Synthesis and Discovery of Ligustrazine-Heterocycle Derivatives as Antitumor Agents

Abstract

Ligustrazine (TMP) is a natural pyrazine alkaloid extracted from the roots of Ligusticum Chuanxiong Hort, which has the potential as an antitumor agent. A series of 33 ligustrazine-heterocycle (TMPH) derivatives were designed, synthesized, and investigated via antitumor screening assays, molecular docking analysis, and prediction of drug-like properties. TMP was attached to other heterocyclic derivatives by an 8-12 methylene alkyl chain as a linker to obtain 33 TMPH derivatives. The structures were confirmed by ¹H-NMR, ¹³C-NMR, and high-resolution mass spectroscopy spectral (HR-MS) data. The antiproliferative activity against human breast cancer MCF-7, MDA-MB-231, mouse breast cancer 4T1, mouse fibroblast L929, and human umbilical vein endothelial HUVEC cell lines was evaluated by MTT assay. Compound 12-9 displayed significant inhibitory activity with IC₅₀ values in the low micromolar range (0.84 ± 0.02 µM against the MDA-MB-231 cell line). The antitumor effects of compound 12-9 were further evaluated by plate cloning, Hoechst 33 342 staining, and annexin V-FITC/PI staining. The results indicated that compound 12-9 inhibited the proliferation and apoptosis of breast cancer cells. Furthermore, molecular docking of compound 12-9 into the active site of the Bcl-2, CASP-3, and PSMB5 target proteins was performed to explore the probable binding mode. The 33 newly synthesized compounds were predicted to have good drug-like properties in a theoretical study. Overall, these results indicated that compound 12-9 inhibited cell proliferation through PSMB5 and apoptosis through Bcl-2/CASP-3 apoptotic signaling pathways and had good drug-like properties. These results provided more information, and key precursor lead derivatives, in the search for effective bioactive components from Chinese natural medicines.

Keywords: antitumor; apoptosis; ligustrazine–heterocyclic (TMPH) derivatives; proliferation; triple-negative breast cancer.

SCHEME 1

Synthetic route for the TMPH derivatives.

FIGURE 1

Effect of TMPH 12–9 on the colony formation in MDA-MB-231 cells. (A) Representative images of MDA-MB-231 colonies; (B) percentage of colonies compared with that of the control group, n = 3. ^∗∗: p < 0.05; ^∗∗∗: p < 0.001 versus control.

FIGURE 2

Fluorescence microscopy images of MDA-MB-231 cells with Hoechst staining: the control group and treatment group with 1.0 μM TMPH 12–9.

FIGURE 3

Annexin V-FITC and PI staining to evaluate apoptosis in MDA-MB-231 cells following compound 12–9 treatment. MDA-MB-231 cells were treated with MDA-MB-231 (0, 0.5, 1.0, and 2.0 μM, for 48 h), stained by annexin V-FITC and PI, and analyzed using flow cytometry.

FIGURE 4

Visual presentation of the interaction of the crystal structure of compound 12–9 (shown as stick) with its docked pose of Bcl-2, CASP-3, and PSMB5 and (represented as a secondary structure in green) performed by the Schrodinger. [(A–C) represented as the ligand interaction diagram with Bcl-2, CASP-3, and PSMB5 respectively. (D–F) represented as binding interactions with Bcl-2, CASP-3, and PSMB5, respectively.].