Design, synthesis and antiproliferative evaluation of novel sulfanilamide-1,2,3-triazole derivatives as tubulin polymerization inhibitors

Article abstract of DOI:10.1007/s10637-018-0632-7

Summary: Microtubule as an important target in the cancer therapy was used to design novel tubulin polymerization inhibitors. Sulfanilamide-1,2,3-triazole hybrids were designed by a molecular hybridization strategy and their antiproliferative activity aga

Full text of DOI:10.1007/s10637-018-0632-7

Investigational New Drugs
https://doi.org/10.1007/s10637-018-0632-7
SHORT REPORT
Design, synthesis and antiproliferative evaluation of novel
sulfanilamide-1,2,3-triazole derivatives as tubulin
polymerization inhibitors
Shewei Guo1 & Yingwei Zhen1 & Mengguo Guo1 & Longzhou Zhang1 & Guosheng Zhou1
Received: 1 May 2018 /Accepted: 27 June 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2018
Summary
Microtubule as an important target in the cancer therapy was used to design novel tubulin polymerization inhibitors.
Sulfanilamide-1,2,3-triazole hybrids were designed by a molecular hybridization strategy and their antiproliferative activity
against three selected cancer cell lines (BGC-823, MGC-803 and SGC-7901) were evaluated. All sulfanilamide-1,2,3-triazole
hybrids displayed potent inhibitory activity against all cell lines. In particular, compound 10b showed the most excellent
inhibitory effect against MGC-803 cells, with an IC50 value of 0.4 μM. Cellular mechanism studies elucidated that 10b induced
apoptosis by decreasing the expression level of Bcl-2 and Parp and increasing the expression level of BAX. 10b inhibited the
epithelial-mesenchymal transition process by up-regulating E-cadherin and down-regulating N-cadherin. Furthermore, the tubu-
lin polymerization inhibitory activity in vitro of 10b was 2.4 μM. In vivo anticancer assay, 10b effectively inhibited MGC-803
xenograft tumor growth without causing significant loss of body weight. These sulfanilamide-1,2,3-triazole hybrids as potent
tubulin polymerization inhibitors might be used as promising candidates for cancer therapy.
.
.
.
.
Keywords Sulfanilamide-1,2,3-triazole Apoptosis Epithelial-mesenchymal transition Tubulin Xenograft tumor growth
Introduction
Sulfanilamide skeleton as a aprivileged bioactive unit
displayed an important role in antitumour drug discovery [4–6].
Ureido-substituted benzenesulfonamide 1 could inhibit the me-
tastases formation of 4T1 cancer cells at pharmacologic concen-
trations of 45 mg/kg [7]. N-(7-indazolyl)benzenesulfonamide de-
rivative 2 is a promising antitumor agent containing a sulfon-
amide moiety, which induced cell cycle arrest and cell apoptosis
[8]. 4-((6-([1,1′-biphenyl]-3-yl)-9H-purin-2-yl)amino)
benzenesulfonamide 3 as a potent antitumor agent exhibited high
activity toward CDK2 (IC50 = 0.044 μM) [9] (Fig. 1).
Triazoles as a pharmacological fragement display an ample
spectrum of biological activities and are widely employed as
pharmaceuticals [10]. They have been regarded as an interest-
ing unit in terms of biological activity and some of them have
also shown significant anticancer activity in vitro [11].4-
Methyl-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)benzenesulfonamide 4 (Fig. 2) showed the excel-
lent inhibitory effect against PC-3 cell line with an IC50 value
of 4.08 uM and the tubulin polymerization inhibitory activity
with an IC50 value of 2.41 uM [12]. 1,2,3-Triazole linked
Microtubule as one of the most important targets in the cancer
therapy accomplishes its physiologic function by the dynamic
equilibrium of the polymerization or depolymerization of
α,β-heterodimer [1]. Antitumor drugs targeting to tubulin in-
cluding vincristine, vinblastine, paclitaxel, and docetaxel are
one of the major classes of cytotoxic drugs in clinical use for
treatment of cancers [2]. However, neurotoxicity and cardio-
vascular toxicity of these drugs currently represent the main
obstacles to broad their clinical application [3]. Therefore,
there is still an urgent need to search for novel tubulin poly-
merization inhibitors for cancer therapy.
* Shewei Guo
gswfeicui@163.com
1
The First Affiliated Hospital of Zhengzhou University,
Zhengzhou 450052, China

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Fig. 1 Sulfanilamide derivatives
as as potent anticancer agents
aminocombretastatin conjugate 5 displayed the potent tubulin
polymerization activity with an IC50 value of 0.95 uM [13]. 2-
Methoxy-5-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5-
yl)aniline 6 as a tubulin polymerization inhibitor displayed
potent antiproliferative activity against several cancer cells
with IC50 values in the nanomolar range [14].
stir for 8 h at room temperature. Upon completion, the
precipitated product was filtered to afford the products
10a~10l, which were purified with column chromatogra-
phy on silica gel (hexane/EtOAc = 9/1).
Based on the results of 1,2,3-triazole and sulfanilamide
as antitubulin or anticancer skeletons, we envisioned that a
sulfanilamide ring linked a 1,2,3-triazole unit would serve
as novel and potent tubulin polymerization inhibitors (Fig. 3).
Herein, we report the synthesis and anticancer activity of
sulfanilamide-1,2,3-triazole analogues, as well as the in-
hibitory effect of tubulin polymerization for the most
active compound.
4-(4-(((2-Oxo-2H-chromen-4-yl)oxy)methyl)
-1H-1,2,3-triazol-1-yl)benzenesulfonamide (10a)
o
1
Yield 87%. Yellow solid. Mp: 197~199 C. H NMR (400
MHz, DMSO-d6) δ 9.12 (s, 1H), 8.22 – 8.10 (m, 2H), 8.04
(d, J = 8.9 Hz, 2H), 8.00 (s, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.55
(s, 2H), 7.22 (d, J = 2.3 Hz, 1H), 7.07 (dd, J = 8.6, 2.4 Hz, 1H),
6.33 (d, J = 9.5 Hz, 1H), 5.40 (s, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 160.98, 160.21, 155.29, 144.24, 143.96, 143.51,
138.48, 129.56, 127.49, 123.38, 120.46, 112.87, 112.78,
101.63, 61.54. HR-MS (ESI): Calcd. C18H15N4O5S, [M+
H]+m/z: 399.0763, found: 399.0768.
Experimental
Chemistry
4-(4-(((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)
-1H-1,2,3-triazol-1-yl)benzenesulfonamide (10b)
All reagents and solvents were purchased from Shanghai
Aladdin Bio-Chem Technology Co., LTD. All NMR spec-
tra were recorded with a Bruker DPX 400 MHz spectrom-
eter (TMS as internal standard). Mass spectra (MS) were
recorded on Esquire 3000 mass spectrometer by
electrospray ionization (ESI).
1
Yield 76%. Yellow solid. Mp: 149~150oC. H NMR (400
MHz, DMSO-d6) δ 9.12 (s, 1H), 8.26 – 8.10 (m, 2H), 8.10
– 7.95 (m, 2H), 7.72 (d, J = 8.8 Hz, 1H), 7.55 (s, 2H), 7.20
(d, J = 2.5 Hz, 1H), 7.09 (dd, J = 8.8, 2.5 Hz, 1H), 6.24 (d,
J = 1.1 Hz, 1H), 5.40 (s, 2H), 2.41 (d, J = 0.9 Hz, 3H). 13
C
General method to synthesize
NMR (100 MHz, DMSO-d6) δ 160.88, 160.07, 154.64,
153.34, 143.96, 143.55, 138.47, 127.49, 126.54, 123.35,
120.44, 113.50, 112.56, 111.38, 101.66, 61.53, 18.10. HR-
MS (ESI): Calcd. C19H17N4O5S, [M+H]+m/z: 413.0920,
found: 413.0928.
phenothiazole-1,2,3-triazole hybrids 10a~10l
Alkyne intermediates 7a~7l (3mmol), azide derivative 9 (3
mmol), CuSO4.5H2O (0.6 mmol) and sodium ascorbate
(0.3 mmol) were dissolved in THF/H2O (10 ml/10 ml) to
Fig. 2 1,2,3-Triazole derivatives as as novel microtubule-interacting agents

Invest New Drugs
Fig. 3 Rational design of target
compounds.
4-(4-(((2-Oxo-2H-chromen-6-yl)oxy)methyl)
-1H-1,2,3-triazol-1-yl)benzenesulfonamide (10c)
117.54, 107.48, 52.67, 43.34, 27.93. HR-MS (ESI): Calcd.
C17H18N5O2S, [M+H]+m/z: 356.1181, found: 356.1188.
1
Yield 88%. Yellow solid. Mp: 134~136oC. H NMR (400
4-(4-((6-Methoxyindolin-1-yl)methyl)
MHz, DMSO-d6) δ 9.20 (s, 1H), 8.19 (d, J = 8.8 Hz, 2H),
8.06 (d, J = 8.8 Hz, 2H), 7.85 (dd, J = 7.9, 1.4 Hz, 1H), 7.74 –
7.62 (m, 1H), 7.56 (s, 2H), 7.43 (d, J = 7.9 Hz, 1H), 7.39 –
7.32 (m, 1H), 6.22 (s, 1H), 5.56 (s, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 164.29, 161.49, 152.77, 144.04, 142.63, 138.46,
132.84, 127.49, 124.19, 123.56, 123.00, 120.54, 116.44,
115.02, 91.49, 62.72. HR-MS (ESI): Calcd. C18H15N4O5S,
[M+H]+m/z: 399.0763, found: 399.0768.
-1H-1,2,3-triazol-1-yl)benzenesulfonamide (10f)
1
Yield 80%. Yellow solid. Mp: 200~201oC. H NMR (400
MHz, DMSO-d6) δ 8.86 (s, 1H), 8.13 (d, J = 8.7 Hz, 2H),
8.01 (d, J = 8.7 Hz, 2H), 7.52 (s, 2H), 6.72 (s, 1H), 6.70 – 6.55
(m, 2H), 4.36 (s, 2H), 3.64 (s, 3H), 2.85 (t, J = 8.0 Hz, 2H),
2.51 (d, J = 1.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ
145.62, 144.91, 143.68, 138.60, 131.44, 127.45, 121.91,
120.13, 111.58, 108.18, 55.42, 53.42, 44.48, 28.28. HRMS
(ESI) calcd for C18H20N5O3S [M + H]+: 386.1287, found:
386.1289.
4-(4-(((2-oxo-2H-chromen-6-yl)oxy)methyl)
-1H-1,2,3-triazol-1-yl)benzenesulfonamide (10d)
4-(4-((3,4-dihydroquinolin-1(2H)-yl)methyl)
-1H-1,2,3-triazol-1-yl)benzenesulfonamide (10g)
1
Yield 90%. Yellow solid. Mp: 237~238oC. H NMR (400
MHz, DMSO-d6) δ 9.11 (s, 1H), 8.19 – 8.10 (m, 2H), 8.09 –
7.95 (m, 2H), 7.81 (d, J = 9.0 Hz, 1H), 7.54 (s, 2H), 7.27 (d, J
= 2.5 Hz, 1H), 7.14 (dd, J = 8.9, 2.5 Hz, 1H), 5.41 (s, 2H),
2.55 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 160.89,
156.41, 152.56, 148.81, 143.97, 143.45, 138.46, 127.49,
127.11, 123.40, 120.45, 116.50, 113.27, 113.19, 101.64,
61.62, 16.08. HR-MS (ESI): Calcd. C19H16ClN4O5S, [M+
H]+m/z: 447.0530, found: 447.0537.
1
Yield 84%. Yellow solid. Mp: 219~220oC. H NMR (400
MHz, DMSO-d6) δ 8.81 (s, 1H), 8.12 (d, J = 8.7 Hz, 2H),
8.00 (d, J = 8.7 Hz, 2H), 7.51 (s, 2H), 6.94 (t, J = 7.7 Hz, 1H),
6.87 (d, J = 7.2 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 6.48 (t, J =
7.2 Hz, 1H), 4.60 (s, 2H), 3.49 – 3.44 (m, 2H), 2.70 (t, J = 6.3
Hz, 2H), 1.99 – 1.87 (m, 2H). 13C NMR (100 MHz, DMSO-
d6) δ 145.74, 144.54, 143.65, 138.57, 128.79, 127.43, 126.78,
122.27, 121.26, 120.10, 115.79, 111.05, 49.13, 45.81, 27.42,
21.77. HRMS (ESI) calcd for C18H20N5O2S [M + H]+:
370.1339, found: 370.1342.
4-(4-(Indolin-1-ylmethyl)-1H-1,2,3-triazol-1-yl)
benzenesulfonamide (10e)
1
Yield 68%. Yellow solid. Mp: 217~218oC. H NMR (400
4-(4-((2,3,4,5-tetrahydro-1H-benzo[b]azepin-1-yl)
methyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide
(10h)
MHz, DMSO-d6) δ 8.88 (s, 1H), 8.13 (d, J = 8.8 Hz, 2H),
8.02 (d, J = 8.7 Hz, 2H), 7.53 (s, 2H), 7.02 (dd, J = 13.8, 7.2
Hz, 2H), 6.72 (d, J = 7.8 Hz, 1H), 6.60 (t, J = 7.2 Hz, 1H), 4.45
(s, 2H), 3.41 (t, J = 8.3 Hz, 2H), 2.89 (t, J = 8.2 Hz, 2H). 13
C
Yield 78%. White solid. Mp: 185~187oC. H NMR (400
1
NMR (100 MHz, DMSO-d6) δ 151.42, 144.81, 143.70,
138.60, 129.78, 127.46, 127.01, 124.26, 121.86, 120.14,
MHz, DMSO-d6) δ 8.87 (s, 1H), 8.16 (d, J = 8.7 Hz, 2H),
8.02 (d, J = 8.7 Hz, 2H), 7.52 (s, 2H), 7.15 – 7.04 (m, 3H),

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6.89 – 6.77 (m, 1H), 4.47 (s, 2H), 2.99 – 2.93 (m, 2H), 2.82 –
2.76 (m, 2H), 1.63 (d, J = 3.4 Hz, 2H), 1.55 (d, J = 4.3 Hz,
2H). 13C NMR (100 MHz, DMSO-d6) δ 151.58, 147.20,
143.68, 138.64, 135.32, 129.81, 127.43, 126.57, 121.77,
121.11, 120.13, 117.59, 53.38, 49.28, 34.26, 29.52, 25.39.
HRMS (ESI) calcd for C19H21N5O2S [M + H]+: 384.1495,
found: 384.1494.
3H). 13C NMR (100 MHz, DMSO-d6) δ 166.21, 162.13,
147.39, 144.12, 139.12, 129.66, 127.95, 126.76, 121.82,
120.61, 113.95, 55.82, 35.21. HRMS (ESI) calcd for
C17H17N5O4S Na[M + Na]+: 410.0898, found: 410.0899.
Biology
Antiproliferative activity assay
N-((1-(4-sulfamoylphenyl)-1H-1,2,3-triazol-4-yl)
methyl)pyridine-3-sulfonamide (10i)
BGC-823, MGC-803 and SGC-7901 were maintained in the
mixture of 89% RPMI1640 medium, 10% fetal bovine serum
and 1% penicillinestreptomycin in a humidified atmosphere of
5% CO2 and 95% air at 37 oC. All cell lines were purchased
from the China Center for Type Culture Collection (CCTCC,
Shanghai, China).
Cells were seeded into 96-well plates at a concentration of
5000 cells per well. After 24 h incubation, the culture medium
was removed and replaced with medium containing the can-
didate compounds. The cells were incubated for another 72 h.
Then, 20 uL of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazo lium bromide solution (5 mg/mL) was added
to each well and incubated for 1.5 h. Added 150 uL dimethyl
sulfoxide to each well and shook 30 min; the absorbance was
measured using a microplate reader at awavelength of 570 nm.
Each concentration was analyzed in triplicate and the experi-
ment was repeated three times.
Yield:39%,yellow soild,m.p.:169-170°C 1H NMR (400 MHz,
DMSO-d6) δ 8.94 (s, 1H), 8.75 (s, 1H), 8.69 (s, 1H), 8.57 (t, J
= 5.8 Hz, 1H), 8.15 (d, J = 8.1 Hz, 1H), 8.08 – 7.97 (m, 4H),
7.58 (dd, J = 7.9, 4.8 Hz, 1H), 7.53 (s, 2H), 4.26 (d, J = 5.7 Hz,
2H). 13C NMR (100 MHz, DMSO-d6) δ 152.88, 147.11,
144.37, 143.81, 138.38, 136.94, 134.55, 127.50, 124.04,
121.92, 120.23, 37.76. HRMS (ESI) calcd for
C14H14N6O4S2Na[M + Na]+: 417.0414, found: 417.0416.
N-((1-(4-sulfamoylphenyl)-1H-1,2,3-triazol-4-yl)
methyl)thiophene-2-carboxamide (10j)
1
Yield: 72%,white soild,m.p.: 226-228°C. H NMR (400
MHz, DMSO-d6) δ 9.14 (s, 1H), 8.81 (s, 1H), 8.15 (d, J =
8.4 Hz, 2H), 8.00 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 4.6 Hz, 1H),
7.52 (s, 2H), 7.16 (s, 1H), 4.60 (d, J = 5.2 Hz, 2H). 13C NMR
(100 MHz, DMSO-d6) δ 161.64, 146.93, 144.16, 140.06,
139.09, 131.46, 128.85, 128.42, 127.95, 121.93, 120.64,
35.04. HRMS (ESI) calcd for C14H13N5O3S2Na[M + Na]+:
386.0354, found: 386.0358.
Apoptosis analysis
MGC-803 cells were seeded into a 6-well plate (6.0 X 106
cells/well). After incubation for 24 h with the test compound
10b, Hoechst 33258 was added to the culture mediumto give
final concentrations of 5 mg/mL. The cells were incubated
2-Chloro-N-((1-(4-sulfamoylphenyl)
-1H-1,2,3-triazol-4-yl)methyl)benzamide (10k)
o
with the staining mixture for 30 min at 37 C and then the
Yield: 64%,white soild,m.p.:197-198°C. 1H NMR (400 MHz,
DMSO-d6) δ 9.05 (t, J = 5.4 Hz, 1H), 8.79 (s, 1H), 8.16 (d, J =
8.7 Hz, 2H), 8.03 (d, J = 8.7 Hz, 2H), 7.67 – 7.23 (m, 6H),
4.61 (d, J = 5.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ
166.86, 146.64, 144.23, 139.10, 136.93, 131.38, 130.46,
130.10, 129.55, 128.01, 127.51, 121.80, 120.71, 35.12.
HRMS (ESI) calcd for C16H14ClN5O3S Na[M + Na]+:
414.0401, found: 414.0404.
cells were observed under a fluorescence microscope (Leica
Microsystems AG, Wetzlar, Germany). Apoptotic cells were
examined and identified according to the condensation and
fragmentation of their nuclei by fluorescence microscopy.
Western blot
MGC-803 cells were cultured and colleted with different con-
centrations of compound 10b after treating 24 h, and then
Western blot analysis was performed. Briefly, Cells were lysed
in cell lysis buffer [1% NP-40, 0.1% sodium dodecyl sulfate
(SDS), 150 mM NaCl, 25 mM TriseHCl, 1% deoxycholic acid
sodium salt, 1% PMSF] for 60 min on ice. Total protein con-
tents were measured by protein assay reagent. The membrane
was indicated primary antibodie overnight at 4 oC. After that,
the membrane was incubated with secondary antibodies.
4-Methoxy-N-((1-(4-sulfamoylphenyl)
-1H-1,2,3-triazol-4-yl)methyl)benzamide (10l)
Yield:46%,white soild,m.p.:192-194°C 1H NMR (400 MHz,
DMSO-d6) δ 8.96 (s, 1H), 8.78 (s, 1H), 8.15 (d, J = 8.4 Hz,
2H), 8.01 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 8.5 Hz, 2H), 7.52 (s,
2H), 7.01 (d, J = 8.4 Hz, 2H), 4.61 (d, J = 5.1 Hz, 2H), 3.81 (s,

Invest New Drugs
Tubulin polymerization inhibitory activity assay
approximately 100 mm3, the mice were randomly divided
into corresponding NaCl, 10b (60mg/kg) treatment groups
(n = 10 mice in each group). The treatment groups re-
ceived intragastric administration of 10b per day for a
period of 21 days.
Tubulin polymerization in vitro was assyed with 100 uL sam-
ples and monitored by absorbance at 350 nm (Filter A00019x)
in 96-well plates with an Appliskan plate reader (Thermo
Fisher Scientific, Waltham, MA, USA). An amount of 6 mg/
mL tubulin was resuspended in PEM buffer (80 mM PIPES, 1
mM EGTA, 0.5 mM MgCl2, 1 mM ATP, 10.2% (v/v) glycer-
ol) and then was preincubated with 1,2,3-triazole hybrid 10b
or DMSO on ice. Data were exported using the Thermo
Scientific SkanIt software of the Appliskan and Excel soft-
ware was used to generate plots.
Results and discussion
Chemistry
Alkynes 7a~7l used in this work were purchased from
Shanghai Aladdin Bio-Chem Technology Co., LTD (Fig. 4).
4-Aminobenzenesulfonamide 8 was treated with NaN3 to pro-
vide 4-azidobenzenesulfonamide 9.
Cell scarification assay
The scarification test performed by MGC-803 cells were
seed in 6-well plate which was used to make a scratch in
cells. Control and 1,2,3-triazole hybrid 10i culture medium
without fetal bovine serum were added subsequently after
washed 3 times by PBS. Then the cells were cultured at
37°C and take photos.
Cu(I)-catalyzed azide–alkyne 1,3-dipolar cycloaddition
popularly known as the “click reaction” serves as the
highly dependable tool for the facile construction of var-
ious architectures at the molecular level [15, 16]. The
sulfanilamide-1,2,3-triazole hybrids 10a~10l were syn-
thesized through a click reaction between alkyne inter-
mediates 7a~7l and 4-azidobenzenesulfonamide 9 using
copper sulfate and sodium ascorbate in THF/H20
(Scheme 1). This synthetic strategy resulted in high
yields (78%~92%) of target sulfanilamide-1,2,3-triazole
hybrids 10a~10l.
In vivo anti-tumor activity
Animals were treated according to methods established by
the ethics committee of Zhengzhou University and these
experiments were carried out in accordance with the ap-
proved guidelines and approved by the ethics committee
of Zhengzhou University. BALB/c nude mice (18 g, aged
4-5 weeks) were purchased from Human SJA Laboratory
Animal Co. Ltd. (Hunan, China). Mice were subcutane-
ously implanted with MGC-803 cells (6 × 106 cells per
mouse) in the back right flank. Once the volumes reached
Antiproliferative activity and SARs
Sulfanilamide-1,2,3-triazole hybrids 10a~10l were evalu-
ated their antiproliferative activity against three cancer
cell lines (BGC-823, MGC-803 and SGC-7901) using a
MTT (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-
Fig. 4 Azide derivatives 7a~7l
used in this study

Invest New Drugs
Scheme 1 Synthesis of
sulfanilamide-1,2,3-triazole
hybrids 10a~10l. Reagents and
conditions: (a) NaN3, THF/
H2O=5:1, rt; (b) CuSO4.5H2O,
sodium ascorbate, alkyne
intermediates 7a~7l, THF:H2O
(1:1), rt.
tetrazolium bromide) assay, and the well-known tubulin
polymerization inhibitor colchicine was used as a control
[17, 18]. The antiproliferative results were summarized
in Table 1.
IC50 values ranging from 0.4 μM to 10.7 μM. This result
suggested that 1,2,3-triazole unit displayed a synergistic
role for antiproliferative activity.
In addition, the coumarin substituent attaching the
1,2,3-triazole ring affected the antiproliferative activity.
Replacement of the 6-hydroxy-2H-chromen-2-one group
of compound 10c with a 4-hydroxy-2H-chromen-2-one
group of compound 10a led to a decrease of the antipro-
liferative activity. However, changing the 3-chloro-7-hy-
droxy-4-methyl-2H-chromen-2-one (compound 10d) to
7-hydroxy-2H-chromen-2-one (compound 10b) improved
the inhibitory activity, indicating the significance of cou-
marin substituent attaching the 1,2,3-triazole ring in their
antiproliferative activity. When the coumarin substituent
(7-hydroxy-2H-chromen-2-one) of compound 10b was
In order to investigate the different inhibitory effect
between 1,2,3-triazoles and non-1,2,3-triazoles, non-
1,2,3-triazole-sulfanilamide 9 and 1,2,3-triazole-sulfanil-
amide hybrids 10a~10l were investigated their antiprolif-
erative activity. Removal of the 1,2,3-triazole fragement
was clearly detrimental for the inhibitory activity against
all the cancer cell lines. Compared with compound 9, the
introduction of 1,2,3-triazole scaffold to hybrids 10a~10l
resulted in a significant improvement of inhibitory activ-
ity. Especially, compound 10b, 10d, 10g and 10i showed
the excellent inhibitory effect against all cell lines with

Invest New Drugs
Table 1 Antiproliferative results of sulfanilamide-1,2,3-triazole hybrids 10a~10l
Compound
IC50 ˄uM˅
MGC-803
>100
11. 9 0.7
BGC-823
SGC-7901
>100
9
>100
16.2 1.6
3.4 0.3
8.1 1.0
4.7 1.4
56.3 0.8
29.1 1.3
10.7 1.2
18.1 1.7
3.6 1.3
20.7 0.2
49.1 1.5
20.3 1.0
10a
10b
10c
10d
10e
10f
10g
10h
15.3 0.5
f
0.4 0.1
3.5 0.1
9.1 1.2
6.0 1.8
9.4f0.2
5.1 0.3
10.5 1.3
8. 7 0.4
3.8 0.3
12.8 0.1
0.7 0.1
25.1 0.4
18. 8 0.2
23.4 0.3
45.9 0.7
30.7 0.7
7.2 1.1
19.8 1. 8
4.7 0.1
48.2 0.5
26.3 0.1
19.2 1.0
0.027 0.004
26.3 1.0
10i
10j
10k
10l
Colchicine
0.142 0.008
6.9 1.6
0.089 0.007
14.1 1.5
5-Fu
a
Inhibitory activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50%
(IC50). Data are presented as the means SDs of three independent experiments
b
Not determined
replaced by N-heterocycles (indoline, 6-methoxyindoline,
1,2,3,4-tetrahydroquinoline, and 2,3,4,5-tetrahydro-1H-
benzo[b]azepine) of 10e~10h, the antiproliferative activ-
ity was decreased.
Among all these compounds, compound 10b displayed
the best inhibitory activity result against MGC-803 cells.
In addition, compound 10b was further examined for
possible inhibitory effect against GES-1 cell line (normal
human gastric epithelial cell line). As a result, compound
10b exhibited no inhibitory effect against GES-1 (>100
μM). However, 10b exhibited potent antiproliferative ac-
tivity against MGC-803 cell line with an IC50 value of 0.4
μM, indicating that compound 10b had good selectivity
between cancer cells and normal cells.
Fig. 5 Representative images of
MGC-803 cells colonies after
treatment with various
0
0.5 uM
1uM
concentrations for 7 days

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Fig. 6 MGC-803 cells treated by
10b showed typical apoptotic
morphologies.
0
0.5 uM
1uM
Effect of clone formation
Compound 10b inhibited cell EMT via scarification
test and regulated the expression of EMT-related
proteins.
Based on the most excellent activity against MGC-803 cells,
10b was chosen to perform its detailed antiproliferative
mechainsms. As shown in Fig. 5, colony formation assay
measures the ability of MGC-803 cells to grow and form foci,
which represents an indirect estimation of neoplastic transfor-
mation. MGC-803 cells treated with 10b exhibited fewer and
smaller colonies compared to the control with a colony forma-
tion at 1 μM, which indicated that 10b could significantly in-
hibit the proliferation of MGC-803 cells as a concentration-
dependent manner.
Epithelial-mesenchymal transition (EMT) process was unique
for the phenotypic changes of tumor cells characterized by a
transition from polarized rigid epithelial cells to migrant mes-
enchymal cells, thus conferring the ability of tumor invasion
and metastasis [23]. In this study, we found that compound
10b could inhibit the epithelial-mesenchymal transition prog-
ress (Fig. 8).
In the scarification test, the distance of scratch obviously
changed in a concentration-dependent manner compared with
control (Fig. 8a). At the same time, we detected the EMT-
related proteins expression such as E-cadherenin, and N-
cadherenin. The results showed that E-cadherin was up-regu-
lated, and N-cadherin was down-regulated (Fig. 8b), indicat-
ing 10i could inhibit the EMT process against MGC-803 cells.
Effect of morphological changes
In addition, 10b was explored the cell apoptosis by Hoechst
33258 staining [19]. After 24 h incubation with MGC-803
cells at indicated concentrations (0, 0.5uM, and 1uM), char-
acteristic apoptotic morphological changes were observed by
fluorescence microscope, including cell rounding, chromain
shrinkage and formation of apoptotic bodies (Fig. 6). The
studies revealed that hybrid 10b induced morphological
changes of MGC-803 cells possibly by apoptosis.
Compound 10b displayed potent tubulin
polymerization inhibitory activity
Based on the obtained IC50 values against the cancer cell lines,
1,2,3-triazole hybrid 10b was evaluated the tubulin polymer-
ization inhibitory effect. Colchicine was used as a reference
drug and the results are summarized in Fig. 9 [24–27]. When
tubulin was incubated with 10b at indicated concentrations
Compound 10b regulated apoptosis-related proteins
Cell shrinkage, chromatin condensation, and nuclear mem-
brane blebbing as the changes of morphological features are
the characteristics of apoptotic cells [20]. Based on the above
results, we elplored the effect of compound 10b on celluar
apoptosis. The BCl-2 family proteins such as BCl-2 dictate
cellular survival or death decisions by regulating the mito-
chondrial outer membrane [21]. Along with the fact that acti-
vation of the BCl-2-controlled apoptotic pathway seems crit-
ical for the efficacy of most chemotherapeutics, BCl-2 family
members have emerged as the attractive targets for cancer
therapy [22]. We can see from the result in Fig. 7, compound
10b increased the expression level of Bax and reduced the
expression level of Bcl-2 and Parp which could promote apo-
ptosis of MGC-803 cells.
0
0.5 uM 1uM 2uM
PARP
Bcl-2
Bax
GAPDH
Fig. 7 Expression of apoptosis-related proteins in MGC-803 cells after
treatment by compound 10b for 24 h.

Invest New Drugs
Fig. 8 (a) The scarification test of
10b on MGC-803 cells at 24h. (b)
Western blot of EMT-related
proteins at 24h.
a
0
0.5 uM
1uM
b
0
0.5 uM 1uM 2uM
E-cadherin
N-cadherin
GAPDH
(1uM, 2uM and 5uM), the increased tendency of the fluores-
cence intensity was obviously slowed down. The inhibitory
concentration that reduced the polymerized tubulin by 50%
(IC50) of compound 10b was 2.4 μM. It revealed hybrid 10b
was a novel tubulin polymerization inhibitor.
control and 60 mg/kg 10b groups were 1.34 0.20 g, and 0.48
0.22 g (inhibitory rate: 64.18%), respectively (Fig. 10c).
These results demonstrated the potent antitumor activity of
10b against gastric cancer cells in vivo and low toxicity to-
ward mice.
Compound 10b exhibited potent antitumor activity
in a xenograft model
Conclusion
There is no significant difference in mean body weights over
time between control and 10i treated group (Fig. 10a). The
growth rate of MGC803 xenograft tumors from mice which
were treated with 10b was lower than those from the vehicle
control-treated mice (Fig. 10b). The average tumor weights of
In summary, novel sulfanilamide-1,2,3-triazole hybrids were
designed, synthesized and evaluated their anticancer activity
against three selected cancer cell lines (BGC-823, MGC-803
and SGC-7901). All compounds exhibited potent inhibitory
activity against the cancer cell lines selected. Particularly,
Fig. 9 Effect of compound 10b
IC 5 0 = 2 .4 0 .1 u M
on the tubulin polymerization.
2 5 0 0
2 0 0 0
1 5 0 0
1 0 0 0
C o n tr o l
1 u M
2 u M
5 u M
5 0 0
0
2 0
4 0
6 0
8 0
1 0 0
T im e /m in

Invest New Drugs
b
c
a
Normal
60mg/kg
Fig. 10 10b inhibited MGC803 tumor growth in vivo. (a) The body weight-time bar charts. Statistical analyses demonstrated that the average volume (b)
and weight (c) of MGC803 xenografts from 10b treated mice were significantly reduced. ***P < 0.001.
3. Fu D-J, Fu L, Liu Y-C, Wang J-W, Wang Y-Q, Han B-K, Li X-R,
compound 10i showed the most excellent anticancer activity
with an IC50 value of 0.4 μM against MGC-803 cancer cells.
Zhang C, Li F, Song J, Zhao B, Mao R-W, Zhao R-H, Zhang S-Y,
Zhang L, Zhang Y-B, Liu H-M (2017) Structure-activity relation-
The mechanism displayed that 10b induced morphological
change against MGC-803 cells by increasing the expression
level of Bax and decreasing the expression level of Bcl-2 and
Parp. Importantly, in vitro tubulin polymerization assay re-
vealed 10b was a novel tubulin polymerization inhibitor with
an IC50 value of 2.4 μM. Compound 10b inhibited EMT
process and regulated the expression level of EMT-related
proteins. In vivo study, the hybrid 10b could inhibit MGC-
803 xenograft tumor growth without causing significant loss
of body weight. Highlight of structure activity relationship
was the importance of 1,2,3-triazol scaffold for these sulfanil-
amide-1,2,3-triazole hybrids. Efforts to optimize the structure
of compound 10b to further improve its potency are ongoing.
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Informed consent Informed consent was obtained from all individual
participants included in the study.
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