Synthesis and biological evaluation of 1,2,4-triazole and 1,3,4-thiadiazole derivatives as potential cytotoxic agents

Article abstract of DOI:10.1248/cpb.c13-00242

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 be

Full text of DOI:10.1248/cpb.c13-00242

November 2013
Regular Article
Chem. Pharm. Bull. 61(11) 1099–1104 (2013)
1099
Synthesis and Biological Evaluation of 1,2,4-Triazole and
1,3,4-Thiadiazole Derivatives as Potential Cytotoxic Agents
Yao-Jie Shi, Xue-Jiao Song, Xiao Li, Ting-Hong Ye, Ying Xiong, and Luo-Ting Yu*
State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University; Chengdu
610041, China.
Received March 28, 2013; accepted July 23, 2013; advance publication released online August 10, 2013
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 rang-
ing from 0.04 to 23.6µM against nine human cancer cell lines. Further biological evaluation indicated that
DNA replication was blocked by treatment with compound 5m in HCT116 cells.
Key words 1,2,4-triazole; 1,3,4-thiadiazole; cytotoxicity; DNA replication
Cancer has been a great challenge to medical science and dichloromethane to give compounds 3a–n.13) Compounds 4a–
the chemotherapy has been one of the most commonly used k/5a–n were prepared by reacting 3a–n and 2-amino-5-sul-
treatment options for cancer treatment. The development of fanyl-1,3,4-thiadiazole or 3-amino-5-sulfanyl-1,2,4-triazole
more novel small molecule compounds as antitumor drug is under reflux using ethanol as solvent for about 4–6h in the
needed urgently.1)
presence of 2M aqueous sodium hydroxide solution. The crude
Some derivatives of 1,2,4-triazole and 1,3,4-thiadiazole products were purified to yield 4a–k/5a–n.14) The structures
have potential biological activities and have been applied in of all compounds were determined by 1H-NMR, and MS
medicine, such as benatradin (diuretic), letrozole (antineoplas- (electrospray ionization (ESI)).
tic, aromatase inhibitor),2) and etoperidone (antidepressant).3)
Cytotoxicity The cytotoxicities of the synthesized com-
In recent years, the synthesis and biological evaluation of pounds were tested on five human tumor cell lines, includ-
1,2,4-triazole and 1,3,4-thiadiazole derivatives have become ing human pancreatic cancer cell line (BxPC-3), human lung
hot spots. Numerous of them possess antifungal,4) antiglau- adenocarcinoma cell line (H1975), human ovarian carcinoma
coma,5) anti-inflammatory,6) antioxidant,7) and anticancer8–12) cell line (SKOV-3), human melanoma cell line (A875) and
activities and are of particular interest for medicinal chemists. human colorectal adenocarcinoma cell line (HCT116) by the
In view of these observations, we designed and synthesized a MTT assay with adriamycin (ADM) as a positive control. The
series of new 1,2,4-triazole and 1,3,4-thiadiazole analogs and results were expressed as the IC50 and summarized in Table 1.
1
the structures of them were confirmed by H-NMR, MS and
Through the structure–activity relationship study, we found
elemental analysis. The cytotoxicities of these compounds that when R1, R2 was H, R3 were electron withdrawing groups
were tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- (ditrifluoropropan-2-ol and cyano), compounds displayed more
tetrazolium bromide (MTT) assay. The most potent compound potency than R3 were electron donating groups (hydrogen and
(5m) was selected to test DNA replication on HCT116 cells by morpholino). Such as compounds 4d/5d and 4f/5f showed
the 5-ethynyl-2ʹ-deoxyuridine (EdU) incorporation assay.
better activities than compounds 4a/5a and 4e/5e. Noteworthi-
ly, compound 5f (IC50=0.05µM) showed a significant improve-
ment in potency than 4f (IC50=4.1 µM) on BxPC-3. Addition-
Results and Discussion
Chemistry The synthesis of compounds 4a–k/5a–n was ally, bulky group (butyl, 4c) contributed much more potency
showed in Chart 1. Commercially available 4-(chloromethyl)- compared with small groups (hydrogen and fluoro, 4a,b). In
benzoyl chloride (1) was first reacted with various substituted contrast, compound 5c which R3 was butyl had no activity
amines 2a–n in the presence of triethylamine as the base in against the tested cell lines except HCT116. Maybe the hydro-
Reagents and conditions: a) dichloromethane, triethylamine, reflux, 4–6h, 87–96%; b) ethanol, 3-amino-5-sulfanyl-1,2,4-triazole or 2-amino-5-sulfanyl-1,3,4-thiadiazole,
2M NaOH, reflux, 3–4h, 63–91%.
Chart 1. Synthetic Route for Compounds 4a–k and 5a–n
The authors declare no conflict of interest.
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: yuluot@scu.edu.cn

1100
Vol. 61, No. 11
Table 1. The Antiproliferative Activities for Compounds 4a–k and 5a–n
IC50 (µM)
Compd.
X
R1
R2
R3
BxPC-3
>100
>100
1.7
7.6
>100
H1975
>100
>100
24.3
SKOV-3
A875
>100
>100
22.7
31.7
>100
28.9
>100
HCT116
4a
4b
4c
S
S
H
H
H
H
H
>100
>100
28.9
11.6
>100
29.4
>100
25.1
8.8
>100
84.3
F
S
H
H
C(CH3)3
>100
12.3
4d
4e
S
H
H
CN
>100
>100
S
H
H
Morpholino
>100
>100
>100
37.6
4f
S
H
H
C(CF3)2OH
4.1
12.1
2.2
27.1
42.2
>100
27.2
>100
>100
>100
>100
>100
>100
>100
4g
4h
4i
S
H
CF3
CF3
CF3
H
OCH3
S
H
CN
36.6
S
H
F
4.1
38.4
85.6
4j
S
CF3
CF3
H
H
64.3
18.7
>100
56.3
>100
>100
>100
36.1
>100
8.7
>100
38.5
>100
>100
>100
>100
>100
>100
>100
58.4
4k
5a
5b
5c
S
H
CN
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
—
H
H
>100
>100
>100
51.3
>100
0.05
18.1
14.9
5.24
H
H
F
H
H
C(CH3)3
5d
5e
H
H
CN
68.1
>100
26.3
>100
>100
12.0
H
H
Morpholino
5f
H
H
C(CF3)2OH
19.6
5g
5h
5i
H
CF3
CF3
CF3
H
OCH3
CN
F
41.7
3.8
15.3
44.9
19.2
88.7
7.6
67.9
28.3
29.0
71.8
H
12.1
H
23.4
5.4
5j
CF3
CF3
H
H
86.6
32.6
>100
3.6
>100
>100
>100
12.8
5k
5l
H
CN
H
65.9
CF3
H
>100
0.04
16.6
0.31
6.3
25.8
>100
1.2
5m
5n
ADM
H
CF3
NO2
—
3.2
19.4
1.4
9.7
0.68
CN
—
H
15.3
1.1
18.9
0.8
12.1
—
4.9
phobic decrease the activities of the 1,2,4-triazole derivatives.
human cancer cell lines: human glioma cell line (U251),
Furthermore, introduction of a trifluoromethyl group, there human colorectal adenocarcinoma cell line (SW620), human
was a clearly increase in the potency of compounds. When R1 breast cancer cell line (MDA-MB-231) and human colon
or R2 was trifluoromethyl, compounds 4g–k/5g–l exhibited cancer cell line (LS174T) as shown in Table 2. Compound
moderate activities. Meanwhile, when R3 was trifluoromethyl, 5m, especially, showed the most potent cytotoxicities against
the activities of compounds 5m had an obviously increase BxPC-3, A875, HCT116 and LS174T (IC50=0.04, 0.68, 1.2,
against the five cell lines. It indicated that trifluoromethyl was 0.08µM, respectively) and were better than positive control
essential for activity. Moreover, compounds 5h, 5k and 5n, ADM (IC50=0.31, 0.8, 4.9, 1.3µM, respectively).
which have two electron withdrawing groups (trifluoromethyl,
cyano and nitro) in similar of 4h and 4k, showed good activi- used for labeling of proliferating cells, can incorporate into
ties. replicating DNA during S phase. We used the EdU incorpora-
The EdU Incorporation Assay EdU, a thymidine analog
These results revealed that 1,2,4-triazole derivatives and tion assay to investigate the cell proliferation after treatment
1,3,4-thiadiazole derivatives had similar structure–activ- with compound 5m on HCT116 cells (Fig. 1A). The red nuclei
ity relationship. However, some 1,2,4-triazole derivatives represented the cells in S phase.15) These data showed that the
exhibited much better potency and were worth being further number of proliferating cells clearly decreased with increas-
investigated. Besides, from Table 1 we can find that most of ing concentrations of compound 5m (0, 2.5, 5.0, 10.0µM), and
the compounds have cytotoxicity against BxPC-3 cell line. It indicated that DNA replication was blocked. The histogram
revealed BxPC-3 maybe a sensitivity cytotoxicity cell to these showed a significant decrease of EdU labeled cells and the
compounds, so we did not chose this cell line to further study. proportions were 41.30%, 38.08%, 34.05%, and 28.11%, re-
And compared with the cytotoxicity of compounds 4c and spectively (Fig. 1B).
5c, we found that HCT116 cell line is very worthy to further
investigation.
Conclusion
To further study, the most potent analog 5m was selected
In summary, a series of 3-amino-5-sulfanyl-1,2,4-triazole
for evaluation of its inhibitory activities against other four and 2-amino-5-sulfanyl-1,3,4-thiadiazole analogues were

November 2013
1101
Table 2. The Antiproliferative Activities for Compound 5m
IC50 (µM)
Compd.
BxPC-3
H1975
SKOV-3
A875
HCT116
U251
SW620
MDA-MB-231
LS174T
5m
0.04
0.31
3.2
1.4
9.7
1.1
0.68
0.8
1.2
4.9
23.5
0.12
1.3
0.9
23.6
2.7
0.08
1.3
ADM
Fig. 1. (A) The Fluorescence Microscopic Appearance of Hoechst and EdU on HCT116 Cells at Different Concentrations of Compound 5m for 24h
(B) Statistic results of the EdU incorporation assay.
synthesized and their cytotoxicities against a panel of human
General Experimental Procedure The compound 1
cancer cell lines in vitro were evaluated. Among them, (4-(chloromethyl)benzoyl chloride, 12mmol) was added
3-amino-5-sulfanyl-1,2,4-triazole derivatives showed higher dropwise to a mixture of 2a–n (10mmol) and triethylamine
cytotoxic than 2-amino-5-sulfanyl-1,3,4-thiadiazole deriva- (12mmol) in dichloromethane (20mL) at room temperature
tives. The most potent compound 5m exhibited highest cyto- with stirring. Then the reaction mixture was refluxed for
toxicity against BxPC-3, H1975, A875, HCT116, SW620 and 4–6h. After cooling to room temperature, the mixture was
LS174T (IC50=0.04, 3.2, 0.68, 1.2, 1.3, 0.08µM, respectively). slowly poured to ice water (50mL). The solution was extracted
The EdU incorporation assay exhibited that DNA replication with dichloromethane (3×60mL) and the organic phase was
was blocked clearly by treatment with compound 5m on a dried over with sodium sulfate anhydrous. After removal of
concentration-dependent manner.
the dichloromethane, the solid residue was purified by recrys-
These results indicated that 3-amino-5-sulfanyl-1,2,4- tallization with petroleum ether–ethyl acetate to give com-
triazole derivatives serve as promising nucleus for subsequent pounds 3a–n.
modification in the search for novel antitumor agent, and fur-
ther studies are ongoing in our laboratory.
General Procedure for Preparing Compounds 4a–k
The 2-amino-5-sulfanyl-1,3,4-thiadiazole (5mmol) was sus-
pended in 15mL ethanol and treated with 5mL aqueous solu-
tion of sodium hydroxide (2mol/L). The mixture was stirred at
Experimental
All solvents and reagents were analytical grade pure. Melt- room temperature for 30min. Then the compounds 3a–k were
ing points were measured using a Kofler hot stage apparatus added and the mixture was heated to reflux for 3–4h. After
1
and were uncorrected. H-NMR spectra were carried out on the reaction mixture was cooled to room temperature, the
a Bruker Avance (Varian Unity Inova) 400MHz 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 spectrom-
eter. Column chromatography was carried out on silica gel
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-phenyl-
1H-NMR
benzamide (4a): Yield 81%; mp 231.4–233.6°C.
(200–300 mesh, Qingdao Marine Chemical Ltd., Qingdao, (DMSO-d6) δ: 10.22 (1H, s), 7.91 (2H, d, J=8.4Hz), 7.77 (2H,
China). Thin layer chromatography (TLC) was performed on d, J=8.0Hz), 7.50 (2H, d, J=8.0Hz), 7.36 (2H, t, J=8.0Hz),
TLC silica gel 60 F254 plates. The human cancer cell lines 7.29 (1H, s), 7.10 (2H, d, J=7.2Hz), 4.38 (2H, s). (ESI-MS)
were purchased from the American Type Culture Collection m/z: 343.1 (M+H)+. Anal. Calcd for C16H14N4OS2: C, 56.12; H,
(ATCC, Rockville, MD, U.S.A.) and the China Center for 4.12; N, 16.36. Found: C, 56.14; H, 4.19; N, 16.32.
Type Culture Collection (CTCCC, Wuhan, China). Dulbecco’s
4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4-fluoro-
modified Eagle’s medium (DMEM) and RPMI 1640 were pur- phenyl)benzamide (4b): Yield 69%; mp 196.3–198.1°C.
chased from Gibco (Grand Island, New York, U.S.A.). Fetal 1H-NMR (DMSO-d6) δ: 10.29 (1H, s), 7.90 (2H, d, J=8.0Hz),
bovine serum (FBS) was purchased from Hyclone (Logan, 7.76 (2H, d, J=8.0Hz), 7.50 (2H, d, J=8.0Hz), 7.31 (2H, s),
Utah, U.S.A.).
7.17 (2H, d, J=8.4Hz),4.37 (2H, s). (ESI-MS) m/z: 361.1 (M+

1102
Vol. 61, No. 11
H)+. Anal. Calcd for C16H13FN4OS2: C, 53.32; H, 4.3.64; N, H, 3.19; N, 13.65. Found: C, 49.78; H, 3.24; N, 13.62.
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-cyano-2-
4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4-tert- (trifluoromethyl)phenyl)benzamide (4k): Yield 86%; mp
1
butylphenyl)benzamide (4c): Yield 71%; mp 204.9–206.1°C. 205.9–207.8°C. H-NMR (DMSO-d6) δ: 10.67 (1H, s), 8.37
1H-NMR (DMSO-d6) δ: 10.19 (1H, s), 7.92 (2H, d, J=8.4Hz), (1H, s), 8.29 (1H, s), 8.16 (1H, s), 7.87 (2H, d, J=8.4Hz), 7.67
7.67 (2H, d, J=8.4Hz), 7.47 (2H, d, J=8.0Hz), 7.34 (2H, d, (2H, d, J=8.4Hz), 6.12 (2H, s), 4.37 (2H, s). (ESI-MS) m/z:
J=8.0Hz), 7.03 (2H, s), 4.37 (2H, s), 1.28 (9H, s). (ESI-MS) 436.0 (M+H)+. Anal. Calcd for C18H12F3N5OS2: C, 49.65; H,
m/z: 399.1 (M+H)+. Anal. Calcd for C20H22N4OS2 : C, 60.27; 2.78; N, 16.08. Found: C, 49.62; H, 2.82; N, 16.13.
H, 5.56; N, 14.06. Found: C, 60.21; H, 5.49; N, 14.11.
General Procedure for Preparing Compounds 5a–n
4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4- The 3-amino-5-sulfanyl-1,2,4-triazole (5mmol) was suspended
cyanophenyl)benzamide (4d): Yield 83%; mp 231.4–233.6°C. in 15mL ethanol and treated with 5mL aqueous solution of
1H-NMR (DMSO-d6) δ: 10.39 (1H, s), 7.96 (2H, d, J=8.8Hz), sodium hydroxide (2mol/L). The mixture was stirred at room
7.85 (2H, d, J=8.4Hz), 7.80 (2H, d, J=8.8Hz), 7.51 (2H, d, temperature for 30min. Then the compounds 3a–n were
J=8.4Hz), 6.08 (2H, s), 4.37 (2H, s). (ESI-MS) m/z: 368.1 (M+ added and the mixture was heated to reflux for 3–4h. After
H)+. Anal. Calcd for C17H13N5OS2 : C, 55.57; H, 3.57; N, 19.06. the reaction was cooled to room temperature, the precipitate
Found: C, 55.60; H, 3.51; N, 19.04.
was collected by filtration and washed with water to yield the
4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4-morpho- crude products. Compounds 5a–n were purified by column
linophenyl)benzamide (4e): Yield 89%; mp 244.1–246.2°C. chromatography on silica gel using petroleum ether/ethyl
1H-NMR (DMSO-d6) δ: 10.04 (1H, s), 7. 89 (2H, d, J=8.4Hz), acetate as eluent.
7.63 (2H, d, J=8.4Hz), 7.48 (2H, d, J=8.0Hz), 7.29 (2H, s),
4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-phenyl-
6.94 (2H, d, J=8.0Hz), 4.37 (2H, s), 3.74 (2H, t); 3.07 (2H, t). benzamide (5a): Yield 78%; mp 175.3–176.9°C. 1H-NMR
(ESI-MS) m/z: 428.1 (M+H)+. Anal. Calcd for C20H21N5O2S2: (DMSO-d6) δ: 12.02 (1H, s), 10.16 (1H, s), 7.88 (2H, d,
C, 56.18; H, 4.95; N, 16.38. Found: C, 56.14; H, 4.91; N, 16.43. J=8.4Hz), 7.74 (1H, t, J=8.0Hz), 7.46 (2H, d, J=8.0Hz),
4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4-(1,1,1, 7.28 (2H, t, J=8.4Hz), 7.19 (2H, s), 7.08 (2H, d, J=7.2Hz),
3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)benzamid (4f): 4.38 (2H, s). (ESI-MS) m/z: 326.4 (M+H)+. Anal. Calcd for
1
Yield 75%; mp 264.7–266.1°C. H-NMR (DMSO-d6) δ: 10.01 C16H15N5OS: C, 59.06; H, 4.65; N, 21.52. Found: C, 59.13; H,
(1H, s), 7.80 (2H, d, J=8.0Hz), 7.74 (2H, d, J=8.0Hz), 7.66 4.62; N, 21.49.
(2H, d, J=8.0Hz), 7.58 (2H, d, J=8.0Hz), 6.91 (2H, s), 6.16
4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4-fluoro-
(1H, s), 4.38 (2H, s). (ESI-MS) m/z: 509.0 (M+H)+. Anal. phenyl)benzamide (5b): Yield 74%; mp 233.5–235.7°C.
Calcd for C19H14F6N4O2S2: C, 44.88; H, 2.78; N, 11.02. Found: 1H-NMR (DMSO-d6) δ: 12.13 (1H, br), 10.29 (1H, s), 7.87 (2H,
C, 44.83; H, 2.81; N, 11.01.
d, J=8.0Hz), 7.77 (2H, m), 7.51 (2H, d, J=8.0Hz), 7.16 (2H,
4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4- dd, J=8.4Hz), 5.98 (2H, s), 4.28 (2H, s). (ESI-MS) m/z: 344.1
methoxy-3-(trifluoromethyl)phenyl)benzamide (4g): Yield (M+H)+. Anal. Calcd for C16H14FN5OS: C, 55.96; H, 4.11; N,
1
63%; mp 186.9–188.8°C. H-NMR (DMSO-d6) δ: 10.36 (1H, 20.40. Found: C, 55.91; H, 4.09; N, 20.38.
s), 8.11 (1H, s), 8.02 (1H, d, J=2.0Hz), 7.90 (2H, d, J=8.0Hz),
4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4-tert-
7.51 (2H, d, J=8.4Hz), 7.32 (3H, t, J=8.4Hz), 4.38 (2H, s), butylphenyl)benzamide (5c): Yield 86%; mp 197.3–199.1°C.
3.88 (3H, s). (ESI-MS) m/z: 441.1 (M+H)+. Anal. Calcd for 1H-NMR (DMSO-d6) δ: 12.02 (1H, br), 10.14 (1H, s), 7.89 (2H,
C18H15F3N4O2S2: C, 49.08; H, 3.43; N, 12.72. Found: C, 49.11; d, J=8.4Hz), 7.70 (2H, d, J=8.4Hz), 7.52 (2H, d, J=8.0Hz),
H, 3.40; N, 12.69.
7.36 (2H, d, J=8.0Hz), 7.16 (2H, s), 4.37 (2H, s), 1.28 (9H, s).
4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4-cyano-3- (ESI-MS) m/z: 382.2 (M+H)+. Anal. Calcd for C20H23N5OS: C,
(trifluoromethyl)phenyl)benzamide (4h): Yield 77%; mp 62.97; H, 6.08; N, 18.36. Found: C, 62.94; H, 6.05; N, 18.31.
1
244.1–246.7°C. H-NMR (DMSO-d6) δ: 10.93 (1H, s), 8.46
4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4-
(1H, s), 8.28 (1H, d, J=8.0Hz), 8.17 (1H, d, J=8.0Hz), 7.94 cyanophenyl)benzamide (5d): Yield 82%; mp 216.5–217.7°C.
(2H, d, J=8.0Hz), 7.55 (2H, d, J=8.0Hz), 7.31 (2H, s), 1H-NMR (DMSO-d6) δ: 11.98 (1H, s), 10.59 (1H, s), 7.98 (2H,
4.39 (2H, s). (ESI-MS) m/z: 436.0 (M+H)+. Anal. Calcd for d, J=8.8Hz), 7.87 (2H, d, J=8.4Hz), 7.80 (2H, d, J=8.8Hz),
C18H12F3N5OS2: C, 49.65; H, 2.78; N, 16.08. Found: C, 49.61; 7.58 (2H, d, J=8.4Hz), 6.08 (2H, s), 4.30 (2H, s). (ESI-MS)
+
H, 2.75; N, 16.10.
m/z: 349.1 (M−H) . Anal. Calcd for C17H14N6OS: C, 58.27; H,
4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(4-fluoro-3- 4.03; N, 23.98. Found: C, 58.31; H, 4.01; N, 23.94.
(trifluoromethyl)phenyl)benzamide (4i): Yield 72%; mp 4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4-
1
196.9–198.6°C. H-NMR (DMSO-d6) δ: 10.63 (1H, s), 8.36 morpholinophenyl)benzamide (5e): Yield 71%; mp 183.1–
1
(1H, s), 8.11 (1H, d, J=8.0Hz), 7.95 (2H, d, J=8.4Hz), 185.4°C. H-NMR (DMSO-d6) δ: 12.13 (1H, s), 10.12 (1H, s),
7.73 (1H, s, J=8.0Hz), 7.53 (2H, d, J=8.4Hz), 7.31 (2H, s), 7.88 (2H, d, J=8.4Hz), 7.65 (2H, d, J=8.4Hz), 7.50 (2H, d,
4.38 (2H, s). (ESI-MS) m/z: 429.0 (M+H)+. Anal. Calcd for J=8.0Hz), 7.31 (2H, s), 6.92 (2H, d, J=8.0Hz), 4.37 (2H, s),
C17H12F4N4OS2: C, 47.66; H, 2.82; N, 13.08. Found: C, 47.68; 3.74 (2H, t); 3.07 (2H, t). (ESI-MS) m/z: 411.2 (M+H)+. Anal.
H, 2.85; N, 13.12.
Calcd for C17H14N6OS: C, 58.27; H, 4.03; N, 23.98. Found: C,
4-((5-Amino-1,3,4-thiadiazol-2-ylthio)methyl)-N-(2-(tri- 58.31; H, 4.05; N, 23.99.
fluoromethyl)phenyl)benzamide (4j): Yield 81%; mp 249.3–
252.0°C. H-NMR (DMSO-d6) δ: 10.01 (1H, s), 7.89 (2H, d, 1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)benzamide
J=8.0Hz), 7.76 (1H, m), 7.62 (2H, d, J=8.4Hz), 7.57 (2H, d, (5f): Yield 68%; mp 203.2–204.5°C. H-NMR (DMSO-d6) δ:
4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4-(1,1,
1
1
J=8.0Hz), 7.43 (1H, m), 6.10 (2H, s), 4.30 (2H, s). (ESI-MS) 12.01 (1H, s), 10.07 (1H, s), 7.85 (2H, d, J=8.0Hz), 7.77 (2H,
m/z: 411.0 (M+H)+. Anal. Calcd for C17H13F3N4OS2: C, 49.75; d, J=8.0Hz), 7.68 (2H, d, J=8.0Hz), 7.54 (2H, d, J=8.0Hz),

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6.92 (2H, s), 6.19 (1H, s), 4.37 (2H, s). (ESI-MS) m/z: 492.1 d, J=8.0Hz), 7.68 (1H, d, J=8.0Hz), 7.58 (2H, d, J=8.8Hz),
(M+H)+. Anal. Calcd for C19H15F6N5O2S: C, 46.44; H, 3.08; N, 7.50 (1H, d, J=8.0Hz), 7.40 (1H, s), 7.03 (2H, s), 4.61 (2H, s).
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- 51.64; H, 3.31; N, 24.80. Found: C, 51.65; H, 3.29; N, 24.83.
methoxy-3-(trifluoromethyl)phenyl)benzamide (5g): Yield Cell Proliferation Assay Cell lines BxPC-3 and H1975
(ESI-MS) m/z: 394.1 (M−H)+. Anal. Calcd for C17H13N7O3S: C,
69%; mp 235.1–237.4°C. 1H-NMR (DMSO-d6) δ: 12.14 were maintained in RPMI 1640 containing 10% FBS, penicil-
(1H, br), 10.40 (1H, s), 8.12 (1H, s), 8.03 (1H, d, J=8.0Hz), lin (100U/mL) and streptomycin (100U/mL). Cell lines U251,
8.00 (1H, d, J=8.0Hz), 7.92 (2H, d, J=8.4Hz), 7.53 (2H, d, HCT116, SW620, A875, MDA-MB-231, LS174T and SKOV-3
J=8.4Hz), 7.27 (2H, s), 4.43 (2H, s), 3.88 (3H, s). (ESI-MS) were maintained in DMEM containing 10% FBS, penicillin
m/z: 424.1 (M+H)+. Anal. Calcd for C18H16F3N5O2S: C, 51.06; (100U/mL) and streptomycin (100U/mL). Cells were grown
H, 3.81; N, 16.54. Found: C, 51.02; H, 3.79; N, 16.51.
under humidified conditions with 5% CO2 at 37°C.
4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4-
The tested compounds were prepared as 20µM stock solu-
cyano-3-(trifluoromethyl)phenyl)benzamide (5h): Yield 89%; tion in dimethyl sulfoxide (DMSO) and diluted in the relevant
mp 107.5–109.8°C. 1H-NMR (DMSO-d6) δ: 11.96 (1H, s), assay media. DMSO was used as a negative control and adria-
10.89 (1H, s), 8.46 (1H, s), 8.26 (1H, d, J=8.4Hz), 8.16 (1H, mycin (ADM) was used as a positive control. Cells (3–5×103/
d, J=8.4Hz), 7.91 (2H, d, J=8.4Hz), 7.56 (2H, d, J=8.4Hz), well) were seeded in 200µL of medium/well in 96-well plates
6.08 (2H, s), 4.30 (2H, s). (ESI-MS) m/z: 419.1 (M+H)+. Anal. (Costar Corning, Rochester, NY, U.S.A.) for 24h, followed by
Calcd for C18H13F3N6OS: C, 51.67; H, 3.13; N, 20.09. Found: C, compounds treatment for 72h. A volume of 20µL of 5mg/mL
51.66; H, 3.15; N, 20.13.
MTT was added per well and incubated for another 2–4h at
4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4- 37°C, then the supernatant fluid was removed and 150µL/well
fluoro-3-(trifluoromethyl)phenyl)benzamide (5i): Yield 67%; DMSO was added. The 96-well plates were vibrated on micro-
mp 209.9–211.4°C. 1H-NMR (DMSO-d6) δ: 11.96 (1H, s), vibrator for additional 10min. The light absorptions (OD)
10.52 (1H, s), 8.26 (1H, d, J=8.0Hz), 8.07 (1H, d, J=8.0Hz), were measured at 570nm with SpectraMAXM5 microplate
7.89 (2H, d, J=8.4Hz), 7.62 (2H, d, J=8.4Hz), 7.53 (1H, d, spectrophotometer (Molecular Devices). The effect of com-
J=8.0Hz), 6.54 (2H, s), 4.37 (2H, s). (ESI-MS) m/z: 412.1 pounds on tumor cells viability was expressed by IC50 (con-
(M+H)+. Anal. Calcd for C17H13F4N5OS: C, 49.63; H, 3.19; N, centration causing 50% loss in cell viability) of each cell line.
17.02. Found: C, 49.61; H, 3.17; N, 17.06.
The EdU Incorporation Assay HCT116 cells growing in
4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(2- a 96-well plate (3×103 cells/well) were treated with compound
(trifluoromethyl)phenyl)benzamide (5j): Yield 69%; mp 5m for 24h. Then, the proliferation cells were assayed with an
1
211.5–214.6°C. H-NMR (DMSO-d6) δ: 11.98 (1H, s), 10.12 EdU-ApolloTM 567 DNA Proliferation Detection kit (Invitro-
(1H, s), 7.86 (2H, d, J=8.0Hz), 7.74 (2H, d, J=8.0Hz), 7.66 gen, Carlsbad, CA, U.S.A.) according to the manufacturer’s
(1H, m), 7.51 (2H, d, J=8.0Hz), 7.36–7.42 (1H, m), 6.07 (2H, instructions. Briefly, the cells were fixed with formaldehyde
s), 4.29 (2H, s). (ESI-MS) m/z: 394.1 (M+H)+. Anal. Calcd for for 15min and washed with PBS, then permeablized with
C17H14F3N5OS: C, 51.90; H, 3.59; N, 17.80. Found: C, 51.88; H, 0.2% Triton-100 for 5min, followed by incubation with the
3.61; N, 17.83.
EdU for 30min in dark. After incubation, cells were washed
4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(4- with 0.5% Triton-100 in PBS for 10min. For subsequent DNA
cyano-2-(trifluoromethyl)phenyl)benzamide (5k): Yield 73%; staining, sections were incubated with Hoechst 33342 (5µg/
mp 263.6–265.3°C. 1H-NMR (DMSO-d6) δ: 11.98 (1H, s), mL) for 10min. The plates were then washed with PBS twice
10.87 (1H, s), 8.47 (1H, s), 8.26 (1H, s), 8.12 (1H, s), 7.91 (2H, and observed with Thermo Array scan VTI (Thermo Scien-
d, J=8.4Hz), 7.58 (2H, d, J=8.4Hz), 6.09 (2H, s), 4.29 (2H, s). tific).
(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.
Acknowledgment This work was supported by Shijiaz-
4-((5-Amino-4H-1,2,4-triazol-3-ylthio)methyl)-N-(3-(tri- huang Pharmaceutical Group Co., Ltd.
fluoromethyl)phenyl)benzamide (5l): Yield 87%; mp 228.1–
1
229.7°C. H-NMR (DMSO-d6) δ: 11.96 (1H, s), 10.09 (1H,
s), 7.92 (1H, d, J=8.0Hz), 7.81 (2H, d, J=8.6Hz), 7.54 (1H,
d, J=8.0Hz), 7.43 (2H, d, J=8.4Hz), 7.26 (2H, d, J=8.0Hz),
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.
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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. H-NMR (DMSO-d6) δ: 11.96 (1H, s), 10.09
1
(1H, s), 7.87 (2H, d, J=8.0Hz), 7.80 (2H, d, J=8.0Hz), 7.74
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