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FAAH inhibition by the 2-(piperazin-1-yl)ethyl and 3-(piperazin-1-yl)propyl derivatives <jats:bold>24<\/jats:bold> and <jats:bold>30<\/jats:bold> was confined to the periphery in mice (30 mg\/kg), whereas hepatic FAAH activity was inhibited by over 90%.<\/jats:p>","DOI":"10.1515\/pac-2016-0104","type":"journal-article","created":{"date-parts":[[2016,4,24]],"date-time":"2016-04-24T00:58:01Z","timestamp":1461459481000},"page":"341-347","source":"Crossref","is-referenced-by-count":4,"title":["Synthesis and structure\u2013activity relationships of ionizable 1,3,4-oxadiazol-2(3<i>H<\/i>)-ones as peripherally selective FAAH inhibitors with improved aqueous solubility"],"prefix":"10.1515","volume":"88","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0957-4137","authenticated-orcid":false,"given":"Alexandre","family":"Beliaev","sequence":"first","affiliation":[{"name":"Laboratory of Chemistry , Department of Research and Development , BIAL \u2013 Portela and C\u00aa., S.A., Coronado (S. 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The reaction mixture was then cooled to 0\u20135\u00b0C using an ice bath, whereupon diethyl azodicarboxylate (DEAD) (3.88 mL, 24.5 mmol) in THF (5 mL) was charged dropwise at such a rate that the yellow colouration on addition of DEAD dissipated before the next addition. The reaction was then allowed to warm up naturally and stirred at 20\u201325\u00b0C until judged complete by TLC (typically 12 h), concentrated under reduced pressure and purified by column chromatography using an appropriate mixture of petroleum ether \u2013 ethyl acetate as eluent."},{"key":"2024030511044865785_j_pac-2016-0104_ref_027","unstructured":"Typical procedure: 5-(2,4-difluorophenoxy)-3-(4-(2-(piperazin-1-yl)ethoxy)phenyl)-1,3,4-oxadiazol-2(3H)-one dihydrochloride (24). tert-Butyl 4-(2-(4-(5-(2,4-difluorophenoxy)-2-oxo-1,3,4-oxadiazol-3(2H)-yl)phenoxy)ethyl)piperazine-1-carboxylate (5) (6 g, 11.6 mmol) was added in portions to TFA (280 mL) with stirring at 20\u201325\u00b0C. The mixture was stirred for 1 h and TFA was removed under reduced pressure. The oily residue was re-evaporated twice with toluene (ca. 100 mL), then dissolved in EtOAc (120 mL), and cooled to 0\u20135\u00b0C, whereupon 2 M ethereal hydrogen chloride solution (25.5 mL, 51 mmol) was slowly added causing precipitation. The solid was collected, washed with diethyl ether and anhydrous EtOH and dried under vacuum at 45\u00b0C to give a white powder. Yield 3.62 g (64%)."},{"key":"2024030511044865785_j_pac-2016-0104_ref_028","unstructured":"In vivo assay (animal treatment): Male NMRI mice, obtained from Envigo (Spain), were maintained under controlled environmental conditions in a colony room (12 h light\/dark cycle, room temperature 22\u00b11\u00b0C and humidity 55\u00b115%) with food and water provided ad libitum. Animals were quarantined for 1 week before dosing and the experiments were all carried out during daylight hours. Animals were fasted overnight before compound administration (30 mg\/kg), which was performed by single intragastric bolus at a volume of 8 ml\/kg and using carboxymethylcellulose (CMC) 0.5% as vehicle. Fifteen minutes before sacrifice animal were anaesthetised with pentobarbital (60 mg\/kg) administered intraperitoneally. One hour after compound administration animals were sacrificed. A fragment of liver and brain without cerebellum were removed and put in plastic vials containing membrane buffer (3 mM MgCl2, 1 mM EDTA, 50 mM Tris HCl pH 7.4). Tissues were stored at -30\u00b0C until analysis. All animal procedures were conducted in accordance with the 2010\/63\/EU European Directive on the protection of animals used for scientific purposes and the Portuguese law on animal welfare (Decreto\u2013Lei 113\/2013). The number of animals used was the minimum possible in compliance with current regulations and scientific integrity. Enzymatic activity determination: FAAH assay on collected tissues was performed as described previously [8]. In short, tissues were homogenized in membrane buffer with either Potter\u2013Elvejhem (brains) or Heidolph Diax (livers). Total protein was determined with the BioRad Protein Assay (BioRad) using a standard curve of BSA (50\u2013250 \u03bcg\/mL). Reaction mix (total volume of 200 \u03bcL) contained: 2 \u03bcM AEA (2 \u03bcM AEA+5 nM 3H-AEA), 0.1% fatty acid free BSA, 15 \u03bcg (brain) or 5 \u03bcg (liver) protein, in 1 mM EDTA, 10 mM Tris pH 7.6. After a 15 min pre-incubation period at 37\u00b0C, reaction was started with the addition of the substrate solution (AEA) and then carried out for 10 min (brain) or 7 min (liver) before termination by the addition of 400 \u03bcL activated charcoal suspension. After a 30 min incubation period at room temperature with agitation, charcoal was sedimented by centrifugation in microfuge (10 min at 16000 g). 200 \u03bcL of the supernatant were added to 800 \u03bcL Optiphase Supermix scintillation cocktail previously distributed in 24-well plates. Counts per minute (cpm) were determined in a MicrobetaTriLux scintillation counter. In each assay blanks (without protein) were prepared. The percentage of remaining enzymatic activity was calculated with respect to controls (no compound) and after blank subtraction."},{"key":"2024030511044865785_j_pac-2016-0104_ref_029","doi-asserted-by":"crossref","unstructured":"M. Mor, S. Rivara, A. Lodola, P. V. Plazzi, G. Tarzia, A. Duranti, A. Tontini, G. Piersanti, S. Kathuria, D. Piomelli. J. Med. Chem.47, 4998 (2004).","DOI":"10.1021\/jm031140x"},{"key":"2024030511044865785_j_pac-2016-0104_ref_030","doi-asserted-by":"crossref","unstructured":"J. Z. Patel, J. van Bruchem, T. Laitinen, A. A. Kaczor, D. 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