Synthesis, molecular docking and biological evaluation of 1,3,4-oxadiazole derivatives as potential immunosuppressive agents

Article abstract of DOI:10.1016/j.bmc.2012.01.023

A series of novel 1,3,4-oxadiazole derivatives (5a-5s) have been designed, synthesized and evaluated for their immunosuppressive activity. Most of these synthesized compounds were proved to have potent immunosuppressive activity and low toxicity. Among th

Full text of DOI:10.1016/j.bmc.2012.01.023

Bioorganic & Medicinal Chemistry 20 (2012) 1373–1379
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, molecular docking and biological evaluation of 1,3,4-oxadiazole
derivatives as potential immunosuppressive agents
⇑
Ru Yan, Zhi-Ming Zhang, Xian-Ying Fang, Yang Hu, 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 novel 1,3,4-oxadiazole derivatives (5a–5s) have been designed, synthesized and evaluated for
their immunosuppressive activity. Most of these synthesized compounds were proved to have potent
immunosuppressive activity and low toxicity. Among them, compounds (5m–5r) showed the most
potent biological activity against lymph node cells. The results of flow cytometry (FCM) and western blot-
ting demonstrated that compound 5q induce cell apoptosis by the inhibition of PI3 K/AKT pathway.
Received 20 December 2011
Revised 11 January 2012
Accepted 12 January 2012
Available online 25 January 2012
Molecular docking was performed to position compound 5q into PI3Kc binding site in order to explore
the potential target.
Keywords:
Oxadiazoles
Immunosuppressive activity
Molecular docking
Ó 2012 Published by Elsevier Ltd.
PI3K
c
1. Introduction
O
O
O
O
Immunosuppressant is a kind of drugs that can reduce the
body’s immune reaction by inhibiting the immune cells prolifera-
tion, that is commonly used to transplant rejection and the
treatment of autoimmune diseases such as systemic lupus erythe-
matosus, rheumatoid arthritis and psoriasis.1–3 Phosphoinositide
3-kinases (PI3 Ks), key components of the PI3 K/AKT pathway
which plays essential roles in various cellular activities, are lipid
kinases that phosphorylate the 3-hydroxyl group of phosphoinosi-
tides.4 PI3 Ks are grouped into three classes (I, II, and III) according
to their structure preference and substrate specificity, class I PI3Ks
are separated into two subfamilies (IA and IB) depending on the
OH
O
O
A
B
O
O
OH
O
O
O
O
O
O
receptors to which they couple.5,6 Among these isoforms, PI3K
c
C
D
(the only isoform of class IB) plays a pivotal role in inflammation,
and it is involved in allergy, development of chronic inflammation,
autoimmune diseases.7,8 Therefore, there is important significance
Figure 1. The structure of compounds A–D.
as hypoglycemic,14 anti-viral,15 anti-cancer,16 anti-bactericidal17
effects. In particular, a few substituted 1,3,4-oxadiazoles have been
found to exhibit immunosuppressive activities.18–21 Therefore,
oxadiazole derivatives have raised considerable attention to
medicinal research, and a large number of investigations on their
synthesis and biological activities have been reported during the
last 10 years.22–25
A series of novel oxadiazoles compounds containing a 1,4-ben-
zodioxan template were firstly synthesized and anti-inflammatory
activities were detected. Among them, the compounds 5m–5r
displayed potent immunosuppressive activity by inhibiting ConA
co-stimulated lymph node cells proliferation and had low toxicity.
Preliminary study on the immune mechanism of the compounds
to discover a kind of drug that can influence PI3Kc.
Compounds containing a 1,4-benzodioxan template (Fig. 1)
with acetic acid substituent on C6 or C2 (A and B) have anti-inflam-
matory activity.9,10 However, compound B have a better activity, it
can be used as a nonsteroidal anti-inflammatory drug (NSAID).9
A
serious of derivatives containing a 1,4-benzodioxan template were
designed and synthesized by Harrak, and among them, compounds
C and D (Fig. 1) showed good anti-inflammatory activity.11,12
Oxadiazoles are an important class of heterocyclic compounds,
which have been reported to have many various activities,13 such
⇑
Corresponding author. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Ó 2012 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2012.01.023

1374
R. Yan et al. / Bioorg. Med. Chem. 20 (2012) 1373–1379
Table 1
found that compounds induced cell apoptosis by the inhibition of
PI3 K/AKT pathway. From the results, we can conclude that some
of the synthesized compounds are potent immunosuppressant.
Structure of oxadiazole derivatives
O
R4
N
N
2. Results and discussion
2.1. Chemistry
O
O
R3
O
R1
R2
R3
Nineteen 1,3,4-oxadiazole derivatives (5a–5s) were firstly syn-
thesized to screen for the anti-inflammatory activity. The synthetic
route was based on the following sequence of reactions,26–28 as
shown in Scheme 1. As an example, the reaction of 2,3-dihydro-
benzo[b][1,4]dioxine-6-carboxylic acid with concentrated H2SO4
was refluxed in ethanol for overnight to produce the corresponding
ester. Secondly, a mixture of the ester and hydrazine hydrate in
ethanol was refluxed for overnight, by which the corresponding
hydrazide was formed. Thirdly, the hydrazone products were syn-
thesized by refluxing the mixture of hydrazide and the different
substituted benzaldehyde in ethanol for 5 h. The hydrazone and
glacial acetic acid in acetic anhydride were refluxed for 1 h, after
which the mixture was poured into ice water and oxadiazoline
analogs were collected. When needed, oxadiazoline analogs were
purified by recrystallization in aqueous methanol. All structure of
the synthetic compounds were shown in Table 1.
Compound
R1
R2
R4
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
H
Cl
H
H
H
H
F
H
H
Cl
H
H
H
H
H
H
H
OMe
H
Br
H
H
F
H
H
H
H
H
Cl
Br
H
H
NO2
OMe
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Br
Cl
Br
H
H
OCH2Ph
Cl
H
F
H
OCOCH3
OCOCH3
OCOCH3
OCOCH3
OCOCH3
OCOCH3
H
H
H
H
H
2.2. Crystal structure of compound 5r
5s
H
H
Among these compounds, a crystal structure of compound 5r
was determined by X-ray diffraction analysis. The crystal data
are presented in Table 2 and Fig. 2 give perspective views of 5r
with the atomic labeling system.
Table 2
Crystal data for compound 5r
O
O
Crystal parameters
Compound 5r
20H16Br2N2O6
O
Formula
C
O
OH
O
i
Crystal size (mm)
0.2 ꢀ 0.2 ꢀ 0.1
MW (g molꢁ1
)
537.94
Crystal system
(°)
b (°)
(°)
a (Å)
b (Å)
Monoclinic
90.00
111.259(6)
90.00
12.094(8)
12.410(8)
14.889(9)
2082(2)
O
O
1
a
2
O
c
O
NH
ii
c (Å)
NH2
V (Å3)
O
Z
4
3
h limits(°)
hkl limits
F(000)
Data/restraints/parameters
Absorption coefficient(mm-1
Reflections collected
Independent reflections
R1/wR2 [I > 2&Gs(I)]
R1/wR2 (all data)
GOF
2.20 6 h 6 29.03
O
ꢁ16 6 h 6 15, ꢁ16 6 k 6 16, ꢁ18 6 l 6 20
R1
R2
1482
O
O
5200/0/271
12.565
17597
5200 [Rint = 0.1082]
0.0759/0.1457
0.1786/0.1877
1.024
N
NH
iii
)
R3
4a-4s
R4
R4
O
N
N
O
R3
2.3. Immunosuppressive activity
O
iv
R1
R2
2.3.1. Cytotoxicity test
O
5a-5s
Generally speaking, the inhibitory activity of the compounds is
due to cell apoptosis or toxic effect, so we firstly performed
cytotoxicity test before detecting biological activity. The oxadiaz-
ole compounds were detected for their cyotoxicity on lymph node
Scheme 1. General synthesis of compounds 5a–5s.Reagents and conditions: (i)
ethanol, concentrated sulfuric acid; reflux; (ii) hydrazine hydrate, ethanol; reflux;
(iii) substituted benzaldehyde, ethanol, reflux; (iv) acetic anhydride, glacial acetic
acid, reflux.
cells with cyclosporin
A (CsA) as the positive control. The

R. Yan et al. / Bioorg. Med. Chem. 20 (2012) 1373–1379
1375
the potential drug which has high activity and low toxicity. Consid-
ering the two factors, compound 5q may be a potential anti-
inflammatory drug with high efficacy (IC50 = 1.83
icity (CC50 = 363.36 M).
lM) and low tox-
l
2.3.3. PI3K
The PI3K
examined and the results were presented in Table 4. The tested
compounds showed potent PI3K inhibitory. The results of PI3K
c
enzymatic activity
c
inhibitory potency of oxadiazole derivatives was
c
c
inhibitory activity of the tested compounds were corresponding
to the structure relationships (SARs) of their inhibitory effects on
lymph node cells. The results suggested that inhibitory activity
on lymph node cells of the compounds may by inhibiting PI3K
enzymatic activity.
c
Figure 2. Crystal structure diagrams of compound 5r.
2.3.4. Apoptosis assay
Table 3
Cytotoxicity assay and inhibitory activity of the compounds on lymph node cells
In order to study the preliminary anti-inflammatory mechanism
of the compounds, we performed flow cytometry (FCM) (Fig. 3). As
shown in Figure 3, lymph node cells were treated with 0, 3, 10 and
Compound
CC50 SD (
l
M)
IC50 SD (
l
M)
SI
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
209.99 25.04
343.16 36.30
363.72 21.21
412.36 34.13
258.59 18.16
293.17 24.78
304.19 27.35
275.68 18.14
248.25 21.63
268.74 22.86
210.51 19.53
298.08 18.35
491.43 37.86
288.37 21.52
225.63 37.34
261.89 27.45
363.36 28.34
332.28 25.68
323.71 23.67
13.78 1.87
36.06 0.39
52.35 5.64
>100
5.82
6.56
<3.63
4.27
15.15
4.07
8.97
30.36
4.98
8.67
4.02
3.59
65.88
133.51
184.94
183.14
198.56
163.68
<3.23
196.86
30 lM of compounds 5n and 5q for 24 h. The compounds can in-
crease the percentage of apoptosis in a dose-independent manner.
The results indicated that compounds 5n and 5q can induce apop-
tosis of ConA stimulated lymph node cells, exceptionally com-
pound 5q obviously induced apoptosis.
95.36 6.78
17.06 0.60
72.08 3.87
33.90 1.36
9.08 0.31
49.83 4.32
30.98 6.32
52.42 5.43
82.83 7.56
7.46 0.37
2.16 0.12
1.22 0.09
1.43 0.13
1.83 0.18
2.03 0.23
>100
2.3.5. Western blotting
To determine whether target compounds suppress activation of
AKT (a key downstream effector of PI3K and important node in the
PI3K/AKT/mTOR signaling pathway) and Caspase 3 (an important
enzyme in apoptosis), selected compound 5q were tested for its
suppression on p-AKT and Cleaved Caspase 3. As shown in Figure
4, p-AKT (pS473) was dose-dependent lower regulated upon 5q
treated for 24 h. The activity of Cleaved Caspase 3 was dose-depen-
dent up-regulated upon 5q treated. It can be conferred from the re-
sult that the apoptosis induced by 5q may be mediated by the
inhibition of PI3K/AKT pathway.
5s
CsA
0.07 0.01
2.4. Binding model of compounds 5q into PI3Kc structure
pharmacological results of these compounds were summarized in
Table 3. What we can see from the data is that most of the com-
pounds were low toxic.
To gain better understanding on the potency of the synthesized
compounds and guide further structure–activity relationships
(SARs) studies. The molecular docking was performed by potent
2.3.2. Inhibitory activity on lymph node cells
inhibitor 5q into binding site of PI3K
plied Discovery Studio3.1 (DS. 3.1). The binding modes of com-
pound 5q and PI3K were depicted in Figures 5 and 6. In the
binding mode, compound 5q is nicely bound to the PI3K via
two hydrogen bonds and Pi-sigma. Cl atom on the benzene ring
and amino hydrogen of LYS 890 form hydrogen bond (H–Clꢂ ꢂ ꢂH:
2.47 Å, 158.4°), the oxygen atom of the benzene group also form
hydrogen bond (H–Oꢂ ꢂ ꢂH: 2.46 Å, 157.3°) with hydrogen atom of
VAL 882. Pi-sigma (2.92 Å) is formed by SER 806 with benzene ring.
The results of molecular docking and western blotting show that
compound 5q influence the expression of downstream protein by
c. All docking runs were ap-
All the compounds were tested in vitro for the inhibition activ-
ity on ConA stimulated lymph node cells. The results were summa-
rized in Table 3. The table showed that most of the synthesized
compounds showed potent inhibitory activity with the IC50 value
at low micromolar.
c
c
Structure–activity relationships (SARs) were inferred from Ta-
ble 3. In general, compounds with electronic-withdrawing substit-
uents (halogen or acetoxy) on the benzene ring showed more
potent inhibitory activities than compounds only contained elec-
tronic-donating substituents (methyl or methoxy) on the benzene
ring. Exceptionally compounds (5m–5r) with acetoxy on the ben-
zene ring exhibited higher immunosuppressive activity compared
with other compounds. Among the compounds of acetoxy substit-
uents, the activities of Cl substituent is a little higher activity than
that of Br substituent (5o > 5p, 5q > 5r). The position of substitu-
ents on benzene ring also influenced the activities. For example,
the order of the activities is that substituent at the ortho (5g) posi-
tion > substituent at the meta (5k) position > substituent at the
para (5l) position.
bonding to PI3Kc. These results, along with the date of inhibitory
Table 4
PI3K inhibitory activity of the selected compounds
c
Compound
PI3Kc IC50 (lM)
5n
5o
5p
5q
2.06
0.98
1.31
1.75
1.98
7.26
Among them, compound 5o displayed the best immunosup-
pression activity, as it has the lowest IC50 (IC50 = 1.22 lM). How-
ever, the cytotoxicity and SI (SI = 198.56) of the compound 5q
have absolute advantage compared to others. Our goal is to explore
5r
LY294002a
a
Reported value, Ref. 29.

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R. Yan et al. / Bioorg. Med. Chem. 20 (2012) 1373–1379
120
100
80
60
40
20
0
0 uM
3 uM
10 uM
30 uM
5n
5q
Figure 3. Lymph node cells were stimulated with ConA for 24 h, and then various concentrations of compounds 5n and 5q were added to incubate for 24 h. Cells were stained
by Annexin V-FITC and datas were analyzed by flow cytometry (FCM).
3. Conclusions
A series of novel oxadiazole compounds have been designed
and firstly synthesized and their biological activities were also
evaluated as potent anti-inflammatory activity. Among them,
compound 5q demonstrated the potent inhibitory activity (IC50
=
1.83 M, SI = 198.56) compared to positive control (IC50 = 0.07 M,
l
l
SI = 196.86). The results of flow cytometry (FCM) and western blot-
ting demonstrated that compound 5q induced cell apoptosis by the
inhibition of PI3K/AKT pathway. Molecular docking showed that
Figure 4. Western blotting was performed to detect the effect of compound 5q on
p-AKT (pS473) and Cleaved Caspase3.
compound 5q bound to PI3Kc by two hydrogen bonds and a
activity assay indicated that compound 5q would be a kind of
Pi-sigma. This study showed that compound 5q may be a potential
potential inhibitor.
anti-inflammatory drug.
Figure 5. The molecular docking of compound 5q into PI3K. Compound 5q is nicely bound to the PI3K via two hydrogen bonds (H–Clꢂ ꢂ ꢂH: 2.47 Å, 158.4°, H–Oꢂ ꢂ ꢂH: 2.46 Å,
157.3°) and Pi-sigma (2.92 Å).

R. Yan et al. / Bioorg. Med. Chem. 20 (2012) 1373–1379
1377
by recrystallization in anhydrous ethanol, giving compound 4 as
a white solid.
4.1.4. Synthesis of 1-(5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-
1,3,4-oxadiazol-3(2H)-yl)ethanone (5)
Compound 4 (0.001 mol) and glacial acetic acid (2 mL) were
added in Ac2O (5 mL), and the resulting solution was heated to re-
flux for 1 h. The reaction mixture was poured into ice water, which
was collected using suction filtration and dried, followed by recrys-
tallization in anhydrous ethanol, followed by drying to yield 5a–5s.
4.1.4.1.
1-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-phenyl-
1,3,4-oxadiazol-3(2H)-yl)ethanone (5a). White solid, yield 73%,
mp: 129–130 °C. 1H NMR (CDCl3, 300 MHz): 2.33 (s, 3H), 4.26–
4.32 (m, 4H), 6.89–6.92 (m, 1H), 7.05 (s, 1H), 7.37–7.40 (m, 5H),
7.42–7.48 (m, 2H). MS (ESI): 325.1 (C18H17N2O4, [M+H]+). Anal.
Calcd for C18H16N2O4: C, 66.66; H, 4.97; N, 8.64; O, 19.73. Found:
C, 66.68; H, 4.99; N, 8.63.
4.1.4.2. 1-(2-(2-Chlorophenyl)-5-(2,3-dihydrobenzo[b][1,4]di-
oxin-6-yl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (5b). White solid,
yield 69%, mp: 171–172 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.26
(s, 3H), 4.29–4.30 (m, 4H), 6.98 (d, J = 8.4 Hz, 1H), 7.22 (d,
J = 2.0 Hz, 1H), 7.28–7.31 (m, 2H), 7.41–7.51 (m, 3H), 7.56 (d,
J = 7.9 Hz, 1H). MS (ESI): 359.8 (C18H16ClN2O4, [M+H]+). Anal. Calcd
for C18H15ClN2O4: C, 60.26; H, 4.21; N, 7.81. Found: C, 60.24; H,
4.21; N, 7.83.
Figure 6. 3D model of the interaction between compound 5q and PI3 K bonding
site. The protein is represented by molecular surface. Compound 5q is depicted by
balls.
4. Experimental section
4.1. Methods of synthesis
4.1.4.3. 1-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-(4-nitro-
phenyl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (5c). Yellow solid,
Yield 60%; mp: 151–152 °C. 1H NMR (DMSO-d6, 300 MHz): 2.25
(s, 3H), 4.28–4.31 (m, 4H), 7.01 (d, J = 8.40 Hz, 1H), 7.27–7.31
(m,2H), 7.32–7.36 (m,1H), 7.76 (d, J = 8.79 Hz, 2H), 8.29 (d,
J = 8.61 Hz, 2H). MS (ESI): 370.10 (C18H16N3O6, [M+H]+). Anal. Calcd
for C18H15N3O6: C, 58.54; H, 4.09; N, 11.38; O, 25.99. Found: C,
58.53; H, 4.09; N, 11.40.
Melting points (uncorrected) were determined on an XT4 MP
apparatus (Taike Corp., Beijing, China). ESI mass spectra were ob-
tained on a Mariner System 5304 mass spectrometer, and 1H
NMR spectra were recorded on a Bruker PX500 or DPX300 spec-
trometer at 25 °C with TMS and solvent signals allotted as internal
standards. Chemical shifts were reported in ppm (d). Elemental
analyses were performed on a CHN–O–Rapid instrument and were
within 0.4% of the theoretical values.
4.1.4.4. 1-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-(4-meth-
oxyphenyl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (5d). White so-
lid, yield 70%, mp: 153–154 °C. 1H NMR (300 MHz, DMSO-d6) d:
2.22 (s, 3H), 3.76 (s, 3H), 4.29–4.30 (m, 4H), 6.96–7.00 (m, 3H),
7.09 (s, 1H), 7.23 (d, J = 2.0 Hz, 1H), 7.29–7.33 (m, 1H), 7.36 (d,
J = 8.6 Hz, 2H). MS (ESI): 355.4 (C19H19N2O5, [M+H]+). Anal. Calcd
for C19H18N2O5: C, 64.40; H, 5.12; N, 7.91. Found: C, 64.42; H,
5.10; N, 7.90.
4.1.1. Synthesis of 2,3-dihydrobenzo[b][1,4]dioxine-6-carbox-
ylate (2)
The 2,3-dihydrobenzo[b][1,4]dioxine-6-carboxylic acid (9.0 g,
50 mmol) was dissolved in anhydrous ethanol (50 mL) containing
concentrated H2SO4 (5 mL), which were refluxed overnight. The
solvent was evaporated until no longer liquid outflow. Water
(20 mL) was added, extracting by adding respectively 40, 30 and
20 mL ethyl acetate. The organic phases were sequentially washed
with saturated NaHCO3, brine, and dried over MgSO4. The solvent
was removed under reduced pressure to yield compound 2.
4.1.4.5. 1-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-(p-tolyl)-
1,3,4-oxadiazol-3(2H)-yl)ethanone (5e). White solid, yield 62%,
mp: 115–116 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.22 (s, 3H),
2.31 (s, 3H), 4.28–4.30 (m, 4H), 6.99 (d, J = 8.4 Hz, 1H), 7.10 (s,
1H), 7.22–7.24 (m, 3H), 7.30–7.33 (m, 3H). MS (ESI): 339.4
(C19H19N2O4, [M+H]+). Anal. Calcd for C19H18N2O4: C, 67.44; H,
5.36; N, 8.28. Found: C, 67.40; H, 5.35; N, 8.30.
4.1.2. Synthesis of 2,3-dihydrobenzo[b][1,4]dioxine-6-carbo-
hydrazide (3)
To compound 2 (0.05 mol) dissolved in anhydrous ethanol
(35 mL), hydrazine hydrate (15 mL) was added and the mixture
was refluxed for 24 h. The reaction mixture was evaporated and
until no longer liquid outflow. Then water was added and com-
pound 3 was precipitated.
4.1.4.6. 1-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-(3-meth-
oxyphenyl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (5f). White so-
lid, yield 64%, mp: 124–125 °C. 1H NMR (300 MHz, DMSO-d6) d:
2.22 (s, 3H), 3.74 (s, 3H), 4.28–4.29 (m, 4H), 6.96–6.99 (m, 4H),
7.09 (s, 1H), 7.23 (d, J = 2.0 Hz, 1H), 7.29–7.33 (m, 2H). MS (ESI):
355.4 (C19H19N2O5, [M+H]+). Anal. Calcd for C19H18N2O5: C, 64.40;
H, 5.12; N, 7.91. Found: C, 64.45; H, 5.10; N, 7.92.
4.1.3. Synthesis of N0-benzylidenebenzohydrazide (4)
To a stirred solution of hydrazide (1 mmol) and appropriate
benzaldehyde in ethanol (25 mL), water (5 mL) was added fol-
lowed by dropwise addition of glacial acetic acid (0.2 mL). The
mixture was refluxed for 5 h, after which the solution was poured
into ice water. The mixture was stirred until precipitate formed,
which was collected using suction filtration and dried, followed
4.1.4.7. 1-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-(2-fluoro-
phenyl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (5g). White solid,
yield 76%, mp: 127–128 °C. 1H NMR (300 MHz, CDCl3) d: 2.35 (s,
3H), 4.26–4.33 (m, 4H), 6.88–6.91 (m, 1H), 7.07–7.18 (m, 2H),

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R. Yan et al. / Bioorg. Med. Chem. 20 (2012) 1373–1379
7.23 (s, 1H), 7.34–7.40 (m, 4H). MS (ESI): 343.3 (C18H16FN2O4,
[M+H]+). Anal. Calcd for C18H15FN2O4: C, 63.15; H, 4.42; F, 5.55;
N, 8.18. Found: C, 63.11; H, 4.43; N, 5.57.
DMSO-d6) d: 2.12–2.17 (m, 6H), 4.30–4.31 (m, 4H), 7.00 (d,
J = 8.4 Hz, 1H), 7.12 (s, 1H), 7.20–7.31 (m, 3H), 7.55–7.59 (m, 1H),
7.63 (s, 1H). MS (ESI): 417.8 (C20H18ClN2O6, [M+H]+). Anal. Calcd
for C20H17ClN2O6: C, 57.63; H, 4.11; N, 6.72. Found: C, 57.60; H,
4.12; N, 6.74.
4.1.4.8. 1-(2-(3-Bromophenyl)-5-(2,3-dihydrobenzo[b][1,4]di-
oxin-6-yl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (5h). White solid,
yield 64%, mp: 142–143 °C. 1H NMR (300 MHz, CDCl3) d: 2.34 (s,
3H), 4.28–4.32 (m, 4H), 6.90–6.93 (m, 1H), 7.00 (s, 1H), 7.24–7.29
(m, 1H), 7.38–7.44 (m, 3H), 7.51 (d, J = 7.9 Hz, 1H), 7.59 (d,
J = 1.7 Hz, 1H). MS (ESI): 404.2 (C18H16BrN2O4, [M+H]+). Anal. Calcd
for C18H15BrN2O4: C, 53.62; H, 3.75; N, 6.95. Found: C, 53.66; H,
3.74; N, 6.93.
4.1.4.16. 2-(3-Acetyl-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-
2,3-dihydro-1,3,4-oxadiazol-2-yl)-4-bromophenyl acetate (5p).
Light yellow, Yield 67%, mp: 204–206 °C. 1H NMR (300 MHz,
DMSO-d6) d: 2.11–2.17 (m, 6H), 4.28–4.31 (m, 4H), 7.01 (d,
J = 8.4 Hz, 1H), 7.12 (s, 1H), 7.19–7.22 (m, 2H), 7.30 (dd,
J = 8.43 Hz,1H), 7.68–7.72 (m, 1H), 7.77 (d, J = 2.4 Hz, 1H). MS
(ESI): 462.3 (C20H18BrN2O6, [M+H]+). Anal. Calcd for C20H17BrN2O6:
C, 52.08; H, 3.71; N, 6.07. Found: C, 52.04; H, 3.73; N, 6.06.
4.1.4.9. 1-(2-(4-(Benzyloxy)phenyl)-5-(2,3-dihydrobenzo[b][1,4]-
dioxin-6-yl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (5i). White solid,
yield 69%, mp: 131–132 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.22 (s,
3H), 4.29–4.30 (m, 4H), 5.12 (s, 2H), 6.99 (d, J = 8.4 Hz, 1H), 7.03–
7.08 (t, J = 7.4 Hz, 3H), 7.23 (d, J = 2.0 Hz, 1H), 7.29–7.45 (m, 8H).
MS (ESI): 540.2 (C25H23N2O5, [M+H]+). Anal. Calcd for C25H22N2O5:
C, 69.76; H, 5.15; N, 6.51. Found: C, 69.73; H, 5.12; N, 5.54.
4.1.4.17. 2-(3-Acetyl-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-
2,3-dihydro-1,3,4-oxadiazol-2-yl)-4,6-dichlorophenyl
acetate
(5q). Brown solid, Yield 66%, mp: 156–157 °C. 1H NMR
(300 MHz, DMSO-d6) d: 2.14–2.18 (m, 6H), 4.29–4.30 (m, 4H),
6.99 (d, J = 8.6 Hz, 1H), 7.14 (s, 1H), 7.19 (d, J = 2.0 Hz, 1H), 7.28
(dd, J = 8.4 Hz,1H), 7.70 (d, J = 2.6 Hz, 1H), 7.91 (d, J = 2.6 Hz, 1H).
MS (ESI): 452.3 (C20H17Cl2N2O6, [M+H]+). Anal. Calcd for
C20H16Cl2N2O6: C, 53.23; H, 3.57; N, 6.21. Found: C, 53.20;
H,3.58; N, 6.22.
4.1.4.10. 1-(2-(2,4-Dichlorophenyl)-5-(2,3-dihydrobenzo[b][1,4]-
dioxin-6-yl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (5j). White solid,
yield 65%, mp: 136–138 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.25 (s,
3H), 4.29–4.30 (m, 4H), 6.98 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 2.1 Hz,
1H), 7.28–7.31 (m, 2H), 7.46–7.53 (m, 2H), 7.75 (d, J = 1.62 Hz,
1H). MS (ESI): 394.2 (C18H15Cl2N2O4, [M+H]+). Anal. Calcd for
4.1.4.18. 2-(3-Acetyl-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-
2,3-dihydro-1,3,4-oxadiazol-2-yl)-4,6-dibromophenyl
acetate
C
18H14Cl2N2O4: C, 54.98; H, 3.59; Cl, 18.03; N, 7.12. Found: C,
(5r). Light yellow solid, Yield 61%, mp: 177–178 °C. 1H NMR
(300 MHz, DMSO-d6) d: 2.16–2.17 (m, 6H), 4.30–4.32 (m, 4H), 7.02
(d, J = 8.4 Hz, 1H), 7.14 (s, 1H), 7.21 (s, 1H), 7.30 (d, J = 8.4 Hz, 1H),
7.86 (d, J = 2.2 Hz, 1H), 8.13 (d, J = 1.3 Hz, 1H). MS (ESI): 540.2
(C20H17Br2N2O6, [M+H]+). Anal. Calcd for C20H16Br2N2O6: C, 44.47;
H, 2.99; N, 5.19. Found: C, 44.44; H, 2.98; N, 5.21.
54.95; H, 3.61; N, 7.10.
4.1.4.11. 1-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-(3-fluo-
rophenyl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (5k). White solid,
yield 69%, mp: 129–130 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.24
(s, 3H), 4.28–4.31 (m, 4H), 7.01 (d, J = 8.6 Hz, 1H), 7.17 (s, 1H),
7.25–7.35 (m, 5H), 7.46–7.53 (m, 1H). MS (ESI): 343.3
(C18H16FN2O4, [M+H]+). Anal. Calcd for C18H15FN2O4: C, 63.15; H,
4.42; F, 5.55; N, 8.18. Found: C, 63.12; H, 4.40; N, 8.19.
4.1.4.19. 1-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-(naph-
thalen-1-yl)-1,3,4-oxadiazol-3(2H)-yl)ethanone
(5s). White
crystal, Yield 66%, mp: 167–168 °C. 1H NMR (300 MHz, CDCl3) d:
2.45 (s, 3H), 4.24–4.27 (m, 4H), 6.84–6.91 (m, 1H), 7.35–7.38 (m,
2H), 7.44–7.63 (m, 4H), 7.75 (s, 1H), 7.90 (d, J = 7.7 Hz, 2H), 8.21
(d, J = 8.6 Hz, 1H). MS (ESI): 375.4 (C22H19N2O4, [M+H]+). Anal.
Calcd for C22H18N2O4: C, 70.58; H, 4.85; N, 7.48. Found: C, 70.56;
H, 4.86; N, 7.46.
4.1.4.12. 1-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-(4-fluo-
rophenyl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (5l). White solid,
yield 69%, mp: 123–124 °C. 1H NMR (300 MHz, CDCl3) d: 2.32 (s,
3H), 4.27–4.32 (m, 4H), 6.89–6.92 (m, 1H), 7.03–7.09 (m, 3H),
7.37–7.40 (m, 2H), 7.43–7.48 (m, 2H). MS (ESI): 343.3
(C18H16FN2O4, [M+H]+). Anal. Calcd for C18H15FN2O4: C, 63.15; H,
4.42; N, 8.18. Found: C, 63.18; H, 4.40; N, 8.15.
4.2. Cytotoxicity test
The cytotoxic activity in vitro was measured using the MTT as-
say. Cells were cultured in a 96-well plate at a density of 5 ꢀ 105
cells and different concentrations of compounds were respectively
added to each well. The incubation was permitted at 37 °C, 5% CO2
4.1.4.13. 4-(3-Acetyl-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-
2,3-dihydro-1,3,4-oxadiazol-2-yl)phenyl acetate (5m). White
solid, yield 66%, mp: 143–144 °C. 1H NMR (300 MHz, DMSO-d6)
d: 2.24–2.27 (m, 6H), 4.29–4.31 (m, 4H), 6.99 (d, J = 8.6 Hz, 1H),
7.17–7.20 (t, J = 5.78 Hz, 3H), 7.26 (d, J = 2.0 Hz, 1H), 7.33 (dd,
J = 8.4 Hz,1H), 7.49 (d, J = 8.6 Hz, 2H). MS (ESI): 383.4
(C20H19N2O6, [M+H]+). Anal. Calcd for C20H18N2O6: C, 62.82; H,
4.74; N, 7.33. Found: C, 62.85; H, 4.72; N, 7.32.
atmosphere for 24 h before the cytotoxicity assessments. 20 lL
MTT reagent (4 mg/mL) was added per well 4 h before the end of
the incubation. Four hours later, the plate was centrifugaled at
1200 rcf for 5 min and the supernatants were removed, each well
was added with 200 lL DMSO. The absorbance was measured at
a wavelength of 570 nm (OD570 nm) on an ELISA microplate read-
er. Three replicate wells were used for each concentration and each
assay was measured three times, after which the average of CC50
and SD were calculated. The cytotoxicity of each compound was
expressed as the concentration of compound that reduced cell via-
bility to 50% (CC50). The results were summarized in Table 3.
4.1.4.14. 2-(3-Acetyl-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-
2,3-dihydro-1,3,4-oxadiazol-2-yl)phenyl acetate (5n). White so-
lid, yield 60%, mp: 158–160 °C. 1H NMR (300 MHz, DMSO-d6) d:
2.12–2.18 (m, 6H), 4.26–4.29 (m, 4H), 6.98 (d, J = 8.6 Hz, 1H),
7.13–7.19 (m, 3H), 7.26–7.32 (m, 2H), 7.46–7.51 (m, 2H). MS
(ESI): 383.4 (C20H19N2O6, [M+H]+). Anal. Calcd for C20H18N2O6: C,
62.82; H, 4.74; N, 7.33. Found: C, 62.85; H, 4.72; N, 7.32.
4.3. Cell proliferation assay
4.1.4.15. 2-(3-Acetyl-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-
2,3-dihydro-1,3,4-oxadiazol-2-yl)-4-chlorophenyl acetate (5o).
Light yellow, Yield 65%, mp: 192–193 °C. 1H NMR (300 MHz,
Cells were activated by Con A (5
cells were plated into 96-well plate per well with 5 lg/mL Con A.
After that, different concentrations of compounds were added to
l
g/mL) for 24 h. Then, 5 ꢀ 105

R. Yan et al. / Bioorg. Med. Chem. 20 (2012) 1373–1379
1379
each well, respectively. The cells were incubated for 72 h. 20
l
L
docking procedure. Types of interactions of the docked enzyme
with ligand were analyzed after end of molecular docking. Ten
docking poses were saved for each ligand and the final docked con-
formation was scored.
MTT reagent (4 mg/mL) was added per well 4 h before the end of
the incubation. Four hours later, the plate was centrifugaled at
1200 rcf for 5 min and the supernatants were removed, each well
was added with 200 lL DMSO. The absorbance was read at a wave-
length of 570 nm (OD570 nm) on an ELISA microplate reader. IC50
was calculated and the results were summarized in Table 3. The
immunosuppressive activity of each compound was expressed as
the concentration of the compound that inhibited ConA stimulated
T cell proliferation to 50% (IC50) of the control value. The selective
index (SI) value was used to evaluate the bioactivity of the com-
pounds. The selective index (SI) is determined as the ratio of the
concentration of the compound that reduced cell viability to 50%
(CC50) to the concentration needed to inhibit the proliferation to
50% (IC50) of the control value.
4.8. Crystal structure determination
Crystal structure determination of compound 5q were carried
out on a Nonius CAD4 diffractometer equipped with graphite-
monochromated Mo K
a (k = 0.71073 Å) radiation (Fig. 2). The
structure was solved by direct methods and refined on F2 by full-
matrix least-squares methods using SHELX-97.31 All non-hydrogen
atoms of compound 5q were refined with anisotropic thermal
parameters. All hydrogen atoms were placed in geometrically ide-
alized positions and constrained to ride on their parent atoms.
4.4. PI3Kc inhibitory assay
Acknowledgments
Nineteen 1,3,4-oxadiazole derivatives containing 1,4-benzodi-
This work was financed by National Natural Science Foundation
of China (No. J1103512).
oxan were tested in a search for small molecule inhibitors of PI3K
c,
which was purchased from R&D Systems (Minneapolis, MN). PI3K
c
was incubated for 4 h at room temperature with or without the
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4.7. Molecular docking
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ID: 3L54) was chosen as the template to compare the docking
mode between compound 5q bound to PI3K.30 The molecular dock-
ing procedure was performed by using LigandFit protocol within
Discovery Studio 3.1. For ligand preparation, the 3D structures of
compound 5q were generated and minimized using Discovery Stu-
dio 3.1. For enzyme preparation, the hydrogen atoms were added,
and the water and impurities were removed. The whole PI3K en-
zyme was defined as a receptor and the site sphere was selected
based on the ligand binding location of LXX, then the LXX molecule
was removed and compound 5q was placed during the molecular
31. Sheldrick, G. M. SHELX-97. Program for X-ray Crystal Structure Solution and
Refinement; Göttingen University: Germany, 1997.