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Tesi etd-03212014-113804

Thesis type
Tesi di laurea specialistica LC5
Design, synthesis and biological evaluation of heterocyclic derivatives as potential MAGL inhibitors
Corso di studi
relatore Prof.ssa Manera, Clementina
relatore Dott.ssa Arena, Chiara
Parole chiave
  • neuroprotection
  • neuroinflammation
  • MAGL
  • endocannabinoid system
  • cancer
Data inizio appello
Riassunto analitico
Monoacylglycerol lipase (MAGL) is a membrane-associated cytosolic serine hydrolase with the highest expression levels in the brain, white adipose tissue and liver. MAGL catalyses the hydrolysis of monoacylglycerols into fatty acids and glycerol and in particular the endocannabinoid 2-arachidonoylglycerol (2-AG) is one of its main substrates The lipid messenger 2-AG is synthesized by phospholipase C (PLC)-mediated cleavage of membrane phospholipids, resulting in the release of diacylglycerol (DAG) that is then hydrolysed by diacylglycerol lipase (DAGL). 2-AG acts primarily by binding to both cannabinoid G protein-coupled receptors CB1R and CB2R; 2-AG is either synthesized on demand or likely associated to binding proteins in spatially separated pools. In the brain, MAGL is responsible for about 80% of the hydrolysis of 2-AG while the remaining 20% arises mainly from the activity of other two serine hydrolases, α,β-hydrolase-6 (ABHD-6) and -12 (ABHD-12). The pharmacological inhibition of MAGL activity in vivo leads to a strong accumulation of 2-AG, in particular in the brain which triggers a higher activation of CB1R and CB2R resulting in analgesic, anxiolytic, antidepressant, sleep-enhancing and anti-inflammatory effects. Recently, it has been shown that in some tissues, such as brain, liver and lung, 2-AG acts as a precursor for the arachidonic acid production. Arachidonic acid is mainly oxygenated by cyclooxygenase-2 to generate pro-inflammatory eicosanoids such as prostaglandin-E2 and –D2. Therefore, the analgesic and anti-inflammatory effects induced by MAGL inhibition can be dependent on two underlying mechanisms, the first by directly increasing 2-AG levels and the second, by lowering the arachidonic acid formation as previously described in brain. Thus, MAGL could represent the link between two lipids signalling pathways: the endocannabinoid and the eicosanoid systems. For these reasons, the modulation of MAGL activity can lead to several beneficial effects through either enhancing the tone of the endocannabinoid system or lowering eicosanoids production. Furthermore in cancer, MAGL plays a distinct role in controlling global FFAs levels that serve as the building blocks for synthesis of pro-tumorigenic signaling lipids such as PGE2 and lysophosphatidic acid (LPA).
Figure 1. Lipid signaling pathways coordinately regulated by MAGL.
Therefore, MAGL inhibitors may represent an attractive therapeutic approach for the treatment of pain, inflammation, anxiety, neurodegeneration, cancer and other disorders. Furthermore, because MAGL inhibitors do not exert control over AA and prostaglandin pathways in the gastrointestinal system, they also do not induce the gastrointestinal bleeding associated with the use of dual COX1/COX2 inhibitors.
Since mechanistically MAGL is a serine hydrolase, the first wide group of inhibitors are the general serine-hydrolase inhibitors that bind in either a reversible or an irreversible covalent manner to the nucleophilic serine. Chemically, they are distinguished in three main reactive groups: fluorophosphonates, trifluoromethylketones and sulfonylfluorides.
The second group of inhibitors was inspired by the endogenous substrate 2-AG and then several 1-AG derivatives were synthesized.
Finally, de novo inhibitors were studied, whose structures do not resemble any endogenous cannabinoid. Their general structure is characterized by flat and hydrophobic pharmacophoric points containing a linker group connected with polar pharmacophore. One of these compounds the carbamate derivative URB 602 was reported as a selective MAGL inhibitor with relatively low potency. However, URB602 administration has been shown to attenuate nociception in rodent models of acute, inflammatory, and neuropathic pain.
In my thesis work, we designed and synthetized compounds as analogues of URB602. The new compounds were obtained following the general scheme of synthesis reported below.
These novel compounds were tested using a HPLC-based assay, previously developed in our research laboratory, employing human recombinant MAGL as enzyme and 4-nitrophenylacetate (4-NPA) as enzymatic substrate; The assay allows to evaluate MAGL enzymatic activity trough chromatographic separation and subsequent absorbance measurement of p-nitrophenol (PNP), obtained as metabolite after MAGL-mediated cleavage of 4-NPA. The inhibitory activity of compounds tested was related to the residual enzymatic activity, and evaluated by quantitative detection of PNP formation, at the optimal wavelength of 315 nm.