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Innate and adaptive resistance to cancer therapies, such as chemotherapies, molecularly targeted therapies, and immune-modulating therapies, is a major issue in clinical practice. Subpopulations of tumor cells expressing the receptor tyrosine kinase AXL become enriched after treatment with antimitotic drugs, causing tumor relapse. Elevated AXL expression is closely associated with drug resistance in clinical samples, suggesting that AXL plays a pivotal role in drug resistance. Although several molecules with AXL inhibitory activity have been developed, none have sufficient activity and selectivity to be clinically effective when administered in combination with a cancer therapy. Here, we report a novel small molecule, ER-851, which is a potent and highly selective AXL inhibitor. To investigate resistance mechanisms and identify driving molecules, we conducted a comprehensive gene expression analysis of chemoresistant tumor cells in mouse xenograft models of genetically engineered human lung cancer and human triple-negative breast cancer. Consistent with the effect of AXL knockdown, cotreatment of ER-851 and antimitotic drugs produced an antitumor effect and prolonged relapse-free survival in the mouse xenograft model of human triple-negative breast cancer. Importantly, when orally administered to BALB/c mice, this compound did not induce retinal toxicity, a known side effect of chronic MER inhibition. Together, these data strongly suggest that AXL is a therapeutic target for overcoming drug resistance and that ER-851 is a promising candidate therapeutic agent for use against AXL-expressing antimitotic-resistant tumors.

Supplementary Data Figure S1-S7

Supplementary Table S1

IC50 values for the inhibition of 93 human kinases by E7090.

Western blot analysis of FGFR1 in human lung cancer cell lines harboring FGFR1 amplifications.

The FGFR signaling pathway has a crucial role in proliferation, survival, and migration of cancer cells, tumor angiogenesis, and drug resistance. FGFR genetic abnormalities, such as gene fusion, mutation, and amplification, have been implicated in several types of cancer. Therefore, FGFRs are considered potential targets for cancer therapy. E7090 is an orally available and selective inhibitor of the tyrosine kinase activities of FGFR1, -2, and -3. In kinetic analyses of the interaction between E7090 and FGFR1 tyrosine kinase, E7090 associated more rapidly with FGFR1 than did the type II FGFR1 inhibitor ponatinib, and E7090 dissociated more slowly from FGFR1, with a relatively longer residence time, than did the type I FGFR1 inhibitor AZD4547, suggesting that its kinetics are more similar to the type V inhibitors, such as lenvatinib. E7090 showed selective antiproliferative activity against cancer cell lines harboring FGFR genetic abnormalities and decreased tumor size in a mouse xenograft model using cell lines with dysregulated FGFR. Furthermore, E7090 administration significantly prolonged the survival of mice with metastasized tumors in the lung. Our results suggest that E7090 is a promising candidate as a therapeutic agent for the treatment of tumors harboring FGFR genetic abnormalities. It is currently being investigated in a phase I clinical trial. Mol Cancer Ther; 15(11); 2630–9. ©2016 AACR.

Supplementary Table legends and Supplementary Figure legends.

Innate and adaptive resistance to cancer therapies, such as chemotherapies, molecularly targeted therapies, and immune-modulating therapies, is a major issue in clinical practice. Subpopulations of tumor cells expressing the receptor tyrosine kinase AXL become enriched after treatment with anti-mitotic drugs, causing tumor relapse. Elevated AXL expression is closely associated with drug resistance in clinical samples, suggesting that AXL plays a pivotal role in drug resistance. Although several molecules with AXL inhibitory activity have been developed, none have sufficient activity and selectivity to be clinically effective when administered in combination with a cancer therapy. Here, we report a novel small molecule, ER-001259851-000, which is a potent and highly selective AXL inhibitor. To investigate resistance mechanisms and identify driving molecules, we conducted a comprehensive gene expression analysis of chemo-resistant tumor cells in mouse xenograft models of genetically engineered human lung cancer and human triple-negative breast cancer. Consistent with the effect of AXL knockdown, co-treatment of ER-001259851-000 and antimitotic drugs produced an anti-tumor effect and prolonged relapse-free survival in the mouse xenograft model of human triple-negative breast cancer. Importantly, when orally administered to BALB/c mice, this compound did not induce retinal toxicity, a known side effect of chronic MER inhibition. Together, these data strongly suggest that AXL is a therapeutic target for overcoming drug resistance and that ER-001259851-000 is a promising candidate therapeutic agent for use against AXL-expressing anti-mitotic-resistant tumors.

Axl and Mer are members of the TAM (Tyro3-Axl-Mer) family of receptor tyrosine kinases. Previously, we reported that enzyme-mediated inhibition of Mer by an Axl/Mer dual inhibitor led to retinal toxicity in mice, whereas selective Axl inhibition by compound 1 did not. On the other hand, compound 1 showed low membrane permeability. Here, we designed and synthesized a novel series of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine derivatives and evaluated their Axl and Mer inhibitory activities, leading to identification of ER-001259851-000 as a potent and selective Axl inhibitor with drug-likeness and a promising pharmacokinetic profile in mice.

Axl and Mer are a members of the TAM (Tyro3-Axl-Mer) family of receptor tyrosine kinases, which, when activated, can promote tumor cell survival, proliferation, migration, invasion, angiogenesis, and tumor-host interactions. Chronic inhibition of Mer leads to retinal toxicity in mice. Therefore, successful development of an Axl targeting agent requires ensuring that it is safe for prolonged treatment. Here, to clarify whether enzyme inhibition of Mer by a small molecule leads to retinal toxicity in mice, we designed and synthesized Axl/Mer inhibitors and Axl-selective inhibitors. We identified an Axl/Mer dual inhibitor 28a, which showed retinal toxicity at a dose of 100 mg/kg in mice. Subsequent derivatization of a pyridine derivative led to the discovery of a pyrimidine derivative, 33g, which selectively inhibited the activity of Axl over Mer without retinal toxicity at a dose of 100 mg/kg in mice. Additionally, the compound displayed in vivo anti-tumor effects without influencing body weight in a Ba/F3-Axl isogenic subcutaneous model.

Hypoxia-inducible factor-1 (HIF-1) is the chief transcription factor regulating hypoxia-driven gene expression. HIF-1 overexpression is associated with poor prognosis in several cancers and therefore represents an attractive target for novel antitumor agents. We explored small molecule inhibitors of the HIF-1 pathway. Using high-throughput-screening, we identified benzanilide compound 1 (IC50=560 nM) as a seed. Subsequent extensive derivatization led to the discovery of compounds 43a and 51d, with anti-HIF-1 activities in vitro (IC50=21 and 0.47 nM, respectively), and in vivo. Additionally, 43a (12.5-100mg/kg) also displayed in vivo anti-tumor efficacy, without influencing body weight.

The FGFR signaling pathway has a crucial role in proliferation, survival, and migration of cancer cells, tumor angiogenesis, and drug resistance. FGFR genetic abnormalities, such as gene fusion, mutation, and amplification, have been implicated in several types of cancer. Therefore, FGFRs are considered potential targets for cancer therapy. E7090 is an orally available and selective inhibitor of the tyrosine kinase activities of FGFR1, -2, and -3. In kinetic analyses of the interaction between E7090 and FGFR1 tyrosine kinase, E7090 associated more rapidly with FGFR1 than did the type II FGFR1 inhibitor ponatinib, and E7090 dissociated more slowly from FGFR1, with a relatively longer residence time, than did the type I FGFR1 inhibitor AZD4547, suggesting that its kinetics are more similar to the type V inhibitors, such as lenvatinib. E7090 showed selective antiproliferative activity against cancer cell lines harboring FGFR genetic abnormalities and decreased tumor size in a mouse xenograft model using cell lines with dysregulated FGFR. Furthermore, E7090 administration significantly prolonged the survival of mice with metastasized tumors in the lung. Our results suggest that E7090 is a promising candidate as a therapeutic agent for the treatment of tumors harboring FGFR genetic abnormalities. It is currently being investigated in a phase I clinical trial. Mol Cancer Ther; 15(11); 2630–9. ©2016 AACR.

Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes that play crucial roles in the adaptation of cancer cells to hypoxia. HIF-1α overexpression has been associated with poor prognosis in patients with various types of cancer. Here, we describe ER-400583-00 as a novel HIF-1 inhibitor. ER-400583-00 suppressed the production of HIF-1α protein in response to hypoxia, with a half-maximal inhibitory concentration value of 3.7 nM in human U251 glioma cells. The oral administration of 100 mg/kg ER-400583-00 to mice bearing U251 tumor xenografts resulted in a rapid suppression of HIF-1α that persisted for 24 h. Immunohistochemical analysis revealed that ER-400583-00 suppressed the proliferation of cancer cells most prominently in areas distal to the region of blood perfusion, where HIF-1α-expressing hypoxic cancer cells were located. These hypoxic cancer cells were resistant to radiation therapy. ER-400583-00 showed a synergistic interaction with radiation therapy in terms of antitumor activity. These data suggest that HIF-1 blockade by small compounds may have therapeutic value in cancer, especially in combination with radiation therapy.

Asymmetric trifluoromethylation of ketones with (trifluoromethyl)trimethylsilane catalyzed by cinchonidine-derived quaternary ammonium phenoxides proceeded smoothly to afford the trifluoromethylate...

Chiral quaternary ammonium phenoxides are readily prepared from commercially available cinchona alkaloids and are employed as useful new asymmetric organocatalysts. The cinchonidine-derived catalyst is highly effective on Michael addition and successive lactonization between glycine-derived silyl enolates and α,β-unsaturated ketones. According to this asymmetric reaction, the corresponding 3-amino-3,4-dihydropyran-2-ones are formed in high yields with complete diastereoselectivities and excellent enantioselectivities.

Chiral quaternary ammonium phenoxides were readily prepared from commercially available cinchona alkaloids and proved to be useful new asymmetric organocatalysts. Among various chiral quaternary ammonium phenoxides, a cinchonidine-derived catalyst that bears both a sterically hindered N1-9-anthracenylmethyl group and a strongly electron withdrawing 9-O-3,5-bis(trifluoromethyl)benzyl group were found to be highly effective for the Michael addition of ketene silyl acetals (derived from phenyl carboxylates) and alpha,beta-unsaturated ketones followed by lactonization. Optically active 3,4-dihydropyran-2-one derivatives were obtained in high yields with excellent control of enantio- and diastereoselectivity. This catalyst can be handled in air and stored at room temperature in a sealed bottle without decomposition for at least one month.

The first efficient synthesis of 3-amino-3,4-dihydropyran-2-ones from glycine-derived silyl enolates and α,β-unsaturated ketones was successfully performed by using quaternary ammonium phenoxide pr...

The fibroblast growth factor (FGF) signaling pathway comprises 18 ligands and 4 FGF receptor subtypes, FGFR1, 2, 3 and 4, which are known as receptor-type tyrosine kinases corresponding to those ligands. Upon ligand binding, FGFRs activate an array of downstream signaling pathways, such as the mitogen activated protein kinase (MAPK) and the phosphoinositide-3-kinase (PI3K)/Akt pathways. Genetic abnormalities (gene fusion, mutation and amplification) of FGFRs are known to cause constitutive activation of their signaling pathways, which play an important role in proliferation, survival and migration of cancer cells, tumor angiogenesis, drug resistance, etc. These abnormalities have also been reported to be involved in different cancer types including lung, breast, endometrial, gastric, and bladder cancer so far. Therefore, FGFRs are considered as one of potential targets for cancer therapy. E7090 is an orally available, selective and potent inhibitor of FGFR1, 2 and 3 tyrosine kinase activities. E7090 displayed inhibition of FGFR1, 2, and 3 kinase activities with the IC50 values of approximately 1 nmol/L. E7090 also inhibited the growth of SNU-16, human gastric cancer cell line harboring FGFR2 amplification with an IC50 value of 3 nmol/L. This activity was about 60-fold stronger than that against VEGF-stimulated HUVEC growth. Kinase profiling assay consisting of 93 kinases including non-receptor and serine/threonine kinases, also demonstrated that E7090 inhibited limited kinases including FGFR-1, -2 and -3. In addition, E7090 inhibited proliferation of human cancer cell lines harboring various types of FGFRs gene abnormalities such as amplification, mutation, or translocation in vitro, which are confirmed by the inhibition of FGFR signaling. E7090 also showed significant antitumor activities on various human xenografts harboring FGFRs gene abnormalities in a dose dependent manner and demonstrated tumor shrinkage at several doses in some models. Furthermore, pharmacodynamics analysis revealed that E7090 inhibited phosphorylation of FGFRs in SNU-16 xenograft tumors in a dose-dependent manner. Overall, the in vitro and in vivo studies confirm that E7090 is a potent and selective FGFRs inhibitor, showing promising antitumor activities with wider therapeutic windows in preclinical cancer models harboring FGFRs gene abnormalities. E7090 has been advanced through preclinical development and we disclose here the first details of its preclinical profile. Citation Format: Saori Watanabe Miyano, Yuji Yamamoto, Kotaro Kodama, Setsuo Funasaka, Satoshi Nagao, Naoko Hata Sugi, Hiroko Kuramochi, Katsuyuki Ishikawa, Kiyoshi Okamoto, Yukinori Minoshima, Takayuki Nakagawa, Yusuke Nakatani, Yuki Karoji, Isao Ohashi, Yoshinobu Yamane, Keigo Tanaka, Toshimi Okada, Tomohiro Matsushima, Junji Matsui, Masao Iwata, Akihiko Tsuruoka, Toshimitsu Uenaka. E7090: A potent and selective FGFR inhibitor with activity in multiple FGFR-driven cancer models with distinct mechanisms of activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 770. doi:10.1158/1538-7445.AM2015-770

Diastereo- and enantioselective tandem Michael addition and lactonization between various silyl enolates derived from phenyl carboxylates and α,β-unsaturated ketones were successfully carried out b...

Chiral quaternary ammonium phenoxides derived from cinchona alkaloids were easily synthesized and were effectively employed in the tandem Michael addition and lactonization between α,β-unsaturated ...

The use of tetraalkoxysilanes, particularly tetrakis(perfluoroalkoxy)silanes, in dehydration condensation of carboxylic acids with amines was investigated. Tetrakis(1,1,1,3,3,3-hexafluoro-2-propoxy...

Phenoxide ion-catalyzed tandem Michael addition and lactonization between α,β-unsaturated ketones and silyl enolates derived from phenyl carboxylates proceeded smoothly to afford the corresponding ...

Various nitrogen heterocycles were produced by the reactions of ω-bromoalkyldiphenylsulfonium or diphenylvinylsulfonium triflates with nitrogen nucleophiles. Further, the reactions of diphenylstyrylsulfonium triflates with N-metalated phthalimide and imidazoles afforded α-phthalimidostyrenes and α-(imidazol-1-yl)styrenes in good yields, respectively.

A vinylogous aldol-type reaction between 4-methyl-2-(trimethylsiloxy)furan and aldehydes catalyzed by cinchonidine-derived quaternary ammonium phenoxide proceeded smoothly to afford the correspondi...

Tetrakis(pyridin-2-yloxy)silane [Si(OPy)4] is easily prepared by trans-silylation between silicon tetrachloride and trimethyl(pyridin-2-yloxy)silane in the molar ratio of 1:4. This novel reagent is...

Various imidazolylsilane derivatives were synthesized by a trans-silylation procedure between 1-(trimethylsilyl)imidazoles and chlorosilanes. Among them, tris- and tetrakis(imidazol-1-yl)silane com...

Phenoxide ion-catalyzed tandem Michael addition and lactonization between α,β-unsaturated ketones and silyl enolates derived from phenyl carboxylates proceeded smoothly to afford the corresponding ...

Tetrakis(2-methylimidazol-l-yl)silane [Si(2-Me-Im) 4 ], a new dehydrating reagent having silicon-imidazole linkage, reacted readily with carboxylic acids at room temperature to form the corresponding 1-acyl-2-methylimidazole intermediates, which smoothly underwent subsequent condensation with nucleophiles such as amines or thiols to afford the corresponding carboxamides or thioesters in good to excellent yields, respectively.