Synthesis, biological evaluation, and molecular docking studies of 1,3,4-thiadiazol-2-amide derivatives as novel anticancer agents

Article abstract of DOI:10.1016/j.bmc.2012.03.040

A series of 1,3,4-thiadiazol-2-amide derivatives (5a-5y) have been designed and synthesized, and their biological activities were also evaluated as potential antiproliferation and FAK inhibitors. Among all the compounds, 5h showed the most potent activity

Full text of DOI:10.1016/j.bmc.2012.03.040

Bioorganic & Medicinal Chemistry 20 (2012) 2789–2795
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation, and molecular docking studies
of 1,3,4-thiadiazol-2-amide derivatives as novel anticancer agents
Xian-Hui Yang a, , Lu Xiang a, , Xi Li a, Ting-Ting Zhao a, Hui Zhang a, Wen-Ping Zhou a,
b,
a,
Xiao-Ming Wang a, , Hai-Bin Gong , Hai-Liang Zhu
⇑
⇑
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b Xuzhou Central Hospital, Xuzhou 221009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,3,4-thiadiazol-2-amide derivatives (5a–5y) have been designed and synthesized, and their
biological activities were also evaluated as potential antiproliferation and FAK inhibitors. Among all
the compounds, 5h showed the most potent activity in vitro, which inhibited the growth of MCF-7
Received 15 February 2012
Revised 18 March 2012
Accepted 19 March 2012
Available online 24 March 2012
and B16-F10 cell lines with IC50 values of 0.45 and 0.31
lM, respectively. Compound 5h also exhibited
significant FAK inhibitory activity (IC50 = 5.32 M). Docking simulation was performed to position com-
l
pound 5h into the FAK structure active site to determine the probable binding model. The results of anti-
proliferative and Western-blot assay demonstrated that compound 5h possessed good antiproliferative
activity. Therefore, compound 5h with potent FAK inhibitory activity may be a potential anticancer agent.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
1,3,4-Thiadiazol-2-amide derivatives
Focal adhesion kinase
Structure–activity relationship
Molecular docking
1. Introduction
C-terminal domain of FAK, FAK-CD or FRNK12,13 or FAK siRNA,14,15
caused decreased cellular viability, growth inhibition, or apoptosis.
Cancer is a class of diseases in which cell, or a group of cells
display uncontrolled growth, invasion, and sometimes metastasis.
During the last few decades, anticancer therapy has progressed sig-
nificantly, but the management of malignancies in humans still
constitutes a major challenge for contemporary medicine.1–4
Focal adhesion kinase (FAK) was first discovered in 1992 and
was implicated in integrin signaling.5–7 It is a 125 kDa protein tyro-
sine kinase recruited at an early stage to focal adhesions and is
phosphorylated in response to cell attachment and mediates focal
adhesion formation.8,9 Focal adhesions are found at the cell mem-
brane where the cytoskeleton interacts with the proteins of the
extra-cellular matrix. The clustering of integrins at these sites
attracts a large complex of proteins which regulate processes such
as anchorage-dependent proliferation and cell migration. Signal
transduction mediated by interactions between cells and the extra-
cellular matrix (ECM) at focal adhesions is an important determi-
nant of cell fate. Focal adhesion kinase is involved in multiple
cellular functions such as cell proliferation, survival, motility, inva-
sion, metastasis, and angiogenesis.10 Different approaches to inhi-
bit FAK with FAK antisense oligonucleotides,11 dominant-negative
Recently, FAK was proposed to be a new potential therapeutic
target in cancer.16,17
Phenyl-1,3,4-thiadiazoles are a class of small molecules that
have received much interest in the fields of chemistry and biology
due to their broad spectrum of activity. The 1,3,4-thiadiazole
scaffold is an interesting building block that has been used to
synthesize a variety of useful bioactive compounds. Phenyl-1,3,4-
thiadiazole derivatives have been reported to be anticancer,18
antimicrobial,19 anti-tubercular,20 anti-convulsant,21 anti-inflam-
matory,22 analgesic,22 anti-anxiety, anti-depressant23 and anti-vir-
al24 agents. For this type of derivatives, a different mechanism of
action is assigned, depending on the type of modification of
1,3,4-thiadiazole ring.25–27 The action connected with the apopto-
tic mechanisms and angiogenesis, which is a crucial step in
the tumorigenesis, seems to be very promising in anticancer
therapy.28,29
In our previous publication we described synthesis and in vitro
antiproliferative activity against some human cancer cell lines of
compounds of 1,3,4-thiadiazole derivatives containing 1,4-benzo-
dioxan.30 The significantly antiproliferative and FAK inhibitory
effect of 1,3,4-thiadiazole derivatives were found.
As you know, amide derivatives were associated with broad
spectrum of biological activities including antitumor properties.31
Herein, in continuation to extend our research on antitumor com-
pounds with FAK structure inhibitory activity, we report in the
present work the synthesis and structure–activity relationships
of a series of 1,3,4-thiadiazol-2-amide derivatives as antitumor
⇑
Corresponding authors. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail addresses: yangxianhui1988@126.com (X.-H. Yang), kaka_bb@163.com
(L. Xiang), 553289134@qq.com (X. Li), changbiyuan7@163.com (T.-T. Zhao),
zhanghui.805@163.com (H. Zhang), wenping_zhou@163.com (W.-P. Zhou),
xmwang@nju.edu.cn (X.-M. Wang), ghbxuzhou@yahoo.com.cn (H.-B. Gong),
zhuhl@nju.edu.cn (H.-L. Zhu).
These two authors equally contributed to this paper.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.040

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X.-H. Yang et al. / Bioorg. Med. Chem. 20 (2012) 2789–2795
agents. Biological evaluation indicated that some of the synthe-
sized compounds were potent inhibitors of FAK.
more potent activities than those with electron-donating substitu-
ents (5j, 5k) in the A-ring. A comparison of the para substituents on
the A-ring demonstrated that an electron-withdrawing group (5d,
5g) have slightly improved antiproliferative activity, whereas Me
(5j) and OMe (5k) group substituent had minimal effects compared
with 5a, which has no substituent. In the case of constant A ring
substituents, compounds with substituted 5-Cl on salicylic acid
moiety showed better antitumor activity than compounds with
substituted 5-Br.
Among these compounds (5o–5w) with benzoic acid substitu-
ents, compounds with para electron-donating substituents ( 5r,
5s) showed more potent activities than those with electron-with-
drawing substituents (5p, 5q) in the A-ring. A comparison of the
para substituents on the A-ring demonstrated that an electron-
donating group ( 5r, 5s) have slightly improved antiproliferative
activity, whereas F (5p) and Cl (5q) substituent had minimal effects
compared with 5o. In the case of constant A ring substituents,
change of substituents on benzoic acid moiety could also affect
the activities of these compounds. Among the compounds 5t–5w,
these compounds (5v, 5w) with para electron-donating substituted
showed stronger anticancer activity and the strength order was
OMe > Me, followed that 5t and 5u led to a noteworthy poor
activity.
2. Results and discussion
2.1. Chemistry
The synthetic route of the 1,3,4-thiadiazol-2-amide derivatives
5a–5y is outlined in Scheme 1. These compounds were synthesized
from 2-amino-1,3,4-thiadiazoles 3 and different substituted car-
boxylic acids 4. Firstly, the different substituted benzoic acid 1
were treated with thiosemicarbazide 2 in presence of phosphoryl
chloride yielded 2-amino-1,3,4-thiadiazoles. Secondly, the cou-
pling reaction between the obtained different substituted 2-ami-
no-1,3,4-thiadiazoles and different substituted carboxylic acids
was performed by using 1-ethyl-3-(3-dimethylaminopropyl) car-
bodiimide hydrochloride (EDCl) and N-hydroxybenzotriazole
(HOBt) in anhydrous methylene dichloride, and refluxed to give
the desired compounds 5a–5y (Table 1). Among these compounds,
5c–5k and 5m are reported for the first time. All of the synthetic
compounds gave satisfactory elementary analytical and spectro-
scopic data. 1H NMR and ESI-MS spectra were consistent with
the assigned structures.
To examine whether the compounds inhibit FAK, we tested
their FAK inhibitory activity against MCF-7 cell line. The results
were summarized in Table 2. Most of the tested compounds dis-
played potent FAK inhibitory. Among them, compounds 5h showed
2.2. Bioactivity
To test the anticancer activities of the synthesized compounds,
we evaluated antiproliferative activities of compounds 5a–5y
against MCF-7 and B16-F10 cells. The results were summarized
in Table 2. As illustrated in Table 2, the active analogs showed a
distinctive potential pattern of selectivity as well as broad-spec-
trum in antitumor activity. Among them, compound 5h displayed
the most potent inhibitory with IC50 of 5.32
lM (the positive con-
trol Staurosporine with an IC50 of 11.32 M for FAK). The results of
l
FAK inhibitory activity of the tested compounds were correspond-
ing to the Structure–activity relationships (SAR) of their antitumor
activities. This demonstrated that the potent antitumor activities of
the synthetic compounds were probably correlated to their FAK
inhibitory activities.
the most potent inhibitory activity (IC50 = 0.45
lM for MCF-7 and
IC50 = 0.31 M for B16-F10), comparable to the positive control
l
Staurosporine (IC50 = 3.07
B16-F10, respectively).
lM for MCF-7 and IC50 = 2.82 lM for
In an effort to study the preliminary mechanism of the
compound with potent inhibitory activity, the Western-blot
experiment was performed to assay the effect of compound 5h.
The Western-blot results were summarized in Figure 1. The result
indicated that compound 5h possessed good antiproliferative activ-
ity is probably correlated to its excellent FAK inhibitory activity.
To gain better understanding on the potency of the studied
compounds and guide further SAR studies, we proceeded to
examine the interaction of compound 5h with FAK crystal struc-
ture (2ETM pdb). The molecular docking was performed by insert-
ing compound 5h into ATP binding site of FAK. All docking runs
The activity of the tested compounds could be correlated to
structure variation and modifications. By investigating the varia-
tion in selectivity of the tested compounds over the two cell lines,
it was revealed that different acids substitutes led to different anti-
tumor activity, and the potency order was salicylic acid > benzoic
acid > nicotinic acid > phenylacetic acid. Regarding these com-
pounds (5a–5k) with salicylic acid substituents, Structure–activity
relationships in these compounds demonstrated that compounds
with para electron-withdrawing substituents ( 5d, 5g) showed
NH2
S
N
COOH
S
N
a
+
H2N
N
H
NH2
R1
R1
3
1
2
O
NH2
N
NH
C
S
R2
S
N
N
b
N
R2
COOH
+
4
R1
R1
3
5
Scheme 1. General synthesis of 1,3,4-thiadiazol-2-amide derivatives (5a–5y). Reagents and conditions: (a) POCl3, reflux, 8 h, 50%NaOH; (b) EDCl, HOBt, dichloromethane,
reflux, 8 h.

X.-H. Yang et al. / Bioorg. Med. Chem. 20 (2012) 2789–2795
2791
Table 1
Structure of compounds 5a–5y
O
NH
C
R2
S
N
N
A
R1
Compound
R1
H
R2
Compound
R1
R2
5a
5n
OCH3
N
HO
HO
Cl
5b
H
5o
H
Br
5c
5d
5e
H
F
5p
5q
5r
F
HO
HO
Cl
Cl
Br
F
CH3
HO
5f
F
5s
5t
OCH3
HO
HO
F
5g
5h
Cl
Cl
H
Cl
Br
Cl
5u
H
HO
Me
OMe
5i
Cl
5v
H
H
HO
HO
HO
5j
CH3
OCH3
5w
5k
5x
5y
Cl
5l
H
Br
N
5m
CH3
N
were applied LigandFit Dock protocol of Discovery Studio 3.1. The
binding modes of compound 5h and FAK were depicted in Figure 2.
All the amino acid residues which had interactions with FAK were
exhibited in Figure 3. In the binding mode, compound 5h is
potently bound to the ATP binding site of FAK via hydrophobic
interactions and binding is stabilized by one hydrogen bond, one
p–cation interaction and one
p–Sigma interaction. The hydrogen
atom of the hydroxyl group on salicylic acid moiety formed one

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X.-H. Yang et al. / Bioorg. Med. Chem. 20 (2012) 2789–2795
Table 2
along with the biological assay data, suggested that compound 5h
was a potential inhibitor of FAK.
Inhibition (IC50) of MCF-7 and B16-F10 cells proliferation and inhibition of FAK by
compounds 5a–5y
Compound
IC50 SD (
lM)
3. Conclusion
MCF-7a
B16-F10a
FAKb
9.36 0.86
In this study, a series of 1,3,4-thiadiazol-2-amide derivatives
have been synthesized and evaluated for their antitumor activi-
ties. These compounds exhibited potent antiproliferative activities
against MCF-7 and B16-F10 cells and FAK inhibitory activities.
Among all of the compounds, 5h showed the most potent inhibi-
tion activity which inhibited the growth of MCF-7 and B16-F10 cell
5a
2.76 0.21
1.12 0.09
1.28 0.12
1.21 0.07
0.75 0.07
0.86 0.06
1.02 0.08
0.45 0.03
0.67 0.04
2.36 0.12
2.17 0.10
8.22 0.45
4.52 0.23
2.99 0.18
5.87 0.26
5.35 0.27
4.85 0.33
3.57 0.18
1.55 0.09
4.85 0.38
4.49 0.25
3.28 0.23
1.83 0.13
6.47 0.41
7.52 0.46
3.07 0.13
2.02 0.17
1.14 0.11
1.28 0.13
1.16 0.08
0.68 0.03
1.02 0.06
1.15 0.10
0.31 0.04
0.56 0.04
1.97 0.11
1.39 0.09
6.18 0.47
3.88 0.19
2.36 0.15
5.15 0.50
4.75 0.42
4.06 0.38
3.52 0.32
1.58 0.11
4.36 0.45
4.65 0.31
2.76 0.26
1.75 0.13
8.23 0.66
9.06 0.62
2.82 0.22
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
5y
6.89 0.85
7.25 0.81
7.02 0.87
6.13 0.72
6.41 0.59
6.79 0.83
5.32 0.69
5.85 0.65
8.54 0.79
7.47 0.74
18.59 1.07
12.37 0.85
9.75 0.91
16.18 1.13
15.49 0.93
12.68 0.87
11.72 0.96
7.71 0.52
13.97 0.94
11.87 0.88
9.84 0.55
7.89 0.48
19.67 1.09
21.52 1.25
11.32 0.85
lines with IC50 values of 0.45 and 0.31
lM, respectively and inhib-
ited the FAK with IC50 of 5.32 M. Molecular docking was further
l
performed to study the inhibitor-FAK protein interactions. After
analysis of the binding model of compound 5h with FAK, it was
found that several interactions with the protein residues in the
ATP binding site might play a crucial role in its FAK inhibitory
activities and antiproliferative activities. Western-blot results also
showed that compound 5h possessed good antiproliferative activ-
ity is probably correlated to its excellent FAK inhibitory activity.
The information of this work might be helpful for the design and
synthesis of FAK inhibitors with stronger activities.
4. Experiments
4.1. Materials and measurements
Staurosporine
a
Inhibition of the growth of tumor cell lines.
Inhibition of FAK.
All chemicals and reagents used in the current study were of
analytical grade. All the 1H NMR spectra were recorded on a Bruker
DPX300 model Spectrometer in DMSO-d6 and chemical shifts were
reported in ppm (d). ESI-MS spectra were recorded on a Mariner
System 5304 Mass spectrometer. Elemental analyses were per-
formed on a CHN-O-Rapid instrument. TLC was performed on the
glassbacked silica gel sheets (Silica Gel 60 GF254) and visualized
in UV light (254 nm). Column chromatography was performed
using silica gel (200–300 mesh) eluting with ethyl acetate and
petroleum ether.
b
4.2. General procedure for synthesis of 2-amino-1,3,4-
thiadiazoles
Figure 1. Compound 5h was examined by Western blotting. Data are representa-
tive of three independent experiments.
hydrogen bond with the carboxylic oxygen of Glu A:506 (bond
length: 0.952 Å; bond angle: 162.5°). The enzyme surface model
was showed in Figure 2b, which revealed that the molecule was
well embedded in the active pocket. This molecular docking result,
A stirring mixture of substituted benzoic acid (10 mmol),
thiosemicarbazide (10 mmol) and POCl3 (10 mL) was heated at
75–80 °C for 6 h, The reaction was monitored by TLC. After cooling
down to room temperature, water was added. The reaction
Figure 2. (a) Compound 5h (colored by atom: carbons: gray; nitrogen: blue; oxygen: red; sulfur: yellow; Chlorine: green) is bond into FAK (entry 2ETM in the Protein Data
Bank). The dotted lines show the hydrogen bond and the yellow line show the –cation interaction and –Sigma interaction. Glu A:506 is the mean of Glu 506 in A chain. (b)
The surface model structure of compound 5h binding model with FAK complex.
p
p

X.-H. Yang et al. / Bioorg. Med. Chem. 20 (2012) 2789–2795
2793
Figure 3. 2D Ligand interaction diagram of compound 5h with FAK using Discovery Studio program with the essential amino acid residues at the binding site are tagged in
circles. The purple circles show the amino acids which participate in hydrogen bonding, electrostatic or polar interactions and the green circles show the amino acids which
participate in the Van der Waals interactions.
mixture was refluxed for 1 h. After cooling, the mixture was basi-
fied to pH 8–9 by the dropwise addition of 50% NaOH solution
under ice bath. The precipitate was filtered and recrystallized from
ethanol to gain compounds 3.
12.26 (s, 1H). ESI-MS: 350.7 (C15H10ClFN3O2S, [M+H]+). Anal. Calcd
for C15H9ClFN3O2S: C, 51.51; H, 2.59; N, 12.01. Found: C, 51.44; H,
2.61; N, 12.07.
4.3.4. 5-Bromo-N-(5-(4-fluorophenyl)-1,3,4-thiadiazol-2-yl)-2-
hydroxybenzamide(5f)
4.3. General procedure for synthesis of target compounds 5a–5y
White solid, yield 79%, mp: 300 °C. 1H NMR (300 MHz, DMSO-d6,
d ppm): 7.01 (d, J = 8.8 Hz, 1H), 7.40 (d, J = 8.7 Hz, 2H), 7.61–7.65
(m, 1H), 8.02–8.07 (m, 3H), 11.64 (s, 1H), 12.35 (s, 1H). ESI-MS:
395.2 (C15H10BrFN3O2S [M+H]+). Anal. Calcd for C15H9BrFN3O2S: C,
45.70; H, 2.30; N, 10.66. Found: C, 45.61; H, 2.32; N, 10.61.
Compounds 5a–5y were synthesized by coupling substituted
2-amino-1,3,4-thi- adiazoles with substituted carboxylic acids,
using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-
chloride (EDCl) and N-hydroxybenzotriazole (HOBt) as condensing
agent. The mixture was refluxed in anhydrous CH2Cl2 for 8–10 h.
The products were extracted with ethyl acetate. The extract was
washed successively with 10% HCl, saturated NaHCO3 and water,
respectively, then dried over anhydrous Na2SO4, filtered and evap-
orated. The residue was purified by column chromatography using
petroleum ether and ethyl acetate (3:1).
4.3.5. N-(5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl)-2-hydroxy-
benzamide(5g)
White solid, yield 66%, mp: 300 °C. 1H NMR (300 MHz, DMSO-d6,
d ppm): 6.99–7.07 (m, 2H), 7.48–7.54 (m, 1H), 7.62 (t, J = 4.3 Hz,
2H), 7.97–8.02 (m, 3H), 11.69 (s, 1H), 12.28 (s, 1H). ESI-MS: 332.8
(C15H11ClN3O2S [M+H]+). Anal. Calcd for C15H10ClN3O2S: C, 54.30;
H, 3.04; N, 12.67. Found: C, 54.21; H, 3.06; N, 12.62.
4.3.1. 5-Bromo-2-hydroxy-N-(5-phenyl-1,3,4-thiadiazol-2-
yl)benzamide(5c)
4.3.6. 5-Chloro-N-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)-2-
hydroxybenzamide(5h)
White solid, yield 81%, mp: 300 °C. 1H NMR (300 MHz, DMSO-
d6, d ppm): 7.01 (d, J = 8.8 Hz, 1H), 7.56 (t, J = 2.7 Hz, 3H), 7.62–
7.65 (m, 1H), 7.97–7.99 (m, 2H), 8.04 (d, J = 2.4 Hz, 1H), 11.70
(s, 1H), 12.32 (s, 1H). ESI-MS: 377.2 (C15H11BrN3O2S, [M+H]+). Anal.
Calcd for C15H10BrN3O2S: C, 47.89; H, 2.68; N, 11.17. Found: C,
47.82; H, 2.69; N, 11.23.
White solid, yield 68%, mp: 300 °C. 1H NMR (300 MHz, DMSO-
d6, d ppm): 7.06 (d, J = 9.0 Hz, 1H), 7.52 (d, J = 6.2 Hz, 1H), 7.63
(d, J = 8.6 Hz, 2H), 7.91 (d, J = 2.1 Hz, 1H), 8.00 (d, J = 8.2 Hz, 2H),
11.70 (s, 1H), 12.31 (s, 1H). ESI-MS: 367.2 (C15H10Cl2N3O2S,
[M+H]+). Anal. Calcd for C15H9Cl2N3O2S: C, 49.19; H, 2.48; N,
11.47. Found: C, 49.24; H, 2.46; N, 11.53.
4.3.2. N-(5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl)-2-hydroxy-
benzamide(5d)
4.3.7. 5-Bromo-N-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)-2-
hydroxybenzamide(5i)
White solid, yield 63%, mp: 300 °C. 1H NMR (300 MHz, DMSO-
d6, d ppm): 6.99–7.08 (m, 2H), 7.37–7.43 (m, 2H), 7.48–7.54
(m, 1H), 7.97–8.07 (m, 3H), 11.74 (s, 1H), 12.27 (s, 1H). ESI-MS:
316.3 (C15H11FN3O2S, [M+H]+). Anal. Calcd for C15H10FN3O2S: C,
57.14; H, 3.20; N, 13.33. Found: C, 57.06; H, 3.31; N, 13.28.
White solid, yield 76%, mp: 300 °C. 1H NMR (300 MHz, DMSO-
d6, d ppm): 6.98 (d, J = 8.8 Hz, 1H), 7.59–7.63 (m, 3H), 7.97–8.02
(m, 3H), 11.65 (s, 1H), 12.34 (s, 1H). ESI-MS: 411.7 (C15H10BrN3O2S
[M+H]+). Anal. Calcd for C15H9BrN3O2S: C, 43.87; H, 2.21; N, 10.23.
Found: C, 43.78; H, 2.23; N, 10.28.
4.3.3. 5-Chloro-N-(5-(4-fluorophenyl)-1,3,4-thiadiazol-2-yl)-2-
hydroxybenzamide(5e)
4.3.8. 2-Hydroxy-N-(5-(p-tolyl)-1,3,4-thiadiazol-2-yl)benzamide
(5j)
White solid, yield 66%, mp: 300 °C. 1H NMR (300 MHz, DMSO-d6,
d ppm): 7.06 (d, J = 8.7 Hz, 1H), 7.40 (t, J = 8.5 Hz, 2H), 7.52
(d, J = 8.5 Hz, 1H), 7.91 (s, 1H), 8.04 (t, J = 6.7 Hz, 2H), 11.73 (s, 1H),
Whitepowders, yield81%, mp:300 °C. 1HNMR(300 MHz, DMSO-
d6, d ppm): 2.39 (s, 3H), 6.99–7.07 (m, 2H), 7.37 (d, J = 7.86 Hz, 2H),

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X.-H. Yang et al. / Bioorg. Med. Chem. 20 (2012) 2789–2795
7.51 (t, J = 7.14 Hz, 1H), 7.87 (d, J = 8.07 Hz, 2H), 7.99 (d, J = 6.60 Hz,
1H), 11.71 (s, 1H), 12.30 (s, 1H). ESI-MS: 312.4 (C16H14N3O2S,
[M+H]+). Anal. Calcd for C16H13N3O2S: C, 61.72; H, 4.21; N, 13.50.
Found: C, 61.76; H, 4.22; N, 13.54.
subsequently electrotransferred onto a polyvinylidene difluoride
membrane (Millipore, Bedford, MA, USA). The membrane was
blocked with 5% nonfat milk for 2 h at room temperature. The
blocked membrane was probed with the indicated primary anti-
bodies overnight at 4 °C, and then incubated with a horse radish
peroxidase (HRP)-coupled secondary antibody. Detection was per-
formed using a LumiGLO chemiluminescent substrate system
(KPL, Guildford, UK).
4.3.9. 2-Hydroxy-N-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)
benzamide(5k)
White powders, yield 70%, mp: 300 °C. 1H NMR (300 MHz,
DMSO-d6, d ppm): 3.84 (s, 3H), 6.98–7.06 (m, 2H), 7.10 (d,
J = 8.04 Hz, 2H), 7.50 (t, J = 7.68 Hz, 1H), 7.91 (d, J = 8.04 Hz, 2H),
7.99 (d, J = 7.86 Hz, 1H), 12.24 (s, 2H). ESI-MS: 328.4 (C16H14N3O3S,
[M+H]+). Anal. Calcd for C16H13N3O3S: C, 58.70; H, 4.00; N, 12.84.
Found: C, 58.63; H, 3.98; N, 12.86.
4.7. Docking simulations
The three-dimensional X-ray structure of tubulin (PDB code:
2ETM) was chosen as the template for the modeling study of com-
pound 5h bound to FAK. The crystal structure was obtained from
the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/
home.do).
The molecular docking procedure was performed by using
LigandFit protocol within Discovery Studio 3.1. For ligand prepara-
tion, the 3D structures of 5h was generated and minimized using
Discovery Studio 3.1. For protein preparation, the hydrogen atoms
were added, and the water and impurities were removed. The
whole FAK was defined as a receptor and the site sphere was
selected based on the ligand binding location of ATP, then the
ATP molecule was removed and 5h was placed during the molecu-
lar docking procedure. Types of interactions of the docked protein
with ligand were analyzed after the end of molecular docking.
4.3.10. N-(5-(p-Tolyl)-1,3,4-thiadiazol-2-yl)nicotinamide(5m)
White powders, yield 85%, mp: 300 °C. 1H NMR (300 MHz,
DMSO-d6, d ppm): 2.39 (s, 3H), 7.37 (d, J = 7.68 Hz, 2H), 7.61
(t, J = 6.12 Hz, 1H), 7.88 (d, J = 7.32 Hz, 2H), 8.46 (d, J = 7.86 Hz,
1H), 8.82 (d, J = 4.11 Hz, 1H), 9.25 (s, 1H), 13.31 (s, 1H). ESI-MS:
297.3 (C15H13N4OS, [M+H]+). Anal. Calcd for C15H12N4OS: C,
60.79; H, 4.08; N, 18.91. Found: C, 60.73; H, 4.10; N, 18.95.
4.4. Antiproliferation assay
The antiproliferative activities of the prepared compounds
against MCF-7 and B16-F10 cells were evaluated as described else-
where with some modifications.32 Target tumor cell lines were
grown to log phase in RPMI 1640 medium supplemented with
10% fetal bovine serum. After diluting to 2 Â 104 cells mLÀ1with
Acknowledgements
the complete medium, 100 lL of the obtained cell suspension
The work was financed by National Natural Science Foundation
of China (No. J1103512).
was added to each well of 96-well culture plates. The subsequent
incubation was permitted at 37 °C, 5% CO2 atmosphere for 24 h
before the cytotoxicity assessments. Tested samples at pre-set con-
centrations were added to six wells with Staurosporine co-assayed
References and notes
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