Synthesis and biological evaluation of novel dimethyl[1,1′-biphenyl]- 2,2′-dicarboxylate derivatives containing thiazolidine-2,4-dione for the treatment of concanavalin A-induced acute liver injury of BALB/c mice

Article abstract of DOI:10.1016/j.ejmech.2011.10.005

In this paper, we reported the synthesis of bifendate derivatives and evaluation of anti-inflammatory activity by detecting the production of the Nitric Oxide (NO) in the lipopolysaccharide(LPS)-stimulated RAW 264.7 cell lines. Among the newly derivatives

Full text of DOI:10.1016/j.ejmech.2011.10.005

European Journal of Medicinal Chemistry 46 (2011) 5941e5948
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel dimethyl[1,10-biphenyl]-2,20-
dicarboxylate derivatives containing thiazolidine-2,4-dione for the treatment
of concanavalin A-induced acute liver injury of BALB/c mice
,
c
,
,
,
Guangcheng Wang a 1, Chongyang Deng a 1, Caifeng Xie a, Liang Ma a, Jincheng Yang a c, Neng Qiu a,
Qinyuan Xu a, Tao Chen a, Fei Peng a, Jinying Chen a, Jingxiang Qiu a, Aihua Peng a , Yuquan Wei ,
,
b,
a
*
Lijuan Chen a
,b,
*
a State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Keyuan Road 4, Gaopeng Street, Chengdu 610041, People’s Republic of
China
b State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu,
Sichuan, People’s Republic of China
c Pharmacy College of West China of Sichuan University, Chengdu, Sichuan 610065, 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:
In this paper, we reported the synthesis of bifendate derivatives and evaluation of anti-inflammatory
activity by detecting the production of the Nitric Oxide (NO) in the lipopolysaccharide(LPS)-stimulated
RAW 264.7 cell lines. Among the newly derivatives, compound 7k was the most potent one and two
other compounds (7e and 7f) also exhibited greater anti-inflammatory activity than bifendate. Further
in vivo studies confirmed that 7k significantly and dose-dependently inhibited carrageenan-induced paw
edema and decreased the serum levels of alanine aminotransaminase, and aspartate aminotransaminase
in concanavalin A-induced hepatitis model. Histopathological evaluation demonstrated that 7k has
better hepatoprotective effect on acute liver injury induced by concanavalin A than bifendate, suggesting
that 7k is a potential drug candidate for the treatment of hepatic injuries.
Received 29 June 2011
Received in revised form
26 September 2011
Accepted 2 October 2011
Available online 8 October 2011
Keywords:
Anti-inflammation
Hepatoprotective
Thiazolidine-2,4-dione
Bifendate
Ó 2011 Published by Elsevier Masson SAS.
1. Introduction
[6,7], although its analog bicyclol, was reported to have better
hepatoprotective profile.
Hepatitis is a major public health concern affecting almost 10%
of the Chinese population. Schizandrin C, isolated from Chinese
medicinal herb Schizandra Chinese (Chinese wuweizi), has shown
a variety of pharmacological activities, such as anticancer [1], anti-
inflammatory [2], anti-HBV [3], especially anti-hepatotoxic activity
against live injury induced by CCl4 [3]. Bifendate (DBD), a synthetic
intermediate of Schizandrin C, which similar pharmacological effect
as Schizandrin C [4,5], has still clinically used for the treatment of
chronic hepatitis for its low cost and minimal side effects in China
Thiazolidine-2,4-dione (TZDs) and their derivatives are found to
be associated with various biological activities. TZDs, such as tro-
glitazone [8], pioglitazone [9], and rosiglitazone, are therapeutic
agents for the treatment of type 2 diabetes [10]. Apart from their
known anti-diabetic activity, TZDs have also been shown to inhibit
the production of pro-inflammatory cytokines and the expression
of inducible nitric oxide synthase in vascular cells [11] and mono-
cytes/macrophages [12,13]. TZDs were found to significantly
decrease serum levels of ALT and AST during treatment in patients
with type 2 diabetes [14].
Our previous studies has shown that (Z)-5-(4-methoxybe-
nzylidene)-thiazolidine-2,4-dione (SKLB 010) [15e17], exhibited
potent anti-inflammation activity. We hypothesized that bifendate
derivatives containing thiazolidine-2,4-dione might have better
hepatoprotective effects than bifendate. In present studies, we
designed and synthesized a series of bifendate derivatives con-
taining (Z)-5-benzylidene-thiazolidine-2,4-dione, which were
* Corresponding authors. State Key Laboratory of Biotherapy, West China
Hospital, West China Medical School, Sichuan University, Keyuan Road 4, Gaopeng
Street, Chengdu 610041, People’s Republic of China. Tel.: þ86 28 85164063;
fax: þ86 28 85164060.
E-mail addresses: peng_aihua@163.com (A. Peng), lijuan17@hotmail.com
(L. Chen).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2011 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2011.10.005

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G. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 5941e5948
linked by ester moiety (Fig. 1). These newly bifendate derivatives
were first evaluated for the anti-inflammatory effects by measuring
the production of the Nitric Oxide (NO) on the lipopolysaccharide
(LPS)-induced RAW 264.7 cell lines. Subsequently, compound 7k,
which showed higher inhibitory effect compared with bifendate
was further evaluated using two inflammatory models from
carrageenan-induced paw edema in ICR mice and concanavalin A-
induced acute liver injury in BALB/c mice.
2. Chemistry
A general synthesis of bifendate derivatives is exhibited in
Scheme 1. Bifendate 1 was hydrolyzed with KOH solution and then
acidified with conc. HCl to give the dicarboxylic acid 2. Compound 2
was treated with anhydrous acetic anhydride to give an intra
Fig. 1. Structure of bifendate, SKLB 010, and a design for synthesis of Dimethyl[1,10-
Biphenyl]-2,20-dicarboxylates Bearing thiazolidine-2,4-dione derivatives.
Scheme 1. Reagents and conditions: (a) (i) 5% KOH, 6 h; (ii) conc. HCl (89.5%); (b) Ac2O, reflux, 12 h (73.6%); (c) MeOH, reflux 9 h (96.8%); (d) SOCl2, reflux, 2 h; (e) Et3N, CH2Cl2, r.t.,
overnight (52%e88.4%); (f) -alanine, thiazolidine-2,4-dione, AcOH, reflux, 6 h (41.8 %e91%).
b

G. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 5941e5948
5943
Fig. 2. The results of compound 7ae7l inhibited LPS-induced NO production in RAW 264.7 macrophages. Each bar represents mean ꢀ SE of 3e5 independent experiments.
Statistical significance relative to LPS is indicated, *P < 0.05, **P < 0.01, ***P < 0.001.
molecular anhydride 3. Treatment of 3 with methanol yielded the
monoester 4 [18]. Compound 4 reacted with SOCl2 to yield corre-
sponding acyl chloride 5, which was treated with substituted
phenols to yield ester 6ae6l. Knoevenagel condensation of thia-
The thickness of right hind was measured every 1 h for five
consecutive hours. Under the conditions used for carrageenan-
induced inflammation, swelling of the paw occurred rapidly after
the injection of carrageenan with an increase rate of 34.64% at 1 h,
which reached the maximum at 3 h (52.90%). Notably, edema was
reduced by 54.88% at 3 h in the 7k-treated group (50 mg/kg)
reduced, whereas it was only reduced by 20.98% at 3 h in the
bifendate-treated group (Fig. 4). These results show that compound
7k displays stronger anti-inflammatory activity than bifendate in
a dose-dependent manner.
zolidine-2,4-dione with 6ae6l in the presence of
b-alanine in
refluxing acetic acid afforded the desired target compounds 7ae7l.
Based on literature precedence the isomeric ratio was presumed to
be mainly the Z-stereoisomer [19,20].
3. Biological results and discussion
3.3. Hepatoprotective activity
3.1. Nitrite assay
3.3.1. Serum levels of ALT and AST analysis
After treatment of RAW 264.7 macrophages with LPS for 24 h,
NO production was assessed by measuring the accumulation of
nitrite, a stable metabolite of NO in the media, based on Griess [21].
As shown in Fig. 2, LPS-induced NO production was significantly
inhibited by all the tested compounds, indicating that these
compounds possessed potent anti-inflammatory activity. It was
notable that compound 7e, 7f and 7k exhibited higher anti-
inflammatory activities than bifendate. Compound 7k, the most
potent one, was investigated in subsequent research. Compound 7k
concentration-dependently inhibited NO production. (Fig. 3).
Serum ALT and AST are two marker enzymes which indicate the
extent of liver injury. Compound 7k (50 mg/kg) was orally
administered to BABL/c mice 20 h prior to Con A (12.5 mg/kg) i.v.
injection. Con A induction increased serum levels of ALT and AST. As
shown in Table 1, 7k decreased the serum levels of ALT and AST by
3.2. Carrageenan-induced paw edema in rats
Carrageenan-induced edema in the hind foot pad was used as
a model to determine the anti-inflammatory effect of compound
7k. Thirty ICR mice were divided into 5 groups (6 mice per group).
Fig. 4. Effects of 7k and bifendate on hind-paw edema induced by carrageenan in
mice. The results are expressed as mean (SEM, where n ¼ 6 rats. *P < 0.05, **P < 0.01,
***P < 0.001).
Table 1
Effects of 7k and bifendate on ALT and AST. The results are expressed as mean (SEM,
where n ¼ 6 rats. *P < 0.05, **P < 0.01, ***P < 0.001).
Treatment ALT (U/L)
Treatment AST (U/L)
Normal
Con A
DBD
39.57 ꢀ 7.99
99.17 ꢀ 55.04
2008.33 ꢀ 773.91
1016.62 ꢀ 461.57*
620.13 ꢀ 561.16**
Fig. 3. Compound 7k inhibited LPS-induced NO release in a dose-dependent manner
in RAW 264.7 macrophages. Each bar represents mean ꢀ SE of three-independent
experiments. Statistical significance relative to LPS is indicated, *P < 0.05; **P < 0.01,
***P < 0.001, ##P < 0.01.
3343.23 ꢀ 822.19
1490.50 ꢀ 950.28**
168.42 ꢀ 110.77***
7k

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G. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 5941e5948
Fig. 5. The photomicrography of liver sections from rats treated with Con A, the post-doses of compound 7k, bifendate; (A) Liver section of normal rat; (B) liver section of the
control rat treated with Con A; (C) liver section of the Con A -treated rat post-dosed by compound 7k (50 mg/kg); (D) liver section of the Con A -treated rat post-dosed by bifendate
(50 mg/kg).
94.96% (n ¼ 6, ***P < 0.001) and 69.12% (n ¼ 6, **P < 0.01) in Con A-
induced hepatitis model compared with untreated group. However,
the serum levels of ALT and AST were only reduced by 44.58%
(n ¼ 6, **P < 0.01) and 50.62% (n ¼ 6, *P < 0.05) in bifendate-treated
group. The results demonstrate that compound 7k exhibit potent
hepatoprotective activity.
inflammatory and hepatoprotective. Among the tested deriva-
tives, compound 7k shows potent anti-inflammatory and hep-
atoprotective activities. However, further structural modification is
planned to increase anti-hepatitis activity.
5. Experimental section
3.3.2. Histopathological analysis
5.1. Chemistry
In an effort to test whether compound 7k could prevent liver
cell damage. Microscopic examination revealed extensive infiltra-
tion of monocytes in the liver of Con A-induced mice was observed.
Livers exhibited congestion and dilatation of blood vessel, as well as
widespread hepatocellular necrosis in liver lobules. Liver lesions
were slightly improved in bifendate-treated mice. In contrast,
compound 7k-treated mice obviously accompanied with reduction
of inflammatory cell infiltration and less necrotic areas. Liver cells
were arranged in rows, and nucleus was clearly visible (Fig. 5). The
results indicate that 7k exerts striking hepatoprotective effect
against Con A-induced hepatitis in mice.
Chemistry reagents of analytical grade were purchased from
Chengdu Changzheng Chemical Factory, Sichuan, PR China. TLC was
performed on 0.20 mm Silica Gel 60 F254 plates (Qingdao Ocean
Chemical Factory, Shandong, China). Nuclear magnetic resonance
spectra (NMR) were recorded at 400 MHz on a Varian spectrometer
(Varian, Palo Alto, CA) model Gemini 400 and reported in parts per
million. Mass spectra (MS) were measured by Q-TOF Priemier mass
spectrometer (Micromass, Manchester, UK).
5.1.1. 7,70-Dimethoxy-4,40-bibenzo[d][1,3]dioxole-5,50-dicarboxylic
acid (2)
4. Conclusion
A mixture of 1 (10 g) and 5% aq. KOH solution (300 mL) was
stirred at 100 ꢁC for 6 h. After cooling to room temperature, the
mixture was filtered, and the filtrate was acidified with concentrate
HCl. The white precipitate was collected by filtration to give 2 (8.4 g,
89.5%). The solid was dried in vacuum at 60 ꢁC for 24 h. 1H NMR (d6-
In summary, we have synthesized a series of dimethyl[1,10-
Biphenyl]-2,20-dicarboxylate derivatives containing thiazolidine-
2,4-dione and examined for their biological activities of anti-

G. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 5941e5948
5945
DMSO, 400 MHz)
d
: 3.88 (s, 6H), 5.96 (s, 2H), 5.99 (s, 2H), 7.26 (s,
191.2; IR (KBr): 3010, 2947, 1734, 1704, 1639, 1588, 1414, 1318,
1225, 1168, 1100, 1039, 932, 781 cmꢂ1; MS (ESI, m/z): 531.23
[MþNa]þ.
2H), 12.36 (s, 2H); MS (ESI, m/z): 389.19 [MꢂH]ꢂ.
5.1.2. 7,70-Dimethoxy-4,40-bibenzo[d][1,3]dioxole-5,50-dicarboxylic
acid anhydride (3)
5.1.7. 5-(4-Formyl-2,6-dimethoxyphenyl) 50-methyl 7,70-
To a suspension of 2 (8.3 g) in Ac2O (50 mL) was heated to reflux
for 12 h. After cooling to room temperature, the mixture concen-
trated under reduced pressure. The residue was dissolved in
toluene (40 mL), and the white precipitate was filtered off to yield 3
(6.1 g, 73.6%), which was used in the next step without further
purification.
dimethoxy-4,40-bibenzo-[d][1,3]dioxole-5,50-dicarboxylate (6d)
The compound 6d was prepared in a similar manner to the
procedure described for the preparation of 6a to yield a solid
(79.2%): 1H NMR (CDCl3, 400 MHz)
d
: 3.69 (s, 3H), 3.83 (s, 6H), 3.91
(s, 3H), 4.01 (s, 3H), 5.97e6.04 (m, 4H), 7.10 (s, 2H), 7.36 (s, 1H), 7.63
(s, 1H), 9.88 (s, 1H); 13C NMR (CDCl3, 100 MHz)
: 51.8, 56.3, 56.3,
d
56.5, 56.6, 102.4, 102.5, 106.1, 106.7, 111.2, 111.7, 112.0, 113.1, 122.1,
123.3, 134.0, 134.2, 138.3, 138.8, 142.4, 147.3, 147.3, 147.4, 153.1, 153.1,
162.9, 166.2, 191.1; IR (KBr): 3016, 2944, 1733, 1695, 1638, 1602,
1462, 1418, 1326, 1240, 1171, 1132, 1037, 931, 770 cmꢂ1; MS (ESI, m/
z): 591.29 [MþNa]þ.
5.1.3. 7,70-Dimethoxy-50-(methoxycarbonyl)-4,40-bibenzo[d][1,3]
dioxole-5-carboxylic acid (4)
A mixture of 3 (6.1 g) and methanol (200 mL) was stirred at
65 ꢁC for 9 h. The most of the solvent was removed under reduced
pressure. The white precipitate was collected by filtration to obtain
4 (5.9 g, 96.8%): 1H NMR (d6-DMSO, 400 MHz)
d: 3.58 (s, 3H), 3.89
5.1.8. 5-(2,4-Dichloro-6-formylphenyl) 50-methyl 7,70-dimethoxy-
4,40-bibenzo-[d][1,3]dioxole-5,50-dicarboxylate (6e)
(s, 6H), 5.97e6.01 (m, 4H), 7.24 (s, 1H), 7.27 (s, 1H), 12.34 (s, 1H); MS
(ESI, m/z): 427.16 [MþNa]þ.
The compound 6e was prepared in a similar manner to the
procedure described for the preparation of 6a to yield a solid
5.1.4. 5-(2-Ethoxy-4-formylphenyl) 50-methyl 7,70-dimethoxy-4,40-
bibenzo[d][1,3]dioxole-5,50-dicarboxylate (6a)
(55.7%): 1H NMR (CDCl3, 400 MHz)
d: 3.71 (s, 3H), 3.91 (s, 3H), 4.03
(s, 3H), 5.98e6.06 (m, 4H), 7.35 (s, 1H), 7.64 (d, 1H, J ¼ 2.8 Hz), 7.70
To a suspension of 4 in dioxane (10 mL) was added SOCl2
(1.46 mL) and DMF (1 drop), and the mixture heated at reflux for
2 h, then concentrated under reduce pressure to give 5 as a white
solid. The residue 5 was dissolved in CH2Cl2 (20 mL), then Et3N
(1 mL) and 2-ethoxy-4-hydroxybenzaldehyde (0.80 g) were added
at room temperature. The mixture stirred at room temperature for
12 h, and washed with water, brine, dried over anhydrous
magnesium sulfate, and concentrated under reduce pressure. The
residue was purified by silica gel column chromatography to
(s, 1H), 7.74 (d, 1H, J ¼ 2.8 Hz), 9.97 (s, 1H); 13C NMR (CDCl3,
100 MHz) d: 52.0, 56.5, 56.7, 102.5, 102.7, 111.3, 111.3, 112.4, 113.5,
120.4, 123.0, 127.2, 129.7, 130.9, 132.7, 135.0, 138.5, 139.8, 142.6,
142.6, 147.2, 147.6, 147.9, 162.9, 166.1, 186.6; IR (KBr): 3010, 2951,
1749, 1713, 1637, 1594, 1424, 1321, 1163, 1103, 1044, 932, 750 cmꢂ1
;
MS (ESI, m/z): 599.26 [MþNa]þ.
5.1.9. 5-(2-Bromo-4-formylphenyl) 50-methyl 7,70-dimethoxy-4,40-
bibenzo-[d][1,3]dioxole-5,50-dicarboxylate (6f)
provide a solid 6a (71.3%): 1H NMR (CDCl3, 400 MHz)
d: 1.33 (t, 3H,
The compound 6f was prepared in a similar manner to the
procedure described for the preparation of 6a to yield a solid
J ¼ 6.8 Hz), 3,70 (s, 3H), 3.92 (s, 3H), 4.01 (s, 3H), 4.07 (q, 2H,
J ¼ 7.2 Hz, J ¼ 14.0 Hz), 5.99e6.05 (m, 4H), 7.16 (d, 1H, J ¼ 8.0 Hz),
7.37 (s, 1H), 7.41e7.43 (m, 2H), 7.64 (s, 1H), 9.91 (s, 1H); 13C NMR
(74.7%): 1H NMR (CDCl3, 400 MHz)
d: 3.71 (s, 3H), 3.92 (s, 3H),
4.02 (s, 3H), 6.00e6.06 (m, 4H), 7.29 (d, 1H, J ¼ 8.0 Hz), 7.37 (s,
(CDCl3, 100 MHz) d: 14.5, 51.9, 56.5, 56.6, 64.6, 102.4, 102.5, 111.2,
1H), 7.71 (s, 1H), 7.80 (dd, 1H, J ¼ 8.0 Hz, 2.0 Hz), 8.11 (d, 1H,
111.7, 111.8, 111.8, 113.1, 122.0, 123.3, 123.4, 124.5, 135.0, 138.3, 139.0,
142.5, 142.5, 145.4, 147.3, 147.4, 151.4, 163.0, 166.2, 191.1; IR (KBr):
3014, 2943, 1721, 1698, 1637, 1595, 1433, 1320, 1167, 1103, 1041, 969,
873, 748 cmꢂ1; MS (ESI, m/z): 575.26 [MþNa]þ.
J ¼ 2.0 Hz), 9.92 (s, 1H); 13C NMR (CDCl3, 100 MHz)
d: 52.0, 56.5,
56.7, 102.4, 102.7, 111.2, 111.5, 112.2, 113.4, 117.5, 121.1, 123.2, 124.5,
129.8, 134.5, 135.1, 138.4, 139.5, 142.6, 142.6, 147.2, 147.5, 153.1,
162.5, 166.2, 189.6; IR (KBr): 3010, 2900, 1744, 1706, 1637, 1592,
1421, 1320, 1167, 1102, 1042, 941, 757 cmꢂ1; MS (ESI, m/z): 609.09
[MþNa]þ.
5.1.5. 5-(4-Formyl-2-methoxyphenyl) 50-methyl 7,70-dimethoxy-
4,40-bibenzo-[d][1,3]dioxole-5,50-dicarboxylate (6b)
The compound 6b was prepared in a similar manner to the
procedure described for the preparation of 6a to yield a solid
5.1.10. 5-(2-Fluoro-4-formylphenyl) 50-methyl 7,70-dimethoxy-4,40-
bibenzo-[d][1,3]dioxole-5,50-dicarboxylate (6g)
(71.3%): 1H NMR (CDCl3, 400 MHz)
d
: 3.71 (s, 3H), 3.83 (s, 3H), 3.92
The compound 6g was prepared in a similar manner to the
procedure described for the preparation of 6a to yield a solid
(s, 3H), 4.01 (s, 3H), 5.99e6.05 (m, 4H), 7.15 (d, 1H, J ¼ 8.0 Hz), 7.36
(s, 1H), 7.43e7.45 (m, 2H), 7.61 (s, 1H), 9.92 (s, 1H); 13C NMR (CDCl3,
(87.6%): 1H NMR (CDCl3, 400 MHz)
d
: 3.71 (s, 3H), 3.93 (s, 3H), 4.02
(s, 3H), 6.00e6.06 (m, 4H), 7.27e7.30 (m, 1H), 7.37 (s, 1H), 7.60 (s,
1H), 7.64e7.67 (m, 2H), 9.93 (s, 1H); 13C NMR (CDCl3, 100 MHz)
100 MHz) d: 51.9, 56.0, 56.5, 56.6, 102.4, 102.6, 110.9, 111.2, 111.7,
111.9,113.0,122.0,123.3,123.5,124.7,135.1,138.3,139.0,142.5,142.5,
145.1, 147.3, 147.4, 152.2, 163.1, 166.2, 191.1; IR (KBr): 3013, 2943,
2839, 1723, 1698, 1638, 1595, 1416, 1317, 1170, 1191, 1036, 923, 854,
760 cmꢂ1; MS (ESI, m/z): 561.21 [MþNa]þ.
d:
52.0, 56.5, 56.7, 102.5, 102.7, 111.2, 111.5, 112.1, 113.3, 116.7, 116.9,
121.1,123.2,124.7,126.6,138.4,139.4,142.6,143.3,147.2,147.5,153.4,
155.9, 162.6, 166.2, 189.8; IR (KBr): 3013, 2950, 1745, 1701, 1638,
1593, 1436, 1321, 1168, 1103, 1042, 932, 752 cmꢂ1; MS (ESI, m/z):
549.24 [MþNa]þ.
5.1.6. 5-(3-Formylphenyl) 50-methyl 7,70-dimethoxy-4,40-bibenzo
[d][1,3]dioxole-5,50-dicarboxylate (6c)
The compound 6c was prepared in a similar manner to the
procedure described for the preparation of 6a to yield a solid
5.1.11. 5-(2,4-Dibromo-6-formylphenyl) 50-methyl 7,70-dimethoxy-
4,40-bibenzo-[d][1,3]dioxole-5,50-dicarboxylate (6h)
(88.4%): 1H NMR (CDCl3, 400 MHz)
d
: 3.72 (s, 3H), 3.94 (s, 3H),
The compound 6h was prepared in a similar manner to the
procedure described for the preparation of 6a to yield a solid
4.01 (s, 3H), 6.01e6.05 (m, 4H), 7.72 (d, 1H, J ¼ 8.0 Hz), 7.38 (s,
1H), 7.50e7.54 (m, 2H), 7.58 (s, 1H), 7.71 (d, 1H, J ¼ 8.0 Hz), 9.97
(52.0%): 1H NMR (CDCl3, 400 MHz)
d
: 3.71 (s, 3H), 3.91 (s, 3H), 4.03
(s, 3H), 5.99e6.06 (m, 4H), 7.35 (s, 1H), 7.73 (s, 1H), 7.93e7.95 (m,
2H), 9.93 (s, 1H); 13C NMR (CDCl3, 100 MHz)
: 52.0, 56.5, 56.7,
102.5, 102.7, 111.3, 111.3, 112.5, 113.5, 119.2, 120.3, 120.4, 123.0,
(s, 1H); 13C NMR (CDCl3, 100 MHz)
d: 52.0, 56.6, 56.7, 102.4, 102.6,
111.2, 111.8, 111.9, 112.8, 122.1, 122.2, 123.2, 127.3, 127.7, 130.1,
137.7, 138.3, 139.1, 142.6, 142.6, 147.2, 147.4, 151.4, 164.1, 166.2,
d

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G. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 5941e5948
130.9, 131.3, 138.5, 139.8, 140.7, 142.6, 142.6, 147.2, 147.6, 149.6,
162.8, 166.0, 186.7; IR (KBr): 3004, 2899, 1748, 1741, 1637, 1592,
1422, 1321, 1168, 1102, 1043, 932, 779 cmꢂ1; MS (ESI, m/z): 588.97
[MþNa]þ.
extract was washed with water, brine, dried over anhydrous
magnesium sulfate, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography to give
7a (64.3%): 1H NMR (d6-DMSO, 400 MHz)
d
: 1.23 (t, 3H, J ¼ 7.2 Hz),
3.62 (s, 3H), 3.87 (s, 3H), 3.95 (s, 3H), 4.04 (q, 2H, J ¼ 6.8 Hz,
J ¼ 14.0 Hz), 6.03e6.09 (m, 4H), 7.17 (s, 2H), 7.25 (s, 1H), 7.34 (s, 1H),
7.48 (s, 1H), 7.79 (s, 1H), 12.65 (s, 1H); 13C NMR (d6-DMSO, 100 MHz)
5.1.12. 5-(5-Formyl-2-methoxyphenyl) 50-methyl 7,70-dimethoxy-
4,40-bibenzo-[d][1,3]dioxole-5,50-dicarboxylate (6i)
The compound 6i was prepared in a similar manner to the
procedure described for the preparation of 6a to yield a solid
d: 14.5, 52.0, 56.6, 56.7, 64.4, 102.6, 102.7, 110.6, 111.2, 111.3, 112.5,
115.8, 122.1, 122.3, 123.5, 123.9, 123.9, 131.5, 132.1, 137.9, 138.6, 141.2,
142.2, 142.2, 147.3, 147.4, 150.7, 163.1, 165.8, 167.5, 168.0; IR (KBr):
3226, 2940, 1746, 1707, 1637, 1506, 1420, 1321, 1167, 1041, 917,
751 cmꢂ1; MS (ESI, m/z): 650.32 [MꢂH]ꢂ; HPLC purity ¼ 98.7%.
(87.9%): 1H NMR (CDCl3, 400 MHz)
d: 3.71 (s, 3H), 3.85 (s, 3H), 3.91
(s, 3H), 4.01 (s, 3H), 6.00e6.04 (m, 4H), 7.02 (d, 1H, J ¼ 8.4 Hz), 7.37
(s, 1H), 7.52 (d, 1H, J ¼ 2.0 Hz), 7.61 (s, 1H), 7.70 (dd, 1H, J ¼ 8.4 Hz,
2.0 Hz), 9.83 (s, 1H); 13C NMR (CDCl3, 100 MHz)
d: 51.9, 56.2, 56.5,
56.7, 102.4, 102.5, 111.2, 111.8, 111.8, 112.1, 113.0, 122.1, 123.3, 124.5,
129.9, 130.1, 138.3, 138.9, 140.3, 142.5, 142.5, 147.3, 147.4, 156.6,
163.5, 166.2, 190.1; IR (KBr): 3015, 2945, 1729, 1709, 1688, 1638,
1604, 1510, 1438, 1323, 1127, 1100, 1036, 931, 780 cmꢂ1; MS (ESI, m/
z): 561.23 [MþNa]þ.
5.1.17. (Z)-5-(4-((2,4-dioxothiazolidin-5-ylidene)methyl)-2-
methoxyphenyl) 50-methyl-7,70-dimethoxy-4,40-bibenzo[d][1,3]
dioxole-5,50-dicarboxylate (7b)
The compound 7b was prepared in a similar manner to the
procedure described for the preparation of 7a toyield a solid (84.1%):
1H NMR (d6-DMSO, 400 MHz)
d: 3.62 (s, 3H), 3.78 (s, 3H), 3.86 (s, 3H),
5.1.13. 5-(2-Formylphenyl) 50-methyl 7,70-dimethoxy-4,40-bibenzo
[d][1,3]dioxole-5,50-dicarboxylate (6j)
3.95 (s, 3H), 6.02e6.09 (m, 4H), 7.14e7.17 (m, 2H), 7.24 (s, 1H),
7.35e7.35 (m,1H), 7.45 (s,1H), 7.80 (s,1H),12.65 (s,1H); 13C NMR (d6-
The compound 6j was prepared in a similar manner to the
procedure described for the preparation of 6a to yield a solid
DMSO, 100 MHz) d: 52.1, 56.2, 56.6, 56.7, 102.5, 102.7, 110.6, 111.4,
112.5,115.0,122.0,122.2,123.4,123.9,123.9,131.5,132.2,137.9,138.7,
140.9,142.2,142.2,143.5,147.2,147.4,151.6,163.3,165.8,167.5,168.0;
IR (KBr): 3209, 2928, 1748, 1706, 1638, 1417, 1322, 1166, 1103, 1037,
925, 740 cmꢂ1; MS (ESI, m/z): 636.30 [MꢂH]ꢂ; HPLC purity ¼ 98.7%.
(88.2%): 1H NMR (CDCl3, 400 MHz)
d: 3.72 (s, 3H), 3.91 (s, 3H), 4.02
(s, 3H), 5.98e6.05 (m, 4H), 7.13 (d,1H, J ¼ 8.0 Hz), 7.33e7.36 (m, 2H),
7.57e7.62 (m, 1H), 7.65 (s, 1H), 7.87e7.89 (m, 1H), 10.06 (s, 1H); 13C
NMR (CDCl3, 100 MHz) d: 52.0, 56.5, 56.7, 102.4, 102.6, 111.3, 111.7,
111.9, 113.1, 121.7, 123.1, 123.2, 126.3, 128.4, 129.3, 135.3, 138.4, 139.3,
142.6, 142.6, 147.1, 147.5, 152.6, 164.1, 166.2, 188.5; IR (KBr): 3018,
2951, 1736, 1709, 1636, 1605, 1415, 1316, 1169, 1102, 1036, 935,
754 cmꢂ1; MS (ESI, m/z): 531.23 [MþNa]þ.
5.1.18. (E)-5-(3-((2,4-dioxothiazolidin-5-ylidene)methyl)phenyl)
50-methyl 7,70-dimethoxy-4,40-bibenzo[d][1,3]dioxole-5,50-
dicarboxylate (7c)
The compound 7c was prepared in a similar manner to the
procedure described for the preparation of 7a to yield a solid (91%):
5.1.14. 5-(4-Formylphenyl) 50-methyl 7,70-dimethoxy-4,40-bibenzo
[d][1,3]dioxole-5,50-dicarboxylate (6k)
1H NMR (d6-DMSO, 400 MHz)
d: 3.63 (s, 3H), 3.87 (s, 3H), 3.96 (s,
3H), 6.05e6.09 (m, 4H), 7.13e7.16 (m, 1H), 7.19 (s, 1H), 7.26 (s, 1H),
The compound 6k was prepared in a similar manner to the
procedure described for the preparation of 6a to yield a solid
7.47 (s, 1H), 7.49e7.58 (m, 2H), 7.78 (s, 1H), 12.68 (s, 1H); 13C NMR
(d6-DMSO, 100 MHz) d: 52.2, 56.6, 56.8, 102.6, 102.8, 110.7, 111.4,
(0.81 g, 79.7%) as a solid. 1H NMR (CDCl3, 400 MHz)
d
: 3.72 (s, 3H),
111.4, 112.1, 122.6, 122.7, 123.4, 123.7, 125.1, 127.7, 130.7, 130.8, 134.8,
138.0, 138.6, 142.3, 142.3, 147.2, 147.3, 151.0, 164.2, 165.9, 167.4,
167.9; IR (KBr): 3237, 2898, 1745, 1705, 1636, 1486, 1437, 1315, 1169,
1099, 1035, 931, 760 cmꢂ1; MS (ESI, m/z): 606.25 [MꢂH]ꢂ; HPLC
purity ¼ 99.2%.
3.94 (s, 3H), 4.01 (s, 3H), 5.99e6.06 (m, 4H), 7.17 (d, 2H, J ¼ 8.8 Hz),
7.38 (s,1H), 7.57 (s,1H), 7.87 (d, 2H, J ¼ 8.8 Hz), 9.97 (s,1H); 13C NMR
(CDCl3, 100 MHz) d: 52.0, 56.6, 56.7, 102.4, 102.6, 111.3, 111.9, 111.9,
112.8, 122.1, 122.1, 122.2, 123.3, 131.2, 131.2, 133.9, 138.3, 139.1, 142.6,
142.6, 147.2, 147.2, 155.6, 163.7, 166.2, 190.9; IR (KBr): 3010, 2945,
1720, 1637, 1595, 1418, 1320, 1203, 1165, 1098, 1039, 930, 861,
754 cmꢂ1; MS (ESI, m/z): 531.24 [MþNa]þ.
5.1.19. (Z)-5-(4-((2,4-dioxothiazolidin-5-ylidene)methyl)-2,6-
dimethoxyphenyl) 50-methyl-7,70-dimethoxy-4,40-bibenzo[d][1,3]
dioxole-5,50-dicarboxylate (7d)
5.1.15. 5-(4-Chloro-2-formylphenyl) 50-methyl 7,70-dimethoxy-4,40-
bibenzo-[d][1,3]dioxole-5,50-dicarboxylate (6l)
The compound 7d was prepared in a similar manner to the
procedure described for the preparation of 7a to yield a solid (82%):
The compound 6l was prepared in a similar manner to the
procedure described for the preparation of 6a to yield a solid
1H NMR (d6-DMSO, 400 MHz)
d
: 3.62 (s, 3H), 3.77 (s, 6H), 3.86 (s,
3H), 3.94 (s, 3H), 5.91e6.10 (m, 4H), 6.95 (s, 2H), 7.24 (s, 1H), 7.44 (s,
1H), 7.77 (s,1H),12.66 (s,1H); 13C NMR (d6-DMSO,100 MHz)
: 52.0,
(63.9%): 1H NMR (CDCl3, 400 MHz)
d: 3.71 (s, 3H), 3.91 (s, 3H), 4.02
d
(s, 3H), 5.97e6.02 (m, 4H), 7.10 (d, 1H, J ¼ 8.8 Hz), 7.26 (s, 1H), 7.52
56.4, 56.4, 56.6, 56.7, 102.5, 102.7, 107.0, 107.0, 110.6, 111.3, 111.4,
112.7, 121.9, 123.4, 124.2, 129.7, 131.6, 131.8, 138.0, 138.7, 142.2, 147.3,
147.4, 152.5, 152.5, 152.5, 162.7, 165.7, 167.5, 168.1; IR (KBr): 3151,
2940, 1746, 1710, 1639, 1608, 1588, 1416, 1320, 1164, 1044, 935,
780 cmꢂ1; MS (ESI, m/z): 666.30 [MꢂH]ꢂ; HPLC purity ¼ 99.9%.
(dd, 1H, J ¼ 8.8 Hz, 2.8 Hz), 7.62 (s, 1H), 7.83 (d, 1H, J ¼ 2.8 Hz), 9.98
(s, 1H); 13C NMR (CDCl3, 100 MHz)
d: 52.0, 56.6, 56.8, 102.4, 102.6,
111.4, 111.6, 112.1, 113.1, 121.4, 123.1, 124.8, 128.7, 129.4, 132.2, 134.9,
138.4, 139.5, 142.6, 142.6, 147.1, 147.6, 151.1, 163.9, 166.2, 187.2; IR
(KBr): 3010, 2948, 1716, 1637, 1594, 1479, 1422, 1321, 1168, 1104,
1043, 933, 764 cmꢂ1; MS (ESI, m/z): 565.19 [MþNa]þ.
5.1.20. (E)-5-(2,4-dichloro-6-((2,4-dioxothiazolidin-5-ylidene)
methyl)phenyl) 50-methyl-7,70-dimethoxy-4,40-bibenzo[d][1,3]
dioxole-5,50-dicarboxylate (7e)
5.1.16. (Z)-5-(4-((2,4-dioxothiazolidin-5-ylidene)methyl)-2-
ethoxyphenyl) 50-methyl-7,70-dimethoxy-4,40-bibenzo[d][1,3]
dioxole-5,50-dicarboxylate (7a)
The compound 7e was prepared in a similar manner to the
procedure described for the preparation of 7a to yield a solid
A mixture of 6a (0.552 g, 1 mmol),
b
-alanine (0.105 g, 1.2 mmol),
(50.3%): 1H NMR (d6-DMSO, 400 MHz)
d: 3.62 (s, 3H), 3.85 (s, 3H),
thiazolidine-2,4-dione (0.35 g, 3 mmol) and AcOH (10 mL) was
stirred at 120 ꢁC for 6 h. After cooling to room temperature, the
mixture was diluted with water and extracted with CH2Cl2. The
3.97 (s, 3H), 5.97e6.11 (m, 4H), 7.24 (s, 1H), 7.45 (s, 1H), 7.46 (d, 1H,
J ¼ 2.0 Hz), 7.62 (s, 1H), 7.91 (d, 1H, J ¼ 2.0 Hz), 12.86 (s, 1H); 13C
NMR (d6-DMSO, 100 MHz) d: 52.1, 56.5, 56.8, 102.6, 103.0, 110.6,

G. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 5941e5948
5947
112.0, 113.2, 120.0, 122.5, 123.3, 126.8, 129.1, 130.1, 130.4, 131.1, 131.7,
5.1.25. (E)-5-(2-((2,4-dioxothiazolidin-5-ylidene)methyl)phenyl)
50-methyl 7,70-dimethoxy-4,40-bibenzo[d][1,3]dioxole-5,50-
dicarboxylate (7j)
138.0, 139.6, 142.2, 142.3, 143.5, 144.8, 147.6, 147.9, 163.0, 165.5,
166.8,167.2; IR (KBr): 3245, 2946,1753,1712,1636,1593,1421,1320,
1165, 1102, 1043, 935, 783 cmꢂ1; MS (ESI, m/z): 674.21 [MꢂH]ꢂ;
HPLC purity ¼ 99.4%.
The compound 7j was prepared in a similar manner to the
procedure described for the preparation of 7a to yield a solid
(72.5%): 1H NMR (d6-DMSO, 400 MHz)
d: 3.63 (s, 3H), 3.84 (s, 3H),
5.1.21. (Z)-5-(2-bromo-4-((2,4-dioxothiazolidin-5-ylidene)methyl)
phenyl) 50-methyl-7,70-dimethoxy-4,40-bibenzo[d][1,3]dioxole-5,50-
dicarboxylate (7f)
3.98 (s, 3H), 5.99e6.10 (m, 4H), 7.21e7.23 (m, 2H), 7.41e7.45 (m,
1H), 7.52e7.56 (m, 3H), 7.61 (s, 1H), 12.69 (s, 1H); 13C NMR (d6-
DMSO, 100 MHz) d: 52.1, 56.5, 56.7, 102.6, 102.8, 110.6, 111.3, 111.4,
The compound 7f was prepared in a similar manner to the
procedure described for the preparation of 7a to yield a solid
112.6, 121.7, 123.4, 124.5, 126.2, 126.6, 126.9, 128.4, 131.8, 137.9,
139.0, 142.2, 142.3, 143.5, 147.0, 147.5, 149.7, 163.8, 165.9, 167.2,
167.9; IR (KBr): 3229, 2945, 1745, 1709, 1636, 1597, 1420, 1320, 1167,
1099, 1042, 931, 759 cmꢂ1; MS (ESI, m/z): 606.26 [MꢂH]ꢂ; HPLC
purity ¼ 98.2%.
(60.8%): 1H NMR (d6-DMSO, 400 MHz)
d: 3.63 (s, 3H), 3.86 (s, 3H),
3.96 (s, 3H), 6.07e6.10 (m, 4H), 7.25 (s, 1H), 7.33 (d, 1H, J ¼ 8.8 Hz),
7.57 (s, 1H), 7.60 (d, 1H, J ¼ 8.8 Hz), 7.76 (s, 1H), 7.96 (s, 1H), 12.72 (s,
1H); 13C NMR (d6-DMSO, 100 MHz)
d: 52.1, 56.6, 56.8, 102.7, 102.9,
110.7, 111.1, 111.6, 112.8, 116.7, 121.3, 123.4, 125.0, 126.1, 129.1, 129.8,
133.2,135.1,138.0,139.1,142.2,142.2,147.2,147.5, 148.8,162.8,165.8,
167.4, 168.1; IR (KBr): 3240, 2947, 1747, 1708, 1637, 1591, 1422, 1321,
1166, 1102, 1043, 915, 765 cmꢂ1; MS (ESI, m/z): 684.16 [MꢂH]ꢂ;
HPLC purity ¼ 97.4%.
5.1.26. (Z)-5-(4-((2,4-dioxothiazolidin-5-ylidene)methyl)phenyl)
50-methyl 7,70-dimethoxy-4,40-bibenzo[d][1,3]dioxole-5,50-
dicarboxylate (7k)
The compound 7k was prepared in a similar manner to the
procedure described for the preparation of 7a to yield a solid
(0.392 g, 64.6%): 1H NMR (d6-DMSO, 400 MHz)
d: 3.63 (s, 3H), 3.87
5.1.22. (Z)-5-(4-((2,4-dioxothiazolidin-5-ylidene)methyl)-2-
fluorophenyl) 50-methyl-7,70-dimethoxy-4,40-bibenzo[d][1,3]
dioxole-5,50-dicarboxylate (7g)
(s, 3H), 3.96 (s, 3H), 6.06e6.09 (m, 4H), 7.18 (d, 2H, J ¼ 8.4 Hz), 7.25
(s, 1H), 7.48 (s, 1H), 7.63 (d, 2H, J ¼ 8.4 Hz), 7.80 (s, 1H), 12.65 (s, 1H);
13C NMR (d6-DMSO, 100 MHz)
d: 52.2, 56.6, 56.7, 102.7, 102.8, 110.7,
The compound 7g was prepared in a similar manner to the
procedure described for the preparation of 7a to yield a solid
111.4, 111.4, 112.2, 122.3, 122.8, 122.8, 123.5, 123.8, 130.9, 131.1, 131.6,
131.6, 138.0, 138.7, 142.3, 142.3, 147.1, 147.3, 151.8, 164.0, 166.0, 167.6,
168.1; IR (KBr): 3225, 2948, 1706, 1637, 1595, 1501, 1416, 1322, 1211,
1169, 1100, 1042, 934, 754 cmꢂ1; MS (ESI, m/z): 606.32 [MꢂH]ꢂ;
HPLC purity ¼ 99.4%.
(89.6%): 1H NMR (d6-DMSO, 400 MHz)
d: 3.63 (s, 3H), 3.87 (s, 3H),
3.96 (s, 3H), 6.02e6.10 (m, 4H), 7.26 (s, 1H), 7.36e7.40 (m, 1H),
7.44e7.46 (m, 1H), 7.52 (s, 1H), 7.60e7.63 (m, 1H), 7.78 (s, 1H), 12.72
(s, 1H); 13C NMR (d6-DMSO, 100 MHz)
d: 52.1, 56.6, 56.8, 102.6,
102.9, 110.7, 111.1, 111.7, 112.7, 118.4, 118.6, 121.1, 123.3, 125.2, 125.4,
126.4,129.9,138.0,139.1,142.3,143.5,147.2,147.4,152.6,155.1,162.8,
165.8, 167.4, 167.8; IR (KBr): 3276, 2951, 1750, 1708, 1637, 1507, 1420,
1322, 1167, 1109, 1037, 917, 781 cmꢂ1; MS (ESI, m/z): 624.26
[MꢂH]ꢂ; HPLC purity ¼ 98.9%.
5.1.27. (E)-5-(4-chloro-2-((2,4-dioxothiazolidin-5-ylidene)methyl)
phenyl)50-methyl 7,70-dimethoxy-4,40-bibenzo[d][1,3]dioxole-5,50-
dicarboxylate (7l)
The compound 7l was prepared in a similar manner to the
procedure described for the preparation of 7a to yield a solid
(67.0%): 1H NMR (d6-DMSO, 400 MHz)
d: 3.62 (s, 3H), 3.84 (s, 3H),
5.1.23. (E)-5-(2,4-dibromo-6-((2,4-dioxothiazolidin-5-ylidene)
methyl)phenyl) 50-methyl 7,70-dimethoxy-4,40-bibenzo[d][1,3]
dioxole-5,50-dicarboxylate (7h)
3.97 (s, 3H), 5.98e6.10 (m, 4H), 7.20 (s, 1H), 7.29 (d, 1H, J ¼ 8.8 Hz),
7.47 (d, 1H, J ¼ 2.4 Hz), 7.51 (s, 1H), 7.55 (s, 1H), 7.58 (dd, 1H,
J ¼ 8.8 Hz, 2.4 Hz), 12.79 (s, 1H); 13C NMR (d6-DMSO, 100 MHz)
d:
The compound 7h was prepared in a similar manner to the
procedure described for the preparation of 7a to yield a solid
52.1, 56.5, 56.7, 102.6, 102.9, 110.6, 111.1, 111.6, 112.6, 121.4, 123.4,
123.4, 125.3, 127.7, 128.2, 128.5, 130.8, 131.3, 137.9, 139.1, 142.2,
142.3, 147.0, 147.5, 148.2, 163.5, 165.9, 167.0, 167.5; IR (KBr): 3213,
2941, 1744, 1703, 1637, 1585, 1480, 1416, 1318, 1162, 1107, 1043, 921,
748 cmꢂ1; MS (ESI, m/z): 640.23 [MꢂH]ꢂ; HPLC purity ¼ 98.5%.
(41.8%): 1H NMR (d6-DMSO, 400 MHz)
d: 3.62 (s, 3H), 3.85 (s, 3H),
3.97 (s, 3H), 5.98e6.11 (m, 4H), 7.23 (s, 1H), 7.42 (s, 1H), 7.61 (d, 1H,
J ¼ 2.0 Hz), 7.64 (s, 1H), 8.12 (d, 1H, J ¼ 2.0 Hz), 12.85 (s, 1H); 13C
NMR (d6-DMSO, 100 MHz) d: 52.1, 56.5, 56.8, 102.6, 103.0, 110.6,
111.0, 112.1, 113.3, 119.2, 119.8, 120.1, 122.8, 123.3, 130.1, 130.2, 130.9,
136.6, 138.0, 139.6, 142.2, 142.3, 146.4, 147.2, 147.6, 162.1, 165.5,
166.8, 167.3; IR (KBr): 3243, 2945, 1752, 1712, 1636, 1594, 1424,
1320, 1164, 1102, 1043, 929, 780 cmꢂ1; MS (ESI, m/z): 764.00
[MꢂH]ꢂ; HPLC purity ¼ 99.3%.
5.2. Biological assays
5.2.1. Cell culture
RAW 264.7 macrophages were cultured in RPMI 1640 at 37 ꢁC in
a humidified atmosphere in the presence of 5% CO2.
5.1.24. (E)-5-(5-((2,4-dioxothiazolidin-5-ylidene)methyl)-2-
methoxyphenyl) 50-methyl-7,70-dimethoxy-4,40-bibenzo[d][1,3]
dioxole-5,50-dicarboxylate (7i)
5.2.2. Nitrite assay
RAW 264.7 cells were seeded into 96-well plate at density of
The compound 7i was prepared in a similar manner to the
procedure described for the preparation of 7a to yield a solid
1 ꢃ104 per well with 100
m
L culture medium and cultured for 24 h.
The cells were then treated with or without LPS (1
mg/mL) in the
(81.6%): 1H NMR (d6-DMSO, 400 MHz)
d: 3.64 (s, 3H), 3.80 (s, 3H),
absence or presence of different concentrations of compounds for
22 h. Bifendate was used as a positive control. The nitrite concen-
tration in the culture medium was measured as an indicator of NO
3.86 (s, 3H), 3.96 (s, 3H), 6.01e6.09 (m, 4H), 7.19 (d, 1H, J ¼ 2.0 Hz),
7.26e7.28 (m, 2H), 7.47 (s, 1H), 7.49e7.52 (m, 1H), 7.74 (s, 1H), 12.58
(s, 1H); 13C NMR (d6-DMSO, 100 MHz)
d: 52.1, 56.4, 56.6, 56.7, 102.6,
production according to the Griess reaction. Each 50 mL of culture
102.7, 110.7, 111.3, 111.4, 112.4, 113.8, 121.8, 122.2, 123.4, 124.3, 126.0,
129.9,131.2,137.9,138.6,139.7,142.2,143.5, 147.3,147.4,153.0,163.3,
165.8, 167.5, 168.0; IR (KBr): 3218, 2946, 1743, 1705,1637, 1598, 1511,
1422, 1321, 1273, 1168, 1099, 1041, 931, 780 cmꢂ1; MS (ESI, m/z):
636.29 [MꢂH]ꢂ; HPLC purity ¼ 99.2%.
supernatant was mixed with an equal volume of Griess reagent and
incubated at room temperature for 10 min. he absorbance of the
mixture at 540 nm was measured with a SpectraMax M5 (Sunny-
vale, CA, USA). The values obtained were compared with those of
standard concentrations of sodium nitrite dissolved in RPMI 1640,

5948
G. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 5941e5948
and the concentrations of nitrite in the conditioned media of
sample-treated cells were calculated.
5.2.7. Histological examination
Small pieces of liver tissues were collected in 4% buffered
formalin and embedded in paraffin. Samples were cut 4
mm and
5.2.3. Animal treatment
stained with hematoxylin and eosin [15].
Male ICR mice and female BALB/c, purchased from the animal
breeding laboratories of Sichuan University Animal Center
(Sichuan, Chengdu, China), were breeded at a constant temperature
of 22 ꢁC, relative humidity 55.5% with 12 h dark-light cycles for at
least 2 weeks before the experiment. All experimental procedures
were performed according to the NIH Guide for the Care and Use of
Laboratory Animals.
Acknowledgments
The authors greatly appreciate the financial support from
National Key Technologies R&D Program of China (2009ZX09102-
045).
References
5.2.4. Paw edema induced by carrageenan
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Male ICR mice weighting 22e25 g were treated with carra-
geenan for determination of anti-inflammatory activity. Bifendate
and compound 7k dissolved into 5% Tween 80. Mice were
randomly divided into three groups, and each group consisted of six
animals. All animals were injected with 1% freshly prepared
suspension of carrageenan (Sigma, St. Louis, Missouri, USA) into
subplantar tissue of the right hind paw. Before 30 min, one groups
were orally administered with bifendate at dose of 50 mg/kg, other
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10 mg/kg, 25 mg/kg, 50 mg/kg, respectively. Another control group
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5.2.5. Acute liver injury mice model
Female BALB/c mice weighing 20e22 g, were administrated by
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30 min challenge, animals were orally administered with bifendate
or compound 7k at dose of 50 mg/kg, dissolved into 5% Tween 80.
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collected to determine the levels of ALT and AST. Liver tissues were
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5.2.6. Assay for serum aminotransferase levels
The serum levels of ALT and AST were measured by an auto-
mated enzyme assay, commercial kits and Roche automated
analyzers (Roche Diagnostics GmbH, Mannhein, Germany).