HRP20010833A2 - Short-chain peptide dye conjugates used as contrast agents for optical diagnostics - Google Patents

Abstract

Conjugates (I) of vasoactive intestinal peptide (VIP), somatostatin or neurotensin peptides and polymethine dyes are new. Peptide-dye conjugates of formula (I) are new. X = a sequence of 5-30 alpha -, beta - or gamma -amino acids (D or L) corresponding to the amino acid sequence of VIP, somatostatin or neurotensin or a fragment, derivative or analog thereof, optionally cyclized by a disulfide bridge between two cysteines or by an amide bridge between the N and C termini; A<1> = H, acetyl, 1-10C alkyl (optionally substituted by 1-3 COOH groups and/or 1-6 OH groups), a polyoxyethylene group comprising 2-30 CH2CH2O units, or a polymethine dye having at least one absorption maximum in the 380-1200 nm range; and A<2> = OH, NH2 or a polymethine dye having at least one absorption maximum in the 380-1200 nm range; provided that at least one of A<1> and A<2> is a polymethine dye, that when A<1> is a polymethine dye, it is bonded to the N-terminal amino group and that when A<2> is a polymethine dye, it is bonded to a lysine amino group or serine hydroxy group at any position within X. Independent claims are also included for the following: (1) a diagnostic method comprising administering (I) to a patient, either intravenously or to the bronchi by inhalation or to the gastrointestinal tract, esophagus or bladder by spraying and then washing out excess (I), and then performing an endoscopic investigation by local excitation of fluorescence at an excitation wavelength of 350-1200 nm and site-specific detection of the fluorescence emitted by the dye; and (2) cyanine dyes of formula (II). p = 1-3; n = 1-4 or 10; R<1>, R<2> = 4-sulfobutyl, 3-sulfopropyl, 2-sulfoethyl, 3-methyl-3-sulfopropyl, methyl, ethyl or propyl; R<3> = H, COOE<1>, CONE<1>E<2>, NHCOE<1>, NHCONHE<1>, NE<1>E<2>, OE<1>, OSO3E<1>, SO3E<1> or SO2NHE<1>; and E<1>, E<2> = H, CH3, ethyl, or 3-6C alkyl optionally interrupted by 1-2 O atoms and/or a CO group and/or optionally substituted by 1-5 OH groups.

Description

The invention relates to compounds, which are used in tumor diagnostics, and consist of dye conjugates with short-chain peptides, which are derived from vasoactive intestinal peptides, from somatostatin or from neurotensin, the use of these compounds as optical diagnostics and diagnostic agents, which contain such connections.

Changes caused by diseases are reflected at the cellular level, often as a state opposite to normal, and manifests itself in a changed distribution of receptors or repeated expression. These differences can be both of a quantitative nature (eg the amount of transferrin receptors on proliferating cells) and of a qualitative nature (eg the expression of vascular endothelial growth factor, VEGF). Until now, the use in the display of one pathological receptor expression or distribution, due to the necessary sensitivity of the detection procedure, was used exclusively in radiodiagnostics.

Heptahelical receptors are target molecules for many pharmacologically active substances (eg ß-blockers, H2 acid blockers, antihistamines). In addition to pharmaceutical application, only radioactively labeled agonistic ligands of these receptors are used, which are used diagnostically for the so-called receptor scintigraphy for in-vivo detection and localization of tumors. In doing so, the mechanism of receptor-mediated endocytosis is used, for example via the somatostatin receptor, which is strongly expressed on neuroendocrine tumors. Clinically approved for Scintigraphic routine diagnostics is the somatostatin analog 111In-DTPA-pentetreotide (Octreoscan®);

Literature: J. Steroid. Biochem. Mole. Biol. 37, 1079-82, 1990, J. Nucl. Honey. 32, 1184-9, 1991; J. Nucl. Honey. 33, 652-8, 1992; Digestion 3, 54-9, 1994, J. Clin. Invest. 93, 1321-5, 1994, Metabolism 45, 21-3, 1996.

Another way of using radioactively labeled VIP and VIP-analogues, which bind to the VIP-receptor. The VIP-receptor will be overexpressed in a wide range of tumors (including adenocarcinoma).

WO 96/30055 describes radiodiagnostic and radiotherapeutic reagents, in particular VIP-receptor binding peptides, which are radioactively labeled and can be used for radiodiagnosis and therapy. VIP-receptor-binding, Tc-99m-labeled peptides for scintigraphy have been described with particular advantage.

Further reading: Cancer Research 54, 690-700, 1994; Endocrinology 136, 2662-80, 1994, J. Nucl. Honey. 40, 353-361, 1999.

All described diagnostic methods of application, which are based on the somatostatin receptor, are radiodiagnostic methods of application (scintigraphy with 123I, 125I, 111In or 99mTc-labeled peptides).

Literature: EP 588754, US 5650134; US 6520675; US 5225180; WO 96/23527; J. Steriod. Biochem. Mole. Biol. 37, 1083-87, 1990, Lancet 242-4, 1989, J. Nucl. Honey. 39, 1913-17, 1998.

Until now, fluorescently labeled peptides, which are conjugated with dyes, have not been known, which enable an in-vivo-fluorescence detection of tumors (Photochem. Photobiol. 68, 603-632, 1998).

The purpose of the invention is to present new compounds that enable sensitive diagnosis of tumors, through the detection of fluorescent radiation, through the use of receptor-specific binding of compounds to target organs. In doing so, special dye molecules, which are attached to biomolecules, should give a highly sensitive fluorescent signal.

The task will be solved by preparing compounds, which are covalently bound to short-chain peptides. These conjugates have a high affinity for binding to heptahelical receptors, especially somatostatin receptor, VIP-receptor (vasoactive intestinal receptor) and neurotensin receptor and will also be received intracellularly via receptor-mediated endocytosis. The compounds presented by the invention are suitable for technically simple, harmless optical diagnostics of tumor cells and tumor tissue, which express somatostatin receptors, VIP-receptors or neurotensin receptors more strongly compared to healthy cells. Compounds for fluorescent diagnostics are especially suitable for this, and they are of particular advantage for fluorescent-endoscopic diagnostics of internal organs, such as the esophagus, cervix, colon and bronchi of various types of tumors, such as adenocarcinoma, neuroendocrine tumors or ductal pancreatic tumors.

Particular preference is given to colors that are specific in that they meet special photophysical and chemical requirements. From the photo-physical aspect, dyes must have a high absorption coefficient, in order to be able to give an effective signal, even at low tissue concentrations. Absorption maxima must, by free selection, cover a wide spectral range. Thus, for detection in deeper tissue layers (several centimeters below the surface of the skin), the spectral range between 600 and 900 nm is essential, while absorption wavelengths from 400 to 600 nm are sufficient for surface detection. From a chemical point of view, the colors must have high photosensitivity and no phenomena of decay (photobleaching) during stimulation (recording). Dyes must be compatible, as building blocks in the synthetic solid phase synthesis process of peptides and thus be stable under normal synthesis conditions, in order to satisfy a simple, affordable production process of structurally defined conjugates of peptides and dyes with a solid stoichiometric relationship between dye and peptide. The set requirements are best met with polymethine dyes, especially cyanine, merocyanine, oxonol and squarilium.

The subject of the invention are therefore

Peptide-polymethine-dye conjugates of the general formula (I)

A1 – (X)m – A2 (I)

whereby

X for α, β or γ-amino acids or D or L-configuration i

m stands for the number from 5 to 30,

where the resulting amino sequence (X)m is straight-chain or via a disulfide bond between cysteine or homocysteine or amido, between the N and C-terminus cyclized and for the amino sequence of vasoactive intestinal peptide (VIP), somatostatin or neurotensin or for fragments, partial sequences, derivatives or analogues of VIP, somatostatin or neurotensin stands,

A1 for one hydrogen atom, one acetyl residue or one alkyl residue with up to 10 C-atoms, which can also be substituted with 1 to 3 carboxyl groups and/or 1 to 6 hydroxy groups or one poly(oxyethylene) residue with 2 up to 30 -CH2CH2O-units or one dye molecule from the polymethine dye class, which shows one absorption region from 380 to 1200 nm,

A2 for a hydroxy group, one amino group or one dye molecule from the polymethine dye class, which shows at least one absorption maximum in the range from 380 to 1200 nm, reads

provided that at least one of the residues A1 or A2 represents one dye molecule from the class of polymethine dyes, which show at least one absorption maximum in the range from 380 to 1200 nm,

where, in the event that A1 and/or A2 one dye molecule from the class of polymethine dyes, which show at least one absorption maximum in the range from 380 to 1200 nm, represents, A1 is connected to one N-terminal amino group, and A2 to one amino group of the amino acid lysine or to one hydroxy group of the amino acid serine at any position within the amino acid sequence (X)m,

and their physiologically tolerable salts.

Fragments, parts of the sequences, derivatives or analogs of the mentioned peptides stand, among other things, for shortened sequences of amino acids, changes of individual or several amino acids against the corresponding D-amino acids, inverted sequences and combinations of the mentioned labels.

Fragments, parts of the sequences, derivatives or analogs of the mentioned peptides can also contain unnatural amino acids, such as naphthalanine, cyclohexylalanine, norleucine, norvaline, α-aminoadipic acid, α-amino butyric acid, β-alanine, β-cyclohexylalanine, ornithine, sarcosine or bhydroxylysine.

Particularly preferred derivatives of the invention are compounds of the general formula I, characterized by the fact that the color molecule A1 and/or A2 stands for one cyanine, squarilium, croconium, merocyanine or oxonol. These colors belong to the class of polymethine colors and have the above-mentioned advantages.

Further preferred compounds of the general formula I presented by the invention are characterized in that,

is a molecule of color A1 and/or A2 for one cyanine or squarilium (color) of the general formula II

[image]

whereby

D for one fragment corresponding to the general formula III to VI, wherein the asterisked position indicates linkage to B, reads,

[image]

B for the fragment corresponding to the general formula VII to XII

[image]

R1 and R2 for E1, R3 for one fluorine, chlorine, bromine, iodine atom or one nitro group or for one residue –COOE1, -CONE1E2, -NHCOE1, -NHCONHE1, -NE1E2, -OE1, -OSO3E1, SO3E1, - SO2NHE1, -E1,

wherein E1 and E2 independently of each other for one hydrogen atom, one C1-C4-sulfoalkyl chain, one saturated or unsaturated, branched or unbranched C1-C50-alkyl chain, wherein the chain or parts of the chain also have one or more aromatic or saturated cyclic C5-C6- or bicyclic units to form

can, provided that the C1-C50 alkyl chain of 0 to 15 oxygen atoms and/or 0 to 3 carbonyl groups are interrupted and/or substituted with 0 to 5 hydroxyl groups, provided that

R4 for one hydrogen atom, for one fluorine, chlorine, bromine, iodine atom or for one branched or straight-chain C1-C10-alkyl chain, it says,

b one number 2 or 3 means,

X and Y are independently O, S, Se, -CH=CH- or C(CH3)2

denotes,

L for one group corresponding to the following formula

[image]

where n denotes a number from 1 to 10,

stands.

(In the above formula, the solid line indicated in the part of the molecule on the left indicates the connection to the basic skeleton of the dye, and the broken line in the part of the molecule on the right, the connection to the peptide.)

From the class of polymethine dyes are cyanine dyes, for example those based on the indole structure, indocarbo-, indodicarbo- and indotricarbacyanine are particularly advantageous. Such structures are characterized by high chemical and photochemical stability. Derivatives can be obtained via favorable synthesis, which arbitrarily absorb between 400 and 1000 nm and fluoresce, through substitution with similar links and functional groups, for example with carboxyl groups, can be attached to peptides and have high solubility, for example via sulfonate groups. In contrast to the cyanine dyes known in the literature, the compounds presented by the invention have only one reactive group, which enables one stoichiometrically defined connection to the peptide during the synthesis of the conjugate resin.

Particularly valued embodiments of the compounds of the general formula I presented by the invention are characterized by the fact that,

- dye molecule A1 and/or A2 for one indocarbocyanine-, one indodicarbocyanine- or one indotricarbocyanine dye stand,

- dye molecule A1 and/or A2 for one indocarbocyanine-, one indodicarbocyanine- or one indotricarbocyanine dye of the general formula XIII or XIV

[image]

[image]

whereby

p for 1,2 or 3

n stands for a number of 1,2,3,4 or 10,

R1 and R2 independently of each other for one 4-Sulfobutyl-, 3-sulfopropyl-, 2-sulfoethyl-, 3-methyl-3-sulfopropyl-, methyl-, ethyl- or propyl residue is

AND

R3 for hydrogen, one chlorine, bromine, iodine atom or one nitro group or for one residue –COOE1, -CONE1E2, -NHCOE1, -NHCONHE1, -NE1E2, -OE1, -OSO3E1, -SO3E1, ---SO2NHE1,

wherein E1 and E2, mutually independent, for one hydrogen atom or for one methyl, ethyl or one C3-C6-alkyl residue, which from 0 to 2 oxygen atoms and/or from 0 to 1 carbonyl group is interrupted and/or with 0 to 5 hydroxyl groups substituted, or for one poly(oxyethylene)glycol residue with 2 to 30 –CH2CH2O-units it stands,

stands,

- that the dye molecule A1 and/or A2 for one indocarbocyanine-, one indodicarbocyanine- or one indotricarbocyanine dye of the general formula XIII or XIV

[image]

[image]

whereby

p for 1, 2 or 3

n for 1, 2 or 4,

R1 and R2 independently of each other for one 4-sulfobutyl- or 3-sulfopropyl residue,

R3 for a hydrogen atom or for one –COOE1 residue or -CONHE1,

where E1 is one hydrogen atom or one methyl-, ethyl- or one C3-C6-alkyl residue, which is terminated from 0 to 2 oxygen atoms and/or from 0 to 1 carboxyl group and/or substituted with 0 to 5 hydroxyl groups , has the meaning,

stands,

- that one molecule of dye A1 and/or A2 for one indotricarbocyanine dye of the general formula XV or XVI reads:

[image]

[image]

whereby

n stands for 2 or 3,

R1 and R2 independently represent one 4-sulfobutyl-, 3-sulfopropyl- or 2-sulfoethyl residue,

R3 for one residue –CONH-peptide, -CONH-(CH2)m-CONH-peptide, -CONH-(CH2)n-NH-CS-NH-peptide or -CONH-(CH2)n-NHCO-CH2-peptide with m=1 to 10 and n=2 or 3, it stands,

or the following group is represented by:

[image]

R4 and R5 independently of each other for one hydrogen atom, one methyl residue or one hydroxylated alkyl residue such as, for example, 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 1,3-dihydroxy-2-propyl, 2,3,4 -trihydroxybutyl, 1,3,4-trihydroxy-2-butyl, 2,3,4,5,6-pentahydroxyhexyl, stands

R6 stands for one of the following groups:

-(CH2)m-CONH-peptide with m=0 to 2,

-(CH2)m-NH-CS-NH-peptide with m=0 to 2,

and X stands for one oxygen atom or one sulfur atom;

- that the dye molecule A1 and/or A2 for one indotricarbocyanine dye of the general formula XVII reads:

[image]

whereby

R1 and R2 independently represent one 4-sulfobutyl- or 3-sulfopropyl residue,

R3 for one residue –CONH-peptide, CONH-(CH2)m-CONH-peptide, -CONH-(CH2)n-NH-CS-NH-peptide or –CONH-(CH2)n-NHCO-CH2-peptide with m =1 to 10 and n=2 or 3, it says

or the following group is represented by:

[image]

and R4 and R5 independently of each other for one hydrogen atom or one methyl residue or one hydroxylated alkyl residue, such as, for example, 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 1,3-dihydroxy-2-propyl, 2,3 ,4-trihydroxybutyl, 1,3,4-trihydroxy-2-butyl, 2,3,4,5,6-pentahydroxyhexyl, it says.

The use of color-labeled antibodies in tumor detection is known in the literature (J. Cell. Pharmacol. 3, 141-145, 1992; Cancer Immunol. Immunother. 41, 257-63, 1995; Cancer Research 54, 2643-9, 1994, Biotechnol. Prog. 13, 649-658, 1997).

In contrast to this, the compounds presented by the invention contain as biomolecules low-molecular peptides and peptide derivatives, which demonstrate the advantages of antibodies, such as a high possibility of binding to target molecules, without the diagnostic potential exceeding unfavorable pharmacokinetics (long time of blood sedimentation, allergic adverse effects - immunogenicity) limited.

Biological and pharmacological requirements for peptide sequences are correspondingly satisfactory plasmability with faster collection in the target tissue and at the same time rapid elimination from the rest of the body, for example via the renal excretion route.

Surprisingly, it was discovered that the peptide sequence, which contains at least 5 amino acids on the C-terminus side of the VIP-sequence and to which a dye for fluorescent diagnostics is attached, has one with native VIP comparable recordings in tumor cells. In view of this, it was discovered that through the incorporation of at least one D-amino acid or by changing at least one D-amino acid, plasma stability can be significantly increased. On the example of a complete exchange of all L- with D-amino acids in one VIP-binding peptide dye conjugate, it could be proven that the property of binding and penetration into cells remained unchanged.

Further particularities of the compounds of general formula I presented by the invention, which are preferred, are characterized by,

that (X)m for the amino acid sequence of the native, known in nature, human vasoactive intestinal peptide, corresponds to

[image]

or for fragments, parts of sequences, derivatives or analogues of vasoactive intestinal peptides, consisting of 5 to 30 amino acids,

What is it,

- yes (X)m for the amino sequence corresponding to somatostatin

[image]

or for fragments, parts of sequences, derivatives or analogs of somatostatin, consisting of 5 to 20 amino acids

What is it,

- yes (X)m for the amino acid sequence corresponding to neotensin

pyroglutamic acid-LYENKPRPYIL

or for fragments, parts of sequences, derivatives or analogs of neurotensin, consisting of 5 to 20 amino acids,

What is it,

- that as fragments, parts of sequences, derivatives or analogues of vasoactive intestinal peptide (VIP) the following sequence of amino acids was selected:

[image]

- that the following sequence was selected as a VIP analogue:

FSDAVFTDNY TRLRKQMAVK KYLNSILN

[image]

[image]

[image]

- that as an analogue of VIP, one compound was selected according to the following formula:

[image]

where X1, X2 and X3 can represent any amino acid,

that 2 to m amino acids independently of each other, opposite any amino acid or opposite another L- or D-amino acid can be changed, whereby the same has the above-mentioned meaning,

- that at least one amino acid (X)m can be independently substituted for another, unnatural amino acid or amino acid derivative,

- that at least one amino acid (X) m independently of each other, for another, unnatural amino acid or amino acid derivative, as. eg naphthalene, cyclohexylalanine, norleucine, norvaline, α-aminoadipic acid, α-aminobutyric acid, β-alanine, β-cyclohexylalanine, ornithine, sarcosine or b-hydroxylysine, may be substituted

- that as an analogue of VIP, one compound, according to the following formula, was selected:

[image]

where X1, X2, X3 and X4 are not natural amino acids or amino acid derivatives, such as naphthalanine, cyclohexylalanine, norleucine, norvaline, α-aminoadipic acid, α-aminobutyric acid, β-alanine, β-cyclohexylalanine, ornithine, sarcosine or b -hydroxylysine, can represent,

- that some amino acids (X)m are substituted for their specific D-amino acid forms

- that as fragments, parts of sequences, derivatives or analogues of vasoactive intestinal peptides of retrosynthetic amino acid sequences, in which 2 to m amino acids are changed for some D-amino acids,

are selected, having the above meaning,

- that the following amino acid sequences were selected as fragments, parts of sequences, derivatives or analogues of vasoactive intestinal peptides:

[image]

- that the following amino acid sequences were selected as fragments, parts of sequences, derivatives or analogs of somatostatin:

[image]

- that the following amino acid sequences were selected as fragments, parts of sequences, derivatives or analogues of neurotensin:

[image]

The terminology of general formula I contains the usual way of notation of amino acid sequences. The N-terminus is always on the left and the C-terminus on the right (unsubstituted corresponds to H-(X)m-OH or, in the case of an amide, H-(X)m-NH2. The one-letter abbreviations used for amino acids can be read in M .Bodanszky, Peptide chemistry – A practical textbook, 2nd edition, Springer-Verlag Heidelberg 1993, p 3. uppercase letters denote amino acids with L-configuration (natural amino acids), lowercase letters denote D-amino acids, disulfide bonds (cyclic peptides) are over dashes indicating the connections between the corresponding letters (C = cysteine or homocysteine) marked Sequences are marked as retrosynthetic, in which, during synthesis, the sequence of amino acids compared to the given native sequence is inverted, which means that the synthesis begins with the initial N-terminal amino acid and produces a sequence according to the original C-terminal amino acid.

An analogue of VIP will be shown by substitution analysis (see example 43). The following analogues of VIP have a special advantage:

[image]

[image]

The substances presented by the invention have different advantages compared to radioactively labeled substances. Fluorescent dyes can be referred to fluorescent emission countless times. There does not lie any continuous signal, which would lead to a decay with a corresponding half-life, which lies in the essence. Correspondingly, the diagnosis time can be selected as desired and also repeated as often as desired and is not limited by the half-life of one isotope. The patient is not exposed to any ionizing radiation, the applied light beam is harmless in the applied amounts. The optical detection technique enables high-sensitivity detection, fewer photons, and is therefore comparable to the sensitivity of radiodiagnostics.

One method for diagnostics using a near-infrared light beam (NIR-light beam) using dye conjugates of biomolecules has already been described (WO 96/17628). As a special advantage, in the case of the presented invention, it was shown that peptide conjugates in a more favorable quantity and higher purity, through the process of automated synthesis of the solid phase, are presentable. It is surprising that it was discovered that various compounds of indolcyanine, which carry carboxylic groups, as analogues of carboxylic acids, can be used as N-terminus, as they can be attached to the amino group of lysine, on a solid component (resin).

After separation from the resin and chromatographic purification, dye and peptide conjugates are obtained, purity level > 95%. One new mode of attachment allows the attachment of halogenacetyl dye compounds to the amino acids cysteine and homocysteine, which can replace any desired amino acid of the native VIP sequence.

A more significant problem with the use of light to stimulate fluorescence is the limited depth of light penetration, which in the VIS range is within a sub-millimeter, and in the NIR range can be as much as a centimeter. Considering the depth of penetration, detection procedures in the surface layer of diseased tissue, as well as soft tissues, are unproblematic.

The subject of the invention is therefore mammographic procedures, endoscopic procedures and intraoperative procedures, in which, under the application of the compounds presented by the invention, diseased tissue areas can be diagnosed through detection based on the principle of fluorescence or non-absorbing radiation. A special subject of the invention is the application of the compounds presented by the invention in endoscopic procedures, such as colonoscopy, bronchoscopy, esophageal endoscopy, in which changes in the surface layers of tissue are diagnosed. The use of white light with direct visual assessment is very widespread in endoscopic diagnostics. The compounds presented by the invention contribute, by inducing signals specific for individual tissues, to a significant improvement of the procedure, especially when diagnosing early, visually invisible changes in tissues (eg colonic dysplasia).

It was discovered that after dispersing the compounds presented by the invention chemically related to dyes, in the intestines of rats with chemically induced dysplasias and colon cancer, by washing and performing an endoscopic fluorescent diagnosis (initiation 740 nm, detection 760 nm), diseased parts of the colon tissue can be proven. using increased fluorescence.

The subject of the invention is therefore also a procedure for endoscopic diagnostics, especially of the gastrointestinal tract, with the use of the compounds presented by the invention. In doing so, one or more substances are injected into the tissues, first of all intravenously or topically by spraying, and then with the help of light from the appropriate spectral range, electronic initiation (stimulus) and illumination of the applied colored substance is performed. The reflected or emitted fluorescence of the colored substance is registered. Preference is given to methods in which tissues on large surfaces are exposed to a light substimulus, and fluorescence is locally induced, which is recorded with a single CCD camera, or tissue areas that are in the stage of decomposition can be recorded in a raster and the received signals can be transferred by computer into one synthetic image. At the same time, fluorescence can be detected and interpreted spectrally and/or phase selectively, as well as stationary and/or separated in time. The fluorescent images obtained can be produced simultaneously with the images obtained with white light and used for data interpretation in any form.

The synthesis of the compounds presented by the invention follows relying on methods already known in the literature. Peptides are made by synthesis on solid phase, on polymer resin. The details are already known in the field.

Literature: Peptide chemistry – A practical textbook (M. Bodanszky), 2nd edition, Springer-Verlag Heidelberg 1993; Anti-Cancer Drug Design 12, 145-167, 1997; J. Am. Chem. Soc. 117, 11821-2, 1995.

Colored substances are prepared separately and then, during the synthetic preparation, the solid phase of the peptide is connected to it, and the compounds presented by the invention are obtained as highly pure compounds after separation of the resin and purification. Such colored substances, which contain carboxyl groups, are preferred, and after activation with the help of reagents, they are joined with amino groups of peptides, especially ε-amino groups of lysine or N-terminal peptide-amino groups. Furthermore, preference is given to dye compounds with haloalkyl or haloacetyl residues, which are linked to the thiol groups of the peptide, especially to the amino acid cysteine or homocysteine.

Literature for the synthesis of polymethine dyes: 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, 1966; 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; Anal. Biochem. 217, 197-204, 1994; US 4981977; US 5688966; US 5808044; WO97/42976; WO 97/42978; WO98/22146; WO 98/26077; EP 0800831.

Dyes that contain exactly one carboxyl group, with a particular advantage of cyanine dyes of the general formula XVIII, are especially suitable for coupling with peptides in the solid phase.

[image]

whereby

p for 1,2 or 3,

n stands for 1,2,3,4 or 10,

R1 and R2 are mutually independent for one 4-sulfobutyl-3-sulfopropyl-, 2-sulfoethyl-, 3-methyl-3-sulfopropyl-, methyl-, ethyl-, or propyl residue and

R3 for hydrogen or for one residue –COOE1, -CONE1E2, -NHCOE1, -NHCONHE1, -NE1E2, -OE1, -OSO3E1, -SO3E1, -SO2NHE1,

where E1 and E2 are mutually independent for one hydrogen atom

or for one methyl-, ethyl-, or one C3-C6-alkyl residue, which is substituted by 0 to 2 oxygen atoms and/or 0 to 5 hydroxyl groups, it says

or cyanine dyes of the general formula XIX or XX

[image]

[image]

whereby

n stands for 2 or 3

R1 and R2 independently represent one 4-sulfobutyl-, 3-sulfopropyl- or 2-sulfoethyl residue,

R3 for one –COOH-group or one of the following residues is:

-CONH-(CH2)n-COOH with n=2 or 3,

-CONH-(CH2)n-NCS with n=2 or 3,

-CONH-(CH2)n-NHCO-CH2-X1 with n=2 or 3 and X1 = Cl, Br, J

[image]

R4 and R5 independently of each other for one hydrogen atom, one methyl residue or one hydroxylated alkyl residue, such as, for example, 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 1,3-dihydroxy-2-propyl, 2,3, 4-trihydroxybutyl, 1,3,4-trihydroxy-2-butyl, 2,3,4,5,6-pentahydroxyhexyl stands,

R6 stands for one of the following groups:

-(CH2)m-COOH with m= 0 to 2,

-(CH2)m-NCS with m= 0 to 2

and X stands for one oxygen atom or one sulfur atom;

or cyanine dyes of the general formula XXI

[image]

whereby

R1 and R2 independently represent one 4-sulfobutyl-, 3-sulfopropyl- or 2-sulfoethyl residue,

R3 for one –COOH-group or one of the following residues is:

-CONH-(CH2)n-COOH with n=2 or 3,

-CONH-(CH2)n-NCS with n=2 or 3,

-CONH-(CH2)n-NHCO-CH2-X1 with n=2 or 3 and X1 = Cl, Br, J

[image]

R4 and R5 independently of each other for one hydrogen atom, one methyl residue or one hydroxylated alkyl residue, such as, for example, 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 1,3-dihydroxy-2-propyl, 2,3, 4-trihydroxybutyl, 1,3,4-trihydroxy-2-butyl, 2,3,4,5,6-pentahydroxyhexyl stands.

The advantage of only one group that can be activated, such as one carboxyl group or one already activated group, such as one isothiocyanate, one haloalkyl group or one halogenacetyl group, consists in the fact that one chemically unique connection can follow. The haloacetyl group has one special advantage, that a chemically unique connection to the mercapto group of cysteine or homocysteine follows. Such binding can take place in solution on the unbound and deprotected peptide. Through the activated groups, one connection to the peptide is possible, without the consequent reactions following. To increase solubility in water, peptide-dye conjugates show an increased number of hydroxy groups on the dye. Through the position of the linker on the indole system of the dye, an additional satisfactory hydrophilicity can be produced through the residues on the sulfonate groups on the oxygen atoms of the indole system. Thus, a structurally unique coupling reaction with peptides (see examples 4 to 38 and 44 to 49) can be carried out.

A further object of the invention is an optical diagnostic for in-vivo diagnosis of diseased tissue parts, which is characterized by the fact that it contains at least one compound of the general formula I together with the usual auxiliary and/or carrier substances as well as a diluting agent.

The following examples illustrate the invention:

Examples 1 to 3:

Synthesis of indocyanine dye 1-3 for synthetic connection of solid phases on amino groups (N-terminal or ε-lysine) of peptides

The synthesis follows generally starting from 1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolene and 1-(4-Sulfobutyl)-2,3,3-trimethyl-5-carboxy-3H-indolene (Cytometry 10, 11-19, 1989, Talanta 39, 505-510, 1992).

Example 1:

Synthesis of 1,1`-bi(4-sulfobutyl)indocarbocyanine-5-carboxylic acid, sodium salt (1)

0.8 g (4.0 mmol) of N,N-diphenylformamidine will be placed in 15 ml of acetic anhydride at room temperature. in smaller portions with 1.4 g (4.2 mmol) of 1-(4-sulfobutyl)-2,3,3-trimethyl-5-carboxy-3H-indolenine mixed, 30 min, at room temp. chilled. Next will be 1.2 g (4.1 mmol) of 1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine, 1.2 g (14.6 mmol) of dry sodium acetate, 15 ml of acetic anhydride acid and 6 ml of acetic acid added. The reaction mixture is then heated for 1 hour at 120ºC, the dark red solution is cooled and mixed with 100 ml of ether. The precipitated solid is filtered off. This is followed by one chromatographic purification on RP-Kieselgel EUROPREP 60-30 C18, 60A, 20-45 m (eluent: water/MeOH, step gradient from 0% to 70% MeOH). Fractions containing the product will first be freed from methanol in a rotary evaporator and subsequently lyophilized, yield: 1.5 g (58%), red lyophilizate.

Example 2:

Synthesis of 1,1`-bi(4-sulfobutyl)indodicarbocyanine-5-carboxylic acid, sodium salt (2)

1.2 g (4.1 mmol) of 1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine and 1.0 g (3.9 mmol) of malonaldehyde-bi-phenylimine-hydrochloride will be in 15 ml of acetic anhydride for 30 min, mixed at 120ºC and then in a water bath until room temp. chilled. Next will be one after the other 1.4 g (4.2 mmol) of 1-(4-sulfobutyl)-2,3,3-trimethyl-5-carboxy-3H-indolenine, 1.2 g (14.6 mmol) of dry sodium acetate, 15 ml of acetic anhydride and 6 ml of acetic acid added. The reaction mixture is heated for 1 hour at 120ºC, and then the blue solution is cooled and mixed with 100 ml of ether. Work-up and purification follows as described in example 1, yield: 1.8 g (66%), blue lyophilisate.

Example 3:

Synthesis of 1,1`-bi(4-sulfobutyl)indotricarbocyanine-5-carboxylic acid, sodium salt (3)

1.2 g (4.1 mmol) of 1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine and 1.1 g (3.9 mmol) of glutaconaldehyde-dianylhydrochloride will be in 15 ml of acetic anhydride acid for 30 min at 120ºC, mixed and then in a water bath at room temperature. chilled. Next, 1.4 g (4.2 mmol) of 1-(4-sulfobutyl)-2,3,3-trimethyl-5-carboxy-3H-indolenine, 1.2 g (14.6 mmol) of dry sodium acetate, 15 ml of acetic anhydride and 6 ml of acetic acid added. The reaction mixture will be heated for 1 hour at 120ºC, the blue solution will be cooled and mixed with 100 ml of ether. Processing and purification follows as described in example 1, yield: 1.8 g (60%), blue lyophilisate.

The structures of compounds from examples 1-3 are shown in description 1.

Examples 4 to 6:

Synthesis of indocyanine dyes 4-6 from 1-3 to the synthetic connection of the solid phase to the amino groups (N-terminus or ε-lysine) of the peptide

The synthesis follows through amidation of dyes 1-3 with β-alanine-t-butylester and "acidic" cleavage of the t-butylester group.

One solution of 0.5 mmol of dye 1-3 and 0.1 g (1.0 mmol) of triethylamine in 20 ml of dimethylformamide will be mixed at 0ºC with 0.5 mmol of TBTU in 10 ml of dimethylformamide and stirred for 15 min at 0ºC. Next, a solution of 0.11 g (0.6 mmol) of β-alanine-t-butyl ester hydrochloride and 0.6 mmol of triethylamine in 5 ml of dimethylformamide will be added dropwise, and the reaction mixture will be stirred for 2 hours at room temperature. After adding 100 ml of diethyl ether, the precipitated solid will be filtered, dissolved in 20 ml of dichloromethane, mixed with 10 ml of trifluoroacetic acid and at room temperature. 24 hours mixed. The mixture will be reduced in a vacuum, and the residue as described in example 1, chromatographically purified and lyophilized: utilization: 0.21 g (58%) 4, 0.29 g (76%) 5, 0.28 g (72%) 6.

Examples 7 to 9:

Synthesis of indocyanine dyes 7-9 from 1-3 to the synthetic connection of the solid phase to the amino groups (N-terminus or ε-lysine) of the peptide

The preparation follows analogously to examples 4-6 with the use of 0.1 g (0.6 mmol) glycine-t-butyl ester hydrochloride, utilization: 0.25 g (68%) 4, 0.30 g (80%) 5, 0 ,32 g (83%) 6.

Examples 10 to 12:

Synthesis of indocyanine dyes 10-12 from 1-3 to synthetic coupling of the solid phase to peptides via binding to cysteine thiol groups

The synthesis follows the amidation of dyes 1-3 with 3-aminopropanol and the conversion of alcohol groups into one bromide.

One solution of 0.5 mmol of dye 1-3 and 0.1 g (1.0 mmol) of triethylamine in 20 ml of dimethylformamide will be mixed at 0ºC with 0.5 mmol of TBTU in 10 ml and stirred for 15 min at 0ºC. Next, a solution of 850 mg (1.2 mmol) of 3-aminopropanol and 1.2 mmol of triethylamine in 5 ml of dimethylformamide will be added dropwise, and the reaction mixture will be stirred for 6 hours at room temperature. After the addition of 100 ml of diethyl ether, the precipitated solid substance will be filtered and, as described in example 1, chromatographically purified and lyophilized.

Conversion to bromide 10-12 follows by mixing 0.3 mmol of the intermediate with 55 mg (0.5 mmol) of N-bromosuccinide and 130 mg (0.5 mmol) of triphenylphosphine in a mixture of 4 ml of dichloromethane and 4 ml of dimethylformamide for 48 hours at 4ºC. . With the addition of 3 ml of ether, the products are precipitated, filtered and added as a raw material for connection with the peptide.

Examples 14-16 and 18-27:

Synthesis of resin peptide conjugates from VIP-receptor binding peptides and dyes 1-9 and 13.

a) Solid phase synthesis: On 50 µmol of TentaGel-Sram-resin (Rapp Polymere, Tubingen) there will be peptides according to the Fmoc-strategy, analogous to the Standard-Fmoc-Maschinen-Protokoll (Pept. Res., 36 (1990) 225) for application of reagents for linking PyBOP (benzotriazol-1-yloxy-tris-pyrrolidionophosphonium hexafluorophosphate) and N-methylmorphine, synthesized. The following secondary protective groups are used: trityl for Cys, His, Asn, Gln: t-butyl for Asp, Glu, Ser and Thr; t-butyloxycarbonyl for Lys and Trp and pentamethylchloramsulfonyl for Arg.

Binding to the ε-amino group of one lysine (example 24) will be achieved through the orthogonal technique of protecting groups. Fmoc-Lys(Dde)-OH will be used as the building block of lysine, and the peptide will be synthesized as described above. After the acetylation of the N-terminus, the removal of the Dde-protecting group is followed selectively, via 3% hydrazine hydrate.

b) Binding of the dye: The peptides that are still attached to the solid phase will have the dye attached to the N-terminus or to the lysine. For this, 75 µmol of the specified dye 1-9 and 13 (1.5 eq) will be dissolved in 600 µmol of dimethylformamide and with 83 µmol of TBTU (2-(1Hbenzotriazol-1-yl)-1,1,3,3-tetramethyluronium- tetrafluoroborate) as well as 150 µmol of N,N-diisopropylethylamine mixed. After 2 minutes, the same reaction mixture will be added to the corresponding resin that can carry the peptide, and left overnight in the reaction. Finally, the resin will be washed 5x with dimethylformamide and 3x with dichloromethane and air-dried.

c) Removal of protective groups and separation of the dye-peptide conjugate: 1.5 ml of a mixture consisting of 750 mg of phenol, 250 µl of ethanedithiol, 500 µl of thioanisole and 500 µl of water in 10 ml of trifluoroacetic acid will be on a resin that can carry peptide conjugate added and allowed to act for 4 hours. The dye and peptide conjugates will be precipitated with cold t-butyl methyl ether, washed 6 times with cold diethyl ether, dissolved in 5% acetic acid and dried by freeze-drying. Purity analysis and purification of the conjugate follows by means of RP-HPLC (reversed phase high-pressure liquid chromatography) on a Vydac-C18-column (gradient: water +0.05% TFA/acetonitrile, 5% to 60% acetonitrile in 20 min, detection: 214 and 750 nm.)

The structures of the synthesized conjugates of dyes and peptides are summarized in the following review:

Dye conjugates with VIP-receptor-binding peptide I

Example 14 to 16:

color conjugates 1-3 with VIP (1-28)

[image]

n = 1: indocarbocyanine-VIP(1-28)-conjugate 14

n = 2: indodicarbocyanine-VIP(1-28)-conjugate 15

n = 3: indotricarbocyanine-VIP(1-28)-conjugate 16

Example 17:

Conjugate of dye 12 with Cys17-VIP(1-28)

[image]

Indotricarbocyanine-Cys17-VIP(1-28)-conjugate 17

Example 18 to 20:

Conjugate colors 1-3 with VIP(14-28)

[image]

n = 1: indocarbocyanine-VIP(14-28)-conjugate 18

n = 2: indodicarbocyanine-VIP(14-28)-conjugate 19

n = 3: indotricarbocyanine-VIP(14-28)-conjugate 20

Example 21 to 23:

Conjugate colors 4-6 with VIP(14-24)

[image]

n = 1: indocarbocyanine-β-alanine-VIP(14-28)-conjugate 21

n = 2: indodicarbocyanine-β-alanine-VIP(14-28)-conjugate 22

n = 3: indotricarbocyanine-β-alanine-VIP(14-28)-conjugate 23

Example 17:

Synthesis of resin peptide conjugates consisting of VIP-receptor binding peptides and dyes 10-12.

In order for dyes 10-12, which carry bromine, to chemoselectively bind to the peptide through a thioether bond, one cysteine or homocysteine with an orthogonal protective group must be incorporated into the peptide. The synthesis of the solid phase of the peptide will be carried out as described in examples 14-16/18-27, and the building element Fmoc-cys(Mmt)-OH will be added. The monomethoxytrityl group (Mmt) can be cleaved off via 1% TFA/5% triisobutylsilane in dichloromethane as a result of the established side protective groups. For this, the resin will be incubated 3 times with 1 ml of the above solution for 10 minutes. After washing the resin with dichloromethane (3x), DMF (5x) and ethanol (3x), the resin will be incubated for 2 min with a 20% solution of cesium carbonate (1 ml) and finally with water (2x), ethanol (2x) and DMF - (2x) washed. In conclusion, 75 µmol of a specific color 10-12 (1.5 eq) will be dissolved in 600 µl of dimethylformamide and added to the specific resin, and the procedure will be repeated after 30 min. Finally, the resin will be washed 5x with dimethylformamide and with dichloromethane (2x). After drying the resin in air, the protective groups are removed and separated from the "support", as described above.

The structures of further synthesized conjugates of peptides and dyes are summarized in the following review:

Dye conjugates with VIP-receptor-binding peptides II

Example 24:

Conjugate of dye 6 with Lys25-VIP(14-25)

[image]

(ε-amino-indotricarbocyanine)-Lys25-VIP(14-25)-conjugate 24

Example 25:

Color 3 conjugate with D-VIP(14-24)

[image]

Indotricarbocyanine-D-VIP(14-24)-conjugate 25

Example 26:

Conjugate of color 13 with D-VIP(14-24)

[image]

Indotricarbocyanin-5-glucamide of carboxylic acid-D-VIP(14-24)-conjugate 26

Example 27:

Conjugate of color 13 with retro-D-VIP(14-24)

[image]

Indotricarbocyanine-5-glucamide of carboxylic acid-retro-D-VIP(14-24)-conjugate 27

Example 28 to 32:

Synthesis of peptide conjugate resin consisting of somatostatin-binding peptides and dyes 1-9 and 13

General provisions:

The synthesis will be carried out on 500 mg of TCP-Thr(But)-fmoc-resin (Pepchem Tubingen) with one addition (specific groups) of 0.49 mmol/g. The peptide will be synthesized "step by step" using the following temporary protecting groups: tert.-butyl for Thr and Ser, trityl for Cys and Asp, Boc for trp and Lys. HBTU will be used as the condensation reagent.

After the separation of the N-terminus of the Fmoc-protecting group of dyes 1-9, 13 will be condensed in a special synthesis step. For that there will be 255 mg of resin in ca. 2 ml DMF suspended and with 0.5 mmol dye, 0.5 mmol HBTU and 0.17 ml DIEA mixed. The mixture will be at room temperature for the next 18 hours. mixed, the resin will be removed after the flow, the residue washed with dichloromethane and dried. Separation of the dye-peptide conjugate from the resin is followed by 95% trichloroacetic acid, with the addition of triisopropylsilane, and subsequent lyophilization from 10% acetic acid. The crude product will be cyclized using activated carbon and chromatographically purified (50x300 mm VYDAC RP-18, gradient: water/acetonitrile).

The structures of synthesized dye-peptide conjugates are summarized in the following review:

Dye conjugates with somatostatin binding peptides

Examples 28 to 30:

Conjugate dye 1-3 with pentetreotide

[image]

n = 1: Indocarbocyanine-pentetreotide 28

n = 2: Indodicarbocyanine-pentetreotide 29

n = 3: Indotricarbocyanine-pentetreotide 30

Example 31:

Dye 3 conjugate with somatostatin-14

[image]

Indotricarbocyanin-somatostatin-14-conjugate 31

Example 32:

Conjugate of dye 13 with somatostatin-14

[image]

Carbonic acid indotricarbocyanin-5-glutamide-somatostatin-14-conjugate 32

Examples 33 to 38:

Synthesis of peptide conjugates from neurotensin-peptide and dye 3

Synthesis of substances follows analogously as described for Examples 14-27, general protocol.

The structures of synthesized conjugates of peptides and dyes are summarized in the following review:

Dye conjugates with neurotensin binding peptides

Examples 33 to 35:

Conjugate dye 7-9 with D-Tyr11-neurotensin (7-13)

[image]

n = 1: Indocarbocyanine-D-Tyr11-neurotensin (7-13)-conjugate 33

n = 2: Indodicarbocyanine-D-Tyr11-neurotensin (7-13)-conjugate 34

n = 3: Indotricarbocyanine-D-Tyr11-neurotensin (7-13)-conjugate 35

Example 36:

Conjugate of dye 2 with D-Tyr11-neurotensin

[image]

(ε-amino-Lys6-indodi-carbocyanine)-D-Tyr11-neurotensin-conjugate 36

Example 37 to 38:

Conjugate of color 10-11 with D-Tyr11-neurotensin (5-13) - Cys

[image]

n = 1: Indocarbocyanin-D-Tyr11-neurotensin (5-13)-Cys-conjugate 37

n = 2: Indodicarbocyanine-D-Tyr11-neurotensin (5-13)-Cys-conjugate 38

Example 39:

Absorption and fluorescence properties of synthesized dye and peptide conjugates

Absorption maxima and coefficients of specific wavelengths characteristic of the mentioned compounds (Extinktionskoeffizient) were determined in PBS and bovine plasma (Perkin Elmer Lambda 2 device). The fluorescence emission spectrum was obtained after excitation on the short-wave side (ca. 40 nm from the absorption maximum) (SPEX Fluorolog, R928 PMT)

Absorption and fluorescence data are combined in Table 4. Table 5 shows typical absorption and fluorescence-emission spectra on examples.

Example 40:

Determination of cell images using fluorescence microscopy

The binding and uptake (absorption into tissues) of the compounds presented by the invention were investigated in vitro on human tumor cells, which receptors for vasoactive intestinal peptide and/or somatostatin and/or neurotensin are expressed.

For this, 5x105 tumor cells were incubated in 1.5 ml of medium containing the test substance. For this, different concentrations of test substances (10 nM - 10 µM) were used, the incubation time was varied (1 min - 24 hours). After incubation, the cells were fixed and microscopic preparations were prepared. Examination was performed on an Axiovert 135 fluorescence microscope, which was equipped with one Cy 7-(Exciter HQ 710/70nm, Emitter 810/90nm, Beamsplitter 750nm LP), Cy5-(Exciter 575-625 nm , Emitter 660-710nm BP, Beamsplitter 645nm) and Cy3- filter group (Exciter 546/12 nm, Emitter 590 nm LP, Beamsplitter 580 nm). Photographs of all preparations were taken using a white light source and fluorescence, using one CCD-camera (Visitron RTE/CCD – 576), and digitally stored.

The selected results are described below:

Microscopic images were taken with white light and a fluorescent source (Cy7-filters) of HT29 cells, after 30 minutes of incubation with 10 µM Indotricarbocyanine-VIP(1-28)-conjugate 16. On the "fluorescent" images, fluorescence was further homogeneously distributed over the cells. detected. Additionally, areas with increased signal intensity are visible, which may be associated with vesicular compartments. The cells on the "fluorescent" images correlate in their spatial arrangement with those recorded using a white light source.

With the following compounds, "white" and "fluorescent" recordings (Cy7-filters) were obtained in an analog sequence:

Indotricarbocyanine-VIP(14-28)-conjugate 20, 10 µM, HT29-cells. Homogeneously distributed fluorescence over the cells, good correlation with "white" images.

Indotricarbocyanine-retro-D-VIP(24-14) 25, 10 µM, HT29-cells. Homogeneously distributed fluorescence over the cells, good correlation with "white" images.

Indotricarbocyanine-pentetreotide-conjugate 30, 10 µM, RIN38-VIP1-cells. Fluorescent areas with a vesicular pattern in the area of the cell membrane, good correlation with "white" images.

Furthermore, fluorescent images were obtained in an analog version with the following compounds:

(Amino-indotricarbocyanine-Lys25-VIP(14-25)-conjugate 24, 10 µM, RIN38-VIP1-cell, Cy7-filters. The fluorescence image shows one homogeneous, intracellular fluorescence, near the cell nucleus.

Indocarbocyanine-VIP(1-28)-conjugate 15, 10 µM, RIN38-VIP1-cell, Cy5-filters. The fluorescent image shows intracellular fluorescent areas with a vesicular pattern in the area of the cell membrane.

Indocarbocyanine-VIP(1-28)-conjugate 14, 10 µM, RIN38-VIP1-cell, Cy3-filters. The fluorescent image shows intracellular fluorescent areas with a vesicular pattern in the area of the cell membrane.

Example 41:

Examination of tumor accumulation using in-vivo-fluorescence photographs in tumor-bearing mice

The properties that give an image of the compounds presented by the invention were tested in-vivo, after injection in mice (hairless), tumor carriers. For this, 0.1 µmol/kg to 2 µmol/kg of the substance was administered intravenously, and the accumulation in the tumor area was monitored in the time interval from 0 to 48 hours. The fluorescence of the substance was induced by irradiating the animals with near-infrared light of wavelength 640 nm (indodicarbocyanins) or 740 nm (indotricarbocyanins), which was produced using a Nd:YAG laser. Fluorescent emission at a wavelength of >700 nm or >800 nm was detected by an intensified CCD-camera, and the fluorescent images were digitally stored.

The selected results are described below:

Fluorescence images of the whole body before and 1 hour after the application of 0.1 µmol/kg indotricarbocyanine-VIP(14-28)-conjugate 20 were taken from one "naked" mouse, the carrier of a tumor disease (HT29-tumor in the right hind loin) . Fluorescence intensity before application is negligible (weak autofluorescence). 1 hour after the application resulted in a 2-fold signal intensity in the tumor, relative to the contralateral loin, in an otherwise homogeneous distributed fluorescence emission over the rest of the body.

Fluorescence images of the whole body before and 1 hour after the application of 0.1 µmol/kg indotricarbocyanine-pentetreotide-conjugate 30 were taken from one tumor-bearing mouse (RIN38-SSTR2-tumor in the right posterior loin). Fluorescence intensity before application is negligible (weak autofluorescence). 1 hour after the application resulted in a 3-fold signal intensity in the tumor, relative to the contralateral loin, in an otherwise homogeneous distributed fluorescence emission over the rest of the body.

From one mouse bearing a tumor disease (HT29-tumor in the right back loin) there were fluorescence images of the whole body before and 1 hour after the application of 0.1 µmol/kg (ε-amino-indotricarbocyanine)-Lys25-VIP(14-25)- of conjugate 25, recorded. Fluorescence intensity before application is negligible (weak autofluorescence). 1 hour after the application resulted in a 1.5-fold signal intensity in the tumor, relative to the contralateral loin. Additionally, an increased fluorescent signal was detected in the kidneys.

Fluorescence images of the whole body before and 5 min after the application of 0.2 µmol/kg indodicarbocyanine)-VIP(1-28)-conjugate 15 were recorded from one mouse bearing a tumor disease (HT29-tumor in the right posterior loin). Fluorescence intensity before application is negligible (weak autofluorescence). 1 hour after application resulted in a 1.4-fold signal intensity in the tumor, towards the contralateral flank. In addition, an increased fluorescent signal was detected in the kidneys.

Example 42:

Investigation of the stability of peptide and dye conjugates in bovine plasma

The chemical stability of the compounds presented by the invention in plasma was investigated in vitro, depending on the time, by means of HPLC. For this, a 1mM peptide solution in PBS was added to bovine plasma (Graeber company, frozen, for heparin analysis), a concentration of 30µM was pipetted, and the solution was incubated at 37ºC.

At different time intervals (0.5; 1; 2; 4; 6; 24 hours) the samples were processed, during which 1 ml of the plasma solution was mixed with 1 ml of MeOH, and the precipitated proteins were centrifuged.

The analysis of the remaining liquid part followed by means of HPLC, through the determination of the amount at 750 nm, which refers to the content after 1 minute of incubation at 0ºC (control test).

HPLC: Beckmann, diode array detector TIDAS (J&M company) 350-1000 nm;

Column: Chromasil 5µ, 250 mm x 4.5 mm

Eluent: A: 90% H2O(+0.5% TFA) / 10% MeOH

B: 10% H2O(+0.5% TFA) / 90% MeOH

Gradient: 10% B to 100% B in 20 min

Examples are summarized in Table 6.

Example 43:

Substitution analysis of VIP moves "spot" syntheses

1. Peptide synthesis on cellulose

Peptide synthesis on cellulose (spot-synthesis) was first published in 1988 by R. Frank and R. Doring, and described in detail by R. Frank in 1992. Here was the method established at AG Schneider-Mergener, used.

The cellulose membrane was chemically modified in order to prepare suitable anchor functions for the subsequent peptide synthesis. In doing so, one mercapto-function was introduced on one amino-functionalized cellulose membrane (CAPE-membrane). The first amino acid in the form of a bromopropyl ester could be attached to this mercaptofunction. Subsequently, all amino acids of the peptide were successively upgraded according to the Fmoc strategy. At the end, the indodicarboyanin-dye of the N-terminus was attached to the peptide, and then all side protective groups were cleaved off.

In order for the peptides to be tested as receptor compounds, the peptide had to be cleaved from the cellulose. For this, a method was developed, which for the first time succeeded in cleaving peptides with an authentic C-terminus from cellulose.

1a. Cellulose membrane modification

One 20x30 cm cellulose membrane (Whatman 50) was on 2 min. incubated with methanol/1.2% perchloroacetic acid and further dried.

After a 3-hour incubation with 10% epibromohydrin in dioxane/1.2% perchloric acid, it was left for 30 min. in the reaction with methanol, and after that it was washed twice with methanol.

It was subsequently washed 3x with dimethylformamide (DMF) and incubated overnight with 50% 1,3-diaminopropane (v/v) in DMF. After that, it was washed as follows: 3x DMF, 2x ethanol, 2x aqua dest., 2x ethanol for 15 min with 5M sodium methanolate, 3x methanol, 4x aqua dest., 3x ethanol, 1x diethyl ether.

1b. Definition of "spots".

To define the spots, 1.3 µl of 0.6 M Fmoc-ß-alanine-Opfp-solution was double-pipetted with an auto-spot robot 222 XL (Abimed, Langenfeld) at a reaction time of 15 min to certain points of the cellulose membrane.

The membrane was acetylated for 2 minutes with 2% acetic anhydride solution and 30 minutes with 20% acetic anhydride/10% diisopropylethylamine.

To separate the Fmoc-protecting groups, the membrane was then washed 3x with DMF, 2x for 10 min. incubated with 20% piperidine solution, washed 5x with DMF and 1x with ethanol. Free amino groups could be visualized with bromphenol blue. After repeated washing with ethanol, the membrane was dried.

1c. Binding of Mmt-mercaptopropionic acid and bromopropyl ester

0.6 M mercaptopropionic acid was in the reaction time of 15 min. double on the defined spot pipetted. It was then washed 3 times with DMF and 3 times with dichloromethane (DCM).

Separation of the Mmt-protecting groups was followed by incubation for 2 min. with 10% dichloroacetic acid/0.5% trifluoroacetic acid and 3x5 min. with 10% dichloroacetic acid/0.5% trifluoroacetic acid/5% triisobutylsilane. The following washing steps were performed:

1x DCM, 2x ethanol, 1x aqua dest., 1-2 min. with 10% cesium carbonate, 1x aqua dest., 2x ethanol, 1x diethylether.

The determined Fmoc-bromopropyl-esters of amino acids were 3x, at a concentration of 0.6 M and a reaction time of 15 min. connected. Removal of the Fmoc-protecting groups was carried out in 1b.

1 d. Linking of amino acids

Peptides were built through repeated pipetting of a 0.6 M solution of amino acids in N-methylpyrrolidone onto the spots and final removal of the Fmoc-protecting groups.

1e. Binding of indodicarbocyanine dye

One solution of 0.3 M indodicarbocyanine dye with 0.3 M TBTU and 0.6 M diisopropylethylamine was activated and pipetted 4 times with a reaction time of 15 min onto the spots.

1 f. Cleavage of side protecting groups

Removal of side protective groups follows through membrane treatment in regular sequences with 90% trifluoroacetic acid/3% triisobutylsilane/2% aqua. dest./1% phenol for 30 min and with 50% trifluoroacetic acid/3% triisobutylsilane/2% aqua.dest./1% phenol for 2.5 hours. It was subsequently washed 4x with dichloromethane, 3x with DMF and 1x with ethanol.

2. Separation of the peptide from the cellulose membrane

Spots were drilled and washed with methanol. For separation, it was incubated for 30 min with 70 mM sodium methanolate in methanol. The pH was corrected by adding 37% hydrochloric acid. Next, the peptides were dried in a Speed-Vac (lyophilizer).

After drying, the peptides were dissolved in aqua dest. and analyzed by RP-HPLC and MALDI-TOF.

3. Cell assay and flow cytometry

The concentration of VIP-derivatives was read photometrically via color. In the cell assay, the peptides were used in conc. of 150 mM. In doing so, 1x105 RIN38(VAC1)-cells with VIP-derivatives were incubated for one hour at 37ºC in binding buffer (50 mM Tris/HCl, pH 7.5, 5nM MgCl2, 1mM CaCl2, 100mM NaCl, 4% BSA). .

Subsequently, the cells were washed twice with PBS, transferred to FACS-reagent containers and incubated for 5 min. on 377 g centrifuged. The cell sediment was resuspended in 300 µl of cell fixative, measured on a FACS-Calibur (Bacton Dickinson) with FL4-Optik.

4. Analysis of results

Fluorescence intensity in flow cytometry measured by native, naturally occurring, human VIP-peptides was 100% used. The standard deviations of 28 native VIP-peptides were 11%. Further VIP-peptides were 100% uniform with such.

Figure 7 shows the relative fluorescence intensity of RIN38 VPAC1-cells after incubation in the presence of 150 nM dye-labeled peptides for 1 hour, at 37ºC. Data in percentages refer to the native peptide of a certain order.

Literature for spot-synthesis:

1. Frank, R. (1992) Spot synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 48, 9217-9232

2. Kramer, A., Schneider-Mergener, J. (1998) Synthesis and screening of peptide libraries on continuous cellulose membrane supports. Methods in Molecular Biology 87, 25-39

3. Volkmer-Engert, R., Hoffmann, B., Schneider, Mergener, J. (1997) Tetrahedron Lett. 38, 1029-1032

4. Licha, K., Bhargava, S:, Rheinländer, C., Becker, A., Schneider-Mergener, J., Volkmer-Engert, R. (in press) Highly parallel Nano-synthesis of cleavable peptide-dye conjugates on cellulose membranes. Tetrahedron Lett.

Example 44

a) 5-N-(2,3-dihydroxypropyl)aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine

0.9 g (2.6 mmol) of 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine (Anal. Biochem. 217, 197, 1994) will be in 30 ml of absolute N,N-dimethylformamide and 3 ml of pyridine were added and mixed with 1.35 g (5.3 mmol) of disuccinimidyl carbonate. After 3 hours, 0.965 g (10.6 mmol) of 2,3-dihydroxypropylamine was added. Everything is mixed at room temperature, the mixture is evaporated to dryness, and the residue is mixed with diethyl ether. The solid substance will be sucked off, and purification will be performed on RP-material, chromatographically.

Yield: 0.82 g (76% d. Th.)

Analysis (refers to substances free from solvents).

[image]

b) 4-[2-[4-chloro-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]-3,5-(propane -1,3-diyl)-1,3,5-heptatrien-1-yl]-5-N(dihydroxypropyl)aminocarbonyl-3,3-dimethyl(3H)indolio]butanesulfonate, sodium salt

One solution of 360 mg (1 mmol) N-[5-anilino-3-chloro-2,4-(propane-1,3-diyl) 2,4-pentadien-1-ylidene]anilinum chloride, 825 mg ( 2 mmol) of 5-N-(2,3-dihydroxypropyl)aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine (Example 44a) and 330 mg (4 mmol of dry sodium acetate in 30 ml of ethanol will be boiled for 2 hours, under argon, on the return flow.

Yield: 0.58 g (59% d. Th.)

Analysis (refers to solvent-free substances):

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c) 4-[2-[4-(4-(2-carboxyethyl)phenyloxy)-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indoline-2- ylidene]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-5-N(dihydroxypropyl)aminocarbonyl-3,3-dimethyl(3H)indolio]butanesulfonate, sodium salt, N-hydroxysuccinimide ester

225 mg (1.4 mmol) of 3-(4-hydroxyphenyl)propionic acid was dissolved in 10 ml of dry N,N-dimethylformamide, under a protective gas, and with 65 mg (2.7 mmol) of sodium hydride (60% in oil) mixed. After 30 min. 138 mg (0.14 mmol) of 4-[2-[4-chloro-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene] were added ]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1 yl]-5-N (dihydroxypropyl)aminocarbonyl-3,3-dimethyl (3H)indolio]butanesulfonate, sodium salt (example 44b) and mixed for the next 30 min. The reaction mixture is then mixed with dry ice and evaporated to dryness. The residue is purified via a preparative HPLC. To prepare the active ester, 14 mg (120 µmol) of N-hydroxysuccinimide and 2 mg (2.4 µmol) of carboxylic acid are dissolved in 200 µl of N,N-dimethylformamide. After 10 min. 24 mg (120 µmol) of dicyclohexylcarbodiimide is added and stirred overnight at room temperature. The active ester will be further used in the next stage without further processing.

Analogous to the synthesis according to Example 14-16 and 18-27a (solid phase peptide synthesis) the solid phase VIP analog HSDAVFWDNY TRLRKQMAVK KYLNSILN will be synthesized. Dye 4-[2-[4-(4-(2-carboxyethyl)phenyloxy)-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene ]-3,5-(propane-1,3-diyl)–1,3,5–heptatrien-1yl]-5-N (dihydroxypropyl) aminocarbonyl –3,3-dimethyl (3H) indolio] butanesulfonate, sodium salt,

it will be according to example 14-16 and 18-27 b (binding of dyes) linked to the peptide and according to example 14-16 and 18-27 c (detachment of protective groups and separation of the peptide-dye conjugate) that conjugate is isolated and purified.

d) 4-[2-[4-(4-(2-isothiocyanatoethyl)phenyloxy)-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2- ylidene]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-5-N(dihydroxypropyl)aminocarbonyl-3,3-dimethyl(3H)indolio]butanesulfonate, sodium salt

To a suspension of 28 mg (0.6 mmol) of sodium hydride (60% in oil) in 4 ml of dry N,N-dimethylformamide, 116 mg (0.6 mmol) of 4-[2-[4 -chloro-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]-3,5-(propane-1,3-diyl)-1 ,3,5-heptatrien-1 yl]-5-N (dihydroxypropyl)aminocarbonyl-3,3-dimethyl (3H)indolio]butanesulfonate, sodium salt (Example 44b). The mixture is stirred overnight at room temperature. and mixed with dry ice. It is evaporated to dryness on a rotary evaporator and the residue is preparatively purified on HPLC.

Usability: 85 mg (54% d. Th).

Analysis (refers to solvent-free substances):

[image]

Analogous to the synthesis according to Example 14-16 and 18-27a (solid phase peptide synthesis) the solid phase VIP analog HSDAVFTDNY TRLRFQMAVK KYLNSILN will be synthesized. Color 4-[2-[4-(4-(2-isothiocyanatoethyl)phenyloxy)-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene ]-3,5-(propane-1,3-diyl)–1,3,5-heptatrien-1yl]-5-N (dihydroxypropyl) aminocarbonyl –3,3-dimethyl (3H) indolio] butanesulfonate, sodium salt, it will be attached to the peptide N-terminus according to example 14-16 and 18-27 b (binding of dyes) to the peptide connected and according to example 14-16 and 18-27 c (detachment of protective groups and separation of the peptide-dye conjugate) that conjugate is isolated and purified.

In an analogous way, further synthetic hydrophilic paints can be constructed from the following building elements:

Hydrophilic indolenine derivatives with hydroxyalkyl substituents: (prepared according to example 44a

a) 5-N-(2,3-dihydroxypropyl)-N-methylaminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H) indolenine

b) 5-N-(dihydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine

c) 5-N-(2,3-dihydroxypropyl)-N-(hydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H) indolenine

d) 5-N,N-(bi-dihydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine

e) 5-N-(2,3,4,5,6-pentahydroxyhexyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine

f) 5-N-(1,3,4-trihydroxybutyl-2-yl)-N-methylaminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine

Example 45

A) 5-N-(2,3,4,5,6-pentahydroxyhexyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine

0.6 g (1.8 mmol) of 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine (Anal. Biochem. 217, 197, 1994) will be in 20 ml of absolute N,N-dimethylformamide and 2 ml of pyridine were added and mixed with 0.95 g (3.6 mmol) of disuccinimidyl carbonate. After 2 hours, 1.4 ml (10 mmol) of triethylamine and 322 mg (1.8 mmol) of glucamine are added. Everything is mixed overnight at room temperature, evaporated to dryness, and the residue is mixed with diethyl ether. The solid substance is sucked off and purified on RP-material, chromatographically.

Yield: 0.67 g (74% d.Th.).

Analysis (refers to substances without solvents):

[image]

B) 4-[2-[4-chloro-7-[5-N-(2,3,4,5,6-pentahydroxyhexyl)aminocarbonyl-3,3-dimethyl-1-(4sulfonatobutyl)indolin-2-ylidene ]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl5-[N-(2,3,4,5,6-pentahydroxyhexyl )-aminocarbonyl](3H)indolio]butanesulfonate, sodium salt

Solution of 180 mg (0.5 mmol) N-[5-anilino-3-chloro-2,4(propane-1,3-diyl)-2,4-pentadien-1-ylidene]aniline chloride, 503 mg (1 mmol) 5-N-(2,3,4,5,6-pentahydroxyhexyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine (Example 45a) and 165 mg (2 mmol) of dry sodium acetate in 10 ml of ethanol will be boiled for 2 hours under argon on a reflux condenser. Ethanol is further distilled off and the residue is purified by chromatography.

Usability: 0.31 g (53% d.Th.)

Analysis (refers to substance without solvent):

[image]

C) 4-[2-[4-(4-isothiocyanatothiophenyloxy)-7-[5-N-(2,3,4,5,6-pentahydroxyhexyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl) )indolin-2-ylidene]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl5-[N-(2,3,4 ,5,6-pentahydroxyhexyl)-aminocarbonyl](3H)indolio]butanesulfonate, sodium salt

54 mg (0.4 mmol) of 4-aminothiophenol will be in 10 ml of absolute N,N-dimethylformamide under an atmosphere saturated with argon, dissolved at room temperature with 165 mg (0.14 mmol) of 4-[2-[4-chloro -7-[5-N-(2,3,4,5,6-pentahydroxyhexyl)aminocarbonyl-3,3-dimethyl-1-(4sulfonatobutyl)indolin-2-ylidene]-3,5-(propane-1, 3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl5-[N-(2,3,4,5,6-pentahydroxyhexyl)-aminocarbonyl](3H)indolio] butanesulfonate, sodium salt (example 45b). after 10 minutes, the reaction mixture will be mixed with dry ice and 210 mg (1 mmol) of thiocarbonyldiimidazole will be added. After 45 minutes, the dye will be precipitated with dietether, and the solid substance will be isolated by centrifugation. For purification, RP-material can be chromatographed.

Usability: 78 mg (43% d.Th.)

Analysis (refers to substances without solvents):

[image]

Analogously to the synthesis according to example 14-16 and 18-27 a (solid phase peptide synthesis) the VIP analog HSWAVFTDNY TRLRKQMAVK KYLNSILN on the solid phase will be synthesized. Dye 4-[2-[4-(4-isothiocyanatothiophenyloxy)-7-[5- N-(2,3,4,5,6-pentahydroxyhexyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl) indolin-2-ylidene]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl5-[N-(2,3,4, 5,6-pentahydroxyhexyl)-aminocarbonyl](3H)indolio]butanesulfonate, the sodium salt will be attached to the peptide, the N-terminus and according to examples 14-16 and 18-27 c (removal of protective groups and separation of the conjugate of dye and peptide) will be that conjugate will be isolated and purified.

In an analogous way, further symmetrical hydrophilic paints can be upgraded on the following building elements:

Hydrophilic indolenine derivatives with hydroxyalkyl substituents: (prepared according to example 44 a).

a) 5-N-(2,3-dihydroxypropyl)-N-methylaminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H) indolenine

b) 5-N-(hydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine

c) 5-N-(2,3-dihydroxypropyl)-N-(hydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H) indolenine

d) 5-N,N-(bi-hydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine

e) 5-N,N-(2,3,4,5,6-pentahydroxyhexyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H) indolenine

f) 5-N-(1,3,4-trihydroxybutyl-2-yl)-N-methylaminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine

Example 46

7-[5-N-(2,3-dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]-1,3,5- heptatrien-1-yl]-3 ,3-dimethyl-5-carboxy (3H) aminocarbonyl] (3H) indolio] butanesulfonate, sodium salt

A solution of 2.35 g (5.71 mmol) of 5-N-(2,3-dihydroxypropyl)aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolene (Example 44a) and 1 57 g (5.5 mmol) of glutaconaldehyde dianilide hydrochloride in 25 ml of acetic anhydride will be mixed for 30 minutes at 120ºC. 2.4 g (7.1 mmol) of 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine, 1.71 g of sodium acetate, 22 ml of acetic anhydride and 8 .6 ml of acetic acid. The reaction mixture is stirred for 1 hour at 120ºC, cooled to room temperature and the product is precipitated with diethyl ether. The raw material is chromatographed over RP-material.

Usability: 9.1 g (40% d.Th.)

Analysis (refers to substances without solvents):

[image]

Analogously to the synthesis according to example 14-16 and 18-27 a (solid phase peptide synthesis) the VIP analog HSDAVFTDNY TRLRKKMAVK KYLNSILN on the solid phase will be synthesized. Dye 7- [5- N - (2,3-dihydroxypropyl) aminocarbonyl -3,3-dimethyl -1-(4-sulfonatobutyl) indolin-2-ylidene]-1,3,5-heptatrien-1-yl]- 3,3-dimethyl-5-carboxy (3H)indolio]butanesulfonate, the sodium salt will be attached to the peptide, the N-terminus and according to examples 14-16 and 18-27 c (removal of protective groups and separation of the conjugate of dye and peptide) will be this conjugate will be isolated and purified by HPLC.

In an analogous way, further asymmetric hydrophilic paints can be upgraded on the following building elements:

Hydrophilic indolenine derivatives with hydroxyalkyl substituents: (prepared according to example 44 a).

a) 5-N-(2,3-dihydroxypropyl)-N-methylaminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H) indolenine

b) 5-N-(dihydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine

c) 5-N-(2,3-dihydroxypropyl)-N-(hydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H) indolenine

d) 5-N,N-(bi-dihydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine

e) 5-N,N-(2,3,4,5,6-pentahydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine

f) 5-N-(1,3,4-trihydroxybutyl-2-yl)-N-methylaminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine

Indolenine derivatives with carboxyl groups:

a) 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine

b) 5-carboxymethyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H) indolenine

c) 5-carboxy-1-(3-sulfopropyl)-2,3,3-trimethyl (3H) indolenine

d) 5-carboxymethyl-1-(3-sulfopropyl)-2,3,3-trimethyl (3H)indolenine

e) 5-carboxy-1-(2-sulfoethyl)-2,3,3-trimethyl (3H) indolenine

f) 5-carboxymethyl-1-(2-sulfoethyl)-2,3,3-trimethyl (3H) indolenine

Dialin derivatives for reactions with the aforementioned indolenines to form mono-, di- or tricarbocyanine:

a) glutaconaldehydedianilide hydrochloride

b) malonaldehyde-bi-phenylimine-hydrochloride

c) N,N-diphenylformamide

d) N-[5-anilino-2,4-(propane-1,3-diyl)-2,4-pentadien-1-ylidene]anilinium chloride

e) N-[5-anilino-2,4-(ethane-1,2-diyl)-2,4-pentadien-1-ylidene]anilinium chloride

f) N-[5-anilino-3-chloro-2,4-(propane-1,3-diyl)-2,4-pentadien-1-ylidene]anilinium chloride

g) N-[5-anilino-3-chloro-2,4-(ethane-1,2-diyl)-2,4-pentadien-1-ylidene]anilinium chloride

Example 47

7-[5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]-1,3,5-heptatriene- 1-yl]-3,3-dimethyl-5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl](3H)indolio]butanesulfonate, sodium salt,

N-hydroxysuccinimide ester

a) 4-[2-[4-chloro-7-[5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl-3,3-dimethyl-1-(4sulfonatobutyl)indolin-2-ylidene ]-3,5-[2-(methoxycarbonyl)propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl-5-[N-(1,3, 4-trihydroxybut-2-yl)-aminocarbonyl](3H)indolio]butanesulfonate, sodium salt

To a solution of 0.8 g (5 mmol) of 4-(methoxycarbonyl)-cyclohexanone in 5 ml of dichloromethane, 5.5 ml (10 mmol) of phosphorus oxychloride and 6 ml of N,N-dimethylformamide were added at 0ºC. It is continuously heated for 1 hour in the return flow. Dichloromethane is distilled to it and added, at max. 5ºC aniline and 10 ml methanol. The reaction mixture is poured onto ice, 5 ml of concentrated hydrochloric acid is added and the intermediate product is left to crystallize for 5 hours at 0ºC. The crystals are separated and used without further purification in the next reaction. For this, the crystals are dissolved in dry methanol and 4.4 g (10 mmol) of 5-N-(2,3,4,5,6-pentahydroxyhexyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3 ,3-trimethyl(3H)indolenine (see Example 45) and 0.8 g of dry sodium acetate. Everything is heated for 1 hour on a reflux condenser, the solid substance is filtered off and the filtrate is evaporated to dryness. The residue is chromatographed for purification.

Usability: 3.25 g (58% d.Th.)

Analysis (refers to solvent-free substances):

[image]

b) 7-[5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]–3,5-(2 -carboxypropane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl-5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl](3H )indolio]butanesulfonate, sodium salt

A mixture of 10 mg (0.4 mmol) of sodium hydride and 80 mg (1.3 mmol) of ethanethiol in 10 ml of dry N,N-dimethylformamide will be stirred, under nitrogen, for 30 minutes at room temp. 112 mg (0.1 mmol) of 4-[2-[4-chloro-7-[5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl-3,3-dimethyl-1 -(4sulfonatobutyl)indolin-2-ylidene]-3,5-[2-(methoxycarbonyl)propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl-5 -[N-(1,3,4-trihydroxybut-2-yl)-aminocarbonyl](3H)indolio]butanesulfonate, sodium salt (Example 47 a) in 3 ml of N,N-dimethylformamide. The reaction mixture will be heated for 2 hours at 100ºC and after cooling to room temperature, mixed with carbon dioxide. The mixture will be evaporated to dryness on a rotary evaporator, and the residue will be extracted with hot ethanol. The extract will be evaporated to dryness and chromatographed.

Usability: 75 mg (67% d.Th.)

Analysis (refers to solvent-free substances):

[image]

c) 7-[5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]–3,5-(2 -carboxypropane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl-5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl](3H )indolio]butanesulfonate-N-hydroxysuccinimide ester, sodium salt

To prepare the active ester, 14 mg (0.12 mmol) of N-hydroxysuccinimide and 64 mg (0.06 mmol) of carboxylic acid in 2 ml of N,N-dimethylformamide. After 15 min. 25 mg (0.12 mmol) of dicyclohexylcarbodiimide is added and stirred overnight at room temperature. The active ester was purified by preparative HPLC.

Usability: 60 mg (86% d. Th.)

Analysis (refers to solvent-free substances):

[image]

Analogously to the synthesis according to example 14-16 and 18-27 a (solid phase peptide synthesis) the VIP analog HSDAVFTDNY TRLRKAMAVK KYLNSILN on the solid phase will be synthesized. Dye 7- [5- N - (2,3-dihydroxypropyl) aminocarbonyl -3,3-dimethyl -1-(4-sulfonatobutyl) indolin-2-ylidene]-3,5-(2-carboxypropane-1,3- diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl-5-N-1,3,4-trihydroxybutyl-2-yl)-aminocarbonyl (3H)indolio]butanesulfonate, sodium salt be will according to example 14-16 and 18-27 c (binding of dye) to the peptide N-terminus and according to example 14-16 and 18-27 c (removal of protective groups and separation of the dye-peptide conjugate) that conjugate will be isolated and purified.

Example 48

7-[5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]–3,5-(2-carboxypropane -1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl-5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl](3H)indolio ] butanesulfonate, sodium salt

a) 5-N-(11-aminoundecyl)aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine

340 mg (1 mmol) of 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine (Anal. Biochem. 217,197, 1994) will be in 5 ml of absolute N,N-dimethylformamide and 1 ml of pyridine was added and mixed with 0.5 g (2 mmol) of disuccinimidyl carbonate. After 3 hours, 0.805 g (4 mmol) of 11-aminoundecanoic acid was added. It is stirred overnight at room temperature, evaporated to dryness and the residue mixed with diethyl ether. The solid substance is removed and purified by chromatography on RP-material.

Yield: 0.37 g (71% d. Th.)

Analysis (refers to solvent-free substances):

[image]

b) 7-[5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]–3,5-(2 -carboxypropane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl-5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl](3H )indolio] butanesulfonate, sodium salt

One mixture of 0.35 g (0.67 mmol) of 5-N-(11-aminoundecyl)aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolene (Example 48a) and 0, 18 g (0.645 mmol) of glutaconaldehydedianil in 3 ml of acetic anhydride will be stirred for 20 min at 110ºC. 344 mg (0.83 mmol) of 5-N-(2,3-dihydroxypropyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine (example 44a), 0 ,2 g of sodium acetate, 3 ml of acetic anhydride in 1 ml of acetic acid. The reaction mixture is stirred at 110ºC for 2 hours, cooled to room temperature. and precipitate the product with diethyl ether. The crude product is chromatographed over RP-material.

Yield: 0.41 g (60% d. Th.).

Analysis (refers to solvent-free substances):

[image]

Analogously to the synthesis according to example 14-16 and 18-27 a (solid phase peptide synthesis) the VIP-analog HSDAVFTDNY TRLRKQMVK KYLNSILN on the solid phase will be synthesized. Color 7- [5- N - (1,3,4-trihydroxybutyl-2-yl)-aminocarbonyl -3,3-dimethyl -1-(4-sulfonatobutyl) indolin-2-ylidene]-3,5-(2 -carboxypropane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl-5-N-1,3,4-trihydroxybutyl-2-yl)-aminocarbonyl (3H) indolio]butanesulfonate, the sodium salt will be according to example 14-16 and 18-27 c (linkage of dye) to the peptide N-terminus and according to example 14-16 and 18-27 c (linkage of dye) to the peptide N-terminus, linked according to example 14-16 and 18-27c (removal of protecting groups and separation of dye-peptide conjugate), that conjugate will be isolated and purified.

Furthermore, the colors presented by the invention can be prepared according to example 44a and b, whereby in place of 11-aminoundecanoic acid in example 48a, the following amino acids and in example 46 mentioned indolene derivatives with hydroxyalkyl substituents a)-f) are used:

and). glycine

ii). alanine

iii). β-Alanine

iv). 4-aminobutanoic acid

c). 6-aminohexanoic acid

you). H2N-(CH2CH2O)3CH2COOH (TH 53, 20, 6977)

vii). H2N-(CH2CH2O)4CH2COOH (JOC 63, 5, 1728, 1998)

viii). H2N-CH2CH2COO(CH2CH2O)4-CO-CH2CH2COOH (Let. Pept. Sci. 6, 135, 1999)

ix). HCl*H2N-PEG-COOH (MW 3400 g/mol; Shearwater Polymers Inc., USA)

Example 49

4-[2-[4-(4-(N-(4-aza-6-bromo-5-oxohexyl)aminocarbonyl-ethyl)phenyloxy)-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3- dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-5-N-(dihydroxypropyl) aminocarbonyl-3,3-dimethyl(3H) indolio]butanesulfonate, sodium salt

a) [3-[N-(tert-butoxycarbonyl)amino]propyl]-N'-(bromoacetyl)-amide

2.5 g (14.4 mmol) of [3-[N-(tert.-butoxycarbonyl)amino]propyl-amide will be dissolved in 15 ml of dioxane and after the addition of 4.4 ml of triethylamine, at 0ºC, with 3.2 g (16 mmol) of bromoacetyl bromide mixed. The mixture is stirred overnight at room temperature and then a further 320 mg of bromoacetyl bromide is added. After 2 hours, at room temperature, the precipitate will be removed, the solution will be concentrated, and the residue will be dissolved in the ethyl ester of acetic acid. The solution is washed with water, and the organic phase is dried over sodium sulfate.

Yield: 3.2 g (75% d. Th.).

Analysis (refers to solvent-free substances):

[image]

b) [3-[N-(tert.-bromoacetyl)amino]propyl] amine, hydrochloride

3.1 g (10.5 mmol) of [3-[N-(tert-butoxycarbonyl)amino]-N`-(bromoacetyl)-amide (Example 49a) will be with 50 mmol of 1M hydrochloric acid in ethyl acetic acid ether for 5 hours mixed, at room temp. The precipitate/product will be removed and the residue washed in ethyl acetate.

Yield: 2.3 g (95% d. Th.).

Analysis (refers to solvent-free substances):

[image]

c) 4-[2-[4-(4-(N-(4-aza-6-bromo-5-oxo-hexyl)aminocarbonyl-ethyl)phenyloxy)-7-[5-N-(dihydroxypropyl)aminocarbonyl- 3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-5-N -(dihydroxypropyl)aminocarbonyl-3,3-dimethyl(3H)indolio]butanesulfonate, sodium salt

121 mg (0.1 mmol) 4-[2-[4-(4-(2-carboxyethylphenyloxy)-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl) indolin-2-ylidene]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl(3H ) indolio]butanesulfonate, sodium salt, N-hydroxysuccinimide ester (example 44c) will be dissolved in 0.5 ml of N,N-dimethylformamide, with 0.06 ml of triethylamine and 70 mg (0.3 mmol) [3-[N- (bromoacetyl)amino]propyl]amine, hydrochloride (Example 49b) mixed.

The mixture is stirred for 4 hours at 60ºC, cooled continuously to room temperature, and the product is precipitated with diethyl ether. The solid residue is removed and washed abundantly with diethyl ether.

Usability: 0.11 mg (80% d. Th.)

Analysis (refers to substances without solvents):

[image]

Analogous to the synthesis according to example 14-16 and 18-27 a (solid phase peptide synthesis) the VIP-analog HSDAVFTDNY TRLRKQCAVK KYLNSLLN on the solid phase will be synthesized

Bromide dye 4-[2-[4-(4-(N-(4-aza-6-bromo-5-oxo-hexyl)aminocarbonylethyl)phenyloxy)-7-[5-N-(dihydroxypropyl)aminocarbonyl-3, 3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-5-N-( dihydroxypropyl)aminocarbonyl-3,3-dimethyl(3H)indolio]butanesulfonate, sodium salt, will be attached to the peptide N-terminus according to example 17, and according to examples 14-16 and 18-27 c (detachment of protective groups and separation of peptide conjugate -color) that conjugate isolated and purified.

LIST OF SEQUENCES

GENERAL INFORMATION:

Applicant:

(A) NAME: Institut fur Diagnostikforschung GMBH

an der freien Universitat Berlin

STREET: Spandauer Damm 130

CITY: Berlin

(E) COUNTRY: Germany

POSTAL CODE (ZIP): D-14050

TELEPHONE: (030)-30390412

TELEFAX: (030)-30390499

APPLICATION TITLE: Short-chain conjugates of peptides and dyes as contrast agents in optical diagnostics

NUMBER OF SEQUENCES: 8

COMP. READABLE FORM:

MEDIA FORM: Floppy disk

COMPUTER: IBM PC compatible

OPERATING SYSTEM: PC-DOS/MS-DOS

SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)

(v) CURRENT APPLIED DATA:

APPLICATION NUMBER:

(vi) OLDER APPLICATION DATA

APPLICATION NUMBER

REGISTERED DATA

(2) INFORMATION ABOUT SEQ ID NO: 1

(i) SEQUENCE CHARACTERISTICS:

LENGTH: 28 amino acids

TYPE: peptides

CHAINING: single

TOPOLOGY:

(ii) TYPE OF MOLECULES: peptides

(iii) HYPETHETICAL: NO

(iv) ORIGINAL SOURCE: Synthetic

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1

His-Trp-Asp-Ala-Val-Phe-Trp-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser- Ile-Leu-Assn

(3) INFORMATION FOR SEQ ID NO: 2

(i) SEQUENCE CHARACTERISTICS:

LENGTH: 28 amino acids

TYPE: peptides

CHAINING: single

TOPOLOGY:

(ii) TYPE OF MOLECULES: peptides

(iii) HYPETHETICAL: NO

(iv) ORIGINAL SOURCE: synthetic

SEQUENCE DESCRIPTION: SEQ ID NO: 2

His-Ser-Asp-Ala-Val-Phe-Thr-Phe-Asn-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile- Leu-Asn

(4) INFORMATION FOR SEQ ID NO: 3

(i) SEQUENCE CHARACTERISTICS:

LENGTH: 28 amino acids

TYPE: peptides

CHAINING: single

TOPOLOGY:

(ii) TYPE OF MOLECULES: peptides

(iii) HYPETHETICAL: NO

(iv) ORIGINAL SOURCE: synthetic

SEQUENCE DESCRIPTION: SEQ ID NO: 3

His-Ser-Asp-Ala-Val-Phe-Thr-Lys-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser- Ile-Leu-Assn

(5) INFORMATION FOR SEQ ID NO: 4

(i) SEQUENCE CHARACTERISTICS:

LENGTH: 28 amino acids

TYPE: peptides

CHAINING: single

TOPOLOGY:

(ii) TYPE OF MOLECULES: peptides

(iii) HYPETHETICAL: NO

(iv) ORIGINAL SOURCE: synthetic

SEQUENCE DESCRIPTION: SEQ ID NO: 4

His-Ser-Asp-Ala-Val-Phe-Thr-Gln-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser- Ile-Leu-Assn

(6) INFORMATION FOR SEQ ID NO: 5

(i) SEQUENCE CHARACTERISTICS:

LENGTH: 28 amino acids

TYPE: peptides

CHAINING: single

TOPOLOGY:

(ii) TYPE OF MOLECULES: peptides

(iii) HYPETHETICAL: NO

(iv) ORIGINAL SOURCE: synthetic

SEQUENCE DESCRIPTION: SEQ ID NO: 5

His-Ser-Asp-Ala-Val-Phe-Thr-Arg-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser- Ile-Leu-Assn

(7) INFORMATION FOR SEQ ID NO: 6

(i) SEQUENCE CHARACTERISTICS:

LENGTH: 28 amino acids

TYPE: peptides

CHAINING: single

TOPOLOGY:

(ii) TYPE OF MOLECULES: peptides

(iii) HYPETHETICAL: NO

(iv) ORIGINAL SOURCE: synthetic

SEQUENCE DESCRIPTION: SEQ ID NO: 6

His-Ser-Asp-Ala-Val-Phe-Thr-Trp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser- Ile-Leu-Assn

(8) INFORMATION FOR SEQ ID NO: 7

(i) SEQUENCE CHARACTERISTICS:

LENGTH: 28 amino acids

TYPE: peptides

CHAINING: single

TOPOLOGY:

(ii) TYPE OF MOLECULES: peptides

(iii) HYPETHETICAL: NO

(iv) ORIGINAL SOURCE: synthetic

SEQUENCE DESCRIPTION: SEQ ID NO: 7

His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Arg-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser- Ile-Leu-Assn

(9) INFORMATION FOR SEQ ID NO: 8

(i) SEQUENCE CHARACTERISTICS:

LENGTH: 28 amino acids

TYPE: peptides

CHAINING: single

TOPOLOGY:

(ii) TYPE OF MOLECULES: peptides

(iii) HYPETHETICAL: NO

(iv) ORIGINAL SOURCE: synthetic

SEQUENCE DESCRIPTION: SEQ ID NO: 8

His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Tyr-Arg-Leu-Arg-Lys-Gln-Met-Arg-Val-Lys-Lys-Tyr-Leu-Asn-Ser- Ile-Leu-Assn

Claims (33)

1. Conjugates of peptides and polymethine dyes, general formula (I) A1 – (X)m – A2 I whereby X for one α, β or γ-amino acid with D or L-configuration and m stands for a number from 5 to 30, whereby the resulting amino acid sequence (X)m of straight-chain nature or via a disulfide bridge between 2 cysteines or homocysteines or amide between the N- and C-terminus can be cyclized for the amino acid sequence of vasoactive intestinal peptide (VIP), somatostatin or neurotensin, or for fragments , parts of the sequences, derivatives or analogues of VIP, somatostatin or neurotensin, it says A1 for one hydrogen atom, one acetyl residue or one alkyl residue with up to 10 C atoms, which can also be substituted with 1 to 3 carboxy groups and/or 1 to 6 hydroxy groups, or one poly(oxyethylene) residue with 2 up to 30 –CH2-CH2O-units or one dye molecule from the class of polymethine dyes, which show at least one absorption maximum in the range from 380 to 1200 nm, stands A2 for one hydroxy group, one amino group or one dye molecule from the class of polymethine dyes, which show at least one absorption maximum in the range from 380 to 1200 nm, reads under the conditions that at least one of the residues A1 or A2 is one dye molecule from the class of polymethine dyes, which show at least one absorption maximum in the range from 380 to 1200 nm, represents, where in the case that A1 and/or A2 one dye molecule from the class of polymethine dyes, which show at least one absorption maximum in the range from 380 to 1200 nm, represents A1 on the N-terminal amino group, and A2 on one amino group or the amino acid lysine or to one hydroxyl group of the amino acid serine in any position within the amino acid sequence (X)m is linked, and their physiologically tolerable salts. 2. Compounds according to claim 1, which are indicated by the fact that the dye molecule A1 and/or A2 stands for one cyanine-, squarilium-, croconium-, merocyanine- or oxonol-dye. 3. Compounds according to claim 1 or 2, which are indicated by the fact that the dye molecule A1 and/or A2 is for one cyanine- or squarilium-dye of the general formula II [image] whereby D for one fragment corresponding to the general formula III to IV, where the position marked with an asterisk indicates the point of connection with B, stands, [image] B for one fragment corresponding to the general formula VII to XII [image] R1 and R2 for E1, R3 for one fluorine-, chlorine-, bromine-, iodine- atom or one nitro group or for one residue -COOE1, -CONE1E2, -NHCOE1, -NHCONHE1, -NE1NE2, -OE1, -OSO3E1 , -SO3E1, -SO2NHE1, -E1, where E1 and E2 independently of each other for one hydrogen atom, one C1-C4-sulfoalkyl chain, one saturated or unsaturated C1-C40-alkyl chain, wherein the chain or parts of this chain also have one or more aromatic or saturated cyclic C5-C6 - or bi-cyclic C10-units can form, while the C1-C50-alkyl chain from 0 to 15 oxygen atoms and/or from 0 to 3 carbonyl groups is interrupted and/or substituted with 0 to 5 hydroxy groups , stands, R4 for one hydrogen atom, one fluorine, chlorine, bromine, iodine - atom or one branched or straight-chain C1-C10-alkyl chain, it says, b number 2 or 3 means, X and Y, independently of each other, O, S, Se, -CH=CH- or C(CH3)2 denotes, L for one group corresponding to the following formula [image] whereby n denotes a number from 1 to 10, stands. 4. Compounds according to claims 1 to 3, characterized in that the dye molecule A1 and/or A2 stands for one indocarbocyanine-, one indodicarbocyanine- or one indotricarbocyanine-dye. 5. Compounds according to claim 4, characterized in that the dye molecule A1 and/or A2 for one indocarbocyanine-, one indodicarbocyanine- or one indotricarbocyanine dye of the general formula XIII or XIV [image] whereby p for 1,2 or 3, n stands for 1,2,3,4 or 10, R1 and R2 independent of each other, for one 4-sulfobutyl-, 3-sulfopropyl-, 2-sulfoethyl-, 3-methyl, 3-sulfopropyl-, methyl-, ethyl-, or propyl - residue, are And R3 for one hydrogen, chlorine, bromine, iodine atom or one nitro group or for one residue -COOE1, -CONE1E2, -NHCOE1, -NHCONHE1, -NE1E2, -OE1, -OSO3E1, -SO3E1, -SO2NHE1, -E1, where E1 and E2 are mutually independent, for one hydrogen atom or for one methyl, ethyl or C3-C6-alkyl residue, which is interrupted by 0 to 2 oxygen atoms and/or 0 to 1 carbonyl groups and/or by 0 up to 5 hydroxyl groups substituted, or for one poly(oxyethylene)glycol residue with 2 to 30 -CH2CH2O-units, stands. 6. Compounds according to claim 4, characterized in that the dye molecule A1 and/or A2 for one indocarbocyanine, one indodicarbocyanine or one indotricarbocyanine dye of the general formula XIII or XIV [image] whereby p for 1,2 or 3, n for 1,2 or 4, R1 and R2, mutually independent, for one 4-sulfobutyl or 3-sulfopropyl residue, R3 for hydrogen or for one residue -COOE1 or -CONHE1, where E1 is one hydrogen atom or one methyl, ethyl, or one C3-C6-alkyl residue, which is terminated by 0 to 2 oxygen atoms and/or by 0 to 1 carbonyl group and/or substituted by 0 to 5 hydroxyl groups , has the meaning, stands. 7. Compounds according to claim 4, characterized in that the dye molecule A1 and/or A2 for one indotricarbocyanine dye of the general formula XV or XVI reads: [image] [image] XVI whereby n stands for 2 or 3, R1 and R2, mutually independent, one 4-sulfobutyl, 3-sulfopropyl, or 2-sulfoethyl residue represent, R3 for one residue -CONH-peptide, -CONH-(CH2)m-CONH-peptide, -CONH-(CH2)n-NH-CS-NH-peptide or -CONH-(CH2)n-NHCO-CH2-peptide with m=1 to 10 and n=2 or 3, it says, or represents one of the following groups: [image] R4 and R5, mutually independent, for one hydrogen atom, one methyl residue or one hydroxylated alkyl residue, are R6 for one of the following groups are: -(CH2)m-CONH-peptide with m=0 to 2, -(CH2)m-NH-CS-NH-peptide with m=0 to 2, and X for one hydrogen atom or one sulfur atom, it says. 8. Compounds according to claim 4, characterized in that the dye molecule A1 and/or A2 for one indotricarbocyanine dye of the general formula XVII is: [image] whereby R1 and R2 mutually independent, one 4-sulfobutyl, 3-sulfopropyl or 2-sulfoethyl residue, represent, R3 for one residue –CONH-peptide, -NH-CS-NH-peptide with n=2 or 3 or one of the following groups represents: [image] and R 4 and R 5 , mutually independent, for one hydrogen atom, one methyl residue or one hydroxylated alkyl residue, stand. 9. Compounds according to claim 7 or 8, characterized in that the hydroxylated alkyl residues for 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 1,3-dihydroxy-2-propyl, 2,3,4-trihydroxybutyl, 1,3,4-trihydroxy-2-butyl, 2,3,4,5,6-pentahydroxyhexyl stand. 10. Compounds according to claim 1, characterized in that (X)m for the amino acid sequence of the native vasointestinal peptide corresponds HSDAVFTDNYTRLRKQMAVKKYLNSILN or for fragments, parts of fragments, derivatives or analogs of vasoactive intestinal peptide, consisting of 5 to 30 amino acids, stands. 11. Compounds according to claim 1, characterized in that (X)m for the amino acid sequence of somatostatin corresponding [image] or for fragments, parts of sequences, derivatives or analogs of somatostatin, consisting of 5 to 20 amino acids, stands. 12. Compounds according to claim 1, characterized in that (X)m for the neurotensin amino acid sequence corresponding pyroglutamic acid – LYENKPRPRYIL or for fragments, parts of sequences, derivatives or analogues of neurotensin, consisting of 5 to 20 amino acids, What is it. 13. Compounds according to claim 10, characterized in that the following amino acids are selected as fragments, parts of sequences, derivatives or analogs of vasoactive intestinal peptide: [image] 14. Compounds according to claim 10, characterized in that VIP-peptide analogs are selected from the following group of sequences: [image] [image] [image] [image] 15. Compounds according to claim 10, characterized in that as VIP analog, one compound, according to the following formula, is selected: HSDAVFTX1X2Y X3RLRKQMAVK KYLNSILN, where X1, X2 and X3 can each represent an amino acid. 16. Compounds according to at least one of the preceding claims, characterized in that 2 to m amino acids, independently of each other, can be substituted for all their D-amino acids or for other L- or D-amino acids, where m has the above-mentioned meaning. 17. Compounds according to at least one of the preceding claims, characterized in that at least one of the mutually independent amino acids (X)m can be substituted for another non-natural amino acid or amino acid derivative. 18. Compounds according to claim 17, characterized in that one of the following compounds is selected as non-natural amino acids or amino acid derivatives: naphthalene, cyclohexylalanine, norleucine, norvaline, α-aminoadipic acid, α-amino butyric acid, β-alanine, β- cyclohexylalanine, ornithine, sarcosine or δ-hydroxylysine. 19. Compounds according to at least one of the previous claims, characterized in that one compound according to the following formula is selected as an analogue of VIP: X1SDAVX2TDNX3 TRLRKQMAVK KX4LNSILN, where X1, X2, X3 and X4 represent unnatural amino acids or amino acid derivatives. 20. Compounds according to claim 19, characterized in that non-natural amino acids or amino acid derivatives from the following group are selected: naphthalene, cyclohexylalanine, norleucine, norvaline, α-amino adipic acid, α-amino butyric acid, β-alanine, β-cyclohexylalanine , ornithine, sarcosine or δ-hydroxylysine. 21. Compounds according to at least one of the preceding claims, characterized in that individual amino acids (X)m are substituted for their corresponding D-amino acids. 22. Compounds according to at least one of the preceding claims, characterized in that as fragments, parts of sequences, derivatives or analogs of vasoactive intestinal peptides, retrosynthetic sequences of amino acids are selected. 23. Compounds according to at least one of the preceding claims, indicated by the fact that as fragments, parts of sequences, derivatives or analogs of vasoactive intestinal peptides of retrosynthetic amino acid sequences, in which 2 to m amino acids are replaced by corresponding D-amino acids, which are selected, wherein m has the above meaning. 24. Compounds according to claim 10, characterized in that the following amino acid sequences are selected as fragments, parts of sequences, derivatives or analogues of vasoactive intestinal peptides: [image] AvKKyLNSILN AvKKyLNSIL AvKKyLNSI 25. Compounds according to claim 11, characterized in that the following amino acid sequences are selected as fragments, parts of sequences, derivatives or analogs of somatostatin: [image] 26. Compounds according to claim 12, characterized in that the following amino acid sequences are selected as fragments, parts of sequences, derivatives or analogues of neurotensin: [image] RRPYIL RRPyIL RRPwIL 27. Use of compounds according to at least one of the previous requirements for in-vivo diagnosis of tumors, other diseased tissue areas or adenomas, using optical disease detection procedures or for in-vivo fluorescent diagnosis of tumors, tumor cells and/or inflammatory processes on tissues , using endoscopic procedures for diagnosis in the gastrointestinal tract, esophagus, bronchial tract or bladder or cervix or for in-vivo-fluorescence diagnosis and/or absorption diagnosis of breast tumors, using optical mammography (transillumination or optical breast tomography). 28. Procedures for endoscopic in-vivo-fluorescence diagnostics with the use of compounds according to claim 1, indicated by the fact that the said compounds are administered to the patient intravenously or topically, by spraying in the gastrointestinal tract, esophagus, urinary bladder or by inhalation in the bronchi, in case scattering unbound, the excessive part of the compound can be removed by washing, endoscopic examination using a local stimulus with one selected wavelength in the spectral range from 380 to 1200 nm and through the locally dependent detection of specific, color-emitted fluorescent radiation, will be carried out. 29. Optical diagnostics for in-vivo diagnostics of diseased tissue areas, characterized by the fact that it contains at least one compound according to claim 1, together with the usual auxiliary and/or carrier substances, as well as dilution agents. 30. Cyanine dyes of the general formula XVIII, [image] whereby p for 1,2 or 3, n stands for 1,2,3,4 or 10, R1 and R2, mutually independent, for one 4-sulfobutyl, 3-sulfopropyl, 2-sulfoethyl, 3-methyl-3-sulfopropyl, methyl, ethyl or propyl residue stands and R3 for hydrogen or for one residue -COOE1, -CONE1E2, -NHCOE1, -NHCONHE1, -NE1E2, -OE1, -OSO3E1, -SO3E1, -SO2NHE1, where E1 and E2, independently of each other, for one hydrogen atom or for one methyl, ethyl or one C3-C6-alkyl residue, which is interrupted from 0 to 2 oxygen atoms and/or from 0 to 1 carbonyl group and/or with 0 to 5 hydroxyl groups substituted, stands, stands. 31. Cyanine dyes of the general formula XIX or XX [image] [image] whereby n for 2 or 3 R1 and R2, independently of each other, represent one 4-sulfobutyl, 3-sulfopropyl or 2-sulfoethyl residue, R3 for one -COOH-group or one of the following residues is: -CONH-(CH2)n-COOH with n=2 or 3, -CONH-(CH2)n-NCS with n=2 or 3, -CONH-(CH2)n-NHCO-CH2-X1 with n=2 or 3, and X1 = Cl, Br, I [image] R4 and R5, mutually independent, for one hydrogen atom, one methyl residue or one hydroxylated alkyl residue, such as 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 1,3-dihydroxy-2-propyl, 2, 3,4-trihydroxybutyl, 1,3,4-trihydroxy-2-butyl, 2,3,4,5,6-pentahydroxyhexyl stand, R6 stands for one of the following groups: -(CH2)m-COOH with m=0 to 2, -(CH2)m-NCS with m=0 to 2, -(CH2)m-CONH-peptide with m = 0 to 2, -(CH2)m-NH-CS-NH-peptide with m = 0 to 2, and X for one oxygen atom or one sulfur atom, it says. 32. Cyanine dyes of the general formula XXI [image] whereby R1 and R2, independently of each other, one 4-sulfobutyl or 3-sulfopropyl residue represent, R3 for one -COOH-group or one of the following residues is: -CONH-(CH2)n-COOH with n=2 or 3, -CONH-(CH2)n-NCS with n=2 or 3, -CONH-(CH2)n-NHCO-CH2-X1 with n=2 or 3, and X1 = Cl, Br, I [image] and R4 and R5, independent of each other, for one hydrogen atom, one methyl residue or one hydroxylated alkyl residue, such as, for example, 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 1,3-dihydroxy-2-propyl, 2 ,3,4-trihydroxybutyl, 1,3,4-trihydroxy-2-butyl, 2,3,4,5,6-pentahydroxyhexyl, standing. 33. Analogues of VIP, indicated by the following sequences: [image] [image]