NO309864B1 - Cyclopeptides, processes for their preparation, pharmaceutical preparations, processes for their preparation, and the use of compounds for the preparation of medicinal products - Google Patents

Abstract

The invention relates to novel cyclopeptides of the formula I Cyclo-(A-B-C-D-Arg) I in which A and B in each case independently of one another denote Ala, Asn, Asp, Arg, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, C denotes Asp or Asp(O-C1-4-alkyl) and D denotes Gly or Ala, at least two of the amino acid radicals indicated being present in the D-form, and their salts. These compounds act as integrin inhibitors and can be used in particular for the prophylaxis and treatment of circulation disorders and in tumour therapy.

Description

The invention relates to new cyclopeptides of formula I

where

with at least two of the specified amino acid residues being in the D form,

as well as salts thereof.

Similar compounds are known from Pharmazie, 40 (8), pp. 532-5 (1985).

The invention was based on the task of finding new compounds with valuable properties, especially those that can be used for the production of pharmaceuticals.

It was found that the compounds of formula I and salts thereof have very valuable properties. Above all, they act as integrin inhibitors, as they particularly inhibit the interaction between p3-integrin receptors and ligands. This effect can e.g. is detected by the method indicated by J.W. Smith et al. in J. Biol. Chem., 265, pp. 12267-12271 (1990). In addition, anti-inflammatory effects occur. All these effects can be demonstrated using methods known from the literature.

The compounds can be used as active pharmaceutical compounds in human and veterinary medicine, in particular for prophylaxis and for the treatment of diseases of the circulatory system, thrombosis, heart attack, arteriosclerosis, inflammation, apoplexy, angina pectoris, tumours, osteolytic diseases, especially osteoporosis, angiogenesis and restenosis after angioplasty.

The abbreviations of amino acid residues listed above and below stand for the rest of the following amino acids:

Ala alanine

Asp(OR) aspartic acid (p-ester)

Arg arginine

Gly glycine

Phe phenylalanine

Val valin.

Furthermore, below means:

BOC tert.-butoxycarbonyl

CBZ benzyloxycarbonyl

DCCI dicyclohexylcarbodiimide

DMF dimethylformamide

EDCI N-ethyl-N'-(3-dimethylaminopropyl) -

carbodiimide hydrochloride

An ethyl

FMOC 9-fluorenylmethoxycarbonyl

HOBt 1-Hydroxybenzotriazole

Me methyl

Mtr 4-Methoxy-2,3, 6-trimethylphenyl-sulfonyl OBut tert-butyl ester

OMe methyl ester

OEt ethyl ester

POA phenoxyacetyl

TFA trifluoroacetic acid.

If the above-mentioned amino acids can appear in several enantiomeric forms, e.g. as a component in connection with formula I, above and below all these forms and also mixtures thereof (e.g. the DL forms) are included.

The object of the invention is further a method for producing a compound of formula I according to claim 1 or a salt thereof, characterized in that it is released from one of its functional derivatives by treatment with a solvolysing or hydrogenolysing agent,

or that a peptide of formula II

where

Z is -A-B-C-D-Arg-,

-B-C-D-Arg-A-,

-C-D-Arg-A-B-,

-D-Arg-A-B-C- or

-Arg-A-B-C-D-

or a reactive derivative of such a peptide, is treated with a ring closing agent,

and/or that a basic or acidic compound of formula I is transferred to a salt thereof by treatment with an acid or base.

Above and below, the residues A, B, C, D and Z have the meanings given for formulas I and II, unless otherwise expressly stated.

In the formulas above, alkyl preferably stands for methyl, ethyl, isopropyl or tert-butyl.

Group A is preferably D-Val. B is preferably D-Phe. C is preferably Asp, especially D-Asp. D is preferably Gly.

In accordance with this, the object of the invention is in particular such compounds of formula I where at least one of the mentioned residues has one of the preferred meanings indicated above.

A preferred group of compounds can be expressed through partial formula Ia, which otherwise corresponds to formula I, where however

The compounds of formula I and also the starting compounds for their preparation are otherwise prepared according to known methods, as they are described in the literature (e.g. in such standard works as Houben-Weyl, Methoden der organischen Chemie, Georg-Thieme-Verlag, Stuttgart), and then under reaction conditions which are known and suitable for the aforementioned reactions. Thus, you can also make use of well-known variants, which are not explained in more detail here.

The starting compounds can, if desired, also be formed in situ so that they are not isolated from the reaction mixture, but immediately converted further to the compounds of formula I.

The compounds of formula I can be obtained by freeing them from their functional derivatives by solvolysis, especially hydrolysis, or by hydrogenolysis.

Preferred starting compounds for the solvolysis or hydrogenolysis are those which instead of one or more free amino and/or hydroxy groups contain correspondingly protected amino and/or hydroxy groups, preferably such as instead of an H atom, which is bound to an N- atom, carries an amino protecting group, e.g. those corresponding to formula I, but which instead of an NH2 group contain an NHR' group (where R' means an amino protecting group, e.g. BOC or

CBZ).

Furthermore, starting compounds are preferred which instead of the H atom in a hydroxy group carry a hydroxy protecting group, e.g. such as correspond to formula I, but which instead of a hydroxyphenyl group contain an R''O-phenyl group (where R'' means a hydroxy protecting group).

Several, identical or different, protected amino and/or hydroxy groups may also be present in the starting compound molecule. If the protective groups present are different from each other, they can in many cases be selectively cleaved off.

The term "amino protecting group" is commonly known and refers to groups which are suitable to protect an amino group against chemical reactions (blockers), but which are however easily removable, after which the desired chemical reaction is carried out elsewhere in the molecule. Typical of such groups are particularly unsubstituted or substituted acyl, aryl, aralkyl and aralkyl groups. As the amino protecting groups are removed after the desired reaction (or reaction sequence), their nature and size are otherwise not of decisive importance; however, those with 1-20, especially 1-8, C atoms are preferred. In the context of the present method, the term "acyl group" is to be understood in its broadest sense. It includes acyl groups which are derived from aliphatic, araliphatic, aromatic or heterocyclic carboxylic acids or sulphonic acids, as well as in particular alkoxycarbonyl, aryloxycarbonyl and, above all, aralkylcarbonyl groups. Examples of such acyl groups are alkanoyl such as acetyl, propionyl, butyryl; aralkanoyl such as phenylacetyl; aroyl such as benzoyl or toluyl; aryloxyalkanoyl as POA; alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, BOC, 2-iodoethoxycarbonyl; aralkyloxycarbonyl such as CBZ ("carbobenzoxy"), 4-methoxybenzyloxycarbonyl, FMOC; arylsulfonyl as Mtr. Preferred amino protecting groups are BOC and Mtr, further CBZ, FMOC, benzyl and acetyl.

The term "hydroxy protecting group" is likewise generally known and refers to groups which are suitable to protect a hydroxy group against chemical reactions, but which are however easily removable, after which the desired chemical reaction is carried out elsewhere in the molecule. Typical of such groups are the unsubstituted or substituted aryl, aralkyl and acyl groups mentioned above, and also alkyl groups. The nature and size of the hydroxy protecting groups is not critical as they can again be removed after the desired chemical reaction or reaction sequence; preferred are groups with 1-20, especially 1-10, C atoms. Examples of hydroxy protecting groups are, among others, benzyl, p-nitrobenzoyl, p-toluenesulfonyl and acetyl, benzyl and acetyl being particularly preferred. The COOH groups in aspartic acid and glutamic acid are preferably protected in the form of tert-butyl ester (e.g. Asp (OBut)).

The functional derivatives of the compounds of formula I to be used as starting materials can be prepared according to usual methods in amino acid and peptide synthesis, such as, for example described in the aforementioned standard works and patent applications, e.g. also according to the solid phase method according to Merrifield (B.F. Gysin and R.B. Merrifield, J. Am. Chem. Soc, 94, p. 3102ff

(1972).

The release of the compounds of formula I from their functional derivatives takes place, depending on the protecting group used, e.g. with strong acids, suitably with TFA or perchloric acid, but also with other strong inorganic acids such as hydrochloric or sulfuric acid, strong organic carboxylic acids such as trichloroacetic acid or sulphonic acids such as benzene or p-toluenesulphonic acid. The presence of an additional inert solvent is possible, but not necessary. Organic solvents are preferably suitable as inert solvents, e.g. carboxylic acids such as acetic acid, ethers such as tetrahydrofuran or dioxane, amides such as DMF, halogenated hydrocarbons such as dichloromethane, also alcohols such as methanol, ethanol or isopropanol, as well as water. Furthermore, there are mixtures of the above-mentioned solvents. TFA is preferably used in excess without the addition of a further solvent, perchloric acid in the form of a mixture of acetic acid and 70% perchloric acid in the ratio 9:1. The reaction temperatures for the cleavage are suitably between approx. 0 and 50 °C, preferably working between 15 and 30 °C (room temperature).

The groups BOC, OBut and Mtr can e.g. is preferably cleaved with TFA in dichloromethane or with approx. 3-5 N HC1 in dioxane at 15-30 °C, the FMOC group with an approx. 5-50% solution of dimethylamine, diethylamine or piperidine in DMF at 15-30 °C.

Hydrogenolytically removable protecting groups (e.g.

CBZ or benzyl) can e.g. is cleaved by treatment with hydrogen in the presence of a catalyst (e.g. a noble metal catalyst such as palladium, conveniently on a support such as charcoal). As a solvent, those indicated above are suitable, especially e.g. alcohols such as methanol or ethanol, or amides such as DMF. Hydrogenolysis is usually carried out at temperatures between approx. 0 and 100 °C, and pressure between approx. 1 and 200 bar, preferably at 20-30 °C and 1-10 bar. A hydrogenolysis of the CBZ group takes place e.g. well at 5-10% Pd-C in methanol or with ammonium formate (instead of H2) on Pd-C in methanol/DMF at 20-30 °C.

Compounds of formula I can also be obtained through cyclization of compounds of formula II under the conditions of a peptide synthesis. Thereby, work is carried out appropriately according to usual methods within peptide synthesis, such as e.g. described in Houben-Weyl, vol. 15/11, pp. 1-806 (1974).

The reaction preferably takes place in the presence of a dehydrating agent, e.g. a carbodiimide such as DCCI or EDCI, further propanephosphonic anhydride (cf. Angew. Chem., 92, p. 129

I (1980)), diphenylphosphoryl azide or 2-ethoxy-N-ethoxycarbonyl-

in

1,2-dihydroquinoline, in an inert solvent, e.g. an i

in

in

halogenated hydrocarbon such as dichloromethane, an ether such as tetrahydrofuran or dioxane, an amide such as DMF or dimethylacetamide, a nitrile such as acetonitrile, or in mixtures of these solvents, at temperatures between approx. -10 and 40 °C, preferably between 0 and 30 °C. In order to retain the intramolecular ring closure before the intermolecular peptide bond, it is appropriate to work in diluted solutions (pre-dilution principle).

Instead of II, suitable reactive derivatives of these substances can also be used in the reaction, e.g.

such where the reactive groups are blocked intermediately by protective groups. The amino acid derivatives II can e.g. are used in the form of the activated esters thereof, which are suitably formed in situ, e.g. by addition of HOBt or N-hydroxy-cysuccinimide.

The starting compounds of formula II are usually new. They can be produced according to known methods, e.g. the above-mentioned methods for peptide synthesis and removal of protective groups.

As a rule, protected penta-peptide esters with the formula R'-Z-OR'' are first synthesized, e.g. BOC-Z-OMe or BOC-Z-OEt, which are then saponified into acids of formula R'-Z-OH, e.g. BOC-Z-OH; from these the protective group R' is cleaved off, whereby the free peptide of formula H-Z-OH (II) is obtained.

A base of formula I can be transferred with an acid to the corresponding acid addition salt. For this turnover, acids that give physiologically acceptable salts are particularly relevant. Thus, inorganic acids can be used, e.g. sulfuric acid, nitric acid, halogen hydrogen acids such as hydrochloric acid or hydrobromic acid, phosphoric acids such as orthophosphoric acid, sulfamic acid, further organic acids, especially aliphatic, alicyclic, araliphatic, aromatic or heterocyclic mono- and polybasic carboxylic, sulfonic or sulfuric acids, e.g. formic acid, acetic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, lactic acid, tartaric acid, malic acid, benzoic acid, salicylic acid, 2- or 3-phenylpropionic acid, citric acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methane - or ethanesulfonic acid, ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,

vs

p-toluenesulfonic acid, naphthalene mono- and -disulfonic acids, lauryl-sulphuric acid. Salts with physiologically unacceptable acids, e.g. picrates, can be used for the isolation and/or purification of the compounds of formula I.

On the other hand, an acid of formula I can be converted into one of its physiologically acceptable metal or ammonium salts by reaction with a base. As salts, sodium, potassium, magnesium, calcium and ammonium salts, further substituted ammonium salts, e.g. dimethyl, diethyl or diisopropylammonium salts, monoethanol, diethanol or triethanol ammonium salts, cyclohexyl, dicyclohexyl ammonium salts, dibenzylethylene diammonium salts, also e.g. salts with N-methyl-D-glucamine or with arginine or lysine.

The new compounds of formula I and their physiologically acceptable salts can be used for the preparation of pharmaceutical preparations by bringing them together with at least one carrier or auxiliary substance and, if desired, together with one or more additional active compounds in a suitable dosage form. The preparations thus obtained can be used as pharmaceuticals in human or veterinary medicine. Carrier substances are organic or inorganic substances suitable for enteral (e.g. oral or rectal), parenteral (e.g. intravenous injection) or local (e.g. topical, dermal, ophthalmic or nasal) administration. application, or suitable for an application in the form of an inhalation spray, and which does not react with the new compounds, e.g. water or aqueous, isotonic saline solution, lower alcohols, vegetable oils, benzyl alcohols, polyethylene glycols, glycerol triacetate and other fatty acid glycerides, gelatin, soy lecithin, carbohydrates such as lactose or starch, magnesium stearate, talc, cellulose, petroleum jelly. For oral use, tablets, dragees, capsules, syrups, juices or drops are particularly useful; of particular interest are lacquer tablets and capsules with gastric juice-resistant coatings or capsule sleeves. For rectal application, suppositories serve, for parenteral application solutions, preferably oil or water solutions, further suspensions, emulsions or implants. For topical application, e.g. solutions that can be used in the form of eye drops, further e.g. suspensions, emulsions, creams, ointments or compressed preparations. For application as an inhalation spray, sprays containing the active compound either dissolved or suspended in a propellant gas or propellant gas mixture (e.g. C02 or chlorofluorohydrocarbons) can be used. Appropriately, the active compound is then used in micronized form, as one or more additional physiologically compatible solvents may be added, e.g. ethanol. Inhalation solutions can be administered using ordinary inhalers. The new compounds can also be lyophilized and the lyophilizates obtained are used for the production of injection preparations. The injections can thereby be given as a bolus or as a continuous infusion (eg intravenous, intravascular, subcutaneous or intrathecal). The stated preparations may be sterilized and/or contain auxiliary substances such as preservatives, stabilizers and/or wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffer substances, coloring and/or flavoring substances. If desired, they can also contain one or more additional active compounds, e.g. one or more vitamins.

The compounds according to the invention can, as a rule, be administered analogously to other known peptides that are available in the trade, particularly, however, analogously to the compounds described in US patent document no. 4,472,305, preferably in dosages between approx. 0.05 and 500, especially between 0.5 and 100 mg per dosage unit. The daily dosage is preferably between approx. 0.01 and 2 mg/kg body weight. However, the particular dose for each particular patient depends on a wide variety of factors, e.g. of the activity of the particular compound used, of the age, body weight, general state of health, heredity, of the diet, of the time and route of administration, of the excretion rate, the drug combination and the severity of the individual disease for which the therapy applies. The parenteral application is preferred.

Above and below, all temperatures are given in °C. In the following examples, "usual work-up" means: if necessary, water is added, it is neutralized, extracted with ether or dichloromethane, separated, the organic phase is dried over sodium sulfate, filtered, evaporated and purified by chromatography on silica gel and/or crystallization. RZ = retention time (minutes) by HPLC on a "LiChrosorb RP select B" column (250-4.7 µm), eluent: 0.3% TFA in water, isopropanol gradient 0-80 vol% in 50 minutes at 1 ml/ minute. Flow through and detection at 215 nm. M<+> = molecular peak in the mass spectrum obtained according to the "Fast Atom Bombardment" method.

Example 1

A solution of 30 mg of cyclo-(D-Val-L-Phe-D-Asp-(OBut)-Gly-D-Arg(Mtr)) [which can be obtained by cyclization of H-D-Arg(Mtr)-D- Val-L-Phe-D-Asp(OBut)-Gly-OH according to the method stated in example 2] in 840 ul TFA, 170 ul dichloromethane and 85 ul thiophenol was allowed to stand for 2 hours at 20 °C, then evaporated at 37 °C under reduced pressure and freeze-dried after dilution with water. After gel filtration on "Sephadex G10" in acetic acid/water 1:1 and subsequent purification by preparative HPLC in a "LiChrosorb RP8" column with an isopropanol gradient in 0.3% TFA/water, cyclo-(D-Val-L -Phe-D-Asp-Gly-D-Arg), RZ 17.9; M<+> 575.

Analogously, one got:

from cyclo-(D-Val-D-Phe-D-Asp(OBut)-Gly-D-Arg(Mtr)): cyclo-(D-Val-D-Phe-D-Asp-Gly-D-Arg) , RZ 18.5; M<+> 575; from cyclo-(D-Val-D-Phe-L-Asp(OBut)-Gly-D-Arg(Mtr)): cyclo-(D-Val-D-Phe-L-Asp-Gly-D-Arg) , RZ 19.3; M<+> 575; from cyclo-(D-Val-D-Phe-D-Asp(OBut)-L-Ala-D-Arg(Mtr)): cyclo-(D-Val-D-Phe-D-Asp-L-Ala- D-Arg), RZ 20.3; M<+> 589; from cyclo-(D-Val-D-Phe-D-Asp(OMet-Gly-L-Arg(Mtr)): cyclo-(D-Val-D-Phe-D-Asp(OMe)-Gly-L- Arg), RZ 21.4;

M<+> 589 p

Example 2

A solution of 80 mg of H-L-Arg-D-Val-D-Phe-D-Asp-(OMe)-Gly-ONa [which can be obtained by cleaving the FMOC group from FMOC-L-Arg-D-Val- D-Phe-D-Asp(OMe)-Gly-0-Wang (where O-Wang means the residue used in the Merrifield synthesis of a

-4-oxymethylphenoxymethyl-polystyrene, which is cross-linked to 1% with p-divinylbenzene) with morpholine and cleavage of penta-

the peptide from the polymer with TFA/dichloromethane 1:1] in 8 ml of DMF was diluted with 72 ml of dichloromethane and 34 mg of finely ground NaHCO 3 was added. After cooling in dry ice/acetone, 34 µl of diphenylphosphoryl azide was added. After standing for 16 hours at 20°C, the dichloromethane was removed at 37°C. The remaining solution was gel filtered ("Sephadex G10" column in isopropanol/water 8:2) and then chromatographed on a polymer column ("Mitsubishi MCI CHP-20P") in an isopropanol in water gradient. Cyclo-(D-Val-D-Phe-D-Asp(OMe)-Gly-L-Arg) was obtained, RZ 21.4; M<+> 589.

The following examples relate to pharmaceutical preparations.

Example A

In1ex1onsalass

A solution of 100 g of a cyclopeptide of formula I and 5 g of disodium hydrogen phosphate in 3 L of double-distilled water was adjusted to pH 6.5 with 2 N hydrochloric acid, sterile filtered and filled into injection vials, lyophilized under sterile conditions and closed sterile. Each vial contained 5 mg of active compound.

Example B

Suppositories

A mixture of 20 g of active compound of formula I was melted with 100 g of soy lecithin and 1400 g of cocoa butter, poured into molds and allowed to cool. Each suppository contained 20 mg of active compound.

Example C

Resolution

A solution of 1 g active compound of formula I, 9.38 g NaH 2 PO 4 x 2 H 2 O, 28.48 g Na 2 HPO 4 x 12 H 2 O and 0.1 g benzalkonium chloride in 940 ml double-distilled water was made. It was adjusted to pH 6.8, filled up to 1 1 and sterilized by irradiation. This solution can be used in the form of eye drops.

Example D

Ointment

500 mg of active compound of formula I was mixed with 99.5 g of petroleum jelly under aseptic conditions.

Example E

Pills

A mixture of 100 g of a cyclopeptide of formula I, 1 kg of lactose, 600 g of microcrystalline cellulose, 600 g of corn starch, 100 g of polyvinylpyrrolidone, 80 g of talc and 10 g of magnesium stearate was compressed in the usual manner into tablets so that each tablet contained 10 mg active connection.

Example F

Dragees

Tablets were pressed as indicated in Example E and then coated in the usual manner with a coating of sucrose, corn starch, talc, tragacanth and colorant.

Example G

Capsules

In the usual way, hard gelatin capsules were filled with an active compound of formula I so that each capsule contained 5 mg of active compound.

Example H

Inhalation regulations

14 g of active compound of formula I was dissolved in

10 1 isotonic NaCl solution and the solution filled in commercial syringe containers with a pump mechanism. The solution can be sprayed into the mouth or nose. One syringe stroke (approx. 0.1 ml) corresponds to a dose of approx. 0.14 mg.

Claims (10)

1. Cyclopeptide, characterized in that it has formula I where A is Val, B is Phe, C is Asp or Asp(O-C 1 -alkyl), and D is Gly or Ala, in that at least two of the specified amino acid radicals are present in D-form, as well as salts thereof. 2. Cyclopeptide according to claim 1, characterized in that it is cyclo-(D-Val-L-Phe-D-Asp-Gly-D-Arg). 3. Cyclopeptide according to claim 1, characterized in that it is cyclo-(D-Val-D-Phe-D-Asp-Gly-L-Arg). 4. Cyclopeptide according to claim 1, characterized in that it is cyclo-(D-Val-D-Phe-L-Asp-Gly-D-Arg). 5. Cyclopeptide according to claim 1, characterized in that it is cyclo-(D-Val-D-Phe-D-Asp-L-Ala-D-Arg). 6. Cyclopeptide according to claim 1, characterized in that it is cyclo-(D-Val-D-Phe-D-Asp(OMe)-Gly-L-Arg). 7. Process for producing a compound of formula I according to claim 1 or a salt thereof, characterized in that it is released from one of its functional derivatives by treatment with a solvolysing or hydrogenolysing agent, or that a peptide of formula II where Z is -A-B-C-D-Arg-, -B-C-D-Arg-A-, -C-D-Arg-A-B-, -D-Arg-A-B-C- or -Arg-A-B-C-D- or a reactive derivative of such a peptide, is treated with a ring closing agent, and/or that a basic or acidic compound of formula I is transferred to a salt thereof by treatment with an acid or base. 8. Procedure for the production of pharmaceutical preparations, characterized in that a compound of formula I according to claim 1 and/or a physiologically acceptable salt thereof is brought together with at least one solid, liquid or semi-liquid carrier or auxiliary substance in a suitable dosage form. 9. Pharmaceutical preparation, characterized in that it has a content of at least one compound with the general formula I according to claim 1 and/or a physiologically acceptable salt thereof. 10. Use of compounds of formula I according to claim 1 or of physiologically acceptable salts thereof for the preparation of a medicinal product.