Synthesis and Antiproliferative Insights of Lipophilic Ru(II)-Hydroxy Stearic Acid Hybrid Species

Abstract

Metallodrugs represent a combination of multifunctionalities that are present concomitantly and can act differently on diverse biotargets. Their efficacy is often related to the lipophilic features exhibited both by long carbo-chains and the phosphine ligands. Three Ru(II) complexes containing hydroxy stearic acids (HSAs) were successfully synthesized in order to evaluate possible synergistic effects between the known antitumor activity of HSA bio-ligands and the metal center. HSAs were reacted with [Ru(H)₂CO(PPh₃)₃] selectively affording O,O-carboxy bidentate complexes. The organometallic species were fully characterized spectroscopically using ESI-MS, IR, UV-Vis, and NMR techniques. The structure of the compound Ru-12-HSA was also determined using single crystal X-ray diffraction. The biological potency of ruthenium complexes (Ru-7-HSA, Ru-9-HSA, and Ru-12-HSA) was studied on human primary cell lines (HT29, HeLa, and IGROV1). To obtain detailed information about anticancer properties, tests for cytotoxicity, cell proliferation, and DNA damage were performed. The results demonstrate that the new ruthenium complexes, Ru-7-HSA and Ru-9-HSA, possess biological activity. Furthermore, we observed that the Ru-9-HSA complex shows increased antitumor activity on colon cancer cells, HT29.

Keywords: DNA damage; Ru(II); alkyl chain; anticancer; hydroxy stearic acid; lipophilicity.

Figure 1

General structures of hydroxy stearic acids (HSAs).

Scheme 1

Synthesis of complexes 2–4. (i) PPh₃, EtOH, KOH, reflux, o/n (ii) HSA, 1,2-DME, reflux, 3–4 h.

Figure 2

Molecular structure of 4.

Figure 3

Arbitrary view of the crystal packing of 4. For the sake of clarity only the hydrogens engaged in H bonding (light blue dotted lines) are reported.

Figure 4

The percentage cell viability of human malignant and non-malignant cells analyzed with an MTT viability assay. (A) The cells were treated for 48 h with a concentration range of 0.25–10 μM with compounds 2, 3, and 4 dissolved in DMSO (vehicle control). (B) Compound 1 or CDDP (positive control) at the same concentrations was also included. Error bars are standard deviations. Significant differences are indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 5

Flow cytometric analysis of control and treated cells with 10 µM of 3. (A) (Left) HT29, HeLa, and IGROV1 were treated with 10 μM of complex 3 for 24 h and then were analyzed using a flow cytometer. (Right) The percentage of cells in the different cell cycle phases was calculated at 24 h. (B) (Left) HT29, HeLa, and IGROV1 were treated with 10 μM of complex 3 for 48 h and then were analyzed using a flow cytometer. (Right) The percentage of cells in the different cell cycle phases was calculated at 48 h. Error bars are standard deviations. Significant differences are indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 6

(Above left) Representative Western blot image of γH2AX in HT29 cells treated with 3 (10 µM) for 6 h or exposed to UV for 3 min. (Above right) Representative image of acetylation status of histones H2/H3 and H4. (Below) Relative quantification of γH2AX and histone acetylation. Arbitrary densitometry units (A.U.) were normalized by H1 histone. All data represent mean ± SD (n = 3). *** p < 0.001, compared with controls.