Synthesis and Anti-infective
Studies of Some Novel Pyrazoline Derivatives
S. M. Hipparagi1*, M.
D. Bhanushali2, S. A. Patil3, N. S. Desai2
1Department of
Pharmaceutical Chemistry, KLE University’s, College of Pharmacy, Rajajinagar,
Bangalore-40.
2Department of
Pharmaceutical Chemistry, Maratha Mandal’s College of Pharmacy, Belgaum. 590
016
3Department of
Pharmacognosy, Maratha Mandal’s College of Pharmacy, Belgaum. 590 016
*Corresponding Author E-mail: chemsmh@yahoo.co.in
In the present study a new series of 3,
5-diaryl-1-phenyl-2-pyrazoline were synthesized. Chalcones were synthesized by
treating various substituted acetophenone with different substituted aromatic
aldehydes in the presence of ethanol and sodium hydroxide solution. The
pyrazoline derivatives were synthesized by cyclocondensation of chalcones with
phenyl hydrazine. The structures of the synthesized compound have been
established on the basis of IR and 1HNMR data. The compounds were
evaluated in-vitro for their antimicrobial and anti-tubercular activities.
KEY WORDS: Chalcones, Pyrazoline, Antimicrobial, Anti-tubercular
INTRODUCTION:
The treatment of
infectious disease still remain an important and challenging problem because of
a combination of factors including emerging infectious diseases and the
increasing number of multi-drug resistant microbial pathogens. Inspite of a
large number of antibiotics and chemotherapeutics available for medical use,
the emergence of old and new antibiotic resistance developed in the last
decades , has created a substantial medical need for new classes of
anti-infective agents. A potential approach to overcome the resistance problem
is to design innovative agent with a different mode of action, so that no cross
resistance with the present therapeutical can occur.
Infectious
diseases are one of the leading causes of death worldwide. Hence, discovering a
novel anti-infective agent with better pharmacological profile is still highly
desirable. So in recent decade’s heterocyclic compounds containing N, S and O
have been under investigation because of their important medicinal properties1,
2.
Among them
pyrazoline have been reported to possess a variety of significant and diverse
pharmacological activities such as antibacterial3, antifungal3,
antiviral4, anti-tubercular5, antiamoebic6,
anticancer7, anti-inflammatory8, analgesic9,
antidepressant10 and anticonvulsant11 activity. In light
of these findings it was felt worthwhile to synthesize some new pyrazoline
derivatives and evaluate for their antimicrobial and anti-tubercular activity.
MATERIALS AND METHODS:
Melting points
were determined in open capillary tube and are uncorrected. All the chemicals
were obtained from S.D. Fine make, while the reagents and solvents were of
analytical grade purity. The IR spectra were recorded on a Jasco FTIR-460
spectrometer using KBr disc method. 1HNMR spectra were recorded on
Bruker Advance II 400 NMR spectrometer using DMSO/ CDCl3 as solvent
and TMS as an internal standard. All the chemical shift are expressed in δ
ppm. The purity of synthesized compounds has been checked by TLC.
General procedure
for the synthesis of Chalcones (C1-12)12:
An equimolar
mixture of substituted acetophenone (0.01 M) and substituted aromatic aldehyde
(0.01 M) was stirred for one hour in 50 ml of ethanol and then 10 ml of 10%
sodium hydroxide solution was added slowly to above solution with continuous
stirring. Then the mixture was stirred for 8-10 hours at room temperature and
kept in a refrigerator for an overnight. On the next day, the mixture was
poured into a beaker containing ice-cold water (100 ml). Then the aqueous layer
was acidified with concentrated HCl. The precipitate obtained was filtered at
pump, washed with water, dried and recrystallized from ethanol. The completion
of the reaction was monitored by TLC using Benzene: Methanol (9:1 v/v) as
solvent system.
General procedure
for the synthesis of Pyrazoline (P1-12)13:
A mixture of the
chalcones (0.01 M) and phenyl hydrazine (0.02M) was refluxed in glacial acetic
acid (40 ml) for 6- 8 hours. Then mixture was cooled and poured on to crushed
ice and the solid mass that separated out was filtered, washed free of acid
with cold water, dried and recrystallized from ethanol. The completion of the
reaction was monitored by TLC using Ethyl acetate: Hexane (8:2) as solvent
system. Physical data of synthesized compounds are presented in Table 1.
Scheme
|
Comp. code
|
R
|
R’
|
|
P1
|
H
|
Cl
|
|
P2
|
H
|
OH
|
|
P3
|
H
|
NO2
|
|
P4
|
F
|
Cl
|
|
P5
|
F
|
OH
|
|
P6
|
F
|
NO2
|
|
P7
|
Cl
|
Cl
|
|
P8
|
Cl
|
OH
|
|
P9
|
Cl
|
NO2
|
|
P10
|
Br
|
Cl
|
|
P11
|
Br
|
OH
|
|
P12
|
Br
|
NO2
|
Spectroscopic
data of synthesized compounds:
The infra red
spectra of synthesized chalcones (C1-12) showed a carbonyl
absorption in the region 1626-1681 cm-1 which is the characteristic
of α, β- unsaturated carbonyl group as well as an olfenic C=C band in
the region 1428-1491 cm-1. The 1H NMR spectra showed the
olfenic H-β and H-α two doublets in the region at 
7.56-7.84
ppm. The aromatic protons appeared as multiplets at 
7.07-8.11
ppm.
The infra red
spectra of synthesized pyrazoline (P1-12) are 3389.41 (Ar-OH),
2938.11 (C-H str), 1578.15 (C=N str). 1H NMR ppm
2.40-2.56 (2H, methylene of pyrazoline) 4.17-4.22(1H, methine of pyrazoline),
4.99 (1H of hydroxy) 7.40-7.96 (13H, Ar-H).
Microbiological
Activity
Antimicrobial
Activity14:
The antimicrobial
activity of the synthesized compounds was determined by cup-plate method. The
organisms selected for antibacterial activity were two gram positive strain
(Staphylococcus aureus and E. Faecalis) and two gram negative strain
(Escherichia coli and K. pneumonia). Similarly the antifungal activity was
carried out by using Aspergillus niger and Candida albicans. The concentration
of sample compounds was 75 mcg/ml.Ciprofloxacin and Fluconazole were used as
standard drugs for antibacterial and antifungal activity respectively at 75
mcg/ml. Control test with Dimethyl formamide was formed for every assay but
showed no inhibition of the microbial growth.
The antibacterial
and antifungal activity of synthesized compounds is given in table 2.
Anti-Tubercular
Activity15:
The
anti-tubercular screening was carried out by Middle brook 7H9 broth against Mycobacterium
tuberculosis of H37Rv strain. The minimum
inhibitory concentration (MIC) of each synthesized compound was determined by
microplate Alamar Blue assay (MABA) and is
defined as the lowest concentration of drug, which inhibits ≤99% of
bacterial population present at the beginning of the assay. This method is
non-toxic and shows good correlation with BACTEC radiometric method. Briefly,
200µl of sterile deionzed water was added to all outer perimeter wells of
sterile 96 wells plate to minimized evaporation of medium in the test wells
during incubation. The 96 wells plate received 100 µl of the Middlebrook 7H9
broth and serial dilution of compounds were made directly on plate. The final
drug concentrations tested were 0.01 to 20.0 µl/ml. Plates were covered and
sealed with parafilm and incubated at 37şC for five days. After this time, 25µl
of freshly prepared 1:1 mixture of Almar Blue reagent and 10% tween 80 was added
to the plate and incubated for 24 hrs. A blue color in the well was interpreted
as no bacterial growth, and pink color was scored as growth. The MIC was
defined as lowest drug concentration which prevented the color change from blue
to pink. Isoniazid was used as standard drug. Some of the
synthesized compounds have shown significant activity as compared with the
standard drug. The anti-tubercular activity of synthesized compounds is given
in table 3.
Table3:Anti-tubercular
activity of synthesized compounds (P1-P12)
|
SL.NO
|
Compounds
|
MIC
µg/ml
|
|
1
|
P1
|
100
|
|
2.
|
P2
|
100
|
|
3.
|
P3
|
1.6
|
|
4.
|
P4
|
50
|
|
5.
|
P5
|
100
|
|
6.
|
P6
|
25
|
|
7.
|
P7
|
12.5
|
|
8.
|
P8
|
3.12
|
|
9.
|
P9
|
25
|
|
10.
|
P10
|
100
|
|
11.
|
P11
|
3.125
|
|
12.
|
P12
|
3.125
|
|
13
|
Isoniazid
|
0.2
|
RESULT:
Chalcones were
prepared by treating substituted acetophenone with different aromatic aldehyde.
Further chalcones were reacted with phenyl hydrazine to yield pyrazoline (P1-12).
The assigned structure and molecular formula of the newly synthesized compounds
were confirmed and supported by spectra data and elemental analysis, which was
in full agreement with proposed structure. The compounds were screened in-vitro
for the antibacterial and antifungal activity by cup-plate method against
selected pathogen bacteria and human pathogen fungi. The compounds were also
screened in-vitro for the anti-tubercular activity.
DISCUSSION:
The structures of
the synthesized compounds (P1-12) were confirmed on the basis of
spectra data and elemental analysis. The IR spectrum of P1-12
exhibited a band due to 1578 cm-1 (C=N, pyrazoline ring) and 1H
NMR spectrum the appearance of a signal at 4.17-4.22 (1H of
methine), 2.40-2.56(2H, methylene) confirms the presence of the pyrazoline
ring.
The synthesized
compounds were screened in-vitro for anti-infective activity. The result
revealed that synthesized compounds possessed moderate antibacterial activity
and significant antifungal activity. The compound P7, P9, P10, and P12 showed
promising activity against Staphylococcus aureus where as other compounds
showed poor activity. Compound P7 and P9 showed promising activity against E.
Faecalis and other have shown low activity. Compound P4, P7, P9 and P10 have
shown significant activity against Escherichia coli and K. pneumonia and rest
compounds have shown moderate and low activity. The compounds P3, P7 and P9
have shown good activity against Albican Candida and compound P7 and P9 have
shown excellent activity against Aspergillus Niger where as rest of compounds
have shown moderate to low activity against these fungi.
The synthesized
compounds were screened in-vitro for anti-tubercular activity and compound P3
have shown excellent activity, compound P8, P11 and P12 have shown significant
activity as compared with standard and rest of them have shown moderste to low
activity. The data reported in this paper may be helpful guide for the
medicinal chemists who are working in this area.
ACKNOWLEDGEMENT:
The authors are
thankful to the principal, Maratha Mandal’s College of Pharmacy, Belgaum for
providing the facilities for this research work.
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