DE10251894A1 - New peptide-containing benzophenoxazine or napthoquinone derivatives, useful as chromogenic substrates for enzymes, e.g. for diagnosis, monitoring therapy and detecting pyrogens - Google Patents

Abstract

Peptide-containing benzo(a)phenoxazine and naphthoquinone derivatives (I) - (IV) are new. Peptide-containing benzo(a)phenoxazine and naphthoquinone derivatives of formulae (I) - (IV) are new. R = alkyl, aryl, aralkyl, hydrogen or cyano; R3 - R6, R7, R8 = hydrogen, cyano, nitro, alkyl, aralkyl, halo or alkoxy; A, B = alpha, beta or gamma-amino acid with 2-15C and up to 4 nitrogen, two sulfur and 6 oxygen, and B may also be a dipeptide; D = arginine, lysine, tyrosine, phenylalanine, tryptophan or their homologs; X = hydrogen or a standard protecting group for terminal amino; L = optional linker that forms a peptide bond with D; R1, R2 = hydrogen, cyano, halo, 1-4C alkyl, aryl, aralkyl or alkoxy; and provided that in (III), at least one of R3 to R6 is amino to which X-A-B-D- is linked.

Description

The measurement of enzyme activities is for example for the diagnosis of diseases such as thromboembolism, fibrinolysis, Arthritis, wound healing important or when used for tracking of anticoagulant therapy in laboratories or hospitals in diagnostics. On the other hand, the determination of the enzymatic activity used as a means of detecting pyrogens (endotoxins), the while sterilization in solutions to be injected (LAL test) can. The technology currently used is based on proteolytic activity used enzymes. The proteolytic activity of the enzyme is called an increase the optical density when a peptide bond is cleaved between a specific recognition sequence of the peptide and the dissociable Chromogens measured (Witt, I. 1991). This leads to a change of the spectrum and the absorption at 405 nm during the cleavage (dye label: p-nitroaniline).

The object of the invention was to create peptide sequences especially of course to couple existing ones with new types of chromogens (labeling), the longer ones Wavelengths, preferably absorb more than 630 nm.

wobei R alkyl, aryl, H, aralkyl, CN sein kann, R3, R6, R7 und R8 gleich oder verschieden sein können und H, CN, NO₂, alkyl, aralky, halogen oder alkoxy sein können,
A und B identisch oder verschieden sein können eine alpha, Beta oder gamma Aminosäure sein können, die 2 bis 15 C Atome und bis zu 4 N Atome, 2 Schwefelatome und 6 Sauerstoffatome umfaßt und B gegebenfalls ein Dipeptid ist, das aus diesen Aminosäuren gebildet ist,
C Arginin, Lysin, Tyrosin, Phenylalanin oder Tryptophan und ihre Homologen ist und X ein H Atom oder eine in der Peptidchemie übliche Schutzgruppe für terminale Aminogruppen ist,
und L ein optionaler Linkerbaustein ist, der in der Lage ist, mit C eine Peptidbindung auszubilden.

wobei R1 und R 2 gleich oder verschieden sein können und H, CN, halogen, C1 bis C4 alkyl, aryl oder aralkyl oder alkoxy ist,
R3, R6, R7 und R8 gleich oder verschieden sein können und H, CN, NO₂, alkyl, aralkyl, halogen oder alkoxy sein können,
A und B identisch oder verschieden sein können eine alpha, beta oder gamma Aminosäure sein können, die 2 bis 15 C Atome und bis zu 4 N Atome, 2 Schwefelatome und 6 Sauerstoffatome umfaßt und B gegebenfalls ein Dipeptid ist, das aus diesen Aminosäuren gebildet ist,
C Arginin, Lysin, Tyrosin, Phenylalanin oder Tryptophan und ihre Homologen ist,
und X ein H Atom oder eine in der Peptidchemie übliche Schutzgruppe für terminale Aminogruppen ist,
und L ein optionaler Linkerbaustein ist, der in der Lage ist, mit C eine Peptidbindung auszubilden.

wobei R1, R2 gleich oder v erschieden sind und alkyl, ary1, H, aralkyl, CN, sind, R3, bis R6 R8 gleich oder verschieden sein können und H, CN, NO₂, NH₂, alkyl, aralkyl, halogen oder alkoxy sein können mit der Bedingung daß mindestens ein Rest aus R3 bis R6 eine Aminogruppe ist,
A und B identisch oder verschieden sein können eine alpha, beta oder gamma Aminosäure sein können, die 2 bis 15 C Atome und bis zu 4 N Atome, 2 Schwefelatome und 6 Sauerstoffatome umfaßt und B gegebenfalls ein Dipeptid ist, das aus diesen Aminosäuren gebildet ist,
C Arginin, Lysin, Tyrosin, Phenylalanin oder Tryptophan und ihre Homologen ist,
und X ein H Atom oder eine in der Peptidchemie übliche Schutzgruppe für terminale Aminogruppen ist,
und L ein optionaler Linkerbaustein ist, der in der Lage ist, mit C eine Peptidbindung auszubilden.

wobei R1, R2 gleich oder v erschieden sind und alkyl, aryl, H, aralkyl, CN, sind, R3, bis R6 R8 gleich oder verschieden sein können und H, CN, NO₂, NH₂, alkyl, aralkyl, halogen oder alkoxy sein können,
A und B identisch oder verschieden sein können eine alpha, beta oder gamma Aminosäure sein können, die 2 bis 15 C Atome und bis zu 4 N Atome, 2 Schwefelatome und 6 Sauerstoffatome umfaßt und B gegebenfalls ein Dipeptid ist, das aus diesen Aminosäuren gebildet ist,
C Arginin, Lysin, Tyrosin, Phenylalanin oder Tryptophan und ihre Homologen ist,
und X ein H Atom oder eine in der Peptidchemie übliche Schutzgruppe für terminale Aminogruppen ist,
und L ein optionaler Linkerbaustein ist, der in der Lage ist, mit C eine Peptidbindung auszubilden.This object is achieved by providing compounds of the chemical formulas (I) to (IV):

where R can be alkyl, aryl, H, aralkyl, CN, R3, R6, R7 and R8 can be the same or different and H, CN, NO ₂ , alkyl, aralky, halogen or alkoxy,
A and B can be identical or different can be an alpha, beta or gamma amino acid which comprises 2 to 15 C atoms and up to 4 N atoms, 2 sulfur atoms and 6 oxygen atoms and B is optionally a dipeptide which is formed from these amino acids .
C is arginine, lysine, tyrosine, phenylalanine or tryptophan and their homologues and X is an H atom or a protective group for terminal amino groups which is customary in peptide chemistry,
and L is an optional linker building block capable of forming a peptide bond with C.

where R1 and R 2 can be the same or different and is H, CN, halogen, C1 to C4 alkyl, aryl or aralkyl or alkoxy,
R3, R6, R7 and R8 can be the same or different and can be H, CN, NO ₂ , alkyl, aralkyl, halogen or alkoxy,
A and B can be identical or different can be an alpha, beta or gamma amino acid which comprises 2 to 15 C atoms and up to 4 N atoms, 2 sulfur atoms and 6 oxygen atoms and B is optionally a dipeptide which is formed from these amino acids .
C is arginine, lysine, tyrosine, phenylalanine or tryptophan and their homologues,
and X is an H atom or a protective group for terminal amino groups which is customary in peptide chemistry,
and L is an optional linker building block capable of forming a peptide bond with C.

where R1, R2 are the same or different and are alkyl, ary1, H, aralkyl, CN, R3, to R6 R8 may be the same or different and H, CN, NO ₂ , NH ₂ , alkyl, aralkyl, halogen or alkoxy can be with the condition that at least one radical from R3 to R6 is an amino group,
A and B can be identical or different can be an alpha, beta or gamma amino acid which comprises 2 to 15 C atoms and up to 4 N atoms, 2 sulfur atoms and 6 oxygen atoms and B is optionally a dipeptide formed from these amino acids .
C is arginine, lysine, tyrosine, phenylalanine or tryptophan and their homologues,
and X is an H atom or a protective group for terminal amino groups which is customary in peptide chemistry,
and L is an optional linker building block capable of forming a peptide bond with C.

where R1, R2 are the same or different and are alkyl, aryl, H, aralkyl, CN, R3, to R6 R8 may be the same or different and H, CN, NO ₂ , NH ₂ , alkyl, aralkyl, halogen or alkoxy could be,
A and B can be identical or different can be an alpha, beta or gamma amino acid which comprises 2 to 15 C atoms and up to 4 N atoms, 2 sulfur atoms and 6 oxygen atoms and B is optionally a dipeptide formed from these amino acids .
C is arginine, lysine, tyrosine, phenylalanine or tryptophan and their homologues,
and X is an H atom or a protective group for terminal amino groups which is customary in peptide chemistry,
and L is an optional linker building block capable of forming a peptide bond with C.

Attenuated total reflection spectroscopy (ATRS) was found reflection spectroscopy) using a planar thin-film waveguide chip that has been chemically modified, for example, with a selective chemical polymer, is suitable as optimal optical technology for an enzyme test, in particular for an endotoxin test. The coupled light travels within the planar waveguide and creates an evanescent field along the surface. The evanescent field migrates within the adjacent medium and has a depth of penetration of a few nanometers (approximately 10 nm) in the Z direction of the traveling light. The absorption of light using the ATRS technique within the adjacent layer correlates with the change in the concentration of the specific analyte.

According to the invention, chromophore-labeled substrates can basically be produced using two methods will get. These substrates come with integrated optical Waveguides as a measuring platform in connection with low-cast semiconductor lasers in the emission wavelength range from above 630 nm used.

• 1. Chromophore label, directly or through one suitable spacer on BOC-Ser (Bz) -Gly-Arg be coupled and none or during proteolytic cleavage just a little change the absorption wavelength exhibit. This is in the range of> 630 nm. Such substrates cannot in solution can be used because the proteolysis is principally not spectrophotometric can be tracked. Instead, according to the invention Substrates about suitable spacer N-terminal on the waveguide of the optical chips immobilized and the detection of proteolysis via the change in absorption in the evanescent field of the waveguide, induced by the diffusion of the chromophore out of it becomes. The chromophore only needs a low water solubility feature, as it is related to the volume of the measuring cell in only is released in very small quantities.
• 2. Chromophore label that is directly over a free chromophore amino group acting as a donor of the chromophoric electron system carboxy-terminally coupled as amide to BOC-Ser (Bz) -Gly-Arg and accordingly in proteolysis bathochromic shift of absorption max. As in the first case, such Chromophores in free form at> 630 nm sufficient absorption. Such labeled peptide substrates are for one Immobilization on the waveguide of the optical chip is not optimal, since the detectable at the required wavelength in the evanescent field Species, the free chromophore, largely after proteolysis diffuses out of it and thus the sensitivity of the system is inherently restricted. Detection in solution with conventional Concentrations of labeled substrate, however, are with optical Chips as a measuring platform advantageous, provided that the water solubility of the chromophore is sufficiently high is.

Chromogens have been found to which are bound directly to the substrate have only a limited molecular size allowed to, so as not to hinder the enzymatic attack by the target protease. Known examples are 7-amino-4-methycoumarin (AMC) and 2-aminacridone (2-AA).

The following substrates and coagulagens have been produced:

• 1 . BOC-Ser (Bn) -Gly-Arg (HCl) -pNA: This substrate was used as a reference for enzymatic cleavage test in solutions used. Measurements on a chip were not possible because the wavelength of about 405 nm is not accessible with the laser spectrometer.
• 2. Nile blue as a chromogen which is linked to 4-aminobenzoic acid (as Linker) is coupled in the form of a carboxylamide.
• 3. Meldola blue as a chromogen that binds to 1,4-phenylenediamine (linker) is coupled. this leads to further to a 4 - ((benzyl-L-serinyl-L-glycyl) -L-arginylamindo) -N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) aniline.

Both substrates (in the following also named as LAL substrates) were cleaved by trypsin, although a spectral shift did not occur. However, a change could the absorbance at 641 and 674 nm can be discovered in solution, since the Chromogen was cleaved from the substrate and almost in solution insoluble is. The absorption spectra and the absorption coefficients were in various solvents a pH of 7 and a temperature of 37 ° C characterized.

Surprisingly, it has been found that aminonaphthoquinones meet all the requirements for a chromogen that is coupled to a peptide. Examples of naphthoquinones according to the invention are:

While 4 has the longest wavelength absorption band at 479 nm in ethanol, in comparison, this is shifted bathochromically at 5 nm 33 nm. For 6-amino-2,3-dicyano-l, 4-naphthoquinone (6), for which the absorption band due to the compared to the chlorine substituents stronger in 5 Another bathochromic electron-withdrawing cyano substituent Shift occurs. As a result, the absorption band of 6 is in ethanol at around 630 nm.

These are a class of chromogens, whose spectra change after enzymatic hydrolysis, in particular by changing the absorption maximum to longer wavelengths. at this chromogen is the chromogen via the amino terminal group connected to the peptide (arginine) at the 6-amino position and changes its spectrum after hydrolysis of the amide group. Are here too the amino-1,4-naphthoquinones if necessary via bound a linker to the peptide sequence.

By coupling the chromogen BOC-Ser- (Bz) -Gly-Arg-6ANC, for example, was attached to the peptide generated; the peptide substrate showed an absorbance of A <0.06 in one Wavelength λ = 479 nm and a baseline absorbance at 550 nm. By adding trypsin at a pH of 7.4, an absorption maximum quickly became apparent of free ANC at a wavelength λ = 479 nm and a second corresponding wavelength λ 550 nm. By comparing the Effects of electron-accepting substituents on the absorption spectra of existing chromophores (7-amino-4-methylcoumarin and 2-aminoacridone) the bathochromic effect of electron donor substituents was found in the order of magnitude of about 100 nm.

Sensitive layers, on-chip immobilization:
The chip surface was chemically modified and functionalized using two different methods:

• 1. By applying a selective polymer layer on the waveguide.
• 2. a) By immobilizing specific chromophores the surface of the chip, which is caused by a linker (avidin-biotin, suberic acid, hydrocarbon chains, Hexylenediamine, sulfhydryl groups) are bridged. b) By immobilization chromogenic substrates (specific oligopeptide labeled like dyes mentioned above occurs) on the waveguide.

Showed inside the polymeric membrane the dye has a spectral shift due to different mechanisms. Using chromogenic substrates, the dye is immobilized Cleave the peptide after adding the enzyme (e.g. serine protease). After cleavage, the chromophore changes its spectrum or at least its distribution and diffuses from the detection layer closer to the surface of the waveguide (diffusion-restricted process).

Application of the layers of the detection area was done (using a "Genesis NPS" pipettor) with an active Tip M (volumes) by Tecan. The substrates can as optically active materials in defined areas on the surface of the optical chip are deposited.

biosensors

Biosensors are based on a coupling of biomolecules that recognize specific analytes as receptors in the broadest sense with physicochemical transducers that convert a biologically generated signal into electrical measurement signals. Bioactive recognition components include, but are not limited to, antibodies, enzymes, cell organelles, or whole microorganisms. Peptides and proteins are also referred to as biomolecules. However, peptides are not bioactive in that they can convert other molecules or promote specific bonds to other molecules. A distinction is made between two basic types of biosensors: on the one hand the so-called bioaffinity sensors and on the other hand the so-called metabolism sensors. The bioaffinity sensors generate a signal by complexing bioactive molecules to form an analyte. Here, the initial state is again after the measurement. While the latter are based on a specific recognition and subsequent implementation of substrates. After this This sensor process must either be discarded or the initial state restored.

enzymes

Enzymes are in the sense of the invention highly specialized proteins that catalyze biochemical reactions without being consumed. Ribozymes that come under the term Enzymes can be subsumed include ribonucleic acid molecules. Surpass enzymes synthetic catalysts in their catalytic properties. Enzymes have a high affinity and selectivity to certain substrates. Important among the enzymes are in particular Protestases, the so-called serine proteases I, such as trypsin, bacterial serine proteases II, such as subtilisin, cysteine proteases such as papain, aspartic proteases such as penicilopepsin, metalloproteases I as boron coride oxide peptidase A, bacterial metalloproteases II as Thermolysin. Proteases always digest on a particular one Place on a peptide or protein. Hydrolyze endopeptidases certain amide bonds within a protein, while exopeptidases Cleave one or three amino acids at the C or N terminal of the peptide.

chromogenic

Chromogens are molecules that electromagnetic radiation, for example visible light, UV or IR, can absorb. According to the invention, the term “chromogen” also includes the so-called leuco form, as well as the actual chromophore, which in particular absorbed in the range of visible light. Usually stand out Chromogens characterized by extensive conjugated connection systems, where the Π-Π * transitions into Visible are shifted.

Other suitable chromogens besides the basic bodies Medola blue and Nile blue are, for example, amino-substituted 1,4 Naphthoquinones such as 6-amino-2,3-dichloro-l, 4-naphthoquinone and 6-amino-2,3-dicyano-l, 4-naphthoquinone). You can also Naturally the 4 or 5 amino naphthoquinones can also be used. Further Suitable chromogens are, for example, 2-anilino-3-chloro-l, 4-naphthoquinone, 2- (4-N, N-diethylamino-phenyl) -1,4-naphthoquinone, 2,3-dicyano-1,4-naphtholdiol, 2,3-dicyano-1,4-naphthoquinone, 2,3-dicyano-l, 4-naphthoquinone-l-phenyleneimine, 4-nitro [2.2] paracyclophane or nitrated paracyclophane and simple Cyclophanes.

characters

The invention is illustrated below of figures and embodiments exemplified. It it is clear that the above and below characteristics to be described not only in combination, but also be claimed alone.

1 shows a synthetic scheme for the preparation of a peptide labeled with an eminonaphtoquinone.

2 shows the ¹ H-NMR spectrum of 6-amino-1,4-naphthoquinone.

3 shows the ¹ H NMR spectrum of compound 15.

4 shows the ¹ H NMR spectrum of compound 16.

5 shows the ¹ H-NMR spectrum of 6-nitro-2,3-dichloro-1,4-naphthoquinone (compound 22).

DMF

N, N-dimethylformamide

NEt ₃

triethylamine

EE

ethyl acetate

DC

TLC

IBCF

isobutylchloroformate

AcOH

Eisesssig

iPrOH

2-propanol

BOC

benzyloxycarbonyl

MeOH

methanol

R _F

retention factor

nBuOH

n-butanol

ANC

aminonaphthoquinone

TSCC

tosyl

EtOH

ethanol

DCM

dichloromethane

FC

Flash chromatography

example 1

Nile blue as chromogenic building block

Synthesis of 4 - ((benzyl-L-serinyl-L-glycyl) -L-arginylamido) -N- (9-diethylaminobenzo [a] phenoxazin-5 ylidene) benzoic acid amide

• 1.1. Synthesis of 4-amino-N- (9-diethylamino-benzo (a) phenoxazin-5-ylidene) benzoic acid amide Step A: 4-Nitro-N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide 11.3 g of Nile Blue A was stirred into 140 ml of DMF and 11 g of NEt ₃ were added to the mixture, followed by 7.2 g of N-oxy-succinimidyl 4-nitrobenzoate After stirring overnight, the solvent was removed in vacuo and under high vacuum, the residue on silica gel 60 adsorbed and after application of the same on a chromatography column conditioned with CHCl ₃ / EE 8: 1 (v / v) and filled with silica gel 60, eluted with the same solvent mixture. Yield: 5.6 g purity control: DC R _F = 0.60 (CHCl ₃ / EE 11: 1 v / v) Step B: 4-amino-N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide 4 g of 4-nitro-N- (9-diethylamino-benzo [a] phenoxazine -5-ylidene) benzoic acid amide were dissolved in 70 ml of glacial acetic acid and 10 g of zinc dust were added with stirring largely removed in vacuo and the residue was transferred with CHCl ₃ into a shaking funnel. The organic phase was washed with 2N NaOH and dried over MgSO ₄ . After removing the solvent in vacuo, the remaining residue was purified by chromatography on silica gel 60 (CHCl ₃ / MeOH 40: 1 v / v). Yield: 2.2 g purity control: DC R _F = 0.24
• 1.2 Synthesis of (4 - ((Benzyl-L-serinyl-L-glycyl) -L-arginylamido) -N- (9-diethylaminobenzo [a] phenoxazin-5-ylidene) benzoic acid amide) Step A: 4- (N _α N _δ N _ω tri-ZL-arginylamido) -N- (9-diethylamino-benzo (a) phenoxazin-5-ylidene) benzoic acid amide 2.88 g (N _α N _δ N _ω tri-ZL-arginine were in argon in 8 ml abs. After cooling to room temperature, 0.7 ml of NEt ₃ and 0.65 ml of IBCF were added and the resulting mixture was stirred for 20 minutes, after which 1.32 g of 4-amino-N- (9-diethylamino-benzo [a] phenoxazine were added -5-ylidene) benzoic acid amide and the reaction mixture was stirred for 12 h at room temperature, the solvent was removed under high vacuum and the residue was purified by chromatography on silica gel 60 (CHCl ₃ / EtOH 30: 1 v / v). Yield: 2.89 g Purity control: TLC R _F - 0.34 Step B: 4- (L-arginylamido) - N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide 750 mg 4- (N _α N _δ N _ω tri-ZL -arginylamido) - N- (9-diethylamino-benzo [a ] phenoxazin-5-yliden) benzoic acid amide were dissolved in 20 ml of dioxane and added with rinsing with 30 ml of McOH to a prehydrogenated suspension of 500 mg palladium on activated carbon (10%, about 50% o water content) in 50 ml of McOH. After 3 h of hydrogenation with vigorous stirring under 1.1 bar of H ₂ , 12.5 ml of 1% methanolic HCl were added to the reaction mixture and the same was filtered through Celite. After the filtrate had been freed from the solvent in vacuo, the residue was purified by chromatography on silica gel 60 (iPrOH / AcOH / H ₂ O 3: 2: 2 v / v / v). Yield: 250 mg purity control: TLC R _F = 0.20 Step C: 4 - (((Na BOC- (benzyl-L-serinyl)) - L-glycyl) -L-arginylamido) -N- (9-diethylaminobenzo [a] phenoxazin-5-yliden) benzoic acid amide 453 mg 4- (L-arginylamido) - N- (9-diethylamino-benzo [a] phenoxazin-5-yliden) benzoic acid amide and 433 mg N _α -BOC- (benzyl-L-serinyl) ) -L-glycyl-N-oxysuccinimidyl ester was mixed with a mixture of 7.5 ml DMF and 91 mg NEt ₃ and the resulting solution was stirred for 12 h. After the solvent had been removed under high vacuum, the resulting residue was purified by chromatography on silica gel 60 (iPrOH / AcOH / H ₂ O 4: 1: 1 v / v / v). Yield: 350 mg purity control: TLC R _F = 0.18 Step D: 4 - ((Benzyl-L-serinyl-L-glycyl) -L-arginylamido) -N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene ) benzoic acid amide 222 mg 4 - (((N _α BOC- (benzyl-L-serinyl)) - L-glycyl) -L-arginylamido) -N- (9-diethylaminobenzo [a] phenoxazin-5-ylidene) benzoic acid amide were in a mixture of 10 ml DCM and 10 ml TFA for 2 h at room temperature. The solvent was removed in vacuo and then in a high vacuum and the residue was purified by chromatography on silica gel 60 (CHCl ₃ / MeOH / AcOH 1: 2: 2 v / v / v). Yield: 180 mg purity control: DC R _F = 0.17 (iPrOH / AcOH / H ₂ O 2: 1: 1 v / v / v)

Example 2

meldola as a chromogenic building block

Synthesis of 4 - ((benzyl-L-serinyl-L-glycyl) -L-arginylamido) -N- (9-diethylaminobenzo [a] phenoxazin-5 ylidene) aniline

• 2.1 Synthesis of 4-amino-N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline Step A: 4-nitro-N- (9-dimethylamino-benzo [ajphenoxazin-5-ylidene) aniline (ETH ^T 6005) 7.03 g of Meldola blue and 3.28 g of 4-nitroaniline were placed under argon. Then 172 ml of DCE dried over molecular sieves and 8 ml of NEt ₃ also dried over molecular sieves were added and the resulting suspension was refluxed for 12 h. After cooling, 110 g of neutral aluminum oxide (activity level III) were added to the RG and concentrated in vacuo and then in a high vacuum. The crude product was then chromatographed on 200 g of neutral aluminum oxide (activity level III) (gradient from hexane / DCM 1/1 to pure DCM). The resulting product was recrystallized from DCM / EE. Yield: 2.7 g purity control: DC R _F. Step B: 4-Amino-N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline A suspension of 413 mg of 4-nitro-N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene ) Aniline in 8 g of glacial acetic acid was mixed with 1.1 g of zinc dust at RT with stirring. The previously deep blue suspension turned into a light green solution. After 15 min, the RG was largely freed of AcOH in vacuo and transferred to a shaking funnel using DCM. It was washed with 2 N NaOH and the aqueous phase was extracted again with DCM. After drying over MgSO ₄ and concentration in vacuo, the title compound was obtained in sufficient purity for the subsequent coupling. Yield: 346 mg purity control: DC R _F = 0.45 (MeOH / AcOH 2: 1 v / v)
• 2.2 Synthesis of 4 - ((Benzyl-L-serinyl-L-glycyl) -L-arginylamido) -N- (9-diethylaminobenzo [a] phenoxazin-5-ylidene) aniline Step A: 4- (N _α N _δ N _ω tri-ZL-arginylamido) - N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline 378 mg N _α, N _δ, N _ω- tri-ZL-arginine were dissolved under argon in 1.2 ml abs. DMF dissolved in the heat. After cooling to room temperature, 66 mg abs. NEt ₃ and 90 mg of IBCF were added and the resulting mixture was stirred for 20 min. A solution of 250 mg of 4-amino-N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline in 3.5 ml of DMF was then added and the reaction mixture was stirred at room temperature for 12 h. The solvent was removed in a high vacuum and the residue was purified by chromatography on silica gel 60 (CHCl ₃ / MeOH / AcOH 13: 1: 1 v / v / v). The resulting acetate was taken up in DCM, washed with 2 N NaOH and the organic phase was dried over K ₂ CO ₃ and concentrated in vacuo. Yield: 338 mg purity control: TLC R _F = 0.21 Step B: 4- (L-arginylamido) -N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline 200 mg 4- (N _α, N _{δ ,} N _ω- tri-ZL-arginylamido) - N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline were suspended or partially dissolved in 18 ml of McOH and made into a pre-hydrogenated suspension of 155 mg palladium on activated carbon ( 10%, about 50% water content) in 6 ml of McOH. After 2 hours of hydrogenation with vigorous stirring under 1.1 bar of H ₂ , a few drops of glacial acetic acid were added to the reaction mixture and the same was filtered through cellite. After the filtrate had been freed from the solvent in vacuo, the title compound was obtained in sufficient purity for the subsequent reaction. Yield: 105 mg purity control: DC R _F. Step C: 4 - (((N _α BOC- (benzyl-L-serinyl)) - L-glycyl) -L-arginylamido) -N- (9-dimethylaminobenzo [a] phenoxazin-5-ylidene) aniline 73 mg N _α BOC- (benzyl-L-serinyl)) - L-glycyl-N-oxysuccinimidyl ester and 81 mg of 4- (L-arginylamido) -N- (9-dimethylammo-benzo [a] phenoxazin-5-ylidene) aniline were mixed with a mixture of 1 ml of DMF and 22 mg of NEt _{3 was} added and the resulting solution was stirred for 12 h. After the solvent had been removed under high vacuum, the resulting residue was purified by chromatography on silica gel 60 (nBuOH / AcOH / H ₂ O 5: 1: 1 v / v / v). Yield: 65 mg purity control: TLC R _F = 0.23 Step D: 4 - ((Benzyl-L-serinyl-L-glycyl) -L-arginylamido) -N- (9-diethylamino-benzo [aJphenoxazin-5-ylidene) aniline

Example 3

naphthoquinone as a chromogenic building block

Compound 4 was first synthesized according to the method described in the literature (cf. experimental part) and then coupled to Z-Arg (Z ₂ ) -OH (13).

After deprotection to 16 the synthesis took place of substrate 18 BOC-Ser (Bz) -Gly-Arg-6ANC without Difficulties (see scheme 2).

Schema 2: Schema der Synthesen von 6-Amino-l,4-naphthochinon (4) und BOC-Ser(Bz)-Gly-Arg-6ANC (18)

Scheme 2: Scheme of the syntheses of 6-amino-l, 4-naphthoquinone (4) and BOC-Ser (Bz) -Gly-Arg-6ANC (18)

Synthesis strategy for the representation of the chromogen labels 4, 5 and 6 Since the synthesis of 5 and 21 takes place without difficulty starting from 22, the synthesis of 4 was alternatively carried out as follows:
The following path was chosen for the representation of 6:

Alternatively, the conversion from 5 to 6 took place over 25:

• 3.1 Synthesis of 6-amino-1,4-naphthoquinone (4) 3.1.1 p-toluenesulfonic acid-β-naphthylamide (8) 55.7 g (389 mmol) of β-naphthylamine (7) were added in 111 g (1.4 mol = 113 ml ) Dissolved pyridine and 84.8 g (393 mmol = 1.01 eq.) Of TsCl were added in portions with stirring at RT. The mixture was allowed to stir for about 2 h, then the RG was concentrated to the mixture of 175 ml. HCl and 500 g of ice poured. The RP was filtered off and the filtrate extracted once with ether. The RP was then dissolved in DCM and the solution was combined with the ether phase. You washed with sat. CuSO ₄ (until the color was no longer intensified) and sat. NaCl, dried over MgSO ₄ and concentrated at RV and HV. DC (H / EE 2: 1) showed only slight reddish impurities remaining at the starting spot. Yield: 101.98 g (342.9 mmol = 88%). 3.1.2 1,6-Dinitro p-toluenesulfonyl-2-naphthylamide (9) 29.7 g (100 mmol) of tosylate 8 were dissolved in 89 ml of glacial acetic acid heated to 55 ° C and slowly stirred with 20.8 ml of 72% HNO ₃ (from a mixture of 18 ml of 65% HNO ₃ and 4.5 ml of smoking HNO ₃ ). The RG was allowed to cool to RT with stirring, whereupon a portion of the product crystallized out in the form of a light yellow solid, the RG was mixed with 10 ml of water and, after 10 minutes, suction filtered through a glass suction filter. The title compound was obtained in pure form by washing with a little semi-concentrated HOAc and drying on the HV. Yield: 27.9 g (72 mmol = 72%). 3.1.3 6-Nitro-2-diazo-l-naphthol (10) 13 g (33.6 mmol) 9 were concentrated in 26 ml. H _Z SO ₄ stirred in and the mixture then heated to 40 ° C, until a clear solution was obtained. The RG was then cooled to <20 ° C and concentrated with stirring with the solution of 3.12 g (45 mmol = 1.3 eq.) NaNO ₂ in 14.3 ml. HzSO ₄ offset. 52 ml of glacial acetic acid were then added in portions with stirring and the RG was stirred until no more phase separation was visible. The RG was then slowly added to 1.51 ice water (50% ice) and the whole was left to stand overnight. The product was suctioned off and dried at the HV. Yield: 6.67 g (31 mmol = 92%). 3.1.4 6-nitro-l-naphthol (11) 7.82 g (36.3 mmol) 10 were cooled (∼ 10 ° C) in a mixture of 58.6 ml glacial acetic acid and conc. H ₂ SO ₄ dissolved. This solution was then added over a period of about 15 to the well-stirred suspension of 19.55 g of Cu ₂ O in 156 ml of EtOH, heated to 55 ° C. The RG was then heated to 80 ° C., taking care that this temperature was not exceeded, 5.45 g of Cu ₂ O were added to the RG and the mixture was stirred for a further 30 min. The RG was then filtered through a glass suction filter (D3), the residue was washed with a little boiling EtOH and the filtrate was concentrated on a RV at a bath temperature of 60 ° C. until most of the ethyl acetate formed had been removed. The same was stirred into 780 ml of ice / water mixture (2/3 ice) and the resulting mixture was left to stand at RT overnight. The next day the mixture was extracted with DCM, the extract was dried over MgSO ₄ , concentrated on a RV and the resulting brownish orange-red solid was dried on a HV. The title compound was obtained by filtration over silica gel (∅ = 5 cm, h = 5 cm, hexane / ethyl acetate 3: 1) and subsequent concentration and drying in the form of an orange-red solid. Rf: 0.25 (hexane / ethyl acetate 3: 1) Yield: 2.61 g (13.8 mmol = 38%). 3.1.5 6-Amino-l-naphthol (12) 2.1 g (11.1 mmol) 11 were dissolved in 21 ml iPrOH and added to the well-prehydrated mixture of 5.5 ml Ra-Ni suspension and 5.5 ml iPrOH. The RG was hydrogenated with intensive stirring for 3 h at RT under 3 bar H2, then the catalyst was sucked off through a glass filter and washed with acetone. The RP was dissolved in a little acetone, filtered through silica gel (∅ = 5 cm, h = 10 cm, hexane / ethyl acetate 3: 2), then dissolved in ethyl acetate and 12 were obtained from it in the form of colorless crystals after adding hexane. Rf: 0.28 (hexane / ethyl acetate 3: 2) Yield: 1.64 g (10.3 mmol = 93%).
• 3.1.6 6-Amino-1,4-naphthoquinone (4) To a stirred solution of 400 mg (2.52 mmol) of 6-amino-1-naphthol (12) in 24.8 ml of McOH, cooled to 10 ° C, which is dissolved in a 500 ml two-necked flask, a cooled solution (5-10 ° C) of 1.55 g (5.78 mmol = 2.3 eq.) of potassium nitrodisulfonate (Fremy salt) was added over a 1/2 hour under a gentle Ar stream [14, 15 ] in 25 ml 1/6 M KHP ₂ O ₄ and 88 ml water. After no more educt was present in the RG, approximately after 45 min, the RG was extracted with ethyl acetate, the extract was dried over MgSO ₄ and concentrated. After purification via FC (∅ = 3 cm, h = 20 cm, hexane / ethyl acetate 3: 2), 271 mg (1.56 mmol = 62%) of pure title compound were obtained in the form of dark violet crystals. Rf: 0.32 (DCM / ethyl acetate 10: 1)
• 3.2 Synthesis of BOC-Ser (Bz) -Gly-Arg-6ANC (18) 3.2.1 Z-Arg (Z ₂ ) -6ANC (15) 1,038 g (1.8 mmol) Z-Arg (Z ₂ ) OH (13) were placed with a stirring magnet under Ar in a heated and sealed with 25 septum flask and mixed with 2.7 ml of absolute THF using a syringe. After a clear solution had been obtained by stirring at RT, the RG was cooled to -5 ° C. and with stirring through the septum with 249 μl (181.8 mg = 1.8 mmol, Hamilton syringe) absolute NEt ₃ , followed by 234 μl ( 246.3 mg = 1.8 mmol, Hamilton syringe) freshly distilled isobutyl chloroformate. The mixture was stirred at -5 ° C. for about 1/2 h and then the solution of 156 mg (0.9 mmol = 0.5 eq.) 6-amino-1,4-naphthoquinone (4) in 1.5 ml of dry DMF was added in one shot , After stirring for 1/2 h at -5 ° C, the cooling was removed and the RG was stirred for 6 h at RT. The RG was concentrated on the RV and thoroughly dried on the HV. After purification via FC ((6 = 3 cm, h = 20 cm, DCM / ethyl acetate 11: 1 until after the elution of excess 6ANC (4), then DCM / ethyl acetate 10: 1), 270 mg (369 μmol = 41% ) 7 pure title compound in the form of a light yellow solid Rf: Z-Arg (Z ₂ ) -6ANC (15): 0.25 (DCM / ethyl acetate 10: 1) 6ANC (4): 0.32 (DCM / ethyl acetate 10: 1) P1 (By-product): 0.53 (DCM / ethyl acetate 10: 1 3.2.2 Arg-6ANC (16) 100 mg (137 μmol) Z-Arg (Z ₂ ) -6ANC (15) were added in approx. 4 ml 1,4-dioxane dissolved and added with rinsing with a little McOH to the prehydrogenated suspension of 37 mg Pd / activated carbon (10%, 50% water content) in 10 ml McOH The mixture was stirred with vigorous stirring for 2 1/2 hours under 1.2 bar H ₂ in a 2.28 ml of 1% methanolic HCl (273 μmol = 2 eq.) Were added to the RG and the catalyst was sucked off through a glass filter pad filled with Celite while rinsing with McOH. The filtrate was concentrated to dryness at RV and HV and via FC (∅6 = 1 cm, h = 20 cm, 1 -Butanol / glacial acetic acid / water 3: 1: 1) purified. After concentration on RV and HV, 56.6 mg (126 μmol = 92% o) of the title compound were obtained as diacetate in the form of a greenish-yellow solid. Rf: 0.47 (1-butanol / HOAc / water 3: 1: 1) 3.2.3 BOC-Ser (Bz) -Gly-Arg-6ANC (18) 26 mg (57.8 μmol) Arg-6ANC (16) and 36 mg (80 μmol = 1.38 eq.) BOC-Ser (Bz) -Gly-OSu (17) were dissolved in 0.4 ml dry DMF (brownish yellow solution) and 13.8 μl (10.5 mg = 1.8 eq.) NEt ₃ using a GC syringe added. The RG was shaken overnight and the solvent was then removed at the HV. The after FC (∅ = 1 cm, h = 20 cm, CHCl ₃ / MeOH / HOAc 8: 1: 1 until shortly before (clearly visible) elution of 18, then CHC1 ₃ / MeOH / HOAc 7: 1: 1) product fraction obtained was evaporated to dryness on RV and HV and then suspended in CHC to remove residues of silica gel and filtered. After concentration, 27 mg (37.2 μmol = 64%) of the slightly apolar-contaminated title compound resulted as monoacetate in the form of a brownish-yellow powder. Rf: 0.11-0.25 (CHC1 ₃ / MeOH / HOAc 8: 1: 1)
• 3.3 synthesis of 6-amino-2,3-dichloro-l, 4-naphthoquinone (5) 3.3.1 6-nitro-2,3-dichloro-l, 4-naphthoquinone (22) 5 g (22 mmol) 2, 3-dichloro-l, 4-naphthoquinone were concentrated to 15 ml. H ₂ SO _{4 was} added and the mixture was heated to 80 ° C. with stirring until a clear solution was obtained. 30 ml of smoking HNO ₃ were added and the RG was stirred at 80-90 ° C. for 1 h. The same was then placed in 800 ml of ice water and the precipitate was filtered off with suction. The filtrate was extracted with ether and combined with the extract with the solution of the precipitation in ether. You washed twice with sat. NaHCO ₃ and once with sat. NaCl, dried over MgSO ₄ and concentrated. The RP was drawn onto 3 large polyspoons of silica gel and purified via FC (∅ = 5 cm, h = 20 cm, hexane / ethyl acetate 5: 1). This resulted in 1.12 g (4.13 mmol = 16.5%) of pure title compound in the form of lemon-yellow leaves. Rf: 5-nitro-2,3-dichloronaphthoquinone (22): 0.38 (hexane / ethyl acetate 5: 1) 5-nitro-2,3-dichloronaphthoquinone: 0.16 (hexane / ethyl acetate 5: 1) 3.3.2 6-amino 2,3-dichloro-l, 4-naphthoquinone (5) a.) 135 mg (0.5 mmol) 6-nitro-2,3-dichloro-l, 4-naphthoquinone (22) were dissolved in 0.7 ml of hot glacial acetic acid and under Ar the solution of 450 mg SnCl ₂ dihydrate (2 mmol) in 3-4 drops conc. HCl added. The RG was heated to 80 ° C. while stirring under Ar until the color changed from violet to dirty yellow (a few minutes). By adding 0.7 ml of conc. HCl, the hydrochloride of 6-amino-2,3-dichloro-l, 4-naphthohydro-quinone was precipitated and suction filtered. After drying at the HV, the RP was dissolved in a little water (approx. 5 ml) and acidified with a lot of conc. HCl on. The hydrochloride which crystallized out after about half an hour of cooling in the refrigerator was suction filtered and thoroughly dried at the HV. This resulted in 81 mg (287 μmol = 57%) of 6-amino-2,3-dichloro-l, 4-naphthohydroquinone hydrochloride in the form of a slightly yellowish powder. b.) 22.8 mg (81 μmol) of hydrochloride were dissolved in 2 ml of water and a solution of about 500 mg of FeC ₁₃ in a few ml of water was added with stirring, whereupon the RG immediately turned deep violet. After stirring for about • h, the RG was extracted with ethyl acetate, the extract was dried over MgSO ₄ and concentrated. 19 mg (78 μmol = 97%) of pure title compound resulted in the form of deep violet crystals. Rf: 0.45 (hexane / ethyl acetate 1: 1)

Example 4

Enzymatic cleavage of BOC-Ser (Bz) -Gly-Arg-6ANC (18) with trypsin (bovine pancreas)

Stock solutions:

• TRIS buffer: 4.41 g (30 mmol) CaCl ₂ . 2 H ₂ O and (2x conc.) 1,211 g (10 mmol) TRIS in 100 ml water, adjusted to pH 7.4 with 0.1 M HCl
• Trypsin: 1 mg trypsin (bovine pancreas) in 100 ml 0.001 M HCl solved, 5 ml of which diluted to 100 ml with 0.001 M HCl => 0.5 mg / l.
• Substrate: 4.34 mg (6 μmol) dissolved in 240 ml DMSO, to 2.4 ml TRIS buffer (2x) and 2.16 ml water. Concentration: 1.25 mM The stock solution was filtered twice over a little cotton in a Pasteur pipette. After standing for two days of for the trials of unused residue had turned out to be a fine one, however significant sediment formed.

Measurement solutions:

The measuring solutions were prepared directly in the measuring cuvettes in such a way that 2 ml total volume contained 25 μg / 1 trypsin, 100 μl DMSO (5%), simple TRIS buffer concentration (0.015 M CaC1 ₂ , 0.005 M TRIS) and the indicated ideal substrate concentration , The trypsin solution was added after annealing the solution at 37.2 ° C. for 15 minutes immediately before the start of the measurement.

Claims (4)

Chemical compound of the general formula (I):

where R can be alkyl, aryl, H, aralkyl, CN, R ₃ , R ₆ , R ₇ and R _{8 can be the} same or different and H, CN, NO ₂ , NH ₂ , alkyl, aralky, halogen or alkoxy , A and B can be identical or different can be an alpha, beta or gamma amino acid which comprises 2 to 15 C atoms and up to 4 N atoms, 2 sulfur atoms and 6 oxygen atoms and B is optionally a dipeptide formed from these amino acids C is arginine, lysine, tyrosine, phenylalanine or tryptophan and their homologues and X is an H atom or a protective group for terminal amino groups which is customary in peptide chemistry, and L is an optional linker building block which is capable of binding to C with a peptide train. Chemical compound of the general formula (II)

where R ₁ and R _{2 may be the} same or different and H, CN, halogen, C1 to C4 is alkyl, aryl or aralkyl or alkoxy, R ₃ , R ₆ , R, and R _{8 may be the} same or different and H, CN , NO ₂ , NH ₂ , alkyl, aralkyl, halogen or alkoxy, A and B can be identical or different can be an alpha, beta or gamma amino acid which can have 2 to 15 C atoms and up to 4 N atoms, 2 sulfur atoms and comprises 6 oxygen atoms and B is optionally a dipeptide formed from these amino acids, C is arginine, lysine, tyrosine, phenylalanine or tryptophan and their homologues, and X is an H atom or a protective group for terminal amino groups which is customary in peptide chemistry, and L is an optional linker building block capable of forming a peptide bond with C. Chemical compound of the general formula (III)

where R ₁ , R _{2 are the} same or different and are alkyl, aryl, H, aralkyl, CN, R ₃ , to R _{6 may be the} same or different and H, CN, NO ₂ , NH ₂ , alkyl, aralkyl, halogen or can be alkoxy with the condition that at least one radical from R ₃ to R _{6 is} an amino group, one nitrogen substituent of which has the motif XABC-, where A and B can be identical or different and can be an alpha, beta or gamma amino acid, which comprises 2 to 15 C atoms and up to 4 N atoms, 2 sulfur atoms and 6 oxygen atoms and B is optionally a dipeptide formed from these amino acids, C is arginine, lysine, tyrosine, phenylalanine or tryptophan and their homologues, and X is an H atom or a protective group for terminal amino groups which is customary in peptide chemistry, and L is an optional linker building block which is capable of forming a peptide bond with C. Chemical compound of the general formula (IV)

where R ₁ , R _{2 are the} same or different and are alkyl, aryl, H, aralkyl, CN, R ₃ to R _{6 can be the} same or different and H, CN, NO ₂ , NH ₂ , alkyl, aralkyl, Can be halogen or alkoxy, A and B can be identical or different can be an alpha, beta or gamma amino acid which comprises 2 to 15 C atoms and up to 4 N atoms, 2 sulfur atoms and 6 oxygen atoms and B is optionally a dipeptide, which is formed from these amino acids, C is arginine, lysine, tyrosine, phenylalanine or tryptophan and their homologues, and X is an H atom or a protective group for terminal amino groups which is customary in peptide chemistry, and L is an optional linker building block which is capable of this is to form a peptide bond with C.