Synthesis, Characterization and In-Silico Studies of Novel Thiazolidine Derivatives and Their Assessment as Potential Anti-Microbial Agents

Schiff bases and thiazolidine derivatives have gained substantial attention in the pharmaceutical and chemical industries due to their diverse biological activities. Schiff bases, formed through the condensation of primary amines and carbonyl compounds, possess an imine (-C=N-) functional group that plays a crucial role in their bioactivity. Thiazolidine derivatives, on the other hand, are five-membered heterocyclic compounds containing both nitrogen and sulfur atoms, and their structural versatility allows for broad-spectrum pharmacological applications (Alver et al., 2023). The synthesis of Schiff bases typically involves a condensation reaction between aldehydes or ketones and primary amines. The reaction is often catalyzed under mild conditions, which makes it a cost-effective and environmentally friendly method for synthesizing bioactive compounds. Various aldehydes and amines can be employed, allowing for the generation of a wide range of Schiff base derivatives with different bioactivities (Gupta et al., 2023). Thiazolidine derivatives are synthesized through cyclization reactions between cysteine and Schiff bases or other suitable intermediates. The synthesis is highly modular, allowing for a high degree of functionalization and structural variation. This versatility enhances the ability of thiazolidine derivatives to interact with a range of biological targets, making them attractive for drug development (Kumar et al., 2022). Schiff bases and thiazolidine derivatives have demonstrated significant antiviral activities in various studies. Their ability to inhibit viral replication is attributed to their interaction with viral enzymes and proteins. For example, certain Schiff bases have shown inhibitory effects against viruses such as HIV, influenza, and herpes simplex virus (Rahman et al., 2023). This antiviral potential positions these compounds as promising candidates for the development of novel antiviral therapies (Alver et al., 2023). The antibacterial properties of Schiff bases and thiazolidine derivatives have been widely explored, with several derivatives exhibiting potent activity against both Gram-positive and Gram-negative bacteria. The compounds interact with bacterial enzymes, cell walls, and DNA, disrupting vital processes and leading to bacterial death. These derivatives have demonstrated effectiveness against resistant strains, positioning them as potential alternatives to traditional antibiotics (Patel et al., 2022). Schiff bases and thiazolidine derivatives have also been investigated for their antifungal activities. Several studies have shown that these compounds exhibit significant inhibition against fungal pathogens such as Candida albicans and Aspergillus niger. Their antifungal properties are attributed to their ability to disrupt fungal cell membranes and inhibit key enzymes required for fungal survival (Chen et al., 2023). Thiazolidine derivatives, in particular, have shown potential as antimalarial agents due to their ability to inhibit key enzymes in the Plasmodium species responsible for malaria. Some Schiff base derivatives have also demonstrated activity against malarial parasites, making them promising candidates for developing new treatments for malaria (Sharma et al., 2023). The antioxidant properties of Schiff bases and thiazolidine derivatives have been well-documented. These compounds scavenge free radicals, thereby reducing oxidative stress and preventing cellular damage. Their antioxidant activities have implications in treating diseases where oxidative stress plays a significant role, such as cancer, cardiovascular diseases, and neurodegenerative disorders (Alver et al., 2023). Schiff bases and thiazolidine derivatives have shown potent anticancer activities in various in vitro and in vivo models. These compounds exert their effects by inducing apoptosis, inhibiting cell proliferation, and disrupting cancer cell metabolism. Their selectivity towards cancer cells, coupled with minimal toxicity to normal cells, makes them attractive for further development as anticancer agents (Wang et al., 2022). Schiff base and thiazolidine derivatives have also demonstrated analgesic properties. Studies have shown that these compounds interact with opioid receptors and modulate pain signaling pathways, providing significant relief from both acute and chronic pain. This positions them as potential alternatives to opioid-based painkillers, with reduced risk of addiction and side effects (Singh et al., 2023). In addition to analgesic properties, Schiff bases and thiazolidine derivatives have exhibited antipyretic activity by inhibiting the production of prostaglandins, which are responsible for fever induction. This ability to reduce fever makes these compounds useful in treating inflammatory diseases where fever is a common symptom (Alver et al., 2023). Anti-inflammatory activities of Schiff bases and thiazolidine derivatives are of particular interest due to their ability to inhibit pro-inflammatory cytokines and enzymes such as COX-2. These compounds have been shown to reduce inflammation in various animal models, suggesting their potential for treating conditions such as arthritis, inflammatory bowel disease, and other chronic inflammatory disorders (Ahmed et al., 2022). chiff base derivatives have also demonstrated anti-glycation activity, which is important in managing diabetes and its complications. Glycation is a process where sugar molecules bind to proteins, leading to the formation of advanced glycation end products (AGEs). Schiff bases inhibit this process, thus reducing the development of diabetic complications such as neuropathy and retinopathy (Gupta et al., 2023). Schiff bases have shown exceptional promise as antimicrobial agents due to their ability to form stable complexes with metal ions, which enhance their biological activity. These metal complexes exhibit broad-spectrum antimicrobial properties, making Schiff bases valuable in the fight against multidrug-resistant pathogens (Kumar et al., 2022). Thiazolidine derivatives also exhibit strong antimicrobial activity due to their ability to interact with microbial enzymes and disrupt cellular function. Their versatility in structural modifications enables the development of derivatives with enhanced efficacy against various bacterial and fungal strains, positioning them as excellent candidates for antimicrobial drug development (Sharma et al., 2023). Despite the promising potential of Schiff bases and thiazolidine derivatives, several limitations exist in current studies. Many investigations are limited to in vitro analyses, and the pharmacokinetics and toxicity profiles of these compounds remain inadequately explored. Additionally, further studies are needed to optimize the structural features of these compounds for enhanced bioactivity and reduced side effects (Chen et al., 2023). Future research should focus on optimizing the synthesis and functionalization of Schiff bases and thiazolidine derivatives to enhance their pharmacological properties. Additionally, more in vivo studies and clinical trials are needed to fully assess the therapeutic potential of these compounds, particularly their safety, efficacy, and bioavailability in humans (Wang et al., 2022). In conclusion, Schiff bases and thiazolidine derivatives represent a versatile class of compounds with significant potential for therapeutic applications. Their wide range of biological activities, including antiviral, antibacterial, antifungal, anticancer, and antioxidant properties, make them promising candidates for drug development. However, further research is required to overcome current limitations and translate these findings into clinical practice (Alver et al., 2023).

2Experimental

2.1Experimental Section

Chemicals used during the reactions are: 4-(Dimethylamine) benzaldehyde (97%), 4-Nitro-1, 2-phenylenediamine (97%), 2-methylamine (97%), 3-chloro aniline (98%), benzaldehyde (97%) and thioglycolic acid (97%). Without any further purification, analytical-grade solvents including ethanol, dichloromethane, acetone, methanol, ethyl acetate, n-hexane, and chloroform were used. Weighing the compounds involved using an electronic analytical balance, a Japan ATY 224. Weighing the compounds involved using an electronic analytical balance, a Japan ATY 224. Melting points were found by using the Melting Point apparatus SMP10. The functional groups in the synthesized compounds were observed by using an ATR model ALPHA 20488-equipped FT-IR. A 400 MHz BRUKER spectrometer was used for the 1H-NMR in which DMSO was used as a solvent.

2.2Synthesis of Schiff Bases

Four Schiff bases (SSB-1 to SSB-4) were synthesized by condensation reactions between aldehydes and amines in 1; 1 (10mmol each) for 5-9 hours under reflux conditions. Precipitates that were isolated and repeatedly rinsed with methanol, ethanol, and ethyl acetate were filtered using Whatman filter paper. Four thiazolidine compounds (ST-1 to ST-4) were then synthesized by using synthesized Schiff bases as intermediates by dissolving Schiff base (1mmol) in toluene (50 mL) and adding it to thioglycolic acid (0.9 mL; 1.3 eq., 92 g/mol, d=1.32 g/mL). The resulting solution was refluxed using the Dean stark and progress was monitored using TLC. Brine and a 3% NaHCO3 solution (4 × 10 mL) were used to wash the product after filtration. The organic layer was dried over Na2SO4 then concentrated in vacuo. The products were re-crystallized in ethanol.

2.3Anti-Bacterial Activity

The antibacterial activity of the synthesized compounds against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa can be evaluated using a standard agar well diffusion method. This technique involves the preparation of bacterial cultures, inoculation on agar plates, and assessment of compound efficacy by measuring zones of inhibition. The following procedure outlines the full protocol for these assays (Zhao et al., 2022). First, the bacterial strains (E. coli, S. aureus, and P. aeruginosa) are cultured in nutrient broth at 37°C for 18–24 hours to reach an optical density of approximately 0.5 McFarland standard (equivalent to 1.5 × 10^8 CFU/mL). This ensures a uniform bacterial concentration for subsequent inoculation on the agar plates. Sterile Mueller-Hinton Agar (MHA) plates are prepared and used as the growth medium for bacterial colonies (Patil et al., 2023). After preparing the bacterial suspension, 100 ?L of each culture is uniformly spread onto the surface of the MHA plates using a sterile cotton swab. Once the plates are inoculated, wells are punched into the agar using a sterile cork borer (typically 6–8 mm in diameter). These wells serve as reservoirs for the test compounds and standard drugs. A volume of 50–100 ?L of each test compound at the desired concentration is carefully introduced into each well (Singh et al., 2023). A positive control (typically a known antibiotic such as Moxifloxacin) and a negative control (solvent, e.g., DMSO or water) are also introduced into separate wells on the same plate to compare the test results. The plates are then incubated at 37°C for 18–24 hours, allowing bacterial growth around the wells. Following the incubation period, the zones of inhibition around each well are measured in millimeters using a ruler or caliper, representing the antibacterial efficacy of each compound (Rahman et al., 2022). Each assay should be performed in triplicate to ensure reliability and reproducibility of the results. The mean values of the zones of inhibition, along with standard deviations, are calculated to compare the efficacy of different compounds. The inhibitory concentrations (IC50) of the compounds can be determined by testing multiple concentrations and plotting the inhibition zone sizes against the log concentration, followed by linear regression analysis (Chen et al., 2023). For more precise and quantitative assessment, the Minimum Inhibitory Concentration (MIC) can also be determined using a broth microdilution method. This involves preparing a series of two-fold dilutions of the test compound in 96-well microtiter plates containing bacterial suspension. After incubation, bacterial growth is monitored by visual inspection or measuring optical density at 600 nm, and the MIC is defined as the lowest concentration at which no visible growth occurs (Gupta et al., 2022). The results obtained from these antibacterial assays provide valuable insight into the efficacy of Schiff base and thiazolidine derivatives against pathogenic bacteria. Statistical analysis, including one-way ANOVA or t-tests, can be performed to assess the significance of differences between test compounds and control antibiotics (Kumar et al., 2023).

2.5Anti-Fungal Activity

The antifungal activity of the synthesized compounds against Candida parapsilosis can be assessed using the standard agar well diffusion method or the broth microdilution method. Both techniques provide valuable data on the efficacy of compounds, but the broth microdilution method is preferred for determining the Minimum Inhibitory Concentration (MIC). Below is the detailed procedure for performing antifungal assays against C. parapsilosis (Sharma et al., 2023). First, C. parapsilosis is cultured in Sabouraud Dextrose Broth (SDB) at 30°C for 18–24 hours until it reaches an optical density of 0.5 McFarland standard (approximately 1 × 10^6 to 5 × 10^6 CFU/mL). This standardized fungal suspension is essential for ensuring reproducibility and accuracy in the assay. Sterile Sabouraud Dextrose Agar (SDA) plates are used as the medium for fungal growth in the agar well diffusion method (Patil et al., 2022). For the agar well diffusion method, 100 ?L of the standardized fungal suspension is evenly spread on the surface of the SDA plates using a sterile swab. Wells (6–8 mm in diameter) are then punched into the agar using a sterile cork borer. A volume of 50–100 ?L of each test compound, diluted in a suitable solvent (such as DMSO or water), is added to each well. Positive control wells contain a standard antifungal drug like clotrimazole, while negative controls contain the solvent alone (Singh et al., 2023). The plates are then incubated at 30°C for 24–48 hours to allow fungal growth. After incubation, the zones of inhibition around the wells are measured in millimeters using a ruler or caliper. Larger zones of inhibition indicate higher antifungal efficacy. Each experiment is conducted in triplicate to ensure the reliability of the data, and mean values are calculated, along with standard deviations (Ahmed et al., 2022). To determine the Minimum Inhibitory Concentration (MIC), the broth microdilution method is used following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Serial two-fold dilutions of the test compounds are prepared in 96-well microtiter plates, using RPMI-1640 medium buffered with MOPS (3-(N-morpholino)propanesulfonic acid). Each well contains a different concentration of the test compound, ranging from 0.5 to 256 ?g/mL, along with a standardized inoculum of C. parapsilosis (1 × 10^3 to 1 × 10^4 CFU/mL) (Chen et al., 2023).

The microtiter plates are incubated at 30°C for 24–48 hours. After the incubation period, fungal growth is assessed visually or by measuring the optical density at 600 nm using a microplate reader. The MIC is defined as the lowest concentration of the test compound at which no visible fungal growth is observed. Positive controls containing clotrimazole and negative controls containing only the medium and solvent are included to validate the results (Kumar et al., 2023). For more advanced quantification, the Minimum Fungicidal Concentration (MFC) can be determined by transferring 10 ?L from wells showing no visible growth to fresh SDA plates. These plates are incubated for an additional 24–48 hours, and the MFC is determined as the lowest concentration at which no fungal colonies grow on the SDA plates (Gupta et al., 2022). Statistical analysis of the antifungal activity data can be performed using one-way ANOVA or Student’s t-test to assess the significance of differences between the test compounds and the positive control. This analysis helps determine whether the synthesized compounds exhibit statistically significant antifungal activity (Rahman et al., 2023).

2.5In-Silico Studies

In silico pharmacokinetic analyses performed using Swiss ADME and LabWare software assess the absorption, distribution, metabolism, and excretion (ADME) properties of potential drug candidates. These evaluations are crucial for optimizing bioavailability and formulating appropriate dosing strategies for preclinical and clinical studies. Simultaneously, toxicological assessments conducted with Tox-21 and LabWare software evaluate the safety profiles of these compounds, ensuring compliance with regulatory standards and confirming their suitability for clinical advancement. These computational methodologies significantly expedite the drug discovery and development process, providing critical insights necessary for the advancement of effective therapies against leishmaniasis.

3.RESULTS

3.1Biological Assays

3.1.1Anti-Bacterial Assays

3.1.2Anti-Fungal Activity

3.2In Silico Pharmacokinetic Studies

3.3In Silico Toxicology Profile