DE3108322A1 - Chromogenic enzyme substrate - Google Patents

Abstract

For the purpose of increasing the specificity of such substrates with respect to certain enzymes with simultaneous fitness for use in a physiological pH range and good solubility thereof in aqueous medium, the enzyme substrate contains novel compounds of the general formula <IMAGE> in which R1 denotes hydrogen, alkyl, a sulphonyl radical or an acyl radical, A represents D-Arg, L-Arg, D-Lys, L-Lys, D-Orn or L-Orn, which amino acid component is optionally linked to the substituent R1 via one or more further amino acid components, and Hal denotes halogen.

Description

The invention relates to a chromogenic enzyme substrate based on amino acid derivatives or oligopeptides.

Such chromogenic enzyme substrates are used for the quantitative determination of proteolytic enzymes, the substrates being hydrolytically cleaved and their cleavage products being examined spectrophotometrically. Chromogenic enzyme substrates based on amino acid derivatives are already known which are characterized, for example, according to US Pat. No. 4,070,245 by the amino acid sequence Gly-Pro-Arg or Gly-Pro-Lys, where the N-terminal amino acid can be protected and C- a p-nitroanilino group is bound terminally to Arg or Lys.

A similar structure of a chromogenic substrate is known from AT-PS 349 648, in which a tripeptide is linked peptidically to the chromogenic group p-nitroaniline via the amino acid residues Arg, Lys or Orn.

When using known chromogenic substrates, the difficulties arise that the cleavage reactions take place optimally in a pH range from 8.3 to 8.5, that is, in a higher than the physiological pH range pH range (7.3 to 7.5), which means a deviation from the native conditions, which is undesirable. Furthermore, some of these known substrates have the disadvantage that they are poorly soluble.

Another disadvantage of known chromogenic substrates is that they are not specifically cleaved by a certain enzyme, e.g. thrombin, but are also attacked by various other enzymes, such as trypsin, plasmin, kallikrein, etc.

The invention aims to overcome these disadvantages or difficulties and has the task of creating a chromogenic enzyme substrate with a new chromogenic or fluorescent group, as well as with a novel modified amino acid sequence, which substrates can be used in the physiological pH range, characterized by improved specificity, good solubility and high light absorption of their cleavage products.

This object is achieved according to the invention in that the chromogenic enzyme substrate contains new compounds of the general formula

(I)

or their salts contains, wherein

R [deep] 1 is hydrogen, alkyl, a sulfonyl radical, in particular benzenesulfonyl or p-methoxybenzenesulfonyl (Mbs) or an acyl radical, in particular alkoxycarbonyl, such as t.Butyloxycarbonyl, benzyloxycarbonyl, formyl or benzoyl,

A for one of the following amino acid components:

D-Arg, L-Arg, D-Lys, L-Lys, D-Orn or L-Orn, which optionally has one or more of the following further amino acid components: Gly, D-Glu (OX [deep] 1), L-Glu (OX [deep] 1) [where X [deep] 1 represents an ester residue], D-Glu, L-Glu, D-Ala, L-Ala, D-Val, L-Val, DIle, L- Ile, D-Leu, L-Leu, D-Pip, L-Pip, D-Pro, L-Pro, D-Aze, L-Aze, D-Phe, L-Phe, Phenylglycyl, D-Tyr, L- Tyr, D-Pyr, L-Pyr, the remainder of a c [deep] alpha-branched amino acid, like Aib, a small beta-amino acid, like small beta-Ala, and small beta-cyclohexylalanyl, a small gamma-amino acid, like Gaba , or an N-alkyl-amino acid, such as Sar, is linked to the substituent R [deep] 1, and

Hal means halogen.

The abbreviations used above and in the following description have the following meaning:

amino acids

H-Aib-OH aminoisobutyric acid

H-Ala-OH alanine

H-Arg-OH arginine

H-Aze-OH azetidine-2-carboxylic acid

H-Gabe-OH small gamma-aminobutyric acid

H-Glu-OH glutamic acid

H-Gly-OH glycine

H-Ile-OH isoleucine

H-Leu-OH Leucine

H-Lys-OH lysine

H-Orn-OH ornithine

H-Phe-OH phenylalanine

H-Pip-OH pipecolic acid

H-Pro-OH proline

H-Pyr-OH pyroglutamic acid (2-pyrrolidone-5-carboxylic acid)

H-Sar-OH sarcosine

H-Tyr-OH tyrosine

H-Val-OH valine

miscellaneous

Adoc Adamantyloxycarbonyl

Aoc t. Amyloxycarbonyl

Bpoc Z- (p-biphenylyl) -isopropyloxycarbonyl-

Boc t-butyloxycarbonyl

Bz benzoyl

-Cl-NA chloronitroanilide

-o-Cl-pNA o-chloro-p-nitroanilide

DCCI dicyclohexylcarbodiimide

Ddz small alpha, small alpha-dimethyl-3,5-

dimethoxybenzyloxycarbonyl

EE ethyl acetate

Fmoc 9-fluorenylmethoxycarbonyl-

Foc furfuryloxycarbonyl

HAc CH [deep] 3COOH

HMPT hexamethylphosphoric triamide

HOBt 1-hydroxybenzotriazole

Iboc Isobornyloxycarbonyl-

Mbs p-methoxybenzenesulfonyl-

MW molecular weight

MeOH methanol

pNA p-nitroanilide

Nvoc 6-nitroveratryloxycarbonyl-

PÄ petroleum ether

Pipoc Piperidinooxycarbonyl

Pz p-phenylazobenzyloxycarbonyl-

t. tertiary

TFA trifluoroacetic acid

-ONP p-nitrophenyl ester

-OPCP pentachlorophenyl ester

-OTCP trichlorophenyl ester

-OSu hydroxysuccinimide ester

Z benzyloxycarbonyl

Z (p-Br) - p-bromobenzyloxycarbonyl-

Z (p-Cl) - p-chlorobenzyloxycarbonyl-

Z (OMe) - p-methoxybenzyloxycarbonyl-

Z (p-NO [deep]) 2- p-nitrobenzyloxycarbonyl-

Z (o-NO [deep] 2) - o-nitrobenzyloxycarbonyl-

Z (OMe) [deep] 2- 3,5-dimethoxybenzyloxycarbonyl

According to one embodiment of the invention, an amino acid component representing A, D-Arg, L-Arg, D-Lys, L-Lys, D-Orn or L-Orn, is substituted by R [deep] 1 in the side chain amino function.

The novelty of the chromogenic substrates lies - for all embodiments - in the component of the halogenated p-nitroanilide group; for some specific embodiments in the N-terminal protective group Mbs, for still others in a specific sequence of L- and D-stereomers of the amino acid residues known per se; In addition, some of the amino acid components mentioned in addition to Arg, Lys and Orn, such as Aib, Gaba, small beta-Ala, small beta-cyclohexylalanine and Sar, have not yet been incorporated into such substrates.

According to the invention, a chromogenic enzyme substrate advantageously contains a new compound of the formula (I) in which, in an acyl group X-CO- representing the radical R [low] 1

X denotes an aliphatic araliphatic, cycloaliphatic or aromatic hydrocarbon radical having 1 to 19 carbon atoms, which radical is optionally substituted by alkyl, amino or aminoalkyl.

R [deep] 1 preferably represents Mbs.

Particularly preferred are substrates which, as compounds of the general formula (I), are a compound of the formulas

(II),

(III)

(IV)

(V)

or

(VI)

contain.

The enzyme substrates mentioned have an excellent cleavage capacity in the physiological pH range and can therefore be used under native conditions.

Furthermore, they are highly specific for thrombin. Specificity is understood here as the increase in extinction per unit of time when a given amount of an enzyme acts on a chromogenic substrate, divided by the increase in extinction per unit of time when a given amount of thrombin acts on the same substrate at a given pH.

The enzyme substrates according to the invention are used for the determination of serine proteases, in particular for the determination of thrombin in the presence of other serine proteases in the physiological pH range.

The effectiveness of these substrates is shown below. The following reagents were used for the investigation:

Buffer solution: 6.06 g of tries (hydroxymethyl) aminomethane and 10.6 g of NaCl were dissolved in about 900 ml of distilled water and adjusted to a pH of about 5 M hydrochloric acid

7.5 or 8.5 are set. The volume of the solution was then made up to 1000 ml with distilled water.

Chromogenic substrates, as listed in the following table, were dissolved in a concentration of 1 mmol / l in distilled water.

Bovine thrombin "Topostasin" (La Roche) was dissolved in a 0.15 M NaCl solution and brought to a concentration of 1 IU per ml.

Trypsin: pancreatic protease (Merck) was adjusted to a final concentration of 1 mU / ml in a solution of 0.7% NaCl and 0.7% trisodium citrate × 2H [deep] 2O, which had a pH of 6.5. The units are based on the cleavage of benzoyl-arginyl-p-nitroaniline (BAPNA), with 1 unit of trypsin cleaving 1 μmol of BAPNA in 1 minute at 25 ° C.

Factor Xa (Activated Factor X); "Diagen chromatographed Bovine Factor Xa" (Diagnostic Reagents Ltd., Thame, Oxon-England).

Plasmin: "Plasmin" (Kabi) was dissolved in distilled water and adjusted to a concentration of 1.25 plasmin-casein units per ml.

Kallikrein: This enzyme was generated from human plasma.

The tests with thrombin, trypsin, factor Xa and plasmin were carried out in the following way:

A plastic tube was placed in a water bath thermostated at 37 ° C. and 1.0 ml of buffer solution was pipetted into it. After a warming-up time of 5 minutes, 0.1 ml of the respective enzyme solution was added and mixed. Then successful te the addition of 0.1 ml solution of the respective chromogenic substrate. The solutions were mixed and measured spectrophotometrically after about 10 seconds (Beckman twin-beam photometer No. 25, layer thickness 10 mm, wavelength 405 nm and temperature 37 ° C.).

The tests for kallikrein activity are carried out in the following way:

A plastic tube was placed in a water bath thermostated at 37 ° C. and 0.7 ml of buffer solution was pipetted into it. After a warming-up time of 5 minutes, 0.05 ml of human plasma and 0.05 ml of kaolin suspension with 10 mg of kaolin per ml of buffer solution were pipetted in and mixed. After incubation for 7 minutes at 37 ° C., the kaolin was separated from the solution in a centrifuge for about 3 minutes. The kaolin-free solution was mixed with 0.1 ml of a chromogenic substrate solution at 37 ° C., mixed and, after about 10 seconds, measured spectrophotometrically in the manner described.

The results can be seen from the following tables, with Table I represents the increase in absorbance per 1 minute multiplied by a factor of 1000. In Table II is the so-called quotient of the monospecificity, which is defined as

, and the sum of these quotients for a specific chromogenic substrate at the two pH values 7.5 and 8.5 of the buffer solution used in each case.

Table I.

Table II

It can be seen from Table I that the chromogenic substrates tested have high amidolytic activities. It can also be seen from Table I that the measurements can be carried out properly in the physiological pH range.

Table II shows that the quotient of the monospecificity is low and in some cases even comes very close to or even reaches the ideal value of zero. The sum of the quotients, based on thrombin, of measurements at pH 7.5 and pH 8.5, which is also given in Table II, shows through the low values that the monospecificity for thrombin is excellent compared to all other enzymes investigated.

The invention further comprises a process for the preparation of new compounds of the general formula (I) and their salts, which is characterized in that halo-p-nitroaniline is converted into halo-p-nitrophenyl isocyanate, this with one of the following having a free carboxyl group , but amino acids protected on their other functional groups with the components: D-Arg, L-Arg, D-Lys, L-Lys, D-Orn or L-Orn, whereupon the reaction product after liberation of the terminal amino group with an or several of the following amino acids having a free carboxyl group but protected on their other functional groups with the components: Gly, D-Glu (OX [deep] 1), L-Glu (OX [deep] 1) [where X [deep] 1 represents an ester residue], D-Glu, L-Glu, D-Ala, L-Ala, D-Val, L-Val, D-Ile, L-Ile, D-Leu, L-Leu, D-Pip, L-Pip, D-Pro, L-Pro, D-Aze, L-Aze, D-Phe, L-Phe, Phenylglycyl, D-Tyr, L-Tyr, D-Pyr, L-Pyr, having a free carboxyl group C [t ief] small alpha branched amino acids, like H-Aib-OH, small beta-amino acids, like H-small beta-Ala-OH and small beta-cyclohexylalanine, small gamma-amino acids, like H-Gaba-OH, or N-alkyl -Amino acids, such as H-Sar-OH, is reacted, whereupon, after the protective groups have been split off, a hydrogen atom on the terminal NH [deep] 2 group is replaced by acyl or sulfonyl.

Two or more of the following amino acid components are expedient: Gly, D-Glu (OX [deep] 1), L-Glu (OX [deep] 1) [where X [deep] 1 represents an ester residue], D-Glu, L- Glu, D-Ala, L-Ala, D-Val, L-Val, D-Ile, L-Ile, D-Leu, L-Leu, D-Pip, L-Pip, D-Pro, L-Pro, D-Aze, L-Aze, D-Phe, L-Phe, Phenylglycyl, D-Tyr, L-Tyr, D-Pyr, L-Pyr, a residue of a c [deep] small alpha-branched amino acid, like Aib, a small beta-amino acid such as small beta-Ala and small beta-cyclohexylalanyl, a small gamma-amino acid such as Gaba, or an N-alkyl-amino acid such as Sar, as at the terminal NH [deep] 2 group, if applicable Acyl or sulfonyl substituted dipeptide or oligopeptide are used.

To introduce a Mbs group for R [deep] 1 in the terminal amino group of an oligopeptide of the general formula (I), the terminal amino acid is advantageously reacted with Mbs chloride and the Mbs amino acid obtained either in the presence of a condensing agent directly or after conversion into the corresponding active ester coupled with a dipeptide or oligopeptide.

To prepare the compound of the formula (II), the Mbs-small beta-Ala-OH is advantageously used or an acid addition salt thereof.

To prepare the compound of the formula (III), the Mbs-Gly-OH is preferably used with or an acid addition salt thereof.

To prepare the compound of the formula (IV), the Z-Abi-OH, in which Z is benzyloxycarbonyl, is advantageously used with reacted and from the compound obtained the protective groups are split off.

In detail, the following is also stated with regard to the implementation of the method according to the invention:

In the preparation of the new compounds of the formula (I), the amino functions of the intermediate stages must be blocked by protective groups which are customary in peptide chemistry. Possible protecting groups are: Boc, Z, Z (OMe), Z (p-Br), Z (p-Cl), Z (p-NO [deep] 2), Z (o-NO [deep] 2) , Z (OMe) [low] 2, Ddz, Nvoc, Bpoc, Aoc, Adoc, Iboc, Fmoc, Foc, Pipoc, Pz, small alpha, small alpha-dimethylbenzyloxycarbonyl, 2-iodo-ethoxycarbonyl, 2- (p-tosyl ) -ethoxycarbonyl, 1- (1-adamantyl) -1-methylethoxycarbonyl, p-toluenesulfonyl, phthalyl, trifluoroacetyl, triphenylmethyl, o-nitrophenylthio, formyl, benzylsulfonyl, o-hydroxyarylidene, acetoacetyl, 5,5-dimethylcycyl, ohexadione, Nitrophenoxyacetyl and especially Mbs. The Mbs group proves to be advantageous over many other protective groups, especially for chromogenic substrates.

If a terminal carboxyl group is present, this can either through the remainder of the chromophore (Cl-pNA) or through methyl, ethyl, benzyl, p-nitrobenzyl, benzhydryl, isopropyl, t-butyl, p-methoxybenzyl -, alkylbenzyl, phenacyl, phenyl, 4-picolyl, small beta-methyl thioethyl, 4- (methylthio) phenyl, trimethylsilyl and phthalimidomethyl groups are protected.

If arginine is used, the side chain of arginine, the guanidino radical, can be replaced by N [high] small omega-tosyl, N [small] omega-Boc-, N [high] [small] omega-nitro and N [high] small omega-Z (p-NO [low] 2) -, N [high] small omega-Z-, N [high] small omega-adamantyloxycarbonyl-, N [high] small omega-isobornyloxycarbonyl groups or protected by protonation .

In the process according to the invention, the halo-nitroanilines used as starting materials can be converted into the corresponding halo-nitrophenyl isocyanates. These active compounds are reacted with the corresponding N-protected amino acids. The halo-nitroanilides serve to protect the carboxyl group in the preparation of the chromogenic substrates. During the synthesis of the new chromogenic substrates, both the protective groups of the side chains and the amino functions are split off under conditions under which attack of the anilide bond does not occur.

The chromogenic substrates according to the invention are produced according to the methods customary in peptide chemistry for tying the peptide bonds. The carbodiimide, azide, anhydride, isocyanate, ynamine, phosphorazo, acid chloride method or the methods of activated amides or activated esters (trichlorophenyl ester, pentachlorophenyl ester, p-nitrophenyl ester, thiophenyl ester, Selenophenylester, N-Hydroxyiperidylester) can be used.

The compounds prepared according to the invention can be characterized by elemental analysis, melting point and, if appropriate, by rotation value and thin-layer chromatography.

Silica gel plates (ready-to-use TLC plates, silica gel 60 F 254 and silica gel 60 without fluorescent indicator) from E. Merck AG, Darmstadt, are used throughout for thin-layer chromatography. The following solvent mixtures are used:

A - butanol / glacial acetic acid / water (3: 1: 1)

B - chloroform / methanol / ammonia solution (25%) (60:45:20)

C - methanol / chloroform (2: 1)

D - choroform / methanol / benzene / water (40: 40: 40: 5).

The ninhydrin solution is prepared as follows:

300 mg of ninhydrin are dissolved in 100 ml of n-butanol and 3 ml of glacial acetic acid. This solution is stored in the refrigerator and used as a spray solution.

The chloro-tolidine solution is prepared as follows:

80 mg of o-tolidine, 15 ml of acetic acid and 0.55 g of potassium iodide are made up to 250 ml with water. For development, the moistened plate is placed in a chlorine chamber for 10 minutes, then ventilated and sprayed with the reagent.

The process according to the invention is explained in more detail by the following working instructions and examples, working instructions 1 to 38 explaining the preparation of intermediates and precursors and examples 1 to 33 the preparation of compounds according to the invention.

Working instruction 1:

p-Methoxy-benzenesulfonyl chloride (Mbs-Cl): 80 g of anisole are dissolved in 300 ml of absolute chloroform, the solution is cooled to -10 ° C and a total of 172.4 g of chlorosulfonic acid are added dropwise so that the temperature does not exceed 0 ° C increases. When the reaction has ended, the cold bath is removed and the reaction solution is warmed to room temperature. After the evolution of gas has ceased, the solution is placed on ice. poured. The aqueous phase is separated off in a separating funnel and extracted twice more with chloroform. The combined chloroform phases are washed with water and dried over sodium sulfate. After the chloroform has been stripped off, the oil obtained is distilled in vacuo. The oil obtained crystallizes on rubbing with a glass rod.

Yield: 93.3 g (61% of theory)

Molecular formula: C [deep] 7H [deep] 7O [deep] 3SCl

MG: 206.65

Elemental analysis: C H S Cl

calc .: 40.69% 3.41% 15.52% 17.16%

found: 40.32% 3.68% 15.74% 17.01%

M.p .: 43 ° C, R [low] f [low] D: 0.76

Working instruction 2:

Benzyloxycarbonyl-aminoisobutyric acid (Z-Aib-OH): 5.02 g

Aib-OH are dissolved in 30 ml of 1.67 N NaOH, 25 ml of acetone are then added and the mixture is cooled to 0 ° C .; 16 g of 80% benzyl chloroformate are dissolved within one hour at pH 10.8 to 10.9 and 25 ml of dry acetone are added dropwise with stirring so that the temperature does not rise above + 2 ° C. After 2½ hours, the acetone is removed in vacuo, the aqueous solution is covered with ethyl acetate and acidified to pH 2.5 with 3N HCl. The organic phase is separated off and the aqueous phase is extracted twice more with ethyl acetate. The combined ethyl acetate phases are washed with water and dried over sodium sulfate. Crystallization takes place from ether / petroleum ether or from benzene / petroleum ether.

Yield: 10.5 g (88.5% of theory)

Molecular formula: C [deep] 12H [deep] 15NO [deep] 4

MG: 237.26

Elemental analysis: C H N

calc .: 60.75% 6.4% 5.9%

found: 60.7% 6.6% 5.95%

M.p .: 75 ° C, R [low] f [low] D: 0.46

Working instruction 3:

Benzyloxycarbonyl-D-phenylalanine (Z-D-Phe-OH): 5 g of H-D-Phe-OH are dissolved in 20 ml of 2N sodium hydroxide solution and 7 ml of benzyl chloroformate are added dropwise at 0 ° C. To maintain a pH of 9.5, the reaction mixture thus obtained is mixed with 2N sodium hydroxide solution using an autotitrator (time required about 2 hours). The reaction solution is extracted with ether in order to remove excess benzyl chloroformate. The aqueous phase is then covered with ethyl acetate, cooled to 5 ° C. and acidified to pH 2.5 with 3N HCl. The phases are separated in a separating funnel, and the aqueous phase is extracted twice more with ethyl acetate. The combined organic phases are then washed with saturated sodium chloride solution and water and dried over sodium sulfate. After removing the solvent in vacuo, the compound is taken up in ether and precipitated with petroleum ether.

Yield: 7.3 g (80.6% of theory)

Molecular formula: C [deep] 17H [deep] 17NO [deep] 4

MG: 299.33

Elemental analysis: C H N

calc .: 68.21% 5.72% 4.68%

found: 68.34% 5.75% 4.73%

Melting point: 63 ° C, R [low] f [low] D: 0.31, [small alpha] [low] D [high] 20: + 6.98 ° (c = 1, methanol)

Working instruction 4:

N-p-methoxybenzenesulfonyl-small beta-alanine (Mbs-small beta-Ala-OH): 8.9 g

(0.1 m) H-small Beta-Ala-OH are suspended in 50 ml H [deep] 2O / 50 ml acetone and brought to pH 9 with 2N NaOH, then the solution is cooled to + 5 ° C and at this Temperature with 22.7 g of p-methoxybenzenesulfonyl chloride, dissolved in 50 ml of acetone, added. The pH value is kept constant at 9 by means of an autotitrator. After the reaction has taken place, the acetone is removed on a rotary evaporator and the residue with

Ethyl acetate covered and acidified to pH 2.5. The aqueous phase is extracted twice more with ethyl acetate. The combined ethyl acetate phases are washed neutral with water and dried over sodium sulfate. The crystallization takes place from ethyl acetate / petroleum ether.

Yield: 20.5 (79.1% of theory)

Molecular formula: C [deep] 10H [deep] 13NO [deep] 5S

MG: 259.28

Elemental analysis: C H N S

calc .: 46.32% 5.05% 5.40% 12.37%

found: 46.12% 5.25% 5.21% 12.03%

M.p .: 123 ° C, R [low] f [low] D: 0.24

Working instruction 5:

N-p-Methoxybenzenesulfonyl-small gamma-aminobutyric acid (Mbs-Gaba-OH):

10.31 g of H-Gaba-OH are dissolved in 60 ml of acetone / water (1: 1) and brought to pH 9 with 2N NaOH. By adding dropwise a solution of 22.71 g of p-methoxybenzenesulfonyl chloride and 50 ml of acetone while titrating with 2N NaOH using an autotitrator, the amino acid is converted within 4 hours. The acetone is removed in vacuo and the aqueous phase is extracted twice with ether. The aqueous phase is covered with ethyl acetate, the reaction solution is then brought to a pH of 2.5 with 3N HCl; the phases are separated and the water phase is extracted twice more with ethyl acetate. The combined organic phases are washed neutral with sodium chloride solution and water and dried over sodium sulfate. Crystallization takes place from ethyl acetate / petroleum ether.

Yield: 22.9 g (83.8% of theory)

Molecular formula: C [deep] 11H [deep] 15NO [deep] 5S

MG: 273.31

Elemental analysis: C H N S

calc .: 48.34% 5.53% 5.12% 11.73%

found: 48.25% 5.32% 5.68% 11.51%

M.p .: 108 ° C, R [low] f [low] D: 0.44

Working instruction 6:

N-p-methoxybenzenesulfonyl-glycine (Mbs-Gly-OH): 7.5 g Gly-OH are suspended in 20 ml water / 20 ml dioxane; 22.7 g of p-methoxybenzenesulfonyl chloride are added to the solution and the mixture is titrated with 4N NaOH at pH 9 to 9.5 on an autotitrator (reaction time 3 hours). The dioxane is removed in vacuo, the aqueous phase is covered with a layer of ethyl acetate and acidified to pH 2 with 3N HCl. After the phases have been separated, the aqueous phase is extracted twice more with ethyl acetate. The combined organic phases are washed neutral with water and dried over sodium sulfate. When the ethyl acetate is concentrated, the desired compound crystallizes out. Recrystallization takes place from ethyl acetate.

Yield: 17.1 g (69.4% of theory)

Molecular formula: C [deep] 9H [deep] 11NO [deep] 5S

MG: 245.25

Elemental analysis: C H N

calc .: 44.07% 4.56% 5.68%

found: 44.40% 4.52% 5.71%

Mp .: 124-125 ° C, R [low] f [low] A: 0.60

Working instruction 7:

Np-methoxybenzenesulfonyl-L-valine (Mbs-Val-OH): 11.7 g of Val-OH are suspended in 60 ml of acetone / water (1: 1); after cooling to 5 ° C., a solution of 22 is added dropwise , 71 g of p-methoxybenzenesulfonyl chloride in 50 ml of acetone. The pH value is maintained at 9 on the autotitrator by titration with 4N NaOH. After the reaction has ended, the acetone is removed in vacuo. The aqueous phase is then extracted twice with ether. The watery one

Phase is covered with ethyl acetate and brought to pH 2.5 with 3N HCl. After the phases have been separated in a separating funnel, the aqueous phase is extracted twice more with ethyl acetate. The combined organic phases are washed neutral with saturated sodium chloride solution and water and dried over sodium sulfate. Crystallization takes place from ethyl acetate / petroleum ether.

Yield: 25.4 g (85.4% of theory)

Molecular formula: C [deep] 12H [deep] 17NO [deep] 5S

MG: 287.33

Elemental analysis: C H N S

calc .: 50.16% 5.96% 4.87% 11.16%

found: 49.32% 5.80% 4.79% 10.96%

Melting point: 102 to 105 ° C, R [low] f [low] D: 0.37, [small alpha] [low] D [high] 20: -17.69 ° (c = 1, methanol)

Working instruction 8:

N-p-methoxybenzenesulfonyl-L-proline (Mbs-Pro-OH): 11.5 g

L-Pro-OH are dissolved in 50 ml of 2N NaOH / 50 ml of dioxane, cooled to 0 ° C. and mixed with 22.7 g of p-methoxybenzenesulfonyl chloride (Mbs-Cl), dissolved in 50 ml of acetone, and 4N NaOH, while stirring Autotitrator titrated at pH 9 (reaction time 2½ hours). The dioxane is removed in vacuo, the aqueous phase is covered with a layer of ethyl acetate and acidified to pH 2 (3N HCl). The phases are separated in a separating funnel, and the aqueous phase is extracted twice more with ethyl acetate. The combined organic phases are washed neutral with water and dried over sodium sulfate. Crystallization takes place after the addition of petroleum ether (30 to 50 ° C).

Yield: 25.1 g (88% of theory)

Molecular formula: C [deep] 12H [deep] 15NO [deep] 5S

MG: 285.32

Elemental analysis: C H N

calc .: 50.51% 5.3% 4.91%

found: 50.94% 5.51% 4.94%

Melting point: 107 ° C, R [low] f [low] D: 0.28, [small alpha] [low] D [high] 20: -111.6 ° (c = 1, methanol)

Working instruction 9:

Mbs-L-Phenylalanine (Mbs-L-Phe-OH): 16.52 g (0.1 M) L-Phe are dissolved in 50 ml of 2N NaOH / 50 ml of dioxane, cooled to 0 ° C., with 22.7 g of p-methoxybenzenesulfonyl chloride are added and the pH is kept at 9 on the autotitrator with 2N NaOH. After the reaction has ended, it is concentrated to dryness, taken up in ethyl acetate and acidified to pH 2.5 with 3N HCl. The ethyl acetate phase is separated off, and the H [deep] 2O phase is extracted twice more with EA. The combined EA phases are washed neutral with water and dried over Na [deep] 2SO [deep] 4. The crystallization takes place from EE / petroleum ether.

Yield: 26.53 g (79.1% of theory)

Molecular formula: C [deep] 16H [deep] 17NO [deep] 5S

MG: 335.38

Elemental analysis: C H N S

calc .: 57.30% 5.11% 4.18% 9.56%

found: 57.18% 5.25% 4.13% 10.8%

Fp .: 136 to 137 ° C, R [low] f [low] D: 0.45, [small alpha] [low] D [high] 20: -12.7 ° (c = 2, DMF)

Working instruction 10:

t. Butyloxycarbonyl-L-phenylalanine (Boc-Phe-OH): 16.52 g of L-Phe-OH are suspended in 50 ml of dioxane / 50 ml of water, and 15 ml of Boc-azide are added. It is titrated on an autotitrator with 4N NaOH (reaction time 15 hours) while stirring. The solution obtained in this way is extracted three times with ether, the aqueous phase is covered with ethyl acetate and acidified to pH 2.5. The phases are separated in a separating funnel; the aqueous phase is covered twice with ethyl acetate. The contaminated organic phases are washed neutral with water and dried over sodium sulfate. Crystallization takes place from ether / petroleum ether.

Yield: 23.8 g (89.7% of theory)

Molecular formula: C [deep] 14H [deep] 19NO [deep] 4

MG: 265.31

Elemental analysis: C H N

calc .: 63.88% 7.22% 5.28%

found: 63.25% 7.10% 5.18%

Fp .: 90 ° C, R [low] f [low] D: 0.39, [small alpha] [low] D [high] 20: + 14.0 ° (c = 0.1, HAc)

Working instruction 11:

t. Butyloxycarbonyl-L-isoleucine (Boc-Ile-OH): 6.56 g

L-Ile-OH are suspended in 10 ml water / 10 ml dioxane and titrated with 4N NaOH with the addition of 8 ml Boc-azide with stirring on an autotitrator at pH 9.8 (reaction time 10 hours). The resulting solution is extracted three times with ether in order to remove the excess Boc-azide. The aqueous phase is covered with a layer of ethyl acetate and cooled to 0 ° C. After acidification to pH 2.5, the phases are separated in a separating funnel. The aqueous phase is extracted twice more with ethyl acetate. The contaminated organic phases are washed neutral with water and dried over sodium sulfate. Crystallization takes place after the addition of n-hexane.

Yield: 11 g (96% of theory)

Molecular formula: C [deep] 11H [deep] 21NO [deep] 4

MG: 231.29

Elemental analysis: C H N

calc .: 57.12% 9.15% 6.06%

found: 57.01% 8.93% 6.02%

Fp .: 66 ° C, R [low] f [low] D: 0.45, [small alpha] [low] D [high] 20: + 3 ° (c = 1, HAc)

Working instruction 12:

t. Butyloxycarbonyl-L-leucine (Boc-Leu-OH): 26.2 g of L-leucine are suspended in 70 ml of water / 70 ml of dioxane, and 30 ml of Boc-azide are added. The reaction mixture is stirred on the autotitrator at pH 9.75 overnight at room temperature. The reaction mixture is then shaken out three times with ether in order to remove the excess Boc-azide.

After the dioxane has been drawn off on the rotary evaporator, it is covered with a layer of ethyl acetate and acidified to pH 2.5 with 3N HCl. The organic phase is separated off in a separating funnel, the aqueous phase is extracted three more times with ethyl acetate. The combined organic phases are washed neutral with water and dried over sodium sulfate. After removing the ethyl acetate, the remaining oil is covered with petroleum ether. The substance crystallizes after standing for a long time at -20 ° C.

Yield: 45.1 g (97.6% of theory)

Molecular formula: C [deep] 11H [deep] 21NO [deep] 4

MG: 231.29

Elemental analysis: C H N

calc .: 57.12% 9.15% 6.05%

found: 56.89% 9.01% 5.87%

Fp .: 45 to 48 ° C, R [low] f [low] D: 0.50, [small alpha] [low] D [high] 20: 21.5 ° (c = 1, HAc)

Working instruction 13:

t. Butyloxycarbonyl-L-proline (Boc-L-Pro-OH): 23 g (0.02 mol) of L-proline are suspended in 200 ml of dioxane / water (1: 1), with 31.46 g (0.22 mol ) Boc-azide added and brought to a pH of 8.6 with 4N NaOH according to the instructions of E. Schnabel (Ann.Chem. 702, 188-196 (1967)) on the autotitrator to react.

Yield: 34 g (70% of theory)

Molecular formula: C [deep] 10H [deep] 17O [deep] 4N

MG: 215.24

Elemental analysis: C H N

ber .: 55.75% 7.96% 6.50%

found: 55.93% 8.07% 6.34%

Melting point: 132 to 134 ° C, R [low] f [low] D: 0.43, [small alpha] [low] D [high] 20: -67.3 ° (c = 1, HAc)

Working instruction 14:

t. Butyloxycarbonyl-L-valine (Boc-Val-OH): 11.7 g of L-valine are suspended in a mixture of 50 ml of water / 50 ml of dioxane, and 16 ml of Boc-azide are added. You keep that

Reaction mixture with 4N sodium hydroxide solution on an autotitrator at pH 9.5 (reaction time 12 hours). The solution obtained in this way is extracted by shaking with ether in order to remove excess Boc-azide, then the dioxane is removed in vacuo. The aqueous solution is covered with a layer of ethyl acetate and acidified to pH 2.5 with 3N hydrochloric acid. The phases are separated in a separating funnel; the aqueous phase is extracted three more times with ethyl acetate; the combined ethyl acetate phases are washed with NaCl solution and water and dried over sodium sulfate. The ethyl acetate is removed in vacuo. The remaining oil is taken up in absolute ether, the desired substance is made to crystallize by adding petroleum ether.

Yield: 19.6 g (90% of theory)

Molecular formula: C [deep] 10H [deep] 19NO [deep] 4

MG: 217.27

Elemental analysis: C H N

calc .: 55.28% 8.82% 6.45%

found: 55.53% 8.64% 6.21%

Fp .: 76 to 79 ° C, R [low] f [low] D: 0.66, [small alpha] [low] D [high] 20: + 6.1 ° (c = 1, HAc)

Working instruction 15:

Benzyloxycarbonyl-sarcosine (Z-Sar-OH) 17.8 g of sarcosine are suspended in 50 ml of water / 50 ml of dioxane and brought to pH 9.5 with 2N sodium hydroxide solution. The reaction mixture is cooled to 5 ° C. and 35 ml of benzyl chloroformate are added dropwise while maintaining the pH value 9.5; the reaction mixture is stirred for 4 hours at 5 ° C. and for 1 hour at room temperature. Most of the dioxane is then removed on a rotary evaporator. The aqueous solution is extracted twice with ether in order to remove the excess benzyl chloroformate. After the aqueous phase has been adjusted to a pH of 2.5 with 3N hydrochloric acid, it is extracted three times with ethyl acetate. The combined vinegar ester phases are washed with concentrated sodium chloride solution and water and dried over sodium sulfate. After the solvent has been stripped off, an oil is obtained.

Yield: 18.1 g (81.1% of theory)

Molecular formula: C [deep] 11H [deep] 13NO [deep] 4

MG: 223.23

Elemental analysis: C H N

calc .: 59.19% 5.87% 6.27%

found: 59.17% 5.43% 6.18%

R [deep] f [deep] D: 0.65

Working instruction 16:

N [high] small alpha-t.Butyloxycarbonyl-N [high] small epsilon-benzyloxycarbonyl-L-lysine (Boc-Lys (Z) -OH): 50 g of H-Lys (Z) -OH are dissolved in 200 ml of dioxane / Water (1: 1) suspended and brought to pH 10.2 with 2N sodium hydroxide solution; the suspension is mixed with 30 ml of Boc-azide and automatically titrated with 4N sodium hydroxide overnight while maintaining a pH of 10.2. The solution obtained in this way is largely freed from dioxane in vacuo, and the aqueous solution is extracted three times with ether. The aqueous solution is covered with ethyl acetate, cooled to 0 ° C. and acidified to pH 3 with ice-cold 2N hydrochloric acid. The phases are separated in a separating funnel, the aqueous phase is extracted twice with ethyl acetate. shaken. The combined organic phases are washed with saturated sodium chloride solution and ice water and dried over sodium sulfate. After removing the solvent in vacuo, the product is obtained as an oil.

Yield: 58.14 g (73.7% of theory)

Molecular formula: C [deep] 19H [deep] 28N [deep] 2O [deep] 6

MG: 380.44

Elemental analysis: C H N

calc .: 59.99% 7.42% 7.36%

found: 59.73% 7.54% 7.28%

R [low] f [low] D: 0.41, [small alpha] [low] D [high] 20: -9.0 ° (c = 1, HAc); M.p .: 58 to 60 ° C

Working instruction 17:

N [high] small epsilon-benzyloxycarbonyl-L-lysine (H-Lys (Z) -OH): 36.5 g of H-Lys-OH.HCl and 16 g of sodium hydroxide in 160 ml of water are mixed with 25 g of copper sulfate pentahydrate in 80 ml Water added. After cooling to 0 ° C., 38 ml of benzyl chloroformate are added dropwise to the reaction solution in such a way that the reaction temperature of 0 ° C. is not exceeded. After the addition, the mixture is stirred for a further 4 hours at 0 ° C. and overnight at room temperature. The precipitated copper complex is filtered off and largely sucked dry with 400 ml of water and 200 ml of acetone.

After the complex obtained has been suspended in 400 ml of water, 72 ml of concentrated hydrochloric acid are added while cooling. Hydrogen sulfide is passed into the solution for 1 hour. After brief degassing, the precipitated copper sulfide is filtered off and washed with water. The combined washing waters are adjusted to the isoelectric point (pH 7) with concentrated ammonia and the solution is left in the refrigerator overnight. The precipitated material is filtered off, washed with ice water and methanol and dried at 50 ° C. to constant weight.

Yield: 50 g (90% of theory)

Molecular formula: C [deep] 14H [deep] 20N [deep] 2O [deep] 4

MG: 280.32

Elemental analysis: C H N

calc .: 59.99% 7.19% 9.99%

found: 59.71% 7.43% 9.77%

Melting point: 255 ° C, R [low] f [low] A: 0.35, [small alpha] [low] D [high] 20: + 13.8 ° (c = 2.3N HCl)

Most active N-protected amino acids:

Working instruction 18:

t. Butyloxycarbonyl-L-leucyl-pentachlorophenyl ester (Boc-Leu-OPCP): 4.62 g of Boc-leucine are dissolved together with 5.32 g of pentachlorophenol in 50 ml of methylene chloride and cooled to 0 ° C. After adding 4.12 g of dicyclohexylcarbodiimide, the mixture is stirred for 2 hours at 0 ° C. and overnight at room temperature. After the precipitated dicyclohexylurea has been filtered off, the solvent is removed in vacuo. The residue is taken up in ethyl acetate and washed successively with 1N citric acid, 1% sodium hydrogen carbonate solution and water and dried over sodium sulfate. It is crystallized from methanol.

Yield: 8.5 g (88.7% of theory)

Molecular formula: C [deep] 17H [deep] 20NO [deep] 4Cl [deep] 5

MG: 479.62

Elemental analysis: C H N

ber .: 42.57% 4.31% 2.92%

found: 42.77% 4.17% 2.73%

Melting point: 109 ° C, R [low] f [low] D: 0.80, [small alpha] [low] D [high] 20: -37.2 ° (c = 1, dimethylformamide (DMF))

Working instruction 19:

Benzyloxycarbonyl-D-phenylalanyl-pentachlorophenyl ester (Z-D-Phe-OPCP): 5.99 g of Z-D-Phe-OH are dissolved together with 5.32 g of pentachlorophenol and 100 ml of methylene chloride and 4.12 g of dicyclohexylcarbodiimide are added at 0 ° C; the mixture is stirred for 3 hours at 0 ° C. and overnight at room temperature. After filtering off the dicyclohexyl urea, the compound is precipitated by adding ether / petroleum ether (1: 1). Recrystallization takes place from methylene chloride / ether / petroleum ether.

Yield: 9.1 g (83% of theory)

Molecular formula: C [deep] 23H [deep] 16NO [deep] 4Cl [deep] 5

MG: 547.65

Elemental analysis: C H N Cl

calc .: 50.44% 2.94% 2.56% 32.37%

found: 50.21% 2.73% 2.71% 32.58%

Melting point: 150 ° C, R [low] f [low] D: 0.82, R [low] f [low] A: 0.88, [small alpha] [low] D [high] 20: +5 .39 ° (c = 1.02, DMF)

Working instruction 20:

Benzyloxycarbonyl-small alpha-aminoisobutyric acid pentachlorophenyl ester (Z-Aib-OPCP): 11.86 g of Z-Aib-OH are dissolved together with 13.31 g of pentachlorophenol in 150 ml of methylene chloride and cooled to 0 ° C. After 10.3 g of dicyclohexylcarbodiimide have been added, the mixture is stirred for 2 hours at 0 ° C. and overnight at room temperature. The precipitated dicyclohexylurea is filtered off and the solvent is removed in vacuo. The desired substance is obtained by recrystallizing the residue twice from isopropanol.

Yield: 19.8 g (86.3% of theory)

Molecular formula: C [deep] 18H [deep] 14NO [deep] 4Cl [deep] 5

MG: 485.58

Elemental analysis: C H N Cl

calc .: 44.52% 2.91% 2.88% 36.51%

found: 44.71% 2.76% 2.41% 36.64%

R [deep] f [deep] D: 0.79

Working instruction 21:

Np-methoxybenzenesulfonyl-small gamma-aminobutyryl-pentachlorophenyl ester (Mbs-Gaba-OPCP): 5.46 g of Mbs-Gaba-OH are dissolved together with 5.33 g of pentachlorophenol in 100 ml of methylene chloride and cooled to 0 ° C., then added 4.12 g of dicyclohexylcarbodiimide are added and the mixture is stirred for 2 hours at 0 ° C. and overnight at room temperature. The precipitated dicyclohexylurea is filtered off with suction, the solvent in vacuo removed. The residue is recrystallized from isopropanol.

Yield: 8.2 g (78.8% of theory)

Molecular formula: C [deep] 17H [deep] 14NO [deep] 5Cl [deep] 5S

MG: 521.63

Elemental analysis: C H N

calc .: 39.14% 2.71% 2.69%

found: 39.24% 2.53 2.78%

R [deep] f [deep] D: 0.63; M.p .: 124-127 ° C

Working instruction 22:

t. Butyloxycarbonyl-L-valine-2,4,5-trichlorophenyl ester (Boc-Val-OTCP): 10.08 g of Boc-valine are dissolved in 75 ml of methylene chloride, 9.88 g of 2,4,5-trichlorophenol are added and the solution is added -5 ° C cooled. 10.3 g of dicyclohexylcarbodiimide are added and the mixture is stirred for 2 hours at 0 ° C. and overnight at room temperature. After filtering off the cyclohexylurea and stripping off the solvent in vacuo, the substance crystallizes from petroleum ether at -20.degree.

Yield: 15.8 g (% of theory)

Molecular formula: C [deep] 16H [deep] 20Cl [deep] 3NO [deep] 4

MG: 396.70

Elemental analysis: C H N Cl

ber .: 48.44% 5.08% 3.53% 26.81%

found: 48.62% 5.19% 3.72% 26.21%

Fp .: 52 ° C, R [low] f [low] D: 0.79, [small alpha] [low] D [high] 20: -30.37 ° (c = 1.02, DMF)

Working instruction 23:

Benzyloxycarbonyl-L-pyroglutamyl-p-nitrophenyl ester (Z-Pyr-ONP): 13.15 g of Z-Pyr-OH are dissolved in a mixture of 100 ml of methylene chloride and 80 ml of absolute pyridine and mixed with 16.2 g of di- (p -Nitrophenyl) sulfide was added in portions and then kept at 45 ° C. for 3 hours. The solution obtained in this way is, after cooling, washed successively with 0.5N hydrochloric acid, dilute sodium hydrogen carbonate solution and water and dried over sodium sulfate. To

Removal of the solvent in vacuo, the residue is recrystallized from ethyl acetate.

Yield: 18 g (94% of theory)

Molecular formula: C [deep] 19H [deep] 16N [deep] 2O [deep] 7

MG: 384.3

Elemental analysis: C H N

calc .: 59.37% 4.20% 7.29%

found: 48.96% 4.01% 7.35%

Melting point: 140 ° C, R [low] f [low] D: 0.77, R [low] f [low] A: 0.82, [small alpha] [low] D [high] 20: -50 , 1 ° (c = 1, THF)

Working instruction 24:

N-p-Methoxybenzenesulfonyl-L-phenylalanyl-N-hydroxysuccinimide ester (Mbs-Phe-OSu): 3.36 g of Mbs-Phe-OH are dissolved together with 1.15 g of hydroxysuccinimide in 30 ml of methylene chloride and cooled to -5 ° C. After adding 2.06 g of dicyclohexylcarbodiimide, the mixture is stirred for 3 hours at 0 ° C. and overnight at room temperature. The precipitated dicyclohexylurea is filtered off and the solvent is removed in vacuo. Recrystallization takes place from isopropyl alcohol.

Yield: 3.5 g (80.9% of theory)

Molecular formula: C [deep] 20H [deep] 20N [deep] 2O [deep] 7S

MG: 432.45

Elemental analysis: C H N

calc .: 55.55% 4.66% 6.48%

found: 55.32% 4.42% 6.72%

Fp .: 151 to 152 ° C, R [low] f [low] D: 0.62, [small alpha] [low] D [high] 20: -54.6 ° (c = 1, DMF)

Working instruction 25:

Np-methoxybenzenesulfonyl-small beta-alanyl-N-hydroxysuccinimide ester (Mbs-small Beta-Ala-OSu): 12.96 g of Mbs-small Beta-Ala-OH are dissolved together with 5.75 g of N-hydroxysuccinimide in 150 ml of methylene chloride and then cooled to 0 ° C., then 10.3 g of dicyclohexylcarbodiimide are added and the mixture is stirred for 2 hours at 0 ° C. and overnight at room temperature. After filtering off the dicyclohexylurea formed and removing the solvent, it is taken in a little absolute methylene chloride and cools to -20 ° C; the precipitated residue of dicyclohexylurea is removed and the solvent is stripped off at a bath temperature below 40 ° C. in vacuo. The residue is recrystallized from isopropanol.

Yield: 13.5 g (75.8% of theory)

Molecular formula: C [deep] 14H [deep] 16N [deep] 2O [deep] 7S

MG: 356.35

Elemental analysis: C H N S

calc .: 47.19% 4.53% 7.86% 9.00%

found: 47.28% 4.75% 7.53% 9.22%

M.p .: 108 to 110 ° C, R [low] f [low] D: 0.60

Working instruction 26:

N-p-Methoxybenzenesulfonyl-L-prolyl-N-hydroxysuccinimide ester (Mbs-Pro-OSu): 2.85 g of Mbs-Pro-OH are dissolved together with 1.15 g of hydroxysuccinimide in 30 ml of methylene chloride and cooled to 0 ° C. After adding 2.06 g of dicyclohexylcarbodiimide, the mixture is stirred for 3 hours at 0 ° C. and overnight at room temperature. After the precipitated dicyclohexylurea has been filtered off and the solvent has been removed in vacuo, the residue is recrystallized from isopropyl alcohol.

Yield: 3.8 g (87.9% of theory)

Molecular formula: C [deep] 16H [deep] 18N [deep] 2O [deep] 7S

MG: 432.45

Elemental analysis: C H N

calc .: 50.26% 4.74% 7.33%

found: 50.14% 4.82% 7.54%

Fp .: 85 to 88 ° C, R [low] f [low] D: 0.63, [small alpha] [low] D [high] 20: -114.2 ° (c = 0.92, DMF)

Working instruction 27:

Benzyloxycarbonyl-sarcosyl-pentachlorophenyl ester (Z-Sar-OPCP): 11.16 g of Z-Sar-OH are added together with 13.33 g

Pentachlorophenol dissolved in 75 ml of methylene chloride with the addition of a few drops of dimethylformamide and cooled to 0 ° C; 10.3 g of dicyclohexylcarbodiimide are added and the mixture is stirred at this temperature for 2 hours and then overnight at room temperature. After the mixture obtained has been cooled to -20 ° C., the precipitated dicyclohexylurea is filtered off. Crystallization takes place on addition of ether / petroleum ether.

Yield: 19.8 g (84% of theory)

Molecular formula: C [deep] 17H [deep] 12NO [deep] 4Cl [deep] 5

MG: 471.55

Elemental analysis: C H N Cl

ber .: 43.30% 2.57% 2.97% 37.59%

found: 43.10% 2.51% 2.63% 37.34%

M.p .: 103 ° C, R [low] f [low] A: 0.84

Couplable H-Arg (NO [deep] 2) -o-Cl-pNA

Working instruction 28 a:

o-chloro-p-nitrophenyl isocyanate: In a solution of 20 g o-chloro-p-nitroaniline in absolute dioxane becomes dry

Hydrogen chloride gas introduced until saturation. Phosgene is then passed through the solution at 50 ° C. for one hour until a clear solution has formed. The excess phosgene is removed by a stream of dry nitrogen. The solution obtained is evaporated to dryness on a rotary evaporator. The residue is recrystallized from petroleum ether.

Yield: 20.8 g (90.1% of theory)

Molecular formula: C [deep] 7H [deep] 3N [deep] 2O [deep] 3Cl

MG: 198.57

Elemental analysis: C H N Cl

calc .: 42.34% 1.52% 14.11% 17.85%

found: 42.52% 1.74% 14.33% 17.74%

M.p .: 62 ° C, R [low] f [low] D: 0.77

Working instruction 28 b:

N [high] small omega-nitro-L-arginine: To nitrating acid, consisting of 40 ml nitric acid, 25 ml fuming sulfuric acid and 15 ml concentrated sulfuric acid, add 30.9 g L-arginine within 4 hours. This mixture is stirred until it is completely dissolved and this is poured onto fragmented ice. The pH is then adjusted to 6.8 with concentrated ammonia solution. After standing overnight at 4 ° C., the desired product is obtained. After suctioning off and washing with ice water and ether, it is dried in vacuo over P [deep] 2O [deep] 5.

Yield: 30.8 g (81.5% of theory)

Molecular formula: C [deep] 6H [deep] 13N [deep] 5O [deep] 4

MG: 219.20

Elemental analysis: C H N

calc .: 32.88% 5.98% 31.95%

found: 32.76% 5.74% 31.77%

Melting point: 251 ° C, R [low] f [low] A: 0.26, [small alpha] [low] D [high] 20: + 24.3 ° (c = 1, 2N HCl)

Working instruction 28 c:

N [high] small alpha-benzyloxycarbonyl-N [high] small omega-nitro-L-arginine (Z-Arg (NO [low] 2) -OH): 32.8 g N [high] small omega-nitroarginine are in 150 ml of 1N NaOH dissolved and then cooled to 0 ° C. With simultaneous dropwise addition, 26.8 ml of carbobenzoxychloride and 158 ml of 1N sodium hydroxide solution are added to the solution. The mixture is stirred for 2 hours at 5 ° C. and 2 hours at room temperature. After the reaction mixture has been acidified to pH 2, the benzyloxycarbonyl compound initially separates out as an oil, which immediately crystallizes through. The precipitated product is taken up in potassium hydrogen carbonate solution (4%); this solution is extracted three times with ether and then acidified to pH 2 with 4N hydrochloric acid. The crystalline product is filtered off and dried in vacuo at 35 ° C. over phosphorus pentoxide. Recrystallization takes place from ethanol / water.

Yield: 48 g (90.8% of theory)

Molecular formula: C [deep] 14H [deep] 19N [deep] 5O [deep] 6

MG: 353.34

Elemental analysis: C H N

calc .: 47.59% 5.42% 19.82%

found: 47.39% 5.64% 19.66%

Melting point: 134 to 136 ° C, R [low] f [low] D: 0.38, [small alpha] [low] D [high] 20: -3 ° (c = 1, MeOH)

Working instruction 28 d:

N [high] small alpha-benzyloxycarbonyl-N [high] small omega-nitro-L-arginyl-o-chloro-p-nitroanilide (Z-Arg (NO [low] 2) -o-Cl-pNA): 10, 6 g of Z-Arg (NO [deep] 2) -OH are dissolved in 70 ml of hexamethylphosphoric acid triamide (HMPT) and treated with 3.04 g (4.22 ml) of triethylamine. The mixture is then cooled to 5 ° C., 5.96 g of o-chloro-p-nitrophenyl isocyanate are added in portions and the mixture is stirred at 5 ° C. for 1 hour and at room temperature overnight. The reaction solution is then added dropwise with vigorous stirring to 750 ml of 2.5% NaHCO [deep] 3 solution, the deposited product is filtered off with suction and washed with 2% NaHCO [deep] 3 solution, 0.5N hydrochloric acid and water and then dried over phosphorus pentoxide. Recrystallization takes place from methanol / ether.

Yield: 12.5 g (82.02% of theory)

Molecular formula: C [deep] 20H [deep] 22N [deep] 7O [deep] 7Cl

MG: 507.89

Elemental analysis: C H N Cl

calc .: 47.30% 4.37% 19.31% 6.98%

found: 47.28% 4.24% 19.25% 6.77%

Fp .: 142 to 145 ° C, R [low] f [low] A: 0.78, [small alpha] [low] D [high] 20: -5.2 ° (c = 1, DMF)

Working instruction 28 e:

N [high] small omega-nitro-L-arginyl-o-chloro-p-nitroanilide hydrobromide (H-Arg (NO [low] 2) -o-Cl-pNA.HBr): 11 g N [high] small alpha Benzyloxycarbonyl-N [high] small omega-nitro-L-arginyl-o-chloro-p-nitroanilide are poured over with 50 ml of hydrogen bromide / glacial acetic acid and stirred for 3 hours at room temperature. After the benzyloxycarbonyl group has been split off, the product is precipitated with absolute ether. After the solution has stood for one hour at 0 ° C., the product is filtered off with suction and washed with ether. Recrystallization takes place from methanol / ether.

Yield: 8 g (80.8% of theory)

Molecular formula: C [deep] 12H [deep] 17N [deep] 7O [deep] 5ClBr

MG: 454.67

Elemental analysis: C H N Cl Br

calc .: 31.67% 3.77% 21.56% 7.80% 17.58%

found: 31.32% 3.52% 21.71% 7.98% 17.34%

Mp .: 200 ° C, [small alpha] [low] D [high] 20: + 17.85 ° (c = 1, DMF)

Protected o-Cl-pNA peptides and amino acids

Working instruction 29:

N [high] small alpha-t.Butyloxycarbonyl-N [high] small epsilon-benzyloxycarbonyl-L-lysyl-o-chloro-p-nitroanilide (Boc-Lys (Z) -o-Cl-pNA): 3.11 g Boc-Lys (Z) -OH are dissolved in 25 ml of freshly distilled hexamethylphosphoric acid triamide, and 0.826 g (1.15 ml) of triethylamine are added at room temperature; 3.25 g of para-nitrophenyl isocyanate are then added in portions to this solution, and the batch is stirred for 24 hours at room temperature. The processing is carried out in the same way as the arginine derivative (working regulation 26). Recrystallization takes place here from ethyl acetate / petroleum ether.

Yield: 3.4 g (77.8% of theory)

Molecular formula: C [deep] 25H [deep] 31N [deep] 4O [deep] 7Cl

MG: 534.99

Elemental analysis: C H N Cl

calc .: 56.13% 5.84% 10.47% 6.63%

found: 56.02% 5.93% 10.78% 6.42%

R [low] f [low] A: 0.68, [small alpha] [low] D [high] 20: -7.8 ° (c = 1, DMF); M.p .: 115 to 118 ° C

Working instruction 30:

N [high] small epsilon-benzyloxycarbonyl-L-lysyl-o-Cl-p-nitroanilide hydrochloride (H-Lys (Z) -o-Cl-pNA.HCl): 3 g Boc-Lys (Z) -o-Cl- 50 ml of 1.2N hydrogen chloride in glacial acetic acid are poured over the pNA and the mixture is stirred for 1 hour at room temperature. The solution thus obtained is concentrated to dryness on a rotary evaporator. The residue is taken up in a little methylene chloride / methanol (1: 1) and added dropwise to an excess of ice-cold ether.

Yield: 2.26 g (84.3% of theory)

Molecular formula: C [deep] 20H [deep] 24N [deep] 4O [deep] 5Cl [deep] 2

MG: 478.4

Elemental analysis: C H N Cl

calc .: 50.17% 5.06% 11.71% 14.82%

found: 50.25% 5.33% 11.51% 14.76%

[small alpha] [low] D [high] 20: + 82.6 ° (c = 1, DMF); Mp .: 148 to 150 ° C, R [low] f [low [D: 0.51

Working instruction 31:

t. Butyloxycarbonyl-L-isoleucyl-N [high] small omega-nitro-L-arginyl-o-chloro-p-nitroanilide (Boc-Ile-Arg (NO [low] 2) -o-Cl-pNA): 510 mg Boc -Ile-OH are dissolved together with 298 mg of hydroxybenzotriazole in 30 ml of dimethylformamide, cooled to 0 ° C. and mixed with 455 mg of dicyclohexylcarbodiimide; this reaction mixture is stirred for 3 hours at 0 ° C., then 1.002 g of H-Arg (NO [deep] 2) -o-Cl-pNA.HCl and 0.24 ml of N-methylmorpholine are added and the mixture is stirred for a further 2 hours at 0 ° C and overnight at room temperature. The solvent is de- removed, the residue taken up in ethyl acetate and washed successively with 5% NaHCO [deep] 3 solution, 1N citric acid and water. After drying over sodium sulfate, the substance is crystallized by adding petroleum ether.

Yield: 1 g (77.28% of theory)

Molecular formula: C [deep] 23H [deep] 35N [deep] 8O [deep] 8Cl

MG: 587.03

Elemental analysis: C H N

calc .: 47.06% 6.01% 19.09%

found: 47.32% 5.84% 19.25%

[small alpha] [low] D [high] 20: + 42 ° (c = 1, DMF); M.p .: 195 to 200 ° C, R [low] f [low] D: 0.64

Working instruction 32:

t. Butyloxycarbonyl-L-leucyl-N [high] small omega-nitro-L-arginyl-o-chloro-p-nitroanilide (Boc-Leu-Arg (NO [low] 2) -o-Cl-pNA): 959 mg Boc -Leu-OPCP are dissolved together with 909 mg of H-Arg (NO [deep] 2) -o-Cl-pNA.HBr in 25 ml of dimethylformamide, mixed with 0.22 ml of N-methylmorpholine and stirred for 24 hours at room temperature. The dimethylformamide is removed at 40 ° C. on a rotary evaporator. The residue is partitioned between ethyl acetate and water. The organic phase is washed successively with 5% NaHCO [deep] 3 solution, 1N citric acid and water and dried over sodium sulfate. After removing the solvent, it is crystallized from methylene chloride / ether / petroleum ether.

Yield: 0.85 g (72.4% of theory)

Molecular formula: C [deep] 23H [deep] 35N [deep] 8O [deep] Cl

MG: 587.03

Elemental analysis: C H N

calc .: 47.06% 6.01% 19.09%

found: 47.32% 6.24% 19.34%

[small alpha] [high] 20 [low] D: + 40 ° (c = 1, DMF); Mp .: 175 to 178 ° C, R [low] f [low] A: 0.83

Working instruction 33:

t. Butyloxycarbonyl-L-prolyl-N [high] small epsilon-benzyloxycarbonyl-L-lysyl-

-o-chloro-p-nitroanilide (Boc-Pro-Lys (Z) -o-Cl-pNA):

6.45 g of Boc-Pro-OH are dissolved together with 4.05 g of hydroxybenzotriazole in 75 ml of dimethylformamide and cooled to 0 ° C. At this temperature, 6.18 g of dicyclohexylcarbodiimide are added and the mixture is stirred for one hour. Then, one after the other, 15.35 g of H-Lys (Z) -o-Cl-pNA.HCl and 3.3 ml of N-methylmorpholine are added. The batch is stirred for a further 2 hours at 0 ° C. and overnight at room temperature. It is worked up as usual and recrystallized from ethyl acetate / petroleum ether.

Yield: 14.08 g (74.3% of theory)

Molecular formula: C [deep] 30H [deep] 38N [deep] 5O [deep] 8Cl

MG: 532.11

Elemental analysis: C H N

ber .: 57.00% 6.06% 11.08%

found: 57.21% 6.38% 11.25%

Fp .: 65 to 67 ° C, R [low] f [low] D: 0.70, [small alpha] [low] D [high] 20: -43.0 ° (c = 1, DMF)

Working instruction 34:

t. Butyloxycarbonyl-L-prolyl-N [high] small omega-nitro-L-arginyl-o-chloro-p-nitroanilide (Boc-Pro-Arg (No [low] 2) -o-Cl-pNA): 0.65 g of Boc-L-proline are dissolved together with 0.40 g of hydroxybenzotriazole in 30 ml of dimethylformamide and cooled to 0 ° C., then 0.62 g of dicyclohexylcarbodiimide are added and the mixture is stirred at this temperature for 1 hour. After adding 1.36 g of N [high] small omega-nitro-L-arginyl-o-chloro-p-nitroanilide hydrobromide and 0.33 ml of N-methylmorpholine, the mixture is stirred for 2 hours at 0 ° C. and overnight at room temperature. Dicyclohexylurea which has precipitated is filtered off with suction and the dimethylformamide is removed in vacuo. The remaining oil is taken up in ethyl acetate and washed three times with 5% sodium hydrogen carbonate solution, 1N citric acid solution and water. The ethyl acetate phase is dried over sodium sulfate. The desired product is excreted by adding petroleum ether.

Yield: 0.8 g (51.56% of theory)

Molecular formula: C [deep] 22H [deep] 31N [deep] 8O [deep] 8Cl

MG: 570.99

Elemental analysis: C H N Cl

calc .: 46.28% 5.47% 19.62% 6.21%

found: 46.54% 5.21% 19.84% 6.35%

Melting point: 114 to 118 ° C, R [low] f [low] D: 0.58, [small alpha] [low] D [high] 20: -55.3 ° (c = 1, DMF)

Working instruction 35:

t. Butyloxycarbonyl-L-valyl-N [high] small omega-nitro-L-arginyl-o-chloro-p-nitroanilide (Boc-Val-Arg (NO [low] 2) -o-Cl-pNA):

1.98 g of Boc-Val-OTCP are dissolved together with 2.1 g of nitroarginyl-o-chloro-p-nitroanilide hydrobromide and 30 ml of dimethylformamide. After adding 0.33 ml of N-methylmorpholine, the mixture is stirred for 48 hours at room temperature. After removing the solvent in vacuo, the residue is taken up in ethyl acetate and washed successively with 5% sodium hydrogen carbonate solution, 1N citric acid and water. After drying over sodium sulfate, the ethyl acetate is removed in vacuo. Recrystallization takes place from methylene chloride / ether / petroleum ether.

Yield: 1.3 g (49.06% of theory)

Molecular formula: C [deep] 22H [deep] 33N [deep] 8O [deep] 8Cl

MG: 573.61

Elemental analysis: C H N Cl

calc .: 46.11% 5.81% 19.56% 6.19%

found: 46.33% 5.92% 19.22% 6.48%

[small alpha] [low] D [high] 20: + 80.7 ° (c = 1, DMF); M.p .: 195 to 198 ° C, R [low] f [low] D: 0.64

Working instruction 36:

t. Butyloxycarbonyl-L-phenylalanyl-N [high] small omega-nitro-L-arginyl-o-chloro-p-nitroanilide (Boc-Phe-Arg (NO [low] 2) -o-Cl-pNA): 530 mg Boc -Phe-OH are dissolved together with 270 mg of hydroxybenzotriazole in 25 ml of dimethylformamide and cooled to 0 ° C., then 412 mg of dicyclohexylcarbodiimide are added and the mixture is stirred for 3 hours at this temperature. After adding 850 mg of H-Arg (NO [deep] 2) -o-Cl-pNA.HBR and 0.22 ml of N-methylmorpholine, the mixture is stirred at 0 ° C. for a further 2 hours and overnight at room temperature. After the precipitated dicyclohexylurea has been filtered off and the dimethylformamide has been removed, the residue is taken up in ethyl acetate. The organic phase is washed three times each with 5% Na — HCO [deep] 3 solution, 1N citric acid and water and then dried over sodium sulfate. Crystallization takes place from ethyl acetate / petroleum ether.

Yield: 0.85 g (68.5% of theory)

Molecular formula: C [deep] 26H [deep] 33N [deep] 8O [deep] 8Cl

MG: 621.05

Elemental analysis: C H N

calc .: 50.28% 6.36% 18.04%

found: 50.33% 6.21% 18.54%

R [low] f [low] D: 0.65, [small alpha] [low] D [high] 20: -54.4 ° (c = 1, DMF); M.p .: 172-175 ° C

Working instruction 37:

t.Butyloxycarbonyl-L-leucyl-N [high] small epsilon-benzyloxycarbonyl-L-lysyl-o-chloro-p-nitroanilide (Boc-Leu-Lys (Z) -o-Cl-pNA): 2.31 g Boc -Leu-OH are dissolved together with 1.35 g of hydroxybenzotriazole in 50 ml of absolute dimethylformamide and cooled to 0 ° C. After adding 2.06 g of dicyclohexylcarbodiimide, the mixture is stirred for 1 hour at 0 ° C and then 4.71 g of H is added -Lys (Z) -o-Cl-pNA.HCl and 1.01 g of N-methylmorpholine are added and the mixture is stirred for a further 2 hours at 0 ° C. and overnight at room temperature. The precipitated dicyclohexylurea is filtered off, the solvent is removed in vacuo, the residue is taken up in ethyl acetate and washed three times in succession with 5% sodium hydrogen carbonate solution, 0.5 N citric acid and water. After drying over sodium sulfate, the compound is precipitated by adding petroleum ether. Recrystallization takes place from methylene chloride / ether / petroleum ether.

Yield: 5.6 g (86.4% of theory)

Molecular formula: C [deep] 31H [deep] 42N [deep] 5O [deep] 8Cl

MG: 648.16

Elemental analysis: C H N Cl

calc .: 57.45% 6.53% 10.81% 5.47%

found: 57.48% 6.72% 10.93% 5.55%

R [deep] f [deep] D: 0.73, R [deep] f [deep] A: 0.93, [small alpha] [deep] D [high] 20: + 18.0 ° (c = 1 , DMF); M.p .: 113 to 115 ° C

Working instruction 38:

Benzyloxycarbonyl-aminoisobutyryl-L-prolyl-N [high] small omega-nitro-L-arginyl-o-chloro-p-nitroanilide (Z-Aib-Pro-Arg (NO [deep] 2) -o-Cl-pNA) : 237 mg of Z-Aib-OH are dissolved together with 135 mg of hydroxybenzotriazole in 25 ml of dimethylformamide and cooled to 0 ° C., then 206 mg of dicyclohexylcarbodiimide are added and the mixture is stirred at 0 ° C. for 3 hours. After adding 507 mg of H-Pro-Arg (NO [deep] 2) -o-chloro-pNA.HCl and 0.11 ml of N-methylmorpholine, the mixture is stirred for a further 2 hours at this temperature and overnight at room temperature. After the precipitated dicyclohexylurea has been filtered off and the solvent is stripped off in vacuo, the mixture is partitioned between ethyl acetate and water and washed successively with 5% NaHCO [deep] 3 solution, 1N citric acid and water. The ethyl acetate is removed after drying over sodium sulfate. Crystallization takes place from ethyl acetate / methanol / petroleum ether.

Yield: 500 mg (72.45% of theory)

Molecular formula: C [deep] 29H [deep] 36N [deep] 9O [deep] 9Cl

MG: 690.11

Elemental analysis: C H N

calc .: 50.47% 5.26% 18.27%

found: 50.33% 5.31% 18.10%

Melting point: 104 to 108 ° C, R [low] f [low] A: 0.73, [small alpha] [low] D [high] 20: + 0.4 ° (c = 0.99, DMF)

Working instruction 39:

t. Butyloxycarbonyl-glycyl-N [high] small omega-nitro-L-arginyl-o-chloro-p-nitroanilide (Boc-Gly-Arg (NO [low] 2) -o-Cl-pNA): 1.75 g Boc -Glycine are dissolved together with 1.35 g of hydroxybenzotriazole in 50 ml of dimethylformamide and cooled to 0 ° C .; it is mixed with 2.06 g of dicyclohexylcarbodiimide and 4.55 g of H-Arg (NO [deep] 2) -

-o-Cl-pNA.HBr and 1.1 ml of N-methylmorpholine, and the reaction solution is stirred for 2 hours at 0 ° C. and overnight at room temperature. After the dicyclohexylurea formed has been filtered off, the solvent is removed in vacuo. The residue is partitioned between ethyl acetate and 5% sodium hydrogen carbonate solution. The organic phase is then washed successively with 5% sodium hydrogen carbonate solution, 1N citric acid and water and dried over sodium sulfate. The substance is crystallized from ethyl acetate / petroleum ether.

Yield: 4.5 g (84.9% of theory)

Molecular formula: C [deep] 19H [deep] 27N [[deep] 8O [deep] 8Cl

MG: 530.93

Elemental analysis: C H N Cl

calc .: 42.98% 5.13% 21.11% 6.68%

found: 42.22% 5.32% 21.33% 6.21%

Fp .: 98 to 100 ° C, R [low] f [low] D: 0.51, [small alpha] [low] D [high] 20: + 23.78 ° (c = 0.5, DMF)

Working instruction 40:

t. Butyloxycarbonyl-small beta-alanyl-L-leucyl-N [high] small omega-nitro-L-arginyl-o-chloro-p-nitroanilide (Boc-small beta) -Ala-Leu-arg (NO [low] 2) -o-Cl-pNA): 302 mg of Boc-small Beta-Ala-Leu-OH are dissolved together with 135 mg of hydroxybenzotriazole and cooled to 0 ° C. 206 mg of dicyclohexylcarbodiimide are added and the mixture is stirred for one hour at 0 ° C., and then 455 mg of H-Arg (NO [deep] 2) -o-Cl-pNA.HBr and 0.11 ml of N-methylmorpholine are added. The mixture is stirred for a further 2 hours at 0 ° C. and overnight at room temperature. After filtering off the dicyclohexylurea, the solvent is removed in vacuo. The residue is taken up in ethyl acetate and washed as usual and dried over sodium sulfate. The substance is obtained in a solid state by adding petroleum ether.

Yield: 200 mg (30.4% of theory)

Molecular formula: C [deep] 26H [deep] 40N [deep] 9O [deep] 9Cl

MG: 658.11

Elemental analysis: C H N

calc .: 47.45% 6.13% 19.15%

found: 47.38% 6.02% 19.45%

M.p .: 180 to 182 ° C, R [low] f [low] D: 0.62

Example 1:

Benzyloxycarbonyl-D-phenylalanyl-L-valyl-L-arginyl-o-chloro-p-nitroanilide acetate (Z-D-Phe-Val-Arg-o-Cl-pNA.HAc):

548 mg of Z-D-Phe-OPCP are dissolved together with 548 mg of H-Val-Arg-o-Cl-pNA.2HAc in 30 ml of absolute dimethylformamide and mixed with 0.11 ml of N-methylmorpholine. After adding a spatula tip of hydroxybenzotriazole as a catalyst, the batch is stirred for 36 hours at room temperature. After the solvent has been stripped off in vacuo, the residue is taken up in ethyl acetate and the compound is precipitated by adding petroleum ether. After reprecipitation from the same solvent, the substance is purified by chromatography on Sephadex G 15 (eluent: 10% acetic acid).

Yield: 620 mg (80.6% of theory)

Molecular formula: C [deep] 36H [deep] 45N [deep] 8O [deep] 9Cl

MG: 769.26

Elemental analysis: C H N Cl

calc .: 56.21% 5.90% 14.57% 4.61%

found: 56.35% 5.85% 14.53% 4.70%

Fp .: 98 to 102 ° C, R [deep] f [deep] D: 0.35, R [deep] f [deep] A: 0.72, [small alpha] [deep] D [high] 20: + 55.7 ° (c = 1, DMF)

Example 2:

Benzyloxycarbonyl-small alpha-aminoisobutyryl-L-prolyl-N [high] small epsilon-benzyloxycarbonyl-L-lysyl-o-chloro-p-nitroanilide (Z-Aib-Pro-Lys (Z) .o.-Cl-pNA) : 1.46 g of Z-Aib-OPCP are dissolved together with 1.71 g of H-Pro (Z) -o-Cl-p-NA.HCl in 30 ml of dimethylformamide and, with the addition of 0.34 ml of N-methylmorpholine 36 Stirred for hours at room temperature. After removing the dimethylformamide in vacuo, the residue is dissolved in acetic acid taken up ethyl ester and washed successively with 5% sodium hydrogen carbonate solution, 1N citric acid and water three times each and dried over sodium sulfate. The desired substance is obtained by dripping the resulting solution into a large amount of ice-cold petroleum ether. Recrystallization also takes place from ethyl acetate / petroleum ether.

Yield: 1.25 g (55.3% of theory)

Molecular formula: C [deep] 37H [deep] 43N [deep] 6O [deep] 9Cl

MG: 751.24

Elemental analysis: C H N Cl

calc .: 59.16% 5.77% 11.19% 4.72%

found: 59.32% 5.84% 11.02% 4.97%

Fp .: 58 to 62 ° C, R [low] f [low] A: 0.84, [small alpha] [low] D [high] 20: -13 ° (c = 1, DMF)

Example 3:

Benzyloxycarbonyl-L-pyroglutamyl-glycyl-L-arginyl-o-chloro-p-nitroanilide hydroacetate (Z-Pyr-Gly-Arg-o-Cl-pNA.HAc): 384.5 mg of Z-Pyr-ONP are added together with 505 , 9 mg of H-Gly-Arg-o-Cl-pNA.2HAc dissolved in 30 ml of dimethylformamide and mixed with 0.12 ml of N-methylmorpholine at room temperature. The reaction solution is stirred for 36 hours. After removing the solvent in vacuo, the residue is taken up in methanol and added dropwise to ice-cold ether. The compound obtained in this way is recrystallized again from the same solvent mixture and then subjected to column chromatography on Sephadex G 15 (eluent: 10% strength glacial acetic acid).

Yield: 480 mg (69.4% of theory)

Molecular formula: C [deep] 29H [deep] 35N [deep] 8O [deep] 10Cl

MG: 691.1

Elemental analysis: C H N

calc .: 50.40% 5.10% 16.21%

found: 50.30% 5.18% 16.01%

Fp .: 115 to 120 ° C, R [low] f [low] A: 0.43, [small alpha] [low] D [high] 20: -42 ° (c = 1, DMF)

Example 4:

Benzyloxycarbonyl-D-phenylalanyl-L-isoleucyl-L-arginyl-o-chloro-p-nitroanilide hydroacetate (ZD-Phe-Ile-Arg-o-Cl-pNA.HAc): 548 mg ZD-Phe-OPCP together with 562 mg of H-Ile-Arg-o-Cl-pNA.2HAc dissolved in 30 ml of dimethylformamide, mixed with 0.11 ml of N-methylformamide and a spatula tip of hydroxybenzotriazole as a catalyst and stirred for 36 hours at room temperature. The solvent is removed in vacuo. The residue is taken up in methylene chloride, then a few drops of methanol are added to dissolve completely. The substance is precipitated by adding ether. The substance is obtained in analytically pure form by column chromatography on Sephadex G 15 (eluent: 20% acetic acid).

Yield: 600 mg (76.6% of theory)

Molecular formula: C [deep] 37H [deep] 47N [deep [8O [deep] 9Cl

MG: 783.28

Elemental analysis:

C H N

ber .: 56.74% 6.05% 14.30%

found: 56.51% 6.21% 14.28%

M.p .: 98-104 ° C

R [deep] f [deep] A: 0.71

[small alpha] [low] D [high] 20: -12 ° (c = 1, DMF)

N-terminally unprotected o-Cl-pNA dipeptides

Example 5:

L-Prolyl-L-arginyl-o-chloro-p-nitroanilide dihydroacetate (H-Pro-Arg-o-Cl-pNA.2HAc): 0.8 g Boc-L-Pro-L-Arg (NO [deep] 2 ) -o-Cl-pNA are in a Sakakibara apparatus with the addition of 1 ml of anisole with 15 ml of liquid hydrogen fluoride treated. After stirring for one hour at 0 ° C., the hydrogen fluoride is removed with a stream of nitrogen. The residue is taken up in water and extracted three times with ether to remove the anisole. The organic phase is discarded, the aqueous phase is treated with anion exchange resin Dowex 1 × 10 (acetate form) and finally freeze-dried.

Yield: 500 mg

Molecular formula: C [deep] 19H [deep] 28N [deep] 7O [deep] 6Cl

MG: 485.93

Elemental analysis: C H N Cl

calc .: 46.96% 5.81% 20.18% 7.30%

found: 46.74% 5.92% 20.09% 7.35%

M.p .: 195-199 ° C

R [deep] f [deep] A: 0.18

[small alpha] [low] D [high] 20: + 17.6 ° (c = 1, H [low] 2O)

Example 6:

L-prolyl-N [high] small epsilon-benzyloxycarbonyl-L-lysyl-o-chloro-p-nitroanilide hydrochloride (H-Pro-Lys (Z) -o-Cl-pNa.HCl): 13 g Boc-Pro-Lys (Z) -o-Cl-pNA are poured over with 100 ml of 1.2N HCl / glacial acetic acid and stirred for 1.5 hours at room temperature. The course of the cleavage reaction is monitored by thin-layer chromatography. The solution thus obtained is added dropwise to 1 l of ice-cold ether with vigorous stirring. The precipitated substance is filtered off with suction and dried over potassium hydroxide in a high vacuum. Recrystallization takes place from methanol / ether.

Yield: 10.05 g (86% of theory)

Molecular formula: C [deep] 25H [deep [31N [deep] 5O [deep] 6Cl [deep] 2

MG: 568.46

Elemental analysis: C H N

calc .: 52.82% 5.50% 12.32%

found: 52.74% 5.71% 12.71%

Fp .: 58 to 62 ° C, [small alpha] [low] D [high] 20: -26.29 ° (c = 1, DMF)

Example 7:

L-isoleucyl-L-arginyl-o-chloro-p-nitroanilide dihydroacetate (H-Ile-Arg-o-Cl-pNa.2HAc): 780 mg Boc-Ile-Arg (NO [deep] 2) -o-Cl- pNA are treated in a Teflon apparatus with 10 ml of liquid hydrogen fluoride with the addition of 1 ml of anisole as a cation trap. After a reaction time of one hour, the protective groups have been split off quantitatively; the hydrogen fluoride is blown with dry nitrogen. After the residue has been dried over potassium hydroxide in vacuo, it is taken up in water and the solution is extracted three times with ether in order to remove the anisole. The aqueous phase is treated with Dowex 1 x 4 anion exchange resin (acetate form) and then freeze-dried.

Yield: 680 mg (91% of theory)

Molecular formula: C [deep] 22H [deep] 36N [deep] 7O [deep] 8Cl

MG: 562.02

Elemental analysis: C H N

calc .: 47.02% 6.46% 17.44%

found: 47.21% 6.24% 17.25%

Fp .: 46 to 48 ° C, R [low] f [low] A: 0.33, [small alpha] [low] D [high] 20: -30.9 ° (c = 1, DMF)

Example 8:

Glycyl-L-arginyl-o-chloro-p-nitroanilide dihydroacetate (H-Gly-Arg-o-Cl-pNA.2HAc): 1 g of Boc-Gly-Arg (NO [deep] 2) -o-Cl-pNA at 0 ° C for one hour with liquid hydrogen fluoride to which 1 ml of anisole was added as a cation scavenger. After the hydrogen fluoride has been blown in a stream of dry nitrogen and dried, the residue is taken up in water in vacuo over potassium hydroxide, shaken out three times with ether to remove the anisole, and finally anion exchange resin is added

Dowex 1 x 4 (acetate form) to convert the hydrofluoride formed into the hydroacetate. The aqueous solution thus obtained is freeze-dried.

Yield: 650 mg (68.1% of theory)

Molecular formula: C [deep] 18H [deep] 28N [deep] 7O [deep] 8Cl

MG: 505.92

Elemental analysis: C H N Cl

calc .: 42.73% 5.58% 18.47% 6.68%

found: 42.51% 5.71% 18.32% 6.41%

Fp .: 84 to 88 ° C, R [low] f [low] A: 0.27, [small alpha] [low] D [high] 20: -31.4 ° (c = 0.5 DMF)

Example 9;

L-Valyl-L-arginyl-o-chloro-p-nitroanilide acetate (H-Val-Arg-o-Cl-pNA.HAc): 1 g Boc-Val-Arg (NO [deep] 2) -o-Cl- pNA are mixed with 20 ml of liquid hydrogen fluoride in a Sakakibara apparatus, to which 1.5 ml of distilled anisole has been added as a cation trap. After stirring for one hour at 0 ° C., the hydrogen fluoride is removed with nitrogen. The residue is taken up in water and extracted three times with ether to remove the anisole. The aqueous phase is treated with an ion exchange resin Dowex 1 x 10 (acetate form) and then lyophilized.

Yield: 0.76 g (89.41% of theory)

Molecular formula: C [deep] 19H [deep] 30N [deep] 7O [deep] 6Cl

MG: 487.95

Elemental analysis: C H N Cl

calc .: 46.77% 6.20% 20.09% 7.27%

found: 46.97% 6.30% 20.25% 7.44%

Fp .: 125 to 128 ° C, [small alpha] [low] D [high] 20: + 2 ° (c = 1, DMF)

Example 10:

L-Leucyl-N [high] small epsilon-benzyloxycarbonyl-L-lysyl-o-chloro-p-nitroanilide hydrochloride (H-Leu-Lys (Z) -o-Cl-pNA.HCl): 3 g Boc-Leu-Lys (Z) -o-Cl-pNA are mixed with 30 ml of 1,2N hydrogen chloride in glacial acetic acid and for 1.5 hours at room temperature touched. The compound precipitates after the addition of ether. Recrystallization takes place from methylene chloride / methanol / ether.

Yield: 2.3 g (85.2% of theory)

Molecular formula: C [deep] 26H [deep] 35N [deep] 5O [deep] 6Cl [deep] 2

MG: 584.50

Elemental analysis: C H N Cl

calc .: 53.43% 6.04% 11.98% 12.13%

found: 53.21% 6.24% 11.73% 12.48%

Fp .: 76 to 78 ° C, R [low] f [low] D: 0.48, [small alpha] [low] D [high] 20: + 90 ° (c = 1, DMF)

Example 11:

L-phenylalanyl-L-arginyl-o-chloro-p-nitroanilide (H-Phe-Arg-o-Cl-pNA.2HAc): 800 mg Boc-Phe-Arg (NO [deep] 2) -o-Cl- 10 ml of liquid hydrogen fluoride are added to pNA in a Teflon apparatus with the addition of 1 ml of anisole and the mixture is stirred at 0 ° C. for 1 hour. After the hydrogen fluoride has evaporated in a stream of dry nitrogen, the last traces of HF are removed by drying the residue over potassium hydroxide. The residue is partitioned between ether and water. The aqueous phase is extracted twice with ether in order to remove the added anisole. The water phase is then treated with Dowex 1 × 4 ion exchange resin (acetate form). The solution thus obtained is freeze-dried.

Yield: 570 mg (74.2% of theory)

Molecular formula: C [deep] 25H [deep] 34N [deep] 7O [deep] 8Cl

MG: 596.04

Elemental analysis: C H N Cl

calc .: 50.38% 5.75% 16.45% 5.95%

found: 50.12% 5.53% 16.40% 5.71%

Fp .: 88 to 92 ° C, R [low] f [low] A: 0.24, [small alpha] [low] D [high] 20: -28.6 ° (c = 1.05, DMF)

Example 12:

L-Leucyl-L-arginyl-o-chloro-p-nitroanilide dihydroacetate (H-leu-Arg-o-Cl-pNA.2HAc): 1 g Boc-Leu-arg (NO [deep] 2) -o-Cl- pNA are stirred as usual in a Teflon apparatus with 10 ml of liquid hydrogen fluoride / anisole (10: 1) for one hour at 0 ° C. After removing the hydrogen fluoride, drying is carried out over potassium hydroxide in a stream of nitrogen. The residue is taken up in water and extracted three times with ether to remove the anisole. The aqueous phase is treated with ion exchange resin Dowex 1 x 4 (acetate form) and freeze-dried.

Yield: 780 mg (81.5% of theory)

Molecular formula: C [deep] 22H [deep] 36N [deep] 7O [deep] 8Cl

MG: 562.02

Elemental analysis: C H N

calc .: 57.01% 6.46% 17.45%

found: 56.84% 6.52% 17.14%

Fp .: 48 to 52 ° C, R [low] f [low] A: 0.39, [small alpha] [low] D [high] 20: -45.4 ° (c = 1, DMF)

Mbs-protected o-Cl-pNA peptides

Example 13:

Np-methoxybenzenesulfonyl-small gamma-aminobutyryl-L-prolyl-L-arginyl-o-chloro-p-nitroanilide hydroacetate (Mbs-Gaba-Pro-Arg-o-Cl-pNA.HAc): 392 mg of Mbs-Gaba-OPCP dissolved together with 410 mg of H-Pro-Arg-o-Cl-pNA.2HAc in 25 ml of absolute dimethylformamide; 0.08 ml of N-methylmorpholine are added and the reaction mixture is stirred for 36 hours at room temperature. A spatula tip of hydroxybenzotriazole is added to accelerate the reaction. After the reaction has ended, the solvent is removed in vacuo, the residue is taken up in methanol and precipitated with absolute ether. The substance obtained in this way is purified by column chromatography (Sephadex G 15, eluent 15% acetic acid).

Yield: 500 mg (89.8% of theory)

Molecular formula: C [deep] 30H [deep] 41N [deep] 8O [deep] 10sCl

MG: 741.22

Elemental analysis: C H N S Cl

calc .: 48.61% 5.58% 15.11% 4.33% 4.78%

found: 48.73% 5.92% 15.48% 4.52% 4.55%

Fp .: 95 to 100 ° C, R [low] f [low] A: 0.55, [small alpha] [low] D [high] 20: -37.62 ° (c = 1.01, DMF)

Example 14:

Np-methoxybenzenesulfonyl-L-valyl-L-leucyl-N [high] small epsilon-benzyloxycarbonyl-L-lysyl-o-chloro-p-nitroanilide (Mbs-Val-Leu-Lys (Z) -o-Cl-pNA) : 0.576 g of Mbs-Val-OH are dissolved together with 0.27 g of hydroxybenzotriazole in 30 ml of absolute dimethylformamide and cooled to 0 ° C. After adding 0.41 g of dicyclohexylcarbodiimide, the mixture is stirred for one hour at 0 ° C. and then 1.17 g of H-Leu-Lys (Z) -o-Cl-pNA.HCl and 0.22 ml of N-methylmorpholine are added in succession . The mixture is stirred for a further two hours at 0 ° C. and overnight at room temperature. The dicyclohexylurea formed is filtered off and the solvent is removed in vacuo. The residue is taken up in ethyl acetate and washed successively with 5% sodium hydrogen carbonate solution, 1N citric acid and water three times each. The organic phase is dried over sodium sulfate. The substance is obtained by adding ether / petroleum ether to a solution in methylene chloride.

Yield: 1.17 g (71.6% of theory)

Molecular formula: C [deep] 38H [deep] 49N [deep] 6O [deep] 10SCl

MG: 817.36

Elemental analysis: C H N S

calc .: 55.84% 6.04% 10.28% 3.92%

found: 55.44% 6.25% 10.54% 3.98%

[small alpha] [low] D [high] 20: -3.84 ° (c = 1, DMF); M.p .: 210 ° C, R [low] f [low] A: 0.89

Example 15:

Np-methoxybenzenesulfonyl-small beta-alanyl-L-prolyl-N [high] small epsilon-benzyloxycarbonyl-L-lysyl-o-chloro-p-nitroanilide (Mbs-small Beta-Ala-Pro-Lys (Z) -o- Cl-pNA): 0.78 g of Mbs-small Beta-Ala-OH are mixed with 0.41 g of hydroxybenzotriazole in 40 ml of dimethylformamide dissolves and cooled to 0 ° C., then 0.62 g of dicyclohexylcarbodiimide are added and the mixture is stirred at 0 ° C. for 1 hour. After adding 1.71 g of H-Pro-Lys (Z) -o-Cl-pNA.HCl and 0.34 ml of N-methylmorpholine, the mixture is stirred for a further 2 hours at 0 ° C. and overnight at room temperature. After filtering off the precipitated dicyclohexylurea and removing the solvent in vacuo at a temperature below 40 ° C., the residue is taken up in ethyl acetate and washed three times in succession with 5% sodium hydrogen carbonate solution, 1N citric acid and water each time. After drying over sodium sulfate, the solvent is removed and taken up in a little methylene chloride. The solution thus obtained is added dropwise to much ice-cold ether. The precipitated product is filtered off and dried over calcium sulfate in a high vacuum.

Yield: 1.54 g (66.4% of theory)

Molecular formula: C [deep] 35H [deep] 41N [deep] 6O [deep] 10SCl

MG: 773.26

Elemental analysis: C H N S Cl

calc .: 54.36% 5.34% 10.87% 4.15% 4.58%

found: 54.36% 5.28% 10.53% 4.03% 4.32%

Fp .: 65 to 68 ° C, R [low] f [low] A: 0.82, [small alpha] [low] D [high] 20: -46.3 ° (c = 1, DMF)

Example 16:

Np-methoxybenzenesulfonyl-glycyl-L-prolyl-N [high] small epsilon-benzyloxycarbonyl-L-lysyl-o-chloro-p-nitroanilide (Mbs-Gly-pro-Lys (Z) -o-Cl-pNA): 735 mg of Mbs-Gly-OH are dissolved together with 0.41 g of hydroxybenzotriazole in 30 ml of dimethylformamide and cooled to 0 ° C., then the coupling reagent dicyclohexylcarbodiimide (620 mg) is added and the batch is then stirred for one hour at 0 ° C. 1.71 g of H-Pro-Lys (Z) -o-Cl-pNA.HCl and 0.33 ml of N-methylmorpholine are added, and the mixture is stirred for a further 2 hours at 0 ° C. and overnight at room temperature. After filtering off and removing the solvent, the residue is taken in ethyl acetate ester and washes successively with 5% sodium hydrogen carbonate solution, 1N citric acid and water. After drying the organic phase over sodium sulfate, the ethyl acetate is removed and taken up in a little methylene chloride. The solution obtained in this way is added dropwise to ice-cold ether, whereupon the desired substance precipitates. Recrystallization takes place from methylene chloride / ether.

Yield: 1.38 g (60.7% of theory)

Molecular formula: C [deep] 34h [deep] 39N [deep] 6O [deep] 10SCl

MG: 759.24

Elemental analysis: C H N Cl S

calc .: 53.78% 5.18% 11.07% 4.67% 4.22%

found: 53.53% 5.25% 11.27% 4.91% 4.25%

Fp .: 80 to 82 ° C, R [low] f [low] D: 0.32, R [low] f [low] A: 0.47, [small alpha] [low] D [high] 20: -45.2 ° (c = 1, DMF)

Example 17:

Np-methoxybenzenesulfonyl-small gamma-aminobutyryl-L-prolyl-N [high] small epsilon-benzyloxycarbonyl-L-lysyl-o-chloro-p-nitroanilide (Mbs-Gaba-Pro-Lys (Z) -o-Cl-pNA ): 0.98 g of Mbs-Gaba-OPCP are dissolved together with 1.14 g of H-Pro-Lys (Z) -o-Cl-pNA, HCl and 30 ml of dimethylformamide and mixed with 0.22 ml of N-methylmorpholine. The reaction mixture is stirred for 36 hours at room temperature. After removing the solvent in vacuo, the residue is taken up in ethyl acetate and washed successively with 5% sodium hydrogen carbonate solution, 0.5N citric acid and water three times each and dried over sodium sulfate. Crystallization takes place from the solvent mixture ethyl acetate / petroleum ether.

Yield: 1.29 g (45.7% of theory)

Molecular formula: C [deep] 36H [deep] 43N [deep] 6O [deep] 10SCl

MG: 787.29

Elemental analysis: C H N S

calc .: 54.92% 5.51% 10.67% 4.07%

found: 54.95% 5.71% 10.32% 4.52%

Mp .: 75 to 80 ° C, [small alpha] [low] D [high] 20: -48.7 ° (c = 1, DMF), R [low] f [low] A: 0.81

Example 18:

Np-methoxybenzenesulfonyl-glycyl-L-prolyl-L-lysyl-o-chloro-p-nitroanilide hydrobromide (Mbs-Gly-Pro-Lys-o-Cl-pNA.HBr): 1 g Mbs-Gly-Pro-Lys (Z ) -o-Cl-pNA are poured over with 30 ml of a 15% solution of hydrogen bromide in glacial acetic acid and stirred at room temperature. The cleavage reaction is followed by thin-layer chromatography and is complete after about 45 minutes. The substance is made to separate out by adding ether. The substance can be further purified by reprecipitation from methanol / ether. The substance is obtained analytically pure by column chromatography on Sephadex G 15 (eluent: 20% acetic acid). The eluate is lyophilized.

Yield: 710 mg (76.4% of theory)

Molecular formula: C [deep] 26H [deep] 34N [deep] 6O [deep] 8ClBrS

MG: 706.02

Elemental analysis: C H N

calc .: 44.23% 4.85% 11.90%

found: 44.14% 4.73% 11.71%

Fp .: 80 to 85 ° C, R [low] f [low] A: 0.47, [small alpha] [low] D [high] 20: -45.21 ° (c = 1, DMF)

Example 19:

Np-methoxybenzenesulfonyl-small gamma-aminobutyryl-L-prolyl-L-lysyl-o-chloro-p-nitroanilide hydrobromide (Mbs-Gaba-Pro-Lys-o-Cl-pNA.HBr): 1 g Mbs-Gaba-Pro- Lys (Z) -o-Cl-pNA are poured over with 30 ml of 15% hydrogen bromide, dissolved in glacial acetic acid, and stirred for about 45 minutes at room temperature. The reaction is monitored by thin layer chromatography. Deposition of the substance takes place after the addition of absolute ether. Recrystallization takes place from the solvent system methylene chloride / methanol / ether. After column chromatography on Sephadex G 15 (eluent: 20% acetic acid) the substance is analytically pure. The eluate is freeze-dried.

Yield: 680 g (72.9% of theory)

Molecular formula: C [deep] 28H [deep] 38N [deep] 6O [deep] 8SClBr

MG: 734.07

Elemental analysis: C H N

calc .: 45.81% 5.22% 11.45%

found: 45.32% 5.48% 11.22%

Fp .: 68 to 70 ° C, R [low] f [low] A: 0.42, [small alpha] [low] D [high] 20: -28.7 ° (c = 1, DMF)

Example 20:

Np-methoxybenzenesulfonyl-small beta-alanyl-l-prolyl-L-lysyl-o-chloro-p-nitroanilide hydrobromide (Mbs-small Beta-Ala-Pro-Lys-o-Cl-pNA.HBr): 1 g Mbs-small Beta-Ala-Pro-Lys (Z) -o-Cl-pNA are poured over with 30 ml of 15% HBr / glacial acetic acid and stirred at room temperature. The course of the reaction is followed by thin-layer chromatography. The splitting off of the benzyloxycarbonyl protective group is complete within 40 minutes. The substance is precipitated by adding ether. After filtering off, the compound is dried over potassium hydroxide under high vacuum for 24 hours. The reprecipitation takes place from methanol / ether and the final purification by running through Sephadex G 15 (eluent: 20% acetic acid). The eluate is lyophilized.

Yield: 650 with (69.7% of theory)

Molecular formula: C [deep] 27H [deep] 36N [deep] 6O [deep] 8ClBrS

MG: 720.05

Elemental analysis: C H N

ber .: 45.04% 5.04% 11.67%

found: 44.88% 5.21% 11.43%

R [low] f [low] A: 0.43, [small alpha] [low] D [high] 20: + 5.9 ° (c = 0.5, DMF); M.p .: 88 to 95 °

Example 21:

Np-methoxybenzenesulfonyl-L-valyl-L-leucyl-L-arginyl-o-chloro-p-nitroanilide hydroacetate (Mbs-Val-leu-Arg-o-Cl-pNA.HAc): 144 mg Mbs-Val-OH and 68 mg of hydroxybenzotriazole are dissolved in 25 ml of methylene chloride and 103 mg of dicyclohexylcarbodiimide are added at 0 ° C. After stirring for two hours at 0 ° C., 281 mg of H-Leu-Arg-o-Cl-pNA.2HAc and 0.06 ml of N-methylmorpholine are added. One stirs for a further 2 hours at 0 ° C and then overnight at room temperature. After the dicyclohexylurea formed has been filtered off, the solvent is removed in vacuo. The substance is obtained by dripping its methanolic solution into a lot of cold ether. The substance thus obtained is then chromatographed on Sephadex G 15 (eluent: 20% acetic acid). The eluate is freeze-dried.

Yield: 326 g (83% of theory)

Molecular formula: C [deep] 32H [deep] 47N [deep] 8O [deep] 10SCl

MG: 771.29

Elemental analysis: C H N

calc .: 53.91% 6.44% 15.24%

found: 53.72% 6.31% 15.51%

Fp .: 175 to 180 ° C, R [low] f [low] A: 0.68, [small alpha] [low] D [high] 20: -6.04 ° (c = 1, DMF)

Example 22:

Np-methoxybenzenesulfonyl-L-valyl-L-prolyl-L-arginyl-o-chloro-p-nitroanilide hydroacetate (Mbs-Val-Pro-Arg-o-Cl-pNA.HAc): 287 mg of Mbs-Val-OH are combined dissolved with 135 mg of hydroxybenzotriazole in 30 ml of methylene chloride, cooled to 0 ° C. and treated with 206 mg of dicyclohexylcarbodiimide. The batch is stirred for 2 hours at room temperature and 546 mg of H-Pro-Arg-o-Cl-pNA.2HAc and 0.11 ml of N-methylmorpholine are then added in succession. After stirring for two hours at 0 ° C. and further stirring overnight at room temperature, the dicyclohexylurea is filtered off and the solvent is removed in vacuo. The crude substance is obtained by adding ether to its methanolic solution: Further purification is carried out by column chromatography on Sephadex G 15 (eluent: 20% acetic acid). The eluate is freeze-dried.

Yield: 600 mg (79.4% of theory)

Molecular formula: C [deep] 31H [deep] 43N [deep] 8O [deep] 10SCl

MG: 755.25

Elemental analysis: C H N

calc .: 49.31% 5.74% 14.84%

found: 49.25% 5.54% 14.54%

Mp .: 120 to 125 ° C, R [low] f] low] A: 0.60, [small alpha] [low] D [high] 20: -25.4 ° (c = 1, DMF)

Example 23:

Np-methoxy benzenesulfonyl-L-phenylalanyl-L-valyl-L-arginyl-o-chloro-p-nitroanilide acetate (Mbs-Phe-Val-Arg-o-Cl-pNA.HAc): 216 mg Mbs-Phe-OSu be dissolved together with 275 mg of H-Val-Arg-o-Cl-pNA.2HAc in 25 ml of dimethylformamide, then 0.05 ml of N-methylmorpholine is added and the mixture is stirred for 36 hours at room temperature. A spatula tip of hydroxybenzotriazole is added to catalytically accelerate the reaction. After the reaction has ended, the solvent is removed in vacuo and the residue is taken up in absolute methanol and the desired substance is precipitated with ether. The final purification is carried out by means of column chromatography (Sephadex G 15, eluent 10% acetic acid).

Yield: 310 mg (77.11% of theory)

Molecular formula: C [deep] 35H [deep] 45N [deep] 8O [deep] 10SCl

MG: 805.31

Elemental analysis: C H N

calc .: 52.20% 5.63% 13.91%

found: 52.44% 5.77% 13.71%

Mp .: 115 to 118 ° C, [small alpha] [low] D [high] 20: + 31.6 ° (c = 1, DMF), R [low] f [low] A: 0.62

Example 24:

Np-methoxybenzenesulfonyl-small beta-alanyl-L-prolyl-L-arginyl-o-chloro-p-nitroanilide (Mbs-small Beta-Ala-Pro-Arg-o-Cl-pNA): 855 mg Mbs-small Beta Ala -OH are dissolved together with 345 mg of hydroxysuccinimide in 30 ml of dimethylformamide and cooled to -5 ° C. After 618 mg of dicyclohexylcarbodiimide have been added, the mixture is stirred for 3 hours at this temperature, then 1.46 g of H-Pro-Arg-o-Cl-pNA.HAc and 0.33 ml of N-methylmorpholine are added. Stir for an additional hour at -5 ° C and let stand overnight at room temperature. After the dicyclohexylurea has been filtered off with suction, the solvent is removed in vacuo. The residue is chromatographed on a Sephadex LH 20 column which is equilibrated with 20% strength acetic acid.

Yield: 0.75 g (78.45% of theory)

Molecular formula: C [deep] 30H [deep] 35N [deep] 8O [deep] SCl

MG: 703.18

Elemental analysis: C H N

ber .: 51.20% 5.01% 22.33%

found: 51.43% 5.21% 22.35%

Fp .: 115 to 120 ° C, R [low] f [low] D: 0.19, R [low] f [low] A: 0.41, [small alpha] [low] D [high] 20: -46.5 ° (c = 1, DMF)

Example 25:

Np-Methoxybenzenesulfonyl-L-prolyl-L-phenylalanyl-L-arginyl-o-chloro-p-nitroanilide (mbs-Pro-Phe-Arg-o-Cl-pNA): 307.9 mg of Mbs-Pro-OSu are combined with 480 mg of H-Phe-Arg-o-Cl-pNA, 2HAc dissolved in 25 ml of dimethylformamide, mixed with 0.09 ml of N-methylmorpholine and stirred for 24 hours at room temperature. After the solvent has been stripped off in vacuo, the residue is placed on a chromatography column with dimensions of 1.6 × 100 cm packed with Sephadex G 15 and eluted with 10% acetic acid.

Yield: 570 mg (74.2% of theory)

Molecular formula: C [deep] 25H [deep] 34N [deep] 7O [deep] 8Cl

MG: 596.04

Elemental analysis: C H N

calc .: 50.38% 5.75% 16.45%

found: 50.55% 5.97% 16.45%

Fp .: 107 to 110 ° C, R [low] f [low] A: 0.24, [small alpha] [low] D [high] 20: -74.9 ° (c = 1.07, DMF)

Example 26:

Np-methoxybenzenesulfonyl-glycyl-L-prolyl-L-arginyl-o-chloro-p-nitroanilide acetate (Mbs-Gly-Pro-Arg-o-Cl-pNA.HAc): 183.7 mg of Mbs-Gly-OH are combined with 101 mg of hydroxybenzotriazole dissolved in 25 ml of dimethylformamide and brought to 0 ° C cooled, after adding 154.5 mg of dicyclohexylcarbodiimide, the mixture is stirred for 3 hours at this temperature and then 409.1 mg of H-Pro-Arg-o-Cl-pNA.2HAc and 0.08 ml of N-methylmorpholine are added. After further stirring at 0 ° C. (2 hours) and finally overnight at room temperature, the precipitated dicyclohexylurea is filtered off and the solvent is removed in vacuo. Crystallization takes place on methanol / ether. The last traces of by-products are removed by column chromatography (Sephadex G 15, eluent: 10% acetic acid).

Yield: 470 mg (87.9% of theory)

Molecular formula: C [deep] 28H [deep] 37N [deep] 8O [deep] 10SCl

MG: 713.17

Elemental analysis: C H N

calc .: 47.16% 5.23% 15.71%

found: 47.33% 5.12% 15.55%

R [deep] f [deep] A: 0.37; Fp .: 121 to 123 ° C, [small alpha] [low] D [high] 20: -38 ° (c = 1, DMF)

Example 27:

Np-methoxybenzenesulfonyl-L-valyl-L-leucyl-L-lysyl-o-chloro-p-nitroanilide hydrobromide (Mbs-Val-Leu-Lys-o-Cl-pNA.HBr): 1 g Mbs-Val-Leu-Lys (Z) -o-Cl-pNA are poured with 30 ml of 15% hydrogen bromide in glacial acetic acid and stirred for about an hour at room temperature. The cleavage reaction is followed by thin layer chromatography. After the benzyloxycarbonyl protective group has been split off, the substance is deposited by adding ether. Reprecipitation takes place from methylene chloride / methanol / ether. Final cleaning takes place on Sephadex G 15 (eluent: 20% acetic acid).

Yield: 700 mg (74.9% of theory)

Molecular formula: C [deep] 30H [deep] 44N [deep] 6O [deep] 8SClBr

MG: 764.11

Elemental analysis: C H N

ber .: 47.15% 5.80% 11.00%

found: 47.21% 5.34% 11.32%

Fp .: 170 to 175 ° C, R [low] f [low] A: 0.66, [small alpha] [low] D [high] 20: + 54.4 ° (c = 1, DMF)

Example 28:

Benzyloxycarbonyl-D-phenylalanyl-prolyl-arginyl-o-chloro-p-nitroanilide hydroacetate (ZD-Phe-Pro-Arg-o-Cl-pNA.HAc): 547.7 mg of ZD-Phe-OPCP together with 545.5 mg of H-Pro-Arg-o-Cl-pNA.2HAc dissolved in 30 ml of absolute dimethylformamide and mixed with 0.11 ml of N-methylmorpholine. After adding a spatula tip of 1-hydroxybenzotriazole as a catalyst, the reaction mixture is stirred for 36 hours at room temperature. After removing the solvent in vacuo, the residue is allowed to crystallize from methylene chloride / ether. The substance is obtained analytically pure by chromatography on Sephadex G-15 (eluent: 20% acetic acid).

Yield: 400 mg (52.1% of theory)

Molecular formula: C [deep] 36H [deep] 43N [deep] 8O [deep] 9Cl

MG: 767.24

Elemental analysis: C H N

calc .: 56.36% 5.65% 14.61%

found: 56.31% 5.48% 14.43%

Fp .: 117 to 120 ° C, R [low] f [low] A: 0.59, [small alpha] [low] D [high] 20: -5.8 ° (c = 0.95, DMF)

Example 29:

Benzyloxycarbonyl-sarcosyl-prolyl-arginyl-o-chloro-p-nitroanilide hydroacetate (Z-Sar-Pro-Arg-o-Cl-pNA.HAc): 471.5 mg Z-Sar-OPCP together with 545.5 mg H. -Pro-Arg-o-Cl-pNA.2HAc dissolved in 30 ml of absolute dimethylformamide and mixed with 0.11 ml of N-methylmorpholine and a spatula tip of 1-hydroxybenzotriazole as a reaction accelerator. The batch is stirred for 24 hours at room temperature and then freed from the solvent. Reprecipitation takes place from methylene chloride / ether. The final purification is carried out by chromatography on Sephadex G-15 (eluent: 20% acetic acid).

Yield: 580 mg (83.9% of theory)

Molecular formula: C [deep] 30H [deep] 39N [deep] 8O [deep] 9Cl

MG: 691.14

Elemental analysis: C H N Cl

calc .: 52.14% 5.69% 16.21% 5.13%

found: 52.12% 5.23% 16.01% 5.04%

Fp .: 96 to 98 ° C, R [low] f [low] A: 0.50, [small alpha] [low] D [high] 20: -1.6 ° (c = 1.03, DMF)

N-terminally unprotected o-Cl-pNA tripeptides

Example 30:

Aminoisobutyryl-L-prolyl-L-arginyl-o-chloro-p-nitroanilide diacetate (H-Aib-Pro-Arg-o-Cl-pNA.2HAc): 460 mg Z-Aib-Pro-Arg (NO [deep] 2 ) -o-Cl-pNA are treated with 10 ml of liquid hydrogen fluoride in a Teflon apparatus with the addition of 1 ml of anisole (for 1 hour at 0 ° C.). After the hydrogen fluoride has been displaced by a stream of dry nitrogen, the last traces of HF are removed by drying the residue in a high vacuum over potassium hydroxide. The residue is dissolved in water. The aqueous solution is extracted three times with ether to remove the anisole, then the aqueous solution is mixed with an anion exchange resin (acetate form). The solution thus obtained is freeze-dried. The product thus obtained is purified by column chromatography on Sephadex G-15 (eluent: 10% acetic acid).

Yield: 310 mg (73.7% of theory)

Molecular formula: C [deep] 25H [deep] 39N [deep] 8O [deep] 9Cl

MG: 631.09

Elemental analysis: C H N

calc .: 47.58% 6.23% 17.76%

found: 47.33% 6.14% 17.92%

Fp .: 86 to 90 ° C, R [low] f [low] A: 0.29, [small alpha] [low] D [high] 20: + 74.5 ° (c = 1, DMF)

Example 31:

L-Pyroglutamyl-glycyl-L-arginyl-o-chloro-p-nitroanilide dihydrobromide (H-Pyr-Gly-Arg-o-Cl-pNA.2HBr): 250 mg Z-Pyr-Gly-Arg-o-Cl-pNA .HAc are poured over with 20 ml of HBr in glacial acetic acid and stirred for one hour at room temperature. The substance is obtained by dripping the reaction solution into 250 ml of ether. By recrystallization from methanol / ether and column chromatography on Sephadex G-15

(Eluent: 10% glacial acetic acid) the substance is obtained in analytically pure form.

Yield: 200 mg (84.4% of theory)

Molecular formula: C [deep] 19H [deep] 26N [deep] 8O [deep] 6ClBr [deep] 2

MG: 657.74

Elemental analysis: C H N

ber .: 34.72% 3.98% 17.04%

found: 34.34% 4.02% 17.01%

Fp .: 115 to 118 ° C, R [low] f [low] A: 0.36, [small alpha] [low] D [high] 20: + 13.33 ° (c = 1, DMF)

Example 32:

D-phenylalanyl-L-valyl-L-arginyl-o-chloro-p-nitroanilide dihydrobromide (HD-Phe-Val-Arg-o-Cl-pNA.2HBr): 400 mg ZD-Phe-Val-Arg-o-Cl -pNA.HAc are poured over with 50 ml of 3N HBr / glacial acetic acid and stirred for about an hour at room temperature. The reaction of the splitting off of protective groups is monitored by thin layer chromatography. The solution thus obtained is added dropwise to 560 ml of ether with stirring. The precipitated substance is filtered off with suction and dried over potassium hydroxide in a high vacuum for 24 hours. The compound is obtained analytically pure by chromatography on Sephadex G 15 (eluent: 20% glacial acetic acid).

Yield: 340 mg (88.8% of theory)

Molecular formula: C [deep] 26H [deep] 37N [deep] 8O [deep] 5ClBr [deep] 2

MG: 736.90

Elemental analysis C H N

ber .: 42.38% 5.06% 15.21%

found: 42.14% 5.21% 15.34%

Fp .: 205 to 210 ° C, R [low] f [low] A: 0.42, [small alpha] [low] d [high] 20: -36.2 ° (c = 0.5, DMF)

Example 33:

small beta-alanyl-L-leucyl-L-arginyl-o-chloro-p-nitroanilide dihydroacetate (H-small Beta-Ala-Leu-Arg-o-Cl-pNA.2HAc): 140 mg Boc-small beta-Ala- Leu-Arg (NO [deep] 2-o-Cl-pNA) are treated as usual with liquid hydrogen fluoride in a Teflon apparatus

Working up is carried out analogously to the described splitting off of protective groups with hydrogen fluoride. The lyophilized substance is purified by column chromatography on Sephadex G 15 (eluent: 20% acetic acid).

Yield: 90 mg (62.4% of theory)

Molecular formula: C [deep] 25H [deep] 41N [deep] 8O [deep] 9Cl

MG: 633.10

Elemental analysis: C H N

calc .: 47.43% 6.53% 17.70 5

found: 47.24% 6.48% 17.84%

Fp .: 56 to 58 ° C, R [low] f [low] A: 0.39, [small alpha] [low] D [high] 20: -29.1 ° (c = 1, DMF)

Example 34:

D-phenylalanyl-isoleucyl-arginyl-o-chloro-p-nitroanilide-dihydrobromide (H-D-Phe-Ile-Arg-o-Cl-pNA.2HBr):

500 mg of Z-D-Phe-Ile-Arg-o-Cl-pNA.HAc are poured over with 30 ml of 3N HBr in glacial acetic acid and stirred for one hour at room temperature. The solution thus obtained is added dropwise to 500 ml of ice-cold ether while stirring. The precipitate is washed with a lot of ether and dried over potassium hydroxide in vacuo. Recrystallization takes place from methanol / methylene chloride / ether. After chromatography on Sephadex G 10 (eluent: 20% acetic acid) the substance is analytically pure.

Yield: 450 mg (93.88% of theory)

Molecular formula: C [deep] 27H [deep] 39N [deep] 8O [deep] 5ClBr [deep] 2

MG: 750.93

Elemental analysis: C H N

calc .: 43.19% 5.24% 14.92%

found: 43.12% 5.04% 14.73%

M.p .: 230 ° C; R [deep] f [deep] A. 0.36; [small alpha [[low] D [high] 20: -22.2 ° (c = 1, DMF)

Example 35:

Sarcosyl-prolyl-arginyl-o-chloro-p-nitroanilide-dihydrobromide (H-Sar-Pro-Arg-o-Cl-pNA.2HBr): 240 mg Z-Sar-Pro-

-Arg-o-Cl-pNA.HAc are poured over 30 ml of a 3N solution of hydrogen bromide in glacial acetic acid at room temperature and stirred for one hour. The solution thus obtained is added dropwise to 500 ml of ice-cold ether with vigorous stirring. The precipitate is filtered off with suction, washed well with ether and dried over potassium hydroxide in vacuo. Recrystallization takes place from methylene chloride / methanol / ether. In order to remove the last traces of by-products, the substance obtained in this way is chromatographed on Sephadex G 10 (eluent: 20% acetic acid).

Yield: 120 mg (55.43% of theory)

Molecular formula: C [deep] 20H [deep] 31N [deep] 8O [deep] 5Br [deep] 2

MG: 623.34

Elemental analysis: C H N

ber .: 38.54% 5.01% 17.98%

found: 38.21% 5.03% 17.04%

Fp .: 150 to 153 ° C, R [low] f [low] A: 0.11, [small alpha] [low] D [high] 20: -53.6 ° (c = 1, DMF)

Claims (16)

1. Chromogenic enzyme substrate based on amino acid derivatives or oligopeptides, characterized in that there are new compounds of the general formula (I) or their salts contains, wherein R [deep] 1 hydrogen, alkyl, a sulfonyl radical, in particular benzenesulfonyl or p-methoxybenzenesulfonyl (Mbs) or an acyl radical, especially alkoxycarbonyl, such as t. Means butyloxycarbonyl, benzyloxycarbonyl, formyl or benzoyl, A for one of the following amino acid components: D-Arg, L-Arg, D-Lys, L-Lys, D-Orn or L-Orn, which optionally has one or more of the following further amino acid components: Gly, D-Glu (OX [deep] 1), L-Glu (OX [deep] 1) [where X [deep] 1 represents an ester residue], D-Glu, L-Glu, D-Ala, L-Ala, D-Val, L-Val, DIle, L- Ile, D-Leu, L-Leu, D-Pip, L-Pip, D-Pro, L-Pro, D-Aze, L-Aze, D-Phe, L-Phe, Phenylglycyl, D-Tyr, L- Tyr, D-Pyr, L-Pyr, the remainder of a c [deep] alpha-branched amino acid, like Aib, a small beta-amino acid, like small beta-Ala, and small beta-cyclohexylalanyl, a small gamma-amino acid, like Gaba , or an N-alkyl-amino acid, such as Sar, is linked to the substituent R [deep] 1, and Hal means halogen. 2. Enzyme substrate according to claim 1, characterized in that an amino acid component standing for A, D-Arg, L-Arg, D-Lys, L-Lys, D-Orn or L-Orn, in the side chain amino function by R [deep] 1 is substituted. 3. Enzyme substrate according to claims 1 and 2, characterized in that in an acyl group X-CO- representing the radical R [deep] 1 X denotes an aliphatic, araliphatic, cycloaliphatic or aromatic hydrocarbon radical having 1 to 19 carbon atoms, which radical is optionally substituted by alkyl, amino or aminoalkyl. 4. Enzyme substrate according to claim 1, characterized in that R [deep] is 1 Mbs. 5. enzyme substrate according to claims 1 and 4, characterized in that the new compound (II) is. 6. enzyme substrate according to claims 1 and 4, characterized in that the new compound (III) is. 7. enzyme substrate according to claim 1, characterized in that the new compound (IV) is. 8. enzyme substrate according to claim 1, characterized in that the new compound (V) is. 9. enzyme substrate according to claim 1, characterized in that the new compound (VI) is. 10. A process for the preparation of the new compounds according to claim 1 and their salts, characterized in that halo-p-nitroaniline is converted into halo-p-nitrophenyl isocyanate, this with one of the following, having a free carboxyl group, but protected at its other functional groups Amino acids with the components: D-Arg, L-Arg, D-Lys, L-Lys, D-Orn or L-Orn is reacted, whereupon the reaction product after releasing the terminal amino group with one or more of the following, having a free carboxyl group , amino acids with the components: Gly, D-Glu (OX [deep] 1) L-Glu (OX [deep] 1) [where X [deep] 1 represents an ester residue], D-Glu , L-Glu, D-Ala, L-Ala, D-Val, L-Val, D-Ile, L-Ile, D-Leu, L-Leu, D-Pip, L-Pip, D-Pro, L -Pro, D-Aze, L-Aze, D-Phe, L-Phe, Phenylglycyl, D-Tyr, L-Tyr, D-Pyr, L-Pyr, C [deep] small alpha-branched amino acids with a free carboxyl group , like H-Aib-O H, small beta-amino acids, like H-small beta-Ala-OH and small beta-cyclohexylalanine, small gamma-amino acids, like H-Gaba-OH, or N-alkyl-amino acids, like H-Sar-OH to- is set, whereupon, after the protective groups have been split off, a hydrogen atom on the terminal NH [deep] 2 group is replaced by acyl or sulfonyl. 11. The method according to claim 10, characterized in that two or more of the following amino acid components: Gly, D-Glu (OX [deep] 1), L-Glu (OX [deep] 1) [where X [deep] 1 is an ester residue represents], D-Glu, L-Glu, D-Ala, L-Ala, D-Val, L-Val, D-Ile, L-Ile, D-Leu, L-Leu, D-Pip, L-Pip , D-Pro, L-Pro, D-Aze, L-Aze, D-Phe, L-Phe, Phenylglycyl, D-Tyr, L-Tyr, D-Pyr, L-Pyr, a residue of a C [deep] small alpha-branched amino acid, like Aib, a small beta-amino acid, like small beta-Ala and small beta-cyclohexylalanyl, a small gamma-amino acid, like Gabe, or an N-alkyl-amino acid, like Sar, as at the terminal NH [deep] 2 group, dipeptide or oligopeptide optionally substituted by acyl or sulfonyl can be used. 12. The method according to claim 11, characterized in that to introduce an Mbs group for R [deep] 1 in the terminal amino group of an oligopeptide of the general formula (I), the terminal amino acid is reacted with Mbs chloride, the resulting Mbs amino acid is coupled either in the presence of a condensing agent directly or after conversion into the corresponding active ester with a dipeptide or oligopeptide. 13. The method according to claims 10 to 12 for the preparation of the compound according to claim 5, characterized in that Mbs-small beta-Ala-OH with or an acid addition salt thereof is reacted. 14. The method according to claims 10 to 12 for the preparation of the compound according to claim 6, characterized in that Mbs-Gly-OH with or an acid addition salt thereof is reacted. 15. The method according to claims 10 and 11 for the preparation of the compound according to claim 7, characterized in that Z-Aib-OH, wherein Z is benzyloxycarbonyl, with is reacted and from the compound obtained the protective groups are split off. 16. Use of enzyme substrates according to claims 1 to 9 for the determination of serine proteases, in particular of thrombin in the presence of other serine proteases in the physiological pH range.