This invention relates generally to peptides having unnatural amino acids and to the preparation of new unnatural amino acids, which may be derived from diamino acids, such as Lys, Orn, ^' Dpr and Dbu. More particularly, it relates to GnRH analogs having such unnatural amino acids which can be prepared either in such fully assembled peptides or for incorporation into such peptides as a part of the usual chain elongation synthesis process.

In one more particular aspect, the present invention relates to peptides which inhibit gonadal function and the release of the steroidal hormones, progesterone and testosterone and also to peptides which promote the release of such steroids, as well as to methods of promoting or preventing 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 hypothala ic hormones acts as a factor that

triggers the release of the gonadotropic hormones, particularly I-H, and this hormone is referred to herein as GnRH although it has also been referred to as LH-RH and as LRF. GnRH has been isolated and characterized as a decapeptide having the following structure: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ 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(NH ₂ ) 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 serihe, 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 endo etriosis. 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. In the female, they can also be used for hirsutism.

On the other hand, GnRH agonists function in the same manner as GnRH in promoting the release of LH and FSH, and agonists which exhibit greater biopotency and/or longer duration of action are considered valuable.

In one aspect, it is desired to provide improved peptides which either are strongly antagonistic to endogenous GnRH and prevent secretion of LH and the release of steroids by the gonads of mammals or are strong agonists of GnRH of particular interest are compounds which are more effective in vivo when administered orally.

SUMMARY OF THE INVENTION The present invention provides unnatural amino acids can be prepared de novo or by modifying a previously prepared peptide or protected peptide-resin containing the desired overall sequence which includes one or more amino acid residues which are to be modified. Preferred amino acids of the invention contain a modified guanidino group.

In another particular aspect, the invention provides peptides which inhibit the release of

gonadotropins in mammalians, including humans, and it also provides methods for inhibiting the release of steroids by the gonads of male and female mammalians. The invention also provides improved GnRH analogs which are strong agonists of GnRH and can be used to promote the reproduction processes of mammalians. As mentioned above, these GnRH antagonists may be used to inhibit the production of gonadotropins and sex hormones under various circumstances, including precocious puberty, hormone dependent neoplasia, dysmenorrhea, endometriosis and steroid-dependent tumors.

The invention provides unnatural amino acids having the following formula U ^* : o y

where n is an integer from 1 to 6 and is preferably 1,2,3 or 4; Y = N-CN, N-CONHR,, S, O or CH-N0 ₂ , where R, is H, Ac, alkyl (preferably C, to C ₄ ) , naphthyl, pyridyl, pryrimidyl, pyrazinyl, indolyl, quinolinyl or imidazolyl, which alkyl and cyclic groups are unsubstituted or substituted (preferably by chloro, fluoro, bromo, amino, nitro, alkyl (C, to C ₄ ) and alkoxy (C, to C ₄ )) ; X = NH, O, S, N ₃ , M _r (CH _q ) _p -M ₂ or M^CH^-M^CH^^, where M, is NR ₁₀ , N, O, S or CHR ₃ wherein ₃ is methyl, ethyl, propyl, phenyl, pyridinyl, pyrimidinyl or purinyl; q=l or 2? p, p' and p" are integers between 0 and 6; R ₁₀ is H, methyl, ethyl, propyl, phenyl or substituted phenyl (preferably by Cl, F, N0 ₂ or NH ₂ ) ; and M ₂ and M ₃ = M COOH, CONH ₂ , COOR _j or CN (preferably X is NH, O or S) j R, = H, alkyl (preferably C _t to C ₆ and most preferably C, to C ₄ ) , modified alkyl (preferably C, to C ₅ , the terminal carbon of which is either substituted with NH ₂ , OH, Cl, Br or F

or is replaced with CF ₃ or CF ₂ CF ₃ ) , alkenyl (preferably C ₂ to C ₄ ) , such as CH ₂ CH=CHR ₃ , alkenyl (preferably C ₂ to C ₄ ) , such as CH ₂ C≡CR ₃ , aryl such as benzyl, tolyl, p-amino- benzyl (analinyl) and pCl-benzyl or a direct bond to X; R^ = R,, OH, NH ₂ , NHR,, heterocycle (preferably as illustrated hereinafter) or desR ₂ , with R ₂ being desR ₂ when X = N ₃ . Optionally ₂ and X can be interconnected, or R, and R ₂ can be connected to each other via a branched or unbranched methylene bridge of type -(CH^- or -(CH ₂ ) _m -M-(CH ₂ ) _(n ,-. In such an R ₁ -R ₂ moiety, m and m' are integers from 1 to 6 and preferably from 1 to 3; and M - NH, O, S or CHR ₄ , wherein R _; is lower alkyl or aryl and is preferably methyl, ethyl, propyl, phenyl or pCl-phenyl, with M preferably being 0 or S. Most preferably, when R, and R ₂ are interconnected, they form a 5, 6, or 7-member heterocyclic ring with the "N-C-X" portion of the formula U ^* . If desired to form a cyclic peptide, XR ₂ can contain a part of another diamino acid within the same peptide, e.g., the omega amino group of the 5-position residue can be so linked to such an unnatural amino acid residue in the 8-position.

Modification of the specified primary amino function of a given amino acid or peptide is carried out by treatment of either the appropriately protected peptide or the amino acid with an appropriate reagent(s).

Peptides or amino acids where Y is N-CN (herein referred to as cyanoguanidines) are prepared by reaction of an amino group with diphenyl cyanocarboni idic acid (I):

N-CN N-CN [ [

Q-NH, + PhO-C-OPh > Q-NH-C-OPh

(I) (II) wherein "Q" is used to broadly represent either the major portion of a peptide or an amino acid having a primary

amino group (such as the amino acid which is depicted above) as a part of formula U ^* .

The peptide or amino acid having the N-substituted-N'-cyano-O-phenylisόurea moiety (II) can then be either isolated or further functionalized by reaction with a second nucleophile HXR ₂ to produce cyanoguanidine-containing peptides or amino acids having the formula (III) : N-CN N-CN

I I

Q-NH-C-OPh + HXR ₂ > Q-NH-C-XR _j ,

(II) (III) For example,

N-CN N-CN (aminomethyl

I I cyanoguanidino Q-NH-C-OPh + NH ₂ CH ₃ > Q-NH-C-NHCH ₃ moiety)

(ID (HI)

For example, where HXR ₂ •= H ₂ N-CH ₂ -pyridine, the result is: N-CN (3 aminomethyl pyridyl cyanoguanidino moiety)

This group may also be referred to (IUPAC nomenclature) as N-g-cyano-N-g'-3-methylpyridylguanidino. Such compounds can be hydrolyzed under acidic conditions to produce compounds which are also biopotent—*for example:

The hydrolyzed versions, referred to herein as including the N-g'-amido group, can also be synthesized directly by reacting phosgene derivates with moieties having a guanidino function.

If HXR ₂ is an amino group of another peptide or protein, one will obtain a peptide-peptide dimer or peptide-protein dimer conjugated via the cyanoguanidine moiety. If HXR ₂ is the N-terminal primary amino group or the side chain amino group of another amino acid in the ^* same peptide, one will obtain a cyclic peptide (IV) linked via the cyanoguanidine moiety:

N-CN I

Q.-NH-C

I (iv) Q ₂ NH wherein Q ₁ and Q ₂ represent the remainders of two amino acid residues in the same peptide. Cyclization via the cyanoguanidine derivative is preferably effected while a part of the peptidoresin, as opposed to subsequently cyclizing the linear peptide.

A special case arises when -XR ₂ contains a second nucleophilic site and X has the general form: ^M r( ^CH _q ) _p - ^M ₂ ^or M _r (CH ₂ ) _pl -M ₂ -(CH ₂ ) _pll -M ₃ , where M,, M ₂ and M ₃ are individually NH, N, 0, S, or CHR ₃ , with p, p', p" being 0,1,2 or 3 and q being 1 or 2. Examples of such nucleophiles include H ₂ NNH ₂ , CH ₃ HNNH ₂ , CH _j HNNHCH _j , H ₂ NOH, and H ₂ N-CH ₂ -CH ₂ OH. In this case, the cyanoguanidine moiety that is formed can be converted into the corresponding heterocycle (V) which forms from the

initial intermediate by reaction of the omega amino group with the cyano group εuch as:

Q-NH- (V)

For example, where -XR ₂ = -HNNH ₂ ,

NH,

/ N-CN N—C (1,2,4 triazole

I I I moiety) Q-NH-C-HNNH ₂ > Q-NH-C N

\ /

N H

Furthermore, where -XR _j = -CH ₃ NNHCH ₃

NH

/ N-CN ^• — C

I I I

Q-NH-C-CH ₃ NNHCH ₃ > Q-NH-C N-CH ₃

N I

CH ₃

Where XR _j contains a carboxylic acid group or the equivalent, particularly a carboxylic ester or carboxylic amide, a heterocyclic moiety, such as a saturated pyri idine-like moiety (VI), is formed, by reaction of the carboxylic group with the secondary amino group (R _j , when M ₁ is N, and similar 6-membered heterocyclic moieties are formed when M _t is O or S. For example, R ₂ may be M ₁ -(CH ₂ ) _p -M ₂ with M ₂ = COOH, COOCH ₃ or C0NH ₂ and p being an

integer between 1 and . For instance in εuch a case where an aliphatic carboxylic acid group is present and p=2:

N-CN

I

N-CN c

I / \ 3-peptide-2-

Q-NH-C-N-(CH ₂ ) _p -C00H -> Q-N NH (VI) (cyanoimino)-4 oxohexahydro- H CH, pyrimidino

\ /

CH,

If R ₂ includes an ortho-substituted aromatic carboxylic acid, e.g. benzoic acid (q=l and p=6) , the corresponding quinazoline-like species (VII) is formed:

N-CN

I

Q-NH- (VII)

Such benzoic acid may be further substituted, and such substitutions may in any of the other 4 ring positions, as shown, creating the corresponding substituted quinazoline-like moiety which is considered to be equivalent to the unsubstituted. X' may be H, Cl, Br, F, NHCH ₃ or SCH ₃ , and ₇ and R _j may be H, CH ₃ or CH ₂ CH ₃ .

The molecules wherein X - N ₃ and R ₂ is deεR _j (i.e. deleted) are useful for photolabeling because of the activity of the -N ₃ group and are formed by reacting the moiety (II) with sodium azide (NaN _s ) .

Peptides wherein Y is 0 (herein referred to as ureas) or S (herein referred to as thioureas) are prepared by the well known procedure in which the desired side chain amino group is treated with an appropriate isocyanate or isothiocyanate to obtain such ureas or thioureas, respectively. •

0 I

Q-NH ₂ + 0=C=NR ₂ > Q-NH-C-NH^ or S

I

Q-NH ₂ + S=C=NR ₂ > Q-NH-C-NHR _j

Peptides or amino acids wherein Y is CH-N0 ₂ (herein referred to as diaminonitroethylenes) are prepared by conversion of the corresponding urea to a carbodiimide: O

followed by treatment with nitromethane anion (prepared by the action of sodium hydride on nitromethane in dry DMF) as disclosed generally in F. Meimas, et al.. Synthesis. 509-510 (1985):

H NO,

\ / C

Q-NH=C= R ₂ + CH ₃ N0 ₂ + NaH • -> |

Q-NH-C-NH-^

An alternative synthesis that may be used is as follows:

Generally, in accordance with the present invention, peptides are synthesized which are antagonists or agonists of GnRH, i.e., they either strongly inhibit the secretion of gonadotropins by the pituitary gland of mammalians, including humans, and/or inhibit the release of steroids by the gonads, or they strongly promote such secretion or release. These peptides are analogs of GnRH containing one or more unnatural amino acids of the formula U ^* in the 5-position and/or the 6-position and/or the 8-position. An antagonist should have a 1-position substitution, preferably dehydroPro or -(l-or 2-naphthyl)-D-alanine (hereinafter ,9-D-lNAL or S-D-2NAL) , a 2-position substitution in the form of a modified D-Phe and a 3-position substitution, preferably in the form of substituted or unsubstituted D-Trp, D-3PAL or 0-D-NAL. The 5-position may be occupied by (a) Tyr, (b) a halogenated or methylated Phe or Tyr, (c) Arg, (d) Lys in which the side chain amino group is acylated by 3-carboxypyridine (nicotinic acid) or by 2 or 4-carboxypyridine, i.e. Lys(cpd), preferably Lys(3cpd) which is also referred to as Lys(Nic), (e) His or (f) the residue of the amino acid U ^* . Agonists have a 6-position substitution which is U ^* , and the antagonists may have U ^* or εuch a substituted or acylated Lys in the 6-position. Instead of Leu in the 7-position, both may have Nle,.NML, Phe, Nva, Met, Tyr, Trp or PAL, of which the Phe or Trp

may be εubstituted. The antagonists may also have an optional substitution in the 8-position, which preferably may be U ^* or isopropyl Lys, i.e., (ILys) or Lys(Ipr), wherein the εide chain amino group iε substituted by isopropyl, and a substitution in the 10-position such as D-Ala. At least one residue of an amino acid of the formula U ^* is present in each peptide of the invention.

Modified D-Phe in the 2-position provides increased antagonistic activity as a result of the specific modifications present in the benzene ring. Single substitutions for hydrogen in the ring are preferably made in the para- or 4-position, but might be in either the 2- or 3-position also; the substitutions are selected from chloro, fluoro, bromo, methyl, methoxy and nitro, with chloro, fluoro and nitro being preferred. Dichloro substitutions are in the 2,4 or 3,4 positions in the ring. The α-carbon atom may also be methylated, e.g. (C ^a Me/ Cl)Phe. The 1-position substituent is preferably modified so that its a amino group contains an acyl group, such as for yl(For), acetyl(Ac), acrylyl(Acr) , vinylacetyl(Vac) or benzoyl(Bz), with acetyl and acrylyl being preferred. PAL and D-PAL represent the L- and D-isomers of pyridylalanine where the ,9-carbon of Ala is linked to the 2-, 3- or 4-position, preferably to the 3-position, on the pyridine ring. When ,5-D-NAL is present in the 1-position and R _j is not Arg, a hydrophilic D-amino acid residue, such as 4-NH ₂ -D-Phe, 4-guanidino- D-Phe, D-His, D-Lys, D-Orn, D-Arg, D-Har(Homoarginine) or D-PAL is preferably present in the 6-position if U ^* is not present. When dehydroPro is present in the 1-position, D-PAL or a D-isomer of a lipophilic amino acid, such as D-Trp, D-Phe, For-D-Trp, N0 ₂ -D-Trp, D-Leu, D-Ile, D-Nle, D-Tyr, D-Val, D-Ala, dialkyl Arg, dialkyl Har,

D-Ser(OtBu), ,3-D-NAL or (i Bzl)D-His is preferably in the

6-position, if ϋ ^* is not present.

These GnRH analogs are very soluble at a pH just below physiological pH, i.e. about 4.5 to about 6, and thus can be formulated and administered in concentrated form, greatly facilitating administration at a pH of about 5 to 7.4 which is presently preferred. The antagonists inhibit ovulation of female mammals when administered at low levels at proeεtruε and are also effective to cause resorption of fertilized eggs if administered shortly after conception. The antagonists are also effective for the contraceptive treatment of male mammals and"the treatment of steroid-dependent tumors. The agonists are substantially more potent than native GnRH.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As previously mentioned, the unnatural amino acids are represented by the formula U ^* :

0 Y

and there is at least one such residue in each peptide ^" of the invention, wherein X, Y, R, and R _j are as defined previously.

More specifically, the GnRH antagonists of the present invention are represented by the following Formula (F,) :

G-AA,-(A)D-Phe-AA ₃ -Ser-AAs-AA ₆ -AA ₇ -AA ₈ -Pro-AA ₁₀ wherein G is hydrogen or an acyl group having 7 or less carbon atoms; AA, is dehydroPro, D-pGlu, (A)D-Phe, (B)D-Trp, Pro, or ,3-D-NAL; A is H, Cl, F, N0 ₂ , CH ₃ , OCH ₃ , C ^β Me/4Cl, Cl ₂ or Br; B is H, N0 ₂ , NH ₂ , 0CH ₃ , F, Cl, Br, CH ₃ , N ⁱⁿ For or N ⁱⁿ Ac; AA ₃ is U ^* , D-PAL, ,9-D-NAL.or (B)D-Trp; A^

is U ^* , Tyr, (C)Arg, Lys(cpd), Orn(cpd), Dbu(cpd), Dpr(cpd), (A)Phe, (3I)Tyr or His; AA ₆ is U ^* , ,9-D-NAL, (B)D-Trp, (A')D-Phe, (D)D-Orn, (D)D-Lys, (D)D-Dbu, (D)D-Dpr, D-Har, D-Tyr, (E)D-Hiε, D-PAL, (C)D-Arg or a suitable lipophilic D-isomer; A' is A, NH ₂ , NHCH ₃ or gua; C is H or lower alkyl; D is G, cpd or an aryl group; E is H, imBzl or dinitrophenol; AA ₇ is Nle, Leu, NML, (A)Phe, Met, Nva, Tyr, (B)Trp or PAL; AA ₈ is u\ (C')Arg, (C ^» )Har or ILys; C is H or di-lower alkyl; AA ₁₀ is D-Ala-NH ₂ , Gly-NH ₂ , AzaGly-NH ₂ or NH(R) ; R is lower alkyl, preferably CH ₂ CH ₃ ; and U ^* is as defined above. When AA _t is S-D-NAL and AA _j is not Arg, then AA ₆ is preferably U ^* , 4-NH ₂ -D-Phe, D-Lys, D-Orn, D-Har, D-His, 4-gua-D-Phe, D-PAL or D-Arg. By dehydroPro is meant 3,4 dehydroproline, C _j H _y O _j N. By 5-D-NAL is meant the D-isomer of alanine which is substituted by naphthyl on the /3-carbon atom, i.e., also 3-D-NAL. Preferably 3-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. PAL represents alanine which is substituted by pyridyl on the β-carbon atom; preferably the linkage is to the 3-position on the pyridine ring. When subεtituted 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, e.g. with formyl (N ⁱⁿ For- or lFor-) or with acetyl. N ⁱⁿ For-D-Trp and 6N0 ₂ -D-Trp are the preferred substituted residues. By NML is meant N*CH ₃ -L-Leu. By Dbu is meant alpha, gamma diamino butyric acid, and by Dpr is meant a,β diamino propionic acid. When dehydroPro is present in the 1-position, Tyr or ϋ ^* is preferably present in the 5-position and a lipophilic residue is in

the 6-position. By 4-gua-D-Phe is meant a residue of D-Phe having guanidine substituted in the para-position. By AzaGly-NH ₂ is meant NHNHCONH ₂ . The guanidino group of an Arg residue in the 5- or 6-position may be substituted by lower alkyl, i.e. 1 to 4 carbon atoms, e.g., propyl(Pr). When D-Lys, D-Dbu, D-Dpr or D-Orn is present in the 6-position and it is not a part of an unusual amino acid U ^* , its side-chain-amino group may be acylated by an acyl group which may be aliphatic, heterocyclic or aromatic, e.g. nicotinic acid, or may be substituted by an aryl group having not more than 1 phenyl ring. More specifically the GnRH agonists of the invention are represented by the following Formula (F ₂ ) : pGlu-His-Trp-Ser-Tyr-(U)-AA ₇ -Arg-Pro-AA ₁₀ , wherein AA ₇ and AA, ₀ are as defined hereinbefore; preferably AA ₇ is Leu or NML and AA ₁₀ is NHCH ₂ CH ₃ .

Overall, the invention thus provides GnRH analogs having the Formula (F ₃ ) :

G-AA-AA ₂ -AA ^, -Ser-AA _s -AA ₆ -AA ₇ -AA _g -Pro-AA ₁₀ wherein AA is pGlu or AA,; AA _j is His or (A)D-Phe; AA' is Trp or AA ₃ ; and all others are as defined hereinbefore.

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 hydroxy ethylated 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 when such a C-terminus is desired. 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 stepwiεe 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 intermediateε made generally in accordance with the invention may be represented by the formula: X ¹ -AA-AA ₂ (X ⁵ )-U ₃ -Ser(X ³ )-U ₅ ~U ₆ -AA ₇ (X ² or X ⁷ )-U _β -Pro-X ⁸ wherein: U ₃ is either U' or AA' (X ² ) ; U ₅ is either U' or ^' AA^X* or X ⁵ ); U ₆ is either U' or AA ₆ (X* or X ⁵ or X ⁶ ); U ₈ is either U' or AA _g (X ⁵ or X ⁶ ); U' is either Lys(X ^β ), Orn(X'), Dbu(X*) or Dpr(X ^a ) ; X ¹ is an α-amino protecting group of the type known to be useful in the art in the stepwiεe εyntheεiε 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 X ¹ 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, εuch aε allyl(Aly), triphenylmethyl(trityl) and benzyl(Bzl) ; (7) trialkylsilane groups, such as trimethylsilane. The preferred α-amino protecting group is Boc when X iε hydrogen.

X ² is hydrogen or a protecting group for the indole nitrogen of Trp, such as Bz, Ac or For. In many syntheεes,there is no need to protect Trp, and such protection is not used if acylated D-Trp is present elsewhere in the peptide.

X ³ is a protecting group for the hydroxyl side chain of Ser or Thr, 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-dichlorobenzyl(DCB) . 2BrZ is preferred. X ^s is a protecting group for a εide chain guanidino group, εuch as that in Arg or Har, or for the imidazole group of His, εuch as nitro, Tos, trityl, adamantyloxy¬ carbonyl, Z and 2,4-dinitrophenol(Dnp) , or X ⁵ may be hydrogen, which means there is no protection on the side chain group atoms. Tos is generally preferred.

X* is a protecting group for an amino side chain group such as Z or 2C1Z; X" is a subclaεs of X ⁶ comprising such protecting groups that can be removed without removing other side chain protecting groups so as to

allow the omega-amino group to thereafter take part in the reactions to build the unnatural amino-acid residue. Preferably a base-labile group, such as Fmoc, methylsulfonylethyloxycarbonyl(Msc) or trifluoroacet l(Tfa) , is used; however, it may also be possible to use a hydrazine-labile group such as phthaloyl.

X ⁷ is hydrogen or a protecting group for Met, such as oxygen; Met is generally left unprotected. X ⁸ may be Gly-NH-[resin εupport], D-Ala-NH- [resin support] or N(A)-[resin support]; X ⁸ may also be an amide either of Gly or of D-Ala or a substituted amide attached directly to Pro or NHNHC0NH ₂ .

The criterion for selecting side chain protecting groupε for X ² -X ⁷ iε that the protecting group εhould 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. Protecting groups generally should not be split off under coupling conditions but εhould be removable upon completion of the synthesis of the desired amino acid sequence under reaction conditions that will not alter the peptide chain.

When the X ⁸ group is Gly-NH-[resin support] or D-Ala-NH-[reεin εupport], an amide bond connects Gly or D-Ala to a BHA resin or to a MBHA resin. When the X ⁸ group is N(A)-[resin support], a substituted amide bond

connects Pro to an N-alkylaminomethyl (NAAM) resin. When X ⁸ is AzaGly-NH ₂ , the peptide iε 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 X ¹ protecting group for the α-amino group of 3-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 in the presence of dicyclohexyl carbodiimide (DCC) or preferably with acetic anhydride or by another εuitable reaction aε known in the art.

Thus, the invention also provides a method for making a peptide, said peptide having the formula:

G-AA-AA _j -AA'-Ser-AA _j -AA^AA _y -AA _g -Pro-AA^, wherein at least one of AA ¹ , AA _j , AA ₆ and AA _g is U ^* and the symbols are as set forth hereinbefore, which method comprises (a) forming an intermediate peptide having the formula: X ¹ -AA-AA ₂ (X ⁵ )-U ₃ -Ser(X ³ )-U ₅ -U ₆ - AA ₇ (X ² or X ⁷ )-U _β -Pro-X ⁸ wherein: U ₃ is either U' or AA' (X ² ) ; U ₅ is either U' or AA _j (X* or X ⁵ ); U ₆ is either U' or AA ₆ (X* or X ⁵ or X ⁶ ); U _β iε either U' or AA ₈ (X ⁵ or X ⁶ ); U' iε either Lys(X'), 0rn(X ⁸ ), Dbu(X') or Dpr(X ^s ) ; X ¹ iε hydrogen or an α-amino protecting group; X ² is hydrogen or a protecting group for an indole nitrogen; X ³ is a protecting group for a hydroxyl group of Ser or Thr; X* is hydrogen or a protecting group for a phenolic hydroxyl group of Tyr; X ⁵ is either hydrogen or a protecting group for a guanidino or imidazole side chain; X ⁶ is a protecting group for a primary amino side chain of which X* is a subgroup that iε

removable without removing other protecting groups; X ⁷ is hydrogen or a protecting group for Met; X ⁸ 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

NHNHC0NH ₂ ; provided however that at least one of U ₃ , U ₅ , U ₆ and U ₈ is either Lys(X*), Orn(X ^β ), Dbu(X ^β ) or Dpr(X*) ; (b) removing at leaεt one X ^a to deprotect a εide chain primary amino group of at leaεt one amino acid residue of said intermediate peptide; (c) reacting said deprotected side chain primary amino group to build said residue into one having the formula U ^* ; and (d) splitting off any remaining groups X ¹ to X ⁷ and/or cleaving from any resin support included in X ⁸ . Purification of the peptide iε effected by ion exchange chromatography on a CMC column, followed by partition chromatography using the elution εyεtem: 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-r328.

The antagonists of the invention are effective at levels of lesε than 100 microgramε 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 thuε can be prepared aε relatively concentrated solutionε for administration. The antagonists are also effective to arrest spermatogenesis when adminiεtered to male mammalε on a regular baεis 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 reasonable 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 steroidε for other purpoεeε aε indicated hereinbefore.

In the following formulas, the U ^* residueε are 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 parentheseε. Preferably, the original residue is incorporated in the main peptide chain, for example Lys or D-Lys or Orn, Dbu, Dpr or a 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 of the amino acid U ^* . However, as indicated hereinbefore, the suitably protected unnatural amino acid U ^* can be added as a part of the usual chain elongation process.

With respect to modified side chain amino groups of the amino acids Lys, Orn, Dbu and Dpr, the following abbreviations are used: act=acetyl aminotriazole 2amp=2 aminomethyl pyridyl cyanoguanidino 3amp=3 aminomethyl pyridyl cyanoguanidino 4amp=4 aminomethyl pyridyl cyanoguanidino bcg=aminobutyl cyanoguanidino bzcg=aminobenzyl cyanoguanidino bur=N-g-amido, N-g'-butylguanidino chcg=aminocyclohexyl cyanoguanidino ecg=aminoethyl cyanoguanidino icg=aminoisopropyl cyanoguanidino hcg=aminohexyl cyanoguanidino hicg=hiεtaminyl cyanoguanidino mcg=aminomethyl cyanoguanidino

ncg=aminoethyl(l or 2)naphthyl cyanoguanidino mncg ^' =aminomethyl(1 or 2)naphthyl cyanoguanidino

Ocg=0-phenyl cyanoguanidino pcg=aminopropyl cyanoguanidino

Sbcg=thiobutyl cyanoguanidino tcg=3-amino 1,2,4 triazole trcg=indole ethylamino cyanoguanidino(tryptamino cyanoguanidino) mpcg= methylpyridyl cyanoguanidino

EXAMPLE I Peptides as indicated in TABLE I having the formula: Pro-D-Ala-NH ₂ are prepared by the solid-phase procedure referred to above. E

For purposes of an example, a representative solid phase synthesis of Peptide No. 1 above, which is referred to as [Ac-,3-D-2NAL ¹ , (4C1)D-Phe ² , D-3PAL ³ , Lys(icg) ⁵ , D-Lys(icg) ⁶ , ILys ⁸ , D-Ala ¹⁰ ]-GnRH is εet forth hereinafter. Thiε peptide has the following formula:

Ac-0-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-Lys(iεopropyl cyanoguanidino)-D-Lys(isopropyl cyanoguanidino)-Leu- - Lyε(iεopropyl)-Pro-D-Ala-NH ₂ .

An MBHA reεin is used, and Boc-protected D-Ala is coupled to the resin over a 2-hour period in CH ₂ C1 ₂ using a 3-fold excess of Boc derivative and DCC as an activating reagent. The D-Ala residue attaches to the MBHA reεidue by an amide bond.

Following the coupling of each amino acid reεidue, washing, deblocking and coupling of the next amino acid residue is carried out in accordance with the following schedule uεing an automated machine and beginning with about 5 grams of resin:

STEP REAGENTS AND OPERATIONS MIX TIMES MIN.

1 CH ₂ C1 ₂ wash-80 ml. (2 times) 3

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

3 CH ₂ C1 ₂ wash-80 ml. (3 times) 3

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

5 Isopropyl alcohol + 1% ethanedithiol wash-80 ml. (2 times) 3

6 TEA 12.5 percent in CH ₂ C1 ₂ -70 ml.

(2 times). 5 7 MeOH wash-40 ml. (2 times) 2

8 CH ₂ C1 ₂ wash-80 ml. (3 times) 3

9 Boc-amino acid (10 mmoles) in 30 ml. of either dimethylformamide(DMF) or CH ₂ C1 ₂ , depending upon the

24 εolubility of the particular protected amino acid, (1 time) pluε DCC (10 mmoles) in CH ₂ C1 ₂ 30-300 10 MeOH wash-40 ml. (2 times) 3

11 Triethylamine(TEA) 12.5 percent in CH ₂ Cl ₂ -70 ml. (1 time) 3

After step 3, an aliquot may be taken for a ninhydrin test as well known in the art: if the test is negative, proceed to step 4 for removal of BOC-group prior to coupling of the next amino acid; if the test is positive or slightly poεitive, repeat steps 9 through 11. The above schedule is used for coupling of each of the amino acids of the peptide of the invention after the first amino acid haε been attached. N ^β Boc protection is used for each of the remaining amino acids throughout the synthesis. N*Boc-,S-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 or commercially available from Synthe Tech, Oregon, U.S.A. The side chains of Lys in the 5-position and of D-Lys in the 6-position are protected with Fmoc. Bzl (benzyl ether) is used as a εide chain protecting group for the hydroxyl group of Ser. Boc-Lyε(Ipr) iε uεed for the 8-poεition. After deblocking the α-amino group at the N-terminal using trifluoroacetic acid(TFA), acetylation iε achieved uεing a large excess of acetic anhydride in dichloromethane. Following completion of the assembly of the peptide and acetylation of the N-terminus, the following intermediate is present: Ac-£-D-2NAL-(4Cl)D-Phe-D-3PAL- Ser(Bzl)-Lys(Fmoc)-D-Lys(Fmoc)-Leu-Lys(Ipr)-Pro-D-Ala-

NH-[MBHA resin support]. The unnatural amino acids in the 5- and 6-positions are formed by εimultaneously carrying out the following reactions with the deprotected side chains of the Lys residues. The Fmoc protecting group is removed from both by treatment of the peptidoresin with 20 percent piperidine in DMF for 5 minutes, then wash with DMF, then treatment with more piperidine/DMF for 20 minutes. After washing the reεin with DMF, CH ₃ OH, CH ₂ C1 ₂ , and finally DMF, the newly freed amino group is treated with a large excess (>10 fold) of diphenyl cyanocarbonimidate(PCI) in DMF. Thereafter, the peptide is then subjected to the standard wash (see Stepε 10-11) and then treated with isopropylamine disεolved in DMF for 24 hourε at about 22*C. to complete the formation of the aminoisopropyl cyanoguanidino moiety; for εome of ^' the more hindered reactants, this step may be repeated.

The cleavage of the peptide from the resin and deprotection of the Ser side chain takeε place very readily at 0"C. with HF. Aniεole is added as a scavenger prior to HF treatment. After the removal of HF under vacuum, the resin is extracted with 50% acetic acid, and the washings are lyophilized to provide a crude peptide powder.

Purification of the peptide is then effected by high performance liquid chromatography (HPLC) , as known in the art and specifically set forth in J. Rivier, et al. Jj_ Chromatography _f 288 (1984) 303-328.

The peptide is judged to be homogeneous uεing thin layer chromatography and εeveral different εolvent εystems, aε well aε by using reversed-phase high pressure liquid chromatography and an aqueous triethylammonium phosphate solution plus acetonitrile. Amino acid analysis of the resultant, purified peptide is conεiεtent with the formula for the prepared εtructure, εhowing

substantially integer-values for each amino acid in the chain; mass εpectral analysis is also consiεtent. The optical rotation iε meaεured on a photoelectric polarimeter aε

20 [α] = -2.8±0.5(c=l, 50% acetic acid). D The other peptides in Table I are similarly syntheεized and purified. The peptideε are aεεayed in vivo and may alεo be teεted in vitro. If performed, in vitro teεting is carried out using dissociated rat pituitary cells maintained in culture for 4 days prior to the asεay. The levelε of LH mediated in response to the application of peptides is aεsayed by specific radioimmunoassay for rat LH. Control diεheε of cells only receive a measure which is 3 nanomolar in GnRH; experimental disheε receive a measure 3 nanomolar in GnRH plus a measure having either the present standard antagonist for comparison purposes i.e. [Ac-dehydro Pro ¹ , (4F)D-Phe ² , D-Trp ³ '°]-GnRH or the test peptide, in concentrations ranging from 0.01 t 10 nanomolar. The amount of LH secreted in the sa pleε treated only with GnRH iε compared with that secreted by the εamples treated with the peptide plus GnRH. The ability of the test peptide to reduce the amount of LH released by 3 nanomolar GnRH is compared to that of the present standard peptide. The in vivo testing determines 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, is injected with a specified microgram dosage of peptide in either saline, bacteriostatic water, polyethylene glycol, corn oil or mixtures of the above

with ethanol at about noon on the day of proestrus. Proeεtruε iε the afternoon of ovulation. A separate female rat group is used as a control to which the peptide is net administered. Each of the control female rats ovulates on the evening of proestrus; of the rats treated, the number of them which ovulate is recorded. Each of the peptides is considered to be totally effective to prevent ovulation of female rats at a dose of about 500 micrograms.

All peptides listed in Table I are considered effective to block GnRH-induced LH secretion -in vitro at some reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low doεageε. The following Table A εhows the results of .in vivo testing of various of these GnRH antagonists:

TABLE A

28

EXAMPLE II Peptides as indicated in TABLE II having the formula: Ac-dehydroPro-(A)D-Phe-AA^Ser-AA _j -ø-D-ΣNAL- Leu-AA ₈ -Pro-D-Ala-NH ₂ are prepared by the solid-phaεe procedure referred to above.

TABLE II

All peptides listed in Table II are conεidered effective to block GnRH-induced LH εecretion in vitro at εome reaεonable concentration. All of the peptideε are conεidered to be effective to prevent ovulation of female mammals at low dosages.

29

EXAMPLE III Peptideε as indicated in TABLE III having the formula: G-0-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-Tyr-AA ₆ -Leu- AA _β -Pro-AA ₁₀ are prepared by the solid-phaεe procedure referred to above.

TABLE III

D-Arg Lys(beg) D-Ala-NH, D-Lys (beg) Arg D-Tyr Lys(icg) Gly-NH, (Et) D-Arg orn " D-Lys ^« (ecg) D-Har " (meg)

(4gua) D-Phe Dpr(2ncg) AzaGly-NH ₂ D-Orn " (chcg) D-Ala-NH, D-His ^» (teg) II (Bu) D-Arg Dbu(Ironeg) tt (Bz) D-Orn " (2amp) ( NH ₂ ) D-Phe " (4amp) (Ac) D-Lys '• (trcg) AzaGly-NH ₂

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

EXAMPLE IV Peptideε as indicated in TABLE IV having the formula: Ac-AA,-(4C1)D-Phe-D-3PAL-Ser-Tyr-D-Arg-AA ₇ - AA ₈ -Pro-D-Ala-NH ₂ are prepared by the solid-phaεe procedure referred to above.

TABLE IV

All peptideε liεted in Table IV are considered effective to block GnRH-induced LH secretion in vitro at some reasonable concentration. All of the peptides are conεidered to be effective to prevent ovulation of female mammals at low dosages.

31

EXAMPLE V Peptides aε indicated in TABLE V having the formula: Ac-AA,-(4C1)D-Phe-AA^Ser-AA _j -AA^Leu- ILys-Pro-D-Ala-NH ₂ are prepared by the solid-phase procedure referred to above.

TABLE V.

The peptides listed in Table V 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 VI Peptides aε indicated in TABLE VI having the formula: G-AA,-(4C1)D-Phe-D-Trp-Ser-Tyr-AA^Leu-AA _g - Pro-D-Ala-NH ₂ are prepared by the εolid-phase procedure referred to above.

TABLE VI

AA, AA _* AAJJ

dehydroPro D-Lys(beg) Lys(beg)

0-D-2NAL S-D-2NAL " (3amp) 9-D-2NAL D-Val " (teg)

Pro D-Ser(OtBu) " (chcg) dehydroPro (imBzl)D-His " (ecg)

(* Br)D-Phe (5C1)D-Trp Orn "

D-pGlu (6Br)D-Trp " (bzcg)

/5-D-1NAL (Me)D-Arg " (Ocg) dehydroPro D-Har " (4amp)

/S-D-2NAL (Bz)D-Lys " (chcg)

" (Arg ⁵ ) 0-D-2NAL Lys(Ocg) dehydroPro D-Ala " (teg)

The peptides listed in Table VI 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.

33

EXAMPLE VII Peptides as indicated in TABLE VII having the formula: AC-3-D-2NAL-(4C1)D-Phe-D-SPAL-Ser-AA^AA^ Leu-AA ₈ -Pro-D-Ala-NH ₂ are prepared by the solid-phase procedure referred to above.

TABLE VII

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

34

EXAMPLE VIII Peptides aε' indicated in TABLE VIII having the formula: Ac-/3-D-2NAL-(4C1)D-Phe-D-SPAL-Ser-AA _j -AA^ Leu-AA _g -Pro-D-Ala-NH ₂ are prepared by the εolid-phase procedure referred to above.

TABLE VIII

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

EXAMPLE IX

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

Results of -in vivo testing of selected of these antagoniεtε are εhown in the following Table B:

TABLE B

9

37

EXAMPLE X Peptides as indicated in TABLE X having the formula: pGlu-His-Trp-Ser-Arg-AA ₆ -AA ₇ -Arg-Pro-AA, ₀ are prepared by the solid phase procedure referred to above.

TABLE X Leu Gly-NH ₂

II

AzaGly-NH ₂ NHCH ₂ CH ₃

It

NML

II

Leu Gly-NH ₂

NML AzaGlyNH ₂ n NHCH ₂ CH ₃ H NHCH _j

Leu NHCH ₂ CH ₂ CH ₃

II Gly-NH ₂ II

NML ti NHCH ₂ CH ₃

The peptides described in TABLE X are considered to be effective to cause the release of LH and FSH in female rats. All of them are considered to be substantial more effective than native GnRH.

EXAMPLE 145 A peptide intermediate having the formula: AC-/S-D-2NAL-(4C1)D-Phe-D-3PAL-Ser(Bzl)-Lys(Fmoc)-D- 3PAL-NML-Lyε(Dts)-Pro-D-Ala-NH-[resin support] is prepared by the solid phase procedure generally referred to above. The intermediate is then treated with piperidine to remove the Fmoc protecting group and is thereafter reacted with PCI as hereinbefore deεcribed. Next the Dtε protecting group is removed from the amino side chain of the Lys residue in the 8-position using a suitable thiol, such as jS-mercaptoethanol or thiophenol(PhSH) , in DMF, and the peptidoresin is given the standard wash. Thereafter, it iε maintained at 22*C. for 10-60 minuteε or until the ninhydrin test is negative to allow the reaction to proceed to completion, effecting cyclization of the side-chain primary amino group of Lyε ⁸ and the cyanoguanidino moiety which was earlier formed upon the Lys ⁵ side chain. Deprotection and cleavage are then carried out as previously described. Following HPLC purification as previously described, the GnRH antagonist is tested. The peptide ^' is considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE 146 A peptide intermediate having the formula: Ac-/3-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Orn(Fmoc)-D-Trp-

Leu-Lyε(Npε)-Pro-D-Ala-NH-[reεin support] is prepared by - the solid phase procedure referred to above. The peptide intermediate is treated with a suitable thiol as described in Example 145 to form the cyanoguanidino moiety by reaction of PCI with the side chain amino group of the Lys residue in the 8-position, and it is then cyclized with the deprotected side chain amino group of Orn in the 5-position following removal of the Fmoc protecting group. Following cleavage and HPLC

purification as previously described, the GnRH antagonist is tested. The peptide is considered to be effective to prevent ovulation of female mammals at low doεageε.

EXAMPLE 147 A peptide intermediate having the formula:

Ac-0-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Tyr(2BrZ)- D-Lys(Fmoc)-Leu-Lys(Ipr)-Pro-D-Ala-NH-[resin support] is prepared by the solid phase procedure referred to above. Following the removal of the Fmoc protection, the peptide intermediate is reacted as generally described in Example I using naphthyl isocyanate instead of PCI to form the napthylurea moiety with the side chain amino group of the D-Lys residue in the 6-position. Following cleavage and HPLC purification as previously described, the GnRH antagonist is tested. The peptide is considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE 148 A peptide intermediate having the formula: AC-/3-D-2NAL-(4C1)D-Phe-D-3PAL-Ser(Bzl)-Tyr(2BrZ)-

D-Lyε(Fmoc)-Leu-Lyε(Ipr)-Pro-D-Ala-NH-[resin support] is prepared by the solid phase procedure referred to above. Following the removal of the Fmoc protection, the peptide intermediate is reacted as generally described in Example I using naphthyl isothiocyanate instead of PCI to form the napthylthiourea moiety with the εide chain amino group of the residue in the 6-position. Following cleavage and HPLC purification as previously described, the GnRH antagonist is tested. The peptide is considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE 149 A peptide intermediate having the formula: AC-3-D-2NAL-(4C1)D-Phe-D-3PAL-Ser(Bzl)-Tyr(2BrZ)- D-Lys(Fmoc)-Leu-Lyε(Ipr)-Pro-D-Ala-NH-[resin support] is prepared by the solid phase procedure referred to above. Following the removal of the Fmoc protection, the peptide intermediate is first reacted using 2-bromoethyl,2' (Boc- amino)ethyl ether disεolved in DMF for 1 hour or until the ninhydrin teεt is negative to link the carbon atom to the side-chain amino group by the removal of the halogen to form: Q-NH-(CH ₂ ) ₂ -0-(CH ₂ ) ₂ -NH(Boc) . This compound is then reacted as generally described in Example I using PCI to form the cyanoguanidino moiety with the side chain secondary amino group of the residue in the 6-position. Next the Boc protection group is removed, and the primary amino group reacts with the -OPh group to give the compound:

N-CN

Following cleavage and HPLC purification as previouεly deεcribed, the GnRH antagoniεt is tested. The peptide is considered to be effective to prevent ovulation of female mammals at low doεageε.

EXAMPLE 150 (Boc)Dpr iε reacted with PCI aε in Example I to produce

which iε then reacted with

hydrazine dissolved in DMF for 1 to 2 days at room temperature washed with DMF and then repeated to replace the -OPh group with concomminent formation of the heterocycle: NH ₂

NH(Boc) N C^

I I I

H-C-CH,-NH-C. _^ ,N COOH H which is particularly useful as a subεtitute for Hiε in peptide syntheses.

Following purification of the peptides, various of them are further characterized by subjection to high performance liquid chromatography on C, ₈ silica (Vydac 0.46 x 25 cm) using a flow rate of 1.7 ml/min and a gradient of from 35% to 85% by volume of Buffer B over a time span of 50 minutes, with the remainder being Buffer A. Buffer A is a solution of 0.3% triethylamine (v/v) and 0.1% phosphoric acid in water at pH 7.0; Buffer B is 60% by volume acetonitrile in Buffer A. The following Table C showε when the specific peptides elute from the C, ₈ silica having a particle size of about 5μ and a pore size of 30θA when subjected to a gradient of from 35% Buffer B to 85% Buffer B (by volume with the remainder being Buffer A) over 50 minutes at a flow rate of 1.7 ml. per minute, and thereafter at 85% isocratically for 10 min.:

The peptides of the invention are often administered in the form of pharmaceutically acceptable, nontoxie εaltε, 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 saltε for purpoεeε of thiε application) , or of combinationε of the two. Illuεtrative of εuch acid addition εalts are hydrochloride, hydrobromide, sulphate, phosphate, nitrate, oxalate, fumarate, gluconate, tannate, maleate, acetate, citrate, benzoate, εuccinate, alginate, malate, aεcorbate, tartrate and the like. For example, an aqueouε εolution 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 diεintegrating agent, εuch as alginic acid; and a lubricant, such as magnesium εtearate. 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 solutionε or the like may be effected.

The pharmaceutical compoεitionε will uεually contain the peptide in conjunction with a conventional, pharmaceutically-acceptable carrier. Uεually, the doεage will be from about 10 microgramε to about 2.5 milligrams of the peptide per kilogram of the body weight of the host when given intravenously; although oral dosageε will be higher, it. iε anticipated that the nature of theεe compoundε will 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 or agonists of GnRH using a suitable carrier in which the peptide is soluble.

It may also be deεirable 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 εlow releaεe, depot or implant doεage forms may be utilized. For example, a dosage form may contain a pharmaceutically acceptable non-toxic salt of the compound which has a low degree of εolubility in body fluids, for example, an acid addition salt with a polybasie acid; a salt with a polyvalent metal cation; or combination of the two salts. A relatively insoluble salt may also be formulated in a gel, for example, an aluminum εtearate gel. A εuitable, slow-release depot

formulation for injection may alεo contain the GnRH analog 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. intranaεally or intravaginally to achieve fertility inhibition and/or control and alεo in applicationε calling for reverεible 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' dosageε will vary with the form of adminiεtration and the particular εpecies of mammal being treated. An example of one typical dosage form is a bacteriostatic water solution 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. Oral administration of the peptide may be given in either solid form or liquid form.

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 subεtitutionε known in the art which do not εignificantly 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. The 6-position substitutes set forth in Table VI are considered to be

equivalents Icnown in the prior art and can be included in the peptides of the invention. Substituted Phe, such as (4F)Phe, can be used instead of Phe in the 7-position. Both butyl Lyε and diethyl Lys are considered to be equivalents of ILys; however, ILys is preferred when neither U ^* or Arg is in the 8-position. Other hydrophobic amino acid residues can alεo be employed in the 1-poεition, preferably in D-isomer form, and are conεidered equivalentε of those εpecified. Moreover, the analogs can be administered in the form of their pharmaceutically or vetinarially acceptable, nontoxie salts, as indicated hereinbefore, which are considered equivalents.

Particular features of the invention are emphasized in the claims which follow.