Oxadiazole derivatives containing 1,4-benzodioxan as potential immunosuppressive agents against RAW264.7 cells

Article abstract of DOI:10.1016/j.bmc.2011.06.061

A series of oxadiazole derivatives containing 1,4-benzodioxan (4a-4s) have been first synthesized for their potential immunosuppressive activity. Among the compounds, compound 4i showed the most potent biological activity against RAW264.7 cells (inhibition = 37.66 ± 2.34% for NO overproduction and IC₅₀= 0.05 μM for iNOS). Docking simulation was performed to position compound 4i into the iNOS structure active site to determine the probable binding model. RT-PCR experiment results demonstrated that some of these compounds possessed good immunosuppressive activity against iNOS, especially for compound 4i. Therefore, compound 4i with potent inhibitory activity may be a potential agent.

Full text of DOI:10.1016/j.bmc.2011.06.061

Bioorganic & Medicinal Chemistry 19 (2011) 4895–4902
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Oxadiazole derivatives containing 1,4-benzodioxan as potential
immunosuppressive agents against RAW264.7 cells
⇑
⇑
Juan Sun, Ning Cao, Xiao-Min Zhang, Yu-Shun Yang, Yan-Bin Zhang, Xiao-Ming Wang , Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR 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 oxadiazole derivatives containing 1,4-benzodioxan (4a–4s) have been first synthesized for
their potential immunosuppressive activity. Among the compounds, compound 4i showed the most
potent biological activity against RAW264.7 cells (inhibition = 37.66 ± 2.34% for NO overproduction and
Received 27 May 2011
Revised 22 June 2011
Accepted 22 June 2011
Available online 28 June 2011
IC50 = 0.05 lM for iNOS). Docking simulation was performed to position compound 4i into the iNOS struc-
ture active site to determine the probable binding model. RT-PCR experiment results demonstrated that
some of these compounds possessed good immunosuppressive activity against iNOS, especially for com-
pound 4i. Therefore, compound 4i with potent inhibitory activity may be a potential agent.
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
Keywords:
Oxadiazole
Benzodioxan
Immunosuppressive activity
Inducible NOS
1
. Introduction
substituted 1,4-benzodioxan (B) is known as a nonsteroidal anti-
6
inflammatory drug (NSAID). WB 4104 (C) is recognized as a selec-
7
–11
Immunosuppressant is an important class of clinical drugs for an
tive a-adrenoceptor antagonist.
array of medical processes, including transplant rejection and treat-
ment of autoimmune diseases such as systemic lupus erythemato-
sus, rheumatoid arthritis, and psoriasis.1 Nitric oxide (NO), an
endogenous free radical is an important signaling molecule involved
ina widerangeofphysiologicalfunctions, aswellaspathophysiolog-
Besides, 1,3,4-oxadiazoles are an important class of heterocyclic
compounds. The widespread use of them as a scaffold in medicinal
chemistry establishes this moiety as a member of the privileged
,2
1
2
structures class. They possess a variety of biological activi-
1
3–17
ties.
In particular, a few differently substituted 1,3,4-oxadiaz-
18–20
ical states. NO is generated from
L-arginine by a family of nitric oxide
oles have been found to exhibit immunosuppressive activities.
synthases (NOSs) including major of isozymes, endothelial NOS
Further, 1,3,4-oxadiazole heterocycles are very good bioisosteres of
amides and esters, which can contribute substantially in increasing
pharmacological activity by participating in hydrogen bonding
3
(
eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). Both
eNOS and nNOS are constitutively expressed and produce NO at a
low level. However, iNOS is often expressed at high level and is
essentially unregulated once expressed. If activated by many immu-
nological stimuli such as lipopolysaccharide (LPS), interferon (IFN-c)
and a varietyof proinflammatory cytokines, iNOS produces a high le-
2
1,22
interactions with the receptors.
Recently, it was reported that a number of other compounds (D,
E and F) containing the 1,4-benzodioxan template showed potent
2
3–25
anti-inflammatory activity (Fig. 1).
However, to our knowledge,
4
vel of NO to exert defense against pathogens. It is well known that
few reports have been dedicated to the synthesis and iNOS inhibi-
tory activity of oxadiazole derivatives containing 1,4-benzodioxan.
Herein, in continuation to extend our research on compounds with
NO plays a major role in anti-inflammatory and immune reactions,
however, an extremely high level of NO induced by iNOS causes
inflammatory diseases such as rheumatoid arthritis. Therefore, as
drug development targets, inhibitors of NO overproduction and over
expression of iNOS might be beneficial for treatment of inflamma-
tory disorders caused by excessive production of NO.
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.5 The 6-position
2
6
iNOS inhibitory activity, we reported in the present work the syn-
thesis and structure-activity relationships of a series of oxadiazole
derivatives containing 1,4-benzodioxan as potential immunosup-
pressive agents. Biological evaluation indicated that some of the
synthesized compounds were potent inhibitors of iNOS.
2
2
. Results and discussion
.1. Chemistry
Nineteen oxadiazole derivatives containing 1,4-benzodioxan
4a–4s) were synthesized to screen for the immunosuppressive
⇑
Corresponding authors. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
(
0
968-0896/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.061

4
896
J. Sun et al. / Bioorg. Med. Chem. 19 (2011) 4895–4902
O
O
O
N
O
O
COOH
N
N
OCH3
OCH3
N
A
B
NH2
OCH3
O
O
N
H
O
H CO
3
C
O
O
O
O
O
O
COOH
COOH
COOH
N
E
D
F
Figure 1. The structure of compounds A–F.
activity. They were prepared in a four step process (Scheme 1)
and all were reported for the first time. Firstly, the 2,3-dihydro-
benzo[b][1,4] dioxine-6-carboxylic acid on treatment with
2.2.2. Inhibitory activity on NO production
To evaluate the inhibitory effects on the excessive NO produc-
tion, all the compounds were assayed using LPS-activated murine
macrophage-like RAW264.7 cells culture systems. The results were
summarized in Table 2. Among them, compound 4i (inhibi-
tion = 37.66%) exhibited significant immunosuppressive activity.
Structure–activity relationships in these oxadiazole derivatives
containing 1,4-benzodioxan demonstrated that compounds with
different acids substituents led to different immunosuppressive ac-
tivity, and the order was benzoic acid (4k) > phenylacetic acid (4d).
However, when introduced a withdrawing group, the situation was
changed. The compounds with Cl substituted in the phenylacetic
acid group (4m, 4l) exhibited excellent immunosuppressive activ-
ity compared with that in the benzoic acid group (4c), and substi-
tuent Cl at the para (4l) position showed a little stronger activity
than that at the meta (4m) position in the benzene ring. Mean-
2 4
methanol containing concentrated H SO was refluxed overnight.
This step can yield the corresponding ester. Secondly, the ester
was treated with hydrazine hydrate in ethanol overnight, reflux-
ing. Thirdly, the coupling reaction between the obtained com-
pound 3a and the different substituted phenyl acetic acid or
benzoic acid was performed by using carbodiimide hydrochlor-
2 2
ide and N-hydroxybenzotriazole in anhydrous CH Cl ,then re-
fluxed in phosphoryl chloride afforded the target compounds.
Or the coupling reaction between the obtained compound 3a
and the different substituted phenyl acetic acid or benzoic acid
was directly refluxed in phosphoryl chloride afforded the corre-
sponding target compounds 2-(2,3-dihydrobenzo[b][1,4]dioxin-
6
-yl)-5-substituted-1,3,4-oxadiazole. Then compounds 4a–4s
were obtained by subsequent purification with recrystallisation.
All of the synthetic compounds gave satisfactory analytical and
spectroscopic data, which were full accordance with their de-
picted structures.
while, a significant loss of activity was observed when the NO
2
substituent was introduced at the benzoic acid group (4e, 4f, 4o),
and the order of the activities is that substituent at the ortho (4o)
position > substituent at the para (4f) position > substituent at
the meta (4e) position. However, compound 4a (p-Br in the pheny-
lacetic acid group) displayed poor activity. On the contrary, when
introduced a donor group in the benzoic acid (4b, 4g) or phenyla-
cetic acid group (4n), a remarkable increase in the immunosup-
pressive activity was observed. Compound 4i with substituent of
pyridine group showed the most potent activity in these com-
pounds, interestingly, introduction of Br moiety, afforded 4j with
a remarkable decrease in the immunosuppressive activity. Besides,
compound 4h with definic bond substituted on benzene ring
showed better immunosuppressive activity than compound 4s
with the same chain length, however, a significant loss of activity
was observed when the carbon atom of the carbon chain was re-
placed by oxygen atom (4p).
2
2
.2. Biological activity
.2.1. Acute toxicity test
The inhibitory activity of the compounds is sometimes a result
of their toxic effect and consequently might cause an erroneous
2
7–29
conclusion,
prior to the following bioactivity analyses, all the
synthesized oxadiazole derivatives containing 1,4-benzodioxan
were evaluated using LPS-activated murine macrophages
30
RAW264.7 for acute toxicity test. The pharmacological results
were summarized in Table 1. The data showed that most of the ox-
adiazole derivatives containing 1,4-benzodioxan moiety having a
quite low toxicity.

J. Sun et al. / Bioorg. Med. Chem. 19 (2011) 4895–4902
4897
O
O
O
O
O
CH3
OH
i
O
O
1
2
ii
O
O
H
N
O
O
R
O
O
NH2
iv
N
H
N
H
O
3
b
3a
v
iii
N
N
R
O
O
O
4a-4s
NO2
Br
4f R=
4k R=
4p R=
4
4
a R=
O
CH3
OCH3
OCH3
OCH3
Br
4q R=
b R=
4l R=
4g R=
Cl
Cl
Cl
Cl
4
4
c R=
4h R=
4m R=
4n R=
4
4
r R=
O
N
d R=
4i R=
s R=
OCH3
Br
O N
2
4
e R=
4j R=
4o R=
NO2
N
Scheme 1. General synthesis of compounds 4a–4s. Reagents and conditions: (i) methanol, concentrated sulfuric acid; 90 °C; (ii) NH
aromatic carboxylic acids, POCl ; 100 °C.
; 110 °C; iv) EDCꢀHCl, HOBt, dichloromethane, rt; (v) POCl
2
NH
2 2
ꢀH O, ethanol; 90 °C; (iii) aliphatic or
3
3
2
.2.3. iNOS inhibitory assay
The iNOS inhibitory potency of the oxadiazole derivatives con-
tent inhibitory with IC50 of 0.05 lM. The results of iNOS inhibitory
activity of the tested compounds were corresponding to the struc-
ture relationships (SAR) of their inhibitory effects on the excessive
NO production. This demonstrated that the potent inhibitory ef-
fects on the excessive NO production activities of the synthetic
taining 1,4-benzodioxan were examined and the results were sum-
marized in Table 3. Most of the tested compounds displayed potent
iNOS inhibitory. Among them, compound 4i showed the most po-

4
898
J. Sun et al. / Bioorg. Med. Chem. 19 (2011) 4895–4902
Table 1
The acute toxicity test, most of the compounds exhibited nontoxic
Compound
OD540
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
0.99
1.03
1.45
0.45
0.73
0.57
1.02
1.15
1.31
1.22
1.50
0.20
0.63
0.76
0.30
0.36
0.94
0.89
0.10
1.17
0.83
s
LPS
Control
compounds were probably correlated to their iNOS inhibitory
activities.
Table 2
The effect of compounds on LPS-induced NO
production in RAW264.7 cells
2
.2.4. RT-PCR experiment
Compound
Inhibition (%)
The high level of NO is mainly generated by iNOS, so iNOS ex-
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
ꢁ8.53 ± 5.52
21.58 ± 3.71
ꢁ57.48 ± 8.32
ꢁ3.63 ± 2.17
ꢁ33.82 ± 6.25
1.34 ± 1.12
20.03 ± 2.14
25.54 ± 1.98
37.66 ± 2.34
ꢁ42.39 ± 7.92
5.08 ± 0.35
23.70 ± 2.84
28.11 ± 1.94
21.66 ± 4.32
3.65 ± 3.12
6.98 ± 2.15
6.57 ± 2.41
pression is one of the key steps during the process of LPS-activated
NO production. In an effort to study the preliminary mechanism of
the compounds with potent inhibitory activity, the RT-PCR experi-
Table 3
iNOS inhibitory activity of synthetic
compounds
Compound
IC50 (lM)
4
4
4
4
4
4
4
b
g
h
i
k
q
r
0.10 ± 3.03
0.12 ± 2.12
0.09 ± 0.78
0.05 ± 1.14
2.35 ± 2.32
2.02 ± 2.01
2.16 ± 0.12
>100
10.49 ± 1.95
3.71 ± 3.03
s
LPS
LPS
Control
ꢁ30.97 ± 4.13
Control
0.00
0.00 ± 5.76

J. Sun et al. / Bioorg. Med. Chem. 19 (2011) 4895–4902
4899
LPS+100 µM
4g 4h
Ctrl LPS 4b
4i
iNOS
Actin
Figure 4. The RT-PCR experiment was performed to assay the effect of selected
compounds on mRNA expression of iNOS.
group project towards the amino hydrogens of GLY 406 and GLY
o
3
72, forming two H-bond interactions, H–OꢀꢀꢀH: 1.81 Å, 137.81
o
Figure 2. Molecular docking modeling of compound 4i with inducible NOS:
compound 4i was nicely bound to the iNOS with its nitrogen atom of pyridine group
project towards the amino hydrogen of GLY 391, with the hydroxyl group forming a
and H–OꢀꢀꢀH: 2.09 Å, 144.55 , respectively. Meanwhile,
p-cation in-
teraction was shown as yellow column. This molecular docking re-
sult, along with the biological assay data, suggesting that
compound 4i was a potential inhibitor of iNOS.
o
more optimal H-bond (H–NꢀꢀꢀH: 1.95 Å, 167.83 ) interaction, and the oxygen atom
o
of benzene group of 4i also forms hydrogen bond (H–OꢀꢀꢀH: 2.08 Å, 156.72 ) with
hydrogen atom of GLN 190. Besides, the oxygen atom of oxadiazole group project
towards the amino hydrogens of GLY 406 and GLY 372, forming two H-bond
3
. Conclusions
o
o
interactions, H–OꢀꢀꢀH: 1.81 Å, 137.81 and H-O...H: 2.09 Å, 144.55 , respectively.
Meanwhile, -cation interactions were shown as yellow column.
p
A series of new oxadiazole derivatives containing 1,4-benzo-
dioxan moiety were synthesized. Preliminary results showed that
most of the compounds displayed enhanced inhibitory activities
and low toxicity. Compound 4i demonstrated the most potent in-
hibitory activity that inhibited the excessive NO production of
RAW264.7 cells with inhibition of 37.66 ± 2.34% and inhibited
ment was performed to assay the effect of four selected com-
pounds on mRNA expression of iNOS.31 The RT-PCR results were
summarized in Figure 4. Among them, compound 4i strongly in-
hibited the expression of iNOS mRNA. However, the other com-
pounds expressed little inhibitory activity, suggesting that they
might inhibit NO production through other mechanisms.
the activity of iNOS with IC50 of 0.05 lM.
In order to gain more understanding of the structure–activity
relationships observed at the iNOS, molecular docking of the most
potent inhibitor 4i into the binding site of iNOS was performed on
the binding model based on the iNOS complex structure. Analysis
of the compound 4i’s binding conformation demonstrated that
compound 4i was stabilized by hydrogen bonding interaction with
2
.3. Binding model of compounds 4i into iNOS structure
In an effort to elucidate the possible mechanism by which the
GLN190, GLY 372, GLY 391 and GLY 406, as well as four
p-cation
title compounds can induce immunosuppressive activity against
RAW264.7 cells and guide further SAR studies, molecular docking
of the potent inhibitor 4i into binding site of iNOS were performed
on the binding model based on the iNOS complex structure
interactions. RT-PCR results showed the compound 4i was a poten-
tial agent, as well.
(
1M9T.pdb). The binding models of compound 4i and iNOS were
depicted in Figure 2 and Figure 3. In the binding model, compound
i was nicely bound to the iNOS with its nitrogen atom of pyridine
4. Experimental section
4.1. Methods of synthesis
4
group project towards the amino hydrogen of GLY 391, with the
hydroxyl group forming a more optimal H-bond (H–NꢀꢀꢀH: 1.95 Å,
All chemicals used were purchased from Aldrich (USA). The elu-
ates were monitored using TLC. Melting points (uncorrected) were
determined on a XT4MP apparatus (Taike Corp., Beijing, China).
ESI mass spectra were obtained on a Mariner System 5304 mass
o
1
67.83 ) interaction, and the oxygen atom of benzene group of 4i
o
also forms hydrogen bond (H–OꢀꢀꢀH: 2.08 Å, 156.72 ) with hydro-
gen atom of GLN 190. Besides, the oxygen atom of oxadiazole
Figure 3. 3D model of the interaction between compound 4i and the inducible NOS binding site. The inducible NOS was represented by molecular surface. Compound 4i was
depicted by sticks and balls.

4
900
J. Sun et al. / Bioorg. Med. Chem. 19 (2011) 4895–4902
spectrometer, and 1H NMR spectra were recorded on a Bruker
DPX300 spectrometer at 25 °C with TMS and solvent signals allotted
as internal standards, Chemical shifts are reported in ppm (d). Ele-
mental analyses were performed on a CHN–O–Rapid instrument
and were within 0.4% of the theoretical values.
(300 MHz, CDCl
1H); 7.28–7.33 (m, 1H); 7.34–7.36 (m, 4H); 7.48–7.51 (m, 2H).
3
): 4.25 (s, 2H); 4.27–4.32 (m, 4H); 6.91–6.94 (m,
+
MS (ESI): 295.10 (C17
H
15
N
2
O
3
,
[M+H] ). Anal. Calcd for
C H
17 14
N
2
O
3: C, 69.38; H, 4.79; N, 9.52. Found: C, 69.64; H, 4.99;
N, 9.36.
4
.1.1. Synthesis of 2,3-dihydrobenzo[b][1,4]dioxine-6-carboxy-
late (2)
The 2,3-dihydrobenzo[b][1,4]dioxine-6-carboxylic acid (9.0 g,
0 mmol) in methanol (50 mL) containing concentrated H SO
5 mL) was refluxed overnight. Water (100 mL) was added, the or-
ganic phases were washed with saturated NaCl (100 mL) and dried
over Na SO , and the solvents were evaporated.
4
.1.4.5.
2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(3-nitro-
phenyl)-1,3,4-oxadiazole (4e).
2
White powder. Mp: 236–
1
37 °C. H NMR (300 MHz, CDCl
3
): 4.33–4.36 (m, 4H); 7.00–7.03
5
(
2
4
(
m, 1H); 7.65–7.68 (m, 2H); 7.72–7.75 (m, 1H); 8.39–8.41 (d,
J = 5.7 Hz, 1H); 8.47–8.50 (d, J = 7.9 Hz, 1H); 8.92 (s, 1H). MS
+
(
ESI): 326.07 (C16
12 3 5 11 3 5:
H N O , [M+H] ). Anal. Calcd for C16H N O
2
4
C, 59.08; H, 3.41; N, 12.92. Found: C, 59.33; H, 3.69; N, 12.76.
4
.1.2. Synthesis of 2,3-dihydrobenzo[b][1,4]dioxine-6-carbo-
4
.1.4.6. 2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(4-nitrophe-
hydrazide (3a)
neyl)-1,3,4-oxadiazole (4f).
White powder. Mp: 273–275 °C.
To compound 2 (0.01 mol) dissolved in dry ethanol (50 mL) 99%
hydrazine hydrate (1 mL) was added and the mixture was refluxed
for 8–10 h. The reaction mixture was cooled and the solid obtained
was filtered, washed with small quantity of ethanol to give 3a.
1
H NMR (300 MHz, CDCl
3
): 3.48–3.50 (m, 4H); 4.32–4.35 (m,
3
H); 6.91–7.04 (m, 1H); 7.60–7.67 (m, 1H); 8.30–8.43 (m, 2H).
+
MS (ESI): 326.07 (C16
C H N O
16 11 3
H
12
N
3
O
5
,
[M+H] ). Anal. Calcd for
5: C, 59.08; H, 3.41; N, 12.92. Found: C, 59.36; H, 3.11;
N, 12.65.
4
.1.3. Synthesis of N-substituted-2,3-dihydrobenzo[b][1,4]
dioxine-6-carbohydrazide (3b)
4
.1.4.7. 2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(3,4,5-trime-
2 2
A stirred solution of compound 3a (0.1 mol) in CH Cl (50 mL)
thoxyphenyl)-1,3,4-oxadiazole (4g).
2
4
White powder. Mp:
): 3.93–3.97 (m, 9H); 4.21–
was treated with the appropriate substituted phenyl acetic acid
or benzoic acid, EDCꢀHCl (0.15 mol), HOBt (0.05 mol) and refluxed
overnight. Then purification with recrystallisation afforded the
corresponding compound as white powder.
1
05–206 °C. H NMR (300 MHz, CDCl
3
.34 (m, 4H); 6.97–7.02 (m, 1H); 7.26–7.35 (m, 2H); 7.64–7.65
+
(
m, 2H). MS (ESI): 371.12 (C19
H
19
N
2
O
6
, [M+H] ). Anal. Calcd for
C
19 18 2
H N O6: C, 61.62; H, 4.90; N, 7.56. Found: C, 61.93; H, 4.68;
N, 7.78.
4
.1.4. Synthesis of 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-
substituted-1,3,4-oxadiazole. (4a–4s)
4
.1.4.8. 2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-styryl-1,3,4-
General method. An equimolar compound 3a (0.001 mol) and
substituted carboxylic acid in phosphoryl chloride was refluxed
for 10–15 h. Or compound 3b in phosphoryl chloride was refluxed
for 5–7 h. Then reaction mixture was cooled, poured into ice-cold
water and neutralized with 20% NaHCO solution. The resultant so-
3
lid was filtered, washed with water and recrystallized from ethanol
to give the title compounds.
1
oxadiazole (4h).
Yellow powder. Mp: 131–132 °C. H NMR
(
300 MHz, CDCl
3
): 4.33(s, 4H); 6.98–7.00 (d, J = 8.9 Hz, 1H); 7.06–
7
.11 (d, J = 16.8 Hz, 1H); 7.38–7.43 (m, 3H); 7.57–7.64 (m, 5H).
+
MS (ESI): 307.10 (C18
C H N O
18 14 2
N, 8.89.
H
15
N
2
O
3
,
[M+H] ). Anal. Calcd for
3: C, 70.58; H, 4.61; N, 9.15. Found: C, 70.77; H, 4.87;
4
.1.4.9. 2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(pyridin-3-
4
6
.1.4.1. 2-(4-Bromophenyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-
-yl)-1,3,4-oxadiazole (4a). White powder. Mp: 140–141 °C.
): 4.30 (s, 2H); 4.32–4.33 (m, 4H); 6.92–
yl)-1,3,4-oxadiazole (4i).
White powder. Mp: 170–171 °C.
1
H NMR (300 MHz, CDCl
3
): 4.32–4.36 (m, 4H); 7.00–7.03 (m,
1
H NMR (300 MHz, CDCl
3
1
8
H); 7.46–7.50 (m, 1H); 7.63–7.66 (m, 2H); 8.39–8.43 (m, 1H);
6
.95 (m, 1H); 7.21–7.24 (d, J = 5.4 Hz, 2H); 7.46–7.50 (m, 4H). MS
+
12 3 3
.78 (s, 1H); 9.34 (s, 1H). MS (ESI): 282.08 (C15H N O , [M+H] ).
+
(
ESI): 373.01 (C17
H14BrN
O
2 3
,
[M+H] ). Anal. Calcd for
11 3
Anal. Calcd for C15H N O3: C, 64.05; H, 3.94; N, 14.94. Found: C,
6
C
17
2
H13BrN O3: C, 54.71; H, 3.51; N, 7.51. Found: C, 54.43; H,
4.40; H, 3.82; N, 14.69.
3
.90; N, 7.26.
4
.1.4.10. 2-(5-Bromopyridin-3-yl)-5-(2,3-dihydrobenzo[b][1,4]
4
.1.4.2. 2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-o-tolyl-1,3,4-
dioxin-6-yl)-1,3,4-oxadiazole(4j).
White powder. Mp: 222–
): 4.34 (s, 4H); 7.07–7.10 (d,
1
oxadiazole (4b).
White powder. Mp: 116–117 °C. H NMR
1
2
23 °C. H NMR (300 MHz, DMSO-d
6
(
300 MHz, CDCl
3
): 2.76 (s, 3H); 4.34 (s, 4H); 6.98–7.01 (m, 1H);
J = 9.0 Hz, 1H); 7.65–7.67 (m, 2H); 8.74 (s, 1H); 8.93–8.94 (d,
J = 2.0 Hz, 1H); 9.25–9.26 (d, J = 1.5 Hz, 1H); MS (ESI): 359.99
7
1
C
.34–7.42 (m, 3H); 7.63–7.65 (m, 2H); 8.01–8.03 (d, J = 7.5 Hz,
H). MS (ESI): 295.10 (C17
+
H
15
N
2
O
3
, [M+H] ). Anal. Calcd for
+
(
C
15
H
11BrN
3
O
3
, [M+H] ). Anal. Calcd for C15
H
10BrN
3
O3: C, 50.02;
17
14 2
H N O3: C, 69.38; H, 4.79; N, 9.52. Found: C, 69.14; H, 4.99;
H, 2.80; N, 11.67. Found: C, 50.34; H, 2.49; N, 11.97.
N, 9.21.
4
1
.1.4.11.
,3,4-oxadiazole (4k).
2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-phenyl-
4
.1.4.3. 2-(2-Chlorophenyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-
1
Yellow powder. Mp: 162–161 °C.
H
6
-yl)-1,3,4-oxadiazole (4c).
White powder. Mp: 121–123 °C.
NMR (300 MHz, CDCl
3
): 4.33–4.34 (m, 4H); 6.98–7.01 (m, 1H);
1
H NMR (300 MHz, DMSO-d
6
): 4.35–4.35 (m, 4H); 7.09–7.12 (d,
7
.52–7.54 (m, 3H); 7.63–7.66 (m, 2H); 8.10–8.13 (m, 2H). MS
J = 8.4 Hz, 1H); 7.59–7.60 (m, 1H); 7.60–7.61 (m, 2H); 7.62–7.68
+
(
ESI): 281.08 (C16
H
13
N
2 3 12 2 3:
O , [M+H] ). Anal. Calcd for C16H N O
(
(
C
m, 1H); 7.72–7.75 (d, J = 7.8 Hz, 1H); 8.11–8.14 (m, 1H). MS
C, 68.56; H, 4.32; N, 9.99. Found: C, 68.79; H, 4.66, N, 10.21.
+
ESI): 315.05 (C16
11ClN 3: C, 61.06; H, 3.52; N, 8.90. Found: C, 61.35; H, 3.83;
N, 8.68.
H12ClN
2
O
3
,
[M+H] ). Anal. Calcd for
16
H
2
O
4
6
.1.4.12. 3-(3-Chlorobenzyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-
1
-yl-4H-pyrazole (4l).
): 4.22 (s, 2H); 4.27–4.33 (m, 4H); 6.92–6.95 (m,
H); 7.21–7.23 (m, 1H); 7.27–7.29 (m, 2H); 7.35 (s, 1H); 7.48–
White powder. Mp: 75–76 °C. H NMR
(
300 MHz, CDCl
3
4
.1.4.4. 2-Benzyl-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-
1
7
1
oxadiazole (4d).
White powder. Mp: 82–83 °C. H NMR
+
2 3
.51 (m, 2H). MS (ESI): 329.06 (C17H14ClN O , [M+H] ). Anal. Calcd

J. Sun et al. / Bioorg. Med. Chem. 19 (2011) 4895–4902
4901
for C17
H
13ClN
2
O
3: C, 62.11; H, 3.99; N, 8.52. Found: C, 62.37; H,
was prepared in dimethyl sulphoxide (DMSO) followed by dilution
with culture medium to desired concentrations, and DMSO final
concentration was 0.1%. DMSO at 0.1% was added into control
(RAW264.7 cells were treated with LPS only) and blank
(RAW264.7 cells only, without any treatments) groups and showed
no effects on cells.
4
.21; N, 8.77.
4
6
1
4
7
.1.4.13. 2-(4-Chlorobenzyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-
-yl)-1,3,4-oxadiazole (4m).
10 °C. H NMR (300 MHz, CDCl
H); 6.92–6.95 (m, 1H); 7.01–7.06 (m, 2H); 7.29–7.34 (m, 2H);
.47–7.50 (m, 2H). MS (ESI): 329.06 (C17
13ClN 3: C, 62.11; H, 3.99; N, 8.52. Found: C, 62.33;
H, 4.24; N, 8.33.
Yellow powder. Mp: 109–
3
): 4.22 (s, 2H); 4.27–4.32 (m,
1
4
Cells were plated at a density of 5 ꢂ 10 cells in a 96-well plate,
+
H14ClN
2
O
3
, [M+H] ). Anal.
and compounds were added to each plate at the indicated concen-
Calcd for C17
H
2
O
trations. After a 24 h incubation period, 20 lL MTT reagent (5 mg/
mL) was added, and the cells were incubated for 4 h. The superna-
tants were aspirated, and the formazan crystals in each well were
4
.1.4.14. 2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(3-methoxy
benzyl)-1,3,4-oxadiazole (4n) White powder. Mp: 82–83 °C.
): 3.80 (s, 3H); 4.22 (s, 2H); 4.27–4.31 (m,
H); 6.82–6.84 (m, 1H); 6.85 (s, 1H); 6.91–6.94 (m, 2H); 7.23–7.28
dissolved in 200 lL of dimethyl sulfoxide for 30 min at 37 °C. The
absorbance value was monitored by microplate reader at 540 nm.
.
1
H NMR (300 MHz, CDCl
3
4
4.3. Inhibitory activity on NO production
+
(
m, 1H); 7.48–7.51 (m, 2H). MS (ESI): 325.11 (C18
H
17
2
N O
4
, [M+H] ).
Anal. Calcd for C18
H
16
N
2
O
4: C, 66.66; H, 4.97; N, 8.64. Found: C,
The activity of the prepared compounds 4a–4s against
32
6
6.91; H, 4.74; N, 8.79.
RAW264.7 cells was evaluated as described in the literature with
some modifications. Target cells were grown to log phase in RPMI
1640 medium supplemented with 10% fetal bovine serum. Cells
4
.1.4.15. 2-((4-Chlorophenoxy)methyl)-5-(2,3-dihydrobenzo[b]
5
[
1
1,4]dioxin-6-yl)-1,3,4-oxadiazole (4o).
45–146 °C. H NMR (300 MHz, CDCl
White powder. Mp:
were incubated in a 96-well plate at a density of 5 ꢂ 10 cells
1
3
): 4.31–4.35 (m, 4H); 5.30
per well and were left untreated or treated with compounds at var-
(
s, 2H); 6.97–7.02 (m, 3H); 7.30–7.31 (m, 2H); 7.56–7.59 (m, 2H).
ious concentrations (0.5–100 lM) for 24 h. For the proliferation as-
+
MS (ESI): 345.06 (C17
13ClN 4: C, 59.23; H, 3.80; N, 8.13. Found: C, 59.46; H, 3.62;
N, 8.42.
H14ClN
2
O ,
4
[M+H] ). Anal. Calcd for
say, 20 mL MTT (Sigma, 4 mg/mL in PBS) was added per well 4 h
before the end of incubation. After removing the supernatant,
200 mL DMSO was added to dissolve the formazan crystals. The
absorbance at 540 nm (OD540) was read on an ELISA reader (Tecan,
Austria).
C
17
H
2
O
4
1
(
.1.4.16. 2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-phenethyl-
1
,3,4-oxadiazole (4p). White powder. Mp: 79–80 °C. H NMR
NO production was determined by measuring the accumulation
of nitrite in the culture supernatant using the Griess reagent
300 MHz, CDCl
3
): 3.18–3.25 (m, 4H); 4.31–4.33 (m, 4H); 6.94–
6
.96 (d, J = 8.4 Hz, 1H); 7.23 (s, 1H); 7.31–7.34 (m, 3H); 7.48–7.49
according to manufacturer’s instructions. Briefly, 100
supernatant was transferred into a new 96-well plate and mixed
with 100 L of Griess reagent. After 10 min, the absorbance value
lL culture
+
(
m, 1H); 7.52 (s, 2H). MS (ESI): 309.12 (C18
17
H N
2
O
3
, [M+H] ). Anal.
Calcd for C18
H
16
N
2
O3: C, 70.12; H, 5.23; N, 9.09. Found: C, 70.36; H,
l
5
.03; N, 9.35.
at 540 nm was collected by microplate reader. NO inhibitory activ-
ity was calculated using the following formula:
4
.1.4.17. 2-(2,3- Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(2-nitro-
Mp: 122–
): 4.29–4.35 (m, 4H); 6.96–6.99
m, 1H); 7.54–7.57 (m, 2H); 7.70–7.81 (m, 2H); 8.00–8.09 (m, 2H).
NOinhibitoryactivity¼½ControlðOD540nm
Þ
phenyl)-1,3,4-oxadiazole (4q). Yellow powder.
1
1
24 °C. H NMR (300 MHz, CDCl
3
ꢁCompoundsðOD540nmÞ=ðControlðOD540nmÞꢃ
ꢂ100%
(
+
MS (ESI): 326.07 (C16
12 3 5 11 3 5:
H N O , [M+H] ). Anal. Calcd for C16H N O
C, 59.08; H, 3.41; N, 12.9. Found: C, 59.33; H, 3.74; N, 12.69.
4
.1.4.18. 2-(2,3- Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(phenoxy-
4.4. INOS inhibitory assay
methyl)-1,3,4-oxadiazole (4i).
White powder. Mp: 186–
): 4.27–4.32 (m, 4H); 4.68 (s,
1
1
87 °C. H NMR (300 MHz, CDCl
3
Mouse RAW 264.7 cells were grown in RPMI 1640 medium
supplemented by 10% fetal bovine serum under 5% CO atmo-
2
H); 6.91–6.93 (d, J = 8.2 Hz, 1H); 6.96–6.99 (m, 2H); 7.02–7.07
2
(
(
m, 1H); 7.31–7.37 (m, 3H); 7.39–7.40 (m, 1H). MS (ESI): 311.10
sphere at 37 °C. Then, LPS and interferon- were added to this
medium, the final concentrations were 0.2 lg/mL and 100 unit/
c
+
C
17
H
15
N
2
O
4
, [M+H] ). Anal. Calcd for C17
H
14
N
2
O
4: C, 65.80; H,
4
.55; N, 9.03. Found: C, 65.99; H, 4.31; N, 8.83.
mL, respectively. Tris and dithiothreitol was added to this cell
culture after the grown cells were collected from this medium,
4
.1.4.19. 3-(Bromometyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-
with the final concentration of 50 mM and 100 lM, respectively.
1
yl-4H-pyrazole (4s).
300 MHz, CDCl ): 4.33–4.34 (m, 4H); 4.59 (s, 1H); 4.77 (s, 1H);
.98–7.01 (m, 1H); 7.57–7.64 (m, 2H). MS (ESI): 295.00
White powder. Mp: 118–120 °C. H NMR
And then the culture was centrifuged at 10,000g for 10 min.
After that Dowex HCRW2 added to the obtained supernatant
and stirred for 30 min at 4 °C. The obtained supernatant was
(
6
3
+
(
C
12
2
H12BrN O
2
, [M+H] ). Anal. Calcd for C12
2
H11BrN O
2: C, 48.84;
used as crude enzyme. 20
of NADPH, 10 L of compounds, 20
nine (0.5 M) and 80 L of Tris (pH 7.5) were added into the
70 mL obtained crude enzyme. After incubated for 30 min at
7 °C, 200 L of 0.1 M (pH 5.0) including 2 mM EDTA and
lL of Tris (pH 7.5) including 1 mM
3
H, 3.76; N, 9.49. Found: C, 48.65; H, 3.98; N, 9.80.
l
lL of 10 lCi/mL L-[H ]-argi-
l
l
4
.2. Acute toxicity test
RAW264.7 cells were maintained in Dulbecco’s modified Eagle’s
3
l
2 mM EGTA were added. To the resulting was added Dowex
medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 5%
fetal bovine serum (FBS) (Invitrogen), 100 U/mL penicillin, and
50 W-8X and stirred for 5 min. iNOS activity was measured by
monitoring the conversion level of
L
-citrulline from
-[H ]-citrulline in the supernatant was monitored
by scintillation counting.
L-arginine.
3
1
00
l
g/mL streptomycin and incubated at 37 °C in a humidified
. In all cell cultures, each compound
Therefore, L
atmosphere containing 5% CO
2

4
902
J. Sun et al. / Bioorg. Med. Chem. 19 (2011) 4895–4902
4
.5. Reverse transcriptase-polymerase chain reaction (RT-PCR)
References and notes
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1
2
3
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1
[
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2
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2
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2
2
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Acknowledgments
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This work was supported by Jiangsu National Science Founda-
tion (No. BK2009239) and the Fundamental Research Funds for
the Central Universities (No. 1092020804 & 1106020824).
3
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