DE10302787A1 - New indotricarbocyanine dyes are conjugated to biomolecules, used in fluorescence diagnosis of e.g. tumors - Google Patents

Abstract

Indotricarbocyanine dyes (I) are new. Indotricarbocyanine dyes of formula (I) and their salts and solvates are new. [Image] X : O, S or C (di-substituted by methyl, ethyl, propyl, isopropyl and/or butyl); Y' : CH 2-CH 2 or CH 2-CH 2-CH 2; Z' : 1-5C alkyl (substituted by O or S) or -(1,4-phenylene)-O-; R 1- R 4SO 3H or H; R 5-CO-NH-R 8-R 9, -NH-CS-NH-R 8-R 9, -NH-CO-R 8-R 9, COOH or NH 2; R 8unbranched 2-13C alkyl (having C optionally replaced by O or S); R 9-NH-C(=O)-CH 2-Br, 2,5-dioxo-pyrrolyl, chloroacetyl, bromoactyl, iodoacetyl, chloroacetamido, iodoacetamido, chloroalkyl, bromoalkyl, iodoalkyl, pyridyl disulfide or vinyl sulfonamide; and R 6, R 7CH or connected to a hexyl ring (optionally substituted in para-position by 1-4C alkyl) by 1-3C alkyl. Provided that: at least three of R 1-R 4 are SO 3H. Independent claims are included for the following: (1) prepararion of (I); (2) a conjugate (II) of (I) with a biomolecule(s); and (3) a diagnostic kit comprising (I) and/or (II) together with additional adjuvants for implementing an in vivo diagnosis of e.g. tumors.

Description

The present invention relates to new fluorescent dyes from the class of cyanine dyes, in particular indotricarbocyanines with an absorption and fluorescence maximum in the spectral range 700 to 900 nm, a thiol-specific reactive Group and three, preferably four sulfonate groups, to increase the Water as well as the production of the dyes. The present invention also affects the conjugates of these dyes with biomolecules and their uses.

background the invention

The use of light in medical Diagnostics has become increasingly important in recent years (see z. B. Biomedical Photonics Handbook (Editor: T. Vo-Dinh), CRC Press). A large number of diagnostic methods are in the experimental Testing for an application in various medical disciplines, e.g. B. the Endoscopy, mammography; Surgery or gynecology. Light-based process have a high instrumental sensitivity and enable the molecular detection and imaging of the smallest amounts of chromophores and / or fluorophores (Weissleder et al. (2001) Molecular Imaging. Radiology 219, 316-333).

Of particular interest for in vivo use are therefore dyes that are used as an exogenous contrast agent for fluorescence diagnosis and imaging delivered to the tissue and especially fluorescent dyes with an absorption and fluorescence maximum in the spectral range from 700 - 900 nm (diagnostic Window of tissue). Photons of this wavelength become comparative little absorbed by tissue and can therefore be several inches penetrate tissue before the process of absorption (predominantly by oxy- and deoxyhemoglobin) ended the light transport. The absorption can go beyond that by fluorescent dyes introduced into the tissue, but the absorbed energy in the form of longer-wave fluorescent radiation emit. This fluorescence radiation can be detected spectrally separated be made possible the location of the dye and correlation with molecular Structures to which the dye has bonded (see also Licha K (2002) Contrast agents for optical imaging (Review). In: Topics in Current Chemistry - Contrast Agents II (Editor: W. Krause), Volume 222, Springer Heidelberg, p. 1 - 31.).

To be a diagnostically significant one Differentiation between diseased structures and healthy tissue to achieve, the supplied Dye to one if possible high concentration difference between the tissues. This due to tumor physiological properties (blood circulation, Distribution kinetics, delayed Removal). For molecular labeling of disease-specific Structures can Conjugates from fluorescent dyes with target-affine biomolecules, such as Proteins, peptides, antibodies, be used. A certain proportion of these binds after injection Conjugates to molecular target structures, such as receptors or matrix proteins, while the unbound portion is excreted from the body. Thereby the result is a high concentration difference and thus a high one Image contrast when performing fluorescence diagnostics.

Fluorescent dyes from the class of cyanine dyes to the most promising representatives and have been structured in a variety of ways Wide synthesized. In particular carbocyanines with indocarbo-, indodicarbo- and indotricarbocyanine backbone have high extinction coefficients and good fluorescence quantum yields [Licha K (2002) Contrast agents for optical imaging (Review). In: Topics in Current Chemistry - Contrast Agents II (Editor: W. Krause), Volume 222, Springer Heidelberg, p. 1 - 31, and the references cited therein].

This is what WO 96/17628 describes in-vivo diagnostic procedure using near infrared radiation. there become water soluble Dyes and their biomolecule adducts with specific photophysical and pharmacochemical properties, as contrast media for fluorescence and transillumination diagnostics.

The synthesis of cyanine dyes with indocarbo, indodicarbo and indotricarbocyanine backbones is well described in the prior art. Relevant literature on this is, for example: Bioconjugate Chem. 4, 105-111, 1993; Bioconjugate Chem. 7, 356-62, 1996; Bioconjugate Chem. 8, 751-56, 1997; Cytometry 10, 11-19, 1989 and 11, 418-30, 1990; J. Heterocycl. Chem. 33, 1871-6, 1996; J. Org. Chem. 60, 2391-5, 1995; Dyes and Pigments 17, 19-27, 1991, Dyes and Pigments 21, 227-34, 1993; J. Fluoresc. 3, 153-155, 1993; and anal. Biochem. 217, 197-204, 1994. Other methods are in the patent publications US 4,981,977 ; US 5,688,966 ; US 5,808,044 ; WO 97/42976; WO 97/42978; WO 98/22146; WO 98/26077; and EP 0 800 831 described.

In addition, indotricarbocyanines with modified substituents were synthesized and coupled to biomolecules (described, inter alia, in Photochem. Photobiol. 72, 234, 2000; Bioconjugate Chem. 12, 44, 2001; Nature Biotechnol. 19, 237, 2001; J. Biomed. Optics 6 , 122, 2001; J. Med. Chem. 45, 2003, 2002). Further examples can be found in particular in publications WO 00/61194 (“Short-chain peptide dye con jugates as contrast agents for optical diagnostics "), WO 00/71162, WO 01/52746, WO 01/52743 and WO 01/62156.

The previously used in the prior art However, known indotricarbocyanines still have disadvantages that affect their efficient use.

So there are still low fluorescence quantum yields of indotricarbocyanines after coupling to biomolecules. Describe like this Gruber et al. (Bioconjugate Chemn. 11, 696-704, 2000) for the commercially available dye Cy7, that after coupling to a biomolecule there is a loss of fluorescence efficiency. Becker et al. (Photochem.Photobiol. Photobiol. 72, 234, 2000) describe conjugates of an indotricarbocyanine HSA and transferrin with fluorescence quantum yields of 2.9% and 2.8%, and deformed absorption spectra in physiological medium.

The dye conjugates also tend for aggregation. For example, Becker et al. (Photochem. Photobiol. 72, 234, 2000) for Conjugates of an indotricarbocyanine with HSA and transferrin, and Licha et al. (Bioconjugate Chem. 12, 44-50, 2001) for receptor-binding Peptides that this have deformed absorption spectra in physiological medium. These deformations indicate the formation of aggregates and the resulting ones fluorescence quenching out. A similar problem exists when the dyes are not sufficiently soluble in water.

There is also still an inefficient one Access to derivatives with a reactive group. This is how Gruber describes it et al. (Bioconjugate Chem. 11, 696-704, 2000) the use of Cy7 bisfunktionellem NHS esters with two reactive groups, making them potentially covalent Cross-linking of two biomolecules could be done. However, due to two carboxy groups in the molecule, the synthetic one Access to derivatives with only one reactive group is difficult and leads to by-products (e.g. contains monoreactive NHS ester of Cy7 portions of the non-activated and the double activated Cy7 molecule).

So there is a continuous one Need for efficient and easy to manufacture cyanine dyes for the Fluorescence diagnostics that reduce the disadvantages mentioned above or not. Furthermore, these dyes should look good for the Production of conjugates with biomolecules are suitable.

gelöst, worin X O, S oder zweifach substituiertes C ist, wobei die Substituenten ausgewählt sein können aus Methyl, Ethyl, Propyl, Isopropyl und/oder Butyl, Y CH₂-CH₂ oder CH₂-CH₂-CH₂ ist, Z C₁ bis C₅ Alkyl ist, wobei die C-Atome gegebenenfalls durch O oder S substituiert sind oder

ist, R₁ bis R₄ unabhängig voneinander SO₃H oder H sind, mit der Voraussetzung, daß mindestens drei von R₁ bis R₄ SO₃H sind, R₅ -CO-NH-R₈-R₉, -NH-CS-NH-R₈-R₉ oder -NH-CO-R₈-R₉ ist, worin R₈ ausgewählt ist aus der Gruppe bestehend aus unverzweigtem C₂-C₁₃ Alkyl, in dem gegebenenfalls C-Atome durch O oder S ersetzt sind, und R₉ ausgewählt ist aus

oder Chloracetyl, Bromacetyl, Iodacetyl, Chloracetamido, Iodacetamido, Chloralkyl, Bromalkyl, Iodalkyl, Pyridyldisulfid und Vinylsulfonamid, und worin R₆ und R₇ CH ist oder durch ein C₃-Alkyl zu einem Hexylring verbunden sind, der gegebenenfalls in para-Position mit einem C₁ bis C₄-Alkylrest substituiert sein kann, und Salze und Solvate dieser Verbindung.A first object of the invention is to provide an indotricarbocyanine dye of the general formula (I)

dissolved, in which X is O, S or doubly substituted C, where the substituents can be selected from methyl, ethyl, propyl, isopropyl and / or butyl, Y is CH ₂ -CH ₂ or CH ₂ -CH ₂ -CH ₂ , ZC ₁ to C _{5 is} alkyl, the C atoms being optionally substituted by O or S or

R ₁ to R _{4 are} independently SO ₃ H or H, provided that at least three of R ₁ to R _{4 are} SO ₃ H, R ₅ -CO-NH-R ₈ -R ₉ , -NH- CS-NH-R ₈ -R ₉ or -NH-CO-R ₈ -R ₉ , wherein R _{8 is} selected from the group consisting of unbranched C ₂ -C ₁₃ alkyl, in which C atoms are optionally substituted by O or S. are replaced, and R _{9 is} selected from

or chloroacetyl, bromoacetyl, iodoacetyl, chloroacetamido, iodoacetamido, chloroalkyl, bromoalkyl, iodoalkyl, pyridyl disulfide and vinylsulfonamide, and wherein R ₆ and R _{7 are} CH or are linked by a C ₃ alkyl to form a hexyl ring, optionally in the para position with a C ₁ to C ₄ alkyl radical can be substituted, and salts and solvates of this compound.

Preferred is an indotricarbocyanine dye of the present invention, wherein Y is CH ₂ -CH ₂ ; ZC ₁ to C _{5 is} alkyl, where the C atoms are optionally substituted by O or S, and where R ₆ and R _{7 is} CH, and salts and solvates of this compound.

More preferred is an indotricarbocyanine dye of the present invention wherein ZC ₁ -C ₅ alkyl.

ist, und R₆ und R₇ über C₃-Alkyl zu einem Hexylring verbunden ist.Even more preferred is an indotricarbocyanine dye of the present invention, wherein

and R ₆ and R _{7 are} linked via C ₃ alkyl to form a hexyl ring.

An object of the present invention are therefore fluorescent dyes from the class of cyanine dyes, in particular indotricarbocyanines with an absorption and fluorescence maximum in the spectral range 700 to 900 nm, a thiol-specific reactive Group and three, preferably four, sulfonate groups. These serve to increase of water solubility.

Surprisingly could now be found that the indotricarbocyanines according to the invention the above structure (compact position of 3-4 sulfonate groups over sulfonatoethyl residues) have a high fluorescence quantum yield of> 15% and that the fluorescence quantum yield after coupling to biomolecules nearly unchanged remains (maximum loss to approx. 10%). The absorption spectra the conjugates also show no deformation of the dye absorption in the NIR range at approx. 750 nm. This results in good hydrophilicity, decreased aggregation and increased Fluorescence quantum yield versus usual Indotricarbocyanines or Cy7 derivatives with less than three sulfonate groups, especially in the case of Cy7 and other known structures.

Another essential aspect in the provision of the cyanine dyes according to the invention for fluorescence diagnostics relates to derivatives which have reactive functional groups, to enable covalent coupling to target-specific biomolecules. Suitable derivatives are e.g. B. NHS esters and isothiocyanates (bioconjugates Chem. 4, 105-111, 1993; Bioconjugate Chem. 8, 751-56, 1997), with amino groups react, such as maleimides, alpha-haloketones or alpha-haloacetamides (Bioconjugate Chem. 13, 387-391, 2002; Bioconjugate Chem. 11, 161-166, 2000), which react with thiol groups. Further bifunctional linkers can from the group comprising arylene diisothiocyanate, alkylene diisothiocyanate, bis-N-hydroxy succinimidyl esters, hexamethylene diisocyanate and N- (γ-maleimidobutyryloxy) succinimide esters come.

WO 01/77229 describes cyanine dyes with a combination of sulfoaryl groups, alkyl substituents in meso position of the methine chain and at least one reactive group, which bind to biomolecules allows. The working examples however concern indodicarbocyanines (polymethine chain of 5 carbon atoms), and the compounds have no reactive group in the meso position.

WO 00/16810 ("Near infrared fluorescent contrast agent and fluorescence imaging ") describes indotricarbocyanines, i.a. with substituents in the meso position of the C7 polymethine chain. However, it is not derived how reactive groups can be introduced or generated.

Another aspect of the present invention relates to an indotricarbocyanine dye, wherein R _{5 is} COOH or NH ₂ .

The published derivatives are mainly based on the Cy3, Cy5, Cy5.5 and Cy7 basic structure (commercially available from Amersham Pharmacia Biotech; US 5,268,486 ; Cy3 = indocarbo-, Cy5 = indodicarbo-, Cy7 = indotricarbocyanine). Derivatives reactive with thiol groups are of particular interest because they allow directed conjugation with biotechnologically targeted cysteines in biomolecules. The prior art here mainly focuses on Cy3 and Cy5 derivatives.

Particularly preferred indotricarbocyanine dyes according to the invention are selected from the dyes having the formulas (II) to (XX), which are listed in Table 1 below: TABLE 1: Preferred dyes according to the invention

Another aspect of the present The invention relates to a method for producing an indotricarbocyanine dye of the present invention. Here an easy access via 4-substituted Pyridines found. Surprisingly could different 4-substituted Pyridines in high yields using the Zincke reaction (Zincke-König reaction, see Römpps Chemie-Lexikon, 10th edition, page 5067) in meso-substituted glutaconaldehyde dianilide (Precursor to cyanine dye) are transferred.

The symmetrical structure of the dyes of the present invention additionally to the simple and efficient synthesis from 4-substituted pyridines the possibility a defined derivatization with a thiol group selective Reactive group in the symmetrical meso position of the molecule.

So the further derivatization took place to thiol group-reactive compounds. Functionalities that are reactive with thiol groups z. B. maleimide (maleimide), chloroacetyl, bromoacetyl; iodoacetyl, Chloroacetamido, bromoacetamido, iodoacetamido, chloroalkyl, bromoalkyl, Iodoalkyl, pyridyl disulfide and vinyl sulfonamide.

Yet another aspect of the present invention relates to a method of making a conjugate comprising coupling an indotricarbocyanine dye of the present invention to a biomolecule. In the context of the present invention, “biomolecule” should be understood to mean any molecule of biological origin which has a biological activity, in particular enzymatic activity or binding of substances of synthetic or biological origin, such as, for example, pharmaceuticals, peptides, proteins, receptors or nucleic acids are in turn proteins such as enzymes, peptides, antibodies and antibody fragments (such as single chain, Fab, F (ab) ₂ , diabodies, etc.), lipoproteins, nucleic acids such as oligo- or polynucleotides from DNA or RNA, aptamers , PNA, and sugars such as mono-, di-, tri-, oligo- and polysaccharides.

The synthesis and biological characterization of cyanine dye conjugates with biomolecules such as peptides, antibodies and fragments thereof and proteins for the in vivo fluorescence diagnosis of tumors is described in the prior art in various publications. The above-mentioned Cy3, Cy5, Cy5.5 and Cy7 were mainly used (see, inter alia, Nature Biotechnol. 15, 1271, 1997; Cancer Detect. Prev. 22, 251, 1998; J. Immunol. Meth. 231, 239, 1999 ; Nature Biotechnol. 17, 375, 1999; Nature Medicine 7, 743, 2001).

Another aspect of the present The invention relates to a conjugate of an indotricarbocyanine dye according to the invention with a biomolecule, made by a method of the invention. This Conjugate can be characterized as being one as defined above biomolecule comprises being as a biomolecule at least one biomolecule, selected from peptides, proteins, lipoproteins, antibodies or antibody fragments, nucleic acids, such as oligo- or polynucleotides from DNA or RNA, aptamers, PNA, and sugars, such as mono-, di-, tri-, oligo- and Polysaccharides is more preferred. So the coupled protein characterized in that it selected is the group of structural proteins or soluble proteins of the body. All serum proteins (e.g. HSA), antibodies / antibody fragments, such as. an scFv fragment or F (ab) and peptides derived from it, BSA, ovalbumin or a peroxidase.

For example, from the state of the art already antibodies known against molecules are directed, which are strong in the angiogenetically active tissue and in adjacent tissues are expressed at a very low level (see WO 96/01653). Antibodies that are of particular interest against the receptors for vascular growth factors, Receptors on endothelial cells to which inflammatory mediators bind, receptors on endothelial cells to which matrix molecules bind and matrix proteins, which are specific in new vessel formation be expressed. Antibodies or antibody fragments are also preferred, which are directed against the matrix protein EDB fibronectin and conjugates according to the invention it. EDB fibronectin, also known as oncofetal fibronectin, is a splicing variant of fibronectin that is specific to newly formed vessels in the process of angiogenesis forms. Antibodies L19, E8, AP38 and AP39 are particularly preferred against EDB fibronectin (Cancer Res 1999, 59, 347; J Immunol Meth 1999, 231, 239; Protein Expr Purif 2001, 21, 156).

A conjugate according to the invention is preferred that thereby is marked that the Indotricarbocyanine dye about an SH group, especially about an SH group on a cysteine that is coupled to the biomolecule. From this if appropriate, such antibodies and their fragments, which are produced by recombinant techniques so that they are C-terminus or at the N-terminus (within the outer 1-10 amino acids) Contain cysteine, which does not form intramolecular S-S bridges and therefore for the coupling to the dyes of the invention can be used can.

Another aspect of the present Invention relates to a diagnostic kit containing an indotricarbocyanine dye of the present invention and / or a conjugate of the present Invention includes. additionally the kit can be used to carry out in vivo diagnostics of tumors in particular. These tools are for example suitable buffers, tubes, detection reagents or instructions for use. The kit preferably contains all materials for one intravenous Application of the dyes according to the invention. Special embodiments such kits according to the invention are for example as follows.

A first vessel, the antibody (biomolecule) with free SH group and common Contains buffers / additives, either as a solution or freeze-dried material. Another vessel that the dyes according to the invention as a solution (usual Additions) or freeze-dried material in a molar ratio of 0.1 up to 1 (10-fold deficit to equimolar Amount). The dye-containing vessel is optionally mixed with buffer or distilled water and the biomolecule-containing Vessel added, 1-10 min incubated and used directly as a solution for injection.

In another embodiment the kit is in a two-chamber system (e.g. a syringe) that the antibody solution in one chamber and over a destructible Wall spatial separate the dye as a solution or in a second chamber contains solid material. After breaking the wall, the solution for injection is mixed and generated.

A final aspect of the present The invention then relates to the use of a conjugate according to the invention as fluorescent contrast agent for the in vivo diagnosis of tumors. The absorption maximum of Cy7 is in the range of 745nm and is therefore particularly suitable for the in vivo detection of fluorescence from deeper tissue layers (see above). Are Cy7 derivatives with thiol group-selective reactive groups so far not described. Furthermore, the use of Antibody Conjugates for the detection of tumor edge areas already in WO 01/23005 (Antibody dye conjugates for binding to target structures of angiogenesis described in order to intraoperatively depict tumor peripheries), but not using the advantageous dyes according to the invention.

The invention is now intended to be further developed in Reference to the following examples and figures will be described without however limited to this to become.

1 shows the normalized absorption and fluorescence spectrum of conjugate K11 (A) and K15 (B) (see Table 2) in PBS, and

2 the results show the imaging properties of the conjugates according to the invention according to Example 24 with: substance: conjugate K15; Tumor: F9 teratocarcinoma in the right rear flank of the mouse; Dose: 50 nmol / kg body weight (information based on dye); Excitation: 740 nm (diode laser); detection: CCD camera (Hamamatsu) with bandpass filter 802.5 ± 5 nm and the times: before injection and 1h, 6h and 24h after injection. The position of the tumor is indicated by arrows.

Examples

Examples 1-16: Synthesis of indotricarbocyanine dyes with maleimide groups

Example 1: Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-l- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (2 - {[ 2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) ethyl] carbamoyl} ethyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl ) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula II)

a) 1- (2-Sulfonatoethyl) -2,3,3-trimethyl-3H-indolenine-5-sulfonic acid, internal salt

10 g (0.04 mol) of 2,3,3-trimethyl-3H-indolenine-5-sulfonic acid (bioconjugates Chem 1993, 4, 105), 6.8 g (0.04 mol) of 2-chloroethanesulfonic acid chloride and 4.2 g (0.04 mol) of triethylamine are placed in 200 ml of acetonitrile for 6 hours Heated to reflux. The precipitate is filtered off and dried. Yield 5.0 g (35 % d. Th.). Anal Biochem 1994, 217, 197

b) 3-pyridin-4-yl-propionic acid tert-butyl ester

20 g (89 mmol) t-butyl-P, P-dimethylphosphonoacetate in 50 ml THF at 0 ° C to a suspension of 3.9 g (98 mmol) sodium hydride (60% in mineral oil) in 250 ml of THF dropped. After stirring for 1 h at 0 ° C will be a solution of 10 g (93 mmol) pyridine-4-carbaldehyde in 50 ml tetrahydrofuran added dropwise and the reaction mixture 1 h at 0 ° C and 18 h at room temperature. touched. The precipitated solid is removed by filtration and the solution is concentrated. The residue is dissolved in hot isopropanol, insoluble parts are filtered off and the solution for crystallization to 0 ° C cooled. The resulting solid is filtered off, stirred with hexane, filtered and dried. The intermediate (15.3 g) is dissolved in 150 ml of ethanol hydrogenated with 0.15 g of 10% palladium / activated carbon for 6 hours. The catalyst is filtered off, the solution concentrated and the residue on silica gel filtered (eluent diethyl ether). 13.0 g of a light yellow oil (71% d. Th.)

c) 3- [2- (tert-Butyloxycarbonyl) ethyl] glutaconaldehyde dianilide hydrobromide

A solution of 10 g (48 mmol) of tert-butyl 3-pyridin-4-yl-propionate in 150 ml of diethyl ether, 8.9 g (96 mmol) of aniline are added, and subsequently at 0 ° C with a solution of 5.4 g (48 mmol) cyanogen bromide in 20 ml diethyl ether. To 3h stirring at 0 ° C the red solid formed is filtered off and washed with ether and vacuum dried. Yield: 20.3 g (92% of theory).

d) Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (2-carboxyethyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indol-5-sulfonate, inner salt

A suspension of 1.0 g (2.2 mmol) of 3- [2- (tert-butyloxycarbonyl) ethyl] glutaconaldehyde dianilide-hy drobromide (Example 1c)) and 1.5 g (4.4 mmol) of 1- (2-sulfonatoethyl) -2,3,3-trimethyl-3H-indolenine-5-sulfonic acid (Example 1a)) in 20 ml of acetic anhydride and 5 ml of acetic acid are mixed with 0.75 g (9.1 mmol) of sodium acetate and stirred at 120 ° C. for 1 h. After cooling, diethyl ether is added, the precipitated solid is filtered off and purified by chromatography (RP-C18 silica gel, mobile phase water / methanol) and the product is freeze-dried (0.5 g). The protective group is split off by stirring the intermediate in 4 ml of dichloromethane / 1 ml of trifluoroacetic acid for 1 h. After concentration, chromatographic purification (RP-C18 silica gel, mobile phase water / methanol), 0.45 g (23% of theory) of a blue lyophilizate is obtained.

e) Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (2 - {[2nd - (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) ethyl] carbamoyl} ethyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indol-5-sulfonate, inner salt

0.4 g (0.45 mmol) of the title compound from Example 1 d) and 45 mg (0.45 mmol) of triethylamine are dissolved in 10 ml of dimethylformamide, at 0 ° C. with 0.15 g (0.45 mmol) of TBTU added and stirred for 10 min. A solution of 0.17 g (0.68 mmol) of N- (2-aminoethyl) maleimide trifluoroacetate (Int J Pept Protein Res 1992, 40, 445) and 68 mg (0.68 mmol) of triethylamine in 0, 5 ml of dimethylformamide added and 1 h at room temperature. touched. After adding 10 ml of diethyl ether, the solid is centrifuged off, dried and purified by chromatography (RP C-18 silica gel, gradient methanol / water). Yield: 0.30 g of a blue lyophilizate (65% of theory).

Example 2: Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (2 - {[ 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexyl] carbamoyl} ethyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl ) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula III)

The synthesis is carried out analogously to Example 1e) from 0.4 g (0.45 mmol) of the title compound from Example 1d) and 0.21 g (0.68 mmol) of N- (6-aminohexyl) maleimide trifluoroacetate (Int J Pept Protein Res 1992, 40, 445). Yield: 0.38 g of a blue lyophilizate (81% of theory).

Example 3: Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (2 - {[ 13- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -4,7, 10-trioxatridecyl] carbamoyl} ethyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula IV)

The synthesis is carried out analogously to Example 1e) from 0.4 g (0.45 mmol) of the title compound from Example 1d) and 0.28 g (0.68 mmol) of N- (13-amino-4,7,10-trioxatridecyl) ) maleimide trifluoroacetate (Int J Pept Protein Res 1992, 40, 445). Yield: 0.27 g of a blue lyophilizate (51% of theory).

Example 4: Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (4 - {[ 2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) ethyl] carbamoyl} butyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl ) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula V)

a) (3-tert-Butoxycarbonyl-propyl) -triphenyl-phosphonium bromide

50 g (0.30 mol) of 4-bromobutyric acid in 400 ml THF at –40 ° C dropwise treated with 187 g (0.89 mol) of trifluoroacetic anhydride. To Stir for 30 min at -40 ° C 400 ml of tert-butanol / 30 ml of THF were added dropwise within 1 hour. To 16h stirring at room temp. the reaction mixture is poured onto an ice-cold sodium carbonate solution, the aqueous phase extracted three times with diethyl ether and the organic phases over sodium sulfate dried and concentrated. The residue is distilled in vacuo (bp 72 ° C / 0.9 mbar; yield: 41 g). The conversion to the phosphonium salt is carried out by heating 41 g (0.18 Mol) of the intermediate, 44.6 g (0.17 mol) triphenylphosphine and 32.5 g (0.36 mol) sodium hydrogen carbonate in 250 ml acetonitrile for 20h under reflux. The reaction mixture is filtered, concentrated and the residue by stirring with Diethyl ether brought to crystallization. Yield: 58.5 g (40 % d. Th., Based on 4-bromobutyric acid) of a white solid.

b) 5-pyridin-4-yl-pentanoic acid t-butyl ester

A solution of 14 g (28 mmol) (3-tert-butoxycarbonyl-propyl) -triphenylphosphonium bromide (Example 4a)) in 100 ml of anhydrous THF is stirred at -40 ° C with exclusion of air within 20 min with 17.5 ml (28 mmol ) Butyllithium (1.6 M in hexane) was added and the mixture was stirred at -40 ° C. for 1 h. A solution of 2.78 g (26 mmol) of 4-pyridinecarbaldehyde in 20 ml of THF is added dropwise and the mixture is stirred at room temperature for 16 hours. stirred, then poured onto ice water, the aqueous phase extracted three times with diethyl ether and the organic phases dried over sodium sulfate and concentrated. After chromatographic purification (silica gel, eluent hexane / ethyl acetate), the product is obtained as an E, Z mixture (4: 1 by ¹ H NMR; 5.0 g). To hydrogenate the double bond, the intermediate is dissolved in 200 ml of methanol and with 100 mg of PtO ₂ catalyst at room temperature. stirred over hydrogen. After filtration and concentration, a yellow oil is obtained. Yield: 4.9 g (74% of theory).

c) 3- [4- (tert-Butyloxycarbonyl) butyl] glutaconaldehyde dianilide hydrobromide

A solution of 4.0 g (17 mmol) of 5-pyridin-4-yl-pentanoic acid t-butyl ester in 35 ml of diethyl ether with 3.2 g (34 mmol) of aniline and then 0 ° C with a solution of 1.9 g (17 mmol) of cyanogen bromide in 8 ml of diethyl ether. To Stir for 3 hours at 0 ° C the red solid formed is filtered off and washed with ether and vacuum dried. Yield: 7.8 g (95% of theory).

d) Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (4-carboxybutyl) hepta -2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indol-5-sulfonate, inner salt

The synthesis is carried out analogously to the example 1d) from the title compound of Example 4c) (2.5 mmol) and 1- (2-sulfonatoethyl) -2,3,3-timethyl-3H-indolenine-5-sulfonic acid (5 mmol). Yield: 0.85 g (37% of theory) of a blue lyophilizate.

e) Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (4 - {[2nd - (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) ethyl] carbamoyl} butyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indol-5-sulfonate, inner salt

The synthesis is carried out analogously to Example 1 e) from 0.4 g (0.43 mmol) of the title compound from Example 4d). Yield: 0.31 g (69% of theory) of a blue lyophilizate.

Example 5: Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (4 - {[ 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexyl] carbamoyl} butyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl ) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula VI)

The synthesis is carried out analogously to Example 1e) from 0.4 g (0.43 mmol) of the title compound from Example 4d) and 0.20 g (0.66 mmol) of N- (6-aminohexyl) maleimide trifluoroacetate. Yield: 0.35 g of a blue lyophilizate (74% of theory).

Example 6: Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (4 - {[ 13- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -4,7, 10-trioxatridecyl] carbamoyl} butyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula VII)

The synthesis is carried out analogously to Example 1e) from 0.4 g (0.43 mmol) of the title compound from Example 1d) and 0.30 g (0.72 mmol) of N- (13-amino-4,7,10-trioxatridecyl) ) maleimidtrifluoracetat. Yield: 0.27 g of a blue lyophilizate (52% of theory).

Example 7: Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (6 - {[ 2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) ethyl] carbamoyl} hexyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl ) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula VIII)

a) (3-tert-Butoxycarbonyl-pentyl) -triphenyl-phosphonium bromide

The preparation takes place as described in Example 4a), with the intermediate 6-bromohexanoic acid re-tert-butyl ester is implemented as a crude product. 79 g of product (69% of theory) are obtained from 50 g of 6-bromohexanoic acid as a viscous, colorless oil.

b) 7-pyridin-4-yl-heptanoic acid t-butyl ester

The display is as in the example 4b). From 25 g (48.7 mmol) (3-tert-butoxycarbonyl-pentyl) -triphenyl-phosphonium bromide (Example 7a)) 7.5 g of 7-pyridin-4-yl-heptanoic acid t-butyl ester (65% of theory) as a yellow oil.

c) 3- [6- (tert-Butyloxycarbonyl) hexyl] glutaconaldehyde dianilide hydrobromide

A solution of 5.0 g (19 mmol) of 7-pyridin-4-yl-heptanoic acid t-butyl ester in 30 ml of diethyl ether with 3.6 g (38 mmol) of aniline and then 0 ° C with a solution of 2.1 g (19 mmol) of cyanogen bromide in 5 ml of diethyl ether. To 2.5 hours stirring at 0 ° C the resulting red solid is filtered off and washed with ether and vacuum dried. Yield: 8.9 g (91% of theory).

d) Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (6-carboxyhexyl) hepta -2,4,6-trien-l-ylidene} -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indol-5-sulfonate, inner salt

The synthesis is carried out analogously to the example 1d) from the title compound of Example 7c) (3 mmol) and 1- (2-sulfonatoethyl) -2,3,3-trimethyl-3H-indolenine-5-sulfonic acid (6 mmol). Yield: 1.5 g (54% of theory) of a blue lyophilizate.

e) Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (6 - {[2nd - (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) ethyl] carbamoyl} hexyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indol-5-sulfonate, inner salt

The synthesis is carried out analogously to Example 1e) from 0.4 g (0.43 mmol) of the title compound from Example 7d). Yield: 0.31 g (69% of theory) of a blue lyophilizate.

Example 8: Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (6 - {[ 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexyl] carbamoyl} hexyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl ) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula IX)

The synthesis is carried out analogously to Example 1e) from 0.5 g (0.53 mmol) of the title compound from Example 7d) and 0.23 g (0.75 mmol) of N- (6-aminohexyl) maleimide trifluoroacetate. Yield: 0.42 g of a blue lyophilizate (70% of theory).

Example 9: Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (6 - {[ 13- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -4,7, 10-trioxatridecyl] carbamoyl} hexyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula X)

The synthesis is carried out analogously to Example 1e) from 0.5 g (0.53 mmol) of the title compound from Example 7d) and 0.44 g (1.06 mmol) of N- (13-amino-4,7,10-trioxatridecyl) ) maleimidtrifluoracetat. Yield: 0.24 g of a blue lyophilizate (37% of theory).

Example 10: Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (5 - {[ 2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) ethyl] carbamoyl} -3-oxa-pentyl) hepta-2,4,6-trien-1-ylidene} -1 - (2-sulfonatoethyl) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula XI)

a) 3-Oxa-6- (4-pyridinyl) hexanoic acid tert-butyl ester

A solution of 75 g (0.4 mol) of 3- (4-pyridnyl) -1-propanol in 400 ml of toluene / 50 ml of THF is mixed with 10 g of tetrabutylammonium sulfate and 350 ml of 32% sodium hydroxide solution. Then 123 g (0.68 mol) of bromoacetic acid tert-butyl ester are added dropwise and 18 h at room temperature. touched. The organic phase is separated off and the aqueous phase is extracted three times with diethyl ether. The combined organic phases are with Washed NaCl solution, dried over sodium sulfate and concentrated. After chromatographic purification (silica gel; eluent hexane: ethyl acetate), 56 g of product (41% of theory) are obtained as a brownish oil.

b) 3- [4-Oxa-5- (tert-Butyloxycarbonyl) pentyl] glutaconaldehyde dianilide hydrobromide

A solution of 5.0 g (20 mmol) of tert-butyl 3-oxa-6- (4-pyridinyl) hexanoate in 60 ml of diethyl ether with 3.7 g (40 mmol) of aniline and then 0 ° C with a solution 2.2 g (20 mmol) of cyanogen bromide in 8 ml of diethyl ether. To Stir for 1 hour at 0 ° C is mixed with 50 ml of diethyl ether and the resulting red solid filtered off, with ether washed and vacuum dried. Yield: 8.5 g (85% of theory) of one violet solid.

c) Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (6-carboxy-4 -oxahexyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indol-5-sulfonate, inner salt

A suspension of 3.0 g (6 mmol) 3- [2- (tert-butyloxycarbonyl) ethyl] glutaconaldehyddianilid hydrobromide (Example 10b)) and 4.2 g (12 mmol) of 1- (2-sulfonatoethyl) -2,3,3-trimethyl-3H-indolenine-5-sulfonic acid (Example 1a)) in 50 ml of acetic anhydride and 10 ml acetic acid 2.5 g (30 mmol) of sodium acetate are added and the mixture is stirred at 120 ° C. for 50 min. To cooling down is mixed with diethyl ether, the precipitated solid is filtered off, stirred in acetone and dried in a high vacuum. After chromatographic purification (RP-C18 silica gel, eluent Water / methanol), removal of the methanol in vacuo and freeze-drying are obtained directly the title link. Yield: 2.3 g (41% of theory) of a blue Lyophilisate.

d) Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (5 - {[2nd - (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) ethyl] carbamoyl} -3-oxa-pentyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indol-5-sulfonate, inner salt

The synthesis is carried out analogously to Example 1e) from 1.0 g (1.1 mmol) of the title compound from Example 10c). Yield: 0.85 g (73% of theory) of a blue lyophilizate.

Example 11: Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (5 - {[ 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexyl] carbamoyl} -3-oxa-pentyl) hepta-2,4,6-trien-1-ylidene} -1 - (2-sulfonatoethyl) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula XII)

The synthesis is carried out analogously to Example 1e) from 0.5 g (0.55 mmol) of the title compound from Example 10c) and 0.23 g (0.75 mmol) of N- (6-aminohexyl) maleimide trifluoroacetate. Yield: 0.42 g of a blue lyophilizate (68% of theory).

Example 12: Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (5 - {[ 13- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -4,7, 10-trioxatridecyl] carbamoyl} -4-oxapentyl) hepta-2,4,6-triene-1 -ylidene} -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula XIII)

The synthesis is carried out analogously to Example 1e) from 0.5 g (0.55 mmol) of the title compound from Example 10c) and 0.46 g (1.06 mmol) of N- (13-amino-4,7,10-trioxatridecyl) ) maleimidtrifluoracetat. Yield: 0.34 g of a blue lyophilizate (56% of theory).

Example 13: Trisodium 3,3-dimethyl-2- [2- (1 - {[3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] vinylene} -2 - [4- (2 - {[2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) ethyl] carbamoyl} ethyl) -phenoxy] cyclohex-1-en-3-ylidene ) ethylidene] -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula XIV)

a) Trisodium 3,3-dimethyl-2- [2- (1 - {[3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] vinylene} -2- chloro-cyclohex-1-en-3-ylidene) ethylidene] -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indole-5-sulfonate, inner salt

5.0 g (14.4 mmol) of 1- (2-sulfonatoethyl) -2,3,3-trimethyl-3H-indolenine-5-sulfonic acid (example 1a)) and 2.6 g (7.2 mmol) of N - [(3- (anilinomethylene) -2-chloro-1-cyclohexen-1-yl) methylene] aniline hydrochloride (Aldrich) together with 2.5 g (30 mmol) of anhydrous Sodium acetate in 100 ml of methanol heated under reflux for 1 h, cooled, with 150 ml of diethyl ether were added and the mixture was stirred overnight. The The precipitate is filtered off, dried and purified by chromatography (Silica gel, gradient: dichloromethane / methanol). Yield: 3.8 g (58 % d. Th.) Of a blue solid.

b) Trisodium 3,3-dimethyl-2- [2- (1 - {[3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] vinylene} -2- [4- (2-carboxyethyl) phenoxy] cyclohex-1-en-3-ylidene) ethylidene] -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indole-5-sulfonate, inner salt

0.37 g (2.2 mmol) of 3- (4-hydroxyphenyl) propionic acid in 30 ml dimethylformamide are mixed with 0.18 g (4.5 mmol) sodium hydride (60% Mineral oil dispersion) added. After stirring for 30 min at room temp. is cooled to 0 ° C, a solution of 2.0 g (2.2 mmol) of the title compound from Example 12a) in 100 ml of dimethylformamide added dropwise and 2 h at room temperature. touched. The The mixture is quenched with dry ice and the solvent is removed in vacuo. The residue is dissolved in methanol, with 200 ml ether stirred and the precipitated solid is filtered off. There is a chromatographic Purification (silica gel, gradient: ethyl acetate / methanol). Yield: 1.9 g of a blue solid (83% of theory).

c) Trisodium 3,3-dimethyl-2- [2- (1 - {[3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] vinylene} -2- [4- (2 - {[2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) ethyl] carbamoyl} ethyl) -phenoxy] cyclohex-1-en-3-ylidene) ethylidene] -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indol-5-sulfonate, inner salt

0.1 mg (0.10 mmol) of the title compound from Example 12b) are reacted with TBTU and N- (2-aminoethyl) maleimide trifluoroacetate in the presence of triethylamine as described in Example 1e) and the product obtained is purified by chromatography. Yield: 93 mg of a blue lyophilizate (81% of theory).

Example 14: Trisodium 3,3-dimethyl-2- [2- (1 - {[3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] vinylene} -2 - [4- (2 - {[6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexyl] carbamoyl} ethyl) phenoxy) cyclohex-1-en-3-ylidene ) ethylidene] -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula XV)

The synthesis is carried out analogously to Example 1e) from 0.7 g (0.68 mmol) of the title compound from Example 14a) and 0.53 g (1.22 mmol) of N- (13-amino-4,7,10-trioxatridecyl) ) maleimidtrifluoracetat. Yield: 0.56 g of a blue lyophilizate (68% of theory).

Example 15: Trisodium 3,3-dimethyl-2- [2- (1 - {[3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] vinylene} -2 - [4- (2 - {[13- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -4,7, 10-trioxatridecyl] carbamoyl} ethyl) phenoxy] cyclohex-1 -en-3-ylidene) ethylidene) -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula XVI)

The synthesis is carried out analogously to Example 1e) from 0.7 g (0.68 mmol) of the title compound from Example 14a) and 0.59 g (1.36 mmol) of N- (13-amino-4,7,10-trioxatridecyl) ) maleimide trifluoroacetate. There are two chromatographic purifications. Yield: 0.67 g of a blue lyophilizate (75% of theory).

Example 16: Trisodium 3,3-dimethyl-2- [2- (1 - {[3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] vinylene} -2 - [4- (2 - {[2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) ethyl] carbamoyl} ethyl) -phenoxy] -5-tert-butyl-cyclohex- 1-en-3-ylidene) ethylidene] -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula XVII)

a) N - [(3- (anilinomethylene) -2-chloro-5-tert-butyl-1-cyclohexen-1-yl) methylene] aniline hydrochloride

6.7 ml (73.4 mmol) of phosphorus oxychloride are at 0 ° C added dropwise to 8 ml of dimethylformamide. Then a solution of 5.0 g (32.4 mmol) of 4-tert-butylcyclohexanone in 30 ml of dichloromethane added dropwise and the reaction mixture was stirred under reflux for 3 h. After cooling down 0 ° C 6 g (64.8 mmol) of aniline in 5.5 ml of ethanol were slowly added dropwise The mixture is poured onto 200 g of ice and 5 ml of conc. Acid added while stirring. The precipitated solid is filtered off, washed with ether and dried. Yield: 6.8 g (50% of theory) of a red solid.

b) Trisodium 3,3-dimethyl-2- [2- (1 - {[3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] vinylene} -2- chloro-5-tert-Butyl-cyclohex-1-en-3-ylidene) ethylidene] -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indol-5-sulfonate, inner salt

5.0 g (14.4 mmol) of 1- (2-sulfonatoethyl) -2,3,3-timethyl-3H-indolenine-5-sulfonic acid (example 1a)) and 3.0 g (7.2 mmol) of N - [(3- (anilinomethylene) -2-chloro-5-tert-butyl-1-cyclohexen-1-yl) methylene] aniline hydrochloride (Example 16a)) together with 2.5 g (30 mmol) anhydrous sodium acetate in 100 ml of methanol heated under reflux for 1.5 hours, cooled, mixed with 200 ml of diethyl ether and stirred overnight. The The precipitate is filtered off, dried and purified by chromatography (Silica gel, gradient: dichloromethane / methanol). Yield: 4.7 g (68 % d. Th.) Of a blue solid.

c) Trisodium 3,3-dimethyl-2- [2- (1 - {[3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] vinylene} -2- [4- (2-carboxyethyl) phenoxy] -5-tert-Butyl-cyclohex-1-en-3-ylidene) ethylidene] -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indol-5-sulfonate, inner salt

The implementation takes place from the 2.0 g (2.1 mmol) of the title compound from Example 16b) as in Example 13b) described. Yield: 1.5 g (66% of theory).

d) Trisodium 3,3-dimethyl-2- [2- (1 - {[3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] vinylene} -2- [4- (2 - {[2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) ethyl] carbamoyl} ethyl) -phenoxy] -5-tert-butyl-cyclohex-1 -en-3-ylidene) ethylidene] -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indol-5-sulfonate, inner salt

The reaction is carried out from 1.0 g (0.92 mmol) of the title compound from Example 16c) as described in Example 13c). The chromatographic purification is carried out twice with RP C-18 silica gel (Mobile solvent: acetonitrile / water). Yield: 0.24 g (22% of theory).

Examples 17-19: Synthesis of indotricarbocyanine dyes with bromoacetylamide groups

Example 17: Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (5 - {[ 6- (bromoacetylamino) hexyl] carbamoyl} -4-oxapentyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indole-5-sulfonate , inner salt (formula XIX)

a) Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (5 - {(6 aminohexyl) carbamoyl} -4-oxapentyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indol-5-sulfonate, inner salt

The synthesis is carried out analogously to Example 1e) from 0.5 g (0.55 mmol) of the title compound from Example 10c) and 0.15 g (0.70 mmol) of N-Boc-hexanediamine (Fluka). The reaction product is purified by chromatography (RP C18 chromatography, gradient: methanol / water) and, after freeze-drying in 2 ml of trifluoroacetic acid / 8 ml of dichloromethane, is stirred for 15 minutes while cooling with ice. After spinning in a vacuum, it turns back was dissolved in methanol, precipitated with diethyl ether and isolated. Yield: 0.26 g of a blue solid (41% of theory).

b) Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (5 - {[6 - (bromoacetylamino) hexyl] carbamoyl} -4-oxapentyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indol-5-sulfonate, inner salt

0.26 g (0.23 mmol) of the title compound from Example 18a) are cooled in 5 ml of dimethylformamide to -20 ° C., with 28 mg (0.28 mmol) of triethylamine and a solution of 0.10 g (0.46 mmol) of bromoacetyl bromide in 0.2 ml of dimethylformamide. After stirring for 5 hours at a maximum of 0 ° C., the product is precipitated by adding diethyl ether and obtained by repeated reprecipitation from dimethylformamide / diethyl ether and subsequent drying. Yield: 0.23 g (86% of theory) of a blue solid.

Example 18: Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl) -4- (3 - {[ 3- (bromoacetylamino) propyl] carbamoyl} ethyl) hepta-2,4,6-trien-1-ylidene} -1- (2-sulfonatoethyl) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (Formula XVIII)

The synthesis takes place starting from the title compound from example 1d) (0.5 g; 0.56 mmol) and N-Boc-propylenediamine analogously to example 17. Yield over all stages: 0.22 g (37% of theory) ,

Example 19: Trisodium 3,3-dimethyl-2- [2- (1 - {[3,3-dimethyl-5-sulfonato-1- (2-sulfonatoethyl) -3H-indolium-2-yl] vinylene} -2 - [4- (2 - {[3- (bromoacetylamino) propyl] carbamoyl} ethyl) -phenoxy] cyclohex-1-en-3-ylidene) ethylidene] -1- (2-sulfonatoethyl) -2,3-dihydro- 1H-indole-5-sulfonate, inner salt (formula XX)

The synthesis takes place from the title compound from Example 13b) (0.5 g; 0.49 mmol) and N-Boc-propylenediamine analogous to Example 17. Yield over all levels: 0.31 g (53% of theory).

Examples 20-23: Synthesis of conjugates with biomolecules and photophysical characterization of the conjugates

Example 20: Marking from BSA (bovine serum albumin) with the title compounds from example 1-16

General instructions: A solution of 5 mg (0.074 μmol) BSA (Sigma) in 5 ml phosphate buffer (0.1 M Na ₂ HPO ₄ / NaHP ₂ O ₄ , pH 6.8) is mixed with 0.74 μmol each Title compounds from Example 1-16 were added (stock solutions of 0.5 mg / ml in PBS) and incubated at 25 ° C. for 30 min. The conjugate is purified by gel chromatography (column: Sephadex G50, diameter 1.5 cm, Pharmacia, eluent: PBS).

Example 21: Marking from BSA with the title compounds from Examples 17-19

General rule: A solution from 5 mg (0.074 µmol) BSA (Sigma) in 5 ml phosphate buffer (0.1 M borate buffer, pH 8.5) is with 1.10 μmol of the title compounds from Example 17-19 added (stock solutions from 0.5 mg / ml in PBS) and 5 h at 25 ° C incubated. The conjugate is purified by means of gel chromatography (Pillar: Sephadex G50, diameter 1.5 cm, Pharmacia, Eluens: PBS).

Example 22: Marking of anti-ED-B fibronectin scFv antibody AP39 (Single chain fragment) with the title compounds from Examples 1-16

AP39 is a scFv with a C-terminal Cysteine and is a covalent S-S dimer with a molecular weight of approx. 56000 g / mol before (Curr Opin Drug Discov Devel. 2002 Mar; 5 (2): 204-13). By Reduction of the disulfide bridge two monomers with accessible SH group generated (molecular weight 28000 g / mol).

General instructions: 0.3 ml of a solution of AP39 in PBS (conc. 0.93 mg dimer / ml) is mixed with 60 μL of a solution of tris (carboxyethyl) phosphine (TCEP) in PBS (2.8 mg / ml) and incubated under nitrogen at 25 ° C for 1 h. Excess TCEP is separated by gel filtration on a NAP-5 column (eluent: PBS). The amount of AP39 monomer (OD _{280 nm} = 1.4) obtained by means of photometry is 230-250 μg (volume 0.5-0.6 ml). The solution is mixed with 0.03 μmol of the title compounds from Examples 1-16 (stock solutions of 0.5 mg / ml in PBS) and incubated at 25 ° C. for 30 min. The conjugate is purified by gel chromatography on a NAP-5 column (eluent: PBS / 10% glycerin). The immunoreactivity of the conjugate solution is determined by means of affinity chromatography (ED-B fibronectin resin) (J Immunol Meth 1999, 231, 239). The immunoreactivities of the conjugates obtained were> 80% (AP39 before conjugation> 95%).

Example 23: Marking of anti-ED-B fibronectin scFv antibody AP39 (Single chain fragment) with the title compounds from Examples 17-19

General instructions: 0.3 ml of a solution of AP39 in PBS (conc. 0.93 mg dimer / ml) is mixed with 60 μL of a solution of tris (carboxyethyl) phosphine (TCEP) in PBS (2.8 mg / ml) and incubated under nitrogen at 25 ° C for 1 h. Excess TCEP is separated by gel filtration on a NAP-5 column (eluent: 50 mM borate buffer pH 8.5). The amount of AP39 monomer (OD _{280 nm} = 1.4) obtained by means of photometry is 230-250 μg (volume 0.5-0.6 ml). The solution is mixed with 0.06 μmol of the title compounds from Examples 17-19 (stock solutions of 0.5 mg / ml in PBS) and incubated at 25 ° C. for 4 h. The conjugate is purified by gel chromatography on a NAP-5 column (eluent: PBS / 10% glycerin). The immunoreactivity of the conjugate solution is determined by means of affinity chromatography (ED-B fibronectin resin) (J Immunol Meth 1999, 231, 239). The immunoreactivities of the conjugates obtained were> 75% (AP39 before conjugation> 95%).

Photophysical characterization the dye-BSA conjugates from Examples 21 and 22 and the dye scFv Antibody conjugates from Examples 23 and 24.

The degree of loading (molar dye / antibody ratio) is determined photometrically and is based on an extinction coefficient of 75000 L mol ^-1 cm ^-1 in the short-wave absorption shoulder (approx. 690 - 710 nm), the antibody absorption (AP39) with an OD _280nm of 1 , 4 or protein _absorption (BSA) with an OD _277nm of 0.58. The fluorescence quantum yield is determined using a SPEX fluorolog (wavelength-dependent Sensitivity of lamp and detector calibrated) determined relative to indocyanine green (Q = 0.13 in DMSO, J Chem Eng Data 1977, 22, 379, Bioconjugate Chem 2001, 12, 44). Table 2: Properties of conjugates according to the invention

Example 24:

The imaging properties of the conjugates according to the invention were examined in vivo after injection into tumor-bearing nude mice. For this purpose, the conjugates were administered intravenously and the accumulation in the tumor region was observed over a period of 0 to 24 hours. The fluorescence of the substances was stimulated by irradiating the animals with near-infrared light at a wavelength of 740 nm, which was generated with a laser diode (0.5 W power). The fluorescence radiation was detected by an intensified CCD camera and the fluorescence images were stored digitally. In 2 the in vivo effectiveness of the dye conjugates is shown using an example.

Claims (34)

Indotricarbocyanine dye of the general formula (I),

wherein X is O, S or doubly substituted C, where the substituents can be selected from methyl, ethyl, propyl, isopropyl and / or butyl, Y is CH ₂ -CH ₂ or CH ₂ -CH ₂ -CH ₂ , Z is C ₁ to C _{5 is} alkyl, where the C atoms are optionally substituted by O or S or

R ₁ to R _{4 are} independently SO ₃ H or H, provided that at least three of R ₁ to R _{4 are} SO ₃ H, R ₅ -CO-NH-R ₈ -R ₉ , -NH- CS-NH-R ₈ -R ₉ or -NH-CO-R ₈ -R ₉ , wherein R _{8 is} selected from the group consisting of unbranched C ₂ -C ₁₃ alkyl, in which C atoms are optionally substituted by O or S. are replaced, and R _{9 is} selected from

or chloroacetyl, bromoacetyl, iodoacetyl, chloroacetamido, iodoacetamido, chloroalkyl, bromoalkyl, iodoalkyl, pyridyl disulfide and vinylsulfonamide, and wherein R ₆ and R _{7 are} CH or are linked by a C ₃ alkyl to form a hexyl ring, optionally in the para position with a C ₁ to C ₄ alkyl radical can be substituted, and salts and solvates of this compound. The indotricarbocyanine dye according to claim 1, wherein Y is CH ₂ -CH ₂ , Z is C ₁ to C ₅ alkyl, the C atoms are optionally substituted by O or S, and wherein R ₆ and R _{7 is} CH, and salts and solvates thereof Connection. Indotricarbocyanine dye according to claim 1 or 2, wherein ZC is ₁ -C ₅ alkyl. The indotricarbocyanine dye according to claim 1 or 2, wherein

is, and R ₆ and R _{7 are} connected via C ₃ alkyl to form a hexyl ring. Indotricarbocyanine dye according to any one of claims 1 to 4, wherein R _{5 is} COOH or NH ₂ . Indotricarbocyanine dye according to claim 3 of the formula (II)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 3 of the formula (III)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 3 of the formula (IV)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 3 of the formula (V)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 3 of the formula (VI)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 3 of the formula (VII)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 3 of the formula (VIII)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 3 of the formula (IX)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 3 of the formula (X)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 2 of the formula (XI)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 2 of the formula (XII)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 2 of the formula (XIII)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 4 of the formula (XIV)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 4 of the formula (XV)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 4 of the formula (XVI)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 4 of the formula (XVII)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 3 of the formula (XVIII)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 2 of the formula (XIX)

and salts and solvates of this compound. Indotricarbocyanine dye according to claim 4 of the formula (XX)

and salts and solvates of this compound. Process for producing an indotricarbocyanine dye according to one of the claims 1 to 24 comprising a) Providing one or more 4-substituted pyridines, b) transferring the one or more 4-substituted pyridines in meso-substituted glutaconaldehyde dianilides as precursors to cyanine dyes, using the Zincke-König reaction, and c) Obtaining the meso-substituted glutaconaldehyde dianilide as precursors to cyanine dyes. A method of making a conjugate comprising Coupling an indotricarbocyanine dye according to any one of claims 1 to 24 with one biomolecule. Conjugate of an indotricarbocyanine dye with a biomolecule, produced according to claim 26. Conjugate according to claim 27, characterized in that it as a biomolecule at least one biomolecule, selected from peptides, proteins, lipoproteins, antibodies or antibody fragments, nucleic acids, such as oligo- or polynucleotides from DNA or RNA, aptamers, PNA, and sugars, such as mono-, di-, tri-, oligo- and Polysaccharides. Conjugate according to claim 28, characterized in that this Protein selected is the group of structural proteins or soluble proteins of the body. Conjugate according to claim 28 or 29, characterized in that that this soluble Protein a serum protein, such as HSA, BSA, ovalbumin, an enzyme such as a peroxidase or an antibody is scFv fragment or F (ab). Conjugate according to one of claims 27 to 30, characterized in that that this soluble Protein an affinity against ED-B fibronectin. Conjugate according to one of claims 27 to 31, characterized in that that the Indotricarbocyanine dye about an SH group, especially about an SH group on a cysteine to which the biomolecule is coupled. Diagnostic kit comprising an indotricarbocyanine dye according to one of the claims 1 to 24 and / or a conjugate according to any one of claims 27 to 31, together with other aids for carrying out a in vivo diagnosis of tumors in particular. Use of a conjugate according to any one of claims 27 to 31 as a fluorescent contrast agent for the in vivo diagnosis of Tumors.