Polypharmacological profile of 1,2-dihydro-2-oxo-pyridine-3-carboxamides in the endocannabinoid system

Cannabis sativa L. belongs to the family of Cannabaceae and it has been used for centuries for medical or recreational purposes. The first reference to its use dates back to the Neolithic age in China and Japan. Cannabis consumption has raised various medical and social questions, many of which have been under continuing scientific investigation, especially since the mid-1960s, when delta-9-THC (tetrahydrocannabinol) was first isolated and produced synthetically. This compound is one of 113 cannabinoids identified in cannabis and it is the principal psychoactive constituent of this plant. In 1964 studies of the delta-9-THC action led to the hypothesis that exogenous cannabinoids had to act through a system of specific cell receptors and in 1990 a specific receptor was identified in the rat brain, which is G-protein coupled, called CB1R. Later the first endogenous substance, called anandamide (AEA), and another receptor, CB2R, were discovered. At this point, many studies were performed in order to understand the biological role of the endocannabinoid system (ECS). This system is 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 AEA uptake and the enzymes for ECs biosynthesis and inactivation (MAGL, FAAH, ABHD6 and ABHD12). Nowadays we know that the ECS is involved in different physiological and pathological processes including cancer, appetite, memory, neuropathic and inflammatory pain, obesity, neuroprotection and neurodegenerative diseases. Numerous CB1R and CB2R ligands have been developed and tested in vitro and in vivo as a new approach in the context of drug discovery, but none of them reached an advanced stage of clinical development due to central or peripheral side effects that are mainly associated to chronic activation or blocking of CB1R. Alternative strategies were based on the increase of AEA and 2-AG levels by inhibiting their enzymatic degradation. With this strategy the cannabinoid receptors were activated directly by their endogenous ligands. However, in preclinical animal models, the repeated administrations of covalent MAGL inhibitors rapidly induced CB1R desensitization in the brain, while chronic ablation of FAAH activity may be associated to potential side effects on the cardiovascular and metabolic system. Recent findings highlighted the emerging role of each target of the ECS to control symptoms and progression of several diseases. For this reason, it is reasonable to assume that the simultaneous modulation of more targets of the ECS might offer a safer and more effective pharmacological strategy as compared to the single target modulation. The main objective of our research project consists in studying the role of multi-target modulators of the ECS that exerts “pro-cannabinoid” activities by acting with different mechanisms of action. Some previous studies were performed to test the behaviour of 4-substituted and 4,5-disubstituted 1,2-dihydro-2-oxo-pyridine-3-carboxamide derivatives of general structures reported in Figure 1. These compounds showed a broad spectrum of affinity towards both CBR subtypes and some of them selectively inhibited the endocannabinoid degrading enzymes FAAH, ABHD6, ABHD12 and AEA uptake. The new compounds, studied during my thesis work, are characterized by substituents in 5,6 position of the pyridine nucleus and by a p-fluorobenzyl moiety at the N-1 position of the 1,2-dihydro-2-oxo-pyridine ring (R1) which was chosen on the basis of the best results obtained with the previous series. This substituent was also shifted to the oxygen in position 2 of the heterocyclic nucleus. Furthermore, they are characterized by various carboxamides in position 3. The new compounds, synthesized by the research group of Professor Clementina Manera, were evaluated for their binding affinity and functional activity for both cannabinoid receptors (CB1R, CB2R) and for their effects on the main components of the ECS (like FAAH, MAGL, ABHDs and AEA uptake), during my thesis period spent at the Biochemistry and Molecular Medicine in the University of Bern under supervision of Prof. Andrea Chicca. The binding affinities (Ki values) of target compounds were evaluated in competitive radioligand displacement assays against [3H]CP-55,940 using membrane preparations obtained in-house from stable transfected CHO-hCB1 and CHO-hCB2 cells. The compounds which showed the most potent CBRs affinity values were investigated for their functional activity using a [35S]GTPgS assay. In order to evaluate the inhibitory potency and the selectivity on FAAH, MAGL and ABHDs, enzyme activity assays were also performed using the corresponding radiolabelled substrates to measure the amounts of hydrolytic product. For FAAH, MAGL and AEA uptake assay the cell homogenate used in the experiments was obtained from U937 cells. Regarding ABHD12 or ABHD6 assay, the homogenate was prepared from HEK293T-ABHD12 or HEK293T-ABHD-6 respectively using non-transfected HEK293T cells as control. All experiments were performed at least three times in triplicate. In this master thesis all the data obtained from polypharmacological studies of these compounds are discussed and reported in detail