Lucia M. Balsa

Introduction: Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults, with poor prognosis due to relapse, metastasis, and chemoresistance. The search for novel metal-based therapeutics has highlighted copper complexes as promising candidates. Here, we report the in vitro and in vivo antitumor activity of a tetranuclear Cu(II)-hydrazone complex (Cu₄L₄) derived from (E)-5-chloro-N′-(2-hydroxy-3-methoxybenzylidene)thiophene-2-carbohydrazide. Results: Cytotoxic assays on MG-63 OS cells revealed potent activity with an IC₅₀ of 0.50 ± 0.04 µM, significantly surpassing its free ligand (IC₅₀ = 13.9 ± 1.6 µM) and cisplatin (IC₅₀ = 39.0 ± 1.8 µM). This tetranuclear complex outperforms mononuclear Cu-hydrazones analogs (e.g., 4-fold vs. CuHL1, 2-fold vs. CuHL2, 5-fold vs. CuHL3, 17-fold vs. CuHL4,), and Cu₄L₄ also exhibits reduced clonogenic survival, induces reactive oxygen species production, and promotes late apoptosis as a main mechanism, being the main mechanism of action involved in anticancer activity. In multicellular tumor spheroids, the complex maintained strong cytotoxicity (IC₅₀ = 4.11 ± 0.12 µM), impaired spheroid integrity, and markedly inhibited cell migration at sub-IC₅₀ concentrations. The tetranuclear architecture confers markedly enhanced antitumor activity relative to the corresponding mononuclear Cu–hydrazone complexes (e.g., 2-fold vs. CuHL1, 4-fold vs. CuHL2, 2-fold vs. CuHL3). In a xenograft model, sustained administration of Cu₄L₄ (2 mg/kg, i.p., twice weekly) inhibited tumor growth by 43.6%, reduced mitotic index, and increased necrotic area without significant systemic toxicity. Conclusions: Overall, Cu₄L₄ displayed potent and selective antitumor activity against OS cells in 2D, 3D, and in vivo models, underscoring copper–hydrazone complexes as promising scaffolds for the development of new therapies against OS.

We report the synthesis, characterization and anticancer activity of a new Schiff base (H₂L) derived from the condensation of pyridoxamine with pyridoxal and its novel copper(II) and oxidovanadium(IV) complexes: [Cu(HL)Cl] (1), [Cu(LH₂)(phen)]Cl₂ (2), [Cu(LH₂)(amphen)]Cl₂ (3), [V^IVO(HL)Cl] (4), and [V^IVO(LH₂)(phen)]Cl₂ (5), where phen is 1,10-phenanthroline and amphen is its 5-amino derivative. All compounds were characterized by analytical and spectroscopic techniques, namely FTIR, UV-vis and EPR spectroscopy. Their stability in aqueous media was evaluated, revealing that the presence of the phen co-ligand significantly increases the stability. The ternary Cu(II) complexes (2 and 3) impaired cell viability of osteosarcoma cells (MG-63) (IC₅₀ values of 3.6 ± 0.6 and 7 ± 1.9 μM for 2 and 3), while 1 and the V^IVO complexes did not show relevant anticancer activity. Complexes 2 and 3 are also more active than cisplatin (CDDP). Synergistic studies between 2 and sorafenib showed significant synergism on MG-63 cells for the following combinations: 2 (2.0 μM) + sorafenib (10.0 μM) and 2 (2.5 μM) + sorafenib (12.5 μM), whilst the combination of 2 and CDDP did not show synergy. Complex 2 interacts with DNA, inducing significant genotoxic effects on MG-63 cells from 1.0 to 2.5 μM and it increases the ROS levels 880% over basal. Moreover, 2 induces apoptosis at 1.0 and 2.0 μM, while its combination with sorafenib induces apoptosis and necrosis. Finally, compound 2 reduces the cell viability of MG-63 spheroids showing an IC₅₀ value 7-fold lower than that of CDDP (8.5 ± 0.4 μM vs. 65 ± 6 μM). The combination of 2 and sorafenib also showed synergism on spheroids, suggesting that the combination of these drugs improves the anticancer effect against bone cancer cells.

Thiosemicarbazone moiety is a valuable scaffold for the synthesis of metallic complexes with anticancer purposes, when bearing N‐heterocyclic the final compounds possess diverse biological activities. Using thiazole as bioisosteres, the resulting thiosemicarbazones can afford synthetic drugs with a variety of pharmacological effects. Trying to elucidate, if metal complexes from α‐N‐heterocyclic thiosemicarbazones can achieve more selectivity versus special tumor lines, we have developed new metal complexes with 1H imidazole‐4‐carboxaldehyde 4 N‐p‐tolyl‐ and 4 N‐phenylthiosemicarbazone (HL¹and HL²respectively). The solution studies of these ligands showed a tautomeric equilibria in solution and their reaction with Li₂PdCl₄proved the stability of both forms affording two mononuclear Pd(II) complexes. Both tautomeric forms are clearly coordinated to palladium center acting as two different bidentate ligands. Palladium complexes’ stability in biological buffers was investigated to stablish the optimal conditions for the evaluation of cytotoxicity, that on the triple negative adenocarcinoma cell line showed IC₅₀values in the low micromolar range. Complexes were also studied with CT DNA (UV‐Visible spectroscopy and viscosity) and with the pBR322 plasmid supercoiled models, indicating non covalent interaction.

From the interaction of the vanadyl ion with a ligand (HL), obtained by the condensation reaction of o-vanillin and 2-thiophenemethylamine, an oxidovanadium(IV) complex (VOL₂) was obtained. It was characterized by spectroscopic techniques, including solid state FTIR, Raman, EPR and diffuse reflectance, and UV-vis and EPR, in solution. Its thermal behavior was analyzed by means of TGA and DTA. Theoretical studies based on DFT computational methods aided in the interpretation and assignment of spectroscopic data. Cytotoxicity assays (48 h) against bone cancer cells (MG-63, IC₅₀ = 50.4 ± 8.7), breast cancer cells (MCF-7, IC₅₀ = 42.3 ± 4.7), (MDA-MB-231, IC₅₀ = 29.0 ± 1.7) and a normal fibroblast (L929, IC₅₀ = 71.0 ± 3.5) demonstrated the enhancement of the effectiveness and selectivity of the complex as compared with both the ligand and the free metal ion. Besides, the compound inhibits cell migration, increases ROS levels and facilitates apoptosis. On a whole, these results show the main mechanisms of the deleterious effects of [VOL₂] in a triple negative breast cancer cell line (MDA-MB-231), demonstrating that this complex is a promising therapeutic agent for this kind of breast cancer.

New ML₂ complexes were extensively characterized showing stability in solution and promising anti-tumoral results, particularly for CuL2.

Two Pd(II) and Pt(II) complexes with two pyrrol-2-carbaldehyde N-p-chlorophenylthiosemicarbazone ligands are designed and characterized showing mononuclear structures. An important pharmacological property for both compounds is the high selectivity for tumor cells and a lack of activity in healthy cells. The Pd(II) compound shows a higher antitumor activity and selectivity than the Pt(II) compound. Both complexes present a variety of biological interactions: with DNA models (pBR322 and CT DNA), proteins (lysozyme and RNase) and other biological targets like proteosome. Our results show that the Pd(II) complex is a more interesting candidate for potential anticancer therapies than the Pt(II) complex, and we provide new insight into the design and synthesis of palladium compounds as potential antitumor agents.