The melting points were taken in open capillary tube method. The IR spectra of the compounds were record on ABB Bomem FT-IR spectrometer MB 104 IR spectra recorded with potassium bromide pellets. The 1H-NMR spectra of synthesized compounds were recorded on instrument BRUKER NMR spectrometer in DMSO. The Mass spectra of synthesized compound were recorded JEOL GCmate. TLC method was used to check purity of compounds. TLC plates are Pre -coated Silica gel (HF254-200 mesh) aluminum plates using ethyl acetate: n-hexane was used as eluent and visualized under UV- chamber. The IR, 1H-NMR and MASS spectra were consistent with assigned structure of synthesized compounds.
4.2. SYNTHETIC SCHEME
4.3. SYNTHESIS STEPS OF TARGET COMPOUNDS
Step: 1
Step: 2
Step: 3
Title compounds (G1-G12)
Step: 1
Ethyl -4-acetamido phenoxy acetate61
A mixture of p-acetamido phenol (0.01 mol) and ethyl chloroacetate (0.01 mol) was refluxed by using dry acetone in presence of anhydrous potassium carbonate (K2CO3) for 6hrs. The reaction mixture was cooled and then poured in to crushed ice. The solid product obtained, these product was filtered, dried and recrystallized using ethanol.
Step: 2
4-Acetamido phenoxy acetyl hydrazide
The ethyl-4-acetamido phenoxy acetate (0.01 mol), hydrazine hydrate (0.01 mol) was refluxed in the presence of ethanol for 5 hrs. The reaction mixture was cooled and then poured in to crushed ice. The solid product obtained, these product was filtered, dried and recrystallized using ethanol.
Step: 3
2-(4-Acetamido phenoxy methyl) -5-aryl substituted - 1, 3, 4-oxadiazole62
A mixture of 4-Acetamido phenoxy acetyl hydrazide (0.01 mol) and various aromatic acids (0.01 mol) in phosphorus oxychloride (10 ml) was exposed to microwaves at 350 W. intermittently at 2min. intervals for 8-15 min. Completion of the reaction process, monitored by TLC plates. The contents were cooled and poured into the crushed ice. It was neutralized with sodium bicarbonate solution and the solid product obtained, these product was filtered, dried and recrystallized using ethanol.
4.3. PHYSICO-CHEMICAL PROPERTIES AND SPECTRAL DATA OF SYNTHESIZED COMPOUNDS
COMPOUND
G1
IUPAC NAME
4-((5-(3-aminophenyl)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide
Molecular formula
C17H16N4O3
Molecular weight
324.33
Melting point (0C)
1160C
TLC solvent ratio
Ethyl acetate:Hexane
2 : 3
Rf value
0.77
Solubility
Insoluble- Water
Slightly soluble- Chloroform, Ethanol
Freely soluble- DMF, DMSO
Yield (%)
54 %
IR (KBr) ν (cm-1)
3393.16 cm-1 (Ar-NH), 1633.67 cm-1 (C=N), 1575.88 cm-1 (C=C), 1069.05 cm-1 (-C-O-C-), 3132.54 cm-1 (Ar-CH)
1H-NMR δ (ppm)
6.45-7.4 (s, 8H, Ar-H), 5.17 (s, 2H,-CH2), 4.1(s, 2H, -NH2), 2.05 (s,1H, -CH3), 8.05(s, 1H, -NH).
Mass (m/e value)
% relative abundance
324.03 (M+) (5.1) , 310.87 (4) , 296.22 (8.25), 282.76 (2.2), 272.38(2.32), 262.6432 (7.3), 248.34 (11), 217.12 (15), 207.14 (7), 116.67 (18), 58.33(B)
Indian Institute of Technology, Chennai
File: G1 Date Run: 12-20-2010 (Time Run: 10:13:54)
Sample:
Instrument: JEOL GCmate
Inlet: Direct Probe Ionization mode: EI+
COMPOUND
G2
IUPAC NAME
4-((5-(2-aminophenyl)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide
Molecular formula
C17H16N4O3
Molecular weight
324.33
Melting point (0C)
1230C
TLC solvent ratio
Ethyl acetate: Hexane
2 : 3
Rf value
0.76
Solubility
Insoluble- Water
Slightly soluble- Chloroform, Ethanol
Freely soluble- DMF, DMSO
Yield (%)
56%
IR (KBr) ν (cm-1)
3381.92 cm-1 (Ar-NH), 1677.76 cm-1 (C=N), 1530.51 cm-1 (C=C), 1089.81 cm-1 (-C-O-C-), 3118.42 cm-1 (Ar-CH)
1H-NMR δ (ppm)
6.46-7.45 (s,8H, Ar-H), 5.21(s, 2H, -CH2), 4.05(s, 2H, -NH2),2.12 (s, 1H, -CH3), 7.95(s, 1H, -NH),
Mass (m/e value)
% relative abundance
324.57 (M) (6), 284.34 (12.3),262.64 (7.8), 284.34 (10.1), 216.62 (22.3), 174.45 (8), 156.98 (7), 80.10(B)
Indian Institute of Technology, Chennai
File: G2 Date Run: 12-20-2010 (Time Run: 10:20:34)
Sample:
Instrument: JEOL GCmate
Inlet: Direct Probe Ionization mode: EI+
COMPOUND
G3
IUPAC NAME
4-((5-(2-chloro-5-nitrophenyl)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide
Molecular formula
C17H13ClN4O5
Molecular weight
388.76
Melting point (0C)
1350C
TLC solvent ratio
Ethyl acetate: Hexane
3: 2
Rf value
0.64
Solubility
Insoluble- Water
Slightly soluble- Chloroform, Ethanol
Freely soluble- DMF, DMSO
Yield (%)
60%
IR (KBr) ν (cm-1)
3381.95 cm-1 (Ar-NH), 1684.44 cm-1 (C=N), 1586.2 cm-1 (C=C), 1064.25 cm-1 (-C-O-C-), 1365.57 cm-1(N=O), 801.35 cm-1 (C-Cl), 3130.43 cm-1 (Ar-CH)
1H-NMR δ (ppm)
6.74-8.36(m, 7H, Ar-CH) ,5.31(s, 2H, -CH2),2.31 (s, 1H, -CH3), 8.16(s, 1H, -NH),
Mass (m/e value)
% relative abundance
390.26 (M) (4), 388.71 (8.1), 362.27 (4.2), 233.28 (5), 217.31 (8.9), 182.52 (5), 96.79 (7), 78.82(B)
Indian Institute of Technology, Chennai
File: G3 Date Run: 12-20-2010 (Time Run: 10:26:25)
Sample:
Instrument: JEOL GCmate
Inlet: Direct Probe Ionization mode: EI+
COMPOUND
G4
IUPAC NAME
4-((5-(4-chloro-3-nitrophenyl)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide
Molecular formula
C17H13ClN4O5
Molecular weight
388.76
Melting point (0C)
2030C
TLC solvent ratio
Ethyl acetate:Hexane
1:1
Rf value
0.68
Solubility
Insoluble- Water
Slightly soluble- Chloroform, Ethanol
Freely soluble- DMF, DMSO
Yield (%)
62%
IR (KBr) ν (cm-1)
3382.02 cm-1 (Ar-NH), 1677.79 cm-1 (C=N), 1530.6 cm-1 (C=C), 1089.68 cm-1 (-C-O-C-), 1372.45 cm-1 (N=O), 813.038 cm-1 (C-Cl), 3117.5 cm-1 (Ar-CH)
1H-NMR δ (ppm)
6.83-8.42(m, 8H, Ar-CH), 5.35(s, 2H,-CH2), 2.07 (s, 1H,- CH3), 8.24(s, 1H, -NH),
Mass (m/e value)
% relative abundance
390.27(M+) (5), 388.76 (13), 380.25 (8), 261.63 (8), 182.52 (5), 167.62 (17), 156.56 (19), 81.97(B).
Indian Institute of Technology, Chennai
File: G4 Date Run: 12-20-2010 (Time Run: 10:32:48)
Sample:
Instrument: JEOL GCmate
Inlet: Direct Probe Ionization mode: EI+
COMPOUND
G5
IUPAC NAME
4-((5-(2-fluorophenyl)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide
Molecular formula
C17H14FN3O3
Molecular weight
327.31
Melting point (0C)
1870C
TLC solvent ratio
Ethyl acetate: Hexane
1:1
Rf value
0.75
Solubility
Insoluble- Water
Slightly soluble- Chloroform, Ethanol
Freely soluble- DMF, DMSO
Yield (%)
64%
IR (KBr) ν (cm-1)
3392.09 cm-1 (Ar-NH), 1617.53 cm-1 (C=N), 1528.16 cm-1 (C=C), 1093.52 cm-1 (-C-O-C-), 1371.78 cm-1 (C-F), 3114.61 cm-1 (Ar-CH)
1H-NMR δ (ppm)
2.21 (s, 1H, -CH3), 8.09(s, 1H, -NH), 5.21(s, 1H, -CH2), 6.7-8.01(m, 8H, Ar-CH).
Mass (m/e value)
% relative abundance
327.37 (M+) (6.3), 310.37 (2.3), 299.57 (3), 282.87 (3.9), 266.22 (5), 249.61 (1.2), 232.72 (4), 104.86 (8.1), 75.50 (B)
Indian Institute of Technology, Chennai
File: G5 Date Run: 12-20-2010 (Time Run: 10:40:51)
Sample:
Instrument: JEOL GCmate
Inlet: Direct Probe Ionization mode: EI+
COMPOUND
G6
IUPAC NAME
4-((5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide
Molecular formula
C18H17N3O4
Molecular weight
353.35
Melting point (0C)
1740C
TLC solvent ratio
Ethyl acetate:Hexane
3:4
Rf value
0.76
Solubility
Insoluble- Water
Slightly soluble- Chloroform, Ethanol
Freely soluble- DMF, DMSO
Yield (%)
56%
IR (KBr) ν (cm-1)
3457.55 cm-1 (Ar-NH), 1625.05 cm-1 (C=N), 1585.42 cm-1 (C=C), 1094.6 cm-1 (-C-O-C-), 3123.84 cm-1 (Ar-CH)
1H-NMR δ (ppm)
6.8-7.56(m, 8H, Ar-CH), 2.22 (s, 1H, -CH3),3.70 (s, 1H, , -CH3), 5.21(s, 2H, -CH2) ,8.14(s, 1H, NH)
Mass (m/e value)
% relative abundance
339.84 (M+) (6), 324.91 (2.2), 307.71 (3.9), 296.22 (1.5), 249.64 (16), 160.35 (17),132.55 (24), 116.67 (18), 87.74(B).
Indian Institute of Technology, Chennai
File: G6 Date Run: 12-20-2010 (Time Run: 10:46:27)
Sample:
Instrument: JEOL GCmate
Inlet: Direct Probe Ionization mode: EI+
COMPOUND
G7
IUPAC NAME
4-((5-(4-ethoxyphenyl)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide
Molecular formula
C19H19N3O4
Molecular weight
353.37
Melting point (0C)
1750C
TLC solvent ratio
Ethyl acetate:Hexane
1:1
Rf value
0.73
Solubility
Insoluble- Water
Slightly soluble- Chloroform, Ethanol
Freely soluble- DMF, DMSO
Yield (%)
64%
IR (KBr) ν (cm-1)
3430.18 cm-1 (Ar-NH), 1630.28 cm-1 (C=N), 1589.85 cm-1 (C=C), 1091.04 cm-1 (-C-O-C-), 3123.31 cm-1 (Ar-CH)
1H-NMR δ (ppm)
6.74-7.37(m, 8H, Ar-CH), 5.24(s, 2H, -CH2), 2.25 (s, 1H, -CH3), 8.07(s, 1H, -NH),1.31 (s,1H, -CH3),
Mass (m/e value)
% relative abundance
353.16 (M+) (4.9), 335.13 (3.9), 325.22 (2), 302.39 (2.7), 249.58 (6.8), 204.96 (4.9), 183.26 (5),160.32 (5.8), 115.64 (7.1), 91.31 (B)
Indian Institute of Technology, Chennai
File: G7 Date Run: 12-20-2010 (Time Run: 10:52:34)
Sample:
Instrument: JEOL GCmate
Inlet: Direct Probe Ionization mode: EI+
COMPOUND
G8
IUPAC NAME
4-((5-o-tolyl-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide
Molecular formula
C18H17N3O3
Molecular weight
323.35
Melting point (0C)
2110C
TLC solvent ratio
Ethyl acetate:Hexane
1:1
Rf value
0.65
Solubility
Insoluble- Water
Slightly soluble- Chloroform, Ethanol
Freely soluble- DMF, DMSO
Yield (%)
63%
IR (KBr) ν (cm-1)
3354.23 cm-1 (Ar-NH), 1632.99 cm-1 (C=N), 1584.68 cm-1 (C=C), 1092.53 cm-1 (-C-O-C-), 3123.84 cm-1 (Ar-CH)
1H-NMR δ (ppm)
7.41(m, 4H, Ar-CH), 2.31 (s, 1H, -CH3), 8.16(s, 1H, -NH), 5.31(s, 1H, -CH2),
Mass (m/e value)
% relative abundance
323.38 (M+) (8), 277.92 (7), 266.92 (6.2), 249.72 (1.1), 232.7228 (11.8), 158.60 (16.0), 140.88 (14), 132.84 (40), 80.40 (B)
Indian Institute of Technology, Chennai
File: G8 Date Run: 12-20-2010 (Time Run: 10:59:56)
Sample:
Instrument: JEOL GCmate
Inlet: Direct Probe Ionization mode: EI+
COMPOUND
G9
IUPAC NAME
4-((5-(3-nitrophenyl)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide
Molecular formula
C17H14N4O5
Molecular weight
354.32
Melting point (0C)
1690C
TLC solvent ratio
Ethyl acetate:Hexane
2:2
Rf value
0.72
Solubility
Insoluble- Water
Slightly soluble- Chloroform, Ethanol
Freely soluble- DMF, DMSO
Yield (%)
64%
IR (KBr) ν (cm-1)
3382.43 cm-1 (Ar-NH), 1703.01 cm-1 (C=N), 1592.32 cm-1 (C=C), 1088.54 cm-1 (-C-O-C-), 1378.11 cm-1 (N=O), 3112.69 cm-1 (Ar-CH)
1H-NMR δ (ppm)
6.41-7.8(m, 8H, Ar-CH), 2.42 (s, 3H, -CH3), 8.13(s, 1H, -NH), 5.21(s, 2H, CH2).
Mass (m/e value)
% relative abundance
354.16(M) (3.8), 335.16 (4.8), 302.39 (3.1), 287.43 (3.7), 249.58 (7.1), 226.00 (5.8), 204.96 (6.7), 127.56 (13.1), 103.69 (9), 89.93 (B).
Indian Institute of Technology, Chennai
File: G9 Date Run: 12-20-2010 (Time Run: 11:07:26)
Sample:
Instrument: JEOL GCmate
Inlet: Direct Probe Ionization mode: EI+
COMPOUND
G10
IUPAC NAME
4-((5-(2,6-difluorophenyl)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide
Molecular formula
C17H13F2N3O3
Molecular weight
345.3
Melting point (0C)
1800C
TLC solvent ratio
Ethyl acetate:Hexane
2:3
Rf value
0.74
Solubility
Insoluble- Water
Slightly soluble- Chloroform, Ethanol
Freely soluble- DMF, DMSO
Yield (%)
53%
IR (KBr) ν (cm-1)
3368.58 cm-1 (Ar-NH), 1673.42 cm-1 (C=N), 1545.03 cm-1 (C=C), 1085.04 cm-1 (-C-O-C-), 1378.04 cm-1 (C-F), 3115.62 cm-1 (Ar-CH)
1H-NMR δ (ppm)
6.6-7.82(s, 8H, Ar-CH), 2.5 (s, 3H, -CH3), 8.03(s, 1H, -NH), 5.22(s, 2H, -CH2),
Mass (m/e value)
% relative abundance
345.13 (M+) (2.8), 333.16 (1.5), 325.42 (2.7), 286.43 (2.6), 183.26 (6), 160.62 (7), 140.65 (16), 115.64 (33), 95.53 (B)
Indian Institute of Technology, Chennai
File: 10 Date Run: 12-20-2010 (Time Run: 11:16:13)
Sample:
Instrument: JEOL GCmate
Inlet: Direct Probe Ionization mode: EI+
COMPOUND
G11
IUPAC NAME
4-((5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide
Molecular formula
C17H15N3O4
Molecular weight
325.32
Melting point (0C)
2150C
TLC solvent ratio
Ethyl acetate:Hexane
2:3
Rf value
0.72
Solubility
Insoluble- Water
Slightly soluble- Chloroform, Ethanol
Freely soluble- DMF, DMSO
Yield (%)
68%
IR (KBr) ν (cm-1)
3382.83 cm-1 (Ar-NH), 1654.42 cm-1 (C=N), 1541.89. cm-1 (C=C), 1090.01 cm-1 (-C-O-C-), 3118.84 cm-1 (Ar-CH)
1H-NMR δ (ppm)
6.7-7.6(s, 6H, Ar-CH), 2.11 (s, H, -CH3), 8.00(s, 1H, -NH), 5.12(s, 1H, -CH2)
Mass (m/e value)
% relative abundance
325.42 (M) (11.1), 318.68 (16), 292.76 (7), 276.89 (20), 249.99 (8.2), 236.0277 (28.1), 203.2266 (76), 182.2587 (8), 134.4966 (32), 116.55 (B)
Indian Institute of Technology, Chennai
File: 11 Date Run: 11-20-2010 (Time Run: 11:20:34)
Sample:
Instrument: JEOL GCmate
Inlet: Direct Probe Ionization mode: EI+
COMPOUND
G12
IUPAC NAME
4-((5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide
Molecular formula
C16H14N4O3
Molecular weight
310.31
Melting point (0C)
2100C
TLC solvent ratio
Ethyl acetate : Hexane
2: 3
Rf value
0.71
Solubility
Insoluble- Water
Slightly soluble- Chloroform, Ethanol
Freely soluble- DMF, DMSO
Yield (%)
62%
IR (KBr) ν (cm-1)
3482.34 cm-1 (Ar-NH), 1654.26 cm-1 (C=N), 1528.16. cm-1 (C=C), 1093.52 cm-1 (-C-O-C-), 3138.86 cm-1 (Ar-CH)
1H-NMR δ (ppm)
7.31-7.82(s, 8H, Ar-CH), 2.1 (s, 1H, -CH3), 8.13(s, 1H, -NH), 5.2(s, 1H, -CH2)
Mass (m/e value)
% relative abundance
310.36 (M+) (2.8), 289.96 (0.9), 248.58 (1.5), 231.61 (0.8), 157.07 (37), 131.35 (62.1), 115.46 (3.8), 103.53 (62.2), 96.13 (B)
Indian Institute of Technology, Chennai
File: 12 Date Run: 12-20-2010 (Time Run: 11:27:44)
Sample:
Instrument: JEOL GCmate
Inlet: Direct Probe Ionization mode: EI+
CHAPTER-5
PHARMACOLOGICAL EVALUATION
Material and Method
The Various animals and materials used for present study and there resources as follows,
Wister rats
1% Carboxy Methyl Cellulose (Rolax Laboratory)
Pentazocin (Abbott)
Diclofenac sodium ( Rolax Laboratory)
Water for injection (Global Pharma)
Carrageenan ( Sigma )
Synthesized compounds (G1- G12)
Plethysmograph (Indigenous)
The animals used in present study such as Wister rats weighing 150-200 gm were kept in colony cages at 28  20C, relative humidity of 50-55% under 12hrs light and dark cycle. All animals were feed with standard animal feed, water and labium. The test compounds were administered orally, in the form of suspension using 1%CMC as suspending agent. The experimental dose was selected between minimum effective dose and maximum non lethal non lethal dose. All animal experimentation was performed according to protocols and recommendation of animal ethics committee.
IAEC Reference No: IAEC/XXXI/09/CLBMCP dated 22/09/2010.
ANOVA followed by dunnet's test was performed to ascertain the significance of exhibited analgesic and anti-inflammatory activities of synthesized compounds.
Following activities were carried out to the synthesized compounds by bellow mentioned methods.
Acute oral toxicity (acute toxic class method in mice)
Analgesic activity (Tail immersion Method)
Anti-inflammatory activity ( carrageenan induced acute paw edema in rats)
5.1 EVALUATION OF ACUTE ORAL TOXICITY
Introduction
Acute oral toxicity defines to those adverse effects occurring following oral administration of a single dose of a substances or multiple dose given within 24 h. The various methods used to evaluate the acute oral toxicity as follows.
Fixed dose procedure(OECD guideline-420)
Acute toxic class method(OECD guideline -423)
Up and down procedure (OECD guideline -425)
OECD guideline -42363
a) OECD Guidelines for the Testing of Chemicals are periodically reviewed in the light of scientific progress or changing assessment practices. The original Guideline 423 was adopted in March 1996 as the second alternative to the conventional acute toxicity test, described in Test Guideline 401.
i) International agreement has been reached on harmonized LD50 cut-off values for the classification of chemical substances, which differ from the cut-offs recommended in the 1996 version of the Guideline. ii) Testing in one sex (usually females) is now considered sufficient.
b) The acute toxic class method set out in this Guideline is a stepwise procedure with the use of 3 animals of a single sex per step. Depending on the mortality and/or the moribund status of the animals, on average 2-4 steps may be necessary to allow judgment on the acute toxicity of the test substance. This procedure is reproducible, uses very few animals and is able to rank substances in a similar manner to the other acute toxicity testing methods. (Test Guidelines 420 and 425). The acute toxic class method is based on biometric evaluations with fixed doses, adequately separated to enable a substance to be ranked for classification purposes and hazard assessment. The method as adopted in 1996 was extensively validated in vivo against LD50 data obtained from the literature, both nationally and internationally.
c) Guidance on the selection of the most appropriate test method for a given purpose can be found in the Guidance Document on Acute Oral Toxicity Testing.
This Guidance Document also contains additional information on the conduct and interpretation of Test Guideline 423.
Experimental Protocol (acute toxic class method in rats)
In the present study acute oral toxicity of the synthesized compounds were performed by acute toxic class method. In this method the toxicity of synthesized compounds were tested using a step wise procedure, each step using three mice of single sex (female). The mice were fasted prior to dosing (food but water should be with held) for three to four hours. Following the period of fasting the animal should be weighted and synthesized compound were administered orally at a dose 2000 mgkg body weight. Animals were observed individually after dosing at least once during the first 30min;periodically during the first 24 h with special attention giving during the first 4 h and daily thereafter, for total of 14 days. As know mortality observed with the above dose. Test compound dose reduced by specific intervals. The mortality not observed at the dose 1000 mgKg. So a series of doses 100 and 200 mgKg body weight were selected for their pharmacological evaluation. The test procedure with the starting dose of 2000 mgKg body weight as per OECD-423 guidelines was shown as follows.
Fig.1
Flow chart for acute toxic class method (OECD guideline 423) starting dose of 2000 mg Kg body weight p.o
Table: 5.1
Dose Selection by Acute toxicity class method (OECD) guide lines 423 of EECS
Sr. No.
Treatment group
Dose mg/kg
Sign of toxicity
Onset of toxicity
Duration
1
G1
200
400
No
No
14 days
2
G2
200
400
No
No
14 days
3
G3
200
400
No
No
14 days
4
G4
200
400
No
No
14 days
5
G5
200
400
No
No
14 days
6
G6
200
400
No
No
14 days
7
G7
200
400
No
No
14 days
8
G8
200
400
No
No
14 days
9
G9
200
400
No
No
14 days
10
G10
200
400
No
No
14 days
11
G11
200
400
No
No
14 days
12
G12
200
400
No
No
14 days
Results and discussion
Acute oral toxicity studies were performed according to the OECD guideline 423 method.
This method has been designed to evaluate the substance at the fixed doses and provide information both for hazard assessment and substance to be ranked for hazard classification purposes.
The synthesized compounds were administered initially at a dose of 2000mg/kg b.w and 1% CMC (p.o) and observed 14 days mortality due to acute toxicity.
Careful observation were made at least thrice a day for the effect on CNS, ANS, motor activity, salivation and other general signs of toxicity were also observed and recorded.
Since no sign of toxicity observed at 2000 mg/kg b.w. to the group of animals, the LD50 value of the title compounds (G1-G12) expected to exceed 2000 mg/kg b. w. and represented as class 5 (2000 mg/kg < LD50 < 2500 mg/kg)
From the toxicity studies the data revealed that all the synthesized compounds proved to be non toxic at tested dose levels and well tolerated by the experimental animals as there LD50 cut of values > 2000 mg/kg b. w.
5.2 EVALUATION OF ANTI-INFLAMMATORY ACTIVITY
Introduction
Anti-inflammatory agents are substance which modifies the inflammatory reaction. The main anti-inflammatory agents are the glucocorticoids and the non steroidal anti-inflammatory drugs (NSAIDS)
Evaluation method
The three important aspects of inflammation that render themselves readily to measurement are erythema, edema and formation of granulation tissue. Compounds claimed to posses anti-inflammatory activity can be evaluated either by their ability to reduce one one or more of these phenomena in experimentally induced inflammation or by testing their anti-inflammatory activity in experimental arthritis produced in animals. The commonly employed methods are,
Erythematic assay
Granuloma assay
Edema assay
Experimental arthritis assay
Miscellaneous
Erythema assay
In this method irradiation of shaven back skin of guinea pig with UV light cause erythema which can be reduced by anti-inflammatory agents.
Granuloma assay
There are two types of granuloma assays such as cotton wool pellet and granuloma pouch method.
Edema assay
The edema can be produced in experimental animals by the local injection of substance like formaldehyde, carrageenan, histamine, dextrin ovalbumin
Experimental arthritis assay
Poly arthritis induced in rats by injection of dead tubercle bacilli suspended in liquid paraffin is frequently used method kaolin; talc and even mercury have also been injected directly into joints of rats and pigeons to induce arthritis.
Miscellaneous
Localized inflammatory reaction can be produced in rats by
Intra pleural injection of turpentine
Intra peritoneal injection of formaldehyde
Experimental Procedure:-
Carrageenan Induced Paw Oedema Method in Rats64
Anti-inflammatory activity was performed by carrageenan induced paw oedema method in rats. Diclofenac sodium (20 mg/kg i.p) was administered as standard drug for comparison. The synthesized compounds were administered at two dose level (200 and 400 mg/kg).orally 30 minutes prior to the administration of 0.1ml/kg body weight of carrageenan used in saline (1%w/v) into the lateral malleolus of sub-planter region of the rats left hind paw. The paw volumes were measured using the mercury displacement technique with the help of a plethysmonograph immediately before and 30 minutes, 1, 2 and 3 hour after carrageenan injection. The percentage inhibition of paw oedema was calculated by using the following formula.
Percentage protection = [(control-test)/control]Ã-100
Table 5.2.1
Anti Inflammatory Activity of the Synthesized Compounds (200 mg/kg)
Compounds
30 min
60 min
120 min
180 min
MEAN +SEM
%
MEAN +SEM
%
MEAN +SEM
%
MEAN +SEM
%
G1
0.70± 0.13
9.09
0.77±0.02NS
18.94
0.96±0.09
35.57
0.85±0.11
23.42
G2
0.65±0.05*
15.68
0.68±0.04
28.43
0.82±0.11**
44.96
0.79±0.13*
28.82
G3
0.62 ± 0.06
19.48
0.74±0.05
22.10
0.88±0.32
40.93
0.62±0.23*
44.14
G4
0.58± 0.04
24.67
0.64±0.11*
32.63
0.76±0.14**
50.33
0.63±0.12
43.24
G5
0.70 ±0.08
9.09
0.78±0.07
17.89
0.95±0.34
36.24
0.89±0.04
19.81
G6
0.70± 0.05
9.09
0.77±0.05
18.94
0.80±0.13*
46.30
0.64±0.06*
42.34
G7
0.56± 0.09*
27.27
0.68±0.06*
28.42
0.77±0.09*
48.32
0.65±0.11*
41.44
G8
0.75± 0.05
2.59
0.82±0.12
13.68
0.92±0.11
38.25
0.76±0.21
29.72
G9
0.59±0.11*
23.37
0.78±0.03
17.89
0.90±0.05
39.59
0.68±0.09
38.73
G10
0.74± 0.09
3.89
0.85±0.07
10.5
0.98±0.14
34.22
0.79±0.01
28.82
G11
0.64± 0.05
16.88
0.69±0.7*
27.36
0.84±0.21*
43.62
0.74±0.15
33.33
G12
0.63± 0.05
18.18
0.71±0.05
25.26
0.81±0.04NS
45.63
0.75±0.11
32.43
Diclofenac Sodium
0.56±0.09**
0.62±0.13**
0.70±0.17**
0.58±0.05**
Significant differences with respect to control was evaluated by (ANOVA), Dunnet's t test * P<0.05, **P<0.01, NS (Non significant), % (Percentage reduction of edema).
Table 5.2.2
Anti Inflammatory Activity of the Synthesized Compounds (400 mg/kg)
Compounds
30 min
60 min
120 min
180 min
MEAN +SEM
%
MEAN +SEM
%
MEAN +SEM
%
MEAN +SEM
%
G1
0.587± 0.12
40.40
0.411±0.02NS
39.45
0.586±0.11
35.60
0.451±0.09
28.66
G2
0.553±0.04*
43.85
0.407±0.04
40.14
0.566±0.13*
37.80
0.442±0.11**
29.13
G3
0.502 ± 0.06
49.03
0.355±0.05
47.79
0.502±0.23*
44.33
0.412±0.32
33.33
G4
0.501± 0.04
49.13
0.341±0.11*
49.85
0.498±0.12
45.27
0.404±0.14**
34.30
G5
0.530 ±0.08
46.19
0.385±0.07
43.38
0.530±0.04
41.09
0.428±0.34
30.40
G6
0.549± 0.05
44.26
0.392±0.05
42.35
0.593±0.06*
37.83
0.435±0.13*
29.26
G7
0.592± 0.04*
39.89
0.426±0.06*
37.35
0.608±0.11*
33.18
0.455±0.09*
26.01
G8
0.666± 0.05
32.38
0.444±0.12
34.70
0.643±0.21
31.53
0.478±0.11
22.27
G9
0.696±0.15*
29.34
0.457±0.03
32.79
0.629±0.09
30.87
0.473±0.05
23.08
G10
0.525± 0.09
46.70
0.514±0.07
45.00
0.526±0.01
42.19
0.422±0.14
31.38
G11
0.493± 0.05
49.74
0.529±0.7*
51.61
0.492±0.15
45.73
0.402±0.21*
34.63
G12
0.489± 0.1
50.35
0.504±0.05
55.29
0.469±0.11
48.46
0.392±0.04NS
36.37
Diclofenac Sodium
0.498±0.09**
0.319±0.13**
0.480±0.05**
0.397±0.17**
Significant differences with respect to control was evaluated by (ANOVA), Dunnet's t test * P<0.05, **P<0.01, NS (Non significant), % (Percentage reduction of edema).
RESULTS AND DISCUSSION
Anti-inflammatory activity of the synthesized compounds was evaluated by carrageenan induced paw edema method. The activity was studied at 200 and 400 mg/kg b.w. p.o and their effects were measured at 30, 60, 120 and 180 min.
From the data shown in tables, the following observations we made:
Mild to good anti-inflammatory activity was observed for all the synthesized compounds. Graded dose response was also observed.
All compounds exhibited highest activity at 120 min.
When compared with standard drug diclofenac sodium (20 mg/kg i.p) G1, G2, G7 and G8 exhibited equipotent anti-inflammatory activity.
Compounds G2, G4, G7 and G9 exhibited moderate anti-inflammatory activity.
These compound shows anti-inflammatory activity because these compounds having NH2, NO2, Cl groups.
5.3 EVALUATION OF ANALGESIC ACTIVITY
Introduction
Definition:
"Analgesics are agents which relieve or decrease pain sensation by increasing threshold to painful stimuli without causing loss of consciousness".
Pain means of many diseases requiring treatment with analgesics. The addiction liability of opioids leads to intensive research for compounds without side effect.
Many approaches have been used to differentiate the various actions of strong analgesics by developing animal models not only for analgesic activity. Analgesic effects in animals are comparable with the therapeutic effects in man. Painful stimuli can consist of direct stimulation of the efferent sensory nerves or stimulation of pain receptors by various means such as heat or pressure. The role of endogenous peptides such as endorphins gives more insight into brain processes and the action of central analgesics.
Mechanism of action
Cellular action-
Action site is protein coupled Opioid receptors. The inhibition of adenocyclase.
Reducing the intracellular cAMP content affecting protein phosphorylation Pathways.
Exert effects on ion channels through a direct G-protein coupling to the channel. Promote the opening of potassium channels and inhibit the opening of voltage-gated calcium channels.
These membrane effects reduce both neuronal excitability (because of the increasing K+ conductance causes hyper polarization of the membrane) and transmitter release (due to inhibition of the Ca2+ entry).
The overall effect is therefore inhibitory at the cellular level. Increase activity in some neuronal pathways by suppressing the firing of inhibitory interneuron.
The other analgesic mechanism produces by effect on nociceptive pathway.
Evaluation methods
The various methods used to evaluate anticonvulsant activity are as follows,
Hoppners tail clip method
Hot plate method
Radiant heat method
Tail immersion method
Grid shock test
Electric stimulation method
Formalin test in rats
Experimental protocol (Tail immersion method) 65
The analgesic activity was determined by popular method like tail-immersion method. Wister rat (n=6) of either sex selected by random sampling technique was used for the study. Pentazocine at the dose of 20 mg/kg (i.p.) was administered as standard drug for comparison. The test compounds at 2 dose levels (200 and 400 mg/kg) were administered orally. The animals were held in position by a suitable restrainer with the tail extending out and the tail (up to 6 cm) was taken dipped in a beaker of water maintained at 56 ± 40C. The time in sec taken to withdraw the tail clearly out of water was taken as the reaction time. The first reading (0 min) was taken immediately after the administration of the test compound and subsequent reaction time was recorded at 0, 15, 30, 60 and120 min after the administration of compounds. A cut off point of 15 sec was observed to prevent the tail damage. The percentage analgesic activity was calculated using the following formula and the results are presented in table 5.3.1.
PAA = [(B-A)/B] X 100
Where,
B is the reaction time (in sec) before treatment.
A is the reaction time (in sec) after treatment.
PAA is the percentage analgesic activity.
Table 5.3.1 ANALGESIC ACTIVITY OF THE SYNTHESIZED COMPOUNDS
COMP.
DOSE
(mg/k)
0 min
15min
30 min
60 min
120 min
MEAN + SEM
MEAN + SEM
%
MEAN + SEM
%
MEAN + SEM
%
MEAN + SEM
%
G1
200
5.25±0.44
11.08±0.33*
48.5
12.58±0.37
62.6
10.10±0.40*
53.0
9.340.24
48.48
G2
200
5.00±0.50
11.91±0.20**
44.6
14.16±0.30**
57.9
10.08±0.35*
53.1
11.570.27
83.94
G3
200
4.00±0.28
10.66±0.49
50.4
13.16±0.60
60.8
10.33±0.24
51.9
10.350.23*
64.54
G4
200
4.83±0.24
9.00±0.36*
58.1
14.00±0.36**
58.3
10.58±0.27**
50.8
11.770.21*
87.12
G5
200
3.66±o.21
9.83±0.30*
54.3
12.33±0.21**
63.3
9.83±0.30*
54.3
10.120.27*
60.89
G6
200
4.16±0.42
10.75±0.44*
50.0
13.41±0.45*
60.1
10.66±0.33*
50.4
11.740.19*
86.64
G7
200
4.75±0.31
11.25±0.31
47.7
13.66±0.71NS
59.4
11.33±0.33
47.3
9.350.25
48.64
G8
200
4.25±0.35
11.58±0.23*
46.1
13.33±0.33*
60.3
11.00±0.25*
48.8
11.590.27
84.26
G9
200
4.75±0.38
9.16±0.30
57.4
12.91±0.15
61.6
10.91±0.37
49.3
11.210.26**
78.21
G10
200
4.83±0.47
11.16±0.42*
48.1
13.58±0.21**
59.6
10.71±0.45*
50.2
11.830.27**
88.07
G11
200
3.58±0.23
10.00±0.51*
53.3
12.83±0.30*
61.8
10.41±0.35*
53.6
8.820.26
40.22
G12
200
4.58±0.37
9.50±0.76NS
55.8
13.25±0.47*
60.6
10.83±0.30*
49.6
11.230.25
78.53
Control
-
5.33±0.21
12.16±0.30**
63.3
14.50±0.36**
73.2
11.83±0.30**
56.3
6.290.51
-
Standard
Pentazocin
20
5.25±0.44
11.08±0.33*
48.5
12.58±0.37
62.6
10.10±0.40*
53.0
7.410.32
17.80
RESULTS AND DISCUSSION
Synthesized compounds were evaluated for analgesic activity by tail-immersion method. The activity was studied at 100 and 200 mg/kg b.w. (p.o) and effect was measured at the time interval of 15, 30, 60 and 120 min.
Most of the synthesized compounds showed significant analgesic activity and also the graded dose response was observed.
Highest analgesic activity was observed at 60 min for all the compounds (100 and 200 mg/kg).
When compared with standard drug (Pentazocin 20 mg/kg, i.p) the compounds G6, G10, G2 and G4 exhibited comparable analgesic activity at 100 and 200 mg/kg b.w.
Compounds G1, G5 and G3 exhibited moderate analgesic activity.
Among the compounds synthesized compound G11 exhibited lowest analgesic activity.
The order of analgesic activity of the synthesized compounds was shown
G10>G4>G6>G8>G2>G12>G9>G3>G5>G1>G7>G11
Compounds G10 exhibited a statistically significant analgesic activity in the tail immersion method test in the mice, being however less active than the equitoxic dose of pentazocin (20 mg/kg).
It appears reasonable to suggest that the presence of o-hydroxy, methoxy substituted on particularly 2, 5 position of 1, 3, 4-oxadiazole exhibited better activity.
