# US 7,745,413 B2 · Collection of traceable compounds and uses thereof

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- Number: US 7,745,413 B2
- Document ID: US7745413B2
- Status: Granted
- Kind: B2
- Application number: 11/597,891
- Filed: 2005-06-15
- Published: 2008-07-24
- Granted: 2010-06-29
- Earliest claimed: 2004-06-15
- Assignees: Centre National de la Recherche Scientifique (CNRS); Universite Louis Pasteur
- Inventors: Marcel Francois Louis Hibert; Christel Anne Franchet; Jean-Luc Galzi; Franc Emile Jean Pattus; Fabrice Yves Guillier
- Web page: https://patentgrep.com/patent/US7745413B2

## CPC

- C07D 497/10: Spiro-condensed systems
- Y02P 20/55: Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

The use of a collection of compounds of general formula (I), wherein: n is 0 or 1; p represents an integer between 1 and 6; r represents an integer between 1 and 12; R1 and R′1 represent in particular a hydrogen atom; R2 represents an amino acid side chain or an amino acid derivative; R3 represents a group derived from a carboxylic acid, bearing a basic entity; R4 represents in particular an alkyl group containing 1 to 10 carbon atoms; and A represents a hydrogen atom, a protecting group or a tracing group, in particular a fluorophor, a coloring agent or a quencher, for determining, through binding studies, ligands of receptors whose ligand is unknown or whose ligand useful for carrying out specific affinity binding assays is unknown.

## Claims

### Claim 1

A compound of the following formula (I):
wherein:
n is equal to 0 or 1,
p represents an integer varying from 1 to 6,
r represents an integer varying from 1 to 12,
R1 and R′1 represent independently of each other an atom or a group selected from the group consisting of: a hydrogen atom, an alkyl group comprising from 1 to 6 carbon atoms, and an aromatic group,
or
and the
is optionally substituted with one group selected from the group consisting of: an alkyl group comprising from 1 to 6 carbon atoms and an aromatic group,
R2 represents a side chain of an amino acid or an amino acid derivative,
R3 represents a group derived from a carboxylic acid, carrying a basic entity, selected from the group consisting of:
R4 represents an alkyl group comprising from 1 to 10 carbon atoms, a phenyl group substituted by an alkyl group comprising from 1 to 10 carbon atoms, and
A represents a hydrogen atom, a protective group or a tracer group selected from the group consisting of: a fluorophore, a dye or a “quencher”.

### Claim 2 (depends on 1)

The compound of claim 1, wherein R2 represents the side chain of an amino acid or amino acid derivative selected from the group consisting of: alanine, glutamine, leucine, glycine, tryptophan, β-alanine, phenylalanine, 4-chloro-phenylalanine, isonipecotinic acid, 4-aminomethylbenzoic acid, 3-tetrahydroisoquinolinic acid and free or benzylated histidine.

### Claim 3 (depends on 1)

The compound of claim 1, wherein n is equal to 0.

### Claim 4 (depends on 1)

The compound of claim 1, wherein n is equal to 1.

### Claim 5 (depends on 3)

The compound of claim 3, wherein A represents a hydrogen atom or a protective group.

### Claim 6 (depends on 5)

The compound of claim 5, corresponding to one of the following formulae:
wherein R2, R3, R4 and p are as previously defined.

### Claim 7 (depends on 1)

The compound of claim 1, wherein A represents a fluorophore group selected from the group consisting of: Bodipy, and lissamine derivatives.

### Claim 8 (depends on 1)

The compound of claim 1, wherein A represents one of the following groups:

### Claim 9 (depends on 1)

The compound of claim 1, corresponding to the following formula (III):
wherein:
R2, R3, R4 and p are as previously defined,
the piperazine group is optionally substituted with one group selected from the group consisting of: an alkyl group comprising from 1 to 6 carbon atoms and an aromatic group, and
A represents a fluorophore group selected from the group consisting of: Bodipy, and lissamine derivatives.

### Claim 10 (depends on 9)

The compound of claim 9, wherein p=4 and corresponding to the following formula:

### Claim 11 (depends on 9)

The compound of claim 9, corresponding to the following formula (III-a):

### Claim 12 (depends on 1)

The compound of claim 1, corresponding to the following formula (IV):
wherein:
R2, R3, R4, p and r are as previously defined,
the piperazine group is optionally substituted with one group selected from the group consisting of: an alkyl group comprising from 1 to 6 carbon atoms and an aromatic group, and
A represents a fluorophore group selected from the group consisting of: Bodipy, and lissamine derivatives.

### Claim 13 (depends on 12)

The compound of claim 12, wherein p=4 and corresponding to the following formula:

### Claim 14 (depends on 12)

The compound of claim 12, wherein r=4 and corresponding to the following formula:

### Claim 15 (depends on 12)

The compound of claim 12, wherein p and r are equal to 4 and corresponding to the following formula:

### Claim 16 (depends on 12)

The compound of claim 12, corresponding to the following formula (IV-a):

### Claim 17 (depends on 1)

A collection comprising a plurality of compounds of formula (I) as defined in claim 1.

### Claim 18 (depends on 11)

A collection comprising a plurality of compounds of formula (III-a) as defined in claim 11.

### Claim 19 (depends on 16)

A collection comprising a plurality of compounds of formula (IV-a) as defined in claim 16.

### Claim 20 (depends on 1)

A method for screening ligands of receptors no ligand of which is known or no useable ligand of which is known, said method comprising the following stages:
bringing a collection of traceable compounds of formula (I) according to claim 1, together with cells transfected by a construction containing the fusion of the sequence coding for a fluorescent protein with the nucleotide sequence coding for a receptor no ligand of which is known or no useable ligand of which is known, and the mixture of said cells and of said collection,
detecting the fluorescence of said mixture, by exciting said fluorescent protein and measuring the emission fluorescence of said fluorescent protein, and determining the fluorescence extinction percentage by comparing the emission fluorescence of said fluorescent protein to the average fluorescence of said fluorescent protein, the average fluorescence of said fluorescent protein being measured by control tests corresponding to the measurement of the fluorescence of the fluorescent protein in the absence of the collection of compounds, and
determining the compounds which produce a fluorescence extinction percentage of the fluorescent protein of at least 5% and identifying said compounds as ligand.

### Claim 21 (depends on 1)

A method for the preparation on solid support of a compound of formula (I) of claim 1, comprising the following stages:
coupling a solid support of formula
to a compound of formula
in order to obtain a compound of the following formula (1):
R1 and R′1 being as previously defined,
deprotecting the compound of formula (1) followed by coupling of said deprotected compound to the compound of formula
in order to obtain a compound of the following formula (2):
R2 being as previously defined,
deprotecting the compound of formula (2) followed by coupling of said deprotected compound to the compound of formula
in order to obtain a compound of the following formula (3):
p being as previously defined,
deprotecting the Fmoc group of the compound of formula (3) followed by coupling of said deprotected compound to the compound R3COOH in order to obtain a compound of the following formula (4):
R3 being as previously defined,
deprotecting the compound of formula (4) in order to obtain a compound of the following formula (5):
optionally, reacting the compound of formula (5) with p-nitrophenylchloroformate then with the diamine of formula
in order to obtain the compound of the following formula (6):
r being as previously defined,
reacting the compound of formula (5) or the compound of formula (6) with electrophilic tracers, in particular with A-Cl, in order to obtain the compound of the following formula (7):
n being equal to 0 or 1,
A representing Bodipy in the form of activated acid or lissamine in the form of activated sulphonic acid, and
cleaving the compound of formula (7) with the compound R4X, R4 being as defined above, and X representing a halogen atom, in particular I or Br, in order to obtain the compound of formula (I).

### Claim 22

A method for the in vitro determination of ligands of receptors no ligand of which is known or no ligand of which is known that can be used to carry out specific affinity binding studies, comprising:
providing a collection of compounds corresponding to the following general formula (I):
wherein:
n is equal to 0 or 1,
p represents an integer varying from 1 to 6,
r represents an integer varying from 1 to 12,
R1 and R′1 represent independently of each other an atom or a group selected from the group consisting of: a hydrogen atom, an alkyl group comprising from 1 to 6 carbon atoms, and an aromatic group,
or
and the
is optionally substituted with one group selected from the group consisting of: an alkyl group comprising from 1 to 6 carbon atoms and an aromatic group,
R2 represents a side chain of an amino acid or an amino acid derivative,
R3 represents a group derived from a carboxylic acid, carrying a basic entity, selected from the following groups:
is selected from the group consisting of: an alkyl group comprising from 1 to 10 carbon atoms, a phenyl group substituted by an alkyl group comprising from 1 to 10 carbon atoms, and
A represents a hydrogen atom, a protective group or a tracer group, in particular a fluorophore, a dye or a “quencher”, and
performing binding studies with said collection of compounds.

### Claim 23 (depends on 22)

The method of claim 22, wherein the collection of compounds correspond to the following general formula (III-a)

### Claim 24 (depends on 22)

The method of claim 22, wherein the collection of compounds correspond to the following general formula (IV-a)