Synthesis and Anticancer Activity of Mitotic-Specific 3,4-Dihydropyridine-2(1 H)-thiones

Abstract

Most anticancer drugs target mitosis as the most crucial and fragile period of rapidly dividing cancer cells. However the limitations of classical chemotherapeutics drive the search for new more effective and selective compounds. For this purpose structural modifications of the previously characterized pyridine aalog (S1) were incorporated aiming to obtain an antimitotic inhibitor of satisfactory and specific anticancer activity. Structure-activity relationship analysis of the compounds against a panel of cancer cell lines allowed to select a compound with a thiophene ring at C5 of a 3,4-dihydropyridine-2(1H)-thione (S22) with promising antiproliferative activity (IC₅₀ equal 1.71 ± 0.58 µM) and selectivity (SI = 21.09) against melanoma A375 cells. Moreover, all three of the most active compounds from the antiproliferative study, namely S1, S19 and S22 showed better selectivity against A375 cells than reference drug, suggesting their possible lower toxicity and wider therapeutic index. As further study revealed, selected compounds inhibited tubulin polymerization via colchicine binding site in dose dependent manner, leading to aberrant mitotic spindle formation, cell cycle arrest and apoptosis. Summarizing, the current study showed that among obtained mitotic-specific inhibitors analogue with thiophene ring showed the highest antiproliferative activity and selectivity against cancer cells.

Keywords: anticancer agents; colchicine-binding site; dihydropyridine; mitotic spindle; tubulin inhibitors.

Figure 1

Compounds S1-S22 obtained and tested within a three-step optimisation process.

Scheme 1

Sequence of the synthesis of targeted compounds S1-S4 and S8-S22.

Figure 2

The effect of indicated compounds (at 10 µM) on the induction of apoptosis in A375 (A) and HEM (B) cells after 48 h treatment, subsequently stained with Annexin V-PE and 7-AAD and analyzed by flow cytometry. Data are expressed as the mean and SD from at least three independent experiments; * p < 0.05 vs. ctrl; # p < 0.05 vs. CA-4.

Figure 3

Flow cytometry analysis of cell cycle in A375 cells using PI staining after exposure to indicated compounds (at 10 µM) for 6 h (A) and 24 h (B). Data are expressed as the mean and SD from three independent experiments; * p < 0.05 vs. ctrl; # p < 0.05 vs. CA-4.

Figure 4

Representative confocal microscopy images of A375 cells after 6 h incubation with medium and 0.2% DMSO (A), compound S1 (B), S19 (C), S22 (D) at concentration of 10 µM, with stained α-tubulin (green) and chromosomes (blue); magnification ×20 plus zoom ×3 during acquisition.

Figure 5

Quantification of mitotic spindle phenotypes of A375 cells after 6 h incubation with indicated compounds at 10 µM. Data are expressed as the mean and SD from two independent experiments.

Figure 6

Representative confocal microscopy images of A375 cells after 24 h incubation with compound S1 (A), S19 (B), S22 (C) at concentration of 10 µM, with stained α-tubulin (green) and chromosomes (blue); magnification ×40 plus zoom ×2 during acquisition.

Figure 7

Effects of S1 (A), S19 (B) and S22 (C) derivatives on tubulin polymerization in cell free conditions. Increasing relative fluorescence units is indicative of tubulin polymerization. Paclitaxel (PTX) and combretastatin (CA-4) were used as references and DMSO (0.2%) as a vehicle control. Curves of tested compounds represent the mean from at least three independent experiments. Error bars are not shown for the sake of clarity.

Figure 8

(A) Western blot analysis of polymerized microtubules (P) and soluble tubulin dimers (S) fraction in the cells after 6 h treatment with indicated compounds. Original images for blots in Supplementary Materials (Figure S6). (B) Percentage of α-tubulin in the pellet fraction after densitometric analysis of western blot gel. Each densitometric value of tubulin was normalized to the densitometric value of β-actin. Data are expressed as the mean and SD from four independent experiments; * p < 0.05 vs. ctrl; # p < 0.05 vs. CA-4).

Figure 9

Colchicine competitive binding assay. Results have been shown as intrinsic fluorescence of colchicine-tubulin complex normalized to control (0.2% DMSO). CA-4 was used as a positive control whereas vinblastine (VBL), which binds at vinblastine site, was used as negative control. Data are expressed as the mean and SD from three independent experiments for S1, S19, S22, CA-4 and two independent experiments for VBL; * p < 0.05 vs. ctrl; # p < 0.05 vs. CA-4.