Federica Vacondio

Anandamide suppresses pain initiation through a peripheral endocannabinoid mechanism

Peripheral cannabinoid receptors exert a powerful inhibitory control over pain initiation, but the endocannabinoid signal that normally engages this intrinsic analgesic mechanism is unknown. To address this question, we developed a peripherally restricted inhibitor (URB937) of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the endocannabinoid anandamide. URB937 suppressed FAAH activity and increased anandamide levels outside the rodent CNS. Despite its inability to access brain and spinal cord, URB937 attenuated behavioral responses indicative of persistent pain in rodent models of peripheral nerve injury and inflammation and prevented noxious stimulus–evoked neuronal activation in spinal cord regions implicated in nociceptive processing. CB1 cannabinoid receptor blockade prevented these effects. These results suggest that anandamide-mediated signaling at peripheral CB1 receptors controls the access of pain-related inputs to the CNS. Brain-impenetrant FAAH inhibitors, which strengthen this gating mechanism, might offer a new approach to pain therapy.

A Second Generation of Carbamate-Based Fatty Acid Amide Hydrolase Inhibitors with Improved Activity in vivo

The fatty acid ethanolamides are a class of signaling lipids that include agonists at cannabinoid and α type peroxisome proliferator‐activated receptors (PPARα). In the brain, these compounds are primarily hydrolyzed by the intracellular serine enzyme fatty acid amide hydrolase (FAAH). O‐aryl carbamate FAAH inhibitors such as URB597 are being evaluated clinically for the treatment of pain and anxiety, but interactions with carboxylesterases in liver might limit their usefulness. Here we explore two strategies aimed at overcoming this limitation. Lipophilic N‐terminal substitutions, which enhance FAAH recognition, yield potent inhibitors but render such compounds susceptible to attack by broad‐spectrum hydrolases and inactive in vivo. By contrast, polar electron‐donating O‐aryl substituents, which decrease carbamate reactivity, yield compounds, such as URB694, that are highly potent FAAH inhibitors in vivo and less reactive with off‐target carboxylesterases. The results suggest that an approach balancing inhibitor reactivity with target recognition produces FAAH inhibitors that display significantly improved drug‐likeness.

Synthesis and Structure–Activity Relationships of N-(2-Oxo-3-oxetanyl)amides as N-Acylethanolamine-hydrolyzing Acid Amidase Inhibitors

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.

Creatine as a compatible osmolyte in muscle cells exposed to hypertonic stress.

Exposure of C2C12 muscle cells to hypertonic stress induced an increase in cell content of creatine transporter mRNA and of creatine transport activity, which peaked after about 24 h incubation at 0.45 osmol (kg H2O)−1. This induction of transport activity was prevented by addition of either cycloheximide, to inhibit protein synthesis, or of actinomycin D, to inhibit RNA synthesis. Creatine uptake by these cells is largely Na+ dependent and kinetic analysis revealed that its increase under hypertonic conditions resulted from an increase in Vmax of the Na+‐dependent component, with no significant change in the Km value of about 75 μmol l−1. Quantitative real‐time PCR revealed a more than threefold increase in the expression of creatine transporter mRNA in cells exposed to hypertonicity. Creatine supplementation significantly enhanced survival of C2C12 cells incubated under hypertonic conditions and its effect was similar to that obtained with the well known compatible osmolytes, betaine, taurine and myo‐inositol. This effect seemed not to be linked to the energy status of the C2C12 cells because hypertonic incubation caused a decrease in their ATP content, with or without the addition of creatine at 20 mmol l−1 to the medium. This induction of creatine transport activity by hypertonicity is not confined to muscle cells: a similar induction was shown in porcine endothelial cells.

Novel irreversible epidermal growth factor receptor inhibitors by chemical modulation of the cysteine-trap portion.

Irreversible EGFR inhibitors can circumvent acquired resistance to first-generation reversible, ATP-competitive inhibitors in the treatment of non-small-cell lung cancer. They contain both a driver group, which assures target recognition, and a warhead, generally an acrylamide or propargylamide fragment that binds covalently to Cys797 within the kinase domain of EGFR. We performed a systematic exploration of the role for the warhead group, introducing different cysteine-trapping fragments at position 6 of a traditional 4-anilinoquinazoline scaffold. We found that different reactive groups, including epoxyamides (compounds 3-6) and phenoxyacetamides (compounds 7-9), were able to irreversibly inhibit EGFR. In particular, at significant lower concentrations than gefitinib (1), (2R,3R)-N-(4-(3-bromoanilino)quinazolin-6-yl)-3-(piperidin-1-ylmethyl)oxirane-2-carboxamide (6) inhibited EGFR autophosphorylation and downstream signaling pathways, suppressed proliferation, and induced apoptosis in gefitinib-resistant NSCLC H1975 cells, harboring the T790M mutation in EGFR.

N-(2-Oxo-3-oxetanyl)carbamic Acid Esters as N-Acylethanolamine Acid Amidase Inhibitors: Synthesis and Structure–Activity and Structure–Property Relationships

The β-lactone ring of N-(2-oxo-3-oxetanyl)amides, a class of N-acylethanolamine acid amidase (NAAA) inhibitors endowed with anti-inflammatory properties, is responsible for both NAAA inhibition and low compound stability. Here, we investigate the structure-activity and structure-property relationships for a set of known and new β-lactone derivatives, focusing on the new class of N-(2-oxo-3-oxetanyl)carbamates. Replacement of the amide group with a carbamate one led to different stereoselectivity for NAAA inhibition and higher intrinsic stability, because of the reduced level of intramolecular attack at the lactone ring. The introduction of a syn methyl at the β-position of the lactone further improved chemical stability. A tert-butyl substituent in the side chain reduced the reactivity with bovine serum albumin. (2S,3R)-2-Methyl-4-oxo-3-oxetanylcarbamic acid 5-phenylpentyl ester (27, URB913/ARN077) inhibited NAAA with good in vitro potency (IC(50) = 127 nM) and showed improved stability. It is rapidly cleaved in plasma, which supports its use for topical applications.

Irreversible inhibition of epidermal growth factor receptor activity by 3-aminopropanamides.

Irreversible epidermal growth factor receptor (EGFR) inhibitors contain a reactive warhead which covalently interacts with a conserved cysteine residue in the kinase domain. The acrylamide fragment, a commonly employed warhead, effectively alkylates Cys797 of EGFR, but its reactivity can cause rapid metabolic deactivation or nonspecific reactions with off-targets. We describe here a new series of irreversible inhibitors containing a 3-aminopropanamide linked in position 6 to 4-anilinoquinazoline or 4-anilinoquinoline-3-carbonitrile driving portions. Some of these compounds proved to be as efficient as their acrylamide analogues in inhibiting EGFR-TK (TK = tyrosine kinase) autophosphorylation in A549 lung cancer cells. Moreover, several 3-aminopropanamides suppressed proliferation of gefitinib-resistant H1975 cells, harboring the T790M mutation in EGFR, at significantly lower concentrations than did gefitinib. A prototypical compound, N-(4-(3-bromoanilino)quinazolin-6-yl)-3-(dimethylamino)propanamide (5), did not show covalent binding to cell-free EGFR-TK in a fluorescence assay, while it underwent selective activation in the intracellular environment, releasing an acrylamide derivative which can react with thiol groups.

Qualitative structure-metabolism relationships in the hydrolysis of carbamates

The aims of this review were 1) to compile a large number of reliable literature data on the metabolic hydrolysis of medicinal carbamates and 2) to extract from such data a qualitative relation between molecular structure and lability to metabolic hydrolysis. The compounds were classified according to the nature of their substituents (R3OCONR1R2), and a metabolic lability score was calculated for each class. A trend emerged, such that the metabolic lability of carbamates decreased (i.e., their metabolic stability increased), in the following series: Aryl-OCO-NHAlkyl >> Alkyl-OCO-NHAlkyl ∼ Alkyl-OCO-N(Alkyl)2 ≥ Alkyl-OCO-N(endocyclic) ≥ Aryl-OCO-N(Alkyl)2 ∼ Aryl-OCO-N(endocyclic) ≥ Alkyl-OCO-NHAryl ∼ Alkyl-OCO-NHAcyl >> Alkyl-OCO-NH2 > Cyclic carbamates. This trend should prove useful in the design of carbamates as drugs or prodrugs.

Epidermal Growth Factor Receptor Irreversible Inhibitors: Chemical Exploration of the Cysteine-Trap Portion

Covalent EGFR irreversible inhibitors showed promising potential for the treatment of gefitinib-resistant tumors and for imaging purposes. They contain a cysteine-reactive portion forming a covalent bond with the protein. Irreversible kinase inhibitors have been advanced to clinical studies, mostly characterized by an acrylamide or butynamide warhead. However, the clinical usefulness of these compounds has been hampered by resistances, toxicity and pharmacokinetic problems. Investigation on the structure-activity and structure-reactivity relationships may provide useful information for compounds with improved selectivity and pharmacokinetic properties. This review focuses on the exploration of the cysteine-trap portions able to irreversibly inhibit EGFR and other erbB receptors.

Structure-property relationships of a class of carbamate-based Fatty Acid Amide Hydrolase (FAAH) inhibitors: chemical and biological stability

Cyclohexylcarbamic acid aryl esters are a class of fatty acid amide hydrolase (FAAH) inhibitors, which includes the reference compound URB597. The reactivity of their carbamate fragment is involved in pharmacological activity and may affect their pharmacokinetic and toxicological properties. We conducted in vitro stability experiments in chemical and biological environments to investigate the structure–stability relationships in this class of compounds. The results show that electrophilicity of the carbamate influences chemical stability, as suggested by the relation between the rate constant of alkaline hydrolysis (log kpH9) and the energy of the lowest unoccupied molecular orbital (LUMO). Introduction of small electron‐donor substituents at conjugated positions of the O‐aryl moiety increased the overall hydrolytic stability of the carbamate group without affecting FAAH inhibitory potency, whereas peripheral non‐conjugated hydrophilic groups, which favor FAAH recognition, helped decrease oxidative metabolism in the liver.