Compounds, Compositions, and Methods of Use of Activity-Based Probes

Abstract

To provide activity-based probe compounds, compositions, and methods of use.SOLUTION: Provided are activity-based probe compounds for use in labeling a cysteine protease. The compounds are targeted to the protease through a specific targeting element. The compounds additionally include a detectable element, such as a fluorescent label, a radiolabel, or a chelator. In some cases, the compounds additionally include a quenching element that is released upon reaction with the protease. Also provided are compositions comprising the compounds and methods for using the compounds, for example in labeling a protease in an animal and in visualizing a tumor in an animal.SELECTED DRAWING: None

Claims

1. A composition for use in labeling cysteine protease, wherein the composition comprises a compound having the formula (I): 【Chemical 35】 comprising a compound having, wherein L is, 【Chemical 36】 wherein, Here, Q is a quencher that regulates the emission of the fluorophore, and L ３ is an optionally substituted alkyl linker, each carbon atom is optionally replaced by a heteroatom, and Y is a halogen or H, wherein D is a detectable fluorescent label, radiolabel or chelator, T is a targeting group, and together D-T- is, 【Chemical 37】 wherein, Here, L １ is an alkyl linker that has been replaced as necessary, and each carbon atom has been replaced by a heteroatom as necessary, AA １ is an aralkyl amino acid side chain substituted with 1 to 3 A groups as required, U is O, R １ is alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl, or a protecting group, optionally substituted with 1 to 3 A groups, each A is independently alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryl, aryloxy, arylamino, aralkyl, aralkoxy, aralkanoyl, aralkamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl, heteroaralkoxy, heteroaralkanoyl, heteroaralkamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkamino, heterocyclyl, heterocyclyloxy, heterocyclylamino, heterocyclylalkyl, heterocyclylalkoxy, heterocyclylalkanoyl, heterocyclylalkamino, hydroxyl, thio, amino, alkanoylamino, aroylamino, aralkanoylamino, alkylcarboxy, carbonate, carbamate, guanidinyl, urea, halo, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfonamide, or azide, composition.

2. The composition according to claim 2, wherein D is a fluorescent label.

3. The composition according to claim 2, wherein the fluorescent label is fluorescein, Oregon Green, Bora-diaza-indecene, rhodamine, or a cyanine label.

4. The composition according to claim 3, wherein the fluorescent label is a cyanine label.

5. The composition according to claim 4, wherein the cyanine label is Cy5.

6. The composition according to claim 2, wherein the fluorescent label is selected from the group consisting of IRDye 800CW, IRDye 680RD, IRDye 680LT, IRDye 750, IRDye 700DX, IRDye 800RS, and IRDye 650.

7. L is, 【Chemical 38】 The composition according to any one of claims 1 to 6.

8. L is, 【Chemical 39】 wherein R is a QSY quencher and n is an integer from 1 to 16. The composition according to claim 7.

9. The compound has the formula (III) 【Chemical 40】 The composition according to claim 8, having the structure, wherein m is an integer from 1 to 16.

10. The composition according to claim 9, wherein R is QSY21 or sulfo-QSY21, and D is Cy5.

11. The composition according to claim 1, wherein the quencher is IRDyeQC-1.

12. A composition for use in labeling proteases in animals, comprising a compound and a pharmaceutically acceptable carrier, wherein the compound has the formula (I): 【Chemical Formula 41】 having wherein L is 【Chemical 42】 being Here, Q is a quencher that regulates the emission of the fluorophore, and L ３ is an optionally substituted alkyl linker, each carbon atom of which is optionally replaced by a heteroatom, and Y is a halogen or H, wherein D is a detectable fluorescent label, radiolabel or chelator, T is a targeting group, and together D-T- is 【Chemical 43】 being Here, L １ is an alkyl linker optionally substituted, and each carbon atom is optionally replaced by a heteroatom, AA １ is an aralkyl amino acid side chain substituted with 1 to 3 A groups as required, U is O, R １ is alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl, or a protecting group, optionally substituted with 1 to 3 A groups, each A is independently alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryl, aryloxy, arylamino, aralkyl, aralkoxy, aralkanoyl, aralkamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl, heteroaralkoxy, heteroaralkanoyl, heteroaralkamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkamino, heterocyclyl, heterocyclyloxy, heterocyclylamino, heterocyclylalkyl, heterocyclylalkoxy, heterocyclylalkanoyl, heterocyclylalkamino, hydroxyl, thio, amino, alkanoylamino, aroylamino, aralkanoylamino, alkylcarboxy, carbonate, carbamate, guanidinyl, urea, halo, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfonamide, or azide, composition.

13. The composition according to claim 12, wherein the pharmaceutically acceptable carrier comprises mannitol.

14. The composition according to claim 12, wherein the pharmaceutically acceptable carrier comprises an aqueous solution.

15. The composition according to claim 12 for use in a method of visualizing a tumor in an animal, the method comprising: administering the composition to the animal; and measuring a detectable signal generated in the animal from the reaction of the composition with a cathepsin cysteine protease wherein the detectable signal is related to a tumor in the animal.

16. The composition for use according to claim 15, wherein the detectable signal is a fluorescence signal. **Claim 17** The composition for use according to claim 16, wherein the detectable signal is generated at the tumor margin.

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