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    • 专利摘要:
    Compounds and their uses as fluorescent probes. Organic dyes based on F-BODIPY and carnitine derivatives, its obtaining procedure and its application for the labeling or specific fluorescent labeling of mitochondria in living cells. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2695754A1
    申请号:ES201730885
    申请日:2017-07-04
    公开日:2019-01-10
    发明作者:Romero Jose Luís Chiara；Gonzalo Inmaculada García-Moreno；Moraleja Alberto Blázquez；Romero Maria Dolores Chiara；De Santa María Fernández Inés Sáenz
    申请人:Fundacion Para El Fomento En Asturias de la Investig Aplicada Y La Tecnologia Ficyt；Fundacion Para La Investig E Innovacion Biosanitaria En El Principado De Asturias Finba；Consejo Superior de Investigaciones Cientificas CSIC；
    IPC主号:
    专利说明:

    [0001]
    [0002] Compounds and their uses as fluorescent probes
    [0003]
    [0004] The present invention relates to compounds based on F-BODIPY derivatives and carnitine as organic dyes. It also refers to its obtaining procedure and its application for the specific fluorescent labeling of mitochondria in living cells. Therefore, the invention is encompassed in the field of fluorescent probes for biological labeling.
    [0005]
    [0006] BACKGROUND OF THE INVENTION
    [0007]
    [0008] Mitochondria are the organisms present in almost all eukaryotic cells responsible for the production of energy in the form of ATP through cellular respiration. In addition, they are involved in the metabolism of heme groups, calcium homestasis, the production of reactive oxygen species (ROS), inflammation, cell proliferation and apoptosis, thus fulfilling an essential role in the survival of the cell. These organelles also contain their own mitochondrial DNA (mtDNA), which is independent of the DNA of the cell nucleus. The set of cell mitochondria (cellular chondrioma) has a dynamic character that is regulated by complex mechanisms of cellular communication, both intra- and extracellular, to respond to variations in the cell's energy demands through fusion processes. fission, autophagy (mitophagy) and mitochondrial biogenesis. The damage or deregulation of these mechanisms is implicated in numerous pathological processes, either directly, as in genetic mitochondrial diseases, or secondarily in neurodegenerative, inflammatory, cardiovascular diseases and in some metabolic disorders (Suliman, HB, Piantadosi, CA Pharmacol. Rev. 2016, 68, 20). It has been known for some time that there is also a relationship between the changes in the cell condrioma and aging processes, either as a cause or as an effect (Harman, J Gerontol 1956, 11, 298, Balaban, RS, Nemoto, S. ; Finkel, T. Cell 2005, 120, 483). Recent studies are beginning to show that mitochondrial metabolism can also be an important therapeutic target for the treatment of cancer (Weinberg, SE, Chandel, NS Nat. Chem. Biol. 2015, 11, 9).
    [0009] Although great efforts have been devoted to the study of biology of mitochondria, we are still far from knowing in detail many of its basic functions and activities. The development of new technologies that facilitate this study is essential to unravel the complex role played by these organelles in cellular metabolism and its effects on aging and the development and possible treatment of many diseases. Compared to traditional analytical techniques, such as colorimetric assays, fluorescent probes (Li, X., Gao, X., Shi, W., Ma, H. Chem. Rev. 2014, 114, 590) are currently the tools molecular systems more efficient and versatile for the study of biological systems thanks to its high sensitivity, selectivity and ease of use, providing a variety of information in real time and non-destructively. For use in live cell microscopy, a fluorescent probe must ideally fulfill a series of basic requirements: 1) high luminescence (high quantum performance) in aqueous medium when illuminated in the near-infrared visible range; 2) high specificity and affinity for its cellular structure or objective biomolecule; 3) high chemical stability and photostability in the cell medium; 4) ability to penetrate the cell; 5) good solubility in water; and 6) low or no toxicity.
    [0010]
    [0011] Although a wide variety of fluorescent probes are currently known for the specific visualization of mitochondria, some of them commercially available such as for example tetramethylrhodamine ethyl ester (TMRE), 3,3'-dihexyloxacarbocyanine iodide (CiOC6 (3)), MitoTracker® red or MitoTracker® green, none meets all the above-mentioned requirements (Xu, Z .; Xu, L. Chem. Commun.
    [0012] 2016, 52, 1094).
    [0013]
    [0014] A common characteristic of all these probes is the presence of a cationic group (ammonium or phosphonium) that promotes its incorporation into the mitochondria thanks to the high gradient of negative characteristic potential of this organ. Furthermore, all of them are based on xanthine or cyanine structures as a chromophoric group, both incorporating amines in their skeleton, which significantly reduces their photostability, especially in drastic pumping conditions such as those involved in high resolution microscopes (Alvarez, M .; Amat, F., Costela, A., Garcia-Moreno, I., Gomez, C., Liras, M., Sastre, R. Appl. Phys. B. 2005, 80, 993; Cerdan, I., Enciso, E, Martin, V., Banuelos, J., Lopez Arbeloa, I. Costela, A., Garcia-Moreno, I. Nature Photonics, 2012, 6, 621).
    [0015] Therefore, it is of great interest the development of new fluorescent probes as specific markers of living cell mitochondria that can overcome the limitations of the systems currently marketed for this application.
    [0016]
    [0017] DESCRIPTION OF THE INVENTION
    [0018]
    [0019] The present invention describes new fluorescent probes as specific markers of living cell mitochondria based on a compound comprising an F-BODIPY unit as a fluorophore group and an L-carnitine unit as a locator group, whose system overcomes the limitations of currently commercialized systems for this application. Both units have been linked through the boron atom of the fluorophore, which greatly simplifies the final structure and the synthesis procedure. When using an F-BODIPY directly as a starting product, the method has a general character and can provide other similar probes with tunable absorption and emission frequencies throughout the visible spectrum depending only on the F-BODPY used.
    [0020]
    [0021] Staining tests of live human cells of tumor origin with the new fluorescent markers have shown the selectivity for mitochondria demonstrated with co-staining tests with commercial red MitoTracker®. The efficiency and selectivity of the label is independent of the substitution pattern in the chromophore as well as the cell line tested. The new probes show interesting properties with respect to the commercial red MitoTracker®. First, the red MitoTracker® seems to concentrate on the edges of the mitochondria, while the staining with the new probes is preferably located inside it (the mitochondrial matrix). In addition, the intensity of the staining with the new probes increases progressively with the incubation time of the cells, while the commercial dye appears to have these organelles almost instantaneously. These characteristics are revealing, in the case of the new probes and unlike the commercial marker, the intervention of a mechanism of active transport of the same towards the interior of the mitochondria.
    [0022]
    [0023] The methodology described in the present invention provides a new and effective synthesis protocol for the development of new dyes with properties advanced to be applied as selective, stable, effective and cheap biomarkers.
    [0024]
    [0025] Therefore, a first aspect of the present invention relates to a compound of
    [0026] general formula (I) (hereinafter composed of the invention):
    [0027]
    [0028]
    [0029]
    [0030] where:
    [0031] Z is selected from a nitrogen atom (N) or a group C (R7);
    [0032] R1 to R7 are each independently selected from hydrogen, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted C2-C18 alkenyl, substituted or unsubstituted C2-C18 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, halogen, -OR ', -COOR', -SR ', -SOR', -SOOR 'and -NR'R' '; or each two radicals R1 to R7, which are continuous with one another, such as, for example, R1 and R2, R2 and R3, R4 and R5 R6, form a cycloalkyl, aryl or heteroaryl;
    [0033] R 'and R "are each independently selected from hydrogen, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, aryl or heteroaryl; Y
    [0034] X- is a counterion,
    [0035] or any of its isomers.
    [0036]
    [0037] In a preferred embodiment, Z is C (R7) and the compound will be the following compound
    [0038] of formula (II):
    [0039]
    [0040] where R1 to R7 and X "are those defined above.
    [0041]
    [0042] The term "alkyl" refers, in the present invention, to saturated, linear or branched hydrocarbon chains, having from 1 to 18 carbon atoms, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl , tert-butyl, sec-butyl, n-pentyl, n-hexyl, etc. Preferably the alkyl group has between 1 and 6 carbon atoms. Alkyl groups may be optionally substituted by one or more substituents such as alkynyl, alkenyl, halo, hydroxyl, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro or mercapto.
    [0043]
    [0044] The term "alkenyl" refers, in the present invention, to unsaturated, linear or branched hydrocarbon chains, having from 2 to 18 carbon atoms, preferably from 2 to 6, and containing one or more double carbon-carbon bonds and which optionally may contain some triple bond, for example, vinyl, 1-propenyl, allyl, isoprenyl, 2-butenyl, 1,3-butadienyl, etc. The alkenyl radicals may be optionally substituted by one or more substituents such as alkyl, alkynyl, halo, hydroxyl, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro or mercapto.
    [0045]
    [0046] The term "alkynyl" refers to straight or branched hydrocarbon chain residues, of 2 to 18 carbon atoms, preferably 2 to 6, and containing at least one or more triple carbon-carbon bonds and optionally containing some double bond, for example, ethyl, propynyl, butynyl, etc. The radicals Alkynyl may be optionally substituted by one or more substituents such as alkyl, alkenyl, halo, hydroxyl, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro or mercapto.
    [0047]
    [0048] The term "aryl", in the present invention, refers to aromatic rings, single or multiple, having from 5 to 18 carbon atoms in the ring part, such as but not limited to, phenyl, naphthyl, diphenyl, indenyl, fenantrile, fluorenyl or anthracyl. Preferably the aryl group has from 5 to 7 carbon atoms and more preferably the aryl group is a phenyl. The aryl radicals may be optionally substituted at any one of their positions by one or more substituents or two substituents forming a fused ring to the aryl and are independently selected from such as alkyl, alkenyl, alkynyl, O-alkyl, O, halogen, hydroxyl, amino or carboxylic acid. In a preferred embodiment the aryl is an optionally substituted phenyl and more preferably a phenyl optionally substituted by more than one alkyl group, as defined above.
    [0049]
    [0050] The term "heteroaryl" refers to an aryl, as defined above, that contains at least one atom other than carbon, such as S, N, or O, forming part of the aromatic ring.
    [0051]
    [0052] By "cycloalkyl" is meant the present invention to a 3 to 10 membered monoclonal or bicyclic stable radical, which is saturated or partially saturated, and which consists of carbon and hydrogen atoms, such as cyclopentyl, cyclohexyl or adamantyl. The cycloalkyl radicals may be optionally substituted at any one of their positions by one or more substituents or two substituents forming a cycloalkyl fused cycle and are independently selected from such as alkyl, alkenyl, alkynyl, O-alkyl, O, halogen, hydroxyl, amino or carboxylic acid.
    [0053]
    [0054] By "halogen" is meant in the present invention an atom of bromine, chlorine, iodine or fluorine.
    [0055]
    [0056] In another preferred embodiment, R7 is an optionally substituted aryl, more preferably R7 is an optionally substituted phenyl and more preferably phenyl substituted by C1-C6 alkyl, even more preferably it is a trimethylphenyl.
    [0057] In another preferred embodiment, R 2 and / or R 5 are hydrogen or C 1 -C 6 alkyl, more preferably hydrogen or methyl and even more preferably are hydrogen. In a still more preferred embodiment R2 and R5 are hydrogen.
    [0058]
    [0059] In another preferred embodiment, R1, R3, R4 and R6 are each independently hydrogen or C1-C6 alkyl, more preferably R1, R3, R4 and R6 are each independently hydrogen or methyl. More preferably R1, R3, R4 and R6 are hydrogen or methyl.
    [0060]
    [0061] The counterion (X-) is any pharmaceutically acceptable anion known to a person skilled in the art and preferably can be selected from the list that
    [0062]
    [0063] preferably X- can be selected from a halogenide which can be selected from Cl-, Br-, I-, F-; SO4-, BF4-, PF4-, HSO4- and (SO42-) 1/2. More preferably X- is a halide, even more preferably Cl-.
    [0064]
    [0065] By "isomer" is meant in the present invention also any racemic mixture.
    [0066] In an even more preferred embodiment, the compound of the invention is selected from:
    [0067]
    [0068]
    [0069]
    [0070]
    [0071] Another aspect of the present invention relates to a method for obtaining a compound of the invention comprising the reaction between a compound of formula (III) and carnitine:
    [0072]
    [0073]
    [0074] where Z, R1 to R6 and X "are those defined above.
    [0075]
    [0076] In a preferred embodiment of the process of the invention, carnitine can be selected from L-carnitine, D-carnitine and racemic mixture D / L-carnitine, preferably it is L-carnitine.
    [0077]
    [0078] The reaction described can be carried out in the presence of an excess of reagent, where this reagent can be selected from trimethylsilyl chloride (TMSCl), BCl3, BBr3, AlCl3, AlBr3, SnCl4, SnBr4, SiCl4, SiBr4, trimethylsilyl trifluoromethylmethanesulfonate ( Me3SiOTf), triethylsilyl trifluoromethylmethanesulfonate (Et3SiOTf) and triisopropylsilyl trifluoromethylmethanesulfonate (/ -Pr3SiOTf). Preferably the reaction is carried out in the presence of excess trimethylsilyl chloride, approximately between 20-50 mol-equiv.
    [0079]
    [0080] In another preferred embodiment of the process of the invention, the reaction is carried out in a polar aprotic solvent capable of dissolving carnitine. The solvent may be selected from acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, sulfolane, or mixtures of this aprotic polar solvent with other less polar aprotic solvents, such as, for example, not limited to dichloromethane, 1,2-dichloethane, tetrahydrofuran, 2-methyl tetrahydrofuran, 1,4-dioxane, 1,2-dichlorobenzene, ethyl acetate or acetone. In a preferred embodiment the solvent is acetonitrile.
    [0081]
    [0082] On the other hand, the solubility of carnitine in organic solvents can be improved by changing the anion of the salt to the following anions: BF4-, PF6-, BPh4-, p-toluenesulfonate, mesylate, trifluoromethanesulfonate, acetate, benzoate, trichloroacetate or trifluoroacetate.
    [0083]
    [0084] In another embodiment of the present invention the process is carried out at a temperature between 50-150 ° C, in a preferred embodiment the reaction is carried out under thermal conditions by microwave irradiation.
    [0085]
    [0086] In a preferred embodiment, the inventive method is carried out in the presence of an excess of trimethylsilyl chloride in acetonitrile and under thermal conditions by microwave irradiation.
    [0087]
    [0088] Subsequent to the reaction the compound obtained of formula (I) can be recrystallized or purified chromatographically, after evaporating the reaction mixture under reduced pressure.
    [0089]
    [0090] Another aspect of the present invention relates to the use of the compound of the invention as a fluorescent label or probe. More preferably for cellular labeling and even more preferably for specific cellular labeling of mitochondria, more preferably of living cells, and even more preferably in living cells of mammals, preferably human. The cells to be labeled will preferably be cells of tumoral origin.
    [0091]
    [0092] Another aspect of the present invention relates to the use of the compound of the invention, for the manufacture of markers for the diagnosis of mitochondrial diseases. These diseases can be selected from translocase-carnitine-acylcarnitine deficiency, metabolic decompensation in childhood, cardiomyopathy in childhood, fatigability in adulthood, disorders in metabolism and carnitine transport, in addition to diseases such as cancer, type-2 diabetes , neurological diseases, such as Parkinson's and cardiovascular diseases, such as atherosclerosis.
    [0093]
    [0094] Throughout the description and the claims the word "comprises" and its variants do not intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be apparent in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and they are not intended to be limiting of the present invention.
    [0095]
    [0096] DESCRIPTION OF THE FIGURES
    [0097]
    [0098] Fig. 1. Labeling of SCC38 cells in vivo with compound 4 at different concentrations (50, 100 and 500 nM). Scale bar: 10 pm
    [0099]
    [0100] Fig. 2. Flow cytometry studies in cells marked with compound 4 and red MitoTracker® in lines SCC38 and HeLa. Histograms in which the intensity of the marking is represented against the number of events, for each of the staining times (5, 15 and 30 min) and the control (Ctrl), for the SCC38 and HeLa cells, and for each one of the two markers.
    [0101]
    [0102] EXAMPLES
    [0103]
    [0104] In the following the invention will be illustrated by means of tests carried out by the inventors where the obtaining of fluorescent probes of the invention is described, thus as its evaluation as markers of mitochondria in living cells of tumoral origin, which highlights the effectiveness of the compound of the invention by monitoring its activity by both confocal fluorescence microscopy and flow cytometry.
    [0105]
    [0106] A. SYNTHESIS OF COMPOUNDS 3 and 4
    [0107]
    [0108] The general method for the preparation of these fluorescent probes derived from l- carnitine is based on the rational functionalization of the target molecule shown generically in scheme 1:
    [0109]
    [0110] 3, R = H (63%)
    [0111] 4, R = Me (88%) Scheme 1 - Synthesis of two BODIPY-carnitine probes (compounds 3 and 4 ).
    [0112]
    [0113] By this synthesis, carnitine binds directly to the boron atom of F-BODIPY in the form of B-spiro-derivative through the hydroxyl and carboxyl groups to generate the corresponding O-BODIPY derivative. In this way, the process of synthesis is simplified by avoiding processes of prior functionalization of the fluorophore or the introduction of connectors, thereby reducing the size and complexity of the final probe to the minimum possible. Although several methods of synthesizing O-BODIPYs are known from the corresponding F-BODIPYs in the present invention, the one described in Manzano, H. et al., Adv. Funct. Mater. 2016, 26, 2756.
    [0114]
    [0115] All reagents used in the preparation of the above compounds are commercial or were prepared following methods previously described in the literature. The anhydrous solvents were treated by the usual drying techniques or with a PureSol model 400-3-MD solvent purification system for the drying of the following solvents (THF, CH2Cl2, MeCN, toluene, diethyl ether and DMF) and were used in the reaction immediately. Reactions with microwave irradiation (MW) were carried out in a closed tube in a focused CEM Discover or Anton Parr Monowave 300 microwave reactor, using standard pyrex tubes of 10 mL capacity closed with a septum plug. All experiments were carried out at a maximum power of 300 W and a maximum pressure of 20 bar. The monitoring of the reactions was carried out by thin layer chromatography (TLC) using silica gel chromatofolios type 60 F254 (230-400 mesh) with aluminum support and a layer thickness of 0.2 mm (Merck). The CCF plates were visualized under a UV lamp 254/365 nm.
    [0116] Compound 3. To a suspension of L-carnitine hydrochloride (20 mg, 0.100 mmol) in anhydrous MeCN (5 mL) in a 10 mL microwave vial with stir bar was added compound 1 (Nepomnyashchii, AB; et al. , J. Am. Chem. Soc. 2011, 133, 8633) (30 mg, 0.097 mmol) and trimethylsilyl chloride (TMSCl) (491 pL, 3.87 mmol). The reaction was heated with the following temperature gradient in a focused microwave reactor: 25-80 ° C in 30 minutes, 80-100 ° C in 5 minutes, 100 ° C for 30 minutes, 100-120 ° C in 10 minutes , 120 ° C for 30 minutes and 120-150 ° C in 1 hour. The reaction mixture was evaporated under reduced pressure and the reaction crude was dissolved in the minimum amount of CH2Cl2 and re-precipitated by adding tert- butyl methyl ether, to give compound 3 (26.5 mg, 63%) as a solid red with green fluorescence in solution.
    [0117]
    [0118]
    [0119]
    [0120] 1 H NMR (CDCl 3, 400 MHz): 5 = 8.14 (s, 1 H, C 5 H), 7.83 (s, 1 H, C 3 H), 6.95 (s, 2 H, C 3 'H, C 5' H), 6.70 (m, J = 4.15 Hz, 2H, C1H, C7H), 6.49 (dd, J = 8.40, 4.15 Hz, 2H, C2H, C6H), 4.98 (t, J = 10.75 Hz, 1H, C3 "H), 4.33 (d, J = 13.49 Hz, 1H, C4 "H), 3.62 -3.52 (m, 1H, C4" H), 3.41 (s, 9H, C5 "H3, C6" H3, C7 "H3), 2.98 (d, J = 16.3 Hz , 1H, C2 "H), 2.74-2.59 (m, 1H, C2" H), 2.36 (s, 3H, CH3-C4 '), 2.05 (s, 6H, CH3-C2', CH3-C6 '). 13C NMR (101 MHz, CDCU): 5 = 168.80 (C1 "), 148.00 (C8), 145.25 (C3), 144.45 (C5), 139.23 (C4 '), 136.44 (C2'), 135.91 (C6 '), 135.53 (C7a), 135.49 (C8a), 131.10 (C7H), 130.78 (C1), 129.54 (C1 '), 128.51 (C3'), 128.34 (C5 '), 119.20 (C6), 119.16 (C2), 69.53 ( C4 "), 63.06 (C3"), 55.01 (C5 ", C6", C7 "), 36.95 (C2"), 21.28 (CH3-C4 '), 20.11 (CH3-C2'), 20.04 (CH3-C6 ' HRMS (ESI +) m / z: calculated for C25H31BN3O3 + [M +]: 432.2453, found 432.2453, IR (KBr): v = 1719.76 (C = O), 1560.24, 1411.23, 1383.73, 1257.88, 1108.77, 1070.62 cm-1 .
    [0121]
    [0122] Compound 4. To a suspension of L-carnitine hydrochloride (14.5 mg, 0.072 mmol) in anhydrous MeCN (5 mL) in a 10 mL microwave vial with stir bar, compound 2 was added (Kee, HL; et al., J. Phys. Chem. B 2005, 109, 20433) (32.3 mg, 0.088 mmol) and TMSCI (466 pL, 3.67 mmol). The reaction was heated with the following temperature gradient in a focused microwave reactor: 25-80 ° C in 30 minutes, 80-100 ° C in 5 minutes, 100 ° C for 30 minutes, 100-120 ° C in 10 minutes and 120 ° C for 2 hours. The reaction mixture was evaporated under reduced pressure and the reaction crude was dissolved in the minimum amount of CH2Cl2 and reprecipitated by adding tert-butyl methyl ether, to give compound 4 (31.8 mg, 88%) as a red solid with green fluorescence in solution.
    [0123]
    [0124]
    [0125]
    [0126] 1 H NMR (CDCl 3, 400 MHz): 5 = 6.97 (s, 1 H, C 3 H), 6.95 (s, 1 H, C 5 H), 5.99 (s, 1 H, C 6 H), 5.97 (s, 1 H, C 2 H) , 4.55 (s, 1H, C3 "H), 4.17 (s, 1H, C4" H), 3.32 (s, 9H, C5 "H, C6" H, C7 "H), 3.26 (s, 1H, C4" H), 2.87 (d, J = 14.45 Hz, 1H, C2 "H), 2.54 (s, 3H, CH3-C5), 2.47 (d, J = 14.45 Hz, 1H, C2" H), 2.39 (s, 3H, CH3-C3), 2.34 (s, 3H, CH3-C4 '), 2.08 (s, 3H, CH3-C2'), 1.98 (s, 3H, CH3-C6 '), 1.39 (s, 3H, CH3 -C7), 1.38 (s, 3H, CH3-C1) .13C NMR (101 MHz, CDCl3): 5 = 168.85 (C1 "), 155.71 (C3), 154.12 (C5), 143.83 (C8a), 143.52 (C7a ), 142.52 (C8), 139.23 (C4 '), 135.03 (C6'), 133.90 (C2 '), 131.52 (C1), 131.39 (C7), 130.83 (C1'), 129.57 (C5 '), 129.25 (C3) '), 122.46 (C6), 122.43 (C2), 69.45 (C4 ",), 62.31 (C3"), 55.07 (C5 ", C6", C7 "), 37.06 (C2"), 21.36 (CH3-C4') ), 19.76 (CH3-C2 '), 19.42 (CH3-C6'), 17.39 (CH3-C5), 15.67 (CH3-C3), 13.85 (CH3-C7), 13.72 (CH3-C1), HRMS (ESI +) m / z: calculated for C29H39BN3O3 + [M +]: 488.3079, found 488.3090.RTM (KBr): v = 1720.45 (C = O), 1546.29, 1505.72, 1470.57, 1295.12 , 1187.69, 1155.63, 1083.56, 1043.53, 982.88 cm-1.
    [0127]
    [0128] B. Characterization of the new fluorescent probes (compounds 3 and 4) as markers of mitochondria
    [0129] The cellular assays were carried out on two human cell lines: SCC38 and HeLa. The SCC38 line comes from epidermoid carcinoma of the larynx of human patients, while HeLa, the first human cell line established in culture, derives from cervical cancer. The culture medium used for both cell lines was DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% serum fetal bovine (FBS), 100 U / mL of penicillin, 200 pg / mL of streptomycin, 100 pmol / L of non-essential amino acids and 2 mmol / L of glutamine. The cultures were incubated at 37 ° C and 5% CO2.
    [0130]
    [0131] These cell lines were stained with the fluorescent probes synthesized following the protocol described: 30000-50000 SCC38 cells were plated in 24-well, glass bottom plates that were grown to a confluence of 70%. The culture medium was then removed from the well and at least 3 washes were performed with DMEM medium without additives. After this, 1 mL of DMEM and the corresponding fluorescent compound were added to the well. The cells were incubated at 37 ° C for 30 minutes, after which the medium was removed and new washes were made with DMEM. Finally, complete medium was added and the cells were observed under a microscope. Microscopic analysis was performed using a Zeiss AxioObserver Z1 with AxioCam MRM and ApoTome 2 (Carl Zeiss, Germany) with different objectives of 40X 63X immersion oil. The cells were also stained with MitoTracker® Red CMXRos (Thermo Fisher) according to the manufacturer's instructions, in order to carry out comparative studies with the new fluorescent markers here synthesized. Initially, both compounds 3 and 4 were used at a concentration of 50 nM, and the staining time was also 30 minutes.
    [0132]
    [0133] By way of example, the behavior of compound 4 is described by labeling SCC38 cells and analyzed by fluorescence cell microscopy. In Fig. 1 the intensity of labeling according to the concentration of dye in the medium is presented.
    [0134]
    [0135] To confirm the specificity of the labeling by the mitochondria, co-localization studies were carried out by co-staining with the commercial MitoTracker® red mitochondrial marker in the cell line. Compound 4 was used at a concentration of 50 nM and the MitoTracker® Red at 25 nM. Both compounds were added at once and incubated for 30 minutes. The cells marked with both compounds were observed both with the Zeiss AxioObserver Z1 microscope and with a Leica TCS-SP8X spectral confocal laser microscope (Servicios Cientlfico-Tecnicos, University of Oviedo).
    [0136]
    [0137] The cell microscope studies showed, in the case of co-staining with both compounds at the same time, a good co-localization of the two probes without interferences or cytotoxic effects. It should be noted that in many cases it was observed that red MitoTracker® staining concentrated on the edges of the mitochondria, while the green staining of compound 4 was preferably located inside it (the mitochondrial matrix).
    [0138]
    [0139] The kinetics of cell labeling with 4 was studied by flow cytometry with the SCC38 and HeLa cell lines, using red MitoTracker® as a control marker, both at a concentration of 50 nM. The staining times studied were 5, 15, 30 and 45 minutes. After staining, the medium was removed, washed with PBS, trypsinized and the pellet re-suspended in 500 pL of PBS and introduced into the cytometer after filtration. A flow cytometer Cytomics FC500 Cellular Analyzer of Beckman Coulter (Scientific-Technical Services, University of Oviedo) was used. Part of the resuspended cells were seeded and also observed under a microscope. As can be seen in Fig. 2, staining with red MitoTracker® hardly varied by increasing the incubation time with the marker, while the labeling with compound 4 clearly increased in intensity with the incubation time. A new cytometry experiment was carried out in which a longer staining time (45 min) was included, and in which it was possible to see that the marking was even more intense than with the time of 30 minutes. This fact seems to point to the actuation of an active transport mechanism of compound 4 in the mitochondria. On the other hand, in the cotincion experiments it was observed that the red MitoTracker® seems to concentrate on the edges of the mitochondria, while the green staining of the compound 4 is preferably located inside it (the mitochondrial matrix). These facts indicate that the internalization of probe 4, unlike the commercial marker, implies the presence of an active transport mechanism of compound 4 in the mitochondria.
    权利要求:
    Claims (17)
    [1]
    1. Compound of general formula (I)

    [2]
    2. Compound according to claim 1, wherein Z is C (R7).
    [3]
    3. Compound according to any of claims 1 or 2, wherein R7 is an optionally substituted aryl.
    [4]
    4. Compound according to claim 3, wherein R7 is a trimethylphenyl.
    [5]
    5. The compound according to any of claims 1 to 4, wherein R2 and / or R5 are hydrogen.
    [6]
    6. Compound according to any of claims 1 to 5, wherein R1, R3, R4 and R6 are each independently hydrogen or methyl.
    [7]
    7. The compound according to any of claims 1 to 6, wherein R1, R3, R4 and R6 are hydrogen or methyl.
    [8]
    Compound according to any one of claims 1 to 7, wherein X "is selected from a halogenide, SO4", BF4 ", PF4", HSO4 "and (SO42") 1/2.
    [9]
    9. Compound according to any of claims 1 to 8, wherein X "is Cl".
    [10]
    10. Compound according to claim 1, wherein the compound is selected from:

    [11]
    11. Process for obtaining the compound of formula (I) described according to any of claims 1 to 10, comprising the reaction of a compound of formula (III) with carnitine:

    [12]
    12. Use of the compound according to any of claims 1 to 10 as a fluorescent label or probe.
    [13]
    13. Use according to claim 12, for cellular marking.
    [14]
    14. Use according to claim 13, for the cellular labeling of mitochondria.
    [15]
    15. Use according to claim 14, in living cells.
    [16]
    16. Use of the compound according to any of claims 1 to 10, for the manufacture of markers for the diagnosis of mitochondrial diseases.
    [17]
    17. Use according to claim 16, wherein the mitochondrial diseases are selected from translocase-carnitine-acylcarnitine deficiency, metabolic decompensation in childhood, cardiomyopathy in childhood, fatigability in adulthood, disorders in metabolism, cancer, diabetes type- 2, neurological diseases and cardiovascular diseases.
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    同族专利:
    公开号 | 公开日
    EP3650451A1|2020-05-13|
    ES2695754B2|2019-05-17|
    WO2019008209A1|2019-01-10|
    EP3650451A4|2020-11-04|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2013144405A1|2012-03-30|2013-10-03|Consejo Superior De Investigaciones Científicas |Novel chlorinated derivatives of bodipy|
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