DK171845B1 - Tripeptide derivatives and a process for preparing them, and a process for determining serine proteases or components which can co-operate with serine proteases - Google Patents

Description

in DK 171845 B1

Technical area

The present invention relates to novel tripeptide derivatives useful for the determination of serine proteases. The new derivatives are particularly suitable for the determination of factor X. (EC3.4.21.6) or for studies of reactions in which FX_, FX are generated, inhibited or consumed, allowing other enzymes and / or inhibitors to be quantified, who participate in the reactions.

Background of the Invention

Factor X, is a key substance in the reaction cascade leading to d blood coagulation. Several methods based on the determination of FX with chromogenic substrates have been found to be valuable in the clinical diagnosis and explanation of disorders of the blood clotting system (Hemker HC - Handbook of Synthetic Substrates, 1983, Martinus Nijhoff Publishers, Boston) .

State of the art 20

Chromogenic tetrapeptide derivatives are described by Aurell, L., et al. (Haemostasis vol. 7, 1978, 92-94) and have been found to be of significant value in the present case. These substrates are based on the amino acid sequence (-Ile-Glu-Gly-Arg-) which precedes the cleavage sites in the natural substrate, prothrombin. The tetrapeptide substrates are characterized by high selectivity, i.e. that other enzymes, in particular thrombin, do not interfere with the determination of factor X.

α

However, the limited solubility and the many synthetic steps of the preparation present significant disadvantages. In the disclosure of EP 30 34,122, it is disclosed that tripeptide derivatives may also be useful as FX substrates. However, these tripeptides are also α sensitive to thrombin, and the high selectivity of tetrapeptides is not achieved with these tripeptides.

In EP 170,797, compounds which are different from the compounds of the present invention are described in that D-Arg is substituted with D-Lys. The inventive idea according to EP 170.797 is constituted by a new marker, namely 3-carboxy-4-hydroxyanilide, as the chromogenic group. The best compound among the substrates (PS 2000) described has a lower activity compared to S-2222. The substrates of the present invention exhibit a 6 to 13 times higher activity compared to S-2222 against FXa.

5

Description of the Invention

The Tripeptide Derivatives According to the present invention exhibit high sensitivity and solubility at the same time as selectivity properties comparable to those of the previously described tetrapeptides. The tripeptide derivatives of the invention are peculiar in that they have the general formula:

R 1 - X - D-Arg - A - Arg - NH - R 2 wherein R 2 represents hydrogen, an α- or β-naphthyl residue, a lower alkyl residue which may be substituted by a carboxyl group, an unsubstituted or substituted phenyl - or phenylalkyl radical, wherein the alkyl group has 1 to 4 carbon atoms, the substitution is preferably 20 in para position in the phenyl ring, and the substituents are lower alkyl, lower alkoxy, halogen or a nitro group; 0 0 0 NN «X represents -O-C-, -C-, -S- or a single bond on the condition that

N

0 when R 1 is hydrogen, then, and only then X is a single bond; 3q A represents Gly or Sar;

Rg represents an aromatic or heterocyclic residue which, together with the -NH group, by enzymatic hydrolysis of the tripeptide, gives the compound R2-NH2, which can be quantitatively determined by its chromogenic, fluorogenic or electrochemical properties; or di- and tri-salts of inorganic or organic acids thereof.

The compounds R2-NH2 are previously known compounds with chromogenic, fluorogenic or electrochemical properties which enable the quantification of serine proteases by determination of cleaved marker, directly or after derivatization (Hemker HC, loc. Cit. And the references cited therein). .

Examples of the compounds Rg-NHg are: p-nitroaniline, 3-carboxy-4-hydroxyaniline, 3-sulfo-4-nitroaniline, 3-alkoxy-4-nitroaniline, 3-carboxy-4-nitroaniline, 4-methoxy- J-Naphthylamino, 4- (N-ethyl-N-hydroxyethyl) aminoaniline, 5-amino noisophthalic acid dimethyl ester, 5-amino-8-nitroquinoline, 7-amino-4-tr1-fluoromethyl coumarin, 7 -amino-4-methylcoumarin and 4-aminodiphenylamine.

The novel derivatives containing two amino acids of highly basic side chain can form disalts or, when R 1 is hydrogen, trisalts with inorganic or organic acids. Preferred salts are disalted by inorganic or organic acids since they have a high water solubility. Particularly preferred salts are hydrochlorides.

In the determination, the cleavage of the substrates by the appropriate enzymes takes place on the carboxyl side of arginine in the 1-strand, which is why the N-terminal arginine must be present as D-isomer. Surprisingly, at negatively charged or neutral lipophilic portion of the tetrapeptide substrate at biologically pH, it has been possible to exchange with a positively charged, strongly alkaline amino acid while maintaining high selectivity and increased sensitivity.

The invention also relates to a method for determining serine proteases, in particular factor Xa, which is characterized by using a peptide derivative according to the invention.

The invention further relates to a method for determining components which may interact with serine proteases or their zymogenic forms, the method being characterized by using a peptide derivative according to the invention.

Oe new factor X substrates, with their sensitivity, high solubility and good selectivity, are a valuable addition to the existing diagnostic analysis methods based on the determination of factor X .. A good selectivity is an important criterion in determination of FX. in plasma, since otherwise there is a risk of thrombin action, which may be present from the beginning or may form during the assay. Examples of important applications are the determination of FX. in human plasma after activation with a protease β from Russel's Viper Venom (RVV-X) of antifactor X., of heparin and of α FVIIIrC in haemophilia (Hemker HC, loc. cit., S. Rosén, Scand. J. Heamatol. Suppl 40, Vol. 33, 1984, 139-145). Particularly important applications are assays 1 in association with anticoagulation treatments with low molecular weight heparin fractions where traditional blood clotting-based methods cannot be used (Walenga JM et al., Seminars in Thrombosis and Hemostasis, Vol. 11, No. 2, 1985, 100-107 ). Specifically included are methods based on the determination of FXa activity and used for the direct determination of FX and FXa or for the direct determination of FVII, FVII, FVIIIrC, FVIIIrC ,, FIX, FIX., Antithrombin III, platelet factor 4 , heparin, low molecular weight heparins and heparinoids. The properties of the new substrates make them particularly suitable for use in automatic analysis devices.

The invention finally relates to a process for preparing the tri peptide derivatives according to the invention, characterized in that a stepwise synthesis is performed by coupling to 25 Arg which has an -NH-Rg group or a removable carboxyl protecting group which is then replaced. with -NH-Rg, or by coupling Rj-XD-Arg-A to Arg-NH-R2 ·

Description of synthesis 30

In the synthesis of the new FX substrates, the commonly used protecting groups and coupling methods are used in the peptide chemistry (M. Bodansky: "Principles of Peptide Synthesis", Springer Verlag 1984), e.g. stepwise addition of amino acids to the labeled C-terminal amino acid or synthesis of the N-terminal peptide fragment, which is then coupled to the labeled C-terminal amino acid.

Applicable amino protecting groups are benzyloxycarbonyl, fluorenylmethyloxycarbonyl or t-butyloxycarbonyl groups. To protect the guanidino group 1 arginine, protonation, a nine-faith protecting group or a p-toluenesulfonyl protecting group are used. The coupling between the amino acids is effected by activating the α-carboxyl group (eg with active esters, symmetric or asymmetric anhydrides, azide, DCCI or similar reagents).

The invention will now be described in more detail in the form of exemplary embodiments showing the preparation of various substrates according to the invention. Purification of intermediates and end products is performed by precipitation, crystallization or gel filtration chromatography. The purified end products are freeze-dried. Pre-fabricated silica gel glass sheets are used for TLC analysis. After chromatography, the plates are examined in UV light 15 (254 nm) and then developed with ninhydrin as well as with chloro / dicarboxidine reagent. The indicated R ^ values are the result of single chromatography.

The solvent systems used for TLC are listed in the table below.

Designation Solvent System Volume Ratio A - n-Butanol: AcOH: Water (3: 2: 1)

Pa6 - chloroform: MeOH: AcOH: water (34: 4: 9: 2) 25 M n-butanol: AcOH: water: pyridine (15: 3: 12: 10) "3" - EtOAc: AcOH: water: pyridine ( 30: 6: 11: 20) HPLC analysis was performed on a Merck RP column (Hibar LiChra-Cart) with 40% MeOH in 0.5% triethylaminophosphate, pH 2.35, as eluent (1 ml / ml). mine). The optical activity of the final products is determined at 589 nm in 50% acetic acid at a concentration of 0.4-1.0 g / 100 ml and + 25 ° C.

The abbreviations below have the following meaning (IUPAC-35 designations have been used where available):

amino acids:

Arg - arginine D-Arg - D-arginine DK 171845 B1 6

Gly = glycine

Sar = sarcosine (N-methylglycine)

All amino acids in the substrates have L-configuration unless otherwise stated 5.

The free amino acid or peptide is indicated by H- in the N-terminal group and with -OH in the carboxyl-terminal group. The amino group is always listed on the left and the carboxyl group on the right.

10

Abbreviations Ac = acetyl

AcOH = acetic acid AMC = 7-amino-4-methylcoumarin Boc * t-butyloxycarbonyl

Bs * benzenesulfonyl

Bz = benzoyl

Bzls = benzyl sulfonyl CHA = 3-carboxy-4-hydroxyanilide 4-ClBs = 4-chlorobenzenesulfonyl DCCI = dicyclohexylcarbodiimide DCU = dicyclohexylurea DMF = dimethyl formamide

Eoc = Ethyloxycarbonyl EtOAc = Ethyl Acetate

EtOH = ethanol

Et 2 N = triethylamine

Ets = Ethanesulfonyl HOBT = 1-hydroxybenzotriazole HPLC = High Efficiency Liquid Chromatography I = Ionic Strength

Mbs = 4-methoxybenzenesulfonyl

MeOH = methanol

Mes = methanesulfonyl Moz = 4-methoxybenzyloxycarbonyl 4-Nbs = 4-nitrobenzenesulfonyl NEM = 4-ethylmorpholine 4-Nz = 4-nitrobenzyloxycarbonyl

Ns = N-naphthalenesulfonyl DK 171845 B1 7 ON? P-nitrophenyl ester pNA - p-nitroaniline

Sue «succinyl TFA« tri fluoroacetic acid 5 TLC thin layer chromatography

Tos p-toluenesulfonyl

Tris (trix (hydroxymethylJaminomethane Z - benzyloxyearbonyl 10

Example 1

Not-ZD-Ara-Glv-Ara-pNA »2 HCl Molecular Weight - 714.62 1a) Boc-G1v-Ara-pNA» HBr Molecular Weight - 532.40 43 mmol H-Arg-pNA »2HBr dissolved in 120 ml DMF, was neutralized cold (-10 ° C) with Et 2 N. The Et3 N + HBr formed was filtered off and 43 mmol Boc-Gly-OH, 45 mmol HOBT and 50 mmol DCCI were added. The reaction is continued cold with stirring for 1 hour and then at room temperature overnight. The resulting DCU is filtered off and the solution is evaporated in vacuo to an oil which is dissolved in 160 ml of EtOAc, washed with 2% NaHCO 3, HgO, 2% KHSO 4 and HgO.

After drying with Na 2 SO 4, the EtOAc phase is evaporated and precipitated with diethyl ether.

Yield: 71% TLC: Rf - 0.23 (Pa6).

30 lb) H-Glv-Arg-pNA »TFA« HBr Molecular Weight - 546.36 55 ml of TFA is added to 30 mmol of Boc-Gly-Arg-pNA »HBr (prepared according to Example 1a) dissolved in 55 ml of methylene chloride. The mixture is stirred for 30 minutes at room temperature and then precipitated with diethyl ether.

Yield: -100% TLC: Rf - 0.28 (A).

1. Ntt-ZD-Ara-G1v-Arq-pNA «2HC1 Molecular Weight = 714.62 DK 171845 B1 8 2.5 mmol ZD-Arg-OH» HCl, 3 mmol HOBT and 3 mmol DCCI are added to 2.5 mmol H -Gly-Arg-pNA * TFA® HBr (prepared according to Example 1b), which is dissolved in 25 ml of DMF and is neutralized cold (-10 ° C) with Et 2 N.

The mixture is stirred cold for 1 hour and then 48 hours at room temperature. The resulting DCU is filtered off and the solution is evaporated in vacuo to an oil, 30 ml of water is added and the solution is washed with 10 x 20 ml of EtOAc. The aqueous phase is evaporated in vacuo, the product ion exchanged on a Sephadex QAE-25 column in chloride form with 50% EtOH as p eluent and purified on a Merck Lobar prepacked column p (LiChroprep. RP-8-B) with 30% MeOH as eluent (2ml / min) at the end. The purified product is freeze-dried.

15

Yield: 37% TLC: Rf - 0.11 (Pa6) HPLC: 98.5% purity [s] - -18, Γ (c - 0.5%)

Example Z

Ntt-Boc-D-Ara-G1v-Ara-pNA »2HC1 Molecular Weight - 680.59 2.5 mmol H-Gly-Arg-pNA» TFA * HBr and 2.5 mmol Boc-D-Arg-OH »HCl is treated with the same coupling procedure and under the same reaction conditions as in Example 1.

Yield: 35% TLC: Rf - 0.55 (M) HPLC: 96% purity [s] - -25.5 * (c - 0.4%)

Example 3 HD-Aro-Glv-Ara pNA »3HCl Molecular Weight - 616.96 22 ml of TFA is added to 12 mmol of Na-Boc-D-Arg-Gly-Arg-pNA * 2HCl (prepared according to Example 2), which is dissolved in 22 ml of DK 171845 B1 9 methylene chloride. The mixture is stirred for 30 minutes at room temperature and then precipitated with diethyl ether. The product is ion exchanged and purified in the same manner as in Example 1.

Yield: 38% TLC: Rf - 0.30 (M) HPLC: 98% Purity [α] - -62.4 * (c - 0.5%) Example 4

Nft-Ets-D-Arq-G1v-Arq-pNA »2HCl Molecular Weight 672.62 0.5 mmol of ethanesulfonyl chloride and 80 beers of EtgN are added to 0.5 mmol of HD-Arg-Gly-Arg-pNA» 3HCl (prepared according to Example 3), which is dissolved in 10 ml DMF and neutralized cold (-10 * C) with 80 μΐ Et ^ N. The reaction is continued cold with stirring for 1 hour and for 2 hours at room temperature. The mixture is evaporated in vacuo. The product is ion exchanged and purified in the same manner as in Example 1.

20

Yield: 44% TLC: Rf - 0.5 (M) HPLC: 97% purity [is] - -15.6 * (c - 0.65%)

Example 5

Nft-Bs-D-Arq-Glv-Arq-pNA »2HCl Molecular Weight - 720.66 0.5 mmol HD-Arg-Gly-Arg-pNA * 3HCl (prepared according to Example 3) and 0.5 mmol of benzenesulfonyl chloride are treated on in the same manner and under the same reaction conditions as in Example 4.

Yield: 37% 33 TLC: Rf - 0.53 (M) HPLC: 98% Purity [α] + 11.6 * (c - 0.45%)

Ntt-4-Nz-D-Aro-Glv-Arq-pNA «2HC1 Molecular Weight« 759,64 DK 171845 B1

Example 6 0.5 mmol of H-D-Arg-Gly-Arg-pNA * 3HCl (prepared according to Example 3) and 0.5 mmol of 4-nitrobenzyloxycarbonyl chloride are treated in the same manner and under the same reaction conditions as in Example 4.

Yield: 57% TLC: Rf - 0.3 (A) HPLC: 99% Purity [α] - -20.2 ° C (c - 0.45%)

Example 7 15

Ntt-4-Nbs-D-Arq-Glv-Arq-pNA «2HCl Molecular Weight« 765.66 0.5 mmol HD-Arg-Gly-Arg-pNA »3HCl (prepared according to Example 3) and 0.5 mmol 4- nitrobenzenesulfonyl chloride is treated in the same manner and under the same reaction conditions as in Example 4.

Yield: 25% TLC: Rf - 0.28 (A) HPLC - 97% Purity [a] - -0.8 * (c - 0.6%)

Example 8 NOf-Tos-D-Arg-G1v-Arq-pNA »2HCl Molecular Weight * 734.69 0.5 mmol HD-Arg-Gly-Arg-pNA» 3HCl (prepared according to Example 3) and 0.5 mmol p -toluene sulfonyl chloride is treated in the same manner and under the same reaction conditions as in Example 4.

Yield: 45% TLC: Rf - 0.25 (A) HPLC: 98% Purity [<*] - + 23, Γ (c - 0.45%)

Ntt-Moz-D-Aro-Glv-Arq-pNA »2HCl Molecular Weight - 744.66 DK 171845 B1

Example 9 11 5 0.5 mmol of H-D-Arg-Gly-Arg-pNA * 3HCl (prepared according to Example 3) and 0.5 mmol of 4-methyloxybenzyloxycarbonylazide are treated in the same manner and under the same reaction conditions as in Example 4.

Yield: 31% TLC: Rf - 0.22 (A) HPLC: 97% Purity [or] - -15.9 * (c - 0.4%)

Example 10 15

Ntt-Mbs-D-Arq-Glv-Ara-pNA »2HCl Molecular Weight - 750.69 0.5 mmol HD-Arg-Gly-Arg-pNA * 3HCl (prepared according to Example 3) and 0.5 mmol of 4-methoxybenzenesulfonyl chloride are treated in the same manner and under the same reaction conditions as in Example 4.

Yield: 45% TLC: Rf 0.31 (A) HPLC: 99% Purity [a] - + 22.8 * (c - 0.4%)

Example 11

Na-4-ClBs-D-Aro-Glv-Ara-nNA «2HC1 Molecular Weight * 755.12 0.5 mmol HD-Arg-Gly-Arg-pNA» 3HCl (prepared according to Example 3) and 0.5 mmol 4 -chlorobenzenesulfonyl chloride is treated in the same manner and under the same reaction conditions as in Example 4.

Yield: 31% TLC: Rf - 0.38 (A) HPLC: 99% Purity [or] - + 10.9 * (c - 0.4%)

Example 12

Ntt-Ns-D-Ara-61v-Ara DNA »2HCl Molecular Weight - 770.73 DK 171845 B1 12 5 0.5 mmol HD-Arg-Gly-Arg-pNA» 3HCl (prepared according to Example 3) and 0.5 mmol / i-naphthalenesulfonyl chloride is treated in the same manner and under the same reaction conditions as in Example 4.

Yield: 25% TLC: Rf - 0.29 (A) HPLC: 99% Purity [α] = -21.5 * (c + 0.4%)

Example 13 15

Ntt-ZD-Ara-Glv-Ara-AMC »2HCl Molecular Weight - 751.70 2 mmol ZD-Arg-Gly-OH * HCl, 2.5 mmol HOBT and 2.5 mmol DCCI are added to 2 mmol H-Arg-AMC 2HCl, dissolved in 30 ml of DMF and neutralized 20 cold (-10 ° C) with Et 2 N. The reaction is carried out cold with stirring for 1 hour and then for 24 hours at room temperature. The resulting DCU is filtered off and evaporated in vacuo to an oil which is ion exchanged and purified in the same manner as in Example 1.

Yield: 39% TLC: Rf = 0.7 ("3") HPLC: 99% Purity [a] - -28.6 * (c - 0.5%) Example 14

Ntt-Ac-P-Arg-Gly-ArQ-pNA * 2HCl Molecular Weight * 622.54 0.5 mmol HD-Arg-Gly-Arg-pNA »3HCl (prepared according to Example 3) and 0.5 mmol acetic anhydride are treated on in the same manner and under the same reaction conditions as in Example 4.

Yield: 43% TLC: Rf 0.45 (M) DK 171845 B1 13 HPLC: 97% purity [a] - -27.9 * (c - 0.4%)

Example 15 5

Ntt-Suc-D-Arq-Glv-Arg-pNA «2HC1 Molecular Weight« 680.57 0.5 mmol HD-Arg-Gly-Arg-pNA »3HCl (prepared according to Example 3) and 0.5 mmol succinic anhydride are treated in the same manner. and under the same reaction conditions as in Example 4.

Yield: 40% TLC: Rf - 0.23 (A) HPLC: 98% Purity [a] - -21.4 * (c - 0.5%)

Example 16

Ntt-Bzls-D-Arg-GIv-Aro-pNA »2HC1 Molecular Weight - 734.69 0.5 mmol HD-Arg-Gly-Arg-pNA» 3HCl (prepared according to Example 3) and 0.5 mmol of benzyl sulfonyl chloride are treated in the same manner and under the same reaction conditions as in Example 4.

Yield: 38% TLC: Rf - 0.26 (A) HPLC: 98% Purity [a] - -7.8 * (c - 0.4%) Example 17

Na-Bz-D-Arq-Glv-Arq-pNA »2HCl Molecular Weight - 684.61 17a) Z-Glv-Arg (O ^) - pNA Molecular Weight - 530.51 0.35 mol of H-Arg-fNOgJpNA'HBr , which is dissolved in 750 ml of DMF, is neutralized cold (-10 ° C) with Et 2 N. The EtjN * HBr formed is filtered off and 0.35 mole of Z-Gly-OH, 0.35 mole of HOBT and 0.38 mole of DCCI are added. The reaction is carried out cold with stirring for 1 hour and then at room temperature overnight. The resulting DCU is filtered off and the solution is evaporated in vacuo to an oil which is treated with 3x400 ml of 2% NaHCO 3 and 500 ml of water. The product is recrystallized from 3.5 L MeOH.

5

Yield: 80% TLC: Rf - 0.6 (Pag) 17b) H-Glv-ArqfNO1-1-pNAtHCl Molecular Weight - 432.72 0.3 moles of Z-Gly-Arg (NO2) pNA (prepared according to Example 17a ) is suspended in 630 ml of AcOH. 415 ml of 5.6 M HBr in AcOH are added with stirring. The mixture is stirred for 45 minutes at room temperature and poured into 7.5 liters of dry diethyl ether with stirring. The precipitate is filtered off, washed with diethyl ether and dried in vacuo.

Yield: 90% 3 g of the product is ion exchanged in the same manner as in Example 1.

20

Yield: 73% TLC: Rf - 0.15 (Pag) 17c) Ntt-Boc-D-Arq (NO2) -Glv-Arq (NO2l DNA Molecular Weight * 697.69 4 mmol H-Gly-Arg (NO2) -pNA »HCl (prepared according to Example 17b) dissolved in 25 ml of DMF is cold neutralized (-10 ° C) with Et 3 N. The resulting Et 1 HCl is extracted for 1 hour, followed by 4 mmol or-Boc-D -Arg (NO2) -OH, 4 mmol HOBT and 4.2 mmol DCCI are added, the reaction is carried out cold with stirring for one hour and then at room temperature overnight.

The resulting DCU is filtered off and the solution is evaporated in vacuo to an oil which is suspended with 2% NaHCO 3, water, 2% KHSO 4, water and diethyl ether.

Yield: 73% TLC: Rf - 0.32 (Pag) 17d) HD-Ara (NO, 1-G1v-Ara (N (L) -DNA) HCl Molecular Weight - 634.04 DK 171845 B1 3 mmol I -Boc-D-Arg-iNOgJ-Gly-Argt1M ^ J-pNA (prepared according to Example 17c), suspended in 11 ml of AcOH, 11 ml of TFA added and the mixture stirred for 4 hours at room temperature. and ion exchanged in the same manner as in Example 1.

Yield: 72% TLC: Rf - 0.61 (M) 10 17e) Ntt-Bz-D-Arq (NO2) -G1y-Arq (NO2) pNA Molecular Weight - 701.68 0.6 mmol benzoic anhydride and 70 μΊ EtgN is added to 0.5 mmol HD-ArgiNOgJ-Gly-ArgiNOgJ-pNA'HCl (prepared according to Example 17d), which is dissolved in 20 ml of DMF and neutralized cold (-10 ° C) with 70 μΐ EtgN. The reaction is carried out cold with stirring for 1 hour and then for 2 hours at room temperature. The solution is evaporated in vacuo and precipitated with water. The product is suspended with 30 ml of warm MeOH, filtered and dried.

20

Yield: 68% TLC: Rf - 0.31 (Pa6) 17. Ntt-Bz-D-Arq-Glv-Arq-pNA * 2HC1 Molecular Weight * 684.61 0.35 mmol N 2 Bz-D-ArgiNOgJ-Gly -ArgJNOgJ pNA (prepared according to Example 17e) is deprotected by reaction with 15 ml of dry HF in the presence of 0.5 ml of anolsol in a suitable apparatus according to Sakakibara (S. Sakaki -bara et al., Bull. Chem. Soc., Japan , vol. 240, pp. 7164-67, 1967) for 30 minutes at 0 ° C. After the reaction is complete, all HF is distilled off and the crude product ion exchanged and purified in the same manner as in Example 1.

Yield: 36% TLC: Rf - 0.55 (M) HPLC: 98% Purity [α] - -24.4 * (c - 0.6%)

Ntt-Eoc-D-Ara-Glv-Ara-oNA «2HC1 Molecular Weight - 652.57 DK 171845 B1

Example 18 16 5 18a) Ntt-Eoc-D-Year (N0.1-G1y-Arq (NCU-PNA Molecular Weight - 669.63 0.6 mmol ethyl chloroformate and 70 μΐ EtgN is added to 0.5 mmol HD-ArgiNOg Gly-ArgiNOgJ-pNA'HCl (prepared according to Example 17d), dissolved in 20 ml DMF and neutralized cold (-10 * 0) with 70 μΐ 10 EtjN. The reaction is continued cold with stirring for 1 hour and then for 2 hours at The mixture is evaporated in vacuo and precipitated with water, filtered and washed with water and diethyl ether.

Yield: 89% TLC: Rf - 0.26 (Pag) 18. Ntt-Eoc-D-Aro-Glv-Arq-pNA + 2HCl Molecular Weight - 652.57 0.45 mmol Na-Eoc-D-Arg (NO2 ) -Gly-Arg (NO2) pNA (prepared according to Example 18a) is deprotected by reaction with 20 ml of dry HF in the presence of 0.5 ml of anisole in the same manner as in Example 17.

Yield: 41% TLC: Rf - 0.55 (M) HPLC: 98% Purity [or] - -25.2 * (c - 0.35%)

Example 19 N0f-Mes-D-Arq-Glv-Arq-pNA 2HCl Molecular Weight - 658.60 19a) Ntt-Mes-D-Aro (NO: 1-Glv-Arq (NQ2l-DNA Molecular Weight - 675.66) 5 mmol of HD-ArgiM ^ J-Gly-ArgiM ^ pNA ^ HCl (prepared according to Example 17d) dissolved in 20 ml of DMF and 0.6 mmol of methanesulfonyl chloride are treated in the same manner and under the same reaction conditions as in Example 17e.

Yield: 70% DK 171845 B1 17 TLC: Rf - 0.47 (A) 19. Ntt-Mes-D-Arg-Glv-Arq-pNA «2HC1 Molecular Weight - 658.60 5 0.35 mmol Ntt-Mes-D -Arg (NO2) -Gly-Arg (NO2) pNA (prepared according to Example 19a) is deprotected and purified in the same manner as in Example 17.

Yield: 43.5% TLC: Rf - 0.48 (M) HPLC: 97% Purity [or] - 17.6 * (c - 0.5%)

Example 20 Nft-ZD-Aro-Sar-Aro-pNA »2HCl Molecular Weight» 728.66 20a) H-Ar-Arq-pNA.2HCl Molecular Weight «438.33 5 mmol DCCI is added to 5 mmol of 20 H-Arg pNA »2HCl, which is dissolved in 25 ml of DMF and neutralized cold (-10 ° C) with Et 2 N. The mixture is stirred cold for 1 hour and then at room temperature overnight. The resulting DCU is filtered off and the solution is evaporated in vacuo to an oil which is dissolved in 100 ml of n-butanol, washed with 2% NaHCO 3, H 2 O, 2% KHSO 2 and H 2 O. After drying with Na 2 SO 4, the n-butanol phase is evaporated and precipitated with diethyl ether. The precipitate is filtered, washed with diethyl ether and dried. The substance is suspended in 25 ml of HCl solution (1.5 M in ACOH) and stirred for 2 hours at room temperature, precipitated with diethyl ether and ion exchanged in the same manner as in Example 1.

30

Yield: 63.5% TLC: Rf - 0.15 (A) 20. Ntt-ZD-Arq-Sar-Arq-pNA 5 mmol DCCI is added to 3 mmol Sar-Arg-pNA »2HCl (prepared according to Example 20a), which is dissolved in 30 ml of DMF and neutralized cold (-10 ° C) with EtjN. The mixture is stirred cold for 1 hour and then for 72 hours at room temperature.

DK 171845 B1 18

The resulting DCU is filtered off and the solution is evaporated in vacuo. The product is ion exchanged and purified in the same manner as in Example 1.

Yield: 43% TLC: Rf - 0.2 (A) HPLC: 99% Purity [α] - -24.7 * (c - 0.55%)

Example z \ 10

Ntt-ZD-Arq-Glv-Ara-CHA »2HCl Molecular Weight - 729.65 21a) Boc-Ara-CHA.HCl 15 3.2 mmol of isobutyl chloroformate is added to 3.0 mmol of Boc-Arg-CHA in 10 ml DMF and neutralized cold (-10 ° C). The reaction mixture is stirred cold for 15 minutes and a mixture of 3 mmol of 3-carboxy-4-hydroxyaniline, 3 mmol of NEM and 10 ml of DMF is added. The reaction is continued cold with stirring for 2 hours and then at 20 room temperature overnight. The solution is evaporated in vacuo to an oil. 50 ml of n-butanol is added and the mixture is stirred for 3 hours at room temperature. The resulting Boc-Arg-CHA HCl is filtered off.

Yield: 50% TLC: Rf - 0.5 (A) 21b) Boc-Aro-CHA + HCl Molecular Weight - 382.25 8 ml of 2N HCl in AcOH is added to 2 mmol of Boc-Gly-Arg-CHA * HCl. The reaction proceeds for 45 minutes at room temperature. The reaction mixture is evaporated in vacuo to an oil which is dissolved in isopropanol and precipitated with EtOAc.

Yield: 97% TLC: Rf - 0.15 (A) 21c) Boc-Glv-Arq-CHA + HCl Molecular Weight 502.95 2 mmol H-Arg-CHA * 2HCl dissolved in 12 ml DMF is neutralized 171845 B1 19 cold (-10 ° C) with NEM and then 2.1 mmol Boc-Gly-ONp is added. The reaction is continued cold with stirring for 1 hour and then at room temperature overnight. The solution is evaporated in vacuo to an oil which is dissolved in 10 ml of MeOH and precipitated with EtOAc.

5

Yield: 88% TLC: Rf - 0.4 (A) 21d) H-Glv-Aro-CHA + 2HCl Molecular Weight - 439.30 10 ml of 2N HCl in AcOH is added to 2 mmol of Boc-Gly-Arg-CHA * HCl . The reaction mixture is stirred for 45 minutes at room temperature and then precipitated with EtOAc.

Yield: 98% TLC: Rf - 0.15 (A) 21. Ntt-ZD-Arq-Glv-Ara-CHA »2HC1 Molecular Weight - 729.65 20 mmol H-Gly-Arg-CHA« 2HCl dissolved in 10 ml of DMF is neutralized cold (-10 ° C) with NEM and then 1.1 mmol of ZD-Arg-0Np »HNO3 is added. The reaction is continued cold with stirring for one hour and then for 2 hours at room temperature. The solution is evaporated in vacuo to an oil which is precipitated with EtOAc. The product is purified on p 25 a Sephadex column with 10% AcOH as eluent and ion exchanged on a Sephadex1 * QAE-25 column in chloride form with 50% EtOH as eluent.

Yield: 57% TLC: Rf - 0.21 (A) HPLC: 99% Purity [α] - -18.6 * (C - 0.3%)

Experiments 35

Determination of enzyme kinetic properties

The kinetic data for the substrates are determined by measuring the initial rate of hydrolysis (Vi) at different substrate concentrations (Si) and constant enzyme concentrations (Eo). The binding constant (K ^) and maximum velocity (Vmax) are obtained in a known manner from a Lineweaver-Burk plot, after which Kcat is calculated (Hemker H.C. loc. Sit.).

5

methods

Enzymes and substrates are mixed in a buffer solution at 37 ° C. The absorbance change per per minute at 405 nm in a spectrophoto-10 meter (pNA substrate) or in a spectrofluorometer at an excitation wavelength of 380 nm and an emission wavelength of 440 nm (AMC substrate).

In the tests, the following commercially available reference substrates are used:

Reference substrates S-2222: Bz-Ile-Glu- (7-0R) -Gly-Arg-pNA.HCl 20 R: H - 50%; CH3 - 50%

KabiVitrum AB, Stockholm, Sweden.

CBS 31.39: CH3SO2-D-Leu-Gly-Arg-pNA.Ac0H Diagnostica Stago, Asnieres s / Seine, France.

25

Test 1. Determination of kinetic constants for bovine £ JSa 30 FX. bovine (KabiVitrum) with a constant enzyme concentration cl

Eq - 4 nmol / l (pNA substrate)

Eq - 2 nmol / l (AMC substrate) and the substrate at concentrations of 0.01-0.4 mmol / l (Si) 35 are mixed with a tris buffer solution of 0.05 mol / l, pH * 8.3 , -0.25 (NaCl) at 37 ° C. The absorbance change per per minute at 405 nm in a spectrophotometer for pNA substrate and in a spectro-fluorometer at an emission wavelength of 440 nm for the AMC substrate, respectively. From these values are then calculated 1 and Kcat listed in Table I.

Table I shows that the tripeptide substrates of the invention exhibit a high affinity for the enzyme bovine FX. while most 5 substrates have a high turnover rate. The selectivity constant indicates that the new substrates have a better selectivity to bovine FX. than S-2222, and that most of the new substrates are even superior to CBS 31.39.

10 Test 2. Determination of kinetic constants for human FX

9

The substrates were 1 concentrations of 0.1-0.7 mmol / L (Si) mixed with human FX. plasma and treated by that of Aurell, L., et al. described procedure (Perspectives 1n Hemostasis, Ed. Fareed, J.

15, et al., New York, Pergamon Press 1981, pp. 382-388).

Mix and incubate 10 μΐ sample plasma, control or standard, and the 200 μΐ tris buffer solution (0.05 mol / l tris, pH 7.0, I - 0.25 (NaCl)), polybrene (0.1 g / l) at 37 # C for 2-4 minutes.

Add 200 μΐ (37 ° C) 1 concentrations of 0.1-0.7 mmol / L and dissolved in tris buffer solution prepared according to the above. The mixture is well stirred and, within 30 seconds, 200 μΐ RVV + CaClg (20-25 ° C) is added and the mixture is stirred. The absorbance change is measured immediately 1 a spectrophotometer at 405 nm and 25 37 ° C. - The results are listed in Table II.

Table II shows that the new tripeptide substrates exhibit a higher affinity for the human FX enzyme. in comparison with the reference substrates while also having a high rate of turnover, which causes them to outperform the commercially obtainable substrates.

Testing 3. Determination of Thrombin Sensitivity 35 When determining FX 1 plasma, there is always a risk of active thrombin formation that will interfere with FX determination.

9

Bovine thrombin (KabiVitrum) 1 at a constant concentration of Eq * 1.3 nmol / l and substrates 1 concentrations of 0.1-0.4 mmol / l (Si) is mixed in a tris buffer solution (0.05 mol / l). 1, pH «8.3, I» 0.25 (NaCl)) at 37 ° C. The test shall be carried out in accordance with test 1.

5 The change in absorbance per read at 405 nm in a spectrophotometer and the Km and Kcat values are calculated and listed in Table III.

Table III shows that the ratio of the selectivity-10 constants to bovine FX. and for thrombin for the new tripeptide substrates can be compared with those for the tetrapeptides and are superior to the known tripeptides in terms of sensitivity to bovine FX relative to the thrombin sensitivity.

d 15 Test 4. Solve! iohed

The solubility in water and tris buffer solution (0.05 mol / l, pH - 8.3, I - 0.25 (NaCl), respectively) at 25 ° C is listed for some of the new substrates in Table IV in comparison with the for the tetra-peptide substrate $ -2222.

The table shows that the new tri peptide substrates have a significantly higher solubility in water as well as in buffer solution compared to that of tetrapeptide S-2222.

25 30 35 DK 171845 B1 23

Tab 1 I

Substrate Km Kcat, Kcat / Km 5 Ex. No. mrnol / l sec. 1 1 / umol »sec.

S-2222 0.51 180 0.35 1 0.11 250 2.27 2 0.22 180 0.82 4 0.19 270 1.42 10 5 0.26 280 1.07 6 0.21 350 1, 66 7 0.45 245 0.55 8 0.22 230 1.04 9 0.15 310 2.06 15 10 0.20 210 1.05 11 0.27 260 0.96 12 0.12 140 1.14 13 0.19 170 0.89 16 0.04 110 2.75 20 18 0.16 225 1.41 19 0.17 200 1.17 20 0.16 220 1.37 CBS 31.39 0.29 290 1.00

Table II

Substrate K_ V V / K

m max max '! / m

Ex. No. ttmol / l itmol / l min. Min. "1 S-2222 1970 46 0.023 30 1 380 73 0.192 4 340 46 0.135 9 210 51 0.242 16 50 15 0.300 CBS 31.39 1060 61 0.057 35 DK 171845 B1 24

Table III

Substrate Km Kcat Kcat / Km Kcat / Km FXa bovine 5 Ex.no ^ nnnoiZL ssSlL. l / mh & K κ Jk thrombin cat S-2222 0.71 13 0.018 19.4 1 0.13 13 0.100 22.7 4 0.41 23 0.056 25.4 10 9 0.073 11 0.151 13.6 16 0.16 17 0.106 25.9 CBS 31.39 0.23 72 0.313 3.2

Table IV 15

Substrate Relatively soluble!

Eks.no. Water Tris Buffer S-2222 1 1 1> 7> 5 20 4> 7> 10 9> 3> 3 16> 5> 10 25 30 35

Claims (5)

1. Tripeptide derivatives, characterized in that they have the general formula: R 1 - X - D-Arg - A - Arg - NH - R 2 wherein R 2 represents hydrogen, an ar or O naphthyl residue, a lower all cooling residue which can be substituted by a carboxyl group, an unsubstituted or substituted phenyl or phenyl alkyl residue, wherein the alkyl group has 1 to 4 carbon atoms, the substitution is preferably paraposition in the phenyl ring, and the substituents are lower alkyl, lower alkoxy, halogen or a nitro group; X represents O-C-, -C-, -S- or a single bond on the condition that H 0 2q when R 1 is hydrogen, then, and only then X is a single bond; A represents Gly or Sar; Rg represents an aromatic or heterocyclic residue which, together with the 25 -NH group, by enzymatic hydrolysis of the tripeptide, gives the compound Rg-Nt 2, which can be quantitatively determined by virtue of its chromogenic, fluorogenic or electrochemical properties; or di- and tri salts of inorganic or organic acids thereof. 30 Tripeptide derivatives according to claim 1, characterized in that A is Gly. Tripeptide derivatives According to claim 1 or 2, characterized in that X is an oxycarbonyl or sulfonyl group. Tripeptide derivatives according to any one of claims 1-3, characterized in that -NH-R2 is a chromogenic or fluorogenic group. s DK 171845 B1 Tri peptide derivatives according to claim 4, characterized in that NH 2 -R 2 is p-nitroaniline, 7-amino-4-methylcoumarin or 3-carboxy-4-hydroxyaniline. Tripeptide derivatives according to any one of claims 1-5, characterized in that R 1 is ethyl, benzyl or tert-butyl. Tripeptide derivative according to any one of claims 1-6, characterized in that it is in the form of a disalt. 10 Tripeptide derivative according to claim 1, characterized in that it is Na-benzylsulfonyl-D-Arg-Gly-Arg-p-nitroanilide * 2HCl. Tripeptide derivative according to claim 1, characterized in that it is Na-4-methoxybenzyloxycarbonyl-D-Arg-Gly-Arg-p-nitroanilide-2HCl. Tripeptide derivative according to claim 1, characterized in that it is Na-ethanesulfonyl-D-Arg-Gly-Arg-p-nitroanilide 2HCl. 20 Tripeptide derivative according to claim 1, characterized in that it is Na-benzyloxycarbonyl-D-Arg-Gly-Arg-p-nitroanilide · 2HCl. Tripeptide derivative according to claim 1, characterized in that it is Na-t-butyloxycarbonyl-D-Arg-Gly-Arg-p-nitroanilide-2HCl. Process for the preparation of tripeptide derivatives according to any one of claims 1-12, characterized in that a stepwise synthesis is performed by coupling to Arg having a 30 -NH-R2 group or a removable carboxyl protecting group which then is replaced by -NH-R2, or by coupling R 1 -XD-Arg-A to Arg-NH-R2. A method for determining serine proteases, in particular factor 35 X, characterized in that a peptide derivative according to any one of claims 1-12 is used. A method for determining components which may interact with serine proteases or their zymogenic forms, characterized by using a peptide derivative according to any one of claims 1-12. The method of claim 14 or 15 for determining FX, 5 FVII, FVII, FVIII: C, FVIII: Ca, FIX, FIX ,, antithrombin III, blood α α α plate factor 4, heparin, low molecular weight heparins and heparinoids. 10 15 20 25 30 35