synthesis, characterization and biological...
TRANSCRIPT
SYNTHESIS, CHARACTERIZATION AND
BIOLOGICAL EVALUATION OF SOME
NEW 1, 3, 5-TRISUBSTITUTED-2-
PYRAZOLINES
CHAPTER - 3
175
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
176
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
177
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.
178
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).
179
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).
180
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
181
7. Mannich bases on reaction with phenylhydrazine and aqueous
ethanolic NaOH at reflux temperature yield substituted 2-
pyrazolines17 (Scheme 27).
Ph1COCH2CH2NR1R2 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).
182
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.
183
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).
184
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
185
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
186
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
187
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
188
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
189
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)
190
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.
191
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)
192
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.
193
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)
194
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)
195
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)
196
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.
197
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)
198
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.
199
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)
200
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.
201
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)
202
Insecticidal and Herbicidal activities:
Grosscurt et al.63 synthesized 3,4-diphenyl-1-phenylcarbamoyl-
2-pyrazolines (151) possessing insecticidal activity.
R
R1
NN
CNR2R3X
(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
R1R
NN
C O
NH
(153)
203
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)
204
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)
205
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)
206
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)
207
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)
208
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)
209
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
210
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
211
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
212
DRUG ACTIVITY
N
C2H5
CH3
O
N
NN
CN
Zaleplon (185)
Hypnotic and Sedative
213
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.
214
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'-methoxy- 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.
215
The chalcones that were used in the synthesis of 2-pyrazolines:
1. 1-(3'–methoxy -4'-hydroxyphenyl)-3-(2''-pyridinyl)- 2-propen-1-one (B1)
2. 1-(3'–methoxy -4'-hydroxyphenyl)-3-(3''-pyridinyl)-
2-propen-1-one (B2)
3. 1-(3'–methoxy -4'-hydroxyphenyl)-3-(4''-pyridinyl)-
2-propen-1-one (B3)
4. 1-(3'–methoxy -4'-hydroxyphenyl)-3-(2''-furyl)-
2-propen-1-one (B4)
5. 1-(3'–methoxy -4'-hydroxyphenyl)-3-(2''-pyrrolyl)-
2-propen-1-one (B5)
6. 1-(3'–methoxy -4'-hydroxyphenyl)-3-(2''-thienyl)-
2-propen-1-one (B6)
7. 1-(3'–methoxy -4'-hydroxyphenyl)-3-(2''-indolyl)-
2-propen-1-one (B7)
8. 1-(3'–methoxy -4'-hydroxyphenyl)-3-(2''-quinolinyl)-
2-propen-1-one (B8)
9. 1-(3–methoxy -4'-hydroxyphenyl)-3-(9''-anthracenyl)-
2-propen-1-one (B9)
10. 1-(3'–methoxy -4'-hydroxyphenyl)-3-(4''-fluorophenyl)-
2-propen-1-one (B10)
11. 1-(3'–methoxy -4'-hydroxyphenyl)-3-(4''-chlorophenyl)-
2-propen-1-one (B11)
12. 1-(3'–methoxy -4'-hydroxyphenyl)-3-(4''-bromophenyl)-
2-propen-1-one (B12)
13. 1-(3'–methoxy -4'-hydroxyphenyl)-3-(4''-methylphenyl)-
2-propen-1-one (B13)
14. 1-(3'–methoxy -4'-hydroxyphenyl)-3-(4''-methoxyphenyl)-
2-propen-1-one (B14)
15. 1-(3'–methoxy -4'-hydroxyphenyl)-3-(3'',4'', 5''- trimethoxyphenyl)-
2-propen-1- one (B15)
216
HO
H3COAr
O
Ethanol
Pyridine2-4 hoursReflux
HO
H3CO
NN
NHNH2 HCl
Ar
Chalcone Phenylhydrazine 1, 3, 5- tri-substituted-
hydrochloride 2-pyrazoline
Scheme 36
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
217
CH3
OCH3
OCH3
OCH3
OCH3
B13 B14 B15
Procedure:
Synthesis of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-
(2''-pyridinyl)-2-pyrazoline (B1PY1):
1-(3'–methoxy -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
C21H19N3O2, m.p. 2080C, supported by a [M+H]+ ion at m/z 346
(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,
218
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 3.81 integrating for 3 protons is due to the methoxyl group present
on the aromatic ring.
The results of Elemental analysis were also in close agreement
with those of the calculated values.
Based on the above spectral data and elemental analysis, the
structure of the compound B1PY1 was confirmed as 1-phenyl-3-(3'-
methoxy-4'-hydroxyacetophenone)-5-(2"-pyridyl)-2-pyrazoline.
By adopting the above synthetic procedure, pyrazolines of 3'-
methoxy-4'-hydroxyacetophenone chalcones (Chapter–1) were
synthesized and the physical and spectral characteristics of these new
pyrazolines (B1PY1-B15PY15) were presented separately in detail.
219
The new 1, 3, 5-trisubstituted 2- pyrazolines synthesized:
1. 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(2"-pyridinyl)-
2-pyrazoline (B1PY1)
2. 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(3"-pyridinyl)-
2-pyrazoline (B2PY2)
3. 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(4"-pyridinyl)-
2-pyrazoline (B3PY3)
4. 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(2"-furyl)-
2-pyrazoline (B4PY4)
5. 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(2"-pyrrolyl)-
2-pyrazoline (B5PY5)
6. 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(2"-thienyl)-
2-pyrazoline (B6PY6)
7. 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(2"-indolyl)-
2-pyrazoline (B7PY7)
8. 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(2"-
quinolinyl)-2-pyrazoline (B8PY8)
9. 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(9"-
anthracenyl)-2-pyrazoline (B9PY9)
10. 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(4"-
fluorophenyl)-2-pyrazoline (B10PY10)
11. 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(4"-
chlorophenyl)-2-pyrazoline (B11PY11)
220
12. 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(4"-
bromophenyl)-2-pyrazoline (B12PY12)
13. 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(4"-
methylphenyl)-2-pyrazoline (B13PY13)
14. 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(4"-
methoxyphenyl)-2-pyrazoline (B14PY14)
15. 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(3'',4'',5''-
trimethoxyphenyl)-2-pyrazoline (B15PY15)
221
Characterization of the new 1, 3, 5- trisubstituted- 2-pyrazolines
Table 16. Physical characterization data of 1, 3, 5- trisubstituted-
2-pyrazolines
HO
H3CO
NN Ar
Compound
Ar
Molecular
Formula
Relative
Molecular
Mass
(RMM)
Melting
Point
(oC)
Yield
(%)
B1PY1
N
C21H19 N3O2
345
208
78
B2PY2
N
C21H19 N3O2
345
213
75
B3PY3
N
C21H19 N3O2
345
202
77
B4PY4 O
C20H18 N2O3
334
187
72
222
B5PY5 N
H
C20H19 N3O2
333
243
70
B6PY6 S
C20H18 N2SO2
350
205
76
B7PY7 N
H
C24H21 N3O2
383
226
65
B8PY8
N
C25H21 N3O2
295
216
68
B9PY9
C30H24 N2O2
444
198
67
B10PY10
F
C22H19 N2FO2
362
235
70
B11PY11
Cl
C22H19 N2ClO2
378
242
71
B12PY12 Br
C22H19 N2BrO2
423
249
69
223
B13PY13
CH3
C23H22 N2O2
358
204
67
B14PY14 OCH3
C23H22 N2O3
374
220
66
B15PY15
OCH3
OCH3
OCH3
C25H26 N2O5
434
255
72
224
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
225
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)
B2PY2 3404 (O-H), 1595 (C=N), 1495 (C=C Quadrant of Ar),
1436 (CH=CH)
B3PY3 3405 (O-H), 1593 (C=N), 1496 (C=C Quadrant of Ar),
1435 (CH=CH)
B4PY4 3403 (O-H), 1596 (C=N), 1493 (C=C Quadrant of Ar),
1437 (CH=CH), 1205 (-C-O-)
B5PY5 3401 (O-H), 1594 (C=N), 1492 (C=C Quadrant of Ar),
1436 (CH=CH)
B6PY6 3406 (O-H), 1597 (C=N), 1495 (C=C Quadrant of Ar),
1435 (CH=CH), 690 (C-S)
B7PY7 3403 (O-H), 1596 (C=N), 1494 (C=C Quadrant of Ar),
1438 (CH=CH)
B8PY8 3407 (O-H), 1595 (C=N), 1492 (C=C Quadrant of Ar),
1437 (CH=CH)
B9PY9 3401 (O-H), 1597 (C=N), 1495 (C=C Quadrant of Ar),
1439 (CH=CH)
B10PY10 3405 (O-H), 1593 (C=N), 1491 (C=C Quadrant of Ar),
1439 (CH=CH), 1122 (C-F)
226
B11PY11 3403 (O-H), 1596 (C=N), 1496 (C=C Quadrant of Ar),
1438 (CH=CH), 853 (C-Cl)
B12PY12 3402 (O-H), 1594 (C=N), 1493 (C=C Quadrant of Ar),
1436 (CH=CH), 1024 (C-Br)
B13PY13 3409 (O-H), 1591 (C=N), 1492 (C=C Quadrant of Ar),
1433 (CH=CH)
B14PY14 3403 (O-H), 1592 (C=N), 1497 (C=C Quadrant of Ar),
1435 (CH=CH), 1072 (-O-CH3)
B15PY15 3401 (O-H), 1598 (C=N), 1495 (C=C Quadrant of Ar),
1438 (CH=CH), 1070 (-O-CH3)
227
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)
3.81 (3H, s, C-3'-OCH3)
5.36 (1H, dd, HX)
5.50 (1H, s, Ar-OH)
6.79 – 8.15 (12H, Ar-H)
B2PY2 3.15 (1H, dd, HA)
3.81 (3H, s, C-3'-OCH3)
3.90 (1H, dd, HM)
5.40 (1H, dd, HX)
5.50 (1H, s, Ar-OH)
6.79 – 7.68 (12H, Ar-H)
B3PY3 3.25 (1H, dd, HA)
3.75 (1H, dd, HM)
3.81 (3H, s, C-3'-OCH3)
5.40 (1H, dd, HX)
5.50 (1H, s, Ar-OH)
6.79 – 7.75 (12H, Ar-H)
B4PY4
3.15 (1H, dd, HA)
3.81 (3H, s, C-3'-OCH3)
3.85 (1H, dd, HM)
5.30 (1H, dd, HX)
5.50 (1H, s, Ar-OH)
6.21 – 7.65 (11H, Ar-H)
B5PY5 3.20 (1H, dd, HA)
3.79 (1H, dd, HM)
3.81 (3H, s, C-3'-OCH3)
5.30 (1H, dd, HX)
5.50 (1H, s, Ar-OH) 6.70 – 7.62 (12H, Ar-H)
228
B6PY6 3.25 (1H, dd, HA)
3.77 (1H, dd, HM)
3.81 (3H, s, C-3'-OCH3)
5.50 (1H, dd, HX)
5.50 (1H, s, Ar-OH)
6.89 – 7.67 (11H, Ar-H)
B7PY7 3.15 (1H, dd, HA)
3.81 (3H, s, C-3'-OCH3)
3.85 (1H, dd, HM)
5.30 (1H, dd, HX)
5.50 (1H, s, Ar-OH)
6.79 – 7.68 (14H, Ar-H)
B8PY8 3.15 (1H, dd, HA)
3.81 (3H, s, C-3'-OCH3)
3.85 (1H, dd, HM)
5.38 (1H, dd, HX)
5.50 (1H, s, Ar-OH)
6.45 – 7.95 (14H, Ar-H)
B9PY9 3.10 (1H, dd, HA)
3.81 (3H, s, C-3'-OCH3)
3.92 (1H, dd, HM)
5.60 (1H, dd, HX)
5.50 (1H, s, Ar-OH)
6.79 – 7.69 (17H, Ar-H)
B10PY10 3.15 (1H, dd, HA)
3.81 (3H, s, C-3'-OCH3)
3.85 (1H, dd, HM)
5.40 (1H, dd, HX)
5.50 (1H, s, Ar-OH)
6.79 – 7.67 (12H, Ar-H)
229
B11PY11 3.14 (1H, dd, HA)
3.81 (3H, s, C-3'-OCH3)
3.85 (1H, dd, HM)
5.30 (1H, dd, HX)
5.50 (1H, s, Ar-OH)
6.79 – 7.44 (12H, Ar-H)
B12PY12 3.40 (1H, dd, HA)
3.81 (3H, s, C-3'-OCH3)
3.90 (1H, dd, HM)
5.40 (1H, dd, HX)
5.50 (1H, s, Ar-OH)
6.79 – 7.48 (12H, Ar-H)
B13PY13
3.15 (1H, dd, HA)
3.75 (1H, dd, HM)
3.81 (3H, s, C-3'-OCH3)
5.30 (1H, dd, HX)
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)
3.74 (3H, s, C-4''-OCH3)
3.81 (3H, s, C-3'-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)
3.81 (3H, s, C-3'-OCH3)
3.85 (9H, s, 3 x -OCH3)
5.50 (1H, s, Ar-OH)
6.51 – 7.67 (10H, Ar-H)
230
Fig.36. IR spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(2"-pyridinyl)-2-pyrazoline (B1PY1)
HO
H3CO
NN
N
231
Fig.37. 1H NMR spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(2"-pyridinyl)-2-pyrazoline (B1PY1)
232
Fig.38. Mass spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(2"-pyridinyl)-2-pyrazoline (B1PY1)
HO
H3CO
NN
N
233
Fig.39. 1H NMR spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(3"-pyridinyl)-2-pyrazoline (B2PY2)
234
Fig.40. 1H NMR spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(4"-pyridinyl)-2-pyrazoline (B3PY3)
235
Fig.41. 1H NMR spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(2"-furyl)-2-pyrazoline (B4PY4)
236
Fig.42. 1H NMR spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(2"-pyrrolyl)-2-pyrazoline
(B5PY5)
237
Fig.43. 1H NMR spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(2"-thienyl)-2-pyrazoline
(B6PY6)
238
Fig.44. 1H NMR spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(2"-indolyl)-2-pyrazoline
(B7PY7)
239
Fig.45. 1H NMR spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(2"-quinolinyl)-2-pyrazoline (B8PY8)
240
Fig.46. 1H NMR spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(9"-anthracenyl)-2-pyrazoline (B9PY9)
241
Fig.47. 1H NMR spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(4"-fluorophenyl)-2-pyrazoline (B10PY10)
242
Fig.48. 1H NMR spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(4"-chlorophenyl)-2-pyrazoline (B11PY11)
243
Fig.49. 1H NMR spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(4"-bromophenyl)-2-pyrazoline (B12PY12)
244
Fig.50. 1H NMR spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(4"-methylphenyl)-2-pyrazoline (B13PY13)
245
Fig.51. 1H NMR spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(4"-methoxyphenyl)-2-pyrazoline
(B14PY14)
246
Fig.52. 1H NMR spectrum of 1-phenyl-3-(3'-methoxy-4'-hydroxyacetophenone)-5-(3'', 4'', 5''-trimethoxyphenyl)-2-pyrazoline (B15PY15)
247
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.
248
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.
249
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.
250
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.
251
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.
252
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.
253
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
254
DISCUSSION ON THE RESULTS:
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.
255
3.3.3 ANTIBACTERIAL ACTIVITY
The same protocols and procedures that have been followed in
Chapter-1 are used to study antibacterial activity of newly synthesized
2- pyrazolines (B1PY1-B15PY15). The results are presented in Table 22.
256
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) - - - - - - - - - -
257
DISCUSSION ON THE RESULTS:
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.
258
3.3.4 ANTIFUNGAL ACTIVITY
The same protocols and procedures that have been followed in
Chapter-1 are used to study antifungal activity of 2-pyrazolines
(B1PY1-B15PY15). The results are presented in Table 23.
259
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) - - - - - -
260
DISCUSSION ON THE RESULTS:
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.
261
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