DK145799B - TRIPEPTIDES OR SALTS THEREOF USED AS DIAGNOSTIC CHROMOGENT SUBSTRATE WITH HIGH SPECIFICITY ABOVE THROMBIN AND THROMBIN SIMILAR ENZYMES - Google Patents

Description

(19) DENMARK (J

||| 02) PUBLICATION MANUAL OR 145799 B

DIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Application No. 3126/76 (51) Int.CI.3 C 07 C 103/52 (22) Filing day 9 · Jul. 1976 C12Q1 / 56 (24) Race day 9 Jul. 1976 (41) Aim. available Jan 12 I977 (44) Submitted Mar 7 1983 (86) International application # - (86) International filing day - (85) Continuation day - (62) Master application no -

(30) Priority 11 Jul. 1975 * 7507975, SE

(71) Applicant SHARE COMPANY KABI, S-104 25 Stockholm, SE.

(72) Inventor Bo Thuresson of Ekenstam, SE: Leif Erik Aurell, SE:

Karl Goeran Claeson, SE: Birgitta Gunilla Karlsson, SE.

(74) Associate Engineer Lehmann & Ree.

(54) Tripeptides or salts thereof for use as a high specificity diagnostic chromium substrate for thrombin and thrombin-like enzymes.

The present invention relates to tripeptides or salts thereof for use as a high specificity diagnostic chromogenic substrate for thrombin and thrombin-like enzymes.

The tripeptides of the invention are particularly useful for the quantitative determination of thrombin or for studying the reaction.

ISLAND ISLAND

tions in which thrombin is formed, inhibited or degraded, or for T) studies of factors that influence or participate in such reactions, e.g. for the study of antithrombin, pro- thrombin or heparin.

f-

Synthetic substrates for enzyme studies have great Mi advantages over the natural ones, provided they meet certain conditions, such as high sensitivity and specificity to the enzyme, good solubility in water or in the biological test solution, and easy detectability of some of the cleavage products.

One of the best substrates for thrombin study to date is described in Swedish Patent Specification No. 380,257 and consists of the chromogenic tripeptide derivative

Bz-Phe-Val-Arg-pNA (S-2160) A

(for abbreviations: see page 4-5).

This compound has high sensitivity to thrombin and, by enzymatic hydrolysis, gives the chromophore product p-nitroaniline, which can be readily determined spectrophotometrically. However, S-2160 has a limitation due to its relatively low solubility (1 mg / ml).

A low solubility has the disadvantage of having to work very close to the saturation concentration of the substrate to obtain a sufficient substrate concentration. By enzyme determination in various biological systems, a precipitation of the substrate or a combined protein / substrate precipitation may occur. The precipitates mentioned will cause misleading spectrophotometric measurements and thus erroneous enzyme determinations. The enzyme substrate S-2160 becomes significantly more soluble if the benzoyl group is replaced by H:

H-Phe-Val-Arg-pNA B

The now free protonated amino group on Phe increases solubility, but at the same time causes the rate at which thrombin cleaves the substrate to decrease sharply (about 30 times, see Table 1). Furthermore, the substrate in the biological test solution may be undesirably degraded by aminopeptidases from the N-terminal end.

Swedish Patent Specification No. 380,257 also discloses thrombo-substrates of the formulas Bz-D-Phe-Val-Arg-pNA and H-D-Phe-Val-Arg-pNA. The former is also known from Thrombosis Research, Vol. (1972) 267-278, but is stated herein to have a very slow rate of hydrolysis to thrombin.

The tripeptides of the invention are peculiar in that they have the general formula: D-NH 2 - CH - CO - N - CH - CO - NH - CH - CO - NH - R 2 CH 2 CH 2 - ('cH 2) n (CH 2) Wherein R is hydrogen or hydroxy, R is nitrophenyl, naphthyl or methoxynaphthyl, and n is 1, 2 or 3.

With the compounds of the present invention, substrates are obtained which, like the substrate of formula B, are highly soluble, but quite surprisingly, the activity against thrombin is not diminished, but instead 30-50 times higher than the activity of the corresponding substrate with exclusively L-amino acids (Table 1). Furthermore, the N-terminal D-amino acid prevents unwanted attack of aminopeptidases since the latter are specific to L-amino acids. Finally, the new substrates are about $ 400 more active than S-2160 and HD-Phe-Val-Arg pNA. and have a smaller Km value so that they bind better to the thrombin enzyme, which is why a smaller amount of substrate is required to obtain optimal reaction conditions. K is the so-called Michaelis constant, which is the substrate concentration at which the enzyme-catalyzed reaction rate is equal to half the maximum reaction rate under all other equal reaction conditions. In addition to the substrate concentration, optimal reaction conditions also depend on such conditions as the pH of the reaction environment, ionic strength and buffer composition, as is also described in widely available textbooks in enzyme kinetics.

The substrate according to the invention differs from the substrate known from Swedish Patent No. 380,257 HD-Phe-Val-Arg-pNA in that Val is replaced by one of the cyclic imino acids Aze, Pro or Pip, and this gives the substrate of the invention far exceeds sensitivity to thrombin (cf. Table 2).

For the synthesis of the novel tripeptides, protecting groups and coupling methods are commonly used, all of which are known from the peptide chemistry.

As the α-amino protecting group, carbobenzoxy or t-butyloxycarbonyl groups can be advantageously used or any related group such as e.g. p-methoxy-, p-nitro- or p-methoxyphenylazocarbobenzoxy.

il 145799

To protect the δ-guanido group of the arginyl group, it is advantageous to use protonization, an NC 2 group or a p-toluenesulfonyl group.

To protect the hydroxy group in the tyrosine group, it is advantageous to use a t-butyl or benzyl group. As a leaving carboxy protecting group, it is advisable to use methyl, ethyl or benzyl ester.

The coupling between two amino acids or a dipeptide and an amino acid is achieved through activation of the α-carboxy group. The activated derivative may be either isolated or formed in situ and may e.g. be p-nitrophenyl, trichlorophenyl, pentachlorophenyl or other N-hydroxyacetic acid imide ester, symmetric or asymmetric anhydride, acid azide or N-hydroxybenzotriazole ester.

The principle of the synthesis may be a stepwise coupling of the amino acids to the C-terminal arginyl group which is either already provided with a coupled chromophore group having properties as a carboxy protecting group or provided with a leaving carboxy protecting group, and the chromophore group is then coupled to it. protected tripeptide derivatives; alternatively, it is possible to synthesize the N-terminal dipeptide fragment which is then coupled to the arginyl group with or without a chromophore group as described above.

Regardless of the method chosen, a purification of the intermediate and end products by gel filtration chromatography is appropriate, as this method enables rapid synthesis work and provides maximum yields.

TLC analyzes have been performed partly on eluates from GPC and partly on evaporated dried end and intermediate products.

The invention will be described in more detail in the following non-limiting specific examples.

abbreviations

Amino acids: (unless otherwise mentioned is meant the L-form)

Tyr = Tyrosine

Arg = arginine

Aze = 2-Acetidine carboxylic acid

Phe = Phenylalanine

Pip = Pipecolinic acid

Pro = Prolin

Selected = Selected

AcOK = Acetic Acid

Bz = Benzoyl

Cbo- = Carbobenzoxy- DMF = Dimethylformamide

EtgN = Triethylamine 145799

EtOAc = Ethyl acetate HMPTA = N, N, N ,, N ,, N ", N" -hexamethylphosphoric acid amide GPC = Gel filtration chromatography

MeOH = Methanol -OpNP = p-nitrophenoxy -pNA = p-nitroanilide SNA = S-naphthylamide SNA (4-OMe) = 4-methoxy-S-naphthylamide TLC = Thin Layer Chromatography.

Thin layer chromatography;

For the TLC analysis, glass plates prepared with silica gel * 254 (Merck) are used as absorbent. The solution systems used are:

Px = chloroform: MeOH, 9: 1 (volume ratio) A = n-butanol: AcOH water, 3: 2: 1 (volume ratio)

After chromatography, the plates are examined under UV light (254 nm) and then developed with chloro-o-toluidine reagent according to normal practice. When "homogeneous product according to TLC" is obtained, the analysis is carried out in an amount of the order of µg. The indicated R 2 values are the results of separate chromatographs.

The Sephadex® G-15 used for gel filtration is a cross-linked dextrangel. Sephadex® LH-20 is a hydroxypropylated cross-linked dextrangel. The ion exchange moiety Sephadex® QAE-25 is a cross-linked dextran with diethyl- (2-hydroxy-propyl) amino-ethyl as a functional group.

These gels are from Pharmacia Fine Chemicals, Uppsala, Sweden. Description of the syntheses I. Cbo-Arg (NO ^). -pNA

Dissolve 35.3 g (10 mmol) of dry Cbo-Arg (NO2) -OH in 200 ml of dry freshly distilled HMPTA at room temperature, after which 10.1 g (100 mmol)

EtgN and 24.6 g (150 mmol) of p-nitrophenyl isocyanate are added under stirring and under anhydrous conditions. After a reaction time of 24 hours, the solution is poured into 2 1 2% sodium bicarbonate solution with stirring. The precipitate formed is removed by filtration and washed with 2% bicarbonate solution, water, 0.5 N HCl (aqueous) and water and finally dried. The crude product is extracted with hot MeOH, and heavily soluble by-products are filtered off. The filtrate is purified by chromatography on a column of Sephadex's LD-20 which is swollen and eluted with MeOH. Yield: 29.8 g (63%)

Analyzes: Homogeneous by TLC in P 1 (R 2: 0.34) 24 n [a) D + 20.5 (c 1.9 DMF) 6 145799

II. Cbo-Pro-Arg (NC ^) - pNA

4.8 g (10 mmol) of Cbo-Arg (NO2) pNA is suspended in 25 ml of dry AcOH and then 15 ml of 5.6 N HBr in AcOH is added. After a reaction time of 50 minutes at room temperature, the solution is poured into 300 ml of dry ether with vigorous stirring. The ether phase is sucked from the formed precipitate, after which the precipitate is washed with two portions of ether of 100 ml. The HBr H-Arg (NO2) pNA thus formed is dried in vacuo over NaOH at 40 ° C for 16 hours. Then it is dissolved in 25 ml of DMF and the solution is cooled to -10 ° C. A ^ N is now added in an amount sufficient for a wetted pH paper held immediately above the surface of the solution to show weak basic reaction (1.9 ml). Precipitated Et 2 N HBr is removed by filtration and 4.1 g (11 mmol) of Cbo-Pro-OpNP is added. After 1 hour, an additional 0.7 ml of Et3N is added and also after 4 hours. The solution is left overnight to adjust to room temperature. To avoid excess Cbo-Pro-OpNP contaminating the final product during GPC, since they have comparable elution volumes in the chromatographic system used, 0.5 ml (5 mmol) of n-butylamine is added. After 30 minutes, 10 mmol of dilute HCl is added, the reaction solution is evaporated on a rotary evaporator, stirred with a few portions of water, which is removed by decantation. The residue is dissolved in MeOH and chromatographed on a column of Sephadex ® LH-20 which is swollen and eluted with MeOH. The product obtained is homogeneous according to TLC.

Yield: 5.5 g (96%)

Analyzes: TLC in P ^ (R ^: 0.28) [α] 44 -33.0 ° (c 1.0 DMF)

Ilf. Cbo-Pip-Arg (N02) -pNA

Performed as described in II.

Yield: 5.1 g (86%)

Analyzes: Homogeneous by TLC in P 1 (R 2: o, 30) [α] 4 -4 -26.2 ° (c 1.0 DMF)

IV. Cbo-D-Phe-Pip-Arg (N02) -pNA

2.9 g (5 mmol) of Cbo-Pip-Arg (NO2) pNA is decarbobenzoxylated in HBr in AcOH, precipitated and washed with ether and dried as described in II. HBr.H-Pip-Arg (NO2) pNA is then dissolved in 15 ml of DMF. Cool the solution to -10 ° C, make slightly basic with 0.9 ml of Et3N and filter. 3.0 g of 7 145793 (7.15 mmol) of Cbo-D-PheHDpNP are added and then 0.65 g (5 mmol) of N-hydroxybenzotriazole as catalyst. After 1 hour, an additional 0.35 ml of EtgN is added and the same is repeated after 4 hours. The reaction solution is left overnight to adjust to room temperature. The solution is evaporated to dryness in a rotary evaporator. The residue is dissolved in EtOAc and treated with 2% sodium bicarbonate solution and water, then evaporated. The residue is now dissolved in MeOH and chromatographed on Sephadex ^ LH-2o which is swollen and eluted with MeOH. The product obtained is homogeneous according to TLC.

Yield: 2.6 g (70%)

Analyzes: TLC in P ^ (R ^: 0.44) [ct] 4 4 -38.4 ° (c: 1.0 DMF)

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

Performed as described in IV.

Yield: 3.1 g (86%)

Analyzes: Homogeneous by TLC in P1 (R4: 0.46) [a] p4 -6.2 ° (c 1.0 DMF) VI. H-D-Phe-Pro-Arg-pNA 2HC1

From 175 mg (0.246 mmol) of Cbo-D-Phe-Pro-Arg (NO2) pNA, the protecting groups are removed by reaction with 5 ml of dry HF in the presence of 0.3 ml of anisole in an apparatus designed for this purpose according to Saka-kibara in 60 min. at 0 ° C. After completion of reaction and distillation of all HF, the crude product is purified and subjected to ion exchange in two steps: a) GPC on a column of Sephadex® G-15 swollen in 33% AcOH in water with the same medium used for solution and elution. The pure product is freeze-dried from AcOH (aqueous).

/ r \ b) Ion exchange on a column of Sephadex QAE-25 in chloride form swollen in MeOH: water (95: 5) with the same medium used for solution and elution. The pure product is freeze-dried from water.

Yield: 120 mg (80%)

Analyzes: Homogeneous by TLC in A (Rf: 0.29)

Chloride content 11.53% (theoretically 11.6%) 24 n [u] p -122 (c 0.5, 50% AcOH (aqueous)).

145799 8 VII.H-D-Phe-Pip-Arg-pNA.2HC1 Performed as described in VI.

Yield: Homogeneous according to TLC in A (R 2: 0.44)

Chloride content 11.4% (theoretically 11.3%) [a] p4 -109 ° (c 0.4, 50% AcOH (aqueous)).

VIII.Cbo-D-Tyr (Bz) -Pip-Arg (NO2) pNA Performed as described in IV Yield: 3.2 g (75%)

Assays: Homogeneous by TLC in P 1 (R 2: 0.50)

IX. H-D-Tyr-Pip-Arg-pNA

Performed as described in VI.

Yield: (68%)

Assays: Homogeneous by TLC in P 1 (R 2: 0.44)

Chloride content 10.8% (theoretical 11.1%) 24 o [a] D -75.2 (c 0.5, 50% AcOH (aqueous)).

X. Cbo-Aze-Arg (NO2) pNA

Performed as described in II.

Yield: 4.2 g (75%)

Analyzes: Homogeneous by TLC in P 1 (R f: 0.27)

. XI. Cbo-D-Phe-Aze-Arg (NO2) -pNA

Performed as described in IV.

Yield: 2.4 g (69%)

Analyzes: Homogeneous according to TLC in P1 (Rf: 0.47) XII. H-D-Phe-Aze-Arg-pNA.2HC1 Performed as described in VI.

9 1A579S

Yield: (71%)

Analyzes: Homogeneous according to TLC in A (Rf: 0.21)

Chloride content 11.6% (theoretically 11.9%) [a] p4 -130 ° (c 0.5, 50% AcOH) XIII. Cbo-Arg (NO2) -βΝΑ 7.2 g (20 mmol) of dry Cbo-Arg (NO2) -OH are dissolved in 400 ml of THF.

2.0 g (20 mmol) of Et ^ N is added and the solution is cooled to -10 ° C in completely anhydrous environment. Dissolve 2.7 g (20 mmol) of isobutyl chloroformate in 20 ml of THF and add to the cooled solution over 10 min, and after a further 10 min. 3.44 g (20 mmol) of β-naphthylamine is added. The reaction mixture is allowed to adjust to room temperature and left at room temperature for 24 hours. The reaction mixture is evaporated to dryness in vacuo, treated 3-5 times with distilled water, 3-5 times with a 5% sodium bicarbonate solution and again 3-5 times with distilled water, then dried in vacuo. The product is dissolved in MeOH and chromatographed on a column of Sephadex ® LD-20, swollen and eluted with MeOH.

The product obtained is homogeneous according to TLC in p Yield: 8.1 g (84%)

Assays: Homogeneous by TLC in P 1 (R 2: 0.40) [α] 33 + 7.4 ° (c 1.0 DMF)

XIV. Cbo-Pip-Arg (N02) -8NA

Performed as described in II.

Yield: 4.8 g (82%)

Analyzes: Homogeneous according to TLC in P1 (Rf: 0.36) XV. Cbo-D-Phe-Pip-Arg (NQ2) -βΝΑ

Performed as described in IV.

Yield: 2.6 g (71%)

Analyzes: Homogeneous by TLC in P 1 (R 2: 0.48).

10 14579S

XVI. H-D-Phe-Pip-Arg-SNA -2HC1

Done as described in VI ·

Yield: (68%)

Assays: Homogeneous by TLC in A (R 2: 0.44)

Chloride content 11.2% (theoretically 11.3%).

[α] D 4 -105 ° (c 0.5, 50% AcOH (aqueous)).

XVII. cbo-Arg (NO2) -βΝΑ (4-OMe)

Performed as described in XIII,

Yield: 7, lg (70%)

Analyzes: Homogeneous according to TLC in P1 (Rf: 0.42) XVIII. Cbo-Pip-Arg (NQ2) -βΝΑ (4-OMe)

Performed as described in II.

Yield: 4.9 g (79%)

Analyzes: Homogeneous according to TLC in ρχ (Rfi 0.38) XIX. Cbo-D-Phe-Pip-Arg (NQ0) -βΝΑ (4-OMe)

Performed as described in IV.

Yield: 2.7 g (70%)

Analysis: Homogeneous by TLC in P P (R (: 0.51) XX. H-D-Phe-Pip-Arg-βΝΑ (4-OMe) .2HC1

Done as described in VI ·

Yield: (64%)

Assays: Homogeneous by TLC in A (R 2: 0.44)

Chloride content 10.4% (theoretically 10.7%) [cc] +4 -102 ° (c 0.5, 50% AcOH (aqueous)).

11 145799

Determination of thrombin by chromogenic substrates:

The substrates synthesized in accordance with Examples VI and VII are used to study thrombin as described below.

The principle of the study is based on the fact that the cleavage product formed by the enzymatic hydrolysis has a UV spectrum which is substantially different from that of the substrate. Thus, the substrate of Example VII, H-D-Phe-Pip-Arg-pNA has an absorption maximum at 315 nm and a molar extinction coefficient of 12500. At 405 nm, the absorption of the substrate has almost completely ceased. p-Nitroaniline cleaved off the substrate during enzymatic hydrolysis has an absorption maximum at 380 nm and a molar extinction coefficient of 13200, which at 405 nm is only reduced to 9620. Thus, by spectrophotometric determination at 405 nm following the amount of p-nitroaniline formed which is proportional to the degree of enzymatic hydrolysis, which in turn is determined by the active amount of thrombin. For other substrates according to the invention, similar conditions exist, and because of this, the determinations have consistently been made at 405 nm.

Table 1 shows a comparison of the relative reaction rates of the previously mentioned substrate S-2160, its non-benzylated form, as well as modifications thereof containing cyclic imino acids and two of the substrates of the invention. This table clearly shows the advantages of the substrates according to the invention. They react four times faster with thrombin than the best substrate S-2160 to date and are also approx. 10 times more soluble in water than S-2160.

Table 1

Relative reaction rates between thrombin (0.4 NIH / ml) and substrate (0.1 µmol / ml).

Substrate. Relative reaction- Solubility in rate of reaction. water (mg / ml) A (S-2160) Bz-Phe-Val-Arg-pNA 100 0.1 B H-Phe-Val-Arg-pNa 3 1 C * H-Phe-Pro-Arg-pNA 15 1 VI HD-Phe-Pro-Arg-pNa 400 3 D * H-Phe-Pip-Arg-pNA 9 1 VII HD-Phe-Pip-Arg-pNA 420 3 sk

Compounds C and D were prepared analogously to the tripeptides of Examples VI and VII.

12 145799

The substrate of Example VI of this specification has also been tested in relation to the substrates known from Swedish Patent Specification No. 380,257, HD-Phe-Val-Arg-pNA (F) and Bz-D-Phe-Val-Arg pNA (E). The resulting test results are shown in Table 2 below.

Table 2

Thrombin / Relative K (M) Optimal substrate conductivity with concentration (2 x Km)

S-2160 100 1-10-4 0.2 mM

Bz-D-Phe-Val-Arg-pNA (E) 5? ? -4

H-D-Phe-Val-Arg-pNA (F) 95 2 * 10 0.4 mM

H-D-Phe-Pro-Arg pNA (VI) 400 1 * 10 "5 0.02 mM

It is evident from the above test results that the substrate of Example VI of the present invention is more than 400 # more sensitive than the most sensitive of the D-amino acid-containing compounds, substrate (F) known from Swedish Patent No. 380,257, and which is required to obtain optimal concentration only 1/10 of the substrate according to the invention relative to substrate S-2160 and 1/20 to substrate (F).