Synthèse et évaluations pharmacologiques de nouveaux inhibiteurs β-lactamiques de la Fatty Acid Amide Hydrolase humaine (hFAAH) mise en évidence d’un mécanisme d’inhibition réversible et compétitif

Research on cannabis effects has evolved in a medicinal context towards the discovery and the understanding of the endocannabinoid system. Most of the effects, beneficial or psychotropic, produced by the cannabis consumption have been ascribed to the activation of two receptors, called cannabinoid receptors CB1 and CB2, by a liposoluble molecule the tetrahydrocannabinol (THC). Displaying the existence of these two receptors has accelerated the research about an eventual endogenous cannabinoid or “endocannabinoid” system which might regulate similar effects (e. g. inflammation modulation, nociception, appetite stimulation). Later, two derivatives of arachidonic acid, the arachidonoylethanolamide (anandamide) and the arachidonylglycerol (2-AG), were isolated and shown to play this role. Indeed, these two neurotransmitters produced “on demand” both activate the cannabinoid receptors which induce various similar physiological effects to those produced by THC, without inducing the psychotropic ones. This last property signs the originality and the advantages of the endocannabinoid versus cannabinoid system. However, to take advantage of endocannabinoid system for therapeutic use (against inflammation or pain), the regulation system of the endogenous ligands, which is composed of degradation enzymes, must be counteract. Fatty Acid Amide Hydrolase (FAAH) and Monoacylglycerol lipase (MAGL) are both responsible of the degradation of anandamide and 2-AG respectively, limiting their action. Our project took place in a highly competitive context where the discovery and synthesis of new inhibitors of FAAH constitutes a real challenge. Successfully, our efforts drove us to the discovery of a novel reversible β-lactamic inhibitor of FAAH. This manuscript is presented as a compilation of a review and three articles published in scientific journals.C’est dans un contexte médicinal que la recherche sur les effets du cannabis a évolué vers la découverte puis la compréhension du système endocannabinoïde. La plupart des effets, bénéfiques ou psychotropes, engendrés par la consommation de cannabis a été attribuée à l’activation de deux récepteurs, appelés récepteurs cannabinoïdes CB1 et CB2, par une molécule liposoluble le tétrahydrocannabinol (THC). La mise en évidence de ces deux récepteurs, jusqu’alors orphelins, a été le point de départ concernant l’hypothèse qu’un système cannabinoïde endogène ou « endocannabinoïde » puisse réguler le même genre d’effets (e. g. modulation de l’inflammation, perception de la douleur, stimulation de l’appétit). Plusieurs années se sont écoulées avant d’isoler et de démontrer que deux dérivés de l’acide arachidonique biosynthétisés dans l’organisme, l’arachidonoyléthanolamide (anandamide) et l’arachidonylglycérol (2-AG), jouaient effectivement ce rôle. En effet, ces deux neurotransmetteurs produits « à la demande » activent tous les deux les récepteurs cannabinoïdes entraînant ainsi divers effets physiologiques similaires à ceux du THC, sans toutefois engendrer les effets psychotropes. Cette dernière propriété signe l’originalité et les avantages du système endocannabinoïde versus cannabinoïde. Afin d’exploiter le système endocannabinoïde à des fins thérapeutiques (anti-inflammatoire, anti- douleur), il faut cependant contrer le système de régulation des ligands endogènes mettant en jeu plusieurs enzymes de dégradation. La Fatty Acid Amide Hydrolase (FAAH) et la Monoacylglycerol lipase (MAGL) sont les deux enzymes responsables de la dégradation de l’anandamide et du 2-AG respectivement, limitant ainsi leur action. A l’heure actuelle, de nombreux inhibiteurs de ces deux enzymes ont été décrits comme actifs in vitro et in vivo, augmentant ainsi localement la concentration d’endocannabinoïdes et l’intensité des effets mesurés. C’est dans un contexte très compétitif que se situe notre projet de thèse, à savoir la découverte de nouveaux inhibiteurs de la FAAH humaine. Connaissant la structure de la FAAH et sachant qu’elle appartient à la grande famille des protéases à sérine, nous avons utilisé le savoir-faire du laboratoire dans le domaine des β-lactames (antibiotiques) pour adapter un précurseur de carbapénèmes (inhibiteur de DD-peptidases bactériennes) à notre objectif. Pour ce faire, nous avons tout d’abord synthétisé un panel de composés lipophiles que nous avons testés in vitro sur la FAAH et la MAGL humaine. Cette première étape nous a permis de dégager une structure « lead » active sélectivement sur la FAAH vis-à-vis de la MAGL. Après avoir montré pour la première fois que des β-lactames pouvaient être également de bons inhibiteurs de la FAAH humaine, nous avons optimisé cette structure et réalisé une étude relation structure-activité (RSA) jusqu’à obtenir une inhibition nanomolaire (IC50 = 5,3 nM). Par la suite, le mode d’action de nos β-lactames a été étudié par des méthodes originales développées dans cette thèse. Nous avons pu ainsi mettre en évidence une inhibition compétitive réversible, mécanisme tout à fait unique dans la famille des inhibiteurs d’hydrolases à sérine de type β-lactame. Ce manuscrit est présenté sous la forme d’un recueil composé d’une revue et de trois articles publiés dans des revues scientifiques.(CHIM 3) -- UCL, 201

Inhibitors of the Endocannabinoid-Degrading Enzymes, or how to Increase Endocannabinoid's Activity by Preventing their Hydrolysis

Endocannabinoids are lipid transmitters binding and activating the cannabinoid receptors. Both cannabinoid receptors and endocannabinoids, such as 2-arachidonoylglycerol and anandamide, have been shown to control numerous physiological and pathological processes, including in the central nervous system. Thus regulating endocannabinoid levels in-vivo represents an interesting therapeutic perspective in several CNS-related diseases. To date four enzymes - Fatty Acid Amide Hydrolase (FAAH), N-Acylethanolamine-hydrolyzing Acid Amidase (NAAA), Monoacylglycerol Lipase (MAGL), α/β-Hydrolase Domain 6 (ABHD6) - were shown to control endocannabinoid levels in tissues or in intact cells. While the searches for NAAA and ABHD6 inhibitors are still in their beginning, a growing number of selective and potent inhibitors are now available to inhibit FAAH and MAGL activities. Here, based on the literature and patent literature, we review the compounds of the different chemical families that have been developed to inhibit these enzymes, with a special emphasis on FAAH and MAGL inhibitors

An unprecedented reversible mode of action of β-lactams for the inhibition of human fatty acid amide hydrolase (hFAAH)

A series of compound was prepared to clarify the reversible mechanism of β-lactamic hFAAH inhibitors on the one hand, and to modulate some of their physicochemical parameters on the other hand. In particular, two compounds (4b and 4e) were designed to display a potential good leaving group on the crucial carbonyl with a view to possibly acylating the active serine of the hFAAH catalytic triad. Reversibility studies showed that these two compounds retain the reversible mode of inhibition, suggesting a noncovalent interaction between our β-lactams and hFAAH. Finally, pharmacological evaluations of bioisosteres of the lead compound (4a, IC(50) = 5.3 nM) revealed that log P values and PSA could be optimized without altering the FAAH inhibition (IC(50) values from 3.65 nM to 70.9 nM)

Vibrational Circular Dichroism versus Optical Rotation Dispersion and Electronic Circular Dichroism for diastereomers: the stereochemistry of 3-(1′-hydroxyethyl)-1-(3′-phenylpropanoyl)-azetidin-2-one

The absolute configuration of a relatively large and conformationally flexible chiral compound, 3-(1′-hydroxyethyl)-1-(3′-phenylpropanoyl) -azetidin-2-one, is determined using Vibrational Circular Dichroism (VCD) spectroscopy, Optical Rotation Dispersion (ORD) and Electronic Circular Dichroism (ECD). To that end a state of the art experimental VCD spectrum is compared to a theoretical spectrum and the absolute configuration is assigned. ORD and ECD are also used in the assignment to investigate the complementarity of the three techniques. VCD spectroscopy is found to have important advantages over ORD and ECD for diastereomers. The concept of robust modes is applied to this conformationally flexible molecule, showing that its use is limited for such large and flexible molecules

Beta-lactams derived from a carbapenem chiron are selective inhibitors of human fatty acid amide hydrolase versus human monoacylglycerol lipase

A library of 30 beta-lactams has been prepared from (3R,4R)-3-[(R)-1'-(tbutyldimethylsilyloxy)-ethyl]-4-acetoxy-2-azetidinone, and the corresponding deacetoxy derivative, by sequential N- and O-functionalizations with various omega-alkenoyl and omega-arylalkanoyl chains. All compounds were selective inhibitors of hFAAH versus hMGL, and IC(50) values in the nanomolar range (5-14 nM) were recorded for the best representatives. From time-dependent preincubation and rapid dilution studies, and from docking analyses in a homology model of the target enzyme, a reversible mechanism of inhibition of hFAAH is proposed

β-Lactams as inhibitors of human Fatty Acid Amide Hydrolase: synthesis and pharmacological testing

A series of lipophilic ester derivatives (2a-g) of (S)-1-(pent-4’-enoyl)-4-(hydroxymethyl)-azetidin-2-one has been synthesized in three steps from (S)-4-(benzyloxycarbonyl)-azetidin-2-one and evaluated as novel, reversible, -lactamic inhibitors of endocannabinoid-degrading enzymes (hFAAH and hMAGL). The compounds showed IC50 values in the micromolar range and selectivity for hFAAH versus hMAGL. The unexpected thousandfold decrease of activity of 2a comparatively to the known regioisomeric structure 1a (i.e. lipophilic chains placed on N1 and C3 positions of the -lactam core) could be explained on the basis of docking studies into a revisited model of hFAAH active site, considering one or two water molecules in interaction with the catalytic triad

(S)-1-(Pent-4’-enoyl)-4-(hydroxymethyl)-azetidin-2-one derivatives as inhibitors of human fatty acid amide hydrolase (hFAAH): synthesis, biological evaluation and molecular modelling

A series of lipophilic ester derivatives (2a-g) of (S)-1-(pent-4’-enoyl)-4-(hydroxymethyl)-azetidin-2-one has been synthesized in three steps from (S)-4-(benzyloxycarbonyl)-azetidin-2-one and evaluated as novel, reversible, ?-lactamic inhibitors of endocannabinoid-degrading enzymes (hFAAH and hMAGL). The compounds showed IC50 values in the micromolar range and selectivity for hFAAH versus hMAGL. The unexpected thousandfold decrease of activity of 2a comparatively to the known regioisomeric structure 1a (i.e. lipophilic chains placed on N1 and C3 positions of the ?-lactam core) could be explained on the basis of docking studies into a revisited model of hFAAH active site, considering one or two water molecules in interaction with the catalytic triad