Design, synthesis and structure-activity relationship of 3,6-diaryl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines as novel tubulin inhibitors

Abstract

A novel series of 3,6-diaryl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines were designed, synthesized and biologically evaluated as vinylogous CA-4 analogues, which involved a rigid [1,2,4]triazolo[3,4-b][1,3,4]thiadiazine scaffold to fix the configuration of (Z,E)-butadiene linker of A-ring and B-ring. Among these rigidly vinylogous CA-4 analogues, compounds 4d, 5b, 5i, 6c, 6e, 6g, 6i and 6k showed excellent antiproliferative activities against SGC-7901, A549 and HT-1080 cell lines with IC₅₀ values at the nanomolar level. Compound 6i showed the most highly active antiproliferative activity against the three human cancer cell lines with an IC₅₀ values of 0.011-0.015 µM, which are comparable to those of CA-4 (IC₅₀ = 0.009-0.013 µM). Interestingly, SAR studies revealed that 3,4-methylenedioxyphenyl, 3,4-dimethoxyphenyl, 3-methoxyphenyl and 4-methoxyphenyl could replace the classic 3,4,5-trimethoxyphenyl in CA-4 structure and keep antiproliferative activity in this series of designed compounds. Tubulin polymerization experiments showed that 6i could effectively inhibit tubulin polymerization, which was corresponded with CA-4, and immunostaining experiments suggested that 6i significantly disrupted microtubule/tubulin dynamics. Furthermore, 6i potently induced cell cycle arrest at G₂/M phase in SGC-7901 cells. Competitive binding assays and docking studies suggested that compound 6i binds to the tubulin perfectly at the colchicine binding site. Taken together, these results revealed that 6i may become a promising lead compound for new anticancer drugs discovery.

Figure 1

Structures of known tubulin inhibtors and design strategy for the target compounds.

Figure 2

Reagents and conditions: (a) MeOH, conc. H₂SO₄, MW, 70 °C.; (b) N₂H₄·H₂O, MeOH, reflux; (c) CS₂, KOH, MeOH, 25 °C; (d) N₂H₄·H₂O, H₂O, MW, 100 °C.; (e) HCl, 0 °C; (f) CuBr₂, CHCl₃, EtOAc, reflux; (g) EtOH, MW, 80 °C.

Figure 3

(A) CA-4 inhibits microtubule polymerization in vitro. (B) Compound 6i inhibits microtubule polymerization in vitro. Tubulin was mixed with reaction buffer and incubated with CA-4 (0.5, 1, 2, 4 µM), paclitaxel (5.0 µM), 6i (0.1, 0.5, 2.0, 8.0 µM) or vehicle DMSO (control). The reaction was monitored at 37 ^oC.

Figure 4

Effects of 6i on tubulin network of SGC-7901 cells by immunofluorenscence. SGC-7901 cells were treated with DMSO (vehicle control), CA-4 (0.022 µM) or 6i (0.022 µM) for 12 h. The left and middle panels represent the tubulin assembly stained with DAPI and FITC and the right panel represents a merge of the corresponding left and middle panels.

Figure 5

Compound 6i affects the cell cycle progression in SGC-7901 cells. SGC-7901 cells were treated with compound 6i (0.011, 0.022 and 0.044 µM, respectively) or CA-4 (0.022 µM) for 12 h.

Figure 6

Competitive binding of 6i to colchicine-binding site on tubulin. F/F₀ represents inhibition rate (IR = F/F₀) whereas F₀ refers to fluorescence of the 5.0 µM colchicine-tubulin complex, and F describes the fluorescence of a given concentration (1.6 µM, 5.0 µM, and 15.0 µM) of CA-4, compounds 6i and paclitaxel competition with the 5.0 µM colchicine-tubulin complex.

Figure 7

(A) Overlay of 6i (pink), CA-4 (green) and vinylogous CA-4 (yellow) in the colchicine binding site. (B) The hydrogen bond interactions are displayed as green dotted lines and the amino acids are displayed as stick models.