DE2625843A1 - NEW PEPTIDES, THEIR MANUFACTURING AND MEDICINAL PRODUCTS - Google Patents

Description

PATENT LAWYERS O Γ * OCO / O!

PROF. DR. DR. J. REITSTÖTTER ΔΌΔΟΟΗΟ j

DR.-ING. WOLFRAM BUNTE!

DR. WERNER KINZEBACH,

D-8OOO MUNICH 4O. BAUERSTRASSE 22 ■ FERNRUF (Ο89) 37 OS 83 · TELEX 8215208 I8AR D I POSTAL ADDRESS: D-800O MÜNCHEN 43. POST BOX 7βΟ

M / 17 132

ANDREW VICTOR SCHALLY, -q HlNl 1976

2500 Whitney Place, Apt. 319 ^Λ
Metairie, Louisiana / USA

DAVID HOWARD COY

4319 Perrier Street, New Orleans

Louisiana / USA

New peptides, their production
and medicaments containing them

The invention relates to peptides of the formula I.

(pyro) -Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z I j

a) X = Ser, Y = D-Trp and Z = GIy-NH ₂ ; or

b) X = Ser, Y = D-Phe and Z = GIy-NH ₂ ; or ;

c) X = D-Ser, Y = D-Leu and Z = NHR ¹ , wherein
R = lower alkyl,

and intermediate products for their synthesis. . j

The peptides of formula I, wherein

a) X = Ser, Y = D-Trp and Z = GIy-NH ₃ ; or

b) X = Ser, Y = D-Phe and Z = GIy-NH ₂ ; or J

c) X = D-Ser, Y = D-Leu and Z = NHR ¹ '

i are also denoted by ^!

a) L-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-;

tyrosyl-D-tryptophyl-L-leucyl-L-arginyl-L-prolylglycine amide; or

b) L-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-,

tyrosyl-D-phenylalanyl-L-leucyl-L-arginyl-L-prolyl- | glycinamide; or

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c) L-pyroglutamyl-L-histidyl-L-tryptophyl-D-seryl-L-tyrosyl-D-leucyl-L-leucyl-L-arginyl-L-prolyl-lower alkylamide

and can with the abbreviation a) [D-Trp ⁶ ] -LH-RH;

b) [D-Phe ⁶ ] -LH-RH; respectively.

c) [D-Ser, D-Leu, desGly-NH-] -LH-RH- (lower alkyl) amide are designated.

Both luteinizing hormone (LH) and follicle stimulating hormone (FSH) are gonadotropic hormones produced by the pituitary glands of humans and mammals. LH, together with FSH, stimulates the release of estrogens from the maturing follicles in the ovaries and induces the ovulation process in the female organism. In the male organism, LH stimulates the interstitial cells or the Leydig ¹ see intermediate cells and is therefore also called interstitial cell stimulating hormone (ICSH). FSH induces the maturation of the follicles in the ovaries and, together with LH, plays an important role in the cycle of the female organism. FSH promotes the development of the germ cells of the male testes. Both LH and FSH are released by the pituitary gland through the action of the LH- and FSH-releasing hormone, and there is clear evidence that this releasing hormone is formed in the hypothalamus and reaches the pituitary gland in a neurohumoral way, cf., for example, AV Schally et al., Recent Progress in Hormone Research, 24_, 497 (1968).

The natural LH and FSH releasing hormone was created from the Hypothalamus isolated from pigs, and its structure was reported by A.V. Schally et al., Biochem. Res. Commun., 43_, 393 and 1334 (1971) who investigated the decapeptide structure

(pyro) -GIu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NHp proposed.

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This structure has been confirmed by synthesis, see, for example, H. Matsuo et al., Biochem.Biophys.Res.Comm., 45 , 822 (1971) and R. Geiger et al-, ibid., 45., 767 (1971 ).

The following is the natural LH- and FSH-releasing Called the hormone LH-RH.

Because of the importance of LH-RH in both diagnostic and therapeutic medicine, there is considerable interest in the preparation of new compounds with improved properties compared to the natural hormone. One approach to this goal has been the selective modification or replacement of amino acid residues of the LH-RH with other amino acids. Although in a few cases peptides with such changes have been found to be more active than LH-RH, for example [D-Ala] -LH-RH, A. Arimura et al., Endocrinology, 95, 1174 (1974), [D-Leu ⁶ ] -LH-RH and [D-Leu ⁶ , desGly-NH ₂ ¹⁰ ] -LH-RH-ethylamide, JA Vilchez-Martinez et al., Biochem.Biophys.Res.Commun., 59 , 1226 (1974), modified peptides have proven to be less active in most cases.

It has now been found that

a) the replacement of the glycyl residue in the 6-position of LH-RH by D-tryptophan; or

b) the replacement of the glycyl residue in the 6-position of LH-RH by D-phenylalanine; or

c) the replacement of the L-seryl residue in the 4-position of LH-RH by a D-seryl residue, replacement of the glycyl residue in the 6-position by a D-leucyl residue and replacement of the Glycine amide residues in ΙΟ-position by a lower alkyl amide group,

leads to a peptide of the formula I which is much more active and longer effective than LH-RH.

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According to one embodiment of the invention it has been found that the change in the asymmetry of the seryl radical in the 4-position is related with the introduction of a D-leucyl residue in the 6-position to an increased effectiveness and a longer duration of effectiveness of the peptide of the formula I in which X = D-Ser, Y = D-Leu and Z = NHR, which is surprising, in particular there is generally a change in the asymmetry of the amino acid residues of LH-RH and / or a replacement thereof to a derivative which is much less effective than LH-RH itself; See, for example, Y. Hirotsu, Biochem.Biophys. Res. Cotnmun., 5-9_, 277 (1975). In this context it has [D-Ser j-LH-RH was also shown to have less than 5% of the LH-RH activity of the natural hormone.

The properties of the peptides of the formula I according to the invention are of practical importance. The lesser minimally effective Dose reduces side effects as well as manufacturing costs of the compound, and the longer duration of action reduces the need for multiple administrations.

The compounds according to the invention are selected from the group of compounds of the formula I.

(Pyro) -Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z I wherein

a) X = Ser, Y = D-Trp and Z = GIy-NH ₂ ; or

b) X = Ser, Y = D-Phe and Z = GIy-NH ₉ ; or

c) X = D-Ser, Y = D-Leu and Z = NHR, where

R = lower alkyl,

or a non-toxic, pharmaceutically acceptable or usable salt thereof;

a compound of formula II

R ⁸ - (pyro) -Glu-His (N ^ -R ⁷ ) -ΤΓρ-Χ ^α -αγΓ (R ⁵ ) -Y-Leu-Arg (N ^G -R ⁴ ) -Pro-Z ¹ wherein

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a) X ¹ = Ser (R ⁶ ) [Y = D-Trp and Z ¹ = GIy-R ² ; or

b) X ¹ = Ser (R ⁶ ), Y = D-Phe and Z ¹ = GIy-R ² ; or

c) X ¹ = D-Ser (R ⁶ ), Y = D-Leu and Z ¹ = OR ³ , wherein

2 3

R = amino or O- (lower-alkyl), R = lower-alkyl, R, R, R and R = protecting groups that can be split off by one or more chemical treatments that the molecule (pyro) -Glu-His-Trp-X.-Tyr-Y-Leu-Arg-Pro-Z, wherein X, Y and Z are as defined above, do not attack,

and R = hydrogen or one of the protecting groups; and compounds of the formula

R ^ CpyroJ-Glu-His-Trp-D-SeriR ⁶ ) -Tyr (R ⁵ ) -D-Leu-Leu-Arg (N ^G -R ⁴ ) -Pro-NHR ¹

where R = lower alkyl, R ¹ , R, R and R = protecting groups that can be removed by one or more chemical treatments that affect the molecule
(pyro) -Glu-His-Trp-D-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHR not an-

attack, and R = hydrogen or one of these protecting groups.

According to a preferred embodiment, the compounds of formula II and the compound of formula

R ⁸ - ^> yiX)) - Glu-4iLs-Itp-D-Ser (R ⁶ ) -Tyr (R ⁵ ) -D-Leu-Leu-Arg (N ^G -R ⁴ ) -Pro-NHR ¹

12 3 4

R _1, R and R as defined above, R = a protective group for the N -, Ν ^ω - and N ^ω -nitrogen atoms of arginine, selected from the group of tosyl, nitro, benzyloxycarbonyl and adamantyloxycarbonyl; R = a protecting group for the hydroxyl group of tyrosine, selected from the group of 2-bromobenzyloxycarbonyl, benzyl, acetyl, tosyl, benzoyl, tert-butyl, tetrahydropyran-2-yl, trityl, 2,4-dichlorobenzyl and benzyloxycarbonyl; R = a protective group for the hydroxyl group of serine,

5 7

selected from the group defined above for R; R = a protecting group for the imidazole nitrogen atoms of histidine, selected from the group of tosyl and dinitrophenyl; and R = hydrogen or an α-amino protecting group selected from Group of tert-butyloxycarbonyl, benzyl'oxycarbonyl, cyclopentyloxycarbonyl, tert-amyloxycarbonyl and d-isobornyloxy-

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H 132 carbonyl.

Another feature of the invention relates to intermediates, which are bound to a solid resin support. These intermediates are through the formulas

R ⁸ - (pyro) -GIu-His (N ^Irn -R ⁷ ) -Trp-X ^ Tyr (R ⁵ ) -Y-Leu-Arg (N ^G -R ⁴ ) -Pro-Z ² , R ⁹ -Hi s (N ^Im -R ⁷ ) -Trp-X ^a -Tyr (R ⁵ ) -Y-Leu-Arg (N ^G -R ⁴ ) -Pro-Z ² , R ⁹ -Trp-X ¹ -Tyr (R ⁵ ) -Y-Leu-Arg (N ^G -R ⁴⁾ -Pro-Z ^2, and R ⁹ _X ¹ _Tyr (R shown ⁵⁾ -Y-Leu-Arg (N ^G -R ⁴⁾ -Pro-Z ² wherein

a) X ¹ = Ser (R ⁶ ), Y = D-Trp and Z ² = GIy-A; or

b) X ¹ = Ser (R ⁶ ), Y = D-Phe and Z ² = GIy-A; or

c) X ¹ = D-Ser (R ⁶ ), Y = D-Leu and Z ² = A ¹ ;

wherein R, R, R, R and R are as defined above,

R = a person skilled in the art for the stepwise synthesis of polypeptides known α-amino protecting group - suitable groups are listed below - and A and A = anchoring bonds used in the synthesis in the solid state bound to a solid resin support. A is selected from the class of:

^{Resin 1} carrier)

and 0

and

A ¹ = 0

CH,

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The term "lower-alkyl" as used herein is intended to mean alkyl radicals denote and include methyl, ethyl, propyl and isopropyl.

N = the side chain nitrogen atoms of arginine.

Tm '

N = the imidazole nitrogen atoms of histidine. The symbol f & means "phenyl".

In general, the abbreviations used here to designate the amino acids and protective groups are based on the recommendations of the IUPAC-IUB commission for biochemical nomenclature, cf. Biochemistry 1Λ, 1726 (1972). For example t-Boc = tert-butyloxycarbonyl, Z = benzyloxycarbonyl, Tos = tosyl, 2-Br-Cbz = 2-bromobenzyloxycarbonyl, BzI = benzyl and Dnp = 2,4-dinitrophenyl. The abbreviations used here for the various amino acids are Arg = arginine, GIy = glycine, His - histidine, Leu = leucine, Pro = proline, (pyro) -Glu = 5-oxoproline (pyroglutamic acid), Ser = serine, Trp = tryptophan and Tyr = tyrosine. All amino acids described here belong to the L series, unless stated otherwise, ie D-Ser = a D-Seryl radical, D-Leu = a D-Leucyl radical, D-Phe = a D-phenylalanyl radical and D-Trp = a tryptophyl radical .

The peptides of the formula I according to the invention can be obtained in the form of acid addition salts. Examples of such Salts are the salts with organic acids, e.g. acetic acid, lactic acid, succinic acid, benzoic acid, salicylic acid, Methanesulfonic acid or toluenesulfonic acid, as well as polymeric acids, such as tannic acid or tannin or carboxymethyl cellulose, and salts with inorganic acids such as hydrohalic acids, e.g. hydrochloric acid, or sulfuric acid or Phosphoric acid. Optionally, a special acid addition salt can be converted into another acid addition salt, e.g. a salt with a non-toxic, pharmaceutically acceptable acid, by treatment with the appropriate ion exchanger

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resin in the manner described by RA Boissonnas et al., Helv.Chim.Acta, 43 , 1349 (1960). Suitable ion exchange resins are cellulose-based cation exchangers, for example carboxymethyl cellulose, or chemically modified crosslinked dextran cation exchangers, for example those of the Sephadex C type, and strongly basic anion exchange resins, for example those by JP Greenstein and M. Winitz in "Chemistry of the Amino Acids", John Wiley and Sons, Inc., New York and London, 1961, Volume 2, page 1456.

The peptides of the formula I and their salts have valuable long-term LH and FSH-releasing hormone activity.

The valuable LH- and FSH-releasing hormone activity and the long-lasting activity of the compounds according to the invention can be seen from standard pharmacological studies. For example, these activities can be illustrated by studies described by A. Arimura et al., Endocrinology, 9J5_, 1174 (1974). For example, according to the procedure described therein, LH release results obtained in rats given a dose of (50 ng, subcutaneous) of the compound show that the peptides of the formula I have peak activities about 2 hours after dosing and that there is still considerable effectiveness after up to 6 hours. In contrast, with the same dose of LH-RH (50 ng, subcutaneous), peak activity is achieved after about 15 minutes, and after 1 hour no effects of the injection can be observed. Integrated amounts of LH over a period of 6 hours also show that the compounds of the formula I [D-Ser, D-Leu, des-Gly-NH-J-LH-RH-ethylamide, [D-Phe J-LH- RH and [D-Trp J-LH-RH are 21, 20 and 12 times more active in releasing LH than LH-RH, respectively. The FSH release data after injections of the compounds of the formula I show that [D-Ser ⁴ , D-Leu ⁶ , desGly-NH ₂ ¹⁰ J-LH-RH-ethylamide, [D-Phe ⁶ ] -LH-RH and [D-Trp ⁶ J-LH-RH are about 11, 20 and 20 times more active than LH-RH at the same dose

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tion (50 ng).

It can also reduce the effectiveness of a compound of the formula I demonstrated on people:

Radioimmune tests of serum levels of LH after intranasal Administration show that the minimum effective dose of LH-RH is about 2.0 mg, whereas an equivalent one Efficacy at a dose of 0.5 ng [D-Trp J-LH-RH is achieved. Here, the compounds are administered to humans in a normal saline solution. In terms of Comparative studies show the release of FSH in humans of LH-RH 'and [D-Trp J-LH-RH are particularly noteworthy Results. Intranasal administration of up to 2.0 mg LH-RH has little or no effect on the through FHS serum levels measured by radioimmune testing, H.Go Dahlen et al., Horm. Metab. Res., 6_, 510 (1974). However put under the same conditions 0.5 mg [D-Trp J-LH-RH considerable Amounts of FSH free, i.e. amounts ranging from 0.3 to over 1.5 miu / ml. It can be seen that a connection which is capable of effectively releasing FSH, has many therapeutic uses, cf. for example H.G. Dahlen et al., Loc. Cit.

The LH and FSH release properties of the peptides of the Formula I, which in turn cause ovulation in animals, make the peptides useful for veterinary practice as well valuable for animal husbandry. It can often be desirable to limit the heat period in livestock facilities, for example cattle, Sheep or pigs, to run synchronously, either to all females of a group by a male To inseminate an animal with the desired genetic qualities, or to have an artificial insemination at a maximum To be able to carry out a number of large animals, both within a relatively short period of time. So far has this can be done by administering ovulation-inhibiting agents to the animals, discontinuing the administration of these agents

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shortly before the intended for fertilization or artificial insemination Time and trust in the natural production of LH and FSH to induce ovulation and estrus, or by administering gonadotropins. However, this was not entirely satisfactory as the ovulation increased a predetermined time never in all animals at the same time, but only in a certain proportion of them occurred when not using gonadotropic hormones. on the other hand made this method the high cost of gonadotropins and the side effects caused by their administration not practicable. It is now possible to have an essentially complete synchronization of ovulation and the estrus period by first treating the animals of a certain group with an ovulation inhibitor treated, which is then discontinued, and then a peptide of formula I just before that for fertilization or artificial insemination given for a certain period of time, followed by ovulation and to achieve heat at this point. The time to onset of ovulation and oestrus after administration of the peptide varies with the animal species, and the optimal time intervals are too for the particular species determine. In rodents such as rats or hamsters, for example, ovulation occurs within 18 hours Administration of a peptide according to the invention instead.

The above method to achieve ovulation and heat within a precisely predetermined time interval to ensure successful fertilization is particularly important for breeders of pedigree horses and show animals, where the cost of the services of a particularly outstanding male animal is often very considerable are.

The peptides of Formula I can also be useful for increasing the number of live births per gestation in livestock, for example by cattle, sheep or pigs. For this purpose the peptide is used in

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a range of parenteral dosages, preferably by intravenous or subcutaneous injections, ranging from 0.1 to 10 meg per kg of body weight per day, 96 to 12 hours before the expected heat with subsequent fertilization given. An introductory injection of 1000 to 5000 iU of gonadotropin serum from pregnant mares can also be used 1 or 2 days before the above injection of the peptide. A similar treatment with or without a previous one Treatment is also suitable for initiating puberty or the maturation period of pets.

If a peptide of the formula I is used to initiate ovulation and estrus or to initiate puberty in warm-blooded animals, especially used in rodents such as rats or hamsters, or in livestock, it is used systemically, preferably administered parenterally, in combination with a pharmaceutically acceptable liquid or solid carrier. The proportion of the peptide is determined by its solubility in the given carrier, by the chosen route of administration and determined according to standard biological practice. For parenteral administration to animals, the peptide is in a sterile aqueous Solution used that includes other dissolved materials, such as buffers or preservatives, as well as being sufficiently pharmaceutical compatible salts or glucose to make the solution isotonic. The dosage varies with the form of administration and the particular species of animal to be treated, and is preferably from 0.1 meg to 10 meg per kg of body weight. However, it is particularly desirable to have a dose level in the range of about 1 meg to about Apply 5 meg per kg of body weight for effective results.

The peptides of formula I can also be in one of the long-acting, slow-release or depot dosage forms, the described below, preferably by intramuscular injection or by implantation. Such dosage forms are designed to release about 0.1 meg to about 10 meg per kg of body weight per day.

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The peptides of the formula I are also useful in human medicine. For example, Kuman's chorionic gonadotropin (HCG), which contains mainly LH and some FSH, has been used for the treatment of certain endocrine disorders such as menstrual disorders, amenorrhea, poor development of secondary sexual characteristics and infertility in women, or certain cases of Hypogonadism, delayed puberty, cryptorchidism and non-psychogenic impotence in men, more confusing. Recently, female infertility has also been treated with human henopausal gonadotropin (HMG), which contains mostly FSH, followed by treatment with HCG. One of the disadvantages of this female infertility treatment with HCG or with HMG followed by HCG has been found in the fact that such treatment often results in superovulation and undesirable multiple births, probably because it is not possible to obtain just the exact amounts of FSH and to administer LH, which are necessary for ovulation.

The administration of a peptide according to the invention is the same these disadvantages, since the compound only controls the release of LH and FSH via the pituitary gland in precise amounts, necessary for normal ovulation. For these reasons, a peptide according to the invention is not only suitable for the above purposes, but also for use on women in the treatment of disorders of the Cycle, amenorrhea, hypogonadism and poor development of secondary sexual characteristics.

In addition, the peptides according to the invention are for contraception suitable. For example, if the peptide is given to women early in the menstrual cycle, so at this point LH is released and causes premature ovulation. The unripened ovum is either not suitable for fertilization or, if fertilization should take place anyway, it is very unlikely that the fertilized egg is implanted because the

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Estrogen-progestin balance required for the preparation of the endotetrium does not exist and the endometrium is not in the condition required for implantation. On the other hand, if the peptide is administered towards the end of the cycle, the endometrium is disrupted and the Menstruation occurs.

Also, the peptides of the invention are useful in contraception by the "rhythm" method, which is always relative What was unreliable was that it was not possible to predict the woman's ovulation with the required degree of accuracy. The administration of the peptide in the middle of the cycle, i.e. around the time normally expected ovulation, causes ovulation shortly thereafter and thus makes the "rhythm" method safe and effective.

The peptides of Formula I are also useful as diagnostic agents suitable for distinguishing between the functions or injuries of the hypothalamus or pituitary gland in women. In the Administering the peptide to a patient suspected of having such malfunction or injury and to a subsequently observed increase in LH content one has a good indication that the hypothalamus is the reason is for the malfunction and the pituitary gland is intact. On the other hand, it is used following the administration of the peptide no increase in circulating LH is observed, a diagnosis of malfunction or injury can be made with great certainty the pituitary gland.

In the male, administration of a peptide of Formula I creates the amounts of LH (or ICSH) and FSH that are required for a Normal sexual development is necessary and is necessary in cases of hypogonadism or delayed puberty useful in treating cryptorchidism. Furthermore the FSH released by the administration of the peptide stimulates the development of germ cells in the testes, and the peptide is suitable for the treatment of psychogenic and non-psychogenic impotence.

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If the peptides of the formula I, preferably in the form of a-23 Acid addition salt, used / ends in human medicine it systemically, either intravenously, subcutaneously, or by intramuscular injection, or sublingually, nasally, or vaginally in compositions together with a pharmaceutically acceptable one Vehicle or carrier administered.

For administration by the nasal route in the form of drops or sprays, preference is given to a peptide of the formula I dissolved in a sterile aqueous vehicle that also contains other dissolved ingredients, such as buffers or preservatives, as well as sufficient Amounts of pharmaceutically acceptable salts or glucose to achieve an isotonic solution. Dosages by the intranasal route are in the range of 0.1 to 50 mcg / kg or preferably 0.5 to 10 mcg / kg.

The peptides of the formula I can also be used as nasal or vaginal powders or insufflations. For such purposes the peptide is in finely divided solid form together with a pharmaceutically acceptable solid carrier, for example a finely divided polyethylene glycol ("Carbowax 1540"), finely divided lactose or, preferably for vaginal administration, very finely divided silicon dioxide ("Cab-O-Sil"), administered. Such compositions can also be others Excipients in finely divided solid form, such as preservatives, buffers or surfactants, contain.

For sublingual or vaginal administration, preference is given to formulating the peptides of the formula I in solid dosage forms, such as sublingual tablets or vaginal inserts or suppositories, with sufficient amounts of solid excipients such as starch, lactose, certain types of clay, buffers, lubricants, disintegrating agents or surfactants, or with semi-solid excipients common in the formulation of suppositories. Examples of such excipients can be found in the standard pharmaceutical literature, aB in Remington's Pharmaceutical Sciences, Mack Publishing Company _s Easton, Pa = _s 1970.

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The dosage of the peptides of the formula I depends on the form of administration and the specific patient being treated. In general, treatment is started at low levels Dosages that are well below the optimal dose of the compound. Then the dosages in in small steps until optimal effects are achieved according to the circumstances. In general it is desirable to administer the peptides of the formula I in a concentration which generally releases LH and FSH effectively, without giving harmful or adverse side effects, and preferably at a dosage in the range administered from about 0.01 meg to about 100 mcg per kg of body weight, although the above variations can be made. However, a dosage of the peptide is the Formula I, wherein

a) X = Ser, Y = D-Trp and Z = GIy-NH ₂ or

b) X = Ser, Y = D-Phe and Z = GIy-NH ₀

which range from about 0.5 meg to about 5.0 mcg per kg of body weight is, and the peptide of the formula I, wherein

c) X = D-Ser, Y = D-Leu and Z = NHR ¹ ,

those in the range from about 0.1 meg to about 10 mcg per kg of body weight is particularly preferred to achieve effective results.

It is often desirable to use the peptides of the formula I continuously over longer periods of time in long-acting, slow-releasing To be administered in forms or in depot dosage forms. Such dosage forms can be either pharmaceutical compatible salt of the compound having a low degree of solubility in body fluids, e.g. salts with pamoic acid or tannic acid or tannin or carboxymethyl cellulose, or they can contain the peptides in the form of a water-soluble salt, along with a protective Contain carrier that prevents rapid release. in the

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in the latter case, the peptides can, for example, with a non-antigenic, partially hydrolyzed gelatin in the form given to a viscous liquid; or you can adsorbed on a pharmaceutically acceptable solid carrier, v / ie for example zinc hydroxide with or without protamine and suspended in a pharmaceutically acceptable liquid vehicles are administered; or the peptides can be in gels or suspensions with a non-antigenic Protective hydrocolloid, e.g. sodium carboxymethyl cellulose, Polyvinylpyrrolidone, sodium alginate, gelatin, polygalacturonic acids, for example pectin, or certain mucopolysaccharides, together with aqueous or non-aqueous, pharmaceutically acceptable liquid vehicles, preservatives or surfactants. Examples of such formulations can be found in pharmaceutical Standard literature, e.g. in Remington's Pharmaceutical Sciences, loc. Cit. Long-acting, slow-release preparations the peptides can also be made of a pharmaceutically acceptable coating material by inclusion in microcapsules, for example gelatin, polyvinyl alcohol and ethyl cellulose can be obtained. Further examples of coating materials and the methods of microencapsulation are described by J.A. Herbig in "Encyclopedia of Chemical Technology", Volume 13, 2nd edition, Wiley, New York, 196 7, pages 436-456. Such formulations, as well as suspensions of salts of the Peptides that are only sparingly soluble in body fluids are adjusted to be about 0.1 meg to about Releasing 50 meg of the hormone per kg of body weight per day, and they are preferably administered by intramuscular injection. Alternatively, some of the solid dosage forms listed above may, for example, certain Poorly water-soluble salts or dispersions in or adsorbates on solid supports of salts of the peptides, for example Dispersions in a neutral hydrogel of a polymer of ethylene glycol methacrylate or similar monomers, which are cross-linked, as described in U.S. Patent 3,551,556 dated December 29, 1970, are in the form of pellets,

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which release approximately the same amounts as shown above and are implanted subcutaneously or intramuscularly can.

Alternatively, a slow release effect over long periods of time can also be achieved by administering the invention Peptides in the form of an acid addition salt in an intravaginal "device or in a temporary implant, for example a container made of non-irritating silicone polymers such as polysiloxane, e.g. "Silastic", or a neutral hydrogel of a polymer, as described above, is produced, which has a permeability, those capable of releasing from about 0.1 meg to about 50 meg per kg of body weight per day is required. Such intravaginal or implantable dosage forms for prolonged Administration v / have the advantage that they can be removed when it is desired to interrupt treatment or to quit.

The special side chain protective groups used in the synthesis of the peptides of the formula I according to the invention should be selected according to the following rules:

a) The protecting group should be stable towards the reagent and the reaction conditions at every stage of the synthesis, which are chosen to remove the α-amino protecting group,

b) the protective group should retain its protective effect, i.e. not split off under coupling or reaction conditions be, and

c) the protective group of the side chains should be at the end of the synthesis, which contains the desired amino acid sequence, under reaction conditions that do not change the peptide chain, be cleavable.

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With reference to R, suitable α-amino protecting groups include

1) aliphatic urethane protecting groups, for example tert-butyloxycarbonyl, Diisopropylmethoxycarbonyl, biphenylisopropyloxycarbonyl, isopropyloxycarbonyl, tert-amyloxycarbonyl, Ethoxycarbonyl, allyloxycarbonyl;

2) protecting groups of the cycloalkyl urethane type, e.g. cyclopentyloxycarbonyl, Adamantyloxycarbonyl, d-isobornyloxycarbonyl, Cyclohexyloxycarbonyl, nitrophenylsulfenyl, tritylsulfenyl, α, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl and trityl.

9 The α-amino protective group for R is preferably selected from tert-butyloxycarbonyl, Cyclopentyloxycarbonyl, tert-aryloxycarbonyl, d-isobornyloxycarbonyl, o-nitrophenylsulfenyl, biphenylisopropyloxycarbonyl and α, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl.

The peptides of the formula I according to the invention are used a solid phase synthesis, which is to be illustrated by the following embodiments, in which special peptides of formula I are produced.

a) and b) peptides of the formula I, in which

a) X = Ser, Y = D-Trp and Z = GIy-NH ₂ or

b) X = Ser, Y = D-Phe and Z =

The solid phase synthesis of the peptides of the formula I, which are described under a) and b), begins with the C-terminal End of the peptide using an α-amino protected Resin. Such a starting material is made by binding an α-amino-protected glycine to a benzhydrylamine resin, a chloromethylated resin or a hydroxymethyl resin, the former being preferred. the Production of a benzhydrylamine resin is for example by P. Rivaille et al., Helv ^ Chim. Acta, S4, 2772 (1971), and

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the manufacture of the hydroxymethyl resin is carried out by M. Bodans and J.T. Sheehan, Chem. Ind. (London), 38. » 1597 (1966). A chloromethylated resin is commercially available from Bio Rad Laboratories, Richmond, California. at Using the benzhydrylamine resin, an amide-anchoring bond with the α-amino-protected glycine becomes as follows educated:

H OH ^ -.

I I I / \

9 '™ il Har7- \

This makes it possible to use the C-terminal amide function directly after completion of the synthesis of the amino acid sequence to be obtained by removing the resin support from the bound Peptide with formation of the glycinamide at the C-terminal part of the desired decapeptide. In this case it will be when using hydrogen fluoride to split off the resin support, the protective groups of the side chain are also split off, the corresponding decapeptide of the formula I being obtained in which

a) X = Ser, X = D-Trp and Z = GIy-NH ₂ or

b) X = Ser, Y = D-Phe and Z =

When the other resins are used, the anchoring bond is the benzyl ester group as indicated above. In this case, there is a convenient procedure for Conversion of the bound protected peptide to the C-terminal amide in carrying the protected peptide off the resin Remove ammonolysis and then the protective groups the resulting amide by treating with sodium and liquid ammonia or by cleavage with hydrogen fluoride. An alternative method is cleavage by transesterification with a lower alkanol, preferably methanol or ethanol, in the presence of triethylamine and then converting the resulting ester to an amide

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and finally removal of the protective groups as above described. It is also here on J.M. Steward and J.D. Young, "Solid Phase Peptide Synthesis", W.H. Freeman & Co., San Francisco, 1969, pages 40 to 49.

In particular, according to one embodiment of the present invention, an α-amino-protected glycine, preferably tert-butyloxycarbonylglycine, with benzhydrylamine resin with the aid of the compound which activates carboxyl groups, preferably dicyclohexylcarbodiimide, coupled. Subsequent to the coupling of the a-amino-protected glycine to the resin, the α-Amino protective group, for example by trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone or hydrogen chloride acid removed in dioxane. The removal of the protecting group is carried out at a temperature between 0 ° C and room temperature carried out. Other standard cleavage agents and conditions for removing specific α-amino protecting groups can be used, for example by E. Schröder and K. Lübke, "The Peptides", Volume I, Academic Press, New York, 1965, pages 72-75.

After removal of the α-amino protecting group, the remaining a-amino-protected amino acids gradually in the desired Sequence coupled to form the peptide. Every protected amino acid is put into the solid phase reactor introduced in about a three-fold excess, and the coupling is carried out in a medium of methylene chloride or mixtures of Dimethylformamide and methylene chloride carried out. In cases where incomplete coupling occurs, the coupling operation becomes repeated before the α-amino protecting group is split off and before the coupling of the next amino acid to the amino acid bound to the polymer or the peptide he follows. The success of the coupling reaction at any stage of the synthesis is indicated by the ninhydrin reaction, as of E. Kaiser et al., Analyt.Biochem., 214_, 595 (1970), carried out.

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After the synthesis of the desired amino acid sequence the peptide from the resin support by treating with a reagent, such as hydrogen fluoride, which not only removes the peptide from the resin, but all remaining protecting groups of side chains and the a-amino protecting group (if present) on the pyroglutamic acid residue in the case where the Benzhydrylamine resin to obtain the peptide directly Formula I, described above as a) or b), was used.

If a chloromethylated resin is used, the peptide can be separated from the resin by transesterification with a lower alkanol, preferably methanol or ethanol, whereupon the product obtained is chromatographed on silica gel and the fraction obtained is subjected to a treatment with ammonia to convert the lower alkyl ester, preferably the methyl or ethyl ester, into the C-terminal amide. The side chain protecting groups are then protected by procedures as described above, for example by treatment with sodium in liquid ammonia or with hydrogen fluoride.

7 In addition to the protective groups (R) described above

7 for the imidazole nitrogen atoms of histidine, R can be 2,2,2-trifluoro-1-benzoyloxycarbonylaminoethyl and 2,2,2-trifluoro-lt-butyloxycarbonylaminoethyl include.

c) peptides of the formula I, in which

c) X = D-Ser, Y = D-Leu and Z = NHR ¹

Solid phase synthesis of the peptide of formula I wherein X = D-Ser, Y = D-Leu, and Z = NHR, is shielded from the C-terminal end of the peptide using an α-amino shield Proline resin started. Such a starting material is made by attaching an α-amino protected proline to a chloromethylated one Resin or a hydroxymethyl resin, the former being preferred. Making a

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Hydroxymethyl resin is described by M. Bodansky and J. T. Sheehan, Chem. Ind. (London), 3j3, 1597 (1966). A chloromethylated one Resin is commercially available from Bio Rad Laboratories, Richmond, California. When using the chloromethylated Resin, an ester anchor group is formed with the ct-amino protected proline as follows:

R - Pro - 0 - CH ₂ \ carrier

Resin \ .rager /

A convenient method of converting the bound protected Peptide into the C-terminal (lower alkyl) amide consists in cleavage of the protected peptide from the Kars by treatment with a lower alkylamine, cf. D.H. Ccy et al., Biochem., I Biophys. Res. Commun., 5J7_, 335 (1974), where the corresponding protected peptide (lower alkyl) amide receives. The protective groups of the resulting peptide (lower alkyl) amide are then removed by treatment with sodium and liquid ammonia or preferably by splitting hydrogen fluoride with formation of the corresponding peptide according to the invention of the formula I, as described above under c), removed. An alternative method is sapling by transesterification with a lower alkanol, preferably Methanol or ethanol, in the presence of triethylamine and subsequent conversion of the resulting ester into the corresponding (lower alkyl) amide and subsequent removal the protecting groups as described above. Cf. on this J.M. Steward and J.D. Young, "Solid Phase Peptide Synthesis", W.H. Freeman & Co., San Francisco, 1969, pages 40-49.

In particular, according to one embodiment of the present invention, a-amino-protected proline, preferably tert. Butyloxycarbonylproline, with a chloromethylated resin with Using a catalyst, preferably cesium bicarbonate or Triethylamine, coupled. After coupling the cc-amino-protected Prolins to the resin support is the oc-amino protecting group, for example using trifluoroacetic acid

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m / 17 132 - 23 -

re in methylene chloride, trifluoroacetic acid alone, or hydrogen chloride acid in dioxane. The cleavage of the protective group is carried out at a temperature between about 0 C and Carried out at room temperature. Other standard cleavage agents and conditions for removing specific α-amino protecting groups can be used as described by E. Schröder and K. Lübke, "The Peptides", Volume I, Academic Press, New York, 1965, pages 72 to 75 described. After removing the oc-Arnino-Schutsgruppe the remaining a-araino-protected α-amino acids coupled stepwise in the order described to give the compound of formula I, the was described above under c). Any protected amino acid is put into the solid phase reactor in about a three-fold excess introduced, and the coupling is carried out in a medium of methylene chloride or mixtures of dimethylformamide and methylene chloride carried out. In cases where incomplete pairing has occurred, the pairing process will before removing the α-amino protecting group, before coupling the next amino acid to the solid phase reactor. The success of the coupling reaction at each stage of the synthesis is determined by the ninhydrin reaction, as described by E. Kaiser et al., Analyt.Biochem. , 3_4_, 595 (1970).

After synthesis of the desired amino acid sequence, the protected peptide is removed from the resin support by treatment with a (lower alkyl) amine is removed to give the corresponding protected peptide (lower alkyl) amine, and even when using dinitrophenyl or tosyl as Protecting group for the histidyl residue is the dinitrophenyl or tosyl protecting group during treatment with the (low- Alkyl) amine removed. The peptide can also be from the resin be separated by transesterification with a lower alkanol, preferably methanol or ethanol, whereupon the obtained Product purified by chromatography on silica gel and the fraction recovered from treatment with a (lower-alkyl) amine to convert the lower alkyl ester, preferably the methyl or ethyl ester, 'to the C-terminal (lower alkyl) amide is subjected. (It should be noted that,

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m / 17 132 - 24 -

if a dinitrophenyl or tosyl group on the histidyl radical is present, this is also split off.) The remaining Side chain protecting groups of the protected ethylamide v / earth then by the procedures described above, for example by treatment with sodium in liquid ammonia or with hydrogen fluoride with formation of the nonapeptide of the formula I, as described above as c), cleaved off.

The following examples serve to illustrate the invention.

example 1

L-Pyroqlutamyl-L-histidyl (tosyl) -L-tryptophyl-L-seryl (benzyl) -L-tyrosyl (2-bromobenzyloxycarbonyl) -D-tryptor> hyl-L-leucyl-L-

'I H ■ ■■ ■ I I I "I - .1 .Jl IB I -" Ι! Γ Ι Γ "■■■■.! Ill I. I- I. Il

arqinyKtosyl) -L-prolylqlycylbenzhydrylamine resin [R - (pyro) - GIu-His- (N ^Im -R) -Trp-Ser (R ⁶ ) -Tyr (R) -D-Trp-Leu-Arq (N ^G - R ') - Pro-Gly-A; R ⁴ = Tos, R ^D = 2-Br-Cbz, R ⁶ = BzI, R ⁷ = Tos, R = H and A = benzhydrylamine resin]

1.25 g (1.0 mmol) of benzhydrylamine resin are placed in the reaction vessel of an automatic peptide synthesis device (Beckmann model 990), which is programmed to carry out the following '»washing cycles:

a) methylene chloride; b) 33% trifluoroacetic acid in methylene chloride (2 χ for 2.5 and 25 minutes, respectively); c) methylene chloride; d) ethanol; e) chloroform; f) 10% triethylarain in chloroform (2 χ 25 minutes each); g) chloroform; h) methylene chloride.

The washed resin is then with 525 mg (3.0 mmol) tert-Butyloxycarbonylglycine stirred in methylene chloride, and 3.0 mmol of dicyclohexylcarbodiiraxd are added. The mixture is stirred at room temperature (22 to 25 C) for 2 hours, and the amino acid resin is then successively washed with methylene chloride (3 x), ethanol (3 x) and methylene chloride (3 χ). The bound amino acid is treated with 33% trifluoroacetic acid in methylene chloride (2 χ

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split off from the protective group during 2.5 and 25 minutes, and then the washing cycles c) to h) described above are carried out.

The following amino acids (3.0 mmol) then ground
coupled one after the other through the same reaction cycle:
t-Boc-L-proline; t-Boc-L-arginine (Tos); t-Boc-L-leucine;
t-Boc-D-tryptophan; t-Boc-L-tyr-osine (2-Br-Cbz); t-Boc-L-serine (Bzl); t-Boc-L-tryptophan; t-Boc-L-histidine (Tos);
L- (pyro) -glutamic acid.

The finished decapeptide resin is washed 3 with methylene chloride and then 3 with methanol and dried under reduced pressure, 98 % of the theoretical weight increase being obtained.

The benzhydrylamine resin used in this example is a commercially available resin (1 % cross-linked, Bachern Inc., Marina del Rey, California).

Example 2

L-pyroqlutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-tryptophyl-L-leucyl-L-arqinyl-L-prolylqlycine amide; I, X -Ser, Y = D-Trp and Z = GIy-NH2 [(pyro) -GIu-His-Trp-Ser-Tyr- D-Trp-Leu-Arg-Pro-Gly-NH ₂ ]

The removal of the protective groups and cleavage of the decapeptide from the decapeptide resin, described in Example 1, is effected by treating 1.0 g of material with 24 ml of hydrogen fluoride and 6 ml of anisole for 30 minutes at 0 ° C. The hydrogen fluoride is removed under reduced pressure and the anisole is removed by washing with ethyl acetate.

The crude peptide is filtered through gel on a column
(2.5 χ 100 cm) of Sephadex G-25 (a fine-grained resp.
fine, chemically modified cross-linked dextran) purified by eluting with 2m acetic acid, and the frac-

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M / 17 132 - 26 -

tioneri collected and evaporated to dryness in vacuo, the showed a major UV absorption peak at 280 nm.

The remaining oil was on a column (2.5 χ 100 cm) of Sephadex G-25 (fine) applied previously with the lower phase followed by the upper phase of an n-butanol: acetic acid: water (4: 1: 5) solvent system had been equilibrated. Eluting with the upper phase gave a major peak fraction, and the material from this zone was placed on a column (1.4 94 cm) of carboxymethyl cellulose according to the methods of D.H. Coy et al., J. Med. Chem., 16_, 1140 (1973). Corresponding fractions (1050 to 1190 ml) gave D-Trp -LK-RH after lyophilization to constant weight as a white, fluffy powder (80 mg); [α] ™ "" -58.8 ° (c = 0, ^ 3, In-HOAc).

The product was found to be four in the thin-layer chromatogram different solvent systems as homogeneous if amounts applied from 20 to 30 meg and the stains by iodine vapor and then Ehrlich reagent were made visible. Man received the following R ^ values:

1-butanol: acetic acid: water (4: 1: 5, upper phase) 0.25; Ethyl acetate: pyridine: acetic acid: water (5: 5: 1: 3) 0.63; 2-propanol: 1 m-acetic acid (2: 1) 0.38;
1-butanol: acetic acid: water: ethyl acetate (1: 1: 1: 1) 0.51.

Result of the amino acid analysis:

GIu 1.08; His 0.95; Trp 2.00; Ser 0.94; ' Tyr 0.97; Leu 0.93; Arg 0.98; Per 1.00; GIy 1.02; NH ₃ 1.03.

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Example 3

L-pyroqlutamyl-L-histidyl (tosyl) -L-tryptophyl-L-seryl (benzyl) -L-tyrosyKP-brombenzyloxycarbonyD-D-phenylalanyl-L-leucyl-L- arginyKtosyl) -L-prolylglycylbenzhydryl ( pyro) -Glu- His- (N ^Im -R ^/ ) -Trp-Ser (R ⁶ ) -Tyr (R ⁵ ) -D-Phe-Leu-Arg (N ^G -R ⁴ ) -Pro-GIy-A; R ⁴ = Tos, R ⁵ = 2-Br-Cbz, R ⁶ = BzI, R ⁷ = Tps, R = H and A = benzhydrylamine resin]

1.25 g (1.0 mmol) of benzhydrylamine resin was added to the reaction vessel of an automatic peptide synthesis device (Beckmann model 990), programmed to carry out the following washing cycles:

a) methylene chloride, b) 33% trifluoroacetic acid in methylene chloride (2 χ for 2.5 and 25 minutes each), c) methylene chloride, d) ethanol, e) chloroform, f) 10% triethylamine in chloroform (2 for 25 minutes each), g) chloroform, h) methylene chloride.

The washed resin is then stirred with 525 mg (3.0 mmol) of tert-butyloxycarbonylglycine in methylene chloride and mixed with 3.0 mmol of dicyclohexylcarbodiimide. The mixture is stirred at room temperature (22 to 25 ° C) for 2 hours, and the amino acid resin is then successively 3 χ with methylene chloride, 3 χ with ethanol and 3 χ with methylene chloride washed. The bound amino acid is with 33% trifluoroacetic acid in methylene chloride (2 χ during each 2.5 and 25 minutes) freed from the protective group, and then the washing cycles c) to h) described above are carried out.

3.0 mmol of the following amino acids are then used one after the other coupled in the same reaction cycle: t-Boc-L-proline; t-Boc-L-arginine (Tos); t-Boc-L-leucine; t-Boc-D-phenylalanine; t-Boc-L-tyrosine (2-Br-Cbz); t-Boc-L-serine (Bzl); t-Boc-L-tryptophan; t-Boc-L-histidine (Tos); L- (pyro) -glutamic acid.

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The finished decapeptide resin is washed 3 with methylene chloride and then 3 with methanol and dried under reduced pressure, 100 % of the theoretical weight increase being obtained.

The benzhydrylamine resin used in this example is a commercial product (1% cross-linked, Bachern Inc., Marina del Rey, California).

Example 4

L-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D- phenylalanyl-L-leucyl-L-arginyl-L-prolylglycine amide; 1, X = Ser, Y = Phe and Z = Gly-NH- [(pyro) -GIu-His-Trp-Ser-Tyr-D-Phe-Leu-

Arg-Pro-Gly-NH ₂ ]

The removal of the protective groups and cleavage of the decapeptide of the resin described in Example 3 is obtained by treating 1.5 g of material with 24 ml of hydrogen fluoride and 6 ml of anisole carried out at 0 C for 30 minutes. The hydrogen fluoride is removed under reduced pressure and the anisole is removed by washing with ethyl acetate.

The crude peptide is by gel filtration on a column (2.5 χ 100 cm) Sephadex G-25 (fine-grade or fine, chemically modified cross-linked dextran), by eluting Purified with 2m acetic acid, and fractions showing a major UV absorption peak at 280 nm are collected and evaporated to dryness.

The remaining oil is applied to a column (2.5 χ 100 cm) of Sephadex G-25 (feii | i), which was previously mixed with the lower phase, followed by the upper phase of a n-butanol: acetic acid: water (4: 1: 5) solvent system, has been equilibrated. A major peak fraction was obtained by eluting the upper phase, and fractions of this peak were collected and concentrated to dryness. The residue was lyophilized from O, 2N acetic acid

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lized to give 158 mg of [D-Phe J-LH-RH as a fluffy white powder; [ap ^ ⁵ -57.5 ° (c = 0.55,0, In-HOAc).

The product was found to be homogeneous in the thin-layer chromatogram in four different solvent systems when applied from 20 to 30 meg and stains made visible by iodine vapor and then Ehrlich's reagent. The following R _f values were obtained:

1-butanol: acetic acid: water (4: 1: 5, upper phase) 0.14; Ethyl acetate: pyridine: acetic acid: water (5: 5: 1: 3) 0.68; 2-propanol: 1 m-acetic acid (2: 1) 0.41; 1-butanol: acetic acid; water: ethyl acetate (1: 1: 1: 1) 0.47.

The amino acid analysis showed:

GIu 1.01; His 0.97; Trp 0.90; Ser 0.92; Tyr 1.00; Phe 0.96; Leu 1.00; Arg 1.02; Pro 0.95; GIy 1.00; NH ₃ 1.00.

Example 5

L-Pyroglutamyl-L-histidyl (dinitrophenyl) -L-tryptophyl-D-seryl-

(benzyl) -L-tyrosyl (2-bromobenzyloxycarbonyl) -D-leucyl-L-leucyl-

L-arginyl (tosyl) -L-prolyl-0-CH ₂ -Hars, R ⁸ - (pyro) -Glu-His (N ^Irn -R ⁷ ) -Trp-D-Ser (R ⁶ ) -Tyr (R ⁵ ) -D-Leu-Leu-Arg (N ^G -R ⁴ ) -Pro-A ¹ ; R ⁴ = Tos,

Hars- ⁵ = 2-Br-Cbz, R ⁶ = BzI, R ⁷ = Dnp, R ⁸ = H and A ¹ = ⁰ - ^CH 2 "~ \ er ~ /

1.40 g (0.5 mmoles of proline) Boc-proline resin of the formula

Boc-Pro-0-CHp

are placed in the reaction vessel of an automatic peptide synthesis device (Beckmann model 990), which is programmed to carry out the following washing cycles:

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a) methylene chloride, b) 33 % trifluoroacetic acid in methylene chloride (2 χ each 2.5 or 25 minutes), c) methylene chloride, d) ethanol, e) chloroform, f) 10 % triethylamine in chloroform (2 χ each 5 minutes) , g) chloroform and h) methylene chloride.

The washed resin is then stirred with 645 mg (1.5 mmol) of tert-butyloxycarbonyl-tosyl-arginine in methylene chloride, and 1.5 mmol of dicyclohexylcarbodiimide v / earth is added. The mixture is stirred at room temperature (22 to 25 ° C) for 2 hours, and the amino acid resin is then washed successively 3 χ with methylene chloride. The bound amino acid is freed from the protective group with 33% trifluoroacetic acid in methylene chloride (2 × 2.5 or 25 minutes), and then the washing cycles c) to h) described above are carried out.

1.5 mmol of the following amino acids are then used one after the other coupled by the same reaction sequence: t-Boc-L-1eucine; t-Boc-D-leucine; t-Boc-L-tyrosine (2-Br-Cbz); t-Boc-D-serine (BzI); t-Boc-L-tryptophan; t-Boc-L-histidine (Dnp); L- (pyro) -glutamic acid.

The finished nonapeptide resin is washed 3 with methylene chloride and then 3 with methanol and dried under reduced pressure, 96 % of the theoretical weight increase being obtained.

The proline resin used in this example is a commercially available one chloromethylated resin (1% cross-linked, Bio Rad Labs., Richmond, California).

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Example 6

L-Pyroqlutamyl-L-histidyl-L-tryptophyl-D-seryl (benzyl) -L-tyrosyl ^ -brombenzyloxycarbonyD-D-leucyl-L-leucyl-L-arqinyl- (tosyl) -L-prolylethylamide, R - (pyro ) -GIu-His-Trp-D-Ser (R) - ■ Tyr (R) -D-Leu-Leu-Arg (N ^ R ⁴ J-PrO-NHR ¹ ; R ⁴ = Tos, R ⁵ = 2- Br-Cbz, R ⁶ = BzI, R ⁸ = H, and R ¹ = C ₃ H ₅

2.16 g of the protected nonapeptide resin from Example 5 are suspended in 20 ml of ethyl arain at OC and stirred for 6 hours. Excess ethylamine is then allowed to evaporate at room temperature and the cleaved peptide is washed from the resin with dimethylformamide. The protected peptide is then precipitated by the addition of ethyl acetate and filtered to give 672 mg of an off-white powder. R _f on silica gel in 1-butanol: acetic acid: water (4: 1: 5, upper phase) = 0.45. This material is used in Example 7 without further purification.

Example 7

L-Pyroqlutamyl-L-histidyl-L-tryptophyl-D-seryl-L-tyrosyl-D-leucyl-L-leucyl-L-arqinyl-L-prolyl-ethylamide, 1, X = Ser _y Y = D-Leu and Z = NHEt [(pyro) -GIy-His-Trp ~ D-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHE t]

The removal of the protecting groups from the protected nonapeptide, prepared as described in Example 6 is made by treating 670 mg of the material with 50 ml of hydrogen fluoride and 15 ml of anisole for 30 minutes at 0 ° C. The hydrogen fluoride is removed under reduced pressure and the anisole removed by washing with ether.

The crude peptide is purified by gel filtration on a column (2.5 χ 100 cm) of Sephadex G-25 (fine-graded or fine, chemically modified cross-linked dextran) by eluting Purified with O, 2m-acetic acid, and fractions containing a Showing major UV absorption peak at 280 nm are collected and evaporated to dryness.

609852/108 2

The remaining oil is on a column (2.5 χ 100 cm) of Sephadex G-25 (fine) applied previously rcit the lower Phase followed by the upper phase of n-butanol: acetic acid: water (4: 1: 5) was equilibrated as the solvent system. By eluting the upper phase, a main fraction with a high UV absorption at 280 nm is obtained, and this material is chromatography on a column (1.5 94 cm) of silica gel and eluted with a mixture of 1-butanol: acetic acid: water (4: 1: 1). Corresponding political groups (300 to 390 ml) gave after evaporation and lyophilization of water to constant weight [D-Ser, D-Leu, desGly-NH-] -

LH-RH-ethyamide as a white, fluff-like powder of 103 mg; [a] ^ ³ = -29.6 ° (c = 0.54, 0, In-HOAc).

The product proves to be homogeneous in the thin-layer chromatogram in four different solvent systems on silicon dioxide gel plates when 20 to 30 megabytes are applied and the spots are made visible with iodine vapor, followed by Ehrlich's reagent. The following R _f values were obtained:

1-butanol: acetic acid: water (4: 1: 5, upper phase) 0.20; Ethyl acetate: pyridine: acetic acid: water (5: 5: 1: 3) 0.72; 2-propanol: Im-acetic acid (2: 1) 0.43;
1-butanol: acetic acid: water: ethyl acetate (1: 1: 1: 1) 0.50.

The amino acid analysis showed:

GIu 1.03; His 0.93; Trp 1.01; Ser 0.82; Tyr 0.97; Leu 1.98; Arg 1.00; Pro 0.90; Ethylamine 0.98.

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Claims (1)

Claims 11. / Peptides of the formula I (pyro) -GIu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z (I) wherein a) X = Ser, Y = D-Trp, and Z = GIy-NH,; or b) X = Ser, Y = D-Phe and Z = GIy-NPI ₂ ; or c) X = D-Ser, Y = D-Leu and Z = NHR ¹ , wherein 1
R = lower alkyl, or a non-toxic, pharmaceutically acceptable or usable salt thereof. 2. L-Pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-tryptophyl-L-leucyl-L-arginyl-L-prolylglycine amide or a non-toxic, pharmaceutically acceptable addition salt thereof according to claim 1. 3. L-Pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-phenylalanyl-L-leucyl-L-arginyl-L-prolylglycine amide or a non-toxic, pharmaceutically acceptable addition salt thereof according to claim 1 4. L-Pyroglutamyl-L-histidyl-L-tryptophyl-D-seryl-L-tyrosyl-D-leucyl-L-leucyl-L-arginyl-L-prolylethylamide or a non-toxic, pharmaceutically acceptable addition salt thereof according to claim 1. 5. Compound of Formula II R ⁸ - (pyro) -GIu-Hi s (N ^Im -R ⁷ ) -Trp-X ¹ -Tyr (R ⁵ ) -Y-Leu- Arg (N ^ R ⁴¹ 609852/1082 wherein a) X ¹ = Ser (R ⁶ ), Y = D-Trp and Z ¹ = GIy-R ² ; or b) X ¹ = Ser (R ⁶ ), Y = D-Phe and Z ¹ = GIy-R ² ; or c) X ¹ = D-Ser (R ⁶ ), Y = D-Leu and Z ¹ = OR ³ , wherein 2 3 R = amino or O- (lower-alkyl), R = lower-alkyl, R, R, R and R = protecting groups created by one or more chemical treatments that affect the molecule (Pyro) -Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z (I) not attack, can be split off, in which a) X = Ser, Y = D-Trp and Z = GIy-NH ₂ ; or b) X = Ser, Y = D-Phe and Z = GIy-NH ₂ ; or c) X = D-Ser, Y = D-Leu and Z = NHR ¹ , wherein 1 8 R = lower alkyl and R = hydrogen or one of these Protecting groups. 6. Compound of formula II according to claim 5, wherein X = r Ί P? Ser (R), Y = D-Trp and Z = GIy-R, where R = amino. 7. A compound of the formula II according to claim 5, wherein X = Ser- (R ⁶ ), Y = D-Phe and Z ¹ = GIy-R ² , wherein R ² = amino. X = C. Compound of formula II according to claim 5, wherein Ser (R ⁶ ), Y = D-Trp and Z ¹ = GIy-R ² , wherein R ² = amino, 4 5 6 R = tosyl, R = 2-bromobenzyloxycarbonyl, R = benzyl, V 8 R = tosyl and R = hydrogen 9. A compound of the formula II according to claim 5, wherein X is Ser (R ⁶ ), Y = D-Phe and Z ¹ = GIy-R ² , wherein R ² = amino, 4 5 6 R = tosyl, R = 2-bromobenzyloxycarbonyl, R = benzyl, 7 8 R = tosyl and R = hydrogen. 609852/1082 10. Compound of Formula
R ⁸ - (pyro) -Glu-HLS-Trp-D-Ser (R ⁶ ) -Tyr (R ⁵ ) -I> -Leu-Leu-Arg (N ^G -R ⁴ 3- "1 Λ ζ C ^ R where R = lower alkyl, R, R, R and R = protecting groups, which can be cleaved by one or more chemical treatments that affect the molecule (pyro) -Glu-His-Trp-D-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHR do not attack and R = hydrogen or one of these protective groups. 11. A compound according to claim 10, wherein R = ethyl. 12. Compound of Formula II R ⁸ - (pyro) -Glu ^ Üs (N ^Irn -R ⁷ ) -Trp-X ^a ~ Tyr (R ⁵ JY- Leu- Arg (N ^G -R ⁴ J-Pro-Z ¹ wherein a) X ¹ = Ser (R ⁶ ), Y = D-Trp and Z ¹ = GIy-R ² ; or b) X ¹ = Ser (R ⁶ ), Y = D-Phe and Z ¹ = GIy-R ² ; or c) X ¹ = D-Ser (R ⁶ ), Y = D-Leu and Z ¹ = OR ³ , wherein R = amino or O- (lower-alkyl); R ³ = lower alkyl; R ⁴ = a protective group for the N ** ", N ^w and N ^w 'nitrogen atoms of arginine from the group of tosyl, nitro, benzyloxycarbonyl and adamantyloxycarbonyl; R = a protective group for the hydroxyl group of tyrosine from the group of 2-bromobenzyloxycarbonyl, Benzyl, acetyl, tosyl, benzoyl, tert-butyl, tetrahydropyran-2-yl, Trityl, 2,4-dichlorobenzyl and benzyloxycarbonyl; R = a protecting group for the hydroxyl group of serine from the group defined above for R; 7th
R = a protective group for the imidazole nitrogen atoms of histidine from the group of tosyl and dinitrophenyl; 609852/1082 Vi / 11 132 - 36 - R = hydrogen or an α-amino protecting group the group of tert-butyloxycarbonyl, benzyloxycarbonyl, Cyclopentyloxycarbonyl, tert-amyloxycarbonyl and d-isobornyloxycarbonyl. 13. Compound of Formula R ⁸ - (Pyro) -Glu-HLs-Trp-D-Ser (R ⁶ ) -Tyr (R ⁵ ) -D-Leu-Leu-Arg (N ^G -R ⁴ ) -Pro-N HR ¹ wherein 1
R = lower alkyl; R * = a protective group for the N -, N ^ω - and N ^c0 - nitrogen atoms of arginine from the group of tosyl, nitro, benzyloxycarbonyl and adamantyloxycarbonyl; R = a protecting group for the hydroxyl group of tyrosine from the group of 2-bromobenzyloxycarbonyl, Benzyl, acetyl, tosyl, benzoyl, tert-butyl, tetrahydropyran-2-yl, Trityl, 2,4-dichlorobenzyl and benzyloxycarbonyl; R = a protecting group for the hydroxyl group of serine from the group defined above for R; and · R = hydrogen or an α-amino protecting group from the Group of tert-butyloxycarbonyl, benzyloxycarbonyl, Cyclopentyloxycarbonyl, tert-amyloxycarbonyl and d-isobornyloxycarbonyl. 1 4 14. A compound according to claim 13, wherein R = ethyl, R = tosyl, R = 2-brorabenzyloxycarbonyl, R = benzyl and R = hydrogen. 15. Compound selected from the group of -rVttp-X ^a -Tyr (R ⁵ JY-LeU- Arg (N ^G -R ⁴ ) -Pro-Z ² R ⁹ -His (N ^Im -R ⁷ ) -Trp-X ^ Tyr (R ⁵ ) -Y-Leu-Arg (N ^G -R ⁴ ) -Pro-Z ² , R ⁹ -Trp-X ¹ -Tyr ( R ⁵⁾ -Y-Leu-Arg (N ^G -R ⁴⁾ -Pro-Z ^2, and R ⁹ -X ^a -Tyr (R ⁵⁾ -Y-Leu-Arg (N ^G -R ⁴⁾ -Pro-Z ² 609852/1082 M / 17 132 wherein a) X ¹ = Ser (R ⁶ ), Y = D-Trp and Z ² = GIy-A; or b) X ¹ = Ser (R ⁶ ), Y = D-Phe and Z ² = GIy-A; or c) X ¹ = D-Ser (R ⁶ ), Y = D-Leu and Z ² = A ¹ , wherein where R, R, R and R = protecting groups, which are represented by a or several chemical treatments can be split off, which the corresponding compound of formula I (pyro) -GIu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z (I) do not attack in what a) X = Ser, Y = D-Trp and Z = GIy-NH ₂ ; or b) X = Ser, Y = D-Phe and Z = GIy-NH ₂ J or c) X = D-Ser, Y = D-Leu and Z = NHR ¹ , Ί 8 where R = lower alkyl, R = hydrogen or a 9
α-amino protective group, R = an α-amino protective group, A = selected from the group of H I I I N - C ρ and 0 , and A ¹ = 0 CH 1 6 16. A compound according to claim 15, wherein X = Ser (R), Y = D-Trp and Z = GIy-A, where A = a benzhydrylamine resin. 17. A compound according to claim 15, wherein X = Ser (R), Y = 2
D-Phe and Z = GIy-A, where A = a benzhydrylhydrylamine resin. 18. A compound according to claim 15 having the formula R ^ - (pyro) -GIu-HiS (N ^Irn ~ R ⁷ ) -Trp-X ¹ -Tyr (R ⁵ ) -Y-Leu- Arg (N ^G -R ⁴ ) -Pro-Z ² 609852/1082 M / 17 132 - 38 - where X = Ser (R ^ü ), Y = D-Trp and Z = GIy-A, where R ⁴ = 5 fs 7th Tosyl, R = 2-bromobenzyioxycarbonyl, R = benzyl, R = Tosyl, R = hydrogen and A = a benzhydrylamine resin. 19. A compound according to claim 15 having the formula R ⁸ - (pyro) -Glu-His (N ^ -R ^ -Trp-X ^ Tyr (R ⁵ ) -Y-Leu-Arg (N ^G -R ⁴ ) -Pro -Z ² where X ¹ = Ser (R ⁶ ), Y = D-Phe and Z ² = GIy-A, where R ⁴ = tosyl, R = 2-bromobenzyloxycarbonyl, R = benzyl, R = tosyl, R = hydrogen and A = a Benzhydrylamine resin. 20. A compound according to claim 15 having the formula R ⁸ - (pyro) -Glu-His (N ^Im -R ⁷ } -Trp-X ¹ -Tyr (R ⁵ ) -Y-Leu-Arg (N ^G -R ⁴ ) -Pro-Z ² where X ¹ = D-Ser (R ⁶ ), Y = D-Leu and Z ² = A ¹ , where R ⁴ = tosyl, R = 2-bromobenzyloxycarbonyl, R = benzyl, R = 8 1 2,4-Dinitrophenyl, R = hydrogen and A ^- = 0-CHp 21. Process for the preparation of a compound of formula I according to claim 1, characterized in that one i) a compound of the formula
R ⁸ - (pyro) -Glu-His (N ^{: i: tn} -R ⁷ ) -Trp-X ¹ -Tyr (R ⁵ ) -Y-Leu-Arg (N ^G -R ⁴ ) -Pro-Z ² where X = Ser (R), Y = D-Trp and Z = GIy-A, where A = HH S -N ^ I I / resin- \ 4 5 6 7 N-C-p- Iträgeri and R *, R, R and R = protective gravity> - pen that can be cleaved off by one or more chemical treatments that are not appropriate Attack compound of formula I, and R = hydrogen or one of these protective groups, reacts with a reagent, which is suitable for this _s the Sc ^ -r.-grupr.-an E '% R ^, R, R 8th "* and R without / - «riariff ds: * '^; ^ ί ,: r..uvr the formula I under formation the corresponding compound of the formula I 609852/1082 (pyro) -GIu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z wherein X = Ser, Y = D-Trp and Z = GIy-NH ₂ , to be split off; or ii) a compound of formula II R ⁸ - (pyro) -Glu-His (N ^ -R ^ -Trp-X ^ Tyr (R ⁵ ) -Y-Leu-Arg (N ^G -R ⁴ J-Pro-Z ¹ where X ¹ = Ser (R ⁶ ), Y = D-Trp and Z ¹ = GIy-R ² , where R ² = amino and R, R, R and R are protecting groups which can be split off by one or more chemical treatments that the corresponding compound of formula I is not attack, and R = hydrogen or one of these protective groups, with a reagent which protects the groups R, R, R, R and R without attacking the compound of the formula I can split off to form a compound of the formula I (pyro) -GIu-Hi s-Trp-X-Tyr-Y-Leu-Arg-Pro-Z where X = Ser, Y = D-Trp and Z = GIy-NH ₂ , to give; or iii) a compound of the formula
R ⁸ - (pyro J-GIu-HiS (N ¹ ^ -R ⁷ ) -Trp-X ^a -Tyr (R ⁵ ) -Y-Leu-Arg (N ^G -R ⁴ ) -Pro-Z ² where X ¹ = Ser (R ⁶ ), Y = D-Phe and Z ² = GIy-A, where A = and R, R, R and R protecting groups that can be split off by one or more chemical treatments that result in the corresponding compound dung of the formula I do not attack, and R is hydrogen or one of these protective groups, reacts with a reagent, which can split off the protective groups R, R, R, R and R without attacking the compound of formula I, wherein the corresponding compound of formula I. (pyro J-GIu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z (I) 609852/1082 2S2S843 where X = Ser, Y = D-Phe and Z = GIy-NH ₂ ; or iv) a compound of formula II R ⁸ - (pyro) -Glu-His (IT ^ -R ⁷ ) -Trp-X ^ Tyr (R ⁵ ) -Y-Leu-Arg (N ^G -R ⁴ ) -Pro-Z ¹ where X ¹ = Ser (R ⁶ ), Y = D-Phe and Z ¹ = GIy-R ² , where R ² = amino and R, R, R and R are protecting groups which can be split off by one or more chemical treatments , which the corresponding compound of formula I do not attack, and R = hydrogen or one of these protective groups, reacts with a reagent that protects the protective groups R, R, R, R and R can be removed without attacking the compound of formula I, the corresponding Compound of formula I. (pyro) -Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z where X = Ser, Y = D-Phe and Z = GIy-NH ₂ ; or v) a compound of the formula
R ⁸ - (pyro) -G] u-His-Tr.pD- £ er (R ⁶ ) -Tyr (R ⁵ ) -D-Leu-Leu-Arg (N ^G -R ⁴ J-Pro-NHR ¹ where R = lower-alkyl and R, R, R and R are protective groups that can be split off by one or more chemical treatments that result in the corresponding bond of formula I do not attack, and R is hydrogen or one of these protective groups, reacts with a reagent, which can split off the protective groups R, R, R and R without attacking the compound of the formula I, wherein one the corresponding compound of formula I (pyro) -GIu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z where X = D-Ser, Y = D-Leu and Z = NHR, where R = lower alkyl. 22. The method according to claim 21, characterized in that the means for splitting off the protective group is hydrogen fluoride used. 609852/1082 23. Medicaments with customary formulation agents and carriers, characterized in that there are one or more Compounds of claims 1 to 20 contains. 609852/10 82