CA1065859A - Lh-rh analogs and intermediates therefor - Google Patents

Abstract

LH-RH ANALOGS AND INTERMEDIATES THEREFOR

Abstract of the Disclosure [D-Trp6]-LH-RH and [D-Ser4, D-Leu6-desGly-NH210]-LH-RH-(lower alkyl)amides, salts thereof, and intermediates used for the synthesis thereof are disclosed. The compounds have potent LH-and FSH-releasing hormone properties.

Description

. ~~ AHP~6505/6513/6639 ~:

85~ ~
Background of the Disclosure ~;
~a) rl~
This invention relates to peptides of fonmula 1 (pyro)-Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z (1) :~
in which : :
a) X is Ser~ Y is D-Trp and Z is Gly-~12; or b) X is D~Ser, Y is D~Leu a~d Z is NHR wherein R is lower alkyl, and in~ermediates for the synthesis thereof. :- .
The peptides of formula 1 in which a) X is Ser, Y is D-Trp and Z is Gly-NH2; or b) X is D-Ser, Y is D-Leu and Z is NHRl also are called, respectively~
a) L-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-tryptophyl-L-leucyl-L-arginyl-L-prolyl-glycinamide; or b) L-pyroglutamul-L-histidyl-L-tryptophyl-D-seryl-L-tyrosyl-D-leucyl-L-leucyl-L-arginyl-L-prolyl lower alkyl amide, and may be designated by the abbreviation, a) LD-Trp ~-LH-RH or . :~
b) ~D-Ser , D-Leu6~ desGly-NH210~-LH-RH (lower alkyl)amide, respectively. .
~he compound of formula 1 (pyro)-Glu~Uis-Trp-X~Tyr-Y-Leu~rg~Pro-Z
in which a) X is Ser, Y ~5 D-Trp and Z is Gly-NH2~ or b) X is D-Ser, Y is D-Leu and Z is NHR wherein R is :
lower alkyl, or a non-toxic phannaceutically acceptable salt thereof, .
is prepared by-i) reacting a compound of fonnula R8-(pyro)-Glu-His(Nlm-R7)-Trp-xl-Tyr(R5)-y-Leu-Arg(NG-R5)-pro-z2 ~
in which X is Ser(R )~ Y is D-Trp and z2 is Gly-A wherein A is .:
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. _~ AHP-6505/6513/6639 ~C~6~

N -C~ and R4, R5, R6 and R7 are protective gr~ups capable of being removed by one or more chemical treatments which do not affect the corresponding compound of formula 1, and R8 is hydrogen or one of said protective groups with a reagent capable of removing the protective groups, R , R5, R6, R7 and R8 without affec~ing the compound of formula 1 to obtain the corresponding compound of formula 1 (pyro)~Glu-His-Trp~X-lyr-Y-Leu-Arg-Pro-Z
in which X is Ser, Y is D-Trp and Z is Gly-NH2; or .
ii) reacting a compound of formula 11 ~ :
R8-(pyro)-Glu-His(Nlm~R7)-Trp-Xl-'ryr(R5)-Y-Leu-Arg(NG-R4)-Pro-Z
in which X is Ser(R6~, Y is D-Trp and zl is Gly-R wherein R i5 amino ;~
and R4, R5, R6 and R7 are protective groups capable of being removed by one or more chemical treatments which do not affect the corres-ponding compound of formula 1, and R is hydrogen or one of said protective groups with a reagent capable of removing the protective groups R4~ R5, R6, R7 and R8 without affecting the compound of formula 1 to obtain the corresponding compound of formula I
(pyro~-Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z
in which X is Ser, Y is D-Trp and æ i9 Gly-NH2; or iii) reacting a compound of the ormula R -(pyro)-Glu-His-Trp-D-Ser(R ) Tyr(R )-D-Leu-Leu-Arg(N~-R )-Pro-NHR
in which Rl is lowcr alkyl and R4~ R5~ R6 and R~ are protective groups capable of being removed by one or more chemical treatments which do not affect the corresponding compound of formula 1 and R8 is hydrogen or one o~ said protective groups with a reagent capable of removing the protective groups, R4, R5, R6 and R8 without affecting the compound of formula 1 to obtain the corresponding compound of formula 1 (pyro)-Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z
in which X is D-Ser~ Y is D-Leu and Z is NHR wherei~ Rl is lower alkyl;
and when desired converting the compound of formula 1 to a corresponding non-toxic pharmaceutically acceptable salt.

-2a~

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(b) Backqround of the Invention Lutetnizing hormone (LH) and follicle-stimulating hormone (FSH) are both gonadotrophic hormones elaborated by the pituitary gland of humans and of mammals. LH together with FSH stimulates the release of estrogens from the maturing follicles in the ovary and induces the process of ovulation in the female. In the male, LH stimulates the interstitial cells and is for that reason also called interstitial cell stimulating hormone (ICSH). FSH induces maturatlon of the folllcles in the ovary and together with LH, plays an important role tn the cycl7c phenomena in the female.
FSH promotes the development of germinal cells in the testes of the male. ~oth LH and FSH are released from the pituitary gland by the action of LH- and FSH-releasing hormone, and there is good evidence that said releasing hormone i5 elaborated in the hypothalamus ;;

AHP-6505/~j5 13,~66:~;3 6585~
and reaches the pituitary gland by a neurohumoral pathway, see e.g., A.V. Schally, e~ al., Recent Progress in Hormone Research, 24, ~97 (1968).
The natural LH- and FSH-releasing hormone has been isolated from plg hypothalaml and Its constitutTon eluctdated by A.V. Schally, et al., Biochem. Biophys. Res. Commun., 43, 393 and 1334 (1971), who proposed the decapeptide structure (pyro~-Glu-His Trp-Ser-Tyr-GIy-Leu-Arg-Pro-Gly-NH2.
Thls constitution has been confirmed by synthesis;
for example, see H. Matsuo. et al., Biochem. Biophys. Res. Comm., 45, 822 (1971) and R. Geiger, et ai., ibid, 45, 767 (1971).
Herelnafter the natural LH- and FSH-releaslng hormone is called bH-RH.
Because of the importance of LH-RH to both dlagnostlc and therapeutlc medlclne, conslderable Interest has been shown In the p~eparatlon of new compounds havlng Improved properties over the natural hormone. One approach to thls goal has been the selective modification or replacement of amino acid resldues of LH-RH
wlth other amTno aclds, Although in a few tnstances peptides contalntng such alteratlons h~ve been found to be more actlve than LH RH, for example, C~ AIa ~-LH-RH, A. Arimura, fl~ al , Endocrinology, 95, 117~ ~1974)~D-LOU ~-LII RH ~nd ~D-Leu6,desGly-NH210~-LH-RH ~thylamlde, J.A. Vllchez-Mbrtlne7, et al., 810chem. ~lophys. Res. Commun., 59, 1226 (1974), for the most part the modifled peptldes have been less actlve.

" AHP~6505/6513/6639 Now it has been found that~
a) the replacement of the glycyl moiety in position 6 of LH-RH with D-tryptophan; or b) the replacement of the L-seryl moiety in poSitiOn 4 of LH-RH with a D~seryl moiety, replacement of the glycyl moiety in position 6 by a D-leucyl ~oiety, and replacement of the glycinamide moiety in position 10 by a lower alkyl amide group, gives a peptide of formula 1 that is much ~ore active and longer acting than LH-RH.
The present finding, in one embodiment of this invention, that a change in the asymmetry of the seryl residue in position 4 in conjunction with the introduction of a D~leucyl residue at position 6 results in enhanced activity and longer duration of actlon for the peptide of formula 1 in which X is D~Ser, Y is D-Leu and Z i9 NHRl is quite surprising, especially in view of the fact that with respect to LH-RH changes in the asymmetry of its amino acid residues and/or replacement thereof generally lead to a derivative which is far less active than LH-RH itself; for instance see Y. Hirotsu, Biochem. Biophys. Res.
Commun., 59, 277 (1974). Further in keeping with this thought is our finding that ~D-Ser ~-LH-RH has less than 5% of the LH-RH activity of the natural hormone.
The attributes of the present peptides of formula 1 have practlcal signi~lcances the smaller minimum e~ective dose reducing side effects as well as the cost for the preparation of the compound and the longer acting property reducing the need for frequent admlnistration.

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Summar~ of the Invention :~
The compounds of this invention are selected from the ~ -group ConSiSting of formula 1 (pyro)-Glu~His~Trp-X-Tyr-Y-Leu-Arg-Pro-Z (1) in which a) X is Ser, Y is D-Trp and Z is Gly-NH2; or b) X is D-Ser, Y is D-Leu and Z is NHR wherein R is lower alkyl, or a non-toxic pharmaceutically acceptable salt thereof;
a compound of formula (11) R8-(pyro)-Glu-His~Nlm-R7)-Trp-Xl-Tyr(R5)-Y-Leu-Arg(NG-R4)-Pro-Z
in which a) Xl is Ser(R6), Y is D-Trp and Z is Gly-R2; or

2 b) Xl is D-Ser(R6), Y is D~Leu and Z i9 OR wherein R is selected from the group consisting of amino and O-(lower alkyl)~
lS R is lower alkyl, R , R5, R6 and R are protective groups capable of being removed by one or more chemical treatments which do not affect (pyro)-Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z in which X, Y and Z
are as defined herein and R8 is hydrogen or one of said protective groups; and R -(pyro)-Glu-His-Trp-D-Ser(R )-Tyr~R )-D-Leu-Leu-Arg~NG R )-Pro-NHR
in which Rl i8 lower alkyl~ R4~ RS and R6 are protective groups capable of bein~ removed by one or more chemical treatment~ which do not aEfect ~pyro)-C1u~His~D~5qr-Tyr~D~Leu~Leu-Arg-Pro-NURl and R
i~ hydrogen or one of said protective groups.

~ AHP-6505/6513/6639 ~165~3S9 ~

With reference to the compounds of formula 11 and the compound of the formula :
R -(pyro)-Glu-His-Trp-D-Ser(R )-Tyr(R )-D-Leu~Leu-Arg(NG-R )-Pro-NHRl~ ;
in a preferred embodiment Rl and zl are as defined herein, R4 is a protective group for the N~, N , and N nitrogen atoms of arginine seleçted from the group consisting of tosyl~ nitro, benzyloxycarbonyl and~
adamantyloxycarbonyl; R5 is a protective group for the hydroxyl of tyrosine selected from the group consisting of 2~bromobenzyloxycarbonyl, benzyl, ::
acetyl7 tosyl, benzoyl, t~butyl, tetrahydropyran-2-yl, trityl, 2,4- -:
dichlorobenzyl and benzyloxycarbonyl; R6 is a protective group for the ~.
hydroxyl group of serine and is selected from the group defined hereinbeEore for R5; R7 is a protective group for the imidazole nitrogen atoms of ;:
histidine selected from the group of tosyl and dinitrophenyl; and R8 is hydrogen or an ~-amino protective group selected from the group ~5 Consisting of t-butyloxycarbonyl, benzyloxycarbonyl, cyclopentyloxycarbonyl, t-amyloxycarbonyl and d-isobor.nyloxycarbonyl. . .
A further aspect of the present invention relates to intermediates ;.~.
linked to a solid resin support. These intermediates are represented ::
by the formulae:
R8-(pyro)-Glu-His~Nlm-R7)-Trp-xl-TyrtR5)-y-Leu-Arg~NG-R )-Pro-Z ~ ..
R ~His(Nlm-R7)-T~p-xl-Tyr~R5)-y-Leu-Arg(NG-R4)-pro-z2 ~ :
R9~Trp~xl-Tyr(R5)-y-L~u-~rg~NG-R~ pro~z2~ ~lnd R9-Xl_Tyr(R5)-y-Leu~Arg(NG-R4)-pro~z2 in which :
;
a) Xl is Ser(R6)~ Y is D-Trp and z2 is Gly-A; or .

- AHP_6505/6513/6639 b) X is D-Ser(R6), Y is D_Leu and z2 is Al;
wherein R ~ R , R6, R and R are as defined herein, R9 is an ~-amino protective group known to be useful in the art for the stepwise synthesis of polypeptides, suitable groups being listed hereinafter, and A and Al are anchoring bonds used in solid phase synthesis linked to a solid resin support. A is selected from the class consisting of: -N 1 ~ - ~ and 0~ CH2 ~ , and A is: 0 CH - uppor Details of the Invention The term "lower alkyl" contemplates alkyl radicals containing from one to three carbon atoms and includes methyl, ethyl, propyl and isopropyl.
NG means the side chain nitrogen atoms of arginine.
N~m means the imidazole nitrogen atoms of histidine.
The symbol ~ means "phenyl~
In general the abbreviations used herein for designating the amino acids and the protective groups are based on recommendations of the IUPAC~IUB Commission on Biochemical Nonenclature~ See Blochemistry 11~ 1726 (1972). For instance~
~-Boc represents ~-butyloxycarbonyl, Z represents benzyloxycarbonyl Tos represents tosyl, 2-Br Cbs represents 2-bromobenzyloxycarbonyl, Bzl represents benzyl~ and Dnp represents 2~4-dinitrophenyl. The abbrevlations used herein for the various amino acids are ~rg, .

1~6~8S~ AHP-6505/6513/6639 arginine; Gly, glycine; His, histidine; Leu, leucine;
Pro, proline; (pyro)-Glu, 5-oxoproline ~pyroglutamic acid);
Ser, serine; Trp, tryptophan; and Tyr, tyrosine. All amino acids described herein are in the L-series unless stated other-wise, i.e., D-Ser is a D-seryl residue, D-Leu is a D-leucyl residue, and D-Trp is a D-tryptophyl residue.
The peptides of formula 1 of this invention can be obtained in the form of acid addition salts. Examples of sal~s are those with organic acids, e.g. acetic, lactic, succinic, benzoic, salicyclic, methanesulfonic or toluenesulfonic acid, as well as polymeric acids such as tannic acid or carboxymethyl cellulose, and salts with inorganic acids such as hydrohalic acids, e.g.
hydrochloric acid, or sulfuric acid, or phosphoric acid.
If desired a particular acid addition salt is converted into another acid addition salt, e.g. a salt with a non-toxic, pharmaceutically acceptable salt, by treatment with the appropriate ion exchange resin in the manner described by R. A. Boissonnas, et al., ~lelv. Chim. Acta, 43, 1349 (1960). Suitable ion exchange resins are cellulose based cation exchangers, for example carboxy-methylcellulose or chemically modified, cross linked dextran cation exchangers~ for example, those o the Sephadex* C-type~ and strongly basic anlon exchange resins~ for example those listed by ~.P. Greenstoin and M. Winltz ln "~hemlstry o tho Amino Acids"
John Wiley and Sons, Inc., New York and London, 1961l Vol. 2, p. 1~56.
*Trade mark ~ AHP-6505/6513/6639 The peptides of formula I and their salts possess valuable, long-acting LH- and FSH-releasing hormone activity.
The valuable LH- and FSH-releasing hormone activity and long-acting property o~ the compounds of this invention are demonstrated by standard pharmacological procedures. For example, these activities can he demonstrated by tests described by A. Arimura, et al , Endocrinology, 95, 1174 (1974). For ~xample, by following the procedure described therein, LH-release da1a, obtained from rats given a dose (50 ng, subcutaneously) of the compound, show that the peptides of formula I reach peak activity at about two hours after dosing and tha1 significant activity is still present for up to six hours; hwereas after the same dose of LH-RH
(50 ng, subcutaneously), peak activity is reached at about the 15 mlnute mark and no effects of the injection are observed after one ;;15 hour. Also integrated levels of LH over a six hour period indicatetha-i the compounds of formula l; rD-Ser4, D-Leu6, desGly-NH2l0~-LH-RH
ethylarnide, and rD-Trp ~ LH-RH are 21 times and 12 tirnes, respectively, more active in releasing LH than LH-RH. FSH~release data following injections of the compounds of formula I indicate 1hat:
CD-Ser4, D-Leu6, desGly-NH210]-LH-RH ethylamide and [D-Trp6]-lH-RH
~ are about ll times and 20 times, respectively, more active than LH-RH
; at the same dose ~50 n9)~
Mor~ov~r, the actlvi~y of a compound of formula I is delnons~rated in man:
Radioimmunoassay of serum levels of LH after intranasal administration indicates that the minimum effective dose of LH-RH is about 2.0 mg whereas an equivalent de3ree of activity is ob-lained ~ith a 0.5 mg dose of rD-Trp ]-LH-RH. In lhis insiance the compounds are given ," ' .

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-~o hum~ns in a normal saiine ~oluiiorl~ With regard to FSH release In man com?arative studies with LH-RH and [D-Trp ~-L=I-RH give particularly no!eworthy results. Intranasal administration of up to 2.0 mg of LH--R~I has liitle or no effect on FSli serurn levels as measured by radioirnmunoassay, ~i.G. Dahlen, et al., Horm. Metab. Res., 6, 510 (1974); however, at O.S mg under ~he same conditions ~D-Trp6]-LH-RH
reieases signi;icant amounts of FSii, i.e., levels ranging from 0.3 fo greater than 1.5 miu/ml. It will be readily appreciated that a compound that is able to release FSH effectively has many therapeutic ai~plications; see, for example, i-i.G. Dahlen, et al., clted above.

The LH- and FSH-releasing properties of the peptides of fortnu1a ~, which in turn induce ovulation in animals, make the peptides useful in veterinary practice and in animal husbandry.
Is often deslrable to synchronize estrus in livestock, for eYample, càttle, sheep or sw1ne, either in order to be able to mate all .
the females in a given group with a male of the desired genetic ~uallty, or so as to be able to perform artificial insemination on a maximum number of femalr-~s, both within a comparatlvely short perlod of time. In the past, this has been done by administering to the animals an ovulation-inhibiting agent, ~/ithdrawing adm1nlstration of, said agont shortly before the date chosen for matlng or artlflclal Insemtnation, and relying eiiher upon the natural producilon of LH and FSi-l to induce o~ulation and ~o producc estrus or by administering gonadolrophins. Ho~lever, this procedure was not entirely satisfac--ory because ovulation at a predatermined time occurred never in all the animals togeiher but only in a certain propor-tion thereof when gonadotrophir1s were not lJsed. On the other hand, the high cost - of gonadotrophins and side effects encountered in their adrnl~istration made this method impractical. It is now ;~

. .

' posslble to obtain substantially completc synchronization of ovUIatjOn and of Gsttus, by tre~ting the anim~lls in a given group first wlth an ovuiztion Inhibilor whlch Ts subsequently witlldrawn, and then administerin3 a peptide of formula I shortly before the predetermined period of ttme for m3ting or artificial insemination, so as to obtain ovulation and estrus within that time interval.
The delay in the onset of ovulation and estrus following administration of the peptide varies with the species of animals, and the optimal time intervai has to be chosen for each species.
For example, in rodents such as rats or hamsters ovulation takes place within 18 hours following administration of a peptide of thls invention.
The method described above for obtaining ovulation and estrus within a precisely predetermined time interval, so as to be certain of a successful mating, is particularly Importan-t for breeders of race horses and of show animals, where the fees paid for the services of an exceptional male animal often amount to very considerable sums of money.

The peptides of formula L are also useful to Tncrease the number of live births pcr pregnancy in llvestock, for example, cat~le, sheep or swlne, For thls purpose the pep~lde Is glven In a serles of paren~eral dos~s, preferably by Intravenous of subcu~aneous inJections, In the range of 0.1 - 10 mcg. per kilogram of body weight per day, 96 to 12 hours prior to expccted estrus and subsequent mating. A prlming ~InJection of 1000 to ~000 iu of pregnant mares serum gonadotrophin may al~o be glven one to four days prior to the above injection of the peptide A similar treatment, with or without prior priming, is ~;;
aiso ~seful for inducing puberty in farm animals.

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When a peptldc of formula I Is ~mployed for th~ purpose of Induclng ovulation and ~strus or for inducing puberty in warm-blooded animals, especially in rodents such as rats or hamstcrs or in livestock, Tt is adrninistered systemicallyJ preferably parenterally, in combination with a pharmaceutically accepfable llquld or solld carrier. The proportion of the peptide Is determined by its solubility in the given carrier, by the chosen route of adminlstratTon, and by standard biological practice.
For parenteral admtnTstration to animals the peptide Is used In a sterile aqueous soiution which ~ay also con+ain other solutes such as buffers or preservattves, as well as sufficient pharmaceutically acceptable salts or glucose i-o make the solution Isotonic. The dosage will vary with the form of administratlon and w7th the particular species of animal to be treated and is preferably kept at a level of from 0.1 mcg. to 10 mcg. per kilogram body wetght. However, a dosage level in the range vf from about I n~g.
to about 5 r~g. per kilogram body weight ts most desirably employed in order to achiev~ effecti VQ results.
The p~plldesof f~rmula I can also be adminlstored In one of the long-~ actlng, slow-release or depot dosage forms described below, preforably by Intramuscular inJection or by implantailon.
Such dosage forms are destgned to release from about 0.1 mcg~
to about 10 rncg. per kllogram body weighi per day.
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~'\H~j-6505/65 1 3i6G39 :LC3 65iBS9 The peptides of formula I are also useful ln hum3n medtcine. For example, human chorionic gonadotrophin (HCGS
whlch contains mainly Li-l and some FSH has been used for over 30 years to treat certain endocrinological disorders such as disturbances of the cycle, amenorrhea, laclc of development of secondary sex characteristics, and infertility in the female, or certain cases of hypogonadism,delayed puberly, cryptorchidism, and non-psychogenic impotence in the male. Lately, infertility ln the human female has also been`treated with human menopausal gonadotrophln ~HMG) which contains mainly FSH, followed by treatment wlth HCG. One of the dlsadvantages of -the treatrnent of Infert71ity in the human female with HCG or with ~MG followed by HCG has become apparent in that such treatment oftèn results in superovulatlon and unwanted muliiple births, probably because of the irnpossibillty of giving only the exact amounts of FSH and LH which are necessary for ovulation, The admln1strat7on of a peptlde of thls invontlon overcomes ~he above dlsadvantage, because the compound causes release of LH and FSH by Ihe pituttary only in ti)e exact quantllles whtch ;'O , .
are required for normal ovulation. For that reason a :
peptide of this invent10n Ts not only useful for the above purpose, but li Is equally useful In the human female in the treatment of dlsturbances of the cycle, of amenorrhea, of hypogonadism, and of lack of development of secondary sex characteristics.

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~urthermore, the peptides of this invention are useful in contraception. For example, when the peptide is administered to a human female early in the menstrual cycle LH is re~ased at that time and causes premature ow lation. The immature ovum is either not capable of being fertilized, or, if fertiliza~ion should nevertheless have taken place, it is highly unlikely that the fertilized ovum w~ll become implanted because the estrogen-progestin balance required to prepare the endometrium is not present and the endometrium is not in the condition necessary for implantation. On the other hand, when the peptide is administered towards the end of the cycle the endometrium is disrupted and menstruation takes place.
In addition, the peptides of this invention are also useful I in contracept`ion by the "rhythm" method, which has always been 15 ~ relatively unreliable because of the impossibility of predetermining ovulation in the human female with the required degree of accuracy.
Administration of the peptide at mid-cycle, i.e. at about the normally expected time for ovulation, induces ovulation shortly thereafter and makes tho "rhythm" method both safe and effective.
20 ;

AIIP-6505/65i3/6639 65~59 The peptides of formula 1 are useful as a diagnostic tool for distinguishing between hypothalamic and pituitary mal-functions or lesions in the human female. When administering the peptide to a patient suspected of such malfunctions or lesions and a rise in the level of LH is subsequently observed ther~ is good indication to conclude that the hypothalamus is the cause Gf the malfunction and that the pituitary is intact. On the other hand, when no rise in circulating Lll is seen following the administration of the peptide a diagnosis of pituitary malfunction or lesion can be made with a high degree of confidence.
In the human male, administration of a peptide of formllla 1 proYides the amounts of L~l (or ICS~I) and of FSH necessary far normal sexual development in cases of hypogonadism or delayed puberty, and is also useful iTI the treatment of cryptorchidism. Furthermore, the FSH released by the administration of the peptide stimulates the development of germinal cells in the testes, and the peptide is useful in the treatment of psychogenic and non-psychogenic impotence.
When the peptides of formula 1, preferably in the form of an acid addition salt, are employed in h~nan medicine, they ar~ administered systemically, either by i.ntravenous, subcutalleous, or intramuscular injection~ or by sublingual, nasal, or vaginal aclministration, in compositlons iTI con~unction with a pharmaceutically acceptable vehicle or carrier.

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Por a.dmi.r!istration by the nasal route as drops or spray ;.
it is pre~erred to use a peptide of formula 1 in solution in a sterile aqueous vehicle which may also contain other solutes such as buffers or preservatives, as well as sufficient quantities of pharmaceutically acceptable salts or of glucose to make the solution isotonic. Doses by the intranasal route range frum 0.1 to 50 mcg/kg, or preferably 0.5 to 10 mcg/kg. :~
The peptides of formula 1 may also be administered as nasal or vaginal powders or insufflations. For such purposes the pepti.~e is administered in finely di.vided solid fol~n together with a pharmaceutically acceptable solid carrier, for examplc a. finely divided polyethylene glycol ("Carbowax* 1540~'~J finely divide~ lactose, or pre-ferably for vaginal administrationJ vcry finoly dividcd silica ("Cab-~-Sil"*).
Such conlposi.tions may also contain other excipients in fillely divided solid ~orm such as preservativesJ buffe~sJ or surfacc active agents.
For sublingual or vaginal administration it is preferred to formulate the peptides o~ formula 1 in solid dosage forms such as sublingual tablets or vaginal inserts or suppositories wi.th sufficient quantitites of solid excipients such as starch, lactose, cort.ain ~ypes of clay, buffers, and lubricating, di.sill~egratingJ or surface-active agents, o:r with scm:i-sol:id cxcipi.erlts commorlly ~Ised i.n the ornlulation of suppositories. Examples of such exclpients are found in standard pharmaceutica.l textsJ e.g. i.n Remln~ton's Yharmaceutical SciencesJ Mack Publishlng Compully, Easton, Pa., 1970.
*Trade mark X

` ,- AHP-6505/6513/6639 5~
Tho dosage of the peptides of formula I wiil vary with the form of administration and with the particular patient under treatment. Generally, treatment is initiated with small dosages substantially le~s than the optimum dose of the compound. There-after, the dosage is increased by small increments until the optimum effect under the circumstances is reached. In general, the peptides of formula I are most desirably administered at a concentration Ievel that will generally afford effective release of LH and FSH
; without causing any harmful or deleterlous side effects, and ; 10 preferably at a level that is in a range of from about 0.01 mcg.

- to about 100 mcg. per kilogram body weight, although as afore-mentioned variat10ns will occur. However, a dosage level of the peptide of formllîa I in which X is Ser, Y is D-Trp and Z is Gly-NH2 that is in the range of from about 0.5 mcg. to about 5.0 mcg. per ~15 kilogram body welght, and the peptide of formula I in which X is D-Ser, Y ts D-Leu and Z is NHR that is in the range of from aboul 0.1 mcg. to about 10 mcg. per kilogram body weight is most desirably employed to achieve effective results.
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. .

'~ AHP-6505/6513/6639 S~ .

It is oftell (lesirable to administer the pepti~es of formula 1 continuously over prolongec1 pcriods of time in long-acting~ slow-release, or depot dvsage forms. Such do.sage foTms may either contain a pharmaceutically acceptable salt of the compound having a lo~.~ degre0 of solubility in body fluids, for example salts with pamoic or tannic acid or carboxymethylcellulose, or they may contain the peptides in the ~orrn of a water-soluble salt together with a protective carrier which prevents rapid release. In the la~er case, for example, ~he peptides may be formulated with a non-antigenic lQ partially hydrolyzed gelatin in the form of a viscous liquid; or they : . .
may be adsorbed on a pharmaceutically acceptable solid carrier, for example zinc hydroxide with or without protamine, and may be aclmi.nistcrocl in suspension in a pharmaceutically acceptable li.ql1id vell.i.cle; or tho peptides may be formulatetl in gels or suspensions wi~h a protective l5 non-aTItigellic hydrocolloid, for examplo sod.iwn carbox~nethylcellulose, . .
polyvinylpyrrolidolle, sodium alginate, gelatine, polygalacturonic acicls, for example, pectin, or certain mucopolysaccharides, together with aqueous or nol1-aqueous pharmaccutically ~ccept~ble liquid vehicles, preserva~ives, or surfactants. Example~ of such formulations are found :in standard p}larm~ceutical texts, c.g. in Remington's ~harmlceutical Sciences, cited above. Long-~ct;n~, slow-release prepara-k.ions o.~ ~ho pept;dos may u.lso be obt~ lecl by m:Lcroonc.l})sulation in n phnrmaceutically acceptable coati.ng mat~rial, for example gelatinc, poly~inyl alcohol or ethyl cellulose. Further examples of coating materials and of th~
~5 processos ~Ised for microencapsulati.oll are descri.bed by J.A.
Herbig in "Encyclopedia of Chemical Technology", Vol. 13, , .

AHP-6505/~:~.5 13/~639 1(1~6~135~

Znd Ed., Wiley, New York, 1967, pp. 436~456. Such formula-tlons, as well as suspensions of salts of the peptides which are only sparingly soluble in body fluids, are designed to release from about 0.1 mcg to about 50 mcg of the-hormone per kilogram body weight per day, and are preferably administered ~-by intramuscular injection. Alternativ~ly, some of the solid dosage forms listed above, for example certain sparingly ~ater-soluble salls or d7spersions in or adsorbates on solid carr;ers of salts of the peptides, for examp~e dispersions in a neutral hydrogel of a polymer of ethylene glycol meth-acry1ate or stmllar monomers cross-linked as described in U.S. Patent 3,551,556 issued December 29, 1970 to K. Kliment, et al., may also be formulated in the form of pellets releasing about the same amounts as shown above and may be implanted subcutaneously or intramuscuiarly.

.~ ............................................................. . !
~ Alternatively, slow-release effects over prolonged periods of time may also be obtained by administering the peptld~s o~ this inventlon as an acld ad~i~ion salt 1n an intra-vaglnal deYlce or In a temporary ~mplant, for example a container made of a non-irrttating slllcone polymer such as a polysiloxano, e.g~ "Siiast1c'*, or of a neutral hydrogel of a polymer as described above, ~ possesslîlg the requlred degree of permeabTlity to release from iabout 0.1 mcg. to about 50 mcg per kilogram body weigh~
... . . . . .
per day. Such intra-vaginal or implant dosage forms for , .
prolonged administration hàve the advantage that they may be removed when if is desired to interrupt or to terminate treat-ment. -* Trade mark ... .i 1~6~85g Process In selecting a particular side chain protect:ive group to be used in the synthesis of the present peptides o~ formula 1, the following rules should be followed: (a) the protective group must be stable to the reagent and under the reaction conditions -selected for removing the ~-amino protective group at each step o~
the synthesis, (b) the protective group must retain its protecting properties ~i.e , not be split off under coupling conditions), and (c) the side chain protective group must be r~movable upon the . :.
completion of the synthesis containing the desired amino acid sequence under reaction conditions that will not alter the peptide chai.n.
With reference to R9, suitable a-amino protective groups include (1) aliphatic urethan protective groups lllustrated by t-butylo~ycarbonyl, diisopropylmethoxycarbonyl, biphenylisopropyloxy-carbonyl, isopropyloxycarbonyl, t-amyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl; (2) cycloalkyl urethan type protective groups illustrated by cyclopentyloxycarbonyl, adamantyloxycarbonyl, d-isobornyloxycarbonyl, cyclohexyloxycarbonyl; nitrophenylsulenyl, tritylsulfonyl, a,a-dimethyl-3,5-dimethoxybenzyloxycarbonyl and trityl. The preferred ~-amino protective group for R9 is selectod from the group consi~ting of t-butyloxycarbonyl, cyclopentyloxycarbonyl, t-amyloxycarbonyl, d-isobornyloxycarbonyl, o-nitropllenylsulfenyl,.bi-phenylisopropyloxycarbonyl, and a,a-dimethyl-3,5-dimethoxybenzyloxy-carbonyl.

f `- AHP-6505/6513/6639 ~ 58~ -The paptides of formula I of this invention are preparcd ~; vsing solid phase synthesis which will be illustrated by the followingembodiments in which specific peptides of formula I are prepared.
a) PePtide of formula I in which X is Ser, Y is D-Trp and Z is Gly-NH2 The solid phase synthesis of the preceding peptide of formula I is commer,ced from the C-terminal end of the peptide using an a-amino protected resin. Such a starttng material is prepared by attaching an ~-amino protected glycine to a benzhydrylamine resin, a chloromethylated resin or a hydroxymethyl resin, the former being preferred. The pre-paration of a benzhydrylamine resin is described by Y. Rivaille, el- al., Helv. Chim. Acta, 54, 2772 (1971) and the preparation of 1he hydroxymethyl resin is described by M. Bodanszky and J.T. Sheehan, Chem. Ind (London) 38, 1597 ~1966). A chloromethylated resin is commercially available from Bio Rad LaboraiorTes, Richmond, California. In using lho ben~hydrylamine resin an amide anchoring bond ;s formed with the ~-amirlo protected glycine as follows:

R9 - ~ - C~2 ~ NH - ~ -~ This permits the C-terminal amidc funciion to be obiatned direcily after the amino acid sequence in the syn-ihesls Is comple~d by cleav1n~ off ~he resin suppor~ of tha linked pcp-i-ide to form i~he glycine amide al the C-terminal portion of the desired decapeptide. In this Tnstance the use o~ -hydrogen fluortde for cleavlng off the resin support also removes ~he sidc chain protectlve groups to give the correspondillg decapeptide -;. '.', '.:

- ~ .

' , . : :, ... ... ., . .. . ` , . .. .. .: .

~ AHP-6505/6513/6639 i , :
of formula I in which X is Ser, Y is D-Trp and Z is Gly-NH2.
Wher7 the other resins are used, the anchoring bond is the benzylester group as illustrated hereinbefore. In this Instancc a convenient procedure for converting the linked protected peptide to the C-terminal amide is to ammonolize .~, the protected peptide off the resin and then remove the protective groups of the resulting amide by treat~ent with sodium and liquid ammonia or by hydrogen fluoride cleavage. An alternative procedure would be to cleave by transesterification with a lower alkanol, preferably t~thanol or ethanol, in the presence of triethylamine and then convert the resulting ester into an amide and subsequently deprotect as described above. See also J.M. Steward and J.D. Young, ~- I'Solid Phase Peptide Synthesis", W.H. Freeman ~ Co., San Francisco, Ig69, pp. 40-~9.
More speciflcally, in an embodiment of the present inven-lion an ~-amino protected glycine, proferably t-butyloxy-carbonylglycine, ~ ~' is coup1ed to benzhydrylamino resin with lhe aid of the carboxyl group activating cornpound, preferably, dicyclohexylcarbodiimide.
Following the coupling of the ~-amino protected glycine to the resin support, the ~-amino protecting group is removed such as by using ; trlfluoroacelic acid in methylene chlorlde, -~rTfluoroacetic acld alone or hydrochlorlc acid in dToxane. The deprotecliorl is cartied out a~ a t~mp~ra-~urc between 0C and room lemperafure, Other standarci ; cleavlng reagents and cond11ions for removal of speciiic t~-amino protectin9 groups may be used as described by E. Schroder and K. Lubke, I ~'The Peptldes", Vol, I, Academic Press, New ~ork, 1965, pp. 72-75.

. , .

.~ :

.

~ AHP-6505/6513/6639 ~6~

After removal of the ~-amino protecting group, 1-he remaining a-amino protected amino acids are coupled step-wise in the desired order to ohtaln the peptide~ Each proteeted arnino acid Ts introduced into the solid phase reactor ir about a three-fold `
excess and the coupling is carried ou-~ in a medium of methylene chloride or mixtures of dirnethylformamide in ~ethylene chloride. In cases where incornplete coupling occ~lrred the coupling procedure is repeated before removal of the ~-amino protecting group, prior to . :~ .
the coupling of the next amino acid to the polymer bound amino acid ~' or peptide. The success of the coupling reaction at each stage of the synthesis is monitored by the ninhydrin reaction as de 5C ri~ed by E. Kaiser, et al., Analyt. Biochem. 34, 595 (1970).
After the deslred arn7no acid sequence has been synthesized, the peptide ls removed from the resin support by treatr~nt with a reagent such as hydrogen fluoride which not only cleaves the peptide from the resin but also cleaves all remaining side chain protecting groups and 1he ~-amino protr-~cting group (if present) on the pyroglutamic acid residue in the case where the benzhydrylamine resin was used to `~
obtain directly the peptide of formula I in which X i5 Ser, Y is D-Trp ~ and Z is Gly-NH2.
'' . . ' .

,~'' .':
,~, .

.'~ ..................................................................... ... .

, -2~-' ." '.
.:~ . . ' .:~ ;,: :. :
,`: ' ' '' .; .
'. ' ~ : ' . ~ X "" `'' ~ AHP-6505/6513/6639 ~L~6513~i~
Where a chloromethylatecl resin is used the peptide may be separated from the resin by transesterification with a lower alkanol, preferc7bly methanol or e-thanol, after which the recovered producl is chromatographed on silica gel and the collected fraction subjected to treatment with ammonia to convert the lower alkyl ester, preferably the methyl or ethyl ester, to the C-terminal - amide. The side chain protecting groups are then cleaved by procedures described above, for example by treatment with sodium in liquid ammonia or by hydrogen fluoride.
In addition to the above described protec-iing groups (R7) for the imidazole nitrogen atoms of histid;ne, R7 can include 2,2,2-trifluoro-1-benzoyloxycarbonylaminoethyl and 2,2,2-trifluoro-1-t-butyloxycarbonylaminoethyl.
b~ Peptide of formula I in which X is D-Ser, Y is D-Leu an~l 7 1_ IIR
The solid phase synthesis of the pep-iicJe of formula I in which X i 5 D-Ser, Y is D-Leu and Z is Ni-lR i 5 commenced from Ihe C-termlnal end of the peptlde uslng an a-amino pro-iected proline resin. Such a starting material is prepared by attaching an ~-arniho ..
protected proline to a chloromethylated resin or a hydroxymethyl rosin, the former being preferred. The preparation of a hydroxy-; methyl resin is described by M. Bodansky and J. r . Sheehan, Ohem.
Ind. (london) 38, 1597 ~1966). A chloromethylate(l resin is commercially avallable From Bio Rad iaborai~ories, Rlchmoncl, Cc71ifornia. In llslnc7 1he chloromethylated resin an es-i-er anchoring group is forrned witl7 tl)e ~-amino protected proline as follows:
g ~
R ~Pro-O--C~i .
.' 5~
A conYerlier,t procedure for convertillg the linked protected peptide to the C-terminal (lower alkyl)amide consists in cleaving t-he pro1-ected p~p1ide off the rosin by treatment with a lower alkylamine, ~;
cf, D.H, Coy, et al., Bioch0m., Biophys. Res. Commun., 57, 335 (1974)~
- 5 tQ obtain the corresponding protected peptide (lower alkyl)amide.
Thereafter, the protective groups of the resulting peptide (lower alkyl)-amide are removed by treatment with sodium and liquid ammonia or preferably by hydrogen fluoride cleavage to give the corresponding peptide of formula I in which X is D-Ser, Y is D-Leu and Z is NHRI.
~lO An alternative procedure consists in cleaving by transesterification with a lower alkanol, preferably methanol or ethanol, in the presence ; of triethylamine and then to convert the resulting ester into the ~ corresponding (lower alkyl)amide and subsequently deprotect as ; :. . ; :
; described above. See also J.M. Stewarl and J. D. Yound, "Solid Phase ~ 15 Pep1ide Synthesis", W, H. Freernan ~ Co., San Francisco, 1969, pp. ~0-49, ,:; ,, More specifically, in an embodiment of the present Invention ~-amino protected proline, preferably t-butyloxy-carbonylprolTne, is coupled to a chlorornethylated resin with the ; aid of a catalyst, preferably, cesium bicarbonate or triethylamine.
Following the coupling of the ~-amino protected proline to the :. .
resin support, the a--amino protecting group is removed, for example by using -Irifluoroacetic actd in m~thylenc chloride, trifluoro-acetlc acid alone or hydrochlorTc acid in dioxane. Tho deprolectlon is carried oul at a lemporalure be~ween aboul 0C
~5 and room temperalure. Other standard cleaving reagents and - condjtions for removal of spectflc a-amtno protecting groups ~ay be used as described by E~ Schroder and K. Lubke, "The Peptides~, Vol. I, Academic Press, New York, 1965, pp. 72-75.
. . .
After removal of the ~-amino protec1-ing group, the remaining a-aminO protected amino acids are coupled step-wise in the ~ 2 ~ RHP-6505/6513/6639 5~
~esired or~'er lo obtain the compound of formula I in which X is D-Ser, Y i5 D-Leu and Z ts NHR 0 Each protected amino acid is introduced in-to the ~olid phase reactor in about a three-fold excess and the coupling is carried ou-t in ~ medium of methylen~ chloride or mixtures of dimethylformamide and methylene chloride. In cases where incomplete coupling has occurred ~-he coupling procedure is repeated before removal of the ~-amino protecting group, prior to ihe - ~ coupling of the next amino acid to the solid phase reactor. The success Or the coupling reaction at each stage of the synthesis is monitored by the ninhydrin reaction as described by E. Kaizer, et al., Analyt. Biochem. 34, 595 (1970).
After the desired amino acid sequence has been synthesized, the protected peptide is removed from the resin support by ~reatment . .
with a (lower alkyl)amine to give the corresponding pr-otected peptlde (lower alkyl~amine and in the case where dinilrophenyl or tosyl has -~ been used as Ihe protec-ting group for the hisl-idyl re~sidue, the dinitrophenyl or tosyl protecting group also is removed during the treatment with the (lower alkyl)amine. The peptide may also be separated from the resin by transesterification wi-th a lower alkanol, preferably ~o methanol or ethanol, after which the recovered product is purified by chromcttograpty on silica gel and the collected fraction subjecled to treatrrtent with a (lower alkyl)amine to converl the lower alkyl es-ler, preferably Ihe methyl or ethyl estrtr, to the C~termlnal (lower alkyl~-amide. (Note that the dinitrophenyl or tosyl group, if present on the histidyl residue, will also be cleaved). lhe remaining side chain pro1ecttng groups of the prolected (lower alkyl~mide ar~ then cleaved by procedures described above, for example by treatment with sodium in liquid ammonia or by hydrogen fluoride to give the nonapeptide of formula I in which X is D-Ser,Y is D-Leu and Z is NHR .
~50 The following examples illustrate further this inven-tion.

X

, 651~S9 EXAMPLF. 1 L-Pyroglutamyl-L-histidyl(tosyl)-L-tryptophyl-L-seryl(benzyl)-L-tyrosyl~2-bromo-benzyloxycarbonyl)-D-tryptophyl-I,-leucyl-L-arginyl~tosyl)-l.-prolylglycylbenzhydrylamine resin (R8-(pyro)-Glu-His-(Nlm-R7)-Trp-Ser(R6)-Tyr(R5)-D-Trp-Leu-Arg~NG-R4~-Pro-Gly-A; R4 = Tos, R5 = 2-Br-Cbz, R6 = Bzl, ~ = Tos, R8 = H and A = benzhydrylamine resin).
Benzhydrylamine resin (1.25 g, 1.O mmole) is placed in the reaction vessel of a Beckman Model 990 automatic peptide synthesizer programmed to carry out the following wash cycle: ~a) methylene chloride; ~b) 33% trifluoroacetic acetic in methylene chloride (2 times for 2.5 and 25 minutes each); (c) methylene chloride; (d) ethanol; ~e) chloroform; ~f) 10% triethylamine in chloroform ~2 times for 25 minutes each); (g) chloroform; (h) methylene chloride, The~washed resin is then stirred with t-butyloxycarbonyl ~lycine (525 mg, 3.0 mmoles) in methylene chloride and dicyclohexyl-carbodiimide (3,0 mMoles) is added. The mixture is stirred at room temperature (22-25C) for 2 hours and the amino acid resin is then washed successively with methylene chloride (3 times), ethanol (3 times), and methylene chloride (3 times). The attached amino acid is deprotected with 33% trifluoroacetic acid in methylene chloride (2 times ~or 2.5 ~md 25 minutes each and then steps (c) through (h) as described in the above wash cycle ar~ por~orlned.
The followlng amino acids (3,0 ~nmoles) are then coupled successively by the same cycle of events: t-Boc-L-protine;
t-Boc-L-arginine(Tos); t-Boc-L-leucine; t-Boc-D-tryptophan;

, ~

~5859 A~IP-6505/6513/6639 t-Boc-L-tyrosine (2-Br-Cbz); t-Boc-L-serine(Bzl); t-Boc-L-tryptophan; t-Boc-L-histidine(Tos); L-(pyro)-glutamic acid.
The completed decapeptide resin is washed with methylene chloride (3 times) followed by methanol (3 times) and dried under reduced pressure whereupon 98% of the theoretical weight gain is obtained.
The benzhydrylamine resin used in this example is a commercially available resin (1% cross linked, Bachem Inc., Marina del Rey, Caliornia).

L-Pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-trypt~phyl-L-leucyl-L-arginyl-L-prolylglycinamide;
_, X = Ser, Y 5 D-Trp and Z ~ Gly-Nll2 ((pyro)-Glu-llis-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-GIy-N~I~
Removal of protecting groups and cleavage of the decapeptide from the decapeptide resin, described in ~xample 1, is carried out by treatment of 1.0 g of material with hydrogen fluoride (24 ml) and anisole (6 ml~ at 0C for 30 minutes.
The hydrogen fluoride is removed under reduced pressure and the anisole removed by washing with ethyl acetate.
The crude peptide is purified by gcl filtratlon on a column (2.5 x 100 cm) of Sephadex* G-25 ( a fine grade, chemically modifiod cross-l;inke~ doxtran) by olution with 2 molar acetic acid and fractiolls shown to contain a major UV
absorption peak at 280 nm were pooled and evaporated to dryness.
The rosidual oil was applied to a column (2.5 x 100 cm) of Sephadex* G-25 (fine), previously equilibrated with the lower phase followed by the upper phase of n-butanol: acetic acid: water (4:1:5) solvent system. Elution with the upper phase gives a major peak fraction, and material from this area was sub-*Trade mark ~ 1:116~359 jected to elution on a column (1.4 x 94 cm) of carboxymethyl cellulose accorcling to the conditions described by D.H. Coy, et al., J. Med. Chem., 16, 1140 (1973). Appropriate fractions (1050 -1190 ml), after lyophilization to constant weight, gave D-Trp6-LH-RH as a white, fluffy powder ~80 mg); ~a)2D
-58.8 ~c = 0.33, lN HOAc).
The product was homogeneous by thin layer chromatogTaphy in four separate solvent systems when loads of 20-30 mcg were applied and spots visualized by exposure to iodine vapour followed by Ehrlich reagent. The following Rf values were obtained:
l-butanol: acetic acid:water (4:1:5: upper phase), 0.2S;
ethyl acetate: pyridine: acetic acid: water ~5:5:1:3), 0.63;
2-propanol: 1 M acetic acid (2:1), 0.38; l-butanol: acet:;c acid: water:~ ethyl acetate (1:1:1:1), 0.51.
Amino acid analysis gave: Glu, 1.08; IIis, 0.95; Trp, 2.00;
Ser, 0.94; TyrJ 0.97; Leu, 0.93; Arg, 0.98; Pro, 1.00; Gly, 1.02;
N~13, 1.03.

' ~ AHP-6505/6513/6639 35~, ~ ~n~ c~ istidyl(dinitroP_e~ trYP-roPhyl-D--~erYl~tenzv~
L-tYrosYI(2-bromobenzyloxycarbonyl) _ e cyl-l-leucy!-L-arqinyl(tosyl-L-prolYI-0-CH2-resin, R -tpyro)-Glu-His(NIm-R7)-Trp-D-Ser(R5)-lyr(R5)-D-Leu-Leu-Arg(NG-R4)-Pro-AI; R4 = Tos, R - 2--Br-Cbz, R6 = Bzl, R7 - Dnp, R8 - H and Al = 0-CH2 ~
~ f~
Boc-Proline resin of the formula Boc-Pro-0-C~ -~resin (1.40 9, 0.5 mmole of proline) is tO placed in the reaction vessel of a Beckman Model 990 au-i-omatic peptide synthesizer programmed to carry out the following wash cycle: (a) methylene chloride; (b) 33% trifluoroacetic in methylene chloride (2 times for 2.5 and 25 minui-es each); (c) methylene chloride; (d) ethanol; ~e) ch!oroform; (f) 10~ triethylamine in chloroform (2 times for 5 minutes each); (9) chloroform; and (h) methylene chloride.
The washed resin is then stirred with t-butyl-oxycarbonyl-tosyl-argine (645 rng, 1.5 mmoles) in methylene chloride and dicyclohexylcarbodiimide (1.5 mmoles) is added. The mixture is stirred at room tempera1ure (22-25C) for ; 2 llours and Ihe amino acid resirl is -Ihen washed successively wiih melhylenc chloride (3 times). The ali-ached amirlo acid is dcprotectcd with 33~ irifluoroaceiic acid in meihylene chlorid~ (2 limes for 2.5 and 25 minutes each and 1hen steps (c) through (h) as described in the above wash cycle ar-e performed.
Th~ followln~ amino acids ~1.5 mmoles) are then coupled successively by the same cycle of events: t-Boc-L leucine; t-Boc-D-leucine;

-3~-~ 5~5~ AHP-6505/6513/6639 FXh~PL~ 3 - Continued t-~oc-L-tyrosine (2-8r-~bz); i-80c-D-serine(8zl~; t-80c-L-tryptophan; t-Boc~L-his-lidlne(Dnp); L-~pyro~-glutamic acid.
The corrlpleted nonapeptide resin is washed ~it-h methylene chloride (3 times) followed by methanol (3 times) and dried under reduced pressure ~hereupon 96~ of the theoreTical wetght gain is obtained.
The proline resin used in this example is made from a commercially available chloromethylated resin (Ig cross linked, Bio Rad Labs, Richmond, California).

' :. ' i ~ .:

.. . ..
' : ' ' '~"

'' ' ' .
. . .

.
.' ' ' ~ .
.
, ;.
,: '~ :

'--` ~:

58~9 L-Pyroqlut m~cl _____St idyl-L-tryptophyl-D-seryl(be~ L~ -tvr~ bromot nzyloxycarbonYI~-D le cyl-L-leucyl-L-arqinyl(tosy_~=
L prol ~ _hylamide, R8-tpyro)-Glu-His-Trp-D-Ser(R6)-7 Tyr(R )-D-Leu-Leu-Arg(N -R )-Pro-NHR ; R = Tos, R5 =
2-Br-Cbz, R = Bzl, R8 = H and Rl = C2H5 The protected nonapeptide resin (2.16 9 described in - Exa~ple 3) is suspended in ethylamine (20 ml) at 0C and stirred for 6 hours. Excess ethylamine Is then allowed to evaporate at room temperature and the cleaved peptide is washed from the resin with dimethylformamide. The protected peptide is then precipitated by the addition of ethyl acetate and filtered to give a cream-colored powder (672 mg). Rf on sitica 9el in l-butanol: acetic acid:
water (4:1:5, upper phase) is 0.45. This material is used in Example 5 without further purification.

., .

:, , . I .

, .' ;'.

~ . .

' . ~

t l: d l~ AHP-6rJ05/6513/6639 i85~
FX ~_LE 5 L-PYroqlu tdmv ~ histidy~ yplophyl- _ eryl-L-tyrosyl-D-I-I-Ie cyl-L-arglnyl-L-~r~y~ ethvl3lnide~ = Ser, Y - D Leu and_Z ~ NHEt r~eyro?-G~u-H s-lrP-D-~er-Tyr-D-Leu-le Arg-Pro-NHEt]
Rem~val of protecting groups from the protected nonapeptide, prepared as described in Example 4, is carried out by treating 670 mg of 1he rr,aferial with hydrogen fluoride ~50 ml) and anisole (15 ml) at 0C for 30 minutes. The hydrogen fluoride is removed under reduced pressure and the anisole removed by washing with ether.
The crude peptide is purified by gel filtration on a column (2.5 x lOO cm) of Sephadex* G-25 (a fine grade, chemically modified cross-linked dextran) by elu-tion with 0.2 molar acetic acid and fractions shown to contain a major lJV ab50rp-tion peak at 280 nm are pooled and evapora-led to dryness.
The resldual oil is applied to a column (2.5 x lOO cm) of Sephadex~ G-25 (fine), previously equilibrated with the lower phase followed by the upper phase of n-butanol; acetic acid;
water (4:1:5) solvent system. Elution with the upper phase gives a major fraction with high u.v. absorption at 280 nm and this material - is subjected to chromatography on a column (1.5 x 94 cm) of 5i lica gel and elution with a l-butanol:acetic acid: wa1er ~4 1:1) mixlure.
Approprlate fractlons ~300 390 ml), af~er evaporation and Iyophiliza~ion lo cons~anl woighl from water, givc ~D-Ser4, D-Leu6, desGly-NI-12l0]-~5 LH-RH elhylarnide as a white, fluffy powder (IO:S rng); r~23 = -29.6 , ~c = 0.5~, O.IN HOAc).

*Trade mark "' ,:
' ::

,"' ~.
; ':

~ 5~5~ AHP-6505/6513/6639 EXA LE 5 -~Contlnued The product ts homogeneous by thin layer chromatography In four separate solvent systems on 5i lica gel plates when loads of 20-30 mcg are applied and spots visualized by exposure to iodine vapour followed by Ehrlich reagent. The following kf values are obtained;
I-hutanol: acetic acid:water (4:1:5: upper phase), 0.20;
ethyl acetate pyridine: acetic acid: water (5:5:1:3), 0.72;
2-propanol: I M acetic acid (2:1), 0.43; I-butanol: acetic acid: water: ethyl acetate (1:1:1:1), 0.50.
Amino acid analysis gives: Glu, 1.03; His, 0.93; Trp, 1.01; Ser, 0.12; Tyr, 0.97; Leu, 1.98; Arg, 1.00; Pro, 0.90i ethylamine, 0.98.
. .
~15 ~ .

. .
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: . . . . ,. - -- - ,, , , :

Claims (15)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing a compound of formula 1 (pyro)-Glu-His-Trp-X Tyr-Y-Leu-Arg-Pro-Z (1) in which a) X is Ser, Y is D-Trp and Z is Gly-NH2, or b) X is D-Ser, Y is D-Leu and Z is NHR1 wherein R1 is lower alkyl, or a non-toxic pharmaceutically acceptable salt thereof, comprising:
i) reacting a compound of formula R8-(pyro)-Glu-HistN1m-R7)-Trp-X1-Tyr(R5)-Y-Leu-Arg(NG-R4)-Pro-Z2 in which X1 is Ser(R6), Y is D-Trp and Z2 is Gly-A wherein A is and R4, R5, R6 and R7 are protcctive groups capable of being removed by one or more chemical treatments which do not affect the correspondinq compound of formula 1, and R8 is hydrogen or one of said protective groups with a reagent capable of removing the protective groups R4, R5, R6, R7 and R8 without affecting the compound of formula 1 to obtain the corresponding compound of formula 1 (pyro)-Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z
in which X is Sor, Y is D-Trp and Z is Gly-NH12; or (i) reacting a compound of formula 11 R8-(pyro)-Glu-His(N1m-R7)-Trp-X1-Tyr(R5)-Y-Leu-Arg(NG-R4)-Pro-Z1 in which X1 is Ser(R6), Y is D-Trp and Z1 is Gly-R2 wherein R2 is amino and R4, R5, R6 and R7 are protective groups capable of being removed by one or rnore chemical treatments which do not affect the corresponding compound of formula 1, and R8 is hydrogen or one of said protective groups with a reagent capable of removing the protective groups R4, R5, R6, R7 and R7 without affecting the compound of formula 1 to obtain the corresponding compound of formula (pyro)-Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z
in which X is Ser, Y is D-Trp and Z is Gly-NH2-; or iii) reacting a compound of the formula R8-(pyro)-Glu-His-Trp-D-Ser(R6)-Tyr(R5)-D-Leu-Leu-Arg(NG-R4)-Pro-NHR1 in which R1 is lower alkyl and R4, R5, R6, and R8 are protective groups capable of being removed by one or more chemical treatments which do not affect the corresponding compound of formula I and R8 is hydrogen or one of said protective groups with a reagent capable of removing he protective groups R4, R5, R6 and R8 without affecting the compound of formula I to obtain the corresponding compound of formula I
(pro)-Clu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z
in when X is desired Y is D-Leu and Z is NHR1 wherein R1 is lower alkyl;
and when desired converting the compound of formula I to a corresponding non-toxic pharmaceutically acceptable salt.

2. The process of Claim 1 in which the protective group removing agent is hydrogen fluoride.

3. The process of Claim 1 for preparing a compound of formula I

(pyro)-Clu-His-Trp-X-Tyr- Y-Leu-Arg-Pro-Z (I) in which X is Ser, Y is D-Trp and Z is Gly-NH2, or a non-toxic pharmaceutically acceptable salt thereof, which comprises:
reacting a compound of formula R8-(pyro)-Glu-His(N1m-R7)-Trp-X1-Tyr(R5)-Y-Leu-Arg(NG-R4)-Pro-Z2 in which X1 is Ser(R6), Y is D-Trp and Z2 is Gly-A wherein A is and R4, R5, R6 and R7 are protective groups capable of being removed by one or more chemical treatments which do not affect the corresponding compound of formula I, and R is hydrogen or one of said protective groups with a reagent capable of removing the protective groups R4, R5, R , R and R3 without affecting the compound of formula I to obtain the corresponding compound of formula I
(pyro)-Glu-His_Trp-X-Tyr-Y-Leu-Arg-Pro-Z
in which X is Ser, Y is D-Trp and Z is Gly-NH2, and when desired converting the compound of formula I to a corresponding non-toxic pharmaceutically acceptable salt.

4. The process of Claim 3 in which the protective group removing agent is hydrogen fluoride.

5. The process of Claim 1 for preparing a compound of formula I
(pyro)-Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z (I), in which X is Ser, Y is D-Trp and Z is Gly-NH2, or a non-toxic pharmaceutically acceptable salt thereof, which comprises:
reacting a compound of formula II
R8-(pyro)-Glu-His(N1m-R7)-Trp-X1-Tyr(R5)-Y-Leu-Arg(NG-R4)-Pro-Z1 in which X1 is Ser(R6), Y is D-Trp and Z1 is Gly-R2 wherein R2 is amino and R4, R5, R6 and R7 are protective groups capable of being removed by one or more chemical treatments which do not affect the corresponding compound of formula I, and R8 is hydrogen or one of said protective groups with a reagent capable of removing the protective groups R4, R5, R6, R7 and R8 without affecting the compound of formula I to obtain the corresponding compound of formula I
(pyro)-Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z
in which X is Ser, Y is D-Trp and Z is Gyl-NHe, and when desired converting the compound of formula I to a corresponding non-toxic pharmaceutically acceptable salt.

6. The process of Claim 5 in which the protective group removing agent is hydrogen fluoride.

38 .

7. The process of Claim 1 for preparing a compound of formula I
(pyro)-Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z (I) in which X is D-Ser, Y is D-Leu and Z is NHR1 wherein R1 is lower alkyl, or a non-toxic pharmaceutically acceptable salt thereof, which comprises:
reacting a compound of formula R8-(pyro)-Glu-His-Trp-D-Ser(R6)-Tyr(R5)-D-Leu-Leu-Arg(NG-R4)-Pro-NHR1 in which R1 is tower alkyl and R4, R5, R6 and R8 are protective groups capable of being removed by one or more chemical treatments which do not affect the corresponding compound of formula I and R8 is hydrogen or one of said protective groups with a reagent capable of removing the protective groups R4, R5, R6 and R8 without affecting the compound of formula I to obtain the corresponding compound of formula I
(pyro)-Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z
in which X is D-Ser, Y 1s D-Leu and Z is NHR1 wherein R1 is lower alkyl; and when desired converting the compound of formula I to a corresponding non-toxic pharmaceutically acceptable salt.

8. The process of Claim 7 in which the protective group removing agent is hydrogen fluoride.

9. The process of Claim 7 in which R1 is ethyl,

10. The process of Claim 9 in which the protective group removing agent is hydrogen fluoride.

11. A compound of formula I
(pyro)-Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z (I) in which a) X is Ser, Y Is D-Trp and Z is Gly-NH2; or b) X is D-Ser, Y is D-Leu and Z is HNR1 wherein R1 is lower alkyl, or a non-toxic pharmaceutically acceptable salt thereof, when prepared by the process of Claim 1 or 2, or a chemical equivalent thereof.

12. The compound of Claim 3 which is L-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-tryptophhyl-L-leucyl-L-arginyl-L-prolylglycinamide, or a non-toxic, pharmaceutically acceptable addition salt thereof, when prepared by the process of Claim 3 or 4, or a chemical equivalent thereof.

13. The compound of Claim 5 which is L-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-tryptophyl-L-leucyl-L-arginyl-L-prolylglycinamide, or a non-toxic, pharmaceutically acceptable addition salt thereof, when prepared by the process of Claim 5 or 6, or a chemical equivalent thereof.

14. The compound of formula I
(pyro)-Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z (I) in which X is D-Ser, Y is D-Leu and Z is NHR1 wherein R1 is lower alkyl, or a non-toxic, pharmaceutically acceptable salt thereof, when prepared by the process of Claim 7 or 8, or a chemical equivalent thereof.

15. The compound of Claim 9 which is L-pyroglytamyl-L
L-tryptophyl-D-seryl--L-tyrosyl-D-leucyl-L-leucyl-L-arginyl-L-prolyl ethylamide, or a non-toxic, pharmaceutically acceptable addition salt thereof, when prepared by the process of Claim 9 or 10, or a chemical equivalent thereof.