Design, synthesis and biological evaluation of N-arylsulfonyl carbazoles as novel anticancer agents

Abstract

In this work, a set of structurally diverse synthetic carbazoles was screened for their anticancer activities. According to structure-activity relationship studies, carbazoles with an N-substituted sulfonyl group exhibited better anticancer activity. Moreover, compound 8h was discovered to show the most potent anticancer effects on Capan-2 cells by inducing apoptosis and cell cycle arrest in G2/M phase. Finally, the in vivo study demonstrated that 8h prevented the tumor growth in PANC-1 and Capan-2 xenograft models without apparent toxicity.

Fig. 1. Representative carbazoles with anticancer activities.

Scheme 1. Synthesis of carbazoles. Reaction conditions: diphenyleniodonium 5 (0.1 mmol), amine 6 (4 equiv.), Na₂CO₃ (3 equiv.), Cu(OAc)₂ (0.2 equiv.), 16 h, in refluxing i-PrOH/(CH₂OH)₂ (1.8/0.2 mL), argon atmosphere. (a) Additional CuI (0.2 equiv.) was added. (b) 7o was obtained from the desulfonylation of 8a.

Scheme 2. Newly synthetic arylsulfonyl N-substituted carbazoles for more potent anticancer agents.

Fig. 2. Compound 8h induced DNA damage and cell cycle arrest in G2/M phase. (A) Protein γ-H2AX was analyzed by western blot. PANC-1 and Capan-2 cells were treated with 1 or 3 μM of compound 8h for 24 h. (B) Effect of compound 8h at 3 μM or 5 μM on the cell cycle of Capan-2 for 24 h. Cellular DNA contents were measured by PI staining using flow cytometry. The numbers indicate the normalization of each phase of cell cycle. (C) Histograms demonstrated the percentage of Capan-2 at different phases of the cell cycle. Error bars represent mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 3. The effect of compound 8h on the level of cyclin B1, p-Tyr15-cdc2. (A) Western blot was used to determine the expression of cyclin B1, p-Tyr15-cdc2 in PANC-1 and Capan-2 cells treated in the absence or presence (1 or 3 μM) of 8h for 24 h. (B) Densitometric analysis of cyclin B1, p-Tyr15-cdc2 proteins normalized with β-actin. The expression ratio of related proteins was quantified against β-actin using ImageJ software. Data were derived from three independent replicates. Bars means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 4. Flow cytometric analysis of 8h induced apoptosis in Capan-2 cells using Annexin V-FITC and PI double staining. (A) Apoptosis rates were determined after Capan-2 cells were treated with 8h at 3 μM or 5 μM for 48 h. Percentage numbers indicate cell death population. (B) Percentage of Annexin V/PI positive cells were derived from three independent replicates. Data shown are mean values ± SD (error bar). **p < 0.01; ***p < 0.001.

Fig. 5. Induction of ROS increase by compound 8h and its partial reversion by an antioxidant NAC. (A) DCF-DA staining showed the effect of 3 μM (blue line) and 5 μM (red line) of 8h on cellular ROS levels after 24 h treatment. (B and C) Pretreatment with NAC effectively reversed the ROS accumulation. Capan-2 cells were pre-treated with 3 mM NAC for 1 h and then incubated with compound 8h (3 or 5 μM) for 24 h. Error bars represent mean ± SD. (D and E) Compound 8h-induced apoptosis in Capan-2 cells was partially prevented by pre-treatment with NAC compared to Fig. 4. Capan-2 cells were pre-treated with 3 mM NAC for 1 h, and then incubated with the indicated concentrations (3 or 5 μM) of 8h for 24 h. Error bars represent mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 6. In vivo antitumor effect of compound 8h in PANC-1 xenograft model. 8h was administered three times per week intraperitoneally at 40 mg kg⁻¹ dosage. (A) Image of all dissected tumors. (B) The effect of 8h on the PANC-1 tumor growth. (C) Tumor weight at day 28 after 4 weeks of treatment. (D) Mice weights were measured twice a week. Bars means ± SD. **p < 0.01; ***p < 0.001.

Fig. 7. In vivo antitumor effect of compound 8h in Capan-2 xenograft model. 8h was administered three times per week intraperitoneally at 40 mg kg⁻¹ dosage. (A) Image of all dissected tumors. (B) The effect of 8 h on the Capan-2 tumor growth. (C) Tumor weight after 25 days of treatment. (D) Mice weights were measured twice a week. Bars means ± SD. *p < 0.05.

Scheme 3. Synthesis of cyclic diphenyliodoniums (5). Reaction conditions: (a) Pd(PPh₃)₄, K₃PO₄, EtOH, reflux, 6 h. (b) (i). NaNO₂, 0 °C THF, 4 M HCl, (ii). KI, rt. (c) mCPBA (85%), TfOH, CH₂Cl₂, rt, 2 h.