Target-Oriented Synthesis of Marine Coelenterazine Derivatives with Anticancer Activity by Applying the Heavy-Atom Effect

Abstract

Photodynamic therapy (PDT) is an anticancer therapeutic modality with remarkable advantages over more conventional approaches. However, PDT is greatly limited by its dependence on external light sources. Given this, PDT would benefit from new systems capable of a light-free and intracellular photodynamic effect. Herein, we evaluated the heavy-atom effect as a strategy to provide anticancer activity to derivatives of coelenterazine, a chemiluminescent single-molecule widespread in marine organisms. Our results indicate that the use of the heavy-atom effect allows these molecules to generate readily available triplet states in a chemiluminescent reaction triggered by a cancer marker. Cytotoxicity assays in different cancer cell lines showed a heavy-atom-dependent anticancer activity, which increased in the substituent order of hydroxyl < chlorine < bromine. Furthermore, it was found that the magnitude of this anticancer activity is also dependent on the tumor type, being more relevant toward breast and prostate cancer. The compounds also showed moderate activity toward neuroblastoma, while showing limited activity toward colon cancer. In conclusion, the present results indicate that the application of the heavy-atom effect to marine coelenterazine could be a promising approach for the future development of new and optimized self-activating and tumor-selective sensitizers for light-free PDT.

Keywords: cancer; chemiluminescence; coelenterazine; heavy-atom effect; photodynamic therapy; self-activating photosensitizers; triplet chemiexcitation.

Scheme 1

Chemical structures of coelenterazine and derivatives (a). Schematic representation of the proposed tumor-selective and self-activating photodynamic therapy based on the chemiluminescent reaction of R-Cla (b).

Figure 1

Potential energy curves (in kcal·mol⁻¹) for the S₀ and T₁ states during the thermolysis of OH-Cla (top) and Br-Cla dioxetanones (bottom), as a function of intrinsic reaction coordinates (in amu^1/2 bohr). Calculations were made with the ωB97XD density functional in implicit water. The bottom figure is reprinted with permission from [12]. Copyright Elsevier 2019.

Figure 2

(a) Normalized chemiluminescence output of OH-, Cl-, and Br-Cla in DMF-acetate buffer pH 5.14 (0.68%). (b) Chemiluminescence intensity of OH-, Cl-, and Br-Cla in the presence of 20 mg of KO₂ in methanol. (c) Normalized chemiluminescence spectra of OH-, Cl-, and Br-Cla in DMF-acetate buffer pH 5.14 (0.68%). (d) Normalized fluorescence intensity of spent chemiluminescent reactions of OH-, Cl-, and Br-Cla coelenteramide after 30 min of reaction.

Figure 3

The 2D excitation-emission matrices (EEMs) of spent chemiluminescent reactions of OH-, Cl-, and Br-Cla after 30 min of reaction in DMSO-acetate buffer pH 5.14 (0.68%) solution.

Figure 4

Effect of OH-, Cl-, and Br-Cla on HT-29 cell viability. Cells were cultured in the presence of increasing concentrations of each compound. After 48 h, an MTT assay was performed to measured cellular viability. Results are presented as mean ± SEM. * Statistically significant vs. control at p < 0.05; **** statistically significant vs. control at p < 0.0001.

Figure 5

Microscopic cellular visualization of HT-29 cells after 48 h of incubation with cell medium and (a) 0.1% DMSO (control), (b) OH-Cla, (c), Cl-Cla, or (d) Br-Cla. Representative images were obtained with a high contrast brightfield objective (10×) (LionHeart FX Automated Microscope), from three independent experiments.

Figure 6

Effects of OH-, Cl-, and Br-Cla on SH-SY5Y cellular viability. Cells were cultivated in the presence of increasing concentrations of each compound. After 48 h, an MTT assay was performed to measured cellular viability. Results are presented as mean ± SEM. * Statistically significant vs. control at p < 0.05; **** statistically significant vs. control at p < 0.0001.

Figure 7

Microscopic cellular visualization of SH-SY5Y cells after 48 h of incubation with cell medium and (a) 0.1% DMSO (control), (b) OH-Cla, (c), Cl-Cla, or (d) Br-Cla. Representative images were obtained with a high contrast brightfield objective (10×) (LionHeart FX Automated Microscope), from three independent experiments.

Figure 8

Relative viabilities of (a) MCF-7 and (b) PC-3 cells after 72 h incubation with several concentrations of Br-Cla, always without light irradiation. Results are presented as mean ± SEM. * Statistically significant vs. control at p < 0.05; ** statistically significant vs. control at p < 0.01; **** statistically significant vs. control at p < 0.0001.