Synthesis, Anti-bacterial, Anti-fungal and Anti-inflammatory Activities of Some Novel 3, 5-Diaryl-1-Aryl Pyrazoline Derivatives.

 

M. D. Bhanushali1*, S. M. Hipparagi2

1Research Scholar, Bhagwant University, Ajmer and Department of Pharmaceutical Chemistry,

Maratha Mandal’s College of Pharmacy, Belgaum. 590 016.

2 Department of Pharmaceutical Chemistry, KLE University’s, College of Pharmacy, Rajajinagar,

Bangalore-40.

*Corresponding Author E-mail: pc_mahe@yahoo.co.in

 

ABSTRACT:

4-substituted acetanilide on treatment with substituted benzaldehyde affords the corresponding chalcones (C13-C24). Further treatment of chalcones with phenylhydrazine undergone cyclocondensation and gave a new series of novel 3, 5-Diaryl-1-aryl-2- pyrazoline (P13-P24). All synthesized compounds were screened for their antibacterial and antifungal activity at 75µg concentration and anti-inflammatory activity at 200mg/ kg body weight. Some of the compounds have shown excellent activity.

 

KEYWORDS: Pyrazoline, Antibacterial, Antifungal, Anti-tubercular activity.

 

INTRODUCTION:

The chemistry of heterocyclic compounds is one of the most complex branch of organic chemistry. It is equally interesting for its theoretical implication, for the diversity, for its synthetic procedure and for the physiological, biological and industrial significance1. In particular nitrogen containing heterocyclic compounds has been studied extensively. Among these pyrazoline have proved to be the most useful framework for biological activities and have been reported for their antibacterial2, antifungal3, anti-tubercular4, analgesic5, anti-inflammatory6, anti-cancer7, anti-convuslant8, anti-depressant9 and anti-histaminic activity10.

 

In normal practice chemotherapeutics and anti-inflammatory drugs are prescribed in microbial infection, as it is always associated with pain and inflammation. The compound possessing all above activities is not common. Prompted by all these observation and in the continuation of our earlier work on the synthesis of some chalcones and pyrazoline11 we report here in the synthesis of some new series of pyrazoline derivatives, which have been screened for their antibacterial, antifungal and anti-inflammatory activity.

 

MATERIALS AND METHODS:

Monitoring of reaction and purity of synthesized compounds has been checked by TLC .Melting points were determined in open capillary tube and are uncorrected. The IR spectra were recorded on a Jasco FTIR-460 spectrometer using KBr disc method. 1HNMR spectra were recorded on Bruker Avance II 400 NMR spectrometer using DMSO/ CDCl3 as solvent and TMS as an internal standard. The entire chemical shift is expressed in  ppm.

 

General procedure for the synthesis of Chalcones (C13-24)12, 13:

An equimolar mixture of substituted acetanilide and substituted benzaldehyde in 25 ml of ethanol were stirred together in presence of 40 ml of 20% NaOH solution for 5-6 hours. The mixture was kept in refrigerator overnight. The crystals obtained were washed with water until the solution become neutral to litmus paper and recrystallized from suitable solvents. Other compounds (C13-C24) were synthesized similarly.

 

General procedure for the synthesis of Pyrazoline (P13-24)14:

An equimolar mixture of chalcone and phenylhydrazine in 30 ml of ethanol, in presence of few drops of glacial acetic acid, were refluxed for 4-6 hours. The reaction mixture was poured into cold water and the product which separated was filtered and recrystallized from suitable solvent. Other compounds (P13-P24) were synthesized similarly.       

 

Scheme 

 

 

Reagents and Conditions:

a) Ethanol, 20% NaOH, stirred 5-6 hours b) Ethanol, Phenyl hydrazine, glacial acetic acid, reflux 4-6 hours.

 

Comp. code

R

R’

Comp. code

R

R’

P13

H

Cl

P19

Cl

Cl

P14

H

OH

P20

Cl

OH

P15

H

NO2

P21

Cl

NO2

P16

F

Cl

P22

Br

Cl

P17

F

OH

P23

Br

OH

P18

F

NO2

P24

Br

NO2

 

All the synthesized compounds (P13-P24) were characterized by IR, 1H NMR and Mass spectral studies. The physical and spectral data of synthesized compounds are as follows.

 

5-(4-chloro-phenyl)-1-phenyl-4, 5-dihydro-1H-pyrazol-3-yl]-phenyl-amine (P13): Yield-60.51%, m.p-111-1120C, Rf: 0.73 (hexane: ethylacetate/8:2), IR (KBr) Vmax cm-1: 3344.84 (NH Str), 3200.20 (-ArC-H Str), 2925.66 (C-H Str), 1600.21(C=N Str of 2-Pyrazoline), 1504.32(-Ar CH=CH- Str), 753.96 (C-Cl Str).

 

4-(2-phenyl-5-phenylamino-3,4-dihydro)-2H-pyrazol-3-yl]-phenol (P14): Yield-57.75%, m.p-113-1150C, Rf: 0.75 (hexane: ethylacetate/8:2), IR (KBr) Vmax cm-1: 3459.31 (OH Str), 3348.61 (NH Str), 31.4.45 (-ArC-H Str), 2928.38 (C-H Str), 1608.65(C=N Str of 2-Pyrazoline), 1558.35(-Ar CH=CH- Str).

 

[5-(4-nitro-phenyl)-1-phenyl-4, 5-dihydro-1H-pyrazol-3-yl]-phenyl-amine (P15): Yield-55.86%, m.p-107-1080C, Rf: 0.70 (hexane: ethylacetate/8:2), IR (KBr) Vmax cm-1: 3342.89 (NH Str), 3066.33 (-ArC-H Str), 2923.67 (C-H Str), 1596.61(C=N Str of 2-Pyrazoline), 1502.94(-ArCH=CH- Str), 1330.55 (C-NO2 Str). 1H NMR (DMSO-d6, δ ppm): 7.93-6.72 (m, 14H, Ar-H), 4.67-4.62 (d, 1Hx, CH of pyrazoline), 4.26 (s, 1H, NH), 3.39-3.32 (d, 1Hb, CH of pyrazoline), 2.10-2.08 (d, 1Ha, CH of pyrazoline). ES MS: m/z-358.58 (M+).

 

[5-(4-chloro-phenyl)-1-phenyl-4, 5-dihydro-1H-pyrazol-3-yl]-(4-fluoro-phenyl)-amine (P16): Yield-64.38%, m.p-190-1920C, Rf: 0.76 (hexane: ethylacetate/8:2), IR (KBr) Vmax cm-1: 3343.07 (NH Str), 3133.43 (-ArC-H Str), 2923.97 (C-H Str), 1597.38 (C=N Str of 2-Pyrazoline), 1502.31(-ArCH=CH- Str), 1096.18 (C-F Str), 756.64 (C-Cl Str). 1H NMR (DMSO-d6, δ ppm): 7.92-6.72 (m, 13H, Ar-H), 4.67-4.62 (d, 1Hx, CH of pyrazoline), 4.25 (s, 1H, NH), 3.39-3.32 (d, 1Hb, CH of pyrazoline), 2.10-2.08 (d, 1Ha, CH of pyrazoline).

 

4-[5-(4-fluoro-phenyl-4-phenylamino) 2-phenyl-3, 4-dihydro)-2H-pyrazol-3-yl]-phenol (P17): Yield-61.95%, m.p-176-1780C, Rf: 0.73 (hexane: ethylacetate/8:2), IR (KBr) Vmax cm-1: 3430.99 (OH Str), 3324.04 (NH Str), 3182.43 (-ArC-H Str), 2882.53 (C-H Str), 1606.00(C=N Str of 2-Pyrazoline), 1499.45(-ArCH=CH- Str), 1099.62 (C-F Str).

 

(4-fluoro-phenyl) - [5-(4-nitro-phenyl)-1-phenyl-4, 5-dihydro-1H-pyrazol-3-yl]-amine (P18): Yield-60.90%, m.p-184-1860C, Rf: 0.74 (hexane: ethylacetate/8:2), IR (KBr) Vmax cm-1: 3350.98 (NH Str), 3006.96 (-ArC-H Str), 2942.10 (C-H Str), 1599.97(C=N Str of 2-Pyrazoline), 1538.55(-ArCH=CH- Str), 1349.44 (C-NO2 Str), 1110.90 (C-F Str). 1H NMR (CDCl3, δ ppm): 7.99-6.65 (m, 13H, Ar-H), 4.53 (s, 1H, NH), 3.86-3.84 (d, 1Hx, CH of pyrazoline), 3.02-2.75 (dd, 1Hb, CH of pyrazoline), 2.55-2.32 (dd, 1Ha, CH of pyrazoline). ES MS: m/z-376.43 (M+).

 

(4-fluoro-phenyl)-[5-(4-chloro-phenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl]- amine (P19): Yield-63.61%, m.p-150-1520C, Rf: 0.68 (hexane: ethylacetate/8:2), IR (KBr) Vmax cm-1: 3354.76 (NH Str), 3060.08 (-ArC-H Str), 2883.57 (C-H Str), 1558.14 (C=N Str of 2-Pyrazoline), 1472.77(-ArCH=CH- Str), 729.09 (C-Cl Str). 1H NMR (DMSO-d6, δ ppm): 7.80-7.10 (m, 13H, Ar-H), 5.51-5.47 (dd, 1Hx, CH of pyrazoline), 4.56 (s, 1H, NH), 3.93-3.85 (dd, 1Hb, CH of pyrazoline), 2.50-2.08 (dd, 1Ha, CH of pyrazoline). ES MS: m/z-382.18 (M+).

 

4-[5-(4-chloro-phenyl-4-phenylamino) 2-phenyl-3, 4-dihydro)-2H-pyrazol-3-yl]-phenol (P20): Yield-59.77%, m.p-128-1290C, Rf: 0.71 (hexane: ethylacetate/8:2), IR (KBr) Vmax cm-1: 3382.52 (OH Str), 3263.39 (NH Str), 3161.87 (-ArC-H Str), 2960.91 (C-H Str), 1613.36 (C=N Str of 2-Pyrazoline), 1517.00(-ArCH=CH- Str), 761.15 (C-Cl Str).

 

(4-chloro-phenyl) - [5-(4-nitro-phenyl)-1-phenyl-4, 5-dihydro-1H-pyrazol-3-yl]-amine (P21): Yield-71.68%, m.p-117-1180C, Rf: 0.768 (hexane: ethylacetate/8:2), IR (KBr) Vmax cm-1: 3249.4 (NH Str), 3086.89 (-ArC-H Str), 2950.07 (C-H Str), 1586.99 (C=N Str of 2-Pyrazoline), 1502.85(-ArCH=CH- Str), 1310.58 (C-NO2 Str), 743.16 (C-Cl Str).

 

(4-Bromo-phenyl)-[5-(4-chloro-phenyl)-1-phenyl-4, 5-dihydro-1H-pyrazol-3-yl] - amine (P22): Yield-69.01%, m.p-212-2140C, Rf: 0.72 (hexane: ethylacetate/8:2), IR (KBr) Vmax cm-1: 3388.49 (NH Str), 3218.84 (-ArC-H Str), 2960.91 (C-H Str), 1613.36 (C=N Str of 2-Pyrazoline), 1517.00(-ArCH=CH- Str), 761.15 (C-Cl Str), 638.41 (C-Br Str). 1H NMR (DMSO-d6, δ ppm): 8.18-6.82 (m, 13H, Ar-H), 4.36 (s, 1H, NH), 4.03-3.93 (d, 1Hx, CH of pyrazoline), 3.33-2.97 (dd, 1Hb, CH of pyrazoline), 2.33-2.06 (d, 1Ha, CH of pyrazoline).

 

4-[5-(4-Bromo-phenyl-4-phenylamino) 2-phenyl-3, 4-dihydro)-2H-pyrazol-3-yl]-phenol (P23): Yield-70.83%, m.p-189-1910C, Rf: 0.80 (hexane: ethylacetate/8:2), IR (KBr) Vmax cm-1: 3363.65 (OH Str), 3267.20 (NH Str), 3172.03 (-ArC-H Str), 2944.48 (C-H Str), 1606.25 (C=N Str of 2-Pyrazoline), 1526.02(-ArCH=CH- Str), 610.60 (C-Br Str). 1H NMR (DMSO-d6, δ ppm): 8.19-6.82 (m, 13H, Ar-H), 5.68 (s, 1H, OH), 4.38 (s, 1H, NH), 4.09-3.98 (d, 1Hx, CH of pyrazoline), 3.23-3.17 (d, 1Hb, CH of pyrazoline), 2.33-2.08 (d, 1Ha, CH of pyrazoline).

 

(4-Bromo-phenyl) - [5-(4-nitro-phenyl)-1-phenyl-4, 5-dihydro-1H-pyrazol-3-yl]-amine (P24): Yield-70.99%, m.p-168-1690C, Rf: 0.69 (hexane: ethylacetate/8:2), IR (KBr) Vmax cm-1: 3362.52 (NH Str), 3256.40 (-ArC-H Str), 2920.77 (C-H Str), 1604.50 (C=N Str of 2-Pyrazoline), 1521.82(-ArCH=CH- Str), 1328.36 (C-NO2 Str), 608.18 (C-Br Str).

 

Microbiological Activity:

Antimicrobial Activity15:

The antimicrobial activity was carried out by cup plate agar diffusion method. All synthesized were screened for antibacterial activity at 75 µg/ ml concentration against Staphylococcus aureus, Enterococcus. Faecalis, Escherichia coli and Klebsiella. pneumonia. Similarly the antifungal activity was carried out against Aspergillus niger and Candida albicans at 75 µg/ ml concentration. Ciprofloxacin and Fluconazole were used as standard drugs for antibacterial and antifungal activity respectively at 75 µg /ml. Control test with Dimethyl formamide was formed for every assay but showed no inhibition of the microbial growth. The antibacterial and antifungal activity of synthesized compounds is given in table 1.

 

Pharmacological Screning:

Acute oral toxicity:

The acute oral toxicity was carried out as per the guidelines set by Organization for Economic Co-operation and Development (OECD), revised draft guidelines 423, received from Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. Animal ethical committee clearance was obtained for carrying out the experiment (IAEC/MMCP/2011-12/06).

 

The LD50 of the compounds was found to be 2000mg/kg body wt. One tenth of the dose was selected as a therapeutic dose for evaluation (i.e. 200mg/kg).

 

 

Table 1:  Antibacterial and Antifungal activity of synthesized compounds. (P13-P24)

Compounds

Zone of inhibition at 75 µg/ml

S. aureus

E. faecalis

E. coli

K. pneumonia

C. albicans

A. niger

P13

13

11

12

12

13

12

P14

14

14

12

13

14

14

P15

15

13

16

14

17

15

P16

11

12

14

11

12

11

P17

11

10

11

12

11

14

P18

17

16

18

17

19

17

P19

10

11

15

15

16

15

P20

21

19

22

20

21

20

P21

18

15

17

16

18

17

P22

13

14

12

14

15

13

P23

12

12

14

12

15

14

P24

19

17

19

18

20

18

Ciprofloxacin

26

26

30

29

-

-

Fluconazole

-

-

-

-

27

25

 

Table 2: Anti-inflammatory activity of synthesized compounds P13-P24  

Comp

Mean paw oedema volume ± SEM

0 hour

1 hour

2 hour

3 hour

4 hour

Ct.

1.503±0.014

1.648±0.015

1.688±0.012

1.702±0.0012

1.720±0.010

Std.

1.483±0.011

1.602±0.014

1.585±0.014***

1.563±0.010***

1.517±0.010***

P13

1.492±0.011

1.597±0.009

1.637±0.011ns

1.643±0.012*

1.608±0.011***

P14

1.502±0.015

1.615±0.013

1.642±0.007ns

1.630±0.009**

1.600±0.007***

P15

1.512±0.012

1.623±0.018

1.613±0.012**

1.613±0.010***

1.575±0.009***

P16

1.462±0.010

1.585±0.012

1.575±0.009***

1.542±0.010***

1.502±0.009***

P17

1.503±0.008

1.622±0.013

1.647±0.007 ns

1.640±0.008**

1.612±0.007***

P18

1.458±0.018

1.587±0.013

1.617±0.017**

1.630±0.011**

1.602±0.010***

P19

1.503±0.019

1.607±0.022

1.587±0.023***

1.553±0.019***

1.527±0.017***

P20

1.483±0.010

1.602±0.017

1.593±0.013***

1.565±0.008***

1.527±0.006***

P21

1.490±0.014

1.593±0.016

1.623±0.016*

1.618±0.013***

1.592±0.014***

P22

1.507±0.016

1.627±0.015

1.620±0.010**

1.603±0.012***

1.575±0.011***

P23

1.470±0.023

1.587±0.019

1.623±0.014*

1.617±0.017***

1.583±0.014***

P24

1.488±0.015

1.602±0.014

1.628±0.014*

1.612±0.013***

1.577±0.013***

All values are expressed as mean±SEMs, ***p<0.001, **p<0.05, *p<0.01, One way ANOYA followed by Dunnett’s‘t’ test 


 

Anti-inflammatory Activity16     

Carrageenan Induced Rat Hind Paw Edema:

Anti-inflammatory activity was determined by carrageenan induced rat hind paw method of winter et al. Wistar rats (150-200 g) were used for the experiment. Rats were selected by random sample technique Diclofenac sodium was administered as standard drug at 100 mg/kg body weight. Test group received 200 mg/kg body weight of synthesized compounds and the control group received 0.5% w/v of sodium CMC. All the drugs were administered orally 30 mins before the carrageenan injection. The initial reading was taken with the help of Plethysmometer by water displacement at 0hr., i.e., immediately after injecting carrageenan and the procedure was repeated at 1, 2, 3, and 4 hours. The anti-inflammatory activity of synthesized compounds is given in table 2.

 

RESULT AND DISCUSSION:

The synthesis of the target compound was according to the illustrated scheme. The chalcones were prepared by conventional Claisen-Schmidht condensation, which further where reacted with phenyl hydrazine to yield 3, 5-diaryl substituted-1-aryl pyrazoline derivatives. IR, 1HNMR and Mass spectra of all synthesized compounds were recorded and found in fully agreement with the proposed structure.

 

All the newly synthesized pyrazoline derivatives were screened for their antibacterial, antifungal and anti-inflammatory activity. The compound P21 and P24 showed promising activity against Staphylococcus aureus where as other compounds showed poor activity. Compound P21 showed promising activity against E. faecalis and other have shown low activity. Compound P18 and P24 have shown significant activity against Escherichia coli and compound P20 and P24 has shown promising activity against K. pneumonia and rest compounds have shown moderate to low activity. The compounds P18 and P24 have shown good activity against Albican candida and compound P20 and P24 have shown excellent activity against Aspergillus niger where as rest of compounds have shown moderate to low activity against these fungi.

 

The compound P16, P19 and P20 have shown potent activity in reducing the local edema induced in the rat paw by injection of an irritant agent. And rest of them has shown moderate to low activity.

 

CONCLUSION:

Among the twelve compound synthesized compounds P18, P20, P21 and P24 have shown promising activity. Structure activity relationship based on the observed result indicated that pyrazoline bearing choro, bromo group along with nitro group has shown the promising activity. So it further deserve an attention in order to develop a new leads with structure activity relationship of this derivatives.

 

ACKNOWLEDGEMENT:

The authors are thankful to the principal, Maratha Mandal’s College of Pharmacy, Belgaum and Research Director, Bhagwant University, Ajmer for providing the facilities for this research work.

 

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Received on 15.05.2013       Modified on 10.06.2013

Accepted on 17.06.2013      © AJRC All right reserved

Asian J. Research Chem. 6(7): July 2013; Page    618-622