Synthesis, Characterization and In Vitro and In Silico Biological Evaluation of New Mannich-Based Rhodanine and Thiazolidine-2,4-dione Derivatives as Potential Anti-Lung-Cancer Agents

Abstract

In this study, 10 new rhodanine and thiazolidine-2,4-dione derivatives based on Mannich-modified vanillin were synthesized, characterized, and evaluated for their anticancer potential against A549 lung cancer and BEAS-2B normal cells. Among them, compound 5c exhibited the most potent anticancer activity, with an IC₅₀ of 2.43 μM and a selectivity index of 10.91, showing higher selectivity than the reference drug sorafenib. Molecular docking studies suggested 5c as a strong potential epidermal growth factor receptor (EGFR) inhibitor, supported by a docking score of –9.827 kcal/mol and key interactions with residues such as Met-793, Leu-788, and Phe-856. Molecular dynamics simulations further confirmed the stability of the 5c-EGFR complex. ADMET predictions indicated favorable pharmacokinetic and safety profiles for 5c, including high permeability, oral absorption, and no significant toxicity. These findings highlight 5c as a promising lead compound for targeted lung cancer therapy, warranting further preclinical studies.

Lung cancer, which is characterized by the uncontrolled proliferation of cells in the lung tissue, is one of the most prevalent and lethal types of cancer worldwide. It is primarily classified into two major subtypes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for approximately 85% of cases and includes adenocarcinoma, squamous cell carcinoma, and large cell carcinoma as its subtypes.[1] SCLC, on the other hand, tends to grow and metastasize more rapidly.[2] Lung cancer poses a significant global health burden due to its high incidence and mortality rates, with limited early detection and effective treatment options contributing to its poor prognosis. Statistically, lung cancer remains a leading cause of cancer-related deaths worldwide. According to the World Health Organization, in 2020, there were approximately 2.2 million new cases of lung cancer, resulting in 1.8 million deaths, which accounts for 18% of all cancer-related fatalities.[3] It is the most common cause of cancer death in men and the second most common in women. The disease often progresses silently, with symptoms typically emerging at advanced stages, leading to poor five-year survival rates, particularly in metastatic cases.

The molecular landscape of lung cancer has been extensively studied and numerous potential therapeutic targets that drive tumor progression have been identified. Among these, the epidermal growth factor receptor (EGFR) plays a pivotal role in the development of NSCLC.[4] EGFR mutations, often found in adenocarcinoma, lead to the continuous activation of downstream signaling pathways that promote uncontrolled cell proliferation, survival, and metastasis.[5] EGFR inhibitors, such as gefitinib, erlotinib, and osimertinib, have demonstrated remarkable efficacy in patients harboring EGFR mutations, significantly improving progression-free survival and quality of life.[6] In addition to EGFR, several other molecular targets have been identified in lung cancer. The vascular endothelial growth factor receptor (VEGFR) family is critical in angiogenesis, a process essential for tumor growth and metastasis.[7] Other key molecular targets include anaplastic lymphoma kinase (ALK) rearrangements, ROS1 fusions, and mutations in the KRAS and BRAF genes. The identification and targeting of these molecular pathways have not only advanced our understanding of lung cancer biology but also paved the way for personalized treatment strategies.[4b] [8]

Thiazolidine-2,4-dione and rhodanine rings are important heterocyclic scaffolds in the development of anticancer agents due to their unique structural and electronic properties, which enable interactions with key molecular pathways involved in cancer progression.[9] Compounds containing these rings exhibit diverse mechanisms of action, including the induction of apoptosis, cell cycle arrest, inhibition of angiogenesis, and disruption of metastasis.[10] Both scaffolds have demonstrated significant potential in modulating critical processes related to tumor growth and progression.[11] These properties make thiazolidine-2,4-dione and rhodanine derivatives promising candidates for anticancer drug development, with opportunities for further optimization to enhance their efficacy and specificity.[12]

The Mannich reaction, a condensation process involving an active hydrogen compound, an aldehyde, and an amine, leads to the formation of versatile compounds known as Mannich bases. In drug design, the Mannich reaction helps optimize the pharmacokinetic properties of drugs, enhancing their distribution in the body. By incorporating a polar group through the Mannich reaction, the hydrophilicity of a drug can be increased, which is beneficial for water-soluble drug profiles. Alternatively, using specific amine reagents can increase lipophilicity, which enhances lipid solubility, depending on therapeutic needs.[13] Aminomethylated drugs resulting from Mannich reactions may also act as prodrugs, releasing their active components through controlled hydrolysis, either by deaminomethylation or diamination.[14] These compounds can be classified into C-, N-, S-, and P-Mannich types, depending on the structure of the active hydrogen compound. Phenolic Mannich bases, part of the C-Mannich class, are particularly significant due to their C–C bond formation and wide range of biological activities. Studies report that these compounds exhibit potent activities, including anticancer,[15] antibacterial,[16] anti-inflammatory,[17] anticholinergic,[18] carbonic anhydrase,[19] and AR inhibition.[20] The broad therapeutic potential of these compounds highlights their value in medicinal chemistry and drug development.

Thiazolidine-2,4-dione and rhodanine scaffolds are valuable in anticancer drug development due to their ability to modulate key pathways such as apoptosis, cell cycle arrest, and metastasis. To enhance their pharmacokinetic properties, we employed the Mannich reaction, a well-established strategy in medicinal chemistry that improves solubility, bioavailability, and metabolic stability. Mannich bases, particularly phenolic C-Mannich compounds, exhibit diverse biological activities, including anticancer effects (Scheme [1]).

In this study, we synthesized and evaluated novel Mannich base derivatives of these scaffolds, leveraging their structural advantages for potential anticancer applications. The aim of this study was to synthesize and characterize novel compounds containing thiazolidine-2,4-dione and rhodanine scaffolds, derived from Mannich bases of vanillin, and to evaluate their anticancer activities in human lung adenocarcinoma epithelial (A549) and human bronchial epithelial (BEAS-2B) cell lines. The anticancer potential was assessed in vitro and in silico through molecular docking, molecular dynamics simulations, and ADME and toxicity predictions. Ten compounds were synthesized and characterized, and their structures were elucidated. This study focused on determining the anti-lung-cancer activity of these compounds and contributed to the development of targeted therapeutic agents for lung cancer.

Chemistry

A novel series of Mannich-based rhodanine and thiazolidine-2,4-dione derivatives was synthesized by following the procedures outlined in Scheme [2].[21] The synthesis commenced with a Mannich reaction, in which vanillin was reacted with five different secondary anilines and formaldehyde to yield Mannich-based aldehydes in high yields. These aldehydes were then subjected to a piperidine-catalyzed Knoevenagel condensation with rhodanine and thiazolidine-2,4-dione, affording the title products in yields ranging from 74 to 80%.

The molecular structures of rhodanine and thiazolidine-2,4-dione compounds 5a–e and 6a–e with Mannich bases were confirmed using ¹H NMR, ¹³C NMR, and ESI-HRMS methods. The olefinic protons of the rhodanine-5-arylidene derivatives exhibited a singlet resonance between 7.50 and 7.24 ppm, whereas those of the thiazolidine-2,4-dione-5-arylidene derivatives displayed a downfield shift, resonating between 7.70 and 7.55 ppm. The aromatic protons attached to the Mannich base gave a doublet resonance signal between 7.16 and 7.01 ppm. The methylenic protons adjacent to the nitrogen and the aromatic ring showed a singlet signal between 3.89 and 3.65 ppm. Methoxy protons resonated at 3.86 to 3.82 ppm. Protons specific to the morpholine or piperazine rings resonated in the aliphatic region, ranging from 3.70 to 2.48 ppm. The C=S carbons of compounds 5a–e resonated between 201.6 and 196.9 ppm, whereas the C=O signals appeared in the range of 180.4 to 168.3 ppm.[9b] [11c] Aromatic carbons were observed between 151.2 and 112.8 ppm. One of the olefinic carbons resonated between 134.1 and 129.7 ppm, while the other was detected between 123.6 and 119.5 ppm. Methylenic carbons showed peaks at 58.2 to 56.7 ppm, and methoxy carbons resonated between 56.3 and 55.8 ppm.

Biological Activity Studies

To evaluate the cytotoxic activity, the target compounds were tested against A549 and BEAS2B cell lines; the results are presented in Table [1]. Compounds 5a and 5b exhibited IC₅₀ values of 4.79 and 2.83 µM, respectively, against A549 cells, indicating potent anticancer activity, while they displayed lower toxicity to BEAS-2B cells (IC₅₀ 23.39 and 25.29 μM, respectively). Compound 5c demonstrated the most potent anticancer activity with an IC₅₀ value of 2.43 μM against A549 cells and an IC₅₀ of 26.51 μM against BEAS-2B cells, resulting in the highest selectivity index (SI) of 10.91. Compounds 5d and 5e also exhibited low IC₅₀ values of 2.10 and 2.11 μM against A549 cells, with corresponding IC₅₀ values of 19.65 and 21.31 µM against BEAS-2B cells, showing good selectivity and strong anticancer potential. Compounds 6a and 6b displayed moderate IC₅₀ values of 3.21 and 2.46 μM, respectively, against A549 cells, and IC₅₀ values of 22.54 and 19.88 μM against BEAS-2B cells. Compounds 6c, 6d, and 6e showed higher IC₅₀ values against A549 cells, with IC₅₀ values of 2.36, 2.40, and 2.74 μM, respectively, and lower selectivity compared to the others, with IC₅₀ values of 13.63, 16.04, and 13.08 μM against BEAS-2B cells.

Sorafenib, used as a reference anticancer drug, exhibited an IC₅₀ value of 2.92 μM against A549 cells and an IC₅₀ value of 15.78 μM against BEAS-2B cells, showing its established anticancer activity. Among the novel compounds, 5c, 5d, and 5e stood out for their potent anticancer activity, particularly 5c, which exhibited the highest selectivity index (SI) of 10.91, indicating a promising therapeutic potential with minimal toxicity to normal cells.

Molecular Docking Studies

The molecular docking analysis of the most active and selective compounds (5c, 5d, and 5e) against the EGFR was performed to assess their potential inhibition mechanism and ligand–protein interactions. The results are summarized in Table [2].

The docking scores from the IFD XP Glide method revealed that 5c exhibited the most favorable binding, with a score of –9.827 kcal/mol, indicating strong interaction with the EGFR active site. Compound 5e also showed significant binding affinity with a score of –9.256 kcal/mol, suggesting its potential as a competitive inhibitor. Compound 5d, with a score of –7.148 kcal/mol, displayed a weaker binding affinity compared to those of 5c and 5e, though it still shows promise as a potential inhibitor.

In terms of binding free energies calculated with MM-GBSA, compound 5c again demonstrated the most potent binding interaction with a ΔG value of –89.21 kcal/mol, reflecting a stable and energetically favorable complex. Compounds 5d and 5e exhibited ΔG values of –69.09 and –53.47 kcal/mol, respectively, indicating less favorable but still significant binding interactions. These results suggest that compound 5c has the strongest potential for binding to EGFR, followed by 5e and 5d. The high binding affinity of 5c, as reflected by both the docking scores and MM-GBSA ΔG values, makes it a promising candidate for further investigation as an EGFR inhibitor in the context of lung cancer therapy. The molecular docking two-dimensional (2D) and three-dimensional (3D) ligand–protein interactions between compound 5c and the active site of EGFR are given in Figure [1].

The 2D ligand–protein interactions of 5c-EGFR complex (Figure [1a]) show that the carbonyl oxygen of the rhodanine ring forms a hydrogen-bond interaction (purple arrows) with Met-793. This hydrogen bond plays a significant role in stabilizing the ligand within the receptor’s active site. There are two additional hydrogen-bond interactions between Leu-788 and the phenolic hydroxyl group and the tertiary nitrogen of piperazine ring. The hydrogen bonding between the ligand and these residues enhances the specificity and affinity of the interaction, suggesting a favorable binding mode for 5c within EGFR. Finally, the phenyl ring of the Mannich base fragment forms a π–π stacking interaction (green line) with Phe-856. Figure [1b] shows the 3D ligand–protein interactions of the 5c-EGFR complex; the yellow dashes depict the hydrogen-bond interactions, and the turquoise dashes represent the π-π stacking interactions. The lengths of hydrogen bonds vary from 1.72 to 2.40 Å and the length of the π–π stacking interaction is 4.94 Å. In Figure [1b], the bluish cloud represents the ligand’s surface binding areas, while the grayish cloud indicates the protein’s binding region on the surface. Both binding surfaces are shown to be perfectly aligned, with the ligand fully occupying the binding region of the protein. This complete overlap between the ligand and protein binding site suggests an optimal fit, demonstrating that the ligand has effectively positioned itself within the receptor’s active site, which is essential for its potential to inhibit the target protein.

The molecular docking studies highlighted the strong potential of compound 5c as an EGFR inhibitor, supported by its superior IFD XP Glide docking score (–9.827 kcal/mol) and MM-GBSA ΔG binding free energy (–89.21 kcal/mol). The binding mode analysis revealed key interactions, including hydrogen bonds with Met-793 and Leu-788 and a π–π stacking interaction with Phe-856, further substantiating its high binding affinity and specificity for the EGFR active site. Compounds 5e and 5d also exhibited significant, though weaker, binding interactions, demonstrating promise as inhibitors. The comprehensive alignment of the ligand and protein binding regions, especially for compound 5c, suggests a favorable and stable interaction, making it a strong candidate as an anticancer agent.

Molecular Dynamics (MD) Studies

In this study, MD simulations were conducted to assess the stability and conformational dynamics of the ligand–protein complexes. The root-mean-square deviation (RMSD) was used to evaluate the overall stability, with lower RMSD values indicating structural integrity. Additionally, the root-mean-square fluctuation (RMSF) was analyzed to identify flexible protein residues, with higher RMSF values reflecting greater mobility. Together, RMSD and RMSF analyses offer insights into the stability and dynamics of the ligand–protein interactions.[22]

In Figure [2], the results of a 100 ns MD simulation of the compound 5c-EGFR complex are presented. Figure [2a] shows the frequency of interactions between the ligand and EGFR residues throughout the simulation. Key hydrogen-bond interactions were observed with residues Met-793 (83% of the simulation time), Lys-728 (96%), Asp-855 (81%), and Leu-788 (61%). Additionally, π–π stacking interactions between the phenyl ring of the ligand and Phe-856 were observed during 22% of the simulation time. These interactions, particularly the hydrogen bonds, contribute to the stability of the ligand in the binding site, maintaining its binding conformation.

The RMSD plots of protein Cα (blue), ligand (pink), and ligand fit on protein (red) are shown in Figure [2b]. The average RMSD of the protein Cα atoms was calculated to be 2.8 Å, indicating a relatively stable protein structure with minor fluctuations during the simulation. The ligand RMSD averaged 1.6 Å, suggesting that the ligand retained its binding mode with slight fluctuations. Moreover, the ligand’s average deviation from its initial position was 0.8 Å, reflecting its stable positioning within the binding site. These observations, supported by the analysis of interactions and RMSD data, demonstrate the stability of the compound 5c-EGFR complex during the 100 ns simulation. This stability, primarily driven by persistent hydrogen-bond interactions and π–π stacking, suggests that compound 5c may exhibit strong binding affinity and inhibitory activity against EGFR.

Figure [2c] and 2d present the RMSF values of protein Cα atoms and ligand atoms, respectively. In Figure [2c], green vertical lines indicate the residues involved in ligand interactions. The average RMSF of protein Cα atoms was calculated to be 1.2 Å, indicating overall stability with minimal fluctuations across the protein backbone. In comparison, the average RMSF of ligand atoms was calculated to be 0.9 Å, reflecting the relatively low flexibility and stable binding of the ligand within the EGFR active site. The alignment of lower RMSF regions with residues in contact with the ligand further highlights the key interactions contributing to the stability of the complex.

Figures [2e] and 2f depict the fractional interaction histograms and the timeline of interactions during the 100 ns simulation, respectively. During the course of the simulation, a functional group can interact with multiple amino acid residues, and a single amino acid residue can interact with multiple functional groups. In such cases, all interactions are aggregated to generate a histogram. In the histogram, green bars represent hydrogen bonds, blue bars indicate water-bridged hydrogen bonds, purple bars correspond to hydrophobic interactions, and red bars denote ionic interactions.

As shown in Figure [2e], the most abundant fractional interactions occur with Met-793, Lys-728, Asp-885, Leu-788, and Phe-856. These amino acids can be considered key residues responsible for inhibition. Figure [2f] provides a timeline of interaction dynamics and continuity throughout the simulation. The residues Met-793, Lys-728, Asp-885, Leu-788, and Phe-856 consistently maintain interactions over the entire simulation period. While other amino acids display transient and less pronounced interactions over time, the stability of the complex is primarily attributed to these key residues.

The 100 ns MD simulation results highlight the stability of the 5c-EGFR complex, primarily driven by persistent interactions with key residues in the EGFR binding site. Hydrogen-bond interactions with Met-793, Lys-728, Asp-885, and Leu-788, along with π–π stacking with Phe-856, play crucial roles in maintaining the compound’s binding conformation. The relatively low RMSD and RMSF values for both the protein and the ligand further support the structural stability and strong binding of the ligand within the active site. These findings suggest that 5c exhibits high binding affinity and the potential for effective inhibitory activity against EGFR, warranting further investigation.

Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) Predictions

ADMET predictions are essential in drug design because they allow pharmacokinetics and safety profiles to be assessed early in development. Poor ADMET properties often cause drug failures, making early evaluation critical. Key factors include hydrogen-bond capacity, solubility, LogP, and rules such as Lipinski’s Rule of Five for oral bioavailability. Studies including Caco-2 and MDCK assess permeability and BBB penetration, while metabolic stability ensures prolonged efficacy. Toxicity predictions evaluate risks such as hepatotoxicity, nephrotoxicity, neurotoxicity, cardiotoxicity, carcinogenicity, and mutagenicity, helping to identify safe, effective drug candidates.[23] ADMET predictions for the most active compounds are presented in Table [3].

The ADMET predictions for the most active compounds (5c, 5d, and 5e) highlight favorable drug-likeness profiles, with all complying with Lipinski’s rule of five and Jorgensen’s rule of three. Compound 5c exhibits the best absorption and permeability (89% human oral absorption, QPPCaco = 207 nm/s, QPPMDCK = 358 nm/s), while 5e also demonstrates good absorption (85%) and permeability. In contrast, 5d has moderate permeability (QPPCaco = 71 nm/s) and lower absorption (75%). Solubility values (QPlogS: –4.8 to –4.5) are within acceptable ranges for all compounds. Toxicity assessments reveal no risks for hepatotoxicity, cardiotoxicity, carcinogenicity, or mutagenicity. However, neurotoxicity is flagged for 5c (64%) and 5e (63%), while nephrotoxicity is noted for 5d (57%). Predicted LD₅₀ values (350–500 mg/kg) place all the compounds in toxicity class 4. Overall, compound 5c exhibits the most balanced ADMET profile, combining high permeability and oral absorption with a favorable safety profile, making it the most promising candidate for further preclinical development.

In conclusion, a novel series of rhodanine and thiazolidine-2,4-dione derivatives derived from Mannich bases of vanillin were synthesized, characterized, and evaluated for their anticancer potential against lung cancer.[21] Ten compounds were synthesized, and their structures were elucidated using techniques such as ¹H NMR, ¹³C NMR, FT-IR, and ESI-HRMS. The in vitro cytotoxicity was assessed on A549 lung cancer and BEAS-2B normal cells, revealing selective anticancer activity. Among them, 5c showed the most potent activity, with an IC₅₀ of 2.43 μM against A549 cells, a selectivity index of 10.91, low toxicity to BEAS-2B cells (IC₅₀: 26.51 μM), and better selectivity than the reference drug sorafenib.

Molecular docking studies indicate that 5c has a strong binding affinity to EGFR, with a high docking score of –9.827 kcal/mol and key interactions with amino acid residues such as Met-793, Leu-788, and Phe-856. Molecular dynamics simulations confirmed the stability of the 5c-EGFR complex, with low RMSD and RMSF values. ADMET predictions reveal favorable drug-like properties for 5c, including high permeability, good oral absorption (89%), and no toxicity, indicating its potential as a drug candidate.

In conclusion, compound 5c has emerged as a promising lead for targeted lung cancer therapy, demonstrating potent anticancer activity, strong EGFR inhibition, and favorable pharmacokinetic properties.

The biological activity[24] and computational studies[25] were conducted following the methodology published in our previous works.

Conflict of Interest

The authors declare no conflict of interest.

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• 25e Kılınç N, Açar M, Tuncay S, Karasakal F. Ö. Lett. Drug Des. Discovery 2022; 19: 996
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• 25f Kılınç N. Int. J. Chem. Technol. 2021; 5: 77
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Corresponding Authors

Publication History

Received: 27 December 2024

Accepted after revision: 17 February 2025

Accepted Manuscript online:
17 February 2025

Article published online:
08 April 2025

© 2025. Thieme. All rights reserved

Georg Thieme Verlag KG
Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany

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• Supplementary Material