The time from the experiment were housed individually and maintained in a temperature-controlled environment on a 12-hour light/dark cycle (off 7:30 AM; on 7:30 PM). Except for the duration of testing, animals have been offered free access to food and water. Animals administered compounds through the oral route had been deprived of food 10 hours ahead of the experiment. For toxicology studies, compound 5 was administered to male Sprague-Dawley rats weighing 30050 g (Harlan). Twenty-four hours soon after the last dose of compound five, animals were killed, blood was obtained and centrifuged, and serum was separated and frozen for analysis of serum clinical chemistry at IDEXX Laboratories (Sacramento, CA). For alcohol self-administration studies, male alcohol-preferring Wistar rats (22549 g) were obtained in the University of Indiana (Indianapolis, IN) and have been housed in groups of two or 3 and maintained von Hippel-Lindau (VHL) Degrader review inside a temperature-controlled atmosphere on a 12-hour light/dark cycle (off 7:30 AM; on 7:30 PM). Except for the duration of behavioral testing, animals were given free access to meals and water.4-CF3-benzoic acid-d4 (113.3 mg, 0.584 mmol, two equiv.), and BOP (258 mg, 0.584 mmol, two equiv.) were placed in anhydrous DCM (four ml) and DIPEA (152 ml, 0.876 mmol, three equiv.) was added and also the reaction was stirred overnight at area temperature to afford the ester-amide. After purification by flash chromatography (100 EtOAc) the ester-amide was dissolved in methanol and potassium carbonate was added. The mixture was stirred at room temperature for three hours, potassium carbonate was removed by filtration, and the item was purified by preparative thin layer chromatography (CHCl3/MeOH) 20/1 to get in quantitative yield the desired item. The purity was .98 around the basis of HPLC and liquid chromatography ass spectrometry (LCMS). 1 H-NMR (CDCl3) d 0.13.18 (m, 2H), 0.53.59 (m, 2H), 0.81.92 (m, 1H), 1.39.62 (m, 3H), 1.66.74 (m, 1H), 1.89.0 (m, 1H), 2.17.26 (m, 2H), 2.39 (d, J 5 six.three Hz, 2H), 2.65 (d, J five 9.9 Hz, 2H), 3.04 (d, J 5 13.4 Hz, 1H), 3.16 (d, J 5 5.2 Hz, 1H), 3.64 (d, J 5 11.0 Hz, 1H), four.08.18 (m, 2H), four.63 (d, J 5 5.24 Hz, 1H), 6.52 (d, J five 8.0 Hz, 1H), six.67 (d, J five eight.0 Hz, 1H), 7.79 (d, J 5 9.0 Hz, 1H). Electrospray ionization/MS m/z 5 518.95 [M1H].Pharmacokinetic StudiesThe evening ahead of the oral pharmacokinetic study, the animals had been fasted. Groups of two jugular cannulated rats were administered compound 5 hydrochloride by the intravenous route of administration (20 or 50 mg/kg, 1 ml/kg) or the oral route of administration (200 mg/kg, 2 ml/kg) in isotonic saline. For the intravenous study of compound 5, blood was taken at 5, 15, 30, 60, 120, 240 minutes and six and 10 hours. For the oral study, blood was taken at 15, 30, 60, 120, and 240 minutes and 6 and ten hours. Blood was combined with 2 IUs of heparin and immediately cooled to 4 . Separated plasma was brought to a pH of 10 with ammonium hydroxide, and 400 pg/ml compound 4 was added as an internal standard and extracted with hexane/methyl-tert-butyl ether (three:1, v:v). After centrifugation at 13,000g for five minutes, the organic fraction was collected along with the solvent was removed with a stream of argon. The residue was reconstituted in water:acetonitrile:formic acid (80:20:0.1, v:v) and run isocratically in 0.1 formic acid in water, 0.1 formic acid in acetonitrile (60:40) using a Waters Acquity instrument and Waters XEVO tandem quadrupole detector (Waters, Milford, MA). An MC4R Agonist Synonyms aliquot was analyzed by reverse-phase HPLC making use of a Synergi Polar.
