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N-Acylethanolamine acid amidase (NAAA) is an N-terminal cysteine hydrolase primarily found in the endosomal-lysosomal compartment of innate and adaptive immune cells. NAAA catalyzes the hydrolytic deactivation of palmitoylethanolamide (PEA), a lipid-derived peroxisome proliferator-activated receptor-α (PPAR-α) agonist that exerts profound anti-inflammatory effects in animal models. Emerging evidence points to NAAA-regulated PEA signaling at PPAR-α as a critical control point for the induction and the resolution of inflammation and to NAAA itself as a target for anti-inflammatory medicines. The present Perspective discusses three key aspects of this hypothesis: the role of NAAA in controlling the signaling activity of PEA; the structural bases for NAAA function and inhibition by covalent and noncovalent agents; and finally, the potential value of NAAA-targeting drugs in the treatment of human inflammatory disorders.

Objectives URB937, a peripheral fatty acid amide hydrolase (FAAH) inhibitor, exerts profound analgesic effects in animal models. We examined, in rats, (1) the pharmacokinetic profile of oral URB937; (2) the compound's ability to elevate levels of the representative FAAH substrate, oleoylethanolamide (OEA); and (3) the compound's tolerability after oral administration. Methods We developed a liquid chromatography/tandem mass spectrometry (LC/MS-MS) method to measure URB937 and used a pre-existing LC/MS-MS assay to quantify OEA. FAAH activity was measured using a radioactive substrate. The tolerability of single or repeated (once daily for 2 weeks) oral administration of supramaximal doses of URB937 (100, 300, 1000 mg/kg) was assessed by monitoring food intake, water intake and body weight, followed by post-mortem evaluation of organ structure. Key findings URB937 was orally available in male rats (F = 36%), but remained undetectable in brain when administered at doses that maximally inhibit FAAH activity and elevate OEA in plasma and liver. Acute and subchronic treatment with high doses of URB937 was well-tolerated and resulted in FAAH inhibition in brain. Conclusions Pain remains a major unmet medical need. The favourable pharmacokinetic and pharmacodynamic properties of URB937, along with its tolerability, encourage further development studies on this compound.

© 2015 American Chemical Society. Fatty acid ethanolamides such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are lipid-derived mediators that potently inhibit pain and inflammation by ligating type-α peroxisome proliferator-activated receptors (PPAR-α). These bioactive substances are preferentially degraded by the cysteine hydrolase, N-acylethanolamine acid amidase (NAAA), which is highly expressed in macrophages. Here, we describe a new class of β-lactam derivatives that are potent, selective, and systemically active inhibitors of intracellular NAAA activity. The prototype of this class deactivates NAAA by covalently binding the enzymes catalytic cysteine and exerts profound anti-inflammatory effects in both mouse models and human macrophages. This agent may be used to probe the functions of NAAA in health and disease and as a starting point to discover better anti-inflammatory drugs.

Many known 5-HT7 ligands contain either a serotonin-like or an arylpiperazine structure that, in published SAR studies, are generally supposed to bind the same receptor pocket. Conversely, we explored the hypothesis that two such moieties can co-exist in the same ligand, binding to different pockets. We thus designed and synthesized a set of compounds including both a 5-hydroxyindol-3-ylethyl and a 1-arylpiperazine moieties connected by a short linker. The compounds were tested for their affinity for human 5-HT7 serotonin receptor. We further prepared a novel series of 5-HT7 ligands, where the 5-hydroxyindol-3-ylethyl moiety was bioisosterically replaced by a 3-hydroxyanilinoalkyl one. Among the newly synthesized compounds, potent ligands at the 5-HT7 receptor, behaving as antagonists in functional tests, were identified, even if they showed limited subtype selectivity. Docking studies within a model of the 5-HT7 receptor showed that the binding site can actually accommodate both moieties, with the serotonin-like one in the putative orthosteric site and the arylpiperazine one occupying an accessory pocket. The present results demonstrate that it is possible to devise and develop new 5-HT7 ligands merging two privileged structures in the same molecule.

N-Acylethanolamine acid amidase (NAAA) is a cysteine hydrolase that catalyzes the hydrolysis of endogenous lipid mediators such as palmitoylethanolamide (PEA). PEA has been shown to exert anti-inflammatory and antinociceptive effects in animals by engaging peroxisome proliferator-activated receptor α (PPAR-α). Thus, preventing PEA degradation by inhibiting NAAA may provide a novel approach for the treatment of pain and inflammatory states. Recently, 3-aminooxetan-2-one compounds were identified as a class of highly potent NAAA inhibitors. The utility of these compounds is limited, however, by their low chemical and plasma stabilities. In the present study, we synthesized and tested a series of N-(2-oxoazetidin-3-yl)amides as a novel class of NAAA inhibitors with good potency and improved physicochemical properties, suitable for systemic administration. Moreover, we elucidated the main structural features of 3-aminoazetidin-2-one derivatives that are critical for NAAA inhibition.

Melatonin (MLT) is a neurohormone implicated in several physiological processes such as sleep. Contrasting results have been produced on whether or not it may act as a hypnotic agent, and the neurobiological mechanism through which it controls the vigilance states has not yet been elucidated. In this study we investigated the effect of MLT (40 mg/kg), a non-selective MT1/MT2 receptor agonist (UCM793, 40 mg/kg), and a selective MT2 partial agonist (UCM924, 40 mg/kg) on the 24-h vigilance states. EEG and EMG sleep-wake patterns were registered across the 24-h light-dark cycle in adult Sprague-Dawley male rats. MLT decreased (-37%) the latency to the first episode of non rapid eye movement sleep (NREMS), enhanced the power of NREMS delta band (+33%), but did not alter the duration of any of the three vigilance states. Differently, UCM793 increased the number of episodes (+52%) and decreased the length of the episodes (-38%) of wakefulness but did not alter the 24-h duration of wakefulness, NREMS and REMS. UCM924 instead reduced the latency (-56%) and increased (+31%) the duration of NREMS. Moreover, it raised the number of REMS episodes (+57%) but did not affect REMS duration. Taken together, these findings show that MLT and non-selective MT1/MT2 receptor agonists do not increase the quantity of sleep but differently influence the three vigilance states. In addition, they support the evidence that selective MT2 receptor agonists increase NREMS duration compared to MLT and non-selective MT1/MT2 agonists.

The design of compounds selective for the MT1 melatonin receptor is still a challenging task owing to the limited knowledge of the structural features conferring selectivity for the MT1 subtype, and only few selective compounds have been reported so far. N-(Anilinoalkyl)amides are a versatile class of melatonin receptor ligands that include nonselective MT1/MT2 agonists and MT2-selective antagonists. We synthesized a new series of N-(anilinoalkyl)amides bearing 3-arylalkyloxy or 3-alkyloxy substituents at the aniline ring, looking for new potent and MT1-selective ligands. To evaluate the effect of substituent size and shape on binding affinity and intrinsic activity, both flexible and conformationally constrained derivatives were prepared. The phenylbutyloxy substituent gave the best result, providing the partial agonist 4 a, which was endowed with high MT1 binding affinity (pKi=8.93) and 78-fold selectivity for the MT1 receptor. To investigate the molecular basis for agonist recognition, and to explain the role of the 3-arylalkyloxy substituent, we built a homology model of the MT1 receptor based on the β2 adrenergic receptor crystal structure in its activated state. A binding mode for MT1 agonists is proposed, as well as a hypothesis regarding the receptor structural features responsible for MT1 selectivity of compounds with lipophilic arylalkyloxy substituents.

The cysteine amidase N-acylethanolamine acid amidase (NAAA) is a member of the N-terminal nucleophile class of enzymes and a potential target for anti-inflammatory drugs. We investigated the mechanism of inhibition of human NAAA by substituted β-lactones. We characterized pharmacologically a representative member of this class, ARN077, and showed, using high-resolution liquid chromatography-tandem mass spectrometry, that this compound forms a thioester bond with the N-terminal catalytic cysteine in human NAAA. © 2012 American Chemical Society.

New derivatives of 4-phenyl-2-propionamidotetralin (4-P-PDOT) were prepared and tested on cloned MT1 and MT2 receptors, with the purpose of merging previously reported pharmacophores for nonselective agonists and for MT2-selective antagonists. A 8-methoxy group increases binding affinity of both (±)-cis- and (±)-trans-4-P-PDOT, and it can be bioisosterically replaced by a bromine. Conformational analysis of 8-methoxy-4-P-PDOT by molecular dynamics, supported by NMR data, revealed an energetically favored conformation for the (2S,4S)-cis isomer and a less favorable conformation for the (2R,4S)-trans one, fulfilling the requirements of a pharmacophore model for nonselective melatonin receptor agonists. A new superposition model, including features characteristic of MT2-selective antagonists, suggests that MT1/MT2 agonists and MT2 antagonists can share the same arrangement for their pharmacophoric elements. The model correctly predicted the eutomers of (±)-cis- and (±)-trans-4-P-PDOT. The model was validated by preparing three dihydronaphthalene derivatives, either able or not able to reproduce the putative active conformation of 4-P-PDOT.

We report the synthesis, binding properties and intrinsic activity at MT(1) and MT(2) melatonin receptors of new dimeric melatonin receptor ligands in which two units of the monomeric agonist N-{2-[(3-methoxyphenyl)methylamino]ethyl}acetamide (1) are linked together through different anchor points. Dimerization of compound 1 through the methoxy substituent leads to a substantial improvement in selectivity for the MT(1) receptor, and to a partial agonist behavior. Compound 3a, with a trimethylene linker, was the most selective for the MT(1) subtype (112-fold selectivity) and compound 3d, characterized by a hexamethylene spacer, had the highest MT(1) binding affinity (pK(iMT1)=8.47) and 54-fold MT(1)-selectivity. Dimerization through the aniline nitrogen of 1 abolished MT(1) selectivity, leading to compounds with either a full agonist or an antagonist behavior depending on the nature of the linker.

5-HT(6) receptor is one of the most recently cloned serotonin receptors, and it might play important roles in Alzheimer's disease, depression, and learning and memory disorders. Availability of only very few 5-HT(6) receptor agonists, however, does not allow examining their contribution in psychopharmacological processes. Therefore, a new 5-HT(6) receptor agonist, ST1936, was synthesized. ST1936 binds to human 5-HT(6) receptors with good affinity (K(i)=28.8 nM). ST1936 also exhibited some moderate binding affinity for 5HT(2B), 5HT(1A), 5HT(7) receptors and adrenergic α receptors. ST1936 behaved as a full 5-HT(6) agonist on cloned cells and was able to increase Ca(2+) concentration, phosphorylation of Fyn kinase, and regulate the activation of ERK1/2 that is a downstream target of Fyn kinase. These effects were completely antagonized by two 5-HT(6) receptor antagonists, SB271046 and SB258585. The other 5-HT(6) receptor agonist, WAY181187 also increased Fyn kinase activity. These results suggest that both ST1936 and WAY181187 mediate 5-HT(6) receptor-dependent signal pathways, such as cAMP, Fyn and ERK1/2 kinase, as specific agonists.

The fatty acid ethanolamides (FAEs) are a family of bioactive lipid mediators that include the endogenous agonist of peroxisome proliferator-activated receptor-alpha, palmitoylethanolamide (PEA). FAEs are hydrolyzed intracellularly by either fatty acid amide hydrolase or N-acylethanolamine-hydrolyzing acid amidase (NAAA). Selective inhibition of NAAA by (S)-N-(2-oxo-3-oxetanyl)-3-phenylpropionamide [(S)-OOPP, 7a] prevents PEA degradation in mouse leukocytes and attenuates responses to proinflammatory stimuli. Starting from the structure of 7a, a series of beta-lactones was prepared and tested on recombinant rat NAAA to explore structure-activity relationships (SARs) for this class of inhibitors and improve their in vitro potency. Following the hypothesis that these compounds inhibit NAAA by acylation of the catalytic cysteine, we identified several requirements for recognition at the active site and obtained new potent inhibitors. In particular, (S)-N-(2-oxo-3-oxetanyl)biphenyl-4-carboxamide (7h) was more potent than 7a at inhibiting recombinant rat NAAA activity (7a, IC(50) = 420 nM; 7h, IC(50) = 115 nM) in vitro and at reducing carrageenan-induced leukocyte infiltration in vivo.

The scope and limitations of using palladium-catalyzed cross-coupling reactions of diverse butyl metal species with two different 2-halopurines were evaluated. While tributylboranes reacted readily and regioselectively with both 2-chloro-6-dibenzylaminopurines and 2-iodo-6-chloropurines, all the other alkyl metal species were much less reactive and gave very poor yield and/or selectivity of the desired product. This protocol was applied to the synthesis of an important adenosine A(2A) receptor antagonist, ST1535.

Melatonin is a modified tryptophan with potent biological activity, exerted by stimulation of specific plasma membrane (MT1/MT2) receptors, by lower affinity intracellular enzymatic targets (quinone reductase, calmodulin), or through its strong anti-oxidant ability. Scattered studies also report a perplexing pro-oxidant activity, showing that melatonin is able to stimulate production of intracellular reactive oxygen species (ROS). Here we show that on U937 human monocytes melatonin promotes intracellular ROS in a fast (

Cannabinoid CB1receptor antagonists reduce body weight in rodents and humans, but their clinical utility as anti-obesity agents is limited by centrally mediated side effects. Here, we describe the first mixed CB1antagonist/CB2agonist, URB447 ([4-amino-1-(4-chlorobenzyl)-2-methyl-5-phenyl-1H-pyrrol-3-yl](phenyl)me thanone), which lowers food intake and body-weight gain in mice without entering the brain or antagonizing central CB1-dependent responses. URB447 may provide a useful pharmacological tool for investigating the cannabinoid system, and might serve as a starting point for developing clinically viable CB1antagonists devoid of central side effects. © 2009 Elsevier Ltd.

Author(s): Valitutti, Giovanni; Duranti, Andrea; Mor, Marco; Piersanti, Giovanni; Piomelli, Daniele; Rivara, Silvia; Tontini, Andrea; Tarzia, Giorgio; Traldi, Pietro

Background: The endocannabinoid anandamide may be involved in the regulation of emotional reactivity. In particular, it has been shown that pharmacological inhibition of the enzyme fatty acid amide hydrolase (FAAH), which catalyzes the intracellular hydrolysis of anandamide, elicits anxiolytic-like and antidepressant-like effects in rodents. Methods: We investigated the impact of chronic treatment with the selective FAAH inhibitor, URB597 (also termed KDS-4103), on the outcomes of the chronic mild stress (CMS) in rats, a behavioral model with high isomorphism to human depression. Results: Daily administration of URB597 (.3 mg·kg-1, intraperitoneal [IP]) for 5 weeks corrected the reduction in body weight gain and sucrose intake induced by CMS. The antidepressant imipramine (20 mg·kg-1, once daily, IP) produced a similar response, whereas lower doses of URB597 were either marginally effective (.1 mg·kg-1) or ineffective (.03 mg·kg-1). Treatment with URB597 (.3 mg·kg-1) resulted in a profound inhibition of brain FAAH activity in both CMS-exposed and control rats. Furthermore, the drug regimen increased anandamide levels in midbrain, striatum, and thalamus. Conclusions: URB597 exerts antidepressant-like effects in a highly specific and predictive animal model of depression. These effects may depend on the ability of URB597 to enhance anandamide signaling in select regions of the brain. © 2007 Society of Biological Psychiatry.

Author(s): Valitutti, Giovanni; Duranti, Andrea; Lodola, Alessio; Mor, Marco; Piersanti, Giovanni; Piomelli, Daniele; Rivara, Silvia; Tontini, Andrea; Tarzia, Giorgio; Traldi, Pietro

N-Acylethanolamine acid amidase (NAAA) is a lysosomal cysteine hydrolase involved in the degradation of saturated and monounsaturated fatty acid ethanolamides (FAEs), a family of endogenous lipid agonists of peroxisome proliferator-activated receptor-alpha, which include oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). The beta-lactone derivatives (S)-N-(2-oxo-3-oxetanyl)-3-phenylpropionamide (2) and (S)-N-(2-oxo-3-oxetanyl)-biphenyl-4-carboxamide (3) inhibit NAAA, prevent FAE hydrolysis in activated inflammatory cells, and reduce tissue reactions to pro-inflammatory stimuli. Recently, our group disclosed ARN077 (4), a potent NAAA inhibitor that is active in vivo by topical administration in rodent models of hyperalgesia and allodynia. In the present study, we investigated the structure activity relationship (SAR) of threonine-derived beta-lactone analogues of compound 4. The main results of this work were an enhancement of the inhibitory potency of beta-lactone carbamate derivatives for NAAA and the identification of (4-phenylpheny1)-methyl-N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl]carbamate (14q) as the first single-digit nanomolar inhibitor of intracellular NAAA activity (IC50 = 7 nM on both rat NAAA and human NAAA).

Inhibition of adenosine A2A receptors has been shown to elicit a therapeutic response in preclinical animal models of Parkinson's disease (PD). We previously identified the triazolo-9H-purine, ST1535, as a potent A(2A)R antagonist. Studies revealed that ST1535 is extensively hydroxylated at the ω-1 position of the butyl side chain. Here, we describe the synthesis and evaluation of derivatives in which the ω-1 position has been substituted (F, Me, OH) in order to block metabolism. The stability of the compounds was evaluated in human liver microsomes (HLM), and the affinity for A(2A)R was determined. Two compounds, (2-(3,3-dimethylbutyl)-9-methyl-8-(2H-1,2,3-triazol-2-yl)-9H-purin-6-amine (3 b) and 4-(6-amino-9-methyl-8-(2H-1,2,3-triazol-2-yl)-9H-purin-2-yl)-2-methylbutan-2-ol (3 c), exhibited good affinity against A(2A)R (Ki =0.4 nM and 2 nM, respectively) and high in vitro metabolic stability (89.5% and 95.3% recovery, respectively, after incubation with HLM for two hours).

Neuropathic pain is an important public health problem for which only a few treatments are available. Preclinical studies show that melatonin (MLT), a neurohormone acting on MT1 and MT2 receptors, has analgesic properties, likely through MT2 receptors. Here, we determined the effects of the novel selective MLT MT2 receptor partial agonist N-{2-([3-bromophenyl]-4-fluorophenylamino)ethyl}acetamide (UCM924) in 2 neuropathic pain models in rats and examined its supraspinal mechanism of action. In rat L5-L6 spinal nerve ligation and spared nerve injury models, UCM924 (20-40 mg/kg, subcutaneously) produced a prolonged antinociceptive effect that is : (1) dose-dependent and blocked by the selective MT2 receptor antagonist 4-phenyl-2-propionamidotetralin, (2) superior to a high dose of MLT (150 mg/kg) and comparable with gabapentin (100 mg/kg), but (3) without noticeable motor coordination impairments in the rotarod test. Using double staining immunohistochemistry, we found that MT2 receptors are expressed by glutamatergic neurons in the rostral ventrolateral periaqueductal gray. Using in vivo electrophysiology combined with tail flick, we observed that microinjection of UCM924 into the ventrolateral periaqueductal gray decreased tail flick responses, depressed the firing activity of ON cells, and activated the firing of OFF cells; all effects were MT2 receptor-dependent. Altogether, these data demonstrate that selective MT2 receptor partial agonists have analgesic properties through modulation of brainstem descending antinociceptive pathways, and MT2 receptors may represent a novel target in the treatment of neuropathic pain.

The O-arylcarbamate URB597 (cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester; also referred to as KDS-4103) is a potent inhibitor of fatty-acid amide hydrolase (FAAH), an intracellular serine hydrolase responsible for the inactivation of the endogenous cannabinoid anandamide. URB597 demonstrates a remarkable degree of selectivity for FAAH over other serine hydrolases (e.g. cholinesterases) or other components of the endocannabinoid system (e.g. cannabinoid receptors). However, in a proteomic-based selectivity screen based on the displacement of fluorophosphonate-rhodamine (FPR) from mouse brain proteins, it was recently shown that URB597 prevents FPR binding to triacylglycerol hydrolase (TGH) with a median inhibitory concentration of 192 nM. To determine whether this effect correlates with inhibition of TGH activity, we investigated the ability of URB597 to inhibit triolein hydrolysis in rat liver and heart tissues, which are rich in TGH, as well as white adipose tissue (WAT), which is rich in adipose triacylglycerol lipase (TGL) and hormone-sensitive lipase. The results show that URB597 does not affect triolein hydrolysis in any of these tissues at concentrations as high as 10 μM, whereas it inhibits FAAH activity at low nanomolar concentrations. Moreover, intraperitoneal (i.p.) administration of URB597 at doses that maximally inhibit FAAH in vivo (0.3-3 mg kg-1) exerts no effect on triolein hydrolysis and tissue triacylglycerol (TAG) levels in rat liver, heart or WAT. The results indicate that URB597, while potent at inhibiting FAAH, does not affect TGH and TGL activities in rat tissues. © 2006.

Herein we report attempts to optimize the pharmacological properties of 5-(2-hydroxyethoxy)-N-acetyltryptamine (5-HEAT), a melatonin receptor ligand previously described by us. Several 5-substituted and 2,5-disubstituted N-acyltryptamines were synthesized and evaluated in vitro for the human cloned MT(1) and MT(2) receptors. From this series of N-acyltryptamines the 2-bromo derivative (5 c) retains the interesting efficacy profile of 5-HEAT and shows increased melatonin receptor affinities; it represents one of the first examples of a high-affinity MT(1) agonist/MT(2) antagonist. Some other full agonists for both melatonin receptors which exhibit similar or increased affinity relative to that of melatonin were obtained.

3-(Acetylaminomethyl)-2-(ethoxycarbonyl)-6-methoxy-1,3,4,5-tetrahydrobenzo[cd]indole (2) is a rigid melatonin analogue that as a racemate displays about the same affinity and intrinsic activity of melatonin (1) in in vitro experiments. We report here the resolution of the racemate by preparative medium pressure liquid chromatography (MPLC) and the X-ray determination of the R absolute configuration of the (-)-enantiomer. The two enantiomers were separately tested as MT1 and MT2 ligands, and the (+)-(S)-2 showed a potency comparable to that of melatonin and about three orders of magnitude greater than that of its enantiomer. The information obtained by crystallographic analysis and NMR studies about the conformational preference for 2 and by the pharmacological characterization of (R)-2 and (S)-2 was employed in a molecular modeling study, aimed at reassessing the melatonin receptor pharmacophore model for agonist compounds. Chiral enantioselective agonists reported in the literature were also included in the study.

Three-dimensional homology models of human MT(1) and MT(2) melatonin receptors were built with the aim to investigate the structure-activity relationships (SARs) of MT(2) selective antagonists. A common interaction pattern was proposed for a series of structurally different MT(2) selective antagonists, which were positioned within the binding site by docking and simulated annealing. The proposed antagonist binding mode to the MT(2) receptor is characterized by the accommodation of the out-of-plane substituents in a hydrophobic pocket, which resulted as being fundamental for the explanation of the antagonist behavior and the MT(2) receptor selectivity. Moreover, to assess the ability of the MT(2) receptor model to reproduce the SARs of MT(2) antagonists, three new derivatives of the MT(2) selective antagonist N-[1-(4-chloro-benzyl)-4-methoxy-1H-indol-2-ylmethyl]-propionamide (7) were synthesized and tested for their receptor affinity and intrinsic activity. These compounds were docked into the MT(2) receptor model and were submitted to molecular dynamics studies, providing results in qualitative agreement with the experimental data. These results confirm the importance of the out-of-plane group in receptor binding and selectivity and provide a partial validation of the proposed G protein-coupled receptor model.

Fatty acid amide hydrolase (FMH) is an intracellular serine enzyme that catalyzes the hydrolysis of bioactive fatty acid ethanolamides such as anandamide and oleoylethanolamide (OEA). Genetic deletion of the faah gene in mice elevates brain anandamide levels and amplifies the effects of this endogenous cannabinoid agonist. Here, we show that systemic administration of the selective FAAH inhibitor URB597 (cyclohexyl carbamic acid 3′-carbamoyl-biphenyl-3-yl ester; 0.3 mg/kg i.p.) increases anandamide levels in the brain of rats and wild-type mice but has no such effect in FAAH-null mutants. Moreover, URB597 enhances the hypothermic actions of anandamide (5 mg/kg i.p.) in wild-type mice but not in FAAH-null mice. In contrast, the FAAH inhibitor does not affect anandamide or OEA levels in the rat duodenum at doses that completely inhibit FAAH activity. In addition, URB597 does not alter the hypophagic response elicited by OEA (5 and 10 mg/kg i.p.), which is mediated by activation of peroxisome proliferator-activated receptor type-α. Finally, exogenously administered OEA (5 mg/kg i.p.) was eliminated at comparable rates in wild-type and FAAH-/- mice. Our results indicate that URB597 increases brain anandamide levels and magnifies anandamide responses by inhibiting intracellular FAAH activity. The results also suggest that an enzyme distinct from FAAH catalyzes OEA hydrolysis in the duodenum, where this lipid substance acts as a local satiety factor. Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics.

Following the disclosure of 3-(1,2,2-trimethylpropyl) 4-[3,5-dimethyl-2-propyloxycarbonyl]pyrrolecarboxylate as a potent and selective mGluR1 non-competitive antagonist, the role and the importance of the pyrrole template were investigated. Different aromatic moieties were investigated as possible bio-isosteric replacement of the original scaffold and some of them were shown to be partially able to mimic the properties of the original pyrrole ring.

Oleylethanolamide (OEA) is a naturally occurring lipid that regulates satiety and body weight. Although structurally related to the endogenous cannabinoid anandamide, OEA does not bind to cannabinoid receptors and its molecular targets have not been defined. Here we show that OEA binds with high affinity to the peroxisome-proliferator-activated receptor-alpha (PPAR-alpha), a nuclear receptor that regulates several aspects of lipid metabolism. Administration of OEA produces satiety and reduces body weight gain in wild-type mice, but not in mice deficient in PPAR-alpha. Two distinct PPAR-alpha agonists have similar effects that are also contingent on PPAR-alpha expression, whereas potent and selective agonists for PPAR-gamma and PPAR-beta/delta are ineffective. In the small intestine of wild-type but not PPAR-alpha-null mice, OEA regulates the expression of several PPAR-alpha target genes: it initiates the transcription of proteins involved in lipid metabolism and represses inducible nitric oxide synthase, an enzyme that may contribute to feeding stimulation. Our results, which show that OEA induces satiety by activating PPAR-alpha, identify an unexpected role for this nuclear receptor in regulating behaviour, and raise possibilities for the treatment of eating disorders.

New penicillin, penicillin sulfone and sulfoxide derivatives bearing a C-6-alkyliden substituent were prepared. Their chemical synthesis, in vitro antibacterial activity and inhibition properties against two selected enzymes representing Class A and C β-lactamases are reported. Compounds 3a – c , 7a – c were able to inhibit either TEM-1 (a Class A enzyme, from Escherichia coli ) or P-99 (a Class C enzyme, from E. cloacae ), or both enzymes, when tested in competition experiments using nitrocefin as the reporter substrate. However, when tested in combination with amoxicillin, the same compounds did not show synergistic effects against E. coli and E. cloacae strains producing TEM-1 and P99 enzymes, respectively. This finding is most likely related to poor penetration through the bacterial cell wall, as shown by using a more permeable isogenic E. coli strain. Interestingly, a synergistic effect against a strain of S. aureus which produces PC1-enzyme (a Class A β-lactamase) was observed for compound 3a when used in combination with amoxicillin.

The blood-brain barrier (BBB) is the main entry route for chemicals into the mammalian central nervous system (CNS). Two transmembrane transporters of the ATP-binding cassette (ABC) family - breast cancer resistance protein (ABCG2 in humans, Abcg2 in rodents) and P-glycoprotein (ABCB1 in humans, Abcb1 in rodents) - play a key role in mediating this process. Pharmacological and genetic evidence suggests that Abcg2 prevents CNS access to a group of highly potent and selective O-arylcarbamate fatty-acid amidohydrolase (FAAH) inhibitors, which include the compound URB937 (cyclohexylcarbamic acid 3′-carbamoyl-6-hydroxybiphenyl-3-yl ester). To define structure-activity relationships of the interaction of these molecules with Abcg2, in the present study we tested various peripherally restricted and non-restricted O-arylcarbamate FAAH inhibitors for their ability to serve as transport substrates in monolayer cultures of Madin-Darby Canine Kidney-II (MDCKII) cells over-expressing Abcg2. Surprisingly, we found that the majority of compounds tested - even those able to enter the CNS in vivo - were substrates for Abcg2 in vitro. Additional experiments in MDCKII cells overexpressing ABCB1 revealed that only those compounds that were dual substrates for ABCB1 and Abcg2 in vitro were also peripherally restricted in vivo. The extent of such restriction seems to depend upon other physicochemical features of the compounds, in particular the polar surface area. Consistent with these in vitro results, we found that URB937 readily enters the brain in dual knockout mice lacking both Abcg2 and Abcb1, whereas it is either partially or completely excluded from the brain of mice lacking either transporter alone. The results suggest that Abcg2 and Abcb1 act together to restrict the access of URB937 to the CNS. © 2014 Elsevier Ltd.

Several indole analogues of melatonin (MLT) were obtained by moving the MLT side chain from C(3) to C(2) of the indole ring. Binding and in vitro functional assays were performed on cloned human MT1 and MT2 receptors, stably transfected in NIH3T3 cells. Quantitative structure-activity relationship studies showed that 4-methoxy-2-(N-acylaminomethyl)indoles, with a benzyl group in position 1, were selective MT2 antagonists and, in particular, N-[(1-p-chlorobenzyl-4-methoxy-1H-indol-2-yl)methyl]propanamide (12) behaved as a pure antagonist at MT1 and MT2 receptors, with a 148-fold selectivity for MT2. We present a topographical model that suggests a lipophilic group, located out of the plane of the indole ring of MLT, as the key feature of the MT2 selective antagonists.

A number of 6-methoxy-1-(2-propionylaminoethyl)indoles, carrying properly selected substituents at the C-2 indole position, were prepared and tested as melatonin receptor ligands. Affinities and intrinsic activities for the human cloned mt 1 and MT 2 receptors were examined and compared with those of some 2-substituted melatonin derivatives recently described by us. A quantitative structure–activity relationship (QSAR) study of the sixteen 2-substituted indole compounds, 5a – k , 1 , 8 – 11 , using partial least squares (PLS) and multiple regression analysis (MRA) revealed the existence of an optimal range of lipophilicity for the C-2 indole substituent. There are also indications that planar, electron-withdrawing substituents contribute to the affinity by establishing additional interactions with the binding pocket. No mt 1 /MT 2 subtype selectivity was observed, with the relevant exception of the 2-phenethyl derivative 5e , which exhibited the highest selectivity for the h-MT 2 receptor among all the compounds tested (MT 2 /mt 1 ratio of ca. 50). Conformational analysis and superposition of 5e to other reported selective MT 2 ligands revealed structural and conformational similarities that might account for the MT 2 /mt 1 selectivity of 5e .

It has been difficult, so far, to obtain melatonin analogs possessing high selectivity for the respective melatonin receptors, mt 1 and MT 2 . In the present work, we report the synthesis and pharmacological characterization of a new compound N-{2-[5-(2-hydroxyethoxy)-1H-indol-3-yl)] ethyl} acetamide or 5-hydroxyethoxy-N-acetyltryptamine (5-HEAT). To assess the activity of the compound, the following tests were performed: affinity determination for the high- and low-affinity receptor states (2-[ 125 I]iodomelatonin binding), potency and intrinsic activity in inducing G protein activation ([ 35 S]GTPγS binding assay). 5-HEAT showed little selectivity for the mt 1 receptor, with pK i values of 7.77 for mt 1 and 7.12 for the MT 2 receptors, respectively. 5-HEAT was able to differentiate between the high- and the low-affinity receptor states in the mt 1 but not in the MT 2 receptor. 5-HEAT induced a high level of G protein activation when acting through the mt 1 receptor, with a relative intrinsic activity of 0.92. On the contrary, it elicited only minimal MT 2 receptor-mediated G protein activation, with a relative intrinsic activity of 0.16, and was also able to inhibit the melatonin-induced MT 2 receptor-mediated G protein activation, with a pK B value of 7.4. In conclusion, it appears that 5-HEAT possesses very different efficacies at the two melatonin receptors, behaving as a full melatonin receptor agonist at the mt 1 and as an antagonist/weak partial agonist at the MT 2 receptor. Therefore, it is a promising ligand for use in functional studies aimed at distinguishing between the effects mediated by the different melatonin receptors in the human.

We review our work towards the design and synthesis of high-affinity melatonin (N-acetyl-5-methoxytryptamine) agonist and antagonist compounds. High affinity melatonergic agonists were obtained by shifting the melatonin side chain from C3 to N1 of the indole ring system. Conversely, by moving the side chain from C3 to C2 it was possible to obtain melatonin antagonist compounds, albeit of moderate affinity.

This paper reviews our progress made in characterizing structure-affinity relationships of indole-based melatonin analogs. Evidence is presented suggesting a preferred folded conformation for the amido side chain, almost orthogonal to the plane of indole. A 3D-QSAR comparative molecular field analysis (CoMFA) model, accounting for the observed differences in binding affinity within different classes of melatonergic ligands, and capable of quantitatively predicting the binding affinity of new compounds, is also reported.

Twenty 4,5-dimethylpyrrole-2-carboxylic acids (5a-t) with different 2-carbamoylvinyl chains in position 3 were prepared to further investigate the relationships between structure and in vitro affinity for the strychnine-insensitive glycine-binding site. None of these compounds was superior to (E)-3-(N-phenyl-2-carbamoylvinyl)-4,5-dimethylpyrrole-2-carb oxylic acid III (pKi = 6.70), which was taken as a reference standard, but overall the results obtained indicate that the N-phenyl-2-carbamoylvinyl substituent of III may be replaced with the N-(1-adamantyl)-2-carbamoylvinyl group as in 5h (pKi = 6.20) without considerable loss of affinity. This finding adds to previous knowledge.

The electron ionization mass spectrometric behaviour of melatonin (N-acetyl-5-methoxytryptamine) and of a series of its isomers has been studied with the aid of metastable ion experiments. The data show that the different positions of the substituents on the indole ring influence the fragmentation patterns and the relative abundances of the generated ions. © 1998 John Wiley & Sons, Ltd.

The CoMFA methodology was applied to melatonin receptor ligands in order to establish quantitative structure-affinity relationships. One hundred thirty-three compounds were considered: they were either collected from literature or newly synthesized in order to gain information about the less explored positions. To this end, various melatonin derivatives were prepared and their affinity for quail optic tecta melatonin receptor was tested. Compounds were aligned on the putative active conformation of melatonin proposed by our previously reported pharmacophore search, and their relative affinities were calculated from the displacement of 2-[125I]-iodomelatonin on different tissues expressing aMT receptors. Compounds were grouped into three sets according to their topology. Subset A: melatonin-like compounds; subset B: N-acyl-2-amino-8-methoxytetralins and related compounds; subset C:N-acyl-phenylalkylamines and related compounds. CoMFA models were derived for each set, using the steric, electrostatic, and lipophilic fields as structural descriptors; the PLS analyses were characterized by good statistical parameters, taking into account the heterogeneity of the binding data, obtained with different experimental protocols. From the CoMFA model for the melatonin-like compounds, besides the well-known positive effect of 2-substitution, a low steric tolerance for substituents in 1, 6, and 7, and a negative effect of electron-rich 4-substituents were observed; the information provided by the newly synthesized compounds was essential for these results. Moreover, a comprehensive model for the 133 compounds, accounting for a common alignment and a common mode of interaction at the melatonin receptor, was derived (Q2 = 0.769, R2 = 0.905). This model validates our previously reported pharmacophore search and offers a clear depiction of the structure-affinity relationships for the melatonin receptor ligands.

The synthesis of several novel indole melatonin analogues substituted at the 2-position with acylaminomethyl (8-11), acylaminoethyl (5a-k), or acylaminopropyl (13) side chains is reported. On the basis of a novel in vitro functional assay (specific binding of [35S]GTPgammaS), which can discriminate agonist from partial agonist, antagonist, and inverse agonist ligands, 5a,g, h,j and 13 were shown to be partial agonists, 5d,e and 8-11 competitive antagonists, and 5b,c,k putative inverse agonists. Binding and functional assays were performed on cloned human MT1 receptor. Structure-activity relationship considerations indicate that N-[1-aryl-2-(4-methoxy-1H-indol-2-yl)(C1-C2)alkyl]alkanamides represent a lead structure for this type of ligands.

The synthesis and biological evaluation of 3-ureido and 3-carbamate derivatives of 1,5-benzodiazepines bearing bridged cycloalkyl substituents at N-1 are reported. Their activity as CCK-B receptor ligands is briefly discussed.

2,3-Dihydro-6,7-dichloro-pyrido[2,3-b]pyrazine-8-oxide was synthesized and evaluated for in vitro/in vivo antagonistic activity at the strychnine insensitive glycine binding site on the NMDA receptor revealing it to be a useful tool to evaluate the effectiveness of glycine antagonists in vivo.

The synthesis and biological evaluation as potential CCK-B receptor ligands of a number of 1-isopentyl-3-aryloxycarbamoyl-5-aryl-1,5-benzodiazepines substituted with halogen atoms on the benzo-fused ring is here briefly discussed.

Many known 5-HT7 ligands contain either a serotonin-like or an arylpiperazine structure that, in published SAR studies, are generally supposed to bind the same receptor pocket. Conversely, we explored the hypothesis that two such moieties can co-exist in the same ligand, binding to different pockets. We thus designed and synthesized a set of compounds including both a 5-hydroxyindol-3-ylethyl and a 1-arylpiperazine moieties connected by a short linker. The compounds were tested for their affinity for human 5-HT7 serotonin receptor. We further prepared a novel series of 5-HT7 ligands, where the 5-hydroxyindol-3-ylethyl moiety was bioisosterically replaced by a 3-hydroxyanilinoalkyl one. Among the newly synthesized compounds, potent ligands at the 5-HT7 receptor, behaving as antagonists in functional tests, were identified, even if they showed limited subtype selectivity. Docking studies within a model of the 5-HT7 receptor showed that the binding site can actually accommodate both moieties, with the serotonin-like one in the putative orthosteric site and the arylpiperazine one occupying an accessory pocket. The present results demonstrate that it is possible to devise and develop new 5-HT7 ligands merging two privileged structures in the same molecule.

The peripherally restricted fatty acid amide hydrolase (FAAH) inhibitor URB937 (3, cyclohexylcarbamic acid 3′-carbamoyl-6-hydroxybiphenyl-3-yl ester) is extruded from the brain and spinal cord by the Abcg2 efflux transporter. Despite its inability to enter the central nervous system (CNS), 3 exerts profound antinociceptive effects in mice and rats, which result from the inhibition of FAAH in peripheral tissues and the consequent enhancement of anandamide signaling at CB1 cannabinoid receptors localized on sensory nerve endings. In the present study, we examined the structure-activity relationships (SAR) for the biphenyl region of compound 3, focusing on the carbamoyl and hydroxyl groups in the distal and proximal phenyl rings. Our SAR studies generated a new series of peripherally restricted FAAH inhibitors and identified compound 35 (cyclohexylcarbamic acid 3′-carbamoyl-5- hydroxybiphenyl-3-yl ester) as the most potent brain-impermeant FAAH inhibitor disclosed to date. © 2013 American Chemical Society.

Carbamate and urea derivatives are important classes of fatty acid amide hydrolase (FAAH) inhibitors that carbamoylate the active-site nucleophile Ser241. In the present work, the reactivation mechanism of carbamoylated FAAH is investigated by means of a quantum mechanics/molecular mechanics (QM/MM) approach. The potential energy surfaces for decarbamoylation of FAAH covalent adducts, derived from the O-aryl carbamate URB597 and from the N-piperazinylurea JNJ1661610, were calculated and compared to that for deacylation of FAAH acylated by the substrate oleamide. Calculations show that a carbamic group bound to Ser241 prevents efficient stabilization of transition states of hydrolysis, leading to large increments in the activation barrier. Moreover, the energy barrier for the piperazine carboxylate was significantly lower than that for the cyclohexyl carbamate derived from URB597. This is consistent with experimental data showing slowly reversible FAAH inhibition for the N-piperazinylurea inhibitor and irreversible inhibition for URB597. © 2013 American Chemical Society.