Abstract:
Peptides which include unnatural amino acides and which either inhibit or promote the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. Administration of an effective amount of such peptides that are GnRH antagonists prevents ovulation of female mammalian eggs and/or the release of steroids by the gonads. The antagonists may be used to treat steroid-dependent tumors, such as prostatic and mammary tumors. The peptides are analogs of the decapeptide GnRH wherein there is at least one residue of an unnatural amino acid in the 3-position, the 5-position, the 6-position and/or the 8-position. Such unnatural amino acids are useful in the synthesis of peptides and have the formula U*: ##STR1## where n is an integer from 1 to 6; Y is N--CN, N--CONHR 9 , S, O or CH--NO 2  ; R 9  is H, Ac, lower alkyl, aromatic or heterocyclic; X is NH, O, S, M 1  (CH q ) p  M 2  or M 1  --(CH 2 ) p&#39;  --M 2  (CH 2 ) p&#34;  --M 3 , where M 1  is NR 10 , O, S or CHR 3  wherein R 3  is methyl, ethyl, propyl, phenyl, pyridinyl, pyrimidinyl or purinyl, q is 1 or 2; p, p&#39; and p&#34; are integers between O and 6; R 10  is H, lower alkyl or the like, and M 2  and M 3  are M 1 , COOH, CONH 2 , COOR 3  or CN; R 1  is H, alkyl, modified alkyl, alkenyl, alkynyl, aryl or a direct bond to X; R 2  is R 1 , OH, NH 2 , NHR 1 , or heterocycle.

Description:
This invention was made with Government support under Grant No. HD-13527 and Contracts N01-HD-9-2903 and N01-HD-0-2906 awarded by the National Institutes of Health. The Government has certain rights in this invention. 
    
    
     This application is a continuation-in-part of U.S. Ser. No. 07/428,827, filed Oct. 30, 1989. 
     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 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 has been isolated and characterized as a decapeptide having the following structure: 
     
         pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH.sub.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 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, Ile is isoleucine, Thr is threonine, Lys is lysine, Asp is aspartic acid, Asn is asparagine, Gln 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 endometriosis. 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 *  : ##STR2## where n is an integer from 1 to 6 and is preferably 1,2,3 or 4; Y=N--CN, N--CONHR 9 , S, O or CH--NO 2 , where R 9  is H, Ac, alkyl (preferably C 1  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 1  to C 4 ) and alkoxy (C 1  to C 4 )); X=NH, O, S, N 3 , M 1  --(CH q ) p  --M 2  or M 1  --(CH 2 ) p&#39;  --M 2  (CH 2 ) p&#34;  --M 3 , where M 1  is NR 10 , N, O, S or CHR 3  wherein R 3  is methyl, ethyl, propyl, phenyl, pyridinyl, pyrimidinyl or purinyl; q=1 or 2; p, p&#39; and p&#34; are integers between 0 and 6; R 10  is H, methyl, ethyl, propyl, phenyl or substituted phenyl (preferably by Cl, F, NO 2  or NH 2 ); and M 2  and M 3  =M 1 , COOH, CONH 2 , COOR 3  or CN (preferably X is NH, O or S); R 1  =H, alkyl (preferably C 1  to C 6  and most preferably C 1  to C 4 ), modified alkyl (preferably C 1  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 , alkynyl (preferably C 2  to C 4 ), such as CH 2  C.tbd.CR 3 , aryl such as benzyl, tolyl, p-aminobenzyl (anilinyl) and pCl-benzyl or a direct bond to X; R 2  =R 1 , OH, NH 2 , NHR 1 , heterocycle (preferably as illustrated hereinafter) or desR 2 , with R 2  being desR 2  when X=N 3 . Optionally R 2  and X can be interconnected, or R 1  and R 2  can be connected to each other via a branched or unbranched methylene bridge of type --(CH 2 ) m  --or --(CH 2 ) m  --M--(CH 2 ) m&#39;  --. In such an R 1  --R 2  moiety, m and m&#39; are integers from 1 to 6 and preferably from 1 to 3; and M=NH, O, S or CHR 4 , wherein R 4  is lower alkyl or aryl and is preferably methyl, ethyl, propyl, phenyl or pCl-phenyl, with M preferably being O or S. Most preferably, when R 1  and R 2  are interconnected, they form a 5, 6, or 7-member heterocyclic ring with the &#34;N-C-X&#34; 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 cyanocarbonimidic acid (I): ##STR3## wherein &#34;Q&#34; 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&#39;-cyano-O-phenylisourea 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): ##STR4## This group may also be referred to (IUPAC nomenclature) as N-g-cyano-N-g&#39;-3-methylpyridylguanidino. 
     Such compounds can be hydrolyzed under acidic conditions to produce compounds which are also biopotent--for example: ##STR5## The hydrolyzed versions, referred to herein as including the N-g&#39;-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: ##STR6## 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 1  --(CH q ) p  --M 2  or M 1  --(CH 2 ) p&#39;  --M 2  --(CH 2 ) p&#34;  --M 3 , where M 1 , M 2  and M 3  are individually NH, N, O, S, or CHR 3 , with p, p&#39;, p&#34; 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 3  HNNHCH 3 , 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 such as: ##STR7## For example, where --XR 2  =--HNNH 2 , ##STR8## 
     Where XR 2  contains a carboxylic acid group or the equivalent, particularly a carboxylic ester or carboxylic amide, a heterocyclic moiety, such as a saturated pyrimidine-like moiety (VI), is formed, by reaction of the carboxylic group with the secondary amino group (R 1 ), when M 1  is N, and similar 6-membered heterocyclic moieties are formed when M 1  is O or S. For example, R 2  may be M 1  --(CH 2 ) p  --M 2  with M 2  =COOH, COOCH 3  or CONH 2  and p being an integer between 1 and 4. For instance in such a case where an aliphatic carboxylic acid group is present and p=2: ##STR9## If R 2  includes an ortho-substituted aromatic carboxylic acid, e.g. benzoic acid (q=1 and p=6), the corresponding quinazoline-like species (VII) is formed: ##STR10## 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&#39; may be H, Cl, Br, F, NHCH 3  or SCH 3 , and R 7  and R 8  may be H, CH 3  or CH 2  CH 3 . 
     The molecules wherein X=N 3  and R 2  is desR 2  (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 3 ). 
     Peptides wherein Y is O (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. ##STR11## 
     Peptides or amino acids wherein Y is CH-NO 2  (herein referred to as diaminonitroethylenes) are prepared by conversion of the corresponding urea to a carbodiimide: ##STR12## 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): ##STR13## 
     An alternative synthesis that may be used is as ##STR14## 
     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 β-(1-or 2-naphthyl)-D-alanine (hereinafter β-D-1NAL or β-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 β-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 such 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 substituted. 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 side chain amino group is 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/4Cl)Phe. The 1-position substituent is preferably modified so that its α-amino group contains an acyl group, such as formyl(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 β-carbon of Ala is linked to the 2-, 3- or 4-position, preferably to the 3-position, on the pyridine ring. When β-D-NAL is present in the 1-position and R 5  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, NO 2  -D-Trp, D-Leu, D-Ile, D-Nle, D-Tyr, D-Val, D-Ala, dialkyl Arg, dialkyl Har, D-Ser(OtBu), β-D-NAL or (imBzl)D-His is preferably in the 6-position, if U 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 proestrus 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 *  : ##STR15## and there is at least one such residue in each peptide of the invention, wherein X, Y, R 1  and R 2  are as defined previously. 
     More specifically, the GnRH antagonists of the present invention are represented by the following Formula (F 1 ): 
     
         G-AA.sub.1 -(A)D-Phe-AA.sub.3 -Ser-AA.sub.5 -AA.sub.6 -AA.sub.7 -AA.sub.8 -Pro-AA.sub.10 
    
     wherein G is hydrogen or an acyl group having 7 or less carbon atoms; AA 1  is dehydroPro, D-pGlu, (A)D-Phe, (B)D-Trp, Pro, or β-D-NAL; A is H, Cl, F, NO 2 , CH 3 , OCH 3 , C a  Me/4Cl, Cl 2  or Br; B is H, NO 2 , NH 2 , OCH 3 , F, Cl, Br, CH 3 , N in  For or N in  Ac; AA 3  is U * , D-PAL, β-D-NAL or (B)D-Trp; AA 5   is U * , Tyr, (C)Arg, Lys(cpd), Orn(cpd), Dbu(cpd), Dpr(cpd), (A)Phe, (3I)Tyr or His; AA 6  is U * , β-D-NAL, (B)D-Trp, (A&#39;)D-Phe, (D)D-Orn, (D)D-Lys, (D)D-Dbu, (D)D-Dpr, D-Har, D-Tyr, (E)D-His, D-PAL, (C)D-Arg or a suitable lipophilic D-isomer; A&#39; 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&#39;)Arg, (C&#39;)Har or ILys; C&#39; 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 1  is β-D-NAL and AA 5  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 5  H 7  O 2  N. By β-D-NAL is meant the D-isomer of alanine which is substituted by naphthyl on the β-carbon atom, i.e., also 3-D-NAL. Preferably β-D-2NAL is employed wherein the attachment to naphthalene is at the 2-position on the ring structure; however, β-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 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, e.g. with formyl (N in  For- or 1For-) or with acetyl. N in  For-D-Trp and 6NO 2  -D-Trp are the preferred substituted residues. By NML is meant N.sup.α CH 3  -L-Leu. By Dbu is meant alpha, gamma diamino butyric acid, and by Dpr is meant α,β diamino propionic acid. When dehydroPro is present in the 1-position, Tyr or U *  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 3 ) pGlu-His-Trp-Ser-Tyr-(U * )-AA;-Arg-Pro-AA 10 , wherein AA 7  and AA 10  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.sub.2 -AA&#39;-Ser-AA.sub.5 -AA.sub.6 -AA.sub.7 -AA.sub.8 -Pro-AA.sub.10 wherein AA 
    
     is pGlu or AA 1  ; AA 2  is His or (A)D-Phe; AA&#39; 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 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 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 U.S. Pat. No. 4,569,967, issued Feb. 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. Pat. 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: X 1  -AA-AA 2  (X 5 )-U 3  -Ser(X 3 )-U 5  -U 6  -AA 7  (X 2  or X 7 )-U 8  -Pro-X 8  wherein: U 3  is either U&#39; or AA&#39;(X 2 ); U 5  is either U&#39; or AA 5  (X 4  or X 5 ); U 6  is either U&#39; or AA 6  (X 4  or X 5  or X 6 ); U 8  is either U&#39; or AA 8  (X 5  or X 6 ); U&#39; is either Lys(X a ), Orn(X a ), Dbu(X a ) or Dpr(X a ); X 1  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 o-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, such as allyl(Aly), triphenylmethyl(trityl) and benzyl(Bzl); (7) trialkylsilane groups, such as trimethylsilane. The preferred o-amino protecting group is Boc when X is hydrogen. 
     X 2  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. 
     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 4  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 5  is a protecting group for a side chain guanidino group, such as that in Arg or Har, or for the imidazole group of His, such as nitro, Tos, trityl, adamantyloxycarbonyl, 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 6  is a protecting group for an amino side chain group such as Z or 2ClZ; X a  is a subclass 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 trifluoroacetyl(Tfa), is used; however, it may also be possible to use a hydrazine-labile group such as phthaloyl, ##STR16## or a thiolabile group such as Nps or Dts. 
     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 support], 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 NHNHCONH 2 . 
     The criterion for selecting side chain protecting groups for X 2  -X 7  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. 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. 
     When the X 8  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 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 is preferably made by classical solution synthesis, as disclosed in U.S. Pat. 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 β-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 o-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 suitable reaction as known in the art. 
     Thus, the invention also provides a method for making a peptide, said peptide having the formula: 
     G-AA-AAz-AA&#39;-Ser-AA 5  -AA 6  -AA 7  -AA 8  -Pro-AA 10  , wherein at least one of AA&#39;, AA 5 , AA 6  and AA 8  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 8  -Pro-X 8  wherein: U 3  is either U&#39; or AA&#39;(X 2 ); U 5  is either U&#39; or AA 5  (X 4  or X 5 ); U 6  is either U&#39; or AA 6  (X 4  or X 5  or X 6 ); U 8  is either U&#39; or AA 8  (X 5  or X 6 ); U&#39; is either Lys(X a ), Orn(X a ), Dbu(X a ) or Dpr(X a ); X 1  is 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 4  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 a  is a subgroup that is 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 NHNHCONH 2  ; provided however that at least one of U 3 , U 5 , U 6  and U 8  is either Lys(X a ), Orn(X a ), Dbu(X a ) or Dpr(X a ); (b) removing at least one X a  to deprotect a side chain primary amino group of at least 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 is effected by ion exchange chromatography on a CMC column, followed by partition chromatography using the elution system: n-butanol;0.1N 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 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 steroids for other purposes as indicated hereinbefore. 
     In the following formulas, the U residues 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 parentheses. 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&#39;-butylguanidino 
     chcg=aminocyclohexyl cyanoguanidino 
     ecg=aminoethyl cyanoguanidino 
     icg=aminoisopropyl cyanoguanidino 
     hcg=aminohexyl cyanoguanidino 
     hicg=histaminyl cyanoguanidino (aminoethyl imidazolyl cyanoguanadino) 
     mcg=aminomethyl cyanoguanidino 
     ncg=aminoethyl(1 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: Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-AA 5  -AA 6  -Leu-AA 8  -Pro-D-Ala-NH 2  are prepared by the solid-phase procedure referred to above. 
     
                       TABLE I______________________________________  AA.sub.5  AA.sub.6     AA.sub.8______________________________________1        Lys(icg)    D-Lys(icg)   ILys2        Lys(mcg)    D-Lys(mcg)   &#34;3        Lys(chcg)   D-Lys(chcg)  &#34;4        Lys(tcg)    D-Lys(tcg)   &#34;5        Lys(pcg)    D-Lys(pcg)   &#34;6        Lys(2amp)   D-Lys(2amp)  &#34;7        Lys(3amp)   D-Lys(3amp)  &#34;8        Lys(4amp)   D-Lys(4amp)  &#34;9        Lys(hcg)    D-Lys(hcg)   &#34;10       Lys(ecg)    D-Lys(ecg)   &#34;11       Lys(Ocg)    β-D-2NAL                             &#34;12       Lys(bcg)    &#34;            &#34;13       Lys(Nic)    D-Lys(Nic)   Lys(bcg)14       Lys(bcg)    β-D-2NAL                             &#34;15       &#34;           D-Lys(bcg)   Arg16       Tyr         β-D-2NAL                             Lys(icg)______________________________________ 
    
     For purposes of an example, a representative solid phase synthesis of Peptide No. 1 above, which is referred to as [Ac-β-D-2NAL1, (4Cl)D-Phe 2 , D-3PAL 3 , Lys(icg) 5  D-Lys(icg) 6 , ILys 8 , D-Ala 10  ]-GnRH is set forth hereinafter. This peptide has the following formula: Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-Lys(isopropyl cyanoguanidino)-D-Lys(isopropyl cyanoguanidino)-Leu-Lys(isopropyl)-Pro-D-Ala-NH 2 . 
     An MBHA resin is used, and Boc-protected D-Ala is coupled to the resin over a 2-hour period in CH 2  Cl 2  using a 3-fold excess 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 5 grams of resin: 
     
         ______________________________________                        MIXSTEP  REAGENTS AND OPERATIONS                        TIMES MIN.______________________________________1     CH.sub.2 Cl.sub.2 wash-80 ml. (2 times)                        32     Methanol(MeOH) wash-30 ml. (2 times)                        33     CH.sub.2 Cl.sub.2 wash-80 ml. (3 times)                        34     50 percent TFA plus 5 percent 1,2-eth-                        10 anedithiol in CH.sub.2 Cl.sub.2 -70 ml. (2 times)5     Isopropyl alcohol + 1% ethanedithiol                        3 wash-80 ml. (2 times)6     TEA 12.5 percent in CH.sub.2 Cl.sub.2 -70 ml.                        5 (2 times)7     MeOH wash-40 ml. (2 times)                        28     CH.sub.2 Cl.sub.2 wash-80 ml. (3 times)                        39     Boc-amino acid (10 mmoles) in 30 ml. of either dimethylformamide(DMF) or CH.sub.2 Cl.sub.2, depending upon the solubility of the particular protected amino acid, (1 time) plus DCC (10 mmoles) in CH.sub.2 Cl.sub.230-30010    MeOH wash-40 ml. (2 times)                        311    Triethylamine(TEA) 12.5 percent                        3 in CH.sub.2 Cl.sub.2 -70 ml. (1 time)______________________________________ 
    
     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 positive, 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 has been attached. N.sup.α Boc protection is used for each of the remaining amino acids throughout the synthesis. N.sup.α Boc-α-D-2NAL is prepared by a method known in the art, e.g. as described in detail in U.S. Pat. No. 4,234,571, issued November 18, 1980 Or commercially available from Synthe Tech, Oreg., 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 side chain protecting group for the hydroxyl group of Ser. Boc-Lys(Ipr) is used for the 8-position. After deblocking the o-amino group at the N-terminal 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, the following intermediate is present: Ac-β-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 simultaneously 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 resin with DMF, CH 3  OH, CH 2  Cl 2 , and finally DMF, the newly freed amino group is treated with a large excess &gt;10 fold) of diphenyl cyanocarbonimidate(PCI) in DMF. Thereafter, the peptide is then subjected to the standard wash (see Steps 10-11) and then treated with isopropylamine dissolved in DMF for 24 hours at about 22° C. to complete the formation of the aminoisopropyl cyanoguanidino moiety; for some 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 takes place very readily 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 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. J. Chromatography, 288 (1984) 303-328. 
     The peptide is judged to be homogeneous using thin layer chromatography and several different solvent systems, as well as 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 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].sub.D.sup.20 =-2.8±0.5(c=1, 50% acetic acid). 
    
     The other peptides in Table I are similarly synthesized and purified. The peptides are assayed in vivo and may also be tested in vitro. If performed, 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 Pro 1 , (4F)D-Phe 2 , D-Trp 3 ,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 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. 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; 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 dosages. The following Table A shows the results of in vivo testing of various of these GnRH antagonists, with the dosages being given in micrograms: 
     
                       TABLE A______________________________________Peptide          Rats              RatsNo.      Dosage  Ovulating   Dosage                              Ovulating______________________________________1.       2.5     0/9         1.0   0/52.       2.5     6/6         2.0   2/103.       2.5     7/9         2.0   1/104.       2.5     0/6         2.0   1/105.       2.0      1/106.       2.5     0/6         1.0   7/177.       2.5      3/208.       2.5      5/19       1.0   5/59.       2.5     0/6         1.0   5/510.      2.5     5/8         2.0   1/1012.      2.5     6/7         5.0   9/1013.      5.0      5/12       10.0  3/515.      2.5     0/4         1.0   8/18______________________________________ 
    
     EXAMPLE II 
     Peptides as indicated in TABLE II having the formula: Ac-dehydroPro-(A)D-Phe-AA 3  -Ser-AA 5  -β-D-2NAL-Leu-AA 8  -Pro-D-Ala-NH 2  are prepared by the solid-phase procedure referred to above. 
     
                       TABLE II______________________________________A          AA.sub.3    AA.sub.5   AA.sub.8______________________________________17   4Cl       β-D-2NAL                      Tyr      Lys(bcg)18   &#34;         &#34;           &#34;        Lys(ecg)19   4F        (1For)D-Trp (2F)Phe  Orn(ecg)20   &#34;         &#34;           Tyr      Dpr(2ncg)21   &#34;         &#34;           (2NO.sub.2)Phe                               Dbu(icg)22   &#34;         (1Ac)D-Trp  (2CH.sub.3)Phe                               Dbu(2amp)23   4Br       &#34;           Tyr      Dbu(3amp)24   &#34;         &#34;           (2Br)Phe Dbu(4amp)25   H         D-Trp       (2Cl)Phe Orn(2mncg)26   4NO.sub.2 (5CH.sub.3)D-Trp                      (3CH.sub.3)Phe                               Orn(hcg)27   &#34;         (5F)D-Trp   His      Lys(tcg)28   2,4Cl.sub.2          (5Cl)D-Trp  (3F)Phe  Dpr(trcg)29   &#34;         (6NO.sub.2)D-Trp                      (3Br)Phe Orn(1ncg)30   C.sup.α e/4Cl          (50CH.sub.3)D-Trp                      (3I)Tyr  Orn(pcg)31   3,4Cl.sub.2          (5NH.sub.2)D-Trp                      (3Cl)Phe Dpr(chcg)______________________________________ 
    
     All peptides listed in Table II 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 III 
     Peptides as indicated in TABLE III having the formula: G-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-Tyr-AA 6  -Leu-AA 8  -Pro-AA 10  are prepared by the solid-phase procedure referred to above. 
     
                       TABLE III______________________________________G        AA.sub.6    AA.sub.8    AA.sub.10______________________________________32   Ac      D-Arg       Lys(bcg)  D-Ala-NH.sub.233   Ac      D-Lys(bcg)  Arg       &#34;34   For     D-Tyr       Lys(icg)  Gly-NH.sub.235   Bz      (Et)D-Arg   Orn(icg)  &#34;36   Ac      D-Lys       Orn(ecg)  &#34;37   Vac     D-Har       Orn(mcg)  &#34;38   Acr     (4gua)D-Phe Dpr(2ncg) AzaGly-NH.sub.239   Ac      D-Orn       Dpr(chcg) D-Ala-NH.sub.240   Acr     D-His       Dpr(tcg)  &#34;41   Ac      (Bu)D-Arg   Dbu(1mncg)                              &#34;42   &#34;       (Bz)D-Orn   Dbu(2amp) &#34;43   Vac     (4NH.sub.2)D-Phe                    Dbu(4amp) &#34;44   Bz      (Ac)D-Lys   Dbu(trcg) AzaGly-NH.sub.2______________________________________ 
    
     All peptides 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 
     Peptides as indicated in TABLE IV having the formula: Ac-AA 1  -(4Cl)D-Phe-D-3PAL-Ser-Tyr-D-Arg-AA 7  -AA 8  -Pro-D-Ala-NH 2  are prepared by the solid-phase procedure referred to above. 
     
                       TABLE IV______________________________________AA.sub.1         AA.sub.7   AA.sub.8______________________________________45     β-D-2NAL Leu        Lys(bcg)46     (1Ac)D-Trp    Met        Lys(2amp)47     (6Br)D-Trp    Tyr        Lys(3amp)48     (5F)D-Trp     Nle        Lys(4amp)49     (6NO.sub.2)D-Trp                Met        Orn(chcg)50     (5Cl)D-Trp    Tyr        Orn(pcg)51     (4Cl)D-Phe    Phe        Dpr(pcg)52     (4F)D-Phe     (4F)Phe    Dpr(tcg)53     (2,4Cl.sub.2)D-Phe                NML        Dbu(mcg)54     dehydroPro    Nle        Dbu(2ncg)55     β-D-2NAL Leu        Lys(Ocg)56     (60CH.sub.3)D-Trp                (1For)Trp  Orn(1ncg)57     (5NH.sub.2)D-Trp                Nva        Orn(ecg)58     (4NO.sub.2)D-Phe                NML        Orn(1mncg)59     dehydroPro    (6NO.sub.2)Trp                           Dbu(hicg)______________________________________ 
    
     All peptides listed in Table IV 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 V 
     Peptides as indicated in TABLE V having the formula: Ac-AA 1  -(4Cl)D-Phe-AA 5  -AA 6  -Leu-ILys-Pro-D-Ala-NH 2  are prepared by the solid-phase procedure referred to above. 
     
                       TABLE V______________________________________AA.sub.1   AA.sub.3   AA.sub.5  AA.sub.6______________________________________60  β-D-2NAL          D-3PAL     Lys(bcg)                             β-D-2NAL61  &#34;          (6NO.sub.2)D-Trp                     &#34;       (Dnp)D-His62  &#34;          D-Trp      Lys(ecg)                             (4gua)D-Phe63  dehydroPro β-D-2NAL                     Orn     (6NO.sub.2)D-Trp64  &#34;          β-D-1NAL                     Orn(mcg)                             D-Val65  β-D-2NAL          (1For)D-Trp                     Orn(tcg)                             (Pr)D-Arg66  &#34;          &#34;          Dbu(bcg)                             (5NH.sub.2)D-Trp67  dehydroPro D-Trp      Dbu(2amp)                             D-Tyr68  &#34;          D-2PAL     Dbu(4amp)                             D-Nle69  &#34;          (1Ac)D-Trp Dbu(hcg)                             (4F)D-Phe70  Pro        D-3PAL     Lys(pcg)                             β-D-1NAL71  (1For)D-Trp          &#34;          Lys(chcg)                             (4NHCH.sub.3)D-Phe72  β-D-2NAL          &#34;          Dpr(hcg)                             (Ac)D-Orn73  &#34;          &#34;          Dpr(Ocg)                             (4NH.sub.2)D-Phe74  β-D-1NAL          (6Br)D-Trp Dpr(tcg)                             (1For)D-Trp75  (6CH.sub.3)D-Trp          D-4PAL     Dpr(bzcg)                             D-4PAL______________________________________ 
    
     All 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 as indicated in TABLE VI having the formula: G-AA 1  -(4Cl)D-Phe-D-Trp-Ser-Tyr-AA 6  -Leu-AA 8  -Pro-D-Ala-NH 2  are prepared by the solid-phase procedure referred to above. 
     
                       TABLE VI______________________________________G        AA.sub.1     AA.sub.6    AA.sub.8______________________________________76   Ac      dehydroPro   D-Lys(bcg)                               Lys(bcg)77   Ac      β-D-2NAL                     β-D-2NAL                               Lys(3amp)78   Ac      β-D-2NAL                     D-Val     Lys(tcg)79   Acr     Pro          D-Ser(OtBu)                               Lys(chcg)80   H       dehydroPro   (imBzl)D-His                               Lys(ecg)81   Bz      (4Br)D-Phe   (5Cl)D-Trp                               Orn(ecg)82   &#34;       D-pGlu       (6Br)D-Trp                               Orn(bzcg)83   For     β-D-1NAL                     (Me)D-Arg Orn(Ocg)84   &#34;       dehydroPro   D-Har     Orn(4amp)85   Vac     β-D-2NAL                     (Bz)D-Lys Orn(chcg)86   Ac      β-D-2NAL(Arg.sup.5)                     β-D-2NAL                               Lys(Ocg)87   H       dehydroPro   D-Ala     Lys(tcg)______________________________________ 
    
     All 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. 
     EXAMPLE VII 
     Peptides as indicated in TABLE VII having the formula: Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-AA 5  -AA 6  -Lue-AA 8  -Pro-D-Ala-NH 2  are prepared by the solid-phase procedure referred to above. 
     
                       TABLE VII______________________________________  AA.sub.5  AA.sub.6     AA.sub.8______________________________________88       Arg         D-Lys(bcg)   Arg89       &#34;           D-Lys(Ocg)   &#34;90       Orn(bcg)    (4gua)D-Phe  (Et.sub.2)Arg91       Lys(2ncg)   D-Lys(2ncg)  ILys92       Orn(bzcg)   D-Lys(bzcg)  Har93       Lys(act)    D-Lys(act)   ILys94       Lys(hicg)   D-Lys(hicg)  ILys95       Lys(trcg)   D-Lys(trcg)  ILys96       Lys(bcg)    D-Lys(bcg)   ILys97       &#34;           D-3PAL       (EtPr)Har98       &#34;           D-Lys(Nic)   (Me.sub.2)Arg99       Orn(tcg)    D-Lys(tcg)   (MeBu)Arg100      Lys(Sbcg)   D-Lys(Sbcg)  ILys______________________________________ 
    
     All peptides listed in Table VII 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 VIII 
     Peptides as indicated in TABLE VIII having the formula: Ac-βD-2NAL-(4Cl)D-Phe-D-3PAL-Ser-AA 5  -AA 6  -Leu-AA 8  -Pro-D-Ala-NH 2  are prepared by the solid-phase procedure referred to above. 
     
                       TABLE VIII______________________________________  AA.sub.5   AA.sub.6     AA.sub.8______________________________________101      Orn(bcg)     D-Orn(bcg)   ILys102      Orn(bcg)     D-Orn(2amp)  ILys103      Orn(tcg)     D-Orn(tcg)   ILys104      Orn(bcg)     β-D-2NAL                              ILys105      Orn(2mpcg)   β-D-2NAL                              ILys106      Lys(Nic)     D-Lys(Nic)   Lys(bur)107      Lys(2ncg)    D-Lys(2ncg)  ILys108      Lys(bzcg)    D-Lys(bzcg)  ILys109      Lys(icg)     D-Lys(icg)   Arg110      Orn(icg)     D-Orn(icg)   ILys______________________________________ 
    
     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 
     Peptides as indicated in TABLE IX having the formula: Ac-βD-2NAL-(4Cl)D-Phe-AA 3  -AA 6  -Leu-AA 8  -Pro-D-Ala-NH 2  are prepared by the solid-phase procedure referred to above. 
     
                       TABLE IX______________________________________AA.sub.3      AA.sub.5  AA.sub.6  AA.sub.8______________________________________111   D-Lys(bcg)  Tyr       D-Arg   Arg112   D-Lys(2amp) Tyr       D-Arg   Arg113   D-Lys(tcg)  Tyr       D-Arg   Arg114   D-Lys(bcg)  Arg       β-D-2NAL                               Arg115   D-Lys(tcg)  Arg       β-D-2NAL                               Arg116   D-Lys(bcg)  Tyr       D-Arg   ILys117   D-Lys(tcg)  Tyr       D-Arg   ILys118   D-Lys(bcg)  Tyr       β-D-2NAL                               ILys119   D-Lys(bcg)  Tyr       D-3PAL  ILys120   D-Lys(2mpcg)             Tyr       D-3PAL  ILys121   D-Lys(bcg)  Tyr       D-Arg   Arg122   D-Lys(bcg)  Lys(bcg)  D-Lys(bcg)                               ILys123   D-3PAL      Lys(bur)  β-D-2NAL                               Arg124   D-Lys(bcg)  Tyr       β-D-2NAL                               Lys(bcg)125   D-Orn(2amp) Tyr       D-3PAL  Lys(2amp)______________________________________ 
    
     All peptides listed in Table IX 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. 
     Results of in vivo testing of selected of these antagonists are shown in the following Table B, with the dosages being given in micrograms: 
     
                       TABLE B______________________________________Peptide          Rats              RatsNo.      Dosage  Ovulating   Dosage                              Ovulating______________________________________17.      10      6/8         15     7/1033.      2.5      4/1755.      10      6/7         25    2/560.      2.5      4/14       1.0    8/1086.      2.5      4/15       5.0   5/588.      2.5     0/4         1.0    4/15    10      0/889.      10       2/1893.      2.5      5/1094.      1.0      7/1196.      1.0      9/12101.     2.5     0/5         1.0   5/8102.     2.5     2/9103.     2.5     5/7104.     2.5     6/8105.     2.5     0/5106.     5.0     0/7111.     2.5      1/11       1.0   0/5                        0.5   5/7112.     1.0     4/8113.     1.0     5/6114.     5.0     4/5115.     2.5116.     2.5     0/6         1.0   2/8117.     10      0/6         5     2/5122.     10      4/6123.     10       1/10       5     0/6______________________________________ 
    
     EXAMPLE X 
     Peptides as indicated in TABLE X having the formula: pGlu-His-Trp-Ser-Arg-AA 6  -AA 7  -Arg-Pro-AA 10  are prepared by the solid phase procedure referred to above. 
     
                       TABLE X______________________________________AA.sub.6       AA.sub.7   AA.sub.10______________________________________126     D-Lys(bcg) Leu        Gly-NH.sub.2127     D-Lys(ecg) &#34;          &#34;128     D-Lys(pcg) &#34;          &#34;129     D-Lys(hcg) &#34;          AzaGly-NH.sub.2130     D-Lys(tcg) &#34;          NHCH.sub.2 CH.sub.3131     D-Lys(4amp)              &#34;          &#34;132     D-Lys(3amp)              NML        &#34;133     D-Lys(2amp)              &#34;          &#34;134     D-Lys(chcg)              Leu        Gly-NH.sub.2135     D-Lys(Ocg) &#34;          &#34;136     D-Orn(bcg) &#34;          &#34;137     D-Orn(tcg) NML        AzaGlyNH.sub.2138     D-Orn(mcg) &#34;          NHCH.sub.2 CH.sub.3139     D-Dbu(2amp)              &#34;          NHCH.sub.3140     D-Dbu(chcg)              Leu        NHCH.sub.2 CH.sub.2 CH.sub.3141     D-Dbu(bzcg)              &#34;          Gly-NH.sub.2142     D-Dpr(ecg) &#34;          &#34;143     D-Dpr(hicg)              NML        &#34;144     D-Dpr(trcg)              &#34;          NHCH.sub.2 CH.sub.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-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Lys(Fmoc)-D-3PAL-NML-Lys(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 described. Next the Dts protecting group is removed from the amino side chain of the Lys residue in the 8-position using a suitable thiol, such as β-mercaptoethanol or thiophenol(PhSH), in DMF, and the peptidoresin is given the standard wash. Thereafter, it is maintained at 22° C. for 10-60 minutes 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 Lys 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-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Orn(Fmoc)-D-Trp-Leu-Lys(Nps)-Pro-D-Ala-NH-[resin 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 dosages. 
     EXAMPLE 147 
     A peptide intermediate having the formula: Ac-β-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-β-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 isothiocyanate instead of PCI to form the napthylthiourea moiety with the side 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-β-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 first reacted using 2-bromoethyl,2&#39;(Bocamino)ethyl ether dissolved in DMF for 1 hour or until the ninhydrin test 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  -O-(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: ##STR17## 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 150 
     (Boc)Dpr is reacted with PCI as in Example I to produce ##STR18## which is 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: ##STR19## which is particularly useful as a substitute for His in peptide syntheses. 
     Following purification of the peptides, various of them are further characterized by subjection to high performance liquid chromatography on C1a silica (Vydac 0.46×25 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 shows when the specific peptides elute from the C 18  silica having a particle size of about 5μ and a pore size of 300Å 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.: 
     
                       TABLE C______________________________________Peptide No.   Time of Elution______________________________________2.            26.563.            47.64.            20.325.            35.66.            31.047.            28.968.            27.749.            54.2410.           30.8611.           56.912.           46.7813.           33.415.           41.117.           49.632.           40.5633.           35.84______________________________________ 
    
     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, hydrobromide, sulphate, phosphate, nitrate, oxalate, fumarate, gluconate, tannate, maleate, acetate, citrate, benzoate, succinate, alginate, malate, acetate, citrate, benzoate, succinate, alginate, malate, ascorbate, tartrate and the like. For example, an aqueous solution of the peptide can be repeatedly treated with 1N 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; although oral dosages will be higher, it is anticipated that the nature of these compounds 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 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 dosage form may contain a pharmaceutically acceptable non-toxic salt of the compound which has a low degree of solubility in body fluids, for example, an acid addition salt with a polybasic 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 stearate gel. A suitable, slow-release depot formulation for injection may also 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. 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 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 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. The 6-position substitutes set forth in Table VI are considered to be equivalents known 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 Lys and diethyl Lys are considered to be equivalents of ILys; however, ILys is preferred when neither 1 or Arg is in the 8-position. 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 analogs 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.