EP0190299A1

EP0190299A1 - NEW p-PHENYLENEDIAMINE PEPTIDES AND REACTANCE CONTAINING THEM FOR THE DETERMINATION OF PROTEASES OF THE BLOOD COAGULATION SYSTEM

Info

Publication number: EP0190299A1
Application number: EP85904101A
Authority: EP
Inventors: Knut Bartl; Udo Becker; Herbert Von Der Eltz; Helmut Lill; Hans-Georg Batz
Original assignee: Roche Diagnostics GmbH; Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1984-08-02
Filing date: 1985-07-31
Publication date: 1986-08-13
Also published as: AU4723885A; JPS61501635A; AU564556B2; WO1986001209A3; WO1986001209A2; DE3428543A1; ZA855814B

Abstract

Dans de nouvelles compositions ayant la formule générale (I), A est l'acide aminé arginine ou lysine, X est un groupe d'acides aminés de protection à terminal N ou un acide aminé D, Y est une liaison simple ou une chaîne composée de 1 à 3 acides aminés, NR1R2 est un groupe en position o ou p où R1 et R2 sont indépendants l'un de l'autre et représentent de l'hydrogène ou de l'alcoyle ayant 1 à 3 atomes C et R3 est un atome d'hydrogène, un groupe carboxyle, un atome d'halogène ou un groupe alcoyle ayant 1 à 3 atomes C. Ces compositions sont utiles comme substrats chromogènes de protéinases. Elles sont obtenues par réduction et le cas échéant par alkylation des nitroanilides correspondantes. Un réactif correspondant pour déterminer des protéïnases du système de coagulation du sang comprend une composition ayant la formule (I), un agent d'oxydation, un dérivé d'aniline ou de phénol qui forme un colorant par oxydation avec de la phénylène-diamine et une substance tampon.In new compositions having the general formula (I), A is the amino acid arginine or lysine, X is a group of N-terminal protective amino acids or a D amino acid, Y is a single bond or a compound chain of 1 to 3 amino acids, NR1R2 is a group in position o or p where R1 and R2 are independent of each other and represent hydrogen or alkyl having 1 to 3 C atoms and R3 is a hydrogen atom, a carboxyl group, a halogen atom or an alkyl group having 1 to 3 C atoms. These compositions are useful as chromogenic substrates of proteinases. They are obtained by reduction and, where appropriate, by alkylation of the corresponding nitroanilides. A corresponding reagent for determining proteinases of the blood coagulation system comprises a composition having formula (I), an oxidizing agent, an aniline or phenol derivative which forms a dye upon oxidation with phenylenediamine and a buffer substance.

Description

New p-phenylenediamine peptides and those containing them

Reagents for the determination of proteases of the

Blood coagulation system ------------------------------------------------- ---------------------------

The determination of proteases of the blood coagulation system is of considerable clinical and scientific importance, especially in the context of the determination and regulation of the ability of the blood to clot.

It is already known to measure the proteases of the blood coagulation system using certain chromophoric peptides which are specifically cleaved by the protease to be determined in each case and thereby cleave off an optically determinable group. Since it is essential here not to disturb the blood coagulation cascade by adding such artificial substrates, i. H. Most of the chromogenic, peptide-like substrates known for proteases from other origins cannot be influenced adversely, for the determination of the proteases of the blood coagulation system. Two p-nitroaniline derivatives which contain the rest of Tos-Gly-Pro-Arg or H-D-Phe-Pip-Arg- have been found to be suitable protease substrates which do not adversely affect the coagulation cascade. However, these substrates have the disadvantage that the measurement of the dye formed when they are cleaved by the protease is disturbed by substances colored red or yellow, and therefore measurement in the blood itself is not possible.

The invention is therefore based on the object of eliminating this disadvantage and of creating a substrate for the proteases of the blood coagulation system which does not adversely affect the coagulation cascade and others on the one hand forms a color during cleavage, the absorption maximum of which is longer-wave than 565 nm and thereby also enables determination in whole blood or in the presence of other interfering colored substances.

According to the invention, this object is achieved by compounds of the general formula I.

in the

A the amino acid arginine or lysine,

X is an N-terminal amino acid protecting group or

D-amino acid, Y is a single bond or a chain consisting of 1 to 3 amino acids, NR ₁ R _{2 is} a group in the o- or p-position with R ₁ and R ₂ independently of one another in each case hydrogen or alkyl having 1 to 3 C atoms and R _{3 represents} a hydrogen atom, a carboxyl group, a halogen atom or an alkyl group having 1 to 3 C atoms.

The new compounds of the invention can be cleaved by the proteases of the blood coagulation system with the liberation of an o- or p-phenylenediamine derivative, which in the presence of an oxidizing agent with a suitable coupling partner, such as. B. N-methylanthranilic acid forms a color, the absorption maximum is longer than 565 mn. Surprisingly, it has been shown that the blood coagulation cascade is not negatively influenced by this reaction and the novel compounds according to the invention are therefore suitable as substrates for the determination of the proteases of the blood coagulation system itself or of other factors of the blood coagulation system which regulate the activity of the proteases of the blood coagulation system.

Preferred compounds of the general formula 1 are those in which R ₁ R ₂ = H. Also preferred are those compounds in which X = Tos and Y = Gly-Pro, X = HD-Phe and Y = Pip, X = HD-Ile and Y = Pro, X = CH ₃ SO ₂ -D-Nle and Y = Gly, X = CH-OCO-D-Nle and Y = Gly.

The compounds according to the invention are particularly preferred Tos-Gly-Pro-Arg-p-phenylenediamine and H-D-PhePip-Arg-p-phenylenediamine.

The new compounds according to the invention can be prepared using the methods of peptide synthesis familiar to the person skilled in the art. The preparation is preferably carried out by using a compound of the general formula II

in which X, Y, A and R _{3 have} the meaning given for formula I, and the NH ₂ group formed is optionally alkylated to prepare those compounds in which R ₁ and R _{2 represent} an alkyl group.

The reduction of the compounds of the general formula II is preferably carried out by catalytic hydrogenation, for example using palladium / carbon as a catalyst in solvents customary for reductive hydrogenation, such as methanol / glacial acetic acid.

As already mentioned above, the compounds according to the invention are particularly suitable as chromophobic substrates for the determination of proteases, in particular proteases of the blood coagulation system, such as thrombin, plasmin, plasminogen, factor Xa, kallikrein, plasminogen activator etc. However, they are also suitable as substrates for other proteases such as B. chymotrypsin, trypsin and the like.

The compounds according to the invention are cleaved by the respective protease to release the phenylenediamine or a phenylenediamine derivative, and this is oxidized in the presence of an oxidizing agent such as K ₃ [Fe (CN) ₆ ] with a suitable coupling component to give a dye with an absorption maximum greater than 565 nm. With p-phenylenediamine itself, the determination can be carried out particularly well at 644 nm. At this wavelength, colored substances present in blood, plasma, serum or other body fluids no longer interfere. The intrinsic color of the blood or of hemolytic (red color) or icteric (yellow color) plasmas or sera therefore has no influence on the measuring system. Therefore, coagulation factors, factors of the fibrinolytic system or the kallikrein-kinin system, can be derived from rows such as B. Leukocyte-released proteolytic enzymes such as granulocyte elastase or cathepsin-G can be determined in body fluids with the aid of the compounds according to the invention. These compounds also enable the analysis of entire enzyme cascades of the coagulation system, as is the case in the test systems of the prothrombin time method (PT or quick test) or the partial thromboplastin time method (PTT test).

The compounds according to the invention are also suitable for determining the inhibitors of certain active proteolytic enzymes by measuring the reduction in activity of these proteases. An example of this is the measurement of the decrease in activity of thrombin due to the presence of antithrombin.

Aromatic amines or phenols are suitable as coupling components for this reaction. Compounds of the formula II are particularly suitable

(II)

in the

X is an NR ₂ R ₃ or -OR group,

R _{1 is} hydrogen or chlorine, if R ₄ and / or R ₆ also

Are chlorine, R ₂ and R _{3 are the} same or different and are hydrogen, an alkyl, aralkyl or aryl group with up to 8 carbon atoms, which may also be hydroxyl, alkoxy with up to 5 carbon atoms, -CO ₂ H , SO ₃ H may be substituted. R ₄ and R _{6 are} hydrogen, low alkyl with up to 5 atoms of chlorine, bromine, iodine, -COOR ₂ , SO ₃ H ₂ ,

OR ₂ , NR ₂ R ₃ where R ₂ and R _{3 have} the above meaning;

R ₅ and R _{7 represent} an alkyl group with up to 5 carbon atoms, where

R ₄ and R ₅ or R ₆ and R ₇ together with the benzene ring can also form a naphthyl and anthryl skeleton.

The alkyl groups in the above compounds have up to 6, preferably 1 to 3, carbon atoms. The methyl group and ethyl group are particularly preferred.

In addition to the already mentioned and particularly preferred N-methylanthrahilic acid, preferred examples of this are xylenols such as 2,3-xylenol, dialkylmethanilic acids such as diethylmethanilic acid and their salts, N-alkyl- (3'sulfobenzyl) anilines, in particular the N-ethyl compound, N-alkyl N- (3'-sulfobenzyl) -3- toluidine, especially the methyl, ethyl and propyl compound and N-biscarboxybenzyl-3-anisidine in the form of the salts, especially the sodium salt.

Another object of the invention is a reagent for determining the proteases of the blood coagulation system, which is characterized in that it contains a compound according to formula 1, an oxidizing agent, an aniline or phenol derivative which forms a dye with phenylenediamine and buffer.

K ₃ Fe (CN) ₆ or peroxide / peroxidase are preferred as the oxidizing agent. In addition to the components necessary for the actual reaction, the reagents according to the invention may also contain: compound according to general formula I, coupling component, oxidizing agent and buffer substance pH 6 to 10, activators (e.g. magnesium salts), wetting agents, stabilizing agents, thickeners and other customary auxiliaries. The reagent can be present either in dried form, dissolved in an aqueous solvent, or impregnated on an absorbent carrier (test strip).

The following examples further illustrate the invention.

Example 1 to 15

0.2 mmol of the respective 4-nitroanilide, which corresponds to the 4-aminoanilides given below, are dissolved in methanol / glacial acetic acid, mixed with 10% paladium / carbon (10%) and hydrogenated at room temperature in the shaker until the reaction is complete ( DC control). It is suctioned off through a Seitz filter, water is added and the methanol is distilled off in vacuo. The aqueous residue is freeze-dried. All R _F values are determined on finished plates from Merck, Kieselgel 60 F ₂₅₄ with the eluent: n-butanol-glacial acetic acid-water / 50-15-25 (development: Cl ₂ / toluidine). The following connections are made in this way:

1 N-tosyl-glycyl-L-prolyl-L-arginyl-4-aminoanilide (xAcOH) R _F = 0, [alpha = -49 ° C (c = 0.5 g in 100 ml MeOH).

2

N-succinyl-L-alanyl-L-prolyl-L-phenylalanyl-4-aminoanilide (hydrogenation, without addition of HOAc)

R _F = 0.49

[alpha] = -90 ° C (c = 0.3 g in 100 ml MeOH).

3 N-tosyl-glycyl-L-prolyl-L-lysyl-4-aminoanilide

(xAcOH) R _F = 0.41 [alpha] = -52 ° (c = 0.3 g in 100 ml MeOH). N-benzoyl-L-prolyl-L-phenylalanyl-L-arginyl-4aminoanilide (xAcOH)

R _F = 0.48

[alpha = -61 ° (c = 0.3 g in 100 ml MeOH).

N-Mesyl-D-norleucyl-glycyl-L-arginyl-4-aminoanilide (xAcOH) R _F = 0.39

[alpha] = -9 ° (c = 0.5 g in 98 ml MeOH + 2 ml HOAc).

N-methoxycarbonyl-D-norleucyl-glycyl-L-arginyl-4aminoanilide (AcOH)

R _F = 0.40

[alpha] = -15.8 ° (c = 0.5 g in 98 ml MeOH + 2 ml

HOAc).

H-D-isoleucyl-L-prolyl-L-arginyl-4-aminoanilide dihydrochloride

R _F = 0.14

[alpha] = -41.9 ° (c = 1 g in 100 ml water)

H-D-Valyl-L-leucyl-L-arginyl-4-aminoanilide dihydroxychloride

R _F = 0.32

N-Acetyl-L-isoleucyl-L-glutamyl-glycyl-L-arginyl4-aminoanilide hydrochloride

R _F = 0.34

H-D-phenylalanyl-L-pipecolyl-L-arginyl-4-aminoanilide dihydrochloride

R _F = 0.33 11 HD-Valyl-L-leucyl-L-lysyl-4-aminoanilide dihydrochloride R _F = 0.35

12 N-Benzoyl-L-isoleucyl-L-gamma-piperidylglutamylglycyl-L-arginyl-4-aminoanilide (xAcOH) R _F = 0.42

13 H-D-but-cyclohexylalanyl-lysyl-4-aminoanilide (xAcOH)

14 succinyl-alanyl-alanyl-prolyl-phenylalanyl- (3carboxy-4-amino) -anilide

15 Succinyl-alanyl-alanyl-prolyl-phenylalanyl- (3halogeno-4-amino) anilide

Example 16

Carrying out a quick test on the substrate Tos-GlyPro-Arg-p-phenylenediamine (compound from Example 1)

Making the necessary solutions:

Solution 1: 10 ml Tris-HCl buffer, pH 8.1 are mixed with 4 ml thromboplastin (Boehringer Mannheim, Order No. 244252) and 1 ml substrate solution (6.83 mmol / l in water) and then 1:10 with a Tris buffer pH 8.1, which additionally contains 6 mmol / l CaCl ₂ and 0.25 mol / l urea. Solution 2:

N-methylanthranilic acid, Na salt 0.25 mol / l in water.

Solution 3:

Potassium hexacyanoferrate-III, K ₃ [Fe (CN ₆ )], 5 mmol / l.

Execution:

Photometer with filter 644 nm and recorder 10 cm / min, thermostatted to 37 ° C.

All solutions are preheated to 37 ° C.

Pipetting scheme: Mix 1 ml solution 1 0.1 ml solution 2 0.1 ml solution 3.

Mix 0.1 ml sample (citrated plasma).

The recorder is started at the same time as the sample is added.

You get a reaction curve, such as. B. is shown in European Patent Application 82 102 773.7. The time from the start of the reaction to reaching an extinction difference of 0.1 is a measure of the quick value of the sample, which can be expressed as a percentage of the norm using a reference curve. Example 17

Carrying out a PTT test (partial thromboplastin time) with Tos-Gly-Pro-Arg-p-phenylenediamine.

Making the necessary solutions:

Solution 1:

1 ml of a PTT reagent, such as is used for optically registering coagulation machines (e.g. actin, Dade or Neothromtin, Behring), is mixed with Tris / HCl buffer 0.1 mol / 1, pH 7.6 Diluted 1:10.

Solution 2:

N-methyl-anthranilic acid, Na salt, 0.25 mol / 1 in water.

Solution 3:

Potassium hexacyanoferrate III (K ₃ Fe (CN) ₆ /, 0.005 mol / l in

Water.

Solution 4:

Tos-Gly-Pro-Arg-p-phenylenediamine and 0.35 mmol / l,

Ca acetate 0.044 mol / l in water.

All solutions are brought to 37 ° C and used as follows:

Photometer equipment and setting as in example 1 (quick test). The following solutions are mixed in a cuvette:

0.1 ml sample (citrated plasma) 1.0 ml solution 1 0.1 ml solution 2 incubation 5 min 37 ° C 0.1 ml solution 3 0.1 ml solution 4

With the addition of solution 4, the recorder is started at the same time. The type of reaction curve is the same as in Example 1. The reaction time is the time from the start (addition of solution 4) to reaching an extinction difference of 0.1.

A search test based on this test principle to determine and determine an inherited deficiency in coagulation factor VIII (hamophilia A) is described in Example 3 below:

Example 18

A reference curve for determining factor VIII is created as follows:

Component 1:

Citrate plasma from 10 normal donors is pooled and represents a factor VIII content of 100% of the

Standard.

Component 2:

Citrate plasma from a donor with hereditary hamophilia A with a residual activity of less than 1% of the norm (e.g. available from George King Biomedical, Overland Park, Kansas, USA). Component 1 and 2 are mixed so that factor VIII activities of 100% (pure component 1), 50 (component 1 and 2 1 + 1 mixed), 25, 10 and 5% of the norm arise. A PTT test analogous to Example 2 is carried out on these samples. The reaction times listed in Table 1 are obtained. If the reaction times are plotted against the% factor VIII activity, a reference curve for factor VIII is obtained, from which the factor VIII content of this patient sample can be determined over the reaction time of a patient sample.

Example 19

N-benzyloxycarbonyl-glycyl-prolyl- (εBoc) lysine

6.0 g of Cbo-Gly-Pro-hydroxysuccinimide ester in 100 ml of acetonitrile are added to 4.9 g of N _ε -Boc-lysine and 3.7 g of NaHCO ₃ in 100 ml of H ₂ O, and the mixture is stirred overnight at room temperature further.

The acetonitrile is distilled off in vacuo and the aqueous residue is acidified to pH 2 with 2N HCl. The precipitate is filtered off and recrystallized twice from 20% methanol. Yield: 7.3 g. Elemental analysis: C = 58.23% (ex _. 58.41%), H = 7.12%

(H _calc. 7.16%), N = 10.68% (N _calc. 10.48%).

Example 20

N, N-dimethyl-2-bromo-4-nitro-aniline

5 g of 2-bromo-4-nitro-aniline are dissolved in 100 ml of DMF and 100 ml of acetonitrile, mixed with 21.5 ml of formalin and 4.5 g of NaBH ₃ CN and stirred for 48 hours at room temperature. Any precipitate that has formed is filtered off. 500 ml of H ₂ O are added and the precipitate formed is filtered off with suction. Yield: 2.5 g. Elemental analysis: C = 39.08% (C _b. 39.21%), H = 3.63%

(H _calc. 3.70%), N = 11.55% (N _calc. 11.43%),

Br = 32.37% (Br _calc. 32.60%). Example 21

N, N-dimethyl-2-methyl-4-nitro-aniline

5 g of 2-methyl-4-nitro-aniline are methylated analogously to the example above.

Yield: 3.2 g

Elemental analysis: C = 59.75% (C _over 59.99%), H = 6.65%

(H _calc. 6.71%), N = 15.74% (N _calc. 15.55%).

Example 22

N, N-dimethyl-2-carboxy-4-nitro-aniline

5 g of 2-carboxy-4-nitro-aniline are methylated analogously to the example above.

Yield: 3.2 g

Elemental analysis: C = 51.22% (C calc _. 51.43%), H = 4.76%

(H _calc. 4.80%), N = 13.53% (N _calc. 13.33%)

Example 23

N-benzyloxycarbonyl-glycyl-prolyl-lysine-4-dimethylamino3-bromo-anilidex2CF ₃ COOH

Solution A: 1 g of N, N-dimethyl-2-bromo-4-nitro-aniline is dissolved in 120 ml of DMF, mixed with 0.1 g of Pd / C and hydrogenated until the calculated amount of hydrogen is absorbed in the shaker. The catalyst is then filtered off under nitrogen. Solution B: 2.2 g of Cbo-gly-pro- (ε-Boc) -lys are dissolved in 50 ml of DMF, treated with 1.3 ml of triethylamine and cooled to -20 ° C. 0.7 ml of xobutyl chloroformate are added and the mixture is stirred for 15 minutes.

Solution A is added dropwise to solution B within 30 minutes. The mixture is stirred for one hour at -20 ° C. and allowed to come to room temperature overnight. The reaction mixture is evaporated to dryness. The residue is taken up in 10 ml of CH ₂ Cl ₂ , the undissolved solution is filtered off, 8 ml of trifluoroacetic acid are added and the mixture is stirred at room temperature for 4 hours. It is evaporated to dryness, the residue is taken up in a little methanol and purified by chromatography on "Sephadex LH 20". Yield: 1.1 g elemental analysis: C = 45.93% (C _over 46.11%), H = 4.75%. (H _calc. 4.81%), N = 10.0% (N _calc. 9.78%)

Example 24

N-benzyloxycarbonyl-glycyl-prolyl-lysine-4-dimethylamino3-methyl-anilidex2CF ₃ COOH

0.75 g of N, N-dimethyl-2-methyl-4-nitro-aniline are reacted analogously to the example above.

Yield: 1.1 g

Elemental analysis: C = 51.15% (C _over 51.38%), H = 5.55%

(H _calc. 5.58%), N = 10.75% (N _over .10,58%) Example 25

N-benzyloxycarbonyl-glycyl-prolyl-lysine-4-dimethylamino -3-carboxy-anilidex2CF ₃ COOH

0.86 g of N, N-dimethyl-2-carboxy-4-nitro-aniline are reacted analogously to the example above.

Yield: 1.0 g

Elemental analysis: C = 49.5% (C _calc . 49.52%), H = 5.12%

(H _calc. 5.13%), N = 10.55% (N _calc. 10.19%).

Claims

Patent claims

1 . Compound of the general formula I

in the

A the amino acid arginine or lysine,

X is an N-terminal amino acid protecting group or a D-amino acid, Y is a single bond or one of 1 to 3

Chain consisting of amino acids, NR ₁ R _{2 is} a group in the o- or p-position with R ₁ and R ₂ independently of one another in each case hydrogen or alkyl having 1 to 3 C atoms and R _{3 is} a hydrogen atom, a carboxyl group, a halogen atom or one Alkyl group with 1 to 3 carbon atoms.

2. Compound according to claim 1 with R ₁ R ₂ = H.

3. A compound according to claim 1 or 2 with X = Tos and Y = Gly-Pro.

4. A compound according to claim 1 or 2 with X = H-D-Phe and Y = Pip.

5. A compound according to claim 1 or 2 with X = HD-Ile and Y = Pro.

6. A compound according to claim 1 or 2 with X = CH ₃ SO ₂ -D-Nle and Y = Gly.

7. A compound according to claim 1 or 2 with X = CH ₃ OCO-D-Nle and Y = Gly.

8. A process for the preparation of a compound according to claim 1 to 7, d a d u r c h g e k e n n z e i c h n e t that a compound of general formula II

in which X, Y, A and R _{3 have} the meaning given in claim 1, and the NH ₂ group formed is optionally alkylated.

9. The method according to claim 8, d a d u r c h g e k e n n z e i c h n e t that the compound of general formula II is catalytically hydrogenated.

10. Reagent for the determination of the proteases of the blood coagulation system, characterized in that it contains a compound according to claim 1, an oxidizing agent, an aniline or phenol derivative which forms a dye with phenylenediamine and buffer.

11. Reagent according to claim 10, characterized in that it contains K ₃ Fe (CN) ₆ or peroxide / peroxidase as the oxidizing agent.

12. Reagent according to claim 10 or 11, d a d u r c h g e k e n n e e c h n e t that it contains N-methylantranilic acid, dimethylantranilic acid, N-ethyl-N (3'-sulfobenzene) aniline or 2,3-xylenol as aniline or phenol derivative.

13. A reagent according to claim 10, 11 or 12, which also means that it is impregnated on an absorbent carrier.