WO2011051484A1

WO2011051484A1 - Fluorescence dyes

Info

Publication number: WO2011051484A1
Application number: PCT/EP2010/066548
Authority: WO
Inventors: Lutz Tietze; Birgit Krewer; Miso Mitkovski
Original assignee: Georg-August-Universität Göttingen Stiftung Öffenlichen Rechts
Priority date: 2009-10-30
Filing date: 2010-10-30
Publication date: 2011-05-05
Also published as: DE102009051438A1

Abstract

The present invention relates to novel compounds suited as fluorescence dyes. The present invention in particular relates to a novel class of pharmacologically active compounds that are derivatized with chromophores. The present invention further relates to applications of the compounds according to the invention.

Description

FLUORESCENT DYES

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 in the field of life sciences. A large number of dyes, in particular fluorescent dyes, have already been described which are used in imaging methods and for the representation of structures, for the labeling of molecules, etc. become.

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.

In WO2009 / 017394 further substituted CC-1065 analogues and corresponding conjugates are described. These are further compounds which are very similar to the molecules described in WO2007 / 089149.

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 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. It has long been known to use corresponding 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. Such 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.

For marking individual areas of 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. Alternatively, there are 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

Compounds, in particular fluorescent dyes, for marking cell structures, cell changes and cell states. Description of the present invention

In a first aspect, compounds of the general formulas I or I I are provided:

with 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 (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 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;

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 the radicals R ^3, R ^4, DB, R ^8, R ^9, R ^10, R ^11, R ^6, and / or R ^7, in particular, either R ⁶ and / or R ^7, independently vonei- Nander represents a group- (C (R _C H) 2) i -6-X7-chromophore,

wherein X _{7 is} selected from C = O, O, NR _C H, wherein RCH is selected from hydrogen and optionally substituted _C _1-8 alkyl or _C _1-8 acyl; 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 π-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

wherein 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 , 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 ^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-soluble group, wherein R ^x, R ^y, and R ^z are independently selected from H and optionally substituted Ci 1 5 alkyl, Ci 1 5 heteroalkyl, C3-15 cycloalkyl, C ₃ 1 5 heterocycloalkyl , C ₄ i ₅ aryl, or C ₅ heteroaryl _4- i

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 ones to form carbon rings or heterocycles, and 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;

or pharmaceutically acceptable salts or solvates thereof.

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.

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 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.

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 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. Thus, with the aid of 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. 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 according to 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 simply and directionally introduced into cells. to be brought. 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 e.g. 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 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.

According to the invention, 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 π-electrons. More preferably, the chromophoric unit is not connected via conjugated ττ electrons with the other units of the compounds of the invention. That is, there is no conjugated π-electron system between the chromophore moiety, such as R ⁶ or R ⁷ , and the tricyclic center, usually the pharmacophore region.

In a preferred embodiment, compounds of general formulas I and II are provided which have a dapoxyl chromophore. That is, as chromophore according to formula I or II is a Dapoxylrest, which may optionally be derivatized before. 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. 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.

In a preferred embodiment, R is hydrogen.

In another preferred embodiment, the

Substrate R to a cleavable substrate. This 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.

It is preferable that the 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. s acyl, 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.

With the aid of the substrate R, targeting of the compounds according to the invention to target structures is possible. That is, a targeted coupling of the compounds according to the invention to target structures and a corresponding incorporation of these compounds according to the invention into, for example, selected cells and cell types is 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 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).

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 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-

(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 present in R ⁶ and / or R ⁷ chromophore is a dapoxyl derivative.

In a further preferred embodiment, 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) ethoxy or hydrogen.

Particularly preferred compounds are those 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 found 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 colourfulness, but 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 , OPCOXOR ^ OR ¹ ), PiOXOR ^ OR ¹ ), OR ^h , NHR ', N (R ^h ) R ', ⁺ N (R ^h ) (R') R ^j , SR ^ R ¹ ) ^, SR ^ XR ¹ ), C (O) R ^h , C (O) OR ^h , C (O) N (R ⁱ ) R ^h , OC (O) R ^h , OC (O) OR ^h , OCCOJNiR ^ R ¹ , N (R ⁱ ) C (O) R ^h , N (R ⁱ ) C (O) OR ^h , N (R ') C (O) N (R ^j ) R ^h , and the thio derivatives of these substituents, or a protonated or deprotonated form of these substituents, wherein R ^h , R ¹ , and R ^{j are} independently selected from H and optionally substituted Ci-15 alkyl, Ci-15 heteroalkyl, C3-15 cycloalk yl, C3-15 heterocycloalkyl, C ₄ is aryl, or C -i thereof ₅ heteroaryl or a combination of two or more of R ^h, R ', and R ^j are optionally joined together to form one or more carbon rings or heterocycles ,

The term "alkyl" as used herein 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.

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 fused rings, and examples of aryl include phenyl, naphthyl and antracenyl.

The term "acyl" as used herein refers to the functional group having the general structure R- (C = O) - wherein R represents a carbon radical, in particular a carbon chain having Ci to Cs carbon atoms.

The term "water-soluble group" as used herein 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 Examples of 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.

The term "heteroaryl" as used herein 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. 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, in particular S, N, O or P. 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 moieties unless otherwise stated, that is, the term "optionally substituted alkyl, heteroalkyl, aryl, acyl" is optionally substituted alkyl substituted heteroalkyl, optionally substituted aryl, optionally substituted acyl "to read.

Pharmaceutically acceptable 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.

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

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.

5 shows fluorescence microscopic images of the two cell types which can be distinguished after incubation of the cells (A549) with the seco drug (1S, 10R) -5, a) vital and dead cells, λ _em = 500 nm (FIG. in the original blue); b) vital cells, _em = 560 nm (yellow in original); c) Overlay of emissions at λ _em = 500 nm (blue) and λ _em = 560 nm (yellow).

FIG. 6: FIG. 6 shows a fluorescence micrograph of vital and dead cells (A549) after incubation with (1S, 10R) -5. a) A _exc = 405 nm, λ _em = 460-500 nm, representation in blue: cell nuclei stained by Hoechst 33342 (8) and cytoplasmic organelles stained by 5; b) _{λ exc} = 514 nm, _λ _em = 560 nm, green representation: mitochondria of vital cells stained by ( _1S , 10R) -5 (upper 2/3 of the cells); c) -exc = 633 nm, _em = 680 nm Ä, display in red: by MitoTracker ^® Deep Red (9) stained mitochondria of viable cells (upper 2/3 of the cells); d) _{λ exc} = 561 nm, em = 630 nm, light blue: cell membranes of vital cells stained by trypan blue (upper 2/3 of the cells) and stained cytosol of dead cells (lower 1/3 the cells); e) Overlapping the fluorescence of (1 S, 10R) -5 (green) and MitoTracker Deep Red 9 (red) to yellow; f) Overlay of ad.

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 ITI M 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: y-D-galactosidase (EC 3.2.1 .23) of Escherichia coli G 5635 (Sigma), activity: 250-600 units (units (U)) per mg protein at pH 7.3 and 37 ° C, 1 U = 1 μιτιοΙ 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: 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.

Fluorescent dye of compounds (-) - (1 S, 10 /?) - 4 and (1 S, 10 /?) - 5: Zur

Coupling of the dye Dapoxyl ^® -3-sulfonamidopropionic acid to the free amino functionality in (-) - (1 S, 10R) -1 was the Dapoxyl ^® -3- Sulfonamidpropionsäure succinimidylester D1 01 62 (3) from Invitrogen. Mitochondria-selective fluorescent dye: MitoTracker ^® Deep Red 633 FM (9) from Invitrogen was used to stain the mitochondria of vital cells.

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 μM) 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.

Fluorimetry: A spectrofluorometer Quanta 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 (-) - (1 S, 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 (250 χ 20 mm, particle size: 5 μιτι) were used as well as a prepacked column of the type Kromasil 1 00 C18 (7 μιτι, 250 χ 20 mm) from Jasco, in conjunction with a guard column of the type Kromasil 100 C18 ( 5 μιτι, 50 χ 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)

1 aliquot of the stock solution (c = 1 ITIM) 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 pipetted into a quartz cuvette. With a fluorimeter, the fluorescence intensity of the emissions was measured at em = 500 nm and λ _em = 560 nm for an excitation wavelength range of λ exc = 200-600 nm.

Parameters: Excitation scan, digital cfg, Exe: 200-600 nm, length: 400 nm, Em 1: 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 ITI M) of the substance to be examined in DMSO (2 .mu.mol, 2 nmol) was dissolved in the chosen solvent (200 .mu.l, solvent: methanol, imaging buffer or water), the solution mixed with a vortexer and a cuvette is pipetted. The fluorescence intensity was measured with a fluorimeter using excitation with laser light of the _{wavelength λ exc} = 350 nm (or 378 nm, 405 nm or 514 nm) for an emission range of em = 400-800 nm.

Parameters: 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.

example 1

Synthesis of the compounds of the invention The compounds of the invention were synthesized according to the scheme shown in FIG.

Synthesis of (-) - {3- [4- (5- (4-Dimethylaminophenyl) oxazol-2-yl) benzenesulfonylamino] propionic acid - [(1S, 10?) - 1- (10-chloro-ethyl) - 3 - [(5- (2- (W, W-dimethylamino) ethoxy) indol-2-yl) carbonyl] -5-0- ^ -D-galactopyranosyl-1,2-dihydro-3H-benz [e ] indol-7-ylmethyl] amide trifluoroacetate}, (-) - (1S, 10R) -4

To a solution of the NHS ester (3) (12.3 mg, 24.0 μηηοΙ, 1.0 eq.) In absolute DMF (200 μί) was added a solution of galactoside (-) - (1S, 10R) -2 (25.0 mg, 27.9 μηηοΙ , 1.2 eq.) And / ^' -Pr ₂ NEt (23.0 [iL, 139 μηηοΙ, 5.8 eq.) Are added dropwise in absolute DMF (150 μί). 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 collection of the eluate, removal of the solvents under reduced pressure and removal of the remaining solvent by lyophilization afforded the fluorescently labeled glycosidic prodrug (-) - (1S, 10R) -4 as a yellow solid (27.8 mg, 21.5 μηηοΙ, 89%). , Purification of the prodrug (-) - (1S, 10R) -4 was carried out by preparative HPLC under the following conditions:

HPLC (preparative):

1 H NMR (600 MHz, DMSO-d ₆ , 80 ° C): δ = 1.63 (d, J = 6.6 Hz, 3 H, 11-H ₃ ), 2.40 (t, J = 7.4 Hz, 2 H, 2 ^S -H _2), 2.93 (s, 6 H, 2 "-NMe _2), 2.98 (s, 6 H, 12 ^# -NMe _2), 3.14 (t, J = 7.4 Hz, 2 H, 3 ^S -H ₂ ), 3.48 (dd, J = 9.6, 3.2 Hz, 1 H, 3 "'- H), 3.52-3.57 (m,

3 H, 5 "'- H, 2" - H ₂ ), 3.60 (dd, J = 10.6, 5.5 Hz, 1 H, 6 "' - H _a ), 3.69 (dd, J = 10.6, 7.2 Hz, 1 H, 6 "'- H _b ), 3.81-3.86 (m, 2 H, 2"' - H, 4 "'- H), 4.20 (dt, J = 9.4, 2.6 Hz, 1 H, 1-H) , 4.36 (t, J = 5.0 Hz, 2 H, 1 "-H ₂ ), 4.42 (m _c , 2 H, 12-H ₂ ), 4.62 (dd, J = 11.0, 2.6 Hz, 1 H, 2 H _a ), 4.70 (dd, J = 11.0, 9.4 Hz, 1 H, 2-H _b ), 4.79 (dq, J = 6.6, 3.0 Hz, 1 H, 10-H), 4.92 (d, J = 7.8 Hz, 1 H, 1 "'- H), 6.83 (d, J = 9.0 Hz, 2 H, 2 Ar-H), 7.01 (dd, J = 9.0, 2.2 Hz, 1 H, 6'-H), 7.12 (d, J = 2.0Hz, 1H, 3'-H), 7.28 (d, J = 2.2Hz, 1H, 4'-H), 7.45-7.48 (m, 2H, 7'-H, 8-H), 7.54-7.56 (m, 2 H, 3 ^S -NH, 8 ^# -H), 7.65 (d, J = 9.0 Hz, 2 H, 2 Ar-H), 7.89 (d, J = 8.5 Hz, 1 H, 9-H), 7.96 (d, J = 8.4 Hz, 2 H, 2 Ar-H), 8.18 (s, 1 H, 12-NH), 8.20-8.23 (m,

4 H, 4-H, 6-H, 2 x Ar-H), 9.64 (s _br , 1 H, NH ⁺ ), 11.5 (s, 1 H, 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 ^m), 61.4 (C-10) 62.7 (C-1 "), 67.5 (C-4 ^m), 70.5 (C-2 '"), 73.1 (C-3 ^m), 75.2 (C-5''), 101.8 (C-9b), 102.1 (C-1 '''), 104.0 (C-4'), 105.4 (C-3 '), 112.1 (2 x 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 χ Ar-C), 126.1 (2 χ Ar-C), 127.4 (C-8), 127.4 (2 χ 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 (2 ^sec . 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) [M- CF ₃ CO ₂ -C ₆ HnO ₅ + H] ²⁺ .

C56H61CIF3N7O14S (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, 10?) - 1- (10 chloroethyl) -3 - [(5- (2- (W, W-dimethylamino) ethoxy) indol-2-yl) carbonyl] -5-hydroxy-1,2-dihydro-3H-benz [e] indole 7-ylmethyl] amide hydrochloride, (1S, 10 /?) - 5 A solution of the fluorescently labeled prodrug (-) - (1 S, 10R) -4 (5.0 mg, 3.9 μιτιοΙ) 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 μηηοΙ, 69%) as a yellow solid.

HPLC (preparative):

1H-NMR (600 MHz, DMSO-d _6): 5 = 1 .61 (d, J = 6.7 Hz, 3 H, 1 1 H _3), 2:37 (t, J = 7.2 Hz, 2 H, 2 ^S -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, 2 H, 3 ^S -H _2), 3:54 (m _c, 2 H, 2 "-H _2), 4.14 (m _c, 1 H, 1 H), 4:33 to 4:41 (m _c, 4 H, 1" - H ₂ , 1 2-H ₂ ), 4.56 (dd, J = 1 1 .0, 2.0 Hz, 1 H, 2-H _a ), 4.68-4.76 (m, 2 H, 2-H _b , 1 0- H), 6.83 (d, J = 8.8 Hz, 2 H, 2 Ar-H), 7.00 (dd, J = 9.0, 2.4 Hz, 1 H, 6'-H), 7.16 (d, J = 1 .8 Hz, 1 H, 3'-H), 7.26 (d, J = 2.4 Hz, 1 H, 4'-H), 7.39 (dd, J = 8.5, 1 .4 Hz, 1 H, 8-H), 7.44 (d, J = 9.0 Hz, 1 H, 7'-H), 7.62 (s, 1 H, 8 ^# -H), 7.67 (d, J = 8.8 Hz, 2 H, 2 Ar-H), 7.79 (t, J = 6.0 Hz, 1 H, 3 ^S -NH), 7.84 (d, J = 8.5 Hz, 1 H, 9-H), 7.94-7.99 (m, 4 H, 4-H, 6-H, 2 Ar-H), 8.23 (d, J = 8.4 Hz, 2 H, 2 Ar-H), 8.45 (t, J = 6.0 Hz, 1 H, 1 2 -NH), 10.2, 10.3 ( 2s _br , 2H, OH, NH ⁺ ), 1 1 .6 (s, 1H, 1'-NH).

MS (ESI): m / z (%) = 904.3 (100) [M-Cl] ⁺ .

C ₄ 8H5i CI ₂ N ₇ 0 ₇ S (940.93). calc .: 904.3254

gef. : 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 ΠΓΙ Μ) of each substance to be tested ((1 S, 10R) -4 or (1 S, 10R) -5 in DMSO (2 μΙ_, 20 nmol) was mixed with a solution of cellular or plasmid DNA in bidistilled water (400 μΙ_) or double-distilled water (400 μΙ_) The concentration of cellular DNA was 30 pg / mL, 1 5 g / mL or 7.5 g / mL and the concentration of plasmid DNA 32 g / mL 1 aliquot (200 μΙ_) of the reaction solution was immediately deep-frozen and the other aliquot (200 μΙ_) 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 μί, 1% DMSO, Concentration of cellular DNA: 30 g / mL, 15 g / mL or 7.5 g / mL, concentration of plasmid DNA: 32 pg / mL) The results for Compound 5 are shown in Figure 3. A decrease is clear to detect the emission at 560nm after stimulation at 525nm.

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 μ ί) and mixed with an aliquot of a solution of the nuclear dye Hoechst 33342 in water (1 μ ί, concentration of the stock solution: 1 8 μ Μ, 18 pmol) (Cfinai = 18 nM). The solution was mixed briefly with the vortexer, pipetted into a 1 cm cuvette with stirrer and immediately (1 -2 min after the batch) with constant stirring the time change of fluorescence emission at λ _em = 500 nm and λ _em = 560 nm for an excitation at lexc = 365 nm, 405 nm and 514 nm recorded. Parameter: Exc. 365 nm: 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; Measurement: 1 sec, every 60 sec; Measuring time: 3661 sec; Integration: 1 sec.

As shown in Figure 4, 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. With 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 μl M NaHCO ₃ and 4 μl M Glutamine) in a concentration of 25 × 10 ³ cells in 500 μl 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 . Alternatively, the cells were seeded in a concentration of 5 × 10 ³ cells in 500 μ ί 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 μΙ_, 1% DMSO) or with a solution of DMSO (in Ultraculture ^® medium 500 μΙ_, 1% DMSO). The concentration of the compounds in the incubation solution was 10 μΜ ((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. With Using 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 the wavelength _{λ exc} = 405 nm, which was quite diffuse visible in the cytosol of the cells, but also in small round structures (Figure 5a). 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 _e xc = 405 nm resulted in a blue-green fluorescence emission with a maximum at λ _em = 500 nm and at / texc = 514 nm a yellow-green fluorescence emission with a maximum at em = 560 nm (FIG. 5 b). So a distinction between dead and living cells is possible.

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

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

To stain the cell nuclei was a solution of the dye

Hoechst 33342 in buffer (1 μ ί, concentration of the stock solution: 36 ITI M, 36 nmol) mixed with the imaging buffer in the chambers (Cfinai = 72 μ Μ) and the cells incubated for several minutes at room temperature before observation.

For staining dead cells and staining the cell membranes, a solution of the dye trypan blue in physiological buffer (5-25 μ ί) was mixed with the imaging buffer in the chambers and the cells were placed in front of the cells Observation incubated for a few minutes at room temperature.

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

FIG. 6 shows the results.

The co-localization experiments with the MitoTracker ^® clearly show that the seco-Drug (1 S, 10R) -5 was enriched in the mitochondria of living cells (Fig. 6b and Fig. 6c, upper 2/3 of the cells, 5: green shown, Mito-Tracker (9): shown in red, Fig. 6e, superposition of 5 and 9: shown in yellow). In these living cells, only the cell membranes were stained by trypan blue (Figure 6d, shown in light blue). In the mitochondria, trypan blue clearly stained cells in the cytosol (Fig. 6b-e, bottom 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 (Figure 6d). 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 (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.

Claims

claims

1 . Compounds of the general formula I or I I:

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 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, lk, 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 , N HR ^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 is selected from H and optionally substitu ierte Ci _-4 alkyl, Ci _-4 heteroalkyl, C _3- 7 cycloalkyl, C _3- 7 heterocycloalkyl, C ₄ i are independently selected from aryl or C ₂ _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;

and / or R ^{5 '} and R ^6' and / or R ^{6 '} and R ^7' and / or R ^{7 '} and R ^14' are not present, ie. they form a double 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 substitu ierte 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 the radicals R ^3, R ^4, DB, R ^8, R ^9, R ^10, R ^{1 1,} R ^6, and / or R ^7, in particular, either R ⁶ and / or R ^7, independently vonei- nander represents a group- (C (R _C H) 2) i -6-X7-chromophore,

wherein X _{7 is} selected from C = O, O, NR _C H, wherein RCH is selected from hydrogen and optionally substituted Ci _-8 alkyl or Ci _-8 acyl; Chromophore is a unit which has a color in the visible or invisible region, preferably in the visible region, optionally after excitation, in particular all colors except white in the visible range;

Χβ 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 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;

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 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 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 d _-8 alkyl or Ci _-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 ^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-soluble group, wherein R ^x, R ^y, and R ^z are independently selected from H and optionally substituted alkyl OM S, S CM heteroalkyl, C _3- 15 cycloalkyl, 03-15 heterocycloalkyl, C ₄ is 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 can optionally, to form one or more optionally substituted carbon cycles or 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 linked to one or more optionally substituted al to form iphatic 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 sen from a monosaccharide, disaccharide or oligosaccharide, in particular hexose, pentoses or heptoses optionally as a deoxy derivative or amino derivative, and optionally substituted with halogen, Ci _-8 alkyl, Ci-8 acyl, Ci -8 heteroalkyl, C3-7 cycloalkyl, C3-7 heterocycloalkyl, C _4- 12 aryl or C ₂ -i heteroaryl, amino or amide groups, or with amino, Amido, or carboxyl moieties, which may be optionally substituted with halogen, alkyl ds, ds acyl, ds heteroalkyl, d-7 cycloalkyl, C _3- 7 heterocycloalkyl, C 4-12 aryl or C 12 heteroaryl, amino or amide groups; Dextran, dipeptide, tripeptide, tetrapeptide, oligopeptide, peptidomimetics, or an antibody, or combinations thereof.

Compounds according to one of the preceding claims, wherein said one of the general formulas I, where DB is selected from a radical of general formula II I, wherein 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- (N, N-dimethylamino) ethoxy. methylamino) acetylamino, 2-aminoethoxy, 2-aminoacetylamino, (piperidin-4-yl) methoxy, 2- (N-methyl-N-

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 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) ethoxy or hydrogen.

6. A compound according to any one of the preceding claims, wherein these are: 32

7. A compound according to any one of the preceding claims, wherein R ⁶ and / or R ⁷ , if they represent a chromophore-containing group, are selected from dapoxyl-3-sulfonamide propionate, Dapoxyl-3-sulfonamidobutyrate, Dapoxyl-3-sulfonamides, 4 ' , 6-diamidino-2-phenylindole, bisbenzimide, 2,5'-bi-1H-benzimidazole, 2 '- (4-ethoxyphenyl) -5- (4-methyl-1-piperazinyl).

8. 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.

9. 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.

10. Use of a compound according to any one of claims 1 to 7 in fluorescence oreszenz-based methods in the dual exitation and dual emission mode.

1 1.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.

12. 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.

13. 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 therapies with drugs.

14. 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.