Discovery of Novel, Thienopyridine-Based Tyrosine Kinase Inhibitors Targeting Tumorigenic RON Splice Variants

Abstract

Herein, we report the identification, structural optimization, and biological efficacy of thieno[2,3-b]pyridines as potent inhibitors of splice variants of the tyrosine kinase recepteur d'origine nantais (RON). Among synthesized compounds, compound 15f exhibited excellent in vitro kinase inhibition and antiproliferative activity, as well as in vivo antineoplastic efficacy against RON splice variant-expressing tumors. Moreover, compound 15f with excellent pharmacokinetics demonstrated significant activity with greater tumor growth inhibition (74.9% at 10 mg/kg) than compounds 2 and 4 in a patient-derived xenograft model. Collectively, 15f represents a promising, novel anticancer agent targeting RON splice variants.

Figure 1

Chemical structure of some representative c-Met/RON inhibitors and compound 6.

Figure 2

Oncogenic potential of RON splice variants.

Figure 3

Cell death rates (%, @ 5 μM) of 7-phenoxythieno[3,2-b]pyridine derivatives. KM12C and HT29 (RONΔ160) cells were treated with several compounds, including 15f for 48 h, and dead cells were counted by trypan blue exclusion. Significance (p values) was determined by an unpaired, two-tailed Student’s t test; ***p < 0.001 vs control.

Figure 4

Docked conformations of 15f (green) and BMS-777607 (4, magenta) binding to the binding pocket in RON homology model.

Figure 5

Cell-based RON kinase activity against RON splice variants (RONΔ155, Δ160, and Δ165) of 15f compared with control (DMSO) and known RON/c-Met inhibitors. Data was quantified using ImageJ, for the expression of phosphorylated RON, as determined by Western blot analysis. Significance (p values) was determined by an unpaired, two-tailed Student’s t test; ***p < 0.001, **p < 0.01 vs control.

Figure 6

In vivo antitumor effect of 15f against KM12C, SW620, and T84 xenograft models (BALB/c-nude female mice). (A) Relative tumor growth of colorectal cancer mouse KM12C (RONΔ160) xenografts treated with vehicle (control) and 15f (1, 3, 10, and 30 mg/kg) for 14 days, as indicated (n = 5 per group; data represents means ± SEMs). (B) Relative growth of SW620 (RONΔ155) colorectal cancer xenograft tumors in mice treated with vehicle (control), 15f (30 mg/kg), and 4 (BMS-777607, 30 mg/kg) for 28 days, as indicated (n = 5 per group; data represents means ± SEMs). (C) Relative growth of T84 (RONΔ165) colorectal cancer xenograft tumors in mice treated with vehicle (control), 15f (30 mg/kg), and 4 (BMS-777607, 30 mg/kg), for 34 days, as indicated (n = 11 per group; data represents mean ± SEM). Drugs were given by oral gavage, once daily. Significance (p values) was determined by unpaired, two-tailed Student’s t tests; ***p < 0.001, **p < 0.01, *p < 0.05, vs control.

Figure 7

In vivo efficacy of 15f against a colorectal cancer patient-derived xenograft (PDX) model (KRAS wt, RONΔ160). Colon tumor specimens were obtained from patients at Asan Medical Center with Institutional Review Board approval (IRB: S2018-0033-0009). Graph of treatment with vehicle (control), 15f (10 mg/kg), 2 (crizotinib, 10 mg/kg), and 4 (BMS-777607, 10 mg/kg) for 28 days, as indicated (n = 6 per group; data represents means ± SEMs). Doses were given by oral gavage, once daily. Significance (p values) was determined by unpaired, two-tailed Student’s t tests; ***p < 0.001, **p < 0.01, *p < 0.05, vs control.

Scheme 1. Synthesis of 1,2-Dihydropyridine-3-carboxylic Acid Intermediates 10a–f

Reagents and conditions: (a) CH₃C(OEt)₃, AcOH, 120 °C, 2 h; (b) DMF-DEA, 70 °C, 2 h, 28%; (c) substituted phenyl boronic acid, Cu(OAC)₂, pyridine, DCM, rt, 12 h, 65%; (d) LiOH·H₂O, EtOH, H₂O, 70 °C, 3 h, 41–84%.

Scheme 2. Synthesis of 7-Phenoxythieno[3,2-b]pyridine Derivatives 15a–p

Reagents and conditions: (a) R₃-Br, n-BuLi, THF, −78 °C, 0.5 h; (b) ZnCl₂, −78 °C to rt, 1 h; (c) Pd(PPh₃)₄, 70 °C, 2 h, 65–75%; (d) 2-fluoro-4-nitrophenol, K₂CO₃, Ph₂O, 160–200 °C, 5–13 h, 40–70%; (e) Fe, EtOH, H₂O, 100 °C, 3 h, 70–85%; (f) 10a, EDC·HCl, HOBt, Et₃N, DCM, rt, 24 h, 75–85%; (g) 4N HCl in dioxane, rt, 1 h, 96%; (h) TFA, acetone, H₂O, 60 °C, 12 h, 80%; (i) NaBH(OAc)₃, 1,2-DCE, rt, 4 h, 73%; (j) SOCl₂, DCM, rt, 3 h, 95%; (k) various substituted cycloalkylamines, K₂CO₃, MeCN, 80 °C, 5 h, 30–50%.

Scheme 3. Synthesis of 7-Phenoxythieno[3,2-b]pyridine Derivatives 23a–n

Reagents and conditions: (a) n-BuLi, THF, −78 °C, 0.5 h; (b) ZnCl₂, −78 °C to rt, 1 h; (c) 4-[(6-bromopyridin-3-yl)methyl]morpholine, Pd(PPh₃)₄, 70 °C, 2 h, 65%; (d) substituted 4-nitrophenol, K₂CO₃, Ph₂O, 200 °C, 12 h, 50–70%; (e) Fe, EtOH, H₂O, 100 °C, 3 h, 70–85%; (f) 10a–e, hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU), i-Pr₂NEt, DCM, rt, 24 h, 43–82%; (g) NaCIO₂, KH₂PO₄, 2-methyl-2-butene, t-BuOH, H₂O, rt, 1 h, 53%; (h) H₂SO₄, MeOH, 75 °C, 3 h, 99%; (i) 4-fluorophenyl boronic acid, Cu(OAC)₂, pyridine, DCM, rt, 3 h, 69%; (j) 6 M NaOH, EtOH, H₂O, 50 °C, overnight, 74%; (k) 22d, HATU, i-Pr₂NEt, DCM, rt, 1 h, 82%; (l) R₆–OH, NaH, THF, rt, 1 h, 65–99%.