molecules Molecules Molecules Molecules 1420-3049 MDPI 10.3390/molecules17043933 molecules-17-03933 Article Synthesis and Biological Evaluation of Novel Benzothiazole-2-thiol Derivatives as Potential Anticancer Agents Shi Xuan-Hong Wang Zhao Xia Yong Ye Ting-Hong Deng Mei Xu You-Zhi Wei Yu-Quan Yu Luo-Ting * State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China Author to whom correspondence should be addressed; Email: yuluot@scu.edu.cn; Tel: +86-28-8550-3817; Fax: +86-28-8516-4060. 30 03 2012 04 2012 17 4 3933 3944 22 02 2012 20 03 2012 26 03 2012 © 2012 by the authors; licensee MDPI, Basel, Switzerland. 2012

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

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.

 anticancer synthesis apoptosis benzothiazole-2-thiol derivatives 1. 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 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.

 2. Results and Discussion 2.1. Chemistry

Twenty novel benzothiazole-2-thiol derivatives linked with heterocyclic rings were designed and synthesized by the route shown in Scheme 1.

molecules-17-03933-f001

Synthetic route for 7a–t.

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 1H-NMR, 13C-NMR and HRMS (ESI).

 2.2. 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.

molecules-17-03933-t001_Table 1

The anti-proliferative activities for compounds 1, 2 and 7a–t.

Compd. R1 R2 IC50 (µM) a
HepG2 SW480 HeLa
1 4-chlorobenzyl 2-methoxyphenyl 0.7 5.6 4.0
2 benzyl chloromethyl 1.0 5.2 4.6
7a benzyl chloroethyl 5.6 15.7 8.6
7b benzyl bromoethyl 6.6 14.9 48.1
7c 2-pyridinyl chloromethyl 0.7 2.3 3.1
7d 5-chloro-2-pyridinyl chloromethyl 0.26 0.46 0.035
7e 5-bromo-2-pyridinyl chloromethyl 0.048 0.68 0.02
7f 5-methyl-2-pyridinyl chloromethyl 0.091 1.0 0.03
7g 3-pyridinyl chloromethyl 6.6 6.4 12.6
7h 2-chloro-4-pyridinyl chloromethyl 5.0 2.9 1.9
7i 2-chloro-4-methyl-3-pyridinyl chloromethyl 0.4 2.4 0.4
7j 2-pyrimidinyl chloromethyl 24.3 54.8 24.3
7k 2-thiazolyl chloromethyl 13.1 7.1 14.5
7l 2-pyridinyl 2-methoxyphenyl >100 >100 >100
7m 5-chloro-2-pyridinyl 2-methoxyphenyl >100 >100 0.6
7n 5-bromo-2-pyridinyl 2-methoxyphenyl 23.5 23.8 23.5
7o 5-methyl-2-pyridinyl 2-methoxyphenyl 45.6 31.1 0.8
7p 3-pyridinyl 2-methoxyphenyl >100 >100 >100
7q 2-chloro-4-pyridinyl 2-methoxyphenyl >100 >100 >100
7r 2-chloro-4-methyl-3-pyridinyl 2-methoxyphenyl 23.1 >100 34.6
7s 2-pyrimidyl 2-methoxyphenyl 27.2 >100 >100
7t 2-thiazolyl 2-methoxyphenyl >100 >100 >100

a Values are means of three experiments.

These novel compounds showed great variation of IC50 values on the three cell lines and compounds 7d, 7e, 7f and 7i exhibited potent inhibitory activities. To further study the cytotoxic profile, the potent analogues 7d, 7e, 7f and 7i were selected for further evaluation of inhibitory activities against other eleven types of human cancer cell lines, including colon cancer cell lines HCT-116 and SW620, lung cancer cell line A549, prostate cancer cell line PC-3, pancreatic cancer cell line BxPC-3, breast cancer cell line BT474, epidermoid cancer cell line A431, ovarian cancer cell line SKOV-3, non-small cell lung cancer cell line H460, breast cancer cell line MDA-MB-468 and SKRB-3.

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 (IC50 = 20 nM) and 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). 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.

molecules-17-03933-t002_Table 2

The anti-proliferative activities for compounds 7d, 7e, 7f, 7i, 1 and 2 against various cancer cell lines.

Compd. IC50(µM) a
HCT116 BT474 SW620 H460 PC-3 BXPC-3 A431 A549 SKOV-3 MDA-MB-468 SKRB-3
7d 0.8 4.3 0.033 3.7 7.9 0.1 0.02 0.2 2.7 1.5 0.5
7e 0.3 0.6 0.0043 0.3 6.0 0.3 0.2 0.044 0.4 0.9 0.0012
7f 0.6 5.3 1.5 4.2 6.8 0.5 0.1 0.3 3.5 2.5 1.9
7i 2.2 5.2 6.5 14.6 8.6 9.3 4.8 8.3 5.8 6.8 11.3
1 1.6 >100 4.1 39.7 4.8 4.7 1.0 2.0 6.0 2.4 1.5
2 1.2 7.8 1.3 10.0 6.0 6.6 4.5 4.4 4.6 4.0 8.0

a Values are means of three experiments.

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.

Compound 7e concentration-dependently induced apoptosis in HepG2 cancer cells.

 3. Experimental 3.1. 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. 1H-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). 13C-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 C18 (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.

 3.2. Preparation of Compounds <italic><bold>4a–j</bold></italic>

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.

 3.3. Preparation of Compounds <italic><bold>6a–j</bold></italic>

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.

 3.4. General Procedure for Preparing Compounds <italic><bold>7a–t</bold></italic>

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.

N-(2-(2-(Benzylamino)-2-oxoethylthio)benzo[d]thiazol-6-yl)-3-chloropropanamide (7a). Yellow powder, yield 72%, purity 99.8%, mp 177.2–178.1 °C; 1H-NMR (DMSO-d6) δ: 2.87 (t, J = 6.2 Hz, 2H), 3.91 (t, J = 6.2 Hz, 2H), 4.19 (s, 2H), 4.33 (d, J = 6.0 Hz, 2H), 7.22–7.28 (m, 5H), 7.55 (dd, J = 2.0, 4.4 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H), 8.40 (d, J = 2.0 Hz, 1H), 8.82 (t, J = 5.8 Hz, 1H), 10.33 (s, 1H); 13C-NMR (DMSO-d6) δ: 36.53, 39.49, 40.76, 42.54, 111.35, 118.49, 121.04, 126.76, 127.10, 128.19, 131.70, 135.45, 135.81, 138.80, 148.68, 148.80, 164.35, 166.43, 168.09; HRMS (ESI) m/z: Calcd. for C19H18ClN3O2S2 420.0602; Found: 420.0592 (M-H+).

N-Benzyl-2-(6-(2-bromoacetamido)benzo[d]thiazol-2-ylthio)acetamide (7b). Yellow powder, yield 75%, purity 98.0%, mp 155.6–159.7 °C; 1H-NMR (DMSO-d6) δ: 4.08 (s, 2H), 4.20 (s, 2H), 4.33 (d, J = 6.0 Hz, 2H), 7.22–7.28 (m, 5H), 7.55 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.8 Hz, 1H), 8.38 (s, 1H), 8.83 (s, 1H), 10.64 (s, 1H); 13C-NMR (DMSO-d6) δ: 28.72, 39.48, 42.54, 111.56, 118.65, 121.10, 126.86, 127.32, 128.29, 131.86, 135.48, 135.59, 138.94, 148.74, 148.81, 165.23, 167.36, 169.70; HRMS (ESI) m/z: Calcd. for C18H16BrN3O2S2 449.9940; Found: 449.9929 (M-H+).

2-Chloro-N-(2-(2-oxo-2-(pyridin-2-ylamino)ethylthio)benzo[d]thiazol-6-yl)acetamide (7c). Yellow powder, yield 70%, purity 92.0%, mp 168.4–172.9 °C; 1H-NMR (DMSO-d6) δ: 4.29 (s, 2H), 4.45 (s, 2H), 7.54–7.58 (m, 2H), 7.78 (d, J = 8.8 Hz, 1H), 8.18–8.19 (m, 2H), 8.37 (d, J = 16.0 Hz, 1H), 8.89 (s, 1H), 10.35 (s, 1H), 10.54 (s, 1H); 13C-NMR (DMSO-d6) δ: 39.46, 43.49, 111.69, 113.46, 118.75, 119.69, 121.12, 135.42, 135.57, 138.32, 148.02, 148.91, 151.59, 164.63, 164.79, 166.20; HRMS (ESI) m/z: Calcd. for C16H13ClN4O2S2 393.0241; Found: 393.0241 (M-H+).

2-Chloro-N-(2-(2-(5-chloropyridin-2-ylamino)-2-oxoethylthio)benzo[d]thiazol-6-yl)acetamide (7d). Yellow powder, yield 70%, purity 96.8%, mp 217.2–218.8 °C; 1H-NMR (DMSO-d6) δ: 4.29 (s, 2H), 4.44 (s, 2H), 7.50–7.54 (m, 1H), 7.77 (d, J = 8.8 Hz, 1H), 8.41 (s, 1H), 7.50–8.41 (m, 3H), 10.54 (s, 1H), 11.10 (s, 1H); 13C-NMR (DMSO-d6) δ: 39.48, 43.54, 111.80, 114.52, 118.77, 121.16, 125.38, 135.49, 135.61, 138.01, 148.79, 148.94, 150.27, 164.62, 165.37, 166.35; HRMS (ESI) m/z: Calcd. for C16H12Cl2N4O2S2 426.9852; Found: 426.9822 (M-H+).

N-(5-Bromopyridin-2-yl)-2-(6-(2-chloroacetamido)benzo[d]thiazol-2-ylthio)acetamide (7e). Yellow powder, yield 71%, purity 96.0%, mp 221.3–222.5 °C; 1H-NMR (DMSO-d6) δ: 4.29 (s, 2H), 4.43 (s, 2H), 7.53 (d, J = 8.8 Hz, 1H), 7.76 (d, J = 8.8 Hz, 1H), 8.47 (s, 1H), 8.38 (s, 1H), 8.02 (m, 2H), 10.54 (s, 1H), 11.09 (s, 1H); 13C-NMR (DMSO-d6) δ: 39.98, 44.05, 112.29, 114.26, 115.56, 119.26, 121.66, 135.79, 135.99, 141.22, 149.12, 149.44, 151.07, 165.11, 165.23, 166.88; HRMS (ESI) m/z: Calcd. for C16H12BrClN4O2S2 472.7864; Found: 472.7853 (M-H+).

2-Chloro-N-(2-(2-(5-methylpyridin-2-ylamino)-2-oxoethylthio)benzo[d]thiazol-6-yl)acetamide (7f). Yellow powder, yield 90%, purity 97.1%, mp 214.0–214.9 °C; 1H-NMR (DMSO-d6) δ: 2.25 (s, 3H), 4.29 (s, 2H), 4.41 (s, 2H), 7.53 (dd, J = 2.0, 4.4 Hz, 1H), 7.61 (dd, J = 2.0, 4.2 Hz, 1H), 7.77 (d, J = 9.2 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 8.18 (s, 1H), 8.37 (d, J = 1.0 Hz, 1H), 10.54 (s, 1H), 10.82 (s, 1H); 13C-NMR (DMSO-d6) δ: 17.22, 39.46, 43.53, 111.75, 113.13, 118.74, 121.14, 128.73, 135.30, 135.47, 139.06,147.22, 148.95, 149.20, 164.70, 164.74, 166.02; HRMS (ESI) m/z: Calcd. for C17H15ClN4O2S2 407.0398; Found: 407.0397 (M-H+).

2-Chloro-N-(2-(2-oxo-2-(pyridin-3-ylamino)ethylthio)benzo[d]thiazol-6-yl)acetamide (7g). Yellow powder, yield 68%, purity 92.0%, mp 160.6–165.4 °C; 1H-NMR (DMSO-d6) δ: 4.29 (s, 2H), 4.45 (s, 2H), 7.55 (d, J = 8.0 Hz, 2H), 7.78 (d, J = 8.8 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H), 8.37 (s, 2H), 8.89 (s, 1H), 10.66 (s, 1H), 11.20 (s, 1H); 13C-NMR (DMSO-d6) δ: 39.43, 43.50, 111.67, 118.52, 118.72, 121.18, 130.46, 135.43, 135.51, 140.30, 148.83, 148.89, 164.36, 164.80, 166.47; HRMS (ESI) m/z: Calcd. for C16H13ClN4O2S2 393.0241; Found: 393.0247 (M-H+).

2-Chloro-N-(2-(2-(2-chloropyridin-4-ylamino)-2-oxoethylthio)benzo[d]thiazol-6-yl)acetamide (7h). Yellow powder, yield 91%, purity 95.7%, mp 192.5–197.1 °C; 1H-NMR (DMSO-d6) δ: 4.29 (s, 2H), 4.43 (s, 2H), 7.47–7.54 (m, 2H), 7.75–7.78 (m, 2H), 8.29 (d, J = 5.6 Hz, 1H), 8.39 (s, 1H), 10.55 (s, 1H), 11.08 (s, 1H); 13C-NMR (DMSO-d6) δ: 39.48, 43.53, 111.81, 112.46, 112.62, 118.76, 121.15, 135.34, 135.49, 147.83, 148.85, 150.47, 151.02, 164.34, 164.75, 166.93; HRMS (ESI) m/z: Calcd. for C16H12Cl2N4O2S2 426.9852; Found: 426.9883 (M-H+).

2-Chloro-N-(2-(2-(2-chloro-4-methylpyridin-3-ylamino)-2-oxoethylthio)benzo[d]thiazol-6-yl)acetamide (7i). Yellow powder, yield 80%, purity 98.0%, mp 186.4–188.3 °C; 1H-NMR (DMSO-d6) δ: 2.22 (s, 3H), 4.29 (s, 2H), 4.41 (s, 2H), 7.34 (d, J = 4.8 Hz, 1H), 7.67 (t, J = 8.8 Hz, 2H), 8.19 (d, J = 4.8 Hz, 1H), 8.55 (s, 1H), 10.24 (s, 1H), 10.37 (s, 1H); 13C-NMR (DMSO-d6) δ: 17.81, 39.54, 43.53, 111.87, 118.83, 121.08, 125.10, 130.49, 135.30, 135.56, 147.02, 148.67, 148.77, 148.99, 164.43, 164.76, 165.62; HRMS (ESI) m/z: Calcd. for C17H14Cl2N4O2S2 441.0008; Found: 440.9992 (M-H+).

2-Chloro-N-(2-(2-oxo-2-(pyrimidin-2-ylamino)ethylthio)benzo[d]thiazol-6-yl)acetamide (7j). Brownish red powder, yield 63%, purity 90.0%, mp 172.6–174.9 °C; 1H-NMR (DMSO-d6) δ: 4.29 (s, 2H), 4.59 (s, 2H), 7.22 (t, J = 4.8 Hz, 1H), 7.54 (d, J = 4.4 Hz, 1H), 7.78 (d, J = 8.8 Hz, 1H), 8.38 (s, 1H), 8.69 (d, J = 2.4 Hz, 2H), 10.58 (s, 1H), 11.04 (s, 1H); 13C-NMR (DMSO-d6) δ: 39.46, 43.52, 109.98, 117.43, 121.60, 136.22, 147.29, 157.33, 157.66, 158.40, 166.22; HRMS (ESI) m/z: Calcd. for C15H12ClN5O2S2 394.0194; Found: 394.0109 (M-H+).

2-Chloro-N-(2-(2-oxo-2-(thiazol-2-ylamino)ethylthio)benzo[d]thiazol-6-yl)acetamide (7k). Yellow powder, yield 78%, purity 95.0%, mp 218.3–224.7 °C; 1H-NMR (DMSO-d6) δ: 4.29 (s, 2H), 4.46 (s, 2H), 7.25 (d, J = 3.6 Hz, 1H), 7.50–7.55 (m, 2H), 7.77 (d, J = 8.8 Hz, 1H), 8.38 (s, 1H), 10.55 (s, 1H), 12.53 (s, 1H); 13C-NMR (DMSO-d6) δ: 39.46, 43.54, 111.81, 113.79, 118.77, 121.18, 135.33, 135.52, 137.71, 148.87, 157.75, 164.32, 164.74, 165.64; HRMS (ESI) m/z: Calcd. for C14H11ClN4O2S3 398.9806; Found: 398.9816 (M-H+).

2-Methoxy-N-(2-(2-oxo-2-(pyridin-2-ylamino)ethylthio)benzo[d]thiazol-6-yl)benzamide (7l). Yellow powder, yield 70%, purity 92.0%, mp 154.2–158.9 °C; 1H-NMR (DMSO-d6) δ: 3.90 (s, 3H), 4.45 (s, 2H), 7.08 (t, J = 7.4 Hz, 1H), 7.14 (t, J = 6.0 Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.49–7.54 (m, 1H), 7.63–7.69 (m, 2H), 7.80 (dd, J = 7.0, 6.4 Hz, 2H), 8.35 (d, J = 3.6 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 8.54 (s, 1H), 10.35 (s, 1H), 10.91 (s, 1H); 13C-NMR (DMSO-d6) δ: 39.46, 111.69, 118.75, 121.12, 135.41, 135.96, 148.91, 164.19, 164.57, 166.22; HRMS (ESI) m/z: Calcd. for C22H18N4O3S2 451.0893; Found: 451.0887 (M-H+).

N-(2-(2-(5-Chloropyridin-2-ylamino)-2-oxoethylthio)benzo[d]thiazol-6-yl)-2-methoxybenzamide (7m). Yellow powder, yield 70%, purity 97.0%, mp 148.5–154.1 °C; 1H-NMR (DMSO-d6) δ: 3.90 (s, 3H), 4.45 (s, 2H), 7.19 (d, J = 8.4 Hz, 1H), 7.2 (t, J = 7.4 Hz, 2H), 7.63–7.75 (m, 2H), 7.77 (d, J = 8.8 Hz, 1H), 7.92 (d, J = 4.4 Hz, 1H), 8.08 (d, J = 8.4 Hz, 1H), 8.41 (d, J = 2.4 Hz, 1H), 8.54 (s, 1H), 10.35 (s, 1H), 11.11 (s, 1H); 13C-NMR (DMSO-d6) δ: 39.46, 55.84, 111.83, 111.93, 114.52, 119.17, 120.46, 120.99, 124.68, 125.38, 129.66, 132,09, 135.41, 135.95, 137.99, 146.43, 148.73, 150.28, 156.46, 164,17, 164.53, 166.37; HRMS (ESI) m/z: Calcd. for C22H17ClN4O3S2 485.0504; Found: 485.0521 (M-H+).

N-(2-(2-(5-Bromopyridin-2-ylamino)-2-oxoethylthio)benzo[d]thiazol-6-yl)-2-methoxybenzamide (7n). Yellow powder, yield 75%, purity 99.0%, mp 178.4–183.5 °C; 1H-NMR (DMSO-d6) δ: 3.90 (s, 3H), 4.45 (s, 2H), 7.07 (t, J = 7.4 Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.66 (d, J = 10.8 Hz, 2H), 7.77 (d, J = 8.8 Hz, 1H), 7.95–8.03 (m, 2H), 8.48 (s, 1H), 8.54 (s, 1H), 10.35 (s, 1H), 11.10 (s, 1H); 13C-NMR (DMSO-d6) δ: 39.49, 55.87, 111.86, 111.95, 113.78, 115.08, 119.19, 120.48, 121.01, 124.69, 129.68, 132.11, 135.41, 135.96, 148.63, 148.74, 150.59, 156.48, 164.17, 164.54, 166.41; HRMS (ESI) m/z: Calcd. for C22H17BrN4O3S2 528.9998; Found: 529.0070 (M-H+).

2-Methoxy-N-(2-(2-(5-methylpyridin-2-ylamino)-2-oxoethylthio)benzo[d]thiazol-6-yl)benzamide (7o). Yellow powder, yield 76%, purity 92.0%, mp 182.7–188.5 °C; 1H-NMR (DMSO-d6) δ: 2.28 (s, 3H), 3.90 (s, 3H), 4.45 (s, 2H), 7.08 (t, J = 7.6 Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.50–7.62 (m, 2H), 7.68 (t, J = 10.2 Hz, 2H), 7.78 (d, J = 8.8 Hz, 1H), 7.95 (s, 1H), 8.18 (s, 1H), 8.52 (d, J = 2.0 Hz, 1H), 10.31 (s, 1H), 10.76 (s, 1H); 13C-NMR (DMSO-d6) δ: 17.24, 39.49, 55.86, 111.84, 111.96, 112.99, 119.18, 120.47, 121.00, 124.71, 128.61, 129.67, 132.10, 135.41, 135.95, 138.57, 147.80, 148.79, 149.49, 156.48, 164.33, 164.55, 165.90; HRMS (ESI) m/z: Calcd. for C23H20N4O3S2 465.1050; Found: 465.1071 (M-H+).

2-Methoxy-N-(2-(2-oxo-2-(pyridin-3-ylamino)ethylthio)benzo[d]thiazol-6-yl)benzamide (7p). Yellow powder, yield 75%, purity 95.0%, mp 154.8–159.5 °C; 1H-NMR (DMSO-d6) δ: 3.90 (s, 3H), 4.45 (s, 2H), 7.08 (t, J = 7.6 Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.50–7.62 (m, 2H), 7.55(d, J = 8.0 Hz, 1H), 7.70 (t, J = 6.6 Hz, 1H), 7.78 (d, J = 8.8 Hz, 1H), 8.29 (d, J = 8.4 Hz, 1H), 8.46 (d, J = 4.4 Hz, 1H), 8.39 (s, 1H), 8.95 (s, 1H), 10.36 (s, 1H), 11.12 (s, 1H); 13C-NMR (DMSO-d6) δ: 39.44, 55.62, 111.90, 112.33, 119.21, 119.95, 120.99, 121.24, 124.69, 126.23, 129.67, 132.97, 135.41, 135.96, 140.75, 144.51, 148.75, 156.47, 158.03, 164.19, 164.57, 167.32; HRMS (ESI) m/z: Calcd. for C22H18N4O3S2 451.0893; Found: 451.0833 (M-H+).

N-(2-(2-(2-Chloropyridin-4-ylamino)-2-oxoethylthio)benzo[d]thiazol-6-yl)-2-methoxybenzamide (7q). Yellow powder, yield 70%, purity 90.0%, mp 177.9–180.8 °C; 1H-NMR (DMSO-d6) δ: 3.90 (s, 3H), 4.44 (s, 2H), 7.07 (t, J = 7.4 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H), 7.47–7.53 (m, 2H), 7.53–7.62 (m, 2H), 7.76 (t, J = 5.8 Hz, 1H), 7.78 (s, 1H), 8.29 (d, J = 5.6 Hz, 1H), 8.55 (s, 1H), 10.35 (s, 1H), 11.06 (s, 1H); 13C-NMR (DMSO-d6) δ: 39.44, 55.84, 111.90, 111.93, 112.47, 112.62, 119.20, 120.46, 120.99, 124.67, 129.65, 132.11, 135.41, 135.97, 148.65, 150.47, 151.03, 156.45, 163.91, 164.57, 166.95; HRMS (ESI) m/z: Calcd. for C22H17ClN4O3S2 485.0504; Found: 485.0491 (M-H+).

N-(2-(2-(2-Chloro-4-methylpyridin-3-ylamino)-2-oxoethylthio)benzo[d]thiazol-6-yl)-2-methoxybenzamide (7r). Yellow powder, yield 77%, purity 90.0%, mp 166.5–168.1 °C; 1H-NMR (DMSO-d6) δ: 2.22 (s, 3H), 3.90 (s, 3H), 4.41 (s, 2H), 7.08 (t, J = 7.2 Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 4.8 Hz, 1H), 7.52 (t, J = 7.8 Hz, 1H), 7.64 (d, J = 7.2 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 8.8 Hz, 1H), 8.19 (d, J = 4.8 Hz, 1H), 8.55 (s, 1H), 10.24 (s, 1H), 10.37 (s, 1H); 13C-NMR (DMSO-d6) δ: 17.84, 39.51, 55.87, 111.91, 111.97, 119.22,120.49, 120.92, 124.76, 129.65, 130.51, 132.11, 135.47, 135.96, 147.05, 148.70, 148.76, 156.48, 164.03, 164.61, 165.67; HRMS (ESI) m/z: Calcd. for C23H19ClN4O3S2 500.0123; Found: 500.0124 (M-H+).

2-Methoxy-N-(2-(2-oxo-2-(pyrimidin-2-ylamino)ethylthio)benzo[d]thiazol-6-yl)benzamide (7s). Yellow powder, yield 70%, purity 90.0%, mp 169.2–171.8 °C; 1H-NMR (DMSO-d6) δ: 3.90 (s, 3H), 4.46 (s, 2H), 7.08 (t, J = 7.4 Hz, 1H), 7.18–7.24 (m, 2H), 7.52 (t, J = 7.6 Hz, 1H), 7.64–7.69 (m, 2H), 7.78 (d, J = 8.8 Hz, 1H), 8.54 (s, 1H), 8.69 (d, J = 8.4 Hz, 2H), 10.35 (s, 1H), 11.04 (s, 1H); 13C-NMR (DMSO-d6) δ: 39.46, 55.86, 111.84, 111.95, 119.17, 120.48, 121.00, 124.67, 129.68, 132.12, 135.38, 135.90, 148.80, 156.48, 157.37, 158.41, 164.45, 164.55, 166.18; HRMS (ESI) m/z: Calcd. for C21H17N5O3S2 452.0846; Found: 452.0821 (M-H+).

2-Methoxy-N-(2-(2-oxo-2-(thiazol-2-ylamino)ethylthio)benzo[d]thiazol-6-yl)benzamide (7t). Yellow powder, yield 68%, purity 92.0%, mp 237.7–239.1 °C; 1H-NMR (DMSO-d6) δ: 3.90 (s, 3H), 4.47 (s, 2H), 7.08 (t, J = 7.4 Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 3.6 Hz, 1H), 7.52 (t, J = 6.4 Hz, 2H), 7.64–7.69 (m, 2H), 7.77 (d, J = 8.8 Hz, 1H), 8.54 (s, 1H), 10.36 (s, 1H), 12.55 (s, 1H); 13C-NMR (DMSO-d6) δ: 39.37, 56.37, 112.39, 112.47, 114.32, 119.71, 120.98, 121.54, 125.24, 130.15, 132.60, 135.95, 136.50, 138.24, 149.19, 156.97, 158.27, 164.38, 165.07, 166.18; HRMS (ESI) m/z: Calcd. for C20H16N4O3S3 457.0458; Found: 457.0457 (M-H+).

 3.5. Cell Culture

Cell lines HepG2, HeLa, HCT116, SW620, SKOV-3 and MDA-MB-468 were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (10 mg/L). Cell lines BT474, A431 and SKRB-3 were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 20% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (10 mg/L). Cell lines H460, PC-3, A549, SW480, and BxPC-3 were maintained in RPMI 1640 containing 10% FBS, penicillin (100 U/mL) and streptomycin (10 mg/L). Cells were grown in a 5% CO2 incubator at 37 °C.

 3.6. Cell Proliferation Assay (MTT Assay)

Cells (3–5 × 103/well) were seeded in 200 µL of medium/well in 96-well plates (Costar Corning, Rochester, NY, USA) and cultured for 24 h. The compounds dissolved in dimethylsulfoxide (DMSO) were added to final concentration of 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, respectively. Compounds 1 and 2 were used as positive control. After 48 h exposure, a volume of 10 μL of 10 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added per well and incubated for another 4 h at 37 °C, then the supernatant fluid was removed and DMSO (150 μL) was added 150 μL/well to dissolve formazan crystals for 15–20 min. The light absorptions (OD) were measured at 570 nm with SpectraMAXM5 microplate spectrophotometer (Molecular Devices). The effect of compounds on tumor cells viability was expressed by IC50 of each cell line. Values shown are the % viability vs. ctrl + SD, n = six independent experiments in triplicate.

 3.7. Apoptosis Analysis by Flow Cytometry (FCM)

Cells were seeded into 1 mL of medium/well cell in 6-well plates (Costar Corning) culture bottles at 10 × 106 cells 24 h before treatment. Then cells were treated with compound 7e for 24 h: 0 µM (control), 0.625 µM, 1.25 µM, 2.5 µM. After 24 h incubation, floating and adherent cells were collected, washed three times with PBS (pH 7.4) and fixed for 24 h with cool alcohol at 4 °C. 1 mL cell suspension (106 /mL) was washed three times with cooled PBS, treated with RNase for 30 min at 37 °C, stained it with PI for 30 min at 37 °C in a dark environment, and taken for flow cytometry analysis.

 4. Conclusions

In conclusion, a series of novel benzothiazole-2-thiol derivatives were synthesized and their anti-proliferative activities were evaluated in vitro. The results showed that the pyridinyl-2-amine linked benzothiazole-2-thiol compounds 7d, 7e, 7f and 7i exhibited increased anticancer activities compared with compounds 1 and 2 on the three human cancer cells (SW480, HeLa and HepG2). Further studies showed that they displayed potent and broad-spectrum anti-proliferative activities against other human cancer cell lines. Among these compounds, 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). The results of flow cytometry analysis indicated that compound 7e induce apoptosis in HepG2 cancer cells on a concentration-dependent manner. These data suggested that compound 7e may be powerful tumor growth inhibitors via apoptosis as novel anticancer agents and be worth being further investigated as a potential of anticancer agent.

 Acknowledgements

This work was supported by National Science and Technology Major Project of China (2009ZX09103-132). The authors gratefully thank Yong-Qiu Mao (State Key Laboratory of Biotherapy, Huaxi Hospital, Sichuan University) for helpful technical assistance and discussion in the flow cytometry analysis.

 References and Notes Quiroga J. Hernandez P. Insuasty B. Abonıa R. Cobo J. Sanchez A. Nogueras M. Low J.N. Control of the reaction between 2-aminobenzothiazoles and Mannich bases. Synthesis of pyrido[2,1-b][1,3]benzothiazoles versus [1,3]benzothiazolo[2,3-b]quinazolines J. Chem. Soc. Perkin Trans. 2002 1 555 559 Kok S.H.L. Chui C.H. Lam W.S. Chen J. Lau F.Y. Wong R.S.M. Cheng G.Y.M. Lai P.B.S. Leung R.W.T. Tang J.C.O. Synthesis and structure evaluation of a novel cantharimideand its cytotoxicity on SK-Hep-1 hepatoma cells Bioorg. Med. Chem. Lett. 2007 17 1155 1159 10.1016/j.bmcl.2006.12.03917240140 Kok S.H.L. Chui C.H. Lam W.S. Chen J. Lau F.Y. Wong R.S.M. Cheng G.Y.M. Tang W.K. Teo I.T.N. Cheung F. Apoptogenic activity of a synthetic cantharimidein leukaemia: Implication on its structural activity relationship Int. J. Mol. Med. 2006 18 1217 1221 17089029 Kok S.H.L. Gambari R. Chui C.H. Yuen M.C.W. Lin E. Wong R.S.M. Lau F.Y. Cheng G.Y.M. Lam W.S. Chan S.H. Synthesis and anti-cancer activity of benzothiazole containing phthalimide on human carcinoma cell lines Bioorg. Med. Chem. 2008 16 3626 3631 10.1016/j.bmc.2008.02.00518295491 Song E.Y. Kau R.N. Park M.Y. Jin Y.L. Lee K. Kim G. Lee K.Y. Yang J.S. Shin J.H. Nam K.Y. Synthesisof amide and urea derivatives of benzothiazole as Raf-1 inhibitor Eur. J. Med. Chem. 2008 43 1519 1524 10.1016/j.ejmech.2007.10.00818023932 Wang Z. Shi X.H. Wang J. Zhou T. Xu Y.Z. Huang T.T. Li Y.F. Zhao Y.L. Yang L. Yang S.Y. Synthesis, structure-activityrelationships and preliminary antitumorevaluation of benzothiazole-2-thiol derivatives as novel apoptosis inducers Bioorg. Med. Chem. Lett. 2011 21 1097 1101 10.1016/j.bmcl.2010.12.12421262571 Guo X.Z. Shi L. Wang R. Liu X.X. Li B.G. Lu X.X. Synthesis and biological activities of novel nonpeptide angiotensin II receptor antagonists based on benzimidazole derivatives bearing a heterocyclic ring Bioorg. Med. Chem. Lett. 2008 16 10301 10310 10.1016/j.bmc.2008.10.040 Liu Y. Gerald N.L. Activationofheterocyclic amines by combinations of prostaglandin Hsynthase-1 and -2 with N-acetyltransferase 1 and 2 Cancer Lett. 1998 133 115 123 10.1016/S0304-3835(98)00217-1 Peng F. Wei Y.Q. Tian L. Yang L. Zhao X. Lu Y. Mao Y.Q. Kan B. Lei S. Wang G.S. Induction of apoptosis by norcantharidin in human colorectal carcinoma cell lines: Involvement of the CD95 receptor/ligand Cancer Res. Clin. Oncol. 2002 128 223 230 10.1007/s00432-002-0326-5 Baraldi P.G. Preti D. Tabrizi M.A. Fruttarolo F. Saponaro G. Baraldi S. Romagnoli R. Moorman A.R. Gessi S. Varani K. Hybrid molecules between distamycin A and active moieties of antitumor agents Bioorg. Med. Chem. 2007 15 17 35 10.1016/j.bmc.2006.07.00417081759 Musso D.L. Cochran F.R. Kelley J.L. McLean E.W. Selph J.L. Rigdon G.C. Orr G.F. Davis R.G. Cooper B.R. Styles V.L. Indanylidenes. 1. Design and synthesis of (E)-2-(4,6-difluoro-1-indanylidene)acetamide, a potent, centrally acting muscle relaxant with antiinflammatory and analgesic activity J. Med. Chem. 2003 46 399 408 10.1021/jm020067s12540239

Sample Availability: Samples of the compounds 7a–t are available from the authors.