Search for ABCB1 Modulators Among 2-Amine-5-Arylideneimidazolones as a New Perspective to Overcome Cancer Multidrug Resistance

Abstract

Multidrug resistance (MDR) is a severe problem in the treatment of cancer with overexpression of glycoprotein P (Pgp, ABCB1) as a reason for chemotherapy failure. A series of 14 novel 5-arylideneimidazolone derivatives containing the morpholine moiety, with respect to two different topologies (groups A and B), were designed and obtained in a three- or four-step synthesis, involving the Dimroth rearrangement. The new compounds were tested for their inhibition of the ABCB1 efflux pump in both sensitive (parental (PAR)) and ABCB1-overexpressing (MDR) T-lymphoma cancer cells in a rhodamine 123 accumulation assay. Their cytotoxic and antiproliferative effects were investigated by a thiazolyl blue tetrazolium bromide (MTT) assay. For active compounds, an insight into the mechanisms of action using either the luminescent Pgp-Glo™ Assay in vitro or docking studies to human Pgp was performed. The safety profile in vitro was examined. Structure-activity relationship (SAR) analysis was discussed. The most active compounds, representing both 2-substituted- (11) and Dimroth-rearranged 3-substituted (18) imidazolone topologies, displayed 1.38-1.46 fold stronger efflux pump inhibiting effects than reference verapamil and were significantly safer than doxorubicin in cell-based toxicity assays in the HEK-293 cell line. Results of mechanistic studies indicate that active imidazolones are substrates with increasing Pgp ATPase activity, and their dye-efflux inhibition via competitive action on the Pgp verapamil binding site was predicted in silico.

Keywords: 5-arylideneimidazolone; ABCB1 modulator; Dimroth rearrangement; Pgp; Pgp docking; T-lymphoma cancer cell; cancer MDR; rhodamine 123 accumulation assay.

Figure 1

5-Arylideneimidazolones able to block mechanism of bacterial multidrug resistance (MDR) (1,2) [21,22].

Figure 2

Previously found (thio)hydantoin derivatives able to modulate ABCB1 in cancer cells (3–5) and the reference modulator, verapamil (6) [23,24].

Scheme 1

Synthesis route for compounds 7–20; (i), CH₃COONa, CH₃COOH, reflux, 5–7 h; (ii) CH₃I, C₂H₅ONa, rt, 24 h; (iii) proper amine, oil bath 120–130 °C, 15 min; C₂H₅OH, reflux 5–7 h (iv) Dimroth rearrangement.

Figure 3

The effect of the ABCB1 substrate verapamil (VER) (200 μM), ABCB1-negative compound caffeine (CFN), and compounds 11, 12, and 18 (100 μM) on ABCB1 basal activity. The compounds are recognized as ABCB1 substrates if they stimulate its basal activity (>100%). Data are presented as the mean ± SD. Statistical significance was evaluated by one-way ANOVA, followed by Bonferroni’s comparison test (* p < 0.05, *** p < 0.001, **** p < 0.0001 compared to the basal activity).

Figure 4

On the left: Position of the top-ranked docking pose of verapamil in human Pgp homology model; On the right: Binding mode and molecular interactions observed for the top-ranked docking pose of verapamil.

Figure 5

(a) Binding mode of compounds 18 and 12 adopting docking pose 1; (b) molecular interactions for the best docking pose 1 represented by compound 18.

Figure 6

(a) Binding mode of compounds 11, 18, and 12 adopting docking pose 2; (b) molecular interactions for the best docking pose 2 represented by compound 11.

Figure 7

(a) Binding mode of compounds 11 and 18 adopting docking pose 3; (b) molecular interactions for the best docking pose 3 represented by compound 11.

Figure 8

The viability of the HEK-293 cell line after incubation with compound 11 or 18 for 72 h. DOX—doxorubicin. Control—1% DMSO in the cell culture media. The statistical significance was evaluated by a one-way ANOVA, followed by Bonferroni’s comparison test (*** p < 0.001, **** p < 0.0001 compared to control).