INTRODUCTION:

These infectious diseases are the principal factors behind global deaths. The world's leading cause of death is infectious diseases. A number of bacteria have improved decreased sensitivity to antimicrobial agents in current use and drug resistance is common in many microorganisms, particularly Gram-positive bacteria. Infections with vancomycin resistant methicillin-resistant Staphylococcus aureus (MRSA) and Enterprocci (VRE) present a severe medical concern. In addition, for patients with immunosuppressive conditions, such as those with HIV, antiretroviral therapy or transplantation, care for infectious diseases is exceedingly difficult. Given the evidence that drug-resistant strains have spread rapidly worldwide and the emergence of drug-resistant strains among community-acquired diseases, the need for the identification or reliability of antimicrobial agents working to combat these resistant strains is important.²

Antibiotics are microbial metabolites or synthetic analogues that, in small doses, prevent the growth and survival of microorganisms without major toxins in the host. Selected toxicity is an important concept. Antibiotics are among the most commonly prescribed drugs today, although the resistance to the bacterium caused by the pressure of evolution and misuse threatens their continued effectiveness. In many cases, the clinical benefits of natural antibiotics have been enhanced by the use of chemicals in the original formulation, resulting in extensive antimicrobial appearance, greater potency, less toxicity, easier handling, and additional pharmacokinetic benefits. Through traditional use, many synthetic substances that are not related to natural products but that inhibit or kill microorganisms are called antimicrobial agents instead.³

Because of a variety of factors, including emerging infectious diseases and an increasing number of multidrug-resistant pathogens with the specific significance of Gram positive pathogens, treatment of infectious diseases remains an significant and challenging problem. For patients in hospitals, immunosuppressive patients with antiretroviral therapy, and organ transplants, the issue of treatment has become increasingly significant. In addition to the vast number of antibiotics and chemotherapeutics available for medicinal use, there has been a great medical need for new types of antibacterial agents at the same time as the emergence in recent decades of old and new antibiotic resistance. The only way to solve the resistance issue is to develop new agents with a different way of operating so that the current therapy does not resist.⁴

The widespread use of antibiotics has contributed to the development of many pathogens that are drug-resistant. This highlights the ongoing need for new groups of antimicrobial agents to be established and existing drugs to be changed in a way that helps them to retain their immune function but decreases their resistance to pathogens. Due to the distinct modes of action that can be prevented by refusing known drugs, the formulation of generic chemotherapeutic agents is especially beneficial.⁵

MATERIALS AND METHODS:

All the chemicals used in the 4-thiazolidinone synthesis were purchased from S. D. Chem Fine. Ltd., Mumbai and Lancaster's Sigma-Aldrich Chemical Co., and used directly without further purification. To track the progress of the reaction and output of the product, thin layer chromatography was used. On a 0.25 mm precoated plate of silica gel 60F254, E., a thin layer of chromatography of the combined chemicals is produced. Merck, Darmstadt, Germany, used a particular medium for solvents. Under UV light and in the Iodine chamber, screening is performed. Spot detection is achieved over short and long distances under a UV light. M.P has been calculated by and is not set by the open capillary system. Infrared spectra (max in cm-1) of synthetic chemicals reported in the potassium bromide Shimadzu FTIR-8400s, Perkin Elmer 881 in the 400-4000 cm^-1 range.

Using the fast atomic bomb device, the Micromass Quattro II tool used the electron spray ionisation process, and the JEOL Accutof-DARTMS using real-time analysis, mass spectra were reported on the JEOL SX 102 / DA-600 metal.

On the Brucker ADVANCE DRX 300 MHz / 200 MHz spectrometer, ¹HNMR spectra (ppm, δ) were registered with Tetra methyl silane as the internal standard.

In the Elementar Vario EL III, basic analysis (carbon, hydrogen and nitrogen) was performed.

Preparation of thiosemicarbazone derivative (3a-3b)

General Procedure:

The isatin (1a) / chloroisatin (1b) and thiosemicarbazide (2) equimolar mixture was dissolved in 90 percent ethanol and refluxed for 1 hour with a few drops of glacial acetic acid. The solution was tested by TLC using the chloroform: methanol solvent method (95: 5). Excess ethanol has been removed and ice water has been added to the residue. They filtered, washed, dried the solid product, and then blended it with ethanol.

Preparation of Thia-3, 4, 9-triaza-fluoren-2-ylamine derivatives (4a-b)

General Procedure:

Equimolar isatin-3-thiosemicarbazone (3a)/5-chloroisatin-3-thiosemicarbazone (3b) and cold con. con. 4-5 decreases. H2SO4 was dissolved and dissolved in ethanol for approximately 8 hours. TLC tested the reverse reaction using chloroform: methanol (98: 2). It cooled the reaction mixture and isolated it from the liquid ammonia. They poured a neutral mixture into ice water. Using air conditioning, it is filtered, dried and reassembled.

Preparation of Imine derivatives (6aI-6bIII):

General Procedure:

In the presence of 5-6 drops of glacial acetic acid, an equal amount of thia-3, 4, 9-triaza-fluoren-2-ylamine derivative and suitable aldehyde were fully dissolved in 20 ml of ethanol and the reaction mixture was then completed. Solution termination has only been checked by TLC using another method. The hot mixture was poured into the crushed ice after completing the reaction. Using ethanol, the impure substance was then purified by recrystallization.

Preparation of thiazolidin-4-one derivatives (7aI-7bIII)

General Procedure:-

In 50 ml of methanol, an equivalent quantity of imine derivative was dissolved. By pulling down the anhydrous zinc chloride, the equimolar level of thioglycolic acid was added and the mixture was stirred until the reaction was complete. Solution termination using a different solvent was checked by TLC. They collected and sealed the ethanol extraction and dumped the remains into ice water. Using ethanol, the solid product was filtered, washed, dried and then stabilized.

Antimicrobial Activity:

Inhibition of bacterial growth under standard conditions can be used to demonstrate the therapeutic efficacy of an antibacterial agent. Any subtle molecular mutations that may not be detected by chemical means will be exposed to changes in antimicrobial activity which is why antimicrobial experiments are very helpful in resolving doubts about possible changes in the potency of antimicrobial agents.

Antimicrobial testing is based on comparisons of small-scale inhibition of growth through small amounts of antimicrobial agents that will be tested by that process with known concentration for standard adjustment of antibiotics with known activity.

Methods for determining antimicrobial activity:-

The antimicrobial activity of synthesized compounds is tested by several methods:-

1. Cylinder- plate or Cup plate Method.

2. Turbidimetric or Tube assay Method.

3. Disc Diffusion Method.

4. Ditch-Plate Technique

5. Solid Dilution Method

Cylinder-Plate or Cup- PlateMethod:-

In cylinder-plate method, a solution of known concentration of the standard preparation and the test sample are prepared and applied to the surface of solid agar medium in a sterile cylinder or in cavities prepared by a borer in the agar medium containing microbial culture. After 18 hours of incubation at 37⁰C, the diameter of circular inhibition zones is measured.

Turbidmetric or Tube Assay Method:-

This tube assay method depends upon inhibition of growth of a microbial culture, in a uniform solution of drug in broth, taken in a tube and a standardized suspension of test organism inoculated. After overnight incubation, the minimum inhibitory concentration is ready by noting the lowest concentration of drug that inhibits growth.¹

Disc Diffusion Method:-

Thedisk diffusion method uses filter paper disk, 6.0 mm in diameter, charged with appropriate concentration of drug. These disks are placed on petri plates containing a standardized amount of test organism over an agar medium. After overnight incubation, the degree of sensitivity is determined by measuring zone of inhibition of growth around disk.

Ditch-Plate Technique:-

The solution is placed in a ditch cut in nutrient agar contained in a petri dish, or it may be mixed with a little agar before pouring into the ditch. The test organisms (as many as six may be tested) are streaked up to the ditch. The plate is then incubated. Organisms growing up to the ditch are considered resistant.

Solid Dilution Method:-

In this method, the dilutions of the substances under test are made in agar instead of broth. The agar containing the substances under test subsequently of the test organism may be tested. Multipoint inoculators enable several organisms to be tested on one plate.

Microorganisms:-

The microorganisms B. pummilus (MTCC 1456), P. fluresceus (MTCC 2421), M. luteus (MTCC 1538), P. aeruginosa (MTCC 424), P. chrysogenum (MTCC 161), E. coli (MTCC 1573), A. niger (MTCC 2546), B. subtilis (MTCC 441) and S. aureus (MTCC 1430) were purchased from Institute of Microbial Technology, Chandigarh.

Microbiological testing of synthesized compounds:-

Procedure-

The sterilised nutrient agar media (cooled at 40^oc) was poured with a definite volume of the microbial suspension (inoculums) and thoroughly mixed. Approximately 20 ml of this suspension was poured aseptically into the petri dishes and remained in place until the media solidified. Using a sterile cork borer, the surface was pierced with agar plates. The prepared wells were filled separately with an equal amount of synthesized solution compounds (7aI-7bIII) and standard drugs. After duration of pre-incubation diffusion, in defined conditions, the plates were incubated face-up for a definite time. The inhibition zone has been calculated and is recorded.

RESULTS AND DISCUSSION:

The exhaustive literature surveyrevealed that 4-thiazolidinone possess diverse biological properties like antifungal, anti-inflammatory, antiproteolytic, antibacterial, anti-viral, anti-depressant, anticonvulsant etc. The objective of the present work was the syntheses, characterization and evaluation of antimicrobial activity of some thiazolidinone derivatives.

The 4-thiazolidinone synthesis was performed in 4 stages. In the first step, in the presence of a few drops of glacial acetic acid in 90 % ethanol, Isatin/5-chloroisatin was condensed with thiosemicarbazide to create various thiosemicarbazones. In the second step, in the presence of sulfuric acid, thiosemicarbazones cyclized to form corresponding Thia-3, 4, 9-triaza-fluoren-2-ylamines in the presence of sulfuric acid.

Thia-3,4,9-triaza-fluoren-2-ylamines then react with different heterocyclic aldehydes to give corresponding imines in absolute ethanol and glacial acetic acid. Finally, in the presence of anhydrous zinc chloride, the corresponding imine was cyclized by thioglycolic acid to supply thiazolidine-4-ones.

Physical, spectral and elemental research have characterized all the synthesized compounds. Thin layer chromatography using precoated silica gel G plates has established the purity of the compounds. The spots of both short and long wavelengths were observed under a UV light. In the iodine chamber, the spots were further identified. The melting range has been calculated and is uncorrected by the open capillary process.

The presence of C = N at 1593-1608 cm-1 and N-H at 3413-3415 cm-1 respectively was shown by the IR spectrum of Isatin/ 5-chloroisatin-3-thiosemicarbazone (3a-b). Thia-3,4,9-triaza-fluoren-2-ylamines (4a-b) IR spectral data showed characteristic bands at 1618-1627 cm-1 C = N stretch and two absorption bands at 3442, 3515 and 3415, 3484 cm-1 10 N-H stretch for 4a and 4b, respectively.

For the multiple compounds synthesized, [M+1]+ peaks were observed in mass spectra. In the range of 3.37-3.75 for methyl proton and 7.26-7.68 for benzyl proton, the δ value was found in the 1H-NMR spectra in the various synthesised compounds. For the measured presence of carbon, hydrogen and nitrogen, elemental analysis was performed. In Table 3.1 and Table 3.2, the IR, MS, NMR and elemental analysis data for all synthesised compounds (3a-7bIII) are shown.

The synthesised derivatives of thiazolidinone were tested by the cup plate method for antimicrobial activity against bacterial and fungal strains. A microorganism called B. Pumilus (MTCC1456), P. fluorescens (MTCC 2421), P. aeruginosa (MTCC 424), M. luteus (MTCC 1538), P. chrysogenum (MTCC 161), E. (MTCC 1573) coli, A. Niger (2546 MTCC), B. (MTCC 441) and S. subtilis. Aureus (MTCC 1430) was collected from the Chandigarh Institute of Microbial Technology, India. Norfloxacin and Fluconazole have been used as standard drugs for antibacterial and antifungal activities respectively.

It was found that the antimicrobial efficacy of all the compounds was concentration dependent. It was found that both compounds 7aI-7bIII were more effective than Gram-positive strains against Gram-negative strains. The Gram-negative bacterial cell wall is high in lipid content and low in peptidoglycan.

The Gram-positive bacteria cell wall, on the other hand, is low in lipid content and high in peptidoglycan. Compounds that were more lipophilic than the Gram-positive bacteria could have penetration into the Gram-negative bacteria. The compounds therefore demonstrate greater activity than Gram-positive strains against Gram-negative strains.

Compounds 7aI demonstrated strong antibacterial activity against Bacillus subtilis, Psedomonas aeruginosa, Pseudomonas fluorescens and Bacillus pumilus, containing MIC 30 μg / mL. Compounds 7aII have been found to be the most effective against Bacilluspumilus with the lowest MIC (20 μg / mL) and strong activity against Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas fluorescens and Escherichia coli with a MIC of 40-50 μg / mL.

Two strains of bacteria (Bacillus pumilus and Pseudomonas aeruginosa) were found to be most susceptible at 20-50 μg / mL to all compounds. The least susceptible strain against all the synthesised compounds was found to be Staphylococcus aureus. The antibacterial activity was found to be greater than antifungal activity for all the compounds.

The compounds had antibacterial activity in the order of 7aI>7aII>7bII>7aIII>7bI>7bI>7bIII. The antifungal activity was 7aIII=7bII>7bIII>7aII>7aII>7aI=7bI.\\\7bI.

Table 1. Mean diameter of zone of inhibition (mm) of synthesized compounds (7aI-7aIII and 7bI-7bIII), standard and control against various microorganisms.

S. No.	Compounds (mol wt.)	Conc. (µg/mL)	Gram +ve strains				Gram –ve strains			Fungal strains
S. No.	Compounds (mol wt.)	Conc. (µg/mL)	BS	SA	BP	ML	PA	EC	PF	AN	PC
1.	7aI (302.37)	100	10	-	7	8	8	7	8	-	-
		250	13	-	9	10	11	10	10	-	-
		500	15	8	12	12	12	13	13	8	10
		750	18	11	18	14	15	15	17	11	12
		1000	20	15	22	17	20	18	21	14	15
		1250	22	17	25	19	24	20	24	17	17
2.	7aII (414.50)	100	8	-	6	-	7	8	7	-	-
		250	10	8	8	-	10	10	10	-	8
		500	13	10	11	8	13	12	14	8	9
		750	15	12	15	11	15	13	16	10	10
		1000	17	14	20	14	17	15	19	15	12
		1250	20	18	26	16	20	18	22	19	15
3.	7aIII (365.43)	100	8	-	8	-	7	7	7	-	-
		250	10	8	10	8	9	9	10	8	9
		500	12	9	15	11	13	13	13	11	12
		750	13	10	17	14	15	15	15	14	13
		1000	15	12	19	15	18	18	18	15	15
		1250	18	15	21	17	22	22	20	17	17
4.	7bI (336.82)	100	8	-	8	-	7	9	-	-	-
		250	10	8	10	-	9	11	7	-	-
		500	15	10	12	10	14	14	8	10	12
		750	17	13	13	14	17	17	12	13	14
		1000	19	15	15	17	18	19	15	15	18
		1250	21	19	18	19	20	21	19	17	20
		100	-	-	8	-	8	7	8	-	-
5.	7bII (448.95)	250	7	-	10	-	10	10	11	8	8
		500	11	6	13	8	12	13	12	11	11
		750	15	8	15	12	13	15	15	13	15
		1000	18	10	17	15	15	17	20	15	15
		1250	20	15	20	20	18	20	24	18	18
6.	7bIII (399.88)	100	-	-	10	-	7	-	7	-	-
		250	7	7	13	7	10	11	8	9	9
		500	9	9	15	8	13	12	11	12	12
		750	14	11	18	12	15	15	15	15	15
		1000	19	13	20	15	18	20	17	18	18
		1250	22	17	23	19	20	23	21	20	20
7.	Norfloxacin (319.34)	10	25	22	30	24	26	25	27	-	-
8.	Fluconazole (306.27)	10	-	-	-	-	-	-	-	22	23
9.	Control	-	-	-	-	-	-	-	-	-	-

BS B. subtilis, SA S. aureus, BPB. pumillus, MLM. luteus, PAP. aeruginosa, EC E coli, PFP. fluoresceus, AN A. niger, PC P. chrysogenum, Control = 10 % v/v DMSO, (-) = no activity