Synthesis of Novel Methyl 3-(hetero)arylthieno[3,2- b]pyridine-2-carboxylates and Antitumor Activity Evaluation: Studies In Vitro and In Ovo Grafts of Chick Chorioallantoic Membrane (CAM) with a Triple Negative Breast Cancer Cell Line

Abstract

A series of novel functionalized methyl 3-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates 2a-2h were synthesized by C-C Pd-catalyzed Suzuki-Miyaura cross-coupling of methyl 3-bromothieno[3,2-b]pyridine-2-carboxylate with (hetero)aryl pinacol boranes, trifluoro potassium boronate salts or boronic acids. Their antitumoral potential was evaluated in two triple negative breast cancer (TNBC) cell lines-MDA-MB-231 and MDA-MB-468, by sulforhodamine B assay. Their effects on the non-tumorigenic MCF-12A cells were also evaluated. The results demonstrated that three compounds caused growth inhibition in both TNBC cell lines, with little or no effect against the non-tumorigenic cells. The most promising compound was further studied concerning possible effects on cell viability (by trypan blue exclusion assay), cell proliferation (by bromodeoxyuridine assay) and cell cycle profile (by flow cytometry). The results demonstrated that the GI₅₀ concentration of compound 2e (13 μM) caused a decreased in MDA-MB-231 cell number, which was correlated with a decreased in the % of proliferating cells. Moreover, this compound increased G0/G1 phase and decreased S phases, when compared to control cells (although was not statistic significant). Interestingly, compound 2e also reduced tumor size using an in ovo CAM (chick chorioallantoic membrane) model. This work highlights the potential antitumor effect of a novel methyl 3-arylthieno[3,2-b]pyridine-2-carboxylate derivative.

Keywords: Suzuki-Miyaura coupling; anticancer activity; thieno[3,2-b]pyridines; triple negative breast cancer.

Figure 1

Effect of compound 2e on MDA-MB-231 viable cell number, determined by trypan blue exclusion assay. Cells were treated for 48 h with medium (Blank), control (DMSO; vehicle), compound 2e (at 13 µM) and Doxorubicin (positive control; 50 nM). Results represent the mean ± S.E.M. of at least three independent experiments. ** p ≤ 0.01 and *** p ≤ 0.001 of Control vs. Treatments.

Figure 2

Effect of compound 2e on MDA-MB-231 cellular proliferation, determined by bromodeoxyuridine (BrdU) incorporation assay. Cells were treated for 48 h with medium (Blank), control (DMSO; vehicle), compound 2e (at 13 µM) and Doxorubicin (positive control; 50 nM). (A) Representative fluorescence microscopy images of BrdU incorporation (green) and DAPI stained nuclei (blue). Amplification = 200×. (B) Percentage of BrdU-incorporating cells. The results are presented as the mean ± S.E.M. of three independent experiments. * p ≤ 0.05 and *** p ≤ 0.001 of Control vs. Treatments.

Figure 3

Effect of compound 2e on MDA-MB-231 cell cycle distribution, analyzed by flow cytometry following incubation with propidium iodide (PI). Cells were treated for 48 h with medium (Blank), control (DMSO; vehicle), compound 2e (at 13 µM) and Doxorubicin (positive control; 50 nM). (A) Representative histograms of the MDA-M-231 cell cycle profile with the different treatments. (B) Percentage of MDA-MB-231 cells in the different phases of the cell cycle. Results represent the mean ± S.E.M. of at least three independent experiments. * p ≤ 0.05 and *** p ≤ 0.001 of Control vs. Treatments.

Figure 4

Effect of compound 2e on tumor size, using the in ovo chick embryo chorioallantoic membrane (CAM) model. TNBC MDA-MB-231 cells were grafted in the chicken eggs, and after 24 h were treated with the vehicle (DMSO) or with the compound 2e at 13 μM, for 48 h. (A) Tumor size of eggs treated with vehicle and compound 2e. Data is from two independent experiments. * p ≤ 0.05 of Vehicle vs. Treatment. (B) Representative images (1.25× amplification) or Hematoxylin & Eosin (H&E) stain of tumor size of cells treated with vehicle (DMSO) and compound 2e.