Structural exploration, synthesis and pharmacological evaluation of novel 5-benzylidenethiazolidine-2,4-dione derivatives as iNOS inhibitors against inflammatory diseases

Article abstract of DOI:10.1016/j.ejmech.2014.12.036

In our previous work, 3I inhibited the LPS-induced iNOS activity and NO production in RAW 264.7 cells and improved joint inflammation and cartilage destruction in inflammatory model. In this study, we synthesized 59 derivatives and bioisosteres on the bas

Full text of DOI:10.1016/j.ejmech.2014.12.036

European Journal of Medicinal Chemistry 92 (2015) 178e190
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structural exploration, synthesis and pharmacological evaluation of
novel 5-benzylidenethiazolidine-2,4-dione derivatives as iNOS
inhibitors against inflammatory diseases
Liang Ma 1, Heying Pei 1, Lei Lei, Linhong He, Jinying Chen, Xiaolin Liang, Aihua Peng,
Haoyu Ye, Mingli Xiang, Lijuan Chen*
State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, West China Medical School,
Sichuan University, Chengdu 610041, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In our previous work, 3I inhibited the LPS-induced iNOS activity and NO production in RAW 264.7 cells
and improved joint inflammation and cartilage destruction in inflammatory model. In this study, we
synthesized 59 derivatives and bioisosteres on the basis of 3I by Knoevenagel condensation and bio-
logically evaluated for the study of structure-activity relationship (SAR). We found that 7e44 suppressed
Received 30 May 2014
Received in revised form
7 October 2014
Accepted 21 December 2014
Available online 26 December 2014
the iNOS activity (IC50 25.2 mM) and LPS-induced NO production (IC50 45.6 mM) in RAW 264.7 cells. As for
the SAR study, the dimethoxylphenyl group of 7e44 was potential for a further modification. At a dose of
10 mg/kg, oral administration of 7e44 possessed protective properties in both carrageenan-induced paw
edema of male ICR mice and adjuvant-induced arthritis of Lewis female rats. Although the activity of 7
e44 was slightly inferior, the PK profiles of 7e44 were superior to those of 3I.
Keywords:
Inflammation
5-Benzylidenethiazolidine-2,4-dione
Nitric oxide
© 2014 Elsevier Masson SAS. All rights reserved.
Inducible nitric oxide synthase
Paw edema
1. Introduction
chronic inflammation [4,5]. NO is generated via the oxidation of L-
arginine by nitric oxide synthase (NOS), with the consumption of
molecular oxygen, NADPH and other cofactors [6]. In mammals,
there are at least three isoforms of the NOS enzymes: endothelial,
neuronal and inducible [7]. The constitutive neuronal NOS (nNOS)
and endothelial NOS (eNOS) are Ca2þ-dependent enzymes that play
key roles in the nervous and cardiovascular systems [8]. The
expression of inducible NOS (iNOS) could be activated by the
Inflammation is a complex process involving numerous medi-
ators of cellular and plasma origins with interrelated biological
functions [1]. Macrophages, neutrophils and lymphocytes play
important roles in the pathogenesis of various types of inflamma-
tory diseases such as rheumatoid arthritis, osteoarthritis, inflam-
matory bowel disease and multiple sclerosis [2]. Proinflammatory
stimuli activate cellular responses and regulate inflammatory im-
mune functions including the recruitment of macrophages and the
production of cytokines [3].
stimulation of cytokines (e.g., TNF-a) or bacterial lipopolysaccha-
ride (LPS) and further induces large quantities of cytotoxic NO
following immunological challenge in macrophages or other cells
[9,10]. Considerable evidences revealed that suppression of over-
production of iNOS-mediated NO might be useful drug target for
the treatment of inflammatory diseases [11,12].
NO, being an endogenous free radical, is a unique mediator in
the process of vasodilation, non-specific host defense and acute or
In our previous work, we developed that the IC50 values of (Z)-
N-(3-chlorophenyl)-2-(4-((2,
4-dioxothiazolidin-5-ylidene)
Abbreviations: NOS, nitric oxide synthase; NO, nitric oxide; LPS, lipopolysac-
charide; NF-kB, nuclear factor-kB; TNF-a, tumor necrosis factor-a; IL-1b, inter-
methyl)phenoxy)acetamide 3I (SKLB023) on LPS-induced iNOS
activity and NO production in RAW 264.7 macrophages were 8.66
and 23.55 respectively [13]. Our results exhibited that 3I signifi-
cantly improved joint inflammation and cartilage destruction in
three inflammatory models of carrageenan-induced paw edema,
adjuvant-induced arthritis (AIA) and collagen-induced arthritis
leukin-1 ; AIA, adjuvant-induced arthritis.
b
* Corresponding author. State Key Laboratory of Biotherapy and Cancer Center/
Collaborative Innovation Center of Biotherapy, West China Hospital, West China
Medical School, Sichuan University, Keyuan Road 4, Gaopeng Street, Chengdu
610041, China.
E-mail address: chenlijuan125@163.com (L. Chen).
These authors contributed equally.
1
http://dx.doi.org/10.1016/j.ejmech.2014.12.036
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

L. Ma et al. / European Journal of Medicinal Chemistry 92 (2015) 178e190
179
(CIA) [14]. Furthermore, 3I inhibited NF-
macrophages by luciferase assay and the number of macrophages
in synovial tissues. The levels of NO, TNF- and IL-1 in plasma and
k
B activity in peritoneal
further converted into iodoalkylamides 3 by NaI. The 4 were
accomplished by employing hydroxybenzaldehydes, 3, and K2CO3
[15]. In Scheme 1, the intermediates 6 was prepared through the
condensation of appropriate halides (R2-X) with thiazolidine-2,4-
dione in three different procedures: (1) method A employed NaH
as base and DMF as solvent; (2) method B used anhydrous K2CO3 as
base and acetone as a valid solvent; (3) method C applied Cs2CO3 as
base and CH3CN as solvent. Derivatives from 7-1 to 7-48 (Scheme 1)
were prepared in good yields via a Knoevenagel condensation be-
tween the corresponding hydroxybenzaldehydes 4 and a range of
substituted thiazolidine- 2,4-dione cores 6 [16,17]. Three conditions
have been developed for the Knoevenagel condensation: (4)
method D was performed in the presence of piperidine (cat.) and
EtOH solvent; (5) method E was refluxed by the application of
a
b
peritoneal macrophages and the expression of iNOS in vivo were
down-regulated by 3I at the different doses. Additionally, 3I sup-
pressed the phosphorylation of components of the mitogen-
activated protein kinases (MAPKs) including p38, ERK1/2 and JNK
by western blotting [15]. However, the oral bioavailability (F%) of 3I
is only 18.1% which is not suitable as a drug-like candidate [13]. In
view of above-mentioned advantages and disadvantages of anti-
inflammatory agent 3I, these encourage us to explore novel de-
rivatives or analogues to inhibit the iNOS-mediated NO production
for the treatment of inflammatory diseases.
There were two purposes to perform the structural explorations
on the basis of lead compound 3I in the study: to obtain the more
potent anti-inflammatory agents and/or to get more drug-like
compounds with the comparable anti-inflammatory activity. So,
by the combination of various chemical methods, fifty-nine 3I de-
rivatives and bioisosteres in the present study have been success-
fully synthesized. Numerous chemical substituents or groups (Fig.1,
such as Saturated or unsaturated aliphatic chain, Polar or hydro-
philic aliphatic chain, Substituted aromatic ring, and Bioisosterism
by the replacement of other heterocycles, etc.) have been intro-
duced to these derivatives and bioisosteres for the improvement in
activity and/or druggability. Furthermore, these compounds were
evaluated for the inhibition of LPS-induced NO production in RAW
264.7 macrophages and the treatment of carrageenan-induced paw
edema in ICR mice and adjuvant-induced arthritis in Lewis female
rats.
glacial acetic acid and b-alanine; (6) method F was heated to reflux
in the mixture of piperidine (cat.), glacial acetic acid (cat.) and
toluene as solvent.
The preparation of compound 7-49 has been obtained in a
tandem six-step sequence from 3-chloroaniline (1) as described in
Scheme 2. Treatment of 1 with BOC-glycine in the presence of EDCI
and DMAP (cat.) in ice bath afforded 2-49 with a good yield. The
chemical reduction of 2-49 using the LiAlH4 in THF solvent yielded
the compound 3-49. The intermediates 4-49 were obtained by the
combination of 3-49 with 2-chloroacetyl chloride in the presence of
triethylamine. The aldehyde 5-49 was accomplished by employing
4-49, 4-hydroxybenzaldehyde, anhydrous K2CO3 and KI in refluxed
acetone. The Knoevenagel condensation of 5-49 with thiazolidine-
2,4-dione to give 6-49 was carried out by employing the piperidine,
benzoic acid, and toluene-EtOH (v/v ¼ 1:1) solvent. Finally, 6-49
was deprotected by trifluoroacetic acid (TFA) and further washed
by aqueous NaHCO3 to give the product 7-49.
2. Chemistry
As for the bioisosteres of thiazolidine-2,4-dione, compound 8a
was synthesized by two-step easy sequence from commercially
available pyridin-2-amine as described in Scheme 2S Of
Supplementary Material [18]. The 8a reacted with aldehyde
The intermediates 2 were obtained by the combination of 3-
chloroaniline (1) with chloroalkynoyl chloride in the presence of
triethylamine (Scheme 1S in Supplementary Material). The 2 were
Fig. 1. Structural explorations based on anti-inflammatory agent 3I (SKLB023).

180
L. Ma et al. / European Journal of Medicinal Chemistry 92 (2015) 178e190
At this stage, all compounds were fully analyzed and charac-
terized by nuclear magnetic resonance (NMR), mass spectrometry
and high-performance liquid chromatography (HPLC) before
entering the biological screening.
3. Biological results and discussion
3.1. Inhibition and cytotoxicity in RAW 264.7 microphages
Lipopolysaccharide (LPS) is an important structural component
of the outer membrane of gram-negative bacteria, and it is also a
well-studied immunostimulator that induced an immune inflam-
matory response, and especially, the expression of NO production
and other proinflammatory cytokines. Here, 59 synthetic 3I de-
rivatives and bioisosteres were evaluated for inhibitory activity
against LPS-induced NO production in RAW 246.7 cells. The mac-
rophages were pre-treated with compounds for 2 h and then
incubated with LPS (1 mg/mL) for 18 h. The amounts of NO released
into culture media was detected according to the Griess method in
the form of nitrite. Here, 3I and indomethacin were chosen as a
positive control.
As depicted in Fig. 1, we found that 12 compounds (7-5, 7-10, 7-
14, 7-16, 7-17, 7-32, 7-40, 7-44, 7-52, 7-56, 7-58, and 7-59) partially
suppressed NO production at a concentration of 100 mM and 7-44
was the more comparable anti-inflammatory effect among fifty-
nine synthetic derivatives and bioisosteres. The positive 3I and
Indomethacin exhibited the most potent inhibitory activities.
Compared to the most potent 3I, the introduction of two methoxyl
groups into the phenyl ring of 5-benzylidenethiazolidine-2,4-dione
moiety led to 7-44. In addition, the other synthetic derivatives and
bioisosteres did not show the significant inhibitory activity.
As for the study of structure-activity relationship (SAR), the
introduction of chain saturated aliphatic groups (e.g., Ethylated in
7-1, Isobutylated in 7-2 and Butylated in 7-6) and unsaturated
(Propenylated in 7-3) into the N-atom site of thiazolidine-2,4-dione
failed to improve the inhibitory activity compared to 3I. However,
the high unsaturated propynylated derivative (7-5) and cyclo-
derivative (7-7, 7-16, and 7-17) exhibited the potential inhibitory
potency in contrast to LPS stimulated group. In addition, we could
find that the other compounds of N-substituent in thiazolidine-2,4-
dione moiety (e.g., polar group acetic acid in 7-8, benzoic acid in 7-
31 or big steric effect in 7-35) were non-active to inhibit the NO
production without statistical significance in Table 1. Inspection of
their structural features in Scheme 1 and their inhibitory activities
in Fig. 2 highlighted that the chemical bond of NeH in thiazolidine-
2,4-dione was approved to important for the inhibition.
Scheme 1. Synthesis of 3I-based derivatives 7 a a Reagents and Conditions: (i) Method
.
A: R2-X, NaH, DMF, 0 ꢀC to r.t, overnight; Method B: R2-X, K2CO3, acetone, reflux,
overnight; Method C: R2-X, Cs2CO3, CH3CN, reflux, overnight; (ii) Method D: 4,
piperidine (cat.), EtOH, reflux, 6 h; Method E: 4, b
-alanine, AcOH, 100 ꢀC; Method F: 4,
piperidine (cat.), AcOH (cat.), toluene, reflux, overnight.
Although aforementioned derivatives exhibited less potent ac-
tivities against the NO production than that of 3I and Indomethacin,
our synthetic strategy was further devoted to keep the thiazolidine-
2,4-dione moiety intact (Table 2). With respect to these 8 com-
pounds from 7-41 to 7-46, the substituents such as monomethoxyl
(7-41 and 7-42), monoethoxyl (7-43), dimethoxyl (7-44) and fluoro
(7-45) group of phenyl ring of 5-benzylidenethiazolidine-2,4-dione
moiety were explored. We also change the linker site in compound
7-40 or lengthened the linker in compounds 7-47 and 7-48. We
found that 7-40 (P < 0.05) and 7-44 (P < 0.01) were the more
efficient, while the 7-44 was more potent that 7-40. Between 7-40
and 7-44, we could find that the para-orientation is superior to
meta-substitution and the number of electron-donating group
(EDG) contributed to the inhibition from 7-41 to 7-45. The dime-
thoxylphenyl ring of 7-44 was worth to be a further structural
modification by the introduction of EDG. The inhibitory activity of
7-44 was comparable to that of 3I and positive control Indometh-
acin against the production of NO at the same concentration.
As we all known, the bioisosteric replacement was important
intermediate 4a in NaOAc and AcOH at 100 ꢀC to give the 3I-based
bioisostere 7-50. The condensation of imidazolidine-2,4-dione de-
rivatives (8c-e) with 4a employing pyrrolidine (cat.) and EtOH
solvent afforded 7-51, 7-52, and 7-53, respectively (Scheme 3) [19].
Treatment of 1H-pyrrole-2,5-dione with triphenylphosphine (PPh3)
in refluxed MeOH afforded the compound 8b and further
condensed with 4a via a Witting reaction to give final product 7-54
(Scheme 2S) [20]. Similarly, the Knoevenagel reaction of 4a with
barbituric acid or thiobarbituric acid in a mixture of ethanol/water
as valid solvent was refluxed for 5 h to afford the targeted com-
pound 7-55 (X ¼ O), and 7-56 (X ¼ S) [21]. Finally, the commercially
available heterocyclic cores of rhodanine, rhodanine-N-acetic acid
and 2-iminothiazolidin-4-one were treated with 4a in the presence
of piperidine (cat.) and EtOH to give the bioisosteric derivatives of
thiazolidine-2,4-dione 7-57, 7-58, and 7-59, respectively (Scheme
3).

L. Ma et al. / European Journal of Medicinal Chemistry 92 (2015) 178e190
181
Scheme 2. Synthesis of 3I-based compound 7-49a. a Reagents and Conditions: (i) N-Boc-Gly, EDCI, DMAP (cat.), CH2Cl2, 0 ꢀC to r.t, overnight; (ii) LiAlH4, THF, 0 ꢀC to r.t, overnight;
(iii) 2-chloroacetyl chloride, NaHCO3, CH2Cl2, 0 ꢀC to r.t; overnight; (iv) KI, K2CO3, 4-hydroxybenzaldehyde, acetone, reflux, overnight; (v) thiazolidine-2,4-dione, piperidine, benzoic
acid, toluene-EtOH (v/v ¼ 1:1), reflux, overnight; (vi) TFA/CH2Cl2, 0 ꢀC to r.t, 4 h, then washed by NaHCO3.
a
Scheme 3. Synthesis of bioisosteric derivatives of 3Ia. Reagents and Conditions: (i) 8a, NaOAc, AcOH, 100 ꢀC, 5 h; (ii) 8c-e, pyrrolidine (cat.), EtOH, reflux, 5e15 h; (iii) 8b, MeOH,
reflux, overnight; (iv) EtOHeH2O, reflux, 5 h; (v) piperidine (cat.), EtOH, reflux, 6 h.
theory in structural modification of drug development. There we
have synthesized 10 compounds to evaluate the bioisosteres of
thiazolidine-2,4-dione (2-Iminothiazolidin-4-ones, Imidazolidine-
2,4-diones, Pyrrolidine-2,5-dione, Barbituric acid, Thiobarbituric
acid, and 2-Thioxothiazolidin-4-ones from 7-50 to 7-59 in Scheme
2S). Although the four compounds 7-52, 7-56, 7-58 and 7-59
showed the weak inhibition compared to that of 7-44, the bio-
isosteric replacements was dispensable for the activity to some
content and the thiazolidine-2,4-dione moiety still is the best
choice. Consequently, the dimethoxylphenyl group of 7-44 was
potential for a further modification.
compounds were further exposed to RAW 264.7 macrophages to
explore their inhibitory effects and cytotoxicity. As shown in
Table 1, 7-44 exhibited the most potent inhibitory potencies on
iNOS activity (IC50 ¼ 25.20
mM), and the generation of LPS-
mediated NO (IC50 ¼ 45.60 M), whereas the results were inferior
m
to those of 3I and Indomethacin on LPS-induced RAW 264.7 cells. To
check whether the suppressive effects of these compounds on
iNOS, and NO was related to cell viability, MTT assay was adopted.
As exhibited in Table 1, 7-14, 7-44 and 7-58 showed no cytotoxicity
on RAW 264.7 macrophages (without or with LPS, IC50 > 80.00 mM).
Similarly, the positive 3I and indomethacin did not show any cyto-
The iNOS mediated the NO production and so selected
toxicity in the previus study [13]. However, we found that effects of

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L. Ma et al. / European Journal of Medicinal Chemistry 92 (2015) 178e190
Table 1
IC50 values of NO, iNOS and cytotoxicity and physicochemical property.
Compd.
NO IC50
(
m
M)
iNOS IC50
(
mM)
Cytotoxicity IC50
(m
M)
clog Pc
tPSAc
Solubility (mg/mL)d
ꢁLPS
þLPS
DMSO
EtOH
PBS
3I
21.32
40.30
>80.00
53.20
45.60
75.35
68.90
8.66b
23.47b
e
>80.00
>80.00
68.40
>80.00
>80.00
>80.00
>80.00
>80.00
>80.00
72.50
>80.00
>80.00
46.30
2.44
e
84.50
e
34.0
e
1.5
e
<0.5
e
Indoa
7-11
7-14
7-44
7-52
7-58
2.79
3.26
2.18
3.16
2.48
84.94
78.95
102.96
87.74
95.94
e
e
e
32.10
25.20
43.21
47.80
e
e
e
78.0
e
3.1
e
<0.5
e
>80.00
e
e
e
a
Indomethacin, an anti-inflammatory drug.
Data from Reference No.12.
Calculated with ChemBioDraw 12.0.
b
c
d
Detected maximum soluble concentration by nephelometry.
7-11 and 7-52 were more cytotoxic than the other compounds,
especially in the LPS-induced macrophages. On the basis of cellular
viability and anti-inflammatory activity in vitro, the more potent
derivative 7-44 were further evaluated in the next experimental
process.
the binding site of iNOS by employing a protein-ligand docking
program FRED [25]. Scoring function chemgauss 3 [26] was used for
exhaustive searching, solid body optimizing and interaction
scoring. The pose with the most favorable score was remained.
Compounds 3I, 7-11, 7-14, 7-44 and 7-52 are among the top
structures by computational scoring which almost is consistent
with in vitro iNOS screening (Supplementary material). As depicted
in Fig. 3, the meta-chlorophenyl moiety of 7-44 was buried in a
pocket between Phe363, Trp366 and an HEME. The linker between
the two phenyl groups was enclosed by a polar cavity consisting of
Glu371, Asp376 and Mem349. The thiazolidinedione moiety was
surrounded by Asn348, Arg260, Tyr485 and Arg382. In addition,
two strong hydrogen bonds are formed between 7-44 and Glu371
In addition, at the temperature of 25 ꢀC, the solubility of 7-44
and 3I in DMSO, phosphate buffer saline (PBS, pH 7.4) and ethanol
solution were respectively 78.0 and 43.0 mg/mL, both less than
0.5 mg/mL, 3.1 and 1.5 mg/mL, by detecting maximum soluble
concentration by nephelometry. After the introduction of two
methoxyl group into 5-benzylidenethiazolidine-2,4-dione slightly
improved the solubility profiles compared to 3I, and one possible
reason is that two EDG increase the hydrophily of molecule, which
was in accordance with the predicted cLogP and tPSA (A2) data by
the ChemBioDraw Ultra 12.0 software. These profiles may be a right
guideline for our further structural modification and exploration
for potent and drug-like anti-inflammatory agents.
and Asn348 and a p-p interaction between the meta-chlorophenyl
ring of 7-44 and HEME was also observed. From the docking result,
the dimethoxylphenyl ring of 7-44 was worth to be a further
structural modification.
3.2. Pharmacokinetic profiles
Furthermore, a pharmacokinetic profile of 7-44 was determined
in male SpragueeDawley rats (n ¼ 6) following intravenous and
oral administration at 5.0 mg/kg and 20 mg/kg, respectively. After
an i.p. injection, the results indicated that 7-44 has a high plasma
3.4. Inhibition of carrageenan-induced paw edema and adjuvant-
induced arthritis
concentration of 331.5
while the Cmax and t1/2 were 46.2
m
g/L and a long t1/2 of 7.83 h in this species,
To evaluate the in vivo anti-inflammatory potency of 7-44,
carrageenan-induced paw edema of acute inflammation was
employed. The edema is an important parameter of acute inflam-
mation for evaluating compounds with potential anti-
inflammatory activity. In the carrageenan-induced paw edema
test, the inflammatory response was quantified by increment in
paw size (edema) 2 h after carrageenan was injected and the paw
edema was observed (Fig. 4A). Oral administration of 7-44 at a dose
of 10 mg/kg could suppress carrageenan-induced paw edema.
Treatment with 10 mg/kg 7-44 exhibited less inhibitory activity
than same dose of indomethacin and 3I. 7-44 inhibited edema for-
mation after edema induction with the inhibitory rate of 26.1%
(p < 0.05) at the primary endpoint, while 3I and indomethacin
showed an inhibitory rate of 37.8% (p < 0.01) and 33.5% (p < 0.05) of
edema development, respectively.
mg/L and 5.9 h after oral admin-
istration. Oral bioavailability (F %) was estimated to be 27.2% on the
basis of the AUC0-t ratio and drug dose. In our previous work, we
found that the oral F% and t1/2 were 18.1% and 2.16 h [13]. Intro-
duction of two methoxy groups in 7-44 made it comparable activity
and preferable oral bioavailability to those of 3I because that the
two methoxy groups maybe not change the integrality of 3I. And by
the various structural explorations, we summarized that the thia-
zolidine-2,4-dione was very important to activity and the phenyl
ring of 5-benzylidenethiazolidine-2,4-dione was further worthy of
structural modification to improve the druggability. These profiles
may be a right guideline for our further structural modification and
explorations.
3.3. Docking study of iNOS
Because of the inhibition of carrageen-induced paw edema, 7-
44 has been chosen to further be examined on animal model of
adjuvant-induced arthritis (AIA). AIA is a well-established experi-
mental model of rheumatoid arthritis and is often used for testing
agents for anti-inflammatory activity. In this model, 90e100% of
rats developed arthritis within 14e18 days after adjuvant injection.
As displayed in Fig. 4B, rats treated with 7-44 at a dose of 10 mg/kg
did not develop severe arthritis, indicating that it exhibited po-
tential immune-modulating activity. Arthritic score was reduced in
the therapeutic process.
To gain better understanding on the potency of the studied
compounds, we proceeded to examine the interaction of all com-
pounds with iNOS protein which 3D structure was gained from
Protein Data Bank (PDB ID: 1r35) [22]. Molecule was built with
ChemBio3D and optimized at molecular mechanical and semi-
empirical level by using Hyperchem software [23]. Conformers of
compound were created by the aid of Omega [24] and the up limit
of conformer number was set to 2000. Then they were docked to

L. Ma et al. / European Journal of Medicinal Chemistry 92 (2015) 178e190
183
Fig. 2. The amount of nitrate in LPS-induced RAW 264.7 macrophages. Results are means SD (n ¼ 3). ###P < 0.001 vs. control. *P < 0.05; **P < 0.01 and ***P < 0.001 vs. LPS group.
5. Experimental section
Table 2
Pharmacokinetic Profiles of 7-44 in SD rats.a
5.1. Biological methods
ip
op
46.2
2.27
5.90
421.1
27.2
Cmax
(
m
g/L)
331.5
0.07
7.83
387.2
5.1.1. Materials
tmax (h)
t1/2 (h)
LPS (Escherichia coli serotype 0111:B4), MTT, Carrageenan from
seaweed (a mixture of lambda and kappa-carrageenan) and Indo-
methacin were obtained from Sigma Chemical Co. (St. Louis, MO).
AUC0-t
F (%)
(
m
g/L h)
a
Data were mean concentrations in mouse plasma (n ¼ 6) following a single
5.0 mg/kg intravenous dose or 20 mg/kg oral dose.
5.1.2. Cell culture
RAW 264.7 macrophages were obtained from ATCC (Rockville,
MD, USA). These cells were grown in RPMI 1640 containing 10%
FBS,100 units/mL penicillin, and 100 mg/mL streptomycin in 95% air,
5% CO2 humidified atmosphere at 37 ꢀC.
5.1.3. Nitrite assay
RAW 264.7 cells were seeded into a 96-well culture plate at a
density of 1 ꢂ104 cells per well with 500
mL of culture medium and
incubated for 24 h. The cells were then pre-treated with com-
pounds 7 and Indomethacin at 100
with LPS (1 g/mL) for 18 h. The nitrite concentration in the me-
dium was measured according to Griess reaction by adding 50
Griess reagent (1% sulfanilamide and 0.1% N-(1-naphthyl)ethyl-
enediamine dihydrochloride in 5% phosphoric acid) to 50 L of
mM for 2 h before stimulation
m
m
L
m
medium for 5 min. The OD540 was measured with a microplate
reader. Concentrations were calculated by comparison with OD540
of a standard solution of sodium nitrite prepared in culture
medium.
Fig. 3. The interaction mode of 7-44 within the active site of murine iNOS.
4. Conclusion
5.1.4. Cell cytotoxicity
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 mL DMSO. The op-
tical density was measured using an ELISA plate reader at 570 nm
(OD570).
In this study, we have synthesized 59 derivatives and bio-
isosteres based on 3I and evaluated for their inhibitory activity on
LPS-induced NO production and iNOS activity. We found that 7-44
suppressed the iNOS activity, and the production of NO in RAW
264.7 cells. Furthermore, treatment of 7-44 at a dose of 10 mg/kg
improved the inflammatory response such as decrement of the paw
edema and signs of AIA, while the result was a little inferior to that
of 3I at the same dose. The introduction of two methoxyl group into
phenyl ring of 5-benzylidenethiazolidine-2,4-dione moiety was
slightly inferior to 3I against in vitro NO production and iNOS ac-
tivity. In other side, the limited structural modification improved
the PK profiles to add the plasma concentration and make 7-44
exhibit the approximate in vivo anti-inflammatory activity to 3I.
5.1.5. Assay of iNOS enzymatic activity
After treated with LPS (1 mg/mL) and 7-44 (1e50 mM) for 2 h at
37 ꢀC, the culture supernatant was removed and 100
m
m
L of NOS
L of NOS
assay buffer (1ꢂ) were added to each well. Then 100
assay reaction solution (50% NOS assay buffer, 39.8% MilliQ 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

184
L. Ma et al. / European Journal of Medicinal Chemistry 92 (2015) 178e190
Fig. 4. Inhibition of carrageenan-induced paw edema in ICR mice (A) and adjuvant-induced arthritis in female Lewis rats (B). The results were expressed as the means SD (n ¼ 6).
*P < 0.05; and **P < 0.01 vs. control.
measured with a fluorescence plate reader (Biotek) at excitation of
5.1.8. Statistical analysis
485 nm and emission of 528 nm.
Experimental values are presented as the arithmetic
means SD. GraphPad Prism 5.0 software followed by unpaired t
test with Welch's correction was used to determined statistical
differences. P < 0.05 was considered to be statistically significant.
5.1.6. Carrageenan-induced paw edema test in ICR mice
ICR male mice and female Lewis rats were housed in filtered-
capped polycarbonate cages and allowed food and water ad libi-
tum. Animals were kept on a cycle of 12 h light/darkness at
5.2. Chemistry
22
1
ꢀC and acclimated for at least one week until use. All pro-
Chemical reagents of analytical grade were purchased from
Chengdu Changzheng Chemical Factory (Sichuan, P. R. China). TLC
was performed on 0.20 mm silica gel 60 F254 plates (Qingdao Ocean
Chemical Factory, China). The purity of compounds was determined
to be ꢃ 97% by HPLC analysis with a photodiode array detector
(Waters) and the chromatographic column was a atlantis C18
tocols of in vivo experiments and animals received human cares
were operated according to National Institutes of Health Guide-
lines. The mice were randomly divided into four groups. Doses
(10 mg/kg and 50 mg/kg) of compounds were administered i.p. to
the test groups, respectively. The positive control group was given
access to Indomethacin (10 mg/kg, i.p.) and the vehicle control
group was given access to the same volume of olive oil (10 mL/kg,
i.p.). Thirty minutes after the administration, acute paw edema was
induced in the right hind paw by subplantar injection of 1% freshly
(150 mm ꢂ 4.6 mm, i.d. 5
400 MHz on a Varian spectrometer model Gemini 400 and reported
in parts per million. Chemical shifts ( ) are quoted in ppm relative
to TMS as an internal standard, where (
) TMS ¼ 0.00 ppm. The
m
m) (Waters). 1H NMR was recorded at
d
d
prepared carrageenan suspension in normal saline, 50
mL per a
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. Mass Spectra were measured by Q-
TOF Priemier mass spectrometer utilizing electrospray ionization
(ESI) (Micromass).
mouse. The thickness of the paw was measured pre-injection and at
intervals of 1, 2, 3, 4, 5 and 6 h post-injection, using a Dial Thickness
Gage. The percent increase of paw thickness was calculated based
on the pre-injection thickness of the paw.
5.1.7. Induction and assessment of AIA
5.2.1. General procedure for synthesis of compounds 3
Briefly, 12-week-old, gonad-intact, female Lewis rats with six
rats per group were injected on the ventral side of the base of the
tail with CFA. The degree of arthritis severity was monitored daily
and scored according to the following disease indices: hindpaw
erythema, hindpaw swelling, tenderness of the joints, and move-
ments, and posture. An integer scale of 0 to 3 is used to quantify the
level of erythema (0, normal paw; 1, mild erythema; 2, moderate
erythema; 3, severe erythema) and swelling (0, normal paw; 1, mild
swelling; 2, moderate swelling; 3, severe swelling of the hindpaw).
The maximal possible score per day is 12, and this score was seen in
all rats 8 days after CFA injection. A cohort of Lewis rats not injected
with CFA but receiving daily oral doses of vehicle (2% Tween 80/
0.5% methylcellulose) for 21 days were designated normal controls.
CFA-treated rats were dosed with vehicle for 7 days until full joint
inflammation developed and then were randomized into four
groups and treated for another 14 days. One group continued to be
dosed with vehicle. And the other three groups were treated with
7-44 (10 mg/kg), 3I (10 mg/kg) and indomethacin (10 mg/kg),
respectively.
2-Chloroacetyl chloride (24 mmol) was slowly added to a
mixture of ReNH2 (20 mmol) and Et3N (24 mmol, 3.3 mL) in
anhydrous DCM (20 mL) at 0 ꢀC. The mixture warmed to room
temperature and stirred for 20 h. After the solvent was removed,
the residue was washed by water (3 ꢂ 20 mL) and the precipitate
was collected. The crude product 2 was purified by crystallization
from ether/petroleum. Then, 2 (1.0 mmol) and NaI (8.0 mmol) were
added into acetone (20 mL) and the mixture was refluxed. After the
solvent was removed under reduced pressure, the residue 3 was
afforded without further purification.
5.2.2. General procedure for synthesis of compounds 4
Hydroxybenzaldehyde (11 mmol), K2CO3 (2.76 g, 20 mmol) and
4 (10 mmol) were dissolved in acetone (30 mL). The mixture was
refluxed overnight and then cooled to room temperature. Then
K2CO3 solid was filtered and the acetone was removed under
reduced pressure to obtain the crude products. The residue was
purified by silica gel column chromatography to give the

L. Ma et al. / European Journal of Medicinal Chemistry 92 (2015) 178e190
185
appropriate aldehydes 4.
z: 429.05 [MꢁH]ꢁ.
5.2.3. General procedure for synthesis of compounds 6
5.2.4.3. (Z)-2-(4-((3-Allyl-2,4-dioxothiazolidin-5-ylidene)methyl)
phenoxy)-N-(3-chlorophenyl)acetamide 7-3. Prepared by general
procedure for the synthesis of 6 (Method A) and 7 (Method D). Yield
Method A: Thiazolidine-2,4-one (1.0 mmol) was dissolved into
DMF (10 mL) at 0 ꢀC and NaH (1.5 mmol, 60%) was slowly added
into the solution. R2-X (1.0 mmol) was added into the mixture and
allowed to warm up to room temperature overnight. Then, 25 mL
water was added and the mixture was extracted by EtOAc
(3 ꢂ 15 mL), and the combined organic layer was washed by water
(2 ꢂ 10 mL) and brine (2 ꢂ 10 mL). The solvent was removed to
obtain the crude product without purification.
49.0%; White powder; 1H NMR (400 MHz, DMSO-d6):
d 10.37 (s,
1H), 7.92 (s, 1H), 7.84 (t, 1H, J ¼ 1.6 Hz), 7.64 (d, 2H, J ¼ 8.8 Hz), 7.53
(d, 1H, J ¼ 8.0 Hz), 7.37 (t, 1H, J ¼ 8.0 Hz), 7.18e7.15 (m, 3H),
5.89e5.82 (m, 1H), 5.18e5.13 (m, 2H), 4.84 (s, 2H), 4.26 (d, 2H,
J ¼ 7.2 Hz); MS (ESI), m/z: 427.04 [MꢁH]ꢁ.
Method B: Thiazolidine-2,4-one (1.0 mmol), R2-X (1.0 mmol),
anhydrous K2CO3 (1.5 mmol) were added into acetone (15 mL) and
the mixture was refluxed overnight. Then K2CO3 solid was filtered,
and the solution was collected and further extracted by EtOAc
(3 ꢂ 15 mL), water (2 ꢂ 10 mL), brine (2 ꢂ 10 mL), and dried by
anhydrous Na2SO4. The solvent was removed to obtain the crude
product without purification.
Method C: Thiazolidine-2,4-one (1.0 mmol), R2-X (1.0 mmol),
Cs2CO3 (1.5 mmol) were added into CH3CN (15 mL) and the mixture
was refluxed overnight. Then K2CO3 solid was filtered, and the
solution was collected and further extracted by EtOAc (3 ꢂ 15 mL),
water (2 ꢂ 10 mL), brine (2 ꢂ 10 mL), and dried by anhydrous
Na2SO4. The solvent was removed to obtain the crude product
without purification.
5.2.4.4. (Z)-N-(3-Chlorophenyl)-2-(4-((3-(3-methylbut-2-en-1-yl)-
2,4-dioxothiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-4.
Prepared by general procedure for the synthesis of 6 (Method A)
and 7 (Method D). Yield 54.1%; White powder; 1H NMR (400 MHz,
DMSO-d6):
d 10.36 (s, 1H), 7.89 (s, 1H), 7.84 (s, 1H), 7.61 (d, 2H,
J ¼ 8.8 Hz), 7.53 (d, 1H, J ¼ 8.8 Hz), 7.36 (t, 1H, J ¼ 8.0 Hz), 7.17e7.14
(m, 3H), 5.18 (m, 1H), 4.83 (s, 2H), 4.22 (d, 2H, J ¼ 6.8 Hz), 1.74 (s,
3H), 1.68 (s, 3H); MS (ESI), m/z: 455.01 [MꢁH]ꢁ.
5.2.4.5. (Z)-N-(3-Chlorophenyl)-2-(4-((2,4-dioxo-3-(prop-2-yn-1-yl)
thiazolidin-5-ylidene)methyl)phenoxy)acetamide 7e5. Prepared by
general procedure for the synthesis of 6 (Method A) and 7 (Method
D). Yield 23.1%; White powder; 1H NMR (400 MHz, DMSO-d6):
d
10.37 (s, 1H), 7.96 (s, 1H), 7.84 (t, 1H, J ¼ 2.0 Hz), 7.64 (d, 2H,
J ¼ 8.8 Hz), 7.52 (d, 1H, J ¼ 8.4 Hz), 7.37 (t, 1H, J ¼ 8.0 Hz), 7.19e7.14
(m, 4H), 4.84 (s, 2H), 4.23 (d, 2H, J ¼ 2.4 Hz); MS (ESI), m/z: 425.01
[MꢁH]ꢁ.
5.2.4. General procedure for synthesis of compounds 7
Method D: The appropriate aldehyde 4 (1.0 mmol), thiazolidine-
2,4-one derivatives 6 (1.2 mmol), piperidine (cat.) and 10 mL EtOH
were mixed and heated to reflux for 6 h. Then a portion of water/ice
was added and the precipitated solids were collected by sucking
filtration and washed with distilled water (4 ꢂ 15 mL), EtOH
(2 ꢂ 10 mL), and ether (2 ꢂ 10 mL). The solids obtained were dried
in vacuum at 40 ꢀC for 24 h.
5.2.4.6. (Z)-2-(4-((3-Butyl-2,4-dioxothiazolidin-5-ylidene)methyl)
phenoxy)-N-(3-chlorophenyl)acetamide 7-6. Prepared by general
procedure for the synthesis of 6 (Method A) and 7 (Method D). Yield
78.0%; White powder; 1H NMR (400 MHz, DMSO-d6):
d 10.36 (s,
1H), 7.93 (s, 1H), 7.84 (s, 1H), 7.62 (d, 2H, J ¼ 8.8 Hz), 7.53 (d, 1H,
J ¼ 8.0 Hz), 7.36 (t, 1H, J ¼ 8.0 Hz), 7.17e7.15 (m, 3H), 4.84 (s, 2H),
3.64 (t, 2H, J ¼ 7.2 Hz), 1.54 (q, 2H, J ¼ 7.2 Hz), 1.27 (q, 2H, J ¼ 7.2 Hz),
0.89 (t, 3H, J ¼ 7.2 Hz); MS (ESI), m/z: 443.10 [MꢁH]ꢁ.
Method E: The appropriate aldehyde 4 (1.0 mmol), thiazolidine-
2,4-one derivatives 6 (2.0 mmol), 10 mL glacial acetic acid, and b-
alanine (2.0 mmol) were mixed and heated to reflux for 4 h. Then a
portion of water/ice was added and the precipitated solids were
collected by sucking filtration and washed with distilled water
(4 ꢂ 15 mL), EtOH (2 ꢂ 10 mL), and ether (2 ꢂ 10 mL). The solids
obtained were dried in vacuum at 40 ꢀC for 24 h.
Method F: The appropriate aldehyde 4 (1.0 mmol), thiazolidine-
2,4-one derivatives 6 (1.2 mmol), piperidine (cat.), glacial acetic
acid (cat.) and 10 mL toluene were mixed and heated to reflux
overnight. Then a portion of water/ice was added and the precipi-
tated solid was collected by sucking filtration and washed with
distilled water (4 ꢂ 15 mL), EtOH (2 ꢂ 10 mL), and ether (2 ꢂ 10 mL).
The solids obtained were dried in vacuum at 40 ꢀC for 24 h.
5.2.4.7. (Z)-N-(3-Chlorophenyl)-2-(4-((3-cyclopentyl-2,4-
dioxothiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-7.
Prepared by general procedure for the synthesis of 6 (Method A)
and 7 (Method D). Yield 87.3%; White powder; 1H NMR (400 MHz,
DMSO-d6):
d 10.36 (s, 1H), 7.86e7.84 (m, 2H), 7.60 (d, 2H,
J ¼ 8.8 Hz), 7.53 (d, 1H, J ¼ 8.0 Hz), 7.36 (t, 1H, J ¼ 8.4 Hz), 7.17e7.14
(m, 3H), 4.83 (s, 2H), 4.67 (qt, 1H, J ¼ 8.4 Hz), 1.97e1.57 (m, 8H); MS
(ESI), m/z: 455.05 [MꢁH]ꢁ.
5.2.4.8. (Z)-2-(5-(4-(2-((3-Chlorophenyl)amino)-2-oxoethoxy)ben-
zylidene)-2,4-dioxothiazolidin-3-yl)acetic acid 7-8. Prepared by
general procedure for the synthesis of 6 (Method A) and 7 (Method
D). Yield 29.0%; Yellow powder; 1H NMR (400 MHz, DMSO-d6):
5.2.4.1. (Z)-N-(3-Chlorophenyl)-2-(4-((3-ethyl-2,4-dioxothiazolidin-
5-ylidene)methyl)phenoxy)acetamide 7-1. Prepared by general
procedure for the synthesis of 6 (Method A) and 7 (Method D). Yield
65.3%; White powder; 1H NMR (400 MHz, DMSO-d6):
d 10.34 (s,
d
14.00e13.00 (br-s, 1H), 10.42 (s, 1H), 7.93 (s, 1H), 7.84 (s, 1H), 7.64
(d, 2H, J ¼ 8.8 Hz), 7.54 (d, 1H, J ¼ 8.4 Hz), 7.36 (t, 1H, J ¼ 8.0 Hz),
7.18e7.14 (m, 3H), 4.84 (s, 2H), 4.22 (s, 2H); MS (ESI), m/z: 445.02
[MꢁH]ꢁ.
1H), 7.89 (s, 1H), 7.83 (t, 1H, J ¼ 2.0 Hz), 7.62 (d, 2H, J ¼ 8.8 Hz), 7.53
(d, 1H, J ¼ 8.0 Hz), 7.36 (t, 1H, J ¼ 8.0 Hz), 7.18e7.14 (m, 3H), 4.83 (s,
2H), 3.67 (q, 2H, J ¼ 7.2 Hz), 1.15 (t, 3H, J ¼ 7.2 Hz); MS (ESI), m/z:
415.05 [MꢁH]ꢁ.
5.2.4.9. (Z)-Ethyl 2-(5-(4-(2-((3-chlorophenyl)amino)-2-oxoethoxy)
benzylidene)-2,4-dioxothiazolidin-3-yl)acetate 7-9. Prepared by
general procedure for the synthesis of 6 (Method A) and 7 (Method
5.2.4.2. (Z)-N-(3-Chlorophenyl)-2-(4-((3-isopropyl-2,4-
dioxothiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-2.
Prepared by general procedure for the synthesis of 6 (Method A)
D). Yield 49.5%; White powder; 1H NMR (400 MHz, DMSO-d6): 1H
d
and 7 (Method D). Yield 35.2%; White powder; 1H NMR (400 MHz,
NMR (400 MHz, DMSO-d6): d 10.37 (s, 1H), 7.98 (s, 1H), 7.84 (s, 1H),
DMSO-d6):
d
10.37 (s,1H), 7.85e7.84 (m, 2H), 7.60 (d, 2H, J ¼ 8.4 Hz),
7.66 (d, 2H, J ¼ 8.8 Hz), 7.53 (d,1H, J ¼ 8.0 Hz), 7.37 (t,1H, J ¼ 8.0 Hz),
7.19e7.15 (m, 3H), 4.85 (s, 2H), 4.49 (s, 2H), 4.17 (q, 2H, J ¼ 7.2 Hz),
1.21 (t, 3H, J ¼ 7.2 Hz); MS (ESI), m/z: 473.05 [MꢁH]ꢁ.
7.52 (d,1H, J ¼ 8.0 Hz), 7.36 (t,1H, J ¼ 8.0 Hz), 7.17e7.15 (m, 3H), 4.83
(s, 2H), 4.53 (qt, 1H, J ¼ 6.8 Hz),1.40 (s, 3H), 1.38 (s, 3H); MS (ESI), m/

186
L. Ma et al. / European Journal of Medicinal Chemistry 92 (2015) 178e190
5.2.4.10. (Z)-Methyl 3-(5-(4-(2-((3-chlorophenyl)amino)-2-
oxoethoxy)benzylidene)-2,4-dioxothiazolidin-3-yl)propanoate 7-10.
Prepared by general procedure for the synthesis of 6 (Method A)
and 7 (Method D). Yield 69.3%; White powder; 1H NMR (400 MHz,
1H, J ¼ 8.0 Hz), 7.19e7.15 (m, 3H), 4.85 (s, 2H), 3.98 (s, 2H),
3.56e3.34 (m, 4H), 3.11 (s, 2H), 1.97e1.69 (m, 4H); MS (ESI), m/z:
484.10 [MꢁH]ꢁ.
DMSO-d6):
d
10.36 (s, 1H), 7.90 (s, 1H), 7.83 (s, 1H), 7.63 (d, 2H,
5.2.4.17. (Z)-N-(3-Chlorophenyl)-2-(4-((2,4-dioxo-3-(2-(piperidin-1-
J ¼ 8.8 Hz), 7.52 (d, 1H, J ¼ 8.0 Hz), 7.36 (t, 1H, J ¼ 8.0 Hz), 7.18e7.15
(m, 3H), 4.84 (s, 2H), 3.88 (t, 2H, J ¼ 7.2 Hz), 3.59 (s, 3H), 2.68 (t, 2H,
J ¼ 7.2 Hz); MS (ESI), m/z: 473.01 [MꢁH]ꢁ.
yl)ethyl)thiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-17.
Prepared by general procedure for the synthesis of 6 (Method A)
and 7 (Method D). Yield 36.0%; Yellow powder; 1H NMR (400 MHz,
DMSO-d6):
d 8.20 (s, 1H), 7.85 (s, 1H), 7.72 (s, 1H), 7.53 (d, 2H,
5.2.4.11. (Z)-N-(3-Chlorophenyl)-2-(4-((3-(methoxymethyl)-2,4-
J ¼ 8.8 Hz), 7.45 (d, 1H, J ¼ 8.0 Hz), 7.29 (t,1H, J ¼ 8.4 Hz), 7.15 (d, 1H,
J ¼ 8.0 Hz), 7.09 (d, 2H, J ¼ 8.8 Hz), 4.67 (s, 2H), 3.88 (t, 2H,
J ¼ 6.8 Hz), 2.58 (t, 2H, J ¼ 6.8 Hz), 2.45 (m, 4H), 1.54e1.52 (m, 4H),
1.42e1.40 (m, 2H); MS (ESI), m/z: 498.05 [MꢁH]ꢁ.
dioxothiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-11.
Prepared by general procedure for the synthesis of 6 (Method A)
and 7 (Method D). Yield 45.9%; Yellow powder; 1H NMR (400 MHz,
DMSO-d6):
d 10.36 (s, 1H), 7.94 (s, 1H), 7.84 (s, 1H), 7.65 (d, 2H,
J ¼ 8.0 Hz), 7.30 (d, 1H, J ¼ 8.4 Hz), 7.36 (t, 1H, J ¼ 8.0 Hz), 7.19e7.14
(m, 3H), 4.98 (s, 2H), 4.84 (s, 2H), 3.30 (s, 3H); MS (ESI), m/z: 431.10
[MꢁH]ꢁ.
5.2.4.18. (Z)-N-(3-Chlorophenyl)-2-(4-((3-(2-morpholinoethyl)-2,4-
dioxothiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-18.
Prepared by general procedure for the synthesis of 6 (Method A)
and 7 (Method D). Yield 81.1%; Light yellow powder; 1H NMR
5.2.4.12. (E)-Ethyl 4-((Z)-5-(4-(2-((3-chlorophenyl)amino)-2-
oxoethoxy)benzylidene)-2,4-dioxothiazolidin-3-yl)but-2-enoate 7-
12. Prepared by general procedure for the synthesis of 6 (Method
A) and 7 (Method D). Yield 78.9%; White powder; 1H NMR
(400 MHz, DMSO-d6): d 8.20 (s, 1H), 7.85 (s, 1H), 7.71 (s, 1H), 7.54 (d,
2H, J ¼ 8.4 Hz), 7.45 (d, 1H, J ¼ 7.6 Hz), 7.31e7.26 (m, 2H), 7.15 (d, 1H,
J ¼ 8.0 Hz), 7.10 (d, 2H, J ¼ 8.8 Hz), 4.67 (s, 2H), 3.88 (t, 2H,
J ¼ 6.4 Hz), 3.65 (t, 4H, J ¼ 4.0 Hz), 2.62 (t, 2H, J ¼ 6.4 Hz), 2.50 (m,
4H); MS (ESI), m/z: 500.01 [MꢁH]ꢁ.
(400 MHz, DMSO-d6):
d 10.37 (s, 1H), 7.92 (s, 1H), 7.83 (s, 1H), 7.64
(d, 2H, J ¼ 8.8 Hz), 7.53 (d, 1H, J ¼ 8.4 Hz), 7.37 (t, 1H, J ¼ 8.0 Hz),
7.19e7.15 (m, 3H), 6.90e6.83 (tt, 1H, J ¼ 4.8 Hz, J ¼ 8.0 Hz),
5.95e5.91 (m, 1H), 4.84 (s, 2H), 4.44 (d, 2H, J ¼ 3.2 Hz), 4.12 (q, 2H,
J ¼ 6.0 Hz), 1.12 (t, 3H, J ¼ 6.0 Hz); MS (ESI), m/z: 517.01 [MꢁH]ꢁ.
5.2.4.19. (Z)-2-(4-((3-Benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)
phenoxy)-N-(3-chlorophenyl)acetamide 7-19. Prepared by general
procedure for the synthesis of 6 (Method B) and 7 (Method E). Yield
56.9%; White powder; 1H NMR (400 MHz, DMSO-d6):
d 10.37 (s,
5.2.4.13. (Z)-N-(3-Chlorophenyl)-2-(4-((3-(2-(dimethylamino)
ethyl)-2,4-dioxothiazolidin-5-ylidene)methyl)phenoxy)acetamide 7-
13. Prepared by general procedure for the synthesis of 6 (Method
A) and 7 (Method D). Yield 55.9%; Yellow powder; 1H NMR
1H), 7.94 (s, 1H), 7.84 (s, 1H), 7.64 (d, 2H, J ¼ 8.8 Hz), 7.53 (d, 1H,
J ¼ 8.4 Hz), 7.39e7.30 (m, 6H), 7.18e7.15 (m, 3H), 4.84 (s, 4H). MS
(ESI), m/z: 477.07 [MꢁH]ꢁ.
(400 MHz, DMSO-d6):
d
8.21 (s, 1H), 7.85 (s, 1H), 7.71 (t, 1H,
5.2.4.20. (Z)-N-(3-Chlorophenyl)-2-(4-((3-(2-methylbenzyl)-2,4-
J ¼ 2.0 Hz), 7.52 (d, 2H, J ¼ 8.8 Hz), 7.45 (dd, 1H, J ¼ 0.8 Hz,
J ¼ 7.2 Hz), 7.29 (t, 1H, J ¼ 8.0 Hz), 7.15 (dd, 1H, J ¼ 0.8 Hz, J ¼ 8.0 Hz),
7.09 (d, 2H, J ¼ 8.8 Hz), 4.66 (s, 2H), 3.87 (t, 2H, J ¼ 6.4 Hz), 2.58 (t,
2H, J ¼ 6.4 Hz), 2.28 (s, 6H); MS (ESI), m/z: 476.05 [MꢁH]ꢁ.
dioxothiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-20.
Prepared by general procedure for the synthesis of 6 (Method B)
and 7 (Method E). Yield 44.3%; White powder; 1H NMR (400 MHz,
DMSO-d6):
d 10.37 (s, 1H), 7.95 (s, 1H), 7.84 (s, 1H), 7.65 (d, 2H,
J ¼ 8.8 Hz), 7.53 (d, 1H, J ¼ 8.0 Hz), 7.36 (t, 1H, J ¼ 8.0 Hz), 7.20e7.15
(m, 6H), 7.03 (d, 1H, J ¼ 6.8 Hz), 4.84 (s, 2H), 4.82 (s, 2H), 2.63 (s,
3H); MS (ESI), m/z: 491.10 [MꢁH]ꢁ.
5.2.4.14. (Z)-N-(3-Chlorophenyl)-2-(4-((3-(3-(dimethylamino)-2-
methylpropyl)-2,4-dioxothiazolidin-5-ylidene)methyl)phenoxy)acet-
amide 7-14. Prepared by general procedure for the synthesis of 6
(Method A) and 7 (Method D). Yield 76.8%; Yellow powder; 1H NMR
5.2.4.21. (Z)-N-(3-Chlorophenyl)-2-(4-((3-(2-fluorobenzyl)-2,4-
(400 MHz, DMSO-d6):
d
8.29 (s, 1H), 7.81 (s, 1H), 7.70 (t, 1H,
dioxothiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-21.
J ¼ 2.0 Hz), 7.50 (d, 2H, J ¼ 8.8 Hz), 7.48 (dd, 1H, J ¼ 0.8 Hz,
J ¼ 7.2 Hz), 7.29 (t, 1H, J ¼ 8.0 Hz), 7.15 (dd, 1H, J ¼ 0.8 Hz, J ¼ 8.0 Hz),
7.03 (d, 2H, J ¼ 8.8 Hz), 4.67 (s, 2H), 3.96 (m, 1H), 3.87 (d, 2H,
J ¼ 6.4 Hz), 2.58 (d, 2H, J ¼ 6.4 Hz), 2.28 (s, 6H), 2.11 (d, 3H,
J ¼ 6.0 Hz); MS (ESI), m/z: 486.10 [MꢁH]ꢁ.
Prepared by general procedure for the synthesis of 6 (Method B)
and 7 (Method E). Yield 21.0%; Light yellow powder; 1H NMR
(400 MHz, DMSO-d6):
d 10.37 (s, 1H), 7.94 (s, 1H), 7.84 (t, 1H,
J ¼ 2.0 Hz), 7.63 (d, 2H, J ¼ 9.2 Hz), 7.54e7.51 (m, 1H), 7.39e7.35 (m,
3H), 7.21e7.14 (m, 5H), 4.84 (s, 2H), 4.20 (s, 2H); MS (ESI), m/z:
495.05 [MꢁH]ꢁ.
5.2.4.15. (Z)-N-(3-Chlorophenyl)-2-(4-((3-(2-(diethylamino)ethyl)-
2,4-dioxothiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-15.
5.2.4.22. (Z)-N-(3-Chlorophenyl)-2-(4-((3-(3-fluorobenzyl)-2,4-
Prepared by general procedure for the synthesis of 6 (Method A)
dioxothiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-22.
and 7 (Method D). Yield 47.8%; Yellow powder; 1H NMR (400 MHz,
Prepared by general procedure for the synthesis of 6 (Method B)
DMSO-d6):
d
8.30 (s, 1H), 7.85 (s, 1H), 7.71 (t, 1H, J ¼ 2.0 Hz), 7.52 (d,
and 7 (Method E). Yield 33.9%; White powder; 1H NMR (400 MHz,
2H, J ¼ 8.8 Hz), 7.45 (dd, 1H, J ¼ 0.8 Hz, J ¼ 7.2 Hz), 7.29 (t, 1H,
J ¼ 8.0 Hz), 7.15 (dd, 1H, J ¼ 0.8 Hz, J ¼ 8.0 Hz), 7.09 (d, 2H,
J ¼ 8.8 Hz), 4.66 (s, 2H), 3.87 (t, 2H, J ¼ 6.4 Hz), 2.58 (t, 2H,
J ¼ 6.4 Hz), 2.28 (s, 6H); MS (ESI), m/z: 486.05 [MꢁH]ꢁ.
DMSO-d6):
d
10.37 (s, 1H), 7.95 (s, 1H), 7.84 (t, 1H, J ¼ 2.0 Hz), 7.64
(d, 2H, J ¼ 8.8 Hz), 7.53 (d, 1H, J ¼ 8.0 Hz), 7.43e7.35 (m, 2H),
7.18e7.13 (m, 5H), 4.89 (s, 2H), 4.84 (s, 2H); MS (ESI), m/z: 495.03
[MꢁH]ꢁ.
5.2.4.16. (Z)-N-(3-Chlorophenyl)-2-(4-((2,4-dioxo-3-(2-(pyrrolidin-
5.2.4.23. (Z)-N-(3-Chlorophenyl)-2-(4-((3-(4-fluorobenzyl)-2,4-
1-yl)ethyl)thiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-16.
dioxothiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-23.
Prepared by general procedure for the synthesis of 6 (Method A)
Prepared by general procedure for the synthesis of 6 (Method B)
and 7 (Method D). Yield 55.9%; Light yellow powder; 1H NMR
and 7 (Method E). Yield 43.5%; White powder; 1H NMR (400 MHz,
(400 MHz, DMSO-d6):
d
10.38 (s, 1H), 9.04 (br-s, 1H), 7.94 (s, 1H),
DMSO-d6):
d 10.37 (s, 1H), 7.94 (s, 1H), 7.84 (s, 1H), 7.64 (d, 2H,
7.84 (s, 1H), 7.65 (d, 2H, J ¼ 8.4 Hz), 7.51 (d, 1H, J ¼ 8.0 Hz), 7.37 (t,
J ¼ 8.8 Hz), 7.53 (d, 1H, J ¼ 8.8 Hz), 7.39e7.31 (m, 2H), 7.25e7.15 (m,

L. Ma et al. / European Journal of Medicinal Chemistry 92 (2015) 178e190
187
5H), 4.89 (s, 2H), 4.84 (s, 2H); MS (ESI), m/z: 495.06 [MꢁH]ꢁ.
DMSO-d6): d 12.98 (s, 1H), 10.36 (s, 1H), 7.95e7.91 (m, 3H), 7.83 (t,
1H, J ¼ 1.6 Hz), 7.64 (d, 2H, J ¼ 8.8 Hz), 7.30 (d, 1H, J ¼ 8.0 Hz), 7.42
(d, 2H, J ¼ 8.8 Hz), 7.36 (t, 1H, J ¼ 8.0 Hz), 7.19e7.14 (m, 3H), 4.90 (s,
3H), 4.84 (s, 3H); MS (ESI), m/z: 522.02 [MꢁH]ꢁ.
5.2.4.24. (Z)-2-(4-((3-(2-Chlorobenzyl)-2,4-dioxothiazolidin-5-
ylidene)methyl)phenoxy)-N-(3-chlorophenyl)acetamide7-24.
Prepared by general procedure for the synthesis of 6 (Method B)
and 7 (Method E). Yield 45.9%; White powder; 1H NMR (400 MHz,
5.2.4.32. (Z)-N-(3-Chlorophenyl)-2-(4-((2,4-dioxo-3-(2-oxo-2-(phe-
nylamino)ethyl)thiazolidin-5-ylidene)methyl)phenoxy)acetamide 7-
32. Prepared by general procedure for the synthesis of 6 (Method
C) and 7 (Method F). Yield 34.0%; White powder; 1H NMR
DMSO-d6):
d 10.37 (s, 1H), 7.96 (s, 1H), 7.84 (s, 1H), 7.65 (d, 2H,
J ¼ 8.4 Hz), 7.51 (t, 1H, J ¼ 8.4 Hz), 7.39e7.31 (m, 3H), 7.24e7.14 (m,
4H), 4.90 (s, 2H), 4.85 (s, 2H); MS (ESI), m/z: 511.03 [MꢁH]ꢁ.
(400 MHz, DMSO-d6):
d 10.23 (s, 1H), 9.96 (s, 1H), 7.85 (s, 1H), 7.75
5.2.4.25. (Z)-2-(4-((3-(3-Chlorobenzyl)-2,4-dioxothiazolidin-5-
(s, 1H), 7.65e7.56 (m, 2H), 7.34e7.26 (m, 3H), 6.90 (s, 1H), 7.39e7.30
ylidene)methyl)phenoxy)-N-(3-chlorophenyl)acetamide
7-25.
(m, 5H), 4.66 (s, 2H), 4.43 (s, 2H); MS (ESI), m/z: 520.05 [MꢁH]ꢁ.
Prepared by general procedure for the synthesis of 6 (Method B)
and 7 (Method E). Yield 57.5%; White powder; 1H NMR (400 MHz,
5.2.4.33. (Z)-N-(3-Chlorophenyl)-2-(4-((2,4-dioxo-3-(2-oxo-2-(p-
tolylamino)ethyl)thiazolidin-5-ylidene)methyl)phenoxy)acetamide 7-
33. Prepared by general procedure for the synthesis of 6 (Method
C) and 7 (Method F). Yield 10.9%; White powder; 1H NMR
DMSO-d6):
d 10.36 (s, 1H), 7.94 (s, 1H), 7.84 (s, 1H), 7.63 (d, 2H,
J ¼ 8.4 Hz), 7.52 (d, 1H, J ¼ 8.0 Hz), 7.39e7.34 (m, 4H), 7.27 (m, 1H),
7.18e7.14 (m, 3H), 4.84 (s, 4H); MS (ESI), m/z: 511.00 [MꢁH]ꢁ.
(400 MHz, DMSO-d6): d 10.37 (s, 1H), 10.31 (s, 1H), 7.76 (s, 1H), 7.84
5.2.4.26. (Z)-2-(4-((3-(4-Chlorobenzyl)-2,4-dioxothiazolidin-5-
(t,1H, J ¼ 2.0 Hz), 7.66 (d, 2H, J ¼ 8.8 Hz), 7.53 (d,1H, J ¼ 9.2 Hz), 7.43
(d, 2H, J ¼ 8.4 Hz), 7.37 (t, 1H, J ¼ 8.0 Hz), 7.29 (d, 1H, J ¼ 8.0 Hz), 7.19
(d, 2H, J ¼ 9.2 Hz), 7.13 (d, 2H, J ¼ 8.4 Hz), 4.85 (s, 2H), 4.49 (s, 2H),
2.25 (s, 3H); MS (ESI), m/z: 552.05 [MꢁH]ꢁ.
ylidene)methyl)phenoxy)-N-(3-chlorophenyl)acetamide
7-26.
Prepared by general procedure for the synthesis of 6 (Method B)
and 7 (Method E). Yield 69.0%; White powder; 1H NMR (400 MHz,
DMSO-d6):
d 10.36 (s, 1H), 7.93 (s, 1H), 7.83 (s, 1H), 7.63 (d, 2H,
J ¼ 8.4 Hz), 7.52 (d, 1H, J ¼ 8.4 Hz), 7.43e7.33 (m, 5H), 7.18e7.14 (m,
5.2.4.34. (Z)-N-(4-Acetylphenyl)-2-(5-(4-(2-((3-chlorophenyl)
amino)-2-oxoethoxy)benzylidene)-2,4-dioxothiazolidin-3-yl)acet-
amide 7-34. Prepared by general procedure for the synthesis of 6
(Method C) and 7 (Method F). Yield 43.1%; Brown solid; 1H NMR
3H), 4.84 (s, 2H), 4.82 (s, 2H); MS (ESI), m/z: 511.03 [MꢁH]ꢁ.
5.2.4.27. (Z)-2-(4-((3-(4-Bromobenzyl)-2,4-dioxothiazolidin-5-
ylidene)methyl)phenoxy)-N-(3-chlorophenyl)acetamide
7-27.
(400 MHz, DMSO -d6): d 10.78 (s, 1H), 10.38 (s, 1H), 7.97 (s, 2H), 7.84
Prepared by general procedure for the synthesis of 6 (Method B)
(s,1H), 7.45 (s,1H), 7.71e7.66 (m, 4H), 7.53 (d,1H, J ¼ 8.4 Hz), 7.37 (t,
1H, J ¼ 8.0 Hz), 7.20e7.15 (m, 3H), 4.85 (s, 2H), 4.56 (s, 2H), 2.54 (s,
3H); MS (ESI), m/z: 562.06 [MꢁH]ꢁ.
and 7 (Method E). Yield 56.0%; White powder; 1H NMR (400 MHz,
DMSO-d6):
d 10.36 (s, 1H), 7.93 (s, 1H), 7.83 (s, 1H), 7.63 (d, 2H,
J ¼ 8.4 Hz), 7.56e7.51 (m, 3H), 7.36 (t, 1H, J ¼ 8.0 Hz), 7.27 (d, 2H,
J ¼ 8.0 Hz), 7.18e7.14 (m, 3H), 4.84 (s, 2H), 4.80 (s, 2H); MS (ESI), m/
z: 554.99 [MꢁH]ꢁ.
5.2.4.35. (Z)-N-(3-Chlorophenyl)-2-(4-((3-(2-(naphthalen-1-
ylamino)-2-oxoethyl)-2,4-dioxothiazolidin-5-ylidene)methyl)phe-
noxy)acetamide 7-35. Prepared by general procedure for the syn-
thesis of 6 (Method C) and 7 (Method F). Yield 45.0%; Brown solid;
5.2.4.28. (Z)-N-(3-Chlorophenyl)-2-(4-((3-(4-cyanobenzyl)-2,4-
dioxothiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-28.
1H NMR (400 MHz, DMSO-d6):
d 10.41 (s, 1H), 10.37 (s, 1H), 8.11 (d,
Prepared by general procedure for the synthesis of 6 (Method B)
1H, J ¼ 8.0 Hz), 7.98e7.95 (m, 2H), 7.83 (s, 1H), 7.80 (d, 1H,
J ¼ 8.0 Hz), 7.68e7.64 (m, 3H), 7.61e7.56 (m, 2H), 7.56e7.48 (m, 2H),
7.36 (t, 1H, J ¼ 8.0 Hz), 7.19e7.14 (m, 3H), 4.84 (s, 2H), 4. 69 (s, 2H);
MS (ESI), m/z: 570.10 [MꢁH]ꢁ.
and 7 (Method E). Yield 80.0%; Yellow solid; 1H NMR (400 MHz,
DMSO-d6):
d 10.37 (s, 1H), 7.94 (s, 1H), 7.83 (m, 3H), 7.64 (d, 2H,
J ¼ 8.8 Hz), 7.53e7.49 (m, 3H), 7.36 (t, 1H, J ¼ 8.0 Hz), 7.18e7.14 (m,
3H), 4.92 (s, 2H), 4.84 (s, 2H); MS (ESI), m/z: 502.05 [MꢁH]ꢁ.
5.2.4.36. (Z)-N-Benzyl-2-(5-(4-(2-((3-chlorophenyl)amino)-2-
oxoethoxy)benzylidene)-2,4-dioxothiazolidin-3-yl)acetamide 7-36.
Prepared by general procedure for the synthesis of 6 (Method C)
and 7 (Method F). Yield 55.0%; White powder; 1H NMR (400 MHz,
5.2.4.29. (Z)-N-(3-Chlorophenyl)-2-(4-((3-(4-nitrobenzyl)-2,4-
dioxothiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-29.
Prepared by general procedure for the synthesis of 6 (Method B)
and 7 (Method E). Yield 50.2%; White powder; 1H NMR (400 MHz,
DMSO-d6):
d
10.37 (s,1H), 8.01 (t,1H, J ¼ 6.0 Hz), 7.94 (s,1H), 7.84 (s,
DMSO-d6):
d
10.37 (s, 1H), 8.22 (d, 2H, J ¼ 9.2 Hz), 7.96 (s, 1H), 7.84
1H), 7.65 (d, 2H, J ¼ 8.8 Hz), 7.53 (d, 1H, J ¼ 8.0 Hz), 7.37e7.34 (m,
3H), 7.26 (m, 3H), 7.19e7.15 (m, 3H), 4.84 (s, 2H), 4.34 (s, 2H), 4.31
(d, 2H, J ¼ 6.0 Hz); MS (ESI), m/z: 552.04 [MꢁH]ꢁ.
(s, 1H), 7.65 (d, 2H, J ¼ 8.8 Hz), 7.59 (d, 2H, J ¼ 8.8 Hz), 7.52 (d, 1H,
J ¼ 9.6 Hz), 7.37 (t, 1H, J ¼ 8.0 Hz), 7.19e7.15 (m, 3H), 4.98 (s, 2H),
4.85 (s, 2H); MS (ESI), m/z: 522.04 [MꢁH]ꢁ.
5.2.4.37. (Z)-2-(5-(4-(2-((3-Chlorophenyl)amino)-2-oxoethoxy)ben-
zylidene)-2,4-dioxothiazolidin-3-yl)-N-methyl-N-phenylacetamide 7-
37. Prepared by general procedure for the synthesis of 6 (Method
C) and 7 (Method F). Yield 34.0%; White powder; 1H NMR
5.2.4.30. (Z)-methyl 4-((5-(4-(2-((3-Chlorophenyl)amino)-2-
oxoethoxy)benzylidene)-2,4-dioxothiazolidin-3-yl)methyl)benzoate
7-30. Prepared by general procedure for the synthesis of 6 (Method
B) and 7 (Method E). Yield 32.9%; White powder; 1H NMR
(400 MHz, DMSO-d6):
d 10.37 (s, 1H), 7.90 (s, 1H), 7.84 (t, 1H,
(400 MHz, DMSO-d6):
d
10.37 (s,1H), 7.95e7.93 (m, 3H), 7.84 (s,1H),
J ¼ 2.0 Hz), 7.64 (d, 2H, J ¼ 9.2 Hz), 7.55e7.47 (m, 5H), 7.37 (t, 2H,
J ¼ 8.0 Hz), 7.18e7.14 (m, 3H), 4.84 (s, 2H), 4. 12 (s, 2H), 3.20 (s, 3H);
MS (ESI), m/z: 552.07 [MꢁH]ꢁ.
7.64 (d, 2H, J ¼ 8.4 Hz), 7.53 (d, 1H, J ¼ 9.2 Hz), 7.45 (d, 1H,
J ¼ 8.4 Hz), 7.37 (t, 1H, J ¼ 8.0 Hz), 7.19e7.14 (m, 3H), 4.91 (s, 2H),
4.84 (s, 2H), 3.84 (s, 3H); MS (ESI), m/z: 535.07 [MꢁH]ꢁ.
5.2.4.38. (Z)-N-(3-Chlorophenyl)-2-(4-((2,4-dioxo-3-(pyridin-2-
5.2.4.31. (Z)-4-((5-(4-(2-((3-Chlorophenyl)amino)-2-oxoethoxy)ben-
ylmethyl)thiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-38.
zylidene)-2,4-dioxothiazolidin-3-yl)methyl)benzoic
acid
7-31.
Prepared by general procedure for the synthesis of 6 (Method C)
Prepared by general procedure for the synthesis of 6 (Method C)
and 7 (Method F). Yield 44.5%; White powder; 1H NMR (400 MHz,
and 7 (Method F). Yield 75.0%; White powder; 1H NMR (400 MHz,
DMSO-d6):
d
10.37 (s, 1H), 8.56 (s, 1H), 8.52 (d, 1H, J ¼ 4.4 Hz), 7.95

188
L. Ma et al. / European Journal of Medicinal Chemistry 92 (2015) 178e190
(s, 1H), 7.84 (s, 1H), 7.73 (d, 1H, J ¼ 8.0 Hz), 7.64 (d, 2H, J ¼ 8.4 Hz),
7.53 (d, 1H, J ¼ 8.0 Hz), 7.38 (q, 2H, J ¼ 8.0 Hz), 7.19e7.15 (m, 3H),
4.88 (s, 2H), 4.84 (s, 2H); MS (ESI), m/z: 478.00 [MꢁH]ꢁ.
5.2.4.46. (Z)-N-(3-Chlorophenyl)-2-(4-(1-(2,4-dioxothiazolidin-5-
ylidene)ethyl)phenoxy)acetamide 7-46. Prepared by general proce-
dure for the synthesis of 7 (Method F). Yield 50.5%; Brown solid; 1H
NMR (400 MHz, DMSO-d6): d 12.36 (s, 1H), 9.96 (s, 1H), 7.76 (s, 1H),
7.75 (s, 1H), 7.41 (d, 2H, J ¼ 8.4 Hz), 7.36 (d, 1H, J ¼ 8.0 Hz), 7.33 (t,
1H, J ¼ 8.2 Hz), 7.18 (d, 2H, J ¼ 8.4 Hz), 4.66 (s, 2H), 2.43 (s, 3H); MS
(ESI), m/z: 401.03 [MꢁH]ꢁ.
5.2.4.39. (Z)-N-(3-Chlorophenyl)-2-(4-((2,4-dioxo-3-(pyridin-3-
ylmethyl)thiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-39.
Prepared by general procedure for synthesis of 6 (Method C) and 7
(Method F). Yield 31.0%; Light yellow powder; 1H NMR (400 MHz,
5.2.4.47. (Z)-N-(3-Chlorophenyl)-3-(4-((2,4-dioxothiazolidin-5-
ylidene)methyl)phenoxy)propanamide 7-47. Prepared by general
procedure for the synthesis of 7 (Method D). Yield 31.9%; White
DMSO-d6):
d
8.55 (d, 1H, J ¼ 4.8 Hz), 8.20 (s, 1H), 7.90 (s, 1H), 7.72 (s,
1H), 7.67 (t, 1H, J ¼ 6.8 Hz), 7.54 (d, 2H, J ¼ 8.4 Hz), 7.45 (d, 1H,
J ¼ 8.0 Hz), 7.31e7.28 (m, 2H), 7.22e7.14 (m, 2H), 7.10 (d, 2H,
J ¼ 8.4 Hz), 5.07 (s, 2H), 4.67 (s, 2H); MS (ESI), m/z: 478.10 [MꢁH]ꢁ.
powder; 1H NMR (400 MHz, DMSO-d6):
d 12.27 (s,1H),10.00 (s,1H),
7.94 (s, 1H), 7.72 (s, 1H), 7.62e7.31 (m, 3H), 7.15 (s, 1H), 7.00 (d, 1H,
J ¼ 8.0 Hz), 6.97 (d, 2H, J ¼ 8.0 Hz), 4.32 (t, 2H, J ¼ 6.0 Hz), 2.64 (t,
2H, J ¼ 6.0 Hz); MS (ESI), m/z: 401.01 [MꢁH]ꢁ.
5.2.4.40. (Z)-N-(3-Chlorophenyl)-2-(3-((2,4-dioxothiazolidin-5-
ylidene)methyl)phenoxy)acetamide 7-40. Prepared by general pro-
cedure for the synthesis of 7 (Method E). Yield 59.0%; White
powder; 1H NMR (400 MHz, DMSO-d6):
d 12.54 (s, 1H), 11.97 (s, 1H),
5.2.4.48. (Z)-N-(3-Chlorophenyl)-4-(4-((2,4-dioxothiazolidin-5-
ylidene)methyl)phenoxy)butanamide 7-48. Prepared by general
procedure for the synthesis of 7 (Method D). Yield 59.0%; White
10.35 (s, 1H), 7.84 (s, 1H), 7.76 (s,1H), 7.58 (d, J ¼ 8.8 Hz, 2H), 7.52 (m
3H), 7.17 (m, 1H), 7.15 (m, 1H), 4.83 (s, 2H); MS (ESI), m/z: 387.04
[MꢁH]ꢁ.
powder; 1H NMR (400 MHz, DMSO-d6):
d 10.31 (s, 1H),10.03 (s, 1H),
7.97 (s, 1H), 7.75 (s, 1H), 7.65 (d, 1H, J ¼ 1.6 Hz), 7.34 (s, 1H), 7.02 (d,
1H, J ¼ 1.6 Hz), 4.11 (t, J ¼ 5.6 Hz, 2H), 2.80 (t, 2H, J ¼ 5.6 Hz),
2.23e2.15 (m, 2H); MS (ESI), m/z: 415.00 [MꢁH]ꢁ.
5.2.4.41. (Z)-N-(3-Chlorophenyl)-2-(5-((2,4-dioxothiazolidin-5-
ylidene)methyl)-2-methoxyphenoxy)acetamide 7-41. Prepared by
general procedure for the synthesis of 7 (Method E). Yield 84.1%;
5.2.5. Synthesis of compound 7-49
Yellow powder; 1H NMR (400 MHz, DMSO-d6):
d 12.50 (s,1H),10.40
Step 1: 3-Chloroaniline (6.38 g, 50 mmol), N-Boc-Gly (9.28 g,
53 mmol), EDCI (11.50 g, 60 mmol) was dissolved into DCM
(100 mL) at 0 ꢀC and allowed to warm up to room temperature
overnight. Then, the solvent was evaporated and extracted by
EtOAc (3 ꢂ 50 mL) and water (3 ꢂ 50 mL). The organic layers was
combined and washed by 0.5 N HCl (2 ꢂ 15 mL), NaHCO3
(2 ꢂ 15 mL), water (2 ꢂ 15 mL), brine (2 ꢂ 10 mL), and dried by
anhydrous Na2SO4. The solvent was removed to obtain the crude
product 2-49 without purification. Yield 95.0%. 1H NMR (400 MHz,
(s, 1H), 7.84 (s, 1H), 7.71 (s, 1H), 7.50 (d, 1H, J ¼ 8.0 Hz), 7.36 (t, 1H,
J ¼ 8.0 Hz), 7.27 (d, 1H, J ¼ 8.4 Hz), 7.18 (d, 1H, J ¼ 8.4 Hz), 7.14 (br-s,
2H), 4.79 (s, 2H), 3.88 (s, 3H); MS (ESI), m/z: 417.03 [MꢁH]ꢁ.
5.2.4.42. (Z)-N-(3-Chlorophenyl)-2-(4-((2,4-dioxothiazolidin-5-
ylidene)methyl)-2-methoxyphenoxy)acetamide 7-42. Prepared by
general procedure for the synthesis of 7 (Method E). Yield 69.0%;
Light yellow powder; 1H NMR (400 MHz, DMSO-d6):
d 12.55 (s, 1H),
10.38 (s, 1H), 7.82 (s, 1H), 7.76 (s, 1H), 7.48 (d, 1H, J ¼ 7.9 Hz), 7.36 (t,
1H, J ¼ 8.0 Hz), 7.26 (s,1H), 7.17e7.14 (m, 2H), 7.07 (d,1H, J ¼ 8.4 Hz),
4.82 (s, 2H), 3.83 (s, 3H); MS (ESI), m/z: 417.02 [MꢁH]ꢁ.
DMSO-d6):
d
8.60 (s, 1H), 7.63 (s, 1H), 7.36 (d, 1H, J ¼ 8.0 Hz), 7.22 (t,
1H, J ¼ 8.0 Hz), 7.07 (d, 1H, J ¼ 8.0 Hz), 5.47 (s, 1H), 3.95 (d, 2H,
J ¼ 6.4 Hz), 1.48 (s, 9H); MS (ESI), m/z: 283.00 [MꢁH]ꢁ.
Step 2: In ice bath, 2-49 (14.2 g, 50 mmol) was dissolved into
anhydrous THF (60 mL) and LiAlH4 (4.9 g, 125 mmol) was slowly
added into the mixture at 0 ꢀC and allowed to warm up to room
temperature overnight. Then, 250 mL water added and the solvent
was filtered. The layer was washed by EtOAc (3 ꢂ 50 mL), water
(3 ꢂ 50 mL) and brine (2 ꢂ 30 mL), dried by anhydrous Na2SO4. The
solvent was removed to obtain the white solid 3-49. Yield 91.2%. 1H
5.2.4.43. (Z)-N-(3-Chlorophenyl)-2-(4-((2,4-dioxothiazolidin-5-
ylidene)methyl)-2-ethoxyphenoxy)acetamide 7-43. Prepared by
general procedure for the synthesis of 7 (Method E). Yield 39.0%;
Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 12.55 (s, 1H), 10.37 (s,
1H), 7.50 (s, 1H), 7.48 (s, 1H), 7.36 (t, 1H, J ¼ 8.0 Hz), 7.24 (d, 1H,
J ¼ 1.2 Hz), 7.17e7.14 (m, 2H), 7.06 (d, 1H, J ¼ 8.4 Hz), 4.83 (s, 2H),
4.11 (q, 2H, J ¼ 7.2 Hz), 1.38 (t, 3H, J ¼ 7.2 Hz); MS (ESI), m/z: 431.05
[MꢁH]ꢁ.
NMR (400 MHz, DMSO-d6):
d
7.06 (t, 1H, J ¼ 8.0 Hz), 6.65 (d, 1H,
J ¼ 8.0 Hz), 6.56 (s, 1H), 6.46 (dd, 1H, J ¼ 8.0 Hz, J ¼ 3.0 Hz), 4.83 (s,
1H), 4.21 (s, 1H), 3.37 (t, 2H, J ¼ 5.8 Hz), 3.24 (t, 2H, J ¼ 6.0 Hz), 1.45
(s, 9H); MS (ESI), m/z: 269.02 [MꢁH]ꢁ.
5.2.4.44. (Z)-N-(3-Chlorophenyl)-2-(4-((2,4-dioxothiazolidin-5-
ylidene)methyl)-2,6-dimethoxyphenoxy)acetamide
Prepared by general procedure for the synthesis of 7 (Method E).
Yield 20.0%; Yellow powder; 1H NMR (400 MHz, DMSO-d6):
12.64
7-44.
Step 3: In ice bath, 3-49 (5.6 g, 20.6 mmol) and NaHCO3 (2.6 g,
30.9 mmol) was dissolved into DCM (50 mL) and stirred for 10 min.
Then, 2-chloroacetyl chloride (3.49 g, 30.9 mmol) was added slowly
and allowed to warm up to room temperature overnight. Then,
100 mL water added and the solvent was filtered. The layer was
washed by EtOAc (3 ꢂ 20 mL), water (3 ꢂ 20 mL) and brine
(2 ꢂ 20 mL), dried by anhydrous Na2SO4. The solvent was removed
to obtain the white solid 4-49. Yield 79.6%. 1H NMR (400 MHz,
d
(s, 1H), 10.05 (s, 1H), 7.90 (t, 1H, J ¼ 2.0 Hz), 7.77 (s, 1H), 7.55 (d, 1H,
J ¼ 8.0 Hz), 7.38 (t,1H, J ¼ 8.0 Hz), 7.17 (dd,1H, J ¼ 0.8 Hz, J ¼ 8.0 Hz),
6.98 (s, 2H), 4.59 (s, 2H), 3.87 (s, 6H); 13C NMR (100 MHz, DMSO-
d6): d 168.2, 167.7, 166.7, 159.6, 140.0, 132.2, 131.9, 130.7, 126.4, 123.7,
121.0, 119.4, 118.2, 115.8, 67.2, 57.2; MS (ESI), m/z: 447.03 [MꢁH]ꢁ.
DMSO-d6): d 7.42e7.13 (m, 2H), 7.30e7.27 (m,1H), 7.21 (m,1H), 4.91
5.2.4.45. (Z)-N-(3-Chlorophenyl)-2-(4-((2,4-dioxothiazolidin-5-
ylidene)methyl)-2-fluorophenoxy)acetamide 7-45. Prepared by
general procedure for the synthesis of 7 (Method E). Yield 40.0%;
(s, 2H), 3.85 (s, 2H), 3.87e3.83 (m, 2H), 3.35 (d, 2H, J ¼ 6.4 Hz), 1.42
(s, 9H); MS (ESI), m/z: 345.01 [MꢁH]ꢁ.
Step 4: 4-49 (1.5 g, 4.26 mmol), 4-hydroxybenzaldehyde (0.52 g,
4.26 mmol), K2CO3 (0.88 g, 6.39 mmol) and KI (1.06 g, 6.39 mmol)
were dissolved in anhydrous acetone (100 mL). The reaction
mixture was refluxed overnight and then cooled to room temper-
ature. Then the K2CO3 solid was filtered and the acetone solution
was removed under reduced pressure to obtain the crude products.
Light yellow powder; 1H NMR (400 MHz, DMSO-d6):
d 12.62 (s, 1H),
10.45 (s, 1H), 7.81 (s, 1H), 7.74 (s, 1H), 7.55 (dd, 1H, J ¼ 1.6 Hz,
J ¼ 11.2 Hz), 7.47 (d, 1H, J ¼ 7.6 Hz), 7.40e7.33 (m, 2H), 7.27 (t, 1H,
J ¼ 8.8 Hz), 7.15 (d, 1H, J ¼ 8.0 Hz), 4.94 (s, 2H); MS (ESI), m/z: 405.01
[MꢁH]ꢁ.

L. Ma et al. / European Journal of Medicinal Chemistry 92 (2015) 178e190
189
The residue was purified by silica gel column chromatography to
give the white solid 5-49. Yield 82.0%. 1H NMR (400 MHz, DMSO-
DMSO-d6): d 11.19 (s, 1H), 10.46 (s, 1H), 10.31 (s, 1H), 7.85 (t, 1H,
J ¼ 2.0 Hz), 7.61 (d, 2H, J ¼ 8.8 Hz), 7.54 (dd, 1H, J ¼ 1.8 Hz,
J ¼ 8.4 Hz), 7.36 (t,1H, J ¼ 8.0 Hz), 7.15 (dd,1H, J ¼ 2.0 Hz, J ¼ 8.0 Hz),
7.03 (d, 2H, J ¼ 8.8 Hz), 4.78 (s, 2H); MS (ESI), m/z: 370.05 [MꢁH]ꢁ.
d6):
d
9.88 (s, 1H), 7.80 (d, 2H, J ¼ 8.0 Hz), 7.44 (d, 2H, J ¼ 8.0 Hz),
7.41 (s, 1H), 7.21 (s, 1H), 6.87 (d, 2H, J ¼ 8.0 Hz), 4.90 (s, 1H), 4.50 (s,
2H), 3.86 (t, 2H, J ¼ 5.2 Hz), 3.35 (d, 2H, J ¼ 5.2 Hz), 1.43 (s, 9H); MS
(ESI), m/z: 431.01 [MꢁH]ꢁ.
5.2.7.3. (Z)-2-(4-((1-Benzyl-2,5-dioxoimidazolidin-4-ylidene)
methyl)phenoxy)-N-(3-chlorophenyl)acetamide 7-52. Prepared by
procedure for the synthesis of 7-51 using 8d. Yield 19.5%; White
Step 5: 5-49 (230 mg, 0.5 mmol), thiazolidine-2,4-dione
(115 mg, 0.8 mmol), piperidine (83 mg, 0.8 mmol), benzoic acid
(98 mg, 0.8 mmol), and 10 mL toluene/EtOH (1:1) were heated to
reflux overnight. The mixture was filtered and washed by EtOH
(2 ꢂ 10 mL), water (2 ꢂ 10 mL), and dried in vacuum at 40 ꢀC for
24 h to afford light yellow solid 6-49. Yield 48.9%. 1H NMR
powder; 1H NMR (400 MHz, DMSO-d6):
d 10.80 (s,1H),10.32 (s,1H),
7.84 (s,1H), 7.64 (d, 2H, J ¼ 8.8 Hz), 7.54 (d,1H, J ¼ 8.4 Hz), 7.38e7.28
(m, 6H), 7.15 (d, 1H, J ¼ 7.6 Hz), 7.04 (d, 2H, J ¼ 8.4 Hz), 4.79 (s, 2H),
4.66 (s, 2H); MS (ESI), m/z: 460.10 [MꢁH]ꢁ.
(400 MHz, DMSO-d6): d 8.86 (s, 1H), 7.69 (s, 1H), 7.51e7.32 (m, 5H),
7.23 (m,1H), 6.85 (d, 2H, J ¼ 8.0 Hz), 4.99 (s,1H), 4.48 (s, 2H), 3.86 (t,
2H, J ¼ 6.4 Hz), 3.36 (t, 2H, J ¼ 6.4 Hz), 1.44 (s, 9H); MS (ESI), m/z:
530.10 [MꢁH]ꢁ.
5.2.7.4. (Z)-N-(3-Chlorophenyl)-2-(4-((1,3-dibenzyl-2,5-
dioxoimidazolidin-4-ylidene)methyl)phenoxy)acetamide
Prepared by procedure for the synthesis of 7-51 using 8e. Yield
40.0%; White powder; 1H NMR (400 MHz, DMSO-d6):
10.30 (s,
7-53.
Step 6: In ice bath, 6-49 (53 mg, 0.1 mmol) was added in 5 mL
TFA/DCM (1:1) and stirred for 2 h. The mixture was evaporated to
dryness and dissolved into EtOAc and washed by NaHCO3
(2 ꢂ 15 mL), water (2 ꢂ 15 mL), brine (2 ꢂ 15 mL) and dried by
anhydrous Na2SO4. The solvent was evaporated to give the white
solid 7-49.
d
1H), 7.94 (s, 1H), 7.92 (s, 1H), 7.83 (s, 1H), 7.53 (d, 1H, J ¼ 8.0 Hz),
7.37e7.26 (m, 10H), 7.14 (dd, 1H, J ¼ 1.2 Hz, J ¼ 6.8 Hz), 6.98 (d, 2H,
J ¼ 8.8 Hz), 6.52 (s, 1H), 4.97 (s, 2H), 4.74 (s, 2H), 4.73 (s, 2H); MS
(ESI), m/z: 550.10 [MꢁH]ꢁ.
5.2.7.5. (E)-N-(3-Chlorophenyl)-2-(4-((2,5-dioxopyrrolidin-3-
ylidene)methyl)phenoxy)acetamide 7-54. Procedure: 4a (1.0 mmol),
8b (1.0 mmol), and 10 mL MeOH were heated to reflux for 5 h. The
mixture was filtered and washed by EtOH (2 ꢂ 10 mL), water
(2 ꢂ 10 mL), and dried in vacuum at 40 ꢀC for 24 h to afford 7-54.
5.2.5.1. (Z)-N-(2-Aminoethyl)-N-(3-chlorophenyl)-2-(4-((2,4-
dioxothiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-49.
12.52
Yield 40.0%; White powder; 1H NMR (400 MHz, DMSO-d6):
d
(s, 1H), 7.74 (s, 1H), 7.70 (s, 1H), 7.51 (m, 5H), 6.96e6.95 (m, 2H) 4.51
(s, 2H), 3.66 (d, J ¼ 6.0 Hz, 2H), 3.08 (d, J ¼ 6.0 Hz, 2H); MS (ESI), m/
z: 430.00 [MꢁH]ꢁ.
Yield 23.4%; White powder; 1H NMR (400 MHz, DMSO-d6):
d 11.38
(s, 1H), 10.34 (s, 1H), 7.84 (s, 1H), 7.60 (d, 2H, J ¼ 8.8 Hz), 7.54 (d, 1H,
J ¼ 8.0 Hz), 7.38e7.34 (m, 2H), 7.15 (d, 1H, J ¼ 8.0 Hz), 7.08 (d, 2H,
J ¼ 8.8 Hz), 4.80 (s, 2H), 3.61 (s, 2H); MS (ESI), m/z: 369.02 [MꢁH]ꢁ.
5.2.6. Synthesis of compounds 8
Synthesis of 8a [15]: 2-chloro-N-(pyridin-2-yl)acetamide
(1 mmol), KSCN (2 mmol), and 10 mL acetone were refluxed for 5 h.
The mixture was filtered and washed by water. The crude product
was recystallized by EtOH to afford yellow solid 8a (Yield 76%).
Synthesis of 8b [17]: 1H-pyrrole-2,5-dione (1.0 mmol), PPh3
(1.0 mmol) and 5 mL CH3OH was heated to reflux for 1.5 h. The
mixture was filtered and washed by CH3OH (2 ꢂ 10 mL) to afford
the 8b (Yield 89.9%).
Synthesis of 8d-e [16]: In ice bath, imidazolidine-2,4-dione
(1.0 mmol) was dissolved in 10 mL DMF and NaH (1.5 mmol, 60%)
was slowly added into the mixture. Then, (bromomethyl)benzene
(1.1 mmol) was added and stirred overnight. The mixture was
evaporated to dryness. The residue was purified by silica gel col-
umn chromatography to give 8d and 8e.
5.2.7.6. N-(3-Chlorophenyl)-2-(4-((2,4,6-trioxotetrahydropyrimidin-
5(2H)-ylidene)methyl)phenoxy)acetamide 7-55. Procedure: alde-
hyde 4a (0.5 mmol), ethanol (3 mL), distilled water (3 mL), and
barbituric acid (3.0 mmol) were refluxed at 60 ꢀC for 3e4 h. The
formed solids were collected by filtration and washed with boiling
water (3 ꢂ 15 mL), ethanol (3 ꢂ 15 mL), and ether (3 ꢂ 15 mL). The
obtained solids were dried in vacuo for 24 h. Yield 43.9%; Yellow
powder; 1H NMR (400 MHz, DMSO-d6):
d 11.33 (s, 1H), 11.20 (s, 1H),
10.37 (s, 1H), 8.36 (d, 2H, J ¼ 8.8 Hz), 8.25 (s, 1H), 7.83 (t, 1H,
J ¼ 1.8 Hz), 7.54e7.51 (m, 1H), 7.37 (t, 1H, J ¼ 8.4 Hz), 7.17e7.14 (m,
1H), 7.11 (d, 2H, J ¼ 9.2 Hz), 4.88 (s, 2H); MS (ESI), m/z: 430.13
[MþMeOHeH]ꢁ.
5 . 2 . 7 . 7 . N - ( 3 - C h l o r o p h e n y l ) - 2 - ( 4 - ( ( 4 , 6 - d i o x o - 2 -
thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)phenoxy)acet-
amide 7-56. Prepared by procedure for the synthesis of 7-55 using
thiobarbituric acid. Yield 49.9%; Yellow powder; 1H NMR (400 MHz,
5.2.7. Synthesis of bioisosteric derivatives of thiazolidine-2,4-dione
5.2.7.1. (Z)-N-(3-Chlorophenyl)-2-(4-((2-imino-4-oxo-3-(pyridin-2-
yl)thiazolidin-5-ylidene)methyl)phenoxy)acetamide
7-50.
Procedure: 4a (0.3 mmol), 8a (0.36 mmol), anhydrous NaOAc
(0.6 mmol) and 5 mL acetic acid were heated to 100 ꢀC for 5 h. The
mixture was filtered and washed by EtOH (2 ꢂ 10 mL), water
(2 ꢂ 10 mL), and dried in vacuum at 40 ꢀC for 24 h to afford 7-50.
DMSO-d6): d 12.41 (s, 1H), 12.31 (s, 1H), 10.38 (s, 1H), 8.41 (d, 2H,
J ¼ 9.2 Hz), 8.27 (s, 1H), 7.83 (t, 1H, J ¼ 2.0 Hz), 7.53 (d, 1H,
J ¼ 9.2 Hz), 7.37 (t,1H, J ¼ 8.0 Hz), 7.17e7.12 (m, 3H), 4.90 (s, 2H); MS
(ESI), m/z: 446.09 [MþMeOHeH]ꢁ.
Yield 21.0%; Brown solid; 1H NMR (400 MHz, DMSO -d6):
d 12.37
(br-s, 1H), 10.37 (s, 1H), 8.53 (s, 1H), 7.88e7.84 (m, 2H), 7.66 (d, 2H,
J ¼ 8.4 Hz), 7.62 (s, 1H), 7.54 (d, 1H, J ¼ 8.4 Hz), 7.37 (t, 1H,
J ¼ 8.0 Hz), 7.22e7.12 (m, 5H), 4.83 (s, 2H), 4.11 (q, 2H, J ¼ 7.2 Hz),
1.38 (t, 3H, J ¼ 7.2 Hz); MS (ESI), m/z: 463.05 [MꢁH]ꢁ.
5.2.7.8. (Z)-N-(3-Chlorophenyl)-2-(4-((2-imino-4-oxothiazolidin-5-
ylidene)methyl)phenoxy)acetamide 7-57. Procedure: aldehyde 4a
(1.0 mmol), 2-thioxothiazolidin-4-one (1.2 mmol), piperidine (cat.),
and 10 mL EtOH were mixed and heated to reflux for 6 h. Then a
portion of water/ice was added and the precipitated solids were
collected by sucking filtration and washed with distilled water
(4 ꢂ 15 mL), EtOH (2 ꢂ 10 mL), and ether (2 ꢂ 10 mL). The solids
obtained were dried in vacuum at 40 ꢀC for 24 h. Yield 30.0%;
5.2.7.2. (Z)-N-(3-Chlorophenyl)-2-(4-((2,5-dioxoimidazolidin-4-
ylidene)methyl)phenoxy)acetamide 7-51. Procedure: 4a (1.0 mmol),
8c (1.0 mmol), pyrrolidine (cat.), and 10 mL EtOH were heated to
reflux for 5 h. The mixture was filtered and washed by EtOH
(2 ꢂ 10 mL), water (2 ꢂ 10 mL), and dried in vacuum at 40 ꢀC for
24 h to afford 7-51. Yield 43.0%; Yellow powder; 1H NMR (400 MHz,
Yellow powder; 1H NMR (400 MHz, DMSO-d6):
d 13.80 (s, 1H),
10.358 (s,1H), 7.84 (s,1H), 7.62e7.59 (m, 3H), 7.53 (d,1H, J ¼ 8.4 Hz),
7.37 (t, 1H, J ¼ 8.0 Hz), 7.18e7.15 (m, 3H), 4.84 (s, 2H); MS (ESI), m/z:

190
L. Ma et al. / European Journal of Medicinal Chemistry 92 (2015) 178e190
[6] G. Cirino, E. Distrutti, J.L. Wallace, Nitric oxide and inflammation, Inflamm.
402.98 [MꢁH]ꢁ.
Allergy Drug Targets 5 (2006) 115e119.
[7] S.M. Morris, Enzymes of arginine metabolism, J. Nutr. 134 (2004)
2743Se2747S.
5.2.7.9. (Z)-2-(5-(4-(2-((3-Chlorophenyl)amino)-2-oxoethoxy)ben-
[8] M. Colasanti, H. Suzuki, The dual personality of NO, Trends Pharmacol. Sci. 21
(2000) 249e252.
[9] J. MacMicking, Q.W. Xie, C. Nathan, Nitric oxide and macrophage function,
Annu. Rev. Immunol. 15 (1997) 323e350.
zylidene)-4-oxo-2-thioxothiazolidin-3-yl)acetic
acid
7-58.
Prepared by procedure for the synthesis of 7-57 using 2-(4-oxo-2-
thioxothiazolidin-3-yl)acetic acid. Yield 25.4%; Yellow powder; 1H
[10] P. Tripathi, P. Tripathi, L. Kashyap, V. Singh, The role of nitric oxide in in-
flammatory reactions, FEMS Microbiol. Immunol. 51 (2007) 443e452.
[11] V. Trajkovic, Modulation of inducible nitric oxide synthase activation by
immuno- suppressive drugs, Curr. Drug Metab. 2 (2001) 315e329.
[12] F. Wang, J.R. Sun, M.Y. Huang, H.Y. Wang, P.H. Sun, J. Lin, W.M. Chen, Design,
synthesis and anti-inflammatory evaluation of novel 5-benzylidene-3,4-
dihalo-furan-2-one derivatives, Eur. J. Med. Chem. 72 (2013) 35e45.
[13] L. Ma, C.F. Xie, Y.H. Ma, J. Liu, M.L. Xiang, X. Ye, H. Zheng, Z.Z. Chen, Q.Y. Xu,
T. Chen, J.Y. Chen, N. Qiu, G.C. Wang, X.L. Liang, A.H. Peng, S.Y. Yang, Y.Q. Wei,
NMR (400 MHz, DMSO-d6):
d 10.62 (s, 1H), 9.05 (br-s, 1H), 7.85 (s,
1H), 7.76 (s, 1H), 7.62 (d, 2H, J ¼ 8.0 Hz), 7.55 (d, 1H, J ¼ 8.0 Hz), 7.35
(t, 1H, J ¼ 8.0 Hz), 7.17e7.13 (m, 3H), 4.85 (s, 2H), 4.42 (s, 2H); MS
(ESI), m/z: 461.00 [MꢁH]ꢁ.
5.2.7.10. (Z)-N-(3-Chlorophenyl)-2-(4-((2-imino-4-oxothiazolidin-5-
ylidene)methyl)phenoxy)acetamide 7-59. Prepared by procedure for
the synthesis of 7e57 using 2-iminothiazolidin-4-one. Yield 35.3%;
L.J. Chen,
Synthesis
and biological
evaluation
of
novel
5-
benzylidenethiazolidine-2, 4-dione derivatives for the treatment of inflam-
matory diseases, J. Med. Chem. 54 (2011) 2060e2068.
Yellow powder; 1H NMR (400 MHz, DMSO-d6):
d 10.34 (s, 1H), 9.38
(s, 1H), 9.13 (s, 1H), 7.84 (s, 1H), 7.62e7.52 (m, 4H), 7.36 (t, 1H,
J ¼ 8.0 Hz), 7.15e7.13 (m, 3H), 4.81 (s, 2H); MS (ESI), m/z: 386.02
[MꢁH]ꢁ.
[14] C.F. Xie, L. Ma, J. Liu, X.X. Li, H.Y. Pei, M.L. Xiang, L.J. Chen, SKLB023 Blocks joint
inflammation and cartilage destruction in arthritis models via suppression of
nuclear factor-kappa B activation in macrophage, PloS One 8 (2013) e56349.
[15] L. Ma, J.Y. Chen, X.L. Liang, C.F. Xie, C.Y. Deng, L. Huang, A.H. Peng, Y.Q. Wei,
L.J. Chen, Synthesis and evaluation of 5-benzylidenethiazolidine-2,4-dione
derivatives for the treatment of non- alcoholic fatty liver disease, Arch.
Pharm. Weinh. 345 (2012) 517e524.
5.3. Molecular docking assay
[16] A.C. Tripathi, S.J. Gupta, G.N. Fatima, P.K. Sonar, A. Verma, S.K. Saraf, 4-
Thiazolidinones: the advances continue…, Eur. J. Med. Chem. 72 (2014)
52e77.
[17] S. Nazreen, M.S. Alam, H. Hamid, M.S. Yar, S. Shafi, A. Dhulap, P. Alam,
M.A. Pasha, S. Bano, M.M. Alam, S. Haider, Y. Ali, C. Kharbanda, K.K. Pillai,
Design, synthesis, in silico molecular docking and biological evaluation of
The iNOS was chosen as the target receptor. The 3D structure of
the receptor was gained from Protein Data Bank (PDB ID: 1r35).
Conformers of molecules were created by the aid of Omega and the
up limit of conformer number for each molecule is set to 2000.
Compound 7-44 was docked to the active site of iNOS by employing
a protein-ligand docking program FRED. Scoring functions Chem-
gauss version two, shapegauss and screenscore were used for
exhaustive searching, solid body optimizing and interaction
scoring. This Fig. 3 was generated by the software Virtual Molecular
Dynamics (VMD).
novel oxadiazole based thiazolidine-2,4-diones bis-heterocycles as PPAR-
g
agonists, Eur. J. Med. Chem. 87 (2014) 175e185.
[18] A. Mobinikhaledi, N. Foroughifar, S. Faghihi, Synthesis of some novel 5-(Ary-
lidene)-2- imino-3-(pyridin-2-yl)thiazolidin-4-one Derivatives, Phosphorus
Sulfur 184 (2009) 1837e1842.
[19] J.C. Thenmozhiyal, P.T.-H. Wong, W.K. Chui, Anticonvulsant activity of phe-
nylmethylene- hydantoins: a structure-activity relationship study, J. Med.
Chem. 47 (2004) 1527e1535.
[20] H. Mizufune, M. Nakamura, H. Mitsudera, Process research on arylnaph-
thalene lignan aza- analogues: a new palladium-catalyzed benzannulation of
Acknowledgment
a,b-bisbenzylidenesuccinic acid derivatives, Tetrahedron 62 (2006)
8539e8549.
We are grateful to the National Key Programs of China during
the 12th Five-Year Plan Period (2012ZX09103101-017) and China
Postdoctoral Science Foundation (2014M552373).
[21] L. Ma, C.F. Xie, Y. Ran, X.L. Liang, L. Huang, H.Y. Pei, J.Y. Chen, J. Liu, Y. Sang,
H.J. Lai, A.H. Peng, M.L. Xiang, Y.Q. Wei, L.J. Chen, Synthesis and biological
evaluation of 5-benzylidenepyrimidine-2,4,6(1H,3H,5H)-trione derivatives for
the treatment of obesity- related nonalcoholic fatty liver disease, J. Med.
Chem. 55 (2012) 9958e9972.
[22] E.A. Hallinan, S.W. Kramer, S.C. Houdek, W.M. Moore, G.M. Jerome,
D.P. Spangler, A.M. Stevens, H.S. Shieh, P.T. Manning, B.S. Pitzele, 4-Fluori-
nated L-lysine analogs as selective i-NOS inhibitors: methodology for intro-
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.12.036.
ducing fluorine into the lysine side chain, Org. Biomol. Chem.
1 (2003)
3527e3534.
ꢀ
[23] L.C. Menikarachchi, J.A. Gascon, QM/MM approaches in medicinal chemistry
research, Curr. Top. Med. Chem. 10 (2010) 46e54.
References
[24] R.O. Hynes, Integrins: bidirectional, allosteric signaling machines, Cell 110
(2002) 673e687.
[25] J. Gao, P. Amara, C. Alhambra, M.J. Field, A generalized hybrid orbital (GHO)
method for the treatment of boundary atoms in combined QM/MM calcula-
tions, J. Phys. Chem. A 102 (1998) 4714e4721.
[26] M.D. Eldridge, C.W. Murray, T.R. Auton, G.V. Paolini, R.P. Mee, Empirical
scoring functions: I. The development of a fast empirical scoring function to
estimate the binding affinity of ligands in receptor complexes, J. Comput.
Aided Mol. Des. 11 (1997) 425e445.
[1] R. Medzhitov, Inflammation 2010: new adventures of an old flame, Cell 140
(2010) 771e776.
[2] R. Medzhitov, Origin and physiological roles of inflammation, Nature 454
(2008) 428e435.
[3] C. Nathan, Points of control in inflammation, Nature 420 (2002) 846e852.
[4] R.M. Clancy, A.R. Amin, S.B. Abramson, The role of nitric oxide in inflammation
and immunity, Arthritis Rheum. 41 (1998) 1141e1151.
[5] C. Bogdan, Nitric oxide and the immune response, Nat. Immunol. 2 (2001)
907e916.