Conventional and microwave assisted synthesis of pyrazolone Mannich bases possessing anti-inflammatory, analgesic, ulcerogenic effect and antimicrobial properties

Article abstract of DOI:10.1016/j.bmcl.2014.04.067

In the present study, an efficient synthesis of some Mannich base of 5-methyl-2-[(2-oxo-2H-chromen-3-yl)carbonyl]-2,4-dihydro-3H-pyrazol-3-one (4a-j) have been described by using conventional and non-conventional (microwave) techniques. Microwave assisted

Full text of DOI:10.1016/j.bmcl.2014.04.067

Accepted Manuscript
Conventional and Microwave Assisted Synthesis of Pyrazolone Mannich Bases
Possessing Anti-Inflammatory, Analgesic, Ulcerogenic Effect and Antimicro-
bial Properties
Kullampalayam Krishnasamy Sivakumar, Aiyalu Rajasekaran, Palaniappan
Senthilkumar, Prasad P Wattamwar
PII:
S0960-894X(14)00424-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.04.067
BMCL 21560
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 December 2013
26 March 2014
18 April 2014
Please cite this article as: Sivakumar, K.K., Rajasekaran, A., Senthilkumar, P., Wattamwar, P.P., Conventional and
Microwave Assisted Synthesis of Pyrazolone Mannich Bases Possessing Anti-Inflammatory, Analgesic,
Ulcerogenic Effect and Antimicrobial Properties, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://
dx.doi.org/10.1016/j.bmcl.2014.04.067
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1
CONVENTIONAL AND MICROWAVE ASSISTED SYNTHESIS OF PYRAZOLONE
MANNICH
BASES
POSSESSING
ANTI-INFLAMMATORY,
ANALGESIC,
ULCEROGENIC EFFECT AND ANTIMICROBIAL PROPERTIES
Kullampalayam Krishnasamy Sivakumar*, Aiyalu Rajasekaran, Palaniappan
Senthilkumar, Prasad P Wattamwar
Department of Pharmaceutical Chemistry, KMCH College of Pharmacy, Coimbatore-641 048,
Tamilnadu, India.
*Address for correspondence
K.K.Sivakumar,
Asst Professor,
Department of Pharmaceutical Chemistry,
KMCH College of Pharmacy, Kalapatti road,
Kovai estate, Coimbatore-641 048,
Tamilnadu, India.
Tel Phone: +91 9626164116; Fax: +91 4222628645
Email: sivaveni2004@gmail.com

2
Abstract
In the present study, an efficient synthesis of some mannich base of 5-methyl-2-[(2-oxo-
2H-chromen-3-yl) carbonyl]-2,4-dihydro-3H-pyrazol-3-one (4a-j) have been described by using
conventional and non-conventional (Microwave) techniques. Microwave assisted reactions
showed that require shorter reaction time and good yield. The newly synthesized compounds
were screened for their anti-inflammatory, analgesic activity, antioxidant, and antibacterial
effects were compared with standard drug. Among the compounds studied, compound (4f)
showing nearly equipotent anti-inflammatory and analgesic activity than the standard drug
(Indomethacin), along with minimum ulcerogenic index. Compounds (4b and 4i) showing 1.06
times more active than ciprofloxacin against tested Gram-negative bacteria.
Keywords: Pyrazolone, Coumarins, anti-inflammatory, analgesic, antioxidant, and
antibacterial.

3
Inflammation is a protective mechanism employed by tissues against endogenous and
exogenous antigens. Chronic inflammation causes cancers1 and exerts its cellular side effects
mainly through excessive production of free radicals and depletion of antioxidants. Free radical’s
role in acute or chronic inflammation has been well established by different studies2-3. A free
radical is defined as any chemical species that contains unpaired electrons. This unpaired
electron usually produces a highly reactive free radical. The most abundant radical in biological
systems is molecular oxygen (O2), particularly reactive oxygen species (ROS) and reactive
nitrogen species (RNS) which play a vital role in body: deleterious and beneficial effects4.
Usually the beneficial effects of ROS involve defense against microbial pathogens. This role
occurs by low concentration of these molecules. However, overproduction of ROS or RNS can
damage and inhibit the normal functions of lipids, proteins and DNA. This effect is due to
intracellular reduction of O2 into ROS or free radicals, which is toxic to cells and tissues5.
Coumarins have attracted intense interest in recent years because of their diverse
pharmacological properties particularly to reduce tissue edema and inflammation by reducing the
formation and scavenging of ROS involved in free radical-mediated injury. Among other
properties of coumarins, their antioxidant effects have been extensively examined6. Since it is
well-known that coumarins are powerful nontoxic, natural antioxidants able to quench active free
˙
radicals such as O2 , OH˙, or lipid peroxyl radicals LOO˙. On the other hand, the core pyrazolone
structure generally attracted widespread attention because of the diversity of biological activity
as antitumor7, analgesic8, anti-inflammatory9, antipyretic10, antioxidant11, antiviral12,
antitubercular13 and antibacterial14 activities. In addition, antipyrine15 (a) (2, 3-dimethyl-1-
phenyl-3-pyrazolin-5-one) was the first pyrazolone derivative used in the management of pain
and inflammation. Further, pyrazole derivatives such as dipyrone, aminopyrine,

4
isopropylantipyrine, phenyl butazone, oxyphenbutazone, celecoxib and deracoxib are potent anti-
inflammatory and analgesic agents (Fig 1). However their use became restricted due to their GI
side effects. In view of these observations and in continuation of our research programme on the
synthesis of pyrazolone ring containing heterocyclic moiety16-17. We report herein the synthesis
of some Mannich base pyrazolone derivatives by ecofriendly (Microwave irradiation) and
traditional method, which have been found to possess an interesting profile of anti-inflammatory
and analgesic activity, with significant reduction in their ulcerogenic potential and their
antimicrobial potency.
The synthetic pathway for the synthesis of the targeted compounds is illustrated in
Scheme 1. The key intermediate compound 5-methyl-2-[(2-oxo-2H-chromen-3-yl) carbonyl]-
2,4-dihydro-3H-pyrazol-3-one (3) and titled compounds (4a-j) was synthesized by Conventional
and Microwave method according to previously reported procedures with little modification18-22
.
Ethyl 2-oxo-2H-chromene-3-carboxylate (1) was prepared by cyclization of salicylaldehyde with
diethylmalonate in presence of catalytic amount of piperidine. Reaction of this ester compound 1
with hydrazine hydrate in ethanol formed 2-oxo-2H-chromene-3-carbohydrazide (2). The key
intermediate (3) was prepared by cyclization of compound (2) with ethyl acetoacetate in
presence of glacial acetic acid. The key intermediate pyrazolone (3) considered as a cyclic amide
and hydrogen atom attached to C4 atom should be appreciably labile to participate in the
Mannich condensation. Therefore, the condensation of pyrazolone (3) with formaldehyde (60%)
and various aromatic primary amines resulted in the formation the corresponding Mannich base
derivatives. The titled compounds were achieved by both microwave (ecofriendly) and
conventional (Traditional) methods18-22. The physicochemical data are described in Table 1.
Microwave assisted techniques are found to be more effective in perspective of environment,

5
reaction time, high yields, ease of work-up and isolation of products. More over microwave
irradiation offers several advantages:23 solvents are often expensive, toxic, difficult to remove in
the case of aprotic dipolar solvents with high boiling point, and are environmentally polluting
agents. Time and yield data of newly synthesized compounds by microwave and conventional
methods were given in Table 2. Structure of the synthesized compounds (4a-j) was established
1
on the basis of physicochemical, elemental analysis and spectral data (IR, HNMR and Mass).
The IR spectrum of compound (1) shows an absorption band at 1724 cm-1, corresponding to the
vibration of the lactone of coumarin, a band at 1773 cm-1, characteristic of the carboxylic ester
moiety (COOC2H5), while the compound (2) spectra showed the disappearance of the
characteristic bands of the carboxylic acid ester and the appearance of strong bands in the 3225
cm-1 region, attributed to NH group stretching. Structures of key intermediates (3) showed that
the disappearance of the characteristic bands of NH group stretching and further cyclization of
(3) was confirmed by the IR spectra which showed a single C=O band of pyrazolone (1675 cm-1)
assigned to the amide function. Mannich bases of pyrazolones (4a-j) confirmed the presence of
–NH, –N=C and –NC=O fused ring system present in the synthesized compounds by the
presence of IR stretching bands at 3330, 1685, 1560 cm-1, respectively. Proton assignments in
1H-NMR spectra for compound (1) showed signals at δ 7.02-8.20 (m, 5H, Ar-H), 4.23 (q, 2H,
CH2), and 1.31 (t, 3H, CH3 for carboxylic ester), while compound (2) showed the disappearance
of the characteristic signals for the ethyl group, and appearance of signals at δ 11.60 (s, 1H, NH-
NH2). The key intermediate (3) showed the absence of the signals for the NHNH2 group, while
the pyrazolinone CH3 signal appeared at δ 2.45 - 2.69 ppm. Formation of Mannich bases (-CH2-
NH-) of pyrazolones (4a-j) confirmed doublet signals in the range 4.34-4.56 (NH) and 3.34-3.62
(CH2) δ ppm. Compounds 4b, 4d and 4e showed the appearance of the characteristic signals for

6
aromatic OH at δ 5.23 ppm. Sulfonic acid (-SO3H) containing titled compound 4e showed the
appearance of the characteristic signals at δ 1.92 ppm. Compound 4g containing methoxy group
signal appeared at δ 3.12 ppm. Further, the formation of title compounds was confirmed by
recording their mass spectrums which were in full agreement with their molecular weights and
the results of elemental analysis (carbon, hydrogen and nitrogen) were ± 0.4 % of the theoretical
values. In conclusion, we have synthesized some Mannich base pyrazolone derivatives using
microwave assisted techniques and are more convenient, environmentally safe as they require
less volume of solvent, short reaction span and better yields as compared to conventional
techniques.
To assess the anti-inflammatory activity, the compounds were evaluated by carrageenan
induced paw oedema in albino rats. The paw edema was employed as a model for acute
inflammation, each test compound was dosed orally (at 0.03 mmol/kg), 1 h prior to induction of
inflammation by carrageenan injection24. The anti-inflammatory activity was expressed as %
inhibition of oedema and was calculated by the following equation24:
% Inhibition = 100× [1-Vt/Vc]
Where ‘Vt’ is the mean increase of paw thickness of rat after administration of the tested
compounds or the reference drug. ‘Vc’ is the mean increase in paw thickness in rat after
administration of carrageenan in the positive control group.
The compounds showed anti-inflammatory activity ranging from 21.64 % to 68.86 % and
45.28 % to 79.91 %, inhibition after 4 and 6 h respectively (Table 3), whereas standard drug
Indomethacin showed 67.8 % and 75.98 % inhibition. Substitution of withdrawing group at the
para/ortho position of phenyl ring imparted significant anti-inflammatory activity to the resulting
pyrazolone (4a-j). Compounds 4f and 4g contain sulfonic group at para- or meta- position

7
showed better anti-inflammatory activity compared to Indomethacin at 4th and 6th h. Compound
4f is 1.1 times more active than standard and compound 4g is 1.2 times more active than
standard. All the synthesized compounds demonstrated significant reduction in oedema after 6th h
compared to 4th h. This difference in activity between 4th h and 6th h can be attributed to the
bioavailability and pharmacokinetic parameters of the drug. The candidate molecules might get
ionized after 4 h, which enhances the drug absorption and distribution thereby increasing the
bioavailability (Cmax). Among the titled pyrazolones, possessing electron withdrawing
substituents at 4th position in the mannich base phenyl ring (4a, 4c, 4f and 4g), the highest anti-
inflammatory activity was obtained with substituent having lowest lipophilicity, lowest electron
withdrawing power and highest polarizability. The presence of p-chloro substituent (compound
4j) favoured anti-inflammatory activity at 6th h has highest lipophilicity. On the other hand,
introduction of disubstituent (4d) or bulky groups (4e) resulted in drastic decrease anti-
inflammatory activity. The remaining pyrazolone derivatives showed weak anti-inflammatory
activity. Presence of hetero atom (4h) in the phenyl ring decreased anti-inflammatory activity.
All newly synthesized compounds were evaluated for their analgesic activity by applying
the acetic acid-induced writhing test in mice using indomethacin as a standard drug (Table 4).
Percentage protection was calculated using the following formula25,
% Protection= [(a-b)/a] ×100.
Where ‘a’ is average number of writhing in control group and ‘b’ is average number of writhing
in treated group. Potency of the tested compounds was calculated according to the following
equation25:
Potency % = [% inhibition of the tested compound / % inhibition of Indomethacin] ×100.

8
The analgesic activity data (Table 4) showed that compounds having 4-chloro group (4j)
in the phenyl ring at 4th position in the mannich base pyrazoline nucleus possess highest
percentage of protection (103.78%), greater than the standard drug indomethacin. It was
observed that compound (4f) showing nearly equipotent analgesic, anti-inflammatory activity
also exhibited better antioxidant activity 120% with improved GI safety profile ulcerogenic
effect compared to other tested compounds. Introduction of a 3-SO3H group (4g) in the phenyl
ring at 4th position in the mannich base pyrazoline nucleus, led to a 1.92 fold decrease in
analgesic activity compared to compound 4f. Compounds 4a, 4c and 4e bearing electron
withdrawing group in the phenyl ring at 4th position in the mannich base pyrazoline nucleus
showed significant analgesic activity compared to compounds bearing electron donating group.
Gastric-ulcerogenic effect was evaluated by the following method23, five hours after the
oral treatment of rats with the tested compounds and standard drugs, they were killed under deep
ether anesthesia and their stomachs were removed. The stomach of each rat was opened through
great curvature and examined for lesions or bleedings (Table 3). It was observed that
pyrazolones 4a, 4c, 4d, 4f, and 4g had gastric safety profile better than other synthesized
compounds. Other hand, compounds with electron donating group at 4th position in the mannich
base phenyl ring (4b, 4h, 4i and 4j) revealed certain ulcerogenic effect.
The effects of various substituents on the phenyl ring of pyrazolones (4a–j) in producing
antioxidant activity in the descending order were found to be: 4f, > 4g & 4e, > 4d, > 4a, > 4b, >
4c, > 4h, > 4i and > 4j. In general, electron rich atoms present in phenyl ring showed significant
antioxidant activity. Antioxidant activity results were well correlated with anti-inflammatory
activity of the synthesized compounds. Compounds 4a, 4c, 4f, 4g and 4j bearing electron
withdrawing group in the phenyl ring at 4th position in the mannich base pyrazoline nucleus with

9
highest molecular weight, polarizability and lowest log P value showing significant antioxidant
activity also GI safety profile compared to other synthesized compounds.
The compounds were also screened for there in vitro antimicrobial activity against two
Gram-positive (Micrococcus luteus, Staphylococcus aureus) and Gram-negative (Escherichia
coli, Klebsiella pneumoniae) in triplicates using Agar-diffusion method26. Ciprofloxacin was
used as reference drug. The results were recorded for each tested compound as the average
diameter of inhibition zones (IZ) of bacterial or fungal growth around the disks in mm. The
minimum inhibitory concentration (MIC) determined for compounds showed significant growth
inhibition zones (> 8 mm) using twofold serial dilution method26. The MIC (µM/mLx10-3) and
inhibition zone diameters values are recorded in Table 5. In general, most of the tested
compounds revealed better activity against the Gram-negative rather than the Gram-positive
bacteria. Among the pyrazolones at 4th position having activating substituents in the mannich
base phenyl ring compounds (4b and 4i) showed 1.06 times more active than ciprofloxacin
against tested Gram-negative bacteria. All other synthesized compounds showed reasonable anti-
bacterial activity against tested Gram-negative strains with the percentage zone of inhibition
ranging 71 - 88. All the compounds demonstrate weak to moderate MIC activity compared to
Ciprofloxacin. In view of these observations, we conclude that this series (4a-j) could be
developed as a novel class of NSAIDs. However, further detailed pharmacological screening is
required to identify the potent molecule without severe side effects.
ACKNOWLEDGEMENT
The authors are thankful to Dr. Thavamani D. Palaniswami, Managing Trustee, Kovai
Medical Research and Education Trust, Coimbatore. The authors are grateful to Indian Institute

10
of Science, Bangalore and Indian Institute of Technology, Madras for providing NMR and Mass
spectral data.
Reference
1. Allavena, P.; Sica, A.; Solinas, G.; Porta, C.; Mantovanii, A. Crit Rev Oncol Hematol.2008,
66(1), 1.
2. Rekka, E.; Chrysselis, M.; Siskou, I.; Kourounakis, A. Chem. Pharm. Bull.2002, 50(7), 904.
3. Cuzzocrea, S.; Thiemermann, C.; Salvemini, D. Curr. Med. Chem.2004, 11(9), 1147.
4. Valko, M.; Rhodes, C. J.; Moncol, J.; Izakovic, M.; Mazur, M. Chem. Biol. Interact.2006,
160(1), 1.
5. Wickens, A. P. Respir Physiol.2001, 128(3), 379.
6. Shashwat, M.; Gaurav. S.; Ajit, K.; Barbara, W. V.; Ashok, L. C.; Hanumantharao, G. R.;
Luciano, S.; Balaram, G.; Marc, E. B.; Ashok, K. P.; Shyam, B.; Virinder, S. P. ARKIVOC.
2008 (vi), 119
7. Elisabeth, C.; Bettina, A.; Roy, M. G.; Francisco, C.; Thomas, E.; Ste´phane, L.; Bruno, P.;
Michelle, P. Eur. J. Med. Chem. 2006, 41, 1470.
8. Fatma, A. F. R.; Nagwa, M. A. G.; Hanan, H. G.; Mona, F. S. Chem. Pharm. Bull.2013,
61(8), 834.
9. Roland W. B.; Han Xu.; Xiaoming, Z.; Kristine, M.; Jennifer, G.; Mike, F.; Matthew, A.;
Matthew, H. P.; Min, W.; Michele, M. E.; Kelly, R.; Vellarkad, N. V.; Philip, T.; Randall, H.
Bioorg. Med. Chem. Lett.2006, 16, 3713.
10. Soad, A.M. E.H.; El-Sayed, A.M. B.; Ibrahim, M. E. A. Eur. J. Med. Chem.2006, 41,155.
11. Hidehiko, N.; Ryo, O.; Ayako, K.; Takayoshi, S.; Naoki, M. Bioorg. Med. Chem. Lett.2006,
16, 5939.

11
12. Ramajayam, R.; Kian-Pin, T.; Hun-Ge, L.; Po-Huang, L. Bioorg. Med. Chem.2010, 18,
7849.
13. Ahsan, M. J.; Govindasamy, J.; Bhushan Jain, C.; Rajeswar Dutt, K.; Khalilullah, H.;
Nomani, M. S. Bioorg. Med. Chem.Lett.2012, 22, 969.
14. Moreau, F.; Desroy, N.; Genevard, J. M.; Vongsouthi, V.; Gerusz, V.; Le Fralliec, G.;
Oliveira, C.; Floquet, S.; Denis, A.; Escaich, S.; Wolf, K.; Busemann, M.; Aschenbrenner, A.
Bioorg. Med. Chem. Lett.2008,18, 4022.
15. Sherif A. F. Rostom, Ibrahim M. El-Ashmawy, Heba A. Abd El Razik, Mona H. Badr,
Hayam M. A. Ashour. Bioorg. Med. Chem. 2009, 17, 882.
16. Sivakumar, K. K.; Rajasekaran, A. J. Pharm. Bioall. Sci.2013, 5(2), 126.
17. Sivakumar, K.K.; Rajasekaran, A; Ramalingeswararao, K.; Narasimhan, B. Indian. J.
Pharm. sci.2013, 75(4), 463.
18. Dariusz, B. Coumarins: Fast Synthesis by Knoevenagel Condensation under Microwave
Irradiations J. Chem. Research (S).1998, 468.
19. Suresh, K.; Veeresh, M.; Prashant, A.; Mahesh, P.; Pradeep, K.; Ronad.; Shivalingarao, M.;
Thippeswamy, A.H.M.; Satyanarayana, D. Eur. J. Med. Chem.2009, 44, 1682.
20. Atul, M.; Alpeshkumar, M.; Jitender, V.; Vijay, V.; Arun, M.; Kuldip, U.; Hrishikesh, A.;
Evans, C.; Anamik, S. Eur. J. Med. Chem.2008, 43, 2395.
21. Rishikesh, V. A.; Cendilkumar, A.; Divakar, G.; Ganesh, S. A.; Rajesh, J. O. Saudi
Pharm. J.2011, 19, 233.
22. Sriram, D.; Yogeeswari, P.; Pobba Reddy, S. Bioorg. Med. Chem. Lett.2006, 16, 2113.
23. Brittany L. Hayes. Recent advances in microwave-assisted synthesis. Aldrich mica ACTA.
2004, 37(2), 66.

12
24.Abraham Gómez-Rivera, Hidemí Aguilar-Mariscal, Nancy Romero-Ceronio, Luis F. Roa-de
la Fuente, Carlos E. Lobato-García. Synthesis and anti-inflammatory activity of three nitro
chalcones. Bioorg. Med. Chem. Lett.2013, 23, 5519.
25. Mona, M. H. Eur. J. Med. Chem.2012, 55, 12.
26. Hala, M. A.; Nashwa, M. S.; Heba, A. E. Eur. J. Med. Chem.2011, 46, 4566.

Table(s)
Table 1. Physicochemical data of synthesized compounds (4a-j)
Melting
Point
(°C)
Cpd
code
Molecular
Formula
Molecular
Weight
Rf
Value
Polariz
ability
Ar
C log P
4a
4b
4c
C22H17N3O6
C21H17N3O5
C21H16N4O6
419.39
391.38
420.37
276
350
205
0.658
0.614
0.865
2.11
2.15
2.39
41.17
39.41
40.72
4d
4e
C22H17N3O7
435.39
521.09
278
288
0.664
0.626
2.46
2.32
41.82
51.53
C25H19N3O8S
4f
C21H18N4O6S
C21H18N4O6S
454.46
454.46
286
212
0.749
0.782
1.64
1.64
43.48
43.48
4g
4h
4i
C20H16N4O4
C22H19N3O5
376.37
405.40
260
253
0.812
0.728
1.24
2.30
37.91
41.31
4j
C21H16 ClN3O4
409.52
276
0.892
3.06
40.67

Table 2. Time and yield data of newly synthesized compounds 4a-j using conventional and
microwave irradiation techniques
Conventional
Method
Microwave Irradiation
Method
Cpd
code
Power
(W)
Time
Percentage
Time
(min)
Percentage
Yield
(h)
Yield
4a
4b
4c
4d
4e
4f
5.30
58
5
5
5
5
5
5
5
5
5
5
5
300
300
300
300
300
300
300
300
300
300
300
88
81
85
87
79
86
87
82
86
78
81
5.00
4.50
6.00
6.15
5.30
5.30
5.00
4.55
5.15
5.20
61
66
65
55
68
65
69
69
61
67
4g
4h
4i
4j
4a

Table 3. In-vivo acute anti-inflammatory and in-vitro antioxidant activity of synthesized compounds (4a-j) in carrageenan-induced paw edema,
ulceration and DPPH Radical scavenging assay
Cpd
code
Anti-inflammatory activity
% Inhibition Oedema thickness
Ratio of
ulceration
Anti-oxidant
activity
IC50 (µg/µl)
Oedema thickness
(mm) ±SEM 4 h
% Inhibition
after 6 h
(mm) ±SEM 6h
after 4 h
4a
4b
4c
4d
1.260 ± 0.172***
2.231 ± 0.114*
1.320 ± 0.201***
2.396 ± 0.220***
63.63
35.61
61.90
30.85
1.081 ± 0.257***
1.947 ± 0.101***
1.107 ± 0.147***
1.939 ± 0.214***
72.61
50.68
71.96
50.88
1/8
3/8
1/8
2/8
155
220
150
190
4e
4f
4g
4h
4i
4j
2.715 ± 0.185*
1.079 ± 0.154*
1.116 ± 0.147***
2.389±0.219*
2.215 ± 0.131*
1.956±0.255**
1.144±0.127*
3.465±0.13**
-
21.64
68.86
67.79
31.05
36.07
43.54
67.84
-
2.160 ± 0.292***
0.873 ± 0.118*
0.793 ± 0.153*
1.961 ± 0.218***
1.821 ± 0.120*
1.125±0.132*
0.985 ± 0.127***
3.948±0.136**
-
45.28
77.88
79.91
50.32
55.06
71.50
75.98
-
5/8
0/8
2/8
3/8
3/8
3/8
5/8
0/8
-
125
120
125
240
250
150
-
Indomethacin
Control
Ascorbic acid
-
95
-
-
The results are expressed as mean ±SEM (n=5). Significance was calculated by using one-way ANOVA with Dunnet’s t- test. The difference in results was considered significant when p<0.05. *p<0.05 vs control at
0.03mmol/kg b.w; **p<0.01 vs control at 0.03mmol/kg b.w; ***p< 0.001 vs control at 0.03mmol/kg b.w.

Table 4. In-vivo analgesic activity of synthesized compounds by acetic acid induced
writhing method in mice.
Cpd
code
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
Control
Dose (0.03 mM/Kg) Writhing reflex % Inhibition % Potency
qty in mg/kg
12.58
11.74
12.61
13.06
15.63
13.63
13.63
11.29
12.16
12.29
-
(mean ± SEM)
30±36*
45±41*
31±25**
50±26**
35±47*
27±12**
51±52***
54±23*
61.04
41.56
59.74
35.06
54.55
64.93
33.77
29.87
32.47
71.43
-
88.68
60.38
86.79
50.94
79.25
94.33
49.06
43.40
47.17
103.78
-
52±34**
22±31**
77±23*
Indomethacin 10.34
24±41***
68.83
100
The result are expressed as mean ± SEM (n=5). Significance was calculated by using one-way ANOVA with Dunnet`s t-test. The
different in result was considered significant When p < 0.05, *p < 0.05 vs control at .03mmol/kg, **p < 0.01 vs control at .03mmol/kg;
***p < 0.001 vs control at 0.03mmol/kg.

Table 5. In-vitro antibacterial activitya against Gram-positive and Gram-negative bacteria for
compounds 4a-j
Cpd
code
Zone of inhibition in mm (ZI), Percentage of inhibition (%) & Minimum
Inhibition Concentration (MIC=µM/mLx10-3)
M.luteus
S.aureus
ZI (%) MIC ZI (%) MIC
50 59.61 12 71 119.22 14
50 63.88 18 106 63.88 18
88 118.96 12
E. coli
K. pneumoniae
ZI (%) MIC
ZI
(%)
MIC
4a
18
18
15
12
-
-
-
12
-
18
72 119.22 12
82
106
71
71
106
119.22
63.88
118.96
114.84
47.98
4b
4c
4d
4e
4f
4g
4h
72 127.75 12
60 118.96 12
48 114.84 12
-
-
-
50 118.96 15
50 114.84 12
63 47.98 15
63 55.01 12
71 57.42
88 23.99
71 27.51
12
18
15
NT
NT
NT
15
15
88
55.01
18
15
15
-
75 55.01 12
63 66.42 15
71 55.01
88 132.85 15
12
71
88
106
71
100
-
55.01
66.42
61.67
61.05
0.59
-
48 66.42
NT
72 61.05
4i
4j
CPN
DMSO
-
63 61.67 18 106 30.83
NT 12 71 30.52
17 100 04.72
18
12
17
-
-
25 100 0.59
24 100 9.43
-
-
-
-
-
-
-
-
-
a Mean value of three experiment; - Indicate no inhibition; NT - Not tested;

Figure(s)
Synthetic pathway for the synthesis of the targeted compounds (4a-j)

Figure 1: Pyrazole derivatives used as anti-inflammatory and analgesic agents

Structural requirements for antiinflammatory, analgesic, antioxidant and antibacterial
activity of synthesized compounds