Antitumor evaluation of novel phenothiazine derivatives that inhibit migration and tubulin polymerization against gastric cancer MGC-803 cells

Article abstract of DOI:10.1007/s10637-018-0682-x

Two novel series of 1,2,3-triazole?phenothiazine hybrids and dithiocarbamate?phenothiazine hybrids were designed and synthesized by molecular hybridization strategy. Their antiproliferative activity against three gastric cancer cell lines (MKN28, MGC-803

Full text of DOI:10.1007/s10637-018-0682-x

Investigational New Drugs
https://doi.org/10.1007/s10637-018-0682-x
SHORT REPORT
Antitumor evaluation of novel phenothiazine derivatives that inhibit
migration and tubulin polymerization against gastric cancer
MGC-803 cells
Nan Liu1 & Zhe Jin2 & Jing Zhang2 & Jianjun Jin2
Received: 30 August 2018 /Accepted: 23 September 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2018
Summary
Two novel series of 1,2,3-triazole−phenothiazine hybrids and dithiocarbamate−phenothiazine hybrids were designed and syn-
thesized by molecular hybridization strategy. Their antiproliferative activity against three gastric cancer cell lines (MKN28,
MGC-803 and MKN45) were evaluated. Among them, hybrid 13h displayed the most potent inhibitory activity against gastric
cancer MGC-803 cells with an IC50 value of 1.2 μM. Hybrid 13h could inhibit migration by regulating the expression level of N-
cadherin, E-cadherin, Vimentin, and actived-MMP2. Furthermore, it could regulate wnt/β-catenin signaling pathway on MGC-
803 cells in a concentration-dependent manner by decreasing the expression level of Wnt5α, β-catenin and TCF4. From the
tubulin polymerization assay results in vitro, hybrid 13h was a novel tubulin polymerization inhibitor. By oral administration
assay, compound 13h could effectively inhibit MGC-803 xenograft tumor growth in vivo without obvious side effects. In
summary, compound 13h might be an orally active antitumor agent with clinical applications to the treatment of gastric cancer.
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Keywords Molecular hybridization strategy Migration Tubulin polymerization Wnt/β-catenin MGC-803 xenograft tumor
growth
Introduction
However, the limitations of their dose-limiting toxicities and
drug resistance prompt the development of novel classes of
microtubule-targeting agents [5].
Microtubules as dynamic polymers of α,β-tubulin displayed
important roles in the cell ranging from cell morphology
maintenance to subcellular transport, cellular signalling and
cell motility [1]. Because of the multiple functions in cancer
cells, microtubulin was a highly attractive target for antitumor
drugs discovery [2]. Recently, various microtubule-targeting
agents were designed [1]. Paclitaxel and its second-generation
analogue docetaxel as microtubule-targeting agents were suc-
cessfully used to treat various cancers in the clinic [3, 4].
Phenothiazine skeleton was used to design and synthesize
novel anticancer agents in medicinal chemistry [6].
Phenothiazine analogue 1 (Fig. 1) exhibited potent cell growth
inhibitory avtivity on human colon Duke’s type D, colorectal
adenocarcinoma COLO 205 cells and human kidney adeno-
carcinoma A498 cells [7]. Thiazolo[5,4-b]phenothiazine 2
displayed antiproliferative activity against THP-1 human
monocytic leukaemia and HL-60 human promyelocytic cell
lines [8]. Phenothiazine carrying a sulfonamide moiety 3 as a
potent aromatase inhibitor displayed the potent antitumor ac-
tivity in vitro against T47D cell line by activation of both
intrinsic and extrinsic pathways [9].
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10637-018-0682-x) contains supplementary
material, which is available to authorized users.
Molecular hybridization is a novel strategy in drug de-
sign based on the combination of different bioactive
fragements to produce a new hybrid with improved affinity
and efficacy, when compared to the parent drugs [10].
1,2,3-Triazole as a anticancer fragement was usually used
in molecular hybridization strategy to obtain new antican-
cer hybrids [11]. Quinoline- 1,2,3-triazole hybrid 4 (Fig. 2)
demonstrated the significant activity on the C-32 and SNB-
* Nan Liu
zdu581121@163.com
1
Department of pharmacy, The First Affiliated Hospital of Zhengzhou
University, Zhengzhou 450000, China
2
First Affiliated Hospital of Henan University of Science and
Technology, Luoyang 471000, China

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Fig. 1 Phenothiazine derivatives as potent anticancer agents
19 cell lines [12]. Pyrazolo[3,4-d]pyrimidin-4(5H)-one
tethered to 1,2,3-triazole 5 arrested cell cycle at S phase
and induced apoptosis in U87 GBM cell lines [13].
Theophylline containing 1,2,3-triazole 6 displayed potent
anticancer activity by targeting human epidermal growth
factor receptor 2 [14]. In addition, dithiocarbamate was
also used to design anticancer hybrids by molecular hy-
bridization strategy. Dithiocarbamate-nitrostyrene hybrid
7 could inhibit the cancer cell proliferation by inducing
apoptosis through caspase-3 activation [15]. Quinoline-
dithiocarbamate hybrid 8 exhibited potent anticancer activ-
ities on human non-small cell lung cancer cell line H460
[16]. Quinazolin-4(3H)-one-dithiocarbamate hybrid 9
inhibited proliferation of A549, MCF-7, HeLa, HT29 and
HCT-116 cells with IC50 values of 5.44, 7.15, 12.16, 10.35
and 11.44 μM, respectively [17].
Experimental
Chemistry
Chemical reagents and solvents were purchased from Beijing
Yinuokai company. NMR spectra were obtained by a Bruker
DPX 600 MHz spectrometer from Shnaghai. For 1,2,3-tri-
azole-phenothiazines, mass spectrawere recorded on Esquire
3000 mass spectrometer by electrospray ionization (ESI).
General synthesis of compounds 13a-13i
10H-phenothiazine (3 mmol), acetone (15 mL), propargyl
bromide (3 mmol) and NaOH (3 mmol) were added carefully
and the reaction mixture was stirred at room temperature for
5 h. Upon completion, the reaction mixture was concentrated
under vacuum to afford crude intermediate 12, which were
used in the next reaction without further purification. Crude
intermediate 12 (1 mmol), azides (1 mmol), CuSO4.5H2O
(0.3 mmol) and Na2HPO4.12H2O (0.15 mmol) were dissolved
in acetone/H2O (10 ml/10 ml) to stir for 7 h at room
In this work, we designed and synthesized novel phenothi-
azine hybrids bearing 1,2,3-triazole unit and dithiocarbamate
unit as tubulin polymerization inhibitors by molecular hybrid-
ization strategy. In addition, these phenothiazines were
evluated their anticancer mechanisms against gastric cancer
MGC-803 cells.
Fig. 2 Anticancer 1,2,3-triazole
unit and dithiocarbamate unit
used in molecular hybridization
strategy

Invest New Drugs
temperature. Upon completion, the crude products were puri-
fied with column chromatography on silica gel (hexane/
EtOAc = 9/1).
10-((1-(3,4-Dimethoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)
-4a,10a-dihydro-10H-phenothiazine (13d)
10H-phenothiazine (4 mmol), acyl chloride derivatives
(4 mmol), acetone (10 ml) and NaHCO3 (5 mmol) were added
carefully and the reaction mixture was stirred at room temper-
ature for 3 h. Crude intermediates 14a~14d were obtained by
recrystallization. 14a~14d (2 mmol), carbon disulfide
(2 mmol), piperazine derivatives (3 mmol) and Et3N
(3 mmol) were dissolved in acetone (7 ml) to stir for 4 h at
room temperature. Upon completion, the crude products were
purified with column chromatography on silica gel (hexane/
EtOAc = 11/1) to obtain 15a~15d.
Yield:86%, yellow solid, m.p:201~202 °C. 1H NMR
(400 MHz, DMSO-d6) δ 8.60 (s, 1H), 7.37 (d, J = 2.5 Hz,
1H), 7.31 (dd, J = 8.7, 2.5 Hz, 1H), 7.11–7.04 (m, 4H), 7.01
(d, J = 8.8 Hz, 1H), 6.94 (dd, J = 5.9, 2.8 Hz, 2H), 6.86 (td,
J = 7.5, 1.0 Hz, 2H), 5.14 (s, 2H), 3.76 (s, 3H), 3.73 (s, 3H).
13C NMR (100 MHz, DMSO-d6) δ 149.30, 148.83, 144.35,
144.10, 129.98, 127.51, 126.73, 122.69, 122.65, 121.54,
115.76, 111.99, 104.50, 55.87, 55.79, 43.85. HR-MS (ESI)
calcd for C23H23N4O2S [M + H]+: 419.1542, found:
419.1549.
10-((1-(2-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)
-4a,10a-dihydro-10H-phenothiazine (13a)
(3-(4-((4,10-Dihydro-10H-phenothiazin-10-yl)methyl)
-1H-1,2,3-triazol-1-yl)phenyl)methanol(13e)
Yield:73%, white solid, m.p:171~172 °C. 1H NMR
(400 MHz, DMSO-d6) δ 8.32 (s, 1H), 7.62 (dd, J = 7.9,
1.6 Hz, 1H), 7.55–7.45 (m, 1H), 7.28 (d, J = 7.7 Hz, 1H),
7.20–7.04 (m, 7H), 6.95 (td, J = 7.6, 1.0 Hz, 2H), 5.24 (s,
2H), 3.78 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ
151.25, 144.19, 143.22, 130.54, 127.45, 126.79, 125.61,
125.47, 125.28, 122.94, 122.68, 120.89, 115.94, 113.08,
56.07, 43.54. HR-MS (ESI) calcd for C22H21N4OS [M +
H]+: 389.1436, found: 389.1439.
Yield:68%, yellow solid, m.p:186~188 °C. 1H NMR
(400 MHz, DMSO-d6) δ 8.69 (s, 1H), 7.84 (s, 1H), 7.74 (d,
J = 7.9 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.41 (d, J = 7.6 Hz,
1H), 7.14 (t, J = 7.0 Hz, 4H), 7.02 (d, J = 8.0 Hz, 2H), 6.93 (t,
J = 7.4 Hz, 2H), 5.38 (t, J = 5.7 Hz, 1H), 5.23 (s, 2H), 4.59 (d,
J = 5.7 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 144.77,
144.59, 144.05, 136.40, 129.54, 127.52, 126.73, 126.35,
122.65, 122.64, 121.51, 118.11, 117.68, 115.73, 62.27,
43.81. HR-MS (ESI) calcd for C22H21N4OS [M + H]+:
389.1436, found: 389.1438.
10-((1-(2,4-Dimethoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)
-10H-phenothiazine (13b)
1-(4-(4-((4,10-Dihydro-10H-phenothiazin-10-yl)methyl)
-1H-1,2,3-triazol-1-yl)phenyl)ethan-1-one (13f)
Yield:77%, yellow solid, m.p:157~158 °C. 1H NMR
(400 MHz, CDCl3) δ 7.66 (s, 1H), 7.51 (d, J = 8.7 Hz, 1H),
7.12–6.90 (m, 4H), 6.91–6.65 (m, 4H), 6.59–6.35 (m, 2H),
5.21 (s, 2H), 3.75 (s, 3H), 3.60 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 161.26, 152.52, 144.36, 143.80, 127.34, 127.12,
126.32, 124.98, 123.90, 122.79, 119.88, 115.47, 104.72,
99.51, 55.85, 55.69, 45.18. HR-MS (ESI) calcd for
C23H23N4O2S [M + H]+: 419.1542, found: 419.1548.
Yield:65%, white solid, m.p:185~186 °C. 1H NMR
(400 MHz, DMSO-d6) δ 8.80 (s, 1H), 8.06 (d, J = 8.8 Hz,
2H), 8.00 (d, J = 8.8 Hz, 2H), 7.07 (t, J = 7.4 Hz, 4H), 6.94
(d, J = 7.8 Hz, 2H), 6.87 (t, J = 7.4 Hz, 2H), 5.18 (s, 2H), 2.55
(s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 145.01, 144.02,
139.45, 130.00, 127.54, 126.74, 122.69, 121.76, 119.65,
115.74, 112.43, 43.75, 26.78. HR-MS (ESI) calcd for
C23H21N4OS [M + H]+: 401.1436, found: 401.1438.
10-((1-(3,5-Dimethoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)
-4a,10a-dihydro-10H-phenothiazine(13c)
10-((1-(3-Nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)
-4a,10a-dihydro-10H-phenothiazine(13g)
Yield:83%, yellow solid, m.p:140~142 °C. 1H NMR
(400 MHz, DMSO-d6) δ 8.33 (s, 1H), 7.24 (d, J = 3.1 Hz,
1H), 7.21 (d, J = 9.2 Hz, 1H), 7.16 (ddd, J = 7.5, 6.2, 1.4 Hz,
4H), 7.11–7.04 (m, 3H), 6.95 (td, J = 7.5, 1.0 Hz, 2H), 5.24 (s,
2H), 3.76 (s, 3H), 3.72 (s, 3H). 13C NMR (100 MHz, DMSO-
d6) δ 153.18, 145.02, 144.19, 143.28, 127.46, 126.80, 125.89,
125.41, 122.95, 122.69, 115.95, 115.61, 114.48, 110.46,
56.55, 55.76, 43.47. HR-MS (ESI) calcd for C23H23N4O2S
[M + H]+: 419.1542, found: 419.1546.
Yield:91%, white solid, m.p:149~150 °C. 1H NMR
(400 MHz, DMSO-d6) δ 8.80 (s, 1H), 8.06 (d, J = 8.8 Hz,
2H), 8.00 (d, J = 8.8 Hz, 2H), 7.07 (t, J = 7.4 Hz, 4H), 6.94
(d, J = 7.8 Hz, 2H), 6.87 (t, J = 7.4 Hz, 2H), 5.18 (s, 2H), 2.55
(s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 145.01, 144.02,
139.45, 130.00, 127.54, 126.74, 122.69, 121.76, 119.65,
115.74, 112.43, 43.75, 26.78. HR-MS (ESI) calcd for
C21H18N5O2S [M + H]+: 404.1181, found: 404.1189.

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10-((1-(4-Methoxy-2-nitrophenyl)-1H-1,2,3-triazol-4-yl)
methyl)-4a,10a-dihydro-10H-phenothiazine (13h)
(p, J = 6.8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ
170.77, 138.83, 132.80, 128.43, 128.03, 127.78, 127.55,
45.07, 31.37, 28.23. HR-MS (ESI) calcd for C16H15ClNOS
[M + H]+: 304.0563, found: 304.0567.
Yield:72%, white solid, m.p:149~150 °C. 1H NMR
(400 MHz, DMSO-d6) δ 8.51 (s, 1H), 8.03 (s, 1H), 8.00 (s,
2H), 7.17 (dd, J = 10.9, 4.3 Hz, 4H), 7.06 (d, J = 8.0 Hz, 2H),
6.95 (t, J = 7.4 Hz, 2H), 5.27 (s, 2H), 3.95 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 151.18, 148.00, 144.13, 143.80,
130.43, 127.46, 126.82, 125.55, 125.53, 123.01, 122.74,
116.25, 115.95, 108.38, 57.04, 43.38. HR-MS (ESI) calcd
for C22H20N5O3S [M + H]+: 434.1287, found: 434.1289.
5-Chloro-1-(10H-phenothiazin-10-yl)pentan-1-one (14d)
White solid, yield:67%, mp:99-103 °C. 1H NMR (400 MHz,
CDCl3) δ 7.42 (d, J = 7.8 Hz, 2H), 7.37 (dd, J = 7.7, 1.1 Hz,
2H), 7.30–7.21 (m, 2H), 7.21–7.11 (m, 2H), 3.37 (t, J =
6.1 Hz, 2H), 2.42 (s, 2H), 1.79–1.55 (m, 4H). 13C NMR
(100 MHz, CDCl3) δ 170.57, 137.73, 132.29, 127.02,
126.24, 125.99, 125.87, 43.56, 32.35, 30.69, 21.55. HR-MS
(ESI) calcd for C17H17ClNOS [M + H]+: 318.0719, found:
318.0723.
10-((1-(2-Methoxy-4-nitrophenyl)-1H-1,2,3-triazol-4-yl)
methyl)-4a,10a-dihydro-10H-phenothiazine (13i)
Yield:72%, white solid, m.p:191~192 °C. 1H NMR
(400 MHz, DMSO-d6) δ 8.54 (s, 1H), 8.03 (s, 1H), 7.70 (dt,
J = 19.2, 4.6 Hz, 2H), 7.14 (ddd, J = 6.4, 4.0, 1.5 Hz, 4H),
7.06–6.86 (m, 4H), 5.25 (s, 2H), 2.48 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 144.21, 143.98, 143.72, 141.78,
134.54, 127.49, 127.31, 126.71, 125.44, 124.93, 122.68,
122.57, 115.73, 43.85, 20.36. HR-MS (ESI) calcd for
C22H20N5O3S [M + H]+: 434.1287, found: 434.1288.
2-Oxo-2-(10H-phenothiazin-10-yl)ethyl-4-(pyridin-2-
yl)piperazine-1-carbodithioate (15a) Light yellow solid,
1
yield:6%, mp:185-188 °C. H NMR (400 MHz, DMSO-d6)
δ 8.12 (dd, J = 4.9, 1.3 Hz, 1H), 7.70 (d, J = 7.1 Hz, 2H),
7.63–7.50 (m, 3H), 7.42 (td, J = 7.8, 1.2 Hz, 2H), 7.33 (t,
J = 7.2 Hz, 2H), 6.79 (d, J = 8.6 Hz, 1H), 6.67 (dd, J = 6.8,
5.0 Hz, 1H), 4.44 (s, 2H), 4.22 (s, 2H), 4.01 (s, 2H), 3.62 (s,
4H). 13C NMR (100 MHz, DMSO-d6) δ 194.60, 166.26,
158.60, 148.04, 138.45, 138.16, 128.40, 127.84,127.66,
113.79, 107.45, 44.06. HR-MS (ESI) calcd for
C24H23N4OS3 [M + H]+: 479.1034, found: 479.1039.
2-Chloro-1-(10H-phenothiazin-10-yl)ethan-1-one (14a)
1
Light yellow solid, yield:61%, mp:120–122 °C. H NMR
(400 MHz, DMSO-d6) δ 7.70 (d, J = 7.8 Hz, 2H), 7.60 (dd,
J = 7.7, 1.1 Hz, 2H), 7.43 (td, J = 7.7, 1.4 Hz, 2H), 7.35 (td,
J = 7.6, 1.1 Hz, 2H), 4.51 (s, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 171.16, 165.47, 138.07, 132.75, 128.55,
128.47, 127.98, 127.85, 127.71, 127.56, 127.39, 43.22. HR-
MS (ESI) calcd for C14H11ClNOS [M + H]+: 276.0250,
found: 276.0256.
3-Oxo-3-(10H-phenothiazin-10-yl)propyl 4-(pyridin-2-
yl)piperazine-1-carbodithioate (15b) White solid, yield:80%,
1
mp:114-117 °C. H NMR (400 MHz, DMSO-d6) δ 8.13 (d,
J = 3.4 Hz, 1H), 7.71–7.47 (m, 5H), 7.41 (dd, J = 10.9, 4.3 Hz,
2H), 7.31 (t, J = 7.5 Hz, 2H), 6.80 (d, J = 8.6 Hz, 1H), 6.68
(dd, J = 6.8, 5.1 Hz, 1H), 4.28 (s, 2H), 3.96 (s, 2H), 3.63 (s,
4H), 3.48 (t, J = 6.5 Hz, 2H), 2.91 (s, 2H). 13C NMR
(100 MHz, DMSO-d6) δ 194.91, 169.60, 158.13, 147.55,
138.12, 137.63, 127.89, 127.44, 127.28,127.09, 113.25,
106.91, 43.53, 33.48, 31.65. HR-MS (ESI) calcd for
C25H25N4OS3 [M + H]+: 493.1190, found: 493.1197.
3-Chloro-1-(10H-phenothiazin-10-yl)propan-1-one (14b)
Green solid, yield:26%, mp:140-143 °C. 1H NMR (400 MHz,
DMSO-d6) δ 7.64 (d, J = 7.9 Hz, 2H), 7.58 (d, J = 7.8 Hz,
2H), 7.42 (t, J = 7.6 Hz, 2H), 7.33 (t, J = 7.6 Hz, 2H), 3.63
(t, J = 6.3 Hz, 2H), 2.51 (d, J = 1.3 Hz, 2H). 13C NMR
(100 MHz, DMSO-d6) δ 168.86, 138.46, 128.47, 127.86,
127.79, 127.67, 37.46, 28.80. HR-MS (ESI) calcd for
C15H13ClNOS [M + H]+: 290.0406, found: 290.0409.
3-Oxo-3-(10H-phenothiazin-10-yl)propyl-4-(2-
hydroxyethyl)piperazine-1-carbodithioate (15c) Yellow solid,
yield:14%, mp:162–164 °C. 1H NMR (400 MHz, DMSO-d6)
δ 7.61 (d, J = 7.8 Hz, 2H), 7.56 (d, J = 7.6 Hz, 2H), 7.40 (t, J =
7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 4.45 (t, J = 5.2 Hz, 1H),
4.16 (s, 2H), 3.82 (s, 2H), 3.51 (dd, J = 11.3, 5.6 Hz, 2H), 3.44
(t, J = 6.3 Hz, 2H), 2.89 (s, 2H), 2.47 (s, 4H), 2.42 (t, J =
6.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 162.12,
156.89, 156.20, 143.80, 138.11, 131.93, 130.36, 130.09,
128.71, 123.70, 123.42, 122.75, 118.77, 117.99,
4-Chloro-1-(10H-phenothiazin-10-yl)butan-1-one (14c)
Gray green solid, yield:53%, mp:95-99 °C. 1H NMR
(400 MHz, DMSO-d6) δ 7.64 (d, J = 7.4 Hz, 2H), 7.57 (dd,
J = 7.7, 1.2 Hz, 2H), 7.41 (td, J = 7.7, 1.4 Hz, 2H), 7.32 (td,
J = 7.6, 1.2 Hz, 2H), 3.58 (t, J = 6.5 Hz, 2H), 2.60 (s, 2H), 1.93

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117.91,114.23, 55.60, 52.26, 38.08. HR-MS (ESI) calcd for
C22H26N3O2S3 [M + H]+: 460.1187, found: 460.1189.
1 mM PMSF, 1 mM MgCl2, 1 mM orthovanadate, 2 mM
EGTA, 0.5% NP-40, and protease inhibitor (pH 6.8). The
experimental results were repeated three times.
5-Oxo-5-(10H-phenothiazin-10-yl)pentyl-4-(2-
hydroxyethyl)piperazine-1-carbodithioate (15d) Orange liq-
uid, yield:79%. H NMR (400 MHz, CDCl3) δ 7.43 (d, J =
Animal studies
1
7.8 Hz, 2H), 7.37 (dd, J = 7.7, 1.2 Hz, 2H), 7.29–7.22 (m, 2H),
7.16 (td, J = 7.6, 1.2 Hz, 2H), 4.26 (s, 2H), 3.88 (s, 2H), 3.68–
3.41 (m, 2H), 3.16 (t, J = 7.0 Hz, 2H), 2.55–2.47 (m, 6H), 2.44
(t, J = 6.7 Hz, 2H), 1.71–1.55 (m, 4H). 13C NMR (100 MHz,
CDCl3) δ 196.39, 170.77, 137.74, 132.25, 127.00, 126.29,
125.98, 125.83, 58.12, 56.78, 51.29, 35.65, 32.79, 28.25,
27.06, 23.30. HR-MS (ESI) calcd for C24H30N3O2S3 [M +
H]+: 488.1500, found: 488.1507.
Animals were obtained from First Affiliated Hospital of
Henan University of Science and Technology and these ex-
periments were carried out in accordance with the approved
guidelines and approved by the ethics committee. MGC-803
xenograft models were established based on reported papers
[22, 23]. Mice were divided into control groups and treatment
groups (1,2,3-triazole-phenothiazine hybrid 13h, 50 mg/kg).
The treatment groups received intragastric administration to
do in vivo assay.
Biology
Results and discussion
MTT assay
Chemistry
Human gastric cancer cells were cultured by RPMI 1640 medi-
um with 10% FBS and 100 U/ml penicillin and 0.1 mg/ml strep-
tomycin in the 37 °C in an atmosphere containing 5% CO2. MTT
assay was used to examine the antiproliferative activity of 1,2,3-
triazole-phenothiazines following the reported paper [18].
Azides were obtained by classical diazotation−azidation reac-
tion from the reported papers [24, 25]. The structures of azides
10a~10i used in this work were listed in Fig. 3.
In order to synthesize phenothiazine-1,2,3-triazole hybrids,
10H-phenothiazine 11 was used as starting material. 10H-phe-
nothiazine 11 reacted with propargyl bromide at the alkaline
condition to form intermediate 12. Without any purification,
the crude system reacted with azides 10a~10i in the presence
of disodium phosphate dodecahydrate by copper-catalyzed
azide-alkyne cycloaddition reaction to synthesize hybrids
13a~13i (Scheme 1).
Migration assay
24-Well cell migration assay was used in the migration by
reported papers [19, 20]. MGC-803 cells were treated with
hybrid 13h at different concentrations for 24 h. Then,
10,000 cells were added in each well in 24-Well cell migration
plate and cultured for 24 h. Data are presented as mean SD
from three independent experiments.
Western blot analysis
MGC-803 cells were treated with compound 13h at different
concentrations for 48 h, the cells were collected and lysed. The
total protein extracts were boiled with 5 × loading buffer, sep-
arated and transferred by PVDF membrane. The membranes
were blocked with 5% milk at room temperature for 1 h, and
then incubated overnight at 4 °C with primary antibodies.
After washing the membrane with the secondary antibody
(1:2000) at room temperature for 2 h.
Tubulin polymerization assay in vitro
MGC-803 cells were treated with compound 13h for 6 h and
lysed to do tubulin polymerization assay in vitro by previous
method [21]. The buffer was the mixture of 20 mM Tris-HCl,
Fig. 3 The structures of azides 10a~10i used in this work

Invest New Drugs
Scheme 1 Synthesis of 1,2,3-triazole−phenothiazine hybrids. Regents and conditions: a NaOH, acetone, propargyl bromide, room temperature; b
Na2HPO4.12H2O, CuSO4.5H2O, azides 13a~13i, acetone/H2O, room temperature
To synthesize phenothiazine-dithiocarbamate hybrids,
10H-phenothiazine 11 reacted with acyl chloride deriva-
tives in the precence of sodium bicarbonate to form inter-
mediate 14a~14d. Piperazine derivatives, carbon disulfide
and intermediate 14a~14d were stirred to obtaoin com-
pounds 15a~15d (Scheme 2) in the precence of
triethylamine.
−phenothiazine hybrids were evaluated their anticancer activ-
ity in vitro against three gastric cancer cell lines (MKN28,
MGC-803 and MKN45) in this work. In the previous papers,
5-fluorouracil was used as a reference drug to compare the
antiproliferative activity with 1,2,3-triazoles [32, 33]. So, we
also used 5-fluorouracil as a control drug in the antiprolifera-
tive activity evaluation.
The anticancer activity results in vitro was listed in Table 1.
Compound 11 and compound 12 displayed weak antiprolifer-
ative activity with IC50 values >100 μM. However, com-
pounds 13a~13i exhibited potent antiproliferative activity
with IC50 values from 1.2 μM to 20.2 μM. These results
revealed that 1,2,3-triazole played the pivotal effect for anti-
cancer activity in vitro.
Antiproliferative activity evaluation
Gastric cancer as one of the leading causes of death in the
world is a heavy burden to public health [26–30]. Many
1,2,3-triazoles displayed the potent anticancer activity against
gastric cancer cells [31]. Thus, all 1,2,3-triazole
Scheme 2 Synthesis of
phenothiazine-dithiocarbamate.
Regents and conditions: a
NaHCO3, acetone, acyl chloride,
room temperature; b Et3N,
CuSO4.5H2O, piperazine
derivatives, acetone, room
temperature

Invest New Drugs
Table 1 Antiproliferative activity of phenothiazine hybrids 13a~13i
Compound 13h was further examined for possible cytotox-
icity against GES-1 (normal human gastric epithelial cell line).
We found that compound 13h exhibited no cytotoxicity
against GES-1 (>40 μM). The results indicated that com-
pound 13h had good selectivity between the selected cancer
cell line (MGC-803) and a normal cell line (GES-1).
Compound
IC50 (μM)a
MKN28
MGC-803
MKN45
11
>100
>100
>100
12
>100
>100
>100
13a
17.5 0.6
8.3 0.6
9.2 1.4
11.3 0.5
7.6 0.8
3.5 0.3
4.5 0.5
2.7 0.4
5.3 0.2
>100
20.1 0.9
12.4 1.0
17.0 0.6
13.7 1.1
12.3 1.2
8.6 0.9
7.2 1.7
1.2 0.1
4.1 1.3
>100
16.8 1.2
20.2 1.7
12.0 1.5
9.8 0.6
14.2 0.7
9.2 1.3
8.2 1.6
3.5 0.3
6.4 1.2
>100
Phenothiazine hybrids inhibited cell proliferation
against MGC-803 cells
13b
13c
13d
13e
To investigate antiproliferation ability, MGC-803 cells
were added and treated by different 1,2,3-triazole−pheno-
thiazine hybrids in the 24-well plates. From the Fig. 4,
1,2,3-triazole−phenothiazine hybrids exhibited moderate
to good anti-proliferation ability against MGC-803 cells.
Notably, compounds 13b, 13e, 13f, 13g, 13h and 13i
displayed cell proliferation efficiency of 72%, 72%, 52%,
49%, 2 and 17% respectively at 4 μM concentration for
72 h. Among them, compound 13h showed the most potent
antiproliferation efficiency with percentages of 49 and 23%
for 48 h and 72 h at 1 μM concentration.
13f
13g
13h
13i
14a
14b
15a
>100
>100
>100
16.5 1.6
23.1 3.4
8.7 0.6
28.0 2.4
6.9 0.7
17.2 0.8
18.3 2.4
10.2 1.6
24.2 2.3
9.4 1.6
17.2 0.3
25.7 3.2
9.0 1.5
17.4 1.8
19.4 1.4
15b
15c
15d
5-Fluorouracil
Compound 13h inhibited the migration against MGC-803
cells
a Antiproliferative activity was assayed by exposure for 48 h. The data are
presented as the means of three independent experiments
From the antiproliferative activity results, we selected the phe-
nothiazine hybrid 13h to do antiproliferative mechanisms.
The effect of migration on MGC-803 cell line with compound
13h treatment was explored by previous paper using a 24-well
migration plate [34]. With the treatment of phenothiazine hy-
brid 13h at 2 μM and 4 μM (Fig. 5), the migration rate was
decreased to 26 and 4%, respectively. These results showed
that phenothiazine hybrid 13h inhibited the migration of
MGC-803 cell line in a concentration-dependent manner.
To explore the structure activity relationship, various
1,2,3-triazole−phenothiazine hybrids with different sub-
stituent groups on phenothiazine ring were synthesized.
Replacement of the 2,6-diOCH3 unit on the phenyl ring
(13c) with a 2-OCH3 unit (13a) led to an decrease in
activity, but changing to a 2-OCH3–4-NO2 group (13i)
led to a significant increase in activity against all of the
tested cell lines. Importantly, 1,2,3-triazole-phenothiazine
hybrid 13h containing a 2-NO2–4-OCH3 group on the
phenyl ring exhibited the best antiproliferative activity
with an IC50 value of 1.2 μM against MGC-803 cells
among all hybrids. These results indicated that the sub-
stituents on the phenyl ring attaching 1,2,3-triazole skel-
eton may play a significant role in their inhibitory
activity.
For the phenothiazine-dithiocarbamate hybrids 15a~15d,
all the hybrids exhibited moderate to potent antiproliferative
activity with IC50 values from 8.7 μM to 28.0 μM. However,
phenothiazine derivatives 14a~14b without dithiocarbamate
fragement displayed too weak antiproliferative activity with
IC50 value >100 μM. These results revealed that dithiocarba-
mate unit played the important role for anticancer activity of
phenothiazine-dithiocarbamate hybrids in vitro. This finding
might be useful to design more powerful anticancer drugs in
the future.
Compound 10h regulated the epithelial-mesenchymal
transition process
Epithelial-mesenchymal transition (EMT) described the mo-
lecular reprogramming and phenotypic changes involved in
the conversion of polarised immotile epithelial cells to motile
mesenchymal cells [35]. E-cadherin and N-cadherin were
calcium-dependent cell adhesion molecules, and the loss of
E-cadherin–mediated adhesion played a key role in the tran-
sition of epithelial tumors [36].
Based on the migration results, we tested the epithelial-
mesenchymal transition related markers (N-cadherin, E-
cadherin, Vimentin, and actived-MMP2). As the results
shown in Fig. 6, phenothiazine hybrid 13h could upregulate
the expression level of E-cadherin and downregulate the
expression level of N-cadherin, vimentin and actived-
MMP2. These results revealed that phenothiazine hybrid

Invest New Drugs
Compound 13b
Compound 13e
120
100
80
60
40
20
0
120
100
80
60
40
20
0
48h
72h
48h
72h
Control
1μM
2μM
4μM
Control
1μM
2μM
4μM
Compound 13g
Compound 13f
120
100
80
60
40
20
0
120
100
80
60
40
20
0
48h
72h
48h
72h
Control
1μM
2μM
4μM
Control
1μM
2μM
4μM
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Compound 13i
Compound 13h
48h
72h
48h
72h
Control
1μM
2μM
4μM
Control
1μM
2μM
4μM
Fig. 4 Cell proliferation efficiency for phenothiazine hybrids against MGC-803 cells
13h could regulated the EMT process in a concentration-
dependent manner.
triazole-phenothiazine hybrids could bind to tubulin, pheno-
thiazine hybrid 13h was investigated for tubulin polymeriza-
tion activity in vitro. As shown in Fig. 7, the fluorescence
intensity was decreased in the presence of compound 13h in
a concentration-dependent manner. The IC50 value of tubulin
polymerization activity was 2.87 μM. These results indicated
that phenothiazine hybrid 13h was a novel tubulin polymeri-
zation inhibitor.
Compound 10h inhibited tubulin polymerization
Recently, some papers have revealed that 1,2,3-triazole group
was a promsing skeleton to design tubulin polymerization
inhibitors [37, 38]. To confrm whether these designed 1,2,3-
Fig. 5 The migration effect on
Control
2μM
4μM
MGC-803 cell line with com-
pound 13h treatment. *: p < 0.05
verse control
120
100
80
60
40
20
0
*
*
Control
2μM
4μM
Concentration

Invest New Drugs
Control 1μM
2
μM
4
μM
Control 1μM
2μM
4μM
β
-catenin
N-cadherin
E-cadherin
Wnt5α
TCF4
Vimentin
GAPDH
actived-MMP2
Fig. 8 Compound 13h regulated the Wnt/β-catenin signaling pathway
GAPDH
Fig. 6 Compound 13h regulated the EMT related markers
compared with the normal group. The average tumor weights
of control and 50 mg/kg drug treatment group were 1.440
0.200 g, and 0.430 0.250 g (inhibitory rate: 70.14%) respec-
tively. Importantly, there was no apparent body weight loss in
drug treatment group, indicating the low side effects of com-
pound 13h in vivo.
Compound 10h regulated the Wnt/β-catenin signaling
pathway
Wnt/β-catenin signaling pathway plays an important role in
the regulation of embryonic development and tumorigenesis
[39]. Since its deregulation results in severe human diseases,
especially cancer, the wnt signaling pathway constitutes a
promising platform for pharmacological targeting of cancer
[40, 41]. In this work, we tested the expression level of wnt/
β-catenin signaling pathway related markers (Wnt5α, β-
catenin and TCF4). As shown in Fig. 8, phenothiazine hybrid
13h decreased the expression level of Wnt5α, β-catenin and
TCF4 in a concentration-dependent manner, suggesting that
hybrid 13h could regulate wnt/β-catenin signaling pathway.
a
b
Compound 10h inhibited tumor growth against a xenograft
model
To further determine whether phenothiazine hybrid 13h is a
potential candidate drug for clinical application against gastric
cancer, it was selected for in vivo testing against MGC-803
xenograft tumor growth in severe combined immunodeficien-
cy (SCID) mice. From the antitumor results in Fig. 9, tumor
volumes of drug treatment group were obviously decreased
**
c
2500
control
2uM
2000
4uM
1500
6uM
1000
500
0
20
40
60
80
Tinme/min
Fig. 9 1,2,3-triazole-phenothiazine hybrid 10h inhibited MGC-803 tu-
mor growth in vivo. **P < 0.01
Fig. 7 Compound 13h inhibited tubulin polymerization in vitro

Invest New Drugs
7. Ghinet A, Moise I-M, Rigo B, Homerin G, Farce A, Dubois J
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Conclusions
In summary, novel phenothiazine hybrids were designed and
synthesized by molecular hybridization strategy. Among
them, compound 13h displayed the most potent activity
against MGC-803 cells with an IC50 value of 1.2 μM. Based
on the mechanisms, we found that compound 13h could in-
hibit MGC-803 cells migration by regulating wnt/β-catenin
signaling pathway. Importantly, compound 13h was revealed
as a novel tubulin polymerization inhibitor and an orally ac-
tive antitumor agent.
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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
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