WO1994019370A1

WO1994019370A1 - Gnrh antagonists

Info

Publication number: WO1994019370A1
Application number: PCT/US1994/001891
Authority: WO
Inventors: Jean E. F. Rivier; Catherine L. Rivier
Original assignee: The Salk Institute For Biological Studies
Priority date: 1993-02-22
Filing date: 1994-02-18
Publication date: 1994-09-01
Also published as: AU6246894A; ZA94896B; IL108509A0

Abstract

Peptides having significantly modified amino acids inhibit the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. Administration of an effective amount of such GnRH antagonists prevents ovulation of female mammalian eggs and/or the release of steroids by the gonads. These antagonists may be used to treat steroid-dependent tumors, such as prostatic and mammary tumors. A particularly effective analog of the decapeptide GnRH which is highly soluble in water at physiologic pH has the formula: Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-NαCH3Aph(3-amino 1,2,4 triazole)-D-Aph(3-amino 1,2,4 triazole)-Leu-Lys(isopropyl)-Pro-D-Ala-NH2.

Description

GNRH ANTAGONISTS.

This invention relates generally to peptides which are antagonists of human gonadotropin releasing hormone (GnRH) and more particularly to GnRH antagonists which include significantly modified amino acids which have advantageous physical, chemical and biological properties. In a more particular aspect, the present invention relates to decapeptides which inhibit the gonadal function and the release of the steroidal hormones progesterone and testosterone, and to methods of administering such decapeptides for such purpose and particularly to prevent ovulation.

BACKGROUND OF THE INVENTION The pituitary gland is attached by a stalk to the region in the base of the brain known as the hypothalamus. In particular, follicle stimulating hormone (FSH) and luteinizing hormone (LH) , sometimes referred to as gonadotropins or gonadotropic hormones, are released by the pituitary gland. These hormones, in combination, regulate the functioning of the gonads to produce testosterone in the testes and progesterone and estrogen in the ovaries, and they also regulate the production and maturation of gametes.

The release of a hormone by the anterior lobe of the pituitary gland usually requires a prior release of another class of hormones produced by the hypothalamus. One of the hypothalamic hormones acts as a factor that triggers the release of the gonadotropic hormones, particularly LH, and this hormone is referred to herein as GnRH although it has also been referred to as LH-RH and as LRF. GnRH was isolated and characterized as a decapeptide some 20 years ago, and it was found shortly thereafter that analogs of GnRH having a D-isomer instead of Gly in the 6-position, such as [D-Ala⁶]-GnRH (U.S. Patent No. 4,072,668) having the following formula: pGlu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH₂, have greater binding strength to the receptor and greater biological potency than the native hormone.

Peptides are compounds which contain two or more amino acids in which the carboxyl group of one acid is linked to the amino group of the other acid. The formula for GnRH, as represented above, is in accordance with conventional representation of peptides where the amino terminus appears to the left and the carboxyl terminus to the right. The position of the amino acid residue is identified by numbering the amino acid residues from left to right. In the case of GnRH, the hydroxyl portion of the carboxyl group of glycine at the C-terminus has been replaced with an amino group(NH₂) i.e. the C-terminus is amidated. The abbreviations for the individual amino acid residues above are conventional and are based on the trivial name of the amino acid, e.g. pGlu is pyroglutamic acid, Glu is glutamic acid. His is histidine, Trp is tryptophan, Ser is serine, Tyr is tyrosine, Gly is glycine. Leu is leucine, Nle is norleucine, Orn is ornithine, Arg is arginine, Har is homoarginine. Pro is proline, Sar is sarcosine, Phe is phenylalanine, Ala is alanine, Val is valine, Nva is norvaline, lie is isoleucine, Thr is threonine, Lys is lysine. Asp is aspartic acid, Asn is asparagine. Gin is glutamine, and Met is methionine. Except for glycine, amino acids of the peptides of the invention are of the L-configuration unless noted otherwise.

There are reasons for desiring to prevent ovulation in female mammalians, and the administration of GnRH analogs that are antagonistic to the normal function of GnRH have been used to suppress or delay ovulation. For this reason, analogs of GnRH which are antagonistic to GnRH are being investigated for their potential use as a contraceptive or for regulating conception periods. GnRH antagonists may also be used for the treatment of precocious puberty and endometriosiε. Such antagonists have also been found useful to regulate the secretion of gonadotropins in male mammals and can be employed to arrest spermatogenesis, e.g. as male contraceptives for treatment of male sex offenders, and for treatment of prostatic hypertrophy. More specifically, GnRH antagonists can be used to treat steroid-dependent tumors, such as prostatic and mammary tumors, and for the control of the timing of ovulation for in vitro fertilization. In the female, they can also be used to treat hirsutism.

There are a number of peptides that are known to cause histamine to be released from mast cells which cells are found in the skin, the gingiva and other locations throughout the body. As a result, inflammation is caused, often resulting in edema of the face and elsewhere on the skin. It was earlier found that certain GnRH antagonists that were effective in preventing ovulation had the undesirable adverse side effect of stimulating histamine release, generally rendering such GnRH analogs unacceptable for administration to humans. As a result, the design of GnRH analogs was directed to providing peptides that would retain the biological efficacy but would not have such undesirable histamine release, see J. Rivier et al., J. Med. Chem.. 29. 1846- 1851 (1986) . In addition, it is felt to be important that the peptide analog should have good duration of action upon LH secretion, a property which is considered to be enhanced by resistance to proteolytic enzyme degradation in the body, and thus this property was also kept in focus in analog design. In addition to facilitate administration of these compounds to mammals, particularly humans, it is considered extremely advantageous for the GnRH decapeptide to have high solubility in water at normal physiologic pH, i.e. about pH 5 to about pH 7.4.

In J. Rivier et al., J. Med. Chem.. 35. 4270-4277 (1992) , the design and synthesis of GnRH antagonists having improved properties in various of these respects are described. Despite the attractive properties of these GnRH analogs, the search has continued for still further improved GnRH antagonists.

SUMMARY OF THE INVENTION

It has now been found that GnRH antagonist deca¬ peptides having the following formula, and closely related analogs thereof, have particularly improved properties in all of the aforementioned respects: Ac-,9-D-2NAL-(4C1)D-Phe-D-3PAL-Ser-N^βCH₃Aph(3-amino 1,2,4 triazole)-D-Aph(3-amino 1,2,4 triazole)-Leu- Lys(isopropyl)-Pro-D-Ala-NH₂.

These antagonists are particularly useful as fertility regulators in humans for they are extremely biopotent and long-acting. They exhibit negligible side effects of stimulating histamine release, and they have excellent solubility in aqueous buffers at physiologic pHs. The presence of the lower alkyl, e.g. methyl, substitution at the alpha-nitrogen of the modified phenylalanine residue in the 5-position, in combination with the side chains of the 5- and 6-position residues which include the amino triazole-substituted phenyl moieties, is believed to be responsible for the substantial improvements exhibited, including high solubility and biopotency of long-acting duration. As a result, this decapeptide finds particular use in administration to mammals, especially humans, as a fertility regulator and for the treatment of pathological conditions such as precocious puberty, hormone dependent neoplasia, dysmenorrhea, endometriosis and steroid- dependent tumors. These GnRH antagonists are extremely soluble in the physiologic pH range of about 5 to about 7.4, and therefore they can be formulated and administered in concentrated form, particularly at a pH between about 5 and about 7. The antagonists are also effective for the contraceptive treatment of male mammals and for the treatment of steroid-dependent tumors. The antagonists are surprisingly long-acting in their suppression of LH levels following administration and have a particularly low side effect in respect of histamine release.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS U.S. Patent No. 5,169,932, issued December 8, 1992, discloses the design and synthesis of a number of GnRH antagonists wherein the side chains of selected residues are reacted to create cyanoguanidino moieties, some of which subsequently spontaneously convert to a desired heterocycle, e.g. a 3-amino, 1,2,4 triazole. The cyanoguanidino moieties are built upon the omega-amino group in an amino acid side chain such as lysine, ornithine or 4-amino phenylalanine (Aph) or an extended chain version thereof, such as 4-amino homophenylalanine (Ahp) . GnRH antagonists having such significantly modified or unnatural amino acids in the 5- and 6- positions have good biological potency, and it has now been found that, in combination with a lower alkyl substitution to the alpha-amino group of the 5-position residue in the decapeptide, particularly advantageous overall properties are obtained. Over the last decade, the particular properties of each of the 10 residues in the sequence of GnRH, from the standpoint of creating an effective antagonist, have been studied in depth, and as a result of these studies, it has been discovered that there are various equivalent residues that can be chosen and that substitutions of one of these equivalents for another does not significantly detract from the biological potency of the decapeptide GnRH antagonist. The N-terminus is preferably N- acylated. Generally in the 1-position at the N-terminus, it has been shown that an N-acylated D-isomer having an aromatic ring structure in its side chain is suitable, such as substituted D-Phe, substituted or unsubstituted D-Trp, or 3-D-Nal, and these 3 residues can generally be interchangeably employed at the N-terminus without substantially altering biological efficacy. It has also become generally accepted that the inclusion of a para-substituted or a 2,4 chloro- disubstituted or a pentamethyl(Me₅)D-Phe residue in the 2- position adds significantly to GnRH antagonist activity and that the specific identity of the ring substituent is of only relatively minor importance when selected from among the following: chloro, fluoro, bro o, nitro, methyl and alkoxy. With respect to the 3-position of the molecule, it has been well accepted that a D-isomer having a multiple-ring side chain is preferred, and therefore D-Pal, D-Nal or D-Trp (unsubstituted or with a substitution such as 6N0₂ or N'ⁿFor) can be alternatively employed without significantly changing biological antagonist activity. Although equivalents which are tolerated have also been disclosed for serine in the 4- position, such have not significantly improved biological activity, and the native residue is preferred in this position.

It is disclosed in U.S. Patent No. 4,652,550, issued March 24, 1987, that a number of substitutions can be made for Leu in the 7-position without significantly detracting from biological potency, and thus these potential substituents are now considered to be equivalents. However, generally Leu or N^βCH₃Leu(NML) has been used, and one of these two or Nle or Phe is generally preferred. In the 8-position, the naturally- occurring residue Arg can be employed, or it can be substituted by Har, isopropyl lysine (ILys) or isopropyl ornithine (IOrn) which are considered to be equivalents for this position. In the 9-position, the native residue Pro is preferred; however, N-methylalanine can be used as an equivalent. Although the amidated native residue Gly can be employed at the C-terminus, D-alanine having its alpha-carboxyl group amidated is preferred. Other equivalents either use AzaGly-NH₂, or omit the 10-position residue and instead use the ethylamide of proline in the 9-position.

In view of the foregoing, the present invention is broadly considered to provide a family of GnRH antagonists represented by the following formula: G-AA,-(A)D-Phe-AA₃-Ser-(R,)AA_j-AA^AA^AA_g-Pro-AA^ wherein G is an acyl group having 7 or less carbon atoms ; AA.. is (A) D-Phe , (B) D-Trp or 0-D-NAL; A is Cl , F, N0₂ , Br, CH₃, OCH₃ , Me₅ or Cl₂ ; B is H, N0₂, OCH₃, F, Cl , Br, CH₃ or N^ιnFor ; AA₃ is D-PAL, ,5-D-NAL or (B) D-Trp ; kA^ is Xaa ; R, is N"CH₃ or N'Oi-.CH_j ; AA₆ is D-Xaa ; AA₇ is Leu, NML, Nle, Phe, Met, Nva , Tyr, Trp or PAL; AA_g is Arg, Har, ILys or IOrn; AA₁₀ is D-Ala-NH₂, Gly-NH₂, AzaGly-NH₂ or NH R^ ; R₂ is lower alkyl , preferably CH₂CH₃ ; and Xaa is Aph , Ahp , Lys or Orn having its ω-amino group substituted by

NH.

H

By /S-D-NAL is meant the D-isomer of alanine which is substituted by naphthyl on the /9-carbon atom, i.e., also 3-D-NAL. Preferably _/9-D-2NAL is employed wherein the attachment to naphthalene is at the 2-position on the ring structure; however, 3-D-1NAL may also be used. The preferred 1-position residues are 0-D-NAL, substituted D- Phe and optionally substituted D-Trp. PAL represents alanine which is substituted by pyridyl on the 3-carbon atom; preferably the linkage is to the 3-position on the pyridine ring. When substituted D-Trp is employed, single substitutions for hydrogen are preferably made in either the 5- or 6-position, which are selected from chloro, fluoro, bromo, methyl, amino, methoxy and nitro, with chloro, fluoro and nitro being preferred. Alternatively, the indole nitrogen may be acylated with formyl (N¹ⁿFor- or lFor-) . D-3PAL, NⁱⁿFor-D-Trp and 6N0₂-D-Trp are the preferred residues for the 3-position although ,3-D-2Nal and D-Trp are also often used. By dehydroPro is meant 3,4 dehydroproline, C₅H₇0₂N (Δ³Pro) . By NML is meant .WCH-_j-L-Leu. By Aph is meant 4NH₂Phe; by Ahp is meant 4NH₂-homophenylalanine. By AzaGly-NH₂ is meant NHNHCONH₂. The 7-position residue is preferably Leu, NML, Nle or Phe, and the 8-position residue is preferably ILys. A preferred subgenus of GnRH antagonists has the formula:

AC-_/S-D-2NAL-(4C1)D-Phe-D-3PAL-Ser-(R,)AA_jj-AA^AA^Lys (isopropyl)-Pro-AA₁₀ wherein AA_j is Xaa, R.. is N"CH₃ or N^βCH₂CH₃, AA₆ is D-Xaa, AA₇ is Leu or N°CH₃Leu; AA₁₀ is D-Ala-NH₂, Gly-NH₂, or NHCH₂CH₃; Xaa is Aph(atz) , Ahp(atz) , Lys(atz) or Orn(atz) ; and atz is

An additional preferred subgenus of GnRH antagonists has the formula:

AC-0-D-2NAL-(4C1)D-Phe-D-3PAL-Ser-(R,)AA_j-AA^AA^Lys (isopropyl)-Pro-AA₁₀ wherein AA_j is Xaa; R., is N^βCH₃ or n^aC¥₂CE₃ ; AA₆ is D-Xaa ; AA₇ is Leu or NML; AA₁₀ is D-Ala-NH₂ , Gly-NH₂ or NHCH₂CH₃ ; and Xaa is

HO

^? n is 1 or 2.

Ano er pre erre subgenus of GnRH antagonists has the formula :

Ac-,3-D-2NAL- (4C1) D-Phe-D-3PAL-Ser-N^αCH₃AA₅-AA₆-Leu-Lys ( isopropyl ) -Pro-D-Ala-NH₂, wherein AA_j is Xaa ; AA₆ is D-Xaa and Xaa is

The peptides of the present invention can be synthesized by classical solution synthesis, but are preferably synthesized by a solid phase technique. A chloromethylated resin or a hydroxymethylated resin may be used; however, a methylbenzhydrylamine(MBHA) resin, a benzhydrylamine (BHA) resin or some other suitable resin known in the art which directly provides a C-terminal amide or substituted amide upon cleavage is preferably employed. For example, peptides having a substituted amide at the C-terminus are preferably synthesized using an N-alkylamino methyl resin as taught in United States Patent No. 4,569,967, issued February 11, 1986. Solid phase synthesis is conducted in a manner to stepwise add amino acids in the chain in the manner set forth in detail in the U.S. Patent No. 4,211,693. Side-chain protecting groups, as are well known in the art, are preferably included as a part of any amino acid which has a particularly reactive side chain and optionally in the case of others, such as Trp, which amino acids are to be coupled in the chain being built upon the resin. Such synthesis provides the fully protected intermediate peptidoresin.

Chemical intermediates made generally in accordance with the invention may be represented by the formula: X¹-AA,(X²)-(A)D-Phe-AA₃(X²)-Ser(X³)-(R,)AA_JJ- AA₆-AA₇(X² or X^A)-AA₈(X⁵ or X⁶)-Pro-X⁷ wherein X¹ is an α-amino protecting group of the type known to be useful in the art in the stepwise synthesis of polypeptides and when G in the desired peptide composition is a particular acyl group, that group may be used as the protecting group. Among the classes of α-amino protecting groups covered by X¹ are (1) acyl-type protecting groups, such as formyl(For), trifluoroacetyl, phthalyl, p-toluene- sulfonyl(Tos) , benzoyl(Bz), benzenesulfonyl, dithiasuccinoyl(Dts) o-nitrophenylsulfenyl(Nps) , tritylsulfenyl, o-nitrophenoxyacetyl, acrylyl(Acr) , chloroacetyl, acetyl(Ac) and γ-chlorobutyryl; (2) aromatic urethan-type protecting groups, e.g., benzyloxycarbonyl(Z) , fluorenylmethyloxycarbonyl(Fmoc) and substituted benzyloxycarbonyl, such as p-chlorobenzyloxy-carbonyl(ClZ) , p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl and p-methoxybenzyloxycarbonyl; (3) aliphatic urethan protecting groups, such as tertbutyloxycarbonyl(Boc) , diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl and allyloxycarbonyl; (4) cycloalkyl urethan-type protecting groups, such as cyclopentyloxycarbonyl, adamantyloxycarbonyl and cyclohexyloxycarbonyl; (5) thiourethan-type protecting groups, such as phenylthiocarbonyl; (6) alkyl-type protecting groups, such as allyl(Aly), triphenylmethyl(trityl) and benzyl(Bzl) ; (7) trialkylsilane groups, such as trimethylsilane. The preferred α-amino protecting group is Boc when X is hydrogen. X² is hydrogen or a protecting group for the indole nitrogen of Trp, such as Bz, Ac or For. In many syntheses there is no need to protect Trp, and such protection is not used if acylated D-Trp is present elsewhere in the peptide. X³ is a protecting group for the hydroxyl side chain of Ser, e.g. Ac, Bz, trityl, DCB or benzyl ether(Bzl) and is preferably Bzl.

X* is hydrogen or a protecting group for the phenolic hydroxyl group of Tyr selected from the group consisting of tetrahydropyranyl, tert-butyl, trityl, benzyl, Z, 2-bromobenzyloxycarbonyl(2BrZ) and 2,6-dichloro- benzyl(DCB). 2BrZ is preferred.

Met, if used, may be protected by oxygen, but Met is generally left unprotected. X^s is a protecting group for a side chain guanidino group in Arg or Har, such as nitro, Tos, trityl, adamantyloxycarbonyl, Z and 2,4-dinitrophenol(Dnp) , or X⁵ may be hydrogen, which means there is no protection on the side chain group atoms. Tos is generally preferred. X⁶ is a protecting group for an amino side chain group, primary or secondary amino, such as Z or 2C1Z.

X⁷ may be Gly-NH-[resin support], D-Ala-NH-[resin support] or N(A)-[resin support]; X⁷ may also be an amide either of Gly or of D-Ala or a substituted amide attached directly to Pro or NHNHC0NH₂. The criterion for selecting certain side chain protecting groups for X²-X⁶ is that the protecting group should be stable to the reagent under the reaction conditions selected for removing the α-amino protecting group (preferably Boc) at each step of the synthesis.

These protecting groups generally should not be split off under coupling conditions but should be removable upon completion of the synthesis of the desired amino acid sequence under reaction conditions that will not alter the peptide chain. Other protecting groups initially employed for the 5- and 6-position residues are removed prior to cleavage from the resin, as explained hereinafter.

When the X⁷ group is Gly-NH-[resin support] or D-Ala-NH-[resin support], an amide bond connects Gly or D-Ala to a BHA resin or to a MBHA resin. When the X⁷ group is N(A)-[resin support], a substituted amide bond connects Pro to an N-alkylaminomethyl (NAAM) resin. When X⁷ is AzaGly-NH₂, the peptide is preferably made by classical solution synthesis, as disclosed in U.S. Patent No. 4,234,571.

When G is acetyl, for example, in the final formula, it may be possible to employ it as the X¹ protecting group for the α-amino group of 9-D-NAL or whatever amino acid is used in the 1-position by adding it before coupling this last amino acid to the peptide chain. However, a reaction is preferably carried out with the peptide on the resin (after deblocking the α-amino group while the side-chain groups remain protected) , e.g. by reacting with acetic acid, or preferably with acetic anhydride, in the presence of diisopropyl or dicyclohexyl carbodiimide (DIC or DCC) or by some other suitable reaction as known in the art. Thus, the invention also provides a method for making a GnRH peptide having the formula:

G-AA,-(A)D-Phe-AA₃-Ser-(R,)AA_j-AA^AA^AA_g-Pro-AA^ , wherein AA.., AA₃, AA_j, AA₆, AA₇, AA_β and AA₁₀ are as set forth hereinbefore, which method comprises (a) forming an intermediate peptide having the formula:

X¹-AA,(X²)-(A)D-Phe-AA_j(X²)-Ser(X³)-(R,)AA_;;(X^a)-AA₆(X^a)-AA₇(X² or X⁴)-AA₈(X⁵ or X⁶)-Pro-X⁷ wherein X¹ is hydrogen or an α-amino protecting group; X² is hydrogen or a protecting group for an indole nitrogen; X³ is a protecting group for a hydroxyl group of Ser; X⁴ is hydrogen or a protecting group for a phenolic hydroxyl group of Tyr; X⁵ is either hydrogen or a protecting group for a guanidino side chain; X⁶ is a protecting group for an amino side chain, of which X* is a subgroup that is removable without removing other protecting groups; X⁷ is Gly-NH-[resin support], D-Ala-NH-[resin support], N(A)-[resin support], an amide either of Gly or of D-Ala or a substituted amide attached directly to Pro or NHNHCONH₂; (b) removing X⁸ from AA₅ and AA₆ to deprotect a side chain primary amino group of these amino acid residues of said intermediate peptide; (c) reacting said deprotected side chain primary amino groups to build said residue into one having an animo-triazole moiety; and (d) splitting off any remaining groups X¹ to X⁶ and/or cleaving from any resin support included in X⁷.

Purification of the peptide is effected by ion exchange chromatography on a CMC column, followed by partition chromatography using the elu ion system: n-butanol;0.IN acetic acid (1:1 volume ratio) on a column packed with Sephadex G-25, or by using HPLC, as known in the art and specifically set forth in J. Rivier, et al. J. Chromatography. 288 (1984) 303-328.

The antagonists of the invention are effective at levels of less than 100 micrograms per kilogram of body weight, when administered subcutaneously at about noon on the day of proestrus, to prevent ovulation in female rats. For prolonged suppression of ovulation, it may be necessary to use dosage levels in the range of from about 0.1 to about 2.5 milligrams per kilogram of body weight. These analogs are particularly soluble at physiological pHs and thus can be prepared as relatively concentrated solutions for administration. The antagonists are also effective to arrest spermatogenesis when administered to male mammals on a regular basis and can thus be used as contraceptives. Since these compounds will reduce testosterone levels (an undesired consequence in the normal, sexually active male) , it may be desirable to administer replacement dosages of testosterone along with the GnRH antagonist. These antagonists can also be used to regulate the production of gonadotropins and sex steroids for other purposes as indicated hereinbefore.

In the following formulas, the residues for positions 5 and 6 are sometimes defined in terms of the original amino acid residue having a side chain amino group plus the modification in question which is set forth in the accompanying parentheses. Preferably, the original residue is incorporated in the main peptide chain, for example Aph, Ahp, Lys, Orn or the respective D-isomer thereof, and is modified while a part of the peptide chain that is still attached to the resin to form the desired residue AA_j or AA₆. However, the suitably protected significantly modified amino acid can be added to the growing peptide chain as a part of the usual chain elongation process, if desired.

The following Examples illustrate a number of GnRH antagonist peptides embodying various features of the invention. All of these peptides include at least one D- isomer amino acid residue. The N-alkyl modification of the amino acid to be included in the 5-position can be ade prior to its linkage to the peptidoresin (as described below) or the α-amino group can be alkylated after the residue has been attached to the peptidoresin during the solid-phase synthesis using steps known in the art.

EXAMPLE 1 Preparation Of N°Boc-N^βmethyl(4-Fmoc-amino)L-phenylanaline

40 grams of Boc-L-Phe(0.152 mole) were dissolved in 100 roilliliters of tetrahydrofuran (THF) and added dropwise to 13.58 grams of NaH(0.452 mole) (3eq) which had been dissolved in 500 milliliters of THF at 0"C. Thereafter, 42.6 grams of methyl iodide (2eq) , dissolved in THF, were added dropwise over 30 minutes, and stirring in a beaker was continued for about 36 hours at room temperature. The reaction was stopped by placing the beaker in an ice bath and by the slow addition of 20 milliliters of glacial acetic acid and 20 milliliters of distilled water. The THF was removed by rotary evaporation, and the remaining oil was diluted with 300 milliliters of water. After adjusting to a pH of about 8 with sodium carbonate, the mixture was extracted twice, using 200 milliliters of ethyl ether each time. Following extraction, the remainder is acidified to a pH of about 2, with sulfuric acid, and it is then extracted twice with 300 milliliters of ethylacetate. After drying over anhydrous magnesium sulfate, solvent removal is effected by evaporation, giving about 40 grams of an oil which crystallizes. The crystals of Boc-N^αMePhe have a melting point of 71°C and, when dissolved in CH₃OH, have an [α]_D equal to -93.6 ±l(c = 1.1, methanol) .

31 grams of this Boc-protected amino acid (0.11 mole) were cooled to 0^βC and stirred together with 30 milliliters of trifluoroacetic acid (TFA) and 1 milliliter of anisole for 30 minutes at 0*C. The mixture was then diluted with 600 milliliters of t-butylmethyl ether and 1.4 liters of ethyl ether to precipitate the amino acid, which is collected by filtering. The amino acid is then dissolved in 50 milliliters of water, and the pH is adjusted to about 6 with ammonium hydroxide. Drying overnight over high vacuum produced 12 grams of N°MePhe having a melting point of about 253"C and [α]_D = +15.4° ±l(c = 0.96, IN HC1) . The N-alpha-methyl-Phe is then substituted with nitrate in the 4- or para-position as follows. A mixture of 40 milliliters of 95% sulfuric acid and 4 milliliters of 70% nitric acid was cooled to about -2*C, and 11.6 grams of the amino acid (0.065 mole) were added slowly over 30 minutes with stirring, which was continued for about one hour. The mixture was then poured onto 350 grams of ice, and the pH was adjusted to about 6 with ammonium hydroxide. The amino acid was recrystallized three times from water at 90*C to assure purification. Then drying was carried out under high vacuum, which produced 8.7 grams (0.039 mole) of N^αMe4N0₂Phe having a melting point of about 255*C and an [α]_D = +32.8° ±l(c = 1, IN HC1) . 8.5 grams of the N^βMe4N0₂Phe (0.036 mole) were reacted with Boc-carbonate under standard conditions to produce (Boc-N^βMe4N0₂Phe in an amount of about 11.5 grams and in the form of a yellow oil, which crystallizes and has an [α]_D = -73.6° + l(c = 0.8, 50% acetic acid).

This compound was then reduced using a palladium catalyst on a carbon substrate in a mixture of equal parts of ethanol and acetic acid by bubbling hydrogen gas through the mixture until uptake of H₂ ceased. The solvent was then removed by rotary evaporation, and the remaining compound was dissolved in water and its pH adjusted to about 3. Extraction was twice carried out with 50 milliliters of ethyl ether and then twice with 50 milliliter portions of ethylacetate. The pH is then adjusted to 9.4 with ammonium hydroxide.

The side chain amino group is next protected with Fmoc by reaction with 9-fluorenylmethyl- succinimidylcarbonate (Fmoc-Osu) under standard conditions. The resultant Boc-N^βMe4NH₂Phe(Fmoc) having an [α]_D = -43.8° ±l(c = 1.2, ethylacetate). This N^β-Boc- protected and Fmoc-side chain-protected Phe is then used in the peptide synthesis which follows.

EXAMPLE 2 Peptide No. 1 in the form of the decapeptide [Ac-β- D-2NAL¹, (4C1)D-Phe², D-3PAL³, N^βMeAph(atz)⁵, D-Aph(atz)⁶, ILys⁸, D-Ala¹⁰]-GnRH is synthesized by solid-phase synthesis. This peptide has the following formula: [AC-/9-D-2NAL-(4C1)D-Phe-D-3PAL-Ser-N^aCH₃Aph(3-amino 1,2,4 triazole)-D-Aph(3-amino 1,2,4 triazole)-Leu- Lys(isopropyl)-Pro-D-Ala-NH₂.

About 1.5 grams (0.76 mM/g) of MBHA resin are used, and Boc-protected D-Ala is coupled to the resin over a 2- hour period in CH₂C1₂ using about 2 millimoles of Boc derivative and DCC as an activating reagent. The D-Ala residue attaches to the MBHA residue by an amide bond. Following the coupling of each amino acid residue, washing, deblocking and coupling of the next amino acid residue is carried out in accordance with the following schedule using an automated machine and beginning with about 1.5 grams of resin: -ιε

STEP REAGENTS AND OPERATIONS MIX TIMES MIN.

1 CH₂C1₂ wash-80 ml. (2 times) 3

2 Methanol(MeOH) wash-30 ml. (2 times) 3 3 CH₂C1₂ wash-80 ml. (3 times) 3

4 50 percent TFA plus 5 percent 1,2-eth- anedithiol in CH₂Cl₂-70 ml. (2 times) 10

5 Isopropyl alcohol + 1% ethanedithiol wash-80 ml. (2 times) 3 6 TEA 12.5 percent in CH₂Cl₂-70 ml. 5

7 MeOH wash-40 ml. (2 times) 2

8 TEA 12.5 percent in CH₂Cl₂-70 ml. 5

9 CH₂C1₂ wash-80 ml. (3 times) 3

10 Boc-amino acid (10 m oles) in 30 ml. of either dimethylformamide(DMF) :

CH₂C1₂ or DMF alone, depending upon the solubility of the particular protected amino acid, plus DIC or DCC (10 mmoles) in CH₂C1₂ 30-300 11 MeOH wash-40 ml. (2 times) 3

12 Triethylamine(TEA) 12.5 percent in CH₂Cl₂-70 ml. 3

13 MeOH wash-30ml. (2 times) 3

14 DCM wash-80 ml. (2 times) 3

The above schedule is used for coupling of each of the amino acids of the peptide of the invention after the first amino acid has been attached. N*Boc protection is used for each of the remaining amino acids throughout the synthesis. N^βBoc-9-D-2NAL is prepared by a method known in the art, e.g. as described in detail in U.S. Patent No. 4,234,571, issued November 18, 1980; it is also commercially available from SyntheTech, Oregon, U.S.A. The side chain primary amino groups of ^MeAph in the 5- position and of D-Aph in the 6-position are protected by F oc, with the amino acid for the 5-position being prepared by the process set forth in Example 1. Bzl(benzyl ether) is used as a side chain protecting group for the hydroxyl group of Ser. Boc-Lys(Ipr,Z) is used for the 8-position. After deblocking the α-amino group at the N-terminus using trifluoroacetic acid (TFA) , acetylation is achieved using a large excess of acetic anhydride in dichloromethane. Following completion of the assembly of the peptide and acetylation of the N-terminus, about 3.2 grams of the following intermediate are present: Ac-/3-D-2NAL-(4Cl)D- Phe-D-3PAL-Ser(Bzl)-N^βCH₃Aph(Fmoc)-D-Aph(Fmoc)-Leu- Lys(Ipr,Z)-Pro-D-Ala-NH-[MBHA resin support]. The side chains on the amino acid residues in the 5- and 6- positions are modified by simultaneously carrying out the following reactions with the deprotected side chains of the Aph residues. The Fmoc protecting group is removed from both residues by treatment of the peptidoresin with 20 percent piperidine in DMF for about 15 minutes; it is preferably washed with DMF and then treated with more piperidine/DMF for 15 minutes. After preferably washing the peptidoresin with DMF, the newly freed amino groups are treated with a large excess (>10 fold) of diphenyl cyanocarbonimidate(PCI) in 5 ml. of DMF and left to react overnight at room temperature. Thereafter, the peptide is washed with DMF, CH₃OH and CH₂C1₂, and this standard wash is repeated. Then, the peptidoresin is treated with 5 ml. of hydrazine dissolved in 5 ml. of DMF, for 12 hours at about 22"C. to complete the formation of the cyanoguanidino moiety; this step is preferably repeated. The cyanoguanidino moieties that are formed spontaneously convert to the corresponding heterocycle, i.e. 3-amino, 1,2,4 triazole. The standard wash is then carried out and repeated. The peptidoresin is dried, and then cleavage of the peptide from the resin and deprotection of the Ser and the Lys side chains is carried out at 0^*C. with HF. Anisole is added as a scavenger prior to HF treatment. After the removal of HF under vacuum, the resin is washed twice with 100 ml. of ethyl ether. The cleaved peptide is extracted from the resin with equal parts of CH₃CN and H₂0, repeating the process and using 150 ml. each time. The extracts are pooled and lyophilized, and they provide about 762 mg of a crude peptide powder.

Purification of the peptide is then effected by preparative high performance liquid chromatography (HPLC) , as known in the art and specifically set forth in J. Rivier, et al. £. Chromatography. 288. 303-328 (1984) . The first preparative RP-HPLC separation uses a TEAP (triethylammonium phosphate) buffer system. This separation is repeated using the same buffer system with a slightly different gradient, and the final separation is carried out using a 0.1% TFA (trifluoroacetic acid) gradient, all as described in detail in the J.

Chromatography article. About 204 milligrams of the decapeptide are obtained.

The peptide is judged to be homogeneous using capillary zone electrophoresis (CZE) , as well as by using reversed-phase high pressure liquid chromatography and an aqueous triethylammonium phosphate buffer plus acetonitrile. The purity is estimated to be about 99.2%. Amino acid analysis of the resultant, purified peptide is consistent with the formula for the prepared structure, showing substantially integer-values for each amino acid in the chain; mass spectral analysis is also consistent. The optical rotation is measured on a photoelectric polarimeter as [α]^° = -43.1° ±1.0(c=0.63, 50% acetic acid). The peptide is assayed .in vivo to determine its effectiveness to prevent ovulation in female rats. In this test, a specified number of mature female Sprague-Dawley rats, e.g. five to ten, each having a body weight from 225 to 250 grams, are injected with a specified microgram dosage of the peptide in saline, bacteriostatic water, polyethylene glycol, corn oil or a mixture of one of the above with ethanol, at about noon on the day of proestrus. Proestrus is the afternoon of ovulation. A separate female rat group is used as a control to which the peptide is not administered. Each of the control female rats ovulates on the evening of proestrus; the number of the rats treated which ovulate is recorded. In vivo testing of the peptide shows that, at a dosage of 2.5 micrograms, 0 out of 8 rats treated ovulate, and at a dosage of 1 microgram, only 2 out of 9 rats ovulate.

In vitro testing is carried out using dissociated rat pituitary cells maintained in culture for 4 days prior to the assay. The levels of LH mediated in response to the application of peptides is assayed by specific radioimmunoassay for rat LH. Control dishes of cells only receive a measure which is 3 nanomolar in GnRH; experimental dishes receive a measure 3 nanomolar in GnRH plus a measure having either the present standard antagonist for comparison purposes i.e. [Ac-dehydro Pro¹, (4F)D-Phe², D-Trp³'⁶]-GnRH or the test peptide, in concentrations ranging from 0.01 to 10 nanomolar. The amount of LH secreted in the samples treated only with GnRH is compared with that secreted by the samples treated with the peptide plus GnRH. The peptide is twice as potent as our standard [Ac-Δ³Pro¹, D-4FPhe², D-Trp^3,6]- GnRH.

The peptide is considered to be particularly useful because of its substantially greater solubility in aqueous buffers at a pH of from about 5 to about 7, compared to other compounds of generally comparable biological efficacy. This solubility is believed to be due to the fact that the lower alkyl, e.g. CH₃, group on the α-nitrogen of the amino acid in position 5 breaks the hydrogen bond responsible for ,9-sheet formation in combination with the effect provided by the particular side-chains on the 5- and 6-position residues which incorporate triazole-substituted phenylalanine moieties.

EXAMPLE 3 The following peptides indicated in TABLE A, having the general formula: Ac-3-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser- (N^JAA_j-AA^Leu-AA_g-Pro-D-Ala-NH_g are prepared by the solid-phase procedure generally referred to above.

TABLE A

_έ s

By atz is meant 3-amino, 1,2,4 triazole. The peptides listed in Table A are considered effective to block GnRH-induced LH secretion .in vitro at a reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE 4 Peptides as indicated in TABLE B having the formula: G-AA.,-(A)D-Phe-AA₃-Ser-N^βCH₃Aph(atz)-D-Aph(atz)-AA₇-ILys- Pro-AA₁₀ are prepared by the solid-phase procedure generally referred to above.

TABLE B ^ ___ G , A. ₃ AA_{7 10}

15 AC D-Trp 4F D-Trp 16 Acr 6N0₂D-Trp 4C1 0D-2NAL 17 For Me₅D-Trp 4Br 6FD-Trp 18 Bz 4FD-Phe 4N0₂ ForD-Trp 19 Acr 3D-1NAL 4CH₃ 6N0₂D-Trp 20 Ac 4ClD-Phe 4F ,9D-1NAL 21 Bz 5D-2NAL 40CH₃ D-3PAL 22 Acr 4N0₂D-Phe 4F _/SD-1NAL 23 Ac 9D-2NAL 2,4C1₂ 6BrD-Trp 24 For 4CH₃D-Phe 4F 6CH₃D-Trp 25 Ac 6ClD-Trp 4Br 6C1D-Trp 26 Acr 6FD-Trp 4F D-2PAL

The peptides listed in Table B are considered effective to block GnRH-induced LH secretion in vitro at a reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

The peptides of the invention are often administered in the form of pharmaceutically acceptable, nontoxic salts, such as acid addition salts, or of metal complexes, e.g., with zinc, barium, calcium, magnesium, aluminum or the like (which are considered as addition salts for purposes of this application) , or of combinations of the two. Illustrative of such acid addition salts are hydrochloride, hydrobro ide, sulphate, phosphate, nitrate, oxalate, fumarate, gluconate, tannate, aleate, acetate, citrate, benzoate, succinate, alginate, alate, ascorbate, tartrate and the like. For example, an aqueous solution of the peptide can be repeatedly treated with IN acetic acid and then lyophilized to yield the acetic acid salt thereof. If the active ingredient is to be administered in tablet form, the tablet may contain a pharmaceutically- acceptable diluent which includes a binder, such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate. If administration in liquid form is desired, sweetening and/or flavoring may be used as part of the pharmaceutically-acceptable diluent, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.

The pharmaceutical compositions will usually contain the peptide in conjunction with a conventional, pharmaceutically-acceptable carrier. Usually, the dosage will be from about 10 micrograms to about 2.5 milligrams of the peptide per kilogram of the body weight of the host when given intravenously. Oral dosages would be higher; however, the nature of these compounds should permit effective oral administration. Overall, treatment of subjects with these peptides is generally carried out in the same manner as the clinical treatment using other antagonists of GnRH using a suitable carrier in which the peptide is soluble.

It may also be desirable to deliver the GnRH analog over prolonged periods of time, for example, for periods of one week to one year from a single administration, and slow release, depot or implant dosage forms may be utilized. For example, a suitable, slow-release depot formulation for injection may contain the GnRH antagonist or a salt thereof dispersed or encapsulated in a slow degrading, non-toxic or non-antigenic polymer such as a polylactic acid/polyglycolic acid polymer, for example, as described in U.S. Pat. No. 3,773,919. These compounds may also be formulated into silastic implants. These peptides can be administered to mammals intravenously, subcutaneously, intramuscularly, orally, percutaneously, e.g. intranasally or intravaginally to achieve fertility inhibition and/or control and also in applications calling for reversible suppression of gonadal activity, such as for the management of precocious puberty or during radiation- or chemotherapy. They are also useful for treatment of steroid-dependent tumors. Effective dosages will vary with the form of administration and the particular species of mammal being treated. An example of one typical dosage form is a bacteriostatic water solution at a pH of about 6 containing the peptide which solution is administered parenterally to provide a dose in the range of about 0.1 to 2.5 mg/kg of body weight per day. Although the invention has been described with regard to its preferred embodiments, it should be understood that changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims which are appended hereto. For example, other substitutions known in the art which do not significantly detract from the effectiveness of the peptides may be employed in the peptides of the invention. D-2PAL and D-4PAL are considered to be equivalents of D-3PAL. Other equivalent acylating groups can be used instead of acetyl at the N-terminus. Substituted Phe, such as (4F)Phe, can be used instead of Phe in the 7-position. Har is considered the equivalent of Arg in the 8-position, and both butyl Lys and diethyl Lys are considered to be equivalents of ILys. However, ILys is most preferred. Other hydrophobic amino acid residues can also be employed in the 1-position, preferably in D-isomer form, and are considered equivalents of those specified. Moreover, the antagonists can be administered in the form of their pharmaceutically or vetinarially acceptable, nontoxic salts, as indicated hereinbefore, which are considered equivalents. Particular features of the invention are emphasized in the claims which follow.

Claims

WHAT IS CLAIMED IS:

1. A GnRH antagonist peptide, or a nontoxic salt thereof, having the formula: Ac-_/9-D-2NAL-(4Cl)D-Phe-D- 3PAL-Ser-N^βCH₃AA_s(3-amino 1,2,4 triazole)-AA₆(3-amino 1,2,4 triazole)-Leu-Lys(isopropyl)-Pro-D-Ala-NH₂, wherein AAg is

Aph or Lys and AA₆ is D-Aph or D-Lys.

5

2. A GnRH antagonist peptide, or a nontoxic salt thereof, according to Claim 1 having the formula: c-β- D-2NAL-(4C1)D-Phe-D-3PAL-Ser-N^βCH₃Aph(3-amino 1,2,4 triazole)-D-Aph(3-amino 1,2,4 triazole)-Leu- ° Lys(isopropyl)-Pro-D-Ala-NH₂.

3. A GnRH antagonist peptide, or a nontoxic salt thereof, according to Claim 1 having the formula: Ac-,3-D-2NAL-(4C1)D-Phe-D-SPAL-Ser-^CH_jLys(3-amino 1,2,4 triazole)-D-Lys(3-amino 1,2,4 triazole)-Leu- Lys(isopropyl)-Pro-D-Ala-NH₂.

4. A method for inhibiting the secretion of gonadotropins in mammals comprising administering to a mammal an effective amount of a GnRH antagonist peptide or a nontoxic salt thereof as defined in Claim 1.

5. A GnRH antagonist peptide or a nontoxic salt thereof, said peptide having the formula:

G-AA.4- (A) D-Phe-AA₃-Ser- (R, ) AA₅-AA₆-AA₇-AA_β-Pro-AA₁₀ wherein G is an acyl group having 7 or less carbon atoms ; AA, is (A) D-Phe , (B) D-Trp or ,9-D-NAL; A is Cl , F, N0₂, Br, CH₃, OCH₃, Me₅ or Cl₂; B is H, N0₂, OCH₃ , F, Cl , Br, CH₃ or NⁱⁿFor; AA₃ is D-PAL, ,3-D-NAL or (B) D-Trp; AA_j is Xaa ; R, is N^CH_j or N^βCH₂CH₃ ; AA₆ is D-Xaa ; AA₇ is Leu, NML, Nle , Phe , Met , Nva , Tyr, Trp or PAL; AA_g is ILys , Arg , Har, or IOrn; AA₁₀ is D-Ala-NH₂, Gly-NH₂, AzaGly-NH₂ or NHfR_j,) ; R_j is lower alkyl; and Xaa is Aph, Ahp, Lys or Orn having its ω-amino group substituted by

NH₂

I

N c n II c N

N

I H

6. A GnRH antagonist peptide according to Claim 5 wherein AA, is J-D-2NAL.

7. A GnRH antagonist peptide according to Claim 5 wherein AA₃ is D-3PAL.

8. A GnRH antagonist peptide according to Claim 5 wherein AA₃ is 6N0₂-D-Trp.

9. A GnRH antagonist peptide according to Claim 5 wherein AA_j is Aph(atz).

10. A GnRH antagonist peptide according to Claim 9 wherein R, is N^αCH₃.

11. A GnRH antagonist peptide according to Claim 10 wherein AA₆ is D-Aph(atz) .

12. A GnRH antagonist peptide according to Claim 11 wherein AA₇ is Leu, NML, Nle or Phe.

13. A GnRH antagonist peptide according to Claim 12 wherein AA_g is ILys or Arg.

14. A GnRH antagonist peptide according to Claim 13 wherein AA_1fl is D-Ala-NH,. ^{10 2}