PEG mediated synthesis and biological evaluation of asymmetrical pyrazole curcumin analogues as potential analgesic, anti-inflammatory and antioxidant agents

Article abstract of DOI:10.1111/cbdd.12416

The new series of asymmetrical pyrazole curcumin analogues 4a-g were synthesized by using polyethylene glycol (PEG-400) as a green reaction medium and evaluated for their in vivo analgesic and in vitro antioxidant (H₂O₂, DPPH, Ferrous reducing power and Nitric oxide scavenging activity) and anti-inflammatory activities. All the compounds synthesized 4a-g showed the potential to demonstrate analgesic activity as compared to the standard ibuprofen. Among the tested series, compounds 4e and 4b exhibited good hydrogen peroxide scavenging activity as compared to the standard butylated hydroxy toluene (BHT). Compounds 4b, 4d, 4f, and 4g showed good DPPH free radical scavenging activity. Compounds 4b, 4c, 4d, 4e and 4g showed excellent ferrous-reducing power activity, whereas all the compounds showed better nitric oxide scavenging activity than standard ascorbic acid. Additionally, all the synthesized compounds were also screened for their in vitro anti-inflammatory activity. Compounds 4b, 4d, 4f and 4g showed good anti-inflammatory activity as compared to standard diclofenac sodium.

Full text of DOI:10.1111/cbdd.12416

Received Date : 21-Jan-2014
Revised Date : 24-May-2014
Accepted Date : 30-Jul-2014
Article type : Research Article
PEG mediated synthesis and biological evaluation of asymmetrical
pyrazole curcumin analogues as potential analgesic, anti-
inflammatory and antioxidant agents
Shravan Y. Jadhava*, Raghunath B. Bhosalea, Sachin P. Shiramea, Sandeep B. Patilb,
Suresh D. Kulkarnic
aOrganic Chemistry Research Laboratory, School of Chemical Sciences, Solapur University, Solapur-
413255, Maharashtra, India.
bPharmacology Department, Appasaheb Birnale College of Pharmacy, Sangli-416416, Maharashtra,
India
cPharmacology Department, Luqman College of Pharmacy, Gulberga- 584102, Karnataka, India
*Corresponding author: Tel./fax: 091-217-2744770
E-mail: shravanjadhav.chem@gmail.com bhosale62@yahoo.com
The new series of asymmetrical pyrazole curcumin analogues 4a-g were synthesized by using
polyethylene glycol (PEG-400) as a green reaction medium and evaluated for their in vivo
analgesic and in vitro antioxidant (H2O2, DPPH, Ferrous reducing power and Nitric oxide
scavenging activity) and anti-inflammatory activities. All the compounds synthesized 4a-g
showed the potential to demonstrate analgesic activity as compared to the standard ibuprofen.
Among the tested series, compounds 4e and 4b exhibited good hydrogen peroxide
scavenging activity as compared to the standard butylated hydroxyl toluene (BHT).
Compounds 4b, 4d, 4f and 4g showed good DPPH free radical scavenging activity.
Compounds 4b, 4c, 4d, 4e and 4g showed excellent ferrous reducing power activity whereas
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an 'Accepted Article', doi: 10.1111/cbdd.12416
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all the compounds showed better nitric oxide scavenging activity than standard ascorbic acid.
Additionally, all the synthesized compounds were also screened for their in vitro anti-
inflammatory activity. Compounds 4b, 4d, 4f and 4g showed good anti-inflammatory activity as
compared to standard diclofenac sodium.
Keywords: Asymmetrical Pyrazole Curcumin, PEG-400, Analgesic activity, Antioxidant activity,
Anti-inflammatory activity.
Curcumin is a β-diketone constituent of the turmeric that is obtained from the powdered rhizome of
Curcuma longa. Curcumin has a wide range of interesting biological activities such as anti-
inflammatory, antioxidant, antiviral, cutaneous wound healing, hypocholesterolemic effects in diabetic
patients, anti-angiogenic and stimulatory response to stress-induced biological activity (1,2). Curcumin
has been demonstrated to possess preventative activity against Aβ-aggregation in Alzheimer’s model
(3). Evidence from both in vitro and in vivo studies show that β-diketone moiety is responsible for
instability and weak pharmacokinetic profiles of curcumin. Recently, synthetic modifications of
curcumin, which were aimed at enhancing its bioactivities, suggested that the stability and metabolic
profile of curcumin could be enhanced by deleting β-diketone moiety. Recent studies from several
independent groups demonstrated that the curcumin analogues without β-diketone either retained or
increased various biological activities such as anticancer (4, 5), antibacterial (6) and anti-inflammatory
(7).
The survey of literature reveals that many curcumin analogues have been used as lead
molecules for the design and development of array of therapeutic agents including anti-inflammatory,
anticancer, antioxidant, antiviral, and antimicrobials (8). Several analogues of curcumin have been
synthesized and evaluated for multifunctional pharmacological application in a variety of diseases
such as liver fibrosis, inflammation, cardiovascular diseases, and cancer (9-11). Several curcuminoid
pyrazoles have been synthesized and described as new therapeutic agents in inflammatory bowl
diseases targeting the matrix metalloproteinases(12). Novel series of curcumin analogues were
synthesized and tested as anticancer and antiangiogenic agents (13).
Variety of traditional non-steroidal anti-inflammatory drugs (NSAIDs), such as naproxen,
ibuprofen, diclofenac, flurbiprofen, indomethacin and aspirin are available in the market which have
some limitations in clinical use like gastrointestinal (GI) haemorrhage and ulceration, addiction,
tolerance especially for opiates(14). The present day NSAIDs are having side effects. The
development of alternatives to NSAIDs is being attempted all over the world.
Reactive oxygen species (ROS) are formed and degraded by all aerobic organisms, leading
to either physiological concentrations required for normal cell function, or excessive quantities, the
state called oxidative stress. These species react indiscriminately with almost every type of
biomolecules found in living cell such as, sugars, amino acids, phospholipids, DNA bases, organic
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acids and may deviate the cells from its normal physiological functions(15). Oxidative stress is also
proposed to be involved in the process of aging both by including damage to mitochondrial DNA and
by other mechanisms (16, 17), and leads to the initiation of various diseases such as cancer (18),
ischemia-reperfusion injury (19), atherosclerosis (20), cardiovascular (21), inflammation (22), and
neurodegenerative disorders such as Alzheimer’s and Parkinson's disease (23,24). Antioxidants are
the compounds capable of scavenging the free radicals; for this antioxidant therapy is one of the
recent options.
On the other hand, PEG solvents are known to be inexpensive, easily available, thermally
stable, recyclable, biological compatible, non-toxic and water soluble compound that does not
hydrolyze on long storage (25, 26). Due to these advantages, PEGs of different molecular weights are
extensively used as solvents or vehicles in various pharmaceutical industries. However, PEG polymer
of low molecular weight differs significantly from polymers of high molecular weight in their physico-
chemical properties, higher toxicity and possibly genotoxicity (27). The toxicity data of different
molecular weight PEG’s are also available and some of them are already approved for internal
consumption by the USA FDA (28). The use of PEG as a green and alternative reaction medium in
organic reactions is relatively recent (29).
The researcher’s recent success on the development of new selective eco-friendly
methodologies using polyethylene glycol (PEG-400) (30, 31) as a green solvent for the preparation of
biologically active compounds, they report the synthesis of some new series of asymmetrical pyrazole
curcumin analogues (APCAs) using aq.NaOH in PEG-400 as green alternative reaction medium and
evaluate them as analgesic agent along with assessing their antioxidant and anti-inflammatory
potential. To the best of our knowledge this is the first report on the in vivo analgesic study of newly
synthesized of asymmetrical pyrazole curcumin analogues (APCAs).
Methods and Materials
Instrumentation
Melting points were determined with digital thermometer and were uncorrected. IR spectra
1
were recorded on FT-IR spectrometer (Perkin Elmer) using KBr disc method. HNMR spectra were
recorded on 1HNMR (Varian-NMR-mercury 300 MHz) spectrometer in CDCl3 as solvent. All chemical
shifts (δ) are quoted in parts per million downfield from TMS and coupling constants (J) are given in
hertz. Abbreviations used in the splitting pattern were as follows: s = singlet, d = doublet, t= triplet, q =
quintet, m = multiplet. The mass spectra were obtained with a Shimadzu LCMS-2010 EV. All the
reagents and solvents used were of analytical grade and were used as supplied unless otherwise
stated. Thin layer chromatography was performed on pre-coated silica plates (Merks kiesegel
60F254, 0.2 mm thickness) sheet. The spots could be visualized easily under UV light.
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Chemistry
Although, curcumin is non-toxic and has promising biological activities, preclinical and clinical
studies have indicated that poor bioavailability and pharmacokinetic profile are due to its instability
under physiological condition which has limited its application in anti-cancer therapies (32-34).To
overcome these barriers, several research groups have synthesized curcumin analogues. Robinson
et al. (4) have synthesized enone and dienone analogues, Ahn et al. (35) have synthesized the bis-
alkynyl/alkyl, pyridine and thiophene derivatives as curcumin analogues. Woo et al. (36) have
synthesized curcumin mimics containing enone and amide ring which were shown to possess
increased anti-angiogenic activity. Bandgar et al. (37) have synthesized novel curcumin mimics
incorporating olefin as well as aromatic, alicyclic or heteroaromatic amide moities and evaluated for
antioxidant, cytotoxic and antimicrobial activity. Bandgar et al. (38) have also synthesized novel
curcumin analogues and evaluated as anti-inflammatory, anti-cancer and anti-oxidant agents.
Herein, we synthesized the new curcumin analogues containing enone and amide including
pyrazole moiety. The title compounds asymmetrical pyrazole curcumin analogues (APCAs) were
prepared by the acylation of 3-aminoacetophenone (1) with fluoro acyl chloride to afford
corresponding amide (2), which on Claisen-Schmidt condensation with various substituted 1,3-
diphenyl-1H-pyrazole-4-carbaldehydes (3a-g) and NaOH in PEG-400 furnished APCAs (4a-g) in good
to excellent yields (Scheme-1). The completion of the reaction was monitored by TLC
. The
substituted 1,3-diphenyl-1H-pyrazole-4-carbaldehydes (3a-g) were prepared by the Vilsmeir-Haack
reaction (39) of various substituted aryl hydrazones from various substituted acetophenones. All the
synthesized compounds were characterized by IR, 1HNMR and Mass spectroscopy.
General procedure for the preparation N-(3-Acetyl-phenyl)-4-fluoro-benzamide (2)
1-(3-Amino-phenyl)ethanone 1 (1 g, 7.40 mM) was suspended in 20 mL of 5 % of sodium
hydroxide solution in round bottom flask and added 2 mL 4-fluorobenzoyl chloride, 0.5 mL at a time,
with constant shaking and stirred vigorously for 10 min, reaction mixture was heated under reflux on
water bath at 70-80oC for 30 min until the odor of the 4-fluorobenzoyl chloride was disappeared. Make
sure that the mixture has an alkaline pH. Filter off the solid benzoyl derivative and recrystallized it
from petroleum ether and ethyl acetate to obtain compound 2.
General procedure for the preparation of asymmetrical pyrazolecurcumin analogues (4a-g)
The substituted 1,3-diphenyl-1H-pyrazole-4-carbaldehydes 3a-g were prepared by Vilsmeier-
Haack reaction on acetophenone hydrazones obtained from various substituted acetophenones
according to literature method (39).
A mixture of substituted 1,3-diphenyl-1H-pyrazole-4-
carbaldehydes 3a-g (1 mM) and N-(3-Acetyl-phenyl)-4-fluoro-benzamide 2 (1 mM) was dissolved in
15 mL PEG-400. To this mixture, NaOH (20%, 1mL) was added and the reaction mixture was stirred
at 40-50oC temperature for 1h. The reaction mixture was then poured into 100 mL ice cold water. The
product was separated out; it was filtered and processed out. The products obtained were purified by
recrystallization from ethanol to afford pure compounds 4a-g.
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Analgesic activity
The analgesic activity was evaluated using the acetic acid induced writhing method (40) using
groups of six animals each. A solution of acetic acid (1% v/v) in distilled water was prepared and
injected intraperitoneally in a volume of 0.1 mL. One group was kept as control, and the animals of
the other group were pretreated with the test compounds, 1 h before the acetic acid (1% v/v)
treatment. The writhing episodes were recorded for 15 min; stretching movements consisting of
arching of the back, elongation of body, and extension of hind limbs were counted.
Preparation of test compounds
After suspending the test compounds in 1.0% aq. solution of gum acacia, the test samples were
administered to the test animals orally (5 mg/kg). The positive and negative control group animals
received the same experimental handling as those of the test groups except that the drug-treated
control group animals received only appropriate volumes of vehicle and of the reference drug,
Ibuprofen (15 mg/kg, p.o).
Pharmacology
Swiss albino mice of either sex weighing 20–25 g were obtained from Animal house, Luqman
College of Pharmacy, Gulberga, Karnataka, India. All the animals were housed under standard
ambient conditions of temperature (25 2oC) and relative humidity of 50 5%. A 12/12-h light/dark
cycle was maintained. All the animals were allowed to have free access to water and standard
palletized laboratory animal diet 24 h before pharmacological studies. The experimental procedures
and protocols used in this study were reviewed and approved by the Institutional Animal Ethical
Committee (IAEC) of Luqman College of Pharmacy, Gulberga, constituted in accordance with the
guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA/ 346), Government of India.
Antioxidant activity:
Hydrogen Peroxide (H2O2) Scavenging activity
The hydrogen peroxide scavenging assay was performed by the reported method (41). A
solution of hydrogen peroxide (40 mM) was prepared in phosphate buffer (pH 7.4). The 1 mM
concentrations of various compounds (4a-g) were added to a hydrogen peroxide solution (0.6 mL, 40
mM). Absorbance of hydrogen peroxide at 230 nm was determined after 10 min. against a blank
solution containing phosphate buffer without drug. The percentage scavenging of hydrogen peroxide
of synthetic compounds and standard compounds was calculated using the following formula:
% scavenged [H2O2] = [(A0-A1)/A0] x 100
Where,
A0 = the absorbance of the control
A1 = the absorbance in the presence of the sample of MO and standards.
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DPPH radical scavenging activity
The ability of compounds to scavenge DPPH radical was assessed using literature method
(42) with slight modification. Briefly, 1 ml of synthesized compounds (4a-g) as 100mM was mixed with
3.0 mL DPPH (0.5 mmol/L in methanol), the resultant absorbance was recorded at 517 nm after 30
min. incubation at 37OC. The percentage of scavenging activity was derived using the following
formula,
Percentage of inhibition (%) = [(Acontrol - Asample) / Acontrol] x 100
Where,
A control - absorbance of DPPH
A sample - absorbance reaction mixture (DPPH with Sample).
Ferrous reducing power
The reducing ability of compounds was measured according to the reported method [43].
100mM of the synthesized compounds (1.0 mL) was mixed with 2.5 mL of phosphate buffer (0.2 M,
pH 6.6) and 2.5 mL of potassium ferricyanide (1%). The mixture was incubated at 50 OC for 20 min.
with TCA (10%: 2.5 mL). Then mixture was centrifuged at 3 000 rpm for 10 min. The supernatant (2.5
mL) was mixed with 2.5 mL of distilled water and 0.5 mL of ferric chloride (1%) and the absorbance
was measured at 700 nm. Higher absorbance of the reaction mixture indicated greater reducing
power. The reducing power of compounds was compared with that of standard antioxidant.
Nitric oxide radical scavenging activity
Nitric oxide radical scavenging was carried out as per the reported method [42]. Nitric oxide
radicals were generated from sodium nitroprusside solution. 1 mL of 10 mM sodium nitroprusside was
mixed with 1 mL of 100 mM synthesized compounds in phosphate buffer (0.2 M pH 7.4). The mixture
was incubated at 25 OC for 150 min. After incubation the reaction mixture mixed with 1.0 mL of pre-
prepared Griess reagent (1% sulphanilamide, 0.1% napthylethylenediamine dichloride and 2%
phosphoric acid). The absorbance was measured at 546 nm and percentage of inhibition was
calculated using the same formula as above. The decreasing absorbance indicates a high nitric oxide
scavenging activity.
Percentage of inhibition (%) =[(Acontrol - Asample) / Acontrol] x 100
Where,
A control - absorbance of Nitric oxide
A sample - absorbance reaction mixture (Nitric oxide with Sample).
In vitro anti-inflammatory activity by Protein denaturation method [44]
The reaction mixture (10 mL) consisted of 0.4 mL of egg albumin (from fresh hen’s egg), 5.6 mL of
phosphate buffered saline (PBS, pH 6.4) and 4 mL of synthesized compounds (100mM). Similar
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o
volume of double-distilled water served as control. Then the mixtures were incubated at (37 C 2) in
o
a incubator for 15 min and then heated at 700 C for 5 min. After cooling, their absorbance was
measured at 660 nm by using vehicle as blank. Diclofenac sodium at the 100 mM was used as
reference drug and treated similarly for determination of absorbance. The percentage inhibition of
protein denaturation was calculated by using the following formula,
% inhibition = 100 x ( Vt / Vc - 1)
Where, Vt = absorbance of test sample,
Vc = absorbance of control.
Results and discussion
Spectral analysis
All the synthesized compounds were characterized by IR, 1H NMR, and MS. The IR spectrum
of the titled compounds showed absorption due to –NH stretching at ~3350 cm-1, amide carbonyl
group at ~1645 cm-1.1H NMR spectrum (300 and 400 MHz) recorded in DMSO-d6 showed a typical
singlet at δ ~ 9-10 (for -NH) and a typical 1H-1H coupling constant in between 12-16 Hz showing
trans stereochemistry of the double bond.
N-(3-Acetyl-phenyl)-4-fluoro-benzamide (2)
Yield: 78%; MP: 115-116oC; IR (KBr): 3476, 3296, 3077, 2968, 1709, 1609, 1499, 1281, 1178, 1061,
1
862 cm -1. H NMR (300 MHz, CDCl3, δ in ppm): 8.36 (broad s, 1H, -NH), 8.16 (s, 1H, ArH), 8.10 (d,
1H, ArH,J = 8.1 Hz), 7.72 (d, 1H, ArH,J = 7.8 Hz), 7.47 (t, 1H, ArH,J = 7.8 Hz), 7.96-7.7.13 (m, 4H,
ArH), 2.60 (s, 3H, CH3). MS: m/e 258 (M+1).
4-Fluoro-N-(3-{3-[3-(4-fluoro-phenyl)-1-phenyl-1H-pyrazol-4-yl]-acryloyl}-phenyl)-benzamide
(4a)
Yield: 82%; mp: 210-2120C; IR (KBr): 3744, 3292, 3117, 3058, 1649, 1595, 1529, 1409, 1241, 842,
1
751cm-1. H NMR (300 MHz, CDCl3, δ in ppm): 7.17-7.98 (m, 17H, Ar-H); 7.39 (d, 1H, J=15.0 Hz, -
CH=CH-); 7.88 (d, 1H, J=15.0 Hz, -CH=CH-); 8.18 (s, 1H, pyrazole-H); 8.39 (s, 1H, N-H, D2O
exchangeable); MS: m/e 506 (M+1).
N-(3-{3-[3-(4-Chloro-phenyl)-1-phenyl-1H-pyrazol-4-yl]-acryloyl}-phenyl)-4-fluoro-benzamide
(4b)
Yield: 80%; mp: 217-2180C; IR (KBr): 3821, 3743, 3314, 3114, 1651, 1593, 1527, 1418, 1239, 841,
757 cm-1. 1H NMR (300 MHz, CDCl3, δ in ppm): 7.18 (t, 2H, ArH, J = 8.4 Hz); 7.65 (d, 2H, ArH, J = 8.4
Hz); 8.02 (s, 1H, ArH); 7.16-8.0 (m, 12H, Ar-H); 7.37 (d, 1H, J=15.0 Hz, -CH=CH-); 7.81 (d, 1H,
J=15.0 Hz, -CH=CH-); 8.19 (s, 1H, pyrazole-H); 8.37 (s, 1H, N-H, D2O exchangeable). 13C NMR (100
MHz, DMSO-d6, δ in ppm): 188.8, 164.5, 151.6, 151.6, 139.5, 138.8, 138.1, 134.0, 133.5, 130.9,
130.7, 130.0, 129.0, 127.2, 124.8, 123.7, 121.9, 119.9, 118.7, 117.7, 115.4, 115.2. MS: m/e 522
(M+1).
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N-(3-{3-[3-(4-Bromo-phenyl)-1-phenyl-1H-pyrazol-4-yl]-acryloyl}-phenyl)-4-fluoro-benzamide
(4c)
Yield: 79%; mp: 248-2500C; IR (KBr): 3821, 3743, 3314, 3114, 1651, 1593, 1527, 1501, 1418, 239,
841,757 cm-1. 1H NMR (300 MHz, CDCl3, δ in ppm): 7.38-8.40 (m, 17H, Ar-H); 7.39 (d, 1H, J=15.0 Hz,
-CH=CH-); 7.79 (d, 1H, J=15.0 Hz, -CH=CH-); 9.4 (s, 1H, pyrazole-H); 10.5 (s, 1H, N-H, D2O
exchangeable); MS: m/e 568 (M+2).
4-Fluoro-N-{3-[3-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)-acryloyl]-phenyl}-benzamide (4d)
Yield: 86%; mp: 140-1410C; IR (KBr): 3853, 3743, 3392, 3245, 3123, 2924, 1645, 1577, 1533, 1422,
1239, 845, 749 cm-1. 1H NMR (300 MHz, CDCl3, δ in ppm): 2.41 (s, 3H, Methyl-H); 7.19 (t, 2H, ArH, J
= 8.4 Hz), 7.28-8.03 (m, 15H, Ar-H); 7.30 (d, 1H, J=15.0 Hz, -CH=CH-); 7.90 (d, 1H, J=15.0 Hz, -
CH=CH-); 8.14 (s, 1H, pyrazole-H); 8.37 (s, 1H, N-H, D2O exchangeable); MS: m/e 502 (M+1).
4-Fluoro-N-(3-{3-[3-(4-methoxy-phenyl)-1-phenyl-1H-pyrazol-4-yl]-acryloyl}-phenyl)-benzamide
(4e)
Yield: 88%; mp: 209-2110C; IR (KBr): 3853, 3743, 3392, 3245, 3123, 2924, 1645, 1577, 1533, 1422,
1
1239, 845, 749 cm-1. H NMR (300 MHz, CDCl3, δ in ppm): 2.91 (s, 3H, Methoxy-H); 7.16-8.03 (m,
17H, Ar-H); 7.29 (d, 1H, J=15.0 Hz, -CH=CH-); 7.89 (d, 1H, J=15.0 Hz, -CH=CH-); 8.14 (s, 1H,
pyrazole-H); 8.37 (s, 1H, N-H, D2O exchangeable); MS: m/e 518 (M+1).
4-Fluoro-N-(3-{3-[3-(3-nitro-phenyl)-1-phenyl-1H-pyrazol-4-yl]-acryloyl}-phenyl)-benzamide (4f)
Yield: 78%; mp: 211-2130C; IR (KBr): , 3739, 3290, 3108, 1644, 1590, 1526, 1500, 1409, 1352, 844,
757 cm-1. 1H NMR (300 MHz, CDCl3, δ in ppm): 7.69 (t, 1H, ArH, J = 8.1 Hz); 8.06 (d, 1H, ArH, J = 7.2
Hz); 8.18 (s, 1H, ArH); 8.29 (d, 1H, ArH, J = 8.1 Hz); 7.18-8.31 (m, 13H, Ar-H); 7.42 (d, 1H, J=15.0
Hz, -CH=CH-); 7.87 (d, 1H, J=15.0 Hz, -CH=CH-); 8.42 (s, 1H, pyrazole-H); 8.66 (s, 1H, N-H, D2O
exchangeable). MS: m/e 533 (M+1).
N-{3-[3-(1,3-Diphenyl-1H-pyrazol-4-yl)-acryloyl]-phenyl}-4-fluoro-benzamide(4g)
Yield: 86%; mp: 224-2250C; IR (KBr): 3819, 3739, 3313, 3116, 3062, 1652, 1593, 1526, 1500, 1426,
1
1226, 843, 756cm-1. H NMR (300 MHz, CDCl3, δ in ppm): 7.17-8.02 (m, 18H, Ar-H); 7.37 (d, 1H,
J=15.0 Hz, -CH=CH-); 7.77 (d, 1H, J=15.0 Hz, -CH=CH-); 8.13 (s, 1H, pyrazole-H); 8.39 (s, 1H, N-H,
D2O exchangeable). MS: m/e 488 (M+1).
Biological studies
In vivo analgesic activity
The seven asymmetrical pyrazole curcumin analogues shown in Scheme-1 were tested for in
vivo analgesic activity by using the acetic acid induced writhing method. The assays were carried out
as per the reported methods and the results are summarized in Table 1. The analgesic activity result
revealed that all the tested compounds (4a-g) showed potent analgesic activity in the range of 80-
96% as compared to the standard Ibuprofen (62%). The entire series of APCAs (4a-g) exhibited
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excellent analgesic activity at 5 mg/kg p.o. When their activities were compared with Ibuprofen, it was
determined that all the compounds showed analgesic activity more than standard Ibuprofen. The
compound carrying methoxy- and chloro- substituent at p-position of phenyl ring (4e and 4b)
appeared as the most active compound in this series. However, all other compounds were also the
most notable ones because they showed a remarkable analgesic activity as compared to the
reference drug Ibuprofen. All the data were analyzed by one-way ANOVA test followed by Dunnet’s
test in the acetic acid induced writhing model.
In vitro anti-inflammatory activity
All the synthesized compounds were evaluated for anti-inflammatory activity by protein
denaturation method and results are presented in Table 1. The results revealed that the synthesized
compounds 4b, 4g, 4d and 4f showed good inhibition (85.19 – 79.31%) as compared with standard
diclofenac sodium (90.21%) whereas rest of the compounds showed moderate inhibition.
In vitro antioxidant activity
Reactive oxygen species and nitrogen species contribute to the pathophysiology of anti-
inflammatory (45) conditions. Taking into the account of multifactorial character of oxidative stress
which is involved in many pathological states, we have evaluated antioxidant activity of synthesized
asymmetrical pyrazole curcumin analogues (APCAs) against reactive oxygen species such as
hydrogen peroxide, DPPH, ferrous reducing power and nitric oxide radical scavenging activity and
results are presented in Table 1. All the synthesized compounds showed good to moderate
scavenging activity against hydrogen peroxide. The antioxidant activity result revealed that the
compound 4e (88.45%) was found to possess excellent inhibition of H2O2 scavenging activity followed
by compound 4b (71.12%) as compared to the BHT (88.42%). The remaining compounds 4a, 4d, 4f
and 4g showed (46-61%) moderate inhibition of H2O2 scavenging activity except compound 4c (22%).
In case of DPPH free radical scavenging activity, compounds 4f, 4b, 4d and 4g exhibited
good activity (42.32 – 36.80%) as compared to standard ascorbic acid (42.98) whereas rest of the
compounds showed moderate activity. Compounds 4d, 4e, 4g, 4b and 4c showed excellent ferrous
reducing power activity (55.55 – 44.44%) as compared to standard ascorbic acid (42.44%), whereas
the remaining compounds 4a and 4f showed moderate activity (33.33%).
The nitric oxide radical scavenging activity result revealed that all the synthesized curcumin
analogues (77.94 – 32.35%) were found showning excellent activity than standard ascorbic acid
(32.32%). Among the series, the compounds 4b, 4a, 4c and 4f showed more potent nitric oxide
radical scavenging activity. The wide variation in the free radical scavenging potential for the tested
compounds may be due to the variation in the proton–electron transfer by the derivatives due to
difference in their structures
As the reactive oxygen species are involved in pain and inflammation, the analgesic agents
should have both antioxidant and analgesic activity. The NSAIDs like Ibuprofen and Aspirin have both
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analgesic and antioxidant activities (46). In case of our synthesized compounds, compounds 4b, 4d,
4e, 4f and 4g showed potent analgesic as well as good antioxidant and anti-inflammatory activity,
suggesting that the analgesic activity of the compound might be partly connected to its antioxidant
and anti-inflammatory activity.
Conclusion
In conclusion, a series of new asymmetrical pyrazole curcumin analogues (APCAs) (4a-g)
have been synthesized with excellent yields without formation of any detectable side products by
1
using PEG-400 as an alternative reaction medium and were characterized by IR, H NMR and mass
spectrometry. All the newly synthesized compounds (4a-g) were evaluated for their analgesic,
antioxidant and anti-inflammatory potential. The result of analgesic activity revealed that all the
compounds were found to be shown better activity than standard Ibuprofen, suggesting that all the
synthesized APCAs were found to be superior over the standard Ibuprofen. In case of antioxidant
activity, compounds 4e was found to be potent whereas compound 4b was found to be comparable
H2O2 scavenging activity to standard BHT. The compounds 4f, 4b, 4d and 4g were found to be good
DPPH free radical scavenging activity as compared to standard ascorbic acid. In ferrous reducing
power activity, compounds 4d, 4e, 4g, 4b and 4c were found to be superior than standard ascorbic
acid, whereas all the synthesized compounds were found potent nitric oxide radical scavenging
activity than standard ascorbic acid. The result of anti-inflammatory activity revealed that the
compounds 4b, 4g, 4d and 4f showed pronounced anti-inflammatory activity which was comparable
to diclofenac sodium. In general, compounds 4b, 4d, 4e, 4f and 4g were found to be the most active
analgesic agents in addition to having potential antioxidant as well as anti-inflammatory activity. Thus,
these compounds constitute an interesting template for the evaluation of new analgesic agents and
may be helpful for the design of new therapeutic tools.
Acknowledgments
The author SYJ is thankful to the University Grants Commission (WRO), Pune for the award Teacher
Fellowship under XI plan and also thankful to The Principal, D.B.F Dayanand College of Arts and
Science, Solapur for their constant encouragement and guidance.
Conflict of Interest
Authors have no conflict of interest.
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R
F
O
O
i
H
N
O
NH2
+
H
N
N
O
3a-g
1
2
ii
R
3a, 4a, R=4-F
F
O
3b, 4b, R=4-Br
3c, 4c, R=4-Cl
3d, 4d, R=4-CH3
3e, 4e, R=4-OCH3
3f, 4f, R=3-NO2
3g, 4g, R=4-H
H
N
N
N
O
4a-g
Scheme1. Reagents and conditions: (i) 4-Fluorobenzoyl chloride, NaOH, 70-80oC,30 min.
(ii) NaOH, PEG-400, stirr, 40-50oC, 1 h.
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Entry
Analgesic activity
Anti-
inflammator
y activity
(in vitro)
%
Antioxidant activity
(in vivo)
(in vitro)
No.of
%
inhibitio
n
H2O2
(%)
DPPH
Ferrous Nitric oxide
reducin radical
g power scavenging
Writhings
SEM
inhibition
(%)
(%)
activity
(%)
--
Control
26.00 0.68 --
--
--
--
--
--
--
--
Ibuprofen
10.00 0.57* 62
*
--
--
4a
4b
4c
4d
4e
4f
5.16 0.70**
1.05 0.22**
2.16 0.30**
2.16 0.30**
1.00 0.25**
2.00 0.44**
3.83 0.40**
80
96
92
92
96
92
85
--
65.51
85.19
60.89
79.31
55.17
79.31
82.75
90.21
51.03
71.12
22.75
46.75
88.45
61.79
53.10
--
34.84
40.85
34.84
39.29
30.30
42.32
36.80
--
33.33
44.44
44.44
55.55
55.55
33.33
55.55
--
69.11
77.94
67.74
44.08
32.35
62.5
4g
38.29
--
Diclofenac --
sodium
BHT
--
--
--
--
--
88.42
--
--
--
--
Ascorbic
acid
--
42.98
42.44
32.32
Table 1. Analgesic, anti-inflammatory and antioxidant activity of compounds 4a-g.
SEM – standard error mean; N = 6; *P< 0.05, **P<0.01, ***P<0.001;
BHT –butylatedhydroxytoluene; --, Not determined;
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