Synthesis and biological evaluation of novel thiazolidinone derivatives as potential anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2013.04.010

The modulation of pro-inflammatory cytokines provides a target for controlling inflammatory diseases and attracts much attention in current anti-inflammatory drug development. Here, four series of thiazolidinone derivatives were synthesized and screened for anti-inflammatory activities. A majority of these compounds showed excellent inhibition on the expression of TNF-α and IL-6 in LPS-stimulated macrophages. Discussions are given regarding the structure-activity relationships. Compounds 12d and 12h inhibited LPS-induced TNF-α and IL-6 release in a dose-dependent manner. Furthermore, 12d exhibited a significant protection against LPS-induced septic death in mouse model. Together, these data present a series of new thiazolidinones with potential therapeutic effects in acute inflammatory diseases and they could be important leads in the continuing anti-inflammatory drug research.

Full text of DOI:10.1016/j.ejmech.2013.04.010

Accepted Manuscript
Synthesis and biological evaluation of novel thiazolidinone derivatives as potential
anti-inflammatory agents
Jie Hu, Yi Wang, Xiaoyan Wei, Xixi Wu, Gaozhi Chen, Gaozhong Cao, Xueqian
Shen, Xiuhua Zhang, Qinqin Tang, Guang Liang, Xiaokun Li
PII:
S0223-5234(13)00242-0
10.1016/j.ejmech.2013.04.010
EJMECH 6120
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 November 2012
Revised Date: 2 April 2013
Accepted Date: 2 April 2013
Please cite this article as: J. Hu, Y. Wang, X. Wei, X. Wu, G. Chen, G. Cao, X. Shen, X. Zhang,
Q. Tang, G. Liang, X. Li, Synthesis and biological evaluation of novel thiazolidinone derivatives as
potential anti-inflammatory agents, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.04.010.
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ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel thiazolidinone derivatives as
potential anti-inflammatory agents
Jie Hu 1, #, Yi Wang 1, #, Xiaoyan Wei 2, Xixi Wu 1, Gaozhi Chen 1, Gaozhong Cao 2, Xueqian Shen 1,
Xiuhua Zhang 2, Qinqin Tang 1, Guang Liang 1, *, Xiaokun Li 1,
*
1
School of Pharmaceutical Sciences, Wenzhou Medical College, University Town, Wenzhou, Zhejiang
325035, China
2
Department of Pharmacy, The 1st Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang
325035, China
Graphical abstract:
Four series of thiazolidinone derivatives were synthesized and evaluated for anti-inflammatory activities.
O
N
R1
R3
S
R1
R2
S
N
N
S
R3
CHO
R2
Thiazolidinone derivatives
N
O
N
O
EtOOC
O
12d
Keywords: Thiazolidinones; Anti-inflammation; Macrophages; Sepsis

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel thiazolidinone derivatives as
potential anti-inflammatory agents
Jie Hu 1, #, Yi Wang 1, #, Xiaoyan Wei 2, Xixi Wu 1, Gaozhi Chen 1, Gaozhong Cao 2, Xueqian Shen 1,
Xiuhua Zhang 2, Qinqin Tang 1, Guang Liang 1, *, Xiaokun Li 1,
*
1
School of Pharmaceutical Sciences, Wenzhou Medical College, University Town, Wenzhou, Zhejiang
325035, China
2
Department of Pharmacy, The 1st Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang
325035, China
# These authors contribute equally to this work.
* Corresponding author:
Guang Liang, Ph.D, Associate Professor
Director, Bioorganic & Medicinal Chemistry Research Center
School of Pharmaceutical Sciences, Wenzhou Medical College
Tel: (+86)-577-86699524; Fax: (+86)-577-86699527
E-mail: cuiliang1234@163.com
And Xiaokun Li, Ph.D, Professor
School of Pharmaceutical Sciences, Wenzhou Medical College
Tel: (+86)-577-86689983; Fax: (+86)-577-86689983
E-mail: proflxk@163.com
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Abstract
The modulation of pro-inflammatory cytokines provides a target for controlling inflammatory diseases and
attracts much attention in current anti-inflammatory drug development. Here, four series of thiazolidinone
derivatives were synthesized and screened for anti-inflammatory activities. A majority of these compounds
showed excellent inhibition on the expression of TNF-α and IL-6 in LPS-stimulated macrophages.
Discussions are given regarding the structure-activity relationships. Compounds 12d and 12h inhibited
LPS-induced TNF-α and IL-6 release in a dose-dependent manner. Furthermore, 12d exhibited a significant
protection against LPS-induced septic death in mouse model. Together, these data present a series of new
thiazolidinones with potential therapeutic effects in acute inflammatory diseases and they could be
important leads in the continuing anti-inflammatory drug research.
Key words: Thiazolidinones; Anti-inflammation; Macrophages; Sepsis
1. Introduction
Inflammation is a hallmark of many diseases and the persistence of this process may lead to various
diseases associated with acute or chronic inflammation including sepsis, arthritis, atherosclerosis, diabetes
and even cancer [1]. Pro-inflammatory cytokines, whose expression can be induced by endotoxin
(lipopolysaccharide, LPS), share many cellular and molecular features in common and are deeply involved
in the inflammation-mediated diseases by amplifying inflammatory signal in multiple pathways [2-3].
Interleukin (IL)-6 and tumor necrosis factor (TNF) are important pro-inflammatory cytokines, which are
significantly involved in fever, inflammation, diabetic complications, and septic shock and death [4-6].
Hence the modulation of these products provides a target for controlling inflammatory diseases and attracts
much attention in current anti-inflammatory drug development [7-8]. In addition, due to the resistance of
such diseases to conventional treatments, as well as the side-effects of presently available
anti-inflammatory drugs, there is a pressing need for the development of novel anti-inflammatory drugs [9].
Small molecule inhibitors of cytokine expression are emerging, and a few of them are having
advanced into clinical trials [8, 10]. In the past five years, our group has designed and evaluated a lot of
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synthetic compounds as anti-inflammatory agents targeting pro-inflammatory cytokines, including IL-6 and
TNF-α, some of which are underwent the preclinical assessment as anti-inflammatory and anti-sepsis
candidates [11-13]. It is well-known that a number of heterocyclic compounds containing nitrogen and
sulphur exhibit a wide variety of biological activities [14]. Thiazolidinones are derived from the
thiazolidine structure containing sulfur and nitrogen in a five member ring. It is contained in a variety of
bioactive compounds, especially anti-HIV, anti-bacterial, anti-tuberculosis, anti-tumor, anti-histaminic, or
anti-convulsant agents [15-18]. Out of these biological activities, the anti-inflammatory activity of
thiazolidinone-containing compounds is of particular interest recently [19]. For example, Sharma et al [20]
reported a series of thiazolidinone derivatives and found 2-hydroxyphenyl thiazolidinone as a potential
anti-inflammatory and analgesic agent. In addition, thiazolidinone has been considered as an effective lead
of
anti-inflammatory
COX-2
inhibitors.
Ottana
et
al
[21]
evaluated
3,3'-(1,2-ethanediyl)bis[2-(4-methoxyphenyl)-thiazolidin-4-one] as a new COX-2 inhibitor and showed its
ability to attenuate the carrageenan-induced lung injury in experimental models. Some
5-arylidene-2-imino-4-thiazolidinones were recently designed and evaluated for COX-2 inhibition and
anti-inflammatory activity [22]. Thus, previous evidences prompt us to study the anti-inflammatory activity
of newly synthetic thiazolidinone derivatives both in vitro and in vivo.
2. Chemistry
According to the designed structures, 32 thiazolidinones are divided into four series, which are
prepared from four intermediates 10-13, respectively (Figure 1). The common synthetic route of the
intermediates thiazolo[3,2-a]benzimidazol-3(2H)one (10) and 5,6-dihydroimidazo[2,1-b]thiazol-3(2H)-one
(11) is outlined in Schemes 1. Imidazole-2-thiols (2a) and (2b) were synthesized by the conventional
refluxing ethanol-water solution of carbon disulphide and diamines, respectively, with a yield ranging
74.2-90.5%. The substituted-thiol acetic acids (3a and 3b) were obtained through William’s reaction of
substituted imidazole-2-thiols (2a and 2b) with chloroacetic acid in the presence of sodium hydroxide.
Further, heating acetic acids 3a and 3b with acetic anhydride in pyridine medium on steam bath afforded
the intermediates 10 and 11, respectively.
Please insert Figure 1
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Please insert Scheme 1
For
the
synthesis
of
ethyl
7-methyl-3-oxo-5-phenyl-3,5-dihydro-2H-thiazolo[3,2-a]
pyrimidine-6-carboxylate (12), ethyl-2-mercapto-4-methyl-6-phenyl-1,6-dihydropyrimidine-5-carboxylate
(6) was firstly prepared using an one pot Bi-ginelli reaction of ethyl acetylacetate (4), thiourea (5), and
benzaldehyde in the presence of a catalytic amount of sulfamic acid according to the known procedure [23],
and subsequently, the product 6 was refluxed with ethyl 2-chloroacetate in the presence of pyridine as
catalyst to afford the cyclization product 12 (Schemes 2). The synthesis of
thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (13) is shown in Scheme 3. Compound 8 was prepared by the
reaction of commercial available thiosemicarbazide 7 and formic acid according to the reported method
[24]. The ring closure of intermediate 8 occurred in 20% NaOH solution to afford 2H-1,2,4-triazole-3-thiol
(9) in a yield of 30.8%. Subsequently, 13 was obtained by the cyclization procedure with ethyl
2-chloroacetate.
Please insert Scheme 2
Please insert Scheme 3
Finally, four intermediates 10-13 were allowed to undergo a Knoevenagel condensation with different
aryl aldehydes in CH3COONa/CH3COOH medium to afford the target thiazolidinone derivatives (10a-10o,
11a-11d, 12a-12h and 13a-13e), respectively. The products were isolated by conventional work-up in
satisfactory yields. Analytical and spectral data of all synthesized compounds are in full agreement with the
proposed structures.
3. Pharmacology
3.1 Inhibitory screening against LPS-induced TNF-α and IL-6 release
The stimulation by microbial endotoxins may induce an inflammatory process in the macrophages
through cytokine signaling pathways. Lipopolysaccharide (LPS), an endotoxin from the walls of
Gram-negative bacteria, is a potent stimulator of inflammatory cytokines in macrophages. Target
compounds (10a-10o, 11a-11d, 12a-12h and 13a-13e) were evaluated for their anti-inflammatory activity
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of inhibiting the TNF-α and IL-6 release in LPS-stimulated mouse macrophage cell line RAW 264.7 cells.
The cells were pre-incubated with thiazolidinones at 10 ꢀM, curcumin (as a positive control at 10 ꢀM) or
control medium (DMSO) for 60 min. After that, cells were treated with LPS (0.5 ꢀg/ml) for 22 hours at 37
oC. The amount of TNF-α and IL-6 in medium was detected through enzyme-linked immunosorbant assay
(ELISA) and normalized by protein concentration of cells harvested in homologous culture plates.
Please insert Figure 2
The inhibitory screening results are shown in Figure 2. Curcumin, a natural anti-inflammatory
compound, was used as a positive control for comparison. A majority of 32 tested compounds exhibited
significant anti-inflammatory activity in inhibition against LPS-induced TNF-α and IL-6 expression in vitro.
Among these compounds, 12d, 12h, 13a, 13c, 11c, and 11d exhibited strong inhibitory abilities in TNF-α
expression (inhibitory rates>50%, Figure 2A). Meanwhile, nine compounds, including 10k, 12d, 12g, 12h,
13a, 13c, 13e, 11b, and 11d showed the inhibitory effect on IL-6 expression in an inhibition rate that
ranging from 50% to 87% (Figure 2B). Thiazolidinones 12d and 12h, especially, showed the strongest
inhibitory effect on LPS-induced cytokine releases and their inhibitory rates reached 62% and 60%
(anti-TNF-α) and 92% and 87% (anti-IL-6), respectively, compared to the LPS control.
To our knowledge, it is the first time to report the cytokine-inhibitory effects of thiazolidinone
derivatives and discuss their structure activity relationship (SAR). It is observed that substituted pyrimidine
derivatives (12a-12h) showed stronger anti-inflammatory activity than other three thiazolidinone series.
SAR of substituted pyrimidine derivatives also indicated that the arylidene moieties at R3 position could
significantly influence the cytokine-inhibitory ability. The introduction of heterocycles or heterocyclic
groups at R3 position could enhance the anti-inflammatory activity, such as active compounds 12d, 12g and
12h. On the other hand, when remaining the same arylidene moiety, the replacement of the
pyrimidine-thiazolidinone core with benzo[d]imidazole-thiazolidinone (10i, 10m), triazole-thiazolidinone
(13c, 13d) or imidazole-thiazolidinone (11b, 11c) led to slightly decrease in the ability of cytokine
inhibition. A similar rule was observed in thiazolidinones with an arylidene moiety of o-halogenated phenyl
or 5-methylthiophenyl. Regarding to the arylidene moiety itself, the electron-withdrawing groups and
heterocyclic groups seemed to be favorable to the anti-inflammatory activity than electron-donating ones.
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3.2 Compound 12d inhibit TNF-α and IL-6 release in a dose-dependent manner
Among the active compounds above, two compounds, 12d and 12h, demonstrated the highest
anti-inflammatory activities and low cytotoxicity (Figure S1 in supplemental file) in mouse macrophages.
Thus, they were chosen for further evaluation of their dose-dependent inhibitory effects against
LPS-induced TNF-α and IL-6 release. RAW 264.7 macrophages were pre-treated with 12d and 12h in a
series of concentrations (0.3, 1.1, 3.3 and 10 ꢀM) for 2 h and were subsequently incubated with LPS (0.5
ꢀg/ml) for 22 h. As shown in Figure 3A, pretreatment with 12d exhibited a dose-dependent inhibition on
both TNF-α and IL-6 release induced by LPS. However, 12h did not show a good dose-dependent result in
inhibiting TNF-α expression (Figure 3B). Accordingly, the IC50 values of these two compounds are
calculated and shown in Figure 3. 12d exhibited the lowest IC50 value (0.83 ꢀM) when inhibiting IL-6
release. The dose-dependent inhibition by 12d suggests its potential as an anti-inflammatory agent.
Please insert Figure 3
3.3 Effect of 12d pretreatment on survival rate in mice after LPS administration
As a major endotoxin, LPS from Gram-negative bacteria has been implicated as a major cause of
sepsis. Inflammatory shock as a consequence of LPS release remains a serious clinical concern, which can
cause a variety of pathologies ranging from mild (fever) to lethal (septic shock, organ failure, and death)
[25]. Our data have demonstrated the inhibitory effects of 12d on LPS-induced cytokine release in vitro. To
determine whether 12d are able to attenuate endotoxin shock through inhibiting LPS-induced inflammatory
response, 15-18 week old mice were injected with LPS at the dosage of 20 mg/kg intravenously (i.v.) in the
presence or absence of 12d administration, and their survival rates were monitored for seven days,
respectively. As shown in Figure 4, eighty percent of animals treated with LPS alone died within 7 days as
a result of the septic shock. In animals receiving 12d at 15 mg/kg intraperitoneally (i.p.) 15-min after LPS
injection (for therapeutic effect), the survival rates were significantly increased compared to that of the
control group (70 % survivals in the treatment group, p<0.01 v.s. LPS group). However, the preventive
administration group, in which 12d was i.p. injected 15-min prior to LPS injection, did not show significant
attenuation on LPS-induced septic death. This may be ascribed to a possible fast metabolism of 12d in vivo.
Thus, our data provide novel evidence for 12d’s therapeutic potential in sepsis.
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Please insert Figure 4
4. Conclusion
In summary, four types of thiazolidinone derivatives were synthesized and their anti-inflammatory
activities were evaluated. A majority of the synthetic compounds exhibited significant inhibitory activities
against LPS-induced TNF-α and IL-6 expression in RAW 264.7 macrophages. Discussion and conclusions
were made regarding structure-activity relationships. 12d and 12h, belonging to pyrimidine- thiazolidinone
series, are two of most potent compounds, and especially, 12d exhibited anti-inflammatory abilities in a
dose-dependent manner with relatively low IC50 values. In vivo, intraperitoneal administration with 12d
markedly protected mice from LPS-induced septic death. These present the possibility that thiazolidinones
might serve as potential agents for the treatment of various inflammatory diseases. On the other hand, the
thiazolidinone derivatives have been reported to show anti-tumor properties. However, the molecular
mechanism by which thiazolidinones exert their biological functions are still unclear. Taking in account the
association between inflammatory cytokine and tumor pathogenesis, it is indicated that their anti-tumor and
anti-inflammatory functions may derived from the same targets, for example, IKK-β and NF-κB. Further
study is under investigation in our lab to demonstrate the possible crosstalk and complementation
between their anti-inflammation and anti-tumor properties using 12d as a probe. Although the
anti-inflammatory mechanism and underlying targets are still unknown, the beneficial effects of these
compounds on LPS-induced inflammation make thiazolidinones one of important leads in the continuing
drug development and research.
5. Experimental section
5.1 Chemical synthesis
5.1.1 Materials and methods
Solvents were distilled and dried by standard methods. N,N-dimethylformamide (DMF) was prepared
by drying over barium oxide or 4Å molecular sieves overnight. Thiosemicarbazide, acetoacetate and
thiourea (5) were purchased from Alfa Aesar and Sigma-Aldrich, respectively. Other chemicals were
obtained from local suppliers and were used without further purification. All reactions were monitored by
1
thin-layer chromatography (250 silica gel 60 F254 glass plates). H-NMR spectra were recorded on Bruker
600 MHz instruments, and the chemical shifts were presented in terms of parts per million with TMS as the
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internal reference. Electron-spray ionization mass spectra in positive mode (ESI-MS) data were obtained
with a Bruker Esquire 3000+ spectrometer. Chromatographic purification was carried out on Silica Gel 60
(E. Merck, 70-230 mesh).
5.1.2 Synthesis of 2a and 2b
A mixture of ortho-phenylenediamine (1a) or ethane-1,2-diamine (1b) (0.033 mol), carbon disulfide
(0.33 mol) and a solution of 95% ethanol-water (10:1, 33.0 ml) in a flask were heated under reflux for 3 h.
Norit (1.2 g) was then added cautiously, the mixture was heated at the reflux temperature for 10 min, and
then norit was removed by filtration soon. The filtrate was heated to 60-70 °C, followed by the addition of
30 mL warm water (60-70 °C) and 2.5 mL acetic acid with good stirring. After cooling, the mixture was
separated to give glistening white crystals (2a or 2b) with yields of 90.5% and 74.2%, respectively.
5.1.3 Synthesis of 3a and 3d
The mixture of midazoles 2a or 2b (0.01 mol) and sodium monochloroacetate (0.011 mol) in methanol
(20 mL) was added and refluxed for 5 h. The reaction mixture was cooled and poured into 20 mL of cold
water to yield a colored precipitate, which was then separated by filtration and washed by water to furnish
3a or 3b in white flakes solid.
5.1.4 General Procedure for Synthesis of 10 and 11
Compound 3a or 3b (0.005 moL) was dissolved in a solution of pyridine (3.0 mL) and acetic anhydride
(1.0 moL). The reaction mixture was then added acetone (4.0 mL) slowly, followed by a heating mantle for
30 min. The colored solid that separated by cooling was filtered and washed with water or crystallized from
acetic acid to give final bright colored product 10 or 11 with yields of 48.0% or 63.0%, respectively.
5.1.5 Synthesis of ethyl 2-mercapto-4-methyl-6-phenyl-1,6-dihydropy rimidine-5-carboxylate (6)
Sulfamic acid (0.4 moL) was added to a solution of benzaldehyde (0.5 moL), ethyl acetylacetate (0.6
moL), and thiourea (0.75 moL) in ethanol (10 mL) and reflux for 2 h. The mixture was cooled to room
temperature, the residue was separated and washed with water to give a white solid (6) with 70.3% yield.
5.1.6 Synthesis of 12
This compound was synthesized using the same method described for the synthesis of compound 10 with
a yield of 58.5%.
5.1.7 Synthesis of 1-formylthiosemicarbazide (8)
Formic acid (4.0 ml) in a round-bottomed flask was heated on a steam bath for 15 min, and commercial
available thiosemicarbazidean (7) 1.82 g (20 mmoL) was added. The mixture was then stirred until the
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thiosemicarbazide dissolved completely. The boiling water (60 mL) was added after 2-h reaction. The
milky solution was filtered and the filtrate was cooled in an ice bath for 2 h. Compound 8 was collected by
suction filtration with 29.0% yield.
5.1.8 Synthesis of 2H-1,2,4-triazole-3-thiol (9)
2-formylhydrazinecarbothioamide (8) (10.0 mmoL) was added to a solution of 20% NaOH (2.0 mL), and
then refluxed for 1 h. The reaction mixture was cooled, poured into crushed ice, and neutralized with
conc.HCl. The product was filtered, dried, and recrystallized from ethanol to give intermediate
2H-1,2,4-triazole-3-thiol (9) with 30.8% yield.
5.1.9 Synthesis of 13
This compound was synthesized using the same method described for synthesis of compound 10 with a
yield of 62.5%.
5.1.10 General Procedure for Synthesis of 10a-10o, 11a-11d, 12a-12h and 13a-13e.
A reaction mixture of 10-13 (1.0 equiv), various aromatic aldehydes (1.0 equiv) and anhydrous sodium
acetate (1.0 equiv) in glacial acetic acid (25.0 mL) was refluxed on a heating mantle for 3 h. The colored
solid that separated on cooling was filtered, washed with water, and crystallized from acetic acid or
separated by column chromatography to give 10a-10o, 11a-11d, 12a-12h and 13a-13e with 19.8-79.7%
yields.
5.1.10.1 (2Z)-2-(2-Bromobenzylidene)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one (10a):
Yellow powder, m.p: 189.2-193.2 oC. 1H NMR (600 MHz, DMSO-d6) δ(ppm): 8.167 (s, 1H), 7.942 (d, J
= 7.8 Hz, 1H), 7.863 (d, J = 7.8 Hz, 1H), 7.723 (d, J = 7.8 Hz, 1H), 7.680 (d, J = 7.2 Hz, 1H), 7.629-7.654
(m, 1H), 7.467-7.495 (m, 1H), 7.385-7.441 (m, 2H). 13C NMR (600 MHz, DMSO-d6) δ(ppm): 112.37,
119.34, 124.30, 125.51, 125.84, 128.85, 128.91, 129.34, 130.16, 132.33, 132.51, 133.25, 133.83, 148.23,
153.16, 158.60. ESI-MS m/z: 390.9 (M+CH3OH+H)+, 410.9 (M+CH3OH+Na)+, calcd for C16H9BrN2OS:
357.22.
5.1.10.2 (2Z)-2-(2-Chlorobenzylidene)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one (10b):
o
Yellow powder, m.p: 230.2-232.4 C, [235 oC, lit.[26]]. ESI-MS m/z: 354.3 (M+CH3OH+H)+, 367.0
(M+CH3OH+Na)+, calcd for C16H9ClN2OS: 312.77.
5.1.10.3 (2Z)-2-(2-Fluorobenzylidene)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one (10c):
Yellow powder, m.p: 207.6-210.7 oC. 1H NMR (600 MHz, DMSO-d6) δ(ppm): 8.079 (s, 1H), 7.956 (d, J
= 7.2 Hz, 1H), 7.728-7.756 (m, 1H), 7.693 (d, J = 7.2 Hz, 1H), 7.620-7.658 (m, 1H), 7.459-7.484 (m, 2H),
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7.389-7.435 (m, 2H). 13C NMR (600 MHz, DMSO-d6) δ(ppm): 112.37, 116.36, 116.50, 119.35, 124.29,
125.67, 125.83, 126.65, 127.97, 129.17, 130.15, 133.41, 133.47, 148.27, 153.12, 158.74. ESI-MS m/z:
329.1 (M+CH3OH+H)+, 351.0 (M+CH3OH+Na)+, calcd for C16H9FN2OS: 296.32.
5.1.10.4 (2Z)-2-(2-Methoxybenzylidene)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one (10d):
o
Yellow powder, m.p: 212.1-214.0 C, [211 oC, lit.[27]]. ESI-MS m/z: 341.1 (M+CH3OH+H)+, 363.0
(M+CH3OH+Na)+, calcd for C17H12N2O2S: 308.35.
5.1.10.5 (2Z)-2-(3-Hydroxy-4-methoxybenzylidene)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one (10e):
o
Yellow powder, m.p: 257.3-259.5 oC, [260-262 C, lit.[28]]. ESI-MS m/z: 322.8 (M-H)-, 354.8
(M+CH3OH-H)-, calcd for C17H12N2O3S: 324.35.
5.1.10.6 (2Z)-2-(4-Hydroxy-3-methoxybenzylidene)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one (10f):
Yellow powder, m.p: 238.2-240.1 oC [238-239 oC, lit.[29]]. ESI-MS m/z: 322.8(M-H)-, 354.8
(M+CH3OH-H)-, calcd for C17H12N2O3S: 324.35.
5.1.10.7 (2Z)-2-[(2,4-Dimethylphenyl)methylidene]-[1,3]thiazolo[3,2-a]benzimidazol-1-one (10g):
Yellow powder, m.p: 170.1-171.9 oC. 1H NMR (600 MHz, DMSO-d6) δ(ppm): 8.169 (s, 1H), 7.949 (d, J
= 7.2 Hz, 1H), 7.677 (d, J = 7.2 Hz, 1H), 7.481 (d, J = 7.8 Hz, 1H), 7.376-7.436 (m, 2H), 7.242 (d, J = 8.4
Hz, 1H), 7.232 (s, 1H), 2.436 (s, 3H), 2.343 (s, 3H). 13C NMR (600 MHz, DMSO-d6) δ(ppm): 19.35, 21.01,
112.32, 119.24, 124.10, 125.41, 125.72, 127.50, 127.56, 128.88, 130.15, 131.92, 133.67, 139.16, 141.20,
148.22, 153.22, 158.99. ESI-MS m/z: 307.6 (M + H)+, 339.2 (M + CH3OH+H)+, calcd for C18H14N2OS:
306.38.
5.1.10.8 (2Z)-2-(2-Nitrobenzylidene)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one (10h):
o
Yellow powder, m.p: 234.9-237.0 C, [240 oC, lit.[26]]. ESI-MS m/z: 356.1 (M+CH3OH+H)+, 378.0
(M+CH3OH+Na)+, calcd for C16H9N3O3S: 323.33.
5.1.10.9 (2Z)-2-[4-(Morpholin-4-yl)benzylidene][1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one (10i):
Yellow powder, m.p: 234.9-237.0 oC, [231 oC, lit.[30]]. ESI-MS m/z: 363.0 (M+CH3OH+H)+, calcd for
C20H17N3O2S: 363.43.
5.1.10.10 (2Z)-2-(Naphthalen-2-ylmethylene)benzo[4,5]imidazo[2,1-b]thiazol-3(2H)-one (10j):
1
Yellow powder, m.p: 266.2-267.7 oC. H NMR (600 MHZ, DMSO-d6) δ(ppm): 8.374 (s, 1H), 8.275 (s,
1H), 8.122-8.137 (m, 2H), 7.999-8.030 (m, 2H), 7.842 (d, J = 7.2 Hz, 1H), 7.713 (d, J = 7.8 Hz, 1H),
7.637-7.676 (m, 2H), 7.408-7.459 (m, 2H). ESI-MS m/z: 361.2 (M+CH3OH+H)+, calcd for C20H12N2OS:
328.39.
10

ACCEPTED MANUSCRIPT
5.1.10.11 (2Z)-2-[(1H-Indol-3-yl)methylene]benzo[4,5]imidazo[2,1-b]thiazol-3(2H)-one (10k):
o
1
Yellow powder, m.p: 320-321.6 C. H NMR (600 MHz, DMSO-d6) δ(ppm): 12.364 (s, 1H), 8.380 (s,
1H), 8.044 (s, 1H), 7.976-7.995 (m, 2H), 7.686 (d, J = 7.2 Hz, 1H), 7.540 (d, J = 7.8 Hz, 1H), 7.365-7.425
(m, 2H), 7.280-7.307 (m, 1H), 7.238-7.264 (m, 1H). 13C NMR (600 MHz, DMSO-d6) δ(ppm): 110.49,
112.13, 112.84, 110.06, 118.41, 119.07, 121.37, 123.21, 123.65, 125.27, 127.02, 128.95, 130.22, 130.68,
136.98, 148.26, 152.77, 159.39. ESI-MS m/z: 350.2 (M+CH3OH+H)+, calcd for C18H11N3OS: 317.34.
5.1.10.12 (2Z)-2-[(Thiophen-2-yl)methylidene]-[1,3]thiazolo[3,2-a]benzimidazol-1-one (10l):
Yellow powder, m.p: 263.4-266.2 oC, [261 oC, lit.[30]]. ESI-MS m/z: 317.1 (M+CH3OH+H)+, calcd for
C14H8N2OS2:284.36.
5.1.10.13 (2Z)-2-[(5-Methylthiophen-2-yl)methylidene]-[1,3]thiazolo[3,2-a]benzimidazol-1-one (10m):
Yellow powder, m.p: 203.6-207.5 oC. 1H NMR (600 MHz, DMSO-d6) δ(ppm): 8.305 (s, 1H), 7.940 (d, J
= 7.8 Hz, 1H), 7.663-7.688 (m, 2H), 7.362-7.424 (m, 2H), 7.075-7.083 (m, 1H), 2.601 (s, 3H). 13C NMR
(600 MHz, DMSO-d6) δ(ppm): 15.63, 112.24, 119.21, 120.44, 123.99, 125.55, 128.12, 129.52, 130.15,
134.67, 136.53, 148.20, 149.22, 152.05, 159.02. ESI-MS m/z: 466.1 (M+H)+, calcd for C15H10N2OS2:
298.38.
5.1.10.14 (2Z)-[ (5-Bromo-2-ethoxy)benzylidene]-[1,3]thiazolo[3,2-a]benzimidazol-1-one (10n):
Yellow powder, m.p: 213.7-217.9 oC. 1H NMR (600 MHz, DMSO-d6) δ(ppm): 8.141 (s, 1H), 7.938 (d, J
= 7.2 Hz, 1H), 7.682 (d, J = 9.0 Hz, 2H), 7.651 (s, 1H), 7.378-7.481 (m, 2H), 7.184 (d, J = 9.0 Hz, 2H),
4.216-4.228 (m, 2H), 1.406-1.429 (m, 3H). 13C NMR (600 MHz, DMSO-d6) δ(ppm): 14.36, 64.69, 112.06,
112.32, 115.26, 119.31, 123.46, 124.20, 125.78, 126.87, 129.32, 130.03, 131.13, 135.15, 148.25, 153.14,
156.23, 158.94. ESI-MS m/z: 293.3 (M+H)+, 331.2 (M+CH3OH+H)+, 353.0 (M+CH3OH+Na)+, calcd for
C18H13BrN2O2S: 401.28.
5.1.10.15 (2Z)-2-[2,3-dichlorobenzylidine]-[1,3]thiazolo[3,2-a]benzimidazol-1-one (10o):
Yellow powder, m.p: 244.1-246.0 oC. 1H NMR (600 MHz, DMSO-d6) δ(ppm): 8.191 (s, 1H), 7.956 (d, J
= 7.8 Hz, 1H), 7.682 (d, J = 8.4 Hz, 1H), 7.695-7.735 (m, 2H), 7.611-7.638 (m, 1H), 7.397-7.453 (m, 2H).
13C NMR (600 MHz, DMSO-d6) δ(ppm): 112.41, 119.39, 124.40, 125.91, 127.84, 128.72, 129.21, 130.53×
2, 132.36, 133.22, 148.35, 150.87, 158.91×2, 184.66. ESI-MS m/z: 350.3 (M+H)+, 379.1 (M+CH3OH+H)+,
401.0 (M+CH3OH+Na)+, calcd for C16H8Cl2N2OS: 347.22.
5.1.10.16
Ethyl(Z)-2-(2-fluorobenzylidene)-7-methyl-3-oxo-5-phenyl-3,5-dihydro-2H-thiazolo
[3,2-a]pyrimidine-6-carboxylate (12a):
11

ACCEPTED MANUSCRIPT
Yellow powder, m.p: 174.7-177.6 oC. 1H NMR (600 MHz, DMSO-d6) δ(ppm): 7.856 (s, 1H), 7.799 (d, J
= 7.8 Hz, 1H), 7.575-7.606 (m, 2H), 7.409-7.435 (m, 1H), 7.347-7.380 (m, 2H), 7.312-7.367 (m, 3H),
6.049 (s, 1H), 4.030-4.057 (m, 2H), 2.390 (s, 3H), 1.099-1.122 (m, 3H). 13C NMR (600 MHz, DMSO-d6)
δ(ppm): 13.83, 22.34, 55.15, 60.23, 109.06, 123.45, 125.35, 127.57×2, 128.63, 128.72×2, 128.90, 130.33,
132.23, 132.47, 133.70, 140.02, 140.24, 150.77, 155.00, 163.84, 164.78. ESI-MS m/z: 485.0 (M+H)+, calcd
for C2312DBrN2O3S: 483.38.
5.1.10.17
Ethyl(Z)-2-(2-fluorobenzylidene)-7-methyl-3-oxo-5-phenyl-3,5-dihydro-2H-thiazolo
[3,2-a]pyrimidine-6-carboxylate (12b):
Yellow powder, m.p: 138.6-144.6 oC, [137 oC, lit. [31]]. ESI-MS m/z: 423.4 (M+H)+, 445.2 (M+Na)+,
calcd for C2312DFN2O3S: 422.47.
5.1.10.18
Ethyl(Z)-2-(2,3-dimethylbenzylidene)-7-methyl-3-oxo-5-phenyl-3,5-dihydro-2H-thiazolo-
[3,2-a]pyrimidine-6-carboxylate (12c):
o
Yellow powder, m.p: 165.9-167.8 C. 1H NMR (600 MHz, DMSO-d6) δ(ppm): 7.843 (s, 1H),
7.345-7.367 (m, 3H), 7.302-7.314 (m, 3H), 7.168 (m, 2H), 6.040 (s, 1H), 4.015-4.061 (m, 2H), 2.384 (s,
3H), 2.327 (s, 3H), 2.307 (s, 3H), 1.101-1.125 (m, 3H). 13C NMR (600 MHz, DMSO-d6) δ(ppm):13.85,
19.26, 20.96, 22.40, 54.93, 60.16, 108.66, 119.86, 127.09, 127.39, 127.50×2 ,128.55, 128.69×2, 128.91,
130.42, 131.82, 139.01, 140.33, 140.86, 151.12, 155.73, 164.16, 164.85. ESI-MS m/z: 433.2 (M+H)+,
455.1 (M+Na)+, calcd for C25H24N2O3S: 432.53.
5.1.10.19
Ethyl(Z)-2-(4-morpholinbenzylidene)-7-methyl-3-oxo-5-phenyl-3,5-dihydro-2H-thiazolo
[3,2-a]pyrimidine-6-carboxylate (12d):
Yellow powder, m.p; 179.8-181.0 oC. 1H NMR (600 MHz, DMSO-d6) δ(ppm): 7.679 (s, 1H), 7.460 (d, J
= 9.0 Hz, 2H), 7.332-7.360 (m, 3H), 7.273-7.302 (m, 2H), 7.211 (d, J = 7.2 Hz, 1H), 7.053 (d, J = 7.8 Hz,
1H), 6.043 (s, 1H), 3.992-4.072 (m, 2H), 3.713-3.730 (m, 4H), 3.286-3.309 (m, 4H), 2.385 (s, 3H),
1.089-1.389 (m, 3H). 13C NMR (600 MHz, DMSO-d6) δ(ppm): 174.23, 165.10, 156.14, 144.99, 143.47,
133.66, 132.04, 128.64×2, 128.53, 127.64, 127.32, 126.35×2, 122.10, 114.13, 100.69, 65.78, 59.55×2,
54.01×2, 46.66, 22.68, 17.13, 13.98. 13.87. ESI-MS m/z: 490.1 (M+H)+, 512.1 (M+Na)+, calcd for
C27H27N3O4S: 489.59.
5.1.10.20 Ethyl(Z)-2-(2-Hydroxy-3-methoxybenzylidene)-7-methyl-3-oxo-5-phenyl-3,5-dihydro-2H-
thiazolo[3,2-a]pyrimidine-6-carboxylate (12e):
12

ACCEPTED MANUSCRIPT
Yellow powder, m.p: 232.1-234.4 oC. 1H NMR (600 MHz, DMSO-d6) δ(ppm): 9.816 (br.s, 1H), 7.984 (s,
1H), 7.306-7.350 (m, 2H), 7.291-7.295 (m, 3H), 7.083 (d, J = 9.0 Hz, 1H), 6.909-6.983 (m, 2H), 6.038 (s,
1H), 4.029-4.053 (m, 2H), 3.828 (s, 3H), 2.380 (s, 3H), 1.109-1.133 (m, 3H). 13C NMR (600 MHz,
DMSO-d6) δ(ppm): 13.86, 22.41, 54.84, 55.99, 60.12, 108.52, 114.28, 118.57, 119.68, 119.78, 127.38×2,
128.35, 128.50, 128.67×2, 140.43, 146.66, 148.04, 151.30, 155.98, 164.53, 164.87. ESI-MS m/z: 451.2
(M+H)+, 438.1 (M+Na)+, calcd for C24H22N2O5S: 450.51.
5.1.10.21 Ethyl(Z)-2-(2-naphthalen-1-ylmethylene)-7-methyl-3-oxo-5-phenyl-3,5-dihydro-2H-thiazolo
[3,2-a]pyrimidine-6-carboxylate (12f):
o
1
Yellow powder, m.p: 202.3-204.6 C. H NMR (600 MHz, DMSO-d6) δ(ppm): 8.202 (s, 1H),
8.032-8.060 (m, 2H), 7.965 (d, J = 7.8 Hz, 1H), 7.930-7.933 (m, 1H), 7.698 (d, J = 7.8 Hz, 1H),
7.589-7.631 (m, 2H), 7.293-7.378 (m, 5H), 6.067 (s, 1H), 4.043-4.066 (m, 2H), 3.828 (s, 3H), 2.403 (s, 3H),
1.118-1.142(m, 3H). 13C NMR (600 MHz, DMSO-d6) δ(ppm): 13.86, 22.42, 54.98, 60.18, 108.78, 119.96,
125.92, 127.19, 127.43×2, 127.71, 128.69×2, 128.13, 128.55, 128.76, 129.04, 130.41, 130.93, 132.70,
132.97, 133.36, 140.30, 151.14, 155.58, 164.26, 164.94. ESI-MS m/z: 455.1 (M+H)+, calcd for
C27H22N2O3S: 454.54.
5.1.10.22 Ethyl(Z)-2-[(5-methylthiophen-2-yl)methylene]-7-methyl-3-oxo-5-phenyl-3,5-dihydro-2H-
thiazolo[3,2-a]pyrimidine-6-carboxylate (12g):
Yellow powder, m.p: 180.1-182.2 oC. 1H NMR (600 MHz, DMSO-d6) δ(ppm): 7.966 (s, 1H), 7.498 (d, J
= 3.6 Hz, 1H), 7.333-7.358 (m, 2H), 7.277-7.300 (m, 3H), 7.010 (d, J = 3.6 Hz, 1H), 6.029 (s, 1H),
4.011-4.070 (m, 2H), 2.558 (s, 3H), 2.383 (s, 3H), 1.101-1.132 (m, 3H). 13C NMR (600 MHz, DMSO-d6)
δ(ppm): 13.86, 15.59, 22.39, 54.96, 60.14, 108.55, 115.47, 126.36, 126.52, 127.35×2, 128.01, 128.67×2,
134.98, 135.71, 140.40, 148.40, 151.34, 155.44, 164.03, 164.86. 13C NMR (600 MHz, DMSO-d6) δ(ppm):
13.86, 15.59, 22.39, 54.96, 60.14, 108.55, 115.47, 126.36, 127.35, 128.01×2, 128.51, 128.67×2, 134.98,
135.71, 140.40, 148.40, 151.34, 155.44, 164.30, 164.86. ESI-MS m/z: 425.1 (M+H)+, 447.1 (M+Na)+,
calcd for C22H20N2O3S2: 424.54.
5.1.10.23 Ethyl(Z)-2-[(1-methyl-1H-pyrrol-2-yl)methylene]-7-methyl-3-oxo-5-phenyl-3,5-dihydro-2H-
thiazolo[3,2-a]pyrimidine-6-carboxylate (12h):
Yellow powder, m.p: 221.2-224.5oC. 1H NMR (600 MHz, DMSO-d6) δ(ppm): 7.625 (s, 1H),
7.343-7.355 (m, 2H), 7.281-7.299 (m, 3H), 7.218 (m, 1H), 6.579 (d, J = 2.4 Hz, 1H), 6.321 (d, J = 2.4 Hz,
13

ACCEPTED MANUSCRIPT
1H), 6.035 (s, 1H), 4.030-4.056 (m, 2H), 3.730 (s, 3H), 2.380 (s, 3H), 1.111-1.135 (m, 3H). 13C NMR (600
MHz, DMSO-d6) δ(ppm): 13.98, 22.46, 33.79, 54.02, 59.55, 108.07, 115.28, 126.35×2, 127.30, 127.65,
128.53×2, 128.63, 140.73, 143.47, 144.99, 151.72, 156.06, 164.29, 165.10, 174.23. ESI-MS m/z: 408.4
(M+H)+, 430.3 (M+Na)+, calcd for C22H21N3O3S: 407.49.
5.1.10.24 (5Z)-5-(2-bromobenzylidene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (13a):
1
Yellow powder, m.p: 193.5-195.7 oC. H NMR (600 MHz, DMSO-d6) δ(ppm): 8.519 (s, 1H), 8.277 (s,
1H), 7.876 (d, J = 7.8 Hz, 1H), 7.713 (d, J = 7.8 Hz, 1H), 7.629-7.655 (m, 1H), 7.494-7.523 (m, 1H). 13C
NMR (600 MHz, DMSO-d6) δ(ppm): 125.60, 128.64, 128.91, 129.57, 132.01, 133.03, 133.91, 136.81,
155.73, 159.44, 159.79. ESI-MS m/z: 308.5 (M+H)+, 340.4 (M+CH3OH+H)+, 362.1 (M+CH3OH+Na)+,
calcd for C11H6BrN3OS: 308.15.
5.1.10.25 (5Z)-5-(2,3-dimethoxybenzylidene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (13b):
1
Yellow powder, m.p:196.3-198.8 oC. H NMR (600 MHz, DMSO-d6) δ(ppm): 8.496 (s, 1H), 8.289 (s,
1H), 7.284-7.327 (m, 2H), 7.201-7.216 (m, 1H), 3.879 (m, 3H), 3.851 (m, 3H). 13C NMR (600 MHz,
DMSO-d6) δ(ppm): 56.01, 61.36, 116.71, 120.64, 125.04, 125.83, 125.94, 134.30, 148.44, 152.78, 156.26,
159.32, 159.91. ESI-MS m/z: 288.9 (M)+, 319.9 (M+CH3OH+H)+, calcd for C13H11N3O3S: 289.31.
5.1.10.26 (5Z)-5-(4-morpholinobenzylidene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (13c):
Yellow powder, m.p: 216.3-218.8 oC. 1H NMR (600 MHz, DMSO-d6) δ(ppm): 8.474 (s, 1H), 8.151 (s,
1H), 7.643 (d, J = 8.4 Hz, 2H), 7.115 (d, J = 9.0 Hz, 2H), 3.731-3.747 (m, 4H), 3.368-3.384 (m, 4H). 13
C
NMR (600 MHz, DMSO-d6) δ(ppm): 46.45×2, 65.76×2, 114.02×2, 117.46, 121.35, 133.19×2, 140.43,
152.95, 156.46, 158.94, 159.04. ESI-MS m/z: 315.3 (M+H)+, 347.2 (M+CH3OH+H)+, calcd for
C15H14N4O2S: 314.36.
5.1.10.27 (5Z)-5-[(5-methylthiophen-2-yl)methylene]thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (13d):
1
Yellow powder, m.p: 206.3-208.8 oC. H NMR (600 MHz, DMSO-d6) δ(ppm): 8.481 (s, 1H), 8.470 (s,
1H), 7.129 (d, J = 3.6 Hz, 1H), 7.104 (d, J = 3.6 Hz , 1H), 2.610 (s, 3H). 13C NMR (600 MHz, DMSO-d6)
δ(ppm): 15.73, 119.64, 128.40, 133.14, 134.27, 138.09, 150.89, 156.11, 158.58, 159.12. ESI-MS m/z: 250.2
(M+H)+, 282.0 (M+CH3OH+H)+, 303.9 (M+CH3OH+Na)+, calcd for C10H7N3OS2: 249.31.
5.1.10.28 (5Z)-5-[(1H-indol-3-yl)methylene]thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (13e):
Yellow powder, m.p: 315.4-317.3 oC. 1H NMR (600 MHz, DMSO-d6) δ(ppm): 9.470䟉s, 1H), 8.520 (s,
1H), 8.465 (s, 1H), 8.380 (d, J = 7.8 Hz, 1H), 8.162 (s, 1H), 8.162 (d, J = 8.4 Hz, 1H), 7.492-7.497 (m, 1H),
14

ACCEPTED MANUSCRIPT
7.440-7.453 (m, 1H). 13C NMR (600 MHz, DMSO-d6) δ(ppm): 114.81, 116.21, 119.48, 123.96, 124.51,
126.38, 129.43, 130.00, 134.96, 155.90, 158.69, 159.31, 170.02. ESI-MS m/z: 269.1 (M+H)+, 301.1
(M+CH3OH+H)+, calcd for C13H8N4OS: 268.29.
5.1.10.29 (2Z)-2-(2-chlorobenzylidene)-5,6-dihydroimidazo[2,1-b]thiazol-3(2H)-one (11a):
o
o
Yellow powder, m.p: 172.0-173.9 C, [169 C, lit.[32]]. ESI-MS m/z: 265.3 (M+H)+, calcd for
C12H9ClN2OS: 264.73.
5.1.10.30 (2Z)-2-(4-morpholinobenzylidene)-5,6-dihydroimidazo[2,1-b]thiazol-3(2H)-one (11b):
Yellow powder, m.p: 212.2-124.9 oC. 1H NMR (600 MHz, DMSO-d6) δ(ppm): 7.545 (s, 1H), 7.444 (d, J
= 9.0 Hz, 2H), 7.052 (d, J = 9.0 Hz, 2H), 4.264-4.292 (m, 2H), 3.775-3.802 (m, 2H), 3.726-3.742 (m, 4H),
3.230-3.260 (m, 4H). 13C NMR (600 MHz, DMSO-d6) δ(ppm): 41.59, 47.02×2, 60.73, 65.84×2, 114.38×2,
121.88, 122.56, 129.89, 131.07×2, 151.70, 156.52, 159.83. ESI-MS m/z: 316.4 (M+H)+, calcd for
C16H17N3O2S: 315.39.
5.1.10.31
(11c):
(2Z)-2-[(5-methylthiophen-2-yl)methylene]-5,6-dihydroimidazo[2,1-b]thiazol-3(2H)-one
Yellow powder, m.p: 182.1-183.9 oC. 1H NMR (600 MHz, DMSO-d6) δ(ppm): 7.819 (s, 1H), 7.423 (d, J
= 3.6 Hz, 1H), 6.980 (d, J = 3.0 Hz, 1H), 4.267-4.295 (m, 2H), 3.775-3.802 (m, 2H), 2.470 (s, 3H). 13C
NMR (600 MHz, DMSO-d6) δ(ppm): 15.41, 41.71, 60.79, 123.35, 123.50, 127.42, 133.75, 134.93, 146.13,
156.22, 159.50. ESI-MS m/z: 251.0 (M+H)+, calcd for C11H10N2OS2: 250.34.
5.1.10.32
(11d):
(2Z)-2-[(1-methylpyrrol-2-yl)methylidene]-5,6-dihydroimidazo[2,1-b][1,3]thiazol-3-one
o
1
Yellow powder, m.p: 221.4-223.3 C. H NMR (600 MHz, DMSO-d6) δ(ppm): 7.496 (s, 1H),
7.118-7.124 (m, 1H), 7.458(d, J = 3.6 Hz, 1H), 6.262(d, J = 3.6 Hz, 1H), 4.261-4.288 (m, 2H), 3.767-3.794
(m, 2H), 3.739 (s, 3H). 13C NMR (600 MHz, DMSO-d6) δ(ppm): 32.78, 41.46, 60.70, 109.71, 113.19,
117.70, 120.61, 127.10, 127.98, 156.75, 159.95. ESI-MS m/z: 234.0 (M+H)+, calcd for C11H11N3OS:
233.29.
5.2 Biological Assays
5.2.1 Cell line and reagents
Mouse RAW 264.7 macrophages were obtained from the American Type Culture Collection (ATCC,
Manassas, VA). Cell culture reagents were obtained from Gibco (Eggenstein, Germany). Fetal bovine
15

ACCEPTED MANUSCRIPT
serum was from HyClone (Logan, UT) and was heat-inactivated for 30 min at 65 oC. LPS purchased from
Sigma (St. Louis, MO) was dissolved in PBS. Omeprazole and compounds were dissolved in DMSO for in
vitro experiments.
5.2.2 Cell treatment and ELISA assay
Mouse RAW 264.7 macrophages were cultured in DMEM media (Gibco) supplemented with 10% FBS,
o
100 U/mL penicillin, and 100 mg/mL streptomycin at 37 C with 5% CO2. Cells were pretreated with
compounds or vehicle control for 2 h, then treated with LPS (0.5 ꢀg/ml) for 22 h. After treatment, the
culture media and cells were collected separately. The culture media collected were centrifuged at 1000
rpm for 10 min. The levels of TNF-α and IL-6 in the media were determined by ELISA using mouse
TNF-α and mouse IL-6 ELISA Kits (eBioscience, San Diego, CA). After centrifugation, the supernatant
o
was separated and stored at -80 C until use. Cells were washed with PBS and harvested with cell lysis
buffer (Tris-HCl 20 mM, NP40 1%, NaCl 150 mM, EDTA 2 mM, Na3VO4 200 mM, SDS 0.1%, NaF 20
mM). The mixed liquor was shaken vigorously for 10 min in lysis buffer at 0 oC. After being centrifuged at
12,000 rpm for 5 min at 4 oC, the total protein was collected and the concentrations were determined using
Bio-Rad protein assay reagents. The total amount of the inflammatory factor in the media was normalized
to the total protein amount of the viable cell pellets.
5.2.3 Compound 12d attenuated LPS-induced inflammatory mortality in C57BL/6 mice
Compound 12d was firstly dissolved with macrogol 15 hydroxystearate (a nonionic solubilizer for
injection from BASF) with or without medium chain triglycerides (MCT, from BASF) in water bath at 37
oC. The concentration of 12d was 2 mg/mL. The concentration of solubilizer was ranged 5䠟10%, and
MCT 0.5䠟2% in final solution. For the vehicle, the mixture of solubilizer and MCT was prepared at 10%
and 2% respectively. Male C57BL/6 mice were obtained from the Animal Center of Wenzhou Medical
College (Wenzhou, China). Animals were housed at a constant room temperature with a 12:12 hour
light-dark cycle, and fed with a standard rodent diet and water. The animals were acclimatized to the
laboratory for at least 7 days before used in experiments. Protocols involving the use of animals were
approved by the Wenzhou Medical College Animal Policy and Welfare Committee (Approval documents:
2009/APWC/0031). Mice weighing 18-22 g were treated with 12d solution (150 ꢀL, 15 mg/kg) by i.p.
injection either 15 min before or 15 min after the i.v. injection of LPS (20 mg/kg). Control animals received
a similar volume (200 ꢀL) of vehicle. Body weight change and mortality were recorded for 7 days.
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Acknowledgement
Financial support was provided by the National Natural Science Funding of China (21272179 &
81173140), High-level Inovative Talent Funding of Zhejiang Department of Health (G. L.), National “863”
project (2011AA02A113), Zhejiang Natural Science Funding (LY12H16003), and Zhejiang Key Group
Project in Scientific Innovation (2010R50042).
References:
[1] H. Tilg, A.R. Moschen, Adipocytokines: mediators linking adipose tissue, inflammation and
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Figure Legends
Figure 1. A brief illustration for the design of intermediates and target thiazolidinones.
Scheme 1. General synthesis and chemical structures of thiazolidinone derivatives (10a-10o and 11a-11d)
Scheme 2. General synthesis and chemical structures of thiazolidinone derivatives (12a-12h).
Scheme 3. General synthesis and chemical structures of thiazolidinone derivatives (13a-13e).
Figure 2. Thiazolidinone derivatives inhibited LPS-induced TNF-α and IL-6 secretion in RAW 264.7
o
macrophages. Macrophages were plated at a density of 4.0×105/plate for overnight in 37 C and 5% CO2.
Cells were pre-treated with curcumin (cur, 10 ꢀM) or compounds (10 ꢀM) for 2 h, then treated with LPS
(0.5 ꢀg/ml) for 22 h. TNF-α and IL-6 levels in the culture medium were measured by ELISA and were
normalized by the total protein. The results were expressed as the percentage of LPS control. Each bar
represents mean ±SEM of 4 independent experiments. Statistical significance relative to LPS is indicated,
*p < 0.05; **p < 0.01.
Figure 3. 12d and 12h inhibited LPS-induced TNF-α and IL-6 release in a dose-dependent manner in
o
RAW264.7 macrophages. Macrophages were plated at a density of 1.2×106/plate overnight in 37 C and
5% CO2. Cells were pre-treated with 12d and 12h at indicated concentrations for 2 h, followed by LPS (0.5
20

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ꢀg/ml) treatment for 22 h. TNF-α and IL-6 levels in the culture medium were measured by ELISA and were
normalized to the total protein amount. The results are expressed as percent of LPS control. Each bar
represents mean ±SEM of 3-5 independent experiments. Statistical significance relative to LPS is indicated,
*p < 0.05; **p < 0.01.
Figure 4. 12d improves survival of mice subjected to a lethal dose of LPS. Mice were treated with 15
mg/kg 12h (i.p.) either 15 min before or 15min after the injection of 20 mg/kg of LPS (i.v.). Survival rats
were recorded for 7 days after the LPS injection at the interval of 1 day. N=12 animals in each group (**
p<0.01 vs LPS group).
21

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Highlights
ꢀ Synthesis of four series of thiazolidinone derivatives.
ꢀ thiazolo[3,2-a]pyrimidine derivatives showed strongest anti-inflammatory
activity among 32 compounds.
ꢀ Presenting an important lead in the anti-inflammatory drug research.

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S
HN
Thiazolidinone
O
CH3COONa,
Acetic acid
R1
R2
N
S
R1
S
R3
S
R3
CHO
N
N
N
reflex, 3h
19.8-79.7%
R2
O
O
O
10
Thiazolidinone derivatives
10a-10o, 11a-11d, 12a-12h and 13a-13e
N
S
N
S
N
EtOOC
N
N
S
N
O
N
O
11
12
O
13
Figure 1
Scheme 1

ACCEPTED MANUSCRIPT
Scheme 2
ClCH2COOEt
EtOH, Pyridine
N
N
N
H2N
H2N
S
H2N
S
20% NaOH
HCOOH
S
SH
N
N
reflux, 1h
71%
reflux, 3h
62.5%
NH
reflux, 2h
100%
OHC NH
N
H
N
13
H
O
7
9
8
CH3COONa,
Acetic acid,
R4-CHO
Br
reflex, 3h
40.8-79.7%
13a, R4 =
(79.7% yield)
(40.8% yield)
N
S
13d, R4 =
13e, R4 =
(48.6% yield)
(76.9% yield)
CH3
OCH3
S
13b, R4 = H3CO
N
N
R4
O
O
13a-13e
NH
N
13c, R4 =
(59.4% yield)
Scheme 3

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A
200
150
100
50
TNF-α
*
*
**
**
*
*
*
*
*
*
**
**
**
0
LPS 0.5µg/ml
150
100
50
B
IL-6
*
*
**
*
*
*
*
*
**
*
*
**
**
*
**
**
**
**
**
**
**
**
**
0
LPS 0.5ug/ml
Figure 2

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A
150
100
50
IL-6
TNF-α
IC50 = 0.83 µM
IC50 = 4.17 µM
*
*
*
**
**
**
**
**
0
LPS 0.5µg/mL + 12d
LPS 0.5µg/mL + 12d
B
150
100
50
TNF-α
IL-6
IC50 = 6.39µM
IC50 = >10µM
**
**
**
*
**
0
LPS 0.5µg/mL + 12h
LPS 0.5µg/mL + 12h
Figure 3
100
Vehicle
Vehicle+LPS(20mg/kg)
80
60
40
20
0
LPS(20mg/kg) + 12d
(15mg/kg) (for treatment)
**
12d (15mg/kg)+LPS
(20mg/kg) (for prevention)
0
1
2
3
4
5
6
7
Days
Figure 4

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Cytotoxicity assay for synthetic thiazolidinone derivatives
Method: MTT (methyl thiazolyl tetrazolium) assay
RAW264.7 macrophages were seeded into 96-well plates at a density of 5000 cells per well in 1640
medium, supplemented with 5% heat-inactivated serum, 100 U/ml penicillin, and 100 µg/ml
o
streptomycin. The cells were maintained at 37 C in a humidified atmosphere containing 5% CO2. All
experiments were carried out 24 h after cells were seeded. Tested compounds were dissolved in DMSO
and diluted with 1640 medium to the final concentrations of 20 µM. The tumor cells were incubated with
test compounds for 48 h before the MTT assay. A fresh solution of MTT (5 mg/ml) prepared in NaCl
solution (0.9%) was added to each single well of the 96-well plate. The plate were then incubated in a
CO2 incubator for 3 h, cells dissolved with 100 µl DMSO, and then analyzed in a multi-well-plate reader
at 570 nM.
Result:
Before the in vitro anti-inflammatory evaluation, synthetic thiazolidinone derivatives were tested for
their cytotoxicity and safety in RAW264.7 macrophages. Cell viability was determined by MTT method
after treatment with compounds for 48 h. As shown in Figure S1, these compounds at 20µM showed low
toxicity or non-toxicity.
Figure S1. Compounds showed low toxicity or non-toxicity on RAW264.7 macrophages.
S2