Synthesis and Biological Evaluation of 1,2,4-Triazole and 1,3,4-Thiadiazole Derivatives as Potential Cytotoxic Agents

Abstract

A series of new 3-amino-5-sulfanyl-1,2,4-triazole and 2-amino-5-sulfanyl-1,3,4-thiadiazole derivatives have been synthesized and their cytotoxicities were evaluated on a panel of human cancer cell lines (BxPC-3, H1975, SKOV-3, A875, HCT116, etc.). The best one (compound 5m) exhibited activities with IC₅₀ values ranging from 0.04 to 23.6 µM against nine human cancer cell lines. Further biological evaluation indicated that DNA replication was blocked by treatment with compound 5m in HCT116 cells.

Cancer has been a great challenge to medical science and the chemotherapy has been one of the most commonly used treatment options for cancer treatment. The development of more novel small molecule compounds as antitumor drug is needed urgently.¹⁾

Some derivatives of 1,2,4-triazole and 1,3,4-thiadiazole have potential biological activities and have been applied in medicine, such as benatradin (diuretic), letrozole (antineoplastic, aromatase inhibitor),²⁾ and etoperidone (antidepressant).³⁾ In recent years, the synthesis and biological evaluation of 1,2,4-triazole and 1,3,4-thiadiazole derivatives have become hot spots. Numerous of them possess antifungal,⁴⁾ antiglaucoma,⁵⁾ anti-inflammatory,⁶⁾ antioxidant,⁷⁾ and anticancer^8–12) activities and are of particular interest for medicinal chemists. In view of these observations, we designed and synthesized a series of new 1,2,4-triazole and 1,3,4-thiadiazole analogs and the structures of them were confirmed by ¹H-NMR, MS and elemental analysis. The cytotoxicities of these compounds were tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The most potent compound (5m) was selected to test DNA replication on HCT116 cells by the 5-ethynyl-2ʹ-deoxyuridine (EdU) incorporation assay.

Results and Discussion

Chemistry

The synthesis of compounds 4a–k/5a–n was showed in Chart 1. Commercially available 4-(chloromethyl)benzoyl chloride (1) was first reacted with various substituted amines 2a–n in the presence of triethylamine as the base in dichloromethane to give compounds 3a–n.¹³⁾ Compounds 4a–k/5a–n were prepared by reacting 3a–n and 2-amino-5-sulfanyl-1,3,4-thiadiazole or 3-amino-5-sulfanyl-1,2,4-triazole under reflux using ethanol as solvent for about 4–6 h in the presence of 2 M aqueous sodium hydroxide solution. The crude products were purified to yield 4a–k/5a–n.¹⁴⁾ The structures of all compounds were determined by ¹H-NMR, and MS (electrospray ionization (ESI)).

Chart 1. Synthetic Route for Compounds 4a–k and 5a–n

Reagents and conditions: a) dichloromethane, triethylamine, reflux, 4–6 h, 87–96%; b) ethanol, 3-amino-5-sulfanyl-1,2,4-triazole or 2-amino-5-sulfanyl-1,3,4-thiadiazole, 2 M NaOH, reflux, 3–4 h, 63–91%.

Cytotoxicity

The cytotoxicities of the synthesized compounds were tested on five human tumor cell lines, including human pancreatic cancer cell line (BxPC-3), human lung adenocarcinoma cell line (H1975), human ovarian carcinoma cell line (SKOV-3), human melanoma cell line (A875) and human colorectal adenocarcinoma cell line (HCT116) by the MTT assay with adriamycin (ADM) as a positive control. The results were expressed as the IC₅₀ and summarized in Table 1.

Table 1. The Antiproliferative Activities for Compounds 4a–k and 5a–n

Compd.	X	R1	R2	R3	IC50 (µM)
					BxPC-3	H1975	SKOV-3	A875	HCT116
4a	S	H	H	H	>100	>100	>100	>100	>100
4b	S	H	H	F	>100	>100	>100	>100	84.3
4c	S	H	H	C(CH3)3	1.7	24.3	28.9	22.7	>100
4d	S	H	H	CN	7.6	>100	11.6	31.7	12.3
4e	S	H	H	Morpholino	>100	>100	>100	>100	>100
4f	S	H	H	C(CF3)2OH	4.1	27.1	29.4	28.9	>100
4g	S	H	CF3	OCH3	12.1	42.2	>100	>100	>100
4h	S	H	CF3	CN	2.2	>100	25.1	36.6	37.6
4i	S	H	CF3	F	4.1	27.2	8.8	38.4	85.6
4j	S	CF3	H	H	64.3	>100	>100	>100	>100
4k	S	CF3	H	CN	18.7	>100	56.3	38.5	>100
5a	NH	H	H	H	>100	>100	>100	>100	>100
5b	NH	H	H	F	>100	>100	>100	>100	>100
5c	NH	H	H	C(CH3)3	>100	>100	>100	>100	58.4
5d	NH	H	H	CN	51.3	>100	36.1	68.1	>100
5e	NH	H	H	Morpholino	>100	>100	>100	>100	>100
5f	NH	H	H	C(CF3)2OH	0.05	19.6	8.7	26.3	12.0
5g	NH	H	CF3	OCH3	18.1	41.7	15.3	67.9	71.8
5h	NH	H	CF3	CN	14.9	3.8	44.9	28.3	12.1
5i	NH	H	CF3	F	5.24	23.4	19.2	29.0	5.4
5j	NH	CF3	H	H	86.6	>100	88.7	>100	>100
5k	NH	CF3	H	CN	32.6	3.6	7.6	65.9	12.8
5l	NH	H	CF3	H	>100	>100	6.3	25.8	>100
5m	NH	H	H	CF3	0.04	3.2	9.7	0.68	1.2
5n	NH	CN	H	NO2	16.6	19.4	15.3	18.9	12.1
ADM	—	—	—	—	0.31	1.4	1.1	0.8	4.9

Through the structure–activity relationship study, we found that when R₁, R₂ was H, R₃ were electron withdrawing groups (ditrifluoropropan-2-ol and cyano), compounds displayed more potency than R₃ were electron donating groups (hydrogen and morpholino). Such as compounds 4d/5d and 4f/5f showed better activities than compounds 4a/5a and 4e/5e. Noteworthily, compound 5f (IC₅₀=0.05 µM) showed a significant improvement in potency than 4f (IC₅₀=4.1 µM) on BxPC-3. Additionally, bulky group (butyl, 4c) contributed much more potency compared with small groups (hydrogen and fluoro, 4a, b). In contrast, compound 5c which R₃ was butyl had no activity against the tested cell lines except HCT116. Maybe the hydrophobic decrease the activities of the 1,2,4-triazole derivatives.

Furthermore, introduction of a trifluoromethyl group, there was a clearly increase in the potency of compounds. When R₁ or R₂ was trifluoromethyl, compounds 4g–k/5g–l exhibited moderate activities. Meanwhile, when R₃ was trifluoromethyl, the activities of compounds 5m had an obviously increase against the five cell lines. It indicated that trifluoromethyl was essential for activity. Moreover, compounds 5h, 5k and 5n, which have two electron withdrawing groups (trifluoromethyl, cyano and nitro) in similar of 4h and 4k, showed good activities.

These results revealed that 1,2,4-triazole derivatives and 1,3,4-thiadiazole derivatives had similar structure–activity relationship. However, some 1,2,4-triazole derivatives exhibited much better potency and were worth being further investigated. Besides, from Table 1 we can find that most of the compounds have cytotoxicity against BxPC-3 cell line. It revealed BxPC-3 maybe a sensitivity cytotoxicity cell to these compounds, so we did not chose this cell line to further study. And compared with the cytotoxicity of compounds 4c and 5c, we found that HCT116 cell line is very worthy to further investigation.

To further study, the most potent analog 5m was selected for evaluation of its inhibitory activities against other four human cancer cell lines: human glioma cell line (U251), human colorectal adenocarcinoma cell line (SW620), human breast cancer cell line (MDA-MB-231) and human colon cancer cell line (LS174T) as shown in Table 2. Compound 5m, especially, showed the most potent cytotoxicities against BxPC-3, A875, HCT116 and LS174T (IC₅₀=0.04, 0.68, 1.2, 0.08 µM, respectively) and were better than positive control ADM (IC₅₀=0.31, 0.8, 4.9, 1.3 µM, respectively).

Table 2. The Antiproliferative Activities for Compound 5m

Compd.	IC50 (µM)
	BxPC-3	H1975	SKOV-3	A875	HCT116	U251	SW620	MDA-MB-231	LS174T
5m	0.04	3.2	9.7	0.68	1.2	23.5	1.3	23.6	0.08
ADM	0.31	1.4	1.1	0.8	4.9	0.12	0.9	2.7	1.3

The EdU Incorporation Assay

EdU, a thymidine analog used for labeling of proliferating cells, can incorporate into replicating DNA during S phase. We used the EdU incorporation assay to investigate the cell proliferation after treatment with compound 5m on HCT116 cells (Fig. 1A). The red nuclei represented the cells in S phase.¹⁵⁾ These data showed that the number of proliferating cells clearly decreased with increasing concentrations of compound 5m (0, 2.5, 5.0, 10.0 µM), and indicated that DNA replication was blocked. The histogram showed a significant decrease of EdU labeled cells and the proportions were 41.30%, 38.08%, 34.05%, and 28.11%, respectively (Fig. 1B).

Fig. 1. (A) The Fluorescence Microscopic Appearance of Hoechst and EdU on HCT116 Cells at Different Concentrations of Compound 5m for 24 h

(B) Statistic results of the EdU incorporation assay.

Conclusion

In summary, a series of 3-amino-5-sulfanyl-1,2,4-triazole and 2-amino-5-sulfanyl-1,3,4-thiadiazole analogues were synthesized and their cytotoxicities against a panel of human cancer cell lines in vitro were evaluated. Among them, 3-amino-5-sulfanyl-1,2,4-triazole derivatives showed higher cytotoxic than 2-amino-5-sulfanyl-1,3,4-thiadiazole derivatives. The most potent compound 5m exhibited highest cytotoxicity against BxPC-3, H1975, A875, HCT116, SW620 and LS174T (IC₅₀=0.04, 3.2, 0.68, 1.2, 1.3, 0.08 µM, respectively). The EdU incorporation assay exhibited that DNA replication was blocked clearly by treatment with compound 5m on a concentration-dependent manner.

These results indicated that 3-amino-5-sulfanyl-1,2,4-triazole derivatives serve as promising nucleus for subsequent modification in the search for novel antitumor agent, and further studies are ongoing in our laboratory.

Experimental

All solvents and reagents were analytical grade pure. Melting points were measured using a Kofler hot stage apparatus and were uncorrected. ¹H-NMR spectra were carried out on a Bruker Avance (Varian Unity Inova) 400 MHz spectrometer using tetramethylsilane as internal reference; Chemical shifts are showed in δ values (ppm) and the coupling constants are expressed in J values (Hz). Low resolution ESI-MS spectra were carried out on a Waters triquadrupole mass spectrometer. 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. The human cancer cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD, U.S.A.) and the China Center for Type Culture Collection (CTCCC, Wuhan, China). Dulbecco’s modified Eagle’s medium (DMEM) and RPMI 1640 were purchased from Gibco (Grand Island, New York, U.S.A.). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, Utah, U.S.A.).

General Experimental Procedure

The compound 1 (4-(chloromethyl)benzoyl chloride, 12 mmol) was added dropwise to a mixture of 2a–n (10 mmol) and triethylamine (12 mmol) in dichloromethane (20 mL) at room temperature with stirring. Then the reaction mixture was refluxed for 4–6 h. After cooling to room temperature, the mixture was slowly poured to ice water (50 mL). The solution was extracted with dichloromethane (3×60 mL) and the organic phase was dried over with sodium sulfate anhydrous. After removal of the dichloromethane, the solid residue was purified by recrystallization with petroleum ether–ethyl acetate to give compounds 3a–n.

General Procedure for Preparing Compounds 4a–k

The 2-amino-5-sulfanyl-1,3,4-thiadiazole (5 mmol) was suspended in 15 mL ethanol and treated with 5 mL aqueous solution of sodium hydroxide (2 mol/L). The mixture was stirred at room temperature for 30 min. Then the compounds 3a–k were added and the mixture was heated to reflux for 3–4 h. After the reaction mixture was cooled to room temperature, the precipitate was collected by filtration and washed with water to yield the crude products. Compounds 4a–k were purified by dissolved in glacial acetic acid and precipitated by dilute ammonium hydroxide at pH 7–8.

4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-phenylbenzamide (4a): Yield 81%; mp 231.4–233.6°C. ¹H-NMR (DMSO-d₆) δ: 10.22 (1H, s), 7.91 (2H, d, J=8.4 Hz), 7.77 (2H, d, J=8.0 Hz), 7.50 (2H, d, J=8.0 Hz), 7.36 (2H, t, J=8.0 Hz), 7.29 (1H, s), 7.10 (2H, d, J=7.2 Hz), 4.38 (2H, s). (ESI-MS) m/z: 343.1 (M+H)⁺. Anal. Calcd for C₁₆H₁₄N₄OS₂: C, 56.12; H, 4.12; N, 16.36. Found: C, 56.14; H, 4.19; N, 16.32.

4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4-fluorophenyl)benzamide (4b): Yield 69%; mp 196.3–198.1°C. ¹H-NMR (DMSO-d₆) δ: 10.29 (1H, s), 7.90 (2H, d, J=8.0 Hz), 7.76 (2H, d, J=8.0 Hz), 7.50 (2H, d, J=8.0 Hz), 7.31 (2H, s), 7.17 (2H, d, J=8.4 Hz),4.37 (2H, s). (ESI-MS) m/z: 361.1 (M+H)⁺. Anal. Calcd for C₁₆H₁₃FN₄OS₂: C, 53.32; H, 4.3.64; N, 15.54. Found: C, 53.29; H, 4.3.61; N, 15.58.

4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4-tert-butylphenyl)benzamide (4c): Yield 71%; mp 204.9–206.1°C. ¹H-NMR (DMSO-d₆) δ: 10.19 (1H, s), 7.92 (2H, d, J=8.4 Hz), 7.67 (2H, d, J=8.4 Hz), 7.47 (2H, d, J=8.0 Hz), 7.34 (2H, d, J=8.0 Hz), 7.03 (2H, s), 4.37 (2H, s), 1.28 (9H, s). (ESI-MS) m/z: 399.1 (M+H)⁺. Anal. Calcd for C₂₀H₂₂N₄OS₂ : C, 60.27; H, 5.56; N, 14.06. Found: C, 60.21; H, 5.49; N, 14.11.

4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4-cyanophenyl)benzamide (4d): Yield 83%; mp 231.4–233.6°C. ¹H-NMR (DMSO-d₆) δ: 10.39 (1H, s), 7.96 (2H, d, J=8.8 Hz), 7.85 (2H, d, J=8.4 Hz), 7.80 (2H, d, J=8.8 Hz), 7.51 (2H, d, J=8.4 Hz), 6.08 (2H, s), 4.37 (2H, s). (ESI-MS) m/z: 368.1 (M+H)⁺. Anal. Calcd for C₁₇H₁₃N₅OS₂ : C, 55.57; H, 3.57; N, 19.06. Found: C, 55.60; H, 3.51; N, 19.04.

4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4-morpholinophenyl)benzamide (4e): Yield 89%; mp 244.1–246.2°C. ¹H-NMR (DMSO-d₆) δ: 10.04 (1H, s), 7. 89 (2H, d, J=8.4 Hz), 7.63 (2H, d, J=8.4 Hz), 7.48 (2H, d, J=8.0 Hz), 7.29 (2H, s), 6.94 (2H, d, J=8.0 Hz), 4.37 (2H, s), 3.74 (2H, t); 3.07 (2H, t). (ESI-MS) m/z: 428.1 (M+H)⁺. Anal. Calcd for C₂₀H₂₁N₅O₂S₂: C, 56.18; H, 4.95; N, 16.38. Found: C, 56.14; H, 4.91; N, 16.43.

4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)benzamid (4f): Yield 75%; mp 264.7–266.1°C. ¹H-NMR (DMSO-d₆) δ: 10.01 (1H, s), 7.80 (2H, d, J=8.0 Hz), 7.74 (2H, d, J=8.0 Hz), 7.66 (2H, d, J=8.0 Hz), 7.58 (2H, d, J=8.0 Hz), 6.91 (2H, s), 6.16 (1H, s), 4.38 (2H, s). (ESI-MS) m/z: 509.0 (M+H)⁺. Anal. Calcd for C₁₉H₁₄F₆N₄O₂S₂: C, 44.88; H, 2.78; N, 11.02. Found: C, 44.83; H, 2.81; N, 11.01.

4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4-methoxy-3-(trifluoromethyl)phenyl)benzamide (4g): Yield 63%; mp 186.9–188.8°C. ¹H-NMR (DMSO-d₆) δ: 10.36 (1H, s), 8.11 (1H, s), 8.02 (1H, d, J=2.0 Hz), 7.90 (2H, d, J=8.0 Hz), 7.51 (2H, d, J=8.4 Hz), 7.32 (3H, t, J=8.4 Hz), 4.38 (2H, s), 3.88 (3H, s). (ESI-MS) m/z: 441.1 (M+H)⁺. Anal. Calcd for C₁₈H₁₅F₃N₄O₂S₂: C, 49.08; H, 3.43; N, 12.72. Found: C, 49.11; H, 3.40; N, 12.69.

4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4-cyano-3-(trifluoromethyl)phenyl)benzamide (4h): Yield 77%; mp 244.1–246.7°C. ¹H-NMR (DMSO-d₆) δ: 10.93 (1H, s), 8.46 (1H, s), 8.28 (1H, d, J=8.0 Hz), 8.17 (1H, d, J=8.0 Hz), 7.94 (2H, d, J=8.0 Hz), 7.55 (2H, d, J=8.0 Hz), 7.31 (2H, s), 4.39 (2H, s). (ESI-MS) m/z: 436.0 (M+H)⁺. Anal. Calcd for C₁₈H₁₂F₃N₅OS₂: C, 49.65; H, 2.78; N, 16.08. Found: C, 49.61; H, 2.75; N, 16.10.

4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4-fluoro-3-(trifluoromethyl)phenyl)benzamide (4i): Yield 72%; mp 196.9–198.6°C. ¹H-NMR (DMSO-d₆) δ: 10.63 (1H, s), 8.36 (1H, s), 8.11 (1H, d, J=8.0 Hz), 7.95 (2H, d, J=8.4 Hz), 7.73 (1H, s, J=8.0 Hz), 7.53 (2H, d, J=8.4 Hz), 7.31 (2H, s), 4.38 (2H, s). (ESI-MS) m/z: 429.0 (M+H)⁺. Anal. Calcd for C₁₇H₁₂F₄N₄OS₂: C, 47.66; H, 2.82; N, 13.08. Found: C, 47.68; H, 2.85; N, 13.12.

4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(2-(trifluoromethyl)phenyl)benzamide (4j): Yield 81%; mp 249.3–252.0°C. ¹H-NMR (DMSO-d₆) δ: 10.01 (1H, s), 7.89 (2H, d, J=8.0 Hz), 7.76 (1H, m), 7.62 (2H, d, J=8.4 Hz), 7.57 (2H, d, J=8.0 Hz), 7.43 (1H, m), 6.10 (2H, s), 4.30 (2H, s). (ESI-MS) m/z: 411.0 (M+H)⁺. Anal. Calcd for C₁₇H₁₃F₃N₄OS₂: C, 49.75; H, 3.19; N, 13.65. Found: C, 49.78; H, 3.24; N, 13.62.

4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4-cyano-2-(trifluoromethyl)phenyl)benzamide (4k): Yield 86%; mp 205.9–207.8°C. ¹H-NMR (DMSO-d₆) δ: 10.67 (1H, s), 8.37 (1H, s), 8.29 (1H, s), 8.16 (1H, s), 7.87 (2H, d, J=8.4 Hz), 7.67 (2H, d, J=8.4 Hz), 6.12 (2H, s), 4.37 (2H, s). (ESI-MS) m/z: 436.0 (M+H)⁺. Anal. Calcd for C₁₈H₁₂F₃N₅OS₂: C, 49.65; H, 2.78; N, 16.08. Found: C, 49.62; H, 2.82; N, 16.13.

General Procedure for Preparing Compounds 5a–n

The 3-amino-5-sulfanyl-1,2,4-triazole (5 mmol) was suspended in 15 mL ethanol and treated with 5 mL aqueous solution of sodium hydroxide (2 mol/L). The mixture was stirred at room temperature for 30 min. Then the compounds 3a–n were added and the mixture was heated to reflux for 3–4 h. After the reaction was cooled to room temperature, the precipitate was collected by filtration and washed with water to yield the crude products. Compounds 5a–n were purified by column chromatography on silica gel using petroleum ether/ethyl acetate as eluent.

4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-phenylbenzamide (5a): Yield 78%; mp 175.3–176.9°C. ¹H-NMR (DMSO-d₆) δ: 12.02 (1H, s), 10.16 (1H, s), 7.88 (2H, d, J=8.4 Hz), 7.74 (1H, t, J=8.0 Hz), 7.46 (2H, d, J=8.0 Hz), 7.28 (2H, t, J=8.4 Hz), 7.19 (2H, s), 7.08 (2H, d, J=7.2 Hz), 4.38 (2H, s). (ESI-MS) m/z: 326.4 (M+H)⁺. Anal. Calcd for C₁₆H₁₅N₅OS: C, 59.06; H, 4.65; N, 21.52. Found: C, 59.13; H, 4.62; N, 21.49.

4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4-fluorophenyl)benzamide (5b): Yield 74%; mp 233.5–235.7°C. ¹H-NMR (DMSO-d₆) δ: 12.13 (1H, br), 10.29 (1H, s), 7.87 (2H, d, J=8.0 Hz), 7.77 (2H, m), 7.51 (2H, d, J=8.0 Hz), 7.16 (2H, dd, J=8.4 Hz), 5.98 (2H, s), 4.28 (2H, s). (ESI-MS) m/z: 344.1 (M+H)⁺. Anal. Calcd for C₁₆H₁₄FN₅OS: C, 55.96; H, 4.11; N, 20.40. Found: C, 55.91; H, 4.09; N, 20.38.

4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4-tert-butylphenyl)benzamide (5c): Yield 86%; mp 197.3–199.1°C. ¹H-NMR (DMSO-d₆) δ: 12.02 (1H, br), 10.14 (1H, s), 7.89 (2H, d, J=8.4 Hz), 7.70 (2H, d, J=8.4 Hz), 7.52 (2H, d, J=8.0 Hz), 7.36 (2H, d, J=8.0 Hz), 7.16 (2H, s), 4.37 (2H, s), 1.28 (9H, s). (ESI-MS) m/z: 382.2 (M+H)⁺. Anal. Calcd for C₂₀H₂₃N₅OS: C, 62.97; H, 6.08; N, 18.36. Found: C, 62.94; H, 6.05; N, 18.31.

4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4-cyanophenyl)benzamide (5d): Yield 82%; mp 216.5–217.7°C. ¹H-NMR (DMSO-d₆) δ: 11.98 (1H, s), 10.59 (1H, s), 7.98 (2H, d, J=8.8 Hz), 7.87 (2H, d, J=8.4 Hz), 7.80 (2H, d, J=8.8 Hz), 7.58 (2H, d, J=8.4 Hz), 6.08 (2H, s), 4.30 (2H, s). (ESI-MS) m/z: 349.1 (M−H) ⁺. Anal. Calcd for C₁₇H₁₄N₆OS: C, 58.27; H, 4.03; N, 23.98. Found: C, 58.31; H, 4.01; N, 23.94.

4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4-morpholinophenyl)benzamide (5e): Yield 71%; mp 183.1–185.4°C. ¹H-NMR (DMSO-d₆) δ: 12.13 (1H, s), 10.12 (1H, s), 7.88 (2H, d, J=8.4 Hz), 7.65 (2H, d, J=8.4 Hz), 7.50 (2H, d, J=8.0 Hz), 7.31 (2H, s), 6.92 (2H, d, J=8.0 Hz), 4.37 (2H, s), 3.74 (2H, t); 3.07 (2H, t). (ESI-MS) m/z: 411.2 (M+H)⁺. Anal. Calcd for C₁₇H₁₄N₆OS: C, 58.27; H, 4.03; N, 23.98. Found: C, 58.31; H, 4.05; N, 23.99.

4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)benzamide (5f): Yield 68%; mp 203.2–204.5°C. ¹H-NMR (DMSO-d₆) δ: 12.01 (1H, s), 10.07 (1H, s), 7.85 (2H, d, J=8.0 Hz), 7.77 (2H, d, J=8.0 Hz), 7.68 (2H, d, J=8.0 Hz), 7.54 (2H, d, J=8.0 Hz), 6.92 (2H, s), 6.19 (1H, s), 4.37 (2H, s). (ESI-MS) m/z: 492.1 (M+H)⁺. Anal. Calcd for C₁₉H₁₅F₆N₅O₂S: C, 46.44; H, 3.08; N, 14.25. Found: C, 46.41; H, 3.05; N, 14.30.

4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4-methoxy-3-(trifluoromethyl)phenyl)benzamide (5g): Yield 69%; mp 235.1–237.4°C. ¹H-NMR (DMSO-d₆) δ: 12.14 (1H, br), 10.40 (1H, s), 8.12 (1H, s), 8.03 (1H, d, J=8.0 Hz), 8.00 (1H, d, J=8.0 Hz), 7.92 (2H, d, J=8.4 Hz), 7.53 (2H, d, J=8.4 Hz), 7.27 (2H, s), 4.43 (2H, s), 3.88 (3H, s). (ESI-MS) m/z: 424.1 (M+H)⁺. Anal. Calcd for C₁₈H₁₆F₃N₅O₂S: C, 51.06; H, 3.81; N, 16.54. Found: C, 51.02; H, 3.79; N, 16.51.

4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4-cyano-3-(trifluoromethyl)phenyl)benzamide (5h): Yield 89%; mp 107.5–109.8°C. ¹H-NMR (DMSO-d₆) δ: 11.96 (1H, s), 10.89 (1H, s), 8.46 (1H, s), 8.26 (1H, d, J=8.4 Hz), 8.16 (1H, d, J=8.4 Hz), 7.91 (2H, d, J=8.4 Hz), 7.56 (2H, d, J=8.4 Hz), 6.08 (2H, s), 4.30 (2H, s). (ESI-MS) m/z: 419.1 (M+H)⁺. Anal. Calcd for C₁₈H₁₃F₃N₆OS: C, 51.67; H, 3.13; N, 20.09. Found: C, 51.66; H, 3.15; N, 20.13.

4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4-fluoro-3-(trifluoromethyl)phenyl)benzamide (5i): Yield 67%; mp 209.9–211.4°C. ¹H-NMR (DMSO-d₆) δ: 11.96 (1H, s), 10.52 (1H, s), 8.26 (1H, d, J=8.0 Hz), 8.07 (1H, d, J=8.0 Hz), 7.89 (2H, d, J=8.4 Hz), 7.62 (2H, d, J=8.4 Hz), 7.53 (1H, d, J=8.0 Hz), 6.54 (2H, s), 4.37 (2H, s). (ESI-MS) m/z: 412.1 (M+H)⁺. Anal. Calcd for C₁₇H₁₃F₄N₅OS: C, 49.63; H, 3.19; N, 17.02. Found: C, 49.61; H, 3.17; N, 17.06.

4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(2-(trifluoromethyl)phenyl)benzamide (5j): Yield 69%; mp 211.5–214.6°C. ¹H-NMR (DMSO-d₆) δ: 11.98 (1H, s), 10.12 (1H, s), 7.86 (2H, d, J=8.0 Hz), 7.74 (2H, d, J=8.0 Hz), 7.66 (1H, m), 7.51 (2H, d, J=8.0 Hz), 7.36–7.42 (1H, m), 6.07 (2H, s), 4.29 (2H, s). (ESI-MS) m/z: 394.1 (M+H)⁺. Anal. Calcd for C₁₇H₁₄F₃N₅OS: C, 51.90; H, 3.59; N, 17.80. Found: C, 51.88; H, 3.61; N, 17.83.

4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4-cyano-2-(trifluoromethyl)phenyl)benzamide (5k): Yield 73%; mp 263.6–265.3°C. ¹H-NMR (DMSO-d₆) δ: 11.98 (1H, s), 10.87 (1H, s), 8.47 (1H, s), 8.26 (1H, s), 8.12 (1H, s), 7.91 (2H, d, J=8.4 Hz), 7.58 (2H, d, J=8.4 Hz), 6.09 (2H, s), 4.29 (2H, s). (ESI-MS) m/z: 419.1 (M+H)⁺. Anal. Calcd for C₁₈H₁₃F₃N₆OS: C, 51.67; H, 3.13; N, 20.09. Found: C, 51.70; H, 3.11; N, 20.06.

4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(3-(trifluoromethyl)phenyl)benzamide (5l): Yield 87%; mp 228.1–229.7°C. ¹H-NMR (DMSO-d₆) δ: 11.96 (1H, s), 10.09 (1H, s), 7.92 (1H, d, J=8.0 Hz), 7.81 (2H, d, J=8.6 Hz), 7.54 (1H, d, J=8.0 Hz), 7.43 (2H, d, J=8.4 Hz), 7.26 (2H, d, J=8.0 Hz), 6.09 (2H, s), 4.29 (2H, s). (ESI-MS) m/z: 394.1 (M+H)⁺. Anal. Calcd for C₁₇H₁₄F₃N₅OS: C, 51.90; H, 3.59; N, 17.80. Found: C, 51.90; H, 3.57; N, 17.84.

4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4-(trifluoromethyl)phenyl)benzamide (5m): Yield 79%; mp 193.1–194.2°C. ¹H-NMR (DMSO-d₆) δ: 11.96 (1H, s), 10.09 (1H, s), 7.87 (2H, d, J=8.0 Hz), 7.80 (2H, d, J=8.0 Hz), 7.74 (2H, d, J=8.0 Hz), 7.52 (2H, d, J=8.0 Hz), 6.09 (2H, s), 4.29 (2H, s). (ESI-MS) m/z: 394.0 (M+H)⁺. Anal. Calcd for C₁₇H₁₄F₃N₅OS: C, 51.90; H, 3.59; N, 17.80. Found: C, 51.94; H, 3.62 N, 17.83.

4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(2-cyano-4-nitrophenyl)benzamide (5n): Yield 79%; mp 213.8–215.6°C. ¹H-NMR (DMSO-d₆) δ: 12.23 (1H, s), 10.30 (1H, s), 7.83 (2H, d, J=8.0 Hz), 7.68 (1H, d, J=8.0 Hz), 7.58 (2H, d, J=8.8 Hz), 7.50 (1H, d, J=8.0 Hz), 7.40 (1H, s), 7.03 (2H, s), 4.61 (2H, s). (ESI-MS) m/z: 394.1 (M−H)⁺. Anal. Calcd for C₁₇H₁₃N₇O₃S: C, 51.64; H, 3.31; N, 24.80. Found: C, 51.65; H, 3.29; N, 24.83.

Cell Proliferation Assay

Cell lines BxPC-3 and H1975 were maintained in RPMI 1640 containing 10% FBS, penicillin (100 U/mL) and streptomycin (100 U/mL). Cell lines U251, HCT116, SW620, A875, MDA-MB-231, LS174T and SKOV-3 were maintained in DMEM containing 10% FBS, penicillin (100 U/mL) and streptomycin (100 U/mL). Cells were grown under humidified conditions with 5% CO₂ at 37°C.

The tested compounds were prepared as 20 µM stock solution in dimethyl sulfoxide (DMSO) and diluted in the relevant assay media. DMSO was used as a negative control and adriamycin (ADM) was used as a positive control. Cells (3–5×10³/well) were seeded in 200 µL of medium/well in 96-well plates (Costar Corning, Rochester, NY, U.S.A.) for 24 h, followed by compounds treatment for 72 h. A volume of 20 µL of 5 mg/mL MTT was added per well and incubated for another 2–4 h at 37°C, then the supernatant fluid was removed and 150 µL/well DMSO was added. The 96-well plates were vibrated on micro-vibrator for additional 10 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 (concentration causing 50% loss in cell viability) of each cell line.

The EdU Incorporation Assay

HCT116 cells growing in a 96-well plate (3×10³ cells/well) were treated with compound 5m for 24 h. Then, the proliferation cells were assayed with an EdU-ApolloTM 567 DNA Proliferation Detection kit (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. Briefly, the cells were fixed with formaldehyde for 15 min and washed with PBS, then permeablized with 0.2% Triton-100 for 5 min, followed by incubation with the EdU for 30 min in dark. After incubation, cells were washed with 0.5% Triton-100 in PBS for 10 min. For subsequent DNA staining, sections were incubated with Hoechst 33342 (5 µg/mL) for 10 min. The plates were then washed with PBS twice and observed with Thermo Array scan VTI (Thermo Scientific).

Acknowledgment

This work was supported by Shijiazhuang Pharmaceutical Group Co., Ltd.

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