Design of Quinoline-Derived Schiff Base Metal Complexes as Bioactive Drug Candidates: Structural Elucidation, Stability Determination, DFT, and Docking Studies with DNA-Targeting Potential Profiles

Abstract

Three novel metal complexes of the tridentate ligand 4-nitro-2-(quinolin-8-yliminomethyl)phenol (NQP) were synthesized and fully characterized using elemental analysis, TGA, magnetic susceptibility, FT-IR, NMR, and UV–Vis spectroscopy. Stoichiometric studies and characterization data proposed square-planar Pd(II), tetrahedral Zn(II), and octahedral Fe(III) geometries. Density functional theory calculations (B3LYP and B3LYP/6-311G(d,p) with LANL2DZ for metals) showed good agreement with experimental findings and revealed enhanced nonlinear optical properties, as evidenced by increased polarizability and hyperpolarizability values. Biological studies demonstrated significant antimicrobial activity, with the Pd–NQP complex exhibiting superior efficacy against bacterial and fungal strains compared to ofloxacin and fluconazole, following the order NQP < Zn < Fe < Pd. Cytotoxicity assays against Hep-G2, MCF-7, and HCT-116 cell lines revealed strong anticancer activity, particularly for the Pd(II) complex (IC₅₀ = 6.35–12.95 μ_g/μL), comparable to cisplatin. All complexes showed higher DPPH radical scavenging activity than ascorbic acid and strong DNA-binding affinity. Antimicrobial activity was further validated experimentally, while molecular docking studies elucidated favorable binding interactions with microbial proteins and cancer-related targets.

1. Introduction

The exploration of metal coordination compounds has become one of the most dynamic areas of modern inorganic and bioinorganic chemistry [1,2,3]. The chemical versatility, structural diversity, and wide range of biological properties exhibited by these complexes have driven continuous research into their synthesis and applications. Among the ligands used to design new metal-based systems, Schiff bases occupy a prominent position because of their ease of preparation, rich donor characteristics, and structural flexibility [4,5]. Their azomethine (–C=N–) linkage plays a vital role in chelation, promoting the formation of thermally and kinetically stable complexes with transition metals [6,7]. Owing to these features, Schiff base complexes have shown promising potential in catalysis, photochemical processes, and medicinal chemistry, particularly as antibacterial, anticancer, and antioxidant agents [8,9]. Integrating a quinoline moiety into a Schiff base framework further enhances its coordination and biological characteristics. Quinoline and its derivatives are widely recognized pharmacophores known for their ability to interact with nucleic acids and enzymes through π–π stacking and hydrogen bonding [10,11]. This heteroaromatic system not only improves lipophilicity and electron delocalization within the ligand structure but also enhances its metal–ligand charge transfer (MLCT) behavior. Consequently, Schiff bases derived from quinoline derivatives often display enhanced redox activity, improved stability, and significant bioactivity, making them ideal candidates for the development of biologically relevant coordination compounds [12,13]. The choice of metal center plays a critical role in determining the physicochemical and biological profiles of Schiff base complexes. Transition metals such as zinc(II), palladium(II), and iron(III) play essential and diverse roles in both biological and chemical systems [14]. Zinc(II) is a non-redox metal ion that contributes to enzyme catalysis, gene regulation, and cellular defense mechanisms, and its complexes are generally regarded as biologically safe [15]. Palladium(II) complexes share coordination similarities with platinum drugs and have demonstrated notable antitumor and antimicrobial properties, often with lower toxicity profiles [16]. Iron(III), on the other hand, is central to redox biology, participating in electron-transfer and oxidative processes that are crucial for metabolic balance [17]. The ability of these metals to form stable complexes with multidentate Schiff base ligands allows for precise tuning of structural, electronic, and biological properties [18,19]. The ligand 4-nitro-2-(quinolin-8-yliminomethyl)-phenol offers an attractive scaffold for coordination chemistry due to its mixed donor atoms (N and O) and the presence of a nitro substituent, which acts as a strong electron-withdrawing group. This substituent can modulate the electronic environment of the metal center, influencing both the stability and reactivity of the complexes. Moreover, the combination of nitro, phenolic, and quinoline functionalities creates a conjugated system capable of engaging in efficient DNA binding through electrostatic, groove, or intercalative interactions, which are vital for understanding their pharmacological potential [20,21]. Recent studies on Schiff base transition metal complexes have demonstrated that variations in ligand environments significantly influence coordination geometry, electronic structure, and spectroscopic behavior, particularly for Zn(II), Fe(III), and related metal ions [22,23].

Moreover, contemporary reviews emphasize that systematic comparisons of synthetic strategies and structure–property relationships are essential for rationalizing the reactivity and functional performance of newly designed metal complexes [24,25].

In the present investigation, new Zn(II), Pd(II), and Fe(III) complexes of the 4-nitro-2-(quinolin-8-yliminomethyl)-phenol Schiff base ligand were synthesized and systematically characterized. Structural elucidation was achieved using UV–Vis, IR, NMR, and mass spectroscopic analyses, complemented by thermal and elemental studies to assess their geometry and composition. The stability and DNA interaction properties of these complexes were further examined to gain insights into their binding mechanisms and possible biological roles. Additionally, the biomedical relevance of the synthesized complexes was explored through antibacterial, antioxidant, and cytotoxicity assessments. This study provides a comprehensive understanding of how ligand design, metal selection, and structural features influence the physicochemical and biological properties of Schiff base complexes. The findings contribute to the broader goal of developing new, multifunctional coordination compounds with potential applications in therapeutics, diagnostics, and biomolecular engineering. The selection of Zn(II), Fe(III), and Pd(II) ions was deliberately designed to probe the coordination adaptability of the NQP ligand toward metal centers with contrasting electronic configurations, geometries, and chemical functionalities. This set of metal ions allows a systematic comparison of how the same ligand framework interacts with different metal centers, oxidation states, and electronic configurations, enabling discussion of the resulting physicochemical behavior and tentative structure–property correlations

2. Results and Discussion

2.1. Physicochemical Characterization of the NQP Ligand and Its Corresponding Metal Complexes

Analytical characterization of the ligand and its synthesized complexes (Table 1) proposed structural consistency across tested parameters. The stoichiometric ratios observed were 1:1 metal-to-ligand for NQP-Pd and NQP-Zn, and 1:2 for NQP-Fe. These compounds exhibited strong stability under ambient atmospheric conditions and in solid-state form. Solubility assessments revealed broad miscibility across diverse media, including polar aprotic solvents (e.g., DMSO, DMF), organic phases like methanol and ethyl acetate, aqueous solutions, acetone, and common laboratory solvents.

Table 1.

The synthesized ligand, denoted as NQP, and its corresponding metal complexes, chemical formulas, and CHN analyses.

Compounds	Empirical Formula (Formula Weight)	Color	µeff (B.M.)	Λm (Ω−1 cm2 mol−1)	Analysis (%) Found (Calc.)
					C	H	N
NQP	C16 H11 N3O3 (293.28)	Pale red	-	-	(65.45) (65.53)	3.85 (3.78)	14.40 (14.33)
NQP-Pd [Pd(NQP)(CH3COO)].2H2O	C18H17N3O7Pd (493.76)	Orange	Dia Magnetic	10.75	43.71 (43.78)	3.55 (3.47)	8.57 (8.51)
NQP-Zn [Zn(NQP)(H2O)].H2O.NO3	C16H14N4O8Zn (455.69)	Light yellow	Dia Magnetic	59.60	42.23 (42.17)	3.18 (3.10)	12.21 12.29)
NQP-Fe [Fe(NQP)2].H2O.NO3	C32H22N7O10Fe (720.40)	Deep brown	5.50	63.10	53.44 (53.35)	3.02 (3.08)	13.68 (13.61)

2.1.1. Elemental Analyses and Molar Conductance Values for the Inspected Compounds

The elemental (CHN) microanalytical data for the synthesized NQP–Pd, NQP–Zn, and NQP–Fe complexes were found to be in excellent agreement with the theoretically calculated values, confirming the proposed stoichiometric formulations. The slight deviations observed between the calculated and experimental percentages fall within acceptable analytical limits (±0.3%), indicating high purity and successful coordination of the ligands to the respective metal centers (Table 1). These findings are consistent with previously reported coordination behaviors of analogous Schiff base and quinoline-derived transition metal complexes [26,27]. The molar conductance values of the complexes were measured in 10⁻³ M DMF solutions at 25 °C. The recorded conductance values are 10.75 Ω⁻¹ cm² mol⁻¹ for NQP–Pd, 59.60 Ω⁻¹ cm² mol⁻¹ for NQP–Zn, and 63.10 Ω⁻¹ cm² mol⁻¹ for NQP–Fe [28,29,30]. This observation suggests that the complexes behave as mono-electrolytes in solution in case of NQP-Zn and NQP-Fe complexes, implying the possible presence of anionic counter ions (such as NO₃⁻) outside the coordination sphere. The NQP-Pd complex value is consistent with the non-electrolytic nature of the complex, suggesting that on the exterior of the coordinating sphere, there are no anions.

These conductivity findings are in good agreement with those reported for structurally related Pd(II), Zn(II), and Fe(III) chelates bearing N,O- and N,N-donor ligands. As a result, the convergence of experimental CHN data, solubility profiles, and molar conductance values validates the proposed molecular formulations and supports the successful synthesis of the target NQP–Pd, NQP–Zn, and NQP–Fe complexes.

2.1.2. FTIR Spectrum

Infrared spectroscopy serves as a powerful tool for probing metal complex structures by examining vibrational modes associated with metal-ligand bonds. Analysis of transition metal complexes reveals characteristic shifts in ligand absorption frequencies, providing evidence for coordination sites (Figure 1 and Table S1).

Figure 1.

IR spectra for the prepared complexes.

Key diagnostic vibrations include those from OH/H₂O groups, carbonyl (C=O) functionalities, and Salen (CH-N) units, which collectively illuminate structural properties and coordination behavior. The free ligand exhibits an imine CH-N vibration at 1624 cm⁻¹. Upon metal binding, this band undergoes a hypsochromic shift: appearing at 1611 cm⁻¹ for NQP-Pd complex, 1602 cm⁻¹ for NQP-Zn complex, and 1605 cm⁻¹ in NQP-Fe compex. This consistent downward displacement confirms azomethine nitrogen participation in coordination [31,32,33]. A distinct hydroxyl stretching vibration appears at 3419 cm⁻¹ in the unbound ligand spectrum. Complex formation produces broadened absorptions at 3449 cm⁻¹ (NQP-Pd), 3405 cm⁻¹ (NQP-Zn), and 3437 cm⁻¹ (NQP-Fe), indicative of hydrated water molecules, a finding supported by elemental analysis data (Table S1). The ligand’s C-O absorption at 1281 cm⁻¹ shifts to lower frequencies following complexation: observed at 1255 cm⁻¹ (Pd complex), 1248 cm⁻¹ (Zn complex), and 1257 cm⁻¹ (NQP-Fe compex). This displacement suggests hydroxy group involvement in C-O-M bonding after deprotonation [34,35]. New vibrational bands emerge in the complexes’ spectra: Metal-oxygen stretches appear at 523 cm⁻¹ (NQP-Pd), 566 cm⁻¹ (NQP-Zn), and 558 cm⁻¹ (NQP-Fe). Additional distinct absorptions at 474 cm⁻¹ (Pd-N), 468 cm⁻¹ (Zn-N), and 497 cm⁻¹ (Fe-O) confirm coordination through both nitrogen and oxygen donor atoms [29,30,36,37]. On the other hand, the vibrational band of acetate group υ (COO) was noticed at 1543 and 1428 cm⁻¹ in spectrum of NQP-Pd complex. The vibrational difference (Δ = υ_as-υ_s) is close to monodentate binding of acetate group [8,36]

2.1.3. ¹H-NMR & ¹³C-NMR Spectral Evaluations of NQP Ligand and Its NQP-Pd and NQP-Zn Complexes

The NQP ligand underwent detailed characterization via ¹H-NMR spectroscopy in deuterated dimethyl sulfoxide (DMSO-d₆), with tetramethylsilane (TMS) providing the chemical shift reference. As depicted in Figure S1, the spectrum reveals a sharp singlet at 14.12 ppm, indicative of the phenolic hydroxyl proton. A second well-defined singlet emerges at 9.11 ppm, assigned to the aromatic proton adjacent to the quinoline nitrogen (s, d, 1H, CH_arm). The imine proton (CH=N) registers as a singlet at 8.95 ppm. Within the aromatic region, complex splitting patterns appear: a doublet spanning 8.46–8.43 ppm (d, 3H, pyridine CH_arm), overlapping signals between 7.89 and 7.94 ppm (d, 5H, CH_arm), and a distinct doublet at 6.97–6.99 ppm (d, 2H, CH_arm), collectively mapping the proton topology. Complementary ¹³C-NMR data (Figure S2) in DMSO-d₆ exhibit key resonances: the imine carbon (CH=N) at 160.50 ppm, the hydroxyl-bound carbon (C–OH) at 162.10 ppm, and the pyridine ring carbon (C=N) at 148.80 ppm. Additional carbon signatures emerge at 139.72, 137.10, 135.60, 133.30, 130.70, 127.10, 126.70, 125.70, 122.10, 120.10, 119.00, 118.20, and 117.30 ppm, delineating the ligand’s carbon framework. These spectroscopic signatures collectively validate NQP’s structural architecture.

In the ¹H NMR spectrum of the NQP-Pd complex, the influence of the Pd(II) ion is evident from the systematic downfield displacement of proton resonances. A sharp singlet at δ 8.81 ppm corresponds to the aromatic proton adjacent to the quinoline nitrogen (CH_arm), while the imine proton (–CH=N) appears as a distinct singlet at δ 8.57 ppm, confirming the integrity of the azomethine moiety after coordination. Within the aromatic region, multiple well-defined signals are observed: a doublet between δ 8.28–8.11 ppm (3H) assigned to pyridyl protons, a cluster of overlapping resonances in the δ 7.84–7.62 ppm range attributed to five aromatic protons, and an additional doublet at δ 7.18 ppm (2H) corresponding to protons on the substituted benzene ring. A signal at δ 2.87 ppm represents the methyl protons (–CH₃) in methoxy group attached to the ligand framework, completing the proton mapping pattern (Figure S3).

The ¹³C NMR spectrum of NQP–Pd exhibits numerous characteristic resonances at δ 162.89, 161.15, 155.46, 152.32, 149.17, 139.38, 138.21, 136.18, 133.42, 130.58, 126.35, 122.75, 120.11, 119.23, 117.78, 115.14, 114.23, and 82.68 ppm (Figure S4).

The ¹³C NMR spectrum of the NQP–Pd complex confirms the coordination of the ligand to the palladium center. The phenolic carbon appears at δ 161.15 ppm, significantly deshielded due to coordination of the phenolic oxygen to Pd(II). The azomethine (C=N) carbon resonates at δ 155.46 ppm, reflecting a downfield shift caused by coordination of the imine nitrogen. Carbons of the quinoline ring, including the carbon adjacent to the ring nitrogen, appear in the range δ 152.32–114.23 ppm, while the aromatic carbon attached to the nitro group is observed at δ 149.17 ppm, consistent with electron-withdrawing effects. Signals corresponding to the coordinated acetate are also evident: the carbonyl carbon appears in the expected downfield region 162.89 ppm, while the methyl carbon resonates at δ 82.68 ppm, influenced by coordination. Overall, the ¹³C NMR data support the formation of the NQP–Pd complex with the ligand bound via the phenolic oxygen and azomethine nitrogen, and the acetate group coordinated to the metal center, confirming the proposed structure (Figure S4).

Similarly, the ¹H NMR spectrum of the NQP-Zn complex displays resonances consistent with coordination to the Zn(II) ion. The aromatic proton adjacent to the quinoline nitrogen appears as a singlet at δ 8.01 ppm, while the imine proton (–CH=N) resonates at δ 7.83 ppm, slightly upfield relative to the Pd(II) analogue—reflecting the weaker deshielding influence of Zn(II). The aromatic region exhibits characteristic splitting patterns: a doublet at δ 7.67 ppm (3H, pyridyl protons), overlapping multiplets between δ 7.32–7.25 ppm (5H, aromatic protons), and a doublet at δ 6.83 ppm (2H, aromatic protons), collectively defining the proton environment within the complex (Figure S5).

The ¹H NMR spectrum of the Zn(II) complex shows the expected resonances of the coordinated ligand framework, confirming successful complex formation. A few additional low-intensity signals are also observed, which can be reasonably attributed to trace residual solvent molecules and/or minor amounts of unreacted starting materials remaining after isolation.

Importantly, these weak resonances are negligible compared to the main signals of the Zn(II) complex, indicating that the isolated product is predominantly the desired coordination species. Furthermore, the high purity of the Zn(II) complex is supported by the agreement between the spectroscopic results (FTIR and NMR) and the elemental analysis/mass spectrometric data, confirming the proposed composition and suitability of the complex for subsequent biological evaluation.

The ¹³C NMR spectrum of NQP–Zn shows signals at δ 160.15, 152.64, 149.24, 136.76, 135.48, 132.13, 131.37, 129.35, 126.18, 123.65, 122.45, 119.56, 117.14, 115.36, 114.43, and 113.82 ppm (Figure S6).

The ¹³C NMR spectrum of the NQP–Zn complex confirms coordination of the ligand to the zinc center. The phenolic carbon appears at δ 160.15 ppm, significantly deshielded due to coordination of the phenolic oxygen to Zn(II). The azomethine (C=N) carbon resonates at δ 152.64 ppm, indicating a downfield shift resulting from coordination of the imine nitrogen. Carbons of the quinoline ring, including those adjacent to the ring nitrogen, appear in the range δ 149.24–113.82 ppm, with the aromatic carbon attached to the nitro group observed at δ 149.24 ppm, consistent with electron-withdrawing effects of the nitro substituent. The overall pattern of chemical shifts, particularly the deshielded phenolic and azomethine carbons, supports the formation of the NQP–Zn complex with the ligand coordinated via the phenolic oxygen and azomethine nitrogen, in agreement with the proposed structure (Figure S6).

2.1.4. Electronic Absorption Spectra (EAS)

UV–Vis electronic spectral characteristics of the uncoordinated NQP Ligand and its metal coordination Complexes NQP–Fe, NQP–Zn, and NQP–Pd were recorded in (EtOH) solution in the wavelength range of 200–800 nm (Figure 1), and the corresponding absorption bands and molar absorptivity values are summarized in Table S2. The free ligand (NQP) exhibits two prominent high-intensity absorption bands at 243, that can be ascribed to π→π* transitions within the aromatic system. Additional medium-intensity bands observed at 293 and 304 nm are assigned to n→π* transitions involving the lone pair of the azomethine nitrogen and the π-system of the C=N chromophore, and an intra-ligand transition near 395 and 470 nm [38]. Upon complexation with metal ions, distinct spectral shifts and the appearance of new bands were observed, confirming the coordination of the ligand to the metal centers. The spectrum of the NQP–Pd complex revealed absorption peaks at 250 and 293 nm due to π→π* and n→π* transitions, respectively. Additional moderate-intensity bands appeared at 307 and 399 nm, assignable to LMCT transitions, while a weak broad band at 467 nm was attributed to a d–d transition corresponding to a square-planar geometry around the Pd(II) ion [39]. The NQP–Zn complex displayed intense bands at 255 and 329 nm, corresponding to π→π* and n→π* transitions, respectively. Additional lower-intensity absorptions were observed at 341 and 448 nm, which can be ascribed to LMCT transitions from the ligand orbitals to the vacant 4s and 4p orbitals of Zn(II). The absence of any d–d transition band is consistent with the d¹⁰ electronic configuration of Zn(II), confirming its tetrahedral geometry [40]. In the NQP–Fe complex, absorption maxima appeared at 277, 295, and 314 nm corresponding to π→π* and n→π* transitions, similar to those of the free ligand but slightly red-shifted due to metal–ligand interaction. Additional weak bands at 371 nm and 482 nm with are assigned to ligand-to-metal charge transfer (LMCT) with molar absoptivity (1351 and 1741 dm³ Mol⁻¹ cm), respectively (Table S2). In high-spin Fe(III) complexes, weak spin- and Laporte-forbidden d–d transitions can be easily masked by the intense LMCT bands in the UV–Vis spectrum supports an octahedral high-spin configuration for the Fe(III) center as seen in Figure 2 [41].

Figure 2.

Electronic absorption spectra of NQP ligand and its metal complexes in Ethanol at 298 K. Minor negative absorbance values result from instrumental baseline correction and solvent/reference mismatch.

2.1.5. Magnetic Moment

Magnetic susceptibility measurements were performed for the NQP–Pd, NQP–Zn, and NQP–Fe complexes to elucidate their electronic structures and coordination geometries. The NQP–Pd complex showed diamagnetic behavior (μ_eff ≈ 0.00 B.M.), consistent with a low-spin d⁸ Pd(II) center in a square-planar geometry, confirming complete electron pairing and non-electrolytic character [41]. Similarly, the NQP–Zn complex was diamagnetic, as expected for a d¹⁰ Zn(II) configuration, indicating a mononuclear species with a likely tetrahedral coordination geometry [40,42]. In contrast, the NQP–Fe complex exhibited paramagnetic with a magnetic moment of 5.50 B.M., characteristic of a high-spin d⁵ Fe(III) ion in an octahedral environment, corresponding to five unpaired electrons and a ligand field insufficient to induce spin pairing [43].

2.1.6. Thermal Analysis

TGA combined with microanalytical data was used to evaluate the composition, thermal stability, and stepwise decomposition of the synthesized NQP–Pd, NQP–Zn, and NQP–Fe complexes [44,45,46]. Measurements were performed under N₂ from 50 to 800 °C at a heating rate of 10 °C min⁻¹ (Table 2). The NQP–Pd(II) complex exhibits a multistep decomposition profile, reflecting the progressive degradation of its coordination framework. The initial mass loss of approximately 7.3%, observed below 130 °C, is reasonably attributed to the release of two coordinated water molecules. A second weight loss of about 11.9% occurring in the 135–245 °C range may be associated with the partial degradation of a low-molecular-weight organic fragment of the ligand backbone. Further mass losses recorded between 250 and 395 °C and 400–670 °C correspond to successive decomposition of the remaining organic moieties of the Schiff base ligand, leading to the collapse of the chelate structure. Above 670 °C, a stable residue amounting to approximately 24.7% of the initial mass remains; this value is consistent with the theoretical mass expected for PdO and is therefore assigned to palladium oxide on the basis of mass balance considerations and literature precedent.

Table 2.

Pyrolytic Breakdown Pathways: Mass Flux Profiles, Volatilized Fragments, Residual Products, and Step-Specific Kinetic Parameters in Synthesized Coordination Compounds.

Complexes	Temp °C	Fragment Loss %		Weight Loss %		E* (kJ mol−1)	A (S−1)	∆H* (kJ mol−1)	∆G* (kJ mol−1)	∆S* (Jmol−1K−1)
		Molecular Formula	M. Wt.	Found	(Calc)
NQP-Pd Residue	30–130	2H2O	36	7.32	(7.29)	35.17	1.95	33.75	133.81	−283.45
	135–245	C2H3O2	59	11.89	(11.95)			30.55	164.22	−288.70
	250–395	C7H4NO2	134	27.18	(27.13)			28.15	201.76	−291.54
	400–670	C9H6N2	142	28.68	(28.76)			27.80	267.89	−297.14
	>670	PdO	122.4	24.73	(24.79)			-	-	-
NQP-Zn Residue	30–165	H2O + NO2 + 1/2O2	80	17.60	(17.55)	31.78	1.27	31.78	131.37	−280.15
	170–230	H2O	18	3.89	(3.96)			29.25	163.71	−284.27
	235–470	C7H4NO2	134	29.46	(29.41)			27.10	206.93	−287.50
	475–650	C9H6N2	142	31.14	(31.17)			26.35	269.63	−291.18
	>650	ZnO	81.39	17.81	(17.86)			-	-	-
NQP-Fe Residue	30–145	H2O + NO2 + 1/2O2	80	11.04	(11.10)	28.45	0.85	28.17	127.51	−275.56
	150–245	C7H4NO3	150	20.89	(20.82)			26.20	157.53	−279.20
	250–360	C7H4NO2	134	18.13	(18.16)			24.09	187.49	−282.70
	365–490	C9H6N2	142	19.78	(19.71)			22.68	222.74	−285.60
	495–620	C9H6N2O	158	21.89	(21.93)			21.75	261.07	−288.16
	>620	FeO	71.84	10.02	(9.97)			-	-	-

The NQP–Zn(II) complex undergoes thermal decomposition through four overlapping stages. The first step, associated with a mass loss of about 17.6% below 170 °C, can be attributed to the removal of lattice water molecules accompanied by the thermal decomposition of crystallized nitrate ions present in the lattice, released as nitrogen dioxide (NO₂). The relatively low decomposition temperature confirms the non-coordinated nature of the nitrate group. A small subsequent mass loss of approximately 3.9% between 170 and 230 °C is consistent with the completion of dehydration processes. The major decomposition of the organic ligand framework takes place between 235 and 470 °C, followed by a final degradation step in the 475–650 °C range, reflecting the gradual breakdown of the Schiff base backbone. The final residue obtained after 650 °C corresponds closely to the calculated value for ZnO, supporting its assignment as the terminal decomposition product.

The NQP–Fe(III) complex displays a distinct decomposition pattern consistent with its 1:2 metal-to-ligand stoichiometry. An initial mass loss of approximately 11.04% below 150 °C is attributed to the decomposition of hydrated water molecules followed by decomposition of nitrate-containing moieties (NO₂ + 1/2O₂). Subsequent weight losses observed between 150 and 245 °C and further extended up to 490 °C are associated with stepwise degradation of the coordinated Schiff base ligands. The final decomposition stage occurring between 495 and 620 °C corresponds to the removal of the remaining organic fragments, resulting in the formation of a stable inorganic residue. The residual mass is consistent with the formation of iron oxide, as inferred from the experimental mass and comparison with the reported thermal behavior of related Fe(III) complexes.

Kinetic Aspects

The kinetic and thermodynamic parameters for the thermal decomposition of the NQP–Pd, NQP–Zn, and NQP–Fe complexes (Table 2), calculated using the Coats–Redfern and Horowitz–Metzger methods [47,48], reveal activation energies consistent with stable coordinated metal chelates undergoing multistep decomposition. The relatively high activation energy (E*) values reflect strong metal–ligand bonding and agree with the enhanced thermal stability observed in the TGA profiles. Positive enthalpy (ΔH*) values confirm the endothermic nature of the decomposition processes, while the large positive Gibbs free energy (ΔG*) values indicate non-spontaneous reactions with slow kinetics under the applied conditions. All complexes exhibit negative entropy (ΔS*), suggesting the formation of more ordered, rigid transition states during the rate-determining step. A compensation effect between E* and the pre-exponential factor (A) was observed, with moderate to low A values indicating sluggish pyrolysis typical of metal chelates. Overall, these results support a complex decomposition mechanism and corroborate the high structural robustness of the synthesized complexes, in agreement with their magnetic and spectroscopic features [49].

2.1.7. Assessment of the Stoichiometric Ratios of the Complexes in Solution

Determining the stoichiometric composition of coordination compounds in solution provides essential insight into their mode of complexation and binding behavior. The metal-to-ligand ratios of the synthesized NQP–Pd, NQP–Zn, and NQP–Fe complexes were established using Job’s method of continuous variation [49] in combination with the mole ratio method [50]. These complementary techniques enable accurate evaluation of the coordination stoichiometry between the central metal ion and the NQP ligand. In Job’s method, equimolar solutions of the ligand and each metal salt were mixed in varying proportions while ensuring that the overall molar concentration remained unchanged. The absorbance of each mixture was measured at the wavelength corresponding to the maximum complex formation band. As illustrated in Figure 3, the absorbance mole fraction curves for NQP–Pd and NQP–Zn complexes exhibit distinct maxima at a ligand mole fraction X ligand of approximately 0.50, which clearly indicates a 1:1 metal-to-ligand stoichiometric ratio in solution. This observation suggests that both Pd(II) and Zn(II) ions coordinate with a single NQP ligand molecule through its azomethine nitrogen and phenolic oxygen donor sites, leading to the formation of stable mononuclear chelates. In contrast, the absorbance curve for the NQP–Fe complex displayed a maximum at X ligand ≈ 0.61, signifying a 1:2 metal-to-ligand ratio. This finding implies that each Fe(III) center coordinates with two NQP ligand molecules, forming a six-coordinate octahedral complex. Such stoichiometric behavior is consistent with the strong coordination tendency of Fe(III) toward multiple donor atoms, which stabilizes the high-spin octahedral geometry typically observed for trivalent iron complexes. The molar ratio method further corroborated these results (Figure S7). The absorbance of solutions containing fixed metal ion concentrations and increasing ligand concentrations exhibited distinct inflection points at metal-ligand ratios corresponding to those obtained from Job’s method 1:1 for Pd(II) and Zn(II) complexes, and 1:2 for the Fe(III) complex. The convergence of results from both analytical approaches provides strong evidence for the stoichiometric formulations of these complexes in solution. Thus, the combined spectrophotometric data confirm the formation of mononuclear NQP–Pd and NQP–Zn complexes with a 1:1 metal–ligand ratio, and a binuclear-type NQP–Fe complex characterized by a 1:2 coordination ratio, reflecting the distinct electronic configurations and coordination preferences of the respective metal ions.

Figure 3.

Continuous variation plot for the prepared NQP-complexes.

The Estimated Stability (Formation) Constants of the Synthesized Complexes

The stability constants of the metal–ligand complexes were determined spectrophotometrically under identical experimental conditions to ensure reproducibility. Each measurement was repeated at least three times using independently prepared solutions, and the resulting data were analyzed by nonlinear fitting of the absorbance changes as a function of concentration. The reported stability constants correspond to the average values obtained from these independent experiments. The associated uncertainties were estimated from the standard deviation of the calculated constants and were found to be within ±5–8%, depending on the metal ion. This level of uncertainty is typical for spectrophotometric determinations of stability constants in ethanol and does not affect the relative comparison of complex stabilities or the conclusions drawn from the study. The stability and strength of interaction between the NQP ligand and metal ions in solution were quantitatively assessed through spectrophotometric evaluation using Job’s method of continuous variation. The formation constants (K_f) of the synthesized complexes NQP-Pd, NQP-Zn, and NQP-Fe were determined based on absorbance data corresponding to maximum complexation (Table 3). The calculated K_f values were 2.7 × 10⁴, 1.8 × 10⁴, and 7.1 × 10⁸ for the Pd(II), Zn(II), and Fe(III) complexes, respectively. These results reveal a pronounced stability trend following the order Fe(III) > Pd(II) > Zn(II), reflecting the stronger electrostatic and coordination interactions associated with the higher oxidation state and greater charge density of the Fe(III) ion. Complementary thermodynamic parameters were derived to further interpret the energetics of the complexation process. The standard Gibbs free energy changes (ΔG°) were calculated from the relationship ΔG° = –RT ln K_f, yielding values of –25.28, –24.76, and –50.49 kJ mol⁻¹ for NQP–Pd, NQP–Zn, and NQP–Fe, respectively. The negative ΔG° values confirm that the complexation reactions proceed spontaneously under the experimental conditions, signifying favorable thermodynamics and the predominance of complex species in equilibrium. The significantly higher magnitude of ΔG° and K_f for the Fe(III) complex suggests that its formation is particularly favorable, likely due to strong ligand-to-metal charge transfer (LMCT) contributions and enhanced chelate stabilization through bidentate coordination of the azomethine nitrogen and phenolic oxygen donor atoms. In contrast, the lower K_f values of the Zn(II) and Pd(II) complexes are consistent with their moderate Lewis acidity and lower field stabilization effects. The dissociation constants (pK) derived from these data further support this trend, indicating that all complexes possess considerable solution stability, with the Fe(III) derivative exhibiting exceptional thermodynamic robustness [50,51].

Table 3.

The K_f and log K_f stability parameters, along with the calculated Gibbs free energy (ΔG°) values, for the investigated complexes at 298 K.

Complex	Kf	Log Kf	ΔG° kJ mol−1
NQP-Pd	2.7 × 104	4.43	−25.28
NQP-Zn	1.8 × 104	4.26	−24.76
NQP-Fe	7.1 × 108	8.85	−50.49

2.2. pH Profile of the Investigated NQP-M Complexes

The influence of pH on the stability and structural integrity of the synthesized NQP–Pd, NQP–Zn, and NQP–Fe complexes was investigated spectrophotometrically to elucidate their behavior under varying acid–base conditions. Understanding pH-dependent stability is essential for predicting complex performance in catalytic, environmental, and biological systems, where proton concentration can significantly affect coordination equilibria. As depicted in Figure 4, the absorbance pH profiles of all complexes display distinct stability regions across the investigated pH range (1–13). Each complex exhibits a gradual increase in absorbance with rising pH, reaching a well-defined plateau region between pH 4 and 11, indicating optimal structural stability and minimal dissociation within this interval. The persistence of constant absorbance throughout this wide pH window demonstrates the robustness of the metal-ligand coordination bonds and suggests the absence of major hydrolysis or protonation events that could otherwise disrupt the chelation equilibrium. At pH values below 3, a marked decrease in absorbance is observed, attributable to ligand protonation and subsequent weakening of metal–donor interactions. Conversely, at highly alkaline conditions (pH > 11), a similar decline occurs due to possible hydroxide precipitation or partial dissociation of the complex species. The overall stability trend follows the order NQP–Fe > NQP–Pd > NQP–Zn, in agreement with their respective formation constants and coordination strengths. The enhanced resistance of the Fe(III) complex to pH variation may result from its higher charge density and stronger ligand field stabilization, which reinforce metal–ligand bonding even under extreme conditions. This broad pH stability range is particularly advantageous for applications in biochemical catalysis, environmental remediation, and pharmaceutical systems, where pH fluctuations are common. The ability of these complexes to retain their coordination framework under diverse pH environments suggests potential utility in aqueous media and biological matrices without significant structural degradation. Furthermore, the demonstrated pH tolerance implies improved bioavailability and functional durability, enhancing their potential role in drug delivery, sensor development, and catalytic reaction mechanisms [52].

Figure 4.

Stability curves of the prepared NQP-Pd, NQP-Zn, and NQP-Fe complexes in Ethanol.

2.3. Molecular Orbital Treatment

The optimized geometry of the synthesized ligand NQP was achieved using the B3LYP/6-311G(d, p) level of Density Functional Theory calculations, as illustrated in Figure 5. The energy-minimized structure represents the most stable conformation of the molecule under gas-phase conditions. Analysis of the optimized coordinates revealed that the dihedral angle values (Table S3) were either 0° or 180°, confirming the planarity of the ligand framework. This coplanar arrangement facilitates extensive π-electron delocalization across the aromatic system, thereby enhancing the ligand’s ability to coordinate with transition metal centers through its conjugated donor sites. The natural charge distribution obtained from Natural Population Analysis calculations further identifies the potential chelating atoms. As shown in Table S4, the atoms N6, N17, and O28 exhibit the most significant negative charge densities, with calculated natural charges of –0.487, –0.551, and –0.659, respectively. These negative charge values indicate strong electron-donating tendencies, designating these atoms as the primary coordination centers involved in metal binding. Consequently, the computational findings strongly support experimental observations, confirming that coordination with Pd(II), Fe(III), and Zn(II) ions predominantly occurs through the azomethine nitrogen (–CH=N), quinoline nitrogen, and phenolic oxygen donor sites of the NQP ligand.

Figure 5.

The molecular configurations, atomic labeling conventions, and dipole moment orientations for the NQP ligand and its palladium, iron, and zinc complexes.

2.3.1. Geometry of Solid Chelates

The B3LYP/6-311G (d, p)-LANL2DZ hybrid basis sets were employed to refine the lowest-energy configurations of palladium, iron, and zinc solid-state chelates. Figure 5 displays the geometrically optimized structures, atomic labeling scheme, and dipole moment orientations for these metal complexes. Dihedral angles, bond distances, and angular parameters surrounding the central metallic ions appear documented in Supplementary Table S3. Notably, the N6-C5 linkage exhibits elongation in Pd, Fe, and Zn chelates relative to the unbound ligand. This lengthening arises from coordination bonds formed between metal cations and the electron-donating N6/N17 atoms. Concurrently, the C13-N17 bond extends, confirming N17’s role as a chelation site. Conversely, contraction occurs in the C23-O28 bond compared to precursor compounds, designating O28 as an additional coordination center. Metal-nitrogen coordinative bonds display greater lengths than typical covalent M-N interactions, diminishing ionic characteristics within the chelates [53]. Within Pd-, Fe-, and Zn-NQP complexes, N6, N17, and O28 atoms serve as primary coordination sites. Iron chelate bond angles—∠C6-N13-Fe33 (121.874°), ∠N13-Fe33-O62 (63.630°), and ∠N13-Fe33-N50 (116.353°)—reveal a distorted octahedral geometry [54]. Stability analyses confirm distorted octahedral (Fe), square planar (Pd), and tetrahedral (Zn) configurations for NQP chelates, governed by metal-ligand coordination at N6, N17, and O28 positions.

2.3.2. Global Reactivity Descriptors

Frontier molecular orbital (HOMO–LUMO) analyses were employed to elucidate electron distribution and charge-transfer behavior in the NQP ligand and its Pd(II), Fe(III), and Zn(II) complexes (Figure S8). Accurate determination of these orbital energies is essential for evaluating redox properties. The HOMO–LUMO energy gap (ΔE) serves as a key descriptor of chemical reactivity and hardness/softness, where smaller ΔE values indicate higher reactivity and greater global softness, while larger gaps reflect increased hardness and reduced reactivity. The calculated quantum chemical parameters, including total energy, ΔE, ionization potential, electron affinity, electronegativity, chemical potential, chemical hardness, and global softness, are summarized in Table 4.

Table 4.

Quantum chemical parameters including frontier molecular orbital energies, (HOMO and LUMO energies), energy gap, ionization energy (I), electron affinity (A), absolute electronegativity (χ), absolute hardness (η), global softness (S), and chemical potential (V)—were evaluated for the NQP ligand and its Pd, Fe, and Zn complexes using B3LYP/6-311G(d,p) and B3LYP/6-311G(d,p)-LANL2DZ methods.

Parameter	Ligand (NQP)	Fe-NQP	Pd-NQP	Zn-NQP
ET, a.u.	−1006.42	−2134.49	−1361.16	−1147.91
EHOMO, a.u.	−0.2346	−0.2244	−0.2204	−0.1292
ELUMO, a.u.	−0.0978	−0.1253	−0.1238	−0.0769
Eg, eV	3.7225	2.6959	2.6286	1.4237
I, eV	6.3838	6.1049	5.9974	3.5160
A, eV	2.6613	3.4093	3.3688	2.0923
χ, eV	4.5226	4.7571	4.6831	2.8041
η, eV	1.8613	1.3478	1.3143	0.7118
S, eV	0.2686	0.3710	0.3804	0.7024
V, eV	−4.5226	−4.7571	−4.6831	−2.8046

Chelation markedly influences the frontier orbital energies of the Pd–, Fe–, and Zn–NQP complexes, leading to HOMO destabilization relative to the free ligand, as reflected by increased ionization potentials (−0.2204 eV for Pd, −0.2244 eV for Fe, and −0.1292 eV for Zn vs. −0.2346 eV for NQP), while simultaneously stabilizing the LUMO levels. HOMO electron density is primarily localized over the phenyl rings and donor O/N atoms in the ligand, whereas LUMO density is delocalized over the ligand framework excluding nitro groups; upon coordination, HOMO density shifts toward the metal-binding sites and LUMO density extends over the metal centers. The reduced HOMO–LUMO gaps of the metal complexes (Table 4) indicate enhanced chemical reactivity, lower hardness (η), and higher softness (S), favoring polarization and efficient charge-transfer processes.

2.3.3. Mapping of the Molecular Electrostatic Potential (MEP) of the Investigated Compounds

The molecular electrostatic potential (MEP) maps of the NQP ligand and its Pd, Fe, and Zn complexes (Figure 6), generated from B3LYP/6-311G(d,p) calculations for the ligand and B3LYP/6-311G(d,p)-LANL2DZ for the complexes, illustrate the spatial charge distribution and reactive sites. The color scale ranges from red (most negative) to blue (most positive). Strong negative potentials are localized on the nitrogen and oxygen atoms, particularly at N6, N17, and O28, reflecting the presence of lone pairs involved in metal coordination, while positive regions are mainly associated with hydrogen and carbon atoms. Oxygen atoms exhibit the highest negative potential, whereas hydrogen centers show the greatest positive character. These electrostatic features indicate that regions of negative potential are the most susceptible to electrophilic attack, governing the preferred reactivity patterns of the complexes [55,56].

Figure 6.

Visualizing Charge Landscapes: Electrostatic Potential Maps (a) and Surface Contours (b) for NQP Ligand and Its Palladium, Iron, and Zinc Complexes via Hybrid DFT Methods.

2.3.4. Natural Charge Distribution and Population Analysis

The electrochemical characteristics of molecular complexes are heavily influenced by charge distribution across constituent atoms. Table 4 details natural atomic charges and electronic configurations for Pd(II), Fe(II), and Zn(II) cations. Calculated results reveal metal ion charges below their formal +2 oxidation state due to electron donation from ligand atoms N6, N17, and O28. As Table S4 illustrates, these nitrogen and oxygen sites exhibit maximum negative charge density, confirming their role as primary electron donors. Analysis indicates reduced electropositivity values of 0.694 (Pd), 1.085 (Fe), and 1.398 (Zn), correlating with respective orbital occupancies: Pd 4d^8.75, Fe 3d^6.44, and Zn 3d^9.97. Table S5 quantifies electron transfer from ligands to metal centers as 1.306e (Pd-NQP complex), 1.915e (Fe-NQP), and 0.602e (Zn-NQP)

2.3.5. Nonlinear Optical Characteristics (NLO)

The distribution of atomic charges enables reliable estimation of molecular dipole orientation and strength. Nonlinear optical (NLO) materials have attracted considerable interest due to their applications in optical computing, data storage, and information processing systems [57,58,59]. Table 5 summarizes the calculated dipole moments (μ), static polarizabilities (α), and first-order hyperpolarizabilities (β) of the NQP ligand and its Pd, Fe, and Zn complexes, compared with urea as a standard reference [59], since experimental NLO data for the investigated compounds are unavailable. The obtained values show noticeable differences from previous reports [60]. Polarizability and hyperpolarizability were calculated in atomic units and converted into esu using 0.1482 × 10⁻²⁴ (α) and 8.6393 × 10⁻³³ (β). The dipole moments confirm the polar nature of all systems, following the order: Zn-complex > Pd-complex > NQP ligand > Fe-complex, with all exceeding that of urea. Importantly, β values indicate significant NLO enhancement, where NQP is ~13× urea, Fe–NQP ~6×, Pd–NQP ~23×, and Zn–NQP ~25×. These results suggest that the NQP ligand and its metal complexes are promising candidates for advanced NLO material applications.

Table 5.

Novel Computational Characterization of NQP Metal Complexes: Dipole Moments, Polarizability Profiles, and Hyperpolarizability Trends via Hybrid DFT Approaches.

Property	Urea	Ligand (NQP)	Fe-NQP	Pd-NQP	Zn-NQP
µ, D	1.3197	7.9087	2.4383	11.0015	11.6909
XX, a.u.	-	−139.2427	−379.5483	−192.1049	−179.9274
YY	-	−107.3217	−186.6038	−171.8473	−126.6059
ZZ	-	−128.749	−273.6689	−170.2038	−137.5542
XY	-	−8.6322	7.3339	−43.5795	−3.3077
XZ	-	0	−9.0564	0.2611	−2.6322
YZ	-	0	0.5411	0.21	−9.618
<α> esu	-	−1.8541 × 10−23	−4.1487 × 10−23	−2.6387 × 10−23	−2.1938 × 10−23
Δα, esu	-	4.1762 × 10−24	24.802 × 10−24	3.1311 × 10−24	7.2289 × 10−24
XXX	-	−159.4917	124.24	530.7301	−404.4384
XXY	-	−122.6108	39.2529	−1.1457	−129.0398
XYY	-	−71.1415	7.1966	−35.3387	−54.8563
YYY	-	−117.1665	−2.9909	−118.9548	−122.9424
XXZ	-	0	4.1844	−0.2149	−0.8265
XYZ	-	0	−0.2367	0.3462	8.7902
YYZ	-	0	0.6056	0.4538	40.7942
XZZ	-	21.7179	4.0556	6.6482	4.4549
YZZ	-	22.9552	−3.1423	32.9372	−60.3864
ZZZ	-	0	2.4088	−0.2513	28.4488
<β>, esu	0.1947 × 10−30	2.6012 × 10−30	1.2066 × 10−30	4.4022 × 10−30	4.8034 × 10−30

2.4. DNA Interaction with the Synthesized Complexes

2.4.1. Electronic Spectra of Interaction with DNA

The interaction between the synthesized NQP–Pd, NQP–Zn, and NQP–Fe complexes and calf thymus DNA (CT-DNA) was explored spectrophotometrically to elucidate their binding behavior and possible interaction modes. The titration experiments were performed by gradually adding buffered CT-DNA solutions to fixed concentrations of each metal complex, and the corresponding electronic absorption spectra were recorded at room temperature (Figure 7, Figures S9 and S10). The resulting spectral variations were analyzed to determine the mode and strength of binding, with key spectroscopic parameters summarized in Table 6.

Figure 7.

(a) Electronic spectral scans for the binding of CT-DNA with NQP-Zn complex (10⁻³ M) in 0.01 M tris buffer (pH = 7.2, 298 K) with CT-DNA (0–100 μM, from top to bottom). (b) Plot of [DNA]/(ε_a-ε_f) versus [DNA] for the interaction of CT-DNA with NQP-Zn complex.

Table 6.

Optical signatures revealing DNA engagement by engineered molecular constructs.

Complex	λmax Free (nm)	λmax Bound (nm)	∆n	Chromism (%)	Type of Chromism	∆G kJ mol−1	Binding Constant $\times$ 105
NQPPd	266 353 480	258 342 474	−8 −11 −6	43 34 50	Hypo Hypo Hypo	−33.63	7.85
NQPZn	257 322 444	254 324 432	−3 2 −12	42 34 40	Hypo Hypo Hypo	−32.56	5.10
NQPFe	260 346 466	258 343 461	−2 −3 −5	17 15 14	Hypo Hypo Hypo	−31.77	3.7

Upon progressive addition of DNA, a gradual decrease in absorbance intensity (hypochromism) was observed for all three complexes, accompanied by a small blue shift in the λ_max values (Δ_λ = 3–11 nm). These spectral changes are characteristic of a non-covalent interaction mode, primarily involving electrostatic attraction or partial intercalation between the complex and DNA base pairs [61]. The observed hypochromism results from reduced transition probabilities as the electronic transitions of the aromatic chromophore couple weakly with the π orbitals of the DNA bases, thereby restricting π–π* excitation within the ligand framework [61,62].

The magnitude of hypochromism varied slightly among the complexes, decreasing in the order NQP–Pd > NQP–Zn > NQP–Fe, suggesting stronger interaction affinity of the Pd(II) complex toward the DNA helix (Scheme 1).

Scheme 1.

The proposed mechanism for DNA interaction via electrostatic and minor groove binding modes for the NQP-Zn complex.

This can be attributed to the enhanced planarity and higher polarizability of the Pd(II) center, which facilitates stronger electrostatic and stacking interactions. Conversely, the Fe(III) complex exhibited the weakest hypochromism, consistent with its more compact coordination sphere and reduced aromatic overlap. The calculated binding constants (K_b) obtained from absorption titration data followed the same sequence: 7.85 × 10⁵ (NQP–Pd) > 5.10 × 10⁵ (NQP–Zn) > 3.70 × 10⁵ (NQP–Fe), indicating moderate to strong DNA-binding affinity through an electrostatic or groove-binding mechanism rather than complete intercalation. The corresponding negative ΔG° values (–33.63, –32.56, and –31.77 kJ mol⁻¹) further confirm that the complex DNA binding processes are spontaneous and thermodynamically favorable. Taken together, the spectral and thermodynamic results reveal that these complexes associate with DNA through partial intercalation assisted by electrostatic interactions, where the extended π-conjugated system of the ligand facilitates limited stacking within the DNA base pairs. The stronger interaction of the NQP–Pd complex highlights its potential in DNA-targeted applications, such as anticancer or diagnostic agents, where moderate DNA affinity and reversible binding behavior are essential for biological compatibility [63,64].

2.4.2. Viscosity Measurements

To clarify the DNA-binding mode of the synthesized complexes (NQP–Fe, NQP–Pd, and NQP–Zn), viscosity measurements were performed. DNA viscosity is highly sensitive to changes in helix length and flexibility, making it a useful indirect probe for binding interactions. Classical intercalation (e.g., ethidium bromide) typically causes a marked viscosity increase due to DNA helix elongation, whereas groove or electrostatic binding usually produces minor changes [4,63,64]. Partial intercalation may induce weaker viscosity enhancement due to DNA bending or distortion [4,63,64].

As shown in Figure 8, the relative viscosity of CT-DNA increased gradually upon addition of each complex, suggesting partial intercalation of the aromatic imine moiety into the DNA base pairs. The viscosity increase followed the order NQP–Fe > NQP–Pd > NQP–Zn, consistent with spectrophotometric binding results. The pronounced effect of NQP–Fe indicates a stronger interaction, likely involving partial intercalation combined with electrostatic attraction. This may result from nitrate substitution by water in aqueous solution, generating a partially cationic species that can interact with the DNA phosphate backbone (Scheme 2), explaining its higher binding constant.

Figure 8.

Dynamic viscosity measurements of the synthesized NQP complexes were performed at [DNA] = 0.5 mM, [complex] and [ethidium bromide] ranging from 25 to 250 μM, and at 298 K.

Scheme 2.

The proposed mechanism for DNA interaction via replacement and Intercalative modes for the NQP-Pd complex.

2.4.3. Gel Electrophoresis

Agarose gel electrophoresis was employed to further examine the interaction of the synthesized Salen-based metal complexes (NQP–Pd, NQP–Zn, and NQP–Fe) with calf thymus DNA, providing qualitative evidence of DNA binding and cleavage behavior (Figure 9). Upon incubation of the DNA samples with the tested complexes, noticeable alterations were observed in the migration and intensity of the DNA bands compared with the control (untreated DNA).

Figure 9.

DNA binding results of the prepared complexes based on gel electrophoresis lane: Lane 1: NQP-Zn complex + DNA; lane 2: NQP-Fe complex + DNA; lane 3: NQP-Pd complex + DNA; lane 4: NQP-Zn complex; lane 5: NQP-Fe complex; lane 6: NQP-Pd complex.

The control DNA exhibited a distinct, sharp band with no observable fragmentation, indicating its intact structure. In contrast, the DNA samples treated with the metal complexes displayed partial reduction in band intensity and minor smearing effects, which suggest interaction between the complexes and DNA strands. The observed decrease in band sharpness indicates partial cleavage or structural modification of the DNA backbone, likely resulting from the coordination of the metal centers to nucleophilic sites within DNA. These results clearly imply that the metal ions play a crucial role in mediating the binding and partial cleavage of DNA. The observed activity may be attributed to the ability of the complexes to facilitate electron transfer or reactive oxygen species (ROS) generation, leading to limited cleavage at specific sites along the DNA strand. Among the studied chelates, NQP–Fe exhibited slightly greater DNA interaction, consistent with its higher binding constant obtained from UV–Vis spectroscopic studies. The gel electrophoresis results corroborate the partial intercalative binding nature of the NQP complexes. The metal centers, in concert with the extended π-conjugated imine ligands, enable insertion between DNA base pairs or coordination to the phosphate backbone. Consequently, these complexes can disrupt the supercoiled structure of DNA, supporting their potential as DNA-active agents capable of modulating genetic material and inhibiting microbial proliferation [65].

2.5. Biological Evaluation of the Prepared NQP Ligand and Its Complexes

2.5.1. Antimicrobial Activity

The in vitro antimicrobial properties of the synthesized NQP Salen ligand and its corresponding meta complexes (NQP–Pd, NQP–Zn, and NQP–Fe) were evaluated against three bacterial strains, Serratia marcescens (Gram-negative), Escherichia coli (Gram–negative), and Micrococcus luteus (Gram-positive), and three fungal species, Aspergillus flavus, Geotrichum candidum, and Fusarium oxysporum. The inhibition of microbial growth by chemotherapeutic agents generally proceeds through microstatic mechanisms. All the examined compounds exhibited pronounced antimicrobial activity against the tested organisms. Comparison of the biological activities of the free NQP ligand and its metal complexes with those of standard bactericidal and fungicidal agents revealed that the metal complexes displayed higher inhibition zones and thus greater biological efficacy (Table S6). Notably, all complexes demonstrated significantly enhanced antimicrobial potential relative to the uncoordinated ligand. The increased activity of the transition–metal complexes can be rationalized based on Overtone’s concept and chelation theory. Upon chelation, the polarity of the metal ion decreases markedly owing to orbital overlap between the metal center and donor atoms of the ligand. This process allows partial sharing of the metal’s positive charge with the ligand, increasing π–electron delocalization throughout the chelate ring. Consequently, the overall lipophilicity of the complex rises, facilitating its penetration through microbial lipid membranes and thereby promoting blockage of metal–binding sites within microbial enzyme systems [65,66].

The minimum inhibitory concentration (MIC) values (mg/mL) and activity indices were determined using the serial dilution method and are summarized in Table 7 and Table S7.

Table 7.

Evaluation of antimicrobial activity via minimum inhibitory concentration for NQP azomethine and its metal complexes.

Compound	(MIC) Minimum Inhibition Concentration µg/mL
	Bacteria			Fungi
	S. marcescence	E. coli	M. luteus	A. flavus	G. candidum	F. oxysporum
NQP	8.00	7.25	6.25	8.75	6.50	7.25
NQPPd	3.00	2.50	1.75	3.00	2.00	2.50
NQPFe	4.00	3.25	2.50	3.75	3.00	3.25
NQPZn	3.50	2.75	2.25	3.50	2.50	2.75
Ofloxacin	2.50	2.25	1.25
Fluconazole				2.50	1.50	2.25

Among the investigated species, the NQP–Pd complex exhibited the most pronounced antimicrobial effect, showing the lowest MIC values against M. luteus and G. candidum (Figure 10 and Figure 11), calculated according to the following relationship [67,68].

$$\mathrm{A}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y} \mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x}\left(\mathrm{A}\right)={\displaystyle \frac{\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{o}\mathrm{m}\mathrm{p}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}\left(\mathrm{m}\mathrm{m}\right)}{\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a} \mathrm{f}\mathrm{o}\mathrm{r} \mathrm{s}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{d} \mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}\left(\mathrm{m}\mathrm{m}\right)}}\times 100$$ (1)

Figure 10.

Assessment of antibacterial potential of the studied compounds toward Micrococcus luteus at different concentrations.

Figure 11.

Assessment of antifungal potential of the studied compounds toward G. candidum at different concentrations.

The observed variations in biological activity among the complexes can be attributed to differences in several physicochemical parameters such as solubility, dipole moment, conductivity, redox potential, coordination geometry, and membrane permeability, all of which are influenced by the nature of the coordinated metal ion. Furthermore, discrepancies in microbial cell wall permeability and ribosomal structure may also affect compound uptake and binding efficiency. Complexes exhibiting relatively low lipid solubility tend to show diminished antimicrobial action, possibly due to limited diffusion through the microbial membrane and reduced access to intracellular targets. Although chelation significantly enhances antibacterial behavior, the overall activity of the complexes is governed by a synergistic interplay of multiple factors, including solubility, steric effects, redox potential, coordination number, metal–ligand bond length, hydrophobicity, and overall molecular geometry [68,69].

2.5.2. Anticancer Activity

Cancer research remains one of the most actively pursued scientific fields worldwide due to the complex nature and high mortality rate of this disease. Cancer arises when normal body cells lose their regulatory control and begin to proliferate abnormally. Following cardiovascular disorders, cancer is the second leading cause of death globally. Although several therapeutic strategies are available, most current anticancer agents are cytotoxic in nature, and their administration forms the basis of chemotherapy.

The anticancer mechanisms of various chemotherapeutic and metal-based agents can be summarized as follows:

• i.Enzymatic or chemical inactivation: Certain carcinogens exhibiting high reactivity toward cellular macromolecules, including DNA, protein structures, and enzymatic systems, can undergo direct neutralization. A subset of chemicals operates by suppressing or activating cytochrome P₄₅₀ enzymes, thereby facilitating carcinogen detoxification pathways.
• ii.Prevention of formation of active species: Many genotoxic carcinogens require metabolic activation in the liver to form reactive intermediates capable of binding to DNA. Anticancer agents that inhibit this bioactivation prevent carcinogen-DNA adduct formation.
• iii.Scavenging mechanisms: Certain anticancer compounds can adsorb or bind dietary carcinogens, keeping them intact and preventing their interaction with DNA.
• iv.Antioxidant and free radical scavenging: Reactive oxygen species (ROS) and free radicals play a major role in carcinogenesis by inducing DNA damage, mutagenesis, and cytotoxicity. They can inhibit DNA repair and inactivate tumor suppressor genes. Many medicinal plants and synthetic ligands possess antioxidant properties that neutralize ROS, thus reducing oxidative stress–induced tumor initiation and progression [70].

Novel imine compound NQP and its coordinated complexes (NQP-Pd, NQP-Zn, NQP-Fe) underwent cytotoxicity screening across 0–10 μM concentrations targeting malignant cell lines: hepatic carcinoma HepG-2, mammary carcinoma MCF-7, and colorectal carcinoma HCT-116. Half-maximal inhibitory concentrations (IC₅₀) were derived for all test agents, with quantitative outcomes visualized in Figure 12 and Table S8. The results demonstrated that all complexes exhibit significant cytotoxic potency, exceeding that of the free ligand and comparable to the standard drug Cisplatin. The variation in biological behavior among the complexes can be attributed to differences in metal ion nature and coordination environment, both of which influence their interaction with cellular biomolecules.

Figure 12.

Potency Benchmarks (IC₅₀): NQP Ligand and Complexes in Colon (HCT-116) vs. Breast (MCF-7) Cancer Cell Assays.

According to Tweedy’s chelation theory [71,72,73], the enhanced cytotoxicity of the metal complexes arises from chelation-induced reduction in the metal ion’s polarity and increased electron delocalization across the chelate ring. This facilitates membrane permeability and cellular uptake, leading to improved biological activity. Cytotoxicity results indicated that all tested complexes showed potent antiproliferative effects, with IC₅₀ values ranging between 12.95 and 17.50 μg/μL for HCT-116, 7.45–12.56 μg/μL for HepG-2, and 6.35–10.11 μg/μL for MCF-7 cell lines. The sensitivity of the cancer cells towards the tested compounds followed the order: MCF-7 > HepG-2 > HCT-116.

Among all tested complexes, NQP–Pd exhibited the highest cytotoxicity with an IC₅₀ value of (6.35 μg/μL) against MCF-7 cells, followed by NQP–Zn (8.25 μg/μL) and NQP–Fe (10.11 μg/μL). Notably, all complexes were more active than the free NQP ligand, confirming the enhancement of antitumor potency upon complexation. The improvement in cytotoxic activity can be ascribed to an increase in the acidity of coordinated sites within the ligand upon complexation, allowing stronger hydrogen bonding interactions with biomolecular targets. Moreover, variation in coordination geometry and the nature of the metal ion notably influence the binding affinity of the complexes toward DNA and proteins, thereby altering their biological profiles [74]. According to Gaetke and Chow [75,76], metal ions may also mediate oxidative stress through Fenton-like pathways, generating reactive oxygen species (ROS) capable of inducing oxidative damage in cancer cells. Hence, the observed cytotoxicity may result from a combination of chelation-driven membrane permeability enhancement and ROS-mediated cellular damage, both contributing to the overall anticancer activity of the NQP metal complexes.

2.5.3. Antioxidant Activity

The DPPH radical scavenging assay is among the most widely used and reliable methods for evaluating the antioxidant potential of chemical compounds, owing to the ability of the stable DPPH radical to accept either an electron or a proton. The reaction between DPPH and an antioxidant compound results in a decrease in the characteristic absorption band intensity of DPPH, reflecting the reduction of the radical species and its subsequent stabilization through electron transfer or hydrogen atom donation. The antioxidant performance of the synthesized NQP ligand and its metal complexes (NQP–Pd, NQP–Zn, and NQP–Fe) was evaluated using this method, and the results are summarized in Figure 13 and Table S9. The degree of discoloration of the DPPH solution upon treatment with the tested samples was used as a quantitative measure of their radical-scavenging efficiency [77]. The calculated IC₅₀ values were 61.2 μg/mL for the free NQP ligand, 18.7 μg/mL for NQP–Pd, 22.35 μg/mL for NQP–Zn, and 26.4 μg/mL for NQP–Fe. These results clearly demonstrate that the metal complexes possess markedly higher antioxidant capacities than the uncoordinated ligand and even surpass the activity of the standard reference antioxidant, ascorbic acid. Among all the investigated species, the NQP–Pd complex exhibited the most potent DPPH radical scavenging effect, as indicated by its lowest IC₅₀ value (18.7 μg/mL). This strong antioxidant behavior can be attributed to the facilitation of electron transfer from the Pd(II) center and the extended π-conjugation within the imine ligand framework, which together stabilize the radical intermediates formed during the scavenging process.

Figure 13.

DPPH suppression by the investigated compounds.

2.6. Computational Binding Analysis Targeting Microbial Pathogens and Mammary Carcinoma

Antibacterial agents primarily function by disrupting cellular processes such as cell wall assembly, protein production, nucleic acid replication, and metabolic pathways [71,72,73]. Molecular docking analyses validated inhibitor effectiveness. Researchers selected binding pocket residues from bacterial and breast cancer protein structures in the Protein Data Bank (PDB), assessing interaction profiles against target proteins based on experimental bioactivity data. Synthesized compounds underwent docking simulations with Micrococcus luteus (PDB: 3IF5), Geotrichum candidum (PDB: 1THG), and breast cancer cells (PDB: 3HB5). Binding free energies and receptor interactions were quantified, with results cataloged in Table S10 (showing binding energies in kcal/mol). Visualizations in Figure 14 and Figure S11 depict 2D/3D interaction models across all tested entities, where dashed lines indicate hydrogen bonds. Compound-receptor interfaces consistently involved hydrogen donors/acceptors, π-hydrogen bonds, and π-π stacking. These configurations demonstrated stability, reinforced by hydrogen bonding within docked complexes. The study mimics actual docking dynamics, recording interaction energies between ligands and target proteins. Negative binding affinities confirmed spontaneous interactions, with lower (more negative) energies correlating with stronger binding. Table S10 data revealed NQP-1THG exhibited superior negative binding energy compared to Pd-, Zn-, and Fe-NQP-1THG complexes. Similarly, NQP-3IF5 outperformed its metal-complexed counterparts. For breast cancer target 3HB5, Pd-NQP-3HB5 showed markedly higher affinity than NQP-3HB5, Zn-, or Fe-NQP-3HB5 chelates, suggesting enhanced inhibitory potential. Binding efficacy rankings for Pd/Fe/Zn-NQP chelates against receptors were: Pd-NQP-1THG > Pd-NQP-3HB5 > Pd-NQP-3IF5.

Figure 14.

Binding dynamics revealed: computational analysis of NQP Ligand and metal chelate interactions within microbial and oncogenic receptor sites.

3. Methods and Materials

3.1. Materials

All chemical reagents were sourced commercially from Fluka and Sigma-Aldrich Chemie GmbH. These included quinolin-8-amine, 2-hydroxy-5-nitrobenzaldehyde, palladium(II) acetate, zinc nitrate hexahydrate, and iron(III) nitrate nonahydrate. Dimethylformamide and absolute ethanol were utilized without additional purification procedures.

3.2. Instrumentation Employed for Methods of Charachterization

The research employed a comprehensive suite of methodological tools and approaches, carefully designed to enhance the accuracy and dependability of its findings are available in Supporting Information.

3.3. Salen Schiff Base Synthesis Protocol

The Schiff base ligand 4-Nitro-2-(quinolin-8-yliminomethyl)-phenol (NQP) was synthesized via a condensation reaction between equimolar quantities of 8-aminoquinoline and 5-nitrosalicylaldehyde. Specifically, 8-aminoquinoline (1.44 g, 10 mmol) dissolved in 15 mL of absolute ethanol was mixed with 5-nitrosalicylaldehyde (1.67 g, 10 mmol) in 15 mL ethanol. The reaction mixture was refluxed under continuous stirring for 2 h. Upon completion, the mixture was allowed to cool to room temperature, leading to the formation of a pale red precipitate. The solid product was separated by filtration, recrystallized from ethanol, and dried over a desiccator to yield the pure Schiff base ligand (Scheme 3).

Scheme 3.

The schematic illustration outlines the synthesis route of the NQP ligand and the formation of its corresponding metal complexes.

¹H NMR (400 MHz, DMSO-d₆, δ ppm): 6.97–6.99 (d, 2H/aromatic CH), 7.89–7.94 (d, 5H, aromatic CH), 8.43–8.46 (d, 3H/aromatic CH of quinoline ring), 8.95 (s, 1H/CH=N azomethine proton), 9.11 (d, 1H/aromatic CH adjacent to quinoline nitrogen), 14.12 (s, 1H/phenolic –OH).

¹³C NMR (DMSO-d₆, δ ppm): 162.10 (C=H–N), 160.50 (C–OH), 148.80 (C=N of quinoline ring), 117.30, 118.20, 119.00, 120.10, 122.10, 125.70, 126.70, 127.10, 130.70, 133.30, 135.60, 137.10, 139.72 (aromatic carbons).

3.4. Metal Chelates Synthesis Protocol

The synthesized Schiff base ligand NQP (4 mmol, 1.31 g) was dissolved in 15 mL of absolute ethanol and subsequently reacted with the corresponding metal salts. Separate ethanolic solutions containing Zn(NO₃)₂·4H₂O (4 mmol, 1.19 g), Pd(CH₃COO)₂ (4 mmol, 0.488 g), and Fe(NO₃)₃·9H₂O (2 mmol, 0.81 g) were prepared individually and added dropwise to the ligand solution under continuous stirring. The reaction mixtures were refluxed for 2 h to ensure complete complexation, during which gradual evaporation led to the formation of solid products. The resulting precipitates were collected by filtration, thoroughly washed with ethanol to remove unreacted residues, and dried in a desiccator over anhydrous calcium chloride. For convenience, the obtained complexes were abbreviated as NQP–Pd, NQP–Zn, and NQP–Fe, corresponding to the palladium(II), zinc(II), and iron(III) coordination products, respectively (Scheme 3).

3.5. Inspection of Magnetic Moment Measurements

Magnetic moment measurements for the prepared complexes are shown in supporting information [68,71,78].

3.6. Thermo-Gravimetric Analysis and Kinetic Studies

The detailed information for thermo-gravimetric analysis and kinetic studies are provided in the supporting information according to literature [71,73,79].

3.7. Establishing Chelate Ligand Stoichiometry Using Job’s Analysis and Molar Ratio Techniques

The stability constants and composition of the examined metal complexes were investigated in aqueous media using Job’s method of continuous variation and the mole ratio technique [80,81]. Transmittance measurements were obtained through sequential addition of both ligand and metal ion solutions. After sonication and equilibration of all samples, the recorded transmittance values were plotted against two parameters: the ligand mole fraction ([L]/([L] + [M])) and the ligand-to-metal concentration ratio ([L]/[M]).

Determination of Measurable Stability Parameters for Coordinated Molecular Assemblies

The stability constants (K_f) for newly synthesized complex solutions were measured using UV-Vis spectroscopy, aligning with Job’s continuous variation approach. This quantification employed the formula below [81];

$${\mathrm{k}}_{\mathrm{f}}={\displaystyle \frac{\frac{\mathrm{A}}{{\mathrm{A}}_{\mathrm{M}}}}{\mathrm{C}(1{-\mathrm{A}/{\mathrm{A}}_{\mathrm{m}})}^2}}$$ (2)

where there is 1:1 molar ratio.

$${\mathrm{k}}_{\mathrm{f}}={\displaystyle \frac{\frac{\mathrm{A}}{{\mathrm{A}}_{\mathrm{M}}}}{4{\mathrm{C}}^2(1{-\mathrm{A}/{\mathrm{A}}_{\mathrm{m}})}^3}}$$ (3)

where there is 1:2 molar ratio.

In the spectrophotometric evaluation of metal–ligand complexation, the initial metal ion concentration is expressed as [C], while A denotes the measured absorbance at any selected point within the absorption spectrum. The maximum absorbance (A_m) corresponds to the condition of complete complex formation, where all metal ions are bound to ligand molecules. The formation constant (K_f) is derived from absorbance data obtained through methods such as Job’s continuous variation or mole ratio analysis, providing quantitative insight into the equilibrium stability of the complexes.

The standard Gibbs free energy change (ΔG) associated with complex formation is calculated using the thermodynamic relationship:

ΔG = −RT ln K_f (4)

where R is the universal gas constant (8.314 J·mol⁻¹·K⁻¹), T is the absolute temperature (K), and K_f is the formation constant of the metal–ligand complex. The negative value of ΔG indicates a spontaneous and thermodynamically favorable complexation process.

3.8. DFT and Docking Studies

3.8.1. DFT Perspective

Computational investigations were conducted using Gaussian 09W software (Revision C.01) to perform geometry optimization and energy minimization of the synthesized ligand and its corresponding metal complexes. These studies were undertaken to elucidate the molecular geometries in the absence of single-crystal X-ray diffraction data [82]. The Density Functional Theory (DFT) framework was employed, with calculations carried out at the B3LYP/6-311G (d, p) level of theory under gas-phase, ground-state conditions for the free ligand [83,84,85]. For the metal complexes, a mixed basis set approach was adopted: the B3LYP/6-311G (d, p) basis set was applied to nonmetal atoms, while LANL2DZ effective core potentials (ECPs) were used for the metal centers (Pd, Fe, and Zn) to appropriately account for relativistic effects and core valence electron interactions [86,87]. Geometry optimization was performed without imposing symmetry constraints, allowing unrestricted variation in bond lengths, bond angles, and dihedral angles to achieve global energy minima. The optimized geometries were subsequently analyzed to extract key quantum chemical descriptors, including total electronic energy, dipole moment, frontier molecular orbital (HOMO–LUMO) energies, and electrostatic potential (ESP) surfaces, providing insight into the electronic distribution and reactivity profiles of the investigated systems [88,89]. Molecular orbital visualizations were generated using GaussView 5.0, while nonlinear optical (NLO) properties, such as the mean polarizability (<α>), first hyperpolarizability (<β>), anisotropy of polarizability (Δα), and dipole moment (μ), were computed directly from the Cartesian coordinate outputs of the optimized structures [89,90].

3.8.2. Docking Perspective

The followed protocol in docking studies is shown in the supporting information [91,92,93].

3.9. Biological Studies

3.9.1. DNA Interaction with Studied Chelates

Binding affinity assessments for novel chelate compounds with DNA were conducted in a Tris-HCl buffer system (10 mM, pH 7.3) supplemented with 100 mM NaCl. CT-DNA concentration was quantified spectrophotometrically at 260 nm, with purity verified by an (A 245/A 262) absorbance ratio of 1.8, indicating the absence of protein contamination. Freshly prepared DNA stock solutions stored at 4 °C were utilized within 4 days. Binding experiments involving the chelates were performed in DMSO. Ethidium bromide concentrations were calibrated at 480 nm (ε = 5860 M⁻¹ cm⁻¹) using established methodology [94]. Absorbance measurements at 260 nm (A₂₆₀) targeted the peak DNA absorption wavelength for concentration estimation. To maintain accuracy, A₂₆₀ values were confined to the instrument’s linear detection range (typically 0.1–1.0). DNA concentration was calculated by correcting A₂₆₀ readings for turbidity (via A₃₂₀ subtraction), applying dilution factors, and applying the standard conversion where A₂₆₀ = 1.0 corresponds to 50 µg/mL of pure double-stranded DNA. Post-electrophoresis validation of concentration and yield was achieved by comparing sample band intensity against a DNA quantitation ladder.

Spectrophotometric Titration Techniques

This method relies on maintaining a constant chelate concentration while varying DNA doses from 0 to 100 μM. After combining DNA with the chelate solution, the mixture was equilibrated at room temperature (25 °C) for 15 min. This allowed clear observation of absorbance changes in the ligand-to-metal charge transfer (LMCT) peak. CT-DNA dissolved in Tris-HCl buffer (pH 7.3) was added to both the chelate solution and a reference blank solution (containing all components except the chelate) to correct for DNA absorption spectra. Absorbance measurements were made using a 1 cm quartz cuvette across the 200–500 nm range, referenced against the blank. The binding affinity [K_b] for every chelate to DNA; may estimate through the following equation [94,95].

$${\displaystyle \frac{[DNA]}{A}}={\displaystyle \frac{[DNA]}{(\in b-\in f)}}+{\displaystyle \frac{A}{Kb}}$$ (5)

Sketch a relationship with both [DNA]/A, [where A = (є_a − є_f)] against [DNA] besides the following proportion for ${\displaystyle \frac{Slope}{intercept}}$; K_b may be evaluated. The term [DNA] denotes the molar concentration of DNA present in the system. The parameter є_a quantifies the degree of fluorescence suppression, calculated as the ratio A_obs/[tested molecule] at defined DNA concentrations. Separately, є_f reflects the suppression efficiency for unbound chelates, while є_b corresponds to suppression observed for DNA-bound chelates. To determine the standard Gibbs free energy change (ΔG^#) associated with DNA binding, the following equation is applied: ΔG^# = −RT ln (Kb).

Viscosity Technique

Hydrodynamic testing was conducted at ambient temperature (25.00 °C) across multiple concentrations. While maintaining a constant CT-DNA concentration, compounds were evaluated at concentrations ranging from 0 to 100 μM. Flow times were recorded using a digital stopwatch in triplicate, with mean values derived from these measurements [82]. Results were analyzed by plotting (η/η⁰)^1/3 against the inverse binding ratio 1/B (where B = [DNA]/[compound]). Here, η and η₀ represent the viscosities of DNA solutions with and without the test compounds, respectively. Viscosity values were calculated using the equation: η = (t − t°)/t°, where t denotes flow time in the presence of the studied complex and t₀ indicates flow time in buffer solution [96,97].

Agarose Gel Electrophoresis

The detailed protocol for agarose gel electrophoresis is shown in supporting information based in literature [97,98].

3.9.2. Antibacterial Activity

This study evaluated the in vitro antimicrobial performance of organic ligands and their metal coordination compounds against selected microorganisms. Test organisms included Gram-negative bacteria (Escherichia coli and Serratia marcescens), Gram-positive bacteria (Micrococcus luteus), and fungi (Fusarium oxysporum, Getrichm Candidum, and Aspergillus flavus). Bacterial cultures were grown on nutrient agar at 37 °C for 24 h, while fungal strains were cultivated on malt extract agar at 28 °C for 48 h. The agar diffusion method [99,100,101,102] was employed for assessment. Stock solutions (1.0 × 10⁻³ M) of ligands and complexes were prepared in a DMSO: water mixture (1:9 ratio), then serially diluted to 1.0 × 10⁻⁶ M using deionized water. Microbial suspensions standardized to 0.80 g dm⁻³ NaCl concentration were blended with cooled agar medium (0.5 mL suspension per 10 mL agar) and uniformly layered in Petri dishes. Aliquots (100 μL) of diluted solutions were introduced into 10 mm diameter wells bored in the inoculated agar. Control experiments accounted for solvent interference and metal ion effects. Prior to main incubation, plates underwent a 4-h equilibration phase at 15 °C to facilitate antimicrobial diffusion. Subsequently, plates were incubated at appropriate growth temperatures. Post-incubation inhibition zones, reflecting antimicrobial efficacy, were measured quantitatively.

3.9.3. Cytotoxicity

Three cancer cell lines—HCT-116 (human colorectal carcinoma), MCF-7 (human breast adenocarcinoma), and HepG-2 (human hepatocellular carcinoma) were acquired from the American Type Culture Collection (ATCC). These models were utilized to assess the anticancer activity of the synthesized complexes. Cells underwent 24-h compound exposure at 37 °C, followed by fixation with 50% trichloroacetic acid (TCA) during a 1-h incubation at 4 °C. Cell proliferation inhibition was measured spectrophotometrically using sulforhodamine B (SRB) according to literature protocols [103,104,105]. Post-fixation, plates were rinsed, air-dried, and stained for 30 min at ambient temperature with 0.4% SRB dissolved in 1% acetic acid. After removing excess dye via 1% acetic acid washes, plates were thoroughly dried. Protein-bound SRB was subsequently solubilized in 10 mM Tris base solution, and absorbance readings were taken at 564 nm. Compound efficacy was determined through calculation of half-maximal inhibitory concentration (IC₅₀) values (Equation (6)).

$${\mathrm{I}\mathrm{C}}_{50}(\mathrm{\%})={\displaystyle \frac{{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}_{OD}-{Compound}_{OD}}{{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}_{OD}}}\times 100$$ (6)

Absorbance measurements for all wells were recorded at 564 nm. To quantify tumor cell line sensitivity to the chemical agent, the Sulforhodamine B (SRB) assay was utilized. Dose–response relationships were visualized through survival curves plotting chemical concentration against cell viability percentages for each cancer cell line. IC₅₀ values were derived by applying a four-parameter logistic (4PL) model to fit sigmoidal curves to the experimental data. This nonlinear regression analysis was executed in GraphPad Prism 10.6.1, which computes IC₅₀ through interpolation from the fitted curve rather than linear extrapolation of inhibition data.

3.9.4. Antioxidant Activity Using the DPPH Technique

The antioxidant activity of the ligand and its metal complexes was assessed using the DPPH radical scavenging assay [105]. A freshly prepared 0.1 mM methanolic DPPH solution was used, and different concentrations of each compound were mixed with DPPH-saturated DMSO. Then, 1.0 mL of each sample was added to 2.0 mL DPPH solution (1:2 v/v) and incubated in the dark for 30 min at room temperature, followed by 30 min at 37 °C. The absorbance decrease was measured at 517 nm against a control. Radical scavenging activity was calculated using the following relation

$$\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H} \mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{e}\mathrm{f}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{t} (\mathrm{\%})={\displaystyle \frac{{\mathrm{A}}_O-A_S}{{\mathrm{A}}_O}}\times 100$$ (7)

where A_o and A_s represent the absorbance of the control and test samples, respectively.

The IC₅₀ values, denoting the compound concentration required to achieve 50% inhibition of DPPH radicals, were subsequently calculated following the methodology reported in the literature [106,107,108,109]. These values served as quantitative indicators of the antioxidant strength of each compound.

4. Conclusions

A tridentate Schiff base ligand, 4-nitro-2-(quinolin-8-yliminomethyl)phenol (NQP), was synthesized and used for the preparation of Pd(II), Zn(II), and Fe(III) complexes. Elemental analysis together with spectroscopic techniques (NMR, UV–Vis, and IR) and thermal analysis confirmed the formation of well-defined metal–ligand systems with metal-to-ligand stoichiometries of 1:1 for the Pd(II) and Zn(II) complexes and 1:2 for the Fe(III) complex. Job’s method and mole-ratio studies reliably established these stoichiometric relationships in solution. Molar conductivity measurements revealed non-electrolytic behavior for the Pd(II) complex, while the Zn(II) and Fe(III) complexes exhibit 1:1 electrolytic behavior in solution due to the presence of nitrate counter ions. Thermogravimetric analysis demonstrated multistep thermal degradation processes for all complexes. The onset decomposition temperatures extracted from the TG profiles indicate appreciable thermal stability, while the final residual masses are consistent with the formation of metal oxide species, as inferred from mass balance considerations and literature comparisons. Correlation of all obtained data with DFT calculations established square-planar, tetrahedral, and octahedral geometries for Pd(II), Zn(II), and Fe(III), respectively, with coordination occurring through the azomethine nitrogen, quinoline nitrogen, and phenolic oxygen atoms. DNA-binding studies revealed effective interaction of the complexes with CT-DNA, occurring through electrostatic contributions with partial intercalative character. Biological investigations showed that metal coordination enhances the antimicrobial and antioxidant activities compared to the free ligand, with the Pd(II) complex exhibiting the most pronounced antimicrobial efficacy against Micrococcus luteus and Geotrichum candidum and superior DPPH radical scavenging activity relative to ascorbic acid. Cytotoxic screening against HCT-116, HepG-2, and MCF-7 cancer cell lines demonstrated significant anticancer potential for all complexes, particularly the Pd(II) derivative against MCF-7 cells. Overall, the improved biological performance of the metal complexes is attributed to chelation-induced modifications in physicochemical properties, which likely enhance their interaction with biological targets.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27041828/s1.

Author Contributions

Conceptualization, A.M.A.-D. and S.K.A.; methodology A.M.A.-D., I.O., S.K.A., F.S.A., S.M.A., M.M.K. and I.O.B.; data curation, A.M.A.-D., S.A.A.-L., M.F., M.S., I.O. and F.S.A.; writing—original draft preparation, A.M.A.-D., I.O., S.K.A., F.S.A., S.M.A., S.A.A.-L., M.M.K.; writing—review and editing, A.M.A.-D., I.O., S.K.A. and S.A.A.-L.; funding acquisition, I.O., S.K.A., F.S.A., S.M.A., M.M.K. and M.F. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research received no external funding.