DE102009051438A1 - fluorescent dyes - Google Patents

Abstract

The present invention relates to new compounds suitable as fluorescent dyes. In particular, the present invention relates to a new class of pharmacologically active compounds derivatized with chromophores. In a further aspect, the present invention is directed to applications of the compounds according to the invention.

Description

The present invention relates to novel, suitable as fluorescent dyes compounds. In particular, the present invention relates to a new class of chromophore derivatized, pharmacologically active compounds. In a further aspect, the present invention is directed to applications of the compounds of the invention.

State of the art

Dyes, in particular fluorescent dyes, represent an important tool for the representation and detection of structures, states and changes, especially in the field of life science. A large number of dyes, in particular fluorescent dyes, have already been described in imaging methods and for the preparation of structures, be used for marking molecules, etc.

These dyes may be covalently coupled to other structures, such as proteins, nucleic acids or other chemical or biological entities. Such compounds are z. B. then used in diagnostic applications. Many fluorescent dyes build z. B. on fluorescein or rhodamine structures. In the meantime, however, a large number of other fluorescent dyes with fluorophore groups have also been described. For this purpose, z. B. on the book: The Handbook, A Guide to Fluorescent Probes and Labeling Technologies, Molecular Probes - Invitrogen, 10th Edition , referenced. 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 US Pat. B. in the WO 02/059122 . WO 98/11101 . WO 97/12862 . WO 01/83448 or WO 2007/089149 , The molecules described there, z. B. in the WO 2007/089149 , incorporated herein by reference, include corresponding DNA-binding moieties in association with pharmacophore moieties.

Thus, it has been shown that 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 for. B. in the WO 01/83448 and the WO 2007/089149 described. 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. It has long been known to use corresponding prodrugs, which are then converted at the destination into pharmacologically active compounds. This conversion can z. B. be acid-catalyzed or carried out enzymatically or otherwise. Such prodrugs and drugs based on bases of various CC-1065 analogs have been described, see e.g. B. WO 2007/089149 and WO 01/83448 . WO 98/11101 ,

To mark individual areas of a cell, z. As cellular organelles of endocytosis and exocytosis, various dyes are known. These are z. As described in the above-mentioned manual for fluorescence probes and labeling technologies. A variety of different molecules are shown. These are z. B. as a lyso tracker, ER tracker, etc. by the company Invitrogen, USA, sold commercially. Alternatively, there are molecular biology methods, eg. For example, plasmids or vectors that introduce appropriate fluorosensin-based markers in the target areas.

The present invention is directed to novel compounds useful as dyes, in particular fluorescent dyes, for labeling cell structures, cell alterations and cell states.

Description of the present invention

mit R¹ ausgewählt aus einem Halogen und OSO₂R^u, wobei R^u ausgewählt ist aus gegebenenfalls substituierten C₁-C₆ alkyl, C_1-6 Perhaloalkyl, Benzyl und Phenyl;
R² ist ausgewählt aus Wasserstoff und gegebenenfalls substituierten C_1-8 Alkyl; R³, R^3', R⁴ und R^4' sind unabhängig voneinander ausgewählt aus Wasserstoff und gegebenenfalls substituierten C_1-8 Alkyl, wobei zwei oder mehrere von R², R³, R^3', R⁴ und R^4' gegebenenfalls miteinander verbunden sind, um ein oder mehrere gegebenenfalls substituierte Kohlenstoffzyklen oder Heterozykien auszubilden;
X² ist ausgewählt aus O, C(R¹⁴)(R^14') und NR^14', wobei R¹⁴ ausgewählt ist aus Wasserstoff und gegebenenfalls substituierten C_1-8 Alkyl oder C_1-8 Acyl und wobei R^14' nicht vorhanden ist oder ausgewählt ist aus Wasserstoff und gegebenenfalls substituierten C_1-8 Alkyl oder C_1-8 Acyl;
R⁵, R^5', R⁶, R^6', R⁷ und R^7' sind unabhängig voneinander ausgewählt aus H, OH, SH, NH₂, N₃NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, Halogen, R^k, SR^k, S(O)R^k, S(O)₂R^k, S(O)OR^k, S(O)₂OR^k, OS(O)R^k, OS(O)₂R^k, OS(O)OR^k, OS(O)₂R^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^kR^LR^m, C(O)R^k, C(O)OR^k, C(O)N(R^L)R^k, OC(O)R^k, OC(O)R^k, OC(O)OR^k, OC(O)N(R^k)R^L, N(R^L)C(O)R^k, N(R^L)C(O)OR^k, und N(R^L)C(O)N(R^m)R^k, wobei R^k, R^L und R^m unabhängig voneinander ausgewählt sind aus H und gegebenenfalls substituierte C_1-4 Alkyl, C_1-4 Heteroalkyl, C_3-7 Cycloalkyl, C_3-7 Heterocycloalkyl, C_4-12 Aryl oder C_4-12 Heteroaryl Gruppen, zwei oder mehr von R^k, R^L und R^m bilden gegebenenfalls miteinander ein oder mehrere gegebenenfalls substituierte aliphatische oder aromatische Kohlenstoffzyklen oder Heterozyklen aus;
und/oder R^5' und R^6' _und/oder R^6' und R^7' und/oder R⁷ und R^14' sind nicht vorhanden, d. h., sie bilden eine im Ring vorhandene Doppelbildung zwischen den mit den entsprechenden substituierten Atomen aus, nämlich R^5' und R^6', und/oder R^6' und R^7', und/oder R^7' und R^14';
zwei oder mehrere von R⁵, R^5', R^6', R⁷, R¹⁴, und R^14' können gegebenenfalls miteinander verbunden sein, um ein oder mehrere gegebenenfalls substituierte aliphatische oder aromatische Kohlenstoffzyklen oder Heterozyklen auszubilden;
und/oder R⁵ + R^5', und/oder R⁶ + R^6' und/oder R⁷ + R^7' sind unabhängig voneinander =O, =S oder =NR¹², wobei R¹² ausgewählt ist aus H und gegebenenfalls substituierten C_1-6 Alkyl;
unter der Voraussetzung, dass entweder R⁶ und/oder R⁷ unabhängig voneinander eine Gruppe-(C(R_CH)₂)_1-6-X₇-Chromophor darstellt,
wobei X₇ ausgewählt ist aus C=O, O, NR_CH, wobei R_CH ausgewählt ist aus Wasserstoff und gegebenenfalls substituierten C_1-8 Alkyl oder C_1-8 Acyl;
X₆ ist ausgewählt aus C oder N;
R_DB ist ausgewählt aus Wasserstoff und gegebenenfalls substituierten C_1-8 Alkyl oder C_1-8 Acyl;
X₁ ist ausgewählt aus O, S, und NR¹³, wobei R¹³ ausgewählt ist aus H und gegebenenfalls substituierten C_1-8 Alkyl;
R ist ausgewählt aus Wasserstoff oder ein enzymatisch abspaltbares Substrat;
a und b sind unabhängig voneinander ausgewählt aus 0 und 1;
c ist ausgewählt aus 0, 1 und 2;
d ist ausgewählt aus 0 oder 1;
DB ist Wasserstoff oder eine mit DNA in Wechselwirkung tretende Verbindung, insbesondere eine der allgemeinen Formel III

wobei X³ ausgewählt ist aus O, S, und NR¹⁵, wobei R¹⁵ ausgewählt ist aus H und gegebenenfalls substituierten C_1-8 Alkyl oder C_1-8 Acyl; oder -X³- ist X^3a und X^3b, wobei X^3a verbunden ist mit dem Kohlenstoff mit dem X⁴ verbunden ist und X^3b ist verbunden mit dem Phenylring in ortho-Position zu R¹⁰, wobei X^3a ausgewählt ist aus Wasserstoff und gegebenenfalls substituierten C_1-8 Alkyl oder Acylrest und X^3b ist aus der gleichen Gruppe ausgewählt wie die Reste definiert für R⁸, wobei X^3a und X^3b keine kovalente Verbindung untereinander ausbilden;
X⁴ ist ausgewählt aus N und CR¹⁶, wobei R¹⁶ ausgewählt ist aus Wasserstoff und gegebenenfalls substituierten C^1-8 Alkyl oder C^1-8 Acyl;
X⁵ ist ausgewählt aus O, S, und NR¹⁷, wobei R¹⁷ ausgewählt ist aus H und gegebenenfalls substituierten C_1-8 Alkyl oder C_1-8 Acyl; R⁸ _, R⁹, R¹⁰, und R¹¹ werden unabhängig ausgesucht aus H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, Halogen, R^x, SR^x, S(O)R^x, S(O)₂R, S(O)OR^x, S(O)₂OR^x, OS(O)R^x, OS(O)₂R^x, OS(O)OR^x, OS(O)₂OR^x, OR^x, NHR^x, N(R^x)R^y, ⁺N(R^x)(R^y)R^z, P(O)(OR^x)(OR^y), OP(O)(OR^x)(OR^y), SiR R^yR^z, C(O)R^x, C(O)OR^x, C(O)N(R^y)R^x, OC(O)R^x, OC(O)OR^x, OC(O)N(R^x)R^y, N(R^y)C(O)R^x, N(R^y)C(O)OR^x, N(R^y)C(O)N(R^z)R^x, und einer Wasser-Gruppe, wobei R^x, R^y, und R^z unabhängig ausgewählt sind aus H und optional substituierten C_1-15 Alkyl-, C_1-15 Heteroalkyl-, C_3-15 Cycloalkyl-, C_3-15 Heterocycloalkyl-, C_4-15 Aryl-, oder C_4-15 Heteroaryl-Resten, wobei einer odermehrere der optionalen Substituenten in R^x, R^y, und R^z optional eine wasserlösliche Gruppe ist, und zwei oder mehr der R^x, R^y, und R^z können optional verbunden sein, um einen oder mehrere optional substituierte Kohlenstoffzyklen oder Heterozyklen zu bilden, und mindestens einer der Reste von R⁸, R⁹, R¹⁰, und R¹¹ umfasst eine wasserlösliche Gruppe, zwei oder mehr der R⁸, R⁹, R¹⁰, R¹¹, oder X^3b sind optional verbunden, um einen oder mehrere optional substituierte aliphatische oder aromatische Kohlenstoffzyklen oder Heterozyklen zu bilden;
oder pharmazeutische annehmbare Salze oder Solvate hiervon.In a first aspect, compounds of the general formulas I or II are provided:

wherein R ^{1 is} selected from a halogen and OSO ₂ R ^u , wherein R ^{u is} selected from optionally substituted C ₁ -C ₆ alkyl, C _1-6 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 linked 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 C _1-8 alkyl or C _1-8 acyl and where R ^{14 '} is absent is or is selected from hydrogen and optionally substituted C _1-8 alkyl or C _1-8 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, halogen, 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 (O) OR ^k , C (O) N (R ^L ) R ^k , OC (O) R ^k , OC (O) R ^k , OC (O) OR ^k , OC (O) N (R ^k ) R ^L , N (R ^L ) C (O) R ^k , N (R ^L ) C (O) OR ^k , and N (R ^L ) C (O) N (R ^m ) R ^k , where R ^k , R ^L and R ^{m are} independently selected from H and optionally substituted C _1-4 alkyl, C _1-4 heteroalkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, C _4-12 aryl or C _4-12 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;
and / or 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 ring present in the ring between those with the corresponding substituted atoms namely R ^{5 '} and R ^6' , and / or R ^{6 '} and R ^7' , and / or R ^{7 '} and R ^14' ;
two or more of 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;
and / or R ⁵ + R ^{5 '} , and / or R ⁶ + R ^6' and / or R ⁷ + R ^{7 '} are independently = O, = S or = NR ¹² , wherein R ^{12 is} selected from H and optionally substituted C _1-6 alkyl;
with the proviso that either R ⁶ and / or R ⁷ independently represent a group (C (R _CH ) ₂ ) _1-6 -X ₇ chromophore,
wherein X _{7 is} selected from C = O, O, NR _CH , wherein R _{CH is} selected from hydrogen and optionally substituted C _1-8 alkyl or C _1-8 acyl;
X ₆ is selected from C or N;
R _DB is selected from hydrogen and optionally substituted C _1-8 alkyl or C _1-8 acyl;
X ₁ is selected from O, S, and NR ¹³ , wherein R ^{13 is} selected from H and optionally substituted C _1-8 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 III

wherein X ^{3 is} selected from O, S, and NR ¹⁵ , wherein R ^{15 is} selected from H and optionally substituted C _1-8 alkyl or C _1-8 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 C _1-8 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 C ^1-8 alkyl or C ^1-8 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, 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 , OR ^x , NHR ^x , N (R ^x ) R ^y , ⁺ N (R ^x ) (R ^y ) R ^z , P (O) (OR ^x ) (OR ^y ), OP (O) (OR ^x ) (OR ^y ), SiR R ^y R ^z , C (O) R ^x , C (O) OR ^x , C (O ) N (R ^y ) R ^x , OC (O) R ^x , OC (O) OR ^x , OC (O) N (R ^x ) R ^y , N (R ^y ) C (O) R ^x , N (R ^y ) C (O) OR ^x , N (R ^y ) C (O) N (R ^z ) R ^x , and a water group wherein R ^x , R ^y , and R ^{z are} independently selected from H and optionally substituted C _1-15 alkyl, C _1-15 heteroalkyl, C _3-15 cycloalkyl, C _3-15 heterocycloalkyl, C _4-15 aryl, or C _4-15 heteroaryl, wherein one or more of the optional substituents in R ^x , R ^y , and R ^{z is} optionally a water-soluble group, and two or more of R ^x , R ^y , and R ^z may be optionally joined to form one or more optionally substituted carbon cycles or He and at least one of R ⁸ , R ⁹ , R ¹⁰ , and R ¹¹ 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;
or pharmaceutically acceptable salts or solvates thereof.

These compounds are preferably those CC-1065 analogs, as described, for. B. in the WO 2007/089149 and WO 01/83448 are fully incorporated herein by reference. The present compounds are characterized by having a DNA-binding region, a pharmacophore moiety and a fluorophore moiety.

Surprisingly, it has been found that 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 of the invention, the nucleus shows no expected color, while a fluorescence labeling could be observed in particular in cellular organelles of endocytosis and exocytosis but also the mitochondria. The compounds of the invention are therefore particularly suitable for imaging methods. These imaging methods are in particular light microscopic and fluorescence-based methods.

By the term "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 determination of the effectiveness of mitochondria-associated diseases and for the determination of the diagnosis, prognosis, course and determination of the effectiveness of therapies Agents. The compounds of the invention further allow the use of these in combination with other known fluorescent dyes, so as to achieve a multiple labeling of various organelles. Thus, with the aid of the compounds of the invention in combination with z. As DNA intercalating compounds but also in combination with corresponding molecules, the z. B. specific lysosomes, endosomes or other subcellular structures mark, multiple staining done. By way of example, a combination of the compounds according to the invention with systems from Invitrogen, USA, such as Lyso-Tracker, ER-Tracker or Mito-Tracker may be mentioned. Furthermore, the compounds according to the invention can be used in combination with Hoechst dyes or propidium iodide.

Thus, 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 of the invention allow labeling with a short incubation time.

Depending on the substituents, in particular as a function of the substituent R, the compounds can be introduced into cells in a simple and directed manner. R is preferably hydrogen.

Especially in the study of changes in mitochondrial morphology compounds of the invention are particularly suitable. 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.

By way of example, due to the presence of the z. B. enzymatically cleavable substrates 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, for. B. on the known based on plasmids or vectors systems such. For example, 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.

According to the invention, the chromophore is a component or a unit which ensures the basic presence of the color. This coloration can be done 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 another wavelength. The light can be both visible and non-visible light.

(4-[5[4-(dimethylamino)phenyl]-2-oxazolyl]-benzen) oder auch benannt als 2-Phenyl-5-(4-N,N-dimethylaminophenyl)oxazol. Dieser Dapoxylrest kann entsprechend derivatisiert sein, um eine Kopplung mit anderen chemischen Resten zu ermöglichen. Typische Derivate schließen ein: -3-Sulfonamidpropionat-Dapoxylderivate, -3-Sulfonamidbutyrat-dapoxylderivate, -3-sulfonamid-Dapoxylerivate, etc.In a preferred embodiment, compounds of general formulas I and II are provided which have a dapoxyl chromophore. In this context, the term "dapoxyl" means the following unit:

(4- [5 [4- (dimethylamino) phenyl] -2-oxazolyl] -benzene) or also named as 2-phenyl-5- (4-N, N-dimethylaminophenyl) oxazole. This dapoxyl radical may be derivatized accordingly to allow coupling with other chemical moieties. Typical derivatives include: -3-sulfonamidopropionate-dapoxylderivatives, -3-sulfonamidobutyrate-dapoxylderivatives, -3-sulfonamide-dapoxylerivatives, etc.

These 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.

As a result, the compounds according to the invention are particularly suitable for processes in the dual excitation and dual emission mode, for. B. using a fluorescence microscope. This allows z. B. qualitatively and quantitatively determine the cell vitality.

In a preferred embodiment, R is hydrogen.

In another preferred embodiment, the substrate R is a cleavable substrate. This cleavable substrate is preferably one which can be produced 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.

The radical R is preferably one selected from the group comprising monosaccharide, disaccharide or oligosaccharide, in particular hexoses, pentoses or heptoses, optionally as the deoxy derivative or amino derivative and optionally substituted by halogen, C _1-8 alkyl, C _{1 -8} acyl, C _1-8 heteroalkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, C _4-12 aryl or C _4-12 heteroaryl, amino or amide groups, or with amino, amido or carboxyl moieties, the optionally substituted with halogen, C _1-8 alkyl, C _1-8 acyl, C _1-8 heteroalkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, C _4-12 aryl or C _4-12 heteroaryl, amino - or amide radicals; Dextran, dipeptide, tripeptide, tetrapeptide, oligopeptide, peptidomimetics, or an antibody or antibody fragment, or combinations thereof.

With the aid of the substrate R, targeting of the compounds according to the invention to target structures is possible. D. h., It is a targeted coupling of the compounds of the invention to target structures and a corresponding introduction of these compounds of the invention in z. B. selected cells and cell types possible. 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 compound of the invention with R except H can be carried out by chemical, photochemical, physical, biological or enzymatic processes under the appropriate conditions. These conditions include, for. B. the provision of appropriate enzymes, the change of the surrounding environment, the action of z. As radiation, such as UV light, etc. The skilled person are known methods. The substrate is suitably accessible to known methods such as ADEPT (Antibody Directed Enzyme Prodrug Therapy) or MDEPT (Macromolecular-Directed Enzyme Prodrug Therapy), VDEPT (Virus-Directed Enzyme Prodrug Therapy) or GDEPT (Gen -Directed Enzyme Prodrug Therapy).

In a preferred embodiment, R is further a dextran. In particular, for a temporal and spatial detection of endocytosis, a dextran-containing compound according to the invention is provided.

In a further preferred embodiment, the compound according to the invention is one of the general formula I, wherein DB is selected from a radical of the general formula III, where R ⁸ , R ⁹ , R ¹⁰ , or R ^{11 are} 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) ethoxy or hydrogen and the chromophore present in R ⁶ and / or R ⁷ is a dapoxyl derivative.

In a further preferred embodiment, the compound of the invention is one of the general formula II, wherein the chromophore of R ⁶ and / or R ^{7 is} a dapoxyl derivative and wherein DB is a radical of the general formula III with R ⁸ , R ⁹ , R ¹⁰ or R 11 is 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- (methyl N-methyl-N- (2-methoxy-2-oxoethyl) amino) ethoxy or hydrogen.

Particularly preferred compounds are the compounds IV, V, VI, VII shown below:

In particular, the dapoxyl derivatives allow the use of the compounds of the invention in various methods due to their large Stokes shift.

Surprisingly, it has been shown that 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 color, whereas they emit (fluoresce) in other sub-cellular regions.

Accordingly, 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.

The term "substituted", as used herein in particular with reference to alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, acyl, refers to those groups having one or more substituents selected from the group comprising OH, = O, = S, = NR ^h , = N-OR ^h , S ^h , NH ₂ , NO ₂ , NO, N ₃ , CF ₃ , CN, OCN, SCN, NCO, NCS, C (O) NH ₂ , C (O ) H, C (O) OH, halo, R ^h , SR ^h , S (O) R ^h , S (O) OR ^h , S (O) ₂ R ^h , S (O) ₂ OR ^h , OS (O R ^h , OS (O) OR ^h , OS (O) ₂ R ^h , OS (O) ₂ OR ^h , OP (O) (ORh) (OR ^h ), P (O) (ORh) (OR ⁱ ) , OR ^h , NHR ⁱ , N (R ^h ) R ⁱ , ⁺ N (Rh) (R ⁱ ) R ^j , Si (R ^h ) (R ⁱ ) R ^j , Si (R ^h ) (R ⁱ ) (R ^j ), C (O) R ^h , C (O) OR ^h , C (O) N (R ⁱ ) R ^h , OC (O) R ^h , OC (O) OR ^h , OC (O) N (R ^h ) R ⁱ , N (R ⁱ ) C (O) R ^h , N (R ⁱ ) C (O) OR ^h , N (R ⁱ ) C (O) N (R ⁱ ) R ^h , and the thio derivatives thereof Substituents, or a protonated or deprotonated form of these substituents, wherein R ^h , R ⁱ , and R ^{j are} independently selected from H and optionally substituted C _1-15 Alk yl, C _1-15 heteroalkyl, C _3-15 cycloalkyl, C _3-15 heterocycloalkyl, C _4-15 aryl, or C _4-15 heteroaryl or a combination thereof, two or more of R ^h , R ⁱ , and R ^j are optionally linked together to form one or more carbon cycles or heterocycles.

The term "alkyl" as used herein refers to straight-chained or branched, saturated or unsaturated hydrocarbon substituents, examples of the alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, decyl, isopropyl, sec Butyl, isobutyl, tert-butyl, iso-pentyl, vinyl, allyl, 1-butenyl, 2-butenyl, isobutenyl, pentenyl and the like.

The term "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.

The term "heteroalkyl" as used herein 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.

The term "aryl" as used herein refers to aromatic substituents which may be one or more rings fused together. Examples of aryl include phenyl, naphthyl and antracenyl.

The term "heteroaryl" as used herein refers to aromatic substituents which may consist of one or more rings fused together. Here, at least one carbon atom of the aromatic R group is replaced by a heteroatom. Examples of heteroaryl groups include pyridinyl, furanyl, pyrrolyl, triazolyl, pyrazolyl, imidazolyl, thiophenyl, indolyl, benzofuranyl, benzimidazolyl, indazolyl, benzotriazolyl, benzisoxazolyl, and quinolinyl.

The term "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. Examples of heterocycloalkyls include: tetrahydrofuranyl, pyrrolidinyl, piperidinyl, 1,4-dioxanyl, morpholinyl, piperazinyl, oxyzolidinyl, decahydroquinolinyl.

When terms such as "optionally substituted" are used, these terms refer to all subsequent residues unless otherwise specified. That is, the expression " optionally substituted alkyl, heteroalkyl, aryl, acyl "is to be read as" optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, optionally substituted acyl ".

Pharmaceutically acceptable salts are in particular acid addition salts, which are formed according to amine groups. Likewise, base addition salts are possible or corresponding zwitter addition salts.

The term "pharmaceutically acceptable solvates" refers to the association of one or more solvent molecules and a compound of the invention. Examples of such solvent molecules that form pharmaceutically acceptable solvates include water, isopropyl alcohol, ethanol, methanol, DSMO, ethyl acetate, and acetic acid.

The compounds of the invention, combinations of CC-1065 analogs and chromophores, 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.

Brief description of the figures:

1 Figure 6 shows a scheme of coupling galactoside (-) - (1S, 10R) -2 with the NHS-activated fluorescent dye D10162 (3) to the fluorescently labeled prodrug (-) - (1S, 10R) -4.

2 shows a scheme of the cleavage of the sugar moiety in the fluorescently labeled prodrug (-) - (1S, 10R) -4 to the corresponding fluorescently labeled seco-Drug hydrochloride (1S, 10R) -5.

3 shows the absorption spectra of the fluorescently labeled seco drug (1S, 10R) -5 with respect to a fluorescence emission of λ _em = 500 nm and λ _em = 560 nm after 24 h incubation in a) water or b-d) of an aqueous solution of isolated cellular DNA Concentration 7.5 μg / mL (b), 15 μg / mL (c) or 30 μg / mL (d).

4 shows the temporal change in the intensity of fluorescence emission of Hoechst 33342 after addition to an aqueous solution of isolated cellular DNA (concentration: 3 μg / ml) without or with prior incubation of the DNA with the fluorescently labeled seco-drug (1S, 10R) -5.

5 provides fluorescence micrographs of the two cell types that can be differentiated after incubation of the cells (A549) with the seco-Drug (1S, 10R) -5. a) vital and dead cells, λ _em = 500 nm (originally blue); b) vital cells, λ _em = 560 nm (yellow in original); c) superposition of the emissions at λ _em = 500 nm (blue) and λ _em = 560 nm (yellow).

6 shows fluorescence micrograph of vital and dead cells (A549) after incubation with (1S, 10R) -5. a) λ _exc = 405 nm, λ _em = 460-500 nm, blue image: cell nuclei stained by Hoechst 33342 (8) and cytoplasmic organelles stained by 5; b) λ _exc = 514 nm, λ _em = 560 nm, representation in green: by (1S, 10R) -5 stained mitochondria of vital cells (upper 2/3 of the cells); c) λ _exc = 633 nm, the = 680 nm, displayed in red: by MitoTracker ^® Deep Red (9) stained mitochondria of viable cells (upper 2/3 of the cells); d) λ _exc = 561 nm, λ _em = 630 nm, representation in light blue: trypan blue stained cell membranes of vital cells (upper 2/3 of the cells) and stained cytosol of dead cells (lower 1/3 of the cells); e) Overlapping the fluorescence of (1S, 10R) -5 (green) and MitoTracker Deep Red 9 (red) to yellow; f) superposition of a-d.

In the following, the invention will be explained in more detail with the aid of the examples, without the invention being restricted to these.

example 1

Materials:

The cell lines used are available via ATCC and were cultured in the recommended media.
Culture medium for A549: DMEM (Dulbecco's Modified Eagles Medium) with 4.5 g / l glucose (Biochrom, T043-10). The medium was supplemented with 4 mM L-glutamine and 3.7 g / l sodium bicarbonate.
Medium additives: 10% FBS (fetal calf serum) from Biochrom, inactivated for 30 min at 56 ° C.
Enzyme: β-D-galactosidase (EC 3.2.1.23) from Escherichia coli G 5635 (Sigma), activity: 250-600 units (units (U)) per mg protein at pH 7.3 and 37 ° C, 1 U = 1 μmol Substrate turnover per minute.
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: 137.0 mM NaCl, 2.7 mM KCl, 8.1 mM 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.
Fluorescent dye of compounds (-) - (1S, 10R) -4 and (1S, 10R) -5: Used to couple the dye Dapoxyl ^® -3-sulfonamidopropionic acid to the free amino functionality in (-) - (1S, 10R) -1 the dapoxyl ^® -3-Sulfonamidpropionsäure succinimidyl ester D10162 (3) from Invitrogen.
Mitochondria-selective fluorescent dye: For staining the mitochondria of vital cells was MitoTracker ^® Deep Red 633 FM (9) from Invitrogen.
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: with NaOH to pH 7.4 titrated solution of NaCl (135 mM), KCl (5:37 mM), MgCl ₂ (1.66 mM), CaCl ₂ (1.8 mM), HEPES (10 mM) and glucose (5.55 mM) in redistilled 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.
Fluorimetry: A spectrofluorometer Quants Master from PTI (Photon Technology International) equipped with 2 photomultiplier detectors 814 from PTI was used to record the excitation and emission spectra. The FeliX32 program and a precision cuvette (layer thickness: 10 mm) from Hellma were used. To record the absorption spectrum of the prodrug (-) - (1S, 10R) -4 also served a spectrometer Cary 5E Varian and for recording the emission spectrum, a spectrometer Fluorolog Jabin Yvon.
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 were used (250 x 20 mm, particle size: 5 .mu.m) and a prepacked column of the type Kromasil 100 C18 (7 .mu.m, 250 x 20 mm) from Jasco 100 C18 (in conjunction with a guard column of the type Kromasil 5 μm, 50 × 20 mm) from Jasco. N-heptane, n-hexane and dichloromethane were used as solvents of HPLC grade (Chiralpak ^® IA) and bidistilled water (addition of 0.1 vol .-% trifluoroacetic acid for peptide synthesis) and acetonitrile or methanol in HPLC grade (Kromasil 100 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)

1 aliquot of the stock solution (c = 1 mM) of the substance to be examined in DMSO (2 .mu.l, 2 nmol) was dissolved in the chosen solvent (200 .mu.l, solvent: methanol, imaging buffer or water), the solution mixed with a vortexer and in a Pipette quartz cuvette. The fluorescence intensity of the emissions was measured with a fluorimeter at λ _em = 500 nm and λ _em = 560 nm for an excitation wavelength range of λ _exc = 200-600 nm.
Parameters: Excitation scan, digital cfg, Exc .: 200-600 nm, length: 400 nm, Em1: 500 nm, Em2: 560 nm, Step size: 5 nm (or 1 nm), Integration: 1 sec, Averages: 1.

General procedure for measuring the fluorescence emission spectrum at a fixed excitation wavelength (emission scan)

1 aliquot of the stock solution (c = 1 mM) of the substance to be examined in DMSO (2 .mu.l, 2 nmol) was dissolved in the chosen solvent (200 .mu.l, solvent: methanol, imaging buffer or water), the solution mixed with a vortexer and in a Cuvette pipetted. Mixed with a vortex device and pipetted into a cuvette. With a fluorimeter, the fluorescence intensity was excited by excitation Laser light of wavelength λ _exc = 350 nm (or 378 nm, 405 nm or 514 nm) measured for an emission range of λ _em = 400-800 nm.
Parameters: Emission scan, digital cfg, Exc .: 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.

example 1

Synthesis of the compounds of the invention

The compounds of the invention were prepared according to the in 1 Synthesized Scheme synthesized.

Synthesis of (-) - {3- [4- (5- (4-Dimethylaminophenyl) oxazol-2-yl) benzenesulfonylamino] propionic acid - [(1S, 10R) -1- (10-chloro-ethyl) -3- [ (5- (2- (N, N-dimethylamino) ethoxy) indol-2-yl) carbonyl] -5-O-β-D-galactopyranosyl-1,2-dihydro-3H-benz [e] indol-7- ylmethyl] amide trifluoroacetate), (-) - (1S, 10R) -4

To a solution of NHS ester (3) (12.3 mg, 24.0 μmol, 1.0 eq.) In absolute DMF (200 μL) was added a solution of galactoside (-) - (1S, 10R) -2 (25.0 mg, 27.9 μmol , 1.2 eq.) And i-Pr ₂ NEt (23.0 μL, 139 μmol, 5.8 eq.) In absolute DMF (150 μL). After stirring for 9 h at room temperature, the reaction solution was diluted with bidistilled water (0.1% TFA, 3.0 mL) and CH ₃ CN (2.5 mL). 2 mL each of this solution was injected into the preparative HPLC system. Fractional capture of the eluate, removal of the solvents under reduced pressure and removal of the residual solvent by lyophilization afforded the fluorescently labeled glycosidic prodrug (-) - (1S, 10R) -4 as a yellow solid (27.8 mg, 21.5 μmol, 89%). Purification of the prodrug (-) - (1S, 10R) -4 was carried out by preparative HPLC under the following conditions: HPLC (preparative): Pillar: Kromasil 100 C18 Gradient: Time [min] H ₂ O (0.1% TFA) / CH ₃ CN 0 83/17 0-60 83/17 → 50/50 60-70 50/50 → 83/17 River: 12 ml min ^-1 t _R : 38.5 min Fraction: 38.0-41.0 min ¹ H-NMR (600 MHz, DMSO-d ₆ , 80 ° C): δ = 1.63 (d, J = 6.6 Hz, 3H, 11-H ₃ ), 2.40 (t, J = 7.4 Hz, 2H, 2 ^S -H _2), 2.93 (s, 6H, 2 '' - NMe _2), 2.98 (s, 6H, 12 ^# -NMe _2), 3.14 (t, J = 7.4 Hz, 2H, ^S 3 -H _2), 3.48 (dd, J = 9.6, 3.2 Hz, 1H, 3 '''- H), 3.52-3.57 (m, 3H, 5''' - H, 2 '' - H ₂ ), 3.60 (dd, J = 10.6, 5.5 Hz, 1H, 6 '''- H _a ), 3.69 (dd, J = 10.6, 7.2 Hz, 1H, 6''' - H _b ), 3.81-3.86 (m, 2H, 2 ''' -H, 4 '''- H), 4.20 (dt, J = 9.4, 2.6 Hz, 1H, 1-H), 4.36 (t, J = 5.0 Hz, 2H, 1''- H ₂ ), 4.42 ( m _c , 2H, 12-H ₂ ), 4.62 (dd, J = 11.0, 2.6 Hz, 1H, 2-H _a ), 4.70 (dd, J = 11.0, 9.4 Hz, 1H, 2-H _b ), 4.79 (dq, J = 6.6, 3.0 Hz, 1H, 10-H), 4.92 (d, J = 7.8 Hz, 1H, 1 '''- H), 6.83 (d, J = 9.0 Hz, 2H, 2 × Ar -H), 7.01 (dd, J = 9.0, 2.2 Hz, 1H, 6'-H), 7.12 (d, J = 2.0 Hz, 1H, 3'-H), 7.28 (d, J = 2.2 Hz, 1H , 4'-H), 7:45 to 7:48 (m, 2H, 7'-H, 8-H), 7:54 to 7:56 (m, 2H, 3 ^S -NH, 8 ^# -H), 7.65 (d, J = 9.0 Hz, 2H, 2 × Ar-H), 7.89 (d, J = 8.5 Hz, 1H, 9-H), 7.96 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 8.18 (s, 1H, 12-NH), 8.2 0-8.23 (m, 4H, 4-H, 6-H, 2 x Ar-H), 9.64 (s _br , 1H, NH ⁺ ), 11.5 (s, 1H, 1'-NH).
¹³ C-NMR (125 MHz, DMSO-d ₆ ): δ = 23.4 (C-11), 35.5 (C-2 ^S ), 38.7 (C-3 ^S ), 40.1 (12 ^# -NMe ₂ ), 42.5 ( C-12), 42.9 (2 "-NMe ₂ ), 46.0 (C-1), 52.0 (C-2), 55.7 (C-2"), 59.5 (C-6 "'), 61.4 ( C-10), 62.7 (C-1 ''), 67.5 (C-4 '''), 70.5 (C-2'''), 73.1 (C-3 '''), 75.2 (C-5'''), 101.8 (C-9b), 102.1 (C-1 '''), 104.0 (C-4'), 105.4 (C-3 '), 112.1 (2 × Ar-C), 113.3 (C-) 7 '), 114.6 (CF ₃ CO ₂ ), 115.6 (C-6'), 118.9 (C-5a), 121.4 (C-8 ^# ), 121.7 (C-4, C-6), 122.7 (C-). 7), 123.3 (C-9), 125.5 (2 × Ar-C), 126.1 (2 × Ar-C), 127.4 (C-8), 127.4 (2 × Ar-C), 128.5 (C-4 ^# ), 130.2 (C-9 ^# ), 131.2 (C-9a), 132.0 (C-3a '), 134.7 (C-7a'), 141.1 (C-2 ', C-1 ^{#) ,} 141.6 (C-) 3a), 150.1 (C-7 ^# ), 152.0 (C-12 ^# ), 152.7 (C-5 '), 153.4 (C-5), 157.5 (C-5 ^# , 1'-C = O), 160.0 (CF ₃ CO ₂ ), 169.6 (2S-C = O).
MS (ESI): m / z (%) = 1066.4 (100) [M-CF ₃ CO ₂ ] ⁺ , 533.7 (80) [M-CF ₃ CO ₂ + H] ²⁺ , 452.7 (41) CF ₃ CO ₂ -C ₆ H ₁₁ O ₅ + H] ²⁺ C ₅₆ H ₆₁ ClF ₃ N ₇ O ₁₄ S (1180.64). calc .: 1066.3782 Found .: 1066.3778, [M-CF ₃ CO _2] ⁺ (ESI-HRMS).

Example 2

Conversion of the prodrug compounds into seco-drug compounds using the example of 3- [4- (5- (4-dimethylaminophenyl) oxazol-2-yl) benzenesulfonylamino] -propionic acid - [(1S, 10R) -1- (10-chloro ethyl) -3 - [(5- (2- (N, N-dimethylamino) ethoxy) indol-2-yl) carbonyl] -5-hydroxy-1,2-dihydro-3H-benz [e] indol-7 -ylmethyl] amide hydrochloride, (1S, 10R) -5

A solution of the fluorescently labeled prodrug (-) - (1S, 10R) -4 (5.0 mg, 3.9 μmol) in methanol (3 mL) was treated with conc. HCl (0.3 mL) and the reaction mixture stirred for 3 days at room temperature. The solvent was removed under reduced pressure, the residue was dissolved in methanol and the product was separated from the educt by preparative HPLC. The remaining starting material was dissolved in methanol (4 ml), treated with conc. HCl (4 mL) and stirred for 4 days at room temperature. The solvent was removed and again the remaining starting material was separated from the product by preparative HPLC. Combination of the product fractions yielded the target molecule (1S, 10R) -5 (2.6 mg, 2.7 μmol, 69%) as a yellow solid.
HPLC (preparative): Pillar: Kromasil 100 C18 Gradient: Time [min] H ₂ O (0.06% conc. HCl) / MeOH 0-20 50/50 → 25/75 20-23 0/100 23-25 0/100 → 50/50 25-30 50/50 River: 18 ml min ^-1 t _R : 15 minutes Fraction: 14.2-17.0 min ¹ H-NMR (600 MHz, DMSO-d _6): δ = 1.61 (d, J = 6.7 Hz, 3H, 11-H _3), 2:37 (t, J = 7.2 Hz, 2H, ^S 2 -H ₂₎ , 2.88 (d, J = 5.0 Hz, 6H, 2 "- NMe ₂ ), 2.98 (s, 6H, 12 ^# -NMe ₂ ), 3.07 (dt, J = 7.2, 6.0 Hz, 2H, 3 ^S -H _2), 3:54 (m _c, 2H, 2 '' - H _2), 4.14 (m _c, 1H, 1H), 4:33 to 4:41 (m _c, 4H, 1 '' - H _2, 12-H ₂ ), 4:56 (dd, J = 11.0, 2.0 Hz, 1 H, 2-Ha), 4.68-4.76 (m, 2H, 2H _b, 10-H), 6.83 (d, J = 8.8 Hz, 2 H, 2 × Ar-H), 7.00 (dd, J = 9.0, 2.4 Hz, 1H, 6'-H), 7.16 (d, J = 1.8 Hz, 1H, 3'-H), 7.26 (d, J = 2.4 Hz , 1H, 4'-H), 7.39 (dd, J = 8.5, 1.4 Hz, 1H, 8-H), 7.44 (d, J = 9.0 Hz, 1H, 7'-H), 7.62 (s, 1H, 8 ^# -H), 7.67 (d, J = 8.8 Hz, 2H, 2 × Ar-H), 7.79 (t, J = 6.0 Hz, 1H, 3 ^S -NH), 7.84 (d, J = 8.5 Hz, 1H, 9-H), 7.94-7.99 (m, 4H, 4-H, 6-H, 2 x Ar-H), 8.23 (d, J = 8.4 Hz, 2H, 2 x Ar-H), 8.45 ( t, J = 6.0 Hz, 1H, 12-NH), 10.2, 10.3 (2 x s _br , 2H, OH, NH ⁺ ), 11.6 (s, 1H, 1'-NH).
MS (ESI): m / z (%) = 904.3 (100) [M-Cl] ⁺ . C ₄₈ H ₅₁ Cl ₂ N ₇ O ₇ S (940.93). calc .: 904.3254 Found: 904.3250, [M-Cl] ⁺ (ESI-HRMS).

Example 3

Determination of the absorption spectra of the compounds according to the invention when interacting with DNA

1 aliquot of the stock solution (c = 10 mM) of each substance to be tested ((1S, 10R) -4 or (1S, 10R) -5 in DMSO (2 μL, 20 nmol) was mixed with a solution of cellular or plasmid DNA in bidistilled The concentration of cellular DNA was 30 μg / mL, 15 μg / mL or 7.5 μg / mL and the concentration of plasmid DNA 32 μg / mL 1 aliquot (200 μL). The reaction solution was immediately frozen and the other aliquot (200 μL) was incubated for 24 h at 25 ° C. and then frozen in. For comparison, the solutions of pure cellular or plasmid DNA with DMSO (200 μL, 1% DMSO, concentration of cellular DNA: 30 μg / mL, 15 μg / mL or 7.5 μg / mL, Concentration of plasmid DNA: 32 μg / mL). The results for compound 5 are in the 3 shown. Significantly, a decrease in the emission at 560 nm after excitation at 525 nm can be seen.

Example 4

Determination of the competitive interaction of the compounds of the invention with known DNA dyes

The solutions investigated according to Example 3 were diluted with water (800 μL) and mixed with an aliquot of a solution of the nuclear dye Hoechst 33342 in water (1 μL, concentration of the stock solution: 18 μM, 18 pmol) (c _final = 18 nM). The solution was mixed briefly with the vortex apparatus, pipetted into a 1 cm cuvette with stirrer and immediately (1-2 min after the batch) with constant stirring the temporal change of the fluorescence emission at λ _em = 500 nm and λ _em = 560 nm for an excitation at λ _exc = 365 nm, 405 nm and 514 nm recorded.
Parameter: Exc. 365 nm: Em1: 500 nm, Em2: 560 nm; Exc. 405 nm: Em1: 500 nm, Em2: 560 nm; Exc. 514 nm: Em1: 500 nm, Em2: 560 nm; Measurement: 1 sec, every 60 sec; Measuring time: 3661 sec; Integration: 1 sec.

Like in the In a solution of untreated DNA in water, the characteristic fluorescence for the highest dye increased to twice the initial intensity. When the DNA solution was previously incubated with Compound 5 for five hours, the increase in intensity was slower and did not reach the same intensity. In a 24-hour preincubation only a very small increase could be detected.

Example 5

Differentiated staining of dead and living cells with the compounds according to the invention

As a cell line, 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 mM NaHCO ₃ and 4 mM glutamine) in a concentration of 25 × 10 ³ cells in 500 μL of medium per chamber of the coverslip (Nunc ^® Lab-Tek ^® Chamber Slide with 4 wells on Permanox ^® ). The cells were adhered for 24 h at 37 ° C and 7.5% CO ₂ . Alternatively, the cells were seeded at a concentration of 5 x 10 ³ cells in 500 μL of media per well of the coverslip and adhered for 3 days at 37 ° C and 7.5% CO ₂ .

The prepared cells were washed (2 x 1 mL) and treated with a solution of the test substance in DMSO and Ultraculture ^® medium (500 .mu.l, 1% DMSO) or with a solution of DMSO in Ultraculture ^® medium (500 with serum-free Ultra Culture ^® medium μL, 1% DMSO). The concentration of the compounds in the incubation solution was 10 μM ((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 (2 x 1 mL) and examined in the imaging buffer by means of a confocal fluorescence microscope.

In the 5 the results for Compound 5 are shown. With the help of a trypan blue staining two cell populations could be distinguished (not shown):
The first cell type were dead cells that could be stained with trypan blue. They showed a blue-green fluorescence emission only when excitation with laser light of wavelength λ _exc = 405 nm, which was quite diffuse visible in the cytosol of the cells, but also in small round structures ( ). These cells appeared bluish in transmitted light, had a reddish cell nucleus and tended to form membrane protrusions. Cells of the second cell type were vital and appeared brownish in transmitted light. Here, excitation with λ _exc = 405 nm _{resulted in} a blue-green fluorescence emission with a maximum at λ _em = 500 nm and at λ _exc = 514 nm a yellow-green fluorescence emission with a maximum at λ _em = 560 nm ( ). So a distinction between dead and living cells is possible.

Multiple staining with Hoechst 33342, Mito-Tracker and compound 5

To clarify the question of whether it is actually a question of mitochondria in the elongated organelles = showed a yellow-green fluorescence emission of λ _em 560 nm, was a co-Lolokalisationsexperiment with the mitochondria-selective dye MitoTracker ^® Deep Red (9) of the Company Invitrogen performed. Due to its strongly red-shifted fluorescence emission, this not only allows simultaneous observation with the seco-Drug (1S, 10R) -5, which is in the blue-green and yellow-green range Fluorescent, the blue fluorescent Hoechst and the red fluorescent trypan blue, but also the detection of vital cells.

To stain the cell nuclei, a solution of the dye Hoechst 33342 in buffer (1 μL, concentration of the stock solution: 36 mM, 36 nmol) was mixed with the imaging buffer in the chambers (c _final = 72 μM) and the cells for a few minutes before observation Room temperature incubated.

To stain dead cells and to stain the cell membranes, a solution of the dye trypan blue in physiological buffer (5-25 μL) was mixed with the imaging buffer in the chambers and the cells incubated for a few minutes at room temperature before observation.

For co-localization of the seco-drug (1S, 10R) -5 (incubation: 2-10 μM, 30 min to 2.5 h) with the mitochondrial dye Mito-Tracker, a solution of the Mito-Tracker in DMSO (1-1.25 μL, concentration of the stock solution: 10 μM) was mixed with the imaging buffer in the chambers (c _final = 20-25 nM) and the cells were incubated for 30 min-1.5 h at room temperature before observation.

In the 6 the results are shown.

The co-localization experiments with the MitoTracker ^® clearly show that the seco-Drug (1S, 10R) -5 was enriched in the mitochondria of living cells ( and , upper 2/3 of the cells, 5: green, mito-tracker (9): shown in red, , Superposition of 5 and 9: shown in yellow). In these living cells, trypan blue ( , shown in light blue) only the cell membranes stained. In the mitochondria by trypan blue clearly cytosol-stained dead cells ( -E, lower 1/3 of the cells) 9 and 5 could instead be detected only with very weak intensities.

Remarkably, an increasing proportion of cells showed signs of apoptosis a few hours after a seco-drug incubation ( ). Membrane protuberances formed and collapsed membrane potential in the mitochondria of affected cells, so that the mitochondria of these cells could no longer be stained by the MitoTracker ^® 9.

The blue-green fluorescence of the seco drug (1S, 10R) -5 ( , shown in blue) could be detected in cytosolic organelles of living and dead cells, especially near the nucleus. Since the fluorescence of the DNA dye Hoechst 33342 (8) was excited and detected at wavelengths similar to the blue-green fluorescence of the seco drug, staining of the cell nuclei by 8 ( , blue) can be distinguished only by the localization of the staining in the nucleus of the blue-green fluorescence of the seco-drug in the cytosolic organelles.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 02/059122 [0004]
• WO 98/11101 [0004, 0005]
• WO 97/12862 [0004]
• WO 01/83448 [0004, 0005, 0005, 0009]
• WO 2007/089149 [0004, 0004, 0005, 0005, 0009]

Cited non-patent literature

• The Handbook, A Guide to Fluorescent Probes and Labeling Technologies, Molecular Probes - Invitrogen, 10th Edition [0003]

Claims (14)

Compounds of the general formula I or II:

wherein R ^{1 is} selected from a halogen and OSO ₂ R ^u , wherein R ^{u is} selected from optionally substituted C ₁ -C ₆ alkyl, C _1-6 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 linked 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 C _1-8 alkyl or C _1-8 acyl and where R ^{14 '} is absent is or is selected from hydrogen and optionally substituted C _1-8 alkyl or C _1-8 acyl; R ⁵ and 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, halogen, 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 (O) OR ^k , C (O) N (R ^L ) R ^k , OC (O) R ^k , OC (O) R ^k , OC (O) OR ^k , OC (O) N (R ^k ) R ^L , N (R ^L ) C (O) R ^k , N (R ^L ) C (O) OR ^k , and N (R ^L ) C (O) N (R ^m ) R ^k , where R ^k , R ^L and R ^{m are} independently selected from H and optionally substituted C _1-4 alkyl, C _1-4 heteroalkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, C _4-12 aryl or C _4-12 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; and / or 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' ; two or more of 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; and / or R ⁵ + R ^{5 '} , and / or R ⁶ + R ^6' and / or R ⁷ + R ^{7 '} are independently = O, = S or = NR ¹² , wherein R ^{12 is} selected from H and optionally substituted C _1-6 alkyl; with the proviso that either R ⁶ and / or R ⁷ independently represents a group (C (R _CH ) ₂ ) _1-6 -X ₇ chromophore wherein X _{7 is} selected from C = O, O, NR _CH wherein R _{CH is} selected from hydrogen and optionally substituted C _1-8 alkyl or C _1-8 acyl; X ₆ is selected from C or N; R _DB is selected from hydrogen and optionally substituted C _1-8 alkyl or C _1-8 acyl; X ₁ is selected from O, S, and NR ¹³ , wherein R ^{13 is} selected from H and optionally substituted C _1-8 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 III

wherein X ^{3 is} selected from O, S, and NR ¹⁵ , wherein R ^{15 is} selected from H and optionally substituted C _1-8 alkyl or C _1-8 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 C _1-8 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 C _1-8 alkyl or C _1-8 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 , OR ^x , NHR ^x , N (R ^x ) R ^y , ⁺ N (R ^x ) (R ^y ) R ^z , P (O) (OR ^x ) (OR ^y ), OP (O) (OR ^x ) (OR ^y ), SiR ^x R ^y R ^z , C (O) R, C (O) OR ^x , C ( O) N (R ^y ) R ^x , OC (O) R ^x , OC (O) OR ^x , OC (O) N (R ^x ) R ^y , N (R ^y ) C (O) R ^x , N ( R ^y ) C (O) OR ^x , N (R ^y ) C (O) N (R ^z ) R ^x , and a water group wherein R ^x , R ^y , and R ^{z are} independently selected from H and optionally substituted C _1-15 alkyl, C _1-15 heteroalkyl, C _3-15 cycloalkyl, C _3-15 heterocycloalkyl, C _4-15 aryl, or C _4-15 heteroaryl radicals wherein one or more of the optional substituents in R ^x , R ^y , and R ^{z is} optionally a water-soluble group, and two or more of R ^x , R ^y , and R ^z may be optionally linked to one or more optionally substituted carbon cycles Heterocycles and at least one of R ⁸ , R ⁹ , R ¹⁰ , and R ¹¹ comprises a water-soluble group, two or more of R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , or X ^3b are optionally joined together to form one or more optionally substituted aliphatic or aromatic carbon cycles or heterocycles; or pharmaceutically acceptable salts or solvates thereof. A compound according to claim 1, wherein R is selected from a substrate represented by proteolytic enzymes, plasmin, cathepsin, cathepsin B, beta-glucuronidase, galactosidase, mannosidase, glucosidase, neuramidase, sucrose, 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. A compound according to claim 1 or 2, wherein R is selected from a monosaccharide, disaccharide or oligosaccharide, especially hexoses, pentoses or heptoses optionally as a deoxy derivative or amino derivative and optionally substituted with halogen, C _1-8 alkyl, C _1-8 Acyl, C _1-8 heteroalkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, C _4-12 aryl or C _4-12 heteroaryl, amino or amide groups, or with amino, amido or carboxy moieties optionally substituted may be substituted with halogen, C _1-8 alkyl, C _1-8 acyl, C _1-8 heteroalkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, C _4-12 aryl or C _4-12 heteroaryl, amino or amide radicals; Dextran, dipeptide, tripeptide, tetrapeptide, oligopeptide, peptidomimetics, or an antibody, or combinations thereof. Compounds according to any one of the preceding claims, which is one of the general formulas I, wherein DB is selected from a radical of general formula III, wherein R ⁸ , R ⁹ , R ¹⁰ , or R ^{11 are} 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) ethoxy or hydrogen and the chromophore present in R ⁶ and / or R ⁷ is a dapoxyl derivative. A compound according to any one of claims 1 to 3, which is a compound of general formula II, wherein the chromophore of R ⁶ and / or R ^{7 is} a dapoxyl derivative and wherein DB is a radical of general formula III with R ⁸ , R ⁹ , R ¹⁰ , or R ¹¹ 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) ethoxy or hydrogen. A compound according to any preceding claim, wherein these are:

A compound according to any one of the preceding claims, wherein R ⁶ and / or R ⁷ , when they are a chromophore-containing group, are selected from dapoxyl-3-sulfonamidopropionate, dapoxyl-3 Sulfonamidobutyrate, dapoxyl-3-sulfonamides, 4 ', 6-diamidino-2-phenylindole, bisbenzimide, 2,5'-Bi-1H-benzimidazoles, 2' - (4-ethoxyphenyl) -5- (4-methyl-1) piperazinyl). Use of a compound according to any one of claims 1 to 7 as a fluorescent dye, in particular as a marker in imaging methods. Use of a compound according to any one of claims 1 to 7 in combination with at least one second dye in fluorescence-based methods. Use of a compound according to any one of claims 1 to 7 in fluorescence-based methods in the dual-exitation and dual-emission modes. Use of a compound according to any one of claims 1 to 7 for distinguishing living, apoptotic and dead cells and for cell sorting and cell separation. Use of a compound according to any one of claims 1 to 7 for the selective labeling of organelles in cells, in particular organelles of endo- and exocytosis, preferably lysosomes, endosomes; as well as mitochondria. Use of a compound according to any one of claims 1 to 7 in the imaging diagnosis, prognosis, course and drug effectiveness determination of mitochondrial-associated diseases or drug therapies. Use of a compound according to one of claims 1 to 7 for the qualitative and quantitative determination of nucleic acids, in particular double-stranded nucleic acids.

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