Synthesis, biological evaluation and molecular docking studies of 1,3,4-thiadiazole derivatives containing 1,4-benzodioxan as potential antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2011.08.039

A series of 1,3,4-thiadiazole derivatives containing 1,4-benzodioxan (2a-2s) have been synthesized to screen for FAK inhibitory activity. Compound 2p showed the most potent biological activity against HEPG2 cancer cell line (EC₅₀ = 10.28 μg/mL

Full text of DOI:10.1016/j.bmcl.2011.08.039

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6116–6121
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, biological evaluation and molecular docking studies
of 1,3,4-thiadiazole derivatives containing 1,4-benzodioxan as potential
antitumor agents
⇑
Juan Sun, Yu-Shun Yang, Wei Li, Yan-Bin Zhang, Xiao-Liang Wang, Jian-Feng Tang, Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of 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-thiadiazole derivatives containing 1,4-benzodioxan (2a–2s) have been synthesized to
screen for FAK inhibitory activity. Compound 2p showed the most potent biological activity against
HEPG2 cancer cell line (EC50 = 10.28 lg/mL for HEPG2 and EC50 = 10.79 lM for FAK), which was compa-
rable to the positive control. Docking simulation was performed to position compound 2p into the FAK
structure active site to determine the probable binding model. The results of antiproliferative and Wes-
tern-blot assay demonstrated that compound 2p possessed good antiproliferative activity against HEPG2
cancer cell line. Therefore, compound 2p with potent FAK inhibitory activity may be a potential antican-
cer agent against HEPG2 cancer cell.
Received 15 June 2011
Revised 25 July 2011
Accepted 8 August 2011
Available online 16 August 2011
Keywords:
Thiadiazole
Benzodioxan
Antitumor activity
Focal adhesion kinase
Ó 2011 Elsevier Ltd. All rights reserved.
Cancer, second cause of mortality in the world, is continuing to
be a major health problem in developing as well as undeveloped
countries.1 Despite the progress achieved in medicine during cen-
tury, the continued commitment to the laborious task of discover-
ing new anticancer agents remains critically important. Therefore,
there is an increasing need for new therapies, especially those that
are based on current knowledge of cancer biology as well as that
taking advantage of the cancer cells phenotype, described by Hana-
han and Weinberg.2
Focal adhesion kinase is a 125 kDa protein that localizes to focal
adhesions3 and is activated and tyrosine phosphorylated in re-
sponse to integrin clustering.4 These result in signal transmission
to the cell nucleus to trigger cell division and motility. FAK is in-
volved in multiple cellular functions such as cell proliferation, sur-
vival, motility, invasion, metastasis, and angiogenesis.5 Different
approaches to inhibit FAK with FAK antisense oligonucleotides,6
dominant-negative C-terminal domain of FAK, FAK-CD or FRNK7,8
or FAK siRNA9,10 caused decreased cellular viability, growth inhibi-
tion, or apoptosis. Recently, FAK was proposed to be a new poten-
tial therapeutic target in cancer.11,12
substituted 1,4-benzodioxan (B) is known as a non-steroidal anti-
inflammatory drug.14 WB 4104 (C) is recognized as a selective
a
nucleus constitutes the active part of several biologically active
compounds (D), including antitumor,20–22 antimycotic23,24 and
anti-inflammatory agents.25–27
-adrenoceptor antagonist.15–19 In addition, 1,3,4-thiadiazole
Recently, it was reported that a number of other compounds
containing the 1,4-benzodioxan template showed potent antitu-
mor activity.28 However, to our knowledge, few reports have been
dedicated to the synthesis and focal adhesion kinase structure
inhibitory activity of 1,3,4-thiadiazole derivatives containing 1,4-
benzodioxan. Herein, in continuation to extend our research on
antitumor compounds with FAK structure inhibitory activity, we
report in the present work the synthesis and structure–activity
relationships of a series of 1,3,4-thiadiazole derivatives containing
1,4-benzodioxan as antitumor agents. Biological evaluation indi-
cated that some of the synthesized compounds were potent inhib-
itors of FAK structure.
Nineteen 1,3,4-thiadiazole derivatives containing 1,4-benzodi-
oxan were synthesized to screen for the antitumor activity. All of
them were synthesized for the first time. The synthesis of com-
pounds 2a–2s followed the general pathway outlined in Scheme
1. They are prepared in two steps. Firstly, the 2,3-dihydro-
benzo[b][1,4] dioxine-6-carboxylic acid on treatment with
thiosemicarbazide in presence of phosphoryl chloride yielded
Compounds containing a 1,4-benzodioxan template have re-
ceived significant attention in chemical, medicinal and pharmaceu-
tical research as this structural scaffold is found in a variety of
drugs. For example (Fig. 1), the mesylate salt of doxazosin (A) is
an effective drug for treatment of hypertension.13 The 6-position
N-(5-(2,3-dihydrobenzo[b][1,4]
dioxin-6-yl)-1,3,4-thiadiazol-2-
amine. Secondly, the coupling reaction between the obtained col-
orless solid and the different substituted phenyl acetic acid or
⇑
Corresponding author. Tel.: +86 25 8359 2672; fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.039

J. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6116–6121
6117
O
O
O
N
O
COOH
N
N
OCH3
O
N
O
OCH3
O
A
B
NH2
OCH3
O
O
N
H
NH
N
N
H3CO
N
S
R
C
O
F
O
D
Figure 1. The structure of compounds A, B, C and D.
N
O
N
NH2
H
O
O
O
O
N
NH2
OH
S
H2N
i
S
1
Cl
O
R
COOH
R
ii
N
S
N
N
N
NH2
NH
O
O
O
O
R-COOH
iii
S
O
R
2a-2s
H3CH2CO
H3CH2CO
O
2eR=
2aR=
2iR=
2jR=
2qR=
2rR=
2mR=
Cl
Cl
N
CH3
NH2
2fR=
2gR=
2hR=
2bR=
2cR=
2nR=
2oR=
2pR=
N
N
Cl
N
CH3
2kR=
2lR=
2sR=
CH3
Br
2dR=
F
Br
H3CO
Scheme 1. General synthesis of compounds (2a–2s). Reagents and conditions: (i) POCl3, reflux, 30 min, KOH; (ii) SOCl2, 90 °C; (iii) EDC, HOBT, dichloromethane, rt.
benzoic acid was performed by using carbodiimide hydrochloride
and N-hydroxybenzotriazole in anhydrous CH2Cl2, what afforded
the corresponding target compounds N-(5-(2,3-dihydrobenzo
[b][1,4]dioxin-6-yl)-1,3,4-thiadiazol-2-yl)-substituted-acetamide.
Then compounds 2a–2s were obtained by subsequent purification
with recrystallisation. All of the synthetic compounds gave satis-
factory analytical and spectroscopic data, which were full accor-
dance with their depicted structures.
All the synthesized derivatives 2a–2s were evaluated for their
ability to antiproliferative activity against HEPG2, HELA, SW1116
and BGC823. The results were summarized in Table 1. As illus-
trated in Table 1, the active analogs showed a distinctive potential

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J. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6116–6121
Table 1
pattern of selectivity as well as broad-spectrum antitumor activity.
Besides, compound 2p showed the most potent biological activity
Antiproliferative activity of the synthesized compounds (2a–2s)
against HEPG2 cancer cell line with EC50 value of 10.28 lg/mL.
Compound
EC50
HELA
(l
g/mL)
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 four cell lines,
it was revealed that among the tested compounds, compound 2j
was the most active members with EC50 values of 14.21–
HEPG2
SW1116
BGC823
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
18.28
34.78
>100
>100
18.43
13.78
45.78
42.15
>100
15.33
>100
23.78
>100
>100
15.35
10.28
>100
>100
>100
23.31
21.74
49.89
>100
>100
>100
34.86
26.76
54.22
51.21
>100
32.45
>100
>100
>100
>100
>100
>100
>100
>100
>100
31.02
29.12
51.29
67.64
>100
>100
47.62
40.69
>100
>100
>100
16.16
43.57
>100
>100
41.21
30.35
60.17
>100
>100
>100
28.52
24.92
27.38
46.15
>100
>100
>100
>100
>100
>100
>100
14.21
67.87
>100
>100
>100
>100
45. 36
35.76
41.02
>100
17.37
26.83
32.45
logical activity against HEPG2 cancer cell line with EC50 value of
10.28 g/mL. Regarding the nineteen compounds, different acids
lg/mL. Besides, compound 2p showed the most potent bio-
l
substitutes led to different antitumor activity, and the potency or-
der was phenylpropinic acid >phenylacetic acid >benzoic acid. As
shown in Table 1, compounds 2e and 2f with substituted Cl group
on benzene ring showed better antitumor activity with EC50 of
13.78–47.62
lg/mL than compounds 2g and 2h with substituted
Br group with EC50 of 42.15–54.22
lg/mL against HEPG2 and HELA
cancer cell lines. Among these compounds, compounds with p-
substituted Cl group (2f) exhibited more potent activity than com-
pounds with m-substituted Cl group (2e). Besides, there was the
same rule with the compounds substituted by Br. Based on the
data obtained, compounds 2q and 2r with pyridine ring were
2q
2r
2s
5-Fluorouracil
Staurosporine
Figure 2. Molecular docking modeling of compound 2p with FAK: compound 2p is nicely bound to the FAK with its nitrogen atom of thiadiazole ring project toward the
amino hydrogen of GLY 505, with the hydroxyl group forming a more optimal H-bond (H–NÁ Á ÁH: 2.164 Å, 153.1°) interaction, and the hydrogen atom of imine group of 2p
also forms hydrogen bond (N–HÁ Á ÁO: 2.010 Å, 121.8°) with oxygen atom of ILE428.
Figure 3. 3D model of the interaction between compound 2p and FAK binding site. FAK is represented by molecular surface. Compound 2p is depicted by sticks and balls.

J. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6116–6121
6119
Table 2
FAK inhibitory activity of selected compounds
Compound
FAK (EC50, lM)
2a
2b
2e
2f
2g
2h
2j
2l
17.74
21.04
18.11
12.15
26.15
23.73
13.98
19.02
13.69
10.79
11.32
Figure 5. Compound 2p was examined by Western blotting. Data are representa-
tive of three independent experiments.
2o
2p
Staurosporine
found to be inactive against HEPG2, HELA and SW1116 cancer cell
lines. However, introduction of NH2 or Cl moieties, afforded 2n and
2o with a remarkable increase in the antitumor activity against
SW1116 and HEPG2 cancer cells, EC50 values, 41.21 and
Table 3
The docking calculation of the synthesized compounds (2a–2s)
15.35
with pyridine ring showed moderate activity toward BGC823 can-
cer cell line with EC50 values, 35.76 and 41.02 g/mL, respectively.
lg/mL, respectively. Interestingly, compounds 2q and 2r
Compound
Binding energy
IC units
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
À8.58
À7.84
À8.31
À8.37
À7.39
À8.52
À6.97
À6.53
À8.16
À7.73
À8.27
À9.56
À7.11
À7.43
À8.08
À8.67
À6.45
À8.72
À7.58
nM
nM
l
The addition of CH3 group (2s), however, led to decrease in cyto-
toxic activity against all cell lines.
l
l
M
M
In an effort to elucidate the possible mechanism by which the
title compounds can induce antitumor activity in the four cells
and guide further SAR studies, molecular docking of the potent
inhibitor 2p into ATP binding site of FAK were performed on the
binding model based on the FAK structure (2ETM.pdb). The binding
models of compound 2p and FAK were depicted in Figures 2 and 3.
In the binding model, compound 2p is nicely bound to the FAK
with its nitrogen atom of thiadiazole ring project toward the amino
hydrogen of GLY 505, with the hydroxyl group forming a more
optimal H-bond (H–NÁ Á ÁH: 2.164 Å, 153.1°) interaction, and the
hydrogen atom of imine group of 2p also forms hydrogen bond
(N–HÁ Á ÁO: 2.010 Å, 121.8°) with oxygen atom of ILE428. The molec-
ular docking results suggested that compound 2p was a potential
nM
nM
nM
nM
lM
nM
lM
nM
l
l
M
M
nM
nM
lM
lM
lM
0.5
M
1.0
M
control
104
103
102
101
104
103
102
101
32.8%
8.0%
24.49%
13.17%
58.3%
0.8%
_
58.85%
3.49%
_
100
101
102
FL3-H
103
104
100
101
102
FL3-H
103
104
4.0
M
2.0
M
104
103
102
101
104
103
102
101
26.23%
27.21%
18.00%
23.25%
39.52%
11.00%
50.76%
4.03%
100
101
102
FL3-H
103
104
100
101
102
FL3-H
103
104
Figure 4. HEPG2 cells isolated from naïve mice were cultured with anticancer and various concentrations of 2p for 24 h. Cells were stained by Annexin VeFITC/PI and
apoptosis was analyzed by flow cytometry.

6120
J. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6116–6121
Figure 6. Compound 2p was examined by Western blotting. Data are representative of three independent experiments.
inhibitor of FAK. The docking calculation of the other compounds
were also depicted in Table 3.
Western-blot results were summarized in Figures 5 and 6. The re-
sult indicated that compound 2p showed excellent inhibitory
activity.
In addition, we also selected the top 10 compounds which had
better antiproliferative activity to test their FAK inhibitory activity
against HEPG2 cell line. The results were summarized in Table 2.
Most of the tested compounds displayed potent FAK inhibitory.
Among them, compound 2p showed the most potent inhibitory
In conclusion, a series of N-(5-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)-1,3,4-thiadiazol-2-yl)-substituted-acetamide
substituted
derivatives have been synthesized and evaluated for their antitu-
mor activities. Compound 2p demonstrated the most potent inhib-
itory activity that inhibited the growth of HEPG2 cells with EC50 of
with EC50 of 10.79 lM. The results of FAK inhibitory activity of
the tested compounds were corresponding to the structure rela-
tionships (SAR) of their antitumor activities. This demonstrated
that the potent antitumor activities of the synthetic compounds
were probably correlated to their FAK inhibitory activities.
Apoptosis is an essential mechanism used to eliminate acti-
vated HEPG2 cells during the shut down process of excess immune
responses and maintain proper immune homeostasis, while defi-
cient apoptosis of activated HEPG2 cell is associated with a wide
variety of immune disorders. We detected the mechanism of com-
pound 2p inhibition effects by flow cytometry (FCM) (Fig. 4), and
found that the compound could induce the apoptosis of activated
HEPG2 cells in a dose dependent manner. The result indicated that
compound 2p induced apoptosis of antitumor stimulated HEPG2
cells.
10.28
10.79
l
l
M and inhibited the activity of FAK kinase with EC50 of
M, which was comparable to the positive control stauro-
sporine. Molecular docking study indicated that compound 2p
was nicely bound to the FAK with two hydrogen bonds. Apoptosis
assay and Western-blot results also showed the compound 2p was
a potential antitumor agent.
Acknowledgments
This work was supported by Jiangsu National Science Founda-
tion (No. BK2009239) and the Fundamental Research Funds for
the Central Universities (No. 1092020804 & 1106020824).
Supplementary data
In an effort to study the preliminary mechanism of the com-
pound with potent inhibitory activity, the Western-blot experi-
ment was performed to assay the effect of compound 2p. The
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.08.039.

J. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6116–6121
6121
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