DESIGN AND SYNTHESIS OF NEW 2-OXO-1,2-DIHYDROPYRIDINE-3-CYCLOHEPTANECARBOXAMIDE DERIVATIVES AS ALLOSTERIC MODULATORS OF CB2R

The endocannabinoid system (ECS) is a neuromodulatory system, able to modulate different physiological functions. It is composed by cannabinoid receptors, (CB1Rs and CB2Rs), endogenous cannabinoids, Anandamide (AEA) and 2-Arachidonoylglycerol ( 2-AG ), so far the best characterized and enzymes for their synthesis and degradation such as fatty acid amide hydrolase (FAAH), the monoacylglycerol lipase (MAGL) and two α/β hydrolase 6 and 12 (ABHD 6, ABHD12). CB1Rs and CB2Rs belong to the superfamily of G protein-coupled receptors and they present different distribution; CB1Rs are abundant mostly in the central nervous system (CNS), whereas CB2Rs are located mainly in immune tissues, such as macrophages microglia and B and T cells even if, recently, they have been reported to be present also in the CNS. The direct activation of CBRs results in several beneficial effects, in different physiological and pathological processes such as neurodegenerative diseases, cancer, neuropathic pain, inflammation and nausea. For this reason, several CB1R and CB2R ligands have been developed and tested in vitro and in vivo. Unfortunately, none of them reached an advanced stage of clinical development due to several side effects especially on the CNS such as mood alteration (euphoria, anxiety, panic), acute psychoses, impaired cognition and motor performance mostly due to CB1Rs. Consequently, in order to reduce these unwanted side effects generally caused by a direct modulation of CBRs, allosteric modulators have been developed as a new therapeutic strategy. Allosteric modulators bind to allosteric binding sites, which are topographically distinct from orthosteric binding sites, modulating the receptors’ functional state. They show many advantages compared to orthosteric ligands, such as, reduction of side effects, higher specificity of binding, because of the highly conserved sequences of orthosteric binding site rather than the allosteric binding site. Furthermore, they modulate the activity of CBRs only when the orthosteric ligand is present and they might show synergizing effects if co-administrated with other modulators of ECS. They are classified in PAM, NAM and SAM depending on the type of modulation on the affinity and / or efficacy of the orthosteric ligand. PAMs (Positive Allosteric Modulators) and NAMs (Negative Allosteric Modulators) respectively activate or inhibit the receptor response, while SAMs (silent allosteric modulators), do not affect the response of endogenous agonist ligands. Moreover, there are bitopic ligands, that bind to both the orthosteric and allosteric sites. Recently, in the laboratory where I performed my thesis, compound A was synthesized as new CBRs ligand and, unexpectedly, it resulted to behave as CB2R PAM. Its activity was demonstrated through [3H]CP55940 binding assays, in which it enhanced the binding of the unselective CB1R/CB2R agonist CP55940, and [35S]GTPγS assays which confirmed its allosteric behavior. Since the ECS is implicated in neuropathic pain, compound A has been tested for neuropathic pain in a cold allodynia test with mice previously treated with high doses of the chemotherapeutic oxaliplatin. Compound A showed a very interesting activity. During my thesis, in order to define the structural requirements need for the CB2R allosteric modulation I change the substituents at the positions C3, C4 and C5 of the 2-hydroxy-pyridine nucleus of compound A, synthesizing compounds B, C and D. These compounds will be tested, for their binding and functional activity at CBRs, at the University of Aberdeen in professor Pertwee’s laboratory