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Abstract

An oxothiazolidinylidene derivative, Ethyl (Z)-2-((Z)-3-(4-acetamidophenyl)-2-(((E)-1-(4-acetamidophenyl)ethylidene)hydrazono)-4-oxothiazolidin-5-ylidene)acetate (Z-EAHT) was rationally designed and evaluated as a potential epidermal growth factor receptor inhibitor targeting both wild-type and T790M mutant forms. Computational analyses demonstrated favorable electronic properties, strong ATP-binding pocket compatibility, and stable protein–ligand interactions supported by 200 ns molecular dynamics simulations and Molecular Mechanics/Generalized Born Surface Area (MM-GBSA) binding energy calculations. In silico ADME-Tox profiling predicted acceptable solubility, moderate absorption, and a favorable safety profile. Experimentally, Z-EAHT exhibited potent epidermal growth factor receptor inhibition (IC₅₀ = 0.012 µM for wild-type epidermal growth factor receptor; 0.013 µM for T790M mutant epidermal growth factor receptor) and demonstrated selective cytotoxicity against A549, MCF-7, and HepG-2 cancer cells while sparing normal WI-38 cells. Mechanistic investigations confirmed apoptosis induction and epidermal growth factor receptor downregulation. Overall, Z-EAHT emerged as a promising mutation-tolerant epidermal growth factor receptor inhibitor warranting further preclinical investigation.

Keywords

ADME-Tox anti-cancer epidermal growth factor receptor inhibitors molecular dynamics simulations principal component analysis of trajectories

Introduction

Although epidermal growth factor receptor (EGFR) is a validated therapeutic target,^1,2 resistance mutations – particularly T790M – limit the long-term efficacy of first-generation tyrosine kinase inhibitors (TKIs). Therefore, the development of mutation-tolerant inhibitors remains a significant challenge.^3,4

Computational drug design has become an essential component of modern drug discovery, integrating structure-based design, molecular docking, density functional theory (DFT), and molecular dynamics (MD) simulations to optimize structural and electronic properties.^5–8

In our recent work, computational approaches were employed to rationalize the design strategy in the context of EGFR inhibition. DFT calculations were utilized to evaluate electronic reactivity and molecular stability, while molecular docking was applied to assess compatibility within the ATP-binding pocket. MD simulations were conducted to examine conformational stability and potential mutation tolerance, particularly in resistant EGFR variants. In addition, Molecular Mechanics/Generalized Born Surface Area (MM-GBSA) analysis provided quantitative estimation of binding energetics to support structure–activity interpretation.^9–13 When paired with in silico ADME-Tox evaluations, these methods allow the identification of promising drug-like candidates while reducing experimental time, cost, and effort.^14,15

In this context, oxothiazolidinylidene derivatives have attracted growing attention due to their versatile biological activities, including antitumor,¹⁶ anti-inflammatory,¹⁷ and antimicrobial effects.^18–20 The oxothiazolidinylidene core, when linked with heteroaromatic or hydrazono moieties, has been shown to enhance molecular flexibility and facilitate key hydrogen bonding and π–π stacking interactions within the ATP-binding cleft of kinases, thereby improving inhibitory potency and mutation tolerance.^21,22 Although oxothiazolidinylidene derivatives had previously been explored for various biological activities, their application as EGFR-targeting inhibitors remained limited. Most reported EGFR inhibitors (EGFRIs) relied on classical hinge-binding heterocycles and established pharmacophoric arrangements. In contrast, Z-EAHT introduced a hybrid oxothiazolidinylidene framework that was strategically designed to maintain hinge-region anchoring while extending hydrophobic interactions within the ATP-binding pocket.

This structural configuration represented a deviation from conventional EGFRI scaffolds and offered a distinct interaction profile that may have contributed to improved binding stability.

Rationale

EGFRIs share four key pharmacophoric features essential for binding to the EGFR active site. Using Erlotinib I as an example: (1) a planar heteroaromatic system that fits into the adenine-binding pocket for π–π stacking, (2) a terminal hydrophobic head occupying hydrophobic region I to enhance affinity, (3) a linker NH group forming hydrogen bonds in the linker region, and (4) a hydrophobic tail extending into hydrophobic region II for complex stabilization^23–27 (Figure 1(a)).

Figure 1.

(a) Core pharmacophoric characteristics of known EGFR inhibitors. (b) Strategy for developing a Z-EAHT as a novel EGFR inhibitor.

Our group previously developed a thiadiazole-based EGFRI (N-ASTPC) that exhibited encouraging activity, motivating further structural refinement toward improved mutation-tolerant EGFR inhibition.¹⁰ Based on its promising results, N-ASTPC was selected as the lead compound for developing a potential anticancer oxathiazolidine derivative targeting EGFR. The molecule was designed to incorporate the key structural features characteristic of EGFR inhibitors (Figure 1(b)). In our design strategy, the oxothiazolidinylidene scaffold (Z-EAHT) was employed as a heterocyclic core to occupy the adenine-binding pocket of EGFR, with 4-acetamidophenyl groups on both sides serving as hydrophobic moieties that interact with hydrophobic regions I and II. To optimize the molecular fit and expand the Structure–Activity Relationship (SAR), the benzene sulfonamide of the lead scaffold (N-ASTPC) was replaced with an acetamidophenyl group, retaining hydrophobicity for terminal pocket interactions while introducing an additional hydrogen-bond donor-acceptor through the acetamide functionality. This modification was intended to enhance ATP-pocket accommodation and maintain key interactions required for effective inhibition of both wild-type and mutant EGFR forms. Also, provided a favorable balance between hydrophobicity and polarity, enhancing π–π stacking with aromatic residues and directional hydrogen-bonding. Moreover, incorporation of an α,β-unsaturated ethyl ester fused to the heterocyclic core adds structural rigidity and extends the π-system, further stabilizing binding within the EGFR active site.

To the best of our knowledge, this study represented an oxothiazolidinylidene-based scaffold demonstrating comparable sub-nanomolar inhibitory potency against both wild-type EGFR (EGFR-WT) and the resistant EGFR-T790M mutation. Unlike previously reported EGFR inhibitors, this work integrated long-timescale MD simulations, free energy decomposition, ProLIF interaction profiling, principal component analysis of trajectories (PCA-T), and free energy landscape (FEL) analyses with experimental validation, providing comprehensive mechanistic insight and establishing Z-EAHT as a mutation-tolerant EGFR inhibitor candidate.

Results and discussion

In silico investigations

DFT calculations

DFT were conducted using the B3LYP/6-31 G + (d, p) level of theory within the Gaussian 09 software. The molecular structure of Z-EAHT was comprehensively optimized to ascertain the global minimum energy configuration, and the results are presented in Figure 2(a).

Figure 2.

Optimized structure (a), HOMO and LUMO distributions (b), the plot of TDOS (c), Mulliken charge distribution (d), and ESP (e) at the B3LYP/6-31G+ (d, p) level for Z-EAHT.

The analysis of frontier molecular orbitals (FMO) sheds light on the electronic configuration and reactivity of molecules, which is essential for comprehending their interactions with biological targets. FMO analysis examines the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) to reveal reactive regions. The energy gap between these orbitals indicates chemical stability, with smaller gaps suggesting higher reactivity. FMO analysis helps predict molecular interactions by identifying nucleophilic or electrophilic sites, aiding drug design in biochemical systems.²⁸

This is depicted in Figure 2(b), the HOMO is predominantly located on the electron-rich nitrogens of hydrazine, nearby aromatic rings, and the acetamidophenyl group, suggesting that these areas serve as electron donors when interacting with the target protein. The LUMO is distributed over the thiazolidinone moiety and carbonyl, indicating their roles as electron acceptors. This specific HOMO and LUMO distribution facilitates charge transfer throughout the molecular structure and predicts significant chemical interactions with the biological target. The HOMO/LUMO gap is shown in Figure 2(b) was calculated to be relatively small (3.318 eV), indicating efficient electron mobility across Z-EAHT and favorable interactions with protein residues. A low HOMO/LUMO gap (E_gap) enhances both the chemical stability of the molecule against spontaneous excitation and its selectivity in biological interactions

Table 1 presents the calculated global chemical parameters for Z-EAHT. The ionization potential (IP) is moderate at 6.26 eV, indicating both chemical reactivity and rational stability, which enhances the selective binding with EGFR. The electron affinity (EA) value of 2.94 eV is balanced, allowing the molecule to function as either an electron donor or acceptor, depending on the interaction requirements with the EGFR. A negative chemical potential (μ) value suggests resistance to the spontaneous escape of electrons. The electronegativity value (χ, 4.60 eV) is consistent with the charge transfer and hydrogen bond formation. The balanced hardness and softness (η and σ) values support both kinetic stability and chemical reactivity, which are desirable characteristics of cancer-inhibiting agents. The high electrophilicity (ω = 17.61 eV) indicates an efficient capacity to accept electrons, thereby enhancing the reactivity of Z-EAHT toward nucleophiles of EGFR amino acid residues. These findings underscore the balanced chemical reactivity and electronic stability, supporting the efficacy of Z-EAHT as a potent cancer-inhibiting drug. The Total density of states (DOS) spectrum is shown in Figure 2(c) and offers a detailed perspective on Z-EAHT’s electronic structure. The electronic states are not sharply isolated but are distributed across a wide range of energies. This balanced electronic density suggests moderate localization, which facilitates the molecule’s interaction with the protein target and supports the electronic dynamic response necessary for biological functions.

Table 1.

The DFT calculated global reactivity parameters for Z-EAHT.

IP	EA	μ (eV)	χ (eV)	η (eV)	σ (eV^-1)	ω (eV)	Dm (Debye)	TE (eV)	∆N_max	∆E (eV)
6.264	2.938	−4.601	4.601	1.663	0.601	17.606	2.436	−54,855.691	2.766	−17.606

As shown in Figure 2(d), the electron density is distributed throughout Z-EAHT, emphasizing the strong electrophilic and nucleophilic centers. This distribution allows the compound to interact with the protein target via aromatic stacking and direct hydrogen bond formation. The acetamidophenyl moiety, along with carbonyl oxygen, nitrogen, and C29, C31, and C23, carries negative charges, making them susceptible to electrophilic attack and serving as electron-donating centers. Conversely, the C30 and C25 sites are positive centers poised for nucleophilic interactions with the protein target through electron density acceptance. The thiazolidine ring as shown in Figure 2(d), contains both electrophilic and nucleophilic centers, which are crucial for binding to the target protein. This unique distribution of the Mulliken charge effectively facilitates electronic transitions and enables binding with the target through electron donation and electron acceptance. Consequently, electrophilic centers can bind to the nucleophilic residues of the EGFR pocket, whereas nucleophilic centers interact with electrophilic amino acids, thereby enhancing the inhibitory performance.

The mapping of the Electrostatic potential (ESP) surface of Z-EAHT is illustrated in Figure 2(e). The ESP map provides a visualization of the charge distribution and reactivity of heterocyclic drugs. The regions of electron deficiency (positive values in blue) are localized over the hydrogen atoms of the acetamido and hydrazono groups, which can form hydrogen bonds and are involved in nucleophilic attack. The electron-rich zones, which have negative values (red zones), are concentrated over the nitrogen and carbonyl oxygen atoms. These red zones serve as nucleophilic sites for the binding of proteins. The balanced electrostatic potential surface indicates that the designed molecule has both reactive zones and relatively inert regions, which support selective interactions with the target proteins.

Molecular docking

To investigate the potential mechanism of EGFR inhibition by Z-EAHT, molecular docking simulations were conducted. The EGFR X-ray crystal structure bound to Erlotinib (PDB ID: 4HJO) was retrieved from the Protein Data Bank. The re-docking of Erlotinib successfully reproduced its reported binding mode within the EGFR active site, showing close alignment with the crystallographic pose and a low RMSD value. The ligand preserved the key hydrogen bond with Met769, consistent with previously reported data, confirming the validity of the docking protocol (Figure 3(a)). Docking results showed that Z-EAHT fits snugly into the EGFR binding pocket, adopting a stable conformation that differs from conventional hinge-binding inhibitors. The ligand formed one critical hydrogen bonds with Met769, anchoring it securely within the active site. Moreover, extensive hydrophobic contacts with Leu820, Lys721, Ala719, Val702, Cys773, Leu768, and Arg817 further stabilized the complex (Figure 3(b)). This network of hydrogen-bonding and hydrophobic interactions indicates a strong and favorable binding of Z-EAHT to EGFR. Such a binding pattern may underlie its observed in vitro inhibitory activity and highlights the compound’s potential to engage EGFR active site. To validate the docking results, the binding mode and docking score of the synthesized compound were directly compared with the standard drug (Erlotinib). Z-EAHT demonstrated a docking score of –20.99 kcal/mol, which was better than that of the reference drug (−20.21 kcal/mol), suggesting a favorable binding affinity toward the target protein. While both ligands preserved the essential hinge-region interaction with Met769, Z-EAHT established additional hydrophobic contacts, suggesting enhanced stabilization within the binding pocket (Table 2).

Figure 3.

(a) 3D and 2D interactions of Erlotinib with EGFR active site. (b) 3D and 2D interactions of Z-EAHT with EGFR active site.

Table 2.

Comparative docking analysis of Z-EAHT and Erlotinib against EGFR (PDB ID: 4HJO).

Parameter	Z-EAHT	Erlotinib
Docking score (kcal/mol)	–20.99	–21.10
Number of hydrogen bonds	1	1
Key H-bond residues	Met769	Met769
pi-alkyl interactions	9	5
pi-Sigma interactions	–	1
pi-cation interactions	–	1
pi-sulfur interactions	1	–

MD simulations

MD simulations demonstrated notable conformational stability and distinct dynamic behavior of the EGFR protein upon complexation with Z-EAHT. As shown in Figure 4(a), the backbone of EGFR in the EGFR_Z-EAHT complex maintained a steady root mean square deviation (RMSD) of approximately 2.5 Å toward the end of the simulation, suggesting limited structural deviations from its initial conformation. Similarly, the RMSD trajectory of Z-EAHT (Figure 4(b)) exhibited consistent stability, fluctuating minimally within the range of 3–3.5 Å throughout the simulation period.

Figure 4.

Molecular dynamics analyses of the EGFR–Z-EAHT complex: (a) RMSD profile of the EGFR backbone, (b) RMSD of Z-EAHT, (c) RoG of EGFR, (d) SASA of EGFR, (e) fluctuations in the number of hydrogen bonds, (f) RMSF of EGFR residues, and (g) center-of-mass distance between Z-EAHT and EGFR.

Further structural evaluations using the radius of gyration (ROG) (Figure 4(c)) and solvent-accessible surface area (SASA) (Figure 4(d)) indicated no substantial alterations in the protein’s compactness or solvent exposure, implying that ligand binding did not induce major conformational rearrangements in EGFR. Hydrogen bond analysis (Figure 4(e)) revealed the presence of an average of two persistent hydrogen bonds between Z-EAHT and EGFR, with occasional transient increases to three or four bonds in specific simulation frames, suggesting stable yet dynamic intermolecular interactions.

Residue-level flexibility, as depicted by the root mean square fluctuation (RMSF) of C-alpha atoms (Figure 4(f)), showed minimal deviations ranging from 1 to 1.8 Å across most residues, reflecting overall rigidity of the protein-ligand complex. Notably, residue ALA847:HB2 exhibited a higher RMSF value of 8.29 Å, possibly indicating localized flexibility or transient conformational movement in that region. The center-of-mass distance analysis (Figure 4(g)) confirmed consistent ligand positioning, maintaining a stable average separation of approximately 12 Å until the end of the simulation.

Collectively, these results confirm that the EGFR_Z-EAHT complex remains dynamically stable under simulated physiological conditions, with minimal structural changes and persistent intermolecular interactions, supporting the hypothesis of a robust and well-maintained binding conformation.

Figure 5 summarized the MM-GBSA binding free energy analysis of the EGFR–Z-EAHT complex. The calculated total binding free energy was −40.01 kcal/mol, indicating a strong and favorable interaction. Van der Waals (−54.9 kcal/mol) and electrostatic contributions (−29.8 kcal/mol) were identified as the primary stabilizing forces, highlighting the synergistic role of nonpolar and polar interactions within the EGFR binding pocket (Figure 5(a)).

Figure 5.

(a) Individual MM-GBSA energy components and their corresponding values for the EGFR–Z-EAHT complex. (b) Per-residue binding free energy analysis of the EGFR–Z-EAHT complex.

Per-residue energy decomposition analysis of amino acids located within 1 nm of the ligand further identified key contributors to binding stabilization (Figure 5(b)). Notably, Thr766, Gln767, Lys692, Ile765, Leu764, Asn818, and Gly700 exhibited favorable energetic contributions, while ILE829 showed a minor unfavorable contribution, possibly due to steric or electrostatic effects.

Overall, the MM-GBSA results confirmed that Z-EAHT achieved stable binding within the EGFR active site, predominantly driven by van der Waals and electrostatic interactions.

Protein–ligand interaction fingerprint (ProLIF) analysis provided additional insight into the residue-level interactions governing Z-EAHT recognition within the EGFR binding pocket. As presented in Supplementary Data (Fig. S2.2.1 and S2.2.2), several residues exhibited high-frequency hydrophobic contacts (>90%) throughout the simulation, including Leu694, Val702, Ala719, Cys791, and Met769. Notably, Met769 also demonstrated significant hydrogen bond acceptor and van der Waals interactions, indicating its key stabilizing role. These findings indicate that persistent hydrophobic contacts substantially contributed to complex stabilization, complementing the energetic profile obtained from the MM-GBSA analysis.

PCA-T was performed to investigate the dominant collective motions of the EGFR–Z-EAHT complex. The first principal component accounted for approximately 73.5% of the total variance, indicating that the essential dynamics were largely confined within a reduced conformational subspace. Detailed scree plot and cosine content analyses are provided in Supplementary Data (Fig. S2.2.3).

Projection of the trajectory onto the PC1–PC2 plane revealed well-defined and recurrent low-energy basins (Figure 6), indicating stable conformational sampling throughout the simulation. The presence of shallow energy barriers between adjacent minima suggested smooth transitions between closely related metastable states without structural destabilization. For completeness, additional projections onto the PC1–PC3 and PC2–PC3 planes are provided in Supplementary Data (Fig. S2.2.4).

Figure 6.

Two- and three-dimensional FEL projections of the EGFR–Z-EAHT trajectory mapped onto PC1-PC2. The accompanying energy scale represents free-energy values calculated using the gmx sham algorithm.

Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) profiling study

The comparative ADMET analysis between Z-EAHT and the reference drug Erlotinib highlights several pharmacokinetic distinctions that may influence their therapeutic profiles (Table 3). Z-EAHT exhibited very low predicted blood–brain barrier (BBB) permeability, suggesting limited central nervous system (CNS) exposure. While reduced CNS penetration may potentially minimize CNS-related adverse effects in systemic indications, it could also represent a limitation in the context of CNS malignancies or brain metastases, where adequate brain exposure is therapeutically desirable. In contrast, Erlotinib exhibited higher predicted BBB permeability, which may contribute to its CNS activity but may also be associated with increased neurotoxicity.²⁹ The solubility of Z-EAHT is predicted to be good, indicating better aqueous compatibility and formulation potential compared to Erlotinib, which displays low solubility. While Z-EAHT shows moderate absorption, slightly lower than Erlotinib’s good absorption, this could potentially be improved through formulation optimization due to its favorable solubility. Both compounds are predicted to be non-inhibitors of CYP2D6, indicating a low likelihood of drug–drug interactions mediated by this enzyme. In addition, Z-EAHT has plasma protein binding (PPB) below 90%, suggesting a higher proportion of free drug available for biological activity, whereas Erlotinib’s PPB exceeds 90%, implying stronger protein association and potentially reduced free drug concentration. Overall, the ADMET profile of Z-EAHT indicates a promising balance between solubility, bioavailability, and safety, supporting its further evaluation as a potential EGFR inhibitor.

Table 3.

Predicted ADMET properties of Z-EAHT compared with the reference EGFR inhibitor Erlotinib.

Comp.	BBB level	Solubility level	Absorption level	CYP2D6 prediction	Plasma protein binding
Z-EAHT	Very low	Good	Moderate	Non inhibitor	Less than 90%
Erlotinib	High	Low	Good	Non inhibitor	More than 90%

In silico toxicity studies

The predicted toxicity parameters of Z-EAHT were compared with those of the reference EGFR inhibitor Erlotinib to evaluate its preliminary safety profile (Table 4). Both compounds were predicted as non-toxic according to the DTP model and non-mutagenic in the Ames test, suggesting a low genotoxic risk. However, notable differences were observed in carcinogenic potency and tolerability. Z-EAHT exhibited a higher carcinogenic potency TD₅₀ value (124.82 mg/kg/day) compared to Erlotinib (8.05 mg/kg/day), indicating a significantly lower potential for carcinogenicity in rats. The rat maximum tolerated dose (R-MTD) for Z-EAHT (0.0376 g/kg) was lower than that of Erlotinib (0.0828 g/kg), suggesting Z-EAHT may elicit pharmacological effects at lower doses, potentially enhancing its therapeutic index.

Table 4.

In silico toxicity and safety profile of Z-EAHT and Erlotinib.

Comp.	DTP	(TD₅₀)^a	Ames M	R-MTD^b	LD₅₀^b	R-LOAEL^b	SI	OI
Z-EAHT	Non-Toxic	124.82	Non-Mutagen	0.037	0.613	0.0293	Non-Irritant	Mild
Erlotinib	Non-Toxic	8.05	Non-Mutagen	0.082	0.662	0.0359	Non-Irritant

mg/kg/day.

g/kg.

Furthermore, the rat oral LD₅₀ of Z-EAHT (0.613 g/kg) and Erlotinib (0.662 g/kg) were comparable, indicating similar acute oral toxicity. The chronic R-LOAEL (lowest observed adverse effect level in Rats) of Z-EAHT (0.0293 g/kg) was slightly lower than that of Erlotinib (0.0359 g/kg), implying that prolonged exposure to Z-EAHT remains within an acceptable safety margin. Both compounds were predicted to be non-irritant to skin (SI) and mild ocular (OI) irritants, suggesting a favorable local tolerability profile.

Overall, the toxicity assessment supports Z-EAHT as a relatively safe EGFR inhibitor candidate, with lower predicted carcinogenic potential and comparable systemic tolerance to Erlotinib, making it a promising scaffold for further preclinical development. The ADMET and toxicity predictions were performed using the validated QSAR-based ADMET module in Discovery Studio 4. Although these models provide reliable early-stage estimations, the results remain predictive and require further experimental validation.

Synthesis

The design and synthesis of the EGFR (epidermal growth factor receptor) enzyme inhibitor were achieved through a systematic synthetic route. Initially, 4-aminoacetophenone 1 was acetylated with acetic anhydride to afford derivative 2.^11,30–32 Subsequent treatment of derivative 2 with methyl hydrazinecarbodithioate 3 in anhydrous ethanol under reflux furnished the key intermediate 4.^11,30–32 Condensation of intermediate 4 with N-(4-aminophenyl)acetamide 5 produced the (E)-N-(4-(1-(2-((4-acetamidophenyl)carbamothioyl)hydrazono)ethyl)phenyl)acetamide 6. Finally, derivative 6 was treated with diethyl acetylene dicarboxylate DEAD in ethanol under stirring for 8 h, affording ethyl (Z)-2-((Z)-3-(4-acetamidophenyl)-2-(((E)-1-(4-acetamido phenyl) ethylidene)hydrazono)-4-oxothiazolidin-5-ylidene)acetate 8 (Z-EAHT) as presented in Scheme 1).

Scheme 1:

General Route to Synthesis of Ethyl Acrylate-Thiazolidinone Derivatives 7, Reagents and Conditions: 1.

The structural integrity of the synthesized compound was confirmed through comprehensive spectroscopic and analytical characterization. The FTIR spectrum displayed characteristic absorption bands corresponding to NH stretching (3328 and 3204 cm^-1), carbonyl (C=O) stretching (1684 cm^-1), and imine (C=N) functionality (1593 cm^-1), consistent with the proposed structure. The ¹H NMR spectrum showed two distinct singlets attributable to NH protons along with well-resolved aromatic and aliphatic signals matching the expected proton environments. The ¹³C NMR spectrum further confirmed the presence of carbonyl, aromatic, and aliphatic carbons at the anticipated chemical shifts. Elemental analysis values were in close agreement with the calculated composition, confirming molecular integrity. In addition, high-performance liquid chromatography (HPLC) analysis indicated 100% purity, demonstrating the absence of detectable impurities.

Biological evaluation

Enzyme inhibition

The EGFR inhibitory potency of the newly designed compound Z-EAHT was evaluated against both EGFR-WT and the T790M mutant form, with Erlotinib serving as the reference standard (Table 5). Z-EAHT demonstrated potent inhibitory activity with an IC₅₀ of 0.012 ± 0.001 µM against EGFR-WT, which is more than twofold stronger than Erlotinib (0.029 ± 0.001 µM). Against the drug-resistant EGFR-T790M mutant, Z-EAHT retained its potency (IC₅₀ = 0.013 ± 0.001 µM), again outperforming Erlotinib (0.030 ± 0.004 µM).

Table 5.

EGFR inhibitory activity (IC₅₀, µM) of Z-EAHT and Erlotinib against wild-type (EGFR-WT) and mutant (EGFR-T790M) forms.

	Z-EAHT	Erlotinib
EGFR -WT IC₅₀ µM	0.012 ± 0.001	0.029 ± 0.001
EGFR-T79 IC₅₀ µM	0.013 ± 0.001	0.03 ± 0.004

The enhanced potency of Z-EAHT may be attributed to the presence of its hydrazono-oxothiazolidinylidene core, which likely enables additional hydrogen bonding and π–π stacking interactions within the EGFR active site, stabilizing the ligand–receptor complex. In contrast, Erlotinib’s planar quinazoline ring system, while effective, can suffer from reduced affinity toward mutant forms due to decreased flexibility and steric constraints.

Overall, these results indicate that Z-EAHT is a more potent and mutation-tolerant EGFR inhibitor compared to Erlotinib. Its low nanomolar IC₅₀ values for both EGFR-WT and EGFR-T790M, combined with its favorable ADMET and toxicity profile, highlight Z-EAHT as a promising candidate for further in vitro and in vivo evaluation as a potential next-generation EGFR inhibitor capable of overcoming resistance associated with existing TKIs.

Cytotoxicity

The in vitro cytotoxicity evaluation of Z-EAHT and Erlotinib was performed against a panel of human cancer cell lines—HepG-2 (hepatocellular carcinoma), MCF-7 (breast adenocarcinoma), and A549 (lung carcinoma)—as well as the WI-38 normal lung fibroblast line to assess selectivity and safety (Table 6). Z-EAHT exhibited potent anticancer activity with IC₅₀ values of 7.12 ± 0.98 µM (HepG-2), 5.66 ± 0.16 µM (MCF-7), and 3.78 ± 0.25 µM (A549), all of which are comparable to or stronger than Erlotinib (7.80 ± 0.72, 7.30 ± 0.32, and 6.65 ± 0.78 µM, respectively). Notably, Z-EAHT demonstrated superior potency in A549 cells, a lung cancer line known to overexpress EGFR, with nearly a twofold improvement in cytotoxic activity compared to Erlotinib.

Table 6.

Cytotoxic activity (IC₅₀, µM) of Z-EAHT and Erlotinib against cancer and normal cell lines.

	WI-38	HepG-2	MCF-7	A549
Erlotinib	31.10± 1.18	7.80± 0.72	7.30± 0.32	6.65 ± 0.78
Z-EAHT	96.5± 1.25	7.117± 0.98	5.66± 0.16	3.78± 0.25

In contrast, Z-EAHT displayed much lower cytotoxicity toward normal WI-38 cells (IC₅₀ = 96.5 ± 1.25 µM) than Erlotinib (31.10 ± 1.18 µM), indicating enhanced selectivity and a wider therapeutic window. This improved selectivity index (SI) suggests that Z-EAHT may exert stronger tumor-specific effects while minimizing toxicity to normal cells. The reduction in off-target cytotoxicity may be attributed to the oxothiazolidinylidene scaffold, which could improve receptor specificity and reduce nonspecific interactions with normal cellular proteins.

Collectively, these findings confirm that Z-EAHT exhibits potent and selective anticancer activity across multiple EGFR-expressing cell lines, surpassing Erlotinib in both efficacy and safety toward normal cells. This profile supports Z-EAHT as a promising candidate for further in vivo antitumor and mechanistic studies, particularly in EGFR-driven lung and breast cancers.

Immunofluorescence analysis

The immunofluorescence analysis (Figure 7) illustrates the effect of Z-EAHT on EGFR protein expression in A549 cells compared with untreated controls. DAPI staining (blue) revealed normal nuclear morphology, while the green fluorescence from the anti-EGFR antibody indicated the abundance and localization of EGFR protein within the cells. In the control group, intense and widespread EGFR staining was observed, confirming high basal receptor expression typical of A549 cells. In contrast, cells treated with Z-EAHT displayed a notable reduction in EGFR-associated fluorescence intensity, indicating suppressed EGFR protein expression following treatment. The merged images confirm that Z-EAHT effectively downregulates EGFR without significantly altering overall cell morphology or nuclear integrity.

Figure 7.

Immunofluorescence analysis showing the effect of Z-EAHT on EGFR protein expression in A549 cells. (Cells were stained with DAPI (nuclei, blue) and anti-EGFR antibody (green); merged images indicate EGFR localization and suppression post-treatment.)

Quantitative analysis further supports these observations: EGFR expression in the Z-EAHT-treated group decreased significantly (p < 0.05) from approximately 80% in control cells to about 65% post-treatment. This downregulation is consistent with Z-EAHT’s potent in vitro EGFR inhibitory activity (IC₅₀ = 0.012 µM) and correlates with its strong cytotoxic effects. Overall, the immunofluorescence confirms that Z-EAHT effectively suppresses EGFR expression in lung cancer cells, providing further mechanistic evidence that its anticancer activity is mediated through target-specific EGFR inhibition and signaling attenuation. These findings reinforce the potential of Z-EAHT as a next-generation EGFR-targeted anticancer candidate with dual inhibitory and downregulatory properties.

Quantification of apoptosis-related proteins using enzyme-linked immunosorbent assay

The apoptotic induction potential of Z-EAHT was further investigated by quantifying the expression levels of key apoptosis-related markers—Bax, Bcl-2, and Caspase-3—in A549 lung carcinoma cells (Table 7). Treatment with Z-EAHT resulted in a remarkable upregulation of pro-apoptotic proteins and suppression of the anti-apoptotic factor, confirming the compound’s ability to trigger apoptosis via the intrinsic mitochondrial pathway.

Table 7.

Effect of Z-EAHT on apoptotic biomarkers in A549 cells.

Cod.	Bax conc.	FLD	Bcl-2 conc.	FLD	Casp. 3 conc.	FLD
Z-EAHT	283.99	5.594	2.07	0.3778	308.9	5.797
Cont. A549	50.77	1	5.48	1	53.29	1

Concentrations and fold-level differences (FLD) of Bax, Bcl-2, and Caspase-3 relative to untreated control.

Specifically, Bax expression increased from 50.77 ng/mL in control cells to 283.99 ng/mL following Z-EAHT treatment, representing an approximate 5.6-fold elevation (fold-level difference (FLD) = 5.59). This strong induction of Bax promotes mitochondrial membrane permeabilization, leading to cytochrome c release and activation of downstream caspases. In contrast, the Bcl-2 concentration decreased drastically from 5.48 ng/mL in control to 2.07 ng/mL, corresponding to a 0.38-fold reduction. The significant decrease in the Bcl-2/Bax ratio confirms a shift toward a pro-apoptotic state, which is widely recognized as a hallmark of apoptosis induction in EGFR-driven tumor cells.

Furthermore, Caspase-3, the major executioner caspase, showed a substantial increase from 53.29 to 308.9 ng/mL (5.80-fold elevation), supporting that Z-EAHT effectively activates the caspase cascade leading to programmed cell death. The combined upregulation of Bax and Caspase-3 with concurrent suppression of Bcl-2 strongly indicates that Z-EAHT induces apoptosis through a mitochondria-dependent mechanism.

Taken together, these findings suggest that Z-EAHT triggers apoptotic cell death in A549 cells by modulating the Bax/Bcl-2 ratio and activating Caspase-3, thereby validating its mechanism of action as a potent EGFR inhibitor with pro-apoptotic potential comparable or superior to standard agents such as Erlotinib. This dual effect—EGFR inhibition coupled with intrinsic apoptosis activation—reinforces Z-EAHT’s promise as a selective and mechanistically robust anticancer candidate.

Correlation between in silico and experimental findings

A comparison between the in silico predictions and the experimental findings revealed a strong correlation. Z-EAHT exhibited a favorable docking score (−20.99 kcal/mol) and stable binding interactions within the EGFR-WT active site, which is consistent with its potent in vitro inhibitory activity (IC₅₀ = 0.012 µM). The preservation of the critical hinge-region hydrogen bond and extensive hydrophobic interactions may account for the observed biological potency. Furthermore, the predicted ADMET profile supported acceptable drug-like properties, aligning with the experimental performance of the compound. Overall, the computational findings successfully rationalize and support the experimental results.

Materials and methods

Chemistry

Chemicals, reagents, and instrumentation

All reagents and solvents employed in the synthesis of Z-EAHT were of analytical grade and obtained from Al-Nasr Company (Egypt). The instruments and equipment utilized for analytical procedures are detailed in Supplementary Information.

Synthesis of Z-EAHT

A general procedure for the synthesis of intermediates 2 and 4

Intermediates 2 and 4 were achieved using the procedures promulgated in the literature.^11,30–32

Synthesis of methyl hydrazinecarbodithioate 3

The reagent was prepared using a typical technique authenticated in literature.^11,30–33

Synthesis of ethyl (Z)-2-((Z)-3-(4-acetamidophenyl)-2-(((E)-1-(4-acetamido phenyl) ethylidene)hydrazono)-4-oxothiazolidin-5-ylidene)acetate (Z-EAHT)

In a 100-mL oven‑dried round‑bottom flask equipped with a magnetic stir bar, the intermediate (0.01 mol) was dissolved in methanol (10 mL), followed by the addition of Diethyl acetylenedicarboxylate (DEAD, 0.01 mol, 1 mmol). The reaction mixture was stirred at room temperature for 8 h. Upon completion, the precipitated product was collected by filtration, purified through recrystallization from an appropriate solvent, and obtained as Z-EAHT in a good yield.

Color: yellow powder; Yield: 78%; m.p.: 195-197°C, FTIR (KBr, νmax/cm⁻¹); 3328, 3204 (2NH), 3061, 3003 (CH-aromatic), 2789 (CH-aliphatic), 1684 (C=O), 1593 (C=N), and 1566 (C=C); HPLC purity 100%; ¹H NMR (400 MHz, DMSO-d₆) δ_H = 10.17 (s, 1H, NH₍₂₄₎), 10.16 (s, 1H, NH₍₃₄₎), 7.82 (d, J = 8.4 Hz, 2H, ArH_{(18, 20)}), 7.70 (dd, J = 18.9, 8.4 Hz, 4H, ArH_{(17,19,27,29)}), 7.43 (d, J = 8.5 Hz, 2H, ArH_(26,30)), 6.77 (s, 1H, C=CH(₂), 4.28 (q, J = 7.1 Hz, 2H, OCH₃CH₂₍₃₅₎), 2.22 (s, 3H, CH₃₍₁₄₎), 2.08 (d, J = 5.9 Hz, 6H, 2CH_3(22,32)), 1.29 (t, J = 7.1 Hz, 3H, OCH₂CH₃₍₃₆₎); ¹³C NMR (101 MHz, DMSO) δ_C = 169.14 (C₃₁), 166.01 (C₂₁), 164.58 (C₁), 164.14 (C₈), 158.88 (C₁₃), 142.31 (C₆), 141.93 (ArC₁₈), 140.09 (C₉), 131.88 (ArC₁₈), 129.25 (ArC₁₅), 128.74 (ArC₂₅), 127.85 (ArC_16,20), 119.55 (ArC_26,30), 119.02 (ArC_27,29), 115.55 (C₂), 61.91 (C₃₅), 24.55 (C₂₂), 24.51 (C₃₂), 15.15 (C₁₄), 14.50 (C₃₆); Anal. calcd for C₂₅H₂₅N₅O₅S (507.57): C, 59.16; H, 4.96; N, 13.80. Found: C, 59.43; H, 5.12; N, 14.03%.

DFT calculations

The molecular geometry of Z-EAHT was optimized using Gaussian 09 with DFT at the B3LYP/6–311++G(d, p) level. At this level, the Z-EAHT’s energetic, structural, and electronic properties were calculated. Global reactivity descriptors—ionization potential (I), electron affinity (A), chemical hardness (η), softness (σ), chemical potential (µ), electronegativity (χ), and electrophilicity index (ω)—were determined using Koopman’s theorem.³⁴ Additional data are provided in Supplementary Information.

Molecular docking studies

Molecular docking of Z-EAHT against EGFR-WT was conducted using validated protocols. Protein structures were prepared by removing crystallographic water molecules, adding hydrogens, and performing energy minimization. The ligand and Erlotinib were pre-optimized and assigned proper charges prior to docking. Binding poses, docking scores, and 2D/3D interaction maps are detailed in Supplementary Information.

MD simulations

The EGFR–Z-EAHT complex was simulated for 200 ns using GROMACS 2021,³⁵ following system preparation in CHARMM-GUI with the CHARMM36m force field for the protein, ions, and TIP3P water.^36,37 The system was solvated in a 10 nm cubic box with a 1 nm buffer, neutralized with 0.154 M NaCl, and simulated under periodic boundary conditions. After energy minimization via the steepest descent method, equilibration was carried out under NVT (310 K) and NPT (1 atm) ensembles using the Velocity Rescale thermostat and Berendsen barostat, followed by production with the Nose–Hoover thermostat and Parrinello–Rahman barostat.^38,39 LINCS constraints were applied to hydrogen bonds, and electrostatics were treated using the Particle Mesh Ewald (PME) method with a 1.2 nm cutoff.⁴⁰ Simulation trajectories were recorded every 0.1 ns (2,000 frames) and analyzed in VMD for RMSD, RMSF, Rg, SASA, hydrogen bonding, and center-of-mass distances. Detailed simulation parameters are provided in Supplementary Information.

Binding free energy analysis (MM-GBSA)

Binding free energies were calculated using the MM-GBSA approach implemented in gmx_MMPBSA. A per-residue energy decomposition analysis was performed to identify amino acid residues within 1 nm of the ligand that significantly contribute to the stability of the EGFR–Z-EAHT complex. The simulations were conducted at an ionic strength of 0.154 M, with a solvation model parameter (igb) = 5, and dielectric constants set to 1.0 (internal) and 78.5 (external). Individual free energy components were evaluated based on the standard MM-GBSA equation, incorporating molecular mechanics, solvation, and surface energy terms to quantify total binding affinity.^41,42 Details are included in Supplementary Information.

ProLIF interaction analysis

Interaction profiling was performed using the ProLIF Python package to quantitatively and qualitatively assess ligand–residue interactions throughout the 200 ns simulation. Key interaction types—including hydrogen bonding, π–π stacking, π–cation contacts, hydrophobic interactions, and salt bridges—were identified, and their occupancy frequencies were quantified across all trajectory frames.⁴³ Complete analysis parameters are provided in Supplementary Information.

PCA-T

EGFR α-carbon mobility was analyzed through PCA-T based on the mass-weighted covariance matrix (C). Trajectories were aligned to the equilibrated reference structure of the EGFR–Z-EAHT complex. Dominant motions were extracted from eigenvectors computed using gmx covar, and their magnitude was determined from eigenvalues. The essential subspace was defined via cumulative variance, scree plot inflection, and deviations from Gaussian eigenvector distributions. Cosine content (ci) values (0–1) were calculated to assess motion convergence.^44–46

FEL analysis

FEL mapping was performed to visualize the conformational states sampled during MD simulation. The landscape was projected onto two principal reaction coordinates, reflecting the dominant motions. The free energy (Gα) associated with each conformation was computed from the Boltzmann distribution, linking conformational probability to thermodynamic stability.⁴⁷ Detailed FEL plots are provided in Supplementary Information.

In Silico ADMET and toxicity prediction

ADMET and toxicity profiles were predicted using BIOVIA Discovery Studio. Parameters assessed included Ames mutagenicity, carcinogenicity, rat LD₅₀, rat LOAEL, maximum tolerated dose, DTP toxicity, and skin/ocular irritancy. Comparative ADMET data for Z-EAHT and Erlotinib are summarized in Supplementary Information.

In vitro EGFR enzyme inhibition assay

EGFR inhibition assays for Z-EAHT were conducted using an EGFR Kinase Assay Kit (BPS Bioscience, USA) according to the manufacturer’s instructions. IC₅₀ values for EGFR-WT and EGFR-T790M were determined from concentration–response curves.⁴⁸ Immunofluorescence assays preceded as described earlier.⁴⁹ In brief, EGFR expression in A549 lung carcinoma cells was analyzed using immunocytofluorescence staining. Cells (1 × 10⁴ per well) were seeded on 8-well chamber slides and treated with Z-EAHT at its IC₅₀ concentration for 48 h. After treatment, cells were fixed with 4% paraformaldehyde, permeabilized using 0.1% Triton X-100, and blocked with 1% bovine serum albumin (BSA). Subsequently, they were incubated overnight at 4 °C with a rabbit anti-EGFR primary antibody (ab52894, Abcam; 1:250), followed by a goat anti-rabbit IgG Alexa Fluor® 488 secondary antibody (ab150077, Abcam; 1:1000). After thorough PBS washing, cells were mounted with DAPI-containing antifade medium (ab188804, Abcam) and examined using a Zeiss Axio Imager.Z2 fluorescence microscope. Fluorescence intensity was measured and analyzed using Zen 11 Blue software. Raw data and fluorescence images are available in Supplementary Information.

In vitro cytotoxicity and selectivity assays

Cytotoxicity was assessed using the MTT assay against HepG-2, MCF-7, HCT-116, PC-3, and A549 cancer cell lines, along with the normal WI-38 fibroblast line. Cells were treated with increasing Z-EAHT concentrations for 48 h, followed by MTT incubation and absorbance measurement at 570 nm.^50,51 Details of the used method are described in the Supplementary Information.

Apoptosis-related protein quantification (enzyme-linked immunosorbent assay)

The levels of Bax, Bcl-2, and Caspase-3 proteins in A549 cells treated with Z-EAHT were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits following the manufacturers’ protocols. Fold change (FLD) was calculated relative to untreated controls. Experimental replicates and raw absorbance data are provided in Supplementary Information.

Conclusion

In this study, a novel oxothiazolidinylidene hybrid, Z-EAHT, was rationally designed and comprehensively evaluated as a potential EGFR inhibitor. DFT analyses confirmed its structural and electronic suitability for receptor binding, while molecular docking demonstrated high affinity toward the EGFR-WT form. The 200 ns MD simulations, supported by MM-GBSA, ProLIF, PCA-T, and FEL analyses, revealed a dynamically stable complex with favorable conformational energetics, confirming strong and persistent interactions within the EGFR active site. In silico ADME-Tox profiling predicted good solubility, moderate absorption, low blood–brain barrier permeability, and a non-toxic, non-mutagenic profile, suggesting acceptable drug-like behavior. Experimentally, Z-EAHT was successfully synthesized and exhibited superior EGFR inhibitory activity (IC₅₀ = 0.012 µM for EGFR-WT and 0.013 µM for EGFR-T790M) compared to Erlotinib. Moreover, it demonstrated selective cytotoxicity toward cancer cell lines while sparing normal cells, accompanied by apoptosis induction through Bax upregulation, Bcl-2 suppression, and Caspase-3 activation, as well as a measurable reduction in EGFR expression. Collectively, these computational and experimental findings establish Z-EAHT as a promising, mutation-tolerant EGFR inhibitor with a favorable pharmacokinetic and safety profile. Although the present findings demonstrated promising in vitro potency and favorable computational validation, further in vivo pharmacokinetic evaluation and efficacy studies are necessary to fully establish the therapeutic potential of Z-EAHT. Future structural optimization may enhance selectivity, metabolic stability, and resistance-overcoming capability, supporting its advancement as a next-generation EGFR-targeted anticancer agent.

Supplemental Material

sj-pdf-1-chl-10.1177_17475198261442267 – Supplemental material for An EGFR-Targeting Oxothiazolidinylidene Derivative: From Computational Design to Experimental Validation

Supplemental material, sj-pdf-1-chl-10.1177_17475198261442267 for An EGFR-Targeting Oxothiazolidinylidene Derivative: From Computational Design to Experimental Validation by Eslam B Elkaeed, Hazem Elkady, Walid E Elgammal, Ahmed Nofal, Hazem A Mahdy, Dalal Z Husein, Fatma G Amin, Aisha A Alsfouk, Ibrahim H Eissa and Ahmed M Metwaly in Journal of Chemical Research

Footnotes

ORCID iDs

Hazem Elkady

Walid E Elgammal

Ahmed Nofal

Hazem A Mahdy

Fatma G Amin

Ahmed M Metwaly

Ethical considerations

Not Applicable.

Consent to participate

Not Applicable.

Consent for publication

Not Applicable.

Author contributions

A.A.A. contributed to funding acquisition as well as manuscript writing and revision. H.A.M., H.E., W.E.E., and A.N. performed the chemical synthesis as well as contributed to manuscript writing and revision. E.B.E. contributed to manuscript writing and revision. F.G.A. carried out the MD simulations, while D.Z.H. conducted the DFT calculations. I.H.E. also contributed to the experimental methodology, ensuring the availability of essential synthesis materials. A.M.M. supervised the project, designed the study, and prepared the initial manuscript draft. All authors reviewed and approved the final manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R116), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

The data that support the findings of this study are enclosed in the manuscript and the supplementary materials.

Supplemental material

Supplemental material for this article is available online.

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