Synthesis and Biological Evaluation of Oxazine and Thiazine derivatives as Potent microbial agents

J.S. Rudra Bhavani, Farath Sulthana, Kalva Swapna, Tadikonda Rama Rao, Mugdivari Sangeetha*

CMR College of Pharmacy, Kandlakoya, Medchal, Hyderabad, Telangana,India-501401

*Correspondence: Sangeetha.kodiganti@gmail.com

DOI: https://doi.org/10.71431/IJRPAS.2025.4603   

INTRODUCTION

CHALCONES Chalcone is a simple chemical substance naturally occurring in vegetables, fruits and other plants. The word chalcos means bronze, which results from the colours of most natural chalcones. The name chalcones was given by kostanecki and Tambor. Chalcones have been used as intermediate for the preparations of compounds having therapeutic value. Chalcone is an aromatic ketone that serves as the building block of numerous significant biological molecules referred to as chalcones. The 1,3-diphenyl-2-propen-1-one derivative, or chalcone core, is made up of two aromatic rings connected by a three carbon-α, β-unsaturated carbonyl system. Literature review reveals that chalcone derivatives exhibit diverse pharmacological activities. 

The chemical compound chalcone is C6H5C(O)CH=CHC6H5. It is a ketone that is α, βunsaturated. Chalcones, also referred to as chalconoids, are a group of diverse biologically significant chemicals.

APPLICATIONS:

1.  Anticancer activity:

The mechanism of action of anticancer action of chalcone compounds has been extensively studied. Tumor protein p53 suppresses tumor growth and alters the cell cycle. p53 is essential for both stopping the growth of precancerous cells and preserving the integrity of cells and the genome.

The nuclear factor Kappa B(NF-kB) is a important transcription factor that plays a major role in inflammation, innate immunity and carcinogenesis. Because angiogenesis plays a significant role in the development and spread of cancer, it is a prospective target for cancer therapy.

2.  Anti-inflammatory activity:

Cox is a membrane bound enzyme, which is responsible for anti-inflammatory activity.  NSAIDS such as aspirin, ibuprofen, nimesulide etc are the best-known cyclooxygenase inhibitors but they show many side effects.so sulfonyl tricyclic compounds of chalcones were synthesised. They are cox 2 inhibitors at both enzymatic and gene level. Sulphonyl group enhance the anti-inflammatory activity.

3.  Anti-malarial activity:

The two main parasites that cause malaria in humans are Plasmodium falciparum and Plasmodium vivax. P. falciparum is the primary cause of most fatalities. The antimalarial properties of licochalcone A, a naturally occurring substance derived from Chinese liquorice root, were initially documented due to its strong antimalarial properties.  Some chalcones inhibit falcipain cysteine proteases, but it is unclear if the anti-malarial activity of this class is primarily due to protease inhibition. Several oxygenated chalcones and bischalcones were reported to have antimalarial activity. The synthesis and antimalarial activity of several quinolinyl chalcone analogues have been described in recent research. There are several compounds of phenylurenyl chalcone with antimalarial properties.

4.  Anti-viral activity:

Naringenin-chalcone is a naturally present in citrus fruits which possess an anti-viral property. Another natural chalcone, Myrigalone also exhibit anti-viral activity against the herpes simplex virus. Introduction of purine ether and addition of diethyl malonate or nitromethane in double bond are beneficial to antiviral activity of chalcone compound.

These compounds were exhibited good anti-viral activity, better than that of ribavirin.

5.  Anti-diabetic activity:

Chalcones bearing electron donating or electron withdrawing substitutions are exhibiting glucose uptake activity. chalcones with chloro, bromo, iodo and hydroxy substitutions at 2 positions on A ring exhibit the highest activity. chalcone possess a unique skeleton which is totally different from other anti-diabetic drugs in market.

6.  Anti-HIV:

There are a few well-known chalcones that are active against the human immunological virus and can be derived from both natural and synthetic sources. Xanthohumol, a naturally occurring chalcone, has anti-HIV characteristics. In 2006, Nakagawa and Lee identified a distinct β- hydroxy chalcone demonstrates strong anti-HIV properties and belongs to the genus Desmos. A different chalcone that was isolated from Maclura tinctoria (Moraceae) leaves shown inhibitory efficacy against AIDS-related infections Candida albicans and Cryptococcus neoformans.

AIM

•         In the present work the effort is made to develop a convenient method for the synthesis of novel oxazine and thiazine derivatives by conventional method. 

•         Understanding the importance of oxazine and thiazine for antimicrobial activity, some novel oxazine and thiazine derivatives were synthesized by structural modification on the oxazine and thiazine ring.  

OBJECTIVES

•         To synthesize the novel oxazine and thiazine derivatives by using reported methodology.

•         To purify the intermediates and final compounds by recrystallization/ chromatographic techniques using suitable solvents.

•         To characterize the synthesized compounds by the help of physical (MP, R_f values), TLC and spectral data (FT-IR, ¹H-NMR, Mass spectroscopy). 

•         Finally, to evaluate the synthesized compounds for their possible antimicrobial activity.

•         To identify the potent compounds, if any for their specific activity and for future exploitation.

MATERIALS AND METHODOLOGY

PRINCIPLE

The principle behind the synthesis and biological evaluation of oxazine and thiazine derivatives from chalcone as potent microbial agents lies the design and synthesis of novel compounds with structural features that confer antimicrobial activity. Common methods include for Synthesis Claisen-Schmidt condensation, aldol condensation, and heterocyclization reactions to form oxazine and thiazine rings. The synthesized compounds are characterized using spectroscopic techniques such as FTIR to confirm their chemical structures and purity. The synthesized oxazine and thiazine derivatives are subjected to comprehensive biological evaluation to assess their antimicrobial potency. This involves testing the compounds against a panel of clinically relevant microorganisms.

METHODOLOGY:

Preparation of chalcone 

1.Take 5ml of benzaldehyde and add 6ml of acetophenone and 5 ml of ethanol. 

2.Prepare NaOH solution (1.5 grams of NaOH in 10ml of water).

3.The NaOH solution is added to the above mixture drop by drop and kept for 2-3 hours of stirring on the magnetic stirrer

4. Filter out the sticky paste like texture which is formed.

Benzaldehyde                                   Acetophenone                                      Chalcone

Preparation of Oxazine derivative.

1.A mixture of chalcone (1gram) and urea (1gram), was stirred for about 2-3 hours with a magnetic stirrer by adding ethanolic NaOH solution drop by drop (1.5g NaOH and 10Ml ethanol). 

2.Then 200ml of cold water was poured in to the above mixture and kept for continuous stirring for an hour, and after that, we kept the mixture in a refrigerator for 24 hours. 

3.The precipitate obtained was filtered and washed.

Preparation of thiazine derivative.

1.A mixture of chalcone (1gram) and thiourea (1gram), was stirred for about 2-3 hours with a magnetic stirrer by adding ethanolic NaOH solution drop by drop (1.5g NaOH and 10Ml ethanol).

2.Then 200 ml of cold water was poured in to the above mixture and kept for continuous stirring for an hour, and after that, we kept the mixture in a refrigerator for 24 hours. 

3.The precipitate obtained was filtered and washed.     

IDENTIFICATION AND CHARACTERISATION

The identification and characterization of the prepared compound were carried out by the following procedure- 

1.. Solubility 

2.  Thin layer chromatography 

3.  Biological activity   

SOLUBILITY:

The solubility of synthesized compounds wastested in various solvents. The solubility is the ability of a solid, liquid, or gaseous chemical substance to dissolve in solvent and form a solution in specific a homogenous solution.

THIN LAYER CHROMATOGRAPHY:

·         Cleaned and dried glass plates were taken. 

·         Uniform slurry of silica Gel-G in water was prepared in the ratio of 1:2. 

·         The slurry was then poured into the chamber of the TLC applicator, which was fixed and thickness was set to 0.5 mm 

·         Glass plates were moved under the applicator smoothly to get a uniform coating of slurry on the plates. 

·         The plates were dried at room temperature and then kept for activation at 110°C for 1 hour. The compound was taken in a small bored capillary tube and spotted at 2 cm from the base end of the plate.

·         Then the plate was allowed to dry at room temperature and plates were transferred to chromatographic chamber containing solvent system for development. 

·         The developed spots were detected by exposing them to iodine vapours. Then the Rf values of compounds were calculated using the formula. 

Rf value = distance moved by sample/ distance moved by solvent

Biological Evaluation (Antimicrobial Activity) 

PRINCIPLE:

Nutrient agar is used for making plates for the growth of micro- organisms. Agar plates provide maximum surface area and it is easy to study colony characteristics. 

LIMITATIONS OF AGAR:

PROCEDURE:

Follow the procedure as nutrient broth, but add 1-2percent of agar powder to nutrient broth.  Weigh all the additives separately by physical balance and add all the additives in a suitable container. Dissolve with the help of stirrer and adjust the pH using sodium hydroxide, sterilize the media in autoclave at 15lb pressure at 120°c for 20 min. 

Preparation of sample solution: 

·         Prepare different concentrations of sample solution (i.e.)10 mg/ml, 20, 30, 40, solutions. 

·         Take 10 mg of sample and dissolve in solvent and make up to 10 ml to get 1 mg/ml or 1000 mg/ml solutions. 

·         From the above solution take 0.1, 0.2, 0.3 and 0.4 and makeup to 10ml respectively to get 10, 20, 30,40 μg/ml. 

EXPERIMENTAL PROCEDURE BY CUP PLATE METHOD

·         Prepare nutrient media and transfer 20 ml into boiling tube, plug and sterile them. 

·         After cooling inoculate each boiling tube with 0.1ml of test organism (Bacillus subtilis) and (E. coli).

·         The inoculated agar media is poured into Petri plate and solidified. 

·         Make holes in the solidified media at the center by using sterile borer. Add 0.1ml of prepared antibiotic solution into the holes. 

·         Incubate the Petri plate at 37°C for 24hrs.

THIN LAYER CHROMATOGRAPHY:

Rf = Distance travelled by solute / Distance travelled by solvent

Table-1 TLC Calculations for synthesised compounds

Table-2 Solubility test for synthesized compounds

Table-3 Zone of inhibition of standard streptomycin against E. coli

Table-4 Zone of inhibition of standard streptomycin against B. subtilis

Table-5 Zone of inhibition of Oxazine Derivative against E. coli

Table-6 Zone of inhibition of Oxazine Derivative against B. subtilis

Table-7 Zone of inhibition of Thiazine Derivative against E. coli

Table-8 Zone of inhibition of Thiazine Derivative against B. subtilis

Table-9 FTIR Spectral Characterization of Chalcone

Table-10 FTIR Spectral Characterization of Oxazine

Table-11 FTIR Spectral Characterization of Thiazine

Figure 2 FTIR Graph of Chalcone

Figure 3 FTIR Graph of Oxazine

Figure 4 FTIR Graph of Thiazine

CONCLUSION:

•      Chalcones and their derivatives possess a wide spectrum of pharmacological properties. 

•      Recent development in chemistry have led to the synthesis of chalcone derivatives which have been biologically investigated to find out the pharmacological activities.

•      The major aspect of the project is for preparation of chalcone and its derivatives and revealing their biological activity.

•      molecule possess capabilities for designing bioactive potential agents. 

•      Derivatives of thiazine their nanoparticles and complexes with transition metals can be synthesized that may possess effective pharmacological actions.

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