Investigation of chalcones as selective inhibitors of the breast cancer resistance protein: critical role of methoxylation in both inhibition potency and cytotoxicity

Abstract

ABCG2 plays a major role in anticancer-drug efflux and related tumor multidrug resistance. Potent and selective ABCG2 inhibitors with low cytotoxicity were investigated among a series of 44 chalcones and analogues (1,3-diarylpropenones), by evaluating their inhibitory effect on the transport of mitoxantrone, a known ABCG2 substrate. Six compounds producing complete inhibition with IC(50) values below 0.5 μM and high selectivity for ABCG2 were identified. The number and position of methoxy substituents appeared to be critical for both inhibition and cytotoxicity. The best compounds, with potent inhibition and low toxicity, contained an N-methyl-1-indolyl (compound 38) or a 6'-hydroxyl-2',4'-dimethoxy-1-phenyl (compound 27) moiety (A-ring) and two methoxy groups at positions 2 and 6 of the 3-phenyl moiety (B-ring). Methoxy substitution contributed to inhibition at positions 3 and 5, but had a negative effect at position 4. Finally, methoxy groups at positions 3, 4, and 5 of the B-ring markedly increased cytotoxicity and, therefore, should be avoided.

Figure 1

General structures of targeted chalcones and analogues.

Figure 2

3D-QSAR analyses. (Left) The 44 molecules were modeled, aligned on the central core, and inserted in the databank for a computed 3D-QSAR study. The calibration and validation coefficients were, respectively, 0.924 and 0.651. Two highly active compounds, chalcone 27 and its indolyl analogue, 38, are displayed in capped stick, and the other molecules are represented in lines. (Right) CoMFA contribution volumes with all molecules. Volumes were plotted with 20 for positive contribution (yellow for steric and red for electrostatic fields) and 70 for negative contribution (green for steric and blue for electrostatic fields).

Figure 3

Inhibition specificity for ABCG2 relative to ABCB1 and ABCC1. (A) The inhibition on ABCB1-mediated mitoxantrone efflux was determined by flow cytometry as described under Experimental Section and normalized using control cells transfected by the empty vector (taken as 100% inhibition). (B) The percent inhibition on ABCC1-mediated calcein transport was determined using the same approach. Data are the mean ± SD of two independent experiments.

Figure 4

Inhibition of ABCG2 efflux activity and intrinsic cytotoxicity. (A) The IC₅₀ values (concentrations for half-maximal inhibition) on ABCG2-mediated mitoxantrone efflux were determined by flow cytometry, as described under Experimental Section. The curves were fitted using GraphPad Prism 5 software. (B) Cell viability of ABCG2-transfected HEK293 cells and control cells upon treatment with compounds 27, 28, 31, and 37–39, up to 100 µM, for 72 h. The values represent the mean ± SD of percent cell viability with respect to the untreated control. Data correspond to at least two independent experiments performed in triplicate.

Scheme 1^a

^a(a) KOH (50% in H₂O), EtOH, 60 °C.