Note: Descriptions are shown in the official language in which they were submitted.
AHP-6497
~7~35
Back~round o~ th~ ~nv~ti~
1. Field of Invention
This invention relates to a novel process for preparing a
nonapeptide having luteinizing hormone (LH)- and follicle
stimulating hormone tFSH)-releasing activity. In particular, the
present invention relates to a novel process for preparing the
nonapeptide of formula (1), H-Pyr-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-
Pro-NHEt tl~ to salts thereof with pharmaceutically acceptable
acids, to pharmaceutical compositions containing said LH- and
FSH-releasing nonapeptide, and to intermediates obtained in
said process.
LH and FSHare both gonadotrophic hormones elaborated by
the pituitary gland of humans and of animals~ LH together with
FSH stimulates the release of estrogens from the maturing follicles
1~ in the ovary and induces the process of ovulation in the female.
In the male, LH stimulates the interstitial cells and is for that
reason also called interstitial cell stimulating hormone (ICSH).
The follicle-stimulating hormone tFSH) induces maturation of the
fol!icles in the ovary and together with LH, plays an important
role in the cyclic phenomena in the female. FSH promotes the
development of germinal cells in the testes of the male. Both LH
and FSH are released rrom the pituitary gland by the action of LH-
and FSH-releasing hormone, and there is good evidence that said re-
leasing hormone is elaborated in the hypothalamus and reaches the
~5 pituitary gland by a neurohumoral pathway, see e.g. A.V. Schally
et al ., Recent Progress in Hormone Research, 24, 497 t1968).
The natural LH- and FSH-releasing hormone has bsen isolated
from pig hypothalami and its constitution elucidated by A.V. Schally
et al., Biochem. Biophys. Res. Commun., 43, 393 and 1334 (1971), who
' \
. 2
AIIP-6497
~7~S
proposed the decapeptide structure
(pyro)-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2.
The LH- and FSH-releasing hormone may also be represented
by the formula ~~
H-Pyr-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. Comm., 45,
ô22 (1~71) and R. Geiger et al., ibid, 45, 767 (1971).
2. Description of the Prior Art
;
The nonapeptide of formula (I) having LH- and FSH-releasing
activity has been synthesized by D.H. Coy, et al., Biochem. Biophys.
Res. Commun., 57, 335 ~1974) using solid-phase peptide synthesis
methods; the same hormone has also been synthesized by M.Fujino,
et al.~ Arch. Biochem. Biophys., 154, 488 (1973) and M. Fujino,
et al., Biochem. Biophys. Res. Commun., 57, 1248 (1974). In con-
tradistinction to the processes of the references cited above the
process of this invention is simpler and more efficient in giving
considerably better over-all yields than any of the known procedures.
It is a particular advantage of the process of this invention that
it reqùires only a minimum of protective groups for the intermediates,
especially where secondary functions are concerned. Thus, the hydroxyl
group in serine does not have to be protected; the NH-groups in
tryptophan and in histidine do not require protection; and no pro-
tection for the guanidino function in arginine and for the hydroxyl
group of tyrosine is necessary in the later s-tages of the process.
The process of this invenTion is thus also more convenient and less
cumbersome than the processes of Prior Art. An added advantage of
the process of this invention is the fact that the final step thereof
consists in the condensation of two unprotected fragments, each of
which is well defined~ easy to purify, and each obtainable in a high
--3--
,! ~ .. , .. . . ' . `
AHP- 6~97
7~
STate of purity. Also, the final step of the process of -rhis invention
utilizes the az;de coupling method for the condensation of the two
peptide fragments. A noteworthy advantage of the azide coupling method
is that this method gives the least amount o~ racemization compared to
the other methods of peptide condensations, or no detectable racemization
at all. Thus, the important amino acid, D-alanine, is subjected to the
minimum of racemization during the coupling of the peptide fragments. T~e
final product is obtained in a high degree of purity and in good
yields. The product of the process of this invention thus obtained
is thc free, unprotected nonapeptide which does not require any
further deprotective steps, in contradistinction to the processes of
the references cited above where rather severe deprotection conditions
are used to give the final product.
In keeping with the need for a practical synthesis of the
IS nonapeptide of formula (1), the present invention discloses a new
practical process for the large scale preparation of the nonapeptide.
Furthermore the present process has additional advantages in that it
starts from readily available materials, avoids noxious reagents~ is
executedf~cilely,utilizes easily removable protecting groups and
provides a pure product having a high degree of physiological potency.
Summary of the_lnvention ~
The method for preparing tbe biologically active nonapeptide of
formula 1 H-Pyr-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt (1),
comprises condensing the hexapeptide hydrazide 5-oxoprolyi-histidyl-
tryptophyl-seryl-tyrosyl-D-alanine hydrazide (IV) by means of the
azide coupling method with the tripeptide,leucyl-arginyl-proline
ethylamide (V) and isolating said nonapeptide as its acid addition salt
which is converted to the base or a pharmaceutically acceptable acid
, .
AHP-6497
~7D~7~5
addition salts. The above mentioned hexapeptide hydrazide, 5-oxo-
prolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine hydrazide, is
prepared by condensing 5-oxoprolyl-histidyl-tryptophane hydrazide
by means of the azide coupling method with tne protected hydrazide
of seryl-tyrosyl-D-alanine, preferably seryl-tyrosyl-D-alanine
carboxyhydra~ide t-butyl ester (Il), and isolating the protected
hydrazide of the hexapeptide 5-oxoprolyl-h,stidyl-tryptophyl-seryl-
tyrosyl-D-alanine, preferably S-oxoprolyl-histidyl-tryptophyl~seryl-
tyrosyl-D-alanine carboxyhydrazide t-butyl ester (111), which is
treated under moderately acidic conditions to obtain the hexapeptide
hydrazide named above.
The tripeptide fragments 11 and IV and the above sequence
of reactions, omitting the successive introduction and removal of
protecting groups as well as the methods of condensation and the
condensing agents used, is shown below in Fig. 1,
FIGURE I
5-Oxoproline~
~ Histidine ~ _ ..
Tryptophane IV.
Serine - I _
Tyrosine~
l Nonapeptide of
D-Alanine ~ formula (I)
~5 Leucjne -
Arginine ~ . `
Proline
. 5
AHP-64s7
~7~5
Details of the-Invention
In general the abbreviations used herein for designating the
amino acids and the protective groups are based on recommendations
of the IUPAC-IUB Commission on Biochemical Nomenclature, see J. Biol.
Chem., 241, 2~91 (1966). For instance, Pyr, His, Trp, Ser, Tyr, D-Ala,
Leu, Arg and Pro represent "residues" of L-pyroglutamic acid or
5-oxo-L-proline, L-histidine, L-tryptophane, L-serine, L-tyrosine,
D-alanine, L-leucine, L-arginine and L-proline, respectively.
"Residue" means a radical derived from the corresponding amino
acid by eliminating the OH portion of the carboxyl group and the
H portion of the amino group. Except for alanine, which has the
unnatural D-configuration, all the other amino acids have the natural
L-configuraticn.
A number of procedures or techniques for the preparation of
peptides have hitherto been well established. For instance, the
functional group or groups which are not involved in the peptide
bond formation reaction areoptjonally protected by a protecting
group or groups prior to the condensation reaction. For example,
protecting groups for an amino function of a peptide or amino
acid not involved in lhe peptide bond formation are the alkoxy-
carbonyls which include benzyloxycarbonyl (represented by Z),
t-butyloxycarbonyl (represented by Boc),or ~,~-dimethyl-3,5-dimethoxy~
benzyloxycarbonyl trepresented by Ddz). Protecting groups
for the hydroxyl of tyrosine include such ether-forming groups
as benzyl (represented by 8zl) or t-butyl (represented by Bu~
Protecting groups for the guanidino group of arginine include
nitro (represented by N02)or benzyloxycarbonyl
AHP-6497
7~7~5
(represented by Z), or salt ~ormation with a strong acid.
The carboxylic acid function of a peptide or amino acid can be con-
sidered protected by a lower alkyl or lower aralkyl este, which includes
methyl ~represented by OMe), ethyl (represented by OEt) or benzyl
~represented by OBzl), and also by substituted hydrazides which
include t-butyloxycarbonyl hydrazide (represented by NHNH-Boc),
benzyloxycarDonyl hydrazide (represented by NHNH-Z) or ~,~-dimethyl-
3J5-dimethoxybenzyloxycarbonyi hydrazide (represented by NHN~I-Ddz).
To promote facile condensation of the peptide carboxyl group
iO with a free amino group of another peptide to form a new peptide
bond, the terminal carboxyl group must be activated. Examples of
tlle activated form of the terminal carboxyl are acid chlorideJanhydride,
azTde, activated ester, or an o-acyl urea of a dialkylcarbodiimide. Des-
crtptions of these and other protecting and carboxyl-activating groups
are found in general textbooks of peptide chemistry; for example
K.D. KoppleJ "Peptides and Amino Acids", W. A. BenjaminJ Inc., New
York, 1966, pp. 45 - 51 and E. Schr~der and K. LUbke, "The Peptides";
Vol. 1, Academic PressJ New York, 1965, pp. 77 - 128. The following
activated esters have proved to be particularly suitable in the process
of this invention: 2,4,5-trichlorophenyl (represented by OTcp),
pentachlorophenyl (represented by OPcp), p-nitrophenyl (represented
by ONp), succinimido and l-benzotr~iazolylo
The abbreviation Me represents a methyl group, Et represents
an ethyl gro!~p, NHNH2 represents a hydrazide group, and NH2 or NHEt
represents the amide group or the ethylamide group, respectively.
Th~ terms "peptide, polypeptide, tripeptide, hexapeptide,
and the like" às used herein are not limited to refer to the
respective parent peptides but are also used in reference to
.. . .
AHP-6497
~7~ 35
modified peptides having functionalized or protecting groups. In
addition the term "peptide" as used herein is used in reference to
a peptide with one to nine amino acid residues.
The term "lower alkyl" as used herein contemplates hydro-
carbon radicals having one to three carbon atoms and includes methyl,
ethyl and propyl.
The term "mineral acid" as used herein contemplates the
strong inorganic acids and includes hydrochloric, hydrobromicJ
sulfuric, phosphoric and the like. When the term 7s used in con-
junction with an anhydrous system, hydrogen chloride is the
preferred mineral acid.
The term "miIdly acid conditions" as used herein contemplates
conditions in which a dilute aqueous solution of an organic acid,
for example 30 - 90~ aqueous formic, acetic or propionic acid, pre-
ferably 70 - 80%, or I to 10% aqueous trifluoroacetic acid, is a
principal component of the reaction medium.
The term "moderately acidic conditions" as used herein
contemplates conditions in which concentrated organic acids or
aqueous solutions of the mineral acids are used as a principal
component of the reaction medium at temperatures ranging from
about -30 to 30C. Examples of preferred conditions in this
case include the use of 50 to 100~ trifluoroacetic acid at 0 to
30C or of 0.1 - 12N hydrochloric acid at -20 to 10C.
The term "organic nitrite" includes the commercially
available alkyl nitrites, for example, t~butyl nitrite, isoamyl
nitrite~ and the like.
The term "organic base" as used herein includes triethyl-
amine, N-ethylmorpholine, N-methylpiperidine, pyridine, N-ethyl-
diisopropylamine and 1-he like.
AHP-6497
~7~
The term "strong base" as used herein contemplates both
organic bases, as described above, and strong inorganic bases
including the hydroxides and carbonates of sodium and potassium.
The term "azide method" as used herein refers to the
method of coupling two pep-tide fragments which comprises the
reaction of a peptîde hydrazide with a reagent which furnishes nitrous
acid in sjtu. Suitable reagents for this purpose include a lower
alkyl nitrite (e.g. t-butyl nitrite,isoamyl nitrite) or an alkali
metal nitrite salt (e.g. sodium nitrite, or potassium nitrite) in the
presence of a strong acid such as hydrochloric, sulfuric or phosphoric,
acid. The corresponding peptide azide thus obtained is then reacted
wlth a peptide having a free amino group to obtain the desired
pcptide.
Preferred conditions for the azide method of coupling
comprise reacting the peptide hydrazide with the organic nitrite
in the presence of a mineral acid in an anhydrous inert organic
solvent, for example, dimethylformamide, dimethyl sulfoxide, ethyl
acetate, methylene dichloride, tetrahydrofuran, dioxane, and the
like, at -30 to 20C, preferably at about -15C, for 10 - 60
minutes to obtain the coresponding azide, rendering the resulting
mixture alkaline by the addition of a strong base, preferably an
organic base, for example N-ethyldiisopropylamjne, N-ethylmorpholine
or triethylamine, and thereafter reacting the azide in ~he said
mixture with the peptide unit having the free amino group at tempera-
tures ranging from -30C to 20C for about one to two hours and
then at 0 to 30C for 10 to 24 hours. See also the a~ove cited
textbooks of Kopple and Schr~ber and LUbke for additional descriptions
of this method.
.
,.- ~ .
AHP- 6497
71~5
The nonapeptide of formula I obTained by the process of th;s
invention in the form of an acid addition salt possesses LH- and
FSH-releasing properties and is more potent than the natural hormone
when tested in the radioimmunoassay described by Niswender et al.,
Proc. Soc. Exp. Biol. Med., 128, 807 (1968). It is also more potent
in the assay determining induction of ovulation in the ha~ster
described by Arimura et al., Science 174, 511 (1971), and in a
modification of the similar assay in the rat dèscribed by Arimura
et ai., in Endrocinology 80, 515 (1967).
The LH- and FS~-releasing propèrties of the present nonapeptide
and its corresponding pharmaceutically accepta~le salts, which in
turn induce ovulation in animals, render the nonapeptide an useful
agent in veterinary practice and in animal husbandry. It is often
desirable ~o 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 quality, 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 administering to the animals an
ovutation-inhibiting agent, withdrawing administration of said
agent shortly before the date chosen for mating or artificial
insemination, and relying either u~pon the natural production of
~H and FSH to induce ovulation and to produce estrus or by
administering gonadotrophins. However, this procedure was nol
entirely satisfactory because ovulation at a predetermined time
occured never in all the animals together but only in a certain
proportion thereof when gonadotrophins were not used~ On
the other hand, the high cost of gonadotrophins and side effects
\,
-- i û~
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AHP-6~97
4~S
encountered in -~heir administration made this method impractical.
It is now possible to obtain substantially complete synchronization
of ovulation and of estrus, by treating the animals in a given group
first with an ovulation inhibitor which is subsequently withdrawn,
and then administering the LH- and FSH-releasing agent produced by
the process of this invention shortly before the predetermined period
of time for mating or artifical insemination, so as to obtain ovulation
and estrus within that time interval. The delay in the onset of
ovulation and estrus following administration of the nonapeptide produced
by the process of this invention varies with the species of animal,
and the optimal time interval has to be chosen for each species.
For example, in rodents such as rats or hamsters ovulation takes
place within 18 hours following administration of the LH- and FSH-
releasing agent of this invention.
The method described above for obtaining ovulation and
estrus within a precisely predetermined time interval so as to be
certain of a successful mating is particularly important for breeders
of race horses and of show animals, where the fees paid for the services
of an exceptional male animal often amount to very considerable sums
~0 of money. For example, following adminis~ration of the nonapeptide
produced by the process of th~s Invention to cycllng mares the
duration of estrus is shortened by about two days and ovulation takes
place within ~ - 5 days after the onset of estrus. In this manner
the time of ovulation becomes more accurately predictable and the
chances of achieving a successful mating are much better than has
been possible in the past.
The LH- and FSH-releasing agent produced by the process of
this invention is also useful to increase the number of live births
per pregnancy in livestock, for example, cattle, sheep or swine~
For this purpose the nonapeptide of this invention is given in
3 series of parenteral doses, preferably by intravenous or sub-
cutaneous injections, in the range of 10 ~9 to 2000 ~9 per kilogram
A~IP-6~97
~3'7~ 35
of body weight per day, 9~ to 12 hours prior -~o expecte~ estrus and
subsequent mating. A priming injection of 1000 to 5000 IU o~
pregnant mares serum gonadotrophin may also be given one to four
days prior to the above injection of LH- and FSH-releasing hormone.
A similar treatment, with or without prior priming, is also useful
for inducing puberty in farm animals.
When the nonapeptide produced by ~he process of this invention
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 nonapeptide
is determined by its solubility in the given carrier, by the chosen
route of administration, and by standard biological practice. For
parenteral administration to animals the nonapeptide may be used in
a sterile aqueous solution which may also contain other solutes such
as buffers or preservatives, as well as sufficient pharmaceutically
acceptaDle salts or glucose to malce the solution isotonic. The
dosage will vary with the form of administration and with the particular
spec7es of animal to be treated and is preferably kept at a level of
from 50 ~9 to 2000 ~9 per kilogram body weight-. However, a dosage
level in the range of from about IC0 ug to about 1000 ~9 per kilo-
gram body weight is most desirably employed in order to achieve
effective resuIts.
2~ The nonapeptide may also be administered in one of the long
acting, slow-release or depot dosage forms described below, preferably
by intramuscular injection or by implantation. Such dosage forms
are designed to release from about 5 ~9 to about lOO ug per
kilogram body weight ~er day.
-12-
: . , ~ :
, . . : .
AHP-6~97
~'7~713~
The nonapeptide produced by the process of this invention
is also useful in human medicine. For example, human chorionic
gonadotrophin (~G) which contains mainly LH and some FSH has been
used for over 30 years to treat certain endocrinological disorders
such as disturbances of the cycle, amenorrhea, lack of development
of secondary sex characteristics, and infertility in the female, or
certain cases of hypogonadism, delayed puberty, cryptorchidism) 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 1-reatment 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 irnpossibility of giving only the exact
amounts of FSH and LH which are necessary for ovulation. The
administration of the nonapeptide produced by the process of this invention
overcomes the above disadvantage, because the nonapeptide induces release
of LH and FSH by the pituitary only in the exact quantitites which are
required for normal ovulation. For that reason the nonapeptide
produced by the process of this invention is not only useful for the
above purpose, but it is equally useful in the human female in the
treatment of disturbances of the cycle, of amenorrhea, of hypogonadism,
and of lack of development of secondary sex characteristicsO
Furthermore, the LH- and FSH-releasing agent produced by
~5 the process of this invention is also useful in contraception. For
example, when the hormone 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,
or, if fertilization should nevertneless 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
~7~L7~35 AHP-6497
not present and the endometrium is not in Ihe condition necessary for
implantation. On the other hand, when the agent is administered
towards the end of the cycle the endometrium is disrupted and
menstruation takes place.
In addition, the LH- and FSH-releasing agent produced by the
process of this invention is also useful in contraception by the
"rhythm" method, which has always been relatively unreliable
because of the impossibility of predetermining ovulation in the
human female with the required degree of accuracy. Administration
of the agent at mid-cycle, i.e. at about the normally expected time
for ovulation, induces ovulation shortly thereafter and makes the
"rhythm" method both safe and effective.
The LH- and FSH-releasing agent produced by the process
of this invention is also useful as a diagnostic tooi for dis-
tinguishing between hypothalamic and pituitary malfunctions or
lesions in humans. When administering the agent to a patient suspected
of such malfunctions or lesions and if a rise in the level of LH is
subsequently observed, it constitutes good presumptive evidence to
conclude that the hypothalamus is the cause of the malfunction and
that the pituitary is intact. On the other hand, when no rise ir
circulating LH is seen following the administration of the agent
a diagnosis of pituitary malfunction or lesion can be made with
a hiqh degree of confidence. ¦~
In the human male, administration of the LH- and FSH- ¦
releasing agent obtained by the process of this invention provides
the amounts of LH and of FSH necessary for promoting sexual
development in cases of hypogonadism or delayed puberty, and is also
useful In the treatment of cryptorchidism. Furthermore, the FSH
released by the administration of the agent stimulates the deve-
.. ~ .
:
~/~ .
. '
. . ,
AHP-6497
~74~
lopment of germinal cells in the testes, and the agent is useful in
the treatment of non-psychogenic impotence.
When the LH- and FSH-releasing agent obtained by the process
of this invention in the form of an acid addition salt is employed
in human medicine, it is administered systemically, either by
intravenous, subcutaneous, or intramuscular injection, or by
sublingual J nasal, or vaginal administration, in compositions in
conjunction with a pharmaceutically acceptable vehicle or carrier.
For administration by injection or by the nasal route
as drops or spray it is preferred to use the agent in solution in
a sterile aqueous vehicle which may also contain other solutes such
as buffers or preservatives, as well as sufficient quantities of
pllarn~aceutically acceptable salts or of glucose to make the solution
isotonic.
IS The LH- and FSH-releasing agent produced by the process of
this invention may also be administered as nasal or vaginal powders
or insufflations. For such purposes the hormone is administered in
finely divided solid form together with a pharmaceutically accpetable
solid carrier, for example, a finely divided polyethylene glycol
2~ ("Carbowax 1540"), finely divided lactose, or, preferably oniy for
vaginal administration, very finely divided silica ("Cab-O-Sil").
Such composi-tions may also contain~other excipients in finely
divided solid form such as preservatives, buffers,or surface active
agonts. 1;
For sublinguai or vaginal administration it is preferred
to formulate the agent in solid dosage forms such as sublingual
tablets or vaginal inserts or suppositories with sufficient
quantitites of solid excipients such as starch, lac~lose) certain
types of clay, buffers, and lubricating, disintegrating, or surface-
,-- .
-15-
AHP-6497
~379L~35
active agents, or witn semi-solid excipients commonly used in the
formulation of suppositories~ Examples of such excipients are found
in standard pharmaceutical texts, e.g. in Remington's Pharmaceutical
Sciences, Mack Publishing Company, Easton, Pennsylvania, 1970.
The dosage of the LH- and FSH-releasing agent obtained by the
process of this invention will vary with the form of administration
and with the particular subject under treatment. Generally, treatment
is initiated with small dosages substantially less than the optimum
dose of the hormone. Thereafter, the dosage is increased by small
increments until the optimum effect under the circumstances is reachedO
In general, the agent obtained by the process of tnis invention is most
desirably 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 level that is in a range
of from about 10 ~9 to about 2000 ~9 per kilogram body weight, although
as aforementioned variations will occur. However, a dosage level
that is in the range of from about 50 ~9 to a~out IOOO ug per kilo-
gram body weight is most desirably employed in order to achieve
effective results.
; 20 It is often deslrabl~ to administer the agent continuously
over prolonged periods of time in long-acting, slow-release, or
depot dosage forms. Such dosage forms may either contain a pharmaceuti- ¦
cal Iy acceptable salt of the agent having a low degree of solubility in
body fluids, for example one of those salts described below, or they
~5 may contain the agent in the form of a water-soluble salt together
with a protective carrier which prevents rapid releaseO In the
latter case, for example, the agent may be formulated with a non-
antigenic partially hydrolyzed gelatin in the form of a viscous -
liquid; or the agent may be adsorbed on a pharmaceutically acceptable
. .
-16-
. .' ' "
-
- :
AHP-6497
S
solid carrier, for example zi llC hydroxide, and may be administered in
suspension in a pharmaceutically acceptable liquid vehicle; or the
agent may be formulated in gels or suspensions with a protective
non-antigenic hydrocolloid, for example sodium carboxymethylcellulose,
polyvinylpyrrolidone, sodium 31ginate, gelatine, polygalacturonic
acids, for example pectin, or certain mucopolysaccharides, together
with aqueous or non-aqueous pharmaceutically liquid vehicles, pre-
servaTives, 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
agent produced according to the process of this invention may also be
obtained by microencapsulation in a pharmaceutically acceptable
coating material, for example gelatine, polyvinyl alcohol or ethyl
cellulose. Further examples of coating materials and of the pro-
1~ cesses used for microencapsulation are described by J.A. Herbig in
Encyclopedia of Chemical Technology, Vol. 13, 2nd Ed,, Wiley, New
York 1967, pp. 436- 456. Such formulations, as well as suspensions of
salts of the agent which are only sparingly soluble in body fluids,
for example salts with pamoic,alginic or tannic acid are designed
to r^elease from about I ~9 to about 1000 ug of the hormone per
krlogram body weight per day, and are preferably administered by
intramuscular injection. Alternatively, some of the solid dosage
forms list`ed above~ for example certain sparingly water-soluble
salts or dispersions in or adsorbates on solid carriers of salts
~5 of the agent, for example dispersions in a neutral hydrogel of a polymer
of ethylene glycol mothacrylate or similar monomers cross-linked as
described in U.S. Patent 3,5519556 may also be fot^mulated in the
form of pellets releasing about the same amounts as shown above
and may be implanted subcutaneously or intramuscularly.
.
i7-
.
AHP-6497
7~'7~3~
Alternatively, slow-release effec-~s over prolonged periods of
time may also be obtained by administering the agent obtained by the
process of thi~ invention as an acid addition salt i an intra-vaginal
device or in a temporary implant, for example a container made of a
non-irritating sil7cone polymer such as 3 polysiloxane, e.g. "Silastic",
or of a neutral hydrogel of a polymer as described above, possessing
the required degree of permeability to release from about I ~9 to
about 1000 ~9 per kilogram body weight per day. Such intra-vaginal
or implant dosage forms for prolonged administration have the
advan~age that they may be removed when it is desired to interrupt
or to terminate treatment.
Process
The process of this invention is carried out in the following
manner.
A solution of an amino protected and hydroxyl protected
activated ester of tyrosine, preferably benzyloxycarbonyl-0-benzyl-
tyrosine 2,4,5-trichlorophenyl ester, prepared as described by
J~S~ Morley, J. ~hem. Soc., (C), 2410, (1947~, in an inert anhydrous
solvent, preferably dimethylformamide, is added to a solution containing
substantially one molar equivalent of a protected hydrazide of
D-alanine, preferably D-alanine carboxyhydrazide t butyl ester
(prepared as described for the cor~responding '~-isomer by M. Felix
and R.B. Merrifield, J. Amer. Chem. Soc., '~,2, 1385 (1970), in an
inert anhydrous solvent, preferably dimethylformamide, cooled to a
temperature of from about -10C to about 5C. The mixture is kept
at~a temperature of from about -10C to about 5C for about one to
three days and evaporated under reduced pressure. The residue is
taken up in an inert anhydrous solvent, preferably ethyl acetate and
; -i8-
.
AHP-6497
addea to an anhydrous non-polar solvent, preferably diethyl ether. The
precipitate is collected by -fiItration ancl crystallization yields the
corresponding fully protected dipeptide tyrosyl-D-alanine, preferably
benzyloxycarbonyl-O-benzyl-tyrosyl-D-alanine carboxyhydrazide _-butyl
ester, Said last-named compound is dissolved in an inert solvent system,
preferably a mixture of methanol and dimethylformamide, a noble metal
catalyst, e.g. palladium on charcoal~ is added and the mixture is
agitated in an atmosphere of hydrogen at room temperature until
substantially two molar equivalents of hydrogen have been taken up,
FiItration of the catalyst, evaporation of the fiItrate, trituration
of tne residue with a non-polar solvent, preferably diethyl ether,
yields the corresponding protected hydrazide of the dipeptide tyrosyl-D-
alanine, preferably tyrosyl-D~alanine carboxyhydrazide t-butyl ester.
Said last-named compound is dissolved in an inert solvent,
preferably dimethylformamide, cooled to a temperature of about 0C
and mixed with a molar excess, preferably 1.1 to 1.4 molar equivalents,
of an activated ester of a protected serine, preferably benzyloxy-
carbonyl-serine pentachlorophenyl ester, prepared as described by
J. Kovacs et al., J. Org. Chem., 32, 3696 (19~7), in an inert solvent,
preferably dimethylformamide. The mixture is kept at about 0C for
one to three days and the soi~ent is evaporated under reduced pressure.
The residue is taken up in a substantially water-immiscible organic
solvent, preferably ethyl acetate and the solution is dried and
evaporated. The residue is taken up in a mixture of organic solvents
of suitable polarity and an organic base, preferably methanol-ethyl
acetate-pyridine, and the solution îs subjected to chromatography, I
preferab!y on silica. Elution, evaporation of the eluates, and
crystallization yields the corresponding protected hydrazide
\
.. ,, --19--
.
~7~ AHP-6497
of the tripeptjde seryl-tryrosyl-D-alanine in which the terminal
amino ~rou~ is a!so protected, preferably benzyloxy-
carbonyl-seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester.
Said last-named compound is dissolved in an inert solvent,
preferably methanol, a noble metal catalyst, e.g~ palladium on
charcoal, is added and the mixture i5 agitated in an atmosphere of
hydrogen at room temperature until hydrogen is no longer absorbed,
usually -~wo to five hours. FiItration of the catalyst, andevaporation
of the fiItrate yields the corresponding protected hydrazide of the
tr;peptide seryl-ty~osyl-D-alanine, preferably seryl-tyrosyl-D-alanine
carboxyhydrazide t-butyl ester (Il),
A solution of 5-oxoprolyl-histidyl-tryptophan hydrazide,
described by H. Immer et al., J. Med. Chem., 17, 1060 (1974), in an
inert anhydrous solvent, preferably a mixture of dimethylformamide
tS and dimethyl sulfoxide, is cooled to a temperature of from about
-15C to about 5C, mixed with a solution of about two to five
molar equivalents, preferably three molar equivalent of a strong
mineral acid, preferably hydrogen chloride, in an anhydrous organic
solvent, preferably ethyl acetate. The mixture is cooled to about t
-12C and an organic nitrite, preferably t-butyl ~itrîte (1.0 to 1.5
molar equivalents, preferably 1.3 molar equivalents), is added to
the stirred solution. After about 10 to 20 minutes at -15 to -10C
the mixture is rendered alkaline by the addition of an organic base,
preferably three to five equivalents of N-ethyldiisopropylamine,
2S followed by the dropwise addition of substantially one molar equivalent
of a protected hydrazide of the tripeptide seryl-tyrosyl-D-alaniner
preferably seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester,
prepared as described above, in an inert organic solvent, preferably
dimethylformamide. Stirring is continued for 30 to 90 minutes at about
'~ .
r
. _ .
-20-
.-: .-..... : . . :
-~ - A~IP-6497
~7~7~5
0C, and finally for 20 to 30 hours at about 20 lo 30 C. Evaporation
of the solvent, taking up the residue in a lowor alkanol, preferably
methanol, and addition to a non-polar solvent, preferably diethyl ether
or petroleum ether, collection OT the precipitate, ~ollowed by crystalliza-
tion yields the corresponding protected hydra~ide of the hexapeptide5-oxoprolyl-histidyl-tryptophyl-scryl-tyrosyl-D-alanine carboxyhydra~ide
t-butyl ester (111). Said last-named compound is dissolved in tri-
tluoroacetic acid at a temperature of about 0 to 5C, and the
solution Is stirred at about 0 to 5C, for 20 to ~0 minutes and then
at 20 to 30C for another 20 to 40 minutes. Evaporation of the
solvent, taking up the residue in a lowor alkanol, preferably methanol,
addltlon to a non-polar solvent, preferably diethyl ether, and
collection of the precipitate yields the hexapeptide 5-oxoprolyl-
histidyl-tryptophyl-seryl-tyrosyl- ~ alanine hydrazide (IV) as the
trifluoroacetic acid addltion salt. Said last-nar~d compound Is
Isolated In the form of the free base by lon exchange chromatography on
a strongly baslc anionic resih; preferred resins are cross-linked
polystryene resins substituted with strongly bas7c groups, for example
Amberlite* IRA-400 or IRA-410.
A lowcr alkyl ester of arglnyl~prollne In wl~lch tho tormlnal
amlno and the guanldTno groups are protected, preferably t-butyloxy-
carbonyl-nitroarginylproline methyl ester (prepared as described in
U.S. Patent No. 3,835,108, issued September 10, 197~) is dissolvad in
trifluoroacetic acid and the soiution is allowed to stand at room
temperature for 20 to 40 minutes. Evaporation of the solvent,
taking up the residue in a lower alkanol, preferably methanol, addition to
a non-polar solvent, preferably dTethyl ether, and collection of the
preclpitate ylelds the guanldlne proteclod lower alkyl es~er o~ arglnyl-
proline, preferably nitroarginyl-proline methyl ester as the trlfluoro-
acetic acid addition salt. Said last-narned coMpound is dissolved in an
inert organic solvent, preferably dimcthylformamide, containing a
: substantially equlmolar amount of an organic base, preferably
~Trade Mark
~3'
. ~ -21-
A~IP-6497
7~7~5
triethylamine, cooled to a temperature of about 0C and mixed with
a solution containing a substantially equimolar amount of a protected
activated ester of !eucine, preferably benzyloxycarbonyl-leucine
2,4,5-trichlorophenyl ester, in an inert organic solvent, preferably
dimethylformamide, cooled to a temperature of about 0 to 10C. The
mixture is allowed to stand at 0 to 5C for two to four days and
evaporated. The residue is taken up in a substantially water
immiscible organic solvent, preferably ethyl acetate, washed, dried,
and evaporated. The residue is taken up in a solvent of suitable
polarity, preferably a mixture of methanol and ethyl acetate, and sub-
jected to chromatography on silica. Evaporation of the eluate,
dissolving the residue in a lower alkanol, preferably methanol,
addition to a non-polar solvent, preferably diethyl ether, and collection
of the precipitate yields the corresponding protected lower alkyl ester
of leucyl-arginyl-proline, preferably benzyloxycarbonyl-leucyl-
nitroarginyl-proline methyl ester. Said last-named compound is
taken up in a lower alkanol or alkoxyalkanol, preferably methoxy-
ethanol~ an excess (1.1 to 1.5 molar equivalents preferably 1~2
equivalents) of an aqueous alkali metal hydroxide, preferably
sodium hydroxide or potassium hydroxide, is added and tne mixture is
stirred at room temperature for one-half to two hours, preferably
for about one hour. A substantially equimolar amount of the aqueous
alkali metal hydroxide is added and the mixture stirred at 20 to
30C for 20 to 30 hours. The mixture is cooled to a temperature of
2S about 0C, acidified with a strong mineral acid, preferably hydro- il
chloric acid, and evaporated almost to dryness. After addit70n of
water the residue is collected and dried to yield the corresponding pro-
tected tripeptide leucyl-arginyl-proline, preferably benzyloxycarbonyl-
22
.
AHP-64s7
~7~
leucyl-nitroarginyl-proline, Said las-~-named compound and a compound
having a hydroxyl group which is capable of -rorming an activated ester,
pre.'erably l-hydroxybenzotriazole, (0.1 to 2.5 molar equivalents,
preferably about two molar equivalents) in an inert organic solvent,
preferably dimethylformamide is cooled to a temperature of about 0C
and treated with an excess,;preferably of about 1.2 molar equivalents,
of dicyclohexylcarbodiimide, The mixture is stirred at a temperature
of about 0C for about 45 to 75 minutes, then at about 20 to 30C for
about 45 to 75 minutes, and cooled to a temperature of about 0C. Sub-
stantially two molar equivalents of a lower alkylamine, preferably
ethylamine, is added and the mixture is stirred at 20 to 30C for 20 to
30 hours. After removal of tne precipitate, the fiItrate is evaporated.
The residue is dissolved in a substantially water-immiscible organic
solvent, preferably ethyl acetate, washedJ dried and evaporated. The
residue is taken up in an organic solvent of suitable polari~y, pre-
ferably a mixture of methanol and ethyl acetate, and subjected to
chromatography on silica gel. After evaporation of the eluate, the
residue is taken up in a lower alkanol, preferably methanol, and
added to a non-polar solvent9 preferably diethyl ether. The pre-
cipitate is collected and dried to yield the corresponding protected
lower alkylamide of leucyl-arginyl-proline9 preferably benzyloxy-
carbonyl-leucyl-nitroarginyl-proli~ne ethylamide (the latl-er compound
has also been prepared in a different manner as described in the UOS.
Patent No. 3,853,837, issued December 10, 1974). Said last-named
compound is dissolved in an inert solvent, preferably a mixture of
methanol and acetic acid, a noble metal catalyst, e.g. palladium on
charcoal, is added and the mixture is agitated in an atmosphere of
hydrogen at room temperature until hydrogen is no longer absorbed,
preferably for about 15 to 30 hours. FiItration of the catalyst
- -23-
AHP-64g7
3~7~
and evaporation of the filtrate yields a residue of the corresponding
lower alkylamide of the tripeptide leucyl-arginyl-proline, pre-
ferably leucyl-arginyl-proline ethylamide (V), isolated as the
, acetic acid addition salt.
A solution of 5-oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-
alanine hydrazide trifluoroacetate (IV), obtained as described above,
in an inert anhydrous solvent, prefèrably a mixture of dimethylformamide
and dimethyl sulfoxide, is cooled to a temperature of from about
-20C to about -10C, mixed with a solution of about three to seven
molar equivalents, preferably five molar equivalents of a strong
mineral acid, preferably hydrogen chloride, in an inert organic
solvent, preferably ethyl acatate, and the mixture is cooled to a
temperature of from about -30C to about -20C. An organic nitrite,
preferably t-butyl nitrite or isoamyl nitrite (1.0 to 1.5 molar
equivalents, preferably 1.2 molar equivalents), is added with
stirring and the mixture is stirred for 15 to 30 minutes, preferably
for about 20 minutes at a temperature of from about -25C to about
-10C. Sufficient quantities of an organic base, preferably
N-ethyldiisopropylamine, are added with stirring to make the
mixture alkaline, preferably pH 8-9. Keeping the mixture at a
temperature of from about -30C to about -15C, a solution of
leucyl-arginyl-proline ethylamjde ~ diacetate (V) (1~0 to 1.5 molar
equivalents, preferably 1.1 molar equivalents), prepared as described
above.and substantially 2 molar equivalents of an organic base9
preferably N-ethyldiisopropylamine, in an inert solvent, preferably
dimethylformamide,is added with stirring. The mixture is agitated
at a temperature of from about -25C to about -10C for ~5 to 75
minutes, at a temperature of about 0 to 5C for about ~5 to 75
2~
AI~P-6~97
~7~7~3~
minutes, and at a temperature of about 20 to 30C for about 20 to
30 hours. After evaporation, the residue is dissolved in an organic
solvent, preferably methanol, added to a non-polar solvent, preferably
diethyl ether, and the precipitate is collected. The precipitate is
purified by column chromatography in which a variety of supports may
be used, for example, carboxymethylcellulose or chemically modified
cross-linked dextrans such as "Sephadex-~G-25 or L~1-20". Dependlng upon
tl)e support used, the above desTred compound is obtained in the form of
an acid addition salt or as the free base. The preferred method
comprises purlfication of the precipitate by gel tiItration chromato-
graphy on a chemically modified cross-linked dextran ("Sephadex LH-20")
usTng a suitable solvent, preferably methanol, and yields the sub-
stantially pure nonapeptide 5-oxoprolyl-histidyl-tryptophyl-seryl-
tyrosyl-D-alanyl-leucyl-arginyl-proline ethylamide (1), isolated
as the hydrochlortde sal-t. If the acetic acid addition salt of the
nonapeptida is desired, the precipitate is purified by ion exchange
chromatography, for example on carboxymethylcellulose, using an
aqueous solution of ammonium acetate as eluant. Purlficatlon by
partition chromatography on a chemically modified cross-linked dextran
( "Sephadex G-25") using acetic acid as the eluting solvent gives ~he
substantially pure nonapeptido as the acetic acid addition salt. The
free base of the above nonapepttde is obtained by subjecting an acid
addition salt to ion exchange chromalography on a strongly basic
anionic resin, for example one of those listed in Greenstein and
Winit2,"Chemistry of the Amino Acids", John Wiley and Sons, Inc.,
New York and London 1961, Vol. 2, p 1456. Preferred anion exchange
resins are cross-linked polystyrene resins substituted with strongly
basic groups such as Amberlite*lRA - 400 or IRA - 410.
..
c~ -25
AHP-6~97
~74~
It it is desired to obtain a pharmaceutically acceptable acid 1
addition salt ofthe nonapeptide of formula 1, the free base of the non-
apeptide is conYer~ed to pharmaceutlcally accep~ab~a ~cld addltlon
satts by reacting it with acids which are pharmaceutically acceptable.
Examples of such acids are inorganic acids, for example hydrochloric
acid, sulfuric acid or phosphoric acid and organic acids, for example
formtc acid, acetic acid, propionic acid,maleic acid or lactic
acid.
If it is desired to obtain a salt of the nonapeptide of
formula I which is sparingly soluble in water or in body fluids, the
base or an acid addition salt tnereof as obtained by the process of
this invention is treated in aqueous solution with a pharmaceutically
acceptable sparingly water-soluble acid, for example tannic, alginic,
or pamoic acid, preferably in the form of one of their salts, for
example the alkali metal salts. The hormone precipitates as the salt
with the respective sparingly water-soluble acid and is isolated, for
example by filtration or centrifugation.
The above sequence of reactions constituting an embodiment of
the process of this invention is shown in Fig. 2, using the con-
ventional abbreviations for the various amino acids and protective
groups.
The followirg E~amples illustrate this invertion.
-26-
AHP- 6fi97
S
. , . . .... ... . - .~ ,.
_
Z
--_r ~ I ~
A ~
~1^ ~
e ~. j~
~ ~ ~ ~ / _. . ~
I_ N N N N N
_ . . _ __ O
. . 3 . C ~ 3 3 3 I
O
O
I
--27-
ANP-6497
~7~7B~
EXAMPL~ I
Banzyloxycarbonyl-O-benzvl-tyrosvl-D-alan;ne Carboxvhydrazide-t-Butyl Ester
(~-Tyr-D-Ala-NHNH-Boc)
~ Bzl
To a cooled (ice bath), stirred solution of D-alanine
carboxyhydrazide t-butyl ester (6 9, 29.6 mmoles) in dimethylformamide
(50 ml) is added a solution of benzyloxycarbonyl--benzyl-tyrosine
2,4,5-trichlorophenyl ester (17 9, 28.3 mmoles) in dimethyl-formamide
(50 ml). The mixture is kept in an ice bath fôr 2 days. The solvent
is evaporated, the residue dissolved in e-~hyl acetate and precipitated
with ether. The precipitate is crystallized from ethyl acetate giving
the title compound: mp 154 - 156, [~]D5 ~9-9 (c - 1%,dimethylformamide).
-2a-
AHP- 6497
S
EXAMPLE 2
Tyrosyl-D-alanine Carboxyhydrazide t-BUtYl Ester (I-l-Tyr-D-Ala-N~INH-Boc
Benzyloxycarbonyl- ~benzyl-tyrosyl-D-alanine carboxyhydrazide
t-butyl ester ~described in Example 1, 12 9, 20.4 mmoles) is dissolved
in methanol (250 ml~ and dimethylformamide (25 ml) and 5% palladium-
charcoal ca~-alyst (2.5 9) is added unclsr nitrogcn. The hydrogcnation
is carrisd out With th~ hydrog~nation vessel connected to a stirred
solution of sodium hydroxide to absorb carbon dioxide. The mixture
is hydrogenated overnight. The catalyst is removed by filtration
through diatomaceous earth ~ Celite ~, the fiItrate evaporated under
reduced pressure, and the oily residue triturated with ether. The
solid is collected and dried under reduced pressure to give the title
compound.
*Celite
-29-
.
AHP-6497
~37~
~XAMP~E 3
Benzyloxvcarbonyl-seryl-tyrosyl-n-alanjne Carboxyhydrazide t- Butyl Ester
(Z-Ser-Tyr-D-Ala-NHNH-Boc
To a cold (ice bath) solution of tyrosyl-D-alanine carboxy-
hydrazide ~-butyl ester (described in Example 2, 20.4 mmoles in dimethyl-
formamide (50 ml) is added with stirring a solution of benzyloxycarbonyl-
serine pentachlorophenyl ester (11.6 g, 23.8 mmoles) in dimethylformamide
(40 ml). The mixture is kept in an ice bath for 2 days. The solvent
is evaporated, the residue dissolved in ethyl acetate and the solution
washed with 10% citric acid solution, saturated sodium bicarbonate
solution and saturated sodium chloride solution. After drying over
anhydrous magnesium sulfate the solvent is evaporated and the residue
subjccted to column chromatography on silica gel using 1% methanol
1~ and 1% pyridine in ethyl acetate. Crystallization from ethyl acetate-
methanol-benzene-hexane gives the title compound mp 178, [~]D5 -18.4
(c = ~, dimethylformamide1.
~0
-
_
-30
.,
AHP-6~97
~7~7~5
EXAMPLE 4
Seryi-tyrosyi-D-alanine Carboxyhydra~ide t-Butvl-Ester (ll)
(H-Ser-Tyr-D-Ala-NHNH-Boc
Benzylocycarbonyl-5eryl-tyrosyl-D-alarline carboxyhydra.zTde
t-butyl ester (described in Example 3, 5 9, 8.52 mmoles) is dissolved
in methanol (250 ml), 5% palladium-charcoal catalyst (600 mg) is
added and the mixture is agitated under hydroyen in tne same manner as
described in Example 2 for 3 hr. The reaction mixture is filtered
through diatomaceous earth ("Celite")and the flltrate Is evaporated
to dryness under reduced pressure to give the title compound;
amino acid analysis: Ser, 0.92; Tyr, 1.05; Ala, 1~00.
*Trade Mark
-31
AHP-6497
7~S
EXAMPLE 5
5-Oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine Carboxyhydrazide
t-Butyl-Ester tlll) (H-Pyr-His-Trp-Ser-Tyr-D-Ala-NHNH-Boc)
To a stirred solution of 5-oxoprolyl-histidyl-tryptophane
hydrazide (3.71 9, 8.0 mmoles) in dimethyl sulfoxide (24 ml) is added
dimethylformamide (50 ml) followed by a 2.04 N solution of hydrogen
chloride in ethyl acetate (9.5 ml, 19.4 mmoles) at -10C. The mixture is
cooled to -12C and t-butyl nitrite (1.2 ml, 10.4 mmoles) is added. The
solution is stirred at -10C f~r 17 min, cooled to -15C and neutralized
with N-ethyldiisopropylamine (3.32 ml, 19 mmoles). To the mixture is
added dropwise a solution of seryl-tyrosyl-D-alanine carboxyhydrazide
t-butyl ester (described in Example 4, 3.5 9, 7.75 mmoles) in dimethyl-
formamide (45 ml). The solution is stirred at -10 for I hr., at 0C
for I hr. and at room temperature overnight. The solvent is evaporated
under reduced press~re, the oily residue dissolved in methanol (40 ml) and
tne product precipitated with ether (1000 ml). The precipltate is cry-
stallized from methanol giving the t7tle compound mp. 178C
(dec.), [~]D25 _9.4o (c = ~, dimethylformamide), amino acid analysis:
His 1.04, NH3 0.34, Ser 0.90, Glu 1.04, Ala 1.0, Tyr 1.02.
32
AHP-6497
~7~5
~XAMPLE 6
5-Oxoprolyl-histidyl-tryptophy-l-servl-tyrosyl-D-alanine HYdrazide
Trif!uoroacetate (IV) rH-Pyr-His-Trp-Ser-Tyr-~-Ala-NllNH ~ ,CO~H (IV~
S-Oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine
carboxyhydrazide t-butyl ester ~described in Example 5, 1.07 9, 1.14
mmole) is dissolved in cold (ice bath)trifluoroacetic acid (30 ml) with
stirring, The solution is stirred at 0C for ~0 min and then at room
tcmperature for 30 min. The solvent is evaporaled at reduced pressure,
the residue is dTssolved in methanol and the product is precipitated with
ether. The precipitate is collected by fiItration and dried under reduced
pressure to yield the title compound; amino acid analysis: His, 0.9~;
Ser, 0.84; Glu, 1.05; Ala, 1.00; Tyr, 0.97; thin layer chromato9raphy
on slllca gel from n-butanol-water-ethyl acetate-acetic acid (1:1:1:1) gives
Rf 0.55; single spot with ~ and Cl-tolidine reagentsO The tTtle compound
as the trtfluoroacetic acid salt is subjected to ion exchange chromatography
on a strongly baslc anionic resin ("Amberlite*lRA-400") using methanol-water
as the eluant to yield the tftle compound as the free base, amino acid
analysTs: His, 1.01; Ser, 0.81; Glu, 1.03; Ala, 1.00; Tyr, 1.06.
* Trade Mark
. . .. . - . , . . :
AHP-6497
7~
EXAMPLE 7
Nitroarqinvl-proline methyl ester trifluoroacetate,(H-Arlq-Pro-OMe-CF3C02H)
N02
t-Butyloxycarbonyl-nitroarglnyl-proline methyl ester (10.7 9,
25 mmoles) is dissolved in trifluoroacetic acid (125 ml) and the solution is
S left to stand at room temperature for 30 min. Trifluoroacetic acid is
evaporated and the residue is dissolved in methanol (75 ml). The methanolic
solution is added to ether (3000 ml) dropwise under stirring. The pre-
cipitate is collected by fiitration and dried under reduced pressure to
give the title compound.
. 34
AHF-6497
47~S
EXAMPLE 8
Benzyloxvcarbon~l-leucyl-nitroarqinyl-proline Methyl Ester
(;~-Leu-AIrq-Pro-CMe
N02
To a cooled (0~) stirred solution of nitroarginyl-proline
methyl ester (described in Example 7, 2.3 9, 5 mmoles) and dry
triethylamine ~0.8 ml) in dry dimethylformamide (10 ml) is added a
cold solution of benzyloxycarbonyl-leucine 2,4,5-trichlorophenyl ester
(2.3 9, 5 mmoles) in dry dimethylformamide (10 ml). The mixture is
left to stand at 0C for 3 days. The solvent is evaporated under
reduced pressure and th~ residue dissolved in ethyl acetate (150 ml).
The solution is washed with water (3 x 30 ml), IN hydrochloric acid
~2 x 30 ml), 5g sodium bicarbonate solution (2 x 30 ml) and
s~turated sodium chloride solution (2 x 30 ml). After drying over
anhydrous magnesium sulfate, the soivent is evaporated under reduced
pressure, and the residue is subjected to chromatography on silica gel
using 9~ methanol in ethyl acetate as the eluant. After evaporation of
the eluate, the residue is taken Up in methanol and added to ether. The
precipitate is collected by filtration and dried under reduced pressure toyieId
the title compound, nmr (CDC13) ~ 0.90 (d J = 5Hz, 6H), 3.70 (s, 3H),
5.13 (s, 2H), 7.40 (s, 5H). ~-
.
.. . .
- : ~
AHP-6497
7~
EXAMPLE 9
Benzyloxycarbonyl-leucyl-nitroarqinyl-proline (Z-leu-AIrq-Pro-OH)
N02
To a solution of benzyloxycarbonyl-nitroarginyl-proline methyl
ester ~described in Example 8, 2 9, 3.46 mmoles) in methoxyethanol
(20 ml) is added under vigorous stirring IN sodium hydroxide solution
~ ml). After I hr more IN sodium hydroxide (3 ml) is added and the
mixture is stirred overnight. The mixture is cooled in an ice bath and
IN hydrochloric acid solution is added (7O5 ml). The solvent is
evaporated under reduced pressure and the residue precipitated by
addition of water. The precipitate is collected by fiItration and
dried under reduced pressure to give the title compound, nmr (CDC13)
A 0.95 ~d,J = 5Hz, 6H), 5.05 (s, 2H), 7.35 (s, 5H).
-36-
- ,, . : ,
AHP-5497
~7~7~35
EXAMPLE I0
Benzyloxycarbonvl-leucyl-nitroarqinyl-proline Ethylamide
(~-Leu-A~q-Pro-NHEt)
N 2
To a cooled (ice bath) stirred soiution of benzyloxycarbonyl~
leucyl-nitroarginyl-proline (described in Example 9, 7.0 9, 12.4 mmoles)
and l-hydroxybenzotriazole (3.3 9, 24.5 mmoles) in dry, distilled
dimethylformamide is added dicyclohexylcarbodiimide (3.07 9, 14.9 mmoles).
The solution is stirred for I hr a~ 0C and for I hr at room temperature.
The mix1ure is cooled to 0~ and ethylamine ~I.l 9, 1.6 ml, 24.5 mmoles)
is added. The mixture is stirred overnight at room temperature. The
pr~cipitate is removed by fiItration, the fiItrate evaporated and the
r~sidue dissolved in ethyl acetate. The solution is washed with 10%
sodium carbonate solution, water, IN aqueous hydrochloric acid and
saturated sodium chloride solution. The solution is dried over
magnesium sulfate and the solvent evaporated. The residue is subjected
to chromatography on silica gel using 9% metharol in ethyl acetate.
After evaporation of tne eluate, the residue is taken up in methanol and
added to ether. The residue is collected by filtration and dried under
reduced pressure to yield the title compound: [~]2D5 = -33.8~ (c = I~,
dimethylformamide).
-- -37-
AHP-6~97
~t7~Lt7~
EXAMPLE 11
Leucyi-arqinyl-pr-o-!-ine Ethylamide Diacetate (V)
[H-Leu-Arq-Pro-NHEt~2 CH ~ ~H (V)l
Benzyloxycarbonyl-leucyl-nitroarginyl-proline ethylamide
S (described in Example 10, 2.76 9, ~.67 mmoles~ is dissolved in methanol
(69 ml) and glacial acetic acid (69 ml) followed by hydrogenation over 5
palladium-charcoal catalyst (276 mg) in the same manner as de~cribed
in Example 2. After 24 hr., the ca~alyst is removed by filtration
through diatomaceous earth ("Celite") and the filtrate evaporated under
reduced pressure to give the title compound.
Trade Mark
-38-
,~ .
AHP-6497
'1~7~7B5
EXAMPLE 12
5-Oxoprolyl-histidyl-tryptophyl-seryl-tvrosvl-D-alanYl-leucYl-ar-linYl-
proline EthYlamide (I) [H-PYr-His-Trp-Ser-Tyr-D-Ala-Leu-Arq-Pro-NHEt (l)
5-Oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine
hydrazide trifluoroacetate (IV) (descrlbed in Example 6, 4.3 9, 4.25 mmolesl
is dissolved in dimethyl sulfoxide t21 ml), dimethylformamide (21 ml) is
added and the mixture is cooled to -15C. To this mixture is added a solutio
of hydrogen chloride in ethyl acetate (2.69 N, 7.9 ml, 21.2 me~). The
mixture is cooled to -20oc and t-butylnit~ite (527 mg, 0.6 ml), S.l mmoles)
is added. The mixture is stirred at -15C for 20 min, cooled to -20C and
N-ethyldiisopropylamine (5 ml, pH 8) is added. Leucyl-arginyl-proline
ethylamide diacetate (V . described in Example 11, 4.67 mmoles) is
dissolved in dimethylformamide (15 ml) and N-ethyldiisopropylamine
(2.8 ml pH 8), the solution is subjected to reduced pressure in a rotary
evaporator for 30 min and added to the solution of the azide prepared
above at -20Co The mixture is kept at -15C for I hr., at 0C for I hr
and at room temperature overnightO The solvent is evaporated, ~he
residue dissolved in methanol and the crude product precipitated from
4000 ml ether. The precipitate is separated by filtration and dried under
reduced pressure. The precipitate is purified by gel fiItration
chromatography on a chemically modified cross-linked dextran ("Sephadex*
LH-20") using methanol as the eluant. The combtned fracttcnsare concentrated
to dryness under reduced pressure to yield the title compound as the
hydrochloric acid addition salt, ~max 289 (~ 4530)~ 279 (~ 5920)J
220 nm (~ 40,500), amino acid analysis His, 1.02; Arg, 1.15; Ser,
0~89; Glu, 1.06; Pro, 1.24; Ala, 1.00; Leu, 1.01; Tyr, 1.03.
Alternatively, the precipitate is purified by ion exchange
chromatography on carboxymethylcellulose ("Whatman~-CM-23") using
* Trade Mark
-39-
A~IP-6~97
~7~7~3~
aqueous ammonium acetate as eluant. After removal of the solvent trom
the eiuates the ti-tle compound is obtained as the acetic acid addition
sall, amino acid analysis: Ilis, 1.05; Arg, 1.10; Ser, O.B5; GIU~ 1.04;
Pro, 1.19; Ala, 1.00; Leu, 0.95; Tyr, 1.01.
All-ernatively, an acid addition salt is subjected to ion
exchange chromatography on a strongly basic anionic resin (''AmberliteX
I RA - 400~) to yield the title compound as the free base, arnino a^id
analys;s: His, 1.03; Arg, 1.06; Ser, 0.91; Glu, 1.01; Pro, 1.22;
Ala, 1.00; Leu, 1.01; Tyr, 0.93.
Tracle Mark
-40-
.
