KR910002702B1 - Method for producing peptide - Google Patents

Abstract

No content.

Description

[Name of invention]

Preparation of Peptides

Detailed description of the invention

The present invention relates to a peptide for inhibiting gonadotropin secretion by the pituitary gland and a peptide for preventing ovulation or inhibiting steroid secretion in mammals including humans. More specifically, the present invention relates to the preparation of peptides that inhibit the function of the gonads and the secretion of steroid hormones, progesterone and testosterone.

The pituitary gland is connected by a pituitary gland to a site known as the hypothalamus in the base of the brain. Especially follicle-stimulating hormone (FSH) and luteinizing hormone (LH), these hormones, sometimes called gonadotropin or gonadotropin, are secreted by the pituitary gland. Together, these hormones regulate the gonad's ability to produce testosterone in the testes and progesterone and estrogen in the ovaries, which also regulate the production and maturation of the gametes.

Hormonal secretion by the anterior pituitary gland requires that another class of hormones be released before the hypothalamus. One of the hypothalamic hormones acts as a factor that triggers the secretion of gonadotropins, especially LH, which is called GnRH, although it may be referred to as LH-RH and LRF. GnRH was isolated and identified as a decapeptide having the structure

p-Glu-His-Trp-Ser-Thr-Gly-Leu-Arg-Pro-Gly-NH ₂

Peptides are compounds that contain two or more amino acids and one carboxyl group of amino acids is linked to an amino group of another acid. The structure of GnRH as shown above follows the usual peptide notation, where the amino terminus is on the left and the carboxyl terminus is on the right. The positions of amino acid residues were identified by numbering amino residues from left to right. In the case of GnRH, the hydroxyl portion of the carboxyl group of glycine was substituted with an amino group (NH ₂ ). The abbreviations for the individual amino acid residues are common and are based on the generic names of amino acids. For example, p-Glu is pyroglutamic acid, His is histidine, Trp is tryptophan, Ser is serine, Tyr is tyrosine, Gly is glycine, and Leu is Leucine, Orn is Ornithine, Arg is Arginine, Pro is Proline, Sar is Salcosine, Phe is Phenylalanine and Ala is Alanine. These amino acids, along with valine, isoleucine, threonine, lysine, aspartic acid, asparagine, glutamine, cysteine, methionine, phenylalanine and proline, are usually considered to be naturally occurring amino acids or amino acids derived from proteins. The amino acids of the peptides of the invention, except glycine, are L-type unless otherwise noted.

There is a reason for wanting to suppress ovulation in mammalian females and GnRH analogs that antagonize normal function of GnRH have been used to inhibit or delay ovulation. For this reason GnRH analogs that antagonize GnRH have been investigated for use as contraceptives or for control of conception. GnRH antagonists can also be used to treat precocious adolescence or endometritis. These antagonists have also been shown to be useful for inhibiting gonadotropin secretion in mammalian males and have been found to be used, for example, as male contraceptives to inhibit spermatogenesis and to treat prostatic hyperplasia. It is desirable to obtain improved peptides that strongly antagonize endogenous GnRH and that inhibit steroid release and LH secretion by mammalian gonads.

The present invention provides a peptide that inhibits the secretion of gonadotropin in a mammal, including humans, which also provides a method for inhibiting the secretion of steroids by the gonads of male and female mammals. Advanced GnRH analogs strongly antagonize GnRH and have an inhibitory effect on mammalian reproduction. These analogs can be used to inhibit the production of gonadotropins and sex hormones in a variety of situations, including precocious adolescence, hormone dependent nephropathy, dysmenorrhea, endometritis.

In general, peptides are synthesized according to the present invention that strongly inhibit the secretion of gonadotropin by the pituitary gland of mammals, including humans, and / or inhibit steroid secretion by the gonads. These peptides are analogs of GnRH and they are substituted at the 1-position, preferably dehydro-Pro or β- (1- or 2-naphthyl) -D-alanine (hereinafter β-D-1NAL or (subject to β-D-2NAL) and the substitution in the 2-position of the D-Phe form, the substitution in the 3-position, preferably of the form D-Trp, D-3PAL or β-D-NAL, 4- Optional substitutions such as diamino acids containing up to 5 carbon atoms in position, any substitution of either (a) halogenated or methylated L-Phe or L-Tyr or (b) Arg in the 5-position; Substituents at the 6-position and optional substitutions at the 7-position, such as Nla, NML, Phe, Nva, Met, Tyr, Trp, Cys, PAL, and 4F-D-Phe. The modified D-Phe at the 2-position shows increased antagonistic activity by the specific modifications present in the benzene ring. Monosubstitution of hydrogen in the ring takes place in the para- or 4-position, the substitution being selected from chloro, bromo, fluoro, nitro and chloro, fluoro, and nitro are preferred. The dichloro substituent is at the 2, 4 or 3, 4 position of the ring. α-carbons may also be methylated, for example (C ^α Me / 4Cl) Phe. When unsubstituted D-Trp or β-D-NAL is present in the 3-position, R ₇ is preferably Tyr, PAL, Phe or 4-D-Phe, and D-3PAL is present in the 3-position In this case R ₅ is preferably Arg. The 1-position substitution may modify the alpha amino group to contain an acyl group such as formyl (For), acetyl, acrylyl, vinylacetyl (Vac) or benzoyl (Bz), and the acetyl (Ac) and acrylyl (Acr Is preferred. PAL and D-PAL represent the L- and D-isomers of pyridyl-alanine, wherein the β-carbon of Ala is preferably linked to the 3-position of the pyridine ring. When β-D-NAL is in the 1-position and R ₅ is not Arg, 4-4-NH ₂ -D-Phe, 4-guanido-D-Phe, D-His, D-Lys, D- Preferably, a hydrophilic D-amino acid residue such as Orn, D-Arg, D-Har (homoarginine) or D-PAL is present at the 6-position. When dihydro-Pro is in the 1-position, D-Trp, D-Phe, For-D-Trp, NO ₂ -D-Trp, D-Leu, D-Ile, D-Nle, D-Tyr, Although the D-isomers of lipophilic amino acids, such as D-Val, D-Ala, D-Ser (OtBu), β-D-NAL or (imBzl) D-His, are preferably in the 6-position, D-PAL may also be used. have. It is optional to replace Gly at 10-position with D-Ala with other substitutions.

These peptides are often called GnRH antagonists because they have a very high effect of inhibiting LH secretion. The peptide inhibits ovulation in mammalian females at very low doses of estrus and is effective in causing resorption of fertilized eggs when administered immediately after conception. These peptides are also effective as contraceptives for mammalian males.

More particularly, the peptide of the present invention is represented by the following structural formula (I).

XR _1- (W) D-Phe-R ₃ -R ₄ -R ₅ -R ₆ -R ₇ -Arg-Pro-R ₁₀

Wherein X is hydrogen or an acyl group of C _1-7 ; R ₁ is dihydro-Pro, D-pGlu, D-Phe, 4Cl-D-Phe, D-Trp, Pro or β-D-NAL; W is Cl, F, NO ₂ , C ^α Me / 4Cl, Cl ₂ or Br; R ₃ is D-3PAL, β-D-NAL or (Y) D-Trp, where Y is H, NO ₂ , NH ₂ , OCH ₃ , F, Cl, Br, CH ₃ , N ⁱⁿ For or N ⁱⁿ Ac; R ₄ is Ser, Orn, AAL or aBu; R ₅ is Tyr, Arg, (3F) Phe, (2F) Phe, (3I) Tyr, (3CH ₃ ) Phe, (2CH ₃ ) Phe, (3Cl) Phe or (2Cl) Phe; R ₆ is a D-isomer of a lipophilic amino acid or 4-NH ₂ -D-Phe, D-Lys, D-Orn, D-Har, D-His, 4-gua-D-Phe, D-PAL or D -Arg; R ₇ is Nle, Leu, NML, Phe, Met, Nva, Tyr, Trp, Cys, PAL or 4F-D-Phe; R ₁₀ is Gly-NH ₂ , D-Ala-NH ₂ or NH-Y ′, wherein Y ′ is lower alkyl, cycloalkyl, fluorolowalkyl or NHCONH-Q, and Q is H or lower alkyl; Provided that when R ₃ is D-3PAL R ₅ is Arg and when R ₃ is β-D-NAL or D-Trp, R ₇ is Tyr, PAL Phe or 4F-D-Phe. R ₆ is preferably 4-NH ₂ -D-Phe, D-Lys, D-Orn, D-Har, D-His, 4-gua when R ₁ is β-D-NAL and R ₅ is not Arg -D-Phe, D-PAL or D-Arg.

β-D-NAL is a D-isomer of alanine in which β-carbon atom is substituted by naphthyl, also called 3-D-NAL. It is preferable to use β-D-2NAL having naphthalene attached to the 2-position of the ring structure, but β-D-1NAL may also be used. PAL represents alanine in which β-carbon is substituted by pyridyl and it is preferable that the 3-position of the pyridine ring is connected. For D-Trp in the 3-position, a single substitution of hydrogen in the 5- or 6-position can be made, which is selected from chloro, fluoro, bromo, methyl, amino, methoxy and nitro, chloro, fluoro And nitro are preferred. Indole nitrogen may also be acylated with, for example, formyl (N ⁱⁿ For- or 1For-) or acetyl. N ⁱⁿ For-D-Trp and 6NO ₂ -D-Trp are preferred substituted residues. When unsubstituted D-Trp is used in the 3-position, certain residues such as Phe, 4F-D-Phe, preferably Tyr or PAL, are introduced at the 7-position. NML means N ^α CH ₃ -L-Leu. AAL means β-amino-Ala, aBu means α, Y diaminobutyric acid and one or Orn may be present at the 4-position. If Ser is not present in the 4-position, it is preferred to have Dihydro Pro in the 1-position. 4-gua-D-Phe means a residue of D-Phe containing guanidine substituted in the para position.

The peptides of the present invention are synthesized by conventional solution synthesis or by solid phase methods using chloromethylated resins, methylbenzhydrylamine resins (MBHA), benzhydrylamine (BHA) resins or other suitable resins known in the art. Can be. The solid phase method is performed by stepwise addition of amino acids in the chain, as detailed in US Pat. No. 4,211,693. It is desirable to add side chain protecting groups well known in the art to Ser, Tyr, and Arg (if they are present) and some substitutions, which may optionally be added to Trp before they are coupled to the chain and immobilized on the resin. Can be. This method provides a fully protected intermediate peptide resin.

The intermediate of the present invention can be represented by the following structure.

X ¹ -R _1- (W) D-Phe-R ₃ (X ² ) -R ₄ (X ³ ) -R ₅ (X ⁴ X ⁵ ) -R ₆ (X ⁵ ) -R ₇ (X ⁶ )- Arg (X ⁵ ) -Pro-X ⁷

Wherein X ¹ is an α-aminoprotecting group in a form known to be useful in the field in the step-fitting of the polypeptide and when X is a specific acyl group in the desired peptide composition, the hetero group may be used as a protecting group. Β-aminoprotecting groups belonging to X ¹ include (1) acyl protecting groups such as formyl (For), trifluoroacetyl, phthalyl, p-toluenesulfonyl (Tos), benzoyl (Bz), benzenesulfonyl, o-nitrophenylsilphenyl (Nps), tritylsulphenyl, o-nitrophenoxyacetyl, acrylyl (Acr), chloroacetyl, acetyl (Ac) and β-chlorobutyryl, (2) aromatic urethane type protecting groups such as Benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, ie p-chloro-benzyloxycarbonyl (Clz), p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl and p-methoxybenzyloxycarbon Neil; (3) aliphatic urethane type protecting groups such as tetrabutyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl and allyloxycarbonyl; (4) cycloalkylurethane type protecting groups such as cyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl; (5) thiurethane type protecting groups such as phenylthiocarbonyl; (6) alkyl type protecting groups such as allyl (Aly), triphenyldecyl (trityl) and benzyl (Bzl); (7) trialkylsilane groups such as trimethylsilane; There is this. Preferred α-aminoprotecting groups are Boc when X is hydrogen.

X ² is a protecting group for Trp's indole nitrogen such as hydrogen or benzyl, but in many synthesis, Trp does not need to be protected. X ³ is selected from the group consisting of acetyl, benzoyl, tetrahydropyranyl, t-butyl, trityl, benzyl and 2,6-dichlorobenzyl as the protecting group of an alcoholic hydroxyl group of hydrogen or Ser, with benzyl being preferred. .

Alternatively, when Ser is substituted, X ³ may be a protecting group of a branched chain amino group, such as Tos, Z or ClZ.

X ⁴ is a protecting group of the phenolic hydroxyl group of Tyr when hydrogen or Tyr is present, tetrahydropyranyl, t-butyl, trityl, benzyl, z, 4-bromobenzyloxycarbonyl and 2,6- Dichlorobenzyl. Preference is given to 2,6-dichlorobenzyl (DCB).

X ⁵ is, for example, a nitro, Tos, trityl, benzyloxycarbonyl, adamantyl oxycarbonyl, a protecting group of an imidazole group or an amino group or side chain guanidino group such as Arg, Lys, His such as Z and Boc or ⁴ may be a protecting group of the same Tyr or a protecting group of a Trp such as X ² or X ⁵ may be hydrogen which means that there is no protecting group at the branched chain atom. Tor is generally preferred.

X ⁶ is hydrogen, a Tyr protecting group such as X ⁴ , or a Cys protecting group, preferably selected from the group consisting of p-methoxybenzyl (MeOBzl), p-methylbenzyl, acetamidomethyl, trityl and Bzl. Most preferred protecting group is p-methoxybenzyl.

X ⁷ is Gly-O-CH _2- [resin support]; O-CH _2- [resin support]; D-Ala-O-CH _2- [resin support]; It may be Gly-NH- [resin support] or D-Ala-NH- [resin support] which may be hydride, amide, ester or OH of either Gly or D-Ala and may be directly bonded to Pro. .

The criterion for selecting a side chain protecting group of X ² -X ⁶ is that the protecting group must be safe for the reagent under the reaction conditions chosen for the α-amino protecting group removal at each synthesis step. The protecting group must not be released under coupling conditions and the protecting group must be able to be removed under reaction conditions that do not alter the peptide chain when the desired amino acid sequence is completed.

An ester of one of the many functional groups of the polystyrene resin support when the X ⁷ group is Gly-O-CH _2- [resin support], D-Ala-O-CH _2- [resin support] or O-CH _2- [resin support] It may be represented as. If the X ⁷ group is Gly-NH- [resin support] or D-Ala-NH- [resin support], the amide linkage connects Gly or D-Ala to the BHA resin or MBHA resin.

If X is, for example, acetyl in the final structure, it can be used as the X ¹ protecting group for any amino acid used in the 1-position or the α-amino group of D-NAL by adding it before the last amino acid is coupled to the peptide chain. have. However, the reaction may, for example, be carried out with acetic acid in the presence of dicyclohexylcarbodiimide (DCC) or preferably with acetic anhydride or by any suitable reaction known in the art (whether the side chain group is protected while After deprotection of the amino group), preferably with the resinous peptide).

Fully protected peptides can be separated from chloromethylated resin supports by ammonolysis known in the art to produce fully protected amide intermediates. Separation of the peptide from the benzhydryl amine resin as well as deprotection of the peptide can be performed at 0 ° C. with hydrofluoric acid (HF). It is desirable to add anisole to the peptide prior to treatment with HF. After removal of HF in vacuo, the separated deprotected peptides are usually treated with ether, decanted and dissolved in dilute acetic acid and lyophilized.

Accordingly, the present invention also provides a method for preparing a peptide or non-toxic salt thereof consisting of the following (a)-(c), wherein the peptide has the following structural formula.

XR _1- (W) D-Phe-R ₃ -R ₄ -R ₅ -R ₆ -R ₇ -Arg-Pro-R ₁₀

X is hydrogen or an acyl group of C ₇ or less; R ₁ is dihydro-Pro, D-pGlu, D-Phe, 4Cl-D-Phe, D-Trp, Pro or β-D-NAL; W is Cl, F, NO ₂ , C ^α Me / 4Cl, Cl ₂ or Br; R ₃ is D-3PAL, β-D-NAL or (Y) D-Trp, where Y is H, NO ₂ , NH ₂ , OCH ₃ , F, Cl, Br, CH ₃ , N ⁱⁿ For or N ⁱⁿ Ac; R ₄ is Ser, Orn, AAL or aBu; R ₅ is Tyr, Arg, (3F) Phe, (2F) Phe, (3I) Tyr, (3CH ₃ ) Phe, (2CH ₃ ) Phe, (3Cl) Phe or (2Cl) Phe; R ₆ is a D-isomer of a lipophilic amino acid, 4-NH ₂ -D-Phe, D-Lys, D-Orn, D-Har, D-His, 4-gua-D-Phe, D-PAL or D- Arg; R ₇ is Nle, Leu, NML, Phe, Met, Nva, Tyr, Trp, Cys, PAL or 4F-D-Phe; R ₁₀ is Gly-NH ₂ , D-Ala-NH ₂ or NH-Y ′, wherein Y ′ is lower alkyl, cycloalkyl, fluorolowalkyl or NHCONH-Q, and Q is H or lower alkyl; Provided that when R ₃ is D-3PAL R ₅ is Arg and when R ₃ is β-D-NAL or D-Trp, R ₇ is Tyr, PAL Phe or 4F-D-Phe.

(a) after producing the intermediate compound of formula

X ¹ -R _1- (W) D-Phe-R ₃ (X ² ) -R ₄ (X ³ ) -R ₅ (X ⁴ or X ⁵ ) -R ₆ (X ⁵ ) -R ₇ (X ⁶ ) Arg (X ⁵ ) -Pro-X ⁷

Wherein X ¹ is hydrogen or an α-amino protecting group; X ² is a protecting group of hydrogen or indole nitrogen; X ³ is a protecting group of an alcoholic hydroxyl group protecting group or a side chain amino group of hydrogen or Ser; X ⁴ is hydrogen or Tyr Is a protecting group of a phenolic hydroxyl group of X ⁵ and X ⁶ are each a hydrogen or a protecting group of each side chain present; X ⁷ is Gly-O-CH _2- (resin support), O-CH _2- (resin support) ), D-Ala-O-CH _2- (resin support), Gly-NH- (resin support) D-Ala-NH- (resin support), Gly-NH ₂ and ester amide and hydrazide do.

(b) remove one or more of the X ¹ -X ⁶ groups and / or separate from any resin support contained in X ⁷ , and (c) convert the resulting peptides to their non-toxic salts as desired.

Purification of the peptide was performed by ion exchange chromatography on a CMC column followed by n-butanol; Disperse chromatography on a column packed with Sephadex G-25 using an elution system with a 0.1 N acetate (1: 1) volume ratio, or by a patent document known in the art (J. Rivier, et al. J. Chromatography, 288 ( 1984) 303-328) using HPLC.

Peptides of the present invention are effective at doses of up to 100 μg / kg body weight when administered to estrus about noon to suppress ovulation in female rats. It may be necessary to use doses in the range of about 0.1-2.5 mg / kg body weight for long-term ovulation suppression. These antagonists are effective in inhibiting spermatogenesis when administered to male mammals and thus can be used as contraceptives. Because these compounds reduce testosterone levels (usually undesirable for men who are sexually active), it is reasonable to administer GnRH antagonists along with alternative amounts of testosterone. These antagonists can also be used to modulate gonadotropin and sex steroids for the other purposes mentioned above.

Example I

Peptides as in Table 1 having the structural formula below are prepared by the solid phase method described above.

Ac-R _1- (4F) D-Phe-R ₃ -Ser-Tyr-R ₆ -Leu-Arg-Pro-R ₁₀

TABLE 1

Representative peptide No. 1 (hereinafter referred to as [Ac-β-D-2NAL ¹ , (4F) D-Phe ² , (6NO ₂ ) D-Trp ³ , D-Arg ⁶ ] -GnRH) for illustrative purposes) The solid phase synthesis of is described below. This peptide has the following structural formula;

Ac-β-D-2NAL- (4F) D-Phe- (6NO ₂ ) D-Trp-Ser-Tyr-D-Arg-Leu-Arg-Pro-Gly-NH ₂ .

Other peptides are similarly synthesized and purified.

A BHA resin was used and Boc-protected Gly was coupled to the resin over 2 hours in CH ₂ Cl ₂ using a 3 fold excess of Boc derivative and DCC as activator. Glycine residues are attached to the BHA resin by amide bonds.

Coupling, washing, deprotection of each amino acid residue, coupling of the next amino acid residue is carried out using an automated device according to the following schedule starting with about 5 g of resin.

After step 13, a portion of the liquid is taken and tested for ninhydrin, if the test is negative, sent to step 1 for coupling of the next amino acid; If the test is positive or slightly positive, repeat steps 9-13.

The schedule is used to couple each amino acid of the peptide of the invention after attaching the first amino acid. N ^α Boc protection is performed on each of the remaining amino acids in the synthesis. N ^α Boc-β-D-2NAL is prepared according to methods known in the art. (USPatent No. 4,234,571, November 18, 1980) Arg side chains are protected by Tos.

OBzl is used as a side chain protecting group of the hydroxyl group of Ser. (6NO ₂ ) Leave D-Trp unprotected. N ^α Boc-β-D-2NAL is introduced as the final amino acid. Boc-Arg (Tos) and Boc- (6NO) D-Trp having low solubility in CH ₂ Cl ₂ are selected from DMF; Coupling using a CH ₂ Cl ₂ mixture.

After deprotection of the N-terminal α-amino group, acetylation is carried out using excess acetic anhydride in dichloromethane. Separation of the peptide from the resin and complete deprotection of the side chains occur very easily with HF at 0 ° C. Anisole is added as a scavenger prior to HF treatment. After removal of HF in vacuo, the resin is extracted with 50% acetic acid and the liquor is lyophilized to obtain the crude peptide powder.

Ion exchange chromatography was then performed on CMC (Whatman CM32,50 / 50) using a gradient of 0.05-0.3 M NH ₄ OAc in methanol / water. Elution system: n-butanol: 0.1-acetic acid (1: 1 Dispense chromatography was performed in a gel filtration column using a volume ratio.

Thin layer chromatography and several other solvent systems were also used to determine if the peptide was homogeneous using reverse phase high pressure liquid chromatography and aqueous triethylammonium phosphate solution and acetonitrile. The amino acid analysis of the resulting pure peptides was consistent with the structural formula of the prepared structure and showed that each amino acid in the chain had a substantially integer value. The photoluminescence was determined to be [α] _D22 = -31.8 ° ± 1 (c = 1.50% acetic acid) on the photoelectric photometer.

Peptides were assayed in vivo and in vitro. In vitro tests were performed using the excised rat pituitary cells maintained in culture 4 days prior to testing. LH levels sensitive to peptide use were measured for mouse LH using a special radioimmunoassay. The cells of the control plate supplied only 3 nanomolar GnRH and the experimental plate, in addition to the 3 nanomolar GnRH, the comparative standard antagonist of the present invention [Ac-dehydro Pro ¹ , (4-F) D-Phe ² , D- Trp ^3.6 ] -GnRH or test peptides were supplied at a concentration of 0.01-10 nanomolar. The amount secreted in the sample treated only with GnRH was compared with the amount secreted in the sample treated with peptide + GnRH. The ability of the test peptide to reduce the amount of LH liberated by 3 nanomolar GnRH was compared to the standard foam peptide. In vivo testing determined the effect of inhibiting ovulation in female rats. In this trial, a certain number of ripe female Sprague-Dawley rats, weighing 225-250 g each, were injected with corn oil in a specific dose (μg) at about midday estrus. The estrus is ovulation afternoon. A separate group of mice was used as a control without the peptide. Each control cancer rat ovulated in the evening of estrus and recorded the ovulated number of treated mice. Each peptide appears to be very effective at inhibiting ovulation in rats at very low doses, and each peptide appears to be effective at about 5 μg doses. Another test was conducted at lower doses and the results are shown in Table A below.

TABLE A

All peptides listed in Table 1 have been shown to be effective in blocking GnRH-induced LH secretion at several moderate concentrations in in vitro tests. Many of these peptides have been shown to have even higher titers in vivo than this standard. All peptides are believed to be effective at inhibiting ovulation in female mammals at very low doses and some choices have been shown to have titers of at least two times that of any of the known and tested GnRH antagonists.

Example II

Peptides described in Table II having the following structural formula were prepared by the solid phase method as described above.

Ac-β-D-2NAL- (W) D-Phe-R ₃ -R ₄ -R ₅ -D-Arg-R ₇ -Arg-Pro-D-Ala-NH ₂

TABLE II

In vitro and / or in vivo testing of the peptides listed in Table II showed that the peptides listed in Table II were effective at blocking GnRH-induced LH secretion in vitro at appropriate doses. Many of these peptides have higher titers in vivo than the present standard. All peptides have been shown to be effective at preventing ovulation of female mammals at very low doses.

Example III

Peptides as described in Table III having the following structural formula were prepared by the solid phase method described above.

X-β-D-2NAL- (4CL) D-Phe- (6NO ₂ ) D-Trp-Ser- R ₅ -R ₆ -NML-Arg-Pro-R ₁₀

TABLE III

In vitro and / or in vivo testing of the peptides listed in Table III showed that the peptides listed in Table III were effective at blocking GnRH induced LH secretion in vitro at moderate concentrations. Many of these peptides have higher titers in vivo than this standard. All peptides have been shown to be effective at inhibiting ovulation of female mammals at very low doses.

Example IV

Peptides as described in Table IV having the structural formula below were prepared by the solid phase method described above.

Ac-R ₁ -4Cl-D-Phe-D-Trp-Ser-Tyr-R ₆ -R ₇ -Arg-Pro-R ₁₀ -NH ₂

Table IV

The peptide No. 45 [also referred to as [Ac-β-D-2NAL ¹ , 4Cl-D-Phe ² , D-Trp ³ , D-Arg ⁶ , Nle ⁷ , D-Ala ¹⁰ ] -GnRH] has the following structural formula .

Ac-β-D-2NAL-4Cl-D-Phe-D-Trp-Ser-Tyr-D-Arg-Nle-Arg-Pro-D-Ala-NH ₂

The photoluminescence of the various synthetic peptides was measured in 50% acetic acid (c = 1) on a photophotometer and reported in Table B below.

All peptides listed in Table IV have been shown to be effective at blocking GnRH-induced LH secretion in vitro at appropriate doses. Many of these peptides have higher titers in vivo than this standard. All peptides have been shown to be effective at inhibiting ovulation in female mammals at very low doses and some have been shown to have a titer of at least twice the GnRH antagonist previously known and tested.

Example V

Peptides as described in Table V having the following structural formula were prepared by the solid phase method described above.

Ac-R _1- (W) D-Phe-R ₃ -Ser-Tyr-R ₆ -R ₇ -Arg-Pro-D-Ala-NH ₂

TABLE V

In vitro and / or in vivo testing of the peptides specified in Table V showed that the peptides listed in Table V were effective at blocking GnRH-induced LH secretion in vitro at appropriate concentrations. Many of these peptides have higher in vivo titers than this standard. All peptides have been shown to be effective at inhibiting ovulation of female mammals at very low doses.

Example VI

Peptides / peptides as described in Table VI having the following structural formula were prepared by the solid phase method described above.

XR ₁ -4F-D-Phe-D-Trp-Ser-Tyr-R ₆ -R ₇ -Arg-Pro-D-Ala-NH ₂

Table VI

In vitro and / or in vivo testing of the peptides listed in Table VI showed that the peptides listed in Table VI were effective at blocking GnRH-induced secretion in vitro at moderate concentrations.

Many of these peptides have higher in vivo titers than this standard. All peptides have been shown to be effective at inhibiting ovulation of female mammals at very low doses.

EXAMPLE VII

Peptides as described in Table VII having the structural formula below were prepared by the solid phase method described above.

Ac-R _1- (W) D-Phe-R ₃ -R ₄ -Tyr-R ₆ -Leu-Arg-Pro-D-Ala-NH ₂

[Table VII]

In vitro and / or in vivo testing of the peptides listed in Table VIII showed that the peptides listed in Table VIII were effective at blocking GnRH-induced LH secretion in vitro at appropriate concentrations. Many of these peptides have higher in vivo titers than this standard. All peptides have been shown to be effective at inhibiting ovulation in female mammals at very low doses.

In vivo testing was performed at different doses for the various peptides and the results are shown in Table B.

TABLE B

EXAMPLE VII

Peptides as described in Table VII having the structural formula below were prepared by the solid phase method described above.

Ac-R ₁ -R ₂ -D-3PAL-Ser-Arg-R ₆ -R ₇ -Arg-Pro-R ₁₀

[Table VII]

In vivo tests were performed on the peptides listed in Table VII to assess the effect of inhibiting ovulation in female rats. All of them have been shown to inhibit ovulation in female rats at very low doses and have been shown to be effective at doses of about 10 μg. Special evaluation of many of these peptides was performed at low doses and the results are shown in Table C below.

TABLE C

Peptides of the invention are often in the form of pharmaceutically acceptable non-toxic salts, such as acid addition salts, or metal complexes such as, for example, zinc, barium, calcium, magnesium, aluminum (referred to as addition salts in this application), or mixed forms thereof. Administered. Examples of such acid addition salts include hydrochloride, hydrochloride, sulphate, phosphate, nitrate, oxalate, fumarate, gluconate, tannin, maleate, acetate, citrate, benzoate, succinate, alginate, malate, There are such as ascorbates and tartarates.

For example, an aqueous solution of the peptide may be repeated with 1N acetic acid and then lyophilized to form its acetate. If the active ingredient is administered in tablet form, the tablet may contain a pharmaceutically acceptable diluent, which may include binders such as tragacanth rubber, corn starch or gelatin; Disintegrants such as alginic acid; Lubricants such as magnesium stearate are included. If it is desired to administer in liquid form, a pharmaceutically acceptable diluent may be used as a fragrance and / or sweetener, and intravenous administration is effective by dissolving in isotonic saline, phosphate buffer, and the like.

Pharmaceutical compositions usually contain a conventional pharmaceutically acceptable carrier with the peptide. Generally, the dose will be about 1-100 μg per kg of body weight administered intravenously and higher when administered orally. Treatment of patients with these peptides is carried out in the same manner as clinical treatment with other GnRH antagonists.

These peptides can be administered intravenously, subcutaneously, intramuscularly, orally, transdermally, e.g., nasal, in order to inhibit and / or control fertilization, and to require reversible inhibition of gonadal activity, such as modulating precocious adolescence or during radiation or chemotherapy. It can be administered to mammals by intranasal or vaginal route. The effective amount depends on the dosage form and the particular mammal species being treated. One typical dosage form is an antimicrobial aqueous solution containing a peptide and the solution is administered at a dose of about 0.1-2.5 mg / kg body weight per kilogram. Oral administration of the peptide may be administered in solid or liquid form.

Although the invention has been described in terms of preferred embodiments, modifications and variations apparent to those skilled in the art can be made without departing from the scope of the invention as set forth in the appended claims. Other substitutions known in the art can be used in the peptides of the invention, for example, which do not significantly impair the effects of the pupide.

Substitution of the phenyl ring of the D-Phe ² residue may also be made at the 3- and 2,4 positions, which is considered equivalent and similarly considered equivalent to D-2PAL and D-4PAL. At the C-terminus Pro ⁹ may be bound to the following component which is generally regarded as equivalent; Gly-OCH ₃ , Gly-OCH ₂ CH ₃ , Sar-NH ₂ or NH-Y 'wherein Y' is lower alkyl, in particular ethyl, cycloalkyl, fluorolowalkyl or NHCONHQ and Q is H or lower alkyl Represents salcosine.

The features of the invention are highlighted in the following claims.

Claims (11)

Peptides having the formula or non-toxic salts thereof XR _1- (W) D-Phe-R ₃ -R ₄ -R ₅ -R ₆ -R ₇ -Arg-Pro-R ₁₀ wherein X is hydrogen or C _1- An acyl group of ₇ ; R ₁ is dihydro-Pro, D-pGlu, D-Phe, 4Cl-D-Phe, D-Trp, Pro or β-D-NAL; W is Cl, F, NO ₂ , C ^α Me / 4Cl, Cl ₂ or Br; R ₃ is D-3PAL, β-D-NAL or (Y) D-Trp, where Y is H, NO ₂ , NH ₂ , OCH ₃ , F, Cl, Br, CH ₃ , N ⁱⁿ For or N ⁱⁿ Ac; R ₄ is Ser, Orn, AAL or aBu; R ₅ is Tyr, Arg, (3F) Phe, (2F) Phe, (3I) Tyr, (3CH ₃ ) Phe, (2CH ₃ ) Phe, (3Cl) Phe or (2Cl) Phe; R ₆ is a D-isomer of a lipophilic amino acid or 4-NH ₂ -D-Phe, D-Lys, D-Orn, D-Har, D-His, 4-gua-D-Phe, D-PAL or D -Arg; R ₇ is Nle, Leu, NML, Phe, Met, Nva, Tyr, Trp, Cys, PAL or 4F-D-Phe; R ₁₀ is Gly-NH ₂ , D-Ala-NH ₂ or NH-Y ′, wherein Y ′ is lower alkyl, cycloalkyl, fluorolowalkyl or NHCONH-Q, and Q is H or lower alkyl; Provided that when R ₃ is D-3PAL R ₅ is Arg and when R ₃ is β-D-NAL or D-Trp, R ₇ is Tyr, PAL Phe or 4F-D-Phe. The peptide of claim 11, wherein W is 4Cl or 4F. The method according to claim 11 or 12, wherein R ₁ is β-D-2NAL and R ₆ is 4-NH ₂ -D-Phe, D-Lys, D-Orn, D-Har, D-His, 4-gua A peptide that is -D-Phe, D-PAL or D-Arg. The peptide of claim 13, wherein R ₆ is D-Arg. The peptide of claim 11 or 12, wherein R ₇ is Tyr or 3PAL. The peptide of claim 11 or 12, wherein R ₃ is (Y) D-Trp and Y is 6NO ₂ or N ⁱⁿ For. The peptide according to claim 11 or 12, wherein R ₁ is β-D-2NAL, R ₃ is D-PAL, and R ₅ is Arg. 18. The peptide of claim 17, wherein R ₆ is D-Trp, D-PAL, β-D-NAL, (imBzl) D-His or (6NO ₂ ) d-Trp. The peptide of claim 18, wherein R ₆ is (imBzl) D-His or (6NO ₂ ) D-Trp and R ₇ is Leu. The peptide of claim 18 or 19, wherein R ₄ is Ser, X is Ac or Acr and R ₁₀ is D-Ala-NH ₂ or Gly-NH ₂ . A pharmaceutical composition for regulating the secretion of gonadotropin in a mammal, comprising as an active ingredient a combination of an effective amount of a peptide as defined in any one of claims 11-20 and a large amount of non-toxic diluent.