Rational design and synthesis of 2-oxo-1,2-dihydropyridine-3-carboxamide derivatives as novel endocannabinoid system modulators

The Endocannabinoid System (ECS) is a complex lipid signaling system composed by at least two cannabinoid receptors (CBRs), CB1R and CB2R, endogenous bioactive lipids known as endocannabinoids (ECs), the most studied of which are anandamide (AEA) and 2-arachidonoylglycerol (2-AG), the EC transporter and the enzymes for ligand biosynthesis and inactivation. CBRs are G protein-coupled receptors modulated by ECs or exogenous ligands. CB1R is localized mainly in the central nervous system (CNS) and it is the most abundant receptor in the mammalian brain, while CB2R is expressed primarily in cells of the immune and hematopoietic systems, but recently was found to be present also in the brain. The ECS was reported to be implicated in a wide range of disparate diseases and pathological conditions, such as mood and anxiety disorders, movement disorders (Parkinson's and Huntington's disease), neuropathic pain, multiple sclerosis, spinal cord injury, cancer, atherosclerosis, myocardial infarction, stroke, hypertension, glaucoma, obesity/metabolic syndrome and osteoporosis. Therefore, soon after the discovery of CBRs, drugs directly targeting these receptors have been studied. Unfortunately, this approach was found to be responsible of numerous side effects, such as anxiety, depression, suicidal ideation, psychotropic effects or immune dysfunction, that have limited the clinical development of these new drugs. In order to overcome these adverse effects, two different approaches for modulating ECS can be developed: allosteric modulation of the CBRs and multi-target strategy. Allosteric ligands bind to CBR allosteric sites, which are topographically distinct from orthosteric sites, altering the receptor signaling properties of the orthosteric ligand, changing ligand affinity, functional efficacy, and functional potency. Indeed allosteric modulation offers several advantages: subtype specificity, because of the higher sequence divergence at allosteric binding sites; tissue selectivity, because the effects are exerted only where endogenous ligands are present; dependence on endogenous ligands for signaling since their effect is saturable; biased signaling/probe-dependence towards orthosteric ligands that preferentially engage certain signaling interactions rather than other. Regarding multi-target compounds, they might exert their “pro-cannabinoid” activities by acting via multiple mechanisms of action within the ECS and represent a promising pharmacological modulation of the ECS, since the simultaneous modulation of two or more targets might offer a safer and more effective pharmacological strategy if compared to a single target modulation. In the laboratory where I performed my thesis the first CB2R positive allosteric modulator (PAM) A was synthetized and tested in vitro and in vivo. In order to better understand the structural requirements for the binding to CB2Rs allosteric site, during my thesis work, analogs of compound A with general structure B differently substituted in position 1, were synthesized. Moreover, another part of my thesis concerned the design and synthesis of bitopic compounds. Bitopic compounds bind to both allosteric and orthosteric sites simultaneously. These new compounds may have some important advantages since the allosteric portion provides receptor subtype and tissue selectivity, while the orthosteric portion promotes receptor binding and activation. Bitopic ligands may demonstrate to be particularly useful in situations where endogenous agonist tone is progressively lost, such as in neurodegenerative disorders, since the agonist ligand and the allosteric modulator are administered simultaneously. Also, the allosteric modulator may enhance the affinity of the orthosteric ligand demonstrating additive or synergistic therapeutic effects, leading to the administration of a lower dose. Our aim was to synthesize bitopic ligands obtained linking the pharmacophoric portion of allosteric compound B with that of orthosteric agonists. In a previous research program conducted in the laboratory where I performed my thesis the unselective CBRs orthosteric agonist compound C was synthesized and tested in vitro and in vivo. During my thesis, C was modified in position 1. All these new compounds will be tested for their functional activity