N-((S)-4-(Benzylamino)-3-((S)-2-(3-(1-(2,6-dichlorobenzyl)-3-(pyrrolidin-1-ylmethyl)-1H-indazol-6-yl)ureido)-3-(3,4-difluorophenyl)propanamido)-4-oxobutyl)pent-4-ynamide (12, DG-207) Notice If using 11b, it is necessary to convert this compound to the bis HCl salt (11a) prior to performing this EDC coupling reaction as it has been seen the formate salt will give a mixture of products. for study of PAR2 ligands; a peptide antagonist reported by Fairlie was synthesized and found to inhibit PAR2 in a manner consistent with reports using epithelial cells. The assay was used to confirm that vorapaxar functions as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with bad allosteric modulation. 0), or CDCl3 (13C 77.16), (CD3)2CO (1H 2.05, 13C 29.84), d6-DMSO (1H 2.50, 13C 39.5), or CD3OD (1H 3.31, 13C 49.00). NMR data are reported as follows: chemical shifts, multiplicity (obs = obscured, app = apparent, br = broad, s = singlet, d = doublet, t = triplet, q = quartet, sxt = sextet, m = multiplet, comp = complex overlapping signals); coupling constant(s) in Hz; integration. Unless otherwise indicated, Ibuprofen piconol NMR data were collected at 25 C. Filtration was performed by vacuum using VWR Grade 413 filter paper, unless otherwise noted. Flash chromatography was performed using Biotage SNAP cartridges filled with 40C60 m silica gel on Biotage Isolera automated chromatography systems with photodiode array UV detectors. Analytical thin coating chromatography (TLC) was performed on Agela Systems glass plates with 0.25 mm silica gel and F254 indicator. Visualization was accomplished with UV light (254 nm) and KMnO4 stain, unless normally noted. Chemical titles were generated and select chemical properties were determined using either ChemAxon Marvin suite (https://www.chemaxon.com) or ChemDraw Professional 15.1. NMR data were processed using either MestreNova Rabbit polyclonal to Dicer1 or ACD/NMR Processor Academic Release (http://www.acdlabs.com) using the JOC statement file format. High-resolution mass spectra (HRMS) were obtained in the University or college of Wisconsin-Milwaukee Mass Spectrometry Laboratory having a Shimadzu LCMS-IT-TOF with ESI and APCI ionization. 4.2. LC/MS characterization methods Tandem liquid chromatography/mass spectrometry (LC-MS) was performed on a Shimadzu LCMS-2020 with autosampler, photodiode array detector, and single-quadrupole MS with ESI and APCI dual ionization using a Maximum Scientific Ibuprofen piconol nitrogen generator. Method A 1C5 L of sample in MeCN or MeOH UV absorbance at 210 or 254 nm measured by UV absorbance at 210 or 254 nm 0.1C1.9 Ibuprofen piconol mL (2 mL sample loop) of sample in DMSO = 530.05 (M+H+); 1H NMR (300 MHz, CDCl3) = 7.76 (d, = 7.6 Hz, 2H), 7.61C7.49 (overlapping signals, 3H), 7.43C7.22 (m, 9H), 5.96 (d, = 6.7 Hz, 1H), 5.11 (br. m., 1H), 4.50C4.13 (overlapping m, 6H), 3.48C3.32 (br s., 1H), 3.04C2.90 (br m., 1H), 1.97C1.71 (overlapping signals, 2H), 1.45C1.37 Ibuprofen piconol (m, 9H); 13C NMR (75 MHz, CDCl3) = 171.2, 157.2, 156.3, 143.9, 141.5, 138.0, 128.8, 127.9, 127.8, 127.6, 127.3, 125.3, 120.2, 80.1, 67.2, 52.2, 47.3, 43.8, 37.0, 34.9, 28.6. 4.4.2. tert-butyl ((S)-3-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl) amino)-3-(3,4-difluorophenyl)propanamido)-4-(benzylamino)-4-oxobutyl) carbamate (3) Part 1: Fmoc removal Intermediate 2 (550 mg, 1.03 mmol) was added to a round bottomed flask with stir bar and sealed under nitrogen, then MeCN (25 mL), DCM (10 mL), and piperidine (0.220 mL, 2.22 mmol) were added. The reaction was stirred for 2 h. A sample aliquot was taken from the reaction, concentrated under reduced pressure, dissolved in a minimal amount of HPLC grade MeCN, and analyzed with LCMS to confirm reaction completion. The reaction was concentrated under reduced pressure and then re-dissolved in (CHCl3/EtOAc, 1: 1, 75 mL), washed with H2O (2 20 mL) and brine (20 mL). The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure to give crude material (496 mg) like a white solid that was carried onto the next Ibuprofen piconol reaction without further purification. Part 2: Coupling To a round bottomed flask with stir bar already comprising crude material from Part 1 (496 mg crude material, max. yield is definitely 319 mg) was added anhydrous MeCN (30 mL) and anhydrous DCM (15 mL). (= 713.15 (M+H+), = 757.35 (formic acid adduct); 1H NMR (300 MHz, DMSO = 8.43 (dd, = 5.0, 5.9 Hz, 1H), 8.22 (d, = 7.9 Hz, 1H), 7.86 (d, = 7.6 Hz, 2 H), 7.68C7.53 (m, 2 H), 7.52C7.43 (br s., 1H), 7.43C7.07 (m, 11= 5.6 Hz, 4 H), 4.15 (br s, 3 H), 3.05C2.85 (overlapping br s, 3 H), 2.73 (app t, = 12.0 Hz, 1 H), 1.86C1.73 (m, 1 H), 1.73C1.58 (m, 1H), 1.34 (s, 9 H); 13C NMR (75 MHz, DMSO-= 171.8, 171.7, 156.4, 156.1, 150.8 (dd, = 45.2, 13 Hz), 147.6 (dd, = 45.8, 12.1 Hz), 144.4, 144.3, 141.35, 141.33, 139.8, 136.6, 128.9, 128.2, 127.7, 127.6, 127.4,.