New hydrazone derivatives of pyrazole-4-carboxaldehydes exhibited anti-inflammatory properties

Article abstract of DOI:10.2174/1570180816666190731113441

Background: Several series of hydrazone derivatives of pyrazole-4-carboxaldehydes (4-11) were designed and synthesized to screen their inflammatory activity. Methods: The products were characterized by¹H NMR,¹³C NMR and HRMS. In vitro LPS-induced TNF-α model and in vivo xylene-induced ear-edema model were used to evaluate their anti-inflammatory activity. Results and Conclusion: Bioassays indicated that most of the compounds markedly inhibited the expression of TNF-α at the concentration of 10 μg/mL. Compounds 7b and 11c, two of the most potent compounds, exhibited TNF-α inhibitory ability in a dose-dependent manner with IC₅₀ values of 5.56 and 3.69 μM, respectively. In vivo, intraperitoneal administration with 7b and 11c markedly inhibited the ear edema at the doses of 20 and 50 mg/kg. Compound 11c, inhibited edema by 49.59 % at the dose of 20 mg/kg, was comparable to the reference drug dexamethasone at the same dose (with an inhibition of 50.49 %). To understand the binding pattern, molecular docking of representa-tive 7b and 11c was performed, which demonstrated that both compounds have a forceful binding with the TNF-α, and that the phenyl and hydrazide moieties of them play a significant role in binding with the target site.

Full text of DOI:10.2174/1570180816666190731113441

502
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Letters in Drug Design & Discovery, 2020, 17, 502-511
RESEARCH ARTICLE
ISSN: 1570-1808
eISSN: 1875-628X
New Hydrazone Derivatives of Pyrazole-4-carboxaldehydes Exhibited
Anti-inflammatory Properties
Impact
Factor:
0.953
Mingxia Song1, #, Bing Liu2, #, Shengwang Yu1, Shihui He1, Yuqiu Liang1, Sifan Li1, Qiuyan Chen1
and Xianqing Deng1,*
2
1Medical College, Jinggangshan University, No 28, Xueyuan Road, Ji'an, 343009, Jiangxi, China; Affiliated Hospital
of Jinggangshan University, No 110, Jinggangshan Avenue, Ji'an, Jiangxi, 343000, China
Abstract: Background: Several series of hydrazone derivatives of pyrazole-4-carboxaldehydes (4-
11) were designed and synthesized to screen their inflammatory activity.
Methods: The products were characterized by 1H NMR, 13C NMR and HRMS. In vitro LPS-induced
TNF-α model and in vivo xylene-induced ear-edema model were used to evaluate their anti-
inflammatory activity.
A R T I C L E H I S T O R Y
Received: April 18, 2019
Revised: May 31, 2019
Accepted: June 24, 2019
Results and Conclusion: Bioassays indicated that most of the compounds markedly inhibited the
expression of TNF-α at the concentration of 10 μg/mL. Compounds 7b and 11c, two of the most
potent compounds, exhibited TNF-α inhibitory ability in a dose-dependent manner with IC50 values
of 5.56 and 3.69 μM, respectively. In vivo, intraperitoneal administration with 7b and 11c markedly
inhibited the ear edema at the doses of 20 and 50 mg/kg. Compound 11c, inhibited edema by 49.59 %
at the dose of 20 mg/kg, was comparable to the reference drug dexamethasone at the same dose (with
an inhibition of 50.49 %). To understand the binding pattern, molecular docking of representative
7b and 11c was performed, which demonstrated that both compounds have a forceful binding with
the TNF-α, and that the phenyl and hydrazide moieties of them play a significant role in binding
with the target site.
DOI:
10.2174/1570180816666190731113441
ꢀ
Keywords: Anti-inflammatory, hydrazine, pyrazole, TNF-α, docking, antiinflammatory activity.
1. INTRODUCTION
approval of celecoxib as an anti-inflammatory agent in the
1990s, many medicinal chemists have focused on the design
and synthesis of pyrazole derivatives as anti-inflammatory
drugs [10-13]. Hydrazones are a class of organic compounds
in the Schiff base family, which have also been extensively
investigated for their uses in medicinal chemistry. Hydra-
zone and their derivatives are known to exhibit interesting
diverse biological activities like antioxidant [14], anti-
inflammatory [15-17], anticonvulsant [18, 19], antimicrobial
[20-22], and anticancer [23-25]. Mohamed et. al. described
some pyrazole-hydrazone derivatives, which showed potent
anti-inflammatory agents via inhibiting COX-2 or 5-LOX
enzymes [26-28]. These findings indicated that the combina-
tion of pyrazole and hydrazone moieties into one molecule is
an alternation strategy to obtain potent anti-inflammatory
agents.
Inflammation is part of the complex biological response
of body tissues in response to the damage resulting from
microbial pathogen infection, chemical stimuli, and physical
trauma [1]. Although acute inflammation is required as a
defense mechanism, persistent inflammation is harmful and
should be suppressed. Dysregulation of inflammation may
lead to various diseases such as arthritis, heart attacks, ather-
osclerosis, Alzheimer’s disease, diabetes and even cancer [2-
4]. Tumor Necrosis Factor alpha (TNF-α) has been well rec-
ognized as pro-inflammatory cytokine, which plays an im-
portant role in inflammatory-related diseases by amplifying
inflammatory signals via multiple pathways [5, 6]. Hence,
the inhibition of this cytokine provides a target for control-
ling and treating inflammatory diseases and attracts much
attention in current anti-inflammatory drug discovery [7-9].
In our previous work, a skeleton of pyrazole-hydrazone was
established to evaluate their anti-inflammatory and antimi-
crobial activities [29]. Most of these compounds showed
excellent TNF-α inhibitory activity, and some of them were
shown to have comparable in vivo anti-inflammatory activity
to the reference drug dexamethasone. As a continuation of
the previous work, in this study, twenty-four muti-substituted
Pyrazoles are significant double nitrogen, five-
membered, heterocyclic compounds. Since the discovery and
*Address correspondence to this author at the Medical College, Jinggang-
shan University, No 28, Xueyuan Road, Ji'an, Jiangxi, 343009, China;
E-mail: dengxianqing1121@126.com
#These authors contributed equally to this work.
1875-628X/20 $65.00+.00
©2020 Bentham Science Publishers

Pyrazole Hydrazones as Anti-inflammatory Agents
Letters in Drug Design & Discovery, 2020, Vol. 17, No. 4 503
ꢀ
Scheme 1. The synthesis route of intermediates 3a-3d.
Cl
H
N
R3
N
N
N
O
4a-4c
a
Cl
CHO
H
Cl
N
R3
a
H
N
a
R1
N
R3
Cl
N
N
N
O
N
N
N
N
R2
O
5a-5c
7a-7c
a
4a, 5a, 6a, 7a: R3 = H
4b, 5b, 6b, 7b: R3 = CH3
4c, 5c, 6c, 7c: R3 = phenyl
Cl
H
N
R3
N
N
N
O
Conditions: Hydrazides, C2H5OH, CH3COOH
6a-6c
ꢀ
Scheme 2. The synthesis of target compounds 4a-c, 5a-c, 6a-c, and 7a-c.
pyrazole-hydrazone derivatives were synthesized and
screened for their anti-inflammatory properties.
Compounds 8a-8c, 9a-9c, 10a-10c, and 11a-11c were ob-
tained using the same conditions via reacting compounds 3a-
3d with N-phenylhydrazinecarboxamides (Scheme 3). The
structures of the desired products were confirmed by 1H
NMR, 13C NMR, and mass spectrometry.
2. CHEMISTRY
According to the designed structures, twenty-four pyra-
zole-hydrazone derivatives were divided into eight series,
which were prepared from the intermediates 3. The interme-
diates (3a-3d) were prepared according to Scheme 1. The
preparation of 1,3-substituted pyrazole-2-one (2a-2d) was
carried out at 90 °C by reacting the ethyl acetoacetate (or
ethyl benzoacetate) with methylhydrazine (or phenylhydra-
zine) in alcohol. Under Vilsmeier-Haack (DMF-POCl3) con-
ditions, compounds 2a-2d were transformed into the corre-
sponding 5-chloro-4-carboxaldehyde functionalized pyra-
zoles (3a-3d). The intermediates (3a-3d) reacted with formyl
hydrazide, acetylhydrazide or benzoyl hydrazide in alcohol
at room temperature in the presence of acetic acid to produce
compounds 4a-4c, 5a-5c, 6a-6c, and 7a-7c (Scheme 2).
Compound 4c was used as an example of the structure
1
confirmation. In the H-NMR spectrum, two singlets, due to
the CH3 on the pyrazole ring, was observed at 2.40 and 3.77
ppm. The aromatic protons of the benzene ring revealed
peaks in the 7.50-7.92 ppm range. The absorption peak of C-
H in imine was found at 8.38 ppm. The absorption peak of
NH in the amide was observed at 11.65 ppm as a singlet. The
absorption peak in the hydrogen spectrum was in complete
conformity with the hydrogen signal in the structure. The 13C
NMR spectra also gave accurate information about the struc-
ture of compound 4c, which has 11 types of carbons in dif-
ferent chemical environments. Moreover, the high-resolution
mass spectrometry of 4c displayed an [M + H]+ signal at m/z

504 Letters in Drug Design & Discovery, 2020, Vol. 17, No. 4
Song et al.
R3
N
O
N
N
N
H
N
H
Cl
8a-8c
CHO
R3
R3
N
N
N
O
O
N
R1
Cl
N
N
N
H
N
H
N
H
N
H
N
N
Cl
Cl
R2
9a-9c
11a-11c
8a, 9a, 10a, 11a: R3 = H
8b, 9b, 10b, 11b: R3 = Cl
8c, 9c, 10c, 11c: R3 = CH3
R3
N
O
N
N
Conditions: N-phenylhydrazinecarboxamides,
N
H
N
H
C2H5OH, CH3COOH
Cl
10a-10c
ꢀ
Scheme 3. The synthesis of target compounds 8a-c, 9a-c, 10a-c, and 11a-c.
277.0848, corresponding to its molecular weight of
277.0851.
this study. The positive drug DXMS exhibited an inhibition
rate of 63.89% at the same concentration (10 μg/mL).
Some Structure-Activity Relationships (SAR) were ob-
tained in comparison to the activities of the compounds with
different substituents. The series of compounds 4a-4c and
8a-8c, with the methyl group in the 1- and 3- positions of
pyrazole ring, showed relatively weaker anti-inflammatory
activity than the other series of compounds. In the series of
4-7, the activity order is 4<5<6<7, which suggested that the
phenyl group is more beneficial than methyl group in the 1-
and 3- positions. The substituents on the hydrazide in com-
pounds 4-7 are hydrogen (4a, 5a, 6a, 7a), methyl (4b, 5b,
6b, 7b), and phenyl (4c, 5c, 6c, 7c), respectively. There
seems to be no regularity for the effects of these substituents
on the cytokine-inhibitory ability, though the formyl hydra-
zine seems to be favorable to the anti-inflammatory activity,
which gives four compounds (4a, 5a, 6a, 7a) with relatively
higher cytokine-inhibitory ability. In the series of 8-11, the
activity order is 8<9≈10≈11. In these series, compounds 8c,
9c, 10c, 11c containing methyl on phenyl ring, exhibited
better inhibitory ability compared to the others.
3. RESULTS AND DISCUSSION
3.1. Anti-inflammatory Activity
3.1.1. In Vitro Inhibition LPS-induced TNF-a Release
Lipopolysaccharide (LPS), an endotoxin of gram-
negative bacterial cell walls, is thought to be a major risk
factor in initiating the inflammatory processes by stimulating
the release of inflammatory cytokines, such as tumor necro-
sis factor (TNF-α) [30]. The LPS-induced TNF-α release
model was considered to be an effective screening method
for new anti-inflammatory agents in vitro [31]. In the current
study, compounds 4-11 were evaluated for their anti-
inflammatory activity based on their ability to inhibit TNF-α
release in LPS-stimulated RAW264.7 mouse macrophages.
Dexamethasone was used as the standard, which was a corti-
costeroid that prevents the release of substances in the body
that cause inflammation such as TNF-α [32, 33]. The cells
were seeded and pretreated with the test compounds or dex-
3.1.2. Compound 7b and 11c Inhibit TNF-α Release in a
Dose-dependent Manner
amethasone (DXMS) at a concentration 10 μg/mL for 4 h
before treatment with LPS (1 μg/mL) for 24 h at 37 °C. Cell-
free supernatants were collected and analyzed for levels of
TNF-α using an ELISA kit. The screen results of target
compounds and positive control DXMS are shown in Fig. (1).
Among the active compounds discussed above, com-
pounds 7b and 11c exhibited the highest cytokine-inhibitory
ability. Thus, they were chosen for further evaluation of their
dose-dependent inhibitory effects against LPS-induced TNF-
α release. RAW 264.7 macrophages were pre-treated with 7b
and 11c in a series of concentrations (30, 10, 3.3, 1.1 and
0.37 μM) for 4 h and were subsequently incubated with LPS
(1 μg/mL) for 24 h. As shown in Fig. (2), pretreatment with
7b and 11c both exhibited a dose-dependent inhibition on
TNF-α release induced by LPS. Accordingly, the IC50 values
were calculated and shown in Fig. (2). Compound 7b and
As shown in Fig. (1), expect compounds 4b and 8a, all of
the tested compounds significantly inhibited LPS-induced
TNF-α generation in vitro. Among these compounds, 6a, 6c,
7a-7c, 9-9c, 10a-10c, and 11a-11c exhibited strong inhibi-
tion with inhibitory rates above 50%. Compounds 7b, and
11c, showing 67.18% and 70.73% TNF-α inhibitory activity,
respectively, were two of the most promising compounds in

Pyrazole Hydrazones as Anti-inflammatory Agents
Letters in Drug Design & Discovery, 2020, Vol. 17, No. 4 505
ꢀ
Fig. (1). Anti-inflammatory activity of the compounds 4-11 in the LPS-induced TNF-α release test in RAW264.7 macrophage. Cells
were pre-treated with test compounds or dexamethasone (DXMS) at 10 μg/mL for 4 h, followed by LPS (1 μg/mL) treatment for 24 h. TNF-
α levels in the culture medium were measured by ELISA. The results are expressed as the level of TNF-α. Each bar represents mean SEM of
three parallel experiments. Values are significant at *p < 0.05, **p < 0.01, ***p < 0.001 as compared to the LPS group (all comparisons were made
by ANOVA followed by Dunnett’s test).
ꢀ
Fig. (2). Compounds 7b and 11c inhibited LPS-induced TNF-α release in a dose-dependent manner in RAW 264.7 macrophages.
Cells were pre-treated with test compounds at designated concentrations for 4 h, followed by LPS (1 μg/mL) treatment for 24 h. TNF-α levels in
the culture medium were measured by ELISA. The results are expressed as the level of TNF-α. Each bar represents mean SEM of three paral-
lel experiments. Values are significant at *p < 0.05, **p < 0.01, ***p < 0.001 as compared to the LPS group (all comparisons were made by
ANOVA followed by Dunnett’s test).
11c showed the IC50 values of 5.56 and 3.69 μM, respective-
ly. The dose-dependent inhibition by 7b and 11c further sug-
gested their potential as anti-inflammatory agents.
(DMSO) was used as the vehicle, and DXMS was used as
positive control. Anti-inflammatory activity was defined by
the ability of each compound to prevent edema. As shown in
Fig. (3), both tested compounds and DXMS inhibited the ear
edema significantly in a dose-dependent manner. At the dose
of 20 mg/kg, compound 11c inhibited edema by 49.59%,
which was comparable to DXMS at inhibiting xylene-
induced ear edema (50.49%). The in vivo activities of the
compound 11c was superior to that of 7b, which was in line
with the previous in vitro data.
3.1.3. In vivo Inhibition of Xylene-induced Ear Edema
Xylene-induced edema leads to an acute inflammatory
response and is known to cause severe vasodilation and
edematous skin changes, partially associated with phospho-
lipase A2 (PLA2) [34, 35]. Xylene treatment in mice in-
creases the release of inflammatory mediators, such as his-
tamine, serotonin, bradykinin, and TNF-α. Based on the in
vitro inhibitory effects of our test compounds toward TNF-α
expression, the in vivo anti-inflammatory activity of com-
pounds 7b and 11c were evaluated using the xylene-induced
ear edema mouse model. In this test, dimethyl sulfoxide
3.1.4. In silico Docking Studies
In the above data, the in vivo and in vitro anti-
inflammatory ability of the target compounds have been de-
scribed. To further confirm whether the anti-inflammatory

506 Letters in Drug Design & Discovery, 2020, Vol. 17, No. 4
Song et al.
activity of the synthesized compounds mediates directly
through TNF-α, and to assess their interactions with the tar-
get protein, the molecular docking of representative 7b and
11c with the TNF-α protein was performed by Discovery
Studio 2019 version [36].
π-π, π-alkyl, and π-charge interaction with VAL91, TYR87,
and ARG82. In addition to the above, the phenyl group in
the 1- position and chlorine atom in 5- position also showed
π-alkyl interactions with LEU93 and PHE124, respectively.
As seen in Fig. (4) (right), LYS98, TYR119, LEU55,
LEU197, PRO12, LYS11, and ALA156 were the critical
residues in the binding mode of compound 11c. The NH
group of compound 11c, as an H-bond donor, was involved
in an interaction with carbonyl group of LEU55 and
LEU197. The phenyl group in the 1- position was responsi-
ble for π-alkyl interaction with LYS98, while the phenyl
group of N-phenylhydrazinecarboxamide moiety and the
methyl on it exhibited π-charge and π-alkyl hydrophobic
binding with LYS98, TYR87, and ARG82. These docking
results indicated that phenyl group in 1- and 3- position of
pyrazole ring and hydrazide moiety in the side chain may
play a significant role in binding to the target site of TNF -α.
4. EXPERIMENTAL PROTOCOLS
4.1. Chemistry
Melting points were determined in open capillary-tubes
ꢀ
1
and were uncorrected. H NMR and 13C NMR spectra were
Fig. (3). Anti-inflammatory activity of the compounds 7b and 11c
in the xylene-induced ear-edema test in mice. The test compounds
and DXMS were administered i.p. at dose of 20 and 50 mg/kg, and
edema was quantified after 30 min. **p<0.01 and ***p<0.001 com-
pared with the control group (all comparisons were made by ANOVA
followed by Dunnett’s test).
recorded using an AV-300 spectrometer (Bruker, Switzer-
land), and all chemical shifts were given in parts per million
relative to tetramethylsilane. High-resolution mass spectra
were measured on an MALDI-TOF/TOF mass spectrometer
(Bruker Daltonik, Germany). All the reagents and solvents
were purchased from Aladdin (Shanghai, China) or Si-
nopharm Chemical Reagent Co. Ltd. (Shanghai, China), and
were used as received.
Interactions of the compounds 7b and 11c with amino
acids TNF-α protein were illustrated and showed in Fig. (4).
As seen in Fig. (4) (left), the most important residues in the
binding mode of compound 7b are GLN125, VAL91,
TYR87, ARG82, LEU93, PHE124 and LEU93. The hydra-
zide group of compound 7b, as an H-bond donor, was in-
volved in an interaction with the carbonyl group of GLN125,
while the phenyl group in the 3- position was responsible for
4.1.1. General Procedure for the Preparation of Com-
pounds 2a-2d
To a cooled (ice-bath) and stirred solution of methyl hy-
drazine (1.84 g, 0.04mol), the solution of ethyl acetoacetate
(1.30 g, 0.01 mol) or ethyl benzoacetate (1.92 g, 0.01 mol)
with 30 mL of absolute alcohol was added dropwise. Then,
the mixture was refluxed and monitored by TLC (developing
ARG82
TYR87
GLN125
LYS98
PHE124
PRO12
LYS11
VAL91
LEU93
ALA156
LEU55
LEU83
LEU157
PHE124
TYR199
VAL91
ꢀ
Fig. (4). Images of compounds 7b and 11c in the TNF-α active site (PDB: 2AZ5).

Pyrazole Hydrazones as Anti-inflammatory Agents
Letters in Drug Design & Discovery, 2020, Vol. 17, No. 4 507
solvent: CH2Cl2/CH3OH = 30/1). After completion of the
reaction, the mixture was evaporated under reduced pressure
to obtain products 2a and 2c.
= 9.0 Hz, NH). 13C-NMR (DMSO-d6, 75MHz): δ 164.88,
146.58, 137.91, 127.56, 111.14, 36.31, 14.46. ESI-HRMS
calcd for C7H10ClN4O+ ([M + H]+): 201.0538; found:
201.0544.
To a cooled (ice-bath) and stirred solution of phenylhy-
drazine (4.32 g, 0.04 mol), the solution of ethyl acetoacetate
(1.30 g, 0.01 mol) or ethyl benzoacetate (1.92 g, 0.01 mol)
with 30 mL of absolute alcohol was added dropwise. Then,
the mixture was refluxed and monitored by TLC (developing
solvent: CH2Cl2/CH3OH = 30/1). After completion of the
reaction, the solid formed was filtrated and washed with
small amount of cooled alcohol to give products 2b and 2d.
4.1.5.2. N'-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)meth-
ylene)acetohydrazide (4b)
1
Mp 206-207ºC, yield 62%. H-NMR (CDCl3, 300MHz):
δ 2.32 (s, 3H, CH3), 2.42 (s, 3H, COCH3), 3.80 (s, 3H,
NCH3), 7.65 (s, 1H, CH=N), 9.25 (s, 1H, NH). 13C-NMR
(CDCl3, 75MHz): δ 173.23, 147.68, 135.41, 128.21, 111.16,
36.01, 20.46, 14.56. ESI-HRMS calcd for C8H12ClN4O+
([M + H]+): 215.0694; found: 215.0685.
4.1.2. General Procedure for the Preparation of Com-
pounds 3a-3d
4.1.5.3. N'-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)methy-
lene)benzohydrazide (4c)
To a cold, stirred solution of dimethylformamide (3.65 g,
0.05 mol), phosphorus oxychloride (23 g, 0.15 mol) was
added dropwise. Then, compound 2 (0.05 mol) was added in
Mp 179-181ºC, yield 56%. 1H-NMR (DMSO-d6,
300MHz): δ 2.40 (s, 3H, CH3), 3.77 (s, 3H, NCH3), 7.50-
7.92 (m, 5H, Ph-H), 8.38 (s, 1H, CH=N), 11.65 (s, 1H, NH).
13C-NMR (DMSO-d6, 75MHz): δ 163.05, 146.97, 140.75,
134.05, 132.05, 128.90, 127.95, 127.89, 111.62, 36.37,
14.57. ESI-HRMS calcd for C13H14ClN4O+ ([M + H]+):
277.0851; found: 277.0848.
o
batches. The mixture was stirred at 90 C and monitored by
TLC (developing solvent: CH2Cl2/CH3OH = 30/1). After
completion of the reaction, the mixture was cooled and
poured into ice-cold water followed by extracting with
CH2Cl2, which was dried to afford compounds 3a-3d.
4.1.5.4. N'-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)formohydrazide (5a)
4.1.3. General Procedure for the Preparation of Com-
pounds 4a-4c, 5a-5c, 6a-6c, and 7a-7c
1
Mp 165-167 ºC, yield 51%. H-NMR (CDCl3, 300MHz):
To a solution of compound 3a (1.59 g, 0.01 mol) in 20
mL of ethanol, an equimolar quantity of formyl hydrazide or
acetylhydrazide or benzoyl hydrazide was added. Then, 0.5
mL of acetic acid was added. The mixture was stirred at
room temperature and accompanied with TLC monitoring
(developing solvent: CH2Cl2/CH3OH = 30/1). After comple-
tion of the reaction, the solid formed was collected by filtra-
tion to give crude product, which was recrystallized from
dichloromethane/ethanol (1:2) to afford the purified products
(4a-4c). The compounds 5a-5c, 6a-6c, and 7a-7c were pre-
pared using the same procedures with the compounds 3b, 3c,
and 3d as the reactants, respectively.
δ 2.53 (s, 3H, CH3), 7.43-7.56 (m, 5H, Ph-H), 7.84 (s, 1H,
CH=N), 8.78 (d, 1H, J = 7.8 Hz, CHO), 9.64 (d, 1H, J = 7.8
Hz, NH). 13C-NMR (CDCl3, 75MHz): δ 165.06, 149.28,
138.15, 137.62, 129.17, 128.61, 128.17, 124.92, 112.57,
14.73. ESI-HRMS calcd for C12H12ClN4O+ ([M + H]+):
263.0694; found: 263.0701.
4.1.5.5. N'-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)acetohydrazide (5b)
1
Mp 223-225 ºC, yield 57%. H-NMR (CDCl3, 300MHz):
δ 2.36 (s, 3H, COCH3), 2.53 (s, 3H, CH3), 7.43-7.56 (m, 5H,
Ph-H), 7.79 (s, 1H, CH=N), 9.70 (s, 1H, NH). 13C-NMR
(CDCl3, 75MHz): δ 173.57, 149.14, 137.69, 135.50, 129.15,
128.53, 127.79, 124.91, 113.06, 20.50, 14.83. ESI-HRMS calcd
for C13H14ClN4O+ ([M + H]+): 277.0851; found: 277.0854.
4.1.4. General Procedure for the Preparation of Com-
pounds 8a-8c, 9a-9c, 10a-10c, and 11a-11c
To a solution of compound 3a (1.59 g, 0.01 mol) in 20
mL of ethanol, an equimolar quantity of N-
phenylhydrazinecarboxamides was added. Then, 0.5 mL of
acetic acid was added. The mixture was stirred at room tem-
perature and accompanied by TLC monitoring (developing
solvent: CH2Cl2/CH3OH = 30/1). After completion of the
reaction, the solid formed was collected by filtration to give
crude product, which was recrystallized from dichloro-
methane/ethanol (1:2) to afford the purified products (8a-8c).
The compounds 9a-9c, 10a-10c, and 11a-11c were prepared
using the same procedures with the compounds 3b, 3c, and
3d as the reactants, respectively.
4.1.5.6. N'-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)benzohydrazide (5c)
Mp 169-171ºC, yield 54%. 1H-NMR (DMSO-d6,
300MHz): δ 2.51 (s, 3H, CH3), 7.49-7.94 (m, 10H, Ph-H),
8.49 (s, 1H, CH=N), 11.78 (s, 1H, NH). 13C-NMR (DMSO-
d6, 75MHz): δ 163.16, 148.87, 140.39, 137.75, 133.98,
132.14, 129.77, 129.14, 128.94, 128.00, 127.74, 125.41,
113.69, 14.86. ESI-HRMS calcd for C18H16ClN4O+ ([M +
H]+): 339.1007; found: 339.1012.
4.1.5.7. N'-((5-Chloro-1-methyl-3-phenyl-1H-pyrazol-4-yl)
methylene)formohydrazide (6a)
1
Mp 160-161 ºC, yield 40%. H-NMR (CDCl3, 300MHz):
4.1.5. Characterization for the Target Compounds
δ 3.94 (s, 3H, NCH3), 7.42-7.59 (m, 5H, Ph-H), 7.77 (s, 1H,
CH=N), 8.68 (d, 1H, J = 7.8 Hz, CHO), 9.21 (d, 1H, J = 7.8
Hz, NH). 13C-NMR (CDCl3, 75MHz): δ 164.77, 151.08,
137.71, 131.94, 128.84, 128.59, 128.57, 128.05, 110.03,
36.59. ESI-HRMS calcd for C12H12ClN4O+ ([M + H]+):
263.0694; found: 263.0699.
4.1.5.1. N'-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)methy-
lene)formohydrazide (4a)
Mp 177-178 ºC, yield 60%. 1H-NMR (DMSO-d6,
300MHz): δ 2.30 (s, 3H, CH3), 3.73 (s, 3H, NCH3), 7.91 (s,
1H, CH=N), 8.59 (d, 1H, J = 9.0 Hz, CHO), 11.48 (d, 1H, J

508 Letters in Drug Design & Discovery, 2020, Vol. 17, No. 4
Song et al.
4.1.5.8. N'-((5-Chloro-1-methyl-3-phenyl-1H-pyrazol-4-yl)
methylene)acetohydrazide (6b)
4.1.5.14. 2-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)meth-
ylene)-N-(4-chlorophenyl)hydrazine- carboxamide (8b)
Mp 144-147ºC, yield 56%. 1H-NMR (DMSO-d6,
300MHz): δ 2.05 (s, 3H, COCH3), 3.89 (s, 3H, NCH3), 7.44-
7.63 (m, 5H, Ph-H), 8.16 (s, 1H, CH=N), 11.05 (d, 1H, J =
7.8 Hz, NH). 13C-NMR (CDCl3, 75MHz): δ 171.62, 149.18,
138.17, 134.46, 132.19, 128.40, 128.33, 126.87, 110.59,
36.50, 20.18. ESI-HRMS calcd for C13H14ClN4O+ ([M +
H]+): 277.0851; found: 277.0844.
Mp 223-224ºC, yield 78%. 1H-NMR (DMSO-d6,
300MHz): δ 2.40 (s, 3H, CH3), 3.75 (s, 3H, NCH3), 7.34 (d,
2H, J = 9.0 Hz, Ph-H), 7.60 (d, 2H, J = 9.0 Hz, Ph-H), 7.86
(s, 1H, CH=N), 8.50 (s, 1H, NH), 10.67 (s, 1H, NH). 13C-
NMR (DMSO-d6, 75MHz): δ 153.13, 146.74, 138.39,
134.13, 128.94, 127.20, 126.44, 121.05, 111.43, 36.31,
14.62. ESI-HRMS calcd for C13H14Cl2N5O+ ([M + H]+):
326.0570; found: 326.0568.
4.1.5.9. N'-((5-Chloro-1-methyl-3-phenyl-1H-pyrazol-4-yl)
methylene)benzohydrazide (6c)
4.1.5.15. 2-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)meth-
ylene)-N-(p-tolyl)hydrazinecarbox- amide (8c)
Mp 214-216ºC, yield 43%. 1H-NMR (DMSO-d6,
300MHz): δ 3.92 (s, 3H, NCH3), 7.42-7.91 (m, 10H, Ph-H),
8.49 (s, 1H, CH=N), 11.69 (s, 1H, NH). 13C-NMR (DMSO-
d6, 75MHz): δ 163.15, 150.20, 140.65, 134.01, 132.43,
132.09, 128.98, 128.95, 128.89, 128.81, 128.00, 127.51,
111.05, 37.04. ESI-HRMS calcd for C18H16ClN4O+ ([M +
H]+): 339.1007; found: 339.1010.
1
Mp 213-215ºC, yield 69%. H-NMR (CDCl3, 300MHz):
δ 2.32 (s, 3H, Ph-CH3), 2.46 (s, 3H, CH3), 3.81 (s, 3H,
NCH3), 7.13 (d, 2H, J = 6.3 Hz, Ph-H), 7.40 (d, 2H, J = 6.3
Hz, Ph-H), 7.69 (s, 1H, CH=N), 7.99 (s, 1H, NH), 8.95 (s,
1H, NH). 13C-NMR (CDCl3, 75MHz): δ 153.48, 147.20,
135.43, 133.53, 132.94, 129.54, 127.78, 119.36, 111.18,
36.06, 20.82, 14.48. ESI-HRMS calcd for C14H17ClN5O+
([M + H]+): 306.1116; found: 306.1112.
4.1.5.10. N'-((5-Chloro-1,3-diphenyl-1H-pyrazol-4-yl)meth-
ylene)formohydrazide (7a)
4.1.5.16.
2-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-
Mp 171-172ºC, yield 84%. 1H-NMR (DMSO-d6,
300MHz): δ 7.47-7.72 (m, 10H, Ph-H), 8.13 (s, 1H,
CH=N), 8.58 (d, 1H, J = 7.5 Hz, CHO), 11.62 (d, 1H, J =
7.5 Hz, NH). 13C-NMR (DMSO-d6, 75MHz): δ 164.90,
151.49, 137.75, 137.41, 132.01, 129.83, 129.60, 129.36,
129.00, 128.97, 127.16, 125.87, 112.31. ESI-HRMS calcd
yl)methylene)-N-phenylhydrazinecarbox -amide (9a)
1
Mp 193-194ºC, yield 77%. H-NMR (CDCl3, 300MHz):
δ 2.58 (s, 3H, CH3), 7.09-7.57 (m, 10H, Ph-H), 7.80 (s, 1H,
CH=N), 8.09 (s, 1H, NH), 8.94 (s, 1H, NH). 13C-NMR
(CDCl3, 75MHz): δ 153.26, 148.63, 137.96, 137.65, 133.53,
129.18, 129.10, 128.63, 127.48, 124.96, 123.47, 119.24,
112.91, 14.78. ESI-HRMS calcd for C18H17ClN5O+ ([M +
H]+): 354.1116; found: 354.1122.
for C17H14ClN4O+ ([M
325.0849.
+
H]+): 325.0851; found:
4.1.5.11. N'-((5-Chloro-1,3-diphenyl-1H-pyrazol-4-yl)met-
hylene)acetohydrazide (7b)
Mp 184-186ºC, yield 81%. 1H-NMR (DMSO-d6,
300MHz): δ 2.05 (s, 3H, COCH3), 7.47-7.72 (m, 10H, Ph-
H), 8.07 (s, 1H, CH=N), 11.15 (s, 1H, NH). 13C-NMR
4.1.5.17.
2-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-
yl)methylene)-N-(4-chlorophenyl) hydrazinecarboxamide
(9b)
1
Mp 220-221 ºC, yield 65%. H-NMR (CDCl3, 300MHz):
(DMSO-d6, 75MHz):
δ
172.18, 151.21, 137.78,
δ 2.57 (s, 3H, CH3), 7.28-7.55 (m, 9H, Ph-H), 7.79 (s, 1H,
CH=N), 8.08 (s, 1H, NH), 8.84 (s, 1H, NH). 13C-NMR
(CDCl3, 75MHz): δ 153.00, 148.59, 137.59, 136.58, 133.82,
129.20, 129.07, 128.68, 128.40, 127.56, 124.94, 120.37,
112.75, 14.77. ESI-HRMS calcd for C18H16Cl2N5O+ ([M +
H]+): 388.0726; found: 388.0725.
134.54, 132.25, 129.82, 129.55, 129.24, 129.06, 128.85,
127.28, 125.83, 112.75, 20.65. ESI-HRMS calcd
for C18H16ClN4O+ ([M
339.1011.
+
H]+): 339.1007; found:
4.1.5.12. N'-((5-Chloro-1,3-diphenyl-1H-pyrazol-4-yl)meth-
ylene)benzohydrazide (7c)
4.1.5.18.
2-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-
yl)methylene)-N-(p-tolyl)hydrazine- carboxamide (9c)
Mp 230-231ºC, yield 80 %. 1H-NMR (DMSO-d6,
300MHz): δ 7.48-7.93 (m, 15H, Ph-H), 8.59 (s, 1H, CH=N),
11.80 (s, 1H, NH). 13C-NMR (DMSO-d6, 75MHz): δ 163.22,
151.71, 140.30, 137.81, 133.91, 132.18, 132.04, 129.84,
129.60, 129.33, 129.08, 129.04, 128.92, 128.04, 127.47,
125.92, 112.77. ESI-HRMS calcd for C23H18ClN4O+ ([M +
H]+): 401.1164; found: 401.1152.
1
Mp 221-222ºC, yield 73%. H-NMR (CDCl3, 300MHz):
δ 2.32 (s, 3H, Ph-CH3), 2.58 (s, 3H, CH3), 7.14 (d, 2H, J =
6.0 Hz, Ph-H), 7.41 (d, 2H, J = 6.0 Hz, Ph-H), 7.49-7.58 (m,
5H, Ph-H), 7.78 (s, 1H, CH=N), 8.00 (s, 1H, NH), 8.83 (s,
1H, NH). 13C-NMR (CDCl3, 75MHz): δ 153.28, 148.63,
137.66, 135.35, 133.31, 133.05, 129.58, 129.18, 128.61,
127.42, 124.96, 119.39, 112.94, 20.82, 14.78. ESI-HRMS
calcd for C19H19ClN5O+ ([M + H]+): 368.1273; found:
368.1279.
4.1.5.13. 2-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)meth-
ylene)-N-phenylhydrazinecarboxamide (8a)
1
Mp 204-205ºC, yield 71%. H-NMR (CDCl3, 300MHz):
4.1.5.19.
2-((5-Chloro-1-methyl-3-phenyl-1H-pyrazol-4-
δ 2.47 (s, 3H, CH3), 3.82 (s, 3H, NCH3), 7.06-7.53 (m, 5H,
Ph-H), 7.69 (s, 1H, CH=N), 8.07 (s, 1H, NH), 8.79 (s, 1H,
NH). 13C-NMR (CDCl3, 75MHz): δ 153.27, 147.21, 138.01,
133.62, 129.07, 127.84, 123.39, 119.21, 111.11, 36.08,
14.49. ESI-HRMS calcd for C13H15ClN5O+ ([M + H]+):
292.0960; found: 292.0963.
yl)methylene)-N-phenylhydrazine- carboxamide (10a)
1
Mp 221-222ºC, yield 69%. H-NMR (CDCl3, 300MHz):
δ 3.95 (s, 3H, NCH3), 7.06-7.63 (m, 10H, Ph-H), 7.76 (s, 1H,
CH=N), 7.85 (s, 1H, NH), 9.11 (s, 1H, NH). 13C-NMR
(CDCl3, 75MHz): δ 153.42, 150.46, 138.04, 132.75, 132.64,

Pyrazole Hydrazones as Anti-inflammatory Agents
Letters in Drug Design & Discovery, 2020, Vol. 17, No. 4 509
128.91, 128.76, 128.71, 128.56, 127.86, 123.21, 119.20,
110.63, 36.56. ESI-HRMS calcd for C18H17ClN5O+ ([M +
H]+): 354.1116; found: 354.1120.
5. PHARMACOLOGY
5.1. Cell Line and Reagents
4.1.5.20.
2-((5-Chloro-1-methyl-3-phenyl-1H-pyrazol-4-
Mouse RAW264.7 macrophage cell line was obtained
from the China Cell Bank (Beijing, China). Fetal bovine
serum was purchased from Biological Industries (Kibbutz
Beit-Haemek, Israel). LPS was purchased from Sigma (St.
Louis, MO, USA), and was dissolved in PBS. DXMS and
compounds were dissolved in DMSO for in vitro experi-
ments.
yl)methylene)-N-(4-chlorophenyl)- hydrazinecarboxamide
(10b)
Mp 237-238ºC, yield72%. 1H-NMR (DMSO-d6,
300MHz): δ 3.91 (s, 3H, NCH3), 7.33-7.61 (m, 9H, Ph-H),
7.96 (s, 1H, CH=N), 8.16 (s, 1H, NH), 10.74 (s, 1H, NH).
13C-NMR (DMSO-d6, 75MHz): δ 152.87, 149.58, 138.07,
133.48, 132.83, 129.05, 129.03, 128.97, 128.81, 127.47,
126.54, 120.78, 110.90, 36.97. ESI-HRMS calcd for
5.2. Cell Treatment and ELISA Assay (TNF-α)
C18H16Cl2N5O+ ([M
388.0721.
+
H]+): 388.0726; found:
Mouse RAW264.7 macrophages were cultured in RPMI
1640 medium (Zhejiang Tianhang Biotechnology Co., Ltd.)
supplemented with 10% FBS, 100 U/mL penicillin, and 100
4.1.5.21.
2-((5-Chloro-1-methyl-3-phenyl-1H-pyrazol-4-
o
μg/mL streptomycin at 37 C with 5% CO2. Cells were pre-
yl)methylene)-N-(p-tolyl)hydrazine- carboxamide(10c)
treated with test compounds, DXMS or vehicle control for 4
h, then treated with LPS (1μg/ml) for 24 h. After treatment,
the supernatant was separated and used for the determination
of levels of TNF-α by ELISA using mouse TNF-α ELISA
Kits Biolegend (San diego, CA, USA).
Mp 222-223 ºC, yield 60 %. 1H-NMR (CDCl3, 300MHz):
δ 2.32 (s, 3H, Ph-CH3), 3.94 (s, 3H, NCH3), 7.09 (d, 2H, J =
6.3 Hz, Ph-H), 7.25 (d, 2H, J = 6.3 Hz, Ph-H), 7.44-7.64 (m,
5H, Ph-H), 7.75 (s, 1H, CH=N), 7.76 (s, 1H, NH), 9.09 (s,
1H, NH). 13C-NMR (CDCl3, 75MHz): δ 153.51, 150.41,
135.44, 132.71, 132.67, 132.56, 129.40, 128.71, 128.53,
127.84, 126.8, 119.34, 110.69, 36.55, 20.81. ESI-HRMS
calcd for C19H19ClN5O+ ([M + H]+): 368.1273; found:
368.1280.
5.3. Xylene-Induced Ear-Edema Model
Animal experiments were carried out on KunMing mice
(half male and female) weighing 20-26 g. The mice were
housed collectively in polycarbonate cages in groups of ten,
where they were maintained on a 12 h light/dark cycle in a
temperature-controlled (25 2 ºC) laboratory with free ac-
cess to food and water. Each animal was used only once.
Procedures involving animals and their care were conducted
in accordance with the Guide for the Care and Use of Labor-
atory Animals, 8th Edition, National Academies Press,
Washington, DC. Local ethical committee approval was also
obtained. In this screening, selected compounds were evalu-
ated for their anti-inflammatory activity via in vivo inhibition
assay of xylene-induced ear edema in mice [37]. Compounds
and DXMS were dissolved in DMSO and administered to
mice intraperitoneally at a dose of 50 mg/kg and 20 mg/kg
(0.05 mL/20 g body weight). Control group received vehicle
only. Thirty minutes after administration, mice were used in
the xylene-induced ear-edema test (20 μL xylene was daubed
by a micropipette on the surface of the right ear of each
mouse). After 30 min, mice were sacrificed, and a cylindrical
tissue plug (7 mm diameter) was excised from both ears of
mice. Edema was quantified by measuring the difference in
weight between the plugs from the right and left ears. Anti-
inflammatory activity was expressed as the percent reduction
in edema in comparison with the control group. DXMS was
tested in parallel as a reference drug.
4.1.5.22. 2-((5-Chloro-1,3-diphenyl-1H-pyrazol-4-yl)meth-
ylene)-N-phenylhydrazinecarboxamide (11a)
Mp 220-222ºC, yield 80%. 1H-NMR (DMSO-d6,
300MHz): δ 6.99-7.74 (m, 15H, Ph-H), 8.03 (s, 1H, CH=N),
8.05 (s, 1H, NH), 10.08 (s, 1H, NH). 13C-NMR (DMSO-d6,
75MHz): δ 152.89, 151.13, 138.97, 137.76, 132.71, 132.56,
129.85, 129.56, 129.36, 129.23, 129.07, 129.02, 127.34,
125.78, 123.00, 119.18, 112.74. ESI-HRMS calcd for
C23H19ClN5O+ ([M
416.1275.
+
H]+): 416.1273; found:
4.1.5.23. 2-((5-Chloro-1,3-diphenyl-1H-pyrazol-4-yl)meth-
ylene)-N-(4-chlorophenyl)hydrazine- carboxamide (11b)
Mp 240-241ºC, yield 75%. 1H-NMR (DMSO-d6,
300MHz): δ 7.32-7.73 (m, 14H, Ph-H), 8.04 (s, 1H, CH=N),
8.17 (s, 1H, NH), 10.85 (s, 1H, NH). 13C-NMR (DMSO-d6,
75MHz): δ 152.85, 151.17, 138.04, 137.75, 133.05, 132.49,
129.86, 129.59, 129.52, 129.40, 129.05, 128.95, 127.41,
126.60, 125.79, 120.81, 112.70. ESI-HRMS calcd for
C23H18Cl2N5O+ ([M
450.0889.
+
H]+): 450.0883; found:
4.1.5.24. 2-((5-Chloro-1,3-diphenyl-1H-pyrazol-4-yl)met-
hylene)-N-(p-tolyl)hydrazinecarbox- amide (11c)
5.4. In Silico Docking Studies
Mp 201-202ºC, yield 78 %. 1H-NMR (CDCl3, 300MHz):
δ 2.31 (s, 3H, Ph-CH3), 7.09 (d, 2H, J = 6.3 Hz, Ph-H), 7.24
(d, 2H, J = 6.3 Hz, Ph-H), 7.47-7.71 (m, 10H, Ph-H), 7.72 (s,
1H, CH=N), 7.83 (s, 1H, NH), 9.06 (s, 1H, NH). 13C-NMR
(CDCl3, 75MHz): δ 153.41, 151.60, 137.68, 135.37, 132.79,
132.48, 132.25, 129.41, 129.21, 129.03, 128.95, 128.91,
128.52, 127.63, 125.27, 119.36, 112.28, 20.81. ESI-HRMS
calcd for C24H21ClN5O+ ([M + H]+): 430.1429; found:
430.1431.
The docking studies were carried out using Discovery
Studio (release 2019) and the 3D crystal structure of 2AZ5
(Crystal Structure of TNF-alpha with a small molecule inhib-
itor) was downloaded from Protein Data Bank. The three-
dimensional structures of 7b and 11c were constructed using
Chem. 3D ultra 12.0 software [Chemical Structure Drawing
Standard; Cambridge Soft corporation, USA (2010)], then it
was energetically minimized by using MMFF94 with 5000

510 Letters in Drug Design & Discovery, 2020, Vol. 17, No. 4
Song et al.
iterations and minimum RMS gradient of 0.10. For protein
preparation, the hydrogen atoms were added, and the water
and ligands were removed. Types of interactions of the
docked protein with 7b and 11c were analyzed after the end
of molecular docking. Several docking patterns were ob-
tained and ranked and by DOCKER_INTERACTION_
ENERGY. The lowest energy conformation of ligand-
protein complex was analyzed.
REFERENCES
[1]
Ferrero-Miliani, L.; Nielsen, O.H.; Andersen, P.S.; Girardin, S.E.
Chronic inflammation: Importance of NOD2 and NALP3 in inter-
leukin-1beta generation. Clin. Exp. Immunol., 2007, 147(2), 227-
235.
[PMID: 17223962]
[2]
[3]
Tilg, H.; Moschen, A.R. Adipocytokines: Mediators linking adi-
pose tissue, inflammation and immunity. Nat. Rev. Immunol., 2006,
6(10), 772-783.
[http://dx.doi.org/10.1038/nri1937] [PMID: 16998510]
Philip, M.; Rowley, D.A.; Schreiber, H. Inflammation as a tumor
promoter in cancer induction. Semin. Cancer Biol., 2004, 14(6),
433-439.
[http://dx.doi.org/10.1016/j.semcancer.2004.06.006]
15489136]
Grivennikov, S.I.; Greten, F.R.; Karin, M. Immunity, inflamma-
tion, and cancer. Cell, 2010, 140(6), 883-899.
[http://dx.doi.org/10.1016/j.cell.2010.01.025] [PMID: 20303878]
Syggelos, S.A.; Giannopoulou, E.; Gouvousis, P.A.; Andonopou-
los, A.P.; Aletras, A.J.; Panagiotopoulos, E. In vitro effects of non-
steroidal anti-inflammatory drugs on cytokine, prostanoid and ma-
trix metalloproteinase production by interface membranes from
loose hip or knee endoprostheses. Osteoarthritis Cartilage, 2007,
15(5), 531-542.
CONCLUSION
In summary, several types of pyrazole-hydrazone deriva-
tives were synthesized and their anti-inflammatory activities
were evaluated. Majority of the synthetic compounds exhib-
ited significant inhibitory activities against LPS-induced
TNF-α expression in RAW 264.7 macrophages. Compounds
7b and 11c, two of most potent compounds, exhibited TNF-α
inhibitory ability in a dose-dependent manner with IC50 val-
ues in µM level. In vivo, intraperitoneal administration with
7b and 11c markedly inhibited the ear edema at the doses of
20 and 50 mg/kg. To understand the binding pattern, mo-
lecular docking of representative 7b and 11c was performed,
which demonstrated that both compounds have a forceful
binding with the TNF -α, and that the phenyl and hydrazide
moieties of them may play a significant role in binding with
the target site.
[PMID:
[4]
[5]
[http://dx.doi.org/10.1016/j.joca.2006.11.003] [PMID: 17188523]
Calixto, J.B.; Campos, M.M.; Otuki, M.F.; Santos, A.R. Anti-
inflammatory compounds of plant origin. Part II. modulation of
pro-inflammatory cytokines, chemokines and adhesion molecules.
Planta Med., 2004, 70(2), 93-103.
[http://dx.doi.org/10.1055/s-2004-815483] [PMID: 14994184]
Liu, Z.; Tang, L.; Zou, P.; Zhang, Y.; Wang, Z.; Fang, Q.; Jiang,
L.; Chen, G.; Xu, Z.; Zhang, H.; Liang, G. Synthesis and biological
evaluation of allylated and prenylated mono-carbonyl analogs of
curcumin as anti-inflammatory agents. Eur. J. Med. Chem., 2014,
74, 671-682.
[http://dx.doi.org/10.1016/j.ejmech.2013.10.061]
24321865]
Hu, J.; Wang, Y.; Wei, X.; Wu, X.; Chen, G.; Cao, G.; Shen, X.;
Zhang, X.; Tang, Q.; Liang, G.; Li, X. Synthesis and biological
evaluation of novel thiazolidinone derivatives as potential anti-
inflammatory agents. Eur. J. Med. Chem., 2013, 64, 292-301.
[http://dx.doi.org/10.1016/j.ejmech.2013.04.010]
23644212]
Malaviya, R.; Laskin, J.D.; Laskin, D.L. Anti-TNFα therapy in
inflammatory lung diseases. Pharmacol. Ther., 2017, 180, 90-98.
[PMID: 28642115]
[6]
[7]
ETHICS
APPROVAL
AND
CONSENT
TO
PARTICIPATE
[PMID:
This study was approved by Medical ethics committee of
Jinggangshan University (approval number: 20180558).
[8]
HUMAN AND ANIMAL RIGHTS
No humans were used in this study. Procedures involving
animals and their care were conducted in accordance with
the Guide for the Care and Use of Laboratory Animals, 8th
Edition, National Academies Press, Washington DC.
[PMID:
[9]
[10]
Tageldin, G.N.; Fahmy, S.M.; Ashour, H.M.; Khalil, M.A.; Nassra,
R.A.; Labouta, I.M. Design, synthesis and evaluation of some py-
razolo[3,4-d]pyrimidines as anti-inflammatory agents, Bioorg.
Chem., 2018, 78, 358-371.
CONSENT FOR PUBLICATION
Not applicable.
[https://doi.org/10.1016/j.bioorg.2018.03.030]
[11]
[12]
[13]
Duncan, S.A.; Baganizi, D.R.; Sahu, R.; Singh, S.R.; Dennis, V.A.
SOCS proteins as regulators of inflammatory responses induced by
Bacterial Infections: A Review. Front. Microbiol., 2017, 8, 2431.
[http://dx.doi.org/10.3389/fmicb.2017.02431] [PMID: 29312162]
Bekhit, A.A.; Abdel-Aziem, T. Design, synthesis and biological
evaluation of some pyrazole derivatives as anti-inflammatory-
antimicrobial agents. Bioorg. Med. Chem., 2004, 12(8), 1935-1945.
[http://dx.doi.org/10.1016/j.bmc.2004.01.037] [PMID: 15051061]
Hassan, G.S.; Abdel Rahman, D.E.; Abdelmajeed, E.A.; Refaey,
R.H.; Alaraby Salem, M.; Nissan, Y.M. New pyrazole derivatives:
Synthesis, anti-inflammatory activity, cycloxygenase inhibition as-
say and evaluation of mPGES. Eur. J. Med. Chem., 2019, 171, 332-
342.
AVAILABILITY OF DATA AND MATERIALS
The authors confirm that the data supporting the findings
of this study are available within the article.
FUNDING
This work was supported by the National Natural Science
Foundation of China (No. 21562028, No. 81560561).
CONFLICT OF INTEREST
[http://dx.doi.org/10.1016/j.ejmech.2019.03.052]
30928706]
[PMID:
The authors declare no conflict of interest, financial or
otherwise.
[14]
[15]
Belkheiri, N.; Bouguerne, B.; Bedos-Belval, F.; Duran, H.; Bernis,
C.; Salvayre, R.; Nègre-Salvayre, A.; Baltas, M. Synthesis and an-
tioxidant activity evaluation of a syringic hydrazones family. Eur.
J. Med. Chem., 2010, 45(7), 3019-3026.
ACKNOWLEDGEMENTS
[http://dx.doi.org/10.1016/j.ejmech.2010.03.031]
20403645]
[PMID:
The school of life sciences, Jinggangshan University was
appreciated for providing the BIOVIA Discovery Studio
software in this study.
Kaplancikli, Z.A.; Altintop, M.D.; Ozdemir, A.; Turan-Zitouni, G.;
Khan, S.I.; Tabanca, N. Synthesis and Biological Evaluation of

Pyrazole Hydrazones as Anti-inflammatory Agents
Letters in Drug Design & Discovery, 2020, Vol. 17, No. 4 511
Some Hydrazone Derivatives as Anti-inflammatory Agents. Lett.
Drug Des. Discov., 2012, 9, 310-315.
[http://dx.doi.org/10.1016/j.bioorg.2017.08.014]
28865292]
El-Sayed, M.A.; Abdel-Aziz, N.I.; Abdel-Aziz, A.A.; El-Azab,
A.S.; Asiri, Y.A.; Eltahir, K.E. Design, synthesis, and biological
evaluation of substituted hydrazone and pyrazole derivatives as se-
lective COX-2 inhibitors: Molecular docking study. Bioorg. Med.
Chem., 2011, 19(11), 3416-3424.
[http://dx.doi.org/10.1016/j.bmc.2011.04.027] [PMID: 21570309]
Mohammed, K.O.; Nissan, Y.M. Synthesis, molecular docking, and
biological evaluation of some novel hydrazones and pyrazole de-
rivatives as anti-inflammatory agents. Chem. Biol. Drug Des.,
2014, 84(4), 473-488.
[http://dx.doi.org/10.1111/cbdd.12336] [PMID: 24720475]
Liang, Z.C.; Huang, Y.P.; Wang, S.B.; Deng, X.Q. Synthesis and
Biological Evaluation of Some Pyrazole derivatives, Containing
(Thio) Semicarbazide, as Dual Anti-Inflammatory-Antimicrobial
Agents. Lett. Drug Des. Discov., 2019, 16
[http://dx.doi.org/10.2174/1570180816666190325163117]
Waseem, T.; Duxbury, M.; Ito, H.; Ashley, S.W.; Robinson, M.K.
Exogenous ghrelin modulates release of pro-inflammatory and anti-
inflammatory cytokines in LPS-stimulated macrophages through
distinct signaling pathways. Surgery, 2008, 143(3), 334-342.
[http://dx.doi.org/10.1016/j.surg.2007.09.039] [PMID: 18291254]
Guruvayoorappan, C.; Kuttan, G. (+)-Catechin inhibits tumour
angiogenesis and regulates the production of nitric oxide and TNF-
α in LPS-stimulated macrophages. Innate Immun., 2008, 14(3),
160-174.
[http://dx.doi.org/10.1177/1753425908093295] [PMID: 18562575]
Meng, A.; Wang, B.; Zhang, X.; Qi, N.; Liu, D.; Wu, J. Additive
Suppression of LPS-Induced IL-10 and TNF-α by Pre-treatment of
Dexamethasone and SB203580 in a Murine Alveolar Macrophage
Cell Line (MH-S). Inflammation, 2015, 38(3), 1260-1266.
[http://dx.doi.org/10.1007/s10753-014-0093-x] [PMID: 25563207]
Xu, B.G.; Li, S.H.; Song, M.X.; Deng, X.Q. Study on Antibacterial
and Anti-inflammatory Activity of Rhododendron Pulchrum and Its
Active Fraction. Journal of Liaocheng University. Nat. Sci., 2018,
31, 86-92.
Luber-Narod, J.; Austin-Ritchie, T.; Hollins, C., III; Menon, M.;
Malhotra, R.K.; Baker, S.; Carraway, R.E. Role of substance P in
several models of bladder inflammation. Urol. Res., 1997, 25(6),
395-399.
[http://dx.doi.org/10.1007/BF01268854] [PMID: 9443648]
Kim, H.D.; Cho, H.R.; Moon, S.B.; Shin, H.D.; Yang, K.J.; Park,
B.R.; Jang, H.J.; Kim, L.S.; Lee, H.S.; Ku, S.K. Effects of β-glucan
from Aureobasidium pullulans on acute inflammation in mice.
Arch. Pharm. Res., 2007, 30(3), 323-328.
[PMID:
[http://dx.doi.org/10.2174/157018012799129828]
[27]
[16]
Freitas, R.H.C.N.; Cordeiro, N.M.; Carvalho, P.R.; Alves, M.A.;
Guedes, I.A.; Valerio, T.S.; Dardenne, L.E.; Lima, L.M.; Barreiro,
E.J.; Fernandes, P.D.; Fraga, C.A.M. Discovery of naphthyl-N-
acylhydrazone p38α MAPK inhibitors with in vivo anti-
inflammatory and anti-TNF-α activity. Chem. Biol. Drug Des.,
2018, 91(2), 391-397.
[http://dx.doi.org/10.1111/cbdd.13085] [PMID: 28815968]
Kajal, A.; Bala, S.; Sharma, N.; Kamboj, S.; Saini, V. Therapeutic
potential of hydrazones as anti-inflammatory agents. Int. J. Med.
Chem., 2014, 2014761030
[28]
[29]
[30]
[31]
[32]
[17]
[18]
[http://dx.doi.org/10.1155/2014/761030] [PMID: 25383223]
Dehestani, L.; Ahangar, N.; Hashemi, S.M.; Irannejad, H.; Honar-
chian Masihi, P.; Shakiba, A.; Emami, S. Design, synthesis, in vivo
and in silico evaluation of phenacyl triazole hydrazones as new an-
ticonvulsant agents. Bioorg. Chem., 2018, 78, 119-129.
[http://dx.doi.org/10.1016/j.bioorg.2018.03.001]
29550532]
[PMID:
[19]
Kaushik, D.; Khan, S.A.; Chawla, G.; Kumar, S. N′-[(5-chloro-3-
methyl-1-phenyl-1H-pyrazol-4-yl)methylene] 2/4-substituted hy-
drazides: synthesis and anticonvulsant activity. Eur. J. Med. Chem.,
2010, 45(9), 3943-3949.
[http://dx.doi.org/10.1016/j.ejmech.2010.05.049]
20573423]
[PMID:
[20]
[21]
Rasras, A.J.M.; Al-Tel, T.H.; Al-Aboudi, A.F.; Al-Qawasmeh,
R.A. Synthesis and antimicrobial activity of cholic acid hydrazone
analogues. Eur. J. Med. Chem., 2010, 45(6), 2307-2313.
[http://dx.doi.org/10.1016/j.ejmech.2010.02.006]
20181416]
[PMID:
Popiołek, Ł.; Biernasiuk, A.; Berecka, A.; Gumieniczek, A.; Malm,
A.; Wujec, M. New hydrazide-hydrazones of isonicotinic acid:
Synthesis, lipophilicity and in vitro antimicrobial screening. Chem.
Biol. Drug Des., 2018, 91(4), 915-923.
[33]
[34]
[http://dx.doi.org/10.1111/cbdd.13158] [PMID: 29220872]
Polovic, S.; Bilic, V.L.; Budimir, A.; Kontrec, D.; Galic, N.;
Kosalec, I. Antimicrobial assesment of aroylhydrazone derivatives
in vitro. Acta Pharm., 2019, 67, 277-285.
[22]
[23]
[24]
[http://dx.doi.org/10.2478/acph-2019-0020]
Kumar, D.; Maruthi Kumar, N.; Ghosh, S.; Shah, K. Novel
bis(indolyl)hydrazide-hydrazones as potent cytotoxic agents.
Bioorg. Med. Chem. Lett., 2012, 22(1), 212-215.
[35]
[36]
[37]
[http://dx.doi.org/10.1016/j.bmcl.2011.11.031] [PMID: 22123320]
Hayat, M.; Mohammed, K.; Saeed, K.S.; Salar, U.; Khan, M. Baig,
T.; Ahmad, A.; Parveen, S.; Taha, M. Antimicrobial Activities of
Synthetic Arylidine Nicotinic and Isonicotinic Hydrazones. Lett.
Drug Des. Discov., 2018, 15, 1057-1067.
[http://dx.doi.org/10.1007/BF02977613] [PMID: 17424938]
Kharbanda, C.; Alam, M.S.; Hamid, H.; Javed, K.; Bano, S.; Dhu-
lap, A.; Ali, Y.; Nazreen, S.; Haider, S. Synthesis and evaluation of
pyrazolines bearing benzothiazole as anti-inflammatory agents.
Bioorg. Med. Chem., 2014, 22(21), 5804-5812.
[http://dx.doi.org/10.2174/1570180814666170914120337]
Altıntop, M.D.; Ozdemir, A.; Ilgın, S.; Atli, O. Synthesis and Bio-
logical Evaluation of New Pyrazole-based Thiazolyl Hydrazone
Derivatives as Potential Anticancer Agents. Lett. Drug Des. Dis-
cov., 2014, 11, 833-839.
[http://dx.doi.org/10.2174/1570180811666140226235350]
Abdelgawad, M.A.; Labib, M.B.; Abdel-Latif, M. Pyrazole-
hydrazone derivatives as anti-inflammatory agents: Design, synthe-
sis, biological evaluation, COX-1,2/5-LOX inhibition and docking
study. Bioorg. Chem., 2017, 74, 212-220.
[25]
[26]
[http://dx.doi.org/10.1016/j.bmc.2014.09.028] [PMID: 25311566]
Li, S.X.; Deng, X.D.; Jiang, F.L.; Zhao, Y.J.; Xiao, W.S.; Kuang,
X.Z.; Sun, X.M. Design and Synthesis of Novel Diaryl Heterocy-
clic Derivatives as Selective Cyclooxygenase-2. Lett. Drug Des.
Discov., 2008, 5, 127-133.
[http://dx.doi.org/10.2174/157018008783928517]