Synthesis and Biological Evaluation of Novel Benzothiazole-2-thiol Derivatives as Potential Anticancer Agents

A series of novel benzothiazole-2-thiol derivatives were synthesized and their structures determined by 1H-NMR, 13C-NMR and HRMS (ESI). The effects of all compounds on a panel of different types of human cancer cell lines were investigated. Among them, pyridinyl-2-amine linked benzothiazole-2-thiol compounds 7d, 7e, 7f and 7i exhibited potent and broad-spectrum inhibitory activities. Compound 7e displayed the most potent anticancer activity on SKRB-3 (IC50 = 1.2 nM), SW620 (IC50 = 4.3 nM), A549 (IC50 = 44 nM) and HepG2 (IC50 = 48 nM) and was found to induce apoptosis in HepG2 cancer cells.


Introduction
A number of benzothiazole derivatives have exhibited interesting biological activities [1][2][3] and attracted continuing interest for further molecular exploration as useful anticancer agents [4,5]. Our preceding studies had found that two benzothiazole-2-thiol compounds (compounds 1 and 2) displayed good anticancer activities and induced HepG2 cell apoptosis in vitro [6]. In order to develop more potent tumor growth inhibitors as novel anticancer agents, we designed and synthesized a series of novel benzothiazole-2-thiol derivatives through incorporation of heterocyclic rings (pyridine, pyrimidine and thiazole) to benzothiazole-2-thiol derivatives with the activity and safety advantages of the OPEN ACCESS heterocyclic ring structures [7,8]. The effects of all the novel compounds on a panel of different types of human cancer cell lines were investigated by the MTT assay and compound 7e was selected to examine apoptosis on HepG2 cell cells by flow cytometry. As a result, the pyridinyl-2-amine linked benzothiazole-2-thiol compounds exhibited potent anticancer activities and compound 7e inhibited the proliferation of HepG2 cell via inducing apoptosis.

Chemistry
Twenty novel benzothiazole-2-thiol derivatives linked with heterocyclic rings were designed and synthesized by the route shown in Scheme 1.
Commercially available amines (compounds 3a-j) were first reacted with 2-chloroacetyl chloride in the presence of potassium carbonate as the base in dichloromethane to give crude compounds 4a-j. The raw products 4a-j were purified by recrystallization from ethyl acetate/petroleum ether. The compounds 6a-j were prepared by reacting 4a-j with 6-aminobenzothiazole-2-thiol (compound 5) and triethylamine (TEA) as the base in tetrahydrofuran (THF) under reflux. Compounds 6a-j respectively were thus obtained and could be used directly for the next step without further purification. The reaction mixtures of compounds 6a-j were further reacted with 3-chloropropyl chloride, 2-bromoacetyl bromide, 2-chloroacetyl chloride and 2-methoxybenzoyl chloride in the presence of triethylamine (TEA), respectively. The precipitates were collected by filtration and washed with water to yield the crude products (compounds 7a-t). Each compound was purified by column chromatography on silica gel using petroleum ether/ethyl acetate as eluent. The structures of all compounds were determined by 1 H-NMR, 13 C-NMR and HRMS (ESI).

Biological Activities
The twenty novel synthesized benzothiazole-2-thiol derivatives were investigated for anticancer activity in vitro on cancer cell lines by the MTT assay with compounds 1 and 2 as positive controls. Compounds 1 and 2 showed good activity against human colon adenocarcinoma cell line (SW480), human cervical cancer cell line (HeLa) and human hepatocellular carcinoma cell line (HepG2) in our preceding studies. Herein, we selected firstly the three human cell lines to test these compounds and the results were presented in Table 1.  As shown in Table 2, compounds 7d, 7e, 7f and 7i exhibited potent and broad-spectrum anticancer activities which were much better than compounds 1 and 2. Among them, compound 7d showed the most potent antitumor activities against A431 (IC 50 = 20 nM) and compound 7e displayed the most potent anticancer activity on SKRB-3 (IC 50 = 1.2 nM), SW620 (IC 50 = 4.3 nM), A549 (IC 50 = 44 nM) and HepG2 (IC 50 = 48 nM). The antitumor activities of compounds 7d and 7e were about 10-1,000 times greater than compounds 1 and 2 (SW620, A549, SKRB-3 and HepG2). These results suggested that pyridin-2-amine linking benzothiazole-2-thiol derivatives have potent and broad-spectrum anti-cancer activities and be worth being further investigated as candidate of anticancer agent. In order to investigate the apoptosis effects of the benzothiazole-2-thiol derivatives, we selected compound 7e to examine apoptosis effects on HepG2 cells by flow cytometry. Flow cytometric analysis was performed to measure the apoptotic cells and the cell cycle after propidium iodide (PI) staining [9]. From Figure 1, the percentages of apoptotic cells were 34.2%, 46.2% and 53.3%, respectively, with 0.625 µM, 1.25 µM, and 2.5 µM compound 7e treatment for 24 h. The results indicated that compound 7e inhibited the proliferation of HepG2 cell via inducing apoptosis on a concentration-dependent manner. The exact and further biological mechanism of compound 7e is under investigation in our laboratory.

General
The human cancer cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Dulbecco's modified Eagle medium (DMEM) and RPMI 1640 were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). Column chromatography was carried out on silica gel (200-300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China). Thin layer chromatography (TLC) was performed on TLC silica gel 60 F254 plates. Melting points were measured using a on a Kofler hot stage apparatus and were uncorrected. 1 H-NMR spectroscopy was performed using a Varian Unity Inova-400 spectrometer. The chemical shift values are reported in d units (ppm) relative to internal standard tetramethylsilane (TMS). 13 C-NMR spectra were recorded on a Bruker AV II-600 MHz spectrometer. Mass spectrometry was carried out on a Waters Q-TOF Premier mass spectrometer. All solvents were dried and freshly distilled prior to use according to standard procedures. All the chemicals used were of analytical grade and commercially available. The purity of compound screened in biological assays was determined to be ≥90% by HPLC analysis with a photodiode array detector (Waters, Milford, MA, USA). An Atlantis C 18 (150 mm × 4.6 mm, i.d. 5 μm) (Waters) was used with a gradient elution of methanol and HPLC-grade water as mobile phase at a flow rate of 1 mL/min.

Preparation of Compounds 4a-j
Compounds 4a-j were prepared following the literature procedure [10]. 2-Chloroacetyl chloride (0.75 mol) was added dropwise to a mixture of 3a-j (0.5 mol) and potassium carbonate (1.0 mol) in dichloromethane (550 mL) at 0 °C with stirring. After removal of the dichloromethane and vacuum filtration, the solid was washed with water and dried under vacuum for 12 h at 25-30 °C. The title compounds 4a-j were purified by recrystallization with petroleum ether/ethyl acetate.

Preparation of Compounds 6a-j
Compounds 6a-j were synthesized according to a literature method [11] with some modifications. Briefly, 6-aminobenzothiazole-2-thiol (35.0 mmol) was added to a mixture of 4a-j (38.5 mmol) and triethylamine (TEA, 70.0 mmol) in tetrahydrofuran (THF, 480 mL) at room temperature with stirring for 5 h. The title compounds 6a-j were obtained, respectively, and could be used directly for the next step without further purification.

General Procedure for Preparing Compounds 7a-t
The reaction mixtures of compounds 6a-j (3 mmol) were further reacted with acyl chloride (3.6 mmol) in the presence of triethylamine (TEA, 4.5 mmol) using tetrahydrofuran (THF, 30 mL) as solvent. The completion of the reaction was monitored by TLC and took 4-26 h. The precipitate was collected by filtration and washed with water to yield the crude product. Compounds 7a-t were purified by column chromatography on silica gel using petroleum ether/ethyl acetate as eluent.