INTRODUCTION:

Heterocyclic compounds are widely distributed in nature and are essential for life activities of plants and animals. They play a vital role in the metabolism of all living cells, for example the vitamins and co-enzyme precursors thiamine, riboflavin etc. Some of these are natural products for example antibiotics such as penicillin and cephalosporin etc. However, the large majority of them are synthetic heterocycle, which find their widespread use as anticancer agents, analeptics, analgesics, hypnotics, pesticides, insecticides, weedicides and rodenticides.

There are large numbers of synthetic heterocycles having practical applications as dyestuffs, co-polymers, solvents, photographic sensitizers and developers, antioxidants and vulcanization accelerators in the rubber

industry and most of them are used as valuable intermediates in synthesis. Such large number of heterocyclic compounds are known and this number is increasing very rapidly. Nearly six million compounds are recorded in chemical abstract and approximately half of these are heterocycles^7,8

The five-membered heterocycles containing two nitrogen atoms have two isomers,

1) Pyrazole(1,2-diazole)

2) Imidazole(1,3-diazole)

(1) (2)

Figure 1. Pyrazole

In 1883 Knorr et. al1 gave the generic name ‘pyrazole’ to above class of compounds, which is a five membered unsaturated ring containing compounds with two adjacent nitrogen³

Pyrazolone:

There are three possible heteropyrazolines [3], [4], [5] in which cabonyl is adjacent to nitrogen.

Figure 2. Pyrazolone

The carbonyl at position five leads to 5-hydroxyl pyrazoles, since the 5-hydroxy compounds exihibits pronounced enol characters. Toutomerid forms shown below for the 1-phenylderivatives are the fundamental structures involved in the pyrazolone⁹

Derivatives of Pyrazole:

Most of the drugs in this chemical category are structurally related to the aromatic compound pyrazole, which are shown below. If one of the double bonds in pyrazole is saturated then that results pyrazoline, however, if both are saturated, then compound is known as pyrazolidine^3,9.

Pyrazole pyrazoline pyrazolidine

Figure 3 .Derivatives of Pyrazole:

Chalcone:

Chalcone is an aromatic ketone and an enone that forms the central core for a variety of important biological compounds, which are known collectively as chalcones or chalconoids. Alternative names for chalcone include benzylideneacetophenone, phenyl styryl ketone, benzalacetophenone, β- phenylacrylophenone, γ-oxo-α,γ-diphenyl- α-propylene, and α-phenyl-β- benzoylethylene⁸.

Properties:

1. Chemical formula-C₁₅H₁₂O

2. Molar mass- 208.260g·mol⁻¹

3. Density- 1.071g/cm³

4. Melting point- 55 to 57°

5. Boiling point 345 to348

Structure:

Figure 4. Chalcone

Chemicals:

1. Acetophenone

2. 4-methylactophenone

3. Phenylhydrazine

4. Dimethulformide

5. Glacial acetic acid

6. Silicagel

7. Potassiumcarbonate

METHOD OF PREPARATION:

Step -1:

Actic acid 1ml And phenyl hydrazine hydrochloride 20mmol were added to solution of substituted acetophenone in 90ml of ethanol. Then the reaction mixture was refluxed for 1hrs the precipitation was filtered and washed with ethanol. after drying in vaccum product was obtained²

Step-2:

Vilsmler-hack reagent was prepared from previously from seperately cooled Dimethylformide (2.8g, 35, 3mmol) and POCL₃. A solution (3g) in DMF 3ml was added dropwise to the Vilsmer hack reagent was the warmed at room temp and refluxed for 4-5 hrs .After cooling at room temp the mixture was basified with a cool saturated k₂co₃ The precipited was filtred, strongly washed with water and recrysallised fromEthanol¹

Step-3:

0.01mole of ethanolic solution of acetophenone and 0.01 mole of 1,3-diphenyzoles-4-carboxaaldehyde were mixed was kept overnight at room temp. The content was poured over Crushed ice and acidified with Dil.hydrochrolic Acid. The solid was dried and recrystallised with ethanol¹

Dilution Of Compound:

All the synthesized compounds i.e. dissolved in dimethylsulfoxide (DMSO) so concentration of 400mg/ml of DMSO. Amoxicillin and Clotrimazole were dissolved in dimethylsulfoxide (DMSO) so as to get concentration of DMSO and used as standard drug for antibacterial and antifungal screening separately dissolved 10mg/ml of DMSO and use respectively⁴

Sterilization of equipments and chemicals:

MacConkey agar (M081), Nutrient Agar medium (Nutrient broth N011) normal saline solution were sterilized in autoclave at 15 lbs pre sure (120°C) for 15mins.

Preparation of MacConkey agarslant:

206mg of MacConkey agar was dissolved in 4ml of distilled water, boiled and then poured it in the test tube and the test tube was plugged with cotton and then sterilized in autoclave at 15 lbs pressure (121°C) for 15 min. After the sterilization the tubes containing the MacConkey agar were kept in inclined position for 2 hrs. Then on the solid surface of these slants the pure culture of the test bacteria i.e. Klebsiella penumoniae and Escherichia coli were streaked in aseptic condition and then incubated 37°C for 24hrs.All the synthesized compounds i.e. dissolved in dimethylsulfoxide (DMSO) so concentration of 400ng/ml of DMSO. Amoxicillin, Clotrimazole were dissolved in dimethylsulfoxide (DMSO) so as to get concentration of of DMSO and used as standard drug for antibacterial and antifungal screening separately dissolved 10mg/ml of DMSO and userespectively.⁵

Preparation of Nutrient Agar mediumslant:

112mg of Nutrient Agar medium and 100mg of agar agar powder was dissolved in 4ml of distilled water, boiled and then poured it in the test tube and the test dissolved in was plugged with cotton and then sterilized in autoclave at 15 lbs pressure (121°C) 5 min. After sterilization the tube containing soybean casein digest medium were for 15mins. Aft in inclined position for 30mins. Then on solid surface of these slants pure culture kept in inclined fungi i.e. Bacillus Subtilis, , and of test bacteria E.coli were streaked in aseptic condition and then incubated at 37°C for 24hrs.⁵

Preparation of suspension of test bacteria and test fungi (standardized inoculum):

By using the 24hrs old growth of the test bacteria/fungi from the slant, suspension of the bacteria/fungi were made separately in sterile normal saline solution (0.85% NaCl in distilled water) in aseptic condition, to get moderate turbidity. The turbidity of each suspension was compared and adjusted with the turbidity of the solution resulting by mixing 0.5ml of 1.175% of barium chloride and 99.5ml of 0.36N H₂SO₄ acid.⁶

Antimicrobial Sensitivity test:

Antimicrobial sensitivity test have been carried out by using disc- diffusion method, performed in nutrient agar for bacterial and saboraud's agar for fungi.

Preparation of culture media for antibacterial sensitivity test:

Soybean casein digest agar was prepared by weighing 3gms of soybean casein digest medium and 2.5gms of agar agar powder in 100ml of distilled water. Then it was sterilized in autoclave at 15 lbs pressure (121°C) for 15mins. After sterilization the media was cooled up to 45°C and then it was poured in sterile Petri plates in aseptic condition. Approximately 20-25ml of media was poured in each plate. Then the media from the plate was allowed to getsolidified.⁴

Inoculation of suspension of bacteria and fungi on culture media:

Sterile, non-toxic cotton swab were dipped in to the standardized inoculum (turbidity as adjusted as to obtained confluent growth on the Petri plate) and then the entire agar surface of the plate was streaked with the swab three times, turning the plate at 60° angle between streaking. Then the streaked inoculum was allowed to dry for 5- 15mins with lid in place. Sterile paper disc made by punching Whatman (No.41) paper were dipped separately in to the solutions containing synthesized drug (500µg/ml of DMSO) and standard drug amoxicillin (10mg/ml of DMSO.) and Clotrimazole (10mg/ml of DMSO) in aseptic condition with help of sterile forcep and were then placed on the surface of inoculated culture media after which the plates were kept in refrigeration for 30 min. For the diffusion of drug from the paper disc in to the culture media. After 30mins. The plates were incubated at 37ºC^4,5

OBSERVATION TABLE:

(Observation was recorded by measuring the zone of inhibition in mm)

AntibacterialActivity:

Chart 1 : Std Amoxicilline(10mg/1ml)

Name of organism

Control

DMSO

Std

Bacillus substilis

+++

Highly Active = +++ (inhibition zone >18mm) Moderately Active= ++(Inhibition zone 12 -18mm), Slightly Active = +(inhibition zone 6-12mm} Inactive= - (Inhibition Zone <6mm)

Antifungal Activity:

Chart 2: Std clotrimazole(10mg/1ml of DMSO)

Control

DMSO

Std

Candida albicanes

+++

Highly Active = +++ (inhibition zone >18mm) Moderately Active= ++(Inhibition zone 12 -18mm), Slightly Active = +(inhibition zone 6-12mm} Inactive= - (Inhibition Zone <6mm)

RESULT:

Antibacterial Activity:

Amoxicillin used as standard drug for synthesis derivatives and compound 3c show high antibacterial activity against E.Coli

Antifungal Activity:

Clotrimazole used as standard drug for synthesis derivatives and compound 3c show High antifungal activity against Candida albicans

CONCLUSION:

From observation table And Result it was concluded that all sythesised compound was used for Antibacterial And Antifungal Activity and 3c compound show high activity against E.Coli and Candida albicanes

REFERENCES:

1. Kost A. N, Grandberg L.L Adv; Heterocycalic Chem 6,347(1966)

2. Pawar R.A, Patil A.A, Indian Jornal of Chemistry 33 b156-158(1994)

3. Sammes M.P, Kartiki A.R: Adv. Heterocyclic Chem33,38(1883)

4. Kulkarni S.K, Handbook of Experimental Pharmacology, 3^rd Editoon, 127-128(2005)

5. Ghosh M.N, Fundamental of Experimental Pharmacolgy, Scientific Book Agency Culcutta 155(1951)

6. Z. Arnold G. Collin, Czech Commun,28, 863 (1963)

7. S. Selvi, P.T. Permunalorg.Prop.Int.33,194(2001), Chemistry of heterocyclic compound 33,23(1999)

8. Pelczar, M.J., chan, E.C.S. and Krieg N.R., Microbiology, TATA McGra Whill, 5^th edition,3- 17(1993)

9. Canetti G., et al: Advance in techniques of testing mycobactrial drug sensitivity and useof sensitivity tests in Tuberculosis control programmes, Bulletin of the WHO, 41,21-43(1969)

Received on 08.07.2022 Modified on 29.12.2022

Res. J. Pharmacology and Pharmacodynamics.2023;15(3):109-111.

DOI: 10.52711/2321-5836.2023.00020