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

Abstract

&These two authors contributed equally to this work. ABSTRACTA 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 [(η 6 -p-cym)RuCl(PPh 3 ) 2 ] + , allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N'-di-Boc-N"-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semi-preparative HPLC and characterized by ESI and MALDI-TOF MS and NMR spectroscopy. The cytotoxicity of the compounds was tested in 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, neomycin-ruthenium conjugate (2) displayed moderate anti-proliferative activity in both cancer cell lines (IC 50 ≈ 80 µM), whereas that of the neamine conjugate (4) was inactive (IC 50 ≈ 200 µM).However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC 50 = 7.17 µM in DU-145 and IC 50 = 11.33 µM in MCF-7). Although the same ranking in anti-proliferative activity was found in the non-tumorigenic cell line (3 >> 2 > 4), IC 50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g. IC 50 = 49.4 µM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC 50 = 12.75 µM for 3) 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 2 and 3 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 anti-proliferative activity of 3.Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular 3 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.

Metal-based glycoconjugates and their potential in targeted anticancer chemotherapy

“…neomycin, paromomycin, kanamycin A) have shown high selectivity and affinity toward RNA structures and proved effective in modulating RNA functions [136,137]. In the attempt to combine the potential RNA-targeting properties of aminoglycosides with the anticancer activity of ruthenium(II)-arene scaffolds, guanidinylated neomycin-ruthenium conjugates were prepared [138]. In particular, compound Ru-6 showed significant anticancer activity in vitro toward human MCF7 breast adenocarcinoma and DU145 prostate carcinoma cells (IC 50 = 11.33 and 7.17 µM, respectively), and increased cellular uptake upon guanidylation (compared to the non-guanidinylated metal analogue).…”

Covalent and non-covalent binding of metal complexes to RNA

“…In the sections below some recent advances in non-covalent binding of metal complexes to RNA are described and discussed. In particular, the following aspects are covered: (i) the use of metal complexes as RNA structural probes [40,107,108] (Section 4.1); (ii) the structure of a metal complex interacting with an RNA three-way junction [41] (Section 4.2); (iii) the development of metal complexes targeting viral RNA [109][110][111][112] (Section 4.3); (iv) the combination of metal complexes with small RNA-binding organic molecules (Section 4.4) [113][114][115][116][117]; (v) the use of RNA-targeting metal complexes in cell imaging [42,43,[118][119][120][121] (Section 4.5); (vi) comparison between DNA and RNA binding properties of some metal complexes [122][123][124][125] (Section 4.6); (vii) the RNA binding ability of some metal surfactants and metal polymers [126][127][128][129] (Section 4.7) and (viii) the binding of metal complexes to RNA triplexes [46,[130][131][132][133] (Section 4.8).…”

Synthesis of Janus Compounds for the Recognition of G-U Mismatched Nucleobase Pairs

“…As shown in Scheme 3, the guanidinylated ligands (24)(25)(26)(27) were prepared by direct guanidinylation of their precursors (20)(21)(22)(23) following previously reported procedures. n=2 JanusB-Nea2 (23) JanusB-Nea2G4 (27) Scheme 3.…”

“…Our next objective was to study the interaction of Janus-Neamine ligands (20)(21)(22)(23) and their guanidinylated derivatives, Janus-Guanidinoneamine (24)(25)(26)(27), with Tau RNA, and in particular to evaluate their ability to stabilize Tau RNA. Besides wt we choose two of the mutated sequences associated with the development of tauopathies, +3 and +16.…”

“…These results prompted us to evaluate the ability of Janus-Guanidinoneamine ligands (24)(25)(26)(27) to stabilize the +3 mutated sequence. As shown in Table 2, guanidinylation of the aminoglycoside moiety in Janus-Neamine ligands had always a positive effect on the thermal stability of the RNA upon complexation.…”