PEPTIDES WHICH INHIBIT GONADAL FUNCTION

PEPTIDE INHIBITEUR DE LA FONCTION DES GONADES

English Abstract

PEPTIDES WHICH INHIBIT GONADAL FUNCTION
ABSTRACT
Peptides which inhibit the secretion of gonado-
tropins by the pituitary gland and inhibit the release of
steroids by the gonads. The peptides have the structure:
R1-R2-R3-ser-Tyr-R4 -R5-Arg-R6
wherein R1 is selected from the group consisting of D-pGlu,
D-Pro,D-Trp, D-His, D-Arg,D-Leu, Formyl D-Pro, Acetyl
D-Pro, Benzoyl D-Pro and .beta.-Ala; R2 is selected from the
group consisting of D-Phe, Phe, N.alpha. Me-Phe, His, D-His,
d-Trp, Trp and N.alpha. Me-Leu; R3 is selected from the group
consisting of D-Trp, Trp; D-Phe, Phe, Pro and D-His; R4 is
selected from the group consisting of Gly, D-Trp, D-Phe
and D-Tyr; R5 is selected from the group consisting of Leu
and N.alpha. Me-Leu; and R6 is selected from the group consist-
ing of Pro-Gly-NH2 and Pro-NH-CH2-CH3.

Claims

The embodiments of the invention in which an
exclusive property or privilege is claimed are defined
as follows:
1. A method for making a peptide useful as a
pharmaceutical comprising the steps of preparing an
ester of N.alpha. protected Pro or Gly amino acid and a resin
selected from the group consisting of chloromethylated
resins and benzhydrylamine resins, deprotecting said
amino acid, stepwise coupling N.alpha. and side chain
protected amino acids to said amino acid to form a
resin-coupled peptide having the structure:
R1-R2-R3-Ser-Tyr-R4-R5-Arg-R6
wherein R1 is selected from the group consisting of
D-pGlu, D-Pro, D-Trp, D-His, D-Arg, D-Leu, formyl D-Pro,
acetyl D-Pro, benzoyl D-Pro and .beta.-Ala, R2 is selected
from the group consisting of D-Phe, D-His and D-Trp;
R3 is selected from the group consisting of D-Trp,
D-Phe, and D-His; R4 is selected from the group
consisting of D-Trp, D-Phe and D-Tyr; R5 is selected
from the group consisting of Leu and N.alpha. Me-Leu and R6
is selected from the group consisting of Pro-Gly-NH2
and Pro-NH-CH2-CH3.
2. A method in accordance with Claim 1 where
R1 is D-pGlu, R2 is D-Phe, R3 is D-Trp, R4 is
D-Trp, R5 is Leu and R6 is Pro-Gly-NH2.
3. A method in accordance with Claim 1 wherein
R1 is D-pGlu, R2 is D-Phe, R3 is D-Trp, R4 is
D-Trp, R5 is N.alpha. Me-Leu and R6 is Pro-Gly-NH2.
4. A method in accordance with Claim 1 wherein
R1 is D-pGlu, R2 is D-Phe, R3 is D-Trp, R4 is
ID-Trp, R5 is Leu and R6 is Pro-NH-CH2-CH3.
5. A method in accordance with Claim 1 wherein
R1 is D-pGlu, R2 is D-Phe, R3 is D-Trp, R4 is
D-Trp, R5 is N.alpha. Me-Leu and R6 is Pro-NH-CH2-CH3.

6. A method in accordance with Claim 1 wherein
R1 is D-Trp, R2 is D-Phe, R3 is D-Trp, R4 is
D-Trp, R5 is Leu and R6 is Pro-Gly-NH2.
7. A method in accordance with Claim 1 wherein
R1 is D-Arg, R2 is D-Phe, R3 is D-Trp, R4 is
D-Trp, R5 is Leu and R6 is Pro-Gly-NH2.
8. A method in accordance with Claim 1 wherein
R1 is D-Leu, R2 is D-Phe, R3 is D-Trp, R4 is
D-Trp, R5 is Leu and R6 is Pro-Gly-NH2.
9. A method in accordance with Claim 1 wherein
R1 is D-His, R2 is D-Phe, R3 is D-Trp, R4 is
D-Trp, R5 is Leu and R6 is Pro-Gly-NH2.
10. A method in accordance with Claim 1
wherein R1 is D-Pro, R2 is D-Phe, R3 is D-Trp,
R4 is D-Trp, R5 is Leu and R6 is Pro-Gly-NH2.
11. A method in accordance with Claim 1
wherein R1 is formyl D-Pro, R2 is D-Phe, R3 is
D-Trp, R4 is D-Trp, R5 is Leu and R6 is
Pro-Gly-NH2.
12. A method in accordance with Claim 1
wherein R1 is acetyl D-Pro, R2 is D-Phe, R3 is
D-Trp, R4 is D-Trp, R5 is Leu and R6 is
Pro-Gly-NH2.
13. A method in accordance with Claim 1
wherein R1 is benzoyl D-Pro, R2 is D-Phe, R3 is
D-Trp, R4 is D-Trp, R5 is Leu and R6 is
Pro-Gly-NH2.
14. A method in accordance with Claim 1
wherein R1 is .beta.-Ala, R2 is D-Phe, R3 is D-Trp,
R4 is D-Trp, R5 is Leu and R6 is Pro-Gly NH2.
15. A method in accordance with Claim 1
wherein R1 is D-pGlu, R2 is D-Trp, R3 is D-Trp,
R4 is D-Trp, R5 is Leu and R6 is Pro-Gly-NH2.
16

16. A peptide of the formula:
R1-R2-R3-Ser-Tyr-R4-R5-Arg-R6
wherein R2, R3, R4, R5 and R6 are defined as
in Claim 1, when prepared by the process of Claim 1 or
by an obvious chemical equivalent thereof.
17. A peptide in accordance with Claim 16
wherein R1 is D-pGlu, R2 is D-Phe, R3 is D-Trp,
R4 is D-Trp, R5 is Leu and R6 is Pro-Gly-NH2,
when prepared by the process of Claim 2 or by an obvious
chemical equivalent thereof.
18. A peptide in accordance with Claim 16
wherein R1 is D-pGlu, R2 is D-Phe, R3 is D-Trp,
R4 is D-Trp, R5 is N.alpha. Me-Leu and R6 is
Pro-Gly-NH2, when prepared by the process of Claim 3
or by an obvious chemical equivalent thereof.
19. A peptide in accordance with Claim 16
wherein R1 is D-pGlu, R2 is D-Phe, R3 is D-Trp,
R4 is D-Trp, R5 is Leu and R6 is
Pro-NH-CH2-CH3, when prepared by the process of
Claim 4 or by an obvious chemical equivalent thereof.
20. A peptide in accordance with Claim 16
wherein R1 is D-pGlu, R2 is D-Phe, R3 is D-Trp,
R4 is D-Trp, R5 is N.alpha. Me-Leu and R6 is
Pro-NH-CH2-CH3, when prepared by the process of
Claim 5 or by an obvious chemical equivalent thereof.
21. A peptide in accordance with Claim 16
wherein R1 is D-Trp, R2 is D-Phe, R3 is D-Trp,
R4 is D-Trp, R5 is Leu and R6 is Pro-Gly-NH2,
when prepared by the process of Claim 6 or by an obvious
chemical equivalent thereof.
22. A peptide in accordance with Claim 16
wherein R1 is D-Arg, R2 is D-Phe, R3 is D-Trp,
R4 is D-Trp, R5 is Leu and R6 is Pro-Gly-NH2,
when prepared by the process of Claim 7 or by an obvious
chemical equivalent thereof.
17

23. A peptide in accordance with Claim 16
wherein R1 is D-Leu, R2 is D-Phe, R3 is D-Trp,
R4 is D-Trp, R5 is Leu and R6 is Pro-Gly-NH2,
when prepared by the process of Claim 8 or by an obvious
chemical equivalent thereof.
24. A peptide in accordance with Claim 16
wherein R1 is D-His, R2 is D-Phe, R3 is D-Trp,
R4 is D-Trp, R5 is Leu and R6 is Pro-Gly-NH2,
when prepared by the process of Claim 9 or by an obvious
chemical equivalent thereof.
A peptide in accordance with Claim 16
wherein R1 is D-Pro, R2 is D-Phe, R3 is D-Trp,
R4 is D-Trp, R5 is Leu and R6 is Pro-Gly-NH2,
when prepared by the process of Claim 10 or by an
obvious chemical equivalent thereof.
26. A peptide in accordance with Claim 16
wherein R1 is formyl D-Pro, R2 is D-Phe, R3 is
D-Trp, R4 is D-Trp, R5 is Leu and R6 is
Pro-Gly-NH2, when prepared by the process of Claim 11
or by an obvious chemical equivalent thereof.
27. A peptide in accordance with Claim 16
wherein R1 is acetyl D-Pro, R2 is D-Phe, R3 is
D-Trp, R4 is D-Trp, R5 is Leu and R6 is
Pro-Gly-NH2, when prepared by the process of Claim 12
or by an obvious chemical equivalent thereof.
28. A peptide in accordance with Claim 16
wherein R1 is benzoyl D-Pro, R2 is D-Phe, R3 is
D-Trp, R4 is D-Trp, R5 is Leu and R6 is
Pro-Gly-NH2, when prepared by the process of Claim 13
or by an obvious chemical equivalent thereof.
29. A peptide in accordance with Claim 16
wherein R1 is .beta.-Ala, R2 is D-Phe, R3 is D-Trp,
R4 is D-Trp, R5 is Leu and R6 is Pro-Gly-NH2,
when prepared by the process of Claim 14 or by an
obvious chemical equivalent thereof.
18

30. A peptide in accordance with Claim 16
wherein R1 is D-pGlu, R2 is D-Trp, R3 is D-Trp,
R4 is D-Trp, R5 is Leu and R6 is Pro-Gly-NH2,
when prepared by the process of Claim 15 or by an
obvious chemical equivalent thereof.
19

Description

;5~3t4
The present invention relates to peptides
which inhibit the release of gonadotropins by the pitui-
tary gland in mammalians, including humans. More parti-
cularly, the present invention is directed to peptides
which inhibit gonadal function and the release of the
steroidal hoxmones, progesterone and testosterone.
The pituitary gland is attached by a stalk to
the region in the base of the brain known as thy hypo-
thalamus. The pitui~ary gland has two lobes, the anter-
ior and the posterior lobes. The posterior lobe of the
: pituitary gland stores and passes onto the general cir- -
~ culation two hormones manufactured in~the hypothalamus,
: these being vasopressin and oxytocin. The anterior lobe
of the pituitary gland secretes a numb,er of hormones,.
which are complex protein or glyco-protein molecules
that travel through the blood stream to various organs
and which, in turn, stimulate the secretion into the
hlood stre~m of other hormones from the peripheral or-
gans. In particul~r, follicle stimulating hormone and
luteinizing hormone, sometimes re~erred to as gonadotro-
pins or gonadotropic hormones, are relaesed by the pitui-
tary gland. These hormones, in combination t regulate
the functioning of the gonads to produce testosterone in

s~
the testes and progesterone an~l estrogen in the ovaries, '
as well as regulating the production and maturation of
gametes.
~he release of a hormone by the anterior lobe
of the pituitary gland usually requires a prior release
of another class of hormones produced by the hypothala
mus. One of the hypothalamic hormones acts as a factor
that triggers the release of the gonadotropic hormones,
particularly luteinizing hormone. For convenience, lu-
teni~ing hormone is hereinafter referred to as LH. Thehypothalamic hormone which acts as a releasing factor for
LH is referred to herein as LRF wherein RF stands for
"releasing factor" and the L signifies that the hormone
releases LH. LRF has been isolated and identified.
It has been demonstrated that some female mam-
malians who have no ovulatory cycle and who show no pi-
tuitary or ovarian defect begin to secrete normal amounts
of the gonadotropins. LH and FSH (follicle stimulating
hormone) after the administration of LRF. The adminis-
tration of LRF is suitable for the treatment of those
cases of infertility where the function defect resides in
the hypothalamus. Ovulation can be induced in female ma-
mmalians by the administration of LRF. However, the
dosage level of LRF required to influence ovulation may
sometimes be high. Recent reports have also indicated
that the administration of large and frequent dosages of
LRF actually inhibit gonadal function in female rats by
disruption of the hormonal network. For this reason, LRF
and analogs of LRF which are more potent than LRF to pro-
mote release of LH have been investigated for its poten-
tial use as a contraceptive. The principal disadvantage
to the use of these peptides as a potential contracep-
tive is, of course, the requirement for large and fre-
quent dosages. It would be desirable to provide peptides
which are antagonistic to endogenous LRF and which pre-
vent secretion of LH.
Accordingly, it is a principal object of the

~651~4
present invention to provide peptides which inhibit the
release of gonadotropins in mammalians, including humans.
Another object of the present invention is to
provide peptides which inhibit the release of steroids
5 by the gonads of male and female mammalians, including
humans. A further object of the present invention is to
provide peptides which have an inhibitory effect on the
reproduction processes of mammalians, including humans~
These and other ob~`ects of the present inven-
10 tion will become more apparent from the followingdetailed description.
In one particular aspect, the invention provides
a method for making a peptide useful as a pharmaceutical
comprising the steps of preparing an ester of N~Cprotected
15 Pro or Gly ~mino acid and a resin selected from the group
consisting of chloromethylated resins and benzhydrylamine
resins, deprotecting said amino acid, stepwise coupling
N ~and side chain protected amino acids to said amino acid
to form a resin-coupled peptide having the structure;
Rl-R2 R3~Ser~TYr-R4-Rs-Arg-R6
wherein Rl is selected from the group consisting of
D-pGlu,D-Pro, D-Trp, D-His, D-Arg, D-Leu, formyl D-Pro,
acetyl D-Pro, benzoyl D-Pro and ~ -Ala, R2 is selected
from the group consisting of D-Phe, D-His and D-Trp;
25 R3 is selected from the group consisting of D-Trp~
D-Phe, and D-His; R4 is selected from the group consisting
of D-Trp, D-Phe and D-Tyr; R5 is selected from the group
consisting of Leu and N~ Me-Leu; and R6 is selected from
the group consisting of Pro-Gly-NH2 and Pro-NH-CH2-CH3.
~enerally, in accordance with the present in-
vention, peptides have been synthesized which inhibit the
secretion of gonadotropins by the pituitary gland of
mammalians, including humans and/or inhibit the release
of steroids by the gonads. The peptides act to inhibit
35 the release of gonadotropins.
B

S~L
-3a-
LRF has been characterized as a decapeptide
having the following struc-ture.
p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
Peptides are compounds which contain two or
5 more amino acids in which the carboxyl group of one acid
is linked to the amino group of the other acid. The for-
mula 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
10 right. The position of the amino groups is identified by
numbering the amino groups from left to right. In the
case of L~F, the hydroxyl portion of the carboxyl group
has been replaced with an amino group ~NH2). The abbre-
viations for the individual amino acid groups above are
15 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 arginine
and Pro is proline. Except for glycine, amino acids of the
20 peptides of the invention are of the I.-configuration

s~
--4--
unless not~d otherwise.
It is known that the substitution of D-amino
acids for Gly in the ~-position or the LRF decapeptide
provides a peptide material having from about 1 to 30
times greater potency than does LRF to effect the re-
lease of luteinizing hormone and other gonadotropins by
the pituitary gland of mammalians. The releasing effect
is obtained when the substituted peptide is introduced
into the blood stream of a mammalian.
It is also known that substitution of various
amino acids for His (or the deletion of His) at the
2-position of the LRF decapeptide produces peptide ma-
terials having an i ~ bitory effect on the release of
luteinizing hormone and oth~r gonadotropins by the pi-
tuitary gland of m~mmalians. In particular, varying de-
grees of inhibition of the release of luteinizing hor-
mone are obtained when His is delèted or replaced by Asp,
Cyc, D-Ala,-dca IIi~, ~-Phe and Gly. It has been further
discovered that the inhibitory effect Gf those peptides
modified at the 2-position can be greatly 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-~la-Leu-Arg-Pro-Gly-NH2 is 3 times more po-
tent as an inhibitor for the release of gonadotropins
than is the same peptide where Gly is present in the
6-position rather than D-Ala.
In accordance with the present invention, pep-
tides have been synthesized which are highly potent to
inhibit release of LH. These peptides have been found
to inhibit ovulation of female mammals when administered
at very low levels at proestrous. The peptides are also
effective to cause resorption of fertilized eggs if ad-
ministered shortly after conception.
The peptides of the present invention are rep-
resented by the following formula:
Rl~R2~R3~Ser-l`Yr-R4-R5-Arg-R6
wherein Rl is selected from the group consisting of

i59~
D-pGlu, D-Pro, D-Trp, D-His, D~rg, D-Leu, ~ormyl D-Pro,
Acetyl D-Pro, Benzoyl D-Pro and ~-Ala, R2 is selected
from the group consisting of D-Phe, Phe, N~ Me-Phe, His,
D-His, D-Trp, Trp and N~ Me-Leu; R3 is selected from the
group consisting of D-Trp, Trp; D-Phe, Phe, Pro and D-His; R4
0 is selected from the group consisting of Gly, D-Trp,
D-Phe, and D-Tyr; R5 is selected from the group consist-
ing of Leu and N~Me Leu; and R6is selected from the
group consisting of Pro-Gly-NH2 and Pro-NH-CH2-CH3.
The peptides of the present invention were syn-
thesized by a solid phase technique. The synthesis was
conducted in a step wise manner o~ chloromethylated
resin for those peptides wherein ~ 6is Pro-NH-CH2-CH3 and
on benzhydrylamine resin for those peptides wherein R5 is
Pro-Gly-NH2. The resin was composed of fine beads (20 -
70 microns in diameter) of a synthetic resin prepared by
copolymerization of styrene with 1 to 2~ divinylbenzene.
For chloromethylated resin, the benzene rings in the
resin were chloromethylated in a Friedel-Crafts reaction
with chloromethyl ether and stannic chloride. The
chlorine thus introduced is a reactive benzyl chloride
type of linkage. The Friedel-Crafts reaction is con-
tinued until the resin contains 0.5 to 2 millimoles of
chlorine per gram of resin. The benzhydrylamine resin
was prepared in accordance with the following
(Step A.) FRIEDEL-CRAFTS reaction:
101
C6H5-C-Cl+C6H5-resin AlC13 ~6 5 6 4 (I)
C6H5N2
(Step B.) LEUKART'S reductive amination:
o
NH-CH
O
(I) NH40-~H C6H5-CH-C6H4-resin (II)
..--- ~
160C

;tj~
(Step C.) Hydrolysis, neutralization:
NH2
~II) 1) 6N HCl/8hr _~ 6 5 ~H C6H4-resin
2) triethylamine
As described hereinbelow, the reagents used
will be first listed by their chemical name and their
common abbreviation. Thereafter, the reagent will some-
times be referred to by the common abbreviation.
` In the preparation of peptides wherein ~ is
10 Pro-NH-CH2-CH3, the triethylammonium salt of N Boc pro-
tected Pro is esterified onto the chloromethylated resin
by refluxing in ethanol for about 48 hours. Also pos-
sible is theUSe of the resin or potassium salts in di-
methylformamide (DMF) or dimethylsulfoxide (DMS) respec
15 tively or temperatures ranging from 40 to 80C. After
deprotection and neutralization, the N~ Boc derivative of
next amino acid, Arg is added along with a coupling agent
which is dicyclohexylcarbodiimide (DCC). The side chain
of Arg is protected with tosyl (Tos). Deprotection, neu-
20 tralization and addition of successive amino acids is per-
formed in accordance with the fol:Lowing schedule:
Schedule for coupling of amino acids in solid
phase synthesis of D-pGlu-D-Phe-D-Trp-Ser-Tyr-
D-Trp-Leu-Arg-Pro-NH-CH2-CH3 on 10 grams of
resin.

65i9~
Step Reagents and operations Mix times
_ min.
1 CH2C12 wash 80 ml (2 times) 3
2 ~thanol (~0l;) wash 30 ml (2 times) 3
3 CII~C12 wash 80 ml (3 times) 3
4 50 percent trifluoroacetic acid (TFA)
plus 5 percent 1,2 - ethanedithiol in
CH2C12 70 ml (2 times) 10
CH2C12 wash 80 ml (2 times) 3
6 Triethylamine (Et3N) 12.5 percent in
dimethylfonmamide (DMF) 70 ml
(2 times) 5
7 MeOII wash 40 ml (2 times) 2
8 CH2C12 wash 80 ml (3 times) 3
9 Boc-amino acid (10 mmoles) in 30
ml D~ (1 time) plus dicyclohexylcar-
kodiimide (DCC) (10 mmoles) in DMF 30
M~OH wash 40 ml (2 times) 3
11 Et3N 12.5 percent in DMF 70 ml (1 time) 3
12 M~0~ wash 30 ml (2 times) 3
13 CH2C12 wash 80 ml (2 times) 3
After step 13, an aliquot is taken for a nin-hydrin test: if the
test is negative, go back to step 1 for coupling of the next amuno acid;
if the test is positive or slightly positive, go ack to steps 9
through 13.
The above schedule is used for coupling of each of the amino acids
of the peptide of the invention. ~ Boc protection is used for each of
the remaining amino acids throughout the synthesis. Bzl is used as a
side chain protecting group for Ser and Tyr. 2-6 dichlorobenzyl can be
used as the side chain protecting group for Tyr. p-Toluenesulfonyl (Tbs),
dinitrophenyl (Dnp) or Boc can ~e used as the side chain pro-tecting
group for His. pGlu is introduc~d as benzyloxycarbonyl (z) protected
amino acid.
The above methcd is used to provide the ~ully protected pepti-
doresin Xl~RllR2-R3-Ser (X2)-~yr(X3)-
cg~ Ir
~X .

65g~
R~-R5-~rg-Pro-O~resin in which each oE the protective ~roups are as
defined hereinbelow. If desired the fully protected peptide can be
removed from the resin support by aminolysis employing dimethyLamine,
methylamine, ethylamine, n-propylamine, i-propylamine, butylanune, iso-
butylamine, pen-tylamine~ or phenethylamine to yield the fully protected
alkyl amide intermediate. For example, cleavage of the peptide from
the resin is performed by stirring the resin overnight in distilled
ethylamine to o& in a pressure bottle. After removal of excess
ethylamine by distillation under nitrogen or vacuum, the resin, suspended
in methanol, is removed from the slurry by filtration. The resin is
further washed successively with DMF, methanol, and a mixture of DMF
and methanol. The recovered solution of cleaved, protected peptide
is evaporated to dryness on a rotary vacuum evaporatQr at room temp-
erature. The peptide is taken in a minimum amount of methanol to dissolve
the peptide. The solution is added dropwise to a 50 times volume excess
of dry ether with stirring. A flocculent precipitate appears which is
recovered by filtration or centrifugation. The recovered precipitate is
dried to provide the intermediates which are part of the invention
The inte~rmediates of the invention may be representel as:
Xl-Rl-R2- R3-Ser(X2)-Tyr(~3)-R -R -Arg-Pro-X where X represents
hydrogen or an a-amino protecting group; x2 is a protecting group for
Ser c~nd preferably is benzyl ether (O~zl); X3 is a protecting group for
Tyr selected from the group consisting of OBzl and 2-6 dichlorobenzyl;
and X4 is selected from dimethylamine, alkylamino of 1 to 5 carbon atoms,
phenethylamine, O-CH2-1resin support~ or Gly-O-CM2[resin support] or
Gly-N~I[resin support~-
The criterion for selecting side chain protecting groups for Xl-X
are that the protecting group must be stable to the reagent under the
reaction conditions se-
-8-
cg/~C~

Sg4
_9_
lected for re~oving the -amino protecting group at each
step of the synthesis, the protecting group must not be
split off under coupling conditions and the protecting
group must be removable upon completion of the synthesis
of the desired amino acid sequence under reaction condi-
tions that will not alter the peptide chain.
The group -O-CH2[resin support] or Gly-O-CH2-
[resin support] defining X4 in the intermediates of the
invention represents the ester moiety of one of the many
functional groups of the polystyrene resin support. The
group Gly-NH-[resin support] defining X4 in the inter-
mediates of the invention represents the amide bond of
- Cly to benZhydrylamine resin.
Deprotection of the peptides as well as cleav-
age of the peptide from the benzhydrylamine resin takesplace at 0C with hydrofluoric acid (HF). Aniso~e 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 is effected by ion
exchange chromotography on a CMC column, followed by
partition chromotography using the elution system:
n-butanol; acetic acid; water (4:1:5; volume ratio). The
partition chromotography column packing is Sephadex G 25.
The preparation of those peptides wherein R5 is
Pro-Gly-NH2 on benzhydrylamine resin was in accordance
with the following; N~ and side chain protecting groups
were the same as defined above during the synthesis:
Coupling of a residue was carried out for 1 to
5 hours in methylenechloride, dimethylformamide (DMF) or
mixtures thereof using a 3-5 fold excess of BOC protected
amino acids and dicyclo~thylcarbodiimide (DCC) activating
reagent. The first residue is attached to the benzhydry-
lamine resin by an amide bond. The coupling reaction
through the synthesis was monitored by a ninhydrin test,
as reported by Kaiser et al. Anal. Biochemi. 34 (1970) 595.

~1~6594
--10--
- The deblocking proced~re consisted of a 20 minutes treat-
ment in TFA containing 5 percent, 1,2-ethaneditniol, fol-
lowed by neutralization with triethylamine (Et3N) in DMF
or methylene chloride. Numerous washes with MeOH and
CH2C12 followed each step.
The cleavage of the peptide from the resin and
complete deprotection of the peptide takes place very
readily at 03C with hydrofluoric acid (HF). Anisol was
- added to the resin prior to treatment with HF. After the
removal of HF, under vacuum, the resin was treated with
ether, filtered and the peptide was then eluted with
acetic acid and water. The combined acetic acid-water ex-
tracts were evaporated and subjected to purification.
Purification of the peptide was effected by par-
tition chromatography in a gel filtration column using theelution system; n-Butanol; 0.1N acetic acid; (1:1; volume
ratio). This was followed by a simple gel filtration
using 0.5N acetic acid as eluent.
The peptides are used at a level effective to
prevent ovulation of female mammalian eggs. The peptide
of the invention is effective at levels of 5 milligrams
pex kilogram of body weight when administered at about
noon on the day of proestrous to prevent ovulation. It is
preferred to use dosage levels in the range of from about
.1 to about 10 milligrams per kilogram of body weight.
Higher levels can be used but no significant benefit is
attained through use of higher levels.
The following examples further illustrate var-
ious features of the invention, but are intended to in no
way limit the scope of the invention which is defined in
the appended claims.
EXAMPLE I
The following peptides of the invention having
the indicated formula ware prepared by the solid phase
procedure described hereinabove. For those peptides
wherein R6 is Pro-NH-CI-I2-CH3 a chloromethylated resin is
used. A benzhydrylamine resin is used for those peptides

S9~
wherein R6 is Pro-Gly-NH2.
D-pGlu-R2-R3-Ser-Tyr-R4 R5 Arg R6
Peptide
Composition ~ R3 R4 ~ R6
1 D-Phe D-Trp D-Trp L~u Pro-Gly-NH2
2 Na Me-Phe D-Trp D-Trp Leu Pro-Gly-NH2
3 D-Phe D-Trp D-Trp N Me-Leu Pro-Gly-NH2
4 D-Phe Phe D-Trp Leu Pro-Gly-NH2
D-Phe Pro D-Trp Leu Pro-Gly-NH2
6 D-Phe D-Trp D-Trp Leu Pro-N-CH2-CH3
7 D-Phe D-Trp D-Trp W Me-eu Pro-N-~12-CH3
The peptides set forth in the foregoing table
were assayed in vitro and in vivo. The in vitro assay was
_ _
made using a four day old primary culture of dispersed rat
pituitary cells. The levels of LH mediated in response to
the application of peptides was assayed by specific radio-
immunoassay for rat LH. Control dishes of cells received
only 3 x 10 9 M of LRF: experimental dishes received
_g
3 x 10 M LRF and a concentration of test peptide ranging
from 10 9 M to 10 7 M. Results as expressed in Table I
~In Vitro Column) are expressed as the molar ratio of test
peptide required to reduce the amount of LH released by
3 x 10 M LRF to 50 percent of the control value.
Thereafter, the culture was a~ain innoculated
with 3 x 10 5 M of LRF and the molar ratio of the pep-tide of
the invention required to sup~ess secretion of LH generated
by the LRF was determined. This molar ratio is set forth
hereinbelow in Table I.
- The effectiveness of the peptide compositions of
the invention was also determined in vivo, as follows:
Twenty-seven day old rats were injected with 50 nonograms
of LRF. Thereafter the molar ratio of the peptide re-
quired to suppress secretion of LH generated by the LRF
was determined. This value is set forth hereinbelow in
Table I.

6~9~L
-12-
T~BLE I
In Vitro In Vivo
Molar Ratio Necessary
to Produce Inhibition Molar Ratio to
5 Peptide of LRF (3 x 10 M) Suppress Secre-
Composition Mediated LH Secretion tion of LH
1 3:1 S0:1
2 30:1
3 2:1
10 4 4~1
5:1
6 3:1
7 2:1
Each of the peptides described hereinabove was
15 used to determine its effectiveness to prevent ovulation
in female rats. In this test, ten female rats were in-
jected with one milligram of peptide in corn oil at about
noon on the day of proestrous. Proestrous is the afternoon
before esterous (ovulation). In each case, the peptide was
2~ found to be 100~ effective to prevent ovulation of the fe-
male rats. A separate ten female rat group was used as a
control to which the peptide was not administered. Each of
the ten control rat females had ovulation at estrous.
EXAMPLE II
The following peptides of the inventio~ having
the indicated formula were prepared by the solid phase pro-
cedure described hereinabove.
R -R -R3-ser-Tyr-R4-Leu-Arg-pro-Gly-NH

~l~165~
.
~13-
Peptide Rl R2 R3 R~
Com~o_ition
~ D-Trp D-Phe D-Trp D-Trp
9 D-Arg D-Phe D-Trp D-Trp
510 D-Leu D-Phe D-Trp D-Trp
11 D-His D-Phe D-Trp D-Trp
12 D-pGlu D-Phe D-Trp D-Trp
13 D-Pro D-Phe D-Trp D-Trp
1~ Formyl D-Pro D-Phe D-Trp D-Trp
1015 Acetyl D-Pro D-Phe D-Trp D-Trp
16 Benzoyl D-Pro D-Phe D-Trp D-Trp
17 ~-Ala D-Phe D-Trp D-Trp
18 D-pGlu D-Trp D-Trp D-Trp
Each of the peptides described hereinabove was
15 used to determine its effectiveness to prevent ovulation
in female rats. In this test, ten female rats having an
average weight of 2 kilograms were injected with the level
of the peptide indicated hereinbelow in Table II. The pep-
tide was injected at about noon on the day of proestrous.
2~ The effectiveness of the peptide to prevent ovulation at
the indicated level of injection is indicated hereinbelow
in Table II for the indicated level of use. A separate ten
female rat group was used as a control to which the peptide
was administered. Each of the ten control rat females had
25 ovulation at estrous.

5~4
-14-
TABLE II
.
Peptide Level of Use Number of Rats
Compositlon Micrograms Havlng Ovulation
8 750 9
9 750
500 9
11 750 3
12 150 0
~3 100 4
13 . 250 0
14 500 4
lS --_ ___
16 --_ ___
17 750
18 500 4