Design, synthesis and anticancer activity of 5-aryl-4-(4-arylpiperazine-1-carbonyl)-1,2,3-thiadiazoles as microtubule-destabilizing agents

Article abstract of DOI:10.1016/j.bioorg.2020.104199

Hereby, we report our efforts on discovery and optimization of a new series of 5-aryl-4-(4-arylpiperazine-1-carbonyl)-1,2,3-thiadiazoles as new microtubule-destabilizing agents along our previous study. Guided by docking model analysis, we introduced the 1,2,3-thiadiazole moiety containing the hydrogen-bond acceptors as B-ring of XRP44X analogues. Extensive structure modifications were performed to investigate the detailed structure and activity relationships (SARs). Some compounds exhibited potent antiproliferative activities against three human cancer cell lines (SGC-7901, A549 and HeLa). The compound 5m exhibited the highest potency against the three cancer cell lines. The tubulin polymerization experiments indicated that compound 5m effectively inhibited the tubulin polymerization, and immunostaining assay revealed that it significantly disrupted microtubule dynamics. Moreover, cell cycle studies revealed that compound 5m dramatically arrested cell cycle progression at G2/M phase.

Full text of DOI:10.1016/j.bioorg.2020.104199

Journal Pre-proofs
Design, synthesis and anticancer activity of 5-aryl-4-(4-arylpiperazine-1-car-
bonyl)-1,2,3-thiadiazoles as microtubule-destabilizing agents
Chao Wang, Zeyu Wang, Minghuan Gao, Yuelin Li, Yujing Zhang, Kai Bao,
Yingliang Wu, Qi Guan, Daiying Zuo, Weige Zhang
PII:
S0045-2068(20)31496-6
DOI:
https://doi.org/10.1016/j.bioorg.2020.104199
YBIOO 104199
Reference:
To appear in:
Bioorganic Chemistry
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Accepted Date:
13 May 2020
14 June 2020
30 July 2020
Please cite this article as: C. Wang, Z. Wang, M. Gao, Y. Li, Y. Zhang, K. Bao, Y. Wu, Q. Guan, D. Zuo, W.
Zhang, Design, synthesis and anticancer activity of 5-aryl-4-(4-arylpiperazine-1-carbonyl)-1,2,3-thiadiazoles as
microtubule-destabilizing agents, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2020.104199
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Design, synthesis and anticancer activity of 5-aryl-4-(4-arylpiperazine-
1-carbonyl)-1,2,3-thiadiazoles as microtubule-destabilizing agents
Chao Wanga, Zeyu Wangb, Minghuan Gaoc, Yuelin Lia, Yujing Zhanga, Kai Baob, Yingliang
Wuc, Qi Guana*, Daiying Zuoc*, Weige Zhanga*
aKey Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education,
Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016,
China.
bWuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe
District, Shenyang, 110016, China.
cDepartment of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road,
Shenhe District, Shenyang 110016, China.
E-mail addresses: guanqi@syphu.edu.cn (Q. Guan), zuodaiying@163.com (D. Zuo),
zhangweige2000@sina.com (W. Zhang).

Abstract
Hereby, we report our efforts on discovery and optimization of a new series of 5-aryl-
4-(4-arylpiperazine-1-carbonyl)-1,2,3-thiadiazoles as new microtubule-destabilizing
agents along our previous study. Guided by docking model analysis, we introduced the
1,2,3-thiadiazole moiety containing the hydrogen-bond acceptors as B-ring of XRP44X
analogues. Extensive structure modifications were performed to investigate the detailed
structure and activity relationships (SARs). Some compounds exhibited potent
antiproliferative activities against three human cancer cell lines (SGC-7901, A549 and
HeLa). The compound 5m exhibited the highest potency against the three cancer cell
lines. The tubulin polymerization experiments indicated that compound 5m effectively
inhibited the tubulin polymerization, and immunostaining assay revealed that it
significantly disrupted microtubule dynamics. Moreover, cell cycle studies revealed
that compound 5m dramatically arrested cell cycle progression at G2/M phase.
Keywords: 1,2,3-thiadiazole, antiproliferative activity, microtubule-destabilizing
agent, molecular docking.

1. Introduction
Microtubules are cylindrical polymers composed of α- and β-tubulin, which are a major
component of the cytoskeleton and mediate essential cellular functions including cell
proliferation, trafficking, signaling and migration in eukaryotic cells. Interfering with
the microtubule dynamic equilibrium, by either inhibiting tubulin polymerization or
blocking microtubules disassembly, prevents proper microtubule function and
ultimately leads to cell death [1-3]. Therefore, microtubules are regarded as an excellent
target for development of clinically useful anticancer drugs [4,5]. Microtubule targeting
agents are generally categorized into microtubule-stabilizing and microtubule-
destabilizing agents [6]. Compounds such as taxol and epothilone prevent microtubule
disassembly and are referred to as microtubule-stabilizing agents. On the contrary,
vinca alkaloid and colchicine (1) interfere with the assembly of tubulin into
microtubules and are classified as microtubule-destabilizing agents. In recent decades,
a large number of outstanding microtubule-destabilizing agents have been reported,
such as combretastatin A-4 (CA-4, 2) and TN16 (3) (Fig. 1) [7,8].
MeO
MeO
O
NHCOCH3
NH
NH
MeO
MeO
MeO
OMe
OH
O
O
OMe
OMe
3
1
TN16,
Colchicine,
2
CA-4,
Figure 1. Chemical structures of some microtubule destabilizers.
XRP44X (4), a novel prominent microtubule-destabilizing agent (Wasylyk et al., 2008),
consists of four rings (denoted as rings A, B, C and D in Figure 2) with a carbonyl
linkage between B-ring and C-ring [9]. The replacement of the B-ring of XRP44X with

a bioisosteric five-membered heterocycle nucleus yields several analogues, which have
similar potency and mechanism of actions compared to XRP44X [10,11]. Based on the
reported molecular modeling of XRP44X or its analogues with tubulin, the binding
domain can be divided into three pockets (P-1, P-2 and P-3 in Figure 2) [10]. The
carbonyl group, as a linkage, provokes an orientation for a hydrogen bond with the
amide nitrogen of Alaβ317, and serves as an anchor to place XRP44X or its analogues
in the proper orientation within the binding site. The A-ring fosters van der Waals
interactions with the hydrophobic P-1 pocket consisting of Lysβ254 and Leuβ248,
while the C- and D-ring extends deeper to strand S6 of the N-terminal nucleotide
binding domain and interacts with the hydrophobic P-3 pocket formed by Cysβ241,
Leuβ242 and Leuβ252. The B-ring in the hydrophobic P-2 pocket enhances the van der
Waals interactions with it. However, we noted that some amino acid residues in P-2
pocket, such as Asnβ258 and Lysβ352, as hydrogen-bond donors might also form
additional interactions with B-ring, which would facilitate the development of new
XRP44X analogues.

Containing hydrogen-bonding
acceptors
S
N
N
N
N
B
B
Structure-based design
R1
A
A
O
N
N
O
C
C
N
N
Cl
R2
D
D
4
5
XRP44X,
5m: R1=2-fluoro; R2=3,5-dimethoxy
5t: R1=3-fluoro; R2=3,5-dimethoxy
5a': R1=4-fluoro; R2=3,5-dimethoxy
5h': R1=2-chloro; R2=3,5-dimethoxy
etc.
P-2 pocket
5m
5h'
P-1 pocket
P-3 pocket
4
Figure 2. Design of the target compounds.
As part of our ongoing effort to develop new tubulin inhibitors, we herein attempt to
replace the pyrazole moiety of XRP44X with a 1,2,3-thiadiazole, one of the significant
structural fragments containing several hydrogen-bond acceptors. Based on the
previous docking model, we conducted docking simulations of the designed target
compounds, and the preliminary results indicated that compounds 5m (R1 = 2-fluoro,
R2 = 3,5-dimethoxy) and 5h’ (R1 = 2-chloro, R2 = 3,5-dimethoxy) might interact with
tubulin in a similar way that XRP44X does. Moreover, the 1S of 1,2,3-thiadiazole of
compounds 5m and 5h’ and the 3N of 1,2,3-thiadiazole of compound 5m might enable
additional hydrogen bonding interactions with Asnβ258 and Lysβ352 in P-2 pocket. To
the best of our knowledge, this new binding mode in P-2 pocket has not been explored
in the design of XRP44X analogues.
2. Results and discussion

2.1 Chemistry
The target compounds 5-aryl-4-(4-arylpiperazine-1-carbonyl)-1,2,3-thiadiazols (5)
were prepared as outlined in Scheme 1. The substituted aromatic aldehydes (6) reacted
with ethyl diazoacetate, DBU and IBX in DMSO to afford the corresponding ethyl 2-
diazo-3-oxo-3-arylpropanoates (7) via a one-pot procedure in 60-90% yields [12].
Subsequently, 7 reacted with Lawesson’s reagent in toluene under reflux conditions to
afford the key intermediates ethyl 5-aryl-1,2,3-thiadiazole-4-carboxylates (8) [13].
Finally, 8 were treated with corresponding arylpiperazines to afford the target
compounds 5 in the presence of trimethylaluminium in 42-70% yields [14,15].
O
O
S N
N
CHO
a
b
OEt
R1
R1
R1
N2
O
OEt
6
7
8
R1
H
S N
6a~8a
6b~8b
6c~8c
6d~8d
6e~8e
6f~8f
B
N
2-fluoro
R1
3-fluoro
4-fluoro
2-chloro
3,4-dichloro
4-chlorol
A
c
O
N
C
N
R2
6g~8g
D
5
R1
R2
3-fluoro
4-methoxy
2,5-dimethoxy
3,5-dimethoxy
H
R1
R2
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
H
H
H
H
3-fluoro
3-fluoro
4-fluoro
4-fluoro
4-fluoro
4-fluoro
4-fluoro
4-fluoro
4-fluoro
2-chloro
2,5-dimethoxy
3,5-dimethoxy
H
3-chloro
3-methyl
3-methoxy
4-methoxy
3,4-dimethyl
3,5-dimethoxy
3-chloro
5u
5v
5w
5x
5y
5z
2-fluoro
2-fluoro 3-fluoro
2-fluoro 3-chloro
2-fluoro 3-methyl
2-fluoro 3-methoxy
2-fluoro 3-trifluoromethyl
2-fluoro 4-methoxy
2-fluoro 2,5-dimethoxy
2-fluoro 3,5-dimethoxy
5a'
5b'
5c' 2-chloro
5d' 2-chloro
3-methyl
3-methoxy
3-trifluoromethyl
4-methoxy
2,5-dimethoxy
3,5-dimethoxy
2-chloro
2-chloro
2-chloro
2-chloro
3,4-dichloro 3,5-dimethoxy
4-chloro
4-chloro
5e'
5f'
5g'
5h'
5i'
3-fluoro
H
3-fluoro 3-chloro
3-fluoro 3-methyl
3-fluoro 3-methoxy
3-fluoro 4-methoxy
4-methoxy
3,5-dimethoxy
5j'
5k'
Scheme 1. Reagents and conditions: (a) N2CHCO2Et, DBU, IBX, DMSO, rt., 10 h; (b) Lawesson's

reagent, toluene, reflux, 4 h; (c) arylpiperazine, AlMe3 (1.0 M solution in heptane), DCM, rt., overnight.
2.2 Biological Evaluation
2.2.1 In Vitro Antiproliferative Activity
In vitro antiproliferative activities against three human cancer cell lines, including
SGC-7901 (human gastric carcinoma cell line), A549 (human lung carcinoma cell line)
and HeLa (Human cervical carcinoma cell line), were determined using a standard MTT
assay with colchicine and CA-4 as the positive controls. As shown in Table 1, all
halogenated compounds showed significant change on the activity compared with
corresponding compounds without halogens. Compared with 5d, 5m and 5h’ showed
greatly improved activity, which indicated electronegativity is crucial on the A-ring.
Moreover, halogens on the A-ring exhibited an order of potency being ortho- > meta- >
para-substituted in generally (5g vs. 5o vs. 5v, 5i vs. 5q vs. 5x, 5m vs. 5t vs. 5a’, etc.).
It was noted that compounds (5u ~ 5z and 5i’ ~ 5k’) with a substituent on para-
substitution of A-ring almost lost antiproliferative activity. Then, we investigated the
effect of substituents on the D-ring and found that the volume and lipophilicity of
substituents, rather than electronegativity, may be the important factors affecting
activity (5f ~ 5j, 5o ~ 5q and 5b’ ~ 5e’). The compounds with OCH3 on meta-
substitution of the D-ring gave higher activity than those on para-substitution (5i vs.
5k, 5q vs. 5r, 5d’ vs. 5f’), suggesting that the substituents’ positions had greater effects
on activity. In addition, di-meta-substituted compounds showed increased activity,
which could be confirmed by compounds 5l, 5m, 5s and 5t. Among the target
compounds, compound 5m (R1 = 2-fluoro, R2 = 3,5-dimethoxy) showed the best
antiproliferative activity against the HeLa cell lines with IC50 value of 0.092 ± 0.005
μM.

Table 1. Antiproliferative activity of all compounds. The experiments were performed three times,
and the results of representative experiments are shown.
(IC50 ± SD, μM)a
Compound
R1
R2
SGC-7901
A549
HeLa
5a
5b
H
3-fluoro
4-methoxy
2,5-dimethoxy
3,5-dimethoxy
H
>30
>30
25.30 ± 2.6
>30
>30
H
>30
>30
5c
H
7.25 ± 0.27
5.62 ± 0.20
>30
8.97 ± 0.19
3.954 ± 0.21
>30
5d
H
4.96 ± 0.29
>30
5e
2-fluoro
2-fluoro
2-fluoro
2-fluoro
2-fluoro
2-fluoro
2-fluoro
2-fluoro
2-fluoro
3-fluoro
3-fluoro
3-fluoro
3-fluoro
3-fluoro
3-fluoro
3-fluoro
4-fluoro
4-fluoro
4-fluoro
4-fluoro
4-fluoro
4-fluoro
4-fluoro
2-chloro
2-chloro
2-chloro
2-chloro
2-chloro
2-chloro
2-chloro
3,4-dichloro
4-chloro
4-chloro
5f
3-fluoro
27.8 ± 1.6
7.09 ± 0.47
8.20 ± 0.61
3.76 ± 0.20
20.1 ± 1.8
>30
23.0 ± 1.4
8.51 ± 0.39
9.17 ± 0.76
4.32 ± 0.14
>30
25.6 ± 1.7
6.76 ± 0.31
10.5 ± 0.64
5.60 ± 0.23
8.65 ± 0.50
>30
5g
3-chloro
5h
3-methyl
5i
3-methoxy
3-trifluoromethyl
4-methoxy
2,5-dimethoxy
3,5-dimethoxy
H
5j
5k
>30
5l
0.92 ± 0.094
0.97 ± 0.088
0.82 ± 0.052
5m
5n
0.103 ± 0.019 0.276 ± 0.022 0.092 ± 0.005
>30
15.1 ± 0.87
17.6 ± 0.90
6.67 ± 0.24
>30
>30
>30
5o
3-chloro
20.5 ± 1.19
14.9 ± 0.67
10.2 ± 0.59
>30
16.6 ± 0.77
12.6 ± 0.68
9.13 ± 0.31
>30
5p
3-methyl
5q
3-methoxy
4-methoxy
2,5-dimethoxy
3,5-dimethoxy
H
5r
5s
3.93 ± 0.24
2.54 ± 0.083
>30
6.67 ± 0.39
3.94 ± 0.11
5t
3.07 ± 0.096 0.812 ± 0.018
5u
>30
>30
>30
>30
5v
3-chloro
>30
5w
5x
3-methyl
>30
>30
>30
3-methoxy
4-methoxy
3,4-dimethyl
3,5-dimethoxy
3-chloro
>30
>30
>30
5y
28.9 ± 2.9
>30
>30
>30
5z
>30
>30
5a’
5b’
5c’
5d’
5e’
5f’
5g’
5h’
5i’
13.7 ± 1.2
4.27 ± 0.60
5.11 ± 0.31
7.41 ± 0.72
18.6 ± 2.4
>30
19.2 ± 1.4
>30
3.48 ± 0.20
3.93 ± 0.13
4.09 ± 0.29
5.72 ± 0.50
28.3 ± 3.2
>30
3-methyl
10.2 ± 0.43
9.21 ± 0.80
>30
3-methoxy
3-trifluoromethyl
4-methoxy
2,5-dimethoxy
3,5-dimethoxy
3,5-dimethoxy
4-methoxy
3,5-dimethoxy
>30
4.23 ± 0.54
0.96 ± 0.10
>30
6.25 ± 0.47
1.02 ± 0.32
>30
3.34 ± 0.18
0.89 ± 0.077
26.7
5j’
5k’
CA-4b
Colchicineb
>30
>30
>30
>30
>30
>30
0.049 ± 0.004 0.080 ± 0.006 0.043 ± 0.002
0.112 ± 0.011 0.124 ± 0.013 0.086 ± 0.008

a IC50: the half maximal inhibitory concentration.
b Used as positive controls.
2.2.2 Effect on Tubulin Polymerization
In order to shed light on the biological mechanism of this series of compounds, the most
potent compound 5m was selected to examine the antitubulin activity, meanwhile CA-4
was used as positive control and paclitaxel, the first compound known to interact with
and stabilize tubulin, was utilized as negative control. As shown in Fig. 3, compound
5m effectively inhibited tubulin polymerization (IC50 = 36.8 μM). The experimental
results showed that compound 5m caused a dose-dependent inhibition of tubulin
polymerization; In contrast, paclitaxel, a microtubule-stabilizing agent, could distinctly
promote this process. Therefore, the results indicated that compound 5m was a tubulin
inhibitor.
Figure 3. Effects of compound 5m on tubulin polymerization. Tubulin had been pre-incubated for 1
min with compound 5m at 7.5 µM, 15 µM, 30 µM and 60 µM, CA-4 at 5 µM, Paclitaxel at 5 µM or

vehicle DMSO at room temperature before GTP was added to start the tubulin polymerization reactions.
The reaction was monitored at 37
℃.
2.2.3 Analysis of Immunofluorescence Staining
To further investigate the impacts of compound 5m on intracellular microtubules,
HeLa cells were utilized for immunofluorescence assay to directly observe the changes
in morphology of microtubules and CA-4 was utilized as reference. As illustrated in
Fig. 4, the control cells displayed well-organized microtubule network throughout the
cells. After treatment with compound 5m or CA-4, (at their respective 2-fold IC50
concentrations, respectively) microtubules became irregular arrangement and
organization, and the microtubule network showed a disruption. The study results
suggested that compound 5m disrupted cytoskeleton similarly to CA-4.

Figure 4. Effects of compound 5m and CA-4, at their respective 2-fold IC50 concentrations, on the
cellular microtubule network and microtubule reassemble by immunofluorescence. HeLa cells were
treated with compound 5m or CA-4 for 24 h, and then direct microscopy detection of the fixed and
stained cell was performed. The cellular microtubules were stained with anti-a-tubulin-FITC specific
antibodies (green). DNA was stained by 4’,6-diamidino-2-phenylindole (DAPI, blue).
2.2.4 Cell Cycle Analysis
To evaluate the effects of compound 5m on the cell mitosis, the effect of compound
5m on the cell cycle of HeLa cells was analyzed by flow cytometry (Fig. 5) (CA-4 was
also comparatively examined as a positive control). Similar as CA-4, compound 5m
arrested the cell cycle in G2/M phase significantly, increasing the percentage of cells
in G2/M phase at 12 h. At the same time, polyploidy was induced increasingly by
compound 5m. The proportion of G2/M declined while the proportion of Sub-G1
increased in 24 h, 36 h, 48 h and 72 h. This indicated that part of the cells underwent
mitotic catastrophe. Compound 5m was demonstrated to clearly cause G2/M phase
arrest in a time-dependent manner.
Figure 5. Effects of CA-4 and compound 5m on cell cycle. HeLa cells lines treated with CA-4 and
compound 5m, at their respective 2-fold IC50 concentrations, for 0, 12, 24, 36, 48 and 72 h.

3. Conclusion
Through the detailed analysis of the binding mode of XRP44X and its analogues, we
introduced 1,2,3-thiadiazole, a hydrogen-bond acceptor, as the B-ring of XRP44X
analogue to design a series of 5-aryl-4-(4-arylpiperazine-1-carbonyl)-1,2,3-thiadiazole
derivatives as novel tubulin inhibitors. This work reveals that the B-ring of XRP44X
analogues can form additional hydrogen-bond interactions besides foster van der Waals
interaction with amino acid residues in P-2 pocket. The most potent compound 5m (R1
= 2-fluoro, R2 = 3,5-dimethoxy) exhibited noteworthy potency against a set of cancer
cell lines (IC50 value, 0.092-0.276 μM) and significant inhibition of tubulin
polymerization. The compound 5m arrested most cells in the G2/M phase of the cell
cycle and disrupted cellular microtubules, thus providing evidence that compound 5m
is a new kind of microtubule-destabilizing agent.
4. Experimental
4.1 Chemistry
4.1.1 Materials and methods
All of reagents and solvents were purchased from chemical company. 1H NMR and 13C
NMR spectra were tested in CDCl3 or DMSO-d6 with TMS as the internal reference on
a Bruker AVANCE 400 or 600 (1H at 400 or 600 MHz, 13C at 150 MHz). Mass spectra
(MS) were measured on an Agilent 1100-sl mass spectrometer with an electrospray
ionisation source from Agilent Co. Ltd. High resolution accurate mass determinations
(HRMS) for all of the final target compounds were obtained on a Bruker Micromass
Time of Flight mass spectrometer equipped with electrospray ionisation (ESI). TLC
analysis was used for determining the extent of reactions under UV light (wavelength:
365 nm and 254 nm). Melting point was measured (uncorrected) on hot-stage
microscope (Beijing Taike, X-4). The microwave reactions were carried out in a single

mode cavity microwave synthesizer (CEM Corporation, NC, USA).
4.1.2 General synthetic procedures for arylpiperazines
A solution of arylamines (1 mmol), bis(2- chloroethyl)amine hydrochloride (1.1 mmol)
and K2CO3 (3 mmol) in n-BuOH were stirred at irradiated in a microwave reactor for
30 min at 150°C. The reaction mixture was cooled to room temperature and dissolved
in methanol (4 mL), followed by the addition of diethyl ether (150 mL). The precipitate
formed was recovered by filtration and washed with diethyl ether to obtain
arylpiperazines as HCl salt. The HCl salt was used for the next reaction without further
purification [16,17].
4.1.3 General synthetic procedures for ethyl 2-diazo-3-oxo-3-arylpropanoates 7
To a solution of ethyl diazoacetate (1.2 mmol) in DMSO at room temperature were
added in succession DBU (0.1 mmol), corresponding aromatic aldehydes (1 mmol),
and a solution of IBX (2 mmol) in DMSO. After being stirred for 10 h at room
temperature, the reaction mixture was quenched with aqueous NaHCO3 and then
extracted with ethyl acetate (3 × 20 mL), the combined organic layers were copiously
washed with aqueous NaHCO3 and finally with water, and the mixture was then dried
with Na2SO4, filtered, and concentrated in vacuo. The resulting crude product purified
by flash column chromatography on silica gel (ethyl acetate/petroleum ether = 1:3)
afforded the pure products [12].
4.1.4 General synthetic procedures for ethyl 5-aryl-1,2,3-thiadiazole-4-carboxylates (8)
A solution of various ethyl 2-diazo-3-oxo-3-arylpropanoates (7) (1 mmol) and
Lawesson’s reagent (1.2 mmol) in toluene were heated at reflux for 4 hours, then cooled
to room temperature and concentrated in vacuo. The resulting oil was purified by

column chromatography (ethyl acetate/petroleum ether = 1:2) to give the pure products.
4.1.4.1 Ethyl 5-phenyl-1,2,3-thiadiazole-4-carboxylate (8a)
Yellow Solid; yield: 56%; 1H NMR (600 MHz, CDCl3): δ =7.51 (5H, m), 4.42 (2H, q,
J = 6.9 Hz), 1.34 (3H, t, J = 7.0 Hz). ESI-MS: m/z = 235.0 [M+H]+.
4.1.4.2 Ethyl 5-(2-fluorophenyl)-1,2,3-thiadiazole-4-carboxylate (8b)
Red Solid; yield: 62%; 1H NMR (600 MHz, CDCl3): δ = 7.53 (1H, m), 7.45 (1H, m),
7.28 (1H, m), 7.23 (1H, t, J = 8.9 Hz), 4.42 (2H, q, J = 6.9 Hz), 1.31 (3H, t, J = 7.1 Hz).
ESI-MS: m/z = 253.1 [M+H]+.
4.1.4.3 Ethyl 5-(3-fluorophenyl)-1,2,3-thiadiazole-4-carboxylate (8c)
1
Light yellow Solid; yield: 50%; H NMR (600 MHz, CDCl3): δ = 7.47 (1H, m), 7.30
(2H, m), 7.23 (1H, m), 4.44 (2H, q, J = 6.9 Hz), 1.36 (3H, t, J = 7.3 Hz). ESI-MS: m/z
= 253.0 [M+H]+.
4.1.4.4 Ethyl 5-(4-fluorophenyl)-1,2,3-thiadiazole-4-carboxylate (8d)
Yellow Solid; yield: 60%; 1H NMR (600 MHz, CDCl3): δ =7.55 (2H, q), 7.19 (2H, t, J
= 9.5 Hz), 4.44 (2H, q, J = 7.2 Hz), 1.37 (3H, t, J = 7.1 Hz). ESI-MS: m/z = 253.1
[M+H]+.
4.1.4.5 Ethyl 5-(2-chlorophenyl)-1,2,3-thiadiazole-4-carboxylate (8e)
Light yellow Solid; yield: 66%; 1H NMR (600 MHz, CDCl3): δ = 7.54 (1H, d, J = 7.9
Hz), 7.47 (1H, m), 7.38 (2H, m), 4.35 (2H, q, J = 7.3 Hz), 1.24 (3H, t, J = 7.0 Hz). ESI-
MS: m/z = 269.0 [M+H]+.
4.1.4.6 Ethyl 5-(3,4-dichlorophenyl)-1,2,3-thiadiazole-4-carboxylate (8f)
Light yellow Solid; yield: 67%; 1H NMR (600 MHz, CDCl3): δ = 7.66 (1H, d, J = 2.1
Hz), 7.57 (1H, d, J = 8.4 Hz), 7.39 (1H, dd, J = 2.1, Hz, J = 8.2 Hz), 4.45 (2H, q, J =
7.4 Hz), 1.38 (3H, t, J = 7.1 Hz). ESI-MS: m/z = 302.9 [M+H]+.
4.1.4.7 Ethyl 5-(4-chlorophenyl)-1,2,3-thiadiazole-4-carboxylate (8g)

Light yellow Solid; yield: 69%; 1H NMR (600 MHz, CDCl3): δ = 7.48 (4H, q, J = 9.3
Hz), 4.44 (2H, q, J = 7.3 Hz), 1.37 (3H, t, J = 7.1 Hz). ESI-MS: m/z = 269.0 [M+H]+.
4.1.5 General synthetic procedures for (5-aryl-1,2,3-thiadiazol-4-yl)(4-arylpiperazin-
1-yl)methanones (5)
To a solution of arylpiperazines (0.1 mmol) in anhydrous DCM was added
trimethylaluminum (0.5 ml, 1 M in heptane). The reaction was stirred at RT under N2
for 15 min. A solution of an appropriate ethyl 5-aryl-1,2,3-thiadiazole-4-carboxylates
(8) (0.1 mmol) in anhydrous DCM was added and the reaction was stirred at RT under
N2 for 16 h. The reaction was quenched with 5 ml of 1 M HCl and diluted with DCM.
The combined organic layer was washed with water and brine and then dried over
Na2SO4, filtered and concentrated to yield the crude product. The crude product was
purified by column chromatography (n-hexane/ EtOAc = 1:1) on silica gel to afford
pure products.
4.1.5.1 5-Phenyl-4-(4-(3-fluorophenyl)piperazine-1-carbonyl)-1,2,3-thiadiazol (5a)
1
Yellow Solid; yield: 61%; Mp: 46.9-48.6°C; H NMR (600 MHz, CDCl3): δ = 7.52
(2H, m), 7.41 (3H, m), 7.12 (1H, q), 6.55 (1H, dd, J = 1.9 Hz, J = 8.8 Hz), 6.50 (1H,
m), 6.46 (1H, m), 3.92 (2H, t, J = 5.2 Hz), 3.33 (2H, t, J = 5.1 Hz), 3.19 (2H, t, J = 5.2
Hz), 2.87 (2H, t, J = 5.3 Hz). 13C NMR (150 MHz, CDCl3): δ = 162.7 (d, J = 246.7 Hz),
160.3, 154.6, 151.3 (d, J = 10.1 Hz), 150.5, 129.9, 129.3 (d, J = 8.0 Hz), 128.5 (2C),
128.0 (2C), 125.2, 110.8 (d, J = 2.0 Hz), 105.9 (d, J = 21.2 Hz), 102.5 (d, J = 24.7 Hz),
48.0, 47.8, 45.7, 41.0; HRMS calcd for C19H17FN4NaOS [M+Na]+ 391.1005, found
391.1009.
4.1.5.2 5-Phenyl-4-(4-(4-methoxyphenyl)piperazine-1-carbonyl)-1,2,3-thiadiazol (5b)
1
Yellow Solid; yield: 52%; Mp: 66.5-68.2°C; H NMR (600 MHz, CDCl3): δ = 7.59
(2H, m), 7.48 (3H, m), 6.83 (4H, s), 4.00 (2H, t, J = 4.7 Hz), 3.76 (3H, s), 3.39 (2H, s),

3.12 (2H, t, J = 5.2 Hz), 2.81 (2H, s). 13C NMR (150 MHz, CDCl3): δ = 160.3, 154.4,
153.5, 150.7, 143.9, 129.9, 128.5 (2C), 128.0 (2C), 125.3, 118.1 (2C), 113.5 (2C), 54.5,
50.1, 50.0, 46.1, 41.4; HRMS calcd for C20H20N4NaO2S [M+Na]+ 403.1205, found
403.1219.
4.1.5.3 5-Phenyl-4-(4-(2,5-dimethoxyphenyl)piperazine-1-carbonyl)-1,2,3-thiadiazol
(5c)
1
Deepred Solid; yield: 52%; Mp: 36.3-37.9°C; H NMR (600 MHz, CDCl3): δ = 7.59
(2H, m), 7.49 (3H, t, J = 6.1 Hz), 6.77 (1H, d, J = 8.1 Hz), 6.51 (1H, dd, J = 2.7 Hz, J
= 8.4 Hz), 6.40 (1H, d, J = 3.0 H), 4.02 (2H, t, J = 5.1 Hz), 3.80 (3H, s), 3.75 (3H, s),
13
3.39 (2H, t, J = 5.1 Hz), 3.10 (2H, t, J = 5.2 Hz), 2.78 (2H, t, J = 4.9 Hz). C NMR
(150 MHz, CDCl3): δ = 161.4, 155.1, 154.0, 151.8, 146.5, 141.3, 130.9, 129.6 (2C),
129.0 (2C), 126.3, 111.9, 106.5, 106.0, 55.9, 55.6, 50.5, 50.2, 47.3, 42.5; HRMS calcd
for C21H22N4NaO3S [M+Na]+ 433.1310, found 433.1309.
4.1.5.4 5-Phenyl-4-(4-(3,5-dimethoxyphenyl)piperazine-1-carbonyl)-1,2,3-thiadiazol
(5d)
1
Brown Black Solid; yield: 47%; Mp: 50.7-52.2°C; H NMR (600 MHz, CDCl3): δ =
7.58 (2H, m), 7.48 (3H, d, J = 7.1 Hz), 6.05 (1H, t, J = 2.2 Hz), 6.02 (2H, d, J = 2.2
Hz), 3.98 (2H, t, J = 5.0 Hz), 3.76 (6H, s), 3.38 (2H, t, J = 5.4 Hz), 3.24 (2H, t, J = 5.4
13
Hz), 2.92 (2H, t, J = 5.4 Hz). C NMR (150 MHz, CDCl3): δ = 160.5 (2C), 160.3,
154.5, 151.6, 150.6, 129.9, 128.5 (2C), 128.0 (2C), 125.2, 94.7 (2C), 91.4, 54.3 (2C),
51.4, 49.9, 45.8, 41.2; HRMS calcd for C21H22N4NaO3S [M+Na]+ 433.1310, found
433.1311.
4.1.5.5 5-(2-Fluorophenyl)-4-(4-phenylpiperazine-1-carbonyl)-1,2,3-thiadiazol (5e)
White Solid; yield: 57%; Mp: 62.1-63.4°C; 1H NMR (600 MHz, CDCl3): δ = 7.56 (1H,
m), 7.42 (1H, m), 7.20 (4H, m), 6.85 (3H, m), 3.92 (2H, t, J = 5.4 Hz), 3.94 (2H, t, J =

5.3 Hz), 3.23 (2H, t, J = 5.3 Hz), 3.03 (2H, t, J = 5.1 Hz). 13C NMR (150 MHz, CDCl3):
δ = 160.2, 157.8 (d, J = 258.9 Hz), 152.3, 149.7, 148.0 (d, J = 2.2 Hz), 131.7 (d, J = 8.6
Hz), 129.7, 128.3 (2C), 124.1 (d, J = 3.2 Hz), 119.7, 115.8 (2C), 115.5 (d, J = 22.2 Hz),
113.8 (d, J = 14.1 Hz), 48.7, 48.5, 46.1, 41.3; HRMS calcd for C19H17FN4NaOS
[M+Na]+ 391.1005, found 391.1012.
4.1.5.6
5-(2-Fluorophenyl)-4-(4-(3-fluorophenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5f)
1
Yellow Solid; yield: 59%; Mp: 53.2-55.1°C; H NMR (600 MHz, CDCl3): δ = 7.62
(1H, m), 7.49 (1H, m), 7.25 (3H, m), 6.67 (1H, dd, J = 1.9 Hz, J = 8.8 Hz), 6.59 (2H,
m), 4.00 (2H, t, J = 5.2 Hz), 3.60 (2H, t, J = 5.3 Hz), 3.31 (2H, t, J = 5.4 Hz), 3.13 (2H,
t, J = 5.1 Hz). 13C NMR (150 MHz, CDCl3): δ = 162.7 (d, J = 250.8 Hz), 160.2, 157.8
(d, J = 254.8 Hz), 152.2, 151.4 (d, J = 8.6 Hz), 148.3 (d, J = 2.2 Hz), 131.8 (d, J = 7.0
Hz), 129.8, 129.3 (d, J = 9.1 Hz), 124.1 (d, J = 3.0 Hz), 115.5 (d, J = 20.2 Hz), 113.8
(d, J = 13.1 Hz), 110.8 (d, J = 2.3 Hz), 105.9 (d, J = 21.2 Hz), 102.4 (d, J = 23.2 Hz),
48.1, 47.8, 45.9, 41.1; HRMS calcd for C19H16F2N4NaOS [M+Na]+ 409.0911, found
409.0916.
4.1.5.7
5-(2-Fluorophenyl)-4-(4-(3-chlorophenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5g)
1
Light yellow Solid; yield: 54%; Mp: 70.9-72.9°C; H NMR (600 MHz, CDCl3): δ =
7.62 (1H, m), 7.50 (1H, m), 7.27 (2H, m), 7.19 (1H, t, J = 8.5 Hz), 6.88 (2H, d, J = 7.3
Hz), 6.79 (1H, d, J = 8.8 Hz), 4.01 (2H, t, J = 5.2 Hz), 3.60 (2H, t, J = 5.1 Hz), 3.31
(2H, t, J = 5.3 Hz), 3.13 (2H, t, J = 5.3 Hz). 13C NMR (150 MHz, CDCl3): δ = 160.2,
157.8 (d, J = 254.8 Hz), 152.2, 150.8, 148.4 (d, J = 2.2 Hz), 134.1, 131.8 (d, J = 8.0
Hz), 129.8, 129.2, 124.1 (d, J = 3.2 Hz), 119.4, 115.6, 115.5 (d, J = 22.2 Hz), 113.8 (d,
J = 14.1 Hz), 113.6, 48.2, 48.0, 45.9, 41.1; HRMS calcd for C19H16ClFN4NaOS

[M+Na]+ 425.0615, found 425.0623.
4.1.5.8
5-(2-Fluorophenyl)-4-(4-(3-methylphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5h)
1
Light yellow Solid; yield: 58%; Mp: 34.7-36.5°C; H NMR (600 MHz, CDCl3): δ =
7.63 (1H, m), 7.50 (1H, m), 7.28 (1H, m), 7.24 (1H, d, J = 7.6 Hz), 7.19 (1H, t, J = 7.8
Hz), 6.79 (3H, s), 4.04 (2H, s), 3.62 (2H, s), 3.31 (2H, s), 3.13 (2H, s), 2.33 (3H, s). 13C
NMR (150 MHz, CDCl3): δ = 160.2, 157.9 (d, J = 245.9 Hz), 152.3, 148.1, 138.1, 131.7
(d, J = 9.2 Hz), 129.7, 128.9 (d, J = 7.1 Hz), 128.1, 124.1 (d, J = 4.1 Hz), 120.9, 116.8,
115.5 (d, J = 23.5 Hz), 113.8 (d, J = 13.0 Hz), 113.1, 48.9 (2C), 46.0, 41.2, 20.7; HRMS
calcd for C20H19FN4NaOS [M+Na]+ 405.1161, found 405.1176.
4.1.5.9
5-(2-Fluorophenyl)-4-(4-(3-methoxyphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5i)
Light yellow Solid; yield: 56%; Mp: 131.5-133.4°C; 1H NMR (600 MHz, CDCl3): δ =
7.62 (1H, m), 7.48 (1H, m), 7.26 (2H, m), 7.19 (1H, d, J = 8.8 Hz), 6.53 (1H, dd, J =
2.3 Hz, J = 8.1 Hz), 6.46 (2H, m), 4.00 (2H, t, J = 5.2 Hz), 3.79 (3H, s), 3.57 (2H, t, J
13
= 5.1 Hz), 3.29 (2H, t, J = 5.3 Hz), 3.10 (2H, t, J = 5.3 Hz). C NMR (150 MHz,
CDCl3): δ = 161.2, 160.6, 158.9 (d, J = 251.1 Hz), 153.3, 152.1, 149.1 (d, J = 3.2 Hz),
132.8 (d, J = 10.0 Hz), 130.7, 130.0, 125.1 (d, J = 3.8 Hz), 116.5 (d, J = 21.6 Hz), 114.8
(d, J = 14.2 Hz), 109.4, 105.4, 103.3, 55.2, 49.6, 49.4, 47.1, 42.3; HRMS calcd for
C20H19FN4NaO2S [M+Na]+ 421.1110, found 421.1125.
4.1.5.10 5-(2-Fluorophenyl)-4-(4-(3-(trifluoromethyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5j)
Brown Solid; yield: 49%; Mp: 42.9-43.5°C; 1H NMR (600 MHz, CDCl3): δ = 7.62 (1H,
m), 7.50 (1H, m), 7.39 (1H, t, J = 8.2 Hz), 7.28 (1H, m), 7.24 (1H, m), 7.16 (1H, d, J =
7.6 Hz), 7.13 (1H, s), 7.11 (1H, d, J = 8.5 Hz), 4.04 (2H, t, J = 5.3 Hz), 3.65 (2H, t, J =

5.3 Hz), 3.36 (2H, t, J = 5.4 Hz), 3.19 (2H, t, J = 5.2 Hz). 13C NMR (150 MHz, CDCl3):
δ = 161.2, 158.8 (d, J = 245.7 Hz), 153.1, 150.8, 149.5 (d, J = 3.0 Hz), 132.8 (d, J = 9.2
Hz), 131.5, 130.9, 129.8, 125.2 (d, J = 3.1 Hz), 123.2, 119.7, 117.1 (d, J = 3.0 Hz),
116.5 (d, J = 21.5 Hz), 114.8 (d, J = 13.3 Hz), 113.1 (d, J = 4.0 Hz), 49.3, 49.1, 46.9,
42.2; HRMS calcd for C20H16F4N4NaOS [M+Na]+ 459.0879, found 459.0882.
4.1.5.11
5-(2-Fluorophenyl)-4-(4-(4-methoxyphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5k)
Brown Solid; yield: 47%; Mp: 53.7-55.2°C; 1H NMR (600 MHz, DMSO-d6): δ = 7.67
(1H, m), 7.62 (1H, m), 7.48 (1H, t, J = 9.6 Hz), 7.41 (1H, t, J = 7.9 Hz), 6.89 (2H, d, J
= 7.9 Hz), 6.83 (2H, d, J = 8.4 Hz), 3.84 (2H, t, J = 5.6 Hz), 3.68 (3H, s), 3.41 (2H, t, J
13
= 5.2 Hz), 3.07 (2H, t, J = 5.1 Hz), 2.79 (2H, t, J = 5.0 Hz). C NMR (150 MHz,
DMSO-d6): δ = 160.8, 158.9 (d, J = 249.7 Hz), 153.9, 148.2, 148.2, 145.3, 133.7 (d, J
= 9.2 Hz), 131.2, 130.1 (d, J = 7.1 Hz), 126.0 (d, J = 3.5 Hz), 118.6 (2C), 117.0 (d, J =
22.0 Hz), 114.8 (2C), 54.6, 50.5, 50.2, 47.0, 42.2; HRMS calcd for C20H19FN4NaO2S
[M+Na]+ 421.1110, found 421.1121.
4.1.5.12 5-(2-Fluorophenyl)-4-(4-(2,5-dimethoxyphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5l)
Deepred Solid; yield: 51%; Mp: 46.2-7-47.1°C; 1H NMR (600 MHz, CDCl3): δ = 7.64
(1H, m), 7.50 (1H, m), 7.26 (2H, m), 6.79 (1H, d, J = 9.5 Hz), 6.53 (1H, d, J = 8.8 Hz),
6.48 (1H, s), 4.03 (2H, t, J = 5.0 Hz), 3.82 (3H, s), 3.76 (3H, s), 3.57 (2H, t, J = 5.2 Hz),
13
3.15 (2H, t, J = 5.1 Hz), 2.96 (2H, t, J = 4.9 Hz). C NMR (150 MHz, CDCl3): δ =
161.3, 158.9 (d, J = 245.7 Hz), 154.0, 153.4, 148.6, 146.5, 141.5, 132.7 (d, J = 8.7 Hz),
130.6, 125.1 (d, J = 3.0 Hz), 116.5 (d, J = 21.5 Hz), 114.9 (d, J = 13.3 Hz), 111.9, 106.6,
106.0, 55.9, 55.6, 50.6, 50.3, 47.4, 42.5; HRMS calcd for C21H21FN4NaO3S [M+Na]+
451.1216, found 451.1221.

4.1.5.13 5-(2-Fluorophenyl)-4-(4-(3,5-dimethoxyphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5m)
Brown Solid; yield: 55%; Mp: 91.5-93.4°C;1H NMR (600 MHz, CDCl3): δ = 7.62 (1H,
t, J = 7.4 Hz), 7.49 (1H, m), 7.25 (2H, m), 6.07 (3H, d, J = 5.7 Hz), 3.99 (2H, t, J = 5.2
Hz), 3.77 (6H, s), 3.56 (2H, t, J = 5.1 Hz), 3.28 (2H, t, J = 4.8 Hz), 3.08 (2H, t, J = 5.2
13
Hz). C NMR (150 MHz, CDCl3): δ = 161.5 (2C), 160.2, 157.8 (d, J = 245.7 Hz),
152.2, 151.7, 148.0, 131.8 (d, J = 9.2 Hz), 129.7, 124.1 (d, J = 2.6 Hz), 115.5 (d, J =
21.5 Hz), 113.8 (d, J = 14.8 Hz), 94.7 (2C), 91.4, 54.3 (2C), 48.6, 48.4, 46.0, 41.2;
HRMS calcd for C21H21FN4NaO3S [M+Na]+ 451.1216, found 451.1223.
4.1.5.14 5-(3-Fluorophenyl)-4-(4-phenylpiperazine-1-carbonyl)-1,2,3-thiadiazol (5n)
1
Yellow Solid; yield: 59%; Mp: 92.3-94.0°C; H NMR (600 MHz, CDCl3): δ = 7.45
(1H, m), 7.38 (1H, d, J = 7.6 Hz), 7.34 (1H, dd, J = 2.3 Hz, J = 9.2 Hz), 7.27 (2H, t, J
= 8.4 Hz), 7.19 (1H, m), 6.91 (3H, m), 4.02 (2H, t, J = 5.4 Hz), 3.46 (2H, t, J = 5.2 Hz),
13
3.27 (2H, t, J = 5.4 Hz), 3.02 (2H, t, J = 5.2 Hz). C NMR (150 MHz, CDCl3): δ =
161.8 (d, J = 254.8 Hz), 159.9, 153.2 (d, J = 3.0 Hz), 151.0, 149.6, 130.2 (d, J = 10.1
Hz), 128.3 (2C), 127.1 (d, J = 7.0 Hz), 124.0 (d, J = 2.5 Hz), 119.9, 116.9 (d, J = 21.2
Hz), 115.8 (2C), 115.1 (d, J = 23.2 Hz), 48.8, 48.5, 46.0, 41.3; HRMS calcd for
C19H17FN4NaOS [M+Na]+ 391.1005, found 391.1014.
4.1.5.15
5-(3-Fluorophenyl)-4-(4-(3-chlorophenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5o)
1
Yellow Solid; yield: 60%; Mp: 69.3-70.7°C; H NMR (600 MHz, CDCl3): δ = 7.39
(1H, q), 7.31 (1H, d, J = 8.4 Hz), 7.27 (1H, d, J = 9.3 Hz), 7.12 (2H, m), 6.79 (2H, t),
6.69 (1H, d, J = 8.4 Hz), 3.94 (2H, t, J = 5.1 Hz), 3.40 (2H, t, J = 5.2 Hz), 3.21 (2H, t,
J = 5.2 Hz), 2.98 (2H, t, J = 5.4 Hz). 13C NMR (150 MHz, CDCl3): δ = 161.8 (d, J =
258.9 Hz), 159.9, 153.53 (d, J = 3.0 Hz), 150.9, 150.7, 134.1, 130.2 (d, J = 8.1 Hz),

129.2, 127.0, 124.0 (d, J = 3.4 Hz), 119.5, 116.9 (d, J = 22.2 Hz), 115.6, 115.2 (d, J =
24.3 Hz), 113.7, 48.3, 48.0, 45.9, 41.2; HRMS calcd for C19H16ClFN4NaOS [M+Na]+
425.0615, found 425.0620.
4.1.5.16
5-(3-Fluorophenyl)-4-(4-(3-methylphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5p)
Yellow Solid; yield: 61%; Mp: 119.8-121.0°C; 1H NMR (600 MHz, CDCl3): δ = 7.45
(1H, m), 7.38 (1H, m), 7.34 (1H, m), 7.17 (2H, m), 6.72 (3H, m), 4.01 (2H, t, J = 5.2
Hz), 3.44 (2H, t, J = 5.3 Hz), 3.25 (2H, t, J = 5.4 Hz), 3.01 (2H, t, J = 5.2 Hz), 2.31
(3H, s). 13C NMR (150 MHz, CDCl3): δ = 161.8 (d, J = 253.8 Hz), 159.9, 153.2 (d, J =
3.1 Hz), 151.0, 149.7, 138.0, 130.2 (d, J = 8.7 Hz), 128.1, 127.1 (d, J = 8.7 Hz), 123.9
(d, J = 3.5 Hz), 120.7, 116.9 (d, J = 20.9 Hz), 116.7, 115.1 (d, J = 23.7 Hz), 112.9, 48.9,
48.6, 46.1, 41.4, 20.7; HRMS calcd for C20H19FN4NaOS [M+Na]+ 405.1161, found
405.1176.
4.1.5.17
5-(3-Fluorophenyl)-4-(4-(3-methoxyphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5q)
Yellow Solid; yield: 64%; Mp: 123.6-124.8°C; 1H NMR (600 MHz, CDCl3): δ = 7.45
(1H, m), 7.38 (1H, d, J = 8.1 Hz), 7.34 (1H, m), 7.19 (2H, m), 6.50 (1H, dd, J = 2.3 Hz,
J = 8.2 Hz), 6.47 (1H, dd, J = 2.3 Hz, J = 8.1 Hz), 6.42 (1H, t), 4.00 (2H, t, J = 5.2 Hz),
3.78 (3H, s), 3.44 (2H, t, J = 5.1 Hz), 3.27 (2H, t, J = 5.2 Hz), 3.02 (2H, t, J = 5.2 Hz).
13C NMR (150 MHz, CDCl3): δ = 161.8 (d, J = 251.1 Hz), 159.9, 159.6, 153.3, 153.3,
151.0, 130.2 (d, J = 8.2 Hz), 129.0, 127.1 (d, J = 8.2 Hz), 123.9 (d, J = 3.4 Hz), 117.0
(d, J = 21.3 Hz), 115.1 (d, J = 23.1 Hz), 108.4, 104.5, 102.3, 54.2, 48.7, 48.4, 46.0,
41.3; HRMS calcd for C20H19FN4NaO2S [M+Na]+ 421.1110, found 421.1130.
4.1.5.18
5-(3-Fluorophenyl)-4-(4-(4-methoxyphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5r)

1
Yellow Solid; yield: 60%; Mp: 46.3-47.6°C; H NMR (600 MHz, CDCl3): δ = 7.56
(1H, m), 7.49 (1H, d, J = 7.4 Hz), 7.45 (1H, m), 7.30 (1H, m), 6.98 (2H, d, J = 8.4 Hz),
6.94 (2H, d, J = 9.1 Hz), 4.12 (2H, t, J = 5.2 Hz), 3.87 (3H, s), 3.55 (2H, s), 3.25 (2H,
t, J = 4.7 Hz), 3.01 (2H, s). 13C NMR (150 MHz, CDCl3): δ = 161.8 (d, J = 262.0 Hz),
159.9, 153.6, 153.2, 151.0, 143.9, 130.2 (d, J = 7.6 Hz), 127.1 (d, J = 9.2 Hz), 124.0 (d,
J = 3.3 Hz), 118.2 (2C), 116.9 (d, J = 21.5 Hz), 115.1 (d, J = 23.5 Hz), 113.5 (2C), 54.5,
50.3, 50.0, 46.2, 41.8; HRMS calcd for C20H19FN4NaO2S [M+Na]+ 421.1110, found
421.1121.
4.1.5.19 5-(3-Fluorophenyl)-4-(4-(2,5-dimethoxyphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5s)
Brown Solid; yield: 51%; Mp: 35.2-37.1°C; 1H NMR (600 MHz, CDCl3): δ = 7.46 (1H,
q), 7.38 (1H, d, J = 8.1 Hz), 7.34 (1H, d, J = 9.5 Hz), 7.20 (1H, m), 6.78 (1H, d, J = 8.4
Hz), 6.52 (1H, dd, J = 2.7 H, J = 8.4 Hz), 6.4 (1H, d, J = 2.0 Hz), 4.03 (2H, t, J = 5.0
Hz), 3.81 (3H, s), 3.75 (3H, s), 3.44 (2H, t, J = 5.4 Hz), 3.13 (2H, t, J = 5.0 Hz), 2.88
(2H, t, J = 4.7 Hz). 13C NMR (150 MHz, CDCl3): δ = 161.8 (d, J = 245.7 Hz), 160.0,
153.0, 152.9, 151.2, 145.5, 140.2, 130.2 (d, J = 8.2 Hz), 127.1 (d, J = 9.2 Hz), 123.9 (d,
J = 3.0 Hz), 116.9 (d, J = 20.5 Hz), 115.1 (d, J = 23.5 Hz), 110.9, 105.5, 105.1, 54.9,
54.6, 49.7, 49.3, 46.3, 41.5; HRMS calcd for C21H21FN4NaO3S [M+Na]+ 451.1216,
found 451.1216.
4.1.5.20 5-(3-Fluorophenyl)-4-(4-(3,5-dimethoxyphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5t)
Brown Solid; yield: 42%; Mp: 46.8-48.2°C; 1H NMR (600 MHz, CDCl3): δ = 7.46 (1H,
m), 7.38 (1H, m), 7.34 (1H, m), 7.20 (1H, m), 6.05 (3H, q), 3.99 (2H, t, J = 5.4 Hz),
3.76 (6H, s), 3.43 (2H, t, J = 5.4 Hz), 3.25 (2H, t, J = 5.4 Hz), 3.00 (2H, t, J = 4.7 Hz).
13C NMR (150 MHz, CDCl3): δ = 161.8 (d, J = 257.9 Hz), 160.5 (2C), 159.9, 153.3,

151.6, 150.9, 130.2 (d, J = 8.1 Hz), 127.0 (d, J = 9.4 Hz), 123.9 (d, J = 3.5 Hz), 116.9
(d, J = 21.5 Hz), 115.1 (d, J = 23.7 Hz), 94.7 (2C), 91.5, 54.2 (2C), 48.7, 48.4, 45.9,
41.3; HRMS calcd for C21H21FN4NaO3S [M+Na]+ 451.1216, found 451.1211.
4.1.5.21 5-(4-Fluorophenyl)-4-(4-phenylpiperazine-1-carbonyl)-1,2,3-thiadiazol (5u)
1
Yellow Solid; yield: 70%; Mp: 53.5-54.8°C; H NMR (600 MHz, CDCl3): δ = 7.61
(2H, m), 7.28 (2H, m), 7.17 (2H, t, J = 8.8 Hz), 6.91 (3H, m), 4.01 (2H, t, J = 5.3 Hz),
13
3.46 (2H, t, J = 5.2 Hz), 3.27 (2H, t, J = 5.1 Hz), 3.04 (2H, t, J = 5.2 Hz). C NMR
(150 MHz, CDCl3): δ = 163.1 (d, J = 253.9 Hz), 160.1, 153.8, 150.6, 149.6, 130.2 (d, J
= 9.2 Hz) (2C), 128.3 (2C), 121.4 (d, J = 3.2 Hz), 119.9, 115.9 (d, J = 4.6 Hz) (2C),
115.7 (2C), 48.9, 48.6, 46.1, 41.3; HRMS calcd for C19H17FN4NaOS [M+Na]+
391.1005, found 391.1014.
4.1.5.22
5-(4-Fluorophenyl)-4-(4-(3-chlorophenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5v)
1
Yellow Solid; yield: 59%; Mp: 37.1-39.1°C; H NMR (600 MHz, CDCl3): δ = 7.61
(2H, m), 7.18 (3H, t, J = 7.6 Hz), 6.86 (2H, t), 6.76 (1H, d, J = 9.0 Hz), 4.00 (2H, t, J =
5.0 Hz), 3.47 (2H, t, J = 5.3 Hz), 3.28 (2H, t, J = 5.2 Hz), 3.06 (2H, t, J = 5.1 Hz). 13C
NMR (150 MHz, CDCl3): δ = 163.1 (d, J = 242.7 Hz), 160.1, 154.1, 150.7, 150.5, 134.1,
130.2 (d, J = 9.1 Hz) (2C), 129.2, 121.4 (d, J = 4.5 Hz), 119.5, 115.9, 115.7 (d, J = 10.1
Hz) (2C), 113.6, 48.4, 48.0, 45.9, 41.2; HRMS calcd for C19H16ClFN4NaOS [M+Na]+
425.0615, found 425.0629.
4.1.5.23
5-(4-Fluorophenyl)-4-(4-(3-methylphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5w)
Yellow Solid; yield: 61%; Mp: 112.6-114.1°C; 1H NMR (600 MHz, CDCl3): δ = 7.61
(2H, m), 7.16 (3H, q), 6.72 (3H, m), 4.00 (2H, t, J = 5.3 Hz), 3.44 (2H, t, J = 5.2 Hz),
13
3.25 (2H, t, J = 5.1 Hz), 3.02 (2H, t, J = 5.3 Hz), 2.31 (3H, s). C NMR (150 MHz,

CDCl3): δ = 163.1 (d, J = 255.9 Hz), 160.1, 153.7, 150.7, 149.7, 138.0, 130.2 (d, J =
8.2 Hz) (2C), 128.1, 121.4 (d, J = 3.8 Hz), 120.8, 116.7, 115.8 (d, J = 21.9 Hz) (2C),
113.0, 48.9, 48.6, 46.1, 41.4, 20.7; HRMS calcd for C20H19FN4NaOS [M+Na]+
405.1161, found 405.1183.
4.1.5.24
5-(4-Fluorophenyl)-4-(4-(3-methoxyphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5x)
1
Yellow Solid; yield: 65%; Mp: 81.6-82.7°C; H NMR (600 MHz, CDCl3): δ = 7.61
(2H, m), 7.18 (3H, m), 6.50 (1H, dd, J = 2.3 Hz, J = 8.2 Hz), 6.47 (1H, dd, J = 2.3 Hz,
J = 8.4 Hz), 6.42 (1H, m), 4.00 (2H, t, J = 5.2 Hz), 3.78 (3H, s), 3.45 (2H, t, J = 5.3
Hz), 3.27 (2H, t, J = 5.4 Hz), 3.03 (2H, t, J = 5.2 Hz). 13C NMR (150 MHz, CDCl3): δ
= 163.1 (d, J = 251.1 Hz), 160.1, 159.6, 153.8, 151.0, 150.6, 130.2 (d, J = 8.9 Hz) (2C),
129.0, 121.4 (d, J = 3.2 Hz), 115.8 (d, J = 22.5 Hz) (2C), 108.5, 104.5, 102.3, 54.2,
48.7, 48.41, 46.0, 41.3; HRMS calcd for C20H19FN4NaO2S [M+Na]+ 421.1110, found
421.1121.
4.1.5.25
5-(4-Fluorophenyl)-4-(4-(4-methoxyphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5y)
1
Yellow Solid; yield: 67%; Mp: 62.3-63.7°C; H NMR (600 MHz, CDCl3): δ = 7.61
(2H, m), 7.18 (2H, t, J = 8.8 Hz), 6.85 (4H, m), 4.00 (2H, s), 3.77 (3H, s), 3.45 (2H, s),
3.14 (2H, s), 2.91 (2H, s). 13C NMR (150 MHz, CDCl3): δ = 163.1 (d, J = 253.9 Hz),
160.1, 153.7, 153.6, 150.7, 143.9, 130.2 (d, J = 10.2 Hz) (2C), 121.4 (d, J = 3.6 Hz),
118.1 (2C), 115.8 (d, J = 22.5 Hz) (2C), 116.5 (2C), 54.5, 50.3, 50.0, 46.2, 41.5; HRMS
calcd for C20H19FN4NaO2S [M+Na]+ 421.1110, found 421.1119.
4.1.5.26 5-(4-Fluorophenyl)-4-(4-(3,4-dimethylphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5z)
1
Yellow Solid; yield: 66%; Mp: 82.9-84.0°C; H NMR (600 MHz, CDCl3): δ = 7.61

(2H, m), 7.17 (2H, t, J = 8.5 Hz), 7.03 (1H, d, J = 8.5 Hz), 6.70 (2H, d, J = 43.7 Hz),
13
4.01 (2H, s), 3.45 (2H, s), 3.21 (2H, s), 2.98 (2H, s), 2.23 (3H, s), 2.19 (3H, s). C
NMR (150 MHz, CDCl3): δ = 164.1 (d, J = 261.6 Hz), 161.6, 154.8, 151.7, 148.9, 137.5,
131.2 (d, J = 8.9 Hz) (2C), 130.3 (2C), 122.4 (d, J = 3.8 Hz), 119.0, 116.8 (d, J = 20.7
Hz) (2C), 114.7, 50.5, 50.3, 47.1, 42.4, 20.2, 18.9; HRMS calcd for C21H21FN4NaOS
[M+Na]+ 419.1318, found 419.1327.
4.1.5.27 5-(4-Fluorophenyl)-4-(4-(3,5-dimethoxyphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5a’)
1
Light brown Solid; yield: 54%; Mp: 55.5-57.2°C; H NMR (600 MHz, CDCl3): δ =
7.53 (2H, m), 7.10 (2H, t, J = 8.8 Hz), 5.97 (3H, m), 3.91 (2H, t, J = 5.1 Hz), 3.69 (6H,
s), 3.36 (2H, t, J = 5.0 Hz), 3.18 (2H, t, J = 4.7 Hz), 2.94 (2H, t, J = 5.0 Hz). 13C NMR
(150 MHz, CDCl3): δ = 163.1 (d, J = 250.2 Hz), 160.5 (2C), 160.1, 153.8, 151.6, 150.6,
130.2 (d, J = 8.5 Hz) (2C), 121.4 (d, J = 5.2 Hz), 115.8 (d, J = 22.2 Hz) (2C), 94.7 (2C),
91.4, 54.3 (2C), 48.7, 48.4, 45.9, 41.3; HRMS calcd for C21H21FN4NaO3S [M+Na]+
451.1216, found 451.1218.
4.1.5.28
5-(2-Chlorophenyl)-4-(4-(3-chlorophenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5b’)
Brown Solid; yield: 63%; Mp: 96.3-97.4°C; 1H NMR (600 MHz, CDCl3): δ = 7.53 (2H,
m), 7.44 (1H, m), 7.37 (1H, m), 7.18 ( 1H, d, J = 8.0 Hz), 6.86 ( 2H, d, J = 8.5 Hz),
6.77 (1H, t), 3.91 (2H, t, J = 5.1 Hz), 3.64 (2H, t, J = 5.1 Hz), 3.23 (2H, t, J = 5.1 Hz),
3.08 (2H, t, J = 5.1 Hz). 13C NMR (150 MHz, CDCl3): δ = 159.5, 153.2, 152.2, 150.8,
134.1, 131.8 (2C), 130.7, 129.3, 129.2, 126.4, 124.7, 119.3, 115.5, 113.6, 48.3, 47.9,
45.9, 41.1; HRMS calcd for C19H16Cl2N4NaOS [M+Na]+ 441.0320, found 441.0324.
4.1.5.29
5-(2-Chlorophenyl)-4-(4-(3-methylphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5c’)

1
Deepred Solid; yield: 61%; Mp: 89.2-90.7°C; H NMR (600 MHz, CDCl3): δ = 7.52
(2H, m), 7.43 (1H, m), 7.38 (1H, m), 7.16 (1H, t, J = 7.6 Hz), 6.72 (3H, m), 3.91 (2H,
t, J = 5.3 Hz), 3.61 (2H, t, J = 5.3 Hz), 3.20 (2H, t, J = 5.2 Hz), 3.04 (2H, t, J = 5.2 Hz),
2.32 (3H, s). 13C NMR (150 MHz, CDCl3): δ = 159.5, 153.3, 151.9, 149.8, 138.0, 131.8,
130.7, 130.6, 129.3, 128.1, 126.4, 124.7, 120.6, 116.7, 112.9, 48.8, 48.5, 46.1, 41.3,
20.7; HRMS calcd for C20H19ClN4NaOS [M+Na]+ 421.0866, found 421.0891.
4.1.5.30
5-(2-Chlorophenyl)-4-(4-(3-methoxyphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5d’)
Red Solid; yield: 54%; Mp: 60.5-62.3°C; 1H NMR (600 MHz, CDCl3): δ = 7.52 (2H,
m), 7.43 (1H, m), 7.38 (1H, m), 7.18 (1H, d, J = 8.5 Hz), 6.51 (1H, d, J = 7.6 Hz), 6.46
(1H, d, J = 8.5 Hz), 6.44 (1H, s), 3.91 (2H, t, J = 5.3 Hz), 3.79 (3H, s), 3.62 (2H, t, J =
5.2 Hz), 3.21 (2H, t, J = 5.1 Hz), 3.05 (2H, t, J = 5.2 Hz). 13C NMR (150 MHz, CDCl3):
δ = 159.6, 159.5, 153.3, 151.9, 151.0, 131.8, 130.7 (2C), 129.3, 129.0, 126.4, 124.7,
108.4, 104.4, 102.3, 54.2, 48.7, 48.3, 46.0, 41.2; HRMS calcd for C20H19ClN4NaO2S
[M+Na]+ 437.0815, found 437.0837
4.1.5.31 5-(2-Chlorophenyl)-4-(4-(3-(trifluoromethylphenyl)piperazine-1-carbonyl)-
1,2,3-thiadiazol (5e’)
1
Light yellow Solid; yield: 58%; Mp: 42.9-42.6°C; H NMR (600 MHz, CDCl3): δ =
7.53 (2H, m), 7.44 (1H, m), 7.38 (2H, m), 7.14 (1H, d, J = 8.2 Hz), 7.09 (1H, s), 7.05
(1H, d, J = 9.4 Hz), 3.94 (2H, t, J = 5.1 Hz), 3.67 (2H, t, J = 5.3 Hz), 3.28 (2H, t, J =
5.2 Hz), 3.14 (2H, t, J = 5.4 Hz). 13C NMR (150 MHz, CDCl3): δ = 160.5, 154.2, 153.3,
150.9, 132.8, 131.7, 131.7, 131.5, 130.3, 129.8, 127.4, 125.7, 125.0, 119.5, 116.9 (d, J
= 4.0 Hz), 113.0 (d, J = 3.5 Hz), 49.3, 49.1, 46.9, 42.2; HRMS calcd for
C20H16ClF3N4NaOS [M+Na]+ 475.0583, found 475.0613.
4.1.5.32
5-(2-Chlorophenyl)-4-(4-(4-methoxyphenyl)piperazine-1-carbonyl)-1,2,3-

thiadiazol (5f’)
Brown Solid; yield: 43%; Mp: 57.9-59.1°C; 1H NMR (600 MHz, CDCl3): δ = 7.53 (2H,
m), 7.44 (1H, m), 7.39 (1H, m), 6.85 (4H, d, J = 8.4 Hz), 3.93 (2H, s), 3.78 (3H, s),
3.62 (2H, s), 3.10 (2H, s), 2.94 (2H, s). 13C NMR (150 MHz, CDCl3): δ = 159.5, 153.6,
153.3, 151.8, 144.0, 131.8, 130.7, 130.7, 129.3, 126.4, 124.7, 118.1 (2C), 113.5 (2C),
54.5, 50.3, 49.9, 46.2, 41.3; HRMS calcd for C20H19ClN4NaO2S [M+Na]+ 437.0815,
found 437.0817.
4.1.5.33 5-(2-Chlorophenyl)-4-(4-(2,5-dimethoxyphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5g’)
Brown Solid; yield: 66%; Mp: 96.8-97.9°C; 1H NMR (600 MHz, CDCl3): δ = 7.53 (2H,
m), 7.44 (1H, m), 7.39 (1H, m), 6.78 (1H, d, J = 8.8 Hz), 6.52 (1H, dd, J = 3.0 Hz, J =
8.8 Hz), 6.44 (1H, d, J = 3.0 H), 3.94 (2H, t, J = 5.1 Hz), 3.81 (3H, s), 3.76 (3H, s), 3.60
13
(2H, t, J = 5.1 Hz), 3.06 (2H, t, J = 5.1 Hz), 2.90 (2H, t, J = 4.8 Hz). C NMR (150
MHz, CDCl3): δ = 159.6, 153.4, 153.0, 151.4, 145.5, 140.4, 131.9, 130.7 (2C), 129.3,
126.4, 124.8, 110.9, 105.5, 105.9, 54.9, 54.6, 49.7, 49.3, 46.3, 41.5; HRMS calcd for
C21H21ClN4NaO3S [M+Na]+ 467.0921, found 467.0926.
4.1.5.34 5-(2-Chlorophenyl)-4-(4-(3,5-dimethoxyphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5h’)
Brown Solid; yield: 68%; Mp: 44.7-46.2°C; 1H NMR (600 MHz, CDCl3): δ = 7.52 (2H,
m), 7.43 (1H, m), 7.38 (1H, m), 6.05 (3H, s), 3.90 (2H, t, J = 5.2 Hz), 3.77 (6H, s), 3.61
13
(2H, t, J = 5.0 Hz), 3.20 (2H, t, J = 4.9 Hz), 3.04 (2H, t, J = 5.0 Hz). C NMR (150
MHz, CDCl3): δ = 160.5 (2C), 159.5, 153.3, 151.9, 151.7, 131.8, 130.7, 130.7, 129.3,
126.4, 124.7, 94.7 (2C), 91.4, 54.3 (2C), 48.6, 48.3, 46.0, 41.2; HRMS calcd for
C21H21ClN4NaO3S [M+Na]+ 467.0921, found 467.0922.
4.1.5.35 5-(3,4-Dichlorophenyl)-4-(4-(3,5-dimethoxyphenyl)piperazine-1-carbonyl)-

1,2,3-thiadiazol (5i’)
Brown Solid; yield: 68%; Mp: 87.7-89.2°C; 1H NMR (600 MHz, CDCl3): δ = 7.71 (1H,
d, J = 2.1 Hz), 7.55 (1H, d, J = 8.4 Hz), 7.47 (1H, dd, J = 2.3 Hz, J = 8.1 Hz), 6.06 (3H,
s), 3.99 (2H, t, J = 5.0 Hz), 3.77 (6H, s), 3.49 (2H, t, J = 5.2 Hz), 3.28 (2H, t, J = 4.9
13
Hz), 3.09 (2H, t, J = 5.0 Hz). C NMR (150 MHz, CDCl3): δ = 161.5 (2C), 160.6,
153.7, 152.6, 152.1, 135.5, 133.8, 131.4, 130.9, 128.2, 126.1, 95.8 (2C), 92.6, 55.3 (2C),
49.8, 49.5, 47.1, 42.4; HRMS calcd for C21H20Cl2N4NaO3S [M+Na]+ 501.0531, found
501.0548.
4.1.5.36
5-(4-Chlorophenyl)-4-(4-(4-methoxyphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5j’)
1
Yellow Solid; yield: 63%; Mp: 43.1-45.0°C; H NMR (600 MHz, CDCl3): δ = 7.55
(2H, d, J = 7.5 Hz), 7.46 (2H, d, J = 8.3 Hz), 6.85 (4H, t, J = 8.8 Hz), 4.02 (2H, s), 3.77
(3H, s), 3.65 (2H, s), 3.16 (2H, s), 2.94 (2H, s). 13C NMR (150 MHz, CDCl3): δ = 160.0,
153.6, 153.6, 150.8, 143.8, 136.3, 129.4 (2C), 128.8 (2C), 123.7, 118.3 (2C), 113.6 (2C),
54.5, 50.2, 50.2, 46.2, 41.5; HRMS calcd for C20H19ClN4NaO2S [M+Na]+ 437.0815,
found 437.0824.
4.1.5.37 5-(4-Chlorophenyl)-4-(4-(3,5-dimethoxyphenyl)piperazine-1-carbonyl)-1,2,3-
thiadiazol (5k’)
Brown Solid; yield: 63%; Mp: 43.5-45.2°C; 1H NMR (600 MHz, CDCl3): δ = 7.55 (2H,
d, J = 7.8 Hz), 7.45 (2H, d, J = 8.4 Hz), 6.06 (3H, s), 3.99 (2H, t, J = 5.1 Hz), 3.77 (6H,
s), 3.45 (2H, t, J = 5.2 Hz), 3.27 (2H, t, J = 4.8 Hz), 3.05 (2H, t, J = 5.1 Hz). 13C NMR
(150 MHz, CDCl3): δ = 160.5 (2C), 160.0, 153.8, 151.5, 150.7, 136.3, 129.3 (2C), 128.8
(2C), 123.7, 94.8 (2C), 91.6, 54.3 (2C), 48.8, 48.5, 45.9, 41.3; HRMS calcd for
C21H21ClN4NaO3S [M+Na]+ 467.0921, found 467.0930.
4.2 Biological evaluation

4.2.1 Cell culture
The human gastric adenocarcinoma SGC-7901 cells, lung adenocarcinoma A549 cells
and cervical carcinoma HeLa cells were cultured in RPMI-1640 medium containing 10%
FBS, 100 U/mL streptomycin and 100 U/mL penicillin at 37°C in a humidified
atmosphere containing 5% CO2. All cell lines were purchased from the American Type
Culture Collection (ATCC, Manassas, VA).
4.2.2 In vitro antiproliferative activity
The antiproliferative activities of target compounds 5, CA-4 and colchicine were
determined by a standard MTT assay. In brief, cells were seeded into 96-well plates at
a density of 1-3×104/well (depending on the cell growth rate). 24 h later, cells were
incubated with various concentrations of the test compounds for 72 h. Then the drug-
containing medium was removed and replaced with 100 mL of fresh medium
containing 5 mg/mL MTT solution. After 4 h of incubation, the medium with MTT was
removed, and 100 mL of dimethyl sulphoxide (DMSO) was added to each well. The
plates were gently agitated until the purple formazan crystals were dissolved, and the
absorbance was measured at 570 nm by a microplate spectrophotometer (MK3, Thermo,
Germany). The data were calculated and plotted as the per cent viability compared to
the control. The 50% inhibitory concentration (IC50) was defined as the concentration
that reduced the absorbance of the untreated wells by 50% of the vehicle in the MTT
assay [18].
4.2.3 Effect on tubulin polymerization
The effects of 5m and CA-4 on the polymerization of tubulin were determined by
employing a fluorescence-based tubulin polymerization assay kit (BK011P,
Cytoskeleton, USA) according to the manufacturer’s protocol. The tubulin reaction mix
contained 2 mg/mL porcine brain tubulin (> 99% pure), 2 mM MgCl2, 0.5 mM EGTA,