metastatic breast cancer are planned.

The Discovery of Macrocyclic XIAP Antagonists from a DNA-Programmed Chemistry Library, and Their Optimization To Give Lead Compounds with in Vivo Antitumor Activity.

Affinity selection screening of macrocycle libraries derived from DNA-programmed chemistry identified XIAP BIR2 and BIR3 domain inhibitors that displace bound pro-apoptotic caspases. X-ray cocrystal structures of key compounds with XIAP BIR2 suggested potency-enhancing structural modifications. Optimization of dimeric macrocycles with similar affinity for both domains were potent pro-apoptotic agents in cancer cell lines and efficacious in shrinking tumors in a mouse xenograft model.