SYNTHESIS, CHARACTERIZATION AND EVALUATION OF BIOACTIVE MOLECULES LINKED TO PHOTOREMOVABLE PROTECTING GROUPS FOR SPATIO-TEMPORAL CONTROL OF THEIR ACTIVITY THROUGH PHOTOACTIVATION

Biological processes are highly complex phenomena governed by a series of precise spatio-temporal events. Among the most innovative approaches developed over the last decade to investigate such processes, photopharmacology has emerged as a powerful tool, as it exploits light to control biological functions. In particular, optopharmacology enables the regulation of pathophysiological processes through the use of molecules that become bioactive upon light stimulation. Initially applied mainly to biological investigations, optopharmacology is now increasingly recognized for its great potential in the clinical and medical fields. Among the various photosensitive systems, molecular switches, molecular motors, photodynamic therapy (PDT), and photoremovable protecting groups (PPGs) play important roles in pharmaceutical research. The focus of this doctoral research lies on PPGs: these are chromophores covalently linked to approved drugs or bioactive molecules at functional groups essential for their activity. The resulting species, generally referred to as photo-prodrugs or caged compounds, are able to release the native drug upon light irradiation at a specific wavelength, which cleaves the bond between the PPG and the bioactive molecule. In the literature, several classes of PPGs have been described, including o-nitrobenzyl, coumarin, and BODIPY derivatives, all of which can cage and release a variety of functional groups. However, when focusing on alcohols, phenols, and primary or secondary amines, these are typically linked through carbonate or carbamate linkers, which are intrinsically unstable under physiological conditions due to the presence of esterases. Moreover, the photorelease of tertiary amines has been only rarely reported for such PPGs. A notable exception is represented by derivatives of 8-cyano-7-hydroxyquinolin-2-ylmethyl (8CyHQ), which have been specifically developed and optimized for the release of tertiary aliphatic amines and tertiary anilines upon both one-photon excitation (1PE, 365–405 nm) and two-photon excitation (2PE, 740 nm). The interest of this thesis is focused on the photorelease of tertiary amines through 1PE. Consequently, the first objective of this work was the optimization study of the quinoline core to achieve both absorption maxima shifted toward safer irradiation wavelengths (>420 nm) and higher chemical yield of payload release. Starting from 8-bromo-7-hydroxyquinolin-2-ylmethyl (8BHQ), the first quinoline based PPG described, we first demonstrated the ability of it in releasing tertiary amines, exploiting a residual absorption tail at 440 nm. From this starting point, we synthesized novel quinoline-based PPGs bearing various electron-withdrawing substituents at position 8 with the aim of improving 8BHQ properties. Unfortunately, among all the newly synthesized derivatives, only 8BHQ proved to be the most efficient quinoline-based PPG for tertiary amine release at 440 nm, even outperforming the well-established 8CyHQ. However, upon 365 nm light irradiation all new derivatives showcase excellent photoreactivity, in particular the compound containing a CF3 which demonstrated to have a significant superior quantum yield (Qu) value. We subsequently explored substitution at position 3, introducing different substituents of diverse electronic and steric nature while keeping the 8BHQ substitution pattern otherwise constant. Although this study is still ongoing, several derivatives have already shown improved performance compared to the 8BHQ reference, in particular the derivative bearing a cyano group in C3 significantly improved 8BHQ performance dramatically increasing photoreactivity and the yield of released leaving amine upon blue light irradiation. In parallel with this photochemical optimization, two photopharmacological projects were also pursued. The first project involved the photocaging of ivabradine in order to achieve selective antagonism of different subtypes of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Ivabradine is a clinically used bradycardic agent indicated for the treatment of stable angina pectoris. Its mechanism of action involves antagonism of the HCN4 channel; however, the drug itself is unable to discriminate between the four HCN subtypes. Through a photocaging strategy, ivabradine was covalently linked to 8BHQ, and its effective photorelease was demonstrated in vitro, ex vivo on neuronal HCN channels, and in vivo on cardiac HCN channels in zebrafish. This study has been completed and published, and it opens the way to new perspectives for the use of caged ivabradine in diseases involving dysregulation of non-cardiac HCN channels, such as HCN2-related neuropathic pain. The second project focused on the photocaging of the antitubercular drug bedaquiline, a highly potent agent against Mycobacterium tuberculosis due to its ability to deplete ATP resources by inhibiting the enzyme ATP synthase (ATPe). However, its clinical use is limited by severe cardiac and hepatic toxicities. The rationale behind designing 8BHQ-caged bedaquiline was therefore to reduce the systemic side effects of the native drug. Unlike the ivabradine project, however, we observed residual anti-mycobacterial activity of caged bedaquiline in the dark, particularly against M. tuberculosis in macrophages. To address this issue, a series of novel quinoline-based PPGs was synthesized, introducing polyethylene glycol (PEG) chains of variable length at position C4 of the quinoline chromophore. This modification was hypothesized to hinder enzymatic cleavage of the drug–PPG linkage by activated macrophage enzymes. Among the synthesized derivatives, the compound bearing a triethylene glycol chain proved the most promising, as it displayed complete inactivity in the dark. This project is currently nearing completion, with ongoing experiments to evaluate the light-induced uncaging and anti-mycobacterial activity of these derivatives in infected macrophages.