Rhenium Complexes with Red-Light-Induced Anticancer Activity

Abstract

Rhenium(I) pyridocarbazole complexes with photoinduced antiproliferative activity are reported. The substitutionally inert complexes induce cell death by singlet oxygen generation upon irradiation with red light (λ ≥ 620 nm), while only weak background cytotoxicity is observed in the dark. Due to their ability to inhibit protein kinases (nanomolar IC₅₀ values against Pim1 at 10 μM ATP), this class of rhenium complexes point into the direction of dual function antiproliferative therapy with a single drug in which photodynamic therapy is combined with the inhibition of cancer related protein kinases.

Introduction

Photodynamic therapy (PDT) has become an attractive alternative and complement to conventional cancer chemotherapy due to the ability of spatially controlling drug action and an overall low risk of resistance development.^[1] The method is based on a photosensitizer which, upon irradiation with visible light in the presence of oxygen, generates reactive oxygen species (ROS) such as ¹O₂, thereby resulting in oxidative damage of cellular components and ultimately inducing cell death. An important requirement for a sensitizer useful in the clinic is the activation with long visible light wavelengths (600–850 nm) in order to achieve an optimal tissue penetration. Current photosensitizers used for PDT are mainly based on organic molecules such as porphyrin derivates, chlorines, phthalocyanines, and porphycenes. However, metal complexes should constitute an attractive class of compounds for PDT, since in addition to their well established photochemical properties,^[2,3] they bear attractive additional features such as structural diversity and complexity, tunable ligand exchange kinetics, unusual reactivities, and the availability of radioisotopes, which –potentially in combination with a photoinduced reactivity– render them attractive scaffolds for the development of novel therapeutic and diagnostic agents.^[4]

We recently introduced the first examples of rhenium(I) complexes with visible-light-induced in vitro anticancer activity. Metallo-pyridocarbazole complex 1 (Figure 1) was demonstrated to induce apoptosis upon irradiation with wavelengths λ ≥ 505 nm.^[5] Towards the development of novel PDT drugs, we here wish to report derivatives of this metallo-pyridocarbazole scaffold with improved chemical stability and an ability to induce apoptosis in cancer cells upon irradiation with visible light of longer wavelengths.

Figure 1.

Modifications of the lead structure 1 to derive rhenium(I) pyridocarbazole complexes 2–8 with different absorption maxima. All complexes were used as racemic mixtures.

Results and Discussion

We started a structure-activity-relationship by replacing the monodentate pyridine ligand of 1 against trimethylphosphine (2) or imidazole (3). Figure 2 and Figure 3 display the crystal structures of the rhenium(I) complex 2 and the benzylated derivative of complex 3, respectively, in which rhenium is coordinated in a bidentate fashion to a deprotonated pyrido[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione^[6] ligand, in addition to three CO ligands and one trimethylphosphine or imidazole. Tested for their light-induced anticancer activities in HeLa cells, complexes 2 and 3 displayed similar antiproliferative activities compared to the previous complex 1 when used in combination with a standard LED light source (7 W) (see Supporting Information). However, both complexes turned out to be significantly more hydrolytically stable. When incubated in DMSO-d₆/D₂O (9:1) in the presence of β-mercaptoethanol (5 mM) no signs of decomposition were monitored by ¹H NMR spectroscopy for 2 and 3 after at least 168 h, while only 75% of 1 are retained after just 100 h. Furthermore, irradiation with visible light did not affect the stabilities of complexes 2 and 3 (see Supporting Information for more details).

Figure 2.

Crystal structure of Re complex 2. ORTEP drawing with 50% probability thermal ellipsoids; solvent is omitted for clarity. Selected bond distances (Å) and angles (°): Re1-C21 = 1.941(11), Re1-N1 = 2.223(8), Re1-N4 = 2.173(7), Re1-P1 = 2.440(3), C21-Re1-P1 = 178.3(3), C22-Re1-P1 = 88.4(3), N1-Re1-P1 = 88.9(2).

Figure 3.

Crystal structure of the N-benzylated derivative of Re complex 3. ORTEP drawing with 50% probability thermal ellipsoids; solvent is omitted for clarity. Selected bond distances (Å) and angles (°): Re1-C33 = 1.924(5), Re1-N1 = 2.215(3), Re1-N4 = 2.157(3), Re1-N28 = 2.198(3), C34-Re1-N28 = 94.19(15), C35-Re1-N28 = 175.85(15), N28-Re1-N1 = 80.71(13).

We next turned our attention to the excitation wavelength. A comparison of the absorbance spectra of complexes 1–3 reveals that the monodentate ligand, whether being a strong π-acceptor as pyridine in 1 (λ_max = 512 nm) or a strong σ-donor as PMe₃ in 2 (λ_max = 516 nm) and imidazole in 3 (λ_max = 513 nm) has a negligible influence on the absorbance maxima. We therefore decided to use the Re imidazole complex 3 as a starting point to investigate the influence of substituents within the pyridocarbazole heterocycle on the photophysical properties. A prescreening indicated that π-donating substituents in the indole and σ-accepting substituents in the pyridine moiety exert the most pronounced red shift of the long wavelength absorbance band. Figure 1 displays the most interesting compounds, with a π-donating hydroxyl (4) (Δλ_max = 26 nm) or dimethylamino group (5) (Δλ_max = 49 nm) introduced into the 5-position of the indole moiety, a σ-accepting fluoride (6) (Δλ_max = 15 nm) or trifluoromethyl group (7) (Δλ_max = 24 nm) into the 3-position of the pyridine moiety, and a combination of a π-donating methoxyl group in the indole with a σ-accepting fluoride in the pyridine (8) (Δλ_max = 29 nm) (Figures 1 and 4).

Figure 4.

UV/Vis absorbance of Re complexes 1–8 measured in DMSO (60 μM).

Having achieved a red shift of the long wavelength absorbance band with the complexes 4–8, we continued our study with investigating their ability to produce light-induced singlet oxygen.^[7] As a result, π-donating groups in the indole moiety (4 and 5) completely suppressed the formation of singlet oxygen, whereas the methoxy compound 8 shows a diminished singlet oxygen generation. However, the fluorine (6) and CF₃ (7) derivatives efficiently produce singlet oxygen upon visible light irradiation as demonstrated in Figure 5.^[8] Furthermore, to our delight, a determination of the relative ability to produce singlet oxygen as a function of the wavelength revealed that –in contrast to complex 3 devoid of any modification of the pyridocarbazole moiety– complexes 6 and 7 are able to generate singlet oxygen upon photolysis even with red light of λ ≥ 620 nm (cut-on filter).

Figure 5.

Singlet oxygen production of Re complexes 3, 6, 7 and 8 (50 μM) in PBS/DMSO 1:1. Singlet oxygen was determined according to a method by Kraljić and El Moshni (ref. 7) using the redoxsensitive dye para-nitrosodimethylaniline (50 μM) in the presence of imidazole after 60 min irradiation with cut-on filters of various wavelengths. Bars indicate the average values of six data points corrected by the background reaction in the dark.

Based on their capability to produce singlet oxygen upon irradiation with red light, we selected complexes 6–8 for investigating their red-light-induced antiproliferative properties. As shown in Figure 6, when preincubated with HeLa cells in the dark at 5 μM for 1 h followed by an irradiation for one hour with a conventional household 7 W LED light in combination with a cut-on filter of λ ≥ 620 nm, we observed that all three complexes display an improved cytotoxicity compared to the unmodified Re(I) complex 3. However, complex 6 harboring a fluorine in the 3-position of the pyridine moiety displayed the most pronounced red-light-induced antiproliferative effect with a cell survival of 10% as determined with an MTT assay 24 h after compound administration. At λ ≥ 580 nm complex 6 is significantly more effective. Figure 7 shows the concentration-dependent antiproliferative activity of 6 in HeLa cells providing a half-maximum effective concentration (EC₅₀) of 0.3 μM, which is an improvement over the cytotoxicity in the dark (EC₅₀ = 10 μM) –most likely related to its ability to inhibit protein kinases as discussed below– by more than 30-fold.

Figure 6.

Wavelength-dependent photoactivity of Re complexes 3 and 6–8 in HeLa cells. Complexes (5 μM) were incubated for 1 h before irradiation (60 min), and cell survival was determined by MTT assay 24 h after compound administration. Data are based on the average of 18 data points.

Figure 7.

Visible-light-induced antiproliferative activity of Re complex 6 in HeLa cancer cells. Experimental details: 1 h after addition of 6, cells were irradiated for 30 min at λ ≥ 580 nm. Cytotoxicity was determined 24 h after addition by MTT assay. Standard deviations result from two independent experiments with overall 18 data points for each compound concentration.

The here presented rhenium(I) complexes are based on a metallo-pyridocarbazole scaffold initially developed for the design of metal-containing protein kinase inhibitors.^[9] Towards the goal of using a single compound for combining photodynamic therapy with the inhibition of protein kinases targeted in cancer therapy, we tested the complexes 1–3 and 6 for their inhibition of Pim1 as a model protein kinase involved in cancer. Figure 8 displays the concentration-dependent inhibition, providing half maximal inhibitory concentrations (IC₅₀) in the nanomolar range at 10 μM ATP. The reduced affinity of 2 can be rationalized with the larger bulkiness of the phosphine ligand, thus preventing a proper fit into the ATP binding site of Pim1.

Figure 8.

IC₅₀ curves of Re complexes 1, 2, 3 and 6 against Pim1 at ATP concentrations of 10 μM.

Conclusions

We here presented rhenium(I) pyridocarbazole complexes for applications in photodynamic therapy with improved chemical stability and a red-shifted visible-light-induced anticancer activity. Nanomolar IC₅₀ values determined against the protein kinase Pim1 demonstrate that this class of complexes shows promise as dual function anticancer agents in which PDT is combined with targeted therapy due to protein kinase inhibition.^[10]

Experimental Section

Biological evaluation

Absorption spectra

Absorption spectra for the complexes were determined at a concentration of 60 μM in DMSO using a DU^®800 Spectrophotometer (Beckman Coulter).

Irradiation setup

Irradiation was performed with a 7 W LED reflector (Megaman^® PAR16 GU10 LR0707-SP), which was placed 6 cm above the assay plates. The different light spectra used in the experiments were determined by sorting filters from Newport which set distinct lower transmission borders at 505, 560, 580 and 620 nm. Without any filter a maximum irradiance of 45.8 mW/cm² was calculated for the LED light source.

Singlet oxygen determination

The ¹O₂ production of the compounds was determined using a method based on the work Kraljić and El Moshni.^[7] Accordingly, p-nitrosodimethylaniline (RNO) and imidazole were dissolved in PBS buffer with a concentration of 500 μM for RNO and 80 mM for imidazole. The rhenium(I) compounds were used as 1 mM DMSO stock solutions. RNO (10 μL), imidazole (10 μL), the complexes (5 μL, with a final concentration of 50 μM) and PBS buffer/DMSO (75 μL, 1:1.5) were pipetted in a 96-well plate. To test the ¹O₂ production by the influence of light the absorption maximum of RNO at 440 nm was determined before and after 30–60 min irradiation. Irradiation took place by a 7 W LED reflector without filter, at λ ≥ 505 nm, λ ≥ 560 nm, λ ≥ 580 nm and λ ≥ 620 nm. As a control all complexes were measured in the dark. All measurements were performed two times in triplicate. The ¹O₂ production was determined as the negative difference of absorbance before and after irradiation.

Cell culture

Cells used in this study were the HeLa human cervical cancer cells. They were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin. Cells were maintained in 75 cm² flasks in a 5% CO₂-humidified atmosphere at 37 °C. Passage takes place every 2–3 days. All cell culture ingredients were purchased from Sigma-Aldrich.

Photodynamic treatment

To investigate photoinduced cytotoxicity of the organometallic complexes, cells were seeded at equal concentrations into 96-well microtiter plates in a number of 9 × 10³ cells per well. After overnight cell attachment the designated metal compound was added. A 10 mM stock solution in DMSO was diluted with culture medium to the desired concentration regarding the point that the final DMSO concentration present to the cells is unchanging at 1%. The complex treated cells were incubated for 1 h in the dark before irradiated with a 7 W LED reflector. Following irradiation cells were maintained under normal culture conditions. Cell viability was measured 24 h after complex administration by MTT assay.

Cell viability assays

The toxicity of the photoactivated compounds was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. MTT is a yellow compound that when reduced by functioning mitochondria, produces purple formazan crystals that can be measured spectrophotometrically. For this purpose MTT (Sigma-Aldrich) was dissolved in phosphate buffered saline (PBS) to a concentration of 5 mg/mL and further diluted in culture medium (1:11). Cells were incubated with this MTT-solution for 3 h under normal culture conditions. Afterwards 155 μL of the solution were rejected and 90 μL of DMSO were added. To completely dissolve the formazan salts plates were incubated for 10 min on a shaker and afterwards quantified by measuring the absorbance at 535 nm with a Spectramax M5 microplate reader (Molecular Devices). The cell viability was calculated as percentage of surviving cells compared to untreated and non-irradiated control cells. Control experiments confirm that cell viability is not affected by an irradiation with the LED reflector in the absence of any rhenium complexes (see Supporting Information).

Protein kinase inhibition assays

Inhibition data were obtained by a conventional radioactive assay in which the kinase activity was measured by the degree of phosphorylation of the respective P70 S6 substrate peptide (AnaSpec) with [γ-³³P]ATP (PerkinElmer). Accordingly, different concentrations of the rhenium complexes 1–3 and 6 were preincubated at room temperature for 30 min with Pim1 (Millipore) and P70 S6 substrate peptide and the phosphorylation reaction was subsequently initiated by adding ATP and [γ-³³P]ATP to a final volume of 25 μL, which consisted of MOPS (10 mM, pH 7.0), Mg(OAc)₂ (10 mM), DMSO (5%), Pim1 (1.6 nM), P70 S6 kinase substrate (50 μM), EDTA (0.1 mM), Brij-35 (0.001%), glycerol (0.5%), 2-mercaptoethanol (0.01%), BSA (0.1 mg/mL) and ATP (10 μM) including [γ-³³P]ATP (approximately 0.1 μCi/μL). After incubation for 30 min, the reaction was terminated by spotting 17.5 μL onto circular P81 phosphocellulose paper (diameter 2.1 cm, Whatman), followed by washing with 0.75% phosphoric acid and acetone. The dried P81 papers were transferred to scintillation vials and 5 mL of scintillation cocktail were added. The counts per minute (CPM) were measured with a Beckmann Coulter LS6500 MultiPurpose Scintillation Counter and corrected by the background CPM. The IC₅₀ values were determined in duplicate from sigmoidal curve fits.

Supporting Information

Synthesis of complexes 2–8, proof of purity of the complexes, stability test of complexes 2 and 3, single crystal X-ray diffraction study with 2 and 3Bn, and biological experiments.