N-Alkylaminoferrocene (NAAF)-based prodrugs are activated in the presence of elevated amounts of reactive oxygen species (ROS), which corresponds to cancer specific conditions, with formation of NAAF and p-quinone methide. Both products act synergistically by increasing oxidative stress in cancer cells that causes their death. Though it has already been demonstrated that the best prodrugs of this type retain their antitumor activity in vivo, the effects were found to be substantially weaker than those observed in cell cultures. Moreover, the mechanistic studies of these compounds in vivo are missing. For clarification of these important questions, labeling of the prodrugs with radioactive moieties would be necessary. In this paper, we first observed that the representative NAAF-based prodrugs are hydrolyzed in dilute aqueous solutions to the corresponding arylboronic acids. We confirmed that these products are responsible for ROS amplification and anticancer properties of the parent prodrugs. Next, we developed the efficient synthetic protocol for radiolabeling the hydrolyzed NAAF-based prodrugs by [ 18 F]fluoroglucosylation under the conditions of the copper(I)-catalyzed azide−alkyne 1,3-dipolar cycloaddition and used this protocol to prepare one representative hydrolyzed NAAF-based prodrug radiolabeled with 18 F. Finally, we studied the stability of the 18 F-labeled compound in human serum in vitro and in rat blood in vivo and obtained preliminary data on its biodistribution in vivo in mice carrying pancreatic (AR42J) and prostate (PC3) tumors by applying PET imaging studies. The compound described in this paper will help to understand in vivo effects (e.g., pharmacokinetics, accumulation in organs, the nature of side effects) of these prodrugs that will strongly contribute to their advancement to clinical trials.
Intracellular concentration of reactive oxygen species (e.g., H2O2) in cancer cells is elevated over 10-fold as compared to normal cells. This feature has been used by us and several other research groups to design cancer specific prodrugs, for example, N-alkylaminoferrocene (NAAF)-based prodrugs. Further improvement of the efficacy of these prodrugs can be achieved by their targeting to intracellular organelles containing elevated reactive oxygen species (ROS) amounts. For example, we have previously demonstrated that lysosome-targeted NAAF-prodrugs exhibit higher anticancer activity in cell cultures, in primary cells and in vivo (Angew. Chem. Int. Ed. 2017, 56, 15545). Mitochondrion is an organelle, where electrons can leak from the respiratory chain. These electrons can combine with O2, generating O2−• that is followed by dismutation with the formation of H2O2. Thus, ROS can be generated in excess in mitochondria and targeting of ROS-sensitive prodrugs to these organelles could be a sensible possibility for enhancing their efficacy. We have previously reported on NAAF-prodrugs, which after their activation in cells, are accumulated in mitochondria (Angew. Chem. Int. Ed. 2018, 57, 11943). Now we prepared two hybrid NAAF-prodrugs directly accumulated in mitochondria and activated in these organelles. We studied their anticancer activity and mode of action. Based on these data, we concluded that ROS produced by mitochondria is not available in sufficient quantities for activation of the ROS-responsive prodrugs. The reason for this can be efficient scavenging of ROS by antioxidants. Our data are important for the understanding of the mechanism of action of ROS-activatable prodrugs and will facilitate their further development.
We have studied spectral-luminescent properties of the monomethine cyanine dyes both in their free states and in the presence of either double-stranded deoxyribonucleic acids (dsDNAs) or single-stranded ribonucleic acids (RNAs). The dyes possess low fluorescence intensity in an unbound state, which is increased up to 479 times in the presence of the nucleic acids. In the presence of RNAs, the fluorescence intensity increase was stronger than that observed in the presence of dsDNA. Next, we have performed staining of live and fixed cells by all prepared dyes. The dyes proved to be cell and nuclear membrane permeant. They are photostable and brightly stain RNA-containing organelles in both live and fixed cells. The colocalization confirmed the specific nucleoli staining with anti-Ki-67 antibodies. The RNA digestion experiment has confirmed the selectivity of the dyes toward intracellular RNA. Based on the obtained results, we can conclude that the investigated monomethine cyanine dyes are useful fluorescent probes for the visualization of intracellular RNA and RNA-containing organelles such as nucleoli by using fluorescence microscopy.
The mode of anticancer activity of simple ferrocenes often relies on their intracellular oxidation with the formation of cytotoxic ferrocenium species. The former compounds should be considered as prodrugs and derived from them ferroceniums as drugs. The drugs are 17e − organometallic species. Therefore, they are chemically unstable and decompose with formation of free cyclopentadienyl ligands (which are further transformed to more stable species) and iron ions. The short lifetime of ferrocenium drugs limits the anticancer effect of ferrocene prodrugs. In this paper we prepared a series of acylated aminoferrocene monomers, dimers, and higher oligonuclear complexes Fc N (where N = 1−4). Drugs [Fc N ] + derived from Fc N (N > 1) are more stable than monomeric [Fc] + , provided that the ferrocene units are electronically coupled. Correspondingly, we expected that Fc N species will be more potent anticancer agents in comparison to the related monomers. These assumptions were confirmed for dimer 10. We observed that this prodrug has sufficient solubility in aqueous solution (100 μM) and favorable lipophilicity (log P = 2.2 ± 0.2). Ferrocene units in 10 are efficiently electronically coupled via a −C(O)NH− linker according to cyclic voltammetry, differential pulse voltammetry, and spectroelectrochemistry. We observed that 10 is the most efficient catalyst of the production of reactive oxygen species (ROS) and the most active anticancer agent in comparison to control monomers and their mixtures.
Elevated levels of reactive oxygen species (ROS) and deficient mitochondria are two weak points of cancer cells. Their simultaneous targeting is a valid therapeutic strategy to design highly potent anticancer drugs. The remaining challenge is to limit the drug effects to cancer cells without affecting normal ones. We have previously developed three aminoferrocene (AF)‐based derivatives, which are activated in the presence of elevated levels of ROS present in cancer cells with formation of electron‐rich compounds able to generate ROS and reduce mitochondrial membrane potential (MMP). All of them exhibit important drawbacks including either low efficacy or high unspecific toxicity that prevents their application in vivo up to date. Herein we describe unusual AF‐derivatives lacking these drawbacks. These compounds act via an alternative mechanism: they are chemically stable in the presence of ROS, generate mitochondrial ROS in cancer cells, but not normal cells and exhibit anticancer effect in vivo.
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