DE2621279A1 - Esters and amides of nonapeptide - inhibitors of hypertensive effect of angiotensinI - Google Patents

Abstract

Peptides (I) are new: Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-Z (I) (where Z = or or NH2; R = 1-10 C alkyl). (I) inhibit the conversion of angiotensin I (hypertensin I) to angiotensin II in vivo (but surprisingly are not active in vitro). (I) are thus potent inhibitors of angiotensin I-induced hypertension (essential hypertension) at 0.5-10 mg/kg parenterally in mammals.

Description

n Nonapeptide ester and amide, process for their preparation

and medicinal preparations "The invention relates to esters and the amide des Nonapeptids Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro, process for their preparation and medicinal preparations containing these compounds.

By the action of the enzyme renin on the renin substrate, a pseudoglobulin In the blood plasma, the polypeptide angiotensin I, also known as hypertensin I, is called gets formed. This polypeptide is enzymatically converted into angiotensin IT, also called hypertensin II or called angiotonin, converted.

Angiotensin II, a compound that increases blood pressure occurs in the plasma in hypertensive conditions in quantities sufficient to Exercises to maintain the high blood pressure. By inhibiting the transformation from angiogensin I to angiotensin II enzyme responsible can a cause of high pressure can be eliminated.

U.S. Patent 3,832,337 relates to various peptides and acylated peptides, which inhibit the enzymatic conversion of angiotensin 1 to angiotensin II. there the peptide Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro is also listed.

Studies on the relationships between structure and activity of the peptides disclosed in US Pat. No. 3,832,337 have shown that a free, terminal Carboxyl group is necessary to achieve strong in vitro and in vivo inhibition; See, e.g., Cushman et al., "Inhibition of Angiotensin-Converting Enzyme by Analogs of Peptides from Bothrops jararaca Venom, Experienta, Vol. 29 (1973), p. 1032, Publishing house Birkhäuser, Basel.

It has now been found according to the invention that not only the aforementioned Nonapept1d is an inhibitor for the hypertension induced by angiotension 1, but that esters and amides of this nonapeptide are also suitable for this purpose. This is surprising in that these esters and amides have a very low in vitro or have undetectable enzymatic inhibiting effects. Generally corresponds to in this area the in vitro activity of peptides and in vivo activity. Consequently it is surprising that these esters and amides are effective inhibitors for the by Angiotensin I-induced hypertension.

The invention relates to nonapeptides of the general formula I Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-Y, (I) in which Y is the radical -NH2 or -OR, where R represents an alkyl radical having 1 to 10 carbon atoms.

According to the invention, these compounds are prepared in that corresponding amino acids or peptides or their reactive derivatives according to in the usual methods of peptide synthesis to give the peptide of the formula Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro before coupling or converting and / during or after the formation of this peptide in the terminal Proline residue introduces an alkyl ester or amide group by customary methods.

Unless otherwise stated, all amino acids are in the L configuration before. The short symbols used for the amino acids have the following meanings: Arg = arginine Gln = glutamine Ile = isoleucine Pro = proline Pyr = pyroglutamic acid Trp w Tryptophan The esters of the general formula Ia Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-OR, (Ia) in the right one. 1 \ ikylrest means, and the amide of the formula II Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-NH2 (II) have an inhibitory effect on the high pressure induced by angiotensin I.

Examples of alkyl radicals of the aforementioned esters are straight-chain or branched hydrocarbon radicals with 1 to 10, preferably 1 to 6, carbon atoms, like the methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, Pentyl, hexyl, heptyl, octyl, nonyl and decyl groups, as well as the various Isomers of the latter six groups. The methyl and tert. -Butyl ester.

The esters of the invention are preferably made by reacting a Di-, tri-, tetra-, penta-, hexa-, hepta-, or octapeptide with a terminal one Prolyl radical, which is protected by an alkyl ester radical, with one or more Peptides - depending on the requirements - according to methods customary in peptide synthesis In this way one obtains the nonapeptide alkyl esters of the general Formula Ia.

The esters of the invention can be formed directly without that first the starting peptide of the formula III Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro (III) is formed. The amide of the invention is preferably made by reacting a Di-, tri-, tetra-, penta-, hexa-, hepta- or octapeptide with a terminal, prolyl radical protected by an amide group with one or more peptides - Depending on the requirements - manufactured according to the usual methods of peptide synthesis. This gives the nonapeptide amide of Formula II. This way becomes the amide of the invention directly without prior formation of the parent peptide Formula III formed.

However, it is also possible to prepare the esters and the amide of the invention, by first using the method of US Pat. No. 3,832,337, the starting peptide of Formula III produces and then the terminal prolyl group according to the usual Provides process with an alkyl ester or amide group.

The compounds of the invention inhibit the conversion of angiotensin I in angiotensin II in vivo and act antagonistically against the hypertensive Effect of Angiotensin 1. The compounds of the invention have when administered to mammals such as rats, mice or dogs, in doses of about 0.5 to about 10 mg / kg an inhibitory effect on the hypertensive effect of angiotensin I. To this The compounds of the invention can be used parenterally together with physiological purposes compatible carriers.

The invention thus also relates to medicinal preparations with an inhibiting effect on the hypertensive effect induced by angiotensin I. are characterized by a content of a nonapeptide ester or amide of the invention.

The examples illustrate the invention.

Example 1 Benzyloxycarbonyl-isoeucyl-propyl-proline 4.24 g of prolyl-proline hydrobromide are dissolved in a mixture of 40 ml of dimethylformamide and 2.8 ml of triethylamine. Then 9.27 g of benzyloxycarbonyl-isoleucine-p-nitrophenyl ester and 3 g of 1-hydroxybenzotriazole are obtained admitted. The mixture is kept at room temperature for 16 hours. Afterward 1 ml of dimethylaminopropylamine is added. After 2 hours the solvent will removed under reduced pressure. The residue is dissolved in 200 ml of ethyl acetate dissolved and washed successively with 0.1 N hydrochloric acid and water. The organic Phase is dried over magnesium sulfate and under reduced pressure to dryness evaporated. 13.8 g of solid are obtained. This product is dissolved in ethyl acetate placed on a column charged with 300 g of silica gel and with the same solvent eluted. The fractions containing the desired tripeptide are pooled and evaporated to dryness ne.

Example 2 Benzyloxycarbonyl-isoeucyl-propyl-proline-methyl ester Das tripeptide obtained according to Example 1, 6 g) is dissolved in methanol and with a Solution of diazomethane in diethyl ether treated until a steady yellow color arises.

After 50 minutes, a few drops of acetic acid are added until the yellow color disappears. The solvent is removed under reduced pressure and replaced by ethyl acetate. The solution is successively with 0.1 N hydrochloric acid re, Water, saturated sodium hydrogen carbonate solution and water. The organic Phase is dried over magnesium sulfate and evaporated to dryness. You get the title compound.

Example 3 Benzyloxycarbonyl-glutaminyl-isoleucyl-propyl-proline-methyl ester 1.4 g of the tripeptide from Example 2 are in 30 ml of 95 percent ethanol and 3 ml of 1N hydrochloric acid dissolved. Then 0.3 g of palladium-on-charcoal are added. The suspension is stirred under excess hydrogen pressure until it flows out Gas no more carbon dioxide can be detected. The catalyst is filtered off and the filtrate evaporated under reduced pressure. The residue is in a mixture dissolved from 6 ml of dimethylformamide and 0.5 ml of triethylamine. Then 1, 6 g Benzyloxycarbonyl-glutamine-p-nitrophenyl ester and 0.5 g of 1-hydroxybenzotriazole were added. The reaction is carried out at room temperature until the ninhydrin test negative, then 0.5 ml of dimethylaminopropylamine are added a further hour's reaction time, 200 ml of 3ig5äurecthyle5ter are added.

The solution obtained is washed until it reacts neutrally. The organic phase is dried over magnesium sulfate. After removing the The title compound is obtained using a solvent under reduced pressure.

Example 4 Benzyloxycarbonyl - # - nitroarginyl-propyl-glutaminyl-isoeucylproll-roline methyl ester 14 g of the tetrapeptide from Example 3 are dissolved in 325 ml of anhydrous ethanol and 23 ml of 1N hydrochloric acid dissolved. Then 3.0 g of palladium-on-charcoal are added. The mixture is stirred under hydrogen pressure until no carbon dioxide more is released. The filtrate obtained after filtering off the catalyst is evaporated to dryness.

The residue is dissolved in 43 ml of dimethylformamide and 3.2 ml of triethylamine solved. Immediately thereafter, 15.7 g of benzyloxycarbonyl-nitroacrginyl-proline-2,4,5-trichlorophenyl ester are obtained and 3.1 g of 1-hydroxybenzotriazole were added. The implementation is carried out as long as until the ninhydrin test is negative.

5.4 ml of dimethylaminopropylamine are then added. After 2 hours the solvent is removed under reduced pressure. The residue is in Dissolved ethyl acetate and washed until neutral. Then will the ethyl acetate to a volume of about 70 ml under reduced pressure constricted. The solution obtained is dissolved in 1 liter of diethyl ether with vigorous stirring poured. The precipitate is filtered off and dried. The title compound is obtained.

Example 5 Benzyloxycarbonyl-tryptophyl-propyl-arginyl-propyl-glutaminylisoleucyl-propyl-proline methyl ester 1.8 g of the hexapeptide from Example 4 are in 27 ml of water free Ethanol and 4 ml of 1N hydrochloric acid dissolved. Then 0.38 g of palladium on activated carbon are added admitted. The mixture is stirred under hydrogen pressure until the UV absorption of the chromophoric nitroguanidine residue can no longer be detected. That The filtrate obtained after filtering off the catalyst is reduced under reduced pressure Evaporated pressure. The residue is dissolved in 10 ml of dimethylformamide and 0.28 ml of triethylamine solved. Immediately thereafter, 1.5 g of benzyloxycarbonyltryptophyl-proline-2,4,5-trichlorophenyl ester are added and 0.3 g of 1-hydroxybenzotriazole were added. The implementation will continue as long as until the ninhydrin test is negative. The precipitated triethylamine hydrochloride it is filtered off, the filtrate obtained is dissolved in 250 ml of ethyl acetate with vigorous stirring poured. The precipitate is filtered off and dried. The title compound is obtained.

Example 6 Pyroglutamyl-tryotophyl-prolyl-arginyl-propyl-glutaminyl-isoleucyl-prolyl-proline methyl ester 10 g of the octapeptide from Example 5 are dissolved in 150 ml of anhydrous ethanol and 9 ml / hydrochloric acid dissolved. Then 2 g of palladium-on-charcoal are added. the Suspension is maintained under hydrogen pressure until there is no carbon dioxide more is being developed. The filtrate obtained after filtering off the catalyst is evaporated to dryness under reduced pressure. The residue is in 40 ml of dimethylformamide dissolved. Then 1.2 g 1-hydroxybenzotriazole, 1.2 g of triethylamine and pyroglutamic acid -2,4,5-trichlorophenyl ester admitted. The reaction is continued until the ninhydrin test is negative runs.

The precipitated triethylamine hydrochloride is filtered off.

The filtrate obtained is dissolved in 1.1 liters of ethyl acetate with vigorous stirring poured. The precipitate is filtered off and dried. The title compound is obtained. This product is chromatographed on three-dimensionally crosslinked dextran (Sephadex G-25) cleaned in 0.01 M ammonium hydrogen carbonate solution.

Amino acid analysis: The molar ratios found are given. The numbers in round brackets represent the calculated values.

NH3 0.92 (1.0); Arg 0.86 (1.0); Per 4.0 (4.0); Trp 0.78 (1.0); Gln 2.12 (2.0); Ile 0.96 (1.0).

Dual layer chromatography on silica gel using a mixture from butyl alcohol, pyridine, acetic acid and water in a ratio of 30: 20: 6: 24 as solvent: Rf = 0.53.

Example 7 Benzyloxycarbonyl-isoleucyl-propyl-proline-n-butyl ester A solution of 4.5 g of benzyloxycarbonyl-isoleucyl-prolylproline in 30 ml of chloroform slowly becomes a solution of 1.9 g of 1-n-butyl-3-p-tolyltriazine in 15 ml of chloroform given.

After the reaction is complete, the chloroform solution is neutralized Reaction washed. The organic phase is dried over magnesium sulfate and evaporated under reduced pressure. The title compound is obtained.

Example 8 Benzyloxycarbonyl-glutaminyl-isoleucyl-propyl-proline-n-butyl ester This compound is made according to Example 3 using the tripeptide from Example 7 produced as the starting material.

Example 9 Benzyloxycarbonyl - # - nitroarginyl-propyl-glutaminyl-isoleucylpropyl-proline-n-butyl ester This compound is obtained according to Example 4 using that according to Example 8 Tetrapeptide produced as the starting compound.

Ex. 10 Benzyloxycarbonyl-tryptophyl-propyl-arginyl-propyl-glutaminylisoleucyl-propyl-proline-n-butyl ester This compound is prepared according to Example 5 using that prepared according to Example 9 Hexapoptids produced as the starting compound.

Example 11 Pyroglutamyl-tryptophyl-propyl-arginyl-propyl-glutaminyl isoleucyl-propyl-proline-n-butyl ester This compound is according to Example 6 under Using the octapeptide obtained according to Example 10 as the starting compound.

13 e i 5 p i e 1 12 Benzyloxycarbonyl-isoleucyl-propyl-proline-amide 4.5 g of benzyloxycarbonyl-isoleucyl-prolyl-proline are in a mixture of 30 ml Tetrahydrofuran and 1.4 ml of triethylamine dissolved. The solution is in a cooling belt cooled from -50C and treated with 1.6 ml of isobutyl chloroformate. Then the solution is allowed to come to room temperature within 10 minutes and the mixture is added them with 10 ml of aqueous ammonia solution. After stirring for 4 hours at room temperature the reaction mixture is concentrated under reduced pressure with ethyl acetate diluted and successively with sodium hydrogen carbonate solution, water, 0.1 N hydrochloric acid and washed water. The organic phase is dried over magnesium sulfate and evaporated to dryness under reduced pressure. i4an receives the title compound.

13 e i s p i e 1 13 Benzyloxycarbonyl-glutaminyl-isoleucyl-propyl-proline-amide This compound is obtained according to Example 3 using that according to Example 12 Tripeptide prepared as the starting compound.

Example 14 Benzyloxycarbonyl - # - nitroarginyl-propyl-glutaminyl-isoleucylpropyl-proline-amide This compound is obtained according to Example 4 using that according to Example 13 Tetrapeptide produced as the starting compound.

13 e i 5 p i e 1 15 Benzyloxycarbonyl-tryptophyl-propyl-arginyl-propyl-glutaminylisoleucyl-propyl-proline-amide This compound is prepared according to Example 5 using that according to Example 14 Hexapeptide produced as the starting compound.

EXAMPLE 16 Pyroglutamyl-tryptophyl-prolyl-arginyl-propyl-glutaminylisoleucvl-l) rolvl-proline-amide This compound is obtained according to Example 6 using that according to Example 15 Octapeptide produced.

13 e i 5 p i e 1 17 Benzyloxycarbonyl-tryptophyl-propyl-arginyl-propyl-glutaminyl isoleucyl-propyl-proline-tert-butyl ester 9.42 g (about 10 mmol) benzyloxycarbonyl-nitroarginyl-prolylglutaminyl-isoleucyl-propyl-proline-tert-butyl ester werdon dissolved in 135 ml of anhydrous ethanol and 20 ml of 1N HCl. The solution will be with 1.9 g of 10 percent palladium on activated carbon for 22 hours under a hydrogen pressure gertthrt. It is then filtered through Hyflo and dried to dryness under reduced pressure evaporated. The crude residue is dissolved in 50 ml of dimethylformamide and 1.4 ml of triethylamine dissolved and immediately afterwards with 7.4 g (12 mmol) of benzyloxycarbonyl-tryptophyl-pro lyl-2,4,5-trichlorophenyl ester added. After 30 hours, another 1.5 mmol Benzyloxycarbonyl-tryptophyl-prolyl-2,4,5-trichlorophenyl ester and 1.5 mmol of triethylamine added. After 50 hours the precipitated triethylamine hydrochloride is filtered off. The filtrate is added to 1.2 liters of ethyl acetate with vigorous stirring given. The precipitate is filtered off and washed with ethyl acetate.

10.64 g of the title compound are obtained.

3 e i 5 p i e 1 18 pyroglutamyl-tryptophyl-prolyl-arginyl-propyl-glutaminylisoleucyl-propyl-proline tert-butyl ester 10 g (about 8.5 mmol) of the peptide obtained according to Example 17 are in 150 ml of anhydrous Ethanol and. 8.5 ml of 1N hydrochloric acid dissolved. The solution becomes 10 percent with 2 g Palladium-on-charcoal stirred for 8 hours under a hydrogen pressure. To Filtration through Hyflo, the filtrate is evaporated to dryness under reduced pressure. 9.2 g of crude residue are obtained.

This residue is dissolved in 40 nil dimethalformamide. The solution is with 1.14 g (8.5 mmol) of 1-hydroxybenzotriazole and 1.2 ml (8.5 mmol) of triethylamine and immediately thereafter with 3.14 g (20 percent excess) pyroglutamic acid 2,4,5-trichlorophenyl ester offset. A further 0.6 ml of triethylamine are then added. The reaction will Performed overnight. The precipitated triethylamine hydro chloride is filtered off. The filtrate is poured into 1.1 liters of ethyl acetate with vigorous stirring. The precipitate is filtered off through No. 42 filter paper and washed with ethyl acetate washed. on receives 9.0 g of the title compound.

Amino acid analysis: NH3 0.85 (1.0); Trp 0.66 (1.0); Arg 1.0 (1.0); Glu 2.03 (2.0); Per 4.0 (4.0); Ile 0.9 (1.0).

Thin layer chromatography on silica gel using a mixture from butanol, pyridine, acetic acid and water in the ratio of 30: 20: 6: 24 as Eluent: Rf = 0.58.

The product obtained can be dissolved on diethylaminoethylcellulose (DEAE Purify Sephadex A-25) by column chromatography, eluting with 0.005 m NH4HCO3.

3 e i 5 p i e 1 19 pyroglutamyl-tryptophyl-propyl-arginal-prolyl-glutaminyl-iso leucyl-prolyl-proline methyl ester 1.6 g pyroglutamyl-tryptophyl-prolyl-arginyl-propyl-glutaminyl-isoleucyl-prolyl-proline are dissolved in 80 ml of methanol. The solution is made with excess diazomethane in dimethyl ether and allowed to stand for 7 hours at room temperature. Then acetic acid is added to destroy the excess diazomethane. The received The mixture is evaporated to dryness under reduced pressure. The product will on a column loaded with Sephadex G-25, eluting with 0.01 m (NH4) HCO3 and then purified on DEAE Sephadex A-25, eluting with 0.005 M NH4HCO3.

1.1 g of the title compound are obtained.

B e i 5 p i e 1 20 pyroglutamyl-tryptophyl-prolyl-arginyl-prolyl-glutaminylisoleucvl-rolvl-rolin-amide 1 g pyro glutamyl - tryp tophyl-prolyl-arginyl-prolyl-glutaminylisoleucyl-prolyl-proline is dissolved in 6 ml of dimethyl sulfoxide with gentle warming. The solution will be on Cooled room temperature and then with 0.14 ml of triethylamine and 0.16 ml of isobutyl chloroformate offset. The reaction mixture is stirred for 15 minutes. Then 1 ml is concentrated Ammonia solution added. After 4 hours of reaction, the mixture slowly undergoes added vigorous stirring to 200 ml of ethyl acetate. The precipitate formed is filtered off and washed with ethyl acetate. 1.375 g of solid are obtained.

This product is on a 225 ml, with DEAE Sephadex A-25 loaded column using 0.005 M NH4HCO3 as the eluent by chromatography cleaned. 924 mg of the title compound are obtained.

Amino acid analysis: NH3 1.71 (2.0); Arg 0.88 (1.0); Pro 4.13 (4.0); rrrp 0.80 (1.0); Gln 2.12 (2.0); Ile 0.93 (1.0).

Thin layer chromatography on silica gel using a mixture of butanol, pyridine, acetic acid and water in a ratio of 30: 20: 6: 20: Rf = 0.53.

13 e i 5 p i e 1 21 For determining the I50 values (peptide concentration in µg / ml, which inhibits the angiotensin conversion by 50 percent Enzyme effected) the peptide of Example 19 in different concentrations placed in test tubes measuring 13 x 100 mm, the final volume of which is 0.25 each ml is. The composition of the solution in the test tubes is as follows: 100 millimolar potassium phosphate buffer of pH 7.5, 30 millimolar sodium chloride and 0.3 millimolar angiotensin I. The enzymatic reactions are by the addition of Enzyme started and carried out at an incubation temperature of 37 ° C. The concentration of the peptide of Example 19, which inhibits the conversion of angiotensin by 50 percent I in angiotensin II is 7 µg / ml compared to an I50 value (g / ml) of 0.9 for the starting peptide Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro. This in vitro experiment clearly shows that the peptide of Example 19 is significantly lower in vitro Inhibitory effect on the conversion of angiotensin I to angiotensin II as the starting peptide having.

EXAMPLE 22 The peptide of Example 19 and that as a comparative compound The starting peptide Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro used are intravenous Two male rats anesthetized with atropine are given an infusion of Pentolinium and then an injection of 0.10 µg / kg Angistensin I was given, administered. When determining the degree of hemorrhage of Angiotensin I induced blood pressure-increasing effect can be determined that the in vivo activity of the peptide of Example 19 9 approximately had the effect of that used as the comparative compound Corresponds to the starting peptide.

Claims (7)

Claims Peptides of the general formula I Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-Y (i) in which Y denotes the radical -NH2 or -OR, where R is an alkyl radical with 1 to Represents 10 carbon atoms. 2. Peptides according to claim 1, characterized in that Y is the remainder -OR and R denotes an alkyl radical having 1 to 6 carbon atoms. 3. Peptide according to claim 2, characterized in that R is the methyl group means. 4. Peptide according to claim 2, characterized in that R is the tert. -Butyl group means. 5. Peptide according to claim 1, characterized in that Y is the remainder Means NH2. 6. A method for producing the peptides according to claim 1, characterized characterized in that one has corresponding amino acids or peptides or their reactive Derivatives according to processes known per se in peptic synthesis for 1:> eptid the formula Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro couples or converts and before, during or after the formation of this peptide to the terminal prolyl group after per se customary processes an alkyl ester or amide group. 7. Medicinal preparations for the treatment of hypertension, characterized by a content of a peptide according to claim 1.