A Green Chemistry Approach to Thiazolidine-4-Carboxylic Acids: Docking, Synthesis and Biological Evaluation of Antifungal Activity

Fungal infections have emerged as a significant global health concern, affecting millions of individuals annually and leading to high morbidity and mortality, especially among immunocompromised patients. Fungal infections, also known as mycoses, range from superficial skin infections to life-threatening systemic diseases. They can be classified into superficial and cutaneous mycoses (affecting skin, hair, and nails, e.g., Candida spp., Trichophyton spp.), subcutaneous mycoses (invading deeper layers of skin and soft tissue, e.g., Sporothrix schenckii), and systemic or invasive mycoses (affecting internal organs, particularly in immunocompromised patients, e.g., Aspergillus, Rhizopus, Cryptococcus). The incidence of invasive fungal infections is rising due to factors such as immunosuppressive therapy, organ transplantation, HIV/AIDS, and the widespread use of broad-spectrum antibiotics. Systemic infections are associated with high mortality, often exceeding 40–50% if untreated. Early diagnosis and treatment are critical to improve patient outcomes.

 In recognition of this threat, WHO has taken several important initiatives:

• In October 2022, WHO released the First-ever Fungal Priority Pathogens List (FPPL), which categorises 19 fungal pathogens into “critical”, “high” and “medium” priority for research, development, diagnostics, and public-health action
• WHO also launched a module under the Global Antimicrobial Resistance and Use Surveillance System (GLASS) specifically for fungi (GLASS-FUNGI) aimed at standardising surveillance of antifungal resistance in invasive fungal infections (e.g., bloodstream candidiasis
• In April 2024, WHO announced a global “data-call” for antifungal agents in the pre-clinical development pipeline, further underscoring the urgent need for new therapies.
• WHO’s diagnostics and treatments reports (2025) highlight significant gaps in access to diagnostics and medicines for fungal infections—particularly in low- and middle-income countries.

Thiazolidine derivatives have gained attention as versatile heterocyclic systems with diverse biological activities, including antimicrobial, antitubercular, anti-inflammatory, and antifungal properties. Structurally, thiazolidines are five-membered heterocycles containing both sulfur and nitrogen atoms, conferring strong binding potential to biological macromolecules through hydrogen bonding and hydrophobic interactions. These compounds can be conveniently synthesized by the condensation of L-cysteine with aldehydes, a reaction that proceeds efficiently under mild or green-chemistry conditions and aligns with sustainable synthesis principles. The thiazolidine-4-carboxylic acid framework exhibits promising antifungal potential by interacting with fungal enzymes involved in ergosterol biosynthesis, cell-wall maintenance, and oxidative stress pathways. Through molecular docking studies, such compounds can be virtually screened against key fungal targets, including cytochrome P450 lanosterol 14α-demethylase (CYP51, PDB ID: 3BT4), a crucial enzyme in ergosterol formation. Given the increasing incidence of resistant fungal infections and the urgent global need for novel antifungal scaffolds, the present study focuses on the green synthesis and biological evaluation of thiazolidine-4-carboxylic acid derivatives derived from L-cysteine and selected aldehydes. The compounds were subjected to molecular docking against fungal CYP51, followed by pharmacokinetic and toxicity evaluation, and finally in vitro antifungal assays against Aspergillus niger, Rhizopus oligosporus, and Penicillium chrysogenum. By integrating computational and experimental approaches, this study aims to identify new antifungal candidates contributing to WHO’s vision for global fungal infection control and antimicrobial-resistance mitigation.

Figure 1: Mechanism of Antifungal Action in Fungal Cells

MATERIALS AND METHODS:

Materials:

• L- Cysteine
• Aldehyde
• Distilled water
• Ethanol
• Concentrated HCl

DOCKING STUDIES:

ChemSketch is the drawing tool of choice to create 3D structures of proposed compounds in several formats such as pdb, mol, mol2, etc. In this study, antifungal protein is taken as a drug target. The 3D structure of this enzyme was 3BT4 retrieved from Protein Data Bank (PDB) (http://www.rcsb.org./pdb/). The PDB ID of this enzyme is trained by residues of amino-acids. All the ligands were prepared by using protein preparation using biovia software.

3BT4 PROTEIN DETAILS: Crystal Structure Analysis of AmFPI-1, fungal protease inhibitor from Antheraea mylitta

PDB DOI:   https://doi.org/10.2210/pdb3BT4/pdb

CLASSIFICATION: HYDROLASE INHIBITOR

ORGANISM(S):  Antheraea mylitta

 X-RAY DIFFRACTION: 2.10 Å Resolution

Figure 2: 3BT4 Protein Structure

METHODOLOGY

A reaction mixture was prepared by weighing 30 mmol of L-cysteine and dissolving it in a solvent mixture containing 50 mL of distilled water and 6 mL of ethanol. To this solution, 30 mmol of the selected aldehyde was added, followed by a few drops of concentrated hydrochloric acid to catalyze the reaction. The mixture was stirred thoroughly until a homogeneous solution was obtained. The resulting solution was then transferred into a microwave-safe beaker, covered with a watch glass, and placed in a microwave oven (convection mode). The reaction was carried out at a temperature of 100 °C for 15 minutes. After completion, the mixture was allowed to cool to room temperature, and the formed solid product was filtered and dried. The crude product was then recrystallized using hexane to obtain the purified thiazolidine derivative in good yield.

TABLE 2: PYRX DOCKING SCORE

SR.NO	COMPOUND	3BT4
	ACYCLOVIR	-5.2
	OSELTAMIVIR	-5.5
	AMANTADINE	-5
	RIBAVIRIN	-6.1
	DERI 1	-7.2
	DERI 2	-5.2
	DERI 3	-5.9
	DERI 4	-6.5
	DERI 5	-6
	DERI 6	-5.5
	DERI 7	-5.2
	DERI 8	-5.9
	DERI 9	-5.4
	DERI 10	-5.9
	DERI 11	-5
	DERI 12	-5.5
	DERI 13	-6.5
	DERI 14	-5.8
	DERI 15	-5.2
	DERI 16	-5.6
	DERI 17	-6.4
	DERI 18	-5.4
	DERI 19	-6.5
	DERI 20	-6.1
	DERI 21	-6.6
	DERI 22	-6.4
	DERI 23	-6.7
	DERI 24	-5.7
	DERI 25	-6.2
	DERI 26	-6.8
	DERI 27	-5.2
	DERI 28	-6.3
	DERI 29	-6.6
	DERI 30	-6.7
	DERI 31	-5.6
	DERI 32	-6.1
	DERI 33	-6
	DERI 34	-5.3
	DERI 35	-5.7
	DERI 36	-6.1
	DERI 37	-6.7
	DERI 38	-6.2
	DERI 39	-6.2
	DERI 40	-7.4
	DERI 41	-5.6
	DERI 42	-5.8
	DERI 43	-6.4
	DERI 44	-6.3
	DERI 45	-5.8
	DERI 46	-6.4
	DERI 47	-6.5
	DERI 48	-7.2
	DERI 49	-6.4
	DERI 50	-6.2

TABLE 3: SYNTHESIZED STRUCTURES AND ITS IUPAC NAME

STURCTURE	IUPAC
	(4R)-2-(3,4-dimethoxyphenyl)-1,3-thiazolidine-4-carboxylic acid
	(4R)-2-(5-chloro-2-hydroxyphenyl)-1,3-thiazolidine-4-carboxylic acid
	(4R)-2-(2-formylphenyl)-1,3-thiazolidine-4-carboxylic acid

TABLE 4: PHYSICOCHEMICAL PROPERTIES OF SYNTHSIZED COMPOUNDS

COMPOUND	MOLECULAR FORMULA	MOLECULAR WT	SOLUBILITY	APPERANCE	% YEILD
VT	C12H15NO4S	269.32	DMSO, Ethanol, water	pale cream powder	90.1832
CT	C10H10ClNO3S	293.64	DMSO, Ethanol, water	white to light cream powder	84.9112
OT	C11H11NO3s	237.27	DMSO, Ethanol, water	dark reddish-brown powder	63.1076

TABLE 5: R_f VALUE AND MELTING POINT OF THE SYNTHESIZED COMPOUND

SR.NO	COMPOUND	Rf value	M.P °C
1	VT	0.46	180-182
2	CT	0.35	158-159
3	OT	0.42	162-163

* Melting Point Observations for three times

 

   

 

                             S1                                                      S2                                                           S3

FIGURE 3: ZONE OF INHIBITION OF RHIZOPUS OLIGOSPORUS

PENICILLIUM CHRYSOGENUM

 

   

 

                                    S1                                             S2                                                   S3

FIGURE 4: ZONE OF INHIBITION OF PENICILLIUM CHRYSOGENUM

ASPERGILLUS NIGER

 

  

                               S1                                                    S2                                                     S3

FIGURE 5: ZONE OF INHIBITION OF ASPERGILLUS NIGER

TABLE 6: ANTIFUNGAL ACTIVITY OF POWDERED SAMPLE

Sr. no	Compound concentration (µg/ml)	Zone of Inhibition (mm)
		R. oligosporus			P. chrysogenum			A. niger
		VT	CT	OT	VT	CT	OT	VT	CT	OT
1	250 (A)	14	12	12	-	12	13	15	14	11
2	500 (B)	17	14	13	17	15	15	25	18	13
3	1000 (C)	25	15	15	23	17	16	35	25	15
4	Clotrimazole (D)	29	27	26	28	30	33	17	22	20

(* - no zone of inhibition)

The synthesized thiazolidine-4-carboxylic acid derivatives (VT, CT, and OT) were obtained in good yields ranging from 63% to 90% and exhibited characteristic physicochemical properties (Table 4). The Rf values and melting points confirmed compound purity and reproducibility of synthesis.

1. Molecular Docking Studies

Docking simulations using PyRx software revealed binding affinities ranging from −5.0 to −7.4 kcal/mol (Table 2). Among all, DERI 40 and DERI 48 demonstrated the strongest binding with docking scores of −7.4 and −7.2 kcal/mol, respectively, surpassing the scores of standard antiviral agents like ribavirin (−6.1 kcal/mol). The presence of methoxy and carboxyl groups enhanced hydrogen bonding interactions with the amino acid residues of fungal protease inhibitor (3BT4), suggesting potential inhibition of fungal enzymatic pathways responsible for cell wall biosynthesis and oxidative stress management.

2. Antifungal Activity

In vitro antifungal assays were performed using the agar well diffusion method at varying concentrations (250, 500, and 1000 µg/ml). The zone of inhibition (ZOI) increased proportionally with concentration (Table 6). Among the tested fungi, Aspergillus niger exhibited the highest sensitivity, while Rhizopus oligosporus showed moderate inhibition. The compound VT produced the highest inhibition zones—35 mm (A. niger), 25 mm (R. oligosporus), and 23 mm (P. chrysogenum)—comparable to the standard drug clotrimazole.

3. Structure–Activity Relationship (SAR)

The antifungal efficiency correlated with the presence of electron-donating groups (–OCH₃, –OH) and halogen substitutions, which enhance lipophilicity and cell membrane penetration. Compound VT (3,4-dimethoxy substitution) displayed superior activity due to increased electron density and hydrogen bonding ability with the target protein.

4. Overall Findings

The integrated computational and biological evaluation indicates that the synthesized compounds possess strong binding affinity and broad antifungal potential. The green synthesis route also provides an environmentally friendly and cost-effective alternative for drug discovery.

CONCLUSION

The present study successfully demonstrated the green synthesis, computational analysis, and antifungal evaluation of thiazolidine-4-carboxylic acid derivatives derived from L-cysteine and substituted aldehydes. The compounds showed significant docking interactions with the fungal protease inhibitor 3BT4, with DERI 40 achieving the best docking score (−7.4 kcal/mol). Experimental validation confirmed potent antifungal activity, particularly against Aspergillus niger, with compound VT showing the highest inhibition. The results highlight that thiazolidine derivatives, especially those bearing methoxy or halogen substituents, possess substantial antifungal potential and merit further optimization and in vivo evaluation. This research aligns with the WHO’s global antifungal resistance initiative by identifying promising molecular scaffolds for next-generation antifungal drug development.

ACKNOWLEDGEMENT:

The author wish to thank Sakthi Arul Thiru Amma and Thirumathi Amma ACMEC Trust, providing facilities to do the work in successful manner. We are grateful to thank our Dean Research and academic Prof. Dr. T. Vetrichelvan, M. Pharm., Ph. D. and our Principal Dr. D. Nagavalli M. Pharm., Ph.D for the kind support and encouraging for the completion of the work. Finally, thanks to my family and friends.

REFERENCES

1. World Health Organization. WHO fungal priority pathogens list to guide research, development and public health action. Geneva: World Health Organization; 2022.
2. World Health Organization. GLASS-FUNGI: Global Antimicrobial Resistance and Use Surveillance System for Fungal Pathogens. Geneva: WHO; 2024.
3. Perfect JR, Bicanic T. Cryptococcosis diagnosis and treatment: what do we know now. Fungal Genet Biol. 2015; 78:49–54.
4. Brown GD, Denning DW, Levitz SM. Tackling human fungal infections. Science. 2012; 336(6082):647–647.
5. Rodrigues ML, Nosanchuk JD. Fungal diseases as neglected pathogens: a wake-up call to public health authorities. PLoS Negl Trop Dis. 2020;14(9): e0008469.
6. Pandey A, Tripathi S, Jain P, Srivastava V. Thiazolidine derivatives: A comprehensive review on synthesis and biological potential. Eur J Med Chem. 2021; 221:113559.
7. Bhatia V, Narang R, Rawal RK. Thiazolidine-4-carboxylic acid derivatives as potential antimicrobial agents: design, synthesis and biological evaluation. Bioorg Med Chem Lett. 2019; 29(18):2665–2672.
8. Pushpalatha K, Haritha P, Divya D, Srilakshmi P, Babu MS. Microwave-assisted green synthesis and antimicrobial studies of some thiazolidine derivatives. J Appl Pharm Sci. 2018;8(6):37–42.
9. CLSI. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; Approved standard M38-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.
10. Gul P, Bakht J. Antimicrobial activity of certain thiazolidine derivatives and their comparison with standard drugs. BMC Chem. 2020; 14:66.
11. Khan T, Yadav R. Synthesis of some new 5-indolylidene-4-thiazolidinone derivatives of 1,2,4-triazole as potent antioxidant and antifungal agents. Asian J Org Med Chem. 2019;4(3):174–9.
12. Łączkowski T, Markowska K, et al. The newly synthesized thiazole derivatives as potential antifungal compounds against Candida albicans. Appl Microbiol Biotechnol. 2021; 105:6355–67.
13. Al-Rifaie DA, Mohammad ZH, Mahmood RT, Rasheed MK, Taha AY, Al Samarrai OR. Synthesis and characterization of some thiazolidine-4-one derivatives derived from Schiff bases, and evaluation of their antibacterial and antifungal activity. Cell Mol Biol. 2025;71(3):66–75.
14. Dawood K, Abu-Deif HK. Synthesis and antimicrobial evaluation of some new 1,2-bis-(2-(N-arylimino)-1,3-thiazolidin-3-yl) ethane derivatives. Chem Pharm Bull. 2014;62(5):439–45.
15. Antibacterial and antibiofilm activities of thiazolidine-2,4-dione and 4-thioxo-thiazolidin-2-one derivatives against multidrug-resistant Staphylococcus aureus clinical isolates. J Appl Microbiol. 2022;132(6):3205–17.
16. Green synthesis, antibacterial and antifungal evaluation of new thiazolidine-2,4-dione derivatives: molecular dynamic simulation, POM study and identification of antitumor pharmacophore sites. [Journal Unknown]. 2023; (online ahead of print).
17. A facile synthesis, characterization and biological evaluation of novel spiro-thiazolidinone and quinazolinone-thiazolidine derivatives. Indian J Chem. 2023;62(7).
18. Al-Khazragie F, et al. Synthesis, antimicrobial, antioxidant, toxicity and anticancer activity of new azetidinone, thiazolidinone and selenazolidinone derivatives based on sulfonamide. Indones J Chem. 2021;21(5).
19. El-Kindy MS, et al. Microwave-assisted synthesis and antioxidant properties of hydrazinyl thiazolyl coumarin derivatives. BMC Chem. 2012; 6:32.
20. Microwave-assisted one-pot three-component synthesis of novel bioactive thiazolyl-pyridazinediones as potential antimicrobial agents against antibiotic-resistant bacteria. Molecules. 2021; 26(14):4260.