In Vitro Antibacterial Activities and Calf Thymus DNA–Bovine Serum Albumin Interactions of Tridentate NNO Hydrazone Schiff Base–Metal Complexes

Abstract

Their demonstrable bioactive characteristics, coupled with their wide structural diversity and coordination versatility, render Schiff bases and their coordination complexes biologically active compounds demonstrating outstanding properties. This research describes the synthesis and characterization of new Cu(II) and Ni(II) complexes with an NNO-donor hydrazone ligand (HL). The crystal structure of the HL ligand was determined through single-crystal X-ray diffraction studies. The in vitro antibacterial activities of the HL ligand and its metal(II) complexes against Gram-positive and Gram-negative bacteria demonstrated that the metal(II) complexes displayed greater antimicrobial activities compared to the free Schiff base ligand. Furthermore, the interaction of the ligand and the complexes with calf thymus DNA (CT-DNA) was explored through electronic absorption and viscosity measurements, suggesting intercalation as the most likely mode of binding. The compounds promoted oxidative DNA cleavage, as demonstrated by the strand breaks of the pmChery plasmid under oxidative stress conditions. Finally, fluorescence spectroscopy also revealed the strong binding affinity of these compounds for bovine serum albumin (BSA).

1. Introduction

One of the biggest issues facing modern public health is antibacterial resistance. The effectiveness of traditional antimicrobial treatments is currently under threat from this silent pandemic [1]. In the face of this crisis, the search for innovative alternatives to combat resistant pathogens has gained crucial relevance. In recent years, the use of metallopharmaceuticals has been explored as an alternative to the traditional chemotherapeutics used for the treatment of different diseases. One of the keys to the success of this type of compound depends on the properties of the organic ligands that coordinate with the metal centers. In this sense, Schiff base (SB) ligands have been of interest to researchers seeking new bioactive molecules, since their biological properties and different binding points make them good candidates as ligands for metal ions [2,3,4].

Hydrazones are organic compounds that belong to the family of Schiff bases (SBs) and are characterized by their general structure, R₁R₂C=NNR₃R₄. The chemical properties of their nitrogen (nucleophilic) and carbon (electrophilic–nucleophilic) atoms enable them to take part in a variety of reactions to produce molecules with a wide structural variety and multiple properties, such as antimicrobial activity [5,6] and uses as precursors in dyes, pigments, and catalysts [7]. SB derivatives have shown promise in medicine, potentially helping to treat conditions like mental disorders, cancer, and tuberculosis [8,9,10,11]. Different reports evidence that the formation of coordination compounds with bioactive hydrazones improves its biological activity. The incorporation of heteroatoms like N, O, and S and different functional groups into their structure can contribute to improving the chelating effect of the ligand, increasing their usefulness in chemical, biological, medicinal, and analytical applications [12].

Copper (Cu²⁺) and nickel (Ni²⁺) ions are essential metals that play fundamental roles in numerous physiological processes in living organisms. Copper is known for its role as a cofactor for several enzymes, including superoxide dismutase, which protects cells from oxidative damage, and cytochrome C oxidase, which is part of the mitochondrial respiratory chain and catalyzes the reduction of molecular oxygen to water [13]. The intrinsic properties of copper, combined with the selectivity of different Schiff bases, are promising for applications as antimicrobial agents [14]. Nickel, although less studied, has also been identified as a cofactor for enzymes such as Ni-superoxide dismutase, urease, methyl-coenzyme M reductase (MCR), and CO-dehydrogenase, which play a role in regulating cellular metabolism [15]. The biological properties of nickel and Schiff bases have prompted studies of chemotherapeutic applications, including antimicrobial activity [16,17].

Metal complexes can selectively interact with biological structures in specific and generally controlled ways. These interactions are essential for major biological activities, like enzymatic catalysis and electron transport, and may regulate the stability and activity of essential biomolecules. Metal ions that interact with DNA can affect its structure and, thus, affect genetic replication and gene expression [18]. Bovine serum albumin (BSA) is the most abundant protein in plasma and is considered a model of globular proteins. It is also one of the most studied because its structure is similar to human serum albumin (HSA), making it a biological target for studying the interaction of drugs and potential therapeutic agents [19]. Because coordination compounds may interact with DNA and biomolecules [20], it is essential to understand these interactions to design and develop new drugs for the treatment of various diseases, including cancers, autoimmune disorders, and microbial infections. In our interest in obtaining new compounds with antibacterial activities, the present work addresses the synthesis and comprehensive characterization of 2-((2-(6-bromopyridin-2-yl)hydrazono)methyl)phenol ligand and the copper(II) and nickel(II) complexes (Figure 1, HL). The antibacterial activity of the obtained compounds against Gram-positive and Gram-negative bacterial strains is evaluated, in addition to testing their interaction with DNA chains through different experiments.

2. Results and Discussion

2.1. Synthesis and Characterization of HL

The Schiff base (HL) derived from the condensation reaction between 2-bromo-6-hydrazinylpyridine and benzaldehyde was prepared in a 1:1 molar ratio, based on previous studies [21]. The ligand was isolated as an air-stable pale beige solid that was soluble in DMSO, ethanol, methanol, and DMF but slightly soluble in water. In order to elucidate the molecular structure, the ¹H-NMR spectrum of HL was recorded in DMSO-d₆. The signals corresponding to the protons of the NH and OH groups are observed at 9 and 12 ppm, respectively. The azomethine (CH=N) proton appears as a singlet peak observed at δ = 8 ppm. In addition, in the range of 6–7 ppm, the expected signals appear as multiplet peaks corresponding to the aromatic protons of the organic structure. In the mass spectrum of HL, a peak at m/z = 291 is observed, indicative of the [M]⁺ ion. Another peak at m/z = 274 corresponds to [M-OH]⁺, and a peak at m/z = 197 represents [M-C₆H₅-OH]⁺. The solid was recrystallized in ethanol, obtaining a single crystal available for the single-crystal X-ray analysis. The (E)-2-((2-(6-bromopyridin-2-yl)hydrazono)methyl)phenol compound (HL) was crystallized in the orthorhombic space group Pbca (Figure 2,

Table 1. Summarized crystallographic data.

Compound	(E)-2-((2-(6-Bromopyridin-2-yl)hydrazono)methyl)phenol
Emp. Formula	C12H10BrN3O
FW (g/mol)	292.13
Temp. (K)	293
λ (Å)	MoKα = 0.71073
Crystal system	Orthorhombic
Space group	Pbca
a (Å)	5.7564(1)
b (Å)	12.4294(4)
c (Å)	32.2384(10)
α (°)	90
β (°)	90
γ (°)	90
Volume (Å3)	2306.61(11)
Z	8
ρ calcd (mg/m3)	1.683
Abs.Coeff (mm−1)	3.550
F(000)	1168
θ range (°)	3.3 to 26.4
Reflections collected/unique [R(int)]	18,864/2345 [0.124]
Completeness (%)	99.5
Data/restraints/parameters	2345/0/156
Gof on F2	0.956
R1 [I > 2σ(I)]	0.0476
wR2 [I > 2σ(I)]	0.1342

). The compound presents a conformation, E, with respect to the C=N bond. The molecule is almost planar, with torsion angles in C8–C7–C6–N3 and C4–C5–N2–N3 of 3.01(1) and 0.11(1)°, respectively. The O1–H1···N3 intramolecular interaction, with a distance of 2.644(4) Å, is observed. N2–H2···O1, with a distance of 3.080(4) Å, joins the molecules along the [010] direction (Figure 3a). The 3D supramolecular arrangement is given by π-π slipped-stacking interactions, with distances for N2···C12 and C4···C8 of 3.527(6) and 3.397(6) Å along the [100] and [001] directions (Figure 3b,c).

2.2. Synthesis and Characterization of Metal Complexes

The complexes 1 [Cu(L)(NO₃)] and 2 [Ni(L)₂] were isolated as stable crystalline solids. The elemental analysis showed a significant correlation with the suggested structures, and the molar conductivity values of the complexes in MeOH at 25 °C indicated that the synthesized compounds are non-ionic and non-electrolytic in nature (<76 Ω⁻¹cm²mol⁻¹) [22]. The method of continuous variations (Job’s method) was used to analyze the stoichiometry of the metal complexes. The results suggest a metal–ligand stoichiometry of 1:1 and 1:2 for complexes 1 and 2, respectively (Figure S1, Supplementary Material). Additionally, the amount of metal in compounds 1 and 2 was found by atomic absorption. A 5-point calibration curve was utilized to ascertain the concentration in ppm of Cu(II) and Ni(II), which were very close to the expected values for the proposed structures (Figure S2, Supplementary Material). No significant changes were observed in the absorption bands and stability constant values (K), indicating that the compounds are stable, even after 24 h. This indicates that the metal complexes exhibited stability in solution and that the observed biological activity is not influenced by the decomposition of the compounds. (Figures S3 and S4, Supplementary Material). Unfortunately, after several attempts, it was not possible to isolate any crystals of complexes 1 and 2 to be studied by X-ray diffraction.

The complexes’ formations were evaluated using FT-IR spectroscopy, comparing the spectra of complexes 1 and 2 with the free ligand HL (Figure 4). To identify coordination modes, characteristic peaks have been examined, whose variations in intensity and/or position are interpreted because of their participation in the chelation process. In the FT-IR spectra of complexes 1 and 2, the ν(O–H) band (3200 cm⁻¹) is not observed, indicating the deprotonation of the phenol group and the coordination to the metal. This statement is further supported by the shift to lower wavenumbers of the phenolic stretching frequency ν(C–O) after the formation of the complex (from 1260 cm⁻¹ to ~1245 cm⁻¹). The ν(HC=N) and ν(C=N) vibration frequencies present a shift from 1620 cm⁻¹ to 1610 cm⁻¹ and from 1489 cm⁻¹ to 1454 cm⁻¹ due to the interaction of the nitrogen atom of the pyrimidine ring and azomethine nitrogen atom with the M(II) ions, respectively. Additionally, for complex 1, the bands related to the vibrational modes of the nitrate group are observed at 1434–1540 cm⁻¹ (ν₄), 1306–1316 cm⁻¹ (ν₁), and 1005–1023 cm⁻¹) (ν₂). The vibrations observed in compounds 1 and 2 are comparable to those of other coordination complexes described in the literature [23,24,25].

The analysis of absorption spectra allows for the identification of electronic transitions and the monitoring of metal complex formation, which confirms their synthesis through changes in the absorption bands. Electronic absorption spectra were obtained for HL and its complexes in the wavelength range of 200 to 800 nm (1 × 10⁻³ M in MeOH at 25 °C) (Figure 5). The HL Schiff base ligand’s absorption spectrum showed three strong bands at around 240, 311, and 340 nm. These bands could be caused by π-π* or n-π* transitions [26]. When the absorption spectra of the ligand HL and the M(II) complexes were compared, a hypochromic effect was seen in the absorption peaks and the appearance of new bands. There are broad bands at 413 nm for complex 1 and at 408 nm for complex 2. These bands are caused by the charge transfer between the metal ion and the organic ligand, while the band at 458 nm in the spectrum of complex 1 might be caused by charge transfers from the nitro group to the copper center [27,28,29]. These changes indicate the formation of complexes between the metal cations and the organic ligand HL.

2.3. Biological Studies

2.3.1. Antibacterial Activity

The in vitro antibacterial activity against Gram-positive bacteria (Staphylococcus aureus ATCC 29213, Listeria monocytogenes ATCC 19115, and Bacillus cereus ATCC 10876) and Gram-negative bacteria (Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, and Pseudomonas aeruginosa ATCC 27853) was tested in HL and complexes 1 and 2 by using the broth microdilution method. The minimum inhibitory concentrations (MIC) expressed in µg/mL were validated over three experiments and are listed in

Table 2. MIC values against Gram-positive (S. aureus, L. monocytogenes, and B. cereus) and Gram-negative (E. coli, S. typhimurium, and P. aeruginosa) bacteria (μg/mL).

Compound	S. aureus ATCC25923	L. monocytogenes ATCC19115	B. cereus ATCC10876	E. coli ATCC25922	S. typhimurium ATCC14028	P. aeruginosa ATCC 27853
HL	>1000	>1000	>250	>500	>1000	>2000
1	>250	>500	>62.5	>250	>250	500
2	125	>250	62.5	250	62.5	250
Cp *	<3.9	<3.9	<3.9	<3.9	<3.9	<3.9
AgNO3 *	<5.0	<5.0	<5.0	<5.0	<5.0	<5.0

* Drug control: ciprofloxacin (Cp) and silver nitrate (AgNO₃).

. The results obtained were compared with the reference antibiotics ciprofloxacin (Cp) and silver nitrate (AgNO₃). Complexes 1 and 2 showed antibacterial activity; however, it was less than that of the reference drugs used. The MIC values obtained suggest that M(II) complexes have significant antibacterial behavior compared to the ligand HL, which could be rationalized in terms of changes in the polarity of the metal/ligand system. In the chelation process, the lipophilicity of the metal complex increases, and uncharged molecules could penetrate cell membranes, making their interaction through the bacterial cell wall more effective [30,31]. Interesting antibacterial activity was recorded for the Ni(II) complex in B. cereus and S. typhimurium strains, where it was found to be more active, with an MIC value of 62.5 µg/mL. The Ni(II) complex displayed higher antibacterial activity against all the strains evaluated. This can be accounted for on the basis of a lower polarity in its molecular architecture (NiL₂), which facilitates its interaction with the lipophilic walls of cell membranes compared to the Cu(II) complex. Overall, broad-spectrum antibacterial action was observed in the complexes, although Gram-positive strains were more susceptible to the compounds. This suggests that the action of the compounds could be caused by their effect on cell membranes, which may lead to the inhibition of cellular functions or damage to the fundamental molecular structures of the cell [32].

2.3.2. DNA Interactions

One of the mechanisms that drugs have to inhibit bacterial cell proliferation is their interaction and effect on microbial DNA strands. Therefore, different in vitro assays were carried out to assess the interaction mechanisms between the synthesized compounds and the DNA biopolymer.

• UV-vis studies

Monitoring the electronic absorption spectrum in a DNA titration is one of the methods used to preliminarily evaluate the possible binding modes of compounds with the DNA macromolecule [33]. The conformational modifications of DNA that result from the interaction of such compounds cause alterations in the electronic spectra, such as hypochromic, hyperchromic, hypsochromic (blue-shift), or bathochromic (red-shift) changes [34]. The electronic spectra of M(II) complexes, recorded with the increasing CT-DNA concentration, are given in Figure 6. The absorption spectra of coordination complexes 1 and 2 exhibit a reduction in absorbance, indicating hypochromism. This phenomenon occurs upon the formation of a molecule-DNA complex through intercalation, resulting in a more stable species stabilized by the π-π stacking between the base pairs and the aromatic systems of the intercalating molecule, leading to a decrease in molar absorptivity [35]. The red shift (bathochromism) of the band (413 nm), a characteristic of compound 1, also suggests an intercalation binding mode that changes the electronic environment and stabilizes the DNA molecule [36]. From the above, it can be inferred that the complexes form adducts with DNA in an intercalative form.

In order to quantitatively compare the binding affinity of the compounds with DNA, the intrinsic binding constants (K_b) were determined employing the Wolfe–Shimer equation (Equation (1)). The calculated binding constants were 3.43 × 10³ M⁻¹, 1.49 × 10⁷ M⁻¹, and 6.96 × 10⁴ M⁻¹ for HL, complex 1, and complex 2, respectively. The K_b values obtained for metal complexes 1 and 2 were slightly higher than those of classical intercalation metal complexes, such as cisplatin analogues (6.05 × 10⁴ M⁻¹ to 3.48 × 10⁵ M⁻¹) [37].

• Viscosity measurements and DNA melting

Viscosity measurements are a highly sensitive tool to validate the interaction between compounds and DNA, since they reflect the structural modifications and size alterations resulting from the binding. The viscosity of a CT-DNA solution (600 μM) was determined upon the addition of increasing concentrations of the compounds HL, 1, and 2 (R = [compound]/[DNA]) of between 0 and 1.5 (Figure 7a). The efflux times were recorded by processing the video’s photograms with Camtasia Studio^® software. The data showed that the relative viscosities of DNA increased when the CT-DNA complex adduct was formed with metal complexes 1 and 2, presenting a common behavior of compounds that intercalate inside the nucleobases of DNA, as they induce the elongation of the DNA by unwinding or stretching the double helix [38]. Additionally, the binding mode of the synthesized compounds was studied through DNA melting curves, measuring the electronic absorption at the maximum absorbance of DNA (260 nm) (Figure 7b). It was observed that both complexes 1 and 2 cause an increase in the melting point (Tm) of DNA. Interestingly, the experiment showed that the interaction of complex 1 generated an increase in the Tm (~10 °C) of DNA, which is usually related to an intercalation binding mode that generates greater stability between the strands of the DNA double helix [39]. This result is consistent with the highest value in the intrinsic binding constants (K_b) and viscosity measurements observed for complex 1.

• DNA cleavage

Metal complexes might induce genotoxicity by causing damage or conformational alterations in the DNA of prokaryotic and eukaryotic cells, which can lead to cell death by apoptosis [40]. Therefore, analyzing these interactions is crucial for understanding the mechanisms of action of certain drugs and developing new therapeutic strategies. DNA is a macromolecule that can present different conformations that affect the electrophoretic mobility in the agarose gel. Double-stranded, circular, supercoiled DNA (form I) has a higher electrophoretic mobility than relaxed, circular DNA (form II). Form III is the linear conformation resulting from the cleavage of form II, and its electrophoretic migration is between form I and form II [22,41]. In this study, the effects of compounds HL, 1, and 2 on the DNA conformation of the pmCherry vector isolated from E. coli BL21 were analyzed in the presence and absence of H₂O₂ by monitoring the reaction by electrophoresis in a 1% agarose gel with a 1X TAE buffer at a pH of 7.2 and with Bioline HyperLadder™ 1 kb as a molecular weight marker. Figure 8 and Figure 9 illustrate the displacement patterns of the vector in response to the interaction with the analyzed compounds.

The results obtained showed that both complexes 1 and 2 can alter the conformation of DNA in the absence of an exogenous reagent (H₂O₂, lane 5, Figure 8 and Figure 9), favoring the relaxed, circular form (form II), while the control (free plasmid) and the HL ligand did not generate significant changes in the conformation of the plasmid DNA (lanes 2 and 4, Figure 8 and Figure 9). In the presence of H₂O₂, complexes 1 and 2 showed an increase in oxidative DNA cleavage as the concentration of the complexes increased. This is particularly evident for complex 1, where a reactive oxygen species generator contributed to an increase in oxidative DNA cleavage (lanes 6–10) [23], resulting in the appearance of the linear conformation (form III). At a concentration higher than 50 μmol·L⁻¹, damage was generated in the DNA, and the bands corresponding to the supercoiled form of DNA (form I) were not observed in the gel (Figure 8, lanes 11 and 12).

2.3.3. BSA Interaction

The analysis of the complex-BSA interaction was performed at 25 °C in Tris-HCl/NaCl buffer (100 mM/50 mM, pH 7.4) by electronic absorption and fluorescence assays. BSA shows two characteristic absorption peaks: a low-intensity peak around 280 nm, associated with amino acid residues like tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe), and an intense peak between 220 and 240 nm, related to its secondary structure. The analysis of the variations in these peaks allows the investigation into the interaction mechanisms between the compound and albumin. Absorption and emission spectroscopies also allow the identification of quenching patterns, determining whether the mechanism is dynamic or static. Dynamic quenching affects only the excited state of the fluorophore, while static quenching occurs when a compound–protein complex is formed, altering both absorption and emission.

Figure 10 shows the absorption spectra of BSA after titration with the compounds, where two phenomena stand out: a notable decrease in the intensity of the absorption spectrum and a bathochromic effect on the peak of the protein secondary structure, demonstrating a clear affinity of the compounds for the protein. This change in the absorption spectra suggests a static quenching mechanism [42].

• BSA fluorescence-quenching assays

The fluorescent properties of BSA are due to the Trp, Tyr, and Phe residues; however, of these three amino acid residues, Trp is the most responsible for the intrinsic fluorescence display of this protein, presenting a characteristic emission maximum of around 340 nm [43]. The analysis of the changes in the emission spectrum provides us with valuable information about the complex protein interaction mechanisms, their binding properties, and the interaction forces involved in this binding process [44].

As seen in Figure 11, after successive additions of the synthesized compounds, the emission intensity of BSA is reduced by 50.4%, 49.5%, and 49.2% of the initial fluorescence intensity of BSA for 1, 2, and 3, respectively. A hypsochromic shift of the emission maximum (a shift to shorter wavelengths) for the compounds is also observed. Both phenomena show an interaction between the compounds and BSA; however, hypsochromism also indicates that an increase in hydrophobicity is generated in the microenvironment surrounding the Trp residue [45,46].

The decrease in intensity of fluorophores is known as quenching; this process of fluorescence extinction is due to different mechanisms known as dynamic quenching, static quenching, or simultaneous quenching (static and dynamic). The quenching parameters were determined using the Stern–Volmer equation and modified Stern–Volmer equations (

Table 3. Stern–Volmer quenching constants (K_sv and k_q), binding constant K_b, and binding sites for complex–BSA interactions (n) at room temperature for the BSA-1 and BSA-2 study systems.

	${\mathbf{K}}_{\mathbf{sv}} \left({\times 10}^5{\mathbf{M}}^{-1}\right)$	${\mathbf{k}}_{\mathbf{q}} \left({\times 10}^{14}{\mathbf{M}}^{-1}{\mathbf{s}}^{-1}\right)$	${\mathbf{K}}_{\mathbf{b}} \left({\times 10}^5{\mathbf{M}}^{-1}\right)$	n
BSA-HL	8.92	8.92	6.71	0.89
BSA-1	11.30	11.30	12.20	1.07
BSA-2	9.90	9.90	7.30	0.92

). The assessed compounds exhibit quenching constant K_sv values of order ~10⁵ M⁻¹, indicating a high fluorophore sensitivity to the compounds (quenchers). The bimolecular extinction constant K_q values are significantly greater than the biomolecules maximum K_q (2.0 × 10¹⁰ M⁻¹ s⁻¹), most likely indicating static extinction. The number of binding sites was between 0.89 and 1.07, showing that synthesized compounds bonded to one BSA molecule in a stoichiometry of 1:1.

3. Materials and Methods

3.1. Materials

All the reagents and solvents used were obtained from commercial manufacturers (Sigma-Aldrich, St. Louis, MO, USA and Alfa Aesar, Ward Hill, MA, USA), and used as received. The synthesis of the Schiff base ligand (HL) was carried out using standard research methods [21]. The FT-IR spectra of the synthesized compounds were obtained between the 4000 cm⁻¹ and 600 cm⁻¹ regions on a Shimadzu Affinity 1 (FT-IR) device (Shimadzu, Miyazaki, Japan) with an ATR accessory. Their melting points were recorded on a Mel-Temp II apparatus (Thomas Scientific, Chadds Ford, PA, USA) and are reported without correction. The ¹H-NMR (400 MHz) spectra were obtained in a DMSO-d₆ solution at room temperature (298 K) on a Bruker Ultrashield Avance II 400 spectrophotometer (Bruker Corporation, Billerica, MA, USA). The chemical shifts values(δ) are reported in parts per million (ppm) with respect to tetramethylsilane (TMS, SiMe₄, δ = 0), and the coupling constants (J) are given in Hz. The multiplicities are abbreviated in the following manner: singlet (s), doublet (d), double of doublets (dd), and triplet (t). Elemental (C, H, and N) analyses were determined with the Flash EA 1112 Series CHN Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). A Shimadzu AA-6300 atomic absorption spectrometer was used for copper and nickel analyses. The electronic impact (EI) ionization mass spectrum of the ligand was recorded on a Shimadzu-GCMS-QP2010 (Shimadzu Corporation, Miyazaki, Japan) at 40 eV. A Direct Analysis in Real Time (DART) ionization system was used to obtain the mass spectra of the complexes on an AccuTOF JMS-T100LC (JEOL, Ltd., Tokyo, Japan). The conductivity measurements of the metal complexes were determined in ethanol (1 × 10⁻³ M) using an Orion^TM 131S (Thermo Fischer Scientific, USA). The UV-vis spectroscopy studies were performed using a Thermo Scientific Evolution 201/220 UV-visible spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The stoichiometry of the metal complexes was investigated using the continuous-variation spectrophotometric Job’s method. Fluorescence measurements were obtained at room temperature (298 K) with a JASCO FP-8500 spectrofluorometer (JASCO Corporation, Tokyo, Japan) using quartz cells (1.0 cm). Viscosity measurements were carried out using a Cannon-Ubbelohde semi-micro viscometer (size 75) (CANNON Instrument Company, State College, USA).

3.2. Synthesis of Ligand (HL)

The 2-((2-(6-bromopyridin-2-yl)hydrazono)methyl)phenol was synthesized by adapting a procedure that was reported elsewhere [21] (Figure 12).

Hydrazine monohydrate (18 mmol) was added to an ethanolic solution of 2,6-dibromopyridine (2.95 mmol) and stirred under reflux for 18 h. The progress of the reaction was monitored by TLC. The Schiff base precursor 2-bromo-6-hydrazinylpyridine was extracted with ethyl acetate twice, and the organic phases were combined, dried, and recrystallized from water. Salicylaldehyde (1.10 mmol) and 2-bromo-6-hydrazinylpyridine (1.10 mmol) were mixed and stirred in ethanol (30 mL) at the reflux temperature for 2 h. A pale beige solid was then washed with distilled water (2 × 15 mL) and then recrystallized from a concentrated ethanolic solution to give the final product, HL. Light yellow crystals of good quality were obtained after one week (yield 81%). m.p.: 184–186 °C. C₁₂H₁₀BrN₃O, elemental analysis: C, 49.34 (calc. 49.21); H, 3.45 (3.40); N, 14.38 (14.42)%. IR (selected peaks, ATR cm⁻¹) ν: 3269 (O-H), 1620 (HC=N), 1556 (C=C), 1489 (C=N), 1260 (C–O), 1078 (N-N). The ¹H-NMR (DMSO-d₆, 400 MHz, ppm) δ: 11.28 (s, 1H), 10.28 (s, 1H), 8.30 (s, 1H), 7.64 (d, J = 9.0 Hz, 1H), 7.55 (t, J = 7.9 Hz, 1H), 7.20 (t, J = 7.7 Hz, 1H), 7.07 (d, J = 8.2 Hz, 1H), 6.93 (d, J = 7.4 Hz, 1H), 6.87 (dd, J = 16.7, 7.9 Hz, 2H). MS electron impact (m/z, 40 eV): 291 [M⁺] (21.5), 274 [M⁺-C₁₂H₉BrN₃] (6.74), 197.97 [M⁺-C₆H₅BrN₃] (6.54). λ_Abs = 339 nm, 311 nm, 240 nm. λ_Em = 520 nm, 380 nm.

3.3. Synthesis of Metal(II) Complexes

Coordination complexes were synthesized by modifying a previously reported procedure [21] (Figure 13).

3.3.1. Synthesis of [Cu(L)(NO₃)] (1)

A solution of copper nitrate (0.51 mmol) in EtOH (3 mL) was slowly added to an ethanolic solution (5 mL) of HL (0.51 mmol). The reaction mixture was left under reflux and stirring for 2 h. The green solid obtained was filtered, washed with cold water (2 × 10 mL) and hexane (2 × 10 mL), and then dried at 90 °C (yield 93%). m.p.: >300 °C. C₁₂H₉BrCuN₄O₄, elemental analysis: C, 34.59 (calc. 34.53); H, 2.18 (2.20); N, 13.45 (13.41); Cu, 15.25 (14.82)%. IR (selected peaks, ATR cm⁻¹) ν: 1611 (HC=N), 1526 (C=C), 1453 (C=N), 1478 (asym, −NO₂), 1281 (sym, −NO₂), 1246 (C–O), 1105 (N-N). Λ_M (MeOH, 28.0 °C) (Ω⁻¹cm²mol⁻¹): 76. λ_Abs = 458 nm, 413 nm, 340 nm, 330 nm. λ_Em = 460 nm, 435 nm, 370 nm. MS (DART+) m/z: 415.

3.3.2. Synthesis of [Ni(L)₂] (2)

The synthesis of complex (2) was performed using the same procedure as that for compound (1) to obtain a yellow-colored powder (yield 90%). m.p.: 284–286 °C. C₂₄H₁₈Br₂NiN₆O₂, elemental analysis: C, 44.97 (calc. 44.58); H, 2.83 (2.79); N, 13.11 (13.16); Ni, 9.16 (9.87)%. IR (selected peaks, ATR cm⁻¹) ν: 1608 (HC=N), 1525 (C=C), 1454 (C=N), 1244 (C–O), 1150 (N-N). Λ_M (MeOH, 28.0 °C) (Ω⁻¹cm²mol⁻¹): 45. λ_Abs = 408 nm, 330 nm, 309 nm. λ_Em = 480 nm, 372 nm. MS (DART+) m/z: 640.

3.4. Single-Crystal Structure Determination of HL

Single-crystal X-ray data for HL were collected at room temperature (298 K) on an Enraf-Nonius Kappa-CCD diffractometer using MoKα radiation (0.71073 Å) monochromated by graphite. The cell refinements were performed using the software Collect and Scalepack, and the final cell parameters were obtained on all the reflections. Data reduction was carried out using the software Denzo-SMN and Scalepack [47]. The structure was solved using the software SHELXS-2013 and then refined using SHELXL-2013 [48], included in Olex [48,49,50]. The non-hydrogen atoms of the molecules were clearly resolved, and the full-matrix least-squares refinement of these atoms, with anisotropic thermal parameters, were performed. All the hydrogen atoms were stereochemically positioned and refined with the riding model (with Uiso(H) = 1.2Ueq) [48]. The ORTEP diagram was prepared with Diamond V3.2 [51]. Mercury V2024.2.0 [52] software was used to prepare the artwork of the supramolecular representation. CCDC 2394306 contains the supplementary crystallographic data for HL. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/structures from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge, UK; fax: (+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.

3.5. Antibacterial Activity Test

The effectiveness of the obtained compounds against pathogenic microorganisms was evaluated following the recommended protocols for antimicrobial susceptibility testing by microdilution [53]. The minimum inhibitory concentration was determined in triplicate using the broth microdilution technique on three Gram-positive strains (Staphylococcus aureus ATCC 29213, Listeria monocytogenes ATCC 19115, and Bacillus cereus ATCC 10876) and three Gram-negative strains (Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, and Pseudomonas aeruginosa ATCC 27853). Briefly, bacterial strains were grown in chocolate agar under aerobic conditions and incubated at 37 °C for 24 h. A colony from the bacterial strains was initially resuspended in Mueller–Hinton broth (MHB) to reach a turbidity value of 0.5 McFarland, and the resulting suspension contained approximately 1–4 × 10⁸ colony-forming units (CFU)/mL. Using this suspension, a final solution was obtained that had a concentration of 1–5 × 10⁵ CFU/mL in Mueller–Hinton broth. The bacterial inoculum was incubated in sterile 96-well polypropylene microplates for 20 h in a concentration range of between 3.90 μg/mL and 3000 μg/mL of HL, 1 and 2. A free medium with DMSO (0.01%) and a bacterial medium without an inhibiting agent were used as the positive and negative controls, respectively. Ciprofloxacin (CP) and silver nitrate (AgNO₃) were used as the control antibiotics.

3.6. DNA Interaction

UV-visible absorption titrations were performed to investigate the interaction of compounds HL, 1, and 2 with DNA. In addition, viscosity measurements, thermal denaturation, and agarose gel electrophoresis were carried out using standard methodologies and protocols, which were adapted in our laboratory [54]. Lyophilized calf thymus (CT-DNA), obtained from Sigma Aldrich, was utilized, along with a pmCherry vector that was extracted from E. coli BL21 (DH5α). All the solutions of DNA exhibited an A260/A280 ratio of between 1.8 and 1.9, indicating that the DNA was sufficiently free of RNA and proteins. The UV-vis spectra of HL, 1, and 2 were used to examine the stability of the compounds in solution. The DNA was resuspended in 10 mmol/L Tris and 1 mmol/L EDTA in deionized water, with the pH adjusted to approximately 7.5. The DNA solutions were stored at −5 °C and used within a period of 5 days. Absorption titrations were carried out with a constant amount of the compound, 50 μmol/L, along with enhanced DNA amounts (0–50 µmol/L), and the corresponding DNA solution was used as a reference solution. The solutions of the complexes contained deionized water and a DMSO of approximately 0.2%. The CT-DNA absorbance was eliminated by adding an equal amount of CT-DNA to the sample and the standard solution. The solution mixtures (compound-DNA) were incubated for 5 min, and the absorption spectra were recorded in the range 230–500 nm at room temperature. The binding constants (K_b) of the complexes were calculated according to the literature through the Wolfe–Shimmer Equation (1) [30], where [DNA] is the molar concentration of base pairs, ε_a is the apparent extinction coefficient of the bound complex (A_obs/[compound]), ε_f is the extinction coefficient of the molecule under study, and ε_b is the extinction coefficient of the fully bound compound-DNA complex. Plotting [DNA]/(ε_a − ε_f) vs. [DNA] gives the value of K_b (the ratio of slope to intercept) [30,54].

$${\displaystyle \frac{\left[DNA\right]}{({\epsilon }_a-{\epsilon }_f)}}={\displaystyle \frac{\left[DNA\right]}{({\epsilon }_b-{\epsilon }_f)}}+{\displaystyle \frac{1}{K_b({\epsilon }_b-{\epsilon }_f)}}$$

(1)

3.7. BSA Interaction

The BSA binding studies of the compounds HL, 1, and 2 were performed in a Tris-HCl/NaCl buffer solution (100 mM/50 mM, pH = 7.4) [55]. Stock solutions of the compounds were prepared in a DMSO buffer (1:20). The UV-vis absorption titrations were performed in the range of 200–350 nm at a constant concentration of BSA (2.8 mL, 10 µM) while gradually increasing the concentration of the synthesized compounds (10 µL; 0–10 µM). After each addition, the solutions were kept at room temperature for 5 min to ensure a complete reaction.

The fluorescence emission spectra were measured from 250 to 450 nm at an excitation wavelength of 280 nm. The BSA was titrated against the compounds (0–1.0 mM) at a constant BSA concentration of 1 µM in a Tris–HCl buffer (pH = 7.2). Each spectrum was carried out at room temperature after an incubation time of 5 min. The experimental data was used to draw the Stern–Volmer plot, and the quenching parameters were then calculated from the Stern–Volmer equation (Equation (2)) and modified Stern–Volmer equations (Equation (3)) [56].

$${\displaystyle \frac{F_0}{F}}=1+k_q{\tau }_0\left[Q\right]=1+K_{sv}\left[Q\right]$$

(2)

where F₀ is the BSA fluorescence intensity in the absence of compounds (HL, 1 and 2), F is the BSA fluorescence intensity in the presence of compounds, K_q is the fluorophore quenching rate constant, τ₀ is the average lifetime of the molecule without the quencher (10⁻⁸ s), [Q] is the molar concentration of the compounds (quenchers), and K_SV is the Stern–Volmer (quenching) constant. The K_SV was determined from the slope of the linear plot of F₀/F versus [Q].

$$\log \left({\displaystyle \frac{F_0-F}{F}}\right)=\log \left(K_b\right)+n \mathrm{l}\mathrm{o}\mathrm{g}\left[Q\right]$$

(3)

where K_b is the binding constant, and n is the number of binding sites.

3.8. Stability Studies of the Complexes

The stability of the complexes in the MHB medium was evaluated using a Thermo Scientific Evolution 220 UV-visible spectrophotometer to obtain electronic absorption spectra using a method similar to previously reported studies [57]. The measurements were performed in the range of 300–550 nm in 1 cm quartz cells. Next, 2 mL samples of solutions of the compounds, with concentrations of between 1 and 5 × 10⁻⁶ M, were prepared. The solutions were left at ambient conditions for 24 h, and the UV-vis spectra were measured again. The stability constant (K) can be obtained from the double reciprocal linear graph by the relationship between the slope and the intercept.

4. Conclusions

The present study indicates that metal complexes of Cu(II) and Ni(II) with a new NNO-donor hydrazone ligand contribute significantly to the enhanced antibacterial activity in comparison to the free ligand, thereby demonstrating the advantageous impact of metal coordination on biological activity. In particular, the Ni(II) complex exhibited notable efficacy against both Bacillus cereus and Salmonella typhimurium strains, indicating that the central metal plays a vital role in antimicrobial activity. Additionally, the UV-vis spectroscopy and viscosity assays displayed by the DNA interaction studies reveal that these complexes are capable of intercalating into the DNA double helix, which could contribute to their antibacterial mechanism of action. The DNA oxidative fragmentation ability of these chelates also accords with a possible cytotoxic mechanism based on the production of reactive oxygen species. Moreover, interaction studies with bovine serum albumin (BSA) indicated a high affinity of the metal complexes for the protein, thus suggesting the likelihood of a transport mechanism. The fluorescence analysis showed that quenching is static; thus, it suggests the formation of a stable complex within the BSA. The results point to the undoubted potential of hydrazone-derived Cu(II) and Ni(II) complexes as good candidates for the development of new antimicrobial agents, which may find applications in bacterial chemotherapy. However, it is necessary to conduct some additional studies in better biological models to evaluate their specificity, bioavailability, and specific cytotoxic effects before they can be heralded as potential therapeutic alternatives.