Antiproliferative benzothiazoles incorporating a trimethoxyphenyl scaffold as novel colchicine site tubulin polymerisation inhibitors

Article abstract of DOI:10.1080/14756366.2020.1753721

Tubulin polymerisation inhibitors exhibited an important role in the treatment of patients with prostate cancer. Herein, we reported the medicinal chemistry efforts leading to a new series of benzothiazoles by a bioisosterism approach. Biological testing revealed that compound 12a could significantly inhibit in vitro tubulin polymerisation of a concentration dependent manner, with an IC₅₀ value of 2.87 μM. Immunofluorescence and EBI competition assay investigated that compound 12a effectively inhibited tubulin polymerisation and directly bound to the colchicine-binding site of β-tubulin in PC3 cells. Docking analysis showed that 12a formed hydrogen bonds with residues Tyr357, Ala247 and Val353 of tubulin. Importantly, it displayed the promising antiproliferative ability against C42B, LNCAP, 22RV1 and PC3 cells with IC₅₀ values of 2.81 μM, 4.31 μM, 2.13 μM and 2.04 μM, respectively. In summary, compound 12a was a novel colchicine site tubulin polymerisation inhibitor with potential to treat prostate cancer.

Full text of DOI:10.1080/14756366.2020.1753721

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Antiproliferative benzothiazoles incorporating a
trimethoxyphenyl scaffold as novel colchicine site
tubulin polymerisation inhibitors
Dong-Jun Fu, Si-Meng Liu, Fu-Hao Li, Jia-Jia Yang & Jun Li
To cite this article: Dong-Jun Fu, Si-Meng Liu, Fu-Hao Li, Jia-Jia Yang & Jun Li (2020)
Antiproliferative benzothiazoles incorporating a trimethoxyphenyl scaffold as novel colchicine site
tubulin polymerisation inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1,
1050-1059, DOI: 10.1080/14756366.2020.1753721
To link to this article: https://doi.org/10.1080/14756366.2020.1753721
©
2020 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 17 Apr 2020.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
020, VOL. 35, NO. 1, 1050–1059
2
https://doi.org/10.1080/14756366.2020.1753721
BRIEF REPORT
Antiproliferative benzothiazoles incorporating a trimethoxyphenyl scaffold as
novel colchicine site tubulin polymerisation inhibitors
a
b
b
c
a
Dong-Jun Fu , Si-Meng Liu , Fu-Hao Li , Jia-Jia Yang and Jun Li
a
Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing,
b
People’s Republic of China; Department of Gastroenterology, The Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, People’s
Republic of China; Department of Pharmacy, People’s Hospital of Zhengzhou, Zhengzhou, People’s Republic of China
c
ABSTRACT
ARTICLE HISTORY
Tubulin polymerisation inhibitors exhibited an important role in the treatment of patients with prostate
cancer. Herein, we reported the medicinal chemistry efforts leading to a new series of benzothiazoles by a
bioisosterism approach. Biological testing revealed that compound 12a could significantly inhibit in vitro
tubulin polymerisation of a concentration dependent manner, with an IC50 value of 2.87 lM.
Immunofluorescence and EBI competition assay investigated that compound 12a effectively inhibited
tubulin polymerisation and directly bound to the colchicine-binding site of b-tubulin in PC3 cells. Docking
analysis showed that 12a formed hydrogen bonds with residues Tyr357, Ala247 and Val353 of tubulin.
Importantly, it displayed the promising antiproliferative ability against C42B, LNCAP, 22RV1 and PC3 cells
with IC50 values of 2.81lM, 4.31 lM, 2.13 lM and 2.04 lM, respectively. In summary, compound 12a was a
novel colchicine site tubulin polymerisation inhibitor with potential to treat prostate cancer.
Received 31 December 2019
Revised 26 March 2020
Accepted 2 April 2020
KEYWORDS
Colchicine site tubulin
polymerisation inhibitor;
prostate cancer;
benzothiazole; bioisosterism
2500
Control
Colchicine
1
1
2a-1µM
2a-4µM
1500
500
0
10 20 30 40 50 60 70 80 90 100
Time/minutes
A novel colchicine site tubulin
polymerization inhibitor
Introduction
2–(4-Aminophenyl) benzothiazole analogue 1 (Figure 1) exhibited
1
3,14
the antiproliferative activity against MCF-7 and MDA-468 cells
.
Prostate cancer is the second most commonly diagnosed cancer
and the sixth leading cause of cancer death among men world-
wide, with an estimated 1276000 new cancer cases and 359000
deaths in 2018 . Microtubules play an important role in various
cellular processes, including spindle formation, cellular shape
maintenance, and intracellular transportation . Microtubule has
been an attractive target for chemotherapeutic agents to treat
prostate cancer . Anticancer agents targeting tubulin bind at four
Benzothiazole 2 induced apoptosis and inhibited the growth of
1
5
HL60 and U937 cells . Benzothiazole linked a phenylpyridopyrimi-
1
–3
dinone 3 showed the significant cytotoxicity against human cer-
1
6
vical cancer cell line ME-180 with an IC50 value of 4.01 lM .
Based on these findings, benzothiazole was a promising scaffold
to design anticancer agents. On the other hand, our group
also reported three series of colchicine site tubulin inhibitors: (1)
compound 4 could change the membrane potential of the mito-
4–6
7
binding sites: taxanes, vinca alkaloids, colchicine and laulimalide
1
7
8
,9
chondria against MGC-803 cells ; (2) b-lactam containing a 3,4,5-
trimethoxyphenyl unit 5 exhibited the well antiproliferative activ-
sites . Although some tubulin inhibitors that bind to vinca alka-
loids and taxanes sites have been approved by Food and Drug
Administration (FDA), there are no FDA approved colchicine site ity against MGC-803 cells in vitro and in vivo ; (3) compound 6
1
8
tubulin inhibitors currently. Therefore, it is necessary to develop could inhibit MGC803 cell growth and colony formation, induce
1
9
novel tubulin polymerisation inhibitors targeting the colchicine G2/M phase arrest and promote apoptosis .
binding site to treat prostate cancer.
Bioisosterism is a useful strategy in rational drug design to
Recently, benzothiazole scaffold has been an attractive subject improve pharmacodynamic or pharmacokinetic properties of lead
1
0–12
20
due to its potently anticancer activity in medicinal chemistry
.
compounds . In order to optimise the reported colchicine site
CONTACT Jun Li
drlj666@163.com
Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese
Medicine, Beijing, 100029, People’s Republic of China
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1051
Figure 1. Benzothiazole analogues 1ꢀ3 and colchicine site tubulin inhibitors 4ꢀ6 as anticancer agents.
3
6
1
.53 µM (PC3 cells)
.48 µM (22RV1 cells)
0.02 µM (LNCAP cells)
2.04 µM (PC3 cells)
2.13 µM (22RV1 cells)
4.31 µM (LNCAP cells)
Figure 2. The rational design of benzothiazole analogues by bioisosterism approach.
tubulin inhibitor 4 and discovery more potently anticancer
Benzo[d]thiazole-2-thiol (1 mmol) and sodium hydroxide
2
1–25
agents
, benzothiazole was used as a bioisostere of the ben- (2 mmol) were added to the solution of crude products 8a~8k
ꢁ
zoxazole in the lead structure. Herein, we synthesised a variety of and 11a~11d (1 mmol) in acetone (5 ml). After stirring at 60 C for
benzothiazole derivatives based on the lead compound 4 by the 5 h, the reaction mixture was concentrated to remove acetone.
bioisosterism approach. In addition, the preliminary structure Then, the system was treated with a solution of ethyl acetate
activity relationship (SAR) was explored. The rational design of (30 ml) and H O (30 ml). The organic layer was washed with aque-
2
benzothiazole analogues was described in Figure 2. Compared ous Na
2 3
CO solution, water, and brine. The organic system was
with the reported compound 4, compound 12a showed the bet- concentrated to obtain the residue, which was purified by column
ter antiproliferative activity against prostate cancer cells.
chromatography with n-hexane/ethyl acetate (10:1) to afford the
target compounds 9a~9k and 12a~12d.
Materials and methods
Chemistry
General procedure for preparation of compounds 9aꢀ9k and 2-(Benzo[d]thiazol-2-ylthio)-N-(4-fluorobenzyl)-N-(3,4,5-trimethoxy-
2aꢀ12d phenyl)acetamide (9a)
Imine intermediates 7a~7k and 10a~10d were synthesised Yield: 52%, white solid, m.p:1 4 3 ꢀ 144 C. H NMR (400 MHz,
according to our reported references17,19. A solution of inter- CDCl
) d 7.67 (d, J ¼ 7.9 Hz, 1H), 7.60 (d, J ¼ 8.1 Hz, 1H), 7.37–7.29
mediates 7a~7k and 10a~10d (3 mmol) in dichloromethane (m, 1H), 7.24 – 7.21 (m, 1H), 7.15 (dd, J ¼ 8.5, 5.5 Hz, 2H), 6.87 (t,
20 ml) was reacted with 2-chloroacetyl chloride (6 mmol) at room J ¼ 8.7 Hz, 2H), 6.27 (s, 2H), 4.79 (s, 2H), 3.98 (s, 2H), 3.75 (s, 3H),
1
ꢁ
1
3
(
1
3
3
temperature. After the mixture was stirred for 3 h, the reaction 3.65 (s, 6H). C NMR (100 MHz, CDCl ) d 166.04, 164.53, 152.70,
mixture was diluted with water. The organic layer was washed by 137.06, 135.82, 134.54, 132.04, 129.95, 129.87, 124.95, 123.37,
aqueous NaHCO , water, and brine, dried over anhydrous Na SO . 120.18, 114.33, 114.12, 104.75, 104.72, 59.91, 55.18, 51.88, 35.49.
3
2
4
þ
The system was concentrated to provide the crude products HRMS (ESIþ): m/z [M þ H] calcd for C H FN O S : 499.1162;
2
5
24
2 4 2
8
a~8k and 11a~11d without purification.
found: 499.1167.

1
052
D. -J. FU ET AL.
2
-(Benzo[d]thiazol-2-ylthio)-N-(3-chlorobenzyl)-N-(3,4,5-trimethoxy- 2-(Benzo[d]thiazol-2-ylthio)-N-(3-methoxybenzyl)-N-(3,4,5-trime-
phenyl)acetamide (9 b) thoxyphenyl)acetamide (9 g)
ꢁ
1
ꢁ
1
Yield: 69%, white solid, m.p:1 0 5 ꢀ 106 C. H NMR (400 MHz, Yield: 69%, white solid, m.p:1 2 3 ꢀ 124 C. H NMR (400 MHz,
CDCl ) d 7.72–7.53 (m, 2H), 7.34–7.27 (m, 1H), 7.24–7.20 (m, 1H), CDCl ) d 7.64 (dd, J ¼ 14.7, 8.0 Hz, 2H), 7.36–7.26 (m, 1H), 7.24–7.19
3
3
7
4
.17 (d, J ¼ 1.4 Hz, 2H), 7.12 (dd, J ¼ 9.6, 5.3 Hz, 2H), 6.29 (s, 2H), (m, 1H), 7.10 (t, J ¼ 8.1 Hz, 1H), 6.74 (dd, J ¼ 6.0, 3.2 Hz, 3H), 6.28 (s,
13
.79 (s, 2H), 4.00 (s, 2H), 3.76 (s, 3H), 3.66 (s, 6H).
C NMR 2H), 4.80 (s, 2H), 4.01 (s, 2H), 3.75 (s, 3H), 3.63 (d, J ¼ 7.2 Hz, 9H).
13
(100 MHz, CDCl
3
) d 166.18, 164.45, 152.77, 151.80, 138.14, 137.18,
3
C NMR (100 MHz, CDCl ) d 166.02, 164.57, 158.68, 152.63,
1
1
35.77, 134.54, 133.17, 128.76, 128.17, 126.81, 126.37, 124.98, 151.85, 137.68, 137.04, 135.93, 134.52, 128.36, 124.95, 123.34,
23.37, 120.29, 120.10, 104.73, 59.92, 55.21, 52.12, 35.47. HRMS 120.51, 120.32, 120.05, 113.49, 112.38, 104.82, 59.90, 55.18, 54.17,
þ
þ
(ESIþ): m/z [M þ H] calcd for C H ClN O S : 515.0866; found: 52.58, 35.6. HRMS (ESIþ): m/z [M þ H] calcd for C H N O S :
25
24
2
4
2
26 27 2 5 2
5
15.0870.
511.1361; found: 511.1367.
2
-(Benzo[d]thiazol-2-ylthio)-N-(4-chlorobenzyl)-N-(3,4,5-trimethoxy- 2-(Benzo[d]thiazol-2-ylthio)-N-(4-methoxybenzyl)-N-(3,4,5-trime-
thoxyphenyl)acetamide (9 h)
phenyl)acetamide (9c)
ꢁ
1
ꢁ
1
Yield: 27%, white solid, m.p:1 0 7 ꢀ 109 C. H NMR (400 MHz, Yield: 52%, white solid, m.p:1 2 5 ꢀ 126 C. H NMR (400 MHz,
CDCl ) d 7.67 (d, J ¼ 7.9 Hz, 1H), 7.57 (d, J ¼ 8.0 Hz, 1H), 7.37–7.29 CDCl ) d 7.63 (dd, J ¼ 18.4, 7.9 Hz, 2H), 7.39–7.25 (m, 1H), 7.24–7.15
3
3
(
2
m, 1H), 7.23 (dd, J ¼ 11.1, 4.1 Hz, 1H), 7.19 – 7.05 (m, 4H), 6.29 (s, (m, 1H), 7.09 (d, J ¼ 8.6 Hz, 2H), 6.71 (d, J ¼ 8.6 Hz, 2H), 6.25 (s, 2H),
1
3
13
H), 4.79 (s, 2H), 3.98 (s, 2H), 3.75 (s, 3H), 3.66 (s, 6H). C NMR 4.76 (s, 2H), 3.98 (s, 2H), 3.75 (s, 3H), 3.70 (s, 3H), 3.64 (s, 6H).
C
(
100 MHz, CDCl
3
) d 166.13, 164.48, 152.74, 151.77, 137.10, 135.84, NMR (100 MHz, CDCl ) d 165.83, 164.65, 158.08, 152.60, 151.81,
3
1
1
34.70, 134.55, 132.50, 129.58, 127.56, 124.98, 123.39, 120.18, 136.93, 135.93, 134.52, 129.57, 128.35, 124.89, 123.31, 120.27,
20.14, 104.70, 59.92, 55.22, 52.02, 35.42. HRMS (ESIþ): m/z 120.08, 112.70, 104.83, 59.90, 55.17, 54.24, 51.99, 35.66. HRMS
þ
þ
[M þ H] calcd for C H ClN O S : 515.0866; found: 515.0869.
(ESIþ): m/z [M þ H] calcd for C H N O S : 511.1361; found:
2
5
24
2
4
2
26 27 2 5 2
5
11.1366.
2
-(Benzo[d]thiazol-2-ylthio)-N-(4-bromobenzyl)-N-(3,4,5-trimethoxy-
phenyl)acetamide (9d)
Yield: 82%, white solid, m.p:1 3 0 ꢀ 131 C. H NMR (400 MHz, mide (9i)
2-(Benzo[d]thiazol-2-ylthio)-N-(4-methoxybenzyl)-N-(p-tolyl)aceta-
ꢁ
1
1
CDCl ) d 7.70–7.63 (m, 1H), 7.56 (d, J ¼ 7.8 Hz, 1H), 7.37–7.28 (m, Yield: 78%, yellow liquid. H NMR (400 MHz, CDCl ) d 7.66 (t,
3
3
3
2
H), 7.25–7.20 (m, 1H), 7.07 (d, J ¼ 8.4 Hz, 2H), 6.29 (s, 2H), 4.77 (s, J ¼ 8.1 Hz, 2H), 7.31 (t, J ¼ 7.7 Hz, 1H), 7.21–7.17 (m, 1H), 7.05 (dd,
13
H), 3.97 (s, 2H), 3.75 (s, 3H), 3.65 (s, 6H). C NMR (100 MHz, J ¼ 8.2, 3.6 Hz, 4H), 6.91 (d, J ¼ 8.1 Hz, 2H), 6.69 (d, J ¼ 8.4 Hz, 2H),
13
DMSO-d
6
) d 161.86, 160.43, 148.52, 147.34, 132.89, 131.59, 130.97, 4.76 (s, 2H), 3.92 (d, J ¼ 1.0 Hz, 2H), 3.70 (s, 3H), 2.23 (s, 3H).
C
1
5
30.21, 126.30, 125.71, 120.82, 119.23, 116.40, 115.92, 100.48, NMR (100 MHz, CDCl ) d 165.97, 157.93, 137.75, 137.45, 129.33,
3
þ
5.70, 51.01, 47.87, 31.26. HRMS (ESIþ): m/z [M þ H] calcd for 129.29, 128.20, 127.22, 124.89, 123.26, 120.39, 120.00, 112.70,
þ
C H BrN O S : 559.0361; found: 559.0368.
54.18, 52.12, 35.97, 20.07. HRMS (ESIþ): m/z [M þ H] calcd for
2
5
24
2 4 2
24 23 2 2 2
C H N O S : 435.1201; found: 435.1207.
2
-(Benzo[d]thiazol-2-ylthio)-N-(4-methylbenzyl)-N-(3,4,5-trimethox-
yphenyl)acetamide (9e)
Yield: 43%, white solid, m.p:1 4 4 ꢀ 146 C. H NMR (400 MHz, benzyl)acetamide (9j)
CDCl
2-(Benzo[d]thiazol-2-ylthio)-N-(3,4-dichlorophenyl)-N-(4-methoxy-
ꢁ
1
1
3
) d 7.64 (dd, J ¼ 16.8, 8.0 Hz, 2H), 7.33–7.28 (m, 1H), 7.26–7.20 Yield: 63%, yellow liquid. H NMR (400 MHz, CDCl
3
) d 7.66 (t,
(
4
m, 1H), 7.06 (d, J ¼ 8.0 Hz, 2H), 6.99 (d, J ¼ 7.9 Hz, 2H), 6.26 (s, 2H), J ¼ 6.1 Hz, 2H), 7.37–7.29 (m, 2H), 7.27–7.20 (m, 2H), 7.02 (d,
1
3
.78 (s, 2H), 4.00 (s, 2H), 3.75 (s, 3H), 3.63 (s, 6H), 2.25 (s, 3H).
) d 165.94, 164.76, 152.62, 151.75, 137.02, 4.76 (s, 2H), 3.94 (s, 2H), 3.71 (s, 3H). C NMR (100 MHz, CDCl
36.23, 135.97, 134.49, 133.12, 128.19, 128.04, 124.93, 123.35, 165.73, 164.19, 158.19, 151.46, 139.70, 134.46, 132.48, 131.99,
20.30, 120.07, 104.89, 59.90, 55.17, 52.37, 35.70, 20.11. HRMS 130.24, 129.64, 129.22, 127.34, 127.22, 125.03, 123.44, 120.37,
C
J ¼ 8.4 Hz, 2H), 6.89 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.69 (d, J ¼ 8.4 Hz, 2H),
13
NMR (100 MHz, CDCl
1
1
3
3
) d
þ
þ
(
4
ESIþ): m/z [M þ H] calcd for C H N O S : 495.1412; found: 120.10, 112.94, 54.21, 52.13, 35.30. HRMS (ESIþ): m/z [M þ H]
26 27 2 4 2
95.1417.
2 2 2 2
calcd for C23H19Cl N O S : 489.0265; found: 489.0269.
2
-(Benzo[d]thiazol-2-ylthio)-N-benzyl-N-(3,4,5-trimethoxyphenyl)a-
2-(Benzo[d]thiazol-2-ylthio)-N-(4-methoxybenzyl)-N-phenylaceta-
mide (9k)
cetamide (9f)
ꢁ
1
ꢁ
1
Yield: 69%, white solid, m.p:1 5 0 ꢀ 152 C. H NMR (400 MHz, Yield: 68%, white solid, m.p: 109–110 C. H NMR (400 MHz, CDCl
3
)
CDCl
3
) d 7.65 (dd, J ¼ 14.2, 8.0 Hz, 2H), 7.34–7.27 (m, 1H), 7.24–7.15 d 7.66 (t, J ¼ 8.6 Hz, 2H), 7.29 (dt, J ¼ 15.1, 7.7 Hz, 4H), 7.21 (d,
(
(
m, 6H), 6.25 (s, 2H), 4.83 (s, 2H), 4.00 (s, 2H), 3.74 (s, 3H), 3.62 J ¼ 7.6 Hz, 1H), 7.09–7.01 (m, 4H), 6.68 (d, J ¼ 8.6 Hz, 2H), 4.79 (s,
1
3
13
3 3
s, 6H). C NMR (100 MHz, CDCl ) d 166.00, 164.62, 152.62, 151.85, 2H), 3.92 (s, 2H), 3.69 (s, 3H). C NMR (100 MHz, CDCl ) d 165.80,
1
1
36.99, 136.18, 135.89, 134.55, 128.19, 127.42, 126.59, 124.93, 164.67, 157.97, 151.79, 140.41, 134.50, 129.28, 128.76, 128.05,
23.33, 120.28, 120.09, 104.80, 59.90, 55.15, 52.58, 35.62. HRMS 127.52, 124.87, 123.26, 120.43, 120.02, 112.73, 54.18, 52.13, 36.01.
þ
þ
(ESIþ): m/z [M þ H] calcd for C H N O S : 511.1361; found: HRMS (ESIþ): m/z [M þ H] calcd for C H N O S : 421.1044;
26
27
2
5
2
23 21 2 2 2
5
11.1367.
found: 421.1049.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1053
2
-(Benzo[d]thiazol-2-ylthio)-N-((3,5-dimethylisoxazol-4-yl)methyl)-
In vitro tubulin polymerisation assay
N-(3,4,5-trimethoxyphenyl)acetamide (12a)
Tubulin (5.6 mg/ml) was resuspended in PEM buffer (containing
0 mM PIPES, 1 mM ATP, 1 mM EGTA, 10.2% glycerol, 0.5 mM
ꢁ
1
Yield: 83%, white solid, m.p:1 3 7 ꢀ 138 C. H NMR (400 MHz,
8
CDCl
3
) d 7.67 (d, J ¼ 7.9 Hz, 1H), 7.57 (d, J ¼ 8.1 Hz, 1H), 7.39–7.28
MgCl ) and then was preincubated with compound 12a, colchi-
2
(m, 1H), 7.26–7.21 (m, 1H), 6.31 (s, 2H), 4.65 (s, 2H), 3.92 (s, 2H),
cine or vehicle DMSO on ice. The reaction was monitored by a
spectrophotometer in absorbance at 420 nm (excitation wave-
length is 340 nm).
1
3
3
.76 (s, 3H), 3.69 (s, 6H), 1.95 (s, 3H), 1.90 (s, 3H).
C NMR
(
100 MHz, CDCl ) d 167.43, 165.70, 164.36, 158.95, 153.02, 151.69,
3
1
1
37.51, 134.80, 134.47, 125.14, 123.46, 120.16, 120.05, 109.21,
05.09, 60.05, 55.39, 39.84, 35.19, 9.74, 8.91. HRMS (ESIþ): m/z
þ
[M þ H] calcd for C H N O S : 500.1314; found: 500.1317.
2
4 26 3 5 2
Immunostaining and microscopy
PC3 cells were seeded on the slices and incubated overnight.
Then, cells were treated with different concentrations of 12a.
After 48 h, slices were fixed by 4% paraformaldehyde for 15 min
after washed by PBS for 3 times. 0.5% Triton-X-100 was added
and shaked for 20 min. 0.1% BSA was used to block for 30 min
and then removed. The slices were added b-tubulin antibody
(1:100) and incubated overnight. Then slices were washed by
PBST 3 times, bind with secondary antibody with FITC signal
2
-(Benzo[d]thiazol-2-ylthio)-N-(pyridin-4-ylmethyl)-N-(3,4,5-trime-
thoxyphenyl)acetamide (12 b)
Yield: 65%, white solid, m.p:1 2 9 ꢀ 130 C. H NMR (400 MHz,
CDCl
ꢁ
1
3
) d 8.43 (d, J ¼ 5.5 Hz, 2H), 7.69 (d, J ¼ 7.7 Hz, 1H), 7.63 (d,
J ¼ 8.1 Hz, 1H), 7.38–7.31 (m, 1H), 7.25 (d, J ¼ 7.2 Hz, 1H), 7.20 (dd,
J ¼ 6.5, 4.5 Hz, 2H), 6.39 (s, 2H), 4.85 (s, 2H), 4.02 (s, 2H), 3.75 (s,
1
3
3
1
1
H), 3.67 (s, 6H).
52.92, 151.71, 148.28, 145.96, 137.29, 135.91, 134.61, 125.13,
23.54, 122.71, 120.31, 119.97, 104.41, 59.92, 55.27, 52.00, 35.15.
C NMR (100 MHz, CDCl ) d 166.59, 164.51,
3
(1:500) in a dark. DAPI was used to stain for 3 min and then
þ
removed. After that, images were captured by Laser scanning con-
focal microscope (Nikon, Japan).
HRMS (ESIþ): m/z [M þ H] calcd for C24
24 3 4 2
H N O S : 482.1208;
found: 482.1213.
2
(
-(Benzo[d]thiazol-2-ylthio)-N-((6-chloropyridin-3-yl)methyl)-N-
3,4,5-trimethoxyphenyl)acetamide (12c)
Yield: 73%, white solid, m.p:1 4 5 ꢀ 146 C. H NMR (400 MHz,
CDCl
) d 8.13 (d, J ¼ 2.2 Hz, 1H), 7.73–7.60 (m, 2H), 7.52 (d,
J ¼ 8.1 Hz, 1H), 7.38–7.30 (m, 1H), 7.23 (dd, J ¼ 11.3, 4.0 Hz, 1H),
.12 (d, J ¼ 8.2 Hz, 1H), 6.34 (s, 2H), 4.80 (s, 2H), 3.96 (s, 2H), 3.76
EBI competition assay
ꢁ
1
6-Well plates were seeded with PC3 cells for 24 h. Then, cells were
incubated with compound 12a, colchicine or DMSO for 2 h and
afterward treated with EBI (100 mM) for 2 h. Then, the cells were
harvested and lysed. Cell extracts were used for Western blotting
analysis.
3
7
1
3
(s, 3H), 3.69 (s, 6H). C NMR (100 MHz, CDCl
3
) d 166.47, 164.30,
1
1
4
5
53.00, 151.66, 149.88, 148.93, 138.94, 137.36, 135.60, 134.53,
30.76, 125.06, 123.47, 123.28, 120.20, 120.05, 104.57, 59.94, 55.31,
þ
9.59, 35.13. HRMS (ESIþ): m/z [M þ H] calcd for C H ClN O S : Western blot analysis
24
23
3 4 2
16.0819; found: 516.0826.
ꢁ
Cellular protein lysates were denatured in loading buffer at 100 C
prior to adding into SDS-PAGE. Proteins were transferred to nitro-
cellulose membranes, probed with indicated antibodies, and
visualised by an enhanced chemiluminescence detection system.
The western blotting bands were semi-quantified using Image J
and adjusted for loading control.
2
-(Benzo[d]thiazol-2-ylthio)-N-(naphthalen-2-ylmethyl)-N-(3,4,5-tri-
methoxyphenyl)acetamide (12d)
ꢁ
1
Yield: 52%, white solid, m.p:1 1 8 ꢀ 119 C. H NMR (400 MHz,
CDCl ) d 7.76–7.71 (m, 1H), 7.65 (dt, J ¼ 10.7, 5.0 Hz, 3H), 7.55 (d,
3
J ¼ 8.5 Hz, 2H), 7.42–7.33 (m, 3H), 7.24–7.17 (m, 2H), 6.26 (s, 2H),
1
3
4
.99 (s, 2H), 4.02 (s, 2H), 3.73 (s, 3H), 3.54 (s, 6H).
C NMR
3
(100 MHz, CDCl ) d 166.13, 164.56, 152.63, 151.81, 137.00, 135.89, DAPI staining
1
1
5
34.53, 133.61, 132.16, 131.79, 127.21, 127.19, 126.79, 126.58,
Cells were seeded on a 6-well plate (2 ꢂ 105/well) and treated
26.12, 125.12, 124.97, 124.91, 123.32, 120.29, 120.07, 104.81,
þ
with the test compound after the incubation for 24 h. Then, cells
9.88, 55.11, 52.75, 35.63. HRMS (ESIþ): m/z [M þ H] calcd for
0
were stained by DAPI (4 ,6-diamidino-2-phenylindole) in the dark
C H N O S : 531.1412; found: 531.1415.
2
9 27 2 4 2
for 30 min. The images of cells were observed in a fluorescence
microscope (Nikon, Japan).
Biology
Cell culture and MTT assay
Molecular modelling
PC3, C42B, 22RV1, and LNCAP cell lines were cultured in an
atmosphere containing 5% CO
ꢁ
Molecular modelling was performed using the autodock 4 soft-
2
at 37 C, with RPMI-1640 medium
with 10% foetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml ware from the Scripps Research Institute. Molecules were first gen-
streptomycin. Cells were seeded at a density of 1500 per well in erated using Pymol software. For the receptor preparation, the
9
6-well plates for 72 h. Then, 20 lL MTT (thiazolyl blue tetrazolium PDB code (1SA0) was downloaded from the Protein Data Bank.
bromide) solution was added to each well, and incubated for 4 h Dockings were initially performed using a blind docking method
ꢁ
at 37 C. 150 lL DMSO was added to each well to dissolve the for- on a lower resolution docking. This was followed by a series of
mazan after removing the liquid, the absorbance was determined resolving steps to attempt to fine tune the compound. The final
at 570 nm.
results were analysed by Pymol software.

1
054
D. -J. FU ET AL.
2 3
Scheme 1. Reagents and conditions: (a) K CO , aniline derivatives or various benzyl chlorides, acetone, reflux; (b) Chloroacetyl chloride, dichloromethane, rt; (c) NaOH,
benzo[d]thiazole-2-thiol, acetone, reflux.
Results and discussion
reference drug in the MTT assay to evaluate the antiproliferative
2
6,27
activity of synthetic benzothiazole derivatives
was used as the control drug in this work. In addition, compound
. Therefore, 5-Fu
Synthesis of the target analogues 9aꢀ9k and 12aꢀ12d
The synthetic route of benzothiazoles containing the 3, 4, 5-trime- 4 was also the control drug to compare the antiproliferative
thoxyphenyl fragment was shown in Scheme 1. Aniline derivatives activity.
and various benzyl chlorides were subjected to nucleophilic sub-
stitution reaction to afford imines 7a~7k and 10a~10d in the
presence of potassium carbonate. The imine intermediates were
reacted with chloroacetyl chloride to obtain the amide analogues
From the antiproliferative results of benzothiazole derivatives
in Table 1, benzothiazoles without the 3, 4, 5-trimethoxyphenyl
fragment (9i~9k) displayed very weak activity against 22RV1 cells
with IC50 values of >20 lM. However, benzothiazole-trimethoxy-
phenyl hybrids 9a~9h showed moderate to potent activity with
IC50 values from 5.24 lM to 19.07 lM against 22RV1 cells. Based
on this findings, it revealed that 3, 4, 5-trimethoxyphenyl fragment
exhibited an important role for the antiproliferative activity of
these benzothiazole derivatives.
To obtain potently antiproliferative compounds, trimethoxy-
phenyl fragment as B ring was reserved and the molecular dever-
sity of different substituent groups attaching on the A ring was
explored. Among them, compound 9 h showed the most potent
activity with an IC50 value of 5.24 lM against 22RV1 cells. We
found that the substitution on the A ring was important for the
activity showing an over 3-fold activity loss, when the p-Br group
8a~8k and 11a~11d in the dichloromethane system. The target
analogues 9a~9k and 12a~12d were easily obtained at the reflux
condition with benzo[d]thiazole-2-thiol in the presence of sodium
hydroxide. The chemical structures of all benzothiazole derivatives
9a~9k and 12a~12d were characterised using spectral methods,
and all spectral data corroborated the structures.
Antiproliferative activity of novel benzothiazole derivatives
To discovery novel anti-prostate cancer agents and tubulin poly-
merisation inhibitors, we evaluated the antiproliferative activity of
all benzothiazole derivatives 9a~9k and 12a~12d against a pros- was replaced with the m-Cl group (compounds 9d vs. 9 b). In add-
tate cancer cell line 22RV1 using the 3–(4,5-dimethyl-2-thiazolyl)- ition, compounds 9 g~9h with an electron-donating methoxy
2
,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Based on previ- group on the A ring had a more potent inhibitory effect (7.37 and
ous references, 5-fluorouracil (5-Fu) and was usually selected as a 5.24 lM, respectively) against 22RV1 cells than compounds 9 b~9c

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1055
Table 1. Antiproliferative activity of the synthetic derivatives 9a~9k against Table 2. Antiproliferative activity of the synthetic derivatives 12a~12d against
2RV1 cells. 22RV1 cells.
2
a
Compound
R
1
R
2
22RV1
9
9
9
9
9
9
9
9
9
9
9
4
5
a
a
b
c
d
e
f
g
h
i
4-F
3,4,5-triOCH
3,4,5-triOCH
3,4,5-triOCH
3,4,5-triOCH
3,4,5-triOCH
3,4,5-triOCH
3,4,5-triOCH
3,4,5-triOCH
3
3
3
3
3
3
3
3
16.23 ± 0.84
19.07 ± 1.15
12.17 ± 0.58
6.20 ± 0.71
8.25 ± 0.43
9.06 ± 0.17
7.37 ± 0.56
5.24 ± 0.78
>20
>20
>20
6.48 ± 0.25
18.31 ± 1.27
3-Cl
4-Cl
4-Br
4-CH
H
3-OCH
4-OCH
4-OCH
4-OCH
4-OCH
–
Compound
2a
22RV1
3
1
2.13 ± 0.24
3
3
3
3
3
4-CH
3
j
k
3,4-diCl
H
–
–
12b
5.09 ± 0.62
-FU
–
Antiproliferative activity was assayed by exposure for 72 h.
1
1
2c
11.84 ± 0.91
2d
6.93 ± 0.46
(IC50 >12 lM) with an electron-withdrawing group. From the
biological data of compounds 9a~9h, we can conclude that the
substituents on the A ring had a remarkable effect on their cyto-
toxic activity.
To determine whether the aromatic ring or heterocycles have
an effect on the activity, compounds with a 3, 5-dimethylisoxazole
ring (12a), a pyridine ring (12 b), a 2-chloropyridine ring (12c),
4
5
a
–
–
6.48 ± 0.25
18.31 ± 1.27
-FU
Antiproliferative activity was assayed by exposure for 72 h.
and a naphthalene ring (12d) were synthesised and their antiproli- Compound 12a inhibited tubulin polymerisation by
ferative activity results were shown in Table 2. Replacement of the immunostaining assay
pyridine scaffold of compound 12 b with a 2-chloropyridine frag-
ment (12c) or a naphthalene unit (12d) led to a loss of the activ-
ity. However, changing the pyridine ring of compound 12 b to a
As the microtubule system played an important role in the main-
tenance of cell shape and basic cellular functions, an immuno-
fluorescence staining assay was performed to study whether
benzothiazole hybrid 12a could disrupt the microtubule dynamics
3
, 5-dimethylisoxazole ring of compound 12a led to an improve-
ment of the activity against 22RV1 cells. All these results indicated
that 3, 5-dimethylisoxazole displayed the better inhibitory activity microtubule network in PC3 cells exhibited normal arrangement
2
8
in living cells . As shown in Figure 4, in the control group, the
than other selected heterocycles and phenyl ring.
and organisation, characterised by regularly assembled, normal
microtubules wrapped around the cell nucleus. Benzothiazole
hybrid 12a at the concentration of 1 lM moderately depolymer-
ised interphase microtubules whereas 12a at the higher concen-
tration of 4 lM induced much stronger depolymerisation effects.
These immunofluorescence experiments indicated that compound
Compound 12a inhibited cell proliferation against prostate
cancer
Among all benzothiazole derivatives 9a~9k and 12a~12d, 12a
displayed the most potently antiproliferative activity with an IC50
value of 2.13 lM against 22RV1 cells. Compound 12a was further
examined for possible cytotoxicity against RWPE-1 (normal human
1
2a inhibited tubulin polymerisation in a concentration depend-
ent manner against PC3 cells.
prostatic cell line). We found that compound 12a exhibited no Compound 12a inhibited tubulin polymerisation targeting the
colchicine binding site
cytotoxicity against RWPE-1 (>20 lM). The results indicated that
compound 12a had good selectivity between the selected cancer
cell line (22RV1) and a normal cell line (RWPE-1). To evaluate its
antiproliferative activity against other prostate cancer cell lines,
C42B, LNCAP and PC3 cells were selected to perform MTT assay.
The in vitro tubulin polymerisation inhibition activity of compound
12a was evaluated because of its best antiproliferative activity
results among all benzothiazole hybrids. When tubulin was incu-
bated with the tested compound 12a, the increased tendency of
As shown in the Figure 3, benzothiazole derivative 12a exhibited the fluorescence intensity was obviously slowed down compared
the well antiproliferative activity against C42B, LNCAP and PC3 with the control. The IC50 value of compound 12a was 2.87 lM
cell lines with IC50 values of 2.81 ± 0.43 lM, 4.31 ± 0.27 lM and against tubulin (Figure 5(A)). It revealed thatbenzothiazole hybrid
2
.04 ± 0.51 lM, respectively.
12a was a novel tubulin polymerisation inhibitor.

1
056
D. -J. FU ET AL.
C42B
22RV1
1
50
00
150
100
50
4
7
8 hours
2 hours
48 hours
72 hours
1
*
*
*
*
*
**
*
**
*
*
*
*
*
**
*
**
***
5
0
***
*
**
*
***
*
***
*
***
0
0
0
1
2
3
4
0
1
2
3
4
0
1
2
3
4
0
1
2
3
4
Concentration (µM)
Concentration (µM)
PC3
LNCAP
1
50
00
1
50
00
4
7
8 hours
2 hours
4
7
8 hours
2 hours
1
1
*
*
**
*
*
*
**
*
*
*
*
***
*
**
5
0
50
*
**
*
**
*
***
0
0
0
1
2
3
4
0
1
2
3
4
0
1
2
3
4
0
1
2
3
4
Concentration (µM)
Concentration (µM)
Figure 3. Antiproliferative activity of benzothiazole 12a against four prostate cancer cell lines for 48 h and 72 h.
ꢃp < 0.05 verse control, ꢃꢃp < 0.01 verse control,
ꢃꢃꢃ
ꢃꢃꢃꢃ
p < 0.0001 versus control.
p < 0.001 versus control,
Tubulin
Nucleus
Merged
Control
1
4
µM
µM
Figure 4. Compound 12a showed strong depolymerising effects on the interphase microtubule network.
In order to evaluate whether benzothiazole hybrid 12a directly Cys239 and the Cys354 residues of b-tubulin involved in the col-
3
1
binds to tubulin at the colchicine binding site, we carried out chicine-binding site . The occupancy of colchicine-binding site by
0
N,N -ethylenebis(iodoacetamide) (EBI) competition assay in PC3 tubulin polymerisation inhibitors could inhibit the formation of
29,30
cells according to previously published references
. EBI is an the EBI: b-tubulin adduct. Preincubation of compound 12a at
alkylating agent that has the property to specifically cross-link the 10 lM prevented the formation of the EBI: b-tubulin adduct,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1057
(
A)
resulting in the decrease of the adduct band comparing with
DMSO and EBI treatment. Thus, the assay (Figure 5(B)) indicated
that compound 12a directly bind to the colchicine-binding site of
b-tubulin.
2
500
500
Control
Colchicine
1
1
2a-1µM
2a-4µM
1
Compound 12a induced morphological changes and affected
the expression of apoptosis-related proteins
As most tubulin destabilising agents displayed the ability to
induce morphological changes and affect the expression of apop-
tosis-related proteins , DAPI staining and western blotting experi-
ments were performed to determine the effects of benzothiazole
hybrid 12a. After 48 h incubation with compound 12a (0, 1 lM,
500
0
10 20 30 40 50 60 70 80 90 100
3
2
Time/minutes
(
B)
+
EBI (100 µM)
2
lM and 4 lM), characteristic apoptotic morphological changes
1
2a
DMSO
Colchicine
were observed by fluorescence microscopy, including chromatin
shrinkage and formation of apoptotic bodies (Figure 6(A)). The
studies revealed that compound 12a induced morphological
changes of prostate cancer (22RV1 cells and PC3 cells) possibly by
inducing apoptosis.
0
1µM 10µM 10µM
β-tublin native
EBI: β-tublin adduct
GAPDH
Bcl-2 family proteins are key regulators of apoptosis and over-
Figure 5. (A) Tubulin polymerisation inhibitory activity of 12a. (B) EBI competi- expression of the prominent pro-survival Bcl-2 family members
tion assay of 12a in PC3 cells.
like Bcl-2 is a common feature responsible for deregulation of
apoptosis in prostate cancer cells . In order to investigate the
3
3
(
A)
0
1
2
4 (µM)
2
2RV1
PC3
(
B)
2
2RV1
2
2
2RV1
4
3
3
2
00
50
00
50
*
** ****
Bcl-2
0
1
4 (µM)
Bcl-2
Cleaved-parp
*
*
200
150
Cleaved-parp
100
50
β-tubulin
***
*
**
*
***
4
0
0
1
2
0
1
2
4
Concentration (µM)
PC3
600
500
400
300
200
*
**
Bcl-2
Cleaved-parp
PC3
*
*
0
1
2
4 (µM)
Bcl-2
100
Cleaved-parp
β-tubulin
5
0
***
*
**
*
***
0
0
1
2
4
0
1
2
4
Concentration (µM)
Figure 6. (A) Morphological changes of prostate cancer cells with the treatment of 12a. (B) The expression levels of apoptosis-related proteins (Bcl-2 and Cleaved-
ꢃꢃ ꢃꢃꢃ ꢃꢃꢃꢃ
parp). p < 0.01 versus control, p < 0.001 versus control, p < 0.0001 versus control.

1
058
D. -J. FU ET AL.
(
A)
(
B)
antiproliferative ability against C42B, LNCAP, 22RV1 and PC3 cell
lines with IC50 values of 2.81 lM, 4.31 lM, 2.13 lM and 2.04 lM,
respectively. Molecular mechanisms illustrated that compound
12a could induce morphological changes and affect the expres-
sion levels of apoptosis-related proteins (Bcl-2 and Cleaved-parp)
against prostate cancer cells. From the in vitro tubulin polymerisa-
tion inhibition assay and EBI competition assay, compound 12a
was a novel colchicine site tubulin polymerisation inhibitor. Taken
together, compound 12a might be a lead candidate for its further
development in treatment of prostate cancer.
(
C)
Val177
Asn329
Tyr357
Acknowledgements
Ser178
The authors gratefully thank the support from Zhengzhou
university.
Pro325
Val353
Disclosure statement
Ile355
No potential conflict of interest was reported by the author(s).
Tyr224
Ala247
Funding
Figure 7. Molecular docking studies of compound 12a. (A) Compound 12a
bound to b-tubulin (surface). (B) The complex formed between tubulin and com- This work was supported by the State Key Laboratory of
pound 12a. (C) Hydrogen bonds (red ellipse), hydrophilic interactions (green
ellipse) and hydrophobic interactions (yellow ellipse) observed between com-
pound 12a and residues.
Innovative Natural Medicine and TCM Injections [Grant number
QFSKL2018001] and the Fundamental Research Funds for the
Central Universities [Grant number 2019-JYB-XS-062].
apoptosis effects of compound 12a, the expression of apoptosis-
related proteins (Bcl-2 and Cleaved-parp) were tested. As shown
in the Figure 6(B), the expression level of Bcl-2 was decreased and
the expression level of Cleaved-parp was increased in a concentra-
ORCID
tion-dependent manner against prostate cancer (22RV1 cells and Jun Li
http://orcid.org/0000-0001-8243-5267
PC3 cells).
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