Quinoline‐Conjugated Ruthenacarboranes: Toward Hybrid Drugs with a Dual Mode of Action

Abstract The role of autophagy in cancer is often complex, ranging from tumor‐promoting to ‐suppressing effects. In this study, two novel hybrid molecules were designed, containing a ruthenacarborane fragment conjugated with a known modulator of autophagy, namely a quinoline derivative. The complex closo‐[3‐(η 6‐p‐cymene)‐1‐(quinolin‐8‐yl‐acetate)‐3,1,2‐RuC2B9H10] (4) showed a dual mode of action against the LN229 (human glioblastoma) cell line, where it inhibited tumor‐promoting autophagy, and strongly inhibited cell proliferation, de facto blocking cellular division. These results, together with the tendency to spontaneously form nanoparticles in aqueous solution, make complex 4 a very promising drug candidate for further studies in vivo, for the treatment of autophagy‐prone glioblastomas.


Notes on Materials Storage and Purification
Dulbecco`s phosphate-buffered saline (PBS, 10 mM) was purchased from Biowest (w/o Mg and w/o Ca ions; Product Code: L0615), or freshly prepared in our laboratories according to the technical specification sheet from Dulbecco ® , and stored at room temperature. PBS used for UV-vis experiments was degassed and stored under nitrogen. Magnesium-and fatty acids-free bovine serum albumin (BSA) was purchased from SERVA electrophoresis GmbH, and stored at -20 °C. Thallium(I) ethanolate (Alfa Aesar©) was stored under argon at -20 °C, protected from light. Due to the high instability towards hydrolysis, this compound was used no longer than a month from purchase. Dried and degassed dichloromethane (CH 2 Cl 2 ) and n-hexane were obtained from an MBRAUN solvent purification system (MB SPS-800) and stored under nitrogen over molecular sieves (4 Å). Tetrahydrofuran (THF) was dried over Na/benzophenone, freshly distilled prior to use and stored under nitrogen over molecular sieves (4 Å). Pyridine and dimethylsulfoxide (DMSO) were degassed with nitrogen, dried over CaH 2 , freshly distilled prior to use and stored under nitrogen over molecular sieves (4 Å). Ethanol was degassed with nitrogen, dried over magnesium, freshly distilled prior to use and stored under nitrogen atmosphere over molecular sieve (4 Å), for the synthesis of 2. Alternatively, ethanol was distilled, and the EtOH/H 2 O 3:2 (v/v) mixture for the synthesis of 7, was degassed and stored under argon. BSA stock solutions in PBS were stored at 4 °C in the dark.
Staining Protocols for Flow Cytometric Analysis. For AnnV/PI, AO and ApoStat staining, cells were treated with an IC 50 dose of 3 or 4 for 72 h, then detached with trypsin and washed with PBS. In the case of Ann/PI double staining and Apostat, cells were stained according to the manufacturer's protocols. Cells were incubated with AnnV and PI in Ann-binding buffer (15 µg mL -1 ; 15 min, room temperature)., or ApoStat (ZVAD-FMK-FITC, 1 µL) in PBS (30 min, 37 °C). For detection of acidic vacuoles, upon treatment cells were stained with AO dye (1 μg mL −1 ; 15 min, 37 °C), in PBS. Finally, cells were washed in PBS, resuspended in PBS (or AnnV-binding buffer for AnnV/PI), and analysed. For CFSE staining, detached cells were first stained with a PBS solution of CFSE (1 μM; 10 min, 37 °C), then washed, seeded and then exposed to an IC 50 dose of 3 or 4 for 72 h. Cells were then washed, trypsinised, dissolved in PBS and analysed by CyFlow® Space Partec using the PartecFloMax® software (Partec GmbH, Münster, Germany).

Staining Protocols for Fluorescence Microscopy.
To evaluate the presence of apoptosis, cells were seeded in 8-chamber slides at 1.5 × 10 4 cells/well and treated with an IC 50 dose of 3 and 4 for 72 h. At the end of the incubation period, cells were fixed with 4% (w/v) paraformaldehyde for 15 min at room temperature, washed and covered with DAPI fluoromounth-G (Southern Biotech, Birmingham, AL, USA), before analysis. Slides were analysed with Zeiss AxioObserver Z1 inverted fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany) at 400× magnification. Morphological signs of apoptosis (irregular nuclei shape, condensed chromatin, apoptotic bodies) were examined by three independent observers.
Wound Healing Assay. For evaluation of the cells' motility, wound healing assay, in which cells migrate bidirectionally from the edges of a scratch wound, was used. Cells were seeded in 6-well plates, till reaching the confluence, when scratch over cellular layer was drawn with a sterile pipette tip across the centre of the wells to make a clean wound area. Cells were washed twice with fresh PBS to remove loose or damaged cells, and then exposed to an IC 50 dose of 3 and 4 for 72 h. At the end of the incubation period, cells were washed, fixed with 4% (w/v) paraformaldehyde for 10 min and stained with 2% CV solution for 1 min at room temperature. The wounds were digitally photographed using a Moticam 2000 camera and Nikon Digital Sight Fi2 camera attached to a Nikon SMZ800 stereo zoom microscope.
Western Blot Analysis. Cells were incubated with an IC 50 dose of 3 or 4 for 48 and 72 h and lysed in protein lysis buffer (62.5 mM Tris-HCl (pH 6.8 at 25 •C), 2% (w/v) SDS, 10% glycerol, 50 mM dithiothreitol). Electrophoretical separation was done on 15% SDS-polyacrylamide gels. As a protein molecular weight marker, PageRuler prestained ladder (Thermo Scientific, USA) was used. Electrotransfer to polyvinylidenedifluoride membranes at 5 mA cm -2 was done with a semidry blotting system (Fastblot B43; BioRad, Göttingen, Germany). Membranes were blocked with 5% (w/v) BSA in PBS for 1 h at room temperature. Incubation with specific antibodies for the microtubule-associated protein light chain 3B (LC3B) (Sigma Aldrich, St. Louis, MO, USA) or β-actin (Abcam, Cambridge, UK) was done overnight at 4 °C. As a secondary antibody, goat anti-rabbit IgG-horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA) was used. Bands were detected using a chemiluminescence detection system (ECL; GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK), while the protein levels were determined by densitometry, using ImageJ software (NIH, Bethesda, MD, USA), and expressed relative to β-actin. The results are presented as fold variation in signal intensity relative to the control, that was arbitrarily set to a value of 1.     . 1 H NMR spectra (400.13 MHz) of 4 in wet DMSO-d 6 , in air, measured over one month. No changes in the 1 H NMR spectra were found, signifying that both the ester bond, as well as the [(η 6 -p-cymene)Ru] 2+ -[C 2 B 9 H 10 ] 2fragment, are stable under these conditions. S9 Figure S6. 11 B{ 1 H} NMR spectra (128.28 MHz) of 3 (top) and 4 (bottom) in wet DMSO-d 6 , in air, measured over one month. No changes in the 11 B{ 1 H} NMR spectra were observed, signifying that the [RuC 2 B 9 H 10 ] fragment is stable under these conditions. Figure S7. 1 H NMR spectra (400.13 MHz) of 5 in wet DMSO-d 6 , in air, measured over one month. The second set of signals (second tautomer) for the {4-(7-chloroquinolin-4-yl)oxy}butanol-1-yl group are marked with *. The intensity of these signals is about 5%, with respect to the corresponding signals of 5. Table S1. Crystal data and structure refinement for 1 and 3. Goodness-of-fit on F 2 1.077 1.063