Design, synthesis, and biological evaluation of 1,5-diaryl-1,2,4-triazole derivatives as selective cyclooxygenase-2 inhibitors

Article abstract of DOI:10.1002/ardp.200900227

A series of 1,5-diaryl-1,2,4-triazole derivatives were synthesized and evaluated as cyclooxygenase-2 (COX-2) inhibitors. The results of the preliminary biological assays in vivo showed that eight compounds 5b, 6b, 6c, 7c, 8b, 8d, 9c, and 9d have potent an

Full text of DOI:10.1002/ardp.200900227

500
Arch. Pharm. Chem. Life Sci. 2010, 9, 500–508
Full Paper
Design, Synthesis, and Biological Evaluation of
1,5-Diaryl-1,2,4-triazole Derivatives as Selective
Cyclooxygenase-2 Inhibitors
Bo Jiang1, Yi Zeng1, Meng-Jie Li1, Jin-Yi Xu1, Yong-Na Zhang2, Qiu-Juan Wang2, Ni-Yue Sun3,
Tao Lu3, and Xiao-Ming Wu1
1 Department of Medicinal Chemistry, College of Pharmacy, China Pharmaceutical University, Nanjing,
P.R. China
2 Department of Physiology, College of Pharmacy, China Pharmaceutical University, Nanjing, P.R. China
3 Laboratory of Molecular Design and Drug Discovery, China Pharmaceutical University, Nanjing, P.R. China
A series of 1,5-diaryl-1,2,4-triazole derivatives were synthesized and evaluated as cyclooxygenase-2
(COX-2) inhibitors. The results of the preliminary biological assays in vivo showed that eight
compounds 5b, 6b, 6c, 7c, 8b, 8d, 9c, and 9d have potent anti-inflammatory activity (P < 0.01),
while compounds 6b, 6c, and 9c exhibit marked potency. Compound 6c was then selected for
further investigation. In the COX inhibition assay in vitro, compound 6c was identified as a potent
and selective inhibitor of COX-2 (COX-2 IC50 ¼ 0.37 mM; SI ¼ 0.018), being equipotent to celecoxib
(COX-2 IC50 ¼ 0.26 mM; SI ¼ 0.015). In a rat carrageenan-induced paw edema assay, 6c exhibited
moderate anti-inflammatory activity (35% inhibition of inflammation) at 2 h after administration of
15 mg/kg as an oral dose. A docking study also revealed that compound 6c binds in the active site of
COX-2 in a similar mode to that of the known selective COX-2 inhibitor SC-558.
Keywords: Anti-inflammatory activity / 1,5-Diaryl-1,2,4-triazole / NSAIDs / Selective COX-2 inhibitors
Received: September 21, 2009; Accepted: December 31, 2009
DOI 10.1002/ardp.200900227
Introduction
and is responsible for homeostatic processes such as platelet
aggregation, gastric protection, and renal function. COX-2 is
an inducible isoform that is constitutively present in some
organs, such as brain and kidney, but is significantly up-
regulated in response to a broad range of stimuli in inflam-
matory condition [5, 6]. It has been shown that the biological
functions of COX-1 and COX-2 are much more complicated
than previously recognized. Moreover, the existence of COX-
3, a variant of COX-1, has been proposed and is considered to
be another target for anti-inflammatory agents [7].
Non-steroidal anti-inflammatory drugs (NSAIDs) remain
widely prescribed drugs worldwide and have been used to
treat pain, fever, and symptoms of arthritis like rheumatoid
arthritis (RA), osteoarthritis (OA), etc. However, long-term use
of NSAIDs has been associated with several unwanted effects
such as gastrointestinal mucosal damage, bleeding and renal
toxicity [1–4]. The identification of two different isoforms
of the cyclooxygenase (COX) enzyme known as COX-1 and
COX-2, which are key enzymes in the arachidonic acid metab-
olism, heralded the start of a new era for research in anti-
inflammatory therapy. COX-1 is expressed in many organs
Ever since the first selective COX-2 inhibitor – celecoxib –
has been approved for the market by the FDA for the treat-
ment of inflammation and the management of acute or
chronic pain, diaryl-heterocycles have become the major
class of selective COX-2 inhibitors, such as celecoxib, rofe-
coxib, parecoxib, and valdecoxib (Fig. 1), which display
improved gastrointestinal safety profile compared to the
traditional NSAIDs [8–12]. However, the cardiovascular risk
associated with ‘‘-coxibs’’ has become a concern since
rofecoxib and valdecoxib were withdrawn from the market
[13]. The most plausible mechanism is the suppression of
Correspondence: Jin-Yi Xu, Department of Medicinal Chemistry, College
of Pharmacy, 24 Tongjia Xiang, China Pharmaceutical University, Nanjing
210009, P.R. China.
E-mail: jinyixu@china.com
Fax: þ86 25 8330-2827
Abbreviations: cyclooxygenase-1/2 (COX-1/2); non-steroidal anti-
inflammatory drugs (NSAIDs); selectivity index (SI)
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Selective Cyclooxygenase-2 Inhibitors
501
Figure 2. Structures for celecoxib and 1,5-diaryl-1,2,4-triazoles.
scaffold compounds should be capable of inhibiting the
enzymatic activity of COX-2. Using celecoxib as a lead com-
pound, a series of novel diaryl-1,2,4-triazole derivatives was
designed and synthesized, and preliminary pharmacological
assays in vivo showed that some compounds have certain anti-
inflammatory effects [24]. Based on the above-mentioned
results, different substituents of electron-withdrawing
groups were introduced to the para-position of the N-1 phenyl
ring. Herein, we wish to report the synthesis and biological
evaluation of a series of 1,5-diaryl-1,2,4-triazole derivatives
and their structures which are shown in Fig. 2.
Results and discussion
Figure 1. Selective COX-2 inhibitors.
Chemistry
The synthetic routes of the target compounds are shown in
the Scheme 1. Para-position phenylthiosemicarbazides were
acylated with 4-(methylsulfonyl)benzoyl chloride to afford
compounds 3a to 3h. Followed by cyclization of 3a–h, com-
pounds 4a–h were methylated with iodomethane in good
yield. Then, compounds 5a–h were oxidated with sodium
metaperiodate and 30% hydrogen peroxide to get the desired
compounds 7a–d and 8a–g, respectively. Considering that
there is a sulfonamide moiety in compounds 5e–h and 8e–
g, which may lead to producing by-products in the reaction,
we chose tert-butyl as a protective group and the desired
compounds 6a–d and 9b–d were obtained through deprotec-
tion in trifluoroacetic acid and methylphenyl ether.
COX-2-derived prostacyclin and to some degree prostaglandin
E2, while leaving the platelet COX-1-derived synthesis of pro-
thrombotic thromboxanes unchanged, thereby resulting in
an imbalance among the eicosanoids [14, 15]. Nonetheless,
the concern that COX-2 inhibitors potentiate cardiovascular
events differ among agents, and only with large doses of the
available COX-2 inhibitors do such threats exist [16, 17].
Besides, the clinical use of selective COX-2 inhibitors depends
on the patients’ individual diversity [18]. To date celecoxib
is still available on the market and current evidence suggests
that celecoxib is an important therapeutic option because
of its lower cardiovascular toxicity potential compared to
NSAIDs and other -coxibs [19, 20]. Recent achievements have
also highlighted potential applications of selective COX-2
inhibitors in treatments of cancers [21] and neurodegener-
ative disorders, such as Alzheimer’s disease (AD) and
Parkinson’s disease [22].
Pharmacology
Xylene-induced ear-edema assay in mice [25]
The pharmacological assay of xylene-induced ear edema
in mice was applied. Compounds 5a–d, 6a–d, 7a–d, 8a–d,
and 9b–d were tested for their ability to inhibit ear edema
compared to celecoxib after a single oral dose (Table 1). The
results showed that the target compounds exhibited a broad
range of anti-inflammatory activity. In general, introduction
of a sulfamoyl moiety at the para-position of N-5 at the phenyl
ring showed a more potent anti-inflammatory activity than a
mesyl moiety, indicating that the sulfamoyl moiety is favor-
able to the in-vivo activity. Substituents such as methylthio
Many efforts have been made to develop selective COX-2
inhibitors, and we became interested in exploring new sur-
rogates for the pyrazole moiety of celecoxib. While searching
for replacements of pyrazole with its bioisostere 1,2,4-
triazole, we have noticed a report about 1,5-diaryl-1,2,4-
triazoles displaying certain anti-inflammatory activities,
but without identifying the mechanism of the compounds
[23]. The results led us to propose that such 1,2,4-triazole-
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Scheme 1. The synthesis of 1,5-diaryl-1,2,4-triazole derivatives.
and sulfonyl groups at the 3-position of 1,2,4-triazoles bearing
a sulfamoyl moiety at the para-position of N-5 at the phenyl
ring contributed to good inhibitory potency. It was interest-
ing to note that introduction of a weak electron-withdrawing
group (F, Cl, or Br) at the para-position of the N-1 phenyl ring
provided potent anti-inflammatory activity (6b, 6c, 8d, 9c),
especially the compound possessing Cl (6c) exhibited more a
potent anti-inflammatory (80%) activity than the reference
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Selective Cyclooxygenase-2 Inhibitors
503
Table 1. Inhibitory effect of target compounds on xylene-induced ear edema in mice.
Compound
m (mg)
Inhibition (%)
Thickness (mm)
Inhibition (%)
Model
Celecoxib
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
11.5 ꢁ 1.8
0.448 ꢁ 0.140
0.160 ꢁ 0.098#
0.188 ꢁ 0.033#
0.206 ꢁ 0.030#
0.300 ꢁ 0.034
0.178 ꢁ 0.080#
0.284 ꢁ 0.033§
0.172 ꢁ 0.041#
0.076 ꢁ 0.041#
0.258 ꢁ 0.025§
0.260 ꢁ 0.035§
0.340 ꢁ 0.047
0.176 ꢁ 0.017#
0.234 ꢁ 0.009§
0.196 ꢁ 0.013#
0.178 ꢁ 0.144#
0.202 ꢁ 0.063§
0.182 ꢁ 0.064#
0.304 ꢁ 0.011
0.184 ꢁ 0.074#
0.196 ꢁ 0.044#
4.04 ꢁ 3.05#
8.58 ꢁ 1.70§
7.98 ꢁ 3.10#
9.88 ꢁ 1.90
6.16 ꢁ 4.42§
9.38 ꢁ 1.47
2.88 ꢁ 1.52#
2.30 ꢁ 1.67#
8.92 ꢁ 1.45§
8.48 ꢁ 4.62
9.12 ꢁ 6.29
6.72 ꢁ 2.09#
5.94 ꢁ 2.20#
7.94 ꢁ 1.36#
7.44 ꢁ 3.10§
8.16 ꢁ 2.34§
3.36 ꢁ 3.22#
8.28 ꢁ 3.27
2.62 ꢁ 1.97#
5.22 ꢁ 3.79#
64.9
25.4
30.6
14.1
46.4
18.4
75.0
80.0
22.4
26.3
20.7
41.6
48.4
31.0
35.3
29.0
70.8
28.0
77.2
54.6
64.3
58.0
54.0
33.0
60.3
36.6
61.6
83.0
42.4
42.0
24.1
60.7
47.8
56.3
60.3
54.9
59.4
32.1
59.0
56.3
8d
9b
9c
9d
n ¼ 10, x ꢁ S; § p < 0.05, # p < 0.01.
Table 2. In-vitro COX-1 and COX-2 inhibitory activity data for 6c.
Compound
% inhibition (1 mM)§
COX-2
IC50 (mM)#
Selectivity Index& (SI)
COX-1
COX-1
COX-2
Celecoxib
6c
41.3
40.7
59.4
63.5
16.3$
20
0.26d
0.37
0.015
0.018
§ The results are expressed as the mean %-inhibition value at a concentration of 1 mM of the test compound. The result is the mean of
four determinations; # the test-compound concentration required to produce 50% inhibition of COX-1 or COX-2 in vitro. The result
(IC50, in mM) is the mean of four determinations; & COX-2 selectivity index (IC50 COX-2/IC50 COX-1) in vitro; $ see [27].
Table 3. The effect of compound 6c on carrageenan-induced paw edema in rats.
Compound
Dose&
(mg/kg)
Extent of swelling (mL ꢀ 10) and inhibition (%) at different times
PGE2 (ꢀ102)
1 h
2 h
3 h
4 h
Model
Celecoxib
–
15.0
1.35 ꢁ 0.44
0.80 ꢁ 0.26#
(40.7)
1.97 ꢁ 0.47
1.16 ꢁ 0.29#
(41.1)
2.67 ꢁ 0.92
1.62 ꢁ 0.38#
(39.3)
3.18 ꢁ 1.01
2.05 ꢁ 0.33#
(35.5)
7.3 ꢁ 2.4
4.1 ꢁ 1.2#
6c
30.0
15.0
7.5
0.83 ꢁ 0.27#
1.18 ꢁ 0.23#
1.67 ꢁ 0.44#
2.15 ꢁ 0.57§
4.1 ꢁ 1.0#
4.4 ꢁ 1.3#
4.8 ꢁ 1.4§
(38.5)
(40.1)
(37.5)
(32.4)
0.88 ꢁ 0.23#
(34.8)
1.28 ꢁ 0.28#
(35.0)
1.74 ꢁ 0.33#
(34.8)
2.27 ꢁ 0.62§
(28.6)
1.07 ꢁ 0.32
1.46 ꢁ 0.24#
1.92 ꢁ 0.29§
2.55 ꢁ 0.34
(20.7)
(25.9)
(28.1)
(19.8)
§ p < 0.05; # p < 0.01 vs. model; inhibition values are in (); & the results are expressed as percentage of inhibition following 30, 15,
7.5 mg/kg oral dose of compound 6c.
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drug celecoxib (64.9%). In contrast, those compounds with a
methoxyl group as an electron donor exhibited a weaker
potency, while in our previous work [24] the methyl group
as an electron donor was investigated and the results had
shown that compounds with a methyl group at the para-
position of the N-1 phenyl ring had provided equipotent
inhibitory activity compared to the reference drug celecoxib.
Further studies are needed to address if the lipophilicity of
the compound is beneficial to the inhibitory potency.
COX-1/COX-2 inhibition assay in vitro
Based on the results of preliminary pharmacological tests
in vivo, the best compound 6c has interesting pharmacologi-
cal properties and was selected to be tested in inhibition
assays against COX-1 and COX-2 enzymes, using celecoxib
as a reference drug [26]. As summarized in Table 2, compound
6c exhibited good COX-2 inhibitory activity and selectivity
(63.5% inhibition at 1 mM; COX-1 IC50 ¼ 20.0 mM; COX-2
IC50 ¼ 0.37 mM; SI ¼ 0.018), which is equipotent to celecoxib
(59.4% inhibition at 1 mM; COX-1 IC50 ¼ 16.3 mM; COX-2
IC50 ¼ 0.26 mM; SI ¼ 0.015) [27]. The results suggest that
compound 6c is a selective COX-2 inhibitor.
Figure 3. Conformational superposition of SC-558 from the crystal
structure (gray) and that from the Gold result (light gray).
Carrageenan-induced paw edema and acetic acid-induced
vascular permeability assays [28, 29]
Anti-inflammatory activity evaluations in vivo for 6c were
carried out, and the results are illustrated in Tables 3
and 4. In the rat carrageenan-induced paw edema assay,
compound 6c exhibited a slightly less potent anti-inflamma-
tory activity (34.8% and 35%) than the reference drug cele-
coxib (40.4% and 41.1%) at 1 h and 2 h after administration,
respectively, for a 15 mg/kg oral dose. In addition, it exhib-
ited the ability to decrease the permeability of celiac blood
capillary and to block the release of mediators of inflam-
mation in the acetic acid-induced vascular permeability
assay. These data indicate that compound 6c has certain
potentials equivalent to the reference drug celecoxib in
the acute inflammation model.
Table 4. The effect of compound 6c on acid-induced vascular
permeability in mice.
Compound
Dose (mg/kg)&
A590 (ꢀ10)
Model
Celecoxib
–
2.47 ꢁ 0.52
1.36 ꢁ 0.38#
1.21 ꢁ 0.36#
1.78 ꢁ 0.50#
1.93 ꢁ 0.60§
30
60
30
15
6c
Figure 4. (A) Binding mode of compound 6c (dark gray) and SC-
558 (gray) in the COX-2 active site. (B) Likely binding conformations
of 1,5-diaryl-1,2,4-triazole derivatives.
n ¼ 10, x ꢁ S; § p < 0.05, # p < 0.01 vs. Model; & the results are
expressed as mean ꢁ SEM (n ¼ 10) following 60, 30, 15 mg/kg
oral dose of compound 6c.
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RDCSY-I melting point apparatus; uncorrected. IR spectra:
Shimadzu FTIR-8400S spectrometer (in cmꢂ1; Shimadzu, Japan).
1H-NMR spectra: Bruker ACF-300Q apparatus at 300 MHz (Bruker,
USA), in CDCl3 unless otherwise indicated; d in ppm rel. to Me4Si, J
in Hz. Mass spectrometry (MS): Hewlett-Packard 1100 LC/MSD spec-
Docking study
The superposition between the GOLD-predicted conformation
of the known selective COX-2 inhibitor SC-558 and that of the
X-ray crystallographic complex is shown in Fig. 3. The root
mean square deviation (RMSD) between these two confor-
trometer (Hewlett-Packard, USA); in m/z. Elemental analysis:
Elementar Vario EL III instrument (Elementar, Germany).
˚
mations is 0.84 A, indicating that the parameter set for the
GOLD program is reasonable to reproduce the X-ray structure.
A docking study was carried out to investigate the binding
interactions of compound 6c within the COX-2 binding sites.
As illustrated in Fig. 4, compound 6c binds in the active site of
COX-2 in a similar mode to that of known selective COX-2
inhibitor SC-558 [30]. The chlorophenyl ring is oriented in a
hydrophobic pocket at the apex of the COX-2 binding site
formed by Trp387, Leu384, Tyr381, and Phe381. The methylthio
General procedure for synthesis of compounds 5a–5d
1-(4-Substituted)-5-(4-(methylsulfonyl)phenyl)-1H-1,2,4-triazole-3-
thiols (1 mmol) and K2CO3 (1.1 mmol) were suspended in 10 mL
acetone at 508C for 30 min, then MeI (1.1 mmol) was added, and
the mixture was allowed to reflux for 3 h. Then, the mixture
was filtered and the filtrate was evaporated in vacuo. The residue
was purified by flash chromatography (ethyl acetate/petroleum
ether, 1:2) to get the desired compounds.
group is bound in the vicinity pocket formed by Val349
,
Leu531, Val116, Leu359, and Tyr355. The sulfamoyl group forms
hydrogen bonds with the Arg513, Phe518, and His90, which are
responsible for COX-2 selectivity.
1-(4-Methoxyphenyl)-5-(4-(methylsulfonyl)phenyl)-3-
(methylthio)-1H-1,2,4-triazole 5a
Yield: 53%, white solid, m. p.: 178–1808C; IR (KBr): 2916, 1595,
1
1606, 1513, 1300, 1257, 837 cmꢂ1; H-NMR (DMSO-d6), (d, ppm):
2.61 (3H, s), 3.26 (3H, s), 3.81 (3H, s), 7.05 (2H, d, J ¼ 6.80 Hz), 7.39
(2H, d, J ¼ 6.81 Hz), 7.69 (2H, d, J ¼ 8.43 Hz), 7.95 (2H, d,
J ¼ 8.45 Hz);
ESI-MS:
376
[M þ H]þ.
Anal.
calcd.
Conclusions
for C17H17N3O3S2: C, 54.38; H, 4.56; N, 11.19. Found: C, 54.45;
H, 4.82; N, 10.81.
In search for novel selective COX-2 inhibitors, 1,5-diaryl-1,2,4-
triazole derivatives were synthesized and evaluated. The
results showed that the target compounds displayed a broad
range of anti-inflammatory activities in the xylene-induced
ear edema assay in vivo. Among them, eight compounds 5b,
6b, 6c, 7c, 8b, 8d, 9c, and 9d have potent anti-inflammatory
activity (p < 0.01), while compounds 6b, 6c, and 9c exhibit
marked potency. In the COX inhibition assay in vitro, com-
pound 6c was identified as a potent and selective inhibitor of
COX-2 (COX-2 IC50 ¼ 0.37 mM; SI ¼ 0.018), being equipotent
to celecoxib (COX-2 IC50 ¼ 0.26 mM; SI ¼ 0.015). In a rat
carrageenan-induced paw edema assay, 6c exhibited moder-
ate anti-inflammatory activity (35% inhibition of inflam-
mation) at 2 h after administration of 15 mg/kg oral dose.
A docking study also revealed that compound 6c binds in the
active site of COX-2 in a similar mode to that of the known
selective COX-2 inhibitor SC-558. These results may provide
some useful information for the development of 1,5-diaryl-
1,2,4-triazole derivatives as selective COX-2 inhibitors. As an
interesting substance, compound 6c is currently under fur-
ther investigation.
1-(4-Fluorophenyl)-5-(4-(methylsulfonyl)phenyl)-3-(methyl-
thio)-1H-1,2,4-triazole 5b
Yield: 60%, white solid; m. p.: 185–1888C; IR (KBr): 2916, 1600,
1
1511, 1222, 1310, 1144, 847 cmꢂ1; H-NMR (DMSO-d6), (d, ppm):
2.62 (3H, s), 3.27 (3H, s), 7.37 (2H, m), 7.54 (2H, m), 7.69 (2H, d,
J ¼ 8.60 Hz), 7.96 (2H, d, J ¼ 8.62 Hz); ESI-MS: 364 [M þ H]þ.
Anal. calcd. for C16H14FN3O2S2: C, 52.88; H, 3.88; N, 11.56.
Found: C, 53.24; H, 4.15; N, 11.23.
1-(4-Chlorophenyl)-5-(4-(methylsulfonyl)phenyl)-3-(methyl-
thio)-1H-1,2,4-triazole 5c
Yield: 76%, white solid; m. p.: 154–1568C; IR (KBr): 2996, 1600,
1
1494, 1303, 1151, 1091, 839 cmꢂ1; H-NMR (DMSO-d6), (d, ppm):
2.62 (3H, s), 3.26 (3H, s), 7.49 (2H, d, J ¼ 4.95 Hz), 7.59 (2H, d,
J ¼ 6.74 Hz), 7.71 (2H, d, J ¼ 6.73 Hz), 7.97 (2H, d, J ¼ 5.10 Hz);
ESI-MS: 380 [M þ H]þ. Anal. calcd. for C16H14ClN3O2S2: C, 50.59;
H, 3.71; N, 11.06. Found: C, 50.18; H, 4.09; N, 10.69.
1-(4-Bromophenyl)-5-(4-(methylsulfonyl)phenyl)-3-(meth-
ylthio)-1H-1,2,4-triazole 5d
Yield: 79%, white solid; m. p.: 157–1598C; IR (KBr): 2923, 1639,
1490, 1302, 1152, 833, 535 cmꢂ1 1H-NMR (DMSO-d6), (d, ppm):
;
2.62 (3H, s), 3.26 (3H, s), 7.42 (2H, d, J ¼ 8.37 Hz), 7.71 (4H, m),
7.98 (2H, d, J ¼ 8.02 Hz); ESI-MS: 424 [M þ H]þ. Anal. calcd.
for C16H14BrN3O2S2: C, 45.29; H, 3.33; N, 9.90. Found: C, 45.57;
H, 3.11; N, 9.72.
Experimental
Chemistry
Reagents were purchased from Shanghai Chemical Reagent
Company (China) and were used without further purification.
Column chromatography (CC): silica gel 60 (200–300 mesh).
Thin-layer chromatography (TLC): silica gel 60 F254 plates
(250 mm; Qingdao Ocean Chemical Company, China). M. p.:
General procedure for synthesis of compounds 6a–6d
A solution of the corresponding compounds 5a–d (1 mmol),
CF3COOH (2 mL), and two drops of methylphenyl ether
were stirred at room temperature for 24 h. The mixture was
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evaporated in vacuo and the residue was purified by flash chroma-
tography (ethyl acetate/petroleum ether, 2:3) to get the desired
compounds.
for C17H17N3O4S2: C, 52.16; H, 4.28; N, 10.73. Found: C, 52.51;
H, 4.05; N, 10.30.
1-(4-Fluorophenyl)-3-(methylsulfinyl)-5-(4-(methylsulfo-
nyl)phenyl)-1H-1,2,4-triazole 7b
4-(1-(4-Methoxyphenyl)-3-(methylthio)-1H-1,2,4-triazol-5-
yl)benzenesulfonamide 6a
Yield: 49%, white solid; m. p.: 214–2168C; IR (KBr): 2924, 1633,
1512, 1307, 1149, 1083, 844 cmꢂ1; 1H-NMR (CDCl3), (d, ppm): 3.07
(3H, s), 3.13 (3H, s), 7.18 (2H, m), 7.36 (2H, m), 7.73 (2H, d,
J ¼ 8.52 Hz), 7.95 (2H, d, J ¼ 8.51 Hz); ESI-MS: 380 [M þ H]þ.
Anal. calcd. for C16H14 FN3O3S2: C, 50.65; H, 3.72; N, 11.07.
Found: C, 50.43; H, 3.99; N, 10.67.
Yield: 72%, white solid; m. p.: 245–2498C; IR (KBr): 3446, 3317,
2980, 1607, 1513, 1348, 1164, 1018, 838 cmꢂ1; 1H-NMR (CDCl3),
(d, ppm): 2.67 (3H, s), 3.87 (3H, s), 4.80 (2H, s), 6.95 (2H, d,
J ¼ 4.56 Hz), 7.24 (2H, d, J ¼ 2.21 Hz), 7.65 (2H, d, J ¼ 4.85 Hz),
7.86 (2H, d, J ¼ 1.91 Hz); ESI-MS: 377 [M þ H]þ. Anal. calcd.
for C16H16N4O3S2: C, 51.05; H, 4.28; N, 14.88. Found: C, 51.46;
H, 4.30; N, 14.65.
1-(4-Chlorophenyl)-3-(methylsulfinyl)-5-(4-(methylsulfo-
nyl)phenyl)-1H-1,2,4-triazole 7c
4-(1-(4-Fluorophenyl)-3-(methylthio)-1H-1,2,4-triazol-5-
yl)benzenesulfonamide 6b
Yield: 53%, white solid; m. p. 192–1958C; IR (KBr): 2916, 1632,
1496, 1310, 1148, 1093, 1079, 838 cmꢂ1 1H-NMR (CDCl3), (d,
;
Yield: 67%, white solid; m. p.: 233–2368C; IR (KBr): 3451, 3322,
3075, 1601, 1512, 1341, 1161, 1223, 840 cmꢂ1; 1H-NMR (CDCl3),
(d, ppm): 3.40 (3H, s), 4.87 (2H, s), 7.20 (2H, m), 7.39 (2H, m), 7.69
(2H, d, J ¼ 6.74 Hz), 7.95 (2H, d, J ¼ 7.76 Hz); ESI-MS: 365
[M þ H]þ. Anal. calcd. for C15H13 FN4O2S2: C, 49.44; H, 3.60; N,
15.37. Found: C, 49.21; H, 3.89; N, 15.72.
ppm): 3.08 (3H, s), 3.15 (3H, s), 7.33 (2H, d, J ¼ 8.89 Hz), 7.49
(2H, d, J ¼ 8.90 Hz), 7.75 (2H, d, J ¼ 8.73 Hz), 7.98 (2H, d,
J ¼ 8.72 Hz);
ESI-MS:
396
[M þ H]þ.
Anal.
calcd.
for C16H14ClN3O3S2: C, 48.54; H, 3.56; N, 10.61. Found: C,
48.66; H, 3.90; N, 10.25.
1-(4-Bromophenyl)-3-(methylsulfinyl)-5-(4-(methylsulfo-
nyl)phenyl)-1H-1,2,4-triazole 7d
4-(1-(4-Chlorophenyl)-3-(methylthio)-1H-1,2,4-triazol-5-
yl)benzenesulfonamide 6c
Yield: 61%, white solid; m. p. 179–1828C; IR (KBr): 2924, 1628,
1490, 1309, 1146, 1079, 837, 534 cmꢂ1; 1H-NMR (CDCl3), (d, ppm):
3.08 (3H, s), 3.14 (3H, s), 7.26 (2H, d, J ¼ 8.82 Hz), 7.64 (2H, d,
J ¼ 8.81 Hz), 7.74 (2H, d, J ¼ 8.72 Hz), 7.98 (2H, d, J ¼ 8.68 Hz);
ESI-MS: 442 [M þ H]þ. Anal. calcd. for C16H14BrN3O3S2: C, 43.64;
H, 3.20; N, 9.54. Found: C, 43.32; H, 3.53; N, 9.21.
Yield: 73%, white solid; m. p.: 235–2388C; IR (KBr): 3463, 2921,
1644, 1497, 1335, 1160, 1102, 834 cmꢂ1 1H-NMR (CDCl3), (d,
;
ppm): 2.67 (3H, s), 4.87 (2H, s), 7.28 (2H, d, J ¼ 8.57 Hz), 7.43
(2H, d, J ¼ 8.64 Hz), 7.64 (2H, d, J ¼ 8.30 Hz), 7.91 (2H, d,
J ¼ 8.36 Hz);
ESI-MS:
379
[M ꢂ H]ꢂ.
Anal.
calcd.
for C15H13ClN4O2S2: C, 47.30; H, 3.44; N, 14.71. Found: C,
47.03; H, 3.70; N, 14.98.
General procedure for synthesis of compounds 8a–8d
The corresponding compounds 5a–d and 5f–h (1 mmol) were
added slowly to the solution of glacial acetic acid (2 mL),
30% H2O2 (3 mmol), and one drop of concentrated H2SO4 at
08C with stirring. The mixture was warmed to room temperature
and heated to 408C for 3 h. After cooling down, 1 mL of water
was added and the mixture stirred over 5 min, then filtered, the
filter cake was washed with water (3 ꢀ 1 mL), and then purified
by flash chromatography (ethyl acetate/petroleum ether, 1:1) to
get the desired compounds.
4-(1-(4-Bromophenyl)-3-(methylthio)-1H-1,2,4-triazol-5-
yl)benzenesulfonamide 6d
Yield: 62%, white solid; m. p.: 241–2448C; IR (KBr): 3427, 1586,
1493, 1333, 1162, 830, 618 cmꢂ1; 1H-NMR(CDCl3), (d, ppm): 2.67
(3H, s), 4.84 (2H, s), 7.22 (2H, d, J ¼ 8.79 Hz), 7.58 (2H, d,
J ¼ 8.78 Hz), 7.64 (2H, d, J ¼ 8.70 Hz), 7.92 (2H, d,
J ¼ 8.69 Hz);
ESI-MS:
425
[M þ H]þ.
Anal.
calcd.
for C15H13BrN4O2S2: C, 42.36; H, 3.08; N, 13.17. Found: C,
42.57; H, 3.46; N, 12.83.
General procedure for synthesis of compounds 7a–7d
The corresponding compounds 5a–d (1 mmol) were suspended
in methanol (30 mL), heated to 508C, and then quickly added to
the solution of NaIO4 (3 mmol) in 5 mL of water in one portion.
The mixture was heated to reflux for 5 h and then was filtered.
The filtrate was evaporated in vacuo. The residue was purified by
flash chromatography (acetone/petroleum ether, 1:1) to get the
desired compounds.
1-(4-Methoxyphenyl)-3-(methylsulfonyl)-5-(4-(methyl-
sulfonyl)phenyl)-1H-1,2,4-triazole 8a
Yield: 62%, white solid; m. p.: 189–1918C; IR (KBr): 2924, 1608,
1516, 1259, 1323, 1151, 841 cmꢂ1; 1H-NMR (CDCl3), (d, ppm): 3.08
(3H, s), 3.40 (3H, s), 3.89 (3H, s), 7.00 (2H, d, J ¼ 9.03 Hz), 7.30 (2H,
d, J ¼ 6.87 Hz), 7.77 (2H, d, J ¼ 9.03 Hz), 7.95 (2H, d, J ¼ 6.90 Hz);
ESI-MS: 408 [M þ H]þ. Anal. calcd. for C17H17N3O5S2: C, 50.11; H,
4.21; N, 10.31. Found: C, 49.83; H, 4.64; N, 10.58.
1-(4-Methoxyphenyl)-3-(methylsulfinyl)-5-(4-(methylsulfo-
nyl)phenyl)-1H-1,2,4-triazole 7a
1-(4-Fluorophenyl)-3-(methylsulfonyl)-5-(4-(methylsulfo-
nyl)phenyl)-1H-1,2,4-triazole 8b
Yield: 61%, white solid; m. p.: 189–1918C; IR (KBr): 2931, 1604,
1512, 1333, 1150, 1282, 843 cmꢂ1;1H-NMR (CDCl3), (d, ppm): 3.09
(3H, s), 3.41 (3H, s), 7.22 (2H, d, J ¼ 8.61 Hz), 7.40 (2H, m), 7.75 (2H,
d, J ¼ 8.61 Hz), 7.98 (2H, d, J ¼ 8.61 Hz); ESI-MS: 430 [M þ Cl]ꢂ.
Yield: 61%, white solid; m. p.: 197–1998C; IR (KBr): 2922, 1607,
1515, 1310, 1148, 1071, 1025, 840 cmꢂ1 1H-NMR (CDCl3), (d,
;
ppm): 3.07 (3H, s), 3.14 (3H, s), 3.88 (3H, s), 6.99 (2H, d,
J ¼ 9.02 Hz), 7.28 (2H, d, J ¼ 9.05 Hz), 7.76 (2H, d, J ¼ 8.74 Hz),
7.94 (2H, d, J ¼ 8.72 Hz); ESI-MS: 392 [M þ H]þ. Anal. calcd.
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Arch. Pharm. Chem. Life Sci. 2010, 9, 500–508
Selective Cyclooxygenase-2 Inhibitors
507
Anal. calcd. for C16H14FN3O4S2: C, 48.60; H, 3.57; N, 10.63. Found:
C, 48.39; H, 3.19; N, 10.35.
into twenty-one groups (n ¼ 5). Compounds 5a–5d, 6a–6d, 7a–
7d, 8a–8d, and 9b-9d (24–30 mg/kg), celecoxib (250 mg/kg), and
0.5% CMC-Na solution were administered. One hour later, xylene
(0.01 mL) was applied to the anterior and posterior surfaces
of the right ear lobe and the left ear was regarded as control.
After 30 min, the mice were killed by neck dislocation and
both ears were removed. Circular sections were taken and
weighed, using a cork borer with a diameter of 7 mm. The
thickness of the ear was determined using a micrometer caliper.
The percentage of ear edema was calculated based on the left ear
without xylene.
1-(4-Chlorophenyl)-3-(methylsulfonyl)-5-(4-(methylsulfo-
nyl)phenyl)-1H-1,2,4-triazole 8c
Yield: 60%, white solid; m. p.: 209–2118C; IR (KBr): 2916, 1632,
1495, 1308, 1144, 1084, 839 cmꢂ1; 1H-NMR (CDCl3), (d, ppm): 3.10
(3H, s), 3.40 (3H, s), 7.35 (2H, d, J ¼ 9.03 Hz), 7.51 (2H, d,
J ¼ 9.03 Hz), 7.75 (2H, d, J ¼ 8.58 Hz), 7.99 (2H, d, J ¼ 8.61Hz);
ESI-MS: 446 [M þ Cl]ꢂ. Anal. calcd. for C16H14ClN3O4S2: C, 46.66;
H, 3.43; N, 10.20. Found: C, 46.53; H, 3.80; N, 9.78.
In-vitro cyclooxygenase (COX) inhibition assays
1-(4-Bromophenyl)-3-(methylsulfonyl)-5-(4-(methylsulfo-
nyl)phenyl)-1H-1,2,4-triazole 8d
6-Keto-PGF1a production was measured in order to determine
COX-1 activity. Bovine aortic endothelial cells were obtained
from the thoracic aorta of newborn calves. The cells were main-
tained at 378C and 5% CO2 in DMEM supplemented with 10%
heat-inactivated fetal bovine serum, 2 mM glutamine, 60 mg/L
penicillin, and 100 mg/L streptomycin. Cells were plated into 24-
well plates at a density of 1.0 ꢀ 106 cells/well in 1 mL of DMEM.
After 24 h of incubation, the cells were treated with compound
6c (10ꢂ5, 10ꢂ6, 10ꢂ7, 10ꢂ8 mol/L), DMSO, and the reference drug
celecoxib (10ꢂ6 mol/L). These solutions were incubated for
20 min at 378C and 5% CO2, and then treated with arachidonic
acid (10 mmol/L). After 20 min of incubation, the culture super-
natants were collected and stored at ꢂ708C until required for the
6-keto-PGF1a determination. 6-Keto-PGF1a was measured by using
a radioimmuno assay kit according to the manufacturer’s
instructions.
Yield: 66%, white solid; m. p.: 208–2108C; IR (KBr): 2923, 1639,
1491, 1310, 1148, 836, 550 cmꢂ1; 1H-NMR (CDCl3), (d, ppm): 3.09
(3H, s), 3.39 (3H, s), 7.29 (2H, d, J ¼ 8.70 Hz), 7.66 (2H, d,
J ¼ 8.77 Hz), 7.75 (2H, d, J ¼ 8.41 Hz), 7.99 (2H, d,
J ¼ 8.37 Hz);
ESI-MS:
456
[M þ H]þ.
Anal.
calcd.
for C16H14BrN3O4S2: C, 42.11; H, 3.09; N, 9.21. Found: C, 42.56;
H, 3.43; N, 9.57.
General procedure for synthesis of compounds 9b–9d
The preparation of compounds 9b–d was according to the gen-
eral procedure for the synthesis of compounds 6b–d.
4-(1-(4-Fluorophenyl)-3-(methylsulfonyl)-1H-1,2,4-triazol-
5-yl)benzenesulfonamide 9b
Yield: 68%, white solid; m. p. 236–2408C; IR (KBr): 3450, 2927,
PGE2 production was measured in the culture medium in
order to determine COX-2 activity. Peritoneal macrophages were
gained by lavaging the peritoneal cavity of mice, which were
killed by cervical dislocation for the PGE2 induction assay, cells
were plated into 24-well plates at a density of 1.0 ꢀ 106 cells/well
in 1 mL of RPMI 1640 supplemented with 5% heat-inactivated
fetal bovine serum, 2 mM glutamine, 60 mg/L penicillin, and
100 mg/L streptomycin. After 2 h of incubation, the cells were
washed three times with fresh RPMI 1640 and the culture media
were replaced with fresh RPMI 1640 containing LPS (1 mg/mL)
and compound 6c (10ꢂ5, 10ꢂ6, 10ꢂ7, 10ꢂ8 mol/L), LPS (1 mg/mL)
and celecoxib (10ꢂ6 mol/L), LPS (1 mg/mL) and DMSO, respect-
ively. These solutions were incubated for 6 h at 378C and 5% CO2
and were treated with arachidonic acid (10 mmol/L). After
20 min of incubation, the culture supernatants were collected
and stored at ꢂ708C until required for PGE2 determination. PGE2
was measured by using a radioimmuno assay kit according to the
manufacturer’s instructions.
1601, 1509, 1342, 1143, 1235, 842 cmꢂ1
;
1H-NMR
(CDCl3 þ DMSO), (d, ppm): 3.33 (3H, s), 6.92 (2H, s), 7.17 (2H,
m), 7.37 (2H, m), 7.57 (2H, d, J ¼ 8.51 Hz), 7.87 (2H, d,
J ¼ 8.52 Hz);
ESI-MS:
397
[M þ H]þ.
Anal.
calcd.
for C15H13FN4O4S2: C, 45.45; H, 3.31; N, 14.13. Found: C, 45.88;
H, 3.05; N, 14.58.
4-(1-(4-Chlorophenyl)-3-(methylsulfonyl)-1H-1,2,4-triazol-
5-yl)benzenesulfonamide 9c
Yield: 57%, white solid; m. p.: 254–2568C; IR (KBr): 3389, 3283,
2918, 1559, 1495, 1323, 1144, 1091, 838 cmꢂ1; 1H-NMR (CDCl3),
(d, ppm): 3.40 (3H, s), 4.89 (2H, s), 7.34 (2H, d, J ¼ 9.03 Hz), 7.50
(2H, d, J ¼ 9.02 Hz), 7.70 (2H, d, J ¼ 8.58 Hz), 7.96 (2H, d,
J ¼ 8.58 Hz);
ESI-MS:
413
[M þ H]þ.
Anal.
calcd.
for C15H13ClN4O4S2: C, 43.64; H, 3.17; N, 13.57. Found: C,
43.20; H, 3.58; N, 13.33.
4-(1-(4-Bromophenyl)-3-(methylsulfonyl)-1H-1,2,4-triazol-
5-yl)benzenesulfonamide 9d
Anti-inflammatory activity
The tests of anti-inflammatory activity were adapted from car-
rageenan-induced paw edema and acetic acid-induced vascular
permeability assays with some modifications, respectively.
Male Sprague-Dawley rats (130–170 g) were purchased from
the Animal Centre of Henan Province (Zhengzhou, China). Rats
were divided into five groups (n ¼ 10) and dosed orally with
compound 6c (7.5, 15, and 30 mg/kg), celecoxib (15 mg/kg),
and CMC-Na solution (0.5%). After one hour, 0.1 mL of a 1%
suspension of carrageenan was injected into the plantar surface
of the right hind foot of each rat. The injected (right) and non-
injected (left) foot volumes were measured with a plethysmom-
eter at 1, 2, 3, and 4 h after carrageenan injection. The mean
Yield: 66%, white solid; m. p.: 255–2578C; IR (KBr): 3387, 2917,
1605, 1491, 1323, 1142, 835, 621 cmꢂ1; 1H-NMR (CDCl3), (d, ppm):
3.39 (3H, s), 4.88 (2H, s), 7.28 (2H, d, J ¼ 8.80 Hz), 7.66 (2H, d,
J ¼ 8.81 Hz), 7.70 (2H, d, J ¼ 8.66 Hz), 7.96 (2H, d, J ¼ 8.63 Hz);
ESI-MS: 457 [M þ H]þ. Anal. calcd. for C15H13BrN4O4S2: C, 39.40;
H, 2.87; N, 12.25. Found: C, 39.75; H, 2.56; N, 11.90.
Pharmacology
Xylene-induced ear edema in mice
Swiss albino mice (18–22 g) were purchased from the Animal
Centre of Henan Province (Zhengzhou, China). Mice were divided
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508
B. Jiang et al.
Arch. Pharm. Chem. Life Sci. 2010, 9, 500–508
degree of swelling for each group was calculated and the results
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The mice were divided into groups as described above and
dosed orally with compound 6c (15, 30, and 60 mg/kg), celecoxib
(30 mg/kg), and CMC-Na solution (0.5%). After one hour, 0.5%
Evan’s blue dye solution in saline was injected (0.1 mL/10 g)
through the tail vein immediately followed by the injection of
0.2 mL 0.6% solution of acetic acid intraperitoneally. 30 min
later, the mice were killed by neck dislocation and the viscera
were irrigated with normal saline. This was then collected and
centrifuged at 3000 rpm for 15 min. The supernatants were
increased in volume to 10 mL with normal saline, the absorption
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The authors have declared no conflict of interest.
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ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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