Synthesis, biological evaluation and molecular modeling of dihydro-pyrazolyl-thiazolinone derivatives as potential COX-2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.09.021

A series of dihydro-pyrazolyl-thiazolinone derivatives (5a-5t) have been synthesized and their biological activities were also evaluated as potential cyclooxygenase-2 (COX-2) inhibitors. Among these compounds, compound 2-(3-(3,4-dimethylphenyl)-5-phenyl-4

Full text of DOI:10.1016/j.bmc.2012.09.021

Bioorganic & Medicinal Chemistry 20 (2012) 6648–6654
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and molecular modeling of
dihydro-pyrazolyl-thiazolinone derivatives as potential COX-2 inhibitors
Ke-Ming Qiu a, Ru Yan a, Man Xing a, Hai-Hong Wang a, Hong-En Cui a, Hai-Bin Gong b, , Hai-Liang Zhu a,
⇑
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b Xuzhou Central Hospital, Xuzhou 221009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dihydro-pyrazolyl-thiazolinone derivatives (5a–5t) have been synthesized and their biological
activities were also evaluated as potential cyclooxygenase-2 (COX-2) inhibitors. Among these com-
pounds, compound 2-(3-(3,4-dimethylphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-
Received 20 August 2012
Revised 11 September 2012
Accepted 12 September 2012
Available online 21 September 2012
one (5a) displayed the most potent COX-2 inhibitory activity with IC50 of 0.5 lM, but weak to COX-1.
Docking simulation was performed to position compound 5a into the COX-2 active site to determine
the probable binding model. Based on the preliminary results, compound 5a with potent inhibitory activ-
ity and low toxicity would be a potential and selective anti-cyclooxygenase-2 agent.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Pyrazole
Thiazolinone
COX-2
Anti-inflammatory
1. Introduction
is an important target in order to reduce adverse side effects during
non-steroidal anti-inflammatory treatment, thus improving thera-
Inflammation is a normal response to any noxious stimulus that
threatens the host and may vary from a localized response to a gen-
eralized one.1 Non-steroidal anti-inflammatory drugs (NSAIDs)
such as aspirin, ibuprofen and indomethacin are widely used in
the treatment of acute and chronic inflammatory diseases.2 How-
ever, the long-term clinical employment of NSAIDs is associated
with significant side effects such as gastrointestinal lesions, bleed-
ing and nephrotoxicity.3,4 The clinical efficacy of most NSAIDs is
closely to their inhibition of cyclooxygenase (COXs), which catalyze
the bioconversion of arachidonic acid (AA) to prostaglandins
(PGs).5,6 COXs are membrane-bound hemeprotein which exist in
three distinct isoforms: COX-1, COX-2 and COX-3.7 COX-1 is consti-
tutive form which is present in healthy tissues and responsible for
the thrombogensis and the homeostasis. COX-2 is an inducible
enzyme which is induced in response to the release of several
pro-inflammatory mediators. COX-3 is located in the central
nervous system and could be the pharmacological target of
acetaminophen. Researches showed that the major side effect of
NSAIDs was due to their inhibition of COX-1.8–10 It was thought that
selective COX-2 inhibitors would be effective anti-inflammatory
agents with diminished side effects.11,12 Therefore, the develop-
ment of compounds that would inhibit COX-2 almost exclusively
peutic benefits.
The pyrazole scaffold represents a common motif in many phar-
maceutical actively and remarkable compounds demonstrating a
wide range of pharmacological activities; the most important
activities are anti-tumor,13 anti-bacterial,14 anti-inflammatory,15
the inhibition of cyclooxigenase-216 and CDK2/Cyclin A.17 Litera-
ture survey revealed that many pyrazole derivatives had been
found their clinical application as NSAIDs.18 Among the highly
marketed COX-2 inhibitors that comprise the pyrazole nucleus,
celecoxib is the one which is treated as a safe anti-inflammatory
and analgesic agent.19
As showed in Figure 1, the well-known COX-2 inhibitor cele-
coxib and SC-558 consist of a pyrazole core and coterminous sub-
stitutional benzene rings. Modeled on this structure, we have
designed a series of novel compounds (Fig. 1) by sharing a similar
template with celecoxib, with the presumption that the pyrazole
group is a common nucleus of COX-2 inhibitors which inhibits
COX-2 by binding to its active site.
On the other hand, thiazolinone is an important class of
heterocyclic compounds, which exhibit anti-inflammatory, anti-
proliferative, anti-viral, and anti-bacterial ability.20–22 Synthesis of
the thiazolinones occupies an important place in the realm of
synthetic pharmaceutical chemistry, due to their therapeutic and
pharmacological properties. Athina A. Geronikaki et al. reported that
⇑
Corresponding authors. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail addresses: ghbxzh@yahoo.com.cn (H.-B. Gong), zhuhl@nju.edu.cn
2-(thiazole-2-ylamino)-5-phenylidene-4-thiazolidinone
deriva-
tives (5P–4T) (Fig. 1) were potent inhibitors of COXs.23 Maccari
(H.-L. Zhu).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.021

K.-M. Qiu et al. / Bioorg. Med. Chem. 20 (2012) 6648–6654
6649
Figure 1. Chemical structures of some reported compounds.
et al. reported that 5-(4-methoxyphenylidene)-2-phenylimino-3-
2. Results and discussion
2.1. Chemistry
propyl-4-thiazolidinone (3P–4T) (Fig. 1) had even showed better
COX-2 selectivity than celecoxib and its docking result had given
a proof of the auxiliary.24
On that basis, in this article, a thiazolinone heterocycle was
introduced into the pyrazoline moiety to construct a 2-(3-(phe-
nyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl) thiazolinone template
with the reasoning that this template could integrate potently with
the residues in COX-2 active domain through the hydrophobic
The synthesis of compounds 5a–5t is followed the general path-
way outlined in Scheme 1. Firstly, the chalcones were obtained by
direct condensation by the aromatic aldehydes and the substituted
acetophenone, using 40% potassium hydroxide as catalyst in etha-
nol. Secondly, cyclization of different chalcones with thiosemicar-
bazide under basic condition leads to the formation of pyrazole
derivatives containing thiourea skeleton. Thirdly, dihydro-pyrazol-
yl-thiazolinone derivatives 5a–5t were obtained by reacting com-
pound 4 (1 equiv) with bromoacetic acid (1.2 equiv) in acetic
acid and keeping under stirring at 80 °C for 6–8 h. Moreover, acetic
anhydride (2 equiv) was added as dehydrating agent and sodium
acetate (2 equiv) was used as acid-binding agent. All of the syn-
thetic compounds are reported for the first time and gave satisfac-
tory analytical and spectroscopic data, which were in full
accordance with their depicted structures.
interactions, strengthened by hydrogen bonds and p-cation bonds.
In continuation to extend our research on anti-inflammatory com-
pounds and screen for novel and powerful COX-2 inhibitors, it is
supposed to be worthful to synthesize a series of dihydro-pyrazol-
yl-thiazolinone derivates (5a–5t) as a new class of COX-2 inhibi-
tors combined with pyrazole and thiazolinone considering that
they might exhibit synergistic effect in anti-inflammatory activi-
ties by the inhibition against COX-2. Herein, we reported in the
present work the synthesis, biological evaluation and molecular
modeling of dihydro-pyrazolyl-thiazolinone derivatives as poten-
tial COX-2 inhibitors.
Scheme 1. General synthesis of dihydro-pyrazolyl-thiazolinone derivatives (5a–5t). Reagents and conditions: (i) 40% aqueous potassium hydroxide solution, ethanol, rt; (ii)
thiosemicarbazide, KOH, ethanol, reflux; (iii) bromoacetic acid, acetic anhydride, sodium acetate, acetic acid, 80 °C, 6–8 h.

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K.-M. Qiu et al. / Bioorg. Med. Chem. 20 (2012) 6648–6654
2.2. Cytotoxicity test
2.3. In vitro enzyme inhibition activity
The inhibitory activity of the compounds is sometimes a result
of their toxic effect and consequently might cause an erroneous
conclusion, prior to the following bioactivity analyses, all the syn-
thesized dihydro-pyrazolyl-thiazolinone derivatives were evalu-
ated using LPS-activated murine macrophages RAW264.7 for
cytotoxicity test.25 The pharmacological results were summarized
in Table 1. The data showed that most of dihydro-pyrazolyl-
thiazolinone derivatives having a quite low toxicity.
The synthesized derivatives were evaluated for their ability to
inhibit the COX-1 and COX-2 isozyme using a solid-phase ELISA as-
say. The results were summarized in Table 2. It was observed that
dihydro-pyrazolyl-thiazolinone derivatives containing thiazoli-
none and pyrazole rings showed fairly good inhibiting COX-2 activ-
ities displaying IC50 values from 0.5 to 19.4
Among them, compound 5a revealed the most potent inhibitory
activity (IC50 = 0.5 M for COX-2) and best selectivity (SI = 84.8
lM, but weak to COX-1.
l
for 5a contrast to SI = 100 for celecoxib). The results of western
blotting assay further confirmed that compound 5a with the con-
centration above 1.25 lM could obviously inhibit lipopolysaccha-
Table 1
ride (LPS)-induced COX-2 expression in murine macrophage RAW
Cytotoxicity assay of the compounds in RAW264.7 cells
264.7 cell line (Fig. 2).
Compound
R1
R2
CC50
(l
M)a
SDb
Subsequently SAR studies were performed by modification of
the parent compound to determine how the substituents of the
subunits affect the COX-2 inhibitory activities. Inspection of the
chemical structures of compounds 5a–5t suggested that they could
be divided into two subunits: A and B rings (Scheme 1). As showed
in Table 2, it was obvious that compounds with electron-donating
substituents at para-position and meta-position at A-ring (3, 4-
5a
5b
5c
5d
5e
5f
5g
5h
5i
3,4-2Me
3,4-2Me
3,4-2Me
3,4-2Me
3,4-2Me
3,4-2Me
3,4-2Me
3,4-2Me
3,4-2Me
3,4-2Me
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
4-H
4-F
4-Cl
>450
20.36
20.43
18.97
12.86
24.32
19.52
9.05
12.54
11.37
15.57
14.69
8.34
10.92
14.58
14.17
13.27
15.32
16.25
7.34
246.24
225.63
365.34
383.58
387.46
178.32
236.78
227.30
316.65
295.17
138.98
187.24
279.51
279.35
264.43
165.22
203.59
143.27
178.63
4-Br
4-Me
4-OMe
3-Cl
3-Me
2-Cl
2-Me
4-H
4-F
2Me, IC50 = 0.5–7.2
than that with electron-withdrawing substituents at the same po-
sition (3, 4-2Cl, IC50 = 2.7–19.4 M for COX-2). In the case of con-
lM for COX-2) had better inhibitory activity
5j
5k
5l
l
stant A ring substituents, the groups at B ring also have a great
significance. Compounds 5a–5f, 5k–5p with para substituents on
phenyl ring exhibited significant COX-2 inhibitory activities in
the order of H > Br > Cl > F, H > Me > OMe. The results demon-
strated that an electron-withdrawing halogen group and an elec-
tron donating methyl/methoxyl substituent may show some
relatively negative effects. Faintish electron-withdrawing substitu-
ent (para-Br) (5d, 5n) and faintish electron-donating substituent
(para-Me) (5e, 5o) had relatively minor effects compared
with 5a, whereas strong electron-withdrawing substituent
(para-F/para-Cl) (5b, 5l/5c, 5m) or powerful electron-donating
substituent (para-OMe) (5f, 5p) led to a relatively poor result.
Considering the steric effect of substituents at B-ring, we de-
signed and evaluated the compounds with ortho-Cl-phenyl (5i,
5s), ortho-Me-phenyl (5j, 5t), meta-Cl-phenyl (5g, 5q) and meta-
Me-phenyl (5h, 5r) group substituents in the B-ring. From the
activity datas from Table 2, it can be seen that compounds with
substituents at the para-position (5c, 5m) showed more potent
activities than those with substituents at meta-position (5g, 5q).
Meanwhile, a significant loss of activity was observed when the
halogen substituent was moved from the para-position (4c, 4m)
to the ortho-position (5i, 5s) in the B-ring. The modification of
methyl group in the B ring also apply to this rule with the order
of para > meta > ortho.
5m
5n
5o
5p
5q
5r
5s
5t
4-Cl
4-Br
4-Me
4-OMe
3-Cl
3-Me
2-Cl
2-Me
8.93
The CC50 (cytoxicity) represents the concentration of drug that is required for 50%
cell kill (cytoxicity) of the uninfected cells.
The cytotoxicity of each compound was expressed as the concentration of
compound that reduced cell viability to 50% (CC50).
The data represented the mean of three experiments in triplicate and were
expressed as means SD. SD means The Standard deviation values.
a
b
Table 2
Date of the in vitro COX-1/COX-2 enzyme inhibition assay of the designed compounds
Compound
R1
R2
IC50
COX-1
(l
M)a
COX-2
SIb
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
3,4-2Me
3,4-2Me
3,4-2Me
3,4-2Me
3,4-2Me
3,4-2Me
3,4-2Me
3,4-2Me
3,4-2Me
3,4-2Me
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
4-H
4-F
4-Cl
42.4
40.8
48.6
>50
45.3
>50
>50
>50
44.1
47.2
33.2
34.3
37.7
48.5
44.3
>50
>50
>50
40.2
45.6
10
0.5
84.8
6.4
6.4
4.5
1.8
1.2
2.9
5.2
2.7
7.2
6.3
2.7
19.4
11.7
7.6
4.7
6.8
16.1
7.2
18.4
16.3
0.1
10.8
>27.8
37.8
>17.2
>9.6
>18.5
6.1
7.5
12.3
1.8
3.2
6.4
9.4
>7.4
>3.1
>6.9
2.2
2.8
100
4-Br
4-Me
4-OMe
3-Cl
3-Me
2-Cl
2.4. Molecular docking study
2-Me
4-H
To gain better understanding on the potency of the synthesized
compounds and guide further structure–activity relationships
4-F
4-Cl
4-Br
4-Me
4-OMe
3-Cl
3-Me
2-Cl
5s
5t
2-Me
Celecoxib
a
IC50 value is the compound concentration required to produce 50% inhibition of
COX-1 or COX-2 for means of two determinations and deviation from the mean is
<10% of the mean value.
b
Selectivity index (SI = COX-1 IC50/COX-2 IC50).
Figure 2. Inhibitory effect of compound 5a on COX-2 protein expression.

K.-M. Qiu et al. / Bioorg. Med. Chem. 20 (2012) 6648–6654
6651
(SAR) studies. The molecular docking was performed by putting
inhibitor 5a into binding site of COX-2, the same binding site of
SC-558 (Fig. 1).26 All docking runs were applied by Discovery Stu-
dio3.1 (DS. 3.1). The binding models of compound 5a with COX-2
were depicted in Figure 3A and 3B. The amino acid residues which
had interaction with 5a in the active site were labeled. We can see
that compound 5a was nicely bound to the COX-2 via hydrophobic
interaction and the binding was stabilized by two hydrogen bonds
respectively). In addition, a
p
-cation interaction (3.95 Å) was
formed by Arg 120 with the benzene ring, which enhanced the
binding action between receptor COX-2 and ligand compound 5a.
The electron-donating substituent amino-NH2 strengthened the
p-cation binding and the results indicated that it was primarily
due to direct through-space interaction between the substituent
and the cation. Overall, these results can provide a good explana-
tion for the high potency of compound 5a towards COX-2 protein.
It is known that the main difference between the two COX ac-
tive sites is the replacement of Ile 523 in COX-1 by Val 523, a less
bulky amino acid. This replacement makes COX-2 binding site 25%
larger than the COX-1 and creates an adjunct pocket in the COX-2
active site which may be responsible for selectivity.24
and a p-cation interaction. It was shown that the S and O atoms in
the thiazolinone ring of compound 5a exhibited hydrogen bonds
with His 90 and Ile 517 in COX-2 active site (distance:
N–HÁ Á ÁS = 1.98 Å, 159.46° and N–HÁ Á ÁO = 2.37 Å, 136.44°,
Our docking study showed that compound 5a was oriented so
that its phenyl-pyrazoline ring and thiazolinone ring could fill this
adjunct pocket. But when we docked 5a to the active site of COX-1,
the binding site of flurbiprofen, we found it’s hard to combine
(compound 5a could not get into the active site of COX-1). This
may explain why compound 5a shows significant selective activity
against COX-2 enzyme.
3. Conclusions
In this paper, we have prepared a series of dihydro-pyrazolyl-
thiazolinone derivatives (5a–5t) and evaluated their biological
activities. Preliminary results showed that most of the compounds
displayed enhanced inhibitory activities and low toxicity. Among
them, compound 5a was most active (IC50 = 0.5
as it showed the half maximal inhibitory concentration (IC50) most
close to the positive control celecoxib (IC50 = 0.1 M for COX-2).
lM for COX-2),
l
Moreover, its selectivity between COX-1/COX-2, which was repre-
sented by SI value was very close to celecoxib (SI = 84.8 for 5a and
100 for celecoxib).
Figure 3A. Molecular docking 3D modeling of compound 5a with COX-2: for
Molecular docking was further performed to study the inhibi-
tor-COX-2 protein interactions. After analysis of the binding model
of compound 5a with COX-2, it was found that two hydrogen
clarity, only interacting residues are displayed. The H-bond (green lines) is
displayed as dotted lines, and the p-cation interaction is shown as orange lines.
bonds and a
p-cation interaction with the receptor protein resi-
dues in the active site might play a crucial role in its COX-2 inhibi-
tion and anti-inflammatory activities. Docking result also had
showed that 5a could not even bind into the same active site of
COX-1, probable with the reason that the pocket of COX-1 is 25%
smaller than that of COX-2 by the replacement of Ile 523 in COX-
1 by Val 523, so originating a secondary internal pocket not acces-
sible in COX- 1.27
All of this told us that compound 5a was a favorable and selec-
tive COX-2 inhibitor, which displayed anti-inflammatory via the
COX-2 pathway and was scarcely any toxicity due to little COX-1
inhibition. The results of this study help to find a promising com-
pound with stronger activities and low toxicity toward the devel-
opment of new therapeutic agent to fight against inflammation.
4. Experimental protocols
4.1. Materials and measurements
All chemicals and reagents used in current study were analyti-
cal grade. All the 1H NMR spectra were recorded on a Bruker DPX
300 or DRX 500 model Spectrometer in DMSO-d6 and chemical
shifts were reported in ppm (d). ESI-MS spectra were recorded on
a Mariner System 5304 Mass spectrometer. Elemental analyses
were performed on a CHN-O-Rapid instrument. TLC was performed
on the glass-backed silica gel sheets (silica gel 60 Å GF254) and
visualized in UV light (254 nm). Column chromatography was per-
formed using silica gel (200–300 mesh) eluting with ethyl acetate
and petroleum ether.
Figure 3B. Molecular docking 2D modeling of 5a with COX-2: for clarity, only
interacting residues are displayed. The H-bond (green lines and blue lines) is
displayed as arrow dots, and the p-cation interaction is shown as orange lines.

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K.-M. Qiu et al. / Bioorg. Med. Chem. 20 (2012) 6648–6654
4.2. Synthesis
4.2.3.5. 2-(3-(3,4-Dimethylphenyl)-5-(p-tolyl)-4,5-dihydro-1H-
pyrazol-1-yl)thiazol-4(5H)-one (5e). Yield 82%; mp 219–
4.2.1. General synthetic procedure of chalcones (3a–3t)
221 °C. 1H NMR (CDCl3, 300 MHz): 2.33 (s, 9H), 3.36 (dd,
J1 = 3.48 Hz, J2 = 17.76 Hz, 1H), 3.72 (s, 2H), 3.81 (d, J = 17.01 Hz,
1H), 5.84 (d, J = 7.89 Hz, 1H), 7.10–7.18(m, 4H), 7.21 (d,
J = 7.86 Hz, 1H), 7.51 (d, J = 7.86 Hz, 1H), 7.59 (d, J = 6.78 Hz, 1H).
ESI-MS: 364.48 (C21H22N3OS, [M+H]+). Anal. Calcd for C21H21N3OS:
C, 69.39; H, 5.82; N, 11.56. Found: C, 69.37; H, 5.81; N, 11.59.
Equimolar portions of the appropriately aromatic aldehydes
(3 mmol, 1 equiv) and substituted acetophenone (3 mmol, 1 equiv)
were dissolved in approximately 20 mL of ethanol. The mixture
was allowed to stir for several minutes at 0 °C to let dissolve. Than
a 1 mL aliquot of a 40% aqueous potassium hydroxide solution was
then slowly added dropwise to the reaction flask via a self-equaliz-
ing addition funnel. The reaction solution was allowed to stir at
room temperature for approximately 4–6 h. Most commonly, a
precipitate formed and was then collected by suction filtration.
4.2.3.6.
2-(3-(3,4-Dimethylphenyl)-5-(4-methoxyphenyl)-4,5-
Yield 86%;
dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-one (5f).
mp 223–225 °C. 1H NMR (CDCl3, 300 MHz): 2.33 (s, 6H), 2.38 (s,
3H), 3.35 (d, J = 15.32 Hz, 1H), 3.81 (s, 2H), 3.92 (dd, J1 = 4.38 Hz,
J2 = 18.02 Hz, 1H), 5.86 (d, J = 7.64 Hz, 1H), 6.98 (d, J = 7.38 Hz,
2H), 7.16- 7.18 (m, 3H), 7.54 (d, J = 5.46 Hz, 2H). ESI-MS: 380.48
(C21H22N3O2S, [M+H]+). Anal. Calcd for C21H21N3O2S: C, 66.47; H,
5.58; N, 11.07. Found: C, 66.53; H, 5.61; N, 11.11.
4.2.2. General synthetic procedure of pyrazole derivatives
(4a–4t)
A mixture of chalcone (2 mmol), thiosemicarbazide (3 mmol),
and KOH (2 mmol) was refluxed in ethanol (30 mL) for 12 h. After
cooling, the solution was poured into mass of ice-water and stirred
for a few minutes. The precipitate was filtered and crystallized
from ethanol.
4.2.3.7.
2-(5-(3-Chlorophenyl)-3-(3,4-dimethylphenyl)-4,5-
Yield 78%;
dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-one (5g).
mp 229–231 °C. 1H NMR (CDCl3, 300 MHz): 2.33 (s, 6H), 3.38 (d,
J = 14.84 Hz, 1H), 3.85 (s, 2H), 3.98 (d, J = 16.84 Hz, 1H), 5.82 (d,
J = 8.22 Hz, 1H), 7.14–7.19 (m, 2H), 7.31–7.34 (m, 2H), 7.49 (d,
J = 4.34 Hz, 1H), 7.54 (d, J = 6.34 Hz, 1H), 7.60 (s, 1H). ESI-MS:
384.89 (C20H19ClN3OS, [M+H]+). Anal. Calcd for C20H18ClN3OS: C,
62.57; H, 4.73; N, 10.95. Found: C, 62.62; H, 4.75; N, 10.91.
4.2.3. General synthetic procedure of dihydro-pyrazolyl-
thiazolinone derivatives (5a–5t)
A
mixture of compound 4 (1 mmol), bromoacetic acid
(1.2 mmol), acetic anhydride (2 mmol), and sodium acetate
(2 mmol) was dissolved in acetic acid (20 mL) and kept stirring
in 80 °C for 6–8 h. After cooling, the solution was poured into mass
of ice-water and stirred for a few minutes. The precipitate was fil-
tered and crystallized from methylene dichloride : ethanol = 1:1.
4.2.3.8. 2-(3-(3,4-Dimethylphenyl)-5-(m-tolyl)-4,5-dihydro-1H-
pyrazol-1-yl)thiazol-4(5H)-one (5h).
Yield 74%; mp 215–
218 °C. 1H NMR (CDCl3, 300 MHz): 2.34 (s, 9H), 3.37 (d,
J = 16.84 Hz, 1H), 3.76 (s, 2H), 3.84 (dd, J1 = 7.23 Hz, J2 = 17.04 Hz,
1H), 5.82 (s, 1H), 7.08–7.13 (m, 3H), 7.18 (d, J = 8.65 Hz, 1H), 7.51
(d, J = 8.36 Hz, 2H), 7.56 (d, J = 6.42 Hz, 1H). ESI-MS: 364.48
(C21H22N3OS, [M+H]+). Anal. Calcd for C21H21N3OS: C, 69.39; H,
5.82; N, 11.56. Found: C, 69.43; H, 5.81; N, 11.59.
4.2.3.1.
2-(3-(3,4-Dimethylphenyl)-5-phenyl-4,5-dihydro-1H-
Yield 64%; mp 202–
pyrazol-1-yl)thiazol-4(5H)-one (5a).
204 °C. 1H NMR (CDCl3, 300 MHz): 2.34 (s, 6H), 3.34 (d,
J = 14.32 Hz, 1H), 3.92 (s, 2H), 3.98 (dd, J1 = 6.96 Hz, J2 = 10.89 Hz,
1H), 5.84 (d, J = 8.04 Hz, 1H), 7.16 (d, J = 8.25 Hz, 1H), 7.28 (d,
J = 8.22 Hz, 3H), 7.43 (d, J = 6.84 Hz, 2H), 7.52–7.56 (m, 2H). ESI-
MS: 350.45 (C20H20N3OS, [M+H]+). Anal. Calcd for C20H19N3OS: C,
68.74; H, 5.48; N, 12.02. Found: C, 68.80; H, 5.50; N, 12.06.
4.2.3.9. 2-(5-(2-Chlorophenyl)-3-(3,4-dimethylphenyl)-4,5-dihy-
dro-1H-pyrazol-1-yl)thiazol-4(5H)-one (5i).
Yield 79%; mp
232–233 °C. 1H NMR (CDCl3, 300 MHz): 2.31 (s, 6H), 3.36 (d,
J = 15.34 Hz, 1H), 3.91 (s, 2H), 4.02 (dd, J1 = 4.24 Hz, J2 = 14.38 Hz,
1H), 5.85 (d, J = 8.34 Hz, 1H), 7.16 (d, J = 5.36 Hz, 1H), 7.22–7.26
(m, 3H), 7.52 (d, J = 4.46 Hz, 2H), 7.73 (d, J = 5.46 Hz, 1H). ESI-MS:
384.89 (C20H19ClN3OS, [M+H]+). Anal. Calcd for C20H18ClN3OS: C,
62.57; H, 4.73; N, 10.95. Found: C, 62.63; H, 4.76; N, 10.92.
4.2.3.2.
2-(3-(3,4-Dimethylphenyl)-5-(4-fluorophenyl)-4,5-
Yield 84%;
dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-one (5b).
mp 220–222 °C. 1H NMR (CDCl3, 500 MHz): 2.33 (s, 6H), 3.34 (dd,
J1 = 3.20 Hz, J2 = 17.85 Hz, 1H), 3.94 (s, 2H), 3.96 (dd, J1 = 5.34 Hz,
J2 = 15.84 Hz, 1H), 5.88 (d, J = 8.05 Hz, 1H),7.16 (d, J = 8.26 Hz, 2H),
7.23 (d, J = 7.90 Hz, 1H), 7.45 (d, J = 8.40 Hz, 2H), 7.50 (d,
J = 7.75 Hz, 1H), 7.57 (s, 1H). ESI-MS: 368.44 (C20H19FN3OS,
[M+H]+). Anal. Calcd for C20H18FN3OS: C, 65.38; H, 4.94; N, 11.44.
Found: C, 65.45; H, 4.96; N, 11.47.
4.2.3.10. 2-(3-(3,4-Dimethylphenyl)-5-(o-tolyl)-4,5-dihydro-1H-
pyrazol-1-yl)thiazol-4(5H)-one (5j).
Yield 75%; mp 217–
219 °C. 1H NMR (CDCl3, 300 MHz): 2.34 (s, 3H), 3.37 (d,
J = 18.24 Hz, 1H), 3.87 (s, 2H), 4.08 (dd, J1 = 6.83 Hz, J2 = 15.82 Hz,
1H), 5.82 (d, J = 10.24 Hz, 1H), 6.94 (d, J = 5.34 Hz, 1H), 7.14–7.17
(m, 2H), 7.22 (d, J = 8.62 Hz, 1H), 7.45–7.52 (m, 3H). ESI-MS:
364.48 (C21H22N3OS, [M+H]+). Anal. Calcd for C21H21N3OS: C,
69.39; H, 5.82; N, 11.56. Found: C, 69.46; H, 5.84; N, 11.59.
4.2.3.3.
2-(5-(4-Chlorophenyl)-3-(3,4-dimethylphenyl)-4,5-
Yield 85%;
dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-one (5c).
mp 234–236 °C. 1H NMR (CDCl3, 300 MHz): 2.32 (s, 6H), 3.34 (d,
J = 10.62 Hz, 1H), 3.86 (s, 2H), 3.92 (dd, J1 = 6.26 Hz, J2 = 10.89 Hz,
1H), 5.80 (d, J = 5.22 Hz, 1H), 6.99–7.02 (m, 2H), 7.21–7.26 (m,
2H), 7.51 (d, J = 4.65 Hz, 2H), 7.58 (s, 1H). ESI-MS: 384.89
(C20H19ClN3OS, [M+H]+). Anal. Calcd for C20H18ClN3OS: C, 62.57;
H, 4.73; N, 10.95. Found: C, 62.62; H, 4.71; N, 10.98.
4.2.3.11. 2-(3-(3,4-Dichlorophenyl)-5-phenyl-4,5-dihydro-1H-
pyrazol-1-yl)thiazol-4(5H)-one (5k).
Yield 66%; mp 244–
246 °C. 1H NMR (CDCl3, 300 MHz): 3.36 (d, J = 16.82 Hz, 1H), 3.83
(s, 2H), 4.01 (dd, J1 = 7.13 Hz, J2 = 14.64 Hz, 1H), 5.86 (d,
J = 10.14 Hz, 1H), 7.27–7.29 (m, 3H), 7.41 (d, J = 8.04 Hz, 2H), 7.70
(s, 1H), 7.87 (d, J = 6.85 Hz, 2H). ESI-MS: 391.29 (C18H14Cl2N3OS,
[M+H]+). Anal. Calcd for C18H13Cl2N3OS: C, 55.39; H, 3.36; N,
10.77. Found: C, 55.44; H, 3.35; N, 10.80.
4.2.3.4. 2-(5-(4-Bromophenyl)-3-(3,4-dimethylphenyl)-4,5-dihy-
dro-1H-pyrazol-1-yl)thiazol-4(5H)-one (5d).
Yield 85%; mp
262–264 °C. 1H NMR (CDCl3, 300 MHz): 2.33 (s, 6H), 3.34 (d,
J = 17.19 Hz, 1H), 3.71 (s, 2H), 3.87 (dd, J1 = 6.91 Hz, J2 = 12.89 Hz,
1H), 5.87 (s, 1H), 7.20–7.26 (m, 3H), 7.30 (d, J = 8.04 Hz, 2H), 7.51
(d, J = 8.43 Hz, 1H), 7.59 (d, J = 8.43 Hz, 1H). ESI-MS: 429.35
(C20H19BrN3OS, [M+H]+). Anal. Calcd for C20H18BrN3OS: C, 56.08;
H, 4.24; N, 9.81. Found: C, 56.05; H, 4.24; N, 9.84.
4.2.3.12. 2-(3-(3,4-Dichlorophenyl)-5-(4-fluorophenyl)-4,5-dihy
dro-1H-pyrazol-1-yl)thiazol-4(5H)-one (5l).
Yield 83%; mp
252–254 °C. 1H NMR (CDCl3, 300 MHz): 3.36 (d, J = 14.23 Hz, 1H),

K.-M. Qiu et al. / Bioorg. Med. Chem. 20 (2012) 6648–6654
6653
3.91 (s, 2H), 4.02 (dd, J1 = 4.63 Hz, J2 = 12.24 Hz, 1H), 5.87 (d,
J = 8.84 Hz, 1H), 7.19 (d, J = 6.24 Hz, 2H), 7.28 (d, J = 4.58 Hz, 2H),
7.69 (s, 1H) 7.86 (d, J = 6.24 Hz, 2H). ESI-MS: 409.28
(C18H13Cl2FN3OS, [M+H]+). Anal. Calcd for C18H12Cl2FN3OS: C,
52.95; H, 2.96; N, 10.29. Found: C, 53.02; H, 2.98; N, 10.32.
4.2.3.19.
2-(5-(2-Chlorophenyl)-3-(3,4-dichlorophenyl)-4,5-
Yield 75%;
dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-one (5s).
mp 220–222 °C. 1H NMR (CDCl3, 300 MHz): 3.36 (dd, J1 = 5.08 Hz,
J2 = 15.48 Hz, 1H), 3.86 (s, 2H), 4.03 (dd, J1 = 5.94 Hz,
J2 = 15.38 Hz, 1H), 5.86 (d, J = 8.14 Hz, 1H), 7.22–7.28 (m, 3H),
7.67 (d, J = 5.89 Hz, 1H), 7.72 (d, J = 8.12 Hz, 1H), 7.88 (d,
J = 7.24 Hz, 2H). ESI-MS: 425.73 (C18H13Cl3N3OS, [M+H]+). Anal.
Calcd for C18H12Cl3N3OS: C, 50.90; H, 2.85; N, 9.89. Found: C,
50.96; H, 2.87; N, 9.86.
4.2.3.13.
2-(5-(4-Chlorophenyl)-3-(3,4-dichlorophenyl)-4,5-
(5m). Yield
dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-one
85%; mp 260–262 °C. 1H NMR (CDCl3, 300 MHz): 3.36 (d,
J = 14.26 Hz, 1H), 3.88 (s, 2H), 3.98 (dd, J1 = 4.36 Hz, J2 = 10.45 Hz,
1H), 5.87 (d, J = 8.43 Hz, 1H), 7.44 (d, J = 6.04 Hz, 2H), 7.49 (d,
J = 5.84 Hz, 2H), 7.69 (d, J = 5.34 Hz, 1H), 7.86 (d, J = 14.22 Hz,
2H). ESI-MS: 425.73 (C18H13Cl3N3OS, [M+H]+). Anal. Calcd for
C18H12Cl3N3OS: C, 50.90; H, 2.85; N, 9.89. Found: C, 50.95; H,
2.87; N, 9.92.
4.2.3.20. 2-(3-(3,4-Dichlorophenyl)-5-(o-tolyl)-4,5-dihydro-1H-
pyrazol-1-yl)thiazol-4(5H)-one (5t).
Yield 76%; mp 262–
264 °C. 1H NMR (CDCl3, 300 MHz): 2.34 (s, 3H), 3.36 (dd,
J1 = 4.46 Hz, J2 = 16.94 Hz, 1H), 3.86 (s, 2H), 4.04 (dd, J1 = 6.26 Hz,
J2 = 14.84 Hz, 1H), 5.82 (s, 1H), 6.95 (d, J = 5.34 Hz, 1H), 7.15–7.20
(m, 2H), 7.38–7.41 (m, 1H), 7.69 (d, J = 8.22 Hz, 1H), 7.86 (d,
J = 8.24 Hz, 2H). ESI-MS: 405.31 (C19H16Cl2N3OS, [M+H]+). Anal.
Calcd for C19H15Cl2N3OS: C, 56.44; H, 3.74; N, 10.39. Found: C,
56.49; H, 3.76; N, 10.42.
4.2.3.14.
2-(5-(4-Bromophenyl)-3-(3,4-dichlorophenyl)-4,5-
dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-one
(5n).
Yield
86%; mp 285–287 °C. 1H NMR (CDCl3, 300 MHz): 3.33 (d,
J = 16.32 Hz, 1H), 3.86 (s, 2H), 3.95 (dd, J1 = 6.38 Hz, J2 = 14.04 Hz,
1H), 5.85 (s, 1H), 7.19 (d, J = 7.46 Hz, 2H), 7.69 (d, J = 7.36 Hz,
1H), 7.86 (d, J = 6.82 Hz, 2H), 7.96 (d, J = 8.62 Hz, 2H). ESI-MS:
4.3. Molecular modeling (docking) studies
470.18
18H12BrCl2N3OS: C, 46.08; H, 2.58; N, 8.96. Found: C, 46.03; H,
2.56; N, 8.91.
(C18H13BrCl2N3OS,
[M+H]+).
Anal.
Calcd
for
Molecular docking of compound 5a into the 3D COX-1/COX-2
complex structure (PDB code: 1PGF/1cx2) was carried out using
the Discovery Stutio (version 3.1) as implemented through the
graphical user interface DS-LigandFit protocal.
C
4.2.3.15. 2-(3-(3,4-Dichlorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-
pyrazol-1-yl)thiazol-4(5H)-one (5o). Yield 82%; mp 249–
The three-dimensional structures of the aforementioned com-
pounds were constructed using Chem 3D ultra 11.0 software
[Chemical Structure Drawing Standard; Cambridge Soft corpora-
tion, USA (2009)], then they were energetically minimized by using
MOPAC with 100 iterations and minimum RMS gradient of 0.10.
The crystal structures of COX-2 complex were retrieved from the
RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.
do). All bound water and ligands were eliminated from the protein
and the polar hydrogen was added. The whole COX-2 complex was
defined as a receptor and the site sphere was selected based on the
ligand binding location of SC-558, then the SC-558 molecule was
removed and 5a was placed during the molecular docking proce-
dure. Types of interactions of the docked protein with ligand were
analyzed after the end of molecular docking.
251 °C. 1H NMR (CDCl3, 300 MHz): 2.34 (s, 3H), 3.36 (dd,
J1 = 4.06 Hz, J2 = 14.63 Hz, 1H), 3.87 (s, 2H), 3.98 (dd, J1 = 5.26 Hz,
J2 = 15.42 Hz, 1H), 5.89 (d, J = 6.36 Hz, 1H), 7.15–7.18 (m, 4H),
7.65 (d, J = 6.34 Hz, 1H), 7.85 (d, J = 5.38 Hz, 2H). ESI-MS: 405.31
(C19H16Cl2N3OS, [M+H]+). Anal. Calcd for C19H15Cl2N3OS: C, 56.44;
H, 3.74; N, 10.39. Found: C, 56.49; H, 3.75; N, 10.42.
4.2.3.16. 2-(3-(3,4-Dichlorophenyl)-5-(4-methoxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-one
(5p).
Yield
86%; mp 253–255 °C. 1H NMR (CDCl3, 300 MHz): 3.36 (dd,
J1 = 4.24 Hz, J2 = 17.84 Hz, 1H), 3.82 (s, 3H), 3.88 (s, 2H), 4.02 (dd,
J1 = 5.36 Hz, J2 = 14.44 Hz, 1H), 5.86 (d, J = 7.36 Hz, 1H), 6.94 (d,
J = 8.42 Hz, 2H), 7.20 (d, J = 7.62 Hz, 2H), 7.72 (d, J = 6.42 Hz, 1H),
7.82–7.86 (m, 2H). ESI-MS: 421.31 (C19H16Cl2N3O2S, [M+H]+). Anal.
Calcd for C19H15Cl2N3O2S: C, 54.29; H, 3.60; N, 10.00. Found: C,
54.26; H, 3.62; N, 10.05.
4.4. Cytotoxicity test
The cytotoxic activity in vitro was measured using the MTT
assay. RAW264.7 cells were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented
with 5% fetal bovine serum (FBS) (Invitrogen), 100 U/mL penicillin,
4.2.3.17.
2-(5-(3-Chlorophenyl)-3-(3,4-dichlorophenyl)-4,5-
(5q). Yield
dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-one
78%; mp 256–258 °C. 1H NMR (CDCl3, 300 MHz): 3.34 (dd,
J1 = 3.64 Hz, J2 = 17.54 Hz, 1H), 3.87 (s, 2H), 3.94 (dd, J1 = 6.42 Hz,
J2 = 13.24 Hz, 1H), 5.86 (d, J = 6.42 Hz, 1H), 7.14 (d, J = 5.86 Hz,
1H), 7.32–7.36 (m, 2H), 7.49 (s, 1H), 7.68 (d, J = 5.18 Hz, 1H), 7.86
(d, J = 8.32 Hz, 2H). ESI-MS: 425.73 (C18H13Cl3N3OS, [M+H]+). Anal.
Calcd for C18H12Cl3N3OS: C, 50.90; H, 2.85; N, 9.89. Found: C, 50.90;
H, 2.85; N, 9.89.
and 100 lg/mL streptomycin and incubated at 37 °C in a humidi-
fied atmosphere containing 5% CO2. In all cell cultures, each com-
pound was prepared in dimethyl sulphoxide (DMSO) followed by
dilution with culture medium to desired concentrations, and
DMSO final concentration was 0.1%. DMSO at 0.1% was added into
control (RAW264.7 cells were treated with LPS only) and blank
(RAW264.7 cells only, without any treatments) groups and showed
no effects on cells.
4.2.3.18. 2-(3-(3,4-Dichlorophenyl)-5-(m-tolyl)-4,5-dihydro-1H-
Cells were plated at a density of 5 Â 104 cells in a 96-well plate,
and compounds were added to each plate at the indicated concen-
trations. The incubation was permitted at 37 °C, 5% CO2 atmosphere
pyrazol-1-yl)thiazol-4(5H)-one (5r).
Yield 81%; mp 247–
248 °C. 1H NMR (CDCl3, 300 MHz): 2.34 (s, 3H), 3.33 (d,
J = 10.67 Hz, 1H), 3.90 (s, 2H), 3.95 (dd, J1 = 6.62 Hz, J2 = 12.42 Hz,
1H), 5.87 (d, J = 5.68 Hz, 1H), 7.08 (d, J = 6.04 Hz, 2H), 7.16 (d,
J = 5.43 Hz,1H), 7.58–7.63 (m, 2H), 7.86 (d, J = 6.34 Hz, 2H). ESI-
MS: 405.31 (C19H16Cl2N3OS, [M+H]+). Anal. Calcd for
for 24 h before the cytotoxicity assessments. 20 lL MTT reagent
(4 mg/mL) was added per well 4 h before the end of the incubation.
Four hours later, the plate was centrifugaled at 1200 rcf for 5 min
and the supernatants were removed, each well was added with
C19H15Cl2N3OS: C, 56.44; H, 3.74; N, 10.39. Found: C, 56.49; H,
200 lL DMSO. The absorbance was measured at a wavelength of
3.76; N, 10.43.

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K.-M. Qiu et al. / Bioorg. Med. Chem. 20 (2012) 6648–6654
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4.6. Western blotting
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Acknowledgement
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The work was financed by National Natural Science Foundation
of China (No. J1103512).
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