WO1999019346A1

WO1999019346A1 - Dipeptides for the treatment of diseases related to the biological effect of endothelin

Info

Publication number: WO1999019346A1
Application number: PCT/EP1998/005943
Authority: WO
Inventors: Michael Puhl; Wilhelm Amberg; Heinz Hillen; Andreas Kling; Stefan Hergenröder; Claus Otto Markert
Original assignee: Basf Aktiengesellschaft
Priority date: 1997-10-14
Filing date: 1998-09-18
Publication date: 1999-04-22
Also published as: DE19745151A1; ZA989313B; AU9626498A; JP2001519440A; EP1023318A1

Abstract

New compounds and the use of these compounds for preparing pharmaceutical compositions for the treatment of diseases related to the biological effect of endothelin are disclosed, as well as pharmaceutical compounds suitable for inhibiting the endothelin conversion enzyme (ECE).

Description

DIPEPTIDES FOR TREATING DISEASES ASSOCIATED WITH THE BIOLOGICAL EFFECT OF ENDOTHELINE

5 Description

The invention relates to new compounds and the use of these compounds for the production of pharmaceutical preparations for the treatment of diseases which are associated with the biological action of endothelin. The invention further relates to pharmaceutical compounds suitable for ECE inhibition.

Endothelin is a 21 amino acid peptide that is synthesized and released by vascular endothelium. 5 Endothelin exists in three isoforms, ET-1, ET-2 and ET-3. In the following, "endothelin" or "ET" denotes one or all isoforms of endothelin. Endothelin is a potent vasoconstrictor and has a strong effect on vascular tone. This vasoconstriction is known to be caused by the binding of endothelin 0 to its receptor (Nature, 332, 411-415, 1988; FEBS Letters, 231, 440-444, 1988 and Biochem. Biophys. Res. Co mun., 154, 868-875, 1988).

Increased or abnormal release of endothelin causes persistent vascular contraction in peripheral, renal and cerebral blood vessels, which can lead to disease. As reported in the literature, endothelin is involved in a number of diseases. These include: hypertension, acute myocardial infarction, pulmonary hypertension, Raynaud's syndrome, cerebral vasospasm, stroke, benign prostate hypertrophy, atherosclerosis, asthma and prostate cancer (J. Vascular Med. Biology 2, 207 (1990), J. Am. Med Association 26_4, 2868 (1990), Nature, 114 (1990), N. Engl. J. Med. 212, 205 (1989), N. Engl. J. Med. 222., 1732 (1993), Nephron 5 £, 373 (1994), Stroke 25, 904 (1994), Nature 26_5_, 759 (1993), J. Mol. Cell. Cardiol. 27., A234 (1995); Cancer Research 5JL, 663 5 (1996) , Nature Medicine 1, 944, (1995)).

At least two endothelin receptor subtypes, ET _A and ET _B receptor, are currently described in the literature (Nature 348, 730 (1990), Nature 348, 732 (1990)). Accordingly, substances that inhibit the binding of endothelin to one or both receptors should antagonize the physiological effects of endothelin and should therefore be valuable pharmaceuticals.

A disadvantage of these receptor antagonists, however, is that En5 dothelin has already formed and the action of the endothelin has to be antagonized after its formation. Substances that form an education of the endothelin from its precursor, the so-called "big endothelin", attack at an earlier stage of the endothelin effect and thus represent a sought-after alternative to the endothelin receptor antagonists. Inhibitors based on dipeptide derivatives, the N-terminal of a mercaptocarboxylic acid are described in EP-A-0 524 553, WO 94/17036 and WO 96/22998 as inhibitors for NEP and / or ACE. WO 96/11209 describes such compounds and their preparation as inhibitors for matrix metalloproteases and TNF.

Advantageously, such ECE inhibitors should also have a good effect against other proteases such as ACE.

It was therefore the task of providing inhibitors of the endothelin conversion enzyme (= ECE) which, in addition, inhibit further proteases which are involved in regulating blood pressure, but not unspecific proteases in general.

This object was achieved by compounds of the general formula I;

their physiologically active salts or their combination, in which the substituents have the following meaning:

R ¹ H, substituted or unsubstituted, branched or unbranched Ci-Cs-alkyl, Ci-Cβ-alkylaryl or Ci-Cβ-alkylhetaryl, substituted or unsubstituted aryl or hetaryl,

R ² substituted or unsubstituted 2-thienylmethyl-, ß-naphthylmethyl-, N-Boc-indol-3-ylmethyl _'

R ³ H, substituted or unsubstituted acyl, dissolved.

The invention also relates to a process for the preparation of the abovementioned compounds of the general formula I, characterized in that compounds of the general formula II

with compounds of the general formula III

condensed to compounds of formula IV

and then with compounds of formula V

in the presence of CsSAc and splitting off the protective group SGI to give compounds of the formula I according to claim 1, where the substituents R ¹ , R ² and R ^{3 have} the abovementioned meaning and SGi and SG are a protective group.

The invention further relates to the use of compounds of the general formula I for inhibiting the endothelin conversion enzyme (= ECE), and to pharmaceutical preparations suitable for ECE inhibition, containing the abovementioned compounds of the general formula I, their physiologically active salts or a combination thereof .

The compounds according to the invention are advantageously suitable for the production of pharmaceutical preparations for the treatment of diseases which are associated with the biological action of endothelin. The substituents R ¹ - R ² and R ³ in the above formula I have the following meanings:

R ^{1 is} hydrogen, substituted or unsubstituted, branched or unbranched Ci-Cβ-alkyl, Ci-Cβ-alkylaryl or Cj _. -C ₈ alkylhetaryl, substituted or unsubstituted aryl or hetaryl, wherein

Alkyl branched or unbranched Ci-Cs-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-dimethylb tyl, 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 branched or unbranched Ci-Cs-alkylaryl, such as Ci-Cs-alkyl-phenyl- or Ci-Cβ-alkyl-naphthyl residues such as methylphenyl, ethylphenyl, propylphenyl, 1-methylethylphenyl, butylphenyl, 1-methylpropylphenyl , 2-methylpropylphenyl, 1, 1-dimethylethylphenyl, pentylphenyl, 1-methylbutylphenyl, 2-methylbutylphenyl, 3-methylbutylphenyl, 2,2-dimethylpropylphenyl, 1-ethylpropylphenyl, hexylphenyl, heptylphenyl, octylphenyl, methylnaphthyl , Ethylnaphthyl, propynaphthyl, 1-methylethylnaphthyl, butylnaphthyl, 1-methyl-propylnaphthyl, 2-methylpropylnaphthyl, 1, 1-dimethylethyl-naphthyl, pentylnaphthyl, 1-methylbutylnaphthyl, 2-methylbutylnaphthyl, 2-methyl-naphthyl, 2-methyl-naphthyl, 2-methyl-naphthyl, 2-methyl-naphthyl, 2-methyl-naphthyl, 2-methyl-naphthyl, 2-methyl-naphthyl, 2-methyl-naphthyl, 2-methyl-naphthyl, 2-methyl-naphthyl -Dimethylpropylnaphthyl, 1-ethylpropylnaphthyl hexylnaphthyl, heptylnaphthyl or octylnaphthyl;

Alkylhetaryl branched or unbranched Ci-Cβ-alkylhetaryl residues, the simple or fused aromatic ring systems with one or more heteroaromatic 3- to 8-membered rings, which may optionally contain one or more heteroatoms such as S, N or 0;

Aryl such as phenyl, naphthyl, anthranyl or 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 0, such as thienyl, pyridyl or indoyl. 5

All of the radicals R ^{1 mentioned} can optionally be combined with one or more of the radicals: branched or unbranched C ₁ -C ₄ -alkyl-, aryl-, -COOR, -NH _P (-C-C ₈ -alkyl) ₂ -p, -QH _n (-C-C ₈ alkyl) ι_ _n , -SS-t-butyl, -CN, -N0 ₂ or halogen such as fluorine, chlorine, bromine or 0 iodine, where R = H or Ci-Cs-alkyl, p = 0, 1 or 2, Q = sulfur or oxygen, n = 0 or 1 and Cχ-C ₈ alkyl or aryl has the meaning given above.

R ¹ residues derived from natural or natural amino acids are preferred, functional groups of these residues being protected or unprotected. Since the residues R ^{1 are} advantageously derived from natural or unnatural amino acids, the stereocenters adjacent to the residues can be in both the D and the L configuration (= R or S form). More preferred radicals for R ¹ are substituted or unsubstituted, branched or unbranched C ₁ -C ₄ alkyl, Cι ~ _C4 alkylaryl or Cι-C ₄ -Alkylhetaryl-, substituted or unsubstituted aryl or hetaryl, particularly preferably is C 1 -C ₄ alkylaryl. 5

R ² substituted or unsubstituted 2-thienylmethyl-, α- or ß-naphthylmethyl-, N-Boc-indol-3-ylmethyl _' where the following advantageous radicals may be mentioned as substituents for R ² : branched or unbranched C ₁ -C ₄ alkyl -, aryl-, -COOR, -NH _p (-CC ₈ alkyl) ₂ _ _p , -QH _n (-C ₈ -alkyl) ι- _n , 0 -SS-t-butyl, -CN, - N0 ₂ or halogen such as fluorine, chlorine, bromine or iodine, where R = H or -CC ₈ _alkyl, p = 0, 1 or 2, Q = sulfur or oxygen, n = 0 or 1 and Cι-C ₈ - Alkyl or aryl has the meaning given above. 5

R ³ H, substituted or unsubstituted acyl, in particular in the form of an acetyl or benzoyl radical

The compounds according to the invention can be present as free compounds, or in the form of their physiologically active salts, their renomeric and / or isomeric forms or in the form of the combination of the free compounds and the various salts. The compounds according to the invention are also to be understood as meaning the enantiopure or diastereomerically pure compounds, their salts or mixtures thereof. 5 The enantiomeric or diastereomeric forms of the compounds according to the invention can be purified or prepared in a known manner, for example by forming diastereomer salts, by chiral chromatography processes or by stereoselective synthesis.

The compounds of the formula I were prepared by standard methods of peptide chemistry and protective group chemistry, which the person skilled in the art from, for example, Müller et al. (Houben Weyl Methods of Organic Chemistry, Vol. XV Thieme Verlag Stuttgart),

Fields et al. (Int. J. Peptide Res., 35, 1990, 161) or from the literature cited in the Novabiochem Catalog & Peptide synthesis Handbook 1997 and 1998 by adding a suitably protected amino acid (II) first by activation with a protected D-naphthylalanine -, N ⁱⁿ BocTryptophan- or thienylalanine building block (III) is implemented. Removal of the protective group SG ₂ and reaction with activation with the α-bromocarboxylic acid and csthioacetate (V, see Kellogg, J. Org. Chem., 1986, 51, 3664) then yields (VI). Simple manipulation of the protective groups then makes the compounds of the formula I accessible (see reaction scheme I).

The protective group SGi includes all protective groups known to those skilled in protein synthesis, such as t-butyl, benzyl, benzyloxycarbonyl, trityl, methyl or polymer-bound protective groups in the form of the commercially available polystyrene resins, such as e.g. 2-chlorotrityl chloride resin or Wang resin (Bachern, Nobvabiochem) are suitable. Preferred protective groups are t-butyl and 2-chlorotrityl resins.

Fluorenylmethoxycarbonyl, t-butyloxycarbonyl or benzyloxycarbonyl are suitable as the protective group SG. Activation methods of choice are uronium salts or carbodiimides such as DCC, WSC.

Polymeric protective group 2C1-trityl is preferred for SGi, fluorenylmethoxycarbonyl for SG and DIC in the presence of HOBt as the activation method.

At this point, reference is made to the corresponding specialist literature mentioned above. Reaction Scheme I: Synthesis of the Compounds of Formula I

Remove the protective groups SGi

Compounds of the general formula I inhibit the endothelin conversion enzyme in the μm range. In addition, they also inhibit other relevant metalloproteases such as ACE (= "angiotensin converting enzyme") or NEP 24.11 (= neutral endopeptidase 24.11) and thus enable a very good pharmacological effect at low doses. Matrix metalloproteases (= MMP) such as MMP-1, MMP-3 or MMP-9 are not inhibited by the inhibitors in this concentration range. The advantage of such an inhibition is obvious: on the one hand, other enzymatic processes are not inhibited non-specifically, so that undesirable side effects are also not to be expected, and on the other hand, several proteases involved in the regulation of blood pressure are inhibited. It is therefore highly likely that the inhibitors can be administered in very low doses, which can reduce the likelihood of side effects.

Compounds of the general formula I which are distinguished by the central amino acid D-2-Nal, Trp-Boc or D-Thi thus have a very balanced pharmacology and therefore have advantages in the treatment of diseases in the indication area cardiovascular. The compounds of formula I, their stereomeric, diastereomeric forms and / or physiologically active salts, and their tautomeric or isomeric forms are suitable for the production of pharmaceutical preparations suitable for ECE inhibition for the treatment of diseases, preferably for the production of medicaments that are used to treat diseases associated with the biological effects of endothelin. The enantiomerically pure or diastereomerically pure compounds are preferred as the active ingredient.

The use of the compounds of the formula I for the preparation of pharmaceutical preparations for the treatment of diseases which are associated with the biological action of endothelin offers a new therapeutic potential for the treatment of hypertension, pulmonary hypertension, myocardial infarction, chronic heart failure, angina pectoris, acute / Chronic renal failure, renal insufficiency, cerebral vasospasm, cerebral ischemia, subarachnoid hemorrhage, migraine, asthma, atherosclerosis, endotoxic shock, endotoxin-induced organ failure, intravascular coagulation, restenosis after angioplasty, benign prostatic hyperplasia caused by renal ingestion, ischemia or hypertension, cyclosporin-induced kidney failure, metastasis and growth of mesenchymal tumors, cancer, prostate cancer, contrast agent-induced kidney failure, pancreatitis or gastrointestinal ulcers.

The compounds of formula I are preferably administered in the form of those pharmaceutical preparations in which the release takes place under conditions such as those in certain body compartments, e.g. in the stomach, intestines, bloodstream, liver, predominate.

The invention furthermore relates to combination preparations from pharmaceutical preparations which contain inhibitors of the formula I and inhibitors of the renin-angiotensin system. Inhibitors of the renin-angiotensin system are renin inhibitors, angiotensin II antagonists and, above all, angiotensin converting enzyme (ACE) inhibitors. An advantageous blood pressure lowering effect is thereby achieved through the combination of active substances.

The combinations can be applied in a common galenical form or separately in time and space.

With regard to dosage and type of application, the same factors must be taken into account as for the corresponding individual substances. These combination products are particularly suitable for the treatment and prevention of hypertension and its complications, as well as for the treatment of heart failure.

The compounds according to the invention can be administered in the usual way orally or parenterally (subcutaneously, intravenously, intramuscularly, intraperotonically). It can also be applied with vapors or sprays through the nasopharynx.

The dosage depends on the age, condition and weight of the patient and on the type of application.

Pharmaceutical preparations which contain compounds of the formula I for inhibiting the endothelin conversion enzyme (= ECE) are to be understood in principle as all galenical administration forms, whether solid or liquid, such as tablets, film-coated tablets, capsules, powders, granules, dragees, supposito- Rien, solutions, ointments, creams or sprays. These are manufactured in the usual way. The active ingredients can be processed with the usual pharmaceutical auxiliaries such as tablet binders, fillers, preservatives, tablet disintegrants, flow regulators, plasticizers, wetting agents, dispersants, emulsifiers, solvents, retardants, antioxidants and / or propellants (see H. Sucker et al. : Pharmaceutical Technology, Thieme-Verlag, Stuttgart, 1991). The administration forms obtained in this way normally contain the active ingredient in an amount of 0.1 to 90% by weight.

The combination of a calcium antagonist with the inhibitors of the formula I can be used as an agent for the treatment of diseases which are based on vasoconstriction or are associated with pathological vasoconstriction. Examples are: All forms of high blood pressure (including pulmonary hypertension), coronary heart disease, heart failure, renal and myocardial ischemia, acute and chronic renal failure.

Due to the potentiation of the action of the individual components, the combination of the two action classes is an ideal addition. Another advantage is that the dose reduction reduces undesirable side effects.

The combinations according to the invention are generally administered orally, for example in the form of tablets, coated tablets, dragées, hard and soft gelatin capsules, solutions, emulsions or suspensions. Administration can also be rectal, for example in the form of suppositories, or parenteral, for example in the form of injection solutions, done. The active ingredient can be administered in the form of preparations which contain both active ingredients together, such as tablets or capsules, or separately as an ad-hoc combination of individual substances which can be administered simultaneously or in 5 steps.

A combination according to the invention with pharmaceutically inert, inorganic or organic excipients 10 can be processed to produce tablets, coated tablets, coated tablets and hard gelatin capsules. Lactose, corn starch or derivatives thereof, talc, stearic acid or salts thereof can be used as such excipients for tablets, dragees and hard gelatin capsules. Vegetable oils, waxes, fats, semi-solid and liquid polyols are suitable excipients for soft gelatin capsules.

15

Suitable excipients for the production of solutions and syrups are e.g. Water, polyols, sucrose, invert sugar, glucose and the like. Suitable excipients for injection solutions are water, alcohols, polyols, glycerin, vegetable oils. For 20 suppositories, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols and the like are suitable as excipients.

The pharmaceutical preparations can also contain _e Konservier insurance agents, solubilizers, stabilizing agents. Wetting agents, emulsifying agents, sweetening agents, coloring agents, flavoring agents. Contain salts for changing the osmotic pressure, buffers, coating agents and / or antioxidants.

Examples:

30

Example 1: Synthesis of the Inhibitors (Reaction Scheme I)

500 mg of phenylalanine (II), which was bound to 2Cl-tritylchloride-polystyrene (approx. 0.5 mmol), was after one another in 15 ml of NMP / CH ₂ C1

35 of the 515 ul DIPEA (2 mmol), 305 mg HOBt (2 mmol), 2 mmol of the corresponding D-amino acid in Fmoc-protected form (III) and 645 mg TBTU (2 mmol) were added and shaken for 4 h. After aspirating and washing with NMP and CH2C12, 10 ml of 25% piperidine in NMP were added and the mixture was shaken for two times for 30 min. An¬

Finally, the mixture was suctioned off again and washed with NMP. The further conversion from (IV) to (VI) was carried out without characterizing the intermediates as follows:

45 In 15 ml of NMP, 350 mg (IV) were successively mixed with 241 mg (V) (3 mmol) and 133 mg DIC (3 mmol) and shaken until the ninhydrin test was negative.

After aspirating and washing with NMP, 15 ml of NMP and 94 mg (4.5 mmol) of Cs-thioacetate were again added. After shaking at 23 ° C. for 3 h, the product was filtered off again, washed with NMP, methylene chloride and the compound (VI) dried in vacuo.

Compound (VI) was taken up in a mixture of acetic acid, trifluoroethanol and methylene chloride 1/1/8 and shaken for 1 h. Finally the solid was filtered off and concentrated.

To remove the acetyl protective group to R ³ = H, (VI) 0.5 ml THF / H ₂ 0 3: 1 and 1 ml of a 1 M solution of LiOH in H 0 were added, shaken for 1 h. The pH was then adjusted to 2 using 2N HCl and extracted three times with 5 ml of ethyl acetate each time. The narrowing of the org. Phase gave I (R ³ = H).

The following compounds were obtained by analog synthesis.

Example 2: ECE inhibitor tests, IC ₅₀ determinations

For testing inhibitors of the endothelin conversion enzyme (ECE), recombinant human ECE from CHO cells was used, as described in Schmidt et al. (FEBS Letters 356, 1994: 238-243).

After membrane isolation and solubilization, the enzyme preparations used were further purified by mono-Q chromatography and WGA lectin chromatography. The preparations thus obtained contained no interfering foreign protease activities and had specific activities in the range of 1-20 mU / mg. 5 μl of this enzyme solution were mixed with 495 μl test buffer (100 mM Pi, 500 mM NaCl, 0.1 mg / ml BSA pH 7.2) and each with 5 μl correspondingly concentrated solutions (10 " ³ M, 10" ⁴ M, 10 " ⁵ M etc.) of the inhibitors were pre-incubated in the test buffer for 10 minutes. 50 μl aliquots were mixed with 5 μl 2.10" ³ M Big-ETl solution (= "Big-Endothelinl) in 0.02% acetic acid. The batches were mixed after 1 hour stopped at 37 ° C. by adding 150 μl of 0.5% TFA (= trifluoroacetic acid) in water, centrifuged for 5 minutes at 10000 × g and the enzyme reaction by measuring the endothelin formed by means of reversed phase HPLC, as described in Takada et al (Biochem. Biophys. Res. Comm. 176, 860, 1991), K. Ohnaka et al. (Biochem. Biophys. Res. Commun. 168, 1128, 1990). An inhibition curve was formed from the inhibition values at the various inhibitor concentrations and the half-maximal inhibition (IC ₅₀ value) was read off as a measure of the potency of the inhibitor. Table I shows the ICso values of the various substances compared to ECE, ACE and NEP 24.11.

Table I: IC 50 values of the various inhibitors (in μm)

Claims

Claims:

1. Compounds of the general formula I,

R ¹ H, substituted or unsubstituted, branched or unbranched C 1 -C ₈ alkyl, C 1 -C ₈ alkylaryl or C 1 -C ₈ alkylhetaryl, substituted or unsubstituted aryl or hetaryl,

R ² substituted or unsubstituted 2-thienylmethyl-, ß-naphthylmethyl-, N-Boc-indol-3-ylmethyl '

R ³ H, substituted or unsubstituted acyl.

2. A process for the preparation of compounds of general formula I according to claim 1, characterized in that compounds of general formula II

with compounds of the general formula III

condensed to compounds of formula IV

and then with compounds of formula V

in the presence of CsSAc and splitting off of the protective group SGI to give compounds of the formula I according to claim 1, where the substituents R ¹ , R ² and R ³ have the meaning given in claim 1 and SGi is a protective group.

3. Use of compounds of general formula I, their physiologically active salts or a combination thereof

Claim 1 for the production of pharmaceutical preparations for the treatment of diseases.

4. Use of compounds of general formula I, their physiologically active salts or a combination thereof

Claim 1 for the production of pharmaceutical preparations for the treatment of diseases which are associated with the biological action of endothelin.

5. Use of compounds of general formula I, their physiologically active salts or their combination according to claim 1 for the preparation of pharmaceutical preparations for the treatment of diseases selected from the group of hypertension, pulmonary high pressure, myocardial infarction, chronic heart failure, angina pectoris, acute / chronic kidneys - failure, renal insufficiency, cerebral vasospasm, cerebral ischemia, subarachnoid hemorrhage, migraine, asthma, atherosclerosis, endotoxic shock, endotoxin-induced organ failure, intravascular coagulation, restenosis after angioplasty, benign prostate hypertension, renal intoxication, ischemic inflammation, ischemic inflammation, ischemic inflammation and ischemic inflammation Kidney failure, metastasis and growth of mesenchymal tumors, cancer, prostate cancer, contrast-induced kidney failure, pancreatitis and gastrointestinal ulcers.

6. Use of compounds of general formula I according to claim 1 for inhibiting the endothelin conversion enzyme.

7. Use of compounds of general formula I according to claim 1 in combination with other hypotensive

Active ingredients for the manufacture of pharmaceutical preparations for the treatment of diseases.

8. Pharmaceutical preparations containing compounds of general formula I, their physiologically active salts or their combination according to claim 1.

9. Combination of a pharmaceutical preparation according to claim 8 and at least one other antihypertensive active substances.

10. Combination according to claim 9 containing ACE inhibitors as an antihypertensive active ingredient.