Accepted Manuscript

Anti-inflammatory and antimicrobial activities of novel pyrazole analogues

R. Surendra Kumar, Ibrahim A. Arif, Anis Ahamed, Akbar Idhayadhulla

PII: S1319-562X(15)00167-9
DOI: http://dx.doi.org/10.1016/j.sjbs.2015.07.005
Reference: SJBS 519

To appear in: Saudi Journal of Biological Sciences

Received Date: 4 May 2015

Revised Date: 6 July 2015
Accepted Date: 22 July 2015

Please cite this article as: R. Surendra Kumar, I.A. Arif, A. Ahamed, A. Idhayadhulla, Anti-inflammatory and
antimicrobial activities of novel pyrazole analogues, Saudi Journal of Biological Sciences (2015), doi: http://
dx.doi.org/10.1016/j.sjbs.2015.07.005

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Anti-inflammatory and antimicrobial activities of novel pyrazole analogues
R. Surendra Kumara , Ibrahim A. Arif b, Anis Ahamedb, Akbar Idhayadhullac*
a
Department of Chemistry, Shivani Engineering College, Affiliated to Anna University , Tamil Nadu, India
b
Prince Sultan Research Chair for Environment and Wildlife, Department of Botany & Microbiology,
College of Sciences, King Saud University (KSU), Riyadh, Saudi Arabia.
c*
Department of Chemistry, School of Basic Science, VELS University, Chennai-600117, India.

Abstract: A new sequence of pyrazole derivatives (1-6) was synthesized from condensation technique under

utilizing ultrasound irradiation. Synthesized compounds were characterized from IR, ¹H NMR, ¹³C NMR,

Mass and elemental analysis. Synthesized compounds (1-6) were screened for antimicrobial activity. Among

the compound 3 (MIC : 0.25 µg/mL) was exceedingly antibacterial active against gram negative bacteria of

E.coli and compound 4 (MIC : 0.25 µg/mL) was highly active against gram positive bacteria of

S.epridermidis compared with standard Ciprofloxacin. The compound 2 (MIC : 1 µg/mL) was high

antifungal active against A.niger proportionate to Clotrimazole. Synthesized compounds (1-6) were monitor

for anti-inflammatory activity and the compound 2-((5-hydroxy-3-methyl-1H-pyrazol-4-yl)(4-

nitrophenyl)methyl)hydrazinecarboxamide (4) showed that better activity against anti-inflammatory when

match up to the standard drugs (Diclofenac sodium). Compounds (2, 3 and 4) are most important molecules

and need to develop new drug of antibacterial, antifungal and Anti-inflammatory agents.

Keywords: Ultra sound irradiation; Pyrazole derivatives; Mannich bases; Antimicrobial activity; Anti-
inflammatory activity; Structure-activity Relationships (SAR).

_____________

* Corresponding author at: Department of Chemistry,

School of Basic Science, VELS University, Chennai-600117, Tamil Nadu, India.
Tel.: +91 9994265115; E-mail addresses: [email protected],

1
1. Introduction

The pyrazole moiety is a versatile lead molecule in pharmaceutical development and has a wide range of

biological activities (Goda et al., 2003; El-Emary et al., 2006; Mansour et al., 2003), antibacterial

(Sangapure et al., 2001), antifungal (Gupta et al., 2005; Ashish et al., 2006) and pharmacological activities

such as anti-inflammatory (Makhsumov et al.,1986), antitubercular (Chetan et al., 2000), anticancer

(Nimavat et al., 2007), analgesic (Udupi et al., 1998), antipyretic (Fabiane et al., 2002), anticonvulsant

(Ashok et al., 2001) activities.

CF3
Cl
HO
H
N O
N N
N N
O
O NH2 N

OH
HO
OH
HO

SO2NH2

Pyrazofurin Lonazolac
Celecoxib

N
O
NH

O
N
O
N N N
Cl
NH
N N
Cl

O
Cl

Rimonabant Ramif enazone Phenylbutazone

Fig. 1. The structures of some drugs bearing the pyrazole moiety

Commercial available pyrazole moiety (Figure 1) such as Celecoxib is potent COX-2 inhibitor (Penning et

al., 1997). Some other examples of pyrazole derivatives as NSAID are ramifenazone (Fioravanti et al., 2010),

Lonazolac (NSAID) (Riedel et al.,1981) and Rimonabant (Isidro et al., 2009). Compound (phenylbutazone)

is a non stereoidal drug (Reed et al.,1985; Vennerstorm et al.,1987). Pyrazofurin is potential of antiviral

activity, HCV virus (Rostom et al.,2003; Riyadh et al., 2010; Popovici-Muller et al., 2009; Farghaly et al.,

2011). In current research, Anti-inflammatory drug has been used most prominent research areas. New Anti-

inflammatory drugs are previously used in clinical research, some of the drugs still not efficiently and

intolerable side effects.

2
Based on above study, we need to development of new drugs against Anti-inflammatory and antimicrobial

activities. Therefore, we were led to identifying new approaches of pyrazole derivatives as well as test the

antimicrobial and Anti-inflammatory activity.

2. Methods and Materials

2.1. Chemicals and reagents

All chemicals were acquire from Sigma-Aldrich Chemical Co (Sigma-Aldrich Corp., St. Louis, MO, USA.

The Infrared spectra (KBr) , Proton NMR , Carbon NMR, Mass spectra (EI) , and Elemental analysis (C,

H, N and S) were recorded using by Shimadzu 8201PC (4000-400 cm- 1), Bruker DRX-400 MHz, Jeol JMS

D-300 spectro meter operating , and Elementer analyzer model (Varian EL III).

2.1.1. Synthesis of 2-((5-hydroxy-3-methyl-1H-pyrazol-4-yl)(phenyl)methyl)hydrazinecarboxamide (1)

A mixture of 5-hydroxy-3-methyl-1H-pyrazoles (0.1mol), benzaldehyde (0.1mol) and semicarbazide

hydrochloride (0.1mol) was treated with ultrasound irradiation under ethanol medium. After completion of

reaction, the product was isolated and identified by TLC. The identified product was separated from column

chromatography and recrystallized by suitable solvent. The above experiential procedure was pursuing by

remaining compounds 2-6.

IR (cm-1): 3445 (C-OH), 670(-CH-), 1679 (NH CO), 1569 (NH2). 1 H NMR (DMSO-d6): δ 9.90 (s, 1H, C-

OH), 2.71 (d, J= 4.4 Hz, 1H, CH), 2.23(s, 3H,CH3), 4.45 (dd, J=5.3 Hz, 1H, -CH-), 7.33- 7.49 (m, 5H,

Phenyl ring), 2.36 (d, J=2.0Hz, 1H, NH), 6.81 (d, J=1.4Hz, 1H, NH), 6.25 (s, 2H, NH2). 13C NMR (CDCl3):

δ 167.2(C-OH), 162.6 (C-CH3), 42.2(C-CH-), 52.3(C- CH-NH), 18.7(C-CH3), 155.4(CONH2), 141.7, 112.0,

129.2, 133.8 (Phenyl ring). Mass (m /z): 261.27 (M+, 32%), 244.28, 216.38, 200.27 (100%), 185.26,

170.25, 94.15.

2.1.2. 2-[(4-chlorophenyl)(3-hydroxy-5-methyl-4H-pyrazol-4-yl)methyl]hydrazinecarboxamide(2)

3
 IR (cm-1): 3469 (C-OH), 691(-CH-),1665(NH CO),1554(NH2), 897(C-Cl). 1H NMR (DMSO-d6) : δ 9.96(s,

1H, C-OH), 2.76 (d, J= 4.3 Hz, 1H, CH), 2.29 (s, 3H, CH3), 4.30(dd, J=5.2Hz, 1H, -CH-), 7.21-7.39 (dd, 4H,

Phenyl ring), 2.31 (s, J=2.1Hz, 1H, NH), 6.73 (s, J=1.6Hz,1H,NH), 6.19 (s, 2H, NH2). 13C NMR (CDCl3) : δ

167.2 (C-OH),162.6 (C-CH3),42.2 (C-CH-), 52.3 (C-CH-NH),18.7 (C-CH3),155.4 (CONH2), 141.7, 112.0,

129.2, 133.8 (Phenylring). Mass (m/z): 295.72(M+ , 26%), 277.74, 243.30 (100%), 228.28, 200.27, 185.26,

170.25, 94.15.

2.1.3. 2-((3-hydroxy-5-methyl-4H-pyrazol-4-yl)(4-hydroxyphenyl)methyl)hydrazinecarboxamide (3)

IR(cm-1): 3457(C-OH), 682(-CH-), 1661(NHCO), 1508(NH2),1447(OH).1H NMR(DMSO-d6): δ 9.89 (s,

1H, C-OH), 2.70(d, J= 4.1 Hz, 1H,CH), 2.31 (s,3H,CH3), 4.36(dd, J= 5.6Hz,1H,-CH-), 7.23-7.31 (dd, 4H,

Phenylring), 2.33 (s, J=2.3Hz, 1H, NH), 6.70 (s, J=1.6Hz, 1H, NH), 6.21 (s, 2H, NH2). 13C NMR (CDCl3): δ

167.9 (COH), 163.2 (C2-CH3), 42.6 (C-CH-), 51.9 (C-CH-NH), 18.5 (C-CH3), 155.0 (CONH2), 146.7, 111.5,

128.2, 137.2 (phenylring). Mass (m/z): 277.27(M+, 22%), 259.30, 243.42(100%), 228.28, 200.27, 185.26,

170.25.

2.1.4. 2-[(3-hydroxy-5-methyl-4H-pyrazol-4-yl)(4-nitrophenyl)methyl]hydrazinecarboxamide (4)

IR(cm-1); 3450 (C-OH), 679(-CH-), 1660 (NHCO), 1514(NH2), 1536(C-NO2). 1H NMR (DMSO-d6):

δ9.91 (s, 1H, C-OH), 2.79(d, J=4.6Hz, 1H, CH), 2.32 (s, 3H, CH3), 4.41 (dd, J=5.2Hz, 1H, -CH-), 7.29-7.42

(dd, 4H, Phenyl ring), 2.39 (d, J=2.1Hz, 1H, NH), 6.81 (d, J=1.7Hz, 1H, NH), 6.26 (s, 2H, NH2). 13C NMR

(CDCl3): δ167.1(C-OH), 162.9(C-CH3),42.0 (C-CH-), 52.6 (C-CH-NH), 19.8 (C-CH3), 156.3 (CONH2),

142.9, 112.5, 128.2, 131.2 (phenyl ring). Mass (m /z): 306.27(M+,26%), 289.28,261.27, 245.27(100%),

200.27, 185.26, 170.25.

2.1.5. 2-[(3-hydroxy-5-methyl-4H-pyrazol-4-yl)(4- methoxy phenyl )methyl]hydrazine carboxamide(5)

IR (cm-1): 3460 (C-OH), 682(-CH-),1668 (NHCO),1508 (NH2).1H NMR (DMSO-d6): δ 9.92 (s, 1H, C-

OH), 2.76 (d, J= 4.1Hz, 1H, CH),2.36 (s, 3H, CH3),4.28 (dd, J= 5.5Hz, 1H,-CH-),7.19-7.25 (dd, 4H, Phenyl

4
ring), 2.39 (d, J=2.2Hz, 1H, NH), 6.84(d, J=1.5Hz,1H, NH), 6.15(s,2H, NH2).13C NMR (CDCl3): δ 167.3 (C-

OH),162.0 (C-CH3), 42.9 (C-CH-), 53.1 (C-CH-NH), 19.1 (C-CH3), 156.8 (CONH2),141.7,112.0,129.2,

133.8 (phenyl ring). Mass(1 m/z): 291.30(M+, 41%), 259.30, 243.30, 200.27(100%), 185.26, 170.25, 94.15.

2.1.6. 2-((4-(dimethylamino)phenyl)(3-hydroxy-5-methyl-4H-pyrazol-4-yl)methyl)hydrazinecarboxamide

(6)

IR (cm-1): 3451 (C-OH), 663(-CH-), 1679 (NHCO),1512 (NH2). 1H NMR (DMSO-d6): δ 9.95 (s, 1H, C-

OH), 2.76 (d, J= 4.3Hz, 1H, CH), 2.24 (s, 3H, CH3), 4.41(dd, J= 5.6Hz, 1H, -CH-),7.29-7.41 (dd, 4H,

Phenyl ring), 2.36 (d, J=2.3Hz, 1H, NH), 6.82(d, J=1.9Hz, 1H, NH), 6.17 (s, 2H, NH2). 13C NMR (CDCl3):

δ167.9 (C-OH), 162.5(C-CH3), 43.1(C- CH-), 54.6 (C-CH-NH), 18.9(C-CH3), 157.1 (CONH2), 142.8, 113.0,

127.2, 134.1 (phenylring). Mass (m/z): 304.34(M+,27%), 286.37, 276.30, 243.34(100%), 200.27, 185.26,

170.25, 94.15.

2.2. Pharmacological activity

2.2.1. Anti-inflammatory activity

Isolated compounds (1-6) were evaluated by anti-inflammatory activity, screening method followed from

literature method (Winter et al., 1962). Albino rats of both sexes weighing 150g were divided in to 4 groups,

each group consists of 5 animals. Inflammation was induced by intra planter injection of Histamine (0.1mL

of 1% Histamine for induction of pawedema). The rats are challenged by s.c injection of 0.1mL of 1%

solution of histamine in to the sub-plantar side of the left hind paw. 1h after the administration of the test

compounds (10mg / kg; p.o), one group was kept as control, received only 0.5% carboxy methyl cellulose

solution. The volume was measured before and after 3h of carrageen treatment by means of plethysmometer.

Percentage of anti-inflammatory activity calculated by = (Vc-Vt / Vc) × 100.

Vc=Control, Vt= Test sample

2.2.2. In vitro Antibacterial screening

5
The antibacterial screening for isolated compounds were determined by disc diffusion method (Bauer et al.,

2006) using Mueller–Hinton agar(Hi-Media) medium. The synthesized compounds were evaluated against

gram negative bacteria of Escherichia coli (MTCC-739), Pseudomonas aeruginosa (MTCC-2435),

Klebsiella pneumonia (recultured) and gram positive bacteria of Streptococcus epidermidis, Staphylococcus

aureus (MTCC- 96). Synthesized compounds were initially screened by maximum concentration at

100µg/mL in DMSO. The zone of inhibition was measured after 24h incubation at 37ºC. The MIC was

identified by twofold dilutions of the solution method (64, 32…., 0.5 µg/mL).

2.2.3. In vitro antifungal screening

The antifungal screening for isolated compounds were determined by using an disc diffusion method

(Verma et al., 1998) with Sabouraud’s dextrose agar (Hi-Media).The isolated compounds were estimate

for their in vitro antifungal activity against Aspergillus niger, Candia albicans, Microsporum audouinii

and Cryptococcus neoformans (recultured) . Synthesized compounds were initially screened by maximum

concentration at 100µg/mL in DMSO. The zone of inhibition (mm) was measured after incubation at 37°C.

The MIC was identified by twofold dilutions of the solution method (64, 32…., 0.5 µg/mL).

3. Results

3.1. Synthesis and characterization of pyrazole analogues

Title compounds (1-6) were synthesized from 5(3)-hydroxy-3(5)-methyl-1H-pyrazoles react with aldehyde

and semicarbazide via Ultrasound irradiation under aqueous medium and without catalysis condition, the

synthetic route of pyrazole derivatives represented in Scheme 1. The compounds (1-6) were manufacture by

Mannich base condensation method and the mechanism of the work outline in scheme 1. Physicochemical

data of the compounds (1-6) are given in table 1.

6
 HN N

R
HO CH3
HN N H2N NH
))))))
O CH3
+ + NH 2

NH
O
CHO
HN O
R

H2N

Scheme 1. Synthetic route of the isolated compounds (1-6)

Isolated compounds were characterized by Infra red, Proton NMR, Carbon NMR spectrum, Mass spectra,

and elemental analyses.

The compound 1 was confirmed by IR spectral analysis , which indicates the value of 3445cm-1

corresponding to OH group nearby in pyrazole ring and another absorption bands at 670 cm- 1 corresponding

to -CH- group presented in pyrazole ring, another analysis of ¹H NMR spectrum shows that the signals

observed at δ 13.03, 11.43, 5.45, 2.36, and 6.25 corresponding to NH in pyrazole ring, C-OH, -CH-, NH, CH,

and NH2 protons respectively and ¹³C NMR spectrum shows that peaks at δ 132.2, 51.2, and 12.7

corresponding to C-OH, C-CH-NH, and CH3 carbons respectively. Molecular mass of compound 1 was

confirmed by mass spectral analysis, which is indicated that the molecular ion peak at 261.27(M+ , 32%).

Table 1 Physicochemical data of the compounds (1-6)

Comp. Ar Yield % mpºC m.f m.w Elemental analysis

No. calculated(found)
C H N
1 -H 87 161 C12H15N5O2 261.27 55.16 5.79 26.80
(55.20) (5.71) (26.79)
2 -Cl 78 89 C12H14ClN5O2 295.72 44.74 4.77 23.68
(44.72) (4.76) (23.65)
3 -OH 81 121 C12H15N5O3 277.27 51.98 5.45 25.26
(51.97) (5.40) (25.23)
4 -NO2 72 134 C12H14N6O4 306.27 47.06 4.61 27.44
(47.10) (4.59) (27.43)
5 -OCH3 81 110 C13H17N5O3 291.30 53.60 5.88 24.04
(53.65) (5.86) (24.08)
6 -N(CH3)2 85 97 C14H20N6O2 304.34 55.25 6.62 27.61
(55.30) (6.60) (27.60)
3.2. Anti-inflammatory activity

7
Isolated products (1-6) were evaluated for anti-inflammatory activity match up with diclofenc sodium at oral

dose. Rat albino was used oral dose of the test compound at concentration 10mg/kg, the percentage of the

activity was measured at 3h. Compound (4) has highly active compared with standard. (Figure 2) shows that

activity difference of compounds (1-6), anti-inflammatory activity records are presented in (Table 2).

Figure 2: Anti-inflammatory activity of compounds (1-6)

Table 2: Anti-inflammatory activity of compounds (1-6)

Comp. No Increase in paw Percentage (%) of

volume Activity,
( 3hr – 0hr) Dose (10 mg/ kg)
Control 0.56 -
1 0.22 ± 0.05 66.0*
2 0.18 ± 0.06 67.8*
3 0.32 ± 0.06 42.8*
4 0.10 ± 0.02 82.1*
5 0.38 ± 0.09 32.1*
6 0.41 ± 0.02 26.7*
Standard 0.14 ± 0.01 75.0*

Mean ± SEM, n=6 in each group. Significance levels *P < 0.01 as compared with the respective
control. Diclofenac sodium was used as a standard

3.3. Antibacterial activity

Compound (3) has exceedingly active (MIC: 0.25µg/mL) against gram negative bacteria of E.coli compared

with Ciprofloxacin MIC: 0.5 µg/mL. The compound (4) (MIC: 0.25 µg/mL) is highly active and match up

with standard (MIC : 4µg/mL) against gram-negative bacteria of S.epidermidis. The bacterial zones of

8
inhibition values are presented in table 3 and Fig.3 indicates differentiation of antibacterial activity in

isolated compounds (1-6).

Table 3 Antibacterial activity of isolated products (1-6)

Compounds Gram-negative Gram-positive

E.coli P.aeruginosa K.pneumoniae S.aureus S.epridermidis
1 12 - - - 12
2 16 - 18 - 16
3 28 8 - 10 10
4 20 - 8 12 18
5 18 12 10 - 12
6 15 - - 14 14
Standard 26 17 19 22 15
Ciprofloxacin used as a standard, zone of inhibition measured at (mm).

Figure 3. Antibacterial activity of compound ( 1-6)

3.4. Antifungal activity

The compound (2) MIC : 1 µg/mL has greatly active against A. niger match up to with Clotrimazole MIC : 2

µg/mL. Compound (3) (MIC : 0.5 µg/mL) has equipotent activity against Microsporum audouinii match up

to with standard Clotrimazole (MIC : 0.5 µg/mL) where as compound 5 (MIC : 4 µg/mL) is moderate active

against A.niger. The fungal zones of inhibition values are presented in table 4. Fig. 4 indicates differentiation

of antifungal activity in isolated compounds of compounds (1-6). Minimal inhibitory concentration (MIC)

data are reports in table 5.

9
 Table 4. Antifungal activity of isolated products (1-6)

Compounds A. niger C. albicans C. neoforman s M. audouinii

1 10 - - 12
2 24 12 - 18
3 15 16 8 25
4 14 10 - 10
5 20 12 10 12
6 18 - - 10
Standard 22 24 25 26

Clotrimazole used as a standard, zone of inhibition measured at (mm).

Figure 4. Antifungal activity of compound (1-6)

Table 5. Minimum Inhibition Concentration of isolated products (2, 3and 5)

Comp.No. E.c P.a K.p S.a S.e A. n C. a C. n M. a
2 64 >100 1 >100 16 1 64 >100 16
3 0.25 64 >100 64 64 64 64 >100 0.5
4 16 >100 >100 64 2 64 >100 >100 >100
5 16 32 64 >100 16 4 64 64 32
Ciprofloxacin 0.5 1 2 0.5 4 - - - -
Clotrimazole - - - - - 2 1 0.5 0.5

4. Discussion

The anti-inflammatory and antimicrobial performances of the isolated compounds were confirmed by

structure – activity relationships (Figure 5).

10
The 4-substituted phenyl ring with semicarbazone acts as a lipophilic and hydrogen bonding domains.

Therefore, the above group containing pyrazole ring may be stated that essential pharmacophoric

requirement for Anti-inflammatory activity.

(i) Compound 3 (MIC : 0.25 µg/mL) pyrazole derivative exhibited high activity against gram negative E.coli

compared with standard Ciprofloxacin and also antifungal activity of compound 3 exhibited equipotent

activity (MIC: 0.5 µg/mL) against M. audouinii match up to with standard Clotrimazole (MIC : 0.5

µg/mL), reason of activity due to the presence of 4-OH-phenyl with semicarbazone and pyrazole moiety in

compound 3. (ii) Compound 2 (MIC: 1 µg/mL) has highly of active against gram negative bacteria of

K.pneumoniae and also highly active (MIC: 1 µg/mL) against A.niger compared with standard

Clotrimazole (MIC : 2 µg/mL) in antifungal screening , 4-Cl-phenyl with semicarbazone and pyrazole

moiety were represented to highly active against K.pneumoniae bacterial and A.niger fungal strain.

(iii) Compound 4 is highly active (MIC: 2µg/mL) against gram positive bacteria of S.epridermidis

compared with standard Ciprofloxacin (MIC : 4 µg/mL), reason of activity due to the presence of 4-NO2-

phenyl with semicarbazone and pyrazole moiety in compound 4. The compound 4 is very much active

(82%) against anti-inflammatory in relation to Diclofenac sodium 74.0% of activity.

NH N NH N NH N

HO CH3 CH3 HO CH3

HO

NH NH NH

NH O NH O NH O
HO Cl O2N
NH2 NH2 NH2

Compound 3 Compound 2 Compound 4

Figure 5 Structure activity relationships for compound (3, 2 and 4)

5. Conclusion

In conclusion, synthesized compounds (1-6) were tested with anti-inflammatory and antimicrobial screening.

Among the series, the compound (3) (MIC : 0.25 µg/mL) was found to be most active against gram negative

11
bacterial strain E.coli compared with standard Ciprofloxacin, antifungal activity of compound (2) (MIC:1

µg/mL) was greatly active against A.niger than standard Clotrimazole and the compound (4) showed that

better activity against anti-inflammatory when compared with Diclofenac sodium. Therefore these

derivatives could provide as a high momentous molecule for further development of antimicrobial, anti-

inflammatory agents.

Acknowledgment

We are very grateful to Saudi Biological Society and Prince Sultan Research Chair for Environment and

Wildlife, Department of Botany & Microbiology, College of Sciences, King Saud University (KSU), Riyadh,

Saudi Arabia for encouragement and support.

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