WO2011051484A1 - Colorants fluorescents - Google Patents

Colorants fluorescents Download PDF

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Publication number
WO2011051484A1
WO2011051484A1 PCT/EP2010/066548 EP2010066548W WO2011051484A1 WO 2011051484 A1 WO2011051484 A1 WO 2011051484A1 EP 2010066548 W EP2010066548 W EP 2010066548W WO 2011051484 A1 WO2011051484 A1 WO 2011051484A1
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optionally substituted
alkyl
hydrogen
optionally
ethoxy
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PCT/EP2010/066548
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German (de)
English (en)
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Lutz Tietze
Birgit Krewer
Miso Mitkovski
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Georg-August-Universität Göttingen Stiftung Öffenlichen Rechts
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Publication of WO2011051484A1 publication Critical patent/WO2011051484A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/02—Heterocyclic radicals containing only nitrogen as ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1022—Heterocyclic compounds bridged by heteroatoms, e.g. N, P, Si or B
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom

Definitions

  • the present invention relates to novel, suitable as fluorescent dyes compounds.
  • the present invention relates to a new class of chromophore derivatized, pharmacologically active compounds.
  • the present invention is directed to applications of the compounds of the invention.
  • These dyes may be covalently coupled to other structures, such as proteins, nucleic acids or other chemical or biological entities. Such compounds are then used, for example, in diagnostic applications. For example, many fluorescent dyes are based on fluorescein or rhodamine structures. In the meantime, however, a large number of other fluorescent dyes with fluorophore groups have also been described. Reference is made, for example, to the book: The Handbook, A Guide to Fluorescent Probes and Labeling Technologies, Molecular Probes - Invitrogen, 1 0 th Edition. This work comprehensively depicts the various fluorescent dyes and their conjugation to desired chemical or biological entities.
  • the dyes Hoechst 33342 or Hoechst 33258 are known compounds which bind to DNA or intercalate with the DNA. Many other DNA-binding entities are described in the literature. These compounds, referred to as DNA binders or DNA intercalating compounds, can be found, for example, in WO 02/059122, WO 98/1 1 101, WO 97/1 2862, WO 01/83448 or WO 2007/089149.
  • the molecules described there, for example in WO 2007/089149, to which reference is hereby made, include corresponding DNA-binding units in connection with pharmacophore stood share.
  • pyrroloindole derivatives as analogues of the pharmacological active ingredient CC-1065 in combination with the DNA-binding residue DMAI, 5 '[2' (N, N-dimethylamino) ethoxy] indole, show excellent cytotoxic properties, the DMAI portion shows an excellent DNA intercalating effect or DNA-binding effect.
  • Corresponding compounds are e.g. in WO 01/83448 and WO 2007/089149 or WO2009 / 017394. These documents further describe corresponding seco compounds and prodrugs of these CC-1065 analogs. Prodrugs are necessary, in particular, to make the pharmacological activity of the pharmacophore act only in the target area.
  • prodrugs which are then converted at the destination into pharmacologically active compounds. This conversion can e.g. be acid-catalyzed or carried out enzymatically or otherwise.
  • prodrugs and drugs based on bases of various CC-1065 analogs are described, see, e.g. WO 2007/089149 and WO2009 / 01 7394 and WO 01/83448, WO 98/11111.
  • a cell e.g. cellular organelles of endocytosis and exocytosis
  • various dyes are known. These are e.g. described in the above-mentioned handbook for fluorescence probes and labeling technologies. A variety of different molecules are shown. These are e.g. as a lyso tracker, ER tracker, etc. commercially sold by the company Invitrogen, USA.
  • molecular biology methods e.g. Plasmids or vectors that introduce appropriate fluorosensory-based markers into the target areas.
  • the present invention is directed to new dyes suitable
  • R 1 selected from a halogen and OSO 2 R wherein R u is selected from optionally substituted u, C -I -C east alkyl, Ci-6 perhaloalkyl, benzyl, and phenyl;
  • R 2 is selected from hydrogen and optionally substituted C 1-8 alkyl
  • R 3 , R 3 , R 4 and R 4 are independently selected from hydrogen and optionally substituted C 1-8 alkyl, wherein two or more of R 2 , R 3 , R 3 , R 4 and R 4 are optionally joined together to form one or more optionally substituted carbon cycles or heterocycles;
  • X 2 is selected from O, C (R 14 ) (R 14 ' ) and NR 14' , wherein R 14 is selected from hydrogen and optionally substituted Ci -8 alkyl or Ci -8 acyl and wherein R 14 is absent or is selected from hydrogen and optionally substituted Ci -8 alkyl or Ci -8 acyl;
  • R 5 , R 5 , R 6 , R 6 , R 7 and R 7 are independently selected from H, OH, SH, NH 2 , N 3 NO 2 , NO, CF 3 , CN, C (O) NH 2 , C (O ) H, C (O) OH, halo gen, R k, SR k, S (O) R k, S (O) 2 R k, S (O) OR k, S (O) 2 OR k, OS (O) R k, OS (O) 2 R k , OS (O) OR k , OS (O) 2 R k , OR k , NHR k , N (R k ) R L , + N (R k ) (R L ) R m , P (O) ( OR k ) (OR L ), OP (O) (OR k ) (OR L ), SiR k R L R m , C (O) R k , C
  • R m are independently selected from H and optionally substituted Ci -4 alkyl, Ci -4 heteroalkyl, C 3- 7 cycloalkyl, C 3- 7 heterocycloalkyl, C 4-i 2 aryl, or C 4- i 2 heteroaryl groups, two or more of R k , R L and R m optionally together form one or more optionally substituted aliphatic or aromatic carbon cycles or heterocycles;
  • R 5 ' and R 6' and / or R 6 ' and R 7' and / or R 7 ' and R 14' are absent, ie they form a double ring present in the ring between those with the corresponding substituted atoms from, namely R 5 and R 6 ' , and / or R 6' and R 7 , and / or R 7 ' and R 14' ;
  • R 5, R 5, R 6, R 7, R 14, and R 14 may optionally be connected together to form one or more optionally substituted aliphatic or aromatic carbon rings or heterocycles;
  • Chromophore is a unit which has a color in the visible or invisible range, preferably in the visible range, optionally after excitation, in particular a unit which has all the colors except white in the visible range, eg one which has at least four preferably has at least six conjugated ⁇ -electrons;
  • X 6 is selected from C or N;
  • RDB is selected from hydrogen and optionally substituted C 1-8 alkyl or C 1-8 acyl;
  • Xi is selected from O, S, and NR 13 , wherein R 13 is selected from H and optionally substituted cis alkyl;
  • R is selected from hydrogen or an enzymatically cleavable substrate
  • a and b are independently selected from 0 and 1;
  • c is selected from 0, 1 and 2;
  • d is selected from 0 or 1;
  • DB is hydrogen or a DNA-interacting compound, in particular one of the general formula I II
  • X 3 is selected from O, S, and NR 15 , wherein R 15 is selected from H and optionally substituted Cis alkyl or Cis acyl; or -X 3 - X is X 3a and X 3b , where X 3a is joined to the carbon to which X 4 is joined, and X 3b is joined to the phenyl ring ortho to R 10 , where X 3a is selected from hydrogen and optionally substituted Cis alkyl or acyl radical and X 3b is selected from the same group as the radicals defined for R 8 , wherein X 3a and X 3b do not form a covalent bond with each other;
  • X 4 is selected from N and CR 16 , wherein R 16 is selected from hydrogen and optionally substituted Cis alkyl or Cis acyl;
  • X 5 is selected from O, S, and NR 17 , wherein R 17 is selected from H and optionally substituted C 1-8 alkyl or C 1-8 acyl; R 8 R 9 , R 10 , and R 11 are independently selected from H, OH, SH, NH 2 , N 3 , NO 2 , NO, CF 3 , CN, C (O) NH 2 , C (O) H , C (O) OH, halogen, R x , SR x , S (O) R x , S (O) 2 R x , S (O) OR x , S (O) 2 OR x , OS (O) R x , OS (O) 2 R x , OS (O) OR x , OS (O) 2 OR x , OS (O) 2 OR x , OR x , OR x , NHR X , N (R x ) R y , + N (R x ) (R y
  • R x , R y , and R z are optionally a water-soluble group
  • two or more of R x , R y , and R z may be optionally linked to one or more optionally substituted ones to form carbon rings or heterocycles
  • at least one of R 8, R 9, R 10, and R 1 1 comprises a water-soluble group
  • two or more of R 8, R 9, R 10, R 11, or X 3b are optionally linked to form one or more optionally substituted aliphatic or aromatic carbon cycles or heterocycles;
  • These compounds are preferably those which correspond to the CC-1065 analogues, as disclosed, for example, in WO 2007/089149 and WO 01/83448, which are hereby fully incorporated by reference, and in addition to the radicals R 3 R 4 , DB, R 8 , R 9 , R 10 , R 11 , R 6 and / or R 7 preferably at the radicals R 6 and / or R 7 , a group - (C (R C H) i -6
  • the present compounds are distinguished by having a DNA-binding region, a pharmacophore moiety and a chromophoric, in particular a fluorophore moiety.
  • the compounds of the invention are specifically detectable in certain sub-cellular regions of cells due to their fluorescence. All the more surprisingly, it has been found that the compounds of this invention endowed with a fluorophore component do not color-label DNA-containing structures, such as the cell nucleus of a cell, even though they have DNA-binding domains. Surprisingly, it was found that when using the compounds according to the invention, the nucleus shows no expected staining, while a fluorescence labeling could be observed in particular in cellular organelles of the endocytosis and exocytosis but also of the mitochondria.
  • the compounds according to the invention are therefore particularly suitable for imaging methods. suitable. These imaging methods are in particular light microscopic and fluorescence-based methods.
  • cellular organelles of endocytosis and exocytosis are meant in particular the cellular structures Golgi apparatus, endoplasmic reticulum, lysosomes, endosomes, microbodies, peroxisomes, microsomes, glyoxysomes and cell membranes.
  • the compounds of the invention further show a specific staining of mitochondria. They are therefore also particularly suitable for use in the imaging diagnosis, prognosis, course and drug effectiveness determination of mitochondrial-associated diseases and for the determination of the diagnosis, prognosis, course and drug effectiveness determination of therapies with active substances.
  • the compounds according to the invention furthermore permit the use of these in combination with other known fluorescent dyes in order to achieve a multiple labeling of different organelles.
  • the compounds according to the invention in combination with e.g. DNA intercalating compounds but also in combination with corresponding molecules, e.g. Specifically mark lysosomes, endosomes or other subcellular structures, multiple staining done.
  • the compounds according to the invention can be used in combination with Hoechst dyes or propidium iodide.
  • the compounds according to the invention can be used in investigations with the aid of imaging methods, in particular fluorescence-based methods, such as fluorometers, fluorescence microscopy or flow cytometry, such as FACS. Furthermore, due to the specific staining of mitochondria, specific labeling of mitochondria in combination with other dyes may occur.
  • the compounds according to the invention allow labeling with a short incubation time.
  • the compounds can be simply and directionally introduced into cells. to be brought.
  • R is preferably hydrogen.
  • a change in mitochondrial morphology plays a role in myopathies and neuropathies, various cancers, diabetes, obesity and aging. Accordingly, with the aid of the compounds according to the invention, a detection of such changes in the mitochondrial morphology can take place.
  • R compounds of the invention are used such that they allow a distinction of living, apoptotic cells and dead cells. While living cells and apoptotic cells are stained by the compounds of the invention and these can be detected by a fluorescent signal, no clear signal can be detected in dead cells. This is the case when R is a hydrogen. When R is a substrate other than hydrogen, the compounds of the invention can only slowly penetrate into living cells. In dead cells, these compounds are quickly detectable. According to the invention, the compounds of the general formula I or II are therefore particularly suitable for distinguishing between living, apoptotic and dead cells.
  • the compounds of the invention also allow qualitatively and quantitatively to determine nucleic acids, in particular double-stranded nucleic acids.
  • the present compounds have e.g. above the known ones
  • Plasmid or vector based systems e.g. cloneTech's pEGFP endo system has the advantage that no cell transfection is necessary. As a result, the dyeing procedures are much less labor intensive and faster.
  • the chromophore or the chromophoric "chromophore” is a component or a unit which ensures the basic presence of the colour. This color can be obtained by reflection and scattering of the ambient light, by absorption of a Part of the light or by emission of light of a certain wavelength after excitation of the chromophores by light of a different wavelength.
  • the light may be both visible and non-visible light, in particular light in the visible range, which is optionally emitted after excitation of the chromophore at a different wavelength.
  • Color in the present case means all colors, in particular all colors except white in the visible range.
  • the unit is, for example, one which has at least four, preferably at least six conjugated ⁇ -electrons.
  • the chromophoric unit is not connected via conjugated ⁇ electrons with the other units of the compounds of the invention. That is, there is no conjugated ⁇ -electron system between the chromophore moiety, such as R 6 or R 7 , and the tricyclic center, usually the pharmacophore region.
  • compounds of general formulas I and II which have a dapoxyl chromophore. That is, as chromophore according to formula I or II is a Dapoxylrest, which may optionally be derivatized before.
  • Dapoxyl means the following unit:
  • chromophore moieties especially the dapoxyl chromophores, have a large Stokes shift, with a first excitation at 378 nm and an emission at 500 nm and a second excitation at 514 nm and an emission at 560 nm.
  • the compounds further show a high intensity of emission.
  • the compounds according to the invention are particularly suitable. in the case of methods in dual excitation and dual emission mode, for example with the aid of a fluorescence microscope. In this way, for example, the cell vitality can be qualitatively and quantitatively determined.
  • R is hydrogen
  • Substrate R to a cleavable substrate is preferably one obtained by proteolytic enzymes, plasmin, cathepsin, cathepsin B, beta-glucuronidase, galactosidase, mannosidase, glucosidase, neuramidase, sucroseidase, maltase, fructosidase, glycosylases, prostate specific antigen (PSA), urokinase type Plasminogen activator (u-PA), metalloproteinase, or an enzyme that can be specifically cleaved using targeted enzyme prodrug therapy, such as ADEPT, VDEPT, MDEPT, GDEPT, or PDEPT; or a substituent which can be cleaved or transformed under hypoxic conditions or by reduction by nitroreductase.
  • proteolytic enzymes plasmin, cathepsin, cathepsin B, beta-glucuronidase, galactosidase,
  • radical R is a selected from the group consisting of monosaccharide, disaccharide or oligosaccharide, in particular hexoses, pentoses or heptoses optionally as a deoxy derivative or amino derivative, and optionally substituted with halogen, Ci -8 alkyl, Ci.
  • Ci-8 heteroalkyl Ci -8 heteroalkyl, C3-7 cycloalkyl, C3-7 heterocycloalkyl, aryl or C 4- C 12 -12 heteroaryl, amino or amide groups, or with amino, amido, or carboxyl moieties, which can optionally be substituted with halogen, Ci-8 alkyl, Ci-8 acyl, Ci -8 heteroalkyl, C 3- 7 cycloalkyl, C 3- 7 heterocycloalkyl, C 4- C 12 aryl or 4- 12 heteroaryl, amino or amide groups; Dextran, dipeptide, tripeptide, tetrapeptide, oligopeptide, peptidomimetics, or an antibody or antibody fragment, or combinations thereof.
  • the compounds of the invention wherein R is a substrate other than H have a cleavable or transformable group.
  • the cleavage or transformation of the compounds according to the invention Compounding with R except H can be carried out by chemical, photochemical, physical, biological or enzymatic processes under the appropriate conditions. These conditions include, for example, the provision of appropriate enzymes, the change of the surrounding environment, the action of, for example, radiation, such as UV light, etc.
  • the skilled worker is aware of corresponding methods.
  • the substrate is suitably accessible to known methods such as ADEPT (Antibody Directed Enzyme Prodrug Therapy), PDEPT (Polymer Directed Enzyme Prodrug Therapy) or MDEPT (Macromolecular-Directed Enzyme Prodrug Therapy), VDEPT (Virus-Directed Enzyme Prodrug Therapy) or GDEPT ( Gene-Directed Enzyme Prodrug Therapy).
  • ADEPT Antibody Directed Enzyme Prodrug Therapy
  • PDEPT Polymer Directed Enzyme Prodrug Therapy
  • MDEPT Macromolecular-Directed Enzyme Prodrug Therapy
  • VDEPT Virus-Directed Enzyme Prodrug Therapy
  • GDEPT Gene-Directed Enzyme Prodrug Therapy
  • R is further a dextran.
  • a dextran-containing compound according to the invention is provided.
  • the compound of the invention is one of the general formula I, wherein DB is selected from a radical of general formula II I, wherein R 8, R 9, R 10, or R 1 1 are selected from 2- (morpholino 4-yl) ethoxy, (1-methylpiperidin-4-yl) methoxy, 2- (N, N-dimethylamino) ethoxy, 2- (N, N-dimethylamino) acetylamino, 2-
  • the compound according to the invention is one of the general formula II, wherein the chromophore of R 6 and / or R 7 is a dapoxyl derivative and where DB is a radical of the general formula II with R 8 , R 9 , R 10 , or R 1 1 selected from 2- (morpholino-4-yl) ethoxy, (1-methylpiperidin-4-yl) methoxy, 2- (N, N-dimethylamino) ethoxy, 2- (N, N- dimethylamino) acetylamino, 2- (methylamino) ethoxy, 2- (methylamino) acetylamino, 2-aminoethoxy, 2-aminoacetylamino, (piperidin-4-yl) methoxy, 2- (N-methyl-N- (carboxymethyl) amino) ethoxy , and 2- (N-methyl-N- (2-methoxy-2-oxoethyl) amino)
  • dapoxyl derivatives allow the use of the compounds of the invention in various methods due to their large Stokes shift.
  • the compounds according to the invention when they interact with nucleic acids, in particular double-stranded nucleic acids, are not detectable in visible light due to their sparkleness, but emit (fluoresce) in other sub-cellular regions.
  • the dyes of the invention can be used in particular as markers in imaging processes. These markers are suitable for the selective labeling of various subcellular areas.
  • a qualitative and quantitative determination of nucleic acids, in particular double-stranded nucleic acids, is also possible.
  • a determination of the content or the amount of nucleic acids can be carried out using a competitive method with a second DNA-binding compound.
  • alkyl refers to straight-chain or branched, saturated or unsaturated hydrocarbon radicals. Substituents, examples of the alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, decyl, isopropyl, sec-butyl, isobutyl, tert -butyl, isopentyl, vinyl, allyl, 1-butenyl, 2-butenyl, isobutenyl , Pentenyl and the like.
  • cycloalkyl or “carbon cycles” as used herein refers to saturated or unsaturated, non-aromatic hydrocarbon cycles which may consist of one, two or more rings. Examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexonyl, etc.
  • heteroalkyl refers to straight-chain or branched, saturated or unsaturated hydrocarbon substituents in which at least one carbon is replaced by a heteroatom
  • the heteroatoms are preferably selected from S, N, O, and P.
  • aryl refers to aromatic substituents which may be one or more fused rings, and examples of aryl include phenyl, naphthyl and antracenyl.
  • water-soluble group refers to a functional group that solvates very well in an aqueous environment and imparts improved water-solubility to the compound to which this water-soluble group resides
  • water-soluble groups include, but are not limited to, alcohols and polyhydric alcohols, straight chain or cyclic saccharides, primary, secondary, tertiary or quaternary amines and polyamines, sulfate groups, carboxyl groups, phosphate groups, phosphoryl groups, ascorbate groups, glycols, including polyethylene glycol, and polyethers.
  • heteroaryl refers to aromatic substituents which may consist of one or more rings fused together, where at least one carbon atom of the aromatic ring is present. matic R group replaced by a heteroatom in particular S, N, O or P.
  • heteroaryl groups include pyridinyl, furanyl, pyrrolyl, triazolyl, pyrazolyl, imidazolyl, thiophenyl, indolyl, benzofuranyl, benzimidazolyl, indazolyl, benzotriazolyl, benzisoxazolyl, and quinolinyl.
  • heterocycloalkyl or “heterocycles” as used herein refers to saturated or unsaturated, non-aromatic cyclic hydrocarbon substituents which may consist of one or more fused rings, with at least one carbon in one of the rings replaced by a heteroatom, in particular S, N, O or P.
  • heterocycloalkyls include: tetrahydrofuranyl, pyrrolidinyl, piperidinyl, 1,4-dioxanyl, morpholinyl, piperazinyl, oxyzolidinyl, decahydroquinolinyl.
  • salts are in particular acid addition salts which are formed correspondingly to amine groups. Likewise, base addition salts are possible or corresponding zwitter addition salts.
  • pharmaceutically acceptable solvates refers to the association of one or more solvent molecules and a compound of the invention
  • solvent molecules which form pharmaceutically acceptable solvates include water, isopropyl alcohol, ethanol, methanol, DSMO, ethyl acetate, and acetic acid ,
  • the compounds of the invention allow multiple color labeling of various organelles, with no need for subsequent washing of the samples. Due to the large Stoke-shift, the dyes can be used in different ways. The dyes allow a distinction between living and dead cells.
  • 1 shows a diagram of the coupling of the galactoside (-) - (1 S, 10R) -2 with the NHS-activated fluorescent dye D1 01 62 (3) to the fluorescently labeled prodrug (-) - (1 S, 10R) -4.
  • FIG. 2 shows a diagram of the splitting off of the sugar moiety in the fluorescently labeled prodrug (-) - (1S, 10R) -4 to give the corresponding fluorescently labeled seco-drug hydrochloride (1S, 10R) -5.
  • FIG. 4 shows the temporal change in the intensity of the fluorescence emission of Hoechst 33342 after addition to an aqueous solution of isolated cellular DNA (concentration: 3 pg / ml) without or with prior incubation of the DNA with the fluorescently labeled seco-drug (FIG S, 10R) -5.
  • FIG. 6 shows a fluorescence micrograph of vital and dead cells (A549) after incubation with (1S, 10R) -5.
  • the cell lines used are available via ATCC and were cultured in the recommended media.
  • DMEM Dulbecco 's Modified Eagles Medium
  • the medium was supplemented with 4 ITI M L-glutamine and 3.7 g / L sodium bicarbonate.
  • FBS fetal calf serum
  • PBS buffer solution of the PBS-dry substance (Phosphate Buffered Saline) from Biochrom KG in bidistilled water. Salt concentrations in the buffer solution with pH 7.4: 1 37.0 m M NaCl, 2.7 M M KCl, 8.1 M M Na 2 HPO 4 , 1. 1 mM KH 2 PO 4.
  • Trypan blue ready-to-use solution from Biochrom of 0.5% (w / v) trypan blue in physiological saline.
  • Nuclear Selective Fluorescent Dye Hoechst 33342 (8) from Sigma-Aldrich was used to stain the core DNA. Endoscopic fluorescence labeling: Endocytic vesicles were visualized by expression of the pEGFP-Endo vector (Clontech, catalog number 6935-1) in adherent cells. The transfection was carried out with Lipofectamine 2000 (Invitrogen) according to the supplied protocol.
  • Imaging Buffer NaOH solution (NaCl 135I) (135 ITI M), KCl (5.37 IIT M), MgCl 2 (1.66 IIT M), CaCl 2 (1 .8 IIT M), HEPES (10 ⁇ M) titrated with NaOH to pH 7.4 ) and glucose (5.55 ITI M) in bidistilled water.
  • Fluorescence microscopy A Leica SP2 confocal laser scanning microscope from Leica, an UltraVIEW VoX spinning-disk microscope from Perkin Elmer, and a Zeiss Axiovert 200M inverse wide-field microscope equipped with a Hamamatsu ORCA-ER camera were used the company Zeiss. The processing of the data after the data acquisition took place with the programs ImageJ, Imaris 6.2, Volocity and IgorPro 4.05A Carbon.
  • the detection of the fluorescence emission of the dye Hoechst 33342 on the wide-field microscope was carried out with a 10x objective type Plan-Neofluar 10x / 0.30 Ph1 and the filter set 49 (DAPI) from Zeiss.
  • Preparative HPLC Preparative separations were carried out on a Jasco HPLC system equipped with two solvent pumps model PU-2087 PLUS and a UV detector model UV-2075 PLUS.
  • a finishing column Chiralpak ® IA 250 ⁇ 20 mm, particle size: 5 ⁇ ) were used as well as a prepacked column of the type Kromasil 1 00 C18 (7 ⁇ , 250 ⁇ 20 mm) from Jasco, in conjunction with a guard column of the type Kromasil 100 C18 ( 5 ⁇ , 50 ⁇ 20 mm) of the company Jasco.
  • the solvents used were n-heptane, n- HPLC-quality hexane and dichloromethane (Chiralpak IA) and double-distilled water (addition of 0.1% by volume trifluoroacetic acid for peptide synthesis) and HPLC grade acetonitrile or methanol (Kromasil 1 00 C18). All samples were membrane filtered and the solvents degassed.
  • General procedure for determining the fluorescence intensity at a defined wavelength as a function of the excitation wavelength (absorption scan)
  • Emission scan digital cfg
  • Exe 350 nm (or 378 nm, 405 nm or 514 nm)
  • Em1 and Em2 400-800 nm
  • Step size 5 nm
  • Integration 1 sec
  • Averages 1.
  • Em1 500 nm, Em2: 560 nm
  • Exc. 405 nm Em 1: 500 nm, Em2: 560 nm
  • Exc. 514 nm Em 1: 500 nm, Em2: 560 nm
  • the adherently growing human bronchial carcinoma of the line A549 served.
  • the seeding of the tumor cells was carried out in D10F (DMEM with the addition of 10% fetal calf serum, 44 ⁇ l M NaHCO 3 and 4 ⁇ l M Glutamine) in a concentration of 25 ⁇ 10 3 cells in 500 ⁇ l medium per chamber of the coverslip ( Nunc® Lab -Tek ® Chamber Slide 4 wells on Permanox ®).
  • the cells were adhered for 24 h at 37 ° C and 7.5% CO 2 .
  • the cells were seeded in a concentration of 5 ⁇ 10 3 cells in 500 ⁇ ⁇ medium per chamber of the coverslip and adhered for 3 days at 37 ° C and 7.5% CO 2 .
  • the prepared cells were washed with serum-free Ultra Culture ® medium (2x1 mL) and treated with a solution of the test substance in DMSO and Ultraculture ® medium (500 ⁇ _, 1% DMSO) or with a solution of DMSO (in Ultraculture ® medium 500 ⁇ _, 1% DMSO).
  • the concentration of the compounds in the incubation solution was 10 ⁇ ((1S, 10R) -4 or (1S, 10R) -5 or 25 nM (9) After a certain incubation period (45 min-2.5 h) at 37 ° C and 7.5% CO 2, the cells with Ultraculture ® medium (1 mL) and optinal with imaging buffer (2x1 mL) and examined in the imaging buffer by means of a confocal fluorescence microscope.
  • FIG. 5 shows the results for compound 5.
  • Hoechst 33342 in buffer (1 ⁇ ⁇ , concentration of the stock solution: 36 ITI M, 36 nmol) mixed with the imaging buffer in the chambers (Cfinai 72 ⁇ ⁇ ) and the cells incubated for several minutes at room temperature before observation.
  • FIG. 6 shows the results.
  • the blue-green fluorescence of the seco drug (1 S, 10R) -5 (Fig. 6a, shown in blue) could be detected in cytosolic organelles of living and dead cells, especially near the nucleus. Since the fluorescence of the DNA stain Hoechst 33342 (8) was excited and detected at wavelengths similar to the blue-green fluorescence of the seco drug, staining of the nuclei by 8 ( Figure 6a, shown in blue) was due to localization only the staining in the core of the blue-green fluorescence of seco-drug in the cytosolic organelles to distinguish.

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Abstract

L'invention concerne de nouveaux composés aptes à servir de colorants fluorescents. L'invention concerne en particulier une nouvelle classe de composés pharmaceutiquement actifs dont des dérivés sont formés avec des chromophores. Dans un autre mode de réalisation, l'invention porte sur des utilisations des composés selon l'invention.
PCT/EP2010/066548 2009-10-30 2010-10-30 Colorants fluorescents WO2011051484A1 (fr)

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