# US 8,771,644 B2 · Imaging probes

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- Number: US 8,771,644 B2
- Document ID: US8771644B2
- Status: Granted
- Kind: B2
- Application number: 12/549,017
- Filed: 2009-08-27
- Published: 2010-07-29
- Granted: 2014-07-08
- Earliest claimed: 2007-02-28
- Assignees: Sanofi
- Applicants: Karl-Ulrich Wendt; Maik Kindermann; Anja Globisch
- Inventors: Karl-Ulrich Wendt; Maik Kindermann; Anja Globisch
- Web page: https://patentgrep.com/patent/US8771644B2

## CPC

- C12Q 1/37: involving peptidase or proteinase
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 5/06026: {the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala}
- C07K 5/06078: {and aromatic or cycloaliphatic}
- C07K 5/0812: {and aromatic or cycloaliphatic}
- C07K 5/0827: {containing heteroatoms different from O, S, or N}
- C07K 5/1016: {and aromatic or cycloaliphatic}
- C07K 7/06: having 5 to 11 amino acids
- 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
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present invention relates to molecular probes of the formula (I) {L1-R1-L}n-A-CO—NH—R2-L2 (I) as defined herein that allow for the observation of the catalytic activity of a selected cathepsin in in vitro assays, in cells or in multicellular organisms, a method for their preparation and the use thereof.

## Claims

### Claim 1

A molecular probe for cathepsin protease S of the formula (I):
wherein
X is —CONH—R2-L2
Y is-L-R1-L1, and
R1 and R2 are, independently of each other, each a linker;
L is a bond or a group allowing for a facile conjugation of the group L1;
L1 and L2 are, independent of each other, at least one label optionally bound to a solid support.

### Claim 2 (depends on 1)

The molecular probe according to claim 1, wherein L is a direct bond or a group selected from
—(NRx)-, —O—, —C═N—, —C(═O)—, —C(═O)—NH—, —NH—C(═O)—, —C(═O)H, —CRx=Cry—, —C≡C— and phenyl, wherein Rx and Ry are independently H or (C1-C6)alkyl.

### Claim 3 (depends on 1)

The molecular probe according to claim 1, wherein R1 or R2 is a straight or branched chain alkylene group with 1 to 300 carbon atoms, wherein optionally;
(a) one or more carbon atoms are replaced by oxygen;
(b) one or more carbon atoms are replaced by nitrogen carrying a hydrogen atom, and the adjacent carbon atoms are substituted by oxo, representing an amide function —NH—CO—;
(c) one or more carbon atoms are replaced by an ester function —O—CO—;
(d) the bond between two adjacent carbon atoms is a double or a triple bond; and/or
(e) two adjacent carbon atoms are replaced by a disulfide linkage.

### Claim 4 (depends on 3)

The molecular probe according to claim 3, wherein labels L1 and L2 are independently of each other a spectroscopic probe; a fluorophore; a quencher or a chromophore; a magnetic probe; a contrast reagent; a molecule which is one part of a specific binding pair which is capable of specifically binding to a partner; a molecule covalently attached to a solid support; a biomolecule; or a positively charged linear or branched polymer.

### Claim 5 (depends on 4)

The molecular probe according to claim 4, wherein labels L1 and L2 are independently of each other bound to a positively charged linear or branched polymer.

### Claim 6 (depends on 5)

The molecular probe according to claim 5, wherein one of labels L1 and L2 is a linear poly(arginine) of D- and/or L-arginine with 6-15 arginine residues.

### Claim 7 (depends on 4)

The molecular probe according to claim 4, wherein L1 is one member and L2 is the other member of two interacting spectroscopic probes L1/L2.

### Claim 8 (depends on 7)

The molecular probe according to claim 7, wherein L1/L2 is a FRET pair.

### Claim 9 (depends on 8)

The molecular probe according to claim 8, wherein one of labels L1 and L2 is a fluorophore selected from Alexa 350, dimethylaminocoumarin, 5/6-carboxyfluorescein, Alexa 488, ATTO 488, DY-505, 5/6-carboxyfluorescein, Alexa 488, Alexa 532, Alexa 546, Alexa 555, ATTO 488, ATTO 532, tetramethylrhodamine, Cy 3, DY-505, DY-547, Alexa 635, Alexa 647, ATTO 600, ATTO 655, DY-632, Cy 5, DY-647 and Cy5.5, and the other of labels L1 and L2 is a quencher selected from Dabsyl, Dabcyl, BHQ 1, QSY 35, BHQ 2, QSY 9, ATTO 540Q, BHQ 3, ATTO 612Q, and QSY 21.

### Claim 10 (depends on 1)

A method of using a probe according to claim 1 for imaging a living organism, said method comprising:
(a) administering to said organism a probe of the formula (I);
(b) exposing said organism to electromagnetic radiation which excites non-quenched fluorophore to produce a detectible signal; and
(c) detecting said signal and creating an image thereby.

### Claim 11 (depends on 1)

A method of using a probe according to claim 1 for imaging a living organism, said method comprising:
a) administering to said organism a probe of the formula (I);
(b) exposing said organism to electromagnetic radiation which excites fluorophore to produce a detectible signal; and
(c) detecting said signal and creating an image thereby.

### Claim 12 (depends on 3)

The molecular probe according to claim 3, wherein every third carbon atom of said straight or branched chain alkylene group is replaced by oxygen.

### Claim 13 (depends on 12)

The molecular probe according to claim 12, wherein said straight or branched chain alkylene group comprises a poylethyleneoxy group with 1 to 100 ethyleneoxy units.

### Claim 14 (depends on 4)

The molecular probe according to claim 4, wherein said solid support is a glass slide, a microtiter plate or a polymer.

### Claim 15 (depends on 1)

The probe according to claim 1, selected from the group consisting of