WO2008155593A2 - Composés et compositions pour marquer des gouttelettes de lipide, et procédés pour la visualisation de cellules et/ou d'organelles cellulaires - Google Patents

Composés et compositions pour marquer des gouttelettes de lipide, et procédés pour la visualisation de cellules et/ou d'organelles cellulaires Download PDF

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WO2008155593A2
WO2008155593A2 PCT/HU2008/000074 HU2008000074W WO2008155593A2 WO 2008155593 A2 WO2008155593 A2 WO 2008155593A2 HU 2008000074 W HU2008000074 W HU 2008000074W WO 2008155593 A2 WO2008155593 A2 WO 2008155593A2
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compounds
cells
fluorescent
organelles
dione
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WO2008155593A3 (fr
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Kutato Avicor
Laszlo Puskas
Liliana Feher
Eszter Molnar
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Kutato Avicor
Laszlo Puskas
Liliana Feher
Eszter Molnar
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/44—Iso-indoles; Hydrogenated iso-indoles
    • C07D209/48—Iso-indoles; Hydrogenated iso-indoles with oxygen atoms in positions 1 and 3, e.g. phthalimide

Definitions

  • the present invention relates to compounds for labeling cellular organelles, particularly lipid droplets. Furthermore, this invention includes a method for labeling organelles, especially lipid droplets, and compositions for labeling lipid droplets.
  • DAPI 4-6-diaminido-2-fenilindol
  • Dyes for staining organelles must have specific features, as they need to pass cell membranes to get to the specific organelle. Furthermore, they must have specific binding characters, which enables them to bind selectively to the organelle (to a molecule on its surface or in its inner parts). Preferably the dye should not be toxic to the cells, so that it can be used in live cultures without destroying the cell.
  • Certain fluorescent molecules are suitable for such staining techniques, as they can adsorb light at a specific wavelength and emit this energy at a different spectrum. When this emitted light passes through a filter, which filters light of other wavelengths, the fluorescent compound - or the organelle stained with the compound - can be seen on a black background.
  • This technique is the basis of fluorescent microscopy requiring much less dye for staining compared to conventional light microscopy (also referred to as optical microscopy) staining methods, thus influencing the processes within the observed cell to a lesser extent.
  • the most well-known fluorescent dye is fluorescein, which emits yellowish green light when it is excited by blue light.
  • Another well-known dye is rodamine, which emits deep-red light when it is excited by greenish yellow light.
  • lipid droplets or lipid bodies
  • organelles lipid bodies
  • Morphology and protein contents of the lipid droplets is heterogenous (Denis J. Murphy es mtsai: Mechanism of lipid-body formation, TIBS 24, 1999 March, pages 109-115.) but their formation in plants, in animals and in microorganizms were found to be very similar.
  • Defects in the function of lipid droplets in humans result in numerous diseases, such as fatty liver, obesity, atherosclerosis, II-type diabetes and cancer.
  • the size of lipid bodies vary between 0.1 and 50 ⁇ m.
  • Oil Red O dye has been used now for over 80 years to stain cholesterinesters and triglycerides [French, R.W. Stain Technol. 1, 1926, page 79.). The characterization of the purified dye was performed by Kutt and colleagues (Kutt H, Tsaltas TT. : Staining properties of oil red O and a method of partial purification of the commercial product. Clin Chem. 1959 Apr; 5(2): 149-60). Despite the low solubility of Oil Red O, it is routinely used for staining lipid bodies. It is currently available in solution with isopropanol or propylenglycol for fixed cells (Laughton C.
  • Another dye for staining lipid bodies in fixed cells is BODIPY 493/503 (Gocze PM, Freeman DA.: Factors underlying the variability of lipid droplet fluorescence in MA-IO Leydig tumor cells. Cytometry. 1994 Oct 1; 17(2): 151- 158), or Sudan III which was used in earlier studies (Vigh B, Vigh-Teichmann I, Aros B, Oksche A.: Sensory cells of the "rod-” and "cone-type” in the pineal organ ofRana esculenta, as revealed by immunoreaction against opsin and by the presence of an oil (lipid) droplet. Cell Tissue Res. 1985; 240(1): 143-148).
  • Nile Red or 9- dietilamino-5H-benzo[ ⁇ ]fenoxazin-5-ont, which emits light of approx. 528 nm (yellowish gold color) and approx. 590 nm (red color) depending on the wavelength of the excitation light.
  • Nile red emits light in a range of approx. 60 nm depending on the hydrophobicity of the.
  • the dye showed higher selectivity for lipid droplets (Phillip Greenspan ⁇ s mtsai: Nile Red: A Selective Fluorescent Stain for Intracellular Lipid Droplets, J. Cell. Biol. Vol. 100, pg.
  • Nile Red is a less specific dye for lipid bodies as it has been shown to have a significant number of aspecific bindings to other organelles as well (Gocze PM ⁇ s Freeman DA: Factors underlying the variability of lipid droplet fluorescence in MA- 10 Leydig tumor cells. Cytometry. 1994 Oct 1; 17(2): 151- 158).
  • lipid droplets Although the examination of lipid droplets is highly important as it might help to answer questions related to the previously mentioned diseases, but up until now only a few dyes have been found, that are suitable for selective staining of lipid droplets to a certain extent. More types of lipid droplet dyes would open possibilities for such studies, for example they could give more insights into the formation and functioning of lipid droplets both in healthy and in defective cells. It could be easier to examine the effects of different drug candidate molecules on the function of lipid droplets as well.
  • the objective of the present invention is to provide dyes, which selectively label lipid bodies, and furthermore which are not only applicable on fixed cells but also on live cells.
  • the invention is based on the recognition that compounds with general formula I selectively bind to lipid droplets, and because most of the compounds have fluorescent properties, they provide fluorescent staining of lipid organelles, thus the fluorescent compounds among the compounds having formula I are capable of fluorescent labeling of lipid droplets. Furthermore, we discovered that compounds with formula I can pass cell membranes easily, which enables these molecules to be used for labeling lipid droplets of different multicellular organisms as well in addition to labeling lipid droplets of singular cells. We also realized, that compounds with formula I are generally not toxic making them suitable for labeling lipid droplets in vivo.
  • the present invention provides compounds having the general formula I.
  • the present invention relates to compounds for labeling lipid droplets, the compounds having the general formula I,
  • X are each independently hydrogen, halogen, -Ci-20-alkyl, -C2-2o-alkenyl, -C2- 20-alkinyl, -Cs- ⁇ -cykloalkyl, aryl, aralkyl, adamantyl, heterocyclic, hydroxyl, hydroxyalkyl, or -N-(R 1 , R 2 ) group; n is 0, 1, 2, 3, or 4;
  • R 1 and R 2 may each be independently hydrogen, straight or branching alkyl, cyclo-alkyl, aryl, aralkyl, heterocyclic group, wherein each is un-substituted or halogen substituted; or
  • R 1 and R 2 together with the nitrogen in between them form a 5 or 6 member ring;
  • A is a single bond, -O-, -S-, -CH2-, or -NH-; Y is O or S; Z is O or S;
  • R' and R" are each independently methyl, ethyl, isopropyl, isobuthyl, sec- butyl or terc-butyl;
  • A is a single bond.
  • At least one of Y and Z is O, more preferably both are O.
  • R 1 is hydrogen
  • any three of X(n) is halogen.
  • halogens are fluor.
  • R 1 and/or R 2 are/is un-substituted or halogen substituted ethyl, preferably ethyl or trifluoro-ethyl, especially ethyl group.
  • R 1 and/or R 2 are/is an adamantyl group.
  • at least one of R' and R" is isopropyl, more preferably both are isopropyl.
  • the lipid droplet labeling compound is 2-(2,6- diisopropylphenyl)-5-( ethylamino)- 4,6,7- trifluoroisoindole - 1,3-dione.
  • the compounds with formula I have fluorescent properties.
  • compositions for labeling lipid droplets which compositions contain formula I and other additives or carriers.
  • the invention further relates to a method for visualizing cells and/or organelles with a fluorescent imaging technique, the method comprising: - labeling the cells and/ or organelles with one or more fluorescent compounds preferably being in the form of a solution, and
  • the method is characterized by providing one or more fluorescent compounds having formula I for labeling the cells and/ or organelles.
  • the fluorescent imaging technique is fluorescent microscopy, fluorescent flow-cytometry or fluorescent signal detection based titration, preferably microtitration.
  • the concentration of the solution of the one or more compounds having formula I is between 0.01 to 100 ⁇ M, preferably between 0.1 to 10 ⁇ M, more preferably between 0.5 to 5 ⁇ M.
  • the organelles are lipid droplets.
  • Fig. Ia shows an X-ray diffraction image of the crystal structure of a compound (4a) according to the invention, in which hydrogen atoms and the numbering of heavy atoms are also indicated;
  • Fig. Ib shows an X-ray diffraction image of the crystal structure of a different compound (4b) according to the invention, in which hydrogen atoms and the numbering of heavy atoms are not indicated;
  • Fig. 2a and 2b show images of HepG2 human liver carcinoma cell culture, which is stained with a compound (4a) according to the invention;
  • Fig. 2a being a fluorescent microscopic image
  • Fig. 2b being a light microscope image;
  • Fig. 3a and 3b show images of RVH human melanoma cell culture stained with a compound (4b) according to the invention; Fig. 3a being a fluorescent microscopic image, Fig. 3b being a light microscopic image; Fig. 4a - c show the double -staining of lipid droplets of RVH human melanoma cell culture; Fig. 4a being a light microscope image; Fig 4b being a fluorescent image taken after the cells have been stained with a compound
  • Fig. 4c being a fluorescent image of the cells stained with Oil Red O;
  • Figures 5 show the staining of endoplasmic reticulum of RVH human melanoma cell culture (Fig. 5a) and staining with a compound (4b) according to the invention;
  • Fig. 6 shows the emission and fluorescent spectrum of a compound (4b) according to the invention, where relative intensity is displayed against wavelength (nm);
  • Fig. 7 shows the emission and fluorescent spectrum of a different compound
  • the compounds according to the invention are provided for labeling lipid droplets or organelles binding/ containing lipid droplets, or cells binding/ containing lipid droplets.
  • the compounds according to the invention can bind to the lipid droplets and identification (e.g. visualization) of the location of the compounds allows for identification (e.g. visualization) of the location of the lipid droplets. Accordingly, such compounds can be regarded both as compounds for staining lipid droplets and for labeling lipid droplets. Localization of the compounds can be performed by techniques making use of the fluorescent properties of the compounds or in other known ways, for instance, by isotope labeling. Most of the compounds of the patent, but not all, has fluorescent properties.
  • halogen in the present context denotes a substituent selected from fluorine, chlorine, bromine, or iodine.
  • aryl group in the present context, alone or in combination with any other substituents, denotes one carbocyclic aromatic group or an aromatic ring system with more carbocyclic aromatic system.
  • aryl includes phenyl or naphthyl ring systems.
  • n -C 1- 2o-alkyl in the present context, alone or in combination with any other substituents, denotes a straight chained or branched acyclic hydrocarbon with 1 to 20 carbon atoms.
  • "_,-Ci-2o-alkyl” group can include e.g. methyl, ethyl, propyl, butyl, hexyl, 1 -methyl-ethyl, 1-methyl-propyl, 2- methyl-propyl, or 1 , 1 -dimethyl-ethyl groups.
  • a -,-C5-6-cycloalkyF in the present context means a cycloalkyl substituent, containing 5 or 6 carbon atoms and includes for example cyclopentyl or cyclohexyl groups.
  • aralkyl means an aryl substituted alkyl or cycloalkyl group.
  • Heterocyclyl group designates in the present context substituted or un- substituted alycyclic or aromatic ring containing group that contains one or more heteroatoms selected from nitrogen, oxygen, sulfur or phosphorus.
  • Hydroxyalkyl group means an alkyl group substituted by hydroxyl group.
  • the compounds of the patent are fluorescent dyes, which enables them for use in fluorescent microscopy and in flow cytometry studies.
  • An advantageous property of the compounds according to the invention is that for the most part they are non-toxic to most of the cell types in concentrations in which they can be used in imaging techniques based on the identification of the location of the compounds, e.g. in fluorescent microscopy. This non-toxic character makes these compounds suitable for in vitro studies, so that spatial and/ or temporal plasticity of lipid droplets can be examined in live cells by such compounds and the method according to the invention.
  • the compounds according to the invention are capable of binding to lipid droplets, thereby the cells containing the lipid droplets, or the organelles containing or being bound to such lipid droplets can be visualized, e.g. by fluorescent techniques.
  • the compounds have fluorescent properties, not only is it possible to capture microscope images of single cells or of groups of cells, but cell counting (measuring cell concentration in samples) can be performed as well by measuring the intensity of the fluorescent emission for example by fluorescent flow cytometry analysis.
  • compositions which contain compounds according to the invention.
  • Such compositions might contain well-known additives or carriers, for instance ethylene glycol, propylene glycol, DMSO, ethanol or a mixture of these materials.
  • compositions according to the invention can be applied for example for in vivo diagnosis of atheroma: i) during animal experiments of screening for drug candidate molecules PET analysis can give information on the formation/ size of atheroma without killing the animals; ii) PET analysis or other techniques, such as NMR can give information on the formation/ size of atheroma in human diagnosis of atheroma formation
  • compositions according to the invention are also suitable for detection of transitional zone of a heart attack.
  • a heart attack the central infarction zone is surrounded by a transitional zone, typical of an ischaemic heart muscle. It is a reversibly injured area, which is erupted by the reduced blood flow. Lipid metabolism changes in this area and numerous specific lipid droplets will occur in such cells (Bilheimer DW et ai.: Fatty acid accumulation and abnormal lipid deposition in peripheral and border zones of experimental myocardial infarcts. J. Nucl. Med. 19, 1978, 276-283). Identification and specific separation of this area from the central infarction zone would be relevant in clinical aspects (Ingrid M.
  • compositions of the patent having either carbon- 11 or fluorine- 18 isotopes can be successfully used during PET analyses for example in ischaemia, or in other diseases after local hypoxia (for identification of tumor or prenecrotic tissues). Accordingly, compounds of formula I of the invention also include variants comprising different isotopes, especially carbon- 11 and fluorine- 18 isotopes.
  • Example 2 4-amino-2-(2,6-diisopropylphenil)- 5.6,7-trifluorisoindole-l,3- dione (3a) and 5-amino-2-(2,6-diisopropylphenil)- 4,6,7-trifluorisoindole-l,3- dione (3b)
  • the first fractionated product 3a was evaporated (yield: 5.9 g, 29.5%) and the purity of the product was checked by Thin Layer Chromatography (Rf: 0.19 (chloroform)) and the mass of the product was confirmed by Mass Spectrometry (MW+ 1: 376.9).
  • the second fractionated product 3b was evaporated (yield: 4.6g, 24.2%) and the purity of the product was checked by Thin Layer Chromatography (Rf: 0.09 (chloroform)) and the mass of the product was confirmed by Mass Spectrometry (MW+ 1: 376.9).
  • Example 3 2-(2,6-diisopropylphenyl)-4-(ethylammo)-5,6,7-trifluoroisoindole- 1,3-dione (4a) and 2-(2,6-diisopropylphenyl)-5-(ethylamino)-4,6,7- trifluoroisoindole- 1 ,3-dione (4b)
  • the column was washed with chloroform-cyclohexane 1:3 v/v, then eluted with chloroform-cyclohexane 1:1 v/v solution.
  • the first fractionated product 4a was evaporated (yield: 6.9 g, 30.1%) and the purity of compound 4a was checked by Thin Layer Chromatography (Rf: 0.39 (chloroform: cyclohexane 1 : 1 v/v)) and the mass of the product was confirmed by Mass Spectrometry (MW+ 1: 404.3).
  • the second fractionated product 4b was evaporated (yield: 5.8g, 28.7%) and the purity of compound 4b was checked by Thin Layer Chromatography (Rf: 0.19 (chloroform: cyclohexane 1: 1 v/v)) and the mass of the product was confirmed by Mass Spectrometry (MW+ 1 : 404.2). Crystal structure of compounds 4a and 4b was confirmed by one-crystal X- ray diffraction, and the resulted molecular structures can be seen in Fig Ia (4a) and in Fig Ib (4b). In the figures individual atoms were represented by elliptic rings with sizes proportional to the atomic displacement parameters in the three dimensional space (i.e. the bigger the elliptic ring, the more dynamic the atom in the crystal).
  • Example 4 2-(2,6-diisopropylphenyl)-4-(l-adamanthylamino)-5,6,7- trifluoroisoindole- 1 ,3-dione (5a) and 2-(2,6-diisopropylphenyl)-5-(l- adamanthylamino)-4,6,7-trifluoroisoindole- 1 ,3-dione (5b)
  • the column was washed with chloroform-cyclohexane 1:3 v/v, then eluted with chloroform-cyclohexane 1: 1 v/v solution.
  • the first fractionated product 5a was evaporated (yield: 4.1 g, 27.2%) and the purity of the product was checked by Thin Layer Chromatography (Rf: 0.48 (chloroform-cyclohexane 1: 1)) and the mass of the product was confirmed by Mass Spectrometry (MW+ 1: 511.1).
  • the second fractionated product 5b was evaporated (yield: 3.4g, 25.7%) and the purity of the product was checked by Thin Layer Chromatography (Rf: 0.27 (chloroform-cyclohexane 1: 1)) and the mass of the product was confirmed by Mass Spectrometry (MW+ 1: 51 1.2).
  • Example 5 2-(2,6-diisopropylphenyl)-4,5,6-trifluor-4-(2,2,2- trifluoroethylamino)isoindole-l,3-dione (6a) and 2-(2,6-diisopropylphenyl)- 4,5,7-trifluoro-5-(2,2,2-trifluoroethylamino)isoindole- 1 ,3-dione (6b)
  • the column was washed with chloroform-cyclohexane 1 :3 v/v, then eluted with chloroform-cyclohexane 1: 1 v/v.
  • the first fractionated product 6a was evaporated (yield: 510 mg, 43.3%) and the purity of the product was checked by Thin Layer Chromatography (Rf: 0.45 (chloroform-cyclohexane 1: 1 v/v)) and the mass of the product was confirmed by Mass Spectrometry (MW+ 1 : 458.2).
  • the second fractionated product 6b was evaporated (yield: 204 mg, 17.3%) and the purity of the product was checked by Thin Layer Chromatography (Rf: 0.25 (chloroform-cyclohexane 1: 1 v/v)) and the mass of the product was confirmed by Mass Spectrometry (MW+ 1: 458.2).
  • HepG2 human liver carcinoma cells (10 4 cells/ cm 2 ) were grown in MatTek's Glass Bottom Culture Dish for 24 hours in a 37 0 C/ 5% CO2 incubator in a medium of Dulbecco's Modified Eagle Medium (D-MEM) (high glucose) (Gibco BRL, Carlsbad, CA, USA), penicillin (50 IU/ml)-streptomycin (50mg/ml), 10% fetal bovine serum. After incubation, media was replaced with 200 ⁇ l PBS/dish (0.01 M phosphate buffer, 0.0027 M KCl, 0.137 M NaCl) pH 7.4). Then, 2 ⁇ l of 1 mM 4a, or 4b or Sb solution, or 4 ⁇ l of 1 mM 5a, or 3a, or 3b solution was added, which were previously dissolved in 100% DMSO.
  • D-MEM Dulbecco's Modified Eagle Medium
  • Fluorescent image of the stained cells can be seen in Fig. 2a, light microscopic image in Fig. 2b.
  • the staining of the cells, seen in this image was performed by compound 4a.
  • fluorescent dyes according to the invention stain lipid droplets well (light, well-contoured patches in Fig. 2a), the dye did not diffuse into the nucleus (which is a dark, spherical organelle in the middle of the image).
  • Transmission light microscopic images (Fig. 2b) were taken as follows: excitation was at 543 nm, detection was performed with DIC configuration. Staining shows less contrast as can be seen in Fig. 2b compared to the corresponding fluorescent image.
  • Example 7 Staining of RVH human melanoma cell culture
  • RVH human melanoma cells (10 4 cells /cm 2 ) were grown in MatTek's Glass Bottom Culture Dish for 24 hours in a 37 °C/ 5% CO2 incubator in a medium of Dulbecco's Modified Eagle Medium (D-MEM) (high glucose) (Gibco BRL, Carlsbad, CA, USA), penicillin (50 IU/ml)-streptomycin (50mg/ml), 10% fetal bovine serum. After incubation, media was replaced with 200 ⁇ l PBS/dish (0.01 M phosphate buffer, 0.0027 M KCl, 0.137 M NaCl) pH 7.4). Then, 2 ⁇ l of 1 mM 4a, or 4b or 5b solution, or 4 ⁇ l of 1 mM 5a, or 3a, or 3b solution was added, which were previously dissolved in 100% DMSO.
  • D-MEM Dulbecco's Modified Eagle Medium
  • FIG. 4 shows a double- staining experiment, where RVH human melanoma cells were stained with Oil Red O (Fig. 4c) and a compound (4b) according to the invention (Fig. 4b).
  • the images clearly show that the two fluorescent dyes specifically stained the very same organelles, namely lipid droplets.
  • RVH human melanoma cells were grown on sterile coverslips (10 4 cells/ cm 2 ) for 24 hours in a 37 °C/ 5% CO2 incubator, in a medium of Minimum Essential Medium Eagle (Sigma, St. Louis, MO, USA), Na-pyruvate, glutamine, non-essential aminoacids, (50 IU/ml)-streptomycin (50 mg/ml), 10% fetal bovine serum. Medium was discarded, then coverslips were washed twice with PBS. Cells were fixed with paraformaldehide-containing PME solution (100 mM Pipes, 5 mM MgCb, 5 mM EGTA) for 10 minutes.
  • PME solution 100 mM Pipes, 5 mM MgCb, 5 mM EGTA
  • Oil Red O solution was prepared as follows: isopropanol solution of 600 ⁇ l of 1% Oil Red O (Sigma- Aldrich) was mixed with 400 ⁇ l distilled water. Cells were washed twice with 0.2 ml PBS. Fluorescent microscopic analysis was performed as follows: excitation was at 543 nm, detection was performed with Alexa Fluor 546 configuration. Images were taken of the same cells as in the case of cells stained with compound 4b. The two dyes, used in the experiment, showed colocalization in the cells.
  • Example 9 Double-staining of lipid droplets and endoplasmic reticulum of RVH human melanoma cells
  • Compounds of the patent can stain endoplasmic reticulum when applied in higher (5-10 ⁇ M) concentrations. This finding is not surprising, as lipid droplets are formed in the endoplasmic reticulum (Denis J. Murphy et ah: Mechanism of lipid-body formation, TIBS 24, March 1999, pg. 109-115.).
  • Results of the double- staining with compound 4b and with a well-known endoplasmic reticulum staining dye can be seen in Fig. 5a and 5b.
  • RVH cells After settling of the cells the media was discarded and the cells were washed with HBSS (Hank's Balanced Salt Solution, Gibco BRL, Carlsbad, CA, USA) preheated to 37 0 C, then cells were incubated for 30 minutes in a 37 °C/ 5% CO2 incubator in ER-Tracker Green (glibenclamid BODIPY FL) (Invitrogen, Carlsbad, CA) dye (final concentration 1 ⁇ M) dissolved in DMSO, which specifically stains endoplasmic reticulum.
  • HBSS Hort's Balanced Salt Solution
  • Gibco BRL Carlsbad, CA, USA
  • An advantage of the compounds according to the invention is that they can stain live cells in a few minutes as they can pass cellular membranes practically immediately.
  • Another advantage of the compounds according to the invention is that they are non-toxic - at least with regard to the examined cell cultures - at the given concentrations required for performing selective staining. Thereby, processes taking place in the cell (plasticity of organelles) can be examined by this technique in vitro, which gives a more realistic picture of mechanisms in a live cell than the examination of fixed cells.
  • lipid droplets can be examined simultaneously with other organelles, which can be stained by a fluorescent dye having other color than blue. This enables us to examine intracellular events, which were not yet examined by this type of a fluorescent technique so far.
  • the advantageous feature of most compounds of the invention is their fluorescent property, it must be mentioned that there are other labeling techniques available. It means that an appropriate isotope of any compound according to the invention, i.e. both fluorescent and non-fluorescent compounds, can be used together with isotope detecting techniques. Furthermore, it is to be noted that application together with PEiT and NMR imaging techniques are also possible.

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Abstract

La présente invention porte sur des composés de marquage de gouttelettes de lipide, ayant la structure générale représentée dans la formule I, (I) dans laquelle les X sont chacun indépendamment hydrogène, halogène, alkyle en C1-20, alcényle en C2-20, alcynyle en C2-20, cycloalkyle en C5-6, aryle, aralkyle, adamantyle, un groupe hétérocyclique, hydroxyle hydroxyalkyle, ou un groupe -N-(R1, R2); n vaut 0, 1, 2, 3 ou 4; R1 et R2 peuvent chacun être indépendamment hydrogène, alkyle à chaîne droite ou ramifiée, cycloalkyle, aryle, aralkyle, des composés de hétérocyclique, chacun étant non substitué ou substitué par halogène; ou R1 et R2 conjointement avec l'azote entre eux forment un élément à 5 ou 6 chaînons; A est une liaison simple, -O-, -S-, -CH2- ou -NH-; Y est O ou S; Z est O ou S; R' et R' sont chacun indépendamment méthyle, éthyle, isopropyle, isobutyle, sec-butyle ou tert-butyle. L'invention porte également sur des compositions de marquage de gouttelettes de lipide, qui contiennent des composés de la formule I et autres additifs ou supports. L'invention porte en outre sur un procédé de visualisation de cellules et/ou d'organelles avec une technique d'imagerie fluorescente, le procédé comprenant les opérations consistant à : marquer les cellules et/ou les organelles par un ou plusieurs composés fluorescents, étant de préférence sous la forme d'une solution, et irradier les cellules et/ou organelles par de la lumière d'une longueur d'onde appropriée. Le procédé est caractérisé par le fait qu'il fournit un ou plusieurs composés fluorescents ayant une formule I pour le marquage des cellules et/ou des organelles.
PCT/HU2008/000074 2007-06-21 2008-06-20 Composés et compositions pour marquer des gouttelettes de lipide, et procédés pour la visualisation de cellules et/ou d'organelles cellulaires WO2008155593A2 (fr)

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CN110218186A (zh) * 2018-12-14 2019-09-10 陕西师范大学 一类聚集诱导发光型荧光材料及其制备方法和应用
CN112595698A (zh) * 2020-11-18 2021-04-02 上海中医药大学 油红o的应用及定量检测组织或细胞内脂质的方法
CN113597547A (zh) * 2019-03-19 2021-11-02 国立大学法人群马大学 细胞和组织内脂滴的荧光成像试剂
CN114262272A (zh) * 2021-12-24 2022-04-01 山东大学 萘-茚二酮给受体类化合物及其制备方法与其在脂滴免洗荧光探针中的应用
WO2024191500A1 (fr) * 2023-03-15 2024-09-19 Florida Atlantic University Board Of Trustees Composés lipidiques fluorescents

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WO2012085608A3 (fr) * 2010-12-23 2012-09-07 "Avidin" Kutató, Fejlesztö És Kereskedelmi Korlátolt Utilisation de trifluoro phtalimides pour le traitement de maladies cancéreuses
CN110218186A (zh) * 2018-12-14 2019-09-10 陕西师范大学 一类聚集诱导发光型荧光材料及其制备方法和应用
CN110218186B (zh) * 2018-12-14 2022-12-27 陕西师范大学 一类聚集诱导发光型荧光材料及其制备方法和应用
CN113597547A (zh) * 2019-03-19 2021-11-02 国立大学法人群马大学 细胞和组织内脂滴的荧光成像试剂
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CN114262272A (zh) * 2021-12-24 2022-04-01 山东大学 萘-茚二酮给受体类化合物及其制备方法与其在脂滴免洗荧光探针中的应用
CN114262272B (zh) * 2021-12-24 2023-02-17 山东大学 萘-茚二酮给受体类化合物及其制备方法与其在脂滴免洗荧光探针中的应用
WO2024191500A1 (fr) * 2023-03-15 2024-09-19 Florida Atlantic University Board Of Trustees Composés lipidiques fluorescents

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