Synthesis, structure, and antibacterial activity of novel 5-arylpyrazole derivatives

Article abstract of DOI:10.1071/CH07253

A series of novel 1-(acetyl,carboxamide,carbothioamide)substituted-5- (substituted-phenyl)-3-methy-4,5-dihydropyrazole derivatives have been synthesized and characterized by ¹H NMR, IR, ESI-MS, and elemental analysis. Compounds 6h and 6q were further characterized by single crystal X-ray structural analysis. All of the compounds have been screened for their antibacterial potential in vitro against Bacillus subtilis ATCC 6633, Escherichia coli ATCC 35218, Pseudomonas fluorescens ATCC 13525, and Staphylococcus aureus ATCC 6538. Among the tested compounds, some of them display significant activity against the tested strains, and compounds 5ac and 6h show potent activity with a minimum inhibitory concentration value of 1.562 ?g mL^?1 against B. subtilis ATCC 6633, which is comparable to the positive control penicillin. Structure?effect relationships are also discussed based on the experimental data. CSIRO 2008.

Full text of DOI:10.1071/CH07253

Full Paper
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2008, 61, 223–230
Synthesis, Structure, and Antibacterial Activity of Novel
5-Arylpyrazole Derivatives
Xin-Hua Liu,A Peng-Cheng Lv,B Bo Li,A Hai-Liang Zhu,A,B,D and Bao-An SongC
AKey Laboratory of Anhui Educational Department, Anhui University of Technology,
Maanshan, 243002, P. R. China.
BState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210093, P. R. China.
CKey Laboratory of Green Pesticide and Agriculture Bioengineering, Ministry of Education,
Guizhou University, Guiyang 550025, P. R. China.
DCorresponding author. Email: zhuhl@nju.edu.cn
A series of novel 1-(acetyl,carboxamide,carbothioamide)substituted-5-(substituted-phenyl)-3-methy-4,5-dihydropyrazole
1
derivatives have been synthesized and characterized by H NMR, IR, ESI-MS, and elemental analysis. Compounds 6h
and 6q were further characterized by single crystal X-ray structural analysis. All of the compounds have been screened
for their antibacterial potential in vitro against Bacillus subtilis ATCC 6633, Escherichia coli ATCC 35218, Pseudomonas
fluorescens ATCC 13525, and Staphylococcus aureus ATCC 6538. Among the tested compounds, some of them display
significant activity against the tested strains, and compounds 5ac and 6h show potent activity with a minimum inhibitory
concentration value of 1.562 µg mL−1 against B. subtilisATCC 6633, which is comparable to the positive control penicillin.
Structure–effect relationships are also discussed based on the experimental data.
Manuscript received: 20 July 2007.
Final version: 7 February 2008.
Introduction
ATCC 6633, Escherichia coli ATCC 35218, Pseudomonas flu-
orescens ATCC 13525, and Staphylococcus aureus ATCC 6538
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) colorimetric method.The results show that some
compounds in this series exhibited potent antibacterial activity
against B. subtilis ATCC 6633 and P. fluorescens ATCC 13525,
and compounds 5ac and 6h display potent activity with a mini-
mum inhibitory concentration (MIC) value of 1.562 µg mL−1
against B. subtilis ATCC 6633, which is comparable to the
positive control penicillin.
The identification of novel structures that can be potentially use-
ful in the design of new, potent antibacterial agents is still a major
challenge to medicinal chemistry researchers. Many pyrazole
derivatives are acknowledged to possess a wide range of bio-
activities that include antibacterial activity.[1–6] Much attention
has been directed towards pyrazole derivatives as antimicrobial
agents after the discovery of pyrazofurin, which demonstrated
a broad spectrum of antimicrobial activity.[7] Hoffmann–La
Roche’s group[8,9] has developed a new lead DNA gyrase
inhibitor(compound 1, Fig. 1), which has strong inhibitory activ-
ity against DNA gyrase, and this inhibitory effect can cause
bacterial cell death.[10] Recently, it was reported that compounds
2a, 2b, and 3 (Fig. 1) are selective inhibitors of DNA gyrase with
moderate antibacterial activity.[11,12] Among the compounds
mentioned above, it can be seen that those that contain a diaryl
pyrazole template show weak antibacterial activity. A possible
reason is that these compounds cannot penetrate the membrane
of bacterial cells easily. Motivated by the aforementioned find-
ings, our aim here is to find new, potent DNA gyrase inhibitors
with high antibacterial activity.
Results and Discussion
Synthesis
In order to optimize the reaction conditions for preparation of
compound 4,[13] the synthesis of 4b was carried out under dif-
ferent conditions. The effects of solvent system, base, reaction
time, and temperature are summarized in Table 1. A yield as
high as 75.8% can be achieved when a mixture of sodium hydro-
xide and 4-hydroxybenzaldehyde (2:1, mol ratio) is reacted in
ethanol at 25◦C for 20 h. Compound 5 was obtained by cycliza-
tion of the α,β-unsaturated ketones, in which different a solvent
was selected, such as n-butanol[14] and N,N-dimethylformamide
(DMF). Compound 5bd was obtained on refluxing in DMF but
not in n-butanol. The reaction conditions for the synthesis of
compounds 5 are illustrated in Table 2.
Among the previous research, few reports have been
dedicated to the synthesis and antibacterial activity evalu-
ation of 1-(acetyl,carboxamide,carbothioamide)substituted-5-
(substituted-phenyl)-3-methy-4,5-dihydropyrazole derivatives,
therefore, we have designed and synthesized a series of novel 5-
aryl pyrazole and diaryl pyrazole derivatives that have a similar
structure to compound 3 (Fig. 1).The antibacterial activity of the
prepared compounds have been tested against Bacillus subtilis
Compounds 6g–p were obtained in chloroform by refluxing
for 6 h with pyridine as catalyst. No product was obtained when
the temperature was below 40◦C and the reaction time was less
© CSIRO 2008
10.1071/CH07253
0004-9425/08/030223

224
X.-H. Liu et al.
O
S
H
N
O
N
O
H2N
N
N
CF3
S
O
O
Celecoxib
1
CH3
H
N
MeOOC
H
N
N
H
N
N
2a: R1 ϭ 4-Bu
HN
O
O
2b: R2 ϭ 3,4-diCl
R
2
3
Fig. 1.
Table 1. Yields of 4b at different reaction conditions
C9
—, no product
C5
C11
C14
C16
Entry Solvent system
Base
Time Temp. YieldB
O1
proportionA
[h]
[◦C]
[%]
C17
C8
O2
C20
C18
1
2
3
4
5
6
CH3COCH3-H2O
CH3COCH3-H2O
CH3COCH3-C2H5OH
CH3COCH3-C2H5OH
CH3COCH3-C2H5OH
CH3COCH3-C2H5OH
1:1
1:2
1:1.5
1:2
1:2
1:2
20
24
10
15
24
20
25
30
20
15
28
25
—
—
35.8
69.7
73.2
75.8
N2
C13
O3
C7
C1
N1
C22
C12
C10
C2
C6
Cl
AMol of 4-hydroxybenzaldehyde: mol of sodium hydroxide.
BIsolated yields after recrystallization, yields are based on
4-hydroxybenzaldehyde.
C4
C3
Fig. 2. ORTEP drawing of 6h.
Table 2. The reaction conditions for the transformation to 5
C16
Entry Solvent
Reagent
Time Temp Yield
[h]
[◦C]
[%]
C35
N4
O1
N1
5ac
5bc
5bd
5be
5bf
CH3COOH (98%) N2H4·H2O
CH3COOH (98%) N2H4·H2O
3
3
3
6
3
Reflux 79.8
Reflux 70.5
Reflux 31.2
Reflux 30.1
Reflux 50.2
C1
C9
C3
C11
C24
DMF
Semicarbazide
Thiosemicarbazide
N2H4·H2O
C34
C21
C17
C28
C12
C22
C7
C14
n-butanol
C2H5OH
C31
O2
C19
C27
C37
C5
C33
C13
C29
C38
O4
C36
C26
C23
C18
C20
C30
C32
N3
C10
C4
C6
O3
C25
than 3 h. The effect of the position of the hydroxy group of the
5-arylpyrazole on the yields of compounds 6g–p was studied,
and it can be concluded that para-substituted hydroxy groups
afforded higher yields than ortho-substituted hydroxy groups.
A possible reason may be steric hindrance effects.
To optimize the reaction conditions for the preparation of
compound 6q (6r), the synthesis was carried out with different
bases, such as NaHCO3, KHCO3, Et3N, NaOH, and pyridine.
It was found that the yield was up to 62.1% when the reac-
tion mixture was refluxed for 10 h in acetone catalyzed by
NaOH. In addition, the synthesis conditions of 7s (7t) is in
analogy with 6q, however, amide was obtained instead of ben-
zoate under the similar conditions. The presence of the amide
N2
C8
C2
C15
Fig. 3. ORTEP drawing of 6q.
1
(–CONH–) and hydroxy (–OH) groups were confirmed by H
NMR spectroscopy, the signals of which were easily recognized
at δH 1.61 (br s, 1H) and 5.17 (br s, 1H).
The single-crystal X-ray structure of compounds 6h and 6q
are illustrated in Figs 2 and 3.

Novel 5-Arylpyrazole Derivatives
225
Table 3. Minimum inhibitory concentrations (MIC, µg mL−1) of the synthesized compounds
Compounds
Microorganisms
Gram positive
Gram negative
B. subtilis S. aureus
P. aeruginosa
E. coli
ATCC 6633
ATCC 6538
ATCC 13525
ATCC 35218
5ac
5bc
5bd
5be
5bf
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
1.562
12.5
12.5
12.5
12.5
6.25
1.562
12.5
3.125
12.5
6.25
12.5
12.5
12.5
12.5
3.125
12.5
6.25
6.25
1.562
0.39
0.78
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
1.562
1.562
3.125
3.125
12.5
12.5
12.5
12.5
6.25
12.5
3.125
12.5
12.5
6.25
12.5
12.5
12.5
12.5
6.25
12.5
12.5
6.25
6.25
3.125
1.562
>50
12.5
>50
>50
>50
6.25
>50
6.25
>50
>50
>50
>50
>50
>50
>50
12.5
>50
>50
>50
6.25
3.125
3.125
6r
7s
7t
Penicillin
Kanamycin
Novobiocin
In 6h, the bond length of C(20)–N(1) (1.278(5) Å) is shorter
than that of a typical C=N bond (1.34 Å), which is indicative of
significant double bond character; the C(17)–N(2) (1.468(3) Å)
distance is in line with a normal C–N (1.47 Å) distance, which
is indicative of a single bond. The bond angles of C(17)–N(2)–
N(1), C(8)–C(20)–N(1), and C(20)–N(1)–N(2) are 113.6(3)◦,
114.5(7)◦, and 107.6(3)◦, respectively. The benzene ring I [C(2),
C(3), C(4), C(6), C(10), C(12)] is fairly planar, and the deviation
from the least-squares plane through the ring atoms is 0.0024◦.
While for the second benzene ring II [C(5), C(9), C(11), C(16),
C(18), C(14)], the deviation from the least-squares plane through
the ring atoms is 0.0034◦. The dihedral angles between the plane
of the pyrazole ring [N(1), N(2), C(8), C(17), C(20)] and the
planes of benzene ring I and benzene ring II are 88.4(9)◦ and
86.6(8)◦, respectively.
ATCC 6633 and S. aureus ATCC 6538) and two Gram-negative
bacterial strains (E. coli ATCC 35218 and P. fluorescens ATCC
13525) by the MTT method.The MICs of the compounds against
the four strains are presented inTable 3.The antibacterial activity
of reference compounds kanamycin, penicillin, and novobiocin
are also included.
Compounds 5ac and 6h display potent activity with MIC
values of 1.562 µg mL−1 against B. subtilis ATCC 6633, which
is comparable to the positive control penicillin. Compounds
6g, 6j, 6l, 6q, 7s, and 7t show moderate antibacterial activity
against B. subtilis ATCC 6633 with MIC values of 3.125–
6.25 µg mL−1, while the other compounds display mild antibac-
terial activity against B. subtilis ATCC 6633 with MIC values of
12.5 µg mL−1
.
Compounds 5ac and 6i also exhibit significant antibac-
In 6q, the bond length of C(32)–N(2) (1.281(4) Å) is shorter
than that of typical a C=N bond (1.34 Å), which is indicative of
significant double bond character; the C(38)–N(3) (1.471(3) Å)
distance is in line with a normal C–N (1.47 Å) distance, which is
indicative of a single bond.The bond angles of C(38)–N(3)–N(2)
and N(3)–N(2)–C(32) are 113.1(2)◦ and 107.4(2)◦, respectively.
The benzene ring I [C(3), C(12), C(19), C(31), C(11), C(1)]
is fairly planar, and the deviation from the least-squares plane
through the ring atoms is 0.0039◦. While for the second benzene
ring II [C(27), C(22), C(7), C(14), C(26), C(33)], the deviation
from the least-squares plane through the ring atoms is 0.0111◦.
The dihedral angles between the plane of the pyrazole ring [N(2),
N(3), C(38), C(29), C(32)] and the planes of benzene ring I and
benzene ring II are 129.8(5)◦ and 109.6(4)◦, respectively.
terial activity, it being equal to that of kanamycin against
P. aureus ATCC 13525 with MIC values of 3.125 µg mL−1
,
while compounds 6g, 6l, 6q, and 7t show moderate antibac-
terial activity against P. aureus ATCC 13525 with MIC values
of 6.25 µg mL−1. Other compounds display mild antibacterial
activity against P. aureus ATCC 13525 with MIC values of
12.5 µg mL−1
.
Among all the synthesized compounds, compounds 6g and
6i exhibit high antibacterial activity against E. coli ATCC
35218, while compounds 5bc and 6q display moderate antibac-
terial activity against E. coli ATCC 35218 with MIC values
of 12.5 µg mL−1. Other compounds show mild antibacterial
activity against E. coli ATCC 35218 with MIC values of
50 µg mL−1. However, the compounds prepared were relatively
inactive against S. aureus ATCC 6538 with MIC values of more
than 50 µg mL−1 compared with the three tested strains above.
Based on the experimental data presented inTable 3, it can be
concluded that N-acetyl arylpyrazole derivatives exhibit higher
In-Vitro Antibacterial Assay
All the synthesized compounds were screened for antibacterial
activity against two Gram-positive bacterial strains (B. subtilis

226
X.-H. Liu et al.
O
O
CH3
H
(i)
(ii)
HO
R1
4
H3C
R
1 ϭ o
OH (a), p
OH (b)
O
O
C
S
C
N
R1
N
R
2 ϭ
CH3 (c),
NH2 (d),
NH2 (e),
C
H (f).
R2
5
Scheme 1. Reagent and conditions: (i) CH3COCH3, NaOH, EtOH, 25◦C, 15 h; (ii): (5ac, 5bc) N2H4·H2O,
98% CH3COOH, reflux, 2 h. (5bd) semicarbazide, DMF, reflux, 4 h. (5be) thiosemicarbazide, n-butanol, reflux,
5 h. (5bf) N2H4·H2O, C2H5OH, reflux, 3 h.
antibacterial activity against selected strains than thiosulfate
arylpyrazole and acetamide arylpyrazole derivatives. Further-
more, the fluorine and chlorine substituents play an important
role on the antibacterial activity. In addition, the compounds
that have phenolic hydroxy groups show a higher antibacterial
activity against the tested strains than other compounds.
210002, P. R. China), and penicillin (North China Pharma-
ceutical Co. Ltd, D0211107, Hebei 050015, P. R. China) are
included.
Synthesis
4-(4-Hydroxyphenyl)but-3-en-2-one 4b
The biological activity of a particular substance depends on a
complex sum of individual properties, which includes compound
structure, affinity for the target site, survival in the medium
of application, survival within the biological system, transport
properties, and state of the target organism.[15] Structural analy-
sisofthesecompoundsindicatesthatthe1-acetyl-4,5-dihydro-3-
methyl-5-(2-substitued-phenyl)-1H-pyrazole moiety may have
potentialantibacterialactivityanddeservesfurtherinvestigation.
Aqueous sodium ethanol solution (16.0 mmol) was added to
a solution of salicylaldehyde (8.2 mmol) and acetone (82 mmol)
at room temperature. The resulting yellow solution was then
stirred at 25◦C for 15 h. A brown mixture was obtained that was
acidified by dropwise addition of aqueous HCl (12 mmol) at
25◦C. The resulting solid was dried under vacuum to yield 4b
(1.01 g, 75.8%), mp 45–46◦C. δH (CDCl3) 2.38 (s, 3H), 5.96 (br
s, 1H, OH), 6.58 (d, J 16.1, 1H), 6.87–7.46 (m, 4H, ArH), 7.51
(d, J 13.0, 1H). Compound 4a was synthesized according to the
same method.
Conclusions
In this paper, a series of novel 1-(acetyl,carboxamide,
carbothioamide)substituted-5-(substituted-phenyl)-3-methy-4,5-
dihydropyrazole derivatives were synthesized and screened for
their antibacterial potential in vitro against B. subtilis ATCC
6633, E. coli ATCC 35218, P. fluorescens ATCC 13525, and
S. aureus ATCC 6538. Among the tested compounds, some of
them displayed significant activity against the tested strains, and
compounds 5ac and 6h displayed potent activity with MIC val-
ues of 1.562 µg mL−1 against B. subtilisATCC 6633, which was
comparable to the positive control penicillin. Structural analysis
of these compounds indicated that the 1-acetyl-4,5-dihydro-3-
methyl-5-(2-substitued-phenyl)-1H-pyrazole moiety has poten-
tial antibacterial activity and deserves further investigation.
4-(2-Hydroxyphenyl)but-3-en-2-one 4a
Yield 1.07 g, 80.3%, mp 77–78◦C. δH (CDCl3) 2.29 (s, 3H),
6.08 (br s, 1H, –OH), 6.49 (d, J 16.1, 1H), 6.62–7.20 (m, 4H,
ArH), 7.88 (d, J 13.5, 1H).
1-Acetyl-4,5-dihydro-3-methyl-5-(2-hydroxyphenyl)-
1H-pyrazole 5ac
To
a
solution of 4-(2-hydroxyphenyl)but-3-en-2-one
(10 mmol) in acetic acid (20 mL) was added hydrazine mono-
hydrate (40 mmol) and the reaction mixture was refluxed for
2 h. The mixture was cooled, poured into crush ice, and allowed
to stand at room temperature overnight. The solid product was
collected by filtration and recrystallized from acetone to give the
title compound 5ac as colourless crystals.Yield 58.7%, mp 175–
176◦C. δH (CDCl3) 2.10 (s, 3H, Me), 2.22 (s, 3H, Me), 2.88 (dd, J
18.3 and 3.1, 1H, 4Ha), 3.42 (dd, J 18.2 and 11.2, 1H, 4Hb), 5.56
(dd, J 11.1 and 3.0, 1H, 5H), 6.69–7.10 (m, 4H, ArH), 9.19 (br
s, 1H, OH). νmax (KBr)/cm−1 1648 (C=O), 1627 (C=N), 3430
(–OH). m/z (ESI) 218.0 (C12H14N2O2, [M + H]+). (Found: C
66.12, H 6.21, N 12.59. Calc. for C12H14N2O2: C 66.04, H 6.47,
N 12.84%.)
Experimental
General
1
Melting points were measured uncorrected. H NMR spectra
were recorded on a Varian INOVA300 (300 MHz) pulse Fourier-
transform NMR spectrometer in CDCl3 or (D6)DMSO using
tetramethylsilane as an internal standard. Elemental analyses
were performed with a Vario-III CHN analyzer and were within
0.4% of the theoretical values. Electrospray ionization (ESI)
mass spectra were obtained on a Mariner System 5303 mass
spectrometer. The single crystal structure was determined on a
Bruker SMART 1000 CCD diffractometer.The reagents were all
of analytical grade or chemically pure. The antibacterial activity
of the reference compounds kanamycin, novobiocin (Nanjing
Zhuyan Biotechnology Co. Ltd, Amresco 060D0504, Nanjing
Compounds 5bc–5bf (Scheme 1) were synthesized accord-
ing to the same method. The reagents and solvents are shown in
Table 2.
1-Acetyl-4,5-dihydro-3-methyl-5-(4-hydroxyphenyl)-
1H-pyrazole 5bc
The title compound was obtained as colourless crystals.Yield
61.3%, mp 210–211◦C. δH ((D6)DMSO) 1.98 (s, 3H, Me), 2.11

Novel 5-Arylpyrazole Derivatives
227
H3C
H3C
(iii)
N
N
R3
N
N
OH
Ac
Ac
6
O
C
O
C
O
C
o
R
3 ϭ
o
o
O
(g),
O
O
(i),
Cl
(h),
Cl
O
C
O
O
C
p
o
C
p
O
O
(k),
O
(j),
(l),
Cl
F
O
O
C
O
p
C
p
p
C
O
O
O
(o),
(n),
(m),
CF3
Cl
F
O
H2
H2
p
C
p
o
C
C
O
O
O
(r),
(p),
(q),
N
Scheme 2. Reagents and conditions: (iii): (a) acid chloride, pyridine, CHCl3, reflux, 6 h. (b) 1-(chloromethyl)benzene,
NaOH, CH3COCH3, reflux, 10 h.
(s, 3H, Me), 2.48 (dd, J 18.3 and 3.1, 1H, 4Ha), 3.31 (dd, J 18.2
and 11.2, 1H, 4Hb), 5.44 (dd, J 11.1 and 3.0, 1H, 5H), 6.64–
6.67 (d, J 9.0, 2H), 6.90–6.92 (d, J 8.7, 2H), 9.28 (s, 1H, OH).
νmax (KBr)/cm−1 1644 (C=O), 1620 (C=N), 3427 (OH). m/z
(ESI) 218.2 (C12H14N2O2, [M + H]+). (Found: C 65.82, H 6.42,
N 12.66. Calc. for C12H14N2O2: C 66.04, H 6.47, N 12.84%.)
(dd, J 18.4 and 3.3, 1H, 4Ha), 3.80 (dd, J 18.4 and 11.4, 1H,
4Hb), 5.44 (dd, J 3.3 and 11.4, 1H, 5H), 6.77 (d, J 9.0, 2H), 6.98
(d, J 8.7, 2H), 7.41–9.84 (br, 3H, OH, NH2). νmax (KBr)/cm−1
3435 (–OH), 3347 and 3200 (NH2), 1271 (C=S), 1627 (C=N).
m/z (ESI) 235.0 (C11H13N3OS, [M + H]+). (Found: C 56.29,
H 5.20, N 18.07. Calc. for C11H13N3OS: C 56.15, H 5.57,
N 17.86%.)
1-Acetamide-4,5-dihydro-3-methyl-5-
(4-hydroxyphenyl)-1H-pyrazole 5bd
1H-4,5-dihydro-3-methyl-5-
(4-hydroxyphenyl)pyrazole 5bf
The title compound was obtained as colourless crystals.Yield
31.2%, mp 215–216◦C. δH ((D6)DMSO) 2.01 (s, 3H, Me), 2.80
(dd, J 18.2 and 3.2, 1H, 4Ha), 3.76 (dd, J 18.3 and 11.7, 1H,
4Hb), 5.58 (dd, J 3.2 and 11.3, 1H, 5H), 6.69 (d, J 9.0, 2H), 6.94
(d, J 8.8, 2H), 7.61–9.87 (br, 3H, OH, NH2). νmax (KBr)/cm−1
3432 (–OH), 3340 and 3191 (NH2), 1668 (C=O), 1624 (C=N).
m/z (ESI) 219.4 (C11H13N3O2, [M + H]+). (Found: C 60.44,
H 5.68, N 18.83. Calc. for C11H13N3O2: C 60.26, H 5.98,
N 19.17%.)
The title compound was obtained as colourless crystals.Yield
40.2%, mp 155–156◦C. δH (CDCl3) 2.07 (s, 3H, Me), 2.89 (dd,
J1 10.9, J2 16.1, 1H, 4Ha), 3.97 (dd, J1 10.9, J2 16.1, 1H, 4Hb),
5.37 (dd, J1 3.6, J2 10.8, 1H, 5H), 5.42 (br s, 1H, –OH), 6.44
(br s, 1H, NH), 6.72 (d, J 8.9, 2H), 6.98 (d, J 8.8, 2H). νmax
(KBr)/cm−1 3443 (–OH), 3378 (NH), 1632 (C=N). m/z (ESI)
175.9 (C10H12N2O, [M + H]+). (Found: C 68.40, H 7.02, N
16.04. Calc. for C10H12N2O: C 68.16, H 6.86, N 15.90%.)
1-Carbothioamide-4,5-dihydro-3-methyl-5-
General Procedure for the Preparation of 6g–p
(4-hydroxyphenyl)-1H-pyrazole 5be
To a solution of the 1-(5-(substituted-hydroxyphenyl)-3-
methyl-4,5-dihydropyrazol-1-yl)ethanone (2 mmol), pyridine
(0.010 mmol), andchloroform(30 mL), acidchloride(2.5 mmol)
The title compound was obtained as colourless crystals.Yield
30.1%, mp 208–210◦C. δH ((D6)DMSO) 2.07 (s, 3H, Me), 2.77

228
X.-H. Liu et al.
H3C
H3C
(iv)
N
N
N
N
OH
OH
X
N
H
X
H2N
7
Ph
X ϭ O (s), S (t)
Scheme 3. Reagent and conditions: (iv): 1-(cholormethyl)benzene, NaOH, CH3COCH3, reflux, 10 h.
was added dropwise at 25◦C for 30 min. The reaction mixture
was refluxed for 6 h and washed with 20 mL of H2O and a 5%
NaHCO3 solution, respectively, and then dried over anhydrous
MgSO4. The product was collected by removing the solvent
under vacuum, and the crude residue was purified with a silica
gel column and was eluted with acetone/petroleum ether (4/1)
to afford 6g–p (Scheme 2).
340.8 (C19H17FN2O3, [M + H]+). (Found: C 66.89, H 4.82,
N 8.01. Calc. for C19H17FN2O3: C 67.05, H 5.03, N 8.23%.)
4-(1-Acetyl-3-methyl-4,5-dihydro-1H-pyrazol-5-yl)phenyl
2-Chlorobenzoate 6k
The title compound was obtained as colourless crystals. Yield
50.6%, mp 154–156◦C. δH (CDCl3) 2.07 (s, 3H, Me), 2.31 (s,
3H, Me), 2.74 (dd, J1 4.4, J2 18.1, 1H, 4Ha), 3.37 (dd, J1 11.7,
J2 18.1, 1H, 4Hb), 5.47 (dd, J1 4.4, J2 11.7, 1H, 5H), 7.17–
7.26 (m, 4H), 7.35–7.40 (m, 1H), 7.45–7.52 (m, 2H), 7.99 (d,
J 6.4, 1H). νmax (KBr)/cm−1 1737 (COO), 1652 (C=O), 1592
(C=N). m/z (ESI) 356.2 (C19H17ClN2O3, [M + H]+). (Found: C
64.12, H 5.04, N 7.61. Calc. for C19H17ClN2O3: C 63.96, H 4.80,
N 7.85%.)
2-(1-Acetyl-3-methyl-4,5-dihydro-1H-pyrazol-5-yl)phenyl
Benzoate 6g
The title compound was obtained as colourless crystals. Yield
46.2%, mp 135–136◦C. δH ((D6)DMSO) 1.81 (s, 3H, Me), 2.09
(s, 3H, Me), 2.63 (dd, J1 5.4, J2 18.3, 1H, 4Ha), 3.38 (dd, J1
12.0, J2 18.2, 1H, 4Hb), 5.40 (dd, J1 5.4, J2 12.0, 1H, 5H), 7.18–
7.27 (m, 3H), 7.34–7.39 (m, 1H), 7.60–7.79 (m, 3H), 8.11 (d,
J 7.2, 2H). νmax (KBr)/cm−1 1734 (COO), 1664 (C=O), 1600
(C=N). m/z (ESI) 322.9 (C19H18N2O3, [M + H]+). (Found:
C 71.01, H 5.42, N 8.77. Calc. for C19H18N2O3: C 70.79, H 5.63,
N 8.69%.)
4-(1-Acetyl-3-methyl-4,5-dihydro-1H-pyrazol-5-yl)phenyl
Benzoate 6l
The title compound was obtained as colourless crystals. Yield
57.9%, mp 140–141◦C. δH (CDCl3) 2.04 (s, 3H, Me), 2.31 (s,
3H, Me), 2.74 (dd, J1 4.6, J2 18.1, 1H, 4Ha), 3.37 (dd, J1 11.7,
J2 18.1, 1H, 4Hb), 5.48 (dd, J1 4.6, J2 11.5, 1H, 5H), 7.15 (d, J
8.6, 2H), 7.22 (d, J 8.8, 2H), 7.48–7.65 (m, 3H), 8.16 (d, J 7.1,
2H). νmax (KBr)/cm−1 1730 (COO), 1655 (C=O), 1600 (C=N).
m/z (ESI) 322.0 (C19H18N2O3, [M + H]+). (Found: C 71.1,
H 5.8, N 9.0. Calc. for C19H18N2O3: C 70.8, H 5.6, N 8.7%.)
2-(1-Acetyl-3-methyl-4,5-dihydro-1H-pyrazol-5-yl)phenyl
Chlorobenzoate 6h
The title compound was obtained as colourless crystals. Yield
52.1%, mp 140–141◦C. Crystal data of 6h. C19H17ClN2O3,
M 359.8, orthorhombic, space group Pna2(1), a 9.3230(19), b
15.291(3), c 12.440(3) Å, α 90◦, β 90◦, γ 90◦, V 1773.4(6) nm3,
T 293(2) K, Z 4, Dc 1.336 g cm−3, F(000) 744. Reflections
collected/unique: 1503/1824, Fine, R1 0.0390, wR(F2) 0.0948.
4-(1-Acetyl-3-methyl-4,5-dihydro-1H-pyrazol-5-yl)phenyl
4-Chlorobenzoate 6m
2-(1-Acetyl-3-methyl-4,5-dihydro-1H-pyrazol-5-yl)phenyl
4-Chlorobenzoate 6i
The title compound was obtained as colourless crystals. Yield
61.2%, mp 174–175◦C. δH (CDCl3) 2.07 (s, 3H, Me), 2.31 (s,
3H, Me), 2.74 (dd, J1 4.4, J2 18.1, 1H, 4Ha), 3.37 (dd, J1 11.5,
J2 17.9, 1H, 4Hb), 5.48 (dd, J1 4.6, J2 11.5, 1H, 5H), 7.16 (d, J
8.6, 2H), 7.26 (d, J 8.6, 2H), 7.49 (d, J 8.4, 2H), 8.12 (d, J 8.4,
2H). νmax (KBr)/cm−1 1731 (COO), 1662 (C=O), 1599 (C=N).
m/z (ESI) 356.1 (C19H17ClN2O3, [M + H]+). (Found: C 63.9,
H 5.0, N 7.9. Calc. for C19H17ClN2O3: C 64.0, H 4.8, N 7.9%.)
The title compound was obtained as colourless crystals. Yield
56.1%, mp 158–159◦C. δH ((D6)DMSO) 2.00 (s, 3H, Me), 2.29
(s, 3H, Me), 2.75 (dd, J1 4.4, J2 18.1, 1H, 4Ha), 3.39 (dd, J1
11.5, J2 18.0, 1H, 4Hb), 5.48 (dd, J1 4.6, J2 11.7, 1H, 5H),
7.10–7.19 (m, 4H), 7.42 (d, J 8.4, 2H), 8.07 (d, J 8.4, 2H). νmax
(KBr)/cm−1 1736 (COO), 1652 (C=O), 1610 (C=N). m/z (ESI)
356.9 (C19H17ClN2O3, [M + H]+). (Found: C 63.74, H 5.07,
N 7.71. Calc. for C19H17ClN2O3: C 63.96, H 4.80, N 7.85%.)
4-(1-Acetyl-3-methyl-4,5-dihydro-1H-pyrazol-5-yl)phenyl
2-Fluorobenzoate 6n
2-(1-Acetyl-3-methyl-4,5-dihydro-1H-pyrazol-5-yl)phenyl
2-Fluorobenzoate 6j
The title compound was obtained as colourless crystals. Yield
58.7%, mp 132–133◦C. δH ((D6)DMSO) 2.05 (s, 3H, Me), 2.32
(s, 3H, Me), 2.71 (dd, J1 4.6, J2 18.1, 1H, 4Ha), 3.47 (dd, J1
11.7, J2 18.1, 1H, 4Hb), 5.39 (dd, J1 4.6, J2 11.7, 1H, 5H), 7.12
(d, J 6.4, 2H, ArH), 7.16–7.44 (m, 5H, ArH), 8.14 (d, J 6.4, 1H).
νmax (KBr)/cm−1 1743 (COO), 1658 (C=O), 1598 (C=N). m/z
(ESI) 340.0 (C19H17FN2O3, [M + H]+). (Found: C 67.0, H 5.2,
N 8.4. Calc. for C19H17FN2O3: C 67.1, H 5.0, N 8.2%.)
The title compound was obtained as colourless crystals. Yield
45.2%, mp 151–152◦C. δH (CDCl3) 2.02 (s, 3H, Me), 2.41
(s, 3H, Me), 2.76 (dd, J1 4.6, J2 18.1, 1H, 4Ha), 3.40 (dd, J1
11.7, J2 18.2, 1H, 4Hb), 5.50 (dd, J1 4.6, J2 11.7, 1H, 5H),
7.08–7.20 (m, 6H), 7.47 (t, 1H), 8.10 (d, J 6.4, 1H). νmax
(KBr)/cm−1 1742 (COO), 1647 (C=O), 1599 (C=N). m/z (ESI)

Novel 5-Arylpyrazole Derivatives
229
4-(1-Acetyl-3-methyl-4,5-dihydro-1H-pyrazol-5-yl)phenyl
4-(Trifluoromethyl) Benzoate 6o
J1 11.5, J2 17.6, 1H, 4Hb), 5.05 (s, 2H, CH2), 5.17 (br s, 1H,
OH), 5.34 (dd, J1 5.1, J2 11.5, 1H, 5H), 6.96 (d, 2H, J 8.6,
ArH), 7.17 (d, 2H, J 8.6, ArH), 7.28–7.45 (m, 5H, ArH). νmax
(KBr)/cm−1 3206 (NH), 1676 (C=O), 1629 (C=N). m/z (ESI)
309.0 (C18H19N3O2, [M + H]+). (Found: C 69.8, H 6.0, N 13.2.
Calc. for C18H19N3O2: C 69.9, H 6.2, N 13.6%.)
The title compound was obtained as colourless crystals. Yield
58.5%, mp 167–168◦C. δH (CDCl3) 2.00 (s, 3H, Me), 2.39
(s, 3H, Me), 2.71 (dd, J1 4.4, J2 18.1, 1H, 4Ha), 3.52 (dd,
J1 11.7, J2 18.1, 1H, 4Hb), 5.44 (dd, J1 4.4, J2 11.7, 1H,
5H), 7.12 (d, J 8.6, 2H, ArH), 7.22 (d, J 8.6, 2H, ArH), 7.64
(d, J 8.4, 2H, CF3-ArH), 8.10 (d, J 8.4, 2H, CF3-ArH). νmax
(KBr)/cm−1 1739 (COO), 1665 (C=O), 1621 (C=N). m/z (ESI)
390.8 (C20H17F3N2O3, [M + H]+). (Found: C 61.7, H 4.2,
N 7.1. Calc. for C20H17F3N2O3: C 61.5, H 4.4, N 7.2%.)
N-Benzyl-5-(4-hydroxyphenyl)-3-methyl-4,5-
dihydropyrazole-1-carbothioamide 7t
The title compound was obtained as colourless crystals.Yield
48.2%, mp 185–186◦C. δH (CDCl3) 1.32 (br s, 1H, NH), 2.00
(s, 3H, Me), 2.78 (dd, J1 4.6, J2 17.9, 1H, 4Ha), 3.49 (dd, J1
11.5, J2 17.6, 1H, 4Hb), 5.04 (s, 2H, CH2), 5.12 (br s, 1H,
OH), 5.36 (dd, J1 5.1, J2 11.5, 1H, 5H), 7.01 (d, 2H, J 8.6,
ArH), 7.14 (d, J 8.6, 2H, ArH), 7.28–7.47 (m, 5H, ArH). νmax
(KBr)/cm−1 3154 (NH), 1286 (C=S), 1597 (C=N). m/z (ESI)
324.7 (C18H19N3OS, [M + H]+). (Found: C 66.3, H 6.1, N 12.9.
Calc. for C18H19N3OS: C 66.4, H 5.9, N 12.9%.)
4-(1-Acetyl-3-methyl-4,5-dihydro-1H-pyrazol-5-yl)phenyl
Nicotinate 6p
The title compound was obtained as colourless crystals. Yield
60.1%, mp 201–202◦C. δH (CDCl3), 2.03 (s, 3H, Me), 2.36 (s,
3H, Me), 2.77 (dd, J1 4.6, J2 18.0, 1H, 4Ha), 3.49 (dd, J1 11.7,
J2 18.0, 1H, 4Hb), 5.47 (dd, J1 4.6, J2 11.7, 1H, 5H), 7.00 (d, J
8.6, 2H, ArH), 7.07 (d, J 8.6, 2H, ArH), 7.52 (q, 1H, PyH), 8.20
(d, J 6.8, 1H, PyH), 8.77 (d, J 6.8, 1H, PyH), 9.09 (s, 1H, PyH).
νmax (KBr)/cm−1 1738 (COO), 1670 (C=O), 1621 (C=N). m/z
(ESI) 322.8 (C18H17N3O3, [M + H]+). (Found: C 67.0, H 5.1,
N 13.4. Calc. for C18H17N3O3: C 66.9, H 5.3, N 13.0%.)
Antibacterial Assay
The antibacterial activity of the synthesized compounds was
tested against B. subtilisATCC 6633, E. coliATCC 35218, P. flu-
orescens ATCC 13525, and S. aureus ATCC 6538 using MH
medium (Mueller–Hinton medium: casein hydrolysate 17.5 g,
soluble starch 1.5 g, beef extract 1000 mL). The MICs of the test
compounds were determined by a colorimetric method using
the dye MTT.[16] A stock solution of the synthesized compound
(100 µg mL−1) in DMSO was prepared and graded quantities
of the test compounds were incorporated in a specified quan-
tity of sterilized liquid MH medium. A specified quantity of the
medium that contained the compound was poured into micro-
titration plates. A suspension of the microorganisms was pre-
pared to contain ∼105 cfu mL−1 and applied to microtitration
plates with serially diluted compounds in DMSO to be tested and
incubated at 37◦C for 24 h. After the MICs were visually deter-
mined on each of the microtitration plates, 50 µL of PBS (phos-
phate buffered saline 0.01 mol L−1, pH 7.4, Na2HPO4·12H2O
2.9 g, KH2PO4 0.2 g, NaCl 8.0 g, KCl 0.2 g, distilled water
1000 mL) that contained 2 mg of MTT per mL was added to each
well. Incubation was continued at room temperature for 4–5 h.
The content of each well was removed, and 100 µL of isopropyl
alcohol that contained 5% 1 mol L−1 HCl was added to extract
the dye.After 12 h of incubation at room temperature, the optical
density was measured with a microplate reader at 570 nm.
Procedure for the Preparation of 6q
1-(Chloromethyl)benzene (2.5 mmol) was added dropwise to
a solution of 5a (2 mmol), NaOH (0.010 mmol), and acetone
(30 mL).The reaction mixture was refluxed for 10 h.The mixture
was then cooled, acidified by dropwise addition of aqueous HCl
(0.010 mmol), poured into crushed ice, and allowed to stand at
room temperature overnight. The separated solid was washed by
water, and recrystallized from ethyl acetate to afford compound
6q. The structure was confirmed by X-ray crystallographic data.
Compounds 6r, 7s, and 7t were prepared following the same
method.
1-(5-(2-(Benzyloxy)phenyl)-3-methyl-4,5-
dihydropyrazol-1-yl)ethanone 6q
The title compound was obtained as colourless crystals.Yield
60.1%, mp 116–117◦C. Crystal data for 6q: C19H20N2O2, M
308.47, monoclinic, space group C, a 36.158(2), b 9.2767(11),
c 21.5198(18) Å, α 90.00◦, β 112.17(3)◦, γ 90.00◦, Dc
1.226 g cm−3, Z 8, F(000) 2624. Reflections collected/unique:
6537/2728, Fine, R1 0.0675, wR2 0.1531.
1-(5-(4-(Benzyloxy)phenyl)-3-methyl-4,5-
dihydropyrazol-1-yl)ethanone 6r
Acknowledgements
This work was financed by a grant (Project 070416274X) from Anhui
Natural Science Foundation, Anhui Province, P. R. China. The work was
also supported by the opening foundation of the Key Laboratory of Green
Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou
University, grant No. 2008GDGP0105.
The title compound was obtained as colourless crystals.Yield
65.2%, mp 90–91◦C. δH (CDCl3) 2.06 (s, 3H, Me), 2.29 (s, 3H,
Me), 2.70 (dd, J1 4.4, J2 18.1, 1H, 4Ha), 3.31 (dd, J1 11.5, J2 18.0,
1H, 4Hb), 5.03 (s, 2H, –CH2–), 5.38 (dd, J1 4.4, J2 11.5, 1H, 5H),
6.92 (d, J 8.4, 2H, ArH), 7.11 (d, J 8.4, 2H, ArH), 7.30–7.42 (m,
5H, ArH). νmax (KBr)/cm−1 1660 (C=O), 1612 (C=N), 1236
(–O–C–). m/z (ESI) 307.9 (C19H20N2O2, [M + H]+). (Found:
C 74.2, H 6.2, N 9.5. Calc. for C19H20N2O2: C 74.0, H 6.5,
N 9.1%.)
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