DE2625843C2 - Nona- and decapeptide amides, processes for their preparation and pharmaceuticals containing these compounds - Google Patents

Description

^C)

and their pharmacologically acceptable salts.

2. L-Pyroglutamyl-L-histidyl-L-tryptophyl-D-seryl-L-tyrosyl-D-leucyl-L-Ieucyl-L-arginyl-L-proline ethylamide

and its pharmacologically acceptable salts.

3. A process for the preparation of a compound of the formula I according to claim I or 2, characterized in that a compound of the general formula is used in a manner known per se

with the following substituent meaning and assignment

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

b) X ¹ = SertR "), Y = D-Phe and Z ¹ = GIy-R ² ; in which R ⁴ , R ⁵ , R ⁶ and R ^{7 represent} protecting groups, and

R ^{8 represents} a hydrogen atom or a protecting group;

or of a compound of the general formula R ^ pyroGIu-HisiN ^ -

with the following substituent meaning and assignment

c) X '= Ser (R ⁶ ), Y = D-Trp and Z ² = GIy-A; or d) X ¹ = Ser (R "). Y = D-Phe and Z ² = GIy-A;

wherein R ⁴ . R \ R \ R ⁷ and R * are defined above, and A door

^{H H}

C ₆ H ₃

stands,

or of a compound of the general formula

R'-pyroGlu-His-Trp-D-SertRVTyrlR'j-D-Leu-Lcu-ArgiN ^ -R'VPro-NHR ¹

wherein R 'denotes a CV to Cj-alkyl group and R ⁴ , R \ R "and R ^{7 represent} protective groups,

splits off the protecting groups.

Ni 4. Medicines with customary formulations and carriers, containing one or more compounds

applications of claims 1 to 2.

The invention relates to nona and decapeptide amides of the formula I.

pyroGlu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z (I)

with the following definition and assignment of substituents:

Scr I) IVp GIy-NII, Scr I) -PhC CiIy-NlI, D-Ser D-Leu NH (C ₁ - to CVAIkyl)

and their pharmacologically acceptable sal / c.
These peptides of the formula I are also referred to as:

a) L-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-tryptophyl-L-leucyl-L-arginyl-L-prolylglycine amide,

b) L-pyroglutamyl-L-histidyl-L-tryplophyl-L-scryl-L-tyrosyl-D-phenylalanyl-L-leucyl-L-arginyl-L-prolylglycine amide,

c) L-Pyroglutymal-L-histidyl-L-tryptophyl-D-scryl-L-tyrosyl-D-leucyl-L-leucyl-L-arginyl-L-proline (Ci- to C _r alkyl) amide

: o and can with the abbreviation

a) [D-Trp ^fc | -LH-RH,

b) ID-PhC ¹¹ I-LH-RILbZW.

c) [D-Ser \ D-Lcu \ desGly-NH} "l-LH-RII- (C, to C _r alkyl) amide

are designated.

Both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are gonadotropic Hormones produced by the pituitary gland in humans and mammals. LH stimulates together with FSH induces the release of estrogens from the maturing follicles in the ovaries and in the females Organism the ovulation process. In the male organism, LH stimulates the interstitial cells or the Leydig incidents and is therefore also interstitial cell stimulating hormone (ICSH) called. FSH induces the maturation of the follicles in the ovaries and, together with LH, plays an important role Role in the cycle of the female organism. FSH promotes the development of the male germ cells Testicles. Both LH and FSH are released from the pituitary gland by the action of the LH- and FSH-releasing 3 Hormone is released, and there is clear evidence that this releasing hormone is in the hypcthalamus is formed and reaches the pituitary gland by a neurohumoral route, cf. e.g. A.V. Schally et al .. Recent Progress in Hormone Research, 24, 497 (1968).

The natural LI I and FSH releasing hormone has been isolated from the I lypothalamus of pigs, and its structure has been described by A. V. Schally et al., Hiochcm. Res. Commun., 43,393 and 1334 (1971) who the decapeptide structure

pyroGlu-His-Trp-Ser-Tyr-Gly-Lcu-Arg-Pro-Gly-NH,

proposed. _{4 <}

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).

In the following the natural LH- and FSH-frcisetzcndc hormone LH-RH is called.

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 proven to be more active than LH-RH, for example [D-AIa ⁶ I-LH-RH, A. Arimura et al. Endocrinology, 95, 1174 (1974), (D- LeU ⁶ J-LH-RH and [D-Leu ⁶ , desGly-NH2 "I-LH-RH-ethylamide, JA Vilchez-Martine / et al., Biochem. Biophys. Res. Commun., 59, 1226 (1974) In most cases, modified peptides have proven to be less active.

It has now been found that

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

h) the Krsal /. of the glycyl residue in the 6-position of LII-RH by D-phenylalanine; or _M >

c) the replacement of the L-acryl residue in the 4-position of LH-RH by a D-acryl residue, replacement of the glycyl residue in

6-position by a D-leucyl residue and replacement of the glycine amide residue in 10-position by a C, - to C, -

Alkylamide group,

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

According to one embodiment of the invention, it has been found that the change in the asymmetry of the scryl radical in the 4-position together with the introduction of a D-Lcucylrcsts in the 6-position to an increased effectiveness and prolonged effectiveness of the Pcptidsdcr formula I, wherein X = D-Scr. Y = D-Lcu and

Ser D-Trp GIy-NH ₂ Ser D-Phe GIy-NH ₂ D-Ser D-Le u NH (Cj- to C ₃ -ASkVi)

Z = NHR ¹ , which is surprising, especially since, in general, changing the asymmetry of the amino acid residues of LH-RH and / or replacing it results in a derivative that is significantly less potent than LH-RH itself; See, for example, Y. Hirotsu, Biochem. Biophys. Res. Comm., 59, 277 (1975). In this context, it has also been shown that [D-Ser ⁴ J-LH-RH has less than 5% of the LH-RH activities of the natural hormone.

The properties of the peptides of the formula I according to the invention are of practical importance. The minor one minimal effective dose reduces side effects as well as the cost of making the compound, and the longer duration of action reduces the need for multiple administrations.

The invention relates to nona- and decapeptide amides of the general formula I:
lu

pyroGlu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z (I)

with the following substituent meaning and assignment:

and their pharmacologically acceptable salts.

L-pyroglutamyl-L-histidyl-L-tryptophyl-D-seryl-L-tyrosyl-D-leucyl-L-leucyl-L-arginyl-L-proline ethylamide are preferred and its pharmacologically acceptable salts.

The compounds according to the invention can be obtained by in a manner known per se from a compound of the general formula

R * -pyroGlu-His (N ' ⁿⁱ -R ⁷ ) -Trp-X'-Tyr (R ⁵ ) -Y-Leu-Arg (N ^t: -R -') - Pro-Z ^l

with the following substituent meaning and assignment

a) X '= SeitR "). Y = D-Trp and Z' = GIy-R ² ; or

b) X ¹ = side R "). Y = D-Phe and Z ¹ = GIy-R ² ; wherein
R ⁴ , R \ R "and R ⁷ mean Schulz groups, and

R * denotes a water loft or a protective group;

or of a compound of the general formula
40

with the following substituent meaning and assignment

c) X ¹ = Ser (R "), Y = D-Trp and Z ² = GIy-A; or
d) X ¹ = Ser (R "), Y = D-Phe um! Z ² = GIy-A;

wherein R ⁴ , R ⁵ , R ⁽¹ , R ⁷ and R ⁸ are defined above, and A is
H

C ₁₁ H ₅

stands,

or of a compound of the general formula

R * -pyroGlu-His-Trp-D-Ser (R ") -Tyr (R ⁵ ) -D-Leu-Leu-Arg (N ^t: -R ⁴ ) -ProNHR ^l

wherein R ^{1 is} a C ₁ - to Cj-alkyl group

and
R \ R ⁵ , R "and R ⁷ mean protecting groups,

splits off the protecting groups.

H ^A denotes a protective group for N "-. N" - and N '"- nitrogen atoms of arginine, selected from the group of tosyl, Ni'ry, benzyloxycarbonyl and adamantyloxycurbonyl. R ⁵ denotes a Schulz group Pur, the hydroxyl group of tyrosine, selected from the group of 2-bromobenzyloxycarbonyl, benzyl, acetyl, tosyl, benzoyl, terl-bulyl, tetrahydropyran-2-yl, trityl, 2,4-l) dichlorobenzene and bonyloxycurbonyl; R ^h is a

Protective group for the hydroxyl group of serine, selected from the group defined above for R ". R ⁷ is a protective group for the imidazole nitrogen atoms of histidine, namely a tosyl or dinitrophenyl group. R * denotes a hydrogen atom or σ-amino protective group, selected from the group of tert-butyloxycarbonyl, benzyloxycarbonyl, cyclopentyloxycarbonyl, tert-amyloxycarbonyl and d-isobornyloxycarbonyl.

When using the solid phase method, the connections are connected via the anchorage bond A. bound to a solid resin support.

The term "C | - to C, -Alkyl" denotes alkyl radicals with 1 to 3 carbon atoms and includes methyl, Ethyl, propyl and isopropyl.

N ° = the side chain nitrogen alomc of arginine. N ^lm = the imidazole nitrogen atoms of histidine.

The symbol 0 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, see Biochemistry 11, 1726 (1972). For example t-Boc - tcrt.-Butyloxycarbonyl, Z - Benzyloxycarbonyl.Tos = Tosyl. 2-Br-Cbz = 2-bromobenzyloxycarbonyl, BzI = benzyl and Dnp = 2,4-dinitophenyl. The abbreviations used here for the various amino acids are Arg - Arginine, GIy - Glydir ,, His = Hisiidin, Leu = Leucine. Pro = proline, (pyro) -Glu = 5-oxoproline (pyrolglutamic acid), Scr = serine, Trp = tryptophan and Tyr = tyrosine. All amino acids described here belong to the L series, unless otherwise stated, ti. H. D-Ser = a D-Seryirest, D-Lcu = a D-Leucylrest, D-Phe ·· = a D-Phenylalanylrest and D-Trp = a Tryptophylrest.

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. B. 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 J5 Tannin or carboxymethyl cellulose, and salts with inorganic acids such as hydrohalic acids, e.g. B.!

Hydrochloric acid, or sulfuric acid, or phosphoric acid. Optionally, a special acid addition salt can be converted into another acid addition salt, e.g. B. a salt with a toxic, pharmaceutically acceptable acid, by treatment with the appropriate ion exchange resin according to the by R. A. Boissonnas et al., HeIv. Chim. Ac'a, 43, 1349 (1960). Suitable ion exchanger jo Resins are cation exchangers based on cellulose, 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 J. P. 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-acting LH- and FSH-reducing hormone-J5 Effectiveness.

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, 95, 1174 (1974). For example, according to the procedure described there, LH release l-rgcbniss.dican rats which were administered a dose of (50 ng, subcutaneously) of the compound show that the peptides of the formula I have peak activities about 2 hours after dosing have 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-Lcu \ des-Gly-NHj ⁿ ] -LH-RH-ethylamide, [D-Phe "| -LH -RH and [D-Trp ^ l-LH-RH 21-, 20- and 12 times more active when releasing LH than LH-RH. The FSI I release data after injections of the compounds of the formula I show that [ D-Ser \ D-Leu ", desGly-NH.!" | -LH-RH-ethylamide, [D-PhC ⁶ J-LH-RH and [D-TTp ⁶ I-LH-RH about 11-, 20- and are 20 times more active than LH-RH at the same dose (50 ng).

In addition, the effectiveness of a compound of the formula I can be demonstrated in humans:

Radioimmune studies of studies 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 effectiveness with a dose of 0.5 mg [D-Trp''J- LH-RH is achieved. Here, the compounds are administered to humans in a normal saline solution. With regard to the release of FSH in humans, comparative studies of LH-RH and [D-Trp ^A ] -LH-RH give particularly remarkable results. The intranasal administration of up to 2.0 mg LH-RH has little or no effect on the FHS serum levels measured by radioimmune testing, HG Dahlen et al .. Horm. MeUb. Res., 6,510 (1974). However, under the same conditions, 0.5 mg of [D-Trp ⁶ ] -LH-RH release considerable amounts of FSH, ie amounts ranging from 0.3 to over 1.5 miU / ml. It can be seen that a compound which is capable of effectively releasing FSH has many therapeutic uses, see, for example, HG Dahlen et al., Loc. Cit . _:

The LH and FSH breakdown properties of the peptides of the formula I, which in turn stimulate ovulation in Tic-I

effect, make the peptides valuable for veterinary practice as well as for livestock breeding. It ■ '

can often be desirable nen the Brunstperiode in cattle poses, such as cattle, sheep or pigs 65 ^ i, synchronously to run, either to all females of a group / u hcsamen by a male animal with desirable genetic qualities or to use artificial insemination at a maximum Ϊ--

to be able to carry out a number of female animals, both within a relatively short period of time ':, ^;

Period of time. So far this has been done by administering ovulalionsinhibierer.den agents to the animals, discontinuing the administration of these agents shortly before the intended fertilization or artificial insemination Time and reliance on the natural production of LH and FSH to induce ovulation and estrus, or by administering gonadotropins. However, this was not entirely satisfactory, because ovulation at a predetermined time never occurs in all animals at the same time, but only in some of it occurred when not using gonadotropic hormones. On the other hand, the high cost of the gonadotropic and side effects caused by its administration made it Method not practicable. It is now possible to have an essentially complete synchronization of the To achieve ovation and the period of estrus, by dal.! the animals of a certain group first

in treated with an Ovulatinnsinhinitor, which is then discontinued, and then a peptide of formula I administered shortly before the fertilization or artificial insemination period, in order to avoid ovulation and To achieve heat at this point. The time until the final ovulation and heat after Administration of the peptide varies with the animal species, and dicopiimalcn time intervals are different for each Identify species. In rodents such as rails or hamsters, for example, ovulation takes place within

: ^; \ on 18 hours after administration of a certain peppers.

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

The peptides of the formula can also be used to increase the number of live births per pregnancy in livestock, for example of cattle, sheep or pigs. For this Purpose is using the peptide in a range of parenteral dosages, preferably by intravenous or subcutaneous injections, ranging from 0.1 to 10 meg per day, up to 12 hours before the expected oestrus subsequent fertilization given. An introductory injection of 1000 to 5000 μl of a gonadotropin

:> Serum from pregnant mares can also be given 1 or 2 days before the above injection of the peptide will. Similar treatment with or without previous treatment is also used to initiate the Suitable for puberty or the maturation period of pets.

If a peptide of the formula I is used to initiate ovulation and estrus or to lead to the filling of puberty Warm-blooded animals, especially rodents such as rails or hamsters, or used in livestock, like this

It is administered systemically, preferably parenterally, in combination with a pharmaceutically acceptable liquid or a solid carrier. The proportion of the peplide is given by its solubility in the Tri.j.er, determined by the chosen route of administration and according to standard biological practice. For parenteral administration to animals, the peptide is used in a sterile aqueous solution, as is others dissolved materials, such as pullers or preservatives, as well as sufficiently pharmaceutically acceptable

'· <May contain salts or glucose to make the solution isoionic. The dosage varies with the form the administration and the particular species of animal to be treated, and is preferably 0.1 meg to

H) meg per kg of body weight. However, it is particularly desirable to have a dose range in the range of about 1 meg

up to about 5 meg per body weight must be used to achieve effective results.

The peptides of the formula I can also be used in one of the long-acting, slow-releasing or Depol-Dosic-

JH forms described below, preferably by intramuscular injection or by Implantation. Such dose cylinders are designed to be about 0.1 mcg to about Release 10 meg per hg of body weight per day. The peptides of the formula I are also useful in human medicine. For example, the human chorionic gonadoiropin (UCG) was also used. that mainly LH and something FSH has been used for the treatment of certain endocrine disorders such as menstrual cycle disorders for over 30 years.

• 4> Amenorrhea, development of secondary sexual characteristics and sterility in the Woman, or certain cases of hypogonadism, delayed puberty, cryptorchidism and more non-psychogenic Male impotence. In the last line, the sterility of women also became human Menopausal gonadotropin (HMG) containing primarily FSII followed by treatment with HCG. One of the disadvantages of this infertility treatment is followed by women with HC (J or with I IMG

> u of HCG, it was shown that with such a treatment often a superovular ion and undesirable Multiple births occur, likely because it is not possible to only use the exact amounts of FSH and To administer LH, which are necessary for ovulation.

The administration of a peptide according to the invention compensates for these disadvantages, since the compound > ii> ung son LH and FSH via the hypohysc only in the exact amounts required for normal ovulation.

5> which are caused. For these reasons, a peptide according to the invention is not only for the above

Purposes, but also for use on women in the treatment of menstrual disorders,

amenorrhea, ilypogonadism, and deficient development of secondary sexual characteristics suitable.

In addition, the peptides according to the invention are suitable for contraception. For example, this will be

M) Peptide administered to women / u early in the menstrual cycle, LI I is released at that time and causes premature ovulation. The uncultivated ovum is either unsuitable for Fertilization, or if fertilization does occur, it is very unlikely that that will fertilized egg is implanted because the estrogen-progesin balance required for the preparation of the fndometrium is not present and the fndometrium is not in the state required for the implantation

to is located. On the other hand, if the peptide is given towards the end of the cycle, the endometrium is disrupted and menstruation occurs.

The peptides according to the invention are also useful in contraception by the »Rhythmusu-Melhode, which was always relatively unreliable that it is not possible to ovulate the woman with the required sex

degree of precision to be determined beforehand. The administration of the peptide in the middle of the cycle, i.e. H. about that Normally to be expected cell point of the ovulalion, shortly afterwards causes an ovulation and thus shapes the »RhythmusH method safe and effective.

The peptides of formula I are also useful as a diagnostic means for differentiating between functions or injuries to the I lypothalamus or I! ypophysc of the woman. When administering the peptide to> one patient suspected of having such malfunction or injury, and one afterward observed increase in the Ll l content, one has a good indication that the lypothalamus is the reason for failure and the pituitary gland is intact. On the other hand, if following the administration of the Pcptids no increase in circulating LII is observed, a diagnosis of malfunction or injury to the pituitary gland can be made with a high degree of certainty. in

In the male, the administration of a l'eptide of the formula I creates the amounts of LII (or ICSH) and an FSH, which are necessary for normal sexual development in conditions of hypogonadism or delayed puberty, and is also useful in the treatment of cryptorchidism. In addition, it stimulates through Administration of the FSH peptide released the development of germ cells in the testes, and the peptide is suitable for the treatment of psychogenic and non-psychogenic impotence. : «

If the peptides of the formula I, preferably in the form of an acid addition salt, are used in human medicine, they are administered systemically, either intravenously, subcutaneously or by intramuscular injection or sublingually, nasally or vagina! in compositioneü / ussmmef! with egg pharmaceutically acceptable vehicle or carrier administered.

For administration by the nasal route in the form of drops or sprays, a peptide of the formula I is preferred, dissolved in a sterile aqueous vehicle, which can also contain other dissolved components such as buffers or preservatives, as well as sufficient amounts of pharmaceutically acceptable salts or glucose to achieve an isotonic solution. Dosages by intranasal route are in the range \ onO.l to 50 meg / 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 applied in an oil-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 used by other excipients in divided solid form, such as Preservatives, buffers or surfactants.

For sublingual or vaginal administration, the formulation of the peptides of the formula I in is preferred 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 used in the formulation of Suppositories are common. Examples of such excipients can be found in the standard pharmaceutical literature, e.g. in Remington's Pharmaceutical Sciences, Mack Publishing Compony. Easlon, Pa., 1970.

The dosage of the peptides of the formula I depends on the administration route and on the specific patient to be treated. In general, treatment is started with small doses that are significantly below the optimal dose of the compound. Then the dosages are increased in small slides, until optimal effects appropriate to the circumstances are achieved. In general, it is desirable that the To administer peptides of the formula I in a concentration which is generally effective in releasing LH and FSH, without giving harmful or adverse side effects, and preferably in a dosage im In the range of about 0.01 meg to about 100 meg per kg of body weight, although the above variations can be made. However, a dosage of the peptide of formula 1, wherein

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

b) X = Scr, Y = D-Phe and Z = GIy-NII ,,

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

O X = D-Scr, Y = D-Leu and Z = NH- (C, - to C, -alkyl)

which is in the range from about 0.1 meg to about 10 meg per kg of body weight, particularly preferably used, to achieve effective results.

It is often desirable to use the peptides of the formula I continuously over long periods of time in long-acting, to be administered in slow release forms or in depot dosage forms. Such dosage forms can either be a pharmaceutically acceptable salt of the compound having a low degree of solubility in body fluids, for example 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, together with a contain protective carrier that prevents rapid release. In the latter case, the peptides for example with a non-antigenic, partially hydrolyzed gelatin in the form of a viscous liquid administered; or they can be on a pharmaceutically acceptable solid carrier such as Zinc hydroxide, with or without protamine, adsorbed and suspended in a pharmaceutically acceptable liquid vehicle; or the peptide ": can -n gels or suspensions with a non-antigenic protective hydrocolloid, for example nairium carboxymethylccIIulose, polyvinylpyrrolidone, sodium algi- nat. Gelatin, plygalacturonic 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

to zj

scher standard literature./. See, for example, Remington's Pharmaceutical Sciences, supra, Long-Acting, Slow-Release Preparations of the peptides can also be packed in microcapsules from a pharmaceutically acceptable Überzugmalcrial. for example gelatin, polyvinyl alcohol and ethyl cellulose. Further Examples of coating materials and the methods of microencapsulation are described by J. A. Ilerbig in "Encyclopedia of Chemical Technology". Kand 13, 2. A ullage, Wiley, New York, 1967, ropes 43 * »to 456 described. Such formulations as well as suspensions of salts of the peptides found in body fluids are only sparingly soluble, they are adjusted so that they are about 1 meg to about 50 meg of the hormone per kg of body weight per day, and they are preferably administered by intramuscular injection. Alternatively For example, some of the solid dosage forms listed above may be poor 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 methaerylal or the like Monomers that are cross-linked, as described in US-PS 35 51 556, are in the form of pellets, which release approximately the same amounts as shown above and implanted subcutaneously or intramuscularly can be.

i> Alternatively, a slow-release effect can also be achieved over long periods of time by administration of the Crfindunys-like peptides in the form of an acid addition salt in an intravaginal device or in a temporary implant such as a container made from non-irritating silicone polymers such as Polysiloxane, /. B. "Silastic", or a neutral hydrogel of a polymer, as described above, prepared is, which has a permeability, the / ur release of about 0.1 meg to ei «ä '0 meg per kg of body

: u weight per day is required. Such intravaginal or implantable dosage forms for prolonged administration have the advantage that they can be removed when it is desired to interrupt or terminate the treatment.
With reference to R ″, suitable α-amino groups include

1) aliphatic ^ urethane protecting groups. for example lert.Bulyloxycarbonyl, Diisopmpylmethoxycarbonyl, Biphenylisopropyloxycarbonyl, isopropyloxycarbonyl, lert-amyloxycarbonyl, ethoxycurbonyl, allyloxycarbonyl;

2) Cycloalkyl urethane-type protecting groups e.g. B. cyclopentyloxycarbonyl, adamantyloxycarbonyl, d-isobornyloxyearbonyl, Cyc'ohexyloxycarbonyl, Nitrophenylsulfcnyl, Trilylsulfcnyl, <r. <7-Dimethyl-3,5-Dim-

yo thoxybenzyloxycarbonyl and trityl.

Preference is given to choosing the a-amino protecting group for V? from tert-hulyloxycarbonyl. Cyclopentyloxycarbonyl, lert-amyloxycarbonyl, d-isobornyloxycarbonyl, o-nitrophenylsulfenyl, biphenylisopropyloxycarbonyl and aa-dimethylO.S-dimethoxybenzyloxycarbonyl.

The peptides of the formula I according to the invention are produced using a solid phase synthesis, to be illustrated by the following embodiments.

a) and b) Petide of the formula I. wherein

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

The solid phase synthesis of the peptides of the formula I, which are described under a) and b), is started with the C-terminal end of the peptide using an α-amino-protected resin. Such a starting material becomes a chloromethylated one by binding of a σ-amino-protected glycine to a benzhydrylamine resin Resin or a hydroxymethyl resin, the former being preferred. The production a benzhydrylamine resin is described, for example, by P. Rivaillc et al., HeIv. Chim. Acta, 54, 2772 (1971), and the manufacture of the hydroxymethyl resin is described by M. Bodans and J.T. Sheehan, Chem. Ind. (London), 38, 1597 (1966). A chloromethylated resin is available in the UK from Bio Rad Laboratories. at The use of the benzhydrylamine resin creates an amide-anchored bond with the ff-amino-protected Glycon formed as follows:

R ⁹ - N - CH, - C-NH-C-CH ₄ -

C ₆ H,

This makes it possible to use the C-terminal amide function directly after the synthesis of the amino acid sequence has been completed obtained by cleaving the resin support from the bound peptide to form the glycinamide at the C-terminal part of the desired decapeptide. In this case, when using hydrogen fluoride to split off the resin carrier also split off the protective groups of the side chain, whereby the corresponding decapeptide of formula 1, in which,

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

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

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 converting the protected peptide bound in the C-endständigc amide therein, the protected peptide from the resin by ammonolysis to remove and then Remove the protective groups of the resulting amide by treatment with sodium and liquid ammonia or by cleavage with hydrogen fluoride. An alternative method consists in cleavage by transesterification with a low-alcohol alkanol, preferably methanol or ethanol, in the presence of triethylamine and subsequent conversion of the resulting ester into an amide and finally removal of the protective groups, as described above. Reference is also made here to JM Steward and JD Young, "Solid Phase Peptide Synthesis", W. II. 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, is coupled with benzhydrylamine resin / with the aid of the compound which activates carboxyl groups, preferably dicycloxylcarbodiimide. Subsequent to the coupling of the e-amino-protected cilycine to the compound, the or-amino-Schulz group is removed, for example, by trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone or hydrochloric acid in dioxane. The decision fernung the protecting group is carried out at a temperature of betwee 0 ⁰ C and room temperature. Other standard cleavage agents and conditions for removing specific σ-amino protecting groups can be used, such as, for example, by E. Schröder and K. Lübke, "The Peptides", Volume I, Academic Press, New York, 1965, pages 72 to 75 described.

After removal of the a-amino protecting group, the remaining s-amino-protected amino acids become coupled stepwise in the desired order to form the peptide. Any protected amino acid is introduced into the solid phase reactor in about three-fold excess, and the coupling is in one Medium of methylene chloride or mixtures of dimethylformamide and methylene chloride carried out. In In cases where incomplete coupling occurs, the coupling operation is repeated before the α-amino protecting group is cleaved off and before the coupling of the next amino acid to that on the polymer bound amino acid or the peptide takes place. The success of the coupling reaction at every stage of the synthesis is achieved by the ninhydrin reaction as described by E. Kaiser et al., Analyt. Bioehem., 34,595 (1970).

After the synthesis of the desired amino acid sequence, the peptide is cleaved from the resin support by treatment with a reagent, such as hydrogen fluoride, which not only cleaves the peptide from the resin, but all remaining protective groups of chains and the α-amino protective group (if present) pyroglutamic acid in the case where the benzhydryl amine resin is used to obtain the peptide directly Formula I, described above as a) or b), was used.

If a chloromethylicrtcs resin is used, the peptide can be removed from the Har / by transesterification with a lower alkanol, preferably methanol or ethanol, are separated off, whereupon the product obtained Chromatographed silica gel and the fraction obtained is subjected to a treatment with ammonia in order 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 above described, for example by treatment with sodium in liquid ammonia or with hydrogen fluoride, cleaved.

In addition to the protective groups (R ⁷ ) described above for the imidazole-slickslol talome of histidine, R ⁷ can include 2,2,2-trinuor-1-benzoyIoxycarbonyluminoethyl and 2,2,2-trifluoro-1-tert.-butyloxycarbonylaminoethyl.

c) peptides of formula I, in which c) X = - D-Scr, Y = IM.cu and Z - NII (C, - to C, -alkyl)

Solid phase synthesis of the peptide of formula I, wherein X - D-Ser, Y = D-Leu and /. = NH (C, - to C · - <o alkyl), is started from the C-terminal end of the peptide using a <r-amino-shaped proline resin. Such a starting material is produced by binding an o-amino-coated proline to a chloromethyl resin or a hydroxymethyl ether, the former being preferred. The preparation of a hydroxymethyl resin is described by M. Bodansky and JT Shcchan, Chcm. Ind. (London), .18. 1597 (1966). A chloromethylated Hurz is commercially available from Bio Rad Laboratories, Richmond. California. When using the chloromethylated resin, an ester anchor group is formed with the σ- amino-protected proline as follows:

R «_p _ro _0-CH

A convenient method of converting the bound protected peptide to the C-terminal (Lower-alkyl) -amide consists in the cleavage of <lcs protected peptide from the resin by treatment with a lower alkylamine, see D. H. Coy et al., Uiochem., Biophys. Res. Commun., 57,335 (1974). where one the corresponding protected peptide (lower alkyl) amide is obtained. Then the protecting groups of the resulting Pcptid- (lower-alkyl) -umids by treatment with sodium and liquid ammonia or preferably by hydrofluoric acid cleavage to form the corresponding peptide according to the invention

Form] I, as described above under c), removed. An alternative process consists in cleavage 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 (C ₁ to C ₃ alkyl) amide and subsequent removal of the protective groups, as described above. Cf. on this, JM Steward and JD Young, "Solid Phase Peptide Synthesis", WH Freeman & Co., San Francisco, 1969, pages 40 to 49. <

In particular, according to one embodiment of the present invention, <r-amino-protected proline, preferably tert-butyloxycarbonylproIine. with a chloromethylated resin with the aid of a catalyst, preferably cesium bicarbonate or triethylamine, coupled. After coupling the α-amino-protected pro- The α-amino protecting group is removed from the resin support, for example using trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone or hydrochloric acid in dioxane. The splitting off of the protective group is carried out at a temperature between about 0 ^° C. and room temperature. Other standard cleavage agents and conditions for removing specific ff-amino protecting groups can be used, as described by E. Schröder and K. Lübke, "The Peptides", Volume J, Academic Press, New York, 1965, pages 72 to 75 described. After removal of the α-amino protecting group, the remaining σ-amino-protected amino acids coupled stepwise in the order described to give the compound of formula I, which was described above under c). Every protected amino acid is in the solid phase reactor is introduced in about a three-fold excess, and the coupling is in a medium carried out by methylene chloride or mixtures of dimethylformamide and methylene chloride. In cases where an incomplete pairing has occurred, the Konpeluiigsvorgar: g _before removing the ü-Amino protective group, before coupling the next amino acid to the solid phase reactor, repeated. Of the

Success of the coupling reaction at every stage of the synthesis is determined by the ninhydrin reaction, as described by E. Kaiser et

al., Analyt. Biochem., 34, 5995 (1970).

After synthesis of the desired amino acid sequence, the protected peptide is removed from the resin support

removed by treatment with a (C, - to C, -alkyl) -amine, whereby the corresponding protected peptide- (Cr to Cj-alkyl) -amide is obtained, and also when using dinitrophenyl or tosyl as a protective group for the histidyl radical the dinitrophenyl or tosyl protecting group is removed during treatment with the (C _r to Cy alkyl) amine. The peptide can also be separated from the resin by transesterification with a "lower alkanol", preferably methanol or ethanol, whereupon the product obtained is separated by chromatography Purified on silica gel and the fraction obtained from a treatment with a (C ₁ - to Cj-alkyl) amine to convert the lower-alkyl ester, preferably the methyl or ethyl ester, into the C-terminal (C ₁ - to Cj-alkyl) -amid is subjected. (It should be noted that if a dinitrophenyl or tosyl group is present on the histidyl radical, this is also cleaved off.) The remaining scite chain protecting groups of the protected ethylamide are then removed by the procedures described above, both 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 I.

A) L-Pyroglutamyl-L-histidyl (tosyl) -L-tryptophyl-L-seryl (bcnzyl) -L-tyrosyl (2-bronchinzyloxycarbonyl) -

D-tO'ptophyl-L-leucyl-L-arginylOosyO-L-prolylglycylbenzhydrylamine resin

[R'-ipyroj-Glu-llis-iN ^ -R'j-Trp-SerfR ^ -TyriR ^ -D-Trp-Leu-ArgiN ^ -R-'i-Pro-Gly-A;

R ⁴ = Tos. R ⁵ = 2-Br-Cbz, R "= BzI, R ⁷ = Tos, R * = H and A» benzhydrylamine resin)

1.25 g (1.0 mmol) of benzhydrylamine resin are added to 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 X for 2.5 and 25 minutes respectively); c) methylene chloride; d) ethanol; e) chloroform; 010% triethylamine in chloroform (2 x 25 minutes each); g) chloroform; h) methylene chloride.

The washed resin is then treated with 525 mg (3.0 mmol) of tert-butyloxycarbony / glycine in methylene chloride stirred, and 3.0 mmol of dicyclohexylcarbodiimide are added. The mixture is at room temperature (22 to 25 ° C) stirred for 2 hours, and the amino acid resin is then successively with Methylene chloride (3 x), ethanol (3 x) and methylene chloride (3 x). The bound amino acid will cleaved from the protective group with 33% trilluoroacetic acid in methyl chloride (2 x for 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) are then sequentially through the same reaction cycle coupled: t-Boc-L-proline; t-Boe-L-arginine (Tos); t-Boc-L-lcucine; t-Boc-D-lryplophane; t-Boc-L-tyrosine (2-Br-Cbz); t-Boc-L-scrin (Bzl); l-Boc-L-lryplophane; l-Boc-L-hislidine (Tos); L-pyroglutuminic acid c.

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

The / hydrylaminhar / used in this example is a commercially available liar /. (I% cross-linked).

B) L-pyroglutamyl-L-histidyl-L-tryptophyl-L-scryl-L-tyrosyl-D-tryptophyl-L-leucyl-L-arginyl-L-prolylglycine amide; 1, X = Ser, Y = D-Trp and Z = GIy-NII, | (pyro) -Glu-llis-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NII ₂ ]

The removal of the protective groups and cleavage of the decpcptide from the Decapcplkl-Ilar / ...

under A) is effected by treating 1.0 g 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 through Wash away with ethyl acetate.

The crude peptide is purified by gel filtration on a column (2.5 x 100 cm) of Sephadex ^β G-25, eluting with 2M acetic acid, and the fractions were collected and evaporated to dryness in vacuo, which has a main UV- Showed absorption peak at 280 nm.

The remaining oil was applied to a column (2.5 Χ 100 cm) of Sephadex * G-25 (fine), which was previously treated with the lower phase, followed by the upper phase of a n-butanol: acetic acid: water (4: 1 : 5) solvent system had been equilibrated. Elution with the upper phase gave a major peak fraction and the material from this zone was deposited on a column (1.4 X 94 cm) of carboxymethyl cellulose according to the methods described by DH Coy et al., J. Med. Chem., 16, 1140 (1973) described conditions eluted. Corresponding fractions (1050 to 1190 ml) after lyophilization gave aul'konstantcs weight of D-Trp'-Ll I-RH as a white, fluffy powder (80 mg); [a \% -58.8 ° (r = 0.33, In-HOAc).

The product was found to be homogeneous in the thin-layer chromalograiran in four different solvent systems, when quantities of 20 to 30 megawatts were applied and the stains were made visible by iodine vapor and then Ehrlich-Rcagens. The following R _r values were obtained:

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-propane: I m-acetic acid (2: 1) 0.38;

i-Buianoi: acetic acid: water: alhyiacetal (I 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; 0.93; Arg 0.98; Pro !, 00; GIy 1.02; NH, 1.03. Example 2

A) L-Pyroglutamyl-L-histidyl (tosyl) -L-tryptophyl-L-seryl (benzyl) -L-tyrosyK2-bromobenzyloxycarbonyl) - jo D-phenylalanyl-L-leucyl-L-arginyKtosylJ-L-prolylglycylbenzhydryl resin

[pyK ^ p ^ yi ^ gi ^ y R ⁴ = Tos, R ^s 2-Br-Cb /, R ⁶ = BzI, K ⁷ - Tps, R * = II and A = benzhydrylamine resin]

1.25 g (1.0 mmol) of Bcn / .hydrylamine resin were poured into the reaction vessel of an automatic peptide synthesis device (Beckmann model 990), programmed to carry out the following washing cycles:

a) methyl chloride; b) 33% trifluorocacetic acid in methylene chloride (2 x for 2.5 and 25 minutes each), c) methylene chloride, d) alcohol, c) chloroform, 0.1% triethylamine in chloroform (2 × 25 minutes), g) chloroform, h) methylene chloride.

The washed resin is then stirred with 525 mg (3.0 mmol) tcrt.-Butyloxycarbonylgiycin in methylene w chloride and treated with 3.0 mmol of dicyclohexylcarbodiimide. The mixture is stirred for 2 hours at room temperature (22 to 25 ⁰ C), and the amino acid resin is then successively washed x 3 with X Mcthylenchlorid, 3 x with ethanol and 3 with Methylemhlorid. The bound amino acid is freed from the protective group with 33% trifluoroacetic acid in methylene chloride (2X for 2.5 and 25 minutes in each case), and the washing cycles c) to h) described above are then carried out.

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

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

The benzhydrylamine resin used in this example is a commercial product (I% crosslinked).

B) L-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-lyrosyl-D-phenylalanyl-L-leucyl-L-arginyl-L-prolylglycine amide: 1, X = Ser, Y = D-Phe and Z = GIy-NH ₂ [(pyro) -Glu-His-Trp-Ser-Tyr-D-Phe-Leu-Arg-Pro-Gly-NH ₂ l

The protective groups and cleavage of the decapplide from the resin, described under A), are removed by treating 1.5 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 eluted by gel filtration on a column (2.5 x 100 cm) of Sephadcx * G-25 Purified with 2m-acetic acid, and fractions showing a main UV absorption Pcak 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 (linen), which was previously mixed with the lower phase, followed by the upper phase of an n-Bulanol: acetic acid: water (4: I : 5i solvent system, eluting the upper phase gave a major Pcak-f 'fraction, and fractions of this peak were collected and concentrated to dryness. The residue was lyophilized from O, 2N acetic acid to give 158% mg [D-Phe ^ J-LII-RH obtained as a fluffy white powder; \ a] '"' -57.5 ° (c = 0.55, 0, In-HOAc).

: The product turned out to be in the Dürra layer chromatogram in four different solvent systems

ν homogeneous when applied from 20 to 30 meg and visible through iodine vapor and then Ehrlich reagent

Hj made warriors. The following Rj values were obtained:

■ 5 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.

·: Ίο The amino acid analysis showed:

ψ GIu 1.01; His0.97, Trp0.90, Ser0.92, Tyr 1.00; PheO, 96; Leu 1.00; Arg 1.02; Pro0.95; GIy 1.00; NH, 1.00.

Μ is example 3

(: i A) L-Pyroglutamyl-L-histidyl (dinitrophenyl) -L-tryplophyl-L-seryl (benzyl> -

:? < L-tyrosyl (2-bromobenzyloxycarbonyl) -D-Ieucyl-L-lcucyl-L-arginyl (tosyl) -L-prolyl-0-CH "2-H <> ^{r /} -

% [R ⁸ - (pyro) -Glu-His- (N ^lm -RVTrp-D-Ser (Ryryr (RYD-Leu-Leu-Arg (N ⁽ⁱ -RVPro-A ¹ ;

P 20 R "= Tos, R ⁵ = 2-Br-Cbz, R ⁶ = B / l. R ⁷ = Dnp, R ¹¹ = H

l u " _rt A '= OC _Hj

'".' 25 1.40 g (0.5 mmol of proline) Boc-proline resin of the formula

. BoC-PrO-O-CH ₂ -

|: -ί are placed in the reaction vessel of an automatic Peplid-Synthcsc device (Beckmann model 990)

: filled, which is programmed to carry out the following wash cells:

>! a) methylene chloride; b) 33% trifluoroacetic acid in methylene chloride (2 x 2.5 and 25 minutes each), c) Melhy-

lenchiorid, d) ethanol, e) chloroform, 0 10% triethylamine in chloroform (2 x 5 minutes each), g) chloroform and J? h) methylene chloride.

The washed resin is then stirred with 645 mg (1.5 mmol) of tert-butyloxycarbonyl-tosyl-arginine in methylene chloride, and with 1.5 mmol of dicyclohexylcarbodiimide / ugcscl / .t. The mixture is stirred at room temperature (22 to 25 ^° C.) for 2 hours, and the amino acid resin is then washed successively 3 times with methylene chloride, 3 times with ethanol and 3 times with methylene chloride. The bound amino acid 40 is freed from the protective group with 33% trifluoroacetic acid in methylene chloride (2 × 2.5 b / .w. 25 minutes), and the washing cycles c) to h) described above are then carried out.

1.5 mmol of the following amino acids are then successively followed by the same reaction sequence coupled: t-Boc-L-leucine; t-Boc-L-leucine; NBoc-L-tyrosinf.MJr-Cbz); t-Boe-D-serine (ßzl); t-Boc-L-tryptophan; t-Boc-L-histidine (Dnp); L-pyroglutamic acid c.

45 The finished nonapeptide resin is washed 3 times with methyl chloride and then 3 times with methanol and dried under reduced pressure, with 96% of the theoretical weight gain being obtained. The proline resin used in this example is a commercially available chlorine chloride resin (1% cross-linked).

50 B) L-Pyroglutamyl-L-histidyl-L-tryptophyl-D-seryl (bcn / .yl) -L-tyrosyl (2-bromobenzyloxycarbonyl) -

D-leucyl-L-leucyl-L-arginyKlosylJ-L-prolinälnylarnid, RNpvrol-Glu-His-Trp-D-ScriR ^ -TyrfR ^ -D-Leu-Leu-ArgfN ^ -R ^ -Pro-NHR ¹ ; R ⁴ = Tos, R ⁵ = 2-Br-Cbz, R "= B / 1, R ^K -H and R ¹ = C,. 'K

55 2.16 g of the protected nonapeptide resin from A) are suspended in 20 ml of ethylamine at 0 ° C. and stirred for 6 hours. Excess ethylamine is then allowed to evaporate at room temperature and the cleaved peptide is washed from the shark with dimethylformamide. The schülztc peptide is then eliminated by adding ethyl acetate and filtered to give 672 mg of an off-white powder. R ₁ on silicon dioxide gel in 1-butanol:! Acetic acid: water (4: 1: 5, upper phase) ■ = 0.45. This material

mi is used in C) without further purification.

C) L-Pyroglulamyl-L-histidyl-L-tryptophyl-D-seryl-L-tyrosyl-D-leucy-L-lcucyl-L-arginyl-L-proline-iithylamid, 1.X = Scr, Y = D- Leu and 7th - NHEt [(pyroJ-Gly-llis-Trp-D-Ser-Tyr-D-Leu-Leu-Arg-Pro-Nlint]

65 The protective groups are removed from the protected nonapcptide, as described in B), by treating 670 mg of the material with 50 ml of hydrogen fluoride and 15 ml of anisole at ⁰ ° C. for 30 minutes. The hydrogen fluoride is removed under reduced pressure and the anisole is removed by washing with ether.

The crude peptide is eluted by gel filtration on a column (2.5 x 100 cm) of Sephadex * G-25
Purified with 0.2m acetic acid, and fractions which show a llaupl-1V absorption peak at 280 nm are collected and evaporated to dryness.

The remaining oil is applied to a column (2.5 x 100 cm) of Sephadex "G-25 (fine), the previously
with the untcreji phase, followed by the upper phase of n-Hulunol: acetic acid: water (4: 1: 5) as the solution>
means system was equilibrated. Eluicrcn the upper phase one obtains a main structure with a Ί

high UV absorption at 280 nm, and this painterial is chromatographed at: a column (1.5 x94 cm) Sj

of Siliciumdioxidgcl unler / .ogcn and with a mixture of I-Uutunol: acetic acid: water (4: 1: 1) cluted. it

Corresponding fractions (3 (M) to 390 ml) resulted after evaporation and lyophilization of water to confi f * j

stantcs weight [D-SCr ¹ J) -LeU ^ eSGIy-NIlJ ¹¹ I-LI l-RII-iithylamid as a white, fluffy powder of 103 mg; iu '■ [a \},' = -29.6 ° (<· = 0.54, OJn-IIOAe). ;

The product proves to h in the TLC in four different Lösungsmittclsyslemen

on silicon dioxide gel plates when applying 20 to 30 meg and making the leaks visible with iodine vapor. j

followed by Ehrlich's reagent, as homogeneous. The following «, values were obtained: ^l

I-cutanol: acetic acid: water (4: 1: 5, upper phase) 0.20; i

Ethyl acetate: pyridine: acetic acid: water (5.5: 1: 3) 0.72; I.

2-propanol: Im-acetic acid (2: 1) 0.43;
! ΚυίίίΓίί ', ί: Es:; igsiiurc: water: Aüiyiacewii (I. ί: i: ij ü.Sji.

: ii
The amino acid analysis showed:

GIu 1.03; His 0.93; Irp 1.01; Ser 0.82; Tyr 0.97; Leu 1.98; Arg 1.00; Per 0.90: ethylamine 0.98.

Claims (1)

Claims: 1. Nona and decapeptide amides of the general formula 1: pyroGlu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z (I) with the following substituent meaning and assignment: Ser D-Trp GIy-NII., Ser D-Phe GIy-NH, D-Ser D-Leu NH (C ₁ - to C-alkyl) a) b)