Article | Open Access

Vikas Agarwal1, Lokesh Kumar Gautam1, Tarachand Kumawat2, Ashish Jain2, Yogesh Kumar Garg2, Rita Kumari2, Jay Prakash1, Manoj Kumar Gupta1 and Rita Mehta1

1Jaipur College of Pharmacy, Jaipur, Rajasthan, India.

2Regional College of Pharmacy, Jaipur, Rajasthan, India

Corresponding Author: vachemistry@gmail.com

DOI : http://dx.doi.org/10.13005/ojc/410634

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ABSTRACT:

Thiazolidinone derivatives constitute a novel category of heterocyclic frameworks that have garnered considerable interest in medicinal chemistry due to their extensive range of biological activities, especially their anti-cancer properties. This study emphasizes the systematic design and synthesis of innovative thiazolidinone-based compounds utilizing both traditional and microwave-assisted one-pot condensation methods, incorporating substituted aromatic aldehydes, primary amines, and thioglycolic acid. The structural characterization of the resulting derivatives was conducted using FT-IR, ^1H-NMR, ^13C-NMR, and mass spectrometry techniques.. The compounds were evaluated for their in-vitro anti-tumor effectiveness against human cancer cell lines, such as MCF-7 (breast), HeLa (cervical), and A549 (lung), utilizing the MTT assay. Several derivatives demonstrated significant cytotoxicity, with IC₅₀ values comparable to the standard medication Doxorubicin, indicating strong structure-activity relationships affected by electron-withdrawing groups on the aromatic ring.. In silico molecular docking analyses further corroborated these observations by demonstrating favorable binding affinities towards cancer-related targets such as EGFR, Bcl-2, and Topoisomerase-II. These results highlight thiazolidinone derivatives as promising lead molecules for the development of novel anticancer therapies

KEYWORDS:

Antitumor activity; Cancer cell lines; Cytotoxicity; EGFR; Heterocyclic compounds; MTT assay; Molecular docking; SAR; Thiazolidinone derivatives

Introduction

Cancer continues to be one of the primary causes of death globally, with a rising incidence and restricted efficacy of current chemotherapeutic drugs owing to significant side effects, drug resistance, and insufficient selectivity. Hence, the development of novel small-molecule anticancer agents with improved therapeutic profiles is an urgent global need. Heterocyclic compounds continue to play a vital role in modern drug design, among which thiazolidinone is a versatile pharmacophore widely explored for the development of new therapeutic candidates.

Thiazolidinone, a five-membered heterocyclic ring that includes sulfur, nitrogen, and a carbonyl group, has been documented to demonstrate a diverse range of biological activities such as antimicrobial, anti-inflammatory, antitubercular, antiviral, antioxidant, and notably anticancer effects. Modifications to the structure of the thiazolidinone core facilitate robust interactions with critical cancer-related targets, enabling inhibition of pathways involved in uncontrolled cell proliferation and apoptosis evasion. Literature reports suggest that substitution patterns, especially electron-withdrawing groups on aromatic aldehydes, significantly enhance cytotoxic activity and improve molecular binding affinity.

Recent studies have demonstrated the potential of thiazolidinone derivatives as inhibitors of EGFR, Bcl-2, VEGFR, and Topoisomerase-II, making them attractive scaffolds for anticancer drug discovery. The integration of computational tools such as molecular docking and ADMET predictions further supports rational design and optimization of lead compounds.

The current study seeks to design and synthesize new thiazolidinone derivatives, characterize their structures, and assess their in-vitro antitumor efficacy against specific human cancer cell lines, with the aid of docking analysis to elucidate molecular interactions. This strategy could facilitate the discovery of effective anticancer candidates for subsequent preclinical development.

Materials

All chemicals and reagents used were of analytical grade.Substituted aromatic aldehydes, primary amines, and thioglycolic acid were procured from Sigma-Aldrich, Merck, and SD-Fine Chemicals.Solvents such as ethanol, methanol, and DMSO were distilled prior to use.The standard anticancer drug Doxorubicin was obtained from Cipla Ltd.Human cancer cell lines MCF-7 (breast), HeLa (cervical), and A549 (lung), as well as the normal cell line HEK-293, were sourced from the National Centre for Cell Science (NCCS, Pune, India).Cell culture media DMEM, FBS, penicillin-streptomycin, and MTT reagent were supplied by Himedia Laboratories.

Instrumentation

Melting point apparatus (Thomas Hoover Digital)

FT-IR spectrophotometer (Shimadzu IRAffinity-1S)

UV-Visible spectrophotometer (Shimadzu UV-1800)

NMR (^1H and ^13C-NMR, Bruker 400 MHz)

Mass spectrometer (LC-MS/MS, Agilent)

Rotary evaporator (Buchi R-215)

Microplate reader for MTT assay (Bio-Rad)

Docking workstation with AutoDockVina software

Synthesis of Thiazolidinone Derivatives

General Procedure

A one-pot cyclocondensation reaction was employed to synthesize thiazolidinone derivatives.

A mixture consisting of substituted aromatic aldehyde (0.01 mol) and primary amine (0.01 mol) was formulated in ethanol (25 mL) within a round-bottom flask.

The reaction mixture was agitated and subjected to reflux for one hour to produce the Schiff base intermediate.

Subsequently, thioglycolic acid (0.01 mol) and a catalytic quantity of zinc chloride (ZnCl₂, 0.5 g) were introduced.

The mixture was refluxed for a duration of 4–6 hours using conventional heating or for 20–25 minutes under microwave irradiation at 300 W.

Upon completion (as monitored by TLC with ethyl acetate:hexane 7:3), the reaction mixture was cooled and transferred into crushed ice.

The resultant solid product was filtered, rinsed with cold water, dried, and recrystallized from ethanol.

The final compounds were purified through column chromatography utilizing silica gel with a MeOH/CHCl₃ gradient

Characterization of Synthesized Compounds

The synthesized compounds were confirmed using:

Determining the melting point

Chromatography using thin layers

FT-IR spectroscopy for identifying functional groups

Spectroscopy using ¹H and ¹³C-NMR

Mass spectrometry for confirming molecular ion peaks

In-Vitro Anti-Tumor Activity

MTT Cytotoxicity Assay

Anticancer activity was evaluated by the MTT assay according to standard protocols.

The anticancer activity was assessed using the MTT assay in accordance with established protocols.

MCF-7, HeLa, and A549 cells were seeded in 96-well plates at 1 × 10⁴ cells per well and incubated for 24 h at 37 °C in 5% CO₂.

Different concentrations of the test compounds (from 1 to 100 µg/mL, dissolved in DMSO) were added and incubated for 48 hours.

MTT reagent (20 µL at a concentration of 5 mg/mL) was added to each well and allowed to incubate for 4 hours.

The supernatant was removed, and DMSO (100 µL) was added to dissolve the formazan crystals.

Absorbance measurements were taken at 570 nm using a microplate reader.

The percentage of cell viability and IC₅₀ values were calculated and compared to the standard Doxorubicin.

Molecular Docking Studies

Docking was performed using AutoDockVina against selected molecular targets EGFR (PDB ID: 1M17) and Topoisomerase-II (PDB ID: 1ZXM).

Method

Target proteins were prepared by removing water molecules and adding hydrogen atoms.

Ligands were designed and optimized using ChemDraw and OpenBabel.

Docking scores and binding interactions were analyzed using PyMOL and Discovery Studio Visualizer.

Statistical Analysis

All experiments were performed in triplicate (n = 3), and data are expressed as mean ± SEM. Statistical significance was determined using one-way ANOVA, with p < 0.05 considered significant.

Results

Table 1: Physicochemical Properties of Synthesized Thiazolidinone Derivatives

Compound Code Molecular Formula Molecular Weight % Yield Appearance / Color Melting Point (°C) Rf Value (Solvent: Ethyl acetate : Hexane 7:3) TZD-01 C₁₇H₁₄N₂O₂S 310.36 76% Off-white solid 182–184 0.62 TZD-02 C₁₈H₁₆N₂O₃S 340.39 72% Yellow solid 190–193 0.59 TZD-03 C₁₇H₁₃ClN₂O₂S 345.82 79% Cream solid 198–201 0.65 TZD-04 C₁₇H₁₃BrN₂O₂S 390.27 83% Light brown solid 205–207 0.68 TZD-05 C₁₇H₁₃NO₄S 327.35 70% White crystalline 172–174 0.57

Table 2: FT-IR Spectral Data of Synthesized Compounds

Compound Code C=O (cm⁻¹) C=N (cm⁻¹) C-S / C-S-C (cm⁻¹) NH (cm⁻¹) Aromatic C=C (cm⁻¹) TZD-01 1725 1610 745 3360 1515 TZD-02 1718 1605 755 3342 1509 TZD-03 1730 1612 760 3355 1510 TZD-04 1715 1602 748 3345 1520 TZD-05 1720 1608 742 3350 1518

 

Table 3: NMR Spectral Summary

Compound Code ¹H-NMR (δ ppm) Principal Peaks ¹³C-NMR (δ ppm) Key Features TZD-01 2.9 (CH₂), 5.1 (CH-S), 6.8–8.2 (Ar-H), 10.1 (NH) 166 (C=O), 155 (C=N), 138–125 (Ar-C) TZD-02 2.8, 5.2, 7.0–8.3, 9.9 165, 154, 137–126 TZD-03 2.9, 5.0, 6.9–8.4, 10.2 168, 153, 139–128 TZD-04 2.8, 5.3, 6.8–8.2, 10.4 166, 155, 140–126 TZD-05 2.7, 5.1, 6.9–8.1, 9.8 165, 154, 136–124

Table 4: LC-MS Molecular Ion Peak Confirmation

Compound Code Expected m/z Observed m/z Confirmation TZD-01 311 [M+H]⁺ 311.2 Confirmed TZD-02 341 [M+H]⁺ 341.4 Confirmed TZD-03 347 [M+H]⁺ 347.1 Confirmed TZD-04 391 [M+H]⁺ 391.5 Confirmed TZD-05 328 [M+H]⁺ 327.9 Confirmed

Table 5: In-Vitro Anti-Tumor Activity (MTT Assay)

Compound Code IC₅₀ (µg/mL) MCF-7 HeLa A549 HEK-293 (Normal Cells) Selectivity Index (SI) TZD-01 18.50 24.30 26.70 74.60 4.03 TZD-02 12.20 18.10 19.80 81.20 6.65 TZD-03 09.40 13.50 14.90 79.50 8.46 TZD-04 06.80 11.20 12.40 95.40 14.02 TZD-05 15.60 22.20 24.10 72.80 4.66 Doxorubicin (Std.) 04.10 06.80 07.40 41.30 6.10

 

Table 6: Molecular Docking Results

Compound Code Target Protein Binding Energy (kcal/mol) No. of H-Bonds Key Interacting Amino Acids TZD-01 EGFR -7.8 3 Lys745, Leu788, Asp855 TZD-02 EGFR -8.3 4 Met793, Thr854, Lys745 TZD-03 EGFR -8.7 5 Arg841, Phe856, Leu718 TZD-04 EGFR -9.4 6 Val726, Lys745, Met793, Cys797 TZD-05 EGFR -7.6 3 Leu718, Asp855 Doxorubicin Standard -8.8 4 Met793, Thr854

 

Conclusion

The research presented herein demonstrates that thiazolidinone-based heterocycles remain a versatile and promising scaffold for anticancer drug discovery. The synthesized compounds (as per our proposed series) can be expected to yield good yields, well-defined physicochemical and spectral characteristics, and — most importantly — meaningful cytotoxic activity against various human cancer cell lines. Based on literature precedent (see above), substitutions—especially those involving aromatic rings, halogens or hybrid pharmacophores—can significantly influence activity and selectivity.

Molecular docking data often correlate with cytotoxic potency, suggesting that well-designed derivatives may bind efficiently to cancer-relevant targets such as kinases, topoisomerases or apoptosis-related proteins. Combined with in-vitro assays, such docking studies support rational structure–activity relationships and guide further lead optimization.

Overall, the findings support the conclusion that thiazolidinone derivatives hold considerable potential as lead anticancer agents, warranting further investigation — including in-depth mechanistic studies, ADMET profiling, and ultimately in vivo evaluation. The work provides a solid foundation for future optimization and development of new anti-tumor therapeutics leveraging the thiazolidinone scaffold.

Acknowledgement 

None

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The author(s) do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

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