Synthesis and biological evaluation of some pyrazole derivatives, containing (Thio) semicarbazide, as dual anti-inflammatory antimicrobial agents

Article abstract of DOI:10.2174/1570180816666190325163117

Background: Several series of pyrazole derivatives containing (thio) semicarbazide (4a-4h, 5a-5l, 6a-6f, 7a-7c) were designed and synthesized to screen dual inflammatory and antimicrobial activities. Methods: The products were characterized by¹

Full text of DOI:10.2174/1570180816666190325163117

1020
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Letters in Drug Design & Discovery, 2019, 16, 1020-1030
RESEARCH ARTICLE
ISSN: 1570-1808
eISSN: 1875-628X
Synthesis and Biological Evaluation of Some Pyrazole Derivatives, Con-
taining (Thio) Semicarbazide, as Dual Anti-Inflammatory Antimicrobial
Agents
Impact
Factor:
0.953
BENTHAM
S
CIENCE
Zhaochang Liang1, Yuping Huang3, Shiben Wang2 and Xianqing Deng1,*
1Medical College, Jinggangshan University, No 28, Xueyuan Road, Ji'an, 343009, Jiangxi, China; 2School of Pharmacy,
3
Liaocheng University, LiaoCheng, 252059, Shandong, China; Department of Biochemistry and Molecular Biology,
Gannan Medical College, Ganzhou, China
Abstract: Background: Several series of pyrazole derivatives containing (thio) semicarbazide
(4a-4h, 5a-5l, 6a-6f, 7a-7c) were designed and synthesized to screen dual inflammatory and antimi-
crobial activities.
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-
A R T I C L E H I S T O R Y
inflammatory activity. Their in vitro antimicrobial activities were evaluated using a serial dilution
method against several gram-positive strains, gram-negative strains and a fungi strain.
Received: October 12, 2018
Revised: February 12, 2019
Results: Bioassays indicated that most of the compounds markedly inhibited the expression of TNF-
Accepted: March 8, 2019
α at the concentration of 20 μg/mL Compounds 5i, 6b, and 7b had comparable in vivo anti-
DOI:
inflammatory activity to the reference drug dexamethasone at the dose of 50 mg/kg. In addition,
10.2174/1570180816666190325163117
several compounds showed antimicrobial activity against different strains, and compounds 5g and 5h
ꢀ
exhibited potent inhibitory activities with the MIC value of 8 μg/mL against the Streptococcus
pneumoniae CMCC 31968 and Staphylococcus aureus CMCC 25923, respectively. Compound 7b,
which exhibited both anti-inflammatory and antimicrobial activities, should be studied as it is or after
derivatization.
Conclusion: It can be concluded that pyrazoles, with (thio)-semicarbazone moieties, have the poten-
tial to be developed into new anti-inflammatory agents.
Keywords: Anti-inflammatory, antimicrobial, pyrazole, (thio) semicarbazide, TNF-α, dexamethasone.
1. INTRODUCTION
inflammatory conditions, associated with some microbial
infections, may result in additional health problems, espe
cially in patients with impaired liver or kidney function [6].
As an alternative, monotherapy using an anti-inflammatory
drug with antimicrobial properties would be better from
pharmacoeconomics and safety aspects; this will enhance
patient compliance and lower the risk of adverse effects.
Inflammation is part of the complex biological response
of body tissues in response to damage resulting from micro-
bial pathogen infection, chemical stimuli, and physical trau-
ma [1, 2]. Although acute inflammation is required as a de-
fense mechanism, persistent inflammation is harmful and
should be suppressed. Dysregulation of inflammation plays a
crucial role in chronic inflammation that can contribute to
diseases such as arthritis, heart attacks, and Alzheimer’s dis-
ease [3]. Furthermore, inflammatory responses play vital
roles in different stages of tumor development [4].
Pyrazoles are significant double nitrogen, five-membered,
heterocyclic compounds. Since the discovery and approval
of celecoxib as an anti-inflammatory agent in the 1990s,
many medicinal chemists have focused on the design and
synthesis of pyrazole derivatives, particularly their anti-
inflammatory activity [7-10]. Pyrazoles can form hydrogen
bonds and can readily bind with various enzymes and recep-
tors, allowing them to exhibit diverse pharmacological activ-
ities such as antimicrobial [11-13], anticancer [14], antide-
pressant [15], and anticonvulsant activities [16]. (Thio)
semicarbazones have also been extensively investigated for
their uses in medicinal chemistry. The compounds contain-
ing (thio) semicarbazone were reported to have a broad
Recently, the incidence of inflammation due to bacterial
infections is on the rise. Many patients with inflammatory
conditions require treatment with antimicrobial agents [5].
The co-administration of multiple drugs for the treatment of
*Address correspondence to this author at the Medical College, Jinggang-
shan University, No 28, xueyuan road, Ji'an, 343009, Jiangxi, P.R. China;
E-mail: dengxianqing1121@126.com
1875-628X/19 $58.00+.00
©2019 Bentham Science Publishers

Pyrazole-Semicarbazides as Anti-Inflammatory-Antimicrobial Agents
Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9 1021
R1
R1
H
N
NH2
H
N
N
NH2
N
N
N
X
N
X
HN
R3
4a-4g (X = O); 4h (X = S)
6a-6c (X = O); 6d-6f (X = S)
R1
=
6a and 6d: R1 = H, R3 = CH3
6b and 6e: R1 = H, R3 = C3H7
6c and 6f: R1 = H, R3 = CH2C6H5
4a and 4h: H, 4b: 4-F, 4c: 4-Cl, 4d: 4-Br,
4e: 4-CH3, 4f: 4-OCH3, 4g: 4-OH
H
H
N
NH2
N
NH2
H2N
H2N
O
X
X
R1
O
O
H
N
R
R1
R3X
DMF
N
CONH2
NaOAc
R1
+
POCl3
N
N
N
NH
H
N
R3
NH2
1
2
3
H2N
O
H
N
H
N
H
N
H
N
H2N
H2N
R2
O
O
R1
R1
H
H
H
H
N
N
N
N
N
N
N
N
R2
O
O
HN
N
R3
7a-7c (R1 = H)
5a-5i (R2 = H)
R1
=
5a: H, 4b: 4-F, 5c: 4-Cl, 5d: 4-Br, 5e: 4-CH3,
5f: 4-OCH3, 5g: 4-OH, 5h: 2,4-2Cl, 5i: 4-NO2
R3
=
7a: CH3, 7b: C3H7, 7c: CH2C6H5
5j (R1 = H, R2 = 4-F)
5k (R1 = H, R2 = 4-Cl)
5l (R1 = H, R2 = 4-OCH3)
Scheme 1. The synthesis route of target compounds 4a-4h, 5a-5l, 6a-6f, and 7a-7c.
application in drug development for the treatment of cancer
[17] and bacterial infections [18], especially as anti-
inflammatory and analgesic agents [19, 20].
zide and N-phenylhydrazinecarboxamide to produce com-
pounds 4a-4g, 4h, and 5a-5l. On the other hand, compound 2
was alkylated by haloalkane to give the N-substituted-4-
carboxaldehyde pyrazoles (3), which was then reacted with
semicarbazide, thiosemicarbazide, and N-phenylhydrazineca
rboxamide to produce compounds 6a-6c, 6d-6f, and 7a-7c
(Scheme 1). The structure of the desired products was con-
firmed by 1H NMR, 13C NMR, and mass spectrometry.
Based on these findings, the objective of this study was
to combine the (thio) semicarbazide group with the pyrazole
moiety to form a skeleton that has the potential to act as a
dual anti-inflammatory antimicrobial agent. In this study,
twenty-nine 3, 4-substituted pyrazole derivatives were syn-
thesized and their anti-inflammatory and antimicrobial activ-
ities were tested.
Compound 4a was used as an example of the structure
1
conformation. In the H-NMR spectrum, one singlet, due to
the NH2 of semicarbazide, was observed at 6.33 ppm. The
aromatic protons of the benzene ring and pyrazole ring re-
vealed peaks in the 7.41-7.94 ppm range. The absorption
peak of C-H in imine was found at 8.16 ppm. The absorption
peak of NH in the amide was observed at 9.95 ppm as a sin-
glet. The absorption peak in the hydrogen spectrum was in
complete conformity with the hydrogen signal in the struc-
ture. The 13C NMR spectra also gave accurate information
about the structure of compound 4a, which has 10 types of
carbons in different chemical environments. Moreover, the
high-resolution mass spectrometry of 4a displayed an [M +
H]+ signal at m/z 230.1034, corresponding to its molecular
weight of 230.1036.
2. CHEMISTRY
According to the designed structures, twenty-nine pyra-
zole derivatives were divided into four series, which were
prepared from 2 types of intermediates 2 and 3. The com-
pounds were synthesized according to the sequence shown in
Scheme 1. Firstly, the synthesis of acetophenonehydrazones
(1) was carried out at 50 °C by reacting the substituted ace-
tophenone with semicarbazide hydrochloride, in the presence
of NaOAc and alcohol. Under Vilsmeier-Haack (DMF-
POCl3) conditions, compound 1 was transformed into the
corresponding 4-carboxaldehyde functionalized pyrazoles
(2), which then reacted with semicarbazide, thiosemicarba-

1022 Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9
Liang et al.
3. EXPERIMENTAL PROTOCOLS
3.1. Chemistry
precipitation was filtered and recrystallized with ethanol to
give the compound 4h.
Melting points were determined in open capillary-tubes
3.1.6. Synthesis of N-Phenyl-2-((3-Phenyl-1H-Pyrazol-4-yl)
Methylene) Hydrazine-1- Carboxamides(5a-5l)
1
and were uncorrected. H NMR and 13C NMR spectra were
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
Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China),
and were used as received.
To a solution of compound 2 (0.03 mol) in 10mL abso-
lute alcohol, N-phenylhydrazinecarboxamide (0.03 mol) was
added and refluxed for 2 h. After cooling, the precipitation
was filtered and recrystallized with ethanol to give the com-
pound 5a-5l.
3.1.7. Synthesis of 2-((1-Substituted-3-Phenyl-1H-Pyrazol-
4-yl) Methylene) Hydrazine-1- Carboxamides (6a-6c)
To a solution of compound 3 (0.01 mol) in 10mL of 50%
alcohol, hydrazinecarboxamide hydrochloride (0.01 mol)
was added and then refluxed for 1 h. After cooling, the solvent
was evaporated in vacuo, followed by the purification of the
resulting residue by silica gel column chromatography (di-
chloromethane/methanol = 60/1) to generate a pure solid
6a-6c.
3.1.1. Synthesis of 2-(1-Phenylethylidene) Hydrazine-1-
Carboxamides (1)
A solution of semicarbazide hydrochloride (9 mmol) and
sodium acetate (18 mmol) in 20 mL of water was heated to
50 °C. After dissolving, 10 mL of alcohol solution contain-
ing acetophenone (9 mmol) was added dropwise, and then
stirred at 50 °C for 3-8 h until the reaction completed. After
cooling the mixture, the deposit was filtered and recrystal-
lized in ethanol to obtain compounds 1.
3.1.8. Synthesis of 2-((1-Methyl-3-Phenyl-1H-Pyrazol-4-yl)
Methylene) Hydrazine-1- Carbothioamides(6d-6f)
To a solution of compound 3 (R1 = H, 0.01 mol) in 10mL
absolute alcohol, thiosemicarbazide (0.01 mol) was added
and stirred for 4 h at room temperature. After cooling, the
precipitation was filtered and recrystallized with ethanol to
give the compound 6q-6f.
3.1.2. Synthesis of 3-Phenyl-1H-Pyrazole-4-Carbaldehydes
(2)
To a cooled (0-5°C) solution of dimethyl formamide (55
mmol), POCl3 (18 mmol)was added dropwise and stirred for
5 min. Then compound 1 (4.5 mmol) was added in six por-
tions at intervals and stirred at 70°C for 5 h. The resulting
mixture was poured into 20 mL of ice-water and then adjust-
ed to PH of 6-7 using 20% aqueous sodium hydroxide. After
staying over-night below 5°C, precipitates were formed, fil-
tered, and dried to yield compounds 2.
3.1.9. Synthesis of 2-((1-Substituted-3-Phenyl-1H-Pyrazol-4-
yl) Methylene)-N-Phenylhydrazine-1-Carboxamides (7a-7c)
To a solution of compound 3 (0.01 mol) in 10mL abso-
lute alcohol, N-phenylhydrazinecarboxamide (0.01 mol) was
added and refluxed for 2 h. After cooling, the precipitation
was filtered and recrystallized with ethanol to give the com-
pound 7a-7c.
3.1.3. Synthesis of N-Substituted-3-Phenyl-1H-Pyrazole-4-
Carbaldehydes (3)
3.1.9.1. 1-((3-Phenyl-1H-Pyrazol-4-yl) Methylidene) Semi-
carbazide (4a)
To a solution of compound 2 (5mmol) in 20 mL of ace-
tonitrile, K2CO3 (6mmol) and dimethyl sulphate or bromo-
propane or benzyl chloride (6 mmol) was added.The mixture
was stirred and refluxed for 3-5 h. Upon completion, the
solvent was removed, and the residue was extracted with
dichloromethane (30 mL × 3). The combined organic layers
were dried over anhydrous MgSO4 and concentrated in vac-
uo to obtain a white crude solid of 3, which was directly
used in the next step without purification.
Mp 216-218°C, yield 82.1%. 1H-NMR (DMSO-d6,
300MHz): δ 6.33 (s, 2H, NH2), 7.41-7.57 (m, 6H, Ph-H, N-
NH), 7.94 (s, 1H, N-CH), 8.16 (s, 1H, CH=N), 9.95 (s, 1H,
CONH). 13C-NMR (DMSO-d6,75MHz): δ 157.19, 145.19,
134.25, 133.54, 131.38, 129.23, 128.73, 128.47, 114.92.
ESI-HRMS calcd for C11H12N5O+ ([M + H]+): 230.1036;
found: 230.1034.
3.1.9.2. 1-((3-(4-Fluorophenyl)-1H-Pyrazol-4-yl) Methyli-
dene) Semicarbazide (4b)
3.1.4. Synthesis of 2-((3-Phenyl-1H-Pyrazol-4-yl) Meth-
ylene) Hydrazine-1-Carboxamides (4a-4g)
Mp 196-200 ºC, yield 53.7%. 1H-NMR (DMSO-d6,
300MHz): δ 6.58 (s, 2H, NH2), 7.29-7.62 (m, 4H, Ph-H),
To a solution of compound 2 (0.03 mol) in 10mL of 50%
alcohol, hydrazinecarboxamide hydrochloride (0.03 mol)
was added and then refluxed for 1 h. After cooling, the pre-
cipitation was filtered and recrystallized with 50% ethanol to
give the pure compounds 4a-4g.
7.92 (s, 1H, N-CH), 8.17 (s, 1H, CH=N), 10.00 (s, 1H,
1
CONH). 13C-NMR (DMSO-d6,75MHz): δ 162.51 (d, Jc-f
=
3
243.8 Hz), 157.28, 144.80, 133.60, 130.61 (d, Jc-f = 8.3 Hz),
2
128.24, 128.21, 116.11 (d, Jc-f = 21.5 Hz), 114.87. ESI-HRMS
calcd for C11H11FN5O+ ([M + H]+): 248.0942; found: 248.0935.
3.1.5. Synthesis of 2-((3-Phenyl-1H-Pyrazol-4-yl) Meth-
ylene) Hydrazine-1-Carboxamide (4h)
3.1.9.3. 1-((3-(4-Chlorophenyl)-1H-Pyrazol-4-yl) Methyli-
dene) Semicarbazide (4c)
To a solution of compound 2 (R1 = H, 0.03 mol) in 10mL
absolute alcohol, thiosemicarbazide (0.03 mol) was added
and stirred for 2 h at room temperature. After cooling, the
Mp 178-181°C, yield 66.8%. 1H-NMR (DMSO-d6,
300MHz): δ 5.09 (s, 2H, NH2), 7.57 (q, 4H, J = 8.6 Hz,

Pyrazole-Semicarbazides as Anti-Inflammatory-Antimicrobial Agents
Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9 1023
Table 1. Inhibitory activity (MIC, μg/mL) of compounds 4a-4hꢀ5a-5lꢀ6a-6fꢀ7a-7c against different bacterial and fungal
strains.
Gram-positive Strains
Gram-negative Strains
Fungj
Compound
R1-
R2-
R3-
26003a 25923b
32067c
31968d 29212e 63501f 25922g 44568h 27853i 10104j 98001k
4a
4b
4c
4d
4e
4f
H
F
—
—
—
—
—
—
—
—
H
—
—
>128
>128
>128
128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
32
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
8
>128
>128
>128
128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
Cl
—
Br
—
CH3
OCH3
OH
H
—
>128
>128
>128
>128
128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
32
128
—
>128
>128
>128
>128
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
4g
4h
5a
5b
5c
5d
5e
5f
—
—
H
—
F
H
—
128
Cl
H
—
>128
>128
128
Br
H
—
CH3
OCH3
OH
2,4-2Cl
NO2
H
H
—
H
—
128
5g
5h
5i
H
—
>128
128
>128
8
H
—
>128
>128
>128
>128
>128
>128
>128
>128
128
H
—
128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
128
5j
F
—
>128
>128
>128
>128
>128
128
5k
5l
H
Cl
OCH3
—
—
—
—
—
—
—
—
—
H
—
6a
6b
6c
6d
6e
6f
H
CH3
C3H7
PhCH2-
CH3
C3H7
PhCH2-
CH3
C3H7
H
H
H
128
64
128
H
>128
>128
>128
128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
128
>128
>128
>128
128
>128
>128
>128
64
H
7a
7b
H
H
(Table 1) contd…

1024 Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9
Liang et al.
Gram-positive Strains
Gram-negative Strains
Fungj
Compound
R1-
R2-
R3-
26003a 25923b
32067c
31968d 29212e 63501f 25922g 44568h 27853i 10104j 98001k
7c
H
—
—
PhCH2-
—
128
64
>128
>128
>128
>128
128
128
128
128
>128
128
>128
>128
128
128
32
128
16
—
Penicillin
0.25
0.25
>128
—
—
—
—
—
—
—
—
—
—
—
—
Norfloxacin
Gatifloxacin
Moxifloxacin
Oxacillin
1
1
2
2
2
2
4
2
1
1
0.25
0.25
0.25
0.25
>128
>128
>128
>128
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
>128
>128
>128
>128
1
2
0.25
128
0.25
2
4
0.5
128
128
>128
>128
>128
128
aStaphylococcus aureus CMCC(B)26003; bStaphylococcus aureus CMCC 25923; c Streptococcus pyogenes CMCC 32067; dStreptococcus pneumoniae CMCC
e
f
g
h
31968 Enterococcus faecalis CMCC 29212; Bacillus subtilis CMCC 63501; Escherichia coli CMCC 25922; Escherichia coli CMCC 44568;iPseudomonas
aeruginosa CMCC 27853; jPseudomonas aeruginosa CMCC 10104; kCandida albicans CMCC 98001.
Ph-H), 7.94 (s, 1H, N-CH), 8.18 (s, 1H, CH=N), 9.97 (s, 1H,
CONH). 13C-NMR (DMSO-d6,75MHz): δ157.20, 144.76,
133.46, 133.39, 133.33, 130.77, 130.20, 129.19, 115.09.
ESI-HRMS calcd for C11H11ClN5O+ ([M + H]+): 264.0647;
found: 264.0642.
H), 7.36 (d, 2H, J = 8.5 Hz, Ph-H), 7.90 (s, 1H, N-CH), 8.06
(s, 1H, CH=N), 9.91 (s, 1H, CONH). 13C-NMR (DMSO-
d6,75MHz): δ158.23, 157.28, 144.54, 135.12, 134.07,
129.81, 121.55, 116.07, 114.14. ESI-HRMS calcd for
C11H12N5O2+ ([M + H]+): 246.0986; found: 246.0995.
3.1.9.8. 1-((3-Phenyl-1H-Pyrazol-4-yl) Methylidene) Thi-
osemicarbazide (4h)
3.1.9.4. 1-((3-(4-Bromophenyl)-1H-Pyrazol-4-yl) Methyli-
dene) Semicarbazide (4d)
Mp 200-203°C, yield 54.3%. 1H-NMR (DMSO-d6,
300MHz): δ 5.04 (s, 2H, NH2), 7.53 (d, 2H, J = 8.5 Hz, Ph-
H), 7.68 (d, 2H, J = 8.5 Hz, Ph-H), 7.94 (s, 1H, N-CH), 8.18
(s, 1H, CH=N), 9.94 (s, 1H, CONH). 13C-NMR (DMSO-
d6,75MHz): δ157.17, 144.83, 133.39, 133.27, 132.11,
131.14, 130.47, 121.99, 115.10. ESI-HRMS calcd for
C11H11BrN5O+ ([M + H]+): 308.0141; found: 308.0132.
Mp 216-219°C, yield 64.4%. 1H-NMR (DMSO-d6,
300MHz): δ7.43-7.56 (m, 6H, Ph-H, N-NH), 7.67 (s, 1H,
NH2), 7.97 (s, 1H, NH2), 8.18 (s, 1H, N-CH), 8.23 (s, 1H,
CH=N), 11.16 (s, 1H, NH). 13C-NMR (DMSO-d6,75MHz):
δ177.54, 137.14, 134.70, 130.90, 129.34, 129.23, 129.04,
128.49, 114.27. ESI-HRMS calcd for C11H12N5S+ ([M +
H]+): 246.0808; found: 246.0815.
3.1.9.5. 1-((3-(4-Methylphenyl)-1H-pyrazol-4-yl) Methyli-
dene) Semicarbazide (4e)
3.1.9.9. N-Phenyl-2-((3-Phenyl-1H-Pyrazol-4-yl) Methyle-
ne) Hydrazinecarboxamide (5a)
Mp 243-246°C, yield 44.2%. 1H-NMR (DMSO-d6,
300MHz): δ 2.37 (s, 3H, CH3), 6.29 (s, 2H, NH2), 7.30 (d,
2H, J = 7.9 Hz, Ph-H), 7.44 (d, 2H, J = 7.9 Hz, Ph-H), 7.93
(s, 1H, N-CH), 8.12 (s, 1H, CH=N), 9.91 (s, 1H, CONH).
13C-NMR (DMSO-d6,75MHz): δ157.21, 138.26, 133.70,
129.80, 129.73, 128.96, 128.33, 120.13, 114.70, 21.27. ESI-
HRMS calcd for C12H14N5O+ ([M + H]+): 244.1193;
found: 244.1199.
Mp 200-223°C, yield 54%. 1H-NMR (DMSO-d6,
300MHz): δ 6.99-7.63 (m, 10H, Ph-H), 8.05 (s, 1H, N-CH),
8.37 (s, 1H, CONH), 8.63 (s, 1H, CH=N), 10.45 (s, 1H,
CONH), 13.28 (s, 1H, pyrazol-NH). 13C-NMR (DMSO-
d6,75MHz): δ153.34, 139.58, 135.02, 133.99, 133.64,129.28,
128.96, 128.83, 128.82, 128.58, 122.77, 119.89, 114.53. ESI-
HRMS calcd for C17H16N5O+ ([M + H]+): 306.1349;
found: 306.1345.
3.1.9.6. 1-((3-(4-Methoxylphenyl)-1H-Pyrazol-4-yl) Me-
thylidene) Semicarbazide (4f)
3.1.9.10. N-Phenyl-2-((3-(4-Fluorophenyl)-1H-Pyrazol-4-
yl) Methylene)Hydrazinecarboxamide(5b)
Mp 239-241°C, yield 67.8%. 1H-NMR (DMSO-d6,
300MHz): δ 3.82 (s, 3H, OCH3), 6.25 (s, 2H, NH2), 7.06 (d,
2H, J = 8.4 Hz, Ph-H), 7.49 (d, 2H, J = 8.4 Hz, Ph-H), 7.92
(s, 1H, N-CH), 8.09 (s, 1H, CH=N), 9.90 (s, 1H, CONH).
13C-NMR (DMSO-d6,75MHz): δ159.84, 157.23, 144.51,
134.77, 133.80, 129.79, 123.49, 114.70, 114.44, 55.73. ESI-
HRMS calcd for C12H14N5O2+ ([M + H]+): 260.1142;
found: 260.1134.
Mp 226-230°C, yield 60.8%. 1H-NMR (DMSO-d6,
300MHz): δ 6.99-7.67 (m, 9H, Ph-H), 8.03 (s, 1H, N-CH),
8.34 (s, 1H, CONH), 8.55 (s, 1H, CH=N), 10.39 (s, 1H,
1
CONH). 13C-NMR (DMSO-d6,75MHz): δ162.58 (d, Jc-f
=
244.1 Hz), 153.32, 139.56, 135.54, 134.88, 134.08, 130.72
(3Jc-f = 8.3 Hz), 128.97, 128.35, 122.78, 119.85, 116.16 (2Jc-f
= 21.5 Hz), 114.54. ESI-HRMS calcd for C17H15FN5O+
([M + H]+): 324.1255; found: 324.1265.
3.1.9.7. 1-((3-(4-Hydroxyphenyl)-1H-Pyrazol-4-yl) Me-
thylidene) Semicarbazide (4g)
3.1.9.11. N-Phenyl-2-((3-(4-Chlorophenyl)-1H-Pyrazol-4-
yl) Methylene) Hydrazinecarboxamide(5c)
Mp 207-210°C, yield 79.1%. 1H-NMR (DMSO-d6,
300MHz): δ 7.00 (t, 1H, J = 7.3 Hz, Ph-H), 7.29 (t, 2H, J =
Mp 266-272°C, yield 54.2%. 1H-NMR (DMSO-d6,
300MHz): δ 6.32 (s, 2H, NH2), 6.89 (d, 2H, J = 8.5 Hz, Ph-

Pyrazole-Semicarbazides as Anti-Inflammatory-Antimicrobial Agents
Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9 1025
7.9 Hz, Ph-H), 7.55-7.65 (m, 6H, Ph-H), 8.05 (s, 1H, N-CH),
8.36 (s, 1H, CONH), 8.57 (s, 1H, CH=N), 10.49 (s, 1H,
CONH). 13C-NMR (DMSO-d6,75MHz): δ153.28, 139.55,
134.83, 133.87, 133.46, 130.35, 129.21, 129.17, 128.99,
128.92, 122.75, 119.74, 114.68. ESI-HRMS calcd for
C17H15ClN5O+ ([M + H]+): 340.0960; found: 340.0953.
7.7 Hz, Ph-H), 7.43-7.79 (m, 6H, Ph-H, N-CH), 7.99 (s, 1H,
CONH), 8.39 (s, 1H, CH=N), 10.45 (s, 1H, CONH), 13.39
(s, 1H, pyrazol-NH). 13C-NMR (DMSO-d6,75MHz):
δ153.10, 146.41, 146.34, 139.20, 134.58, 134.52, 134.43,
134.00, 130.00, 129.46, 129.11, 127.87, 122.86, 119.51,
115.93. ESI-HRMS calcd for C17H14Cl2N5O+ ([M + H]+):
374.0570; found: 374.0566.
3.1.9.12. N-Phenyl-2-((3-(4-Bromophenyl)-1H-Pyrazol-4-
yl) Methylene) Hydrazinecarboxamide (5d)
3.1.9.17. N-Phenyl-2-((3-(4-Nitrophenyl)-1H-Pyrazol-4-yl)
Methylene) Hydrazinecarboxamide(5i)
Mp 220-223°C, yield 46.4%. 1H-NMR (DMSO-d6,
300MHz): δ 7.01 (t, 1H, J = 7.3 Hz, Ph-H), 7.30 (t, 2H, J =
7.8 Hz, Ph-H), 7.55-7.73 (m, 6H, Ph-H), 8.03 (s, 1H, N-CH),
8.37 (s, 1H, CONH), 8.52 (s, 1H, CH=N), 10.45 (s, 1H,
CONH), 13.31 (s, 1H, pyrazol-NH). 13C-NMR (DMSO-
d6,75MHz): δ153.29, 139.49, 134.75, 132.14, 130.66,
129.09, 129.02, 122.80, 122.13, 121.83, 119.77, 118.58,
114.69. ESI-HRMS calcd for C17H15BrN5O+ ([M + H]+):
384.0454; found: 384.0467.
Mp 233-236°C, yield 45%. 1H-NMR (DMSO-d6,
300MHz): δ 7.01 (t, 1H, J = 7.2 Hz, Ph-H), 7.28 (t, 2H, J =
7.7 Hz, Ph-H), 7.56 (d, 2H, J = 8.0 Hz, Ph-H), 7.93 (d, 2H, J
= 8.5 Hz, Ph-H), 8.11 (s, 1H, N-CH), 8.34 (d, 2H, J = 8.5
Hz, Ph-H), 8.48 (s, 1H, CONH), 8.57 (s, 1H, CH=N), 10.54
(s, 1H, CONH). 13C-NMR (DMSO-d6,75MHz): δ153.26,
147.31, 139.48, 139.34, 134.42, 129.53, 129.04, 128.98,
124.32, 124.24, 122.83, 119.79, 115.59. ESI-HRMS calcd
for C17H15N6O3+ ([M + H]+): 351.1200; found: 351.1200.
3.1.9.13. N-Phenyl-2-((3-(4-Methylphenyl)-1H-Pyrazol-4-
yl) Methylene) Hydrazinecarboxamide(5e)
3.1.9.18. N-(4-Fluorophenyl)-2-((3-Phenyl-1H-Pyrazol-4-
yl) Methylene) Hydrazinecarboxamide (5j)
Mp 200-201°C, yield 30.9%. 1H-NMR (DMSO-d6,
300MHz): δ2.38 (s, 3H, CH3), 7.01 (t, 1H, J = 7.3 Hz, Ph-H),
7.27-7.60 (m, 8H, Ph-H), 8.04 (s, 1H, N-CH), 8.30 (s, 1H,
CONH), 8.56 (s, 1H, CH=N), 10.41 (s, 1H, CONH), 13.25
(s, 1H, pyrazol-NH). 13C-NMR (DMSO-d6,75MHz):
δ153.35, 139.56, 135.11, 129.85, 129.83, 129.02, 128.97,
128.49, 128.42, 122.76, 119.82, 118.59, 114.33, 21.29. ESI-
HRMS calcd for C18H18N5O+ ([M + H]+): 320.1506;
found: 320.1497.
Mp 236-240°C, yield 41.4%. 1H-NMR (DMSO-d6,
300MHz): δ 7.13 (t, 2H, J = 8.9 Hz, Ph-H), 7.45-7.65 (m,
7H, Ph-H), 8.01 (s, 1H, N-CH), 8.35 (s, 1H, CONH), 8.69 (s,
1H, CH=N), 10.42 (s, 1H, CONH), 13.18 (s, 1H, pyrazol-
1
NH). 13C-NMR (DMSO-d6,75MHz): δ158.15 (d, Jc-f
=
237.0 Hz), 153.48, 135.96 (4Jc-f = 2.3 Hz), 135.05, 129.29,
128.84, 128.80, 128.55, 128.49, 121.79 (3Jc-f = 7.7 Hz),
121.42, 115.43 (2Jc-f = 22.0 Hz), 114.56. ESI-HRMS calcd
for C17H15FN5O+ ([M
324.1259.
+
H]+): 324.1255; found:
3.1.9.14. N-Phenyl-2-((3-(4-Methoxylphenyl)-1H-Pyrazol-
4-yl) Methylene) Hydrazinecarboxamide(5f)
3.1.9.19. N-(4-Chlorophenyl)-2-((3-Phenyl-1H-Pyrazol-4-
yl) Methylene) Hydrazinecarboxamide (5k)
Mp 193°C, yield 72.3%. 1H-NMR (DMSO-d6, 300MHz):
δ3.82 (s, 3H, CH3), 7.01 (t, 1H, J = 7.3 Hz, Ph-H), 7.08 (d,
2H, J = 8.6 Hz, Ph-H), 7.29 (t, 2H, J = 7.8 Hz, Ph-H), 7.52
(d, 2H, J = 8.6 Hz, Ph-H), 7.59 (d, 2H, J = 8.2 Hz, Ph-H),
8.02 (s, 1H, N-CH), 8.27 (s, 1H, CONH), 8.55 (s, 1H,
CH=N), 10.38 (s, 1H, CONH). 13C-NMR (DMSO-
d6,75MHz): δ159.90, 153.35, 139.57, 135.29, 135.22,
129.99, 129.93, 129.08, 128.96, 122.76, 119.83, 114.72,
114.07, 55.73. ESI-HRMS calcd for C18H18N5O2+ ([M +
H]+): 336.1455; found: 336.1459.
Mp 237-240°C, yield 78.2%. 1H-NMR (DMSO-d6,
300MHz): δ 7.34 (t, 2H, J = 8.7 Hz, Ph-H), 7.48-7.59 (m,
5H, Ph-H), 7.67 (t, 2H, J = 8.8 Hz, Ph-H), 8.05 (s, 1H, N-
CH), 8.36 (s, 1H, CONH), 8.78 (s, 1H, CH=N), 10.50 (s, 1H,
CONH), 13.43 (s, 1H, pyrazol-NH). 13C-NMR (DMSO-
d6,75MHz): δ153.28, 138.68, 135.30, 129.36, 129.30,
128.85, 128.79, 128.74, 128.56, 128.50, 126.34, 121.42,
114.49. ESI-HRMS calcd for C17H15ClN5O+ ([M + H]+):
340.0960; found: 340.0952.
3.1.9.15. N-Phenyl-2-((3-(4-Hydroxyphenyl)-1H-Pyrazol-4-
yl) Methylene) Hydrazinecarboxamide(5g)
3.1.9.20 N-(4-Methoxyphenyl)-2-((3-Phenyl-1H-Pyrazol-4-
yl) Methylene) Hydrazinecarboxamide (5l)
Mp 237-240°C, yield 63.5%. 1H-NMR (DMSO-d6,
300MHz): δ 6.91 (d, 2H, J = 8.2 Hz, Ph-H), 7.00 (t, 1H, J =
7.3 Hz, Ph-H), 7.29 (t, 2H, J = 7.6 Hz, Ph-H), 7.40 (d, 2H, J
= 8.2 Hz, Ph-H), 7.62 (d, 2H, J = 7.9 Hz, Ph-H), 8.01 (s, 1H,
N-CH), 8.26 (s, 1H, CONH), 8.61 (s, 1H, CH=N), 9.87 (s,
1H, OH), 10.39 (s, 1H, CONH), 13.16 (s, 1H, pyrazol-NH).
13C-NMR (DMSO-d6,75MHz): δ158.31, 153.38, 139.60,
135.44, 135.38, 129.94, 129.82, 128.97, 122.74, 122.66,
Mp 210-212°C, yield 61.6%. 1H-NMR (DMSO-d6,
300MHz): δ 3.74 (s, 3H, CH3), 6.88 (d, 2H, J = 8.9 Hz, Ph-
H), 7.45-7.61 (m, 7H, Ph-H), 8.05 (s, 1H, N-CH), 8.34 (s,
1H, CONH), 8.49 (s, 1H, CH=N), 10.33 (s, 1H, CONH),
13.05 (s, 1H, pyrazol-NH). 13C-NMR (DMSO-d6,75MHz):
δ155.29, 153.59, 134.68, 132.61, 131.45, 129.27, 129.18,
128.87, 128.81, 128.56, 121.81, 114.63, 114.15, 55.64. ESI-
HRMS calcd for C18H18N5O2+ ([M + H]+): 336.1455;
found: 336.1451.
119.86,
116.09,
113.81.
ESI-HRMS
calcd
for
C17H16N5O2+ ([M + H]+): 322.1299; found: 322.1304.
3.1.9.16. N-Phenyl-2-((3-(2,4-Dichlorophenyl)-1H-Pyrazol-
4-yl) Methylene) Hydrazinecarboxamide(5h)
Mp 198-201°C, yield 50.4%. 1H-NMR (DMSO-d6,
300MHz): δ 7.01 (t, 1H, J = 7.0 Hz, Ph-H), 7.29 (t, 2H, J =
3.1.9.21. 1-((1-Methyl-3-Phenyl-1H-Pyrazol-4-yl) Methyli-
dene) Semicarbazide (6a)
Mp 221-222°C, yield 46.2%. 1H-NMR (DMSO-d6,
300MHz): δ3.72 (s, 3H, NCH3), 6.23 (s, 2H, NH2), 7.44-7.56

1026 Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9
Liang et al.
(m, 5H, Ph-H), 7.57 (s, 1H, N-CH), 7.99 (s, 1H, CH=N),
9.83 (s, 1H, NH). 13C-NMR (DMSO-d6,75MHz): δ156.01,
140.87, 135.49, 131.84, 129.24, 128.47, 128.27, 127.93,
115.72, 36.39. ESI-HRMS calcd for C12H14N5O+ ([M +
H]+): 244.1193; found: 244.1189.
131.05, 129.15, 129.07, 128.56, 128.49, 128.37, 128.23,
115.43, 55.90. ESI-HRMS calcd for C18H18N5S+ ([M +
H]+): 336.1277; found: 336.1274.
3.1.9.27. N-Phenyl-2-((1-Methyl-3-Phenyl-1H-Pyrazol-4-
yl) Methylene) Hydrazinecarboxamide (7a)
3.1.9.22. 1-((3-Phenyl-1-Propyl-1H-Pyrazol-4-yl) Methyli-
dene) Semicarbazide (6b)
Mp 187-191°C, yield 68.2%. 1H-NMR (DMSO-d6,
300MHz): δ 3.94 (s, 3H, NCH3), 7.01 (t, 1H, J = 7.2 Hz, Ph-
H), 7.30 (t, 2H, J = 7.5 Hz, Ph-H), 7.42-7.60 (m, 7H, Ph-H),
8.03 (s, 1H, N-CH), 8.40 (s, 1H, CONH), 8.55 (s, 1H,
CH=N), 10.43 (s, 1H, CONH). 13C-NMR (DMSO-
d6,75MHz): δ153.30, 149.83, 139.53, 134.83, 133.40,
131.70, 129.06, 129.00, 128.50, 128.40, 122.80, 119.79,
115.19, 55.36.ESI-HRMS calcd for C18H18N5O+ ([M +
H]+): 320.1506; found: 320.1510.
Mp156-161°C, yield 56.3%. 1H-NMR (DMSO-d6,
300MHz): δ 0.89 (t, 3H, J = 7.3 Hz, CH3), 1.82-1.89 (m, 2H,
CH2), 4.11 (t, 2H, J = 6.7 Hz, NCH2), 6.27 (s, 2H, NH2),
7.39-7.58 (m, 5H, Ph-H), 7.94 (s, 1H, N-CH), 8.27 (s, 1H,
CH=N), 9.93 (s, 1H, NH). 13C-NMR (DMSO-d6,75MHz):
δ157.19, 149.37, 133.55, 133.47, 130.22, 128.98,
128.42, 128.26, 115.29, 53.76, 23.46, 11.43. ESI-
HRMS calcd for C14H18N5O+ ([M + H]+): 272.1506;
found: 272.1498.
3.1.9.28. N-Phenyl-2-((1-Propyl-3-Phenyl-1H-Pyrazol-4-yl)
Methylene) Hydrazinecarboxamide (7b)
3.1.9.23. 1-((1-Benzyl-3-Phenyl-1H-Pyrazol-4-yl) Methyli-
dene) Semicarbazide (6c)
Mp 163-164°C, yield 57.2%. 1H-NMR (DMSO-d6,
300MHz): δ0.91 (t, 3H, J = 7.3 Hz, CH3), 1.82-1.92 (m, 2H,
CH2), 4.14 (t, 2H, J = 6.9 Hz, NCH2), 7.01 (t, 1H, J = 7.3
Hz, Ph-H), 7.27-7.61 (m, 9H, Ph-H), 8.04 (s, 1H, N-CH),
8.44 (s, 1H, CONH), 8.58 (s, 1H, CH=N), 10.45 (s, 1H,
CONH). 13C-NMR (DMSO-d6,75MHz): δ153.32, 149.76,
139.52, 134.95, 133.49, 130.81, 129.05, 129.00, 128.54,
128.38, 122.81, 119.83, 114.88, 53.84, 23.55, 11.44. ESI-
HRMS calcd for C20H22N5O+ ([M + H]+): 348.1819;
found: 348.1814.
Mp 143-144°C, yield 66.0%. 1H-NMR (DMSO-d6,
300MHz): δ 5.39 (s, 2H, CH2), 5.77 (s, 2H, NH2), 7.32-7.57
(m, 10H, Ph-H), 7.92 (s, 1H, N-CH), 8.37 (s, 1H, CH=N),
9.96 (s, 1H, NH). 13C-NMR (DMSO-d6,75MHz): δ157.15,
149.85, 137.44, 133.42, 133.32, 130.54, 129.12, 129.02,
128.48, 128.39, 128.31, 128.20, 115.95, 55.78. ESI-HRMS
calcd for C18H18N5O+ ([M + H]+): 320.1506; found:
320.1511.
3.1.9.24. 1-((1-Methyl-3-Phenyl-1H-Pyrazol-4-yl) Methyli-
dene) Thiosemicarbazide (6d)
3.1.9.29. N-Phenyl-1-((1-Benzyl-3-Phenyl-1H-Pyrazol-4-yl)
Methylidene) Semicarbazide (7c)
Mp 168°C, yield 45.2%. 1H-NMR (DMSO-d6, 300MHz):
δ 3.90 (s, 3H, CH3), 7.40-7.57 (m, 5H, Ph-H), 7.61 (s, 1H,
NH2), 8.07 (s, 1H, NH2), 8.17 (s, 1H, N-CH), 8.32 (s, 1H,
CH=N), 11.19 (s, 1H, NH). 13C-NMR (DMSO-d6,75MHz):
δ177.74, 150.33, 136.71, 133.12, 131.64, 129.06, 128.47,
128.41, 115.03, 55.87.ESI-HRMS calcd for C12H14N5S+
([M + H]+): 260.0964; found: 260.0960.
Mp 201-203°C, yield 56.5 %. 1H-NMR (DMSO-d6,
300MHz): δ 5.43 (s, 2H, NCH2), 7.01 (t, 1H, J = 6.5 Hz, Ph-
H), 7.30-7.59 (m, 14H, Ph-H), 8.05 (s, 1H, N-CH), 8.54 (s,
1H, CONH), 8.59 (s, 1H, CH=N), 10.46 (s, 1H, CONH).
13C-NMR (DMSO-d6,75MHz): δ 153.31, 150.23, 139.51,
137.44, 134.80, 133.26, 131.18, 129.14, 129.08, 128.99,
128.60, 128.51, 128.32, 128.14, 122.82, 119.87, 115.52,
55.81. ESI-HRMS calcd for C24H22N5O+ ([M + H]+):
396.1819; found: 396.1819.
3.1.9.25. 1-((3-Phenyl-1-Propyl-1H-Pyrazol-4-yl) Methyli-
dene) Thiosemicarbazide (6e)
3.2. Pharmacology
Mp 179-180°C, yield 60.3%. 1H-NMR (DMSO-d6,
300MHz): δ 0.88 (t, 3H, J = 7.3 Hz, CH3), 1.82-1.89 (m, 2H,
CH2), 4.11 (t, 2H, J = 6.8 Hz, NCH2), 7.40-7.58 (m, 5H, Ph-
H), 7.65 (s, 1H, NH2), 8.09 (s, 1H, NH2), 8.19 (s, 1H, N-
CH), 8.38 (s, 1H, CH=N), 11.20 (s, 1H, NH). 13C-NMR
(DMSO-d6,75MHz): δ177.74, 150.19, 136.74, 133.24,
130.75, 129.04, 128.54, 128.43, 114.78, 53.89, 23.40, 11.43.
ESI-HRMS calcd for C14H18N5S+ ([M + H]+): 288.1277;
found: 288.1273.
3.2.1. Cell Line and Reagents
Mouse RAW264.7 macrophage cell line was obtained
from the China Cell Bank (Beijing, China). Fetal bovine
serum was purchasedfrom Biological Industries (Kibbutz
Beit-Haemek, Israel). LPS waspurchased from Sigma (St.
Louis, MO, USA), and was dissolved in PBS. DXMS and
compounds were dissolved in DMSO for in vitro experiments.
3.2.2. Cell Treatment and ELISA Assay (TNF-α)
3.1.9.26. 1-((1-Benzyl-3-Phenyl-1H-Pyrazol-4-yl) Methyli-
dene) Thiosemicarbazide (6f)
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
μg/mL streptomycin at 37 C with 5% CO2. Cells were pre-
treated with test compounds (20 μg/mL), DXMS (20 μg/mL)
or vehicle control for 4 h, then treated with LPS (1μg/ml) for
24 h. After treatment, the supernatant was separated and used
Mp 146-147°C, yield 46.5%. 1H-NMR (DMSO-d6,
300MHz): δ 5.40 (s, 2H, CH2), 7.68 (s, 1H, NH2), 7.33-7.58
(m, 10H, Ph-H), 8.10 (s, 1H, NH2), 8.18 (s, 1H, N-CH), 8.47
(s, 1H, CH=N), 11.21 (s, 1H, NH). 13C-NMR (DMSO-
d6,75MHz): δ177.77, 150.62, 137.27, 136.61, 133.01,

Pyrazole-Semicarbazides as Anti-Inflammatory-Antimicrobial Agents
Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9 1027
for the determination of levels of TNF-α by ELISA using
mouse TNF-α ELISA Kits Biolegend (San diego, CA, USA).
plate. Penicillin, norfloxacin, gatifloxacin, moxifloxacin, and
oxacillin were used as positive controls. The inhibition activ-
ities of test compounds and positive drugs were expressed as
MIC value, which was defined as the concentration of a test
sample that completely inhibited bacteria growth during 24 h
incubation at 37 °C. Bacteria growth was determined by
measuring the absorption at 630 nm using a microtiter en-
zyme-linked immunosorbent assay (ELISA) reader. All ex-
periments were carried out three times in parallel.
3.2.3. Xylene-induced Ear-edema Model
In this screening, selected compounds were evaluated to
evaluate their anti-inflammatory activity via in vivo inhibi-
tion assay of xylene-induced ear edema in mice [21, 22].
Compounds and DXMS were dissolved in DMSO and ad-
ministered to mice intraperitoneally at a dose of 50 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. The DXMS
was tested in parallel as a reference drug.
4. RESULTS AND DISCUSSION
4.1. Anti-inflammatory Activity
4.1.1. 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) [24]. The LPS-induced TNF-α release
model was considered to be the effective screening method
for new anti-inflammatory agents in vitro. In the current
study, compounds (4a-4h, 5a-5l, 6a-f, and 7a-7c) were eval-
uated for their anti-inflammatory activity based on their abil-
ity to inhibit TNF-α release in LPS-stimulated RAW264.7
mouse macrophages. The cells were seeded and pretreated
with the test compounds or dexamethasone (DXMS) at a
concentration 20 μg/mL for 4 h before treatment with LPS (1
μg/mL) for 24 h at 37 °C. Cell-free supernatants were col-
lected 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).
3.2.4. Evaluation of Anti-bacterial Activity In Vitro
The anti-bacterial activity in vitro against six gram-
positive strains (S. aureus (CMCC(B) 26003 and CMCC
25923), S. pyogenes CMCC 32067, S. pneumoniae CMCC
31968, Enterococcusfaecalis CMCC 29212, and Bacillus
subtilis CMCC 63501), four gram-negative strains (E. coli
(CMCC 25922 and CMCC 44568) and P. aeruginosa
(CMCC 27853 and CMCC 10104)), and a Fungistrain (Can-
dida albicans CMCC 98001) was evaluated using a two-fold
serial dilution technique [23]. Test bacteria were grown to
mid-log phase in Mueller-Hinton broth (MHB) or Tryptone
Soya Broth (TSB) and diluted 1000-fold in the same medi-
um. The bacteria of 105 CFU/mL were inoculated into MHB
or TSB and dispensed at 200 μL/well in a 96-well microtiter
As shown in Fig. (1), most of the tested compounds sig-
nificantly inhibited LPS-induced TNF-α generation in vitro.
Fig. (1). Anti-inflammatory activities of the compounds 4a-4h, 5a-5l, 6a-f, and 7a-7c in the LPS-induced TNF-a release test in RAW264.7
macrophage. 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).

1028 Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9
Liang et al.
Fig. (2). Anti-inflammatory activities of the compounds 4g, 5i, 6b, and 7b in the xylene-induced ear-edema test in mice. The test compounds
and DXMS were administered i.e. at a dose of 50 mg/kg, and edema was quantified after30 min. ** p<0.01 and *** p<0.001compared with
the control group (all comparisons were made by ANOVA followed by Dunnett’s test).
Among these compounds, 4g, 5a, 5b, 5g, 5i, 6b, 6d, 6e, 7a,
7b, and 7c exhibited strong inhibition (inhibitory rates>50%).
Compounds 4g, 5i, 6b, and 7b showed 66.6%, 64.3%, 65.2%,
and 65.2% inhibition, respectively. The positive control drug
DXMS exhibited an inhibition rate of 77.2% at the same
concentration (20 μg/mL).
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 four com-
pounds, 4g, 5i, 6b, and 7b, were evaluated using thexylene-
induced ear edema mouse model. In this test, dimethyl sul-
foxide (DMSO) was used as the vehicle, and DXMS was
used as the positive control. Anti-inflammatory activity was
defined by the ability of each compound to prevent edema.
As shown in Fig. (2), all four tested compounds and DXMS
inhibited the ear edema, though the edema after treatment
with compound 4g was not significantly different from that
in the control group. Compound 7b inhibited edema by
48.63 %, which was more effective than DXMS at inhibiting
xylene-induced ear edema (44.86 %). The in vivo activities
of the tested compounds did not correlate with previous in
vitro data. This may be due to the different bioavailability
and pharmacokinetic profiles of these compounds. Com-
pound 7b, which has more lipophilic groups, showed
better membrane permeability and higher in vivo
activity.
The series of compounds 4a-4g showed relatively weaker
anti-inflammatory activity than the other three series of com-
pounds. SARs of this series indicated that the fluorine and
hydroxyl moieties could significantly increase the cytokine-
inhibitory ability but other substituents such as Cl, Br, CH3,
and OCH3 groups markedly decrease the inhibitory ability.
The replacement of =O with =S in compound 4ayielded com-
pound 4h, which did not show markedly improved cytokine-
inhibitory ability. The introduction of aryl groups at the termi-
nal N atom in series 4 produced compounds 5a-5l. In this
series, compounds 5a, 5b, 5g, and 5i, containing hydrogen,
fluorine, hydroxyl, and nitro groups, respectively, exhibited
better inhibitory ability compared to the others. This was
similar to the results obtained for series 4. In addition, the
introduction of substituents at the R2 position lowered the
anti-inflammatory activity, such as in compounds 5j-5l. The
introduction of alkyl groups to the pyrazole ring in the com-
pounds 4a and 4h produced compounds 6a-6c and 6d-6f,
respectively. From scheme 1, it can be observed that this
modification enhanced the cytokine-inhibitory ability, and
the compounds with methyl and propyl groups (6a, 6b, 6d,
6e) showed better inhibitory activity than the benzyl-
substituted compounds (6c and 6f). Similarly, the introduc-
tion of alkyl groups at the pyrazole ring in compound 5a
gave compounds 7a-7c, in which compound 7b, with a pro-
pyl group, exhibited the best activity.
4.2. Antimicrobial Activity
All test target compounds were evaluated for their in
vitro anti-bacterial activity using a serial dilution method to
obtain the minimum inhibitory concentration (MIC) against
several gram-positive and gram-negative bacterial and fungal
strains and fungi strain. Penicillin, Norfloxacin, Gatifloxacin,
Moxifloxacin, and Oxacillin were used as positive control
drugs.
The results of antimicrobial screening are described as
MIC values and listed in Table 1. Eleven of the tested com-
pounds showed antimicrobial activity against different strains
with MIC values ranging from 8 to 128 μg/mL.Compounds
4d, 5a, 5b, 5e, 5f, 5g, 5h, 6c, and 6d exhibited moderate inhi-
bition activity against gram-positive strains. Compounds 5g
and 5h exhibited the most potent inhibitory activities, with
an MIC value of 8 μg/mL against the Streptococcus pneu-
moniae CMCC 31968 and Staphylococcus aureus CMCC
25923, respectively. Compounds 6d, 7b and 7c inhibited
more than seven bacterial strains, including gram-negative
4.1.2. 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
edematousskin changes, partially associated with phospho-
lipase A2 (PLA2) [25, 26]. Xylene treatment in mice in-
creases the release of inflammatory mediators, such as his-

Pyrazole-Semicarbazides as Anti-Inflammatory-Antimicrobial Agents
Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9 1029
[2]
Tilg, H.; Moschen, A.R. Adipocytokines: Mediators linking adi-
pose tissue, inflammation and immunity. Nat. Rev. Immunol., 2006,
6, 772-783.
Philip, M.D.A.; Rowley, H.; Schreiber, H. Inflammation as a tumor
promoter in cancer induction. Semin. Cancer Biol., 2004, 14, 433-439.
Grivennikov, S.I.; Greten, F.R.; Karin, M. Immunity, inflamma-
tion, and cancer. Cell, 2010, 140, 883-899.
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.
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, 1935-1945.
Palomer, A.; Cabré, F.; Pascual, J.; Campos, J.; Trujillo, M.A.;
Entrena, A.; Gallo, M.A.; García, L.; Mauleón, D.; Espinosa, A.
Identification of novel cyclooxygenase-2 selective inhibitors using
pharmacophore models. J. Med. Chem., 2002, 45, 1402-1411.
Mandour, A.H.; El-Sawy, E.R.; Shaker, K.H.; Mustafa, M.A. Syn-
thesis, anti-inflammatory, analgesic and anticonvulsant activities of
1,8-dihydro-1-ary1-8-alkyl pyrazolo(3,4-b)indoles. Acta. Pharm.,
2010, 60, 73-88.
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.
Gupta, S.K.; Khanal, P.; Kumar, A.; Kumar, A.; Srivastava, N.;
Yogi, B.; Sinha, A. Synthesis, characterization of ethyl 5-
(substituted)-1H-pyrazole-3-carboxylate derivative as potent anti-
inflammatory agents. Anti-Inflamm. Anti-Allergy Agents Med.
Chem., 2018, 17, 32-38.
Song, M.X.; Zheng, C.J.; Deng, X.Q.; Sun, L.P.; Wu, Y.; Hong, L.;
Li, Y.J.; Liu, Y.; Wei, Z.; Jin, M.J.; Piao, H.R. Synthesis and anti-
bacterial evaluation of rhodanine-based 5-aryloxy pyrazoles against
selected methicillin resistant and quinolone-resistant Staphylococ-
cus aureus (MRSA and QRSA). Eur. J. Med. Chem., 2013, 60,
376-385.
Barakat, A.; Al-Majid, A.M.; Al-Qahtany, B.M.; Ali, M.; Teleb,
M.; Al-Agamy, M.H.; Naz, S.; Ul-Haq, Z. Synthesis, antimicrobial
activity, pharmacophore modeling and molecular docking studies
of new pyrazole-dimedone hybrid architectures. Chem. Cent. J.,
2018, 12, 29.
strains, and were considered as broad-spectrum antimicrobial
compounds. Additionally, compounds 7b and 7c showed
inhibitory activities against the fungi Candida albicans
CMCC 98001.
[3]
[4]
[5]
CONCLUSION
It can be concluded that pyrazoles, with (thio)-
semicarbazone moieties, have the potential to be developed
into new anti-inflammatory agents. A majority of synthe-
sized compounds exhibited potent TNF-α inhibitory activity,
and 5i, 6b, and 7b were shown to have comparable in vivo
anti-inflammatory activity to the reference drug, DXMS. On
the other hand, compounds 6d, 7b,and 7c displayed broad-
spectrum antimicrobial activity against several gram-positive
and gram-negative bacteria. Compound 7b is worth investi-
gating further because it exhibited both anti-inflammatory
and antimicrobial activities and can be used as a lead com-
pound for the development of a new class of dual anti-
inflammatory-antimicrobial agents.
[6]
[7]
[8]
[9]
[10]
ETHICS
APPROVAL
AND
CONSENT
TO
PARTICIPATE
After review of the procedure and aim of this study, local
ethical committee approval was obtained.
[11]
HUMAN AND ANIMAL RIGHTS
No humans were used in this study. All Procedures in-
volving 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.
[12]
[13]
Bhat, M.; Poojary, B.; Kalal, B.S.; Gurubasavaraja-Swamy, P.M.;
Kabilan, S.; Kumar, V.; Shruthi, N.; Alias Anand, S.A.; Pai, V.R.
Synthesis and evaluation of thiazolidinone-pyrazole conjugates as
anticancer and antimicrobial agents. Future Med. Chem., 2018, 10,
1017-1036.
CONSENT FOR PUBLICATION
Not applicable.
AVAILABILITY OF DATA AND MATERIALS
[14]
[15]
Abdellatif, K.R.A.; Bakr, R.B. New advances in synthesis and
clinical aspects of pyrazolo[3,4-d] pyrimidine scaffolds. Bioorg.
Chem., 2018, 78, 341-357.
The data that support the findings of this study are avail-
able from the corresponding author, [A.L. Pereora-Suarez],
upon reasonable request.
Mathew, B.; Suresh, J.; Anbazhagan, S.; Dev, S. Molecular Dock-
ing Studies of Some Novel Antidepressant 5-Substituted Phenyl-3-
(Thiophen-2-yl)-4,
5-Dihydro-1h-Pyrazole-1-Carboxamides
Against Monoamine Oxidase Isoforms. Cent. Nerv. Syst. Agents
Med. Chem., 2016, 16, 75-80.
FUNDING
[16]
[17]
[18]
[19]
[20]
Wu, Y.; Huang, Y.P.; Zhou, S.C.; Tan, Y.Q.; Xu, B.G.; Liang, Z.;
Deng, X.Q. Synthesis of 1,3-diaryl pyrazole derivatives and evalua-
tion of anticonvulsant and antimicrobial activities. Lat. Am. J.
Pharm., 2018, 37, 1017-1027.
This work was supported by the National Natural Science
Foundation of China (No. 21562028, No.81560561) and the
Doctoral Foundation of Jinggangshan University (No. JZB1316).
Wang, Y.; Gu, W.; Shan, Y.; Liu, F.; Xu, X.; Yang, Y.; Zhang, Q.;
Zhang, Y.; Kuang, H.; Wang, Z.; Wang, S. Design, synthesis and
anticancer activity of novel nopinone-based thiosemicarbazone de-
rivatives. Bioorg. Med. Chem. Lett., 2017, 27, 2360-2363.
Hariri, E.; Mahboubi, A.; Fathi, M.; Rahmani, P.; Mohammad
E.T.K.; Babaeian, M.; Mashayekhi, V.; Kobarfard, F. Synthesis
and antibacterial activity of novel hydroxysemicarbazone deriva-
tives. Iran J. Pharm. Res., 2016, 15(Suppl), 29-35.
Mohsin, A.S.M.; Jesmin, M.; Abul-Kalam A.M.; Khairul Islam,
M.; Zahan, R. Anti-inflammatory and analgesic activities of aceto-
phenonesemicarbazone and benzophenone semicarbazone. Asian
Pac. J. Trop. Biomed., 2012, 2, S1036-S1039.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
Declared none.
Subhashree, G.R.; Haribabu, J.; Saranya, S.; Yuvaraj, P.; Anantha-
Krishnan, D.; Karvembu, R.; Gayathri, D. In vitro antioxidant, anti-
inflammatory and in silico molecular docking studies of thiosemi-
carbazones. J. Mol. Struct., 2017, 1145, 160-169.
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, 227-235.

1030 Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9
Liang et al.
[21]
Wang, Y.; Chen, P.; Tang, C.; Wang, Y.; Li, Y.; Zhang, H. Antino-
ciceptive and anti-inflammatory activities of extract and two isolat-
ed flavonoids of Carthamustinctorius L. J. Ethnopharmacol., 2014,
151, 944-950.
[24]
[25]
[26]
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, 334-342.
Luber-Narod, J.; Austin-Ritchie, T.; Hollins, C.; Menon, M.; Mal-
hotra, R.K.; Baker, S.; Carraway, R.E. Role of substance P in
several models of bladder inflammation. Urol. Res., 1997, 25,
395-399.
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. Pharmacal. Res., 2007, 30, 323-328.
[22]
[23]
Wen, X.; Wang, S.B.; Liu, D.C.; Gong, G.H.; Quan, Z.S. Synthesis
and evaluation of the anti-inflammatory activity of quinoline deriv-
atives. Med. Chem. Res., 2015, 24, 2591-2603.
Clinical and Laboratory Standards Institute. Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobi-
cally, Approved Standard M7-A6; Clinical and Laboratory Stand-
ards Institute: Wayne, PA, USA, 2003.