Metronidazole Interaction with Cu²⁺ and Zn²⁺: Speciation Study in Aqueous Solution and Biological Activity Evaluation

Abstract

Metronidazole (2-methyl-5-nitro-1H-imidazole-1-ethanol, MNZ) is a well-known and widely used drug for its excellent activity against various anaerobic bacteria and protozoa. The purpose of this study is to elucidate the ability of MNZ to form metal complexes with Cu²⁺ and Zn²⁺ and to demonstrate that complexation increases its bioactivity profile against different pathogenic microorganisms. The interaction of MNZ with Cu²⁺ and Zn²⁺ was investigated in NaCl aqueous solution under different conditions of temperature (15, 25, and 37 °C) and ionic strength (0.15, 0.5, and 1 mol L^–1) by potentiometric and spectrophotometric titrations. The obtained speciation models include two species for the Cu²⁺-containing system, namely, CuL and CuL₂, and three species for the Zn²⁺-containing system, namely, ZnLH, ZnL, and ZnLOH. The formation constants of the species were calculated and their dependence on temperature and ionic strength evaluated. Comparison of the sequestering ability of MNZ under physiological conditions revealed a capacity toward Cu²⁺ higher than that toward Zn²⁺. A simulation under the same conditions also showed a significant percentage of the Cu²⁺–MNZ species. The biological assessments highlighted that the complexation of MNZ with Cu²⁺ has a relevant impact on the potency of the drug against two Trypanosoma spp. (i.e., T. b. brucei and T. b. rhodesiense) and one gram-(−) bacterial species (i.e., Escherichia coli). It is noteworthy that the increased potency upon complexation with Cu²⁺ did not result in cytotoxicity against MRC-5 human fetal lung fibroblasts and primary peritoneal mouse macrophages.

1. Introduction

Metronidazole (2-methyl-5-nitro-1H-imidazole-1-ethanol, MNZ), shown in Figure 1,¹ is a 5-nitroimidazole derivative that exhibits great and selective activity against anaerobic bacteria (e.g., Staphylococcus aureus and Escherichia coli)²⁻⁶ and some protozoa (e.g., Entamoeba, Trichomonas, Trypanosoma, and Leishmania spp.).⁷⁻¹⁰ It has also been shown to have excellent antifungal properties.¹¹ Unfortunately, over the years, its overuse has led to the development of MNZ-resistant microorganisms.¹²⁻¹⁴ Among the various methods reported in the literature to overcome the resistance issue,¹² complexation with transition metals has proven to be one of the strategies with the greatest potential to enhance the bioactivity of MNZ¹⁵ and protect it from degradation by gastric juice.¹⁶ The presence of three potential donor atoms (the nitrogen atom of the imidazole ring and the oxygen atoms of the primary alcohol moiety and the nitro group) makes MNZ a good candidate for the coordination with several metal cations such as Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, and Ag⁺.^16,17 In addition, it is noteworthy that Ru(I)-based MNZ metal complexes have shown good antitumor efficacy in the treatment of lung cancer.¹⁸ Therefore, the study of the interaction between MNZ and transitional metal cations is interesting to propose new therapeutic approaches. Zn²⁺ and Cu²⁺ are essential trace elements for all living organisms.^19,20 Both are implicated in numerous biological activities, including modulation of enzymatic and catalytic activities in several oxidation–reduction processes.¹⁶ The structural role of Zn²⁺ is critical in metalloproteins, which are important in transcription and replication processes and in nucleic acid repair.²¹ Zn²⁺ is also involved in several enzymatic activities necessary for various physiological functions, including protection of sulfhydryl groups from oxidation and inhibition of oxidative processes leading to the formation of reactive oxygen species (ROS).²² Cu²⁺ is an essential trace element that functions as a catalytic cofactor in numerous biological processes in both bacteria and humans.²⁰ Furthermore, it has recently been pointed out that Cu⁺ and Cu²⁺ have a significant impact on antibiotic activity.²³ There is growing interest in Cu²⁺ complexes, as it has been shown that they can prevent or even reverse antimicrobial resistance to classical antibiotics.²³⁻²⁶ To date, there appear to be few studies on in vitro to in vivo translation, which is attributed to the lack of knowledge of the molecular mechanisms underlying Cu²⁺–antibiotic interactions under physiological conditions.²³ So far, the exact mechanism for bacterial damage caused by Cu²⁺ has not been fully explained. Therefore, the aim of this study is to verify the ability of MNZ (or L, which indicates “ligand”) to form complexes with Cu²⁺ and Zn²⁺ ions and to demonstrate that complexation allows for increased drug bioactivity. The study of the interaction between MNZ and both metal cations was carried out by potentiometric and UV-spectroscopic titrations in NaCl aqueous solutions of Cu²⁺– and Zn²⁺–MNZ complexes under different temperatures, ionic strengths, and metal/MNZ molar ratio conditions. Measurements performed at different background salt concentrations (NaCl) and temperature values allowed the study of the dependence of stability constants on ionic strength and temperature. The sequestering ability of MNZ toward Zn²⁺ and Cu²⁺ was calculated under physiological conditions by using the pL_0.5 parameter, the cologarithm of the ligand concentration that sequesters 50% of the metal cation in trace. Moreover, a simulation under real conditions of plasma pH, temperature, equilibria, and concentrations of the main inorganic constituents is also reported. In order to test the efficacy of Zn²⁺–MNZ and Cu²⁺–MNZ species, a standard in vitro screening was carried out against a wide panel of pathogenic microorganisms, including one gram-(−) bacterial strain (Escherichia coli), one gram-(+) bacterial strain (Staphylococcus aureus), two species of fungi (Candida albicans and Aspergillus fumigatus), and various protozoa (Trypanosoma cruzi, Trypanosoma brucei brucei, Trypanosoma brucei rhodesiense, and Leishmania infantum). In addition, in vitro cytotoxicity was evaluated on human fetal lung fibroblasts (MCR-5) and primary peritoneal mouse macrophages (PMM).

Figure 1.

Chemical structure of metronidazole (2-methyl-5-nitro-1H-imidazole-1-ethanol, MNZ (L)).

2. Materials and Methods

2.1. Materials

The metal cation solutions were obtained by weighing and dissolving the corresponding salt, zinc(II) chloride monohydrate (Merk, puriss.), and copper(II) sulfate pentahydrate (Fluka, puriss.). All metal solutions were standardized by titration with EDTA (ethylenediamine tetraacetic acid disodium salt, Sigma-Aldrich, purity ≥ 99%). Metronidazole was purchased from Alfa-Aesar/Thermo Fisher (purity ≥ 97%). Solutions of hydrochloric acid and sodium hydroxide used for titrations were prepared by dilution of Fluka ampules and standardized with sodium carbonate (purity ≥ 99.5%, Sigma-Aldrich) and potassium biphthalate (purity ≥ 99.5%, Sigma-Aldrich), respectively. Solutions of sodium chloride were obtained by weighing the salt (Sigma-Aldrich, puriss.), previously dried in an oven at 110 °C for at least 2 h. Distilled water (conductivity <0.1 μS cm^–1) and grade A glassware were employed for the preparation of all solutions.

2.2. Potentiometric Apparatus and Procedure

The potentiometric titrations were performed using a Metrohm model 809 Titrando potentiometer combined with a glass electrode. Each potentiometric system was connected to a PC and the experimental titration data were acquired by the Metrohm TIAMO 2.2 software, which allows control of emf stability, titrant delivery, and data acquisition. The estimated accuracy of the apparatus is ±0.15 and ±0.002 mL for emf and for readings of titrant volume, respectively. Each titration consists of additions of volumes of NaOH standard to 25 mL of the solution containing Cu²⁺ or Zn²⁺, MNZ, HCl, and the supporting electrolyte NaCl. The experimental conditions of the titrations are reported in Table 1. Glass jacket thermostated cells were employed for the measurements performed under different conditions of temperature (15 ≤ t/°C ≤ 37) by bubbling pure N₂ in order to avoid CO₂ and O₂ inside the solutions. For each measurement, an independent titration of HCl with standard NaOH was performed to calculate the standard electrode potential E⁰ and the pK_w value under the same experimental ionic strength and temperature conditions.

Table 1. Experimental Conditions for Potentiometric and Spectrophotometric Titrations.

technique	t (°C)	I (mol L–1)	CM (mmol L–1)	CL (mmol L–1)	M/L ratio	pH range
potentiometry	15, 25, 37	0.15–1	1–2	1–4	0.33–1	2–10
UV spectrophotometry	25	0.15	0.04–0.1	0.05–0.08	0.33–1	2–10

2.3. UV–Vis Apparatus

The spectrophotometric titrations were carried out with a Varian Cary 50 UV–vis spectrophotometer equipped with an optical fiber with a fixed 1 cm path length. The instrument is interfaced to a PC by Varian Cary WinUV software. By using a Metrohm glass electrode and a Metrohm-Titrando 809 potentiometer, the couple data of absorbance (Abs) and pH vs volume of titrant (mL) were recorded simultaneously for each titration point. All titrations were performed using a thermostated cell to keep the desired temperature under N₂ to remove CO₂ and O₂ from the solutions. The experimental conditions of the titrations are reported in Table 1. For each system, at least four measurements were performed in different conditions of the metal–MNZ ratio. The UV spectra were obtained by scanning from 225 to 400 nm, and a baseline containing only HCl, NaCl, and H₂O was recorded to consider the contribution of the matrix.

2.4. Bacteria and Protozoa Cell Cultures

Briefly, S. aureus and E. coli were cultured in MHT (Mueller Hinton Broth) and maintained on TSA (Tryptone Soy Agar). T. cruzi, Tulahuen CL2 galactosidase strain (nifurtimox- sensitive), was maintained on MRC-5_SV2 (human lung fibroblast) cells in MEM medium, supplemented with 200 mmol L^–1l-glutamine, 16.5 mmol L^–1 NaHCO₃, and 5% inactivated fetal calf serum. The T. b. brucei Squib 427 strain (suramine-sensitive) and T. b. rhodesiense STIB-900 strain were maintained in Hirumi’s Modified Iscove’s medium-9 (HMI-9) medium, supplemented with 10% inactivated fetal calf serum. MRC-5_SV2 cells were cultured in MEM + Earle’s salts-medium, supplemented with 2 mmol L^–1l-glutamine, 16.5 mmol L^–1 NaHCO₃, and 5% inactivated fetal calf serum. All cultures were incubated at 37 °C under an atmosphere of 5% CO₂.

2.4.1. Activity against S. aureus and E. coli

The assays were performed at 37 °C in sterile 96-well microtiter plates, each well containing 10 μL of aqueous compound together with a bacterial/fungal/MRC-5 inoculum (190 μL; 5 × 10³ CFU/mL). The compounds were tested at 64, 16, 4, 1, and 0.25 μ-mol L^–1. Bacterial/fungal/MRC-5 growth was compared to untreated-control wells (100% growth) and medium-control wells (0% cell growth). After 3 days of incubation, 50 μL of resazurin was added in each well. After 30 min at 37 °C, the fluorescence was measured at λ_ex = 550 nm and λ_em = 590 nm. The results are expressed as % reduction in cell growth/viability compared to control wells, and IC₅₀ values were determined.

2.4.2. Activity against T. cruzi

The assay was performed at 37 °C under an atmosphere of 5% CO₂ in sterile 96-well microtiter plates, each well containing 10 μL of the aqueous compound together with MRC-5 cell/parasite inoculum (190 μL; 4 × 10³ cells/well + 4 × 10⁴ parasites/well). The compounds were tested at 64, 16, 4, 1, and 0.25 μmol L^–1. Parasite growth was compared to that of untreated-infected controls and uninfected controls. After 7 days of incubation, parasite burdens were assessed after adding the substrate CPRG [chlorophenolred β-d-galactopyranoside; 50 μL/well of a solution of CPRG (15.2 mg); and Nonidet (250 μL) in PBS (100 mL)]. The sample was analyzed spectrophotometrically at 540 nm after 4 h. The results were expressed as % reduction in parasite burden compared to control wells, and an IC₅₀ was calculated.

2.4.3. Activity against T. b. brucei and T. b. rhodesiense

The assay was performed at 37 °C under an atmosphere of 5% CO₂ in sterile 96-well microtiter plates, each well containing the aqueous compound (10 μL) dilution together with the parasite suspension [190 μL; 1.5 × 10³ parasites/well (T. b. brucei) or 4 × 10³ parasites/well (T. rhodesiense)]. The compounds were tested at 64, 16, 4, 1, and 0.25 μmol L^–1. Parasite growth was compared to untreated-infected controls and uninfected controls. After 3 days of incubation, parasite growth was assessed fluorimetrically after addition of resazurin [50 μL; 50 μg/mL in phosphate buffer] to each well. After 6 h (T. b. rhodesiense) or 24 h (T. b. brucei) at 37 °C, fluorescence was measured (λ_ex 550 nm, λ_em 590 nm). The results were expressed as % reduction in parasite growth/viability compared to control wells, and an IC₅₀ was calculated.

2.5. Calculations

Experimental data of potentiometric titrations were processed by using BSTAC4 and STACO4 programs. The most reliable speciation model for each system was obtained taking into account several factors, namely, the statistical parameters standard and mean deviation, the formation percentages of the species, and the simplicity of the model. The LIANA program was used in order to calculate the parameters for the dependence of formation constant values on temperature and ionic strength. More details on software employed in the refinement of the experimental data are reported in ref (27). The HySS program was used to plot the speciation diagrams.²⁸

3. Results and Discussions

3.1. Speciation Model for Zn²⁺–MNZ and Cu²⁺–MNZ Systems

Both the Cu²⁺–MNZ and Zn²⁺–MNZ systems were investigated via potentiometric titrations. For all calculations, protonation constant values of MNZ(L) and hydrolytic constants of Cu²⁺ and Zn²⁺, as well as Cu–Cl^– species formation constant values, were taken into account (see Supporting Information, Tables S1 and S2). Several calculation tests were carried out in order to select the best speciation model for each system, taking into account principles such as model simplicity and improved fit.²⁹ The results evidenced that the Cu²⁺–MNZ(L) speciation pattern includes two species, CuL and CuL₂, and three species for the Zn²⁺–MNZ(L) system, namely, ZnL, ZnLH, and ZnLOH. Formation constant values of Cu²⁺–L and Zn²⁺–L at different temperatures and ionic strength values are reported in Table 2. Unlike the Zn²⁺–L system, the complex species generated by the interaction with Cu²⁺ are characterized by higher stepwise formation constant values, which lead to greater stability of the species and higher formation fractions under the same experimental conditions. More in detail, a comparison between the speciation diagrams under two different temperature values (t = 15 and 37 °C) and I = 0.15 mol L^–1 of both systems is shown in Figure 2. In addition, at t = 25 °C, the ZnLH species, present in the range of 2 ≤ pH ≤ 8, reaches its maximum formation fraction, corresponding to 0.2, in the range 3 ≤ pH ≤ 7 (see Figure 2a). A notable decrease of its formation fraction is evident at t = 37 °C. The two ZnL and ZnLOH species are formed in the ranges 6.5 ≤ pH ≤ 8.5 and 7 ≤ pH ≤ 10, respectively, and reach a maximum of 0.25 at pH = 7.6 (ZnL) and 0.95 at pH = 10 (ZnLOH). Under physiological conditions (t = 37 °C, I = 0.15 mol L^–1), a slight increase of formation fraction and a shift of the maximum fraction toward lower pH are distinguishable for both species. As shown in Figure 2b, at t = 25 °C, CuL species is formed in the range of 4 ≤ pH ≤ 8 with a maximum of 0.4 at pH = 5.8, while CuL₂, present in the range of 5 ≤ pH ≤ 10, reaches a maximum of 0.98 at pH = 10. Both species decrease their formation fractions at t = 37 °C, also shifting the maximum formation percentage toward slightly higher pH values.

Table 2. Experimental Formation Constants of Zn²⁺–MNZ(L) and Cu²⁺–MNZ(L) Species Obtained by Potentiometry.

reaction	t (°C)	I (mol L–1)	log β	reaction	log K
	15	0.15	13.88(9)a		1.88
	25	0.15	13.54(2)		1.87
	25	0.5	14.17(11)		1.66
	25	1	13.96(22)		1.74
	37	0.15	13.02(13)		1.14
	15	0.15	6.36(3)		6.36
	25	0.15	6.57(5)		6.57
	25	0.5	7.40(3)		7.40
	25	1	6.60(8)		6.60
	37	0.15	7.12(2)		7.12
	15	0.15	–0.79(2)		–8.71
	25	0.15	–0.72(2)		–8.44
	25	0.5	0.266(8)		–9.35
	25	1	–0.54(4)		–8.16
	37	0.15	0.09(2)		–8.87
	15	0.15	8.32(10)		8.32
	25	0.15	8.87(4)		8.87
	25	0.5	9.37(4)		9.37
	25	1	8.71(7)		8.71
	37	0.15	8.69(2)		8.69
	15	0.15	16.94(7)		8.62
	25	0.15	17.39(10)		8.52
	25	0.5	18.18(2)		8.02
	25	1	17.65(7)		8.94
	37	0.15	17.12(11)		8.43

^a≥95% of confidence interval.

Figure 2.

a, b Distribution diagrams at I = 0.15 mol L^–1 in NaCl of (a) Zn²⁺–MNZ(L) and (b) Cu²⁺–MNZ(L) systems at C_MNZ = 4 mmol L^–1, C_M = 2 mmol L^–1, t = 25 °C (solid lines), and t = 37 °C (dotted lines).

Both Cu²⁺–L and Zn²⁺–L systems were also analyzed by spectrophotometric titrations to confirm the values of the formation constants and the speciation models. The spectra acquired on the solutions containing Cu²⁺–L and Zn²⁺–L at selected pH in the spectral range 225 ≤ λ ≤ 400 nm are reported in Figure 3a,b. Both systems exhibit a hyperchromic effect as a result of the pH increase. The trend of the spectra referring to solutions containing L and Cu²⁺ or Zn²⁺ is very similar to that of solutions containing only the ligand, as reported in the ref (30). More in detail, an intense UV band with λ_max = 319 nm was observed for free MNZ and for the solutions also containing the metal cations. Moreover, the spectra reported in Figure 3a,b clearly indicate that, under the same concentration conditions, the absorbance of solutions containing L and Cu²⁺ is lower than those containing L and Zn²⁺, confirming the greater extent of the interaction with Cu²⁺ compared to that with Zn²⁺.

Figure 3.

(a, b) Experimental UV spectra at t = 25 °C and I = 0.15 mol L^–1 of (a) Zn²⁺–MNZ(L) and (b) Cu²⁺–MNZ(L) solutions at C_L = 0.05 mmol L^–1 and C_M = 0.05 mmol L^–1.

The trend of the molar extinction coefficients vs λ is shown in Figure 4a,b. CuL and CuL₂ species reach their maximum peak corresponding to 12500 L mol^–1 cm^–1 at λ = 320 nm and 24000 L mol^–1 cm^–1 at λ = 320 nm, respectively.

Figure 4.

(a, b) ε vs λ of (a) Cu²⁺–MNZ(L) and (b) Zn²⁺–MNZ(L) species at t = 25 °C, I = 0.15 mol L^–1.

For data processing, the absorption of the main Cu²⁺ hydrolytic species (CuOH⁺) was taken into account, with an ε trend reported in Figure S1 of the Supporting Information. ZnL, ZnLH, and ZnLOH species showed a maximum peak at λ = 325 nm, corresponding to 18500 L mol^–1 cm^–1 for ZnLH species, and 10000 L mol^–1 cm^–1 for both ZnLOH and ZnL species. The processing of the spectrophotometric experimental data allowed confirmation of both speciation models resulting from the potentiometry and the formation constants of both species relating to the Cu²⁺–L system and of ZnLOH species. The latter is the only species of the Zn²⁺–L system that reaches significant formation percentages under the low concentration conditions employed in the spectrophotometric titrations. Therefore, the formation constants of the other two species, MLH and ML, have been considered in the calculations, and the values obtained by the potentiometry have been kept constant during the process of spectrophotometric data. A comparison between the formation constants gained by potentiometry and spectrophotometry is reported in Table 3.

Table 3. Comparison of Experimental Formation Constants of Zn²⁺–L and Cu²⁺–L Species Obtained by Spectrophotometry and Potentiometry at t = 25 °C and I = 0.15 mol L^–1.

reaction	log βpotentiometry	log βUV	log βavg_value
	8.87(4)a	8.53(1)a	8.70(3)a
	17.39(10)	17.20(6)	17.29(10)
	13.54(2)	13.54	13.54(2)
	6.57(5)	6.57	6.57(3)
	–0.72(2)	–0.90(2)	–0.81(2)

^a≥95% of confidence interval.

3.2. Dependence of Formation Constant Values on the Temperature and Ionic Strength

As is well-known, temperature and ionic strength have a significant impact on formation constant values. For this reason, determining constants under different ionic strengths is crucial to simulate real fluid conditions. Starting from the knowledge of the formation constant values at different temperatures, the enthalpy change values were calculated by using the following van’t Hoff equation:³¹

1

where log β_T is the stability constant at a certain temperature (expressed in kelvin), while log β_θ is the stability constant at t = 298.15 K, and R is the universal gas constant expressed as 8.314 J K^–1 mol^–1. The values of formation enthalpy changes of all of the species of Cu²⁺–L and Zn²⁺–L systems are collected in Table 4, together with entropy and free energy values. As well-known for electrostatic interactions, it is expected that the entropic contributes to the free energy change should be higher than the one given by enthalpy changes, due to the orientation disorder given by the solvation water molecules. This, considering the parameter values referring to partial reactions, is verified for all the species, except for ZnLH.

Table 4. ΔG, ΔH, and TΔS of Cu²⁺–MNZ(L) and Zn²⁺–MNZ(L) Species at t = 25 °C and I = 0.15 mol L^–1 in NaCl.

reaction	ΔGa	ΔHa	TΔSa
	–49.7	29(9)b	79
	–98.7	14(7)	113
	–77.3	–59(11)	18
	–37.5	59(5)	97
	4.6	112(11)	107
	–49.7	29	79
	–49	–15	34
	–77.3	–59(11)	18
	–37.5	59	97
	–47.6	55	103

^aIn kJ mol^–1.

^b≥95% of confidence interval.

The dependence of the formation constants on ionic strength was studied by the following Debye-Hückel-type equation:^32,33

2

where β⁰ is the protonation constant at infinite dilution, z* is the parameter that refers to the charges of the species involved in the formation equilibrium, i.e., ∑z²_reagents – ∑z²_products, and C is an empirical parameter that depends on the stoichiometric coefficients and charges. The formation constants extrapolated to I = 0 mol L^–1, together with the empirical parameter C are reported in Table 5.

Table 5. Formation Constants at Infinite Dilution and Parameters for the Dependence on Ionic Strength of Cu²⁺–MNZ(L) and Zn²⁺–MNZ(L) Species at t = 25 °C in NaCl.

reaction	logT β	C
	9.0(3)a	0.1(4)a
	17.3(7)	1.3(9)
	15.1(2)	–0.3(2)
	7.3(6)	0.3(8)
	0.01(6)	0.4(8)

^a≥95% of confidence interval.

3.3. Sequestering Ability

The tendency of a ligand to complex a free metal cation in solution to form metal–ligand species is expressed as the sequestering ability. To describe the sequestering ability of a ligand toward a certain metal cation, however, the mere knowledge of formation constants is not enough as it is necessary to consider all the possible interactions that both ligand and metal cation may have with all the species present in solution. In order to define it quantitatively, the empirical parameter pL_0.5 was introduced, which represents the cologarithm of the ligand concentration necessary to sequester 50% of the metal cation in traces.^34,35 The calculation is based on the following Boltzmann-type sigmoidal equation:

3

where χ is the sum of molar fractions of the different complex species and pL is the cologarithm of the total ligand concentration.

A comparison between the sequestering ability of MNZ toward Zn², Cu²⁺, and Ca²⁺ (one of the main metal cations present in human body and previously studied in our recent work³⁰) was evaluated under physiological conditions. According to the calculated pL_0.5 values at t = 37 °C, I = 0.15 mol L^–1, and pH plasma mean value (pH = 7.4), the sequestering ability is significantly higher toward Cu²⁺ (pL_0.5 = 4.3) than Zn²⁺ (pL_0.5 = 3.1) and Ca²⁺ (pL_0.5 = 1.9) (Figure 5).

Figure 5.

Comparison of sequestering ability of MNZ(L) toward Cu²⁺, Zn²⁺, and Ca²⁺ at t = 37 °C, I = 0.15 mol L^–1, and pH = 7.4.

3.4. Simulations under Plasma Conditions

The evaluation of the formation percentages of the complex species under physiological conditions can be an intriguing topic to pursue considering that drug complexation can significantly affect cytotoxicity. MNZ(L) with the most significant electrolytes and their respective equilibria (reported in Supporting Information, Table S3) was considered under plasma conditions (t = 37 °C, I = 0.15 mol L^–1, C_Zn = 0.02 mmol L^–1, C_Cu = 0.017 mmol L^–1, C_L = 0.06 mmol L^–1, C_Na = 140 mmol L^–1, C_K = 4.3 mmol L^–1, C_Ca = 1 mmol L^–1, C_Mg = 1 mmol L^–1, C_Cl^- = 102 mmol L^–1, C_HCO3⁻ = 21 mmol L^–1, C_PO4 = 0.4 mmol L^–1).^36,37 As shown in Figure 6a,b, both species of the Cu²⁺–L system reach 10% under plasma conditions, while Zn²⁺–MNZ species are not significant.

Figure 6.

(a, b) Diagrams of MNZ(L) species under plasma conditions (C_L = 0.06 mmol L^–1, C_Cu = 0.017 mmol L^–1, C_Zn = 0.02 mmol L^–1, C_Na = 140 mmol L^–1, C_K = 4.3 mmol L^–1, C_Ca = 1 mmol L^–1, C_Mg = 1 mmol L^–1, C_Cl = 102 mmol L^–1, C_HCO3 = 21 mmol L^–1, C_PO4 = 0.4 mmol L^–1, I = 0.15 mol L^–1, t = 37 °C): (a) speciation diagram; (b) pie chart at pH = 7.4.

3.5. Antimicrobial and Antiprotozoal Activity

Both Cu²⁺–L and Zn²⁺–L complexes were evaluated for their biological activity profiles against an exhaustive panel of pathogenic microorganisms. Specifically, the antimicrobial activity was assessed against the gram-(−) E. coli, the gram-(+) S. aureus, the yeast C. albicans, and the fungus A. fumigatus, whereas the antiprotozoal activity was evaluated against three Trypanosoma spp. (i.e., T. cruzi, T. b. brucei, and T. b. rhodesiense) and one Leishmania sp. (L. infantum). A range of reference compounds (tamoxifen, benznidazole, miltefosine, suramine, doxycycline, flucytosine, and miconazole) were included to demonstrate the validity of the antimicrobial assays (Supporting Information, Table S4). The noncomplexed drug MNZ was used as a main comparator. Salts (chlorides) of Cu²⁺ and Zn²⁺, which can exert their own toxicities, were also used as positive controls. Cytotoxicity assays were performed on both MRC-5 and PMM cells (Table 6). As compared to the pure drug, the complexation with the Zn²⁺ ion did neither affect potency nor selectivity as all IC₅₀ values remained above the highest in-test concentration. The interesting outcome of this study came from the complexation of MNZ with the Cu²⁺ ion. In this latter case, the potency of the drug was increased at least 2.6–2.9-fold against the T. brucei spp. (Cu²⁺–L → IC₅₀ = 2.14 and 1.89 μg/mL, for T. b. brucei and T. b. rhodesiense, respectively) and at least 7.8-fold against E. coli (Cu²⁺–L → IC₅₀ = 0.72 μg/mL). More importantly, this increase in drug potency did not affect cytotoxicity against the two healthy reference cell lines. The activity of Cu²⁺–L against other microorganisms remained unaffected as compared to the reference drug (IC₅₀ > highest in-test concentration), while showing intriguing pathogen-selectivity probably related to different transport mechanisms within cells.

Table 6. In Vitro Antiparasitic Activity and Cytotoxicity (IC₅₀ and CC₅₀ μg/mL, Respectively).

	IC50 or CC50 (μg/mL)
	MNZ(L)	Cu2+	Zn2+	Cu2+–L	Zn2+–L
T. cruzia	>5.64	>2.42	>1.79	>5.33	>6.49
T. b. bruceib	>5.64	0.75	>1.79	1.89	>6.49
T. b. rhodc	>5.64	0.94	>1.79	2.14	>6.49
Sad	>5.64	>2.42	>1.79	>5.33	>6.49
Ece	>5.64	>2.42	>1.79	0.72	>6.49
L. inff	>5.64	>2.42	>1.79	>5.33	>6.49
Cag	>5.64	>2.42	>1.79	>5.33	>6.49
Afh	>5.64	>2.42	>1.79	>5.33	>6.49
MRC-5i	>5.64	>2.42	>1.79	>5.33	>6.49
PMMl	>5.64	>2.42	>1.79	>5.33	>6.49

^aTrypanosoma cruzi Tulahuen CL2 amastigote stage.

^bTrypanosoma brucei brucei Squib-427 strain, suramin-sensitive, trypomastigote stage.

^cTrypanosoma brucei rhodesiense STIB900 strain, drug susceptible, trypomastigote stage.

^dStaphylococcus aureus.

^eEscherichia coli.

^fLeishmania infantum (intracellular amastigotes).

^gCandida albicans.

^hAspergillus fumigatus.

ⁱHuman fetal lung fibroblasts cytotoxicity.

^lPrimary peritoneal mouse macrophages cytotoxicity.

4. Conclusion

An in-depth speciation study of Cu²⁺–L and Zn²⁺–L systems in aqueous solution was carried out in order to point out the thermodynamic behavior of the species and then evaluate their antimicrobial activity. By potentiometric titrations on aqueous solutions, the speciation models and corresponding formation constants of the formed species were determined, which showed the formation of the two species CuL and CuL₂ and ZnLH, ZnL, and ZnLOH. Both models and formation constants obtained were confirmed using spectrophotometric titrations. With the aim of getting a complete thermodynamic picture, the dependence of the formation constant values on the temperature and ionic strength was also investigated. Moreover, the formation percentages of these species were evaluated by simulating the real plasma conditions of temperature, pH, and ionic strength, and over 50 equilibria between the main electrolytes present in the plasma were considered. According to the biological screening, the L complexation with Cu²⁺ has a significant impact on the potency of the drug against T. b. brucei, T. b. rhodesiense, and E. coli; on the contrary, the presence of Zn²⁺ does not lead to enhanced activity.

Glossary

Abbreviations

MNZ or L

Metronidazole

T. cruzi

Trypanosoma cruzi

T. b. brucei

Trypanosoma brucei

T. b. rhod

Trypanosoma brucei rhodesiense

Sa

Staphylococcus aureus

Ec

Escherichia coli

L. inf

Leishmania infantum

Ca

Candida albicans

Af

Aspergillus fumigatus

MRC-5

Human fetal lung fibroblasts cytotoxicity

PMM

Primary peritoneal mouse macrophages cytotoxicity

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c04166.

• Table S1: Hydrolysis constants of Zn²⁺ at different temperatures and ionic strength values; Table S2: Hydrolysis constants of Cu²⁺ and formation constant values of Cu²⁺-Cl^– species at different temperatures and ionic strength values; Figure S1: ε vs λ of CuOH⁺ at t = 25 °C, I = 0.15 mol L^–1; Table S3: Formation constant values of the species considered for simulations under plasma conditions; Table S4: List of reference drug and IC₅₀ values of the screening panel (PDF)

Author Contributions

Conceptualization, O.G., F.C.; Methodology, O.G., F.C., N.V.P., A.M., G.C.; Validation, O.G., F.C., C.F., A.M., G.C.; Investigation, F.C., N.V.P., A.M.; Data curation, O.G., F.C; Writing–original draft preparation, O.G, F.C.; Writing–review and editing, O.G., C.F., F.C., N.M., G.C.; Supervision, O.G., F.C., N. M., G.C. All authors have given approval to the final version of the manuscript.

G.C. is supported by research project grants of the Research Foundation – Flanders (FWO-Vlaanderen) and the Research Fund (BOF) of the University of Antwerp. G.C. participates in COST Action CA21111 (Onehealthdrugs).

The authors declare no competing financial interest.