Novel 2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamides as antioxidant and/or anti-inflammatory compounds

Article abstract of DOI:10.1016/j.ejmech.2013.06.005

A series of novel N-(4-aryl-1,3-thiazol-2-yl)-2-(2,4-dioxo-1,3-thiazolidin- 5-yl)acetamides (4a-k) and N-(1,3-benzothiazol-2-yl)-2-(2,4-dioxo-1,3- thiazolidin-5-yl)acetamide derivatives (4l-o) are synthesized and evaluated for their anti-inflammatory and antioxidant activity (DPPH radical scavenging, superoxide anion scavenging, lipid peroxide inhibition, erythrocyte hemolytic inhibition). Compounds 4k and 4l have exhibited good antioxidant activity in four assays, while compounds 4c, 4d , 4m, 4n and 4o have shown good DPPH radical scavenging efficacy. Compounds 4a, 4h , 4i, 4k, 4m and 4n have possessed excellent anti-inflammatory activity. N-[4-(o-methoxyphenyl)-1,3-thiazol-2-yl]- 2-(2,4-dioxo-1,3-thiazolidin-5-yl) acetamide (4k) and N-(6-nitro-/methoxy-1,3- benzothiazol-2-yl)-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide (4m and 4n) have exhibited both antioxidant and anti-inflammatory activities.

Full text of DOI:10.1016/j.ejmech.2013.06.005

Accepted Manuscript
Novel 2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamides as antioxidant and/or anti-
inflammatory compounds
Satish Koppireddi, Jayaram Reddy Komsani, Sreenivas Avula, Sujitha Pombala,
Satishbabu Vasamsetti, Srigiridhar Kotamraju, Rambabu Yadla
PII:
S0223-5234(13)00370-X
10.1016/j.ejmech.2013.06.005
EJMECH 6230
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 April 2013
Revised Date: 20 May 2013
Accepted Date: 2 June 2013
Please cite this article as: S. Koppireddi, J.R. Komsani, S. Avula, S. Pombala, S. Vasamsetti, S.
Kotamraju, R. Yadla, Novel 2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamides as antioxidant and/or
anti-inflammatory compounds, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.06.005.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
O
O
NH
O
S
NH
O
S
O
O
N
N
R
NH
NH
S
S
R
4
a-k
4
l-o
A new series of N-thiazolyl-thiazolidinedione-2-acetamides
are synthesized and evaluated for DPPH radical scavenging,
SASA, LPI, EHI, IL-1ß and MCP-1 secretion inhibition
activities. Some of them have exhibited good antioxidant
and anti-inflammatory potential.
Anti-inflammatory activity
IC (µM) values
5
0
R
IL-1β MCP-1
4
4
4
h o-Me 7.6
m 6-NO2 6.8
n 6-OMe 7.0
18.7
20.2
39.6

ACCEPTED MANUSCRIPT
Novel 2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamides as antioxidant and/or anti-
inflammatory compounds
a
a
a
b
Satish Koppireddi , Jayaram Reddy Komsani , Sreenivas Avula , Sujitha Pombala , Satishbabu
b
b
a,*
Vasamsetti , Srigiridhar Kotamraju , Rambabu Yadla
a
Fluoroorganics Division, CSIR-Indian Institute of Chemical Technology, Hyderabad-500 607,
b
India; Chemical Biology Division, CSIR- Indian Institute of Chemical Technology, Hyderabad.
*
Corresponding author. Tel.: +91-40-27193171; Fax: +91-40-27193185.
E-mail address: ryadla@yahoo.com (Rambabu Yadla).
ABSTRACT
____________________________________________________________________________
_
A series of novel N-(4-aryl-1,3-thiazol-2-yl)-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamides (4a-k)
and N-(1,3-benzothiazol-2-yl)-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide derivatives (4l-o) are
synthesized and evaluated for their anti-inflammatory and antioxidant activity (DPPH radical
scavenging, superoxide anion scavenging, lipid peroxide inhibition, erythrocyte hemolytic
inhibition). The structure of the compound 4j is confirmed by X-ray crystallography.
Compounds 4k and 4l have exhibited good antioxidant activity in four assays, while compounds
4
4
1
c, 4d, 4m, 4n and 4o have shown good DPPH radical scavenging efficacy. Compounds 4a, 4h,
i, 4k, 4m and 4n have possessed excellent anti-inflammatory activity. N-[4-(o-Methoxyphenyl)-
,3-thiazol-2-yl]-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide (4k) and N-(6-nitro-/methoxy-1,3-
benzothiazol-2-yl)-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide (4m and 4n) have exhibited both
antioxidant and anti-inflammatory activities.
_
_____________________________________________________________________________
Keywords:
Anti-inflammatory activity; Lipid peroxidation; Erythrocyte hemolysis;
Superoxide anion and DPPH radical scavenging;
N-(1,3-Benzothiazol-2-yl)-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide;
N-(4-Aryl-1,3-thiazol-2-yl)-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide.
_
_____________________________________________________________________________
1

ACCEPTED MANUSCRIPT
1
. Introduction
Antioxidant therapies are gaining importance due to their ability to retard disease
progression by reducing the damage caused by free radical oxidative stress in a patient [1].
Physiological levels of reactive oxygen species (ROS) play vital role as signaling molecules to
mediate numerous biological functions causing alterations in cell growth, gene expression and
host defense [2]. Under inflammatory conditions, presence of excess reactive oxygen species
.
-
.
.
-
(
O , OH, H O NO , ONOO ) can initiate damage to nucleic acids, proteins, carbohydrates and
2 2 2,
lipids in many types of cells including macrophages [3]. Increased oxidative stress induced
production of ROS, overwhelming the antioxidant defense system, has been implicated in the
pathogenesis of various disorders including atherosclerosis [4,5], cancer [6,7], asthma [8],
rheumatoid arthritis [9], ischemia-reperfusion injury [10], neurodegenerative diseases [11,12],
inflammation [13], myocardial infarction and also aging [14,15].
Inflammation, on the other hand, is a multifactorial process. In response to infection
stimulus, monocytes/ macrophage lineage cells activate and generate an inflammatory
environment by secreting many pro-inflammatory cytokines. Among the cytokines, IL-1β is a
multipotent, proinflammatory and procoagulant cytokine affecting most cell types causing
alzheimer’s [16], rheumatoid arthritis [17], atherosclerosis [18], diabetes, etc. The onset of
diabetes is believed to open a gateway to several other complications associated with increased
oxidative stress and elevated inflammatory processes. Monocyte chemoattractant protein-1
(MCP-1) is a key regulator of monocyte recruitment to sites of inflammation and plays an
important role in initiating local inflammation at the damaged site. MCP-1 levels are known to
be elevated in various disorders including cardiovascular and neurodegeneration [19, 20]. In
addition, MCP-1 induces several proinflammatory changes including secretion of cytokines and
expression of adhesion molecules like intracellular adhesion molecule (ICAM), Vascular cell
adhesion molecule (VCAM) etc., which in turn facilitates the binding of immune cells to
endothelial cells and may promote an inflammatory environment within the vasculature [19]. It
is desirable to develop small molecules containing two or three biologically active scaffolds
possessing good antioxidant, anti-inflammatory and/or anti-diabetic activities [21,22].
Anti-diabetic drugs and drug candidates such as ciglitazones [23,24], englitazone [25],
pioglitazone [26], rosiglitazone [27] and KRP-297 [28] contain thiazolidinedione scaffold. These
compounds have been reported to be acting as ligands for peroxisome proliferator-activated
2

ACCEPTED MANUSCRIPT
receptors (PPAR) [29]. Many compounds belonging to this class are believed to be potential
anti-inflammatory agents [30]. Pyrazolyl-2,4-thiazolidinediones are reported to exhibit anti-
inflammatory activity [31]. However, the exact mechanism of their action is not completely
understood. Similarly, thiazole ring has been identified as a central feature of myriad natural
products and their synthetic analogues have been pursued by medicinal chemistry researchers
[
32]. 2-Amino-1,3-thiazole based amide compounds have been reported as potential radical
scavengers [33] and P53 inactivators [34]. 2-Amino-5-methyl-1,3-thiazole scaffold is present as
an amide in the nonsteroidal anti-inflammatory drug, meloxicam [35]. By incorporating both
these pharmacophores into a single molecule, we intend to synthesize a new series of 2-(2,4-
dioxo-1,3-thiazolidin-5-yl)acetamides of 2-aminothiazoles and 2-aminobenzimidazoles having
antioxidant and/or anti-inflammatory properties.
2
Results and Discussion
2
.1. Chemistry
2
-(2,4-Dioxo-1,3-thiazolidin-5-yl)acetic acid (1) is obtained in good yield by refluxing
maleic anhydride and thiourea in concentrated hydrochloric acid for 5 h as reported earlier [36].
Iodine catalyzed condensation-cyclisation of thiourea with the respective acetophenone (2a-k) at
o
1
00 C for 8 h [37] has resulted in the formation of the corresponding 4-aryl-1,3-thiazol-2-amine
(3a-k) in good yield. The target molecules viz., N-(4-aryl-1,3-thiazol-2-yl)-2-(2,4-dioxo-1,3-
thiazolidin-5-yl)acetamides 4a-k are synthesized in 65-71% yield by treating a dry
dichloromethane (DCM) solution of 2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetic acid (1), N-
hydroxybenzotriazole (HOBT) and O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU) with the corresponding 4-aryl-1,3-thiazol-2-amine (3a-k) and
N,N-diisopropylethylamine (DIEA) in dry dimethylformamide (DMF) under stirring for 12 h. In
a similar reaction, 2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetic acid (1) is initially activated with
HOBT and HBTU in dry DCM and then reacted in situ with commercially available 2-
aminobenzothiazoles 3l-o in dry DMF in presence of DIEA for 12 h to obtain N-(1,3-
benzothiazol-2-yl)-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide derivatives 4l-o in 54-61% yield.
The detailed reaction sequence is depicted in Scheme 1. The thiazolidinedioneacetic acid and
thiazolamine/ benzothiazolamine scaffolds present in the target molecules 4a-o are attached
through an amide linkage.
3

ACCEPTED MANUSCRIPT
>
Scheme 1 <
The newly synthesized 2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide derivatives 4a-o are well
1
13
characterized by IR, H NMR, C NMR and Mass spectroscopy including HRMS. The IR
-1
spectra of these compounds consisted of absorption bands ranging from 1660 - 1760 cm due to
-
1
secondary amide C=O stretching, 3140 - 3450 cm of N-H stretching of amide group and at 3040
-1
-
3130 cm due to the aromatic C-H stretching frequency. The presence of C-S bond stretching
-1
1
absorption is seen in the range 624 - 757 cm . The H NMR spectra of all the compounds
showed signals for aromatic protons between δ 7.0 - 8.7 ppm. The two N-H protons appeared as
two singlets around δ 11.5 and 12.5 ppm and are D O exchangeable. The thiazole ring proton
2
signal appeared as a singlet at δ 6.9 - 7.9 ppm. Interestingly, the active methylene protons of
acetamide compounds 4a-o are diastereotropic and their signals appeared as ABX pattern. The
CHAHBHX pattern for all the thiazolidinedioneacetic acid derivatives is seen as doublet of
doublet around δ 3.0 - 3.5 ppm and δ 4.6 ppm with coupling constants, JAB = 16.8 - 17.9 H , JAX
Z
=
7.9 - 10.1 H , J = 3.1 - 4.1 H . High coupling constant of J compromises with the data of
Z BX Z AB
Takahashi [38]. The molecular formula and elemental composition are confirmed by high
resolution mass spectral analysis. The structural assignment of these compounds is further
1
3
supported by C NMR data. The physical characteristics of individual compounds, 4a-o is
presented in Table 1.
>
Table 1 <
2
.2. Crystal structure of compound 4j
>
Fig. 1 <
In addition to the spectral characterization, a single crystal of compound 4j is obtained from
ethyl acetate/hexane (80:20). The crystal structure of compound 4j has been determined by X-ray
crystallography. The ORTEP view of compound 4j is displayed in Fig. 1. The crystal data and
4

ACCEPTED MANUSCRIPT
structural refinement parameters are presented in Table 2, while selected bond lengths and bond
angles are listed in Table 3.
>
Table 2 <
>
Table 3 <
2
.3. Biology
2
.3.1. Antioxidant activities
The antioxidant efficacy of the newly synthesized thiazolidinedione derivatives is
assayed by applying DPPH method. Superoxide anion scavenging activity (SASA), inhibition of
lipid peroxidation (LPI) and hemolysis in erythrocyte membrane stabilization or erythrocyte
hemolysis inhibition (EHI) assays are performed only for selected compounds showing good
radical scavenging activity in DPPH method. The sample solutions are prepared at
concentrations of 60, 80, 100 µg/mL and their EC values are obtained. Ascorbic acid and
50
luteolin are employed as standards in these assays. The results of the antioxidant activities of
these compounds are shown in Table 3. The thiazolidinedione-5-acetamides of 4-(p-
fluorophenyl)-thiazol-2-amine
(4c),
4-(p-bromophenyl)-thiazol-2-amine
(4d),
4-(o-
methoxyphenyl)-thiazol-2-amine (4k) and all the benzimidazol-2-amine derivatives (4l-o) have
exhibited good DPPH radical scavenging potential with EC values ranging from 20 - 60 µM as
5
0
compared to ascorbic acid (40.28 µM) and luteolin (44.18 µM) as positive controls. Compounds
k and 4l have also shown good LPI and EHI along with moderate SASA. Among the structural
4
features, the presence of benzothiazole moiety (4l-o) and/or NO or OMe substituent at suitable
2
position as seen in 4h, 4k, 4m and 4n seem to be desirable.
>
Table 4 <
2
.3.2. Anti-inflammatory activity
The thiazolidinedione derivatives 4a-o along with pioglitazone are assayed for their anti-
inflammatory potential in the presence of PMA (phorbol 13-myristate 12-acetate)-induced
5

ACCEPTED MANUSCRIPT
inflammation using THP1 monocyte cell system (Table 5). This method is conventional,
sensitive and well accepted for screening of newer anti-inflammatory agents. Compounds 4b,
4
d, 4f, 4g with IC values of 16.31 µM, 33.12 µM, 18.23 µM and 23.4 µM respectively, showed
50
reasonable anti-inflammatory activities (Table 6). Compounds 4a and 4i showed good activity
with IC50 values of 9.6 µM and 11.72 µM. The thiazolidine-2,4-dione-5-acetamides of 4-(o-
tolyl)-thiazol-2-amine (4h), 4-(o-methoxyphenyl)-thiazol-2-amine (4k), 6-nitro-benzimidazol-2-
amine (4m) and 6-methoxy-benzimidazol-2-amine (4n) have shown excellent IL-1β inhibitory
activity with IC values between 6.83 µM - 7.66 µM with respect to piroxicam (21.33 µM) as
5
0
standard. However, compounds 4c, 4e, 4j, 4l, 4o and pioglitazone (Table 6) did not show
appreciable activity. In terms of MCP-1 inhibition, compounds 4a, 4d, 4e, 4g, 4n and 4o with
IC values of 30.4 µM, 49.13 µM, 31.7 µM, 34.24 µM, 39.6 µM and 63.06 µM, respectively
5
0
showed reasonable activities against MCP-1 (Table 4). Compounds 4c, 4h, 4i, 4j, 4l and 4m
have shown good MCP-1 inhibition activity with IC values between 16.68 µM – 20.29 µM
5
0
which are well comparable to pioglitazone with IC50 of 18.84 µM. MCP-1 levels are
significantly attenuated in pioglitazone treatment animals compared to placebo group in stent
implantation animals [39]. However, neither pioglitazone nor the thiazolidinedione compounds
have shown activity on PMA induced TNF-α production. This result corresponds with the report
of Thieringer, et al., which says that PPAR agonists do not inhibit TNF-α production [40].
>
>
Table 5 <
Table 6 <
3
. Conclusion
We have reported here a simple synthesis of a collection of novel 2,4-thiazolidinedione-
-acetamides of 4-aryl-1,3-thiazol-2-amines and 6-substituted-benzothiazol-2-amines. These
5
compounds are screened in vitro for their DPPH radical scavenging activity along with IL-1β and
MCP-1 secretion inhibition efficacy. The compounds having DPPH radical scavenging potential
are further assayed for their superoxide anion scavenging, lipid peroxidation inhibition and
6

ACCEPTED MANUSCRIPT
erythrocyte hemolysis inhibition activities with reasonable success. Some of the compounds
having methoxy- or nitro- substituent, especially on benzothiazole moiety have exhibited very
good anti-inflammatory activity while showing good antioxidant potential. Further research on
such molecules embedded with two biologically active scaffolds and exhibiting multiple
activities may be useful in development of therapeutic applications where free radical induced
inflammation plays a role in diseases.
4
. Experimental Protocols
4
.1. Synthetic Chemistry
All the reagents and solvents are purchased from commercial suppliers. Solvents are
distilled and dried before use. Melting points are determined on the Veego (VMP-MP) melting
point apparatus and are uncorrected. Merck 60 F254 silica gel coated glass sheets are used for thin
layer chromatography. The compounds are purified by using column chromatography on silica
gel (60-120 mesh) with ethyl acetate/ petroleum ether (1:2) as eluent. Infrared spectra are
1
13
recorded on Perkin-Elmer FT-IR 1600 spectrometer. H NMR and C NMR spectra are recorded
on Bruker Avance 300 MHz and Bruker Inova 400 MHz spectrometer with TMS as internal
standard. Chemical shifts (δ) are given in parts per million and coupling constants are given as
absolute values expressed in Hertz. Mass spectral analysis using electrospray ionization (ESI)
and high resolution mass spectrometry (HRMS) experiments are performed using a quadrupole
time-of-flight mass spectrometer (QSTAR XL, Applied Biosystems/MDS Sciex, Foster City,
CA, USA), equipped with an ESI source.
4
.1.1. Synthesis of 2, 4-dioxo-1,3-thiazolidine-5-acetic acid (1).
A mixture of thiourea (10 g,
1
31 mmol) and of maleic anhydride (12.8 g, 131 mmol) in 33 mL of concentrated hydrochloric
acid is refluxed for 5 h and cooled to room temperature. The precipitate is separated by filtration,
washed with cold water (5 mL) and crystallized from hot water to yield compound 1. Yield 70%;
0
1
mp 167-169 C; H NMR (DMSO-d , 300 MHz) δ (ppm): 2.82-2.91 (dd, J = 17.7, 9.2 Hz, 1H,
6
CH ), 3.05-3.12 (dd, J = 17.5, 3.7 Hz, 1H, CH ), 4.43-4.48 (m, 1H, CH), 11.95 (s, 1H, NH),
2
2
1
2.60 (s, 1H, COOH).
4
.1.2. General procedure for synthesis of 2-amino-4-(substituted-phenyl)-1,3-thiazoles (3a-k).
7

ACCEPTED MANUSCRIPT
A mixture of thiourea (50 mmol), the corresponding acetophenone (25 mmol) and iodine
0
(
25 mmol) is stirred at 100 C for 8 h. Then the reaction mixture is cooled, extracted with diethyl
ether to remove excess of acetophenone, and then washed with aqueous sodium thiosulfate to
remove excess iodine and later with cold water. The crude product is dissolved in hot water,
filtered to remove sulphone, and the filtrate is basified with aqueous Na CO to yield the
2
3
corresponding 2-amino-4-(substituted-phenyl)-1,3-thiazole. The crude product is purified by
recrystallization from alcohol.
o
4
.1.2.1. 4-Phenyl-1,3-thiazol-2-amine (3a). Yield 70%; mp 146-148 C (lit [41] mp 146-148
o
1
C); H NMR (DMSO-d , 300 MHz) δ (ppm): 5.13 (br s, 2H, NH , D O exchangeable), 6.66 (s,
6
2
2
1
H, thiazole-H), 7.23-7.35 (m, 3H, Ar-H), 7.70-7.73 (m, 2H, Ar-H); ESI-MS(positive): 177.0
+
[
M+H] .
o
4
.1.2.2. 4-(p-Chlorophenyl)-1,3-thiazol-2-amine (3b).
Yield 68%; mp 164-166 C (lit [37]
o
1
mp 163-164 C); H NMR (DMSO-d , 300 MHz) δ (ppm): 6.23 (br s, 2H, NH , D O
6
2
2
exchangeable), 6.70 (s, 1H, thiazole-H), 7.30 (d, J = 8.4 Hz, 2H, Ar-H), 7. 71 (d, J = 8.4 Hz, 2H,
+
Ar-H); ESI-MS(positive): 211.0 [M+H] .
o
4
.1.2.3. 4-(p-Fluorophenyl)-1,3-thiazol-2-amine (3c). Yield 69%; mp 108-110 C (lit [42] mp
o
1
1
06 C); H NMR (DMSO-d , 300 MHz) δ (ppm): 5.94 (br s, 2H, NH , D O exchangeable), 6.60
6 2 2
(s, 1H, thiazole-H), 7.01-7.07 (m, 2H, Ar-H), 7.72-7.77 (m, 2H, Ar-H); ESI-MS(positive): 195.0
+
[
M+H] .
o
4
.1.2.4. 4-(p-Bromophenyl)-1,3-thiazol-2-amine (3d).
Yield 65%; mp 178-180 C (lit [37]
o
1
mp 180-181 C); H NMR (DMSO-d , 300 MHz) δ (ppm): 6.32 (br s, 2H, NH , D O
6
2
2
exchangeable), 6.73 (s, 1H, thiazole-H), 7.45 (d, J = 8.4 Hz, 2H, Ar-H), 7. 65 (d, J = 8.4 Hz, 2H,
+
Ar-H); ESI-MS(positive): 256.0 [M+H] .
o
4
.1.2.5. 4-(p-Iodophenyl)-1,3-thiazol-2-amine (3e).
Yield 65%; mp 190-192 C (lit [37] mp
o
1
1
76-177 C); H NMR (DMSO-d , 300 MHz) δ (ppm): 6.85 (s, 1H, thiazole-H), 7.44 (d, J = 8.4
6
+
Hz, 2H, Ar-H), 7.74 (d, J = 8.4 Hz, 2H, Ar-H); ESI-MS(positive): 303.0 [M+H] .
o
4
.1.2.6. 4-(o-Chlorophenyl)-1,3-thiazol-2-amine (3f). Yield 66%; mp 142-144 C (lit [43] mp
o
1
1
26 C); H NMR (DMSO-d , 300 MHz) δ (ppm): 5.15 (br s, 2H, NH , D O exchangeable), 7.03
6 2 2
8

ACCEPTED MANUSCRIPT
(s, 1H, thiazole-H), 7.20-7.30 (m, 2H, Ar-H), 7.41 (d, J = 7.9 Hz, 1H, Ar-H), 7. 81 (d, J = 6.9
+
Hz, 1H, Ar-H); ESI-MS (positive): 211.0 [M+H] .
o
4
.1.2.7. 4-(m-Chlorophenyl)-1,3-thiazol-2-amine (3g).
Yield 65%; mp 126-128 C (lit [43]
o
1
mp 112 C); H NMR (DMSO-d , 300 MHz) δ (ppm): 5.70 (s, 2H, NH , D O exchangeable),
6
2
2
6
.73 (s, 1H, thiazole-H), 7.24-7.37 (m, 2H, Ar-H), 7.63 (d, J = 7.3 Hz, 1H, Ar-H), 7.78 (s, 1H,
+
Ar-H); ESI-MS(positive): 211.0 [M+H] .
o
4
.1.2.8. 4-(o-Tolyl)-1,3-thiazol-2-amine (3h). Yield 68%; mp 81-83 C (lit [37] mp 81-82
o
1
C); H NMR (DMSO-d , 300 MHz) δ (ppm): 2.43 (s, 3H, CH ) 5.14 (br s, 2H, NH , D O
6
3
2
2
exchangeable), 6.45 (s, 1H, thiazole-H), 7.18-7.25 (m, 3H, Ar-H), 7.50-7.53 (m, 1H, Ar-H);
+
ESI-MS(positive): 191.0 [M+H] .
o
4
.1.2.9. 4-(m-Tolyl)-1,3-thiazol-2-amine (3i).
Yield 67%; mp 86-88 C (lit [37] mp 79-92
o
1
C); H NMR (DMSO-d , 300 MHz) δ (ppm): 2.41 (s, 3H, CH ) 6.86 (s, 1H, thiazole-H), 7.24 (d,
6
3
+
J = 7.3 Hz, 1H, Ar-H), 7.32-7.45 (m, 3H, Ar-H); ESI-MS(positive): 191.0 [M+H] .
o
4
.1.2.10. 4-(o-Fluorophenyl)-1,3-thiazol-2-amine (3j).
Yield 66%; mp 98-100 C (lit [44]
o
1
mp 95-96 C); H NMR (DMSO-d , 300 MHz) δ (ppm): 6.25 (s, 2H, NH , D O exchangeable)
6
2
2
6
1
.92 (s, 1H, thiazole-H), 7.06-7.22 (m, 3H, Ar-H), 8.0-8.20 (m, 1H, Ar-H); ESI-MS(positive):
+
95.0 [M+H] .
o
4
.1.2.11. 4-(o-Methoxyphenyl)-1,3-thiazol-2-amine (3k). Yield 58%; mp 180-182 C (lit [45]
1
liquid); H NMR (DMSO-d , 300 MHz) δ (ppm): 3.98 (s, 3H, CH ), 6.96 (s, 1H, thiazole-H),
6
3
7
2
.03-7.07 (m, 2H, Ar-H), 7.39-7.44 (m, 1H, Ar-H), 7.60-7.64 (m, 1H, Ar-H); ESI-MS(positive):
+
07.0 [M+H] .
4
.1.3. General procedure for the synthesis of N-(4-aryl-1,3-thiazol-2-yl)-2-(2,4-dioxo-1,3-
thiazolidin-5-yl)acetamides (4a-k) and N-(1,3-benzothiazol-2-yl)-2-(2,4-dioxo-1,3-thiazolidin-
5
-yl)acetamide derivatives (4l-o).
The general protocol followed by us in obtaining 2-(2,4-dioxo-1,3-thiazolidin-5-
yl)acetamide compounds 4a-o is illustrated for the synthesis of N-(4-phenyl-1,3-thiazol-2-yl)-2-
(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide (4a) as an example. A solution of compound 1 (0.4 g,
o
2
.55 mmol) in DCM (10 mL) is cooled to 0 C and then charged with HOBT (0.36 g, 2.4 mmol),
o
followed by HBTU (0.90 g, 2.4 mmol). The reaction mixture is stirred at 0 C for 45 minutes.
9

ACCEPTED MANUSCRIPT
After that, a solution of 4-phenylthiazol-2-amine, 3a (0.29 g, 1.70 mmol) and DIEA (0.8 mL, 5.1
mmol) in a mixture of DCM (5 mL) and DMF (2.5 mL) is added drop wise over 5 minutes. The
o
reaction temperature is initially maintained at 0 C for 1 h and later at RT for 10 h. Completion of
reaction is evidenced by TLC analysis. After evaporating the DCM solvent on rotavapor, the
reaction mixture is diluted with 40 mL of distilled water and extracted with (3 x 15 mL) of ethyl
acetate. The combined organic layer is washed with saturated aqueous sodium bicarbonate
solution (2 x 10 mL), followed by saturated aqueous sodium chloride solution (2 x 20 mL). After
drying over anhydrous sodium sulfate, the organic layer is filtered and the filtrate is stripped off
the solvent. The crude product thus obtained is purified by column chromatography over silica
gel. The rest of the 2-(thiazolidinedion-5-yl)acetamide derivatives (4b-o) are prepared similarly
by reacting 2,4-dioxo-1,3-thiazolidine-5-acetic acid (1) with the appropriate amine (3b-o).
4
.1.3.1. N-(4-Phenyl-1,3-thiazol-2-yl)-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide ( 4a).
-1
IR (KBr) cm : 3340, 3170 (NH), 3040 (Ar-H), 1750, 1692 (C=O), 1556 (C=C), 1407 (C-N),
1
1
1
060; H NMR (CDCl + DMSO-d , 300 MHz) δ (ppm): 3.06-3.14 (m, 1H, CH ), 3.38 (dd, J =
3
6
2
7.1, 3.5 Hz, 1H, CH ), 4.56 (dd, J = 9.2, 3.5 Hz, 1H, CH), 7.20 (s, 1H, thiazole-H), 7.23-7.38
2
1
3
(
m, 3H, Ar-H), 7.78 (d, J = 7.1 Hz, 2H, Ar-H); C NMR (CDCl + DMSO-d , 75 MHz) δ (ppm):
3 6
3
1
7.5 (CH ), 45.8 (CH), 106.7 (thiazole-C-5), 125.1, 127.0, 127.8, 133.7 (aromatic carbons),
2
49.0 (thiazole-C-4), 157.0 (thiazole-C-2), 167.0 (C=O), 171.4 (C=O), 174.0(C=O).
+
HRMS-ESI m/z [M+H] calcd. for C H O N S : 334.0320; found: 334.0312.
1
4
12
3
3 2
4
.1.3.2. N-[4-(p-Chlorophenyl)-1,3-thiazol-2yl]-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide
-1
(
4b). IR (KBr) cm : 3379, 3136 (NH), 3042 (Ar-H), 1747, 1673 (C=O), 1154, 1075, 747 (C-
1
S-C); H NMR (CDCl + DMSO-d , 300 MHz) δ (ppm): 3.15 (dd, J = 17.1, 8.6 Hz, 1H, CH ),
3
6
2
3
.38-3.40 (m, 1H, CH ), 4.77 (dd, J = 8.6, 3.7 Hz, 1H, CH), 7.47 (d, J = 8.4 Hz, 2H, Ar-H), 7.72
2
1
3
(
s, 1H, thiazole-H), 7.89 (d, J = 8.4 Hz, 2H, Ar-H); C NMR (CDCl + DMSO-d , 75 MHz) δ
3 6
(
ppm): 37.1 (CH ), 45.5 (CH), 107.0 (thiazole-C-5), 126.2, 127.6, 132.1 (aromatic carbons),
2
1
47.0 (thiazole-C-4), 156.8 (thiazole-C-2), 166.8 (C=O), 171.1(C=O), 174.4(C=O). HRMS-ESI
+
m/z [M+H] calcd. for C H O N ClS : 367.9930; found: 367.9929.
1
4
11
3
3
2
4
.1.3.3.
N-[4-(p-Fluorophenyl)-1,3-thiazol-2-yl]-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide
-1
(
4c). IR (KBr) cm : 3240, 3162 (NH), 3041 (Ar-H), 1760, 1680 (C=O), 1560 (C=C), 1410 (C-
1
N), 1090 (C-N); H NMR (CDCl + DMSO-d , 300 MHz) δ (ppm): 3.15-3.23 (m, 1H, CH ), 3.37
3
6
2
1
0

ACCEPTED MANUSCRIPT
(
dd, J = 17.7, 3.7 Hz, 1H, CH ), 4.59-4.62 (m, 1H, CH), 7.06-7.11 (m, 2H, Ar-H), 7.23 (s, 1H,
2
thiazole-H), 7.81-7.85 (m, 2H, Ar-H), 11.96 (br s, 1H, NH, D O exchangeable), 12.37 (br s, 1H,
2
1
3
NH, D O exchangeable); C NMR (CDCl + DMSO-d , 75 MHz) δ (ppm): 35.5 (CH ), 44.5
2
3
6
2
(CH), 105.8 (thiazole-C-5), 113.4, 113.7, 125.7, 129.0 (aromatic carbons), 146.2 (thiazole-C-4),
+
1
55.7 (thiazole-C-2), 166.2 (C=O), 170.4 (C=O), 173.7 (C=O). HRMS-ESI m/z [M+H] calcd.
for C H O N FS : 352.0226; found: 352.0220.
1
4
11
3
3
2
4
.1.3.4.
N-[4-(p-Bromophenyl)-1,3-thiazol-2yl]-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide
-1
(
4d). IR (KBr) cm : 3371, 3140 (NH), 3043 (Ar-H), 1748, 1677 (C=O), 1155, 1065, 600 (C-
1
S-C); H NMR (CDCl + DMSO-d , 300 MHz) δ (ppm): 3.14-3.18 (m, 1H, CH ), 3.36 (dd, J =
3
6
2
1
8
1
7.1, 3.5 Hz, 1H, CH ), 4.58 (dd, J = 8.6, 3.0 Hz, 1H, CH), 7.34 (s, 1H, thiazole-H), 7.49 (d, J =
2
.3 Hz, 2H, Ar-H), 7.73 (d, J = 8.4 Hz, 2H, Ar-H), 11.95 (br s, 1H, NH, D O exchangeable),
2
1
3
2.39 (br s, 1H, NH, D O exchangeable); C NMR (CDCl + DMSO-d , 75 MHz) δ (ppm): 36.3
2
3
6
(
(
[
CH ), 45.0 (CH), 106.9 (thiazole-C-5), 119.8, 126.1, 130.1, 132.1 (aromatic carbons), 146.8
2
thiazole-C-4), 156.3(thiazole-C-2), 166.4 (C=O), 170.7 (C=O), 174.0 (C=O). HRMS-ESI m/z
+
M+H] calcd. for C H O N BrS : 411.9425; found: 411.9425.
1
4
11
3
3
2
4
.1.3.5. N-[4-(p-Iodophenyl)-1,3-thiazol-2-yl]-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide (4e).
-1
1
IR (KBr) cm : 3200, 3112 (NH), 3040 (Ar-H), 1747, 1680 (C=O), 1159, 1065, 625 (C-S-C); H
NMR (CDCl + DMSO-d , 300 MHz) δ (ppm): 3.05 (dd, J = 17.1, 9.6 Hz, 1H, CH ), 3.37 (dd, J
3
6
2
=
17.3, 3.3 Hz, 1H, CH ), 4.55-4.59 (m, 1H, CH), 7.25 (s, 1H, thiazole-H), 7.57 (d, J = 8.3 Hz,
2
1
3
2
3
H, Ar-H), 7.68 (d, J = 8.4 Hz, 2H, Ar-H); C NMR (CDCl + DMSO-d , 75 MHz) δ (ppm):
3 6
6.9 (CH ), 45.4 (CH), 99.2(Ar-C-I), 107.2 (thiazole-C-5), 126.7, 132.9, 136.5 (aromatic
2
+
carbons), 156.8 (thiazole-C-2), 169.9 (C=O), 174.4 (C=O). HRMS-ESI m/z [M+H] calcd. for
C H O N IS : 459.9287; found: 459.9281.
14
11
3
3
2
4
.1.3.6.
N-[4-(o-Chlorophenyl)-1,3-thiazol-2yl]-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide
-1
(
4f). IR (KBr) cm : 3211, 3134 (NH), 3075 (Ar-H), 1756, 1701(C=O), 1138, 1039, 748 (C-S-C);
1
H NMR (CDCl + DMSO-d , 300 MHz) δ (ppm): 3.09 (dd, J = 17.1, 9.2 Hz, 1H, CH ), 3.40
3
6
2
(
dd, J = 17.1, 3.7 Hz, 1H, CH ), 4.61 (dd, J = 9.2, 3.7 Hz, 1H, CH), 7.25-7.37 (m, 2H, Ar-H)
2
1
3
7
7
1
.44-7.47 (m, 2H, Ar-H, thiazole-H), 7.81-7.85 (m, 1H, Ar-H); C NMR (CDCl + DMSO-d ,
3 6
5 MHz) δ (ppm): 37.5 (CH ), 45.8 (CH), 111.9 (thiazole-C-5), 126.0, 128.0, 129.6, 130.3,
2
31.0, 132.6 (aromatic carbons), 145.5 (thiazole-C-4), 156.0 (thiazole-C-2), 167.1 (C=O),
1
1

ACCEPTED MANUSCRIPT
+
1
71.5(C=O), 174.7 (C=O). HRMS-ESI m/z [M+H] calcd. for C H O N ClS : 367.9930;
1
4
11
3
3
2
found: 367.9924.
4
.1.3.7.
N-[4-(m-Chlorophenyl)-1,3-thiazol-2yl]-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide
-1
(
4g). IR (KBr) cm : 3288, 3174 (NH), 3052 (Ar-H), 1753, 1676 (C=O), 1156, 1074, 741 (C-
1
S-C); H NMR (CDCl + DMSO-d , 300 MHz) δ (ppm): 3.16 (dd, J = 17.1, 8.4 Hz, 1H, CH ),
3
6
2
3
.99 (dd, J = 14.1, 6.9 Hz, 1H, CH ), 4.78 (dd, J = 8.6, 3.9 Hz, 1H, CH), 7.37 (d, J = 8.4 Hz, 1H,
2
Ar-H), 7.44 (t, J = 7.7, 7.9 Hz, 1H, Ar-H), 7.82 (s,1H, thiazole-H), 7.85 (d, J = 7.7 Hz, 1H, Ar-
1
3
H), 7.95 (s, 1H, Ar-H); C NMR (CDCl + DMSO-d , 75 MHz) δ (ppm): 37.7 (CH ), 45.9
3
6
2
(
(
[
CH ), 108.0 (thiazole-C-5), 123.3, 125.5, 127.0, 129.4, 133.7, 135.7 (aromatic carbons), 143.0
2
thiazole-C-4), 158.9 (thiazole-C-2), 167.3 (C=O), 171.6 (C=O), 174.8 (C=O). HRMS-ESI m/z
+
M+H] calcd. for C H O N ClS : 367.9930; found: 367.9928.
1
4
11
3
3
2
4
.1.3.8. N-[4-(o-Tolyl)-1,3-thiazol-2-yl]-2-(2,4-dioxo-1,3thiazolidin-5-yl)acetamide (4h).
-1
1
IR (KBr) cm : 3291, 3155 (NH), 3054 (Ar-H), 1739, 1687 (C=O), 1167, 1064, 740 (C-S-C); H
NMR (CDCl + DMSO-d , 300 MHz) δ (ppm): 2.44 (s, 3H, CH ), 3.07(dd, J = 17.5, 9.3 Hz, 1H,
3
6
3
CH ), 3.42 (dd, J = 16.5, 4.1 Hz, 1H, CH ), 4.59-4.61 (m, 1H, CH), 6.92 (s, 1H, thiazole-H),
2
2
1
3
7
.19-7.21 (m, 3H, Ar-H), 7.53 (d, J = 7.2 Hz, 1H, Ar-H); C NMR (CDCl + DMSO-d , 75
3 6
MHz) δ (ppm): 20.1 (CH ), 37.1 (CH ), 45.5 (CH), 109.5 (thiazole-C-5), 124.7, 126.8, 128.5,
3
2
1
1
3
29.8, 133.7, 134.7 (aromatic carbons), 148.8 (thiazole-C-4), 155.9 (thiazole-C-2), 166.7 (C=O),
+
71.2 (C=O), 174.4 (C=O). HRMS-ESI m/z [M+H] calcd. for C H O N S : 348.0477; found:
1
5
14
3
3 2
48.0466.
4
.1.3.9. N-[4-(m-Tolyl)-1,3-thiazol-2-yl]-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide (4i).
-1
1
IR (KBr) cm : 3275, 3154 (NH), 3050 (Ar-H), 1748, 1699 (C=O), 1161, 1091; H NMR
CDCl + DMSO-d , 300 MHz) δ (ppm): 2.39 (s, 3H, CH ), 3.08 (dd, J = 17.1, 9.6 Hz, 1H, CH ),
(
3
6
3
2
3
.44 (dd, J = 17.1, 3.7 Hz, 1H, CH ), 4.58 (dd, J = 9.8, 3.7 Hz, 1H, CH), 7.11 (d, J = 8.1 Hz, 1H,
2
Ar-H), 7.17 (s, 1H, thiazole-H), 7.26 (t, J = 7.5 Hz, 1H, Ar-H), 7.59 (s, 1H, Ar-H), 7.63 (d, J =
1
3
6
4
1
.2 Hz, 1H, Ar-H); C NMR (CDCl + DMSO-d , 75 MHz) δ (ppm): 21.0 (CH ), 36.9 (CH ),
3 6 3 2
6.3 (CH), 108.0 (thiazole-C-5), 122.7, 126.2, 128.4, 128.5, 134.0, 137.7 (aromatic carbons),
48.9 (thiazole-C-4), 157.2 (thiazole-C-2), 168.1 (C=O), 172.4 (C=O), 175.7 (C=O). HRMS-
+
ESI m/z [M+H] calcd. for C H O N S : 348.0477; found: 348.0470.
1
5
14
3
3 2
1
2

ACCEPTED MANUSCRIPT
4
.1.3.10.
N-[4-(o-Fluorophenyl)-1,3-thiazol-2-yl]-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide
-1
(4j).
IR (KBr) cm : 3226, 3153 (NH), 3053 (Ar-H), 1739, 1687 (C=O), 1169, 1062, 739 (C-
1
S-C); H NMR (CDCl + DMSO-d , 300 MHz) δ (ppm): 3.09 (dd, J = 17.1, 9.6 Hz, 1H, CH ),
3
6
2
3
.45 (dd, J = 17.1, 3.5 Hz, 1H, CH ), 4.59 (dd, J = 9.8, 3.5 Hz, 1H, CH), 7.11-7.23 (m, 2H, Ar-
2
H), 7.39 (d, J = 1.8 Hz, 1H, Ar-H),7.56 (s, 1H, thiazole-H), 8.01 (t, J = 7.5, 7.7 Hz, 1H, Ar-H);
1
3
C NMR (CDCl + DMSO-d , 75 MHz) δ (ppm): 37.2 (CH , 45.6 (CH , 108.3 (thiazole-C-5),
3
6
2)
)
1
1
11.1 (d, J = 13.7 Hz), 114.9, 115.2, 123.3, 128.0, (d, J = 8.8 Hz), 128.4 (aromatic carbons),
42.5 (thiazole-C-4), 156.0 (thiazole-C-2), 166.9 (C=O), 171.3 (C=O), 174.6 (C=O). HRMS-
+
ESI m/z [M+H] calcd. for C H O N FS : 352.0226; found: 352.0220.
1
4
11
3
3
2
4
.1.3.11. N-[4-(o-Methoxyphenyl)-1,3-thiazol-2-yl]-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide
-1
(4k).
IR (KBr) cm : 3251, 3184 (NH), 3070 (Ar-H), 1759, 1670 (C=O), 1115, 1056, 750 (C-
1
S-C); H NMR (CDCl + DMSO-d , 400 MHz) δ (ppm): 3.09-3.14 (m, 1H, CH ), 3.45 (dd, J =
3
6
2
1
2
6.8, 3.3 Hz, 1H, CH ), 3.95 (s, 3H, OCH ), 4.59 (dd, J = 10.1, 3.3 Hz, 1H, CH), 6.99-7.02 (m,
2 3
H, Ar-H) 7.26 (t, J = 7.8 Hz, 1H, Ar-H), 7.59 (s, 1H, thiazole-H), 8.08 (d, J = 6.7 Hz, 1H, Ar-
1
3
H) 11.8 (br s, 1H, NH, D O exchangeable), 12.1 (br s, 1H, NH, D O exchangeable); C NMR
2
2
(
CDCl + DMSO-d , 75 MHz) δ (ppm): 36.9 (CH , 46.3 (CH , 55.3(OCH , 111.6 (thiazole-C-
3 6 2) ) 3)
5
2
3
), 111.8, 120.4, 122.4, 128.8, 155.7 (aromatic carbons), 144.7 (thiazole-C-4), 156.5 (thiazole-C-
+
), 168.0 (C=O), 172.5 (C=O), 175.7 (C=O). HRMS-ESI m/z [M+H] calcd. for C H O N S :
1
5
14
4
3 2
64.0426; found 364.0421.
4
.1.3.12. N-(1,3-Benzothiazol-2-yl)-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide (4l).
-1
1
IR (KBr) cm : 3192, 3110 (NH), 2924 (Ar-H), 1742, 1677 (C=O), 1182, 1069, 757 (C-S-C); H
NMR (CDCl + DMSO-d , 300 MHz) δ (ppm): 3.39-3.40 (m, 1H, CH ), 3.44-3.46 (m, 1H, CH ),
3
6
2
2
4
.62-4.67 (m, 1H, CH), 7.23 (t, J = 7.5 Hz, 1H, Ar-H), 7.35-7.40 (m, 1H, Ar-H), 7.68 (d, J = 7.9
+
Hz, 1H, Ar-H) 7.81 (d, J = 7.9 Hz, 1H, Ar-H). HRMS-ESI m/z [M+H] calcd. for C H O N S :
12
10
3
3 2
3
08.0164, found 308.0159.
4
.1.3.13. N-(6-Nitro-1,3-benzothiazol-2-yl)-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide (4m).
-1
1
IR (KBr) cm : 3275, 3193 (NH), 2917 (Ar-H), 1747, 1704 (C=O), 1165, 1044, 749 (C-S-C); H
NMR (CDCl + DMSO-d , 300 MHz) δ (ppm): 3.17 (dd, J = 17.9, 8.9 Hz, 1H, CH ), 3.50 (dd, J
3
6
2
=
17.9, 2.9 Hz, 1H, CH ), 4.62 (dd, J = 8.9, 3.9 Hz, 1H, CH), 7.81 (d, J = 8.9 Hz, 1H, Ar-H),
2
8
.27 (d, J = 8.9 Hz, 1H, Ar-H), 8.77 (s, 1H, Ar-H), 11.96 (brs, 1H, D O exchangeable), 12.84
2
1
3

ACCEPTED MANUSCRIPT
1
3
(
brs, 1H, D O exchangeable); C NMR (CDCl + DMSO-d , 75 MHz) δ (ppm): 37.2 (CH , 46.0
2 3 6 2)
(
CH , 118.8, 120.6, 121.7, 132.1, 143.0, 153.2, 162.8 (benzothiazole), 169.7 (C=O), 172.3
)
+
(
C=O), 175.5 (C=O). HRMS-ESI m/z [M+H] calcd. for C H O N S : 353.0014; found
1
2
9
5
4 2
3
53.0015.
4
.1.3.14. N-(6-Methoxy-1,3-benzothiazol-2-yl)-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide (4n).
-1
1
IR (KBr) cm : 3207, 3094(NH), 3008 (Ar-H), 1738, 1675 (C=O), 1172, 1060, 639 (C-S-C); H
NMR (CDCl + DMSO-d , 400 MHz) δ (ppm): 3.05 (dd, J = 17.1, 9.6 Hz, 1H, CH ), 3.40 (dd, J
3
6
2
=
17.1, 3.5 Hz, 1H, CH ), 3.85 (s, 3H, OCH ), 4.54 (dd, J = 9.4, 3.3 Hz, 1H, CH), 6.92-6.96 (m,
2 3
13
1
H, Ar-H), 7.26 (s, 1H, Ar-H), 7.55 (d, J = 8.6 Hz, 1H, Ar-H); C NMR (CDCl + DMSO-d , 75
3 6
MHz) δ (ppm): 38.8 (CH ), 49.6 (CH), 53.4(OCH ), 102.0, 112.0, 118.0, 130.6, 140.5
2
3
+
(
benzothiazole), 153.9 (C=O), 168.3 (C=O), 188 (C=O). HRMS-ESI m/z [M+H] calcd. for
C H O N S : 338.0269; found 338.0261.
13
12
4
3 2
4
.1.3.15. N-(6-Ethoxy-1,3-benzothiazol-2-yl)-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide (4o).
-1
1
IR (KBr) cm : 3205, 3100 (NH), 2928 (Ar-H), 1743, 1681 (C=O), 1175, 1060, 717 (C-S-C); H
NMR (CDCl + DMSO-d , 300 MHz) δ (ppm): 1.39 (t, J = 6.9 Hz, 3H, CH ), 3.06-3.15 (m, 1H,
3
6
3
CH ), 3.37 (dd, J = 17.1, 3.7 Hz, 1H, CH ), 4.02 (q, J = 6.7, 6.9 Hz, 2H, OCH ), 4.59 (dd, J =
2
2
2
9
.2, 2.8 Hz, 1H, CH), 6.91 (dd, J = 11.1, 2.2 Hz 1H, Ar-H), 7.28 (s, 1H, Ar-H), 7.55 (d, J = 8.8
1
3
Hz, 1H, Ar-H); C NMR (CDCl + DMSO-d , 75 MHz) δ (ppm): 13.21 (CH ), 36.2 (CH ), 44.7
3
6
3
2
(
CH), 62.2(OCH ), 103.0, 113.0, 119.0, 131.6, 141.5, 153.8, 154.1(benzothiazole), 166.7 (C=O),
2
+
1
3
70.5 (C=O), 173.9 (C=O). HRMS-ESI m/z [M+H] calcd. for C H O N S : 352.0426; found
14
14
4
3 2
52.0423.
4
.1.4. X-ray structure analysis
X-ray data for the compound 4j is collected at room temperature using a Bruker Smart
Apex CCD diffractometer with graphite monochromated MoKα radiation (λ=0.71073Å) with ω-
scan method [46]. Preliminary lattice parameters and orientation matrices are obtained from four
sets of frames. Unit cell dimensions are determined using 6121 reflections for 4j data. Integration
and scaling of intensity data are accomplished using SAINT program [46]. The structures are
solved by Direct Methods using SHELXS97 [47] and refinement is carried out by full-matrix
least-squares technique using SHELXL97 [47]. Anisotropic displacement parameters are
included for all non-hydrogen atoms. All H atoms are positioned geometrically and treated as
1
4

ACCEPTED MANUSCRIPT
riding on their parent C atoms, with C-H distances of 0.93--0.97 Å, and with U (H) = 1.2U
iso
eq
(
C) or 1.5U for methyl atoms. CCDC 905873 contains the supplementary crystallographic
eq
data for this paper. These data can be obtained free of charge at
www.ccdc.cam.ac.uk/conts/retrieving.html [or from the Cambridge Crystallographic Data Centre
(CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0) 1223 336 033; email:
deposit@ccdc.cam.ac.uk].
4
4
.2. Biological experiments
.2.1. Experiments in vitro
In the in vitro assays, each experiment is performed at least in triplicate and the standard
deviation of absorbance is less than 10% of the mean.
4
.2.2. DPPH radical scavenging assay
Antioxidant activity of synthesized thiazolidinedione-5-acetamide derivatives is assessed on
the basis of the free radical scavenging effect of the stable 1,1-diphenyl-picrylhydrazyl (DPPH)
following a previously described method [48] with some modifications. The diluted working
solutions of the synthesized compounds are prepared in methanol (5, 10, 20, 40, 60 and 180
ꢀ
g/mL). One mL of methanol solution of DPPH (0.002 %) is mixed with 1 mL solution of test
compound. The mixture is shaken vigorously and left to stand in dark for 30 min. Absorbance of
the resulting solution is measured at 517 nm in a Lambda 25 UV-visible spectrophotometer
(Perkin-Elmer, Shelton, CT, USA). Ascorbic acid and luteolin are used as positive controls. The
radical scavenging ability is measured as a decrease in the absorbance of DPPH. Lower
absorbance of the reaction mixture has indicated higher free radical scavenging activity. DPPH
radical scavenging activity is calculated using the following formula: DPPH radical scavenging
activity (%) = [(absorbance of control – absorbance of test sample)/ (absorbance of control)] x
1
00. Radical scavenging potential is expressed as EC50 value, which represents the test
compound concentration at which 50% of the DPPH radicals are scavenged [49].
4
.2.3. Superoxide radical scavenging assay
The superoxide radical scavenging assay of samples is done according to the protocol
described by Liu, et al., [50].The superoxide radical is generated by phenazine methosulfate -
nicotinamide adenine dinucleotide (PMS/NADH) system, which reduces nitro blue tetrazolium
(NBT) forming a purple coloured formazan. The reaction mixture consisted of 3.0 mL of 16 mM
1
5

ACCEPTED MANUSCRIPT
tris-HCl buffer (pH 8.0) containing 78 mM NADH, 50 µM NBT, 10 µM PMS and various
concentrations (5, 10, 20, 40, 60 and 180 ꢀg/mL) of test compound. After incubation for 5 min at
room temperature the absorbance is read at 560 nm. Ascorbic acid is run in parallel as a positive
control. The scavenging activity of superoxide radical (%) is calculated using the equation:
[
(A560 of control - A560 of sample)/A560 of control] * 100
4
.2.4. Inhibition of lipid peroxidation assay
The inhibition of lipid peroxidation is assayed by measuring malondialdehyde (MDA)
thiobarbituric acid adduct formation using egg yolk as the oxidizable substrate [51]. The yolk is
taken out from an egg and same volume of PBS (0.1 M, pH 7.45) is added to it and stirred
vigorously. The yolk suspension is mixed with 0.5 mL of test sample at various concentrations
(
50, 100, 150 and 300 ꢀg/mL), 0.2 mL of 25 mM FeSO and 1.3 mL of PBS and incubated at 37
4
o
C for 15 min before the reaction is quenched by adding 0.5 mL of 20% trichloroacetic acid and
0
1
mL of 0.8% thiobarbituric acid, and then the mixture is heated at 100 C for 15 min. The
samples are read at 532 nm. Ascorbic acid is used as positive control. The inhibition of lipid
peroxidation is calculated from the following equation:
Inhibition effect (%) = Acontrol – Asample / Acontrol * 100%
4
.2.5. Inhibition of erythrocyte hemolysis assay
The inhibition of erythrocyte hemolysis is measured according to the modified protocol
described by Ng, et al., [52]. The human blood sample is collected in a tube with 3% sodium
citrate. The blood sample is centrifuged at 3,000 g for 10 min to separate erythrocytes from the
plasma and washed with PBS buffer (pH 7.4) and 1.25% erythrocyte suspension is prepared.
Different concentrations of compounds (50, 100, 150 and 300 ꢀg/mL) are added to the mixture
o
containing 1.0 mL erythrocyte suspension and 0.5 mL H O (2.5 mM) and incubated at 37 C for
2
2
o
3
0 min. After incubation, the reaction is terminated by incubating in ice water bath at 4 C and
then centrifuged at 3,000 g for 10 min. The absorbance of the precipitated protein is measured at
40 nm. Ascorbic acid is used as positive control. The inhibition rate of the hemolysis is
calculated using the following equation:
5
Inhibition rate (%) = (AH2O2 – Asample) / (AH2O2 – Anormal) * 100
.2.6. THP1 cell culture
4
1
6

ACCEPTED MANUSCRIPT
THP1 monocytes are obtained from ATCC and grown in RPMI 1640 (Sigma) containing
1
0% FBS (Lonza), non essential amino acids, penicillin (100 U/mL), and streptomycin (100
2
µg/mL) in 75-cm filter vent flasks (Costar), incubated at 37°C in a humidified atmosphere
containing 5% CO and 95% air .
2
4
.2.7. Measurement of cell viability
The effect of synthesized compounds on cell viability is determined by Trypan blue dye
5
exclusion assay. THP1 cells are seeded at a density of 2 x 10 cells/ mL in 24-well plates in
triplicates and are treated with test compounds (10 µM) for 48 h. At the end of the treatments,
cells are harvested and resuspended in 0.4 % Trypan blue (Sigma) and live/dead cells are
counted using cell countess chamber (Invitrogen).
4
.2.8. Enzyme-linked immunosorbent assay for IL-1β and MCP-1 inhibition
To check the inhibitory effects of thiazole derivatives (4a-o) on PMA induced inhibition
5
of inflammation, THP1 monocytes are seeded at a density of 2 x 10 cells/ mL in 24 well plates.
Cells are pre-treated with 5, 10 and 20 µM concentrations of compounds for 1 h before they are
stimulated with 10 ng/mL of PMA. After 48 h of PMA stimulation supernatants are harvested
and assayed for IL-1β , MCP-1 and TNF-α using an enzyme-linked immunosorbent assay
(ELISA) kit following the manufacturer’s instructions (eBiosciences, San Diego, CA, USA). The
absorbance in each well is measured with a microplate reader at 450 nm and corrected at 570 nm.
Concentrations of cytokines released are obtained and the percentage of inhibition of cytokines
production is calculated as compared to control conditions. IC is also obtained as a mean of
5
0
anti-inflammatory potency, expressing the predicted concentration of the correspondent
compound able to reach 50% inhibition. The standard curves are calculated by plotting the
pg/mL concentrations versus absorbance values from the standards, and are used to quantify the
amount of cytokines released by the cells.
Acknowledgments
The authors sincerely thank Jagadeesh Babu Nanubolu (Centre for X-ray Crystallography,
CSIR-IICT) for providing X-ray crystallographic data and Chityal Ganesh Kumar (Medicinal
Chemistry and Pharmacology Division, CSIR-IICT) for his help in obtaining anti-oxidant
1
7

ACCEPTED MANUSCRIPT
activity data. The authors are grateful to the Council of Scientific and Industrial Research
(CSIR), New Delhi, for the financial assistance.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.
References
[
[
[
1] S.R. Maxwell, Drugs 49 (1995) 345- 361.
2] K. Bedard, K.H. Krause, Physiol. Rev. 87 (2007) 245-313.
3] M. Valko, D. Leibfritz, J. Moncol, M.T. Cronin, M. Mazur, J. Telser, Int. J. Biochem.
Cell Biol. 39 (2007) 44-87.
[
[
[
4] M.D. Stringer, P.G. Görög, A. Freeman, V.V. Kakkar, Br. Med. J. 298 (1989) 281-284.
5] R.J. Perry, P. Watson, J.R. Hodges, Neurophychologia 38 (2000) 252-271.
6] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, 3rd Ed,
Oxford University Press, USA, 1999, pp. 192.
[
[
[
7] C.P. Rajneesh, A. Manimaran, K.R. Sasikala, P. Adikappan, Singapore Med. J. 49 (2008)
6
40-643.
8] A.A. Andreadis, S.L. Hazen, S.A. Comhair, S.C. Erzurum, Free Radic. Biol. Med. 35
2003) 213-225.
9] S.B. Abramson, A.R. Amin, R.M. Clancy, M. Attur, Best Pract. Res. Clin. Rheumatol.
5 (2001) 831-845.
(
1
[
[
[
[
[
10] J.M. McCord, New Eng. J. Med. 312 (1985) 159-163.
11] A. Bast, G.R. Haenen, C.J. Doelman, Am. J. Med. 91(suppl.3C) (1991) 2-13.
12] G.B. Bulkley, Surgery 113 (1993) 479-483.
13] N. Sreejayan, M.N. Rao, Arzneimittelforschung 46 (1996) 169-171.
14] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, 2nd Ed,
Oxford University Press, USA, 1989, pp. 416-494.
[
15] T. Finkel, N.J. Holbrook, Nature 408 (2000) 239-247.
1
8

ACCEPTED MANUSCRIPT
[
[
16] H.F. Green, E. Treacy, A.K. Keohane, A.M. Sullivan, G.W. O'Keeffe, Y.M. Nola,
Mol. Cell. Neurosci. 49 (2012) 311-21.
17] D. Lasigliè, E. Traggiai, S. Federici, M. Alessio, A. Buoncompagni, A. Accogli, S.
Chiesa, F. Penco, A. Martini, M. Gattorno, Role of IL-1 beta in the development of
human T(H)17 cells: lesson from NLPR3 mutated patients, PLoS One 6 (2011) e20014.
[
[
[
18] Y. Jiang, M. Wang, K. Huang, Z. Zhang, N. Shao, Y. Zhang, W. Wang, S. Wang,
Biochem. Biophys. Res. Commun. 425 (2012) 121-126.
19] B.C. Tieu, C. Lee, H. Sun, W. Lejeune, A. Recinos, X. Ju, H. Spratt, D.C. Guo,
D. Milewicz, R.G. Tilton, A.R. Brasier, J. Clin. Invest. 119 (2009) 3637-3651.
20] G. Yang, Y. Meng, W. Li, Y. Yong, Z. Fan, H. Ding, Y. Wei, J. Luo, Z.J. Ke,
Brain Pathol. 21 (2011) 279-297.
[
[
21] R.W. Burli, A.F. Haughan, A.J. Hodges, Autoimmunity 43 (2010) 526-538.
22] B.G. Small, B.W. McColl, R. Allmendinger, J. Pahle, G. Lopez-Castejon, N.J. Rothwell,
J. Knowles, P. Mendes, D. Brough, D.B. Kell, Nat. Chem. Biol. 7 (2011) 902-908.
[
[
[
23] T. Yoshioka, T.Fujita, T. Kanai, Y. Aizawa, T. Kurumada, K. Hasegawa, H.
Horikoshi, J. Med. Chem. 32 (1989) 421-428.
24] S.L. Suter, J.J. Nolan, P. Wallace, B. Gumbiner, J.M. Olefsky, Diabetes Care 15 (1992)
1
93-203.
25] D.A. Clark, S.W. Goldstein, R.A. Volkmann, J.F. Eggler, G.F. Holland, B. Hulin,
R.W. Stevenson, D.K. Kreutter, E.M. Gibbs, M.N. Krupp, et al, J. Med. Chem. 34
(
1991) 319-325.
26] Y. Momose, K. Meguro, H. Ikeda, C. Hatanaka, S. Oi, T. Sohda, Chem. Pharm. Bull. 39
1991) 1440-1445.
[
[
[
(
27] B.C.C. Cantello, M.A. Cawthorne, D. Haigh, R.M. Hindley, S.A. Smith, P.L Thurlby,
Bioorg. Med. Chem. Lett. 4 (1991) 1181-1184.
28] K. Murakami, K. Tobe, T. Ide, T. Mochizuki, M. Ohashi, Y. Akanuma, Y. Yazaki,
T. Kadowaki, Diabetes 47 (1998) 1841-1847.
[
29] J. Choi, Y. Ko, H.S. Lee, Y.S. Park, Y. Yang, S. Yoon, Eur. J. Med. Chem. 45 (2010)
1
93-202.
1
9

ACCEPTED MANUSCRIPT
[
[
[
30] M. Zhu, L. Flynt, S. Ghosh, M. Mellema, A. Banerjee, E. Williams, R.A. Panettieri Jr.,
S.A. Shore, Am. J. Respir. Cell Mol. Biol. 45 (2011) 111-119.
31] A.M. Youssef, M.S. White, E.B. Villanueva, I.M. El-Ashmawy, A. Klegeris, Bioorg.
Med. Chem. 18 (2010) 2019-2028.
32] Z. Jin, Muscarine, imidazole, oxazole and thiazole alkaloids, Nat. Prod. Rep. 22 (2005)
1
96-229.
[
33] K.M. Kim, K.H. Kim, T.C. Kang, W.Y. Kim, M. Lee, H.J. Jung, I.K. Hwang, S.B. Ko,
J.Y. Koh, M.H. Won, E. Oh, I. Shin, Bioorg. Med. Chem. Lett. 13 (2003) 2273- 2275.
34] X. Zhu, Q.S. Yu, R.G. Cutler, C.W. Culmsee, H.W. Holloway, D.K. Lahiri, M.P.
Mattson, N.H. Greig, J. Med. Chem. 45 (2002) 5090-5097.
[
[
35] M.Z. Rehman, C.J. Anwar, S. Ahmad, Bull. Korean Chem. Soc. 26 (2005) 1771-1775.
36] B.S. Zimenkovskii, R.V. Kutsyk, R.B. Lesyk, V.S. Matyichuk, N.D. Obushak,
T.I. Klyufinska, Pharm. Chem. J. 40 (2006) 303-306.
[
[
[
[
37] L. Carroll King, R.J. Hlavek, J. Am. Chem. Soc. 72 (1950) 3722-3725.
38] T. Takahashi, Tetrahedron Lett. 5 (1964) 565-572.
39] M. Joner, A. Farb, Q. Cheng, A.V. Finn, E. Acampado, A.P. Burke, K. Skorija,
W. Creighton, F.D. Kolodgie, H.K. Gold, R. Virmani, Arterioscler. Thromb. Vasc. Bol.
2
7 (2007) 182-189.
[
40] R. Thieringer, J.E. Fenyk-Melody, C.B. Le Grand, B.A. Shelton, P.A. Detmers, E.P.
Somers, L. Carbin, D.E. Moller, S.D. Wright, J. Berger, J. Immunol. 164 (2000) 1046-
1
054.
[
[
41] H.L. Liu, Z. Li, T. Anthonsen, Molecules. 5 (2000) 1055-1061
42] W. Nadia, M. Marguerite, T. Bernard, J. Philippe, P. Anne-Francoise, P. Joelle, Eur. J.
Med. Chem. 21 (1986) 59-64.
[
43] S.H. Latif, Z. Muhammad, A. Naveed, W.G. William, L.P. David, Chem. and Pharm.
Bull. 55 (2007) 1014-1017.
[
[
44] J. Renuka, Shipra, J. Indian. Chem. Soc. 74 (1997) 54-55.
45] S.A. Gaikwad, A.A. Patil, M.B. Deshmukh, Phosphorus, Sulfur and Silicon and the
Related Elements. 185 (2010) 103-109.
[
46] SMART & SAINT, Software reference manuals, Versions 6.28a & 5.625, Bruker
Analytical X-ray Systems Inc, Madison, Wisconsin, U.S.A, 2001.
2
0

ACCEPTED MANUSCRIPT
[
47] G.M. Sheldrick, SHELXS97 and SHELXL97, Programs for crystal structure solution
and refinement, University of Gottingen, Germany, 1997.
[
[
[
[
[
48] J.H. Moon, J. Terao, J. Agric. Food Chem. 46 (1998) 5062-5065.
49] W. Bors, W. Heller, C. Michel, M. Saran, Methods Enzymol. 186 (1990) 343-355.
50] F. Liu, V.E. Ooi, S.T. Chang, Life Sci. 60 (1997) 763-771.
51] E.X. Zhang, L.J. Yu, Chinese. J. Marine Drugs 3 (1997) 1-4.
52] T.B. Ng, F. Liu, Z.T. Wang, Life Sci. 66 (2000) 709-723.
2
1

ACCEPTED MANUSCRIPT
Table 1
Structure and physicochemical characteristics of target compounds 4a-o.
O
O
NH
O
S
NH
O
S
O
O
N
N
R
NH
NH
S
S
R
4a-k
4l-o
a
b
o
Entry Product
R
Mol. Formula/
Mol. Wt.
R
f
C log P* mp C
Yield
(%)
1
2
3
4
5
6
7
8
9
4a
4b
4c
4d
4e
4f
H
C H N O S
3 2
0.51 2.28
0.56 3.00
0.54 2.43
0.57 3.15
0.59 3.41
0.55 2.75
0.55 3.00
0.50 2.48
0.51 2.78
0.53 2.43
0.56 1.74
0.57 1.78
0.60 1.62
0.59 2.08
0.58 2.61
b
236-238
>260
71
67
69
67
62
68
68
69
69
68
65
61
56
54
58
14
11
3
3
33
p-Cl
p-F
C H N O S Cl
14
10
3
3 2
3
67
C H N O S F
212-216
252-254
242-244
216-218
212-214
192-194
218-220
220-222
228-230
266-268
272-274
262-264
246-248
14
10
3
3 2
3
51
C H BrN O S
3 2
p-Br
p-I
14
10
3
4
12
C H IN O S
3 2
14
10
3
4
59
o-Cl
m-Cl
o-Me
m-Me
o-F
C H N O S Cl
14
10
3
3 2
3
67
4g
4h
4i
C H N O S Cl
1
4
10
3
3
2
2
2
3
67
C H N O S
15
13
3
3
3
47
C H N O S
15
13
3
3
3
47
1
0
1
2
3
4
5
4j
C H N O S F
1
4
10
3
3
2
2
3
51
1
1
1
1
1
a
4k
4l
o-OMe
H
C H N O S
15
13
3
4
3
63
C H N O S
12
9
3
3
2
2
3
07
4m
4n
4o
6-NO2
6-OMe
6-OEt
C H N O S
12
8
4
5
3
52
C H N O S
13
11
3
4
2
2
3
37
C H N O S
14
13
3
4
3
51
All products are characterized by IR, NMR, ESI-MS and HRMS analysis; Ethyl acetate: Petroleum ether (2:1);
C log P values are calculated using ChemDraw Ultra 10.0.
*

ACCEPTED MANUSCRIPT
Table 2
Crystal data and structure refinement parameters for compound 4j.
Empirical formula
Formula weight
Temperature (K)
Color
Wavelength (Å)
Crystal system
Space group
a(Å)
C H FN O S
14 10 3 3 2
351.37
293
Colorless, needle
0.71073
Monoclinic
C2/c
30.877(4)
5.7127(6)
18.357(3)
117.036(3)
2884.2(6)
8
b(Å)
c(Å)
β(deg)
Volume (Å )
3
Z
3
Dcalc (mg mꢀ )
1.618
0.40
1
Absorption Coefficient (mmꢀ )
F(000)
Crystal size (mm
θ Range for data collection (deg)
1440.0
0.60 × 0.20 × 0.10
2.3-28.0
3
)
Index ranges
h= -36 →36,
k= -6 → 6,
l= -21 →21.
12857
Reflections collected
Independent Reflections(Rint)
Data/restraints/Parameters
No. of parameters
2538 (R = 0.0346)
2538/0/216
216
int
2
Goodness-on-fit on F
1.18
Final R indices [ I > 2σ(I)]
R indices (all data)
Largest diffraction peak and hole (Å e )
CCDC number
R1 = 0.0504, wR2 = 0.1241
R1 = 0.0527, wR2 = 0.1256
0.49 and -0.25
905873
3

ACCEPTED MANUSCRIPT
Table 3
0
Selected bond distances (Å) and bond angles ( ) for the compound 4j.
Bond lengths
S1—C1
S1—C3
O1—C2
O2—C1
N1—C1
C2—N1
O3—C5
N2—C5
N2—C6
S2—C7
S2—C6
N3—C6
N3—C8
F1—C10
Bond angles
O1—C2— N1
O1—C2— C3
C2—N1—C1
N1—C1— S1
O2—C1— N1
O2—C1— S1
C4—C3— S1
O3—C5— N2
O3—C5— C4
N2—C6— S2
N3—C6— N2
N3—C6— S2
F1—C10—C9
F1—C10—C11
C14—C9—C8
1.766(3)
1.824(3)
1.216(3)
1.197(4)
1.383(4)
1.360(4)
1.217(3)
1.360(3)
1.379(4)
1.722(3)
1.729(3)
1.295(3)
1.381(4)
1.361(4)
124.7(3)
111.5(2)
118.5(3)
109.8(2)
125.2(3)
125.0(3)
114.3(2)
112.7(3)
124.3(2)
123.77(19)
120.4(2)
115.9(2)
118.5(3)
118.1(3)
119.8(3)

ACCEPTED MANUSCRIPT
Table 4
Antioxidant activities of the synthesized compounds (4a-o).
a
Compound
EC (µg/ mL)
50
DPPH radical
scavenging
activity
NA
Superoxide anion Lipid peroxidation Erythrocyte
scavenging
activity
NT
inhibition
hemolysis
inhibition
NT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
NT
NA
NT
NT
NT
52.69
49.71
NA
NA
NA
NA
NA
NA
NA
NA
NA
NT
NT
NA
NT
NT
NT
NT
NT
NT
NA
NT
NT
NT
90.16
82.50
58.68
52.18
39.76
51.82
62.90
40.28
44.18
NA
NT
NA
NA
NA
NA
79.94
101.18
NA
131.79
149.70
NA
96.62
107.28
NA
NA
NA
NA
NA
NA
NA
b
Ascorbic acid
Luteolin
21.01
31.01
139.97
149.70
96.625
107.28
b
a
b
EC values are mean of three experiments; Positive control; NA: not active; NT: not tested.
50

ACCEPTED MANUSCRIPT
Table 5
Effect of pioglitazone on PMA-induced IL-1β, MCP-1 levels in THP1 cells.
a
Treatment
conditions
Secreted IL-1β levels
(pg/ mL)
Secreted MCP-1 levels
(pg/ mL)
Without PMA
12 ± 0.38
28.12 ±3.18
PMA (10 ng/mL)
172 ± 0.71
3521.63 ±79.78
2588.33 ±11.64
PMA+ Pioglitazone 165 ± 1.56
10 µM)
(
a
5
2
x 10 /mL THP1 monocytes are seeded in 24 well cell culture plates and stimulated with 10
ng/ mL of PMA for IL-1β, MCP-1 production. Cells are incubated for a period of 48 h. At the
end of the treatment, conditioned media is collected and IL-1β, MCP-1 levels are measured by
ELISA as described in the materials and methods.

ACCEPTED MANUSCRIPT
Table 6
IL-1β and MCP-1 secretion inhibition efficacy of the
a
synthesized compounds (4a-o).
b
Compound
IC (µM)
5
0
IL-1β secretion
MCP-1 secretion
inhibition efficacy inhibition efficacy
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
9.6 ± 2.96
16.31 ± 0.91
NA
33.12 ± 5.57
NA
18.23 ± 1.53
23.4 ± 6.66
7.66 ± 0.46
11.72 ± 4.05
NA
7.23 ± 0.99
NA
6.83 ± 0.24
7.03 ± 0.06
NA
30.4± 3.4
NA
17.58± 1.58
49.13± 3.46
31.7± 1.86
26.49± 2.54
34.24± 1.86
18.79± 2.45
18.94± 0.86
16.68± 1.11
NA
20.8± 2.21
20.29± 3.21
39.6± 1.66
63.06± 4.21
18.84± 2.16
NT
Pioglitazone
Piroxicam
NA
21.33 ± 3.2
c
a
THP1 monocytes are pretreated with 5, 10 and 20 μM concentrations of the above mentioned
thiazole substituents (4a-o) for 1 h before simulation with 10 ng/ mL of Phorbol 13-myristate 12-
acetate (PMA) to induce inflammation for a period of 48 h. At the end of the treatment,
conditioned media is collected and levels of IL-1β and MCP-1 are measured by ELISA as
b
described in the materials and methods. IC50 values are Mean ± SD of three independent
c
experiments; Piroxicam, a known anti-inflammatory agent is used as a positive control; NA:
denotes activity >50 ꢀM; NT: indicates ‘not tested’.

ACCEPTED MANUSCRIPT
Fig. 1. An ORTEP view of the molecular structure and atom-numbering of compound 4j.
Displacement ellipsoids are drawn at the 30% probability level and H atoms are shown as small
spheres of arbitrary radius.