Design, synthesis, and apoptosis-promoting effect evaluation of novel pyrazole with benzo[d]thiazole derivatives containing aminoguanidine units

Article abstract of DOI:10.1080/14756366.2019.1591391

New pyrazole with benzo[d]thiazoles containing hydrazinecarboximidamide substituent was synthesised and evaluated for cytotoxicity and apoptotic activity using the MTT assay, flow cytometry, and Western blot analysis. Among the compounds studied, (E)-2-((1-(6-((4-fluorobenzyl)oxy)benzo[d]thiazol-2-yl)-3-phenyl-1H- pyrazol-4-yl)methylene) hydrazinecarboximidamide (8l) was potent, with IC₅₀ values of 2.41 μM, 2.23 μM, 3.75 μM and 2.31 μM in vitro anti-proliferative activity testing against triple-negative breast cancer cell line MDA-MB-231, non-triple-negative breast cancer MCF-7 cells, and human hepatocarcinoma HepG2 cells, and SMMC-7721 cells, respectively. Especially, the activity against MDA-MB-231 was similar to that of Doxorubicin, which was used as a positive control in this study. Next, the Annexin V/PI flow cytometry assay was used at different concentrations of compound 8l to demonstrate that compound 81 induced apoptosis of MDA-MB-231 cells in a concentration-dependent manner. Finally, these results were further verified by Western blot analysis. Taken together, the results of this study revealed that compound 8l may be a potential anticancer compound play a significant role in the subsequent researches.

Full text of DOI:10.1080/14756366.2019.1591391

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis, and apoptosis-promoting effect
evaluation of novel pyrazole with benzo[d]thiazole
derivatives containing aminoguanidine units
Da Chuan Liu, Mei Jia Gao, Qiang Huo, Tao Ma, Ying Wang & Cheng Zhu Wu
To cite this article: Da Chuan Liu, Mei Jia Gao, Qiang Huo, Tao Ma, Ying Wang & Cheng Zhu
Wu (2019) Design, synthesis, and apoptosis-promoting effect evaluation of novel pyrazole with
benzo[d]thiazole derivatives containing aminoguanidine units, Journal of Enzyme Inhibition and
Medicinal Chemistry, 34:1, 829-837, DOI: 10.1080/14756366.2019.1591391
To link to this article: https://doi.org/10.1080/14756366.2019.1591391
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Published online: 27 Mar 2019.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 01, 829–837
https://doi.org/10.1080/14756366.2019.1591391
RESEARCH PAPER
Design, synthesis, and apoptosis-promoting effect evaluation of novel pyrazole
with benzo[d]thiazole derivatives containing aminoguanidine units
Da Chuan Liua, Mei Jia Gaoa, Qiang Huoa, Tao Maa, Ying Wangb and Cheng Zhu Wua
b
aSchool of Pharmacy, Bengbu Medical College, Bengbu, China; School of Public Foundation, Bengbu Medical College, Bengbu, China
ABSTRACT
ARTICLE HISTORY
Received 18 January 2019
Revised 1 March 2019
Accepted 2 March 2019
New pyrazole with benzo[d]thiazoles containing hydrazinecarboximidamide substituent was synthesised
and evaluated for cytotoxicity and apoptotic activity using the MTT assay, flow cytometry, and Western
blot analysis. Among the compounds studied, (E)-2-((1-(6-((4-fluorobenzyl)oxy)benzo[d]thiazol-2-yl)-3-phe-
nyl-1H- pyrazol-4-yl)methylene) hydrazinecarboximidamide (8l) was potent, with IC50 values of 2.41 mM,
2.23 mM, 3.75 mM and 2.31 mM in vitro anti-proliferative activity testing against triple-negative breast cancer
cell line MDA-MB-231, non-triple-negative breast cancer MCF-7 cells, and human hepatocarcinoma HepG2
cells, and SMMC-7721 cells, respectively. Especially, the activity against MDA-MB-231 was similar to that of
Doxorubicin, which was used as a positive control in this study. Next, the Annexin V/PI flow cytometry
assay was used at different concentrations of compound 8l to demonstrate that compound 81 induced
apoptosis of MDA-MB-231 cells in a concentration-dependent manner. Finally, these results were further
verified by Western blot analysis. Taken together, the results of this study revealed that compound 8l may
be a potential anticancer compound play a significant role in the subsequent researches.
KEYWORDS
Synthesis; benzothiazole;
pyrazoles; aminoguanidine;
anticancer; apoptosis
1. Introduction
inhibition of tumour cells (such as HeLa and Hep3B)10, however,
their activity was not high enough (8.7 mg/mL and 15.3 mg/mL
respectively). Pyrazoles occupy a distinct niche in heterocyclic
chemistry and represent a key motif in medicinal chemistry
because of their capability to exhibit an array of bioactivities,
Despite the continued improvement in cancer therapy, cancer has
remained a major public health problem worldwide, and an
increasing number of patients are being diagnosed with cancer
annually. According to the World Health Organisation (WHO), an
estimated 9.0 million deaths occurred due to cancer in 2018,
accounting for 22% of deaths of all non-communicable diseases
(NCDs), thereby indicating that cancer accounts for nearly one in
every four deaths in the world. Moreover, this percentage is likely
to increase by 50% in the year 2020, which includes 15 million
casualties, and ultimately reaches as many as 22.2 million cases by
20301,2. The lungs, bronchus, breast, prostate, and colon continue
to be the most common causes of cancer and life ends in death3.
Populations in developing countries affected by numbers of differ-
ent types of cancer in Africa, Asia, Central and South America
account for more than 60% of the world’s total cases of cancer,
including antimicrobial11,12 anticancer13,14
,
anti-inflammatory15,
,
antidepressant16, anticonvulsant17 and selective enzyme inhibi-
,
tory18 activities. Furthermore, the benzothiazole nucleus is a
unique scaffold that is promising for further molecular exploration,
and for the synthesis of novel compounds. Literature surveys
revealed that benzothiazole analogues are associated with diverse
pharmacological effects19–21 (Figure 1(c)). Especially, the anticancer
activity of benzothiazole with pyrazole has received increased
attention22 (Figure 1(d)). Therefore, to improve the antitumor
activities of this type of compounds, in this study a series of new
compounds were designed and synthesised by reserving the
which results in about 70% of cancer deaths in the world4. The aminoguanidine moiety, and replacing the chalcone moiety by
number of new cancer cases in China reached 4.29 million in 2015
and the 5-year survival of these patients was 36.9%5. Currently,
there is no effective treatment for cancer patients in the clinic,
however, chemotherapy is still the most widely used type of can-
cer treatment. Identifying novel, effective and safe chemothera-
peutic agents for cancer treatment is one of the key challenges
that are of utmost importance.
1-benzo[d]thiazole-3-phenyl-pyrazole (named 2-(3-phenyl-pyrazol-
1-yl)benzo[d]thiazole). In these compounds, we simultaneously
changed the substituents on the benzo[d]thiazole and investigated
the effects of these compounds on anti-proliferative activity. The
structure of target compounds is shown in Figure 1.
2. Experimental
Aminoguanidine derivatives have recently been the focus of
numerous studies because of their diverse range of biological
properties, including their antibacterial6, antifungal7 anti-inflamma-
tory8, and antimicrobial activities9. In addition, in previous studies,
researchers have reported that the development of a series of chal-
2.1. Chemistry
All reagents and solvents were purchased from commercial sour-
ces. The degree of reactions was monitored by thin layer chroma-
cone with aminoguanidine derivatives showed some activity in the tography (TLC) on Merck pre-coated silica GF254 plates and
CONTACT Ying Wang
Zhu Wu wuchengzhu0611@foxmail.com
dcliu1226@outlook.com
School of Public Foundation, Bengbu Medical College, 2600 Donghai Road, Bengbu 233030, China; Cheng
School of Pharmacy, Bengbu Medical College, 2600 Donghai Road, Bengbu 233030, China
ß 2019 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.

830
D. C. LIU ET AL.
Figure 1. Rational design of the target compounds. (a) Structures of the previously reported compound with aminoguanidine. (b) Examples of pyrazole derivatives
with biological activity. (c) Representative examples of benzothiazole derivatives. (d) A representative example of benzothiazole molecule with pyrazole that exhibits
anticancer activity.
visualised using a combination of UV. Melting points were deter- The remaining precipitate was filtered to yield a russet solid
1
(3a–o), which was used without further purification.
mined in open capillary tubes and were uncorrected. H NMR and
13 C NMR spectra were collected at room temperature on a PX400
spectrometer with TMS and solvent signals allotted as internal
standards. Chemical shifts were reported in ppm (d). Mass spectra
were obtained on an Agilent 1260–6221 TOF mass spectrometer
(Agilent Technologies, USA).
2.1.3. General procedure for the synthesis of 6-alkoxy-2-hydrazino-
benzothiazoles (4a–o)
A mixture of compounds 3a–o (20 mmol) and 0.6 ml of 98%
H2SO4 solution (water solutions) in 20 ml of glycol was refluxed
for 0.5 h at 80 ꢀC. Then, 10 ml of hydrazine hydrate was added,
and the mixture was heated at 140 ꢀC for 5 h. After cooling to the
room temperature, the mixture was added to 50 ml of ice-cold
water. The precipitate formed was filtered and washed with water
to obtain a light green needlelike solid compound 4a–o.
2.1.1. Synthesis of 6-hydroxy-2-aminobenzothiazole (2)
A mixture of 6-methoxy-2, 3-dihydrobenzo[d]thiazol-2-amine (1)
(10 g, 55.56 mmol) and 40 ml of hydrobromic acid (48% water
solution) was refluxed at 126 ꢀC for 20 h. The mixture was allowed
to cool to room temperature and neutralised with NaOH solution
to pH 7–8. Then, the precipitate was filtered and washed with
water. The filtrate was stirred with 100 ml hot water for 0.5 h and
the remaining precipitate was filtered to yield a brown solid, com-
pound 2.
2.1.4. General procedure for the synthesis of 6-alkoxy-2-(2-(1-phe-
nylethylidene) hydrazinyl)benzo[d]thiazole (6a–o)
Hydrazone derivatives were prepared by reacting compounds
4a–o (5 mmol) with acetophenone (compound 5, 5 mmol) in the
presence of glacial acetic acid in ethanol. After that, the resulting
reaction mixture was refluxed for 1 h. On cooling, a solid sepa-
rated out which was filtered, dried and crystallized from ethanol
to afford hydrazones, compound 6a–o (about 80% yields).
2.1.2. General procedure for the synthesis of 6-alkoxy-2-amino-
benzothiazoles (3a–o)
A mixture of compound 2 (2 g, 12 mmol), potassium carbonate
(2 g, 14.4 mmol), appropriate alkyl bromide or benzyl chloride
derivatives (1.32 mmol), and a catalytic amount of benzyltriethyl-
amine chloride (TEBA) was heated in 50 ml acetonitrile under
reflux for 24-48 h. After removing the solvent under reduced pres-
sure, 80 ml of hot water was poured into the flask, and the mix-
2.1.5. General procedure for the synthesis of 1–(6-alkoxybenzo[d]-
thiazol-2-yl)-3-phenyl-1H-pyrazole-4-carbaldehyde (7a–o)
To a cold, stirred solution of dimethylformamide (15 ml) and phos-
phorous oxychloride (40 mmol) was added hydrazone (compound
ture was stirred for 0.5 h to eliminate potassium carbonate excess. 6, 10 mmol). The reaction mixture was stirred at 55–60 ꢀC for 5 h,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
831
then cooled to room temperature, poured into ice cold water and (2C), 128.34, 123.10, 118.75, 116.76, 106.58, 68.63, 28.77, 28.14,
neutralised with saturated aqueous sodium bicarbonate solution 22.33, 14.36. HR-ESI-MS: m/z [M þ H]þ calcd for C23H26N7OS:
448.1914; found: 448.1915.
whereupon a solid separated out that was filtered, washed with
excess of cold water, dried and crystallized from acetic acid to
afford aldehydes 7a–o.
(E)-2-((1-(6-(hexyloxy)benzo[d]thiazol-2-yl)-3-phenyl-1H-pyrazol-4-
yl)methylene)hydrazinecarboximidamide (8e). White powder, yield:
1
76%. mp: 254.4-255.8 ꢀC. H NMR (600 MHz, dmso) d 11.97 (s, 1H),
2.1.6. General procedure for the synthesis of 2-((1–(6-alkoxyben-
zo[d]thiazol-2-yl) -3-phenyl-1H-pyrazol-4-yl)methylene)hydrazine-
carboximidamide (8a–8o)
The compounds 7 (5 mmol) was reacted with aminoguanidine
bicarbonate (5 mmol) in 20 ml refluxing ethanol in the presence of
5 drops of concentrated hydrochloric acid at 60–70 ꢀC for 8–12 h.
The solution was evaporated to dryness under reduced pressure,
and the residue was purified by silica gel column chromatography
with dichloromethane: methanol (50:1) to afforded white
solid 8a–8o.
9.40 (s, 1H), 8.22 (s, 1H), 7.99 (s, 2H), 7.77 (d, J ¼ 8.9 Hz, 1H), 7.71 –
7.63 (m, 3H), 7.58 – 7.49 (m, 3H), 7.10 (dd, J ¼ 8.9, 2.5 Hz, 1H), 4.00
(t, J ¼ 6.5 Hz, 2H), 1.81 – 1.63 (m, 2H), 1.44 – 1.36 (m, 2H), 1.34 –
1.21 (m, 4H), 0.85 (dd, J ¼ 9.2, 4.8 Hz, 3H). 13 C NMR (151 MHz,
dmso) d 157.50, 157.17, 155.64, 153.80, 144.74, 138.66, 134.76,
131.13, 129.81, 129.44 (2C), 128.76 (2C), 128.35, 123.10, 118.75,
116.76, 106.59, 68.65, 31.43, 29.04, 25.62, 22.52, 14.35. HR-ESI-MS:
m/z [M þ H]þ calcd for C23H26N7OS: 462.2070; found: 462.2066.
(E)-2-((1-(6-(heptyloxy)benzo[d]thiazol-2-yl)-3-phenyl-1H-pyrazol-4-
The yield, melting point, analytical data and spectral data of
each compound are given below.
yl)methylene)hydrazinecarboximidamide (8f). White powder, yield:
1
70%. mp: 248.8-250.2 ꢀC. H NMR (600 MHz, dmso) d 11.95 (s, 1H),
9.41 (s, 1H), 8.22 (s, 1H), 7.91 (s, 2H), 7.77 (d, J ¼ 8.9 Hz, 1H), 7.68
(dd, J ¼ 4.6, 1.8 Hz, 3H), 7.53 (ddd, J ¼ 8.5, 7.7, 2.2 Hz, 3H), 7.10 (dd,
J ¼ 8.9, 2.5 Hz, 1H), 4.00 (t, J ¼ 6.5 Hz, 2H), 1.82 – 1.65 (m, 2H), 1.39
(dd, J ¼ 15.2, 7.6 Hz, 2H), 1.34 – 1.18 (m, 6H), 0.84 (t, J ¼ 6.9 Hz,
3H). 13 C NMR (151 MHz, dmso) d 157.51, 157.18, 155.63, 153.80,
144.74, 138.68, 134.77, 131.13, 129.81, 129.44 (2C), 128.76 (2C),
128.35, 123.10, 118.75, 116.77, 106.60, 68.64, 31.69, 29.08, 28.87,
25.92, 22.49, 14.38. HR-ESI-MS: m/z [M þ H]þ calcd for C25H30N7OS:
476.2227; found: 476.2211.
(E)-2-((1-(6-methoxybenzo[d]thiazol-2-yl)-3-phenyl-1H-pyrazol-4-
yl)methylene)hydrazinecarboximidamide (8a). White powder, yield:
1
68%. mp: 283.5-285.5 ꢀC. H NMR (600 MHz, dmso) d 11.97 (s, 1H),
9.40 (s, 1H), 8.22 (s, 1H), 7.92 (s, 2H), 7.79 (d, J ¼ 8.9 Hz, 1H), 7.69
(dd, J ¼ 13.7, 4.6 Hz, 3H), 7.59 – 7.47 (m, 3H), 7.12 (dd, J ¼ 8.9,
2.5 Hz, 1H), 3.82 (s, 3H, -OCH3). 13 C NMR (151 MHz, dmso) d
157.78, 157.56, 155.66, 153.83, 144.85, 138.68, 134.79, 131.13,
129.81, 129.43 (2C), 128.77 (2C), 128.37, 123.13, 118.76, 116.43,
106.03, 56.23. HR-ESI-MS: m/z [M þ H]þ calcd for C19H18N7OS:
392.1288; found: 392.1293.
(E)-2-((1-(6-(octyloxy)benzo[d]thiazol-2-yl)-3-phenyl-1H-pyrazol-4-
yl)methylene)hydrazinecarboximidamide (8g). White powder, yield:
1
50%. mp: 245.0-246.4 ꢀC. H NMR (600 MHz, dmso) d 12.02 (s, 1H),
(E)-2-((3-phenyl-1-(6-propoxybenzo[d]thiazol-2-yl)-1H-pyrazol-4-
yl)methylene)hydrazinecarboximidamide (8b). White powder, yield:
9.40 (s, 1H), 8.22 (s, 1H), 7.92 (s, 2H), 7.77 (d, J ¼ 8.9 Hz, 1H), 7.70 –
7.64 (m, 3H), 7.59 – 7.48 (m, 3H), 7.10 (dd, J ¼ 8.9, 2.6 Hz, 1H), 4.00
(t, J ¼ 6.5 Hz, 2H), 1.75 – 1.67 (m, 2H), 1.38 (dd, J ¼ 15.2, 7.5 Hz,
2H), 1.33 – 1.16 (m, 8H), 0.83 (dd, J ¼ 8.7, 5.2 Hz, 3H). 13 C NMR
(151 MHz, dmso) d 157.50, 157.17, 155.68, 153.79, 144.74, 138.62,
134.76, 131.13, 129.80, 129.44 (2C), 128.76 (2C), 128.35, 123.10,
118.76, 116.77, 106.59, 68.64, 31.68, 29.27 – 28.97 (m), 25.95,
22.52, 14.38. HR-ESI-MS: m/z [M þ H]þ calcd for C26H32N7OS:
490.2384; found: 490.2377.
1
64%. mp: 280.3-282.3 ꢀC. H NMR (600 MHz, dmso) d 12.02 (s, 1H),
9.40 (s, 1H), 8.22 (s, 1H), 7.95 (s, 2H), 7.77 (d, J ¼ 8.9 Hz, 1H), 7.71 –
7.62 (m, 3H), 7.58 – 7.46 (m, 3H), 7.10 (dd, J ¼ 8.9, 2.4 Hz, 1H), 3.97
(t, J ¼ 6.5 Hz, 2H), 1.86 – 1.63 (m, 2H), 0.97 (t, J ¼ 7.4 Hz, 3H). 13 C
NMR (151 MHz, dmso) d 157.51, 157.15, 155.67, 153.78, 144.74,
138.62, 134.76, 131.13, 129.80, 129.44 (2C), 128.76 (2C), 128.34,
123.11, 118.75, 116.76, 106.61, 70.12, 22.44, 10.81. HR-ESI-MS: m/z
[M þ H]þ calcd for C21H22N7OS: 420.1601; found: 420.1606.
(E)-2-((1-(6-butoxybenzo[d]thiazol-2-yl)-3-phenyl-1H-pyrazol-4-
(E)-2-((1-(6-(nonyloxy)benzo[d]thiazol-2-yl)-3-phenyl-1H-pyrazol-4-
yl)methylene)hydrazinecarboximidamide (8c). White powder, yield:
yl)methylene)hydrazinecarboximidamide (8h). White powder, yield:
1
60%. mp: 271.3-272.9 ꢀC. H NMR (600 MHz, dmso) d 11.93 (s, 1H),
1
58%. mp: 242.7-244.2 ꢀC. H NMR (600 MHz, dmso) d 11.96 (s, 1H),
9.40 (s, 1H), 8.22 (s, 1H), 7.91 (s, 2H), 7.78 (d, J ¼ 8.9 Hz, 1H), 7.70 –
7.65 (m, 3H), 7.57 – 7.48 (m, 3H), 7.11 (dd, J ¼ 8.9, 2.5 Hz, 1H), 4.02
(t, J ¼ 6.5 Hz, 2H), 1.77 – 1.65 (m, 2H), 1.43 (dd, J ¼ 14.9, 7.5 Hz,
2H), 0.92 (t, J ¼ 7.4 Hz, 3H). 13 C NMR (151 MHz, dmso) d 157.51,
157.19, 155.63, 153.81, 144.75, 138.72, 134.77, 131.13, 129.81,
129.44 (2C), 128.76 (2C), 128.36, 123.11, 118.74, 116.79, 106.63,
68.37, 31.13, 19.17, 14.11. HR-ESI-MS: m/z [M þ H]þ calcd for
C22H24N7OS: 434.1758; found: 434.1761.
9.40 (s, 1H), 8.22 (s, 1H), 7.90 (s, 2H), 7.77 (d, J ¼ 8.9 Hz, 1H), 7.67
(dd, J ¼ 7.9, 1.5 Hz, 3H), 7.57 – 7.45 (m, 3H), 7.09 (dd, J ¼ 8.9,
2.5 Hz, 1H), 3.99 (t, J ¼ 6.5 Hz, 2H), 1.76 – 1.64 (m, 2H), 1.38 (dd,
J ¼ 15.1, 7.6 Hz, 2H), 1.33 – 1.27 (m, 2H), 1.27 – 1.08 (m, 8H), 0.82
(t, J ¼ 6.9 Hz, 3H). 13 C NMR (151 MHz, dmso) d 157.50, 157.18,
155.66, 153.80, 144.75, 138.67, 134.77, 131.14, 129.80, 129.43 (2C),
128.75 (2C), 128.35, 123.10, 118.75, 116.77, 106.60, 68.65, 31.71,
29.41, 29.28 – 29.00 (m), 25.94, 22.52, 14.37. HR-ESI-MS: m/z
[M þ H]þ calcd for C23H26N7OS: 504.2540; found: 504.2536.
(E)-2-((1-(6-(pentyloxy)benzo[d]thiazol-2-yl)-3-phenyl-1H-pyrazol-4-
(E)-2-((1-(6-(benzyloxy)benzo[d]thiazol-2-yl)-3-phenyl-1H-pyrazol-4-
yl)methylene)hydrazinecarboximidamide (8d). White powder, yield:
1
65%. mp: 264.6-266.2 ꢀC. H NMR (600 MHz, dmso) d 11.98 (s, 1H),
yl)methylene)hydrazinecarboximidamide (8i). White powder, yield:
1
76%. mp: 248.5-249.8 ꢀC. H NMR (600 MHz, dmso) d 11.99 (s, 1H),
9.40 (s, 1H), 8.22 (s, 1H), 7.95 (s, 2H), 7.77 (d, J ¼ 8.9 Hz, 1H), 7.70 –
7.63 (m, 3H), 7.58 – 7.48 (m, 3H), 7.10 (dd, J ¼ 8.9, 2.5 Hz, 1H), 4.00
9.41 (s, 1H), 8.22 (s, 1H), 7.93 (s, 2H), 7.81 (d, J ¼ 9.0 Hz, 2H), 7.70 –
(t, J ¼ 6.5 Hz, 2H), 1.77 – 1.68 (m, 2H), 1.44 – 1.28 (m, 4H), 0.88 (t, 7.65 (m, 2H), 7.53 (dt, J ¼ 7.1, 4.9 Hz, 3H), 7.46 (d, J ¼ 7.6 Hz, 2H),
J ¼ 7.2 Hz, 3H). 13 C NMR (151 MHz, dmso) d 157.50, 157.17, 155.65, 7.39 (t, J ¼ 7.5 Hz, 2H), 7.33 (t, J ¼ 7.3 Hz, 1H), 7.20 (dd, J ¼ 8.9,
153.79, 144.74, 138.64, 134.76, 131.13, 129.80, 129.44 (2C), 128.76 2.5 Hz, 1H), 5.16 (s, 2H). 13 C NMR (151 MHz, dmso) d 157.71,

832
D. C. LIU ET AL.
156.82, 155.67, 153.85, 145.03, 138.67, 137.13, 134.73, 131.12, 138.67, 134.73, 133.59, 131.10, 130.85, 129.82, 129.44 (2C), 128.76
129.82, 129.44 (2C), 128.91 (2C), 128.77 (2C), 128.41, 128.40, 128.25
(2C), 123.18, 118.80, 116.94, 107.27, 70.40. HR-ESI-MS: m/z
[M þ H]þ calcd for C25H22N7OS: 468.1601; found: 468.1603.
(2C), 128.40, 128.32, 127.83, 126.70, 123.22, 118.81, 116.90, 107.36,
69.44. HR-ESI-MS: m/z [M þ H]þ calcd for C25H21ClN7OS: 502.1211;
found: 502.1216.
(E)-2-((1-(6-((2-fluorobenzyl)oxy)benzo[d]thiazol-2-yl)-3-phenyl-1H-
pyrazol-4-yl)methylene)hydrazinecarboximidamide (8j). White pow-
der, yield: 55%. mp: 263.6-264.2 ꢀC. 1H NMR (600 MHz, dmso) d
12.07 (s, 1H), 9.42 (s, 1H), 8.23 (s, 1H), 7.95 (s, 2H), 7.85 (d,
J ¼ 2.6 Hz, 1H), 7.82 (d, J ¼ 8.9 Hz, 1H), 7.68 (dd, J ¼ 8.0, 1.4 Hz, 2H),
7.59 (dd, J ¼ 12.0, 4.5 Hz, 1H), 7.56 – 7.49 (m, 3H), 7.45 – 7.38 (m,
1H), 7.28 – 7.20 (m, 3H), 5.21 (s, 2H). 13 C NMR (151 MHz, dmso) d
161.68, 160.05, 157.86, 156.64, 155.73, 153.87, 145.20, 138.60,
134.75, 131.20 – 131.10 (m), 130.96 (d), 129.82, 129.44 (2C), 128.77
(2C), 128.42, 125.00 (d), 123.96 (d), 123.22, 118.83, 116.90, 115.93,
115.80, 107.31, 64.78. HR-ESI-MS: m/z [M þ H]þ calcd for
C25H21FN7OS: 486.1507; found: 486.1503.
(E)-2-((1-(6-((2,4-dichlorobenzyl)oxy)benzo[d]thiazol-2-yl)-3-phenyl-
1H-pyrazol-4-yl)methylene)hydrazinecarboximidamide (8o). White
powder, yield: 67%. mp: 262.4-263.6 ꢀC. 1H NMR (600 MHz, dmso)
d 11.86 (s, 1H), 9.43 (s, 1H), 8.22 (s, 1H), 7.92 (s, 2H), 7.84 (d,
J ¼ 11.3 Hz, 2H), 7.68 (d, J ¼ 6.9 Hz, 3H), 7.64 (d, J ¼ 8.2 Hz, 1H), 7.60
– 7.50 (m, 3H), 7.48 (d, J ¼ 7.2 Hz, 1H), 7.23 (dd, J ¼ 8.8, 1.9 Hz, 1H),
5.21 (s, 2H). 13 C NMR (151 MHz, dmso) d 157.99, 156.45, 155.57,
153.92, 145.33, 138.76, 134.78, 134.09, 134.08, 133.68, 131.81,
131.10, 129.85, 129.46 (2C), 129.40, 128.78 (2C), 128.44, 128.03,
123.27, 118.83, 116.93, 107.41, 67.49. HR-ESI-MS: m/z [M þ H]þ
calcd for C25H20FN7OS: 536.0822; found: 536.0823.
2.2. Biological evaluation
(E)-2-((1-(6-((3-fluorobenzyl)oxy)benzo[d]thiazol-2-yl)-3-phenyl-1H-
pyrazol-4-yl)methylene)hydrazinecarboximidamide (8k). White pow-
der, yield: 79%. mp: 259.4-260.5 ꢀC. 1H NMR (600 MHz, dmso) d
11.95 (s, 1H), 9.41 (s, 1H), 8.22 (s, 1H), 7.94 (s, 2H), 7.81 (dd,
J ¼ 10.0, 5.7 Hz, 2H), 7.68 (dd, J ¼ 8.0, 1.3 Hz, 2H), 7.57 – 7.50 (m,
3H), 7.47 – 7.41 (m, 1H), 7.35 – 7.26 (m, 2H), 7.22 (dd, J ¼ 8.9,
2.6 Hz, 1H), 7.18 – 7.12 (m, 1H), 5.20 (s, 2H). 13 C NMR (151 MHz,
dmso) d 163.45, 161.84, 157.82, 156.58, 155.64, 153.88, 145.15,
140.09 (d), 138.69, 134.73, 131.18-130.83 (t), 129.83, 129.44 (2C),
128.76 (2C), 128.41, 124.05 (d), 123.22, 118.81, 116.93, 115.15 (d),
114.77 (d), 107.37, 69.52. HR-ESI-MS: m/z [M þ H]þ calcd for
C25H21FN7OS: 486.1507; found: 486.1502.
2.2.1. Cell culture, growth conditions, and treatments
SMMC7721 (human liver carcinoma cells), HepG2 (human liver
hepatocellular carcinoma cells) and SW480 (human colorectal car-
cinoma cells) were cultured in Dulbecco’s modified Eagle’s
medium (DMEM), which was obtained from Gibco (Grand Island,
NY, USA). MDA-MB-231 cells (human breast cancer cells) were cul-
tured in minimum essential medium (MEM, also obtained from
Gibco) supplemented with 10% foetal bovine serum (FBS,
Millipore, USA) and maintained at 37 ꢀC in a humidified incubator
with 5% CO2. All four types of cancer cells were obtained from
Shanghai Cell Bank, Chinese Academy of Sciences (CAS). To estab-
lish a hypoxic condition, the culture medium was supplemented
with 150 mmol/l CoCl2 for 24 h. The compounds were dissolved in
dimethyl sulfoxide (DMSO, BIOSHARP, Hefei, China). The PX-478
(Selleck Chemicals, Houston, USA), a selective HIF-1a inhibitor was
dissolved in ddH2O. The control group was treated with DMSO
only under identical conditions.
(E)-2-((1-(6-((4-fluorobenzyl)oxy)benzo[d]thiazol-2-yl)-3-phenyl-1H-
pyrazol-4-yl)methylene)hydrazinecarboximidamide (8l). White pow-
der, yield: 64%. mp: 253.5-254.7 ꢀC. 1H NMR (600 MHz, dmso) d
11.94 (s, 1H), 9.41 (s, 1H), 8.22 (s, 1H), 7.94 (s, 2H), 7.81 (t,
J ¼ 5.7 Hz, 2H), 7.68 (dd, J ¼ 8.0, 1.3 Hz, 2H), 7.61 – 7.45 (m, 5H),
7.29 – 7.12 (m, 3H), 5.15 (s, 2H). 13 C NMR (151 MHz, dmso) d
163.09, 161.48, 157.75, 156.71, 155.64, 153.87, 145.07, 138.70,
134.73, 133.37 (d), 131.12, 130.53 (d), 129.83, 129.44 (2C), 128.77
(2C), 128.41, 123.19, 118.81, 116.96, 115.81, 115.66, 107.30, 69.68.
HR-ESI-MS: m/z [M þ H]þ calcd for C25H21FN7OS: 486.1507;
found: 486.1507.
2.2.2. In vitro anti-proliferative activity
The 4 kinds of tested cells were seeded in 96-well microtiter
plates at a density of 5 ꢁ 103 cells per well and treated with differ-
ent concentrations of the compounds and adriamycin for 48 h. At
the end of the incubation period, 15 mL of MTT (purchased from
Sigma USA), at a dose of 5 mg/mL in phosphate-buffered saline
(PBS), was added to each well and incubated at 37 ꢀC for 4 h.
After 4 h, the MTT solution was replaced with 150 mL of DMSO to
dissolve the formazan crystals. The plates were further incubated
for 30 min, and the cell viability was determined by measuring
absorbance at a test wavelength of 490 nm with the microplate
reader (Synergy HT, BioTek, Vermont, USA).
(E)-2-((1-(6-((2-chlorobenzyl)oxy)benzo[d]thiazol-2-yl)-3-phenyl-1H-pyr-
azol-4-yl)methylene)hydrazinecarboximidamide (8m). White powder,
yield: 79%. mp: 246.4-247.5 ꢀC. 1H NMR (600 MHz, dmso) d 11.97
(s, 1H), 9.45 (s, 1H), 8.24 (s, 1H), 7.93 (s, 2H), 7.82 (d, J ¼ 5.7 Hz,
2H), 7.69 (dd, J ¼ 7.8, 1.4 Hz, 2H), 7.67 – 7.52 (m, 5H), 7.47 – 7.40
(m, 1H), 7.39 – 7.12 (m, 3H), 5.19 (s, 2H). 13 C NMR (600 MHz,
dmso) d 157.81, 156.34, 155.54, 153.87, 145.26, 139.73, 138.67,
134.73, 133.65, 131.11, 129.82, 129.81, 129.44 (2C), 129.42, 128.77
(2C), 128.71, 128.64, 128.30, 123.27, 118.79, 116.95, 107.21, 68.97.
HR-ESI-MS: m/z [M þ H]þ calcd for C25H21ClN7OS: 502.1211;
found: 502.1215.
2.2.3. Analysis of the apoptosis by flow cytometry
MDA-MB-231 cells were plated in 6-well plates (5.0 ꢁ 105 cells per
well) and incubated at 37 ꢀC for 24 h. Exponentially growing cells
were then incubated with compound 8l at different concentra-
tions (0, 1, 2, 4 and 8 mM). Following 24 h of incubation, untreated
cells (control) and cells treated with compound 8l were centri-
fuged at 1200 rpm for 10 min, then collected and washed twice
with PBS, once with 500 mL Binding Buffer, and stained with 5 mL
annexin V-FITC and 5 mL PI at room temperature in the dark.
(E)-2-((1-(6-((3-chlorobenzyl)oxy)benzo[d]thiazol-2-yl)-3-phenyl-1H-pyr-
azol-4-yl)methylene)hydrazinecarboximidamide (8n). White powder,
1
yield: 78%. mp: 248.0.5-248.8 ꢀC. H NMR (600 MHz, dmso) d 11.95
(s, 1H), 9.41 (s, 1H), 8.22 (s, 1H), 7.93 (s, 2H), 7.80 (dd, J ¼ 11.2,
5.7 Hz, 2H), 7.67 (d, J ¼ 6.7 Hz, 2H), 7.57 – 7.50 (m, 3H), 7.47 – 7.32
(m, 3H), 7.21 (dd, J ¼ 8.9, 2.5 Hz, 1H), 5.18 (s, 2H). 13 C NMR
(151 MHz, dmso) d 157.82, 156.54, 155.64, 153.87, 145.16, 139.73, Apoptotic cells were quantified using
a FACSCalibur flow

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
833
cytometer with the Cell Quest software (Becton-Dickinson, characterised using 1H NMR, 13 C NMR, and HR-ESI-MS.
A
Franklin Lakes, NJ).
detailed overview of physical and analytical data is provided in
the experimental procedures section.
2.2.4. Western blotting assay
3.2. Biological evaluation
MDA-MB-231 cells were collected after being treated with 8l at
different concentrations (1.2 mM, half of the IC50; 2.4 mM, the IC50;
4.8 mM, double of the IC50) for 48 h. Total cell lysates were pre-
pared in RIPA buffer supplemented with protease inhibitors. The
proteins were fractionated with 10% sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS PAGE) and electroblotted
onto nitrocellulose membrane (Bio-Rad). The membranes were
probed with primary antibodies and then probed with relative
secondary antibody. b-Actin was used as a loading control.
3.2.1. In vitro cytotoxicity against human cancer cell lines
All synthesised compounds 8a–o were evaluated for their anti-
cancer activities against four human cancer cell lines, which
included triple-negative breast cancer cell line MDA-MB-231,
non-triple-negative breast cancer cells MCF-7, and two types of
human hepatocarcinoma cell lines, HepG2 and SMMC-7721. The
clinically used antineoplastic drug Doxorubicin was used as the
reference drug. Figure 2 shows that in the preliminary screening
test, synthesised compounds showed moderate to significant
inhibitory activity against all four cell lines at a dose of 10 mM.
Among them, compounds 8a–8i, 8k, 8 l, and 8n had better
inhibitory activities against one or more type of cancer cells.
Next, compounds with a higher inhibition ratio were chosen to
be screened at lower concentrations based on the data pre-
sented in Figure 3. By analogy, we decided to pick some com-
pounds with better anticancer activities. With respect to the
four human cancer cell lines, the IC50 for the chosen com-
pounds was calculated, and the results are summarised in Table
1. The values presented in Table 1 represent the concentration
at which a 50% decrease in cell growth was observed after
48 h of incubation in the presence of the drug, and compared
with control cells that were treated with DMSO. Moreover,
among the compounds tested, compounds 8a and 8l displayed
better activity then others against all four human cancer cell
lines. However, most other compounds, which shared very simi-
lar structures, did not show any significant anticancer activity at
3. Results and discussion
3.1. Chemistry
The synthetic procedure adopted to obtain the target com-
pounds is shown in Scheme 1. The starting material, 6-
methoxy-1, 3-benzothiazol-2-amine (compound 1), reacted with
hydrobromic acid (48% water solutions) to obtain compound
223. Compound 2 reacted further with appropriate alkyl bro-
mides and substituted phenol in acetone to obtain derivative
324. Subsequently, compound 3 was treated with hydrazine
hydrate in the presence of sulphuric acid (98% water solutions)
to create hydrazines derivatives 425. Then hydrazone derivatives
(compound 6) were prepared by reaction of acetophenone 5
with hydrazines derivatives 4 in the presence of glacial acetic
acid in ethanol. Next, compound
6
reacted under
Vilsmeier–Haack (DMF–POCl3) conditions and afforded corre-
sponding pyrazole-4-carbaldehyde derivatives 726. Compounds 7
then reacted with aminoguanidine bicarbonate in the presence low concentrations, thereby suggesting that their anticancer
of catalytic amounts of hydrochloric acid in ethanol to provide activities were sensitive to the structural perturbations. Among
the compounds in series 8a–o13. The chemical structures were them, compound 8a showed better activity against human
HO
O
S
S
O
(a)
(b)
S
R
NH2
NH2
NH2
N
N
N
1
2
3
O
O
NH2
NH
S
O
R
(c)
N
NH
S
(d)
R
+
N
N
6
5
4
O
(e)
N
S
O
(f)
N
NH
NH2
S
R
R
N
N
N
O
N
N
N
H
7
8a-8n
R=
8e: -C6H13
8j: -C6H4 (o-F)
8o: -C6H3(2,4-2Cl)
8a: -CH3
8f: -C7H15
8k: -C6H4 (m-F)
8b: -C3H7
8g: -C8H17
8l: -C6H4 (p-F)
8c: -C4H9
8d: -C5H11
8i: -C6H5
8h: -C9H19
8m: -C6H4 (o-Cl) 8n:-C6H4 (m-Cl)
Scheme 1. Synthesis of compounds 8a–8o. Reagent and conditions: (a) HBr (48% in H2O), reflux, 18h; (b) RBr/RPhCH2Cl, CH3COCH3, K2CO3, reflux, 20 h; (c) 98%H2SO4,
(CH2OH)2, 80 ꢀC, 0.5h, NH2NH2H2O, 140ꢀC, 5 h; (d) EtOH, AcONa, reflux, 1 h; (e) POCl3, DMF, 55–60 ꢀC, 5 h; (f) aminoguanidine bicarbonate, EtOH, 60–70 ꢀC, 8–12 h.

834
D. C. LIU ET AL.
Figure 2. The comparison of the inhibitory activity of synthesised compounds against four cell lines at 10 lM in the preliminary screening test. The ordinate represents
inhibition ratio (%), the abscissa is the synthesised compounds.
Figure 3. The inhibitory activity of the compounds with higher inhibition ratio against four cell lines at 5 lM in the screening test. The ordinate represents inhibition
ratio (%) and the abscissa represents the chosen compounds and the reference drug Doxorubicin.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
835
Table 1. In vitro anticancer activity of the chosen compounds against four can-
cer cell linesa (IC50 lMb).
hepatocarcinoma cell lines, HepG2 and SMMC-7721, with IC50
values of 3.34 mM and 2.03 mM, respectively. In addition, com-
pound 8l also exhibited anticancer activity with IC50 values
against HepG2 and SMMC-7721of 3.75 mM and 2.31 mM, respect-
ively. Notably, compound 8l had a higher activity against MDA-
MB-231 and MCF-7 cells with IC50 values of 2.41 mM and
2.23 mM, respectively. Especially, when triple-negative breast can-
cer cell MDA-MB-231 was evaluated, compound 8l displayed a
remarkable activity, which was as good as the clinically used
Compound
MDA-MB-231
MCF-7
HepG-2
SMMC-7721
8a
8b
8i
3.07 0.30
3.82 0.37
3.71 0.59
3.91 0.77
2.41 0.21
2.24 0.08
3.25 0.45
3.49 0.32
3.62 0.30
3.61 0.55
2.23 0.37
0.34 0.10
3.45 0.28c
4.34 0.34
4.60 0.51
5.09 0.33
3.75 0.19
2.96 0.26
2.03 0.36
3.62 0.49
3.74 0.39
3.14 0.20
2.31 0.34
0.79 0.07
8k
8l
DOXd
Negative control 0.1%DMSO, no activity.
aCytotoxicity as IC50 for each cell line, refers to the concentration of compound antineoplastic drug Doxorubicin. However, compound 8l also
which reduced by 50% the optical density of treated cells with respect to
had toxicity towards normal cells (MCF-10A at 10 mM), thus,
how to reduce the toxicity of compound 8l was an unsolved
problem as well as the future study direction.
untreated cells using the MTT assay.
bThe data represented the mean of three experiments in triplicate.
cBold values signify that the bioactivity of the compound is outstanding.
dUsed as a positive control.
Figure 4. Flow cytometry analyses of apoptosis induction in triple-negative breast cancer cell MDA-MB-231 after treated by different concentrations of compound 8l
(1, 2, 4 and 8 lM) and no treatment (control) as a reference control for 48 h.

836
D. C. LIU ET AL.
that compound 8l induced apoptosis in MDA-MB-231 cells in a
concentration-dependent manner.
3.2.4. Western blot analysis
To verify whether the compound 8l-induced cell death observed
in MDA-MB-231 cells was a result of apoptosis, Western blot analy-
ses were conducted to determine the level of apoptosis proteins.
Apoptosis was induced in MDA-MB-231 breast cancer cells by
compound 8l as confirmed by the downregulation of Bcl-2, and
up-regulation of Bax protein levels (Figure 5), thereby indicating
that compound 8l-induced cell death was related to apoptosis.
Figure 5. Determine the translation of proteins by western blot. MDA-MB-231
cells were treated with compound 8l of 1.2 lM (half IC50), 2.4 lM (IC50), 4.8 lM
(double IC50) and no treatment for 48 h, respectively. b-actin was used for
equal loading.
Conclusion
In this study, a novel series of 2-((1-(6-alkoxybenzo[d]thiazol-2-yl)-
3-phenyl-1H-pyrazol-4-yl)methylene)
hydrazinecarboximidamide
3.2.2. Structure activity relationship
derivatives were synthesised and evaluated for their antitumor
activity. The results showed that all compounds showed different
degrees of actives in the antiproliferative assays against several
cancer cell lines. Compound 8l exhibited potent activity against
triple-negative MDA-MB-231 breast cancer cells, non-triple-nega-
tive MCF-7 breast cancer cells, and two types of human hepato-
carcinoma cell lines, HepG2 and SMMC-7721, with IC50 values of
2.41 mM, 2.23 mM, 3.75 mM, and 2.31 mM. Especially, 8l displayed a
remarkable activity as good as the clinically used antineoplastic
drug Doxorubicin against triple-negative MDA-MB-231 breast can-
cer cell. The more detailed mechanistic study demonstrated that
compound 8l inhibited the proliferation of MDA-MB-231 cancer
cells by inducing apoptosis by downregulating Bcl-2 and upregu-
lating Bax protein levels. Thus, our data showed that compound
8l may represent a potential anticancer lead compound, which
will play an important role in our follow-up study.
Based on the activity profile of the various compounds (Figures 2, 3,
and Table 1), a structure activity relationship (SAR) was developed.
For the eight alkyl chain-substituted derivatives 8a–8h, the length
of the alkyl chain appeared to have a direct impact on the anticon-
vulsant activity of the derivatives. By increasing the alkyl chain
length, the anticancer activity decreased, and ultimately disappeared
at a low concentration, and compound 8a, which bears a methoxyl
group substitution, was the most active compound. This SAR might
be associated with lipid-water partition coefficients of the com-
pounds, which affects drug hydrophobic, drug-receptor interactions,
metabolism of molecules, and especially the ability to pass through
the cell membrane, by which its activity is determined.
Compound 8i was substituted with a benzyloxy group at the 6-
position of the triazolobenzothiazole core, then, F and Cl electron-
withdrawal groups were subsequently added to the benzyloxy
group at different positions, thereby yielding compounds 8j–8o. The
activities of these derivatives with different halogen substitutions on
the benzene ring were determined as follows: when comparing the
derivatives with different F-substitution positions on the benzyl ring,
the activity order was p-F > m-F > o-F. The activities of Cl-substituted
derivatives were mostly weaker when compared to that of F-substi-
tution, except for p-Cl, however, it was still less when compared to
p-F. Compound 8o was replaced by two chlorine atoms on the ben-
zene ring, however, the activity decreased instead of increased.
Interestingly, compound 8i, which does not bear any substitution on
the benzene ring, had good anticancer activity, which was even bet-
ter when compared to that of m-F substitution against HepG 2.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was supported by the Natural Science Fund projects of
Bengbu Medical College and Education Department of Anhui
Natural Science Research Project China under Grant.
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