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Supplementary Figures S1-S3, Tables S1-S7. Suppl. Figure S1: Prolif. Curves, PC3 Cell Cycle & solid tumor apoptosis Suppl. Figure S2: PK/PD Model Performance and eIF4E In Vivo Biomarker Suppl. Figure S3: HCT-116, MBA MD231 & A549 Efficacy Studies Suppl. Table S1: Kinase Selectivity Suppl. Table S2: Cellular Biomarker with SEM Suppl. Table S3: Cellular Proliferation with SEM Suppl. Table S4: Hematological Panel Suppl. Table S5: IRF4 Gene and Protein Data Suppl. Table S6: PK/PD Data PK Data Suppl. Table S7: In Vivo Efficacy Across Tumor Types

mTOR is a serine/threonine kinase that regulates cell growth, metabolism, proliferation, and survival. mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) are critical mediators of the PI3K–AKT pathway, which is frequently mutated in many cancers, leading to hyperactivation of mTOR signaling. Although rapamycin analogues, allosteric inhibitors that target only the mTORC1 complex, have shown some clinical activity, it is hypothesized that mTOR kinase inhibitors, blocking both mTORC1 and mTORC2 signaling, will have expanded therapeutic potential. Here, we describe the preclinical characterization of CC-223. CC-223 is a potent, selective, and orally bioavailable inhibitor of mTOR kinase, demonstrating inhibition of mTORC1 (pS6RP and p4EBP1) and mTORC2 [pAKT(S473)] in cellular systems. Growth inhibitory activity was demonstrated in hematologic and solid tumor cell lines. mTOR kinase inhibition in cells, by CC-223, resulted in more complete inhibition of the mTOR pathway biomarkers and improved antiproliferative activity as compared with rapamycin. Growth inhibitory activity and apoptosis was demonstrated in a panel of hematologic cancer cell lines. Correlative analysis revealed that IRF4 expression level associates with resistance, whereas mTOR pathway activation seems to associate with sensitivity. Treatment with CC-223 afforded in vivo tumor biomarker inhibition in tumor-bearing mice, after a single oral dose. CC-223 exhibited dose-dependent tumor growth inhibition in multiple solid tumor xenografts. Significant inhibition of mTOR pathway markers pS6RP and pAKT in CC-223–treated tumors suggests that the observed antitumor activity of CC-223 was mediated through inhibition of both mTORC1 and mTORC2. CC-223 is currently in phase I clinical trials. Mol Cancer Ther; 14(6); 1295–305. ©2015 AACR.

We report here the synthesis and structure-activity relationship (SAR) of a novel series of triazole containing mammalian target of rapamycin (mTOR) kinase inhibitors. SAR studies examining the potency, selectivity, and PK parameters for a series of triazole containing 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones resulted in the identification of triazole containing mTOR kinase inhibitors with improved PK properties. Potent compounds from this series were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC-3 cancer cells, in vitro and in vivo. When assessed in efficacy models, analogs exhibited dose-dependent efficacy in tumor xenograft models. This work resulted in the selection of CC-115 for clinical development.

We report here the synthesis and structure-activity relationship (SAR) of a novel series of mammalian target of rapamycin (mTOR) kinase inhibitors. A series of 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were optimized for in vivo efficacy. These efforts resulted in the identification of compounds with excellent mTOR kinase inhibitory potency, with exquisite kinase selectivity over the related lipid kinase PI3K. The improved PK properties of this series allowed for exploration of in vivo efficacy and ultimately the selection of CC-223 for clinical development.

mTOR is a serine/threonine kinase that regulates cell growth, metabolism, proliferation, and survival. mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) are critical mediators of the PI3K–AKT pathway, which is frequently mutated in many cancers, leading to hyperactivation of mTOR signaling. Although rapamycin analogues, allosteric inhibitors that target only the mTORC1 complex, have shown some clinical activity, it is hypothesized that mTOR kinase inhibitors, blocking both mTORC1 and mTORC2 signaling, will have expanded therapeutic potential. Here, we describe the preclinical characterization of CC-223. CC-223 is a potent, selective, and orally bioavailable inhibitor of mTOR kinase, demonstrating inhibition of mTORC1 (pS6RP and p4EBP1) and mTORC2 [pAKT(S473)] in cellular systems. Growth inhibitory activity was demonstrated in hematologic and solid tumor cell lines. mTOR kinase inhibition in cells, by CC-223, resulted in more complete inhibition of the mTOR pathway biomarkers and improved antiproliferative activity as compared with rapamycin. Growth inhibitory activity and apoptosis was demonstrated in a panel of hematologic cancer cell lines. Correlative analysis revealed that IRF4 expression level associates with resistance, whereas mTOR pathway activation seems to associate with sensitivity. Treatment with CC-223 afforded in vivo tumor biomarker inhibition in tumor-bearing mice, after a single oral dose. CC-223 exhibited dose-dependent tumor growth inhibition in multiple solid tumor xenografts. Significant inhibition of mTOR pathway markers pS6RP and pAKT in CC-223–treated tumors suggests that the observed antitumor activity of CC-223 was mediated through inhibition of both mTORC1 and mTORC2. CC-223 is currently in phase I clinical trials. Mol Cancer Ther; 14(6); 1295–305. ©2015 AACR.

We report here the discovery of a novel series of selective mTOR kinase inhibitors. A series of imidazo[4,5-b]pyrazin-2-ones, represented by screening hit 1, was developed into lead compounds with excellent mTOR potency and exquisite kinase selectivity. Potent compounds from this series show >1000-fold selectivity over the related PI3Kα lipid kinase. Further, compounds such as 2 achieve mTOR pathway inhibition, blocking both mTORC1 and mTORC2 signaling, in PC3 cancer cells as measured by inhibition of pS6 and pAkt (S473).

We report here the discovery of a novel series of selective mTOR kinase inhibitors and the identification of CC214-2, a compound with demonstrated anti-tumor activity upon oral dosing in a PC3 prostate cancer xenograft model. A series of 4,6-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were discovered through a core modification of our original compound series. Analogs from this series have excellent mTOR potency and maintain selectivity over the related PI3Kα lipid kinase. Compounds such as CC214-2 were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC3 cancer cells, in vitro and in vivo.

The serine/threonine specific protein kinase B-Raf is part of the MAPK pathway and is an interesting oncology target. We have identified thieno[2,3-d]pyrimidines as a core scaffold of small molecule B-Raf inhibitors. The SAR of analogs in this series will be described.

Mantle cell lymphoma (MCL) is a distinct sub-type non-Hodgkin lymphoma characterized by overexpression of cyclin D1 (CCND1) in 95% of patients due to the cytogenetic change of chromosome translocation t(11;14) (q13;q32). It remains one of the most challenging lymphomas associated with shorter response duration to conventional chemotherapy as well as continuous relapses and refractory to current drugs. However, dysregulation of cyclin D1 biology alone is insufficient to develop MCL. The emerging data suggest that the mammalian target of rapamycin (mTOR) plays a crucial role in the proper transmission of proliferative and anti-apoptotic signals through the PI3K/AKT pathway that makes it an attractive therapeutic target for hematological malignances including mantle cell lymphoma. As a single agent, rapamycin analogs such as temsirolimus (CCI-779) achieved 38% overall response rate in heavily pretreated MCL and prolonged progression free survival (PFS) in relapsed and refractory mantle cell lymphoma (4.8 months in temsirolimus vs. 1.9 months in investigator’s choice, ASCO 2008). mTOR regulates two distinct complexes TORC1 and TORC2. TORC1 complex is involved in cell cycle regulation by phosphorylating p70S6K and 4E-BP1, two molecules that are important for translational control of cyclin D1 and c-myc as well as ribosomal biogenesis whereas TORC2 complex mainly regulates phospho- AKT serine 473 leading to cell survival and proliferation. mTOR kinase also negatively regulates autophagy, a process of cellular bulk protein degradation by fusion to lysosomes upon the nutrient deprivation. We have developed mTOR kinase selective inhibitors which exhibit distinct biological profile from rapamycin in many cancer cell lines. Here we demonstrate that a selective mTOR kinase inhibitor displays potent anti-proliferative activity in JeKo-1 and Mino cells associated with decreased phosphorylation of S6, p70S6K, AKT S473, 4E-BP1 as well as decreased cyclin D1 levels leading to G1 arrest. The inhibitor also promotes autophagic cell death at 72h and 96h post-treatment. Furthermore a selective mTOR kinase inhibitor but not rapamycin induces a significant apoptosis in JeKo-1 and Mino cells. The observed apoptosis is correlated with caspases mediated PARP cleavage as well as inhibition of anti-apoptotic protein Mcl-1, suggesting TORC2/AKT S473 complex may provide survival signaling for mantle cell lymphoma. A timecourse study demonstrated that JeKo-1 and Mino cells undergo apoptosis at 24h and 48h followed by significant autophagic cell death at 72h and 96h in a dose dependent manner when exposed to our mTOR kinase inhibitor. In conclusion, mTOR kinase inhibitors are able to induce G1 cell cycle arrest, caspase-dependent apoptosis and autophagic cell death that contribute to the anti-tumor activity. Therefore it may provide a powerful alternative targeted therapy for mantle cell lymphoma.