Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η⁶-<i>p</i>-cym)­RuCl­(PPh₃)₂]⁺, allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with <i>N</i>,<i>N</i>′-di-Boc-<i>N</i>″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption–ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin–ruthenium conjugate (<b>2</b>) displayed moderate antiproliferative activity in both cancer cell lines (IC₅₀ ≈ 80 μM), whereas the neamine conjugate (<b>4</b>) was inactive (IC₅₀ ≈ 200 μM). However, the guanidinylated analogue of the neomycin–ruthenium conjugate (<b>3</b>) required much lower concentrations than the parent conjugate for equal effect (IC₅₀ = 7.17 μM in DU-145 and IC₅₀ = 11.33 μM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (<b>3</b> ≫ <b>2</b> > <b>4</b>), IC₅₀ values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC₅₀ = 49.4 μM for <b>2</b>) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC₅₀ = 12.75 μM for <b>3</b>) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates <b>2</b> and <b>3</b> revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of <b>3</b>. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin–ruthenium conjugate (<b>3</b>) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin–ruthenium conjugate (<b>2</b>). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium­(II) arene derivatives to guanidinoglycosides

Somatostatin Subtype‑2 Receptor-Targeted Metal-Based Anticancer Complexes

Conjugates of a dicarba analogue of octreotide, a potent somatostatin agonist whose receptors are overexpressed on tumor cells, with [PtCl₂(dap)] (dap = 1-(carboxylic acid)-1,2-diaminoethane) (<b>3</b>), [(η⁶-bip)­Os­(4-CO₂-pico)­Cl] (bip = biphenyl, pico = picolinate) (<b>4</b>), [(η⁶-<i>p</i>-cym)­RuCl­(dap)]⁺ (<i>p</i>-cym = <i>p</i>-cymene) (<b>5</b>), and [(η⁶-<i>p</i>-cym)­RuCl­(imidazole-CO₂H)­(PPh₃)]⁺ (<b>6</b>), were synthesized by using a solid-phase approach. Conjugates <b>3</b>–<b>5</b> readily underwent hydrolysis and DNA binding, whereas conjugate <b>6</b> was inert to ligand substitution. NMR spectroscopy and molecular dynamics calculations showed that conjugate formation does not perturb the overall peptide structure. Only <b>6</b> exhibited antiproliferative activity in human tumor cells (IC₅₀ = 63 ± 2 μM in MCF-7 cells and IC₅₀ = 26 ± 3 μM in DU-145 cells) with active participation of somatostatin receptors in cellular uptake. Similar cytotoxic activity was found in a normal cell line (IC₅₀ = 45 ± 2.6 μM in CHO cells), which can be attributed to a similar level of expression of somatostatin subtype-2 receptor. These studies provide new insights into the effect of receptor-binding peptide conjugation on the activity of metal-based anticancer drugs, and demonstrate the potential of such hybrid compounds to target tumor cells specifically