CZ20001326A3 - Compounds, process of their preparation and their use - Google Patents

Abstract

The present invention relates to compounds of the formula I, thereof physiologically active salts or combinations thereof; the substituents have the following meanings: R 1 and R 2 independently of one another; substituted or unsubstituted, branched or unbranched C 1 -C 8 -alkyl C 1 -C 8 -alkyllaryl-, or C 1 -C 6 alkylhetaryl- substituted or unsubstituted aryl or hetaiyl, R 4 substituted or unsubstituted C 4 -C 14 -aryl- C4-C14-hetyryl-, aryl- or more rings containing one or more heteroatoms from the group O, S and N, R 5 C 1 -C 8 -alkyl- C 1 -C 8 -alkylaryl-, aryl- or hetalyl-; and their manufacturing methods. The compounds or combinations thereof are used for production pharmaceutical compositions for treating associated diseases vasoconstrictor or other biological effects of endothelin, eg high blood pressure.

Description

Compounds, their production and their use

Technical field

The invention relates to novel pharmaceutically active compounds, their production and their use.

BACKGROUND OF THE INVENTION

Endothelin is a peptide consisting of 21 amino acids that are synthesized and released by the vascular endothelium. Endothelin exists in three isoforms, ET-1, ET-2 and ET-3. In the following, endothelin or ET refers to one or all isoforms of endothelin. Endothelin is a potential vasoconstrictor and has a potent effect on blood vessel coloration. This vasoconstriction is known to be caused by the binding of endothelin to its receptors (Nature, 331, 411-415, 1988, FEBS Letters, 131, 440-444, 1988 and Biochem. Biophys. Res. Commun., 154, 868-875, 1988).

Increased or abnormal release of endothelin causes arresting of vascular contraction in the peripheral, renal and cerebral vessels, which can lead to disease. As reported in the literature, endothelin is involved in a variety of diseases. These include hypertonia, acute myocardial infarction, pulmonary hypertonia, Raynaud's syndrome, cerebral vascular convulsions, stroke, benign prostate hypertonia, atherosclerosis, asthma and prostate cancer (J. Vascular Med. Biology 1, 207 (1990), J. Am. Med Association 264. 2868 (1990), Nature 144. 114 (1990), N. Engl J. Med 321, 205 (1989), N. Engl J Med 128, 1732 (1993), Nephron 66, 373 (1994), Stroke 25, 904 (1994), Nature 365, 759 (1993), J. Mol Cell Cardiol 27, A234 (1995), Cancer Research 56, 663 (1996), Nature Medicine 1 , 944 (1995)).

At least two endothelin receptor subtypes, the ETα and ETb receptor, have been described in the literature (Nature 348, 730 (1990), Nature 348, 732 (1990)). Accordingly, substances that inhibit endothelin binding at one or both receptors must antagonize the physiological effects of endothelin and therefore constitute a valuable pharmaceutical.

even from endothelin to endothelin, they attack a sought-after alternative of receptors they should

However, a disadvantage of these receptor antagonists is that endothelin has been formed and the effects of its formation must be antagonized. Substances that prevent the formation of its predecessor, the so-called big'-endothelin, earlier stages of entothelin and thus have direct superior efficacy to endothelin antagonists than, for example, ACE inhibitors (ACE = angiotensin converting enzyme, Szelke et al., Nature) 299, 555) or ANP (ANP, Sybertz et al., J. Pharmacol. Exp. Ther. 1989, 250, 624).

Object of the invention

It is therefore a task to prepare an inhibitor of endothelin conversion enzyme (= ECE).

This object is achieved by the compounds of the formula I

their physiologically active salts or combinations thereof, in which the substituents have the following meanings:

R ¹ and R ² are independently substituted or unsubstituted, branched or unbranched Ci-Cs-alky1-, Ci-Ce-alkylaryl, or Ci-Cs-alkylhetaryl-, substituted or unsubstituted aryl or hetaryl, · «♦♦ · • · · _Λ • · · · • · · · • · ···· · ···· ·· * ·· ··> · «► ·""·<

I · ♦ <

·· ··

R ^{3 is} a, b or c

(a) (b)

SY

Y = CH 3 CO-, H

(C)

R ⁴ substituted or unsubstituted C 4 -C 14 -aryl-, C 4 -C 14 -heteryl-, with one or more rings containing one or more heteroatoms from the group O, S and N,

R ^{5 is} C 1 -C 6 -alkyl-, C 1 -C 6 -alkylaryl-, aryl- or hetaryl-.

The invention further relates to a process for the preparation of the above-mentioned compounds of the formula I, characterized in that the compounds of the formula II.

(Π) h ₂ n are condensed with compounds of formula III

fj (lii) and reduce the formula IV with a reducing agent to the compounds of the general formula (II). · * * · * * * * * * *

and reacting the acylatingin with R ⁴ COCl (V) and cleavage of the protecting group SG 1 to the above compounds of formula I, wherein the substituents R ¹ , R ² , R ³ and R ⁴ are as defined above and SG 1 is a protecting group.

The invention furthermore relates to the use of the compounds of the formula I for inhibiting the enzyme endothelin conversion (= ECE), for the manufacture of pharmaceutical preparations for the treatment of diseases and also for the use of these pharmaceutical preparations in combination with at least one other blood pressure lowering agent.

The substituents R ¹ and R ² in the above formulas I, II, III and IV have the following meanings:

R ¹ and R ² independently of each other substituted or unsubstituted, branched or unbranched C 1 -C 6 -alkyl-, C 1 -C 6 -alkylaryl-, or C 1 -C 6 -alkylhetaryl-, substituted or unsubstituted aryl- or hetaryl-, wherein the alkyl is branched or unbranched C 1 -C 6 -alkyl chains such as methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, n-pentyl, 1-methylbutyl, 2-methylbutyl , 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, n-hexyl, 1,1-dimethylpropyl,

1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl,

1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1- methylpropyl, 1-ethyl-2-methylpropyl, n-heptyl or n-octyl;

C 1 -C 6 -alkylC 1 -C 6 -alkylethylphenyl, phenyl, phenyl, propynaphthyl, propylnaphthyl, pentylnaphthyl, branched or unbranched as C 1 -C 6 -alkylphenyl or radicals such as methylphenyl, propylphenyl, 1-ethylethylphenyl, butylphenyl, 1-methylpropyl phenyl, 2-methylpropylphenyl, 1,1-dimethylethylphenyl, pentylphenyl, 1-methylbutylphenyl, 2-methylbutylphenyl, 3-methylbutyl-2,2-dimethylpropylphenyl, 1-ethylpropylphenyl, hexylheptylphenyl, octylphenyl, octylphenyl, octylphenyl, octylphenyl, octylphenyl

1-methylethylnaphthyl, butylnaphthyl, 1-methyl-2-methylpropylnaphthyl 1,1,1-dimethylethylnaphthyl, 1-methylbutylnaphthyl, 2-methylbutylnaphthyl, 3-methylbutylnaphthyl, 2,2-dimethylpropylnaphthyl, 1-ethylpropylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylnaphthylbutyls; , the aromatic ring heteroaromatic optionally may contain S, N or O, or unbranched C 1 -C 6 -alkyl simple or condensed systems. with one or more to 8-membered rings which one or more heteroatoms such as aryl such as phenyl, naphthyl, anthranyl, phenanthryl, hetaryl simple or condensed aromatic ring systems with one or more heteroaromatic 5- to 8-membered rings, which may optionally contain one or more more heteroatoms such as S, N or O such as thienyl, pyridyl or iondyl.

All radicals R ¹ or R ² may be Substituted by one or more radicals -NHP (Ci-Ce-alkyl) 2-p -QHn (Ci-Ce-alkyl) 1-η, -SS-t-butyl - CN, -NO 2 or halogen such as fluorine, chlorine, bromine or iodine, wherein p = 0, 1 or 2, Q represents sulfur or oxygen, n represents 0 or 1 and C 1 -C 6 -alkyl is as defined above.

• · · · · · · · ·

.......

Preference is given to the radicals R ¹ and R ^2, which are derived from natural or unnatural amino acid, the functional groups of these radicality may be protected or unprotected. Since the radicals R ¹ and R ² is preferably derived from natural or unnatural amino acids may be the stereocenter adjacent to the radicals both in the D configuration and in the L configuration (= R- or S-form). Other preferred R ¹ and R ² radicals are substituted or unsubstituted, branched or unbranched C 1 -C 4 -alkyl-, C 1 -C 4 -alkylaryl- or C 1 -C 4 -alkylhetaryl-, substituted or unsubstituted aryl or hetaryl-, particularly preferred is C 1 -C4-alkylaryl-

In principle, R @ ¹ or R @ ² in less preferred form can also be hydrogen. However, compounds with this radical exhibit no or very little biological activity.

In the above formulas I, III and IV, the substituent R ³ has the following meaning:

R ^{3 is} a, b or c

The formulas a, b and c may optionally carry additional substituents. R ⁵ is as defined below in formula b.

The substituent R ⁴ in the above formulas I and V has the following meaning:

R 4 substituted or unsubstituted C 4 -C 14 -aryl-, C 4 -C 14 hetaryl having one or more rings containing one or more heteroatoms from the group O, S and N, wherein:

C4-C14-aryl such as phenyl, naphthyl, anthranyl, phenanthryl,

C 4 -C 14 -hetary 1 simple or condensed aromatic ring systems with one or more heroaromatic 5- to 8-membered rings, which may optionally contain one or more heteroatoms such as S, N or O, such as thienyl-, pyridyl- or iondyl.

All of said R ⁴ radicals may optionally be substituted by one or more of the following radicals: branched or unbranched C 1 -C 4 -alkyl-, C 6 -C 14 -aryl-, COOR ⁶ , -NHp (C 1-6 -alkyl) 2 -p, -QHn (C 1 -C 6 -alkyl), -SS-t-butyl, -CN, -NO 2 or a halogen such as fluorine, chlorine, bromine or iodine, wherein R ⁵ H or C 1 -C 6 -alkyl, p 0, 1 or 2 , Q is sulfur or oxygen, n 0 or 1 and C 1 -C 6 -alkyl-, C 1 -C 4 -alkyl- or C 6 -C 14 -aryl have the meanings given above.

The substituent R ⁵ in formula (b) above has the following meaning:

R ^{5 is} C 1 -C 6 -alkyl-, C 1 -C 6 -alkyl-aryl, aryl- or hetaryl-, branched or unbranched C 1 -C 6 -alkyl chains such as methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethyl-ethyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, n-hexyl, 1,1-dimethylpropyl, 1 , 2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl,

1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl- 1-methylpropyl, 1-ethyl-2-methylpropyl, n-heptyl or n-octyl; alkylaryl aryl, naphthyl branched or unbranched C 1 -C 6 -alkyl such as C 1 -C 6 -alkyl-phenyl or C 1 -C 6 -alkyl radicals such as methylphenyl, ethylphenyl, propylphenyl, 1-methylphenyl, butylphenyl, 1-methylpropylphenyl, 2-methylpropylphenyl, 1,1-dimethylethylphenyl, pentyl1-methylbutylphenyl, 2-methylbutylphenyl, 3-methylbutyl2,2-dimethylpropylphenyl, 1-ethylpropylphenyl, hexylheptylphenyl, hexylheptylphenyl, hexylheptylphenyl, Ethylnaphthyl

1-methylethylnaphthyl, butylnaphthyl, 1-methyl-2-methylpropylnaphthyl, 1,1-dimethylethylnaphthyl, 1-methylbutylnaphthyl, 2-methylbutylnaphthyl, 3-methylbutylnaphthyl, 2,2-dimethylpropylnaphthyl, 1-ethylpropylnaphthylnaphthylnaphthyl, naphthylnaphthyl, naphthylnaphthyl, naphthyl, naphthylnaphthyl, phenyl, propynaphthyl, propylnaphthyl, pentylnaphthyl, aryl such as phenyl, naphthyl, anthranyl, phenanthryl, hetaryl simple or condensed aromatic ring systems with one or more heteroaromatic 5- to 8-membered rings, which may optionally contain one or more heteroatoms such as S, N or O, such as thienyl, pyridyl or iondyl.

The R ⁵ radical may optionally carry additional substituents,

The compounds of the invention may be used as the free compounds, or in the form of their physiologically active salts, their tautomeric or isomeric forms, or in the form of combinations of the free compounds and various salts. The compounds according to the invention also include the pure enantiomeric or pure diasteomeric compounds, their salts or mixtures thereof.

The enantiomeric or diastereomeric forms of the compounds according to the invention can be purified or prepared in a known manner by formation of diastereomeric salts, by chromatography or by stereoselective syntheses.

The preparation of the compounds according to the invention is carried out according to a method known to the person skilled in the art, as for example known from Hruby et al. (J. Med. Chem 1995, 38, 3462), Coy et al. (J. Med. Chem. 1987, 30, 1162) or Coy et al. (Tetrahedron, 44, 1988, 835) and therefore does not need any further explanation. It is preferred to condense the amino acid derivative of the formula II suitably protected for carboxylic acid function by the aminoaldehyde of the formula III to the imine and reduce it to the amine of the formula IV, for example NaBH 3 CN with addition of acid (see Reaction Scheme I).

Reaction scheme

Synthesis of compounds of formula I0

(II)

Suitable protecting groups SGi are all protective groups known to the person skilled in the art, such as t-butyl, benzyl, trytyl, methyl or also polymer bound protecting groups in the form of conventional polystyrene resins, such as 2-chlorotrityl chloride resin or Wang resin (from Bachem, Nobvabiochem) ). Preferred protecting groups are t-butyl and 2-chlorotriyl resin.

. · A · a a ····· «« · ·

The reaction to the imine and the reductions is carried out as described in the literature (VJ Hruby et al. J. Med. Chem 1995, 38, 3462, DH Coy et al. J. Med. Chem. 1987, 30, 1162 and DH Coy. and Tetrahedron, 44, 1988, 835), wherein trimethyl orthoformate may be advantageously added to imine formation, as described by Gallop et al. (J, Am. Chem. Soc. 1995, 117, 7029).

Further derivatization of the compounds of formula IV to the compounds of formula I is preferably carried out with acid chloride with the addition of a base, also this reaction step does not need any further explanation and is known to the person skilled in the art.

The reaction of the compound of formula IV to the compound of formula I is preferably carried out in a mixture of pyridine and methylene chloride (about 1: 1) at temperatures between 0 and 100 ° C, preferably at 20 to 60 ° C with a 2-10 fold excess of acid chloride.

If desired, the compounds of formula I are purified by conventional chromatographic methods after completion of the synthesis, for example, by conventional FPCL- or HPCL chromatography.

The use of the peptide compounds of the invention linkages results in increased stability with respect to and thus exhibits longer biological activity by the CH 2 NH group in that these compounds cleave peptides by enzymes

Since the side chains are not affected by these modifications • · 9 99 9 9 • 9

9 · · · · · · ·

The compounds of the invention resemble very true peptides. They are viewed as synthetic stable analogs of natural substrates, since this slight change in molecules does not alter or conform to the substance conformation.

The compounds of the invention inhibit the endothelin conversion enzyme very selectively with activities in the μηι range and can be used for this. For other metalloproteases such as the ACE enzyme) NEP 24.11 (= neutral matrix metalloproteases (= MMP) inhibition in this range of thrombin do not inhibit.

(= angiotensin converting endopeptidase 24.11) or MMP-1, MMP-3 or MMP-9 could not be observed, also thermolysin, papain and compounds as substrates, respectively

The advantage of such a selective class of inhibitors is that, firstly, it does not interfere with other enzymatic processes, so that undesirable side effects are not envisaged, secondly, they are also very stable due to enzymatic degradation, since they cannot be degraded in non-specific reactions by other proteases. They can therefore be dosed in very small doses, whereby the likelihood of side effects, for example, by degradation products, can be further reduced.

The compounds of the invention, their stereoisomeric forms and / or their physiologically active salts, as well as their tautomeric or isomeric forms, are suitable for the manufacture of pharmaceutical preparations for the treatment of diseases, preferably for the manufacture of medicaments for the treatment of diseases which are associated with vasoconstriction or other biological effects of endothelin. Preferably, the pure enantiomeric or pure diastereomeric compounds are used as active ingredients.

The compounds of the present invention create new therapeutic potential for the treatment of hypertonia, pulmonary high pressure, myocardial infarction, chronic heart insufficiency, angina pectoris, acute or chronic renal failure, renal insufficiency, cerebral vascular cerebral ischemia, subarachnoid haemorrhage, asthma, artherosclerosis, endotoxic endotoxin-induced organ failure, intravascular coagulation, following angioplasty, benign prostatic hyperplasia, ischemic and intoxication-induced renal failure, metastasis and growth of mesenchymal tumors, cancer, prostate cancer, contrast-induced renal failure, pancreas or gastrointestinal ulcer.

The compounds according to the invention are preferably administered in the form of pharmaceutical compositions which are released under conditions such as those found in the specified organs of the body, for example in the stomach, intestines, blood circulation and liver.

A further object of the invention are combination preparations of inhibitors of the invention of formula I and inhibitors of the renin angiotensin system. Inhibitors of the renin angiotensin system are renin inhibitors, angiotensin antagonists II and especially ACE (angiotensin-coverting-enzyme) inhibitors.

These combinations can be applied in a common galenical form or time and spatially separated.

In relation to the dosage and the type of application, the same factors as for the respective individual substances are taken into account.

The compounds of the invention may be administered orally or parenterally (subcutaneously, intravenously, intramuscularly, intraperotoneally) by the usual route. Application can also be by vapor or spray through the nasopharynx cavity.

The dosage depends on the age, condition and weight of the patients as well as the type of administration.

cramps, migraines, • A A ··· • · ··· ·

13, the compositions may be administered as a tablet

The novel compounds of the invention may be used in solid or liquid form in consumer galenic dosage forms, for example, as tablets, capsules, powders, granules, dragees, suppositories, solutions, ointments, creams or sprays. These are produced in a conventional manner. Active substances with conventional galenic auxiliary binders, fillers, preservatives, tablet disintegrants, flow regulators, plasticizers, crosslinkers, dispersants, emulsifiers, solvents, retardants, antioxidants and / or propellants (see H. Sucker et al., Pharmazeutische Technologie, Thieme-Verlag, Stuttgart, 1991). The dosage forms thus obtained contain from 0.1 to 90% by weight of active compound. %.

Combinations of calcium antagonists with inhibitors of the invention can be used as a means to treat diseases that are based on vasoconstriction and occur with pathological vasoconstriction. Examples include some forms of high blood pressure (including pulmonary hypertonia), coronary heart disease, heart insufficiency, renal and myocardial ischemia, acute and chronic renal insufficiency.

Based on the determination of the effect of the individual components, the combination of both active ingredient groups is ideally complemented. Furthermore, it is advantageous that undesirable side effects rarely occur due to dose reduction.

in the form of solutions, rectally, e.g.

The combinations of the invention are administered orally, for example tablets, dragees, hard and soft gelatine capsules, emulsions or suspensions. Administration can also be carried out, for example, in the form of suppositories, or parenterally, by injection. Administration of the active ingredient may be in the form of preparations containing both active ingredients as tablets or capsules, or separately as ad-hoc of the individual substances which may be administered.

• 9 ·· ·

- 14 simultaneously or staggered.

For the production of tablets, dragees and hard gelatine capsules, the combination according to the invention may be incorporated with pharmaceutically inert, inorganic or organic excipients. As such excipients, lactose, corn starch or derivatives thereof, talc, stearic acid or its salts can be used for tablets, dragees and hard gelatine capsules. Vegetable oils, waxes, fats, semi-solid and liquid polyols are suitable as excipients for soft gelatine capsules.

Suitable excipients are, for example, water, polyols, sucrose, invert sugar, glucose, and the like to make solutions and syrups. Suitable excipients for injection solutions are water, alcohols, polyols, glycerin and animal oils. For suppositories, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols and the like are suitable as excipients.

The pharmaceutical preparations may additionally contain preservatives, solvents, stabilizers, crosslinkers, emulsifiers, sweeteners, colorants, flavoring agents, salts for varying the osmotic pressure, buffers, coating agents and / or antioxidants.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Synthesis of compounds Ia to Ik

0.4 mMol phenylalanine, the C-terminal was supported with 2Cl-1-butyl resin (2a), dissolved in 9 ml DMF / HOAc 99: 1 in 0.75 mMol N-Fmoc-phenylalaninal (3a) (R = CH2PH, R3 = Fmoc) and shaken for 0.5 h. NaBH 3 CN was then added in portions until the ninhydrin assay showed no free primary amino groups. Subsequently, it was suctioned off, the solid was washed with DMF, isopropanol and methylene chloride and dried under vacuum.

0.4 mMol of (4a) in about 10 mL of pyridine / methylene chloride 1/1 was dissolved with a catalytic amount of DMAP (= 4-dimethylaminopyridine) and 2 mMol of 2-thiophenecarboxylic acid chloride and shaken until the ninhydrintest showed no secondary amino acid. The reaction was aspirated, washed with DMF and methylene chloride, and the polymeric protecting group was separated as indicated:

(4a) was shaken for 1 hour in about 10 ml of a mixture of acetic acid, trifluoroethanol and methylene chloride 1/1/8, the solution containing 1a was filtered and concentrated (reaction scheme)

II).

Reaction scheme

II: Synthesis of Compound (Ia).

made detected

The following analogous compounds 1b to 1e and method. Reported molar by molecular spectroscopic 1g to 1k were masses were methods.

• φ · ♦ · 4 ·

δ. formula molar mass la O Q chirally 664 O [O] OH ^ 1 O ý -with Cl lb O in /WITH with chirally 708 ^ Y ' O J Ck / OH O J F YY F γΑγ Cl • 1c O SH chirally 620 Yl ^ Y ^OH Y> YO P Ύ> Cl ld O 0 chirally 676 fy ° Y- ° X) iT * r / YY ^X N

-17 • to * · · · · · · · · · · · · · · · · · · · · · · ·

· • 9 9 * 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 99 99

Example 2: ECE inhibitor assay, IC50 determination

Recombinant human ECE from CHO cells as described by Schmidt et al (FEBS Letters 356, 1994, 238-243) was used to test endothelin conversion enzyme (ECE) inhibitors.

The enzyme preparations used were further purified by mono-Q chromatography and WGA lectin chromatography after membrane isolation and solubilization. The preparations thus obtained contained no interfering foreign protease activities and have specific activities in the range of 1 to 20 mU / mg. 5 μΐ of enzyme solution is pre-incubated for 10 minutes 495 μΐ assay buffer (100 mM Pi, 500 mM NaCl, 01 mg / ml BSA, pH 7.2) and 5 μΐ appropriately concentrated solutions (10 ^_3 M and 10 ^-4 M, ΙΟ ⁵ M and so on) inhibitors in assay buffer. The 50 μΐ fraction was mixed with δ μ1 of solution 2. ¹⁰ M Big-ET1 (= big-endothelin 1) in 0.02% acetic acid. The composition was 9999 after 1 hour at 37 ° C

150 μΐ of 0.5% TFA (trifluoroacetic acid) in water was added, the composition was centrifuged at 10000 g for 5 minutes and the endothelin formed was measured by reversed phase HPLC as described in Takada et al. (Biochem. Biophys. Res.Comm. 17 6 , 860 (1991) and K. Ohnak et al. (Biochem. Biophys. Res. Commun. 168, 1128, 1990), the enzyme reaction was determined. The inhibition curve at various inhibitor concentrations was generated and half maximal inhibition (IC 50 value) was subtracted as a measure of inhibitor potency. Table I shows the IC 50 of various substances relative to ECE, ACE and NEP 24.11.

Table I: IC 50 values for various inhibitors

Compounds ICso (ECE) ICso (ACE) ICso (NEP) 1 a 2 pm > 100 µm > 100 µ® lb 3 μηι > 100 µm > 100 µ® 1 e 4 pm > 100 μω > 100 µm

JUDr, Petr Kalensiý attorney at law, enlightened: 0254

Spote & á advokátní kancelář VSetečka Zelený Svorčflc Kalen & Ă a Partners, Hálkova 2 Praha 2,120 00

TM. ·. 02/24942434 »ftE 02 / 24943092-3 φ · · · · · · · ·

φ · · · · · · ««

PV Τοοζο- η £ €

Claims (12)

PATENT CLAIMS Compounds of formula I R ¹ or a physiologically active salt thereof or a combination thereof in which the substituents have the following meanings: R ¹ and R ² are independently substituted or unsubstituted, branched or unbranched Ci-Ce-alky1-, Ci-Cs alkylaryl, or Ci-Cs-alkylhetaryl-, substituted or unsubstituted aryl or hetaryl, R ^{3 is} a, b or c R ⁴ substituted or unsubstituted C 4 -C 14 -aryl-, C 4 -C 14 hetyryl-, with one or more rings containing one or more heteroatoms from the group O, S and N, R ^{5 is} C 1 -C 6 -alkyl 1-, C 1 -C ⁵ -alkylaryl-, aryl- or hetaryl-. Compounds of formula I, their physiologically active salts or combinations thereof according to claim 1, characterized in that R ¹ and R ² independently of each other denote substituted or unsubstituted, branched or unbranched C 1 -C 4 -alkyl-, C1-C4-alkylaryl- or C1-C4-alkylhetaryl-, substituted or unsubstituted aryl- or hetaryl-. • 9 • ··· ···· 9 9999 9 · 9 9 9 9 9 9 3. The method for producing the above compounds of formula I wherein the compound of formula II n ₂ N (Π) condensing a compound of formula III is reduced with a reducing agent to give compounds of formula IV R @ 1 is reacted with an acylating agent R ^{@ 4} COCl (V) and cleavage of the SG1 protecting group to compounds of formula I, wherein R @ ¹ , R @ ² , R @ ³ and R ^{@ 4} are as defined above. Use of the compounds of the formula I, their physiologically active salts or combinations thereof according to claim 1 for the production of pharmaceutical preparations for the treatment of diseases. Use of the compounds of formula I, their physiologically active salts or combinations thereof according to claim 1 for the manufacture of pharmaceutical compositions for the treatment of diseases which are associated with vasoconstriction or other biological effects. endothelin. Use of the compounds of formula I, their physiologically active salts or combinations thereof according to claim 1 for the manufacture of pharmaceutical preparations for the treatment of diseases of the group of hypertonia, pulmonary hypertension, myocardial infarction, chronic heart failure, angina pectoris, acute or chronic renal failure kidney, cerebral vascular spasm, cerebral ischemia, subarachnoidal hemorrhage, migraine, asthma, artherosclerosis, endotoxic shock, endotoxin-induced organ failure, intravascular! coagulation, after angi oplasti, benign prostatic hyperplasia, ischemic and intoxication-induced renal failure or hypertonia, cyclospore-induced renal failure, metastasis and mesenchymal tumor growth, cancer, prostate cancer, contrast-induced renal failure, pancreatis and gastro intestinal ulcer, Use of compounds of formula I according to claim 1 for inhibiting endothelin conversion enzyme. Use of a compound according to claim 7 for the selective inhibition of endothelin conversion enzyme, wherein other proteases from the group ACE, NEP, MMP -1, MMP-3. MMP-9, termolysin, papain and thrombin are not inhibited, Use of the compounds of the formula I according to claim 1 in combination with other blood pressure lowering active substances for the manufacture of pharmaceutical preparations for the treatment of diseases. A pharmaceutical composition comprising the compounds of formula I, their physiologically active salts, or combinations thereof according to claim 1. 44 · 4 · 4 · 9 · ft · ft · ···· fr · 44 4 • 4 4 4 - 23 Combination of a pharmaceutical composition according to claim 10 and at least one additional blood pressure lowering active ingredient. Combination according to claim 11 comprising ACE inhibitors as blood pressure lowering active ingredient.