INTRODUCTION:

Anti-inflammatory agents are compounds that reduce the pain and swelling associated with inflammation¹.

Inflammation is a complex phenomenon involving interrelationship of humoral and cellular reactions through a number of inflammatory mediators; it is a response of the body to injuries such as a blow or a burn. The swelling of the affected region of the body occurs because fluid is directed to that region. The inflammatory response can aid the healing process in conditions such as rheumatoid arthritis. The intake of anti-inflammatory agents can ease the discomfort of arthritis, and other conditions such as asthma.

There are two groups of anti-inflammatory agents, the corticosteroids and the nonsteroidal anti-inflammatory drugs (NSAIDs).

All NSAIDs act by inhibition of the fatty-acid COX enzyme, and thus inhibition of production of prostaglandins and thromboxanes. COX-1 is a constitutive enzyme and COX-2 is induced in inflammatory cells. It is believed that anti-inflammatory action of NSAIDs is related to inhibition of COX-2 and their unwanted effect such as gastric irritation is due to inhibition of COX-1. NSAIDs also exert their antipyretic action through inhibition of prostaglandin production in hypothalamus².

MATERIALS AND METHODS:

Materials:

The chemical used in the present project work were of AR grade and LR grade, purchased from Loba Chemie, Qualigens, Sigma, Ranchem and Merck India. The IR spectra of the synthesize compounds were recorded on a Fourier Transform IR spectrometer (model Shimadzu 8400s) in the range of 400-4000 using KBR pellets and the values of Vmax are reported in cm^-1.¹H-NMR spectra were recorded in Brookfield 200 MHz-NMR spectrometer (Astra Zeneca Pharma India Ltd) using CDCl₃ and chemical shift (s) are reported in parts per million downfield from internal reference Tetramethylsilane (TMS).

Methods:

Step I: preparation of aromatic esters³

In a round bottom flask 30g (0.246mol) of aromatic acid, 145ml (115g, 2.5mol) of absolute ethanol and 5g (2.7ml) of conc. Sulphuric acid was taken.The mixture was refluxed for 4 hours. Distilled off excess of alcohol on a water bath and allowed to cool. Poured it slowly and with stirring on to 200g of crushed ice. Added sufficient ammonia solution to render the resulting solution strongly alkaline. Extract the mixture with five 25 ml portion of ether, dry the combined ethereal extracts over MgSO₄, removed the ether and distilled the residue under pressure.

Step II: preparation of Hydrazides⁴:

A mixture of 0.1 mol of ester and 0.2 mol of hydrazine hydrate were refluxed in 50ml of 95% ethanol for 5 hours. The resultant mixture was concentrated, cooled and poured onto crushed ice. The solid mass thus separated out was filtered, dried and purified by recrystallization from ethanol.

Step III: Preparation of 1, 3, 4-oxadiazole derivatives⁵:

Potassium Hydroxide (0.01 mol) was dissolved in 100ml of alcohol (ethanol) and to this 20 ml carbon disulphide was added in a drop wise fashion for 30 minutes. Then hydrazide (0.01 mol) was added and stirred for 2 hours. After stirring the mixture was refluxed for 4 hours. Later solvent was removed by distillation and the residues was dissolved in crushed ice and acidified with dilute HCl. The solid separated was collected by filtration and dried and recrystallized by ethanol.

IR (KBr) cm^-1: 3147 (NH), 1494 (C=N), 1344 (C-N), 1178 (C-O-C), 1060 (C=S), 759 (Ar, C=C).¹H-NMR, ppm: 7.26-7.58 (m, aromatic), 7.96 (1H, s, SH).

Step IV: Acetylation of oxadiazole⁶:

A mixture of oxadiazole derivatives (0.005mol) and acetic anhydride (10ml) and pyridine (5ml) was refluxed for 4 hours. The excess acetic anhydride was distilled off and residue was poured into ice-cold water, solid was filtered and recrystallised from ethanol - DMF.

IR (KBr) cm^-1: 2946 (C-H), 1755 (C=O), 1612 (C=N), 1182 (C-O-C), 1064 (C=S), 763 (Ar, C=C).

¹H-NMR, ppm: 7.2-7.8, (m, aromatic), 2.7 (s, 3H, CH₃).

Step 3a: Preparation of 2-phenyl 1, 3, 4-oxadiazole derivatives from hydrazide:

A mixture of 0.01 mol of hydrazide and 0.01mol (1.22g) of benzoic acid was dissolved in phosphorous oxychloride and refluxed for about 14 hours. The reaction mixture was slowly poured in crushed ice and kept overnight. The solid mass thus separated out was filtered, dried and purified by recrystallization from ethanol.

IR(KBr) cm^-1: 1479 (C=N), 1273 (C-O-C), 700 (Ar, C=C).¹H-NMR, ppm: 7.2-7.9 (m, aromatic).

Table 1: List of compounds synthesized

SLN.	Structure and Mol. Formula	IUPAC Name	Mol. Wt.	% yield	M.P. (⁰C)
1.	C₁₀H₈O₂N₂S (1R)	S-[5-phenyl-1,3,4-oxadiazol-2-yl] ethanethioate	220	72.72	43-46
2.	C₁₀H₇O₄N₃S (2R)	S-[5-(4'-nitrophenyl)-1,3,4-oxadiazol-2-yl] ethanethioate	265	36.11	48-51
3.	C₁₀H₇O₂N₂SBr (3R)	S-[5-(4'-bromo phenyl)-1,3,4-oxadiazol-2-yl] ethanethioate	289	68.34	120-22
4.	C₁₀H₈O₃N₂S (4R)	S-[5-(4'-hydroxy phenyl)-1,3,4-oxadiazol-2-yl] ethanethioate	236	44.91	52-55
5.	C₁₀H₉O₂N₂S (5R)	S-[5-(4'-amino phenyl)-1,3,4-oxadiazol-2-yl] ethanethioate	235	57.26	90-93
6.	C₁₀H₈O₃N₂S (6R)	S-[5-(2'-hydroxy phenyl)-1,3,4-oxadiazol-2-yl] ethanethioate	236	94.01	67-70
7.	C₁₄H₁₀N₂O (7R)	2,5-diphenyl-1,3,4-oxadiazole	222	75.90	85-90
8.	C₁₄H₉N₃O₃ (8R)	5-(4'-nitrophenyl)-2-phenyl-1,3,4-oxadiazole	267	67.41	110-13
9.	C₁₄H₉N₂OBr (9R)	5-(4'-bromophenyl)-2-phenyl-1,3,4-oxadiazole	291	76.63	67-69
10	C₁₄H₁₀N₂O₂ (10R)	5-(4'-hydroxyphenyl)-2-phenyl-1,3,4-oxadiazole	238	46.21	78-81
11	C₁₄H₁₁N₃O (11R)	5-(4'-aminophenyl)-2-phenyl-1,3,4-oxadiazole	237	80.16	42-44
12	C₁₄H₁₀N₂O₂(12R)	5-(2'-hydroxyphenyl)-2-phenyl-1,3,4-oxadiazole	238	91.59	38-40

PHARMACOLOGICAL STUDIES:

Acute toxicity studies (lD₅₀):

The acute toxicity of acetylated Oxadiazole derivatives was determined by using Albino swiss mice (23-25gm). The animals were fasted for 24 hrs prior to the experiment and up and down procedure (OECD guideline no. 425) method of CPCSEA was adopted for acute toxicity studies. A maximum dose up to 700 mg/kg has been tested for any mortality. From that one dose is selected, 70 mg/kg which is 1/10^th of the LD₅₀ value.

Anti Inflammatory studies^{7, 8}:

The anti-inflammatory activity of the newly synthesized oxadiazole derivatives were carried out using

Carrageenan induced rat hind paw edema method.

Principle:

Inflammation is a tissue-reaction to infection, irritation or foreign substance. It is a part of the host defence mechanism. The inflammatory reaction is readily produced in rats in the form of paw edema with the help of irritants. Substances such as carrageenan, formalin, bradykinin, histamine, mustard when injected to the dorsum of the foot of rats they produce acute paw edema within a few min of injection.Carrageenan is a sulphated polysaccharide obtained from sea weed (Rodophyceae) and by causing the release of histamine, 5-HT, Bradykinin and prostaglandin it produces inflammation and edema.

Procedure:

The adults Wistar albino rats of either sex weighing 200-250 g were selected and were assigned into fourteen groups of 6 animals each. They were marked with picric acid for individual animal identification. The animals were deprived of food overnight. (Water ab libitium) and the synthetic compound were administered once before the injection of carrageenan. First twelve groups received the synthetic compound (1-12) suspended in 0.5% w/v carboxy methyl cellulose sodium salt and administered at dose level of 70 mg/kg in a volume not exceeding 0.5ml/100gms orally. The positive control (50mg/kg) was also administered in a similar manner. The fourteenth group served as solvent control. After 30 min. of test compound administration, 0.1 ml of 1%w/v of carrageenan in normal saline was injected into the sub plantar region of the left hind paw of the rat. Immediately after the carrageenan injection, the volume of its displacement was measured using plethysmometer. The reading was recorded at the end of 180 min. The % inhibition of edema was calculated by using the formula:

% inhibition = 100 x (1-Vt/Vc)

Vt/Vc = edema volume in the rat treated with test drug and control respectively.

Table no.3 Effect of 1, 3, 4-oxadiazole derivatives on carrageenan induced hind paw edema in rats:

Compound

Dose

(mg/kg)

Increased paw volume

% decrease in paw volume after 180 min.

After 60 min.

After 120 min.

After 180 min.

Control

Diclofenac Sod.

Saline sol

0.37±0.02

0.10±0.04***

0.10±0.003***

0.09±0.003***

0.05±0.02***

0.08±0.005***

0.12±0.003***

0.01***

0.09±0.003***

0.20±0.01**

0.15±0.02***

0.16±0.02***

0.22±0.008**

0.01±0.003***

0.39±0.008

0.10±0.04***

0.23±0.003**

0.20±0.003***

0.07±0.05***

0.10±0.005***

0.19±0.005***

0.07±0.006***

0.10±0.003***

0.25±0.003^ns

0.19±0.02***

0.20±0.03***

0.23±0.008**

0.15±0.029***

0.45±0.03

0.12±0.03***

0.26±0.03**

0.10±0.05***

0.20±0.003***

0.20±0.005***

0.10±0.003***

0.12±0.005***

0.32±0.01^ns

0.22±0.02***

0.24±0.03**

0.42±0.01^ns

0.20±0.05***

73.33

42.22

66.67

55.6

55.55

77.77

73.33

28.88

46.7

46.66

55.55

Where, * represents mild significant at P<0.05,

** represents moderate significant at P<0.01,

*** represents highly significant at P<0.001,

ns represents non significant at P>0.05 Vs control.

Values are expressed as Mean ± SEM, [number of animal (n) = 6]. Values were analysed one way ANOVA followed by Tukey-Kramer’s test.

RESULTS AND DISCUSSION:

The titled compounds were synthesized according to the procedures as given in the methodology. The reactions were monitored by TLC. The physical constants like melting point and solubility were determined for all the intermediate and final products. The compounds were further characterized by IR and ¹HNMR. All the titled compounds were evaluated for their antiinflammatory activities. The test compounds were screened for anti-inflammatory activity by carrageenan induced rat hind paw edema method using Diclofenac sodium as standard.

As per results obtained from animal studies it can be concluded that among the 12 synthesized compounds, 4'-Bomo phenyl S-acetylated oxadiazole, 4'-Hydroxy derivative, 4'-Amino derivatives, 2'-Hydroxy derivatives, 2,5-Diphenyl oxadiazole, 4'-Bromo phenyl-2-phenyl oxadiazole, 2'-Hydroxyphenyl-2-phenyl derivatives were found to show significant anti-inflammatory activity at the end of 180 min. (P<0.001) comparable with the standard Diclofenac sodium. The derivatives Phenyl-S-acetylated oxadiazole, 4'-nitro derivative, 4'-Hydroxyphenyl-2-phenyl oxadiazole derivative exhibited moderate anti-inflammatory activity at the end of 180 min. (P<0.01). However, derivative 4'-nitro phenyl-2-phenyl oxadiazole and 4'-amino phenyl-2-phenyl derivative did not show any anti-inflammatory activity (P>0.05).

CONCLUSION:

The main focus of this work was to synthesize, purify, characterize and evaluate anti-inflammatory activities of novel 1, 3, 4-Oxadiazole derivatives. A series of titled compounds, i.e. [1-12] have been synthesized using appropriate synthetic procedure, as per the scheme given in the methodology. The yields of the synthesized compounds were found to be in the range from 36%-94%. Structures of synthesized compounds were characterized and confirmed with the help of analytical data such as IR and ¹HNMR. Anti-inflammatory activity were carried out using carrageenan induced rat hind paw edema method Among the synthesized compounds screened for anti-inflammatory activity 4'-Bomo phenyl S-acetylated oxadiazole, 4'-Hydroxy derivative, 4'-Amino derivatives, 2'-Hydroxy derivatives, 2,5-Diphenyl oxadiazole, 4'-Bromo phenyl-2-phenyl oxadiazole, 2'-Hydroxy phenyl-2-phenyl derivatives were found to show significant anti-inflammatory activity at the end of 180 min. (P<0.001) comparable with the standard Diclofenac sodium. Hence it is concluded that newly synthesized 1, 3, 4-Oxadiazle derivatives do possess considerable anti-inflammatory activity and further lead optimization could be carried out for the better-expected Anti-inflammatory activity.

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Received on 08.01.2019 Modified on 10.02.2019

Research J. Pharm. and Tech. 2019; 12(5):2416-2420.

DOI: 10.5958/0974-360X.2019.00405.0