Search

US-12624020-B2 - Compositions and methods for preparing and using azetidines

US12624020B2US 12624020 B2US12624020 B2US 12624020B2US-12624020-B2

Abstract

The present disclosure provides azetidine compounds of Formula I and their pharmaceutically acceptable salts, their compositions, and methods for their use in determining azetidine compound binding to proteins. The azetidine compounds are useful as probes, for monitoring diacylglycerol kinase activity, and for identifying druggable targets.

Inventors

  • Ku-Lung Hsu
  • Rebecca L. McCloud

Assignees

  • UNIVERSITY OF VIRGINIA PATENT FOUNDATION

Dates

Publication Date
20260512
Application Date
20221012

Claims (18)

  1. 1 . 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 —CH 2 —; wherein when A is —C(O)— or —CH 2 —, then X is CR 2 R 3 and Y is N; and when A is —NH—, then X is NR 4 and Y is CH; B is a bond, C 1 -C 6 -alkylene, or —(C 1 -C 6 -alkylene) (C 6 -C 12 -arylene)-; R 1 is selected from the group consisting of H, C 6 -C 12 -aryl; R 2 and R 3 are independently selected from the group consisting of H, C 1 -C 6 -alkyl, —NRR′ wherein R and R′ are independently selected from H and C 1 -C 6 -alkyl, —(C 1 -C 6 -alkyl) (C 6 -C 12 -aryl) wherein the alkyl is optionally interrupted by one or more —O—; C 6 -C 12 -aryl; —O— (C 6 -C 12 -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 C 1 -C 6 -alkylene, then at least one of R 2 and R 3 is selected from —(C 1 -C 6 -alkyl) (C 6 -C 12 -aryl), C 6 -C 12 -aryl, —O—(C 6 -C 12 -aryl), 5- to 12-membered heteroaryl except triazole and diazole, and 5- to 12-membered-O-(heteroaryl); or R 2 and R 3 in combination with the ring carbon atom to which they are bound form a spiro-fused C 3 -C 8 -cycloalkyl; R 4 is selected from the group consisting of C 6 -C 12 -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.
  2. 2 . The method according to claim 1 , wherein A is —C(O)— or —CH 2 —; X is CR 2 R 3 ; and Y is N.
  3. 3 . The method according to claim 1 , wherein A is —CH 2 —.
  4. 4 . The method according to claim 1 , wherein m is 0 or 1.
  5. 5 . The method according to claim 1 , wherein R 1 is phenyl.
  6. 6 . The method according to claim 1 , wherein R 2 and R 3 are independently selected from the group consisting of H, C 1 -C 6 -alkyl, 5- to 12-membered heteroaryl wherein one or more members is selected from N, O, and S, and 5- to 12-membered-O-(heteroaryl) wherein one or more members is selected from N, O, and S.
  7. 7 . The method according to claim 1 , wherein R 2 is H.
  8. 8 . The method according to claim 1 , wherein A is —NH—; X is NR 4 and Y is CH.
  9. 9 . The method according to claim 8 , wherein R 4 is C 6 -C 12 -aryl or 5- to 12-membered heteroaryl wherein one or more members is selected from N, O, and S.
  10. 10 . The method according to claim 1 , wherein A is —CH 2 —; X is CR 2 R 3 ; Y is N; B is —CH 2 CH 2 —; and R 2 is H.
  11. 11 . The method according to claim 1 , wherein the azetidine compound is selected from the following table: Example Structure TH055 TH056 TH057 RLM- 1451 RLM- 1452 RLM- 1453 RLM- 207 RLM- 266 RLM- 275 RLM- 277 TH058 TH059 TH060 TH061 TH062 TH064 TH065 TH066 TH067 TH068 TH069 TH070 TH071 TH072 TH073 TH074
  12. 12 . The method according to claim 1 , wherein the azetidine compound is selected from the following table: RLM-1451 RLM-1452 RLM-1453 RLM-207
  13. 13 . The method according to claim 1 , wherein the detection of a tag-modified protein in the second product correlates to a determination that the azetidine compound binds to the protein.
  14. 14 . The method according to claim 1 , wherein the test sample comprises a plurality of different proteins.
  15. 15 . The method according to claim 14 , wherein the test sample is selected from live cells and cell lysates.
  16. 16 . The method according to 1 , wherein the tag portion of the azide-tag reagent is a fluorophore.
  17. 17 . The method according to claim 1 , further comprising: D. identifying the azetidine compound binding site or sites on the tagged proteins.
  18. 18 . The method according to claim 1 , wherein the at least one protein in step A is isotopically labeled.

Description

This application is a divisional of U.S. patent application Ser. No. 17/048,500 filed on Oct. 16, 2020, which is a U.S. National Stage of PCT Patent Application No. PCT/US2019/028340, filed under 35 U.S.C. § 371 on Apr. 19, 2019, which claims the benefit of priority to U.S. Provisional Patent Application No. 62/659,892, filed on Apr. 19, 2018, which applications are incorporated by reference as if fully set forth herein. STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under grant No. 2018255830 awarded by the National Science Foundation. The U.S. government has certain rights in the invention. BACKGROUND Azetidine is a saturated heterocyclic organic compound containing three carbon atoms and one nitrogen atom. It is a liquid at room temperature with a strong odor of ammonia and is strongly basic compared to most secondary amines. Azetidines do not occur as frequently in nature and have been studied far less than closely related chemical compounds such as pyrrolidine and β-lactam. Azetidine and its derivatives are relatively rare structural motifs in natural products. Notably, they are a key component of mugineic acids and penaresidins. Perhaps the most abundant azetidine containing natural product is azetidine-2-carboxylic acid, a non-proteinogenic homolog of proline. For improving bioavailability, preventing metabolic degradation, or to add compact functional groups, a significant majority of approved drugs will contain a nitrogen heterocycle1, 2. Piperidines and piperazines still remain the most common heterocycles, while other heterocycles have been largely unexplored. For example, azetidines, four-membered nitrogen heterocycles, are relatively rare in approved drugs. Azetidines possess many desirable properties, such as enhanced solubility, metabolic stability, and 3-D characteristics that could be used to enhance target engagement for inhibitors, yet they have just begun to appear in drug-like molecules. More recently, azetidines have been used to enhance the rate of thiol reactions with acrylamides3, incorporated into antimalarial inhibitors (BRD7929, BRD3914) targeting the parasitic tRNA-synthase4, 5, and appear in the hypertension drug Azelnidipine6, 7. SUMMARY OF THE PRESENT DISCLOSURE The present disclosure addresses these and other needs by providing, in some embodiments, activity-based probes (ABPs) containing azetidine scaffolds as new chemical entities to target druggable binding pockets of proteins, such as human proteins. Without wishing to be bound by any particular theory, it is hypothesized herein that azetidines can be prepared as novel chemical probes useful for academic discovery and for development of drug leads for indications in immunology, cancer, inflammation, and metabolic disease. Other useful azetidines and their preparation can be found in the Examples and in the Appendices. In one embodiment, the present application discloses compositions and methods for tailoring azetidine reactivity. In another embodiment, an azetidine of the present disclosure is useful as a probe and is useful for monitoring diacylglycerol kinase activity and for targeting proteins. In still another embodiment, the diacylglycerol kinase activity is DGKα activity. The present disclosure provides, in various embodiments, azetidine compounds of Formula I and their pharmaceutically acceptable salts: In Formula I compounds, A is —NH—, —C(O)—, or —CH2—. When A is —C(O)— or —CH2—, then X is CR2CR3 and Y is N. When A is —NH—, then X is NR4 and Y is CH. B is a divalent linking group that 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, —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). 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) as defined above. In some embodiments, 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). Subscript m is 0, 1, 2, 3, or 4. In Formula I, any aryl, heterocycle, or heteroaryl is optionally substituted with one to four s