Compositions and methods for preparing and using azetidines

A method for determining the binding of an azetidine compound to a protein, the method comprising:

• A. contacting an azetidine compound of Formula I or a pharmaceutically acceptable salt thereof:
• wherein
• A is —NH—, —C(O)—, or —CH2—;
• wherein
• when A is —C(O)— or —CH2—, then X is CR2R3 and Y is N;
• and
• when A is —NH—, then X is NR4 and Y is CH;
• B is a bond, C1-C6-alkylene, or —(C1-C6-alkylene) (C6-C12-arylene)-;
• R1 is selected from the group consisting of H, C6-C12-aryl;
• R2 and R3 are independently selected from the group consisting of H, C1-C6-alkyl, —NRR′ wherein R and R′ are independently selected from H and C1-C6-alkyl, —(C1-C6-alkyl) (C6-C12-aryl) wherein the alkyl is optionally interrupted by one or more —O—;
• C6-C12-aryl;
• —O— (C6-C12-aryl);
• 5- to 12-membered heteroaryl wherein one or more members is selected from N, O, and S, 5- to 12-membered-O-(heteroaryl) wherein one or more members is selected from N, O, and S;
• wherein when B is C1-C6-alkylene, then at least one of R2 and R3 is selected from —(C1-C6-alkyl) (C6-C12-aryl), C6-C12-aryl, —O—(C6-C12-aryl), 5- to 12-membered heteroaryl except triazole and diazole, and 5- to 12-membered-O-(heteroaryl);
• or R2 and R3 in combination with the ring carbon atom to which they are bound form a spiro-fused C3-C8-cycloalkyl;
• R4 is selected from the group consisting of C6-C12-aryl, 5- to 12-membered heteroaryl wherein one or more members is selected from N, O, and S, 5- to 12-membered-O-(heteroaryl) wherein one or more members is selected from N, O, and S, and 4- to 12-membered heterocycle wherein one or more members is selected from N, O, and S;
• m is 0, 1, 2, 3, or 4;
• and
• any aryl, heterocycle, heteroaryl is optionally substituted with one to four substituents selected from the group consisting of halogen, hydroxy, cyano, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, and carboxy;
• with a test sample comprising at least one protein to yield a first product;
• B. contacting the first product with an azide-tag reagent in a copper-catalyzed azide-alkyne cycloaddition reaction to yield a second product;
• and
• C. detecting the formation of any tag-modified proteins in the second product.