Giorgio Ortar

Novel selective and metabolically stable inhibitors of anandamide cellular uptake

Novel aromatic analogues of N-oleoylethanolamine and N-arachidonoylethanolamine (anandamide, AEA) were synthesized and, based on the capability of similar compounds to interact with proteins of the endocannabinoid and endovanilloid signaling systems, were tested on: (i) cannabinoid CB(1) and CB(2) receptors; (ii) vanilloid VR1 receptors; (iii) anandamide cellular uptake (ACU); and (iv) the fatty acid amide hydrolase (FAAH). The (R)- and, particularly, the (S)-1-(4-hydroxybenzyl) derivatives of N-oleoylethanolamine and AEA (OMDM-1, OMDM-2, OMDM-3, and OMDM-4) inhibited to a varied extent ACU in RBL-2H3 cells (K(i) ranging between 2.4 and 17.7 micro M), the oleoyl analogues (OMDM-1 and OMDM-2, K(i) 2.4 and 3.0 micro M, respectively) being 6- to 7-fold more potent than the arachidonoyl analogues (OMDM-3 and OMDM-4). These four compounds exhibited: (i) poor affinity for either CB(1) (K(i)> or = 5 micro M) or CB(2) (K(i)>10 micro M) receptors in rat brain and spleen membranes, respectively; (ii) almost no activity at vanilloid receptors in the intracellular calcium assay carried out with intact cells over-expressing the human VR1 (EC(50)> or = 10 micro M); and (iii) no activity as inhibitors of FAAH in N18TG2 cell membranes (K(i)>50 micro M). The oleoyl- and arachidonoyl-N-(4-hydroxy-3-methoxybenzyl)hydrazines (OMDM-5 and OMDM-6), inhibited ACU (K(i) 4.8 and 7.0 micro M, respectively), and were more potent as VR1 agonists (EC(50) 75 and 50nM, respectively), weakly active as CB(1) receptor ligands (K(i) 4.9 and 3.2 micro M, respectively), and inactive as CB(2) ligands (K(i)>5 micro M) as well as on FAAH (K(i)> or = 40 micro M). In conclusion, we report two novel potent and selective inhibitors of ACU (OMDM-1 and OMDM-2) and one hybrid agonist of CB(1) and VR1 receptors (OMDM-6). Unlike other compounds of the same type, OMDM-1, OMDM-2, and OMDM-6 were very stable to enzymatic hydrolysis by rat brain homogenates.

Further evidence for the existence of a specific process for the membrane transport of anandamide.

Indirect evidence for the existence of a specific protein-mediated process for the cellular uptake of endocannabinoids has been reported, but recent results suggested that such a process, at least for AEA [ N -arachidonoylethanolamine (anandamide)], is facilitated uniquely by its intracellular hydrolysis by FAAH (fatty acid amide hydrolase) [Glaser, Abumrad, Fatade, Kaczocha, Studholme and Deutsch (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 4269-4274]. In the present study, we show that FAAH alone cannot account for the facilitated diffusion of AEA across the cell membrane. In particular, (i) using a short incubation time (90 s) to avoid AEA hydrolysis by FAAH, AEA accumulation into rat basophilic leukaemia or C6 cells was saturable at low microM concentrations of substrate and non-saturable at higher concentrations; (ii) time-dependent and, at low microM concentrations of substrate, saturable AEA accumulation was observed also using mouse brain synaptosomes; (iii) using synaptosomes prepared from FAAH-deficient mice, saturable AEA accumulation was still observed, although with a lower efficacy; (iv) when 36 AEA and N -oleoylethanolamine analogues, most of which with phenyl rings in the polar head group region, were tested as inhibitors of AEA cellular uptake, strict structural and stereochemical requirements were needed to observe significant inhibition, and in no case the inhibition of FAAH overlapped with the inhibition of AEA uptake; and (v) AEA biosynthesis by cells and sensory neurons was followed by AEA release, and this latter process, which cannot be facilitated by FAAH, was still blocked by an inhibitor of AEA uptake. We suggest that at least one protein different from FAAH is required to facilitate AEA transport across the plasma membrane in a selective and bi-directional way.

Pharmacological modulation of the endocannabinoid system in a viral model of multiple sclerosis

Theilers virus infection of the central nervous system (CNS) induces an immune‐mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). Cannabinoids have been shown to exert beneficial effects on animal models of MS and evidence suggests that the endocannabinoid system plays a role in the tonic control of spasticity. In this study we show that OMDM1 [(R)‐N‐oleoyl‐(1′‐hydroxybenzyl)‐2′‐ethanolamine] and OMDM2 [(S)‐N‐oleoyl‐(1′‐hydroxybenzyl)‐2′‐ethanolamine], two selective inhibitors of the putative endocannabinoid transporter and hence of endocannabinoid inactivation, provide an effective therapy for Theiler murine encephalomyelitis virus‐induced demyelinating disease (TMEV‐IDD). Treatment of TMEV‐infected mice with OMDM1 and OMDM2 enhanced anandamide levels in the spinal cord and ameliorated motor symptoms. This was associated with a down‐regulation of inflammatory responses in the spinal cord. In addition we show that OMDM1 and OMDM2 down‐regulate macrophage function by (i) decreasing the surface expression of major histocompatibility complex (MHC) class II molecules, (ii) inhibiting nitric oxide synthase‐2 (NOS‐2) expression and (iii) reducing the production of the pro‐inflammatory cytokines interleukin‐1beta (IL‐1β) and interleukin‐12 (IL‐12p40). Taken together, these results point to the manipulation of the endocannabinoid system as a possible strategy to develop future MS therapeutic drugs.

A new, palladium-catalyzed synthesis of aromatic mercapturic acid derivatives

Abstract N -Acetyl-L-cysteine methyl ester ( 1 ) reacts under mild conditions with aryl iodides 2 in the presence of a palladium catalyst prepared in situ from Pd 2 dba 3 and dppf to give S -aryl derivatives 3 in moderate to good yields.

Conjugate addition vs. vinylic substitution in palladium-catalysed reaction of aryl halides with β-substituted-α,β-enones and -enals

Abstract The reaction of β-substituted-α,β-enones and -enals with aryl halides in the presence of a palladium catalyst has been investigated. The outcome of the reaction was found to be greatly dependent on the nature of the added base. Tertiary amines tend to favour the formation of conjugate addition-type products while sodium bicarbonate or sodium acetate that of vinylic substitution products. Usually, the use of sodium acetate produced results comparable or better than those obtained with sodium bicarbonate. Furthermore, in the reaction of aryl halides containing strongly electronwithdrawing groups, sodium acetate afforded a dramatic increase in the yield of vinylic substitution products.

A palladium-catalyzed carbonylative route to primary amides

Abstract The palladium-catalyzed reaction of aryl and vinyl iodides and triflates with carbon monoxide in the presence of hexamethyldisilazane followed by hydrolytic work-up affords aromatic and α,β-unsaturated primary amides in good to high yields under relatively mild conditions.

Palladium-catalyzed preparation of exo-aryl derivatives of the norbornane skeleton

Abstract Norbornene and norbornadiene derivatives have been shown to react with aryl and vinyl halides in the presence of a palladium catalyst, formic acid, and tributylamine or piperidine to give hydroarylated and hydrovinylated derivatives in good to high yield. Extension of the reaction to the hindered α,β-unsaturated aldehydic system of myrtenal produced a monocyclic derivative through a palladium-catalyzed ring opening.

Palladium-catalyzed cross-coupling reactions of vinyl and aryl triflates with tetraarylborates

Abstract Sodiumm tetraarylborates 2 have been found to couple efficiently with vinyl and aryl triflates 1 in the presence of a palladium(0) catalyst to afford arylalkenes and biaryls 3 in good yields and under mild conditions.

An efficient synthesis of 3-substituted indoles by palladium-catalyzed coupling reaction of 3-tributylstannylindoles with organic triflates and halides

Abstract The palladium-catalyzed reaction of 1-tosyl-3-tributylstannylindoles 2 with a variety of vinyl, aryl, and heteroaryl triflates and halides ( 1 ) provides a general and efficient method for the synthesis of 3-substituted indoles 3 .

An analytical approach to the forensic identification of different classes of new psychoactive substances (NPSs) in seized materials

RATIONALEnNew psychoactive substances (NPSs) are rapidly spreading worldwide, and forensic laboratories are often requested to identify new substances for which no reference standards or analytical data are available. This article describes an analytical approach that was adopted in Italy by a few collaborative centres of the Italian Early Warning System for Drugs, which has contributed many alerts for the identification of different classes of NPSs in the last 24u2009months.nnnMETHODSnSeized crystals and powders were initially analysed via single quadrupole gas chromatography/mass spectrometry (GC/MS), followed by liquid chromatography/high-resolution mass spectrometry (LC/HRMS) in the positive electrospray ionisation (ESI) mode at 100,000 full width at half maximum resolution (FWHM) without fragmentation to elucidate the elemental compositions of unknown molecules. Different fragmentation voltages during LC/HRMS were applied to study the accurate masses of the obtained characteristic fragments. Nuclear magnetic resonance (NMR) analyses were performed to identify specific isomers when necessary.nnnRESULTSnSome interesting examples of unknown NPSs from seizures later identified in our laboratories are reported, with special focus on those cases where analytical standards were not available during analyses. These cases include cathinones, such as 3-methylmethcathinone (3-MMC), methylone, bk-MBDB (butylone), 4-methylethcathinone (4-MEC), flephedrone, methylenedioxypyrovalerone (MDPV) and pentedrone, methoxetamine, apinaca or AKB48, benzydamine, meta-chlorophenylpiperazine (m-CPP), 5-MeO-N,N-dialkyl tryptamines, such as 5-MeO-DALT and 5-MeOMIPT, benzofurans, such as 6-APB and 4-APB, and diphenidine (identified for the first time in Europe).nnnCONCLUSIONSnThe identification of NPSs in confiscated materials was successfully achieved via GC/MS coupled with LC/HRMS and, in a few cases, NMR analyses. The availability of GC/MS libraries is of great assistance in the identification of new drugs. Alternatively, the study of characteristic molecule fragments combined with the determination of their accurate masses can be a useful approach to identify unknown samples not previously analysed.