Synthesis of novel Schiff base analogues of 4-amino-1,5-dimethyl-2- phenylpyrazol-3-one and their evaluation for antioxidant and anti-inflammatory activity

Article abstract of DOI:10.1016/j.bmc.2012.04.058

4-Aminoantipyrine (4-amino-1,5-dimethyl-2-phenylpyrazole-3-one) and its analogues have been found to be compounds of interest for their anti-inflammatory, analgesic, antiviral, antipyretic, antirheumatic and antimicrobial activities. In the present study,

Full text of DOI:10.1016/j.bmc.2012.04.058

Bioorganic & Medicinal Chemistry 20 (2012) 4103–4108
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of novel Schiff base analogues of 4-amino-1,5-dimethyl-2-
phenylpyrazol-3-one and their evaluation for antioxidant and
anti-inflammatory activity
Mohammad Sayed Alam a, Jung-Hyun Choi b, Dong-Ung Lee c,
⇑
a Department of Chemistry, Jagannath University, Dhaka 1100, Bangladesh
b Institute of Bioconvergence Technology, Dongguk University, Gyeongju 780-714, Republic of Korea
c Division of Bioscience, Dongguk University, Gyeongju 780-714, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
4-Aminoantipyrine (4-amino-1,5-dimethyl-2-phenylpyrazole-3-one) and its analogues have been found
to be compounds of interest for their anti-inflammatory, analgesic, antiviral, antipyretic, antirheumatic
and antimicrobial activities. In the present study, Schiff base analogues of 4-aminoantipyrine were synthe-
sized by the condensation reaction with substituted benzaldehydes and then evaluated for their antioxi-
dant and anti-inflammatory activities. From among the synthesized compounds (3a–m, 4 and 5), 3k and
3f exhibited the highest antioxidant activity followed by 3g, 3l, 3c, 3i, 5, 3m and 3h. The IC50 values for com-
Received 3 March 2012
Revised 27 April 2012
Accepted 28 April 2012
Available online 4 May 2012
Keywords:
4-Aminoantipyrines
Schiff base
pounds 3k and 3f were found to be 0.44 and 0.93
lM, respectively, comparable to that of ascorbic acid (IC50
0.41 M), a standard antioxidant agent. From the comparisons between the hydroxylated and methoxylat-
l
ed compounds, the rank order of antioxidant activity for the products resulting from benzylidene phenyl
ring substitution was 2,4,6-OH > 3,4-OH > 3-OMe-4-OH > 3,5-OMe-4-OH > 2,4-OH > 3-Me-4-OMe > 3,4-
OMe > 4-OMe > 4-OH. The structure–activity relationship study revealed that the position and nature of
the substituted group on the benzylidene phenyl ring of the Schiff base analogues of 4-aminoantipyrine
play an important role in their antioxidant activity. The anti-inflammatory activity of 3f, which also exhib-
ited excellent antioxidant activity, was evaluated in terms of its inhibition of NO production, an inflamma-
tory modulator, in LPS pretreated RAW 264.7 cells using the Griess method. We also examined whether or
not this compound had effect on iNOS and COX-2 mRNA expression in RAW 264.7 cells. It was observed
that compound 3f significantly reduced NO production and inhibited LPS-stimulated iNOS and COX-2
mRNA levels in a dose-dependent manner. Overall, 3f showed promising antioxidant and anti-inflamma-
tory activities and may be used as the lead compound in a future study.
Antioxidant
Anti-inflammation
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Antipyrine has been found to be effective in scavenging peroxy
radicals (ROOÅ), but not effective against other ROS and RNS
The pyrazolone derivative 4-aminoantipyrine (4-amino-1,5-di-
methyl-2-phenylpyrazole-3-one) and its analogues have shown a
wide range of biological activities such as anti-inflammatory,1
analgesic,2 antiviral,3 antipyretic, antirheumatic and antimicrobial
activity.4 These classes of compounds also act as strong inhibitors
of cycloxygenase isoenzymes, platelet thromboxane synthesis
and prostanoid synthesis.1 Oxidative stress generates reactive oxy-
gen species (ROS), including free radicals,5 which attack various
biological macromolecules including proteins, enzymes, DNA,
etc., and give rise to a number of inflammatory and metabolic dis-
orders, cellular aging, reperfusion damage and cancer.6,7 Therefore,
antioxidants have been shown to play an important role in protect-
ing humans against many fatal diseases.
(reactive nitrogen species) or to the oxidative burst from neutro-
phils.1,8,9 Whereas aminoantipyrine (1, Scheme 1) was found to be
a highly efficient scavenger of ROSs, for example, hydroxyl radical
(HOÅ), hypochlorous acid (HOCl), peroxy radical (ROOÅ), singlet oxy-
gen (1O2), RNSs, [e.g. nitric oxide (NO) and peroxynitrite (ONOOꢀ)],
as well as the oxidative burst from neutrophils.1,8–11 Of significance,
in the progression of inflammatory diseases (cancer, coronary artery
disease, etc.), macrophages produce large quantities of NO, an
inflammatory mediator leading to inflammation.12,13
The inducible NO synthase (iNOS) plays an important role in the
production of inflammatory mediators. This enzyme oxidizes the
guanidine moiety of L-arginine, leading to the production of NO
which is associated with various carcinomas and inflammatory
conditions including Type-1 diabetes and arthritis.14 Lipopolysac-
charide (LPS), an endotoxin which is derived from the cell wall of
gram-negative bacteria,15 can induce multiple signaling pathways
⇑
Corresponding author. Tel.: +82 54 770 2224; fax: +82 54 742 9833.
E-mail address: dulee@dongguk.ac.kr (D.-U. Lee).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.058

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M. S. Alam et al. / Bioorg. Med. Chem. 20 (2012) 4103–4108
R4 R3
R5
N
R2
O
O
R4
R1
R5
R3
R2
R1
NH2
CH3
EtOH
N
N
+
N
N
OHC
H3C
H3C
CH3
3
2
1
Comp no.
R1
R2
R3
R4
R5 Comp no.
R1
H
R2
R3
R4
H
R5
3a
3b
3c
3d
3e
3f
3h
3i
OMe OMe
H
H
H
H
H
H
H
H
H
H
H
Cl
OH
OMe
OH
H
H
H
H
OH
H
H
H
H
H
H
H
H
NMe2
OH
H
H
H
H
H
H
H
H
H
H
3j
OMe
OH
H
H
H
3k
3l
OH
H
H
OMe
OH
OMe OH OMe
Me OMe
OH
3m
H
H
H
3g
OMe OH
Scheme 1. Synthesis of 4-aminoantipyrine analogues 3a–m.
to stimulate the production of inflammatory modulators involving
NO, PGE2, TNF-
and interleukins.16 Non-steroidal anti-inflamma-
2. Results and discussion
a
tory drugs (NSAIDs), which are known to inhibit COX enzymes,
are currently used as important therapeutic agents for the treat-
ment of pain and inflammation. Various side effects have been
observed for many commercially available selective COX-2 inhibi-
tors. For example, celecoxib has relatively few gastrointestinal side
effects but its long-term use is related to cardiovascular injury, an
effect which limits its clinical applications.17 Therefore, there is a
critical need for the development of novel drugs with a broad spec-
trum of activities and improved safety profiles that could be used
2.1. Synthesis of the Schiff base analogues of 4-amino-1,5-
dimethyl-2-phenylpyrazol-3-one
Synthesis of the Schiff base analogues was carried out according
to a convenient one-step procedure, that is, by the condensation of
commercially available 4-aminoantipyrine and different substi-
tuted benzaldehydes in ethanol, which provided excellent yields
(80–94%). The synthetic routes of the desired Schiff base analogues
(3a–m, 4 and 5) are outlined in Scheme 1 and 2. All compounds ex-
cept 3b, 3d, 3e and 3g are new. The structures of the compounds
were elucidated by IR, 1H NMR and mass spectral data. In the IR spec-
tra results for the compounds, the characteristic >C@O and –C@N–
stretching absorption bands appeared around 1617–1724 cmꢀ1
and 1579–1596 cmꢀ1, respectively. The 1H NMR spectra showed a
characteristic singlet for the imino proton (–CH@N–) at
9.41–9.83 ppm. The @C–CH3 and –N–CH3 protons were observed
as singlets at 2.33–2.52 and 3.09–3.23 ppm, respectively, and equiv-
alent to three protons each. The aromatic protons were assigned in
the usual way, according to their substitution pattern. The methoxy
protons of compounds 3e, 3g, 3h, 3j, 3l and 3m appeared as singlets
at 3.80–3.96 ppm. The N,N0-dimethyl protons of compound 3c were
observed as a singlet at 3.07 ppm, equivalent to six protons. Com-
pound 4 exhibited a characteristic singlet for the –CH@O proton at
10.84 ppm, which was absent in compound 5, indicating the further
condensation of the formyl group from compound 4 with the amino
group of 4-aminoantipyrine. In addition, the EI-MS spectra of 3a–m,
4 and 5 showed a molecular ion peak with intensities of 88–100%.
on
conditions.
In a previous study, we reported the synthesis, molecular
a long-term basis for mitigating chronic inflammatory
structure and antioxidant activity of 4-[benzylideneamino]-1,5-
dimethyl-2-phenyl-1H-pyrazol-3(2H)-one, a Schiff base ligand of
4-aminoantipyrine.18 The hydroxyl radical scavenging activity19 of
N-alkylated-4-aminoantipyrines and antibacterial property20 of a
series of Schiff base analogues of 4-aminoantipyrine were also
reported. Here, we describe the synthesis and biological evaluation
of a series of novel Schiff base analogues of 4-aminoantipyrine (4-
amino-1,5-dimethyl-2-phenylpyrazole-3-one) designed for use as
antioxidant and anti-inflammatory agents. The syntheses of the
Schiff base analogues of 4-aminoantipyrine were prepared by
condensation reaction with various substituted benzaldehydes in
ethanol. The structures of the new compounds were elucidated by
IR, 1H-NMR, and mass spectrometry. The in vitro DPPH (1,1-diphe-
nyl-2-picrylhydrazyl) free radical scavenging activity of the synthe-
sized compounds was assayed according to the Blois method.21 The
effect of the Schiff base analogue of 4-aminoantipyrine on NO pro-
duction was assayed using the Griess method with LPS-stimulated
RAW264.7 cells. The toxicity profile of the Schiff base analogues
on the RAW264.7 cells was assessed by an MTT assay. We also inves-
tigated whether or not the Schiff base analogues had an effect on the
expression of iNOS and COX-2 proteins using a reverse transcrip-
tion-polymerase chain reaction (RT-PCR) method.
2.2. Antioxidant activity
The newly synthesized Schiff base analogues (3a–m, 4 and 5) of
4-aminoantipyrine were evaluated for their free radical-scavenging
activity using DPPH. The DPPH radical scavenging activities for
the compounds are shown in Table 1. In order to study their

M. S. Alam et al. / Bioorg. Med. Chem. 20 (2012) 4103–4108
4105
O
O
EtOH
N
CHO
N
+
+
NH2
CH3
N
N
H3C
CH3
N
OHC
CHO
H3C
4
1
O
O
N
N
N
N
N
N
H3C
CH3
H3C
CH3
5
Scheme 2. Synthesis of 4-aminoantipyrine derivatives 4 and 5.
aminoantipyrine, based on its electrochemical properties.18 Intro-
duction of a hydroxyl group into the 4-position of the benzylidene
phenyl ring of 3a, yielding 3d, led to a ꢁ2 fold increase in activity,
whereas the 4-methoxy analogue, 3e, showed slightly improved
activity compared to that of compound 3d. Substitution of two hy-
droxyl groups into the 3,4- and 2,4-positions of compound 3a, pro-
duced 3f and 3i, respectively. These substitutions caused a rise in
their antioxidant activity as compared to that of 3a, of 34- (3f)
and 12-fold (3i). Replacement of the hydroxyl groups of 3f with a
methoxy or methyl group, producing 3g, 3h, or 3m, resulted in a
significant decrease in activity. Similar results were also observed
in the case of compound 3i. Introduction of two methoxy groups
into the 3- and 5-position of compound 3d, led to 3l, and an in-
creased antioxidant activity >11-fold greater than that observed
for 3d. Introduction of a chlorine atom or an aldehyde group, a deac-
tivating substituent, onto the benzylidene phenyl ring of 3a re-
sulted in compounds 3b or 4, respectively, and neither showed
any marked change in antioxidant activity. The placement of a
N,N-dimethylamino group, an activating substituent, on the phenyl
ring of 3a, produced 3c and resulted in a ꢁ13-fold increase in anti-
oxidant activity. When the aldehyde group of 4 underwent a con-
densation reaction with another 4-aminoantipyrine molecule to
furnish compound 5, it exhibited ꢁ11-fold higher antioxidant activ-
ity than observed for compound 4. The above structure–activity
relationship study led us to speculate that the structure and posi-
tion of the substituted group on the benzylidene phenyl ring of
the Schiff base analogues of 4-aminoantipyrine play important
roles in their antioxidant activity.
Table 1
DPPH radical scavenging activity of the Schiff base analogues of 4-aminoantipyrine
Compd no.
IC50
(
l
M)
Compd no.
IC50
2.5
(lM)
3a
3b
3c
3d
3e
3f
31.26a
31.68
2.43
18.8
15.27
0.93
3i
3j
3k
3l
3m
4
5
14.53
0.44
1.66
5.22
34.46
3.14
0.41
3g
3h
1.13
7.27
Ascorbic acid
a
Taken from Ref. 18.
Figure 1. Effect of 3f on the viability of RAW264.7 cells.
2.3. Anti-inflammatory activity
structure–activity relationship, we synthesized and tested the anti-
oxidant activity of hydroxylated, non-hydroxylated and methoxy-
lated Schiff base analogues of 4-amino-1,5-dimethyl-2-
phenylpyrazole-3-one. Comparisons of the hydroxylated and non-
hydroxylated Schiff base analogues revealed that the DPPH radical
scavenging activities of the former analogues were generally higher
than those of the latter. Compounds 3a, 3b and 4, bearing no OH
To examine the anti-inflammatory activity of Schiff base
analogues of 4-aminoantipyrine, we arbitrarily selected compounds
3k and 3f, which demonstrated a potent antioxidant activity of
<1.0 lM at IC50. However, only compound 3f could be evaluated
in terms of inhibition of the production of inflammatory modulators
associated with NO because the most active antioxidant compound,
3k was difficult to solubilize, even in DMSO. The cytotoxic effect of
compound 3f on RAW 264.7 cells was determined by MTT assay. At
group, exhibited much higher IC50 values (>30 lM) than those hav-
ing at least one OH group in the aromatic ring. Among this series of
compounds (3a–m, 4 and 5), 3k and 3f showed the highest DPPH
free radical scavenging activity followed by 3g, 3l, 3c, 3i, 5, 3m
and 3h, respectively. The IC50 values of compound 3k and 3f were
concentrations of 6, 12, 25 and 50 lg/mL, the viability of RAW 264.7
cells was not significantly inhibited by compound 3f (Fig. 1).
In order to investigate the effects of 3f on NO production by
RAW 264.7 macrophage cells in the presence or absence of LPS,
we measured the concentration of nitrate and the oxidative metab-
olite of NO in their cell culture medium using the Griess method.
The RAW 264.7 cells were pretreated with 3f for 30 min prior to
stimulation with LPS (200 ng/mL). After 24 h of stimulation by
0.44 and 0.93
lM, respectively, which are comparable to that of
the standard antioxidant agent, ascorbic acid (IC50 0.41
lM). We
have previously reported the proposed antioxidant mechanism
for compound 3a, a non-hydroxylated Schiff base analogue of 4-

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M. S. Alam et al. / Bioorg. Med. Chem. 20 (2012) 4103–4108
4.2. General method for the preparation of Schiff base
analogues of 4-amino-1,5-dimethyl-2-phenylpyrazol-3-one
(3a–m)
An anhydrous ethanol solution (10 mL) of 4-amino-1,5-
dimethyl-2-phenylpyrazol-3-one (203 mg, 1 mmol) was added to
an anhydrous ethanol solution (10 mL) of substituted benzalde-
hyde (1 mmol), and the mixture was refluxed at 80 °C for 4–6 h
under atmospheric conditions (Scheme 1). The progress of the
reaction was monitored by TLC. The precipitates formed were
collected by filtration and purified by recrystallization with
ethanol, and then dried in vacuo to produce the pure compound
with a high yield (80–94%).
4.2.1. 4-Benzylideneamino-1,5-dimethyl-2-phenyl-1H-pyrazol-
3(2H)-one (3a)18
Yield 85%18; mp 178.3 °C (yellow crystal); IR (KBr) 1651 (>C@O),
1597 (C@N) cmꢀ1 1H NMR (DMSO-d6) d ppm: 2.49 (s, 3H, @C–
;
Figure 2. Effect of 3f on NO production and iNOS and COX-2 mRNA expression in
LPS-treated RAW264.7 cells. Cells were pretreated with different concentrations of
3f for 30 min and then stimulated with LPS (200 ng/mL) for 24 h. After incubation,
the NO production levels in the cultured media were measured by Griess reagent.
Cells were harvested, total RNA was isolated, and iNOS and COX-2 mRNA
expression was examined by RT-PCR analysis.
CH3), 3.14 (s, 3H, –N–CH3), 7.31–7.52 (m, 8H, ArH), 7.84–7.89 (m,
2H, ArH), 9.77 (s, 1H, –N@CH); HR-FAB-MS (m/z): 293 [M+H]+, cal-
culated for
+0.4 ppm).
C18H17N3O, 292.1338; found 292.1339 (error:
4.2.2. 4-(4-Chlorobenzylideneamino)-1,5-dimethyl-2-phenyl-
1H-pyrazol-3(2H)-one (3b)20
LPS, the levels of NO in the culture media were determined. As
shown in Figure 2, LPS stimulation resulted in a marked induction
of NO production as compared to untreated cells. However,
pretreatment with 3f, 30 min prior to LPS stimulation significantly
reduced NO production in a dose-dependent manner.
The effects of 3f on iNOS and COX-2 mRNA expression in RAW
264.7 cells was examined by RT-PCR. In order to measure iNOS and
COX-2 mRNA expression levels, the RAW 264.7 cells were pretreated
with 3f for 30 min prior to stimulation with LPS (200 ng/mL). After
24 h of stimulation by LPS, the cells were evaluated for iNOS and
COX-2 mRNA expression. The iNOS and COX-2 mRNA levels were
not detectable in the absence of LPS treatment, but these levels were
significantly induced after LPS exposure. Pretreatment of the cells
with 3f inhibited LPS-stimulated iNOS levels in a dose-dependent
Yield 90%; mp 252.1 °C (yellow solid); IR (KBr) 1649 (>C@O),
1593 (C@N) cmꢀ1 1H NMR (DMSO-d) d ppm: 2.49 (s, 3H, @C–
;
CH3), 3.16 (s, 3H, –N–CH3), 7.35–7.48 (m, 7H, Ar-H), 7.77–7.82 (m,
2H, Ar-H), 9.72 (s, 1H, –N@CH); EI-MS m/z (%): 327 (M+2, 34), 326
(M+1, 21), 325 (M+, 100), 233 (20), 188 (38), 121 (36), 56 (96).
4.2.3. 4-[4-(Dimethylamino)benzylideneamino]-1,5-dimethyl-
2-phenyl-1H-pyrazol-3(2H)-one (3c)
Yield 80%; mp 219.4 °C (yellow solid); IR (KBr) 1650 (>C@O),
1606, 1575 (C@N) cmꢀ1 1H NMR (CDCl3) d ppm: 2.52 (s, 3H,
;
@C–CH3), 3.07 (s, 6H, –N(CH3)2, 3.15 (s, 3H, –N–CH3), 6.77 (d, 2H,
J = 8.2 Hz, Ar-H), 7.33–7.53 (m, 5H, Ar-H), 7.82 (d, 2H, J = 8.2 Hz,
Ar-H), 9.74 (s, 1H, –N@CH); EI-MS m/z (%): 334 (M+, 88), 167
(12), 56 (100).
manner. However, the results indicated that merely 50 lg/mL of 3f
inhibited the LPS-stimulated COX-2 mRNA levels (Fig. 2).
4.2.4. 4-(4-Hydroxybenzylideneamino)-1,5-dimethyl-2-phenyl-
1H-pyrazol-3(2H)-one (3d)20
3. Conclusion
Yield 84%; mp 232.3 °C (yellow powder); IR (KBr) 3591(OH),
1617 (>C@O), 1579 (C@N) cmꢀ1 1H NMR (CDCl3) d ppm: 2.42 (s,
;
In summary, we prepared and evaluated a series of novel Schiff
base analogues of 4-aminoantipyrine (4-amino-1,5-dimethyl-2-
phenylpyrazole-3-one) for their antioxidant and anti-inflammatory
activities. Most of the compounds showed significant antioxidant
activity while compound 3k and 3f showed a potent antioxidant
activity comparable to that of the standard antioxidant agent,
ascorbic acid. Moreover, compound 3f showed promising anti-
inflammatory activity which could be beneficial for use in the
treatment of inflammatory diseases. The results of this study may
lead to the development of a new therapeutic agent useful in fight-
ing diseases caused by oxidative stress and inflammation.
3H, @C–CH3), 3.13 (s, 3H, –N–CH3), 6.84 (d, 2H, J = 8.2 Hz, Ar-H),
7.35–7.52 (m, 5H, Ar-H), 7.65 (d, 2H, J = 8.2 Hz, Ar-H), 9.46 (s, 1H,
–N@CH); EI-MS m/z (%): 307 (M+, 100), 215 (33), 121 (20), 56 (78).
4.2.5. 4-(4-Methoxybenzylideneamino)-1,5-dimethyl-2-phenyl-
1H-pyrazol-3(2H)-one (3e)20
Yield 91%; mp 168.1 °C (yellow crystal); IR (KBr) 1643 (>C@O),
1586 (C@N) cmꢀ1 1H NMR (CDCl3) d ppm: 2.45 (s, 3H, @C–CH3),
;
3.19 (s, 3H, –N–CH3), 3.83 (s, 3H OCH3), 6.81 (d, 2H, J = 8.3 Hz,
Ar-H), 7.29–7.46 (m, 5H, Ar-H), 7.57 (d, 2H, J = 8.3 Hz, Ar-H), 9.41
(s, 1H, –N@CH); EI-MS m/z (%): 321 (M+, 100), 229 (23), 212 (18),
121 (22), 56 (93).
4. Materials and methods
4.1. General
4.2.6. 4-(3,4-Dihydroxybenzylideneamino)-1,5-dimethyl-2-
phenyl-1H-pyrazol-3(2H)-one(3f)
The melting point of the synthesized compounds was deter-
mined using a Stuart SMP3 apparatus, and the results were uncor-
rected. The 1H NMR spectra were recorded on a Varian GEMINI 200
spectrophotometer (200 MHz) using TMS as a reference standard.
FT-IR spectra, using KBr pellets, was obtained with a Bruker Tensor
37 spectrophotometer. EI- and FAB-MS spectra were acquired
using a Jeol JMS-700 mass spectrometer.
Yield 90%; mp 287.2 °C (yellow crystal); IR (KBr) 3491 (OH),
1618 (>C@O), 1586 (C@N)cmꢀ1 1H NMR (DMSO-d6) d ppm: 2.46
;
(s, 3H, @C–CH3), 3.18 (s, 3H, –N–CH3), 6.84 (d, 1H, J = 2.4 Hz, Ar-
H), 7.05 (d, 1H, J = 2.4 Hz, Ar-H), 7.35–7.61 (m, 6H, Ar-H), 9.42 (s,
1H, -N=CH); EI-MS m/z (%): 323 (M+, 100), 231 (37), 121 (15), 56
(76).

M. S. Alam et al. / Bioorg. Med. Chem. 20 (2012) 4103–4108
4107
4.2.7. 4-(4-Hydroxy-3-methoxybenzylideneamino)-1,5-
dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3g)
of the reaction was monitored by TLC. The solvent was evaporated
and then the mixture was subjected to column chromatography on
a silica gel using n-hexane-dichloromethane (2:1) to yield com-
pounds 4 and 5.
Yield 95%; mp 207.5 °C (yellow crystal) (mp. 205 °C)22; IR (KBr)
3542 (OH) 1624 (>C@O), 1581 (C@N) cmꢀ1 1H NMR (DMSO-d6) d
;
ppm: 2.43 (s, 3H, @C–CH3), 3.17 (s, 3H, –N–CH3), 3.80 (s, 3H,
OCH3), 6.88 (d, 1H, J = 2.5 Hz, Ar-H), 7.11 (d, 1H, J = 2.5 Hz, Ar-H),
7.36–7.63 (m, 6H, Ar-H), 9.41 (s, 1H, –N@CH); EI-MS m/z (%): 337
(M+, 100), 245 (34), 228 (17), 121 (16), 56 (84).
4.3.1. 2-[(2,3-Dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-
pyrazol-4-ylimino)methyl]benzaldehyde (4)
Brown oily liquid. Yield 42%; IR (KBr) 1724 (>C@O), 1664
(>C@O), 1596 (C@N) cmꢀ1 1H NMR (CDCl3) d ppm: 2.42 (s, 3H,
;
4.2.8. 4-(3,4-Dimethoxybenzylideneamino)-1,5-dimethyl-2-
phenyl-1H-pyrazol-3(2H)-one (3h)
@C–CH3), 3.09 (s, 3H, -N-CH3), 6.69–6.78 (m, 3H, Ar-H), 7.38–
7.69 (m, 6H, Ar-H), 9.49 (s, 1H, -N@CH), 10.84 (s, 1H, CHO); EI-
MS m/z (%): 319 (M+, 100), 214 (32), 187 (23), 172 (19), 104 (16),
84 (10), 56 (86).
Yield 94%; mp 191.7 °C (yellow crystal); IR (KBr) 1649 (>C@O),
1598, 1579 (C@N) cmꢀ1 1H NMR (CDCl3) d ppm: 2.47 (s, 3H, @C–
;
CH3), 3.10 (s, 3H, –N–CH3), 3.90 (s, 3H, OCH3), 3.96 (s, 3H. OCH3),
6.89 (d, 1H, J = 2.4 Hz, Ar-H), 7.29–7.55 (m, 7H, Ar-H), 9.71 (s, 1H,
–N@CH); EI-MS m/z (%): 351 (M+, 100), 259 (21), 242 (21), 121
(15), 56 (91).
4.3.2. 4-[2-(2,3-Dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-
pyrazol-4-ylimino-methyl)benzylideneamino]-1,2-dihydro-1,5-
dimethyl-2-phenylpyrazol-3-one (5)
Brown oily liquid. Yield 47%; IR (KBr) 1658(>C@O), 1579(C@N)
4.2.9. 4-(2,4-Dihydroxybenzylideneamino)-1,5-dimethyl-2-
phenyl-1H-pyrazol-3(2H)-one (3i)
cmꢀ1 1H NMR (CDCl3) d ppm: 2.45 (s, 3H, @C–CH3), 3.10 (s, 3H,
;
–N–CH3), 6.71–6.85 (m, 6H, Ar-H), 7.49–7.86 (m, 8H, Ar-H), 9.56
(s, 1H, -N@CH); EI-MS m/z (%): 504 (M+, 33), 334 (22), 333 (100),
245 (27), 203 (19), 172 (19), 104 (36), 84 (21), 56 (91).
Yield 91%; mp 231.5 °C (yellow needles); IR (KBr) 3466 (OH),
1624 (>C@O), 1580 (C@N) cmꢀ1 1H NMR (DMSO-d6) d ppm: 2.41
;
(s, 3H, @C–CH3), 3.21 (s, 3H, –N–CH3), 6.42 (m, 2H, Ar-H), 7.28–
7.59 (m, 6H, Ar-H), 9.59 (s, 1H, –N@CH); EI-MS m/z (%): 323 (M+,
100), 231 (40), 203 (18), 121 (14), 56 (64).
4.4. DPPH radical scavenging activity
The free radical-scavenging activity of the synthesized com-
pounds was assayed according to the Blois method with some
modification,21 and using DPPH. To 0.1 ml samples of different con-
centrations (2.5–20 mg/mL) in ethanol, 4 ml of 1.5 ꢂ 10ꢀ5 M DPPH
solution was added, thoroughly mixed, and then left to stand at
room temperature in a dark place. After 30 min of incubation, the
absorbance of the solution was measured at 520 nm, the result of
which was used to calculate DPPH radical scavenging activity (%).
4.2.10. 4-(2, 4-Dimethoxybenzylideneamino)-1,5-dimethyl-2-
phenyl-1H-pyrazol-3(2H)-one (3j)
Yield 83%; mp 183.4 °C (yellow solid); IR (KBr) 1643 (>C@O),
1586 (C@N) cmꢀ1 1H NMR (DMSO-d6) d ppm: 2.47 (s, 3H, @C–
;
CH3), 3.23 (s, 3H, –N–CH3), 3.81 (s, 3H, OCH3), 3.84 (s, 3H, OCH3),
6.62 (m, 2H, Ar-H), 7.18–7.49 (m, 6H, Ar-H), 9.49 (s, 1H, –N@CH);
EI-MS m/z (%): 351 (M+, 100), 259 (56), 121 (20), 56 (97).
4.2.11. 4-(2, 4, 6-Trihydroxybenzylideneamino)-1,5-dimethyl-2-
phenyl-1H-pyrazol-3(2H)-one (3k)
4.5. Cell culture
Yield 82%; mp 243.8 °C (reddish powder); IR (KBr) 3552 (OH),
The RAW264.7 cells were obtained from Korean Cell Line Bank
(Seoul, Korea) and cultured in DMEM supplemented with 10% FBS,
100 U/mL penicillin, 100 g/mL streptomycin and 100 M MEM non-
essential amino acid solution. In all experiments, the cells were
grown to 80–90% confluence and subjected to not more than 20
cell passages.
3490 (OH), 1629 (>C@O), 1579 (C@N) cmꢀ1 1H NMR (CDCl3) d
;
ppm: 2.33 (s, 3H, @C–CH3), 3.13 (s, 3H, –N–CH3), 6.80 (s, 2H, Ar-
H), 7.35–7.60 (m, 5H, Ar-H), 9.83 (s, 1H, –N@CH); EI-MS m/z (%):
339 (M+, 100), 247 (43), 203(24), 121 (14), 84 (15), 56 (68).
4.2.12. 4-(3,5-Dimethoxy-4-hydroxybenzylideneamino)-1,5-
dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3l)
4.6. Measurement of cell viability
Yield 92%; mp 258.9 °C (yellow powder); IR (KBr) 1632 (>C@O),
1588 (C@N) cmꢀ1 1H NMR (DMSO-d6) d ppm: 2.44 (s, 3H, @C–
;
Cell viability was assessed by MTT assay. RAW264.7 cells
seeded onto 96-well plates (5 ꢂ 104 cells/well) were treated with
various concentrations of compound 3f. After 24 h incubation,
CH3), 3.13 (s, 3H, –N–CH3), 3.82 (s, 6H, 2ꢂ–OCH3), 7.01(s, 2H, Ar-
H), 7.36–7.53 (m, 5H, Ar-H), 9.46 (s, 1H, –N@CH); EI-MS m/z (%):
367 (M+, 100), 345 (30), 344 (47), 275 (45), 56 (70).
20 ll of MTT (5 mg/ml) was added and then incubated for 4 h.
The culture medium was removed, and then the cells were dis-
solved in 0.04 N HCl/isopropyl alcohol. The optical densities (OD)
of the resulting cell cultures were measured at 570 nm and
630 nm using a microplate reader.
4.2.13. 4-(3-Methyl-4-methoxybenzylideneamino)-1,5-
dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3m)
Yield 89%; mp 269.8 °C (yellow crystal); IR (KBr) 1624 (>C@O),
1579 (C@N) cmꢀ1 1H NMR (DMSO-d6) d ppm: 2.21 (s, 3H, –CH3),
;
2.42 (s, 3H, @C–CH3), 3.12 (s, 3H, –N–CH3), 3.81 (s, 3H, –OCH3),
7.36–7.57 (m, 7H, Ar-H), 9.43 (s, 1H, –N@CH); EI-MS m/z (%): 335
(M+, 100), 259 (21), 201 (21), 135 (15), 56 (79).
4.7. Measurement of nitric oxide
The RAW264.7 cells (5 ꢂ 105 cells/well) were seeded onto a 24-
well culture plate at 37 °C for O/N in medium. The cells were pre-
incubated with various concentrations of 3f for 24 h. NO production
was monitored by measuring the nitrite levels in the culture media
using Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylenedi-
amine dihydrochloride and 2.5% phosphoric acid). After incubating
for 10 min, the absorbance was measured at 570 nm. Nitrite levels
in the samples were calculated from a standard curve with a known
concentration of sodium nitrite.
4.3. Synthesis of Schiff base analogues of 4-amino-1,5-dimethyl-
2-phenylpyrazol-3-one with phthalaldehyde (4 and 5)
Phthalaldehyde (268 mg, 2 mmol) was added to a solution of 4-
amino-1,5-dimethyl-2-phenylpyrazol-3-one (203 mg, 1 mmol) in
anhydrous ethanol (20 mL) at room temperature. The reaction
mixture was refluxed for 6 h under atmospheric conditions
(Scheme 2) and then cooled to room temperature. The progress

4108
M. S. Alam et al. / Bioorg. Med. Chem. 20 (2012) 4103–4108
3. Evstropov, A. N.; Yavorovskaya, V. E.; Vorob’ev, E. S.; Khudonogova, Z. P.;
Gritsenko, L. N.; Shmidt, E. V.; Medvedeva, S. G.; Filimonov, V. D.; Prishchep, T.
P.; Saratikov, A. S. Pharm. Chem. J. [Khim.-Farm. Zh.] 1992, 26, 426.
4. Ei Ashry, E. S. H.; Awad, L. F.; Ibrahim, E. I.; Bdeewy, O. K. Chinese J. Chem. 2007,
25, 570.
5. Chevion, S.; Roberts, M. A.; Chevion, M. Free Radical Biol. Med. 2000, 28, 860.
6. Ozyurek, M.; Bekasoglu, B.; Guclu, K.; Apak, R. Anal. Chim. Acta. 2009, 636, 42.
7. Nishida, J.; Kawabata, J. Biosci. Biotechnol. Biochem. 2006, 70, 193.
8. Costa, D.; Gomes, A.; Lima, J. L. F. C.; Fernandes, E. Redox Rep. 2008, 13, 153.
9. Costa, D.; Vieira, A.; Fernandes, E. Redox Rep. 2006, 11, 136.
10. Kalyanaraman, B.; Sohnle, P. G. J. Clin. Invest. 1985, 75, 1618.
11. Sayo, H.; Saito, M. Xenobiotica 1990, 20, 957.
12. Ahn, K. S.; Noh, E. J.; Zhao, H. L.; Jung, S. H.; Kang, S. S.; Kim, Y. S. Life Sci. 2005,
76, 2315.
13. Fujihara, M.; Muroi, M.; Tanamoto, K.; Suzuki, T.; Azuma, H.; Ikeda, H.
Pharmacol. Therap. 2003, 100, 171.
14. Surh, Y. J.; Chun, K. S.; Cha, H. H.; Han, S. S.; Keum, Y. S.; Park, K. K.; Lee, S. S.
Mut. Res. 2001, 480–481, 243.
15. Roth, R. A.; Harkema, J. R.; Pestka, J. P.; Ganey, P. E. Toxicol. Appl. Pharmacol.
1997, 147, 300.
4.8. Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA from the RAW264.7 cells was prepared using a TRIzol
reagent (Gibco, Grand Island, NY). The concentration and integrity
of RNA was determined by measuring absorbance at 260/280 nm.
The forward and reverse primers for iNOS were 50-TCTTTGACGCT
CGGAACTGTAGCA-30 and 50-CGTGAAGCCATGACCTTTCGCATT-30,
respectively. The forward and reverse primers for COX-2 were
50-TTGCTGT ACAAGCAGTGGCAAAGG-30 and 50-AGGACAAACAC
CGGAGGGAATC TT-30, respectively. The forward and reverse
primers for the mouse GAPDH mRNA expression (used as a control
for total RNA content for each sample) were 50-AACTTTGGCA
TTGTGGAAGGGCTC-30 and 50-TGGAAGAGTGGGAGTTGCTGTTGA-
30, respectively. RT-PCR was performed using a ONE-STEP RT-PCR
PriMix kit (Invitrogen Co., USA), according to the manufacturer’s
instructions.
16. Kundu, J. K.; Surh, Y. J. Mut. Res. 2005, 591, 123.
17. Frampton, J. E.; Keating, G. M. Drugs 2007, 67, 2433.
18. Alam, M. S.; Lee, D. U. J. Chem. Crystallogr. 2012, 42, 93.
19. Santosa, P. M. P.; Antunesb, A. M. M.; Noronhaa, J.; Fernandesc, E.; Vieiraa, A. J.
S. C. Eur. J. Med. Chem. 2010, 45, 2258.
20. Ali, P.; Meshram, J.; Sheikh, J.; Tiwari, V.; Rajendra Dongre, R.; Ben Hadda, T.
Med. Chem. Res. 2012, 21, 157.
References and notes
1. Costa, D.; Marques, A. P.; Reis, R. L.; Lima, J. L. F. C.; Fernandes, E. Free Radical
Biol. Med. 2006, 40, 632.
2. Burdulene, D.; Palaima, A.; Stumbryavichyute, Z.; Talaikite, Z. Pharm. Chem. J.
[Khim.-Farm. Zh.] 1999, 33, 191.
21. Blois, M. S. Nature 1958, 181, 1199.
22. Suresh, M. S.; Prakash, V. Int. J. Phy. Sci. 2010, 5, 2203.