Light-Activated Carbon Monoxide Prodrugs Based on Bipyridyl Dicarbonyl Ruthenium(II) Complexes

Abstract

Two photoactivatable dicarbonyl ruthenium(II) complexes based on an amide-functionalised bipyridine scaffold (4-position) equipped with an alkyne functionality or a green-fluorescent BODIPY (boron-dipyrromethene) dye have been prepared and used to investigate their light-induced decarbonylation. UV/Vis, FTIR and ¹³ C NMR spectroscopies as well as gas chromatography and multivariate curve resolution alternating least-squares analysis (MCR-ALS) were used to elucidate the mechanism of the decarbonylation process. Release of the first CO molecule occurs very quickly, while release of the second CO molecule proceeds more slowly. In vitro studies using two cell lines A431 (human squamous carcinoma) and HEK293 (human embryonic kidney cells) have been carried out in order to characterise the anti-proliferative and anti-apoptotic activities. The BODIPY-labelled compound allows for monitoring the cellular uptake, showing fast internalisation kinetics and accumulation at the endoplasmic reticulum and mitochondria.

Keywords: anti-apoptotic activity; anti-proliferative; cellular localisation; photoCORM; ruthenium(II).

Scheme 1

Chemical structures of trans‐(Cl),cis‐(CO)‐ruthenium(II) dicarbonyl complexes with bipyridyl ligands substituted at 4,4′‐position (1 a–c) or at 5,5′‐position (2 a,b) and with pyrimidyl ligands (3 a–c).

Scheme 2

Chemical structures of Ru^II dicarbonyl bipyridyl complexes 5 and 10.

Scheme 3

Synthesis of alkyne‐functionalised Ru^II dicabonyl bipyridyl complex 5. Reagents and conditions: i) EDC⋅HCl, HOBt, propargylamine, DIPEA, DMF, r.t., 24 h, yield 61 % of 4; ii) 4, [RuCl₂(CO)₂]_n, DMF, 90 °C, 30 min, darkness, yield 59 % of 5.

Scheme 4

Synthesis of CORM‐BODIPY compound 10. Reagents and conditions: i) NaN₃, KI, DMF, 45 °C overnight; yield >99 % of 7; ii) PPh₃, DMF, r.t., 10 h; iii) 1 b, DCC, HOBt, DMF, r.t., 16 h; iv) DMF, r.t., 16 h; yield 47 % of 9; (iv) [RuCl₂(CO)₂]_n, MeOH, reflux, 2 h, darkness, yield 68 % of 10.

Figure 1

Left: UV/Vis absorption spectra of 5 (50 μm, black line) and 10 (30 μm, red line) in water containing 0.8 % (v/v) DMSO; Right: absorbance (black line) and fluorescence (red line) spectra illustrating the narrow Stokes shift of complex 10.

Figure 2

UV/Vis absorption spectra measured for 5 (50 μm, left) and 10 (30 μm, right) in water containing 0.8 % (v/v) DMSO after different periods of exposure to 350 nm of radiation (E _v≈6 mW cm⁻²) at room temperature.

Scheme 5

Serial mechanisms (model 1 and 2) for the photoreaction of complex 5 and 10. A=starting complex; P1, P2=photoproducts.

Figure 3

Fluorescence spectra of 9 (left, 30 μm in water containing 0.8 % (v/v) DMSO) and 10 (right, 30 μm in water containing 0.8 % (v/v) DMSO) before and after different periods of exposure to 350 nm of radiation (E _v≈6 mW cm⁻²) at room temperature.

Figure 4

MTS assay of 5 using different concentrations (25, 50, 60, 75 and 100 μm) in A431 (left) and HEK293 (right) cell lines measured after 24 and 48 h after 10 min exposure to 350 nm (E _v≈6 mW cm⁻²) at 37 °C. The percentage of cell viability is expressed relative to untreated cells (ANOVA at α=0.05).

Figure 5

MTS assay of 10 using different concentrations (3.15, 6.25, 12.5, 25 and 50 μm) in A431 (left) and HEK293 (right) cell line measured after 24 and 48 h after 10 min exposure to 350 nm (E _v≈6 mW cm⁻²) at 37 °C. The percentage of cell viability is expressed relative to untreated cells (ANOVA at α=0.05).

Figure 6

Time monitoring of cellular uptake of compound 10 (10 μm) by A) A431 cells and B) HEK293 cells.

Figure 7

Cellular colocalisation of compound 10 (10 μm) incubated in A) A431 and B) HEK293 cells for 30 min in culture medium with ER and mitochondria after 30 min incubation in A) A431 and B) HEK293 cells. Fluorescence image of 10 (left). Counter stains were then added for 15 min at 37 °C with 5 % CO_2. Mitotracker^TM (0.25 μm) for mitochondrial staining (mitochondria, merged with compound 10 in bottom right) and ER‐Tracker™ Blue‐White DPX (0.5 μm) for endoplasmic reticulum staining (ER, merged with 10 in bottom right). Scale bars are 10 μm.

Figure 8

Diagram showing the percentage (*P>0.05, **P>0.01, using Dunnett post test as part of one‐way ANOVA analysis) of dead and apoptotic A431 cells treated without (control containing 0.1 % DMSO) or with complexes 5 and 10 (10 μm containing 0.1 % DMSO) in the dark and after irradiation of UV light (350 nm, UV hand lamp, 6 W) for 10 min. The cells were incubated after irradiation 4 h before analysis.