Mitochondrial membrane potential is more negative in cancer cells than in normal cells, allowing cancer targeting by delocalized lipophilic cations (DLCs). However, as the difference is rather small, these drugs affect also normal cells. Now a concept of pro-DLCs is proposed based on an N-alkylaminoferrocene structure. These prodrugs are activated by the reaction with reactive oxygen species (ROS) forming ferrocenium-based DLCs. Since ROS are overproduced in cancer, the high-efficiency cancer-cell-specific targeting of mitochondria could be achieved as demonstrated by fluorescence microscopy in combination with two fluorogenic pro-DLCs in vitro and in vivo. We prepared a conjugate of another pro-DLC with a clinically approved drug carboplatin and confirmed that its accumulation in mitochondria was higher than that of the free drug. This was reflected in the substantially higher anticancer effect of the conjugate.

Because cellular uptake of anticancer Pt and Pt drugs occurs by different mechanisms, the latter ones can exhibit substantial activity towards cells, which have either intrinsic or acquired resistance towards Pt drugs. However, this positive effect is diminished due to reductive activation of Pt drugs in extracellular space that can be one of the reasons why they have not yet been approved for clinical use despite over 60 clinical trials conducted worldwide. Herein, we suggest a solution to this problem by achieving highly specific intracellular versus extracellular prodrug reduction. In particular, we prepared a hybrid Pt prodrug containing two pro-reductants. This hybrid was uptaken by cells, the pro-reductants were activated in the cancer-specific microenvironment (high H O ), and reduced Pt by two one-electron transfers. The drug formed in this way induced cell death both in cisplatin-sensitive and resistant cell lines, but remained nontoxic to normal cells.

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.

We prepared a Pt(iv)-prodrug, which under cancer specific conditions (elevated concentration of reactive oxygen species, ROS) releases a DNA-binding drug oxaliplatin as well as ROS-amplifying drugs p-quinone methide and N-alkylferrocenium. Due to the concerted action of these components, an excellent anticancer effect was achieved: IC50 = 0.4 ± 0.1 μM for human ovarian carcinoma A2780 cells. Importantly, the prodrug was found to be 45-fold less toxic to normal cells (HDFa).

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.

Mitochondrial membrane potential is more negative in cancer cells than in normal cells, allowing cancer targeting by delocalized lipophilic cations (DLCs). However, as the difference is rather small, these drugs affect also normal cells. Now a concept of pro‐DLCs is proposed based on an N‐alkylaminoferrocene structure. These prodrugs are activated by the reaction with reactive oxygen species (ROS) forming ferrocenium‐based DLCs. Since ROS are overproduced in cancer, the high‐efficiency cancer‐cell‐specific targeting of mitochondria could be achieved as demonstrated by fluorescence microscopy in combination with two fluorogenic pro‐DLCs in vitro and in vivo. We prepared a conjugate of another pro‐DLC with a clinically approved drug carboplatin and confirmed that its accumulation in mitochondria was higher than that of the free drug. This was reflected in the substantially higher anticancer effect of the conjugate.

We report the reprogramming of nonheme iron enzymes to catalyze an abiological C(sp3)‒H azidation reaction through iron-catalyzed radical relay. This biocatalytic transformation uses amidyl radicals as hydrogen atom abstractors and Fe(III)‒N3intermediates as radical trapping agents. We established a high-throughput screening platform based on click chemistry for rapid evolution of the catalytic performance of identified enzymes. The final optimized variants deliver a range of azidation products with up to 10,600 total turnovers and 93% enantiomeric excess. Given the prevalence of radical relay reactions in organic synthesis and the diversity of nonheme iron enzymes, we envision that this discovery will stimulate future development of metalloenzyme catalysts for synthetically useful transformations unexplored by natural evolution.

A number of chemical compounds are known, which amplify the availability of reactive oxygen species (ROS) in neutrophils both in vitro and in vivo. They can be roughly classified into NADPH oxidase 2 (NOX2)-dependent and NOX2-independent reagents. NOX2 activation is triggered by protein kinase C agonists (e.g., phorbol esters, transition metal ions), redox mediators (e.g., paraquat) or formyl peptide receptor (FPR) agonists (e.g., aromatic hydrazine derivatives). NOX2-independent mechanisms are realized by reagents affecting glutathione homeostasis (e.g., l-buthionine sulfoximine), modulators of the mitochondrial respiratory chain (e.g., ionophores, inositol mimics, and agonists of peroxisome proliferator-activated receptor γ) and chemical ROS amplifiers [e.g., aminoferrocene-based prodrugs (ABPs)]. Since a number of inflammatory and autoimmune diseases, as well as cancer and bacterial infections, are triggered or enhanced by aberrant ROS production in neutrophils, it is tempting to use ROS amplifiers as drugs for the treatment of these diseases. However, since the known reagents are not cell specific, their application for treatment likely causes systemic enhancement of oxidative stress, leading to severe side effects. Cell-targeted ROS enhancement can be achieved either by using conjugates of ROS amplifiers with ligands binding to receptors expressed on neutrophils (e.g., the GPI-anchored myeloid differentiation marker Ly6G or FPR) or by designing reagents activated by neutrophil function [e.g., phagocytic activity or enzymatic activity of neutrophil elastase (NE)]. Since binding of an artificial ligand to a receptor may trigger or inhibit priming of neutrophils the latter approach has a smaller potential for severe side effects and is probably better suitable for therapy. Here, we review current approaches for the use of ROS amplifiers and discuss their applicability for treatment. As an example, we suggest a possible design of neutrophil-specific ROS amplifiers, which are based on NE-activated ABPs.