Discovery of potential anti-inflammatory drugs: Diaryl-1,2,4-triazoles bearing N-hydroxyurea moiety as dual inhibitors of cyclooxygenase-2 and 5-lipoxygenase

Article abstract of DOI:10.1039/c3ob41936c

A series of hybrids from diaryl-1,2,4-triazole and hydroxamic acid or N-hydroxyurea were synthesized and evaluated as novel anti-inflammatory agents. The biological data showed that (i) all the compounds showed dual COX-2/5-LOX inhibitory activities in vitro, and 15e showed optimal inhibitory activities (COX-2: IC₅₀ = 0.15 μM, 5-LOX: IC₅₀ = 0.85 μM), (ii) 15e selectively inhibited COX-2 relative to COX-1 with selectivity index (SI = 0.012) comparable to celecoxib (SI = 0.015), (iii) 15e exhibited potent anti-inflammatory activity (inhibition: 54.1%) which was comparable to the reference drug celecoxib (inhibition: 46.7%) in a xylene-induced ear edema assay, and (iv) 15e displayed promising analgesic activity in acetic acid-induced writhing response and hot-plate assay. Finally, a molecular modeling study revealed the binding interactions of 15e with COX-2 and 5-LOX. Our findings suggest that 15e may be a promising anti-inflammatory agent for further evaluation.

Full text of DOI:10.1039/c3ob41936c

Organic &
Biomolecular Chemistry
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Discovery of potential anti-inflammatory drugs:
diaryl-1,2,4-triazoles bearing N-hydroxyurea
moiety as dual inhibitors of cyclooxygenase-2 and
5-lipoxygenase†
Cite this: Org. Biomol. Chem., 2014,
12, 2114
Bo Jiang,a,b Xiaojing Huang,a Hequan Yao,a Jieyun Jiang,c Xiaoming Wu,*a
Siyi Jiang,d Qiujuan Wang,d Tao Lue and Jinyi Xu*a
A series of hybrids from diaryl-1,2,4-triazole and hydroxamic acid or N-hydroxyurea were synthesized and
evaluated as novel anti-inflammatory agents. The biological data showed that (i) all the compounds
showed dual COX-2/5-LOX inhibitory activities in vitro, and 15e showed optimal inhibitory activities
(COX-2: IC50 = 0.15 μM, 5-LOX: IC50 = 0.85 μM), (ii) 15e selectively inhibited COX-2 relative to COX-1
with selectivity index (SI = 0.012) comparable to celecoxib (SI = 0.015), (iii) 15e exhibited potent anti-
inflammatory activity (inhibition: 54.1%) which was comparable to the reference drug celecoxib (inhibition:
46.7%) in a xylene-induced ear edema assay, and (iv) 15e displayed promising analgesic activity in acetic
acid-induced writhing response and hot-plate assay. Finally, a molecular modeling study revealed the
binding interactions of 15e with COX-2 and 5-LOX. Our findings suggest that 15e may be a promising
anti-inflammatory agent for further evaluation.
Received 24th September 2013,
Accepted 13th January 2014
DOI: 10.1039/c3ob41936c
www.rsc.org/obc
(ibuprofen, indomethacin and naproxen) and selective COX-2
inhibitors coxibs (celecoxib, rofecoxib and valdecoxib) block AA
1. Introduction
Inflammation is an extremely complex biological process metabolism by inhibiting the COX.2 However, the prolonged use
which implicates a great number of mediators originating of traditional NSAIDs are associated with several serious side
from the over-activation of multiple cascades. Eicosanoids effects such as kidney failure, ulcers and prolonged bleeding
including prostaglandins (PGs) and leukotrienes (LTs) syn- after an injury or surgery.3,4 Furthermore, the withdrawal of rofe-
thesized from arachidonic acid (AA) play an important role in coxib and valdecoxib from the market due to an increased risk
many inflammatory and allergic disorders, e.g. rheumatoid of cardiovascular complications (e.g., myocardial infarction and
arthritis (RA), osteoarthritis (OA), asthma and psoriasis.1 stroke) aroused great concern about coxibs’ safety problem.5–7 In
Following its release from membrane-bound phospholipids, AA spite of advances in the last decades, safe and effective anti-
undergoes further biotransformation via cyclooxygenase (COX) inflammatory drug design remains a great challenge.
and 5-lipoxygenase (5-LOX) pathways. The COX pathway has long
It is generally agreed that drugs acting on an individual
been used as a target for anti-inflammatory drugs. Both tra- target usually exert unwanted therapeutic effects and even
ditional non-steroidal anti-inflammatory drugs (NSAIDs) severe toxicity, whereas multi-target therapeutics which regu-
late multiple nodes of the disease network simultaneously
exhibit a synergistic effect and provide optimal clinical use.8
aState Key Laboratory of Natural Medicines and Department of Medicinal Chemistry,
China Pharmaceutical University, Nanjing 210009, P. R. China.
E-mail: xmwu@cpu.edu.cn; Fax: +86-25-83242366; Tel: +86-25-83242366;
E-mail: jinyixu@china.com; Fax: +86-25-83302827; Tel: +86-25-83271299
bInstitute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, P. R. China
cDepartment of Microbiology, Immunology and Molecular Genetics, University of
Kentucky College of Medicine, Lexington, KY 40536, USA
dDepartment of Physiology, China Pharmaceutical University, Nanjing 210009,
P. R. China
eLaboratory of Molecular Design and Drug Discovery, China Pharmaceutical
5-LOX is another key enzyme in AA metabolism and respon-
sible for the conversion of AA into LTs which are potent
mediators of inflammation and allergic reactions and may
play a role in cardiovascular diseases.9 It has also been well
established that inhibition of the COX enzymes may shunt AA
metabolism to the 5-LOX pathway,10 but when both COX-2 and
5-LOX were blocked the production of inflammatory mediators
could be completely shut off.11 Thus, dual inhibition of COX-2
and 5-LOX constitutes a rational concept for the design of
more efficacious anti-inflammatory agents with an improved
safety profile.12 Besides, both COX-2 and 5-LOX are implicated
University, Nanjing 211198, P. R. China
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ob41936c
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Fig. 1 Structures of zileuton, representative dual COX-2/5-LOX inhibi-
tors and lead compound 1.
in a number of therapeutic areas of interest including
cancers13–15 and age-related degenerative conditions, particu-
larly Alzheimer’s and Parkinson’s diseases.16,17
Various structures of dual COX/5-LOX inhibitors have been
developed, and the representatives are tepoxalin, licofelone
and ZD-2138 (Fig. 1).18–20 Tepoxalin has been approved for the
treatment of OA in dogs in the United States and the European
Union.20,21 Licofelone is a promising dual COX/5-LOX inhibi-
tor. The results of Phase III clinical trials suggested that lico-
felone is effective in the treatment of OA with reduced
gastrointestinal (GI) toxicity and thromboembolic risk compared
to traditional NSAIDs and COX-2 selective inhibitors.22–25
Besides the above-mentioned dual inhibitors, natural products
are also an important source of dual inhibitors,26–29 among
them, flavocoxid (Limbrel®) has been launched to market as an
FDA-regulated medical food for the clinical dietary management
of the metabolic aspects of OA in the United States.30
Recently, we have reported that 1,5-diaryl-1,2,4-triazole
derivatives were identified as selective COX-2 inhibitors,
among which compound 1 exhibited potent and selective
COX-2 inhibitory activity (IC50 = 0.37 μM, SI = 0.018), being
equipotent to celecoxib.31 Meanwhile we have noticed a report
about hydroxamic acid or N-hydroxyurea structures, which have
provided one of the most successful strategies to develop iron-
chelating 5-LOX inhibitors—zileuton (Fig. 1).32 As a part of our
continuing effort to find new NSAIDs candidates, we became
interested in exploring dual COX-2/5-LOX inhibitors with novel
structural characteristics, better anti-inflammatory effects and
lower cardiovascular risk. Accordingly, we designed a series of
novel hybrids in which the selective inhibitory COX-2 moiety
diaryl-1,2,4-triazole of compound 1 was incorporated with
hydroxamic acid or N-hydroxyurea group. Herein we report the
synthesis, biological evaluation and docking studies of these
diaryl-1,2,4-triazoles bearing the hydroxamic acid or hydro-
xyurea moiety as dual COX/5-LOX inhibitors (Fig. 2).
Fig. 2 Diaryl-1,2,4-triazoles bearing the hydroxamic acid or N-hydro-
xyurea moiety.
position substituted phenylhydrazine hydrochloride as initial
material, the critical intermediates 6a–j were synthesized as
described in our previous report.31 The thiols bearing 1,2,4-
triazole were readily converted into the corresponding esters
7a–o by treatment with ethyl 3-bromopropionate in good
yields. The target hydroxamic acid derivatives 8a–j could be
directly prepared by mixing 7a–o with hydroxylamine metha-
nol solution in the presence of KOH at room temperature.
Nevertheless, it was difficult to obtain N-methyl hydroxamic
acid derivatives 10a–o by the above-mentioned procedures, pre-
sumably owing to the steric hindrance of methyl. Thus, the
esters 7a–o were converted to more active acyl chloride 9a–j,
then by reaction with N-methyl hydroxylamine hydrochloride
in the presence of Et3N to give the desired N-methylhydroxa-
mic acid compounds 10a–e. The target compounds bearing
p-NH2SO2 moiety at N-1 phenyl ring were obtained by depro-
tection of tert-butyl in CF3COOH–PhOCH3 solution.
The strategy to synthesize N-hydroxyurea derivatives 15a–j is
shown in Scheme 2. Commercially available bromoacetalde-
hyde diethyl acetal 11 was converted directly to oxime 12,33
which was treated with intermediates 6a–j to yield the requisite
oximes 13a–j in high overall yields. Reduction of the oximes to
the hydroxylamines 14a–j was carried out by treatment of
13a–j with a 3-fold excess of NaBH3CN in the presence of 4 N
HCl. The conversion of 14a–j to the aimed compounds
N-hydroxyureas 15a–j was performed via the addition of
KOCN/HOAc in methanol.
2. Results and discussion
2.1. Chemistry
The target 3,4,5-substituted-1,2,4-triazole derivatives 21a–d
and 22a–d were prepared using the synthetic route illustrated
in Scheme 3. Starting with appropriate benzoic acids 16a–b,
The synthetic route of target compounds 8a–j and 10a–o is
shown in Scheme 1. Using commercially available para-
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Scheme 1 Synthesis of hydroxamic acid derivatives of 1,3,5-substituted-1,2,4-triazoles. Reagents and conditions: (i) KSCN, con. HCl, anhydrous
EtOH, reflux, 5 h; (ii) Et3N, acetone, reflux, 2 h; (iii) a. 10% NaOH, MeOH, reflux, 2 h; b. 10% HCl; (iv) ethyl 3-bromopropionate, K2CO3, acetone, reflux,
3 h; (v) a. 2 mol L−1 NH2OH/MeOH, KOH, r.t., 1 h; b. glacial acetic acid; (vi) TFA/methylanisole, 0 °C–r.t., 24 h; (vii) a. 10% NaOH, MeOH, reflux, 2 h;
b. 10% HCl; c. oxalyl dichloride, dry DCM, r.t., 3 h; (viii) N-methylhydroxylamine hydrochloride, Et3N, dry DCM, r.t., 2 h.
2.2. In vitro COX-2 and 5-LOX inhibitory activities assay
All the target compounds were initially evaluated in vitro to
determine their COX-2 and 5-LOX inhibitory activities by
measuring PGE2 and LTB4 levels from macrophages and leuko-
cytes, respectively. Celecoxib (COX-2 inhibitor), and zileuton
(5-LOX inhibitor) were reference drugs. As summarized in
Table 1, all of the compounds exhibited modest to potent
COX-2/5-LOX inhibitory activities. Among them, 8c, 8d, 8e,
10e, 10k, 10m and 15e showed potent COX-2 inhibitory activity
(IC50 = 0.13–0.19 μM) comparable to that of the reference drug
celecoxib (IC50 = 0.14 μM), meanwhile, 8e, 10e, 10k, 10m, 15c,
15d, 15e, 15k, 15m, 15o, 21a, 21b, 21d, 22b and 22d exhibited
equipotent inhibitory activity of 5-LOX (IC50 = 0.74–0.89 μM) to
that of the reference drug zileuton (IC50 = 0.82 μM). We
Scheme 2 Synthesis of N-hydroxyurea derivatives of 1,3,5-substituted-
1,2,4-triazoles. Reagents and conditions: (i) hydroxylamine hydrochlo-
guessed that the moieties of target compounds in charge of
5-LOX inhibition are hydroxamic acid or N-hydroxyurea, so the
compounds with the same pharmacophore showed small stat-
istical difference in 5-LOX assay.
ride, 1 N HCl, 30 °C, 24 h; (ii) 12, K2CO3, acetone, reflux, 3 h; (iii) sodium
cyanoborohydride, 4 N HCl, 30 °C, 4 h; (iv) potassium cyanate, glacial
acetic acid, MeOH, 30 °C, 4 h; (v) TFA/methylanisole, 0 °C–r.t., 24 h.
It is notable that introduction of electron-withdrawing
group (F, CF3 and Br) at the para-position of the C-5 phenyl
ring provided potent dual COX-2/5-LOX inhibitory activity,
indicating that lipophilicity is critical to the dual COX-2/5-LOX
potency. In the hydroxamic acid substituted series, especially
8e (COX-2: 0.14 μM; 5-LOX: 0.87 μM), 10e (COX-2: 0.17 μM;
5-LOX: 0.86 μM), 10k (COX-2: 0.17 μM; 5-LOX: 0.80 μM) and
benzoic acid hydrazides 18a–b formed by the reaction of
methyl esters 17a–b with para-position substituted phenyl iso-
thiocyanate. 19a–c were cyclized into 4,5-diaryl-4H-l,2,4-tria-
zole-3-thiols (20a–c) in aqueous NaOH/MeOH solution.34 The
thiols 20a–c were further treated with acetaldehyde oxime 12
to form target oximes 21a–d and N-hydroxyureas 22a–d.
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Scheme 3 Synthesis of 3,4,5-substituted-1,2,4-triazole derivatives. Reagents and conditions: (i) iodomethane, K2CO3, DMF, 80 °C, 1 h; (ii) H2NNH2,
H2O, EtOH, reflux, 6 h; (iii) p-fluorophenyl isothiocyanate or p-trifluorophenyl isothiocyanate, anhydrous EtOH, reflux, 5 h; (iv) a. 10% NaOH, MeOH,
reflux, 2 h; b. 10% HCl. (v) 12, K2CO3, acetone, reflux, 3 h; (vi) a. sodium cyanoborohydride, 4 N HCl, 30 °C, 4 h; b. potassium cyanate, glacial acetic
acid, MeOH, 30 °C, 4 h; (vii) TFA/methylanisole, 0 °C–r.t., 24 h.
Table 1 Structures and inhibitory activities against COX-2 and 5-LOX of
Table 1 (Contd.)
target compounds
COX-2
5-LOX
Compds
R1
R2
R3
IC50 a (μM) IC50 a (μM)
COX-2
5-LOX
Compds
R1
R2
R3
IC50 a (μM) IC50 a (μM)
15m
15o
CF3
Br
H2NSO2
H2NSO2
—
—
0.22 0.09 0.80 0.06
0.32 0.14 0.87 0.09
8a
8b
8c
8d
8e
8f
8g
8h
H
CH3SO2
H
H
H
H
H
H
H
H
H
H
1.24 0.11 0.92 0.03
0.66 0.07 1.05 0.10
0.15 0.01 0.93 0.21
0.13 0.03 0.89 0.11
0.14 0.02 0.87 0.12
1.71 0.20 1.08 0.08
1.05 0.22 1.24 0.17
0.72 0.16 0.98 0.05
0.84 0.05 0.97 0.09
0.93 0.07 1.11 0.12
0.94 0.14 0.98 0.04
0.56 0.08 1.02 0.23
0.25 0.05 0.92 0.17
0.21 0.01 0.91 0.05
0.17 0.06 0.86 0.03
0.92 0.13 0.93 0.15
0.51 0.04 0.98 0.07
0.17 0.09 0.80 0.07
0.18 0.05 0.74 0.12
0.44 0.03 0.91 0.18
CH3 CH3SO2
F
CF3
Br
H
CH3 H2NSO2
F
CF3
Br
H
CH3 CH3SO2 CH3
F
CF3
Br
H
CH3 H2NSO2 CH3
F
CF3
Br
21a
22a
F
F
CH3SO2
CH3SO2
0.85 0.11 0.87 0.08
0.97 0.24 0.95 0.15
CH3SO2
CH3SO2
CH3SO2
H2NSO2
21d
22d
F
F
H2NSO2
H2NSO2
1.02 0.31 0.83 0.06
1.15 0.15 0.85 0.04
H2NSO2
H2NSO2
H2NSO2
8i
8j
10a
10b
10c
10d
10e
10g
10i
10k
10m
10o
CH3SO2 CH3
21b
22b
CF3
CF3
CH3SO2
CH3SO2
0.89 0.02 0.78 0.09
0.96 0.08 0.84 0.13
CH3SO2 CH3
CH3SO2 CH3
CH3SO2 CH3
H2NSO2 CH3
Celecoxib
Zileuton
0.14 0.05
—
—
0.82 0.07
H2NSO2 CH3
H2NSO2 CH3
H2NSO2 CH3
a The test compound concentration required to produce 50%
inhibition of COX-2 or 5-LOX in vitro. The result (IC50, μM) is the mean
of four determinations.
10 m (COX-2: 0.18 μM; 5-LOX: 0.74 μM) are much more
effective dual COX-2/5-LOX inhibitors in vitro. While in the N-
hydroxyurea substituted series, compounds with p-CH3SO2
moiety at N-1 phenyl ring and F, CF3 and Br group at para-posi-
tion of C-5 phenyl ring provided more potent COX-2/5-LOX
15a
15b
15c
15d
15e
15g
15i
H
CH3SO2
—
—
—
—
—
—
—
—
0.97 0.16 0.91 0.13
0.53 0.11 0.90 0.08
0.28 0.03 0.89 0.17
CH3 CH3SO2
F
CF3
Br
H
CH3SO2
CH3SO2
CH3SO2
H2NSO2
0.26 0.04 0.87 0.05 inhibition, such as 15c (COX-2: 0.28 μM; 5-LOX: 0.89 μM), 15d
0.15 0.07 0.85 0.03
0.87 0.12 1.03 0.14
0.69 0.06 0.90 0.15
(COX-2: 0.26 μM; 5-LOX: 0.87 μM), 15e (COX-2: 0.15 μM; 5-LOX:
0.85 μM). It is also of interest that shifting the N-1 aromatic
0.26 0.05 0.88 0.12 ring to N-4 position led to a dramatic decrease in COX-2
CH3 H2NSO2
H2NSO2
15k
F
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Table 3 In vivo anti-inflammatory activity of 8d, 10k, 10m and 15e on
xylene-induced ear edema in mice (n = 7, x s)
¯
Swollen extent
(weight, mg)
Groups
Inhibition (%)
Model
Celecoxib
8d
10k
10m
4.70 1.27
2.51 1.44*
4.62 3.10
3.72 1.95
3.68 1.06
2.16 0.47**
—
46.7
1.7
20.9
21.7
54.1
15e
*P < 0.05, **P < 0.01 vs. model.
Fig. 3 The preliminary structure–activity relationships of diaryl-1,2,4-
triazoles.
0.85 µM) to that of reference drug zileuton (IC50 = 0.82 µM, see
Table 1). Thus, the favorable IC50 value of COX-1/COX-2 and
5-LOX as well as reasonable SI value implied that 15e may
have more potent anti-inflammatory activity and relative cardio-
vascular safety profile.
inhibitory activity (COX-2: 0.85–1.15 μM, celecoxib: 0.14 μM),
such as 3,4,5-substituted-1,2,4-triazole derivatives 21a, 21b,
21d, 22a, 22b and 22d, presuming that the “Y-type” structure
of 1,5-diaryl-1,2,4-triazoles is favorable for the compounds
binding in the COX-2 and 5-LOX active site. The structure–
activity relationships are summarized in Fig. 3.
2.4. Xylene-induced ear edema in mice assay
Compounds 8d, 10k, 10m, and 15e were selected for the study
of their anti-inflammatory activity in vivo using xylene-induced
ear edema in mice model and the results are listed in Table 3.
Unfortunately, the hydroxamic acid substituted series with
good dual COX-2/5-LOX inhibitory activity in vitro did not
exhibit potent anti-inflammatory activity (inhibition:
1.7–21.7% at a dose of 30 mg kg−1) in vivo as expected. It is
presumed that the low bioavailability of hydroxamic acid sub-
stituted compounds affects their anti-inflammatory activity.
Nevertheless, N-hydroxyurea substituted compound 15e dis-
played the most potent anti-inflammatory activity (inhibition:
54.1% at a dose of 30 mg kg−1), which is comparable to
the reference drug celecoxib (inhibition: 46.7% at a dose of
30 mg kg−1).
2.3. Assay of COX-1/COX-2 inhibitory and selectivity index of
10e, 10m, 15e and 15m
Based on the favorable results of preliminary pharmacological
tests, compounds 10e, 10m, 15e and 15m were evaluated in
concentration–response studies to determine the COX-1/COX-2
inhibitory IC50. The main problem with the COX-2 drugs is
over-high selectivity to COX-2, leading to an imbalance
between prostacyclin and thromboxane A2 (TXA2), which may
produce potential cardiovascular risks, which is the reason
why rofecoxib was withdrawn from the market. Therefore, the
design of COX-2 “preferential” inhibitors keeping a slight
effect on COX-1 at therapeutic dosage could theoretically limit
the imbalance of prostacyclin/TXA2, while the balance
between prostacyclin and TXA2 is significant to avoid cardio-
vascular side-effects, such as celecoxib, which possess suitable
selectivity for inhibition of COX-2/COX-1, is still available in
clinical. So it is necessary to evaluate selectivity index (SI) of
the compounds to predict the cardiovascular safety of them. As
summarized in Table 2, 15e exhibited potent and selective COX-2
inhibitory activity (COX-1 IC50 = 12.5 µM, COX-2 IC50 = 0.15 µM,
SI = 0.012) which was comparable to that of the reference drug
celecoxib (COX-1 IC50 = 7.7 µM; COX-2 IC50 = 0.12 µM; SI =
2.5. Albumen-induced rat paw edema and acetic
acid-induced mouse vascular permeability assays
As an interesting new entity, 15e was selected for further anti-
inflammatory activity evaluation in vivo and the results are
shown in Tables 4 and 5. In the albumen-induced rat paw
edema assay, the high and medium dosage groups of 15e
exhibited significant anti-inflammatory activity (P < 0.01)
0.015).35 While 15e displayed equipotent 5-LOX inhibition (IC50
=
Table 4 The effect of 15e on albumen-induced paw edema in rats
Table 2 In vitro COX-1 inhibitory activity and selectivity index for 10e,
10m, 15e and 15m
(n = 10, x s)
¯
Swollen extent (×10, mL)
a
COX-1 IC50
(μM)
Selectivity
index b (SI) Groups
Dosea
Compds
(mg kg−1
)
0.5 h
1 h
2 h
3 h
10e
10m
15e
15m
15.5 1.84
20.0 3.61
12.5 0.79
24.4 5.03
7.7c
0.011
0.009
0.012
0.009
0.015c
Model
Celecoxib 30
15e
—
1.40 0.41
1.09 0.44 0.67 0.44 0.63 0.41
0.72 0.38** 0.62 0.29* 0.53 0.34 0.49 0.28
0.69 0.27** 0.65 0.17* 0.63 0.24 0.50 0.24
0.71 0.34** 0.70 0.32* 0.64 0.30 0.56 0.27
0.85 0.53* 0.82 0.47 0.74 0.48 0.65 0.43
60
30
15
Celecoxib
a The result (IC50, μM) is the mean of four determinations. b In vitro *P < 0.05, **P < 0.01 vs. model. a The results are expressed as percentage
COX-2 selectivity index (COX-2 IC50/COX-1 IC50). c See ref. 35.
of inhibition following 60, 30, 15 mg kg−1 oral dose of 15e.
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Table 5 The effect of 15e on acid-induced vascular permeability in
mice (n = 10, x s)
¯
Dosea
Groups
(mg kg−1
)
A590
Model
Celecoxib
15e
—
30
60
30
15
0.375 0.312
0.142 0.098*
0.077 0.069**
0.098 0.111*
0.167 0.191
*P < 0.05, **P < 0.01 vs. model. a The results are expressed as mean
SEM (n = 10) following 60, 30, 15 mg kg−1 oral dose of 15e.
comparable to that of the reference drug celecoxib at 30 min
after compound administration, and lasted for 1 h (P < 0.05) in
high dosage group. On the other hand, 15e dose dependently
decreases the permeability of celiac blood capillary in the
acetic acid-induced mouse vascular permeability assay. These
data indicated that 15e had certain potency equivalent to that
of the reference drug celecoxib in the acute inflammation
model.
Fig. 5 The analgesic activity of 15e on acetic acid-induced writhing
response in mice (n = 10, x s). *P < 0.05, **P < 0.01 vs. model.
¯
2.7. Molecular modeling (docking) studies
Molecular modeling (docking) studies were carried out to
investigate the binding interactions of these derivatives with
the COX-2 and 5-LOX active sites. For clarity, only 15e was dis-
played as it exhibits balanced COX-2/5-LOX inhibitory activity.
Fig. 6 shows the conformational superposition of 15e and
SC-558, the known selective COX-2 inhibitor. Both of the com-
pounds exhibit a Y-type structure and the two phenyl rings
stretch in the same direction indicating that it is preferential
for 15e binding in the active site of COX-2. As illustrated in
Fig. 7, the N-1 bromophenyl ring is oriented towards the apex
2.6. Analgesic activity evaluation
Both chemical (acetic acid-induced writhing response) and
thermal method (hot-plate assay) in mice were employed to
investigate the analgesic activity of 15e. As illustrated in Fig. 4,
15e significantly (P < 0.01) increased the pain threshold
towards the thermal source in a dose-dependent manner after
90 min of administration, indicating that 15e may contribute
to analgesic activity. In the acetic acid-induced writhing
response assay, all dosage groups of 15e dramatically (P < 0.01)
increased the writhe latency and reduced the number of
abdominal constrictions and stretching of hind limbs induced
by the injection of acetic acid (Fig. 5). These results demon-
strated that 15e exhibited potent analgesic activity comparable
to that of the reference drug celecoxib and may involve pro-
inflammatory mediator PGE2 release.
of the COX-2 active site composed of Phe381, Leu384, Tyr385
,
Trp387 and Ser530. The C-5 p-CH3SO2-phenyl moiety undergoes
interactions with hydrophobic residues Tyr355, Phe518, Val523
and Ser353. The SO2CH3 moiety is positioned in a relatively
polar pocket, wherein the two oxygen atoms form hydrogen
bonds with Arg513 and His90, respectively, which are crucial for
Fig. 4 The analgesic activity of 15e on hot-plate assay in mice (n = 10,
Fig. 6 Conformational superposition of 15e (red) and selective COX-2
x
¯
s). *P < 0.05, **P < 0.01 vs. model.
inhibitor SC-558 (green).
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much closer to iron (distance 2.83 Å) than the ligands His367
,
His372 and His550. The results of our docking studies demon-
strated that 15e binds with both active sites of COX-2 and 5-
LOX by hydrophobic interactions with relative amino acid resi-
dues, and stabilizes binding conformation by one or more
hydrogen bonds. The results are in agreement with reported
studies,31 and may validate the potent dual inhibitory activity
of 15e.
3. Conclusions
Fig. 7 Binding mode of 15e in the active site of COX-2. Red lines rep-
In summary, a series of novel hybrids from diaryl-1,2,4-triazole
and hydroxamic acid or N-hydroxyurea were designed, syn-
thesized and evaluated as dual COX-2/5-LOX inhibitors. It was
found that (i) 1,3,5-substituted-1,2,4-triazole scaffold was criti-
resent hydrogen bonds.
COX-2 selectivity. On the other hand, the 5-LOX pharmaco-
phore N-hydroxyurea moiety is oriented in the bottom of the cal for dual COX-2/5-LOX inhibition; (ii) the hydroxamic acid
hydrophobic pocket formed by Val116, Val349, Tyr355, Leu359 hybrid 1,3,5-substituted-1,2,4-triazoles, especially 10k and
and Leu531. The binding mode is similar to that of SC-558.36
10m, were potent dual COX-2/5-LOX inhibitors in vitro; (iii) in
We employed the newly reported human 5-LOX (PDB code the N-hydroxyurea hybrid 1,3,5-substituted-1,2,4-triazoles, 15e
3O8Y) for docking model.37 As shown in Fig. 8, a U-shape con- exhibited optimal dual COX-2/5-LOX inhibitory activities in
formation is found for 15e in the 5-LOX catalytic domain.
vitro and potent oral anti-inflammatory and analgesic activities
1,2,4-Triazole moiety is positioned in the centre of the active in vivo,38 (iv) the molecular modeling study revealed that 15e
site, and the C-5 p-CH3SO2-phenyl moiety is oriented deep into binds with both active sites of COX-2 and 5-LOX by hydro-
the bottom channel of the hydrophobic pocket formed by
phobic interactions and stabilizes binding conformation by
Tyr181, Ala603, Trp599, His600, Asn425, and Phe421, wherein one or more hydrogen bonds. Collectively, 15e might be a
Tyr181, Ala603, Trp599 and His600 are specific to the 5-LOX promising anti-inflammatory agent for further investigations.
sequence. It is noteworthy that one of the oxygen atoms of the
p-CH3SO2 substituent forms a hydrogen bond with Asn425. The
N-1 bromophenyl ring is oriented in a more elongated cleft
4. Experimental section
4.1 Chemistry
and forms a hydrogen bond with Thr364. The non-heme iron,
which plays an important role in 5-LOX cascade, is coordinated
by three conserved histidines (His367, His372 and His550) as Reagents and all solvents were purchased from Shanghai
well as Ile673. The hydroxyl oxygen of N-hydroxylurea moiety
Chemical Reagent Company and used without further purifi-
and the sulphur atom of 15e form hydrogen bonds with His372 cation. All of the experiments were monitored by analytical
and Ile673, respectively. Furthermore, the sulphur atom is thin-layer chromatography (TLC) performed on silica gel
GF254 precoated plates. Column chromatography was carried
out using silica gel (200–300 mesh). The purities of all of the
compounds (>95%) used for biological screening were deter-
mined by high-performance liquid chromatography (HPLC)
(Agilent, 1260 INFINITY) using a Gemini C18 column (4.6 mm
× 150 mm, 5 μm) eluted with a gradient mixture of acetonitrile
and water. Melting points were determined using MEL-TEMP
II melting point apparatus and are uncorrected. 1H-NMR
spectra were recorded on a Bruker-ACF spectrometer (300 or
500 MHz) in DMSO-d6. Chemical shifts are reported in ppm (δ)
relative to tetramethylsilane (TMS) as an internal standard.
Multiplicities are given as s (singlet), d (doublet), dd (double-
doublet), t (triplet), q (quadruplet), m (multiplet) and br s
(broad signal). Mass spectra were recorded on an Agilent LC/
MSD TRAP SL spectrometer equipped with an electrospray ion-
isation (ESI) interface; Elemental analysis: Elementar Vario EL
III instrument. IR (in cm−1) spectra in KBr pellets on a Bruker
FT-IR TENSOR 27 instrument.
Fig. 8 Binding mode of 15e in human 5-LOX active site. Green lines are
General procedure for synthesis of compounds 7a–e,
7f, 7h, 7j, 7l, 7n. Corresponding 1,2,4-triazole-3-thiols 6a–j
distance between non-heme iron to ligands, and the red dashed lines
are hydrogen bonds.
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(4.8 mmol) and K2CO3 (6.8 mmol) were suspended in 10 mL C18H17FN4O4S2: C, 49.53; H, 3.93; N, 12.84. IR (KBr/cm−1):
acetone at 50 °C for 30 min, then ethyl 3-bromopropionate 3423, 2963, 2923, 1657, 1510, 1304, 1149, 844, 779, 564.
(6.8 mmol) was added, the mixture was allowed to reflux for 1H-NMR (DMSO-d6, 500 MHz) δ: ppm 10.51 (s, 1H, -OH), 8.87
3 h, then was filtered and the filtrate was evaporated in vacuo. (s, 1H, -NH), 8.03 (d, J = 8.6 Hz, 2H), 7.75 (d, J = 8.7 Hz, 2H),
The residue was purified by silica gel column chromatography 7.61 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 8.2 Hz, 2H), 3.41 (t, J = 6.9
using ethyl acetate/petroleum ether (1 : 2, v/v) as eluent to Hz, 2H, -CH2-), 3.31 (s, 3H, SO2CH3), 2.56 (t, J = 7.0 Hz, 2H,
afford a white solid.
-CH2-). MS (ESI, m/z): 435 [M − H]−.
N-Hydroxy-3-(5-(4-(methylsulfonyl)phenyl)-1-(4-(trifluoromethyl)-
phenyl)-1H-1,2,4-triazol-3-ylthio)propanamide (8d). The title
compound was obtained as a white solid (0.14 g, 32%), mp
84–86 °C. Found: C, 46.63; H, 3.76; N, 11.90. Calc. for
C19H17F3N4O4S2: C, 46.91; H, 3.52; N, 11.52. IR (KBr/cm−1):
3344, 2925, 1660, 1445, 1326, 1149, 988, 848, 777, 533.
1H-NMR (DMSO-d6, 500 MHz) δ: ppm 10.46 (s, 1H, -OH), 8.82
(s, 1H, -NH), 7.99 (d, J = 8.3 Hz, 2H), 7.91 (d, J = 8.8 Hz, 2H),
7.73 (d, J = 8.2 Hz, 2H), 7.68 (d, J = 8.8 Hz, 2H), 3.37 (t, J =
6.9 Hz, 2H, -CH2-), 3.27 (s, 3H, SO2CH3), 2.52 (t, J = 6.9 Hz, 2H,
-CH2-). MS (ESI, m/z): 485 [M − H]−.
3-(1-(4-Bromophenyl)-5-(4-(methylsulfonyl)phenyl)-1H-1,2,4-
triazol-3-ylthio)-N-hydroxypropanamide (8e). The title com-
pound was obtained as a white solid (0.14 g, 27%), mp
82–84 °C. Found: C, 43.69; H, 3.61; N, 11.60. Calc. for
C18H17BrN4O4S2: C, 43.47; H, 3.45; N, 11.26. IR (KBr/cm−1):
3418, 3228, 3019, 2923, 1660, 1492, 1303, 1150, 987, 780, 534.
1H-NMR (DMSO-d6, 500 MHz) δ: ppm 10.46 (s, 1H, -OH), 8.81
(s, 1H, -NH), 7.98 (d, J = 8.5 Hz, 2H), 7.72 (m, 4H), 7.73 (d, J =
8.2 Hz, 2H), 7.41 (d, J = 8.6 Hz, 2H), 3.35 (t, J = 6.9 Hz, 2H,
-CH2-), 3.26 (s, 3H, SO2CH3), 2.51 (t, J = 6.9 Hz, 2H, -CH2-). MS
(ESI, m/z): 496 [M − H]−.
N-Hydroxy-3-(1-phenyl-5-(4-sulfamoylphenyl)-1H-1,2,4-triazol-
3-ylthio)propanamide (8f). The title compound was obtained
as a white solid (0.04 g, 33.2%), mp 92–95 °C. Found: C, 48.42;
H, 4.35; N, 16.58. Calc. for C17H17N5O4S2: C, 48.68; H, 4.08; N,
16.70. IR (KBr/cm−1): 3345, 3072, 2962, 2922, 1656, 1512, 1451,
1303, 1151, 822, 776, 538. 1H-NMR (DMSO-d6, 500 MHz) δ:
ppm 10.45 (s, 1H, -OH), 8.80 (s, 1H, -NH), 7.82 (d, J = 8.5 Hz,
2H), 7.61 (d, J = 8.5 Hz, 2H), 7.48 (s, 2H, SO2NH2), 7.54 (m,
3H), 7.46 (m, 2H), 3.37 (t, J = 6.9 Hz, 2H, -CH2-), 2.52 (t, J =
6.9 Hz, 2H, -CH2-). MS (ESI, m/z): 418 [M − H]−.
N-Hydroxy-3-(5-(4-sulfamoylphenyl)-1-p-tolyl-1H-1,2,4-triazol-
3-ylthio)propanamide (8g). The title compound was obtained
as a white solid (0.11 g, 29.2%), mp 120–122 °C. Found: C,
50.09; H, 4.68; N, 16.47. Calc. for C18H19N5O4S2: C, 49.87; H,
4.42; N, 16.16. IR (KBr/cm−1): 3442, 2962, 2922, 2851, 1644,
1513, 1335, 1163, 822, 613. 1H-NMR (DMSO-d6, 300 MHz) δ:
ppm 10.46 (s, 1H, -OH), 8.84 (s, 1H, -NH), 7.82 (d, J = 8.5 Hz,
2H), 7.61 (d, J = 8.5 Hz, 2H), 7.48 (s, 2H, SO2NH2), 7.32 (m,
4H), 3.48 (t, J = 6.9 Hz, 2H, -CH2-), 2.50 (t, J = 6.9 Hz, 2H, -CH2-),
2.38 (s, 3H, phenyl-CH3). MS (ESI, m/z): 432 [M − H]−.
General procedure for synthesis of compounds 7g, 7i, 7k,
7m, 7o. A solution of the corresponding compounds 7f, 7h, 7j,
7l, 7n (1 mmol), CF3COOH (2 mL) and two drops of methyl-
phenyl ether was stirred at room temperature for 24 h. The
mixture was evaporated in vacuo, and the residue was purified
by silica gel column chromatography using ethyl acetate/pet-
roleum ether (1 : 3, v/v) as eluent to afford a white solid.
General procedure for synthesis of compounds 8a–j. Hydro-
xylamine hydrochloride (0.025 mol) was suspended in metha-
nol (4.35 mL) at reflux for 1 h, and KOH (0.025 mol) was
refluxed in methanol (8.15 mL) for 1 h. After being cooled to
40 °C, KOH methanol solution was poured into hydroxylamine
hydrochloride suspension, then the precipitated KCl was fil-
tered. The filtrate is 2 mol L−1 hydroxylamine methanol solu-
tion. Corresponding ethyl esters 7a–o (0.76 mmol) were added
(over 30 min) to the 2 mol L−1 hydroxylamine methanol solu-
tion (7 mL, 14 mmol) followed by addition of KOH to adjust to
pH = 10. The mixture was stirred at room temperature for 1 h.
The mixture was poured into stirring cold water, and the pH
was adjusted to 7 by adding acetic acid. The aqueous solution
was extracted with ethyl acetate (3 × 10 mL), and the combined
extracts were dried over anhydrous Na2SO4 and evaporated in
vacuo. The residue was purified by silica gel column chromato-
graphy using CH2Cl2/MeOH (20 : 1, v/v) as eluent to afford a
white solid.
N-Hydroxy-3-(5-(4-(methylsulfonyl)phenyl)-1-phenyl-1H-1,2,4-
triazol-3-ylthio)propanamide (8a). The title compound was
obtained as a white solid (0.12 g, 37.5%), mp 83–85 °C. Found:
C, 52.01; H, 4.03; N, 13.07. Calc. for C18H18N4O4S2: C, 51.66; H,
4.34; N, 13.39. IR (KBr/cm−1): 3214, 3002, 2923, 1661, 1498,
1453, 1303, 1148, 958, 780, 694, 530. 1H-NMR (DMSO-d6,
300 MHz) δ: ppm 10.47 (s, 1H, -OH), 8.83 (s, 1H, -NH), 7.96 (d,
J = 8.5 Hz, 2H), 7.68 (d, J = 8.5 Hz, 2H), 7.54 (m, 3H), 7.46 (m,
2H), 3.33 (t, J = 6.9 Hz, 2H, -CH2-), 3.26 (s, 3H, SO2CH3), 2.52
(t, J = 6.9 Hz, 2H, -CH2-). MS (ESI, m/z): 417 [M − H]−.
N-Hydroxy-3-(5-(4-(methylsulfonyl)phenyl)-1-p-tolyl-1H-1,2,4-
triazol-3-ylthio)propanamide (8b). The title compound was
obtained as a white solid (0.21 g, 39.2%), mp 88–90 °C. Found:
C, 52.44; H, 4.94; N, 12.55. Calc. for C19H20N4O4S2: C, 52.76; H,
4.66; N, 12.95. IR (KBr/cm−1): 3452, 3003, 2924, 1646, 1513,
1304, 1150, 988, 779, 566. 1H-NMR (DMSO-d6, 500 MHz) δ:
ppm 10.44 (s, 1H, -OH), 8.79 (s, 1H, -NH), 7.95 (d, J = 8.5 Hz,
2H), 7.69 (d, J = 8.5 Hz, 2H), 7.33 (s, 4H), 3.33 (t, J = 7.0 Hz, 2H,
-CH2-), 3.25 (s, 3H, SO2CH3), 2.51 (t, J = 7.0 Hz, 2H, -CH2-), 2.38
(s, 3H, phenyl-CH3). MS (ESI, m/z): 431 [M − H]−.
3-(1-(4-Fluorophenyl)-5-(4-sulfamoylphenyl)-1H-1,2,4-triazol-3-
ylthio)-N-hydroxypropanamide (8h). The title compound was
obtained as a white solid (0.17 g, 31.3%), mp 84–86 °C. Found:
C, 46.44; H, 3.85; N, 15.72. Calc. for C17H16FN5O4S2: C, 46.67;
H, 3.69; N, 16.01. IR (KBr/cm−1): 3442, 2962, 2922, 2851, 1644,
1513, 1335, 1163, 822, 613. 1H-NMR (DMSO-d6, 300 MHz) δ:
ppm 10.47 (s, 1H, -OH), 8.83 (s, 1H, -NH), 8.00 (d, J = 8.6 Hz,
3-(1-(4-Fluorophenyl)-5-(4-(methylsulfonyl)phenyl)-1H-1,2,4-
triazol-3-ylthio)-N-hydroxypropanamide (8c). The title com-
pound was obtained as a white solid (0.14 g, 31%), mp
92–94 °C. Found: C, 49.71; H, 4.18; N, 12.48. Calc. for
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2H), 7.77 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 8.7 Hz, 2H), 7.48 (s, 1H, -OH), 7.95 (d, J = 8.5 Hz, 2H), 7.68 (d, J = 8.5 Hz, 2H), 7.53
2H, SO2NH2), 7.32 (d, J = 8.7 Hz, 2H), 3.42 (t, J = 6.9 Hz, 2H, (m, 3H), 7.45 (m, 2H), 3.35 (t, J = 6.8 Hz, 2H, -CH2-), 3.25 (s,
-CH2-), 2.67 (t, J = 6.9 Hz, 2H, -CH2-). MS (ESI, m/z): 436 3H, SO2CH3), 3.10 (s, 3H, -NCH3), 2.52 (t, J = 6.9 Hz, 2H, -CH2-
[M − H]−.
). MS (ESI, m/z): 433 [M + H]+.
N-Hydroxy-N-methyl-3-(5-(4-(methylsulfonyl)phenyl)-1-p-tolyl-
N-Hydroxy-3-(5-(4-sulfamoylphenyl)-1-(4-(trifluoromethyl)phenyl)-
1H-1,2,4-triazol-3-ylthio)propanamide (8i). The title compound 1H-1,2,4-triazol-3-ylthio)propanamide (10b). The title com-
was obtained as a white solid (0.18 g, 26.5%), mp 84–86 °C. pound was obtained as a white solid (0.2 g, 41.7%), mp
Found: C, 44.02; H, 3.69; N, 14.75. Calc. for C18H16F3N5O4S2: 178–180 °C. Found: C, 53.65; H, 4.55; N, 12.81. Calc. for
C, 44.35; H, 3.31; N, 14.37. IR (KBr/cm−1): 3396, 1656, 1454, C20H22N4O4S2: C, 53.79; H, 4.97; N, 12.55. IR (KBr/cm−1): 3443,
1
1325, 1166, 1063, 847, 621, 543. H-NMR (DMSO-d6, 300 MHz) 3093, 2941, 2922, 2802, 1644, 1515, 1318, 1152, 819, 780, 532.
δ: ppm 10.49 (s, 1H, -OH), 8.80 (s, 1H, -NH), 7.91 (d, J = 8.5 Hz, 1H-NMR (DMSO-d6, 300 MHz) δ: ppm 9.66 (s, 1H, -OH), 7.96
2H), 7.79 (d, J = 8.5 Hz, 2H), 7.61 (m, 4H), 7.35 (s, 2H, (d, J = 8.5 Hz, 2H), 7.72 (d, J = 8.5 Hz, 2H), 7.33 (s, 4H), 7.45
SO2NH2), 3.42 (t, J = 6.9 Hz, 2H, -CH2-), 2.59 (t, J = 6.9 Hz, 2H, (m, 2H), 3.32 (t, J = 6.8 Hz, 2H, -CH2-), 3.25 (s, 3H, SO2CH3),
-CH2-). MS (ESI, m/z): 486 [M − H]−.
3-(1-(4-Bromophenyl)-5-(4-sulfamoylphenyl)-1H-1,2,4-triazol-3-
3.12 (s, 3H, -NCH3), 2.89 (t, J = 6.9 Hz, 2H, -CH2-), 2.38 (s, 3H,
phenyl-CH3). MS (ESI, m/z): 481 [M + Cl]−.
ylthio)-N-hydroxypropanamide (8j). The title compound was
3-(1-(4-Fluorophenyl)-5-(4-(methylsulfonyl)phenyl)-1H-1,2,4-
obtained as a white solid (0.17 g, 34.2%), mp 96–98 °C. Found: triazol-3-ylthio)-N-hydroxy-N-methylpropanamide (10c). The
C, 41.35; H, 3.56; N, 14.43. Calc. for C17H16BrN5O4S2: C, 40.97; title compound was obtained as a white solid (0.2 g, 34.7%),
H, 3.24; N, 14.05. IR (KBr/cm−1): 3395, 2373, 2346, 1655, 1492, mp 169–171 °C. Found: C, 50.42; H, 4.41; N, 12.68. Calc. for
1335, 1160, 987, 835, 620. 1H-NMR (DMSO-d6, 300 MHz) δ: C19H19FN4O4S2: C, 50.65; H, 4.25; N, 12.44. IR (KBr/cm−1):
ppm 10.45 (s, 1H, -OH), 8.80 (s, 1H, -NH), 7.84 (d, J = 8.5 Hz, 3454, 2926, 1641, 1511, 1463, 1323, 1156, 850, 781, 567.
2H), 7.72 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 8.5 Hz, 2H), 7.46 (s, 1H-NMR (DMSO-d6, 300 MHz) δ: ppm 9.85 (s, 1H, -OH), 7.98
2H, SO2NH2), 7.40 (d, J = 8.7 Hz, 2H), 3.34 (t, J = 6.9 Hz, 2H, (d, J = 8.5 Hz, 2H), 7.74 (d, J = 8.5 Hz, 2H), 7.53 (d, J = 8.2 Hz,
-CH2-), 2.49 (t, J = 6.9 Hz, 2H, -CH2-). MS (ESI, m/z): 497 2H), 7.43 (d, J = 8.2 Hz, 2H), 3.32 (t, J = 6.9 Hz, 2H, -CH2-), 3.25
[M − H]−.
(s, 3H, SO2CH3), 3.10 (s, 3H, -NCH3), 2.89 (t, J = 6.9 Hz, 2H,
General procedure for synthesis of compounds 9a–j. To a -CH2-). MS (ESI, m/z): 451 [M + H]+.
N-Hydroxy-N-methyl-3-(5-(4-(methylsulfonyl)phenyl)-1-(4-(tri-
solution of the appropriate esters 7a–o (0.01 mol) in 10 mL
methanol, 5 mL 10% NaOH was added, and the mixture was
heated under reflux for 1 h. The solution was cooled and acidi-
fied to pH = 2 by adding 10% HCl. The precipitated yellow
solid was filtered, washed with water and dried to afford
corresponding carboxylic acids. Oxalyl chloride (0.2 mL,
1.97 mmol) and one drop of DMF as catalyst were added to a
solution of appropriate carboxylic acid (0.656 mmol) in dry
CH2Cl2 (10 mL). The resulting mixture was stirred for 3 h at
room temperature and the solvents were evaporated in vacuo.
The residue was used immediately without further purification
for the preparation of the target compounds 10a–o.
fluoromethyl)phenyl)-1H-1,2,4-triazol-3-ylthio)propanamide (10d).
The title compound was obtained as a white solid (0.18 g,
38.6%), mp 186–188 °C. Found: C, 47.82; H, 4.04; N, 11.45.
Calc. for C20H19F3N4O4S2: C, 47.99; H, 3.83; N, 11.19. IR
(KBr/cm−1): 3442, 3091, 2923, 2852, 2814, 1643, 1453, 1315,
1150, 990, 850, 777, 531. 1H-NMR (DMSO-d6, 300 MHz) δ: ppm
9.84 (s, 1H, -OH), 7.99 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 8.4 Hz,
2H), 7.73 (d, J = 8.3 Hz, 2H), 7.68 (d, J = 8.3 Hz, 2H), 3.32 (t,
J = 6.7 Hz, 2H, -CH2-), 3.26 (s, 3H, SO2CH3), 3.12 (s, 3H,
-NCH3), 2.91 (t, J = 6.7 Hz, 2H, -CH2-). MS (ESI, m/z): 501
[M + H]+.
3-(1-(4-Bromophenyl)-5-(4-(methylsulfonyl)phenyl)-1H-1,2,4-
triazol-3-ylthio)-N-hydroxy-N-methylpropanamide (10e). The
title compound was obtained as a white solid (0.11 g, 32.5%),
mp 180–182 °C. Found: C, 44.87; H, 3.48; N, 11.24. Calc. for
C19H19BrN4O4S2: C, 44.62; H, 3.74; N, 10.96. IR (KBr/cm−1):
3453, 3092, 2961, 2921, 2851, 2790, 1643, 1494, 1317, 1150,
824, 781, 531. 1H-NMR (DMSO-d6, 300 MHz) δ: ppm 9.88 (s,
1H, -OH), 7.98 (d, J = 8.4 Hz, 2H), 7.70 (m, 4H), 7.40 (d, J = 8.3
Hz, 2H), 3.32 (t, J = 7.0 Hz, 2H, -CH2-), 3.26 (s, 3H, SO2CH3),
3.09 (s, 3H, -NCH3), 2.88 (t, J = 6.9 Hz, 2H, -CH2-). MS (ESI,
m/z): 513 [M + H]+.
General procedure for synthesis of compounds 10a–o. An
appropriate solution of acyl chloride 9a–j in 10 mL dry CH2Cl2
was added dropwise to the suspension of N-methylhydroxyl-
amine hydrochloride (1.312 mmol) and Et3N (1.312 mmol) in
5 mL dry CH2Cl2. The mixture was stirred for an additional 2 h
at room temperature. Water (30 mL) was added to dilute, and
the CH2Cl2 layer was further washed by 10% HCl and brine.
The organic layer was collected, and dried over anhydrous
Na2SO4, filtered, and evaporated. The residue was purified by
silica gel column chromatography using CH2Cl2/MeOH (60 : 1,
v/v) as eluent to afford a white solid.
General procedure for synthesis of compounds 10g, 10i, 10k,
10m, 10o. A solution of the corresponding compounds 10f,
10 h, 10j, 10 l, 10n (1 mmol), CF3COOH (2 mL) and two drops
of methylphenyl ether was stirred at room temperature for
24 h. The mixture was evaporated in vacuo, and the residue
was purified by silica gel column chromatography using
CH2Cl2/MeOH (40 : 1, v/v) as eluent to afford a white solid.
N-Hydroxy-N-methyl-3-(5-(4-(methylsulfonyl)phenyl)-1-phenyl-
1H-1,2,4-triazol-3-ylthio)propanamide (10a). The title com-
pound was obtained as a white solid (0.25 g, 39.5%), mp
165–167 °C. Found: C, 53.02; H, 4.58; N, 12.73. Calc. for
C19H20N4O4S2: C, 52.76; H, 4.66; N, 12.95. IR (KBr/cm−1): 3440,
3172, 3063, 2947, 2925, 1636, 1501, 1460, 1319, 1265, 1149,
956, 781, 535. 1H-NMR (DMSO-d6, 300 MHz) δ: ppm 9.84 (s,
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N-Hydroxy-N-methyl-3-(1-phenyl-5-(4-sulfamoylphenyl)-1H-1,2,4- which time the reaction mixture was diluted and extracted
triazol-3-ylthio)propanamide (10g). The title compound was with EtOAc (3 × 15 mL). The combined organic extracts were
obtained as a white solid (0.04 g, 38.4%), mp 70–72 °C. Found: dried over anhydrous Na2SO4 and evaporated to give the oxime
C, 49.69; H, 4.58; N, 15.84. Calc. for C18H19N5O4S2: C, 49.87; H, 12 as a volatile liquid, which was used without further
4.42; N, 16.16. IR (KBr/cm−1): 3396, 2924, 2853, 1628, 1499, purification.
1453, 1336, 1161, 769, 619. 1H-NMR (DMSO-d6, 500 MHz)
General procedure for synthesis of compounds 13a–j.
δ: ppm 9.83 (s, 1H, -OH), 7.82 (d, J = 8.6 Hz, 2H), 7.60 (d, J = Corresponding 1,2,4-triazole-3-thiols 6a–j (0.982 mmol) and
8.6 Hz, 2H), 7.52 (m, 3H), 7.44 (m, 4H), 3.32 (t, J = 6.9 Hz, 2H, K2CO3 (1.37 mmol) were suspended in 10 mL acetone at 50 °C
-CH2-), 3.10 (s, 3H, -NCH3), 2.89 (t, J = 6.9 Hz, 2H, -CH2-). MS for 30 min, then bromoacetaldehyde oxime 12 (1.37 mmol)
(ESI, m/z): 434 [M + H]+.
was added. The mixture was allowed to reflux for 3 h, then was
filtered and the filtrate was evaporated in vacuo. The residue
was purified by silica gel column chromatography using ethyl
acetate/petroleum ether (1 : 2, v/v) as eluent to afford a white
solid.
N-Hydroxy-N-methyl-3-(5-(4-sulfamoylphenyl)-1-p-tolyl-1H-1,2,4-
triazol-3-ylthio)propanamide (10i). The title compound was
obtained as a white solid (0.19 g, 47.8%), mp 47–49 °C. Found:
C, 51.28; H, 4.95; N, 15.49. Calc. for C19H21N5O4S2: C, 50.99; H,
4.73; N, 15.65. IR (KBr/cm−1): 3430, 2925, 2854, 1680, 1639,
1541, 1206, 801, 732, 614. 1H-NMR (DMSO-d6, 300 MHz)
δ: ppm 9.84 (s, 1H, -OH), 7.90 (m, 4H), 7.64 (m, 4H), 7.48 (s, 2H,
SO2NH2), 3.34 (t, J = 6.7 Hz, 2H, -CH2-), 3.10 (s, 3H, -NCH3), 2.89
(t, J = 6.7 Hz, 2H, -CH2-). MS (ESI, m/z): 448 [M + H]+.
3-(1-(4-Fluorophenyl)-5-(4-sulfamoylphenyl)-1H-1,2,4-triazol-3-
ylthio)-N-hydroxy-N-methylpropanamide (10k). The title com-
pound was obtained as a white solid (0.1 g, 41.2%), mp
90–92 °C. Found: C, 47.56; H, 4.41; N, 15.33. Calc. for
C18H18FN5O4S2: C, 47.88; H, 4.02; N, 15.51. IR (KBr/cm−1):
3384, 3081, 2931, 2374, 2323, 2248, 1628, 1510, 1337, 1160,
843, 614. 1H-NMR (DMSO-d6, 300 MHz) δ: ppm 9.85 (s, 1H,
-OH), 7.83 (d, J = 8.6 Hz, 2H), 7.66 (d, J = 8.5 Hz, 2H), 7.53 (d,
J = 8.2 Hz, 2H), 7.41 (d, J = 8.2 Hz, 2H), 7.40 (s, 2H, SO2NH2),
3.34 (t, J = 6.7 Hz, 2H, -CH2-), 3.10 (s, 3H, -NCH3), 2.89 (t, J =
6.7 Hz, 2H, -CH2-). MS (ESI, m/z): 450 [M − H]−.
N-Hydroxy-N-methyl-3-(5-(4-sulfamoylphenyl)-1-(4-(trifluoro-
methyl)phenyl)-1H-1,2,4-triazol-3-ylthio)propanamide (10m). The
title compound was obtained as a white solid (0.15 g, 49.3%),
mp 111–113 °C. Found: C, 45.77; H, 3.20; N, 14.15. Calc. for
C19H18F3N5O4S2: C, 45.50; H, 3.62; N, 13.96. IR (KBr/cm−1):
3445, 2377, 2350, 2310, 1617, 1520, 1446, 1325, 1167, 847, 622.
1H-NMR (DMSO-d6, 300 MHz) δ: ppm 9.84 (s, 1H, -OH), 7.90
(m, 4H), 7.64 (m, 4H), 7.48 (s, 2H, SO2NH2), 3.34 (t, J = 6.7 Hz,
2H, -CH2-), 3.10 (s, 3H, -NCH3), 2.89 (t, J = 6.7 Hz, 2H, -CH2-),
2.41 (s, 3H, phenyl-CH3). MS (ESI, m/z): 500 [M − H]−.
3-(1-(4-Bromophenyl)-5-(4-sulfamoylphenyl)-1H-1,2,4-triazol-3-
ylthio)-N-hydroxy-N-methylpropanamide (10o). The title com-
pound was obtained as a white solid (0.1 g, 32.9%), mp
133–135 °C. Found: C, 42.56; H, 3.33; N, 13.49. Calc. for
C18H18BrN5O4S2: C, 42.19; H, 3.54; N, 13.67. IR (KBr/cm−1):
3339, 3200, 2977, 2858, 1729, 1621, 1493, 1164, 766, 544.
1H-NMR (DMSO-d6, 300 MHz) δ: ppm 9.84 (s, 1H, -OH), 7.85
(d, J = 8.6 Hz, 2H), 7.72 (d, J = 8.8 Hz, 2H), 7.62 (d, J = 8.6 Hz,
2H), 7.48 (s, 2H, SO2NH2), 7.40 (d, J = 8.8 Hz, 2H), 3.32 (t, J =
General procedure for synthesis of compounds 15a–
j. NaBH3CN (4.65 mmol) and a trace of methyl orange were
added to a solution of appropriate oximes 13a–j (1.55 mmol)
in 10 mL methanol/THF (1 : 1, v/v). The solution was stirred at
30 °C for 5 min prior to the addition of 4 N HCl that was
added dropwise until the color remained orange red. The
mixture was maintained at 30 °C for an additional 4 h with
stirring. After removal of the organic solvents, water was added
to dilute, the mixture was adjusted to pH = 9 using 10%
NaOH, and extracted with EtOAc (3 × 10 mL). The combined
extracts were washed with brine and dried over anhydrous
Na2SO4, and evaporated in vacuo to provide the hydroxyl-
amines 14a–j as yellowish oils which were used immediately
without further purification for the preparation of N-hydro-
xyureas 15a–j.
To a stirred solution of hydroxylamines 14a–j (0.26 mmol)
in methanol and acetic acid (0.77 mmol) was added a solution
of KOCN (0.77 mmol) in water (2 mL). The mixture was stirred
for 4 h at 30 °C and diluted with 20 mL water, then extracted
with EtOAc (3 × 10 mL) and the combined organic layers were
washed with brine and dried over anhydrous Na2SO4 and con-
centrated to yield a yellowish oil. The residue was purified by
silica gel column chromatography using CH2Cl2/MeOH (50 : 1,
v/v) as eluent to afford a white solid.
1-Hydroxy-1-(2-(5-(4-(methylsulfonyl)phenyl)-1-phenyl-1H-1,2,4-
triazol-3-ylthio)ethyl)urea (15a). The title compound was
obtained as a white solid (0.07 g, 59.6%), mp 73–75 °C. Found:
C, 49.75; H, 4.38; N, 16.44. Calc. for C18H19N5O4S2: C, 49.87; H,
4.42; N, 16.16. IR (KBr/cm−1): 3457, 2922, 2377, 2351, 2320,
1643, 1453, 1302, 1147, 780, 694, 528. 1H-NMR (DMSO-d6,
500 MHz) δ: ppm 9.42 (s, 1H, OH), 7.94 (d, J = 8.5 Hz, 2H), 7.68
(d, J = 8.5 Hz, 2H), 7.53 (m, 3H), 7.45 (m, 2H), 6.34 (s, 2H,
NH2), 3.72 (t, J = 6.8 Hz, 2H, -CH2-), 3.33 (t, J = 6.9 Hz, 2H,
-CH2-), 3.25 (s, 3H, SO2CH3). MS (ESI, m/z): 434 [M + H]+.
1-Hydroxy-1-(2-(5-(4-(methylsulfonyl)phenyl)-1-p-tolyl-1H-1,2,4-
6.9 Hz, 2H, -CH2-), 3.10 (s, 3H, -NCH3), 2.89 (t, J = 6.8 Hz, 2H, triazol-3-ylthio)ethyl)urea (15b). The title compound was
-CH2-). MS (ESI, m/z): 514 [M + H]+.
obtained as a white solid (0.06 g, 62.7%), mp 94–96 °C. Found:
Procedure for synthesis of bromoacetaldehyde oxime C, 50.76; H, 4.44; N, 15.38. Calc. for C19H21N5O4S2: C, 50.99; H,
12. Bromoacetaldehyde diethyl acetal 11 (12.2 mmol) was 4.73; N, 15.65. IR (KBr/cm−1): 3382, 2922, 2351, 2310, 1653,
added to the solution of hydroxylamine hydrochloride 1513, 1303, 1150, 778, 566. 1H-NMR (DMSO-d6, 300 MHz)
(58.4 mmol) in 16 mL H2O and 8 mL 1 N HCl. The biphasic δ: ppm 9.41 (s, 1H, OH), 7.94 (d, J = 8.5 Hz, 2H), 7.68 (d, J = 8.5
solution was stirred at 30 °C until it became homogenous, at Hz, 2H), 7.32 (s, 4H), 6.28 (s, 2H, NH2), 3.74 (t, J = 6.8 Hz, 2H,
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-CH2-), 3.34 (t, J = 6.9 Hz, 2H, -CH2-), 3.24 (s, 3H, SO2CH3), 2.37 Found: C, 45.51; H, 3.95; N, 18.42. Calc. for C17H17FN6O4S2: C,
(s, 3H, phenyl-CH3). MS (ESI, m/z): 448 [M + H]+.
45.12; H, 3.79; N, 18.57. IR (KBr/cm−1): 3460, 2933, 2857, 2355,
2029, 1624, 1577, 1337, 1163, 855, 624. 1H-NMR (DMSO-d6,
300 MHz) δ: ppm 9.43 (s, 1H, OH), 7.81 (d, J = 8.6 Hz, 2H), 7.57
(m, 4H), 7.45 (s, 2H, SO2NH2), 7.43 (d, J = 8.6 Hz, 2H), 6.37 (s,
2H, NH2), 3.71 (t, J = 6.6 Hz, 2H, -CH2-), 3.35 (t, J = 6.2 Hz, 2H,
-CH2-). MS (ESI, m/z): 453 [M + H]+.
4-(3-(2-(1-Hydroxyureido)ethylthio)-1-(4-(trifluoromethyl)phenyl)-
1H-1,2,4-triazol-5-yl)benzenesulfonamide (15m). The title com-
pound was obtained as a white solid (0.03 g, 44.6%), mp
157–159 °C. Found: C, 43.36; H, 3.74; N, 16.89. Calc. for
C18H17F3N6O4S2: C, 43.02; H, 3.41; N, 16.72. IR (KBr/cm−1):
1-(2-(1-(4-Fluorophenyl)-5-(4-(methylsulfonyl)phenyl)-1H-1,2,4-
triazol-3-ylthio)ethyl)-1-hydroxyurea (15c). The title compound
was obtained as a white solid (0.1 g, 48.7%), mp 73–75 °C.
Found: C, 47.50; H, 4.33; N, 15.36. Calc. for C18H18FN5O4S2: C,
47.88; H, 4.02; N, 15.51. IR (KBr/cm−1): 3446, 2923, 1653, 1510,
1
1463, 1305, 1150, 845, 779, 566. H-NMR (DMSO-d6, 300 MHz)
δ: ppm 9.41 (s, 1H, OH), 7.96 (d, J = 8.5 Hz, 2H), 7.69 (d, J =
8.5 Hz, 2H), 7.54 (m, 2H), 7.37 (m, 2H), 6.33 (s, 2H, NH2), 3.73
(t, J = 6.8 Hz, 2H, -CH2-), 3.34 (t, J = 6.9 Hz, 2H, -CH2-), 3.25 (s,
3H, SO2CH3). MS (ESI, m/z): 452 [M + H]+.
1-Hydroxy-1-(2-(5-(4-(methylsulfonyl)phenyl)-1-(4-(trifluoro- 3457, 2930, 2854, 2352, 2027, 1634, 1576, 1335, 1160, 852, 621.
1H-NMR (DMSO-d6, 300 MHz) δ: ppm 9.44 (s, 1H, OH), 7.88
methyl)phenyl)-1H-1,2,4-triazol-3-ylthio)ethyl)urea
(15d). The
(m, 4H), 7.66 (m, 4H), 7.49 (s, 2H, SO2NH2), 6.38 (s, 2H, NH2),
3.73 (t, J = 6.8 Hz, 2H, -CH2-), 3.35 (t, J = 6.9 Hz, 2H, -CH2-). MS
(ESI, m/z): 503 [M + H]+.
title compound was obtained as a white solid (0.07 g, 37.7%),
mp 136–98 °C. Found: C, 45.81; H, 3.24; N, 13.65. Calc. for
C19H18F3N5O4S2: C, 45.50; H, 3.62; N, 13.96. IR (KBr/cm−1):
3473, 2925, 1652, 1446, 1326, 1150, 1062, 849, 777, 533.
1H-NMR (DMSO-d6, 500 MHz) δ: ppm 9.42 (s, 1H, OH), 7.99 (d,
J = 8.5 Hz, 2H), 7.89 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 8.5 Hz, 2H),
7.38 (d, J = 8.3 Hz, 2H), 6.35 (s, 2H, NH2), 3.73 (t, J = 6.8 Hz,
2H, -CH2-), 3.34 (t, J = 6.7 Hz, 2H, -CH2-), 3.26 (s, 3H, SO2CH3).
MS (ESI, m/z): 502 [M + H]+.
4-(1-(4-Bromophenyl)-3-(2-(1-hydroxyureido)ethylthio)-1H-1,2,4-
triazol-5-yl)benzenesulfonamide (15o). The title compound was
obtained as a white solid (0.1 g, 54.2%), mp 155–157 °C.
Found: C, 39.98; H, 3.05; N, 16.61. Calc. for C17H17BrN6O4S2:
C, 39.77; H, 3.34; N, 16.37. IR (KBr/cm−1): 3430, 2926, 2853,
2377, 2347, 2310, 1643, 1493, 1339, 1159, 832, 620. 1H-NMR
(DMSO-d6, 500 MHz) δ: ppm 9.41 (s, 1H, OH), 7.81 (d, J =
8.7 Hz, 2H), 7.62 (d, J = 8.7 Hz, 2H), 7.53 (m, 2H), 7.41 (s, 2H,
SO2NH2), 7.36 (m, 2H), 6.32 (s, 2H, NH2), 3.72 (t, J = 6.9 Hz,
2H, -CH2-), 3.32 (t, J = 6.9 Hz, 2H, -CH2-). MS (ESI, m/z): 514
[M + H]+.
General procedure for synthesis of compounds 17a–b. To a
solution of the appropriate benzoic acids 16a–b (5 mmol) in
10 mL DMF, K2CO3 (5.5 mmol) and CH3I (5.5 mmol) were
added, and the mixture was maintained at 80 °C for 1 h. The
mixture was poured into ice water and the precipitate was fil-
tered to give a white solid.
General procedure for synthesis of compounds 18a–b. A
mixture of the appropriate methyl benzoates 17a–b
(3.27 mmol), hydrazine hydrate (16.3 mmol), and 10 mL of
95% ethanol was heated under reflux for 6 h. Yellow needle
crystals were precipitated as the mixture cooled to room temp-
erature. The precipitate was collected to give yellow needle
crystals.
1-(2-(1-(4-Bromophenyl)-5-(4-(methylsulfonyl)phenyl)-1H-1,2,4-
triazol-3-ylthio)ethyl)-1-hydroxyurea (15e). The title compound
was obtained as a white solid (0.1 g, 41.7%), mp 135–137 °C.
Found: C, 42.46; H, 3.25; N, 13.42. Calc. for C18H18BrN5O4S2:
C, 42.19; H, 3.54; N, 13.67. IR (KBr/cm−1): 3461, 2927, 2854,
1
2346, 2310, 1644, 1493, 1311, 1149, 779, 534. H-NMR (DMSO-
d6, 500 MHz) δ: ppm 9.42 (s, 1H, OH), 7.98 (d, J = 8.5 Hz, 2H),
7.71 (m, 4H), 7.42 (d, J = 8.5 Hz, 2H), 6.33 (s, 2H, NH2), 3.72 (t,
J = 6.8 Hz, 2H, -CH2-), 3.32 (t, J = 6.7 Hz, 2H, -CH2-), 3.25 (s,
3H, SO2CH3). MS (ESI, m/z): 513 [M + H]+.
4-(3-(2-(1-Hydroxyureido)ethylthio)-1-phenyl-1H-1,2,4-triazol-5-
yl)benzenesulfonamide (15g). The title compound was obtained
as a white solid (0.04 g, 46.4%), mp 169–171 °C. Found: C,
46.74; H, 4.58; N, 19.66. Calc. for C17H18N6O4S2: C, 46.99; H,
4.18; N, 19.34. IR (KBr/cm−1): 3448, 2964, 2926, 1644, 1575,
1452, 1340, 1150, 801, 617. 1H-NMR (DMSO-d6, 300 MHz) δ:
ppm 9.42 (s, 1H, OH), 7.82 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.4
Hz, 2H), 7.51 (m, 3H), 7.45 (br, 2H), 7.42 (s, 2H, SO2NH2), 6.33
(s, 2H, NH2), 3.72 (t, J = 6.8 Hz, 2H, -CH2-), 3.33 (t, J = 6.9 Hz,
2H, -CH2-). MS (ESI, m/z): 435 [M + H]+.
General procedure for synthesis of compounds 19a–c. Equi-
molar quantities of the appropriate benzoic acid hydrazide
4-(3-(2-(1-Hydroxyureido)ethylthio)-1-p-tolyl-1H-1,2,4-triazol-5- 18a–b (1.3 mmol) and para-position substituted phenyl iso-
yl)benzenesulfonamide (15i). The title compound was obtained thiocyanate (1.3 mmol) in 10 mL of absolute ethanol were
heated under reflux for 5 h. The precipitate was collected to
give a white solid.
General procedure for synthesis of compounds 20a–c. To a
solution of the appropriate compounds 19a–c (1 mmol) in
5 mL methanol, 5 mL 10% NaOH was added and the mixture
as a white solid (0.1 g, 52.6%), mp 137–139 °C. Found: C,
48.11; H, 4.82; N, 18.96. Calc. for C18H20N6O4S2: C, 48.20; H,
4.49; N, 18.74. IR (KBr/cm−1): 3368, 2925, 1655, 1513, 1451,
1335, 1161, 823, 614. 1H-NMR (DMSO-d6, 300 MHz) δ: ppm
9.42 (s, 1H, OH), 7.82 (d, J = 8.6 Hz, 2H), 7.61 (d, J = 8.4 Hz,
2H), 7.45 (s, 2H, SO2NH2), 7.30 (m, 4H), 6.34 (s, 2H, NH2), 3.71 was heated under reflux for 2 h. The solution was cooled and
acidified to pH = 2 by adding 10% HCl. The precipitated solid
was filtered and washed with water to give a white solid.
General procedure for synthesis of compounds 21a–
d. As described in the procedure for synthesis of compounds
13a–j.
(t, J = 6.9 Hz, 2H, -CH2-), 3.39 (t, J = 7.0 Hz, 2H, -CH2-), 2.37 (s,
3H, phenyl-CH3). MS (ESI, m/z): 449 [M + H]+.
4-(1-(4-Fluorophenyl)-3-(2-(1-hydroxyureido)ethylthio)-1H-1,2,4-
triazol-5-yl)benzenesulfonamide (15k). The title compound was
obtained as a white solid (0.57 g, 49.6%), mp 166–168 °C.
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2-(4-(4-Fluorophenyl)-5-(4-(methylsulfonyl)phenyl)-4H-1,2,4- 7.38 (d, J = 8.3 Hz, 2H), 6.35 (s, 2H, NH2), 3.73 (t, J = 6.8 Hz,
triazol-3-ylthio)acetaldehyde oxime (21a). The title compound
was obtained as a white solid (0.06 g, 43.4%), mp 85–87 °C.
Found: C, 50.51; H, 3.66; N, 13.40. Calc. for C17H15FN4O3S2: C,
50.23; H, 3.72; N, 13.78. IR (KBr/cm−1): 3450, 3074, 2922, 2850,
2358, 1639, 1510, 1310, 1152, 960, 845, 779, 606. 1H-NMR
(DMSO-d6, 300 MHz) δ: ppm 11.34 (s, 0.429H, anti OH), 10.94
(s, 0.563H, syn OH), 7.93 (d, J = 8.5 Hz, 2H), 7.61 (m, 4H), 7.47
(d, J = 8.5 Hz, 2H), 7.42 (t, J = 5.8 Hz, 0.588H, syn –CHvN),
6.93 (t, J = 5.3 Hz, 0.394H, anti –CHvN), 3.95 (d, J = 5.3 Hz,
1H, anti SCH2–), 3.91 (d, J = 5.8 Hz, 1H, syn SCH2–), 3.24 (s,
3H, SO2CH3). MS (ESI, m/z): 407 [M + H]+.
2H, -CH2-), 3.34 (t, J = 6.7 Hz, 2H, -CH2-), 3.26 (s, 3H, SO2CH3).
MS (ESI, m/z): 502 [M + H]+.
4-(4-(4-Fluorophenyl)-5-(2-(1-hydroxyureido)ethylthio)-4H-1,2,4-
triazol-3-yl)benzenesulfonamide (22d). The title compound was
obtained as a white solid (0.06 g, 81.6%), mp 143–145 °C.
Found: C, 45.48; H, 3.98; N, 18.34. Calc. for C17H17FN6O4S2: C,
45.12; H, 3.79; N, 18.57. IR (KBr/cm−1): 3485, 3336, 2377, 2350,
2319, 1634, 1511, 1347, 1158, 847, 621. 1H-NMR (DMSO-d6,
300 MHz) δ: ppm 9.47 (s, 1H, OH), 7.79 (d, J = 8.6 Hz, 2H), 7.55
(m, 4H), 7.43 (s, 2H, SO2NH2), 7.41 (d, J = 8.6 Hz, 2H), 6.35 (s,
2H, NH2), 3.69 (t, J = 6.6 Hz, 2H, -CH2-), 3.35 (t, J = 6.2 Hz, 2H,
2-(5-(4-(Methylsulfonyl)phenyl)-4-(4-(trifluoromethyl)phenyl)- -CH2-). MS (ESI, m/z): 453 [M + H]+.
4H-1,2,4-triazol-3-ylthio)acetaldehyde oxime (21b). The title
compound was obtained as a white solid (0.08 g, 34.1%), mp
70–72 °C. Found: C, 47.08; H, 3.64; N, 12.55. Calc. for
4.2. In vitro COX and 5-LOX inhibition assay
C18H15F3N4O3S2: C, 47.36; H, 3.31; N, 12.27. IR (KBr/cm−1):
3422, 2923, 2852, 1722, 1608, 1439, 1325, 1153, 1066, 958, 779.
1H-NMR (DMSO-d6, 300 MHz) δ: ppm 11.36 (s, 0.42H, anti
OH), 10.96 (s, 0.49H, syn OH), 8.00 (d, J = 8.3 Hz, 2H), 7.93 (d,
J = 8.3 Hz, 2H), 7.63 (m, 4H), 7.43 (t, J = 5.8 Hz, 0.56H, syn
–CHvN), 6.98 (t, J = 5.3 Hz, 0.46H, anti –CHvN), 3.95 (d, J =
5.1 Hz, 1H, anti SCH2–), 3.92 (d, J = 5.7 Hz, 1H, syn SCH2–),
3.24 (s, 3H, SO2CH3). MS (ESI, m/z): 457 [M + H]+.
Stock solutions of test compounds were made by dissolving
each compound in a minimum volume of DMSO. 6-keto-PGF1α
production was measured in order to determine COX-1 activity.
Endotheliocyte was gained from the aorta pectoralis of neogen-
esis calf in vitro, the cells were maintained at 37 °C and 5%
CO2 in DMEM supplemented 10% heat-inactivated fetal
bovine serum, 2 mM glutamine, 60 mg L−1 penicillin and
100 mg L−1 streptomycin. Cells were plated into 24-well plates
at a density of 1.0 × 106 cells per well in 1 mL of DMEM. After
24 h incubation, the cells were treated with test compounds
(10−6 mol L−1), DMSO and celecoxib (10−6 mol mL−1). These
solutions were incubated for 20 min at 37 °C and 5% CO2,
then were treated with arachidonic acid (10 μmol L−1). After
20 min incubation, the culture supernatants were collected
and stored at −70 °C until required for 6-keto-PGF1α determi-
nation. 6-keto-PGF1α was measured by using a radioimmunity
assay kit according to the manufacturer’s instructions.
The activity of the target compounds to inhibit COX-2 was
determined by PGE2 production assay. Macrophages were
gained from the abdominal cavity of rats and plated into 24-
well plates at a density of 1.0 × 106 cells per well in 1 mL of
RPMI 1640 containing 5% FCS. After 24 h incubation at 37 °C
and 5% CO2, aspirin (1 mmol L−1) was added to inactivate
COX-1. Then the culture media were replaced with fresh media
containing LPS (1 μg mL−1) to induce COX-2. After 6 h incu-
bation at 37 °C, the test compounds (10−6 mol L−1), DMSO
and celecoxib (10−6 mol L−1) were added and incubated for
30 min at 37 °C and 5% CO2. The cells were then treated with
arachidonic acid (10 μmol L−1) for 20 min, and the culture
supernatants were collected for the determination of PGE2 pro-
duction using an enzyme immunoassay (EIA) kit (Cat-lot no.
414010, 96-well, Cayman Chemical Company) according to the
manufacturer’s instructions.
4-(4-(4-Fluorophenyl)-5-(2-(hydroxyimino)ethylthio)-4H-1,2,4-
triazol-3-yl)benzenesulfonamide (21d). The title compound
was obtained as a white solid (0.067 g, 76.6%), mp 119–121 °C.
Found: C, 47.55; H, 3.06; N, 17.52. Calc. for C16H14FN5O3S2: C,
47.17; H, 3.46; N, 17.19. IR (KBr/cm−1): 3442, 2919, 2849, 2358,
1639, 1510, 1465, 1334, 1164, 843, 622. 1H-NMR (DMSO-d6,
300 MHz) δ: ppm 11.32 (s, 0.302H, anti OH), 10.92 (s, 0.386H,
syn OH), 7.79 (d, J = 8.6 Hz, 2H), 7.55 (m, 4H), 7.45 (t, J = 8.6
Hz, 2H), 7.42 (s, 2H, SO2NH2), 7.39 (t, J = 5.9 Hz, 0.337H, syn
–CHvN), 6.92 (t, J = 5.3 Hz, 0.313H, anti –CHvN), 3.94 (d, J =
5.3 Hz, 1H, anti SCH2–), 3.89 (d, J = 5.9 Hz, 1H, syn SCH2–). MS
(ESI, m/z): 408 [M + H]+.
General procedure for synthesis of compounds 22a–d. As
described in the procedure for synthesis of compounds 15a–j.
1-(2-(4-(4-Fluorophenyl)-5-(4-(methylsulfonyl)phenyl)-4H-1,2,4-
triazol-3-ylthio)ethyl)-1-hydroxyurea (22a). The title compound
was obtained as a white solid (0.73 g, 76.6%), mp 106–108 °C.
Found: C, 47.59; H, 4.33; N, 15.78. Calc. for C18H18FN5O4S2: C,
47.88; H, 4.02; N, 15.51. IR (KBr/cm−1): 3462, 2963, 2923, 1653,
1510, 1441, 1261, 1090, 801, 510. 1H-NMR (DMSO-d6,
300 MHz) δ: ppm 9.47 (s, 1H, OH), 7.92 (d, J = 8.6 Hz, 2H), 7.58
(m, 4H), 7.42 (t, J = 8.8 Hz, 2H), 6.37 (s, 2H, NH2), 3.69 (t, J =
6.8 Hz, 2H, -CH2-), 3.36 (t, J = 6.9 Hz, 2H, -CH2-), 3.23 (s, 3H,
SO2CH3). MS (ESI, m/z): 452 [M + H]+.
1-Hydroxy-1-(2-(5-(4-(methylsulfonyl)phenyl)-4-(4-(trifluoromethyl)-
phenyl)-4H-1,2,4-triazol-3-ylthio)ethyl)urea (22b). The title com-
pound was obtained as a white solid (0.067 g, 61.2%), mp
132–134 °C. Found: C, 45.82; H, 3.97; N, 13.70. Calc. for
C19H18F3N5O4S2: C, 45.50; H, 3.62; N, 13.96. IR (KBr/cm−1):
3473, 2925, 1652, 1446, 1326, 1150, 1062, 849, 777, 533.
1H-NMR (DMSO-d6, 500 MHz) δ: ppm 9.42 (s, 1H, OH), 7.99 (d,
J = 8.5 Hz, 2H), 7.89 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 8.5 Hz, 2H),
The 5-LOX inhibitor screening assay detects and measures
the LTB4 produced by stimulation of calcium ionophore
A23187. Leukocytes were obtained from the abdominal cavity
of Sprague-Dawley rats injected intraperitoneally with 20 mL
kg−1 of a 0.2% (w/v) glycogen solution. The cell suspensions
were collected with Hanks solution and plated into 24-well
plates at a density of 1.0 × 106 cells per well. After 10 min
This journal is © The Royal Society of Chemistry 2014
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Organic & Biomolecular Chemistry
incubation at 37 °C, L-cysteine (10 mmol), indomethacin (1 mg COX-2 and COX-1 was assigned according to the Amber 4.0 force
L−1), DMSO, celecoxib and test compounds were added in field with Kollmanall-atom charges encoded in Sybyl 6. The
turn, and incubated at 37 °C for 30 min. LTB4 production was crystal structure of 5-LOX was received from the RCSB Protein
initiated by adding calcium ionophore A23187 (5 μmol) and Data Bank (entry code 3O8Y). The geometries of these com-
incubated for 30 min at 37 °C. After being clarified by centrifu- pounds were subsequently optimized using the Tripos force
gation, the supernatant was plated into 96-well plates and field with Gasteiger–Huckel charges. The method of Powell avail-
incubated overnight at 4 °C. Chromogen was added and the able in the Maximin module encoded in Sybyl 6 was used for
plates were retained for 90 min. The 5-LOX activity was deter- energy minimization using an 8 Å nonbond cutoff and an
mined by using an EIA kit (Cat-lot no. 520111, 96-well, energy convergence gradient value of 0.005 kcal (mol Å)−1
Cayman Chemical Company) according to the manufacturer’s Docking studies were performed by the program of GOLD
instructions. Percent inhibition was calculated according to 3.0 running on a Discovery Studio 2.5 workstation. The X-ray
.
the following formula:
coordinates of SC-558 bound to the active site of COX-2 were
used to define active site region with an active site radius of
10 Å. The annealing parameters of van der Waals and hydro-
gen bond interactions were considered within 4.0 and 2.5 Å,
respectively, and other parameters were kept at the default
setting. The procedures for definition of 5-LOX active site were
carried out as described in a previous report.31 The non-heme
iron was added to the model in InsightII and it ligated to three
Inhibition ð%Þ ¼ 100 ꢀ ðOD blank ꢁ OD compÞ=OD blank:
The IC50 values were determined as the maximal inhibition
(100%) by Bliss method.
4.3. Anti-inflammatory assay
histidines (His367, His372, His550) as well as Ile674
.
Three models including xylene-induced ear edema in mice,
albumen-induced paw edema in rats and acetic acid-induced
mouse vascular permeability in mice were carried out to evalu-
ate the anti-inflammatory activity of target compounds as
described previously.31
Competing interest
The authors declare no competing financial interest.
4.4. Analgesic assay
Analgesic activity was determined using acetic acid-induced
writhing response (chemical method) and hot-plate assay
(thermal method) in mice.39,40 Mice were divided into five
groups (n = 10) and administrated orally with 0.5% CMC–Na
solution, celecoxib (30 mg kg−1) or compound 15e (15, 30 and
60 mg kg−1). 1 h post administration, acetic acid (0.7% v/v)
was given intraperitoneally to all the groups at the dose of
0.1 mL per 10 g body weight. Analgesic activity was recorded
by counting the first time of writhe and the number of writhes
after the injection of acetic acid for a period of 15 min. A
writhe is indicated by abdominal constriction and full exten-
sion of hind limb.
To perform hot-plate assay, animals were tested initially for
baseline latency and the mice which showed hind paw-lick
response within 6–8 s were selected for the study. To avoid
tissue damage, animals were exposed to the hot plate for a
maximum of 30 s. 1 h post administration of test and reference
compounds, the animals were individually exposed to the hot
plate maintained at 55 °C and latency was tested at 60, 90 and
120 min post-administration. The experimental protocols of
animal studies were approved by the Animal Research Care
Committee of China Pharmaceutic University.
Abbreviations
PGs
LTs
AA
RA
OA
prostaglandins;
leukotrienes;
arachidonic acid;
rheumatoid arthritis;
osteoarthritis;
COX
5-LOX
cyclooxygenase;
5-lipoxygenase;
NSAIDs non-steroidal anti-inflammatory drugs;
TLC
HPLC
TMS
ESI
thin-layer chromatography;
high-performance liquid chromatography;
tetramethylsilane;
electrospray ionisation;
enzyme immunoassay;
methylthiazolyl tetrazolium;
phosphate-buffered solution;
adenosine diphosphate;
hydroxyl radical;
EIA
MTT
PBS
ADP
OH
NOS
CAT
PRP
PPP
nitric oxide synthase;
catalase;
platelet-rich plasma;
4.5. Molecular modeling (docking) study
platelet-poor plasma.
The crystal structure of murine COX-2 in complex with SC-558
was received from the RCSB Protein Data Bank (entry code
1cx2) and hydrogens were added. The docking experiment on
Acknowledgements
COX-2 was carried out by superimposing the energy-mini- This study was supported by the grants from Key fund of Ministry
mized ligand on SC-558 in the PDB file 1cx2 after which of Education of China (grant no. 108069) and Natural Science
SC-558 was deleted. The potential of the 3D structures of Foundation of Jiangsu Province (grant no. BK2008344).
2126 | Org. Biomol. Chem., 2014, 12, 2114–2127
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Paper
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