CS196314B2 - Process for preparing new chromogenous substrates specific especially for thrombine - Google Patents

Abstract

To determine thrombin or enzymes which cleave the substrates in the same way as thrombin, the material in which the enzymes are to be determined is brought into contact with a novel substrate of the formula I <IMAGE> In the substrate, R2 is a chromogenic radical which is eliminated by enzymatic hydrolysis in the form of the corresponding amine which differs in its absorption spectrum from the substrate of the formula I so that quantitative determination of the enzyme present in the material is possible because the amount of amine which is formed is proportional to the amount of enzyme which is present. In the substrate, the substituents have the meaning stated in Claim 1, and the optionally substituted 1-amino-2-phenylpropionic acid in the peptide chain must be in the D form so that attack of aminopeptidases on the substrate, which would interfere with the determination, is prevented.

Description

The invention relates to a process for the production of novel chromogenic substrates for thrombin - and similar enzymes. The substrates produced by the process of the invention are particularly suitable for the quantitative determination of thrombin or for the investigation of reactions in which thrombin is produced, inhibited or consumed, or for determining factors influencing or participating in such reactions, e.g. antithrombin, prothrombin, and heparin.

Synthetic enzymatic substrates have great advantages over natural substrates, provided that certain conditions are met, such as good sensitivity and good specificity for the enzyme, solubility in the water or the biological liquid being tested and easy to detect some fission products.

One of the best substrates so far - for the determination of thrombin is described in Swedish Patent No. 380 257 and consists of a chromogenic tripeptide derivative of the formula A

Bz — Phe — Val — Arg — ipNA (S-2180)

..... (A).

The abbreviations of amino acid abbreviations and other abbreviations used will be described below.

This substrate has a high sensitivity to thrombin - and - in enzymatic hydrolysis - provides as a chromophoric product -p-nitroaniline, which can be readily determined spectrophotometrically. However, the S-2160 substrate has some limitations because of its relatively low solubility of 1 mg / ml. This low solubility has the drawback of having to work very close to the saturation limit for the substrate in order to achieve a sufficient substrate concentration. In these cases, the determination of the enzyme in the various biological systems may result in a precipitate of the substrate itself or a precipitate of the protein-substrate complex. These precipitates cause erroneous spectrophotometric readings and thus erroneous enzyme assays. Substrate S-2160 is substantially more soluble when a benzoyl group is replaced with a hydrogen atom to form compound B

H-Phe-Val-Arg-pNA (B).

The protonated amino group released on Phe in this way increases the solubility, but at the same time decreases the rate at which the thrombin substrate cleaves approximately 30 times. These facts are shown in Table I. In addition, there is a possibility that the substrate may be undesirably - decomposed - at the N-terminal end in the test biological solution by the action of aminopeptidases.

According to the process of the invention, the substrate of formula B is further modified so that Val is replaced by cyclic imino acid (Aze, Pro or Pip) and at the same time L-Phe is replaced by D-Phe. As expected, the substrates thus obtained are very soluble, but unexpectedly the activity to thrombin, which is usually 30 to 5 times higher than the activity for the corresponding substrate containing only L-amino acids as is, increases or at least does not decrease. also. in Table I. In addition, they are new. substrates · approx. 4Q0%. more sensitive than - S-2160. D-amino acids at the N-terminal end protect the substrate from undesirable degradation by aminopeptidases because these enzymes are specific for L-amino acids.

The object of the invention is, therefore, a process for the production of chromogenic substrates, particularly specific for. thrombin, - for diagnostic purposes with the general formula

where.

R 1 represents a hydrogen atom. . . or. -hydroxy-. group

R 2 is nitrophenyl, naphthyl, nitronaphthyl, methoxynaphthyl, quinolyl or nitroquinolyl; and is - an integer of 1, 2 or - 3, as well as pharmaceutically acceptable salts of these compounds, characterized in that the reaction of amino acids with a C-terminal arginyl residue is carried out which is optionally already provided with a hydrophobic group R2 which concurrently. protecting the carboxyl group, or having a C-terminal arginyl radical, provided with a leaving group ^{on the} carboxyl group, the protected tripeptide derivative, the chromophoric group R 2, and optionally - synthesizes an N-terminus dipeptide fragment which then binds. to an arginyl group, optionally having a chromophore Rz group, the product thus obtained is purified by gel filtration. and optionally. Ion exchange chromatography.

In the synthesis of novel chromogenic substrates, commonly used protecting groups and reaction methods can be used as they are used in chemistry. peptides. ;

Particular preference is given to a carboxybenzoxy or p-butyloxycarbonyl protecting group on a .alpha.-amino group. or a similar group of a related nature, such as p-methoxy, p-nitro or p-methoxyphenylazocarbobenzoxy.

As the protecting group for the α-guanide group of the arginil group, it is preferred. . for-. tonization, the NO 2 - or p-toluenesulfonyl group.

In order to protect the hydroxy group of the tyrosine residue, it is preferred. preferably t-butyl or benzyl. As the leaving group protecting the carboxyl group, the methyl, ethyl or benzyl ester is preferably used.

The reaction between two amino acids or between the dipeptide and the amino acid is performed by activation of the α-carboxyl group. The activated derivative is. isolated or formed directly in the reaction mixture. These include p-nitrophenyl ester, trichlorophenyl ester, or N-hydroxysuccinimide, symmetric or asymmetric anhydride, acid · .azid - or -N .. ⁱ h.ydroxybenzotriazolester.-. . · · '

The synthesis is carried out. so,. The process according to claim 1, characterized in that the reaction of the amino groups with a C-terminal-arginyl radical, which is optionally already present, is carried out gradually. - provided. chromophore group, which. at the same time. it protects the carboxyl group, or this end is provided with a cleavable protective group. group-. the - carboxyl group and the chromophore group - binds. a protected tripeptide derivative, or. is synthesized. - Non-terminal · dipeptide fragment. such . and this fragment then binds to an arginyl group or an already-provided chromophoric group. - as mentioned above.

Irrespective of the reaction method, the intermediate i. resulting - products clean - filtering on. gel, because this method of purification allows. fast execution - - reaction · and ensures maximum yields.

The eluates after gel filtration are analyzed by thin-layer chromatography. layer, the same. Partially evaporated or dried end products and intermediates can also be analyzed.

The invention will be illustrated by the following examples. ..... .....

The following table will list the used. · Abbreviations: amino acids, u. of which. goes-. . o - L196314

-forms, not-11 otherwise noted, and other abbreviations:. .

Abbreviations:

Arg = arginine

Aze .. = 2-azetidine carboxylic acid

Phe = phenylalanine

Plp - plpecollonic acid

Pro = proline

Val valin

AcOH = acetic acid

Bz '= benzoyl

Cbo- = carbobenzoxy

DMF = dimethylformamide

Et 3 N = triethylamine

EtOAc = ethyl acetate

HMPTA = N, N, N ‘, N‘, N,, N--hexamethylphosphoric triamide

GPC = gel filtration

MeOH-methanol

-QpNP = p-nitrophenoxy

-pNA = p-nitronilide

TLC = thin layer chromatography.

Thin-layer chromatography;

For this type of chromatography, previously prepared plain silica gel F144 plates (Merck) as absorbent material were used.

• PI: chloroform: MeOH 9: 1 (volume ratio)

A: n-butanol: AcOH: water 3: 2: 1 (volume ratio). ; in

After chromatography, the plate is scanned in ultraviolet light at 254 µm and then evolved sequentially with a reagent consisting of chlorine and o-toluidines in a conventional manner. When it is stated that the product was homogeneous in thin-layer chromatography, this means that the analysis was performed on a micro-frame amount. Rf values are results from separate chromatographic determinations.

The Sephadex ^(R) G-15 gel used for gel filtration is a dextran gel. with cross bridges. Sephadex gel ^{, R} -LH-20 is a hydroxy propyl dextran cross-linked gel. Sephadex ^(R) QAE-25 ion exchanger is a cross-linked dextran gel with a diethyl- (2-hydroxypropyl) aminoethyl functional group, all of which were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden.

Methods for synthesis

I. CbOA ^ Al’g. (NO 2) pNA '. í '\'

Dissolve 35.3 g (10 inmoles) of anhydrous Cbo-Arg (NO 2 - 4 - OH) in 200 ml of anhydrous, freshly distilled HMPTA at room temperature, then add 10.1 g (100 µmoles) with stirring, in the absence of water. Et 3 N and 24.6 g (150 mmol) of p-nitrophenyl isocyanate After a reaction time of 24 hours, the solution is poured into 2 liters of 2% sodium hydrogen carbonate solution with stirring and the precipitate formed is collected by filtration and washed with 2% hydrogen carbonate solution. The crude product was extracted with warm methanol, and the insoluble material was collected by filtration, and the filtrate was purified by column chromatography on Sephadex ^{, R >} LH-20 in methanol to elute the solvent. The yield was 29.8 g (63%).

Analysis: The product is homogeneous by thin layer chromatography using Pi (Rf: 0.34). ·· '··:' [ajp ²⁴ ; 20.5 ° (c - 1.9, DMF)

II. Cbo — Pro — Arg (NO2) —pNa

4.8 g (10 mmol) of Cbo-Arg (NO2) -pNa are suspended in 25 ml of anhydrous acetic acid, then 15 ml of 5.6 N HBr in acetic acid are added. After a reaction time of 50 minutes at room temperature, the solution is poured into 300 ml of anhydrous solution with vigorous stirring. étheru. The ether phase is filtered off with suction and the precipitate obtained is washed twice with 100 ml of ether. The thus obtained HBr. H -Arg (NOr) -rpNa is dried under vacuum on NaOH at 43 ° C for 16 hours, then dissolved in 25 ml of dimethylformamide and the resulting solution is cooled to -10 ° C. Triethylamine is then added in an amount sufficient to keep the indicator paper at a pH just above the surface still moistened and the reaction to be slightly alkaline. This amount is usually 1.9 ml. The triethylamine-hydrogen bromide complex precipitate was collected by filtration and 4.1 g (11 mmol) of Cbo-Pro-10pNP were added. After a further hour, triethylamine (0.7 ml) was added and the same amount of triethylamine was added after a further 4 hours. The solution was then not allowed to warm to room temperature overnight. 0.5 ml (5 mmol) of n-butylamine was added to the system to prevent excess Cbo-Pro-OPNP from contaminating the resulting product during gel filtration with a similar elution volume. After 30 minutes, 10 mmol of dilute hydrochloric acid solution are added, the reaction mixture is evaporated on a rotary evaporator, then mixed with twice the amount of water and then the water is poured off. The residue was dissolved in methanol and chromatographed on a column of Sephadex ^(R) LH-20, swollen in methanol and eluting with the solvent ^. methanol. The fumed product was homogeneous in thin layer chromatography.

Yield: 5.5 g (96%).

Analysis: thin layer chromatography using Pi (Rf = 0.28).

[α] _D 24 -330 (c = 1.0, DMF).

III. Cbo — Pip — Arg (NO2) —pNA

The method is carried out in a similar manner to method II. ......

Yield: 5.1 g (86%).

Analysis: The product was homogeneous by thin layer chromatography in Pi (Rf: 0.30). [α] _D 24 -28.2 ° (c = 1.0, DMF).

IV. Cbo-Phe-Pip-Arg (NO2) -pNA

2.9 g (5 mmol) of Cbo-Pip-Arg (NO2) -pNA were decarbobenzoxylated in HBr in acetic acid, precipitated and washed with ether and dehydrated as described in Reaction II. The HBr-H-Pip-Arg (NO2) -pNA is then dissolved in 15 ml of dimethylformamide. The solution is cooled to -10 ° C, slightly alkalized with 0.9 ml of triethylamine and filtered. Then 3.0 g (7.15 mmol) of Cbo-Phe-OpNP and 0.65 g of (5 mmol) of N-hydroxybenzotriazole as catalyst are added. After 1 hour an additional 0.35 ml of triethylamine was added and the same was repeated for another 4 hours. The reaction solution was allowed to warm to room temperature overnight. The solution is evaporated to dryness on a rotary evaporator. The residue was dissolved in ethyl acetate and treated with a 2% solution of sodium hydrogen carbonate in water and then evaporated. The residue is - dissolved in methanol and chromatographed on Sephadex ⁽ R) LH-20 in methanol, eluting with methanol. The product obtained - is homogeneous - in thin-layer chromatography.

Yield: 2.7 g (73%).

Analysis: thin layer chromatography using Pi (Rf: 0.35).

[Α] D 24 - 32.9 ° (c = 1.0, DMF).

V. Cbo-D-Phe-Plp-Arg (NO2) -pNa

The procedure is carried out in a similar manner to the reaction scheme - IV.

Yield: 2.6 g (70%).

Analysis: The product is homogeneous by thin-layer chromatography using Pi (Rf: 0.44).

[ _.alpha. ] _D @ 24 -38.4 DEG (c = 1.0, DMF).

VI. Cbo - Phe - Pro - Arg (NO 2) - pNA

The procedure is carried out in a similar manner to Reaction Scheme IV.

Yield: 2.9 g (80%). . Analysis: the product is homogeneous in thin-layer chromatography by using Pi (Rf: 0.38).

[Α] D 24 -39.2 ° (c = 1.0, DMF).

VII. Cbo-D-Phe-Pro-Arg (NO2) -pNA

The procedure is carried out in a similar manner to Reaction Scheme IV.

Yield: 3.1 g - (86%).

Analysis: the product is homogeneous in thin-layer chromatography using Pi (Rf: 0.46).

[]] _Ό 2ΐ_6β2 ° (c = 1.0, DMF).

VIII. H — D — Phe — Pro — Afg — pNA. 2HCl

175 mg (0.246 mmol) of Cbo-D-Phe-Pro-Arg (NO2) -pNA were deprotected by treatment with 5 ml of anhydrous hydrogen fluoride in the presence of 0.3 ml of anisole in a Sakaklbar apparatus designed for this purpose. for 60 minutes at 0 ° C. After the reaction has ended and all hydrogen fluoride has been distilled off, the crude product is purified in vacuo. two stages:

a) Filter through a Sephadex gel column ^{, R)} G-15 after swelling in 33% acetic acid in water, using the same medium to - dissolve the product i - to elute. The pure product is then lyophilized from aqueous acetic acid.

(b) - Sephadex ion exchange column ⁽ R &gt; QAE-25 in chloride form) was chromatographed on swelling in a 95: 5 methanol / water mixture and used to dissolve the product. - is then lyophilized from an aqueous solution of:

Yield: 120 mg (80%). .......

Analysis: - The product is 'homogeneous in thin-layer chromatography' using - A (Rf; 0.29). Chloride content 11.53% (theoretical amount -11.6%).

[α] _D 24 -122 ° - (c '- 0.5, 50% AcOH).

IX H — Phe — Pro — Arg — pNA. 2HCl

The reaction is carried out in a similar manner to Reaction Scheme VIII.

Yield: (71%).

Analysis: The product is homogeneous by thin layer chromatography using A (Rf: 0.22). The chloride content is '11.6%' (theoretical 11.6%).

[.alpha.] D ^{@ 22} = -73.0 DEG (c = 0.5, 50%, AcOH).

X. H — D — Phe — Plp — Arg — pNA. 2HCl

The reaction is carried out in a similar manner to Reaction Scheme VIII.

Yield: (721%).

Analysis: The product is homogeneous in thin layer chromatography using A - (Rf:

0.44). The chloride content is 11.4% - (theoretical amount 11.3%).

[forehead ²⁴ -109 ° (с - 0.4, 50% aq AcOH).

XI. H — Phe — Pip — Arg — pNA, 2ЦС1

The reaction is carried out in a similar manner to Reaction Scheme VIII,

Yield: (55%).

Analysis: The product is homogeneous by thin layer chromatography using A (Rf: 0.41). Chloride content 11,3% (theoretical quantity 11,3%).

[α] D ^{24 -} 80.3 ° (c = 0.5, 50% aqueous AcOH).

XII. Cbo — D — Tyr (OBzl) —Pip — Arg (NO2) —pNA

The reaction is carried out in a similar manner to Reaction Scheme IV.

Yield: 3.2 g (75%).

Analysis: the product is homogeneous in thin layer chromatography using PI (Rf: 0.50).

XIII. H — D — Tyr — Pip — Arg — pNA

The reaction is carried out in a similar manner to Reaction Scheme VIII.

Yield: (68%).

Analysis: The product is homogeneous in thin layer chromatography using Pi (Rf: 0.44). The chloride content is 10.8% (theoretical amount 11.1%).

[ _.alpha. ] _D @ ²⁴ -75.2 DEG (c = 0.5, 50% aqueous AcOH).

XIV. Cbo — Aze — Arg (NO2) —pNA

The reaction is carried out in a similar manner to Reaction Scheme II.

Yield: 4.2 g (75%).

Analysis: The product is homogeneous by thin layer chromatography using Pi (Rf: 0.27).

XV. Cbo-D-Phe-Aze-ArgfNO 2) -pNA

The reaction is carried out in a similar manner to Reaction Scheme XIV.

Yield: 2.4 g (69%).

Analysis: The product is homogeneous by thin layer chromatography using Pi (Rf: 0.47).

XVI. H — D — Phe — Aze — Arg — pNA. 2HCl

The reaction is carried out in a similar manner to Reaction Scheme VIII.

Yield: (71%).

Analysis: The product is homogeneous on chromo-thin-layer matographil using A (Rf: 0.21). The chloride content is 1.6% (theoretical amount 11.9%).

[α] _D ²⁴ -130 ° (c = 0.5, 50% AcOH).

XVII. Cbo — Arg (NO2) —SNA

7.2 g (20 mmol) of anhydrous Cbo-Arg (NO2) -OH are dissolved in 400 ml of tetrahydrofuran. 2.0 g (20 mmol) of triethylamine are added, and the solution is cooled to -10 DEG C. in the complete absence of water. A solution of 2.7 g (20 mmol) of isobutyl chloroformate in 20 ml of tetrahydrofuran was then added over 10 minutes to the cooled solution, and after another 10 minutes 344 g (20 mmol) of N-naphthylamine were added. The reaction mixture was allowed to warm to room temperature and was allowed to stand at this temperature for 24 hours. The reaction mixture is then evaporated to dryness in vacuo, washed 3 to 5 times with distilled water, 3 to 5 x 5% sodium hydrogen carbonate solution and again 3 to 5 times with distilled water, then dried under vacuum. The product is dissolved in methanol and chromatographed on a Sephadex ^{, R)} LH-20 column in methanol, also eluting with methanol. The product obtained is homogeneous in thin layer chromatography.

Yield: 8.1 g (84%).

Analysis: The product is homogeneous in thin layer chromatography 11 using Pt (Rf: 0.40).

[α] _D ²³ + 7.4 ° (c = 1.0, DMF).

XVIII. Cbo — Pip — Arg (NO2) - / JNA

The reaction is carried out in a manner analogous to Reaction Scheme II.

Yield: 4.8 g (82%).

Analysis: The product is homogeneous by thin layer chromatography using Pi (Rf: 0.36).

XIX. Cbo — D — Phe — Pip — Arg (NO2) - /? NA

The reaction is carried out in a similar manner to Reaction Scheme IV.

Yield: 2.6 g (71%).

Analysis: The product is homogeneous by thin layer chromatography in Pi (Rf: 0.48).

XX. H — D — Phe — Pip — Arg — βΝΑ. 2HCl

The reaction is carried out in a similar manner as in Reaction Scheme VIII.

Yield: (68%).

Analysis: The product was homogeneous by thin layer chromatography using Pi (Rf: 0.44). The chloride content is 11.2% (theoretical amount 11.3%).

190314

12 [α] η ²⁴ -105 ° (c = 0.5, 50% aqueous AcOH).

XXI. Cbo-Arg (NOaJ-NA (4-OMe))

The reaction is carried out in a similar manner to Reaction Scheme XVII.

Yield: 7.1 g (70%).

Analysis: The product is homogeneous by thin layer chromatography using Pi (Rf: 0.42).

XXII. Cbo—. Pip — Arg (NO2) - (SNA (4-OMe))

The reaction is carried out in a manner analogous to Reaction Scheme II. ___

--- Уу.ЧУ '';

Yield: 4.9 g (79%). '

Analysis: The product is homogeneous by thin-layer chromatography using -Pi (Rf: 0.38).

XXIII. CbO — D — Phe — Pip — Arg [NO2] - / PNA. 2HCl

The reaction is carried out in a similar manner to Reaction Scheme IV.

Yield: 2.7 g (70%).

Analysis: The product is homogeneous by thin layer chromatography using Pi (Rf: - 0.51).

XXIV. H-D-Phe-Pip-Arg-3NA (4-OMe). 2HCl

The reaction is carried out in a similar manner as in Reaction Scheme VIII.

Yield: (64%).

Analysis: The product is homogeneous by thin layer chromatography using A (Rf: - 0.44). The chloride content is 10.4% (theoretical 10.7%).

[α] 21 D -2 ° -102 ° (c = 0.5, 50% aqueous AcOH).

Determination of thrombin by chromogenic substrates

Substrates, prepared by the method of each example, are used to determine thrombin as follows;

The determination is based on the fact that the same products resulting from enzymatic hydrolysis have a spectrum completely different from that of the substrate in ultraviolet light. For example, the substrate of Example X, H-D-Phe-Pip-Arg-pNA, has an absorption maximum at 315 nm and a molar extinction coefficient of 1250 °. At 405 nm substrate absorption φ is zero. In contrast, p-nitroaniline, which is cleaved from the substrate, has an absorption maximum at 380 nm and a molar extinction o-keftcient of 13-200 at enzymatic hydrolysis, and - this coefficient - already falls at 9620 at 405 nm. by determination at 405 nm, the amount of p-nitroaniline formed is readily determined, this amount being proportional to the degree of enzymatic hydrolysis. Again, this step is directly proportional to the effective amount of thrombin. Similar conditions exist for the other substrates produced by the process of the invention, so that the determination was always performed at 405 nm.

In the following Table I the relative reaction rates of the prior art substrate S-2160, its non-benzoylated form and the substrates produced by the process of the invention are compared. The advantages of substrates according to the invention, which react 4 times faster with thrombin than the best known substrate S-2160, are shown in the table, and are 10 times more soluble in water than this substrate. 0.4 NIH / ml thrombin and 0.1 μmol / ml substrate were used in the reaction.

'TABLE I

Substrate Relative reaction rate Water solubility (mg / ml)

AND [S-2160] Bz — Phe — Vol — Arg — pNA 100 ALIGN! 0.1 (B) H — Phe — Val — Arg — pNA 3 1 IX H — Phe — Pro — Arg — pNA 15 Dec 1 VIII H — D — Phe — Pro — Arg — pNA 400 3 XI 1 H-Phe-Pip-Arg-pNA 9 1 X H — D — Phe — Pip — Arg — pNA 420 3

Claims (1)

OBJECT OF INVENTION A process for the production of novel chromogenic substrates, in particular specific for thrombin, for diagnostic purposes of the general formula wherein R1 is hydrogen or hydroxy; R2 is nitrophenyl, naphthyl, nitronaphthyl, methoxynaphthyl, chiholyl or nitroquinolyl; n is an integer of 1, 2 or 3; optionally having a chromophore group R @ 2 which at the same time protects the carboxyl group or with a C - terminal arginyl residue,. fitted with a removable protective device. group on the carboxyl group, then the protected tripeptide derivative. the chromophoric group R2 binds, optionally synthesizes an N-terminal dipeptide fragment, which then binds to an arginyl group, optionally already provided with the chromophoric group R2, whereupon the product thus obtained is purified by gel filtration and optionally further. ion exchange chromatography.