Microwave Assisted Synthesis of Fluoro-Pyrazole Derivatives for Antiinflammatory Activity.

 

Sahu Sudeep1, Dey Tathagata2*, Khaidem Somila2 and Y. Jyothi2

1SAF Fermion Ltd, Millennium City, Information Technology Park, Kolkata-700 091              2East Point College of Pharmacy, Bidarahalli, Virgonagar Post, Bangalore – 560 049

*Corresponding Author E-mail: tathagatad@gmail.com

 

ABSTRACT:

A series of fluoro-pyrazole derivatives have been synthesized and evaluated for anti-inflammatory screening using formali induce rat paw edema model. The studies of synthesized compounds were characterized by TLC, IR, 1H NMR analysis. These compounds have shown promising anti-inflammatory activity when compared with the standard drug diclofenac sodium.

 

KEYWORDS: Fluoro-pyrazole, Chalcone, Anti-inflammatory activity.

 


INTRODUCTION:

Pyrazoles are well known and important nitrogen-containing five–membered heterocyclic compounds. Derivatives of pyrazoles possess a broad spectrum of biological activities like antibacterial1, antiproliferative2, antitubercular3, antitumor4, antidepressant5, analgesic6, antidiabetic7, cardiovascular8. A number of pyrazole derivatives have been reported to posses interesting biological activities like antiinflammatory9, antimicrobial10 and antiprotozoal11. A classical microwave synthesis was done with several chemical classes of drugs such as substituted benzaldehydes and hydrazines. Microwave assisted organic synthesis (MAOS) has shown to be very effective synthetic procedure in recent times12. The chemical effects of microwave irradiation are diverse and include substantial improvement in both stoichiometric and catalytic chemical reactions. The use of such unconventional reaction conditions reveals several features like: a very short reaction time compared to conventional heating, easy of work-up after a reaction, reduction in the usual thermal degradation and better selectivity13.

 

Protein denaturation is one of the well documented cases of inflammation. Some anti-inflammatory drugs inhibit protein denaturation. Mizushima and others have used protein denaturation as an invitro screening model for anti-inflammatory compounds14. Recent report reveal that dehydrogingerone(4-Hydroxy-3-methyl-benzalacetone) isolated from Zingiber officinale has anti-inflammatory activity.

 

Saturation of the double bond or variation of the aliphatic part results in loss of the anti-inflammatory activity. The presences of fluorine enhance the therapeutic efficiency and lipid solubility15. In view of these observations stimulated us to synthesize a number of analogues of fluoropyrazoles. The phenyl styryl ketones are cyclized to fluoropyrazoles to obtain more active compounds (Scheme I).

The title compounds were characterized by elementary analyses, melting points, IR, 1H NMR measurements.

 

MATERIALS AND METHODS:

All the chemicals were purchased from Aldrich, Fluka, and S.D. Fine Chemical. Melting points were determined by open capillaries and are uncorrected. TLC was carried out by using silica gel. 1H NMR spectra were recorded on Bruker model DRX 300 NMR spectrometer in acetone–d6, CDCl3 or DMSO-d6 using tetramethylsilane (TMS) as an internal standard. Chemical shift values are reported in (ppm). IR spectra were recorded on Perkin Elmer FT-IR spectrometer using KBr pellet.

 

EXPERIMENTAL SECTION:

Step I. Synthesis of Schiff’s base:

3-chloro-4-fluoro aniline (0.01 mol)1 and benzaldehyde (0.01 mol)2 was taken in a beaker along with the 20 ml of ethanol and few drops of glacial acetic acid and irradiated in the microwave oven for 2 mins. The product obtain was poured in to crushed ice which precipitated out. The precipitate 3 was washed with water to removed impurities and then recrystallized from boiling water.

 

Step II Intermediate of chalcone 5

Intermediate product 5 was obtain by the reaction of Schiff’s base 3 (0.01 mol) and 4-amino-acetophenone4 in presence of 20 ml ethanol and irradiated in microwave oven for 2 mins. The product 5 was poured in to crushed ice and precipitate obtained was filtered and dried.

 

Step III. Synthesis of benzaldehyde chalcone 7:

The product 5 (0.01 mol) with benzaldehyde (0.01 mol) 6 was taken in a beaker along with 20 ml of ethanol and irradiated in the microwave oven for 2 mins. The product obtain 7 was poured in to crushed ice and precipitate obtain. The precipitate 7 was filtered and dried.

 

Step IV. Synthesis of pyrazole derivatives 9:

 

Benzaldehyde chalcone 7 (0.01 mol) along with phenyl hydrazine (0.01 mol) 8 was taken in a beaker along with the 20 ml of ethanol and few drops of glacial acetic acid and irradiated in the microwave oven for 2 mins. The product obtain was poured in to crushed ice and precipitate obtain. The precipitate 9 was filtered and dried then recrystallized from petroleum ether and benzene. The physical data of all the synthesized compounds (9a-z) were given in Table-1 and the IR, NMR spectral data of compounds (9b, 9c, 9j, 9k, 9m, 9o, 9q, 9r, 9s, 9u, 9v, 9y and 9z)were summarized in (Table-2).

 

PHARMACOLOGICAL ACTIVITY (Anti-inflammatory activity):16

Three months old Wistar Albino rats of males, weighing 200-250 gm were used. The animals were allowed food and water ad libitum. They were housed in room temperature at 25±2˚C for 24 hrs. The animals were randomly allocated into 9 groups, each group contained 6 animals. The activity was carried out on formalin (2% V/V) induced edema model. The control group received 0.5 ml of DMSO and standard groups of animals were treated with diclofenac sodium (10 mg/kg) as a standard drug. The test groups were treated with different synthesized pyrazole derivatives (9a, 9b, 9l, 9m, 9p, 9x and 9z) with defined dose (10 mg/kg) by intraperitoneal route. After 30 minutes the animals were injected with 0.1 ml of 2% (w/v) formalin in the plantar region of the left paw of control, diclofenac sodium as well as synthesized derivatives -treated groups. Prior to the administration of the formalin, the average volume (V0). The right paw will serve as reference non-inflamed paw for comparison. The paw volume of legs of control and diclofenac-treated rats at 15 min, 30 min 1 hr, 2 hrs, 3 hrs, 4 hrs and 8 hrs after formalin injected subcutaneously was noted with the help of plethysmometer. After injection of the synthesized derivatives (10a-h) the paw volume (Vt) was measured. The oedema was expressed as an increase in the volume of paw and the percentage inhibition of acute oedema was obtained as follows:

% Inhibition = [1− (Vt /Vc)] × 100

Where, ∆V = Vt −V0, V= Mean paw volume. The statistical analysis of data was carried out by one way ANOVA followed by Tukey-Kramer's Multiple Comparisons Test. All the values are expressed as mean paw volume (mg/dl) ± S.E.M, n=6.(Table-3 )

 

RESULT AND DISCUSSION:

Pyrazole derivatives were synthesized by the process of Schiff’s base formation. In this process Schiff’s base were synthesized by the condensation of 3-chloro-4-fluoroaniline with aldehydes. Then obtained Schiff’s base is treated with p-aminoacetophenone to obtain Intermediate of chalcone were condensing with different types of aldehydes to obtained benzaldehyde chalcone. Finally chalcone were treated with different types of phenyl hydrazine to synthesize different of fluoro-pyrazole derivatives (Scheme I). We have found the reaction undergoes very smoothly with the good yield (85.5%). The structures of the synthesized compounds were confirmed by IR and NMR spectroscopy analysis.

 


Table-1: Physical data of synthesized compounds (9a-z.).

 

Compd

R

R'

Mol formula

Mol weight

m.p. (0C)

Yield (%)

Rf value

9a

 

 

C35H27FN4O

538.63

119

68.5

0.52

9b

 

 

C34H25FN4F

524.60

115

83.05

0.54

9c

 

 

C35H27FN4O2

554.63

98

59.20

0.57

9d

 

 

C36H30FN5

551.61

125

55.50

0.74

9e

 

 

C34H25FN4O

524.60

127

85.50

0.55

9f

 

 

C36H29FN4O2

568.66

113

52.20

0.77

9g

 

 

C36H29FN4O3

584.66

134

72.40

0.63

9h

 

 

C36H30FN5O

567.67

120

54.50

0.80

9i

 

 

C35H27FN4O2

554.63

106

76.90

0.71

9j

 

 

C34H25FN4O2

540.60

130

71.50

0.65

9k

 

 

C35H27FN4O3

570.63

124

63.20

0.84

9l

 

 

C36H30FN5O

567.67

133

66.80

0.89

9m

 

 

C34H25FN4O2

540.60

135

78.90

0.66

9n

 

 

C35H27FN4O2

554.63

100

73.10

0.69

9o

 

 

C36H29FN4O3

584.66

190

77.20

0.61

9p

 

 

C35H27FN4O3

570.63

173

61.50

0.79

9q

 

 

C36H29FN4O4

600.66

138

54.10

0.56

9r

 

 

C36H30FN5

551.67

82

67.60

0.62

9s

 

 

C37H32FN5O

581.70

99

75.50

0.60

9t

 

 

C36H30FN5O

567.67

85

74.20

0.67

9u

 

 

C36H30FN5O

567.67

95

79.90

0.73

9v

 

 

C36H25FN4O

524.60

116

70.10

0.85

9w

 

 

C34H25FN4O2

540.60

108

69.20

0.68

9x

 

 

C35H27FN4O

538.63

150

58.50

0.51

9y

 

 

C34H25FN4O

524.60

148

50.50

0.86

9z

 

 

C35H27FN4O2

554.63

114

65.1

0.88

 


Table-2: Spectral data of various synthesized compounds.

Sl. No

Compound Code

Spectral data IR/NMR

1

9b

IR(KBrcm-1): 3317.3(N-H str­), 2954.7(C-H Ar str),2839(C-H-Ali), 1596.7(C=C str),1605(C=O), 1496.7(C=N), 1172.6(C-F str).

2

9c

NMR(300MHz, acetone-d6): δ8.44 (1H, s, NH), δ8.68 (1H, s, N=CH), δ9.08 (1H, s, O-H), δ7.044 -8.13 (H, m, Ar-H).

IR(KBrcm-1): 3294.2(N-H str­), 3055(C-H Ar str), 3380 (O-H-str), 1596.9(C=C str), 1504.4(C=N str), 1373.2(O-H bend), 1126.4(C-F str).

3

9j

IR(KBrcm-1): 3317.3(N-H str­), 3039(C-H Ar str), 1359.5(C=C str), 1473.5(C=N str), 1365.5(O-H bend), 1172.6(C-F str).

4

9k

IR(KBrcm-1): 2839(C-H str Ali), 3600(O-H st), 2931.6(C-H str Ar), 1504.4(C=N), 1172.6(C-F).

5

9m

IR(KBrcm-1): 3363.6(N-H str), 2939.3(C-H str Ar), 1604.7(C=C str), 1504.4(C=N str), 1172(C-F Ar).

6

9o

NMR(300MHz,DMSO-d6): δ8.59 (1H, s, NH), δ8.13 (2H, s, O-H), δ 7.98-6.99 (H, m, Ar-H).

IR(KBrcm-1): 3317(N-H str), 2839(C-H str Ali), 3055(C-H str Ar), 1504.4(C=N).

7

9q

IR(KBr cm-1): 3317(NH), 3031(C-H -Ar), 1357.8(OH-bending), 1596.9(C=C Str), 1488.9(C=N str), 1141.8(C-F str).

8

9r

NMR(300MHz,DMSO-d6): δ8.38 (1H, s, NH), δ2.99 (6H, s, CH3), δ 7.82-6.81(H, m, Ar-H).

IR(KBrcm-1): 3286.5(N-H str), 3047(C-H str Ar), 1496.7(C=N str).1596.9(C=C str)

9

9s

IR(KBrcm-1): 3301.9(N-H str), 3379.1 (O-H str), 3055(C-H str Ali), 1596.9(C-C str), 1488.9(C=N).

10

9u

NMR(300MHz,CDCl3): δ8.14 (1H, s, NH), δ 7.92-7.09 (H, m, Ar-H).

IR(KBrcm-1): 3317(N-H str), 3031(C-H str Ar), 1488.9(C=N), 1596.9(C=C str)

11

9v

NMR(300MHz,CDCl3): δ8.51(1H, s, NH), δ3.82 (3H, s, -OCH3), δ 8.13-6.84 (H, m, Ar-H).

IR(KBrcm-1): 3317(N-H str), 2839(C-H str Ali), 3055(C-H str Ar), 1596.9(C=C str) 1504.4(C=N str).

12

9y

NMR(300MHz,DMSO-d6): δ8.46 (1H, s, NH), δ3.04 (6H, s, -CH3), δ 7.82-6.85 (H, m, Ar-H).

IR(KBrcm-1): 3317(N-H str), 2808(C-H str Ali), 2916.2(C-H str Ar), 1604.7(C=C str) 1504.4(C=N str).

13

9z

IR(KBrcm-1): 3370(N-H str), 2931.6(C-H str Ar), 2839(C-H str Ali) 1604.7(C=C str), 1496.7(C=N str), 1172(C-F Ar).

 

Table-3:  Anti-inflammatory activity of the test compounds (9a, 9b, 9l, 9m, 9p, 9x, 9z) were compared with respect to control and standard.

Treatment

Paw volume in ml due to Formalin at different time intervals (Mean ±SEM)

15 min

30 min

1 Hr

2 Hrs

3 Hrs

4 Hrs

8 Hrs

Control

0.5 ml

0.1866±

0.0413

0.2066 ±

0.0414

0.3066 ± 0.0387a

0.4533 ± 0.o736b

0.6783 ± 0.0855b

0.9233 ± 0.0508b

1.4283 ± 0.1057b

Standard (diclofenac 10 mg/kg)

0.0883 ± 0.0047

0.105 ± 0.0056

0.125 ± 0.0067c

0.1533 ± 0.0080d

0.2200 ± 0.0330d

0.5066 ± 0.0454d

0.8533 ± 0.0422d

9a

0.1266 ± 0.0182

0.1733 ± 0.031

0.1400 ± 0.0193c

0.1100 ± 0.0161d

0.0816 ± 0.0147d

0.0533 ± 0.0128b,d

0.0116 ± 0.0016b,d

9b

0.1033 ± 0.0061

0.125 ± 0.0084

0.1566 ± 0.0091

0.166 ± 0.135d

0.1633 ± 0.0066d

0.1416 ± 0.0074b,d

0.1416 ± 0.0722b,d

9l

0.08 ± 0.0063

0.1033 ± .0066

0.14 ±  0.0068c

0.1716 ± 0.0065d

0.155 ± 0.0076d

0.135 ± 0.0056b,d

0.055 ± 0.0061b,d

9m

0.1783 ± 0.0654

0.2766 ± 0.0760a

0.4966 ± 0.0862b,c

0.635 ± 0.071b,c

0.7 ± 0.0474b

0.5183 ± 0.0575d

0.0966 ± 0.0247b,d

9p

0.0816 ± 0.0230

0.2050 ± 0.0495

0.2950 ± 0.0683a

0.4083 ± 0.0733b

0.2283 ± 0.0437d

0.1583 ± 0.0321b,d

0.035 ± 0.0123b,d

9x

0.1066 ± 0.0160

0.12 ± 0.0152

0.2133 ± 0.0313

0.3483 ± 0.0356a

0.17 ± 0.0327d

0.125 ± 0.0214b,d

0.0333 ± 0.0138b,d

9z

0.0983 ± 0.0218

0.1183 ± .0160

0.1883 ± 0.0304

0.3030 ± 0.0409

0.1766 ± 0.0206d

0.125 ± 0.0290b,d

0.065 ± 0.0183b,d

P< 0.05 when compared with diclofenac. (a). P< 0.01 when compared with diclofenac (b). P< 0.05 when compared with control (c). P< 0.01 when compared with control (d). Where ‘P’ denotes paw volume.

a, b denoting the potency and activity compared with diclofenac and c, d denoting the  potency and activity of the synthesized derivatives when compared with control.

 


Out of the 26(9a-9z) synthesized derivatives, compounds 9a, 9b, 9l, 9m, 9p, 9x and 9z were evaluated for anti-inflammatory activity by formalin induced edema. The configurations a and b denoting the potency and activity of the derivatives at different time intervals when compared with diclofenac, other c and d denoting the  potency and activity of the synthesized derivatives when compared with control. From the pharmacological screening data it appears that all the synthesized derivatives reduced paw edema in comparison to diclofenac at 30 min, 1 hr, 2hrs, 3hrs, 4hrs and 8hrs, in post formalin injection significant (P<0.01) with variable onset of action. While derivatives 9m, 9p exhibited rapid onset of action (>1hr) and showed duration of action (<3hr), derivatives 9a, 9b, 9l, 9x and 9z exhibited delayed onset of action (>3hrs) and activity maintained even at 8th hrs after administration. All the tested derivatives exhibited comparable anti-inflammatory activity with diclofenac and can be used in the formalin induced arthritis.

 

REFERENCES:

1.        Joshi SD, Vagdevi HM, Vaidya VP and Gadaginamath GS. Synthesis of new 4-pyrrol-1-ylbenzoic acid hydrazide analogs and some and some derivative oxadiazole, triazole and pyrrole ring systems: A novel class of potential antibacterial and antitubercular agents. European Journal of Medicinal Chemistry. 2008; 43: 1989-1996.

2.        Chimichi S, Boccalini M, Hassan MMM, Viola G, Dall’Acqua F and Curini M, Synthesis, Structural Determination and Photo-antiproliferative Activity of New 3-Pyrazolyl or -Isoxazolyl Substituted 4-Hydroxy-2(1H)-quinolinonesTetrahedron, 62, 2006, 90-96.

3.        Ali MA, Shaharyar M and Siddiqui AA. Synthesis, structural activity relationship and anti-tubercular activity of novel pyrazoline derivatives. European Journal of Medicinal Chemistry. 2007; 42: 268-275.

4.        Park HJ, Lee K, Park S, Ahn B, Lee JC, Cho HY and Lee KI. Identification of antitumor activity of pyrazole oxime ethers. Bioorganic and Medicinal Cheistry Letter. 2005; 15: 3307-3312.

5.        Palaska E, Aytemir M, uzbay IT and Erol D. Synthesis and antidepression activity of some 3,5-diphenyl-2-pyrazolines. European Journal of Medicinal Chemistry. 2001; 36: 539-543.

6.        Chetan BP, Sreenivasa MT and Bhat AR. Synthesis and evaluation of certain pyrazolines and related compounds for their antitubercular, antibacterial, antifungal activities. Indian Journal of Heterocyclic Chemistry. 2004; 13: 225-228.

7.        Soliman R. Preparation and antidiabetic activity of some sulfonylurea derivatives of 3,5-disubstituted pyrazoles. Journal of Medicinal. Chemistry. 1979; 22 (3):  321-325.

8.        Kumar A, Sexena KK, Gurtu S, Sinha JN and Shanker K. Indolylazetidinylpyrazolines as cardiovascular agents. Indian Drugs. 1988; 24: 1-5.

9.        Makhsumov AD, Dzhuraev, Kilichov G and Nikbaev AT. Antiinflammatory activity of some pyrazole derivatives. Pharmaceutical Chemistry Journal. 1986; 20: 289-291.

10.     Bekhit AA, Ashour Hayam AA, Ghany Yasser SA, Bekhit Alaa Ei-Din A and Baraka A., Synthesis and biological evaluation of some thiazolyl and thiadiazolyl derivatives of 1H-pyrazole as antimicrobial agents, European. Journal of Medicinal Chemistry. 2008; 43: 456-463.

11.     Norma RS and Reto B. Synthesis and Biological Evaluation of Pyrazolylnaphthoquinones as New Potential Antiprotozoal and Cytotoxic Agents. ChemBiochem; 4 (1):  69-72.

12.     Kappe CO and Dallinger D, The impact of Microwave Synthesis on Drug Discovery, Nature. Reviews, 2006, 5, 51-63.

13.     Mavandadi F and Hallberg A. The impact of microwave-assisted organic synthesis in drug discovery. Drug discovery today. 2001; 6(8): 406-416.

14.     Nargund LVG, Hariprasad V and Reddy GRN. Synthesis and anti-inflammatory activity of fluorinated phenyl styryl ketones and N-phenyl-5-substituted aryl-3-p-(fluorophenyl) pyrazolins and pyrazoles. Journal of Pharmaceutical Sciences.  2006; 81 (9): 892-894.

15.     Fyaz MDI. Important fluorinated drugs in experimental and clinical use. Journal of Fluorine Chemistry. 2002; 118(1-2): 27-33.

16.     Vogel GH, Vogel WH. Drug discovery and evaluation-pharmacological assays. 5th ed., New York, Springer, 1996: 555.

 

 

 

 

 

 

 

Received on 04.08.2010          Modified on 03.09.2010

Accepted on 11.09.2010         © RJPT All right reserved

Research J. Pharm. and Tech. 4(3): March 2011; Page 413-419