DK149755B - METHOD OF ANALOGY FOR THE PREPARATION OF LHRH ANALOGUE NONAPEPTIDAMIDE DERIVATIVES OR PHYSIOLOGICALLY ACCEPTABLE SALTS OR METAL COMPLEXES THEREOF - Google Patents

Description

14.9755 i

The present invention relates to an analogous process for the preparation of novel LHRH analogous nonapeptide amide derivatives of the general formula

H- (Pyr) Glu-His-Trp-Ser-A1-Gly-A2-Arg-Pro-Y I

Wherein A represents Tyr or Phe, where A represents Leu, ILe, NLe, Val, NVal or Met and where Y represents a group NHR wherein R is a straight or branched alkyl group having 1-3 carbon atoms, optionally substituted by hydroxy, or physiologically acceptable salts or metal complexes thereof. The process according to the invention is characterized by the characterizing part of claim 1.

In this specification (pyr) represents Glu, His, Trp,

Ser, Tyr, Phe, Gly, Leu, ILe, NLe, Val, NVal, Met, Arg and Pro are the "residues" of L-pyroglutamic acid, L-histidine, L-tryp-tophan, L-serine, L-tyrosine, respectively. L-phenylalanine, glycine, L-leucine, L-isoleucine, L-norleucine, L-valine, L-norvaline, L-methionine, L-arginine and L-proline. By the term "residue" is meant a group derived from the corresponding α-amino acid by the elimination of the OH moiety from the carboxyl group and the H moiety from the α-amino group. In the case of, for example, L-arginine,

HN

NHCHoCHrtCHrtCHCOOH

/ 2 2 2.

H2N NH2 which can be represented by the formula NH2-A-C00H (where NH2 represents the <x -amino group) thus represents the group (-NH-A-CO-) a "residue" of L-arginine and abbreviated to "-Arg - ". Abbreviations for the other α-amino acids mentioned above have the same meaning as the illumination just given for L-arginine.

With respect to the above substituent R, an equal or greater alkyl group having 1-3 carbon atoms which may be hydroxy substituted may be exemplified by methyl, ethyl, n-propyl, i-propyl, hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 2 -hydroxy-n-propyl, 3-hydroxy-n-propyl and 2,2-dihydroxy-i-propyl.

2 149755

It has been known for many years that the hypothalamus contains higher-level factors that control the secretion of tropical hormones from the pituitary gland. Recently, after the isolation of a thyrotropin-releasing hormone (TRH), a hormone that promotes the secretion of the luteinizing hormone (LH) has been extracted in pure form from pigs and sheep, and has been shown to be a decapeptide of structure H- ( Pyr) Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (AV Schally et al, Biochem. Biophys. Res. Commun.

43, 1334, 1971; R.Gnillemin et al Proc. Night. Acad. Sci., USA, 69, 278, 1972). This result is followed by the synthesis of a number of similar peptides and biological tests have also been performed with these analog peptides. However, even a slight modification in the amino acid composition shown above severely diminishes the physiological activity of the peptide, and the chemical structure shown above has been considered essential for the development of maximum physiological activity (AV. Schally et al, Biochem. Biophys. Res. Commun. 4 366, 1972).

Despite this, no-napeptidamide derivatives of formula I and pharmaceutically acceptable salts thereof have now been synthesized, and surprisingly, these compounds have been found to have more potent ovulation-inducing effect than the naturally occurring decapeptide. It has also been found that the compounds of general formula I act on the pituitary gland and here the secretion promotes both luteinizing hormone and follicle stimulating hormone. Furthermore, it has been found that said compounds are useful in both human medicine and veterinary medicine, thus in humans for the treatment of amenorrhea and in the veterinary field in animals for the induction of ovulation and thereby indirectly for the induction of pregnancy in cases of various morbid conditions in the ovary. such as ovarian follicle cysts. Details of this are provided at the end of this specification.

As claimed in claim 1, nonapeptide amide derivatives of general formula I are prepared by producing a reagent (A), L-pyroglutamic acid or peptide fragment containing an L-glutamic acid moiety (i.e., H- (Pyr) Glu-) at the N terminal end, and at the same time calculated therefrom, contains the aforementioned amino acid sequence, condensed with a reagent (B), namely an amine component corresponding to the remaining portion of the aforementioned 1 * 9755 3 nonapeptidamide derivative of general formula I, the two reagents (A) and (B), if desired, are protected by one or more protecting groups, and any protective groups present if desired are removed.

The relationship between reagents A and B is therefore as shown in the diagram below.

Method Reagent A Reagent B

no.

1 H- (Pyr) Glu-OH H-His-Trp-Ser-A1-Gly-A2-Arg

Pro-Y

2 H- (Pyr) Glu-His-OH H-Trp-Ser-A1-Gly-A2-Arg-Pro-Y

3 H- (Pyr) Glu-Hi s-Trp-OH H-Ser-A ^ -Gly-A2 -Arg-Pro-Y

4 H- (Pyr) Glu-His-Trp-Ser-H-A ^ Gly-A2-Arg-pro-Y

OH

5 H- (Pyr) Glu-His-Trp-Ser-H-Gly-A2-Arg-Pro-Y

A -OH

6 H- (Pyr) Glu-His-Trp-Ser-H-A2-Arg-Pro-Y

A-Gly-OH

7 H- (Pyr) Glu-His-Trp-Ser-H-Arg-Pro-Y

A -Gly-A2-OH

8 H- (Pyr) Glu-His-Trp-Ser-H-Pro-Y

A ^ -Gly-A 2-Ar g-OH

9 H- (Pyr) Glu-His-Trp-Ser-H-Y

12

A - A.-Gly-Arg-Pro-OH

When reagent A is used as a compound from reagent A, as reagent B, the compound is used in the right column on the same line, namely containing the remaining part of the desired product and thus needed in the condensation reaction. Reagents A and / or B may be protected prior to the condensation reaction or activated during the condensation reaction, more specifically before the peptide bond formation reaction as described below.

Among those skilled in the art of peptide synthesis, it is well known that all kinds of peptides can be prepared by condensing an amino acid or fragment peptide (i.e., a peptide with a smaller number of units) with an amino acid or other fragment peptide (again a peptide having fewer numbers than the desired one). final product). A number of different kinds of techniques are well known for such condensation reactions.

For example, the functional group (s) (e.g., an amino group, carboxy group, hydroxy group, guanidino group) not involved in the reaction by which the peptide bond (i.e. -CONH-) is formed during the condensation can be protected by one or more protecting groups. before the condensation reaction. In the process of the present invention, reagent A or B can be protected on the functional group (s) not participating in the condensation reaction according to methods known per se.

It is well known that prior to the peptide bond formation reaction, the C-terminal carboxyl group or the N-terminal amino group of an amino acid or peptide fragment involved in the peptide bond formation reaction is activated to enable it to induce peptide bond formation and the peptide bond formation reaction is initiated. a dehydrating agent if not activated. Methods for activating C-terminal carboxyl groups and N-terminal amino groups are well known. In the method of the present invention, such known methods can be used. Ie the condensation of the invention may be carried out by a first step of activating the C-terminal carboxyl group of reagent A or activation of the N-terminal amino group of reagent B, and a second step of the peptide bond formation reaction between the activated reagent A and reagent B or between the reagent Seen A and the activated reagent B; the reaction can also be carried out by a peptide bond formation reaction between reagent A and B in the presence of a dehydrating agent, the reagents A and B being optionally protected.

It is also known that when the above functional group or groups are protected with protecting groups prior to the condensation reaction, then the protecting group or groups may be removed after the condensation reaction. In the present method, removal of protecting groups is performed in accordance with the prior art.

In this context, it is known that a protected L-glutamyl group of the general formula

rco-ch2ch2ch (nh2) co- II

wherein R is an alkoxy group (e.g. methoxy, ethoxy, n-propoxy, i-propoxy or n-butoxy), an aralkoxy group (e.g. benzyloxy) or amino group can be readily converted to the L-pyroglutamyl group itself

T

upon contact with a base such as ammonia or an acid such as acetic acid, and that group II is equivalent to the L-pyroglutamyl group itself in this regard. By the present process, it is to be understood that the L-pyroglutamyl group (i.e., H- (Pyr) Glu-) in reagent A is not only to be perceived as the L-pyroglutamyl group itself, but also the protected L-glutamyl group of the general formula II. In cases where the H- (Pyr) Glu- in reagent A is a group II, the group II is readily converted to the L-pyroglutamyl group itself in accordance with the prior art.

Listed below are some of the procedures that can be advantageously used to carry out the peptide bond formation reaction of the invention.

(l) The method consisting of reacting a protected or unprotected reagent A, whose C-terminal group is a free carboxyl group, with a protected or unprotected reagent B, whose N-terminal amino group is a free amino group, in the presence of a condensing agent.

2) The process of comprising a protected or unprotected reagent A, whose C-terminal carboxyl group has been activated, is reacted with a reagent B whose N-terminal amino group is a free amino group.

3) The method comprising a protected or unprotected reagent A whose C-terminal carboxyl group is a free one. carboxyl group, is reacted with a protected or unprotected reagent B whose N-terminal amino group has been activated.

Examples of a protecting group for the intramolecular amino group of L-pyroglutamic acid are thus benzyloxycarbonyl, t-butoxycarbonyl and isobornyloxycarbonyl; examples of protecting groups for the imino group in L-histidine are benzyl, tosyl, 2,4-dinitrophenyl, t-bu ± oxycarbonyl and carbobenzoxy; examples of protecting groups for the hydroxyl group in L-serine are such ether-forming groups as benzyl and t-butyl; examples of protecting groups for the hydroxyl group in tyrosine are such ether forming groups as benzyl and t-butyl; examples of protecting groups for the guanidino group in L-arginine are nitro, tosyl, carbobenzoxy, isobornyloxycarbonyl and adamantyloxycarbonyl. In addition, the guanidino group of L-arginine can be protected by salt formation with a proton derived from an acid (e.g. hydrochloric or hydrobromic acid) and it will be understood that the proton is included in the term protecting group herein.

The activated form of the C-terminal carboxyl group in Reagent A can be exemplified by the corresponding acid anhydride such as a mixed anhydride with a carboxylic acid monoalkyl ester; or an azide; or an active ester such as the corresponding ester with an alcohol such as pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N-hydroxysuccin imide, N-hydroxy-5-norbornene-2 , 3-dicarboxamide, N-hydroxyphthalimide, N-hydroxybenztriazole. Of these esters, the N-hydroxy-5-norbornene-2,3-dicarboximide ester is preferred. Although N-hydroxy-5-norbornane-2,3-dicarboximide esters of amino acids or peptides are novel, they can be prepared according to the same method as that used for the preparation of N-hydroxysuccin imide esters of amino acids or peptides.

The corresponding phosphoric acid amide is mentioned as an example of an activated form of the N-terminal amino group of the protected or unprotected reagent B.

As a dehydrating agent, any reagent of the species used in peptide syntheses can be used. Particularly preferred are the so-called carbodiimide reagents such as dicyclohexylcarbodiimide.

By carrying out the condensation reaction according to the invention, one can place in a single reactor l) the protected or unprotected reagent A with free c-terminal carboxyl group, 2) the protected or unprotected reagent B with free N-terminal amino group, 3) the above alcohol e.g., N-hydroxy-5-norbornene-2,3-dicarboximide or N-hydroxysuccinimide, and 4) dehydrating agent. In that case, the protected or unprotected reagent A with free C-terminal carboxyl group reacts with the alcohol by means of the dehydrating agent to form the protected or unprotected reagent A with activated C-terminal carboxyl group, after which the protected or unprotected reagent A thus formed activated C-terminal carboxyl group reacts with the protected or unprotected reagent B with free N-terminal amino group. In this embodiment, the activation of the C-terminal carboxyl group and the formation of the peptide bond are carried out together in a single container.

Thus, in the preparation of the above-mentioned peptides of general formula I, one can choose between different embodiments and combinations of the necessary procedures.

A preferred embodiment of the method according to the invention is shown schematically in the following.

8 * 4 * 756 i) H- (Pyr) Glu-His-Trp-Ser-A1-Gly-OK (Reagent A)

+ DCC + HONBI

activation Ψ?

/ \ 1 2 I

H- (Pyr) Glu-His-Trp-Ser-A -Gly-ONBI + H-A, -Arg-Pro-Y

activated reagent A protected reagent B

In formation of J, peptide bond

Z

1 2 I

H- (Pyr) -Glu-His-Trp-Ser-A-Gly-A-Arg-Pro-Y

removal of protection Ψ

Η- (Pyr jGlu-His-Trp-Ser-A1-Gly-A2-Arg-Pro-Y

Z

Il) H- (Pyr) Glu-His-Trp-Ser-A1-Gly-OH + H-A-Arg-Pro-Y

reagent A protected reagent B

+ DCC + HONBI

(l) activation

© peptide bond formation Ψ Z

. 1 2 I

H- (Pyr) Glu-His-ITp-Ser-A-Gly-A-Arg-Pro-Y

removal of protection Ψ

/ \. 1 2 H- (PyrJGlu-His-Trp-Ser-A-Gly-A-Arg-Pro-Y

In this scheme, DCC stands for N, N'-dicyclohexylcarbodiimide, HONBI for N-hydroxy-5-norbornene-2,3-dicarboximide and Z a protecting group on the guanidino group of the L-arginine residue.

Peptide bond formation reactions such as those mentioned above under 1), 2) or 3) are generally carried out in a suitable solvent. Examples of solvents are dry or aqueous dimethylformamide, dimethylsulfoxide, pyridine, chloroform, dioxane, dichloromethane, or tetrahydrofuran or mixtures of such solvents.

Although the reaction temperature is usually in the range of approx. -20 ° C to approx. + 30 ° C the reaction can be carried out at even lower temperature or under heating.

The two starting materials which are to form a peptide bond are generally used in equimolar amounts, although other weight ratios can also be used if desired. Generally, for each mole equivalent of one starting material, one will generally use between 1 and 2 mole equivalents and preferably 1 to 1.4 equivalents of the other of the two starting materials. The amount of the dehydrating agent is usually about 1 to 2 molar equivalents relative to the water eliminated by the peptide bond formation.

Satisfactory results are often obtained when the reaction time is in the range of approx. six to approx. ten hours.

After the peptide bond formation reaction is complete, the reaction product can be isolated, for example, by precipitation with a solvent (in which the compound formed is heavily soluble), and then the precipitate is recovered by filtration.

When reagent A and / or reagent B are protected by one or more protecting groups, the protecting group or groups are generally found in the condensation product. As mentioned above, the protecting group or groups can be removed by conventional processes that do not interfere with the amino acid sequence of the nonapeptidamide derivative obtained as a product, and the removal of the protector leaves the nonapeptidamide derivative I in a state free of protecting groups.

Examples of a conventional process for removing protecting groups include catalytic reduction with such catalysts as palladium black, carbon palladium or platinum; acid hydrolysis with, for example, hydrogen fluoride or ίο

U975S

trifluoroacetic acid; and chemical reduction with, for example, sodium metal in liquid ammonia. Following any of these processes, the desired compound can be isolated in a conventional manner. Suitable methods include the above-mentioned precipitation technique.

The final product thus isolated can be suitably purified such as by column chromatography on, for example, carboxymethyl cellulose or commercially available; polymers for use in purification such as "Sephadex" or "Amberlite" XAD-2 (registered trademarks in Denmark).

Depending on the reaction conditions used, the desired compound is obtained in the form of a base or as a salt, including a physiologically acceptable salt. The base can be liberated from the salt in a conventional manner and the base can be converted to salts by reaction with acid suitable for the production of physiologically acceptable salts. Examples of such acids include inorganic acids such as hydrohalides such as hydrochloric acid, hydrobromic acid, in addition perchloric acid, nitric acid, thiocyanic acid. sulfuric acid or phosphoric acid and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthracanic acid, cinnamic acid, naphthalenesulfonic acid and sulphanilic acid.

The final compound thus obtained can be converted into metal complex salt compound in a manner known per se.

For example, one can react an aqueous solution of the obtained PlPl dam ti dami derivative in the manner described with a salt, hydroxide or oxide of one or more of the metals zinc, nickel, cobalt, copper and iron and then adjust the reaction mixture to a pH 6 to 8, thereby obtaining a heavily soluble adsorption complex salt compound between the metal compound and the nonapeptidamide derivative.

It turns out that the zinc complex salt compounds are the most desirable among the various metal complex salt compounds that can be obtained in this way, namely for extended action after administration.

Due to the extremely low toxicity, the nonapeptidamide derivatives obtained and physiologically acceptable salts thereof obtained in the manner described can be used without risk, thereby obtaining a strong ovulation-inducing effect.

11

14975S

When given rats a very small amount (e.g., 50 to 500 ng / 100 g) of nonapeptidamide derivative I or a physiologically acceptable salt thereof by intravenous, intramuscular or subcutaneous administration, a steep increase in the concentration of luteinizing hormone and follicle-stimulating hormone is observed in the blood; when the rats are infused with 10 to 200 ng / 100 g of such a compound by intravenous or intramuscular route, ovulation is induced in ca. 50% of the rats; and when the rats are infused with 0.1 to 5 μg / 100 g of the compound by intravenous or intramuscular route, ovulation is induced in 100% of the tested rats, even if the rats are in the diestrus (ie, the periods between the rut periods). When the dose is kept constant, intravenous infusion is generally twice as effective as subcutaneous infusion.

For the purposes of this specification, the term ng means nanogram.

These facts show that the subject peptides produced by the present method have more potent hormonal action than the naturally occurring luteinizing hormone releasing hormone.

Injection preparations can be prepared by dissolving a nonapeptide derivative prepared in physiological saline solution according to the invention.

Since the compounds of the present invention have sufficient physiological activity even when used only in very small quantities, it is convenient to use them in the form of lyophilized ampoule containing mannitol as the excipient.

Some examples serve to elucidate the method of the invention. The following abbreviations are used in the examples: Z- * ·: benzyloxycarboxyl; IBOC-: isobornyloxycarbonyl; BOC-: t-butyloxycarbonyl; - One: ethyl ester; -0SU: N-hydroxysuccinimide ester; -OtBu: t-butyl ester; -0NDP: 2,4-dinitrophenol ester; -0NBI: N-hydroxy-5-norbornene-2,3-dicarboximide ester; ΝΒΙ0ΝΒΙ: N-hydroxy-5-norbornene-2,3-dicarboximide; DCC: Ν, Ν'-dicyclohexylcarbodiimide; DMF: Ν, Ν-dimethylformamide; TLC: thin layer chromatography; DCHA: dicyclohexylamine; MeOH: methanol; EtOH: ethanol; n-BuOH: n-butanol.

For thin layer chromatography, the following developing systems are used: 12 149755

Rf 1 = chloroform / methanol / acetic acid 9: 1: 0.5;

Rf 2 = ethyl acetate / pyridine / acetic acid / water 60: 20: 6: 11; R 3 = n-butanol / ethyl acetate / acetic acid / water 1: 1: 1: 1;

Rf =- = n-butanol / acetic acid / water 4: 1: 1;

Rf 5 = n-butanol / pyridine / acetic acid / water 30: 20: 6: 24.

In the examples, weight parts have the same relation to volume parts as g to ml and percentages are weight 0 / unless otherwise stated. "Amberlite" CG-400 is a tertiary amine-styrene-divinylbenzene copolymer from Rohm and Haas Co., USA; "Sephadex" LH-20 is an esterified dextran from Pharmacia Fine Chemicals, Sweden; "Amberlite" XAD-2 is a macroreticular styrene-divinylbenzene copolymer - from Rohm 8c Haas Co., USA, and "Amberlite" IRA-400 is a tertiary amine-styrene-divinylbenzene copolymer from the same company.

Example 1 H- (Pyr) Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-NHC ^ H ^ a) Z-Arg (NO2) -Pro-NHC2H5 In 10 volumes of DMF, 0.901 parts by weight of Z Arg (NO2) -Pro-OH and 0.181 parts by weight of ethylamine hydrochloride, and with cooling to 0 ° C, 0.38 parts by volume of triethylamine are added dropwise. Then 0.43 parts by weight of HONBI and 0.495 parts by weight of DCC are added and the mixture is stirred at 0 ° C for five hours and then at room temperature for ten hours. The urea formed as a by-product is filtered off and the DMF is distilled off. The residue is extracted with chloroform, washed with water and dried over anhydrous magnesium sulfate. The chloroform is distilled off and the residue treated with ether and re-precipitated from methanol / ether. Yield 0.692 part by weight, Rf 1 = 0.5o; mp 141-145 ° C (dec.) = -44.6 ° (c = 1 in methanol).

Calculated for C₂2H310O6N7: C 52.82 H 6.54 N 20.53 Found: C 52.95 H 6.77 N 19.65 ° / 13

t4975S

b) Z-Leu-Arg (NO2) -Pro-NHC2H5 0.572 part by weight Z-Arg (NO2) -Pro-NHC2H5 is dissolved in 5 parts by volume of 25% HBr / acetic acid and the solution is left at room temperature for 30 minutes. Then dry ether is added to the reaction mixture and the resulting precipitate is recovered by filtration and dried. Meanwhile, 0.291 part by weight of Z-Leu-OH is dissolved in 5 parts by volume of dioxane and with cooling 0.247 parts by weight of DCC and 0.215 parts by weight of ΝΒΙ0ΝΒΙ are added. The mixture is stirred for two hours and the urea formed as a by-product is filtered off. To the filtrate is added according to the precipitate formed above, which is dissolved by the addition of 3 volumes of DMF. While cooling, 0.17 volume of triethylamine is added dropwise and then the mixture is stirred at room temperature overnight. The solvent is distilled off and the residue is extracted with chloroform and washed with water. The chloroform layer is dried over anhydrous magnesium sulfate and the chloroform is distilled off. The residue is treated with ether and re-precipitated from methanol / ether. Yield 0.47 part by weight (72%), m.p. 144-146 ° C (dec.), Rf 1 = 0.40, = -58.0 ° (c = 1 in methanol).

c) 0.165 part by weight of Z-Leu-Arg (NO2) -Pro-NHC2H5 is dissolved in 25% s of HBr / acetic acid and the solution is left at room temperature for 30 minutes. Then dry ether is added to the reaction mixture and the resulting precipitate is recovered by filtration and dried well. This precipitate is dissolved in 3 parts by volume of DMF and with cooling and stirring 0.05 parts by volume of N-ethyl morpholine are added dropwise. In this solution, 0.19 parts by volume of H- (Pyr) -Glu-His-Trp-Ser-Tyr-Gly-OH hydrochloride is dissolved, followed by the addition of 0.054 parts by weight of HONBI and 0.062 parts by weight of DCC.

The mixture is stirred at 0 ° C for two hours and then at room temperature overnight. The urea formed as a by-product is removed by filtration and the DMF is distilled off. The residue is treated with ethyl acetate to give a powder weighing 0.32 parts by weight. This powder is placed on a column of "Amberlite" XAD-2 and desorbed in a linear gradient elution system from 5% aqueous ethanol to ethanol. The major fraction is lyophilized to give 0.11 volume of pure H- (Pyr) Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg (NO2) -Pro-NHC2H5 · This product is treated with 4 volumes of hydrogen fluoride at 0 ° C for one hour in the presence of 0.02 volume 14 anisole and 0.02 volume mercaptoethanol. The hydrogen fluoride is distilled off and after drying the residue is dissolved in water. The solution is passed through a column of "Amberlite" IRA-400 in acetate form and then adsorbed onto a column of carboxy-methyl cellulose. The column is eluted in a linear gradient elution system with from 0.005N aqueous ammonium acetate to 0.2N aqueous ammonium acetate and the main fraction is freeze-dried. In this way 0.087 part by weight of pure H- (Pyr) Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-NHC2H5 is obtained. Rf 3 = 0.36, α 4 = -56.2 ° (c = 0.5 in 5% acetic acid). Amino acid analysis: His 0.95 (l)> Arg 0.98 (l), Ser 0.95 (l), Glu 0.98 (1), Pro 1.00 (l), Gly 1.00 (l), Leu 1.00 (l), Tyr 1.00 (l) and ΝΗ ^ Ο ^ Η ^ 1.10 (l). The numbers in brackets indicate the theoretical values.

Example 2

H- (Pyr) Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-NHC₂H ^OH

a) Z-Arg (NO2) -Pro-NHC2H40H

In 5 parts by volume of DMF, 0.9 parts by weight of Z-Arg (NO2) -Pro-OH and 0.43 parts by weight of HONBI are dissolved and, with cooling to 0 ° C, 0.495 parts by weight of DCC are added. The mixture is stirred overnight. The separated urea compound is filtered off and 0.1832 parts by weight of ethanolamine is added to the filtrate and then stirred overnight. The DMF is distilled off under reduced pressure and dioxane is added to the residue. Insoluble material is filtered off. The dioxane is distilled off under reduced pressure and the residue is extracted with n-butanol. The extract is washed with water, dilute hydrochloric acid, water, aqueous sodium bicarbonate solution and water in that order and then the n-butanol is distilled off under reduced pressure. The residue is treated with ether and re-precipitated from methanol / ether. Yield 0.625 part by weight (63.3%), m.p. 124-128 ° C (dec.), Rf 2 = 0.23, α = ° -36.6 ° (c = 0.5 in ethanol).

15 143755

b) Z-Leu-Arg (NO2) -Pro-NHC2H40H

In 2 parts by volume of 25 $ HBr / acetic acid, 0.493 parts by weight of Z-Arg (NO2) -Pr o -NHCgH 2 OH are dissolved and the solution is left at room temperature for 30 minutes. Then, dry ether is added and the resulting precipitate is recovered by filtration and dried to form a powdered product. Meanwhile, 0.282 parts by weight of Z-Leu-OH and 0.215 parts by weight of HONBI are dissolved in 3 parts by volume of DMF and, under cooling to 0 ° C, 0.248 parts by weight of DCC is added followed by stirring for two hours. To the solution is added the aforementioned powdered product and 0.15 by volume of triethylamine is added dropwise. The mixture is stirred overnight. The urea is filtered off and the DMF is distilled off under reduced pressure. The residue is adsorbed on a column of 50 parts by weight of silica gel and desorbed with chloroform / methanol / acetic acid solvent system 9: 1: 0.5. The solvents are distilled off and the residue is crystallized with ether. Yield 0.153 parts by weight, m.p. 110-113 ° C (dec.), Eph 1 = 0.49, α = 0 = -46.6 ° (c = 0.5 in ethanol).

c) In the presence of 0.1 part by volume of anisole, 0.152 part by weight of z-Leu-Arg (NO2) -Pro-NHC2H40H is dissolved in 3 parts by volume of hydrofluoride. The solution is stirred at 0 ° C for one hour. The hydrogen fluoride is distilled off and the residue dried well and dissolved in water. After adding a small amount of concentrated hydrochloric acid, the solution is washed with ether and the aqueous layer is freeze-dried. The resulting powder is dissolved in 5 parts by volume of DMF together with 0.19 parts by volume of H- (Pyr) -Glu-His-Trp-Ser-Tyr-Gly-OH hydrochloride and 0.0493 parts by weight of HONBI. The solution is cooled to 0 ° C and 0.0567 parts by weight of DCC and 0.07 parts by volume of N-ethylmorpholine are added. The whole mixture is stirred overnight. The DMF is distilled off under reduced pressure and the residue is dissolved in water. The insoluble material is filtered off and the filtrate is passed through an "Amberlite" IRA-400 column in acetate form. The effluent is either collected and lyophilized. This product is placed on a column of "Amberlite" XAD-2 and desorbed in a linear gradient elution system of water-methanol. The major fraction is lyophilized to give 0.023 parts by weight of pure H- (Pyr) Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-NHC2H40H. Rf 3 = 0.28, α ° = -54.4 ° (c = 0.5 in 5% aqueous acetic acid).

149756 16

Amino acid analysis: His 1.00 (1), Arg 1.05 (1), Ser 0.95 (1), Glu 1.00 (1), Pro 1.05 (1), Gly 1.00 (1), Leu 0.95 (1), Tyr 0.76 (1).

Example 3 H- (Pyr) Glu-His-Trp-Ser-Tyr-Gly-Eeu-Arg-Pro-NHCH3 a) Z-Arg (NO2) -Pro-NHCH3 In 5 volumes of DMF, 0.675 parts by weight of Z-Arg { NO2) -Pro-OH and 0.101 part by weight of methylamine hydrochloride, and while the solution is cooled to 0 ° C, 0.21 part by volume of triethylamine is added dropwise.

Then 0.322 parts by weight of HONBI and 0.37 parts by weight of DCC are added and the whole mixture is stirred at 0 ° C for five hours and then at room temperature for ten hours. The urea formed as a by-product is filtered off and the DMF is distilled off. The residue is extracted with chloroform, washed with water and dried over anhydrous magnesium sulfate. The chloroform is distilled off and the residue is treated with ethanol and re-precipitated from methanol / ether. Yield 0.612 wt (88%), Eph 1 = 0.30, mp 137-143 ° C (dec.), = -41.5 ° (c = 1 in methanol).

Calculated for C20H2906N7: C 51.82 H 6.31 N 21.15 Found: C 51.84 H 6.54 N 21.57% b) Z-Leu-Arg (NO2) -Pro-NHCH3 In 5 parts by weight 25% s HBr / acetic acid dissolves 0.556 parts by weight of Z-Arg (NO2) -Pro-NHCH3 and the solution is left at room temperature for 30 minutes. Then dry ether is added and the resulting crystals are extracted by filtration and dried. Meanwhile, 0.312 parts by weight of Z-Leu-OH are dissolved in 5 parts by volume of dioxane / ethyl acetate 1: 1, and while the solution is cooled, 0.268 parts by weight of DCC and 0.232 parts by weight of HONBI are added. The whole mixture is stirred for two hours. The urea formed as a by-product is filtered off and the crystals prepared according to the foregoing are added to the filtrate and dissolved by the addition of 3 volumes of DMF. During cooling, 0.17 volume of triethylamine is added. The mixture is stirred at room temperature overnight. The solvent is distilled off. The residue is extracted with chloroform and washed with water. The chloroform layer is dried over anhydrous magnesium sulfate and then the chloroform is distilled off. Then the residue is treated with ether and re-precipitated from methano1 / ether. Yield 0.45 part by weight (78%), Rf 1 = 0.25.

Calcd for Cgg H 2 N 2 C 54.15 H 7.02 N 19.43 found: C 54.37 H 6.98 N 19.52% c) In 5 volumes 25% s HBr / acetic acid dissolve 0.144 part by weight Z-Leu -Arg (NO2) -Pro-NHCH3 and after standing at room temperature for 30 minutes, dry ether is added. The resulting precipitate is collected by filtration and dried thoroughly. Then it is dissolved in 3 parts by volume of DMP and with cooling and stirring 0.05 parts by volume of N-ethylmorpholine are added dropwise.

In this solution, 0.19 parts by weight of H- (Pyr) Glu-His-Trp-Ser-Tyr-Gly-OH hydrochloride is dissolved, followed by the addition of 0.054 parts by weight of HONBI and 0.062 parts by weight of DCC. The whole mixture is stirred at 0 ° C for two hours and then at room temperature overnight. The urea formed as a by-product is filtered off and the DMP is distilled off. The residue is placed on a column of "Amberiite" XAD-2 and desorbed in a linear gradient elution system from 5% aqueous ethanol to ethanol. The main fraction is lyophilized to give 0.137 parts by weight of pure H- (Pyr) Glu-His-ITp-Ser-Tyr-Gly-Leu-Arg (NO2) -Pro-NHCH3 · A portion of this product of 0.09 parts by weight is treated with 4 parts by weight of hydrogenyl fluoride in the presence of 0.02 parts by volume of anisole and 0.02 parts by volume of mercaptoethanol at 0 ° C for one hour. The hydrogen fluoride is distilled off and after drying, the residue is dissolved in water. The solution is passed through "Amberiite" IRA-400 in acetate form and then adsorbed onto a column of carboxymethyl cellulose. The column is eluted in a linear gradient elution system ranging from 0.005N aqueous ammonium acetate to 0.2N aqueous ammonium acetate and the main fraction is freeze-dried. In this way 0.06 part by weight of pure H- (Pyr) Glu-His-ΊΤρ-Ser-Tyr-Gly-Leu-Arg-Pro-NHCH3 is obtained. Rf 3 = 0.37, α | 4 = -55.6 ° (c = 0.5 in 5% aqueous acetic acid).

149755 18

Amino acid analysis: His 0.96 (1), Arg 0.98 (1), Ser 0.96 (1), Glu 1.00 (1), Pro 1.00 (1), Gly 1.00 (1), Leu 0.98 (1), Tyr 0.98 (1), NH 2 CH 3 1.12 (1). The numbers given in brackets are the theoretical values.

Example 4 H- (Pyr) Glu-His-Trp-Ser-Tyr-Gly-NLe-Arg-Pro-NH-CH2CH3 a) Z-NLe-Arg (NO2) -Pro-NH-CH2CH3 In 4 volumes 25% s hydrogen bromide / acetic acid dissolves 0.4775 parts by weight of Z-Arg (NO2) -Pro-NH-CH2CH3 and the solution is left at room temperature for 30 minutes. Then 50 parts by volume of dry ether are added to the reaction mixture and the resulting precipitate is recovered by filtration, washed well with ether and dried over sodium hydroxide under reduced pressure. Meanwhile, 0.2653 parts by weight of Z-NLe-OH is dissolved in a mixture of 2 parts by volume of ethyl acetate and 12 parts by volume of dioxane. While the solution is cooled to 0 ° C, 0.197 parts by weight of ΝΒΙ0ΝΒΙ and 0.226 parts by weight of DCC are added and the whole mixture is stirred for three hours. The urea formed as a by-product which has separated is filtered off and the filtrate is recovered to a solution of it according to the above / precipitate in 1 volume part DMF, followed by dropwise addition of 0.28 volume part triethylamine. The mixture is stirred overnight. The solvent is distilled off and the residue is extracted with 100 volumes of chloroform. The extract is washed with a 5% aqueous solution of sodium hydrogencarbonate, water, 0.5N hydrochloric acid and water and then dried over anhydrous magnesium sulfate. The chloroform is distilled off and the residue is treated with ether and re-precipitated from ethanol / ether. Yield 0.41 parts by weight, mp 109-111 ° C (dec.), Dp2 = -50.4 ° (c = 0.5 in ethanol).

Calculated for C CH ^ONNg, 1/2 H₂O: C 54.07 H 7.22 N 18.68 Found: C 53.79 H 7.09 N 18.24% b) In 2 volumes 25% hydrogen bromide / acetic acid dissolves 0.155 parts by weight of Z-NLe-Arg (NO2) -Pro-NH-CH2CH3 and the solution is left at room temperature for 30 minutes. Then 50 parts by volume of dry ether are added to the reaction mixture and the resulting precipitate is recovered by filtration, washed thoroughly with ether and dried over sodium hydroxide under reduced pressure.

Then it is dissolved in 2 parts by volume of DMF and, under cooling to 0 ° C, 0.19 parts by weight of H- (Pyr) Glu-His-Trp-Ser-Tyr-Gly-OH hydrochloride, 0.09 parts by weight of HONBI, 0.103 parts by weight of DCC are added. and 0.1 by volume of N-ethylmorpholine in said order. The mixture is stirred at 0 ° C for five hours and at room temperature for ten hours.

The separated urea formed by-product is filtered off and the DMF is distilled off under reduced pressure. The residue is dissolved in 4 parts aqueous ethanol with 0.2 parts by weight of urea and the solution is adsorbed on a column of "Amberlite" XAD-2 and desorbed in a linear gradient elution system ranging from 5% aqueous ethanol to 80% aqueous ethanol. The major fraction is lyophilized to give 0.067 part by weight of H- (Pyr) Glu-His-Trp-Ser-Tyr-Gly-NLe-Arg (NO2J-Pro-NH-C2 CH2). A portion of 0.060 part by weight of this product is dissolved in 4 volumes of hydrogen fluoride at -50 ° C in the presence of 0.05 volume of anisole and 0.02 volume of 2-mercaptoethanol, and the solution is stirred at 0 ° C for one hour The hydrogen fluoride is distilled off under reduced pressure and the residue is dried in a desiccator containing sodium hydroxide. The residue is dissolved in 20 parts by volume of water and the solution is passed through "Amberlite" CG-400 in acetate form. lyophilized to give 0.035 parts by weight of the desired product, This product is further placed on a column of "Sephadex" LH-20 and desorbed with 0.1N acetic acid, the homogeneous fraction is lyophilized to give 0.028 part by weight of pure H- (Pyr) Glu-His-Trp-Ser-Tyr-Gly-NLe-Arg-Pro-NH-CH2CH3. cc £ 5 = -52.2 ° (c = 0.5 in 5% acetic acid). Amino acid analysis: His 1.00 (l), Arg 1.02 (l), ΊΤρ 1.00 (l), Ser 0.91 (1), Glu 1.00 (1), Pro 1.05 (l), Gly 0.98 (1), Tyr 1.00 (1), NLe 1.02 (1).

20 149765

Example 5 Η- (Pyr) Glu-His-Trp-Ser-Phe-Gly-Leu-Ar g-Pro-NH-CHr 2 CH 2 a) ZS is -Phe-Gly-OE t 2.5 parts by weight Z-Ehe-Gly -One is dissolved in 50 parts by volume of methanol and the solution is subjected to catalytic reduction with 5% palladium on coal for four hours. The catalyst is removed by filtration and the solvent is distilled off. The residue is dissolved in 20 parts by volume of dimethylformamide and then 2.6 parts by weight of Z-Ser-ODNP are added followed by stirring for eight hours. The solvent is distilled off and the residue is dissolved in ethyl acetate. The solvent is washed with a 5% aqueous solution of sodium hydrogen carbonate, 1N hydrochloric acid and water and then dried over magnesium sulfate. The solvent is distilled off and the solid residue is crystallized from ethyl acetate / petroleum ether. the yield is 1.75 parts by weight (74.2 ° / 0, m.p. 128-129 ° C, α 2 = -26.2 ° (c = 0.6 in ethanol)).

Calcd for 24 H 29 <7> 3: C 61.13 H 6.20 N 8.91 Found: C 60.47 H 6.00 H 8.88% b) Z-Ser-Phe-Gly-NHNHg 1 , 65 parts by weight of z-Ser-Phe-Gly-OEt are dissolved in 20 parts by volume of methanol and then 0.71 part by volume of hydrazine hydrate is added. The mixture is left for eight hours and the resulting crystals are recovered by filtration and washed with methanol / ether 1: 1. The yield is 1.6 parts by weight (100%), mp 191-193 ° C, α 2 = -23.0 ° (c = 2 in DMF).

Calc'd for C 22 H 27 N 5 O 6: C 57.76 H 5.95 N 15.31 found: C 57.32 H 6.21 N 15.53% c) Z-Ser-Phe-Gly-I, eu ~ Arg (N02 Dissolve J-Pro-NH-CHgCHg 0.8 parts by weight of Z-Ser-Phe-Gly-NHNH 2 in 10 volumes of DMF and cool the solution, then add 3.5 volumes of 2N hydrochloric acid, while stirring at -6 ° C. 2N aqueous solution of sodium nitrite and the mixture is allowed to react for ten minutes, meanwhile the precipitate is dissolved by the addition of 10 parts by volume of DMF. The ethyl acetate layer, 80 parts by volume, is dried over sodium sulfate and then added to 20 parts by volume of a solution of 1.04 parts by weight of H-Leu-Arg ( In DMF The ethyl acetate layer is distilled off in an ice bath and the resulting homogeneous solution stirred at 3 ° C for two days. The solvent is distilled off and the residue is purified by chromatography on silica gel. The chromatogram is developed with a solvent system of ethyl acetate / pyridine / acetic acid / water 60: 20: 6: 10 and the active fractions are combined and distilled to remove the solvents. Water is added to the residue and the resulting solid is recovered by filtration. The yield is 0.87 part by weight (61.2%), mp 108 ° C, α 22 = -59.1 ° (c = 0.7 in ethanol).

Calc'd for c41H59011N11, H2O: C 54.72 H 6.83 N 17.12 Found: C 54.95 H 6.84 N 16.74%

d) Dissolve 0.836 part by weight of Z-Ser-Phe-Gly-Leu-Arg (NO2) -Pro-NH-CH2jCH3 in methanol and then add 1 part by volume 1N hydrochloric acid. The solution is subjected to catalytic reduction with palladium black. The catalyst is removed by filtration and the solvent is distilled off. The residue is dissolved in 10 parts by volume of DMF

and then 0.83 parts by weight of H- (Pyr) Glu-His-Trp-OH and 0.36 parts by weight of HONBI are dissolved. The mixture is cooled to -5 ° C and then 0.412 parts by weight of DCC is added. The mixture is allowed to react for two days after which the precipitate is filtered off and the DMF is distilled off. The residue was purified by chromatography using a column of "Amberlite" XAD-2 in a linear gradient elution system ranging from 5% aqueous ethanol to 80% aqueous ethanol. The active fractions are combined and distilled to remove the ethanol, then the remaining aqueous solution is freeze-dried. The resulting powder is further purified by ion exchange chromatography on carboxymethyl cellulose in a linear gradient elution system ranging from 0.005N to 0.1N aqueous solution of ammonium acetate. The active fraction is freeze-dried to form a pure white powder. The yield is 0.424 parts by weight, α = -55 ° (c = 0.5 in 5% acetic acid).

Amino acid analysis: His 1.02 (1), Arg 1.01 (1), Trp 0.91 (1), Ser 0.90 (1), Glu 1.00 (1), Pro 1.00 (1), Gly 0.98 (1), Leu 1.00 (1), Phe 1.00 (1), ethylamine 1.06 (1).

To determine the yield of Trp, the entire amino acid analysis is performed by acid hydrolysis with 5 »7N HCl in the presence of thioglycolic acid.

Example 6 H- (Pyr) Glu-γίs-Trp-Ser-Phe-Gly-ILe-Arg-Pro-NHCgH 2 a) H- (Pyr) Glu-His-Trp-Ser-Phe-Gly-otBu 1.57 parts by weight of H-Gly-OtBu and 4.0 parts by weight of Z-Phe-OSU are dissolved in 20 parts by volume of ethyl acetate and the mixture is stirred at room temperature overnight. The reaction mixture is washed with a 5% aqueous solution of sodium hydrogen carbonate, N hydrochloric acid and water, dried over magnesium sulfate and concentrated under reduced pressure. Petroleum ether is added to the residue and the precipitate is collected by filtration to give 3.5 parts by weight (85%) of Z-Phe-Gly-OtBu, mp 78-79 ° C, ap3 = -16.7 ° (c = 1.0 in ethanol ).

Calculated for C23H2G05N2: C 66.97 H 6.87 N 6.79 found: C 67.14 H 6.64 N 6.88% »7.0 parts by weight of Z-Phe-Gly-OtBu dissolved in 50 parts by volume of methanol . The solution is subjected to catalytic reduction with palladium black as the catalyst and the reduction takes five hours. The reaction mixture is filtered and the filtrate is concentrated by evaporation of the solvent. The residue is dissolved in 10 parts by volume of acetonitrile and then 1.62 parts by weight of Z-Ser-ODNP are added.

The resulting mixture is stirred at room temperature for one day and the acetonitrile is evaporated. The residue is dissolved in ethyl acetate. The solution is washed with 10% ammonia, N hydrochloric acid and water, dried over anhydrous magnesium sulfate and concentrated by evaporation of the solvent. The residue is collected by petroleum ether and recrystallized from ethyl acetate / petroleum ether to give 1.41 parts by weight (70%) of Z-Ser-Phe-Gly-OtBu, mp 105-106 ° C, aj 3 = -26.6 ° (c = 0.74 in ethanol).

Calculated for C26H3307N3: C 62, 50 H 6.66 N 8.41 found: C 62.91 H 6.38 N 8.12% 1.34 parts by weight of Z-Ser-phe-Gly-OtBu dissolved in 50 parts by volume of methanol. The solution is subjected to catalytic reduction using palladium black as a catalyst for five hours. The mixture is filtered and the filtrate concentrated to give a residue. The residue and 1.27 parts by weight of H- (Pyr) Glu-His-Trp-OH and 0.83 parts by weight of HONBI are dissolved in 20 parts by volume of DMF and then cooled with ice. To the mixture is added 0.95 parts by weight of DCC and the resulting mixture is allowed to react overnight. The precipitate is removed by filtration and the filtrate is concentrated. Acetonitrile is added to the residue and then heated. Powders in the mixture are collected by filtration and subjected to re-precipitation from 50% aqueous ethanol to give 1.8 parts by weight (87%) of H- (Pyr) Glu-His-Trp-Ser-Phe-Gly-OtBu, a ^ 3 = -20.6 ° (c = 1.0 in DMF). Calculated for C40H490gN9.2H2O: C 58.59 H 6.52 N 15.38 found: C 59.02 H 6.62 N 15.23% 24 t69755 b) & -ILe-Arg (NO2) -Pro-NHC2H5 1 , 43 parts by weight of Z-Arg (NO 2) -Pro-NHC 2 H 3 are dissolved in 10 parts by volume of 25% HBr / acetic acid and the mixture is left at room temperature for 40 minutes. An additional 80 volumes of dried ether are added and the precipitate is collected by filtration and dried to produce powders.

On the other hand, 0.795 parts by weight of Z-ILe-OH is dissolved in 10 parts by volume of dioxane and then cooled to 0 ° C. To this solution are added 0.59 part by weight ΝΒΙ0ΝΒΙ and 0.68 part by weight DCC and the mixture is stirred for two hours. The mixture is filtered to remove the precipitate and to the filtrate are added the powders prepared in the manner described above. 0.84 volume part of triethylamine is further added and the mixture is stirred at room temperature for 12 hours. The solvent dioxane is removed by evaporation. The residue is dissolved in 100 parts of chloroform. The solution is washed with 0.1N hydrochloric acid, 5% aqueous sodium bicarbonate and water, then dried over anhydrous magnesium sulfate and concentrated by evaporation of the chloroform. The residue is triturated with ether to produce a precipitate which is re-precipitated from ethanol / ether. 1.1 parts by weight of Z-ILe-Arg (NO2) -Pro-NHC2H3 are obtained, mp 103-105 ° C (dec.) = -58.1 ° (c = 1 in methanol).

Calc'd for 02720420 ?8, 1/2 H₂O: C 54.08 H 7.23 N 18.68 Found: C 53.80 H 7.15 N 18.84¾ c) 0.177 part by weight z-ILe-Arg (NO2) - ProNHC2H4 is dissolved in 3 parts by volume 25% HBr / acetic acid and the solution is left at room temperature for 30 minutes followed by the addition of 50 parts by volume of dried ether. The precipitate is collected by filtration, washed well with ether and dried to give an amine component.

On the other hand, 0.221 parts by weight of H- (Pyr) Glu-His-Trp-Ser-Phe-Gly-OtBu is dissolved in 5 parts by volume of trifluoroacetic acid and the solution is allowed to stand at room temperature for 40 minutes. 0.05 volume of 5.7N hydrochloric acid is added to the solution and 50 volumes of ether are added. The precipitate is collected by filtration, dried and dissolved in 3 volumes of DMF and the solution is cooled to 0 ° C. To the solution are added the amine component prepared 25 * 9756 according to the foregoing, 0.097 parts by weight of HONBI, 0.111 parts by weight of DCC and 0.126 parts by weight of triethylamine. The resulting mixture is stirred at 0 ° C for five hours and then at room temperature for another ten hours. The mixture is filtered to remove the dicyclohexylurea thus formed and the filtrate concentrated under reduced pressure. The residue is dissolved in 10 parts by volume of 10% aqueous ethanol and the solution is poured on top of a column packed with "Amberlite" XAD-2. Desorption is carried out in a linear gradient elution system ranging from 10% aqueous ethanol to 80% aqueous ethanol. The fractions containing the product are collected, concentrated by evaporation of the solvent ethanol and lyophilized to give 0.085 part by weight of H- (Pyr) Glu-His-Trp-Ser-Phe-Gly-ILe-Arg (NO2) -Pro-NHC2H2.

0.07 part by weight of this product is dissolved in a mixture of 0.02 part by volume of anisole, 0.02 part by volume of 2-mercaptoethanol and 4 parts by volume of anhydrous hydrogen fluoride. The solution is stirred at 0 ° C for one hour after which the hydrogen fluoride is evaporated under reduced pressure. The residue is dried in a desiccator and then dissolved in 50 parts by volume of water and passed through a column packed with "Amberlite" IRA-400 in acetate form. After passing through the column, the solution is lyophilized to give 0.075 part by weight of crude product. This crude product is adsorbed onto a column of carboxymethyl cellulose and desorbed in a linear gradient elution system from 0.005N ammonium acetate to 0.2N ammonium acetate.

The fractions containing the desired product are freeze-dried.

The product thus obtained is dissolved in 0.1N aqueous acetic acid and passed through a column of "Sephadex" LH-20. After passing through the iblonnen, the solution is lyophilized and 0.043 part by weight of pure product is obtained. Rf 3 = 0.41,? 3 = -59.4 ° (c = 0.5 in 5% acetic acid).

Amino acid analysis: His 1.1 (1), Arg 1.1 (1), Trp 0.87 (1), ethylamine 1.0 (1), Ser 0.81 (1), Glu 1.03 (1), Pro 1.0 (1),

Gly 1.0 (1), ILe 1.06 (1), Phe 0.97 (1).

Example 7 26 149756 H- (Pyr) Glu-His-Trp-Ser-Tyr-Gly-Met-Arg-Pro-NHC2H, - a) BOC-Met-Arg-Pro-NHC2H5 1.91 parts by weight Z-Arg (NO2 ) -PRO-NHC 2 H 5 is dissolved in 10 volumes 25 # s HBr / acetic acid and the solution is allowed to stand for 30 minutes after which dried ether is added. The precipitate is collected by filtration, washed well with ether and dried to give an amine component.

On the other hand, 1.75 parts by weight of BOC-Met-OH.DCHA is dissolved in 100 parts ether, then washed twice with 50 parts by volume 0.2N aqueous sulfuric acid and dried over anhydrous sodium sulfate. The ether is evaporated and the residue is dissolved in 20 parts of dioxane. The solution is cooled to 0 ° C and then added to the solution 0.905 parts by weight of DCC and 0.79 parts by weight of HONBI. The mixture is stirred for two hours. The dicyclohexylurea formed is filtered off and the filtrate is concentrated. The residue is added to 5 parts by volume of DMF and the entire amount of the amine component prepared as described first in the example. The resulting solution is cooled, then 1.12 volumes of triethylamine are added. The mixture is stirred at room temperature for 12 hours and the DMF is evaporated. The residue is dissolved - in 100 volumes of chloroform, washed with 0, 1N hydrochloric acid, 5 # aqueous sodium hydrogen carbonate solution and water, dried over anhydrous magnesium sulfate and concentrated by evaporation of chloroform.

Ether is added to the residue to obtain solid material. The obtained material is re-precipitated from ethyl acetate / ether to give 1.4 parts by weight of BoC-Met-Arg-Pro-NHCoH, - mp 101-104 ° C 22 ° (decomposition), α D = -64.4 ° (c = 1.0 in methanol).

Calcd. For C23H4207NgS, 1/2 HgO: C 47, 33 H 7.42 N 19.20 S 5.49 Found: C 47.67 H 7.23 N 18.94 S 5.48 # b) H- ( Pyr) Glu-His-Trp-Ser-Tyr-Gly-Met-Arg-Pro-NHC 2 H 2,275 parts by weight of BOC-Met-Arg (NO 2) -Pro-NHC is dissolved in a mixture of 0.1 volume part 2-mercaptoethanol and 5 volume parts trifluoroacetic acid and left at room temperature for 30 minutes.

To the mixture is added 0.085 volume 5.7N hydrochloric acid.

149756 27

The resulting mixture is cooled and 50 volumes of ether are added. The precipitate is collected by filtration, washed with ether, dried, dissolved in DMF and cooled to 0 ° 0. To the solution is added 0.318 parts by weight of H- (Pyr) Glu-His-Trp-Ser-Tyr-Gly-OH-hydrochloride, 0.107 parts by weight of HONBI, 0.124 parts by weight of DCC and 0.17 parts by weight of N-ethylmorpholine and then the mixture is stirred for 12 hours. hours. The precipitated dicyclohexylurea is removed by filtration and the filtrate is concentrated under reduced pressure. The residue is dissolved in 5 parts by volume of 5% aqueous ethanol, placed on a column of "Amberlite" XAD-2, and desorbed in a linear gradient elution system ranging from 5% aqueous ethanol to 80% s aqueous ethanol. 0.135 part by weight of H- (pyr) Glu-His-Trp-Ser-Tyr-Gly-Met-Arg (NO2) -Pro-NHC2H2. 0.1 part by weight of this product is dissolved in a mixture of 0.02 part by volume of anisole, 0.02 volumes ethanol and 0.02 volumes 2 mercaptoethanol and 6 volumes hydrogen fluoride, then the mixture is stirred at 0 ° C for one hour, the hydrogen fluoride is removed by evaporation under reduced pressure and the residue is dried in a desiccator. is passed through a column of "Am-berlite" CG-400 in acetate form. After passing through the column, the solution is freeze-dried. The residue is dissolved in 20 parts by volume of water and to the residue is added 0.4 parts by volume of thioglycolic acid. The reaction mixture is diluted with 50 rpm parts are adsorbed on a column of carboxymethyl cellulose and desorbed in a linear gradient elution system ranging from 0.005N ammonium acetate to 0.2N ammonium acetate. The main fractions are freeze-dried to give 0.02 part by weight of the desired product in crude form. The product is adsorbed onto a column of "Sephadex" LH-20 and eluted with 0.1N acetic acid. The eluted solution is freeze-dried to give a purified product, tc: Rf = 0.31 (silica gel; n-butanol / acetic acid / ethyl acetate / water 1: 1: 1: 1) = -43.2 ° (c = 0.25 in 5% acetic acid).

Amino acid analysis: His 0.9 (1), Arg 0.9 (1), Trp 0.8 (1), Ser 0.7 (1), Glu 0.9 (1), Pro 1.1 (1), Gly 1.0 (1), Met 0.9 (1), Tyr 0.7 (1), ethylamine 0.9 (1).

28 149765

Attempt

The compounds prepared by the process of the invention, including the salts, are useful, as mentioned, for the treatment of various ovarian diseases, especially ovarian follicle cysts, and for the induction of pregnancy in abnormal or diseased animals with such diseases.

The therapeutic effect and pregnancy induction are described below on the basis of experiments with the compound H- (Pyr) Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-NHC2H5 acetate (hereinafter designated I ').

1 · QY§tiefollicular cysts

Ca. 700 cows of the Holstein and Japanese Black breeds, diagnosed as having follicular cysts, were given intramuscular compound 1 'at a dose of 25-500 µg per day. subject. 66% of the animals were clinically cured and 55% were fertile.

As a control, 150 cows of the breed Holstein received 5000-30000 I.U. (international units) HCG (human chorigonadotropin) intramuscularly. The percentage of clinically cured animals was 40 and cattle. that became fruitful 33.

As ovarian characters after treatment with compound 1 ', cystic follicle rupture, luteinization or regression of cystic follicles was observed. Concentrations of LH (luteinizing hormone) were measured in some cases. Plasma LH increased rapidly after treatment, reaching peak value 1-2 hours after it. Changes in the plasma LH pattern were similar to the pre-ovulation wave seen in normal-cycle cows.

It is noteworthy that some of the cows conceived by insemination 0-2 days after treatment.

350 cows received 25-500 µg of compound 1 'and were inseminated shortly after treatment.

Ovulations were observed by rectal palpitation in 84% of cases and a conception rate of 67% was achieved at first and second insemination.

149755 29 3 · QYA Ownership Activity

Ca. 230 cows diagnosed as having abnormally resting ovaries received 100-500 µg of compound I '. 65% of the cows came in estrus and 50% of them conceived. Development of the follicle and corpus luteum was noted in cows that had responded.

4 · Experiment_with_rotter

A number of the nonapeptidamide derivatives prepared by the process of the invention were administered to diastrous rat rats. The rats were kept under conditions with light from 7 p.m. 7.30am to 9.30pm on the day of treatment, and 7 p.m. At 14.30 this day, the test compound was administered subcutaneously. The next day, the ovulated eggs in the fallopian tubes were inspected under a differential interference microscope. On this basis, EDgQ was determined for the ovulation-inducing effect and the values are shown in the table below.

These tests can be considered as a criterion as to whether the active compounds will also be effective in the above-described experiments 1-3.

Ai; a2 y ed50, ng / ioo g body weight

Tyr Leu NH-CH3 165.0

Tyr Leu NH-CH2-CH3 32.0

Tyr Leu NH-CH2-CH2-CH3 56.0

Tyr Leu NH-CH2-CH2-OH 170.0

Tyr Leu NH-CH (CH 3) 2 76.0

Tyr NLe NH-CH2-CH3 53.6

Tyr With NH-CH2-CH3 35.0

Phe9 Leu NH-CH2-CH3 84.0

Phe ILe NH-CH2-CH3 115.0

Tyr Leu Gly-NH2 215 + 12 ng of natural decapeptide

Claims (5)

149755 An analogous method for preparing LHRH analogous nonapeptidamide derivatives of the general formula L-pyroglutamyl-L-histidyl-L-tryptophyl-L-12 x seryl-A-glycyl-A-L-arginyl-L-prolyl-Y1. where A represents L-tyrosyl or L-phenylalanyl, where A represents L-leucyl, L-isoleucyl, L-norleucyl, L-valyl, L-norvalyl; or L-methionyl and wherein Y represents the group :: NHR wherein R is a straight or branched alkyl group of 1-3 carbon atoms, optionally substituted with hydroxy, or physiologically acceptable salts or metal complexes thereof, characterized in that a reagent A consisting of L -pyroglutamic acid or a peptide fragment containing an L-pyroglutamic acid moiety at the N-terminal end and calculated therefrom containing part of the amino acid sequence shown in formula I is condensed with a reagent B constituting a residue component corresponding to the remaining portion of non- the peptidamide derivative of the general formula I, which reagents A and B, if desired, are protected by one or more protecting groups, after which any protecting groups present are removed and the resulting product converted, if desired, into a physiologically acceptable salt or metal complex thereof. A process according to claim 1, characterized in that A is L-tyrosyl, A is L-leucyl and Y is NHCI 2 CH 2 or nhch 2 Process according to claim 1, characterized in that A 1 is L-tyrosyl, A 1 is L-leucyl and Y is NHCH 2. Process according to claim 1, characterized in that A is L-phenylalanyl, A is L-isoleucyl and Y is NHCH2CH3. Process according to claim 1, characterized in that A "is" L-tyrosyl, A "is L-leucyl and Y is NHCH2CH2CH3. Process according to claim 1, characterized in that A1 is L-tyrosyl, A2 is L-leucyl and Y is NHCH (CH3) 2 ·