Antiproliferative phenothiazine hybrids as novel apoptosis inducers against MCF-7 breast cancer

Article abstract of DOI:10.3390/molecules23061288

We designed a series of novel phenothiazine-1,2,3-triazole hybrids by the molecular hybridization strategy and evaluated their antiproliferative activity against three cancer cell lines (MDA-MB-231, MDA-MB-468 and MCF-7). For the structure-activity relationships, the importance of 1,2,3-triazole and substituents on phenyl ring was explored. Among these phenothiazine-1,2,3-triazole hybrids, compound 9f showed the most potent inhibitory effect against MCF-7 cells, with an IC₅₀ value of 0.8 μM. Importantly, compound 9f could induce apoptosis against MCF-7 cells by regulating apoptosis-related proteins (Bcl-2, Bax, Bad, Parp, and DR5). These potent phenothiazine-1,2,3-triazole hybrids as novel apoptosis inducers might be used as antitumor agents in the future.

Full text of DOI:10.3390/molecules23061288

molecules
Article
Antiproliferative Phenothiazine Hybrids as Novel
Apoptosis Inducers against MCF-7 Breast Cancer
Jun-Xia Zhang 1,2, Jiao-Mei Guo 2, Ting-Ting Zhang 2, Hong-Jun Lin 2, Nai-Song Qi 2,
Zhen-Guo Li 2, Ji-Chun Zhou 2 and Zhen-Zhong Zhang 1,3,
*
1
School of Pharmaceutical science, Zhengzhou University, Zhengzhou 450001, China; zjx312@163.com
Department of Pharmacology, Henan Provincial Institute of Food and Drug Control,
Zhengzhou 450008, China; ssjsel745288@163.com (J.-M.G.); 18637123006@163.com (T.-T.Z.);
13393723919@163.com (H.-J.L.); 18538293031@163.com (N.-S.Q.); 13298181239@163.com (Z.-G.L.);
18838969948@163.com (J.-C.Z.)
2
3
Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Henan Province,
Zhengzhou 450001, China
*
Correspondence: zhangzhenzhong@zzu.edu.cn; Tel.: +0371-6338-8226
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 18 April 2018; Accepted: 17 May 2018; Published: 28 May 2018
Abstract: We designed a series of novel phenothiazine-1,2,3-triazole hybrids by the molecular
hybridization strategy and evaluated their antiproliferative activity against three cancer cell lines
(MDA-MB-231, MDA-MB-468 and MCF-7). For the structure-activity relationships, the importance of
1,2,3-triazole and substituents on phenyl ring was explored. Among these phenothiazine-1,2,3-triazole
hybrids, compound 9f showed the most potent inhibitory effect against MCF-7 cells, with an IC50
value of 0.8 µM. Importantly, compound 9f could induce apoptosis against MCF-7 cells by regulating
apoptosis-related proteins (Bcl-2, Bax, Bad, Parp, and DR5). These potent phenothiazine-1,2,3-triazole
hybrids as novel apoptosis inducers might be used as antitumor agents in the future.
Keywords: phenothiazine-1,2,3-triazole; MCF-7; apoptosis; DR5
1. Introduction
According to the cancer epidemiology data collected in the cancer statistics 2017, breast cancer
accounts for about 14.4% of cancer related death and 29.6% of new cases in female, and which is the
top type of female new cancer cases and the second cause of female cancer related death worldwide
after lung cancer [1]. Its poor overall prognosis reflects not only its inherent aggressiveness but also
the lack of targeted therapies [
treat breast cancer.
2]. Therefore, it is necessary to develop the potent antitumor agents to
Phenothiazine is a useful scaffold in antitumor drug design [
phenothiazine derivatives were designed as novel anticancer agents. For example, phenothiazine
derivative (Figure 1) proved to be a microtubule-interacting agent with great cell growth
inhibition profile (GI50 in the nanomolar range on human melanoma-adenocarcinoma MDA-MB-435
cells) [ ]. Phenothiazine derivative could arrest tumor cell cycle in the G2/M phase [ ].
The 3-hydroxy-4-methoxyphenylethyl derivative was a potent in vitro tubulin polymerization
3–
5].
Recently, many
1
6
2
7
3
inhibitor but with weak cytotoxicity toward the human colon adenocarcinoma HT29 cell line, in the
micromolar range [8].
Molecules 2018, 23, 1288; doi:10.3390/molecules23061288
www.mdpi.com/journal/molecules

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Figure 1. Phenothiazine derivatives as novel anticancer agents.
Triazole derivatives display an ample spectrum of pharmacological activities and are widely
employed as pharmaceuticals and agrochemicals [ ]. They have been regarded as important
skeletons in terms of biological activity and some of them have also shown significant anticancer
activity [10]. Benzenesulfonamide (Figure 2) showed an IC50 value of 4.08 M against
PC-3 cells and the potent tubulin depolymerization activity in vitro with an IC50 value of
2.41 M. Arylamide-1,2,3-triazole exhibited potent antitumor in vitro against MCF-7 cancer cells
(IC50 = 46 nM) [11]. Picolinamide-1,2,3-triazole could induce apoptosis and colony formation at a
low micromolar concentration against HT-29 cancer cells [12].
9
4
µ
µ
5
6
Figure 2. 1,2,3-Triazole derivatives as novel antitumor agents.
Based on the results of 1,2,3-triazole and phenothiazine as antitumor skeletons, we envisioned that a
phenothiazine ring linked a 1,2,3-triazole unit would serve as a novel and potent anticancer agents. In this
study, the synthesis and antiprolifervative evaluation of phenothiazine-1,2,3-triazole analogues, as well as
the inducing apoptosis properties for the most active compound were explored (Figure 3).
Figure 3. Rational design of target compounds as novel antitumor agents.

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2. Results and Discussion
2.1. Chemistry
Azide derivatives 7a
–7h used in this paper were purchased from Beijing Research Biotechnology
Co., Ltd (Beijing, China) (Figure 4). Commercially available phenothiazine was treated with propargyl
bromide to provide 8a–8b in the presence of potassium carbonate.
Figure 4. Azide derivatives 7a–7h used in this study.
Azide–alkyne 1,3-dipolar cycloaddition reaction popularly known as the “click chemistry”
serves as the highly dependable tool for facile construction of antiproliferative agents [13
These novel phenothiazine hybrids 9a 9k were obtained from alkyne intermediate 8a 8b and azide
derivatives 7a 7h using sodium ascorbate and copper sulfate system in THF/H2O (Scheme 1).
(The spectra of compounds 9a–9k can be seen in the supplementary materials.)
,14].
–
–
–
Scheme 1. Synthesis of phenothiazine-1,2,3-triazole hybrids 9a
–
9k. Reagents and conditions: (
a) K2CO3,
dichloromethane, propargyl bromide, reflux; (b) CuSO4.5H2O, sodium ascorbate, azide derivatives
7a–7h, THF:H2O (1:1), rt.
2.2. Antiproliferative Activity and SARs
All the synthesized and target phenothiazine-1,2,3-triazole hybrids 9a–9k were evaluated for
their antitumor activity in vitro against three breast cancer cell lines (MDA-MB-468, MDA-MB-231
and MCF-7) using a reported MTT assay [15]. In our antiproliferative experiments, 5-fluorouracil

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(5-Fu) was used as a control [16
,17]. Table 1 describes the antiproliferative activity results of
phenothiazine-1,2,3-triazole hybrids 9a–9k and phenothiazine derivatives 8a–8b.
Table 1. Antiproliferative results of phenothiazine-1,2,3-triazole hybrids 9a–9k.
IC50 (µM) a
Compound
MDA-MB-231
MDA-MB-468
MCF-7
8a
8b
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
5-Fu
>100
>100
>100
>100
>100
>100
9.0 ± 0.6
11.4 ± 0.2
14.2 ± 1.3
2.0 ± 0.2
10.5 ± 1.0
1.7 ± 0.1
56.3 ± 0.8
39.1 ± 0.6
11.3 ± 2.6
17.4 ± 1.4
17.3 ± 0.9
10.8 ± 0.3
15.7 ± 0.3
5.5 ± 1.7
9.0 ± 0.9
1.6 ± 0.3
8.4 ± 0.8
1.2 ± 0.2
46.4 ± 2.8
43.3 ± 0.7
11.2 ± 0.8
17.6 ± 2.4
14.3 ± 0.8
7.5 ± 0.2
8.6 ± 1.1
8.8 ± 2.0
11.1 ± 0.5
7.3 ± 1.1
7.2 ± 0.2
0.8 ± 0.1
>100
29.6 ± 1.3
14.2 ± 1.7
15.3 ± 0.4
16.4 ± 1.1
12.7 ± 0.6
a
Inhibitory activity was assayed by exposure for 72 h to substances and expressed as concentration
required to inhibit tumor cell proliferation by 50% (IC50). Data are presented as the means
three independent experiments.
±
SDs of
To explore the inhibitory effect of 1,2,3-triazole unit, phenothiazines 8a
evaluated for their antiproliferative activity. When the 1,2,3-triazole group was removed, the inhibitory
activity against all the cancer cell lines was decreased (IC50 > 100 M), indicating the 1,2,3-triazole
–8b and 9a–9k were
µ
scaffold is necessary for inhibitory activity. Especially, compound 9f showed the most potent inhibitory
effect against all cell lines with IC50 values ranging from 0.8 µM to 1.7 µM.
In addition, the substituent on the phenothiazine ring may be important for antiproliferative
activity. When the hydrogen atom of compounds 9a
compounds 9g 9k, the antiproliferative activity was decreased. Compound 9g displayed the weak
antiproliferative activity against MCF-7 cells (>100 M), indicating the significance of the hydrogen
–9f was replaced by a trifluoromethyl group of
–
µ
atom on the phenothiazine ring for their antiproliferative activity.
The substituent on the phenyl ring also displayed a remarkable effect on the antiproliferative
activity against the breast cancer cell lines. Compound 9c with the 4-bromine atom on the phenyl
ring showed more potent antiproliferative activity (9.0
(15.7 M) against MDA-MB-468 cells, and compound 9f with a 3,4,5-trimethoxy substituent on the
phenyl ring showed more potent antiproliferative activity (0.8 M) than compound 9d with the 4-CH3
substituent (7.3 M) against MCF-7 cell line. When 4-CH3 substituent (9d) as an electron-donating
group was replaced by the electron-withdrawing groups like F, Cl, Br (9a 9b 9c), the antiproliferative
µM) than compound 9a with a 4-F substituent
µ
µ
µ
,
,
activity was decreased against all cancer cell lines. The summary of structure activity relationships is
shown in Figure 5.

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Figure 5. The summary of structure activity relationships.
2.3. Compound 9f Induced Morphological Changes of Mcf-7 Cells
Because of the most potent anticancer activity in vitro against all the selected cancer cell lines
among phenothiazine-1,2,3-triazole hybrids, the antiproliferative mechanism of compound 9f was
explored. To explore cytotoxicity of 9f in MCF-7 cells, cell apoptosis was studied by the reported
Hoechst 33258 staining assay [18]. After 24 h incubation with 9f at 0.5 µM and 1 µM, characteristic
apoptotic morphological changes could be served by fluorescence microscope, including cell rounding,
chromain shrinkage and formation of apoptotic bodies (Figure 6). Therefore, target compound 9f could
cause morphological changes of MCF-7 cancer cells possibly by inducing apoptosis process.
Control
0.5μM
1μM
Figure 6. MCF-7 cells treated by 9f showed typical apoptotic morphologies.
2.4. Compound 9f Induced Apoptosis of Mcf-7 Cells
Based on the biological experiments above, the apoptosis property of compound 9f was
investigated against MCF-7 cells (Figure 7). After treatment with different concentrations of compound
9f (0, 0.5 and 1 µM) for 24 h, MCF-7 cells were stained with propidium Iodide and Annexin V Binding
Buffer, and then analyzed by the flow cytometry. As illustrated in Figure 7, the percentage of apoptotic
cells by treatment of 1 µM compound 9f was changed obviously from the control group.

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0.5 μM
1 μM
Control
5.6%
27.3%
15.6%
6.3%
2.0%
2.4%
9f （μM）
0
0.5
2.0
6.3
1
Early apoptosis (%)
Late apoptosis (%)
2.4
5.6
15.6
27.3
Figure 7. Compound 9f could induce apoptosis of MCF-7 cells.
2.5. Compound 9f Related Apoptosis-Related Proteins
The changes of morphological features, such as cell shrinkage, chromatin condensation,
nuclear membrane blebbing are the characteristics of apoptotic cells [19]. Based on the above results,
we explored the effect of compound 9f on celluar apoptosis. Bcl-2 family proteins could dictate death
decisions or cellular survival by regulating the integrity of the mitochondrial outer membrane [20].
Recent years, Bcl-2 family proteins have emerged as anticancer targets [21].
Due to the potent cytotoxic activity against MCF-7 cell line, compound 9f was evaluated the
related-apoptosis proteins. In western blot assay, compound 9f increased the level of Bad, Bax and
DR5 and reduced the level of Bcl-2 and Parp which could induce apoptosis of MCF-7 cells (Figure 8).
Control 0.5 μM 1 μM
Bad
Bax
Bcl-2
Parp
DR5
β-actin
Figure 8. Expression of apoptosis-related proteins in MCF-7 cells after treatment by compound 9f for 24 h.
3. Experimental Section
3.1. Chemistry
Reagents and solvents used were purchased from Beijing Research Biotechnology Co., Ltd.
(Beijing, China). All nMR spectra were obtained from a Bruker DPX 400 MHz spectrometer
(Agilent, Santa Clara, CA, USA) (internal standard was TMS). Mass spectra (MS) were recorded
on Esquire 3000 mass spectrometer by electrospray ionization (ESI) (Varian, Palo Alto, CA, USA) for all
phenothiazole-1,2,3-triazole hybrids.

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3.1.1. Phenothiazine Derivatives 8a–8b
To a stirred solution of phenothiazine or 2-(trifluoromethyl)-10H-phenothiazine (3 mmol)
in dichloromethane (20 mL), K2CO3 (5 mmol) and propargyl bromide (5 mmol) were added carefully
and the reaction mixture was refluxed for 7 h. After the reaction, the system was concentrated under
vacuum, the residue was dissolved in EtOAc (40 mL) and washed by pure water and brine, and dried
over anhydrous Na2SO4 and concentrated under vacuum to afford crude products 8a–8b, which were
used in the next reaction without further purification.
3.1.2. General Method to Synthesize Phenothiazine-1,2,3-Triazole Hybrids 9a–9k
Alkyne intermediates 8a–8b (3mmol), azide derivatives 7a–7h (6 mmol), CuSO4.5H2O (1.2 mmol)
and sodium ascorbate (0.6 mmol) were dissolved in THF/H2O (20 mL/20 mL). The system was reacted
for 8 h at room temperature. After the reaction, the product was filtered to afford the products 9a–9k,
which were purified with column chromatography on silica gel (hexane/EtOAc = 9/1).
10-{[1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl]methyl}-10H-phenothiazine (9a). Yield: 87%. White solid.
Mp: 166–167 ◦C. 1H nMR (400 MHz, DMSO-d6)
8.01 (s, 1H, CHN), 7.29–7.21 (m, 2H, Ar), 7.19–7.06
(m, 6H, Ar), 6.99–6.85 (m, 4H, Ar), 5.54 (s, 2H, PhCH2), 5.14 (s, 2H, NCH2). 13C nMR (100 MHz,
δ
DMSO-d6) δ 162.96, 160.53, 144.02, 143.77, 132.42, 132.39, 129.87, 129.78, 127.42, 126.74, 123.64, 122.64,
122.62, 115.71, 115.55, 115.34, 51.86, 43.86. HR-MS (ESI): Calcd. C22H18FN4S, [M + H]+ m/z: 389.1236,
found: 389.1239.
10-{[1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl]methyl}-10H-phenothiazine (9b). Yield: 69%. White solid.
Mp: 150–151 ◦C. 1H nMR (400 MHz, DMSO-d6)
δ
8.02 (s, 1H, CHN), 7.57–7.46 (m, 2H, Ar), 7.19–7.04
(m, 6H, Ar), 6.92 (t, J = 7.3 Hz, 4H, Ar), 5.54 (s, 2H, PhCH2), 5.15 (s, 2H, NCH2). 13C nMR (100 MHz,
DMSO-d6) δ 144.01, 143.80, 135.60, 131.53, 129.75, 127.43, 126.75, 123.79, 122.65, 122.63, 121.19, 115.71,
51.91, 43.87. HR-MS (ESI): Calcd. C22H18ClN4S, [M + H]+ m/z: 405.0941, found: 405.0948.
10-{[1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-yl]methyl}-10H-phenothiazine (9c). Yield: 77%. White solid. Mp:
◦
154–155 C. 1H nMR (400 MHz, DMSO-d6)
δ
8.02 (s, 1H, CHN), 7.49–7.28 (m, 2H, Ar), 7.19 (d, J = 8.5 Hz,
2H, Ar), 7.16–7.05 (m, 4H, Ar), 6.93 (t, J = 7.2 Hz, 4H, Ar), 5.56 (s, 2H, PhCH2), 5.15 (s, 2H, NCH2).
13C nMR (100 MHz, DMSO-d6)
δ 144.01, 143.81, 135.18, 132.66, 129.44, 128.61, 127.43, 126.75, 123.78,
122.65, 122.63, 115.71, 51.85, 43.86. HR-MS (ESI): Calcd. C22H18BrN4S, [M + H]+ m/z: 449.0436, found:
449.0440.
10-{[1-(4-Methylbenzyl)-1H-1,2,3-triazol-4-yl]methyl}-10H-phenothiazine (9d). Yield: 90%. White solid.
Mp: 151–152 ◦C. 1H nMR (400 MHz, DMSO-d6)
δ
7.98 (s, 1H, CHN), 7.23–7.00 (m, 8H, Ar), 7.00–6.79
(m, 4H, Ar), 5.49 (s, 2H, PhCH2), 5.13 (s, 2H, NCH2), 2.26 (s, 3H, CH3). 13C nMR (100 MHz, DMSO-d6)
144.03, 143.65, 137.25, 133.15, 129.14, 127.53, 127.41, 126.73, 123.57, 122.61, 115.70, 52.45, 43.86, 20.63.
HR-MS (ESI): Calcd. C23H21N4S, [M + H]+ m/z: 385.1487, found: 385.1489.
δ
◦
10-{[1-Benzyl-1H-1,2,3-triazol-4-yl]methyl}-10H-phenothiazine (9e). Yield: 82%. White solid. Mp: 150–152 C.
1H nMR (400 MHz, DMSO-d6)
8.01 (s, 1H, CHN), 7.37–7.23 (m, 3H, Ar), 7.20–7.05 (m, 6H, Ar),
6.98–6.87 (m, 4H, Ar), 5.55 (s, 2H, PhCH2), 5.15 (s, 2H, NCH2). 13C nMR (100 MHz, DMSO-d6)
144.03,
δ
δ
143.73, 136.18, 128.61, 127.92, 127.45, 127.41, 126.74, 123.75, 122.65, 122.62, 115.72, 52.63, 43.87. HR-MS
(ESI): Calcd. C22H19N4S, [M + H]+ m/z: 371.1330, found: 371.1339.
10-{[1-(3,4,5-Trimethoxybenzyl)-1H-1,2,3-triazol-4-yl]methyl}-10H-phenothiazine (9f).
Yield: 88%.
◦
White solid. Mp: 146–147 C. 1H nMR (400 MHz, DMSO-d6)
δ
8.05 (s, 1H, CHN), 7.19–7.01 (m, 4H, Ar),
7.01–6.83 (m, 4H, Ar), 6.55 (s, 2H, Ar), 5.46 (s, 2H, PhCH2), 5.14 (s, 2H, NCH2), 3.67 (s, 6H, OCH3), 3.62
(s, 3H, OCH3). 13C nMR (100 MHz, DMSO-d6)
152.92, 144.03, 143.66, 137.12, 131.56, 127.39, 126.75,
δ
123.58, 122.62, 122.52, 115.64, 105.12, 59.94, 55.80, 52.87, 43.80. HR-MS (ESI): Calcd. C25H25N4O3S,
[M+Na]+ m/z: 483.1467, found: 483.1468.

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10-{[1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl]methyl}-2-{trifluoromethyl}-10H-phenothiazine (9g). Yield: 93%.
◦
White solid. Mp: 161–162 C. 1H nMR (400 MHz, DMSO-d6)
δ 8.84 (s, 1H, CHN), 7.86 (d, J = 8.9 Hz, 2H,
Ar), 7.78 (d, J = 8.9 Hz, 2H, Ar), 7.35 (d, J = 8.0 Hz, 1H, Ar), 7.26 (d, J = 7.5 Hz, 2H, Ar), 7.21–7.12 (m, 2H,
Ar), 7.04 (d, J = 8.0 Hz, 1H, Ar), 6.98 (t, J = 7.3 Hz, 1H, Ar), 5.30 (s, 2H, NCH2). 13C nMR (100 MHz,
DMSO-d6) δ 144.58, 144.35, 143.29, 135.63, 132.76, 128.40, 128.22, 127.97, 127.32, 126.92, 125.41, 123.36,
122.70, 121.73 (d, J = 14.8 Hz), 121.29, 119.21, 116.16, 112.03, 43.69. HR-MS (ESI): Calcd. C22H15BrF3N4S,
[M + H]+ m/z: 503.0153, found: 503.0158.
10-{[1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl]methyl}-2-{trifluoromethyl}-10H-phenothiazine (9h). Yield: 90%.
◦
White solid. Mp: 153–155 C. 1H nMR (400 MHz, DMSO-d6)
δ 8.84 (s, 1H, CHN), 7.99–7.86 (m, 2H, Ar),
7.73–7.56 (m, 2H, Ar), 7.35 (d, J = 7.7 Hz, 1H, Ar), 7.26 (d, J = 8.4 Hz, 2H, Ar), 7.21–7.12 (m, 2H, Ar), 7.04
(d, J = 7.9 Hz, 1H, Ar), 6.99 (t, J = 7.4 Hz, 1H, Ar), 5.31 (s, 2H, NCH2). 13C nMR (100 MHz, DMSO-d6)
δ
144.58, 144.33, 143.30, 135.23, 132.93, 129.83, 128.41, 128.22, 127.97, 127.91, 127.31, 126.92, 123.36,
121.83, 121.66, 121.56, 119.20, 116.16, 112.03, 43.58. HR-MS (ESI): Calcd. C22H15ClF3N4S, [M + H]+ m/z:
459.0658, found: 459.0662.
10-{[1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl]methyl}-2-{trifluoromethyl}-10H-phenothiazine (9i). Yield: 81%.
White solid. Mp: 134–136 ◦C. 1H nMR (400 MHz, DMSO-d6)
δ
8.07 (s, 1H, CHN), 7.33 (d, J = 7.9 Hz,
1H, Ar), 7.28–7.19 (m, 3H, Ar), 7.19–7.07 (m, 5H, Ar), 7.06–6.90 (m, 2H, Ar), 5.56 (s, 2H, PhCH2), 5.21
(s, 2H, NCH2). 13C nMR (100 MHz, DMSO-d6)
144.44, 143.37, 143.27, 132.41, 132.38, 129.75, 129.66,
δ
127.92, 127.30, 126.90, 125.35, 123.68, 123.31, 121.53, 119.16, 116.11, 115.49, 115.28, 111.93, 51.89, 43.88.
HR-MS (ESI): Calcd. C23H17F4N4S, [M + H]+ m/z: 457.1110, found: 457.1118.
10-{[1-Benzyl-1H-1,2,3-triazol-4-yl]methyl}-2-{trifluoromethyl}-10H-phenothiazine (9j). Yield: 79%. White solid.
◦
1
Mp: 138–139 C. H nMR (400 MHz, DMSO-d6)
(d, J = 8.1 Hz, 1H, Ar), 7.20–7.07 (m, 5H, Ar), 7.03–6.93 (m, 2H, Ar), 5.58 (s, 2H, PhCH2), 5.22 (s, 2H,
NCH2). 13C nMR (100 MHz, DMSO-d6)
144.45, 143.38, 143.21, 136.19, 128.56, 128.24, 128.18, 127.90,
δ 8.07 (s, 1H, CHN), 7.39–7.27 (m, 4H, Ar), 7.24
δ
127.30, 127.24, 126.89, 125.37, 123.82, 123.31, 122.66, 121.51, 119.08, 116.12, 111.97, 52.62, 43.92. HR-MS
(ESI): Calcd. C23H18F3N4S, [M + H]+ m/z: 439.1204, found: 439.1208.
10-{[1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl]methyl}-2-{trifluoromethyl}-10H-phenothiazine (9k). Yield: 86%.
◦
White solid. Mp: 140–142 C. 1H nMR (400 MHz, DMSO-d6)
δ
8.08 (s, 1H, CHN), 7.43–7.28 (m, 3H, Ar),
7.24 (d, J = 8.0 Hz, 1H, Ar), 7.17 (ddd, J = 5.7, 3.8, 2.4 Hz, 4H, Ar), 7.12 (s, 1H, Ar), 7.05–6.85 (m, 2H, Ar),
5.58 (s, 2H, PhCH2), 5.22 (s, 2H, NCH2). 13C nMR (100 MHz, DMSO-d6)
144.44, 143.31, 135.17, 132.66,
δ
129.31, 128.54, 128.27, 128.17, 127.92, 127.85, 127.31, 126.90, 123.82, 123.31, 121.55, 119.13, 119.09. 116.12,
111.89, 51.88, 43.89. HR-MS (ESI): Calcd. C23H17ClF3N4S, [M + H]+ m/z: 473.0815, found: 473.0819.
This compound was also reported in the previous paper [22].
3.2. Biology
3.2.1. Antiproliferative Activity
The all breast cancer cell lines were maintained in the mixture of 89% RPMI1640 medium, 10% fetal
bovine serum and 1% penicillinestreptomycin. All cell lines were purchased from the China Center
for Type Culture Collection (Shanghai, China). The cancer cells were seeded into 96-well plates
(5000 cells per well). After 24 h incubation, the culture medium was removed and replaced with
medium containing the candidate compounds. The cells were incubated for another 72 h. Then, 30
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo lium bromide) solution (5 mg/mL) was added and
incubated for 2 h. Dimethyl sulfoxide (40 L) was added to each well to dissolve the dark blue
formazan; the absorbance was measured using a microplate reader at awavelength of 570 nm.
µL
µ

Molecules 2018, 23, 1288
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3.2.2. Apoptosis Analysis with Hoechst 33258 Staining
Near-confluent MCF-7 cells were seeded into a 6-well plate (5.0
24 h with the test compound, Hoechst 33258 was added to the culture medium to give final concentrations
of 5 mg/mL, respectively. The cells were incubated with the staining mixture for 30 min at 37 C and then
×
106 cells/well). After incubation for
◦
the 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. Hoechst 33258 permeates all the cells and makes the nuclei appear blue.
Apoptosis was revealed by nuclear changes such as chromatin condensation and nuclear fragmentation.
3.2.3. Apoptosis Analysis with Western Blot
MCF-7 cells were cultured with different concentrations of compound 9f for 24 h, both adherent
and floating cells were collected, 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 30 min on ice. Total protein◦contents were measured by
protein assay reagent. The membrane was indicated primary antibodies at 4 C. After that, the membrane
was incubated with secondary antibodies, goat anti-rabbit and goat anti-mouse IgG conjugated.
4. Conclusions
We designed and synthesized a series of novel phenothiazine-1,2,3-triazole hybrids and evaluated their
anticancer activity in vitro against three selected cancer cell lines (MDA-MB-468, MDA-MB-231 and MCF-7).
Among these hybrids, compound 9f showed the most excellent anticancer activity in vitro with an IC50
value of 0.8 µM against MCF-7 cancer cells. Biological mechanism displayed that 9f induced morphological
change and apoptosis against MCF-7 cells by increasing the level of Bad, Bax and DR5 and decreasing the
level of Bcl-2 and Parp.
Supplementary Materials: The spectra of compounds 9a–9k are available online.
Author Contributions: J.-X.Z., J.-M.G., T.-T.Z., H.-J.L., N.-S.Q., and Z.-G.L. performed the synthetic work, J.-X.Z.,
J.-C.Z., and Z.-Z.Z. were responsible for the direction of the biological research. All authors read and approved the
final manuscript.
Funding: This research was funded by the scientific and technological project of Henan Province: 162102310067.
Acknowledgments: This work was supported by the scientific and technological project of Henan Province
(No. 162102310067).
Conflicts of Interest: The authors declare no conflict of interest.
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