FI56525C - NYA KROMOGENA TROMBINSUBSTRAT - Google Patents

Description

[B] (11) ADVERTISEMENT CCCOC 1 J '' UTLÄGGNI NGSSKMFT 3Q3'5 ^ (45) Patent granted II Γ.2 1930 * ^ Sv3 Patent meddelat (51) Kv.lk. * / Lnt.CI. · Q Q? C 103/52 β 01 N 31/1 * FINLAND-FINLAND pi) hw ^ i »ic.i» u.-PK «itwei <ni« i 762010 (22) HakmnitpUvl — AmBkitlngadag O9.O7.76 (23) Alkupttv · —GUtlghatsdag 09.0j.j6 (41) Tullut publikitaksl - Bltvit offwttllf 12.01.77

Patents · ia,,,. „• (44) Date of publication of the publication. -

Patent- och registerstyrelaen 'AniBktn utl * | d och utl. »KrHtan published 31.10.79 (32) (33) (31) Pyy * 1 · ** / atuolkau * - Begird prlorltat H. 07. J 5

Sweden-Sweden (SE) 7507975 “6 (71) Aktiebolaget Kabi, S-lOH 25 Stockholm, Sweden-Svenge (SE) (72) Bo Thuresson af Ekenstam, Mölndal, Leif Erik Aurell, Särö,

Karl Göran Claeson, Särö, Birgitta Gunilla Karlsson, Mölndal,

The present invention relates to novel chromogenic substrates for thrombin and thrombin-like enzymes. formed, inhibited or consumed, or to determine factors that affect or participate in such reactions, for example, antithrombin, prothrombin, and heparin.

Substrates for the determination of enzymes have great advantages over natural substrates, provided that they meet certain conditions, such as high sensitivity and specificity. enzyme, good solubility in water or biological test fluid, and easy detection of some cleavage products.

One of the best growth media to date for the thrombin assay is described in Swedish Patent No. 380257 (corresponding to FI hak. 1299/73) and consists of a chromogenic tripeptide derivative (for page see page 4):

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

2 56525 This has a high sensitivity to thrombin and on enzymatic hydrolysis gives a chromophore p-nitroaniline product which can be easily determined spectrophotometrically. However, S-2160 has the disadvantage of its relatively low solubility (1 mg / ml).

The low solubility has the disadvantage of having to operate very close to the substrate saturation limit in order to achieve a satisfactory substrate concentration. In the determination of the enzyme in different biological systems, precipitation of the substrate as such or combined precipitation of protein / substrate may occur. Said precipitates can cause erroneous spectrophotometer readings and thus erroneous enzyme determinations. The enzyme substrate S-2160 becomes much more soluble if the benzoyl group is replaced by a hydrogen atom, i.e.

H-Phe-Val-Arg-pNA B

The now free proton-containing amino group in the Phe group improves solubility, but also causes the rate at which thrombin cleaves the substrate to decrease sharply, by about 30-fold (cf. Table I). Furthermore, aminopeptidases can cleave the growth substrate in the biological assay solution in an undesired manner from the N-chain terminus.

According to the present invention, the substrate of formula B is modified by exchanging Val for a cyclic imino acid (Aze, Pro or Pip) and L-Phe for the group D-Phe. As expected, the substrates thus obtained are still very soluble, but quite surprisingly the activity against thrombin has not decreased, but instead is 30-50 times higher than that of the corresponding substrate with L-amino acids alone (Table I). Furthermore, these new substrates are about 400% more active than S-2160. The D-amino acid at the N-terminus also blocks the undesired effect of aminopeptidases, as the latter are specific for L-amino acids. The novel chromogenic substrates according to the invention are characterized by the following general formula D-NH "- CH - CO - N - CH - CO - NH - CH - CO - NH - R ~ 2 I I I T 2 CH2 CH2- (CH2) n« JH2> 3

I NH

φ A

H2N NH

R1 3,56525 • or its salts, which may have a hydrogen atom or a hydroxy group.

R 2 is a nitrophenyl, naphthyl or methoxynaphthyl group and n may be 1, 2 or 3.

For the synthesis of substrates for these new chromogenic enzymes, conventional protecting groups and coupling methods are used, all of which are well known in peptide chemistry.

As the amino-protecting group, carbobenzoxy or t-butyloxycarbonyl groups can be preferably used, or any comparable group such as p-methoxy, p-nitro or p-methoxyphenylazocarbobenzoxy group.

It is preferable to use a protonation, an NC4 group or a p-toluenesulfonyl group to protect the tf-guanido group of the arginyl group.

It is preferable to use a t-butyl or benzyl group to protect the hydroxy group in the tyrosine group. As the cleavable carboxy protecting group, it is suitable to use a methyl, ethyl or benzyl ester.

The coupling between two amino acids or a dipeptide and an amino acid is achieved by activating the α-carboxy group. The activated derivative can be either isolated or developed in the mixture itself and can be, for example, p-nitrophenyl, trichlorophenyl, pentachlorophenyl or N-hydroxysuccinimide ester, symmetrical or asymmetric anhydride, acid azide or N-hydroxybenzotriazole ester.

The principle of the synthesis may be the gradual coupling of amino acids to a C-terminal arginyl group which is either already provided with a coupled chromophoric group acting as a carboxy group protecting group or a cleavable carboxy group protecting group and the chromophoric group is then coupled to a protected tripeptate or the dipeptide moiety as such, which is then coupled to the arginyl group with or without the lens of the chromophoric group as described above. The preparation of similar readily soluble chromophoric substrates is described in the above-mentioned Swedish patent, Finnish patent applications 1308/73 4,56525 and 753246 and FEBS Letters 11 (1970) Dec. 249-253 and Bull.Chem. Soc. Japan 46 (1977) No. 2,572-576.

Regardless of the principle chosen, purification of intermediates and final products by gel filtration chromatography is appropriate, as this allows for rapid synthetic work and gives maximum yields.

TLC analysis has been performed in part on eluates from GPC and in part on evaporated and dried end and intermediate products.

The invention is described in more detail in the following examples.

abbreviations

Amino acids unless otherwise indicated refer to the L-form Arg = Arginine

Aze = 2-azetidinecarboxylic acid

Phe = Phenylalanine

Pip = Pipecolic acid

Pro = Proline

Tyr = Tyrosine

Vai = Between me

AcOH = Acetic acid

Bz = Benzoyl-

Cbo- = Carbobenzoxy- DMF = Dimethylformamide

Et 2 N = Triethylamine

EtOAc = Ethyl acetate HMPTA = Ν, Ν, Ν ', N', N ", N" -hexamethylphosphoric triamide GPC = Gel filtration chromatography

MeOH = Methanol -OpNP = p-Nitrophenoxy- -pNA = p-Nitroanilide TLC = Thin layer chromatography £ NA = P-Naphthylamide 0ΝΑ (4-oME) = 4-Methoxy - ^ - naphthylamide

Thin layer chromatography For TLC analysis, prefabricated glass plates with silica gel F254 (Merck) as absorbent are used. The solvent systems used are as follows: 56525 5 P 2: chloroform: MeOH 9: 1 (v / v) A: n-butanol: AcOH: water 3: 2: 1 (v / v)

At the end of the chromatography, the plate is examined under UV light (254 nm) and development is then performed with chlorine / o-toluidine reagent according to general practice. When it is found that "the product is homogeneous by TLC analysis", the analysis is performed with the amount of substance. The R 1 values shown are the results of separate chromatographic procedures.

The gel used for gel filtration, Sephades® G-15, is a crosslinked dextran gel. Sephadei® LH-20 gel is a hydroxypropylated crosslinked dextran gel. The ion exchanger Sephade® ® QAE-25 used is a crosslinked dextran gel with a diethyl (2-hydroxypropyl) aminoethyl group as a functional group. These gels are manufactured by Pharmacia Fine Chemicals, Uppsala, Sweden.

The synthesis of the substrates of the invention is described in the following examples.

Example 1

H-D-Phe-Pro-Arg-pNA · 2 HCl I. Cbo -Arg (NC ^ JpNA

35.3 g (10 mmol) of dry Cbo-Arg (NO 2) -OH are dissolved in 200 ml of dry, freshly distilled HMPTA at room temperature, followed by the addition of 10.1 g (100 mmol) of Et 3 N and 24.6 g (150 mmol) of p-nitrophenyl isocyanate with stirring and under moisture-protected conditions. After a reaction time of 24 hours, the solution is poured into 2 liters of 2% sodium bicarbonate solution with stirring. The precipitate formed is filtered off and washed with 2% bicarbonate solution, water, 0.5 N aqueous HCl and water and finally dried. The crude product is extracted with warm MeOH and the sparingly soluble by-products are filtered off. The filtrate is purified by chromatography on a Sephades® LH-20 column expanded and eluted with MeOH.

Yield: 29.8 g (63%)

Analysis: Homogeneous by TLC analysis in P 2 solvent (R f = 0.34) / Vp 4 + 20 * 5 ° <c? 9 'DMF> 56525 IX. Cbo-Pro-Arg (ΝO 2) -ρΝΑ 4.8 g (10 mmol) of Cbo-Arg (NO 2) -pNA are slurried in 25 ml of dry AcOH, followed by the addition of 15 ml of 5,6-N HBr in AcOH solution. . 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 filtered off with suction and the precipitate is washed with two 100 ml portions of ether. The HBr * H-Arg- (NO 2) -pNA thus obtained is dried in vacuo over NaOH at 40 ° C for 16 hours. It is then dissolved in 25 ml of DMF and the solution is cooled to -10 ° C. A sufficient amount of Et.jN is now added to give a weakly basic reaction (1.9 ml) with a damp pH paper kept immediately above the surface of the solution. The precipitated Et 2 N-HBr is removed by filtration and 4.1 g (11 mmol) of Cbo-Pro-OpNP are added. After one and also four hours, an additional 0.7 ml of Et2N is added. The solution is allowed to settle at room temperature overnight. To prevent excess Cbo-Pro-OpNP from contaminating the final product during GPC because they have similar 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 are added, the reaction solution is evaporated on a rotary evaporator, mixed with a couple of portions of water, which is removed by decantation. The residue is dissolved in MeOH and chromatographed on a Sephade® LH-20 column, which is expanded and eluted with MeOH. The product obtained is homogeneous by TLC analysis. Yield: 5.5 g (96%)

Analysis: TLC in P 2 solvent (R f: 0.28) / k / 1A -33.0 ° (C 1.0, DMF) III. Cbo-Pip-Arg (NO 2) -pNA Carry out according to II Yield: 5.1 g (86%)

Analysis: Homogeneous by TLC analysis in P 2 solvent (R f: 0.30) - 26.2 ° (C 1.0, DMF)

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

2.9 g (5 mmol) of Cbo-Pip-Arg (NO 2) -pNA are dicarbobenzoxylated in HBr dissolved in AcOH and washed with ether and dried according to II. HBr-H-Pip-Arg (NO 2) -pNA is then dissolved in 15 ml of DMF. The solution is cooled to -10 ° C, made slightly basic with 0.9 ml of Et 2 O and filtered. 3.0 g (7.15 mmol) of Cbo-Phe-OpNP are added followed by a further 0.65 g (5 mmol) of N-hydroxybenzotriazole as catalyst. After one hour, 0.35 ml of Et 3 N and the same 7 are added

5652S

the procedure is repeated after 4 hours. The temperature of the reaction solution is allowed to rise to room temperature overnight. The solution is evaporated to dryness on a rotary evaporator. The residue is dissolved in EtOAc and treated with 2% sodium bicarbonate solution and water and then evaporated. The residue is now dissolved in MeOH and chromatographed on a Sephade® LH-20 gel which is swollen and eluted with MeOH. The product obtained is homogeneous by TLC analysis.

Yield: 2.7 g (73%}

Analysis: TLC in P 2 solvent (R f: 0.35) -32.9 ° (c 1.0, DMF) V. Cbo-D-Phe-Pip-Arg (NO 2) -pNA Carry out according to IV Yield: 2.6 g (70%)

Analysis: Homogeneous by TLC analysis in P 2 solvent (R f: 0.44) / * / 4 4 - 38.4 ° (c 1.0, DMF) VI. Cbo-Phe-Pro-Arg (NO 2) -pNA Carry out according to IV Yield: 2.9 g (80%)

Analysis: Homogeneous by TLC analysis in P 2 solvent (R f: 0.38) 1 * 7 - 39.2 ° (c 1.0, DMF) VII. Cbo-D-Phe-Pro-Arg (NO 2) -pNA Carry out according to IV Yield: 3.1 g (86%)

Analysis: Homogeneous, by TLC analysis in P 2 solvent (R f: 0.46) to 6.2 ° (c 1.0, DMF) VIII. HD-Phe-Pro-Arg-pNA · 2HCl 175 mg (0.246 mmol) of Cbo-D-Phe-Pro-Arg (NO 2) -pNA are deprotected by reacting with 5 ml of dry HF in the presence of 0.3 ml of anisole in a Sakakibara apparatus (Sakakibara et al. Bull.Chem.Soc. Japan 40 (9), 2164-67 (1967) for 60 minutes at 0 ° C. After completion of the reaction and when all the HF is distilled, the crude product is purified and treated with an ion exchanger in two steps: a) on a GPC Sephade® G-15 column expanded with a 33% aqueous solution of AcOH with the same medium as the dissolution and elution medium. The pure product is lyophilized from AcOH (aq).

8 5652b b) Ion exchange on a Sephadex "* QAE-25 column in chloride form, expanded in aqueous MeOH (95: 5) with the same medium as solvent and elution medium. The pure product is lyophilized from water.

Yield: 120 mg (80%)

Analysis: Homogeneous by TLC analysis in solvent A (Rf: 0.29) Chloride content 11.53% (theoretical 11.6%) / </ p4 - 122 ° (c 0.5, 50% AcOH (aq.))

Example 2 H-Phe-Pro-Arg-pNA · 2HCl is prepared for reference purposes as described in Example 1 / I-VIII Yield: (71%)

Analysis: Homogeneous by TLC analysis in solvent A (Rf: 0.22) Chloride content 11.0% (theoretically 11.6%) / δc-4-4-7.6 ° (c 0.5, 50% AcOH (aq. )).

Example 3 H-D-Phe-Pip-Arg-pNA · 2HCl

Prepared in a similar manner to that described in Example 1 / I-VIII. Yield: (72%)

Analysis: Homogeneous by TLC analysis in solvent A (Rf: 0.44) Chloride content 11.4% (theoretical 11.3%) / δc / p4 - 109 ° (c 0.4, 50% AcOH (aq.))

Example 4 H-Phe-P ip-Arg-pNA. 2HC1

Prepared for comparison purposes as described in Example 1 / I-VIII.

Yield: (55%)

Analysis: Homogeneous by TLC analysis in solvent A (Rf: 0.41) Chloride content 11.3% (theoretical 11.3%) / Sc7 £ 4 - 80.3 ° (c 0.5, 50% AcOH (aq. ))

Example 5 HD-Tyr-P ip-Arg-pNA · 2HCl I. Cbo-D-Tyr (OBz) -Pip-Arg (NO2) -pNA is prepared according to Example 1 / I-IV Yield: 3.2 g (75% )

Analysis: Homogeneous by TLC analysis in P 2 solvent (Rf: 0.50) 56525 9 II. H-D-Tyr-Pip-Arg-pNA · 2HCl is prepared according to Example 1 / VIII Yield: (68%)

Analysis: Homogeneous by TLC analysis in P 2 solvent (R 2: 0.44) Chloride content 10.8% (theoretical 11.1%) to 75.2 ° (c 0.5, 50% AcOH (aq)).

Example 6 H-D-Phe-Aze-Arg-pNA * 2HCl I. Cbo-Aze-Arg (NO2) -pNA is prepared according to Example 1 / II Yield: 4.2 g (75%)

Analysis: Homogeneous by TLC analysis in P 2 solvent (R 2: 0.27) II. Cbo-D-Phe-Aze-Arg (NO2) -pNA is prepared according to Example 1 / IV Yield: 2.4 g (69%)

Analysis: Homogeneous by TLC analysis in P 2 solvent (R f: 0.47) III. H-D-Phe-Aze-Arg-pNA · 2HCl is prepared according to Example 1 / VIII Yield: (71%)

Analysis: Homogeneous by TLC analysis in solvent A (Rf: 0.21)

Chloride content 11.6% (theoretically 11.9%) / <7 ^ 4 - 130 ° (c 0.5, 50% AcOH)

Example 7 H-D-Phe-Pip-Arg-ftNA * 2HCl

I. Cbo-Arg (NO 2) - £ NA

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.jN are added and the solution is cooled to -10 [deg.] C. under conditions that are completely protected from moisture.

2.7 g (20 mmol) of isobutyl chloroformate dissolved in 20 ml of THF are added to the cooled solution over 10 minutes and after another 10 minutes 344 g (20 mmol) of ε-naphthylamine are added. The reaction mixture is allowed to reach room temperature and left at this temperature for 24 hours. The reaction mixture is evaporated in vacuo 10

S6S2S

dry, treated 3-5 times with distilled water, 3-5 times with 5% sodium bicarbonate solution and again 3-5 times with distilled water, after which it is dried in vacuo. The product is dissolved in MeOH and chromatographed on a Sephades® LH-20 column which is expanded and eluted with MeOH. The product obtained is homogeneous according to TLC analysis in P 2 solvent.

Yield: 8.1 g (84%)

Analysis: Homogeneous by TLC analysis in P. solvent (Rf: 0.40) & Jq + 7.4 ° (C 1.0, DMF) II. Cbo-Pip-Arg (NO 2) -8NA is prepared according to Example 1 / II Yield: 4.8 g (82%)

Analysis: Homogeneous by TLC analysis in P 2 solvent (R 2: 0.36) III. Cbo-D-Phe-Pip-Arg (NO 2) -βΝΑ is prepared according to Example 1 / IV Yield: 2.6 g (71%)

Analysis: Homogeneous by TLC analysis in P 2 solvent (Rf: 0.48) IV. H-D-Phe-Pip-Arg-gNa * 2HCl is prepared according to Example 1 / VIII Yield: (68%)

Analysis: Homogeneous by TLC analysis in solvent A (Rf: 0.44) Chloride content 11.2% (theoretical 11.3%) to 105 ° (c 0.5, 50% AcOH (aq))

Example 8 HD-Phe-Pip-Arg-SNA (4-OMe) · 2HCl I. CbO-Arg (NO 2) -n $ NA (4-OMe) is prepared according to Example 7/1 Yield: 7.1 g (70% )

Analysis: Homogeneous by TLC analysis in P 2 solvent (Rf: 0.42) II. Cbo-Pip-Arg (NO 2) -? NA (4-OMe) is prepared according to Example 1 / II Yield: 4.9 g (79%)

Analysis: Homogeneous by TLC analysis in P 2 solvent (R f: 0.38) 56525 11 III. Cbo-D-Phe-Pip-Arg (NO2) -ONA (4-OMe) is prepared according to Example 1 / IV Yield: 2.7 g (70%)

Analysis: Homogeneous by TLC analysis in P 2 solvent (R 2: 0.51) IV. H-D-Phe-Pip-Arg-ONA (4-OMe) · 2HCl is prepared according to Example 1 / VIII Yield: (64%)

Analysis: Homogeneous by TLC analysis in solvent A (Rf: 0.44) Chloride content 10.4% (theoretically 10.7%) "102 ° (c 0.5, 50% Ac OH (aq.)).

Determination of Thrombin on Chromogenic Substrates The substrates prepared according to the examples are used for the determination of thrombin as follows.

The assay principle is based on the fact that the cleavage product formed by enzymatic hydrolysis has a UV spectrum that is substantially different from that of the substrate. Thus, the substrate HD-Phe-Pip-Arg-pNA of Example X has an absorption maximum at 315 nm and a molar extinction coefficient of 12500. At 405 nm the absorption of the substrate is almost non-existent, p-nitroaniline cleaved from the substrate at nm and a molar extinction coefficient of 13,200, which at 405 nm has dropped to 9620 only. Nearly identical conditions prevail for the other substrates of this invention and therefore the assays have been performed uniformly at 405 nm.

Table 1 shows a comparison of the relative reaction rates between the above-mentioned benzoylated form (B) of the above-mentioned thrombin substrate S-2160 (A) and the substrate according to the invention (Example 1 and 3) and the corresponding L forms (Example 2 and 4). This table clearly shows the superiority of the substrates of this invention.

They react 4 times faster with thrombin than the best previously known substrate S-2160 and are still about 10 times more soluble in water than S-2160.

Claims (2)

56525 12 Table 1 Substrate Relative Solubility _ reaction rate in water (Rog / ml) A (S-2160) Bz-Phe-Val-Arg-pNA 100 0.1 B H-Phe-Val-Arg-pNA 3 1 Eg 2 H -Phe-Pro-Arg-pNA 15 1 Example 1 HD-Phe-Pro-Arg-pNA 400 3 Example 4 H-Phe-Pip-Arg-pNA 9 1 Example 3 HD-Phe-Pip-Arg-pNA 420 3 Claim Novel diagnostically active chromogenic substrates with high specificity for thrombin and thrombin-like enzymes, characterized in that they have the general formula D-NH- - CH - CO - N - CH - CO - NH - CH - CO - NH - R " 2. III 2 CH2 CH2- (CH2) n (CH2) 3 (o) IV / H2N nh * i and its salts, wherein R1 is a hydrogen atom or a hydroxy group, R2 is a nitrophenyl, naphthyl or methoxynaphthyl group and n is 1, 2 or 3. These diagnostically active chromogenic substrates have a specificity for thrombin and a thrombin-binding enzyme, which can be used in combination with 2. I, 1 I 2 φ λ R1 and the same group, color R2 is a hydrogen atom or a hydroxy group, R2 is a nitrophenyl, naphthyl or methoxynaphthyl group and is 1, 2 or 3.