Synthesis and biological evaluation of 4-phenoxy-phenyl isoxazoles as novel acetyl-CoA carboxylase inhibitors

Abstract

Acetyl-CoA carboxylase (ACC) is a crucial enzyme in fatty acid metabolism, which plays a major role in the occurrence and development of certain tumours. Herein, one potential ACC inhibitor (6a) was identified through high-throughput virtual screening (HTVS), and a series of 4-phenoxy-phenyl isoxazoles were synthesised for structure-activity relationship (SAR) studies. Among these compounds, 6g exhibited the most potent ACC inhibitory activity (IC₅₀=99.8 nM), which was comparable to that of CP-640186. Moreover, the antiproliferation assay revealed that compound 6l exhibited the strongest cytotoxicity, with IC₅₀ values of 0.22 µM (A549), 0.26 µM (HepG2), and 0.21 µM (MDA-MB-231), respectively. The preliminary mechanistic studies on 6g and 6l suggested that the compounds decreased the malonyl-CoA levels, arrested the cell cycle at the G0/G1 phase, and induced apoptosis in MDA-MB-231 cells. Overall, these results indicated that the 4-phenoxy-phenyl isoxazoles are potential for further study in cancer therapeutics as ACC inhibitors.

Keywords: Acetyl-CoA carboxylase; antitumor; apoptosis; cell cycle; docking.

Figure 1.

CP-640186 and representative ACC inhibitors with anticancer activity.

Figure 2.

The design strategy of 4-phenoxy-phenyl isoxazoles as novel ACC inhibitors.

Scheme 1.

Synthetic route of key intermediate 5a and 5b. Reagents and conditions: (a) 4-fluorobenzaldehyde, K₂CO₃, DMF, 120 °C, 12 h; (b) hydroxylamine hydrochloride, NaOH, H₂O/EtOH (3:1), 0 °C to r.t., 2 h; (c) NCS, DMF, r.t., 2 h; (d) 2-(but-3-yn-2-yl)isoindoline-1,3-dione, K₂CO₃, toluene, reflux, 12 h.

Scheme 2.

Synthetic route of target compounds 6a–6q. Reagents and conditions: (a) hydrazine hydrate, CH₂Cl₂/EtOH (5:1), reflux, 2 h; (b) appropriate acyl chloride or isocyanate, TEA, CH₂Cl₂, 0 °C to r.t., 6 h; (c) H₂, Pd-C, MeOH, r.t., 3 h; (d) appropriate iodoalkane, Cs₂CO₃, DMF, reflux, 12 h.

Figure 3.

The dose–response curves of selected compounds on hACC1 enzyme.

Figure 4.

(A) Concentration- and time-dependent cytotoxicity of compounds 6g and 6l. The MDA-MB-231 cells were treated with drugs at increasing concentrations for 12, 24, 36, 48, 60, and 72 h, respectively. (B) Effects of 6g and 6l on the levels of malonyl-CoA in MDA-MB-231 cells. MDA-MB-231 cells were treated with diverse concentrations of drugs for 48 h, with the intracellular malonyl-CoA quantified. Data were expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01 versus control group.

Figure 5.

The effect of compounds 6g (A) and 6l (B) on cell-cycle distribution. MDA-MB-231 cells were treated with 6g and 6l at the indicated concentrations for 48 h, and stained with propidium iodide for flow cytometry. Data are expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 versus control group.

Figure 6.

The effect of compounds 6g (A) and 6l (B) on cell apoptosis. MDA-MB-231 cells were incubated with 6g and 6l at the indicated concentrations for 48 h, and stained with Annexin V-FITC/PI for flow cytometry. Data are expressed as mean ± SD (n = 3). **p < 0.01 and ***p < 0.001 versus control group.

Figure 7.

Molecular docking studies for compounds 6a and 6g (PDB ID: 3FF6). (A) Docking model of compound 6a (green) with the active site of ACC. (B) Overlapping conformation of 6a (green) with CP-640186 (cyan). (C) Docking model of compound 6g (yellow) with the active site of ACC. (D) Overlapping conformation of 6g (yellow) with CP-640186 (cyan). All hydrogen bonds are shown with black dashed lines, and related amino acids are highlighted with magenta sticks.