Starting with the parent compound VAEFMK 1 it was identified that bulky, lipophilic moieties are favored in the valine position, while alteration of the alanine position was not tolerated

Starting with the parent compound VAEFMK 1 it was identified that bulky, lipophilic moieties are favored in the valine position, while alteration of the alanine position was not tolerated. the biological evaluation of UCHL1. unless BMS-983970 normally specified as 0.05; **, 0.01; ***, 0.001. Statistical significance determined using college students 0.05; **, 0.01, *** 0.001. NS = not significant. Statistical significance determined using two-tailed trifluoroacetic acid. The mass spectrometer used is BMS-983970 definitely a CMS-L Compact Mass Spectrometer (Advion, Ithaca, NY, USA) with an ESI or an APCI ionization resource. Samples are submitted for analysis using either the atmospheric solids analysis probe (ASAP) or circulation injection analysis (FIA). Compounds were prepared according to the following protocols and are detailed below. Intermediates 13a (Catalog #V4659, AK Scientific, Union City, CA, USA) and 13b (Catalog #7975AH, AK Scientific) were purchased from commercial vendors. These intermediates were left in Plan 3 for continuity. 3.1.2. General Procedure for synthesis of tripeptide halomethylketones Synthesis for analog 21 offered. All other analogs and option synthetic methods reported in Supplementary Materials. Dibenzyl 2-fluoromalonate (3) A mixture of dimethyl 2-fluoromalonate 2 (2.0 g, 13 mmol, 1.0 eq), benzyl alcohol (7.0 g, 65 mmol, 4.9 eq,), WISP1 p-toluenesulfonic acid monohydrate (150 mg, 0.80 mmol, 0.06 eq) in toluene (5.6 mL) were placed in a round bottom flask outfitted having a Dean-Stark apparatus and was heated with stirring to 75 C in vacuo (27 mm of Hg) until all the toluene had distilled. Then (75 mm Hg, 112 C) for an additional 5 h. The combination was cooled to 75 C and isopropanol (15 mL) was added followed by hexanes (30 mL). The combination was placed in the refrigerator and allowed to crystalize overnight. The product was filtered, washed with hexanes (2 30 mL), and dried over night = 48.0 Hz, 1H), 5.25 (s, 4H); APCI-MS: 301.1 [M ? H]?. 3-(Benzyloxy)-2-fluoro-3-oxopropanoic acid (4) Intermediate 3 (2.0 g, 6.6 mmol, 1.0 eq) was suspended in isopropanol (12 mL) and heated to 45 C. 1.0 M aqueous NaOH (6.9 mL, 6.9 mmol, 1.1 eq) was added dropwise over 1 h. After an additional 10 min, the perfect solution is was concentrated to 5 mL and water (2.5 mL) was added. The pH was modified to 9 using saturated sodium bicarbonate and washed with DCM (5 5 mL) to remove benzyl alcohol. The pH of the perfect solution is was modified to 2.2 using 5 M HCl, and di-isopropyl ether (5 mL) was used to extract the product. The pH of the aqueous coating was adjusted to 1 1.9 using 5 M HCl, and further extracted with di-isopropyl ether (5 mL). The combined extracts were washed with brine (5 mL), dried over MgSO4, filtered, and concentrated in vacuo at 35 C to provide an oily residue. This was triturated with hexanes (7 mL) over night with stirring to give a solid. The solid was filtered and dried under vacuum to yield 4 like a white solid (0.42 g, 2.0 mmol, 30%). 1H NMR (500 MHz, Chloroform-= 2.6 Hz, 5H), 5.40 (d, = 47.9 Hz, 1H), 5.32 (d, = 1.1 Hz, 2H). Methyl (S)-4-((tert-butoxycarbonyl)amino)-6-fluoro-5-oxohexanoate (8a) To vial BMS-983970 #1 was added 4 (1.3 g, 6.3 mmol, 1.2 eq) and THF (2 mL/mmol). This was cooled to 0 C before adding 2.0 M isopropylmagnesium chloride in THF (6.30 mL, 12.5 mmol, 2.40 eq). The white suspension was stirred for 1 h at 0 C to generate the magnesium salt 5 to be used for the next reaction. To vial #2 was added (= 14.8, 7.4 Hz, 1H), 1.44 (s, 9H). APCI-MS: 278.0 [M + H]+. Methyl ((benzyloxy)carbonyl)-L-phenylalanyl-L-alaninate (12g) To a solution of ((benzyloxy)carbonyl)-= 7.1 Hz, 1H), 7.49 (d, = 8.8 Hz, 1H), 7.37C7.02 (m, 10H), 4.88 (s, 2H), 4.39C4.10 (m, 2H), 3.58 (s, 3H), 2.96 (dd, = 13.9, 3.7 Hz, 1H), 2.67 (dd, = 13.7, 11.0 Hz, 1H), 1.28 (d, = 7.3 Hz, 3H); APCI-MS: 385.1 [M + H]+. ((Benzyloxy)carbonyl)-L-phenylalanyl-L-alanine (13g) The dipeptide methyl ester 12g (0.61 g, 1.6 mmol, 1.0 eq) was dissolved in THF:H2O (4:1, 10 mL) followed by addition of LiOH (0.046 g, 1.9 mmol, 1.2 eq) at 0 C. The combination was stirred at 0 C for 3 h before acidifying with 1.0 M HCl (2 mL) to pH 3. The aqueous phase was extracted with EtOAc (3 5 mL) and the organic layers were combined and dried over.