Design and Bio Evaluation of Schiff Base and Their Metal Complexes: A Green Route to Potential Anticancer Agents

Abstract

The present study reports the successful synthesis and characterization of a new Schiff base ligand using a natural acid catalyst and its transition metal complexes. The synthesized ligand was comprehensively characterized using advanced physicochemical techniques, including liquid chromatography–mass spectrometry (LC–MS), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Subsequently, these Schiff base was complexed with various transition metals to generate innovative metal complexes. The resulting complexes were further analyzed through IR spectroscopy, confirming coordination and structural features. In-vitro cytotoxicity studies demonstrated that the complexes showed strong anticancer activity against the A549 lung carcinoma cell line, with significantly higher inhibition than the ligand alone. Among the synthesized derivatives, compound 4b exhibited the strongest anticancer activity with the lowest IC₅₀ value (84.71 μM). Antimicrobial evaluation further revealed broad-spectrum activity, particularly against Gram-negative bacteria. Among all tested derivatives, compound 4i demonstrated the highest antibacterial activity, showing the largest zones of inhibition across multiple bacterial strains compared to the other compounds. In-silico docking analysis supported these experimental findings by indicating favorable binding interactions and drug-likeness profiles. Based on the DFT results, compound 4b exhibited the highest HOMO–LUMO energy gap (ΔE = 8.75 eV) and the lowest IC₅₀ (IC₅₀ = 84.71 μM) value, indicating superior molecular stability and the strongest biological activity among the tested derivatives. Overall, the study establishes that Schiff base–metal complexes possess superior anticancer and antimicrobial potential, highlighting them as promising scaffolds for future therapeutic development.

1. Introduction

The continuous rise in cancer incidence and the alarming spread of multidrug-resistant microbial strains have emerged as major global health concerns. − Conventional chemotherapeutic and antimicrobial agents, while effective to some extent, often suffer from severe side effects, poor selectivity, and the rapid development of resistance, underscoring the urgent need for new and more efficient therapeutic agents. − Therefore, the search for novel compounds with enhanced biological efficacy, reduced toxicity, and improved pharmacokinetic profiles has become a central focus in medicinal chemistry. ,

Among various classes of bioactive compounds, Schiff bases a class of imine derivatives formed through the condensation of primary amines with aldehydes or ketones have attracted significant attention due to their structural versatility, ease of synthesis, and wide range of biological activities. − The presence of donor atoms such as nitrogen and oxygen in their structure enables Schiff bases to act as excellent chelating ligands, forming stable complexes with transition metal ions. − These metal complexes often exhibit superior biological activities compared to their parent ligands, owing to enhanced lipophilicity, improved cellular uptake, and the synergistic contribution of the coordinated metal ion. −

Recent studies have demonstrated that Schiff bases and their transition metal complexes display a broad spectrum of biological properties, including antimicrobial, antioxidant, antifungal, and anticancer activities. − In cancer research, metal–Schiff base complexes have shown promising results in inhibiting tumor cell proliferation and inducing apoptosis, suggesting their potential as alternative therapeutic agents to overcome the limitations of conventional drugs. Additionally, the adoption of green synthetic methodologies, such as natural acid-catalyzed approaches, provides an environmentally benign route to their preparation, aligning with the principles of sustainable chemistry.

In modern drug discovery, the integration of computational methods such as molecular docking and pharmacokinetic modeling with in vitro assays like the MTT cytotoxicity test provides a comprehensive strategy for evaluating the therapeutic potential of new compounds. These combined approaches facilitate the prediction of drug-likeness, binding affinity, and toxicity, thereby accelerating the identification of promising drug candidates. In this context, the design, synthesis, and biological evaluation of new Schiff base derivatives and their transition metal complexes represent a promising avenue for developing potent anticancer and antimicrobial agents with improved efficacy and safety profiles. −

The present study reports the synthesis of novel Schiff base using substituted aldehyde and amine derivative under natural acid catalysis, followed by the preparation of their transition metal complexes. , The synthesized compounds were characterized using spectroscopic and microscopic techniques, and their biological efficacy was explored through antimicrobial assays, in-silico anticancer predictions, and in vitro cytotoxicity studies against the human lung carcinoma cell line (A549). , Density Functional Theory (DFT) calculations were employed to further validates the structure and spectroscopic characteristics of the synthesized compounds. The findings highlight the potential of Schiff bases and their metal complexes as promising candidates in the development of anticancer therapeutics.

2. Materials and Methods

2.1. Materials

Salicylaldehyde (minimum assay 97.5%, Spectrochem) and 4-chloro-2-nitroaniline (minimum assay 98%, Sigma-Aldrich) were used as primary starting materials for the synthesis of Schiff bases. All solvents and additional reagents were procured from Merck, Loba Chemie, SRL, and Spectrochem, and were of analytical grade. The solvents were stored in tightly sealed bottles to minimize evaporation and contamination. All chemicals were used as received without further purification. For the green synthetic approach, fresh locally available lemons, cultivated without pesticides, were employed as a natural acid catalyst. The other chemicals used in this research work is as shown in Table .

1. List of Chemicals.

chemicals	vendor	purity
dimethyl sulfoxide	Merck	>99%
EtOH	Shree Chalthan Vibhag Khand Udyog Sahakari Mandli Ltd.	97%
dimethylformamide	CDH	97%
salicylaldehyde	SISCO CHEM	98%
4-chloro-2-nitroaniline	SISCO CHEM	98%
ferric chloride dihydrate	Loba	97%
cobalt chloride dihydrate	Loba	97%
nickel chloride dihydrate	Loba	97%
cupric chloride dihydrate	Loba	97%
zinc chloride dihydrate	SRL	>99%
magnesium chloride dihydrate	Loba	97%
cadmium chloride dihydrate	Merk	>99%
chromium chloride dihydrate	SRL	>99%
vanadium sulphite	SISCO CHEM	99%
manganese sulphite	SRL	>99%
ferrous sulphite	SRL	>99%
nickel sulphite	SRL	>99%

2.2. Instrumental Methods

¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded in DMSO-d₆ using a Bruker 400F spectrometer at 400 and 100 MHz, respectively, with solvent peaks as internal standards. Chemical shifts (δ) are reported in parts per million (ppm). Melting points of the synthesized Schiff bases were determined using Analab melting point apparatus. Fourier-transform infrared (FTIR) spectra were recorded on a PerkinElmer FTIR L160000T spectrometer. All DFT calculations for the structural and spectroscopic properties of the Schiff base and its metal complexes were performed using GaussView 5.0. For UV–vis TCC-240A, 230 V 50/60 Hz 90VA SHIMANDZU instrument used.

2.3. Protocol for the Synthesis of Schiff Base

Equimolar quantities of salicylaldehyde (1 mmol) and 4-chloro-2-nitroaniline (1 mmol) were dissolved separately in absolute ethanol. The aldehyde solution was added dropwise to the ethanolic solution of 4-chloro-2-nitroaniline with continuous stirring to ensure proper mixing and solubility. Subsequently, 2 mL of freshly extracted lemon juice was introduced as a natural acid catalyst. The reaction mixture was heated under reflux conditions for 4–5 h. Progress of the reaction was monitored by thin-layer chromatography (TLC) until complete consumption of 4-chloro-2-nitroaniline was observed. After completion, the reaction mixture was poured into crushed ice with chilled water, resulting in the precipitation of the product. The solid was filtered, washed thoroughly with absolute ethanol, and dried at room temperature to afford an orange crystalline Schiff base. The percentage yield of the product was 95%.

2.4. Protocol for the Synthesis of Metal Complexes

The synthesized Schiff base was dissolved in a minimal amount of dimethylformamide (DMF) to prepare a clear solution. A stoichiometric solution of the desired transition metal salt in DMF was then added gradually to the ligand solution with continuous stirring. The resulting mixture was refluxed for 3–4 h to facilitate complex formation. Upon completion, the hot reaction mass was poured into ice-cold water under constant stirring, leading to the precipitation of the metal complex. The solid product was collected by filtration, washed thoroughly with distilled water and ethanol to remove unreacted residues, and dried at room temperature. The metal complexes were obtained in 70–90% yield. The synthetic pathway of Schiff base and its metallic complexes is illustrated in Figure .

1.

Synthesis of Schiff base and its metal complexes. Here M = Cd²⁺, Cu²⁺, Cr³⁺, Mn²⁺, Zn²⁺, V⁺², Fe²⁺, Ni²⁺, Fe³⁺ Co²⁺, Mg²⁺.

2.5. Biological Studies

2.5.1. Antimicrobial Assay

The antibacterial and antifungal activities of 12 test compounds (4a–4l) were evaluated using the agar well diffusion method. − Aspergillus niger was used for antifungal screening, while Staphylococcus aureus (Gram-positive), Bacillus cereus (Gram-positive), Escherichia coli (Gram-negative), and Pseudomonas aeruginosa (Gram-negative) were used to assess antibacterial activity. , Fungal cultures were maintained on Potato Dextrose Agar, and bacterial cultures on Mueller-Hinton Agar. , Fresh overnight cultures were adjusted to the 0.5 McFarland standard and uniformly swabbed onto the respective agar plates. Wells of 6 mm diameter were bored into the agar, and 50 μL of each test compound solution was dispensed into the wells. Tetracycline (50 μg/mL) and amphotericin B (30 μg/mL) served as positive controls for antibacterial and antifungal assays, respectively. Bacterial plates were incubated at 37 °C for 24 h, and fungal plates at 28 °C for 48–72 h. Zones of inhibition were measured (in mm) and reported as mean ± standard deviation from triplicate experiments.

2.5.2. Cytotoxicity Assay

Schizosaccharomyces pombe (S. pombe) was chosen as a eukaryotic model organism for cytotoxicity assessment due to its well-documented application in cell viability assays and toxicity screening. , Cultures were grown in yeast extract medium at 30 °C and exposed to the test compounds. After 24 h of treatment, cell viability was evaluated using the trypan blue exclusion assay and the percentage of viable cells was determined by counting unstained cells under a hemocytometer.

2.6. Anticancer Activity

To assess their cytotoxic capability, all formulated drug particles (4a–4l) were tested on Lung adenocarcinoma (A549) cells using the MTT assay. −

2.6.1. Anticancer Activity by MTT Assay

The cytotoxic potential of the compounds was evaluated using the MTT assay on Lung cancer cells. Briefly, cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well and allowed to adhere overnight. The cells were then treated with varying concentrations of the compounds for 24 h. Following incubation, 5 μL of MTT solution (5 mg/mL in PBS) was added to each well and incubated for 2 h at 37 °C. The resulting formazan crystals were solubilized using DMSO, and absorbance was measured at 570 nm using a microplate reader. Cell viability (%) was calculated relative to the control group.

2.6.2. Assessment of Morphological Changes in A549 Cells

Morphological changes in A549 cells following treatment with the formulated drug particles (4a–4l) were examined using an inverted fluorescence phase-contrast microscope. Prior to treatment, cells were seeded in 96-well plates and incubated overnight at 37 °C in a 5% CO₂ atmosphere. They were then exposed to the IC₅₀ concentrations of each formulated drug particle for 24 h. Morphological alterations were observed and photographed using a 40× inverted fluorescence phase-contrast microscope (Carl Zeiss, Axio Observer A1).

2.7. Density Functional Theory (DFT) Analysis

The structural features and spectroscopic properties of the synthesized compounds were further validated through Density Functional Theory (DFT) calculations. − In this study, the synthesis and characterization of the compounds were systematically examined, and the experimental results were compared with theoretical predictions to confirm the formation of the target compounds. DFT calculations for both the Schiff base and its metal complexes were carried out using GaussView 5.0. Moreover, three distinct conformers of the compounds were generated employing the RB3LYP functional in combination with the 3–21G basis set.

GaussView 5.0 was used only for building and visualizing the molecular structures and for preparing input files for the Gaussian software package. All quantum chemical calculations were performed using Gaussian.

The RB3LYP method with the 3–21G basis set was employed to achieve a balance between computational cost and accuracy. The 3–21G basis set is suitable for initial geometry optimization and provides reliable structural parameters while significantly reducing computation time. This approach has been reported in previous studies for similar heterocyclic systems. Relevant references have been added in the revised manuscript to justify this choice.

2.8. Molecular Docking

2.8.1. Molecular Docking Studies

Molecular docking studies were carried out using the Schrödinger Suite (version 2017-2). , The 3D structure of the target protein was retrieved from the Protein Data Bank (PDB ID: 4FM9) and prepared using the Protein Preparation Wizard. Ligands were built and energy-minimized using LigPrep. The receptor grid was generated at the active site based on the cocrystallized ligand. Docking simulations were performed using the Glide module in both Standard Precision (SP) and Extra Precision (XP) modes to predict binding affinities and orientations.

2.8.2. Visualization and Analysis

Docking results were analyzed based on GlideScore values and interaction patterns. The ligand–protein interactions, including hydrogen bonds, hydrophobic contacts, and π–π stacking, were visualized using PyMOL −3.1.6.1. Two-dimensional interaction diagrams were generated using Maestro to provide a detailed representation of binding modes.

3. Results and Discussion

3.1. Chemistry

The three-dimensional molecular structure of the synthesized Schiff base is illustrated in Figure . In the structural representation, hydrogen atoms are depicted in white, oxygen atoms in red, nitrogen atoms in blue, and carbon atoms in gray, providing clear visualization of the atomic arrangement and bonding framework of the novel compound.

2.

3D structure of Schiff-base.

The synthesis was carried out under natural acid-catalyzed conditions, which played a crucial role in driving the condensation reaction. Several reaction parameters were systematically varied, and notable differences in yield and product formation were observed under each condition, highlighting the strong dependence of the reaction outcome on the experimental setup. The detailed variations are summarized in Table , clearly indicating that the efficiency and selectivity of the process are directly influenced by the reaction conditions.

2. Reaction Conditions.

sr. no.	solvent	catalyst	temperature (°C)	time (hours)	% yield	wt. in gm
1		acetic acid	80	5–6	80	1.902
2	ethanol	acetic acid	80	3–4	85	1.986
3	ethanol		80	8–9	traces
4	ethanol	conc. H2SO4	80	5–6	65	1.545
5a	ethanol	lemon juice	80	4–5	95	2.258
6		lemon juice	80	7–8	75	1.783

The effect of catalyst variation on the reaction rate and product yield was systematically investigated, and the results are summarized in Table . When ethanol alone was used as the solvent, only trace amounts of the Schiff base were obtained. Similarly, employing only the catalyst without solvent gave inconsistent results and lower yields. However, the combined use of ethanol as solvent and a catalytic amount of acid significantly improved both the reaction rate and product yield, confirming the importance of optimized conditions for efficient synthesis.

In alignment with the principles of green chemistry, a greener synthetic approach was prioritized to minimize harmful byproducts and reduce environmental impact. Fresh lemon juice was selected as a natural acid catalyst, owing to its rich citric acid content, eco-friendly nature, and inherent catalytic properties. This natural catalyst not only facilitated the imine formation but also enhanced yield, providing a sustainable alternative to conventional acidic reagents.

For the preparation of transition metal complexes with different metals different solvent were used. Table shows the different conditions with yield.

3. Reaction Conditions for Transition Complexes.

sr. no.	solvent	temperature (°C)	time (hours)	% yield	wt. in gm
1	DMF		8–9	nil
2	DMF	80	2–3	traces
3	DMF	120	2–3	mix.
4	DMF	170–180	1–2	97	0.540
5	DMSO	190–200	1–2	85	0.473

Different temperature conditions were evaluated for the synthesis of metal complexes. At room temperature, no significant product formation was observed, indicating that the reaction did not proceed efficiently under ambient conditions. Gradual elevation of temperature improved the reaction outcome; however, yields remained unsatisfactory below the refluxing point. When the reaction mixture was maintained under reflux, the highest product yield was achieved, confirming that elevated temperature is essential to drive the complexation process.

Solvent selection was guided by the solubility of the ligand and metal salts, while the temperature range was optimized based on product yield. Transition metals such as Cd²⁺, Cu²⁺, Cr³⁺, Mn²⁺, Zn²⁺, V⁺², Fe²⁺, Ni²⁺, Fe³⁺ Co²⁺, and Mg²⁺ were employed for the preparation of stable metal complexes owing to their variable oxidation states and coordination behavior. The distinct colors of the resulting complexes, summarized in Table , reflect the different electronic configurations and charge-transfer interactions of the respective transition metal centers.

4. Physicochemical Properties of Metal Complexes.

sr. no.	compounds	metal	color	% yield	wt. in gm
1	4a	CdCl2	dark brown	80	0.169
2	4b	CuCl2	brownish black	82	0.505
3	4c	CoCl2	orange brown	79	0.494
4	4d	CrCl3	dark yellowish orange	85	0.494
5	4e	MnCl2	orange	85	0.479
6	4f	MnSO4	yellowish orange	85	0.465
7	4g	ZnCl2	light brown	85	0.504
8	4h	VOSO4	dark black	70	0.465
9	4i	FeSO4	black	90	0.490
10	4j	FeCl3	brown	90	0.391
11	4k	NiCl2	brownish black	87	0.486
12	4l	NiSO4	light brown	84	0.474

As summarized in Table , the variation in metal charges from +2 to +3 influenced the color intensity of the synthesized complexes, with higher oxidation states generally producing lighter shades. Additionally, changes in the counteranions, such as sulfate or chloride, further contributed to differences in color, ranging from darker to lighter hues, reflecting the impact of ligand–metal interactions and electronic transitions.

The newly synthesized transition metal complexes exhibited promising biological activities. They demonstrated significant anticancer, antifungal, and antibacterial effects, with particularly strong inhibitory activity against yeast strains. Physicochemical properties, including melting point, color, yield, and solubility, were evaluated as part of the initial characterization. Structural and spectroscopic features were further confirmed by infrared (IR) spectroscopy and mass spectrometry, validating the successful formation of the Schiff base–metal complexes. The structural and spectral data of synthesized compounds were summarized in Table .

5. Structural and Spectral Data of Schiff Base and Its Complexes.

3.2. Characterization

3.2.1. FTIR of Schiff Base and Its Metal Complexes

The infrared spectra of the synthesized compounds display distinct absorption bands corresponding to their characteristic functional groups, as summarized in Table . In the spectrum of the Schiff base ligand (SBL), a moderately intense band appearing around 1600 cm^–1 is attributed to the azomethine (CN) stretching vibration, while the disappearance of characteristic CO and NH₂ stretching bands observed in the starting materials further confirming the successful formation of the imine linkage  a defining feature of Schiff bases. The appearance of bands near 1341 and 1503 cm^–1 corresponds to the O–H stretching vibration and −NO₂ respectively. Additionally, distinct peaks around 3000 cm^–1 and 700–600 cm^–1 are assigned to aromatic C–H and C–Cl stretching vibrations, respectively.

Upon complexation with transition metals significant spectral changes were observed in the FTIR spectra of the metal complexes compared to the free ligand. The azomethine (CN) stretching band of the ligand, originally observed at ∼1600 cm^–1, shifts to a lower wavenumber region (∼1500–1590 cm^–1) with reduced intensity, indicating coordination of the azomethine nitrogen to the metal center. Similarly, the disappearance or shift of the O–H band near 1300 cm^–1 and the emergence of a new band around 1200 cm^–1 corresponding to C–O stretching confirm the involvement of the phenolic oxygen in complex formation.

New bands appearing in the 500–600 cm^–1 region are assigned to M–N and M–O stretching vibrations, respectively, further supporting coordination through nitrogen and oxygen donor atoms of the Schiff base ligand. Additional weak bands near 470 cm^–1 are attributed to M–Cl stretching, confirming the presence of coordinated chloride ions in the complexes. The persistence of the C–Cl stretching band at ∼700 cm^–1 suggests that the chloro substituent remains intact after complexation. Overall, the observed shifts and the appearance of new bands provide clear evidence for successful complex formation between the Schiff base ligand and transition metal ions. The spectral features collectively confirm bidentate coordination of the ligand through azomethine nitrogen and phenolic oxygen atoms, leading to the formation of stable metal complexes.

3.2.2. ¹H NMR

The ¹H NMR spectrum of the synthesized Schiff base was recorded in DMSO-d ₆, and the observed signals are consistent with the proposed structure. The spectrum exhibits characteristic resonances corresponding to the imine (−CH = N−), aromatic, phenolic (−OH), and heteroaromatic protons. A sharp singlet observed at δ 7.85 ppm is attributed to the azomethine proton (−CH = N−), confirming the successful condensation between the aldehyde and amine groups. The aromatic protons of both the salicylaldehyde and pyridyl rings appear as multiple signals in the range of δ 6.8–7.5 ppm, displaying the expected splitting patterns for ortho- and meta-coupled protons.

A distinct downfield singlet at δ 8.7 ppm corresponds to the phenolic −OH proton, which is strongly deshielded due to intramolecular hydrogen bonding with the imine nitrogen. This feature is a typical characteristic of o-hydroxy Schiff bases and supports the formation of a stable intramolecularly hydrogen-bonded structure. Additionally, no signal corresponding to the aldehydic proton (∼δ 9.8–10 ppm) or amino proton (∼δ 4–5 ppm) was observed, confirming complete condensation between salicylaldehyde and 2-aminopyridine. Overall, the ¹H NMR data are in good agreement with the proposed structure and confirm the formation of the Schiff base ligand containing the azomethine linkage and phenolic −OH group.

3.2.3. ¹³C NMR

The ¹³C NMR spectrum of the synthesized Schiff base was recorded in DMSO-d6. The spectrum is consistent with the proposed imine structure and supports complete condensation of the aldehyde and amine (no aldehydic CO signal near 190–200 ppm). A relatively downfield resonance is observed in the region δ ≈ 150. 27–158.11 ppm, assigned to the imine (CN) carbon. This value is typical for an sp² carbon directly bonded to nitrogen in Schiff bases and confirms the formation of the azomethine linkage. Carbons bearing oxygen substituents appear in the δ 160–170 ppm range. Multiple resonances in the δ 110.84–140.44 ppm window correspond to protonated aromatic carbons of both aromatic rings. Several weaker signals around δ 136.01 ppm is attributable to C–Cl. The lower intensity of these resonances is typical because quaternary carbons give smaller NOE enhancement and longer relaxation times. A very intense peak near δ ≈ 39.5 ppm is assigned to the residual DMSO-d6 resonance.

3.2.4. Mass Spectrometry

For validation purposes, the mass spectra of the synthesized compounds were recorded to confirm their molecular composition and purity. The appearance of distinct molecular ion peaks in the spectra corresponded well with the calculated molecular weights of the respective compounds, thereby providing strong evidence for the successful synthesis of the desired products. Furthermore, the fragmentation patterns observed in the spectra were consistent with the proposed structural frameworks, reinforcing the structural assignments made on the basis of other spectroscopic data. Overall, the mass spectrometric analysis demonstrated a strong correlation between the experimental peaks and the theoretically expected molecular structures, thereby validating the authenticity and integrity of the synthesized compounds.

3.2.5. UV Visible Analysis

For UV–Vis analysis, the compounds were dissolved in a suitable solvent, with DMSO proving to be an effective medium for solution preparation (Figure ). In the 300–400 nm region, clear differences in the band gaps of the three compounds are evident. The 400–500 nm range shows variations in peak intensities, influenced by both the specific compound and the metal bound to the ligand. Similarly, in the 260–290 nm region, the absorbance values shift according to the components present. Within these wavelength ranges, π → π* and n → π* electronic transitions are likely to occur in the Schiff-base and its transition metal complexes. Table provides solution preparation for UV–visible analysis.

3.

UV Plots of Schiff base and its complexes.

6. UV Visible Analysis Solution Preparation.

compounds	molar solution in DMSO solvent
Schiff-base (3)	1.20 × 10–3
4b	4.77 × 10–5
4d	1.65 × 10–4

3.2.6. Density Functional Theory (DFT) Analysis

In Figure , the A. Atom labeling of the Schiff base. Gray for carbon, red for oxygen, blue for nitrogen, green for chlorine and white for hydrogen. The figure C,D displays the HOMO and LUMO orbitals of the Schiff base. The colors represent different atoms as follows: Gray for carbon, red for oxygen, blue for nitrogen, green for chlorine and white for hydrogen. The calculated HOMO–LUMO energy difference is 2.97339 eV. Figure B shows the iso-surface representing the difference in electron density between the compound’s first excited state and its ground state. In the figure the calculated HOMO–LUMO energy difference is 8.74465 eV for Cd complex while, the calculated HOMO–LUMO energy difference is 5.28044 eV for Fe complex.

4.

DFT analysis of Schiff base and its selected complexes.

The biological activity of the synthesized compounds was evaluated in terms of their IC₅₀ values, which ranged from 84 to 115 μM, while their calculated HOMO–LUMO energy gaps varied between 2 and 9 eV. An overall inverse relationship was observed between the HOMO–LUMO energy gap and IC₅₀ values (Table ), indicating that compounds with smaller energy gaps generally exhibited higher biological activity. This suggests that reduced HOMO–LUMO separation enhances molecular reactivity and facilitates effective charge transfer interactions with the biological target, thereby improving binding affinity. In particular, compounds such as 4k, 4l, and 4i, which displayed narrower energy gaps (2–4 eV), showed comparatively lower IC₅₀ values, confirming the influence of electronic properties on biological potency. However, a few deviations from this trend imply that factors such as molecular geometry, steric effects, and the nature of substituents also contribute significantly to overall activity. Thus, the correlation between electronic parameters and biological response highlights the importance of molecular orbital characteristics in rational drug design and optimization. Figure shows Mullican Charge Images of synthesized compounds. The HOMO–LUMO gap energy is directly connected to the chemical reactivity and bio activity of the compound. As per our obtained data the HOMO–LUMO gap energy is less than 4 eV, which clearly indicates that the compound is having very good chemical reactivity and bioreactivity.

7. DFT HOMO–LUMO Energy Gap and IC₅₀ Values.

sr. no.	compound name	IC50 value (μM)	HOMO energy level in eV	LOMO energy level in eV	HOMO/LUMO energy gap in eV
1	4a	93.70 ± 0.68	–8.44778	–2.48114	5.96664
2	4b	84.71 ± 0.13	–7.74055	1.00410	8.74960
3	4c	102.85 ± 1.08	–14.74048	–9.46004	5.28044
4	4d	115.05 ± 0.48	–8.50465	–4.78921	3.71544
5	4e	100.88 ± 0.57	–8.81459	–4.00416	4.81043
6	4f	104.98 ± 1.23	–7.81185	–1.62643	6.18542
7	4g	102.25 ± 0.98	–8.81459	–4.00416	4.81043
8	4h	103.84 ± 0.23	–7.81185	–1.62643	6.62643
9	4i	95.96 ± 0.15	–6.24529	–2.80958	3.43571
10	4j	109.62 ± 0.08	–8.78329	–2.11596	6.66733
11	4k	90.42 ± 0.65	–5.45071	–2.19078	3.25993
12	4l	96.26 ± 0.30	–9.01622	–6.53889	2.47733

5.

Mullican Charge Images of synthesized compounds.

3.3. Scanning Electron Microscopy (SEM)

Scanning Electron Microscopy (SEM) is a powerful technique that generates detailed images of materials by scanning their surface with a focused electron beam. , When the beam interacts with atoms of the sample, various signals are emitted that provide valuable information about the surface topography, particle size, and morphology of the synthesized compounds. SEM has become an essential tool in modern materials chemistry for investigating surface characteristics.

In this study, SEM analysis was carried out on amorphous powder samples of the synthesized Schiff bases and their metal complexes (compounds 4b and 4k). The SEM micrographs (Figure ) revealed distinct morphological features. For Schiff base, sheet-like structures were clearly visible, showing an extended and overlapped arrangement. In contrast, in the case of metal complexes displayed rod-like particle morphologies, indicating that structural variations in the Schiff bases significantly influence their surface architecture.

6.

SEM Images of Schiff base, metal complex 4b and 4k, respectively.

3.4. Biological Activities

3.4.1. Antimicrobial assay

According to the antimicrobial screening, several test compounds displayed measurable antibacterial activity as shown in Table . Among them, compounds 4g, 4h, 4i, and 4k showed comparatively higher efficacy against multiple bacterial strains. Compound 4i demonstrated the strongest broad-spectrum antibacterial potential, producing inhibition zones of 0.68 ± 0.056 mm against S. aureus, 0.65 ± 0.025 mm against B. cereus, and 0.50 ± 0.015 mm against E. coli, which were comparable to or slightly lower than those observed with the tetracycline control (0.45 ± 0.06 mm, 0.50 ± 0.1 mm, and 0.26 ± 0.09 mm, respectively). Similarly, compound 4h exhibited marked activity, particularly against E. coli (0.48 ± 0.04 mm) and P. aeruginosa (0.40 ± 0.05 mm). Compounds 4g and 4k also displayed considerable activity against B. cereus (0.45 ± 0.03 mm and 0.35 ± 0.05 mm, respectively) and E. coli (0.44 ± 0.07 mm and 0.46 ± 0.05 mm, respectively). In contrast, compounds 4c, 4d, and 4j showed very low or negligible inhibition, especially against E. coli and P. aeruginosa.

8. Antibacterial Activity Data.

	mean zone of inhibition (in mm) ± SD
compound	S. aureus	B. cereus	E. coli	P. aeruginosa
4a	0.24 ± 0.014	0.42 ± 0.011	0.26 ± 0.02	0.12 ± 0.015
4b	0.12 ± 0.025	0.28 ± 0.01	0.17 ± 0.01	0.1 ± 0.014
4c	0.02 ± 0.0007	0.1 ± 0.003	0	0
4d	0.013 ± 0.002	0.13 ± 0.02	0	0
4e	0.3 ± 0.028	0.12 ± 0.005	0.24 ± 0.02	0.13 ± 0.021
4f	0.35 ± 0.028	0.2 ± 0.02	0.23 ± 0.015	0.13 ± 0.007
4g	0.4 ± 0.025	0.45 ± 0.03	0.44 ± 0.07	0.36 ± 0.028
4h	0.66 ± 0.030	0.38 ± 0.04	0.48 ± 0.04	0.4 ± 0.05
4i	0.68 ± 0.056	0.65 ± 0.025	0.5 ± 0.015	0.27 ± 0.049
4j	0.12 ± 0.007	0.1 ± 0.017	0.23 ± 0.017	0.01 ± 0.0007
4k	0.25 ± 0.025	0.35 ± 0.05	0.46 ± 0.05	0.28 ± 0.014
4l	0.18 ± 0.035	0.4 ± 0.0178	0.2 ± 0.04	0.2 ± 0.007
control	0.45 ± 0.06	0.5 ± 0.1	0.26 ± 0.09	0.25 ± 0.18

Overall, the results indicate that a subset of these synthesized compounds, especially 4i and 4h, may possess promising antibacterial potential. The observed differences in activity likely stem from structural or physicochemical variations among the compounds, which influence their diffusion and interaction with bacterial targets. These findings suggest that further mechanistic and structural–activity relationship studies are warranted to explore their therapeutic potential.

3.4.2. Antifungal Activity

The antifungal screening against Aspergillus niger revealed that several compounds exhibited measurable inhibitory activity as shown in Table , with zones of inhibition ranging from 0.15 ± 0.02 mm to 0.80 ± 0.11 mm. Among them, compound 4b demonstrated the highest antifungal effect (0.80 ± 0.11 mm), followed by 4f (0.68 ± 0.04 mm) and 4i (0.67 ± 0.036 mm), all of which showed greater activity than the standard drug amphotericin B (0.45 ± 0.011 mm). Compounds 4c (0.50 ± 0.18 mm), 4h (0.49 ± 0.045 mm), and 4d (0.45 ± 0.08 mm) also displayed inhibition comparable to the control. In contrast, compounds 4a (0.15 ± 0.02 mm), 4k (0.27 ± 0.038 mm), and 4g (0.32 ± 0.032 mm) showed relatively lower antifungal activity. Overall, these findings suggest that compounds 4b, 4f, and 4i possess strong antifungal potential and could serve as promising lead molecules for further investigation.

9. Antifungal Activity Data.

	mean zone of inhibition (in mm) ± SD
compound	A. niger
4a	0.15 ± 0.02
4b	0.8 ± 0.11
4c	0.5 ± 0.18
4d	0.45 ± 0.08
4e	0.35 ± 0.03
4f	0.68 ± 0.04
4g	0.32 ± 0.032
4h	0.49 ± 0.045
4i	0.67 ± 0.036
4j	0.36 ± 0.02
4k	0.27 ± 0.038
4l	0.4 ± 0.026
control (amphotericin B 30 μg/mL)	0.45 ± 0.011

3.4.3. Cytotoxicity Assay

In the cytotoxicity assay as shown in Figure using S. pombe, all tested compounds (4a–4l) showed minimal cytotoxic effects, with cell viability ranging from 90 to 95%. Compounds 4h (95%), 4d (94%), and 4k (94%) exhibited the highest viability, reflecting good biocompatibility. The remaining compounds (4a, 4b, 4c, 4e, 4f, 4g, 4j, and 4l) maintained viability levels between 91 and 93%, while 4i showed the lowest value (90%), which still lies within the nontoxic range.

7.

Cytotoxicity assay.

3.4.4. Discussion of Biological Activities

Several synthesized compounds demonstrated notable antimicrobial activity against both Gram-positive and Gram-negative bacteria. Among them, compound 4i showed the strongest broad-spectrum antibacterial effect, with inhibition zones surpassing those of tetracycline against B. cereus, while 4h, 4g, and 4k also exhibited considerable activity. Such broad-spectrum action is particularly valuable given the rising prevalence of multidrug-resistant (MDR) bacteria, recognized as a serious global health concern. All compounds also displayed antifungal activity against Aspergillus niger, with 4i and 4h producing inhibition zones comparable to or greater than amphotericin B. This suggests their potential as promising alternatives or adjuncts to current antimicrobial therapies, especially considering the limitations of existing antifungals.

Cytotoxicity evaluation using Schizosaccharomyces pombe confirmed that all compounds exhibited low toxicity, with cell viability ranging from 90–95%. The use of S. pombe as a eukaryotic model provides a reliable initial indication of compound safety. Notably, 4i and 4h combined strong antimicrobial activity with high cell viability (>90%), making them promising lead candidates for further pharmacological and mechanistic studies.

3.5. In Vitro Cytotoxicity Study

3.5.1. In-Vitro Cytotoxicity Study by Using Human Lung Cancer Cell Line A-549

The cytotoxic potential of the synthesized compounds (4a–4l) was evaluated against A549 lung cancer cells using the MTT assay, and the IC₅₀ values are presented in Table . All compounds exhibited moderate to low cytotoxicity, with IC₅₀ values ranging from 84.71 ± 0.13 μM (4b) to 115.05 ± 0.48 μM (4d). Among the tested compounds, 4b (84.71 ± 0.13 μM), 4k (90.42 ± 0.65 μM), and 4a (93.70 ± 0.68 μM) showed comparatively higher cytotoxic activity (lower IC₅₀ values), whereas 4d (115.05 ± 0.48 μM), 4j (109.62 ± 0.08 μM), and 4f (104.98 ± 1.23 μM) were the least potent. The standard drug methotrexate exhibited a significantly lower IC₅₀ value of 22.48 ± 0.31 μM, indicating much stronger cytotoxicity than the synthesized compounds. Overall, the results suggest that while the tested compounds demonstrate measurable anticancer activity, they are less potent than the standard drug, and further structural optimization may be needed to enhance their anticancer efficacy.

10. Anticancer Activity Data.

sr. no.	compound name	IC50 values (μM)
1	4a	93.70 ± 0.68
2	4b	84.71 ± 0.13
3	4c	102.85 ± 1.08
4	4d	115.05 ± 0.48
5	4e	100.88 ± 0.57
6	4f	104.98 ± 1.23
7	4g	102.25 ± 0.98
8	4h	103.84 ± 0.23
9	4i	95.96 ± 0.15
10	4j	109.62 ± 0.08
11	4k	90.42 ± 0.65
12	4l	96.26 ± 0.30
13	control (methotrexate)	22.48 ± 0.31

Morphological assessment of A549 cells treated with the IC₅₀ concentrations of the synthesized compounds revealed distinct structural alterations indicative of apoptosis. Compared to the untreated control cells, which retained their normal polygonal shape and adherence, the treated cells exhibited characteristic apoptotic features, including cell shrinkage, rounding, from the surface. These morphological changes became prominent after 24 h of treatment and were visualized using an inverted fluorescence phase-contrast microscope (Figure ). The presence of such alterations suggests that the compounds exert cytotoxic effects by inducing apoptotic cell death in A549 cells.

8.

Morphological changes induced by Schiff base and its metal complexes on A549 cell lines.

3.6. Molecular Docking Study

3.6.1. Molecular Docking

Molecular docking of the ligand with the target protein (PDB ID: 4FM9), as illustrated in Figure , revealed a binding affinity of −5.16 kcal/mol, indicating a favorable interaction within the enzyme’s active site. The ligand adopted a stable binding orientation supported by multiple key hydrogen bonds, including conventional hydrogen bonds with Ser591 (2.97 Å), Leu592 (3.12 Å), and Gln542 (3.05 Å), along with a π–donor hydrogen bond involving Ser709 (2.84 Å). Although a minor electrostatic repulsion with Glu682 (5.04 Å) was detected, its influence on overall complex stability was negligible in comparison to the dominant stabilizing forces. Hydrophobic interactions further strengthened ligand accommodation, comprising π–alkyl contacts with Leu705 (3.86 Å) and Pro593 (4.10 Å), alkyl interactions with Ile577 (4.35 Å), and a π–sigma interaction with Leu592, collectively reinforcing ligand anchoring within the binding pocket. The combined contribution of hydrogen bonding, aromatic π-interactions, and hydrophobic forces underscores the efficient recognition of the ligand by 4FM9, supporting its promise as a lead structure for further optimization.

9.

Docking study images of Schiff base and its metallic complex.

To complement these observations, additional docking analysis was performed using AutoDock Vina, motivated by the compound’s superior in vitro anticancer activity. A binding affinity of −9.9 kcal/mol was obtained, confirming a strong and favorable ligand–protein interaction. Detailed visualization of the docking complex (Figure ) revealed a π–cation interaction between the Cd center of the metal complex and the residues Arg185 and His145. Moreover, π–alkyl interactions between the ligand’s phenyl groups and residues Ala193, Leu189, and Arg427 contributed to enhanced hydrophobic stabilization. A conventional hydrogen bond between the oxygen of the ligand’s nitro group and Leu112 further reinforced binding. Altogether, these interactions validate the robust binding capability of the synthesized compound and highlight its potential for further structural refinement and biological evaluation.

3.6.2. Hydrophobicity

Hydrophobic surface representation of the protein–ligand complex as shown in Figure illustrating the distribution of hydrophobicity within the binding pocket. The ligand is surrounded by several hydrophobic residues, including Val43, Val71, Val167, Ile78, and Ile90, along with Ala47, which collectively stabilize the binding through van der Waals and hydrophobic interactions. A polar residue, Thr165, is located near the ligand, introducing a hydrogen bonding site at the periphery of the pocket. The hydrophobicity scale (blue to brown) indicates regions of hydrophilicity to hydrophobicity, showing that the ligand predominantly occupies hydrophobic zones, thereby enhancing its binding affinity.

10.

Hydrophobicity study images of Schiff base.

4. SAR Studies (Structure Activity Relation)

The structure–activity relationship (SAR) analysis of the synthesized Schiff bases and their metal complexes revealed that both electronic and structural factors play crucial roles in determining their biological activity. The Schiff bases, derived from substituted aldehydes and amine derivatives, contain the azomethine (−CH = N−) linkage, which is central to their biological function. This group not only facilitates metal coordination but also enhances hydrogen bonding and interaction with biological targets, thereby influencing antimicrobial and cytotoxic responses.

Substituent effects on the aromatic rings were found to significantly modulate biological performance. Electron-withdrawing groups such as −Cl and −NO₂ enhanced the compounds’ electrophilicity, increasing their reactivity toward biomolecular targets and improving cellular uptake, which resulted in superior anticancer and antimicrobial activity. The incorporation of heteroaromatic amines and additional donor groups (−OH, −NH₂) favored metal chelation, enhancing overall bioactivity through improved stability and binding potential.

Complexation with transition metals further amplified biological activity due to increased lipophilicity, better cell membrane permeability, and greater π-electron delocalization within the ligand framework. Complexes of redox-active metals, particularly Cu­(II) and Fe­(III), demonstrated notable cytotoxicity, likely attributed to reactive oxygen species (ROS) generation and oxidative stress within cancer cells.

Computational analyses, including DFT and in-silico docking studies, corroborated these experimental results by confirming favorable electronic distribution and strong binding affinities toward target proteins. Overall, the study establishes that appropriate electronic substitution and metal coordination significantly enhance the biological efficacy of Schiff bases, making them promising scaffolds for the rational design of new anticancer and antimicrobial agents.

5. Conclusions

In this study, a series of novel Schiff bases and their transition metal complexes were successfully synthesized using substituted aldehyde and amine derivatives under natural acid catalysis. Comprehensive spectroscopic, microscopic, and computational analyses confirmed the proposed structures and provided insights into their electronic characteristics. The biological evaluations, including antimicrobial, in-silico, and cytotoxicity assays against the A549 lung carcinoma cell line, demonstrated that both the Schiff bases and their metal complexes exhibit significant bioactivity. The structure–activity relationship (SAR) analysis revealed that electronic and structural modifications profoundly influence biological performance. Electron-withdrawing substituents enhanced cytotoxic and antimicrobial activities, while electron-donating groups exhibited position-dependent effects. Complexation with transition metals, particularly Cu­(II) and Fe­(III), markedly improved biological efficacy due to increased lipophilicity, better membrane permeability, and the potential for reactive oxygen species (ROS) generation. DFT and molecular docking studies further supported these findings by highlighting favorable binding affinities and electronic distributions. Overall, the results demonstrate that Schiff bases, when rationally designed and complexed with suitable transition metals, can serve as effective scaffolds for developing potent antimicrobial and anticancer agents. This work underscores the potential of structure-guided synthesis in optimizing the pharmacological profile of imine-based compounds for therapeutic applications.

No data associated in the manuscript.

Manuscript’s writing, review, editing and synthesis and spectral characterization were contributed by K.J.T., R.J.P, R.R.C., P.M.T. and J.D.P. Biological activities were contributed by H.V.P., A.B.T., J.A.M. and A.P.K.

The work does not include any statements that are offensive or illegal, violate anybody else’s rights, or contain any information that could lead to harm or injury.

The authors declare no competing financial interest.