New methyl 5-(halomethyl)-1-aryl-1H-1,2,4-triazole-3-carboxylates as selective COX-2 inhibitors and anti-inflammatory agents: Design, synthesis, biological evaluation, and docking study

Article abstract of DOI:10.1016/j.bioorg.2020.104333

A new method was developed for synthesis of 1,2,4-triazole-3-carboxylates 5a–p and 6 from nitrilimines 3a–p through amination and heterocyclization two-steps reactions. All of 1,2,4-triazole-3-carboxylates 5 and 6 were characterized by spectroscopy techni

Full text of DOI:10.1016/j.bioorg.2020.104333

Bioorganic Chemistry 104 (2020) 104333
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
New methyl 5-(halomethyl)-1-aryl-1H-1,2,4-triazole-3-carboxylates as
selective COX-2 inhibitors and anti-inflammatory agents: Design, synthesis,
biological evaluation, and docking study
,
,
,
Sin-Min Lia 1, Shuo-En Tsaib 1, Chia-Yin Chiangb, Cheng-Yen Chungb c, Tsung-Jui Chuangd,
Ching-Chun Tsengb, Wen-Ping Jiange, Guan-Jhong Huange, Chin-Yu Lina, Ya-Chen Yangf,
Mao-Tsu Fuhg, Fung-Fuh Wongb
,c,*
a Institute of New Drug Development, China Medical University, No. 91 Hsueh-Shih Rd., Taichung 40402, Taiwan
b Ph.D. Program for Biotech Pharmaceutical Industry, China Medical University, No. 91, Hsueh-Shih Rd., Taichung 40402, Taiwan
c School of Pharmacy, China Medical University, No. 91, Hsueh-Shih Rd., Taichung 40402, Taiwan
d Master Program for Pharmaceutical Manufacture, China Medical University, No. 91, Hsueh-Shih Rd., Taichung 40402, Taiwan
e Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, No. 91, Hsueh-Shih Rd., Taichung 40402, Taiwan
f Department of Food Nutrition and Health Biotechnology, Asia University, Taichung 41354, Taiwan
g Division of Metabolism, Department of Internal Medicine, China Medical University, Taichung 40402, Taiwan
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new method was developed for synthesis of 1,2,4-triazole-3-carboxylates 5a–p and 6 from nitrilimines 3a–p
through amination and heterocyclization two-steps reactions. All of 1,2,4-triazole-3-carboxylates 5 and 6 were
characterized by spectroscopy technique. Based on the SAR study of anti-inflammation activity, most of these
compounds showed potential anti-inflammatory activity on NO inhibition in LPS-induced RAW 264.7 cells (IC50
< 7.0 µM) compared with Celecoxib and Indomethacin. Several potential compounds 5b–h, 5j, 5l, 5n, and 5o
were subjected to in vitro cyclooxygenase COX-1/COX-2 inhibition assays. Compound 5d showed extraordinary
COX-2 inhibition (IC50 = 17.9 nM) and the best selectivity (COX-1/COX-2 = 1080). Furthermore, 5 mg/kg
compound 5d exhibited better in vivo anti-inflammation and gastric protection results compared to 10 mg/kg
Indomethacin. Docking experiments of 5d into COX-2 binding pocket have been evaluated. Following the bio-
activities experimental data, the potential drug candidate 5d, significantly exhibited better anti-inflammatory
effect than Indomethacin.
Heterocyclization
1,2,4-Triazole
Anti-inflammation
COX-2 inhibitor
1. Introduction
Concomitant use of non-selective NSAIDs act on both COX isoforms,
resulting in an increased risk of gastrointestinal (GI) complications [2].
Inflammation is a complex, defensive process, in which insult to the
body results in the accumulation of local fluid and leukocytes. The in-
flammatory cells work to eliminate the noxious stimulus, preventing
further tissue damage. While inflammation acts as a protective mecha-
nism for the body, it is also associated with pathological disorders,
including atherosclerosis, cancers, infections, neurodegenerative dis-
eases, and rheumatoid arthritis [1].
Long-term use of NSAIDs produce gastrointestinal tract side effects by
inhibiting prostaglandin synthesis through the COX-1 pathway [3].
Whereas, COX-2 selective NSAIDs were shown to exhibit potent anti-
inflammatory activity without causing significant gastrointestinal
injury [4,5]. However, various clinical evidence indicated their impli-
cation in serious cardiovascular accidents [6]. Thus, the investigation of
new anti-inflammatory agents has been raised concerns as major chal-
lenge [7–9].
Most of the clinically employed Non-Steroidal Anti-Inflammatory
Drugs (NSAIDs) are known to inhibit the two isoforms of cyclooxygenase
(COX) enzyme: COX-1 (constitutive form) and COX-2 (inducible form).
Compounds possessing triazole core are known for their wide range
of biological activities and anti-inflammatory properties [10–13].
* Corresponding author at: Ph.D. Program for Biotech Pharmaceutical Industry, China Medical University, No. 91, Hsueh-Shih Rd., Taichung 40402, Taiwan.
E-mail address: ffwong@mail.cmu.edu.tw (F.-F. Wong).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2020.104333
Received 8 July 2020; Accepted 29 September 2020
Available online 2 October 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.

S.-M. Li et al.
Bioorganic Chemistry 104 (2020) 104333
Herein, we give an efficient method for synthesis of multi-substituted
1,2,4-triazoles from hydrazonoyl hydrochlorides (nitrilimines). The re-
action mechanism involved the amination with aqueous ammonium
hydroxide and heterocyclization in two-steps reactions. Furthermore,
the effect of 1,2,4-triazole-3-carboxylates on inflammation activity was
explored in vitro and in vivo to determine the structure–activity rela-
tionship (SAR) and identify the structural fragments responsible for the
biological activity.
procedure involved the treatment of hydrazonamide 4a with 2.0
equivalent of 2-chloroacetyl chloride and NEt3 (5.0 equiv) in toluene
solution at reflux for 5 h. After aqueous work-up and purification by
column chromatography on silica gel, the corresponding methyl 5-
(chloromethyl)-1-aryl-1H-1,2,4-triazole-3-carboxylate product 5a was
obtained in 74% yield (Entry 3 in Table 2).
The designed procedure was applied to hydrazonoyl halides 3b–p
bearing various N1-substituents, including o-, m-, p-F-Ph, o-, m-, p-CF3-
Ph, m-, p-Cl-Ph, m-, p-Me-Ph, p-Br-Ph, p-OMe-Ph, p-CN-Ph, 2,4-di-Cl-Ph,
and quinolinyl with 5.0 equiv of NH4OH to obtain hydrazonamides
4b–p. Subsequently, compounds 4b–p underwent the sequential heter-
ocyclization reaction to give the corresponding 1,2,4-triazole-3-carbox-
ylate derivatives 5b–p in moderate yields (53–83%, Table 2, entries
5–19). This new method was most amenable to a variety of N-1
substituted 1,2,4-triazoles 5b–p bearing N-1 substituents at the ortho,
meta, and para positions of the benzene ring, di-substituted phenyl, and
heterocyclic groups. We supposed that the N1-ortho-substituted steric
hinder would make unfavorable for the heterocyclic process (Table 1,
entries 5 and 8). Furthermore, we assumed the phenyl group with
electron-withdrawing and electron-donating groups at the N-1 sub-
stituents enabled derivatives to undergo heterocyclization. Based on the
above experimental results of Table 1, most transformation yielding
results of this new method with N1 variation were acceptable. All of
1,2,4-triazole-3-carboxylate derivatives 5a–p were successfully isolated
and fully characterized by spectroscopic methods. Compound 1,2,4-tri-
azole product 5e was performed the further structure identification by
X-ray crystallographic analyses and the single-crystal X-ray diffraction
study (ORTEP) and shown in Fig. 2.
2. Results and discussion
2.1. Chemistry
Mono- and di-substituted 1,2,4-triazole derivatives including 1-
phenyl-1H-1,2,4-triazole 1 and methyl 1-phenyl-1H-1,2,4-triazole-3-
carboxylate 2 were prepared by the following reported procedure [14].
They were conceived as the reference standards for demonstrating
comparable structural fragment effects with the designed methyl 5-
(halomethyl)-1-aryl-1H-1,2,4-triazole-3-carboxylate derivatives 5a–p
and 6 on the potential anti-inflammatory activity (Fig. 1). Furthermore,
Indomethacin and Celecoxib was utilized as the inter-reference stan-
dards for the in vitro and in vivo studies (Fig. 1).
2.2. Design and synthesis
To initiate the versatile and efficient synthesis of 1,2,4-triazole 5a–p
and 6, nitrilimines (hydrazonoyl halides) 3a–p were prepared as the
starting material according to literature procedure [15,16]. Recently,
Breinlinger et al. treated nitrilimines 3 with bubbled ammonia (NH3) in
dioxane solution to synthesize hydrazonamides 4 [17]. Herein, we
designed a synthetic approach to multi-substituted 1,2,4-triazoles,
including amination and heterocyclization shown in Scheme 1.
Preliminarily, nitrilimine 3a was carried out in the presence of 5.0
equivalents of NH4OH in H2O/MeOH (1:1) aqueous solution at 0 ◦C for
24 h. The amination reaction was monitored by TLC until the starting
material 3a was fully consumed. After work-up procedure, the corre-
sponding crude hydrazonamide 4a was obtained in satisfactory yields
without purification and isolation. The sequential heterocyclization,
including amination and cyclization, was carried out to synthesize the
multi-substituted 1,2,4-triazoles 5a. Hydrazonamide 4a was also reac-
ted towards 2-chloroacetyl chloride (2.0 Equiv.) with different equiva-
lent amounts of NEt3 (2.0, 3.0, and 5.0 equiv.) in toluene solution at
reflux for 5 h for searching the best reaction condition (Table1, entries
1–3). Notably, a significantly higher yield was obtained in 74% with 2.0
equiv of 2-chloroacetyl chloride in the presence of NEt3 (5.0 equiv, entry
1 in Table 1). Thus, increasing NEt3 resulted in a more efficient reaction
process.
2.3. Biological activity
2.3.1. In vitro inhibition studies of nitric oxide (NO) production
For determining the concentration of test compounds, the MTT assay
[18] was utilized by growing cultured RAW 264.7cells in the presence of
LPS as well as synthesized compounds. The results showed that RAW
264.7 cells survived even when incubated with most of the compounds
at a concentration of 10 μg/mL (Fig. S58). Based on the cell viability
results, compounds 1, 2, 5a–5p, and 6 were examined futher for
inhibitory activity against NO production at 0.625, 1.25, 2.5, 5 or 10 g/
μ
ml via nitrite/nitrate levels in LPS-stimulated RAW 264.7 cells. For the
SAR study, C-3 and C-5 variations on 1,2,4-triazole and various sub-
stituents on N1-phenyl ring were preliminarily evaluated for anti-
inflammatory activity (Table 2). Previously, IC50 values of 1-phenyl-
1H-1,2,4-triazole (1) and 1-phenyl 1H-1,2,4-triazole-3-carboxylate (2)
were significantly poor than alkyl 5-(chloromethyl)-1-aryl-1H-1,2,4-
triazole-3-carboxylates (5a). Therefore, the chloromethyl moiety on C-5
was essential for anti-inflammation activity and the ester moiety on C-3
was considered as assisting group (Table 2, entries 1–3). In comparison
with compounds 5b–g bearing o-, m-, p-F and CF3 (Table 2, entries 4–9),
we found m- or p-substituted substrates on N-1 phenyl ring of 1,2,4-tri-
azoles possessed slightly better activity for NO inhibition than o-
Utilization of 2-chloroacetyl bromide (2.0 equiv) was also tested to
replace 2-chloroacetyl chloride under the same reaction condition. The
corresponding 1,2,4-triazole-3-carboxylate 6 was obtained in 72% yield
(see entry 4 of Table 1). Following the experimental results, the reliable
Me
O
Y
OH
Me
MeO
X
N
N
N
N
X
O
H2N S
O
N
N
N
N
OMe
N
O
Cl
O
CF3
1
2
. X = H,
. X= COOMe
5a-p
6
. X = Cl, . X = Br
Indomethacin
Celecoxib
Fig. 1. Chemical structures of the designed 1,2,4-triazole compounds 1, 2, 5a–p, and 6, and U.S, FDA-approved drugs Indomethacin and Celecoxib.
2

S.-M. Li et al.
Bioorganic Chemistry 104 (2020) 104333
Table 1
The results of study of 1,2,4-triazole derivatives via sequential amidation and cyclization two steps synthesis.
Entry
Nitrilimines
S.M.
NH4OH
Equiv
2-Chloroacetyl halides
NEt3
1,2,4-triazoles 5a–p, 6
Ar
Equiv
X
Equiv
Product
Yields (%)a
1
3a
3a
3a
3a
3b
3c
3d
3e
3f
Ph
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
Cl
Cl
Cl
Br
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
2
3
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5a
5a
5a
6
61
69
74
72
68
74
76
53
68
77
73
78
72
82
83
58
74
77
62
2
Ph
3
Ph
4
Ph
5
o-F-Ph
5b
5c
5d
5e
5f
6
m-F-Ph
p-F-Ph
7
8
o-CF3-Ph
m-CF3-Ph
p-CF3-Ph
m-Cl-Ph
p-Cl-Ph
m-Me-Ph
p-Me-Ph
p-Br-Ph
p-OMe-Ph
p-CN-Ph
2,4-di-Cl-Ph
quinolinyl
9
10
11
12
13
14
15
16
17
18
19
3g
3h
3i
5g
5h
5i
3j
5j
3k
3l
5k
5l
3m
3n
3o
3p
5m
5n
5o
5p
a
The isolated yields were provided.
substituted substrates (Table 2, entries 4–9). Therefore, we focused on
the substrates 5h–n bearing m- and p-substituents with donating or
withdrawing group on N-1 phenyl ring of 1,2,4-triazole for further SAR
study. Based on the IC50 results against NO production, most compounds
Compound 5o with 2,4-di-substituted chloride on the N-1 phenyl ring
also preserved the anti-inflammatory activity (IC50 = 3.59 μM, Table 2,
entry 17). In comparison, compound 5p with quinolinyl-substituted in
N-1 position, significantly lower inhibitory activity against the NO
5h–n exhibited activity between 3.84 and 6.85
μ
M, which indicated that
production (33.0 μM, Table 2, entry 18). However, compound 6 with
the substituted groups on N1-phenyl ring were probably insignificant.
bromomethyl group variation on C-5 elevated the cytotoxicity. Based on
Table 2
IC50 of compounds 1, 2, 5a–p, and 6 and marketed NSAIDs on overproduction of nitric oxide in LPS-stimulated RAW 264.7 cells.
IC50 (µM)a
Entry
Compound
Ar
X
Y
1
1
Ph
H
H
>34.25
>24.51
4.61
6.08
4.19
2.87
7.57
3.88
4.13
4.82
5.87
5.01
6.02
3.84
6.85
4.19
3.59
33.0
2
2
Ph
H
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
3
5a
Ph
CH2Cl
CH2Cl
CH2Cl
CH2Cl
CH2Cl
CH2Cl
CH2Cl
CH2Cl
CH2Cl
CH2Cl
CH2Cl
CH2Cl
CH2Cl
CH2Cl
CH2Cl
CH2Cl
CH2Br
4
5b
o-F-Ph
5
5c
m-F-Ph
p-F-Ph
6
5d
7
5e
o-CF3-Ph
m-CF3-Ph
p-CF3-Ph
m-Cl-Ph
p-Cl-Ph
m-Me-Ph
p-Me-Ph
p-Br-Ph
p-OMe-Ph
p-CN-Ph
2,4-di-Cl-Ph
quinolinyl
Ph
8
5f
9
5g
10
11
12
13
14
15
16
17
18
19
20
21
5h
5i
5j
5k
5l
5m
5n
5o
5p
b
6
–
Indomethacinc
Celecoxibc
166c
22.6c
a
The IC50 values were calculated by FORECAST function in excel on basis of the data acquired in the experiment (n = 4) [18].
The RAW cells were not survival at the test concentration.
b
c
Indomethacin and Celecoxib were used as a reference drug in previous literature [19].
3

S.-M. Li et al.
Bioorganic Chemistry 104 (2020) 104333
fluorinated compounds [20]. Hence, the fluorinated compounds 5b–5g
were selected to investigate the effect of substitution position. The
tendency of COX-2 inhibition on substitution position was meta > para
> ortho (Table 3, entries 1–6). Compounds 5b, 5f, 5h, and 5j with m-
substituents, including F, CF3, Cl, and Me on N-1 phenyl ring presented
remarkably micromolar to nanomolar activity against COX-2 (0.00712,
1.58, 0.773, and 2.69 μM, Table 3, entries 2, 5, and 7–8). The results
indicated compounds with m-haloaryl groups were favorable for COX-2
inhibition compared to m-electron donating groups (Table 3, entry 8).
Surprisingly, the fluorine showed significant contributions towards the
COX-2 inhibitory activity. Compounds 5c (0.00712
μ
M) and 5d (0.0179
μ
M) bearing m- and p-F had about 3–7 times the COX-2 inhibitory ac-
tivity of Celecoxib (0.05
μ
M, Table 3, entry 13). However, di-substituted
compound 5o demonstrated no activity against the COX-2 enzyme (IC50
> 100 μM, Table 3, entry 11). The tendency order of the substitution on
N1-phenyl ring for COX-2 inhibition was observed: F > Cl > CF3 > Me >
CN > Br > 2,4-di-Cl.
Fig. 2. The ORTEP diagram of methyl 5-chloromethyl-1-(4-fluorophenyl)-
1,2,4-triazole-3-carboxylate 5e (CCDC No. 1982110).
On the other hand, the 1,2,4-triazole derivatives 5 universally
revealed inferior inhibition of COX-1 than COX-2 (Table 3), representing
the lower incidence of gastrointestinal toxicity [21]. Based on the
selectivity index results, Compound 5d had both excellent selectivity
(COX-1/COX-2 = 1080, Table 3, entry 3) and activity towards COX-2,
which implied that compound 5d possessed significant inhibition of
gastrointestinal side effect. Therefore, compound 5d could be a safe and
effective potential anti-inflammatory agent. As a result, the animal ac-
tivity model of compound 5d was determined for further in vivo
evaluation.
the results of in vitro inhibition studies of nitric oxide (NO) production,
the series of novel 1,2,4-triazole derivatives 5a–5o possessed greater NO
inhibition than marketed drugs, Indomethacin and Celecoxib (IC50
=
166 and 22.6 µM, Table 3, entries 20 and 21). As such, derivatives 5
would be used as potential anti-inflammatory drugs for the further in-
vitro and in-vivo study.
2.3.2. In vitro inhibition studies of COX-1/COX-2
2.3.3. In vivo anti-inflammation activity
Potential 1,2,4-triazole derivatives 5b–5j, 5l, 5m, and 5o from NO
inhibition results were further subjected to in vitro COX inhibition assay
to explore the COX-1/COX-2 selectivity. It has been reported that the
characteristic effects of fluorine and fluoroalkyl substituents on the
physico-chemical properties are significant to biological activity of
Based on the in vitro results, compound 5d was selected to test for in
vivo anti-inflammatory activity using carrageenan-induced mouse paw
edema protocol as an acute inflammation model. All groups reached the
peak of paw edema after 3 h carrageenan stimulation (Fig. 3). The
Table 3
In vitro COX-1/COX-2 inhibition and selectivity index results.
COX-1
COX-2
Selectivity Indexb
a
a
μ
M IC50
μM IC50
Entry
1
No.
Ar
5b
>100
6.67
–
2
5c
0.109
0.00712
15.3
3
4
5d
5e
19.3
0.0179
2.97
1080
>100
–
5
5f
23.3
1.58
1.91
6
7
5g
5h
>100
>100
>100
–
–
0.773
8
5j
50.6
2.69
18.8
9
5l
29.2
27.7
>100
52.5
7.69
>100
0.556
3.61
–
10
11
5n
5o
12
13
Indomethacinc
Celecoxibc
0.0390
14.8
0.490
0.0700
296
0.0500
a
IC50 is the concentration (μM) needed to cause 50% inhibition of COX-1 and COX-2 enzymatic activity (n = 2).
b
c
Selectivity index = (COX-1 IC50/COX-2 IC50).
Indomethacin and Celecoxib were used as the reference drugs in previous literature [27].
4

S.-M. Li et al.
Bioorganic Chemistry 104 (2020) 104333
sacrificed and the mice serum was centrifuged and removed of the su-
pernatant. The NO level was measured by Griess reagent. In Fig. 4A, the
NO level increased significantly in the edema serum at 5 h after Carra-
geenan injection, which can be markedly reversed by dose-dependent
administration of the compound 5d. It was observed that the treat-
ment with 5 mg/kg of compound 5d significantly reduced the plasma
level of NO up to 86.4%, which was similar to Indomethacin group (10
mg/kg, Fig. 4A). Likewise, both TNF-α and IL-6 levels were increased
significantly in the edema paw at 5 h after Carr injection (p < 0.001).
This effect decreased significantly by treatment with 5d dose-
dependently (Fig. 4B and C). Both mouse hind paw edema and inflam-
matory chemokines experiments showed a half dose of 5 mg/kg of 5d
had similar results with normal dose of Indomethacin (10 mg/kg).
Therefore, the results demonstrated that compound 5d (5 mg/kg)
exhibited significant reduction of inflammatory cytokines compared to
Indomethacin (10 mg/kg), which indicated that 5d possessed better
anti-inflammatory activity than Indomethacin.
Fig. 3. Mouse hind paw edema induced by Carrageenan (n = 6). ** and ***
indicated p < 0.01, p < 0.001 and as compared with the Carr group,
respectively.
2.3.4. Histological examination
The normal paw connective tissue had no cellular infiltration
(Fig. 5A). In contrast, the paw biopsies of Carrageenan-induced model
animals had obvious cellular infiltration in the connective tissue and
accumulation between collagen fibers and intercellular spaces (Fig. 5B).
The marketed NSAID, indomethacin, undeniably prevented the infiltrate
accumulation at a dose of 10 mg/kg, inferring a decreased inflammatory
effect compared to Carrageenan-induced model (Fig. 5C). Particularly,
paw biopsies of mice treated with compound 5d showed a dose-
dependent reduction in Carrageenan-induced inflammatory response
(Fig. 5D–F). In the 5 mg/kg compound 5d group, the inflammatory cells
were significantly reduced, and cellular infiltrations were barely
negative control group, without treatment with the compounds,
increased 0.18 mL of paw volume by injecting carrageenan. In the
positive control group, Indomethacin could effectively reduce paw
edema after 3 h carrageenan stimulation. Interestingly, compound 5d
dose-dependently reduced the paw edema (Fig. 3). More importantly,
compound 5d at concentration of 5 mg/kg displayed more anti-
inflammatory activity than 10 mg/kg Indomethacin (Fig. 3). In other
words, its dosage amount and potency would be relatively better than
Indomethacin.
Following the mice hind paw edema experiment, the mice were
Fig. 4. Effect of compound 5d and Indomethacin on production of (A) NO, (B) TNF-
α
and (C) IL-6 in mice serum (n = 6). Carr, 1% Carrageenan; Indo, Indomethacin.
### indicated p < 0.001 as compared with control group (normal saline treated only). *, **, and *** indicated p < 0.05, p < 0.01, and p < 0.001 as compared with
the Carr group, respectively.
5

S.-M. Li et al.
Bioorganic Chemistry 104 (2020) 104333
Fig. 5. Pathological examinations on Carr-induced edema paw tissues after treatments (n = 6). The pathological changes of paw tissues from (A) control group
(normal saline treated only), (B) Carr group, (C) Indo + Carr group, (D) 1.25 mg/kg 5d + Carr group (E) 2.5 mg/kg 5d + Carr (F) 5 mg/kg 5d + Carr were observed.
observed in intercellular spaces, and restricted to the vascular areas only
(Fig. 5F). Moreover, in the brown site of paw tissue, numerous COX-2
immunoreactive cells were also observed (Fig. 5B–F). Compound 5d
(2.5 and 5 mg/kg) and the reference drug, Indomethacin, were admin-
istered orally 30 min before Carrageenan-induced inflammation. The
obtained results indicated that compound 5d and Indomethacin mark-
edly reduced the increase in COX-2 immunoreactive cells in paws
(Fig. 5C and 5E–F). Based on the results, compound 5d at half dose
exhibited better anti-inflammatory ability compared to Indomethacin.
molecular modelling was performed. The X-ray structure of COX-2 co-
crystallized with Celecoxib (PDB ID 3LN1, resolution 2.40 Å) was used
to perform the docking study using docking program iGEMDOCK v2.1
[22]. Re-docking of the co-crystallized ligand Celecoxib into COX-2
active site via iGEMDOCK was performed, and the RMSD was calcu-
lated by using LigRMSD [23]. Celecoxib docked in the COX-2 binding
site almost at the same position (RMSD = 0.65 Å) as the co-crystallized
data (PDB:3LN1), which validated the iGEMDOCK protocol. The COX-2
enzyme was docked with compounds 5b–5j, 5l, 5m, and 5o in Celecoxib
binding site. The docking model revealed that compound 5d achieved
the lowest docking scores of ꢀ 97.89 kcal/mol (Table S6). As depicted in
Fig. 7, compound 5d was stabilized at COX-2 active site similarly to
celecoxib through three hydrogen bond interactions with His75,
Leu338, and Phe504 and eight van der Waals interactions with Val335,
Leu338, Ser339, Try341, Phe504, Val509, Gly512, and Ala513 [24].
Furthermore, the 1,2,4-triazole scaffold at the N-4 position of compound
5d formed an additional 3.2 Å hydrogen bond with Leu338 and the
carbonyl group at the C-3-position formed two 3.4 Å and 3.3 Å hydrogen
bond interactions with His75 and Phe504, respectively.
2.3.5. Gastric ulcerogenic activity
Histopathological observation of mice gastric mucosa for the pres-
ence of lesions following oral administration of 1.25, 2.5 and 5 mg/kg of
compound 5d as well as reference compound indomethacin was used to
gauge the ulcerogenic potential of the compounds. The normal gastric
mucosa demonstrated absence of epithelial tissue shedding and bacteria
(Fig. 6A). The administration of Carrageenan resulted in several
epithelial tissue shedding and bacteria adhesion (Fig. 6B). The slice of
Indomethacin group was similar with Carrageenan-induced group but
with mild to moderate gastric mucosa erosion (Fig. 6C). Visualization of
the inflammatory reaction in the gastric layers of the mice stomachs
treated with compound 5d revealed an absence of ulceration or any
maceration of gastric mucosa, necrosis, or lymphocytic infiltration. This
is comparable to normal gastric mucosa, which indicated the side effect
of compound 5d was lower than indomethacin (Fig. 6D–F).
One of the important differences between active sites of COX-1 and
COX-2 was resident 523, which showed isoleucine in COX-1 and valine
in COX-2. This difference may be the key contributor to COX-2 selec-
tivity [25,26]. Therefore, the COX-1 enzyme co-crystallized with Cele-
coxib (PDB ID 3KK6 resolution 2.75 Å) was also used to perform the
docking study with compound 5a, 5d, Indomethacin, and Celecoxib in
Celecoxib binding site. Based on the results, compound 5d (-4.4 kcal/
mol) had the worst binding energy to Ile523 compared to compound 5a
(-5.3 kcal/mol), Indomethacin (-15.5 kcal/mol), and Celecoxib (-12.1
kcal/mol). Additionally, compound 5d also correlated well with in vitro
COX-2 inhibitory activity experiment and had a similar docking position
with Celecoxib binding site. Thus, the weak binding energy of Ile523 in
2.4. Molecular modeling and in silico study
2.4.1. Molecular docking study
To gain an insight to the possible binding interactions of 1,2,4-tria-
zoles 5 with the active site of the cyclooxygenase-2 enzyme (COX-2),
6

S.-M. Li et al.
Bioorganic Chemistry 104 (2020) 104333
Fig. 6. Light microscopic picture of gastric mucosa of mice treated with (A) control group (normal saline treated only), (B) Carr group, (C) Indo + Carr group, (D)
1.25 mg/kg 5d + Carr group (E) 2.5 mg/kg 5d + Carr (F) 5 mg/kg 5d + Carr (n = 6).
Fig. 7. Docked pose of Celecoxib (in yellow) and 5d (in magenta) that is generated by iGEMDOCK with COX-2. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
COX-1 and the p-F-phenyl-substituent on N-1 position of 1,2,4-triazole
derivatives may be the key components to the high selectivity of com-
pound 5d.
2.4.2. In silico prediction of physicochemical properties and
pharmacokinetic profile
The emerging concept of “drug-likeness” plays an important role in
the promotion of a lead compound to a marketed drug [27]. Therefore,
we evaluated the physicochemical properties of compound 5d to predict
7

S.-M. Li et al.
Bioorganic Chemistry 104 (2020) 104333
Table 4
In silico physicochemical properties data predicted by Molinspiration.
Code
LogP
M.W.
HBA
HBD
Lipinski’s Violation
NROTB
PSA
%ABSa
5d
1.51
1.62
3.61
269.66
359.81
381.38
5
5
5
0
1
2
0
0
0
4
4
4
57.02
66.84
77.99
89.33
85.94
82.09
Indomethacin
Celecoxib
a
%ABS = 109–0.345 × PSA [28].
Table 5
In silico drug-likeness data (Molsoft) and ADME data (Pre-ADMET) [27].
Compound
Solubility (mg/L)
Model score
CaCo2a (nm/s)
MDCKb (nm/s)
HIAc (%)
BBBd
PPBe (%)
5d
836
211
33.4
ꢀ 1.04
0.680
ꢀ 1.05
21.6
20.1
0.499
228
98.4
98.4
96.7
0.0190
0.0620
0.0270
85.4
87.5
91.1
Indomethacin
0.435
45.1
Celecoxib
a
CaCo2 (permeability through cells derived from human colon adenocarcinoma): < 4 nm/s (low permeability); 4–70 nm/s (medium permeability); > 70 nm/s (high
permeability).
b
MDCK (permeability through Madin-Darby Canin kidney cells tool for rapid permeability): < 25 nm/s (low permeability); 25–500 nm/s (medium permeability); >
500 nm/s (high permeability).
c
HIA(percentage human intestinal absorption): 0–20% (poorly absorbed); 20–70% (moderately absorbed) ; 70–100% (well absorbed).
d
BBB (blood–brain barrier penetration): < 0.1 (low CNS absorption); 0.1–2 (medium CNS absorption); > 2 (high CNS absorption).
e
PPB (plasma protein binding): < 90% (poorly bound); > 90% (strongly bound).
whether it possessed suitable parameters to exhibit drug-likeness by
using Molinspiration, Molsoft and Pre-ADMET software. Compound 5d,
Indomethacin, and Celecoxib were selected to predict the parameters,
including lipophilicity (LogP), molecular weight (M.W.), number of
hydrogen bond donors (HBD) and acceptors (HBA), number of rotatable
bonds (NROTB), polar surface area (PSA), and percentage of oral ab-
sorption (%ABS, Table 5) [24]. Similar to Indomethacin and Celecoxib,
compound 5d expectedly complied with Lipinski’s rule based on mo-
lecular weight (<500), log P (<5), HBA (<10), and HBD (<5, Table 4).
Furthermore, the number of rotatable bonds of compound 5d (NORTB
= 4) was<10 and the PSA value of 5d (PSA = 57.02) was below 140 Å2,
which indicated a good predicted oral bioavailability. The predicted oral
bioavailability of 5d was 89.3%, which was better than marketed drugs,
Indomethacin and Celecoxib (Table 4).
estimation (Table 5). In the HIA values, Compound 5d (98.4%) was
similar to the reference drugs (96.7 and 98.4%), which was considered
to be a reasonable oral absorption pattern (Table 5). Compound 5d
exhibited lower CNS absorption value of 0.019 than reference drugs,
inferring lower brain penetration and CNS side effect (Table 5) [29].
Furthermore, compound 5d showed slightly lower plasma protein
binding (85.4%, Table 5) than Celecoxib and Indomethacin (91.1 and
87.5%, Table 5). This indicated a higher free concentration of 5d would
be detected in serum, and as such, a lower dose could be used in therapy.
Based on the above results, compound 5d significantly exhibited
outstanding physicochemical properties. Thus, compound 5d was po-
tential to be the drug candidate as a selective COX-2 inhibitor for the
treatment of inflammation and will be further investigated for other pre-
clinical trials.
Additionally, the Molsoft software was used to predict drug solubility
and the drug likeness model score, and the Pre-ADMET software was
used to evaluate general pharmacokinetic parameters, including CaCo2
cell permeability coefficient, Madin-Darby Canin kidney (MDCK) cell
permeability coefficient, Human intestinal absorption (HIA), blood-
brain barrier penetration (BBB) and human plasma protein binding
(PPB) [27]. Based on the prediction results of Molsoft software, com-
pound 5d possessed greater solubility (835 mg/L) than Indomethacin
and Celecoxib, and the drug-likeness model score of compound 5d
(ꢀ 1.04) was similar to Celecoxib (ꢀ 1.05, Table 5) Based on the results,
compound 5d followed Lipinski’s rule and possessed exceptional oral
bioavailability, which indicated compound 5d might be promising to be
a drug.
3. Conclusions
An efficient method was developed for synthesis of methyl 5-(hal-
omethyl)-1-aryl-1H-1,2,4-triazole-3-carboxylate derivatives from nitri-
limines, including amination with ammonium hydroxide and
heterocyclization two steps reactions. Obtained 1,2,4-triazoles was
subjected to in vitro NO production and cyclooxygenase COX-1/COX-2
inhibition assays. The structure and activity relationship of 1,2,4-tria-
zole derivatives was established. Interestingly, compound 5d not only
showed favorable inhibition of NO and COX-2 (IC50 of 2.87 and 17.9
nM), it also had a significantly greater selectivity (COX-1/COX-2 =
1080). Additionally, 5 mg/kg compound 5d also exhibited better in vivo
anti-inflammation and gastric protection results, including paw edema,
chemokines, and histological experiments compared to 10 mg/kg
Indomethacin. Docking and other prediction software were conducted.
Moreover, compound 5d showed better cell permeability in the
CaCo2 cell model (21.6 nm/s) and MDCK cell model (228 nm/s) than
Celecoxib and Indomethacin, indicating great oral absorption
O
O
Ar
N
O
X
Cl
Ar
H2N
Ar
X
OMe
OMe
NH4OH
solvents
N
Cl
N
N
N
NEt3, toluene
X = Cl, Br
OMe
NH
NH
O
3a-p
4a-p
5a-p, 6
Scheme 1. Synthesis of 1,2,4-triazole 5a–p and 6.
8

S.-M. Li et al.
Bioorganic Chemistry 104 (2020) 104333
Based on in vivo, in vitro, and prediction studies, compound 5d is a po-
tential drug candidate and needs further investigation in other pre-
clinical trials.
4.1.1.2. Methyl-2-amino-2-(2-(2-(trifluoromethyl)phenyl)hydrazono)ace-
tate (4e). Yield 92%, brown solid, m.p.: 133–135 ◦C, 1H NMR (CDCl3,
400 MHz) δ 3.90 (s, 3H, CH3), 4.63 (br, 2H, NH2), 6.93 (t, J = 8.72 Hz,
1H, ArH), 6.98 (br, 1H, NH), 7.44–7.47 (m, 2H, ArH), 7.69 (d, J = 8.72
Hz, 1H, ArH); 13C NMR (CDCl3, 100 MHz) δ 53.19, 113.87 (q, J = 30.26
Hz), 115.87, 120.08, 124.77 (d, J = 272.29 Hz), 125.97 (q, J = 5.38 Hz),
133.26, 137.76, 142.81; IR (KBr) 3362, 2958, 1721,1639, 1585, 1612,
4. Experimental
4.1. Chemistry
1387,1309, 1276, 1106 cmꢀ 1
.
All chemicals were reagent grade and used as purchased. 1-phenyl-
1H-1,2,4-triazole 1, methyl 1-phenyl-1H-1,2,4-triazole-3-carboxylate 2
and hydrazonoyl chlorides 3a–p were prepared following our reported
procedure [14–16]. All reactions were carried out under nitrogen at-
mosphere and monitored by TLC analysis. Flash column chromatog-
raphy was carried out on silica gel (230–400 mesh). Commercially
available reagents were used without further purification unless other-
wise noted. Dichloromethane, ethyl acetate, and hexanes were pur-
chased from Mallinckrodt Chemical Co. Dimethyl sulfone (DMSO2,
purity = 100%) was purchased from Tokyo Chemical Industry Co.1H
NMR spectra were recorded on Bruker 400 or 500 MHz and 13C NMR
were recorded at 100 or 125 MHz, respectively, using CDCl3 or DMSO‑d6
as solvent. The chemical shifts were given in parts per million and the J
values were given in Hertz. The standard abbreviations s, d, t, q, and m
referred to singlet, doublet, triplet, quartet, and multiplet, respectively.
The purities of the compounds 5 were determined by using the q1HNMR
method (absolute q1HNMR with internal calibration) [30]. Most of pu-
rities were more than >98%, for example, compound 5d was 99.16%
(Fig. S59). The purity of compound 5d was calculated by following
equation:
A solution of crude hydrazonamides 4a–p with 2.0 equivalent of 2-
chloroacetyl chloride (10 mmol, 0.8 mL) or 2-bromoacetyl chloride
(10 mmol, 0.8 mL) in presence of triethylamine (NEt3, 25.0 mmol, 5.0
equiv, 3.5 mL) in anhydrous toluene solution at reflux for 5 h. When the
reaction was completed, worked-up with water (2 × 10 mL), neutralized
by saturated NaHCO3(aq), and extracted with CH2Cl2 (3 × 30 mL). The
organic layer was dried over MgSO4, concentrated under reduced
pressure, and purified by column chromatography on silica gel to give
the corresponding 1,2,4-triazole products 5a–p and 6 in 53–83% yields.
4.1.1.3. Methyl
5-(chloromethyl)-1-phenyl-1H-1,2,4-triazole-3-carbox-
ylate (5a). Yield 74%, yellow solid, m.p.: 60–62 ◦C, 1H NMR (CDCl3,
400 MHz) δ 4.00 (s, 3H, CH3), 4.64 (s, 2H, CH2), 7.53–7.58 (m, 5H, ArH);
13C NMR (CDCl3, 100 MHz) δ 33.81, 52.95, 124.79 (2 × CH), 129.77 (2
× CH), 130.33, 135.96, 152.76, 154.91, 159.83; IR (KBr) 3275, 3026,
2956, 1740, 1595, 1503, 1481, 1440, 1413, 1362, 1225, 1165, 1138
cmꢀ 1; MS (EI) m/z: 253 (M+2, 20), 251 (M+, 56), 220 (36), 159 (16), 193
(55), 186 (19), 184 (69), 176 (58), 152 (15), 117 (24), 107 (72), 92 (22),
91 (100), 77 (90), 65 (31), 64 (40), 63 (15), 59 (15), 51 (56), 49 (17);
HRMS calcd. for C11H10ClN3O2: 251.0462; found: 251.0453.
nic⋅intt⋅MWt⋅m
P(%) =
ic⋅Pic
4.1.1.4. Methyl 5-(chloromethyl)-1-(2-fluorophenyl)-1H-1,2,4-triazole-3-
carboxylate (5b). Yield 68%, yellow solid, m.p.: 102–103 ◦C, 1H NMR
(CDCl3, 400 MHz) δ 4.00 (s, 3H, CH3), 4.63 (s, 2H, CH2), 7.28–7.35 (m,
2H, ArH), 7.52–7.59 (m, 2H, ArH); 13C NMR (CDCl3, 100 MHz) δ 33.61,
52.99, 117.01, 123.96, 125.22, 128.54, 132.73, 154.31, 154.90, 157.60,
159.63; IR (KBr) 3032, 2957, 1743, 1598, 1512, 1482, 1463, 1439,
1414, 1361, 1270, 1220, 1164, 1137 cmꢀ 1; MS (EI) m/z: 269 (M+, 31),
238 (22), 211 (43), 194 (37), 135 (16), 109 (100), 95 (20); HRMS calcd.
for C11H9ClFN3O2: 269.0367; found: 269.0363.
nt⋅intic⋅MWic⋅ms
where MW is the molecular weight, P is the purity of internal calibrant,
mic is the amount of internal calibrant, ms is the amount of sample, int is
integral, and n is the number of protons giving rise to a given NMR
signal. The subscript ic and t represents the internal calibrant and target
analyte, respectively. Infrared spectra (IR) were recorded as neat solu-
tions or solids; and mass spectra were recorded using electron impact or
electrospray ionization techniques. The wavenumbers reported are
referenced to the polystyrene 1601 cmꢀ 1 absorption. Flash column
chromatography purification of compounds was carried out by gradient
elution using hexanes in ethyl acetate (EA) unless otherwise stated.
High-resolution mass spectra were obtained by means of a JEOL JMS-
HX110 mass spectrometer.
4.1.1.5. Methyl 5-(chloromethyl)-1-(3-fluorophenyl)-1H-1,2,4-triazole-3-
carboxylate (5c). Yield 74%, brown solid, m.p.: 95–97 ◦C, 1H NMR
(CDCl3, 400 MHz) δ 4.00 (s, 3H, CH3), 4.67 (s, 2H, CH2), 7.22–7.28 (m,
1H, ArH), 7.35–7.37 (m, 1H, ArH), 7.38–7.39 (m, 1H, ArH), 7.39–7.51
(m, 1H, ArH); 13C NMR (CDCl3, 100 MHz) δ 33.77, 53.01, 112.64,
117.50, 120.29, 131.22, 137.13, 152.76, 159.62, 161.49, 163.98; IR
(KBr) 3080, 3034, 2960, 1742, 1606, 1480, 1409, 1360, 1231, 1206,
1160, 1135, 1025 cmꢀ 1; MS (EI) m/z: 269 (M+, 39), 238 (20), 211 (27),
194 (37), 135 (15), 109 (100), 95 (32), 73 (21); HRMS calcd. for
4.1.1. Standard procedure of synthesis of 1,2,4-triazole 5a–p and 6
A solution of hydrazonoyl chlorides (3a–p, 5.0 mmol, 1.0 equiv) was
treated with 5.0 equivalents of 30% NH4OH solution (2.9 mL) at 0 ◦C for
24 h. When the starting materials 3a–p were fully consumed, the cor-
responding hydrazonamides 4a–p were formed. The resulting solution
was neutralized with saturated NaHCO3(aq) and extracted with CH2Cl2
(3 × 30 mL). The organic layers were dried over MgSO4, filtered, and
concentrated under reduced pressure to give the corresponding crude
hydrazonamides 4a–p without further purification, except for com-
pounds 4a and 4e. The crude compounds 4a and 4e were purified by
column chromatography (n-hexane/EtOAc = 4/1) on silica gel to give
the corresponding hydrazonamide products 4a and 4e in 92% and 86%
yields, respectively.
C11H9ClFN3O2: 269.0367; found: 269.0361.
4.1.1.6. Methyl 5-(chloromethyl)-1-(4-fluorophenyl)-1H-1,2,4-triazole-3-
carboxylate (5d).. Yield 76%, white solid, m.p.: 98–100 ◦C, 1H NMR
(CDCl3, 400 MHz) δ 4.02 (s, 3H, CH3), 4.64 (s, 2H, CH2), 7.25 (t, 2H, J =
8.46, ArH), 7.58–7.61 (m, 2H, ArH); 13C NMR (CDCl3, 100 MHz) δ
33.70, 53.01, 116.91, 127.02, 132.03, 152.90, 154.26, 159.72, 162.04,
164.55, 165.26; IR (KBr) 3464, 3277, 3079, 3009, 2958, 2330, 2044,
1898, 1732, 1606, 1485, 1478, 1363, 1293, 1158 cmꢀ 1; MS (EI) m/z:
271 (M+2, 21), 269 (M+, 66), 238 (28), 211 (36), 202 (38), 194 (64), 135
(21), 125 (27), 109 (100), 95 (55), 83 (18), 82 (17), 75 (20); HRMS
calcd. for C11H9ClFN3O2: 269.0367; found: 269.0369.
4.1.1.1. Methyl 2-amino-2-(2-phenylhydrazono)acetate (4a) [31]. Yield
92%, brown solid, m.p.: 128–130 ◦C, 1H NMR (CDCl3, 400 MHz) δ 2.15
(br, 2H, NH), 3.88 (s, 3H, CH3), 6.69 (br, 1H, NH), 6.89 (t, J = 7.32 Hz,
1H, ArH), 7.09 (d, J = 7.68 Hz, 2H, ArH), 7.24 (t, J = 7.70 Hz, 2H, ArH);
13C NMR (CDCl3, 100 MHz) δ 53.06, 114.15 (2 × CH), 121.20, 129.18 (2
× CH), 135.82, 145.11, 162.75; IR (KBr) 3452, 3343, 2654, 1716, 1600,
4.1.1.7. Methyl 5-(chloromethyl)-1-(2-(trifluoromethyl)phenyl)-1H-1,2,4-
triazole-3-carboxylate (5e). Yield 53%, yellow solid, m.p.: 228–229 ◦C,
1H NMR (CDCl3, 400 MHz) δ 4.01 (s, 3H, CH3), 4.51 (s, 2H, CH2),
7.55–7.58 (m, 1H, ArH), 7.76–7.78 (m, 2H, ArH), 7.87–7.89 (m, 1H,
1439, 1388, 1305, 1249, 1148 cmꢀ 1
.
9

S.-M. Li et al.
Bioorganic Chemistry 104 (2020) 104333
ArH); 13C NMR (CDCl3, 100 MHz) δ 33.26, 53.01, 120.98, 123.65,
127.68, 128.19, 130.33 (2 × CH), 130.77, 133.13, 154.54, 159.60; IR
(KBr) 3030, 2959, 2926, 1744, 1608, 1508, 1458, 1318, 1224, 1182,
1136 cmꢀ 1; MS (EI) m/z: 319 (M+, 39), 290 (20), 287 (67), 263 (34),
261 (100), 244 (44), 220 (30), 185 (22), 159 (87), 145 (67), 139 (17),
132 (22), 109 (18), 59 (16); HRMS calcd. for C12H9F3ClN3O2: 319.0335;
found: 319.0341.
(d, 2H, J = 8.10 Hz, ArH), 7.45 (d, 2H, J = 8.25 Hz, ArH); 13C NMR
(CDCl3, 125 MHz) δ 21.27, 33.87, 52.93, 124.69 (2 × CH), 130.31 (2 ×
CH), 133.56, 140.74, 152.75, 154.16, 159.90; IR (KBr) 3276, 2957,
2926, 1741, 1514, 1482, 1441, 1418, 1363, 1223, 1183,1163, 1138
cmꢀ 1; MS (EI) m/z: 267 (M+2, 36), 266 (15), 265 (M+, 77), 234 (22), 207
(22), 198 (31), 190 (35), 121 (21), 105 (100), 104 (25), 91 (37), 78 (19),
77 (22), 65 (22); HRMS calcd. for C12H12ClN3O2: 265.0618; found:
265.0613.
4.1.1.8. Methyl 5-(chloromethyl)-1-(3-(trifluoromethyl)phenyl)-1H-1,2,4-
triazole-3-carboxylate (5f).. Yield 68%, yellow solid, m.p.: 222–223 ◦C,
1H NMR (CDCl3, 500 MHz) δ 4.03 (s, 3H, CH3), 4.68 (s, 2H, CH2), 7.73 (t,
1H, J = 8.00 Hz, ArH), 7.83 (d, 1H, J = 13.66 Hz, ArH), 7.85 (d, 1H, J =
13.71 Hz, ArH), 7.93 (s, 1H, ArH); 13C NMR (CDCl3, 125 MHz) δ 33.73,
53.13, 124.79 (2 × CH), 129.77 (2 × CH), 130.33, 135.96, 152.76,
154.91, 159.83; IR (KBr) 2958, 1742, 1599, 1498, 1483, 1459, 1362,
1305, 1282, 1224, 1167, 1135 cmꢀ 1; MS (EI) m/z: 319 (M+, 49), 288
(30), 261 (45), 244 (37), 159 (100),145 (42), 59 (16); HRMS calcd. for
4.1.1.14. Methyl 1-(4-bromophenyl)-5-(chloromethyl)-1H-1,2,4-triazole-
3-carboxylate (5l). Yield 83% yield (1.37 g); yellow liquid; 1H NMR
(CDCl3, 500 MHz) δ 4.00 (s, 3H, CH3), 4.64 (s, 2H, CH2), 7.48 (d, 2H, J =
8.45 Hz, ArH) 7.68 (d, 2H, J = 8.50 Hz, ArH); 13C NMR (CDCl3, 125
MHz) δ 33.77, 53.05, 124.56, 126.6 (2 × CH), 133.06 (2 × CH), 135.00,
152.76, 154.43, 159.65; IR (KBr) 2955, 1741, 1496, 1479, 1440, 1417,
1399, 1361, 1223, 1183, 1162, 1137 cmꢀ 1; MS (EI) m/z: 333 (23), 331
(M+2, 99), 329 (M+, 74), 300 (19), 298 (15), 273 (28), 271 (22), 256
(38), 254 (38), 184 (15), 171 (97), 169 (100), 157 (30), 155 (23), 90
(69), 76 (24), 75 (21), 63 (28), 59 (18); HRMS calcd. for C11H9ClBrN3O2:
328.9567; found: 328.9562.
C
12H9F3ClN3O2: 319.0335; found: 319.0330.
4.1.1.9. Methyl 5-(chloromethyl)-1-(4-(trifluoromethyl)phenyl)-1H-1,2,4-
triazole-3-carboxylate (5g). Yield 77%, yellow solid, m.p.: 235–236 ◦C,
1H NMR (CDCl3, 500 MHz) δ 4.03 (s, 3H, CH3), 4.69 (s, 2H, CH2), 7.80
(d, 2H, J = 8.50 Hz, ArH), 7.85 (d, 2H, J = 8.50 Hz, ArH); 13C NMR
(CDCl3, 125 MHz) δ 33.83, 53.14, 124.79 (2 × CH), 129.77 (2 × CH),
132.83, 138.85, 152.89, 154.69, 159.59; IR (KBr) 2959, 1743, 1618,
1439, 1325, 1225, 1169, 1133 cmꢀ 1; MS (EI) m/z: 319 (M+, 45), 288
(36), 263 (16), 261 (51), 252 (26), 244 (46), 185 (16), 159 (100), 145
(53), 139 (26), 59 (22); HRMS calcd. for C12H9F3ClN3O2: 319.0335;
found: 319.0331.
4.1.1.15. Methyl
5-(chloromethyl)-1-(4-methoxyphenyl)-1H-1,2,4-tri-
azole-3-carboxylate (5m). Yield 58%, brown liquid, 1H NMR (CDCl3, 400
MHz) δ 4.01 (s, 3H, CH3), 4.62 (s, 2H, CH2), 7.02 (d, 2H, J = 8.80 Hz,
ArH), 7.49 (d, 2H, J = 8.80 Hz, ArH); 13C NMR (CDCl3, 100 MHz) δ 33.82,
52.94, 55.67, 114.80 (2 × CH), 126.40 (2 × CH), 129.43, 152.84, 154.04,
159.90, 159.89; IR (KBr) 3282, 3007, 2958, 2841, 2248, 2051, 1742,
1609, 1592, 1516, 1482, 1445, 1362, 1302, 1254, 1219, 1181, 1133
cm–1; MS (EI) m/z: 283 (M+2, 17), 281 (M+, 59), 214 (18), 137 (15), 121
(100); HRMS calcd. for C12H12ClN3O3: 281.0567; found: 281.0572.
4.1.1.10. Methyl 5-(chloromethyl)-1-(3-chlorophenyl)-1H-1,2,4-triazole-
3-carboxylate (5h). Yield 73%, yellow solid, m.p.: 101–102 ◦C, 1H NMR
(CDCl3, 400 MHz) δ 4.00 (s, 3H, CH3), 4.66 (s, 2H, CH2), 7.50–7.53 (m,
3H, ArH), 7.64 (t, 1H, J = 1.60 Hz, ArH); 13C NMR (CDCl3, 100 MHz) δ
33.74, 53.06, 122.69, 125.19, 130.68 (2 × CH), 135.68, 136.91, 152.82,
154.40, 159.63; IR (KBr) 3463, 3075, 3030, 2956, 2343, 1738, 1594,
1480, 1470, 1447, 1408, 1360, 1162, 1138, 1103 cmꢀ 1; MS (EI) m/z:
287 (M+2, 30), 285 (M+, 46), 256 (15), 254 (23), 229 (23), 227 (36), 220
(25), 218 (41), 212 (15), 210 (47), 141 (26), 127 (32), 125 (100), 111
(40), 90 (34), 77 (15), 75 (31), 63 (17), 59 (16), 49 (13); HRMS calcd. for
4.1.1.16. Methyl 5-(chloromethyl)-1-(4-cyanophenyl)-1H-1,2,4-triazole-
3-carboxylate (5n). Yield 74%, yellow solid, m.p.: 92–93 ◦C, 1H NMR
(CDCl3, 500 MHz) δ 4.03 (s, 3H, CH3), 4.71 (s, 2H, CH2), 7.82 (d, 2H, J =
8.75 Hz, ArH), 7.88 (d, 2H, J = 8.75 Hz, ArH); 13C NMR (CDCl3, 125
MHz) δ 33.85, 53.18, 114.30, 117.21, 125.14 (2 × ArH), 133.84 (2 ×
ArH), 139.37, 152.86, 154.80, 159.42; IR (KBr) 3105, 2956, 2918, 2850,
2233, 1740, 1607, 1511, 1483, 1440, 1418, 1404, 1369, 1284, 1223,
1182, 1163, 1138 cmꢀ 1; MS (EI) m/z: 276 (M+, 39), 245 (23), 218 (35),
201 (57), 177 (16), 142 (15), 116 (100),102 (29), 59 (19); HRMS calcd.
for C11H10ClN3O2: 276.0414; found: 276.0410.
C
11H9Cl2N3O2: 285.0072; found: 285.0070.
4.1.1.11. Methyl 5-(chloromethyl)-1-(4-chlorophenyl)-1H-1,2,4-triazole-
3-carboxylate (5i). Yield 78%, yellow solid, m.p.: 99–100 ◦C, 1H NMR
(CDCl3, 400 MHz) δ 4.02 (s, 3H, CH3), 4.65 (s, 2H, CH2), 7.52–7.58 (m,
4H, ArH); 13C NMR (CDCl3, 100 MHz) δ 33.75, 53.00, 126.04 (2 × CH),
130.03 (2 × CH), 134.46, 136.49, 152.77, 154.37, 159.63; IR (KBr)
3464, 3098, 3030, 2956, 1741, 1596, 1498, 1479, 1441, 1418, 1361,
1279, 1223, 1183,1162 cmꢀ 1; MS (EI) m/z: 287 (M+2, 38), 285 (M+, 60),
227 (20), 210 (41), 127 (30), 125 (100), 90 (17); HRMS calcd. for
4.1.1.17. Methyl 5-(chloromethyl)-1-(2,4-dichlorophenyl)-1H-1,2,4-tri-
azole-3-carboxylate (5o). Yield 77%, white solid, m.p.: 132–134 ◦C, 1H
NMR (CDCl3, 400 MHz) δ 4.02 (s, 3H, CH3), 4.56 (s, 2H, CH2), 7.46 (s,
2H, ArH), 7.61 (s, 1H, ArH); 13C NMR (CDCl3, 100 MHz) δ 33.40, 52.95,
128.37, 130.03, 130.45, 132.01, 132.41, 137.98, 154.26, 154.58,
159.41; IR (KBr) 3092, 3031, 2956, 1743, 1501, 1472, 1221, 1183,
1164, 1136 cmꢀ 1; MS (EI) m/z: 321 (M+2, 37), 319 (M+, 38), 290 (17),
288 (19), 263 (40), 261 (42), 246 (33), 244 (53), 185 (15), 161 (62), 159
(M+, 100), 145 (20), 133 (15), 126 (19), 124 (18), 124 (43), 109 (17),
59 (18); HRMS calcd. for C11H8Cl3N3O2: 318.9682; found: 318.9684.
C
11H9Cl2N3O2: 285.0072; found: 285.0066.
4.1.1.12. Methyl
5-(chloromethyl)-1-(m-tolyl)-1H-1,2,4-triazole-3-
carboxylate (5j).. Yield 72%, yellow solid, m.p.: 86–87 ◦C, 1H NMR
(CDCl3, 400 MHz) δ 3.97 (s, 3H, CH3), 4.63 (s, 2H, CH2), 7.24–7.41 (m,
4H, ArH); 13C NMR (CDCl3, 100 MHz) δ 21.18, 33.80, 52.86, 121.53,
125.36, 129.40, 131.00, 135.80, 140.12, 152.64, 154.02, 159.79; IR
(KBr) 3463, 3279, 3028, 2956, 2924, 1740, 1612, 1592, 1495, 1482,
1460, 1361, 1308, 1225, 1162, 1136 cmꢀ 1; MS (EI) m/z: 267 (M+2, 18),
265 (M+, 57), 234 (18), 190 (27), 105 (100), 91 (22), 78 (19), 65 (16);
HRMS calcd. for C12H12ClN3O2: 265.0618; found: 265.0627.
4.1.1.18. Methyl 5-(chloromethyl)-1-(quinolin-8-yl)-1H-1,2,4-triazole-3-
carboxylate (5p). Yield 62%, brown solid, m.p.: >286 ◦C, 1H NMR
(CDCl3, 400 MHz) δ 4.01 (s, 3H, CH3), 4.65 (s, 2H, CH2), 7.53 (dd, 1H, J
= 8.40 Hz, j = 4.40 Hz, ArH), 7.71 (t, 1H, J = 7.60, ArH), 7.97 (d, 1H, J
= 7.20, ArH), 8.05 (d, 1H, J = 8.40, ArH), 8.29 (d, 1H, J = 8.00, ArH),
8.87–8.88 (m, 1H, ArH); 13C NMR (CDCl3, 100 MHz) δ 34.99, 52.84,
122.53, 126.24, 128.68, 128.87, 130.89, 133.54, 136.51, 142.29,
151.82, 154.40, 155.21, 160.03; IR (KBr) 3447, 2955, 1738, 1595,
1504, 1469, 1445, 1391, 1221, 1164 cmꢀ 1; MS (EI) m/z: 267 (58), 266
(100), 264 (23), 208 (17), 207 (18), 155 (35), 142 (17); HRMS calcd. for
4.1.1.13. Methyl 5-(chloromethyl)-1-(p-tolyl)-1H-1,2,4-triazole-3-carbox-
ylate (5k). Yield 82%, yellow solid, m.p.: 90–91 ◦C, 1H NMR (CDCl3,
500 MHz) δ 2.43 (s, 3H, CH3), 4.01 (s, 3H, CH3), 4.63 (s, 2H, CH2), 7.33
C14H11ClN4O2: 302.0571; found: 302.0574.
10

S.-M. Li et al.
Bioorganic Chemistry 104 (2020) 104333
4.1.1.19. Methyl 5-(bromomethyl)-1-phenyl-1H-1,2,4-triazole-3-carbox-
ylate (6). Yield 72%, yellow liquid, 1H NMR (CDCl3, 500 MHz) δ 4.02 (s,
3H, CH3), 4.66 (s, 2H, CH2), 7.55–7.56 (m, 3H, ArH), 7.59–7.61 (m, 2H,
ArH); 13C NMR (CDCl3, 125 MHz) δ 33.87, 52.96, 124.79, 129.86 (2 ×
ArH), 130.36 (2 × ArH), 136.07, 152.77, 154.31, 159.84; IR (KBr) 2957,
1738, 1704, 1599, 1500, 1480, 1436, 1412, 1365, 1222, 1164, 1015
cmꢀ 1; MS (EI) m/z: 251 (28), 220 (20), 216 (15), 193 (19), 176 (22), 117
(13), 91 (M+, 100), 77 (43), 65 (14), 64 (16), 51 (18); HRMS calcd. for
C11H10BrN3O2: 294.9956; found: 294.9954.
4.2.3. Animals [31]
Male ICR mice (6–8 weeks) were acquired from the BioLASCO
Taiwan Co., Ltd. (Taipei, Taiwan). The mice were reared in plexiglass
cages at a constant temperature of 22 ± 1 ◦C, and relative humidity of
55 ± 5% with ad libitum access to food and water and 12 h darklight
cycle for at least 2 week before the experiment. All experimental pro-
cedures were performed according to the National Institutes of Health
(NIH) Guide for the Care and Use of Laboratory Animals. In addition, all
tests were conducted under the guidelines of the International Associ-
ation for the Study of Pain (Zimmermann, 1983).
4.2. Biological screening
4.2.3.1. Carrageenan-induced paw edema [32,33]. The mice (about
29–39 g) were divided into 6 groups (n = 6 mice/group) randomly and
orally administrated with a low dose (1.25 mg/kg), medium dose (2.5
mg/kg), and high dose (5 mg/kg) of compound 5d in 1% CMC as test
groups. The positive and vehicle control group were given access to 10
mg/kg Indomethacin in 1% CMC orally and the same volume of 1% CMC
orally, respectively. Thirty minutes after administration, acute paw
4.2.1. Cell culture and reagents [32]
Lipopolysaccharide (endotoxin from Escherichia coli, serotype 0127:
B8), carrageenin, indomethacin, MTT reagent (3-[4,5-dimethylthiazol-
2-yl]- 2,5-diphenyltetrazolium bromide) and other chemicals were
purchased from Sigma Chemical Co. (St. Louis, MO, USA). Murine
macrophage RAW 264.7 cell (BCRC No. 60001) was purchased from the
Bioresources Collection and Research Center (BCRC) of the Food In-
dustry Research and Development Institute (Hsinchu, Taiwan). Cells
were cultured at 37 ◦C in a 5% CO2 humidified atmosphere. The medium
for cell lines was Dulbecco’s Modified Eagle Medium (DMEM, Sigma, St.
Louis, MO, USA) containing 10% fetal bovine serum (FBS) and sub-
cultured every 2 days.
edema was induced by subplantar injection of 50 μL 1% freshly prepared
carrageenan suspension in 0.9% normal saline in the right hind paw. The
volume of the paw was measured pre-injection and at intervals of 1, 2, 3,
4, and 5 h post-injection by using Plethysmometer (model 7159; Ugo
Basile, Varese, Italy).
4.2.3.2. Measurement of serum NO [32]. NO production was indirectly
assessed by measuring the nitrite levels in the mice serum determined by
a colorimetric method based on the Griess reaction.
4.2.1.1. Cell viability assay [32]. Cell viability was assessed by MTT
assay. RAW 264.7 cells were seeded in a 96-well plate at a density of 2 ×
105 cells per well and incubated for 24 h at 37 ◦C. The cells were then
treated with different concentrations of compounds 5a-p and 6 in the
presence of 100 ng/mL LPS (lipopolysaccharide) and incubated at 37 ◦C
for 24 h. After incubation, the cell were washed twice with DPBS and
4.2.3.3. Serum cytokine determination by Enzyme-Linked immunosorbent
assay [32]. The mice serum levels of TNF-α and IL-6 were determined
using commercially available ELISA kit (Biosource International Inc.,
incubated with 100
was then discarded, and 100
μ
L of 0.5 mg/mL MTT at 37 ◦C for 3 h. The medium
Camarillo, CA) according to the manufacturer’s instruction. The con-
μ
L dimethyl sulfoxide (DMSO) was added.
centrations of TNF-α and IL-6 were expressed as pg/mL.
The resulting color was assayed at 540 nm using a microplate reader
(Molecular Devices, Sunnyvale, CA, USA).
4.2.3.4. Histological examination [32–34]. The biopsies of mice hind
paws were taken following 5 h treatment with the interplanetary in-
jection of 1% Carrageenan immediately. The tissue slices were fixed
(1.85% formaldehyde, 1% acetic acid) for 1 week at room temperature,
dehydrated by graded ethanol and embedded in Paraffin (Sherwood
Medical). Tissue sections were deparaffinized with xylene and stained
with hematoxylin and eosin for cell counting. All samples were observed
and photographed with BH-2 Olympus microscopy. The excessive in-
flammatory response was illustrated as massive infiltration of ploy-
morphonuclear leukocytes (PMNs).
4.2.1.2. Measurement of nitrite [32]. Raw 264.7 murine macrophage
were plated in 96-well plates at 2 × 105 cells per well and incubated for
24 h. The cells were then pretreated with LPS (100 ng/mL) with vehicle
or different concentration test compounds for 24 h. Then, NO concen-
tration in the medium was determined using the Griess assay. The su-
pernatant was mixed with the same volume of Griess reagent (1%
sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride and 5%
phosphoric acid) and incubated for 10 min at room temperature; the
absorbance was measured at 540 nm with a Micro-Reader (Molecular
Devices Orleans Drive, Sunnyvale, CA, USA). Sodium nitrite were used
to generate a standard curve, the concentration of NO was measured at
540 nm. The IC50 value indicates the concentration that resulted in a
50% decrease in NO production relative to an untreated control and then
4.2.3.5. Gastric ulcerogenic activity [33]. After measuring the anti-
inflammatory activity the mice were sacrificed and the stomachs were
removed, collected, opened along the greater curvature, washed with
distilled water, and gently cleaned by dipping in saline. Compounds 5d
and indomethacin were evaluated for acute gastric ulcerogenic effect in
mice. Histopathological examination was performed to confirm the de-
gree of inflammatory reaction in the gastric mucosal layers of the treated
mice stomachs.
converted to commonly used unit μM.
4.2.2. In vitro COX-1 and COX-2 inhibitory assay [28]
The ability of the test compounds 5b–h, j, l, n and o to inhibit human
recombinant COX-2 and ovine COX-1 (IC50 value, μM) was determined
using an enzyme immunoassay (EIA) kit (catalog no. 560131, Cayman
Chemical, Ann Arbor, MI) according to the procedure suggested by the
4.3. Molecular modeling and in silico study
manufacturer. The half-maximal inhibitor concentrations IC50 (μM)
were determined and the selectivity index (SI) values were calculated as
IC50 (COX-1)/IC50 (COX-2).
4.3.1. Molecular docking [28,35]
Computational studies were performed with 5b–5j, 5l, 5m and 5o.
The lowest energy conformer for chemical structure was saved in mol2
files by Chemdraw before use in docking studies. The structure of COX-2,
11

S.-M. Li et al.
Bioorganic Chemistry 104 (2020) 104333
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
(d) W.-P. Yen, F.-C. Kung, F.F. Wong, 1,3-Dipolar Cycloaddition of Carbodiimides
and Nitrilimines: Synthesis and Mechanistic Study of 5-Amino-1,2,4-triazoles, Eur.
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Acknowledgments
(e) S.-E. Tsai, W.-P. Yen, Y.-T. Li, Y.-T. Hu, C.-C. Tseng, F.F. Wong, Indium(III)
Chloride Promoted One-Pot Multicomponent Synthesis of 1,2,4-Triazole Carrying
1,2,4-Triazin-6-ones from Nitrile Imines and N, N’-Bis(trimethylsilyl)carbodiimine,
Asian. J. Org. Chem. 6 (2017) 1470–1475.
We are grateful to the China Medical University (CMU108-N–04 and
CMU109-ASIA–08) and the Ministry of Science and Technology of
Taiwan (MOST 109-2113-M-039-003).
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
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