Note: Descriptions are shown in the official language in which they were submitted.
~X6~ i0
NONAPEPTIDE AND DECAPEPTID~ ANALOGS OF L~RH
USEFUL AS LHRH ANTAGONISTS
BAC~GROUND OF_ THF INVENTION
Luteinizing hormone (LH) and follicular stimulating
hormone ~FSH) are released from the anterior pituitary
gland under the control of the releasing hormone LHRH
produced in the hypothalamic region. LH and FSH act on
the gonads to stimulate the synthesis of steroid hormones
and to stimulate gamet~ maturation. The pulsatile
release of LHRH, and thereby the release of LH and FS~,
controls the reproductive cycle in domestic animals and
humans.
LHRH also affects the placenta, and the gonads
indirectly, in causing the release of chorionic
gonadotropin (hCG).
Antagonists of LHRH are useful for the control of
fertility. Such antagonists block ovulation in the
female and suppress spermatogenesis in the male. Related
to these effects is a suppression of normal circulating
levels of sexual steroids of gonadal origin, including
reduction in accessory organ weight in the male and the
female. In domestic animals this effect promotes weight
4157J 23620-FF
~6~7~0
--2--
gain in a feed~lot situation, stimulates abortion in
pregnant animals and in general, acts as a chemical
sterilant.
The natural hormone releasing hormone LHRH is a
decapeptide comprised of naturally occuring amino acids
(which have the L-configuration except for the achiral
amino acid glycine). Its sequence is as follows:
(pyro) Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
1 2 3 4 S 6 7 8 9 10
Many analogs of this natural material have been studied
and the very large majority of them have proven to be of
insufficient biological activity to be clinically
useful. Certain select modifications have proven to have
an agonist effect on biological activity. By far the
most significant enhancement is obtained by changing the
6-position residue from Gly to a D-amino acid.
In addition to agonists, analogs have been prepared
which are competitive antagonists to LHRH; all of which
require deletion or teplacement of the histidine residue
at position 2; Vale, W., et al, Science, 176: 933
(1972). In general, it appears that a D-amino acid
placed in the sequence at that position gives the best
activity; Rees, R. W. A., et al, J. Med. Chem. 17: 1016
(1974).
It has also been shown that adding a modification at
the 6 position, which, without the modification at
position 2, results in the agonist activity cited above,
enhances the antagonist activity of the 2-modified
analogs; Beattie, C. W., et al, J. ~ed. Chem., 18: 1247
(1975); Rivier, J., et al, PePtides 1976 p. 427, Editions
de l'Universite de Bruxelles, Belgium (1976).
Against the background of these two major
alterations, which result in a potent series of LHRH
4157J 23620-FF
760
--3--
antagonists; additional increments in antagonist activity
may be had by modifying positions 1, 3 and/or 10 in the
already 2, 6 modified peptide. Coy, D. H., et al
Peptides 19?6, p. 462, Editions de l'Universite de
Bruxelles, Belgium (1976); Rivier, J. E., et al, Life
Sci. 23: 869 (1978); Dutta, A. S., et al, Biochem
Biophys. Res. ~ommun. 81: 382 (1978), Humphries, J., et
al, Biochem. Biophys. Res. Commun., 85: 709 (1978). It
has also been shown that N-acylation of the amino acid at
position 1 is helpful; Channabasavaia, K., et al,
Biochem. Biophys. Res. Commun. 81: 382 ll91); Coy, D.
H., et al, Peptides. - Structure and Biological Function
p. 775, Pierce Chemical Co. (1979). Additionally,
(N-Ac-D-p-Cl-Phel, D-p-Cl-Phe2, D-Trp3, D-Arg6,
D-Alal0)LHRH has been published by D.H. Coy,
Endocrinology, 110, 1445 (1982). In another instance
D-Ala4 modification to LHRH has been reported to retain
antagonist activity. See E. Pedroza, J.A. Martinez, D.H.
Coy, A. Arimura and A.V. Schally; Int. J. Fert.; 23, 294
(1978).
Since antagonists function by competing with LHRH
for the appropriate receptors, high dosages of these
compounds are re~uired in order to block out the natural
peptide. It is especially desirable, in view of this, to
obtain antagonists with a very high degree of potency and
prolonged activity. The ability to be slowly released
from depot formulations will also be important. The
presently known set of analogs requires comparatively
high levels of compound, with the attendant problems of
increased possibility for toxicity and other side effects.
SUMMARY OF THE INVENTION
The present invention refers to novel, highly potent
nonapeptide and decapeptide analogs of LHRH in which a
replacement at position 2, (thus converting the peptide
to the antagonist series) is made more effective by
4157J 23620-FF
~6~7~0
-4-
replacement of the glycine residue at position 6 by a
novel guanido-substituted, amidine, or tertiary or
quaternary amine water sol~ble amino acid residue which
does not occur in nature. Further enhancements by
substitutions at 1, 2, 3, 4, 7 and/or 10 are also
disclosed. The invention is also directed to various
methods of use of these compounds and to pharm~ceutical
compositions therefor. A further aspect of the invention
involves processes for the preparation of the novel
compounds described above.
DETAIL D_ DESCRIPTI_N OF TH~ INVENTION
Description of the _naloqs
~ he present invention relates to novel nonapeptide
and decapeptide analogs of LHRH which have the formula
A-B-C-D-E-F-G-Arg-Pro-H ~I)
1 2 3 4 5 6 7 8 9 10
and the pharmaceutically acceptable salts thereof,
wherein: ~
A is an amino acyl residue selected from the group
consisting of L-pyroglutamyl, D-pyroglutamyl,
N-acyl-D,L-tr~tophanyl, N-acyl-glycyl,
25 N -Ac-D,L- a3 ~ 4-prolyl, N-Ac-D,L-prolyl,
N-Ac-L-alkylprolyl, N-Ac-D,L-phenylalanyl,
N-Ac-D,L-p-chlorophenylalanyl, N-Ac-D,L-seryl,
N Ac-D,L-threonyl, N-Ac-D,L-alanyl,
3-(1-naphthyl)-D,L-alanyl, 3-(2-naphthyl)-D,L-alanyl,
3-(2,4,6-trimethylphenyl)-D,L-alany~,
3-(4-trifluoromethylphenyl)-D,L-alanyl,
3-(9-anthryl)-D,L-alanyl,
3-(2-fluorenyl)-D,L-alanyl, and
3-(Het)-D~L-alanyl wherein Het is a heterocyclic aryl
35 containing radical selected from
4157J 23620-EF
7~
~5--
~ and ~ ~
wherein A" and A' are independently selected from the
group consisting of hydrogen, lower alkyl, chlorine and
bromine, and G is selected from the group consisting of
oxygen, nitrogen and sulfur;
B is an amino acyl residue selected from the group
consisting of D-phenylalanyl, D-p-Cl-phenylalanyl,
D-p-F-phenylalanyl, D-p-nitrophenylalanyl,
3- ( 3, 4, 5-tr imethoxyphenyl)-D-alanyl, 2,2-diphenylglycine,
D-~-methyl-p-Cl-phenylalanine and
3-(2,4,6-trimethylphenyl)-D-alanyl;
C is an amino acyl residue selected from the group
consisting of L-tryptophanyl, D-tryptophanyl,
D-phenylalanyl, D-Me5phenylalanyl,
3-t3-pyridyl)-D-alanyl, 3-(l-naphthyl)-D-alanyl, and
3-(2-naphthyl)-D-alanyl;3-(2-pyridyl)-D-alanyl and
3-(4-pyridyl)-D-alanyl;
D is an amino acyl residue selected from the group
consisting of L-seryl, and D-alanyl;
E is an amino acyl residue selected from the group
consisting of L-phenylalanyl and L-tyrosyl;
F is an amino acyl selected from the group
consisting of the radicals represented by the following
structural formulas:
a)
HzN-CH-CO2H
( l2)n (II)
NH
F~l -C=NR2
4157J 23620-FF
~;~6~'760
--6--
wherein
n is 1 to 5;
Rl is alkyl of 1 to 12 carbon atoms, -NRR3
wherein R is hydrogen or alkyl of 1 to 4 carbon atoms,
R3 is alkyl of 1 to 12 carbon atoms, cycloalkyl,
phenyl, benzyl, -(CH2~n-morpholino or
-(CH2~nN(R4~2 wherein n i5 1 to 5 and R4 is
lower alkyl;
R2 is hydrogen or R3, or Rl and R2 comprise
a ring represented by the following structural formulas:
!
~5 i~ à:Z)n
\
X X
wherein n is 1 to 7; A is hydrogen, alkyl of 1 to 6
carbon atoms or cycloalkyl; and X is halo or A or
b)
H2N -CH-C02H
(IH2)n (III)
R~- ~R6
R7 R8
wherein R5 is alkyl of 1 to 6 carbon atoms, benzyl,
phenylethyl, cyclohexyl, cyclopentyl;
and R6, R7 and R8 are hydrogen or allvl of 1 to 4 carbon
atoms; and n is the integer 2-5; or
4157J 23620-FF
!
~L~69L760
--7--
c) a substituent of the formula
H2N-CH-C02H '`i~2
CH2 (IV) I ~ CH2CHC02H (V)
~ ! ~
N Rg
wherein ~ is hydrogen, alkyl of l to 12 carbon atoms,
phenyl or phenylloweralkyl;
G is an amino acyl residue selected from the group
consisting of L-leucyl, L-norleucyl and L-norvalyl;
H is D-alaninamide, D-leucinamide, glycinamide or
-NHR5 wherein R5 is lower alkyl, cycloalkyl, fluoro
lower alkyl, or NHCONH-Rlo wherein Rlo is hydrogen or
lower alkyl; and the pharmaceutically acceptable salts
thereof.
The present invention includes novel nonapeptide and
decapeptide analogs of LHRH which have the formula
A-B-C-D-E-F-G-Arg-Pro-H (I)
l 2 3 4 5 6 7 8 9 lO
and the .pharmaceutically acceptable salts thereof,
wherein:
A is an amino acyl residue selected from the group
consisting of N-Ac-D,L-~3'4-prolyl, N-Ac-D,L-prolyl,
N-Ac-L-alkylprolyl, N-Ac-D,L-phenylalanyl,
N-Ac-D,L-p-chlorophenylalanyl, N-Ac-D,L-seryl,
N-Ac-D,L-threonyl, N-Ac-D,L-alanyl,
3-(l-naphthyl)-D,L-alanyl, 3-(2-naphthyl)-D,L-alanyl,
3-(2,4,6-trimethylphenyl)-D,L-alanyl, and
3-(4-tri.fluoromethylphenyl)-D,L-alanyl;
4l57J 23620-FF
J~Z~L760
--8--
B is an amino acyl residue selected from the group
consisting of D-phenylalanyl, D-p-Cl-phenylalanyl,
D-p-F-phenylalanyl, D-p-nitrophenylalanyl,
3-(3,4,5-t-imethoxyphenyl)-D-alanyl, 2,2-diphenylglycine,
D-~-methyl-p-Cl-phenylalanine and
3-(2,4,6-trimethylphenyl)-D-alanyl;
C is an amino acyl residue selected from the group
consisting of D-tryptophanyl, D-phenylalanyl,
D-Me5phenylalanyl, 3-(3-pyridyl)-D-alanyl,
3-(1-naphthyl)-D-alanyl, and 3-(2-naphthyl)-D-alanyl;
D is an amino acyl residue selected from the group
consisting of L-seryl, and D-alanyl;
E is an amino acyl residue selected from the group
consisting of L-phenylalanyl and L-tyrosyl;
F is an amino acyl selected from the sroup
consisting of the radicals represented by the following
structural formulas:
a)
20H2N-CH-CO2H
(CH2)n (II)
IH
Rl -C=NR2
wherein
~ is 1 to 5;
Rl is alkyl of 1 to 12 carbon atoms, -NHR3
wherein R3 is alkyl of 1 to 12 carbon atoms,
cycloalkyl, phenyl, benzyl, morpholino or
-(CH~)nN(R4)2 wherein n is 1 to 5 an~ R4 is
lower alkyl;
R2 is hydrogen or R3; or Rl and R2 comprise
a ring represented by the following structural formulas:
4157J 2362Q-FF
~26~7~0
g
1, i i
H~ ~ HN~,C\~
, ~ \ ~ A-C - ~CH2)n
~ ~ A
/ ~
X X
wherein n is l to 7; A is hydrogen, alkyl of l to 6
0 carbon atoms or cycloalkyl; and X is halo or A or
b)
H2N -CH-C02EI
(IH2)n (III)
R~-N-R6
.~
wherein ~ is alkyl of l to 6 carbon atoms, benzyl,
phenylethyl, cyclohexyl, cyclopentyl;
and R6, R7 and R8 are hydrogen or methyl; and n is
the integer 2-5; or
c) a substituent of the formula
NH2
H2N-CH-CO2H
CH2 (IV) ~ ~,,C~CHC02H !V)
~ Rg
R
4l57J 23620-FF
76~
~10--
wherein Rg is hydrogen, alkyl of 1 to 12 carbon atoms,
phenyl or phenylloweralkyl;
G is an amino acyl residue selected from the group
consisting of L-leucyl, 1-norleucyl and L-norvalyl;
H is D-alaninamide, D-leucinamide, glycinamide or
-NHR5 wherein R5 is lower alkyl or NHCONH2; and the
pharmaceutically acceptable salts thereof.
The replacement of the L-histidyl residue which is
at position 2 in LHRH with one of the residues herein
specified is a requirement to convert the peptide to an
LHRH antagonist. The replacement of the glycyl residue
at position 6 in LHRH with one of the residues specified
as F gives a dramatic enhancement of the antagonist
effect. The substitutions disclosed herein at positions
1, 2, 3, 4, 7 and 10 are further helpful in enhancing the
antagonist activity.
Abbreviations and Definitions
As set forth above, and for convenience i~
describing this invention, the conventional abbreviations
for the various common amino acids are used as generally
accepted in the peptide art as recommended by the
I~PAC-IUB Commission on Biochemical Nomenclature,
Biochemistry, 11, 1726 (1972). These represent L-amino
acids, with the exception of the achiral amino acid
glycine, and with the further exception of any unnatural
or natural amino acids which are achiral, or are
otherwise designated as D-, and of those amino acids
which are substituted herein into positions 1, 2, 3, 4,
6, 7 and 10 for those normally found in LHRH. All
peptide sequences mentioned herein are written according
to the generally accepted convention whereby the
N-terminal amino acid is on the left and the C-terminal
amino acid is on the right.
4157J 23620-FF
7~
Certain other abbreviations will be useful in
describing the invention. The present invention employs
replacements by amino aclds which do not occur in
nature. Particularly commonly employed among these are
the following:
Amino acid residue Abbreviation
3-(2-naphthyl)-D-alanyl D-Nal(2)
3-(p-fluorophenyl)-D-alanyl D-p-F-Phe
3-(p-chlorophenyl)-D-alanyl D-p-Cl-Phe
3-(2,3,4,5,6-pentamethylphenyl)-
D-alanyl D-Me5Phe
3 (2,4,6-trimethylphenyl)-D-alanyl D-Tmp
3-(3,4,5-trimethoxyphenyl)-D-alanyl D-Tmo
3-(4-(trifluoromethylphenyl)-D-alanyl D-Ptf
N, N ' -guanido-dimethyl-D-
homoarginine D-Dmh
N,N'-guanido-diethyl-D-
homoarg.inine D-Deh
N,N'-guanido-dipropyl-D-
homoarginine D-Dph
N,N'-guanido-diisopropyl-D-
homoarginine D-Dih
N,N'-guanido-dihexyl-D-
homoarginine D-Dhh
N-guanido-isopropyl-D-
homoarginine D-Iph
N-guanido-heptyl-D-homoarginine D-Hha
N-guanido-propyl-_-homoarginine D-Prh
N,N'-guanido-dicyclohexyl-D-
homoarginine D-Dch
N,N'~guanido-diisopropyl-D-
arginine D-Dia
N,N'-guanido-dicyclohexyl-D-
arginine D-Dca
N-guanido-(3-dimethylaminopropyl)-
4157J ~3620-FF
~6~76~
-l2-
N'-guanido-ethyl-D-homoarginine D-Aph
N~guanido-(3-dimethylaminopropyl)-
N'-guanido-ethyl-D-arginine D-Apa
3-(3-piperidyl)-D-alanine D-3-Pia
3-(4-piperidyl)-D-alanine D-4-Pia
3-((N -methyl)piperid-4-yl)-D-
alanine D-Mpa
3-((N~-pentyl)piperid-4-yl)-D- -
alanine D-Ppa
3-((NC-benzyl)piperid-4-yl)-D-
alanine D-Bpa
D-N-epsilondihydroimidazolinyllysine D-Dhi
3-(2-pyridyl)-D-alanyl D-2Pal
3-(3-pyridyl)-D-alanyl D-3Pal
3-(4-pyridyl)-D-alanyl D-4Pal
As a further convenience, since the amino acid
sequence of LHRH has been shown to be
(pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2,
l 2 3 4 5 6 7 8 9 lO
nona- and decapeptides in which the amino acid residues
at particular places in the sequence have been replaced
by other amino acid residues or other moieties are
abbreviated by showing the nature of the substitution,
25 superscribed by the location, followed by LHRH as the
parentO
Thus, for example, the sequence,
(pyro)Glu-D-p-F-Phe-Trp-Ser-Tyr-D-Dih-Leu-Arg-Pro-Gly-NH2,
30 l 2 3 4 5 6 7 8 8 10
4157J23620-FF,
76~D
- 12a -
in which the Gly at position 6 ~as been replaced by
D-Dih and the His at position 2 has been replaced by
D-p-F-Phe, is represented [D-p-F-Phe2, D-Dih6]LHRH;
and the sequence
NAc-pro-D~p-F-phe-Trp-ser-Tyr-D-Dih-Leu-Arg-pro-NHEt
1 2 3 4 5 6 7 8 9
.._._ -- /
~5
4157J 23620-FF,
-13-
is represented:
~NAc-Prol, D-p-F-Phe2, D Dih6, Pro9-NHEt]LHRH.
As used hereln, the term "pharmaceutically
acceptable salts" refers to salts that retain the desired
biological activity of the parent compound and do not
impart any undesired toxicological effects. Examples of
such salts are (a) acid addition salts formed with
inorganic acids, for example hydrochloric acid,
hydrobromic acid, sulfuric acid, phosphoric acid, nitric
acid and the like; and salts formed with organic acids
such as, for example, acetic acid, oxalic acid, tartaric
acid, succinic acid, maleic acid, fumaric acid, gluconic
acid, citric acid, malic acid, ascorbic acid, ben20ic
acid, tannic acid, pamoic acid, alginic acid,
polyglutamic acid, naphthalenesulfonic acids,
naphthalenedisulfonic acids, polygalacturonic acid; (b)
salts with polyvalent metal cations such as zinc,
calcium, bismuth, barium, magnesium, aluminum, copper,
cobalt, nickel, cadmium, and the like; or with an organic
cation formed from N,N'-dibenzylethylene-diamine or
ethylenediamine; or (c) combinations, of (a) and (b),
e.g., a zinc tannate salt and the like.
The term "lower alkyl" reers to a straight or
branched chain saturated hydrocarbon group having from 1
to 4 carbon atoms such as, for example, methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and
tert-butyl. "Alkyl of 1 to 6 carbon atoms" encompasses
the same substituents as lower alkyl but in addition may
have 5 or 6 carbon atoms such as, for example, a
n-pentyl, n-hexyl or other branched 5 or 6 carbon
membered moiety. "Alkyl of 1 to 12 carbon atoms"
comprises a radical of 1 to 12 carbon atoms and hvdrogen
only as noted above, except that the radical may have up
to 12 carbon atoms. The term "cycloalkyl" refers to a
cyclic saturated hydrocarbon group having from 3 to 6
4157J 23620-FF
-14-
carbon atoms, for exam~le, cyclopropyl, cyclobutyl,
cyclopentyl and cyclohexyl.
For the purpose of this invention the abbreviation
"alkylPro" refers to cis-5-alkyl-L-prolyl residue wherein
alkyl is the same as ~lower alkyl" defined above. More
specifically "MePro" is cis-s-methyl-L-Prolyl, "EtPro" is
cis-5-ethyl-L-prolyl and "ButPro" is
cis-5-n-butyl-L-Prolyl.
The abbreviation "N-Ac" refers specifically to the
N-acetyl amino acid residue in conformance with generally
10 accepted nomenclature.
Preferred Embodiments of the Com~ounds
Compounds which aré preferred embodiments of the
present invention are those wherein A is N-Ac-L-Pro,
15 N-Ac-D-Ser, N-Ac-D-p-Cl-Phe, N-Ac-D-Nal(2); B is
D-p-F-Phe or D-p-Cl-Phe; C is D-Trp, D-Nal(2), D-3Pal or
D-Phe; D is Ser; E is Tyr; F is the compound of Formula
II wherein n is 3 or 4, Rl is alkyl of 1 to 8 carbon
atoms or cyclohexyl and R2 is hydrogen, or a compound
20 of Formula II wherein Rl is -NHR3 wherein R3 is
methyl, ethyl, n-propyl, isopropyl, n-hexyl or cyclohexyl
and R2 is R3 or hydrogen, or F is a compound of
formula II wherein Rl and R2 are dihydroimidazolinyl
or F is a compound of Formula (IV) or (V) wherein ~ is
25 hydrogen, methyl, pentyl or benzyl; and H is D-AlaNH2,
GlyNH2 or NHEt.
Generally speaking, compounds wherein F is
N,N'-guanido-disubstituted-D-argininyl or D-homoargininyl
are among the preferred embodiments.
More preferred embodiments herein are:
A is N-Ac-L-PrO, N-Ac-D-Nal(2) or N-Ac-D-p-Cl-Phe, B
is D-p-F-Phe or D-p-Cl-Phe, C is D-Nal(2), D-Trp, D-3Pal
4157J 23620-FF~
~64760
-14a-
or D-Pi-e, D is Ser, E is Tyr, F is D-Dmh, D-Deh, D-Dph,
D-Dhh or D-Dhi and H is D-AlaNH2 GlyNH2 or NHEt;
N-Ac-L-Pro-D-p-F-Phe-D-Nal(2)-Ser-Tyr-F-Leu-Arg-Pro-
GlyNH2, wherein F is D Dmh, D-Deh, D-Dph, D-Dhh, D-Dhi,
D-Prh or D-Hha;
1 0
, '
,-
,i
,,
/i
,/ .
,'/
_ _ , ,
4157J 23620-FF,
3L~6~ 0
-15-
N-Ac-L-Pro-D-p-Cl-Phe-D-Trp-Ser-Tyr-F-Leu-Arg-Pro-
NHEt, wherein F is D-Dmh, D-Deh, D-Dph, D-Dhh, D-Dhi,
D-Prh or D-Hha;
N-Ac-D-p-Cl-Phe-D-p-Cl-phe-D-Trp-Ser-Tyr-F-Leu-Arg-
Pro-D-AlaNH2, wherein F is D-Dmh, D-Deh, D-Dph, D-Dhh,
5 D-Dhi, D-Prh or D-Hha;
N-Ac-D-p-Cl-Phe-D-p-C1-Phe-D-Trp-Ser-Tyr-F-Leu-Ar~- -
Pro-GlyNH2, wherein F is D-Dmh, D-Deh, D-Dph, D-Dhh, r
D-Dhi, D-Prh or D-~ha; and
N-Ac-D-Nal(2)-D-p F-Phe-D-Trp-Ser-Tyr-F-Leu-Arg-Pro-
10 GlyNH2, wherein F is D-Dmh, D-Deh, D-Dph, D-Dhh, D-Dhi,
D-Prh, D-Hha or propyl amidine.
N-Ac-D-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-F-Leu-
Arg-Pro-GlyNH2, wherein F is D-Dmh, D-Deh, D-Dph, D-Dhh
or D-Dhi;
N-Ac-D-Nal~2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-F-Leu-
Arg-Pro-D-AlaNH2, wherein F is D-Dmh, D-Deh, D-Dph,
D-Dhh or D-Dhi;
N-Ac-D-Nal(2)-D-p-Cl-Phe-D-3Pal-Ser-Tyr-F-Leu-
Ar~-Pro-GlyNH2, wherein F is D-Dmh, D-Deh, D-Dph, D-Dhh
20 or D-Dhi;
N-Ac-D-Nal(2)-D-p-Cl-Phe-D-3Pal-Ser-Tyr-F-I,eu-
Arg-Pro-D-AlaNH2, wherein F is D-Dmh, D-Deh, D-Dph,
D-Dhh or D-Dhi.
The most particularly preferred embodiments are:
N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-
Pro-NHEt;
N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-Arg-
Pro-NHEt;
N-Ac-_-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-
30 Pro-NHEt;
N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-
Pro-NHEt,
N-Ac-D-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-
Pro-NHEt;
4157J 23620-FF
~6~
--15a-
N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-Arg-
Pro-GlyNH~;
N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser~Tyr-D-Deh-Leu-Arg-
Pro-GlyNH2;
N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dph-Leu Arg-
Pro-GlyNH2;
N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-
Pro GlyNH2;
N-Ac-D Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dhi-Leu-Arg-
10 Pro-GlyNH2;
N-Ac-D-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-Arg-
Pro-GlyNH2;
N-Ac-D-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-
Pro-GlyNH2;
N-Ac-D-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-Arg-
Pro-GlyNH2;
N-Ac-D-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-
Pro-GlyNH2;
N-Ac-D-Nal(2)-D-p-C1-Phe-D-Trp-Ser-Tyr-D-Dhi-Leu-Arg-
20 Pro-GlyNH2;
N-Ac-D-Nal(2)-D-p-F-Phe-D-3Pal-Ser-Tyr-D-Deh-Leu-Arg-
Pro-D-AlaNH2;
N-Ac-D-Nal(2)-D-p-F-Phe-D-3Pal-Ser-Tyr-D-Deh-Leu-Arg-
Pro-D-GlyNH2;
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-3Pal-Ser-Tyr-D-Deh-Leu-
Arg-Pro-D-AlaNH2;
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-3Pal-Ser-Tyr-D-Deh-Leu-
Arg-Pro-D-GlyNH2;
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-
30 Arg-Pro-GlyNH2;
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-
Arg-Pro-GlyNH2;
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-
Arg-Pro-GlyNH2;
4157J 23620-FF
~X6476()
-16-
N-Ac~D-p-Cl-Phe-D-p-Cl-Phe-D-Tr?-Ser-Tyr D-Dhh-Leu-
Arg-pro-GlyNH2;
N-Ac-D-p-cl-phe-D-p-cl-phe-D-Trp-ser-Tyr-D-Dhi-Le
Arg-Pro-GlyNH2;
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Deh Leu-
Arg-Pro-D-AlaNH2;
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-
Arg-Pro-D-AlaNH2;
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp Ser-Tyr-D-Dhh-Leu-
10 Arg-Pro D-AlaNH2;
N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-
Arg-Pro-D-AlaNH2;
N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-
Arg-Pro-D-AlaNH2;
N-Ac-D-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-
Arg-pro-D-AlaNH2;
N-Ac-D-Nal(2)-D-p-C1-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-
Arg-Pro-D-AlaNH2;
N-Ac-D-Nal(2)-D-p-C1-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-
Arg-Pro-D-AlaNH ;
N-Ac-D-Nal(2)-D-p-C1-Phe-D-Trp-Ser-Tyr-D-Dhi-Leu-
Arg-Pro-D-AlaNH2;
N-Ac-D-Nal(2)-D-p-Cl-Phe-D-3Pal-Ser-Tyr-D-Dmh-Leu-
Arg-Pro-D-AlaNH2;
N-Ac-D-Nal(2)-D-p-Cl-Phe-D-3Pal-Ser-Tyr-D-Deh-Leu-
25 Arg-pro-D-AlaNH2;
N-Ac-D-Nal(2)-D-p-Cl-Phe-D-3Pal-Ser Tyr-D-Dph-Leu-
Arg-Pro-D-AlaNH2;
N-Ac-D-Nal(2)-D-p-Cl-Phe-D-3Pal-Ser-Tyr-D-Dhh-Leu-
Arg-Pro-D-AlaNH2;
N-Ac-D-Nal(2)-D-p-C1-Phe-D-3Pal-Ser-Tyr-D-Dhi-Leu-
Arg-Pro-D-AlaNH2;
N-Ac-D-Nal(2)-D-p-C1-Phe-D-3Pal-Ser-Tyr-D-Dmh-Leu-
Arg-Pro-GlyNH2;
4157J 23620-FF~
~6fl~6~
-16a-
N-Ac-D-Nal(2)-D-p-Cl-Phe-D-3Pal-Ser-Tyr-D-Deh-Leu-
Arg-Pro-GlyNH2;
N-Ac-D-Nal~2)-D-p-cl-phe-D-3pal-ser-Tyr-D-Dph-Le
Arg-Pro-GlyNH2;
~ -Ac-D-Nal(2)-D-p-Cl-Phe-D-3Pal-Ser-Tyr-D-Dhh-Leu-
Arg-Pro-GlyNH2; and
N-Ac-D-Nal(2)-~-p-Cl-Phe-D-3Pal-Ser-Tyr-D-Dhi-Leu-
Arg-Pro-GlyNH~.
In all of the above embodiments, the compound may --
0 also be prepared as the corresponding pharmaceutically
acceptable salt.
Assay Proc_dures
The compounds of this invention and, particularly,
the salts thereof, exhibit surprisingly potent and long
lasting LHRH antagonist activity.
Primary measures of potency are ability to inhibit
ovulation in rats, as assayed by the procedure of Corbin,
A. and Beattie, C. W., Endocrine Res. Commun., 2:1 (1975)
and ability to inhibit LH release and ovulation in the
rabbit, as per Phelps, C. P., et al, Endocrinology 100:
1526 (1977).
Other bioassays which are used for LHRH antagonists
and for the compounds of the present invention are:
(a) inhibition of LHRH induced FSH and LH release
in the rat, in vivo; Vilchez-Martinez, J.A.,
et al, Endocrinology, 96: 1130 (1975); and,
(b) inhibition of LH and FSH release by dispersed
anterior pituitary cell cultures as measured
by radioimmuno assay. (Vale, W., et al,
Endocrinology 91: 562 (1972).
Antagonist Effects and Utilities
The following utilities flow from the antagonist
effect of the compounds herein:
4157J ~3620-FF
~;~6~61~)
-17-
- female contraception;
- ovulation suppression or delay;
- induction of parturition;
- synchronization of ovulation;
- estrus suppression;
- growth promotion in female animals;
- luteolysls, menses induction;
- early, first trimester abortifacient;
- therapy for endometriosis;
- therapy for mammary tumors and cysts
- therapy for polycystic ovary syndrome
(Stein-Leventhai);
- therapy for uterine carcinoma;
- therapy for benign prostatic hypertrophy and for
prostatic carcinoma;
- male contraception;
- therapy for diseases which result from excessive
gonadal hormone production in either sex;
- functional castration in male food producing
animals;
- suppression of proestrous bloody discharge in dogs;
- suppression of menopausal symptoms.
The aspect of the present invention which relates to
particular uses for the above-described compounds is
concerned with these utilities, most particularly,
inhibition of ovulation and treatment of endometriosis in
the female, and inhibition of spermatogenesis and
treatment of prostatic tumors in the male.
In the practice of the method of this invention an
effective amount of a compound of the invention or a
pharmaceutical composition containing same is
administered to the subject in need of, or desiring, such
treatment. These compounds or compositions may be
administered by any of a variety of routes depending ~pon
the specific end use, including orally, parenterally
~157J 23620-FF
~26~7~iO
-18-
~including subcutaneouc~ intramuscular and intravenous
administration), vaginally (particularly for
contraception), rectally, buccally (including
sublingually), transdermally or intranasally. The most
suitable route in any given case will depend upon the
use, particular active ingredient, the subject involved,
and the judgment of the medical practitioner. The
compound or composition may also be administered by means
of slow-release, depot or implant formulations as
described more fully herein below.
In general for the uses herein above described, it
is expedient to administer the active ingredient in
amounts between about 0.01 and 10 mg~kg body weight per
day, prefera~ly between about 0.1 and 5.0 mg/kg body
weight per day. This administration may be accomplished
by a single daily administration, by distribution over
several applications or by slow release in order to
achieve the most effective results~
~ he exact dose and regimen for administration of
these compounds and compositions will necessarily be
dependent upon the needs of the individual subject being
treated, the type of treatment, the degree of affliction
or need and, of course, the judgment of the medical
practitioner. In general, parenteral administration
requires lower dosage than other methods of
administration which are more dependent upon absorption.
A further aspect of the present invention relates to
pharmaceutical compositions containing as active
ingredient a compound of the present invention which
compositions comprise such compound in admixture with a
pharmaceutically acceptable, non-toxic carrier. As
mentioned above, such compositions may be prepared for
use for parenteral (subcutaneous, intramuscular or
intravenous) administration particularly in the form of
liquid solutions or suspensions; for use in vaginal or
4157~ ,3620-FF
6~'76~)
-19-
rectal administration particularly in semisolid forms
such as creams and suppositories; for oral or buccal
administration particularly in the form of tablets or
capsules; or intranasally particularly in the form of
powders, nasal drops or aerosols.
The compositions may conveniently be administered in
unit dosage form and may be prepared by any of the
methods well-known in the pharmaceutical art, for example
as described in Remington's Pharmaceutical Sciences, Mack
Publishing Company, Easton, PA., 1970. Formulations for
parenteral administration may contain as common
excipients sterile water or saline, polyalkylene glycols
such as polyethylene glycol, oils of vegetable origin,
hydrogenated naphthalenes and the like. Formulations for
vaginal or rectal administration, e.g. suppositories, may
contain as excipients, for example, polyalkyleneglycols,
vaseline, cocoa butter, and the like. Formulations for
inhalation administration may be solid and contain as
excipients, for example, lactose or may be aqueous or
oily solutions for administration in the form of nasal
drops. For buccal administration typical excipients
include sugars, calcium stearate, magnesium stearate,
pregelatinated starch, and the like.
It is particularly desirable to deliver the
compounds of the present invention to the subject over
prolonged periods of time, for example, for periods of
one week to one year from a single administration.
~arious slow release, depot or implant dosage forms may
be utilized. For example, a dosage form may contain a
pharmaceutically acceptable non-toxic salt of the
compound which has a low degree of sclubility in body
fluids, for example, (a) an acid addition salt with a
polybasic acid such as phosphoric acid, sulfuric acid,
citric acid, tartaric acid, tannic acid, pamoic acid,
alginic acid, polyglutamic acid, naphthalene mono- or
4157J 23620-FF
o~
di-sulfonic acids, polygalacturonic acid, and the like;
(b) a salt with a polyvalent metal cation such as zinc,
calcium, bismuth, barium, magnesium, aluminum, copper,
cobalt, nickel, cadmium and the like, or with an organic
cation formed from e.g., ~,N'-dibenzylethylenediamine or
ethylenediamine; or (c) combinations of (a) and (b) e.g.
a zinc tannate salt. Additionally, the compounds of the
present invention or, preferably, a relatively insoluble
salt such as those just descri~ed, may be formulated in a
gel, for example, an aluminum monostearate gel wi~h, e.g.
sesame oil, suitable for injection. Particularly
preferred salts are zinc salts, zinc tannate salts,
pamoate salts, and the like. Another type of 510w
release depot formulation for in~ection would contain the
compound or salt dispersed or encapsulated in a slow
degrading, non-toxic, non-antigenic polymer such as a
polylactic acid/polyglycolic acid polymer for example as
described in U.S. 3,773,919. The compounds or,
preferably, relatively insoluble salts such as those
described above may also be formulated in cholesterol
matrix pellets, particularly for use in animals.
Additional slow release, depot or implant formulations,
e.g. liposomes, are well known in the literature. See,
for example, Sustained and Controlled Release Drug
Delivery Systems, J. R. Robinson ed., Marcel Dekker,
Inc., New York, 1978. Particular reference with respect
to LHRH type compounds may be found, for example, in U.S.
4,010,125.
SYnthesis of the Pe~tides
..
The polypeptides of the present invention may be
synthesized by any techniques that are known to those
skilled in the peptide art. An excellent summary of the
many techniques so available may be found in J.M. Stewart
and J.D. Young, Solid Phase Peptide Synthesis, W.H.
Freeman Co., San Francisco, 1969, and J. Meienhofer,
4157J 23620-FF
7~i~
-21-
Hormonal Proteins and Peptides, Vol. 2, p. 46., Academic
Press (~ew York), 1973 for solid phase peptide synthesis
and E. Schroder and K. Lubke, The Peptides, vol. 1,
Academic Press (New York), 1965 for classical solution
synthesis.
In general, these methods comprise the sequential
addition of one or more amino acids or suitably protected
amino acids to a growing peptide chain. Normally, either
the amino or carboxyl group of the first amino acid is
protected by a suitable protecting group. The protected
or derivatized amino acid can then be either attached to
an inert solid support or utilized in solution by adding
the next amino acid in the sequence having the
complimentary (amino or carboxyl) group suitably
protected, under conditions suitable for forming the
amide linkage. The protecting group is then removed from
this newly added amino acid residue and the next amino
acid (suitably protected) is then added, and so forth.
After all the desired amino acids have been linked in the
2~ proper sequence, any remaining protecting groups (and any
solid support) are removed sequentially or concurrently,
to afford the final polypeptide. By simple modification
of this general procedure, it is possible to add more
than one amino acid at a time to a growing chain, for
example, by coupling (under conditions which do not
racemize chiral centers) a protected tripeptide with a
properly protected dipeptide to form, after deprotection,
a pentapeptide.
Preferred Embodiments of Synthesis
A particularly preferred method of preparing
compounds of the present invention involves solid phase
peptide synthesis.
In this particularly preferred method the ~-amino
function of the amino acids is protected by an acid or
base sensitive group. Such protecting groups should have
415~J 23620-FF
7~i~
-22-
the properties of being stable to the conditions of
peptide linkage formation, while being readily remsvable
without destruction of the growing peptide chain or
racemization of any of the chiral centers contained
therein. Suitable protecting groups are t-butyloxy-
carbonyl (Boc), benzyloxycarbonyl (Cbz), biphenyl-
isopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxy-
carbonyl, ],]-dimethyl-3,5-dimethoxyben7yloxycarbonyl,
o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl,
9-fluorenylmethyloxycarbonyl and the like, especially
t-butyloxycarbonyl (Boc).
Particularly preferred side chain protecting groups
are, for arginine:nitro, ~-toluenesulfonyl, 4-methoxy-
benzenesulfonyl, Cbz, Boc and adamantyloxycarbonyl; for
tyrosine:benzyl, o-bromobenzyloxycarbonyl, 2,6-dichloro-
benzyl, isopropyl, cyclohexyl, cyclopentyl and acetyl;
for serine:benzyl and tetrahydropyranyl; for
histidine:benzyl, ~-toluenesulfonyl and 2,4-dinitrophenyl.
The C-terminal amino acid is attached to a suitable
solid support. Suitable solid supports useful for the
above synthesis are those materials which are inert to
the reagents and reaction conditions of the stepwise
condensation-deprotection reactions, as well as being
insoluble in the media used. Suitable solid supports are
chloromethylpolystyrene-divinylbenzene polymer, hydroxy-
methyl-polystyrene-àivinylbenzene polymer, and the like,
especially chloromethyl-polystyrene-1% divinylbenzene
pclymer. For the special case where the C-terminus of
the compound will be glycinamide, a particularly useful
support is the benzhydrylamino-polystyrene-divinylbenzene
polymer described by P. Rivaille, et al, _lv. Chim.
Acta., 54, 2772 (1971). The attachment to the
chloromethyl polystyrene-divinylben7ene type of resin is
made by means of the reaction of the N~-protected amino
acid, especially the Boc-amino acid, as its cesium,
4157J 23620-FF
~.2~
23-
tetramethylammonium, triethyla~monium, 1,5-diazabicyclo-
[~.4.01undec-5-ene, or similar salt in ethanol,
acetonitrile, N,N-dimethylformamide ~DMF), and the like,
especially the cesium salt in DMF, with the chloromethyl
resin at an elevated temperature, for example between
about 40 and 60C, preferably about 50C, for from about
12 to 48 hours, preferably abou~ 24 hours~ The N~ Boc-
amino acid is attached to the benzhydrylamine resin by
means of an N 9 N'-dicyclohexylcarbodiimide
(DCC)/l-hydroxybenzotriazole (HBT) mediated coupling for
from about 2 to about 24 hours, preferably about 12 hours
at a temperature of between about lO and 50C, preferably
25C in a solvent such as dichloromethane or DMF,
preferably dichlormethane. The coupling o~ successive
protected amino acids can be carried out in an automatic
polypeptide synthesizer 25 is well known in the art. The
removal o~ the N~-protecting groups may be performed
in the presence of, for example, a solution of trifluoro-
acetic acid in methylene chloride, hydrogen chloride in
dioxane, hydrogen chloride in acetic acid, or other
strong acid solution, preferably ~0% trifluoroacetic acid
in dichloromethane at about ambient temperature. Each
protected amino acid is preferably introduced in
approximately 2.5 molar excess and the coupling may be
carried out in dichloromethane, dichloromethane/DMF
mixtures, DMF and the like, especially in methylene
chloride at about ambient temperature. The coupling
agent is normally DCC in dichloromethane but may be
N,N'-di-~so-propylcarbodiimide (DIC) or other
carbodiimide either alone or in the presence of HBT,
N-hydroxysuccin-imide, other N-hydroxyimides or oximes.
Alternately, protected amino acid active esters (e.g.
p-nitrophenyl, pentafluorophenyl and the like) or
symmetrical anhydrides may be used.
4157J 23620-FF
7~0
-24-
At the end of the solid phase synthesis the fully
protected polypeptide is removed from the resin. When
the linkage to the resin support is of the benzyl ester
type, cleavage is by means of aminolysis with an
alkylamine or fluoroalkylamine for peptides with 2
proline C-terminus, or by aminolysis with, for example,
ammonia/methanol or ammonia/ethanol for peptides with a
glycine C-terminus at a temperature between about 10 and
50C, preferably about 25C, for between about 12 and 24
hours preferably about 18 hours. Alternatively, the
peptide may be removed from the resin by transesteri-
fication, e.g., with methanol, followed by aminolysis.
The protected peptide may be purified at this point by
sillca gel chromatography. The removal of the side chain
protecting groups from the polypeptide is performed by
treating the aminolysis product with, for example,
anhydrous liquid hydrogen fluoride in the presence of
anisole or other carbonium scavenger, treatment with
hydrogen fluoride/pyridine complex, treatment with
tris(trifluoroacetyl)boron and trifluoroacetic acid, by
reduction with hydrogen and palladium on carbon or
polyvinylpyrrolidone, or by reduction with sodium in
liquid ammonia, preferably with liquid hydrogen fluoride,
and anisole at a temperature between about -10 and +10C,
preferably about 0C, for between about 15 minutes and 1
hour, preferably about 30 minutes. For the glycine
terminal peptides on the benzyhydrylamine resins, the
resin cleavage and deprotection steps may be combined in
a single step utilizing liquid hydrogen fluoride and
anisole as described above. The fully deprotected
polypeptide is then purified by a sequence of
chromatographic steps employing any or all of the
following types: ion exchange on a weakly basic resin in
the acetate form; hydrophobic adsorption chromatography
on underivatized polystyrene-divinylbenzene (for example
4157J 23620-FF
~26~7~
-25-
Amberlite*XAD); silica gel adsorption chromatography; ion
exchange chromatography on carboxymethylcellulose;
partition chromatography, e.g~, on Sephadex*G-25, or
countercurrent distributionj high performance liquid
chromatography ~HPLC), especially reverse phase HPLC on
octyl- or octadecylsilyl-silica bonded phase column
packing.
If a racemic amino acid is used in the l, 2~ 3 or 6
position, the diastereomeric nonapeptide or decapeptide
final products are separated, and the desired peptide
containing a D-amino acid in the appropriate position is
isolated and purified, preferably during the
above-described chromatographic process.
The preparation of peptides having C-terminal
azaglycine amides is preferably done using classical
peptide solution synthesis using known peptide
intermediates. This is described in more detail ir.
Example 3.
Thus, in another aspect the present invention
-elates to a method for preparing compounds of the
invention and of the pharmaceutically acceptable salts
thereof which process comprises:
removing protecting groups and, optionally,
covzlently bound solid support from a protected
polypeptide to afford a compound of Formula (I) or a salt
thereof; or coupling together in the required sequence
two fragments of the desired compound of formula (I); or
(2) converting a compound of Formula (I) to a
pharmaceutically acceptable salt, or
(b) converting a salt of a compound of FormLia (I)
to a pharmaceutically acceptable salt, cr
(c) decomposing a salt of a compound of Form~la
(I) to a free polypeptide of Formula (Ij.
It will be ap?reciated that the guanido-substituted,
amidine, and tertiary and auaternarv amine water soluble
* trade mark
4157J 23620-FF
- ~ ,
- . . ~ - .- . - .
~6~6~
-26--
amino acids used in this invention to replace the glycine
residue at position 6 of LHRH are useful intermediates
and as such form an important part of this invention.
Particularly useful intermediates of this invention
include those of formula (II) wherein n is 1 or 2 and the
other variables are as previously defined; those of
formula (II) wherein n is 3, R3 is alkyl of 2 to 12
carbon atoms or as otherwise defined previously, and the
other variables are as previously defined; and those of
formula (II) wherein n is 4 or 5, and the other variables
are as previously defined.
Preferred intermediates include those of
formula (II) wherein n is 4, R is -NRR3 wherein R is
hydrogen and R3 is alkyl of 1 to 12 carbon atoms, and
R2 is alkyl of 1 to 12 carbon atoms.
Such intermediates of formula (II) may be prepared
in any convenient manner, an example of which is by
,uanylating or amidinating the corresponding amine.
The following examples are given to enable those
skilled in the art to more fully understand and practice
the present invention. They should not be construed as a
limitation upon the scope of the invention, but merely as
being illustrative and representative thereof.
Preparation A
N-ACETYL 3-(2-NAPHT~YL)-D,L-ALANINATE
The preparation of 3-(2-naphthyl)-D,L-alanine is
carried out according to the procedureset out in U.S.
Patent 4,341,767.
Preparation of N acetyl 3-(2-naphthyl)-D,L-alinine,
its conversion to methyl N acetyl 3-(2-naphthyl)-D,L-
allnine, and separation of the D isomer is carried out by
the procedure disclosed in U.S. Patent 4,341,767.
4157J 23620-FF
~2~ 76~)
-27-
Preparation B
A mixture of 5.24 g of benzyl N~ -benzyloxy- -
carbonyl-D-lysinate toluenesulfonate (B. Bezus and L.
~ervas, J. Am. Chem. Soc. 83, 719 (1961)) and 1.72 ml
of diisopropylethylamine in 60 ml of dioxane is treated
with 1.89 g of N,N'-diisopropylcarbodiimide. The
reaction mixture is stirred at 100C for 6 hours, cooled
to room temperature and concentrated to a solid. The
solid is suspended in 20 ml of warm DMF, filtered to
remove N,N'-diisopropylurea and the filtrate concentrated
to a solid. Benzyl N~ -benzyloxycarbonyl-N,N'-
guanido-diisopropyl-D-homoargininate tolueneculfonate is
obtained as a white solid by crystallization from
methanol/ethyl acetate [~]D -7.26 (C 0.3, MeOH).
Similarly, by using the above procedure, but
substituting:
N,N'-dicyclohexylcarbodiimide;
N,N'-di-n-hexylcarbodiimide;
N,N'-diethylcarbodiimide;
N,N'-di-n-propylcarbodiimide;
N-i-propylcarbodiimide,
N-propylcarbodiimide;
N,N'-di-n-butylcarbodiimide;
N,N'-dimethylcarbodiimide;
N,N'-di-i-butylcarbodiimide,
N,N'-di-n-pentylcarbodiimide;
N,N'-di-i-pentylcarbodiimide;
N,N'-diphenylcarbodiimide;
~,N'-ditolylcarbodiimide; or
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-~Cl
and the like, there axe obtained:
benzyl N~-benzyloxycarbonyl-N,N'-guanido-
dicyclohexyl-D-homoargininate, [~]D 8.07
(C 0.9 MeOH);
4157J 23620-FF
~IL26~
-28-
benzyl N~-benzyloxycarbonyl-N,N'-guanido-
diethyl D-homoargininate;
benzyl N~-benzyloxycarbonyl-N,N'-guanido-
di-n-propyl-D-homoargininate [a]D 8.07 (C 0.9 MeOH);
benzyl N~-benzyloxycarbonyl-N,N'-guanido-
n-propyl-D-homoargininate;
benzyl N~-benzyloxycarbonyl-N,N'-guanido-
di-n-butyl-D homoargininate;
benzyl N~-benzyloxycaxbonyl-N,N'-guanido-
di-i-butyl-D-homoargininate;
benzyl N~-benzyloxycarbonyl-N,N'-suanido-
di-n-pentyl-D-homoargininate;
benzyl N~-benzyloxycarbonyl-N,N'-guanido-
di-phenyl-D-homoargininate;
benzyl N -benzyloxycarbonyl-N,N'-guanido-
dimethyl-D-homoargininate;
benzyl N~-benzyloxycarbonyl-N,N'-guanido-
di-n-hexyl-D-homoargininate;
benzyl N~-benzyloxycarbonyl-N,N'-~uanido-
di-isopropyl-D-argininate, [a]D -10.5 (C 0.5, MeO~);
benzyl N~-benzyloxycarbonyl, N-guanido-
(3-dimethylaminopropyl)-N'-guanido-ethyl-D-homoargininate
as their benzenesulfonate salts. Similiarly, by
substituting benzyl N~-benzyloxycarbonyl-D-ornithinate
for the D-lysinate there may be obtained the
corresponding arginine analogs as their toluenesulfonate
salts.
Preparation ~
30 (i) Benzyl N~-benzyloxyc ~ l_NG,NG
-ethano-D-homoargininate
To a mixture of 15 ml of toluene and 15 ml of t-~uOH
was added 2.71g of benzyl N~-benzyloxycarbonyl-D-lysinate
and 1.46 g of 2-methylthioimidazoline-HI (available
4157J 23620 FF
75~
29-
from Aldrich). The pH of the mixture was brought to
~8 by the addition of diisopropylethylamine and the
solution heated under reflux for 24 hours.
The solution was concentrated in vacuo and the
residue was loaded on a silica gel column (250 g). The
column was eluted with a gradient from CH2C12/MeOH ~19:1)
to CH2C12/MeoH (7:3~. The fractions containing product
were detected by TLC, pooled, and concentrated to
dryness, 2.9 g of white foam.
A 2 g portion of the above-named product was
dissolved in 50 ml of ~tOH containing 0.8g of 10~ Pd/C.
The solut~on was stirred under H2 for 8 hours. The
mixture was filtered on celite and the filtrate was
concentrated to dryness to give NG,NG-etheno-D-
homoarginine as a white foam, 1.2 g.
(ii) NC~-Boc-NG,NG -ethano-D-homoarglnine
BocN-CH-COOH
(CH2) ~
C
HN/ ~N
`~/
A solution of 2.74 g of D-lysine dihydrochloride and
4.03 g of 2-methylthio-2-imidazoline.hydroiodide in
16.5 ml of 2N NaOH was stirred at room temperataure for
6 days. Analysis of the reaction mixture on an amino
acid analyzer showed that ~70% of the desired
~-dialkylguanido compound had been formed. A further
0.25 g of the 2-methylthio-2-imidazoline.hydroiodide and
1 ml of 2N NaOH were added and the reaction was continued
at room temperature for 3 more days.
4157J 23620-FF
~z~
-30-
The reaction mixture was treated with 0.8 g MgO and
4.36 g of di-tert-butyldicarbonate in 20 ml of dioxane.
The pH was adjusted to 9.5 with lN NaO~. After overnight
reaction some starting material was present, so 1 g of
di-tert-butyldicarbonate was added.
The mixture was filtered and the filtrate was
concentrated to dryness. The residue was dissolved in
H2O and washed with Et2O and the aqueous layer was
adjusted to pH 4 with HOAc. The acidic solution was
washed with EtOAc. The aqueous layer containing the
product was treated with anion exchange resin
(AG-3 acetate, BioRad) and concentrated to dryness.
The crude product was pa~sed through a hydrophobic
chromatography column (~mberlite XAD-2, Rohm & Haas) by
elution with a gradient from H2O to 25% EtOH. The
fractions containing product were pooled to yield 2.7 g
of Na-~oc-NG,NG -ethano-D-homoarginine,
[a]D -19.7 (c 0.l, MeOH).
In a similar fashion, by substituting:
S-methyl-diethyl-iso-thiourea-HI,
S-methyl-dipropyl-iso-thiourea-HI,
S-methyl-dibutyl-iso-thiourea-HI,
S-methyl-dipentyl-iso-thiourea-HI,
S-methyl-dihexyl-iso-thiourea-HI,
S-methyl-diheptyl-iso-thiourea-HI, and
S-methyl-dinonyl-iso-thiourea-HI
for 2-methylthio-2-imidazoline-HI, there are obtained:
Na-Boc-N,N'-guanido-diethyl-D-homoarginine,
Na-Boc-N,N'-guanido-dipropyl-D-homoarginine,
N~-Boc-N,N'-guanido-dibutyl-D-homoarginine,
Na-Boc-N,N'-guanido-dipentyl-D-homoarginine,
Na-Boc-N,N'-guanido-dihexyl-D-homoarginine,
Na Boc-N,N'-guanido-diheptyl-D-homoarginine, and
N -Boc-N,N'-guanido-dinonyl-D-homoarginine.
4157J 23620-FF
~ 2~76~
-31-
Preparation D
This Preparation illustrates the preparation of
N-t-butyloxy carbonyl derivatives of N~Nl-guanido-
disubstituted-D-homoarginines from their toluenesulfonate
precursors.
A mixture of N,N'-guanido-diisopropyl~D-
homoargininate toluenesulfonate (3.25 g) and 100 mg o
10% Pd~C in 50 ml of glacial acetic acid is treated with
hydrogen gas at atmospheric pressure for 4 hours. The
catalyst is filtered on celite and the filtrate is
concentrated to a solid, N,N'-guanido-diisopropyl-
D-homoarginine toluenesulfonate. A solution of this
compound (2.13 g) in 60 ml of 50% dioxane/water is
treated with 10 ml of lN sodium hydroxide and 0.4 g of
magnesium oxide. This mixture is then treated with 1.1 g
of di-t-butyldicarbonate and stirred at room temperature
for 2 hours. The magnesium salt is filtered and the
filtrate is concentrated under vacuum. The basic
solution is ~ashed with ethanol, then brought to pH 2.5
with sodium sulfate. The acidic aqueous solution is
extracted with ethylacetate which is dried over magnesium
sulfate. The drying agent is filtered and the filtrate
is concentrated. Crystallization from ethyl
acetate/hexane affords Na-t-butyloxycarbonyl,N,N'-guanido-
diisopropyl-D-homoarginine toluenesulfonate.
Proceeding in a similiar manner, but substituting
the appropriate toluenesulfonate precursors, other
N~-t-butyloxycarbonyl-N,N'-guanido-disubstituted-D-
homoarginine or D-arginine compounds may be prepared.
PREPARATION E
N~-t-butyloxycarbonyl-3-(4'-(1'-propylpiperidyl))-
D-alanine
A 4.6 g portion of sodium metal was added to 400 ml
3 of absolute ethanol and heated. To the resultant
4157J 23620-FF
~L;26~
solution of sodium ethoxide was added 21 7 g of diethyl
acetamidomalonate and 16.4 g of 4-picolyl chloride
hydrochloride (Aldrich Chem. Co.). The reaction mixture
was heated to 100C for 4 hours, cooled, filtered and
concentrated in vacuo. The mixture was loaded on a
silica gel column in methylene chloride/methanol (19:1)
and eluted with the same mixture. The product was
located as a fast running W positive spot by TLC on
silica gel in methylene chloride/methanol (19:1).
Combined fractions were concentrated to provide the
product.
The product from the foregoing paragraph was
dissolved in 200 ml of ethanol and ~reated with a
solution of 2.72 g of sodium hydroxide in 40 ml of water
15 at 50 C for 6 hours. The solution was acidified with 12
ml of 6N HCl, concentrated to dryness and taken up in 200
ml of dioxane. The suspension was filtered and the
filtrate heated at reflux for 2 hours. The solution was
- cooled and concentrated to dryness to yield ethyl
N~-acetyl-3-(4-pyridyl)-D,L-alanine as a white solid.
A portion of this N-ace~yl ester was resolved by
treatment with 200 ml of the enzyme subtilisin Carlsberg
(Sigma Chem. Co., protease VIII) in a mixture of 300 ml
of dimethyl sulfoxide and 400 ml of 0.01M KCl (pH 7.2).
The pH was maintained by addition of lN NaOH on a pH
stat. After a 6 hour period, the resolution was
complete. The solution was diluted with 400 ml of water
and extracted with 4 X 750 ml of ethyl acetate. The
organic layers were combined and dried over magnesium
sulfate and concentrated to yield ethyl N~-acetyl-3-
(4-pyridyl)-D-alaninate as an oil.
The oil was reacted with 1.22 g of n-propyl bromide
in 50 ml of ethanol after which the solution was
concentrated to dryness to yield ethyl N~ -acetyl-3-
(1-propyl-pyridinium-4-yl)-D-alininate bromide as a white
hygroscopic solid.
4157J 23620-FF
126~7~0
-33-
This white solid was dissolved in 200 ml of ethanol
and was reduced under an atmosphere of hydrogen gas using
100 mg of 10% Pd/C as a catalyst. After an 18 hour
reduction period, the catalyst was filtered out and the
solutin concentrated ~o yield ethyl N~-acetyl-3-(4'-
(l'-propylpiperidyl))-D~alininate as a tan solid. The
free acid was prepared by refluxing the ethyl ester in
100 ml of 6N HCl for 4 hours to yield 3 (4'~ propyl-
piperidyl))-D-alanine as a white solid.
The free acid was dissolved in 100 ml of
dioxane/water (1:1) and treated with 2 g of
di-t-butyldicarbonate. The pH W25 maintained at 9 by
addition of lN NaO~ on a pH stat. After 2 hours the
reaction muxture was concentrated in _acuo, washed wtih
100 ml of ethyl ether and the aqueous laye was loaded on
an Amberlite XAD-2 hydrophobic resin. The column was
eluted with 250 ml of water followed by 250 ml of 50
ethanol/ater. The ethanol eluate was pooled and
concentrated to dryness to yield N~-t-butyloxy-
carbonyl-3-(4l-(1'-propylpiperidyl))-D-alanine as a white
solid.
Proceeding in similiar manner, but substituting
3-picolyl chloride hydrochloride for 4-picolyl chloride
hydrochloride, there is prepared N~-t-butyloxy-
carbonyl-3-(3l-(1l-propylpiperidyl)~-D-alanine.
PREPARATION F
Cis-5-alkylproline compounds may be prepared by the
followin~ method:
To a 200-ml round-bottomed flask is added
(S)-3-(benzyloxycarbonyl)-5-oxo-4-oxazolidinepropionic
acid and 63 ml of anhydrous benzene. To this solution is
added 13.9 g of phosphorus pentachloride at 0C. The
reaction mixture is stirred at 0C for 1 hr during which
time all of the phosphorus pentachloride dissolves. The
4157J 23620-FF
~Z`~i~76~
-34-
benzene solvent is removed under vacuum and coevaporation
with two 25 ml samples of dry benzene, and the residue
dried under vacuum to give a light solid. The light
solid is suspended in 30 ml of hexamethylphosphoramide and
9.4 ml of tetramethyltin and 40 mg of phCH2Pd(PPh3)2Cl is
added. The reaction mixture is heated at 65C ~or 4
hours. An additional 2 ml of tetramethyltin is added at
the end of that period and the reaction mixture is
stirred over night at room temperature.) After dilution
0 with water and extraction with ethyl acetate, the ethyl
acetate layer is washed with water, 5% sodium
bicarbonate, water, 5% sodium bisulfate, water, and
saturated sodium chloride and dried over anhydrous
magnesium sulfate. The solution is filtered and
concentrated to give 16 g of a yellow oil, which is
passed through a silica gel column using ethyl
acetate/hexane~4/6) as eluent. Concentration of the
appropriate fractions gives 15 g of a light yellow oil
which is recrystallized from ethyl acetate-hexane to
20 produce 14.3 g of (5)-3-(benzyloxycarbonyl)-4-
(3-oxobutyl)-5-oxazolidinone as a white solid (74%
yield), having a mp of 64-65C, [~]25= + 102~ (c = 1.1,
CH2C12) -
Anal: Calcd. for ClgH17NOs:
C, 61.85; H, 5.84; N, 4.81.
Found: C, 61.54; H, 5.89; ~, 4.84.
By repeating the above procedure in a similar
manner, and, by replacing the tetramethyltin with a
stoichiometrically equivalent of the appropriate
tetraalkyltinthe following compounds are prepared:
(a) With tetraethyltin:
(S)-3-(Benzyloxycarbonyl)-4-(3-oxopentyl)-5-oxazolidinone
having a mp of 45-46DC;
[`~]D = 82.5(c 0.7, CH30H).
4157J 23620-FF
~2~
-35-
Anal: Calcd. for C16HlgNOs(305.336):
C,62.94; H,6.37; N,4.59.
Found: C,63.02; H,6.15; N,4.48.
(b) ith tetrabutyltin:
(S)-3-(Benzyloxycarbonyl)-4-(3-oxoheptyl)-5-oxazolidinone
as an oil; ~]25 67.9(c 0012, CH30H).
Anal: Calcd. for Cl,3H23NOs EtOAc(421.494):
C,62.69; H,7 41; N,3.32.
Found: C,62.50; H,7.29; N,3.39.
Ten grams of the (S)-3-(benzyloxycarbonyl)-4-
(3-oxobutyl)-5-oxazolidinone from Example 4 is dissolved
in 480 ml of distilled tetrahydrofuran, followed by 160
ml of ammonia at 0C. The reaction mixture is stirred at
0 for 5 hours, then at ambient temperature overnight.
After stripping under vacuum to dryness, the reaction
mixture yields a white solid which is recrystallized from
hoc ethyl acetate to give 8.8 g of (S)-2-(benzyloxy-
carbonylamino)-5-oxo-hexanamide as a white solid
(82% yield), mp 142-144;
[~]D = ~4 0 (c 0.4, CH30H).
Anal.: Calcd. for C7HgNO2:
C, 60.4; H, 6.4; N, 10Ø
Found: C, 60.44; ~, 6.53; N, 10.05.
By repeating the above procedure in a similar manner
and substituting a stoichiometrically equivalent of the
corresponding intermediates from the second previous
paragraph, the following compounds are prepared :
(S)-2-Benzyloxycarbonylamino-5-oxo-heptanamide
having a mp of 133-135C; [~]D -4.17(c 0.8, CH30H)-
Anal: Calcd. for C15H20N24(292-341)
C,61.63; H,6.90; N,9.58.
Found: C,61.51; H,6.75; N,9.16.
(S)-2-Benzyloxycarbonylamino-5-oxo-nonamide having a mp
of 162-163C;[~]D -4.32 (c 0.6, CH3OH).
4157J 236 0-FF
~26~7~
-36-
Anal: Calcd. for C17H24N204(320-395):
C,63.73; H,7.55, N,8.74.
C,63.62; ~,7.56; N,8.82.
To a solution of 2.8 g of (S)-2-benzyloxycarbonyl-
amino)-5-oxohexanamide from the preceeding paragraph in a
mixture of 60 ml of methanol and 7.5 ml of glacial acetic
acid is added, under nitrogen, 1.5 g of palladium
diacetate. This reaction mixture is hydrogenated under
atmospheric pressure for 4 hrs, at which time a thin
layer chromagraphic analysis shows the reaction had gone
to completion. The reaction mixture is then filtered
through Celite and washed with methanol. The reaction
mixture and washings are concentrated to dryness to give
1.7 g of a yellow oil, which is treated with 1 ml of a
mixture of hydrochloric acid and ethyl acetate to produce
the hydrochloride salt. This oil is triturated with
methanol/ethyl ether to produce 1.3 g of a yellow solid;
mp 174-176C;[~]25= -33 (c 0.96, CH30H). The yellow
solid of (S)-cis-5-methylprolinamide (as the
hydrochloride salt) is passed through a Bio-Rex 70 colllmn
(a weakly acid carboxylic acid ion-exchange resin) with
elution first with 300-ml of water, followed by 1%
solution of ammonium hydroxide. Concentration of the
appropriate fractions gives a o.g g Oc a yellow solid,
which is recrystallized from methylene chloride to
produce 0.64 g of (S)-cis-S-methylprolinamide as a yellow
solid (50% yield); mp 55-56C.
By repeating the above procedure in a similar
fashion, and substituting a stoichiometrically equivalent
of the corresponding intermediate, the following
compounds are prepared after reduction:
(S)-cis-5-ethylprolinamide, mp 63-65C; and
(S)-cis-5-butylyprolinamide, mp 74-75C.
4157J 23620-~F
~Z~4760
-37-
4.9 g of Boc-glycine was di~solved in a mixture of
50 ml. ethanol and 50 ml. distilled water. The pH of the
solution was brouqht to 7 with aqueous cesium
bicarbonate. The solvent was ~hen removed under vacuum.
After 18 hours of drying under high vacuum, the
residue was dissolved in 150 ml. dry DMF. 25 g
chloromethylated polystyrene - 1% divinylbenzene
~Merrifield) resin (corresponding to 25 mmole chloride)
was added. The mixture was shaken at 50C for 24 hours,
filtered, and the resin was then washed sequentially with
DMF, water, and ethanol. The resin was dried under
vacuum for 3 days to yield 28.34 g of Boc-Gly-O-Resin.
Example 1
In the reaction vessel of a Beckman 990 Peptide
Synthesizer was placed 7.29 g. (5.5 mmol.) of
Boc-Gly--O-Resin prepared by the reaction of equimolar
ratios of the dry cesium salt of Boc-GlyOH with
chloromethylpolystyrene/1% divinylbenzene. Amino acids
were added sequentially to this resin by means of a
synthesis pro~ram, as follows:
Step 1 CH2C12 wash 1 time 1.5 min
2 50% CF3CO2H/CH2cl2-- 1 time 1.5 min
deprotection
3 50% CF3CO2H/CH2cl2- 1 time 30 min
deprotection
4 CH2C12 wash 3 times 1.5 min
10~ triethylamine/CH2C12 2 times 1.5 min
6 CH2C12 wash 3 times 1.5 min
7 N~-Boc-amino acid 1 time add
solution
B N,N'-dicyclohexylcarbo- 1 time add
diimide solution
4157J 23620-FF
6~
-38~
9 CH2C12 rinse and hold-- 1 time coupling
reaction
coupling 2 hr
10 CH2C12--rinse add 1 time 1.5 min
11 CH2C12 wash 3 times 1.5 min
12 ethanol wash 3 times 1.5 min
13 CH2C12 wash 3 times 1.5 min
Steps 1-13 complete a coupling cycle for one amino
acid and completeness of the reaction is checked by the
ninhydrin method of E. Kaiser, et al., Anal. Biochem.,
10 34, 595 (1970).
The resin was coupled sequentially with a 2.0 to 2.5
molar excess of each protected amino acid and DCC. Thus,
the resin was treated during successive coupling cycles
with
3.01 g. Boc-Pro-OH,
5.99 g. Boc-Arg(Tosyl)-OH,
3.49 9. Boc-Leu-OH H2O
At this point, the resulting tetrapeptide resin ~as
divided into smaller batches. A 1.00 g. batch was
carried for~ard by further coupling in successive cycles
with
0.456 9. Boc-D-Dia-OH toluenesulfonate,
0.44 g. Boc-Tyr(2,6-dichlorobenzyl)-OH,
0.375 g. Boc-Ser(Benzyl)-OH,
0.315 g. Boc-D-Nal(2)-OH,
0.35 g. Boc-D-p-F-Phe-OH,
0.275 g. Boc-L-Proline, and
2.5 ml. acetic anhydride.
The resin was removed from the reaction vessel,
washed with CH2C12, and dried in vacuo to yield
1.66 g. of protected polypeptide resin. The protected
peptide was removed from the resin by treatment at room
temperature for 24 hours with 50 ml. of methanol
saturated at 0C with ammonia. The resin beads were
4157J 23620-FF
~Z6~7~
- 39 -
filtered and washed sequentially with methanol and DME. Solvent
was removed from the filtrate under vacuum to yield the protected
peptide as 0.9 g. of yellow oil.
The protecting groups were removed by treatment with
10 ml. anhydrous liquid HF in the presence of 1 ml. of anisole
(scavenger) in a Kel-F reaction vessel at 0C for 30 minutes. The
HF was evaporated under vacuum and the residue of
N-Ac-L-Pro-D-p-Cl-Phe-D-Nal(2)-Ser-Tyr-D-Dia-Leu-Arg-Pro-GlyNH2,
as its HF salt , was washed with ether. The residue was then
extracted with glacial acetic acid. The acetic acid extract was
lyophilized to yield 0.5 g. of crude material.
The crude material was converted to the acetate salt
by passage in water through a column of a~* (a weakly basic
tertiary amine resin) which had been converted to the acetate
form. Lyophili~ation of the eluate yielded 0.5 g. of the crude
peptide acetate salt as a white solid.
The crude peptide was purified by high performance
liquid chromatography on a 2.5 x 100 cm. column of
LicropreRL* Rp-18 (25-40 micron) equilibrated to the running
buffer 35% CH3 CN/65%H2 0 (0.03 ~ in NH4 OAc, pH 4.5). The major
UV absorbing (280 nm) peak eluting at approximately 3 column
volumes was collected, concentrated to dryness, and lyophilized 3
times from distilled water to yield 75 mg of pure
N-Ac-L-Pro-D-p-F-Phe-D-Nal(2)-Ser-Tyr-D-Dia-Leu-Arg-Pro-GlyNH2,
[a] = -15.4 (C 0.5, HOAc).
D
Proceeding in a similiar manner but substituting the
appropriate A, B, C, D, E, F, G or F amino acid for those recited,
there are prepared the corresponding GlyNH2 decapeptides
exemplified below.
N-Ac-L-Pro-D-p-F-Phe-D-Nal(2)-Ser-Tyr-D-Dih-Leu-
Arg-Pro-GlyNH2,
N-Ac-L-Pro-D-p-F-Phe-D-Nal(2)-Ser-Tyr-D-Iph-Leu-Arg-
Pro-GlyNH2,
Trade-Marks *
~2~6~
-40-
N-Ac-D-Nal(2)-D-p F-Phe-D-Nal(2)-Ser-Tyr-D-Iph-Leu-
Arg-Pro-GlyNH2,
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Phe-Ser-Tyr-D-Iph-Leu-
Arg-GlyNH2~
N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dih-Leu-Arg-
Pro-GlyNH2t
N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-Arg-
Pro-GlyNH2,
N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-
Pro-GlyNH2,
N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D Dph-Leu~Arg-
Pro-GlyNH2,
N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-
Pro-GlyNH2,
N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Prh-Leu-Arg-
Pro-GlyNH2,
N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Hha-Leu-Arg-
Pro-GlyNH2,
N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dpa-Leu-Arg-
Pro-GlyNH2,
N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Mpa-Leu-Arg-
Pro-GlyNH2,
N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Pia-Leu-Arg-
Pro-GlyNH2r
N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Ppa-Leu-Arg-
Pro-GlyNH2,
N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Bpa-Leu-Arg-
Pro-GlyNH2,
N-Ac D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-
Arg-Pro-GlyNH2,
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-
Arg-pro-GlyNH2,
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-
Arg-pro-GlyNH2t
4157J 23620-FF
-41-
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dih-Leu
Arg-Pro-GlyNH2,
N-Ac-D-p-Cl-Phe-D-p-G1-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-
Arg-Pro-GlyNH2,
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Prh-Leu-
Arg-Pro-GlyNH2,
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Hha-Leu-
Arg-Pro-GlyNH2~
N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-Arg-
10 Pro-GlyNH2,
N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-
Pro-GlyNH2,
N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-Arg-
Pro-GlyNH2,
N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dih-Leu-Arg
Pro-GlyNH2,
N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-
Pro-GlyNH2,
N-Ac-Nal(2)-D-p-F-Phe-D-Nal(2)-Ser-Tyr-D-Dph-Leu-Arg-
Pro-GlyNH2,
N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-
Pro-GlyNH2,
N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Prh-Leu-Arg-
Pro-GlyNH2,
N-Ac-Nal(2)-D-p-F-Phe D-Trp-Ser-Tyr-D-Hha-Leu-Arg-
Pro-GlyNH2,
N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-PropylAmidine-Leu-
Arg-Pro-GlyNH2,
N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Mpa-Leu-Arg-
Pro-GlyNH2,
N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Pia-Le~-Arg-
Pro-GlyNH2,
N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Ppa-Leu-Arg-
Pro-GlyNH2,
4157J 23620-FF
47~
42-
N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Bpa-Leu-Arg-
Pro-GlyNH2,
N-Ac-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-Arg-
Pro-GlyNH2,
N-Ac-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr D-Dmh-~eu-Arg-
Pro-GlyNH2,
N-Ac-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-
Pro-GlyNH2, and
N-Ac-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-
~0 Pro-GlyNH2.
In a similar manner, by replacing the
Boc-Gly-O-Resin with Boc-D-Ala-benzhydrylamino-
polystyrene-1%-divinylbenzene resin prepared from
benzhydrylaminopolystyrene-1% di~inylbenzene resin
(Beckman Inst), Boc-D-Ala-OH, DIC and ~BT, was obtained
the corresponding D-Alal0 analo~s of LHRH:
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Nal(2)-Ser-Tyr-D-Iph-Leu-
Arg-D-AlaNH2, and
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dih-Leu-
Arg-Pro-D-AlaNH2,
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-
Arg-Pro-D-AlaNH2,
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-
Arg-Pro-D-AlaNH2,
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-
Ars-Pro-D-AlaNH2,
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Mpa-Leu-
Arg-Pro-D-AlaNH2,
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Pia-Leu-
Arg~PrO-D-AlaNH2,
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Ppa-Leu-
Arg-Pro-D-AlaNH2,
N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Bpa-Leu-
Arg-Pro-D-AlaNH2,
4157J 23620-FF
-43-
N-Ac-D-Nal(2)-D~pCl-Phe-D Trp-Ser-Tyr-D-Deh-Leu-
Arg-Pro-D-AlaNH2, [~]D-21.6~CO.4, HoAc),
N-Ac-D-Nal(2)-D-pC1-Phe-D-Trp-Ser-Tyr-D-Deh-Le~-
Arg-Pro-D-AlaNH2,
N-Ac-D-Nal(2)-D-pCl-Ph~-D-Trp-Ser-Tyr-D-Dmh-Leu-
Arg-Pro-D-AlaNH2,
N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Dhi~-Leu-
Arg-Pro-D-AlaNH2,
N-Ac-D-Nal(2)-D-pCl-Phe-D-~rp-Ser-Tyr-D-Dph~Leu-
Arg-Pro-D-AlaNH~,
N-Ac-D-Nal(2)-D-pF-Phe-D-Trp-Se~Tyr-D-Deh-Leu-
Arg-Pro-D-AlaNH2, and
N-Ac-D-Nal(2)-D-pF-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-
Arg-Pro-D-AlaNH2.
1~ '
: ~D-Dhi is D-~- epsilon dihydroimidazolinyllysine
i~ .
Physical data for N-Ac-D-Nal(2)-D-pF-Phe-D Trp-Ser-Tyr-D-
20 Deh-Leu-Arg-Pro-D-AlaNH2 are as follows:
Melting Point: 160-165~C., ¦~] - 29.0~ (C 1.0, HOAc)
Amino Acid Analysis: Ser 0.9tl)2,0Pro 1.0(1), D-Ala l.0tl),
Leu 1.0(1), Tyr 1.0(1), Trp 0.9(1),
Arg 1.0(1), pF-Phe 0.9(1), D-Deh 0.9(1)
D-Nal(2) 0.9(1), NH3 1.1(1).
~5
' :'- . ' - . '
~z~
- 43a -
_xample 2
For the synthesis of anal~gues with a C-terr.inal
Pro-NHCH2CB3, a svnthesis program identical to tnat
described in ~xample 1 was used. The Beckman 990
Synthesizer reaction vessel was loaded with 2.13 g. of
Boc-Pro-O-~esin, prepared by the reaction of equimolar
ratios of the dry cesium salt of Boc-Pro-OB with
chloromethyl-polystyrene/l~ divinylbenzene (Lab Systems,
Inc.). The quantity of Boc-~ro-O-~esin taken contained
1.4 mmol. of proline.
The resin was co~pled sequen.ially with a 2.0 to 2.5
molar excess of each protected amino acid and DCC. Thus,
the resin was reacted during successive couplins cycles
with
13 1.61 9. Boc-Arg(I~osyl)-OB,
0.93 9. Boc-Leu-OB H2O,
0.94 9. Boc-N,N'-guaniào-diisopropvlhomo2rsinine,
0.49 g. l-hydroxybenzotriazole,
2~ 4157J 23620-FF
~5
. _ _ _ _
. - .
, ,
:: , , . . ., - . , .
76~
-44-
1.75 9. N-~oc-0-2-bromobenzyloxycarbonyl-L-tyrosine,
and
1.11 9. Boc-Ser(Benzyl~-OH~
At this point in the synthesis the quantity of
protected polypeptide resin was spli~ in half and one
half was carried through to completion by sequential
reaction with
0.57 g. Boc-D-Nal~2)-OH~
0.480 g. Boc-D-p-F-Phe-OH
0.21 g. N-Ac-L-proline.
The resin was removed from the reaction vessel,
washed with CH2Cl~, and dried in vacuo to yield 2.26 g.
of protected polypeptide resin.
The protected polypeptide was cleaved from ~he resin
by aminolysis with 25 mL. of ethylamine for 18 hours at
2DC~ The ethylamine was allowed to evaporate anà the
resin was extracted with methanol. The methanol w2S
evaporated to yield 1.39 9. of N-Ac-L-Pro-D-p-Cl-Phe-Trp-
Ser(3enzyl)-Tyr(2,6-dichlorobenzyl)-D-Dih-Leu-
Arg(Tosyl)-Pro-NHCH2CH3 ~his protecSed peptide was mixed
with 3 ml of anisole and 30 mL. redistilled (from CoF3)
anhydrous liquid HF at 0C. for 30 minutes in a Kel-F
reaction vessel. The HF was evaporated under vacuum and
the residue was washed with ether. The residue was
dissolved in 2 M acetic acid and lyophilized to yield
0.82 9. of crude N-Ac-L-Pro-D-p-F-Phe-Trp-Ser-Tyr-D-Dih-
Leu-Arg-Pro-NHCH2CH3 as its acetic acid addition salt.
Final purification was achieved by preparative high
performance liquid chromatography of a 20 mg. sample on a
30 2.5 x 100 mm. column of 40-50 microns. octadecylsilylated
silica (Merck*, Liçhroprep*C18).
Proceeding in a similiar manner, but substit`ting
the appropriate protected amino acid residues whe e
appropriate, there are prepared the following compounds:
* tr~de mark
4157J 23620-FF
,
'' '.,' ', ~ ' - .
:-- ~ .,: - . -
. . ... .. . . . . .
; ~ - -
. - ~ - - , ' - ,
~2~6~
-45-
N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-~ih Leu-
Arg-Pro-NHEt,
N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-Arg-
Pro-NHEt,
5N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-Arg-
Pro-NHEt,
N-Ac-V-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-
Pro-NHEt,
N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-
Arg-Pro-NHEt,
N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Prh-Leu-
Arg-Pro-NHEt,
N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Hha-Leu-
Arg-Pro-NHEt,
N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dpa-Leu-
Arg-Pro-NHEt,
Repeating the above cleavage, substituting a
stoichiometric amount of methylamine and propylamine for
ethylamine there are obtained the corresponding
methylamide or propylamine of the aforementioned
nonapeptide.
Example 3
Compounds of Formula I wherein H is -NH-CONH-R10
may be prepared by classical solution synthesis.
For example, the following approach may be used
wherein ''AzaGlyNHR10'' is -NH-NH-CONH2, to prepare the
peptide as the free peptide or salt.
4157J 23620-FF
~2~6~6~
~-Ac-D-Nal(2)-D-p-Cl-Phe- Cbz-Leu-Ar~-Pro-AzaGlyNH~
D-Trp-Ser-Tyr-OMe NO2
(1) 1 (2)
¦ Boc-D-Deh-Leu-Arg-Pro-
1, AzaGlyNH2 1~+
5 N-Ac-D-Nal(2)-D-p-Cl-Phe- j
D-Trp-Ser~Tyr-N
H-D-Deh-
Leu-Arg-Pro-AzaGlyNH2
~, (3)
N-Ac-D-Nall2)-D-p-Cl-Phe-D-Trp-Ser-~yr-D-Deh-
I,eu-Arg-pro-Aza-GlyNH2
The co~pling of the individual fragments may proceed
by the acyl azide method (J. Honzel, et al, Coll. Czech.
Chem. Comm., 26, 2333 ~1971), by DCC/HBT coupling or
other race~ization free fragment coupling techniques.
Compo~nd (2) is kno~n: A.S. Dutta, et al., J. Chem. Soc.
Perkin I, 1979, 379, and compound (1) may be prepared by
methods analogous to those in Example 1. Compo~nd (3) is
prepared from (2) by removal of the Cbz and ritro groups
by hydrogenolysis, followed by coupling with N-Boc-
N,N'-guanido-diethyl-D-homoarginine using DCC/HET or
other coupling agent known in the art. See Dutta, et al,
supra, for a similar L~RH analogue synthesis.
Often, the fragments coupled in this way will be
peptides or amino acids. Alternatively, the N-terminal
nonapeptide acid may be prepared by the solid phase or
solution method and subsequently coupled to
semicarbazide-HCl by the dicyclohexylcarbodiimidehydroxy-
benzotriazole cr other coupling method.
Physical data for N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-
D-Deh-Leu-Arg-Pro-Aza-GlyNH2 synthesized by the aforesaid
coupling method are as follows:
Melting point: 153-155GC., [~] o-29.6 (C 0.3, HOAc)
Amino Acid Analysis: Ser 0.9(1~, Pro 1.1(1), Leu 1.0(1),
Tyr 0.98(1), Trp 0.9(1), Arg 1.0(1),
FCl-Phe 1.0(1), D-Deh 1.0(1), D-Nal(2)
1.0(1), NH3 1.3(1).
.. - , , .
: - - . . . :
7~0
- 46a -
Example 4
A. A solution of 0.1 g of the hydrogen fluoride
salt of N-Ac-L-Pro-D-p-F-Phe-Trp-Ser-Tyr-D-Dia
Leu-Arg-Pro-GlyNH2 (See Example 1) is dissolved in 50 mL
, 5
.~ , .. . . ~, - .- ' .
-
. . , . -- . .
~2~
-47-
of water and passed through a column of 50 g Dowex 3
anion exchange resin which had previously been
equilibrated with acetic acid and washed with deionized
water. The column is eluted with deionized water and the
effluent is lyophilized to yield the corresponding aceti~
acid salt of N-~c-L-Pro-D-p-F-Phe-Trp-Ser-Tyr-D-Dia-
Leu-Arg-Pro-Gly-NH2, [~]D5-l5.4 (C l, HOAc)
Repeating the above, substituting other acids for
acetic acid during the equilibration of the resin, there
may be obtained, for example, the corresponding salts
with hydrochloric acid, hydrobromic acid, sulfuric acid,
phosphoric acid, nitric acid, benzoic acid, and the like.
Similarly there may be prepared the acid addition
salts of the other peptides ~nalogous to LHRH, described
herein.
B. In the case of salts of low water solubility,
these may be prepared by precipitation from water
utilizing the desired acid. For example:
Zinc tannate salt - a solution of l0 mg of
N-Ac-L-Pro-D-p-Cl-Phe-D-Nal(2)-Ser-Tyr-D-Dih-Leu-Arg-pro-
GlyNH2 acetic acid salt in 0.l mL of water was treated
with a solution of 8 mg of tannic acid in 0.08 mL of
0.25 M NaGH. A solution of 5 mg of ZnSO4 heptahydrate
in 0.l mL of water was immediately added to the solution
of the LXRH analogue.
The resultant suspension was diluted with l mL water
and the precipitate was centrifuged. The supernatant was
decanted and the residue was washed twice with l mL
portions of water by centrifugation of the precipitate
and decantation of the supernatant. The precipitate was
dried in vacuo to yield 15 mg of the mixed zinc tannate
salt of the above named LHRH analogue.
Pamoate salt - to a solution of 50 mg N-Ac-L-Pro-D-
p-F-Phe-D-Nal(2)-Ser-Tyr-D-Dih-Leu-Arg-Pro-Gly-NH2
acetic acid salt in a mixture of l.6 mL of ethanol and
4157J 23620-FF
~ ;~6~7~
-48-
0.1 mL of 0.25 M NaOH was added solution of 11 mg of
pamoic acid in 0.3 mL of 0.25 M NaOH. The solvents were
removed at reduced pressure and the residue was suspended
in 2 mL of water, centrifuged, and the supernatant was
decanted. The precipitate was washed with 1.5 mL H2O,
centrifuged, and the supernatant was decanted. The
precipitate was dried in vacuo to yield 54 mg of the
pamoate salt of the above named L~R~ analogue.
~ n a similar manner other salts of low water
solu~ility may be prepared.
C. Preparation of acid addition salt from free
peptide.
To a solution of 50 mg of N-Ac-L-Pro-D-p-F-Phe-D-
Nal(2)-Ser-Tyr-D-Dia-Leu-Arg-Pro-GlyNH2 as the free
base is added 30 mL of lN acetic acid. The resulting
solution is lyophilized to yield 50 mg. of the acetic
acid salt of the above ~25-15.4 (C 0.5, ~OAc)
Similarly, replacing asetic acid with other acids
(in stoichiometrically equivalent amounts relative to
peptide) there was obtained other acid additon salts of
the peptides herein, for example, the salts with
hydrochloric acid, hydrobromic acid, sulfuric acid,
phosphoric acid, nitric acid.
D. Preparation of salt with metal cation, e.g.,
zinc salt.
To a solution of 50 mg N-Ac-L-Pro-D-p-F-Phe-
D-Nal(2)-Ser-Tyr-D-Dih-Leu-Arg-Pro-Gly-NH2 acetic acid
salt in a mixture of 0.4 mL of 0.25 M NaOH, 0.3 mL water,
and 1 mL ethanol was added a solution of 15 mg of ZnSO4
heptahydrate in 0.2 ml of water. The precipitate was
centrifuged and the supernatant was decanted. The
precipitate was washed with 1 mL of water by
centrifugation and decantation of the supernatant. The
precipitate was dried in vacuo to yield the zinc salt of
the above named LH-RH analogue.
4157J 23620-FF
1~6~7~0
-49-
In a similar manner salts with other multivalent
cations e~g. calcium, bismuth, barium, magnesium,
aluminum, copper, cobalt, nickel, cadmium and the like,
may be prepared.
Example 5
A solution of 50 mg of N-Ac-L-Pro-D-p-F-Phe-D-Nal(2)-
Ser-Tyr-D-Dih-Leu-Arg-Pro-Gly-NH2 acetic acid salt in
25 ml. of water is passed through a 50 g column of Dowex
l (strongly basic, quaternary ammonium anion exchange
resin) which had been equilibrated with NaOH solution to
make the counter ion hydroxide. The column is eluted
with l50 ml of water and the eluant is lyophilized to
yield 45 mg of the corresponding polypeptide as the free
base.
Similarly other acid addition salts of compounds of
the peptides herein, e.g., those mentioned in Example 6,
may be converted to the corresponding free bases.
Example 6
The following are typical pharmaceutical composi-
tions containing, as active insredient, an LHRH
antagonist of the present invention, for example
N-Ac-L-Pro-D-p-F-Phe-D-Nal(2)-Ser-Tyr-D-Dih-Leu-Arg-Pro-
GlyNH2, by itself or as a pharmaceutically acceptable
salt, e.g., the acetic acid addition salt, the zinc salt,
the zinc tannate salt, etc.
A. Tablet formulations for buccal (e.g. sublingual)
administration:
4157J 23620-FF
~Lz6~7~
-50-
1. LHRH Antagonis~ 10.0 mg
Compressible Sugar, USP 86.0 mg
Calcium Stearate 4.0 mg
2. LHRH ~ntagonist 10.0 mg
Compressible Sugar, USP 88.5 mg
Magnesium Stearate 1.5 mg
3. LHRH Antagonist 5.0 mg
Mannitol, USP 83.5 mg
Magnesium Stearate, USP 1.5 mg
Pregelatinized Starch, USP 10.0 mg
4. LHRH Antagonist 10.0 mg
Lactose, USP 74.5 mg
Pregelatinlzed Starch, USP 15~0 mg
Magnesium Stearate, USP 1.5 mg
Method_of Manufacture
a. LH-R~ Antagonist is dissolved in water, a
sufficient quantity to form a wet granulation when mixed
with the sugar portion of the excipients. After complete
mixing, the granulation is dried in a tray or fluid-bed
dryer. The dry granulation is then screened to break up
any large aggregates and then mixed with the remaining
components. The granulation is then compressed on a
standard tabletting machine to the specific tablet weight.
b. In this manufacturing method, all formulations
would include 0.01% gelatin, USP. The gelatin would be
first dissolved in the aqueous granulation solvent
followed by the LH-RH analog. The remaining steps are as
in (a) above.
Formulation 4 could also be used as a tablet for
oral administration.
B. Long Acting intramuscular injectable formulation.
1. Long Actin~ I.M. Injectable - Sesame Oil Gel
LHRH Antagonist 10.0 mg
Aluminum monostearate, USP 20.0 mg
Sesame oil q.s. ad 1.0 ml
4157J 23620-FF
~2~
-51
The aluminum monostearate is combined with the
sesame oil and heated to 125C with stirring until a
clear yellow solution forms. This mixture is then
autoclaved for sterility and allowed to cool. The LH-RH
analogue is then added aseptically with trituration.
Particularly preferred LH-RH analogues are salts of low
solubility, e.g. zinc salts, zinc tannate salts, pamoate
salts, and the like. These exhibit exceptionally long
duration of activity.
10 2. Long Acting I.M. Injectable - Biodegradable
Po ymer Microcapsu es
LHRH Antagonist 1
25/75 glycolide/lactide 99%
copolymer (0.5 intrinsic
viscosity)
Microcapsules (0-150*) of above formulation suspended in:
Dextrose 5.0
CMC, sodium 0.
Benzyl alcohol 0.9%
Tween 80 0.1%
Water, purified q.s. 100.0%
25 mg of microcapsules would be suspended in 1.0 ml of
vehicle.
C. Aqueous Solution for Intramuscular Injection
LHRH Antagonist 500 mg
Gelatin, nonantigenic 5 mg
Water for injection q.s. ad 100 ml
Dissolve gelatin and LHRH antagonist in water for
injection, then sterile filter solution.
D. Formulation for Rectal Administration
Suppository Vehicle for Rectal Administration
_
LHRH Antagonist 5.0 mg
Witepsol H15 20.0 gm
The LHRH antagonist is combined with the molten Witepsol
H15, mixed well and poured into 2 gm molds.
4157J 23620-FF
~Z6~6~
-52-
Blological Activity
The useful activity of the compounds of the invention is
illustrat æ by the following results obtained in the standard
anti-ovulation test of A. Corbin and C.~1. Beattie, Endocr.
Res. Commun. Vol 2, page 1., 1975 ED50in ~s
Propylene
Glycol/saline Corn Oil
Compound Structure noon -24 hrs noon -24 hrs
1 2 3 6 10
10 N-AC
D-pClPhe, D-pClPhe, D-Trp, D-Dph*, D-Ala 2.6 - 2.0 14
N-Ac
D-Nal(2), D-pFPhe, D-Trp, D-Dph*, - 2.4 - 4.7 12
N-Ac
D-Nal(2), D-pFPhe, D-Trp, D-Deh, - 1.6 1.4 3.5
N-Ac-
D-p-Cl-Phe, D-p-Cl-Phe, D-Trp,-D-Deh, - 2.0 2.4 16
N-Ac-
D-p-ClPhe, D-p-ClPhe, D-Trp, D-Dph, - 2.6 2.3
N-AC-
D-p-Cl-Phe,D-p-Cl-Phe,D-Trp,D-Deh,D-Ala10 1.7 1.6 >8
N-A~-
D-p-Cl-Phe, D-p-Cl-Phe, D-Trp, D-Dmh, D-Ala10 1.6 1.3 1.8
N-Ac-
25 D-p-Cl-Phe, D-p-Cl-Phe, D-Trp, D-Dhi, D-Ala10 2.0 2.0 3.6
N-Ac-
D-Nal(2), D-p-F-Phe, D-Trp, D-~mh, - 1.7 0.8 3.3
N-Ac-
D-Nal(2), D-p-F-Phe, D-Trp, D-Dhi, - 1.7 1.0 2.8
30 N-Ac-
D-Nal(2), D-p-Cl-Phe, D-Trp, D-Deh, - 2.0 1.0 1.7
N-Ac-
D-Nal(2), D-p-Cl-Phe, D-Trp, D-Deh, ~-Ala10 0.67 0-5 2.5
4157J 23620-FF
~6~
-5~a-
E~am~le 8
Characterizing data for compounds prepared acc~rding
to the previo~s Exa~les is shown below:
5 Cpd $ Compo~nd
. .
[N-Ac-D-pcl-phel~2fD-Trp3rD-Dih6~D-Alalo]LHRH
(second peak)
2 [N-Ac-Pro ,D-pF-Phe2,D-Nal(2)3,
D-monoisopropylArg6]LHRH
3 [N-Ac-Prol,D-pF-Phe2,D-Nal(2)3,D-Dia6]LHRH
4 IN-Ac-Prol,D-pF-Phe2,D-Nal(2) ,
D-monoisopropylArg6,Pro9-NHE:t]LHRH
5 ~N-Ac-Pro ,D-pF-Phe ,D-Nal(2)3,D-Dia6,
Pro -NHEt]LHRH
6 IN-Ac-D-Nal(2)1~3,D-pF-Phe2~D-Dph ]LHRH
7 ~N-Ac-Pro ,D-pF-Phe ,D-Nal(2) ,D-Dph6]LHRH
~ [N_Ac_D_pcl-phel'2,D-Trp3,D-Dp~6,D-Ala ]LHRH
9 [N-Ac-D-pCl-Phe '2,D-Trp3,D-Dph6jLHRH
20 10 [N-Ac-D-Nal(2) ,D-p~-Phe2,D-Trp3,D-Dph6]LHRH
(second peak)
11 [N-Ac-D-Nal(2) ,D-pF-Phe ,D-Trp3,D-Dph6]LHRH
(third peak)
12 IN-Ac-D-pCl-Phe ' ,D-Trp3,D-Dhh6D-Ala ]LHRH
13 [N-Ac-D-Serl-D-pF-Phe2,D-Trp3,D-Dhh ]LHRH
25 14 [N-Ac-D-Nal(2) -D-pF-Phe2,D-Trp3,D-Dhh6]LHRH
15 [N-Ac-D-pCl-Phel'2,D-Trp3,D-Deh ]LHRH
16 IN-Ac-D-Nal(2)1,D-pF-Phe2,D-Trp3,D-Deh6]LHRH
17 [N-Ac-D-pCl-Phel'2,D-Trp3,D-Deh6D-Ala10]LHRH
18 [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Trp3,D-Deh6 3 -
LHRH
19 [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Trp3,D-Deh6D-Ala
1 ]LHRH
20 [N_AC_D_pcl-phpl~2~D-~rp3 D ~sh6]LHR
4157J 236~0-FF.
.
:. . - .
; ,~ . . .
1;~64'7~
-52~-
21 [N-Ac-D-Nal(2)1,D-pF-Phe2,D-Trp3,D-Wsh ]LHRH
22 I~l-Ac-D-pcl-~hel~2~D-~rp3~D-Dmhfi~D-Alalo]L~R~i
23 ~N-Ac-D-Nal(2) ,D-pF-Phe ,D-Trp3,D-Dmh6]LHRH
24 [N-Ac-D-pcl-phel~2rD-Trp3~D~Dhi6D-Ala ~LHRH
25 IN-Ac-D-Nal(2) 1-D-pF-Phe2 ,D-Trp ,D-Dhi JLHRH
26 ~N-Ac-D-pCl-Phel~2,D-Trp3,D-Dma6D,D-Alal0]-
LHRH[NG,NGl-dimethyl arginine: Dma; NG-ethyl,NG -
dimethylaminopropylhomoarginine: Wsh]
Physical Data
lD 1. mp 157-160. See Note 2. Amino acid analysis
(909): Ser, 0.82 (1); Pro, 1.12 (1); Ala, 1.14 (1),
Le~, 1.16 (1); Tyr, 0.83 (1); NH3, 2.58; Arg, 0.88
(l); D-pCl-Phe + Trp, 2.34 (3); D-Dih, 1.15 (1).
2. mp 144-150; I~]25 -24.0~ (c 0.3, HOAc). Calcd.
for C73H104N17O17F (1510.75): C, 58.04; H
6.94; N, 15.76. Found (143316): C, H low. Amino
acid anaiysis (924): Ser, 0.87 (1); Pro, 2.08 (2);
Gly, 1.01 (1); mono-isopropyl Arg, 0.85 (1); Leu,
1.02 (1); Tyr, 0.90 (1); NH3, 1.33 (1); Arg, 1.06
(1); D-pF-Phe, 0.91 (1); D-Nal(2), 1.09 (1).
3. mp 144-148; f~D5 -15.4 (c 0.32, HOAc). Calcd.
for C76HlloN17O17F (1552.83): C, 58.78; H
7.14; N, 15.33. Fo~nd (144251): C, H low. Amino
acid analysis (923): Ser, 0.94 (1); Pro, 2.05 (2);
Gly, 1.00 (1); D-Dia, 0.85 (1); Leu, 1.03 (1); Tyr,
0.96 (1); NH3, 1.27; Arg, 1.01 (1); D-pF-Phe, 0.g5
(l); D-Nal(2), 1.06 (1).
4. mp 145-150; [~]2 -18.1 (c 0.83, HOAc). See
Note 2.
Amino acid analysis (925): Ser, 0.91 (1); Pro, 2.09
(2); Leu, 1.05 (1); Tyr, 0.84 (1); NH3, 0.41; Arg,
- 1.11 (1); D-pF-Phe, 0.91 (1); D-Nal(2), 0.9S (l);
mono-isopropyl Ars, 0.74 (1).
5. mp 137-142; ~D -16.5 (c 0.2, HOAc). Calcd.
4157J 23620-FF
. ,. . , '
i; 4 ?7;~
-52c-
76 lllN16l6F (l523~83) C, 59.90; H,
7.34; N, 14.71. Found (143315): C, H low. Amino
acid analysis (926): Ser, 0.91 (1); Pro, 2.25 (2);
Leu, 1.05 (1); Tyr, 0.93 (1); NH3, 0.67; Arg,
1.01; D-pF-Phe, 1.00 (1); D-Nal(2), 1.05 (1); D-Dia,
0.86 (1).
6. mp 152-155; ~]25 -15.2 (c 0.2, HOAc). Calcd.
85~116N17O17F (1667): C, 61,24; H,
7.01; N, 14.28. Found (143304): C, H low. Amino
acid analysis (939): Ser, 0.94 (1); Pro, 1.11 (1);
Gly, 1.02 (1); Leu, 1.02 (1); Tyr, 0.90 (1); NH3,
1.45 (1), Arg, 1.06 (1); D-pF-Phe, 0.94 (1);
D-Dph, 1.06 (1); D-Nal(2), 2.32 (2).
7. mp 135-142; ~]D -26.5 (c 0.23, HOAc). Calcd.
H112~17O17F (1566.26): C, 59.02; H,
7.20; N, 15.20. Found (143303): C, H low. Amino
acid analysis (940): Ser, 0.88 (1); Pro, 2.02 (2);
Gly, 1.03 (1); Leu, 1.00 (1); Tyr, 0.88 (1); NH3,
1.61; Arg, 1.08 (1); D-pF-Phe, 0.92 (1); D-Dph,
1.08 (1); D-Nal(2), 0.91 (1).
8. mp 170-172; [~]D5 -17.5 (c 0.37, HOAc). Calcd.
C80H114N18l7l7C12 (1670.82): C,
57.51; H, 6.88; N, 15.09. Found (143294): C, H
low. Amino acid analysis (945): Ser, 0.88 (1);
Pro, 1.09 (1); Ala, 0.88 (1); Leu, 1.01 (1); Tyr,
0.92 (1); NH3, 1.76; Trp, 0.41 (1); Arg, 1.19 (1);
D-pCl-Phe, 1.84 (2); D-Dph, 0.97 (1). Note: Trp
does not separate well from D-pCl-Phe.)
9. mp 165-170; ~]D5 -25 (c 0.43, HOAC). Calcd.
79H112N1817C12 (1656.2) C, 57.27;
H, 6.81; N, 15.22. Found (lg2675): C, H low.
Amino acid analysis (981): Ser, 0.83 (1); Pro, 0.93
(1); Ala, 1.01 (1); Leu, 1.02 (1); Tyr, 0.94 (1);
NH3, 1.18 (1); Trp, 0.97 (1) ; Arg, 1.03 (1);
4157J 23620-FF
~6~7~Q
-52d-
D-pCl-Phe, 1.94 (2)1; D-Dph, 1.16 (1).
10. mp 142-150; [~]D solution opaque in HOAc.
CalCd- for C82H113N1817F (
59.98; H, 6.94; N, 15.35. Found (143302): C, H
low. Amino acid analysis (953~): Ser, 0.95 (1);
Pro, 0.93 (1); Gly, 1.10 (1); Leu, 1.09 (1); Tyr,
0.87 (1); D-pF-Phe, 0.223 (1); NH3, 2.87; Trp,
0.413 (1); Arg, 0.94 (1); D-Dph, 1.25 (1); D-Nal(2),
1.06 (1). (Note: D-pF-Phe values are low.)
11. mp 142-152. See note 20
Amino acid analysis (952): Ser, 0.83 (1); Pro, 0.95
(l); Gly, 0.97 (1); Leu, 1.06 (1); Tyr, 0.96 (1);
NH3, 3.17; Trp, 0.66 (1); Arg, 1.01 (1); D-pF-Phe,
0.78 (1); D-Dph, 1.22 (1); D-Nal~2), 1.27 (1).
12. mp 140-142; [a]25 -14.3 (c 0.14, HOAc). Calcd.
C82H118N18l3C12 4 CH3COOH.3 H2O
(1929.14): C, 56.03; H, 7.31; N, 13.07. Found
(141592): C, 55.75; H, 6.93; N, 12.76. Amino acid
analysis (987): Ser, 0.87 (1); Pro, 1.09 (1); Ala,
1-08 (1); Leui 1.02 (1); Tyr, 0.94 (1); NH3, 1.43;
Trp, 0.88 (1) , Arg, 0.97 (1); D-pCl-Phe, 1.76
(2)1; D-Dhh does not come off column even after
340 min.
13. mp 145-148; [a]25 -4.17 (c 0.11, HOAc). Calcd.
79H121N18l8F (1629-96): C, 58.21; H,
7.48; N, 15.46. Found (142506): C, H low. Amino
acid analysis (963B): Ser, 1.77 (2); Pro, 1.15 (1);
Gly, 1.07 (1); Leu, 1.01 (1); Tyr, 0.96 (1); NH3,
1.26; Trp, 0.73 (1); Arg, 1.01; D-pF-Phe, 0.81 (1).
30 14~ mp 147-151; [~]D5 -27.3 (c 0.51, HOAc). Calcd.
C89H127N1817F (1740-12): C, 61,43; H,
7.36; N, 14.490 Found (142505): C, H low. Amino
acid analysis (964B): Ser, 1.00 (1); Pro, 1.11 (1);
Gly, 1.03 (1); Leu, 1.03 (1); Tyr, 0.95 (1); NH3,
4157J 23620-FF
~l;26~0
-52e-
1.24 (1); Trp, 0.80 (1), Arg, 0.99 (1); D-pF-Phe,
0.87 (1); D-Nal(2), 0.85 (1).
15. mp 155-158; ~]D5 -26 (c 0.44, HOAc). Calcd.
C77H108N18l7C12 (1628-74): C, 56.78;
H, 6.68; N, 15.48. Found (142479): C, H low.
Amino acid analysis (975): Ser, 0.88 (1); Pro, 0.97
(l); Gly, 1.01 (1); Leu, 0.99 (1); ryr, 0.97 (1);
NH3, 1.19; Trp, 0.926 (1) ; Arg, 1.03 (1);
D-pC1-Phe, 1.85 (2)1; D-Deh, 1.28 (1).
16. mp 152-155~; [~]D5 -32.6 (c 0.26, HOAc). Calcd.
81HlllNl~O17F (1628): C, 59.76; H,
6.87; N, 15.49. Found (142481): C, H low. ~mino
acid analysis (966): Ser, 0.93 (1); Pro, 0.99 (1);
Gly, 1.10 (1); Leu, 0.94 (1); Tyr, 0.94 (1); NH3,
1.19; Trp, 0.76 (1); Arg, 1.02 (1); D-pF-Phe, 0.92
(l); D-Deh~ 1.23 (1); D-Nal(2), 0.95 (1).
17. mp 155-157; [~]D5 -18.4 (c 0.27, HOAc). Calcd.
78Hll0N1gl7l7C12 (1642.76) C,
57.03; H, 6.75; N, 15.35~ F~und (143293): C, H
low. Amino acid analysis (997): Ser, 0.89 (1);
Pro, 0.97 ~1); Ala, 0.98 (1); Leu, 1.03 (1); Tyr,
0.99 (1); NH3, 1.34; Trp, 0.93 ; Arg, 1.00 (1);
D-pCl-Phe, 1.86 (2)1; D-Deh, 1.17 (1).
18. mp 158-160; [~]D -29.3 (c 0.41, HOAc). Calcd.
C81HlllN18l7Cl (1644.36): C, 59.16; H,
6.80; N, 15.33. Found (143809): C, H low. Amino
acid analysis (1014): Ser, 0.92 (1); Pro, 1.02 (1);
Gly, 1.02 (1); Leu, 1.02 (1); Tyr, 0.99 (1); NH3,
1.26; Trp, 0.83 (1); Arg, 0.98 (1); D-pCl-Phe, 0.98
(1); D-Deh, 1.16 (1); D-Nal(2), 0.96 (1).
19. mp 154-157; [~]D5 -21.6~ (c 0.25, HOAc).
82 113 18ol7cl (1658-38): C,
58.39; H, 6.87; N, 15.20. Found (144272): C, ~ _
low. Amino acid analysis (1022): Ser, 0.95 (1);
4157J 23620-FF,
~264760
-52f-
Pro, 1.08 (1); Ala, 0.99 (1); Leu, 1.02 (1); Tyr,
1.01 (1); NH3, 1.34; Trp, 0.85 (1); Arg, 0.97 ~1);
D-pCl~Phe, 1.18 (1); D-Deh, 1.17 (1); D-Nal(2), 1.00
(1) -
20. mp 150-155; ~]D5 -17.9 (c 0.25, HOAc). Calcd.
C80H115N19l7C12 ~1685 84): C, 56 99;
H, 6.87; N, 15.79. Found (143292): C, H low.
Amino acid analysis (995): Ser, 0.86 (1); Pro, 1.08
(1); Glyr 1~04 (1); Leu, 0.97 (1); Tyr, 0.91 (1);
NH3, 1.32; Trp, 0.31 (1); Arg, 0.99 (1);
D-pCl-Phe, 1.34 (2); D-Wsh does not come off column
even after 340 min.
21. mp 150-154; [~]D -28.3 5c 0.55, HOAc). Calcd.
g4H118N19O17F (1685): C, 59.88; H,
7.06; N, 15.79. Found (143295): C, H low. Amino
acid analysis (996): Ser, 0.87 (1); Pro, 0.94 (1);
Gly~ 1.05 (1); Leu, 0.99 (1); Tyr, 0.95 (1);
D-pF-Phe, 0.90 (1); NH3, 1.34; Trp, 0.68 (1); Arg,
1.00 (1); D-Nal(2), 0.846 (1); D-Wsh does not come
off column even after 340 minutes.
22. mp 164-166; [~]D5 -12.7 (c 0.3, HOAc). Calcd.
76H106N18O17C12 (1614.72): C, 56.53;
H, 6.62; N, 15.61. Found (143660): C, H low.
Amino acid analysis (1011): Ser, 0.93 (1); Pro,
1.07 (1); Ala, 1.03 (1); Leu, 1.00 (1); Tyr, 0.97
(l); NH3, 1.27; Trp, 0.95 (1)1; Arg, 1.11 (1);
D-pCl-Phe, 1.9 (2)1; D-Dmh, 1.11 (1).
23. mp 160-164; ~]D -26.5 (c 0.29, HOAc). Calcd.
C79H107N18l7F (1600): C, 59.31; H,
6.74; H, 6.74; N, 15.76. Found (143658): C, H
low. Amino acid analysis (1012): Ser, 0.87 (1);
Pro, 1.06 (1); Gly, 1.00 (1); Leu, 1.02 (1); Tyr,
0.98 (1); NH3, 1.31; Trp, 0.87 (1); Arg, 1.08 r
4157J 23620-FF
4~760
-52g-
(1) ; D-pF-Phe, 1.25 (1); D-Dmh 1.08 ; D-Nal(2),
0.89 (1). ,-
24. mp 160-165; [~]D5 -12.2 (c 0.29, HOAc~. Calcd.
76 104N18l7C12 (1612-7): C, 56.60;
H, 6.50; N, 15.60~ Found (143659): C, H low.
Amino acid analysis (1010): Ser, 0.94 (1); Pro,
1.14 (1); Ala, 1.01 (1); Leu, 0.97 (1); Tyr, 0.96
(l); NH3, 1.23; Trp, 1.001; Arg, 1.06 (1);
D-pCl-Phe, 2.00 (2)1; D-Dhi, 0.99 (1).
25. mp 172-174~; [~]DS -29.8 (c 0.62, HOAc). Calcd.
C79H105N18O17F (1598): C, 59,38; H,
6.62; N, 15.78. Found (143657): C, H low. Amino
acid analysis (1013j: Ser, 0.89 (1); Pro, 1.07 (1);
Gly, 1.00 (1); Leu, 0.99 (1); Tyr, 0.97 ~ NH3,
1.24 (1); Trp, 0.85 (1); Arg, 1.04 (1); D-pF-Phe,
0.98 (2); D-Nal(2), 0.92 (1); D-Dhi, 0.98 (1).
26. mp 162-164; ~] -18.7 (c 0.54, HOAc). D5
Calcd- for C75Hlo4Nl8ol7cl2 (
56.28; H, 6.55; N, 15.75. Found (143808): C, H
low. Amino acid analysis (1015): Ser, 0.86 (1);
Pro, 1.14 (1); Ala, 1.01 (1); Leu, 1.01 (1); Tyr,
0.99 (1); NH3, 1.54; Trp, 1.02 (1) ; Arg, 0.99
(l); D-pCl-Phe, 2.04 (2) ; D-Dma, 1.02 (1) .
NOTES
lPeaks do not separate well, and values assigned are
the average Eor the two peaks.
Optical rotation and elemental analysis were not
carried out due to lack of sufficient material.
Also prepared were:
N-Ac-D-Nal(2)-D-p-Cl-Phe-D-Nal(2)-Ser-Tyr-D-Deh-Leu-Arg-
Pro-D-AlaNH2;
N-Ac-D-Nal(2)-D-p-F-Phe-D-Nal(2)-Ser-Tyr-D-Deh-Leu-Arg-
Pro-D-AlaNH ; r
4157J 23620-FF
~264~
-52~
N-Ac-D-Trp-D-p-Cl-Phe-D-Nal(2)-Ser-Tyr-D-Deh-Leu-Arg-
Pro-D AlaNH2;
N-Ac-D Trp-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-
Pro-D-AlaNH2.
- . _
1 0 ~
~/ '
'
/
/ -
_ _ _ _ _ . _ _ _ _
4157J 23620-FF, r
