Synthesis,
Characterization, Anti-tubercular, Analgesic and Anti-Inflammatory Activities
of New 2- Pyrazoline Derivatives
Bindu Sree Koduru*,
Akshay R. Shinde, P. Jaya Preeti, K. Pavan Kumar, R. Rajavel, T. Sivakumar
Department
of Chemistry, Nandha College of Pharmacy and Research
Institute, Erode-638052, Tamil Nadu, India.
*Corresponding
Author E-mail: binduchowdarykoduru@gmail.com
ABSTRACT:
The
compounds of different Pyrazoline derivatives were
synthesized by cyclization of substituted chalcone derivatives in presence of hydrazine hydrate
(a-e). All the synthesized compounds were characterized by spectral analysis
(IR, MS and NMR). The REMA (Resazurin Microtiter Assay) plate
method was used for anti-tuberculosis activity. The minimum inhibitory
concentration (MIC) of the newly synthesized compound was tested against Mycobacterium
tuberculosis H37RV. The Analgesic activity was
performed by Acetic acid induced method. The Anti-inflammatory activity was
performed by Carrageenan induced paw edema method.
KEYWORDS: Chalcone,
Hydrazine Hydrate, Pyrazoline derivatives, Characterization,
Anti-tuberculosis activity.
INTRODUCTION:
Pyrazoline are compounds with noteworthy applications.
Pyrazolines are well known and important nitrogen
containing five membered heterocyclic compounds.
Numerous pyrazoline derivatives have been found to
possess considerable biological activities, which stimulated the research
activity in this field. Pyrazoline and its
derivatives are known to be biologically active compounds and substituted pyrazoles have shown wide range of biological activities
like antioxidant, anti bacterial, antitubercular,
antifungal, antiinflammatory, analgesic, antiartherosclerosis, antidepressant, anticonvulsant,
antitumor, HMG-CoA reductase
inhibition. Pyrazoline derivatives have been
extensively studied during past several years because of their ready
accessibility through synthesis, diverse chemical reactivity and broad spectrum
of biological activities. In the view of these and our continuing interest in
the synthesis of biologically active compounds. We undertook the synthesis of
the title compounds and studied their antitubercular
activity, Analgesic and Anti-inflammatory.
MATERIALS AND
METHODS:
Materials:
The melting
points were taken in open capillary tube and are uncorrected. The IR spectra of
the compounds were recorded on Fourier Transform Infrared Spectrum (Vmax in cm-1) with KBr
pellets. The1H (400 MHz) spectra were recorded on a Bruker
AV III 500MHz FT-NMR Spectrometer (with TMS as internal references). Mass
Spectra were recorded in JOEL GCMAT-II GC-MS Spectrometer. The purity of the
compounds was checked by Thin Layer Chromatography was checked by using silica
gel-G coated plates using chloroform:methanol: water
(6:2:2) and visualized in UV chamber. IR, 1H-NMR, 13C-NMR,
Mass spectroscopy and were consistent with the assigned structures.
Synthetic methods:
Nicotinamide (0.1 M, 12.2 g) was refluxed with (0.1 M,
3.2 g) hydrazine hydrate in presence of 40ml of methanol at 100°-110 °C for six
hours. After the reaction mixture was cooled, filtered, and the separated
product was purified by recrystallization from
ethanol to get a β-picolinoyl hydrazine. The
β-picolinoyl hydrazine (0.1 M, 13.6 g) was
dissolved in 5 ml of water and 5 ml of HCl. After it
was cooled to 0-5 oC in ice salt bath,
cold aq. solution of 6.9 ml sodium nitrite in 8 ml of water was added drop wise
to the above solution, diazonium Salt so formed was
filtered into cold mixture of 13.5 ml of ethyl aceto
acetate and 4 g of Sodium acetate in 25 ml of ethanol. The resulting solid was
filtered and washed with water and it was recrystallized
from ethanol to get a 2-(β-picolinoylaminoazo)-ethyl
aceto acetate. The 2-(β-picolinoylaminoazo)-ethyl
aceto acetate (0.1 M, 27.8 g) was added in different
aromatic aldehydes (a-e) in 20 ml of ethanol
and add 4% sodium hydroxide Solution. The mixture was stirred for 24 hrs at
room tempt. The contents were poured on crushed ice and neutralized with 10% Hcl. The product was filtered, dried and recrystallized from ethanol to get a 1-(β-picolinoylaminoazo)-3-benzylidene propan-2-one (3a-3e). The
mixture of 1-(β-picolinoylaminoazo)-3-benzylidene
propan-2-one (0.01 M, 13.4 g) and sodium hydroxide (0.025 M, 1 g) in 50 ml of
ethanol, thiosemicarbazide (0.12 M, 10 g) was added.
The mixture was refluxed for 8hrs. The products were poured into crushed ice
and the solid mass which separated out was filtered, dried and recrystallized from ethanol to get substituted derivatives
of pyrazole (4a-4e).
Scheme –
Figure-1: Scheme of pyrazoline derivatives
Anti-tubercular activity:
REMA(Resazurin microtiter
assay) plate method:
The Minimum
Inhibitory Concentration (MIC) of the newly synthesized compound was tested
against Mycobacterium tuberculosis H37RV. The MIC was determined by the micro
broth dilution method using Middlebrook 7H9
broth. Serial dilutions such as 1.25 µg,
2.5 µg, 3.75 µg, 5 µg, 6.25 µg, 7.5 µg, 8.75 µg and 10 µg were prepared in
DMSO. The M. tuberculosis strain were
inoculated in nutrient broth and incubated at 37 ºC for overnight. After overnight incubation, the bacterial
culture was swapped in solidified Middlebrook agar
plate and made wells of 6mm diameter were punched in the plate. In each well
the above test solution was added and 100 µml of DMSO added in centre well. The plates were incubated at 37 ºC for 24
hour. After incubation, the diameter of zone of inhibition around the wells
were measured and recorded.
Analgesic activity:
Acetic acid induced writhing method:
The activity was performed in vivo in Albino
mice (25-30 gm) of either sex. Synthesized compounds (20 mg/kg) were administered
orally. The Acetic acid solution (0.1 ml/10 gm) was administered intraperitonially, 30 min after administration of
synthesized compounds. The number of writhing per animal was recorded.
Analgesic activity was expressed as percentage of inhibition of number of writhings. The diclofenac Sodium
(20 mg/kg) used as standard.
Results of
percentage of Analgesic activity of synthesized compounds was calculated using
following formula,
No. of writhing for control – No. of
writhing for test compounds
%Analgesic
Activity= -------------------------------------------------- ×100
No. of
writhing for control
Anti-inflammatory activity:
Carrageenan-Induced Paw
Edema method:
The activity was
performed in male rats (120-150 gm). The Synthesized compounds (30 mg/kg) were
administered. The acute inflammation was produced by sub-planner administration
of 0.1 ml of 1 % Carrageenan in hind paw of the rats.
The thickness of paw was measured at regular time interval after Carrageenan injection. The indomethacin
(100 mg/kg) used as an standard.
The percentage
inhibition of edema was calculated by following formula,
Where,
Vt = mean relative change in paw volume in
test group.
Vc= mean relative change in paw volume in
control group.
RESULTS AND
DISCUSSION:
Chemistry:
[β-picolinoylaminoazomethyl-5-(4-methoxy
phenyl)]-1-thioamide-2-pyrazoline derivative (4a):
IR 1533.13(N=N), 1317.14(C-O), 3262.97(-NH), 1619.91(C=N), 1163.83(C=S),
3367.1(-NH); 1533 (C=N), 1644.98 (C=O), 1001.84 (C-N) cm-1; 1H-NMR
(DMSO): 6.91-9.11 (8H,m,ar,py.), 3.89
(2H,s,Pyrazoline), 2.88-3.54 (2H,dd,pyr.), 3.74 (3H,s,-OCH3), 8.50
(3H,s,pyr.), 5.62-5.64 (1H,d,pyr.); EI MS (m/z): 435.43(M+), 109.13,
133.12, 155.01, 169.02, 225.20, 279.10, 323.13, 338.11.
Table 1: MIC
determination of synthesized compounds against Mycobacterium tuberculosis H37RV.
|
Comp.
|
Conc. of Test Sample/ Minimum Inhibitory
Concentration (MIC) Colour Change
|
|
1.25 µg/ml
|
2.5
µg/ml
|
3.75
µg/ml
|
5.0
µg/ml
|
6.25
µg/ml
|
7.5
µg/ml
|
8.75
µg/ml
|
10.0
µg/ml
|
|
4a
|
-----
|
Blue Colour to Pink
|
-----
|
-----
|
-----
|
-----
|
-----
|
-----
|
|
4b
|
-----
|
-----
|
Blue Colour to Pink
|
-----
|
-----
|
-----
|
-----
|
-----
|
|
4c
|
Blue Colour to Pink
|
-----
|
-----
|
-----
|
-----
|
-----
|
-----
|
-----
|
|
4d
|
-----
|
-----
|
-----
|
-----
|
Blue Colour to Pink
|
-----
|
-----
|
-----
|
|
4e
|
-----
|
Blue Colour to Pink
|
-----
|
-----
|
-----
|
-----
|
-----
|
-----
|
[β-picolinoylaminoazomethyl-5-(2-hydroxy
phenyl)]-1-thioamide-2-pyrazoline derivative (4b):
IR 1533.13(N=N), 1317.14(C-O), 3263.93(-NH), 1163.83 (C=S), 3370.96(OH),
1533 (C=N), 1644.98 (C=O), 999.91 (C-N) cm-1; 1H-NMR
(DMSO): 6.83-8.71
(8H,m,ar,py.), 3.89 (2H,s,Pyrazoline), 2.90-3.56 (2H,m,pyr.), 5.87 (1H,d,pyr.),
8.03 (4H,s,-NH); EI MS (m/z): 421.21 (M+), 122.32, 133.07, 155.01,
169.02, 211.08, 263.07, 304.25, 323.13, 387.13.
[β-picolinoyl amino azo
methyl-5-(phenyl)]-1-thioamide-2-pyrazoline derivative (4c):
IR 1533.13(N=N), 1321.65(C-O), 3263.93(-NH), 1155.76 (C=S), 1533(C=N),
1635.02 (C=O), 1003.41 (C-N) cm-1; 1H-NMR (DMSO): 7.15-9.10(8H,m,ar,py.), 3.89
(2H,s,Pyrazoline), 2.85-3.58 (2H,m,pyr.), 5.60-5.66 (1H,t,pyr.), 2.85-3.58
(2H,m,-NH); EI MS (m/z): 390.13 (M+), 119.10, 133.11, 155.01,
258.011, 274.08, 291.09, 304.34, 323.13.
[β-picolinoyl
aminoazomethyl-5-(4-methyl-5-amino phenyl)]-1-thioamide-2-pyrazoline derivative
(4d):
IR 1509.13(N=N), 1314.28(C-O), 1067.28 (C=S), 1533 (C=N), 3647.70 (-NH2),
1644.98 (C=O), 1941.24 (-CH3), 1067.28 (C-N) cm-1; 1H-NMR
(DMSO): 6.88-9.11(8H,m,ar,py.),
3.89 (2H,s,Pyrazoline), 2.84-3.57 (2H,m,pyr.), 5.69 (1H,d,pyr.), 7.65
(5H,s,-NH); EI MS: 413.43 (M+), 119.10, 133.12, 195.053, 241.21,
291.023, 342.11, 405.99, 433.023.
Table 2: Anti
tubercular activity of synthesized compounds (4a-4e) against Mycobacterium tuberculosis H37RV.
|
Compound
|
MIC
Concentration (µg/ml)
|
|
4a
|
2.5
|
|
4b
|
3.75
|
|
4c
|
1.25
|
|
4d
|
6.25
|
|
4e
|
2.5
|
[β-picolinoylaminoazomethyl-5-(4-hydroxy-3-methoxy
phenyl)]-1-thioamide-2-pyrazoline derivative (4e):
IR 1533.13(N=N), 1318.11(C-O), 3262.97(-NH), 1164.79(C=S), 1533.13
(C=N), 1644.02 (C=O), 997.98(C-N), 3371.92 (-OH) cm-1; 1H-NMR
(DMSO): 6.48-9.11(8H,m,ar,py.),
3.89 (2H,s,Pyrazoline), 2.68-3.51 (2H,m,pyr.), 5.60 (1H,d,pyr.), 7.91
(4H,s,-NH); EI MS: 437.21 (M+), 119.10, 133.12, 155.01, 279.10,
282.28, 304.34, 323.13, 409.04, 437.21.
Figure-2:
Analgesic effect of synthesized compounds on Acetic acid induced
writhing response in mice (% Analgesic activity).
BIOLOGICAL RESULTS:
Anti-tubercular activity:
From Table no. 1
and Table no. 2 the Anti-tubercular activity of synthesized compounds (4a-4e)
was performed by REMA plate method. All the compounds tested illustrated
significant antitubercular. The compounds 4c and 4d
shows good respond against Mycobacterium
tuberculosis.
Analgesic activity:
From Table no. 3
and Figure no. 2 all the Synthesized compounds (4a-4e) showed a significant
decrease in number of writhings. The compounds 4b, 4d
and 4e were found to bring about decrease in number of writhings
comparable to that of standard, (Diclofenac sodium).
Anti-inflammatory activity:
From Table no. 4
all the Synthesized compounds (4a-4e) showed a significant decrease in
inflammation. Moreover, the compounds 4b and 4c were found to also bring about
a decrease in inflammation comparable to that of standard (Indomethacin).
Table 3:
Acetic acid induced writhing response in mice for (4a-4e) for Analgesic activity.
|
Sr. No
|
Derivative
|
Dosage
|
Number of writhings in 20 minutes
(mean ± S.E.M)
|
% Analgesic*
activity
|
|
1
|
Control
|
Vehicle
|
66.5 ± 5.98
|
0
|
|
2
|
Diclofenac
|
20 mg/kg
|
4.66 ± 2.06
|
92.99***
|
|
3
|
4a
|
20 mg/kg
|
22.33 ± 4.30
|
66.32*
|
|
4
|
4b
|
20 mg/kg
|
8.5 ± 1.88
|
87.21*
|
|
5
|
4c
|
20 mg/kg
|
22.66 ± 7.77
|
66.42**
|
|
6
|
4d
|
20 mg/kg
|
10.5 ± 2.57
|
84.21*
|
|
7
|
4e
|
20 mg/kg
|
10.16 ± 1.01
|
84.72**
|
Table 4:
Carrageen induced inflammation in rat model for (4a-4e) for Anti-inflammatory
activity.
|
Sr. No
|
Derivative
|
Dosage
|
% Decrease in inflammation
(mean)
|
|
1
|
Control
|
Vehicle
|
-
|
|
2
|
Indomethacin
|
2 mg/kg
|
66.12***
|
|
3
|
4a
|
20 mg/kg
|
35.1**
|
|
4
|
4b
|
20 mg/kg
|
52**
|
|
5
|
4c
|
20 mg/kg
|
56.32*
|
|
6
|
4d
|
20 mg/kg
|
39*
|
|
7
|
4e
|
20 mg/kg
|
34.5**
|
CONCLUSION:
The present study
was carried out to synthesize and to screen the newly developed compounds for
its pharmacological activity. In this present work an attempt has been made to
synthesize some new 3-(β-Picolinoylaminoazomethyl-5-aromatic
substituted)-1-thioamide-2-pyrazoline derivatives and to study their
anti-tubercular, analgesic and anti-inflammatory activities. Both analytical
and spectral data (IR, MS, 1HNMR) of all the synthesized compounds
were in full agreement with the synthesied structure.
All the synthesized compounds were screened for different biological activities
such as antitubercular activity by REMA plate method,
analgesic activity by acetic acid induced writhing in mice and
anti-inflammatory activity by using carrageenan
induced paw edema in rats.
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