Research paperDesign, synthesis and biological evaluation of second-generation benzoylpiperidine derivatives as reversible monoacylglycerol lipase (MAGL) inhibitors
Graphical abstract
Introduction
The endocannabinoid system (ECS) is composed of two seven-transmembrane G protein-coupled receptors (GPCRs) named cannabinoid receptors type-1 and type-2 (CB1R, CB2R), a family of lipophilic molecules that binds to CB receptors called endocannabinoids (eCBs) and several biosynthetic and degrading enzymes involved in the production and metabolism of eCBs. CB1R is one of the most abundant GPCRs in mammalian brain and it is highly expressed in neuronal cells, where it regulates neurotransmitter release. CB2R is prevalently expressed in immune cells, where it controls the activation state during inflammation. Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are the most important eCBs and are biosynthesized on-demand from phospholipid precursors in the inner leaflet of the plasma membrane and released into the extracellular environment [1]. After activating CB receptors, eCBs are transported into the cytoplasm via facilitated diffusion mediated by a putative eCB membrane transporter. [2,3] Intracellular eCBs degradation is mediated by fatty acid amide hydrolase (FAAH) for AEA, and by monoacylglycerol lipase (MAGL) and α/β hydrolase-6 and -12 (ABHD6 and ABHD12) for 2-AG. MAGL is the main hydrolytic enzyme for 2-AG and it is responsible for approximately 85% of 2-AG hydrolysis in the brain, with a minor contribution of ABHD6 and ABHD12. Modulation of eCB levels represents a promising pharmacological strategy to activate the ECS without the typical side effects associated with direct CB1R agonists [4]. Several studies indicate the therapeutic potential of selective eCB reuptake inhibitors, as well as of selective FAAH and MAGL inhibitors in different animal disease models of inflammation, pain, anxiety and other neuroinflammatory diseases [3,5,6].
In the last decades, many academic research groups and pharmaceutical industries have focused their research on the discovery of new MAGL inhibitors, and some of the most representative inhibitors are reported in Fig. 1 [6,7]. MAGL inhibitors reported in the literature can be classified on the basis of their mechanism of action: 1) irreversible inhibitors, which bind covalently to the enzyme, and usually permanently block its catalytic activity; 2) reversible inhibitors, which interact with MAGL for a limited time, and then the activity of the protein is restored. Irreversible inhibitors are usually highly potent, with inhibition activities in the low nanomolar range; however, they manifested some drawbacks when tested in in vivo studies. Fig. 1 shows three representative irreversible MAGL inhibitors, i.e. derivative 1 (JZL184, 4-nitrophenyl-4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate, Fig. 1) [8], 2 (CAY10499, benzyl(4-(5-methoxy-2-oxo-1,3,4-oxadiazol-3(2H)-yl)-2-methylphenyl)carbamate, Fig. 1) [9], and 3 (ABX-1431, 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-[[2-(pyrrolidin-1-yl)-4-(trifluoromethyl)phenyl]methyl]piperazine-1-carboxylate) [10]. In detail, genetic deletion and irreversible MAGL inhibition determine the loss of CB1R-mediated biological effects and induce cross-tolerance to exogenous CB1R agonists [[11], [12], [13], [14], [15]]. These effects are provoked by prolonged pharmacological blockage or genetic inactivation of MAGL, which leads to an excessive increase of 2-AG concentration (5-10-times over basal levels), which triggers CB1R desensitization. Moreover, MAGL-deficient mice are characterized by an impaired CB1R-dependent synaptic plasticity and physical dependence [11]. These aspects may represent a serious limitation for the therapeutic use of most irreversible MAGL inhibitors, especially for chronic treatments. In the last years, MAGL inhibitors endowed with a reversible mode of action gained more interest in the scientific community as potential alternative strategy to modulate MAGL. Initially, only natural compounds, such as the terpenoid Euphol 4 (Fig. 1), a potent MAGL inhibitor with an IC50 value in the nanomolar range [16], and the triterpenoid β-amyrin 5 (Fig. 1) [17], less potent than Euphol, were identified as reversible MAGL inhibitors. However, they are not selective for MAGL: Euphol was active on other targets, affecting the cell cycle of cancer cells [18], whereas β-amyrin inhibited ABHDs too [17]. Hernández-Torres et al. discovered the first synthetic and selective MAGL inhibitor, benzo[d] [1,3]dioxol-5-ylmethyl 6-phenylhexanoate 6 (Fig. 1): this nanomolar inhibitor was able to slow down the clinical progression and to decrease the symptoms of multiple sclerosis in an experimental autoimmune encephalomyelitis mouse model, without inducing unwanted CB1-mediated side effects [19]. Tabrizi et al. disclosed the 1,5-diphenylpyrazole-3-carboxamide 7 (Fig. 1): this reversible MAGL inhibitor mitigated the neuropathic hypersensitivity induced in vivo by oxaliplatin [20]. A series of long-chain salicylketoxime derivatives was developed as MAGL inhibitors and, among them, 8 (Fig. 1) was the most active compound of this series. Compound 8 proved to be selective for MAGL over other targets of the ECS and to exert antiproliferative activity in a series of cancer cells [21]. Very recently, Takeda Pharmaceuticals identified a series of piperazinyl pyrrolidin-2-ones, exemplified by compound 9 (Fig. 1) [22], which showed high potency in vitro (IC50 value in the subnanomolar range). Some derivatives of this class showed promising efficacy in vivo, decreasing arachidonic acid level and increasing 2-AG level in mouse brain. Compound 10 (Fig. 1) belongs to the class of the benzoylpiperidine-based MAGL inhibitors [[23], [24], [25]], developed by our group since 2014. This compound derives from an optimization process of this scaffold, possessing a phenolic amidic moiety and a 4-isopropyl-benzoyl ring, and these portions are both necessary to reach a nanomolar affinity for MAGL.
Section snippets
Design
In the last years, a series of benzoylpiperidine-based compounds was identified and developed as reversible and selective MAGL inhibitors, reaching inhibition values in the nanomolar range. In the present manuscript, the most potent inhibitor so far discovered within this series of compounds (11, Fig. 2) [24] was further optimized, to find more potent MAGL inhibitors belonging to this chemical class. The structural optimization was focused mainly on the phenolic ring (in blue, Fig. 2) as well
Conclusions
In the present work, the class of benzoylpiperidine-based MAGL inhibitors was expanded and optimized, leading to the identification of compounds 21a,b and 22a,b, which are characterized by a potent MAGL inhibition activity, with Ki values ranging from 11 to 37 nM, a reversible mode of action, as well as a high selectivity for MAGL vs. CB1R, CB2R, FAAH, ABHD6 and ABHD12 (IC50 value > 10 μM in all cases). These four derivatives were also tested in intact human monocytic cell line U937, where they
Synthesis. General procedures and materials
All solvents and chemicals were used as purchased without further purification. Chromatographic separations were performed on silica gel columns by flash chromatography (Kieselgel 40, 0.040–0.063 mm; Merck). Reactions were followed by thin layer chromatography (TLC) on Merck aluminum silica gel (60 F254) sheets that were visualized under a UV lamp. Evaporation was performed in vacuo (rotating evaporator). Sodium sulfate was always used as the drying agent. Proton (1H) and carbon (13C) NMR
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We are grateful to the University of Pisa (Progetti di Ricerca di Ateneo, prog. PRA-2018-18), MIUR (PRIN 2017, project 2017SA5837) and the Italian Ministry of Health – Ricerca Finalizzata 2016 - NET-2016-02363765 for funding.
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