# US 2008/0187947 A1 · Cholesterol-Mimetic Pyrene Probes

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2008/0187947 A1
- Document ID: US20080187947A1
- Status: Published application
- Kind: A1
- Application number: 11/909,476
- Filed: 2006-03-23
- Published: 2008-08-07
- Earliest claimed: 2005-03-23
- Assignees: Centre National De La Recherche Scientifique
- Inventors: Christophe Le Roux; Andre Lopez
- Web page: https://patentgrep.com/patent/US20080187947A1

## CPC

- C07J 9/00: Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
- G01N 33/566: using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds}
- G01N 33/92: involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)}
- G01N 2500/04: Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
- C07J: STEROIDS (seco-steroids C07C)
- 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

The invention concerns compounds called pyrene-cholesterols, of general formula (I), wherein: X represents a CH2 group or an oxygen atom and n is an integer from 2 to 10, as well as their use for implementing a method for detecting and measuring the effects of specific products on biological membranes, and a method for preparing the compounds.

## Claims

### Claim at position 1

1-18. (canceled)

### Claim 19

Compounds hereafter called pyrene-cholesterols, of general formula (I):
wherein X represents a CH2 group or an oxygen atom and n is an integer from 2 to 10, in particular from 2 to 6.

### Claim 20 (depends on 19)

Compound according to claim 19, hereafter called pyrene-ketone-cholesterol, of following formula (4):

### Claim 21 (depends on 19)

Compound according to claim 19, hereafter called pyrene-methylene-cholesterol, of following formula (7):

### Claim 22 (depends on 19)

Method for studying the functional organization of biological membranes, if appropriate in the presence of external stimuli, such as temperature, pressure, modulation by an extrinsic agent of the lipid concentration initially contained in said biological membranes, or the addition of a given product comprising the addition of a pyrene-cholesterol compound defined in claim 19, or a combination of these pyrene-cholesterol compounds.

### Claim 23 (depends on 22)

Method according to claim 22, which is a method of detection, and, if appropriate, a method for measurement of the polarity or the permeability of biological membranes, the concentration of one or more constituents of these membranes, and the spatial and collective organization of the constituents of these biological membranes.

### Claim 24 (depends on 22)

Method according to claim 22, which is a method of detection, and, if appropriate, photophysical measurement of the effects of given products on biological membranes, said method being applied to:
screening bioactive products capable of being agonists or antagonists of receptors present in cell membranes or on the surface of these membranes, said bioactive products capable of being used for the preparation of medicaments intended for the treatment of pathologies involving said receptors;
in vitro diagnosis of pathologies from cells of patients using given products used as markers of these pathologies, or
monitoring the orientation of the therapeutic indication of a medicament comprising a bioactive product as defined above, by determination in vitro of the agonist or antagonist effect of this medicament on the cells of the patient capable of being treated with this medicament.

### Claim 25 (depends on 22)

Method according to claim 22, wherein the biological membranes are chosen from the group constituted by vesicles, unilamellar liposomes, multilamellar liposomes, bilayers or supported lipid multilayers, extracted cell membranes or cell membranes of living cells.

### Claim 26 (depends on 22)

Method according to claim 22, wherein the cell membranes are those of the following eukaryotic cells: CHO, HEK, NRK, AEB, AEH, SHY5Y, 3T3, primary cultures of neurones, fibroblasts, or originating from other organs or tissues.

### Claim 27 (depends on 19)

Method for the study of the functional organization of biological membranes, and the use of a method of detection, and, if appropriate, measurement of the effects of external stimuli, such as temperature, pressure, the modulation by an extrinsic agent of the lipid concentration initially contained in said biological membranes, or the addition of a given product influencing the organization of said biological membranes, characterized in that it comprises:
a stage of incorporation of a compound according to claim 19, or a combination of several of these compounds, into said biological membranes,
if appropriate, the application of external stimuli, to said membranes,
the application to said biological membranes of radiation of wavelength λex characteristic of the absorption of pyrene , λex being comprised between 330 nm and 350 nm,
the measurement of the intensity of the fluorescence radiation emitted by said biological membranes at the emission wavelength of 375 to 385 nm, characteristic of pyrene emission band 1, this intensity being a function of the polarity of the membrane,
the measurement of the intensity of at least one of the two fluorescence radiations emitted by said biological membranes between 390 nm and 425 nm, characteristic of pyrene emission bands 3 and 4, this intensity being a function of the quantity of pyrene, and therefore of compounds, in said membranes,
the determination of the ratio between the intensity of band 1 and that of band 3 and/or 4, namely of the ratios I1/I3 and/or I1/I4,
the correlation between the ratio measured during the previous stage, and the level of polarity of said membranes which is a function of their thickness, thus giving an indication of the functional organization of these membranes in the presence or absence of external stimuli.

### Claim 28 (depends on 27)

Method according to claim 27, applied to screening bioactive products capable of being agonists or antagonists of receptors present in cell membranes or partially inserted into these membranes, said bioactive products being capable of use for the preparation of medicaments intended for the treatment of pathologies in which said receptors are involved, or
monitoring the direction of the therapeutic indication of a medicament comprising a bioactive product as defined above, by in vitro determination of the agonist or antagonist effect of this medicament on the cells of the patient capable of being treated with this medicament,
said method comprising:
a stage of incorporation of said compound according to, or a combination of several of these compounds, into said biological membranes,
the placing said membranes in the presence of a bioactive product, or a bank of bioactive products, under conditions allowing these products to bond with the membrane receptors,
if appropriate, a stage of rinsing said membranes to remove any bioactive product which is not capable of bonding with a membrane receptor,
the application to said biological membranes of a radiation of wavelength of λex characteristic of the absorption of pyrene , λex being comprised between 330 nm and 350 nm,
the measurement of the intensity of bands 1, 3, and/or 4, and the determination of the ratios I1/I3 and/or I1/I4,
the comparison of the ratios I1/I3 and/or I1/I4, obtained in the previous stage with the ratios I′1/I′3 and/or I′1/I′4, measured in the absence of bioactive products, which allows the agonist or antagonist or inverse antagonist nature of the bioactive products tested to be determined, an agonist product being characterized by a ratio I1/I3 and/or I1/I4 respectively greater than or less than the ratio I′1/I′3 and/or I′1/I′4 according to the type of said receptors, an antagonist product being characterized by a ratio I1/I3 and/or I1/I4 equivalent respectively to the ratio I′1/I′3 and/or I′1/I′4, an inverse antagonist product being characterized by a ratio I1/I3 and/or I1/I4 respectively less than or greater than the ratio I′1/I′3 and/or I′1/I′4 i.e. an inverse effect to that of an agonist product .

### Claim 29 (depends on 27)

Method according to claim 27, characterized in that it is carried out in vitro on eukaryotic cells, and comprises a stage of incorporation of said compound, or a combination of several of these compounds, into the membranes of these cells.

### Claim 30 (depends on 27)

Method according to claim 27, wherein the stage of incorporation of said compound, or the combination of several of these compounds, into said membranes is carried out using a transporter intended to exchange cholesterol, a phospholipid, naturally present in said membranes, with said pyrene-cholesterol compound or said combination.

### Claim 31 (depends on 27)

Method according to claim 27, wherein the transporter is selected from the group comprising a methyl-β-cyclodextrin, and a α-, β-, γ-cyclodextrin.

### Claim 32 (depends on 27)

Method according to claim 27, wherein the proportion of said compound, or the combination of several of these compounds, incorporated into said membranes represents about 0.1 to 2% of the surface concentration of the lipids present in the biological membrane.

### Claim 33 (depends on 19)

Composition comprising a compound according to claim 19, or a combination of several of these compounds, in a mixture with a transporter intended to exchange cholesterol or a phospholipid, naturally present in said membranes with said pyrene-cholesterol compound or said combination.

### Claim 34 (depends on 33)

Composition according to claim 33, wherein the transporter is selected from the group comprising a methyl-β-cyclodextrin, and a α-, β-, γ-cyclodextrin.

### Claim 35 (depends on 27)

Method according to claim 27 wherein the stage of incorporation of said compound, or a combination of several of these compounds, into said biological membranes, is realized through the incorporation of a composition comprising said compound or a combination of several of these compounds, in a mixture with a transporter intended to exchange cholesterol or a phospholipid, naturally present in said membranes with said pyrene-cholesterol compound or said combination.

### Claim 36 (depends on 1, 2, 3)

Synthesis method for the compounds of formula (I) according to one of claims 1 to 3, characterized in that it comprises the following stages:
a) reacting the pregnenolone of formula (a) and the pyrene derivative of formula (b) wherein n is an integer from 2 to 10, in particular from 2 to 6, in the presence of EtOK, EtOH, in CH2Cl2 at a temperature between 5° C. and 20° C. and for a duration comprised between 10 and 72 hours, which produces the following compound of formula (1):
b) reacting the compound of formula (1) obtained in the previous stage in the presence of 1.5 equivalents of Ac2O, 10 mol % of DMAP, 1 equivalent of Et3N, in CH2Cl2 for 3 hours, which produces the following compound of formula (2):
c) reacting the compound of formula (2) obtained in the previous stage in the presence of 2 equivalents of TiCl4, 1.5 equivalents of Et3SiH, in CH2Cl2 for 45 minutes at ambient temperature, which produces the following compound of formula (3):
d) reacting the compound of formula (3) obtained in the previous stage in the presence of MeONa, in CH2Cl2 for 4 hours at ambient temperature, leads to the following compound of formula (4):
e) if appropriate, reacting the compound of formula (4) obtained in the previous stage in the presence of TBDMSCl, imidazole, CH2Cl2 for 12 hours at ambient temperature, which produces the following compound of formula (5):
f) reacting the compound of formula (5) obtained in the previous stage in the presence of 2 equivalents of CH2=PPh3, in a THF/DMSO mixture, for 12 hours at 75° C., which produces the following compound of formula (6):
g) reacting the compound of formula (6) obtained in the previous stage in the presence of 5 equivalents of TBAF, in THF, for 12 hours at 65° C., produces the following compound of formula (7):