Synthesis and biological evaluation of novel disulfides incorporating 1,3,4-thiadiazole scaffold as promising antitumor agents

Article abstract of DOI:10.1007/s00044-019-02389-3

In the present study, fourteen 2,5-disubstituted 1,3,4-thiadiazole derivatives containing disulfide group were prepared. The resulting compounds 7a–7n were identified by IR, NMR, MS, and elemental analysis. Their antiproliferative properties in vitro were studied employing standard CCK-8 assay against SMMC-7721, MCF-7, and A549 lines. Bioassay indicated that some compounds showed stronger antitumor effects than reference drugs PX-12 and 5-fluorouracil. Among these screened compounds, compound 7h showed excellent biological activities in inhibiting SMMC-7721 cell proliferation with IC₅₀ at 1.93 ± 0.08 μM. Compounds 7k and 7i manifested highly effective growth inhibitory activity versus MCF-7 cells, with IC₅₀ at 3.04 ± 0.09 and 3.54 ± 0.17 μM, respectively. For A549 cells, compound 7m was found to have the highest antitumor potency with IC₅₀ at 3.67 ± 0.13 μM.

Full text of DOI:10.1007/s00044-019-02389-3

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02389-3
ORIGINAL RESEARCH
Synthesis and biological evaluation of novel disulfides incorporating
1,3,4-thiadiazole scaffold as promising antitumor agents
Sha Li1 Hai-Xin Wang1 Hai-Ying Liu1 Fen Jing1 Xiao-Yun Fu1 Cai-Wen Li1 Yan-Ping Shi1 Bao-Quan Chen1
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Received: 18 March 2019 / Accepted: 25 June 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
In the present study, fourteen 2,5-disubstituted 1,3,4-thiadiazole derivatives containing disulfide group were prepared. The
resulting compounds 7a–7n were identified by IR, NMR, MS, and elemental analysis. Their antiproliferative properties
in vitro were studied employing standard CCK-8 assay against SMMC-7721, MCF-7, and A549 lines. Bioassay indicated
that some compounds showed stronger antitumor effects than reference drugs PX-12 and 5-fluorouracil. Among these
screened compounds, compound 7h showed excellent biological activities in inhibiting SMMC-7721 cell proliferation with
IC50 at 1.93 0.08 μM. Compounds 7k and 7i manifested highly effective growth inhibitory activity versus MCF-7 cells,
with IC50 at 3.04 0.09 and 3.54 0.17 μM, respectively. For A549 cells, compound 7m was found to have the highest
antitumor potency with IC50 at 3.67 0.13 μM.
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Keywords Disulfides 1,3,4-Thiadiazole Synthesis Antitumor activity
Introduction
generally expressed in various biological tissues. The Trx
system has become a hot spot in the research of tumor
molecular biology, and it is a new target of tumor therapy
with important clinical significance (Tonissen and Trapani
2009; Lu and Holmgren 2014; Karlenius and Tonissen
2010). Over the last decades, there have been several
anticancer drugs targeting Trx system in clinical trials
(Hashemy et al. 2006; Mehta et al. 2009; Tan et al. 2010).
Among them, PX-12 (1-methylpropyl 2-imidazolyl dis-
ulfde) is a novel small molecule inhibitor of Trx, which has
antitumor effects for a variety of tumor cells. It is also the
first disulfide compound which has been reported to enter
clinical trials as anticancer drug (Ramanathan et al. 2011;
Diraimondo et al. 2013; Baker et al. 2013) (Fig. 1).
Therefore, the role of disulfides in the antitumor field has
been paid more and more attention (Saiz et al. 2014; Zhu
et al. 2015), and this has also stimulated people’s interest in
the study of disulfide antitumor drugs.
Five-membered heterocyclic compounds, such as 1,3,4-
oxadiazoles, 1,3,4-thiadiazoles, and 1,2,4-triazoles, have a
very wide range of applications in industry, agriculture, and
medicine. In recent years, 1,3,4-thiadiazoles occupies an
important position in research and development in the field
of medicine, which has aroused widespread interest in the
pharmaceutical industry and has conducted a large number
of studies owing to their remarkable biological properties,
including antitumor (Shi et al. 2013; Bhatt et al. 2018;
Malignant tumor is a major disease characterized by
abnormal proliferation and metastasis of cells. Its high
incidence rate and low survival rate have become one of the
greatest threats to human health today. In order to alleviate
the suffering of cancer patients, improve the life quality of
patients, and prolong the lives of patients, medical scientists
from all over the world have made unremitting efforts, but
they have not yet fundamentally solved this thorny problem
which human face. In recent years, targeted treatment has
become an important means of cancer treatment, and has
aroused the attention of medical experts around the world.
Thioredoxin (Trx) is a kind of multifunctional protein that is
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02389-3) contains supplementary
material, which is available to authorized users.
* Cai-Wen Li
lcwlcw66@sina.com
* 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

Medicinal Chemistry Research
intermediates, for example 5a–5n and 6a–6n, was mon-
itored by thin layer chromatography (TLC).
H
N
S
S
General method for the synthesis of 2-substituted
phenyl-5-substituted disulfanyl-1,3,4-thiadiazoles
(7a–7n)
N
Fig. 1 Structure of PX-12
To the solution of intermediate 2 and 2-substituted phenyl-
5-mercapto-1,3,4-thiadiazoles 6a–6n (3.0 mmol) in metha-
nol (15 ml) was added the aqueous solution of NaHCO3
(0.40 g/4.7 mmol in 25 ml water) at ambient temperature
with vigorous stirring. The reaction mixture continued to
stir for 3 h, then it was cooled at 5–10 °C for 12 h. The
precipitated solid was separated, followed by recrystalliza-
tion from mixture of ethanol and water to yield the desired
products.
Polkam et al. 2015; Rezki et al. 2015), antimicrobial (Shetty
et al. 2016; Muǧlu et al. 2018), anticholinesterase (Skrzy-
pek et al. 2013; Altintop et al. 2013), antidiabetic (Pattn
et al. 2011; Lee et al. 2010), antidepressant (Clerici et al.
2001; Khan et al. 2016), Analgesic (Schenone et al. 2006;
Yavuz et al. 2013), anticonvulsant (Luszczki et al. 2015;
Karakus et al. 2009), antiinflammatory (Dekhane et al.
2011; Shkair et al. 2016), antimalarial (Huang et al. 2012),
antitubercular (Patel et al. 2017), antileishmanial (Alipour
et al. 2011; Poorrajab et al. 2009; Foroumadi et al. 2005),
antiglaucoma (Casini et al. 2003), and acetylcholinesterase
inhibitory properties (Xue et al. 2014). Consequently,
people increasingly pay close attention to the development
value and potential application prospects of 1,3,4-thiadia-
zole compounds in the field of medicine. Based on these
scientific facts, in order to screen out new antitumor lead
compound-bearing disulfide core with high efficiency and
low toxicity, hybrid compounds possessing disulfide and
1,3,4-thiadiazole moieties will be formed employing certain
chemical means, and some tumor cells growth inhibitory
effects will be examined in our research.
2-Phenyl-5-(n-butyldisulfanyl)-1,3,4-thiadiazole (7a)
Yellow oil; Yield 60%; IR (cm−1): 3145, 2959, 2939, 2871,
2361, 2342, 1966, 1907, 1694, 1458, 1401, 1281, 1176,
1
1001, 979, 762, 621, 573; H NMR (400 MHz, DMSO-d6)
δ: 7.95 (2H, d, J = 7.8 Hz, Ph-H), 7.58 (2H, t, J = 7.3 Hz,
Ph-H), 7.50 (1H, t, J = 7.6 Hz, Ph-H), 3.06 (2H, t, J =
6.8 Hz, S-S-CH2), 1.65–1.72 (2H, m, S-S-CH2CH2),
1.36–1.44 (2H, m, CH2CH3), 0.88 (3H, t, J = 7.2 Hz, CH3);
13C NMR (100 MHz, DMSO-d6) δ: 168.56, 167.89, 131.75
(2C), 129.69 (2C), 128.77, 127.59, 38.99, 30.95, 21.44,
13.81; MS (ESI, m/z): 283.4 [M + H]+; Calculated for
C12H14N2S3: C, 51.03; H, 5.00; N, 9.92. Found: C, 51.12;
H, 4.97; N, 9.97.
Material and methods
2-(4-Aminophenyl)-5-(n-butyldisulfanyl)-1,3,4-
thiadiazole (7b)
Synthesis
All chemical reagents were purchased from Energy Che-
mical Co. and Tianjin Hengshan Chemical Technology Co.,
Ltd., and they were used as received from commercial
supplies without further purification unless otherwise spe-
cified. The bioassay reagents, including RPMI-1640 and
PBS, were getted from Tianjin Jun Yao Biological Tech-
nology Co., Ltd. Melting points (m.p., uncorrected) were
determined by a X-4 microscope melting point apparatus.
Infrared spectra (IR) were obtained in KBr pellets on a
Yellow solid; m.p.: 73.4–75.3 °C; Yield 69%; IR (cm–1):
3132, 2361, 1635, 1401, 1177, 1107, 991, 826, 619; H
1
NMR (400 MHz, DMSO-d6) δ: 7.60 (2H, d, J = 8.56 Hz,
Ph-H), 6.64 (2H, d, J = 8.5 Hz, Ph-H), 5.92 (2H, s, NH2),
3.02 (2H, t, J = 7.2 Hz, S-S-CH2); 1.64–1.72 (2H, m, S-S-
CH2CH2), 1.35–1.44 (2H, m, CH2CH3), 0.97 (3H, t, J =
7.2 Hz, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 170.75,
168.12, 152.67, 129.47 (2C), 116.73, 114.18 (2C), 38.89,
30.85, 21.34, 13.87; MS (ESI, m/z): 298.5 [M + H]+; Cal-
culated for C12H15N3S3: C, 48.45; H, 5.08; N, 14.13. Found:
C, 48.35; H, 5.11; N, 14.19.
1
Nicolet Avatar 370 spectrometer. Both H NMR and 13C
NMR (400 and 100 MHz, respectively) spectra of the syn-
thesized compounds were acquired on a Bruker Avance III
400 MHz spectrometer using DMSO-d6 as solvent and tet-
ramethylsilane as the internal standard. ESI mass spectra
were recorded on a Waters Xevo G2 QTof (ESI) mass
spectrometer. Elemental analyses (C, H, N) were performed
on a Flash EA1112 analyzer. The reaction progress of some
2-(4-Methylphenyl)-5-(n-butyldisulfanyl)-1,3,4-
thiadiazole (7c)
Yellow oil; Yield 62%; IR (cm−1): 3145, 2962, 2872, 2360,
2342, 2075, 1635, 1398, 1183, 1120, 1077, 976, 817, 760,

Medicinal Chemistry Research
710, 620, 572; 1H NMR (400 MHz, DMSO-d6) δ: 7.81 (2H,
d, J = 8.0 Hz, Ph-H), 7.35 (2H, d, J = 8.0 Hz, Ph-H), 3.03
(2H, t, J = 7.2 Hz, S-S-CH2); 2.36 (3H, s, Ph-CH3),
1.61–1.70 (2H, m, S-S-CH2CH2), 1.33–1.42 (2H, m,
CH2CH3), 0.87(3H, t, J = 4.4 Hz, CH2CH3); 13C NMR
(100 MHz, DMSO-d6) δ: 171.18, 169.92, 142.11, 130.39
(2C), 127.86 (2C), 127.00, 38.86, 30.92, 21.47, 21.36, 13.85;
MS (ESI, m/z): 297.5 [M + H]+; Calculated for C13H16N2S3:
C, 52.67; H, 5.44; N, 9.45. Found: C, 52.79; H, 5.47; N, 9.38.
Calculated for C12H13ClN2S3: C, 45.48; H, 4.13; N, 8.84.
Found: C, 45.59; H, 4.10; N 8.89.
2-(4-Nitrophenyl)-5-(n-butyldisulfanyl)-1,3,4-
thiadiazole (7g)
Yellow solid; m.p.: 94.2–96.1 °C; Yield 70%; IR (cm−1):
3134, 2360, 2074, 1635, 1517, 1401, 1346, 1107, 991, 864,
853, 752, 687, 619; 1H NMR (400 MHz, DMSO-d6) δ: 8.38
(2H, d, J = 8.6 Hz, Ph-H), 8.24 (2H, d, J = 8.6 Hz, Ph-H),
3.10 (2H, t, J = 7.2 Hz, S-S-CH2), 1.67–1.74 (2H, m, S-S-
CH2CH2), 1.37–1.46 (2H, m, CH2CH3), 0.89 (3H, t, J =
7.2 Hz, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 174.69,
167.45, 149.18, 135.23, 129.16 (2C), 124.93 (2C), 38.76,
30.97, 21.35, 13.85; MS (ESI, m/z): 328.4 [M + H]+; Cal-
culated for C12H13N3O2S3: C, 44.02; H, 4.00; N, 12.83.
Found: C, 43.91; H, 3.97; N, 12.90.
2-(4-Methoxyphenyl)-5-(n-butyldisulfanyl)-1,3,4-
thiadiazole (7d)
Yellow solid; m.p.: 55.1–57.0 °C; Yield 73%, IR (cm−1):
3443, 3134, 2360, 2342, 1636, 1401, 1307, 1254, 1131,
1
1108, 1033, 993, 823, 807, 639, 618; H NMR (400 MHz,
DMSO-d6) δ: 7.90 (2H, d, J = 8.6 Hz, Ph-H), 7.10 (2H, d,
J = 8.7 Hz, Ph-H), 3.84 (3H, s, Ph-OCH3), 3.06 (2H, t, J =
7.2 Hz, S-S-CH2); 1.65–1.73 (2H, m, S-S-CH2CH2),
1.35–1.45 (2H, m, CH2CH3), 0.87 (3H, t, J = 7.2 Hz,
CH2CH3); 13C NMR (100 MHz, DMSO-d6) δ: 170.59,
169.57, 162.25, 129.63 (2C), 122.29, 115.27 (2C), 55.94,
38.85, 30.89, 21.33, 13.85; MS (ESI, m/z): 313.5 [M + H]+;
Calculated for C13H16N2OS3: C, 49.97; H, 5.16; N, 8.97.
Found: C, 50.11; H, 5.13; N 8.92.
2-Phenyl-5-(2-butyldisulfanyl)-1,3,4-thiadiazole (7h)
Yellow oil; Yield 70%; IR (cm–1): 3134, 2968, 2930, 2874,
2360, 2342, 1967 1889, 1636, 1457, 1289, 1108, 1001, 978,
762, 689, 622; 1H NMR (400 MHz, DMSO-d6) δ: 7.95 (2H,
d, J = 6.7 Hz, Ph-H), 7.58 (2H, t, J = 7.1 Hz, Ph-H), 7.49
(1H, t, J = 7.2 Hz, Ph-H), 3.18–3.25 (1H, m, S-S-CH),
1.63–1.75 (1H, m, CH2-Ha), 1.54–1.63 (1H, m, CH2-Hb),
1.32 (3H, d, J = 6.8 Hz, CH3CH), 0.95 (3H, t, J = 7.2 Hz,
CH2CH3); 13C NMR (100 MHz, DMSO-d6) δ: 172.51,
169.65, 131.87, 129.81 (2C), 129.75, 127.95 (2C), 49.35,
28.83, 20.10, 11.56; MS (ESI, m/z): 283.4 [M + H]+; Cal-
culated for C12H14N2S3: C, 51.03; H, 5.00; N, 9.92. Found:
C, 51.15; H, 5.05; N, 9.86.
2-(3,4,5-Trimethoxyphenyl)-5-(n-butyldisulfanyl)-
1,3,4-thiadiazole (7e)
White solid; m.p.: 64.0–66.1 °C; Yield 76%; IR (cm−1):
3447, 3145, 2361, 2343, 2075, 1637, 1509, 1401, 1331,
1
1192, 1114, 991, 845, 620; H NMR (400 MHz, DMSO-d6)
δ: 7.22 (2H, s, Ph-H), 3.88 (6H, s, Ph-OCH3), 3.74 (3H, s, Ph-
OCH3), 3.07 (2H, t, J = 7.2 Hz, S-S-CH2), 1.67–1.74 (2H, m,
S-S-CH2CH2), 1.36–1.47 (2H, m, CH2CH3), 0.90 (3H, t, J =
7.6 Hz, CH2CH3); 13C NMR (100 MHz, DMSO-d6) δ:
184.28, 163.35, 153.85 (2C), 140.74, 124.96, 105.29 (2C),
60.61, 56.62 (2C), 38.77, 30.90, 21.33, 13.84; MS (ESI, m/z):
373.5 [M + H]+; Calculated for C15H20N2O3S3: C, 48.36; H,
5.41; N, 7.52. Found: C, 48.26; H, 5.43; N, 7.46.
2-(4-Aminophenyl)-5-(2-butyldisulfanyl)-1,3,4-
thiadiazole (7i)
Yellow solid; m.p.: 99.4–101.3 °C; Yield 75%; IR (cm−1):
1
3151, 2074, 1635, 1401, 1107, 994, 823, 617; H NMR
(400 MHz, DMSO-d6) δ: 7.61 (2H, d, J = 8.6 Hz, Ph-H),
6.64 (2H, d, J = 8.6 Hz, Ph-H), 5.92 (2H, s, NH2), 3.15–3.23
(1H, m, S-S-CH); 1.67–1.76 (1H, m, CH2-Ha), 1.55–1.66
(1H, m, CH2-Hb), 1.33 (3H, d, J = 6.8 Hz, CH3CH), 0.95
(3H, t, J = 7.2 Hz, CH2CH3); 13C NMR (100 MHz, DMSO-
d6) δ: 170.93, 168.23, 152.70, 129.51 (2C), 116.67, 114.15
(2C), 49.28, 28.74, 20.14, 11.59; MS (ESI, m/z): 298.5 [M
+ H]+; Calculated for C12H15N3S3: C, 48.45; H, 5.08; N,
14.13. Found: C, 48.32; H, 5.13; N, 14.07.
2-(4-Chlorophenyl)-5-(n-butyldisulfanyl)-1,3,4-
thiadiazole (7f)
Yellow solid; m.p.: 37.2–39.1 °C; Yield 77%; IR (cm−1):
3443, 3134, 2361, 2341, 1636, 1401, 1132, 1108, 837, 815,
1
639, 616, 569; H NMR (400 MHz, DMSO-d6) δ: 7.98
(2 H, d, J = 8.5 Hz, Ph-H), 7.63 (2H, d, J = 8.5 Hz, Ph-H),
3.07 (2H, t, J = 7.2 Hz, S-S-CH2);1.66–1.73 (2H, m, S-S-
CH2CH2), 1.34–1.45 (2H, m, CH2CH3), 0.90 (3H, t, J =
7.2 Hz, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 173.93,
167.55, 136.57, 129.86 (2C), 129.44 (2C), 128.54, 38.83,
30.94, 21.37, 13.85; MS (ESI, m/z): 317.4 [M + H]+;
2-(4-Methylphenyl)-5-(2-butyldisulfanyl)-1,3,4-
thiadiazole (7j)
Yellow oil; Yield 70%; IR (cm−1): 3134, 2360, 1635, 1507,
1401, 1312, 1289, 1260, 1215, 1183, 1110, 996, 976, 817,

Medicinal Chemistry Research
760, 710, 620; 1H-NMR (400 MHz, DMSO-d6) δ: 7.83 (2H,
d, J = 8.0 Hz, Ph-H), 7.36 (2H, d, J = 8.0 Hz, Ph-H),
3.18–3.26 (1H, m, S-S-CH), 2.37 (3H, s, Ph-CH3),
1.67–1.76 (1H, m, CH2-Ha), 1.55–1.65 (1H, m, CH2-Hb),
1.34 (3H, d, J = 6.8 Hz, CH3CH), 0.95 (3H, t, J = 7.2 Hz,
CH2CH3); 13C NMR (100 MHz, DMSO-d6) δ: 172.24,
169.52, 142.01, 130.38 (2C), 127.84 (2C), 127.07, 49.32,
28.79, 21.47, 20.11, 11.56; MS (ESI, m/z): 297.5 [M + H]+;
Calculated for C13H16N2S3: C, 52.67; H, 5.44; N, 9.45.
Found: C, 52.53; H, 5.49; N, 9.39.
166.98, 154.52, 136.65, 129.92 (2C), 129.59 (2C), 128.51,
49.34, 28.81, 20.12, 11.58; MS (ESI, m/z): 317.4 [M + H]+;
Calculated for C12H13ClN2S3: C, 45.48; H, 4.13; N, 8.84.
Found: C, 45.40; H, 4.16; N, 8.79.
2-(4-Nitrophenyl)-5-(2-butyldisulfanyl)-1,3,4-
thiadiazole (7n)
White solid; m.p.: 114.3–116.5 °C; Yield 74%; IR (cm−1):
3443, 3133, 2359, 1635, 1519, 1401, 1343, 1108, 985, 856,
1
752, 688, 639, 619; H NMR (400 MHz, DMSO-d6,) δ:
2-(4-Methoxyphenyl)-5-(2-butyldisulfanyl)-1,3,4-
thiadiazole (7k)
8.37 (2H, d, J = 8.0 Hz, Ph-H), 8.24 (2H, d, J = 7.9 Hz, Ph-
H), 3.26–3.32 (1H, m, S-S-CH), 1.68–1.75 (1H, m, CH2-
Ha), 1.59–1.68 (1H, m, CH2-Hb), 1.36 (3H, d, J = 6.4 Hz,
CH3CH), 0.98 (3H, t, J = 7.2 Hz, CH2CH3); 13C NMR
(100 MHz, DMSO-d6) δ: 175.41, 167.38, 149.23, 135.27,
129.22 (2C), 124.96 (2C), 49.39, 28.84, 20.13, 11.59; MS
(ESI, m/z): 328.4 [M + H]+; calculated for C12H13N3O2S3:
C, 44.02; H, 4.00; N, 12.83. Found: C, 44.11; H, 4.04;
N, 12.78.
White solid; m.p.: 42.6–44.2 °C; Yield 75%; IR (cm−1):
3145, 2075, 1635, 1401, 1309, 1256, 1109, 992, 837, 811,
1
620; H NMR (400 MHz, DMSO-d6) δ: 7.90 (2H, d, J =
8.1 Hz, Ph-H), 7.10 (2H, d, J = 8.0 Hz, Ph-H), 3.84 (3H, s,
Ph-OCH3), 3.21–3.27 (1H, m, S-S-CH), 1.66–1.73 (1H, m,
CH2-Ha), 1.58–1.65 (1H, m, CH2-Hb), 1.35 (3H, d, J =
6.4 Hz, CH3CH), 0.96 (3H, t, J = 7.2 Hz, CH2CH3); 13C
NMR (100 MHz, DMSO-d6) δ: 171.15, 169.59, 162.28,
129.69 (2C), 122.26, 115.30 (2C), 55.97, 49.31, 28.77,
20.13, 11.58; MS (ESI, m/z): 313.5 [M + H]+; Calculated
for C13H16N2OS3: C, 49.97; H, 5.16; N, 8.97. Found: C,
49.82; H, 5.21; N, 8.90.
Tumor cell growth inhibitory assay
The cell lines (SMMC-7721, MCF-7, and A549) were
cultured in proper medium in a 5% CO2 at 37 °C during the
experiment. The inhibition (IC50) of the selected tumor cells
proliferation by target compounds 7a–7n and reference
drugs was measured by our previous method as described in
literature (Wang et al. 2017).
2-(3,4,5-Trimethoxyphenyl)-5-(2-butyldisulfanyl)-
1,3,4-thiadiazole (7l)
Yellow oil; Yield 69%; IR (cm−1): 3135, 2085, 1634, 1586,
1509, 1403, 1330, 1240, 1129, 998, 845, 821, 759, 732,
618; 1H NMR (400 MHz, DMSO-d6) δ: 7.22 (2H, s, Ph-H),
3.88 (3H, s, Ph-OCH3), 3.87 (3H, s, Ph-OCH3), 3.74 (3H, s,
Ph-OCH3), 3.20–3.29 (1H, m, S-S-CH), 1.68–1.78 (1H, m,
CH2-Ha), 1.57–1.67 (1H, m, CH2-Hb), 1.36 (3H, d, J =
6.8 Hz, CH3CH), 0.96 (3H, t, J = 7.6 Hz, CH2CH3); 13C
NMR (100 MHz, DMSO-d6) δ: 172.13, 169.62, 153.84
(2C), 140.85 (2C), 124.54, 105.44, 60.60, 56.59 (2C),
49.27, 28.79, 20.01, 11.53; MS (ESI, m/z): 373.5 [M + H]+;
Calculated for C15H20N2O3S3: C, 48.36; H, 5.41; N, 7.52.
Found: C, 48.48; H, 5.37; N, 7.58.
Results and discussion
Synthesis
The synthesis pathway of 2-substituted phenyl-5-substituted
disulfanyl-1,3,4-thiadiazoles 7a–7n is shown in Scheme 1,
all of the fourteen 1,3,4-thiadiazole derivatives are unknown
compounds. The reaction of thiourea and n-butylthiol, 2-
butylthiol yielded S-alkyl-thioisothiourea hydrochloride 2
following the literature method (Sirakawa et al. 1970),
which was directly used for the next step reaction without
being purified. The synthesis of 2-substituted phenyl-5-
mercapto-1,3,4-thiadi-azoles 6a–6n was commenced with
benzoic acid by four steps in total yield 45.1–65.0% (Jha
et al. 2010; Li et al. 2013). First, condensation of appro-
priate benzoic acid 3a–3n with ethanol gave ester 4a–4n
(79.3–88.4%). Hydrazidation of ester 4a–4n delivered the
benzohydrazide 5a–5n (80.1–91.4%), which reacted with
CS2 and KOH produced compounds 6a–6n (71.0–80.5%).
Finally, nucleophilic replacement of 6a–6n with 2 in the
presence of NaHCO3 yielded 2-substituted phenyl-5-sub-
stituted disulfanyl-1,3,4-thiadiazoles 7a–7n in moderate
2-(4-Chlorophenyl)-5-(2-butyldisulfanyl)-1,3,4-
thiadiazole (7m)
White solid; m.p.: 52.1–53.9 °C; Yield 72%; IR (cm−1):
3423, 3134, 1634, 1401, 1133, 1108, 996, 851, 821, 639,
615; 1H NMR (400 MHz, DMSO-d6) δ: 7.99 (d, J = 8.4 Hz,
2H, Ph-H), 7.63 (2H, d, J = 8.5 Hz, Ph-H), 3.22–3.30 (1H,
m, S-S-CH), 1.69–1.78 (1H, m, CH2-Ha), 1.57–1.67 (1H,
m, CH2-Hb), 1.36 (3H, d, J = 6.8 Hz, CH3CH), 0.97 (3H, t,
J = 7.2 Hz, CH2CH3); 13C NMR (100 MHz, DMSO-d6) δ:

Medicinal Chemistry Research
S
Scheme 1 Synthetic pathway of
the title compounds 7a–7n.
Reaction conditions and
NH
a
S
R1SH +
HCl
R1
S
NH2
H2N
NH2
reagents: a Concd. HCl, H2O2
(30%), 0–5 °C, 3 h; b EtOH,
concd. H2SO4, reflux, 8–12 h; c
NH2NH2·H2O, EtOH, reflux,
5–8 h; d (i) KOH, CS2, EtOH,
0–5 °C, 2 h; (ii) Concd. H2SO4,
0–5 °C, 2 h; e Compound 2,
MeOH, NaHCO3/H2O, rt, 3 h
1
2
b
c
COOC2H5
COOH
R2
R2
4a-4n
3a-3n
N
S
N
SH
d
CONHNH2
R2
R2
5a-5n
6a-6n
N
N
S
S
R1
e
S
R2
7a-7n
7a
7b
7C
7d
R2 (R1 = n-C4H9): H ( ), 4-NH2 ( ), 4-CH3 ( ), 4-OCH3 ( ), 3, 4, 5-(OCH3)3
7e 7f 7g 7h 7i 7j
(
), 4-Cl ( ), 4-NO2 ( ); R2 (R1 = 2-C4H9): H ( ), 4-NH2 ( ), 4-CH3 ( ),
4-OCH3 (7k), 3, 4, 5-(OCH3)3 (7l), 4-Cl (7m), 4-NO2 (7n)
Table 1 Inhibition (IC50) of SMMC-7721, MCF-7, and A549 cells
proliferation by compounds 7a–7n
yields. The pure form of the compounds 7a–7n was
obtained by recrystallization from the mixture of ethanol
and water.
Compound
IC50 (µM)
SMMC-7721
MCF-7
A549
Pharmacology evaluation
7a
4.64 0.18
8.60 0.33
4.39 0.11
2.94 0.09
2.28 0.12
7.90 0.32
5.14 0.24
1.93 0.08
3.69 0.26
3.89 0.15
6.15 0.31
2.94 0.13
17.83 0.68
6.89 0.29
4.05 0.32
5.62 0.28
8.60 0.38
25.95 1.47
9.66 0.45
8.00 0.37
7.80 0.12
6.74 0.31
8.20 0.33
8.20 0.21
3.54 0.17
4.49 0.16
3.04 0.09
8.25 0.25
6.35 0.18
9.10 0.40
5.12 0.21
14.26 0.66
32.77 1.98
7.80 0.33
9.85 0.42
6.95 0.22
33.37 2.17
8.80 0.29
14.37 0.56
18.73 0.51
8.00 0.18
6.35 0.39
33.37 1.88
5.89 0.24
3.67 0.13
35.63 2.28
10.51 0.30
8.13 0.34
7b
7c
The title compounds consist of two series, which were
known as 7a–7g and 7h–7n, respectively. Compounds
7a–7g all possess a n-butyl group linked with disulfide
bond, whereas, compounds 7h–7n all carry 2-butyl group
bonded to disulfide. Evaluation of the antitumor activity
in vitro for 2-substituted phenyl-5-substituted disulfanyl-
1,3,4-thiadiazoles 7a–7n was carried out by utilizing CCK-
8 assay versus three types of tumor cell lines, namely,
SMMC-7721, MCF-7, and A549. The inhibitory activities
IC50 (μM) are shown in Table 1.
We found from the results that all tested compounds
7a–7n displayed admissible antiproliferative activities for
selected tumor cell lines. For SMMC-7721 cells, 7m
showed moderate antitumor effect (IC50 = 17.83
0.68 μM), whereas, the rest of tested compounds exhibited
good inhibitory efficacy (IC50 = 1.93–8.60 μM). Mean-
while, most title compounds were found to possess higher
antiproliferative activity in comparison with 5-fluorouracil
(IC50 = 5.62 0.28 μM). Among them, six compounds,
including 7d, 7e, 7h, 7i, 7j, and 7l revealed higher anti-
tumor effect than PX-12 (IC50 = 4.05 0.32 μM). By
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
PX-12
5-FU
comparing 7a with 7h, although they have the same phenyl
substituent in position 2 of 1,3,4-thiadiazole, we found that
the difference is obvious in their antitumor efficacy. Com-
pound 7h having 2-butyl group linked to disulfide, showed

Medicinal Chemistry Research
better activity than 7a. Similarly, 7b and 7i, 7d and 7k, 7f
and 7m also emerged obvious differences in antitumor
potency. Especially noteworthy is compound 7h displayed
excellent growth inhibitory activity (IC50 = 1.93
0.08 μM). We also found substitutes at phenyl ring, have
obvious effects on biological activity, but further research
and discussion are needed. For MCF-7 cells, compound 7b
carrying 4-amino group at the phenyl ring, showed mod-
erate antiproliferative activity (IC50 = 25.95 1.47 μM).
The other screened compounds emerged good cytotoxic
efficacy (IC50 < 10 μM), and displayed higher activities than
5-fluorouracil (IC50 = 14.26 0.66 μM). But most com-
pounds did not show obvious advantage in comparison with
PX-12 (IC50 = 5.12 0.21 μM). 7a and 7h, 7e and 7l, 7f
and 7m, 7g and 7n showed similar activity against MCF-7
cells, respectively. but 7b and 7i, 7c and 7j, 7d and 7k
revealed obvious differences in antitumor potency, respec-
tively. Compounds 7k and 7i were manifested promising
growth inhibitory activity with IC50 at 3.04 0.09 and
3.54 0.17 μM, respectively. 7i, 7j, 7k, and 7l carrying
electron-donating groups, including 4-amino, 4-methyl, 4-
methoxy and 3,4,5-trimethoxy, respectively, were superior to
7h in antitumor efficacy. For A549 cells, six compounds,
including 7a, 7e, 7g, 7h, 7k, and 7n revealed moderate
antiproliferative activities (IC50 = 14.37–35.63 μM), whereas,
such as 7h, 7i, 7k, and 7m displayed promising biological
activities. Therefore, the pharmacological results could be
helpful for the development of new anticancer agents.
Acknowledgements This study was financially supported by Tianjin
Municipal Natural Science Foundation (18JCYBJC94900), and
Training Project of Innovation Team of Colleges and Universities in
Tianjin (TD13-5020). The authors greatly thank Prof. Y.M. Liu for his
help in confirming the structure of the newly synthesized compounds,
and we also thank Dr. W. Li for her help in evaluating antitumor
activity of the screened compounds.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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