Synthesis and biological evaluation of 2-(alkoxycarbonyl)-3-anilinobenzo[b]thiophenes and thieno[2,3-b]pyridines as new potent anticancer agents

Abstract

Two new series of inhibitors of tubulin polymerization based on the 2-(alkoxycarbonyl)-3-(3',4',5'-trimethoxyanilino)benzo[b]thiophene and thieno[2,3-b]pyridine molecular skeletons were synthesized and evaluated for antiproliferative activity on a panel of cancer cell lines, inhibition of tubulin polymerization, cell cycle effects, and in vivo potency. Antiproliferative activity was strongly dependent on the position of the methyl group on the benzene portion of the benzo[b]thiophene nucleus, with the greatest activity observed when the methyl was located at the C-6 position. Also, in the smaller thieno[2,3-b]pyridine series, the introduction of the methyl group at the C-6 position resulted in improvement of antiproliferative activity to the nanomolar level. The most active compounds (4i and 4n) did not induce cell death in normal human lymphocytes, suggesting that the compounds may be selective against cancer cells. Compound 4i significantly inhibited in vivo the growth of a syngeneic hepatocellular carcinoma in Balb/c mice.

Figure 1

Binding model of 4n in the colchicine site of tubulin. The hydrogen bond with βAla250 is indicated by a dashed line.

Figure 2

Binding poses of 4h (in gray) and 4k (in magenta) in the colchicine site of tubulin. The hydrogen bond between 4k and βAla250 is indicated by a dashed line.

Figure 3

Percentage of cells in each phase of the cell cycle in Jurkat (A), HT29 (B), and HeLa (C) cells treated with the indicated compounds at the indicated concentrations for 24 h. Cells were fixed and labeled with PI and analyzed by flow cytometry as described in the Experimental Section.

Figure 4

Effect of 4i on G2/M regulatory proteins. HeLa cells were treated for 24 or 48 h with the indicated concentration of 4i. The cells were harvested and lysed for the detection of cyclin B, p-cdc2^Y15, and cdc25C expression by Western blot analysis. To confirm equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody.

Figure 5

Flow cytometric analysis of apoptotic cells after treatment of HeLa cells with 4i (A) or 4n (B) at the indicated concentrations after incubation for 24 or 48 h. The cells were harvested and labeled with annexin-V–FITC and PI and analyzed by flow cytometry. Data are represented as the mean ± SEM of three independent experiments.

Figure 6

Assessment of mitochondrial membrane potential (Δψ_mt) after treatment of HeLa cells with compound 4i (A) or 4n (B). Cells were treated with the indicated concentration of compound for 24 or 48 h and then stained with the fluorescent probe JC-1. Data are presented as the mean ± SEM for three independent experiments.

Figure 7

Mitochondrial production of ROS in HeLa cells following treatment with compound 4i (A, B) or compound 4n (C, D). After 24 or 48 h incubations, cells were stained with H₂-DCFDA (A, C) or HE (B, D) and analyzed by flow cytometry. Data are presented as the mean ± SEM of three independent experiments.

Figure 8

(A) Western blot analysis of caspase-3, cleaved caspase-9, and PARP after treatment of HeLa cells with 4i at the indicated concentrations and for the indicated times. (B) Western blot analysis of Bcl-XL, survivin^Thr32, Mcl-1, and XIAP after treatment of HeLa cells with 4i at the indicated concentrations and for the indicated times. To confirm equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody.

Figure 9

Inhibition of mouse allograft growth in vivo by compound 4i. Male mice were injected subcutaneously at their dorsal region with 10⁷ BNL 1MEA.7R.1 cells, a syngeneic hepatocellular carcinoma cell line. Tumor-bearing mice were administered the vehicle as a control or 5 mg/kg of 4i or CA-4P as a reference compound. Injections were given intraperitoneally daily starting on day 1. The figure shows the average measured tumor volumes (A) and body weights of the mice (B) recorded at the end of the treatments. Data are presented as the mean ± SEM of tumor volume and body weight at each time point for five animals per group. Key: *, p < 0.05 vs control; **, p < 0.01 vs control.

Scheme 1^a

^aReagents and conditions: (a) SHCH₂CO₂CH₃ or SHCH₂CO₂C₂H₅, KOH, H₂O, DMF; (b) tBuONO, CuBr₂, CH₃CN, 65 °C; (c) 3,4,5-trimethoxyaniline, Pd(OAc)₂, BINAP, CsCO₃, PhMe, 120 °C, 16 h.

Chart 1

Chemical Structures of CA-4, 2-(Alkoxycarbonyl)-3-(arylthio)indoles 3, 2-Anilinobenzo[b]thiophenes 2a and 4a–k, and 2-Anilinothieno[2,3-b]pyridines 4l–n