DE2327634A1 - NONAPEPTIDES AND METHOD FOR MANUFACTURING THEREOF - Google Patents

Description

Dr. h- ^ - -irer- ■ * 3 O. May 1973

PATENT LAWYERS

RAK 4103/7

F. Hofimann-La Roche & Co. Aktiengesellschaft, Basel / Switzerland

Uonapeptides and methods for their. Manufacturing

The present invention relates to new nonapeptides of the formula

Mpr — Tyr — lie —Leu — Asp — Cy s — Pro — Q — GIy—

wherein Q is the remainder of the arginine or lysine, Mpr is ß-mercapto-propionyl and

all amino acids with an asymmetric carbon atom have the L-configuration

• and their pharmaceutically usable, non-toxic acid addition salts, as well as processes for the preparation of these compounds.

In the compounds summarized under the formula I. of the formulas

309884/1435

Mez / 13.4.73

2377334

Mpr — Tyr — lie — Leu — Asp — Cy s — Pro — Arg — GIy — NH _? Ia and

Mpr - Tyr - ^ He - Leu - Asp - Cy s - Pro - Ly s - GIy - KH ₂ Ib

they are analogues of those found in nature Neurohypophyseal hormones, e.g. arginine or lysine vasopressin, of the formulas

H-Cy s-Tyr-Phe-Glu-Asp-Cy s -Pro- Arg- GIy-HH ₂ Ha

NH ₉ NH ₀
ι 2 ι 2

H - Cys - Tyr - Phe - GIu - Asp - Cy s - Pro - Lys - GIy - NH ₂ Hb

Differentiate from the naturally occurring vasopressins the compounds according to the invention thus by replacing the amino acids, cysteine, phenylalanine and Glutamine by ß-mercaptopropionic acid, isoleucine and leucine, so they are also called deamino- [lie, Leu] -argininvasopressin or Deamino-LHe, Leu] -lysinvasopressin can be.

The abbreviations used in the present patent application for the individual amino acids and their Protective groups are those which have hitherto been used in peptide chemistry and are generally known to the person skilled in the art [literature: Schröder, E. and Lübke, K.,:. The Peptides, Academic Press * New York & London, Vol. I (1965) and Vol. II (1966) and IUPIC-IUB rulesJ; they therefore do not need any further lexis here.: ..;. tion.

309884 / U36

ORIGINAL INSPEGTSD

In addition, the β-mercaptopropionic acid, which is also derived from cysteine in the present application also considered to be an "amino acid" so that, for example, β-mercaptopropionyl-tyrosine is considered to be a dipeptide, etc. the speech is,'

Unless expressly stated otherwise, the amino acids with an asymmetry center are always to the L configuration.

Examples of pharmaceutically usable, non-toxic acid addition salts are salts with inorganic acids such as Hydrochloric acid, hydrogen bromide, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic, oxalic, Maleic, malic, vinous or citric acid.

The new honapeptides of formula I and their acid addition salts can be prepared in a manner known per se, in particular by the fact that

a) of a peptide of the "formula

HH-R ¹

CH 1 - CO - Tyr - lie - Leu - Asp - HH - CH - CO - Pro - Q - Giy - NH ₂

CH CH

'' III

S - _:. : S,

wherein R is hydrogen or an amide protecting group; Q ^{1 is} a radical of the formula

-HH-CH [- (CH ₂ ) ₃ -HH-C (= HH) -HHR ² ] -CO- or

-HH-CH [- (CH ₀ ), - HH-R ⁵ ] -CO- and, ₂ . ■ 2 -4

R is hydrogen or a guanidine residue

protective group and ■ 5
R is hydrogen or one of the ε-amino group of the

309884 / U3S

Lysin's protective group mean - but

1 2 at least one of the radicals R and R or Br represents a protective group

and all amino acids with an asymmetric carbon atom Have the L-configuration that splits off protecting groups and that free peptide optionally converted into a pharmaceutically usable, non-toxic acid addition salt by reaction with an organic or inorganic acid or

b) a peptide of the formula

I ²

CH ₂ - GO - Tyr - lie - Leu - Asp - HS - GH - CO - Pro - Q - Siy - M ₂ K ₅ - ■ ₄ KI

S T iJ - ■ P ¹ T CJ

wherein Q has the meaning given above and

, IT and R ^ each hydrogen or sulfhydryl

represent protection groups

and all amino acids with an asymmetric carbon atom have L-configuration, oxidized with simultaneous or prior elimination of any protective groups that may be present and the reaction product, if desired, by reaction with an organic or inorganic acid into a pharmaceutically acceptable non-toxic acid addition salt convicted or

c) a peptide of the formula

NH-R ¹

CH ^ - CO - Tyr - Lie - Leu - Asp - M - CH - CO - Pro - Q '- GIy -KH ₂ S - R ^D R ^ -S

wherein R is hydrogen or an amide protecting group; Q * is a remainder of the formula

-BH-CH [- (CH ₂ ) "- KH-C (= NH) -EHR ² ] -CO- or

309884 / U35

t ■

-BH-GH [- (CH ₂ ) -HH-R ⁵ ] -CO-; R is hydrogen or a guanidine residue

■ protective group;
Ir is hydrogen or one of the ε-amino groups

Lysine's protective group and

R ⁴ and R ⁵ each represent hydrogen or sulfhydryl protective groups, but at least one of the radicals R ¹ and R or R ^{5 represents} a protective group

and all amino acids with an asymmetric carbon atom! configuration, oxidized with simultaneous cleavage of the protective group (s) and, if desired, converted or converted the reaction product into a pharmaceutically usable, non-toxic acid addition salt by reaction with an organic or inorganic acid
d) a compound of the formula

Mpr - Tyr - lie - Leu - Asp - Cy s - Pro - Q - GIy - R VI

where Q is the remainder of the lysine or arginine and $ is hydroxy or a residue which activates the carboxyl group,
amidated or

e) a hexapeptide of the formula

¹ ■ ■ fi

CH _O - CO - Tyr - lie - Leu - Asp - NH - CH - CO - R

I ² I

CiH ₀ CH ₀

\ ^z I. VII

with a tripeptide of the formula

H-PrO-Q-GIy-IfH ₂

or a heptapeptide of the formula

3Q9884 / U35

2327534

¹ β

CH _O - CO - Tyr -lie -Leu - Asp -MH - CH - CO - Pro -R

I ² I

CH ₉ CH ₉

\ ^d I ^d IX

s- ^: s

with a dipeptide of the formula

or an ootapeptide of the formula

! ^c ₆

CH ₉ - CO - Tyr - lie - Leu - Asp - KH - CH - CO - Pro - Q - R

r ι

CH ₀ CH ₉

j ² \ ^ά XI

S S

reacted with G-lycinamide and the resulting nonapeptide, if appropriate converted into a pharmaceutically usable, non-toxic acid addition salt, where in the formulas VII-XI R Represents hydroxyl or a group which activates the carboxyl group, Q means the remainder of the arginine or lysine and all amino acids with an asymmetric carbon atom Have an L configuration.

A compound of the formulas IV or V can be oxidized in a known manner (see, for example, Schröder-Lubke, Vol. I, page 275 ff), preferably in an aqueous or aqueous-organic solution by introducing air or oxygen or by means of hydrogen peroxide, iodine, 1,2-diiodoethane or potassium ferricyanide. Any sulfhydryl protective groups present can be removed prior to the oxidation or simultaneously. A compound of the formula IV with R ⁵ = R = · hydrogen, trityl, benzhydryl, acetamidomethyl, benzylthiomethyl and isobutyl

3 09884/1435

ORSGiMAL INSPECTED

oxymethyl can be oxidized, for example with Mrhodan ((SGlT) -) to the cyclic Bep.tid, a compound of the formula IV with R ⁵ = H ⁶ = water:
for example with iodine.

IV with R ^ = R = hydrogen, trityl or acetamidomethyl

Dxe splitting off of protective groups from a peptide of the Formulas III or V can likewise be carried out in a generally known manner and under the reaction conditions applicable to the individual groups.

The amidation of a peptide of formula VI, in particular one in which Q represents the remainder of the arginine can be carried out in a manner known per se, preferably by gentle conversion of the activated ester jpit aqueous ammonia at room temperature.

30988 4/1435

• - 6 -

Anyone can do it in the context of peptide synthesis known. Protecting groups are used.

Examples of amino protecting groups are those of the acyl type (such as pormyl, benzoyl, phthalyl, trifluoroacetyl, p-tosyl, Aryl and alkyl phosphoryl, phenyl and benzylsulfonyl, tritylsulfenyl, o-Nltrophenylsulfenyl, γ-Chlorbutyryl or o-Fitrophenoxyacetyl), of the alkyl type (such as trityl, benzyl, alkylidene) or of the urethane type (such as carbobenzoxy, p-bromine, p-chlorine or p-methoxycarbobenzoxy, tolyloxy, allyloxy, cyclopentyloxy, Cyclohexyloxy- * t-butyloxy- or ljl-dimethylpropyloxy-, 2- (p-biphenylyl) -2-propyloxy-carbpnyl or benzyltmocarbonyl). Amino groups can also be protected by protonation. Examples of amide protecting groups are xanthenyl, 2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl and 4,4'-dimethoxybenzhydryl.

As a special protective groups for the arginine residue may be mentioned are: p-tosyl, carbobenzoxy, p-Nitrocarbobenzoxy, t-butoxy, Adamantyloxy- or isobornyloxycarbonyl. The arginine residue can also be protected by protonation or nitration.

Examples of sulfhydryl protective groups are alkyl or Arylthio groups such as ethylthio, t-butylthio or phenylthio; Alkyl and substituted alkyl groups such as t-butyl, 2-diethoxycarbonyl-ethyl, Benzyl, Trityl, p-Methoxybenzyl, p-Mtrobenzyl, 4-picolyl, benzylthiomethyl, acetamidomethyl or isobutyloxymethyl; Acyl groups such as carbobenzoxy, benzoyl, acetyl, p-methoxybenzyloxycarbonyl or ethylaminocarbonyl or tetrahydropyran-2-yl.

The starting compounds of the formulas III, IV, V, VI, VII, IX and XI are new compounds and are also the subject of the application.

30 9 8 84 / U35

The preparation of the starting compounds can in itself take place in a known manner using the customary, in particular the above-mentioned protective groups.

Examples of carboxyl protecting groups are 0 and S esters (such as methyl, ethyl, tr-butyl, benzyl, cyanomethyl, Phthalimidomethyl, 4-picolyl, 2-p-tosylethyl, phenyl, p-nitrophenyl, thiophenyl or p-nitrobenzyl ester), amides or Hydrazides (such as trityl, phenyl, carbobenzoxy or t-butoxycarbonyl hydrazides). Furthermore, the carboxyl group can be protected by salt formation.

Examples of activated carboxyl groups are esters such as cyanomethyl, p-cyanophenyl, p-nitrophenyl, 2,4,5-trichlorophenyl, Thiophenyl-, p-eritrothiophenylr-, 1-benztriazolylr-, Phthalimidyl, 1-succinimidyl, 1-piperidyl, 8-quinolyl, 5-chloro • 8-quinolyl, 2-pyridyl, 2-thiopyridyl esters or azides.

A compound of the formula IV or V can, for example, by successive chain extension of a dipeptide by one Amino acid unit or can be synthesized from 2 or more fragments. By oxidation in a known manner a compound of the formula V can be converted into a compound of the formula III. A starting compound of the formula III can but also e.g. by implementing a connection

HH-R ¹

I (■

CH-CO-Tyr-lie-leu-Asp-EH-CH-CO-R ^D I ² I

CH ₉ CH ₉

I-. \ ^d XII

S _: S

wherein R is hydroxy or a carboxyl group

represents activating group and e given above

309884/1438

R has the meaning given above,

ORIGINAL INSPECT

with a tripeptide of the formula

Η — Pro- Q '- GIy-M ₀ XIII

■ where Q ^{1 has} the meaning given above, are represented.

A compound of the formula VI can be obtained, for example, by a compound VII in which R is a represents activated ester group, with a tripeptide of the formula

. H-Pro-Q-GIy-OH XIV

converts and the reaction product, if desired, in a known manner Way converted into an activated ester.

A compound of the formula XII can also easily be converted into a hexapeptide by removing the amide protective group Formula VII to be transferred. The heptapeptides (IX) and octapeptides (XI) can be obtained, for example, by reacting the hexapeptide (VII) with a compound of the formula Pro —R or Pro —Q-R will.

The method of preparing nonapeptides of formula I according to the principle 6 + 3, 7 + 2 or 8 + 1 as well as by amidation is preferred for the series of the arginine vasopressin analogs

The compounds of the formula I according to the invention have hormonal activity, qualitatively similar to that of the neurohypophyseal hormones. Particularly noteworthy is the strong natriuretic activity. The compounds according to the invention are natural arginine vasotocin ([lle ^] - arginine vasopressin) and that of VJ Hruby et al. ΓJ.Biol.Chem. 244 . 3890 (1969)] is superior to [leu ^] - oxytocin, a neurohypophyseal hormone analog which has the strongest natriuretic activity known to date. The antihypertensive effect of the? compounds according to the invention is

309884 / U35

ORIGINAL! MSPECTED

wrestler than that of argininvasotocin, so the natriuretic Activity of the compounds I towards the hypertensive Activity is selectively increased.

On the basis of the specified biological activities, the compounds are suitable for the treatment of a wide variety of edema Type and general disorders of the electrolyte alternate, especially those of sodium retention.

The dosage should be regulated according to individual needs and can be between 100 μg to 10 mg per single dose, one to one administered several times per day, vary. ■

The EOnapeptides can be in the form of the free bases or as salts with organic or inorganic acids or with polymers containing acid groups (such as carboxymethyl cellulose or tannic acid) either alone or in Ροπή of suitable medical preparations for, for example, oral, parenteral, enteral or intranasal administration will.

For the production of medical preparations, the Compounds with inorganic or organic auxiliaries that are inert and physiologically acceptable, processed will.

The extraordinary metabolic stability of the present Vasopressin analogs makes them for the. Subcutaneous or pernasal application are particularly suitable.

3Q9884 / U35

. example 1

a) Z-L-Leucyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-N -tosyl-L-arginyl glycine amide.

18, Og ZL-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-N-tosyl-L-arginyl-glycine amide [prepared according to RL Huguenin and RA Boissonnas, HeIv. 4_9, 695 (1966)] were dissolved in 100 ml of glacial acetic acid and treated with 100 ml of a 5 F HBr / glacial acetic acid solution. The mixture was stirred for 45 minutes at room temperature and then added dropwise to ₁ liter of ether. The precipitated hydrobromide of the pentapeptide was washed with ether, dried over KOH and PpO 1 - and dissolved in 100 ml of methanol. The solution was passed over a column from Dowex 2 (OH form) were added, the eluate was concentrated under reduced pressure and the residue was dissolved in 100 ml of DMP. 8.5 g of ZL-Leu-ΟΡΜΓΟρ were added to the solution at 0 °, the mixture was stored at room temperature for 3 days and the protected hexapeptide precipitated by adding 1 l of ethyl acetate, washed with ether and ethyl acetate and dried. Yield: 16.3 g; ^{mp 183-185 °; [a] j 5} = -41.6 ° (c = 0.5, in DMP ).

b) Z-L-isoleucyl-L-leucyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-proly1-F 1-tosyl-L-arginyl-glycine amide.

The Z protective group was cleaved off from 16.0 g of ZL-leucyl-L-asparaginyl-S- ^ benzyl-L-cysteinyl-L-prolyl-F-tosyl-L-arginyl-glycine amide in the manner described under (a) and the free amine obtained was reacted with 6.0 g of ZL-IIe -OPhNOp in 100 ml of DMF. The mixture was stored for 2 days at room temperature, the protected heptapeptide was precipitated by adding 1 l of ethyl acetate and the precipitate was washed with ether, ethyl acetate and isopropanol and dried. Yield: 13.1 g; P. 211-212 °; [a] ^ ⁵ = -41.9 ° (c = 0.5, in DMF).

309884/1435

c) Z-O-Benzyl-I-tyrosyl-L-isoleucyl-L-leucyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-N -tosyl-L ^ arginyl-glycine amide.

From 10.0 g of the ZL-isoleucyl-L-leucyl-L-asparaginyl-S-benzyl-L-cysteinyl-II-prolyl-N-tosyl-L-arginylglycine amide obtained, the Z-protecting group became in the manner described under a) split off and the resulting free amine reacted with 4.5 g of Z-0-benzyl-L-Tyr-0PhN0 ₂ in 100 ml of DMF. After standing for three days at room temperature, the protected octapeptide was precipitated by adding ethyl acetate, washed with ethyl acetate and ethanol, reprecipitated from glacial acetic acid / ethanol, washed with ethanol and dried. Yield: 8.4 g; M.p. 237-238 °; [a] ^ = -36.2 ° (c = 0.5, in DMF).

d) ß-Benzylthiopropionyl-L-tyrosyl-L-isoleucyl-L- -L-asparaginyl-!
L-arginyl glycine amide.

leucyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-proiyl-N -tosyl-

7.0 g of ZO-benzyl-L-tyrosyl-L-isoleucyl-Ij-leucyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-N-tosyl-L-arginylglycine amide were dissolved in 50 ml of glacial acetic acid and 50 ml of a 5 N HBr / glacial acetic acid solution are added. After stirring for one hour, the mixture was added dropwise to 1 liter of ether, the precipitate was filtered off, washed with ether, reprecipitated from ethanol / ether and dried over PpO, - and EOH. 0.5 g of the hydrobromide thus obtained was dissolved in 10 ml of DMF. The solution was brought to pH by adding ethyl diisopropylamine and 0.2 g of β-benzylthiopropionic acid pnitrophenyl ester and a few drops of glacial acetic acid were added at 0 °. After standing for one day at room temperature, the protected peptide was precipitated by adding ethanol, washed with ethanol and dried. This residue was dissolved in a little DMF and reprecipitated by adding water, washed with ethanol and dried. Yield: 0.4 Si

309884/1436

M.p. 224-226 °; [α] ^ ⁵ = -36.6 ° (c = 0.5, in DMP).

1 ^ ζ A

e) Deamino-r [lle, Leu J-argininvasopressin diacetate.

500 mg of ß-benzylthiopropionyl-L-tyrosyl-L-isoleucyl-L-leucyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-N-tosyl-1-arginyl-glycine amide were added to 300 ml of liquid ammonia Sodium reduced. After removing the ammonia, the residue was dissolved in 900 ml of water containing a few drops of glacial acetic acid and the solution was adjusted to pH 6.8 with NaOH. Then 45 ml of a 0.01 molar K, [Pe (CN ) Λ solution were added, the pH being kept at 6.5-7.0 by adding a little NaOH. The reaction mixture was stored at room temperature for 15 hours and passed through a column of Amberlite IR-45 (Cl ~ form). The eluate was acidified with acetic acid, concentrated to about 40 ml under reduced pressure and a mixture of 1-butanol / benzene / pyridine (6: 2: 1) was added dropwise until a second phase began to separate out. The solution was then applied to a column of Sephadex G-25 superfine, which was equilibrated with the sub-y phase of the distribution system l-butanol / benzene / pyridine / 0.2 η acetic acid (6: 2: 1: 7). It was developed with the upper phase of this system. The distribution was according to the 'Folin-Lowry method [OH Lowry et. al. J. Biol. Formerly 1 ° / 5, 265 (1951)]. The central fractions of the main peak were pooled, diluted with water, concentrated under reduced pressure and finally lyophilized (yield 121 mg). This material was subjected to a second partition chromatography in the same system for further purification. The purified deamino- [lie , Leu ^ -argininvasopressin (yield 90 mg) has an Rf value in this system of 0.27; M ^ = * -85.5 ° (c = 0.5, in IN acetic acid).

309884 / U3S

Paper electrophoresis: '' '

Buffer · from 2 ml glacial acetic acid and 20 ml pyridine, made up with -Water to 1: 1 (pH = 6.0): Rf (arginine) = 0.30 ^ 0.05. " Buffer from 37 ml formic acid and 5 ml acetic acid, filled up with water to 1 1 (pH = 1.7) -: Bf (arginine) = 0.25-0.05.

The raw deamino - [He ^, Leu ^] - argininvasopressin can also be cleaned by following removal the perri and perrocyanide ions acidify the eluate using Amberlite IR-45 (Cl ~ r-IOrm.), to Amberlite CG-50 (H -Ροπή) adsorbed, eluted with a mixture of pyridine / glacial acetic acid / water (30: 4: 66) and after intermediate lyophilization Amberlite CG-50 (H -Porm) with a 0.5 M ammonium acetate buffer (pH 6.4) was chromatographed.

Example 2

a) Z-L-Leucyl-L-asparaginyl-S-benzyl-L-cysteine methyl ester.

A solution of 8.4 g of L-asparaginyl-S-benzyl-L-cysteine methyl ester hydrobromide [prepared according to RA Boissonnas et al., HeIv. 3_8, 1491 (1955)] in 30 ml of dimethylformamide (DMP) was "brought to a pH of about 8 at -10 ° by adding 2.8 ml of triethylamine and stirred with 7.8 g of ZL-leucine p-nitrophenyl ester The solidified mixture was diluted with about 70 ml of water and the precipitate filtered off with suction, washed with alcohol, ethyl acetate and ether and dried. P. 195-197 °; [a] p ⁵ = -28.0 ° (c = 1 in DMP ).

b) Z-L-Leucyl-L-asparaginyl-S-benzyl-L-cysteine hydrazide.

A solution of 3 g of the Z-L-leucyl-L-asparaginyl-S-benzyl-L-cysteine methyl ester obtained in a mixture of 20 ml of DMP and 5 ml of dimethy lsulf oxide was at about 4 ° with 1.5 ml

309884 / U35

_ 16 _

Hydrazine hydrate added. The mixture was left to stand for 24 hours at room temperature and filtered. By adding water to the piltrate, a precipitate was obtained, which was filtered off with suction, washed with water and dried. Έ. 230-234 °; [a] ^ ⁵ =. -38, 2 ° (c = 1.1 in DMi ¹ ) ._ ..

c) Z-L-Leucyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-

prolyl-N-tosyl-L-arginyl-glycine amide.

A suspension of 2.25 g of Z-L-leucyl-L-asparaginyl-S-benzyl-L-cysteine hydrazide in 25 ml of DMF was treated with 6 ml of 2.5 N HCl in tetrahydrofuran (THE) at -20 °. 1 ml of isoamyl nitrite was added to the solution. The mixture was stirred for 30 minutes at -20 °, cooled to -30 ° and at this temperature after neutralization with 2.08 ml of triethylamine with a solution of 2.3 g of L-prolyl-N-tosyl-L-arginyl-glycine amide, obtained by hydrogenation of 3 g of ZL-prolyl-N-tosyl-L-arginylglycine amide [according to RL Huguenin and RA Boissonnas, HeIv. 45_, 1629 (1962)] in methanol at normal pressure and room temperature over Pd / C, added to a mixture of 10 ml DMi ¹ and 10 ml THi ¹ . The mixture was then stirred at -10 ° for 1 hour, stored in an ice chest overnight, filtered, and the piltrate was concentrated to remove the THP. After dilution with DMP, the hexapeptide was precipitated by adding water and purified by washing with alcohol and ethyl acetate. P. 180-182 °; [ά] ^ ⁵ = -40.6 ° (c = 0.5, in DMP).

d) ß-Benzylthiopropionyl-L-tyrosyl-L-isoleucyl-L-leucylragine:
glycinamide.

L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-N -tosyl-L-arginyl-

A solution of 1.6 g of Z-L-Leucyl-L-asparaginyl-S-benzyl-

C ¹

L-cysteinyl-L-prolyl-N-tosyl-L-arginyl-glycine amide in 20 ml Glacial acetic acid was mixed with 20 ml of 4 N HBr in glacial acetic acid. That The mixture was stirred at room temperature for 2 hours, then

30 9884 / H3E

then added dropwise to 500 ml of ether and the precipitated hydrobromide washed with ether, dried and dissolved in 5 ml of DMF. This solution was adjusted to pH 7.5 by adding ethyldiisopropylamine and to a solution of β-benzylthiopropionyl-L-tyrosyl-L -isoleucinazid, which was obtained by adding 0.7 g of ß-benzylthiopropionyl-L-tyrosyl-L-isoleucine hydrazide [manufactured by RL Huguenin, HeIv. 49 , 711 (1965)] in a mixture of 5 ml of DMSO, 10 ml of DMi ¹ and 4 ml of 2.4 N HCl in THP, mixed with 0.3 ml of isoamyl nitrite at -20 ° and the G-emisch after 30- minute stirring at -20 ° by adding 1.65 ml of ethyldiisopropylamine neutralized. The mixture was stirred for 30 minutes at -20 ° and 30 minutes at -5 °, then stored for 3 days at + 4 ° and concentrated. The residue was dissolved in 5 ml of DMF, and the peptide was precipitated by adding a mixture of 50 ml of ethanol and 10 ml of water. The precipitate was filtered off, dissolved in 5.ml DIO ¹ and again precipitated by adding a mixture of 150 ml ethyl acetate and 50 ml ether, filtered off, washed with ethyl acetate and ether and dried (yield 0.9 g); M.p. 224-226 °; [a] ^ ⁵ = -36.0 ° (c = 0.5, in DMF).

e) Deamino - [He, Leu J-arginin vasopressin diacetate.

In the manner described in Example 1, β-benzylthiopropionyl-L-tyrosyl-L-isoleucyl-L-leucyl-L-asparaginyl-S-benzyl-L-eysteinyl-L-prolyl-N -tosyl-L-arginyl glycine amide into the deamino - [He, Leu] -argininvasopressin diacetate convicted.

Example 3

a) Boc-L-Leucyl-L-asparaginyl-S-benzyl-L-cysteine- ' benzyl ester.

5.16 g Boc-L-asparaginyl-S-benzyl-L-cysteine-benzyl ester 309884/143 5

_ 18 _

[manufactured according to Μ »P. Perger et al. _f J. Amer. Chem. Soc. f * 4_, 982 (1972)] were dissolved in 50 ml of ethyl acetate saturated with hydrogen chloride. After 30 minutes the hydrochloride was precipitated with dry ether, suction filtered, washed with dry ether and dried over KOH in vacuo. The product (3.74 g) was dissolved in 40 ml of DMP with 3.52 g of Boc-L-leucine dicyclohexylamine salt and 2.02 g of 1-hydroxy-benzotriazole. Tray addition of 1.94 g of dieyclohexylcarbodiimide in 5 ml of DMP at 0 °, the reaction mixture was stirred for 11/2 hours at 0 ° and 5 »1/2 hours at room temperature, filtered, concentrated under reduced pressure, stored at 0 ° for 24 hours and filtered again. The piltrate was further concentrated to approx. 8-10 ml and then 15 ml of water were added. After a few hours, the peptide was filtered off with suction, washed with plenty of water, saturated sodium hydrogen carbonate solution, water, 0.1 M sulfuric acid and water, dried and recrystallized from ethyl acetate and 2-propanol; P. 158-161 ° \ [a] ^ ⁵ = -31.1 ° (c = 1, in DMP). ".

b) Boc-L-Isoleucyl-L-leucyl-L-asparaginyl-S-benzyl-1-eysteine-benzyl ester.

From Boc-L-Leucyl-L-asparaginyl-S-benzyl-L-cysteine benzyl ester the corresponding hydrochloride was prepared in the usual way with ethyl acetate saturated with hydrogen chloride and precipitated with ether. 1.5 g of the hydrochloride obtained, 0.64 g of Boc-L-isoleucine hemihydrate, 0.54 g 1-Hydroxy-benzotriazole and 0.293 ml of N-methylmorpholine were dissolved in 25 ml of DMP and at. 0 ° with 0.605 g of dicyclohexylcarbodiimide added to 2 ml of DMP. After stirring for one hour at 0 ° and 6 hours at room temperature, the mixture was filtered off with suction. The filtrate was admixed with water, the precipitated product was filtered off with suction, with sodium hydrogen carbonate solution, 0.1 M sulfuric acid and water washed and dried. The product was dissolved in 250 ml of methanol at 60 °. The residue was

309884 / U35

-. 13- ■ '

sucked, the filtrate concentrated to about 80 ml. The pure, protected tetrapeptide was obtained by crystallization and work-up of the mother liquors; Mp 218 °; [aj ^ ⁵ = -58.8 °. (c = 1, in MP).

c) Boc-O-Benzyl-L-tyrosyl-L-isoleucyl-L-leucyl-L-asparaginyl-S-benzyl-L-cysteine-benzyl ester.

The crystalline hydrochloride of L-isoleucyl-L-leueyl-Ii-asparaginyl-S-benzyl-L-cysteine-benzyl ester was duYch treatment of the Boc derivative with hydrogen chloride in ethyl acetate and obtained by precipitation with ether. 0.81 g of the hydrochloride, 0.485 g of Boc-0-benzyl-L-tyrosine, 0.27 g of 1-hydroxybenzotriazole and 0.151 ml of N-methylmorpholine were in 30 ml Dissolved DMF and mixed with 0.278 g of dicyclohexylcarbodiimide in 5 ml of DMP at 0 °. The reaction mixture was 1 1/2 hours Stirred at 0 ° and at room temperature for 18 hours, filtered, and the filtrate was concentrated to 10 ml under reduced pressure and the reaction product is precipitated with plenty of water. The precipitate was successively on the suction filter with sodium hydrogen carbonate solution, Water, 0.1 M sulfuric acid and water, dissolved in 200 ml of hot methanol. To concentration to 50 ml allowed to crystallize out and obtained

pe

the protected pentapeptide in pure form; P. 225 °; [a L = -25_, 2 ^Λ (c = 1, in DMP).

d) ß-Benzylthiopropionyl-0-benzyl-L-tyrosyl-L-isoleucyl-Ir-leucyl-L-asparaginyl-S-benzyl-L-cysteine-benzyl ester.

The hydrochloride of O-Benzyl-L-tyrosyl-L-isoleucyl-L-leucyl-L ^ asparaginyl-S-benzyl-L-cysteine-benzyl ester was obtained by treating the Boc derivative with hydrogen chloride in ethyl acetate and precipitation with ether. 0.47 g des Hydrochloride and 0.062 ml of N-methyl imorpholine in 10 ml of DMF

309884 / U35

were treated with a solution of 0.108 g of ß-benzylthiopropionic acid and 0.136 mg of l-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline in 2 ml of THF and stirred for 18 hours "at room temperature. The solvent mixture" was evaporated under reduced pressure and the resultant protected hexapeptide triturated with ethyl acetate, washed with ether, sodium hydrogen carbonate solution, water, 0.1 M sulfuric acid and water and dried; M.p. 240 ° (dec.) I [ ^a lp ⁵ = - ²⁸ ° (c = in DIiF).

e) ß-Mercaptopropionyl-L-tyrosyl-L-isoleucyl-L-leucyl-L-asparaginyl-L-cysteine disulfide.

460 mg of ß-Benzylthiopropionyl-O-benzyl-L-tyrosyl-L-isoleucyl-L-leucyl-L-asparaginyl-S-benzyl-L-cysteine-benzyl ester and 130 mg of urea were reduced in 150 ml of dry ammonia with sodium to one The result was a stable blue coloration for over 30 seconds. After adding 184 mg of ammonium chloride, the reaction mixture was lyophilized. The residue was stored in vacuo over phosphorus pentoxide for 16 hours, dissolved in 170 ml of acid-free water and 60 ml of methanol, filtered and, while flushing with nitrogen, at the same time with a solution of 136 mg of freshly recrystallized diiodoethane in 100 ml of methanol in a mixture of 150 ml of water and 50 ml of methanol was added dropwise, the pH being kept at 7.5 [method according to F. Weygand and G-. Zumach, Z. Na turf or sch. 17b, 807 (1962)]. After the reaction to thiol groups (Ellman reagent) had disappeared, the mixture was concentrated to about 50 ml under reduced pressure, filtered, brought to pH 2-3 with acetic acid, filtered again and lyophilized. and upper phase of the solvent system 1-butanol / O, 25% acetic acid dissolved and desalted and partially purified by 160 distribution steps in this system. The fractions containing the purified product were concentrated and lyophilized and the cyclic

309884 / U35

_ 21 _

Hexapeptide reprecipitated from isopropyl alcohol with diisopropyl ether; [α] ^ ⁵ = -38 ° (c = 0.5 in methanol), thin-layer chromatography on silica gel with i-butanol / acetic acid / water (100: 15: 35): Rf = 0.42.

f) Deamino - [lie, leul arginine vasopressin diacetate.

A solution of 28.5 mg of ß-mercaptopropionyl-L-tyrosyl-L-isoleucyl-L-leucyl-L-asparaginyl-L-cysteine disulfide and 20 mg L-prolyl-L-arginyl-glycinamide dihydrobromide [manufactured according to DT Gish and V. du Vigneaud, J.Am.Chem.Soc. 29: 3579 (1957); RO Studer and V. du Vigneaud, J. Am. Chem. Soc. 82, 1499 (1960)] in 0.4 ml of dimethylformamide, 10.3 mg of dieyclohexylcarbodiimide in 0.2 ml of dimethylformamide were added after the addition of 6 μl of triethylamine and 9 »5 mg of 1-hydroxybenzotriazole at 0 °. After 20 hours at room temperature, the mixture was diluted with water / water, acidified with acetic acid and, after standing for 12 hours at 0 °, filtered. The filtrate obtained was chromatographed on Amberlite CG-50 (H ⁺ -POrIn) with 0.5 M ammonium acetate buffer (pH 6.4).

Example 4

Z-L-Ieucyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-N -tosyl-L-lysyl-glycine amide.

14.0 g of ZL-asparaginyl-S 1-benzyl-L-cysteinyl-L-prolyl-N ^e -tosyl-L-lysyl-glycine amide [prepared according to M. Bodanszky et al. J. Amer. Chem. Soc. 82, 3195 (1960)] were dissolved in 60 ml of glacial acetic acid while warming, and 60 ml of 5N HBr / glacial acetic acid solution were added at room temperature. The mixture was stirred for one hour at room temperature and then added dropwise to 600 ml of ether. The precipitated hydrobromide of the pentapeptide was washed with ether, dried over KOH and? ^Ge ~ 2®5 and dissolved in 100 ml of methanol. The solution was over

3Ü938WU35

given a column of Dowex '2 (OH "* - form), the eluate was concentrated under reduced pressure and the residue was dissolved in 60 ml of DMF. 6.55 g of Z-LeU-OPhNO _{2 were} added to the solution at 0 ° The mixture is stored for 2 days at room temperature and the protected hexapeptide is precipitated by adding 600 ml of ethyl acetate, washed with ether and ethyl acetate and dried. Yield: 11.6 g; mp 223-225 °; M ^ = -43.9 ° ( c = 1.0, in DMF).

b) Z-L-isoleucyl-L-leucyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-N -tosyl-L-lysyl-glycine amide.

The Z-protective group was cleaved from 11.0 g of ZL-leucyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-N -tooyl-L-lysyl-glycine amide in the manner described under a) and the resultant at the terminal amino group, unprotected hexapeptide was _{reacted with 5.0 g of ZL-IIe-OPhNO 2} in 60 ml of DMF. The mixture was stored for 3 days at room temperature, the protected heptapeptide was precipitated by adding 600 ml of ethyl acetate and the precipitate was washed with ether and ethyl acetate and dried. Yield: 9 * 6 g; Ρ; 224-227 °; [a] p ⁵ = -43.3 ° (c = 0.5 in DMF).

-c) Z-O-Be.nzyl-L-tyrosyl-L-isoleucyl-L-leucyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-N -tosyl-L-lysyl-glycine amide.

From 4.5 g of ZL-isoleucyl-L-leucyl-Ir-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-N ^E '-tosyl-L-lysyl-glycine amide in the manner described under a) the Z -Protecting group split off and the heptapeptide obtained unprotected at the terminal amino group was _{reacted with 2.1 g of Z-0-benzyl-L-Tyr_0PhN0 2} * in 60 ml of DMF. After standing for 12 hours at room temperature, the reaction mixture was added dropwise to ethanol, the precipitated, protected octapeptide was filtered off,

30988A / U35

washed with ethanol and ether and dried. Yield 4.2 g; P. 230-232 °; [a] ^ ⁵ = -18.5 ° (c = 1, in DMP).

d) ß-Benzylthiopropionyl-L-tyrosyl-L-isoleucyl-L- -L-asparaginy;
L-lysyl-glycine amide.

leucyl-L-asparaginyl-S-'benzyl-L-cysteinyl-L ^ prolyl-iT ^e -tosyl-

3.0 g Z-0-Benzy1-L-tyrosyl-L-isoleucy1-L-leucyl-L-asparaginyl-Sl> ^{enzyl-L-cysteinyl-I-prolyl-N-tosyl-L-e} lysylglycinamid were dissolved in 25 ml of glacial acetic acid dissolved and mixed with 25 ml of a 5N HBr / glacial acetic acid solution. After stirring for 1 hour, the mixture was added dropwise to 500 ml of ether and the precipitate was filtered off, washed with ether and dried over KOH and PpO ^. The hydrobromide of the octapeptide thus obtained was dissolved in 25 ml of DMF. The solution was brought to pH 7.0 by adding N-methylmorpholine, and 0.7 g of β-benzylthiopropionic acid p-nitrophenyl ester was added. After standing for 12 hours at room temperature, the reaction mixture was added dropwise to a mixture of ethanol-ethyl acetate (1: 1) and the precipitate was filtered off. The protected peptide obtained in this way was dissolved in DICP and reprecipitated by dripping the solution into ethyl acetate. The precipitate was washed with ethyl acetate and ether and dried. Yield: 1.25 g; P. 224-225 °; [a] ^ ⁵ = -40 ° (c = 1.0, in DMP).

e) Deamino - [He ³ , Leu l-lysinvasopressin diacetate.

350 mg of β-benzylthiopropionyl-L-tyrosyl-L-isoleucyl-L-leucyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-N -tosyl-L-lysyl-glycine amide in 350 ml of liquid ammonia were with Sodium reduced. After removal of the ammonia, the residue was dissolved in 500 ml of liquid acetic acid and the Solution adjusted to pH 7.8 with NaOH. Thereupon were

3Ö9884 / U3 5

_ 24 _

52 ml of a 0.01 molar K- [Fe (ClT) ₆ ] solution were added, the pH being kept at 7.2-7.8 by adding a little NaOH. The reaction mixture was stored at 4 ° for 15 hours and passed through a column of Amberlite IR-45 (Cl ^- -FoITn). The eluate was acidified with acetic acid and ^{adsorbed on Amberlite CG-50 (H +} form). After washing with 500 ml of 0.2% acetic acid, it was eluted with a mixture of pyridine / glacial acetic acid / water (30: 4: 66) and the eluate was lyophilized twice with intermediate absorption with water. For further purification, the lyophilizate was dissolved in 3 nil of a 0.5 M ammonium acetate buffer (pH = 6.4) and chromatographed again on a column of Amberlite CG-50 (H form) with this buffer. The eluate was lyophilized several times. Yield: 107 mg; [a] ^ ⁵ = -61.0 ° (c = 1.0 in 95% acetic acid).

Paper electrophoresis:

Buffer consisting of 2 ml of glacial acetic acid and 20 ml of pyridine made up with water to 1 1 (pH = 6.0): Rf (lysine) = .45 to .05. Buffer of 37 ml formic acid and 25 ml acetic acid, made up with water to 1 l (pH = 1.7) Rf (lysine) = 0.28 ± 0.05.

309884/1435

Example 5
Containing sublingual tablet

a) Deamino - [lie?, Leu] -argininvasopressin-

diaceta-t 5 »70 mg

Milk sugar '66.30 ng

Powdered sugar 20.00 mg

Kollidon E 25 x 7.00 mg

Magnesium stearate 1.00 mg

100.00 mg

b) Deamino - [He ^, Leu] -argininvasopressin-

diacetate '11.40 mg

Milk sugar 71.60 mg

Mannitol 60.00mg

Hydroxypropyl methyl cellulose 5 »00 mg

Magnesium stearate 2.00 mg

150.00 mg

Example 6
Solution for injection in ampoules of 5 ml, containing per ml

Deamino - [He, Leu] -argininvasopressin-

diacetate 0.12 mg

KaOl 9.00 mg

HCl 0.1 N ad pH 3.5 q.s.

H ₂ O ad inject. ad 1.0 ml.

309884 / U36

Example 7
Containing lyophilizate of an aqueous solution

Weight parts

Deamino - [He, Leu] arginine invasopressin diacetate 11.40

L-malic acid 0.87

D-mannitol 1 ^ 0.00

162.27

To obtain a ready-to-use injection solution 162.27 mg of the lyophilizate in 10 ml of distilled water. solved.

309884/1435

Claims (1)

Claims 1. Process for the preparation of nonapeptides of the formula Mpr-Tyr-lie-Leu-Asp-Cys-Pro-Q-GIy-NH ₂ I where Q is the remainder of the arginine or lysine, Mpr means ß-mercaptopropionyl and all Amino acids with one asymmetric carbon atom Have an L-configuration, and their pharmaceutically usable, non-toxic acid addition salts, characterized in that a) of a peptide of the formula NH-R ¹
I. CH ₂ - CO —Tyr —lie —Leu — Asp -NH- CH- CO —Pro —Q · - GIy - CH ₉ CH ₀ I- - \. ■ -III wherein R is hydrogen or an amide protecting group, Q ^{1 is} a radical of the formula
-NH-CH [- (CH ₂ ) -NH-C (= NH) -NHR ² ] -CO- or -NH-OH [- (CH _? ) -NH-R ³ J-CO- and R represent hydrogen or one the guanidine residue protective group and
R ^ hydrogen or one of the ε-amino group of the Lysine's protective group mean but one 1 2 "^ of the radicals R and R or R ^ is a protecting group represents and all amino acids with an asymmetric carbon atom L-configuration exhibit, splitting off the protective groups and optionally the free peptide by reaction with an organic or inorganic acid into a pharmaceutically usable, non-toxic acid additive 309884 / U35 2 3 2 7:34 ionization salt transferred, or
"b) a peptide of the formula NH ₂ CH ₀ -CO-Tyr-He-leu-Asp-NH-CH-CO-Pro-Q-GIy-NH ₀ , 2 I-2 CH ₉ CH ₉ Ο — JX JX —Ο wherein Q has the meaning given above and 4 5
R and R each represent hydrogen or sulfhydryl protective groups and all amino acids with an asymmetric carbon atom! »- have configuration, oxidized, with simultaneous or previous cleavage, if necessary existing protecting groups and the reaction product, if desired, by reaction with an organic or inorganic acid converted into a pharmaceutically acceptable, non-toxic acid addition salt, or c) a peptide of the formula NH-R ¹
I. CH ₀ -CO-Tyr-He -leu-Asp-NH-CH-CO -Pro-Q '-GIy-NH ₀ , 2, 2 CH ₉ CH ₉ 5 4 I ² V ΰ — JX It —ü wherein R is hydrogen or an amide protecting group; Q ^{1 is} a radical of the formula -NH-CHf- (CH ₂ ) -NH-CC = NH) -NHR ² ] -CO- or -NH-CH [- (CH ₉ ) -NH-R ³ ] -CO-; 2 C- ⁰ T R is hydrogen or a guanidine radical protecting Group;
R is hydrogen or one of the ε-amino groups of lysine protective group and
4 5
R and R each hydrogen or sulfhydryl chloride ORlGLNAL INGPECTED COPY represent groups, but at least one of the 12 "3 R and R or R represents a protective group and all amino acids with a
asymmetric carbon atom L-configuration ■ have, oxidized with simultaneous elimination of the protective group (s)
and "if desired, converting the reaction product into a pharmaceutically acceptable, non-toxic acid addition salt by reaction with an organic or inorganic acid, or d) a compound of the formula Mpr-Tyr-He -leu -Asp-Cy s -Pro -Q-GIy-R VI where Q is the remainder of the lysine or arginine, R is hydroxy or a residue that activates the carboxyl group and Mpr is ß-mercaptopropionyl, amidated or
e) a hexapeptide of the formula NH ₀ I ² CH _O -CO-Tyr-He-Leu-Asp-NH-CH-CO-R I ² ! CH ₉ CH ₉ I * ■ VII with a tripeptide of the formula H-PrO-Q-GIy-NH ₂ VIII "or a heptapeptide of the formula NH ₀ I * _ß CH _O -CO-Tyr-Ile-Leu-Asp-NH-CH-CO-Pro-R ¹ ϊ ' CH ₀ J CH ₀ \ ^d I ^d IX 3 0 9 8 L - '* / 1 A 3 5 COPY with a dipeptide of the formula - Q - Gly - HH ₂ or an octapeptide of the formula CH _O -CO-Tyr-lie-leu-Asp-NH-CH-CO-Pro-QR ^b , 2 j. CH ₉ CH ₉ I- I ² XI S- _: S reacted with glycine amide and the fonapeptide obtained, if appropriate converted into a pharmaceutically usable, non-toxic acid addition salt, where in the formulas VII-XI R Represents hydroxyl or a group activating the carboxyl group, Q represents the remainder of the arginine or lysine and all amino acids with an asymmetric carbon atom L configuration exhibit. 2. The method of claim 1, wherein a nonapeptide of formula I 'Ia Mpr-Tyr-He-Leu -Asp-Cys-Pro-Arg-G-Iy-NH _p and its pharmaceutically acceptable, non-toxic acid addition salts getting produced. 3. The method of claim 1, wherein a nonapeptide of formula Mpr - Tyr - Lie - Leu - Asp - Cys - Pro - Ly s - GIy - NH ₂ Ib and its pharmaceutically acceptable, non-toxic acid-aadililon salts getting produced. 309884/1435 •. - 31 - ■ 4 · method .according to claim 1, wherein deainino - [He, leu] -arginine-vasopressin-diacetate will be produced. 5. The method according to claim 1, wherein deamino - [He, Leu. ] ■ lysine vasopressin diacetate is produced. 309884/14 35 6. Process for the production of preparations with natriuretic properties, characterized in that one a compound of the formula I or one of its non-toxic acid addition salts as an active ingredient for therapeutic use Administration of suitable, non-toxic, inert, solid or liquid which are usual in such preparations Carriers mixed. 7. Pharmaceutical preparation with natriuretic properties, containing as active component a compound of the formula I or one of its non-toxic acid addition salts. 309884/1435 ORIGINAL 3NSrECTED 8. Use of a compound of the formula I or one of its non-toxic acid addition salts as a natriuretic. el A nonapeptide of the formula
i Mpr-Tyr-Ile-Leu-Asp-Cys-Pro-Q-GIy where Q is the remainder of the arginine or lysine, Mpr means ß-mercaptopropionyl and all amino acids with an asymmetric carbon atom have the L-configuration and its pharmaceutically acceptable, non-toxic acid addition salts. 10. A nonapeptide of the formula \ ^d Mpr-Tyr-lie-Leu-Asp-Cys-Pro-Arg-G-Iy-NH ₂ Ia wherein Mpr is ß-mercaptopropionyl and all amino acids with an asymmetric carbon atom L-configuration exhibit and its pharmaceutically acceptable, non-toxic acid addition salts. 309884/1435 11. A nonapeptide of the formula Mpr-Tyr -lie-LeU-ASp-CyS-PrO-LyS-GIy-KE ₂ Ib wherein Mpr is ß-mercaptopropionyl and all amino acids with an asymmetric carbon atom ! Configuration and its pharmaceutically acceptable, non-toxic acid addition salts. 12. Deamino - [He, Leu] arginine invasopressin diacetate. "1 * 7 A 13. Deamino - [lie, Leu j-lysinvasopressin diacetate. ORiQiMAL INS 309884 / U3 5