Metabolism-guided development of Ko143 analogs as ABCG2 inhibitors

Abstract

ATP-binding cassette subfamily G member 2 (ABCG2), an efflux transporter, is involved in multiple pathological processes. Ko143 is a potent ABCG2 inhibitor; however, it is quickly metabolized through carboxylesterase 1-mediated hydrolysis of its t-butyl ester moiety. The current work aimed to develop more metabolically stable ABCG2 inhibitors. Novel Ko143 analogs were designed and synthesized by replacing the unstable t-butyl ester moiety in Ko143 with an amide group. The synthesized Ko143 analogs were evaluated for their ABCG2 inhibitory activity, binding mode with ABCG2, cytotoxicity, and metabolic stability. We found that the amide modification of Ko143 led to metabolically stable ABCG2 inhibitors. Among these Ko143 analogs, K2 and K34 are promising candidates with favorable oral pharmacokinetic profiles in mice. In summary, we synthesized novel Ko143 analogs with improved metabolic stability, which can potentially be used as lead compounds for the future development of ABCG2 inhibitors.

Keywords: ABCG2 inhibitor; Carboxylesterase; Ko143 analogs; Metabolic stability; Pharmacokinetics.

Fig. 1.

Design of novel ABCG2 inhibitors based on the scaffold of Ko143. Ko143 has poor metabolic stability because of CES1-mediated hydrolysis [16] .The metabolite Ko143 acid has no inhibitory activity toward ABCG2. To bypass the hydrolysis by CES1, the t-butyl ester group in Ko143 was replaced by an amide group to give amide I and II Ko143 analogs.

Fig. 2.

ABCG2 inhibitory activity of Ko143 analogs. (A) Protoporphyrin IX (PPIX)-based assay for the screening of ABCG2 inhibitors. The A549 cells were pretreated with 5-aminolevulinic acid (ALA), a precursor of PPIX, for 4 h. Afterward, an ABCG2 inhibitor was added to the A549 cells and incubated for another 2 h. PPIX was extracted from cell lysates and analyzed by UPLC-QTOFMS. (B) The ABCG2 inhibitory activity of Ko143 and newly synthesized Ko143 analogs (1 μM). A549 cells were treated with PBS control, ALA, or ALA plus aKo143 analog. ****P < 0.0001 vs ALA, one-way ANOVA.

Fig. 3.

Predicted binding modes of Ko143 analogs with ABCG2. (A-B) Chemical structures of K34 and K32. (C-D) The predicted binding poses of K34 and K32 with ABCG2 by molecular docking. The inhibitors are shown in blue. T435 and N436 formed strong hydrogen bonds with the oxygen and nitrogen atom at the indole moiety of K34 and K32, respectively.

Fig. 4.

Metabolic stability and PK of Ko143 analogs. (A) Comparison of the metabolic stability of Ko143, K2 and K34 in HLM (n = 3). (B) PK of Ko143, K2, and K34 in WT mice. (C-E) Comparison of maximum concentration (C_max), area under concentration-time curve (AUC), and apparent clearance (CL/F) of Ko143, K2, and K34. WT mice were treated with Ko143, K2, or K34 (50 mg/kg, p.o.). The concentrations of compounds in sera were analyzed by UPLC-QTOFMS. The PK parameters were calculated by Phoenix WinNonlin. All data are expressed as mean ± SD (n = 4 at each time point). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, vs Ko143, one-way or two-way ANOVA.

Scheme 1.

Synthesis of amide I Ko143 analogs. Reagents and conditions: (a) R₁R₂NH, EDCI, HOBt, CH₂C1₂, rt, overnight; (b) Pd/C, H₂, MeOH, rt, 3-5 h; (c) Fmoc N-hydroxysuccinimide ester, NaHCO₃, 1,4-dioxane, rt, overnight; (d) i) For K2, SOCL₂, DMF, CH₂Cl₂, 0 °C ~ rt, 2 h, then Et₃N, compound 5, rt, overnight; for others, 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate (CIP), compound 5, DIEA, N-methylpyrrolidone. rt, 5 days; ii) piperidine, THF, rt, overnight.

Scheme 2.

Synthesis of amide II Ko143 analogs. Reagents and conditions: (a) i) compound 5, CIP, DIEA, N-methylpyrrolidone, rt, 5 days; ii) piperidine, THF, rt, overnight; (b) Pd/C, HCl, MeOH, rt, 4 h; (c) R₃COCl, Et₃N, CH₂Cl₂, 0 °C~ rt, 4 h; or R₃COOH, EDCI, HOBt, CH₂Cl₂, rt, overnight.