Supplementary Figures 1-9 and Tables 1-3 from Preclinical Characterization of OSI-027, a Potent and Selective Inhibitor of mTORC1 and mTORC2: Distinct from Rapamycin

Supplementary Tables 1-2 from OSI-930: A Novel Selective Inhibitor of Kit and Kinase Insert Domain Receptor Tyrosine Kinases with Antitumor Activity in Mouse Xenograft Models

Supplementary Figures 1-3 from OSI-930: A Novel Selective Inhibitor of Kit and Kinase Insert Domain Receptor Tyrosine Kinases with Antitumor Activity in Mouse Xenograft Models

Supplementary Methods and Figure Legends 1-9 from Preclinical Characterization of OSI-027, a Potent and Selective Inhibitor of mTORC1 and mTORC2: Distinct from Rapamycin

A series of novel 6-aminofuro[3,2-c]pyridines as kinase inhibitors is described, most notably, OSI-296 (6). We discuss our exploration of structure–activity relationships and optimization leading to OSI-296 and disclose its pharmacological activity against cMET and RON in cellular assays. OSI-296 is a potent and selective inhibitor of cMET and RON kinases that shows in vivo efficacy in tumor xenografts models upon oral dosing and is well tolerated.

This letter describes a series of small molecule inhibitors of IGF-1R with unique time-dependent binding kinetics and slow off-rates. Structure-activity and structure-kinetic relationships were elucidated and guided further optimizations within the series, culminating in compound 2. With an IGF-1R dissociative half-life (t 1/2) of >100 h, compound 2 demonstrated significant and extended PD effects in conjunction with tumor growth inhibition in xenograft models at a remarkably low and intermittent dose, which correlated with the observed in vitro slow off-rate properties.

The kinase selectivity and pharmacokinetic optimization of a series of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1 is described. The intersection of insights from molecular modeling, computational prediction of metabolic sites, and in vitro metabolite identification studies resulted in a simple and unique solution to both of these problems. These efforts culminated in the discovery of compound 13a, a potent, relatively selective inhibitor of TAK1 with good pharmacokinetic properties in mice, which was active in an in vivo model of ovarian cancer.

This Letter describes the medicinal chemistry effort towards a series of novel imidazo[1,5-a]pyrazine derived inhibitors of ACK1. Virtual screening led to the discovery of the initial hit, and subsequent exploration of structure-activity relationships and optimization of drug metabolism and pharmacokinetic properties led to the identification of potent, selective and orally bioavailable ACK1 inhibitors.

Abstract Insulin-like growth factor-1 receptor (IGF-1R) has been recognized as a major target in cancer drug discovery due to its strong implications in various stages of tumorigenesis based on accumulated preclinical data over the years. Recent research on compensatory crosstalk between IGF-1R and insulin receptor (IR) signaling pathways suggests that targeting both receptors is critical to fully blocking the IGF signaling axis. Therefore, inhibition of both receptors is anticipated to result in a more therapeutically beneficial response than targeting IGF-1R alone (e.g. IGF-1R specific antibodies). These findings provided the biological rationale as well as set the foundation for the pursuit and ultimate discovery of OSI-906 (linsitinib), a small molecule dual IGF-1R/IR inhibitor currently in clinical development. As part of OSI's ongoing investment in a small molecule drug discovery platform targeting IGF-1R and IR, a new series of potent and selective imidazo[5,1-f][1,2,4]triazine derived inhibitors of IGF-1R and IR have been identified. Structure-activity relationships and optimization driven by structure-based drug design (SBDD) leading to the discovery of FQIT, a potent, highly selective, well-tolerated and orally bioavailable dual inhibitor of IGF-1R and IR with in vivo efficacy in multiple tumor xenograft models will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3900. doi:1538-7445.AM2012-3900

Abstract cMET and RON are receptor tyrosine kinases of the MET proto-oncogene family that are activated by their respective ligands HGF and MSP. Signaling through the cMET/HGF system can be deregulated in cancer by HGF-dependent autocrine activation, gene amplification, and/or the presence of activating mutations, among others, while for RON, constitutively active variants generated by alternative splicing or methylation-dependent promoter usage [short-form RON (sfRON)] have been identified. Approaches to abrogate aberrant cMET and RON signaling that have led to agents in clinical trials include inhibiting their kinase function with small molecules. We report here the discovery and characterization of OSI-296, a dual inhibitor of cMET and RON. The compound exhibited selectivity in a panel of 96 kinases with potent activity against cMET, including common Y1230 mutants, and RON. OSI-296 blocked cMET autophosphorylation in MKN45 cells, resulting in dose-dependent inhibition of downstream ERK, AKT, and STAT3 phosphorylation. It also showed potent cellular activity in ELISA-format sfRON and caRON cell mechanistic assays that we developed, resulting in dose-dependent inhibition of downstream ERK and AKT phosphorylation. OSI-296 showed a PK profile in rodents suitable for oral dosing with >70% bioavailability. In multiple xenografts models (cMET: MKN45, SNU-5, U87MG; RON: caRON), significant tumor growth inhibition was observed upon oral dosing with regression at higher doses. OSI-296 was very well tolerated with little body weight loss and no adverse effects even at the highest tested dose of 300 mg/kg p.o. qdx14. Solid PK/PD/TGI correlations have been established wherein >90% inhibition of cMET or RON phosphorylation sustained over 24 h by OSI-296 translated to 100% TGI. In summary, OSI-296 was shown to be a well tolerated, dual inhibitor of cMET and RON with in vivo activity in mouse xenografts models for both targets upon oral dosing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2915. doi:1538-7445.AM2012-2915

Abstract The phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway is frequently activated in human cancers, and mTOR is a clinically validated target. mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, metabolism, proliferation, and survival. Rapamycin and its analogues partially inhibit mTOR through allosteric binding to mTORC1, but not mTORC2, and have shown clinical utility in certain cancers. Here, we report the preclinical characterization of OSI-027, a selective and potent dual inhibitor of mTORC1 and mTORC2 with biochemical IC50 values of 22 nmol/L and 65 nmol/L, respectively. OSI-027 shows more than 100-fold selectivity for mTOR relative to PI3Kα, PI3Kβ, PI3Kγ, and DNA-PK. OSI-027 inhibits phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1 as well as the mTORC2 substrate AKT in diverse cancer models in vitro and in vivo. OSI-027 and OXA-01 (close analogue of OSI-027) potently inhibit proliferation of several rapamycin-sensitive and -insensitive nonengineered and engineered cancer cell lines and also, induce cell death in tumor cell lines with activated PI3K–AKT signaling. OSI-027 shows concentration-dependent pharmacodynamic effects on phosphorylation of 4E-BP1 and AKT in tumor tissue with resulting tumor growth inhibition. OSI-027 shows robust antitumor activity in several different human xenograft models representing various histologies. Furthermore, in COLO 205 and GEO colon cancer xenograft models, OSI-027 shows superior efficacy compared with rapamycin. Our results further support the important role of mTOR as a driver of tumor growth and establish OSI-027 as a potent anticancer agent. OSI-027 is currently in phase I clinical trials in cancer patients. Mol Cancer Ther; 10(8); 1394–406. ©2011 AACR.

Preclinical and emerging clinical evidence suggests that inhibiting insulin-like growth factor 1 receptor (IGF-1R) signaling may offer a promising therapeutic strategy for the treatment of several types of cancer. This Letter describes the medicinal chemistry effort towards a series of 8-amino-imidazo[1,5-a]pyrazine derived inhibitors of IGF-1R which features a substituted quinoline moiety at the C1 position and a cyclohexyl linking moiety at the C3 position. Lead optimization efforts which included the optimization of structure-activity relationships and drug metabolism and pharmacokinetic properties led to the identification of compound 9m, a potent, selective and orally bioavailable inhibitor of IGF-1R with in vivo efficacy in an IGF-driven mouse xenograft model.

The discovery and optimization of a series of imidazo[1,5-a]pyrazine inhibitors of mTOR is described. HTS hits were optimized for potency, selectivity and metabolic stability to provide the orally bioavailable proof of concept compound 4c that demonstrated target inhibition in vivo and concomitant inhibition of tumor growth in an MDA-MB-231 xenograft model.

This report describes the investigation of a series of 5,7-disubstituted imidazo[5,1-f][1,2,4]triazine inhibitors of insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor (IR). Structure-activity relationship exploration and optimization leading to the identification, characterization, and pharmacological activity of compound 9b, a potent, selective, well-tolerated, and orally bioavailable dual inhibitor of IGF-1R and IR with in vivo efficacy in tumor xenograft models, is discussed.

Abstract The mammalian target of rapamycin (mTOR) protein kinase plays a central role in regulating cell proliferation and cell survival. The PI3K/AKT signaling pathway that activates mTOR is frequently altered in many human cancers. mTOR exists as multi-protein complexes with distinct signaling functions. Complexes containing mTOR and RAPTOR (mTORC1) phosphorylate S6K and 4E-BP1 and are rapamycin sensitive. Whereas, complexes that contain mTOR and RICTOR (mTORC2) phosphorylate AKT and 4E-BP1 and are insensitive to rapamycin treatment. OSI-027 is a selective small molecule dual inhibitor of both mTORC1 and mTORC2 kinase activity with biochemical IC50 values of 22 nM and 65 nM, respectively. In the MDA-MB 231 breast carcinoma xenograft model, a single 65 mg/kg oral dose of OSI-027 resulted in significant inhibition of 4E-BP1 phosphorylation (>70%) up to 16 h in tumor tissue. At 24 h post-dose, 59% inhibition of phospho-4E-BP1 was observed with a corresponding plasma OSI-027 concentration of 2.2 μM. Consistent with its ability to inhibit mTORC2 in vitro, a single dose of 65 mg/kg also resulted in significant inhibition of phospho-AKT (Ser473) for up to 8 hours with slow recovery thereafter. Such extended target suppression at 65 mg/kg was associated with significant efficacy corresponding to 100% tumor growth inhibition (TGI) with 19% regression in the MDA-MB 231 xenograft model when dosed for 14 consecutive days. In comparison, a lower dose of 25 mg/kg resulted in only 39% TGI which corresponded with a shorter duration of pharmacodynamic effects in vivo. In particular, at the 25 mg/kg dose, inhibition of 4E-BP1 phosphorylation was observed only up to 8 h in tumor tissue with return to predose levels by 16 h which corresponded with plasma drug concentrations of <0.1 µM. These data suggest that maximal anti-tumor efficacy may require plasma levels sufficient to chronically suppress phospho-4E-BP1 in tumor tissue. In addition to significant efficacy in MDA-MB 231 breast carcinoma model, OSI-027 demonstrated robust anti-tumor activity in several different human xenograft models representing different histologies, including colon carcinomas, non-small cell lung carcinomas and ovarian carcinomas. These preclinical studies indicate that OSI-027, a dual mTORC1/2 kinase inhibitor, may show broad spectrum anti-tumor activity, including superior efficacy compared to mTORC1-selective rapalogs against some human cancers. OSI-027 is currently being evaluated in Phase I clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4486.

A series of [2-imidazol-1-yl-2-(6-alkoxy-naphthalen-2-yl)-1-methyl-ethyl]-dimethyl-amines were designed and synthesized as CYP26 inhibitors, serving as retinoic acid metabolic blocking agents (RAMBA’s).