# US 10,869,936 B2 · Activity-based probe compounds, compositions, and methods of use

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- Number: US 10,869,936 B2
- Document ID: US10869936B2
- Status: Granted
- Kind: B2
- Application number: 16/338,682
- Filed: 2017-12-23
- Published: 2020-02-06
- Granted: 2020-12-22
- Earliest claimed: 2016-12-23
- Assignees: The Board of Trustees of the Leland Stanford Junior University
- Applicants: The Board of Trustees of the Leland Stanford Junior University
- Inventors: Matthew S. Bogyo; Martijn Verdoes
- Web page: https://patentgrep.com/patent/US10869936B2

## CPC

- A61K 49/0052: {Small organic molecules (oligomers, polymers, dendrimers A61K49/0054)}
- C12Q 1/37: involving peptidase or proteinase
- A61K 49/0032: {Methine dyes, e.g. cyanine dyes}
- G01N 33/575: for cancer
- G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)}
- A61K 49/0034: {Indocyanine green, i.e. ICG, cardiogreen}
- G01N 2333/948: acting on peptide bonds (3.4)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Activity-based probe compounds for use in labeling a cysteine protease are provided. 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.

## Claims

### Claim 1

A compound for use in labeling a protease having the formula (II):
wherein D comprises a benzoindole dye;
L1 is a linker;
AA1 is an amino acid side chain;
U is O, NH, or S;
R1 is alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, or a protecting group, and is 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, sulfonamido, or azido;
L3 is a linker; and
Q comprises a quencher.

### Claim 2 (depends on 1)

The compound of claim 1, wherein the benzoindole dye has the structure:
wherein o is an integer from 1 to 4;
R2 is a C2-C8 alkyl group, optionally substituted with a sulfonate or carbonate;
each R3 is independently a C1-C6 alkyl group; and
L4 is an optionally substituted alkyl linker, wherein each carbon atom is optionally replaced with a heteroatom.

### Claim 3 (depends on 2)

The compound of claim 2, wherein the benzoindole dye has the structure:

### Claim 4 (depends on 1)

The compound of claim 1, wherein L1 is an optionally substituted alkyl linker, wherein each carbon atom is optionally replaced with a heteroatom.

### Claim 5 (depends on 1)

The compound of claim 1, wherein AA1 is an aralkyl amino acid side chain, optionally substituted with 1 to 3 A groups.

### Claim 6 (depends on 1)

The compound of claim 1, wherein U is O.

### Claim 7 (depends on 1)

The compound of claim 1, wherein L3 is an optionally substituted alkyl linker, wherein each carbon atom is optionally replaced with a heteroatom.

### Claim 8 (depends on 1)

The compound of claim 1, wherein L3-Q is
wherein R comprises a QSY quencher or a QC-1 quencher; and
n is an integer from 1 to 8.

### Claim 9 (depends on 8)

The compound of claim 8, wherein the QSY quencher is a hydrophilic QSY quencher.

### Claim 10 (depends on 9)

The compound of claim 9, wherein the hydrophilic QSY quencher is a sulfo-QSY quencher.

### Claim 11 (depends on 8)

The compound of claim 8, wherein the QC-1 quencher has the structure:

### Claim 12 (depends on 1)

The compound of claim 1 having the formula (III):
wherein R comprises a QSY quencher or a QC-1 quencher; and
m and n are independently integers from 1 to 8.

### Claim 13 (depends on 12)

The compound of claim 12, wherein R is
and D is

### Claim 14 (depends on 13)

The compound of claim 13, having the structure:

### Claim 15 (depends on 1)

A composition for use in labeling a protease in an animal comprising a compound of claim 1 and a pharmaceutically acceptable carrier.

### Claim 16 (depends on 15)

A method of labeling a protease in an animal comprising the step of:
administering the composition of claim 15 to the animal.

### Claim 17 (depends on 15)

A method of visualizing a tumor in an animal comprising the steps of:
administering the composition of claim 15 to the animal; and
measuring a detectable signal generated in the animal from a reaction of the composition with a cathepsin cysteine protease; wherein the detectable signal is associated with a tumor in the animal.

### Claim 18 (depends on 17)

The method of claim 17, wherein the detectable signal is a fluorescent signal.

### Claim 19 (depends on 18)

The method of claim 18, wherein the fluorescent signal is generated at a tumor margin.