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Synthesis and in Vitro Antiproliferative Evaluation of Novel Hybrids from 1,3,4-Thiadiazole and Benzisoselenazolone
Fen JingXiaoyun FuSha LiBaolin LiJijun ZhaoXuefeng WangYuming LiuBaoquan Chen
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2015 Volume 63 Issue 6 Pages 431-437

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Abstract

Novel hybrids from 1,3,4-thiadiazole and benzisoselenazolone were designed, synthesized and evaluated for their in vitro antiproliferative activities by CCK-8 assay against three types of human cancer cell lines, SMMC-7721, MCF-7 and A549 cells. The preliminary bioassay results demonstrated that all tested compounds 4a–p showed potent antiproliferative activities, and some compounds exhibited better effects than positive control ethaselen and 5-fluorouracil (5-FU) against various cancer cell lines. Furthermore, compound 4g showed significant antiproliferative activities against SMMC-7721 cells with an IC50 value of 2.08 µM. Compounds 4b and 4m displayed highly effective biological activities against MCF-7 cells with an IC50 values of 2.03 and 2.06 µM, respectively. Compound 4i exhibited the best inhibitory effect against A549 cells with an IC50 value of 1.03 µM.

Cancer is considered to be second most common cause of deaths after cardiovascular diseases in the world. Despite significant progress has been achieved in anticancer therapy, the management of malignancies in humans still constitutes one of the most intractable worldwide health problems.1) Therefore, it is important to identify effective drugs and new targets for the treatment of cancer. Thioredoxin (Trx), thioredoxin reductase (TrxR) and nicotinamide adenine dinucleotide phosphate (NADPH) comprise a thioredoxin system that is involved in many aspects of tumor physiology such as proliferation, apoptosis and metastasis, and has become a novel attractive target for development of new cancer therapeutics.27) Recently, reports of selenium-containing heterocyclic compounds have gradually increased because of their pharmaceutical applications, and have become hot spots in the studies of tumor prevention and therapy.810) Among them, functionalized benzisoselenazolone moieties are present in many pharmacologically active substances. The prominent examples are ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one, Fig. 1] and ethaselen [1,2-(bis-1,2-benzisoselenazol-3(2H)-one) ethane, Fig. 1], which possess potent antioxidant, anti-inflammatory, anticancer and cholinesterase inhibitory activities.4,1114) Particularly, as a novel organoselenium compound targeting thioredoxin reductase (TrxR), ethaselen has been reported to exhibit broad spectrum of antitumor effects with slight toxicity, and has now entered Phase I clinical trials in China.46)

Fig. l. Structures of Ebselen (1) and Ethaselen (2)

1,3,4-Thiadiazole is a privileged scaffold incorporated in many compounds presenting a wide range of pharmacological activities, such as antioxidant,15,16) antibacterial,17,18) antidepressant,19,20) antidiabetic,21,22) antifungal,23,24) anticonvulsant,25,26) antiinflammatory,27,28) antileishmanial29) and acetylcholinesterase inhibitory properties.30) In particular, 1,3,4-thiadiazole derivatives are of significant interested because of their anticancer activities.3135)

Recently, we have reported the synthesis of 1,3,4-thiadiazole derivatives bearing benzisoselenazolone moiety, which contained substituted benzoylmethylthio and phenyl groups on the 1,3,4-thiadiazole ring. The preliminary bioassay results demonstrated that most of compounds showed potent antiproliferative activities against various cancer cell lines, and some compounds revealed stronger antiproliferative effect than positive control ebselen and ethaselen.36,37) In order to obtain compounds with better antiproliferative activities. We report herein the synthesis of some new hybrids from 1,3,4-thiadiazole and benzisoselenazolone. Meanwhile, their in vitro antiproliferative activities were evaluated against SMMC-7721 (human liver cancer cell), MCF-7 (human breast cancer cell) and A549 (human lung cancer cell) cells with the ultimate aim of developing novel potent antitumor agents.

Results and Discussion

Chemistry

As depicted in Chart 1, sixteen hybrids from 1,3,4-thiadiazole and benzisoselenazolone were synthesized and reported for the first time. The preparation of 4-chloromethylbenzoyl chloride 2ap was carried out by reported literature method.34) The reaction of commercially available 2-amino-5-mercapto-1,3,4-thiadiazole with compounds 2a–p in the presence of NaOH in water and ethanol at room temperature yielded the desired intermediates 3a–p in 76–89% yields. 2-Chloroselenobenzoyl chloride was prepared according to reported literature method.4) Finally, the target compounds 4a–p were successfully obtained via the condensation of intermediates 3ap and 2-chloroselenobenzoyl chloride in the presence of triethylamine in dry acetonitrile at room temperature. All newly synthesized compounds 4a–p were purified by silica gel column chromatography and their structures were characterized by IR, 1H-NMR, electrospray ionization (ESI)-MS and elemental analysis.

Chart 1. Synthesis of Target Compounds 4a–p

Reaction conditions and reagents: a) 4-Chloromethylbenzoyl chloride, Et3N, CH2Cl2, rt, 4–6 h; b) 2-Amino-5-mercapto-1,3,4-thiadiazole, NaOH, H2O, EtOH, rt, 7–8 h, 76–89%; c) 2-Chloroselenobenzoyl chloride, Et3N, dry CH3CN, rt, 8–10 h, 45–58%.

Biological Evaluation

The antiproliferative activities of the newly synthesized compounds 4a–p were tested against SMMC-7721, MCF-7 and A549 cell lines using CCK-8[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt] assay, which was used as a substitute for 3-(4,5-dimethylthiazol)-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay. Inhibition of cell proliferation by these active compounds at various concentrations were measured, and their IC50 (the concentration that causes a 50% cell proliferation inhibition) values were calculated and summarized in Table 1. Ethaselen and 5-fluorouracil were used as positive control.

As shown in Table 1, this class of compounds revealed stronger antiproliferative effect than previous reported compounds on the whole.36,37) Most of the newly synthesized compounds 4ap exhibited very good antiproliferative activities against SMMC-7721 cells. Especially noteworthy is compound 4g bearing 4-methoxy substituent at the phenyl ring, showed significant antiproliferative activity with IC50 value of 2.08 µM. Except compounds 4l and 4p possessing 4-chloro and trifluoromethyl substituents, which displayed moderate antiproliferative activities, the other compounds displayed higher activities than positive control ethaselen with IC50 values ranging from 2.08 to 9.21 µM. The substituent groups of phenyl ring on the 1,3,4-thiadiazole play important roles in the potency of biologically active compounds. As compared with compound 4a which has no substituent at the phenyl ring, some compounds showed enhanced antiproliferative effects. But, the electronic effects of substituents on antiproliferative activity against SMMC-7721 cells did not show apparent regularity. In MCF-7 cells, with the exception of compounds 4d, 4l and 4n containing 4-methyl, 4-chloro and 2-nitro substituents, which exhibited moderate antiproliferative activity, the other compounds displayed very good antiproliferative effects with IC50 values ranging from 2.03 to 4.16 µM. Meanwhile all compounds 4a–p showed much better potency than positive control ethaselen, and the majority of them exbibited better activities than 5-fluorouracil. As compared with compound 4a, the majority of substituent groups resulted in a increase in the potency of compounds. Among them, 2-methyl and 4-bromo substituted derivatives, compounds 4b and 4m displayed highly effective biological activities against MCF-7 cells with IC50 values of 2.03 and 2.06 µM, respectively. In A549 cells, all compounds 4ap showed higher activities than positive control 5-fluorouracil. Whereas, most of them, including compound 4a, displayed lower antiproliferative activities than ethaselen. With the exception of compound 4a, the other compounds showed very good antiproliferative activities, and compound 4i bearing 4-fluoro group, exhibited the best inhibitory effect with IC50 value of 1.03 µM. Meanwhile, compounds 4bp carring various substituents all displayed increased activities in comparison to compound 4a. These results suggest that the presence of substituent groups at the phenyl ring is favorable for improving the potency of compounds against A549 cells. Therefore, it is necessary that the further investigation is carried out through structural transformation.

Table 1. The IC50 Values of Compounds 4ap against Three Human Cancer Cell Lines
Compd.IC50 (µM) a)
SMMC-7721MCF-7A549
Ethaselen b)10.68±0.5240.52±2.883.26±0.05
5-FU b)5.62±0.2814.26±0.668.13±0.34
4a4.75±0.126.04±0.4210.54±0.56
4b3.93±0.212.03±0.045.79±0.29
4c4.49±0.144.16±0.264.74±0.24
4d9.21±0.1116.58±1.276.73±0.33
4e6.15±0.193.38±0.122.58±0.09
4f6.04±0.092.87±0.081.58±0.03
4g2.08±0.082.88±0.173.41±0.13
4h3.39±0.253.89±0.242.33±0.03
4i4.19±0.063.38±0.141.03±0.02
4j2.64±0.182.33±0.073.94±0.20
4k3.42±0.272.64±0.164.49±0.16
4l20.15±1.7815.17±1.397.26±0.44
4m3.14±0.092.06±0.041.60±0.07
4n5.24±0.2511.35±1.034.99±0.22
4o3.98±0.153.69±0.081.78±0.06
4p23.12±1.992.86±0.053.37±0.10

a) Each experiment was independently performed three times and expressed as means±standard deviation (S.D.). b) Used as positive control.

Conclusion

In summary, a series of novel hybrids from 1,3,4-thiadiazole and benzisoselenazolone were designed, synthesized and evaluated for their in vitro antiproliferative activities against three types of human cancer cell lines. Some of the compounds inhibited the proliferation better than positive control ethaselen and 5-fluorouracil. In particular, compound 4g showed significant antiproliferative activities against SMMC-7721cells with IC50 value of 2.08 µM. Compounds 4b and 4m displayed highly effective biological activities against MCF-7 cells with IC50 values of 2.03 and 2.06 µM, respectively. Compound 4i exhibited the best inhibitory effect against A549 cells with IC50 value of 1.03 µM. Therefore, the results will be significant in the development of potent antiproliferative agents.

Experimental

Chemistry

Melting points were determined by a X-6 microscope melting point apparatus and are uncorrected. IR spectra were recorded in KBr pellets on a Nicolet Avatar 370 spectrometer. NMR spectra were performed on a Bruker Avance III 400 MHz spectrometer using DMSO-d6 as solvent and tetramethylsilane as internal standard. Chemical shifts are showed in δ values (ppm) and the coupling constants are expressed in J values (Hz). Mass spectra were taken with a Waters Xevo G2 QTof (ESI) mass spectrometer. Elemental analyses (CHN) were performed on a Flash EA1112 instrument. Silica gel (200–300 mesh) for column chromatography and silica GF254 for TLC were produced by Qing dao Marine Chemical Company (China). Unless otherwise noted, all solvents and reagents were obtained from commercial suppliers and used without further purification.

General Procedure for the Synthesis of 3a–p

A solution of sodium hydroxide (6 mmol) in water (10 mL) was added to a mixture of 2-amino-5-mercapto-1,3,4-thiadiazole (6 mmol), appropriate 4-chloromethyl-N-(substituted-phenyl)-benzamide (2a–p, 5 mmol) and ethanol (15 mL). The mixture was stirred for 7–8 h at room temperature (the end of reaction was monitored by TLC), After completion, the mixture was poured into water. The resulting precipitate was collected by filtration, washed well with water and further purified by recrystallization from ethanol to afford target compounds.

2-Amino-5-[4-(phenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3a)

White crystals; mp 223.8–225.6°C; Yield 88%; IR (KBr) cm1: 1638, 1401, 1108, 995, 616, 419; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.23 (s, 1H, Ar-NH), 7.89–7.91 (d, J=7.7 Hz, 2H, Ar-H), 7.76–7.78 (d, J=7.7 Hz, 2H, Ar-H), 7.48–7.50 (d, J=7.7 Hz, 2H, Ar-H), 7.35 (t, J=8.0 Hz, 2H, Ar-H), 7.31 (s, 2H, NH2), 7.10 (t, J=6.8 Hz, 1H, Ar-H), (t, J=6.8 Hz, 1H, Ar-H), 4.38 (s, 2H, CH2); ESI-MS m/z: 343.4 [M+H]+.

2-Amino-5-[4-(2-methylphenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3b)

White crystals; mp 197.0–199.2°C; Yield 87%; IR (KBr) cm1: 1637, 1399, 1107, 991, 617, 419; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 9.87 (s, 1H, Ar-NH), 7.92–7.94 (d, J=7.9 Hz, 2H, Ar-H), 7.48–7.50 (d, J=7.9 Hz, 2H, Ar-H), 7.32–7.34 (d, J=9.1 Hz, 1H, Ar-H), 7.32 (s, 2H, NH2), 7.26–7.28 (d, J=7.1 Hz, 1H, Ar-H), 7.22 (t, J=7.2 Hz, 1H, Ar-H), 7.17 (t, J=7.2 Hz, 1H, Ar-H), 4.38 (s, 2H, CH2), 2.23 (s, 3H, CH3); ESI-MS m/z: 357.5 [M+H]+.

2-Amino-5-[4-(3-methylphenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3c)

White crystals; mp 215.8–217.8°C; Yield 85%; IR (KBr) cm1: 1614, 1401, 1133, 991, 617, 418; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.15 (s, 1H, Ar-NH), 7.88–7.90 (d, J=8.2 Hz, 2H, Ar-H), 7.61 (s, 1H, Ar-H), 7.55–7.57 (d, J=8.2 Hz, 1H, Ar-H), 7.48–7.50 (d, J=8.2 Hz, 2H, Ar-H), 7.31 (s, 2H, NH2), 7.23 (t, J=7.7 Hz, 1H, Ar-H), 6.91–6.93 (d, J=7.7 Hz, 1H, Ar-H), 4.38 (s, 2H, CH2), 2.31 (s, 3H, CH3); ESI-MS m/z: 357.5 [M+H]+.

2-Amino-5-[4-(4-methylphenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3d)

White crystals; mp 244.2–246.8°C; Yield 84%; IR (KBr) cm1: 1641, 1402, 1133, 994, 617, 419; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.15 (s, 1H, Ar-NH), 7.88–7.90 (d, J=8.2 Hz, 2H, Ar-H), 7.63–7.66 (d, J=8.4 Hz, 2H, Ar-H), 7.48–7.50 (d, J=8.2 Hz, 2H, Ar-H), 7.31 (s, 2H, NH2), 7.14–7.16 (d, J=8.4 Hz, 2H, Ar-H), 4.38 (s, 2H, CH2), 2.28 (s, 3H, CH3); ESI-MS m/z: 357.5 [M+H]+.

2-Amino-5-[4-(2-methoxyphenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3e)

White crystals; mp 192.1–193.0°C; Yield 87%; IR (KBr) cm1: 1640, 1401, 1132, 994, 617, 419; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 9.42 (s, 1H, Ar-NH), 7.90–7.92 (d, J=8.3 Hz, 2H, Ar-H), 7.76 (dd, J=1.5, 7.9 Hz, 1H, Ar-H), 7.48–7.50 (d, J=8.3 Hz, 2H, Ar-H), 7.31 (s, 2H, NH2), 7.19 (dt, J=1.5, 8.2 Hz, 1H, Ar-H), 7.10 (dd, J=1.2, 8.2 Hz, 1H, Ar-H), 6.97 (dt, J=1.2, 7.7 Hz, 1H, Ar-H), 4.38 (s, 2H, CH2), 3.84 (s, 3H, CH3); ESI-MS m/z: 373.5 [M+H]+.

2-Amino-5-[4-(3-methoxyphenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3f)

White crystals; mp 189.0–190.8°C; Yield 85%; IR (KBr) cm1: 1637, 1401, 1134, 995, 617, 419; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.20 (s, 1H, Ar-NH), 7.88–7.90 (d, J=8.3 Hz, 2H, Ar-H), 7.48–7.51 (d, J=8.3 Hz, 2H, Ar-H), 7.47 (t, J=2.1 Hz, 1H, Ar-H), 7.36 (dd, J=1.0, 8.1 Hz, 1H, Ar-H), 7.31 (s, 2H, NH2), 7.25 (t, J=8.1 Hz, 1H, Ar-H), 6.68 (dd, J=2.1, 8.1 Hz, 1H, Ar-H), 4.38 (s, 2H, CH2), 3.75 (s, 3H, CH3); ESI-MS m/z: 373.5 [M+H]+.

2-Amino-5-[4-(4-methoxyphenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3g)

White crystals; mp 246.5–248.5°C; Yield 89%; IR (KBr) cm1: 1641, 1401, 1107, 821, 617, 419; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.11 (s, 1H, Ar-NH), 7.88–7.90 (d, J=8.2 Hz, 2H, Ar-H), 7.65 (t, J=2.0 Hz, 1H, Ar-H), 7.68 (t, J=2.0 Hz, 1H, Ar-H), 7.47–7.49 (d, J=8.2 Hz, 2H, Ar-H), 7.31 (s, 2H, NH2), 6.94 (t, J=2.1 Hz, 1H, Ar-H), 6.91 (t, J=2.1 Hz, 1H, Ar-H), 4.37 (s, 2H, CH2), 3.75 (s, 3H, CH3); ESI-MS m/z: 373.5 [M+H]+.

2-Amino-5-[4-(2-fluorophenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3h)

White crystals; mp 203.5–205.7°C; Yield 80%; IR (KBr) cm1: 1637, 1385, 1107, 996, 617, 419; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 9.42 (s, 1H, Ar-NH), 7.90–7.92 (d, J=8.2 Hz, 2H, Ar-H), 7.75 (dd, J=1.3, 7.8 Hz, 1H, Ar-H), 7.48–7.50 (d, J=8.2 Hz, 2H, Ar-H), 7.31 (s, 2H, NH2), 7.19 (dt, J=1.3, 8.3 Hz,, 1H, Ar-H), 7.10 (dd, J=1.0, 7.4 Hz, 1H, Ar-H), 6.97 (dt, J=1.0, 7.7 Hz, 1H, Ar-H), 4.38 (s, 2H, CH2); ESI-MS m/z: 361.4 [M+H]+.

2-Amino-5-[4-(4-fluorophenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3i)

White crystals; mp 242.5–243.9°C; Yield 83%; IR (KBr) cm1: 1641, 1401, 1107, 996, 617, 419; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.29 (s, 1H, Ar-NH), 7.88–7.90 (d, J=8.2 Hz, 2H, Ar-H), 7.77–7.80 (m, 2H, Ar-H), 7.48–7.50 (d, J=8.2 Hz, 2H, Ar-H), 7.31 (s, 2H, NH2), 7.20 (t, J=8.9 Hz, 2H, Ar-H), 4.38 (s, 2H, CH2); ESI-MS m/z: 361.4 [M+H]+.

2-Amino-5-[4-(2-chlorophenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3j)

White crystals; mp 197.1–199.6°C; Yield 88%; IR (KBr) cm1: 1639, 1400, 1132, 995, 617, 419; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.05 (s, 1H, Ar-NH), 7.93–7.95 (d, J=8.3 Hz, 2H, Ar-H), 7.59 (dd, J=1.5, 7.9 Hz, 1H, Ar-H), 7.56 (dd, J=1.4, 8.0 Hz, 1H, Ar-H), 7.50–7.52 (d, J=8.3 Hz, 2H, Ar-H), 7.39 (dt, J=1.5, 7.5 Hz, 1H, Ar-H), 7.30(dt, J=1.4, 7.9 Hz, 1H, Ar-H), 7.31 (s, 2H, NH2),4.39 (s, 2H, CH2); ESI-MS m/z: 377.4 [M+H]+.

2-Amino-5-[4-(3-chlorophenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3k)

White crystals; mp 219.7–221.5°C; Yield 84%; IR (KBr) cm1: 1650, 1401, 1107, 995, 617, 419; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.39 (s, 1H, Ar-NH), 7.96 (t, J=1.9 Hz, 1H, Ar-H), 7.89–7.91 (d, J=8.2 Hz, 2H, Ar-H), 7.70 (dd, J=1.3, 7.1 Hz, 1H, Ar-H), 7.50–7.52 (d, J=8.2 Hz, 2H, Ar-H), 7.39 (t, J=8.2 Hz, 1H, Ar-H), 7.31 (s, 2H, NH2), 7.16 (dd, J=1.3, 7.9 Hz, 1H, Ar-H), 4.38 (s, 2H, CH2); ESI-MS m/z: 377.4 [M+H]+.

2-Amino-5-[4-(4-chlorophenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3l)

White crystals; mp 230.4–232.5°C; Yield 83%; IR (KBr) cm1: 1653, 1399, 1107, 994, 618, 413; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.36 (s, 1H, Ar-NH), 7.88–7.90 (d, J=8.0 Hz, 2H, Ar-H), 7.80–7.82 (d, J=8.1 Hz, 2H, Ar-H), 7.49–7.50 (d, J=8.0 Hz, 2H, Ar-H), 7.40–7.42 (d, J=8.1 Hz, 2H, Ar-H), 7.31 (s, 2H, NH2), 4.38 (s, 2H, CH2); ESI-MS m/z: 377.4 [M+H]+.

2-Amino-5-[4-(4-bromophenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3m)

White crystals; mp 247.9–249.8°C; Yield 79%; IR (KBr) cm1: 1641, 1401, 1133, 995, 617, 419; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.11 (s, 1H, Ar-NH), 7.88–7.90 (d, J=8.2 Hz, 2H, Ar-H), 7.65–7.68 (d, J=9.0 Hz, 2H, Ar-H), 7.47–7.49 (d, J=8.2 Hz, 2H, Ar-H), 7.31 (s, 2H, NH2), 6.91–6.94 (d, J=9.0 Hz, 2H, Ar-H), 4.37 (s, 2H, CH2); ESI-MS m/z: 421.3 [M+H]+.

2-Amino-5-[4-(2-nitrophenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3n)

Pale yellow crystals; mp 191.0–192.9°C; Yield 76%; IR (KBr) cm1: 1638, 1401, 1108, 994, 618, 419; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.75 (s, 1H, Ar-NH), 8.01–8.03 (d, J=7.9 Hz, 1H, Ar-H), 7.90–7.92 (d, J=8.2 Hz, 2H, Ar-H), 7.78 (t, J=6.1 Hz, 1H, Ar-H), 7.76–7.78 (d, J=6.1 Hz, 1H, Ar-H), 7.53–7.55 (d, J=8.2 Hz, 2H, Ar-H), 7.43 (t, J=7.9 Hz, 1H, Ar-H), 7.31 (s, 2H, NH2), 4.39 (s, 2H, CH2); ESI-MS m/z: 388.4 [M+H]+.

2-Amino-5-[4-(4-nitrophenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3o)

Pale yellow crystals; mp 229.0–231.2°C; Yield 79%; IR (KBr) cm1: 1653, 1385, 1108, 995, 637, 413; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.80 (s, 1H, Ar-NH), 8.26–8.29 (d, J=9.2 Hz, 2H, Ar-H), 8.05–8.07 (d, J=9.2 Hz, 2H, Ar-H), 7.92–7.94 (d, J=8.3 Hz, 2H, Ar-H), 7.52–7.54 (d, J=8.3 Hz, 2H, Ar-H), 7.31 (s, 2H, NH2), 4.40 (s, 2H, CH2); ESI-MS m/z: 388.4 [M+H]+.

2-Amino-5-[4-(trifluoromethylphenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3p)

White crystals; mp 249.7–251.9°C; Yield 81%; IR (KBr) cm1: 1641, 1402, 1071, 901, 618, 419; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.36 (s, 1H, Ar-NH), 7.88–7.90 (d, J=8.3 Hz, 2H, Ar-H), 7.75–7.77 (d, J=8.8 Hz, 2H, Ar-H), 7.53–7.55 (d, J=8.8 Hz, 2H, Ar-H), 7.49–7.51 (d, J=8.3 Hz, 2H, Ar-H), 7.31 (s, 2H, NH2), 4.38 (s, 2H, CH2); ESI-MS m/z: 411.4 [M+H]+.

General Procedure for the Synthesis of 4a–p

A solution of 2-chloroselenobenzoyl chloride (5.0 mmol) in dry acetonitrile (25 mL) was added dropwisely to a stirred solution of the corresponding 2-amino-5-[4-(substituted-phenylcarbamoyl)-benzylthio]-1,3,4-thiadiazole (3a–p, 5 mmol) and triethylamine (6 mmol) in dry acetonitrile (25 mL). The reaction mixture was stirred for 8–10 h at room temperature. After completion, the solvent was evaporated under reduced pressure and the residue was washed with water. The insoluble solid was collected and purified by silica gel column chromatography with petroleum ether (60–90°C)/ethyl acetate (1 : 3, volume ratio) as eluent to afford the desired products.

2-{2-[4-(Phenylcarbamoyl)-benzylthio]-1,3,4-thiadiazol-5-yl}-1,2-benzisoselenazol-3(2H)-one (4a)

Yellow solid; mp 276.0–277.9°C; Yield 49%; IR (KBr) cm1: 1639, 1443, 1387, 1020, 752, 623; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.23 (s, 1H, Ar-NH), 8.10–8.12 (d, J=8.1 Hz, 1H, Ar-H), 7.99–8.01 (d, J=7.7 Hz, 1H, Ar-H), 7.90–7.92 (d, J=8.0 Hz, 2H, Ar-H), 7.78 (t, J=8.0 Hz, 1H, Ar-H), 7.75–7.77 (d, J=8.0 Hz, 2H, Ar-H), 7.59–7.61 (d, J=8.0 Hz, 2H, Ar-H), 7.53 (t, J=7.5 Hz, 1H, Ar-H), 7.34 (t, J=7.8 Hz, 2H, Ar-H), 7.10 (t, J=7.4 Hz, 1H, Ar-H), 4.62 (s, 2H, CH2); ESI-MS m/z: 525.4 [M+H]+; Anal. Calcd for C23H16N4O2S2Se: C, 52.77; H, 3.08; N, 10.70. Found: C, 52.65; H, 3.04; N, 10.77.

2-{2-[4-(2-Methylphenylcarbamoyl)-benzylthio]-1,3,4-thiadiazol-5-yl}-1,2-benzisoselenazol-3(2H)-one (4b)

Yellow solid; mp 255.5–257.5°C; Yield 50%; IR (KBr) cm1: 1638, 1457, 1396, 1019, 750, 625; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 9.86 (s, 1H, Ar-NH), 8.10–8.12 (d, J=8.1 Hz, 1H, Ar-H), 7.99–8.01 (d, J=7.6 Hz, 1H, Ar-H), 7.93–7.95 (d, J=8.1 Hz, 2H, Ar-H), 7.78 (t, J=7.5 Hz, 1H, Ar-H), 7.58–7.60 (d, J=8.1 Hz, 2H, Ar-H), 7.53 (t, J=7.5 Hz, 1H, Ar-H), 7.32–7.34 (d, J=7.4 Hz, 1H, Ar-H), 7.26–7.27 (d, J=7.0 Hz, 1H, Ar-H), 7.21 (t, J=7.4 Hz, 1H, Ar-H), 7.16 (t, J=7.4 Hz, 1H, Ar-H), 4.62 (s, 2H, CH2), 2.22 (s, 3H, CH3); ESI-MS m/z: 539.5 [M+H]+; Anal. Calcd for C24H18N4O2S2Se: C, 53.63; H, 3.38; N, 10.42. Found: C, 53.50; H, 3.34; N, 10.38.

2-{2-[4-(3-Methylphenylcarbamoyl)-benzylthio]-1,3,4-thiadiazol-5-yl}-1,2-benzisoselenazol-3(2H)-one (4c)

Yellow solid; mp 242.3–243.8°C; Yield 45%; IR (KBr) cm1: 1639, 1445, 1393, 1019, 735, 623; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.15 (s, 1H, Ar-NH), 8.10–8.12 (d, J=8.1 Hz, 1H, Ar-H), 7.99–8.01 (d, J=7.4 Hz, 1H, Ar-H), 7.89–7.91 (d, J=8.2 Hz, 2H, Ar-H), 7.78 (t, J=8.1 Hz, 1H, Ar-H), 7.60 (s, 1H, Ar-H), 7.58–7.60 (d, J=8.2 Hz, 2H, Ar-H), 7.53–7.55 (d, J=7.7 Hz, 1H, Ar-H), 7.53 (t, J=7.7 Hz, 1H, Ar-H), 7.22 (t, J=7.8 Hz, 1H, Ar-H), 6.91–6.92 (d, J=7.4 Hz, 1H, Ar-H), 4.62 (s, 2H, CH2), 2.30 (s, 3H, CH3); ESI-MS m/z: 539.5 [M+H]+; Anal. Calcd for C24H18N4O2S2Se: C, 53.63; H, 3.38; N, 10.42. Found: C, 53.72; H, 3.37; N, 10.34.

2-{2-[4-(4-Methylphenylcarbamoyl)-benzylthio]-1,3,4-thiadiazol-5-yl}-1,2-benzisoselenazol-3(2H)-one (4d)

Yellow solid; mp 277.3–279.2°C; Yield 53%; IR (KBr) cm1: 1635, 1401, 1108, 991, 731, 616; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.14 (s, 1H, Ar-NH), 8.13–8.15 (d, J=8.1 Hz, 1H, Ar-H), 7.98–8.00 (d, J=6.9 Hz, 1H, Ar-H), 7.89–7.91 (d, J=8.2 Hz, 2H, Ar-H), 7.77 (t, J=8.3 Hz, 1H, Ar-H), 7.62–7.64 (d, J=8.4 Hz, 2H, Ar-H), 7.58–7.60 (d, J=8.2 Hz, 2H, Ar-H), 7.53 (t, J=7.7 Hz, 1H, Ar-H), 7.13–7.15 (d, J=8.4 Hz, 2H, Ar-H), 4.62 (s, 2H, CH2), 2.28(s, 3H, CH3); ESI-MS m/z: 539.5 [M+H]+; Anal. Calcd for C24H18N4O2S2Se: C, 53.63; H, 3.38; N, 10.42. Found: C, 53.55; H, 3.41; N, 10.47.

2-{2-[4-(2-Methoxyphenylcarbamoyl)-benzylthio]-1,3,4-thiadiazol-5-yl}-1,2-benzisoselenazol-3(2H)-one (4e)

Yellow solid; mp 213.5–215.1°C; Yield 48%; IR (KBr) cm1: 1636, 1401, 1108, 994, 745, 615; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 9.42 (s, 1H, Ar-NH), 8.09–8.11 (d, J=8.1 Hz, 1H, Ar-H), 7.99–8.01 (d, J=7.5 Hz, 1H, Ar-H), 7.92–7.94 (d, J=8.1 Hz, 2H, Ar-H), 7.78–7.80 (d, J=7.9 Hz, 1H, Ar-H), 7.76 (t, J=7.1 Hz, 1H, Ar-H), 7.58–7.60 (d, J=8.1 Hz, 2H, Ar-H), 7.53 (t, J=7.4 Hz, 1H, Ar-H), 7.18 (t, J=8.2 Hz, 1H, Ar-H), 7.07–7.09 (d, J=8.1 Hz, 1H, Ar-H), 6.96 (t, J=7.7 Hz, 1H, Ar-H), 4.62 (s, 2H, CH2), 3.82 (s, 3H, CH3); ESI-MS m/z: 555.5 [M+H]+; Anal. Calcd for C24H18N4O3S2Se: C, 52.08; H, 3.28; N, 10.12. Found: C, 52.17; H, 3.26; N, 10.17.

2-{2-[4-(3-Methoxyphenylcarbamoyl)-benzylthio]-1,3,4-thiadiazol-5-yl}-1,2-benzisoselenazol-3(2H)-one (4f)

Yellow solid; mp 193.0–194.2°C; Yield 50%; IR (KBr) cm1: 1637, 1401, 1107, 994, 768, 615; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.19 (s, 1H, Ar-NH), 8.10–8.12 (d, J=8.1 Hz, 1H, Ar-H), 7.99–8.01 (d, J=7.2 Hz, 1H, Ar-H), 7.90–7.92 (d, J=8.2 Hz, 2H, Ar-H), 7.78 (dt, J=1.2, 8.2 Hz, 1H, Ar-H), 7.59–7.61 (d, J=8.2 Hz, 2H, Ar-H), 7.53 (t, J=7.4 Hz, 1H, Ar-H), 7.46 (t, J=1.8 Hz, 1H, Ar-H), 7.34–7.36 (d, J=8.8 Hz, 1H, Ar-H), 7.24 (t, J=8.1 Hz, 1H, Ar-H), 6.68 (dd, J=2.1, 8.2 Hz, 1H, Ar-H), 4.62 (s, 2H, CH2), 3.75 (s, 3H, CH3); ESI-MS m/z: 555.5 [M+H]+; Anal. Calcd for C24H18N4O3S2Se: C, 52.08; H, 3.28; N, 10.12. Found: C, 52.22; H, 3.31; N, 10.06.

2-{2-[4-(4-Methoxyphenylcarbamoyl)-benzylthio]-1,3,4-thiadiazol-5-yl}-1,2-benzisoselenazol-3(2H)-one (4g)

Yellow solid; mp 275.1–277.7°C; Yield 49%; IR (KBr) cm1: 1639, 1401, 1106, 994, 735, 623; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.12 (s, 1H, Ar-NH), 8.10–8.12 (d, J=8.1 Hz, 1H, Ar-H), 7.99–8.01 (d, J=7.2 Hz, 1H, Ar-H), 7.89–7.91 (d, J=8.3 Hz, 2H, Ar-H), 7.77 (t, J=7.5 Hz, 1H, Ar-H), 7.64–7.67 (d, J=9.0 Hz, 2H, Ar-H), 7.47–7.49 (d, J=8.3 Hz, 2H, Ar-H), 7.53 (t, J=7.7 Hz, 1H, Ar-H), 6.92–6.95 (d, J=9.0 Hz, 2H, Ar-H), 4.61 (s, 2H, CH2), 3.74 (s, 3H, CH3); ESI-MS m/z: 555.5 [M+H]+; Anal. Calcd for C24H18N4O3S2Se: C, 52.08; H, 3.28; N, 10.12. Found: C, 51.92; H, 3.25; N, 10.07.

2-{2-[4-(2-Fluorophenylcarbamoyl)-benzylthio]-1,3,4-thiadiazol-5-yl}-1,2-benzisoselenazol-3(2H)-one (4h)

Yellow solid; mp 231.0–233.5°C; Yield 52%; IR (KBr) cm1: 1637, 1395, 1108, 995, 735, 615; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.11 (s, 1H, Ar-NH), 8.10–8.12 (d, J=8.1 Hz, 1H, Ar-H), 7.99–8.01 (d, J=7.6 Hz, 1H, Ar-H), 7.93–7.95 (d, J=8.1 Hz, 2H, Ar-H), 7.78 (t, J=7.5 Hz, 1H, Ar-H), 7.59–7.61 (d, J=8.1 Hz, 2H, Ar-H), 7.59 (t, J=8.1 Hz, 1H, Ar-H), 7.53 (t, J=7.7 Hz, 1H, Ar-H), 7.27–7.29 (d, J=7.3 Hz, 2H, Ar-H), 7.21 (t, J=8.0 Hz, 1H, Ar-H), 4.62 (s, 2H, CH2); ESI-MS m/z: 543.4 [M+H]+; Anal. Calcd for C23H15N4FO2S2Se: C, 51.02; H, 2.79; N, 10.35. Found: C, 50.90; H, 2.82; N, 10.41.

2-{2-[4-(4-Fluorophenylcarbamoyl)-benzylthio]-1,3,4-thiadiazol-5-yl}-1,2-benzisoselenazol-3(2H)-one (4i)

Yellow solid; mp 291.0–293.5°C; Yield 54%; IR (KBr) cm1: 1632, 1401, 1112, 991, 735, 619; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.28 (s, 1H, Ar-NH), 8.09–8.11 (d, J=8.1 Hz, 1H, Ar-H), 7.99–8.01 (d, J=7.2 Hz, 1H, Ar-H), 7.90–7.92 (d, J=8.2 Hz, 2H, Ar-H), 7.78 (t, J=8.0 Hz, 1H, Ar-H), 7.76–7.80 (m, 2H, Ar-H), 7.59–7.61 (d, J=8.2 Hz, 2H, Ar-H), 7.53 (t, J=7.4 Hz, 1H, Ar-H), 7.19 (t, J=8.9 Hz, 2H, Ar-H), 4.62 (s, 2H, CH2); ESI-MS m/z: 543.4 [M+H]+; Anal. Calcd for C23H15N4FO2S2Se: C, 51.02; H, 2.79; N, 10.35. Found: C, 50.95; H, 2.77; N, 10.30.

2-{2-[4-(2-Chlorophenylcarbamoyl)-benzylthio]-1,3,4-thiadiazol-5-yl}-1,2-benzisoselenazol-3(2H)-one (4j)

Yellow solid; mp 255.2–257.0°C; Yield 46%; IR (KBr) cm1: 1638, 1439, 1402, 1022, 755, 623; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.06 (s, 1H, Ar-NH), 8.10–8.12 (d, J=8.2 Hz, 1H, Ar-H), 7.99–8.01 (d, J=7.7 Hz, 1H, Ar-H), 7.94–7.96 (d, J=8.0 Hz, 2H, Ar-H), 7.78 (t, J=7.5 Hz, 1H, Ar-H), 7.59–7.61 (d, J=8.0 Hz, 2H, Ar-H), 7.54–7.57 (d, J=8.8 Hz, 2H, Ar-H), 7.54 (t, J=8.8 Hz, 1H, Ar-H), 7.38 (t, J=7.2 Hz, 1H, Ar-H), 7.30 (t, J=7.4 Hz, 1H, Ar-H), 4.62 (s, 2H, CH2); ESI-MS m/z: 559.4 [M+H]+; Anal. Calcd for C23H15ClN4O2S2Se: C, 49.51; H, 2.71; N, 10.04. Found: C, 49.60; H, 2.68; N, 10.10.

2-{2-[4-(3-Chlorophenylcarbamoyl)-benzylthio]-1,3,4-thiadiazol-5-yl}-1,2-benzisoselenazol-3(2H)-one (4k)

Yellow solid; mp 262.4–264.1°C; Yield 49%; IR (KBr) cm1: 1621, 1446, 1338, 1022, 735, 621; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.40 (s, 1H, Ar-NH), 8.10–8.12 (d, J=8.2 Hz, 1H, Ar-H), 7.98–8.00 (d, J=7.5 Hz, 1H, Ar-H), 7.95 (s, 1H, Ar-H), 7.90–7.92 (d, J=8.1 Hz, 2H, Ar-H), 7.77 (t, J=7.4 Hz, 1H, Ar-H), 7.68–7.70 (d, J=8.8 Hz, 1H, Ar-H), 7.60–7.62 (d, J=8.1 Hz, 2H, Ar-H), 7.53 (t, J=7.5 Hz, 1H, Ar-H), 7.38 (t, J=8.1 Hz, 1H, Ar-H), 7.15–7.17 (d, J=7.8 Hz, 1H, Ar-H), 4.62 (s, 2H, CH2); ESI-MS m/z: 559.4 [M+H]+; Anal. Calcd for C23H15ClN4O2S2Se: C, 49.51; H, 2.71; N, 10.04. Found: C, 49.64; H, 2.74; N, 9.98.

2-{2-[4-(4-Chlorophenylcarbamoyl)-benzylthio]-1,3,4-thiadiazol-5-yl}-1,2-benzisoselenazol-3(2H)-one (4l)

Yellow solid; mp 291.0–293.3°C; Yield 50%; IR (KBr) cm1: 1638, 1401, 1107, 993, 736, 617; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.35 (s, 1H, Ar-NH), 8.13–8.15 (d, J=8.2 Hz, 1H, Ar-H), 7.98–8.00 (d, J=7.9 Hz, 1H, Ar-H), 7.90–7.92 (d, J=8.0 Hz, 2H, Ar-H), 7.79–7.81 (d, J=8.5 Hz, 2H, Ar-H), 7.78 (t, J=8.5 Hz, 1H, Ar-H), 7.59–7.61 (d, J=8.0 Hz, 2H, Ar-H), 7.53 (t, J=8.0 Hz, 1H, Ar-H), 7.39–7.41 (d, J=8.5 Hz, 2H, Ar-H), 4.62 (s, 2H, CH2); ESI-MS m/z: 559.4 [M+H]+; Anal. Calcd for C23H15ClN4O2S2Se: C, 49.51; H, 2.71; N, 10.04. Found: C, 49.59; H, 2.67; N, 10.11.

2-{2-[4-(4-Bromophenylcarbamoyl)-benzylthio]-1,3,4-thiadiazol-5-yl}-1,2-benzisoselenazol-3(2H)-one (4m)

Yellow solid; mp 263.9–265.8°C; Yield 51%; IR (KBr) cm1: 1635, 1401, 1108, 992, 735, 616; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.11 (s, 1H, Ar-NH), 8.09–8.12 (d, J=8.2 Hz, 1H, Ar-H), 7.99–8.01 (d, J=7.5 Hz, 1H, Ar-H), 7.89–7.91 (d, J=8.0 Hz, 2H, Ar-H), 7.77 (t, J=8.0 Hz, 1H, Ar-H), 7.64–7.66 (d, J=8.9 Hz, 2H, Ar-H), 7.57–7.59 (d, J=8.0 Hz, 2H, Ar-H), 7.53 (t, J=7.3 Hz, 1H, Ar-H), 6.91–6.93 (d, J=8.9 Hz, 2H, Ar-H), 4.61 (s, 2H, CH2); ESI-MS m/z: 603.4 [M+H]+; Anal. Calcd for C23H15N4BrO2S2Se: C, 45.86; H, 2.51; N, 9.30. Found: C, 45.97; H, 2.55; N, 9.37.

2-{2-[4-(2-Nitrophenylcarbamoyl)-benzylthio]-1,3,4-thiadiazol-5-yl}-1,2-benzisoselenazol-3(2H)-one (4n)

Yellow solid; mp 254.0–255.5°C; Yield 46%; IR (KBr) cm1: 1638, 1456, 1338, 1018, 741, 621; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.75 (s, 1H, Ar-NH), 8.09–8.11 (d, J=8.0 Hz, 1H, Ar-H), 8.00–8.02 (d, J=7.7 Hz, 1H, Ar-H), 8.00 (t, J=7.7 Hz, 1H, Ar-H), 7.91–7.93 (d, J=7.9 Hz, 2H, Ar-H), 7.76–7.80 (m, 3H, Ar-H), 7.63–7.65 (d, J=7.9 Hz, 2H, Ar-H), 7.51–7.55 (t, J=7.5 Hz, 1H, Ar-H), 7.41–7.44 (t, J=7.7 Hz, 1H, Ar-H), 4.63 (s, 2H, CH2); ESI-MS m/z: 570.4 [M+H]+; Anal. Calcd for C23H15N5O4S2Se: C, 48.59; H, 2.66; N, 12.32. Found: C, 48.70; H, 2.63; N, 12.37.

2-{2-[4-(4-Nitrophenylcarbamoyl)-benzylthio]-1,3,4-thiadiazol-5-yl}-1,2-benzisoselenazol-3(2H)-one (4o)

Yellow solid; mp 291.0–292.5°C; Yield 51%; IR (KBr) cm1: 1638, 1401, 1109, 993, 740, 617; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.79 (s, 1H, Ar-NH), 8.25–8.27 (d, J=9.3 Hz, 2H, Ar-H), 8.15–8.17 (d, J=8.1 Hz, 1H, Ar-H), 8.03–8.06 (d, J=9.3 Hz, 2H, Ar-H), 7.98–8.00 (d, J=7.8 Hz, 1H, Ar-H), 7.93–7.95 (d, J=8.3 Hz, 2H, Ar-H), 7.77 (t, J=8.4 Hz, 1H, Ar-H), 7.62–7.64 (d, J=8.3 Hz, 2H, Ar-H), 7.52 (t, J=7.9 Hz, 1H, Ar-H), 4.63 (s, 2H, CH2); ESI-MS m/z: 570.4 [M+H]+; Anal. Calcd for C23H15N5O4S2Se: C, 48.59; H, 2.66; N, 12.32. Found: C, 48.48; H, 2.67; N, 12.25.

2-{2-[4-(4-Trifluoromethylphenylcarbamoyl)-benzylthio]-1,3,4-thiadiazol-5-yl}-1,2-benzisoselenazol-3(2H)-one (4p)

Yellow solid; mp 289.1–290.9°C; Yield 58%; IR (KBr) cm1: 1635, 1407, 1113, 992, 735, 624; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 10.56 (s, 1H, Ar-NH), 8.09–8.11 (d, J=8.1 Hz, 1H, Ar-H), 7.98–8.00 (d, J=7.6 Hz, 1H, Ar-H), 7.97–7.99 (d, J=8.3 Hz, 2H, Ar-H), 7.91–7.93 (d, J=8.2 Hz, 2H, Ar-H), 7.77 (t, J=8.1 Hz, 1H, Ar-H), 7.70–7.72 (d, J=8.3 Hz, 2H, Ar-H), 7.60–7.62 (d, J=8.2 Hz, 2H, Ar-H), 7.53 (t, J=7.6 Hz, 1H, Ar-H), 4.61 (s, 2H, CH2); ESI-MS m/z: 593.4 [M+H]+; Anal. Calcd for C24H15F3N4O2S2Se: C, 48.73; H, 2.56; N, 9.47. Found: C, 48.81; H, 2.52; N, 9.40.

Biological Assay

The following established in vitro human cancer cell lines were used: SMMC-7721, MCF-7 and A549 which were obtained from Tumor Cell Resources Bank, Chinese Academy of Medical Sciences. Cell counting kit-8 was from Dojindo (Kumamoto, Japan).

Compounds 4a–p, ethaselen and 5-fluorouracil were respectively dissolved in DMSO to make stock solutions at a concentration of 1.0×10−2 mol/L. During the experiment, cell culture medium RPMI-1640 was used to dilute the stock solution to the desired concentration. Cells in the exponential phase were seeded in 96-well culture plates at the confluence of 1×104 cells/ well, kept in 37°C, 5% CO2 incubator for 24 h. Replaced the medium containing different concentrations of compounds in fresh medium, at 37°C, 5% CO2 incubator for 48 h. Added 90 µL of fresh medium and 10 µL CCK-8, kept a 37°C and 5% CO2 for 1h. The sample cell was added into 96-well microplate reader and read the plate at 450 nm, recorded the absorbance value (OD). Cell viability was calculated from the mean values for three wells using the following formula:   

The IC50 value is defined as the concentration that causes a 50% cell proliferation inhibition.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 20971097) and the Tianjin Municipal Natural Science Foundation (No. 13JCYBJC24500).

Conflict of Interest

The authors declare no conflict of interest.

References
 
© 2015 The Pharmaceutical Society of Japan
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