CA1186302A - Lrf antagonists - Google Patents

Abstract

LRF ANTAGONISTS

ABSTRACT

Peptides which inhibit the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. Administration of an effective amount prevents ovulation of female mammalian eggs and/or the release of steroids by the gonads. The peptides have the structure:

X-R1-R2-R3-Ser-Tyr-R4-R5-Arg-Pro-R6 wherein X is hydrogen or an acyl group having 7 or less carbon atoms; R1 is dehydro Pro, dehydro D-Pro, Thz or D-Thz; R2 is D-Phe, D-His, D-Trp, Trp, Cl-D-Phe, dichloro-D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe, AcNH-D-Phe, NO2-D-Phe, dinitro-D-Phe, Br-D-Phe, dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe; R3 is D-Trp, Trp, D-Phe or D-His; R4 is Gly or a D-isomer amino acid; R5 is Leu or N.alpha.Me-Leu;
and R6 is Gly-NH2 or NHCH2CH3.

Description

3~Z

LRF ANTAGON I STS
The present invention relates to peptides which inhihit the release of gona~otropins by the pituitary gland in mammalians, including humans and to methods of preventing ovulation and/or inhibitin~ the release of steroids. More particularly, the present invention is directed to pep~ides which inhibit gonadal function and the release of the steroidal hormones, progesterone and testosterone.
The pituitary gland is attached by a stalk to the region in the base of the brain known as the hypothalamus. The pituitary gland has two lobes, the anterior and the posterior lohes. The posterior lobe of the pituitary gland stores and passes onto the general circulation two hormones manufactured in the hypothalamus, these being vasopre~sin and oxytocin. The anterior lobe of the pituitary gland secretes a number of hormones, which are complex protein or glyco-protein molecules that travel through the bloodstream to various organs and which, in turn, stimulate the secretion into the blood stream of other hormones from the peripheral organs. In part:icular, follicle stlmulatin~ hormon~
~FSH) and lut~iniæing hormone (JJ~I), 30mel-ime~ re~e~red to as gonadotroE~ins or gonaclotropic hoxmones, are 2S released by the pituitary gland. These hormones, in combination, regulate the functioning of the gonads to produce testo~terone in the testes and progesterone and e~trogen in the ovaries, and also regulate the production and maturation o~ yametes.
The release o~ a horrnolle by the anterior lobe of the pituitary gland usua]ly requires a prior release of another clas~ of hormones produced by the hypothalamus. One o~ the hypot}latamic hormones acts as a faator tha~ tri-3ger~ the release of the gonadotropic hormon~3, par~:lcu]arly r.H. ~the hypothalamic hormone ` 1~L8~i302 which acts as a releasing factor for LH is referxed to herein as LRF al~hough it has also been referred to as ~JH-RH and as GnRH. LRF has been isolated and characterized as a decapeptide having the following structure:
p-Glu-His-Trp-Ser-Tyr-Gly~Leu-Arg-Pro-Gly-NH2 Peptides are compollnds which contain two or more amino acids in which the carboxyl group of one acid is linked to the amino group of the other acid7 The ~vrmula for LRF, as represented above, is in accordance with conventional representation of peptides where the amino group appears to the left and the carboxyl group to the right. The position o~ the amino acid r~sidue is identified by numbering the amino acid residues Erom left to right. In the case of LRF, the hydroxyl portion of the carboxyl group of glycine has been replaced with an amino group (NH2). The abbreviations for the individual amino acid residues above are conventional and are based on the trivial name of the amino acid:
where p-Glu is pyroglutamic acid, His is histidine, Trp is tryptophan, Ser is serine, Tyr is tyrosine, Gly is glycine, Leu is Leucine, Arg is argi.nin~ and Pro i~
proline. ~xcept or glycine, amino aaids o~
~eptides o~ the invention are o~ the L-conEiguration unless noted otherwise.
It is known that the substitution of D-amino aclds ~or Gly in the 6 position o~ the LRF Aecape~tide provides a peptifle material having rom about 1 to 35 times greater potency than does LRF to eEect the release o~ LH and other gona~otropin.s by the pituitary gland oE mammalians. The relea~,ing eEEect is obtai.ned when the LRF analog is administered to a mammalian i.ntravenously, subcutaneously, intramuscularly, orally, intrana.sally or intravaginalJ.y.
Tt i~ also known that substitut.ion of various 118~i3~2 amino acids for His (or the deletion of His) at the

2-position of the LRF decapeptide produces analogs having an inhibitory effect on the release of LH and other gonadotropins by the pituitary gland of mammalians. In particular, varying degrees of inhibition of the release o LH are obtained when ~is is deleted (des ~is) or replaced by D-Alar D-Phe or Gly.
rrhe inhibitory effect of such peptides modified at the 2-position can be f~rther enhanced when a D-amino acid is substituted for Gly in the 6-position of the decapeptides. For example, the peptide:
- pGlu-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH2 is 4 ~imes more potent as an inhibitor ~or the release of gonadotropins than is the same peptide where Gly is present in the 6-position rather than D-Ala.
Some female mammalians who have no ovulator~
cycle and who show no pituitary or ovarian de~ec~ begin to secrete normal amounts of the gonadotropins LH and FSH after the appropriate administration of LRF. Thus, the administration of LRF is considered suitable for the treatment o~ those cases of in~ertility where a ~unctional defect resides in the hypothalamus.
There are al~o reason~ ~or d~sirlng ~o prevent ovula~ion i.n ~emal~ mammalians, ~nd the administration of LRF analogs that are antagonistic to the normal ~unction o~ LRF have been used to prevent ovulation.
For this,rea.sont analogs of LRF which are antagoni~tic , tc> L~F are being investigated ~or their potential use as a contraceptive or ~or regulating conception periods.
It is desirect to provide peptides which are strongly antagonistic to endogenous I.RF and which prevent secretion o~ L~ and the release of steroids by the gonads oE mammals.
The present invention provides peptides which inhibit the release o~ gonactotropins ln mammalians, 11i5 ~3~!2 including humans, and also provides methods for inhibiting the release of steroids by the gonads of male and female mammalians The improved LRF analogs are antagonistic to LR~ and have an inhibitory effect on the reproduction processes of mammalians. These analogs may be used to inhibit the production of gonadotropins and sex hormones under various circumstances including precocious puberty, hormone dependen~ neoplasia, dysmenorrhea and endometriosis.
Generally, in accordance with the present invention, peptides have been synthesized which strongly inhibi.t the secretion of gonadotropins by the p;tuit~ry gland of mammalians, including humans, and/or inhibit the release of steroids by the gonads. TheSe peptides are analogs of LKF wherein there is a 1-position substitution in the form of either dehydroproline or meta-thiazolidine-2-carboxylic acid, and preEerably substituents are also present 2-, 3- and 6-positions.
The l-position substituent may be modified so that its alpha amino group contain.s an acyl group, such as formyl, acetyl, acrylyl, vinylacetyl or benæoyl.
Dehydro L-Pro iS preferred in the l~posltion. Modi~ied D-Phe is pre~erably present in the 2-position and provides incr~ased antagonistic activity as a result o~
the specific modi~ications present in the benzene ring.
Single substitutions or hydro~en are pre~erably made in the para- or 4-~osition, and double substitutions are mad~ preferably in the 2,~- or the 3,4-positions. The substitutions are most preferably selected ~rom dichloro, methyl, eluoro, di~luoro, trifluoromethyl, metho~y, bromo, dibromo, nitro, dinitro, acetylamino and methyl mercapto. D-Trp is preerred in the 3-position, and imBzl D-~is or D--Trp or some other lipophilic aromatic D-amino acid is preferred in the 6-position, although Gl~ or any D-isomer am~no acid, e.g. D-Leu and 1~8~i3C~

D-Ser(O-t But), may be used. The substitutions in the - 7- and 10-positions are op1:ional.
Because these peptides are highly potent to inhibit release of LH, they are often referred to as LRF
antagonists. The peptides inhibit ovulation o~ female mammals when administered at very low levels at proestrous and are also effective to cause resorption of ertilized eggs if administered shortly after conception.
More specifically, the peptides of the precent invention are represented by the following formula:
1 2 R3 ser-Tyr-R4-R5-Ar9-pro-R
wherein X is hydrogen or an acyl group having 7 or less carbon atoms Rl is dehydro Pro, dehydro D-Pro, Thz or D-Thz; R2 is D-Phe, D-His, D-Trp, Trp, Cl-D-Phe, dichloro D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe, AcNH-D-Phe, N02-D-Phe, dinitro-D-Phe, Br-D-Phe, dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe; R3 is D-Trp, Trp, D-Phe or D-His; R4 is Gly or a D-isomer amino acid; R5 is Leu or N Me-Leu;
and R6 is Gly-N~I2 or NHCH2CH3.
By dehydro Pro is meant 3,4 dehydroproline, C5I~702N, and when X is an acyl radical, it i~
Attached to the ni~ro~en. By Thz i~ meant meta-thiazolidine-2-carboxylic acid, C4H702NS, which can be prepared by the treatment of cysteine hydrochloride with ~ormaldehyde, and for example, Ac-Thz may be prepared by the reaction o Thz with acetic anhydride.
The peptides of the present invention can be synthesized by a solid phase technique using a chloromethylated resin, a methylbenæhydrylamine resin (MBTIA) or a benzhydrylamine (B~IA) resin. The synthesis is conducted in a manner to step~ise add the amino acids in the chain in the manner set eorth in detail in the

3$ U..S. Patent No. ~,211,G93. Sic~e-chain protecting 3~32 groups, as are well known in the art, are preferably added to Ser, Tyr, Arg and ~is before these amino acids are coupled to the chain being built upon the resin.
Such a method provides the ~ully protected intermediate peptidoresin. The intermediates o the invention may be represented as:
X -R1-R2-R3-Ser(X )-Tyr(X )-R4-R5- Arg(X
)-Pro-X wherein: X is an ~-amino protectiny group of the kype known to be useul in the art in the stepwise synthesis of polypeptides and when X in the desired peptide composition is a particular acyl group, that group may be used as the protecting group. ~mon~
the classes of ~-amino protecting groups covered by X
are (1) acyl-type protecting groups, such as formyl (For), trifluoroacetyl, phthalyl, p-toluenesulfonyl (Tos), benzoyl (Bz), henzensulfonyl, o-nltrophenylsulfen~l (Nps), tritylsulfenyl, o-ni~rophenoxyacetyl, acrylyl (Acr) r chloroacetyl, acetyl ~Ac) and ~-chlorobutyryl; (2) aromatic urethan-type protecting groups, e.g., benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as p-chloro-benzyloxycarbonyl, p-nitrobenæyloxycarhonyl, ~-bromohen%yloxycarbonyl and ~-m~ho~yh~rlzy~ox~c~rbonyl;
(3) aliphatlc ure~han prote~inc3 ~roups, such ~s ter~butyloxycarbonyl (Boc), diisopropylmethoxycarbon~l, isopropyloxyaarbonyl, ethoxYcarbonyl and allyloxycarbonyl; (4) cycloalkyl urethan~type protecting groups, such as cyclopentyloxycarbonyl, adamantyloxycarbonyl and cyclohexyloxycarbonyl; (5) thiourethan-type protectinq groups, such as phenylthiocarbonyl; (~) alkyl-type protecting groups r such as allyl (Aly), triphenylmethyl(trityl) and benzyl ~Bzl); (7) trialkylsilane groups, such as trimethylsilane, The preerred a-amino protecting group is ~oc when X is hydrogen.

~863~2 X is a protecting group for the alcoholic - hydroxyl group of Ser and is selected from the group consisting of acetyl, benzoyl, tetrahydropyrany~, tert-butyl, trityl, benzyl and 2,6-dichlorobenzyl.
Benzyl is preferred.
X3 is a protecting group for the phenolic hydroxyl group of Tyr selected from the group consisting of tetrahydropyranyl, tert-butyl, trityl, benzyl, benzyloxycarbonyl, 4-bromobenzyloxycarbonyl and 2,6-dichlorobenzyl.
X4 is a protecting group for the nitrogen atoms of Arg and is selected from the group consisting of nitro, Tos, benzyloxycarbonyl, adamantyloxycarbonyl, and Boc; alternatively X4 may be hydrogen, which means there are no protecting groups on the side chain nitrogen atoms of arginine.
X is selected ~rom Gly-O-CH2-[resin support]; O-CH2-[resin support] and Gly-NH-~resin support].
The criterion for selecting side chain protecting groups or X2-X4 is that the protecting group must be stable to the reagen~ unc~er ~h~ reaakion condi.tions selected for removing the a-~mino pro~ectillg group at each st:ep o~ the synth~sis. The protec~ing ~roup must not be split of under coupllng conditions, and the protecting group must be removable upon completi~n of the synthesis o the desired amino acid ~equence under reaction condition~ that will not alter the peptide chain.
When the X~ group is Gly-O-C~2-[re3in support], the ester moiety of one of the many fur,ctional groups o~ the polystyrene resin support is being representefl. When the X5 ~roup is Gly-NH-[resin supportl, an amide bond connects Gly to ~E~A resin or to a MBllA resin.

~81~3~2 When X is acetyl, formyl, acrylyl, vinylacetyl, benzoyl or some other acyl group having 7 carbon atoms or ]ess, it may be employed as the X protecting group for the a-amino group of Rl in which case it can be added before coupling of the last amino acid to the peptide chain. Alternatively, a reaction may be carried out with the peptide on the resin, e.g. reacting with acetic acid in the presence of dicyclohexyl carbodiimide (DCC) or preferably with acetic anhydride.
The fully protected peptide can be cleaved from the chloromet~ylated resin support by ammonolysis, as ;s well known in the art, to yield the ully protected amide intermediate. Deprotection of the peptide as well as cleavage of the peptide from the benzhydrylamine resin takes place at 0C with hydrofluoric acid (HF~.
Anisole is added to the peptide prior to treatment with HF. After the removal of HF, under vacuum, the cleaved, deprotected peptide is treated with ether, decanted, taken in dilute acetic acid and lyophilized.
Purification of the peptide i5 effected by ion exchange chroMotography on a CMC column, followed by partition chromo~ography using the elution system:
n-butanol; O.].N acetic aci~ (1:1 v-~ume ra~io) on a column packed with Sephadex G-25 or by using ~PLC as known in the arts. The peptides o the invention are effective at levels of less than 200 micrograms per kilogram o~ body ~eight, when administered at about noon on the day of proestrous, to prevent ovulation in female rats. For prolonged suppres~ion of ovulation, it mav be neceæsary to use dosage levels in the range oE from about 0.1 to about 5 milligrams per kilo~ram of body weight. These antagonists are also efective as contraceptives when administered to male mammals on a regular basi~.
,Sinc~ these compounds will reduce testosterone .

~B6302 levels (an undesired consequence in the normal, sexually active male), it may be reasonable to administer replacement dosages of testosterone along with the LRF
antagonist.
The following examples further illustrate various features of the invention b~t are intended to in no way liMit the scope o~ the invention which is defined in the appended claims.
EXAMPLE I
The following peptides having the formula X-dehydro Pro-R2-D-Trp-5er-Tyr-D-Trp-R5-Arg-Pro-Gly-NH
are prepared by the sol;d pha~e procedure referred to above.
TABLE I

1 Ac 3,4 C12-D-Phe Leu 2 " 4 CF3-D-Phe "
3 " 4F-D-Phe "

4 " 4 AcNH-D-Phe "
" 4 NO2-D-Phe "
6 " 4 Br-D-Phe "
7 " 4 CH3s_D_phe 8 " 4 OCH3-D~Phe "
9 " 4 CM3-D-Ph~ "
ll 2,4 C12-D-Ph~ "
11 ~cr 3,4 C1~-D-Phe 12 Ac 4 ~CH3-D-Phe N MeLeu 13 " 4 CM3-D-Phe "
14 " 3,4 C12-D-Phe "
For purposes o~ an example, a representative solid phase synthesis of Peptide No. 1 above, which is re~erred to as [~c-dehydro Prol, 3,4 C12-D-Phe2,D-Trp3~6~-L~F i3 set forth hereina~ter. This pept~de has the ~ollowlng formula:

~863~Z

Ac-dehydro Pro-3,4 C12-D-Phe-D-Trp-Se~-Tyr-D-Trp -Leu-Arg-Pro-Gly-NH2 A BHA resin is used, and Boc-protected ~ly i~
coupled to the resin over a 2-hour period in CH2C12 using a 3-fold excess of Boc derivative and DCC as an activating reagent. The glycine residue attaches to the BHA residue by an amide bond.
Following the coupling of each amino acid ~esidue, washing, deblocking and coupling of the next amino acid residue is carried out in accordance with the ~ollowing schedule using an automated machine and beginning with about 5 grams of resin:
STEP RE~GENTS AND O ERATIOI~S MIX TIMES MIN.
1 CH2C12 wash-80 ml. (2 times) 3 lS 2 MethanoltMeOH) wash-30 ml. (2 times) 3 3 CH2C12 wash-~0 ml. (3 times) 3 4 50 percent TFA plus 5 percent 1,2-eth-anedithiol in CH2CL2-70 ml. (2 times) 10 CH2C12 wash-80 ml. (2 times) 3 6 TEA 12.5 percent in CH2C12 70 ml.
(2 times) 5 7 MeOH wash-4n ml. ~2 times) 2 8 CH2C12 wash-8n ml. (3 times) 3 9 Boc-amino acid (10 mmoles) in 30 ml. o~ either DME` or CM2C12, depending upon the solubility o the particular protected amino acid, (1 time) plus DCC (10 mmoles) in CH2Cl? 30-300 10 MeOH wash-40 ml. (2 times) 3 11 T~A 12.5 percent in CM2C12-70 ml.
(1 time) 3 12 MeO~ wash-30 ml. (2 times) 3 13 CH2Cla wash-80 ml. (2 times) 3 A~ter step 13, an aliquot is taken ~or a ninhydrin test: i~ the test is negative, go back to step 1 or coupling o~ the next amino acid; if the test is ~L~8~3~

-3].-posi~ive or slightly posi.tive, go back an~ repeat steps g ~hrough l?
The above scheclule is used for coupling of each of the ami.no acids of the peptide of ~he invention a.ter the first amino acid has been attached, N Boc protection is used for each o~ the remaining amino acids throughout the syn'chesis. The side chain o Arg is protecte~ with Tos. OBzl is used as a side chain protecting group ~or the hydroxyl group of Ser, and 2-6 1~ dichlorobenzyl is llsed as the side chain protecting group for the hydroxyl ~roup of 'ryr. N-acetyl dehydro Pro is introduced as t:he final amino acid. Boc-Arc3(Tos) and ~oc-D~Trp, ~hich have low solubility in C~2Cl.2, are coupled using DMF CH2Cl2 mixtures.
Tlle cleavaye o, khe peptide from the resin and complete deprotection o the side chains'takes place very readily at 0C. with HF. Anisole is added as a scavenger prior to HF treatment. After the rernoval of HF under vacuum, the resin is e~tracted with 50~ ace~ic acic~, and the wa.shi.ngs are lyophilized to provide a crude pepticle powder.
Puri~ication of the pepti~e is ~,hen eE~cted b~
ion exchancJe chrom,-ltography on CMC (Whatm~rl CM 3~, U5il1g ~.t ~radi.ent o~ 0,.05 to 0.3M NE~OAc in 50/50 2S Tnethanol/~ater) ~olloweci by partition chromatography in gel ~iltration column using th~ elution system:
n E3~ttanol.; O.lN ~cet:.ic acid (1:1 volurne ratio).
The peptides set forth in the foregoing table are assaye~ n vitro and ln vlvo. 'rhe ln vltro ~est is made using dlssociat,e(l rat pituitary cells rnaintained in culture Eor 4 days ~rior to the assay. The levels of LH
mediated in response to the applicatioll of pept:icles i.s assayed by speci~i.c radioimmuno~ssay For rat LH, Con~rol di~hes o~ cells only receit~e a measure which is 3S 3 nanomol~r i.n L~.E': e~perimental dishes receive a *trade mark ~ ~8~'3~2 measure 3 nanomolar in L~F plus a measure having a concentration of test peptide ranging from 0.01 to 3 nanomolar. The amount of LH secreted in the samples treated only with LRF is compared with that secreted by the samples treated with the peptide plus LRF. Results are calculated and expressed in Table IA ~In Vitro column) as the molar concentration ratio of test peptide to LRF (antagonist/LRF) required to reduce the amount of LH released by 3 nanomolar LRF to 50 percent of the c~ntrol value (ICR50~.
The peptides described hereinabove are used to deterrnine effectiveness to prevent ovulation in female rats. In this test, a specified number of mature female Sprague-Dawley rats, each having a body weight from 225 to 250 grams, are injected with either 5 or 10 micrograms of peptide in corn oil a~ about noon on the day of proestrous. Proestrous is the afternoon before estrous (ovulation)~ A separate female rat group is used as a control to which the peptide is not administered. Each of the control rat females has ovulation a~ estrous. As indicated in the In Vivo column, the peptides are significantly e~ective to pr~vent ovulation o~ ~emale rats a~ A very low dosa~e, and all of the peptide compositions are considered to be 5 ~otally e~fec~ive at a dose of one milligram.
TABLE IA
Jn Vitro In Vivo_(10~) In ViVo ~51ls?
Peptide ICR50 Rats Ovulating Rats Ovulating 1 0.039 0/10 ~/7 2 0.070 7/9 3 0.021 0/10 0/10 0.011 0/10 2/10 6 0.010 5/7 7 0.030 1/10 7/10 63~2 ; .,.

8 0.12~ 3/10 9 0.044 1/7 9/9

5/10 4/7 11 0.048 1/10 . 4/10 12 0.29 ~/10 13 0.11 3/4 14 0.05 1/3 8/10 EXAMPLE II
The following peptides having the formula 10 x-Rl-pcl-D-phe-D-Trp-ser-Tyr-R4-R5-Arg-pro-~6 are prepared by the solid phase procedure as generally described in Bxample I except ~or No. 20 which is prepared on a chloromethylated resin.
, TABI,E II
15 PEPTIDE X Rl R4 R5 R6 Ac Aehydro Pro D-Trp Leu Gly-NH2 16 " ' " N MeLeu "
17 " dehydro D-Pro " Leu "
18 " N MeLeu n 20 19 Acr dehydro Pro 1l . " ,.
Ac dehydro D-Pro " Leu NHCH2CH3 21 AcThz D~Trp Leu Gly-NH2 2 2 nn n N Me I~ u n 23 "D-Thz " " "
24 ~1n 1l ~eu "
" dehydro Pro (imB~l)D-His n 26 n dehydro D-Pro " " "
27 n ThZ n n n 28 " D-Thz rhe peptides set forth in the oregoing table are assayed in vitro and in vivo. The in vitro test is made using dissociated rat pituitary cells maintained in culture for 4 days prior to the assay. The levels o~ LH
mediated in response to the application o~ peptides is as~Ayed by speci~ic radioimmunoassay for rat LH.

363~2 . ~ . ...

Control dishes of cells only receive a measure which is 3 nanomolar in LRF: experimental dishes receive a measure 3 nanomolar in LRF plus a measure having a concentration of test peptide ranging rom 1 to 100 nanomolar. The amount of LH secreted in the samples treated only with LRF is compared with that secreted by the samples treated with the peptide plus LRF. Results are calculated and expressed in Table IIA(In Vitro column) as the molar concentration ratio of test peptide to LRF (antagonist/LRF) required to reduce the amount Gf LH released by 3 nanomolar I,RF to 50 percent of the control value (ICR50).
Several o~ the peptides described hereinabove are used to determine effectiveness ~o prevent ovulation in female rats. In this test, four, seven, nine or ten mature ~emale Sprague-Dawley rats r each having a body weight from 225 to 250 grams, are injected with 0.02 milligram of peptide (unless otherwise indicated) in corn oil at about noon on the day of proestrous.
Proestrous is the afternoon before estrous (ovulation).
A separate ten female rat group is used as a con~rol to which the peptide is not admlnistered. ~ach o~ the ~en control rat female~ has ovulation a~ estrous. ~8 indicated in the In Vivo column, the peptides are significantly effective to prevent ovulation o~ female rat~ at a very low dosage, and all of the peptide compositions are considered to be totally effective at a ~ose o one milligram.
TABLE IIA
In Vitro In Vivo Peptide ICR50 ~ats Ovulating 0.043 0/10*
16 0.05~ 0/7 17 0.19 3/8 -~' 35 1~ 0.27 5/10**

, 19 0.03 0/g - 20 0.2 21 0.14 ~/8 22 0.13 9/10**
23 0.1 24 0.12 0.042 4/10 26 0.2 10/10 27 0.15 7/10**
28 0.1 7/10**
*~0.025 mq. *0.010 mg.
EXAMPLE III
rrhe following peptides having the formula 1 2 R3 Ser-Tyr-D-Trp-R5-Arg-pro R
lS are prepared by a solid phase procedure generally as described in Example I, except for No. 37 where a chloromethylated resin is used.
TABLE III
Pep-tide X Rl R2 R3 R5 R6 2g H dehydro-D,L-Pro D-Phe D-Trp N MeLeu Gly-NH2 30 H " " " I,eu "
31 Ac dehydro-D,L-Pro 32 H " " " ~ MeLeu "
25 33 Ac dehydro-Pro n 1l Leu "
34 Ac dehydro-D-Pro 3S H dehydro-Pro " " " "
36 H dehydro-D-Pro " ~i .. ..
37 ~ dehydro-Pro " " " NHCH2CH3 30 38 Bz dehydrQ-pro D-Trp n 1l Gly-NH2 39 For dehydro-Pro " D-Phe " "
40 For dehydro-Pro D-His Trp " "
41 Acr dehydro-Pro n 42 Bz dehydro-Pro Trp D-His 35 43 Acr dehydro-Pro " D-Trp " "

3C! 2 44 For dehydro-Pro " D-~is " "
Bæ dehydro-Pro D-His " " "
The peptides set rorth in the fore~oing table are assayed in vitro and in vivo. The in vitro assay is made using four day old primary culture of dispersed rat pituitary cells. The levels of L,H mediated in response to the application of peptides is assayed by specific radioimmunoassay for rat LH. Control dishes of cells only receive a measure which is 3 n~nomolar in LR~^
experimental dishes receive a measure 3 nanamoles of LRF
and a concentration of test peptide ranging from 1 to 100 nanomolar. The amount of LH secreted in the samples treated only with LRF is compared with that secreted by the samples treated with t~le peptide plus LRF. Results are calculated and expressed in Table IIIA (In Vitro column) as the molar concentration ratio of test peptide required to reduce the amount of LH released by the 3 nanomolar LRF to 50 percent of the control value ( ICR50 ) -Several of the peptides described hereinabove are used to determine effectiveness to prevent ovulation in female rats. In this test, a number o~ mature female Sprague-Dawley rats, each having a hody wel~ht from 22$
to 250 grams, are injected with 0.02 milli~ram o~
peptide in corn oil at about noon on the day of proestrous. Proestrous is the a~ternoon beore e~trous (ovulation). A separate female rat group is used as a control to which the peptide is not admini~tered. Each o~ the control rat females has ovulation at estrous. As indicated in the In Vivo column, l:he peptides are ~ignificantly effective to prevent ovulation of female rats at a very low dosage, and all of the peptide compositions are considered to be totally ef~ective at a dose of one milligram.

~8~3~2 TA~LE IIIA
In Vitro In Vivo Peptide ICR50Rats Ovulating ~9 0.5:1 ~/10 0.8:1 7/10 31 0.3:1 1/10 3~ 0.4:1 2/10 33 0.6:1 0/4*
*dose of 0.025 mg.
These peptides can be administered to mammals intravenously, subcutaneously, intramuscularly,orally, intranasally or intravaginally to achieve fertility inhibition and/ox control. Effective dosages will vary with the form of administration and the particular species of mammal being treated. An example of one typical dosage form is a physiological saline solution containing the peptide which solution is administered to provide a dose in the range of about 0.1 to S mg/kg of body weight. Oral administration of the peptide may be given in either solid form or liquid form.
Although the invention has been described with regard to its preferred embodimen~s, i~ ~hould ~0 unders~ood that change~ and modlications as would b~
obvious to one having ~he oriAinary skill in ~hi~ art may be made without departing ~rom the scope of the invention which is set forth in the claims which are appended hereto. For example/ other substitutions known in the art which do not signi~lcantly detract from the effectiveness of the peptides may be employed in the peptides oE the invention.

Claims (84)

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

1. A method for the manufacture of a compound having the formula:

X-R1-R2-R3-Ser-Tyr-R4-R5-Arg-Pro-R6 wherein X is hydrogen or an acyl group having 7 or less carbon atoms; R1 is dehydro Pro, dehydro D-Pro, Thz or D-Thz; R2 is D-Phe, D-His, D-Trp, Trp, Cl-D-Phe, dichloro-D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe, AcNH-D-Phe, NO2-D-Phe, dinitro-D-Phe, Br-D-Phe, dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe; R3 is D-Trp, Trp, D-Phe or D-His; R4 is Gly or a D-isomer amino acid; R5 is Leu or N.alpha.Me-Leu;
and R6 is Gly-NH2 or NHCH2CH3.
comprising (a) forming an intermediate compound having the formula:
X1-R1-R2-R3-Ser(X2)-Tyr-(X3)-R4-R5-Arg(X4)-Pro-X5 wherein X1 is an .alpha.-amino protecting group; X2 is a protecting group for the alcoholic hydroxyl group of Ser; X3 is a protecting group for the phenolic hydroxyl group of Tyr; X4 is a protecting group for the nitrogen atoms of Arg; and X5 is selected from Gly-O-CH2[resin support], O-CH2-[resin support], Gly-NH-[resin support], Gly-NH2 and NHCH2CH3;
(b) splitting off one or more of the groups X1 to X5 and, if desired, converting a resulting peptide into a nontoxic salt thereof.

2. A method in accordance with Claim 1 wherein, in step (a), the intermediate compound is formed with R2 representing dichloro-D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe, AcNH-D-Phe, NO2-D-Phe, dinitro-D-Phe, Br-D-phe, dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe.

3. A method in accordance with Claim 2 wherein R1 is dehydro-Pro.

4. A method in accordance with Claim 3 wherein R2 is dichloro-D-Phe.

5. A method in accordance with Claim 3 wherein R2 is 4 CF3-D-Phe.

6. A method in accordance with Claim 3 wherein R2 is 4 AcNH-D-Phe.

7. A method in accordance with Claim 3 wherein R2 is 4 NO2-D-Phe.

8. A method in accordance with Claim 3 wherein R2 is 4 Br-D-Phe.

9. A method in accordance with Claim 3 wherein R2 is 4 CH3S-D-Phe.

10. A method in accordance with Claim 3 wherein R2 is 4 F-D-Phe.

11. A method in accordance with Claim 3 wherein R2 is 4 OCH3-D-Phe.

12. A method in accordance with Claim 3 wherein X is acrylyl.

13. A method in accordance with Claim 3 wherein X is acetyl.

14. A method in accordance with Claim 3 wherein R2 is 2,4 Cl-D-Phe, 3,4 Cl-D-Phe, 4F-D-Phe, 4NO2-D-Phe, 4Br-D-Phe, or 4CH3S-D-Phe.

15. A method in accordance with Claim 14 wherein R3 is D-Trp.

16. A method in accordance with Claim 14 wherein R4 is D-Trp.

17. A method in accordance with Claim 14 wherein R4 is a lipophilic, aromatic D-isomer amino acid.

18. A method in accordance with Claim 14 wherein R5 is Leu.

19. A method in accordance with Claim 14 wherein R6 is Gly-NH2.

20. A method in accordance with Claim 2 wherein R5 is Leu.

21. A method in accordance with Claim 20 wherein X is acrylyl, R3 is D-Trp, R4 is D-Trp and R6 is Gly-NH2.

22. A method in accordance with Claim 20 wherein X is acetyl, R3 is D-Trp, R4 is D-Trp and R6 is Gly-NH2.

23. A method in accordance with Claim 1 wherein, in step (a), the intermediate compound is formed with R2 representing 4 Cl-D-Phe.

24. A method in accordance with Claim 23 wherein R4 is D-Trp.

25. A method in accordance with Claim 23 wherein R4 is imBzl-D-His.

26. A method in accordance with Claim 23 wherein R4 is a lipophilic aromatic D-isomer amino acid.

27. A method in accordance with Claim 26 wherein R5 is Leu.

28. A method in accordance with Claim 26 wherein R6 is Gly-NH2.

29. A method in accordance with Claim 26 wherein X is acetyl.

30. A method in accordance with Claim 26 wherein X is acrylyl.

31. A method in accordance with Claim 26 wherein R1 is dehydro-Pro.

32. A method in accordance with Claim 26 wherein R1 is dehydro-D-Pro.

33. A method in accordance with Claim 26 wherein R1 is Thz.

34. A method in accordance with Claim 26 wherein R1 is D-Thz.

35. A method in accordance with Claim 1 wherein, in step (a), the intermediate compound is formed with R2 representing D-Phe and R3 representing D-Trp.

36. A method in accordance with Claim 35 wherein X is hydrogen.

37. A method in accordance with Claim 35 wherein X is acetyl.

38. A method in accordance with Claim 35 wherein X is acrylyl.

39. A method in accordance with Claim 37 wherein R5 is Leu.

40. A method in accordance with Claim 37 wherein R5 is N MeLeu.

41. A method in accordance with Claim 37 wherein R6 is Gly-NH2.

42. A method in accordance with Claim 37 wherein R6 is NHCH2CH3.

43. A peptide, or a nontoxic salt thereof, having the formula:

X-R1-R2-R3-Ser-Tyr-R4-R5-Arg-Pro-R6 wherein X, R1, R2, R3, R4, R5 and R6 are defined as in Claim 1 when prepared by the method of Claim 1 or by an obvious chemical equivalent thereof.

44. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro and R2 is dichloro-D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe, AcNH-D-Phe, NO2-D-Phe, dinitro-D-Phe, Br-D-Phe, dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe when prepared by the method of Claim 2 or by an obvious chemical equivalent thereof.

45. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro and R2 is dichloro-D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe, AcNH-D-Phe, NO2-D-Phe, dinitro-D-Phe, Br-D-Phe, dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe when prepared by the method of Claim 3 or by an obvious chemical equivalent thereof.

46. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro and R2 is dichloro-D-Phe when prepared by the method of Claim 4 or by an obvious chemical equivalent thereof.

47. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro and R2 is 4 CF3-D-Phe when prepared by the method of Claim 5 or by an obvious chemical equivalent thereof.

48. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro and R2 is 4 AcNH-D-Phe when prepared by the method of Claim 6 or by an obvious chemical equivalent thereof.

49. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro and R2 is 4 NO2-D-Phe when prepared by the method of Claim 7 or by an obvious chemical equivalent thereof.

50. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro and R2 is 4 Br-D-Phe when prepared by the method of Claim 8 or by an obvious chemical equivalent thereof.

51. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro and R2 is 4 CH3S-D-Phe when prepared by the method of Claim 9 or by an obvious chemical equivalent thereof.

52. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro and R2 is 4 F-D-Phe when prepared by the method of Claim 10 or by an obvious chemical equivalent thereof.

53. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro and R2 is 4 CH3-D-Phe when prepared by the method of Claim 11 or by an obvious chemical equivalent thereof.

54. A peptide in accordance with Claim 43 wherein X is acrylyl, R1 is dehydro-Pro and R2 is dichloro-D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe, AcNH-D-Phe, NO2-D-Phe, dinitro-D-Phe, Br-D-Phe, dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe when prepared by the method of Claim 12 or by an obvious chemical equivalent thereof.

55. A peptide in accordance with Claim 43 wherein X is acetyl, R1 is dehydro-Pro and R2 is dichloro-D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe, AcNH-D-Phe, NO2-D-Phe, dinitro-D-Phe, Br-D-Phe, dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe when prepared by the method of Claim 13 or by an obvious chemical equivalent thereof.

56. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro and R2 is 2,4 Cl-D-Phe, 3,4 Cl-D-Phe, 4 F-D-Phe, 4NO2-D-Phe, 4Br-D-Phe or 4CH3S-D-Phe when prepared by the method of Claim 14 or by an obvious chemical equivalent thereof.

57. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro, R2 is 2,4 Cl-D-Phe, 3,4 Cl-D-Phe, 4 F-D-Phe, 4NO2-D-Phe, 4Br-D-Phe or 4CH3S-D-Phe and R3 is D-Trp when prepared by the method of Claim 15 or by an obvious chemical equivalent thereof.

58. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro, R2 is 2,4 Cl-D-Phe, 3,4 Cl-D-Phe, 4 F-D-Phe, 4NO2-D-Phe, 4Br-D-Phe or 4CH3S-D-Phe and R4 is D-Trp when prepared by the method of Claim 16 or by an obvious chemical equivalent thereof.

59. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro, R2 is 2,4 Cl-D-Phe, 3,4 Cl-D-Phe, 4 F-D-Phe, 4NO2-D-Phe, 4Br-D-Phe or 4CH3S-D-Phe and R4 is a lipophilic, aromatic D-isomer amino acid when prepared by the method of Claim 17 or by an obvious chemical equivalent thereof.

60. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro, R2 is 2,4 Cl-D-Phe, 3,4 Cl-D-Phe, 4 F-D-Phe, 4NO2-D-Phe, 4Br-D-Phe or 4CH3S-D-Phe and R5 is Leu when prepared by the method of Claim 18 or by an obvious chemical equivalent thereof.

61. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro, R2 is 2,4 Cl-D-Phe, 3,4 Cl-D-Phe, 4 F-D-Phe, 4NO2-D-Phe, 4Br-D-Phe or 4CH3S-D-Phe and R6 is Gly-NH2 when prepared by the method of Claim 19 or by an obvious chemical equivalent thereof.

62. A peptide in accordance with Claim 43 wherein R2 is dichloro-D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe, AcNH-D-Phe, NO2-D-Phe, dinitro-D-Phe, Br-D-phe, dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe and R5 is Leu when prepared by the method of Claim 20 or by an obvious chemical equivalent thereof.

63. A peptide in accordance with Claim 43 wherein X is acrylyl, R2 is dichloro-D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe, AcNH-D-Phe, NO2-D-Phe, dinitro-D-Phe, Br-D-phe, dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe, R3 is D-Trp, R4 is D-Trp and R6 is Gly-NH2 when prepared by the method of Claim 21 or by an obvious chemical equivalent thereof.

64. A peptide in accordance with Claim 43 wherein X is acrylyl, R2 is dichloro-D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe, AcNH-D-Phe, NO2-D-Phe, dinitro-D-Phe, Br-D-phe, dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe, R3 is D-Trp, R4 is D-Trp and R6 is Gly-NH2 when prepared by the method of Claim 22 or by an obvious chemical equivalent thereof.

65. A peptide in accordance with Claim 43 wherein R2 is 4 Cl-D-Phe when prepared by the method of Claim 23 or by an obvious chemical equivalent thereof.

66. A peptide in accordance with Claim 43 wherein R2 is 4 Cl-D-Phe and R4 is D-Trp when prepared by the method of Claim 24 or by an obvious chemical equivalent thereof.

67. A peptide in accordance with Claim 43 wherein R2 is 4 Cl-D-Phe and R4 is imBzl-D-His when prepared by the method of Claim 25 or by an obvious chemical equivalent thereof.

68. A peptide in accordance with Claim 43 wherein R2 is 4 Cl-D-Phe and R4 is a lipophilic aromatic D-isomer amino acid when prepared by the method of Claim 26 or by an obvious chemical equivalent thereof.

69. A peptide in accordance with Claim 43 wherein R2 is 4 Cl-D-Phe, R4 is a lipophilic aromatic D-isomer amino acid and R5 is Leu when prepared by the method of Claim 27 or by an obvious chemical equivalent thereof.

70. A peptide in accordance with Claim 43 wherein R2 is 4 Cl-D-Phe, R4 is a lipophilic aromatic D-isomer amino acid and R6 is Gly-NH2 when prepared by the method of Claim 28 or by an obvious chemical equivalent thereof.

71. A peptide in accordance with Claim 43 wherein X is acetyl, R2 is 4 Cl-D-Phe and R4 is a lipophilic aromatic D-isomer amino acid when prepared by the method of Claim 29 or by an obvious chemical equivalent thereof.

72. A peptide in accordance with Claim 43 wherein X is acrylyl, R2 is 4 Cl-D-Phe and R4 is a lipophilic aromatic D-isomer amino acid when prepared by the method of Claim 30 or by an obvious chemical equivalent thereof.

73. A peptide in accordance with Claim 43 wherein R1 is dehydro-Pro, R2 is 4 Cl-D-Phe and R4 is a lipophilic aromatic D-isomer amino acid when prepared by the method of Claim 31 or by an obvious chemical equivalent thereof.

74. A peptide in accordance with Claim 43 wherein R1 is dehydro-D-Pro, R2 is 4 Cl-D-Phe and R4 is a lipophilic aromatic D-isomer amino acid when prepared by the method of Claim 32 or by an obvious chemical equivalent thereof.

75. A peptide in accordance with Claim 43 wherein R1 is Thz, R2 is 4 Cl-D-Phe and R4 is a lipophilic aromatic D-isomer amino acid when prepared by the method of Claim 33 or by an obvious chemical equivalent thereof.

76. A peptide in accordance with Claim 43 wherein R1 is D-Thz, R2 is 4 Cl-D-Phe and R4 is a lipophilic aromatic D-isomer amino acid when prepared by the method of Claim 34 or by an obvious chemical equivalent thereof.

77. A peptide in accordance with Claim 43 wherein R2 is D-Phe and R3 is D-Trp when prepared by the method of Claim 35 or by an obvious chemical equivalent thereof.

78. A peptide in accordance with Claim 43 wherein X is hydrogen, R2 is D-Phe and R3 is D-Trp when prepared by the method of Claim 36 or by an obvious chemical equivalent thereof.

79. A peptide in accordance with Claim 43 wherein X is acetyl, R2 is D-Phe and R3 is D-Trp when prepared by the method of Claim 37 or by an obvious chemical equivalent thereof.

80. A peptide in accordance with Claim 43 wherein X is acrylyl, R2 is D-Phe and R3 is D-Trp when prepared by the method of Claim 38 or by an obvious chemical equivalent thereof.

81. A peptide in accordance with Claim 43 wherein R2 is D-Phe, R3 is D-Trp and R5 is Leu when prepared by the method of Claim 39 or by an obvious chemical equivalent thereof.

82. A peptide in accordance with Claim 43 wherein R2 is D-Phe, R3 is D-Trp and R5 is N.alpha.MeLeu when prepared by the method of Claim 40 or by an obvious chemical equivalent thereof.

83. A peptide in accordance with Claim 43 wherein R2 is D-Phe, R3 is D-Trp and R6 is Gly-NH2 when prepared by the method of Claim 41 or by an obvious chemical equivalent thereof.

84. A peptide in accordance with Claim 43 wherein R2 is D-Phe, R3 is D-Trp and R6 is NHCH2CH3 when prepared by the method of Claim 42 or by an obvious chemical equivalent thereof.