Note: Descriptions are shown in the official language in which they were submitted.
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
-2-
~ .
.
)',` / rJ 5 1, ,' f,~
1067~37
., :
.
.
(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
: . . . ,::
: ~ -; : : ;
.:
:
~ ~3~ . ~ . :
:
:
. .
.
i~ ~
~ 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.
--4--
.
.
. ,.. , ., ,., .. , . ,.~, ............... ..
~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.
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AHP-6505/6513/6639
i
1067487
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.
: ~ :
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
'
AHP-6505/6513/6639
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: `
~: :
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".
..
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,
--8--
~ 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.
~ *Trade mark
25 ~
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
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` 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.
;
.
-11-
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.
.
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.
:
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.
:
~ : ,
: : :
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~
~ ~ :
: :
~ , :
-14- ; ~
'
.
~ ' ~
"` :
` A~IP-6505/6513/6639
, _ .
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. ~ ;
~ 20
::: ` ::
,
: ~ :
::
~:
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. ; ~ ~ ~
:' .: ' ~ ~ : : ~ '
::: : :
:
-16-
:
:
~ AHP-6sos/6513/6639
1067487
~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.
~ ~*Trade mark
:
: : ::
:
~ ,
:
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.
..
,
:
~ -18-
.
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,
-19-
~ ~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.
,
~: :
:: :
-21-
'`:
1067487 AHP-6505/6513/6639
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
-22-
` A~IP-6505/6513/6639
1067487
: ~'
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. ~ ~
:
~: ,
-24-
/6~ /C~4
87
'
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
-25-
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.
-27-
~` ~ 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;
~:
-28-
` ~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).
EXAMPLE 2
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
:
-29-
AHP-6505/6513/6639
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~
EXAMPLE 3
... .
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.
-31-
AHP-6505/6513/6639
:: ~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
-32-
.
1067487 AHP-6505/6513/6639
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
EXAMPLE 5
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;
-34-
~ :
AIIP-6505/6513/6639
~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~.
35-
.
.
.
AHP-6505/6513/6639
~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.
,
,
:
-36-
:
~ ~ AHP-6505/6513/6639
~L~6~4~37
BXAMPLE 7
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).
-37-
AHP-6505/6513/6639
~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.
-38-
