CA1067487A - Lh-rh analogs and intermediates therefor - Google Patents

Abstract

LH-RH AHALOGS AND INTERMEDIATES THEREFOR

Abstract of the Disclosure [D-Trp6]-LH-RH,[D-Phe6]-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

106 748!7 f~N R -~505/ri5 1-, i i 3 , .
ack~round of t~,e Disclrs~rr (a) Fie!d of the Invention This invention.relates to peplides 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 Ser, Y is D-Phe and Z is Gly-NH2; or : c) X is D-Ser, Y is D-Leu and~Z is NHRI wherein Rl is lower alkyl~ and intermediates for the synthesis t~lereof.
The peptides of formula I in whjch a) X is Ser, Y is D-Trp and Z is Gly-NH2; or b) X is Ser, Y is D-Phe and Z Is Gly-NH2; or ` c) X is D-Ser, Y is D-Leu and Z is NHRI also are called, respectively, a) L-Pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-tryp~ophyl-L-leucyl-L-arginyl-L-prolylglycinamide; or b~ L-pyroglutamyl-L-histidyl-L-tryphophyl-L-seryl-L-tyrosyl-:~
. . D-phenylalanyl-L-leucyl-L-arginyl-L-prolylglycinamide;:or . c) L-pyroglu~amyl-L-h7stidyl-L--tryptophyl-D-seryl-L-tyrosyl-20 ~ ~ D-leucyl-L-leucyl-L-arginyl-L-prolyi lower alkyl amide, and I may be designated by the abbreviation~
a) ~D-Trp6~-LH-RH;
b) ~D-Phe6]-LH-RH; or ~ :
: c) ~DiSer4,D-Leu ,desGly-NH210~-LH-RH (lower alkyl~amide, ~- 25 -- ~` respectivelyO

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(b) Backqround of the Invent_on.
Lutelnizing hormone (LH) and:foliicle-st7mulating hormone tFSH) are both gonadotrophic hormones elaborated by the pituitary . glond of humans and of mammals. LH together with~FSH stimuiates 5 : the release of estrogens from the maturing follicles in the ovary ::, : : :
; and:induces the process of ovulation in the female. In the male, ,:
UH stimulates the Tntersti~ial cells and is for that reason also called interstit7al cell stimulating hormone ~:ICSH). FSH induces .
~ . maturatton of the follicles in the ovary and together with L~, plays an Importan~ roie tn the cycl7c phenomena in the female.
. FSH~.promotes the development of germinal celis in the testes of the :~ :~
;: male. ~Both LH and FSH~ar~ released from 1he~pi~tuitary gland by th:e actTon of LH and FSH-re~ieasing hormone, and there is good~
evldence that said releasing;hormone is~elàborated in the~hypothalamus : . . . ,::
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~ AHP-6505/6513/6639 i~)67~87 and reaches the pituitary gland by a neurohumoral pathway, see e.g., A.V. Schally, et al.; Recent Progress in Hormone Research, 24, 497 ~1968).
The natural LH- and FSH~releasing hormone has been isolated from pig hypothalami and its constitution elucidated by A.V. Schally, et al., Biochem Biophys. Res. Commun., 43, 393 and 1334 C1971), who proposed the decapeptide structure (pyro)-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2.
This constitution has been confirmed by synthesis; for example~
see H.-Matsuo, et al., Biochem. Biophys. Res. Co~mn., 45, 822 ~1971) and R. Geiger, et al., ibid, 45, 767 ~1971).
Hereinafter the natural LH- and FSH-releasing ~ormone is called LH-RH.
Because of the importance of LH-I~I to both diagnostic and therapeutic medicine, considerable interest has been shown in the preparation of new compounds having improved properties over the/natural hormone. One approach to this goal has been the selective modification -~ or replacemen~ of amino acid residues o~ LH-RH with other amino acids.
Although in a few instances peptides containing such alteratians have ;
been found to be more active than LH-RH, for example, (D-Alafi)-LH-RH, A. Arimura, e~ al., Endocrinology, 95, 1174 (1974), (D-Leu6)-LH-RH and ~D-Leu6,desGly-NH210)-LH-RH ethylamide, I.A. Vilchez-Martinez, et al., Biochem. Biophys, Res. Commun.~ 59, 1226 (1974), for the most part the modified peptides have been less active.

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~067~7 A~IP-6505/6513/6639 Now it has been found that:
a) the replacement of the glycyl moiety in position 6 of LH-RH wi~h D-tryptophan; or b~ the replacement of the glycyl moiety in position 6 of LH-RH with D-phenylalanine; or c) 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 moiety, and replacement of the glycinamide moiety in position 10 by a lower alkyl amide group, gives a peptidc of formula 1 that is much more active and longer acting than LH-~H.
;~ 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 action for the peptide of formulal in which X is D-Ser, Y is D-Leu and Z is NHRl is qulte surprising, especially in view of the fact that wlth 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-Ser4)-LH-RH has less than 5% of the LH-RH activity of the natural hormone.
The attributes of the present peptides of formulal have practical significance: the smaller minimum effective dose reducing side effects as well as the cost for the preparation of the compound and the longer ac~ing property reducing the need for frequeDt adminlstFation.

i Summary 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 (~
in which a~ X is Ser, Y is D-Trp and Z is Gly-NH2; or b) X is SerJ Y is D-Phe and Z is Gly-NH2; or c) X is D-Ser, Y is D-Leu and Z is NHRI where1n Rl is lower alkylJ or a non-toxic pharmaceutlcally acceptable salt thqreof;
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 zl is Gly-R2; or b) Xl is Ser~R6), Y is D-Phe~and Zl~is Gly-R ; or c) .X1 is D-Ser(R6), Y is D-Leu and zl is OR wherein R is selected from the group consisting of nm1no and O-~lower alkyl), R3 is lower alkyl, R4, R5, R6 and R7 are protective groups capable : ~ ~-of belng removed by one or more chemical treatDents whlch do not~
: ~ affect ~pyro~-Glu-His-Trp-X-Tyr-Y-Leu-Arg-Pro-Z in which Xj Y and Z
20~ are as defined~herein and R8~ i9 hydrogen or one of said protective : groups; and:~ ~:
R8-(pyro)-Glu-His-Trp-D-Ser(R6)-Tyr(R ~-D-Leu-Leu-Arg~NG-R4)-Pro-NHR
in which Rl is lower alkyl, R4, R5 and R6 are protoctive groups ~ capable of being removed by one or more chemical treatments which do not affect (pyro)-Glu-His-Trp-D-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHRl and R8 : is hydrogen or one of said protective groups.
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10~7487 AHP-6505/6513/6639 With reference to the compounds of formula lland the compound o~ the formula R -~pyro)-Glu-His-Trp-D-Ser~R6)-Tyr(R )-D-Leu-Leu-Arg~NU-R4)-Pro~NHRl, in a preferred embodiment R and zl are as defined herein, R4 is 5 : a protectiv~ group for the N~, N~, and N~ nitrogen atoms oE arginine selected from the group consisting of tosyl, nitro,:benzyloxycarbonyl and adamantyloxycarbonyl; R is a protecti~e group for the hydroxyl or . tyrosine selected from the gorup consisting of 2-bromobenzyloxycarbonyl, ~: : benzyl, acetyl, tosyl, benzoyl, t-butyl, tetrahydropyran-2-yl, trityl, : 10:::2,4-dichlorobenzyl and benzyloxycarbony; R6 is a protectlve group for the hydroxyl group of ser1ne and is selected from the group defmed here-inbefore for R R7 is a protective group for the imidazole nitrogen ato=s of histidine selected from the group of tosyl and dinitrophenyl; and R8 is hydrogen or an a-amino protective group selected from the group consisting of t-butyloxycarbonyl, benzyloxycarbonyl, cyclopentyloxy-carbonyl, t-amyloxycarbonyl and d-isobornyloxycarbonyl. : :
A further aspect o f the~:present invention relates to inter- :
mediates linked to a~solid resin support. These intermediates are re-:
~ ~ presented by the formulae: :

:~ ~ 20 R8-~pyro)--Glu-His~Nlm-R7)-Trp-XI-Tyr(R5)-Y-Leu-Arg~NG-R4)-Pro-Z2, R9-Hls(Nlm-R7)-Trp-Xl-Tyr~R5)-Y-L~u-Arg(NG-R4)-Pro-Z , R9-Trp-Xl-Tyr~R5)-Y-Leu-Arg(NG-R ~-Pro-Z , and R9-X -Tyr(R5)-Y-Leu-Arg~NG-R4)-Pro-Z2 in which a) Xl is Ser~R6), Y is D-Trp and ~2 is Gly-A; or ' 10674~7 b) Xl ls Ser~R6), Y is D-Phe and Z is Gly-A; or c~ Xl is D-Ser~R ), Y is D-Leu and z2 is Al;
wherein R4, R5, R6, R7 and R8 are as defined herein~ R9 is an ~-amino protective group known to be useful in the art for the ~1 S stepwise synthesis of polypeptides, suitable groups being listed herelnafter, and A and A' are anchoring bonds used ln solid phase synthesis linked to a solid resin support. A is selected from the class consisting of: `
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N - C ~ and O - ~H2 ~ , and Al is : O____ GH _ Z ~7 ~
Details of the Invention The~term "~lower alkyl~ contemplates~alkyl radlca1s cont~alning ;~
from one to three carbon at:oms and in$1udes methyl, ethyl, propyl and isopropyl.

~20 ; ~ ~: NG means the slde chain nitrogen atoms of arginine.
N m means the lmldazole nltrogen atoms of histidlne.
The symbol ~ means t'phenyl".
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In general the abbreviatlons used hereln for designating ~ the amino acids and the protective groups are based on recommendations ; of~the IUPAC-IUB Commission on Blochemical Nomenclature, see Biochemlstry : ~ ~ 11, }726 ~(1972). For instance, t-Boc represents t-butyloxycarbony}, Z
represents benzyloxycarbonylJ Tos represents tosyl, 2-Br-Cbz represents 2-bromobenzyloxycarbonyl, Bzl represents benzyl, and Dnp represents 2,4-di-nltrophenyl. The abbreviation~ used herein for the varlou~ amino aclds are Arg, ~ AHP-6505/6513/6639 'l 06748~
arginine; Gly, glycine; Hi5, histidine; Leu, leucine; Pro, proline;
(pyro)-Glu, 5-oxoproline ~pyroglutamic ~cid); Ser, serine; Trp, tryptophan; and Tyr, tyrosine. All amino acids described herein are in the L-series unless stated otherwise, i.e.~ D-Ser is a D-seryl residue, D-Leu is a D-leucyl residue, D-Phe is a D-phenylalanyl 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 salts are those Wit]
organic acids, e.g. acetic, lactic, succinic, benzoic, salicylic, methanesulfonic or toluenesul~onic 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 sal~, 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., Helv. Chim. Acta, 43, i349 (1960). Suitable ion exchange resins are cellulose based cation exchangers, for example carboxy-~ methylcellulose or chemically modified, cross linked dextran cation ex-changers, for exarnple, those of the Sephadex* C-type, and strongly basic anion exchange resins, for example those listed by J.P. Greenstein and M. Winitz in "Chemistry of the Amino Acids", John Wiley and Sons, Inc., New York and London, 1961, Vol. 2, p. 1456.
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g_ A~IP-6505/6513/6639 :1067487 The peptides of formula 1 and their salts possess valuable, long-acting LH- and FSH-releasing hormone activity.
The valuable LH- and PSH-releasing hormone activity and long-acting property of the compounds of this invention are demonstrated by standard pharmacological procedures. For example, these activities can be demonstrated by tests described by A. Arimura, et al., Endocrinology, 95, 1174 (1974). For example, by following the procedure described therein, LH-release data, obtained from rats given a dose (50 ng, subcutaneously) of the compound, show that the peptides of formula 1 reach pea~ activity at about two hours after dosing and th~t significant activity is still present for up to six hours; whereas after the same dose of LH-RH ~50 ng, subcutaneously), peak activity is raached at about the 15 minute mark and no effects of the injection are observed after one hour. Also integrated levels of LH over a six hour per;od indicate that the compounds of formula 1; (D-Ser4,D-Leu6jdesGly-NH210)-LH-RH ethylamideJ (D-Phe~)-LH-RH
and (D-Trp6)-LH-RH are 21 times, 20 times and 12 times, respectlvely, more active in releasing LH than LH-RH.~ FSH-release data following injections of the compounds of formula 1 indicate tha~:
(D-Ser4~D-Leu6,desGly-NH210)-LH-RH ethylamide, ~D-Phe5)-LH-RH and ~D-Trp6)-LH-RH are about 11 times, 20 times and 20 times, respectively, more active than LH-RH at the same dose (50 ng).
Moreover, the activity of a compound of formula 1 is demonstrated in man:
Radioimmunoassay of serum levels of LH after intranasal administration indicates that the minumum effective dose of LH-RH
is about 2.0 mg whereas an equivalent degree of activity is obtained with a 0.5 mg dose of (D-Trp6)-LH-RH. In this instance the compounds are given ` AHP-6505/6513/6639 la67~s~
to humans in a normal saline solution. With regard to FSH release in man comparative studies with LH-RH and ~D-Trp6)-LH-~I give particularly noteworthy results. Intranasal administration of up to 2.0 mg of LH-RH has little or no effect on FSH serum levels as measured by radioimmunoassay, H.G. Dahlen, et al., ~lorm. Metab. Res. J 6, 510 (1974); however, at 0.5 mg under the same conditions (D-Trp6~-LH-RH
releases slgnificant amounts of FSH, i.e., levels ranging from 0.3 to greater than 1.5 miu/ml. It will be readily appreciated that a compound that is able to release FSH effectively has many therapeutic applications; see, for example, ~.G. Dahlen, et al., cited above.
The LH- and FSH-releasing properties of the peptides of formula 1 which in turn induce ovulation in animals, make the peptides useful in veterinary practive and in animal husbandry is often desirable to synchronize estrus in livestock, for example, cattle, sheep or swine, either in order to be able to mate all the females in a given group with a male of the desired genetic qUallty3 or so as to be able to perform .
artificial insemination on a maximum number of females, both within a comparatively short period of time. In~the past, this has been done by admlnistermg to the~anlmals an bvulatlon-lnlllbiting agent, withdrawing administration of said agent shortly before the date chosen for mating or artificial insemination, and rèlying elther upon the natural pro-duction of LH and FSH to induce ovulation and to produce estrus or by administerlng gonadotrophins. However, this procedure w~s~not entlrely sat-isfac~ory because ovulation at a predetermined~time occurred never ln all ~25 the animals together but only In a certaln proportion thereof when gonadotro-phins were not used. On the other hand, the high cost of gonadotrophins ~ .
side effects encountered in their administration made this method impractical.

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1(~67487 AHP-6505~6513~6639 It is now possible to obtain substantially complete synchronization of ovulation and of estrus, by treating the animals ln a given group ~irst with an ovulation inhibitor whlch is subsequently withdrawn, and then administering a peptide o~ formula 1 shortly before the predetermined , S period of time for mating or artificial insemination, so as to obtain ovulation and estrus within that time ~nterval. The delay in the onset of ovulation and estrus following administration of the peptide varies with the species of animals, and the optimal time interval has to be chosen for each species. For examplel in rodents such as rats or hamsters ovulation :
takes place within 18 hours following administration of a peptide of th.is invention.
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The method described above for obtaining ovulation and estrus w~thin~a precisely~predetermlned time~ mtervsl, so as to be certaln of a~
successful~ mating, is partlcularly~lmportant~ for breeders of race horses 15~ and of show animals, where the fees paid~for;the services~of an exceptional male~animal often amount to very conslderable;~sums of money.
The peptides of formula 1 are~also useful to inCrease the number of live blrths per pregnancy ln livestockl for exampl0l cattle, sheep or : .
swine.~ For this purpose the peptide is given ln a series of~p~renteral doses,~ preferably by intravenous of subcutaneous in~ectîonsl~ in the~;range of 0.1 - 10 mcg. per kilogram of body weight per dayl~96 to l2 hours .
prior to expected estrus and subsequent mating. A priming injection of 1000 to 5000 iu of pregnant mares serum~gonadotrophin may also be given one to four days prior to the above injection of the peptide.
A similar treatment, with or without prior primlng, is also useful for inducing puberty in farm animals.
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A}IP-6505/6513/~639 10674~7 When a peptide of formula 1 is employed for the purpose of inducing ovulation and estrus or for inducing puberty in warm-blooded animals~
especially in rodents such as rats or hamsters or in livestock, it is administered systemically, preferably parenterally, in combination with a pharmaceutically acceptable liquid or solid carrier. The proportion of the peptide is determined by its solubility in thc given carrier, by the chosen route of administrationJ and by standard biological practice. For parenteral administration to animals the peptide is used in a sterile aqueous solution which may also contain other solutes such as buffers or preservatlves, as well as sufficient pharmaceutically acceptable salts or glucose to make the solution isotonic. The dosage will vary with the ;~ form of administration and with the partlcular species o~ animal to be treated and is preferably kept at a level of from 0.1 mcg. to 10 mcg. per kilogram body weight. HoweverJ a dosage level in th0 range o$ from about 1 mcg. to about 5 mcg. per kilogram body weight is most desirably employed in order to achieve effective results.

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The peptides of formulal can also~be administered in one of the long-actingj slow-rel~ase or depot dosage forms described below, pre-ferably by intramuscular in~ection or by lmplantation. Such dosgage forms are designed to release from about 0.1 mcg. to about 10 mcg. per kilo-gram body weight per day.
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0 6 7 ~ 8t7 AHP-6505/6513/6639 The peptides of ormula 1 are also useful in human medicin0.
For example, human chorionic gonadotrophin CHCG) which contains mainly LH and some FSH has been used or over 30 y0ars to treat certain endo-crino~ogical disorders such as disturbances of the cycle, amenorrhea, lack 5 o~ development of secondary sex characteristics, an~ infertility in the female, or certain cases of hypogonadism, delayed puberty, cryptorchidismJ
and non-psychogenic impotence in the male. Lately, infertility in the human female has also been treated with~human menopausal gonadotrophin HMG) whIch contains mainly FSH, followed by treatment with HCG. One of the disadvantages of the treatment of infertility in the human female ~ , ~
with HCG or with HMG followed by HCG has become apparent in that such treatment often results in superovulation and unwanted multiple births, probably because of the impossibility of glving only the exact amoImts ~.
of FSH and~LH -~hich are necessary for ovulation. ~;The adminIstratlon of~
~ a peptide of this invention overcomes the~above dIsadvantage~because;
the compound causes~re~iease of~LH and FSH by the~pituitary only in the :xact quantitite5 which~are required~ for normaI QVUlatiQTI. ~For~that reason a peptide of this~Invention is not only useful for the above ~purpose, but it is equally useful in the human female in the treatment o dlsturbances of the cycle, of amenorrhea, of ~hypogon dism, and~of lack of developm:nt of :econd:ry sex char:ct:ristics~

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1~167487 Furthermore, 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 released at that time and causes premature ovulation. The immature ovum is either not capable of being fertilized, orJ if ~ertilization should neve~theless have taken place, it is highly unlikely that the fertilized ovum will become implanted because the estrogen-progestin balance required to prepare the endometrium IS not present and the endometriu~ is not m the condition necessary for implantation. On the other hand, when the peptide is IO administered towards the end of the cycle the endometrium isdis~upted and menstruation takes place. ~ ~
In addition, the peptides of thls invention are also useful in contraception by the "rhythm" method, which ha.c always been 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 short~ly the~reafter and~makes~the~"rhythm"~
method both safe and effectlve. ~ ;

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1067~87 A~IP-6505/6513/6639 The peptides o~ formula 1 are useful as a diagnostic ~ool for distinguishing betwèen hypothalamic and pituitary malfunctions or lesions in the human female. When administering the peptide to a patient suspec~ed of such malfunctions or lesions and a rise in ~he l~vel o LH is subsequently observed there i5 good Indication to conclude that the hypo-thalamus is the cause of the malfunction and that the pituitary is intact.
On the other hand, when no rise in circulating LH is seen foIIowing the administration of the peptide a diagnosis of pituitary malfunction or lesion can be made with a high degree of confidence.
In the hum:an male, administration of a peptide of armula 1 provides the amounts o LH ~or ICSH) and of FSH necessary ~or normal sexual development in cases of hypogonadism or delayed puberty~ and is also useful in the treatment o cryptorchidIsm. Furthermore, the FSH
released by the administration o the peptide stimulates the development I5 of germinal cells in the testes, and the peptide :i5 useful in the treat-ment of psychogenic~and non-psychogenic impotence.
When the peptides o ormula 1, preferably in the orm of an acid addi~tion salt, are employed in human medicineJ they are administered systemically, either by intravenous, subcutaneous, or intramuscular injection, or by sublingual, nasal, or vaginal administrationj in com-positions in conjunction with a pharmaceutically acceptable vehicle or carrier. ; ~ ~ ~
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~ AHP-6sos/6513/6639 ~or administrat~on by tho nasul routo as:drops or spray it is preferred to use a peptide o formula 1 in~solution in a sterile aqueous vehicle which may also contain other solutes such as buffers or preserva-tives, as~well as sufficient quantlties of pharmaceutically acceptable .
S~ salts or of glucose to make the solution isotonic. Doses by the intra-nasal route range from 0.1 to 50 mcg/kg, or preferably 0.5 to 10 mcg/kg.
; ~ The peptides o~ formula 1 may also be admlnistered as nasal o~ vaginal powders or insu~flations. For such purposes the peptide is ~adminlstered in finely divided solid form together with a pharmaceutically~
o acceptable solid carrier, for example a finely divided polyethylene glycol ("Carbowax* 1540"), finely divided~lactose, or preferably for vaginal ~ ~
administration, very finely divided silica ("Cab-O-Sil"*). Such COmpQSitiOnS
may also contain other excipients in finely divided solid form such as ~ preservatives, buffers, or surface active~agents.
For sublingual or vaglnal admlnlstration it is preferred to formulate the;peptides of formula li m solld~dosage forms~;such as sublingual tab}ets or~vaginal inserts or supposltorles~with~sufflcient quantities of solid excipients such as starch, lact~ose, certain types of clay, buffers and lubrl~cating,~dls mtegratlng, or surface-active~agents, or wlth semi-~ solid~excipients commonly;used in~the formulatl~on of suppositories.
Examples of such excipients are found in standard pharmaceutlcal texts, e.g. in Remington's Pharmaceutlcal~ Sciences, Mack Publishing Company, Easton, Pa., 1970.

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1067487 ~P-65~5/6513/6639 The dosage of the peptides of formula 1 will vary with the form of administration and with the particular patient under treatment.
Generally, treatment is initiated with small dosages substantially less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum efect under the circum-stances is reached. In general, the peptides of formula I are most de-sirably administered at a concentration level that will generally afford effective release of LH and of FSH without causing any harmful or deleterious side effects, and preferably at a lev81 that is in a range of from about 0.01 mcg. to about 100 mcg. per kilogram body weight, although as aforementioned variations will occur. However, a dosage level of the peptide of formula 1 in which a) X is Ser, Y is D-Trp and Z is Gly-N}12 or b) X is Ser, Y is D-Phe and Z is Gly-NH2 that is in the range of from about 0.5 mcg. to about 5.0 mcg. per kilogram body weight and the peptide of formula 1 iD which c) X is D-Ser, Y is D-Leu and Z is NHR
that lS in the range of from about 0.1 mcg. ~o about 10 mcg. per~kilo-gram body weight is most desirably employed to achieve effective results.

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A~lP-6505/6513/663g 1(~674~7 It is oftell desirable to administer the peptides of formula 1 continuously over prolonged periods of time in long-acting, slow-releasa, or depot dosage orms. Such dosage forms may either contain a pharma-ceutically acceptable salt of the compound having a low degree of solubility in body fluids, for example salts with pamoic or tannic acid or carboxymethylcellulose, or they may contain the peptides in the form of a water-soluble salt together w1th a prot0ctive carrier which prevents rapid release. In the latter case, for example, the peptides may be formulated wi~h a non-antigenic 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 pro-.
tamine, and may be administered in suspension in a pharmaceutically acceptable liquid vehicle; or the peptides may be formulated in gels or suspensions with a protective non-antigenic hydrocolloid, for example sodium carboxymethylcellulose, polyvinylpyrrolidone, sodium alginate~
gelatine, polygalacturonic acids, for example, pectin, or certain muco-` polysaccharides, together with aqueous or non-aqueous pharmaceutically acceptable liquid vehicles, preservatives, or surfactants. Examples of such formulations are found in standard pharmaceutical texts, e.g. in Remington's Pharmaceutical Sciences, cited above. Long-acting, slow-release preparations of the peptides may also~be obtained by microen-capsulation in a pharmaceutically acceptable coating material~ for example gelatine, polyvinyl alcohol or ethyl cellulose. Further examples ; of coating materials and of the processes used for microencapsulation are described by J.A. Herbig in "Encycloped1a of Chemical Techno1Oey", Vol. 13, ~ ~067~87 AHP-6505/6513/6639 2nd Ed., Wiley, New York, 1967, pp. 436-456. Such formulations, as ~; well as suspensions of salts of the peptides which are only sparingly soluble in body fluids, ar0 designed to release from about O.l mcg to about 50 mcg of the hormone per kilogram body weight per day, and are preferably adminlstered by lntramuscular injection. Alternatively, some of the solid dosage forms listed above, for example certain sparingly water-soluble salts or dispersions in or ~dsorbates on solid carriers of salts of the pept~ides, for example dispersions in a neutral hydro~el ;of a polymer of ethylene glycol methacrylate or similar monomers cross-l mked as described in U.S. Patent 3,551j556 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 subcùtaneously or intramuscularly.
Alternatively, slow-release effects over~prolonged~periods ~ : , of time may also~be~obtalned by admlnist~ermg the peptldes of thls mventlon~as an acid addltion salt in an~ mtra-vaginal device or~in a ~ ~ ;
temporary~implant, for~exampie a container~made of a non-irritatin ; silicone~polymer such~as a polysiloxane, e.g. "Silastic"*, or of a neutral hydrogel of a polymer as described above, possessing the 20 required degree of permeability to release from about O.l mcg. to about ~;
50 mcg per kilogram body weight per day. Such intra-vaginal or implant : , . ~
dosage forms for prolonged adminis~tration have the advantage~that they ; may be removed ~hen it is desired to interrupt or to terminate treatment.

*Trademark :
~ -20-1~)67~8`7 ~1P-6505t6513/6639 Process In selecting a particular side chain protective group to be used in the synthesls of the present peptides of fo1~ula ~ the following rules should be followed: (a) the protectlve group must be stable to the reagent and under the reaction conditions selected ~or removing the a-amino protective group at each~step of 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 protec~ive group must be rPmovable upon the completion of the synthesis containing the desired amino acid sequence under reaction conditions that will not alter the peptide chain.
With reference to R9, suitable a-amino protective groups include (l) aliphatic urethan protective groups illustrated by t-butyloxycarbonyl, diisopropylmethoxycarbonyl, biphenylisopropyloxycarbonyl, isopropyloxy-carbonyl~ t-amyloxycarbonyl,~ethoxycarbonyl,~allyloxycarbonyl; ~2j cyc1O-alkyl urethan type protective~groups illustrated by cyclopentyloxycarbonyl,~
adamantyloxycarbonyl, d-isobornyloxycarbonyl, cyclohexyloxycarbonyl;
nitrophenylsulfenyl, tritylsulfenyl, a,-dimethyi-3,5-dimethoxybenzyloxy-carbonyl and trityl. The preferred -amino protective group for R9 is selected from the group consisting of t-butyloxycarbonyl, cyclrpentyloxy-carbonyl, t-amyloxycarbonyl, d-isobornyloxycarbonylj o-nitrophenylsulfenyl, biphenylisopropyloxycarbonyl, and a,a-dimethyl-3,5-dimethoxybenzyloxycarbonyI.

, ~: :
:: :

'`:

The peptides of formula 1 of this invention are prepared using solid phase synthesis which will be illustrated by the following cmbodiments in which speci~ic peptides of formuIa 1 are prepared.
b) Peptides of formula 1 in which ~a) X is Ser, Y is D-Trp and Z lS Gly-NH2, or - -- :
(b) X is Ser, Y is D-Phe and Z is Gly-NH2.

The solid phase synthesis of the peptides of formula 1, described above under a and bJ is commenced from the C-terminal end of the peptide using an ~-amino protected~resin. ~Such a starting material is prepared by attaching an a-amino protected glycm e to a benzhydryla:ine ; resin, a chloromethylated resin or a hydroxymethyl resin, the former being preferred. The preparation of a benzhydrylamine resin is described by P. Rivaille, et al., Helv. Chim. Acta, 54, 2772 (1971) and the pre-15~ ~paratlon of the hydroxymethyl res m~ls described by~M. Bodanszky~andJ.T. Sheehan, Ghem. Ind (London)~38,~1597 ~(1966).~ A chloromcthylated~
resin is~commerclally avallable~from Blo~Rad~Laboratorles~, Rlchmond,~
Cal~lfarnla.~ In~uslng the b~enzhydrylam me~res m ~an;amide anchoring bond is formed~with~the ~-amino protected glycinè as follows:

20 ~ d ~ ~ ;
R _ N - ch2 _ _ N

I'his p:er its the C-terminal a:lde functlon~to be obta med directly after the amino acid sequence m the synthesis lS completed by 25~ cleaving off the resin support~of the linked~peptlde to form the glycine amide at thé C-terminal portion of the desired decapeptide. In this instance the use of hydrogen fluoride for cleaving off the resin support also removes the side chain protective groups to give the correspondlng ` A~IP-6505/6513/6639 : ~' decapeptide of ~ormula 1 in which (a~ X is Ser, Y is D-Trp and Z is Gly-NH2, or (b) X is Ser, Y is D-Phe and Z is Gly-NH2.
When the other resins are used, the anchoring bond is the : :
5 benzylester group as illustrated hereinbefore. In this instance a .:
convenient procedure for converting the linked protected peptide to the C-terminal amidc is to a=ono1ize the protected~peptide off the resin and ~: : :
then remove~he protective groups of the resulting amide by treatment with~sodium and~liquid ammonia or bY hydrogen n uoride cleavage. An lO ~ alternative procedure would be to cleave by transesterification with a lower alkanol, preferably methanol or ethanol, in the presence of trie~hylamine and then convert the resulting ester into an am1de and subsequently deprotect as described above. See also J.M.~Steward and J.D. Youn~
;~ ~ "Solld Phase~ Peptlde Synthesls", W.H.~ Freeman~ Co., San Franc1sco, 1969, ~15 pp. 40 49 lore spe~clfica11y, In an émbadlment~of the present mventlon an~`-amino protecting g1ycm e, preferab1y~;t-butyloxy-carbonylglyclne, is coupled to benzhydrylamine resin with the aid of the carboxyl group , activating compound, preferably, dicyclohexylcarbodiimide.; Following the ; 20 coupling of the ~-amino protected glyc me to the resin support, the ~-amlno protectlng group~is removed such as by us mg trifluoroacetic ac1d m~methy1ene chloride~trifluoroacetic acid~alone or hydrochloric acid in dioxane. The deprotection ls carr1ed aut at~a temperature between 0C and room~temperature.
Other standard cleaving reagents and~conditions for removal of speoifio :a-amimo ~25 protectlng~groups may be used as described by E. Schroder and K. Lubke, "The Peptides"~ Vol.. l, Academic Press, New York, 1965, pp. 72 - 75.~ ;
: : ~ :
`: : : : : : : : :

~: ~

.

A~P-6505t6513/6639 1~674l37 After removal of the ~-amino protecting group,-the remaining a-amino protected amino acids are coupled step-wise in the desired order to ~btain the peptide. Bach protected amino acid is introduced into the solid phase reactor in about a three-fold excess and the coupling is carried S out in a medium of methylene chloride of mixtures of dimethylformamide in methylene chloride. In cases where incomplete coupl1ng occurred the coupl mg procedure is repeated before removal o~ 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 described by E. Kaiser, et al., Analyt. Biochem. 34, 595 ~1970).
After the desired amino acid sequence has been synthesized, the peptide is removed from the resin support by treatment with a reagent such~as hydrogen f1uoride which not only cleaves the pept1de from the res1n but also cleaves all remaining side chain protecting groups and the ~-amino protect mg group ~if present) on the pyroglutamic acid `~
residue in the case where the benzhydry~lamine resin was used to obtain ~, directly the peptide of formulal, described above as (a) or (bj. ~ ~

:

~: , /6~ /C~4 ' Where a chloromethylated resin is used the~pepttde may be separated from the resin by transesferification with a lower .
~Ikanol, preferably methanol or ethanol, after wh1ch the recovered product is chromatographed~on sil1ca gel and the collected fraction 5~ ~ subjected to treatment with ammonia to convert the lower alkyl es~er, preferably the methyl or ethgl ester, to the C--~erminal am1de. The slde chain protecttnc groups are then cleaved by procedures described above, for exampie by treatment with sodiu~ in liquid ammonia or by hydrogen fluoride.
~'~ In~addition to the above described protecting groups (R7) for the imida701e nitrogen~atoms of histidine,~R7 can include 2,2,2-trifluoro-1-benzoyloxycarbonylami~noethyl and 2,2,2-trifluoro-1-t-butyloxycarbonylaminoethyl.
; C2 Peptide of formùla l_iQ which ;~
'5 ~ c) X is D-Ser, Y is D-Leu and Z i;s NHR
The solid phase synthesis of the peptide of formula I in which X is D-Ser, Y Is D-Leu~and Z is NHRI is commenced from~
~; the C-terminal end~of the peptide using an~-amino protected proline resin.~ Such a starting~mater~ial is prepare;d by attaching ~20~ an a-amino protected proline to a chloromethylated resin or a ~ hydroxymethyl resin, the former being preferredO The preparation ~i ; ~ of a hydroxymethyl resin is described by M. Bodansky and J.T. Sheehan, Chem. Ind. (Londonj 38, 1597 t1~466). A chloro-methylated resin is commercially available from Bio Rad Laboratories, Richmond, California. In using the~ chloromethy-lated resin an ester anchoring group is formed with the ~-amino protected ~ e as foilows:
9 esin R -Pro-O-CH2 - i~ppor AHP-65~C)/6517,/6~39 ~L06 74~3 7 A convenient procedure for converting the linked protected . .
; peptide to the C-terminal (lower alkyl)amide consists in cleaving ~he protected peptide off the resin by treatment with a lower alkylarnine, cf, D.H. Coy, et al., Biochem., Biophys. Res. Commun., 57~ 335 (1974), to 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 1, described above as(c~ of this invention.
An alternative procedure consists in cleaving by transesterification with a Iower 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 deprofect as descrlbed above. See also J.M. Stewart and J.D. Young, "Solid Phase Peptide Synthesis", W.H. Freeman 1 Co., San Francisco, 1969, pp. 40-49.

More speci~ficaily, in an embodimen~ of the present Invention ~-amino protected proline, preferably t-butyloxy~
carbonylproline, is coupled to a chloromethylated resin with the aTd of a catalyst, preferably, cesium bicarbonate or triethylamine.
Following the coupling of the a-amino protected proline to the resin support, the a-amino protecting group is removed , for example by us7ng trifluoroacetic acid in methylene chloride, trifluoro-acetic acid alone or hydrochloric acid in dioxane. The deprotection is carried out at a temperature between about 0C
and room temperature. Other standard cleavlng reagents and conditions for removal of specific a-am7no protecting groups may be used as described by E. Schroder and K. Lubke, "The Peptides", Vol. 1, Academic Press, New York, 1965, pp. 72-75.
After removal of the a-amino protecting group, the remaining a-amino protected amino acids are coupled step-wtse in the ~06 748 7 AHP_6505/6513/6639 desired order ~o obtain the compound of formula 1 described above under (c). Each protected amino acid is introduced into the solid phase reactor in about a three-fold excess and the coupling is carried out in a medium of methylene chloride or mixtures of dimethylformamide and s methylene chloride. In cases where incomplete coupling has occurred the coupling procedure is repeated before removal of the -amino protecting group, prior to the coupling of the next amino acid to the solid phase reactor. The succeis o~ the coupling reaction at each stage of ~ the synthesis is monitored by the ninhydrin reaction as described by E. Kaiser, et al., Analyt. Biochem. 34, 595` (1970).
After the deslred amino acld sequence has been synthesl~ed, the protected peptide is removed from the resin support by treatment with a (lower alkyl)amine ta give the corresponding protected peptide tlower alkyl~amine and in the case where dinitrophenyl or tosyl has been used as the protecting group for the histidyl residue, the dinitrophenyl or tosyl~protect m g group also lS removed during the treat-ment wlth the (lower al bl)amlne. The~peptide may also be separated from the resin by transesterification wlth a lower alkanol, preferably methanol~or ethanol, a~ter which the recovered product is puri~ied by chromatography on silica gel and the collected fraction subjected to treatment with a (lower alkyl)amine to convert the lower alkyl ester, preferably the methyl or ethyl ester, to the C-terminal (lower alkyl) amide. (Note that the dlnitrophenyl or tosyl group, if present on the histidyl residue, will also be cleaved). The remaining side .
` 25 chaln protecting groups of the protected ~lower;alkyl)amide are 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 1 described above as ~c).
The following examples illustrate further this invention.

~` ~ AHP-6505/6513/6639 ~067~87 EXAMPI.~ 1 L-P~ro~lutamyl-L-histidyl~tosyl)-L-tryptophyl-L-seryl(benzyl)-L-tyrosyl~2-bromo-benzyloxycarbonyll~D-tryptophyl-L-leucyl-L-arginyl(tosyl~-L-prolylglycylbenzhydrylamine resin (R8-~pyro~-Glu-His-~Nlm-R7)-Trp-5er(R6)-Tyr(Rs~-D-Trp-Leu-Arg(N -R ~-Pro-Gly-A; R4 = Tos, R5 _ 2-Br-Cbz~ R6 _ Bzl, R7 - Tos, R8 = ~ and A _ benzhydrylamine resin).
Benzhydrylamine resin (1.25 g, l.0 mmole) is placed ~n the reaction vessel of a Beckman Model 990 automatic peptide synthesizer programmed to carry out the following wash cycle: (a) methylene chloride;
tb) 33% trifluoroacetic acetic in methylene chloride ~2 times for 2.5 and 25 minutes each); ~c) methylene chloride; (d) ethanol; (e) chloro-form; (f) 10% triethylamine in chloroform (2 timos for 25 minutes each);
(g) chloroform; (h) methylcne chloride.

The washed resin is then stirred with t-butyloxycarbanyl glycine (525 mg, 3.0 les) 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 t3 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 and 25 minutes each and then steps (c) through (h) as described in the above wash cycle are performed.
The following amino acids (3.0 mmoles) are then coupled successively by the same cycle of events: t-Boc-L-proline;
t-Boc-L-arglnine(Tos); t-Boc-L-leucine; t-Boc-D-tryptophan;

~:

` ~06748~ A~IP-6505/6513/6639 t-Boc-L-tyrosine t2-Br-Cbs); 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 ben~hydrylamine resln used in this example is a commercially available resin (1% cross linked, Bachem Inc., Marina del Rey, California).

L-Pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-tryptophyl-L-leucyl-L-arginyl-L-prolylglycinamide; 1, X - Ser, Y , D-Trp and Z = Gly-NH~ ((pyro~Glu-Hi`s-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH
Removal of protect mg groups and cleavage of the decapeptide from the decapeptide resin, described in Example 1, is carried out by treatment of 1.0 g of material with hydrogen fluoride ~24 ml) and anisole ~ t6 ml) at 0C for 30 minutes. The hydrogen fluoride is removed under reduced pressure and tha anisole removad by washing with ethyl acetate.
The crude peptide is purifled by gel filtration on a column (Z.5 x 100 cm) of Sephadex* G-25 (a nDe grade, chemically modlfied cross-l mked dextran) by elution with 2 molar acetic acid and fractions shown to contam a major UV absorption peak at 280 nm were pooled and - evaporated to dryness.
The residual oil was applied~to a column (2.5 x 100 cm) of Sephadex* G-25 (fine), prevlously equllibrated with the lower phase followed by the upper phase of n-butanol:acetic acid:water (4:1:5) solvent system. Elution with the uppér phase gives a major peak fractlon, * Trade mark :

10~7487 and material from this area was subjected to elution on a column ~1.4 x 94 c~) of carboxylnethyl cellulose according to the conditions described by D.H. Coy, et al.~ J. Med. Chem., 16, 1140 (1973). Appropriate fractions (1050 - 1190 ml), aft~r lyophilization to constant weight, S gave D-Trp6-LH-~1 as a white, 1uffy powder (80 mg); (~) D -58.8 (c - 0.33, lN HOAc).
The product was homogeneous by thin layer chromatogr?hy in four separate solvent systems when loads of 20-30 mcg were applied and spots visualized by exposure to lodine vapour followed by Ehrlich reagent. The following Rf values were obtained:
:: :
I-butanol. acetic acid:wàter ~4:1:5: upper phase), 0.25;
ethyl acetate: pyri~dine: acetic acid: water ~5:5:1.3), 0.63; 2-propanol:
1 M acetic acid (2:1), 0.38;1-butanol: acetic acid: water: ethyl acetate (1:1:1~:1), 0.51.
Amino acid analysis gave:~Glu, 1.08; His, 0.95; Trp, 2.00;
; Ser, 0.94; Tyr, 0.97; Leu, 0.93; Arg, 0.98; Pro, 1.00; Gly, 1.02;
~NH3, 1.03- ~ ~;

'~
, _30 ~ ~

:: :

: ~ :
:

.

- AHP_6505/6513/G639 106748~

... .
L-Pyroglutamyl-L-histidyl(tosyl)-L-tryptoph L-seryl(benzyl)-L-tyrosyl(2-bromo-ben~yloxyc-rbonyl)-D-phenylalan L-leucyl-L-arginyl(tosyl)-L-prolylglycylbenzhydrylamine resin (R -(pyro)-Glu-His-(Nlm-R7)-Trp-Ser(R6~-TyrtR5)-D-Phe-Leu-Arg(NG- 41-Pro-Gly-A; R4 . Tost R5 ~ 2-Br-Cbs~ R6 _ Bzl, R7 _ Tos, R8 = H and A benzhydrylamine resin?.
Benzhydrylamine resin (1.25 g, 1.0 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 chlor:ide; td~
ethanol; (e) chloroform; (f) 10% triethylamine in chloroform (2 times for 25 minutes each); (g) chloroform; (h) methylene chloride.
The washed resln is then stirred with t-butyloxycarbonyl glycine (525 mg, 3.0 mmoles) in methylene chloride and dlcyclohexyl-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 ~mino acid is deprotected with 33% trifluoroacetic acid in methylene chloride (2 times for 2.5 and 25 minutes each and then steps (c) through (h) as described in the above wash cycle are performed.
The following amino acids (3.0 mmoles) are then coupled successively by the same cycle of events: t-Boc-L-p~oline; t-Boc-L-arginine(Tos); t-Boc-L-leucine; t-Boc-D-phenylalanine; t-Boc-L-tyrosine-(2-Br-Cbz-~oc-L-serine(Bzl); t-Boc-L-tryptophan; t-Boc-L-histidine(Tos);
L-(pyro)-glutamic acid.

:: ~067~37 The completed decapeptide resin is washed with methylene chloride ~3 times~ followed by methanol ~3 times) and dried under reduced pressure whereupon 100~ o~ the theoretical weight gain is obtained.
S The benzhydrylamine res.in used in this example is a commerciallyavailable resin ~1% cross linked, Bachem Inc., Marina del Rey, California).
~XAMPLE 4 L-Yyroglutamyl-L-histldyl-L-tryptophyl-L-seryl-L-tyrosyl-D-phenylalanyl-L-leucyl-L-arginyl-L-prolylglycinamide;
1 X~,~Ser, Y ~ ~-Phe and Z ~ GIy-NH ((pyro)-Glu-His-Trp-Ser-Tyr-D-Phe-Leu-Arg-Pro-Gly-NH

Removal of protécting groups and cleavage of the decapeptide from the resin described in Exampl~e 3 lS carried out by treatment of 1~.5 g of material with hydrogen 1uoride (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;gel filtration on~a column (2.5 x 100 cm~ of Sephadex~ G-25 ~a fine grade, chemically modiied cross-llnked dextran)~by elotion with 2~molar acetlc acid and fractions ~20 shown to contain a major W absorption peak at 280 nm were pvoIed~and evaporated to dryness. ~ ~
The residual oil was applied to a column (2.S 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.
;; ~2S Elution with the upper phase gives a major peak~ fractionand fractions from this peak were pooled and concentrated to dryness. The residue was * Trade mark .

lyophilized from 0.2 N acetic acid to give ~D-Phe )-LH-RH as a fluffy white powder (158 mg); ~?25 -57.5C ~c - 0.55, 0.1 N HOAc).
The yroduct was homogeneous by thin layer chromatography in four separate solvent systems when loads of 20-30 mcg were applied and spots visualized by exposure tO iod.ine vapour followed by ~hrlich reagent. The following Rf values were obtained.
l-butanol: acetic acid: water ~4:1:5: upper phase), 0.14;
ethyl acetate: pyridine: acetic acid: water ~5:5:1:3), 0~68; 2-propanol: 1 M acetic acid (2:1), 0.41; l-butanol: acetic acid:
10 ~ water: ethyl acetate (1:1:1:1), 0.47.
Amino acid analysis gave: Glu, 1.01; His, 0.97; Trp, 0.90;
Ser, 0.92; Tyr, 1.00; Phe, 0.96; Leu~ 1.00; Arg, 1.02; Pro, 0.95;
Gly, 1.00; NH3, 1.00.

:
.
.

' Al~P-65()~5/651316639 ', . ` ~0674~7 L-Pyroglutamyl-L-hi ~ y~(dinitror~henylj.-L--trYptophyl-D-servl~ben7yl~-L-tyrosyl(~-bromobenzyloxycarbonyl)-D-leucyl-L-lcucyl-L-arginyl~tosyl)-L-prolyl-o-cll2-resin~ R -(pyro)-Glu-~lis(N m-R7)-Trp-D-Ser~R6)-; 5 Tyr(R5)-D-Leu-Leu-Arg(NG-R4)-pro-Al;~R4 ~ , R5 _ 2-Br-Cb~, R6 _ Bzl, R : Dnp, R8 _ ll and Al - 0-CII - ~rosin ; ~suppory ~ Boc-Proline resin of the formula Boc-Pro-0-C1l2 - ~resln tl-40 g, 0.5 mmole of proline) lS ' ~ .
: placed in the reaction vessel of a Beckman Model 990 automatic :
peptide synthesizer programmed to carry out the following wash cycle: (a) mcthylcnc chloridc; (b) 33% trifluoroacctlc in mcthylcnc chloride ~2 times for 2.5 and 25 minutes each); (c) methylcne chloride;
(d) ethanol; (e) chloroform; ~f) 10% trlethylamine in chloroform (2 times for 5 minutes each); (g) chloroEorm; and (h) methylene - .
lS chloride. ~ - u - ~ Thc washed resin is then stirred with t-butyloxycarbonyl-tosyl. arginine ~645 mg, 1.5 mmoles3 in methylene chloride and ~~ ~dicyclohexylcarbodiimlde (1.5 mmole~) is added. The mixture is stirred at room temperature (22-25~C) for 2 hours an~ the amino .
20 acid resin is then washed successlvely wlth~methylene chloride (3 ~;
times). The attached amino acid is deprotected with 33% trifluoro-acctic acid in methylene chloridc (2 tlmes for 2.5 and 25 minutes each - ~ ~ and then steps (c) through (h) as described in the above wash cyclc are performed. ~
~ The following amino acids ~l.S mmoles) are then coupled - successively by the same cycle of evcnts: t-Boc-L-lcucine; t-~oc-D-leucine;

~ :

~C)6~4~

t-Boc-L-tyrosine (2-Br-Cbz); t-Boc-D-serine~Bzl); t-Boc-L-tryptophan;
t-Boc-L-histidine~Dnp); L-~pyro)-glutamic acid.
The completed nonapeptide resin is washed with methylene chloride ~3 times) followed by methanol (3 times) and dried under reduced pressure whereupon 96% of the theoretical weight gain is ob~ained.
The proline resin used in this example is made from a commercially available chloromethylated resin ~1% cross linked, Bio Rad Labs, Richmond, California~.

.

.

.

~106~7487 E.YA~IPLE 6 L-Pyroglutamyl-L~histidyl~ tryPtophyl-D-seryl(benzyl)~
tyrosyl~2-bromobenzyloxycarbonyl)-D-leucyl-L-leucyl-L-arginyl(tosyl)_ L-prolylethylamide, R -(pyro)-Glu-His-Trp-D-Ser~R6)-Tyr(R5)-D-Leu-Leu-Ar~tNG-R4)-Pro-N~IRl; R4 - Tos, R5 = 2-Br-Cbzj R6 _ Bzl, R8 =
H and R = C2H5 The protected nonapeptlde resin (2.16 g descrihed in Example 5) is suspended in ethylamine t20 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 silica gel in l-butanol: acetic acid: water ~4:1:5J upper phase) is 0.45. This material is used in Example 7 without further purification.

, , :

:

~ ~ AHP-6505/6513/6639 ~L~6~4~37 L-Pyroglutamyl-L-histidyl-L-tryptophyl-D-seryl-L-tyrosyl-D-leucyl-L-leucyl-B-arginyl-L-prolyl ethylamide;
1, X = Ser, Y ~ D-Leu and Z _ NHEt ((pyro)~lu-His-Trp-D-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt) Removal of protectlng groups from the protected nonapeptide~
prepared as described in Example 6, is carried out by treating 670 mg of the material with hydrogen fluoride (50 ml) and anisole (15 ml) at OC 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 100 cm) of Sephadex* G-25 ~a fine grade, chemically modified cross-linked dextran) by elution with 0.2 molar acetic acid and fractions shown to contain a major UV absorption peak at 280 nm are pooled and evaporated to dryness.
The residual oil i5 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-bu~anol; acetic acid; water (4:1:5) solvent system. ~lution with the upper phase gives a major fraction wlth high u.v. absorption at 280 nm and this material is subjected to chromatography on a column (1.5 x 94 cm) of silica gel and elution with a l-butanol:acetic acid: water ~4:1:1) mixture. Appropriate fractions ~300 - 390 ml), after evaporation and lyophilization to constant weight ~ from water, give (D-Ser4,D-Leu ,desGly-NH210)-LH-RH ethylamide as a white, fluffy powder ~103 mg), (~) 3 _ -29.6~ (c _ 0.54, 0.1N HOAc).

~06~487 EXAMPLE 7 (Continued) The product is homogeneous by thin layer chromotography in four separate solvent systems on silica gel plates when loads of 20-30 mcg are applied and spots visualized by exposure to iodine vapour followed S by Ehrlich reagent. The following Rf values are obtained.
l-butanol: acetic acid:water (4:1:5: upper phase), 0.20;
ethyl acetate: pyridine: acetic acid: water (5:5:1:3), 0.72; 2-propanol:
1 M acetic acid (2:1); 0.43; l-butanol: acetic acid: water: ethyl acetate ~1:1:1:1), O.S0.
Amino acid analysis gives: Glu, 1.03; His, 0.93; Trp, 1.01;
Ser, 0.82; Tyr, 0.97; Leu, 1.98; Arg, 1.00; Pro, 0.90; ethylamine, 0.98.

Claims (18)

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 selected for the group consisting of:
(A) a compound of formula R8-(pyro)-Glu-His(N1m-R7)-Trp-X1-Tyr(R5)-Y-Leu-Arg(NG-R4)-Pro-Z1 in which a) X1 is Ser(R6), Y is D-Trp and Z1 is Gly-R2; or b) X1 is D-Ser(R6), Y is D-Leu and Z1 is OR3 wherein R2 is selected from the group consisting of amlno and O-(lower alkyl), R3 is lower alkyl, R4, R5, R6 and R7 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 (l) 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, respectively; and R8 is hydrogen or one of said protective groups;
(B) 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, R4, R5 and R6 are protective groups capable of being removed by one or more chemical treatments which do not affect (pyro)-Glu-His-Trp-D-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHR1 wherein R1 is lower alkyl, and R8 is hydrogen or an .alpha.-amino protective group; and (e) a compound selected from the group consisting of R8-(pyro)-Glu-His(N1m-R7)-Trp-X1-Tyr(R5)-Y-Leu-Arg(NG-R4)-Pro-Z2, R9-His(N1m-R7)-Trp-X1-Tyr(R5)-Y-Leu-Arg(NG.-R4)-Pro Z2, R9-Trp-X1-Tyr(R5)-Y-Leu-Arg(NG-R4)-Pro-Z2 and R9-X1-Tyr(R5)-y-Leu-Arg(NG-R4)-Pro-Z2 in which a) X1 is Ser(R6), Y is D-Trp and Z2 is Gly-A; or b) X1 is D-Ser(R6), Y is D-Leu and Z2 is A1;
wherein 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 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, respectively, R8 is hydrogen or an .alpha.-amino protective group, R9 is an .alpha.-amino protective group, A is selected from the group consisting of:

, and which comprises:
subjecting a compound of the formula R8-(pyro)-Glu-His(Nim-R7)-Trp-X1-Tyr(R5)-Y-Leu-Arg(NG-R4)-pro-Z2 in which a) X1 is Ser(R6), Y is D-Trp and Z2 is Gly-A, or b) X1 is D-Ser(R6), Y is D-Leu and Z2 is A1 wherein 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 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 NHRI wherein R1 is lower alkyl, respectively, R8 is hydrogen or an .alpha.-amino protective group, R9 is an .alpha.-amino protective group, A is selected from the group consisting of:

, and to ammonolysis, or to trans-esterification with a lower alkanol followed by conversion of the resulting ester by amidetion into the corresponding amide to obtain the compound of formula R8-(pyro)-Glu-His(N1m-R7)-Trp-X1-Tyr(R5)-Y-Leu-Arg(NG-R4) Pro-Z1, as defined hereinbefore in section (A); or reacting a compound of the formula R8-(pyro)-Glu-His-(N1m-R7)-Trp-D-Ser(R6)-Tyr(R5)-D-Leu-Leu-Arg(NG-R4)-Pro-A1 in which R4, R5, R6 and R7 are protective groups capable of being removed by one or more chemlcal/treatments which do not affect (pyro)-Glu-His-Trp-D-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHR1 in which R1 is lower alkyl, R8 is hydrogen or an .alpha.-amino protective group and with a (lower alkyl)amine to obtain the corresponding compound of formula R8-(pyro)-Glu-His-Trp-D-Ser(R6)-Tyr(R5)-D-Leu-Leu-Arg(NG-R4)-Pro-NHR1, as defined herein in section (B); or transforming by solid phase synthesis the .alpha.-amino protected resin of formula R9-Z2 in which R9 and Z2 are as defined hereinbefore in section (C) to obtain the corresponding compound selected from the group consisting of R8-(pyro)-Glu-His(N1m-R7)Trp-X1-Tyr(R5)-Y-Leu-Arg(NG-R4)-Pro-Z2, R9-His(N1m-R7)-Trp-X-tyr(R5)-Y-Leu-Arg(NG-R4)-Pro-Z2, R9-Trp-X1-Tyr(R5)-Y-Leu-Arg(NG-R4)-Pro-Z2 and R9-X1-Tyr(R5)-Y-Leu-Arg(NG-R4)-Pro-Z2 in which a) X1 is Ser(R6), Y is D-Trp and Z2 is Gly-A; or b) X1 is D-Ser(R6), Y is D-Leu and Z2 is A1;
wherein R4, R5, R6, R7, R8 and R9 are as defined hereinbefore in section (C).

2. A compound selected from the group consisting of:
(A) a compound of the formula R8-(pyro)-Glu-His(N1m-R7)-Trp-X1-Tyr(R5)-Y-Leu-Arg(NG-R4)-Pro-Z1 in which a) X1 is Ser(R6), Y is D-Trp and Z1 is Gly-R2; or b) X1 is D-Ser(R6), Y is D-Leu and Z1 is OR3 wherein R2 is selected from the group consisting of amino and O-(lower alkyl), R3 is lower alkyl, R4, R5, R6 and R7 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 (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, respectively; and R8 is hydrogen or one of said protective groups;
(B) 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, R4, R5 and R6 are protective groups capable of being removed by one or more chemical treatments which do not affect (pyro)-Glu-His-Trp-D-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHR1 and R8 is hydrogen or an .alpha.-amino protective group; and (C) a compound selected from the group consisting of , , and in which a) X1 is Ser(R6), Y is D-Trp and Z2 is Gly-A; or b) X1 is D-Ser(R6), Y is D-Leu and Z2 is A1;
wherein 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
(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, respectively; R8 is hydrogen or an .alpha.-emino protective group, R9 is an .alpha.-amino protectlve group, A is selected from the group consisting of:

and , and A1 is , when prepared by the process of Claim 1 or an obvious chemical equivaient thereof.

3. The process of Claim 1 for preparing a compound of the formula in which a) X1 is Ser(R6), Y is D-Trp and Z1 is Gly-R2; or b) X1 is D-Ser(R6), Y is D-Trp and Z1 is OR3 wherein R2 is selected from the group consisting of amino and O-(lower alkyl), R3 is lower alkyl, R4, R5, R6 and R7 are protective groups capable of being removed by one or more chemical treatments which do not affect (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:NHR wherein R1 is lower alhyi, respectiveiy; and R8 is hydrogen or one of said protective groups, which comprises:
subjecting a compound of the formula in which a) X1 is Ser(R6), Y is D-Trp and Z2 is Gly-A, or b) X1 is D-Ser(R6), Y is D-Leu and Z2 is A1 wherein R4, R5, R6 and R7 are protectlve groups capable of being removed by one or more chemical treatments which do not affect the corresponding compound of formula I
(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 NHR1 wherein R1 is lower alkyl, respectively, R8 is hydrogen or an .alpha.-amino protective group, R9 is an .alpha.-amino protective group, A is selected from the group consisting of:

' and , and A1 is to ammonolysis, or to transesterification with a lower alkanol followed by converting the resulting ester by amidation into the corresponding amide to obtain the compound of formula as defined herein.

4. The compound of formula , as defined in Claim 3, when prepared by the process of Claim 3 or an obvious chemical equivalent thereof.

5. The process of Claim I for preparing a compound of the formula in which R1 is lower alkyl, R4, R5 and R6 are protective groups capable of being removed by one or more chemical treatments which do not affect in which R1 is lower alkyi, and R8 is hydrogen or an .alpha.-amino protective group, which comprises:
reacting a compound of the formula Pro-A1 in which R4, R5, R and R7 are protective groups capable of being removed by one or more chemical treetments which do not affect in which R1 is lower alkyl, R8 is hydrogen or an .alpha.-amino protective group and A1 is with a (lower alkyl)amine to obtain the corresponding compound of formula as defined herein.

6. The compound of formula , as defined in Claim 5, when prepared by the process of Claim 5 or an obvious chemical equivalent thereof.

7. The process of Claim I for preparirlg a compound selected from the group consisting of , , and in which a) X1 is Ser(R6), Y is D-Trp and Z2 is Gly-A; or b) X1 is D-Ser(R6), Y is D-Leu and Z2 is A1;
wherein 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

(I) in which a) X is Ser, Y is D-Trp and Z i5 Gly-NH2; or b) X is D-Ser, Y is D-Leu and Z is NHR1 wherein R1 is lower alkyl, respectively; R8 is hydrogen or an .alpha.-amino protective group, R9 is an .alpha.-amino protective group, A is selected from the group consisting of:

and , and A1 is which comprises:

transforming by solid phase synthesis the .alpha.-amino protected resin of formula R9-Z2 in which R and Z are as defined hereinbefore in section (C) to obtain the corresponding compound selected from the group consisting of , , and in which a) X1 is Ser(R6), Y is D-Trp and Z2 is Gly-A; or b) X1 is D-Ser(R6), Y is D-Leu and Z2 is A1, wherein R4, R5, R6, R7, R8, R9, A and A1 are defined herein.

8. The compound selectedfromthe consisting of , , and as defined in Claim 7, when prepared by the process of Claim 7 or an obvious chemical equivalent thereof.

9. The process of Claim 3 for preparing a compound of formula in which a) X1 is Ser(R6), Y is D-Trp and Z1 is Gly-R2; or b) X1 is D-Ser(R6), Y is D-Leu and Z1 is OR3 wherein R2 is amino or 0-(lower alkyl);
R3 is lowere alky;
R4 is a protective group for the N.delta., N.omega. and N.omega. nitrogen atoms of arginine selected from the group consisting of tosyl, nitro, benzyloxycarbonyl and adamantyloxycarbonyl;
R5 is a protective group of the hydroxyl of tyrosine selected from the group consisting of 2-bromobenzyloxycarbonyl, benzyl, acetyl, 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 hereinbefore 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 hydrogenor an .alpha.-amino protective group selected from the group consisting of t-butyloxycarbonyl, benzyloxycarbonyl, cyclopentyloxycarbonyl, t-amyloxycarbonyl and d-isobornyloxycarbonyl.

10. The compound of formula , as dsfined in Claim 9 , when prepared by the process of Claim 9 or an obvious chemical equivaient thereof.

Il, The process of Claim 5 for preparing compound of the formula in which R1 is lower alkyl;
R4 is a protective group for the N.delta., N.omega., and N.omega.1 nitrogen atoms of arginine selected from the group consisting of tosyl, nitro, benzyioxycarbonyl and adamantyloxycarbonyl;
R5 is a protective group of the hydroxyl of tyrosine selected from the group consisting of 2-bromobenzyioxycarbonyl, benzyl, acetyl, tosyl, benzoyl, t-butyl, tetrahydropyran-2-yl, trityl, 2,4-dichlorobenzyl and benzyloxycarbonyl;
R6 is a protective group for the hydroxy group of serine and is selected from the group defined hereinbefore for R5; and R8 is hydrogen or an .alpha.-amino protective group selected from the group consisting of t-butyloxycarbonyl, benzyloxycarbonyl, cyclo-pentyloxycarbonyl, t-amyloxycarbonyl and d-lsobornyloxycarbonyl.

12. A compound of the formula as defined in Claim 11, when prepared by the process of Claim 11 or an obvious chemical equivalent thereof.

13. The process of Claim 7 for preparing a compound selected from the group consisting of , , and in which (a) X1 is Ser(R6), Y is D-Trp and Z2 is Gly-A; or (b) X1 is D-Ser(R6), Y is D-Leu and Z is A1 wherein R4 is a protective group for the N.delta., N.omega. and N.omega.1 nitrogen atoms of arginine selected from the group consisting of tosyl, nitro, benzyloxycarbonyl and adamantyloxycarbonyl;
R5 is a protective group of the hydroxyl or tyrosine selected from the group consisting of 2-bromobenzyloxycarbonyl, benzyl, acetyl, 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 hereinbefore 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 .alpha.-amino protective group selected from the group consisting of t-butyloxycarbonyl, benzyloxycarbonyl, cyclopentyloxycarbonyl, t-amyloxycarbonyl and d-isobornyloxycarbonyl, and R9 is an .alpha.-amino protective group selected from the group consisting of t-butyloxycarbonyl, benzyloxycarbonyl, cyclopentyloxy-carbonyl, t-amyloxycarbonyl and d-isobornyloxycarbonyl, and A and A1 are as defined in Claim 7.

14. A compound selected from the group consisting of , , and , as defined in Claim 13, when prepared by the process of Claim 13 or an obvious chemical equivalent thereof.

15. A process of Claim 11 for preparing a compound of the formula wherein R4 is tosyl, R5 is 2-bromobenzyloxycarbonyl, R6 is benzyl, R8 is hydrogen and R1 is ethyl; which comprises:
reacting a compound of formula in which R4 is tosyl, R5 is 2-bromo-benzylcarbonyl, R6 is benzyl, R7 is 2,4-dinitrophenyl, R8 is hydrogen and A1 is O-CH2 with ethylamine to obtain the compound of formula wherein R4 is tosyl, R5 is 2-bromobenzyloxy-carbonyl, R6 is benzyl, R8 is hydrogen and R1 is ethyl.

16. The compound of formula wherein R1 is ethyl, R4 is tosyl, R5 is 2-bromobenzyloxycarbonyl, R6 is benzyl, R8 is ethyl, when prepared by the process of Claim 15 or an obvious chemical equivalent thereof.

17. The process of Claim 13 for preparing a compound of the formula in which R4 is tosyl, R5 is 2-bromobenzyloxycarbonyl, R6 is benzyl, R7 is tosyl, R8 is hydrogen and A is benzhydrylamine resin in which the transformation by solid phase synthesis includes removing the .alpha.-amino protecting group of the intermediate of the formula wherein R9 is t-butyloxy-carbonyl, R7 is tosyl, X is Ser (R6) wherein R6 is benzyl, R5 is 2-bromobenzyloxycarbonyl, Y is D-Trp, R4 is tosyl and Z2 is Gly-A wherein A is benzyhydrylamine resin and coupling the .alpha.-amino deprotected derivative so obtained with L-(pyro)-glutamic acid.

18. The compound of the formula in which R4 is tosyl, R5 is 2- bromobenzyloxycarbonyl, R6 is benzyl, R7 is tosyl, R8 is hydrogen and A is benzhydrylamine resin, when prepared by the process of Claim 17 or an obvious chemical equivalent thereof.