Design, synthesis, biological evaluation and molecular modeling of novel 1,3,4-oxadiazole derivatives based on Vanillic acid as potential immunosuppressive agents

Article abstract of DOI:10.1016/j.bmc.2012.05.055

In present study, a series of novel 1,3,4-oxadiazole derivatives have been designed, synthesized and purified. All of these compounds are reported for the first time, the chemical structures of these compounds were confirmed by means of ¹H NMR, ESI-MS and elemental analyses. Besides, we evaluated their immunosuppressive activity. Most of these synthesized compounds were proved to have potent immunosuppressive activity and low toxicity. Among them, the bioassay results demonstrated that compounds 5c, 5n, 5p, 5o, 6f and 6g exhibited immunosuppressive activities with IC₅₀ concentration range from 1.25 μM to 7.60 μM against the T cells, and the IC₅₀ of positive control (csa) is 2.12 μM. Moreover, all the title compounds were assayed for PI3K/AKT signaling pathway inhibition using the ELISA assay. We examined the compounds with potent inhibitory activities against IL-1, IL-6 and IL-10 released in ConA-simulated mouse lymph node cells. The results showed compounds 5o and 6f displayed the most potential biological activity against T cells (IC₅₀ = 1.25 μM and 4.75 μM for T cells). The preliminary mechanism of compound 5o inhibition effects was also detected by flow cytometry (FCM). The results of apoptosis and ELISA assay demonstrated that the immunosuppressive activity of compounds 5o and 6f against T cells may be mediated by the inhibition of PI3Kγ/AKT signaling pathway. Molecular docking was performed to position compounds 5o and 6f into PI3Kγ binding site in order to indicate the potential target.

Full text of DOI:10.1016/j.bmc.2012.05.055

Bioorganic & Medicinal Chemistry 20 (2012) 4226–4236
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, biological evaluation and molecular modeling
of novel 1,3,4-oxadiazole derivatives based on Vanillic acid
as potential immunosuppressive agents
Jian-Feng Tang a, , Xian-Hai Lv c, , Xiao-Liang Wang a, Jian Sun a, Yan-Bin Zhang a, Yu-Shun Yang a,
a,
Hai-Bin Gong b, , Hai-Liang Zhu
⇑
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b Xuzhou Central Hospital, Xuzhou 221009, PR China
c College of Science, Anhui Agricultural University, Hefei 230036, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In present study, a series of novel 1,3,4-oxadiazole derivatives have been designed, synthesized and puri-
fied. All of these compounds are reported for the first time, the chemical structures of these compounds
were confirmed by means of 1H NMR, ESI-MS and elemental analyses. Besides, we evaluated their immu-
nosuppressive activity. Most of these synthesized compounds were proved to have potent immunosup-
pressive activity and low toxicity. Among them, the bioassay results demonstrated that compounds 5c,
5n, 5p, 5o, 6f and 6g exhibited immunosuppressive activities with IC50 concentration range from
Received 2 May 2012
Revised 27 May 2012
Accepted 28 May 2012
Available online 5 June 2012
Keywords:
1,3,4-Oxadiazole derivatives
PI3Kc inhibitor
Immunosuppressive activities
1.25 lM to 7.60 lM against the T cells, and the IC50 of positive control (csa) is 2.12 lM. Moreover, all the
title compounds were assayed for PI3K/AKT signaling pathway inhibition using the ELISA assay. We exam-
ined the compounds with potent inhibitory activities against IL-1, IL-6 and IL-10 released in ConA-simu-
lated mouse lymph node cells. The results showed compounds 5o and 6f displayed the most potential
ELISA
biological activity against T cells (IC50 = 1.25 lM and 4.75 lM for T cells). The preliminary mechanism of
Molecular docking
compound 5o inhibition effects was also detected by flow cytometry (FCM). The results of apoptosis and
ELISA assay demonstrated that the immunosuppressive activity of compounds 5o and 6f against T cells
may be mediated by the inhibition of PI3K
to position compounds 5o and 6f into PI3K
c
c
/AKT signaling pathway. Molecular docking was performed
binding site in order to indicate the potential target.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Phosphoinositide 3-kinases (PI3Kc) are lipid kinases that use
the g-phosphate of ATP to 3-phosphorylate phosphoinositides on
the inositol moiety. Their lipid products, the 3-phosphorylated
phosphoinositides, are ubiquitous intracellular messengers that
carry signals relevant to a huge range of cell functions in both
health and disease.9–12 According to their molecular structure, cel-
lular regulation and in vivo substrate specificities, PI3Ks are di-
vided into class I, II and III PI3Ks. The best known PI3Ks are Class
Autoimmune diseases represent a major cause of morbidity and
mortality. More than 70 autoimmune diseases have been de-
scribed, and, although many of these diseases are quite rare, the
collective prevalence of autoimmune diseases is high. The major
autoimmune diseases include rheumatoid arthritis, psoriasis,
Crohn’s disease, systemic lupus erythematosus, and multiple scle-
rosis.1–3 Immunosuppressant is an important class of clinical drugs
for an array of medical processes, including transplant rejection
and the treatment of autoimmune diseases. Although immunosup-
pressive drugs have been used for the treatment of autoimmune
diseases in clinic, their side effects including liver toxicity, nephro
toxicity, infection, cardiovascular toxicity and others cannot be
neglected.4–8 Therefore, there is a clinical need for new and less
toxic therapeutic agents with new mechanisms of action.
I PI3Ks, which include subclass IA (consisting of PI3K
PI3Kd isoforms) and subclass IB (consisting of PI3K
only).13,14 Among these isoforms, PI3Kd and PI3K
are mainly
expressed in the hematopoietic system and mediate immune
responses. PI3K (the only isoform of class IB) plays a pivotal role
a, PI3Kb and
c
isoform
c
c
in inflammation, and it is involved in allergy, development of
chronic inflammation, autoimmune diseases.15,16 Therefore, there
is important significance to discover a kind of drug that can influ-
ence PI3Kc.
1,3,4-Oxadiazoles are an important class of heterocyclic com-
pounds. The widespread use of them as a scaffold in medicinal
chemistry establishes this moiety as a member of the privileged
structures class.17 1,3,4-Oxadiazoles are associated with many
⇑
Corresponding authors. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail addresses: ghbxzh@yahoo.com.cn (H.-B. Gong), zhuhl@nju.edu.cn (H.-L.
Zhu).
These two authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.055

J.-F. Tang et al. / Bioorg. Med. Chem. 20 (2012) 4226–4236
4227
types of biological properties such as anti-inflammatory, hypogly-
cemic, antifungal and antibacterial activities.18–21 Among these, a
few differently substituted 1,3,4-oxadiazoles have exhibited potent
immunosuppressive activities particually.22–24 Therefore, oxadiaz-
ole 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.25–28
compounds and studied their immunosuppressive activities and
PI3K inhibitory activity. Biological evaluation indicated that com-
pounds 5o and 6f were potent inhibitors of PI3Kc.
c
2. Results and discussion
2.1. Chemistry
Vanillic acid (4-hydroxy-3-methoxy benzoic acid), an oxidized
form of vanillin which has been used as a chemical intermediate
in the production of pharmaceuticals, is found in many traditional
Chinese medicines, such as Rhizoma Picrorhizae, Ginseng, Propolis
and BaiHao.29 Vanillic acid has been reported to possess anti-
inflammatory activity.30 Thus, in this paper, we designed, synthe-
sized and purified a series of novel 1,3,4-oxadiazole derivatives
(5a–5q, 6a–6q) derived from Vanillic acid. Docking simulations
were performed to explore the probable molecular target of these
In this study, 34 1,3,4-oxadiazole derivatives (5a–5q, 6a–6q)
derived from 4-hydroxy-3-methoxy benzoic acid were firstly syn-
thesized to screen for the anti-inflammatory activity. The synthetic
route was based on the following sequence of reactions,31 which
was shown in Scheme 1. As an example, the reaction of 4-hydro-
xy-3-methoxybenzoic acid 1 with concentrated H2SO4 was re-
fluxed in ethanol for 10 h to produce the corresponding ester 2.
Secondly, a mixture of the ester and hydrazine hydratein ethanol
was refluxed overnight, by which the corresponding hydrazide 3
OH
OH
i
H CH COOC
OCH
3
3
2
HOOC
OCH
3
2
1
OH
iii
ii
H NHNOC
2
OCH
3
3
OH
R
2
OH
R
1
O
OCH
3
iv
S
R
3
N
O
N
OCH
3
HS
R
4
R
5a-5q
N
5
N
4
v
R
R
2
R
R
1
3
4
O
R
R
2
R
1
O
5
OCH
3
S
R
3
N
N
R
4
R
5
6a-6m
Scheme 1. General synthesis of compounds (5a–6q, 6a–6q).

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J.-F. Tang et al. / Bioorg. Med. Chem. 20 (2012) 4226–4236
Table 1
Structure of 1,3,4-oxadiazole derivatives
R
R
2
R
R
R
1
3
4
O
OH
R
2
R
1
R
2
O
5
R
1
OCH
3
S
O
R
OCH
3
3
N
S
N
R
3
N
N
R
4
R
5
6a-6q
R
4
R
5a-5q
5
Compound
R1
R2
R3
R4
R5
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
H
H
NO2
H
F
H
F
F
H
Br
H
Cl
Me
H
H
H
H
Me
H
Cl
H
F
H
Br
H
H
NO2
H
H
H
H
Cl
H
H
H
H
H
OMe
H
F
H
H
H
H
H
H
H
Br
H
H
H
NO2
H
Cl
H
NO2
H
Me
H
H
H
Cl
H
F
H
H
F
H
H
H
H
Br
H
H
Br
H
Cl
H
H
F
H
H
F
H
H
NO2
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
5j
5k
5l
5m
5n
5o
5p
5q
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
F
H
H
Br
F
H
H
H
H
H
H
6m
6n
6o
6p
6q
OMe
was formed. Thirdly, treatment of the hydrazide 3 with carbon
disulfide in the presence of KOH and 95% ethanol under reflux pro-
vided the key intermediate 4. Finally the synthesis of compounds
5a–5q was accomplished by refluxing 4 with equimolar amount
different substituted benzyl bromides in the presence of NaOH in
acetonitrile. The synthesis of compounds 6a–6q was accomplished
by refluxing 4 with excessive different substituted benzyl bro-
mides in the presence of NaOH in acetonitrile. All target com-
pounds were purified by recrystallisation. All of the synthetic
compounds gave satisfactory analytical and spectroscopic data,
which were in full accordance with their depicted structures. All
structure of the synthetic compounds were shown in Table 1.
conclusion. Prior to the following bioactivity analysis, all the syn-
thesized 1,3,4-oxadiazole derivatives (5a–5q, 6a–6q) were tested
in vitro for their cytotoxicity on lymph node cells. The pharmaco-
logical results were summarized in Table 2. The data showed that
most of the 1,3,4-oxadiazole derivatives had a quite low toxicity.
The potential drug should has a low toxicity. Considering this fac-
tor, compound 5o and 6f may be a potential anti-inflammatory
drug with low toxicity (CC50 = 211.04 lM and 170.84 lM).
2.2.2. Inhibitory activity assay
All the synthesized 1,3,4-oxadiazole derivatives (5a–5q, 6a–6q)
were evaluated for their inhibitory activity on murine lymphocyte
proliferation induced by concanavalin A (ConA). The results were
summarized in Table 2. Among them, compounds 5o and 6f
2.2. Biological activity
(IC50 = 1.25 lM and 4.75 lM for T cells) exhibited the most potent
2.2.1. Cytotoxicity test
immunosuppressive activity.
The inhibitory activity of the compounds is sometimes a result
of their toxic effects and consequently might cause an erroneous
Structure–activity relationships (SARs) were inferred from
Table 2. We can arrive at the conclusion that the activity of the

J.-F. Tang et al. / Bioorg. Med. Chem. 20 (2012) 4226–4236
4229
Table 2
tested compounds may be correlated to structure variation and
modifications. A comparison of the para substituents on the ben-
zene ring demonstrated that compounds (5a–5q) with electronic-
donating substituent (methyl) (5c) showed more potent inhibitory
activities than compounds only contain electronic-withdrawing
substituent (halogen) (5g). Among the compounds with elec-
tronic-withdrawing substituents, the activities of fluorin substitu-
ent (5c) is a little higher activity than that of chlorin substituent
(5g), which indicated that compounds (5a–5q) with small substi-
tuent (F) showed more potent inhibitory activities than com-
pounds with big substituent (Cl). Meanwhile, compound 5o
displayed the best immune suppression activity, as it has the low-
All the synthesized 1,3,4-oxadiazole derivatives (5a–5q, 6a–6q) were tested in vitro
for their cytotoxicity and inhibitory activity on lymph node cells
Compound
CC50 SD (
l
M)
IC50 SD (
l
M)
SI
5a
5b
5c
5d
5e
5f
5g
5h
5i
688.22 60.23
382.15 37.93
549.26 53.87
330.75 33.14
392.24 38.42
473.72 46.84
479.83 46.98
574.98 56.89
520.08 51.97
470.40 46.18
363.31 35.32
684.82 69.24
564.32 56.79
592.43 59.36
211.04 22.56
389.43 39.56
455.46 46.78
265.84 25.85
195.49 18.96
224.53 21.87
144.26 14.34
136.31 14.23
170.84 18.53
178.57 16.78
153.30 14.67
161.06 15.89
150.95 148.65
181.78 17.96
196.67 19.89
168.22 17.05
155.61 16.58
177.59 18.06
275.25 26.84
242.06 23.95
174.24 17.34
14.16 0.21
48.06 0.39
6.21 0.07
9.60 0.09
15.95 0.17
5.79 0.22
11.07 0.14
10.07 0.12
9.69 0.08
8.29 0.09
17.79 0.18
8.19 0.13
8.43 0.07
8.42 0.06
1.25 0.01
7.41 0.03
8.83 0.10
14.37 0.15
18.87 0.17
17.28 0.18
10.64 0.15
17.18 0.28
4.76 0.05
5.22 0.06
12.89 0.13
9.12 0.07
8.71 0.09
12.89 0.10
13.30 0.14
12.23 0.13
7.60 0.06
15.81 0.14
9.06 0.08
10.07 0.11
2.12 0.03
48.60
7.95
10.19
34.45
24.59
81.82
43.35
57.10
53.67
56.74
20.42
83.62
66.94
70.36
168.83
52.55
51.58
18.49
15.19
12.99
8.67
5j
5k
5l
est IC50 (IC50 = 1.25 lM) and low toxicity (CC50 = 211.04 lM). For
5o is the compound with no substituent, we can arrive at the con-
clusion that the size of substituent has a great impact on the activ-
ity of the tested compounds (5a–5q), and the compounds with
smaller substituent showed greater activity. As the compound 5o
showed the best activity, and 5o is the compound with no substi-
tuent. So, we can come to a conclusion, comparing to the electronic
character of substituent, the size of substituent has a greater im-
pact on the activity of the tested compounds (5a–5q).
Compound (6a–6q) with different benzaldehyde substituents
displayed different immunosuppressive activity. A comparison of
the ortho substituents on the benzene ring demonstrated that com-
pounds with halogen (6f) may have more potent inhibitory activi-
ties than compounds with methyl (6b). Furthermore, the inhibitory
activity was governed by the position of substituents. The position
of substituent son benzene ring also influenced the activities. Take
the compounds with Br substituents for example, the order of the
activities is that substituent at the ortho (6j) position > substituent
at the meta (6i) position > substituent at the para (6c) position.
Among them, compound 6f with fluorin group at the ortho position
displayed the best immunosuppression activity, as it has the low-
5m
5n
5o
5p
5q
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
5.00
35.89
34.21
11.89
17.66
17.33
14.10
14.79
13.75
20.48
11.23
30.38
24.04
82.19
6m
6n
6o
6p
6q
Csa
est IC50 (IC50 = 4.76 lM) and low toxicity (CC50 = 170.84 lM).
There are some differences in structure between series 5(5a–
5q) and series 6(6a–6q). The synthesis of compounds (5a–5q) were
accomplished by the combination of 1,3,4-oxadiazole with
equimolar amount benzyl bromides. Meanwhile, the synthesis of
compounds (6a–6q) were accomplished by the combination of
1,3,4-oxadiazole with benzyl bromides and phenolic hydroxyl
group with benzyl bromides. From the inhibitory activity result,
we can find that the compounds of series 5 and the compounds
of series 6 all showed potent inhibitory activities, so we can proof
that the phenolic hydroxyl group of series 5 is not the activity site.
In summary, compounds 5o and 6f displayed the most potential
Table 3
Compound
PI3Kc IC50 (lM)
5c
5n
5p
5o
6f
6g
7.41
6.48
2.87
1.34
5.12
5.74
LY294002
7.26a
biological activity against T cells (IC50 = 1.25
cells), and they may be a potential immunosuppressive agents.
lM and 4.75 lM for T
a
Reported value, Ref. 32.
2.2.3. PI3K
The PI3K
examined and the results were presented in Table 3. The tested
compounds showed potent PI3K inhibitory. The results of PI3K
c
inhibitory assay
c
inhibitory potency of oxadiazole derivatives was
derivatives, compound 5o as studied in vitro. As shown in Figure 1,
lymph node cells stimulated with ConA were treated with 0.25 M,
2.5 M and 25 M of compound 5o for 24 h. The compound in-
l
c
c
l
l
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
creased the percentage of apoptosis by Annexin V-FITC/PI staining
in a dose-dependent manner. The result indicated that compounds
5o could induce apoptosis of ConA stimulated lymph node cells.
on lymph node cells of the compounds may by inhibiting PI3K
enzymatic activity.
c
2.2.5. ELISA assay
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 import
antenzyme in apoptosis). Selected compounds 5c, 5n, 5p, 5o, 6n,
6f, 6g were tested with potent inhibitory activities against IL-1,
IL-6 and IL-10 released in ConA-simulated mouse lymph node cells.
As shown in Figure 6, among these seven compounds, 5o and 6f
exhibited the highest inhibitory effects against ConA-induced IL-
1, IL-6 and IL-10 expression.
2.2.4. Apoptosis assay
Apoptosis is an essential mechanism used to eliminate acti-
vated T cells during the shutdown process of excess immune re-
sponses and maintains proper immune homeostasis, while
deficient apoptosis of activated T cells is associated with a wide
variety of immune disorders. In order to study the preliminary
anti-inflammatory mechanism of the compounds, we performed
flow cytometry (FCM). As representative of these 1,3,4-oxadiazole

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J.-F. Tang et al. / Bioorg. Med. Chem. 20 (2012) 4226–4236
0.25µM
2.5µM
25µM
PI
2.43%
4.34%
4.98%
AnnexinV
FITC
Figure 1. lymph node cells stimulated with ConA were treated with 0.25, 2.5 and 25
lM of compound 5o for 24 h. The compound increased the percentage of apoptosis by
Annexin V-FITC/PI staining in a dose-dependent manner. The result indicated that compounds 5o could induce apoptosis of ConA stimulated lymph node cells.
Figure 2. The molecular docking of compound 5o into PI3K
139.2°).
c. Compound 5o is nicely bound to the PI3K via two hydrogen bonds (H–Oꢀ ꢀ ꢀH: 2.04 Å, 112.2°, H–Oꢀ ꢀ ꢀH: 2.02 Å,
2.3. Binding model of compounds 5o and 6f into PI3K
structure
c
LYS890 (H–Oꢀ ꢀ ꢀH: 1.64 Å, 129.8°), the oxygen atom on the benzene
group forms a hydrogen bond with primary hydrogen atom of
LYS890 (H–Oꢀ ꢀ ꢀH: 1.85 Å, 134.7°), the fluorine atom on the ben-
zene group form hydrogen bond with primary hydrogen atom of
LYS833 (H–Fꢀ ꢀ ꢀH: 2.05 Å, 111.9°), the fluorine atom on the benzene
group form hydrogen bond with hydrogen atom of LYS802 (H–
Fꢀ ꢀ ꢀH: 2.07 Å, 149.5°). This molecular docking result, along with
the biological assay data, suggested that compounds 5o and 6f
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
inhibitor 5o and 6f into binding site of PI3K
were applied Discovery Studio3.1 (DS. 3.1). The binding modes of
compound 5o and PI3K were depicted in Figures 2 and 3. In the
binding mode, compound 5o is nicely bound to the PI3K via three
c. All docking runs
c
was a potential inhibitor of PI3Kc.
c
hydrogen bonds. The oxygen atom of 4-hydroxyl on the benzene
group forms a hydrogen bond with primary hydrogen atom of
LYS890 (H–Oꢀ ꢀ ꢀH: 2.04 Å, 112.2°), the oxygen atom of 3-methox-
yon the benzene group forms a hydrogen bond with primary
hydrogen atom of LYS890 (H-Oꢀ ꢀ ꢀH: 2.02 Å, 139.2°). The binding
3. Conclusions
In summary, a series of 1,3,4-oxadiazole derivatives have been
synthesized and their immunosuppressive activities against ConA
stimulated T cells proliferation were evaluated. Preliminary results
showed that most of the compounds displayed enhanced inhibi-
tory activities and low toxicity. Compounds 5o and 6f demon-
strated the most potent inhibitory activity that inhibited the
modes of compound 6f and PI3Kc were depicted in Figures 4 and
5. In the binding mode, compound 6f is nicely bound to the PI3K
c
via four hydrogen bonds. The oxygen atom of hydroxylon the ben-
zene group form hydrogen bond with primary hydrogen atom of
ConA stimulated T cells with IC50 = 1.25 lM and 4.75 lM for T cells

J.-F. Tang et al. / Bioorg. Med. Chem. 20 (2012) 4226–4236
4231
Figure 3. 3D model of the interaction between compound 5o and PI3Kc bonding
site. The protein is represented by molecular surface. Compound 5o is depicted by
Figure 5. 3D model of the interaction between compound 6f and PI3K
c
bonding
balls.
site. The protein is represented by molecular surface. Compound 6f is depicted by
balls.
and inhibited the activity of PI3K
c
with IC50 of 1.34
l
M. Moreover,
4. Experimental section
4.1. General
the preliminary mechanism of compound 5o inhibition effects was
also detected by flow cytometry (FCM), and the compound exerted
immunosuppressive activity via inducing the apoptosis of acti-
vated lymph node cells in a dose dependent manner. Molecular
docking study indicated that compound 5o and 6f was nicely
All chemicals used were purchased from Aldrich (USA). The
reaction was monitored by thin layer chromatography (TLC). Melt-
ing points (uncorrected) were determined on an XT4 MP apparatus
(Taike Corp., Beijing, China). ESI mass spectra were obtained on a
Mariner System 5304 mass spectrometer, and 1H NMR spectra
bound to the PI3K
the great potential of compounds 5o and 6f as an immunosuppres-
sant targeting PI3K
c with hydrogen bonds. All the results showed
c.
Figure 4. The molecular docking of compound 6f into PI3K
134.7°, H–Fꢀ ꢀ ꢀH: 2.05 Å, 111.9°, H–Fꢀ ꢀ ꢀH: 2.07 Å, 149.5°).
c
. Compound 6f is nicely bound to the PI3K via four hydrogen bonds (H–Oꢀ ꢀ ꢀH: 1.64 Å, 129.8°, H–Oꢀ ꢀ ꢀH: 1.85 Å,

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J.-F. Tang et al. / Bioorg. Med. Chem. 20 (2012) 4226–4236
gave a crude product, which was filtered off and purified by wash-
ing with saturated NaCl (40 mL) together with small quantity of
ethanol to give compound 3.
A
B
4.2.3. General procedure for synthesis of 2-methoxy-4-(1,3,4-
oxadiazol-2-yl)phenol (4)
To a solution of 3 (16 g, 87.9 mmol) in alcohol (100 mL), carbon
disulfide (45 mL), and potassium hydroxide (11.9 g, 21.2 mmol)
were added and the resulting solution was heated to reflux for
20 h. The reaction mixture was concentrated and the residue was
dissolved in water and acidified with hydrochloric acid. The result-
ing solid was filtered, dried and recrystallized from ethanol to af-
ford compound 4 (12 g, 62.5 mmol) as a white solid.
4.2.4. General procedure for synthesis of the target compounds
(5a–5q)
To a solution of 4(0.135 g, 0.7 mmol) in dry acetonitrile (10 mL),
dry potassium hydroxide (0.03 g, 0.59 mmol) and corresponding
benzyl bromides (0.7 mmol) were added and the resulting solution
was heated to 85 °C for 3 h. The mixture was concentrated and ta-
ken in ethyl acetate (50 mL), washed with water (20 mL), saturated
sodium chloride solution (20 mL) and dried over sodium sulfate.
The resulting solution was concentrated and the purification of
the residue by recrystallization from ethanol yielded the desired
compounds 5a–6q.
C
4.2.4.1. 2-Methoxy-4-(5-((4-nitrobenzyl)thio)-1,3,4-oxadiazol-
2-yl)phenol (5a).
Yellow solid. Yield: 68%. Mp: 151–153 °C.
1H NMR (CDCl3, 300 MHz): 3.98 (s, 3H), 4.57 (s, 2H), 5.99 (s, 1H),
7.01 (d, J = 8.25 Hz, 1H), 7.49 (d, J = 10.8 Hz, 2H), 7.68 (d,
J = 8.6 Hz, 2H), 8.21 (d, J = 8.44 Hz, 2H). MS (ESI): 358.26
(C16H14N3O5S, [M+H]+). Anal. Calcd for C16H13N3O5S: C, 53.48; H,
3.65; N, 11.69; O, 22.26; S, 8.92. Found: C, 53.34; H, 3.54; F, 5.75;
N, 11.74; O, 23.06; S, 8.89.
Figure 6. 1,3,4-Oxadiazole derivative inhibited ConA-induced IL-1, IL-6 and IL-10
secretion in Mouse lymph node cells, mouse lymph node cells were incubated in
DMEM media (Gibco) supplemented with 10% FBS, 100 U/mL penicillin, and100 g/
mL streptomycin at 37 °C with 5% CO2. Cells were pre-treated with 5 M of 1,3,4-
oxadiazole derivatives, analogues or vehicle control for 2 h, then treated with ConA
(5 g/mL) for 22 h. The culture media collected were centrifuged at 1000 rpm for
10 min. The levels of IL-1, IL-6 and IL-10 in the media were determined by ELISA
using mouse IL-1, mouse IL-6 and mouse IL-10 ELISA Kits (BOSTER, USA).
4.2.4.2.
methoxyphenol (5b).
4-(5-((3-Bromobenzyl)thio)-1,3,4-oxadiazol-2-yl)-2-
Yellow solid. Yield: 89%. Mp: 129–
131 °C. 1H NMR (300 MHz, CDCl3): 3.99 (s, 3H), 4.47 (s, 2H), 7.01
(d, J = 8.22 Hz, 1H), 7.19–7.27 (m, 1H), 7.40–7.46 (m, 2H), 7.51 (d,
J = 10.06 Hz, 2H), 7.63 (s, 1H). MS(ESI): 392.93 (C16H14BrN2O3S,
[M+H]+). Anal. Calcd for C16H13BrN2O3S: C, 48.87; H, 3.33; Br,
20.32; N, 7.12; O, 12.21; S, 8.15. Found: C, 49.12; H, 3.34; Br,
20.17; N, 7.31; O, 11.67; S, 8.64.
were recorded on a Bruker PX 500 or DPX 300 spectrometer at
25 °C with TMS and solvent signals allotted as internal standards.
Chemical shifts were reported in ppm (d). Element analyses were
performed on a CHN–O–Rapid instrument and were within 0.4%
of the theoretical values.
4.2.4.3.
diazol-2-yl)phenol (5c).
2-Methoxy-4-(5-((4-methylbenzyl)thio)-1,3,4-oxa-
White solid. Yield: 71%. Mp: 164–
166 °C. 1H NMR (300 MHz, CDCl3): 2.35 (s, 3H), 3.99 (s, 3H), 4.49
(s, 2H), 7.01 (d, J = 8.22 Hz, 1H), 7.16 (d, J = 7.86 Hz, 2H), 7.35 (d,
J = 8.04 Hz, 2H), 7.51 (dd, J = 8.22 Hz, J = 2.02 Hz, 1H), 7.54 (d,
J = 1.84 Hz, 1H). MS(ESI): 329.13 (C17H17N2O3S, [M+H]+). Anal.
Calcd for C17H16N2O3S: C, 48.87; H, 3.33; Br, 20.32; N, 7.12; O,
12.21; S, 8.15. Found: C, 49.12; H, 3.34; Br, 20.17; N, 7.31; O,
11.67; S, 8.64.
4.2. Chemistry
4.2.1. General procedure for synthesis of ethyl 4-hydroxy-3-
methoxybenzoate (2)
4-Hydroxy-3-methoxybenzoic acid (20 g, 119 mmol) in ethanol
(50 mL) was treated with concentrated sulfuric acid (3 mL) under
reflux for 10 h. The solvent was evaporated until no longer liquid
outflow. Water (20 mL) was added, extracting by adding respec-
tively 30 mL ethyl acetate. After washing the organic phase with
saturated NaCl solution (40 mL), drying it with anhydrous Na2SO4
and evaporating the solvent under reduced pressure. Crude prod-
uct were purified by column chromatography, product 2 appeared.
4.2.4.4. 2-Methoxy-4-(5-((2-nitrobenzyl)thio)-1,3,4-oxadiazol-
2-yl)phenol (5d).
Yellow solid. Yield: 84%. Mp: 144–146 °C.
1H NMR (300 MHz, CDCl3): 3.96 (s, 3H), 4.84 (s, 2H), 5.99 (s, 1H),
6.99 (d, J = 8.76 Hz, 1H), 7.46–7.50 (m, 3H), 7.57–7.62 (m, 1H),
7.86 (d, J = 6.78 Hz, 1H), 8.14 (d, J = 8.04 Hz, 1H). MS(ESI): 358.64
(C16H14N3O5S, [M+H]+). Anal. Calcd for C16H13N3O5S: C, 53.48; H,
3.65; N, 11.69; O, 22.26; S, 8.92. Found: C, 53.82; H, 3.67; N,
10.94; O, 23.02; S, 8.98.
4.2.2. General procedure for synthesis of 4-hydroxy-3-
methoxybenzohydrazide (3)
A stirred solution of compound 2 (50 mmol) in ethanol (50 mL)
was treated with 85% hydrazine hydrate (20 mL), under reflux for
20 h. After cooling, removal of the solvent under reduced pressure
4.2.4.5. 2-Methoxy-4-(5-((3-nitrobenzyl)thio)-1,3,4-oxadiazol-
2-yl)phenol (5e).
NMR (CDCl3, 300 MHz): 3.97 (s, 3H), 4.57 (s, 2H), 6.99 (d,
Yellow solid. Yield: 90%. Mp: 95–97 °C. 1H

J.-F. Tang et al. / Bioorg. Med. Chem. 20 (2012) 4226–4236
4233
J = 8.02 Hz, 1H), 7.47–7.61 (m, 3H), 7.85 (d, J = 7.68 Hz, 1H), 8.02 (s,
1H), 8.16 (d, J = 8.4 Hz, 1H), 8.35 (s, 1H). MS(ESI): 358.93
(C16H14N3O5S, [M+H]+). Anal. Calcd for C16H13N3O5S: C, 53.48; H,
3.65; N, 11.69; O, 22.26; S, 8.92. Found: C, 54.13; H, 3.64; N,
11.95; O, 22.02; S, 8.91.
(C16H14FN2O3S, [M+H]+). Anal. Calcd for C16H13FN2O3S:C, 57.82;
H, 3.94; F, 5.72; N, 8.43; O, 14.44; S, 9.65. Found: C, 57.79; H,
3.93; F, 5.74; N, 8.39; O, 14.44; S, 9.68.
4.2.4.13.
methoxyphenol (5m).
4-(5-((2-Chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)-2-
White solid. Yield: 91%. Mp: 160–
4.2.4.6.
methoxyphenol (5f).
4-(5-((2-Fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)-2-
White solid. Yield: 81%. Mp: 134–
162 °C. 1H NMR (CDCl3, 300 MHz): 3.97 (s, 3H), 4.52 (s, 2H), 6.97
(d, J = 8.22 Hz, 1H), 7.33–7.39 (m, 4H), 7.42–7.54 (m, 7H). MS(ESI):
348.80 (C16H14ClN2O3S, [M+H]+). Anal. Calcd for C16H13ClN2O3S: C,
55.09; H, 3.76; Cl, 10.16; N, 8.03; O, 13.76; S, 9.19. Found: C, 55.15;
H, 3.73; Cl, 10.22; N, 8.07; O, 13.67; S, 9.15.
136 °C. 1H NMR (CDCl3, 300 MHz): 3.98 (s, 3H), 4.53 (s, 2H), 5.98
(s, H), 7.00 (d, J = 8.04 Hz, 1H), 7.04–7.13 (m, 2H), 7.26–7.32 (m,
1H), 7.49–7.61 (m, 3H). MS(ESI): 331.35 (C16H14FN2O3S, [M+H]+).
Anal. Calcd for C16H13FN2O3S: C, 57.82; H, 3.94; F, 5.72; N, 8.43;
O, 14.44; S, 9.65. Found: C, 57.99; H, 3.93; F, 5.76; N, 8.41; O,
14.48; S, 9.62.
4.2.4.14.
diazol-2-yl)phenol (5n).
2-Methoxy-4-(5-((2-methylbenzyl)thio)-1,3,4-oxa-
White solid. Yield: 82%. Mp: 131–
133 °C. 1H NMR (CDCl3, 300 MHz): 2.47 (s, 3H), 3.96 (s, 3H), 4.56
(s, 2H), 7.01 (d, J = 8.04 Hz, 1H), 7.18–7.24 (m, 3H), 7.41 (d,
J = 6.78 Hz, 1H), 7.51–7.55(m, 2H). MS(ESI): 328.39 (C17H17N2O3S,
[M+H]+). Anal. Calcd for C17H16N2O3S: C, 62.18; H, 4.91; N, 8.53;
O, 14.62; S, 9.76. Found: C, 62.26H, 4.94 N, 8.53; O, 14.65; S, 9.68.
4.2.4.7.
4-(5-((4-Chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)-2-
methoxyphenol (5g).
White solid. Yield: 65%. Mp: 179–
181 °C. 1H NMR (CDCl3, 300 MHz): 3.95 (s, 3H), 4.46 (s, 2H), 5.16
(s, 2H), 6.92 (d, J = 8.4 Hz, 1H), 7.28–7.31 (m, 2H), 7.36–7.41 (m,
6H), 7.47 (dd, J = 8.26 Hz, J = 2.0 Hz, 1H), 7.51 (d, J = 2.04 Hz, 1H).
MS(ESI): 474.12 (C16H14ClN2O3S, [M+H]+). Anal. Calcd for
4.2.4.15. 4-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl)-2-methoxyphe-
C16H13ClN2O3S: C, 58.36; H, 3.83; Cl, 14.98; N, 5.92; O, 10.14; S,
nol (5o).
White solid. Yield: 75%. Mp: 114–116 °C. 1H NMR
6.77. Found: C, 59.03; H, 3.81; Cl, 14.99; N, 5.89; O, 10.25; S, 6.79.
(CDCl3, 300 MHz): 3.97(s, 3H), 4.47 (s, 2H), 6.05 (s, 1H), 7.02 (d,
J = 8.28 Hz, 1H), 7.47 (d, J = 10.8 Hz, 2H); 7.55–7.61 (m, 3H); 8.02
(d, J = 8.44 Hz, 2H). MS(ESI): 314.30 (C16H15N2O3S, [M+H]+). Anal.
Calcd for C16H14N2O3S: C, 61.13; H, 4.49; N, 8.91; O, 15.27; S,
10.20. Found: C,59.07; H, 4.50; N, 8.89.
4.2.4.8. 4-(5-((2,6-Difluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)-2-
methoxyphenol (5h).
White solid. Yield: 73%. Mp: 185–
187 °C. 1H NMR (CDCl3, 300 MHz): 3.98 (s, 3H), 4.54 (s, 2H),
6.89–6.94 (t, J = 7.77 Hz, 2H), 7.01 (d, J = 8.22 Hz, 1H), 7.26–7.30
(m, 2H), 7.51–7.55 (m, 2H). MS(ESI): 350.34 (C16H13F2N2O3S,
[M+H]+). Anal. Calcd for C16H12F2N2O3S: C, 54.85; H, 3.45; F,
10.85; N, 8.00; O, 13.70; S, 9.15. Found: C, 54.78; H, 3.46; F,
10.81; N, 7.95; O, 13.61; S, 9.11.
4.2.4.16.
diazol-2-yl)phenol (5p).
2-Methoxy-4-(5-((3-methoxybenzyl)thio)-1,3,4-oxa-
White solid. Yield: 75%. Mp: 93–
95 °C. 1H NMR (CDCl3, 300 MHz): 3.79 (s, 3H), 3.97 (s, 3H), 4.48
(s, 2H), 6.84 (d, J = 4.83 Hz, 1H), 6.98–7.05 (m, 3H), 7.35 (s, 1H),
7.46–7.51 (m, 3H). MS(ESI): 344.38 (C17H17N2O4S, [M+H]+). Anal.
Calcd for C17H16N2O4S: C, 59.29; H, 4.68; N, 8.13; O, 18.58; S,
9.31. Found: C, 59.19; H, 4.66; N, 8.15; O, 18.63; S, 9.29.
4.2.4.9. 4-(5-((2,4-Difluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)-2-
methoxyphenol (5i).
White solid. Yield: 69%. Mp: 142–
144 °C. 1H NMR (CDCl3, 300 MHz): 3.97 (s, 3H), 4.49 (s, 2H), 5.95
(s, 1H), 6.82–6.85 (m, 2H), 7.03 (d, J = 12.63 Hz, 1H), 7.47–7.55
(m, 3H). MS(ESI): 350.34 (C16H13F2N2O3S, [M+H]+). Anal. Calcd for
4.2.4.17.
4-(5-((4-Bromobenzyl)thio)-1,3,4-oxadiazol-2-yl)-2-
methoxyphenol (5q).
White solid. Yield: 78%. Mp: 186–
C16H12F2N2O3S: C, 54.85; H, 3.45; F, 10.85; N, 8.00; O, 13.70; S,
188 °C. 1H NMR (CDCl3, 300 MHz): 3.97 (s, 3H), 4.44 (s, 2H), 6.01
(s, 1H), 6.99 (d, J = 4.92 Hz, 1H), 7.33–7.36 (m, 2H), 7.45–7.49 (m,
4H). MS(ESI): 344.38 (C17H17N2O4S, [M+H]+). Anal. Calcd for
9.15. Found: C, 54.75; H, 3.43; F, 10.91; N, 8.02; O, 13.67; S, 9.19.
4.2.4.10.
4-(5-((3-Chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)-2-
C17H16N2O4S:C, 59.29; H, 4.68; N, 8.13; O, 18.58; S, 9.31. Found:
methoxyphenol (5j).
White solid. Yield: 79%. Mp: 133–
C, 59.16; H, 4.67; N, 8.15; O, 18.53; S, 9.29.
135 °C. 1H NMR (CDCl3, 300 MHz): 3.96 (s, 3H), 4.46 (s, 2H), 6.99
(d, J = 4.95 Hz, 1H), 7.26–7.27 (m, 2H), 7.34 (s, 1H), 7.46–7.51
(m, 3H). MS(ESI): 348.80 (C16H14ClN2O3S, [M+H]+). Anal. Calcd for
4.2.5. General procedure for synthesis of the target compounds
(6a–6q)
C
16H13ClN2O3S: C, 55.09; H, 3.76; Cl, 10.16; N, 8.03; O, 13.76; S,
To a solution of 4 (0.135 g, 0.7 mmol) in dry acetonitrile
(10 mL), dry potassium hydroxide (0.03 g, 0.59 mmol) and corre-
sponding benzyl bromides (1.4 mmol) were added and the result-
ing solution was heated to 85 °C for 3 h. The mixture was
concentrated and taken in ethyl acetate (50 mL), washed with
water (20 mL), saturated sodium chloride solution (20 mL) and
dried over sodium sulfate. The resulting solution was concentrated
and the purification of the residue by recrystallization from etha-
nol yielded the desired compounds 6a–6q.
9.19. Found: C, 54.99; H, 3.73; Cl, 10.18; N, 8.05; O, 13.73; S, 9.17.
4.2.4.11.
methoxyphenol (5k).
4-(5-((2-Bromobenzyl)thio)-1,3,4-oxadiazol-2-yl)-2-
White solid. Yield: 87%. Mp: 169–
171 °C. 1H NMR (CDCl3, 300 MHz): 3.98 (s, 3H), 4.62 (s, 2H), 5.95
(s, 1H), 6.99 (d, J = 4.95 Hz, 1H), 7.15–7.18 (m, 1H), 7.26–7.28 (m,
1H), 7.48–7.51 (m, 2H), 7.59 (d, J = 4.86 Hz, 1H), 7.63 (d,
J = 4.77 Hz, 1H). MS(ESI): 393.26 (C16H14BrN2O3S, [M+H]+). Anal.
Calcd for C16H13BrN2O3S: C, 48.87; H, 3.33; Br, 20.32; N, 7.12; O,
12.21; S, 8.15. Found: C, 48.91; H, 3.31; Br, 20.40; N, 7.13; O,
12.25; S, 8.22.
4.2.5.1.
2-(4-((3,5-Difluorobenzyl)oxy)-3-methoxyphenyl)-5-
White so-
((3,5-difluorobenzyl)thio)-1,3,4-oxadiazole (6a).
lid. Yield: 79%. Mp: 137–139 °C. 1H NMR (CDCl3, 300 MHz): 3.94
(s, 3H), 4.49 (s, 2H), 5.20 (s, 2H), 6.81–6.93 (m, 4H), 6.99 (d,
J = 8.22 Hz, 1H), 7.46–7.60 (m, 4H). MS(ESI): 476.52
(C23H17F4N2O3S, [M+H]+). Anal. Calcd for C23H16F4N2O3S: C,
57.98; H, 3.38; F, 15.95; N, 5.88; O, 10.07; S, 6.73. Found: C,
56.45; H, 3.36; F, 14.33; N, 5.84; O, 10.36; S, 6.79.
4.2.4.12.
methoxyphenol (5l).
4-(5-((4-Fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)-2-
White solid. Yield: 84%. Mp: 161–
163 °C. 1H NMR (CDCl3, 300 MHz): 3.99 (s, 3H), 4.48 (s, 2H), 6.99
(d, J = 5.04 Hz, 1H), 7.14 (d, J = 4.68 Hz, 2H), 7.33 (d, J = 4.77 Hz,
2H), 7.49 (d, J = 4.92 Hz, 1H), 7.53 (s, 1H). MS(ESI): 332.35

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J.-F. Tang et al. / Bioorg. Med. Chem. 20 (2012) 4226–4236
4.2.5.2.
2-(3-Methoxy-4-((2-methylbenzyl)oxy)phenyl)-5-((2-
4.2.5.9.
2-(4-((3-Bromobenzyl)oxy)-3-methoxyphenyl)-5-((3-
methylbenzyl)thio)-1,3,4-oxadiazole (6b).
Yellow solid.
bromobenzyl)thio)-1,3,4-oxadiazole (6i).
White solid. Yield:
Yield: 75%, Mp: 76–78 °C. 1H NMR (CDCl3, 300 MHz): 2.46(s, 3H),
3.94 (s, 3H), 4.55 (s, 2H), 5.17 (s, 2H), 6.98 (d, J = 8.22 Hz, 1H),
7.17–7.26 (m, 7H), 7.32 (s, 1H), 7.41 (d, J = 7.68 Hz, 2H), 7.49–
7.61 (m, 3H). MS(ESI): 431.92 (C25H25N2O3S, [M+H]+). Anal. Calcd
for C25H24N2O3S: C, 69.42; H, 5.59; N, 6.48; O, 11.10; S, 7.41.
Found: C, 68.89; H, 5.61; N, 6.65; O, 12.16; S, 7.38.
83%. Mp: 127–129 °C. 1H NMR (CDCl3, 300 MHz): 3.96 (s, 3H), 4.45
(s, 2H), 5.17 (s, 2H), 6.93 (d, J = 8.58 Hz, 1H), 7.18–7.26 (m, 2H),
7.36–7.53 (m, 6H), 7.61(s, 2H). MS(ESI): 565.03 (C23H19Br2N2O3S,
[M+H]+). Anal. Calcd for C23H18Br2N2O3S: C, 49.13; H, 3.23; Br,
28.42; N, 4.98; O, 8.54; S, 5.70. Found: C, 49.94; H, 3.25; Br,
27.82; N, 4.73; O, 8.84; S, 5.91.
4.2.5.3.
2-(4-((4-Bromobenzyl)oxy)-3-methoxyphenyl)-5-((4-
(6c). White solid.
4.2.5.10. 2-(4-((2-Bromobenzyl)oxy)-3-methoxyphenyl)-5-((2-
bromobenzyl)thio)-1,3,4-oxadiazole
bromobenzyl)thio)-1,3,4-oxadiazole (6j).
White solid. Yield:
Yield: 83%, Mp: 167–169 °C. 1H NMR (CDCl3, 300 MHz): 3.97 (s,
3H), 4.46 (s, 2H), 5.17 (s, 2H), 6.93 (d, J = 5.68 Hz, 1H), 7.32–7.37
(m, 4H), 7.46–7.54 (m, 6H). MS(ESI): 563.12 (C23H19Br2N2O3S,
[M+H]+). Anal. Calcd for C23H18Br2N2O3S: C, 49.13; H, 3.23; Br,
28.42; N, 4.98; O, 8.54; S, 5.70. Found: C, 49.94; H, 3.25; Br,
27.82; N, 4.73; O, 8.84; S, 5.91.
76%. Mp: 121–123 °C. 1H NMR (CDCl3, 300 MHz): 3.94 (s, 3H), 4.47
(s, 2H), 5.16 (s, 2H), 6.90 (d, J = 8.74 Hz, 1H), 7.16–7.24 (m, 2H),
7.35–7.52 (m, 6H), 7.63 (s, 2H). MS(ESI): 565.03 (C23H19Br2N2O3S,
[M+H]+). Anal. Calcd for C23H18Br2N2O3S: C, 49.13; H, 3.23; Br,
28.42; N, 4.98; O, 8.54; S, 5.70. Found: C, 49.85; H, 3.31; Br,
27.88; N, 4.69; O, 8.81; S, 5.94.
4.2.5.4.
2-(4-((2-Chlorobenzyl)oxy)-3-methoxyphenyl)-5-((2-
4.2.5.11.
2-((2,4-Difluorobenzyl)thio)-5-(4-(2,4-difluorophen-
White solid.
chlorobenzyl)thio)-1,3,4-oxadiazole
(6d).
White
solid.
oxy)-3-methoxyphenyl)-1,3,4-oxadiazole (6k).
Yield: 79%. Mp: 101–103 °C. 1H NMR (CDCl3, 300 MHz): 3.97 (s,
3H), 4.61 (s, 2H), 5.31 (s, 2H), 6.93 (d, J = 8.44 Hz, 1H), 7.22–7.26
(m, 2H), 7.27–7.28 (m, 2H), 7.40 (d, J = 9.34 Hz, 2H), 7.48 (dd,
Yield: 89%. Mp: 135–137 °C.1H NMR (CDCl3, 300 MHz): 3.94 (s,
3H), 4.49 (s, 2H), 6.82–6.87 (m, 3H), 6.88–6.92 (m, 1H), 6.99 (d,
J = 4.95 Hz, 1H), 7.47–7.55 (m, 4H). MS(ESI): 462.42
(C22H15F4N2O3S, [M+H]+). Anal. Calcd for C22H14F4N2O3S: C,
57.14; H, 3.05; F, 16.43; N, 6.06; O, 10.38; S, 6.93 Found:C, 57.19;
H, 3.06; F, 16.38; N, 6.08; O, 10.31; S, 6.96.
J = 8.4 Hz, J = 2.00 Hz, 1H), 7.54–7.55 (m, 2H), 7.62 (dd,
J
= 6.58 Hz, J = 2.56 Hz, 1H). MS(ESI): 472.74 (C23H19Cl2N2O3S,
[M+H]+). Anal. Calcd for C23H18Cl2N2O3S: C, 58.36; H, 3.83; Cl,
14.98; N, 5.92; O, 10.14; S, 6.77. Found: C, 58.67; H, 3.85; Cl,
13.69; N, 5.99; O, 10.44; S, 6.64.
4.2.5.12.
1,3,4-oxadiazole (6l).
2-(4-(Benzyloxy)-3-methoxyphenyl)-5-(benzylthio)-
White solid. Yield: 82%. Mp: 115–
4.2.5.5.
2-(4-((4-Chlorobenzyl)oxy)-3-methoxyphenyl)-5-((4-
White solid. Yield:
117 °C.1H NMR (CDCl3, 300 MHz): 3.97 (s, 3H), 4.52 (s, 2H), 5.23
(s, 2H), 6.97 (d, J = 8.22 Hz, 1H), 7.33–7.37 (m, 4H), 7.39 (m, 7H).
MS(ESI): 404.48 (C23H21N2O3S, [M+H]+). Anal. Calcd for
chlorobenzyl)thio)-1,3,4-oxadiazole (6e).
65%. Mp: 149–152 °C. 1H NMR (CDCl3, 300 MHz): 3.95 (s, 3H), 4.46
(s, 2H), 5.16 (s, 2H), 6.92 (d, J = 8.4 Hz, 1H), 7.28–7.31 (m, 2H),
7.36–7.41 (m, 6H), 7.47 (dd, J = 8.26 Hz, J = 2.0 Hz, 1H), 7.51 (d,
J = 2.04 Hz, 1H). MS(ESI): 474.12 (C23H19Cl2N2O3S, [M+H]+). Anal.
Calcd for C23H18Cl2N2O3S: C, 58.36; H, 3.83; Cl, 14.98; N, 5.92; O,
10.14; S, 6.77. Found: C, 59.03; H, 3.81; Cl, 14.99; N, 5.89; O,
10.25; S, 6.79.
C23H20N2O3S: C, 68.30; H, 4.98; N, 6.93; O, 11.87; S, 7.93 Found:C,
68.25; H, 4.95; N, 6.92; O, 11.91; S, 7.89.
4.2.5.13.
2-(3-Methoxy-4-((3-nitrobenzyl)oxy)phenyl)-5-((3-
nitrobenzyl)thio)-1,3,4-oxadiazole (6m).
White solid. Yield:
79%. Mp:149–151 °C. 1H NMR (CDCl3, 300 MHz): 3.97 (s, 3H), 4.57
(s, 2H), 5.28 (s, 2H), 6.95 (d, J = 5.13 Hz, 1H), 7.48–7.59 (m, 4H),
7.82 (dd, J = 18.93 Hz, J = 18.93 Hz, 2H), 8.18 (dd, J = 12.27 Hz,
4.2.5.6. 2-(4-((2-Fluorobenzyl)oxy)-3-methoxyphenyl)-5-((2-flu-
orobenzyl)thio)-1,3,4-oxadiazole (6f).
White solid. Yield:
J = 12.09 Hz, 2H), 8.34 (d,
J
= 3.75 Hz, 2H). MS(ESI):
81%. Mp: 98–100 °C. 1H NMR (CDCl3, 300 MHz): 3.95 (s, 3H), 4.53
(s, 2H), 5.27 (s, 2H), 6.98–7.18 (m, 5H), 7.26–7.32 (m, 2H),
7.49–7.54 (m, 4H). MS(ESI): 441.12 (C23H19F2N2O3S, [M+H]+). Anal.
Calcd for C23H18F2N2O3S: C, 62.72; H, 4.12; F, 8.63; N, 6.36; O,
10.90; S, 7.28. Found: C, 62.54; H, 4.17; F, 8.93; N, 6.19; O, 10.98;
S, 7.51.
494.48(C23H19N4O7S, [M+H]+). Anal. Calcd for C23H18N4O7S: C,
55.87; H, 3.67; N, 11.33; O, 22.65; S, 6.48 Found:C, 55.95; H,
3.64; N, 11.29; O, 22.71; S, 6.45.
4.2.5.14.
2-(3-Methoxy-4-((4-nitrobenzyl)oxy)phenyl)-5-((4-
White solid. Yield:
nitrobenzyl)thio)-1,3,4-oxadiazol (6n).
86%. Mp: 156–158 °C. 1H NMR (CDCl3, 300 MHz): 3.95 (s, 3H),
4.55 (s, 2H), 5.29 (s, 2H), 6.94 (d, J = 5.13 Hz, 1H), 7.45–7.56 (m,
4H), 7.81 (dd, J = 18.87 Hz, J = 18.96 Hz, 2H), 8.16 (dd,
J = 12.31 Hz, J = 12.04 Hz, 2H), 8.36 (d, J = 3.74 Hz, 2H). MS(ESI):
494.48 (C23H19N4O7S, [M+H]+). Anal. Calcd for C23H18N4O7S: C,
55.87; H, 3.67; N, 11.33; O, 22.65; S, 6.48 Found:C, 55.79; H,
3.69; N, 11.33; O, 22.68; S, 6.51.
4.2.5.7.
2-(4-((2,6-Difluorobenzyl)oxy)-3-methoxyphenyl)-5-
White so-
((2,6-difluorobenzyl)thio)-1,3,4-oxadiazole (6g).
lid. Yield: 73%. Mp: 123–136 °C. 1H NMR (CDCl3, 300 MHz): 3.91
(s, 3H), 4.54 (s, 2H), 5.24 (s, 2H), 6.89–6.96 (m, 4H), 7.11 (d,
J = 8.44 Hz, 1H), 7.26–7.38 (m, 2H), 7.54–7.57 (m, 2H). MS(ESI):
475.82 (C23H17F4N2O3S, [M+H]+). Anal. Calcd for C23H16F4N2O3S:
C, 57.98; H, 3.38; F, 15.95; N, 5.88; O, 10.07; S, 6.73. Found: C,
56.71; H, 3.38; F, 14.29; N, 5.83; O, 10.29; S, 6.69.
4.2.5.15. 2-(4-((3-Chlorobenzyl)oxy)-3-methoxy)phenyl)-5-((3-
chlorobenzyl)thio)-1,3,4-oxadiazole
(6o).
White
solid.
4.2.5.8. 2-(4-((4-Fluorobenzyl)oxy)-3-methoxyphenyl)-5-((4-flu-
Yield: 76%. Mp: 124–126 °C. 1H NMR (CDCl3, 300 MHz): 3.96 (s,
3H), 4.66 (s, 2H), 5.35 (s, 2H), 6.91 (d, J = 8.44 Hz, 1H), 7.26–7.32
(m, 2H), 7.28–7.29 (m, 2H), 7.44 (d, J = 9.34 Hz, 2H), 7.51 (dd,
orobenzyl)thio)-1,3,4-oxadiazole (6h).
White solid. Yield:
85%. Mp: 137–139 °C. 1H NMR (CDCl3, 300 MHz): 3.95 (s, 3H),
4.47 (s, 2H), 5.16 (s, 2H), 6.93–7.10 (m, 5H), 7.39–7.52
(m, 6H). MS (ESI): 442.07 (C23H19F2N2O3S, [M+H]+). Anal. Calcd
for C23H18F2N2O3S: C, 62.72; H, 4.12; F, 8.63; N, 6.36; O, 10.90;
S, 7.28. Found: C, 63.11; H, 4.15; F, 8.89; N, 6.29; O, 10.88; S,
7.34.
J = 8.3 Hz, J = 2.00 Hz, 1H), 7.56–7.57 (m, 2H), 7.65 (dd,
J
= 6.59 Hz, J = 2.52 Hz, 1H). MS(ESI): 472.74 (C23H19Cl2N2O3S,
[M+H]+). Anal. Calcd for C23H18Cl2N2O3S: C, 58.36; H, 3.83; Cl,
14.98; N, 5.92; O, 10.14; S, 6.77. Found: C, 58.62; H, 3.88; Cl,
13.73; N, 5.89; O, 10.43; S, 6.62.

J.-F. Tang et al. / Bioorg. Med. Chem. 20 (2012) 4226–4236
4235
4.2.5.16. 2-(3-Methoxy-4-((4-methylbenzyl)oxy)phenyl)-5-((4-
methylbenzyl)thio)-1,3,4-oxadiazole (6p). Yellow solid.
R&D Systems (Minneapolis, MN). PI3K
c
was incubated for 4 h at
room temperature with or without the presence of the oxadiazoles
derivatives, the final concentration of drug as 3 M, 10 M, 30
and 50 M. The results were summarized in Table 3.
Yield: 75%, Mp: 76–78 °C. 1H NMR (CDCl3, 300 MHz):2.67 (s, 3H),
3.95 (s, 3H), 4.56 (s, 2H), 5.16 (s, 2H), 6.99 (d, J = 8.24 Hz, 1H),
7.16–7.25 (m, 7H), 7.35 (s, 1H), 7.42 (d, J = 7.68 Hz, 2H), 7.48–
7.60 (m, 3H). MS(ESI): 431.92 (C25H25N2O3S, [M+H]+). Anal. Calcd
for C25H24N2O3S: C, 69.42; H, 5.59; N, 6.48; O, 11.10; S, 7.41.
Found: C, 68.69; H, 5.67; N, 6.63; O, 12.18; S, 7.33.
l
l
lM
l
4.7. Cell treatment and ELISA assay
Mouse lymph node cells were incubated in DMEM media
(Gibco) supplemented with 10% FBS, 100 U/mL penicillin, and
4.2.5.17. 2-(3-Methoxy-4-((3-methoxybenzyl)oxy)phenyl)-5-((3-
100
treated with 5
vehicle control for 2 h, then treated with ConA (5
l
g/mL streptomycin at 37 °C with 5% CO2. Cells were pre-
M of 1,3,4-oxadiazole derivatives, analogues or
g/mL) for
methoxybenzyl)thio)-1,3,4-oxadiazole (6q).
White solid.
l
Yield: 79%. Mp: 112–114 °C. 1H NMR (CDCl3, 300 MHz): 3.14 (s,
3H), 3.93 (s, 3H), 4.57 (s, 2H), 5.14 (s, 2H), 6.92 (d, J = 8.79 Hz,
2H), 7.11–7.22 (m, 5H), 7.36 (s, 2H), 7.51 (d, J = 7.64 Hz, 3H),
7.49–7.61 (m, 2H). MS(ESI): 431.92 (C25H25N2O3S, [M+H]+). Anal.
Calcd for C25H24N2O3S: C, 69.42; H, 5.59; N, 6.48; O, 11.10; S,
7.41. Found: C, 68.89; H, 5.61; N, 6.65; O, 12.16; S, 7.38.
l
22 h. The culture media collected were centrifuged at 1000 rpm
for 10 min. The levels of IL-1, IL-6 and IL-10 in the media were
determined by ELISA using mouse IL-1, mouse IL-6 and mouse
IL-10 ELISA Kits (BOSTER, USA). After centrifugation, collect use a
sterile container, centrifugation 20-min at the speed of 2000–
3000 rpm. Remove supernatant, detect the composition of cells, di-
lute cell suspension with PBS (pH 7.2–7.4). Cell concentration
reach 1 million/mL, repeated freeze-thaw cycles, damage cells
and release of intracellular components, centrifugation 20-min at
the speed of 2000–3000 rpm. Remove supernatant, if precipitation
appeared, centrifugal again. The results were shown in Figures 6.
4.3. Cytotoxicity test
Fresh lymph node cells were obtained from BALB/C mice. Cells
were cultured in a 96-well plate at a density of 5 ꢁ 105 cells per
well. Different compounds were added to each well respectively
at the concentration of 10
were used as the control. The incubation was performed in a
humidified, 37 °C, 5% CO2-containing incubator for 24 h. 20 L of
lM. Cells without treated by compounds
4.8. Molecular docking
l
MTT (Sigma, MO; 4 mg/mL in PBS) was added to each well 4 h be-
fore the end of the incubation. MTT formazan production was dis-
solved in 200 lL DMSO replacing the medium at 37 °C for 30 min.
The absorbance was read on an ELISA reader (Tecan, Austria) at
The PI3K
(3L54.pdb) was chosen as the template for the modeling study of
compounds 5o and 6f bound to PI3K . The crystal structure was
c-LXX protein–ligand complex crystal structure
c
obtained from the RCSB Protein Data Bank. The molecular docking
procedure was performed by using LigandFit protocol within Dis-
covery Studio 3.1. For ligand preparation, the 3D structures of 5o
and 6f were generated and minimized using Discovery Studio
3.1. For enzyme preparation, the hydrogen atoms were added,
570 nm (OD570).
4.4. Inhibitory activity assay
Fresh lymph node cells were obtained from BALB/C mice.
5 ꢁ 105 cells per well were cultured in a 96-well plate at the same
conditions to that mentioned above. The cultures were stimulated
and the water and impurities were removed. The whole PI3Kc 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 5o and 6f were placed during the molecular
docking procedure. Types of interactions of the docked enzyme
with ligand were analyzed after the end of molecular docking.
with 5
tive responses for 24 h. After that, cells were left untreated or trea-
ted with compounds at the concentration of 1 M for 72 h. Twenty
lg/mL of concanavalin A (ConA) to induce T cell prolifera-
l
microliters of MTT (Sigma, MO; 4 mg/mL in PBS) was added to each
well 4 h before the end of the incubation. After moving the super-
natant, 200 lL DMSO was added to dissolve the formazan crystals.
Acknowledgment
The absorbance at 570 nm (OD570) was read on an ELISA reader
(Tecan, Austria). Inhibitory activity of each compound was calcu-
lated using the following formula:
This work was financed by National Natural Science Foundation
of China (No. J1103512).
Inhibitory activity ¼ ½ControlðOD570 nmÞ
ꢂ CompoundsðOD570 nmÞꢃ=
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