Design, synthesis and preliminary biological evaluation of modulators of metalloenzymes

The present PhD thesis is focused on the chemical applied to synthesize new molecules as modulators of two metalloenzymes: the Carbonic Anhydrases and the Histone Deacetylases. The thesis reports the design, the preparation, the enzymatic activity and, for selected compounds, the in-vivo characterizations of the new molecules, together with the computational and crystallographic studies performed to investigate their binding modes. The Carbonic Anhydrases (CAs), one of the most efficient enzyme known in nature, evolved in eight genetically different families (α-ι). A large number of isoforms are described among the different organisms, their presence being crucial for pH regulation, secretion of electrolytes and for other essential physiological or pathological processes. For these reasons, CAs are important targets for drugs that can be used for different pathologies, providing that it could be possible to exploit the existent differences between families or isoforms to achieve a selective activity. This may not be an easy task, since the catalytic sites are well conserved, at least among the sixteen human α isoforms (I-XVI); however, variability can be found in hydrophilic and lipophilic accessory sites close to the Zn-binding domain. The Histone Deacetylases (HDACs) is an enzyme family that plays an important role in epigenetic regulation by removing the acetyl groups from the ε-amino moieties of the lysine side chains either in histones, affecting the DNA superhelix, or in non-histone proteins. Based on their homology, the 18 human HDAC isoforms were divided into four major classes (I-IV): three of them (I, II and IV) are classical, zinc-dependent HDACs whereas class III, called sirtuins, require NAD+ to function. By removing the acetyl moieties, the HDACs regulate the gene expression mediated by nuclear receptors; an altered control of this process could therefore lead to an abnormal DNA transcription and the manifestation of several diseases such as neurodegenerative pathologies, viral infections and tumors. Both the inhibition and the activation of some relevant human CA isoforms was investigated in this work of thesis. In particular, from a first series of zinc binders (series I) characterized by a chiral benzylpiperazine scaffold [N. Chiaramonte et al. Eur. J. Med. Chem. (2018), 151, 363-375], two related generations of piperazine-based Carbonic Anhydrases Inhibitors (CAIs) were developed and studied (series II-III) [N. Chiaramonte et al. Bioorg. Chem. (2019), 91, 103130]. As a result of the collaboration with Prof. Bernhard Wünsch of the University of Münster (GE), the preparation of the III series was carried out mainly in Germany. As far as Carbonic Anhydrase activators (CAAs) are concerned, structural modifications of histamine, the first CAA reported in the literature, and Clonidine led to the generation of two series (V-VI) of positive modulators of this enzyme. Additionally, the CA activation profile of a set of commercially available piperazines (series VII) was also assessed [A. Angeli et al. J. Enz. Inhib. & Med. Chem. (2018), 33,1, 303-308]. Through a poly-pharmacological approach, the inhibition of both the CAs and the HDACs was investigated by hybridizing a coumarin moiety (CA inhibitor) with the pan-HDAC inhibitor SAHA (series IV). The contemporary inhibition of these targets could be beneficial in the treatment of several diseases, among which tumors. The biological characterization of these derivatives on some human CAs is described and discussed in this thesis, while the assays against the HDACs are still ongoing.