Skip to content
Frontiers
Search

Original Research

Frontiers in Chemical Biology ·

Volume 5 - 2026 | https://doi.org/10.3389/fchbi.2026.1666012

Comparative reactivity studies of imine-copper(II) and zinc(II) complexes containing both oxindole and benzimidazole scaffolds for biomolecules and tumor cells

  • Aline Monteiro L. Zaballa1
  • Ana Paula Araujo de Oliveira1
  • Rodrigo B. Fazzi1
  • Raphael E. F. de Paiva1
  • Katherine Tsantarlis2
  • Carla Columbano Oliveira2
  • Maciel Santos Luz3
  • Sayuri Miyamoto2
  • Ana M. da Costa Ferreira1*corresponding author
  1. 1Departamento de Química, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
  2. 2Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
  3. 3Laboratório de Processos Metalúrgicos, Materiais Avançados, Instituto de Pesquisas Tecnológicas (IPT), São Paulo, Brazil
Article metrics0 viewsView details
PDFCiteMetrics

Abstract

Metal complexes with oxindole- or benzimidazole-based ligands have been extensively studied regarding their antiproliferative activities. Herein, some copper(II) and analogous zinc(II) complexes with imine ligands containing both oxindole- and benzimidazolescaffolds had their toxicity toward cancerous cells evaluated. These compounds are derived from an endogenous oxindole (isatin), through a condensation reaction with 2-aminomethylbenzimidazole, and their structural and spectroscopic characterization were carried out by using different techniques, including elemental analyses, UV/Vis, FTIR, EPR and Raman spectroscopies, in addition to ESI-MS. The main goal was to verify how different scaffolds (oxindole- and benzimidazole-) could influence the activity of metal complexes. Investigations of these copper compounds interactions with DNA indicated that they bind preferentially at the double-strand grooves, causing oxidative damage by simple- or double-strand cleavages. This damage occurs through the generation of reactive oxygen species (ROS), mostly singlet oxygen, hydrogen peroxide, and superoxide ions, which were also able to damage human albumin (HSA), observed by SDS-PAGE gel, and to cause lipid peroxidation in phosphatidylcholine liposomes, verified by fluorescence measurements. Further, for analogous heteroleptic compounds [Cu(isambz)(phen]ClO 4 4 and [Zn(isambz)(phen)]ClO 4 5, containing also 1,10-phenantroline (phen) as secondary ligand, results signaled the radical hydroxyl as the most important ROS formed. The cytotoxicity of these metal complexes was then verified by assays of cell viability, using the reagent MTT, toward malignant cells (cervical HeLa and melanoma SK-Mel), compared to non-tumor cells (fibroblasts). Corresponding IC 50 values were determined for both cancerous cell lines in the range of a few μM, attesting their remarkable reactivity. Therefore, the herein focused metal compounds, exhibiting diverse targets, could be promising alternatives for metallodrugs against cancer.

Keywords

1 Introduction

Cancer has become one of the most significant public health challenges of the 21st century, although the prevention and early detection of tumors can avoid higher risks. According to the International Agency for Research on Cancer (IARC), one in five people worldwide develop cancer during their lifetime (IARC, 2025), and it has been estimated that by 2040, 28 million new cancer patients will be diagnosed (Plackett, 2025). Anticancer approaches include surgery, radiotherapy, chemotherapies, and targeted therapies. The aim of these is to improve efficiency and, particularly in chemotherapy, prevent the development of multi-drug resistance (Vaidya et al., 2020; Housman et al., 2014). For many decades, researchers have been investigating new medicines for different types of cancer. Most research has sought to understand its cause, find the main favorable targets, and propose novel treatments (Brownlie et al., 2023; Lin and Zhang, 2023; Li et al., 2024). Among the medications developed, metallodrugs deserve special attention, since a synergy between biologically active ligands and metal ions with redox activity often seems to contribute significantly to its efficacy and selectivity (Farrell, 2015; Englinger et al., 2019; Zou et al., 2025).

In addition to platinum, ruthenium, gold, and other metal ions (Adhikari et al., 2024a), copper complexes have been widely studied (Wang et al., 2024) with a great variety of ligands (Santini et al., 2014; Natha et al., 2025; Rogalewicz and Czylkowska, 2025), as well as zinc complexes (Pellei et al., 2021; Adhikari et al., 2024b; Bhattacharjeea et al., 2024). These ligands embrace different compounds, from minerals to inorganic, organic, natural, or synthetic complexes containing promising functional groups, such as imines, oxindoles, and imidazoles, that lead to copper complexes of varied structural features and positive activities regarding cellular cytotoxicity (Bai et al., 2025). Furthermore, a variety of strategies are being developed to improve efficacy and selectivity, mainly based in coordination chemistry concepts involving stability, lability, preferential coordinating atoms, particular geometries, and changes in redox potential (Sadeer et al., 2020; Wang et al., 2019), and speciation inside cells (Levina et al., 2017).

Oxindoles and derivatives have been broadly evaluated as potential pharmaceuticals not only against cancer (de Paiva et al., 2021; Khetmalis et al., 2021) but also as promising scaffolding for antimicrobial (Shin et al., 2019), antiviral (Chander et al., 2018), antitubercular (Velavalapalli et al., 2024), and neglected tropical diseases (Chowdhary et al., 2022; Portes et al., 2023). Benzimidazole groups also seem to be noteworthy scaffolds reported in metallodrugs studies, including anticancer (Roy et al., 2005), antiviral, antimalarial, and antimicrobial agents (Diaconu et al., 2022) among other possible applications (Aroua et al., 2025; Aroua et al., 2023). When metalated, the biological properties of both scaffolds are usually increased, indicating a synergy modulated by the metal as well as by the ligands (Cerchiaro and Da Costa Ferreira, 2006). Additionally, aromatic imine ligands are known to be very cytotoxic, especially when coordinated with copper ions (Serment-Guerrero et al., 2017; Fernandez et al., 2024). Masuri et al. (2022) compared a series of heteroleptic copper (II) phenanthroline-based complexes with a wide range of ligands in terms of different targets and mechanisms of action. Different classes of secondary ligands, inorganic and organic (α-amino acids, carboxylic acids, imines, thiosemicarbazones, etc.), had their anticancer activity discussed regarding diverse malignant cell lines (Moráň et al., 2019).

Herein, some copper (II) and analogous zinc (II) complexes (Figure 1), with a bi- or trinuclear imine ligand termed isambz and containing both oxindole and benzimidazole scaffolds are prepared by different techniques (UV/Vis, FTIR, EPR, Raman spectroscopies, and mass spectrometry) and have their toxicity toward cancerous cells (cervical HeLa and melanoma SK-MEL) evaluated. Moreover, ternary analogous species were obtained by introducing 1,10-phenanthroline (phen) as a secondary ligand to improve their cytotoxicity. The main goal was to verify whether the oxindole- and benzimidazole-imine scaffolds could influence the activity of metal complexes. Comparative reactivity studies have been carried out to identify DNA, protein, and lipid as crucial biomolecules targeted by the complexes.

Proposed structures for metal complexes studied.
Figure 1. Proposed structures for metal complexes studied.

2 Results and discussion

2.1 Syntheses of the studied compounds

Copper (II) complexes with the imine ligand isambz [or (E)-3-(((1H-benzo [d]imidazole-2-yl)methyl)imino)indolin-2-one], 1 and 2, and analogous Zn(II) complex 3 were obtained as 1:1 or 1:2 (M:L) species by metalation in situ of the ligand with copper or zinc perchlorate salts. This ligand was prepared by a condensation reaction between oxindole isatin and 2-(aminomethyl)benzimidazole, in aqueous ethanolic solutions, using generally described procedures and based on our previously reported research (see references in Supplementary Information). For compounds 4 and 5, 1,10-phenanthroline (phen) was added as a second ligand before metalation. Most of these were isolated as the enol form (Figure 1), although in solution they present a tautomeric equilibrium between both enol and keto forms, depending on pH (see Figure 2).

Tautomerical forms of complex 1.
Figure 2. Tautomerical forms of complex 1.

Only complex 2 was isolated in the keto form, in solid state. All these complexes are lipophilic cationic species and are able to enter the cells, as also verified for some similar complexes in the oxindole-based series (de Paiva et al., 2021).

2.2 Spectroscopic characterization

Copper (II) and zinc (II) complexes were characterized by spectroscopic techniques, UV/Vis, FTIR, Raman, NMR, or EPR, in addition to elemental analyses. ESI-MS data are displayed in Supplementary Figures S5A–S5E. The main species identified attested to the proposed structure of these complexes (Figure 1): [Cu(isambz)H2O]ClO4 1, [Cu(isambz)2](ClO4)2 2, [Zn (isambz)H2O]ClO4 3, [Cu(isambz) (phen)]ClO4 4, and [Zn (isambz) (phen)]ClO4 5.

Electronic, FTIR, and Raman spectra are also shown in Supplementary Figures S2 and 3. Additionally, in Supplementary Table S1 the main bands observed in UV/Vis spectra are displayed with corresponding bands assigned. Bands referred to as “d-d bands” were not observed due to the low solubility of these compounds, as they are soluble only in DMSO solvent.

Corresponding main bands in FTIR and Raman spectra are shown in Supplementary Figure S3 and Supplementary Table S2. Typical FTIR bands of imine groups appeared, and bands at 3,447 and 3,345 cm−1 were observed only for complex 2, attesting to the keto form of the coordinated ligand. In the other complexes, the ligand appears in enol form. Complementary Raman bands were also observed (Supplementary Table S2).

Additional EPR spectra were registered to better characterize and compare the geometry around the copper center in complexes 1, 2, and 4 (Supplementary Figure S4). For comparison, EPR parameters in DMSO/water solution, registered at 77 K, are displayed in Table 1.

TABLE 1. EPR parameters for the copper (II) complexes studied, in frozen DMSO/water solution at 77 K, compared to similar oxindole- or benzimidazole-based complexes previously reported.

Complexg┴g//A//, GA//, 10−4 cm−1g///A//, cm
[Cu(isambz)H2O]+ # 12.1072.450186212116
[Cu(isambz)2]2+ # 22.0962.427118133182
[Cu(isambz)phen]+ 42.0652.253158166136
[Cu(phen)2]2+ *2.0662.264​169.5134

From # (Cavichioli et al., 2019) in DMSO/water solution at 77 K, and * (Nunes et al., 2020) measurements in cellular medium at ∼100 K.

These results indicate an axial symmetry with g//>g┴>2.0023, and values G = (g//−2)/(g┴-2) in the range 3.7–4.6 suggest the presence of a dx2-y2 ground state (Hagen, 2006). Complexes 1, 2, and [Cu(phen)2]2+ have a typical planar or tetragonal structure due to the Jahn–Teller effect, while complex 2 presents a more significant tetrahedral distortion, as attested by the g///A// ratio which is frequently used to evaluate a probable distortion around the copper (II) center (Sakaguchi and Addison, 1979), helping to better stabilize the reduced form of the complex. However, complex 4 shows a probably pyramidal square-based geometry. These data complement the spectroscopic and structural characterization of the compounds.

2.3 Biological properties

The reactivity of these metal complexes was then investigated regarding different biomolecules, DNA, albumin (HSA), and lipids to verify their main targets. CD spectra were registered upon the addition of different concentrations of the complexes studied (0–40 µM) to CT-DNA (100 µM) (Figure 3). All curves of CD intensity versus wavelength showed at least two isosbestic points, attesting to the observed interactions. For complexes 1, 2, and 3, a minor decrease in the bands at 220 and 275 nm was verified. In contrast, increasing amounts of complexes 4 and 5 caused more severe modifications in the curves, indicating an increase in intensity at 275 nm and a shift of this band to high wavelength. Additionally, a decrease in the band at 220 nm and the detection of a band at 235 nm were more significant than for the other complexes.

Changes in the CD profiles of CT-DNA (ellipticity vs. wavelength) observed in the presence of the increasing addition of metal complexes **1** to **5**, displayed respectively in **(A-E)**.
Figure 3. Changes in the CD profiles of CT-DNA (ellipticity vs. wavelength) observed in the presence of the increasing addition of metal complexes 1 to 5, displayed respectively in (A-E).

The presence of three isosbestic points in curves D and E may indicate the release of the phenanthroline ligand, since in A, B, and C only two isosbestic points are observed. Many isosbestic points could indicate the presence of more than two detected species. Nunes et al. (2020) and Zheng et al. (2019) describe the separate action of Cu(II) ions and phen molecules in similar complexes.

2.3.1 Assays of DNA/EtBr fluorescence quenching

DNA is usually a target for many metal complexes showing antiproliferative properties. A standard test to verify the interactions of metal complexes with DNA is fluorescence quenching in competitive experiments (Zeng et al., 2021). Therefore, fluorescence assays of EtBr intercalated in CT-DNA in the presence of different concentrations (up to 200 μM) of the metal complexes studied were performed (Figure 4). The data were compared with similar results for [Cu(phen)2)Cl2, a known intercalator in DNA structure (Sigman et al., 1993).

Fluorescence decrease of CT-DNA/EtBr solutions observed with addition of increasing concentrations of the metal complexes investigated: **(A)** copper (II) species; **(B)** zinc (II) species.
Figure 4. Fluorescence decrease of CT-DNA/EtBr solutions observed with addition of increasing concentrations of the metal complexes investigated: (A) copper (II) species; (B) zinc (II) species.

The verified order of reactivity versus CT-DNA/EtBr was complex [Cu(phen)2] > [Cu(isambz)phen] 4 > [Zn (isambz)phen] 5 > [Cu(isambz)2] 2 > [Cu(isambz)H2O] 1. In contrast, complex [Zn (isambz) H2O] 3 was quite inactive. Using the Stern–Volmer equation (Gehlen, 2020) and corresponding curves (Figure 5), the corresponding quenching constants KSV are shown in Table 2 in comparison with the [Cu(phen)2] complex. Interactions with DNA can occur in different modes and are influenced by all ligands coordinated with the metal center. The results indicated that DNA is probably an important target of these metal complexes.

Curves of F<sub>0</sub>/F versus concentration of the studied complexes **1**, **2**, **4**, and **5**.
Figure 5. Curves of F0/F versus concentration of the studied complexes 1, 2, 4, and 5.

TABLE 2. Stern–Volmer constants (KSV) determined for the complexes studied, using CT-DNA (40 μM) with intercalated EtBr (25 μM).

CompoundKSV, M−1
[Cu(isambz)H2O]+ 10.922 × 103
[Cu(isambz)2]2 + 21.9 × 103
[Zn (isambz)H2O]+ 3--
[Cu(isambz)phen]+ 40.52 × 104
[Zn (isambz)phen]+ 51.3 × 103
[Cu(phen)2]2+1.3 × 104

To verify the intracellular concentration and the possible speciation of copper (II) species in HeLa cells after treatment with such complexes, data for complexes 1 and 2 were obtained by ICP-MS (Figure 6A). Despite being larger, complex 2 succeeds better in entering the HeLa cells than complex 1.

**(A)** Amount of copper inserted in HeLa cells after 6 and 24 h incubation with complexes [Cu(isambz)H<sub>2</sub>O]<sup>+</sup> **1** and [Cu(isambz)<sub>2</sub>]<sup>2 +</sup> **2**. Control, no addition of metal; Cu(NO<sub>3</sub>)<sub>2</sub> compound used as reference. **(B)** Speciation of metal complexes **1** and **2** inside HeLa cells, considering nucleus and cytosol, after 3 and 6 h treatment.
Figure 6. (A) Amount of copper inserted in HeLa cells after 6 and 24 h incubation with complexes [Cu(isambz)H2O]+ 1 and [Cu(isambz)2]2 + 2. Control, no addition of metal; Cu(NO3)2 compound used as reference. (B) Speciation of metal complexes 1 and 2 inside HeLa cells, considering nucleus and cytosol, after 3 and 6 h treatment.

As shown in Figure 6B, both complexes 1 and 2 are able to cross the nuclear membrane. Complex 2 shows a more similar distribution between nucleus and cytosol after 3 h incubation, while complex 1 shows some preference for the cytosol. However, this tendency is modified after 6 h incubation, with a higher quantity of complex 1 detected at the nucleus. Therefore, the higher antiproliferative activity of complex 1 seems to be due to its high concentration at the nucleus, earlier damaging the DNA.

Additional experiments indicated that complexes 1 and 2 also enter the nucleus of HeLa cells in higher concentrations than the control after 3 or 6 h incubation (Figure 6B). Complex 2 is almost equally distributed between nucleus and cytosol.

2.3.2 Cell viability in the presence of the complexes studied

The cytotoxicity of the compounds studied was verified for cervical HeLa and melanoma SK-MEL 28 and SK-MEL 147 cells in comparison to non-tumorigenic fibroblast cells (Table 3). Corresponding curves of cell viability versus concentration of these compounds can be found in Figure 7.

TABLE 3. Cell viability data (IC50, µM) of tumor cells (HeLa and SK-MEL) for copper (II) and zinc (II) complexes studied after incubation for 24 h at 37 °C.

ComplexesHeLa cellsFibroblastsSK-MEL 28SK-MEL 147Fibroblasts
[Cu(isambz)H2O]+ 113.1 ± 0.1>10021.70 ± 0.40>50>100
[Cu(isambz)2]2+ 221.2 ± 0.1>10032.2 ± 0.60>50>100
[Zn (isambz)H2O]+ 3>100>100​​​
[Cu(isambz)phen]+ 41.17 ± 0.202.36 ± 0.501.97 ± 0.223.36 ± 0.1842.09 ± 0.40
[Zn (isambz)phen]+ 55.67 ± 1.0>100​​​
Cell viability curves of tumor cells HeLa and melanoma SK-MEL 28 and SK-MEL 147 incubated with the studied metal (II) complexes **1**, **2**, **4**, and **5** for 24 h at 37 °C.
Figure 7. Cell viability curves of tumor cells HeLa and melanoma SK-MEL 28 and SK-MEL 147 incubated with the studied metal (II) complexes 1, 2, 4, and 5 for 24 h at 37 °C.

Copper (II) complexes 1 and 4 were more active than analogous zinc (II) compounds 3 and 5, probably due to their more planar structures that facilitate its binding to DNA, in addition to its ability in generating ROS. When a second ligand (phen) was introduced, a remarkable increase in their reactivity occurred in both cells investigated, as also reported in other examples in the literature. Complex 4, containing the phen ligand would better stabilize the analogous reduced form and so showed better reactivity toward all tumor cells tested, with IC50 values of a few µmoles, in contrast to complexes 1 and 2.

Similar copper (II) complexes have already been compared, especially those containing phen (or derivatives) as a second ligand, showing that the planarity of the aromatic diimine usually enhances the antiproliferative properties of a metal complex, probably because of its intercalation in DNA bases, and in some cases binding to major or minor grooves. In Table 4, some selected copper (II) complexes of different ligands were compared in studies performed mainly with HeLa tumor cells.

TABLE 4. Comparative data for copper (II) complexes with different ligands, and/or analogous complexes containing an aromatic diamine phen as an additional ligand.

CompoundIC50, µMIncubation timeCancer cellRef.
[Cu(isambz)H2O]+ 113.1 ± 0.124 hHeLa and cervicalThis work
[Cu(isambz)2]2 + 240.2 ± 0.124 hHeLaThis work
[Cu(isambz)phen]+ 41.17 ± 0.2024 hHeLaThis work
[Cu(isapn)]ClO480 ± 624 hHeLaVieira et al. (2019)
[Cu(phen)gly] (Cas-gly)1.3324 hHeLaPeña et al. (2019)
[Cu(sal-gly)phen]7.30 ± 0.5924 hHeLaAcilan et al. (2017)
[Cu(L-dipeptide)phen]2.0 to 7.524 hHeLaIglesias et al. (2014)
[Cu(phen)2]Cl21624 hHeLaIglesias et al. (2014)
[Cu(L) (phen)](NO3)L = 4-oxo-4H-chromene-3-carbaldehyde-4(N)-phenylthiosemicarbazone1.83 ± 0.1724 hHeLaMamindla et al. (2024)
[Cu(L) (5,6-dimethyl-1,10-phen)]+ L = 4-oxo-4H-chromene-3-carbaldehyde-4(N)-phenylthiosemicarbazone2.97 ± 0.9224 hHeLaMamindla et al. (2024)
[Cu(bm)2]Cl2 bm = 2-(2-aminophenyl)-1H-benzimidazole87 ± 624 hSiHa and CervicalLoganathan et al. (2012)
[Cu(bba) (phen)]2+ bba = N,N′-bis(benzimidazol-2-ylmethyl)amine7.3 ± 0.224 hHeLaLoganathan et al. (2012)
[Cu(gly-gly)L]L = 5-methyl-2-(2′-pyridyl)-benzimidazole11.67 ± 0.8​HeLaQi et al. (2018)
[Cu(Gly-L-leu)LL = 5-methyl-2-(2′-pyridyl)-benzimidazole7.88 ± 0.3​HeLaQi et al. (2018)
[Cu(ambaf)H2O)](ClO4)2 ambaf= (E)-2-(1-(((1H-benzo [d]imidazole-2-yl)methyl)imino)ethyl)aniline2-(aminoacetophenyl)-methyl-benzimidazole54.1 ± 0.497.1 ± 0.424 h24 hMES-SA and MES-SA/Dx5 and sarcomaVieira et al. (2018)

Indeed, by observing the selected examples in Table 4, we confirm that the insertion of phen as a second ligand caused a remarkable improvement in its anticancer properties against different cells and with diverse types of ligands (Lucaciu et al., 2022). Complex 1 containing two different scaffolds showed high anti-growth activity, even without the phen ligand, better than similar ligands showing just oxindole or benzimidazole groups. Complexes containing only benzimidazole groups seem to be less reactive in confronting oxindole derivatives. By comparing complex [Cu(isambz)H2O]+ 1, containing both focused scaffolds, and complex [Cu(ambaf)H2O)]2+, containing a pyridylimine and a benzimidazole scaffold, the former seems to be more cytotoxic, although the latter is shown to be toxic even to resistant sarcoma cells (Vieira et al., 2018). A more comprehensive study could clarify this strategy of using more than one active scaffold in the same ligand.

2.3.3 Nuclease activity

This activity was determined for all the metal complexes studied, in the range 2.5–20 μM and incubated with DNA for 24 h at 37 °C (Figure 8). Complexes 4 and 5 were also compared to the analogous [Cu(phen)2]2+ compound, extensively reported in pioneer studies (Iglesias et al., 2014). The results showed that complex 1 was more active than complex 2, forming more significant amounts of the DNA form II (NC, nicked circular), while complex 3 was less active up to 20 μM. Interestingly, complex 2 showed no increase of activity at higher concentrations in the range 2.5–20 μM. On the other hand, complex 4 showed similar activity to [Cu(phen)2]2+, and complex 5 was more reactive than analogous [Zn (phen)2]2+, forming high amounts of NC (nicked circular) DNA species at 5 µM. DC (double-strand cleavage) was not observed in any case.

Agarose gel electrophoresis of plasmidial-DNA (pBS) incubated for 24 h at 37 °C in PBS solution with the complexes studied. **(A)** [Cu(isambz)H<sub>2</sub>O]<sup>+</sup> **1,** [Cu(isambz)<sub>2</sub>]<sup>2 +</sup> **2**, [Zn (isambz)H<sub>2</sub>O]<sup>+</sup> **3**. **(B)** [Cu(isambz)phen]<sup>+</sup> **4** and [Zn (isambz)phen]<sup>+</sup> **5,** in concentrations 2.5–20 µM. Ctrl, DNA in PBS. Data for [Cu(phen)<sub>2</sub>]<sup>2+</sup> and [Zn (phen)<sub>2</sub>]<sup>2+</sup> were included for comparison. Ctrl, DNA in PBS; Sc, supercoil DNA; NC, nicked circular DNA.
Figure 8. Agarose gel electrophoresis of plasmidial-DNA (pBS) incubated for 24 h at 37 °C in PBS solution with the complexes studied. (A) [Cu(isambz)H2O]+ 1, [Cu(isambz)2]2 + 2, [Zn (isambz)H2O]+ 3. (B) [Cu(isambz)phen]+ 4 and [Zn (isambz)phen]+ 5, in concentrations 2.5–20 µM. Ctrl, DNA in PBS. Data for [Cu(phen)2]2+ and [Zn (phen)2]2+ were included for comparison. Ctrl, DNA in PBS; Sc, supercoil DNA; NC, nicked circular DNA.

In the presence of the reducing agent ascorbate (AA, 1 mM), both complexes 1 and 2 exhibited an improved and very similar activity when observed in a very low concentration range (0.156–1.25 μM—Figure 9), attesting to the influence of the metal redox catalytic cycle. A single cleavage was observed in this range of concentration for both complexes. However, for compound 2 at concentrations ≥0.625, only the NC species was verified, corresponding to total single DNA cleavage.

Agarose gel electrophoresis of plasmidial-DNA (pBS) incubated for 24 h at 37 °C in PBS solution with the studied complexes [Cu(isambz)]<sup>+</sup> **1** and [Cu(isambz)<sub>2</sub>]<sup>2+</sup> **2,** in concentrations 0.156–1.25 μM in the presence of ascorbate (AA, 1 mM). Ctrl, DNA in PBS; Ctrl + AA, with the addition of ascorbate.
Figure 9. Agarose gel electrophoresis of plasmidial-DNA (pBS) incubated for 24 h at 37 °C in PBS solution with the studied complexes [Cu(isambz)]+ 1 and [Cu(isambz)2]2+ 2, in concentrations 0.156–1.25 μM in the presence of ascorbate (AA, 1 mM). Ctrl, DNA in PBS; Ctrl + AA, with the addition of ascorbate.

Further experiments in the presence of ROS scavengers—azide (NaN3) for singlet oxygen, SOD for superoxide species, DMSO for hydroxyl radical, and catalase for hydrogen peroxide—allowed the identification of the main reactive species responsible for the oxidative damage (Figure 10). Protection of DNA damage was observed in the presence of azide and catalase in the case of complex 2 but only azide for complex 1. Therefore, hydroxyl and superoxide radicals are active species in both cases, and H2O2 is active only when complex 1 was used.

Agarose gel electrophoresis of plasmidial-DNA incubated for 24 h at 37 °C with complexes [Cu(isambz)]<sup>+</sup> **1** and [Cu(isambz)<sub>2</sub>]<sup>2+</sup> **2**. Complexes at 1.25 µM with the addition of different ROS scavengers (NaN<sub>3</sub> 500 mM; SOD 40 U; DMSO 10%; catalase 60 U). Ctrl + AA, DNA in PBS with the addition of ascorbate (AA 1 mM).
Figure 10. Agarose gel electrophoresis of plasmidial-DNA incubated for 24 h at 37 °C with complexes [Cu(isambz)]+ 1 and [Cu(isambz)2]2+ 2. Complexes at 1.25 µM with the addition of different ROS scavengers (NaN3 500 mM; SOD 40 U; DMSO 10%; catalase 60 U). Ctrl + AA, DNA in PBS with the addition of ascorbate (AA 1 mM).

Results with complex 4 were also obtained in the presence of known scavengers of ROS (Figure 11) in comparison with analogous compounds [Cu(phen)2]Cl2.

Agarose gel electrophoresis of plasmidial-DNA incubated for 24 h at 37 °C with complexes [Cu(isambz)phen]<sup>+</sup> **4** and [Cu(phen)<sub>2</sub>]<sup>2+</sup> for comparison. **(A)** Complexes in concentrations 0.019–0.156 µM in the presence of ascorbate (AA, 1 mM). **(B)** Complexes in concentration 1.25 µM with the addition of different ROS scavengers (NaN<sub>3</sub> 500 mM; SOD 40 U; DMSO 10%; catalase 60 U). Ctrl, DNA in PBS, with addition of AA (1 mM). Sc, supercoil DNA; NC, nicked circular; DC, double cleavage.
Figure 11. Agarose gel electrophoresis of plasmidial-DNA incubated for 24 h at 37 °C with complexes [Cu(isambz)phen]+ 4 and [Cu(phen)2]2+ for comparison. (A) Complexes in concentrations 0.019–0.156 µM in the presence of ascorbate (AA, 1 mM). (B) Complexes in concentration 1.25 µM with the addition of different ROS scavengers (NaN3 500 mM; SOD 40 U; DMSO 10%; catalase 60 U). Ctrl, DNA in PBS, with addition of AA (1 mM). Sc, supercoil DNA; NC, nicked circular; DC, double cleavage.

In the case of complex 4, azide, DMSO, and catalase protected against DNA damage, indicating the participation of superoxide anion, hydroxyl radical, and hydrogen peroxide as active ROS.

2.3.4 HSA protein as a possible target

In many proposed chemotherapies, albumin is used as carrier of anticancer agents. The possibility of damage to the HSA protein by the copper (II) complexes under study was monitored by SDS-PAGE gel, incubating the protein (20 μM) for 24 h at 37 °C with different concentrations of complexes 1, 2 and 4 in the range 62.5–50 μM in the absence and presence of ascorbate (AA, 1 mM). The results, shown in Figure 12A, indicate that the complexes showed protease activity at concentrations ≥6.25 µM and in the presence of AA (1 mM), with consequent fragmentation of the protein. Complex 4 and, for comparison, complex [Cu(phen)2]Cl2 showed more extensive protein fragmentation (Figure 12B), while with the analogous zinc (II) complex 5 and corresponding [Zn (phen)2]Cl2 (Figure 12C), the protein band is still observed with concentrations ≥6.25 µM for both compounds, although less intense, in the presence of ascorbate.

SDS-PAGE gel of protein HSA incubated for 24 h at 37 °C without (−) and with (+) the addition of ascorbate (AA, 1 mM) and the complexes: **(A)** [Cu(isambz)H<sub>2</sub>O]<sup>+</sup> **1** and [Cu(isambz)<sub>2</sub>]<sup>2 +</sup> **2**; **(B)** [Cu(isambz)phen]<sup>+</sup> **4** and [Cu(phen)<sub>2</sub>]Cl<sub>2</sub>; **(C)** [Zn (isambz)phen]<sup>+</sup> **5** and [Zn (phen)<sub>2</sub>]Cl<sub>2</sub>.
Figure 12. SDS-PAGE gel of protein HSA incubated for 24 h at 37 °C without (−) and with (+) the addition of ascorbate (AA, 1 mM) and the complexes: (A) [Cu(isambz)H2O]+ 1 and [Cu(isambz)2]2 + 2; (B) [Cu(isambz)phen]+ 4 and [Cu(phen)2]Cl2; (C) [Zn (isambz)phen]+ 5 and [Zn (phen)2]Cl2.

2.3.5 Lipid peroxidation data

Some previous experiments with Raman microscopy verified that oxindole-based copper (II) complexes have lipids in addition to DNA and mitochondria as probable targets (Marin et al., 2023; Marmián-Lopez et al., 2021). Consequently, some experiments were carried out to monitor the release of dye 5 (6)carboxyfluorescein (CF) from liposomes prepared with phosphatidylcholine and incubated with the complexes studied.

These experiments were done by using two concentrations of each complex (1 μg/mL and 10 μg/mL) in the absence and presence of the reducing agent ascorbate (AA, 1 mM) (Figure 13). For comparison, the experiment was done also with [Cu(phen)2]Cl2. In the absence of AA, curves for complexes 1 and 2 were similar and at higher concentration exhibited an inflection at 60,000 s (approximately 16.7 h), indicating a second step faster than the initial one. In the presence of AA, the curve for complex 4 showed an earlier inflection, reaching (at 20,000 s or 5.6 h) a plateau at 80% externalization of CF before complexes 1 and 2 and even complex [Cu(phen)2]Cl2, showing this inflection approximately 30,000 s or 8.3 h. For complexes containing phen ligand, kinetic differences were more remarkable, especially in the presence of ascorbate, when the reaction was faster at low concentration (1 μg/mL) than at high (10 μg/mL).

Externalization of CF in liposomes after incubation with complexes [Cu(isambz)H<sub>2</sub>O]<sup>+</sup> **1**, [Cu(isambz)<sub>2</sub>]<sup>2 +</sup> **2**, [Cu(isambz) (phen)]<sup>+</sup> **4**, and [Cu(phen)<sub>2</sub>]Cl<sub>2</sub> in concentrations 1 μg/mL or 10 μg/mL with and without the addition of ascorbate (AA, 1 mM).
Figure 13. Externalization of CF in liposomes after incubation with complexes [Cu(isambz)H2O]+ 1, [Cu(isambz)2]2 + 2, [Cu(isambz) (phen)]+ 4, and [Cu(phen)2]Cl2 in concentrations 1 μg/mL or 10 μg/mL with and without the addition of ascorbate (AA, 1 mM).

3 Conclusion

Studies on some metal complexes containing the ligand isambz indicated the formation of stable copper (II) and zinc (II) species as perchlorate salts, [Cu(isambz)H2O]+ 1, [Cu(isambz)2]2 + 2, and [Zn (isambz)H2O]+ 3, characterized by different spectroscopic techniques and showing significant activity in oxidative damage to crucial biomolecules such as DNA, proteins and lipids, particularly the copper species. Analogous heteroleptic complexes comprehending an additional phenanthroline ligand, [Cu(isambz)phen]+ 4 and [Zn (isambz)phen]+ 5 were also prepared, and they exhibited significant improvement in their oxidative characteristics, especially in the verified cytotoxicity versus HeLa and SK-MEL tumor cells. Corresponding IC50 values were determined for those cell lines in the range of a few µM, comparable to other similar compounds already reported (Table 4). In previous studies, complexes 2 and 3 also showed moderate cytotoxicity against human hepatoma (HepG2) and neuroblastoma (SH-SY5Y) cells (Cavichioli et al., 2019). Based on our data, different factors have a substantial influence in the reactivity of such complexes, including the stoichiometric relation M:L, the tumor cell type, the geometry around the metal ion provided by the ligands, and the nature of metal ions. The presence of two scaffolds in the ligand seems to increase the observed nuclease activity and cytotoxicity compared to analogous compounds containing only one. Furthermore, all studied copper (II) complexes (1, 2, and 4) exhibited a significant increase in lipid peroxidation process at higher concentrations and in the presence of a reducing agent (Figure 13). The addition of phen as a second ligand remarkably intensified this reactivity toward lipids, indicating a process with two steps, kinetically dependent on the reduction of the copper center. Complex 4, exhibiting isambz and phen ligands, showed higher activity than [Cu(phen)2]2+ in the absence of the reducing agent, ascorbate.

An investigation into copper complexes with 1,10-phen derivatives concluded that, contrary to most reported studies, the interaction of these complexes with cell components and consequent cell degradation involve preferentially separate actions of copper ions coordinated to the main ligand and phen molecules (Nunes et al., 2020).

The presence of more than one scaffold in the same ligand, if carefully selected, could permit fine-tuning of the properties of these complexes to enhance or modulate their potential anticancer activity, combining diverse targets and/or more than one mode of action.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchbi.2026.1666012/full#supplementary-material

Supplementary material Supplementary material at the source

Footnotes

Edited by: Sylvia Mary Draper, Trinity College Dublin, Ireland

Reviewed by: George Barisas, Colorado State University, United States

Jyoti Sharma, Maharishi Markandeshwar University, Mullana, India

Footnotes

CD, circular dichroism; CF, 5(6)-carboxyfluorescein; CT-DNA, calf-thymus DNA; DMEM, Dulbecco modified Eagle medium; DMSO, dimethyl sulfoxide; ICP-MS, inductively coupled plasma mass spectroscopy; isambz, imine ligand, (E)-3-(((1H-benzo[d]imidazole-2-yl)methyl)imino)indolin-2-one; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; phen, 1,10-phenanthroline; ROS, reactive oxygen species.

Statements

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author AC declared that they were an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process and the final decision.

Funding

The author(s) declared that financial support was received for this work and/or its publication. The authors are grateful to Sao Paulo State Research Foundation (FAPESP, grant 2011/50318-1) and Network CEPID Redoxoma–Redox Processes in Biomedicine (FAPESP, grant 2013/07937-8) for financial support. AMLZ thanks Jayr H. Marin for his help with Raman spectroscopy studies and the Brazilian entity Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance code 001) for fellowships during her PhD studies. MSL thanks FAPESP for acquisition of the TQ ICP-MS instrument (Grant 2017/20752-8).

Data availability

The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author.

Ethics approval

Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used. Cells used in these studies, cervical HeLa (ATCC CCL-2), were purchased from the Rio de Janeiro Cell Bank (https://bcrj.org.br/).

Author contributions

AZ: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing. AA: Data curation, Investigation, Methodology, Writing – original draft, Writing – review and editing. RF: Data curation, Investigation, Methodology, Writing – original draft, Writing – review and editing. RP: Data curation, Methodology, Resources, Writing – original draft, Writing – review and editing. KT: Data curation, Investigation, Methodology, Writing – original draft. CO: Data curation, Methodology, Resources, Writing – original draft, Writing – review and editing. ML: Data curation, Investigation, Methodology, Writing – original draft. SM: Data curation, Methodology, Resources, Writing – original draft, Writing – review and editing. AC: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – original draft, Writing – review and editing.

References

  1. Acilan C., Cevatemre B., Adiguzel Z., Karakas D., Ulukaya E., Ribeiro N. (2017). Synthesis, biological characterization and evaluation of molecular mechanisms of novel copper complexes as anticancer agents. Biochim. Biophys. Acta 1861, 218 – 234. doi:10.1016/j.bbagen.2016.10.014
  2. Adhikari S., Nath P., Das A., Datta A., Baildya N., Duttaroy A. K. (2024a). A review on metal complexes and its anti-cancer activities: recent updates from in vivo studies. Biomed. Pharmacother. 171, 116211. doi:10.1016/j.biopha.2024.116211
  3. Adhikari S., Nath S., Kansız S., Balidya N., Paul A. K., Dege N. (2024b). Zinc(II) coordination compound with N′ -(pyridin-2-ylmethylene)nicotinohydrazide: synthesis, crystal structure, computational and cytotoxicity studies. J. Inorg. Biochem. 257, 112598. doi:10.1016/j.jinorgbio.2024.112598
  4. Aroua L. M., Alosaimi A. H., Alminderej F. M., Messaoudi S., Mohammed H. A., Almahmoud S. A. (2023). Synthesis, molecular docking, and bioactivity study of novel hybrid benzimidazole urea derivatives: a promising α-Amylase and α-Glucosidase inhibitor candidate with antioxidant activity. Pharmaceutics 15, 457. doi:10.3390/pharmaceutics15020457
  5. Aroua L. M., Alminderej F. M., Almuhaylan H. R., Alosaimi A. H., Medini F., Mohammed H. A. (2025). Benzimidazole(s): synthons, bioactive lead structures, total synthesis, and the profiling of major bioactive categories. RSC Adv. 15, 7571 – 7608. doi:10.1039/d4ra08864f
  6. Bai D., Nowak M., Lu D., Wang Q., Fitzgerald M., Zhang H. (2025). The outcast of medicine: metals in medicine--from traditional mineral medicine to metallodrugs. Front. Pharmacol. 16, 1542560. doi:10.3389/fphar.2025.1542560
  7. Bhattacharjeea T., Nath S., Baildya N., Das A., Pathak S., Molins E. (2024). Supramolecular assemblies of Zn(II) complex based on dithiolate-amine binary ligands: synthesis, crystal structure, Hirshfeld surface, DFT, molecular docking, and anticancer studies. Inorg. Chem. Commun. 167, 112762. doi:10.1016/j.inoche.2024.112762
  8. Brownlie J., Kulkarni S., Algethami M., Jeyapalan J. N., Mongan N. P., Rakha E. A. (2023). Targeting DNA damage repair precision medicine strategies in cancer. Curr. Op. Pharmacol. 70, 102381. doi:10.1016/j.coph.2023.102381
  9. Cavichioli M., Zaballa A. M. L., de Paula Q. A., Prieto M. B., Oliveira C. C., Civitareale P. (2019). Oxidative assets towards biomolecules and cytotoxicity of new Oxindolimine-Copper(II) and Zinc(II) complexes. Inorganics 7, 12. doi:10.3390/inorganics7020012
  10. Cerchiaro G., Da Costa Ferreira A. M. (2006). Oxindoles and copper complexes of oxindole-derivatives as potential pharmacological agents. J. Braz. Chem. Soc. 17, 1473 – 1485. doi:10.1590/s0103-50532006000800003
  11. Chander S., Tang C.-R., Penta A., Wang P., Bhagwat D. P., Vanthuyne N. (2018). Hit optimization studies of 3-hydroxy-indolin-2-one analogs as potential anti-HIV-1 agents. Bioorg. Chem. 79, 212 – 222. doi:10.1016/j.bioorg.2018.04.027
  12. Chowdhary S., Shalini A. A., Arora A., Kumar V. (2022). A Mini review on Isatin, an anticancer scaffold with potential activities against neglected tropical diseases (NTDs). Pharmaceuticals 15, 536. doi:10.3390/ph15050536
  13. de Paiva R. E. F., Vieira E. G., da Silva D. R., Wegermann C. A., da Costa Ferreira A. M. (2021). Anticancer compounds based on Isatin-Derivatives: strategies to ameliorate selectivity and efficiency. Front. Mol. Biosci. 7, 627272. doi:10.3389/fmolb.2020.627272
  14. Diaconu D., Antoci V., Mangalagiu V., Amariucai-Mantu D., Mangalagiu I. I. (2022). Quinoline–imidazole/benzimidazole derivatives as dual-/multi-targeting hybrids inhibitors with anticancer and antimicrobial activity. Sci. Rep. 12, 16988. doi:10.1038/s41598-022-21435-6
  15. Englinger B., Pirker C., Heffeter P., Terenzi A., Kowol C. R., Keppler B. K. (2019). Metal drugs and the anticancer immune response. Chem. Rev. 119, 1519 – 1624. doi:10.1021/acs.chemrev.8b00396
  16. Farrell N. P. (2015). Multi-platinum anti-cancer agents. Substitution-inert compounds for tumor selectivity and new targets. Chem. Soc. Rev. 44, 8773 – 8785. doi:10.1039/c5cs00201j
  17. Fernandez C. Y., Alvarez N., Rocha A., Mendes L. F. S., Costa-Filho A. J., Ellena J. (2024). Phenanthroline and phenyl carboxylate mixed ligand copper complexes in developing drugs to treat cancer. J. Inorg. Biochem. 260, 112700. doi:10.1016/j.jinorgbio.2024.112700
  18. Gehlen M. H. (2020). The centenary of the Stern-Volmer equation of fluorescence quenching: from the single line plot to the SV quenching map. J. Photochem. Photophys. C. Photochem. Rev. 42, 100338. doi:10.1016/j.jphotochemrev.2019.100338
  19. Hagen W. R. (2006). EPR spectroscopy as a probe of metal centres in biological systems. Dalton Trans., 4415 – 4434. doi:10.1039/b608163k
  20. Housman G., Byler S., Heerboth S., Lapinska K., Longacre M., Snyder N. (2014). Drug resistance in cancer: an overview. Cancers 6, 1769 – 1792. doi:10.3390/cancers6031769
  21. IARC (2025). Int. Agency Res. Cancer, Available online at: (Accessed April 21, 2025).
  22. Iglesias S., Alvarez N., Torre M. H., Kremer E., Ellena J., Ribeiro R. R. (2014). Synthesis, structural characterization and cytotoxic activity of ternary copper(II)–dipeptide–phenanthroline complexes. J. Inorg. Biochem. 139, 117 – 123. doi:10.1016/j.jinorgbio.2014.06.007
  23. Khetmalis Y. M., Shivani M., Murugesan S., Chandra Sekhar K. V. G. (2021). Oxindole and its derivatives: a review on recent progress in biological activities. Biomed. Pharmacother. 141, 111842. doi:10.1016/j.biopha.2021.111842
  24. Levina A., Crans D. C., Lay P. A. (2017). Speciation of metal drugs, supplements and toxins in media and bodily fluids controls in vitro activities. Coord. Chem. Rev. 352, 473 – 498. doi:10.1016/j.ccr.2017.01.002
  25. Li Y., Huang F., Stang P. J., Yin S. (2024). Supramolecular coordination complexes for synergistic cancer therapy. Acc. Chem. Res. 57, 1174 – 1187. doi:10.1021/acs.accounts.4c00031
  26. Lin G., Zhang M. (2023). Ligand chemistry in antitumor theranostic nanoparticles. Acc. Chem. Res. 56, 15781590. doi:10.1021/acs.accounts.3c00151
  27. Loganathan R., Ramakrishnan S., Suresh E., Riyasdeen A., Akbarsha M. A., Palaniandavar M. (2012). Mixed ligand Copper(II) complexes of N,N-Bis(benzimidazol-2-ylmethyl)amine (BBA) with diimine Co-Ligands: Efficient chemical nuclease and protease activities and cytotoxicity. Inorg. Chem. 51, 5512 – 5532. doi:10.1021/ic2017177
  28. Lucaciu R. L., Hangan A. C., Sevastre B., Oprean L. S. (2022). Metallo-drugs in cancer therapy: past, present and future. Molecules 27, 6485. doi:10.3390/molecules27196485
  29. Mamindla A., Murugan D., Varadhan M., Ajaykamal T., Rangasamy L., Palaniandavar M. (2024). Mixed-ligand copper(II)-diimine complexes of 3- formylchromone-N4 -phenyl thiosemicarbazone: 5,6-dmp co-ligand confers enhanced cytotoxicity. RSC Adv. 14, 31704 – 31722. doi:10.1039/d4ra04997g
  30. Marin J. H., Marmián-Lopez M. B., da Silva D. R., Temperini M. L. A., Da Costa Ferreira A. M. (2023). Dual-purpose nanoparticle assembly for identifying preferential intracellular targets of oxindolimine copper(II) complexes through SERS imaging. Front. Chem. Biol. 2, 1254702. doi:10.3389/fchbi.2023.1254702
  31. Marmián-Lopez M. B., Miguel R. B., Araki K., Temperini M. L. A., da Costa Ferreira A. M. (2021). Multivariate probing of antitumor metal-based complexes damage on living cells through Raman imaging. Spectrochim. Acta Part 244, 118838. doi:10.1016/j.saa.2020.118838
  32. Masuri S., Vanhara P., Cabiddu M. G., Moráň L., Havel J., Cadoni E. (2022). Copper(II) phenanthroline-based complexes as potential anticancer drugs: a walkthrough on the mechanisms of action. Molecules 27, 49. doi:10.3390/molecules27010049
  33. Moráň L., Pivetta T., Masuri S., Vašíčková K., Walter F., Prehn J. (2019). Mixed copper(II)–phenanthroline complexes induce cell death of ovarian cancer cells by evoking the unfolded protein response. Metallomics 11, 1481 – 1489. doi:10.1039/c9mt00055k
  34. Natha S., Deb V. K., Baildya N., Roy S., Kaminsky W., Kumar Verma A. (2025). Synthesis, structural analysis, computational investigation, and cytotoxicity studies of new copper(II) coordination polymers. J. Mol. Struct. 1348, 143124. doi:10.1016/j.molstruc.2025.143124
  35. Nunes P., Correia I., Marques F., Matos A. P., Dos Santos M. M. C., Azevedo C. G. (2020). Copper complexes with 1,10 -Phenanthroline derivatives: underlying factors affecting their cytotoxicity. Inorg. Chem. 59, 9116 – 9134. doi:10.1021/acs.inorgchem.0c00925
  36. Pellei M., Del Bello F., Porchia M., Santini C. (2021). Zinc coordination complexes as anticancer agents. Coord. Chem. Rev. 445, 214088. doi:10.1016/j.ccr.2021.214088
  37. Peña Q., Lorenzo J., Sciortino G., Rodríguez-Calado S., Maréchal J. D., Bayón P. (2019). Studying the reactivity of “old” Cu(II) complexes for “novel” anticancer purposes. J. Inorg. Biochem. 195, 51 – 60. doi:10.1016/j.jinorgbio.2019.03.011
  38. Plackett B. (2025). Cancer – a numbers game. Nature 640, S58 – S59. doi:10.1038/d41586-025-01152-6
  39. Portes M. C., Ribeiro G. A., Sabino G. L., de Couto R. A. A., Vieira L. Q., Alves M. J. M. (2023). Antiparasitic activity of Oxindolimine–Metal complexes against chagas disease. Inorganics 11, 420. doi:10.3390/inorganics11110420
  40. Qi Y.-Y., Gan Q., Liu Y.-X., Xiong Y.-H., Mao Z.-W., Le X.-Y. (2018). Two new Cu(II) dipeptide complexes based on 5-methyl-2-(2’-pyridyl) benzimidazole as potential antimicrobial and anticancer drugs: special exploration of their possible anticancer mechanism. Eur. J. Med. Chem. 54, 220 – 232. doi:10.1016/j.ejmech.2018.05.023
  41. Rogalewicz B., Czylkowska A. (2025). Recent advances in the discovery of copper(II) complexes as potential anticancer drugs. Eur. J. Med. Chem. 292, 117702. doi:10.1016/j.ejmech.2025.117702
  42. Roy I., Mathur S., Deb S., Rathnam S. S. V., Tuti N., Shaji U. P. (2005). Benzimidazole-based mononuclear polypyridyl Cu(II) complexes: DNA binding, cleavage, and in vitro antiproliferative studies. Dalton Trans. 54, 6386 – 6401. doi:10.1039/d4dt03379e
  43. Sadeer N. B., Varghese R., Ramamoorthy S., Zengin G. (2020). Coordination and bioinorganic chemistry at the heart of metallodrugs: knowledge gaps and future directions. Inorg. Chim. Acta 572, 122294. doi:10.1016/j.ica.2024.122294
  44. Sakaguchi U., Addison A. W. (1979). Spectroscopic and redox studies of some copper(II) complexes with biomimetic donor atoms: implications for protein copper centres. J. Chem. Soc. Dalton Trans., 600 – 608. doi:10.1039/dt9790000600
  45. Santini C., Pellei M., Gandin V., Porchia M., Tisato F., Marzano C. (2014). Advances in copper complexes as anticancer agents. Chem. Rev. 114 (1), 815 – 862. doi:10.1021/cr400135x
  46. Serment-Guerrero J., Bravo-Gomez M. H., Lara-Rivera E., Ruiz-Azuara L. (2017). Genotoxic assessment of the copper chelated compounds Casiopeinas: clues about their mechanisms of action. J. Inorg. Biochem. 166, 68 – 75. doi:10.1016/j.jinorgbio.2016.11.007
  47. Shin J., Magar B. S., Lee J., sun Kim K., Lee Y. R. (2019). Design, synthesis, and discovery of novel oxindoles bearing 3-heterocycles as species-specific and combinatorial agents in eradicating Staphylococcus species. Sci. Rep. 9, 1 – 18. doi:10.1038/s41598-019-44304-1
  48. Sigman D. S., Bruice T. W., Mazumder A., Sutton C. L. (1993). Targeted chemical nucleases. Acc. Chem. Res. 26, 98 – 104. doi:10.1021/ar00027a004
  49. Vaidya F. U., Chhipa A. S., Mishra V., Gupta V. K., Rawat S. G., Kumar A. (2020). Molecular and cellular paradigms of multidrug resistance in cancer. Cancer Rep., e1291. doi:10.1002/cnr2.1291
  50. Velavalapalli V. M., Kumar G. S., Katari N. K., Gundl R. (2024). Development and evaluation of new oxindole derivatives as potential anti-tubercular agents. J. Mol. Struct. 1304, 137649. doi:10.1016/j.molstruc.2024.137649
  51. Vieira A. P., Wegermann C. A., Da Costa Ferreira A. M. (2018). Comparative studies of Schiff base-copper(II) and zinc(II) complexes regarding their DNA binding ability and cytotoxicity against sarcoma cells. New J. Chem. 42, 13169 – 13179. doi:10.1039/c7nj04799a
  52. Vieira E. G., Miguel R. B., da Silva D. R., Fazzi R. B., de Couto R. A. A., Marin J. H. (2019). New J. Chem. 43, 386 – 398. doi:10.1039/c8nj04654a
  53. Wang X., Wang X., Jin S., Muhammad N., Guo Z. (2019). Stimuli-Responsive therapeutic metallodrugs. Chem. Rev. 119, 1138 – 1192. doi:10.1021/acs.chemrev.8b00209
  54. Wang Y., Tang T., Yuan Y., Wang X., Guan J. (2024). Copper and copper complexes in tumor therapy. ChemMedChem 19, e202400060. doi:10.1002/cmdc.202400060
  55. Zeng Z., Cai J., Li F., Weng Y., Huang Q., Yang H. (2021). Synthesis, crystal structures, DNA interactions, and antitumor activity of two new dinuclear copper(II) complexes with thiazole ligand. RSC Adv. 11, 40040 – 40050. doi:10.1039/D1RA05798G
  56. Zheng P. Y., Eskandari A., Lu C. X., Laws K., Aldous L., Suntharalingam K. (2019). Biophysical analysis of cancer stem cell-potent copper(II) coordination complexes. Dalton Trans. 48, 5892 – 5896. doi:10.1039/c8dt04706e
  57. Zou J. X., Chang M. R., Kuznetsov N. A., Kee J. X., Babak M. V., Ang W. H. (2025). Metal-based immunogenic cell death inducers for cancer immunotherapy. Chem. Sci. 16, 6160 – 6187. doi:10.1039/d4sc08495k

Citation

Zaballa, A. M. L., et al. (2026). Comparative reactivity studies of imine-copper(II) and zinc(II) complexes containing both oxindole and benzimidazole scaffolds for biomolecules and tumor cells. Frontiers in Chemical Biology, Volume 5. https://doi.org/
Received
Accepted
Published
Volume
5 - 2026

Copyright

Copyright © 2026 Zaballa, Araujo de Oliveira, Fazzi, de Paiva, Tsantarlis, Oliveira, Luz, Miyamoto and da Costa Ferreira.

License: https://creativecommons.org/licenses/by/4.0/

*Correspondence: Ana M. da Costa Ferreira

Article metrics

Views
0
Downloads
0
Citations
0
Shares
0

Related articles

Original ResearchNanoprobe XRF analysis reveals the intracellular distribution of organometallic rhenium complexes with different directing ligandC. Rumancev, S. K. Vellas, A. Gräfenstein, D. Obitz, J. Garrevoet, G. Falkenberg, N. Metzler-Nolte, A. RosenhahnPerspectiveAdvances in enhanced sampling simulations of G-protein-coupled receptorsKeya Joshi, Victor Ayo Adediwura, Yinglong MiaoOriginal ResearchA bifunctional carborane-quinazoline combines tumor growth inhibition and boron delivery for boron neutron capture therapy in melanoma and non-small cell lung cancer modelsMaría Alejandra Dagrosa, Marina Carpano, Antonella Pastini, Estanislao Pavlik, Andrés Bechis, María Silvina Olivera, Pablo Vallecorsa, Belén Dávila, Luciano Parodi, Esther Yardena Salem, María Sol Espain, Agustina Mariana Portu, Laura Todaro, Alejandro Urtreger, Pablo Menendez, Hugo Cerecetto, Marcos Couto