CH615662A5 - Process for the preparation of peptides - Google Patents

Abstract

The peptides of formula (pyro)Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z (I> in which X, Y and Z are defined in Claim 1, are prepared by peptide linking of the protected amino acids, according to the so-called solid support technique. Finally, the end peptide is split from its support and the protective groups are removed. The peptides of formula I are hormones capable of releasing luteinising hormone (gonadotropin B) and follicle stimulating hormone (gonadotropin A) and can be used as products inducing ovulation.

Description

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1. Process for the preparation of a peptide of formula (pyro) Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z (I)

in which:

a) X = Ser, Y = D-Trp and Z = Gly-NH2, or b) X = Ser, Y = D-Phe and Z = Gly-NH2, or c) X = D-Ser, Y = D- Leu and Z = NHR1 (R1 = lower alkyl),

as well as their acid addition salts, characterized in that the compounds a) and b) above are obtained by first preparing a protected peptide of formula

R8- (pyro) Glu-His (NIm-R7) -Trp-X1-Tyr (R5) -Y-Leu-Arg (NG-R4) -Pro-Z2 (II)

in which:

a) X1 = Ser (R6), Y = D-Trp and Z2 = Gly-A, or b) X1 = Ser (R6), Y = D-Phe and Z2 = Gly-A,

R4, R5, R6 and R7 representing protective groups, and R8 being hydrogen or a-amino protective group, and A denoting a group of formula reaction with sodium and liquid ammonia or with hydrofluoric acid.

(Al)

(A2)

The present invention relates to the preparation of peptides of formula I:

Ph being a phenyl or phenylene group, respectively, by peptide chain of amino acids whose groups which must not react are protected, according to the solid phase synthesis starting with the glycine protected in α-amino and attached to the support, and in then splitting the protecting groups and the support Al simultaneously or, if the support of formula A2 is used, by first splitting the support and then, in a separate step, the protecting groups;

and, for the preparation of compound c) above, the procedure is carried out using the support of formula A2 above and starting the peptide chain with the proline protected in α-amino and attached to the support, the cleavage of the support by reaction with a lower alkylamine, which introduces the terminal group R1.

2. Method according to claim 1, characterized in that the reagent by which the protective groups are split is hydrofluoric acid.

3. Method according to claim 1, characterized in that the peptide of formula is prepared

(pyro) Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2 (la)

4. Method according to claim 1, characterized in that the peptide of formula is prepared

(pyro) Glu-His-Trp-Ser-Tyr-D-Phe-Leu-Arg-Pro-Gly-NH2 (Ib)

5. Method according to claim 1, characterized in that a peptide of formula is prepared

(pyro) Glu-His-Trp-D-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHR1 (le)

6. Method according to claim 1, in which the solid support of formula A2 is used, characterized in that the peptide of formula I is released by first splitting the group A2 by ammo-nolysis, the removal of the other protecting groups then by reaction with sodium and liquid ammonia or with hydrofluoric acid.

7. Method according to claim 1, in which the solid support of formula A2 is used, characterized in that the peptide of formula I is released by first splitting the group A2 by transesterification with a lower alkanol, then conversion into amide, the removal of the other protective groups then being carried out by

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

(I)

in which:

a) X = Ser, Y = D-Trp and Z = Gly-NH2, or b) X = Ser, Y = D-Phe and Z = Gly-NH2, or c) X = D-Ser, Y = D- Leu and Z = NHR1, where R1 represents a

15 lower alkyl, as well as new intermediates for the synthesis of these peptides.

The peptides of formula I, in which:

a) X = Ser, Y = D-Trp and Z = Gly-NH2, or b) X = Ser, Y = D-Phe and Z = Gly-NH2, or 20 c) X = D-Ser, Y = D -Leu and Z = NHR1

are also called respectively: a) L-Pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-tryptophyl-L-leucyl-L-arginyl-L-prolylglycinamide, or c) L-pyroglutamyl- L-histidyl-L-tryptophyl-D-seryl-L-25 tyrosyl-D-leucyl-L-leucyl-L-arginyl-L-propylalkyl lower amide,

and can be designated respectively by the abbreviations: a) [D-Trp6] -LH-RH,

b) [D-Phe6] -LH-RH, or b) L-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-phenylalanyl-L-leucyl-L-arginyl-L- prolylglycinamide, or c) [D-Ser4, D-Leu6, desGly-NH210] -LH-RH (lower alkyl) amide.

The peptides of formula I are considered to be new.

Luteinizing hormone (LH) and follicle stimulating hormone (FSH) are both gonadotropic hormones secreted by the pituitary gland in humans and mammals in general. The hormone LH and the hormone FSH stimulate the release of estrogen 40 from the ovarian follicles during maturation and cause the process of ovulation in women and females of mammals. In humans and in mammalian males, the hormone LH stimulates the cells of the interstitial glands and, for this reason, is called the hormone of stimulation of the interstitial glands (ICSH). The hormone FSH causes the ovarian follicles to mature and, along with the hormone LH, it plays an important role in cyclical phenomena in women and females. The hormone FSH causes the development of seminal cells in the testicles of male so. The hormones LH and FSH are released from the pituitary gland under the action of the hormone releasing LH and FSH, and it is now almost certain that this releasing hormone is secreted into the hypothalamus and reaches the pituitary gland. a neurohumoral pathway; see, for example, A.V. Schally et al., 55 "Recent Progress in Hormone Research", 24,497 (1968).

The natural hormone releasing LH and FSH from the porcine hypothalamus was isolated and its constitution was demonstrated by A.V. Schally et al., "Biöchem. Biophys. Res. Commun. ”, 43,393 and 1334 (1971), who proposed the structure of decapeptide:

60 (pyro) Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2.

This constitution was confirmed by synthesis; for example, see H. Matsuo et al., "Biochem. Biophys. Res. Comm. ”, 45, 822 (1971) and R. Geiger et al., Ibid., 767 (1971). 65 Hereinafter, the natural hormone releasing LH and FSH is abbreviated as LH-RH.

Because of the importance of the hormone LH-RH both for diagnostics and in therapeutic medicine, there has been much

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interested in the preparation of new compounds with improved properties compared to the natural hormone. One attempt in this direction has been the selective modification or replacement of amino acid residues of the hormone LH-RH with other amino acids. Although in some cases it has been shown that peptides with such modifications are more active than LH-RH, for example: [D-Ala6] -LH-RH, A. Arimura et al., "Endocrinology", 95,1174 (1974), [D-Leu6] -LH-RH and [D-Leu6, desGly-NH210] -LH-RH ethylamide, JA Vilchez-Martinez et al., "Biochem. Biophys. Res. Commun. ”, 59, 1226 (1974), the modified peptides have been shown to be less active in most cases.

We have now found that:

at). replacement of the glycyl moiety in position 6 of LH-RH with D-tryptophan, or b) replacement of the glycyl moiety in position 6 of LH-RH with D-phenylalanine, or c) replacement of L-seryl fragment in position 4 of LH-RH with a D-seryl fragment, replacement of the glycyl fragment in position 6 with a D-leucyl fragment and replacement of the glycinamide fragment in position 10 with a group of lower alkyl amide, give a peptide of formula I which is much more active and of a longer duration of action than the hormone LH-RH.

The present discovery that a change in the asymmetry of the rest of seryl at position 4, together with the introduction of a residue of D-leucyle at position 6, results in enhanced activity and a longer duration of action. for the peptide of formula I, in which X represents D-Ser, Y represents D-Leu and Z represents NHR1, is quite surprising, due to the fact that, as regards LH-RH, changes in the asymmetry of its amino acid residues and / or their replacement generally lead to a derivative which is far less active than LH-RH itself; see, for example, Y. Hirotsu, "Biochem. Biophys. Res. Commun. ”, 59, 277 (1974). Furthermore, taking into account the above, it has been discovered that [D-Ser4] -LH-RH has less than 5% of the activity of the natural hormone LH-RH.

The advantages of the present peptides of formula I are of practical importance: smaller minimum effective dose reducing side effects, as well as the cost of preparation of the compound, and longer lasting action property, reducing the need for frequent administration.

The compounds to be prepared according to the present invention are chosen from the group comprising: the compounds corresponding to formula I:

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

in which:

a) X = Ser, Y = D-Trp and Z = Gly-NH2, or b) X = Ser, Y = D-Phe and Z = Gly-NH2, or c) X = D-Ser, Y = D- Leu and Z = NHR1, where R1 represents a lower alkyl,

as well as the non-toxic salts of these compounds, acceptable in pharmacy. The compounds corresponding to formula II: -

R8- (Pyro) Glu-His (NIm-R7) -Trp-X1-Tyr (R5) -Y-Leu-Arg (NG-R4) -Pro-Z1

in which:

a) X1 = Ser (R6), Y = D-Trp and Z1 = Gly-R2, or b) X1 = Ser (R6), Y = D-Phe and Z1 = Gly-R2, or c) X1 = D- Ser (R6), Y = D-Leu and Z1 = OR3,

formulas in which R2 is chosen from amino and 0- (lower alkyl) radicals, R3 is lower alkyl, R4, R5, R6 and R7 are protective groups which can be separated by one or more chemical treatments which do not affect the (pyro) -Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z, in which X, Y and Z have the definition given above, and R8 represents hydrogen or one precipitated protecting groups; and the compounds corresponding to the formula:

R8- (pyro) Glu-His-Trp-D-Ser (R6) -Tyr (R5) -D-Leu-Leu-Arg- (N ^ R ^ -Pro-NHR1

in which R1 is a lower alkyl, R4, R5, R6 and R8 are protective groups which can be separated by one or more chemical treatments which do not affect the whole (pyro) Glu-His-Trp-D-Ser-Tyr -D-Leu-Leu-Arg-Pro-NHR1, and R8 represents hydrogen or one of the above-mentioned protective groups are substances which are presented as intermediates of the process.

As regards the compounds of formula II and the compounds corresponding to the formula:

R8- (pyro) Glu-His-Trp-D-Ser (R6) -Tyr (R5) -D-Leu-Leu-Arg- (N ^ R ^ -Pro-NHR1,

according to a preferred embodiment, R1, R2 and R3 have the definition given above, R4 pst a protecting group for the nitrogen atoms N6, Nm and Nm of arginine, this protecting group being chosen from the following radicals: tosyle , nitro, benzyloxycarbonyl and adamantyloxycarbonyl; R5 is a protective group for the hydroxyl radical of tyrosine, this protective group being chosen from the 2-bromobenzyloxycarbonyl, benzyl, acetyl, tosyl, benzoyl, t-butyl, tetrahydro-pyran-2-yl, trityl, 2 radicals , 4-dichlorobenzyl and benzyloxycarbonyl; R6 is a protective group for the hydroxyl radical of serine and is chosen from the group defined above for R5; R7 is a protecting group for the imidazole nitrogen atoms of histidine, this protecting group being chosen from tosyl and dinitrophenyl; and R8 is hydrogen or an α-amino protecting group, this group being chosen from the following radicals: t-butyloxy-carbonyl, benzyloxycarbonyl, cyclopentyloxycarbonyl, t-amyl-oxycarbonyl and d-isobornyloxycarbonyl.

In the present process, intermediates are also formed bound to a solid resin support. These intermediaries can be represented by the formulas:

R8- (pyro) Glu-His (NIm-R7) -Trp-X1-Tyr (R5) -Y-Leu-Arg-

(NG-R4) -Pro-Z2, R9-His (NIm-R7) -Trp-X1-Tyr (R5) -Y-Leu-Arg (NG-R4) -Pro-Z ?, R9-Trp-X1 - Tyr (R5) -Y-Leu-Arg (NG-R4) -Pro-Z2, and R9-X1-Tyr (R5) -Y-Leu-Arg (NG-R4) -Pro-Z2,

formulas in which:

a) X1 = Ser (R6), Y = D-Trp and Z2 = Gly-A, or b) X1 = Ser (R6), Y = D-Phe and Z2 = Gly-A, or c) X1 = D- Ser (R6), Y = D-Leu and Z2 = A2.

In these formulas, R4, R5, R6, R7 and R8 have the definition given above, R9 is an α-amino protecting group, known in practice in the gradual synthesis of polypeptides, suitable protecting groups of this kind being listed below. -after, and A and A2 are fixing bonds used in a solid phase synthesis with a solid resin support. A is chosen from:

support support and O — CH2— |

N — C — Ç> -

resin resin

(Ç> = phenyl) (Al) (A2)

The method according to the invention is defined by independent claim 1.

In the present description, the expression lower alkyl designates the alkyl radicals containing from 1 to 3 carbon atoms and it includes methyl, ethyl, propyl and isopropyl.

No. denotes the side chain nitrogen atoms of arginine.

NIra denotes the imidazole nitrogen atoms of histidine.

The symbol Ç) denotes phenyl.

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In general, the abbreviations used in the present case to designate amino acids and protecting groups have been based on the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature, see "Biochemistry", II, 1726 (1972). For example, t-Boc represents t-butyloxy-carbonyl, Z represents benzyloxycarbonyl, Tos represents tosyl, 2-Br-Cbz represents 2-bromobenzyloxycarbonyl, Bzl represents benzyl, and Dnp represents 2,4-dinitrophenyl. The abbreviations used here for the various amino acids are: Arg, arginine; Gly, glycine; His, histidine; Leu, leucine; Pro, proline; (pyro) Glu, 5-oxoproline (pyroglutamic acid); Ser, serine; Trp, tryptophan and Tyr, tyrosine. All the amino acids described here are of the L series, unless otherwise indicated, in particular D-Ser is a residue of D-seryl, D-Leu is a residue of D-leucyl, D-Phe is a residue of D- phenylalanyl and D-Trp is a residue of D-tryptophyl.

The peptides of formula I above can be obtained in the form of acid addition salts. Examples of salts are the salts with organic acids, for example acetic, lactic, succinic, benzoic, salicylic, methanesulfonic or toluenesulfonic acids, as well as with polymeric acids, such as tannic acid or carboxymethylcellulose, and salts with mineral acids, such as hydrohalic acids, for example hydrochloric acid, or sulfuric acid or phosphoric acid. If desired, a particular acid addition salt can be converted to another acid addition salt, for example a salt with a non-toxic acid, pharmaceutically acceptable, by treatment with the appropriate resin. ion exchange, as described by RA Boissonnas et al., "Helv. Chim. Acta ”, 43, 1349 (1960). Suitable ion exchange resins are the cation exchange resins based on cellulose, for example carboxymethylcellulose, or the cation exchange resins based on chemically modified dextran crosslinked, for example resins of the Sephadex-C type, and strongly basic anion exchange resins, for example those listed by JP Greenstein and M. Winitz in "Chemistry of the Amino Acids", John Wiley and Sons, Inc., New York and London, 1961, vol. 2, p. 1456.

The peptides of formula I and their salts exhibit an interesting and long-lasting activity as hormones for releasing LH and FSH.

The activity of interest as a hormone releasing LH and FSH and the long-lasting property of the compounds of the present invention have been demonstrated by traditional pharmacological methods. By way of example, these activities can be demonstrated by the tests described by A. Arimura et al., “Endocrinology”, 95.1174 (1974). By following the method described in this publication, the results of release of LH, obtained on rats having received a dose of the compound (50 ng, subcutaneous route), show that the peptides of formula I reach a maximum activity approximately 2 h after administration of the dose, and that significant activity is still present over a period of up to 6 h; on the other hand, with the same dose of the natural hormone LH-RH (50 ng, subcutaneous route), the maximum activity is reached after approximately 15 min and no effect of the injection is still observed after 1 h . In addition, the integrated levels of LH over a period of 6 h show that the compounds of formula I: [D-Ser4, D-Leu6, desGly-NH210] -LH-RH ethylamide, [D-Phe6] -LH-RH and [D-Trp6] -LH-RH, are respectively 21 times, 20 times and 12 times more active with regard to the release of the hormone LH than the natural hormone LH-RH. The results concerning the release of the hormone FSH, after injections of the compounds of formula I, show that the compounds: [D-Ser4, D-Leu6, desGly-NH210] -LH-RH ethylamide, [D-Phe6] - LH-RH and [D-Trp6] -LH-RH are respectively approximately 11 times, 20 times and 20 times more active than the natural hormone LH-RH at the same dose (50 ng).

In addition, the activity of the compounds of formula I has been demonstrated in humans.

Analyzes (radioimmunization) of LH levels in serum, after intranasal administration, show that the minimum effective dose of LH-RH is approximately 2.0 mg, while an equivalent degree of activity is obtained with a dose of 0.5 mg of [D-Trp6] -LH-RH. In this case, the compounds were administered to humans in normal saline. With regard to the release of FSH in humans, comparative studies between LH-RH and [D-Trp6] -LH-RH give particularly remarkable results. Intranasal administration of up to 2 mg LH-RH has little or no effect on FSH levels in serum, which has been measured by radioimmunization analysis, see HG Dahlen et al., "Horm. Metab. Res. ”, 6, 510 (1974); on the other hand, at 0.5 mg under the same conditions, [D-Trp6] -LH-RH releases significant amounts of FSH, that is to say rates ranging from 0.3 to more than 1.5 μm | i / ml. It will be readily understood that a compound which is capable of releasing FSH effectively has many therapeutic applications; see, for example, H.G. Dahlen et al., above quotation.

The release properties of LH and FSH from the peptides of formula I, which in turn cause ovulation in animals, make these peptides of interest in veterinary medicine and in animal husbandry. It is often desirable to synchronize the estrus in animals, for example in cattle in general, sheep or pigs, either to be able to ensure the fitting of females of a given group with a male of the desired genetic quality, either to be able to carry out an artificial insemination on a maximum number of females, in both cases over a relatively short period of time. Previously, we acted, in the two cases above, by administering to the animals an ovulation inhibiting agent, by suppressing this administration shortly before the date chosen for the artificial apparatus or insemination, and then counting on the production natural LH and FSH to cause ovulation and produce estrus, or by administering gonadotropic hormones. However, this procedure was not entirely satisfactory, since ovulation at a predetermined time never develops in all animals at the same time, but only in a certain proportion of them, when n do not use gonadotropic hormones. On the other hand, the high cost of the latter hormones and the side effects encountered when using them have made this method impractical. It is now possible to achieve almost complete synchronization of ovulation and estrus by treating animals in a given group, first with an ovulation inhibitor which is then removed, and then administering a peptide of formula I shortly before the predetermined period, intended for artificial insemination or insemination, so as to obtain ovulation and oestrus during this time interval. The time before the onset of ovulation and estrus, after administration of such a peptide, varies with animal species, and the optimal time interval should be chosen for each species. For example, in rodents such as rats or hamsters, ovulation occurs within 18 days after administration of a peptide according to the invention.

The method described above for obtaining an ovulation and an oestrus within a precisely predetermined time interval, so as to ensure satisfactory apparatus, is particularly important for breeders of racehorses and show animals , that is to say when the costs to be expected for the services of a particular male often amount to very high sums.

The peptides of formula I are also advantageous for increasing the number of births per litter in animals, in particular in cattle in general, sheep or pigs. To this end, the peptide is administered in a series of parenteral doses, preferably by intravenous or subcutaneous injections, at a rate of approximately 0.1 to 10 μg per kg of body weight per day, and this 96 to 12 h before the expected estrus and the subsequent mating. A

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starting injection of 1000 to 5000 units of gonadotropic hormone of pregnant mare serum can also be scheduled 1 to 4 days before the previous injection of the peptide. A similar treatment, with or without preliminary injection, is also advantageous for causing puberty in farm animals.

When a peptide of formula I is used for the purpose of causing ovulation and estrus or for the purpose of initiating puberty in warm-blooded animals, in particular rodents, such as rats or hamsters, or in cattle, this peptide is administered to the body, preferably parenterally, in combination with a liquid or solid vehicle or support, acceptable in pharmacies. The proportion of the peptide is determined by its solubility in the given vehicle, by the chosen route of administration, and by traditional biological practice. For parenteral administration to animals, the peptide is used in sterile aqueous solution which may also contain other dissolved products, such as buffers or preservatives, as well as a sufficient quantity of glucose or pharmaceutically acceptable salts. to make the solution isotonic. The dose will vary with the form of administration and with the particular species of animal to be treated, and will preferably be maintained at a level of 0.1 to 10 ng per pound of body weight. However, a dose in the range of about 1 to about 5 ng per pound of bodyweight is used particularly advantageously to achieve effective results.

Peptides of formula I can also be administered in one of the long-lasting, slow-release or deposition forms, which will be described below, preferably by intramuscular injection or by implantation. These dosage forms are designed to release about 0.1 to about 10 (xg per pound of bodyweight per day.

The peptides of formula I are also of interest in human medicine. For example, human chorionic gonadostimulin (HCG), which mainly contains the hormone LH and a certain amount of the hormone FSH, has been used for more than 30 years to treat certain endocrinological disorders, such as menstrual cycle, amenorrhea, lack of development of secondary sexual characteristics and infertility in women, or some cases of hypogonadism, delayed puberty, cryptorchidism and non-psychogenic impotence in men. Later, infertility in women was also treated with human gonadostimulins from postmenopausal women (HMG) which mainly contain the hormone FSH, followed by treatment with the hormone HCG. One of the disadvantages of treating infertility in women with HCG or with HMG followed by HCG is that such treatment often results in over-ovulation and unwanted multiple births, possibly due to the inability to administer only the exact amounts of FSH and LH needed for ovulation. Administration of a peptide obtained according to the invention avoids the above disadvantage, since the compound causes the release of LH and FSH from the pituitary gland, only in the exact amounts which are necessary for normal ovulation. For this reason, a peptide according to the invention is not only useful for the above purpose, but it is also interesting in women for the treatment of menstrual cycle disorders, amenorrhea, hypogonadism and l lack of development of secondary sexual characteristics.

The peptides obtained according to the invention are also useful in contraception. For example, when the peptide is administered to a woman very early in the menstrual cycle, the hormone LH is released at this time and causes premature ovulation. The immature egg is incapable of fertilization or, if fertilization should nevertheless occur, it is very unlikely that the fertilized egg is implanted, because the estrogen-progesterone balance, necessary for the preparation of the endometrium, does not exist and the endometrium is not in the state required for implantation.

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On the other hand, when the peptide is administered towards the end of the cycle, the endometrium is ruptured and menstruation occurs.

In addition, the peptides obtained according to the invention are also advantageous in contraception by the method known as rhythms which has always been relatively unreliable due to the impossibility of predetermining ovulation in women with the required degree of precision. The administration of a peptide according to the invention at mid-cycle, that is to say at about the normally expected time of ovulation, causes ovulation soon after and makes the rhythm method both safe and effective.

The peptides of formula I are also useful as diagnostic means for distinguishing between disorders or lesions of the hypothalamus and pituitary gland in women. When the peptide is administered to a patient for whom such disorders or lesions are suspected and then an increase in the level of LH is observed, this is a good indication allowing to conclude that the hypothalamus is the cause of the disorder and that the pituitary gland is intact. On the other hand, when no increase in the level of LH is observed after administration of the peptide, one can then diagnose a disorder or lesion of the pituitary gland, with a high degree of reliability.

In humans, the administration of a peptide of formula I provides the amounts of LH (or ICSH) and FSH necessary for normal sexual development in cases of hypogonadism and delayed puberty, this administration being also interesting in the treatment of cryptorchidism. In addition, the FSH hormone released by the administration of the peptide stimulates the development of the seminal cells of the testes, and the peptide is useful in the treatment of psychogenic impotence and non-psychogenic impotence.

When the peptides of formula I are used in human medicine, preferably in the form of an acid addition salt, they are administered to the body, either by intravenous, subcutaneous or intramuscular injection, or by sublingual, nasal or vaginal administration in the form of combinations formed in conjunction with a pharmaceutically acceptable carrier or carrier.

For administration by the nasal route in the form of drops or sprays, it is preferred to use a peptide of formula I in. solution in a sterile aqueous vehicle which may also contain other dissolved products, such as tampons or preservatives, as well as sufficient amounts of glucose or pharmaceutically acceptable salts to make the solution isotonic. The doses by the intranasal route are from 0.1 to 50 (xg / kg, preferably from 0.5 to 10 ng / kg.

The peptides of formula I can also be administered in the form of powders or breaths by the nasal or vaginal route. To this end, the peptide is administered in a finely subdivided solid form, together with a solid vehicle which is acceptable in pharmacies, for example a finely subdivided polyethylene glycol (Carbowax 1540), finely subdivided lactose, or preferably for the administration, very finely subdivided silica (Cab-O-Sil). Such compositions may also contain other excipients in a finely subdivided solid form, such as preservatives, buffers or surfactants.

For sublingual or vaginal administration, it is preferred to formulate the peptides of formula I in solid doses, for example sublingual tablets or vaginal ova or suppositories, with sufficient quantities of solid excipients, such as starch, lactose, certain types of clays, buffers, and lubricants, disintegrants or surfactants, or with semi-solid excipients commonly used in the formulation of suppositories. Examples of such excipients can be found in conventional pharmaceutical textbooks, for example in "Pharmaceutical Sciences" by Remington, Mack Publishing Company, Easton, Pennsylvania, 1970.

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The dose of the peptides of formula I will vary with the form of administration and with the particular patient being treated. Generally, treatment is started with low doses, significantly lower than the optimal dose of the compound. Then, this dose is increased in small amounts until the optimal effect for the case is reached. In general, the peptides of formula I are administered in the most advantageous manner at a concentration rate which will, in general, bring about an effective release of LH and / or FSH, without however causing harmful or unpleasant side effects, this rate preferably being of the order of approximately 0.01 to approximately 100 ng per kilo of body weight, although this range of doses is subject to variations as mentioned previously. However, a dosage rate of the peptide of formula I, in which:

a) X = Ser, Y = D-Trp and Z = Gly-NH2, or b) X = Ser, Y = D-Phe and Z = Gly-NH2

of the order of about 0.5 ng to about 5.0 ng per kilogram of body weight, and of the peptide of formula I, in which:

c) X = D-Ser, Y = D-Leu and Z = NHR1

on the order of about 0.1 to about 10 ng per pound of body weight is most advantageous for achieving effective results.

It is often desirable to administer the peptides of formula I continuously over extended periods of time in the form of long acting, slow release or depot doses. Such dosage forms may contain a pharmaceutically acceptable salt of the compound having a low degree of solubility in body fluids, for example salts with pamoic or tannic acid or with carboxymethylcellulose, or they may contain peptides in the form of a water-soluble salt, together with a protective vehicle preventing rapid release. In the latter case, one can formulate, for example, the peptides with a partially hydrolyzed gelatin, non-antigen, in the form of a viscous liquid; or these peptides can be adsorbed on a solid pharmacy acceptable vehicle, for example zinc hydroxide with or without protamine, and can be administered in suspension in a liquid pharmacy acceptable vehicle; these peptides can also be formulated in gels or suspensions with a non-antigenic, protective hydrocolloid, for example sodiumcarboxymethyl-cellulose, polyvinylpyrrolidone, sodium alginate,

gelatin, polygalacturonic acids, for example pectin, or certain mucopolysaccharides, together with surface-active agents, preservatives or liquid vehicles, aqueous or non-aqueous, acceptable in pharmacy. Examples of such formulations can be found in traditional pharmaceutical textbooks, for example in the "Pharmaceutical Sciences" manual by Remington, cited above. Long-acting, slow-release preparations of the peptides according to the invention can also be obtained by microencapsulation in a coating material which is acceptable in pharmacies, for example gelatin, polyvinyl alcohol or ethylcellulose. Other examples of coating materials and methods used for microencapsulation are described by J.A. Herbig in "Encyclopedia of Chemical Technology", vol. 13, 2nd ed., Wiley, New York, 1967, pp. 436-456. Formulations of this kind, as well as suspensions of peptide salts, which are only sparingly soluble in body fluids, are designed to release about 0.1 to about 50 ng of the hormone per pound of body weight per daily, and are preferably given by intramuscular injection. As a variant, some of the solid dosage forms, listed above, for example certain sparingly water-soluble salts or certain salt dispersions of the peptides in a solid support or also adsorption products of these salts on these supports, as dispersions in a neutral hydrogel of a polymer of ethylene glycol methacrylate or of crosslinked similar monomers, as described in the patent of United States of America No. 3551556 issued on December 29, 1970 to K. Kliment et al. , can also be

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prepare in the form of implants releasing roughly the same quantities as those mentioned above, these implants can be used subcutaneously or intramuscularly.

Slow-release effects can also be obtained over extended periods of time by administering the peptides of the invention as an acid addition salt in an intravaginal device or in a temporary implant, for example in a receptacle formed of a non-irritating silicone polymer, such as a polysiloxane, for example Silastic, or of a neutral hydrogel of a polymer as described above, having the required degree of permeability to release approximately 0.1 to about 50 ng per pound of body weight per day. Such dosage forms in the form of an intravaginal device or an implant, for very prolonged administration, have the advantage that they can be withdrawn when desired, to interrupt or terminate the treatment.

In the synthesis of the peptides of formula I according to the present invention, the following rules will be followed in the choice of a particular group which protects the side chain:

a) the protective group must be stable with respect to the reagent and under the reaction conditions chosen to remove the α-amino protective group at each phase of the synthesis;

b) the protecting group must retain its protective properties (i.e. not to separate under the conditions of the combination), and c) the side chain protecting group must be separable at the end of the synthesis containing the desired amino acid sequence, under reaction conditions which do not modify the chain of

30 peptide.

With regard to R9, suitable protecting groups for the α-amino radical are: 1) aliphatic urethane protecting groups, illustrated by: t-butyloxycarbonyl, diisopropyl-methoxycarbonyl, biphenylisopropyloxycarbonyl, iso-propyloxycarbonyl, t-amyloxycarbonyl, ethoxycarbonyl , allyl-oxycarbonyl; 2) protective groups of the cycloalkyl-urethane type, illustrated by: cyclopentyloxycarbonyl, adamantyloxy-carbonyl, d-isobornyloxycarbonyl, cyclohexyloxycarbonyl, nitrophenylsulfenyl, tritylsulfenyl, a, a-dimethyl-3,5-dimethoxy-40 and benzyloxycarbonyl. The preferred α-amino protecting group for R9 is selected from the group consisting of: t-butyloxycarbonyl, cyclopentyloxycarbonyl, t-amyloxycarbonyl, d-isobornyloxycarbonyl, o-nitrophenylsulfenyl, biphenylisopropyloxycarbonyl, and a, a-dimethyl-3,5-dimethoxybenzyloxycar -45 bonyle.

The peptides of formula I according to the invention are prepared using a solid phase synthesis, which will be illustrated below by various embodiments, according to which specific peptides of formula I are prepared:

50

a) and b) Peptides of formula I in which:

a) X = Ser, Y = D-Trp and Z = Gly-NH2, or b) X — Ser, Y = D-Phe and Z = Gly-NH2.

The solid phase synthesis of the peptides of formula I, defined 55 under a) and b) above, is initiated at the C-terminal end of the peptide, using a resin protected in α-amino. Such a starting material is prepared by attaching an α-amino protected glycine to a banzhydrylamine resin, a chloro-methylated resin or a hydroxymethylated resin, the former being pre-ferered. The preparation of a benzhydrylamine resin has been described by P. Rivaille et al., "Helv. Chim. Acta ", 54, 2772 (1971) and the preparation of the hydroxymethylated resin has been described by M. Bodanszky and J.T. Sheehan," Chem. Ind. " (London), 38.1597 (1966). A chloromethylated resin can be available on the market 65 from the company Bio Rad Laboratories, Richmond, California. When the benzhydrylamine resin is used, an amide bond is formed with the glycine protected in α-amino, and this in the following way:

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resin support

R9-N-CH2- C-NH- C-0

This allows direct production of the C-terminal amide function after the amino acid sequence has been completed in the synthesis, by separation of the resin support from the bound peptide, to form the glycine amide at the C-terminal part of the desired decapeptide. In this case, the use of hydrofluoric acid to separate the resin support also removes the side chain protecting groups to give the corresponding decapeptide of formula I, in which:

a) X = Ser, Y = D-TrpetZ = Gly-NH2, or b) X = Ser, Y = D-Phe and Z = Gly-NH2.

When the other resins are used, the bonding bond is the benzylester group as illustrated above. In this case, a convenient method for converting the bound protected peptide to the C-terminal amide is to ammonolize the protected peptide from the resin and to separate the protecting groups from the resulting amide by treatment with sodium. and liquid ammonia or by separation with hydrofluoric acid. Another method consists in causing the separation by transesterification with a lower alkanol, preferably methanol or ethanol, in the presence of triethylamine, with then conversion of the resulting ester into an amide and finally removal of the protection, as described previously. See also J.M. Stewart and J.D. Young, "Solid Phase Peptide Synthesis", W.H. Freeman & Co., San Francisco, 1969, pp. 40-49.

More specifically, in one embodiment of the present invention, an α-amino protected glycine, preferably t-butyloxycarbonylglycine, is combined with a benzhydrylamine resin using the carboxyl group activating compound , preferably dicyclohexylcarbodiimide. After combining the α-amino protected glycine with the resin support, the α-amino protecting group is separated, for example using trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone, or hydrochloric acid in dioxane. The protection is removed at a temperature between 0 ° C and room temperature. Other conventional separation reagents and other conditions for the removal of particular protecting groups for α-amino can be used as described by E. Schröder and K. Lubke, "The Peptides", vol. I, Academic Press, New York, 1965, pp. 72-75. After separation of the protecting group into a-amino, the remaining amino acids, protected into a-amino, are combined step by step in the desired order to obtain the desired peptide. Each protected amino acid is brought to the solid phase reaction in an excess of approximately 3 times, and the combination is carried out in a methylene chloride medium or formed by a mixture of dimethylformamide in methylene chloride. In cases where an incomplete combination develops, the combination process is repeated before separation of the protective group into α-amino, before the combination of the next amino acid with the amino acid or the peptide linked to a polymer. The success of the combination reaction at each stage of the synthesis is monitored by the ninhydrin reaction, as described by E. Kaiser et al., "Analyt. Biochem. ", 34,595 (1970).

After synthesis of the desired amino acid sequence, the peptide is separated from the resin support by treatment with a reagent, such as hydrofluoric acid, which not only separates the peptide from the resin, but also separates all the protecting groups side chain residues and the α-amino protecting group, if present, existing on the remainder of pyroglutamic acid, in the case where the banzhydrylamine resin was used to directly obtain the peptide of formula I, such as defined under a) or b).

When using a chloromethylated resin, the peptide can be separated from the resin by transesterification with a lower alkanol, preferably methanol or ethanol, then the recovered product.

is chromatographed on silica gel and the collected fraction is subjected to a treatment with ammonia to convert the lower alkyl ester, preferably the methyl or ethyl ester, to the C-terminal amide. The side chain protecting groups are then separated by the methods described above, for example by treatment with sodium in liquid ammonia or with hydrofluoric acid.

In addition to the protective groups R7 described above for the imidazole nitrogen atoms of histidine, R7 can also consist of 2,2,2-trifluoro-1-benzoyloxycarbonylaminoethyl and 2,2,2-trifluoro- lt-butyloxycarbonylaminoethyl.

c) Peptide of formula I, in which: c) X = D-Ser, Y = D-l £ uetZ = NHRl

Solid phase synthesis of the peptide of formula I, in which X = D-Ser, Y = D-Leu and Z = NHR1, is initiated at the C-terminal end of the peptide, using a proline resin protected in a -amino. Such a starting material is prepared by attaching an α-amino protected proline to a chloromethylated resin or a hydroxymethylated resin, the former being preferred. The preparation of a hydroxymethylated resin has been described by M. Bodansky and J.T. Sheehan, "Chem. Ind. " (London), 38.1597 (1966). A chloromethylated resin is commercially available from Bio Rad Laboratories, Richmond, California. When the chloromethylated resin is used, an ester bonding group is formed with the protected proline in α-amino, and this is as follows:

support

R9 —Pro —O — CH2

resin

A convenient method for converting the bound protected peptide to the C-terminal (lower alkyl) amide is to separate the protected peptide from the resin by treatment with a lower alkyl amine, see DH Coy et al., "Biochem ., Biophys. Res. Commun. ", 57, 335 (1974), to obtain the corresponding protected peptide (lower alkyl) amide. Then, the protecting groups of the resulting peptide (lower alkyl) amide are separated by treatment with sodium and liquid ammonia or preferably with hydrofluoric acid to give the corresponding peptide of formula I, described below. above under c), according to the invention. Another method consists in a separation by transesterification with a lower alkanol, preferably methanol or ethanol, in the presence of triethylamine, with then conversion of the resulting ester into the corresponding (lower alkyl) amide, then removal of protection as described above. See also J.M. Stewart and J.D. Young, "Solid Phase Peptide Synthesis", W.H. Freeman & Co., San Francisco, 1969, pp. 40-49.

More particularly, according to an embodiment of the present invention, an α-amino protected proline, preferably t-butyloxycarbonylproline, is combined with a chloromethylated resin using a catalyst, preferably bicarbonate cesium or triethylamine. After combining the α-amino protected proline with the resin support, the α-amino protecting group is separated, for example using trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone or hydrochloric acid in dioxane. The protection is removed at a temperature between about 0 ° C and room temperature. Other conventional separating agents and other conditions for the removal of protecting groups for α-amino can be used as described by E. Schröder and K. Lubke, "The Peptides", vol. 1, Académie Press, New York, 1965, pp. 72-75. After separation of the protective group into a-amino, the protected amino acids into a-amino are combined step by step in the desired order to obtain the compound

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of formula I illustrated previously under c). Each protected amino acid is brought to the reaction in solid phase in an excess of approximately 3 times, and the combination is carried out in a medium of methylene chloride or a mixture of dimethylformamide and methylene chloride. In cases where incomplete combination occurs, the combination process is repeated before separation of the protecting group into α-amino, before combination of the next amino acid with the solid phase reaction. The development of the combination reaction at each stage of synthesis is monitored by the ninhydrin reaction, as described by E. Kaiser et al., "Analyt. Biochem. ”, 34, 595 (1970).

After synthesis of the desired amino acid sequence, the protected peptide is separated from the resin support by treatment with a (lower alkyl) amine to give the corresponding protected peptide (lower alkyl) amide and, in the case where one has used dinitrophenyl or tosyl as the protecting group for the rest of the histidyl, the dinitrophenyl or tosyl protecting group is also separated during treatment with the (lower alkyl) amine. The peptide can also be separated from the resin by transesterification with a lower alkanol, preferably methanol or ethanol, then the recovered product is purified by chromatography on silica gel and the collected fraction is subjected to a treatment with ( lower alkyl) amine to convert the lower alkyl ester, preferably the methyl or ethyl ester, to the C-terminal (lower alkyl) amide (it should be noted that the dinitrophenyl or tosyl group is it is present on the rest of the histidyle, will also be separated). The remaining side chain protecting groups of the protected ethylamide are then separated by methods described above, for example by treatment with sodium in liquid ammonia or with hydrofluoric acid to give the nonapeptide of formula I illustrated above under c).

The following examples will illustrate the invention even more fully.

Example 1

L-Pyroglutamyl-L-histidyl (tosyl) -L-tryptophyl-L-seryl (benzyl) -L-tyrosyl (2-bromobenzyloxycarbonyl) -D-tryptophyl-L-leucyl-L-arginyl (tosyl) -L-prolylglycyl- benzhydrylamine resin (R8- (pyro) Glu-H is- (N, m-R7) - Trp-Ser (R6) -Tyr (R5) -D-Trp-Leu-Arg (Na-R4) -Pro-Gly -A; R4 = Tos, Rs = 2-Br-Cbz, R6 = Bzl, R7 —Tos, R8 = H and A = benzhydrylamine resin)

A benzhydrylamine resin (1.25 g, 1 mmol) is placed in the reaction vessel of an automatic peptide synthesis device of the Beckman type, model 990, programmed to carry out the following wash cycle: a) methylene chloride ; b) 33% trifluoroacetic acid in methylene chloride (twice for 2.5 and 25 min); c) methylene chloride; d) ethanol; e) chloroform; f) 10% triethylamine in chloroform (2 times for 25 min each time); g) chloroform; h) methylene chloride.

The washed resin is then stirred with t-butyloxycarbonylglycine (525 mg, 3.0 mmol) in methylene chloride and dicyclohexylcarbodiimide (3.0 mmol) is added. The mixture is stirred at room temperature (22-25 ° C) for 2 h and the amino acid / resin mixture is then washed successively with methylene chloride (3 times), ethanol (3 times) and chloride methylene (3 times). The attached amino acid is stripped of its protection with 33% trifluoroacetic acid in methylene chloride (2 times for 2.5 and 25 min), then the washing cycle is carried out for phases c) to h) described above.

The following amino acids (3 mmol) are then combined successively by the same cycle of operations: t-Boc-L-proline; t-Boc-L-arginine (Tos); t-Boc-L-leucine; t-Boc-D-tryptophan; t-Boc-tyrosine (2-Br-Cbz); t-Boc-L-serine (Bzl); t-Boc-L-tryptophane; t-Boc-L-histidine (Tos); L- (pyro) glutamic acid.

The complete decapeptide / resin assembly is washed with methylene chloride (3 times), then with methanol (3 times), and then it is dried under reduced pressure, thereby obtaining 98% of the theoretical weight gain.

The benzhydrylamine resin used in this example is a commercially available resin (1% crosslinking, Bachem Inc., Marina del Rey, California).

Example 2

L-Pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-tryptophyl-L-leucyl-L-arginyl-L-prolylglycinamide;

I, X = Ser, Y = D-Trp and Z = Gly-NH2 [(pyro) Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NHÀ

The removal of the protective groups and the separation of the decapeptide from the decapeptide / resin assembly described in example 1 are carried out by treatment with 1 g of the material with hydrofluoric acid (24 ml) and anisole (6 ml) at 0 ° C for 30 min. The hydrofluoric acid is separated under reduced pressure and the anisole is removed by washing with ethyl acetate.

The crude peptide is purified by gel filtration on a column (2.5 × 100 cm) of Sephadex G-25 (crosslinked, chemically modified dextran, of fine quality) by elution with 2M acetic acid, and the fractions which have a main absorption peak in the ultraviolet at 280 nm are collected and evaporated to dryness.

The residual oil is applied to a column (2.5 x 100 cm) of Sephadex G-25 (fine quality), previously equilibrated with the lower phase, then with the upper phase of a n-butanol / acetic acid solvent system. / water (4/1/5). Elution with the upper phase gives a maximum peak fraction, and the material originating from this zone is subjected to elution on a column (1.4 x 94 cm) of carboxymethylcellulose according to the conditions described by DH Coy et al., J. Med. Chem. ”, 16.1140 (1973). The appropriate fractions (1050-1190 ml), after lyophilization to constant weight, give D-Trp6-LH-RH in the form of a white fluffy powder (80 mg); [a] "= —58.8 ° (c = 0.33,

HOAc IN).

The product is homogeneous during thin layer chromatography in four separate solvent systems, when applying loads of 20-30 ng, and the stains are made visible by exposure to iodine vapor, then to reagent d 'Ehrlich. The following Rf 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; 2M-propanol / acetic acid 1M (2/1): 0.38; 1-butanol / acetic acid / water / ethyl acetate (1/1/1/1): 0.51.

Amino acid analysis gives: Glu, 1.08; His, 0.95, Trp, 2.00; Ser, 0.94; Tyr, 0.97; Leu, 0.93; Arg, 0.98; Pro, 1.00; Gly, 1.02; NH3.1.03.

Example 3

L-Pyroglutamyl-L-histidyl {tosyl) -L-tryptophyl-L-seryl- (benzyï) -L-tyrosyl (2-bromobenzyloxycarbonyl) -D-phenyl-alanyl-L-leucyl-L-arginyl (tosyl) -L benzhydrylamine -prolylglycyl-resin (Rs- {pyro) Glu-His- (NIm-R7) -Trp-Ser (R6) -Tyr (R5) -D-Phe-Ieu-Arg (NG-R4) -Pro-Gly -AT ; R4 = Tos, R5 = 2-Br-Cbz, R6 = Bzl, R7 = Tps, R8 = H and A = benzhydrylamine resin)

A benzhydrylamine resin (1.25 g, 1 mmol) is placed in the reaction vessel of an automatic peptide synthesis device of the Beckman type, model 990, programmed to carry out the following wash cycle: a) methylene chloride ; b) 33% trifluoroacetic acid in methylene chloride (twice for 2.5 and 25 min); c) methylene chloride; d) ethanol; e) chloroform; f) 10% triethylamine in chloroform (2 times for 25 min each time); g) chloroform; h) methylene chloride.

The washed resin is then stirred with t-butyloxycarbonyl-glycine (525 mg, 3 mmol) in methylene chloride and dicyclohexylcarbodiimide (3 mmol) is added. The mixture is stirred at room temperature (22-25 ° C) for -2 h and the amino acid / resin mixture is then washed successively with methylene chloride (3 times), ethanol (3 times) and chloride. methylene (3 times). The attached amino acid is stripped of its protection with trifluoroacetic acid to

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33% in methylene chloride (2 times for 2.5 and 25 min), then the washing cycle is carried out according to phases c) to h) above.

The following amino acids (3 mmol) are then combined successively by the same cycle of operations: t-Boc-L-profine; 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-trypto-phane; t-Boc-L-histidine (Tos); L- {pyro) glutamic acid.

The complete decapeptide / resin assembly is washed with methylene chloride (3 times), then with methanol (3 times), and then it is dried under reduced pressure, to thereby obtain 100% of the theoretical weight gain.

The benzhydrylamine resin used in this example is a commercially available resin (1% crosslinking, Bachem Inc., Marina del Rey, California).

Example 4

L-Pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-phenylalanyl-L-leucyl-L-arginyl-L-propylglycinamide; I, X = Ser, Y = D-PheetZ = Gly-NH2 [(pyro) Glu-H is-Trp-Ser- Tyr-D-Phe-Leu-Arg-Pro-Gly-NH 2]

The removal of the protective groups and the separation of the decapeptide from the decapeptide / resin assembly described in example 3 are carried out by treatment of 1.5 g of the material with hydrofluoric acid (24 ml) and anisole (6 ml) at 0 ° C for 30 min. The hydrofluoric acid is separated under reduced pressure and the anisole is removed by washing with ethyl acetate. The crude peptide is purified by gel filtration on a column (2.5 × 100 cm) of Sephadex G-25 (crosslinked, chemically modified dextran, of fine quality) by elution with 2M acetic acid, and the fractions which have a main absorption peak in the ultraviolet at 280 nm are assembled and evaporated to dryness.

The residual oil is applied to a column (2.5 x 100 cm) of Sephadex G-25 (fine quality), previously equilibrated with the lower phase, then with the upper phase of a n-butanol / acetic acid solvent system. / water (4/1/5). Elution with the upper phase gives a maximum peak fraction, and the fractions of this peak are combined and concentrated until dry. The remainder is lyophilized from 0.2N acetic acid to obtain [D-Phe6] -LH-RH in the form of a fluffy white powder (158 mg); [oc] "= -57.5 ° (c = 0.55, HOAc 0.1N).

The product is homogeneous during thin layer chromatography in four separate solvent systems, when applying loads of 20-30 ng, and the stains can be made visible by exposure to iodine vapor and then to the reagent of Ehrlich. The following Rf 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; 1M 2-propanol / acetic acid (2/1): 0.41; 1-butanol / acetic acid / water / ethyl acetate (1/1/1/1): 0.47.

Analysis of the amino acids gives: Glu, 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; Gly, 1.00; NH3.1.00.

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 — CH2-resin, R8- (pyro) Glu-His (Nlm-R7) -Trp-D-Ser (R6) -Tyr (R5) -D-Leu-Leu-Arg (NG-R4) -Pro-A1 ; R4 = Tos, R5 = 2-Br-Cbz, R6 = Bzl, R7 = Dnp, R8 = H and resin support

Boc-proline-resin of the formula is placed:

support

Boc-Pro-O — CH2

resin

(1.40 g, 0.5 mmol of proline) in the reaction vessel of an apparatus for automatic synthesis of Beckman type peptides, model 990, programmed to carry out the following cycle of washes: a) methylene chloride; b) 33% trifluoroacetic acid in methylene chloride (twice for 2.5 and 25 min); c) methylene chloride; d) ethanol; e) chloroform; f) 10% triethylamine in chloroform (2 times for 5 min each time); g) chloroform, and h) methylene chloride.

The washed resin is then stirred with t-butyloxycarbonyl-tosylarginine (645 mg, 1.5 mmol) in methylene chloride, and dicyclohexylcarbodiimide (1.5 mmol) is added. The mixture is stirred at room temperature (22-25 ° C) for 2 h, and the amino acid / resin mixture is then washed successively with methylene chloride (3 times), ethanol (3 times) and methylene chloride (3 times). The attached amino acid is stripped of its protection with 33% trifluoroacetic acid in methylene chloride (2 times for 2.5 and 25 min), then the washing cycle is carried out according to phases c) to h) previous.

The following amino acids (1.5 mmol) are then combined successively by the same cycle of operations: t-Boc-L-leucine; t-Boc-D-leucine; t-Boc-L-tyrosine (2-Br-Cbz); t-Boc-D-serine (Bzl); t-Boc-L-tryptophan; t-Boc-L-histidine (Dnp); L- (pyro) glutamic acid.

The completed nonapeptide / resin assembly is washed with methylene chloride (3 times), then with methanol (3 times), and then dried under reduced pressure, so that 96% of the theoretical weight gain is obtained. .

The proline / resin assembly used in this example is prepared from a chloromethylated resin available on the market (1% crosslinking, Bio Rad Labs, Richmond, California).

Example 6

L-Pyroglutamyl-L-histidyl-L-tryptophyl-D-seryl (benzyl) -L-tyrosyl (2-bromobenzyloxycarbonyl) -D-leucyl-L-leucyl-L-arginyl (tosyl) -L-propylethylamide, R8- ( pyro) Glu-His-Trp-D-Ser (R6) -Tyr (R5) -D-Leu-Leu-Arg (NG-R4) -Pro-NHR1;

R4 = Tos, R5 = 2-Br-Cbz, R6 = Bzl, R8 = H and R1 = C2H5

The nonapeptide / protected resin assembly (2.16 g, described in Example 5) is suspended in 20 ml of ethylamine at 0 ° C., then the mixture is stirred for 6 h. The excess ethylamine is then allowed to evaporate at room temperature, and the separated peptide is washed to rid it of the resin, with dimethylformamide. The protected peptide is then precipitated by the addition of ethyl acetate and filtered to give a cream-colored powder (672 mg). The Rf value on silica gel in a 1-butanol / acetic acid / water system (4/1/5, upper phase) is 0.45. This material is then used in Example 7 without further purification.

Example 7

L-Pyroç] lutamyl-L-histidyl-L-tryptophyl-D-seryl-L-tyrosyl-D-leucyl-L-leucyl-L-arginyl-L-propylethylamide; I, X = Ser, Y = D-Leu and Z = NHËt [(pyro) Gly-His-Trp-D-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt]

The removal of the protective groups from the protected nonapeptide, prepared as described in Example 6, is carried out by treatment of 670 mg of the material with hydrofluoric acid (50 ml) and anisole (15 ml) at 0 ° C for 30 min. The hydrofluoric acid is separated under reduced pressure and the anisole is removed by washing with ether.

The crude peptide is purified by gel filtration on a column (2.5 x 100 cm) of Sephadex G-25 (crosslinked, chemically modified dextran, of fine quality) by elution with acetic acid.

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0.2M and the fractions exhibiting a significant absorption peak in the ultraviolet at 280 nm are combined and evaporated to dryness.

The residual oil is applied to a column (2.5 x 100 cm) of Sephadex G-25 (fine quality), previously equilibrated with the lower phase, then with the upper phase of a n-butanol / acetic acid solvent system. / water (4/1/5). Elution with the upper phase gives a large fraction having a high absorption in the ultraviolet at 280 nm, and this material is subjected to chromatography on a column (1.5 x 94 cm) of silica gel, providing the elution with a 1-butanol / acetic acid / water mixture (4/1/1). The appropriate fractions (300-390 ml), after evaporation and lyophilization to constant weight from water, give the [D-Ser4, D-Leu6, desGly-NH210] -LH-RH ethylamide, in the form of '' a fluffy white powder (103 mg); [a] è3 = —29.6 ° (c = 0.54, HOAcO.lN).

The product is homogeneous during thin layer chromatography in four separate solvent systems on silica gel plates, when applying loads of 20-30 µg, and the stains are made visible by exposure to iodine vapor, then to Ehrlich's reagent. The following Rf 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 / 1M acetic acid (2/1): 0.43; 1-butanol / acetic acid / water / ethyl acetate (1/1/1/1): 0.50.

Analysis of the amino acids gives: Glu, 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.

R