Specific ally, IL 21 stimulates expansion and cytotoxicity

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Samples were separated by use of a Caliper LC90 system and the remaining substrate and product bands were quantitated. Intact Tyk2 ran in this system at 29 kDa. Addition of thermolysin yielded a partial digestion KU-0063794 product of 27 kDa within 5 minutes, which was unaffected by addition of Compound 2. Subse quent degradation products were strongly influenced by the presence of Compound 2, Therefore the sum of the intensities of the 29 and 27 kDa peaks was used as a measure of inhibitor dependent resist ance to thermolysin digestion. Addition of Compound 2 did not alter the measurable digestion of a BSA control by ther molysin in the same buffer, in dicating that Compound 2 was not an inhibitor of thermolysin protease activity.

Thermolysin amounts were found to be unchanged over the course of the experiment, providing a convenient loading control, Compound synthesis N 3 chloro benzenesulfonamide Step a. 4 aniline A mixture of 5 bromo 2 fluorobenzonitrile, 4 phenylboronic acid, tetrakis palladium and cesium carbonate in 1,2 dimethoxyethane and Lenalidomide Revlimid water was heated at 80 C, under N2 for about 2. 5 hours. to give a dark red solution. The reaction mixture was diluted with ethyl acetate and water then stirred for about 10 minutes. The aqueous layer was separated and re extracted with ethyl acetate, The combined organic phases were washed with water then dried over anhydrous magnesium sulfate and filtered. The solvent was removed to yield a brown solid which was triturated with boiling 30 60 C petroleum ether, cooled and collected.

This solid was further washed with 30 60 C petroleum ether and dried to yield crude N tert butoxycarbonyl 4 aniline as a pale brown solid, The crude protected aniline was dissolved in dichloromethane LY2603618 構造 then treated with trifluoroacetic acid and stirred at ambient temperature for about 3 hours. Water was added and the aqueous acidic layer was separated. The remaining organic layer was further extracted with aqueous hydrochloric acid, The combined acidic aqueous layers were washed with dichloromethane, cooled with ice then basified by the addition of solid sodium hydroxide while maintaining a temperature of below 15 C. The resulting aqueous layer was extracted with ethyl acetate and the combined organic layers were washed with water, dried over anhydrous magnesium sulphate, filtered and concentrated to yield a mauve solid, This was crystallized from ethyl acetate and 30 60 C petroleum ether, filtered, washed with 30 60 C petroleum ether and dried to yield 4 aniline as a pale mauve powdery solid, 1 H NMR 6.

62, 7. 41, 7. 50, 7. 93, 8. 06, LC MS using a Vydac Genesis C8 column with diode array, evaporative light scattering and positive negative electrospray ionization detection Rt 2. 43 min; MS m z: 254, Step b. N 3 chloro benzenesulfonamide 3 Chlorobenzenesulfonyl chloride was added to a mixture of 4 aniline in 1,4 dioxane and N,N diisopropylethylamine, The resulting mixture was stirred at ambient temperature for about 16 hours then dispensed into a microwaveable tube and anhydrous hydrazine was added. The tube was sealed and heated at 140 C for 15 minutes in a microwave.