Supplementary Data from Antitumor Activity and Pharmacology of a Selective Focal Adhesion Kinase Inhibitor, PF-562,271

The synthesis and biological evaluation of novel Tie-2 kinase inhibitors are presented. Based on the pyrrolopyrimidine chemotype, several new series are described, including the benzimidazole series by linking a benzimidazole to the C5-position of the 4-amino-pyrrolopyrimidine core and the ketophenyl series synthesized by incorporating a ketophenyl group to the C5-position. Medicinal chemistry efforts led to potent Tie-2 inhibitors. Compound 15, a ketophenyl pyrrolopyrimidine urea analog with improved physicochemical properties, demonstrated favorable in vitro attributes as well as dose responsive and robust oral tumor growth inhibition in animal models.

Inhibitors of the Hedgehog signaling pathway have generated a great deal of interest in the oncology area due to the mounting evidence of their potential to provide promising therapeutic options for patients. Herein, we describe the discovery strategy to overcome the issues inherent in lead structure 1 that resulted in the identification of Smoothened inhibitor 1-((2R,4R)-2-(1H-benzo[d]imidazol-2-yl)-1-methylpiperidin-4-yl)-3-(4-cyanophenyl)urea (PF-04449913, 26), which has been advanced to human clinical studies.

Abstract Focal adhesion kinase (FAK) is a non-tyrosine kinase that localizes to focal adhesion plaques. It is activated in response to intergin binding to cellular ligands and when phosphorylated inhibits anoikis allowing for anchorage independent cell growth. Recent studies have shown increased FAK expression and phosphorylation status in many types of invasive and aggressive human tumors strongly suggesting FAK is a possible target for anticancer chemotherapy. Literature, in house HTS and de novo studies identified 2, 4-diaminopyrimidines as potent FAK inhibitors. Early SAR efforts quickly determined that smaller substituents, particularly CF3, were optimal in the C5 position. Parallel medicinal chemistry strategies were executed for the C2 and C4 positions. These studies suggested that substituted aryl and fused heteroaryl groups at the C2 position in conjunction with substituted phenyl and heterocycles at the C4 position imparted optimum activity and metabolic stability. Inhibitor-FAK co-crystal structures were utilized to guide in the SAR strategy around the 2, 4-diaminopyrimidine template which afforded several lead compounds. The team's effort culminated in the advancement of PF-562,271 as a potent and reversible inhibitor of FAK (kinase IC50 of 2 nM and cell IC50 of 5 nM) that is > 100x selective against a long list of non-target kinases. In summary, detailed SAR studies were executed on the 2, 4-diaminopyrimidine templates that produced potent inhibitors of FAK with improved ADME properties, and identified a novel and potent series of FAK inhibitors that are selective against most other kinases and have shown activity in clinical trials. This poster will present design, synthesis, challenging chemistry, optimization, and complete inhibitor chemical structures of lead analogs. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A86.

AbstractCancer cells are characterized by the ability to grow in an anchorage-independent manner. The activity of the nonreceptor tyrosine kinase, focal adhesion kinase (FAK), is thought to contribute to this phenotype. FAK localizes in focal adhesion plaques and has a role as a scaffolding and signaling protein for other adhesion molecules. Recent studies show a strong correlation between increased FAK expression and phosphorylation status and the invasive phenotype of aggressive human tumors. PF-562,271 is a potent, ATP-competitive, reversible inhibitor of FAK and Pyk2 catalytic activity with a IC50 of 1.5 and 14 nmol/L, respectively. Additionally, PF-562,271 displayed robust inhibition in an inducible cell-based assay measuring phospho-FAK with an IC50 of 5 nmol/L. PF-562,271 was evaluated against multiple kinases and displays >100× selectivity against a long list of nontarget kinases. PF-562,271 inhibits FAK phosphorylation in vivo in a dose-dependent fashion (calculated EC50 of 93 ng/mL, total) after p.o. administration to tumor-bearing mice. In vivo inhibition of FAK phosphorylation (>50%) was sustained for >4 hours with a single p.o. dose of 33 mg/kg. Antitumor efficacy and regressions were observed in multiple human s.c. xenograft models. No weight loss, morbidity, or mortality were observed in any in vivo experiment. Tumor growth inhibition was dose and drug exposure dependent. Taken together, these data show that kinase inhibition with an ATP-competitive small molecule inhibitor of FAK decreases the phospho-status in vivo, resulting in robust antitumor activity. [Cancer Res 2008;68(6):1935–44]

ND-4 Cancer cells are characterized by the ability to grow in an anchorage-independent manner. This characteristic phenotype is due, in large part, to the activity of the non-receptor tyrosine kinase, focal adhesion kinase. FAK localizes in focal adhesion plaques and has a role as a scaffolding and signaling protein for other adhesion molecules. As such, FAK acts as a signaling molecule for many integrins which do not have intrinsic kinase activity, as well as, complimenting signal transduction through other RTKs (e.g. EGFR, VEGFR).Recent studies demonstrate a strong correlation between increased FAK expression and the invasive phenotype of aggressive human tumors. There has been some controversy regarding the importance of the kinase activity relative to the scaffolding functions of FAK.Potent inhibitors of FAK have been identified based upon a discovery approach that combined compound screening, structure-based drug design and traditional medicinal chemistry. Two different series of compounds, 2,4-di-anilino pyrimidines and 3,5-di-substituted indoles, were found to be modest inhibitors of focal adhesion kinase. Modeling and co-crystal structures of these inhibitors with FAK led to specifically substituted 2,4-diamino pyrimidines, which were found to inhibit FAK in both kinase and cell assays (1 nM-900 nM). Within this particular class of molecules, the anchoring hydrogen bond donor-acceptor motif for kinase activity was identified. Small, seemingly minor changes to inhibitor structure caused major conformational changes in the way the inhibitor bound and co-crystallized with FAK. Structure-based drug design (SBDD) paved the way for design of novel inhibitors with optimal ADME, selectivity, and potency properties by specific substitution at the C2, C4, and C5 positions of the 2,4-diamino pyrimidine core. Replacement of the 3,5-di-substituted indole moiety with a 5-amino oxindole at the pyrimidine C2 position allowed us to retain a key hydrogen bond interaction to Arg 426, while reducing MW and removing a potential metabolic liability in the original dehydropiperidine. Co-crystal structures of C4 derivatives demonstrated the plasticity of the kinase active site in this region, and furthermore suggested a possible route to achieve selectivity over other kinases by regioselective substitution off of the amino-methyl aryl ring with a RSO2R substituent. Finally, SBDD led to the replacement of the original (pyrimidine C5) Br atom with a CF3 group. PF-562,271 is a potent ATP competitive, reversible inhibitor of FAK and Pyk2 kinase with IC50 of 1.5 and 14 nM, respectively. PF-562,271 is potent in an inducible cell based assay* measuring phospho-FAK with an IC50 of 5 nM. PF-562,271 was evaluated in a number of kinase screens and panels and displays [[Unsupported Character - Codename s]]>100x selectivity against a long list of non-target kinases. PF-562,271 inhibits FAK phosphorylation in vivo in a dose dependent fashion (calculated EC50 35 ng/mL, free, 0.5-4 hrs post dose) following oral administration to female athymic (nu/nu) mice bearing human glioblastoma, U87MG subcutaneous tumors. In vivo inhibition of FAK phosphorylation was sustained (56%) for over 4 hours with a single oral dose of 33 mg/kg. The antitumor efficacy of PF-562,271 was evaluated in the following human s.c. xenograft models: PC-3M (prostate), BT474 (breast), BxPc3 (pancreatic), LoVo (colon), U87MG (glioblastoma), and H460 (lung). Regressions were observed in PC-3M, BT474, BxPc3, and LoVo models at doses of 25-50 mg/kg, BID corresponding to Cmax (free) ranges of 77-885 ng/ml, Cave (free) of 14-40 ng/ml, and inhibition of phospho-FAK of 31-76% for >4 hours. Maximum tumor growth inhibition in the U87MG tumor was 51% using an osmotic mini-pump corresponding to a Css of 0.7 ng/mL (free) with a concomitant decrease in phospho-FAK of 45%. No weight loss, morbidity, or mortality were observed in any TGI experiment (up to 50 mg/kg BID x 28 days or 100 mg/kg QD x 25 days). Tumor growth inhibition was dose and drug exposure dependent. BID dosing and mini-pump experiments (Css) resulted in greater tumor growth inhibition in multiple models compared to QD dosing (equivalent total daily dose) suggesting Cave and the time above the Cave are more relevant to efficacy than Cmax. Taken together, these data demonstrate that kinase inhibition with an ATP competitive small molecule inhibitor of FAK results in robust anti-tumor activity. This compound is presently in Phase I clinical trials (clinical data to be presented at 2007 ASCO).

3259 The Tie family of receptor tyrosine kinases plays a crucial role in the development and function of endothelial tissues. Genetic studies have revealed specific roles for the Tie receptors (Tie1 & Tie2) and their ligands (angiopoietins) in promoting the survival, maturation and functional integrity of the vasculature. Inhibition of Tie2 is expected to disrupt angiogenesis and tumor growth via inhibition of vascular modeling. In an effort to develop inhibitors of Tie2, we designed the (4-amino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)(phenyl)methanone template as a new scaffold for Tie2 inhibition. Our initial efforts focused on the elucidation of the structure-activity relationships of the pyrrolopyrimidine series with the goal of enhancing potency and selectivity. This led us to the 5-keto-pyrrolopyrimidine series with either a sulfonamide or urea linker. Sulfonamides are less potent, but selective Tie2 inhibitors, whereas ureas are very potent but non-selective versus Trk. Both series show moderate to high microsomal clearance, possibly due to the metabolically labile N7 tail (e.g. cyclopentyl) and/or the lipophilic nature of these series. Other key issues included low VDss, low apparent microsomal Km (i.e. saturation of metabolism), and/or HERG channel activity. To resolve these PK issues, our chemistry efforts concentrated on extensive analoging at the N7 position and substitution of the phenyl core. We demonstrated that replacement of the N7 tail with a small, less lipophilic group (e.g. iPr) results in significant improvement in the metabolic stability of these series. Furthermore, substitution on the phenyl core with small substituents (e.g. alkyl, halo, ether) or replacement with a heterocycle (e.g. pyridine) was found to enhance Tie2 potency and improve in vitro and in vivo pharmacokinetic properties. These efforts led to the discovery of CE-355774, CE-245,677 and PF-371,989, each dual Tie2/Trk inhibitors. These compounds demonstrate excellent in vitro activity, and CE-245,677 & PF-371,989 were selected for further preclinical evaluation due to their superior ADME properties. In particular, CE-245,677 is a potent reversible inhibitor of Tie2 and TrkA/B kinases with a cellular IC50 of 4.7 and 1 nM, resp., displays >100x selectivity against a number of other angiogenic receptor tyrosine kinases (e.g. KDR, PDGFR, FGFR) and gene family panels, and demonstrates good oral absorption in in vivo rat PK studies (F=80%). In summary, detailed SAR studies were executed on the (4-amino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)(phenyl)methanone template, producing potent inhibitors of Tie2 with IC50s ranging from 5-100 nM, identifying CE-245,677 and PF-371,989 for further preclinical study. Design, synthesis, inhibitor activity, selectivity profile, ADME properties as well as complete inhibitor chemical structures of analogs leading to the identification to CE-245,677 will be presented.