Synthesis and biological evaluation of novel 5-benzylidenethiazolidine-2,4- dione derivatives for the treatment of inflammatory diseases

Article abstract of DOI:10.1021/jm1011534

Twenty-two compounds based on thiazolidine-2,4-dione moiety were synthesized and evaluated for the inhibitory potency on the production of nitric oxide (NO), inducible nitric oxide synthase (iNOS) activity, and the generation of prostaglandin E₂ (PEG₂). (Z)-N-(3-Chlorophenyl)-2-(4- ((2,4-dioxothiazolidin-5-ylidene) methyl) phenoxy) acetamide (3I), superior to the commercial anti-inflammatory drug indomethacin, significantly inhibited iNOS activity (IC₅₀ = 8.66 μM), iNOS-mediated NO, and cyclooxygenase (COX)-2-derived PGE₂ production (IC₅₀ = 4.16 and 23.55 μM, respectively) on lipopolysaccharide (LPS)-induced RAW 264.7 cells. Docking study revealed that 3I was perfectly docking into the active site of murine iNOS and suppressed the expression of iNOS protein as evidenced by Western blot analysis. At the dose of 50 mg/kg, oral administration of 3I possessed protective properties in both carrageenan-induced paw edema and adjuvant-induced arthritis rat models.

Full text of DOI:10.1021/jm1011534

ARTICLE
pubs.acs.org/jmc
Synthesis and Biological Evaluation of Novel
5-Benzylidenethiazolidine-2,4-dione Derivatives for the Treatment of
Inflammatory Diseases
Liang Ma,†,§ Caifeng Xie,†,§ Yinghua Ma,†,§ Juan Liu,† Mingli Xiang,*,† Xia Ye,† Hao Zheng,† Zhizhi Chen,†
Qinyuan Xu,† Tao Chen,† Jinying Chen,† Jincheng Yang,† Neng Qiu,† Guangcheng Wang,† Xiaolin Liang,†
Aihua Peng,† Shengyong Yang,† Yuquan Wei,† and Lijuan Chen*,†
†State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Keyuan Road 4,
Gaopeng Street, Chengdu 610041, China
S Supporting Information
b
ABSTRACT: Twenty-two compounds based on thiazolidine-
2,4-dione moiety were synthesized and evaluated for the
inhibitory potency on the production of nitric oxide (NO),
inducible nitric oxide synthase (iNOS) activity, and the gen-
eration of prostaglandin E2 (PEG2). (Z)-N-(3-Chlorophenyl)-
2-(4-((2,4-dioxothiazolidin-5-ylidene) methyl) phenoxy) acet-
amide (3I), superior to the commercial anti-inflammatory drug
indomethacin, significantly inhibited iNOS activity (IC50 = 8.66
μM), iNOS-mediated NO, and cyclooxygenase (COX)-2-de-
rived PGE2 production (IC50 = 4.16 and 23.55 μM, re-
spectively) on lipopolysaccharide (LPS)-induced RAW 264.7
cells. Docking study revealed that 3I was perfectly docking into the active site of murine iNOS and suppressed the expression of
iNOS protein as evidenced by Western blot analysis. At the dose of 50 mg/kg, oral administration of 3I possessed protective
properties in both carrageenan-induced paw edema and adjuvant-induced arthritis rat models.
’ INTRODUCTION
iNOS produces a high level of NO and induces tissue injury at the
inflammatory site.8 Considerable evidence have shown that
overproduction of NO induced by iNOS has been implicated
in various pathological diseases including septic shock, tissue
damage, and rheumatoid arthritis (RA).9 Prostaglandin E2
(PGE2), one product of the PGs, is a proinflammatory mediator
and derived by the cyclooxygenase-1/2 (COX-1/2) pathway.10
PGE2 regulates vascular permeability, platelet aggregation, and
thrombus formation in the development of inflammation. In-
hibition of PGE2 production reduces the deleterious conse-
quences of inflammatory diseases.11 Therefore, suppression of
overproduction of NO and PGE2 might be useful for the
treatment of inflammatory diseases.
Thiazolidinediones (TZDs), ligands for peroxisome prolifera-
tor-activated receptor-γ (PPAR-γ), exert anti-inflammatory
effects independent of the insulin sensitizing effect.12 Natural
and synthetic ligands of PPAR-γ have been shown to inhibit the
production of several inflammatory cytokines including IL-1β
and TNF-R by macrophages in vitro and blockade inflammatory
developments.13,14 In vivo study has revealed that PPAR-γ
is a modulator of inflammation including the down-regulation
of level of iNOS and NO.15,16 PPAR-γ agonists reduce iNOS
Inflammation is a complex process involving numerous med-
iators of cellular and plasma origin with interrelated biological
effects. Macrophages, neutrophils, and lymphocytes are impor-
tant that have been implicated in the pathogenesis of acute and
chronic inflammatory diseases.1,2
Proinflammatory stimulus activate cellular responses and regu-
late inflammatory and immune functions including the recruit-
ment of macrophages and production of proinflammatory
cytokines.3 Macrophages produce nitric oxide (NO) and
up-regulate prostaglandins (PGs), interleukin-1β (IL-1β),
and tumor necrosis factor-R (TNF-R) in the inflammatory
process. NO, being an endogenous free radical, is an important
and unique mediator in the process of vasodilation, nonspecific
host defense, and acute or chronic inflammation.4-6 NO is
generated via the oxidation of L-arginine to L-citrulline by nitric
oxide synthase (NOS) isoforms in an nicotinamide adenine
dinucleotide phosphate (NADPH)- and O2-dependent process.7
There are at least three isoforms of the NOS enzyme: endothelial
(eNOS), neuronal (nNOS), and inducible (iNOS). Because
iNOS is responsible for the overproduction of NO in inflamma-
tion, it has become a new target for drug development in the
treatment of inflammatory diseases. Normally, NO is sustained at
a low level when stimulated by various inflammatory stimulus
(e.g., proinflammatory cytokines or lipopolysaccharide (LPS)),
Received: September 7, 2010
Published: March 07, 2011
r
2011 American Chemical Society
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Scheme 1. General Synthesis of 3Q-Va
a Reagents and conditions: (a) 2-chloroacetyl chloride, Et3N, CH2Cl2, 0-25 °C, 20 h; (b) KI, K2CO3, 4-hydroxybenzaldehyde, reflux, 24 h; (c)
thiazolidine-2,4-dione, β-alanine, AcOH, reflux, 4 h.
expression and NO production in macrophages, mesangial cells,
and other inflammatory cells.17 A novel small-molecule inhibitor
AS605240,18 one of TZD derivatives, effectively suppressed the
progression of joint inflammation and damage in both lympho-
cyte-independent and lymphocyte-dependent mouse models of
RA. These results encourage us to develop novel agents for the
treatment of NO and iNOS related inflammatory diseases.
In the present study, 22 compounds based on thiazolidine-2,4-
dione moiety were synthesized and evaluated for the biological
studies. Subsequently, (Z)-N-(3-chlorophenyl)-2-(4-((2,4-diox-
othiazolidin-5-ylidene) methyl)phenoxy)acetamide (3I) was
identified as the most potent chemical molecule. The IC50 values
of 3I on iNOS activity, the production of iNOS-mediated NO and
Figure 1. Chemical structure of indomethacin, 1, and 3I.
COX-2-derived PGE2 were 8.66, 23.55, and 4.16 μM, respec-
tively, on LPS-induced RAW 264.7 cells. Western blot analysis
further revealed that 3I inhibited LPS-induced iNOS and COX-2
protein expressions in a dose-dependent manner. Furthermore,
two inflammatory models from carrageenan-induced paw edema
in ICR mice and an adjuvant-induced arthritis in rats demon-
strated that 3I exhibited significantly anti-inflammatory activity.
1H NMR spectrum of 22 compounds exhibited only one signal
for the5-methylidene proton inthe range from7.73 to 7.78ppmin
dimethylsulfoxide (DMSO)-d6 solution, at lower field values
than those expected for the E-isomers.21,22 The Z-configuration
1
of similar compounds also have been identified from the H-
coupled 13C NMR spectrum and X-ray data followed by exam-
ination of the splitting pattern and coupling constant of the signal
of carbonyl group in the heterocyclic system.23-25 These experi-
’ CHEMISTRY
ments confirmed Z-configuration of similar compounds, which
Various amines in this study were converted into chloroace-
tamide derivatives with excellent yields via a reaction employed
2-chloroacetyl chloride in the presence of triethylamine as base
and using dichloromethane as solvent. The pivotal aldehyde
intermediates were synthesized through the use of chloroaceta-
mide derivatives, 4-hydroxybenzaldehyde, potassium carbonate
as base, and potassium iodide as catalyst according to Scheme 1,
and then, to obtain the 22 targeted compounds varying with the
western substituted group quickly, the Knoevenagel reaction
which accomplished by condensation aldehyde intermediate
with thiazolidine-2,4-dione was performed in parallel using
EYELA Personal Organic Synthesizer (Tokyo, Rikakikai) with
a 12-well liquid-phase reaction block.
can be assumed for our compounds.
At this stage, the products were fully analyzed and character-
1
ized by H-nuclear magnetic resonance (NMR), mass spectro-
metry (MS), and high-performance liquid chromatography
(HPLC) before entering the biological screening.
’ RESULTS AND DISCUSSION
Inhibition of NO Production in RAW 264.7 Microphages.
We initially accessed the capacity of our targeted compounds
contained thiazolidine-2,4-dione to inhibit NO production. Here
indomethacin (Figure 1),26 as a nonsteroidal anti-inflammatory
drug, was chosen as a positive control. As depicted in Table 1,
four compounds (3I, 3M, 3O, and 3U) suppressed the LPS-
induced production of NO at a concentration of 10 μM and their
inhibitory rates (IR) were all more than 70% compared to
indomethacin (IR = 63.2%). Among them, 3I (IR = 80.9%)
and 3U (IR = 83.9%) showed the most potent inhibitory effects.
In theory, E and Z geometrical isomers around the exocyclic
double bond (CHdC) are possible for 5-benzylidenethiazoli-
dine-2,4-dione derivatives. However, on the basisof literaturedata
for similar compounds, the Z-configuration has been reported
thermodynamically more stable than the E-configuration.19,20
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Table 1. Inhibitory Effects of 3A-V on NO Production in
15.37 μM for iNOS, 15.37 μM for NO, and 48.59 μM for PGE2)
on LPS-induced RAW 264.7 cells. To check whether the sup-
pressive effects of the four compounds on iNOS, NO, and PEG2
was related to cell viability, MTT assay was adopted. The most
potent one, 3I, showed no cytotoxicity on RAW 264.7 micro-
phages (without or withLPS, IC50 > 100 μM). However, wefound
that effects of 3U (IC50 = 62.26 μM without LPS and 54.97 μM
with LPS) were more cytotoxic than the other three compounds,
3M and 3U were less potent than 3I. On the basis of cellular
viability and anti-inflammatory activity in vitro, 3I were further
evaluated in the next experimental process.
Docking Study of All Molecules with Murine iNOS. All
molecules weredocked to theactive siteof murineiNOSbytheaid
of a protein-ligand docking program FRED. Compounds 3I, 3M,
and 3U are among the top five structures by computational scoring
which almost are consistent with our in vitro screenings. As the
most potent molecule 3I, its interaction mode binding to iNOS
protein data was depicted in Figure 2. The meta-chlorophenyl
moiety of 3I was buried in a pocket wove by Pro344 and Ph363-
Tyr367. The linker between the two phenyl groups was enclosed
by a polar cavity consisting of Gln257, Val346, and Glu371. The
thiazolidinedione moiety was surrounded by Trp457, Met114,
Ans115, Tyr485, and Ans348. In addition, two strong hydrogen
bonds are formed between 3I and Glu371 and Asn115 and a π-π
interaction between the hetero pentacycle of 3I and the side chain
of Tyr485 was also observed. These computational results con-
firmed our hypothesis that 3I was a potent and potential iNOS
inhibitor with perfect binding to the active site of murine iNOS,
and the hypothesis would be tested by Western blot analysis
further. The docking results of all molecules binding to iNOS
protein were shown in Supporting Information.
Detection of iNOS and COX-2 Expression. Because the high
level of NO is mainly generated by iNOS, the expression of iNOS
protein is an important step during the process of LPS-activated
production.28 To investigate the 3I-activated signaling pathway
and verify the docking result of 3I binding to murine iNOS,
Western blot analysis was applied to evaluate the effects on
expression of iNOS protein. As shown in Figure 3, the expression
of iNOS and protein were significantly inhibited by 3I in a good
dose-dependent manner (0-24 μM). Furthermore, COX-2 is
inducible and leads to excess production of prostaglandin (such
as PEG2) during injury, resulting in pain and inflammation.29
The inhibition of COX-2 protein is also thought to be enough to
provide the anti-inflammatory activity. Our result displayed that
the expression of COX-2 protein also was remarkably suppressed
by 3I at concentrations of 12 and 24 μM. Therefore, the data
demonstrated that 3I inhibited NO and PEG2 production
through down-regulation of LPS-induced iNOS and COX-2.
Treatment of Acute Inflammation. To evaluate the In Vivo
anti-inflammatory potency of 3I, two animal models of acute
inflammation were employed including carrageenan-induced
paw edema test and acetic acid-induced vascular permeability
test. The edema is an important parameter of acute inflammation
for evaluating compounds with potential anti-inflammatory
activity. In the carrageenan-induced paw edema test, the inflam-
matory response was quantified by increment in paw size
(edema) 2 h after carrageenan was injected and the paw edema
was observed. Oral administrations of 3I at doses of 10 and 50
mg/kg inhibited carrageenan-induced paw edema (Figure 4A).
Treatment with 50 mg/kg 3I exhibited more inhibitory activity
than dose of 10 mg/kg and 3I suppressed edema formation after
edema induction with the inhibitory rate of 36.2%. Indomethacin
RAW 264.7 Cellsa
NO inhibition
NO inhibition
compds
(%) ( SD
compds
(%) ( SD
indomethacin
63.2 ( 4.0c
55.3 ( 11.1b
38.4 ( 1.8b
0.6 ( 9.7
3L
10.0 ( 3.1
72.0 ( 2.1c
36.0 ( 3.8
83.9 ( 1.3d
51.0 ( 5.4b
47.8 ( 1.2b
41.6 ( 3.3b
14.6 ( 4.1
12.5 ( 6.2
70.9 ( 13.6c
45.1 ( 3.7b
3A
3B
3C
3D
3E
3F
3G
3H
3I
3M
3N
3O
3P
16.2 ( 6.2
26.6 ( 2.9
10.2 ( 1.9
41.5 ( 3.1b
57.9 ( 2.0b
80.9 ( 5.0c
9.7 ( 5.4
3Q
3R
3S
3T
3U
3 V
3J
3K
23.7 ( 3.2
a RAW 264.7 cells were pretreated with 3A-V at the concentration of
10 μM for 2 h and then incubated with LPS (1 μg/mL) for 18 h. The
amount of nitrite with LPS-treated only group was set as 100.0% and the
vehicle control as 0.0%. Inhibition (%) = [LPS (OD540) - compounds
(OD540)]/[LPS (OD540) - control (OD540)] ꢀ 100. Results are means
( SD of at least three independent experiments. b P < 0.05. c P < 0.01.
d P < 0.001 versus the LPS-treated group.
Compared to the unsubstituted phenyl ring compound 3A, the
inhibitory potency decreased dramatically when para substituents
of electron-donating groups (methyl for 3B, methoxyl for 3C,
trifluoromethyl for 3E, and meta- or para-dimethoxy for 3D) were
added, and then, para substituents of electron-withdrawing groups
(acetyl for 3F and para-halogenation including chlorine and
bromine atom for 3J-L and 3N) have clear negative effects on
inhibitory potency. However, the fluorine atom at the para
position (para-fluorinated for 3H and meta-, para-difluorinated
for 3O) improved the inhibitory effects as a result of their inherent
electronegativity and small atomic radius. The properties may
contribute to the stability and also served to block metabolism at
particularly reactive sites and reduced metabolism of the aromatic
group by decreasing its electron density. Our results on inhibitory
activity of NO production indicated that the introduction of meta-
halogenated groups was relevant, as 3G and 3I showed more
activity than the analogues 3H and 3J.
We also examined the replacement of phenyl ring (3A) with
pyridin-2-yl (compound 3S) and 5-methyl pyridine-2-yl (com-
pound 3T) group, the potency of inhibition decreased. The
presence of hydrophilic group seemed to reduce the capacity of
inhibition on NO production. Inclusion of flexible cyclohexyl
(3Q) and benzyl (3R) substituent resulted in a little reduction
in potency and were essentially equipotent with the activity of
3A. Introducing of the steric bulk of R- and β-naphthalene also
improved the inhibition, especially compound 3U (IR = 70.9%).
Inhibition of iNOS Activity, PEG2 Production, and Cyto-
toxicity. Four compounds (3I, 3M, 3O, and 3U) were exposed
to RAW 264.7 cells to investigate the inhibitory effects on iNOS
activity, the production of iNOS-mediated NO, COX-2-derived
PGE2, and cytotoxicity. As shown in Table 2, the four com-
pounds possessed remarkable inhibitory activity on iNOS, PEG2,
and NO. As anticipated, 3I exhibited the most potent inhibitory
potencies on iNOS activity (IC50 = 8.66 μM), the generation of
iNOS-mediated NO and COX-2-derived PGE2 (IC50 = 4.16 and
23.55 μM, respectively) compared to indomethacin (IC50
=
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Table 2. IC50 Values on NO, iNOS, PEG2, and Cytotoxicity in RAW 264.7 Cellsa
cytotoxicity IC50 (μM)
compds
NO IC50 (μM)
iNOS IC50 (μM)
PEG2 IC50 (μM)
without LPS
with LPS
indomethacin
48.89
23.55
57.42
34.08
40.00
23.47
8.66
15.37
4.16
>100
>100
>100
62.26
>100
>100
>100
>100
54.97
>100
3I
3M
3O
3U
26.13
13.92
20.87
14.81
7.12
16.12
a Cytotoxicity = (1 - [compounds (OD570) - background (OD570)]/[control (OD570) - background (OD570)]) ꢀ 100%.
Figure 2. The interaction mode of 3I within the active site of murine iNOS (PDB ID: 1r35). Residues in the active site are displayed in line style and
colored differently. Human erythrocyte membrane protein (HEM) is depicted by gray line and 3I is represented with stick. Hydrogen bonds and π-π
interaction formed between residues within the active site and 3I are highlighted. This figure was generated by the software Virtual Molecular Dynamics
(VMD).27
indomethacin in acetic acid-induced vascular permeability test
(Figure 4B). As suggested above, the consequences of the two
inflammatory models indicated that 3I was a potential anti-
inflammatory agent on inhibition of inflammatory edema.
Treatment of Adjuvant-Induced Arthritis. Because it
inhibited the carrageen in paw edema, 3I has been chosen to be
examined on adjuvant-induced arthritis (AIA). AIA is a well-
established experimental model of rheumatoid arthritis and is
often used for testing agents for anti-inflammatory activity. In this
model, 90-100% of rats developed arthritis within 14-18 days
after adjuvant injection. Histological changes, including leukocyte
(mainly neutrophil) invasion and synovial cell activation, precede
clinical symptoms. As displayed in Figure5A, rats treated with3I at
a dose of 50 mg/kg did not develop severe arthritis, indicating that
it exhibited potential immune- modulating activity. Arthritic score
and body weight loss were significantly reduced in the therapeutic
process. The time course of adjuvant arthritic development,
expressed asarthriticscores, was shownin Figure5A. A continuous
joint swelling was observed in the hind limbs and AIA rats had
Figure 3. Effect of 3I on LPS-induced iNOS and COX-2 protein
expression in RAW 264.7 cells. RAW 264.7 cells were pretreated with
the indicated concentrations of 3I for 2 h and treated with LPS (1 μg/
mL) for 18 h. Equal amounts of total protein were subjected to 8%
(iNOS and COX-2) SDS-PAGE.
showed an inhibitory rate of 33.3-40.7% of edema development.
However, unsatisfactory results of 3I-treated rats on inhibition of
vascular permeability were obtained in contrast to rats’ access to
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Figure 5. Effect of 3I on AIA model. 3I was orally administered once
daily at a dose of 50 mg/kg. (A) Clinical disease activity score of 3I. (B)
Body weight after adjuvant injection. The results were expressed as the
means ( SD (n = 6). *P < 0.05 versus vehicle control.
Figure 4. Effect of 3I on two animal models of inflammation. (A)
Carrageenan-induced paw edema in ICR mice. (B) Acetic acid-induced
vascular permeability in ICR mice. Indo, indomethacin as a positive control.
The results were expressed as the means ( SD (n = 6). ##P < 0.001 versus
blank. *P < 0.05; **P < 0.01; ***P < 0.001 versus vehicle control.
NO, TNF-R, and IL-1β) is regarded as one of the essential
conditions to alleviate a variety of inflammatory diseases trig-
gered by activation of macrophages.
In this study, we have identified that 3I effectively suppressed
the iNOS activity, the production of iNOS-mediated NO, and
COX-2-derived-PEG2 in RAW 264.7 cells induced by LPS. 3I
also inhibited the expression of iNOS and COX-2 protein.
Furthermore, we investigated anti-inflammatory activity of 3I
in mouse model of acute and chronic inflammation. For acute
inflammatory models in ICR mice, treatment of 3I improved the
inflammatory response such as decrement of the paw edema.
In an adjuvant-induced arthritis rat model, oral administration
(50 mg/kg) of 3I resulted in significant reduction of foodpad
swelling and improvement of pathology from joints of rats. These
observations suggest that 3I might be an effective agent for the
treatment of inflammatory diseases.
higher arthritic scores than those treated with 3I between days 17
and 28. 3I were found to improve the body weight loss and the
percent of variation was 15.1% (Figure 5B).
From the results of H&E staining (Figure 6), massive leuko-
cytes infiltrated into the synovial tissue and synovial fluid of joints
with the appearance of synovial cell proliferation in AIA model
group while treatment with 3I at the dosed of 50 mg/kg was
observed with few signs of inflammation and revealed substantial
improvements in histological state of joints.
Having identified potent anti-inflammatory agent with favor-
able in vitro and in vivo results, we selected 3I for further
profiling in rat pharmacokinetics (Table 3). The pharmacoki-
netic data of 3I exhibited that the oral bioavailability was 18.10%,
with the half-time of 2.16 h.
’ EXPERIMENTAL SECTION
’ CONCLUSION
Inflammation is a host response to stimuli and is characterized
by the signs of redness, swelling, heat, and pain. The pathology of
inflammation is initiated by complex processes caused by patho-
gens such as LPS, which is a prototypical endotoxin.30 LPS
activate directly macrophages to produce proinflammatory cyto-
kines, such as NO, TNF-R, and IL-1β.31 Therefore, pharmaco-
logical reduction of LPS-inducible inflammatory mediators (e.g.,
Materials. LPS (Escherichia coli serotype 0111:B4), MTT, Evans
blue, Carrageenan from seaweed (a mixture of λ- and κ-carrageenans),
incomplete Freund’s adjuvant (IFA), and indomethacin were obtained
from Sigma Chemical Co. (St. Louis, MO). The enzyme immunoassay
(EIA) kits for PGE2, TNF-R, and IL-1β were purchased from R&D
Systems (Minneapolis, MN). iNOS rabbit polyclonal antibody (1:1000)
and β-actin monoclonal antibody (1:1000) were obtained from Cell
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Figure 6. H&E staining for histological changes of joints of 3I-treated AIA rats. (A) Normal. (B) Model. (C) 3I-treated model group at the dose of
50 mg/kg.
Table 3. Pharmacokinetic Profiles for 3I in SD Ratsa
times with PBS and were lysed by 30 min incubation on ice in lysis buffer
containing 10 mM Tris-Cl, pH 7.4, 3 mM CaCl2, 2 mM MgCl2, 1% NP-
Cmax (μM) Tmax (h) AUC0-t h (μM h/L) t1/2 (hr) F (%)
3
40, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and protease
inhibitor cocktail. The lysate was centrifuged at 15000g for 10 min
and the supernatant was collected. The protein concentration was
determined using the Bio-Rad protein assay reagent (Bio-Rad, Hercules,
CA). Total protein equivalents were separated by 8% SDS-polyacryla-
mide gel electrophoresis (SDS-PAGE) and were electroblotted onto
polyvinylidene difluoride (PVDF) membrane. The transblotted mem-
brane was washed twice with TBST. After blocking with TBST containing
5% skim milk for 2 h, the membrane was properly cut into three mem-
branes corresponding to iNOS, COX-2, and β-actin. The membranes were
incubated overnight with primary antibodies from rabbit or mouse at 4 °C
with gentle shaking. Blots were washed three times in TBST for 30 min,
followed by incubation with horseradish peroxidase (HRP)-conjugated
secondary antibody for 40 min at room temperature. Immunoreactive
bands were visualized by the enhanced chemiluninescence system.
In Vivo Experiments. ICR male mice and female Lewis rats were
housed in filtered-capped polycarbonate cages and allowed food and
water ad libitum. Animals were kept on a cycle of 12 h light/darkness at
22 ( 1 °C and acclimated for at least one week until use. All progressions
were maintained under controlled conditions and animals received
human care according to National Institutes of Health Guidelines before
and during experiment.
Carrageenan-Induced Paw Edema Test in ICR Mice. The
mice were randomly divided into four groups. Doses (10 mg/kg and 50
mg/kg) of compound 3I were administered ip to the test groups,
respectively. The positive control group and the vehicle control group
were given access to indomethacin (10 mg/kg, ip) and the same volume
of olive oil (10 mL/kg, ip), respectively. Thirty minutes after the
administration, acute paw edema was induced in the right hind paw
by subplantar injection of 1% freshly prepared carrageenan suspension in
normal saline, 0.05 mL per mouse. The thickness of the paw was
measured preinjection and at intervals of 1, 2, 3, 4, and 5 h postinjection,
using a dial thickness gauge. The percent increase of paw thickness was
calculated based on the preinjection thickness of the paw.
Acetic Acid-Induced Vascular Permeability Test in ICR
Mice. The mice were randomly divided into four groups. Doses (10
and 50 mg/kg) of compound 3I were administered ip to the test groups,
respectively. The positive control group and the vehicle control group
were given access to indomethacin (10 mg/kg, ip) and the same volume
of olive oil (10 mL/kg, ip), respectively. After 30 min, 10 mL/kg body
weight of 1% Evans blue in normal saline was injected intravenously into
the tail vein of each animal. Sixty minutes after administration, each
mouse was injected with 10 mL/kg (ip) of 1.2% acetic acid in normal
saline. Fifty minutes after the injection, the mice were sacrificed by
cervical dislocation. Eight milliliters of normal saline was injected into
the peritoneal cavity, and the washing solutions were collected in test
tubes. To clear turbidity due to protein, 1 N NaOH solution to a ratio of
0.1 mL/10 mL was added to each tube, and allowed to stand overnight at
po
iv
15.31
27.0
0.18
0.08
10.0
2.16
0.28
18.10
11.05
a Dose: 50 mg/kg, po; 10 mg/kg, iv.
Signaling Technology, Inc. (Beverly, MA). ICR male mice weighing
18-25 g and female inbred Lewis rats (6-8 weeks old) were purchased
from Tengxin Biotech Company (Chongqing, P. R. China).
Cell Culture. RAW 264.7 murine macrophages were obtained from
American Type Culture Collection (ATCC, Rockville, MD, USA).
These cells were grown in RPMI 1640 containing 10% FBS, 100
units/mL penicillin, and 100 μg/mL streptomycin in a 95% air, 5%
CO2 humidified atmosphere at 37 °C.
Nitrite Assay. RAW 264.7 cells were seeded into a 96-well culture
plate at a density of 1 ꢀ 104 cells per well with 500 μL of culture medium
and incubated for 24 h. The cells were then pretreated with compounds
(3A-V) at a concentration of 10 μg/mL or compounds 3I, 3M, 3O, 3U,
and indomethacin with a series of concentrations for 2 h before
stimulation with LPS (1 μg/mL) for 18 h. The nitrite concentra-
tion in the medium was measured according to the Griess reaction by
adding 50 μL of Griess reagent (1% sulfanilamide and 0.1% N-
(1-naphthyl)ethylenediamine dihydrochloride in 5% phosphoric acid)
to 50 μL of medium for 5 min. The optical density at 540 nm (OD540
)
was measured with a microplate reader. Concentrations were calculated
by comparison with the OD540 values of a standard solution of sodium
nitrite prepared in culture medium.
Cell Cytotoxicity. Cell cytotoxicity was evaluated by MTT and
expressed as IC50. Raw 264.7 cells were treated with compounds alone
or in combination with LPS for 24 h. Cells were washed with PBS and
incubated in 0.5 mg/mL MTT reagent dissolved in RPMI 1640 for 4 h,
and the formazan product dissolved in 150 μL of DMSO. The optical
density was measured using an ELISA plate reader at 570 nm (OD570).
Assay of iNOS Enzymatic Activity. After treated with LPS
(1 μg/mL) and indomethacin, 3I, 3M, 3O, and 3U (1-50 μM) for 2 h at
37 °C, the culture supernatant was removed and 100 μL of NOS assay buffer
(1ꢀ) were added to each well. Then 100 μL of NOS assay reaction solution
(50% NOS assay buffer, 39.8% Milli-Q water, 5% L-arginine solution, 5%
0.1 mM NADPH, 0.2% DAF-FMDA) was added to each well and incubated
for 2 h at 37 °C. Fluorescence was measured with a fluorescence plate reader
(Biotek) at excitation of 485 nm and emission of 528 nm.
Determination of PGE2. RAW 264.7 cells were seeded in wells
and incubated for 24 h. After incubation, the cells were incubated with
compounds (3I, 3O, 3M, and 3U) of different concentrations in the
presence of LPS (1 μg/mL) for 24 h. The PGE2 concentration in the
culture medium was determined by ELISA kit (R&D Systems, Minnea-
polis, MN).
Detection of iNOS and COX-2 Expression. After the treat-
ment with LPS (1 μg/mL) and 3I (0-24 μM), cells were washed three
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Journal of Medicinal Chemistry
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4 °C. The solution was subjected to colorimetry using a UV/vis spectro-
photometer GeneQuant II (Amersham Bioscience, Sweden) at 610 nm.
The vascular permeability effects were expressed according to the
concentration of dye (μg/mL) that had leaked into the peritoneal cavity.
Induction and Evaluation of Adjuvant-Induced Arthritis.
The rat AIA model was introduced in this experiment. The rats were
injected subcutaneously (sc) at the tail base with 0.5 mg heat-killed
inactivated Mycobacterium tuberculosis emulsified in 0.1 mL IFA on day 0,
at multiple sites. 3I was orally administered once daily at a dose of 50
mg/kg, an effective dose according to the previous two animal models of
inflammation experiments. The treatment was initiated from the onset
of disease on day 17 after the vaccination. Animals from the control
group received 0.5% polysorbate 80 only. All animals were treated
continuously up to day 28.
Body weight of rats was observed thrice daily after adjuvant injection.
The clinical disease activity score was initiated from day 13 and assessed
as follows: the animals were inspected every day for symptoms of clinical
arthritis. The score for each limb ranged from 0 to 4; 0, no arthritis; 1,
redness or swelling of one toe/finger joint; 2, redness and swelling of
more than one toe/finger joints; 3, involvement of the ankle and tarsal-
metatarsal joints; 4, redness or swelling of the entire paw. The clinical
score was calculated by adding the scores for four individual legs.
Histopathological Evaluation. Rats were all sacrificed on day
28. The legs reserving knee joints were removed in toto, fixed in 10%
formalin, decalcified in EDTA buffer, subjected in a series progression of
dehydration, and then embedded in paraffin. Samples were serially
sectioned at 4 μm and processed routinely for H&E staining. The
histological changes were examined under microscope.
Pharmacokinetic Study of 3I. Two groups of male and female
Sprague-Dawley rats (180-200 g) were fasted overnight and received
compound 3I as an intravenous (IV) dose (10 mg/kg) or by oral gavage
(50 mg/kg, Dissolved into Tween 80). Blood samples (0.5 mL) were
obtained retro-orbital bleeding at 0.05, 0.25, 0.5, 1, 3, 6, 8, and 24 h post
dose for the IV group and at 0.25, 0.5, 1, 3, 6, 8, and 24 h post dose for the
PO dose group. At each time point, three mice were bled resulting in a
composite pharmacokinetic profile. The tubes were inverted several
times to ensure mixing and placed on ice. Plasma was obtained following
centrifugation at 4 °C (1500-2000g). Plasma samples were stored at -
20 °C until analysis by a UPLC assay.
Chemistry. Chemical reagents of analytical grade were purchased
from Chengdu Changzheng Chemical Factory (Sichuan, P. R. China).
The final compounds were synthesized using an EYELA personal
organic synthesizer with ChemiStation PPS-CTRL and PPW-20A
(Tokyo, Rikakikai) using a 5-well liquid-phase reaction block. TLC
was performed on 0.20 mm silical gel 60 F254 plates (Qingdao Ocean
Chemical Factory, Shangdong, China). 1H NMR were recorded at 400
MHz on a Varian spectrometer (Varian, Palo Alto, CA, USA) model
Gemini 400 and reported in parts per million. Chemical shifts (δ) are
quoted in ppm relative to tetramethylsilane (TMS) as an internal
standard, where (δ) TMS = 0.00 ppm. The multiplicity of the signal
is indicated as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet,
defined as all multipeak signals where overlap or complex coupling of
signals makes definitive descriptions of peaks difficult. MS were mea-
sured by Q-TOF Priemier mass spectrometer utilizing electrospray
ionization (ESI) (Micromass, Manchester, UK). Room temperature
(RT) is within the range 20-25 °C. The purity of compound screened
in biological assays was determined to be g97% by HPLC analysis with a
phodediode array detector (Waters, Milford, MA, USA) and the
chromatographic column was a atlantis C18 (150 mm ꢀ 4.6 mm, I.D.
5 μm) (Waters, Milford, Ireland). All compounds were supplied as 1
mg/mL in DMSO or methanol with 10 μL injected on a partial loop fill
at a flow rate of 1 mL/min for 30 min. Solvent A, 75% methanol; Solvent
B, 25% water with 0.5% trifluoroacetic acid. The detailed HPLC analysis
of 3A-V were shown in the Supporting Information.
General Procedure for Synthesis of 2-Chloro-N-substi-
tuted-acetamide (1A-V). 2-Chloroacetyl chloride (24 mmol)
was slowly added dropwise to a mixture of R-NH2 (20 mmol) and
Et3N (24 mmol, 3.3 mL) in anhydrous CH2Cl2 (20 mL) at 0 °C. The
reaction mixture was warmed to room temperature and stirred for an
additional 20 h. After the solvent was removed under reduced pressure,
the residue was washed with ice water (3 ꢀ 20 mL), and the precipitate
was separated by filtration. The crude product was purified by crystal-
lization from a mixture solvent of Ether/petroleum.
General Procedure for Synthesis of 2-(4-Formylphenoxy)-
N-substituted-phenyl-acetamide and Special Aldehyde In-
termediates (2A-V). 4-Hydroxybenzaldehyde (1.34 g, 11 mmol),
anhydrous K2CO3 (2.76 g, 20 mmol), and the 2-chloro-N-substituted-
acetamide (10 mmol) were dissolved in anhydrous acetone (30 mL),
and then KI (166 mg, 1 mmol) were added into the solution. The
reaction mixture was refluxed for 24 h and then cooled to room
temperature. Then the K2CO3 solid was filtered and the acetone
solution was removed under reduced pressure to obtain the crude
products. The residue was purified by silica gel column chromatography
(eluent:ethyl acetate/petroleum = 1/1.5) to give the appropriate
aldehyde product.
General Procedure for Synthesis of 3A-V. The appropriate
aldehyde (3.0 mmol), 10 mL glacial acetic acid, β-alanine (6.0 mmol),
and 2-thiazolidin-4-one (6.0 mmol) were mixed in parallel in 12 test
tubes with reflux condensers and refluxed at 1000 rpm in tubes for 4 h.
Then a small portion of water was added, and the precipitated solids
were collected by sucking filtration and washed with glacial acetic acid
(3 ꢀ 15 mL), distilled water (4 ꢀ 15 mL), and ether (2 ꢀ 10 mL). The
solids obtained were dried in vacuum at 40 °C for 24 h.
(Z)-2-(4-((2,4-Dioxothiazolidin-5-ylidene)methyl)pheno-
xy)-N-phenylacetamide (3A). Yield 83.4%; analytical HPLC pur-
1
ity = 98.60%; light-yellow solid. H NMR (400 MHz, DMSO-d6):
δ 12.54 (s, 1H), 10.15 (s, 1H), 7.76 (s, 1H), 7.64-7.58 (m, 4H), 7.33
(t, 2H, J = 8.2 Hz), 7.15 (d, 2H, J = 8.4 Hz), 7.09 (t, 1H, J = 7.4 Hz),
4.81 (s, 2H). MS (ESI), m/z: 354.07 [M - H]-.
(Z)-2-(4-((2,4-Dioxothiazolidin-5-ylidene) methyl) pheno-
xy)-N-p-tolylacetamide (3B). Yield 74.3%; analytical HPLC purity
= 98.24%; light-yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 12.54
(s, 1H), 10.06 (s, 1H), 7.76 (s, 1H), 7.59 (d, 2H, J = 8.8 Hz), 7.51 (d, 2H,
J = 8.4 Hz), 7.14 (t, 4H, J = 9.2 Hz), 4.78 (s, 2H), 2.26 (s, 3H). MS (ESI),
m/z: 367.18 [M - H]-.
(Z)-2-(4-((2,4-Dioxothiazolidin-5-ylidene)methyl)pheno-
xy)-N-(4-methoxyphenyl)acetamide (3C). Yield 87.9%; analyti-
1
cal HPLC purity = 98.54%;. light-yellow solid. H NMR (400 MHz,
DMSO-d6): δ 12.54 (s, 1H), 10.01 (s, 1H), 7.76 (s, 1H), 7.59 (d, 2H,
J = 8.4 Hz), 7.53 (d, 2H, J = 8.8 Hz), 7.16 (d, 2H, J = 8.4 Hz), 6.90 (d, 2H,
J = 4.8 Hz), 4.77 (s, 2H), 3.72 (s, 3H). MS(ESI), m/z: 383.10[M - H] -.
(Z)-N-(3,4-Dimethoxyphenyl)-2-(4-((2,4-dioxothiazolidin-
5-ylidene)methyl)phenoxy)acetamide (3D). Yield 86.3%; ana-
lytical HPLC purity = 99.05%; yellow solid. 1H NMR (400 MHz,
DMSO-d6): δ 12.54 (s, 1H), 10.00 (s, 1H), 7.76 (s, 1H), 7.59 (d, 2H, J =
7.6 Hz), 7.33(d, 1H, J = 2.0 Hz), 7.18-7.15 (m, 3H), 6.90 (d, 1H, J = 8.8
Hz), 4.77 (s, 2H), 3.72 (s, 3H), 3.72 (s, 3H). MS (ESI), m/z: 413.10
[M - H]-.
(Z)-2-(4-((2,4-Dioxothiazolidin-5-ylidene)methyl)pheno-
xy)-N-(4-(trifluoromethyl)phenyl)acetamide (3E). Yield 75.8%;
1
analytical HPLC purity = 98.78%; yellow solid. H NMR (400 MHz,
DMSO-d6): δ 12.53 (s, 1H), 10.52 (s, 1H), 7.85 (d, 2H, J = 8.0 Hz), 7.75
(s, 1H), 7.70 (d, 2H, J = 8.4 Hz), 7.58 (d, 2H, J = 8.8 Hz), 7.15 (d, 2H, J =
8.8 Hz), 4.85 (s, 2H). MS (ESI), m/z: 421.01 [M - H]-.
(Z)-N-(4-Acetylphenyl)-2-(4-((2,4-dioxothiazolidin-5-yli-
dene)methyl)phenoxy)acetamide (3F). Yield 84.0%; analytical
HPLC purity = 99.10%; yellow solid. 1H NMR (400 MHz, DMSO-d6):
δ 12.53 (s, 1H), 10.50 (s, 1H), 7.95 (d, 2H, J = 8.4 Hz), 7.77 (d, 1H,
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Journal of Medicinal Chemistry
ARTICLE
J = 10.8 Hz), 7.76 (s, 2H), 7.59 (d, 2H, J = 8.4 Hz), 7.16 (d, 2H, J = 8.4
Hz), 4.86 (s, 2H), 2.53 (s, 3H). MS (ESI), m/z: 395.26 [ M - H]-.
(Z)-N-Cyclohexyl-2-(4-((2,4-dioxothiazolidin-5-ylidene)
methyl)phenoxy)acetamide (3Q). Yield 78.3%; analytical HPLC
purity = 98.61%; light-yellow solid. H NMR (400 MHz, DMSO-d6):
1
(Z)-2-(4-((2,4-Dioxothiazolidin-5-ylidene)methyl)pheno-
xy)-N-(3-fluorophenyl)acetamide (3G). Yield 73.7%; analytical
HPLC purity = 99.11%; white solid. 1H NMR (400 MHz, DMSO-d6):
δ 12.53 (s, 1H), 10.33 (s, 1H), 7.83 (s, 1H), 7.75 (s, 1H), 7.58 (d, 2H, J =
8.4 Hz), 7.52 (d, 1H, J = 8.4 Hz), 7.36 (t, 1H, J = 8.0 Hz), 7.15 (d, 3H, J =
8.4 Hz), 4.82 (s, 2H). MS (ESI), m/z: 371.01 [M - H]-.
(Z)-2-(4-((2,4-Dioxothiazolidin-5-ylidene)methyl)pheno-
xy)-N-(4-fluorophenyl)acetamide (3H). Yield 71.8%; analytical
HPLC purity = 99.40%; yellow solid. 1H NMR (400 MHz, DMSO-d6):
δ 12.54 (s, 1H), 10.21 (s, 1H), 7.76 (s, 1H), 7.67-7.63 (m, 2H), 7.59
(d, 4H, J = 8.8 Hz), 7.17 (t, 2H, J = 8.4 Hz), 4.80 (s,2H). MS (ESI), m/z:
372.06 [M - H]-.
(Z)-N-(3-Chlorophenyl)-2-(4-((2,4-dioxothiazolidin-5-yli-
dene)methyl)phenoxy)acetamide (3I). Yield 92.8%; analytical
HPLC purity = 98.53%; white solid. 1H NMR (400 MHz, DMSO-d6): δ
12.55 (s, 1H), 10.35(s, 1H), 7.84 (s, 1H), 7.77 (s, 1H), 7.59(d, 2H, J = 8.4
Hz), 7.53 (d, 1H, J = 8.0 Hz), 7.37 (t, 1H, J = 8.2 Hz), 7.16 (d, 2H, J = 8.4
Hz), 4.83 (s, 2H). MS (ESI), m/z: 386.98 [M - H]-.
(Z)-N-(4-Chlorophenyl)-2-(4-((2,4-dioxothiazolidin-5-yli-
dene)methyl)phenoxy)acetamide (3J). Yield 73.4%. analytical
HPLC purity = 98.06%; yellow solid. 1H NMR (400 MHz, DMSO-d6):
δ 12.54 (s, 1H), 10.30 (s, 1H), 7.76 (s, 1H), 7.67 (m, 2H, J = 8.8 Hz), 7.59
(d, 2H, J = 8.8 Hz), 7.39 (d, 2H, J = 9.2 Hz), 7.16 (d, 2H, J = 8.8 Hz), 4.81
(s,2H). MS (ESI), m/z: 387.06 [ M - H]-.
δ 12.53 (s, 1H), 7.95 (d, 1H, J = 8.0 Hz), 7.75 (s, 1H), 7.56 (d, 2H, J = 8.8
Hz), 7.09 (d, 2H, J = 8.8 Hz), 4.54 (s, 2H), 3.61 (t,1H, J = 3.6 Hz), 1.74 -
1.64 (m, 4H), 1.58-1.55 (m, 1H), 1.31-1.19 (m, 4H), 1.15-1.06 (m,
1H). MS (ESI), m/z: 359.07 [M - H]-.
(Z)-N-Benzyl-2-(4-((2,4-dioxothiazolidin-5-ylidene)methyl)-
phenoxy)acetamide (3R). Yield 84.3%; analytical HPLC purity =
98.58%; yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 12.54 (s, 1H),
8.70 (t, 1H, J = 6.0 Hz), 7.76 (s, 1H), 7.57 (d, 2H, J = 8.8 Hz), 7.32-7.31
(m, 2H), 7.25-7.21 (m, 3H), 7.12 (d, 2H, J = 8.8 Hz), 4.65 (s, 2H), 4.34
(d, 2H, J = 6.0 Hz). MS (ESI), m/z: 367.15 [M - H]-.
(Z)-2-(4-((2,4-Dioxothiazolidin-5-ylidene)methyl)pheno-
xy)-N-(pyridin-2-yl)acetamide (3S). Yield 65.8%; analytical HPLC
purity = 98.05%; light-yellow solid. 1H NMR (400 MHz, DMSO-d6):
δ 12.53 (s, 1H), 10.62 (s, 1H), 8.35 (d, 1H, J = 8.0 Hz), 8.04 (d, 1H,
J = 8.0 Hz), 7.83-7.75 (m, 2H), 7.75 (s, 1H), 7.58 (d, 2H, J =
8.8 Hz), 7.16-7.12 (m, 3H), 4.89 (s, 2H). MS (ESI), m/z: 354.03
[M - H]-.
(Z)-2-(4-((2,4-Dioxothiazolidin-5-ylidene)methyl)pheno-
xy)-N-(5-methylpyridin-2-yl)acetamide(3T). Yield 74.1%; ana-
lytical HPLC purity = 98.29%; light-yellow solid. 1H NMR (400 MHz,
DMSO-d6): δ 12.54 (s, 1H), 10.53 (s, 1H), 8.18 (q, 1H, J = 3.2 Hz), 7.95
(d, 1H, J = 7.6 Hz), 7.76 (s, 1H), 7.64-7.61 (m, 1H), 7.58 (d, 2H, J = 8.8
Hz), 7.13 (d, 2H, J = 9.2 Hz), 4.88 (s, 2H), 2.26 (s, 3H). MS (ESI), m/z:
368.16 [M - H]-.
(Z)-2-(4-((2,4-Dioxothiazolidin-5-ylidene)methyl)pheno-
xy)-N-(naphthalen-1-yl)acetamide (3U). Yield 68.3%; analytical
HPLC purity = 98.77%; light-yellow solid. 1H NMR (400 MHz, DMSO-
d6): δ 12.54 (s, 1H), 10.21 (s, 1H), 8.00-7.94 (m, 2H), 7.81 (d, 2H, J =
8.0 Hz), 7.78 (s, 1H), 7.66-7.61 (m, 3H), 7.55-7.61 (m, 3H), 7.23 (d,
2H, J = 8.0 Hz), 4.98 (s, 2H). MS (ESI), m/z: 403.05 [M - H]-.
(Z)-2-(4-((2,4-Dioxothiazolidin-5-ylidene)methyl)pheno-
xy)-N-(naphthalen-2-yl)acetamide (3V). Yield 70.0%; analytical
HPLC purity = 99.22%; white solid. 1H NMR (400 MHz, DMSO-d6):
δ 12.54 (s, 1H), 10.38 (s, 1H), 8.32 (s, 1H), 7.90-7.82 (m, 3H), 7.77 (s,
1H), 7.68-7.65 (m, 1H), 7.60 (d, 2H, J = 8.8Hz), 7.48 (t, 1H, J = 7.2 Hz),
7.42 (t, 1H, J = 7.2 Hz), 7.20 (d, 2H, J = 8.8 Hz), 4.88 (s, 2H). MS (ESI),
m/z: 403.10 [M - H]-.
(Z)-N-(4-Bromophenyl)-2-(4-((2,4-dioxothiazolidin-5-yli-
dene)methyl)phenoxy)acetamide (3K). Yield 80.2%; analytical
HPLC purity = 99.45%; light-yellow solid. 1H NMR (400 MHz, DMSO-
d6): δ 12.53 (s, 1H), 10.29 (s,1H), 7.76 (s, 1H), 7.62-7.57 (m, 4H), 7.51
(d, 2H, J= 8.8Hz), 7.15 (d, 2H, J= 8.8Hz), 4.841(s, 2H). MS(ESI), m/z:
431.08 [M - H]-.
(Z)-N-(2,4-Dichlorophenyl)-2-(4-((2,4-dioxothiazolidin-5-
ylidene)methyl)phenoxy)acetamide (3L). Yield 80.3%; analyti-
1
cal HPLC purity = 98.16%; light-yellow solid. H NMR (400 MHz,
DMSO-d6): δ 12.55 (s, 1H), 9.81 (s, 1H), 7.84-7.80 (m, 1H), 7.77
(s, 1H), 7.72-7.71 (m, 1H), 7.60 (d, 2H, J = 8.8 Hz), 7.47-7.44
(m, 1H), 7.18 (d, 2H, J = 8.8 Hz), 4.89 (s, 2H). MS (ESI), m/z: 421.10
[M - H]-.
(Z)-N-(3,5-Dichlorophenyl)-2-(4-((2,4-dioxothiazolidin-5-
ylidene)methyl)phenoxy)acetamide (3M). Yield 72.1%; analy-
’ ASSOCIATED CONTENT
1
tical HPLC purity = 98.64%; light-yellow solid. H NMR (400 MHz,
Supporting Information. 1H NMR and MS analysis of
DMSO-d6): δ 12.55 (s, 1H), 10.47 (s, 1H), 7.77 (s, 1H), 7.74 (d, 2H, J =
1.6 Hz), 7.59 (d, 2H, J = 9.2 Hz), 7.33 (t, 1H, J = 1.6 Hz), 7.16 (d, 2H, J =
8.8 Hz), 4.84 (s, 2H). MS (ESI), m/z: 421.01 [M - H]-.
S
b
1A-V, 2A-V, and 3I; the detailed HPLC analysis of 3A-V;
docking results of all molecules with murine iNOS. This material
is available free of charge via the Internet at http://pubs.acs.org.
(Z)-N-(3-Chloro-4-fluorophenyl)-2-(4-((2,4-dioxothiazoli-
din-5-ylidene)methyl)phenoxy)acetamide (3N). Yield 84.5%;
1
analytical HPLC purity = 98.34%; yellow solid. H NMR (400 MHz,
’ AUTHOR INFORMATION
DMSO-d6): δ 12.54 (s, 1H), 10.36 (s, 1H), 7.96-7.94 (m, 2H), 7.76 (s,
1H), 7.60-7.54 (m, 3H), 7.40 (t, 1H, J = 8.8 Hz), 7.16 (d, 2H, J = 8.4Hz),
4.86 (s, 2H). MS (ESI), m/z: 406.08 [M - H]-.
(Z)-N-(3,4-Difluorophenyl)-2-(4-((2,4-dioxothiazolidin-5-
ylidene)methyl)phenoxy)acetamide (3O). Yield 81.5%; analy-
tical HPLC purity = 99.87%; white solid. 1H NMR (400 MHz, DMSO-
d6): δ 12.53 (s, 1H), 10.37 (s, 1H), 7.82-7.77 (m, 1H), 7.75 (s, 1H), 7.58
(d, 2H, J = 8.8 Hz), 7.45-7.38 (m, 2H), 7.15 (d, 2H, J = 8.4 Hz), 4.81 (s,
2H). MS (ESI), m/z: 389.20 [M - H]-.
Corresponding Author
*For M.-L.X.: phone, þ86-28-85164103; E-mail, tmkxiang@
gmail.com. For L.-J.C.: phone, þ86-28-85164063; fax, þ86-28-
85164060; E-mail, lijuan17@hotmail.com.
Author Contributions
§These authors contributed equally and should be considered co-
first authors.
(Z)-2-(4-((2,4-Dioxothiazolidin-5-ylidene)methyl)pheno-
xy)-N-methyl-N-phenylacetamide (3P). Yield 79.6%; analyti-
cal HPLC purity = 99.91%; yellow solid. 1H NMR (400 MHz, DMSO-
d6): δ 12.52 (s, 1H), 7.73 (s, 1H), 7.52-7.50 (m, 6H), 7.41 (s, 1H),
6.93 (s, 2H), 4.53 (s, 2H), 3.20 (s, 3H). MS (ESI), m/z: 367.20
[M - H]-.
’ ACKNOWLEDGMENT
We are grateful to the National Key Programs of China during
the 11th Five-Year Plan period (2009ZX09102-045 and
2009ZX09501-015).
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Journal of Medicinal Chemistry
ARTICLE
’ ABBREVIATIONS USED
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RA, rheumatoid arthritis; NO, nitric oxide; PEG2, prostaglandin
E2; iNOS, inducible nitric oxide synthase; eNOS, endothelial
nitric oxide synthase; nNOS, neuronal nitric oxide synthase;
COX, cyclooxygenase; IL-1β, interleukin-1β; TNF-R, tumor ne-
crosis factor-R; NADPH, nicotinamide adenine dinucleotide
phosphate; LPS, lipopolysaccharide; TZDs, thiazolidinediones;
PPAR-γ, peroxisome proliferator-activated receptor-γ; IR,
inhibitory rate; IC50, half maximal inhibitory concentration; AIA,
adjuvant-induced arthritis; NMR, nuclear magnetic resonance;
MS, mass spectrometry; HPLC, high performance liquid chro-
matography; DMSO, dimethylsulfoxide; H&E, hematoxylin
and eosin
(22) Tihomir, T.; Nace, Z.; Manica, M. P.; Danijel, K.; Lucija, P. M.
Synthesis and antibacterial activity of 5-ylidenethiazolidin-4-ones and
5-benzylidene-4,6-pyrimidine-diones. Eur. J. Med. Chem. 2010,
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4-benzoxazin-3(4H)-ones and 5-(2-(4-Benzyl-3-oxo-3,4-dihydro-2H-
1,4-benzoxazin-2-yl)ethylidene)thiazolidine-2,4-diones. Acta. Chim.
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