Discovery of a series of 1,3,4-oxadiazole-2(3H)-thione derivatives containing piperazine skeleton as potential FAK inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.03.038

Focal adhesion kinase (FAK) is an important drug target that plays a fundamental role in mediating signal transduction system. We report herein the discovery of a novel class of 1,3,4-oxadiazole-2(3H)-thione derivatives containing piperazine skeleton with

Full text of DOI:10.1016/j.bmc.2017.03.038

Accepted Manuscript
Discovery of a series of 1,3,4-oxadiazole-2(3H)-thione derivatives containing
piperazine skeleton as potential FAK inhibitors
Juan Sun, Shen-Zhen Ren, Xiao-Yuan Lu, Jing-Jing Li, Fa-Qian Shen, Chen
Xu, Hai-Liang Zhu
PII:
S0968-0896(16)31466-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.038
BMC 13636
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
20 December 2016
14 March 2017
Please cite this article as: Sun, J., Ren, S-Z., Lu, X-Y., Li, J-J., Shen, F-Q., Xu, C., Zhu, H-L., Discovery of a series
of 1,3,4-oxadiazole-2(3H)-thione derivatives containing piperazine skeleton as potential FAK inhibitors, Bioorganic
& Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.03.038
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Discovery of a series of 1,3,4-oxadiazole-2(3H)-thione derivatives containing
piperazine skeleton as potential FAK inhibitors
†
†
Juan Suna,b , Shen-Zhen Rena , Xiao-Yuan Lua, Jing-Jing Lia, Fa-Qian Shena, Chen
Xu*a, Hai-Liang Zhu*a,b,
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210093, PR China
b Jiangsu Naique Biological Engineering Technology Company Limited, Zhenjiang
212415, People’s Republic of China
Corresponding author email: zhuhl@nju.edu.cn, xuchn@nju.edu.cn
Abstract: Focal adhesion kinase (FAK) is an important drug target that plays a
fundamental role in mediating signal transduction system. We report herein the
discovery of a novel class of 1,3,4-oxadiazole-2(3H)-thione derivatives containing
piperazine skeleton with improved potency toward FAK. All of the 17 new
synthesized compounds were assayed for the anticancer activities against four cancer
cells, HepG2, Hela, SW116 and BGC823. Because of the combination of
1,4-benzodioxan, 1,3,4-oxadiazole and piperazine ring, most of them exhibited
remarkable antitumor activities. Notably, compound 5m showed the most potent
biological activities (IC50=5.78 µM for HepG2, and IC50=47.15 µM for SW1116), and
its anti-FAK inhibitory activity (IC50=0.78 µM) was also the best. Computational
docking studies also showed that compound 5m has interaction with FAK key
residues in the active site.
Keywords: Benzodioxan; Oxadiazoles; Piperazine; Antitumor activity; FAK
1. Introduction
* Corresponding author. Tel.: +86-25-8968 2572; Fax: 0086+25+8968 2572; E-mail address:
zhuhl@nju.edu.cn, xuchn@nju.edu.cn
† These two authors equally contributed to this paper.
1

Cancer, a major health problem in the world, is the second most common cause of
mortality after heart disease[1]. During the past decades, the progress in medicine was
achieved, but it is still a laborious task to discover new anticancer agents. Therefore,
there is an increasing need to discover new targets and new therapies for the treatment
of cancer[2].
Focal adhesion kinase (FAK) was discovered almost 24 years ago by identification
of an increased phosphorylated protein after v-Src transformation of chicken embryo
cells[3]. It is a 125 kDa protein recruited at an early stage to focal adhesions and is
activated and tyrosine phosphorylated in response to integrin clustering[4-7]. After
years of extensive research, it has now been widely recognized that FAK is a crucial
player in mediating signal transduction system and is involved in multiple cell
biological processes such as cell invasion, metastasis, proliferation and survival[8].
Enhanced FAK signaling may result in uncontrolled proliferation invasion and
metastasis of cells, as observed in cancer development and progression process[8]. A
plethora of studies have provided compelling evidence that FAK inhibitor could act as
chemopreventive agents to induce cancer cells growth inhibition and apoptosis[9-11].
For example, compounds a~d (Figure 1) reported as FAK inhibitors possess a variety
of bioactivities in vitro and cellular studies. These studies indicate that FAK inhibitors
are considered as a promising anti-cancer agents[12].
Figure 1. Structures of previously reported potential FAK inhibitors
2

Recently, it was reported that compounds containing 1,4-benzodioxan template
have received significant attention in chemical, medicinal and pharmaceutical
research[13, 14] as this structural scaffold is widely found in a variety of natural
products, such as Silybin, Purpurenol, 3,4-Dihydroxystyrene dimmers[15-17]
(shown in Figure 2A). After many years research, it could be reported that
1,4-benzodioxan is an important pharmacophore and plays a significant role in
various pharmaceutical applications, some of which are anticancer agents or new
anti-inflammatory compounds[14, 18, 19]. Besides, 1,3,4-oxadiazoles, an important
class of heterocyclic compound, have also attracted significant interest in
pharmaceutical and chemical fields[20, 21]. This particular heterocycle was chosen
because it has favorable properties over 1,2,4-oxadiazoles and other five-membered
heterocycles[22]. In the present study,
a
number of 1,3,4-oxadiazoles
template-containing compounds possess excellent bioavailability and are very good
bioisosteres of amides and esters[23]. Piperazine derivatives exhibit excellent
bioactivities and inhibitory activity on the feeding and growth of various of acarid,
with
low
toxicity
to
human
body,
such
as
1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)quinolone-3-carboxylic acid
derivatives (Figure 2B)[24]. It possess excellent inhibitory activity for Xanthomonas
oryzae and Rhizoctonia solani. Furthermore, epidemiological, clinical, and preclinical
investigations have provided considerable evidence that piperazine derivatives have
been widely used in the treatment of type2 diabetes, articular rheumatism and the
human acute myelocytic leukemia cell line.[24, 25]
In our previous publication, we reported that 1,3,4-oxadiazole derivatives
containing 1,4-benzodioxan are considered as a potential anticancer agents targeted
FAK. For example, compounds e and f (Figure 2C), synthesized by our group in 2011,
exhibited remarkable anticancer activity (IC50=1.27 µM and 10.28 µM against cancer
cells, respectively) and FAK inhibitory activity[14, 26]. Compared with the
conventional drugs, the piperazine ring contained a better balance of nitrogen. The
introduction of the piperazine ring could effectively improve the physicochemical
properties and pharmacodynamics properties[24]. Therefore, we describe the
3

synthesis
and
structure-activity
relationships
of
a
series
of
1,3,4-oxadiazole-2(3H)-thione-substituted 1,4-benzodioxan containing piperazine
derivatives as antitumor agents[27].
Figure 2. (A) Some natural products contained 1,4-benzodioxan, (B) The structure of
1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)quinolone-3-carboxylic acid derivatives, (C)
Some compounds synthesized by our group
Subsequently, in order to validate whether these designed compounds can target
FAK, the molecular docking was performed and the results have been plotted as a
line-scatter graph and presented in Figure 3. As showed in Figure 3, the obtained
results were plotted as a line-scatter graph and it exhibited the corresponding
CDOCKER_INTERACTION_ENERGY of the molecular docking studies. The
estimated interaction energies of these compounds were ranging -48.8565 to -35.2102
kcal/mol, and compound 5m, especially, had a best free energy of -48.8565 kcal/mol.
Compared to the control drugs, all the synthesized compounds possessed lower
interaction energy, suggesting that they are likely to exhibit more potent inhibitory
4

activity against FAK.Therefore, these preliminary analysis served as a stimulant to
synthesize these potential compounds.
60
48.8565
50
40
30
20
10
0
Compounds
Figure 3. The line-scatter graph about CDOCKER_INTERACTION_ENERGY (kcal/mol) of
compounds 5a~5q for FAK. In the binding mode, compound 5m had a best estimated binding free
energy of -48.8565 kcal/mol, 5-Fluorouracil binds to FAK with binding free energy of -35.2102
kcal/mol.
2. Results and discussion
2.1 Chemistry
In this study, seventeen 1,3,4-oxadiazole-2(3H)-thione derivatives containing
piperazine skeleton were synthesized to screen for their antitumor activity, and
they were synthesized for the first time. The structures of compounds 5a~5q were
showed in Table 1 and the synthetic route was showed in Scheme 1a.
5

Table 1. The structures of compounds 5a~5q.
Compounds
5a:
R
Compounds
5g:
R
Compounds
5m:
R
5b:
5h:
5n:
5c:
5d:
5i:
5j:
5o:
5p:
5e:
5f:
5k:
5l:
5q:
As depicted in Scheme 1a, the target compounds 5a~5q were synthesized from
5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazole-2-thiol (4) and various
6

phenylpiperazine. Compound 4 was a significant intermediate and prepared in four
steps. Firstly, esterification of 3,4-dihydroxybenzoic acid with methanol and
concentrated sulfuric acid afforded methyl 3,4-dihydroxybenzoate (1). Secondly,
treatment of compound 1 with dibromoethane in dry acetone gave methyl
2,3-dihydrobenzo[b][1,4]dioxine-6-carboxylate (2). Then, 2,3-dihydrobenzo[b][1,4]-
dioxine-6-carbohydrazide (3) was obtained by reaction of the ester 2 with hydrazine
hydrate (85%) in ethanol. Finally, treatment of the hydrazide 3 with carbon disulfide
in the presence of potassium hydroxide and ethanol under reflux gave the key
intermediate 4. The synthesis of the target compounds 5a~5q were accomplished by
the reaction of compound 4 with various phenylpiperazine in the presence of
formaldehyde in ethanol.
Scheme 1. Synthesis of compounds 5a~5q a
a
Reagents and conditions: (a) methanol, concentrated sulfuric acid, 90°C, overnight; (b)
dibromoethane, K2CO3, dry acetone, 70°C, overnight; (c) hydrazine hydrate, reflux, 2 d; (d)
carbon disulfide, KOH, EtOH, reflux, overnight; (e) formaldehyde, various phenylpiperazine,
EtOH, rt, 5 h
All of the synthetic compounds gave satisfactory analytical and spectroscopic data,
which were in full accordance with their depicted structure.
2.2 Biological activity
2.2.1 Antiproliferation assay.
To test the anticancer activity of the synthesized compounds, we evaluated the
antiproliferative activities of compounds 5a~5q against four cancer cell lines, which
7

were HepG2（human liver cancer cell）, Hela（human cervical cancer cell）, SW1116
（human colorectal carcinoma cell）, BGC823（human gastric cancer）, with the
positive contrast drugs 5-Fluorouracil. In this assay, the IC50 of those compounds
against these cancer cell were summarized up in Table 2.
Table 2. In vitro antiproliferation activities (IC50, µM) of compounds 5a~5q against HepG2, Hela,
SW1116 and BGC823.
IC50 (µM)
Compounds
HepG2a
>50
Helaa
47.18
>50
SW1116a
>50
BGC823a
>50
5a
>50
24.18
>50
18.94
>50
5b
>50
>50
5c
28.38
29.46
>50
>50
>50
28.27
>50
5d
31.65
>50
>50
5e
>50
>50
5f
9.12
>50
21.64
16.89
>50
>50
5g
15.47
11.44
11.35
>50
>50
19.46
>50
5h
37.38
25.08
>50
5i
5j
9.49
25.90
>50
>50
5k
>50
37.28
>50
37.82
47.15
37.12
>50
>50
5l
5.78
>50
5m
>50
>50
22.58
>50
5n
21.35
23.78
17.98
13.31
17.98
>50
5o
33.39
>50
>50
5p
5q
27.08
9.02
28.69
17.37
18.52
5-Fluorouracil
a Cancer cells kindly supplied by State Key Laboratory of Pharmaceutical Biotechnology, Nanjing
University. Errors were in the range of 5–10% of the reported values, from three different assays.
As illustrated in Table 2, the results revealed that most of the target compounds
possess significant anticancer activities with IC50 values ranging from 5.78 to 50 µM.
With regard to selectivity against individual cell lines, a number of 1,3,4-oxadiazole
derivatives possessing 1,4-benzodioxan moiety showed remarkable effectiveness
8

against HEPG2. Among them, compound 5m displayed the most potent
antiproliferative activity (IC50=5.78 µM for HepG2, and IC50=47.15 µM for SW1116),
compared to the positive contrast drugs 5-Fluorouracil (IC50=13.31 µM for HepG2,
IC50=9.02 µM for Hela, IC50=18.52 µM for SW1116 and IC50=17.37 µM for BGC823).
However, compounds 5a, 5f and 5k were proved to be ineffective against those cell
lines.
The subsequent structure-activity relation (SAR) studies were performed by
modification of the parent compound to determine how the substituents affect the
inhibitory activities. As showed in Table 2, the biological results demonstrated the
bioactivity of compounds was greatly affected by the number and position of
substituent group on benzene ring. A comparison of the substitution on benzene ring
were demonstrated as follows: when the compounds were ortho-position-halogen
substituted derivatives, the potency order was Cl > F (e.g., 5i, 5l); For compounds 5a,
5f, 5i, the potency order was found as ortho > meta > para in -Cl substituted benzene
ring while, the activity gradient was found as ortho > para in compounds 5k, 5l,
containing -F substituted benzene ring. Among the compounds (5b, 5h and 5o) with
electron-donating substituted, the potent inhibitory activity order was ortho > meta >
para. With regard to the methyl-substituted compounds, di-substituted compound 5d
has lower activity comparing with mono-substituted compound 5g.
2.2.2 FAK inhibitory assay
To examine whether the compounds inhibit FAK, we selected the top 10
compounds which had better antiproliferative activity to test their FAK inhibitory. The
results were summarized in Table 3. Most of the tested compounds displayed potent
FAK inhibitory activity, and among them compound 5m displayed the most efficient
inhibitory activity with IC50 of 0.78 µM, The results of FAK inhibitory activity of the
tested compounds were in agreement to structure relationships (SAR) of their
antitumor activities. It was demonstrated that the potent antitumor activities of the
synthetic compounds were probably correlated to their FAK inhibitory activities.
9

Table 3. FAK inhibitory activity of compounds 5a~5q.
IC50 (µM)
2.45
Compounds
5b
5e
5g
5h
5i
2.76
1.12
1.46
1.16
5j
2.45
5m
5o
5p
5q
0.78
2.45
3.12
1.97
2.2.3 Binding model of compound 5m into FAK
In an effect to understand the potency of studied compound and guide further
structure-activity relationship studies, molecular docking study was performed to fit
these compounds into ATP binding site of FAK. The results were exhibited in Figure 4.
Docking algorithm utilized: CDOCKER algorithm; Definition of binding site:
-0.92776, 10.8549, 7.55906; Radius: 9; Scoring function: CDOCKER interaction
energy; Rigid receptor: PDB code 2ETM; Flexible ligand docking: YES; cluster
analysis of docking poses: ten optimal poses were retained.
Figure 4. Molecular docking modeling of compound 5m with FAK: (A) 2D Ligand interaction
diagram of compound 5m and PF-573288 with FAK. (B) 3D model of the interaction between
compound 5m and the FAK binding site.
10

a.
b.
Figure 4 showed that the binding model of compound 5m and PF-573288
interacting with FAK protein and the amino acids located in the binding pocket of
protein, such as ASP564 and GLU506. In the binding model, compound 5m was
nicely bound to the ATP binding of FAK with its oxygen of 1,4-benzodioxan group
forms one hydrogen bond with the amino hydrogen atom of ASP564 (angle O…H-N
156.881°, distance 2.02793Å), and the hydrogen atom of the salt of piperazine forms
another hydrogen bond with the oxygen atom of GLU506 (angle N-H…O 157.696°,
distance 1.93673Å). Insight into this picture, we can see that amino acid residues
ASP564 and GLU506 located in the binding pocket also seemed very important for
11

the active conformation of compound PF-573288. According to the above, these
docking results along with the biological assay data suggested that the
1,4-benzodioxan group and piperazine group are significant for these compounds to
bind with FAK and compound 5m might be a potential inhibitor of FAK.
3. Conclusion
In this study, a series of novel 1,3,4-oxadiazole-2(3H)-thione derivatives containing
phenylpiperazine sekelton were designed and synthesized. The biological activities of
these compounds against four different cancer cells (HepG2, Hela, SW1116,
BGC823). The results showed that compound 5m possessed excellent antitumor
activity compared with the 5-Fluorouracil which was widely used in treatment of
cancer in clinical. According to the results of FAK inhibitory assay, we could find that
compound 5m exhibited the most potent FAK inhibitory activity with IC50 of 0.78 µM.
In order to study the structure-activity relationships observed at the FAK, molecular
docking of compound 5m into the binding site of FAK was performed on the binding
model based on the FAK structure. This study showed that compound 5m was nicely
bound into active site of FAK. Consider above analysis, it could concluded that
compound 5m might be a potential inhibitor of FAK.
4. Experimental section
4.1 Methods of synthesis
All chemicals and reagents used in the current study were analytical grade. Thin
layer chromatography (TLC) was performed on glass-backed silica gel sheets (Silica
Gel 60 GF254) and visualized under UV light (254 nm).
All the 1H NMR spectra were recorded on a Bruker DPX 400 model Spectrometer
in CDCl3 and chemical shifts (δ) were reported as parts per million (ppm). Elemental
analyses were performed using a CHN-O-Rapid instrument and were within 0.4% of
the theoretical values. ESI-MS spectra were recorded by Mariner System 5304 Mass
spectrometer. Melting points (uncorrected) were determined on a WRS-1B apparatus
(Jingke Corp., Shanghai, China).
4.1.1 General procedure for the preparation of methyl 3,4-dihydroxybenzoate (1)
12

To a stirred solution of 3,4-dihydroxybenzoic acid (1 mmol) in methanol (30 mL)
was added thionyl chloride (1 mL) dropwise over 1 h at 0 ℃. The mixture was further
stirred overnight at 50 ℃. The solution was cooled to room temperature, and diluted
with water (25 mL). Excess methanol was distilled out and the pH was adjusted to 6
with saturation sodium bicarbonate. Then, the mixture was extracted with ethyl
acetate and washed with brine. After drying the organic layer with anhydrous sodium
sulfate and evaporating the solvent under reduced pressure a solid appeared. The
crude product was recrystallized from ethanol to give compound 1. The completion of
the reaction was monitored on TLC by using and ethyl acetate petroleum ether (1:1)
and observed in UV light.
4.1.2 General procedure for the preparation of methyl 2,3-dihydrobenzo[b] [1,4]
dioxine - 6 -carboxylate (2)
Compound 1 (1 mmol) and potassium carbonate (0.28 g, 2 mmol) were dissolved in
dry acetone (10 mL) and stirred for 15 min at room temperature. Afterward, the
mixture was slowly heated while the 1,2-dibromoethane (1 mmol) was dropwise
added and then refluxed for 24 h. The reaction mixture was diluted with water and
extracted with ethyl acetate. Combined organic layer and dried with anhydrous
sodium sulfate. The solvent was evaporated under reduced pressure and the crude
product was purified by silica gel column chromatography (VDCM/VEtOAc=4:1) to give
compound 2.
4.1.3
General
procedure
for
the
preparation
of
2,3-dihydrobenzo[b][1,4]dioxine-6-carbohydrazide (3)
To a solution of compound 2 (1 mmol) in ethanol (15 mL) was added hydrazine
hydrate (10 mmol), and the mixture was heated at reflux for 2 days. Excessive ethanol
was distilled out and the contents were allowed to cool. The crystals formed, collected
by filtration and washed several times with ethanol to obtain compound 3. The
completion of the reaction was monitored on TLC by using methanol and ethyl
acetate (1:10) and observed in UV light.
4.1.4
General
procedure
for
the
preparation
of
5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazole-2-thiol (4)
13

To a solution of compound 3 (1 mmol) in ethanol (5 mL) were added carbon
disulfide (2 mmol) and Et3N (1.2 mmol), and the mixture was heated at reflux
overnight. The reaction mixture was diluted with ethyl acetate, and the organic layer
was washed with 0.1 M hydrochloric acid and brine and dried over anhydrous sodium
sulfate. The solvent was evaporated under reduced pressure, and compound 4 was
obtained by recrystallized from PE / EtOAc.
4.1.5 General procedure for the preparation of compounds 5a~5q
Formalin (2 mmol, 150 µL) was added to a stirred solution of compound 4 (1 mmol)
in dry ethanol (15 mL), and an ethanol solution (5 mL) of the appropriate
phenylpiperazine (1 mmol) was added portionwise to the reaction mixture. The
mixture was stirred at room temperature for 5 h. The precipitate was collected by
filtration, washed with cold ethanol, dried, and recrystallized from Petroleum
ether/ethyl acetate to give the target compounds 5a~5q.
4.1.5.1
3-(4-(3-Chlorophenyl)piperazin-1-yl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,
4-oxadiazole-2(3H)-thione (5a)
1
White solid, m.p. 151.0~151.1 °C; H NMR (400 MHz, CDCl3) δ: 7.46~7.42 (m,
2H), 7.17~7.13 (m, 1H), 6.97~6.95 (m, 1H), 6.86~6.75 (m, 3H), 5.12 (s, 2H),
4.34~4.30 (m, 4H), 3.21~3.19 (m, 4H), 3.02~2.99 (m, 4H). 13C NMR (400 MHz,
CDCl3) δ: 176.59, 161.02, 152.29, 150.37, 144.32, 135.10, 129.67, 123.97, 123.11,
116.78, 114.18, 112.31, 111.10, 109.22, 71.09, 62.33, 60.28, 56.23, 43.18, 49.96,
47.33. MS (ESI): 445 (C21H22ClN4O3S, [M+H]+). Anal. Calcd for C21H21ClN4O3S: C,
56.69; H, 4.76; N, 12.59%; Found: C, 56.71; H, 4.59; N, 12.72.
4.1.5.2
5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-((4-(4-methoxyphenyl)piperazin-1-yl)
methyl)-1,3,4-oxadiazole-2(3H)-thione (5b)
1
White solid, m.p. 147.2~148.1 °C; H NMR (400 MHz, CDCl3) δ: 7.50~7.47 (m,
2H), 7.01~6.99 (d, J = 8.4 Hz, 1H), 6.94~6.92 (d, J = 9.2 Hz, 2H), 6.89~6.86 (d, J =
9.2 Hz, 2H), 5.17 (s, 2H), 4.39~4.35 (m, 4H), 3.83~3.81 (m, 3H), 3.13~3.08 (m, 8H).
13C NMR (400 MHz, CDCl3) δ: 178.23, 160.97, 160.02, 151.79, 150.04, 148.12,
14

130.23, 126.25, 113.87, 112.74, 111.05, 109.31, 108.90, 103.22, 67.93, 62.04, 60.22,
56.29, 55.03, 53.19, 48.92, 47.38. MS (ESI): 441 (C22H25N4O4S, [M+H]+). Anal.
Calcd for C22H24N4O4S: C, 59.98; H, 5.49; N, 12.72%; Found: C, 60.09; H, 5.33; N,
12.90.
4.1.5.3
5-(2,3 -Dihydrobenzo[b] [1,4]dioxin- 6 -yl)- 3 -(4-phenylpiperazin-1-yl)-
1,3,4-oxadiazole-2 (3H) -thione (5c)
1
White solid, m.p. 165.5~165.7 °C; H NMR (400 MHz, CDCl3) δ: 7.50~7.47 (m,
2H), 7.32~7.31 (d, J = 2.0 Hz, 1H), 7.28 (s, 1H), 7.01~6.89 (m, 4H), 5.16 (s, 2H),
4.35~4.34 (m, 4H), 3.26~3.23 (m, 4H), 3.08~3.05 (m, 4H). 13C NMR (400 MHz,
CDCl3) δ: 177.32, 161.58, 152.18, 149.73, 148.88, 130.32, 128.22, 124.23, 122.10,
117.35, 115.28, 114.93, 110.51, 109.80, 70.22, 62.40, 60.21, 55.73, 53.22, 49.91,
47.03. MS (ESI): 411 (C21H23N4O3S, [M+H]+). Anal. Calcd for C21H22N4O3S: C,
61.44; H, 5.40; N, 13.65%; Found: C, 61.55; H, 5.23; N, 13.72.
4.1.5.4
5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-(4 -(2,4 -dimethylphenyl)piperazin
-1-yl)-1,3,4-oxadiazole-2(3H)-thione (5d)
1
White solid, m.p. 119.0~119.9 °C; H NMR (400 MHz, CDCl3) δ: 7.52~7.49 (m,
2H), 7.02~6.95 (m, 4H), 5.16 (s, 2H), 4.38~4.34 (m, 4H), 3.05~3.03 (m, 4H),
2.95~2.93 (m, 4H), 2.30 (s, 3H), 2.26 (s, 3H). 13C NMR (400 MHz, CDCl3) δ: 179.33,
161.32, 152.38, 150.29, 149.05, 138.10, 126.35, 124.93, 121.73, 118.20, 116.31,
114.29, 110.47, 107.05, 71.05, 63.21, 61.28, 55.36, 53.32, 50.01, 47.79, 20.05, 14.93.
S (ESI): 439 (C23H27N4O3S, [M+H]+). Anal. Calcd for C23H26N4O3S: C, 62.99; H,
5.98; N, 12.78%; Found: C, 63.12; H, 5.79; N, 12.85.
4.1.5.5
5-(
2,3
-Dihydrobenzo[b][1,4]dioxin-6-yl)-
3
-((
4
-(4-nitrophenyl)piperazin-1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione (5e)
1
White solid, m.p. 202.6~202.7 °C; H NMR (400 MHz, CDCl3) δ: 8.16~8.14 (d, J
= 9.2 Hz, 2H), 7.48~7.45 (m, 2H), 7.01~6.99 (d, J = 8.0 Hz, 1H), 6.86~6.83 (d, J =
9.6 Hz, 2H), 5.17 (s, 2H), 4.38~4.35 (m, 4H), 3.51~3.49 (m, 4H), 3.06~3.04 (m, 4H).
15

13C NMR (400 MHz, CDCl3) δ: 175.93, 163.29, 152.18, 150.89, 148.88, 139.39,
126.37, 125.95, 123.19, 116.31, 114.05, 112.93, 111.51, 109.27, 67.83, 63.02, 61.19,
55.46, 53.32, 50.72, 47.58. MS (ESI): 456 (C21H22N5O5S, [M+H]+). Anal. Calcd for
C21H21N5O5S: C, 55.37; H, 4.65; N, 15.38%; Found: C, 55.19; H, 4.82; N, 15.22.
4.1.5.6
3-((4-(4-Chlorophenyl)piperazin-1-yl)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)-1,3,4-oxadiazole-2(3H)-thione (5f)
1
White solid, m.p. 149.2~150.7 °C; H NMR (400 MHz, CDCl3) δ: 7.52~7.49 (m,
2H), 7.38~7.35 (m, 4H), 7.03~7.01 (m, 1H), 5.09 (s, 2H), 4.40~4.36 (m, 4H),
2.92~2.90 (m, 4H), 2.44 (s, 4H). 13C NMR (400 MHz, CDCl3) δ: 177.53, 163.25,
152.04, 149.88, 147.39, 129.83, 126.92, 126.01, 122.39, 116.59, 113.20, 111.92,
110.41, 109.28, 70.25, 63.12, 60.27, 55.37, 53.32, 49.47, 47.38. MS (ESI): 445
(C21H22ClN4O3S, [M+H]+). Anal. Calcd for C21H21ClN4O3S: C, 56.69; H, 4.76; N,
12.59%; Found: 56.80; H, 4.66; N, 12.69.
4.1.5.7
3-((4-Benzhydrylpiperazin-1-yl)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-
1,3,4-oxadiazole-2(3H)-thione (5g)
White solid, m.p. 160.4~161.0 °C; 1H NMR (400 MHz, CDCl3) δ: 7.52~7.49 (d, J =
10.0 Hz, 3H), 7.47 (s, 1H), 7.43~7.41 (d, J = 7.6 Hz, 4H), 7.27 (s, 1H), 7.22~7.20 (m,
2H), 7.04~7.00 (m, 2H), 5.61~5.59 (m, 1H), 5.09 (s, 2H), 4.37~4.36 (m, 4H), 3.26 (s,
13
1H), 2.92 (s, 3H), 2.75 (s, 1H), 2.47 (s, 3H). C NMR (400 MHz, CDCl3) δ: 178.31,
162.18, 151.18, 150.29, 147.72, 132.51, 127.98, 125.31, 125.16, 122.93, 119.56,
115.51, 111.50, 108.25, 69.13, 62.69, 60.33, 55.56, 53.32, 49.94, 48.19, 17.15. MS
(ESI): 501 (C28H29N4O3S, [M+H]+). Anal. Calcd for C28H28N4O3S: C, 67.18; H, 5.64;
N, 11.19%; Found: C, 67.35; H, 5.52; N, 11.03.
4.1.5.8
5-(2,
3
-Dihydrobenzo[b][1,4]dioxin-
6
-yl)-
3
-
((4-(2
-methoxyphenyl)piperazin-1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione (5h)
1
White solid, m.p. 181.0~181.7 °C; H NMR (400 MHz, CDCl3) δ: 7.49~7.47 (m,
2H), 7.07~6.94 (m, 4H), 6.90~6.88 (d, J = 8.0 Hz, 1H), 5.18 (s, 2H), 4.39~4.35 (m,
16

4H), 3.88 (s, 3H), 3.12 (s, 8H). 13C NMR (400 MHz, CDCl3) δ: 178.56, 163.37,
152.15, 150.31, 148.02, 136.01, 128.19, 124.34, 121.53, 118.41, 114.35, 112.56,
110.47, 107.15, 69.76, 63.05, 60.81, 56.25, 55.19, 53.31, 49.63, 47.21. MS (ESI): 441
(C22H25N4O4S, [M+H]+). Anal. Calcd for C22H24N4O4S: C, 59.98; H, 5.49; N, 12.72%;
Found: C, 60.11; H, 5.38; N, 12.89.
4.1.5.9
3-((4-(2-Chlorophenyl)piperazin-1-yl)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)-1,3,4-oxadiazole-2(3H)-thione (5i)
White solid, m.p. 179.1~179.6 °C; 1H NMR (400 MHz, CDCl3) δ: 7.52~7.49 (m,
2H), 7.40~7.37 (m, 1H), 7.39~7.24 (m, 1H), 7.10~7.08 (m, 1H), 7.04~7.00 (m, 2H),
13
5.17 (s, 2H), 4.39~4.35 (m, 4H), 3.12~3.11 (d, J = 4.4 Hz, 8H). C NMR (400 MHz,
CDCl3) δ: 178.32, 164.01, 152.31, 150.83, 147.15, 128.32, 127.71, 126.33, 125.36,
124.83, 122.14, 114.38, 111.05, 109.25, 68.78, 63.03, 62.18, 55.37, 53.41, 49.39,
46.97. MS (ESI): 445 (C21H22ClN4O3S, [M+H]+). Anal. Calcd for C21H21ClN4O3S: C,
56.69; H, 4.76; N, 12.59%; Found: C, 56.52; H, 4.51; N, 12.73.
4.1.5.10
5-(2 , 3 – Dihydrobenzo [b] [1,4]dioxin- 6 -yl)- 3 -((4 -(pyridin- 2
-yl)piperazin-1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione (5j)
White solid, m.p. 171.6~172.4 °C; 1H NMR (400 MHz, CDCl3) δ: 8.22~8.20 (m,
1H), 7.53~7.45 (m, 3H), 7.00~6.98 (d, J = 8.4 Hz, 1H), 6.70~6.65 (m, 2H), 5.17 (s,
2H), 4.38~4.34 (m, 4H), 3.64 (s, 4H), 3.03~3.00 (m, 4H). 13C NMR (400 MHz,
CDCl3) δ: 176.23, 163.07, 158.22, 151.17, 148.72, 145.33, 136.28, 125.23, 117.85,
114.92, 110.81, 108.21, 107.33, 68.35, 62.31, 60.18, 55.39, 53.28, 46.28, 44.39. MS
(ESI): 412 (C20H22N5O3S, [M+H]+). Anal. Calcd for C20H21N5O3S: C, 58.38; H, 5.14;
N, 17.02%; Found: C, 58.51; H, 5.02; N, 17.23.
4.1.5.11
5-(2,3 -Dihydrobenzo[b] [1,4]dioxin-6-yl)- 3 -(( 4 -( 4 –fluorophenyl )
piperazin-1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione (5k)
White solid, m.p. 152.9~153.1 °C; 1H NMR (400 MHz, CDCl3) δ: 7.50~7.47 (m,
2H), 7.03~6.98 (m, 3H), 6.92~6.89 (m, 2H), 5.16 (s, 2H), 4.39~4.34 (m, 4H),
17

3.18~3.15 (m, 4H), 3.08~3.06 (m, 4H). 13C NMR (400 MHz, CDCl3) δ: 177.39,
161.20, 155.81, 151.29, 149.33, 146.39, 126.72, 114.68, 114.21, 113.38, 113.02,
112.38, 111.93, 111.41, 108.37, 68.92, 60.39, 59.93, 55.32, 53.05, 48.73. MS (ESI):
429 (C21H22FN4O3S, [M+H]+). Anal. Calcd for C21H21FN4O3S: C, 58.87; H, 4.94; N,
13.08%; Found: C, 58.69; H, 5.12; N, 13.16.
4.1.5.12
5-(2
,
3
-
Dihydrobenzo[b] [1,4]dioxin-
6
-yl)-
3
-((
4
-
(
2
-fluorophenyl)piperazin-1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione (5l)
White solid, m.p. 147.8~148.9 °C; 1H NMR (400 MHz, CDCl3) δ: 7.50~7.48 (m,
2H), 7.10~7.08 (d, J = 8.0 Hz, 2H), 7.02~6.99 (m, 3H), 5.17 (s, 2H), 4.39~4.36 (m,
4H), 3.16~3.15 (d, J = 9.2 Hz, 4H), 3.11~3.09 (m, 4H). 13C NMR (400 MHz, CDCl3)
δ: 178.33, 163.08, 158.27, 152.09, 150.88, 127.33, 125.83, 125.37, 125.16, 122.32,
114.83, 114.51, 112.49, 109.38, 69.72, 62.29, 55.92, 52.93, 51.29, 50.28, 48.97. MS
(ESI): 429 (C21H22FN4O3S, [M+H]+). Anal. Calcd for C21H21FN4O3S: C, 58.87; H,
4.94; N, 13.08%; Found: C, 58.95; H, 5.08; N, 12.87.
4.1.5.13 5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-((4-(3 (trifluoromethyl)phenyl)
piperazin- 1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione (5m)
White solid, m.p. 132.4~132.8 °C; 1H NMR (400 MHz, CDCl3) δ: 7.50~7.47 (m,
2H), 7.13~6.99 (m, 5H), 5.17 (s, 2H), 4.38~4.34 (m, 4H), 3.31~3.28 (m, 4H),
13
3.09~3.06 (m, 4H). C NMR (400 MHz, CDCl3) δ: 177.52, 163.39, 152.28, 150.33,
149.28, 134.19, 128.56, 127.55, 126.37, 124.39, 118.19, 117.68, 116.04, 112.52,
110.33, 109.29, 68.41, 62.28, 55.33, 53.31, 49.19, 48.22. MS (ESI): 479
(C22H22F3N4O3S, [M+H]+). Anal. Calcd for C22H21F3N4O3S: C, 55.22; H, 4.42;
N,11.71%; Found: C, 55.09; H, 4.59; N, 11.66.
4.1.5.14
3-((4-(2,3-Dichlorophenyl)piperazin-1-yl)methyl)-5-(2,3-dihydrobenzo [b] [1,4]
dioxin -6 -yl)-1,3,4-oxadiazole-2(3H)-thione (5n)
White solid, m.p. 148.5~149.9 °C; 1H NMR (400 MHz, CDCl3) δ: 7.52~7.49 (m,
2H), 7.24~7.19 (m, 2H), 7.04~7.00 (m, 2H), 5.61~5.59 (m, 1H), 5.16 (s, 1H),
13
3.49~3.44 (m, 4H), 4.39~4.34 (m, 4H), 3.10 (s, 4H). C NMR (400 MHz, CDCl3) δ:
18

178.23, 163.32, 152.18, 150.03, 147.19, 135.28, 130.18, 129.23, 126.47, 124.23,
120.13, 114.37, 112.17, 109.23, 69.38, 62.14, 60.55, 55.33, 53.18, 49.39, 47.86. MS
(ESI): 479 (C21H21Cl2N4O3S, [M+H]+). Anal. Calcd for C21H20Cl2N4O3S: C, 52.61; H,
4.21; N, 11.69%; Found: C, 52.73; H, 4.12; N, 11.81.
4.1.5.15
5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)
-
3-
((
4-
(3
-methoxyphenyl)piperazin-1-yl )methyl)-1,3,4-oxadiazole-2(3H)-thione (5o)
White solid, m.p. 155.4~155.8 °C; 1H NMR (400 MHz, CDCl3) δ: 7.50~7.47 (m,
2H), 7.23~7.18 (m, 1H), 7.01~6.99 (m, 1H), 6.58~6.56 (m, 1H), 6.49~6.46 (m, 2H),
5.16 (s, 2H), 4.38~4.34 (m, 4H), 3.85~3.81 (m, 3H), 3.26~3.23 (m, 4H), 3.07~3.05 (m,
4H). 13C NMR (400 MHz, CDCl3) δ: 178.38, 163.28, 152.11, 150.03, 143.22, 137.29,
127.83, 123.98, 116.22, 114.29, 112.04, 110.38, 107.29, 106.83, 68.88, 61.38, 60.39,
55.33, 53.97, 49.39, 48.03, 22.80. MS (ESI): 441 (C22H25N4O4S, [M+H]+). Anal.
Calcd for C22H24N4O4S: C, 59.98; H, 5.49; N, 12.72%; Found: C, 60.12; H, 5.35; N,
12.88.
4.1.5.16
3-((4-(3,4- Dichlorophenyl) piperazin-1-yl)methyl)- 5 -(2 , 3-dihydrobenzo [b] [1,4]
dioxin – 6 -yl)-1,3,4-oxadiazole-2(3H)-thione (5p)
White solid, m.p. 159.2~159.9 °C; 1H NMR (400 MHz, CDCl3) δ: 7.49~7.47 (m,
2H), 7.40~7.37 (d, J = 8.8 Hz, 1H), 7.04~6.97 (m, 2H), 6.77 (s, 1H), 5.61~5.59 (m,
1H), 5.15 (s, 1H), 4.38~4.34 (m, 4H), 3.53~3.52 (m, 2H), 3.43 (s, 2H), 3.24~3.21 (m,
2H), 3.06~3.03 (m, 2H). 13C NMR (400 MHz, CDCl3) δ: 177.89, 163.33, 152.19,
149.98, 143.39, 132.19, 128.56, 125.73, 123.39, 122.90, 116.58, 114.43, 112.25,
109.32, 70.33, 62.29, 60.48, 55.16, 53.32, 49.39, 47.28. MS (ESI): 479
(C21H21Cl2N4O3S, [M+H]+). Anal. Calcd for C21H20Cl2N4O3S: C, 52.61; H, 4.21; N,
11.69%; Found: C, 52.75; H, 4.08; N, 11.76.
4.1.5.17
3-((4-Benzylpiperazin-1-yl)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-
oxadiazole-2(3H)-thione (5q)
White solid, m.p. 97.8~98.2 °C; 1H NMR (400 MHz, CDCl3) δ: 7.48~7.45 (m, 2H),
19

7.39~7.38 (m, 2H), 7.35 (s, 3H), 7.04~6.98 (m, 1H), 5.09 (s, 2H), 4.38~4.34 (m, 4H),
3.59~3.58 (m, 2H), 2.97 (s, 4H), 2.54 (s, 4H). 13C NMR (400 MHz, CDCl3) δ: 178.32,
163.16, 150.83, 148.38, 137.51, 130.05, 128.31, 128.01, 127.11, 126.30, 124.08,
115.39, 110.64, 108.92, 69.28, 63.55, 62.92, 60.28, 55.73, 53.10, 52.97, 51.16. MS
(ESI): 425 (C22H25N4O3S, [M+H]+). Anal. Calcd for C22H24N4O3S: C, 64.24; H, 5.70;
N, 13.20%; Found: C, 64.35; H, 5.62; N, 13.35.
4.2 Anti-proliferation
The antiproliferation activities of the synthesized compounds 5a~5q were evaluated
using a standard MTT-based colorimetric assay. Target tumor cells were grown to log
phase in DMEM medium supplemented with 10% fetal bovine serum. After diluting
to 5×104 cells per mL, 100 µL of the obtained cell suspension was added to each well
of 96-well culture plates, and incubated at 37 ℃, 5% CO2 for 24 h. Then 100 µL a
series concentration of drug-containing medium were added into each well to
maintain the final concentration as 0.1, 1.0, 10 and 100 µM. Each concentration was
in triplicate, and 5-Fluorouracil was used as the positive reference. After 48h
incubation, 20 µL MTT work solution (5 mg/mL MTT dissolved in PBS) was added
to each well. After 4h, the medium was discarded and 100 µL DMSO was added to
each well. The plates were then shaken vigorously for 10min for complete dissolution.
The absorbance at a test wavelength of 570 nm was measured on an ELISA plate
reader (ELX800, BioTek, USA). Each assay was carried out at least three times. IC50
value was defined as the concentration at which 50% of cells could survive.
4.3 FAK inhibitory assay
The ability of the test compounds to inhibit FAK was determined using an enzyme
immuno assay (Bio-Swamp). The experiments were performed according to the
manufacture's instructions.
4.4 Experimental protocol of docking study
Molecular docking of compound 5m into the three-dimensional FAK complex
structure was carried out using the Discovery Studio (version 3.5) as implemented
through the graphical user interface DS-CDOCKER protocol.
Acknowledgments
20

This work is granted by Natural Science Foundation of Jiangsu Province
(BK20160570), and Jiangsu Planned Projects for Postdoctoral Research Funds
(1601041A).
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