DE2307010A1 - METHOD OF MANUFACTURING LHUND FSH RELEASING HORMONE, SALT AND PHARMACEUTICAL AGENTS THEREOF - Google Patents

Description

ΓΑΤΕΝΤΛΝλΥΑΙ, ΤΕ
DR. W. KINZEBACH - DIPL-ING. O. HELLEBRAND

8 Munich 80 13. ΡθΤ> Γ. 1973 Walpurgisstraße 6 Telephone: 0811/47050 34 Telegrams: Hekipit (Munich)

CASB: AHP-5792-1-01

AXERST MC KEMA & HAEHISOET LTD. 1025 Laurentien Boulevard, Saint-Laurent, Province of Quebec, Canada.

Process for the production of the LE- and FSH-releasing hormone, Salts and pharmaceutical agents thereof.

The invention "relates to a process for the production of the Luteinizing hormone (LH) and the 3 ollicle-stimulating hormone (PSH) releasing hormone in 3? Orm of an acid addition salt, it "relates to its salts with pharmaceutically acceptable ones Acids and pharmaceuticals which release LH and PSH Contain hormone.

LH and PSH are gonadotropic hormones * produced by the pituitary gland of the Humans and animals are developed. LH, along with PSH, stimulates the release of estrogens from the maturing ones Pollicles in the ovary and directs the ovulation process "in the female Living being a, In the male living being, LH stimulates the interstitial cells, for this reason it is also interstitial cell-stimulating Called hormone (IGSH). The pollicle

ORtQINAL INSPECTED

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stimulating hormone (FSH) - initiates the maturation of the follicles in the ovary and plays an important role together with the LH. in the cycle of the female living being. TSH promotes the development of the germ cells in the testes of the male living being. LH and I ¹ SH are released from the gonads by the action of LH- and FSH-releasing hormones and there is much to suggest that the releasing hormone is developed in the hypothalamus and reaches the pituitary gland in a neurohumoral way See, for example, Schally et al., Recent Progress in Hormone Research J24, 497 (1968).

This is LH- and FSH-releasing hormone. from the hypothalmus of the Pig and its constitution has been described by Schally et al., Biochem. Biophys. Res. Commun. 43, 393 and 1334 (1971), he proposed the deoapeptide structure:

(pyro) GIu - His - {Drp - Ser - lyr - GIy - Leu - Arg - Pre - ~

This constitution has been confirmed by synthesis (see below), the LH- and FSH-releasing hormone can also be used in something more modern terminology using the formula I: H - Pyr - His - Irp - Ser - 32yr - CrIy - Leu - Arg- Pro - GIy -

State of the art

The LH- and FSH-releasing hormone is described by Sievertsson et al., Biochem. Biophys. Res. Commun. 44, 1566 (1971), using a combination of classical and plague phase (Merrifield) methods; the same hormone is also from Geiger et al., ibid. 45>, 7.67 (197i) _s using a strictly classical method; by Matsuo et al., ibid. 45 ₉ 822 (1971), using a solid-phase method; and by Monahan et al., CR. Acad. Be., Ser. D, 273, No. ₈ 4 508 (1971), with the aid of a "solid phase" method was synthesized »Im

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Contrasted with the methods of the references cited above the method according to the invention is simpler and more efficient, as it is "considerably" better overall yields than any the known method provides. A particular advantage of the method according to the invention is that there is only a minimum of protective groups required for the intermediate products, especially vro secondary punctures are affected. So she must Hydroxyl group in serine cannot be protected; the HH groups in! Tryptophan and in histidine do not need any protection and for the guanidino function in arginine and for the hydroxyl group of the tyrosine, no protection is required in the later stages of the process. The inventive method is therefore cheaper and less cumbersome than the known methods. Another advantage of the method according to the invention is that that its final stage consists in the condensation of two unprotected fragments, each well defined, easy to clean and each is available in high purity. The final product thus obtained is the free, protected decapeptide, none of which Protective groups have to be removed more and that at a higher rate Purity and in good yields.

In the following text, the term "IT-lower alkyl" denotes straight-chain ones or branched alkyl groups with 1-6 carbon atoms and includes methyl, ethyl, propyl, isopropyl, η-butyl, sec, -butyl, t-butyl and the like. The term "low" refers to 1-6 carbon atoms. With the term "strong organic Base "means aliphatic and heterocyclic tertiary nitrogen bases, including triethylamine, dibutylmethylamine, N-methylpyrrolidine, N-methylpiperidine, N-methylpiperazine, H-methylmorpholine and the like; Triethylamine is preferred. The term "halogenated hydrocarbon" is used for those with 1-2 carbon atoms and includes methylene dichloride, Ethylene dichloride, chloroform and the like; Chloroform is j preferred. The term "strong mineral acid" means "when used in conjunction with an anhydrous system;

. - .. 3 .- "..."
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Hydrogen chloride, hydrogen bromide and sulfuric acid ^ chlorine- · hydrogen is preferred; when used in connection with an aqueous system, the term includes any conventional Mineral acid.

! -Pyroglutamic acid is the lactam of L-glutamic acid and has the structure of 5-oxo-L-proline.

Many of the methods used in the synthesis of peptide bonds are commonly referred to by divisional names. The "azide method" relates to the conversion of an amino acid hydrazide, which has a suitably protected amino group with a nitrite, usually t-butyl or xsoamyl nitrite, to obtain the corresponding azide, which then! with an amino acid that has a free amino and one in geeig-ι A protected carboxylic acid group is reacted in order to obtain the desired peptide.

The condensation with dicyclohexylcarbodiimide includes the reaction an amino acid having a suitably protected amino and a free carboxylic acid group with a other amino acid having a free amino and an appropriately protected carboxylic acid group; the peptide bond is released with the escape of the elements of water and formation of ί dicyclohexylurea, which can easily be derived from the reaction j

mixture can be removed, formed. "In the JPaXIe, where the free amino group of the second amino acid reacts difficultly, for example if the free amino group is secondary, as in proline, it is advantageous to give hydroxysuccinimide for reaction in order to form the intermediate hydroxysuecinimide ester of the first amino acid, the smoothly with a secondary amino group · reacts to form the desired peptide bond «In principle, this modification of the gyel © h @ 3sylcarb © imide, <4 $ method an activation of the carboxylic acid groups and © ine

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Activation is also achieved when the 4-nitrophenyl or 2,4-Dinitrophenyl or 2,4,5-! Erichlorphenylester of the carboxylic acid can be used in place of the free acid. Such esters are commonly known as activated esters.

The protective groups used in the process according to the invention and the usual abbreviations with which they and the usual Amino acids are referred to are in Schröder and Lübke, "Ehe Peptides", Academic Press, Hew York and London 1965, described.

Summary of the invention

The method according to the invention can be summarized by the following steps:

N- (5-Oxo-L-prolyl) -L-histidine hydraside as obtained from Gillessen et al., HeIv. Chim. Acta j53, 63 (1970), described, is made using the azide method with a medrigalkyl or Aralkyl esters of L-sryptophane, preferably the benzyl ester, obtained according to Wilcheck et al., J. Org. Chem. 28, 1874 (1963) has been condensed, whereby the N- / N- (5-Oxo-L-prolyl) -L-histidyl7-L-tryptophan-benzyl ester receives. The latter is treated with hydrazine hydrate and gives N- / N- (5-Oxo-L-prolyl) -Ir-histidyl7-L-tryptophan hydrazide (II).

A lower alkyl ester of the K-ZÖ-benzyl-N-carboxy-L-tyrosyl / -glyein-N-benzyl ester, preferably the methyl ester, prepared according to Morley, J. Chem. Soc. (C), 2410 (1967), becomes hydrolyzed corresponding free acid, which is converted into the corresponding mixed anhydride with ethyl chloroformate is and is treated with t-butyl carbazate, whereby the Γr- / 0-benzyl-IT-carboxy - L «tyΓOsyl7glycine-N-bensyl ester-2-carboxyhydrazide-t-butyl ester receives, the latter connection;

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is hydrogenolyzed with the aid of hydrogen and a noble metal catalyst and gives the Nl-iDyrosylglycine-2-carboxyhydrazide-t-butyl ester. Condensation of the last-mentioned compound with an activated ester of the N ~ carboxy-1-seryl-N-benzyl ester, preferably the 2 _y 4-dinitrophenyl ester, prepared according to Marchiori et al., Gazz. öhim. Ital., £ 3, 834 (1963) ,. gives the N- / N- (N-carboxy-I-seryl) »I-tyrosyl-7glycine-N-benzyl ester-2-carboxyhydrazide-t-butyl ester, which is hydrogenolyzed as above and the N- (NL-seryl-L-tyrοsyl ) glycine-2-carboxyhydrazide-t-butyl ester (III) gives.

The N-carboxy-L-proline-N-benzyl ester, prepared according to Berger et al., J. Am. Chem. Soc, J76, 5552 (1954), is condensed with a glycine lower alkyl ester, preferably the ethyl ester, using dicyclohexylcarbodiimide as the condensing agent and the resulting product is treated with ammonia, the 2 - / "(N-carboxy- The latter compound is hydrogenolyzed and treated with N-carboxy-N-nitro-L-arginine-N-butyl ester, obtained according to Hofmann et al., J. Am. Chem. Soc 87 »620 (1965), condensed using N-hydroxysuccinimide and dicyclohexylcarbodiimide as condensing agents, N- / N- (NO arboxy-N-nitro-L-arginyl) -L-prolyl / -glyeinamide-Nt-butyl ester (IV) receives.

The same compound IV is also made in the following alternative route %

A lower alkyl ester of L-proline Sj, preferably the methyl? ester, and N-carboxy-N -nitro-arginine-N-t-butyl ester according to Bpissonas et al., HeIv. Chim. Acta 44, 123 (1961), and Hofmann et. al., quoted above, with the help of dicyclo = » hexylcarbodiimide condenses and give N- (N-carboxy-N -nitro » L-arginyl) -L-proline-N-t-butyl ester “The latter compound is with a glycine lower alkyl ester, preferably

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the ethyl ester, "condensed using dicyclohexylcarbodiimide as the condensing agent and the resulting product is treated with ammonia, N- / N- (N-carboxy-N -nitro-L-arginyl) -I-prolyl / -glycine amide-li- t-butyl ester (IV) which is identical to the product obtained in the manner described above. The protective t-butoxycarbonyl group of the latter compound is removed by treatment with acid and the resulting product is condensed with an activated ester of the N-carboxy- L-leucine-N-benzyl ester, preferably the 2,4,5-trichlorophenyl ester, prepared according to Kenner et al., J. Ohem. Soc, 161 (1968), the N- / N-ZN- (NC arboxy- L-1 eucyl) -N ^G -nitro-L-arginyl / -L-'prolyl7-glycinamide-N-benzyl ester, which is hydrogenolyzed in glacial acetic acid with the aid of hydrogen and a noble metal catalyst to remove the protective nitro and garbobenzoxy groups and the corresponding diacetate salt, N- / N- / N- (NL-Ieucyl) -L-arginylZ-L-prolylZ-gl ycinamide diacetate (V).

N-Z & - (5-Oxo-L-prolyl) -L-histidyl7-L-tryptophan-hydrazid (II) and N- (N-L-seryl-L-tyrosyl) glycine-2-carboxyhydrazine-t-butyl ester (III), both obtained as described above, are condensed by the azide method to give the hexapeptide Ν- / ΪΤ- / ΪΓ- / Ν- / Ν- (5-Oxo-L-prolyl -) - L-histidyl7-L-tryptophyl7-L-seryl / -L-tyrosyl7glycine-2-carboxyhydrazide-t-butyl ester. The same compound is also obtained by condensation of N- ^ f- (5-Oxo-L-prolyl) -L-histidyl7-L-tryptophan, obtained from the corresponding benzyl ester described above by hydrogenolysis , with N- (N-L-Seryl-L-ryrosyl) glyc in-2-carboxyhydrazide butyl ester (III) obtained as described above using dicyclohexylcarbodiimide as a condensing agent, N- / N-Zii- / N-ZN- (5-Oxo-L-prolyl) -L-histidyiy-L-tryptophylZ-L-seryl7-L-tyrosyl7glycine-2-carboxyhydrazide-t-butyl ester, obtained in one of the two ways described above, is treated with g3rifluoroacetic acid and gives the irifuloracetic acid

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salt of hexapeptide hydrazides, namely IT- / N- / U- / N- / N- (5-Oxo-I-prolyl) -L-histidylZ-L-tryptophyl / -I-seryl / -L-tyrosyl / -glycine.-hydrazide-trifluoroacetate (VI).

ι The last-mentioned compound (VI) is made using the azide method with N- / iί- / l · Γ- (lί-Ieucyl) -I-arginyl7-Iι-prolyl / glycinamide diacetate (V) condenses and gives the desired one. Decapeptide, 5-oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosylglycyl-L-leucyl-L-arginyl-L-prolylglycine amide (I), which is isolated in the form of its hydrochloride salt. The latter salt may be desired if necessary converted into another acid addition salt, e.g. a salt with a pharmaceutically acceptable one Acid, by using the appropriate ion exchange resin in the method described by Boissonas et al., HeIv. CMm; Acta, 43, 1349 (1960) described way. Suitable ion exchange resins are strongly basic anion exchange resins such as those of Greenstein and Winitz "Chemistry of the Amino Acids", John. Wiley and Sons, Inc., New York and London 1961, Volume 2, Page 1456, listed. Basically substituted crosslinked polystyrene resins such as Amberlite IRA-400 or IEA-410 are preferred. The above Hydrochloride can also be found in a salt of poor solubility Can be converted into the body fluid by dealing with a poorly water-soluble pharmaceutically acceptable Acid treated.

In addition, the decapeptide of the formula I can also be used in complexes be transferred with pharmaceutically acceptable metals. The acid addition salts and the metal complexes according to the invention Process manufactured LH- and PSH-releasing hormones with pharmaceutically acceptable acids or metals are completely biologically equivalent to the natural hormone.

The above reaction sequence is shown below in Figure 1, the successive introduction and removal of protective groups;

however, as well as the condensation methods and the condensation agents used have been omitted.

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Fig.l

L-pyroglutamic acid
"L-ELetidini

L-tryptophan L-serine

-II.

L-Tyroein— Glycine-

-III.

VI.

L-arginine ~ L-proline-glycine-

odeir.

L-arginine in 'L-proline- glycine

Detailed description of the invention

1. IH and 3? SH releasing activity

The synthetic product of the formula I obtained in 3 form of an acid addition salt by the process according to the invention "has IS and ISH-releasing properties and is as active as the natural hormone" when reading in the radioimmuno test, "described by Niswender et al ., Soc. Exp. Biol. Med. Proc., 128 _t 807 (1968). during the test to determine the induction of ovulation described "the hamster," from Arimura et al., Science 174 "511 (1971), and with a modification of the same

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Testing in the rat, described by Arimura et al. in Endocrinology 80, 515 (1967), it is equally effective.

The IH and FSH relealsing properties of the according to the invention Procedure obtained hormones, which in turn trigger ovulation in animals, make the hormone for the Vet work and livestock farming useful. Often it is desirable to synchronize the livestock oestrus, e.g. in cattle, sheep or pigs, either all female To be able to mate animals of a given group with a male of the desired genetic makeup or to be able to with a maximum number of female animals to be able to carry out artificial insemination, both in a comparatively short time Time. In the past, this was done by giving the animals an ovulation inhibitor, the remedy for short withdrew before the day scheduled for mating or artificial insemination and either switched to natural production left of IH and PSH for induction of ovulation and generation of an oestrus or administered gonadotrophins. However, this procedure was not entirely satisfactory since ovulation at a predetermined time never occurs in all Animals at the same time but only in a certain part of them if no gonadotrophins were used. On the other hand, the high price of gonadotrophins and the side effects associated with their administration made them Method impractical. Now it is possible to achieve practically complete synchronization of ovulation and oestrus, by getting the animals in a given group first treated with an ovulation inhibitor, which is later discontinued, and then that made by the method of the invention IH- and PSH-releasing hormone just before the predetermined one Time of mating or artificial insemination administered so as to ovulation and oestrus in this time interval to reach. The administration of the according to the invention

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according to the method produced hormones following temporal The delay in the onset of ovulation and oestrus will vary with the species, the optimal time interval must be for each Species to be chosen. For example, rodents such as rats or hamsters ovulate within 18 hours following the administration of the IB- and PSH-releasing hormones produced by the method according to the invention.

The method described above to achieve ovulation and oestrus within a precisely predetermined time interval, ■ so that one can be sure of a successful mating is particularly important for breeders of racehorses and show animals, where the fees paid for the service of an excellent male animal are often very substantial sums of money matters.

The LH- and ESH-releasing hormone produced by the method according to the invention is also suitable for increasing the number of live births per pregnancy in cattle, for example cattle: cattle, sheep or pigs. For this purpose, the LH- and FSH-releasing hormone is administered in a series of 3 arenteral doses, preferably by intravenous or subcutaneous injection, 1.0 meg to 100 meg per kg of body weight, per day, 96 to 12 hours before the expected estrus and \ subsequent pairing. A preparatory injection of; 1000 to 5000 IU gonadotrophin from the serum of pregnant mares | can also be given 1 to 4 days before the above injection of LH-I and ESH-releasing hormone. Similar treatment, with or without pre-treatment, can also be used to induce sexual maturity in breeding animals.

When the hormone produced by the method according to the invention for the purpose of initiating ovulation and oestrus or for initiating sexual maturity in warm-blooded animals, in particular

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especially in rodents such as rats or hamsters or, "in cattle
is used, it is administered systemically, preferably
parenterally, in combination with a pharmaceutically acceptable liquid or solid diluent. The amount
The hormone is given by its solubility in the
Diluent, by the chosen route of administration and
determined by standard biological practice. Purely for parenteral administration to living beings, the hormone can be used in a
sterile aqueous solution used as well as others
dissolved components, such as buffer substances or preservatives as well as as much pharmaceutically acceptable salts or
May contain glucose that the solution is made isotonic.
The dose varies with the form of administration and with the. ; particular animal species to be treated and it is preferably maintained at a level of 5 meg to 100 meg per kg of body weight. A dosage in the range of about 10 mcg I to about 50 meg per kg of body weight is most likely to be used. used to achieve good results. The hormone may also be in '■ one of the below-described long-acting, slow-release or depot dosage forms ■, preferably by intramuscular In; ^jek-; tion or by implantation. Such dosage forms are intended to be about 0.5 meg to about 50 meg!

to be given per kg of body weight. , \

The LH- ι and PSH-releasing hormone produced with the aid of the method according to the invention is also suitable for human medicine. i For example, human ghorionic gonadotrophin (HCGr), | which contains mostly LH and some FSH, more than thirty; For years for the treatment of certain endocrinological complaints, v / ie cycle disorders, amenorrhea, lack of elimination! development of secondary sexual characteristics and infertility
in the "woman or in certain cases, of hypogonadism,
delayed sexual maturity, cryptorchidia, and non-psychogenic impotence have been used in men. Lately is

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Infertility "in women with human menopausal gonadotrophin (HMG), which is mainly " SSS. I.

■ · j

contains, with subsequent treatment with HGG. One of the l

Disadvantages of the treatment of infertility in women with j

HCG or with HMG and subsequently with HOG is insofar in Er- j

it seems that such a treatment often leads to j

leads to overovulation and unwanted multiple births, |

probably because of the impossibility only the exact sets ■

To give FSH and IiH, which are necessary for ovulation, j the administration of the according to the method according to the invention ^; produced hormones eliminates the above disadvantages, since that; Hormone the release of LH and FSH by the pituitary gland
are \ conducive only in the exact amounts that ER- for normal ovulation, causes. For this reason it is after
Method according to the invention produced hormone not only for! the above needs, but it is also for the ■ treatment of menstrual disorders, Ämmenorrhöe - hypogonadism \ and lack of development of secondary sexual characteristics of: useful woman.

In addition, according to the method according to the invention, it is; also sired LH- and FSH-releasing hormone for conception. j contraception useful. For example, if a woman's hormone im; administered early in the menstrual cycle
LH is released at this point and causes premature ovulation. The immature egg is either incapable of fertilization, or if fertilization should have taken place anyway, it is extremely unlikely that it will
the fertilized egg becomes embedded because the estrogen / progestin balance required to make the endometrium is absent and the endometrium is not in
is in the condition required for implantation.
On the other hand, if the hormone is towards the end of the cycle
is administered, the endometrium is destroyed and finds it
Menstruation takes place.

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In addition, the IH- and FSH-releasing hormone produced by the method according to the invention is also suitable for contraception using the "rhythm" method, which has hitherto always been relatively unreliable due to the impossibility of predicting ovulation in women with the required accuracy is. Giving the hormone in the middle of the cycle, that is, at about the time normally expected for ovulation, triggers ovulation shortly thereafter and makes the "rhythm" method safe and effective. The IH- and FSH-releasing hormone produced by the method according to the invention is also suitable as a diagnostic tool for differentiating between hypothalamic and pituitary dysfunction or damage in women. Administering the hormone to a patient which is believed that such malfunctions or damage are present, then it is found an increase in the IH-mirror, so this is a good ¹ indication of the conclusion that the hypothalamus is the cause of the malfunction and that the pituitary gland is intact. On the other hand, if there is no increase in circulating IH following administration of the auditory mones, a diagnosis of dysfunction or damage to the pituitary gland may be highly graded! Security must be provided. In men, the administration ensures that IH- and FSH-releasing hormones obtained by the method according to the invention are made available for one; normal sexual development required IH (or ICSH); and FSH levels, in cases of hypogonadism or delayed sexual maturity, and is also useful in the treatment of cryptorchidia. In addition, the FSH released by the administration of the hormone stimulates the development of the germ cells in the testes and the hormone is suitable for the treatment of non-psychogenic impotence.

If the IH- obtained by the process according to the invention and FSH-releasing hormone in the form of an acid addition salt

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used in human medicine, if administered systemically, either by intravenous, subcutaneous or intramuscular injection! tion or by sublingual, nasal or vaginal administration · in compositions in conjunction with a pharmaceutical j compatible carrier or diluent.

administration by injection or for administration By the nasal route as drops or spray, it is preferred to take the hormone in solution in a sterile aqueous vehicle, the also other dissolved components, such as buffer substances or preservatives, as well as sufficient pharmaceutical quantities compatible salts or glucose may contain that the solution is made isotonic to use.

The LH- and FSH-releasing hormone produced by the method according to the invention can also be administered as nasal or vaginal powder or as an insufflation agent. I ^{1 For} such purposes, the hormone is used in finely divided solid form together with a pharmaceutically acceptable solid carrier, e.g. a finely divided polyethylene glycol ("Carbowax 1540"), finely divided lactose or, preferably only for vaginal administration, very finely divided silica ( ¹¹ Such agents can also contain other diluents in finely divided solid form, such as preservatives, buffers or surface-active agents.

For sublingual or vaginal administration it is preferred to use the hormone in fixed dosage forms, e.g. as sublingual tablets or vaginal inserts or suppositories with gnawing amounts of solid diluents such as starch, lactose, certain types of ions, buffer substances, lubricants, disintegration-promoting agents or surface-active agents, or to be formulated with semi-solid diluents commonly used in formulating suppositories.

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Examples of such diluents are in pharmaceutical Found standard books, e.g. in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 1970.

The dosage of the IH- and PSH-releasing hormone obtained by the method according to the invention changes with the form of administration and depending on the particular patient to be treated. In general, treatment is started with small doses, which are much smaller than the optimal hormone dose, after which the dose is increased in small increments until the optimal effect is achieved under the given circumstances. In general, the hormone obtained by the process according to the invention is preferably administered in a concentration level which ^{brings about a generally effective release of IiH and I 1} SH without causing any adverse or harmful side effects, preferably it is administered in a range from about 1 meg to about 100 meg per kg of body weight, as mentioned above, but variations can also be made. Most desirably, however, a dose in the range of about 5 meg to about 50 meg per kg of body weight will be used for good results.

It is often desirable to administer the hormone continuously for extended periods in long-acting or slow-release or depot dosage forms. Such dosage forms can be either a pharmaceutically acceptable salt of the hormone

which is slightly soluble in body fluids, for example one of the salts described below, or they can contain the hormone in the form of a water-soluble salt together with a protective carrier that prevents rapid release. In the latter case _e B "z the hormone can en partially hydrolyzed with a non-antigenic G-Elatine in a viscous Porm

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Liquid formulated or the hormone can be adsorbed on a pharmaceutically acceptable solid carrier, e.g. zinc hydroxide, and it can be administered in suspension in a pharmaceutically acceptable liquid "diluent. Alternatively, the hormone can be in the form of a complex with a pharmaceutically acceptable metal, e.g. zinc , Copper, cobalt, iron or aluminum; the zinc complex is preferred; or the hormone can be administered in gels or suspensions with a protective non-antigenic hydrocolloid, e.g. sodium carboxymethyl cellulose, polyvinylpyrrolidone, sodium alginate, gelatin, polygalacturonic acids, e.g. pectin or certain mucopolysaccharides ^ together with aqueous or non-aqueous pharmaceutically acceptable liquid vehicles, preservatives, or _surface, active agents are formulated Examples of such formulations are to be in standard pharmaceutical texts. find, for example in the above cited Remington's pharmaceutical Sc iences, long-acting, slow-releasing preparations of the hormones produced by the method according to the invention can! can also be obtained by microencapsulation in a pharmaceutically acceptable coating material such as gelatin, polyvinyl alcohol or ethyl cellulose. More examples of coating; materials and for processes leading to microencapsulation; are available from JA Herbig in "Encyclopedia of j Chemical Technology", Volume 13, 2nd Edition, Wiley, New York; 1967, pages 436-456. j

Such formulations and suspensions of salts of the hormone, which are only slightly soluble in body fluids, are for the delivery of about 0.1 meg to about 50 meg des Hormones are determined per kg of body weight per sag and are preferably administered by intramuscular injection. Alternatively, some of the solid dosage forms listed above, e.g., certain slightly water-soluble salts or

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Dispersions in or adsorbates on solid carriers of salts of the hormone, e.g. dispersions in a neutral hydrogel of a polymer made of ethylene glycol methacrylate or similar crosslinked monomers, as described in U.S. Patent 3,551,556, are formulated in the form of pellets, which are about the same amounts as stated above, and which can be implanted subcutaneously or intramuscularly.

Alternatively, slow-release effects can be used over longer periods of time, be achieved by the fact that the hormone obtained by the process according to the invention as an acid addition salt in an intravaginal agent or in a temporary implant, e.g. in one made of a non-irritating silicone polymer, such as polysiloxane, e.g. "Silastic", or from a neutral one Hydrogel of a polymer as described above ', which has the required degree of permeability to about 0.1 meg to about 50 meg per kg of body weight per sag dispense, existing container. Such intravaginal or implant dosage forms for long-term administration have the Advantage that they can be removed as soon as a sub- ' interruption or termination of treatment is desired.

Making the connections ;

The method according to the invention is carried out as follows;

A solution of N- (5-0xo-L-prolyl) -L-histidine hydrazide (cf. G-illessen et al., Cited above) in an inert, anhydrous solvent, preferably a mixture of dimethylformamide and dimethyl isutoxide, is used cooled to a temperature of about -10 C Ms about 5 ⁰ C, with a solution of a strong mineral acid, preferably hydrogen chloride, in an anhydrous ether or cyclic ether _g preferably Eetrahydrofuran,

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mixed and the mixture is cooled to a temperature of about -3O ⁰ C "to about -2O ⁰ G. An organic Mtritol, preferably t-butyl nitrite or isoamyl nitrite, is added with stirring and the mixture is stirred for 20 to 60 minutes, preferably for about 30 minutes, at a! temperature of about -3O ⁰ C to about -20 ⁰ C. This gives a sufficient amount of strong organic base, preferably triethylamine, with stirring added to make alkaline the mixture, preferably to provide a pH 8-9 a. the temperature of the mixture is maintained in the range of about -30 to about -10 ⁰ C, besides, a solution in an inert solvent, preferably dimethylformamide, a lower alkyl or Aralkylesters of L-tryptophan, preferably the benzyl ester (see Wilcheck et al, cited above..), in a 5 -. I5 $ strength by weight excess over the molar equivalency "is preferred to use a molar excess of about 10 $, and the mixture is stirred at about -30 ⁰ C to about -1O ⁰ C for 30 to 60 minutes and then for 16 to 24 hours in an ice bath, preferably for about 18 hours. The precipitate is filtered off, the piltrate is evaporated, the residue is crystallized and the corresponding N- / Si- (5-oxo-L-prolyl) -L-histidyl7-L-tryptophan lower alkyl or aralkyl ester is obtained, the benzyl ester is included preferred for purposes of isolation and purification. The latter compound, preferably in the form of the benzyl ester is dissolved in an anhydrous lower alkanol, preferably methanol, at a temperature of about -20 ⁰ C to about ⁰ ° C, preferably about -10 ⁰ C, cooled, with stirring, with a molar Excess hydrazine hydrate is added and the mixture is stirred first at ice bath temperature for 2-6 hours, preferably for about 3 hours and then for 30-60 hours, preferably for about 40 hours at room temperature (20 to 25 ^{° C.).} The precipitate is filtered off and crystallized from a lower alkanol, preferably methanol, and gives N- / ii- (5-oxo-L-prolyl) -L-histidyl7-L-tryptophylianhydrazide (II).

- 19 309834/1134

AHP-5792-KM ^!

A lower alkyl ester of N-ZÖ-benzyl-N-carboxy-L-tyrosyl / glycine-; N-benzyl ester, preferably the methyl ester (see Morley, cited above), is dissolved in a lower alkanol or alkoxyalkanol, preferably methoxyethanol, a molar excess (5-20 $ excess, preferably about 15 $ excess) of an aqueous alkali metal hydroxide is added, preferably sodium hydroxide, and the mixture is stirred at room temperature for 0.5-2 hours ^; long, preferably about 1 hour. The mix is with! a strong mineral acid, preferably hydrochloric acid, acidified, the precipitate filtered off and crystallized from aqueous lower alkanol, preferably methanol / water, N-ZO-benzyl-N-carboxy-L-tyrosyl / glycine-N-benzyl ester. The latter compound is dissolved in an anhydrous ether or cyclic ether, preferably tetrahydrofuran, the solution is cooled to a temperature in the range from about -2O ⁰ C to about ^{0 0} C, preferably to a temperature of about -1O ⁰ C and practically 1 mol equivalent of ethyl chloroformate is added. The mixture is preferably stirred at about -10 ⁰ C for 5-30 minutes for about 10 minutes, and it is practically 1 mole equivalent of t-butyl carbazate added. The mixture is stirred first 20-60 minutes, preferably for about 30 minutes, at about ⁰ ° C and then .lang about 3-8 hours, preferably about 5 hours, at room temperature (20 to 25 ⁰ C). The solvent is evaporated off, the residue taken up in a practically water-immiscible solvent, preferably a lower alkyl ester of a lower; alkanecarboxylic acid, for example ethyl acetate, the solution is washed, dried and evaporated and the residue crystallized, giving N-ZO-benzyl-N-carboxy-L-tyrosylZ-glycine-IT-benzyl ester-2-carboxyhydrazide-t-butyl ester. The last-mentioned compound is dissolved in an anhydrous lower alkanol, preferably methanol, a noble metal catalyst, for example palladium on carbon, is added and the mixture is shaken in a hydrogen atmosphere at room temperature to essentially

- 20 309834/1134

AHP-5792-1-01

union 2 mol equivalents of hydrogen have been absorbed. The catalyst is filtered off, and the filtrate is evaporated results in a residue of N-L-Iyrosylglyein-2-carboxyhydrazide butyl ester, which is used in the subsequent stage without further purification.

The last-mentioned compound is dissolved in an anhydrous halogenated hydrocarbon, for example chloroform, or preferably in dimethylformamide, and the resulting solution is added, with exclusion of moisture, to a solution of an activated ester of N-carboxy-L-seryl-N-benzyl ester, preferably used the 2,4-dinitrophenyl ester (see Marchiori et al., cited above), which has previously been ^{cooled to about 0} ° C. ^{The mixture is kept at 0} ° C. for several layers, preferably about 3 layers, the solvent evaporated, the residue in a mixture of a halogenated hydrocarbon, a lower alkanol and a small amount of a weak organic base, preferably chloroform / methanol / pyridine , and the solution is chromatographed, preferably on silica. It is eluted, the eluates are evaporated, crystallized and N- ^ N- (N-carboxy-L-seryl) -L-tyrosyl / glycine-N-benzyl ester-2-carboxyhydrazide-t-butyl ester is obtained. The latter compound is dissolved in a lower alkanol, preferably methanol, a noble metal catalyst, preferably palladium on carbon, is added and the mixture is shaken in a hydrogen atomosphere for 5-10 hours, preferably for about 7 hours, at room temperature to substantially 1 mol equivalent of hydrogen has been absorbed. The catalyst is filtered off, the piltrate is evaporated, the residue is crystallized and N- ^ TL-seryl-L-tyrosylJ-glycine ^ -carboxyhydrazide-t-butyl ester (III) is obtained.

A solution of an N-carboxy-L-proline-N-benzyl ester (see Berger et al., Cited above) and an approximately equimolar

- 21 309834/1134

AHP-5792-T-C1

: Amount of a glycine lower alkyl ester acid addition salt, preferably glycine ethyl ester hydrochloride * in a halogenated one; Hydrocarbon solvent, preferably chloroform, j cooled to about 0 ⁰ C and © ine are added essentially ^equidistant: molar amount of a strong organic. Base, preferably 03ri-! ethylamine, and then an essentially equimolar; Volume dicyclohexylcarbodiimide hinsTi _e The mixture is "on:. About ⁰ ° C for 12-24 hours ₉ preferably about 16 hours, stirred ,, filtered, the ^ iltrat washed, dried and evaporated 'The residue is dissolved in a" at about O ⁰ C with ammonia saturated lower alkanol, preferably methanol, added and the solution "left to stand at about ^{0 0} G for 48-72 hours. The solvent is evaporated, crystallized and 2 - /" (N-carboxy-L-prolyl) amino / acetamide-N-benzyl ester. The latter compound is dissolved in acetic acid, a noble metal catalyst, preferably palladium on carbon, is added and the mixture is shaken in a hydrogen atmosphere until essentially 1 mol equivalent of "hydrogen has been absorbed. Filtering and evaporation of the filtrate gives 2- /" (L-Prolyl) amino / acetamide, the "in dimethylformamide, which contains an essentially equimolar amount of a strong organic base, preferably triethylamine, is dissolved, it is cooled and is at a temperature of about -20 ⁰ G to about, O ⁰ C, preferably at about -10 ⁰ C, to a solution of an N-carboxy-F ^ -nitro-L-arginine-IT-lower alkyl ester, preferably the Nt-butyl ester (see Hofmann et al., Cited above), in dimethyl formamide · · = group containing from about 2 mol equivalents ÜT-hydroxysuccinimide and about 1 molar equivalent of dicyclohexylcarbodiimide. the mixture is stirred at about -10 ⁰ C to about 5 ⁰ C, preferably at about ⁰ ° C, first 12-24 For hours at et wa O ⁰ C; and then 24 - ι evaporated for 48 hours at room temperature (20 to 25 ⁰ C), the residue is dissolved in a mixture of: pre halogenated hydrocarbon and a Hiedrigalkanol ^_9: preferably chloroform and methanol was added and ehromatoj graphically on silica purified , you get the corresponding

309834/113 4

AHP-5792-1-G1

^G '

des N "- / N- (N-CarTDOxy-] 5I ^G '-nitro-L-arginyl) -L-

prolyl / glycinamids, preferably the N-t-butyl ester (IV).

Alternatively, the same connection IV is also made as follows:

A lower alkyl ester acid addition salt of L-proline, preferably the methyl ester hydrochloride (see Boissonas et al., Cited above), and an approximately equimolar amount of N-carboxy-N-nitro-L-arginine-Nt-butyl ester (see Hofmann et al ., cited above ) are dissolved in an anhydrous, strongly polar solvent, for example dimethyl sulfoxide, dimethylformamide or acetonitrile or mixtures thereof, preferably in a mixture of acetonitrile and dimethylformamide; an essentially equimolar amount of a strongly organic base, preferably iriethylamine, is added , while stirring. The mixture is heated to a lemperatur preferably cooled from about -20 C to about OC to about -10 ⁰ C., is added a substantially equimolar amount of dicyclohexylcarbodiimide, the mixture is stirred at about O ⁰ C 2-10 hours, preferably for 5 hours, and then 8-24 hours, preferably about 10 hours, at room temperature (20 to 25 ⁰ C), filtered and evaporated the Eiltrat. The residue is taken up in a halogenated hydrocarbon or in a lower alkyl ester of a medrigalkane carboxylic acid, preferably chloroform or ethyl acetate, which is essentially immiscible with water, washed and evaporated, the residue is taken up in a lower alkanol, preferably methanol, and is taken up at room temperature for about 1-3 hours long with about 2 mole equivalents of an aqueous alkali metal hydroxide, preferably sodium hydroxide, stirred. The mixture is filtered, extracted with a halogenated hydrocarbon or a lower alkyl lower alkanoate, preferably chloroform or ethyl acetate, the aqueous phase is acidified, extracted as above, the extracts are evaporated and H- (IT-Carboxy-N ^& -nitro-L-arginyl ) Iiproline Nt-butyl ester. The latter connection will

309834/113 4

AHP-5792-1-C1;

^! dissolved in dimethylformamide, to gi "bt an acid addition salt of a Niedrigalkylesters of glycine, .vorzugsweise Glycinäthyl- ester hydrochlörid, added, the mixture cooled to about ⁰ ° C., i is a substantially equimolar amount of a strong organic; see Base , preferably iriethylamine, and essentially 1 mol \ equivalent of dicyclohexylcarbodiimide and stir the mixture ■ first at about O ⁰ C for 2-5 hours, preferably about: 3 hours and then at room temperature for 12-24 hours, preferably about 17 It is filtered, the solvent is evaporated, the residue is taken up in a mixture of a halogenated hydrocarbon and a lower alkanol, preferably chloroform and methanol ⁰ C with ammonia saturated lower alkanol, preferably in methanol, leaves the solution for several days, preferably for about 3 layers, at about ^{0 0} C stand, the solvent evaporates, crystallizes and receives N- / BM N -nitro-L-arginyli-l-prolyl / glycinamide-Nt-butyl ester (IV), which is identical to the product obtained in the other way described above. The last-mentioned compound of the formula IV, obtained in one of the ways described above, is dissolved in a strong acid such as is usually used for removing protective groups, preferably hydrogen chloride in a lower alkanol or in glacial acetic acid or irifluoroacetic acid, the solution is at about O ⁰ C 20-60 minutes long, preferably about 30 minutes, and then about 5-15 parts by volume, preferably about 10 parts by volume, of an anhydrous, inert water-immiscible ether solvent, for example a lower alkyl ether, preferably diethyl ether , to produce a precipitate of the corresponding salt of N- ^ T- (N -nitro-L-arginyl) -L-prolylglycine amide. The latter is filtered off, in dimethylformamide, which contains an essentially equimolar amount of a strong organic base, preferably triethylamine,

- ■ 24 30 98 3 Λ / 1134

AHP-5792-1-G1

contains, dissolved, the solution is ^{stirred at about 0} ° C. for 15-30 minutes, and an essentially equimolar amount of an activated ester of the N-carboxy-L-leucine-N-benzyl ester, preferably 2,4,5, is added -Trichlorophenyl ester (see Kenner et al., Cited above). The mixture is left to ^{stand first at about 0} ° C. for 24-48 hours and then at room temperature for 24-48 hours. The solvent is evaporated off, the residue is dissolved in a mixture of a halogenated hydrocarbon and a lower alkanol, preferably chloroform / methanol, then it is chromatographed on silica, the eluate is evaporated and N- / F- / N- ( N-carboxy-L-leueyl) -U -nitro-L-arginylZ-L-prolyl / glyeinamide-N-benzyl ester. The latter compound is dissolved in glacial acetic acid, a noble metal catalyst, preferably palladium on carbon, is added and the mixture is shaken at room temperature in a hydrogen atmosphere until essentially 4 mol equivalents of hydrogen have been absorbed. It is filtered, the solvent is evaporated and N- / N- / N- (NL-Leucyl) -L-arginyl7-L-prolyl / glycinamide diacetate (Y) is obtained as an amorphous solid substance.

A solution of N- / N- (5-oxo-L-prolyl) -L-histidyl7-L-tryptophanhydrazide (II), obtained as described above, in an inert anhydrous solvent, preferably in a mixture of dimethylformamide and dimethyl sulfoxide, is cooled to a temperature of about -10 ⁰ C to about 5 ° C, mixed with a solution of a strong mineral acid, preferably hydrochloric acid, in an anhydrous ether or cyclic ether, preferably tetrahydrofuran, and the mixture is heated to a temperature of about - 30 ⁰ C to about -20 ⁰ C cooled. An organic nitrite, preferably t-butyl nitrite or isoamyl nitrite, is added in a practically equimolar amount with stirring and the mixture is stirred for 20 - 60 minutes, | preferably for about 30 minutes, at a temperature of C.Manfiigt, about -30 ⁰ C to about -20 °, a strong organic ausrachende Maige

- 25 309834/1134

AHP-5792-i-eT

Base, preferably triethylamine, is added with stirring in order to make the mixture alkaline, preferably in order to adjust the pH to 8-9. The mixture is kept "at a temperature of about -3O ⁰ C Ms about -1O ⁰ G, an essentially equimolar amount of N- (NL-Seryl-L-tyrosyl) glycine-2-carboxy-hydrazide-t- is added with stirring. butyl ester (III) obtained as described above, in an inert anhydrous solvent, preferably in dimethylformamide, and stir 30 - 60 minutes at about -30 ⁰ C to about -10 ⁰ C on, then a further 30 - 60 minutes at about ⁰ ° C. and finally with cooling in an ice bath for 16-24 hours, preferably about 18 hours The precipitate is filtered off, the piltrate evaporated, the residue taken up in a lower alkanol, preferably methanol, and by adding an ether, preferably diethyl ether , precipitated, then crystallized from a lower alkanol, preferably methanol, and N- / N- / N- / N- / N- (5-Oxo-L-prolyl) -L-hi stidyl7-L-tryptophyl7-L are obtained -seryl7-L-tyrosyl7glycine-2-carboxyhydrazide-t-butyl ester.

Alternatively, the same connection can also be established as follows will:

To a solution of N- / N- (5-Oxo-L-prolyl) -L-histidyl / -L-. tryptophan benzyl ester, obtained as described above, in a lower alkanol, preferably methanol or in glacial acetic acid, a noble metal catalyst, preferably palladium on carbon, is added and the mixture is shaken in a hydrogen atmosphere at room temperature for 10-30 hours, preferably about 16 hours, to substantially 1 mole equivalent Hydrogen has been absorbed. The catalyst is filtered off, the filtrate evaporated, the residue in a lower alkanol, preferably methanol, added, by adding an ether, preferably diethyl ether, is a precipitate is precipitated, which is filtered off ", is obtained in the process man N- / N- (5-0xo-L-prolyl) -L-histidyl7-L-tryptophan, the

- 26 3 0 9 8 3 "i / 113 4

AHP-5792-1-C1

same tripeptide as compound II described above, with the exception that the terminal carboxylic acid is unsubstituted. The latter compound is dissolved in an inert anhydrous solvent, preferably in dimethylformamide, and an essentially equimolar amount of N- (NL-Seryl-L-tyrosyl) glycine-2-carboxyhydrazide-t-butyl ester (III, obtained as described above) is added ), the mixture cools to about OC, an essentially 1 molar equivalent of dicyclohexylcarbodiimide is added and the mixture is stirred, first at about O ⁰ C for about 24 hours and then at room temperature for 4-10 days, preferably about 5 days . It is filtered, the solvent is evaporated, the residue is taken up in a mixture of a halogenated hydrocarbon and a lower alkanol, preferably chloroform and methanol, then chromatographed on silica, the eluate is evaporated, the residue is taken up in a lower alkanol, preferably methanol, a precipitate separates out by adding an ether, preferably diethyl ether, then filtered and receives N- / N- / N- / N- / N- (5-Oxo-L-prolyl) -Ii-histidyl7-L-tryptophyl7-3J- seryl7-l-tyrosyl7glycine-2-carboxyhydrazide-t-butyl ester, which is identical to the same compound prepared as described above.

The latter compound, prepared in one of the ways described above, is in irifluoroacetic acid, preferably in about 90% trifluoroacetic acid, the solution becomes Stirred for 30-60 minutes, first with cooling in an ice bath and then for a further 30-60 minutes at room temperature. Precipitation with an ether, preferably diethyl ether, and subsequent crystallization from a lower alkanol, preferably methanol, gives the hexapeptide N- / N- / N- ^ T- / N- (5-oxo-L-prolyl) -L-histidyl7-L-tryptophyl7-L-seryl7-L-tyrosyl7-glycine hydrazide trifluoroacetate (Vl).

- 27 309834/1 134

AHP-5792-1-C1

The latter compound (VI) in an inert, _water-: serfreien solvent, preferably in a mixture of 'dimethylformamide and dimethyl sulfoxide, dissolved, in addition, is added a solution of a strong mineral acid, preferably chlorine what s acid only off in a anhydrous ether or cyclic: lther, preferably tetrahydrofuran, with stirring at a temperature of -1.0 G ⁰ to 5 ⁰ C, preferably at about ⁰ ° C. The mixture is heated to a temperature of about -30 ⁰ C to about -1O ⁰ C, preferably about -20 ⁰ C, a solution of an essentially equimolar amount of an organic nitrite, preferably t-butyl nitrite or isoamyl nitrite, in dimethylformamide is added and the mixture is stirred at about -30 ⁰ C to about -10 ⁰ C for 30-60 minutes. Sufficiently strong organic base, preferably triethylamine, is added to make the mixture slightly alkaline, preferably a pH of 8-9 is set, and a solution of an essentially equimolar amount Ν- / Ν- / ΪΓ- ( NL-Leucyl) -L-arginyl / -L-prolyl / -glycinamide diacetate (V, obtained as described above) in an anhydrous inert solvent, preferably dimethylformamide, together with so much strong organic base, preferably triethylamine, that the diacetate salt is neutral. is sated. The mixture is stirred at a temperature of about -30 ⁰ C to about -10 ⁰ C for 30-60 minutes, then at about ⁰ ° C for an additional 30 - 60 minutes and finally cooling; stirred in an ice bath for 16-24 hours. The mixture is filtered, the filtrate is evaporated, the residue is taken up in a lower alkanol, preferably methanol, at, precipitated by addition of a ithers, preferably DIAE thy lather, and obtains the crude decapeptide crosslinked Verteilungsehromatographie on a '■ chemically modified dextran ( "Sephadex LH-20") j is purified, the lower phase of an n-butanol / acetic acid / water mixture being used as the solvent. The eluates are evaporated, the residue is taken up in a lower alkanol, preferably methanol, and the substance

- 28 309834/1134

AHP-5792-1-C1

is obtained by adding an ether, preferably diethyl ether,
precipitated, the practically pure decapeptide is obtained: S-Oxo-L-prolyl-l-histidyl-L-tryptophyl-L-seryl-l-tyrosylglycyl-! Ir-leucyl-L-arginyl-L-prolyl-glycine amide (I), which is available as hydro- ^; chloride salt is isolated, which has the same amino acid analysis as the natural product and that in the
biological testing is as effective as the latter.
Palis desired, the above hydrochloride salt can be used in others
Acid addition salts, preferably those with pharmaceutical
■ compatible acids. Purely such purposes
the above hydrochloride salt is treated with a strongly basic anion exchange resin, for example with one of those from G-reenstein
and Winitz in "Chemistry of the Amino Acids", cited above,
named, in the form of its salt with the acid, whose
Hormone salt you want to make. When eluting, one obtains
the IiH- and FSH-releasing hormone in the form of its salt
with the corresponding desired acid. Preferred anions—
Replacement resins are crosslinked polystyrene resins that are strong with
basic groups such as Amberlite IRA-400 _; or IRA-410 ', preferred acids are pharmaceutically _contractual: Liehe acids with which the corresponding one _{pharmaceutically:} receive acceptable salts hormone. ;

If you want a salt of the decapeptide of formula I,
that is only sparingly soluble in water or body fluids
one of its acid addition salts obtained by the process according to the invention is treated in an aqueous solution with a pharmaceutically acceptable solution moderately in water

ι soluble acid, for example tannic acid, alginic acid or emboxylic acid, > preferably in the form of one of its salts, for example in the form of the alkali '. metal salts. The hormone falls as a salt which is accordingly moderate! water-soluble acid and is isolated, for example I by filtering off or centrifuging. |

- 29 309834/1134

AHP-5792-1-C1

The above re ac-fci ons that the process of the present Invention and only the first alternatives Syntheses of the above-described intermediates IY and VI are shown in Pigur 2, the usual Abbreviations used for the various amino acids and protecting groups. · -. .

134/1134

ΛΙ

* I

- Si

NS

(Sl

(\ j

<VY

/ "■

W O Ο, »

. <M

-N

■ &

Λ1

PQ

O O

AHP-5792-1-C1

• Η

ÄHP-5792-1-C1

The decapeptide produced by the method according to the invention can also be converted into a sparingly soluble complex with a pharmaceutically acceptable metal. Such Complexes are made by placing the decapeptide or one of its water-soluble salts in an aqueous dissolves a solution of a salt of the metal and adjusts the pH of the mixture by adding a water-soluble base, e.g. sodium hydroxide, slowly increased until essentially all of the decapeptide has precipitated as a metal complex, then the metal complex is isolated by filtering off or centrifuging off. The .zinc complex is preferred and it is the decapeptide pharmacologically equivalent. The following examples serve to further illustrate the invention, all compounds were identified by elemental analysis.

example 1

3Üf ~ / N- (5-OxQ-I) -PrOIyI) -Ii-hi s tidyl7-L-trypt ophane-benzyl ester

N- (5-0xo-L-prolyl) -L-histidine-hydrazide (0.840 g, 3 mmol) is dissolved at ⁰ ° C. in a solution of dry dimethylformamide (14 ml), dry dimethyl sulfoxide (10.5 ml) and 2 , 4 η of anhydrous gaseous hydrochloric acid dissolved in dry [tetrahydrofuran (7.5 ml). The solution is cooled auf'-20 ⁰ C and added with stirring isoamyl nitrite (0.57 ml). The solution is stirred for 30 minutes at -20 ⁰ C and then cooled to -24 ⁰ C. Triethylamine (3.2 ml) is slowly added until the solution is weakly alkaline (pH 8-9). With stirring gibt'man at -20 ⁰ C a solution of L-2ryptophan ~ benzyl ester (0.970 g, 3.3 mmol) in dry dimethylformamide (3 _s 0 mL). The solution is for 30 minutes at -20 ⁰ C, "G ⁰ stirred for 18 hours at O for 30 minutes and then at ice-bath" the solution is filtered, the precipitate is washed with dry

32 -

0 9 8 3 4/11 "

AHP.-5792-1-C1

Dimethylformamide (2 χ 2 ml), the combined filtrates concentrated under reduced pressure at 4O ⁰ C, the residue is dissolved in methanol (10 ml), are slowly Diathyläther (300 mL) and the precipitate is isolated by filtration. The precipitate is redissolved in methanol (10 ml) and precipitated by adding diethyl ether (300 ml). Crystallizing the precipitate of methanol, and the QJitelverbindung as fei ^

(c = 1.0, DMi ¹ ).

Binding as a fine white powder, Pp = 250 - 252 ⁰ C, M ^ + 1.6 °

If t-butyl nitrite is used instead of isoamyl nitrite or is used if the methyl, ethyl, isopropyl or n-butyl ester of Iryptophane is used, the corresponding one is obtained in the same way Methyl, ethyl, isopropyl or n-butyl esters of the compound mentioned in the Eitel.

example N-Zn- (5-Oxo-L-prolyl) -L-histidyl7-l »-tryptophan-hydrazid (II)

Hydrazine hydrate ($ 99, 1.5 ml) is slowly added with stirring to a solution of N- / N- (5-Oxo-L-prolyl) -L-histidyl7 ~ L-tryptophan benzyl ester (Example 1, 0.868 g) in anhydrous methanol (150 ml) with stirring for 3 hours at -10 ⁰ C. for Bisbadtemperatur the solution is stirred for 40 hours at room temperature. The mixture is filtered and crystallized and the crystalline precipitate from methanol, thereby obtaining the ÖJitelverbindung as fine needles, Pp = 165-169 ⁰ C, [<x] ^ m -24.6 ° (c = 1.0, DMP) .

If you use the methyl, ethyl, isopropyl or n-butyl ester of the starting material instead of the Benzyles.ter, the compound named in the iDitel is also obtained in the same way.

- 33 -309834/1134

AHP-5792-1-C1

example

-ZIr- (^ -Oxo-L-pr olyl) -LM st idyl / -L- tryp t ophan

A mixture of N- / N- (5-Oxo-L-prolyl) -L-histidyl7-L - tryptophan benzyl ester (Example 1, 0.300 g, 0.555 µmol) and 5 # Palladium on charcoal (0.060 g) in glacial acetic acid (30 ml) is rapidly under a hydrogen atmosphere for 16 hours at room temperature touched. The mixture is through diatomaceous earth ("Celite") filtered and the filtrate is concentrated under reduced pressure. The residue is dissolved in methanol (10 ml) and added slowly add diethyl ether (100 ml) with vigorous stirring. The precipitate is isolated and dissolved in methanol (10 ml), 100 ml of diethyl ether are slowly added. The precipitation is isolated, dissolved in methanol, treated with charcoal (0.20 g) and filtered through diatomaceous earth ("Celite"). That The filtrate is evaporated and gives the title compound as a hard white glassy mass, which I will use later as such. will.

Example 4

N- / O-Benzyl-Nc arboxy-L- tyr ο s ο XjjQ ^ J p in-Nb enzyl est he

Aqueous sodium hydroxide (1N, 1N, 5 ml). As soon as the precipitate has completely dissolved (after about 1 hour), aqueous hydrochloric acid (1N, 5.25 ml) is added with stirring. The precipitate is washed with water and recrystallized from methanol / to give the title compound having a melting point of 166 - 167 ° has 0, M ⁰ -22.6 C (c = 1.0, DMF).

; - 34.-

.309834 / 1134

AHP-5792-1-C1

If you use the ethyl, propyl, isopropyl or n-butyl ester of the starting material instead of the methyl ester or "one uses methanol, ethanol, propanol, butanol or 2-ethoxyethanol ("Cellosolve") as a solvent instead of methoxyethanol, the title compound is obtained in the same way,

example

hydrazide t-butyl ester

A solution of N-ZO-benzyl-N-carboxy-L-tyrosyl / glycine-Ni-benzyl ester (Example 4, 2.312 g, 0.005 mol) in dry tetrahydrofuran (12.5 ml) and triethylamine (0.7 ml, 0.005 mol) is cooled to -10 ⁰ C. With stirring, ethyl chloroformate (0.48 ml, 0.005 mol) and, after 10 minutes, t-butyl carbazate (725 mg, 0.005 mol) are added and the mixture is kept at ⁰ ° C. for 30 minutes and kept at room temperature for 5 hours. After the solvent has evaporated, the residue is taken up in ethyl acetate (100 ml), filtered and the piltrate is washed successively with water (2 × 20 ml), aqueous ammonia (3 × 20 ml) and saturated sodium chloride solution (2 × 25 ml). The ethyl acetate layer is dried, evaporated under reduced pressure and the residue was recrystallized from benzene / hexane to give the title compound having a melting point of 88 - 90 ⁰ C is obtained.

Example 6 ^ -L-Tyrosylglycine-2-carboxyhadrazide-t-butyl ester

To a solution of N-ZO-Benzyl-N-carboxy-L-tyrosyl / glycine N-benzyl ester-2-carboxyhydrazide-t-butyl ester (Example 5, 2.88 g, 0.005 mol) in dry methanol (40 ml) are added

- 35 309834/1134

AHP-5792-1-C1 '■

Palladium on carbon (50 °, 200 mg). The hydrogenation will be like this

carried out that the hydrogenation vessel with a stirred!

Sodium hydroxide solution (4 n _r 40 ml) communicates to;

To absorb carbon dioxide. After 4 hours the theoretical amount of hydrogen has been consumed. The catalyst is'über diatomsen-;

earth ("Gelite") filtered off, the Eiltrat v / ird under reduced ■

evaporated tem pressure at 30 ⁰ C and one receives the titlever- ·!

binding as an oily residue, which without cleaning in the next:

Stage is used. If you use platinum instead of palladium; as a catalyst, it is also obtained in the same way the title compound.

example

N- / N- (NC axboxy-l-seryl) -l-tyrosy] J ^r glycine-N-benzyl ester-2- carboxyhydrazifl-t-butyl ester __ »« __ "__-____« »» «._ — _ -

To a solution of N-carboxy-l-seryl-lT-benzyl ester-2,4-dinitrophenyl ester (1.6 g, 0.004 mol) in dry dimethylformamide (10 ml), which has ^{been cooled to 0} ° C. and protected from moisture is, a solution of N-1-lyrosylglycine-2-carboxyhydrazide-t-butyl ester (Example 6, 1.689 g, 0.005 mol) in dry dimethylformamide. The solution is kept at ⁰ ° C. for 3 2 days. After the solvent has evaporated, the residue is taken up in chloroform / methanol / pyridine (100: 25: 1) and transferred to a. 100 times the amount of silica chromatographed. The pure material is taken up in a small amount of methanol and precipitated by the addition of Ither to give the pure (Ditelverbindung having a melting point of 65 - 95 ⁰ C is obtained, [*] ψ = -13.1 ° (c = 1.0 , DMP).

309834/1134

AHP-5792-1-C1

Example 8 IT ~ (IT-] J-Seryl-L-tyro3yl) glycine-2-arboxyIydrazide-t-butyl ester (III)

A mixture of N-carboxy-L-seryl-L-tyrosylglycine-N-benzyl-ester-2-carboxy-hydrazide-t-butyl ester (Example 7, 0.96 g) and 5 $> palladium on carbon (0.090 g) in methanol (35 ml) becomes | stirred vigorously under a hydrogen atmosphere for 7 hours at room temperature, in the same way as described in Example 6 (absorbed hydrogen 49 ml). The mixture is filtered through diatomaceous earth ("Celite") and the Piltrat ^! concentrated under reduced pressure. The residue is crystallized from methanol / diethyl ether and afforded the title compound as fine white prisms having a melting point of 132 - ⁰ / oc / 25 _m _ _5A o _(c _ _no>

If platinum is used as a catalyst instead of palladium, the title compound is likewise obtained in the same way.

Example 9 2- / H-Oarboxy-II-prolyl) amine Q7acetamide-IT-benzyl ester

Add to a solution of N-carboxy-L-proline-H-benzyl ester (12.5 g, 0.05 mol) and glycine ethyl ester hydrochloride (7.26 g, 0.052 mol) in chloroform (100 ml) at O ⁰ G one ethylamine (7.28 ml, 0.052 mol) and then dicyclohexylcarbodiimide (10.7 g, 0.052 mol) with stirring. After stirring for 16 hours at ⁰ ° C., the precipitate is filtered off and the piltrate is washed successively with water (50 ml), hydrochloric acid (1N, 50 ml), water (20 ml) and saturated sodium chloride solution (50 ml), with magnesium sulfate dried and evaporated. The residue is saturated with ammonia ^{at 0 ° C.}

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Methanol (150 ml) was taken up and left to stand at this temperature for 48 hours. After evaporation of the solvent under reduced pressure, the residue from methanol / ether is recrystallized to yield the title compound having a melting point of 112-115 ⁰ C, further recrystallization from methanol / I sopropyläther gives a melting point of 116 - 12O ⁰ C, βχ] ψ = -43.8 ° (c = 1.0, MeOH).

If you use the methyl, propyl, isopropyl or n-butyl ester of G-lycin instead of the ethyl ester, one obtains the same Way also the title compound.

Example 10 1- / Tli-Prolyl) amine Q7 acetamide hydrochloride

A solution of 2 - / (N-carboxy-L-prolyl) amino / acetamide-IT-benzyl ester (Example 9, 610 mg, 2 mmol) in glacial acetic acid (8 ml) containing hydrogen chloride (2 mmol) is mixed with palladium on Eohle ($ 5, 50 mg) in a hydrogen atmosphere ± n in the same manner as described in Example 6, until 1 mol equivalent of hydrogen has been consumed. The mixture is ^filtered: the Piltrat evaporated, the residue is dried under reduced pressure to yield the title compound which is used as such in the subsequent step.

If platinum is used as the catalyst instead of palladium, the title compound is likewise obtained in the same way.

If you use hydrogen bromide or sulfuric acid instead of hydrogen chloride, the corresponding hydrobromide or sulfate salts of the title compound are obtained in the same way.

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Example 11

N- / N- (NC arboxy-N -nitro-L-arginyl) -L-prolylJ-glycinamide-N-. t-butyl ester (IV)

To a solution of N-carboxy-N-nitro-arginine-Nt-butyl ester; (607 mg, 1.9 mmol) and N-hydroxysuccinimide (437 mg, 3.8 mmol) in dimethylformamide (5 ml) are added at -1O ⁰ C, dicyclohexylcarbodiimide (388 mg, 1.9 mmol). The mixture is stirred 1 STUN ■ de at -10 ⁰ C for 2 hours at ⁰ C and 2 hours at room temperature. 2 - / "(L-prolyl) amiao7acetamide hydrochloride, obtained as residue in Example 10, is dissolved in dimethylformamide (4 ml) at ⁰ ° C., 0.38 ml of triethylamine are added and this solution is added to the solution of the active substance described above The ester is at about ⁰ ° C. The mixture is stirred for 14 hours at 0 ° C. and 24 hours at room temperature and then evaporated to dryness under reduced pressure The residue is taken up in chloroform / methanol (100:15) and chromatographed on 100 g of silica . The pure title compound ". separates from absolute ethanol as a gelatinous precipitate, which sinters at approximately 165 ⁰ C, ^[0 HQ = -25.80 (c = 1.0, DMP).

If you use the hydrobromide or the sulfate salt of 2 - / "(L-Prolyl) -amino_ / acetamides instead of the hydrochloride salt, the title compound is obtained in the same way.

Example 12

N- (N-Carboxy-N -nitro-L-arginyl) -L-proline-H-t-butyl ester

To a solution of L-proline methyl ester hydrochloride (5.85 g, 35.4- mmol) and N-carboxy-N -nitro-arginine-N-t-butyl ester (9.4 g,

- 39 -309834/1134

AHP-5792-1-C1

29.4 mmol) in acetonitrile (10 ml) and dimethylformamide (25 ml) are added 4.95 ml of triethylamine. The mixture is cooled to -1O ⁰ C and treated with dicyclohexylcarbodiimide (7.25 g, 35.2 mmol). After 5 hours at ⁰ ° C. and 10 hours at room temperature, the mixture is filtered and the piltrate is evaporated to dryness under reduced pressure. The resulting residue is taken up in ethyl acetate (300 ml) and successively with citric acid solution (10 $, 2 × 90 ml ), Ammonia solution (1N, 3 × 90 ml) and saturated sodium chloride solution (90 ml). The oily residue that remains after drying and evaporation of the organic layer is taken up in methanol (88 ml). Aqueous sodium hydroxide solution (1N , 88 ml) and the mixture is stirred for 1 hour at room temperature. After filtering, the piltrate is extracted with ethyl acetate (3 × 150 ml). The aqueous phase is acidified with hydrochloric acid (4 N, 30 ml) and with Ethyl acetate (3 × 150 ml) extracted The combined ethyl acetate extracts are washed with saturated sodium chloride solution, dried with magnesium sulfate and evaporated under reduced pressure g is obtained as an amorphous residue which, on electrophoresis in an aqueous buffer at pH 5.3, which contains pyridine ($ 1) and acetic acid ($ 0.32), shows a major spot at 4000 volts. It is used in the next stage without further purification.

The ethyl, isopropyl or n ^ -butyl ester of 1-proline is used instead of the methyl ester, the title compound is likewise obtained in the same way.

B e i s ρ i e 1; 13th

N- / N- (NC arboxy-N -nitro-L-arginyl) -L-prolyl7glycine amide-N-1- butyl ester (IV)

j ρ

j N- (N-carboxy-N -nitro-L-arginyl) -L-proline-N-t-butyl ester (Example 12, 7.11 g, 17, -1 mmol) and glycine ethyl ester hydro

- 40 - ■ ..

309834 / 113A

AHP-5792-1-C1

chloride (2.5 g, 17.1 mmol) are dissolved in dimethylformamide (45 ml). While stirring, ^{triethylamine (2.4 ml) is added at 0} ° C. and, after 10 minutes, dicyclohexylcarbodiimide (3.52 g, 17.1 mmol) is added. The mixture is ^{stirred for 3 hours at 0} ° C. and for 17 hours at room temperature, then filtered and the residue is evaporated to dryness under reduced pressure. After chromatography on silica (500 g) with chloroform / methanol (100: 8) as solvent and evaporation, an oily product is obtained. It is dissolved in ^{methanol saturated with ammonia at 0} ° C. and left to stand at this temperature for 3 days. The solvents are evaporated and the residue is triturated with ethyl acetate. The resulting title compound precipitated from absolute ethanol as a gelatinous product having a melting point of 153 - 163 has ⁰ C, [<χ] ψ - -26 ° (c = 1.0, DMF), the product is identical with the compound obtained according to Example 11.

If you use the methyl, isopropyl or n-butyl ester of G-lycins instead of the ethyl ester are obtained in the same way Way also the title compound.

Example 14

N- / N- / N- (NC arboxy-L-leucyl) -N -nitro-L-arginyl7-L-prolyl / -glycinamide-N-benzyl ester

A solution of N- / N- (N-Carboxy-N -nitro-L-arginyl) -L-prolyl7-glycinamide-t-butyl ester (Example 11 or 13, 817 mg, 1.76 mmol) in trifluoroacetic acid (5 ml ) is kept at ⁰ ° C. for 30 minutes. The solution is added dropwise to cold absolute ether (50 ml), the precipitate of N- / N- (N -Ni trol-arginyl) -L-prolyl / glycinamide trifluoroacetate is filtered off and washed well with dry ether. After drying under comparable

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The Pe st substance is taken up under reduced pressure in dimethylformamide (6 ml), which contains triethylamine (0.24-5 ml), and the mixture is stirred ^{at 0-0 for 15 minutes.} A solution of IMJarboxy-L-leucine-lil-benzyl ester ^^ iS-trichlorophenyl ester (860 mg, - 1.96 mmol) and a pot of acetic acid are added to this solution and the mixture is left for 24 hours at O ⁰ O and 24 ' Stand at room temperature for hours. After evaporation to dryness under reduced pressure, the residue is taken up in chloroform / methanol (100: 15) and over 150 g of silica. chromatographed. The chromatographically pure title compound is obtained as an amorphous pest substance; it is dissolved in methanol (3 ml) and precipitated by adding this solution to cold isopropyl ether (50 ml). The title compound is obtained with a rotation value M ^ = -38.4 ° (c = 1.0, DMP).

In the same way as described above, but instead of of trifluoroacetic acid hydrogen chloride in a lower alkanol or used in glacial acetic acid, the hydrochloride of ΪΓ- / Ν- (Η -Mtro-L-arginyl ^ L-prolyl / glycinamides, which is found in is converted into the title compound in the manner described above. '

The title compound is obtained in the same way even if the 4-nitrophenyl or 2,4-dinitrophenyl ester of N-carboxy-L-leucine-N-butyl ester instead of the 2,4,5-triehlophenyl ester is used.

Example 15 "·,

N-Zi-Z! ^; ! i- (KL-Leucyl) -L-arginyl7 - L-prolyl7g: lycinamide diacetate (Y)

A solution of N- / N- / N- (N-Carboxy-L-leucyl) -N -nitro-L-axginyl7-L-proly: l / glycinamide-ϊ ^ -benzyl ester (Example 14, 805 mg, 1.3 mmol) in glacial acetic acid (5 ml), the palladium on charcoal as a catalyst

309834/1134

AHP-5792-1-C1

(5 °, 300 mg) is stirred under hydrogen in the manner described in Example 6 ". The reaction mixture is filtered through diatomaceous earth (" Celite "), the piltrate is concentrated under reduced pressure and dry ither is added dropwise with stirring O ⁰ C. The amorphous precipitate is filtered off, washed well with ether and dried under reduced pressure, whereby the titanium compound is obtained, which is obtained in thin layer chromatography on silica using ethyl acetate / acetic acid / n-butanol / water (1: 1: 1: 1) is homogeneous as a solvent and also in electrophoresis at pH 1.6 in 8% aqueous formic acid at 4000 volts or at pH 5.3 in an aqueous buffer containing 1% pyridine and 0.32% acetic acid , is homogeneous at 4000 volts.

When using platinum instead of palladium as a catalyst the title compound is also obtained in the same way.

Example 16

iT- / N- / N- / N- / N- (5-0xo-I-prolyl) -L-hist seryl7 ~ I ~ tyrosyl7glycine-2-carboxyhydrazide-t-butyl ester

Method a

N- / N- (5-Oxo-Ii-prolyl) -Ii-histidyl7-L-tryptophan hydrazide (II, Example 2, 0.655 g, 1.40 mmol) is added at O ⁰ O in a solution of dry dimethylformamide ( 7 ml), dry dimethyl sulfoxide (5.9 ml) and 2.4 η of anhydrous gaseous hydrogen chloride dissolved in dry tetrahydrofuran (3.5 ml). The solution is cooled to -20 ⁰ C and are Isöamylnitrit (0.206 ml, 1.53 mmol) under stirring. The solution is maintained at -20 ⁰ C for 30 minutes and at -25 ° C.abgekühlt. Triethylamine (1.3 ml) is slowly added until the solution is weak

- 43 30983A / 1134

AHP-5792-1-C1

\ "Is alkaline, pH = 8 - 9. While stirring" at -2O ⁰ C a solution of N- (li-L-seryl-L-tyrosyl) glycine-2'-carboxy-

I add hydraziä-t-butyl ester (III, Example 8, 0.617 g, 1.40 mmol) I in dry dimethylformamide (6 ml). The solution will be! Stirred at -20 ⁰ C for 30 minutes, at ^{0 0} C for 30 minutes and at \ lis bath temperature for 18 hours. You filter the! ! solution and washes the precipitate with dry dimethyl; formamide (2x2 ml). The combined filtrates are under ^; reduced pressure at 40 ⁰ C and concentrated. The residue is in; Dissolve methanol (5 ml), slowly add diethyl ether (500 ml) and filter off the precipitate. The precipitation; is dissolved in methanol (5 ml) and precipitated by adding dietary ether (500 ml). The isolated precipitate is crystallized from methanol and gives the title compound in the form of a fine white powder with a melting point of 182 ^° C. (decomposition).

. Using t-butyl nitrite is obtained in the same way i instead of isoamyl nitrite likewise the title compound.

^: Method B "

\ Dissolve N- / N- (5-Oxo-L-prolyl) -L-histidyl7-L-tryptophan (example: 3, 0.133 g, 0.294 mmol) and N- (NL-Seryl-L ~ tyrosyl) glycine-; 2-carboxy-hydrazide-t-butyl ester (III, Example 8, 0.129 g, 0.294 mmol) in anhydrous dimethylformamide (5 ml). ^At: 'stirring at ⁰ Dicyclohexylearbodiimid O (0.061 g, j 0.294 mmol). The solution is ^{stirred for 24 hours at 0} ° C. and for 5 positions at room temperature, then filtered and the piltrate is evaporated to dryness under reduced pressure. After chromatography on silica (20 g) with Chlorofacm / methanol (2; 1) as solvent, the combined; th. Fractions concentrated under reduced pressure. The residue is dissolved in methanol (2 ml), diethyl ether (100 ml) is slowly added and the precipitate is filtered off. The resulting product is identical to the title compound obtained as described above.

_ 44 -

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AHP-5792-1-C1

Example 17

N-ZN-ZN-ZN-ZN- (5-Oxo-L-pr oly ^
seryl7-L-tyrosyl7glycine-hydrazide-trif luorac etat (YI)

A solution of N- / N- ^ r- / N-ZN- (5-Oxo-I-prolyl) -I-histidyl7-L · - tryptophyl7-Iι-seryl7-L-tyrosyl7glycine-2-car ^^ oxy} Lyrazide t-butyl ester (Example 16, 0.859 g, 0.982 mmol) in 90% trifluoroacetic acid (27 ml) is stirred at ice bath temperature for 30 minutes and then at room temperature for 30 minutes. The solution is slowly added to a solution of diethyl ether (700 ml) with stirring. The Fiederschlag is isolated and crystallized from methanol to thereby obtain the title compound as a fine white powder having a melting point of 200 ⁰ C (decomposition), / ÖC / ^ ⁵ = -21.2 ° (c = 1.0, water).

Example 18

5-0xo-L-prolyl-L-histidyl-L-tryptophyl-Ls euyl-L-tyrοsylglycyl- L-leucyl-L-affginyl-L-prolylglyoinamide (I) dihydrate. Hydrochloride

N-ZN-ZF-ZN-ZN- (5-0xo-L-prolyl) -L-hi stidyl7-L-tryptophyl7-L-seryl7-L-tyrosyl / glycine hydrazide trifluoroacetate (VI, example 17, 0.840 g, 0.762 mmol) is dissolved in a mixture of dry dimethylformamide (3.7 ml), dry dimethyl sulfoxide (3.3 ml) and 1.65 η of anhydrous gaseous hydrochloric acid in dry tetrahydrofuran (2.77 ml) ^{at 0 0 O} . The solution is cooled to -2O ⁰ C and added under stirring with a solution of isoamyl nitrite 1Obigen in dry dimethylformamide (1.12 ml, 0.83 mmol). The solution is stirred for 30 minutes at -20 ⁰ C and then cooled to -25 ⁰ C. Are slowly added triethylamine (0.7 ml ") was added until the solution is slightly alkaline, pH = 8 - 9. While stirring at -20 ⁰ C a solution

- 45 309834/1134

AHP-5792-1-C1

of N- / N- / N- (N-1-leucyl) -L-arginy] J-III-prolylZ-glycine amide diacetate (V, Example I5, 0.427 g, 0.762 mmol) in dry dimethylformamide (4.5 ml ) and iriethylamine (0.28 ml) are added. The resulting solution is ^{stirred "at -20 0} C for 30 minutes," at ^{0 0} O for 30 minutes and at ice bath temperature for 18 hours. The solution is filtered, the precipitate is washed with dry dimethylformamide (2 × 2 ml), the combined filtrates are concentrated under reduced pressure at 40 G, the residue is dissolved in methanol (5 ml) and diethyl ether (500 ml ). The precipitate is isolated, dried and separated by partition chromatography. a chemically modified crosslinked dextran ("Sephadex IH-20 ¹¹ ), wherein the lower phase of a n-butanol / acetic acid / water mixture (8: 4: 40) is used. The combined fractions are reduced under reduced pressure at 45 concentrated ⁰ C for ¹ dryness, dissolved in methanol (5 ml) and added to diethyl ether (250 ml). the Fiederschlag is filtered off, dried and yields the compound Eitel, £ <χ] ψ _B -53.50 ° (c = 1, 0.1 # aqueous acetic acid), ¹ isolated as the hydrochloride salt.

Analysis calculated for G ₅₅ H ₇₅ N ₁₇ O ₁₅ «2H ₂ O - HGl;

Ct 52.67; H: 6.43? N: 18.99; 0: 19.14; Cl: $ 2.83; Found: C: 52.25; B: 6 _sec 23 sec N: 19.32; Os 19.10; Cl-. $ 2.95.

The above-described precipitation is carried out under strictly anhydrous conditions Conditions and in the presence of hydrogen chloride, the eluent compound is obtained as the dihydrochloride salt.

Analysis calculated for C ₅₅ H ₇₅ N ₁₇ O ₁₅ 2 HCl:

Ci 52.62; H: 6.18; N: 18.96; 0: 16.57; Cl: $ 5.64; Found: C: 52.10; H: 5.92; N: 18.85; 0: 17.20; Cl: 5.75 #.

- 46 309834/1134

AHP-5792-1-C1

The amino acid analysis shows the following composition:

Histidine 1.0 1.04 Proline 0.96 1.03 Arginine 0.95 0.93 Glycine 2.01 1.98 Serine 0.90 0.86 leucine 0.86 1.02 Glutamic acid 1.07 1.04 ! Tyrosine 1.12 1.04

Electrophoresis at pH 1.6 in 8% aqueous formic acid and at 3500 volts it gives a single peck and shows that Uniformity of connection.

Using t-butyl nitrite is obtained in the same way instead of isoamyl nitrite also the title compound.

Use other inorganic acids such as hydrobromic acid or sulfuric acid in tetrahydrofuran instead of hydrogen chloride, the corresponding hydrobromide or sulfate salts of the oil compound are obtained in the same way.

If desired, the above hydrochloride salt is mixed with Amberlite IRA-400 or IRA-410 previously used with a pharmaceutically acceptable Acid has been converted to a salt thereof, treated. When eluting, the corresponding salt is obtained Title compound. Alternatively, the above hydrochloride salt is used in aqueous solution with an alkali metal sal ζ der Ϊ annin-, algin- or embonic acid and the corresponding! Dannat-, Alginate or embonate salt of the hormone isolated by filtering or centrifuging.

Example 19

S-Oxo-l-prolyl-L-histidyl-l-tryptophyl-L-seryl-l-tyrosylglycyl- L-leucyl-l-arginyl-L-prolylglycine amide (I) zinc complex

100 mg of the dihydrochloride salt of the decapeptide are dissolved

- 47 -309834/1 134

AHP- ^ 792-1-01

Ponnel I, obtained as described in Example 18, in water (0.8 ml) at room temperature and gives a zinc acetate solution / "0.19 ml, Zn (OCOOH ₃ ) ₂ -2H ₂ O, 0.1 g / nüj A 2 η NaOH solution (0.1 ml) is added dropwise to the mixture while stirring. Each drop of sodium hydroxide solution causes precipitation (in lumps), while stirring is continued until the precipitate disappears and the solution is clear again »The process is brought to an end by adding 0.1 η NaOH solution. until the pH rises to 8.0 - 8.2 and the precipitate can no longer be dissolved .: The end of the precipitation is checked by the Add a drop of NaOE solution to the clear supernatant. After having allowed to stand in the refrigerator overnight _9, the 'title compound (Qj, 1 ml) is abfiltriertg, washed with water and dried in a high vacuum, TTV (EtOH) s 289 nm (ß \ ^ _m 42), 279 nm (E ^ _m 56) and 222 nm (E ^ 574).

- 48 -. j

30983 A / 1134

Claims (3)

GASES; AKP-5792-1-C1 PAiDEUTANSPRUOHE 1. Procedure for establishing LH and FSH leasing Hormones, a decapeptide of the formula I: H- Pyr - His - Irp - Ser - Tyr - GIy - Leu - Arg - Pro - GIy - NH, in the form of its hydrochloride salt, characterized in that the hexapeptide: N- / N- / N- / N- / N- (5-Oxo-L-prolyl) - ^ L-histidyl / -L-tryptophylZ-L-serylZ- L-tyrosyl / glycine hydrazide trifuloracetat is treated in an anhydrous inert solvent with a strong mineral acid and an organic nitrite at a (temperature in the range of about -30 ⁰ C to about -1O ⁰ C the mixture by adding a strong organic Base is made alkaline, a solution of N- / N- / N- (NL-Leucyl) -L-arginyl7-L-prolyl7-glycine amide diacetate in an anhydrous inert solvent is added to the mixture at a temperature in the range of about -30 ⁰ C is stirred to about O ⁰ C and the decapeptide of formula I is isolated as the hydrochloride salt. 2. The method according to claim 1, characterized in that the decapeptide of the formula I by partition chromatography is purified and is isolated in essentially pure form as the diacetate salt. -49- 309834/113 / » ABP-5792-1-C1 3. The method according to claim 2 _S da, characterized in that the decapeptide of formula 1 is purified by partition chromatography on a chemically modified crosslinked dextran using the lower phase of an n-butenol / Bssigsäure / Wasser ™ mixture as a solvent. 4e method according to claim 1 ,. characterized, that the hydrochloride salt of the decapeptide of the formula I with a strongly "basic anion exchange resin in the form of its salt is treated with a pharmaceutically acceptable acid "and the corresponding salt of the deodorant peptide is isolated. 5. The method according to claim 1, characterized in that the hydrochloride salt of the decapeptide of the formula I with an acid which is only sparingly soluble in water "and the corresponding salt of the decapeptide, which is only sparingly soluble in water is separated. 6 " Process according to claim 5" characterized in that tannic acid, alginic acid or enibonic acid is used as the acid that is moderately soluble in water ", 7 «The method according to claim 1 _S, characterized in that the anhydrous inert solvent used is dimethylformamide or a semi-sch of dimethylformamide and dimethyl sulfoxide. 8. The method according to claim 1, characterized in that hydrogen chloride is used as the strong mineral acid. 9. The method according to claim 1, characterized in that <that the strong organic base used is tiräthylamine. _. "- .. 50 - 309834/1134 AHP-5792-1-C1 10. Procedure according to. Claim 1, characterized in that that t-butyl nitrite or isoamyl nitrite is used as the organic nitrite. 11. The method according to claim 1, characterized in that the hexapeptide: N- ^ i- / N- / K- / N- (5-Oxo-L-proIyl) -Ii-histidy3 _t / -L-tryptophylZ-I- serylZ-L-tyrosyl / glycine-hydrazide-trifluoroacetate is prepared by adding a solution of N- / N- (5-0xo-L-prolyl) -L-histidyl7-L-tryptoph.an-hydrazide in an inert anhydrous solvent treated with a strong mineral acid and an organic nitrite at a temperature of about -30 ⁹ G Ms about -20 ⁰ C ", the mixture is made alkaline by adding a strong organic base, a solution of N- (NL-Seryl- L-tyrosyl) -glycine-2-carboxy-hydrasid t-butyl ester is added in an inert anhydrous solvent, the mixture is stirred ⁰ C at a temperature in the range of about -30 ⁰ C to about O, so that N- / N- / N - ^ - / N- (5-Oxo-L-prolyl) -L-histidyl7-L-tryptophyl7-L-seryl7-L-tyrosyl7glycine-2-carboxyhydrazide-t-butyl ester, treated the latter compound with irifluoroacetic acid and that above-mentioned 'hexapeptide isolated. 12. The method according to claim 11, characterized by that as an anhydrous inert solvent dimethylformamide or a mixture of dimethylformamide and dimethyl sulfoxide is used. 13. The method according to claim 11, characterized in that Hydrogen chloride is used as a strong mineral acid. H. The method according to claim 11, characterized in that triethylamine is used as the strong organic base. - 51 309834/1134 AHP-5792-1-C1 15. The method according to claim 11, characterized in that that t-butyl nitrite or isoamyl nitrite is the organic nitrite is used. , ■■ ·; ' 16. The method according to claim 11, characterized in that ;; that the Ή- / Έ- (5-Oxo-L-prolyl) -L-histidyl7-L-tryptophan hydrazide is produced by adding a solution of U- (5--0xo-L-prolyl) <«* L-histidine hydrazide treated in an inert anhydrous solvent with a strong mineral acid and an organic nitrite at a temperature of about -3O ⁰ C Ms about -20 ⁰ C, the mixture is made alkaline by adding a strong organic base, a solution of a lower alkyl - or Aralkylesters of L-tryptophan added in an inert anhydrous solvent, the mixture is stirred ⁰ C at a temperature in the range of about -30 ⁰ C to about O, the appropriate N- / N- (5-0xo-L-prolyl) -L-histidyl7-L-rtryptophan-lower alkyl or aralkyl ester is isolated, the latter compound is treated with hydrazine hydrate and the N ~ / N- (5-0xo-L-prolyl) -L-histidyl7-L-trypt'ophan hydrazide is isolated . · 17. The method according to claim 16, characterized in that that as an anhydrous inert solvent dimethylformamide or a mixture of dimethylformamide and dimethyl sulfoxide is used. 18. The method according to claim 16, characterized in that that hydrogen chloride is used as a strong mineral acid. 19. The method according to claim 16, characterized in that j that triethylamine is used as a strong organic base. 20. The method according to claim 16, characterized in that that t-butyl nitrite or isoamyl nitrite is used as the organic nitrite. - 52 ..- 309834/1 134 ÄHP-5792-1-C1 21. The method according to claim 16, characterized in that that the benzyl ester is used as the ester of L-sryptophane. 22. The method according to claim 11, characterized in that the H- (N-seryl-I-tyrosyl) -glycine-2-car'boxyhydrazid ~ t- "butyl ester is prepared by a lower alkyl ester of Nj / Ö-benzyl- N-carbcciy-L-tyrosyl / glycine-K-benzyl ester treated with an aqueous alkali metal hydroxide, then the mixture is acidified and the N-ZÖ-benzyl-N-carboxy-L-tyrosyl / -glyein-N-benzyl ester is isolated; the last-mentioned compound in solution in an anhydrous ether or cyclic ether first treated with ethyl chloroformate and then with t-butyl carbazate and the n- / Ö ~ benzyl-K-car "boxy-L-tyrosyl7-glyein-N-benzylester-2- carbo: xyhydrazide t-butyl ester isolated; the latter compound treated with hydrogen and a noble metal catalyst "and NL-iDyrosylglycine-2-carboxyhydrazide-t-butyl ester isolated; the latter compound in solution in a halogenated hydrocarbon to an activated ester of the N-carboxy-L-seryl-N-benzyl ester , the mixture is kept at around O ⁰ O for several days and the N- / N- (NC axboxy-L-seryl) -L-tyrosyl7-glycine-N-benzyl ester-2-carboxyhydrazide-t-butyl ester is isolated; the latter compound treated with hydrogen and a noble metal catalyst and isolated the N- (NLS eryl-L-tyrosyl) -glycine-2-carboxyhydrazide-t-butyl ester. 23. The method according to claim 22, characterized in that the lower alkyl ester of N-ZÖ-benzyl-N-carboxy-L-tyrosylJ-glycine-N-benzyl ester the methyl ester is used. 24. The method according to claim 22, characterized in that palladium is used as the noble metal catalyst. - 53 -309834/1134 ΔΗΡ-5792-1-01 25. The method according to claim 22, characterized in that that chloroform is used as the halogenated hydrocarbon. ; ■ ■ j 26. The method according to claim 22, characterized in that I that as an activated ester of N-Carhoxy-L-seryl-ir-'benzylesters I the 2,4-Dinitro.phenylester is used. i 27. The method according to claim 1, characterized in that j that the hexapeptide N- / N- / N- / N - # - (5-Oxo-L-prolyl) -L-histidyl / - [ Iι-tryptophyl7-L- seryl7-L-tyrosyl7-glycine-hydrazide-trifluoro-L acetate is prepared by adding IT- / N- (5-Oxo-L-prolyl) -L- histidyl7-L-tryptophan benzyl ester treated with hydrogen and a 'Bdelmetallkatalysator and the N- / N- (5-0xo-L-prolyl.) -; I-histidyl7 ~ L-tryptophan isolated; the last-mentioned compound ^: in solution in an anhydrous "inert solvent with]! J- (l · ί-L-seryl-L-tyrosyl) -glycine-2-carboxyhydrazide-t-butyl- treated ester and dicyclohexylcarbodiimide at a temperature in the range of about O ⁰ C to room temperature and Ν- / ΪΤ- / Ν-: / Ή- / Έ- ( 5-Oxo-L-prolyl) -L-histidyl / -L- tryptophyl7-L-seryl7-L-tyrosyl7glycine-2-carboxyhydrazide ~ t-butyl ester isolated; treated the latter compound with trifluoroacetic acid and isolated the above-mentioned hexapeptide., 28. The method according to claim 27, characterized in that palladium is used as the soft metal catalyst. 29. The method according to claim 27, characterized in that dimethylformamide is used as the inert solvent. 30. The method according to claim 27, characterized in that the N - ^ -. (5-Oxo-L-prolyl) -L-histidyl7-L-tryptophan benzyl ester is prepared by making a solution of N- (5-0xol-prolyl) -L-histidine-hydrazide in an inert anhydrous - ■ 54 30983A / 1134 '*. AHP-5792-1-C1 Solvent treated with a strong mineral acid and an organic nitrite "at a (temperature of about -3O ⁰ C Us about -2O ⁰ C, the mixture is made alkaline by adding a strong organic base, a solution of 1-tryptophan benzyl ester in an inert anhydrous solvent admitting the mixture at a temperature in the range of about -30 ⁰ C to about O ⁰ C and stirred for the appropriate Ν- / ΪΤ- (5-0χο-1-prolyl) -1-histidyl7-L-tryptophan benzyl ester was isolated. 31. The method according to claim 30, characterized in that that as an anhydrous inert solvent dimethylformamide or a mixture of dimethylformamide and dimethyl sulfoxide is used. 32. The method according to claim 30, characterized in that hydrogen chloride is used as the strong mineral acid will. 33. The method according to claim 30, characterized in that triethylamine is used as the strong organic base. r. 34. The method according to claim 30, characterized in that the organic nitrite is t-butyl nitrite or isoamyl nitrite is used. 35. The method according to claim 27, characterized in that the N- (N-1-seryl-1-tyrosyl) -glycine-2-carbo3cyhydrazide-t-butyl ester is prepared by a lower alkyl ester of the N-ZO-benzyl-N-carboxy-1-tyrosyl / glycine-N-benzyl ester treated with an aqueous alkali metal hydroxide, then acidified the mixture and N-ZÖ-benzyl-N-carboxyl-tyrosyl7glycine-N-benzyl ester isolated; the latter compound in solution in an anhydrous ether or cyclic ether Ether first with ethyl chloroformate and then with - 55 309834/1 AHP-5792-1-C1 Treated t-butyl carbazate and N - ^ - Benzyl-N - carboxy - Ij-tyrosyl7 - glycine-N-benzylester-2-caxbQxyhydrazid-t ~ butylester isolated; the latter compound with hydrogen and a noble metal catalyst treated and H ~ l-Iyrosylglycine - 2 - carboxy-hydrazide-t-butyl ester isolated; the latter compound in solution in a halogenated hydrocarbon to form a activated ester of N-carboxy-L-seryl-N ^ benzyl ester, the mixture is kept at about 0 C for several days and the H- / fr-earboxy-L ~ seryl-L-tyrosyl7glycine - IT-benzyl ester-2-carboxyhydrazide-t-butyl ester isolated; the latter compound is treated with hydrogen and a noble metal catalyst, and the N- (N-L-seryl-L-tyrosyl) glycine-2-carbQxyhydrazide-t-butyl ester isolated. 36. The method according to claim 35, characterized in that that as the lower alkyl ester of N-Zö-benzyl-N-carboxy-L'-tyrosyl / -glycine-R-benzyl ester the methyl ester is used. 37 · The method according to claim 35 »characterized ·, that palladium is used as a noble metal catalyst, 38. The method according to claim 35 »characterized in that that. he uses chloroform as the halogenating hydrocarbon will, 39. The method according to claim 35, characterized in that that as an activated ester of the! ■ "Carboxy-'L-seryl -'- ii-benzyl ester the 2,4-dinitrophenyl ester is used. 40. The method according to claim 1, characterized in that the K - ^ - / N - (^ - I-leucyl) -Ii-arginy ^ -I-proly3Jglycinamiddiacetat is prepared by the N-Oarboxy-L-proline-i ^ benzyl ester in Lösung'in * a halogenated hydrocarbon at about ⁰ O O _r a Glycinniedrigalkylester-acid addition ~ 56 30 983Λ / 1134 AHP-5792-1-C1 salt treated in the presence of a strong organic base and of dicyclohexylearbodiimide, then treated with ammonia and 2 - / ^ carboxy-L-prolyl) amineq7acetainid-l! T-'benzyl ester isolated; the latter compound treated with hydrogen and a noble metal catalyst and 2 - / "(l-Prolyl) -amino / acetamide isolated; the latter compound in solution in dimethylformamide and in the presence of a strong organic base treated with an H-carboxy-N-nitro-1-arginine-H-lower alkyl ester and N-hydroxysuccinimide and dicyclohexylcarbodiimide at a temperature in the range from about -20 C to room temperature and the corresponding N-lower alkyl ester of H- / Er- (ir-Carboxy-N -nitro-1-arginyl) -l-prolyl7-g-tycinamide isolated; the last-mentioned compound is treated with a strong acid and the corresponding salt of the N- / N- (IT-nitro-l-arginyl) -l-prolyl / glycinamides is separated off; the last-mentioned compound in solution in dimethylformamide is treated with an activated ester of the N-carboxy-1-leucine-N-benzyl ester in the presence of a strong organic base at a temperature in the range from about ⁰ ° C. to room temperature and the F- / IT - / N- (IT-carboxy-1-leucyl) -U -nitro-1-arginyl / -! - prolyl7-glycinamide-N-benzyl ester isolated; the last-mentioned compound was treated in solution in glacial acetic acid with hydrogen and a noble metal catalyst and the N- / Ef- / N- (li-l-leueyl) -l-arginyl / -l-prolyl7glyeinamide diacetate was solubilized. 41. The method according to claim 40, characterized in that chloroform is used as the halogenated hydrocarbon will. 42. The method according to claim 40, characterized in that glycine ethyl ester hydrochloride is used as the glycine lower alkyl ester acid addition salt is used. 43. The method according to claim 40, characterized in that triethylamine is used as the strong organic base. - 57 -3 0 9 8 3 k 1 1 1 3 4 AHP-5792-1-C1 44. The method according to claim 40, characterized in that that palladium is used as a noble metal catalyst .. 45. The method according to claim 40, characterized in that that as the N-lower alkyl ester of N-Caxboxy-N -nitro-L-arginine the N-t-butyl ester is used and the N- / N- (N-Carboxy-N nitro-L-arginyl) -L-prolyl7-glycinamidrN-t- "butyl ester is obtained. 46. The method according to claim 40, characterized in that the strong acid is hydrogen chloride or trifluoroacetic acid ■ is used. ■ 47. The method according to claim 40, characterized in that that as an activated ester of N-carboxy-L-leucine-N-benzyl ester the 2,4,5-trichlorophenyl ester is used. 48. The method according to claim 1, characterized in that the N- / N- / N- (NL-leucyl) -L-arginyl7-L-prolyl7-glycinamide diacetate is prepared by adding a lower alkyl ester acid addition salt of L-proline in solution "treated in, an anhydrous, strongly polar solvent with N-carboxy-N -nitro-L-argininli-t-butyl ester in the presence of a strongly organic base and of dicyclohexylcarbodiimide at a temperature in the range from about -2O ⁰ C to room temperature, then treated with an aqueous alkali metal hydroxide and isolated the N- (N-carboxy-N -nitro-L-arginyl) -L-proline-Nt-butyl ester; the latter compound in solution in dimethylformamide with an acid addition salt of a G-lycin lower alkyl ester in the presence of a strong organic base and treated by dicyclohexylcarbodiimide at a temperature in the range from about OC to room temperature, then treated with ammonia and N- ^ f- (N-carboxy-N-nitro-L-arginyl) -L-prol · yl7glycinamid-Nt -butyl ester isolate rt; the latter connection with - 58 ^.
30 983 4 / V AHP-5792-1-C1 a strong acid "treated and the corresponding salt of the li- / N - (^ - Nitro-l-arginyl) -l-prolyl7glycinamides separated; the last-mentioned compound in solution in dimethylformamide with an activated ester of N-carboxy-II-leucine- Treated IT-benzyl ester in the presence of a strong organic base at a temperature in the range from about O ⁰ O to room temperature and N- / ft- / H- (N-carboxy-L-leucyl) -: N -nitro-L-arginylJ -Ir-prolyl / -glycine amide-H-benzyl ester isolated; the latter compound in solution in glacial acetic acid treated with hydrogen and a noble metal catalyst and the K- / N- / ir- (Hl-Leucyl) -L-arginyl / -L-prolyl7glycine amide -diacetate isolated. 49. The method according to claim 48, characterized in that the Medrigalkylester acid addition salt of 1-proline the Ii-proline methyl ester hydrochloride is used, 50. The method according to claim 48, characterized in that triethylamine is used as the strong organic base. 51. The method according to claim 48, characterized in that the aqueous alkali metal hydroxide used is sodium hydroxide will. · 52. The method according to claim 48, characterized in that that as an acid addition salt of a glycine lower alkyl ester the G-lycin ethyl ester hydrochloride is used. 53. The method according to claim 48, characterized in that the strong acid is hydrogen chloride or trifluoroacetic acid is used. 54- The method according to claim 48, characterized in, that palladium is used as the noble metal catalyst. - 59 -.
309834/1134 AHP-5792-1-C1 55 «Method according to claim. 48, characterized in, that as an activated ester of N-carboxy-L-leucine-N-benzyl ester the 2,4,5-trichlorophenyl ester is used. 56 »N-ZN- (5-Oxo-L-proiyl) -L-histidyl7-L-tryptophan-hydrazide, 57. ir-CF-L-Seryl-1-tyrosyl-glycine-Z-carboxyhadrazide-t- "butyl ester. ■ · ■ .58. . N-ZIT- (N-Carboxy-N Hnitro-L-arginyl) -L-prolyl7glyeinamide-N-t- "butyl ester. . , 59. ■ IT- / N ■ - / ϊί ■ ⁱ - (ljί-L-Leucyl) -L-arginyl7-L · -prolyl7glycine amide diacetate. ''. '"-, ■ - ■ ': ■ · ■.: ■■ - - 60. N-Zn-ZN-Zn ^ Zn- (S-Oxo-L-prolylO-L-histidylJ-L-trypto-. phylZ-L-serylZ-L-tyrosyl / glycine-hydrazide-trifluoroacetate.,. 61. Pharmaceutical agent for nasal or parenteral Application containing a pharmaceutically acceptable liquid Carrier and that made by the method of claim 1 Decapeptide.of]? Ormel I. .. ■,.,: 62. Pharmaceutical agent for par ent er al, ■ sublingual or vaginal application, containing a pharmaceutically acceptable, solid carrier and the decapeptide of formula I prepared by the process of claim 1. ■ .- .. "· - 63. ■ Pharmaceuticals. Means for nasal or vaginal isufflation, containing a pharmaceutically acceptable finely divided solid carrier and the finely divided decap peptide produced by the process of claim 1. . . 'Pormel I. - 60 -.
3 0 9834/11 3rd U-. AHP-5792-1-C1 64. Pharmaceutical agent for vaginal use as a suppository, containing a pharmaceutically acceptable semi-solid carrier and that according to the method of claim 1 produced decapeptide of formula I. 65. A pharmaceutical agent for parenteral use, containing that prepared by the method of claim .1 Decapeptide of Foremi I, encapsulated in microcapsules with a pharmaceutically acceptable II "coating material, 66. A pharmaceutical agent for implantation comprising a pharmaceutically acceptable solid carrier or container and the decapeptide of the formula I prepared by the process of claim 1. 67. Pharmaceutical agent for vaginal use, characterized by a pharmaceutically acceptable container and the decapeptide of the formula I prepared by the process of claim 1. 68. 5-Oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosylglycyl-L-leucyl-L-arginyl-1-prolylglycine amide dihydrate hydrochloride. 69. 5-Oxo-I-prolyl-L-histidyl-L-tryptophyl-If-seryl-ltyrosylglycyl-L-leucyl-L-arginyl-L-prolylglycine amide dihydrochloride. 70. Inorganic acid addition salt of 5-oxo-L-prolyl-L-histidyl-1-tryptophyl-L-seryl-L-tyrosylglycycl-L-leucyl-L-arginyl-L-prolylglycine amide namely the hydrochloride, hydro- "bromide or sulfate. - 61 3098 3 4/1 ΑΗΡ-5792-1-C1 71. Complex of 5-oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosylglycyl-L-leucyl-I-arginyl-L-prolylglycine amide with a pharmaceutically acceptable metal. 72. Zinc complex of 5-oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-I-tyrosylglycyl-I-leuyl-L-arginyl-L-p-rolylglycine amide. .- 62 -3 0 9 8: U / 1 1 3 4