SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL EVALUATION...

194

SYNTHESIS, CHARACTERIZATION AND

BIOLOGICAL EVALUATION OF SOME

NEW 1, 3, 5 - TRISUBSTITUTED - 2 -

PYRAZOLINES

CHAPTER - 3

195

3.1 LITERATURE REVIEW

Pyrazole is a π-excessive aromatic monocyclic heterocycle

containing two nitrogen atoms in a five membered 1,2-diazole ring. It

was in the late nineteenth century that Fischer and Knovenagel

described the reaction of acrolein with phenylhydrazine1 to provide a

2-pyrazoline type compound (117). Their experiment seems to be the

first example of pyrazoline formation by the reaction of an α, β-enone

with a hydrazine derivative. Later, Auwers et al.2,3 corraborated that

the product of this reaction was 1-phenyl 2-pyrazoline. During the last

century, after these pioneering studies, numerous 2-pyrazolines were

synthesized by the reaction of α, β-enones with hydrazines. This

simple and convenient procedure has remained one of the most

popular method for the preparation of 2-pyrazolines.

(117)

Pyrazoles exhibit aromatic character with properties resembling

those of both pyrrole and pyridine. 1-pyrazoline, 2-pyrazoline and 3-

pyrazoline are the three partially reduced forms of the pyrazole

structure with different positions of the double bonds and exists in

equilibrium one with the other (Scheme 19). 2-pyrazoline exhibits the

NN

H

12

34

5

196

monoimino character and hence more stable than the rest eventhough

all the three types have been synthesized.4

NN1

2

34

5N

N

H

1

2

34

5 NN

H

1

2

34

5

Scheme 19. All the three partially reduced forms of pyrazoline

Pyrazole is feebly basic and forms salts with inorganic acids.

The imino hydrogen may be replaced by an acyl group. Pyrazole is

very resistant to oxidation and reduction, but may be hydrogenated

catalytically, first to pyrazoline and then to pyrazolidine (Scheme 20).

Both of these compounds are stronger bases than pyrazole.

NN

H

H2

catalyst NN

H

NN

H

H

H2

catalyst

Scheme 20

Pyrazoline derivatives differ considerably in their properties from

those of pyrazole, owing to their much lower stability. The pyrazolines

give the reactions of aliphatic derivatives, resembling unsaturated

compounds in their behavior towards permanganate and nascent

hydrogen. They resemble hydrazones in the manner in which they are

hydrolyzed by mineral acids, and aldazines in their decomposition into

gaseous nitrogen and nitrogen-free substances. Pyrazoline and its

homologues are weak bases. In general they only dissolve in

197

concentrated acids, forming unstable salts which dissociate on the

addition of water. The parent substance, pyrazoline, an oil of boiling

point 114oC, is the most stable of all these compounds. The

pyrazolidines possess strong reducing properties and readily giveup

hydrogen to form pyrazolines.

GENERAL METHODS OF SYNTHESIS OF PYRAZOLINES:

1. α, β-unsaturated carboxylic acid esters reacts with

diazomethane to give 2-pyrazolines. The mechanism of this

reaction was correctly anticipated by Pechmann5 in which the

primary product of this reaction is a 1-pyrazoline, formed by 1,

3-dipolar cyclo addition, which spontaneously isomerizes into

its thermodynamically more stable 2-pyrazoline isomer by a 1,

3-H shift (Scheme 21).

C

COOMeH

C

HMeOOC

CH2N2N

N

COOMeMeOOC

NN

H

COOMeMeOOC

1

2

34

5

1

2

3

4

5

Scheme 21

2. Benzylideneacetone on reaction with diazomethane by 1,3-

dipolar cycloaddition yield 2-pyrazolines ( Scheme 22). This is

probably the first example of the synthesis of a pyrazoline from

the reaction of an α,β-unsaturated ketone and diazomethane

and was published by Azzarello6 in 1906. Later, this reaction

was reinvestigated by Smith and Howard7 and by Raju and Rao8

and the assumption made by Azzarello were corroborated.

198

Me

O

CH CHCH2N2

NN

H

Ph

O

Me

Scheme 22

3. 1,3-Dipolar cycloaddition of chalcones and diazomethane was

first investigated by Smith and Pings9 and 3-benzoyl- 4-phenyl-

1-pyrazoline was prepared as a primary product which was

then isomerized into the 3-benzoyl-4-phenyl-2-pyrazoline on

gentle heating ( Scheme 23).

O

CH CH CH2N2 NN

Ph

O

Ph

NN

Ph

O

Ph

H

Scheme 23

4. The reaction of 2-arylidene-3-phenyl-1-indanones with

diazomethane performed by Mustafa and Hilmy10 can be

considered as the first example of pyrazoline formation by the

cycloaddition of an exocyclic α,β- unsaturated ketone and

diazomethane (Scheme 24).

199

O

Ph

CH

R

CH2N2

O

Ph

N N

R

R

O

Ph

N N

H

Scheme 24

5. α,β-unsaturated aldehydes or ketones do react with

phenylhydrazine to form hydrazones as intermediates. These

hydrazone intermediates on treatment with acetic acid or

hydrochloric acid in ethanol isomerizes to Δ2-pyrazolines. The

reaction scheme is given below (Scheme 25).

200

CH2 CH

CH

N H

NH

Ph

H

PhNHNH2CH2 CHCHO

CH2 CH

CH

N

HN

Ph

CH2 CH

CH

NH

N

HPh

CH2 CH2

CH

NH

N

Ph

CH2 CH2

CH

NN

Ph

NN

Ph

Scheme 25

6. Pijewska et al.11,12 studied the reaction of 3-

arylideneflavanones and diazomethane to yield pyrazolines. The

structure and stereochemistry of the pyrazolines formed have

been elucidated by various NMR techniques. This detailed

spectroscopic investigation13-16 unambiguously proved that the

(E) isomers of flavanones provided trans- spiro-1-pyrazolines,

which were then isomerized to trans-spiro-2-pyrazolines

(Scheme 26).

O R

CAr

HO

O R

ON

N

H

Ar

O R

ON

N

H

Ar

H

CH2N2

Scheme 26

201

7. Mannich bases on reaction with phenylhydrazine and aqueous

ethanolic NaOH at reflux temperature yield substituted 2-

pyrazolines17 (Scheme 27).

Ph1COCH2CH2NR1R

2 Ph2NHNH2

NaOHN

NR2

R1 Ph1

Ph2

Scheme 27

8. The synthesis of tricyclic 2-pyrazolines by an intramolecular

1,3-dipolar cycloaddition of nitrile imines is well documented in

the literature.18-20 2, 3, 3a, 4-tetrahydro- 2-aryl [1] benzopyrano

[4,3-c] pyrazolines have been prepared by the intramolecular

1,3-dipolar cycloaddition of nitrile imines generated either from

1-( o-allyloxyphenyl)- N-( arylhydrazidoyl) chloride on treatment

with triethyl amine or by the irradiation of 2-aryl-5-( o-

allyloxyphenyl) tetrazole (Scheme 28).

202

OCH2CH

C

CH2

Cl

N NH Ar

OCH2CH CH2

N N

NN

Ar

O

N N

Ar1

2

3

a3

4

56

7

8

9

Scheme 28

9. The reaction of chalcones with hydrazines is probably the most

popular procedure for the synthesis of 2-pyrazolines. The most

commonly used method is the reaction of hydrazine and the

chalcones in acetic acid solution to prepare 2-pyrazolines in

high yield21-23 (Scheme 29). This method is used with or

without the isolation of the hydrazone intermediate. Synthesis

of 2-pyrazolines can also be achieved under alkaline conditions

by using pyridine as catalyst in ethanolic solution24. In some

cases the two reactants were refluxed in alcoholic solution

without a catalyst to provide 2-pyrazolines25,26.

203

NH2

NH

R1

R2

C O

CH

CH

R3

NN

R1

R3

R2

Scheme 29

10. 2-arylcarbonylethylthiosulfates when heated with two

equivalents of phenylhydrazine in water for 0.5-3 hours under

reflux yield 1-phenyl-3-aryl-2-pyrazolines27 (Scheme 30).

NHNH2H CH CH2 SSO3Na

CO R

R COCH CH2

NHNH2

R C

N

CH=CH2

NH

R C

N

CH2CH2

N

Scheme 30

11. 1,2-disubstituted hydrazine react with formalin and a carbonyl

compound (Hinmann synthesis) to yield pyrazolines28

(Scheme 31).

204

N

R1

NHR2X

NN

R4 R3

NN

R4 R3

-HXR3COCH2R4

H2CHCHOR1NHNHR2

R1

R2

R1R2X-

HX

+ -..

Scheme 31

12. Cycloaddition reaction of substituted styrenes with p-

anisyldiazomethane at low temperature yield trans-3, 5-bis-(p-

anisyl)-1-pyrazoline29 (Scheme 32).

CH=CH2MeO MeO CHN2 NN

MeO

Me

Scheme 32

205

SPECTRAL FEATURES OF PYRAZOLINES:

NMR SPECTRA OF PYRAZOLINES:

A pyrazoline ring is identified by characteristic spectral

features30 in its 1H NMR spectrum. The three protons in the pyrazoline

ring (118) will show AMX splitting pattern, HA proton appearing at δ

2.98 (dd), JAM= 7.6 Hz and JAX = 12 Hz, HM proton resonating at δ 3.64

(dd), JAM= 12 Hz and JMX=12 Hz and HX proton appearing at δ 5.2 (dd),

JAX= 7.6 Hz and JMX=12 Hz.

NN

H

R1HM

HX

R2

HA

(118)

MASS SPECTRA OF PYRAZOLINES:

Srzic et al.31 studied the fragmentation pathway of 1,3-diphenyl-

2-pyrazoline employing ion kinetic energy spectrometry ( I K E S) and

mass analyzed ion kinetic energy spectrometry (M I K E S) of the

native compound and specifically of isotope ( 2H, 13C, 15N) labelled

compounds, combined with high resolution mass determinations. The

results clearly demonstrated that the large majority of cleavages had

the molecular ion as their precursor.

Sayed et al.32 studied the mass spectrometric fragmentations of

the 3, 5-bisaryl-2- pyrazoline derivatives by high resolution mass

spectrometry. The observed ions may be arranged in three main

206

groups according to their assumed mechanistic formation, viz. (a) by

1,2-elimination processes (Scheme 33), (b) by α-cleavage (Scheme 34)

and (c) by assumed cyclo-reversion ( Scheme 35) .

In 1, 2-elimination, as given below, molecular hydrogen and one

aryl substituent as anisole were lost from the molecular ion.

m/z

250

OCH3

108

m/z 252m/z

M

N NHOCH3

H2

N NH

HHH

OCH3

Scheme 33. Mass fragmentation of 3, 5- disubstituted 2-pyrazoline

involving 1, 2-elimination process

207

The α-cleavage involves the radical loss of the N-1 substituent

and this cleavage may be rationalized as one electron transfer with

and without hydrogen transfer in either direction initiated by cation

radical locations somewhere in the Ar-C=N-N-π orbital system.

N N

HHH

OCH3

RM

NN

HH H

OCH3 R

N N

HH

OCH3 RH

Scheme 34. Mass fragmentation of 3,5- disubstituted 2-

pyrazoline involving α-cleavage

208

A cyclo-reversion process involves the cleavage of the pyrazoline

ring structure and gives rise to a majority of the middle to low mass

range fragments.

N N

HHH

OCH3

RM

M

N N

HH

OCH3

R

C NNR

H3CO

H3CO CH CH C N RH3CO

C NH H3CONR

C CH2

NH

NRH3CO

Scheme 35. Mass spectral fragmentation of substituted 2-

pyrazolines involving cyclo-reversion

209

BIOLOGICAL ACTIVITIES OF 2-PYRAZOLINES:

The pyrazoline nucleus is a ubiquitous feature of various

compounds possessing many pharmacological and physiological

activities and therefore they are useful materials in drug research. It

was reported in the literature that different substituted 2-pyrazolines

possess antimicrobial, anti-inflammatory, analgesic, antipyretic,

antidepressant, antitubercular, antiamoebic, anthelmintic,

anticonvulsant, antihypertensive, antidiabetic, antitumor, anti-HIV,

local anaesthetic, antioxidant, insecticidal and tranquilizing activities.

Given below is a brief account of various modifications reported on 2-

pyrazoline nucleus, which showed a variety of biological and

pharmacological activities.

Antimicrobial activity:

Deshmukh et al.33 synthesized chloro substituted 2-pyrazolines

(119) that showed antibacterial activity when assayed against some

human pathogens.

NN

Ph

OH

Cl

Cl

OMe

(119)

210

Sim et al.34 synthesized 1-acyl-3-naphthyl-5-substitutedphenyl-

2- pyrazolines (120) ; the antimicrobial activity of these compounds

was determined by using ampicillin and clotrimazole as standards.

NN

R

C

CH3

O

(120)

Desai et al.35 synthesized 1H-3-(2-hydroxy-3-nitro-5-

methylphenyl)-5-aryl-2-pyrazolines (121) that exhibited antimicrobial

activity against S. aureus and E. coli.

NN

HO

H

R

O2N

C6H4CH3

(121)

Roda et al.36 synthesized 5-aryl-1-phenyl-3-(3-isopropyl-4-

hydroxy-6-methyl phenyl)-2-pyrazolines (122); these were tested for

antimicrobial activity.

211

NN

HO

(Me)2HC

PhR

Me

(122)

Davood et al.37 synthesized 3,5-dinaphthalene-1-yl-substituted

-2-pyrazolines (123) that showed antibacterial activity.

R2

R1

N N

R3

R4

R4=H,Ph,CONH2,COCH3

R3=H,NMe2

R2=H,Cl,CH3

R1=H,OH

(123)

Saundane et al.38 synthesized a novel substituted 2- pyrazoline

(124), which was screened for antimicrobial activity against S. aureus,

E. coli and A. niger.

N

NN

N

N

N

O

H

Br

Br

H

(124)

212

Naik et al.39 synthesized 3-(2-hydroxy-5-methyl-4,6-

dibromophenyl)-5-(substituted phenyl)-2-pyrazolines (125) possessing

antibacterial activity.

N NCOR1

R

Br OH

Br

Me

R= Ph, C6H4X

R1=Me,Ph

(125)

Karthikeyan et al.40 synthesized 1-aryloxy-3-aryl-5-hydroxy-5-

arylpyrazolines (126) which showed very good antimicrobial activity.

NN

OO

F

Cl

ClOH

R

R1

R = H, OCH3, CN

R1 = Cl, CH3

(126)

Thakare et al.41 synthesized 3-coumaryl-4-aroyl-5-aryl-2-

pyrazolines (127) that showed antimicrobial activity against

pathogenic bacteria.

213

N

CO

NN R1

COH

O

ROO

CH3

R1= H,OCH3,OH

R= H,OCH3

(127)

Balakrishna, et al.42 synthesized 1-nicotinoyl-3,5-diaryl-5-

hydroxy-2-pyrazolines (128) that showed significant antimicrobial

activity.

NN

N

O

Ar1

Ar

HO

Ar = Ar1= Ph, C6H4X

(128)

Akbas et al.43 reported the synthesis of some new 1 H- pyrazole-

3-carboxylic acids (129) and evaluated for their antibacterial activities

against Bacillus cereus 7064, Staphylococcus aureus ATCC 6538,

Escherichia coli ATCC 4230 and Pseudomonas putida using tube

dilution method. The minimal inhibitory concentration (MIC)

214

experiments revealed that all the compounds showed inhibitory effects

on the growth of the test microorganisms.

NN

COOH

O

Ph

Ph

R

(129)

Anti-inflammatory, Analgesic and Antipyretic activities:

Gurar et al.44 synthesized 1-aryl -2-pyrazolines (130) exhibiting

anti-inflammatory and analgesic activity.

NN

R

NHY

R= CF3,H, Me, CH2Ph, PhY= CSNHR, CSNH2, =CHR

1

(130)

Manna et al.45 synthesized N-acetyl-2-pyrazolines (131) that

showed anti-inflammatory, analgesic and antipyretic activities.

N NAc

OHR

R1

(131)

215

Huang et al.46 synthesized 1-arylalkoxy and 1-arylalkyl thioaryl-

2-pyrazolines (132) that showed anti-inflammatory and antiallergic

activity.

N

NNH2

PhCH2O

(132)

Fredrick et al.47 synthesized 3-N-substituted amino-1-[3-

(trifluoromethyl) phenyl]-2-pyrazolines (133) which showed anti-

inflammatory activity.

NNRHN

CF3

R = H, Me, Pro, Bu,PhMe, 2-butenyl

(133)

Bansal et al.48 synthesized 1-acetyl-5 (substitutedaryl)-3-(β-

aminonaphthyl)-2-pyrazolines (134) exhibiting anti-inflammatory

activity against standard drugs phenyl butazone and indomethacin.

N

N N

R

COCH3

H

(134)

216

Amir et al.49 synthesized 3,5-disubstituted pyrazolines (135),

which showed analgesic activity.

N N

CH3CH3

C

O

Ar

(135)

Gursoy et al.50 reported the synthesis of some novel pyrazolines

(136) possessing analgesic activity on acetic acid induced writhing.

The analgesic activity was superior to that antipyrine and

aminopyrine.

NN

C6H5

H3C

H3C

O

N

OR

R1

R2

(136)

Antidepressant activity:

Palaska et al.51,52 synthesized 1,3,5-triphenyl-2-pyrazolines

(137) and these compounds showed significant antidepressant activity

when compared to the standard antidepressant drugs clomipramine

and tranylcypromine.

217

R1

NN

R2OCH3

OCH3

R1= R2= H, Cl, Br, CH3, OCH3

(137)

Prasad et al.53 synthesized 1, 3, 5-triphenyl-2-pyrazolines (138)

and 3-(2″-hydroxynaphthalene-1″-yl)-1,5-diphenyl-2-pyrazlolines

(139) which showed significant antidepressant activity.

NN

R2

R4

R1

R3

(138)

NN

R3

R2

OH

R4

R1

(139)

218

Nesrin et al.54 synthesized 1-N-substituted thiocarbamoyl -3-

phenyl-5-thienyl-2-pyrazolines (140) ; these compounds showed

antidepressant activity.

NN

S

MeO

S NH

R

(140)

Palaska et al.55 prepared 3,5-diphenyl-2-pyrazolines (141)

possessing antidepressant activity.

NN

R1

H

R2 OMe

OMe

(141)

Prasad et al.56 synthesized 3-(3''-coumarinyl)-1, 5-diphenyl-2-

pyrazolines (142) and 3-(2''-hydroxynaphthalen-1''-yl)-1, 5-diphenyl-2-

pyrazolines (143) that showed significant antidepressant activity.

219

OO

NN

HHH R1

R2

R3

R4H

H

H

H

H

H

OH NN

HHH H

H

H

H

H

H

H

H

H

H

(142) (143)

Antitubercular activity:

Kumar et al.57 synthesized substituted 2-pyrazolines (144)

which showed antitubercular activity.

NN

O

N

R3

R2

H

R1

(144)

Babu et al.58 prepared 1, 3, 5-trisubstituted pyrazolines (145)

having antitubercular activity.

O

N N

OC

OH

F

Et

NHNN

O

(145)

220

Shaharyar et al.59 synthesized 1-N-nicotinyl -3-(4′-hydroxy-3′-

methylphenyl)-5 [(sub) phenyl]-2-pyazolines (146) showing

antimycobacterial activity against Mycobacterium tuberculosis.

N

O NN

R

OH

CH3

R=phenyl.4-methoxyphenyl,4-chlorophenyl

(146)

R = phenyl, 4-methoxyphenyl,

4-chlorophenyl

Antiamoebic activity:

Budakoti et al.60 synthesized 1-N-substituted thiocarbamoyl-3,

5-diphenyl-2-pyrazolines (147) which exhibited better antiamoebic

activity than the standard drug metronidazole.

NN

Ph

Ph

CS R

R = 2- adamantylamino,

4- methylpiperidino

(147)

Abid et al.61 synthesized 1-N-substituted thiocarbamoyl-3-

phenyl-2-pyrazolines (148) having antiamoebic activity when tested

against standard drug metronidazole.

221

NN

R

S

R1

(148)

Abid et al.62 synthesized 1-(thiazolo [4,5-b] quinoxaline-2-yl)-3-

phenyl-2-pyrazolines (149) and thiocarboxamide-3-phenyl-2-

pyrazolines (150), which showed antiamoebic activity.

N

NS

N

NN

R

X

(149)

NH2

SN

N

R

X

(150)

222

Insecticidal and Herbicidal activities:

Grosscurt et al.63 synthesized 3,4-diphenyl-1-phenylcarbamoyl-

2-pyrazolines (151) possessing insecticidal activity.

R

R1

NN

CNR2R

3X

(151)

Sirrenberg et al.64 synthesized phenylcarbamoyl-2-pyrazolines

(152) having insecticidal activity.

NCONHN

R1R

R2

R3

(152)

Rolf et al.65 synthesized 1-phenylcarbamoyl-2-pyrazolines (153)

and 3, 5-diphenyl-1-phenylcarbamoyl-2-pyrazolines (154) which

showed insecticidal properties.

R2

R1

R

NN

C O

NH

(153)

223

NN

Cl

Cl

RR1NC X

(154) X = O

Kobus et al.66 synthesized 1-phenylcarbamoyl-2-pyrazolines

(155) and 3-phenyl-1-phenylcarbamoyl-2-pyazolines (156) having

insecticidal activity.

NN

C=O

HN

R

(155)

R1

R

NN

C O

NH

(156)

Kiyomi et al.67 synthesized 3-difluoromethoxyphenyl-1-

phenylcarbamoyl-2-pyazolines (157) that showed insecticidal activity.

F2HCO

NN

R

R1

R2

CONH

(157)

224

Jac et al.68 synthesized 2-pyrazolines (158) and screened for

their herbicidal activity. All the compounds showed good to moderate

activity.

N N

R1

R2

CONHSO2

(158)

Vergiya et al.69 synthesized 3,5-diaryl-1-phenyl/isonicotinoyl-2-

pyrazolines (159) exhibiting herbicidal activities.

NN

O

N

Br

HO Br

OMe

(159)

225

Miscellaneous activities:

Nawal et al.70,71 synthesized some new 2-pyrazolines (160-162)

exhibiting mollucidal activity.

N N

R R1

NHR2

X

N N

R R1

R2

N N

R R1

Ac

(160) (161) (162)

Soliman et al.72 prepared 3,5-diarylpyrazoline sulfonylurea

derivatives (163) having antidiabetic activity.

NN

Y

Z

SO2NHCXNHR

R = (CH2)2Me, cyclohexyl,PhX = O, SY = H, Cl, Br, Mez = H, Me

(163)

Tripathi et al.73 synthesized substituted 2-pyrazoline derivatives

(164) that showed hypoglycemic activity.

NN

R1R2

R4

R3

R

(164)

226

Archana et al.74 prepared 1-acetyl-5-arylidenyl-3-(2′-

oxo/thiobarbituinyl)-2-pyrazolines (165) that showed anticonvulsant

activity.

HN

HN

N NCOMe

O

O

X

R

R=H, 4-MeO, 3-MeO, 4-OHX=S,O

(165)

Pandey et al.75 synthesized 1, 3-disubstituted-5-(2-arylindol-3-

yl)-2-pyrazolines (166) which exhibited CNS depressant activity.

R1 NN

N

H

R2

(166)

Malhotra et al.76 synthesized substituted pyrazolines (167)

possessing cardiovascular activity.

NN

Ph

N SR2

H

R1

(167)

227

Alleheiligen et al.77 synthesized 3-phenyl-2-pyrazolines (168)

which are used in the treatment of cardiovascular and

thromboembolic diseases.

R

N

NNR1

o

(168)

Marayam et al.78 synthesized 1-(2-thiazolyl)-3,5-disubstituted-2-

pyrazolines (169) exhibiting antihypertensive activity when compared

with clonidine as standard.

NN

N

N S

O2N

H3CO

(169)

228

Shivarama et al.79 synthesized aryl-furyl 2-pyrazolines (170)

that showed anthelmintic activity.

MeOO

HN N

NO2

R

(170)

Suthakaran et al.80 synthesized 7-methoxybenzofuran-2-

pyrazolines (171) which showed antioxidant activity.

OCH3

O

NN

COAr

R

R1

(171)

Sook et al.81 synthesized 3, 5-diarylpyrazolines (172) which

showed antioxidant activity and was found as an inhibitor of LDL

oxidation.

t-Bu

OH

t-Bu

N NHR1 R2

OH

R3

(172)

229

Therapeutically important drugs 82-85 containing pyrazole or pyrazoline moiety along with their structures are given below:

DRUG ACTIVITY

CF3

H2NO2S

N N

H3C

Celecoxib (173)

NSAID

NNO

OH9C4

Phenylbutazone (174)

NSAID

NNO

OH9C4

OH

Oxyphenbutazone (175)

NSAID

NNO

CH3

CH3

Antipyrine (176)

Analgesic

230

DRUG ACTIVITY

NNO

CH3

CH3

N

CH3

H3C

Aminopyrine (177)

Analgesic, Antipyretic

NN

CH3

CH3N

O

CH3

NaO3SH2C

Dipyrone (178)

Analgesic, Antipyretic,

Antirheumatic

NNN

N

H

OH

Allopurinol (179)

Treatment of gout

NN

OHH2N

C

CH3

Cl

Cl

H

Muzolimine (180)

Diuretic

231

DRUG ACTIVITY

OHMe

Me

Me

H H

H

N

N

H

Stanozolol (181)

Anabolic and Androgenic

activity

NNHO

COO-Na

+NNNa

+O3S

SO-3Na

+

Amaranth (182)

Used in ulcerative colitis

N

N

O2N

NH2H

7-Amino-5-nitroindazole (183)

Antibacterial

NN

O2N

NO2 H

5,7-Dinitroindazole (184)

Antibacterial

232

DRUG ACTIVITY

N

C2H5

CH3

O

N

NN

CN

Zaleplon (185)

Hypnotic and Sedative

233

3.2 EXPERIMENTAL WORK

Aims and objectives:

It is evident from the literature that 2-pyrazolines and their

derivatives possess important biological activities. A number of 1, 3, 5-

trisubstituted-2-pyrazolines earlier synthesized in our laboratory

possessed significant anti-inflammatory, analgesic and antimicrobial

activities and in continuation of that work it is aimed to synthesize

some more new substituted 1, 3, 5- trisubstituted-2-pyrazolines.

1. To condense the chalcones described in Chapter-1 with

Phenylhydrazine hydrochloride in absolute ethanol to obtain 2-

pyrazolines and to purify these 2- pyrazolines by chromatographic and

crystallization methods.

2. To characterize the compounds using spectral (IR, 1H NMR and Mass)

methods and Elemental analysis. The data related to structural

characterization are given in the form of tables.

3. To screen the synthesized 2-pyrazolines for their toxicity and possible

biological activities like anti-inflammatory, analgesic, antibacterial and

antifungal activities.

4. To identify the active compounds for further exploitation.

234

Materials and Methods:

The same chemicals, solvents, procedures and instruments that

were mentioned in chapter-1 were also used here. Phenylhydrazine

hydrochloride used in the synthesis was purchased from Merck,

Mumbai. The chalcones that were used in the reaction with

phenylhydrazine hydrochloride were prepared from 3'-methyl- 4'-

hydroxyacetophenone as described in chapter-1.

General Procedure for the synthesis of 1, 3, 5- trisubstituted- 2-

pyrazolines 86,87

The chalcones were condensed with phenylhydrazine in

absolute ethanol in the presence of pyridine at reflux temperature (2

to 6 hours). The solvent was completely evaporated and the residue

was poured into ice cold water, which resulted in the formation of the

corresponding 2-pyrazolines (Scheme 36). Reaction completion was

identified by TLC using silica gel - G. After completion of the reaction,

the reaction mixture was poured into crushed ice with constant

stirring. The separated solid was filtered and dried. It was purified by

column chromatography on silica gel, using ethyl acetate and hexane

mixture as the mobile phase. After purification, the 2-pyrazolines were

obtained as light or bright coloured powders.

235

The chalcones that were used in the synthesis of 2-pyrazolines:

1. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-pyridinyl)-2-propen-1-one (B1) 2. 1-(3'–methyl -4'-hydroxyphenyl)-3-(3''-pyridinyl)-2-propen-1-one (B2)

3. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-pyridinyl)-2-propen-1-one (B3)

4. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-furyl)-2-propen-1-one (B4)

5. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-pyrrolyl)-2-propen-1-one (B5)

6. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-thienyl)-2-propen-1-one (B6)

7. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-indolyl)-2-propen-1-one (B7)

8. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-quinolinyl)-2-propen-1-one (B8)

9. 1-(3–methyl -4'-hydroxyphenyl)-3-(9''-anthracenyl)-2-propen-1-one

(B9)

10. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-fluorophenyl)-2-propen-1-one

(B10)

11. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-chlorophenyl)-2-propen-1-one

(B11)

12. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-bromophenyl)-2-propen-1-one

(B12)

13. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-methylphenyl)-2-propen-1-

one (B13)

14. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-methoxyphenyl)-2-propen-1-

one (B14)

15. 1-(3'–methyl -4'-hydroxyphenyl)-3-(3'', 4'', 5''- trimethoxyphenyl)-2-

propen-1- one (B15)

HO

H3C

O

C C C Ar

HH

+ NHNH2 .HCl

NN

Ar

HO

H3C

Ethanol

Pyridine

2 - 6 hours

Reflux

1

2

3 4

5

Chalcone Phenylhydrazine 1, 3, 5- tri- hydrochloride substituted-2-pyrazoline

Scheme 36

236

Where Ar =

N

N

N

B1 B2 B3

O

N

H

S

B4 B5 B6

N

H

N

B7 B8 B9

F

Cl

Br

B10 B11 B12

CH3

OCH3

OCH3

OCH3

OCH3

B13 B14 B15

237

Procedure:

Synthesis of 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(2''-

pyridinyl)-2-pyrazoline (B1PY1):

1-(3' –methyl -4'-hydroxyphenyl)-3-(2''-pyridinyl)-2-propen-1-one

(B1) ( 0.001 mol ) was condensed with phenylhydrazine hydrochloride

(0.001 mol) in the presence of pyridine (0.002 mol) in absolute ethanol

(5 ml) at reflux temperature on a water bath for 2 to 6 hours. The

solvent was evaporated in vacuo and crushed ice was added to the

residue while mixing thoroughly, whereupon a bright yellow solid

separated out. This solid was filtered under vacuum, dried and purified

by column chromatography to give a pure pale yellow solid.

The compound B1PY1 was analyzed for molecular formula as

C21H19N3O, m.p. 2080C, supported by a [M+H]+ ion at m/z 330 (Fig.38).

The IR (KBr disc, cm-1) spectrum (Fig. 36) showed the

characteristic bands at 3406 (O-H), 1594 (C = N) and 1437 (CH=CH).

The 1H NMR spectrum ( 400 MHz, CDCl3, Fig. 37 ) of compound

B1PY1 showed the characteristic HA, HM and HX protons at δ 3.05, 3.60

and 5.36 respectively as doublet of doublets (dd) with J AM =16.5 Hz,

J AX =7.30 Hz and J MX = 9.5 Hz. The spectrum also accounted for all the

12 aromatic protons which appeared in between 6.79 – 8.15. A singlet

at δ 2.31 integrating for 3 protons is due to the methyl group present on

the aromatic ring.

The results of Elemental analysis were also in close agreement

with those of the calculated values.

238

Based on the above spectral data and elemental analysis, the

structure of the compound B1PY1 was confirmed as 1-phenyl-3-(3'-

methyl-4'-hydroxyacetophenone)-5-(2"-pyridyl)-2-pyrazoline.

By adopting the above synthetic procedure, pyrazolines of 3'-

methyl-4'-hydroxyacetophenone chalcones (Chapter–1) were

synthesized and the physical and spectral characteristics of these new

pyrazolines (B1PY1-B15PY15) were presented separately in detail.

239

The new 1, 3, 5-trisubstituted 2- pyrazolines synthesized:

1. 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(2"-pyridinyl)-

2-pyrazoline (B1PY1)





4. 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(2"-furyl)-


5. 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(2"-pyrrolyl)-


6. 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(2"-thienyl)-


7. 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(2"-indolyl)-


8. 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(2"-quinolinyl)-


9. 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(9"-

anthracenyl)-2-pyrazoline (B9PY9)


fluorophenyl)-2-pyrazoline (B10PY10)


chlorophenyl)-2-pyrazoline (B11PY11)

240


bromophenyl)-2-pyrazoline (B12PY12)


methylphenyl)-2-pyrazoline (B13PY13)


methoxyphenyl)-2-pyrazoline (B14PY14)

15. 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(3'',4'',5''-

trimethoxyphenyl)-2-pyrazoline (B15PY15)

241

Characterization of the new 1, 3, 5- trisubstituted- 2-pyrazolines

Table 16. Physical characterization data of 1, 3, 5- trisubstituted-

2-pyrazolines

NN

Ar

HO

H3C

Compound

Ar

Molecular

Formula

Relative

Molecular

Mass

(RMM)

Melting

Point

(oC)

Yield

(%)

B1PY1

N

C21H19 N3O

329

208

78

B2PY2

N

C21H19 N3O

329

213

75

B3PY3

N

C21H19 N3O

329

202

77

B4PY4 O

C20H18 N2O2

318

187

72

242

B5PY5 N

H

C20H19 N3O

317

243

70

B6PY6 S

C20H18 N2SO

334

205

76

B7PY7 N

H

C24H21 N3O

367

226

65

B8PY8

N

C25H21 N3O

379

216

68

B9PY9

C30H24 N2O

428

198

67

B10PY10

F

C22H19 N2FO

346

235

70

B11PY11

Cl

C22H19 N2ClO

362.5

242

71

243

B12PY12 Br

C22H19 N2BrO

407

249

69

B13PY13

CH3

C23H22 N2O

342

204

67

B14PY14 OCH3

C23H22 N2O2

358

220

66

B15PY15

OCH3

OCH3

OCH3

C25H26 N2O4

418

255

72

244

Table 17. Elemental analysis data of 1, 3, 5- trisubstituted- 2-

Pyrazolines

Compound ( % Calculated)

C H N

( % Found)

C H N

B1PY1 76.57 5.81 12.76 76.53 5.78 12.72

B2PY2 76.57 5.81 12.76 76.54 5.79 12.73

B3PY3 76.57 5.81 12.76 76.53 5.77 12.71

B4PY4 75.40 5.67 8.81 75.35 5.63 8.78

B5PY5 75.68 6.03 13.24 75.64 6.00 13.21

B6PY6 71.83 5.42 8.38 71.79 5.40 8.41

B7PY7 78.45 5.76 11.44 78.38 5.72 11.39

B8PY8 79.15 5.54 11.08 79.12 5.50 11.11

B9PY9 84.05 5.65 6.54 84.01 5.67 6.51

B10PY10 76.03 5.49 8.09 76.00 5.45 8.10

B11PY11 72.80 5.20 3.80 72.76 5.17 3.76

B12PY12 64.86 4.66 6.88 64.82 4.61 6.85

B13PY13 80.57 6.48 8.16 80.55 6.45 8.11

B14PY14 77.07 6.19 7.82 77.04 6.17 7.80

B15PY15 71.75 6.26 6.69 71.71 6.21 6.65

245

Table 18. IR spectral data of 1, 3, 5- trisubstituted- 2-pyrazolines

Compound Position of absorption band (cm-1)

B1PY1 3406 (O-H), 1594 (C=N), 1494 (C=C Quadrant of Ar),

1437 (CH=CH)


1436 (CH=CH)


1435 (CH=CH)


1437 (CH=CH), 1205 (-C-O-)


1436 (CH=CH)


1435 (CH=CH), 690 (C-S)


1438 (CH=CH)


1437 (CH=CH)


1439 (CH=CH)


1439 (CH=CH), 1122 (C-F)

246


1438 (CH=CH), 853 (C-Cl)


1436 (CH=CH), 1024 (C-Br)


1433 (CH=CH)


1435 (CH=CH), 1072 (-O-CH3)


1438 (CH=CH), 1070 (-O-CH3)

247

Table 19. 1 H NMR spectral data of 1, 3, 5- trisubstituted- 2-

pyrazolines

Compound Chemical shift ( δ ) in ppm

B1PY1 3.05 (1H, dd, HA)

3.60 (1H, dd, HM)

5.36 (1H, dd, HX)

2.31 (3H, s, C-3'-CH3)

5.50 (1H, s, Ar-OH)

6.79 – 8.15 (12H, Ar-H)

B2PY2 3.15 (1H, dd, HA)

3.90 (1H, dd, HM)

5.40 (1H, dd, HX)

2.31 (3H, s, C-3'-CH3)

5.50 (1H, s, Ar-OH)

6.79 – 7.68 (12H, Ar-H)

B3PY3 3.25 (1H, dd, HA)

3.75 (1H, dd, HM)

5.40 (1H, dd, HX)

2.31 (3H, s, C-3'-CH3)

5.50 (1H, s, Ar-OH)

6.79 – 7.75 (12H, Ar-H)

B4PY4

3.15 (1H, dd, HA)

3.85 (1H, dd, HM)

5.30 (1H, dd, HX)

2.31 (3H, s, C-3'-CH3)

5.50 (1H, s, Ar-OH)

6.21 – 7.65 (11H, Ar-H)

B5PY5 3.20 (1H, dd, HA)

3.79 (1H, dd, HM)

5.30 (1H, dd, HX)

2.31 (3H, s, C-3'-CH3)

5.50 (1H, s, Ar-OH) 6.70 – 7.62 (12H, Ar-H)

248

B6PY6 3.25 (1H, dd, HA)

3.77 (1H, dd, HM)

5.50 (1H, dd, HX)

2.31 (3H, s, C-3'-CH3)

5.50 (1H, s, Ar-OH)

6.89 – 7.67 (11H, Ar-H)

B7PY7 3.15 (1H, dd, HA)

3.85 (1H, dd, HM)

5.30 (1H, dd, HX)

2.31 (3H, s, C-3'-CH3)

5.50 (1H, s, Ar-OH)

6.79 – 7.68 (14H, Ar-H)

B8PY8 3.15 (1H, dd, HA)

3.85 (1H, dd, HM)

5.38 (1H, dd, HX)

2.31 (3H, s, C-3'-CH3)

5.50 (1H, s, Ar-OH)

6.45 – 7.95 (14H, Ar-H)

B9PY9 3.10 (1H, dd, HA)

3.92 (1H, dd, HM)

5.60 (1H, dd, HX)

2.31 (3H, s, C-3'-CH3)

5.50 (1H, s, Ar-OH)

6.79 – 7.69 (17H, Ar-H)

B10PY10 3.15 (1H, dd, HA)

3.85 (1H, dd, HM)

5.40 (1H, dd, HX)

2.31 (3H, s, C-3'-CH3)

5.50 (1H, s, Ar-OH)

6.79 – 7.67 (12H, Ar-H)

249

B11PY11 3.14 (1H, dd, HA)

3.85 (1H, dd, HM)

5.30 (1H, dd, HX)

2.31 (3H, s, C-3'-CH3)

5.50 (1H, s, Ar-OH)

6.79 – 7.44 (12H, Ar-H)

B12PY12 3.40 (1H, dd, HA)

3.90 (1H, dd, HM)

5.40 (1H, dd, HX)

2.31 (3H, s, C-3'-CH3)

5.50 (1H, s, Ar-OH)

6.79 – 7.48 (12H, Ar-H)

B13PY13

3.15 (1H, dd, HA)

3.75 (1H, dd, HM)

5.30 (1H, dd, HX)

2.31 (3H, s, C-3'-CH3)

2.15 (3H, s, C-4''-CH3)

5.50 (1H, s, Ar-OH)

6.79 – 7.44 (12H, Ar-H)

B14PY14 3.15 (1H, dd, HA)

3.50 (1H, dd, HM)

5.40 (1H, dd, HX)

2.31 (3H, s, C-3'-CH3)

3.74 (3H, s, C-4''-OCH3)

5.50 (1H, s, Ar-OH)

6.79 – 7.67 (12H, Ar-H)

B15PY15 3.20 (1H, dd, HA)

3.65 (1H, dd, HM)

5.20 (1H, dd, HX)

2.31 (3H, s, C-3'-CH3)

3.85 (9H, s, 3 x -OCH3)

5.50 (1H, s, Ar-OH)

6.51 – 7.67 (10H, Ar-H)

250

Fig.36. IR spectrum of 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(2"-pyridinyl)-2-pyrazoline (B1PY1)

251

Fig.37. 1H NMR spectrum of 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(2"-pyridinyl)-2-pyrazoline (B1PY1)

252

Fig.38. Mass spectrum of 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(2"-pyridinyl)-2-pyrazoline (B1PY1)

253


254


255

Fig.41. 1H NMR spectrum of 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(2"-furyl)-2-pyrazoline (B4PY4)

256

Fig.42. 1H NMR spectrum of 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(2"-pyrrolyl)-2-pyrazoline (B5PY5)

257

Fig.43. 1H NMR spectrum of 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(2"-thienyl)-2-pyrazoline (B6PY6)

258

Fig.44. 1H NMR spectrum of 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(2"-indolyl)-2-pyrazoline (B7PY7)

259

Fig.45. 1H NMR spectrum of 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(2"-quinolinyl)-2-pyrazoline (B8PY8)

260

Fig.46. 1H NMR spectrum of 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(9"-anthracenyl)-2-pyrazoline (B9PY9)

261

Fig.47. 1H NMR spectrum of 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(4"-fluorophenyl)-2-pyrazoline (B10PY10)

262

Fig.48. 1H NMR spectrum of 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(4"-chlorophenyl)-2-pyrazoline (B11PY11)

263

Fig.49. 1H NMR spectrum of 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(4"-bromophenyl)-2-pyrazoline (B12PY12)

264

Fig.50. 1H NMR spectrum of 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(4"-methylphenyl)-2-pyrazoline (B13PY13)

265

Fig.51. 1H NMR spectrum of 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(4"-methoxyphenyl)-2-pyrazoline (B14PY14)

266

Fig.52. 1H NMR spectrum of 1-phenyl-3-(3'-methyl-4'-hydroxyacetophenone)-5-(3'', 4'', 5''-trimethoxyphenyl)-2-pyrazoline (B15PY15)

267

3.3 BIOLOGICAL EVALUATION

PRESENT WORK

A number of 2- pyrazolines were reported to possess diverse

biological activities like antimicrobial, antidepressant, analgesic, anti-

inflammatory, antiviral, antileishmanial, antitubercular, anti-HIV and

antimalarial activities. In view of the varied biological and

pharmacological importance of different series of 2-pyrazoline

derivatives, it is felt worthwhile to evaluate them for possible activities.

These compounds therefore were screened for anti-inflammatory,

analgesic, antibacterial and antifungal activities.

EXPERIMENTAL METHODS

ACUTE TOXICITY

The same protocols and procedures that were followed in

Chapter-1 were used to study the acute toxicity of 2- pyrazolines.

All the 2-pyrazolines employed in the pharmacological screening

have been found to be free form toxicity as well as toxic symptoms

even at a high dose of 1000 mg/kg body weight and hence they were

considered safe.

268

3.3.1 ANTI-INFLAMMATORY ACTIVITY

The same protocols and procedures that have been followed in

Chapter-1 are used to study anti-inflammatory activity of 2-

pyrazolines (B1PY1-B15PY15). The results are presented in Table 20.

269

Table 20. Anti-inflammatory activity of 1, 3, 5- trisubstituted- 2-

pyrazolines

Compound

Ar

% inhibition ± SEM at

various time intervals

0.5h 1.0h 2.0h 3.0h 4.0h 6.0h

B1PY1 2''-pyridinyl 5±1 6±1 36±1 43±1 75±1 76±1

B2PY2 3''-pyridinyl 5±1 6±1 35±1 44±1 74±1 75±1

B3PY3 4''-pyridinyl 6±1 8±1 34±1 45±1 73±1 77±1

B4PY4 2''-furyl 5±1 6±1 32±1 43±1 70±1 74±1

B5PY5 2''-pyrrolyl 12±1 14±1 50±1 64±1 92±1 95±1

B6PY6 2''-thienyl 6±1 8±1 37±1 48±1 76±1 78±1

B7PY7 2''-indolyl 13±1 16±1 53±1 65±1 94±1 97±1

B8PY8 2''-quinolinyl 6±1 7±1 34±1 45±1 75±1 79±1

B9PY9 9''-anthracenyl 9±1 11±1 44±1 54±1 83±1 89±1

B10PY10 4''-fluorophenyl 8±1 9±1 42±1 53±1 80±1 86±1

B11PY11 4''-chlorophenyl 7±1 10±1 40±1 51±1 79±1 84±1

B12PY12 4''-bromophenyl 6±1 9±1 39±1 50±1 76±1 82±1

B13PY13 4''-methylphenyl 10±1 11±1 47±1 54±1 87±1 90±1

B14PY14 4''-methoxyphenyl 11±1 13±1 48±1 57±1 89±1 91±1

B15PY15 3'',4'',5''-trimethoxyphenyl

13±1 14±1 50±1 59±1 90±1 93±1

Aceclofenac (standard) 22±1 23±1 70±1 85±1 90±1 98±1

All values are represented as mean±SEM (n=6). *P<0.01 compared to

reference standard Aceclofenac. Student’s t-test.Dosage: Aceclofenac-

2 mg/kg and test compounds-10 mg/kg body weight of rat.

270

DISCUSSION ON THE RESULTS:

The anti-inflammatory activities of all the 2-pyrazolines

synthesized have been evaluated by using carrageenan-induced rat

paw oedema method.

The results clearly revealed the potential anti-inflammatory

activity of all these 2-pyrazolines when compared with the standard

drug aceclofenac, but not at an identical dose level. Of all the

compounds tested, compound B7PY7 having the indolyl substitution at

the 5-position of the 2-pyrazoline ring showed maximum activity and

this is followed by compounds having a pyrrolyl ring (compound

B5PY5) on the pyrazoline nucleus. These results of anti-inflammatory

activity for the 2-pyrazolines is consistent with the literature reports.

However, the contributing physico-chemical properties of these

substituents need to be established by a proper QSAR study. Such a

study also may provide useful information in the design and synthesis

of 2-pyrazolines with promising anti-inflammatory activity.

271

3.3.2 ANALGESIC ACTIVITY

A number of pyrazolines were reported to possess significant

analgesic activity and infact some of the drugs currently used in

therapy possessed pyrazoline structure and hence it was felt

worthwhile to screen these compounds synthesized in the present

study for analgesic activity by tail flick method. The working

procedure is described separately and the results are given in

Table 21.

Experimental:

Tail immersion test method / tail flick method88,89 was adopted

for evaluation of analgesic activity of the test compounds. The tail of

the control, standard and test group animals (rats) was dipped in a

beaker of water maintained 55 1oC and the time taken to withdraw

the tail clearly out of water is taken as the reaction time.

Requirements:

Animals: Albino rats of either sex

Standard drug: Ibuprofen suspension in 2% v/v Tween 80 solution

administered orally at the dose of 100 mg/kg body weight.

Samples: Test compounds were suspended in 2% v/v Tween 80

solution and administered orally at the dose of 100 mg/kg body

weight. Water was heated in a beaker and the temperature was

maintained at 55 1 oC.

272

Working procedure:

85 albino rats of either sex weighing between 150-200 grams

were divided into 17 groups of 5 animals each and they were

numbered individually. The animals were fasted for 24 hours before

administering the drug with water ad libitum.

Group I was administered with only 2% v/v Tween 80 solution,

which served as control. Group II was administered with 100 mg/kg

body weight of ibuprofen suspension orally, which served as a

standard. Group III to group XVII were administered with test

compounds respectively, the dose being 100 mg/kg body weight

selected on the basis of the standard drug used. All the animal tails

were dipped into a beaker containing water maintained at 55 1 oC

and the time taken for the animals to flick the tail from the hot water

completely is recorded at 15 minutes, 30 minutes, 1 hour, 2 hours

and 3 hours respectively.

The percentage of protection in the control, standard and drug

treated animals were recorded and calculated by using the formula.

% Analgesic activity (PAA) = [ (Rt/Rc) - 1] x 100

Where Rt and Rc are the reaction time in test and control respectively.

The results of analgesic activity of ibuprofen and the

compounds tested are shown in Table 21.

273

Table 21. Analgesic activity of 1, 3, 5- trisubstituted- 2-

pyrazolines

Com-pound

Ar

% Analgesic activity (PAA)

15 min

30 min

1 h 2 h 3 h

B1PY1 2''-pyridinyl 75±1 86±1 101±1 113±1 125±1

B2PY2 3''-pyridinyl 74±1 82±1 96±1 107±1 121±1

B3PY3 4''-pyridinyl 74±1 81±1 93±1 105±1 118±1

B4PY4 2''-furyl 80±1 90±1 108±1 117±1 134±1

B5PY5 2''-pyrrolyl 81±1 93±1 112±1 120±1 136±1

B6PY6 2''-thienyl 78±1 88±1 105±1 115±1 130±1

B7PY7 2''-indolyl 82±1 95±1 116±1 123±1 141±1

B8PY8 2''-quinolinyl 70±1 78±1 89±1 102±1 115±1

B9PY9 9''-anthracenyl 63±1 75±1 85±1 100±1 113±1

B10PY10 4''-fluorophenyl 92±1 105±1 127±1 135±1 154±1

B11PY11 4''-chlorophenyl 89±1 102±1 123±1 131±1 150±1

B12PY12 4''-bromophenyl 85±1 97±1 120±1 128±1 145±1

B13PY13 4''-methylphenyl 94±1 106±1 130±1 136±1 156±1

B14PY14 4''-

methoxyphenyl

96±1 108±1 133±1 139±1 158±1

B15PY15 3'',4'',5''-tri methoxyphenyl

99±1 111±1 137±1 143±1 162±1

Ibuprofen (standard) 103±1 114±1 149±1 160±1 174±1

Control - - - - -

Values are expressed as mean SEM (n=5). *p<0.05; **p<0.01;

***p<0.001 compared to controls. Students’s t-test

274


All the 2-pyrazolines tested for analgesic activity showed

considerable activity when compared to the standard drug ibuprofen. It

is interesting to note that compound B15PY15 having 3, 4, 5-

trimethoxyphenyl, compound B14PY14 having 4-methoxyphenyl,

compound B13PY13 having 4-methylphenyl ring at the 5-position of the

2-pyrazoline ring possessed the maximum activity. It clearly indicates

the favorable effect of electron releasing substituents on the analgesic

activity of the 2-pyrazolines. 2-Pyrazolines having these substituents

both on the aromatic and the heteroaromatic rings, if synthesized and

tested, may possess significant analgesic activity. Literature reports

also indicated the necessity of electron releasing groups in enhancing

the analgesic activity. 2-Pyrazolines with a fluorine substituent

(compound B10PY10) on the aromatic ring also enhanced the activity.

Hence, compounds having fluorine and other halogens at one or more

positions of the aromatic rings can be synthesized to have compounds

with much better activity.

275

3.3.3 ANTIBACTERIAL ACTIVITY


Chapter-1 are used to study antibacterial activity of newly synthesized

2- pyrazolines (B1PY1-B15PY15). The results are presented in Table 22.

276

Table 22. Antibacterial activity of 1, 3, 5- trisubstituted- 2-

pyrazolines

Com-

pound

Ar

Zone of inhibition in mm

Quantity in µg/ml

B.subtilis B.pumilis S. aureus E. coli P.vulgaris

50 100 50 100 50 100 50 100 50 100

B1PY1 2''-pyridinyl 12 15 11 14 09 12 10 11 11 13

B2PY2 3''-pyridinyl 12 14 10 12 09 11 09 10 10 12

B3PY3 4''-pyridinyl 11 13 10 12 08 10 08 09 09 12

B4PY4 2''-furyl 18 21 17 19 16 18 15 18 17 20

B5PY5 2''-pyrrolyl 15 17 14 16 13 15 12 14 13 15

B6PY6 2''-thienyl 13 15 12 16 11 13 11 12 12 14

B7PY7 2''-indolyl 17 19 16 19 14 18 13 17 15 17

B8PY8 2''-quinolinyl 20 22 19 21 17 20 17 19 18 21

B9PY9 9''-anthracenyl 10 12 08 11 08 09 07 09 06 08

B10PY10 4''-fluorophenyl 23 25 24 25 21 24 21 23 20 23

B11PY11 4''-chlorophenyl 21 24 22 23 19 21 18 20 19 21

B12PY12 4''-bromophenyl 20 21 18 20 17 19 16 19 17 19

B13PY13 4''-methylphenyl 09 11 07 09 06 07 06 08 07 09

B14PY14 4''-methoxyphenyl 08 10 06 08 05 06 04 05 05 06

B15PY15 3'',4'',5''-

trimethoxyphenyl 06 08 05 06 03 04 03 04 04 05

Benzyl penicillin (standard) 28 30 27 30 26 28 26 29 28 30

Control (DMSO) - - - - - - - - - -

277


All the 2-pyrazolines (B1PY1-B15PY15) have been evaluated for

their antibacterial activity against Bacillus subtilis, Bacillus pumilis,

Staphylococcus aureus (Gram-positive) and Escherichia coli, Proteus

vulgaris (Gram-negative), using cup-plate method. The results of this

evaluation have been compared by taking benzyl penicillin as

standard. The antibacterial activity data of 2-pyrazolines (B1PY1-

B15PY15, Table 22) indicated that the compounds have significant

inhibitory activity on all the bacteria at both 50 µg (0.05 ml) and 100

µg (0.1 ml) dose levels when compared with benzyl penicillin.

Among all the compounds tested, compounds B10PY10, B11PY11

and B8PY8 possessed maximum activity. The first two compounds

possessed the halogens on the aromatic ring and thus reveal the

positive contribution of electron withdrawing groups to the

antibacterial activity, while the third compound possessed quinoline

nucleus responsible for antibacterial activity. The results are

consistent with the literature reports. Compounds having these

substituents on the hetero aryl ring can also be synthesized and

screened for antibacterial activity, with a hope to get better

compounds in this series. A QSAR study on a large data bank of 2-

pyrazolines may further provide insights into the structural

requirements and the contributing physico-chemical properties in

enhancing the antibacterial activity.

278

3.3.4 ANTIFUNGAL ACTIVITY


Chapter-1 are used to study antifungal activity of 2-pyrazolines

(B1PY1-B15PY15). The results are presented in Table 23.

279

Table 23. Antifungal activity of 1, 3, 5- trisubstituted- 2-

pyrazolines

Com-pound

Ar

Zone of inhibition (in mm)

Quantity in µg/ml

A. niger C.albicans R. oryzae

50 100 50 100 50 100

B1PY1 2''-pyridinyl 13 15 10 13 11 14

B2PY2 3''-pyridinyl 12 14 10 12 10 13

B3PY3 4''-pyridinyl 11 13 09 10 10 11

B4PY4 2''-furyl 17 19 16 18 14 17

B5PY5 2''-pyrrolyl 15 17 12 15 13 15

B6PY6 2''-thienyl 14 16 11 13 12 13

B7PY7 2''-indolyl 16 18 14 16 13 17

B8PY8 2''-quinolinyl 18 20 17 19 17 20

B9PY9 9''-anthracenyl 10 12 08 10 08 10

B10PY10 4''-fluorophenyl 22 24 20 23 21 23

B11PY11 4''-chlorophenyl 20 22 19 21 19 22

B12PY12 4''-bromophenyl 19 22 18 21 17 21

B13PY13 4''-methylphenyl 08 11 07 09 07 09

B14PY14 4''-methoxyphenyl 07 10 06 07 06 08

B15PY15 3'',4'',5''-

trimethoxyphenyl 06 08 05 06 06 07

Fluconazole (standard) 26 29 25 30 24 29

Control (DMSO) - - - - - -

280


The antifungal activity of the substituted 2-pyrazolines was

evaluated against A.niger, C.albicans and R. oryzae, employing

fluconazole as the standard drug and using the cup- plate method.

A close examination of the Table 23 pertaining to the antifungal

activity data of 2-pyrazolines revealed that all the compounds in this

series have been found to be effective against all the fungi at both the

dose levels tested, when compared with the reference standard. Like

in the case of antibacterial activity, here also compounds with electron

withdrawing groups enhanced the activity and it is much more than

what is observed in the case of antifungal activity of pyrimidines.

Compounds having electron releasing groups also contributed

favorably to the antifungal activity. The contributing physico-chemical

properties of these compounds, however, need to be established by

QSAR studies. 2-Pyrazolines having electron withdrawing and

releasing substituents on the hetero aryl ring can also be synthesized

and screened for antifungal activity in order to get compounds with

promising activity.

281

3.4 REFERENCES

1. Fischer, E. and Knovenagel, O., Ann. Chem., 239, 194 (1887).

2. Auwers, K. V. and Muller, K., Ber. Dtsch. Chem. Ges., 41, 4230

(1908).

3. Auwers, K. V. and Kreuder, A., Ber. Dtsch. Chem. Ges., 58, 1974

(1925).

4. Loudon, J.D., in: Chemistry of Carbon Compounds, Elseveier

Publishing Company, New York (1957).

5. Pechmann, H.V., Ber. Dtsch. Chem. Ges., 27, 1890 (1894).

6. Azzarello, J., Gazz. Chim. Ital., 36, 50 (1906).

7. Smith, L.I. and Howard, K.L., J. Am. Chem. Soc., 65, 165 (1943).

8. Raju, G.V.S. and Rao, K.S., Curr. Sci., 58, 1030 (1989).

9. Smith, L.I. and Pings, W.B., J. Org. Chem., 2, 23 (1937).

10. Mustafa, A. and Hilmy, M.K., J. Chem. Soc., 3254 (1951).

11. Pijewska, L., Kamecki, J. and Perka, W., Polish J. Chem., 59, 285

(1985).

12. Pijewska, L., Kamecki, J. and Perka, W., Pharmazie, 48, 254

(1993).

13. Toth, G., Levai, A., Dinya, Z. and Snatzke, G., Tetrahedron, 47,

8119 (1991).

14. Levai, A. and Patonay, T., J. Heterocyclic Chem., 36, 747 (1999).

15. Neudeck, H. K., Monatsh. Chem., 127, 417 (1996).

282

16. Huisgen, R., Seidel, M., Sauer, J., McFarland, W. and Wallibillich,

G., J. Org. Chem., 24, 892 (1959).

17. Sammour, A.E.A., Tetrahedron, 20, 1067 (1967).

18. Smith, G. and Laude, B., Tetrahedron Lett., 3727 (1978).

19. Padwa, A., Nahm, S. and Sato, E., J. Org. Chem., 43, 1664 (1978).

20. Meier, H. and Heimgartner, H., Helv. Chim. Acta., 68, 1283 (1985).

21. Raiford, L. C. and Entrikin, J. B., J. Am. Chem. Soc., 55, 1125

(1933).

22. Auwers, K. V. and Voss, H., Ber. Dtsch. Chem. Ges., 42, 4411

(1909).

23. Auwers, K. V. and Lammerhirt, E., Ber. Dtsch. Chem. Ges., 54,

1000 (1921).

24. Anjaneyulu, A.S.R., Sudha Rani, G., Gowri Annapurna, K.,

Mallavadhani, U.V. and Murthy, Y.L.N., Indian J. Chem., 34B,

933 (1995).

25. Auwers, K. V. and Heimke, P., Ann. Chem., 458, 186 (1927).

26. Habib, O.M.O., Khalil, A.M., Kandeel, E.M. and Abdalla, E.B., Rev.

Roum. Chim., 31, 629 (1986).

27. Furukawa, M., Yuki, T. and Hayashi, S., Chem. Pharm. Bull., 22,

1990 (1974).

28. Ibrahim, Y.A., Abdou, S. and Selim, S., Heterocycles, 19, 819

(1982).

283

29. Overberger, C.G., Weinshenker, N. and Anselme, J.P., J. Am.

Chem. Soc., 87, 4119 (1965).

30. Howell, W.C., Ktenas, M. and MacDonald, J.M., Tetrahedron

Lett., 26, 1719 (1964).

31. Srzic, D., Klasinc, L., Noppel, H. E. and Gusten, H., Org. Mass

Spectrom., 13, 30 ( 1978).

32. Sayed, G.H. and KjФsen, H., Indian J. Chem., 20B, 640 (1981).

33. Deshmukh, M.S., Rajput, P.R. and Chincholkar. N.M., Asian J.

Chem., 9, 848 (1997).

34. Sim, S. R., Safak, C., Aabbasoglu, U. and Oxcelik B., Int. J. Chem.,

7, 89 (1986).

35. Desai, J.K. and Ankhiwala, M.D., J. Inst. Chemists., 69, 27 (1997).

36. Roda, K.P., Vansdadia, R.N. and Parekh, H., J. Inst. Chemists., 61,

51 (1989).

37. Davood, A. and Shaabanzadeh, M., Molecules, 11, 370 (2000).

38. Saundane, A.R., Badiger, J. and Sharma, P.M.V., Ind. J. Het.

Chem., 14, 331 (2005).

39. Naik, V.R. and Naik, H.B., Asian J. Chem., 11, 1522 (1999).

40. Karthikeyan, M.S., Bantwal, S.H. and Sucheta, N.K., Eur. J. Med.

Chem., 42, 30 (2007).

41. Thakare, N.R. and Jamode, V.S., Asian J. Chem., 19, 3633 (2007).

284

42. Balakrishna, K., Ramesh, C., Ganesh, R., Gururaja, R. and

Shalini, S., J. Ind. Council of Chemists., 18, 39 (2001).

43. Akbas, E., Berber, I., Sener, A. and Hasnaov, B., FARMACO, 60,

23 ( 2005).

44. Gurar, N.I., Gulko, L.I., Gorodelskova, N.R. and Klebanov, B.M.,

Khimiko-Farmatsevtivhesskii zhurnal, 28, 34 (1994).

45. Manna, F., Chimenti, F., Bolasco, A., Cenicola, M.L. and Marmo,

E., Eur. J. Med. Chem., 27, 633 (1992).

46. Huang, F.C., US Patent, 8 (1987).

47. Fredrick, C., Peter, I.J. and Tateson, J.E., Forschung., 53, 44

(2003).

48. Bansal, E., Srivastava, V.K. and Kumar, A., Eur. J. Med. Chem.,

36, 81 (2001).

49. Amir, M. and Shikha, K., Indian J. Chem., 44B, 2532 (2005).

50. Gursoy, A., Seref, D., Gultaze, C. and Kevser, E. and Kamil, V.,

Eur. J. Med. Chem., 35, 359 (2000).

51. Palaska, E., Erol, D. and Demirdamar, R.B., Eur. J. Med. Chem.,

31, 43 (1996).

52. Palaska,E., Bilgin, A.A., Sunal, R. and Gumesel, B., Pharmazie.,

49, 67 (1994).

53. Prasad, Y. R., Rao, L. A., Prasoona, L., Murali, K. and Ravi

Kumar, P., Bioorg Med. Chem. Lett., 15, 5030 (2005).

285

54. Nesrin, G., Yesilada, A., Ucal, G., Erol, K. and Bilgin, A., Archiv.

Der. Pharmazie., 336, 362 (2003).

55. Palaska, E., Aytemir, M. and Uzbay, I.T., Eur. J. Med. Chem., 36,

539 (2001).

56. Prasad, Y.R., Ravi Kumar, P., Asha Deepthi, Ch. and Venkata

Ramana, M., Asian J. Chem., 19, 4790 (2007).

57. Kumar, P. M. , Kumar, R.A. and Ravi, T.K., Indian J. Pharm.

Sci., 67, 755 (2005).

58. Babu, V. H., Manna, S.K., Srinivasan, K.K. and Bhat, G. V., Ind.

J. Het. Chem., 13, 253 (2004).

59. Shaharyar, M., Siddiqui, A.A., Ali, M.A., Sriram, D. and Yogeswari,

P., Bioorg. Med. Chem. Lett., 16, 3947 (2006).

60. Budakoti, A., Mahammad, A. and Amir, A., Eur. J. Med. Chem.,

41, 63 (2006).

61. Abid, M. and Azam, A., Eur. J. Med. Chem., 40, 935 (2005).

62. Abid, M. and Azam, A., Bioorg. Med. Chem. Lett., 16, 2812 (2006).

63. Grosscurt, A.C., Vanher, R. and Wellinga, K., J. Agri. Food Chem.,

27, 406 (1979).

64. Sirrenberg, W., Klauke, E., Hammann, I. and Stendel, W., Ger., 25

(1978).

65. Rolf, V.H., Wellinga, K. and Grosskurt, A.C., J. Agri. Food Chem.,

26, 915 (1978).

286

66. Kobus, W., Grosskurt, A.C. and Van Hes, R., J. Agri. Food Chem.,

25, 987 (1977).

67. Kiyomi, O., Nakajima, Y., Tsugeno, M., Ishii, S. and Hatanaka,

M., US Patent, 14 (1984).

68. Jac, H.H.E., Thus, J.G. and Wellinga, K.S., Pesticide Sci., 29, 101

(1990).

69. Vergiya, V. D., Kothari, S. and Verma, B.L., Ind. J. Het. Chem.,

13, 105 (2003).

70. Nawal, F.A.M., Ibrahim Y.A. and Girgis A.S., Indian J. Chem.,

35B, 935 (1996).

71. Nawal, F.A. M., Ibrahim, Y.A., Girgis, A.S. and Su, F., Pharmazie,

51, 544 (1996).

72. Soliman, R., Faid, A., Hassan, M. and Sam Sik, E., J. Pharm. Sci.,

176, 626 (1987).

73. Tripathi, S., Pandey, B.R., Bathwal, J.P., Kishor, K. and Bhargava,

K.P., Ind. J. Pharmacol., 24, 155 (1980).

74. Archana, V., Kishore, S. and Kumar, A., Arzneimittel Forschung.,

52, 787 (2002).

75. Pandey, B.R., Raman, K., Barthwal, J.P. and Parmar, S.S.,

National Acad. Sci. Lett., 2, 98 (1979).

76. Malhotra, V., Pathak, S., Nath, R., Mekerjee, D. and Shanker, K.,

Indian J. Chem., 41B, 1310 (2002).

287

77. Alleheiligen, S., Brohm, D., Diedrichs, N., Froehlen, B., Gerdes, C.

and Voehringer, V., PCT Int. Appli., 183 (2005).

78. Marayam, B., Shekarchi, M., Jorjani, M. and Shafiee, A., Archiv.

Der. Pharmazie., 337, 25 (2004).

79. Shivarama, B., Veerendra, B., shivananda, M.K., Latha, K.P. and

Vaidya, V.P., Ind. J. Het. Chem., 12, 385 (2003).

80. Suthakaran, R., Somasekhar, G., Sridevi, Ch., Marikannan, M.,

Suganthi, K. and Nagarajan, G., Asian J. Chem., 19, 3353 (2007).

81. Sook, J. T., Kyung, S.K., Ju, R.K., Kyung, H.C., Sangku, L. and

Woo, S.L., Bioorg. Med. Chem. Lett., 14, 2719 (2004).

82. Burger, A., in: Burger’s Medicinal Chemistry and Drug Discovery,

John Wiley and Sons, 6th Edition (2003).

83. Foye, O.W., David, A. W. and Thomas, L. L., in: Foye’s Principles

of Medicinal Chemistry. Lippincott, Philadelphia, 5th edition (2002).

84. Block, J.H. and Beale, J. M., in: Wilson and Gisvold’s. Text Book

of Organic Medicinal and Pharmaceutical Chemistry, Lippincott ,

Philadelphia, 11th edition (2004).

85. Gringauz, A., in: Introduction to Medicinal Chemistry, Wiley-VCH

Inc, Canada (1997).

86. Shivarama, H.B., Shivananda, M.K. and Veerendra, B., Ind. J. Het.

Chem., 12, 135 (2002).

87. Jones, D.G., Liang, X., Stewart, E.L. and Noe, R.A., Bioorg. Med. Chem.

Lett., 15, 3203 (2005).

288

88. Armarego, W.L.F., in: Physical Methods in Hetrocyclic Chemistry, .

Academic Press, New York, 3, 90 (1971).

89. Vanderhaeghe, H. and Claesen, M., Bull. Sci. Chim. Belg., 66, 276

(1957).

SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL EVALUATION...

Documents

Transcript of SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL EVALUATION...