J. Med. Chem. 2003, 46, 4373-4376
Esters, Retroesters, and a Retroamide of Palmitic Acid: Pool for the First Selective Inhibitors of N-PalmitoylethanolamineSelective Acid Amidase
4373
Chart 1. Structures of Anandamide (1), 2-Arachidonoylglycerol (2), N-Oleoylethanolamine (3), and N-Palmitoylethanolamine (4)
Se´verine Vandevoorde,† Kazuhito Tsuboi,‡ Natsuo Ueda,‡ Kent-Olov Jonsson,§ Christopher J. Fowler,§ and Didier M. Lambert*,† Unite´ de Chimie Pharmaceutique et de Radiopharmacie, Universite´ Catholique de Louvain, Avenue Mounier, 73, UCL-CMFA 73.40, B-1200 Brussels, Belgium; Department of Biochemistry, Kagawa Medical University, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan; and Department of Pharmacology and Clinical Neuroscience, Umeå University, SE-901 87 Umeå, Sweden Received April 14, 2003 Abstract: Cyclohexyl hexadecanoate, hexadecyl propionate, and N-(3-hydroxypropionyl)pentadecanamide, respectively ester, retroester, and retroamide derivatives of N-palmitoylethanolamine, represent the first selective inhibitors of “Npalmitoylethanolamine hydrolase” described so far. These compounds are devoid of affinity for CB1 and CB2 receptors and characterized by high percentages of inhibition of Npalmitoylethanolamine-selective acid amidase (84.0, 70.5, and 76.7% inhibition at 100 µM, respectively) with much lower inhibitory effect on either fatty acid amide hydrolase or the uptake of anandamide.
Introduction. Endogenous fatty acid amides and esters are the subject of growing interest in pharmacology since the discovery of anandamide (Chart 1), the ethanolamide of arachidonic acid that has been proposed as an endogenous ligand of cannabinoid receptors.1 Anandamide displays, as a result of its actions upon cannabinoid and vanilloid receptors, a number of interesting pharmacological properties including effects on nociception, memory processes, lung function, spasticity, appetite, and cell proliferation.2 Among the endocannabinoids (i.e., the endogenous agonists of cannabinoid receptors), several polyunsaturated fatty acid ethanolamides have been described but also esters such as 2-arachidonoylglycerol (Chart 1). All these molecules are rapidly inactivated by uptake into cells followed by enzymatic hydrolysis. In the case of the polyunsaturated fatty acid ethanolamides, the enzyme responsible is fatty acid amide hydrolase (FAAH).3 This key hydrolase is abundant in the central nervous system, the liver, and the kidney. FAAH activity is characterized by an optimal pH value at 8.5-104,5 and by high sensitivity to phenylmethylsulfonyl fluoride (PMSF)6 and methyl arachidonoyl fluorophosphonate (MAFP).7,8 In contrast to soluble hydrolases of the same family, FAAH is able to integrate into cell membranes and establish direct access to the bilayer membrane from its active site. Its 2.8 Å crystal structure was recently reported.9 * To whom correspondence should be addressed. E-mail: lambert@ cmfa.ucl.ac.be. † Universite ´ Catholique de Louvain. ‡ Kagawa Medical University. § Umeå University.
Other related endogenous lipid derivatives such as oleamide, N-oleoylethanolamine (Chart 1), and N-palmitoylethanolamine (PEA, Chart 1) induce pharmacological responses10 albeit without significant binding either to cannabinoid receptors11 or to vanilloid receptors. Oleamide induces physiological sleep in animals, analgesia, and cannabinoid-like behavioral responses. NPalmitoylethanolamine exhibits antiinflammatory and analgesic effects, both suppressed by the administration of the CB2 cannabinoid receptor antagonist SR144528.12 N-Oleoylethanolamine has been recently proposed as an endogenous anorexic lipid messenger acting peripherally in the control of appetite.13 These three molecules are also substrates for FAAH,14,15 although they are hydrolyzed at a lower rate than anandamide.4,5,16,17 Recently, another fatty acid amide hydrolyzing enzyme, which is catalytically distinct from the previously reported FAAH, was found in a human megakaryoblastic leukemia cell line (CMK) and rat tissues.18,19 This enzyme is abundant in the lung, the spleen, and the small intestine, and it is characterized by an optimal pH value at 5. It is also much less sensitive to nonselective compounds such as PMSF and MAFP. Among the N-acylethanolamines, PEA is the most active substrate for this enzyme.19 This enzyme, highly purified but not yet cloned,19 is termed “N-palmitoylethanolamine-selective acid amidase (NPAA)” in this paper. Characterization of the physiological role of NPAA would greatly be aided by the discovery of selective inhibitors of this enzyme. Analogues of N-palmitoylethanolamine, the most active substrate, would be a reasonable starting point. In this report, we describe a comparative study on the inhibitory potentials of some esters, retroesters, and a retroamide of palmitic acid, toward FAAH and NPAA. Affinity of the compounds for the CB1 and CB2 cannabinoid receptors was also evaluated by competitive binding experiments. Among the tested compounds, cyclohexyl hexadecanoate, hexadecyl propionate, and N-(3-hydroxypropionyl)pentadecana-
10.1021/jm0340795 CCC: $25.00 © 2003 American Chemical Society Published on Web 09/04/2003
4374
Journal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Letters
Scheme 1. Syntheses of Esters (5-12), Retroesters (13 and 14), and Retroamide 15a
a Reagents: (a) room temperature, 2 h; (b) HCl (cat.), dichloromethane, reflux 4 h (75%); (c) dichloromethane, reflux 3 h (86%); (d) pyridine, mesyl chloride (2 equiv), room temperature 5 h; sodium azide (4 equiv), DMF, 90 °C overnight; (e) diethyl ether, lithium aluminum hydride (1 equiv), reflux 3 h; (f) dichloromethane, trimethylaluminum (1 equiv), β-propiolactone (1 equiv), reflux 6 h.
Table 1. FAAH, NPAA Inhibitory Potentials and Cannabinoid Receptor Recognition of Esters 5-12, Retroesters 13 and 14, Retroamide 15, and Amides 4 and 16-18 of Palmitic Acid
5, OCH3 6, OCH(CH3)2 7, OC(CH3)3 8, OCH2CHdCH2 9, OCH2CH2Cl 10, OCH2CH2Br 11, O-cyclohexyl 12, O-benzyl
CB1 receptor affinitya (% inhibition of specific binding of [3H]-CP55,940)
CB2 receptor affinityb (% inhibition of specific binding of [3H]-Win55,212-2)
10 µM
10 µM
11 ( 0.3 8 ( 0.4 14 ( 0.9 2 ( 0.1 14 ( 0.5 2 ( 0.1 14 ( 0.4 5 ( 0.3 10 ( 0.2
13, hexadecyl acetate 14, hexadecyl propionate
12 ( 0.2 4 ( 0.4
Retroamide
NPAAd % inhibition
10 µM
100 µM
100 µM
18 ( 7
