Design, synthesis, and apoptosis-promoting effect evaluation of novel pyrazole with benzo[d]thiazole derivatives containing aminoguanidine units

Abstract

New pyrazole with benzo[d]thiazoles containing hydrazinecarboximidamide substituent was synthesised and evaluated for cytotoxicity and apoptotic activity using the MTT assay, flow cytometry, and Western blot analysis. Among the compounds studied, (E)-2-((1-(6-((4-fluorobenzyl)oxy)benzo[d]thiazol-2-yl)-3-phenyl-1H- pyrazol-4-yl)methylene) hydrazinecarboximidamide (8l) was potent, with IC₅₀ values of 2.41 µM, 2.23 µM, 3.75 µM and 2.31 µM in vitro anti-proliferative activity testing against triple-negative breast cancer cell line MDA-MB-231, non-triple-negative breast cancer MCF-7 cells, and human hepatocarcinoma HepG2 cells, and SMMC-7721 cells, respectively. Especially, the activity against MDA-MB-231 was similar to that of Doxorubicin, which was used as a positive control in this study. Next, the Annexin V/PI flow cytometry assay was used at different concentrations of compound 8l to demonstrate that compound 81 induced apoptosis of MDA-MB-231 cells in a concentration-dependent manner. Finally, these results were further verified by Western blot analysis. Taken together, the results of this study revealed that compound 8l may be a potential anticancer compound play a significant role in the subsequent researches.

Keywords: Synthesis; aminoguanidine; anticancer; apoptosis; benzothiazole; pyrazoles.

Figure 1.

Rational design of the target compounds. (a) Structures of the previously reported compound with aminoguanidine. (b) Examples of pyrazole derivatives with biological activity. (c) Representative examples of benzothiazole derivatives. (d) A representative example of benzothiazole molecule with pyrazole that exhibits anticancer activity.

Scheme 1.

Synthesis of compounds 8a–8o. Reagent and conditions: (a) HBr (48% in H₂O), reflux, 18 h; (b) RBr/RPhCH₂Cl, CH₃COCH₃, K₂CO₃, reflux, 20 h; (c) 98%H₂SO₄, (CH₂OH)₂, 80 °C, 0.5 h, NH₂NH₂H₂O, 140 °C, 5 h; (d) EtOH, AcONa, reflux, 1 h; (e) POCl₃, DMF, 55–60 °C, 5 h; (f) aminoguanidine bicarbonate, EtOH, 60–70 °C, 8–12 h.

Figure 2.

The comparison of the inhibitory activity of synthesised compounds against four cell lines at 10 μM in the preliminary screening test. The ordinate represents inhibition ratio (%), the abscissa is the synthesised compounds.

Figure 3.

The inhibitory activity of the compounds with higher inhibition ratio against four cell lines at 5 μM in the screening test. The ordinate represents inhibition ratio (%) and the abscissa represents the chosen compounds and the reference drug Doxorubicin.

Figure 4.

Flow cytometry analyses of apoptosis induction in triple-negative breast cancer cell MDA-MB-231 after treated by different concentrations of compound 8l (1, 2, 4 and 8 μM) and no treatment (control) as a reference control for 48 h.

Figure 5.

Determine the translation of proteins by western blot. MDA-MB-231 cells were treated with compound 8l of 1.2 μM (half IC₅₀), 2.4 μM (IC₅₀), 4.8 μM (double IC₅₀) and no treatment for 48 h, respectively. β-actin was used for equal loading.