Synthesis and biological evaluation of disulfides bearing 1,2,4-triazole moiety as antiproliferative agents

Article abstract of DOI:10.1007/s00044-017-2029-0

A series of novel nonsymmetrical disulfides bearing 1,2,4-triazole moiety were designed, synthesized, and evaluated for their in vitro antiproliferative activities against human cancer cell lines SMMC-7721, Hela, A549, and normal cell lines L929 by CCK-8 assay. The preliminary bioassay results demonstrated that most of the tested compounds 8a–r exhibited good antiproliferative activities, and some compounds showed better effects than positive control 5-fluorouracil against various cancer cell lines. Among these compounds, compound 8l showed significant antiproliferative activity against SMMC-7721 cells with IC₅₀ value of 2.97 μM. Compound 8f displayed highly effective biological activity against Hela cells with IC₅₀ value of 3.51 μM. Compound 8d exhibited the best inhibitory effect against A549 cells with IC₅₀ value of 2.79 μM. Furthermore, some of the tested compounds showed weak cytotoxic effect against the normal cell lines L929. The pharmacological results suggest that the substituent groups are vital for improving the potency and selectivity of this class of compounds.

Full text of DOI:10.1007/s00044-017-2029-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2029-0
ORIGINAL RESEARCH
Synthesis and biological evaluation of disulfides bearing
1,2,4-triazole moiety as antiproliferative agents
Xue-Feng Wang1 Shuai Zhang1 Bao-Lin Li1 Ji-Jun Zhao1 Yu-Ming Liu1
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Rui-Lian Zhang1 Bo Li1 Bao-Quan Chen1
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Received: 19 June 2017 / Accepted: 5 August 2017
© Springer Science+Business Media, LLC 2017
Abstract A series of novel nonsymmetrical disulfides
bearing 1,2,4-triazole moiety were designed, synthesized,
and evaluated for their in vitro antiproliferative activities
against human cancer cell lines SMMC-7721, Hela, A549,
and normal cell lines L929 by CCK-8 assay. The pre-
liminary bioassay results demonstrated that most of the
tested compounds 8a–r exhibited good antiproliferative
activities, and some compounds showed better effects than
positive control 5-fluorouracil against various cancer cell
lines. Among these compounds, compound 8l showed sig-
nificant antiproliferative activity against SMMC-7721 cells
with IC50 value of 2.97 μM. Compound 8f displayed highly
effective biological activity against Hela cells with IC50
value of 3.51 μM. Compound 8d exhibited the best inhibi-
tory effect against A549 cells with IC50 value of 2.79 μM.
Furthermore, some of the tested compounds showed weak
cytotoxic effect against the normal cell lines L929. The
pharmacological results suggest that the substituent groups
are vital for improving the potency and selectivity of this
class of compounds.
Introduction
Cancer is considered to be the second most common cause
of deaths after cardiovascular diseases in the world. Despite
significant progress has been achieved in anticancer ther-
apy, the discovery and development of effective anticancer
drugs remain one of the most intractable worldwide health
problems (Anand et al. 2008). During recent years, some
small molecules containing various azoles were synthesized
as potential anticancer agents and have become the subject
of considerable growing interest for the treatment of cancer
(Li et al. 2016; Wang et al. 2015; Yadagiri et al. 2015;
Wang et al. 2011).
1,2,4-Triazole is a versatile lead molecule for designing
potential bioactive agent, and its derivatives are associated
with many types of biological properties, including anti-
oxidant (Padmaja et al. 2015; Aktas-Yokus et al. 2015),
antibacterial (Li et al. 2015; Sokmen et al. 2013), anti-
inflammatory (Sarigol et al. 2015; El-Serwy et al. 2013; Li
et al. 2016), antifungal (Yu et al. 2013; Li et al. 2014),
anticonvulsant (Kahveci et al. 2014; Botros et al. 2013),
analgesic (Ashour et al. 2013; Vijesh et al. 2013), anti-
depressant (Patel et al. 2010; Chelamalla et al. 2017),
anticoagulant (Sun et al. 2017), and anti-thymidine phos-
phorylase effects (Bera et al. 2013). In particular, a great
number of 1,2,4-triazole derivatives have been proved to
show potent antitumor activities (Nagender et al. 2016;
Abdo and Kamel 2015; Kamel and Abdo 2014; Popiołek
et al. 2014; Hou et al. 2011; Wang et al. 2015; Zhao et al.
2012; Shi et al. 2013). Thus, the importance of 1,2,4-tria-
zole heterocyclic in the fields of medicinal chemistry has
drawn widespread attention during the last few decades.
Meanwhile, disulfide derivatives are also known to display
a wide spectrum of biological activities because the dis-
ulfide group is an important core found in many biologically
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Keywords Disulfides 1,2,4-Triazole Antiproliferative
activity
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-2029-0) contains supplementary material,
which is available to authorized users.
* Bao-Quan Chen
chenbaoquan66@126.com
1
School of Chemistry and Chemical Engineering, Tianjin Key
Laboratory of Organic Solar Cells and Photochemical Conversion,
Tianjin University of Technology, 300384 Tianjin, China

Med Chem Res
active compounds with many types of biological activities
including antibacterial (Park et al. 2009; Turos et al. 2008),
anti-HIV-1 (Cesarini et al. 2008), antitumor (Ryu et al.
2004; Hunter et al. 2008; Diraimondo et al. 2013; Rubino
et al. 2017), and β-glucuronidase inhibition properties (Teha
et al. 2016). Particularly, the antitumor character of disulfide
derivatives attracts great interests from medicinal chemists
in recent years.
Considering the biological significance of 1,2,4-triazole
and disulfide, we report herein the synthesis of non-
symmetrical disulfides bearing 1,2,4-triazole moiety.
Meanwhile, their in vitro antiproliferative activities were
evaluated against human cancer cell lines SMMC-7721
(human liver cancer cell), Hela (human cervix adenocarci-
noma cell), A549 (human lung cancer cell), and normal cell
lines L929 (mouse fibroblast cell) with the ultimate aim of
developing novel potent antitumor agents (Fig. 1).
Results and discussion
Chemistry
As depicted in Scheme 1, eighteen nonsymmetrical disulfides
bearing 1,2,4-triazole moiety 8a–r were synthesized and
reported for the first time. The preparation of S-alkyl-thioi-
sothiourea hydrochloride 2 was carried out by reported lit-
erature method (Sirakawa et al. 1970). 3-Substitutedphenyl-
4-amino-5-mercapto-1,2,4-triazole 7a–r were synthesized
from substituted benzoic acid in four steps including ester-
ification, hydrazidation, salt formation, and cyclization
according to reported literature method (Qiao et al. 2001;
Aswathanarayanappa et al. 2013). Finally, the target com-
pounds 8a–r were successfully obtained by the reaction of
intermediate 2 and compounds 7a–r in the presence of
NaHCO3 in ethanol and water at 0–10 °C. All newly syn-
thesized compounds 8a–r were purified by recrystallization
from ethanol/water and their structures were characterized by
1
IR, H NMR, 13C NMR, and High Resolution-Electrospray
Ionization-Mass Spectrometer (HR-ESI-MS).
N
N
Pharmacology and discussion
S
S
R1
N
The antiproliferative activities of the newly synthesized
compounds 8a–r were tested against SMMC-7721, Hela,
A549, and L929 cell lines using CCK-8 [2-(2-methoxy-4-
nitro-phenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-
tetrazolium monosodium salt] assay. Inhibition of cell
proliferation by these active compounds at various
NH2
8a-r
R2
Fig. 1 The structure of target compounds 8a–r
Scheme 1 Synthesis of the
target compounds 8a–r.
Reagents and conditions: a
Concd. HCl, H2O2(30%), 0–5 °C,
3 h; b SOCl2, ethanol, reflux,
4–6 h; c NH2NH2·H2O (85%),
ethanol, reflux, 6–8 h; d KOH,
CS2, ethanol, 0–5 °C, 4–6 h;
e NH2NH2·H2O (85%), ethanol,
reflux, 6–8 h; f Compound 2,
ethanol, NaHCO3/H2O, 0–10 °C,
6–8 h

Med Chem Res
Table 1 In vitro antiproliferative activities of compounds 8a–r against
various cell lines
concentrations was measured, and their IC50 (the con-
centration that causes a 50% cell proliferation inhibition)
values were calculated and are summarized in Table 1. 5-
Fluorouracil (5-FU) was used as positive control.
a
Compound
IC50 (µM)
SMMC-7721
Hela
A549
L929
As shown in Table 1, the newly synthesized compounds
8a–r displayed very good antiproliferative effects against
SMMC-7721 cells with IC50 values ranging from 2.97 to
6.62 μM. Meanwhile, the majority of them showed better
activities than positive control 5-fluorouracil.
8a
8b
8c
6.62 0.18
3.80 0.16
4.64 0.17
4.36 0.21
3.80 0.18
4.64 0.24
4.09 0.20
3.79 0.12
4.37 0.09
3.24 0.14
3.24 0.10
2.97 0.07
3.24 0.19
3.79 0.08
5.22 0.23
5.49 0.09
3.79 0.11
3.52 0.14
5.62 0.28
17.31 1.19
7.75 0.27
7.74 0.18
6.34 0.32
7.17 0.41
3.51 0.13
6.05 0.25
7.10 0.38
7.17 0.15
6.62 0.36
5.21 0.17
4.37 0.13
7.74 0.29
7.75 0.34
19.84 1.78
42.92 3.36
6.05 0.29
9.15 0.45
17.21 0.67
13.38 1.28
8.87 0.39
8.30 0.30
2.79 0.09
6.60 0.34
6.62 0.28
5.77 0.29
7.47 0.40
7.17 0.35
4.37 0.22
5.49 0.27
5.59 0.19
4.92 0.23
6.05 0.38
8.30 0.45
37.59 2.88
8.30 0.48
8.02 0.38
8.13 0.34
9.44 0.43
8.94 0.35
8.91 0.26
4.65 0.12
7.11 0.56
5.95 0.32
15.46 1.29
7.07 0.37
7.26 0.22
5.84 0.15
7.37 0.23
8.11 0.48
8.49 0.39
17.60 1.27
26.92 2.05
32.77 2.37
9.87 0.47
8.80 0.39
2.98 0.15
8d
8e
8f
Especially noteworthy is compound 8l possessing i-
propyl group while R2 is chlorine atom, which showed
significant antiproliferative activity with IC50 value of 2.97
μM. But, no matter R2 is hydrogen or chlorine atom, the
effects of R1 substituents on antiproliferative activity
against SMMC-7721 cells did not show apparent regularity.
In Hela cells, except compounds 8a, 8o, and 8p which
showed moderate antitumor activities, the other compounds
exhibited good antiproliferative effects with IC50 values
ranging from 3.51 to 9.15 μM, and displayed higher activ-
ities than positive control 5-fluorouracil. In particular,
compound 8f carrying n-pentyl substituent while R2 is
hydrogen atom displayed highly effective biological activity
with IC50 value of 3.51 μM. However, compound 8p pos-
sessing n-hexyl substituent while R2 is chlorine atom
showed poor activity with IC50 value of 42.92 μM. In A549
cells, most of the newly synthesized compounds exhibited
good antiproliferative activities and showed better effects
than positive control 5-fluorouracil. Among them, com-
pound 8d possessing n-butyl substituent while R2 is
hydrogen atom exhibited the best inhibitory effect with IC50
value of 2.79 μM. With the exception of compound 8d,
while R2 is hydrogen atom, on the whole the shorter alkyl
chain-substituted derivatives, including 8a, 8b, and 8c,
showed weaker activities than the longer alkyl chain-
substituted derivatives, including 8e, 8f, and 8g. But, while
R2 is chlorine atom, on the whole the shorter alkyl chain-
substituted derivatives, including 8j, 8k, and 8l, exhibited
better activities than the longer alkyl chain-substituted
derivatives, including 8n, 8o, and 8p. These results indi-
cated that the influence of substituents on antiproliferative
activity against A549 cells is complex and unexplained at
present. These results also suggest that this class of com-
pounds displayed stronger antiproliferative effects against
SMMC-7721 cells than Hela and A549 cells on the whole.
We also found from the results that compounds 8h and 8i
possessing benzyl and 4-cholobenzyl substituents, respec-
tively, while R2 is hydrogen atom, showed comparable
activities against Hela and A549 cells, and compounds 8q
and 8r possessing benzyl and 4-cholobenzyl substituents,
respectively, while R2 is chlorine atom, displayed similar
effects against SMMC-7721 and A549 cells. Furthermore,
some of the tested compounds showed weak cytotoxic
effect against the normal cell lines L929. In particular, as
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
5-FUb
Each experiment was independently performed three times and
expressed as mean standard deviation (SD)
a
IC50 is defined as the concentration that causes a 50 % cell
proliferation inhibition
b
Used as positive control
compared with SMMC-7721 cells, all compounds 8a–r
exhibited weaker cytotoxic activity against L929 cells, and
most of the compounds showed highly lower cytotoxic
effect than 5-fluorouracil. Therefore, the results indicate
ample scope for screening hitherto relatively unexplored
disulfides bearing 1,2,4-triazole moiety as antiproliferative
agents.
Conclusion
In summary, a series of novel nonsymmetrical disulfides
bearing 1,2,4-triazole moiety were designed, synthesized,
and evaluated for their in vitro antiproliferative activities
against SMMC-7721, Hela, and A549 human cancer cell
lines by CCK-8 assay. The majority of tested compounds
8a–r inhibited the proliferation better than positive control
5-fluorouracil. In particular, compound 8l showed sig-
nificant antiproliferative activity against SMMC-7721 cells
with IC50 value of 2.97 μM. Compound 8f displayed highly
effective biological activity against Hela cells with IC50
value of 3.51 μM. Compound 8d exhibited the best inhibi-
tory effect against A549 cells with IC50 value of 2.79 μM.
Furthermore, some of the tested compounds showed weak
cytotoxic effect against the normal cell lines L929.

Med Chem Res
Therefore, the results will be significant in the development
of potent antitumor agents.
128.97, 128.46, 127.24, 41.13, 21.96, 13.19. HR-ESI-MS
(m/z): Calcd for C11H14N4S2: 267.0738 [M + H]+, Found
267.0743.
Experimental
Chemistry
3-Phenyl-4-amino-5-(i-propyldisulfanyl)-1,2,4-triazole
(8c) White solid, Yield 49%, m.p.: 105.3–106.8 °C. IR
(KBr, cm–1): 3464, 3415, 3134, 2360, 2342, 1638, 1619,
1452, 1401, 1284, 1242, 1109, 977, 769, 725, 710, 693. 1H
NMR (DMSO-d6, 400 MHz) δ (ppm): 8.03–8.05 (m, 2H,
Ar–H), 7.53–7.56 (m, 3H, Ar–H), 6.25 (s, 2H, NH2), 3.39
(m, 1H, SCH), 1.34 (d, J = 6.8 Hz, 6H, CH3). 13C NMR
(DMSO-d6, 100 MHz) δ (ppm): 154.79, 153.20, 130.39,
128.97, 128.45, 127.21, 41.64, 22.47. HR-ESI-MS (m/z):
Calcd for C11H14N4S2: 267.0738 [M + H]+, Found
267.0741.
Melting points were determined by a X-6 microscope
melting point apparatus and are uncorrected. Infrared
spectra were recorded in KBr pellets on a Nicolet Avatar
370 spectrometer. NMR spectra were performed on a Bru-
ker Avance III 400 MHz spectrometer (1H, 400 MHz; 13C,
100 MHz) using DMSO-d6 as the solvent and tetra-
methylsilane as the internal standard. Chemical shifts are
showed in and 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. Unless
otherwise noted, all solvents and reagents were obtained
from commercial suppliers and used without further
purification.
3-Phenyl-4-amino-5-(n-butyldisulfanyl)-1,2,4-triazole
(8d) White solid, Yield 50%, m.p.: 102.4–104.1 °C. IR
(KBr, cm–1): 3472, 3413, 3132, 2360, 1637, 1401, 1111,
767, 691. 1H NMR (DMSO-d6, 400 MHz) δ (ppm):
8.03–8.05 (m, 2H, Ar–H), 7.53–7.56 (m, 3H, Ar–H), 6.26
(s, 2H, NH2), 2.99 (t, J = 7.2 Hz, 2H, SCH2), 1.68–1.75 (m,
2H, CH2), 1.34–1.43 (m, 2H, CH2), 0.89 (t, J = 7.2 Hz, 3H,
CH3). 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 154.85,
153.02, 130.39, 128.98, 128.46, 127.23, 38.37, 30.64,
21.36, 13.96. HR-ESI-MS (m/z): Calcd for C12H16N4S2:
281.0895 [M + H]+, Found 281.0900.
General procedure for the preparation of compounds (8a–r)
S-Alkyl-thioisothiourea hydrochloride 2 (2.0 mmol) and 3-
substitutedphenyl-4-amino-5-merca-pto-1,2,4-triazole 7a–r
(2.0 mmol) were dissolved in 15 ml ethanol. A solution of
NaHCO3 (4.0 mmol) in 15 ml water was added dropwise
with vigorous stirring at 0–10 °C. The mixture was stirred
for additional 6–8 h. The insoluble solid was collected and
further purified by recrystallization from ethanol/water to
afford the desired products.
3-Phenyl-4-amino-5-(i-butyldisulfanyl)-1,2,4-triazole
(8e) White solid, Yield 47%, m.p.: 104.2–105.3 °C. IR
(KBr, cm–1): 3472, 3413, 3133, 2361, 1638, 1618, 1401,
1
1110, 990, 767, 712, 692. H NMR (DMSO-d6, 400 MHz)
3-Phenyl-4-amino-5-ethyldisulfanyl-1,2,4-triazole (8a)
White solid, Yield 50%, m.p.: 120.8–122.5 °C. IR (KBr,
cm–1): 3474, 3413, 3134, 2362, 1638, 1618, 1472, 1453,
1401, 1284, 1242, 975, 769, 690. 1H NMR (DMSO-d6, 400
MHz) δ (ppm): 8.03–8.05 (m, 2H, Ar–H), 7.53–7.56 (m,
3H, Ar–H), 6.26 (s, 2H, NH2), 2.97–3.02 (q, J = 7.2 Hz,
2H, SCH2), 1.36 (t, J = 7.2 Hz, 3H, CH3). 13C NMR
(DMSO-d6, 100 MHz) δ (ppm): 154.86, 153.06, 130.39,
128.97, 128.47, 127.23, 32.74, 14.44. HR-ESI-MS (m/z):
Calcd for C10H12N4S2: 253.0582 [M + H]+, Found
253.0590.
δ (ppm): 8.03–8.05 (m, 2H, Ar–H), 7.53–7.56 (m, 3H,
Ar–H), 6.26 (s, 2H, NH2), 2.90 (d, J = 6.8 Hz, 2H, SCH2),
1.98–2.09 (m, 1H, CH), 0.97 (d, J = 6.8 Hz, 6H, CH3). 13
C
NMR (DMSO-d6, 100 MHz) δ (ppm): 154.86, 152.98,
130.40, 128.98, 128.45, 127.22, 47.80, 27.79, 21.83. HR-
ESI-MS (m/z): Calcd for C12H16N4S2: 281.0895 [M + H]+,
Found 281.0898.
3-Phenyl-4-amino-5-(n-pentyldisulfanyl)-1,2,4-triazole
(8f) White solid, Yield 46%, m.p.: 99.9–101.7 °C. IR
(KBr, cm–1): 3474, 3413, 3134, 1638, 1453, 1401, 1136,
1
3-Phenyl-4-amino-5-(n-propyldisulfanyl)-1,2,4-triazole
(8b) White solid, Yield 50%, m.p.: 109.6–111.4 °C. IR
(KBr, cm–1): 3471, 3413, 3130, 1638, 1618, 1401, 1110,
975, 769, 690. H NMR (DMSO-d6, 400 MHz) δ (ppm):
8.03–8.05 (m, 2H, Ar–H), 7.53–7.56 (m, 3H, Ar–H), 6.25
(s, 2H, NH2), 2.99 (t, J = 7.2 Hz, 2 H, SCH2), 1.70–1.77
(m, 2H, CH2), 1.28–1.36 (m, 4H, CH2), 0.87 (t, J = 7.2 Hz,
3H, CH3). 13C NMR (DMSO-d6, 100 MHz) δ (ppm):
154.85, 153.01, 130.39, 128.97, 128.46, 127.23, 38.69,
30.36, 28.23, 21.14, 14.29. HR-ESI-MS (m/z): Calcd for
C13H18N4S2: 295.1051 [M + H]+, Found 295.1053.
1
993, 766, 691. H NMR (DMSO-d6, 400 MHz) δ (ppm):
8.03–8.05 (m, 2H, Ar–H), 7.53–7.56 (m, 3H, Ar–H), 6.25
(s, 2H, NH2), 2.97 (t, J = 7.2 Hz, 2H, SCH2), 1.72–1.81 (m,
2H, CH2), 0.91 (t, J = 7.2 Hz, 3H, CH3). 13C NMR
(DMSO-d6, 100 MHz) δ (ppm): 154.86, 153.01, 130.39,

Med Chem Res
1
3-Phenyl-4-amino-5-(n-hexyldisulfanyl)-1,2,4-triazole
(8g) White solid, Yield 51%, m.p.: 103.8–104.5 °C. IR
(KBr, cm–1): 3474, 3413, 3134, 2362, 1638, 1618, 1453,
1275, 1243, 1232, 1014, 974, 904, 828, 767, 683. H NMR
(DMSO-d6, 400 MHz) δ (ppm): 8.09 (d, J = 8.4 Hz, 2H,
Ar–H), 7.63 (d, J = 8.4 Hz, 2H, Ar–H), 6.28 (s, 2H, NH2),
2.96 (t, J = 7.2 Hz, 2H, SCH2), 1.71–1.80 (m, 2H, CH2),
0.96 (t, J = 7.2 Hz, 3H, CH3). 13C NMR (DMSO-d6, 100
MHz) δ (ppm): 153.88, 153.34, 135.29, 130.13, 129.13,
126.04, 41.11, 21.94, 13.19. HR-ESI-MS (m/z): Calcd for
C11H13ClN4S2: 301.0349 [M + H]+, Found 301.0348.
1
1401, 975, 769, 690. H NMR (DMSO-d6, 400 MHz) δ
(ppm): 8.03–8.05 (m, 2H, Ar–H), 7.53–7.55 (m, 3H, Ar–H),
6.25 (s, 2H, NH2), 2.99 (d, J = 7.2 Hz, 2H, SCH2),
1.69–1.76 (m, 2H, CH2), 1.25–1.37 (m, 6H, CH2), 0.86 (t, J
= 6.8 Hz, 3H, CH3). 13C NMR (DMSO-d6, 100 MHz) δ
(ppm): 154.56, 153.57, 129.40, 128.95, 128.45, 127.36,
38.65, 31.26, 28.47, 27.80, 22.44, 14.32. HR-ESI-MS (m/
z): Calcd for C14H20N4S2: 309.1208 [M + H]+, Found
309.1210.
3-(4-Chlorophenyl)-4-amino-5-(i-propyldisulfanyl)-1,2,4-
triazole (8l) Pale yellow solid, Yield 51%, m.p.:
115.6–116.9 °C. IR (KBr, cm–1): 3474, 3413, 3134, 2863,
2360, 2027, 1638, 1618, 1474, 1447, 1401, 1154, 1014,
1
3-Phenyl-4-amino-5-benzyldisulfanyl-1,2,4-triazole (8h)
White solid, Yield 51%, m.p.: 129.3–130.6 °C. IR (KBr,
cm–1): 3475, 3413, 3144, 2360, 1638, 1618, 1469, 1453,
975, 950, 931, 832, 768, 749, 725, 695, 682. H NMR
(DMSO-d6, 400 MHz) δ (ppm): 8.09 (d, J = 8.8 Hz, 2H,
Ar–H), 7.63 (d, J = 8.4 Hz, 2H, Ar–H), 6.27 (s, 2H, NH2),
3.38 (m, 1H, SCH), 1.34 (d, J = 6.8 Hz, 6H, CH3). 13C
NMR (DMSO-d6, 100 MHz) δ (ppm): 153.82, 153.51,
135.26, 130.12, 129.13, 126.05, 41.68, 22.46. HR-ESI-MS
(m/z): Calcd for C11H13ClN4S2: 301.0349 [M + H]+, Found
301.0350.
1
1401, 1238, 1137, 970, 863, 773, 764, 715, 695. H NMR
(DMSO-d6, 400 MHz) δ (ppm): 8.04–8.07 (m, 2H, Ar–H),
7.54–7.57 (m, 3 H, Ar–H), 7.28–7.43 (m, 5H, Ar–H), 6.25
(s, 2H, NH2), 4.27 (s, 2H, SCH2). 13C NMR (DMSO-d6,
100 MHz) δ (ppm): 154.88, 153.05, 137.07, 130.45, 129.99,
129.01, 128.97, 128.50, 128.02, 127.19, 42.55. HR-ESI-MS
(m/z): Calcd for C15H14N4S2: 315.0738 [M + H]+, Found
315.0742.
3-(4-Chlorophenyl)-4-amino-5-(n-butyldisulfanyl)-1,2,4-
triazole (8m) White solid, Yield 53%, m.p.: 104.5–105.9 °C.
IR (KBr, cm–1): 3475, 3414, 3126, 2956, 2929, 2360, 2027,
1638, 1618, 1452, 1401, 1273, 1243, 1220, 1114, 996, 834,
747, 730, 681. 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.09
(d, J = 8.8 Hz, 2H, Ar–H), 7.63 (d, J = 8.4 Hz, 2H, Ar–H),
6.27 (s, 2H, NH2), 2.99 (t, J = 7.2 Hz, 2H, SCH2), 1.68–1.75
(m, 2H, CH2), 1.34–1.43 (m, 2H, CH2), 0.89 (t, J = 7.2 Hz,
3H, CH3). 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 153.88,
153.34, 135.29, 130.12, 129.13, 126.05, 38.40, 30.63, 21.36,
13.95. HR-ESI-MS (m/z): Calcd for C12H15ClN4S2: 315.0505
[M + H]+, Found 315.0504.
3-Phenyl-4-amino-5-(4-chlorobenzyldisulfanyl)-1,2,4-tria-
zole (8i) White solid, Yield 48%, m.p.: 148.4–149.8 °C.
IR (KBr, cm–1): 3475, 3413, 3147, 1656, 1595, 1489, 1465,
1
1402, 991, 837, 825, 804, 772, 739, 718, 693. H NMR
(DMSO-d6, 400 MHz) δ (ppm): 8.04–8.06 (m, 2H, Ar–H),
7.54–7.56 (m, 3H, Ar–H), 7.41–7.47 (AB × 2, JAB = 8.8
Hz, 4H, Ar–H), 6.26 (s, 2H, NH2), 4.25 (s, 2H, SCH2). 13
C
NMR (DMSO-d6, 100 MHz) δ (ppm): 155.04, 153.93,
136.18, 134.55, 131.71, 130.89, 129.40, 128.96, 128.49,
126.26, 41.38. HR-ESI-MS (m/z): Calcd for C15H13ClN4S2:
349.0349 [M + H]+, Found 349.0352.
3-(4-Chlorophenyl)-4-amino-5-(i-butyldisulfanyl)-1,2,4-
triazole (8n) Pale yellow solid, Yield 48%, m.p.:
101.2–103.1 °C. IR (KBr, cm–1): 3473, 3413, 3137, 2966,
2870, 2360, 1638, 1618, 1472, 1449, 1401, 1242, 1014,
3-(4-Chlorophenyl)-4-amino-5-ethyldisulfanyl-1,2,4-tria-
zole (8j) Pale yellow solid; Yield 48%, m.p.: 107.6–109.2
°C. IR (KBr, cm–1): 3474, 3414, 3146, 2361, 1638, 1618,
1474, 1448, 1401, 1276, 1244, 1012, 974, 925, 833, 782,
768, 751, 724, 681. 1H NMR (DMSO-d6, 400 MHz) δ
(ppm): 8.09 (d, J = 8.4 Hz, 2H, Ar–H), 7.63 (d, J = 8.8 Hz,
2H, Ar–H), 6.28 (s, 2H, NH2), 2.96–3.02 (q, J = 7.2 Hz,
2H, SCH2), 1.35 (t, J = 7.2 Hz, 3H, CH3). 13C NMR
(DMSO-d6, 100 MHz) δ (ppm): 153.89, 153.39, 135.28,
130.13, 129.13, 126.05, 32.74, 14.43. HR-ESI-MS (m/z):
Calcd for C10H11ClN4S2: 287.0192 [M + H]+, Found
287.0195.
1
946, 829, 767, 750, 725, 695, 682. H NMR (DMSO-d6,
400 MHz) δ (ppm): 8.09 (d, J = 8.4 Hz, 2H, Ar–H), 7.63 (d,
J = 8.4 Hz, 2H, Ar–H), 6.28 (s, 2H, NH2), 2.90 (d, J = 6.8
Hz, 2H, SCH2), 1.98–2.10 (m, 1H, CH), 0.97 (d, J = 6.8
Hz, 6H, CH3). 13C NMR (DMSO-d6, 100 MHz) δ (ppm):
153.89, 153.30, 135.28, 130.12, 129.14, 126.06, 47.81,
27.78, 21.83. HR-ESI-MS (m/z): Calcd for C12H15ClN4S2:
315.0505 [M + H]+, Found 315.0502.
3-(4-Chlorophenyl)-4-amino-5-(n-pentyldisulfanyl)-1,2,4-
triazole (8o) White solid, Yield 50%, m.p.: 107.7–109.3 °C.
IR (KBr, cm–1): 3473, 3414, 3128, 2926, 2857, 2361, 2342,
2027, 1638, 1618, 1452, 1401, 1273, 1244, 1093, 995, 952,
833, 747, 730, 690. 1H NMR (DMSO-d6, 400 MHz) δ (ppm):
3-(4-Chlorophenyl)-4-amino-5-(n-propyldisulfanyl)-1,2,4-
triazole (8k) White solid, Yield 48%, m.p.: 110.4–112.3 °C.
IR (KBr, cm–1): 3473, 3413, 3133, 2360, 1638, 1618, 1401,

Med Chem Res
8.09 (d, J = 8.8 Hz, 2H, Ar–H), 7.63 (d, J = 8.4 Hz, 2H,
Ar–H), 6.27 (s, 2 H, NH2), 2.98 (t, J = 6.8 Hz, 2H, SCH2),
1.71–1.75 (m, 2H, CH2), 1.24–1.37 (m, 4H, CH2), 0.88 (t, J
= 6.8 Hz, 3H, CH3). 13C NMR (DMSO-d6, 100 MHz) δ
(ppm): 153.87, 153.35, 135.27, 130.12, 129.15, 126.04, 38.65,
30.35, 28.21, 22.15, 14.30. HR-ESI-MS (m/z): Calcd for
C13H17ClN4S2: 329.0661 [M + H]+, Found 329.0667.
Compounds 8a–r and 5-fluorouracil were respectively
dissolved in DMSO to make stock solutions at a con-
centration 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 expo-
nential 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. The medium containing different con-
centrations of compounds was replaced with fresh medium,
at 37 °C, 5% CO2 incubator for 48 h. Then 90 μL of fresh
medium and 10 μL of CCK-8 were added into culture plates
and cultivated at the same conditions for 1 h. The sample
cell was added into 96-well microplate reader and the plate
was read at 450 nm and the absorbance value (OD) was
recorded. Cell viability was calculated from the mean values
for three wells using the following formula:
3-(4-Chlorophenyl)-4-amino-5-(n-hexyldisulfanyl)-1,2,4-
triazole (8p) White solid; Yield 55%, m.p.: 110.4–112.1 °C.
IR (KBr, cm–1): 3474, 3414, 3126, 2957, 2926, 2855, 2360,
2342, 2027, 1638, 1618, 1451, 1401, 1243, 1095, 998, 836,
749, 731, 679. 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.09
(d, J = 8.8 Hz, 2H, Ar–H), 7.63 (d, J = 8.4 Hz, 2H, Ar–H),
6.27 (s, 2H, NH2), 2.98 (t, J = 7.2 Hz, 2H, SCH2), 1.67–1.76
(m, 2H, CH2), 1.26–1.37 (m, 6H, CH2), 0.87 (t, J = 6.4 Hz,
3H, CH3). 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 153.86,
153.32, 135.27, 130.11, 129.13, 126.07, 38.76, 31.24, 28.49,
27.82, 22.44, 14.33. HR-ESI-MS (m/z): Calcd for
C14H19ClN4S2: 343.0818 [M + H]+, Found 343.0817.
Relative cell viability ¼ ðOD value for the test group À
blank ODÞ=ðcontrol OD value À blank OD valueÞ Â 100%
3-(4-Chlorophenyl)-4-amino-5-benzyldisulfanyl-1,2,4-tria-
zole (8q) White solid, Yield 52%, m.p.: 152.9–154.2 °C.
IR (KBr, cm–1): 3474, 3413, 3125, 2260, 2341, 1638, 1618,
1599, 1399, 1013, 971, 914, 862, 834, 766, 750, 731, 697,
1H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.11 (d, J = 8.4
Hz, 2H, Ar–H), 7.64 (d, J = 8.8 Hz, 2H, Ar–H), 7.28–7.42
The IC50 value is defined as the concentration that causes
a 50% cell proliferation inhibition.
Acknowledgements We are grateful to the National Natural Science
Foundation of China (20971097) and Tianjin Municipal Natural Sci-
ence Foundation (13JCYBJC24500) for financial support.
(m, 5H, Ar–H), 6.27 (s, 2H, NH2), 4.27 (s, 2H, SCH2). 13
C
NMR (DMSO-d6, 100 MHz) δ (ppm): 153.88, 153.33,
137.03, 135.31, 130.16, 129.97, 129.16, 128.96, 128.02,
126.05, 42.63. HR-ESI-MS (m/z): Calcd for C15H13ClN4S2:
349.0349 [M + H]+, Found 349.0341.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
3-(4-Chlorophenyl)-4-amino-5-(4-chlorobenzyldisulfanyl)-
1,2,4-triazole (8r) White solid; Yield 48%, m.p.:
125.3–126.7 °C. IR (KBr, cm–1): 3472, 3415, 3133, 2361,
2342, 1638, 1618, 1490, 1401, 1109, 994, 866, 831, 732,
References
1
Abdo NYM, Kamel MM (2015) Synthesis and anticancer evaluation
of 1,3,4-oxadiazoles, 1,3,4-thiadiazoles, 1,2,4-triazoles and
mannich bases. Chem Pharm Bull 63:369–376
Aktas-Yokus O, Yuksek H, Gursoy-Kol O, Alpay-Karaoglu S (2015)
Synthesis and biological evaluation of new 1,2,4-triazole deri-
vatives with their potentiometric titrations. Med Chem Res
24:2813–2824
Anand P, Kunnumakara AB, Sundaram C, Harikumar KB, Tharakan
ST, Lai OS, Sung B, Aggarwal BB (2008) Cancer is a pre-
ventable disease that requires major lifestyle changes. Pharm Res
25:2097–2116
719. H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.10 (d, J =
8.4 Hz, 2H, Ar–H), 7.64 (d, J = 8.4 Hz, 2H, Ar–H),
7.41–7.46 (q, J = 8.8 Hz, 4H, Ar–H), 6.28 (s, 2H, NH2), 4.24
(s, 2H, SCH2). 13C NMR (DMSO-d6, 100 MHz) δ (ppm):
153.88, 153.16, 136.26, 135.32, 132.73, 131.80, 130.16,
129.15, 128.98, 126.02, 41.53. HR-ESI-MS (m/z): Calcd for
C15H12Cl2N4S2: 382.9959 [M + H]+, Found 382.9960.
Ashour HM, Shaaban OG, Rizk OH, El-Ashmawy IM (2013)
Synthesis and biological evaluation of thieno[2′,3′:4,5]pyrimido
[1,2-b][1,2,4]triazines and thieno[2,3-d][1,2,4]triazolo[1,5-a]
pyrimi-dines as anti-inflammatory and analgesic agents. Eur J
Med Chem 62:341–351
Aswathanarayanappa C, Bheemappa E, Bodke YD, Krishnegowda PS,
Venkata SP, Ningegowda R (2013) Synthesis and evaluation of
antioxidant properties of novel 1,2,4-triazole-based schiff base
heterocycles. Arch Pharm Chem Life Sci 346:922–930
Biological assay
The following established in vitro cell lines were used:
SMMC-7721, Hela, A549, and L929, which were obtained
from theTumor Cell Resources Bank, Chinese Academy of
MedicalSciences. Cell counting kit-8 was from Dojindo
(Japan).

Med Chem Res
Bera H, Lee MH, Sun LY, Dolzhenko AV, Chui YK (2013) Synthesis,
anti-thymidine phosphorylase activity and molecular docking of
Patel NB, Khan IH, Rajani SD (2010) Pharmacological evaluation and
characterizations of newly synthesized 1,2,4-triazoles. Eur J Med
Chem 45:4293–4299
5-thioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin
-7-ones. Bioorg
Chem 50:34–40
Popiołek Ł, Rzymowska J, Kosikowska U, Hordyjewska A, Wujec M,
Malm A (2014) Synthesis, antiproliferative and antimicrobial
activity of new Mannich bases bearing 1,2,4-triazole moiety. J
Enzyme Inhib Med Chem 29:786–795
Botros S, Khalil NA, Naguib BH, El-Dash Y (2013) Synthesis and
anticonvulsant activity of new phenytoin derivatives. Eur J Med
Chem 60:57–63
Chelamalla R, Akena V, Manda S (2017) Synthesis of N′-arylidene-2-
(5-aryl-1H-1, 2, 4-triazol-3-ylthio)acetohydrazides as anti-
depressants. Med Chem Res 26:1359–1366
Qiao RZ, Hui XP, Xu PF, Zhang ZY, Cheng DL (2001) Microwave
induced synthesis of 3-aryl-6-(6-/8-substituted 4-chloroquinoline-
3-yl)-s-triazolo [3,4-b]-1,3,4-thiadiazoles. Chin
J
Chem
Cesarini S, Spallarossa A, Ranise A, Schenone S, Bruno O, Colla PL,
Casula L, Collu G, Sanna G, Loddo R (2008) Parallel one-pot
synthesis and structure-activity relationship study of symmetric
formimidoester disulfides as a novel class of potent non-
nucleoside HIV-1 reverse transcriptase inhibitors. Bioorg Med
Chem 16:6353–6363
19:800–806
Rubino S, Busà R, Attanzio A, Alduina R, Stefano VD, Girasolo MA,
Orecchio S, Tesoriere L (2017) Synthesis, properties, antitumor
and antibacterial activity of new Pt(II) and Pd(II) complexes with
2,20-dithiobis(benzothiazole) ligand. Bioorg Med Chem
25:2378–2386
Diraimondo TR, Plugis NM, Jin X, Khosla C (2013) Selective inhi-
bition of extracellular thioredoxin by asymmetric disulfides. J
Med Chem 56:1301–1310
El-Serwy WS, Mohamed NA, Abbas EM, Abdel-Rahman RF (2013)
Synthesis and anti-inflammatory properties of novel 1,2,4-triazole
derivatives. Res Chem Intermed 39:2543–2554
Hunter R, Kaschula CH, Parker IM, Caira MR, Richards P, Travis S,
Taute F, Qwebani T (2008) Substituted ajoenes as novel anti-
cancer agents. Bioorg Med Chem Lett 18:5277–5279
Hou YP, Sun J, Pang ZH, Lv PC, Li DD, Yan L, Zhang HJ, Zheng
EX, Zhao J, Zhu HL (2011) Synthesis and antitumor activity of
1,2,4-triazoles having 1,4-benzodioxan fragment as a novel class
of potent methionine aminopeptidase type II inhibitors. Bioorg
Med Chem 19:5948–5954
Ryu EK, Choe YS, Byun SS, Lee KH, Chi DY, Choi Y, Kim BT
(2004) Synthesis of radioiodine labeled dibenzyl disulfide for
evaluation of tumor cell uptake. Bioorg Med Chem 12:859–864
Sarigol D, Uzgoren-Baran A, Tel BC, Somuncuoglu EI, Kazkayasi I,
Ozadali-Sari K, Unsal-Tan O, Okay G, Ertan M, Tozkoparan B
(2015) Novel thiazolo[3,2-b]-1,2,4-triazoles derived from
naproxen with analgesic/anti-inflammatory properties: Synthesis,
biological evaluation and molecular modeling studies. Bioorg
Med Chem 23:2518–2528
Shi YJ, Song XJ, Li X, Ye TH, Xiong Y, Yu LT (2013) Synthesis and
biological evaluation of 1,2,4-triazole and 1,3,4-thiadiazole
derivatives as potential cytotoxic agents. Chem Pharm Bull
61:1099–1104
Sirakawa K, Aki O, Tsujikawa T, Tsuda
T
(1970) S-
Kahveci B, Menteşe E, Akkaya E, Yılmaz F, Doğan IS, Özel A (2014)
Synthesis of some novel 1,2,4-triazol-3-one derivatives bearing
the salicyl moiety and their anticonvulsant activities. Arch Pharm
Chem Life Sci 347:449–455
Kamel MM, Abdo NYM (2014) Synthesis of novel 1,2,4-triazoles,
triazolothiadiazines and triazolothiadiazoles as potential antic-
ancer agents. Eur J Med Chem 86:75–80
Li BL, Li B, Zhang RL, Zhao JJ, Wang XF, Liu YM, Shi YP,
Liu JB, Chen BQ (2016) Synthesis and antiproliferative
evaluation of novel 1,2,4-triazole derivatives incorporating
benzisoselenazolone scaffold. Bioorg Med Chem Lett 26:
1279–1281
Li C, Liu JC, Li YR, Gou C, Zhang ML, Liu HY, Li XZ, Zheng CJ,
Piao HR (2015) Synthesis and antimicrobial evaluation of 5-aryl-
1,2,4-triazole-3-thione derivatives containing a rhodanine moiety.
Bioorg Med Chem Lett 25:3052–3056
Alkylthioisothioureas. I. Chem Pharm Bull 18:235–242
Sokmen BB, Gumrukcuoglu N, Ugras S, Ugras HI, Yanardag R
(2013) Synthesis, antibacterial, antielastase, antiurease and anti-
oxidant activities of new methoxy substituted bis-1,2,4- triazole
derivatives. J Enzyme Inhib Med Chem 28:72–77
Sun XQ, Hong ZX, Liu MY, Guo S, Yang D, Wang Y, Lan T, Gao
LY, Qi HX, Gong P, Liu YJ (2017) Design, synthesis, and bio-
logical activity of novel tetrahydropyrazolopyridone derivatives
as FXa inhibitors with potent anticoagulant activity. Bioorg Med
Chem 25:2800–2810
Teha M, Ismail NH, Imran S, Wadood A, Rahim F, Muqarrabin
LMRA, Ahmat N (2016) Synthesis of novel disulfide and sulfone
hybrid scaffolds as potent β-glucuronidase inhibitor. Bioorg
Chem 68:15–22
Turos E, Revell KD, Ramaraju P, Gergeres DA, Greenhalgh K, Young
A, Sathyanarayan N, Dickey S, Lim D, Alhamadsheh MM,
Reynolds K (2008) Unsymmetric aryl-alkyl disulfide growth
inhibitors of methicillin-resistant staphylococcus aureus and
bacillus anthracis. Bioorg Med Chem 16:6501–6508
Li LJ, Ding H, Wang BJ, Yu SC, Zou Y, Chai XY, Wu QY (2014)
Synthesis and evaluation of novel azoles as potent antifungal
agents. Bioorg Med Chem Lett 24:192–194
Li YF, Zhang HJ, Quan ZS (2016) Synthesis and anti-inflammatory
activity evaluation of novel 3-alkyl-6-(4H-1,2,4-triazol-4-yl)-3,4-
dihydro-2H-benzo[e][1,3]oxazine derivatives. Med Chem Res
25:2280–2288
Vijesh AM, Isloor AM, Shetty P, Sundershan S, Fun HK (2013) New
pyrazole derivatives containing 1,2,4-triazoles and benzoxazoles
as potent antimicrobial and analgesic agents. Eur J Med Chem
62:410–415
Nagender P, Kumar RN, Reddy GM, Swaroop DK, Poornachandra Y,
Kumar CG, Narsaiah B (2016) Synthesis of novel hydrazone
and azole functionalized pyrazolo[3,4-b]pyridine derivatives as
promising anticancer agents. Bioorg Med Chem Lett
26:4427–4432
Wang F, Yang Z, Liu YB, Ma L, Wu YZ, He L, Shao MF, Yu K, Wu
WS, Pu YZ, Nie CL, Chen LJ (2015) Synthesis and biological
evaluation of diarylthiazole derivatives as antimitotic and anti-
vascular agents with potent antitumor activity. Bioorg Med Chem
23:3337–3350
Padmaja A, Pedamalakondaiah D, Sravya G, Reddy GM, Kumar MVJ
(2015) Synthesis and antioxidant activity of a new class of sul-
fone/sulfonamide-linked bis (oxadiazoles), bis (thiadiazoles), and
bis (triazoles). Med Chem Res 24:2011–2020
Park HB, Kwon HC, Lee CH, Yang HO (2009) Glionitrin
A, an antibiotic-antitumor metabolite derived from competive
interaction between abandoned mine microbes. J Nat Prod 72:
248–252
Wang XM, Mao S, Cao L, Xie XX, Xin MH, Lian JF, Cao YX, Zhang
SQ (2015) Modification of N-(6-(2-methoxy-3-(4-fluor-
ophenylsulfonamido)pyridin-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-
2-yl)acetamide as PI3Ks inhibitor by replacement of the acet-
amide group with alkylurea. Bioorg Med Chem 23:5662–5671
Wang Z, Shi XH, Wang J, Zhou T, Xu YZ, Huang TT, Li YF, Zhou
YL, Yang L, Yang SY, Yu LT, Wei YQ (2011) Synthesis,
structure–activity relationships and preliminary antitumor

Med Chem Res
evaluation of benzothiazole-2-thiol derivatives as novel apoptosis
inducers. Bioorg Med Chem Lett 21:1097–1101
the novel triazole derivatives containing 1,2,3-triazole fragment.
Arch Pharm Res 36:1215–1222
Yadagiri B, Gurrala S, Bantu R, Nagarapu L, Polepalli S, Srujana G,
Jain N (2015) Synthesis and evaluation of benzosuberone
embedded with 1,3,4-oxadiazole, 1,3,4-thiadiazole and 1,2,4-
triazole moieties as new potential antiproliferative agents. Bioorg
Med Chem Lett 25:2220–2224
Zhao PL, Duan AN, Zou M, Yang HK, You WW, Wu SG (2012)
Synthesis and cytotoxicity of 3,4-disubstituted-5-(3,4,5-tri-
methoxyphenyl)-4H-1,2,4-triazoles and novel 5,6-dihydro-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole derivatives bearing 3,4,5-tri-
methoxyphenyl moiety. Bioorg Med Chem Lett 22:4471–4474
Yu SC, Wang N, Chai XY, Wang BG, Cui H, Zhao QJ, Zou Y, Sun
QY, Meng Q, Wu QY (2013) Synthesis and antifungal activity of