DEVELOPMENT OF NOVEL GPR55 LIGANDS AND GPR55/CB2R MULTITARGET MODULATORS FOR NEURODEGENERATIVE DISEASES

Neurodegenerative diseases lead to the loss of specific neuronal populations, with the consequent development of several impairments in cognitive functions, movement and memory. [1] Despite specific pathological triggers, all these disorders are characterized by a common microglia-mediated neuroinflammation. [2] [3] [4] Thus, functional modulation of microglia could represent a novel therapeutic option to counteract neurodegenerative processes. In the last years, the endocannabinoid system (ECS) has emerged as an innovative target, involved in the regulation of various pathological processes, including neurodegenerative disorders. In particular, the "type 2" cannabinoid receptor (CB2R) is mostly expressed in immune cells and tissues, [5] but it has also been detected in microglia cells, especially under neuroinflammatory conditions. [6] [7] At this level, its activation through agonists shifts the microglia from the M1 neurodegenerative state to the M2 neuroprotective state, promoting the resolution of the neuroinflammation. [8] GPR55 is a G-protein coupled receptor initially included among the orphan receptors and later accompanied to the endocannabinoid system, being proposed as the "type 3" cannabinoid receptor (CB3R). [9] It recently emerged as another innovative therapeutic target implicated in the modulation of microglia-mediated neuroinflammation, even if its precise role has not been yet completely understood. [7] [10] Thus, the development of potent and selective GPR55 ligands represents a key step to validate this receptor as pharmacological target and to explore its role in neuroinflammation. However, up to date their design remains challenging, given the lack of information about the GPR55 3D crystal structure and the limited number of ligands reported in the literature, most of which are poorly selective. [11] Thus, in the framework of the development of novel molecules useful in neurodegenerative disorders, my PhD thesis can be divided in two main topics: • Design and synthesis of novel GPR55 ligands, which also represents a key step to understand the role of GPR55 in neuroinflammation, as well as providing potential tool compounds to investigate the structural requirements for the interaction with its binding site. • Design and synthesis of novel multitarget compounds, by connecting two pharmacophoric portions able to modulate both GPR55 and CB2 receptors. Concerning the design and synthesis of novel GPR55 ligands, we developed a novel class of 3-benzylquinolin-2(1H)-one derivatives, starting from 3-benzylcoumarins, recently reported as GPR55 antagonists, with IC50 values in the low micromolar range. [12] In particular, we changed the stereo-electronic properties of the central coumarin scaffold through the replacement of the lactone group with an amide moiety, introducing also additional modifications on the substituents at certain positions of the 3-benzylquinolin-2(1H)-one central core. Some of the newly synthesized compounds displayed excellent binding properties, with Ki values included in the low nanomolar range, resulting to be among the most potent and selective GPR55 agonists developed to date. Among them, the 7-butyl-5-methoxy-3-(2-methoxybenzyl)quinolin-2(1H)-one 1B showed also complete selectivity over both cannabinoid receptors. [13] Based on results obtained from computational studies, [13] a second series of 3-benzylquinolin-2(1H)-ones has been designed and synthesized, in order to deepen a structure-activity relationship for this class of molecules. During my study abroad experience at University of Saskatchewan, Canada, I worked on the evaluation of the affinity and the activity of these synthesized compounds toward hGPR55, as well as selectivity over hCB1R and hCB2R. Some of them showed high affinity toward GPR55 receptor, with Ki values included in the low nanomolar or in the sub-nanomolar range and good selectivity over one or both cannabinoid receptors. In particular, the 5-hydroxy-3-(2-hydroxybenzyl)-1,7-dimethylquinolin-2(1H)-one 3C and the 5-hydroxy-3-(2-hydroxybenzyl)-1-methyl-7-octylquinolin-2(1H)-one 4D resulted as the most affine of the set, with Ki value of 0.51 nM and 0.29 nM, respectively. Interestingly, structural changes shifted the activity from pure agonism to inverse agonism and among them, the 5-methoxy-3-(2-methoxybenzyl)-1,7-dimethylquinolin-2(1H)-one 1C resulted as the most potent inverse agonist of the set. Final compounds were characterized not only via proton and carbon-13 nuclear magnetic resonance (1H-NMR and 13C-NMR), but also through high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS), that I learned during my training in Toscana Life Sciences (TLS), Siena. Moreover, during my training in TLS, we selected derivatives 1B and 1C as the most promising compounds of the first and the second series, respectively, and I performed metabolic assays, in order to provide preliminary information regarding in vivo behaviour. The development of novel multitarget compounds was achieved by connecting two pharmacophoric portions able to modulate both GPR55 and CB2 receptors through alkyl-triazole linkers of different length. Regarding GPR55, we selected a 3-benzylcoumarine derivative KIT17, which proved to be a GPR55 antagonist in the β-arrestin recruitment assay [12] and showed neuroprotective properties in LPS-activated primary microglia cells. [10] Concerning CB2 receptor, a class of naphthyridine-2-ones, which resulted as potent and selective CB2R agonists, [14] has been selected. Among the developed compounds, the 1-(4-(4-(((3-benzyl-2-oxo-2H-chromen-6-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)butyl)-N-((1s,4s)-4-methylcyclohexyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide 18 and the 1-(5-(4-(3-((3-benzyl-5,7,8-trimethyl-2-oxo-2H-chromen-6-yl)oxy)propyl)-1H-1,2,3-triazol-1-yl)pentyl)-N-((1s,4s)-4-methylcyclohexyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide 22 proved to be able to bind both GPR55 and CB2R, with Ki values included in the low nanomolar range. In order to investigate other innovative therapeutic targets useful in the treatment of neurodegenerative disorders, I also took part on a study focused on the development of new ligands of CB1 receptor [15] and on a project concerning a virtual screening for the identification of novel inhibitors of cyclin dependent kinase 5 (Cdk5). [16] During my abroad experience at University of Saskatchewan, Canada, I also took part on a study concerning the evaluation of the effect of some cannabinoid derivatives reported in literature in an in vivo mouse model of anhedonia, which seems to be associated with a down-regulation of both ECS and orexin system. This work demonstrated that combined stimulation of endocannabinoid and orexin systems provided an increase in consumption of sucrose because of an attenuation of chronical stress, confirming their involvement in anhedonia regulation. [17] REFERENCES [1] T.H. Bak, S. 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