Supplementary material:
Water-soluble platinum phtalocyanines as potential antitumor agents
By:
Giuseppina Bologna, Paola Lanuti, Primiano D’Ambrosio, Lucia Tonucci, Laura Pierdomenico, Carlo D’Emilio, Nicola Celli, Marco Marchisio, Nicola d’Alessandro,Eugenio Santavenere, Mario Bressan and Sebastiano Miscia
Supplementary figure 1: Schematic synthetic procedure of -tetrapentylcarboxylated ruthenium (II) phthalocyanine.
Synthetic procedure for -tetrapentylcarboxylated ruthenium (II) phthalocyanine
Heck synthesis of 4-(3,4-phenyldicarbonitrile)-4-pentenoic acid benzyl ester
To a 100 mL reaction flask containing 25 mL of dimethylformamide were added 2.0 g of 4-iodo-1,2-benzenedicarbonitrile (Strem; 7.9mmol), 2.8 g of potassium carbonate (20 mmol), 2.4 g of tetrabutylammonium bromide (8 mmol), 2.8 g of 4-pentenoicacid benzyl ester (15 mmol) and90 mg of palladium acetate (0.4 mmol); the reaction mixture was then magnetically stirred for 6 hr at 60° C. After cooling down to room temperature, the raw mixture was dissolved in 100 mL of dichloromethane and filtrated on a thin silica dish. The obtained clear solution was treated in a separatory funnel with, in sequence, 0.1 M of HCl, water, a saturated solution of NaHCO3 and finally again with water. The final dichloromethane solution was then dried the addition of anhydrous sodium sulphate for one night. After filtration and evaporation of the solvent, the residue was purified by column chromatography,packed with silica gel (60 Å, 230-400 mesh; Sigma-Aldrich)and used hexane/ethyl acetate as eluent (from 9:1 to 1:1). We obtained 2.250 g of4-(3,4-phenyldicarbonitrile)-4-pentenoic acid benzyl ester(90% of yield, referred to the initial 4-iododicarbonitrile).
Synthesis of 4-(3,4-phenyldicarbonitrile)-pentanoic acid
To a 100 mL of THF solution containing 2.054 g (6.5 mmol) of the above synthesized alkenyldicarbonitrilederivative, was added200 mg di Pd/C (Aldrich; 10%) and the obtained suspension was bubbled, by a syringe needle, by a moderate flux of H2 (about 100 bubbles per min).Periodic checks of the reaction were performed by thin layer chromatography on silica using hexane/ethyl acetate 9:1. When the alkenylreagent was totally converted to its respective saturated and debenzylated derivative (after about 72 hr), the reaction mixture was filtered on paper filter to remove the catalysts and, after removing the solvent,the residue was purified by column chromatography,packed with silica gel (60 Å, 230-400 mesh; Sigma-Aldrich) and using hexane/ethyl acetate as eluent (from 9:1 to 1:1). We obtained 1.375 g of 4-(3,4-phenyldicarbonitrile)-pentanoic acid (97% of yield, referred to the initial alkenylcompound).
Supplementary figure 2:1H NMR spectrum of 4-(3,4-phenyldicarbonitrile)-pentanoic acid in CDCl3
Synthesis of -tetrapentylcarboxylated ruthenium (II) phthalocyanine
A 50 mL round flask equipped with a refrigerator and a magnetic stirrer, was filled with 5 mL of pentanol, 600 mg of 4-(3,4-phenyldicarbonitrile)-pentanoic acid (2.64mmol), 100 mg of RuCl3·xH2O and a catalytic amount of 1,8-diazabiciclo[5.4.0]undec-7-ene (DBU). The reaction mixture was heated at 200 °C through an oil batch for 48 hr. After cooling at room temperature, the solvent was removed and the resulting residue was resuspended in 50 mL of saturated solution leading to the whole mixture to reflux for a couple of hours. Always keeping the reflux, the pH of the media was made strongly alkaline by addition of solid NaOH(4 g) and the resulting mixture was refluxed for 46 h. After cooling to room temperature, the mixture was transferred to a flask, leading to the entire volume to 200 mL with deionized water. The precipitation of phthalocyanine (dark-blue) was induced by addition of concentrated HCl, until the pH becamestrongly acidic. To better purify the phthalocyanine, an additional sequence of solubilization with NaOH and re-precipitation with HClwas repeated. At the end, we obtained 55 mg of -tetrapentylcarboxylated ruthenium (II) phthalocyanine(8 % of yield, referred to the initial dinitrile).The phthalocyaninewas characterized by UV-vis and MALDI-TOF spectra.
Supplementary figure 3:Uv-vis spectrum of -tetrapentylcarboxylated ruthenium (II) phthalocyaninein pentanol (max = 634 nm)
Supplementary figure 4:MALDI-TOF spectrum of -tetrapentylcarboxylated ruthenium (II) phthalocyanine. Inset: isotopic pattern of molecular ionat m/z 1014.
SupplementaryFigure 5. Impairment of cell cycle profiles induced by cis-platinum.Flow cytometry analysis of cell cycle profiles were performed on MDA-MB-231 and MCF10A cell lines, following the DNA staining by PI. Representative DNA distributions (histograms) and representative brightfield (Ch1) and PI (Ch4) 40X images, obtained by image flow cytometry (ImageStream) are shown. Data are representative of four separate experiments performed in duplicate.