SYNTHESIS, CHARACTERIZATION AND ANTIINFLAMMATORY ACTIVITY (in vitro) OF NEW OXADIAZOLE INCORPORATED WITH IMIDAZOLE AND PYRAZOLE

About Authors:
KALPESH PATEL, JAYACHANDRAN E.*, SREENIVASA G.M.
* P.G. Department of Pharmaceutical Chemistry,
S.C.S. College of Pharmacy, Harapanahalli-583131,
Karanataka, India.
*drjc_2006@rediffmail.com, kalpesh.m.pharm@gmail.com

Abstract
Various substituted 3-amino-1-(2,4-dinitro phenyl)-5-[(5-substituted-1,3,4-oxadiazol-2-yl)amino]-1-H-pyrazole-4-carboxyamide and (5E)-5-[4-(dimethylamino) benzylidene]-3-(5-substituted-1,3,4-oxadiazol-2-yl)-2-phenyl-3,5-di hydro-4H-imidazol-4-one have been synthesised and evaluated for antiinflammatory activity. Structure of these products has been established by IR, ¹H NMR data. Significant activity were observed for some members of the series.

Reference Id: PHARMATUTOR-ART-1276

INTRODUCTION
Oxadiazole are cyclic compounds containing one oxygen and two nitrogen atom in a five membered ring. The sequence of these atoms may be different as 1,2,4-oxadiazole(a), 1,2,5-oxadiazole(b), 1,2,3-oxadiazole(c) and 1,3,4-oxadiazole(d).

1,3,4-oxadiazoles have been known for about eighty years and investigations in field have been intensified due to the large number of uses and applications in most diverse area. Moreover, ring cleavage reactions of 1,3,4-oxadiazoles have also accelerated interest in various field like medicinal chemistry, polymer industry etc.
The oxadiazole drugs were the first effective chemotherapeutic agents to be employed systematically for the prevention and cure of bacterial infection in human beings.Oxadiazole derivatives are well known to have number of biological activities like Analgesic¹,  Anthelmintic¹, antimicrobial¹, Antiinflammatory^2-5, Antitubercular⁶, Anticonvulsant⁷, Diuretic, CNS depressant, Antiviral, Herbicidal⁸, Insecticide⁸, Anticancer⁹, Antipyretic, Antimitotic, Antiemetic activities.

Imidazole was Planar, Five membered heteroaromatic molecule having two nitrogen. Named first as gluoxaline (first synthesis with glyoxal and ammonia). Amphoteric nature, susceptible to electrophilic and nucleophilic attack. High stability to thermal, acid, base, oxidation and reduction conditions. Extensive intramolecular hydrogen bonding. The reduction products, named  as are other rings of five atoms, are imidazoline and imidazolidine  and their derivatives structure given below,

Imidazole exhibit diverse biological activities like Antimicrobial¹⁰, Analgesic¹¹, Antiinflammatory^12-14, Anthelmintic, Anti-HIV, Treatment of hypoxic tumour cells, CNS depressants¹⁵, Antiviral, Antitubercular¹⁶,Anticancer¹⁷, Antihistamine.
Pyrazole is the name given by “LUDWIG KNORR” to this class of compounds in 1883.  The simple doubly unsaturated compound containing two nitrogen and three carbon atoms in the ring, with the nitrogen atoms neighboring, is known as pyrazole.  The reduction products, named as are other rings of five atoms, are pyrazoline and pyrazolidine.

Several pyrazoline substitution products are used in medicine as Anti-inflammatory^18-19 Analgesic, Antioxidant²¹, Antipyretic, Diuretic, Bovine anaplasmosis, Treatment of rheumatic disorders, Antimicrobial²⁰, Antidepressant, Anticonvulsant, Anticancer²⁰, Antiparasitic activity, Antimalerial, Antimycotics, Antidiabetic.

MATERIAL AND METHODS
Bovine serum albumin (Loba Chem) diclofenac sodium (standard) and all other chemicals were of analytical grade.
Melting point was determined by open capillary tube method and are uncorrected. T.L.C. was run on silica gel G plates using ethyl acetate:pet.ether (10:1) as developing solvent for the purity of the compounds. I.R. Spectra were recorded on SHIMADZU FTIR-8400S spectrophotometer by using KBr pellets technique.

SCHEME -I
1^st Step :
Synthesis of 2-(Substituted methylidene) hydrazine carboxamide (aldehyde semicarbazone) :
Take 0.1 mol of  semicarbazide HCl and 0.1mol of different aromatic aldehydes, sodium acetate as catalyst  and stirr for 3 hrs with glass rod and added alcohol then  heated on hot plate to concentrate it . Add little alcohol if  mixture become solid then heat on a hot plate until dissolve with clicking sound.  Kept it over night. Then  added alcohol (50ml) & heated on a hot plate, cool at room temperature. Add cold water  and filter the product under the vacuum & dry.

2^nd Step :
General synthesis of NaOI solution:
NaOI solution prepared by dissolving 8.6 g Sodium Carbonate , 17 g  Iodine and 15 g potassium Iodide in Distilled Water.  

Synthesis of 5-substituted-1,3,4-oxadiazol-2-amine :
Into aqueous suspension of 4.2 g of  2-(Substituted methylidene) hydrazine carboxamide (aldehyde semicarbazone) add 100 ml of  NaOI solution . The combination mixture was heated at 80-85 ⁰c with continuous stirring for 3 hours. Allow stand and  cool   at room temperature. The precipitated 5-substituted-1,3,4-oxadiazol-2-amine was collected by suction filter. Washed with cold water and dried. The product was recrystalised from ethanol.

3^rd Step :
General synthesis of bis-s-methyl ethylene cyanoacetamide :

Synthesis of cyanoacetamide :
In a 1 litre glass stopped conical flask 50ml of concentrated ammonia was taken, 60ml of ethylcyanoacetate (1.77m) is added drop by drop in ice cold condition. After the complete addition the solution is allowed to stand for 2hrs. in ice cold condition. During this period a white needle shaped crystals were precipitated and are filtered, washed with cold solution of ethanol.

bis-s-methyl ethylene cyanoacetamide :
To a solution of 13.2g of KOH in a minimum quantity of water, cooled in ice bath, added 30ml dimethyl formamide (DMF) and 0.1mol of cyanoacetamide (8.4g). The mixture was treated drop wise with cooling and stirring, 0.1mol of carbondisulphide was added drop wise below 0⁰C and stirred for an hour, then the reaction mixture was allowed to stand for one hour at room temperature then 20ml of dimethylsulphate was added drop by drop with stirring at 10 to 15⁰C. The mixture was allowed to stand for 12hrs. at room temperature and was poured on to ice water. The solid obtained was filtered and washed with water to get bis-s-methyl ethylene cyanoacetamide.

Synthesis of (2Z)-2-cyano-3-(methylsulfanyl)-3-[(5-substituted-1,3,4-oxadiazol-2-yl) amino]prop-2-enamide :
A mixture of 5-substituted-1,3,4-oxadiazol-2-amine (0.1 mol)and bis-s-methyl ethylene cyanoacetamide (0.1 mol) were refluxed in ethanol for 2 hrs. and later excess of ethanol was distilled off and poured onto crushed ice. The product obtained was filtered and recrystallised from ethanol.

4^th Step :
Synthesis of 3-amino-1-(2,4-dinitro phenyl)-5-[(5-substituted-1,3,4-oxadiazol-2yl) amino]-1-H-pyrazole-4-carboxyamide :
A mixture of   (2Z)-2-cyano-3-(methylsulfanyl)-3-[(5-substituted-1,3,4-oxadiazol-2-yl) amino]prop-2-enamide  (0.1 mol) and 2,4-dinitro phenyl hydrazine HCl (0.1 mol) in equimolar concentration wererefluxed in ethanol for 2 hrs. and later excess of ethanol was distilled off and poured onto crushed ice. The product obtained was filtered and recrystallised from mixture of ethanol and DMF.

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SCHEME –II

3^rd Step :
Preparation of oxazolone : (2-phenyl-4-p-dimethylaminobenzylidenyl-5-oxazolone)
A mixture of 37.3 g (26ml, 0.25 mol) of N N`-dimethyl aminobenzaldehyde, 45g (0.25mol) of benzoyl glycine (hippuric acid), 77g (71.5ml, 0.75 mol) of dry acetic acid and 20.5g (0.25mol) of anhydrous sodium acetate was placed in 500ml conical flask and heated on electric hot plate with constant shaking. As soon as the mixture has liquefied completely, the flask was transferred to water bath and refluxed for 2 hrs. 100ml of ethanol was added slowly to the contents of the flask. The mixture was allowed to stand overnight. The crystalline product obtained was filtered by suction. It was washed with two 25ml portions of boiling water and recrystallisation from benzene dried at 100^oC. the dry material melted at 167-168^oC.

4^th Step :
Synthesis of (5Z)-5-[4-(dimethylamino)benzylidene]-3-(5-substituted -1,3,4-oxadiazol -2-yl)-2-phenyl-3,5-dihydro-4H-imidazol-4-one :
5-substituted-1,3,4-oxadiazol-2-amine (0.01 mol) and 4-[4-(dimethylamino) benzylidene]-2-phenyl-1,3-oxazol-5(4H)-one (oxazolone) (0.01 mol) was refluxed in pyridine for 6-8 hrs. The excess of pyridine was distilled off and resulting mass was poured into crushed ice. Filter and recrystalised using mixture of ethanol and DMF.

The denaturation of proteins as one of the causes of inflammation is well documented^22-24. Production of auto-antigens in certain rheumatic diseases may be due to in vivo denaturation of proteins. A number of antiinflammatory drugs are known to inhibit the denaturation of proteins. Muzushima²⁵ and other have employed protein denaturation as an in vitro screening model for antiinflammatory compounds. Bovine serum albumin (Loba Chem) diclofinac sodium (standard) and all other chemicals of analytical grade.

Inhibition of albumin denaturation was studied according to Muzushima and Kabayashi with significant modification. The test compounds were dissolved in minimum amount of dimethyl formamide (DMF) and diluted with phosphate buffer (0.2 M Ph 4 - 7.4). Final concentration of  DMF in all solutions was less than 2.5%. The test solution (1 ml) containing different concentrations of drug was mixed with 1mM albumin (1 ml) in phosphate buffer and incubated at 27⁰±1⁰C in incubator for 15 min.  Denaturation was induced by keeping the reaction mixture at 60⁰±1⁰C in water bath for 10 min.  After cooling the turbidity was measured at 660 nm.  Percentage of inhibition of denaturation was calculated from control where no drug was added.  Each experiment was done in triplicate and average was taken²⁶( Table no. 4).  

RESULTS AND DISCUSSION
Anti-inflammatory activity (in-vitro):
Synthesis and pharmacological screening of 3-amino-1-(2,4-dinitro phenyl)-5-[(5-substituted-1,3,4-oxadiazol-2yl)amino]-1-H-pyrazole-4-carboxyamide and (5E)-5-[4-(dimethylamino)benzylidene]-3-(5-substituted-1,3,4-oxadiazol-2-yl)-2-phenyl-3,5-di hydro-4H-imidazol-4-one were tested for the antiinflammatory activity by using inhibition of albumin denaturation technique compared to standard diclofinac sodium  significant antiinflammatory activity.

Among compounds tested P₁ (50%), P₂ (46.87%), P₄ (69.79%), P₆ (54.86%), P₈ (46.87%),  I₁ (63.10%), I₄ (69.82%), I₈ (50.01%) showed significant antiinflammatory activity compare to standard Diclofenac (95.52%).The tested compounds P_1, P_2, P_4, P_6, P_8, I_1, I_4, I₈ showed significant antiinflammatory activity (in vitro) at low and high concentration compared to standard.

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TABLE NO. 1

ANALYTICAL DATA

Characteristics IR absorption bands of different synthesised compounds are tabulated below:

TABLE NO. 2

TABLE NO. 3

NMR Spectral Data

Table No. 4

Anti-inflammatory activity

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