Synthesis, biological evaluation, and molecular docking studies of novel 1,3,4-oxadiazole derivatives possessing benzotriazole moiety as FAK inhibitors with anticancer activity

Article abstract of DOI:10.1016/j.bmc.2013.04.043

1,3,4-Oxadiazole derivatives have drawn continuing interest over the years because of their varied biological activities. In order to search for novel anticancer agents, we designed and synthesized a series of new 1,3,4-oxadiazole derivatives containing benzotriazole moiety as potential focal adhesion kinase (FAK) inhibitors. All the synthesized compounds were firstly reported. Among the compounds, compound 4 shows the most potent inhibitory activity against MCF-7 and HT29 cell lines with IC₅₀ values of 5.68 μg/ml and 10.21 μg/ml, respectively. Besides, all the compounds were assayed for FAK inhibitory activity using the TRAP-PCR-ELISA assay. The results showed compound 4 exhibited the most potent FAK inhibitory activity with IC₅₀ values of 1.2 ± 0.3 μM. Docking simulation by positioning compound 4 into the FAK structure active site was performed to explore the possible binding mode. Apoptosis which was analyzed by flow cytometry, demonstrated that compound 4 induced apoptosis against MCF-7 cells. Therefore, compound 4 may be a potential anticancer agent against MCF-7 cancer cell.

Full text of DOI:10.1016/j.bmc.2013.04.043

Accepted Manuscript
Synthesis, biological evaluation, and molecular docking studies of novel 1,3,4-
oxadiazole derivatives possessing benzotriazole moiety as FAK inhibitors with
anticancer activity
Shuai Zhang, Yin Luo, Liang-Qiang He, Zhi-Jun Liu, Hai-Liang Zhu
PII:
S0968-0896(13)00369-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.04.043
BMC 10783
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
14 March 2013
7 April 2013
13 April 2013
Please cite this article as: Zhang, S., Luo, Y., He, L-Q., Liu, Z-J., Zhu, H-L., Synthesis, biological evaluation, and
molecular docking studies of novel 1,3,4-oxadiazole derivatives possessing benzotriazole moiety as FAK inhibitors
with anticancer activity, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.
2013.04.043
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Synthesis, biological evaluation, and molecular docking studies of
novel 1,3,4-oxadiazole derivatives possessing benzotriazole moiety as
FAK inhibitors with anticancer activity
Shuai Zhang a, †, Yin Luo a, †, Liang-Qiang He a, Zhi-Jun Liu a, Hai-Liang Zhu a,*
aState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, People’s Republic of China
*Corresponding authors. Tel:+86-25-8359 2572; Fax: +86-25-8359 2672
E-mail address: zhuhl@nju.edu.cn
†These two authors equally contributed to this paper
1

Abstract: 1,3,4-Oxadiazole derivatives have drawn continuing interest over the years
because of their varied biological activities. In order to search for novel anticancer
agents, we designed and synthesized a series of new 1,3,4-oxadiazole derivatives
containing benzotriazole moiety as potential focal adhesion kinase (FAK) inhibitors.
All the synthesized compounds were firstly reported. Among the compounds,
compound 4 shows the most potent inhibitory activity against MCF-7 and HT29 cell
lines with IC50 values of 5.68 μg/ml and 10.21 μg/ml, respectively. Besides, all the
compounds were assayed for FAK inhibitory activity using the TRAP-PCR-ELISA
assay. The results showed compound 4 exhibited the most potent FAK inhibitory
activity with IC50 values of 1.2 0.3 μM. Docking simulation by positioning
compound 4 into the FAK structure active site was performed to explore the possible
binding mode. Apoptosis which was analyzed by flow cytometry, demonstrated that
compound 4 induced apoptosis against MCF-7 cells. Therefore, compound 4 may be a
potential anticancer agent against MCF-7 cancer cell.
Keywords: benzotriazole; 1,3,4-oxadiazole derivatives; FAK; molecular docking;
anticancer activity
1. Introduction
According to the statistics, cancer is the second most common cause of death
after heart disease.1 During the past decades, cancer continued to be a worldwide
killer.2 Despite the mass research was devoted and rapid progress was achieved, there
is a growing demand for new therapies as usual. It is important to identify new agents
and new targets for the treatment of cancer.
As we know, the invasive growth of tumor cells contains complicated progress
steps, involving a variety of biological chemical factors.3,4 Tumor cells must adhere to
the extracellular matrix, and then affect cell adhesion, motility and migration by
promoting the extracellular matrix signal transduction.5-7 Focal adhesion kinase (FAK)
mediated signal transduction system is one of the most important cell signal
transduction pathways.8 FAK is a non-receptor tyrosine kinase that play an important
2

role in cell proliferation, survival, motility, invasion, metastasis, and angiogenesis.9
Enhanced FAK signaling may result in uncontrolled proliferation, survival or
migration of cells, as observed in cancer development and progression process.10
Given the role of FAK in tumorigenesis and metastasis, FAK may be a promising
target for an anticancer drug.
The derivatives of 1,3,4-oxadiazole have high potential for biological activity and
have drawn much attention during the past decades.11-13 The wide array of biological
activities include anti-diabetic,14 anti-tubercular,15 anti-cancer,16 anti-fungal,17
anti-inflammatory,18 anti-bacterial activities.19,20 Also, benzotriazole derivatives have
been found to exhibit potential anti-mycobacterial,21 anti-tubercular,22 antitumor23 and
anti-inflammatory activities.24
Based on the above analysis, we designed and synthesized a series of new
1,3,4-oxadiazole derivatives containing benzotriazole moiety as potential antitumor
agents. Biological evaluation was also carried out for screening potential FAK
inhibitors of the synthesized compounds. We not only evaluated the anticancer
activities and FAK inhibitory activities, but also explored the preliminary mechanism
of the synthesized compounds in cell apoptosis by flow cytometry. Docking
simulations were performed to investigate the inhibitor interaction with FAK and
explore the binding mode of the compound at the active site.
2. Results and discussion
2.1 Chemistry
In this study, nineteen 1,3,4-oxadiazole derivatives containing benzotriazole
moiety were synthesized. All of them were reported for the first time.The synthetic
route of compounds 4-22 was outlined in Scheme 1.
As depicted in Scheme 1, benzotriazole was esterified with ethyl chloroacetate in
acetone in the presence of potassium carbonate to give compound 1. The compound 2
was obtained by compound 1 reacting with 85% hydrazine hydrate in methanol.
Treatment of compound 2 with carbon disulfide in the presence of potassium
hydroxide and anhydrous ethanol under reflux gave compound 3.25The synthesis of
3

compounds 4-22 were accomplished by refluxing compound 3 with halogen
substituted benzyl bromide in the presence of sodium hydroxide in anhydrous
ethanol.26 All of the compounds 4-22 gave satisfactory elementary analyses. 1H NMR
and ESI-MS spectra were consistent with the assigned structures.
2.2 Biological activity
2.2.1 Antiproliferation assay
All the synthesized compounds 4-22 were evaluated for their anticancer activity
against MCF-7 (human breast cancer) and HT29 (human colorectal cancer) cell lines.
Cisplatin is one of the most effective broad-spectrum anticancer drugs. Its
effectiveness seems to be due to the unique properties of cisplatin, which enters cells
via multiple pathways and forms multiple different DNA-platinum adducts while
initiating a cellular self-defense system by activating or silencing a variety of different
genes.27 Hence, we chose cisplatin as reference drug. The results were summarized in
Table 1.
As exhibited in Table 1, it was obvious that compound 4 showed best activity
against MCF-7 cells with the IC50 value of 5.68 μg/ml, much better than reference
drug cisplatin with the IC50 value of 11.20 μg/ml, however, the result of the
compounds against HT29 cells seemed to be less effective. Herein, compound 4 also
exhibited better activity than other compounds against HT29 cell with the IC50 value
of 10.21 μg/ml.
The activity of the tested compounds could be correlated to structure variations.
We investigated the selectivity of the tested compounds against MCF-7 cell and HT29
cell lines, and it was clear that the tested compounds showed better activities against
MCF-7 cell line than HT29 cell line. Among the tested compounds, compound 4
showed the most potent inhibitory activity against MCF-7 cell line with the IC50 value
of 5.68 μg/ml. Structure-activity relationship (SAR) analysis indicated that
compounds with electron-withdrawing group showed stronger activity than those with
electron-donating group. In further study of compounds with electron-withdrawing
group, it was clear that different substituent could lead to different anticancer activity,
4

and the potency order was F (fluorine) > Cl (chlorine) > Br (bromine) > NO2
(nitro-group) > OCH3 (methoxy group) > CH3 (methyl), which showed in Table 1
with compounds 4-9 exhibiting the trend of declining inhibitory activity against
MCF-7 cells. Besides, with regard to the F- substituted compounds, compound 4 has
better inhibitory activity comparing with compounds 10 and 16. Substituents in
different positions also led to different anticancer activities (ortho- > meta- > para-).
2.2.2 FAK inhibitory assay
The FAK inhibitory potency of the 1,3,4-oxadiazole derivatives containing
benzotriazole moiety was examined and the results were summarized in Table 2. Most
of the tested compounds displayed potent FAK inhibitory activities. Among them,
compound 4 displayed the best inhibitory activity with IC50 value of 1.2 0.3 μM
comparable to reference drug cisplatin with IC50 value of 8.6 0.2 μM. The SAR
analysis result of FAK inhibitory activity of the tested compounds was consistent with
that of their anticancer activities. This consistency suggested that the potent anticancer
activities of the synthesized compounds were likely related to their FAK inhibitory
activities.
2.2.3Apoptosis assay
Apoptosis is the normal pathway for clearance of defective or aged cells in the
body. It is an essential mechanism used to eliminate activated MCF-7 cells during the
shut-down process of excessive immune responses and maintain proper immune
homeostasis, while deficient apoptosis of activated MCF-7 cells is associated with a
wide variety of immune disorders. As a representative of these 1,3,4-oxadiazole
derivatives, compound 4 was studied in vitro. We detected the mechanism of
compound 4 inhibitory activity by flow cytometry. As shown in Figure 1, we found
that compound 4 could induce the apoptosis of activated MCF-7 cells in a
dose-dependent manner. To explain the inhibitory activity of compound 4 against
MCF-7 cells, MCF-7 cells were treated with 1, 5, 10, 20 μg/ml of compound 4 for 24h.
The compound 4 increased the percentage of apoptosis by Annexin V-FITC/PI
5

staining in a dose-dependent manner. The percentages of cell apoptosis (14.27%,
28.09%, 46.91%, and 61.29%) are corresponding to the concentration of compound 4.
The results indicated that compound 4 induced apoptosis of MCF-7 cells.
2.3 Binding mode of compound 4 into FAK structure
Molecular docking is an application wherein molecular modeling techniques are
used to predict how protein receptors interact with small molecules. In an effort to
elucidate the possible mechanism by which the title compounds can induce anticancer
activity in MCF-7 and HT29 cells, molecular docking of the potent inhibitor 4 into
active binding site of FAK was performed to simulate a binding mode derived from
FAK structure (2ETM.pdb). All docking runs were applied CDOCKER protocol of
Discovery Studio 3.1.
The docking calculation of the synthesized compounds was showed in Table 3.
The interaction energy between compounds 4-22 and FAK showed the corresponding
results with FAK inhibitory activity. Among the synthesized compounds, compound 4
showed the lowest interaction energy. The 2D and 3D binding modes of compound 4
and FAK were depicted in Figure 2, Figure 3 and Figure 4. In the binding modes ,
compound 4 was nicely bound to the FAK protein catalytic subunit with two
interaction bonds. Visual inspection of the pose of compound 4 into the active site
revealed that compound 4 was nicely bound to FAK with its nitrogen atom of
oxadiazole ring toward the amino hydrogen of CYS502, forming a H-bond interaction.
Also the benzene ring formed a π-cation interaction with LYS454. The molecular
docking results, along with the biological assay data, suggested that compound 4 was
a potential inhibitor of FAK.
3. Conclusion
In this paper, a series of 1,3,4-oxadiazole derivatives containing benzotriazole
moiety were synthesized and evaluated for their anticancer activity. The bioactivity
assay results showed that compound 4 exhibited the most potent inhibitory activity for
FAK with IC50 value of 1.2 0.3 μM, which was comparable to the positive control
6

cisplatin, and good activity against human breast cancer cell MCF-7 with IC50 value
of 5.68 μg/ml, better than the reference drug cisplatin. Molecular docking of the most
potent inhibitor 4 into binding site of FAK was performed, and the results showed
compound 4 could bind well with the FAK active site. Also apoptosis assay results
showed that compound 4 induced apoptosis of stimulated MCF-7 cells. The above
results provided theoretical basis for further structural optimization of
1,3,4-oxadiazole derivatives as FAK inhibitors and showed that compound 4 was a
potential anticancer agent.
4. Experiments
4.1 Materials and measurements
All chemicals and reagents used in current study were of analytical grade. The
reactions were monitored by thin layer chromatography (TLC) on Merck pre-coated
silica GF254 plates. Melting points (uncorrected) were determined on a X-4 MP
apparatus (Tai ke Corp., Beijing, China). 1H NMR spectra were collected on a Bruker
DPX300 spectrometer at room temperature with TMS and solvent signals allotted as
internal standards. Chemical shifts are reported in ppm (δ). Elemental analyses were
performed on a CHN–O-Rapid instrument.
4.2 General procedure for synthesis of compound 1
Benzotriazole (50 mmol) was dissolved in dry acetone (100 ml), then added
anhydrous potassium carbonate (50 mmol). After that, ethyl chloroacetate (50 mmol)
was added then refluxed for 8h. The reaction was monitored by TLC. Afterwards
filtered reaction solution was evaporated under reduced pressure distillation to give
the crude product. The crude product is purified by column chromatography [eluent:
V(petroleum ether) : V(ethyl acetate) = 9 : 1] to give white solid (compound 1).
4.3 General procedure for synthesis of compound 2
To a solution of compound 1(20 mmol) in methanol, 85% hydrazine hydrate (80
mmol) was added and the solution was stirred at 4 ºC for 12h. Then the appearing
7

solid was filtered. The residue was recrystallized from ethanol to obtain compound 2.
4.4 General procedure for synthesis of compound 3
To an ethanol solution of compound 2 (20 mmol) was added potassium hydroxide
(20 mmol) under stirring. Then, carbon disulfide (30 mmol) was added and the
mixture was refluxed for 24h. The reaction solution was evaporated under reduced
pressure distillation. After removal of excess solvent, the residue was poured into
ice-cold water and acidified with dilute hydrochloric acid to obtain the white solid.
The crude product was filtered, washed with ice-cold water and recrystallized from
ethanol to get white crystals (compound 3).
4.5 General procedure for synthesis of the target compounds 4-22
To a stirred solution of compound 3 (1 mmol) and sodium hydroxide (1 mmol) in
acetonitrile (30ml) was added dropwise acetonitrile containing halogen substituted
compounds (1 mmol). The mixture was refluxed for 10-24h and the reaction was
monitored by TLC. Afterwards the solution was cooled to room temperature and the
reaction solution was evaporated under reduced pressure distillation. The residue was
dissolved in ethyl acetate and the organic layer was washed with saturated brine. Then
the organic phase was dried over anhydrous sodium sulfate, filtered, and removed
under reduced pressure distillation. The purification of the residue by recrystallization
from acetonitrile yielded the desired compounds 4-22.
4.5.1
oxadiazole (4)
Clear crystal: Yield 83%; mp 85-86 ºC. 1HNMR (DMSO-D6, 300MHz): 4.45 (s,
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(2-fluorobenzylthio)-1,3,4-
2H), 6.38 (s, 2H), 7.01-7.06 (s, 1H), 7.09-7.15(s, 1H), 7.27-7.35 (m, 2H), 7.44-7.49
(m, 1H), 7.59-7.64 (m, 1H), 7.87 (d, J = 8.40Hz, 1H), 8.12 (d, J = 8.40Hz, 1H).
ESI-MS: 342.36 (C16H13FN5OS, [M+H]+ ). Anal.Calcd for C16H12FN5OS: C, 56.30; H,
3.54; N, 20.52%. Found: C, 56.36; H, 3.54; N, 20.53%.
8

4.5.2
oxadiazole (5)
White powder: Yield 75%; mp 109-110 ºC. 1HNMR (DMSO-D6, 300MHz): 4.51
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(2-chlorobenzylthio)-1,3,4-
(s, 2H), 6.39 (s, 2H), 7.18 (t, J = 7.50Hz, 1H), 7.29 (t, J = 6.03Hz, 1H), 7.38-7.49 (m,
3H), 7.62 (t, J = 7.14Hz, 1H), 7.87 (d, J = 8.25Hz, 1H), 8.11 (d, J = 8.40Hz, 1H).
ESI-MS: 358.82 (C16H13ClN5OS, [M+H]+ ). Anal.Calcd for C16H12ClN5OS: C, 53.71;
H, 3.38; N, 19.57%. Found: C, 53.70; H, 3.34; N, 19.54%.
4.5.3
oxadiazole (6)
Yellow powder: Yield 71%; mp 86-88 ºC. 1HNMR (DMSO-D6, 300MHz): 4.50
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(2-bromobenzylthio)-1,3,4-
(s, 2H), 6.42 (s, 2H), 7.20-7.23 (m, 2H), 7.25-7.44 (m, 2H), 7.46-7.49 (m, 2H), 7.91
(d, J = 9.12Hz, 1H), 8.15 (d, J = 8.93Hz, 1H). ESI-MS: 403.27 (C16H13BrN5OS,
[M+H]+ ). Anal.Calcd for C16H12BrN5OS: C, 47.77; H, 3.01; N, 17.41%. Found: C,
47.78; H, 3.04; N, 17.36%.
4.5.4
oxadiazole (7)
Yellow crystal: Yield 80%; mp 95-97 ºC. 1HNMR (DMSO-D6, 300MHz): 2.30 (s,
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(2-methylbenzylthio)-1,3,4-
3H), 4.45 (s, 2H), 6.40 (s, 2H), 6.95-7.01 (m, 1H), 7.13-7.17 (m, 3H), 7.44-7.49 (m,
1H), 7.59-7.65 (m, 1H), 7.88 (d, J = 8.43Hz, 1H), 8.12 (d, J = 8.43Hz, 1H). ESI-MS:
338.40 (C17H16N5OS, [M+H]+ ). Anal.Calcd for C17H15N5OS: C, 60.52; H, 4.48; N,
20.76%. Found: C,60.51; H, 4.44; N, 20.75%.
4.5.5
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(2-methoxybenzylthio)-
1,3,4-oxadiazole (8)
Yellow powder: Yield 80%; mp 55-56 ºC.1HNMR (DMSO-D6, 300MHz): 3.73 (s,
3H), 4.46 (s, 2H), 6.40 (s, 2H), 6.89-7.01 (m, 1H), 7.19-7.30 (m, 3H), 7.44-7.48 (m,
1H), 7.59-7.66 (m, 1H), 7.87-7.92 (m, 1H), 8.03-8.11 (m, 1H). ESI-MS: 354.40
(C17H16N5O2S, [M+H]+ ). Anal.Calcd for C17H15N5O2S: C, 57.78; H, 4.28; N, 19.82%.
9

Found: C, 57.81; H, 4.33; N, 19.83%.
4.5.6
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(2-nitrobenzylthio)-1,3,4-
oxadiazole (9)
Yellow crystal: Yield 85%; mp 115-116 ºC. 1HNMR (DMSO-D6, 300MHz): 4.72
(s, 2H), 6.37 (s, 2H), 7.43-7.49 (s, 1H), 7.56-7.65 (m, 4H), 7.85-7.88 (m, 1H),
8.04-8.13 (m, 2H). ESI-MS: 369.37 (C16H13N6O3S, [M+H]+ ). Anal.Calcd for
C16H12N6O3S: C, 52.17; H, 3.28; N, 22.81%. Found: C, 52.13; H, 3.24; N, 22.84%.
4.5.7
oxadiazole (10)
White powder: Yield 82%; mp 69-70 ºC. 1HNMR (DMSO-D6, 300MHz): 4.50 (s,
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(3-fluorobenzylthio)-1,3,4-
2H), 6.38 (s, 2H), 7.11-7.16 (s, 1H), 7.29-7.35(s, 1H), 7.48 (d, J= 6.62Hz, 1H),
7.59-7.64 (m, 3H), 7.84 (d, J = 8.40Hz, 1H), 8.12 (d, J = 8.40Hz, 1H). ESI-MS:
342.36 (C16H13FN5OS, [M+H]+ ). Anal.Calcd for C16H12FN5OS: C, 56.30; H, 3.54; N,
20.52%. Found: C, 56.34; H, 3.52; N, 20.57%.
4.5.8
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(3-chlorobenzylthio)-1,3,4-
oxadiazole (11)
1
Yellow powder: Yield 85%; mp 78-80 ºC. HNMR (DMSO-D6, 300MHz): 4.45
(s, 2H), 6.38 (s, 2H), 7.24-7.31 (m, 3H), 7.44-7.49 (m, 2H), 7.59-7.64 (t, J = 6.96Hz,
1H), 7.87 (d, J = 8.40Hz, 1H), 8.11 (d, J = 8.22Hz, 1H). ESI-MS: 358.82
(C16H13ClN5OS, [M+H]+ ). Anal.Calcd for C16H12ClN5OS: C, 53.71; H,3.38; N,
19.57%. Found: C, 53.73; H, 3.41; N, 19.58%.
4.5.9
oxadiazole (12)
Yellow powder: Yield 75%; mp 86-88 ºC. 1HNMR (DMSO-D6, 300MHz): 4.50
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(3-bromobenzylthio)-1,3,4-
(s, 2H), 6.42 (s, 2H), 7.30-7.33 (m, 2H), 7.40-7.44 (m, 2H), 7.46-7.50 (m, 2H), 7.89
(d, J = 8.72Hz, 1H), 8.15 (d, J = 8.98Hz, 1H). ESI-MS: 403.27 (C16H13BrN5OS,
10

[M+H]+ ). Anal.Calcd for C16H12BrN5OS: C, 47.77; H, 3.01; N, 17.41%. Found: C,
47.79; H, 3.01; N, 17.38%.
4.5.10
oxadiazole (13)
Yellow crystal: Yield 85%; mp 95-97 ºC. 1HNMR (DMSO-D6, 300MHz): 2.30 (s,
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(3-methylbenzylthio)-1,3,4-
3H), 4.47 (s, 2H), 6.42 (s, 2H), 7.02-7.04 (m, 1H), 7.18-7.20 (m, 3H), 7.49-7.52 (m,
1H), 7.59-7.65 (m, 1H), 7.90 (d, J = 8.44Hz, 1H), 8.13 (d, J = 8.73Hz, 1H). ESI-MS:
338.40 (C17H16N5OS, [M+H]+ ). Anal.Calcd for C17H15N5OS: C, 60.52; H, 4.48; N,
20.76%. Found: C,60.54; H, 4.49; N, 20.70%.
4.5.11
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(3-methoxybenzylthio)-
1,3,4-oxadiazole (14)
Yellow powder: Yield 70%; mp 55-56 ºC.1HNMR (DMSO-D6, 300MHz): 3.70 (s,
3H), 4.40 (s, 2H), 6.40 (s, 2H), 6.90-7.01 (m, 1H), 7.20-7.33 (m, 3H), 7.44-7.48 (m,
1H), 7.59-7.64 (m, 1H), 7.90-7.92 (m, 1H), 8.03-8.11 (m, 1H). ESI-MS: 354.40
(C17H16N5O2S, [M+H]+ ). Anal.Calcd for C17H15N5O2S: C, 57.78; H, 4.28; N, 19.82%.
Found: C, 57.76; H, 4.33; N, 19.84%.
4.5.12
oxadiazole (15)
Yellow crystal: Yield 75%; mp 103-104 ºC. 1HNMR (DMSO-D6, 300MHz): 4.62
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(3-nitrobenzylthio)-1,3,4-
(s, 2H), 6.37 (s, 2H), 7.49 (t, J= 4.52Hz, 1H), 7.66-7.75 (m, 3H), 7.89-7.94 (m, 2H),
8.14-8.18 (m, 2H). ESI-MS: 369.37 (C16H13N6O3S, [M+H]+ ). Anal.Calcd for
C16H12N6O3S: C, 52.17; H, 3.28; N, 22.81%. Found: C, 52.18; H, 3.27; N, 22.82%.
4.5.13
oxadiazole (16)
White powder: Yield 85%; mp 87-89 ºC. 1HNMR (DMSO-D6, 300MHz): 4.42 (s,
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(4-fluorobenzylthio)-1,3,4-
2H), 6.41 (s, 2H), 7.25-7.34 (m, 4H), 7.44-7.49 (m, 1H), 7.59-7.64 (m, 1H), 7.85 (d, J
= 8.23Hz, 1H), 8.13 (d, J = 8.42Hz, 1H). ESI-MS: 342.36 (C16H13FN5OS, [M+H]+ ).
Anal.Calcd for C16H12FN5OS: C, 56.30; H, 3.54; N, 20.52%. Found: C, 56.28; H, 3.52;
11

N, 20.56%.
4.5.14
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(4-bromobenzylthio)-1,3,4-
oxadiazole (17)
Yellow crystal: Yield 71%; mp 99-104 ºC. 1HNMR (DMSO-D6, 300MHz): 4.41
(s, 2H), 6.38 (s, 2H), 7.26 (d, J = 8.40Hz, 2H), 7.39-7.49 (m, 3H), 7.62 (t, J = 7.11Hz,
1H), 7.86 (d, J = 8.25Hz, 1H), 8.12 (d, J = 8.43Hz, 1H). ESI-MS: 403.27
(C16H13BrN5OS, [M+H]+ ). Anal.Calcd for C16H12BrN5OS: C, 47.77;H, 3.01; N,
17.41%. Found: C, 47.71;H, 3.07; N, 17.42%.
4.5.15
oxadiazole (18)
Yellow crystal: Yield 80%; mp 96-97 ºC. 1HNMR (DMSO-D6, 300MHz): 2.22 (s,
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(4-methylbenzylthio)-1,3,4-
3H), 4.38 (s, 2H), 6.39 (s, 2H), 7.00 (d, J = 7.89Hz, 2H), 7.15 (d, J = 8.07Hz, 2H),
7.44-7.50 (m, 1H), 7.60-7.65 (m, 1H), 7.87 (d, J = 8.40Hz, 1H), 8.12 (d, J = 8.22Hz,
1H). ESI-MS: 338.40 (C17H16N5OS, [M+H]+ ). Anal.Calcd for C17H15N5OS: C, 60.52;
H, 4.48; N, 20.76%. Found: C, 60.55; H, 4.45; N, 20.79%.
4.5.16
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(4-methoxybenzylthio)-
1,3,4-oxadiazole (19)
Yellow powder: Yield 80%; mp 57-58 ºC.1HNMR (DMSO-D6, 300MHz): 3.70 (s,
3H), 4.38 (s, 2H), 6.40 (s, 2H), 6.76 (d, J = 5.13Hz, 2H), 7.19-7.30 (m, 2H), 7.44-7.49
(m, 1H), 7.59-7.64 (m, 1H), 7.87-7.92 (m, 1H), 8.08-8.13 (m, 1H). ESI-MS: 354.40
(C17H16N5O2S, [M+H]+ ). Anal.Calcd for C17H15N5O2S: C, 57.78; H, 4.28; N, 19.82%.
Found: C, 57.80; H, 4.33; N, 19.85%.
4.5.17
oxadiazole (20)
Yellow crystal: Yield 70%; mp 105-107 ºC. 1HNMR (DMSO-D6, 300MHz): 4.57
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(4-nitrobenzylthio)-1,3,4-
(s, 2H), 6.37 (s, 2H), 7.46 (t, J = 4.48Hz, 1H), 7.58-7.69 (m, 3H), 7.84-7.89 (m, 1H),
12

8.07-8.16 (m, 3H). ESI-MS: 369.37 (C16H13N6O3S, [M+H]+ ). Anal.Calcd for
C16H12N6O3S: C, 52.17; H, 3.28; N, 22.81%. Found: C, 52.22; H, 3.24; N, 22.82%.
4.5.18
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(3,4-dichlorobenzylthio)-
1,3,4-oxadiazole (21)
Clear crystal: Yield 81%; mp 94-95 ºC. 1HNMR (DMSO-D6, 300MHz): 4.46 (s,
2H), 6.38 (s, 2H), 7.31-7.33 (m, 1H), 7.40-7.48 (m, 2H), 7.55-7.62 (m, 1H), 7.63-7.69
(m, 1H), 7.85-7.89 (m, 1H), 8.08-8.12 (t, J = 5.4Hz, 1H). ESI-MS: 393.26
(C16H12Cl2N5OS, [M+H]+ ). Anal.Calcd for C16H11Cl2N5OS: C, 48.99; H, 2.83; N,
17.85%. Found: C, 49.02; H, 2.86; N, 17.88%.
4.5.19
2-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-5-(3,4-difluorobenzylthio)-
1,3,4-oxadiazole (22)
White powder: Yield 85%; mp 84-86 ºC. 1HNMR (DMSO-D6, 300MHz): 4.56 (s,
2H), 6.43 (s, 2H), 7.41-7.43 (m, 1H), 7.50-7.55 (m, 2H), 7.62-7.66 (m, 1H), 7.68-7.79
(m, 1H), 7.90-7.95 (m, 1H), 8.12-8.22 (t, J = 6.80Hz, 1H). ESI-MS: 359.35
(C16H12F2N5OS, [M+H]+ ). Anal.Calcd for C16H11F2N5OS: C, 53.48; H, 3.09; N,
19.49%. Found: C, 53.49; H, 3.10; N, 19.46%.
4.6 Antiproliferative assay
After three days of incubation, the antiproliferative activity of the prepared
compounds 4-22 against MCF-7 human breast cancer cells and HT29 human
colorectal cancer cells were evaluated as following protocol. Target tumor cells
(MCF-7 and HT29) were grown to log phase in RPMI 1640 medium supplemented
with 10% fetal bovine serum. After reaching a dilution of 3×104 cells mL-1 with the
medium, 100 μL of the obtained cell suspension was added to each well of 96-well
culture plates. Subsequently, incubation was performed at 37oC in 5% CO2
atmosphere for 24 h before the cytotoxicity assessment. Tested samples at pre-set
concentrations were added to six wells with 5-fluorouracil being employed as a
positive reference. After 48 h exposure period, 25 μL of PBS containing 2.5 mg mL-1
13

of MTT was added to each well. After 4 h, the medium was replaced by 150 μL
DMSO to dissolve the purple formazan crystals produced. The absorbance at 570 nm
of each well was measured on an ELISA plate reader. The data represented the mean
of three independent experiments in triplicate. The IC50 value was defined as the
concentration at which 50% of the cells could survive. The results were summarized
in Table 1.
4.7 FAK inhibitory assay
To evaluate the effect of the compounds on FAK assembly in vitro, varying
o
concentrations were preincubated with 10 μM FAK in glutamate buffer at 30 C and
o
then cooled to 0 oC. After addition of GTP, the mixtures were transferred to 0 C
o
cuvettes in a recording spectrophotometer and warmed up to 30 C and the assembly
of FAK was observed turbid metrically. The IC50 was defined as the compound
concentration that inhibited the extent of assembly by 50% after 20 min
incubation.28,29
4.8 Apotosis assay
MCF-7 cells were treated with various concentrations of compound 4 for 24h and
then stained with both Annexin V-FITC (fluorescein isothiocyanate) and propidium
iodide (PI). Then samples were analyzed by FACSCalibur flow cytometer.
4.9 Docking simulations
Molecular docking of compounds 4 into the three dimensional X-ray structure of
FAK catalytic subunit (PDB code: 2ETM) was carried out using the Discovery Stutio
(version 3.1) as implemented through the graphical user interface DiscoveryStudio
CDOCKER protocol.
The three-dimensional structures of the aforementioned compounds were
constructed using ChemBio 3D Ultra 11.0 software [Chemical Structure Drawing
Standard; Cambridge Soft corporation, USA (2008)], then they were energetically
minimized by using MMFF94 with 5000 iterations and minimum RMS gradient of
14

0.10. The crystal structures of FAK catalytic subunit were retrieved from the RCSB
Protein Data Bank (http://www.rcsb.org/pdb/home/home.-do). All bound water and
ligands were eliminated from the protein and the polar hydrogen was added. The
whole 2ETM was defined as a receptor and the site sphere was selected based on
active centre of 2ETM according to previous report, then compounds 4 were placed
during the molecular docking procedure. Types of interactions of the docked protein
with ligand were analyzed after the end of molecular docking.
Acknowledgements
The work was financed by a grant (No. J1103512) from Natural Science Foundation
of China.
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17

Legends for figures and schemes
Table 1. Antiproliferative activity of the synthesized compounds (4-22)
Table 2. FAK inhibitory activity of the selected compounds (4-22)
Table 3. The docking calculation of the synthesized compounds (4-22)
Figure 1. MCF-7 cells were cultured with anticancer and various concentrations of 4
for 24h. Cells were stained by Annexin VeFITC/PI and apoptosis was analyzed by
flow cytometry. The lower left quadrants showed the vital cells; the upper right
quadrants shows the dead cells, containing the secondary necrotic cells and late
apoptotic cells; the lower right quadrants showed the early apoptotic cells. Inhibition
includes early and late apoptosis.
Figure 2. Ligand interaction diagram of compound 4 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.
Figure 3. The 3D model structure of compound 4 binding mode with FAK:
compound 4 was nicely bound to FAK with its nitrogen atom of oxadiazole ring
toward the amino hydrogen of CYS502, forming H-bond interaction. Also the
benzene ring formed π-cation interaction with LYS454.
Figure 4. The surface model structure of compound 4 binding mode with FAK
Scheme 1. General synthesis of compounds 4-22. Reagents and conditions: (I) ethyl
chloroacetate, acetone, K2CO3; reflux, 8h; (II) NH2NH2·H2O (85%), methanol; 4
,
12h; (III) (i) CS2/KOH, ethanol; reflux 24h; (ii) HCl, PH 5-6; (IV) acetonitrile, NaOH;
reflux 10-24h.
18

Table 1.
IC50(μg/ml)
Compounds
R
MCF-7
5.68
HT29
10.21
15.27
14.30
26.81
25.10
19.35
15.47
15.60
31.20
38.50
36.22
35.10
17.62
33.86
42.30
37.62
34.80
40.24
38.30
15.83
4
5
6
7
8
2-F
2-Cl
2-Br
2-CH3
2-OCH3
2-NO2
3-F
3-Cl
3-Br
3-CH3
3-OCH3
3-NO2
4-F
10.75
11.81
18.89
16.24
15.60
8.25
12.46
16.30
28.92
24.22
20.80
8.70
17.66
30.23
25.82
21.49
45.16
33.75
11.20
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Cisplatin
4-Br
4-CH3
4-OCH3
4-NO2
3,4-2Cl
3,4-2F
Table 2.
Compounds
IC50(μM)
Compounds
IC50(μM)
19

4
5
6
7
8
1.2 0.3
9.5 0.1
9.8 0.2
12.1 1.3
7.1 0.2
7.6 0.5
7.1 0.3
8.3 0.7
14.2 0.4
15.8 1.1
14
15
16
17
18
19
20
21
22
7.5 0.2
7.0 0.9
9.1 0.5
23.7 2.1
33.8 1.4
8.6 0.3
9.2 0.5
28.5 0.9
33.6 0.4
8.6 0.2
9
10
11
12
13
Cisplatin
Table 3.
CDOCKER
INTERATION ENERGY
△Gb(kcal/mol)
CDOCKER
INTERATION ENERGY
Compounds
Compounds
△Gb(kcal/mol)
4
5
6
7
8
-37.7739
-33.5530
-33.0835
-32.3942
-35.7090
-35.2066
-35.5875
-33.7163
-31.8194
-31.6473
14
15
16
17
18
19
20
21
22
-35.4762
-35.4690
-33.7178
-31.2003
-29.4891
-33.8951
-33.4284
-30.4152
-30.4910
9
10
11
12
13
Figure 1.
20

Figure 2.
21

Figure 3.
Figure 4.
Scheme 1.
22

2
23

Synthesis, biological evaluation, and molecular docking studies of
novel 1,3,4-oxadiazole derivatives possessing benzotriazole moiety as
FAK inhibitors with anticancer activity
Shuai Zhang a, †, Yin Luo a, †, Liang-Qiang He a, Zhi-Jun Liu a, Hai-Liang Zhu a,*
aState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, People’s Republic of China
A series of 1,3,4-oxadiazole derivatives have been designed and synthesized, and
their biological activities were also evaluated for FAK inhibitory activity. Compound
4 possessed the most potent enzyme inhibition activities (IC50 = 1.2 0.3 μM for FAK)
and anticancer activities (IC50 = 5.68 μg/ml for MCF-7 and IC50 = 10.21 μg/ml for
HT29). Structure-activity relationship was also analysed to provide more
pharmacophore understanding that could be used to design new agents with more
potent FAK inhibitory activity. Docking simulation was performed to explore the
binding model of compound 4 with FAK.
24