Synthesis of 2-methoxybenzamide derivatives and evaluation of their hedgehog signaling pathway inhibition

Abstract

Aberrant hedgehog (Hh) signaling is implicated in the development of a variety of cancers. Smoothened (Smo) protein is a bottleneck in the Hh signal transduction. The regulation of the Hh signaling pathway to target the Smo receptor is a practical approach for development of anticancer agents. We report herein the design and synthesis of a series of 2-methoxybenzamide derivatives as Hh signaling pathway inhibitors. The pharmacological data demonstrated that compound 21 possessed potent Hh pathway inhibition with a nanomolar IC₅₀ value, and it prevented Shh-induced Smo from entering the primary cilium. Furthermore, mutant Smo was effectively suppressed via compound 21. The in vitro antiproliferative activity of compound 21 against a drug-resistant cell line gave encouraging results.

Fig. 1. Structure of hedgehog pathway inhibitors.

Fig. 2. Design of 2-methoxybenzamide derivatives.

Fig. 3. (A) Overlay of compounds 3 (yellow), 4 (green), 10 (blue) and 17 (violet) in binding pocket of Smo. (B) Docking conformation of compound 3 at the binding site. (C) Docking conformation of compound 17 at the binding site. Hydrogen bonds are represented by the dashed green lines. Surrounding amino acid are shown in grey stick format and labelled.

Scheme 1. Reagents and conditions: (a) (1) SOCl₂, toluene, 80 °C, 3 h; (2) methyl 4-amino-2-methoxybenzoate, TEA, DMF, rt, overnight, yield 85%; (b) NaOH, aqueous alcohol, 40 °C, 5 h, yield 83–92%; (c) 3-(1H-benzo[d]imidazol-2-yl)-4-chloroaniline, HATU, DIPEA, DCM, rt, 24 h, yield 71–86%; the specific position of R group directed at Table 1.

Scheme 2. Reagents and conditions: (a) CH₃ONa, CH₃OH, −20 °C, 2 h; NH₄Cl, 40 °C, 3 h, yield 46%; (b) 2-bromoacetophenone, NaHCO₃, THF, reflux, 40 °C, 24 h, yield 65%; (c) SnCl₂·2H₂O, ethanol, HCl, 80 °C, reflux, 8 h, yield 86%; (d) 5a–g, HATU, DIPEA, DCM, rt, 24 h, yield 76–84%; the specific position of R group directed at Table 1.

Fig. 4. Benzamide analogs inhibit Hh pathway through regulating Smo. Error bars represent standard deviation of three parallel groups.

Fig. 5. The inhibition of drug-resistant Smo mutant for compound 21. The inhibition of Gli-Luc reporter activity by vismodegib (A) and compound 21 (B) in NIH3T3-Gli-Luc cells overexpressing wild-type Smo or Smo D477G. Error data point represents the mean ± SD of three determinations.

Fig. 6. Cell viability of Daoy cells incubated with the indicated doses of vismodegib and compound 21 for 48 h. Data represent mean ± SD of experiments performed in triplicate.

Fig. 7. (A) Docking conformation of compound 1 (orange) at the binding site. (B) Docking conformation of compound 21 (pink) at the binding site. Hydrogen bonds are represented by the dashed green lines. Surrounding amino acid are shown in grey stick format and labelled.