Design, synthesis, characterization and biological evaluation of novel pyrazole integrated benzophenones

Article abstract of DOI:10.1016/j.bmcl.2012.10.031

A series of novel pyrazole integrated benzophenones (9a-j) have been designed, synthesized from 1-methyl-5-(2,4,6-trimethoxy-phenyl)-1H-pyrazole 6. The structures of the regioisomers 6 and 7 were determined by 2D ¹H-¹H COSY, ¹H-¹³C HSQC and ¹H-¹³C HMBC experiments. The newly synthesized compounds (9a-j) were evaluated for in vivo anti-inflammatory activity by carrageenan paw edema in rats and in vitro COX-1/COX-2 inhibition and antioxidant potential. Among the synthesized compounds, compounds 9b, 9d and 9f, were found to be active anti-inflammatory agents in addition to having potent antioxidant activity.

Full text of DOI:10.1016/j.bmcl.2012.10.031

Bioorganic & Medicinal Chemistry Letters 23 (2013) 912–916
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, characterization and biological evaluation of novel
pyrazole integrated benzophenones
Babasaheb P. Bandgar a, , Hemant V. Chavan a, Laxman K. Adsul a, Vishnu N. Thakare b,
⇑
Sadanand N. Shringare a, Rafik Shaikh c, Rajesh N. Gacche c
a Medicinal Chemistry Research Laboratory, School of Chemical Sciences, Solapur University, Solapur 413 255, Maharashtra, India
b Department of Pharmacology, Sinhgad Institute of Pharmaceutical Sciences, Kusgaon (Bk), Lonavala 410 401, India
c Biochemistry Research Laboratory, School of Life Sciences, S.R.T.M. University, Nanded 431 606, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2012
Revised 17 September 2012
Accepted 5 October 2012
Available online 13 October 2012
A series of novel pyrazole integrated benzophenones (9a–j) have been designed, synthesized from
1-methyl-5-(2,4,6-trimethoxy-phenyl)-1H-pyrazole 6. The structures of the regioisomers 6 and 7 were
determined by 2D 1H–1H COSY, 1H–13C HSQC and 1H–13C HMBC experiments. The newly synthesized
compounds (9a–j) were evaluated for in vivo anti-inflammatory activity by carrageenan paw edema in
rats and in vitro COX-1/COX-2 inhibition and antioxidant potential. Among the synthesized compounds,
compounds 9b, 9d and 9f, were found to be active anti-inflammatory agents in addition to having potent
antioxidant activity.
Keywords:
Benzophenones
Pyrazole
Ó 2012 Elsevier Ltd. All rights reserved.
HMBC
HSQC
COSY
Anti-inflammatory
Antioxidant activity
Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used
for the treatment of pain, inflammation and fever. They produce
their therapeutic effects by inhibiting the activity of cyclooxygen-
ase (COX) enzymes. COX is the key enzyme which catalyses the
conversion of arachidonic acid to prostaglandins and thrombox-
anes.1–4 There are two types of cyclooxygenase enzymes, COX-1
and COX-2. COX-1 is a constitutive enzyme, produced in many tis-
sues such as the kidney and the gastrointestinal tract, while COX-2
is inducible and is expressed during inflammation at a site of in-
jury.5 Steroidal anti-inflammatory drugs (Glucocorticoids; dexa-
methasone, betamethasone and prednisolone) are used in
immunological mediated inflammation, however, their major side
effects such as heart, liver, and kidney damages6 limits the use in
acute and chronic inflammation. Presently, NSAIDs are the pre-
ferred agents for the treatment of pain, inflammation and fever
and characterized as dual COX-1 and COX-2 inhibitors.7 The
chronic use of NSAIDs of for pain and inflammation is often accom-
panied by side effects such as gastric ulceration, bleeding and renal
function suppression, due to their inhibitory activity against COX-
1, however COX-2 inhibitors devoid such effect.8,9 Thus, the devel-
opment of COX-2 selective inhibitors has led attention to improve
the therapeutic potency and to reduce the classical side effects
associated with the use of conventional NSAIDs. Reactive oxygen
species (ROS) are implicated in the induction and prolongation of
inflammatory processes.10 Interestingly, a number of therapeuti-
cally useful NSAID’s have been shown to act by virtue of their free
radical scavenging activity through their antioxidant potential.11
Antioxidants are the compounds that prevent oxidative damage in-
duced by free radicals and ROS. Thus, antioxidant therapy has also
gained immense importance in the treatment of the above-men-
tioned diseases.12
Many pharmaceuticals are synthetic compounds and a large
number of them are heterocyclic in nature. Among which, Pyrazole
are an important class of compounds for new drug development
that attracted much attention due to their broad spectrum of bio-
logical activities, such as anti-inflammatory,13 antifungal,14 anti-
cancer,15 and antiviral16 activities. Pyrazole derivatives also act as
antiangiogenic agents,17 kinase inhibitor for treatment of type 2
diabetes, hyperlipidemia, obesity,18 and thrombopiotinmimetics.19
Recently, urea derivatives of pyrazoles have been reported as po-
tent inhibitors of p38 MAP kinase.20 Among the highly marketed
COX-2 inhibitors that comprise of pyrazole nucleus, celecoxib is
the one which is treated as a safe anti-inflammatory and analgesic
agent. It is considered as a typical model of the diaryl heterocyclic
template that is known to selectively inhibit the COX-2 enzyme.
Some other examples of pyrazole derivatives such as deracoxib,
⇑
Corresponding author. Tel./fax: +91 217 2351300.
E-mail address: bandgar_bp@yahoo.com (B.P. Bandgar).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.031

B. P. Bandgar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 912–916
913
H2NO2S
H2NO2S
CF3
H2NO2S
research groups have been reported as effective anti-inflammatory
agents.23 Recently, synthesis and structure–activity relationship of
benzophenones as novel class of p38 MAP kinase inhibitors with
high anti-inflammatory activity have been reported.24 The litera-
ture survey revealed that no efforts were aimed towards the
designing of pyrazole scaffold integrated with benzophenone
framework to verify the importance of this scaffold on the pharma-
cological activity. Based on these interesting biological activity
profiles of pyrazoles and benzophenone analogues, we were
inspired and made an attempt to synthesize a series of novel
pyrazole integrated benzophenone analogues as potent anti-
inflammatory and as antioxidant agents.
N
N
N
N
N
N
CF3
CHF2
F
H3C
Br
O
Deracoxib
Celecoxib
N
SC-558
H3C HN
N
O
N
H3C
O
N
N
N
N
O
O
Synthesis of title compounds [2-hydoxy-4,6-dimethoxy-3-(2-
methyl-2H-pyrazol-3-yl)-phenyl]-phenyl-methanone derivatives
9a–j was achieved by the Friedel-Crafts acylation of 1-methyl-5-
(2,4,6-trimethoxy-phenyl)-1H-pyrazole 6 with various acid chlo-
rides in the presence of anhydrous AlCl3 in dichloromethane under
nitrogen atmosphere at room temperature in good to excellent
yields (Scheme 1).31 All the synthesized compounds were charac-
terized by IR, 1H NMR, and mass spectral data.
Famprofazone
O
Phenylbutazone
Ramifenazone
O
N
N
N
N
O
N
Antipyrine
Figure 1. Structures of some known pyrazole NSAIDs.
Morazone
The regioselective synthesis of key intermediate 1-methyl-5-
(2,4,6-trimethoxyphenyl)-1H-pyrazole 6 was carried out using
the synthetic strategies illustrated in Scheme 2. Xanthoxyline 2
was synthesized by the Friedel-Craft acetylation of 1,3,5-trime-
thoxybenzene 1 by adopting the literature precedent, and succes-
SC-558, mefobutazone, ramifenazone, famprofazone (Fig. 1) have
been reported as potent NSAIDs.21
On the other hand, the proficiency of benzophenone analogues
as chemotherapeutic agent especially as anti-inflammatory is well
documented.22 Benzophenone analogues synthesized by several
sive O-methylation of the resulting xanthoxyline 2 using
dimethyl sulphate and fused potassium carbonate in acetone un-
der reflux condition gave 1-(2,4,6-trimethoxyphenyl)ethanone 3
in excellent yield.25 2-Formylation of acetophenone 3 with ethyl
formate and sodium hydride gave 3-oxo-3-(2,4,6-trimethoxy-
phenyl) propanal 4, and without further purification subsequent
treatment of 4 with hydrazine hydrate afforded the corresponding
5-(2,4,6-trimethoxyphenyl)-1H-pyrazole 5 in 96% yield.29 Treat-
ment of compound 5 with methyl iodide and sodium hydride in
dry DMF under nitrogen atmosphere afforded a mixture of 1-
methyl-5-(2,4,6-trimethoxyphenyl)-1H-pyrazole 6 and 1-methyl-
3-(2,4,6-trimethoxyphenyl)-1H-pyrazole 7, which were easily
seperated by silica gel coloumn chromatography.30
The structures of the regioisomers 6 and 7 were determined
using 1D 1H, 13C NMR spectra, 135-DEPT and 2D NMR experiments
(1H–1H COSY, 1H–13C HSQC and 1H–13C HMBC and NOESY), which
were scanned on Bruker AV-400 and 500 MHz instrument. These
two regioisomers were only differ in the position of methyl group
on pyrazole nucleus, therefore, the 1H and 13C NMR chemical shifts
were found to be almost similar. Thus, the structures of these two
regioisomers were only assigned with the help of HMBC, HSQC and
O
O
O
O
Cl
a
+
R
R
O
O
O
OH
N
N
N
N
6
8
9a-j
F
F
F
Br
CF3
(a)
(f)
(b)
(c)
(d)
(i)
(e)
(j)
R =
Cl
Cl
Cl
Cl
Cl
(g)
OMe
(h)
F
Cl
Scheme 1. Reagents and conditions: (a) AlCl3, CH2Cl2, N2, 0 °C-rt, 24 h.
O
O
O
a
b
c
O
OH
O
O
O
O
O
O
1
2
3
O
O
O
O
d
e
+
O
H
O
O
O
NH
O
O
O
O
O
N
N
N
N
N
O
4
5
7
6
(18%)
(82%)
Scheme 2. Reagents and conditions: (a) CH3COCl, AlCl3, Et2O, N2, 0 °C-rt; (b) Me2SO4, K2CO3, acetone, reflux, 12 h; (c) HCOOEt, NaH, THF, N2, rt; (d) NH2NH2.H2O, rt; (e) MeI,
NaH, DMF, N2, 0 °C.

914
B. P. Bandgar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 912–916
11
11
O
O
7
7
8
8
6
5
6
5
9
9
10
12
12
10
4
4
O
O
O
O
Symbol
Correlations
HMBC
13
3
3
N
N
2
2
1
1
N
N
COSY
13
6
7
Figure 2. HMBC (H to C) and 1H–1H COSY correlations.
COSY correlations. The most important HMBC and COSY correla-
tions are summarized in Figure 2 and the complete chemical shift
assignment is shown in Table 4 and Table 5 (Supplementary data).
According to a Heteronuclear Multiple Bond Correlation (HMBC)
experiment performed with regioisomer 6, the HMBC correlation
observed between H-13 (dH 3.65) and C-3 (dC 135.5) and with reg-
ioisomer 7, the HMBC correlation observed between H-13 (dH 3.97)
and C-1 (dC 130.12) allowing to unambiguous identification of the
regioisomer 6 and 7. Additionally, Heteronuclear single Quantum
Coherence (HSQC) experiment was used to assign proton signals
to the corresponding carbon signals. There is a strong 1H–1H COSY
correlation between H-1 and H-2.
iting COX-1 enzyme less than indomethacin (Table 1). However, all
other compounds were also found to possess comparable inhibitory
profile against both COX-1 and COX-2 with the reference drug indo-
methacin. The SAR study of the compounds revealed that the sub-
stitution of methoxy-, bromo- and fluoro- groups on phenyl ring
had developed the active compounds.
In carrageenan paw edema, there was significant (p <0.01) in-
crease in paw volume of carrageenan control rats when compared
with normal rats.34 Increase in paw edema indicates the inflamma-
Table 2
Anti-inflammatory activity of the target compounds against the carrageenan-induced
paw edema in rats with different time intervals
All the newly synthesized benzophenone analogues (9a–j) were
screened for their in vitro inhibitory potential against the COX-1
Treatment and dose Paw volume (ml) Mean SEM at various time interval
(100 mg/kg.p.o.)
(n = 6)
and COX-2 enzymes at 100 lM by using Colorimetric COX (ovine)
Inhibitor Screening assay kit.32 Indomethacin was used as a refer-
ence compound. The percentage inhibition of COX-1/COX-2 is sum-
marized in Table 1. The results showed that most of the synthesized
compounds had an inhibitory profile against both COX-1 and COX-
2. The entire series of benzophenone analogues 9a–j exhibited con-
1 h
3 h
6 h
Normal group
0.40 0.04
0.35 0.04
1.17 0.1#
0.95 0.07**
1.11 0.06
1.06 0.04
1.20 0.3
0.38 0.06
1.69 0.08##
0.78 0.05**
1.22 0.2
Carrageenan control 0.94 0.05#
Indomethacin
0.86 0.05
0.81 0.05
1.10 0.03
0.92 0.02
0.92 0.04
0.93 0.03
0.89 0.05
1.12 0.03
0.59 0.1
9a
9b
9c
9d
9e
9f
9g
9h
9i
0.96 0.02**
1.63 0.6
siderable inhibition of COX-2 (44–60%) at 100 lM. When their
1.00 0.4
1.44 0.2
0.91 0.8**
1.55 0.7
activities were compared with indomethacin, it was determined
that all the compounds inhibited COX-2 enzyme more than indo-
methacin. The compound carrying methoxy substituent at the
fourth position of phenyl ring, 9f, appeared as the most active com-
pound in this series against COX-2 enzyme. Moreover, its inhibitory
activity against COX-1 enzyme was found to be lower than that of
indomethacin. Among the series compounds having bromo and flu-
oro substituents at m- and p-position of the phenyl ring (9d and 9b
respectively) were also the most notable ones because they showed
a remarkable inhibitory activity against COX-2 enzyme with inhib-
0.89 0.2**
1.05 0.04
1.22 0.07
1.39 0.5
0.99 0.2**
1.17 0.2*
1.62 0.2
0.90 0.04
1.01 0.04
1.29 0.8
1.11 0.2
9j
1.10 0.04
#
p <0.05 when compared with normal control group.
p <0.01 when compared with normal control group.
p <0.01 when compared with carrageenan control group.
p <0.05 when compared with carrageenan control group.
##
**
*
Table 1
In vitro COX-1 and COX-2 enzyme inhibitory activities of pyrazole integrated
benzophenones
Table 3
Antioxidant activities of pyrazole integrated benzophenones
Compound
% Inhibition of COX (100 l
M)a
Compound
% Inhibition at 100
SOR
lM
COX-1
COX-2
DPPH
OH
9a
9b
9c
9d
9e
9f
9g
9h
57.47
58.31
61.67
59.15
55.78
61.35
55.78
61.67
58.31
52.42
70.07
44.38
51.17
48.59
57.33
47.01
60.62
45.38
48.06
48.06
47.54
43.33
9a
9b
9c
9d
9e
9f
9g
9h
9i
36.25
31.54
34.53
40.25
27.98
48.85
43.58
26.13
29.64
31.21
81.52
26.06
22.54
17.61
26.77
25.36
52.24
51.41
21.13
27.47
24.65
51.95
35.49
47.60
26.98
41.63
35.43
35.92
36.54
29.67
38.12
31.86
78.34
9i
9j
9j
AA
Indomethacin
a
The determination was performed in duplicate for two independent
experiments.
Standard: AA = Ascorbic acid; data represent mean of two replicates.

B. P. Bandgar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 912–916
915
90
80
70
60
50
40
30
20
10
0
DPPH SOR OH
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
AA
Compounds
Figure 3. Antioxidant activities of benzophenone derivatives.
tion due to release of inflammatory mediators (serotonin, hista-
mine, prostaglandins etc.) at various time intervals. The synthe-
sized compounds viz. 9b, 9d and 9f are found to significantly
inhibit (p <0.01) the edema formation (Table 2). Inhibition of
paw edema formation at 6 h is greater due to the inhibition of
prostaglandins, which is known to be released at 4-6 h after the
carrageenan injection. Therefore, the compounds possessing signif-
icant COX-2 inhibitory potential (9b, 9d and 9f) also exhibit potent
anti-inflammatory activity in carrageenan induced paw edema in
rats.
template for the evaluation of new anti-inflammatory agents and
may be helpful for the design of new therapeutic tools against
inflammation.
Acknowledgment
Author H.V.C. thankful to Council of Scientific and Industrial Re-
search (CSIR), New Delhi, Govt. of India for financial support in the
form of SRF.
Free radicals especially the reactive oxygen species are pro-
posed to be the key players in the pathophysiological mechanisms
associated with various inflammatory disorders.26 These radicals
react indiscriminately with high rate constant with almost every
type of biomolecules found in living cell such as, sugars, amino
acids, phospholipids, DNA bases, organic acids and may deviate
the cells from its normal physiological functions.27 Antioxidants
are the compounds capable of scavenging the free radicals; for this
antioxidant therapy is one of the recent options.28 Taking into the
account of multifactorial character of oxidative stress; involved in
many pathological states, the entire series of benzophenone ana-
logues containing pyrazole scaffold (9a–j) were evaluated for their
direct scavenging activity against a variety of reactive oxygen and
nitrogen species such as hydroxyl (OH),38 superoxide (SOR)40 and
2,2-diphenyl-2-picrylhydrazyl (DPPH)36 stable free radical. Free
radical scavenging activity was measured in terms of percent inhi-
bition and results are presented in Table 3. All the synthesized
compounds have shown good to moderate scavenging activity
against DPPH, SOR and OH radicals (Fig. 3). The compounds 9f,
9g and 9d showed good DPPH free radical scavenging activity
(40–49%). All other compounds showed moderate DPPH reducing
potential (26–36%). Compounds 9f and 9g were found to possess
significant SOR scavenging activity (52.24%, 51.41% respectively)
as compared to standard ascorbic acid (AA) (51.95%). All other
compounds were weak SOR scavengers (17–27%). Compounds 9b,
9d, 9i and 9g showed 36–47% inhibition as compared to standard
AA (78%). The wide variation in the free radical scavenging poten-
tial for the tested compounds may be due to the variation in the
proton–electron transfer by the derivatives due to difference in
their structures and stability.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
10.031.
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In conclusion, a series of novel pyrazole integrated benzophe-
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were characterised by IR, 1H NMR and mass spectral data. All the
newly synthesized compounds (9a–j) were evaluated for their
anti-inflammatory and antioxidant potential. Among the synthe-
sized compounds, compounds 9f, 9d and 9b were found to be
the most active anti-inflammatory agents in addition to having po-
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20. Duma, J.; Hatoum-Mokdad, H.; Sibley, R.; Riedl, B.; Scott, W. J.; Monahan, M. K.;
Lowinger, T. B.; Brennan, C.; Natero, R.; Turner, T.; Johnson, J. S.; Schoenlebar,
R.; Bhargava, A.; Wilhelm, S. M.; Housley, T. J.; Ranges, G. E.; Shrikhande, A.
Bioorg. Med. Chem. Lett. 2000, 10, 2051.
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Pharmaceutical Press: London, 1993. p. 1; (b) Amir, M.; Kumar, S. Indian J.
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K. Indian J. Chem., Sect B 2003, 42B, 1979.
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phenyl)-1H-pyrazole 6 (1.24 g, 5 mmol) in dry CH2Cl2 (10 ml) under anhydrous
condition. The mixture was cooled to 0 °C, stirred vigorously, and solution of
the benzoyl chlorides (6 mmol) in dry CH2Cl2 (10 ml) was added during 1 h.
The stirring was continued for 2 h at 0 °C and mixture was left at room
temperature for 48 h. After completion of reaction (TLC), reaction mixture was
poured over 50 g crushed ice and treated with concentrated HCl (2.5 ml). The
mixture was heated in water bath for 30 min, CH2Cl2 being allowed to distill
away, cooled and the aqueous layer was extracted with diethyl ether
(2 ꢀ 25 ml). The extracted organic layer was washed with sodium carbonate
(Na2CO3) solution (5%, 15 ml) and then extracted with NaOH (5%, 2 ꢀ 25 ml).
The cooled clear alkaline solution was acidified with HCL and the separated
solid was filtered off, washed with water, dried and recrystallized from
methanol to obtain title compounds 9a–n in pure form.
2-Hydoxy-4,6-dimethoxy-3-(2-methyl-2H-pyrazol-3-yl)-phenyl-phenyl-
methanone (9a): Yield: 86.5%; mp: 148–150 °C; IR (KBr, cmꢁ1): 3435, 2982,
2946, 1622, 1594, 1578, 1470, 1287, 1141; 1H NMR (300 MHz, CDCl3): d 3.60 (s,
3H, NCH3), 3.72 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 6.08 (s, 1H, Pyr-H), 6.30 (s, 1H,
Ar-H), 7.38-7.52 (m, 3H, 3xAr-H), 7.56–7.62 (m, 3H, 2xArH, Pyr-H); MS (ESI):
m/e 339 (M+1).
32. In vitro COX inhibition assay: The assay was performed by using Colorimetric
COX (ovine) inhibitor Screening assay kit.33 Briefly, the reaction mixture
contains, 150
ll of assay buffer, 10
ll of heme, 10 ll of enzyme (either COX-1
or COX-2), and 50
ll of sample (0.1 mM). The assay utilizes the peroxidase
24. Ottosen, E. R.; Sorensen, M. D.; Bjorkling, F.; Skak-Nielsen, T.; Fjording, M. S.;
Aaes, H.; Binderup, L. J. Med. Chem. 2003, 46, 5651.
25. Ahluwalia, V. K. Intermediates for Organic Synthesis; I. K. International
Publisher: New Delhi, 2005.
26. Trouba, K. J.; Hamadeh, H. K.; Amin, R. P.; Germolec, D. R. Antioxid. Redox Signal.
2002, 4, 665.
27. Barry, H.; John, M. C. G. Biochem. J. 1984, 219, 1.
component of the COX catalytic domain. The peroxidase activity can be
assayed calorimetrically by monitoring the appearance of oxidized N,N,N,N0-
tetramethyl-p-phenylenediamine (TMPD) at 590 nm.Indomethacin (0.1 mM)
was used as a standard drug. The percent COX inhibition was calculated using
following equation,
T
COX inhibition activity (%)=1 ꢁ C ꢀ 100
Where T = Absorbance of the inhibitor well at 590 nm.
28. Velavan, S.; Naghlendran, K.; Mahesh, R.; Hazeena B.V. PHCOGMAG 1998, ISSN:
0973–1296.
C = Absorbance of the 100% initial activity without inhibitor well at 590 nm.
33. Maurias, M. Bioorg. Med. Chem. 2004, 12, 5571.
29. Preparation of 5-(2,4,6-trimethoxy-phenyl)-1H-pyrazole (5): Ethyl formate
(2.96 g, 3.21 ml, 30 mmol) was added to a suspension of sodiun hydride (60%
in oil, 0.8 g, 20 mmol) in tetrahydrofuran (3 ml) at room temperature. After
34. Carrageenan induced hind paw edema in rats: Rats were divided into various
groups (n = 6) and allowed to free access to water ad libitum. Different groups
of rats administered with indomethacin (100 mg/kg, b.w.) and various
synthesized compounds 9a–9j (50 mg/kg,) orally. One group of rats served as
a control and administered with gum acacia (1%, w/v; 10 ml/kg, b.w., p.o.). One
hour after the drug administration, to all groups of rats, hind paw edema was
induced by the method of winter et al.35 by injecting 0.1 ml of 1% (w/v)
solution of carrageenan subcutaneously into the subplanter region of hind
paw. The hind paw edema volume was measured by volume displacement
method using plethysmometer (UGO, Besile 7140, Italy) by immersing the paw
till the level lateral malleolus at various time intervals (1, 3 and 6 h) after
carrageenan injection.
35. Winter, C. A.; Risley, E. A.; Nuss, G. W. Proc. Soc. Exp. Biol. Med. 1962, 111, 544.
36. DPPH radical scavenging assay: DPPH (1,1-diphenyl-2-picrylhydrazyl) radical
scavenging assay was carried out as per reported method with slight
modifications.37 Briefly, 1 ml of test solution (0.1 mM) was added to equal
quantity of 0.1 mM solution of DPPH in ethanol. After 20 min of incubation at
room temperature, the DPPH reduction was measured by reading the
absorbance at 517 nm. Ascorbic acid (0.1 mM) was used as reference
compound.
stirring for 10 min,
a solution of 1-(2,4,6-trimethoxy-phenyl)-ethanone 3
(2.1 g, 10 mmol) in THF (15 ml) was added, and the mixture was stirred for 1 h.
To the reaction mixture was then added 1 M HCl (20 ml), and extracted with
diethyl ether (3 ꢀ 25 ml). Organic layer was concentrated under reduced
pressure. To this hydrazine hydrate (5 g, 4.84 ml, 100 mmol) was added, and
the mixture was stirred for 30 min. The reaction mixture was made alkaline by
adding of 6 N NaOH (20 ml) and extracted with ethyl acetate. The organic layer
was washed with brine, dried over anhydrous magnesium sulphate,
evaporated in vacuo, and recrystallized from ethanol to obtain colorless
crystals of title compound 5. Yield: 85%; mp: 138–140 °C; IR (KBr, cmꢁ1): 3293,
3018, 2969, 2939, 2344, 1752, 1607, 1586, 1451, 1204; 1H NMR (400 MHz,
CDCl3): d 3.85 (s, 3H, OCH3), 3.92 (s, 6H, OCH3), 6.22 (s, 2H, ArH), 6.82 (d, 1H,
J = 1.6 Hz, C@CH), 7.6 (d, 1H, J = 1.6 Hz, N@CH), 11.38 (bs, 1H, NH); MS (ESI): m/
e 235 (M+1).
30. Preparation of 1-methyl-5-(2,4,6-trimethoxy-phenyl)-1H-pyrazole (6): 5-(2,4,6-
Trimethoxy-phenyl)-1H-pyrazole 5 (2.34 g, 10 mmol) was dissolved in dry
DMF (15 ml) under N2 atmosphere. Cooled the flask in an ice bath and methyl
iodide (2.84 g, 1.25 ml, 20 mmol) was added to it. To this solution, sodium
hydride (60% in oil, 0.48 g, 12 mmol) was added in portions and the resulted
solution was then allowed to stir at 0 °C for 15 min. The reaction mixture was
poured over crushed ice and the resulted solid was filtered off, recrystallized
from ethanol to afford the title compound 6 in pure form. The filtrate was then
extracted three times with ethyl acetate. The combined extracts were washed
with water. After drying over anhydrous MgSO4, the solvent was distilled off
under reduced pressure. The resulting residue was purified by silica gel column
chromatography to obtain compound 7 in pure form.
37. (a) Roberta, R.; Luciana, G. M.; Luciana, C. C.; Glaucia, P. J. Food Sci. 2006, 71,
102; (b) Blois, M. S. Nat. New Biol. 1958, 181, 1199.
38. Hydroxyl (OH) radical scavenging assay: The OH radicals scavenging activity was
demonstrated with Fenton reaction.39 The reaction mixture contained, 60
l
l of
l of 1-10 phenanthroline (1 mM), 2.4 ml of phosphate buffer
(0.2 M, pH 7.8), 150 l of H2O2 (0.17 M) and 1.5 ml of individual samples
FeCl2 (1 mM), 90
l
l
(0.1 mM). The reaction was started by adding H2O2. After 5 min incubation at
room temperature, the absorbance was recorded at 560 nm. Ascorbic acid
(0.1 mM) was used as reference compound.
1-Methyl-5-(2,4,6-trimethoxy-phenyl)-1H-pyrazole (6): Yield: 82%; mp: 147–
149 °C; IR (KBr, cmꢁ1): 3064, 2962, 2943, 2841, 1612, 1584, 1549, 1473, 1458,
1234, 1161; 1H NMR (500 MHz, CDCl3): d 3.65 (s, 3H, NCH3), 3.75 (s, 6H,
39. Rollet-Labelle, E.; Gragne, M. S.; Elbim, C.; Marquetty, C.; Gougerot-Pocidalo,
M. A.; Pasquier, C. Free Radical Biol. Med. 1998, 24, 563.
40. Superoxide radical (SOR) scavenging assay: The superoxide anion scavenging
assay was performed by the reported method.41 Superoxide anion radicals
were generated in a non-enzymatic Phenazine methosulphate–Nicotinamide
Adenine Dinucleotide (PMS–NADH) system through the reaction of PMS, NADH
and Oxygen. It was assayed by the reduction of Nitroblue tetrazolium (NBT). In
this experiment superoxide anion was generated in 3 ml of Tris HCL buffer
2xOCH3), 3.87 (s, 3H, OCH3), 6.20 (m, 3H, 2xArH, Pyr-H), 7.55 (s, 1H, Pyr-H); 13
C
NMR (125 MHz, CDCl3): 36.66 (m, CH3), 55.40 (m, CH3), 55.75 (s, CH3), 90.54 (s,
CH), 100.69 (w, C), 107.44 (m, CH), 135.52 (w, C), 137.91 (m, CH), 159.32 (m, C),
162.25 (w, C); 135-DEPT: 36.65 (+), 55.40 (+), 55.75 (+), 90.53 (+), 107.44 (+),
137.89 (+); MS (ESI): m/e 249 (M+1).
1-Methyl-3-(2,4,6-trimethoxy-phenyl)-1H-pyrazole (7): Yield: 18%; mp: 104–
106 °C; IR (KBr, cmꢁ1): 3130, 2992, 2956, 2834, 1613, 1586, 1505, 1474, 1224,
1161; 1H NMR (400 MHz, CDCl3): d 3.76 (s, 6H, 2xOCH3), 3.85 (s, 3H, OCH3),
3.97 (s, 3H, NCH3), 6.20 (s, 2H, 2xArH), 6.31 (d, J = 2.8 Hz, 1H, Pyr-H), 7.41 (d,
J = 2.8 Hz, 1H, Pyr-H); 13C NMR (100 MHz, CDCl3): 38.92 (m, CH3), 55.27 (m,
CH3), 55.91 (s, CH3), 90.55 (s, CH), 104.51 (w, C), 107.81 (m, CH), 130.13 (m,
(100 mM, pH 7.4) containing 0.75 ml of NBT (300
(936 M), and 0.3 ml of sample (0.1 mM). The reaction was initiated by adding
0.75 ml of PMS (120 M) to the mixture. After 5 min of incubation at room
temperature the absorbance at 560 nm was measured in spectrophotometer.
Ascorbic acid (0.1 mM) was used as reference compound.
lM), 0.75 ml of NADH
l
l
41. Liu, F.; Ooi, V. E. C.; Chang, S. T. Life Science 1997, 60, 763.