Note: Descriptions are shown in the official language in which they were submitted.
~3~v~ ~
-
The present invention involves the design, synthesis
and use of synthetic analogs of the luteinizing hormone
releasing hormone (LHRH). An important achievement
involved synthesis of analogs which functioned as
antagonists of LHRH, were adequately potent to inhibit
ovulation and allowed the release of only negligible
amounts of histamine. Since there was no way of reliably
forecasting the structure of an antagonist having high
potency and véry low histamine release, it was necessary
o to explore diverse approaches to discover a combination of
structural features which would yield an antagonist of
r t 1 3 ~ 'J ~
~ -2-
'~
LHRH having high potency for ovulation inhibition and very
low activity for histamine release.
Various peptides such as substance P, vasoactive
intestinal peptide, gastrin, somatostatin, as well as
others, are well known to cause the release of histamine
from mast cells. These cells are in many tissues, such as
skin, lung and mesentery, gingiva, etc. Most cells have
granules containing histamine and other mediators of
inflammation which can be released by peptides to cause
capillary dilation and increased vascular permeability.
When it was noted that an antagonist of LHRH, for example
[Ac-D-2-Nall,D-4-F-Phe2,D-Trp3,D-Arg6]-LHRH, caused edema
of the face and extremities when it was administered to
rats, it appeared likely that such antagonists, if
administered to human subjects as a contraceptive agent,
would cause serious edema of the face and elsewhere in the
human body. Such side effects would likely prevent the
administration of such antagonists to human subjects.
The histamine-containing leukocyte is a basophile
which can also release histamine when stimulated by many
of the same peptides mentioned above. Basophiles differ
biochemically from mast cells and such differences may
allow for both predictable and unpredictable histamine
release in response to antagonists of LHRH. An antagonist
of LHRH, to be used clinically to prevent ovulation,
should not significantly release amounts of histamine from
either mast cells or basophiles.
The discovery of the side effects such as the
edematogenic and anaphylactoid actions of LHRH antagonists
made desireable the discovery of new LHRH antagonists
which prevented ovulation but did not release significant
histamine. These undesireable side effects have been
observed in rats, and it is likely that the Food and Drug
~ 13~ 6i9
Administration would not allow the testing of such
antagonists in human subjects.
Karten et al. (4), have reviewed available knowledge
on the structural characteristics for potent histamine
release by antagonists of LHRH. Some of the most
important findings are as follows. A most potent LHRH
antagonist in triggering histamine release in vitro
involved a combination of strongly basic D-amino acid side
chains (Arg or Lys) at position 6 and in close proximity
to Arg , and a cluster of hydrophobic aromatic amino acids
at the N-terminus. Thus, there is no specific amino acid
of the ten amino acids which is solely responsible for
histamine release. On the contrary, structural features
ranging from the N-terminus (the amino acids in the first
few positions, 1-4, etc.), and basic amino acids toward
the C-terminus (positions 6 and 8) somehow participate in
histamine release. Even D-Ala in position 10 has some
influence on histamine release, the rationale for which is
unclear. By themselves, two basic side chains in close
proximity, as in positions 6 and 8, are insufficient alone
to impart high release of histamine. The cluster of
hydrophobic amino acids at the N-terminus is insufficient
alone for high histamine releasing activity. Even a
hexapeptide fragment has revealed moderate histamine
releasing potency. There seems to be no correlation
between antiovulatory potency and histamine release of
these antagonists, in vitro.
In perspective, much of the entire chain of such
decapeptide antagonists may have influence on histamine
release. The same perspective appears to be true, but to
different degrees, for high antiovulatory activity. These
LHRH antagonists are usually decapeptides which indicates
that there are ten variables to adjust for a desired
anti-ovulatory activity and ten variables to adjust for
-4- 13~3~
eliminating histamine releasing activity. There are even
further variations for each of these twenty variables, the
number of possible peptides to design, synthesize and
assay becoming incalculable. Presumably, some of the ten
variables may be independent for anti-ovulatory activity
and histamine releasing activity while some variables may
overlap for these two biological activities. This
situation poses extraordinary difficulties to solve before
an antagonist of high potency for anti-ovulation and very
low potency for histamine release could be produced.
Diverse structural changes and combinations of the
ten amino acids followed by assays of both anti-ovulation
and histamine release activities should be performed in
the hope that a potent antagonist essentially free of side
effects would be discovered. The synthesis of new amino
acids to introduce into the decapeptide chains should also
be explored since the commonly available amino acids might
not suffice.
In the antagonists prepared according to the present
invention, arginine and its derivatives were not utilized.
Lysine was converted into derivatives with acyl groups or
with alkyl groups on the E-amino group. The amino acid
ornithine was acylated or alkylated on the d-amino group.
Both the L- and D- forms of lysine and the L-form of
ornithine were used in synthesizing these acyl and alkyl
derivatives. Structurally related intermediates were also
synthesized. All together, many new peptides were
synthesized by the basic and minimal concepts of ten
variables for anti-ovulation activity and ten variables
for histamine release, which may be independent or
partially overlapping. On such a basis, the number of
such peptides that can be designed becomes overwhelming,
and every reasonable priority must be considered to reduce
" ~5- 13~9659
the number of peptides to be synthesized in the hope that
a discovery will be realized.
Certain peptides were synthesized, tested and found
to demonstrate advantageous peptides. Among these
desireable peptides were the following two.
[N-Ac-D-2-Nall,D-pClPhe ,D-3-Pal ,NicLys ,D-
NicLys6,ILys8,D-Ala10]-LHRH was effective to prevent
ovulation and released remarkably little histamine.
[N-Ac-D-2-Nal ,D-pClPhe2,D-3-Pal3,PicLys5,D-
PicLys ,ILys8,D-Ala ]-LHRH was twice as effective as the
above peptide, and released no more histamine than do
"super agonists" of LHRH, which are presently being
marketed by several pharmaceutical companies.
These two new peptides, and yet additional related
peptides described herein provide acceptable balances of
high anti-ovulatory activity and low histamine release for
full potential clinical utility.
The present invention involves the preparation and
use of decapeptides having antiovulatory activity and with
minimal histamine-releasing effects. These decapeptides
includes those comprising:
Ser , PicLys5 and D-PicLys ;
N-Ac-D-2-Nall, D-pClPhe2, Ser4, D-PicLys5 and Pro9;
N-Ac-D-2-Nall, D-pClPhe2, D-3-Pal3, Ser4, D-PicLys , Pro
and D-Ala10;
N-Ac-D-2-Nall, D-pClPhe2, D-3-Pal3, Ser4, NicLys5, Pro9
and D-Alal0;
1339G~9
--6--
N-Ac-D-2-Nall, D-pClPhe2, D-3-Pal3, Ser4, Leu7, Pro9 and
D-Ala
N-Ac-D-2-Nall, D-pClPhe2, D-3-Pal3, Ser4, Leu7, Pro9 and
D-Ser ~;
D-pClPhe , Pro and D-Ala
D-pClPhe2, Pro9 and Serl~;
N-Ac-D-2-Nall, D-pClPhe2, D-3-Pal3, NicLys5, D-NicLys6,
ILys8 and D-Ala10;
N-Ac-D-2-Nal , D-pClPhe2, D-3-Pal3, NicLys5, D-NicLys6,
ILys8 and D-Ala ~;
N-Ac-D-2-Nall, D-pClPhe2, D-3-Pal3, PicLys5, D-PicLys6,
ILys8 and D-Ala10;
N-Ac-D-2-Nal , D-pClPhe , D-3-Pal3, NicLys5, D-NicLys ,
IOrn8 and D-Ala10;
N-Ac-D-2-Nall, D-pClPhe2, D-3-Pal3, PicLys5, D-PicLys6,
IOrn8 and D-Ala10;
N-Ac-D-2-Nall, D-pClPhe2, D-3-Pal3, MNicLys5, D-MNicLys6,
IOrn8 and D-Ala10;
N-Ac-D-2-Nall, D-pClPhe2, D-3-Pal3, PzcLys , D-PzcLys ,
IOrn8 and D-Ala10;
~ ~7~ 1~96~9
N-Ac-D-pClPhe , D-3-Pal3, Tyr5, D-NicLys6 and ILys8;
N-Ac-D-C12Phel, D-3-Pal3, Tyr5, D-NicLys6 and ILys8;
acylated Lys5, D-acylated Lys6 and N-alkylated diamino
acid8;
NicLys5, D-NicLys6 and ILys ;
PicLys5, D-PicLys6 and ILys ;
NicLys5, D-NicLys6 and IOrn8;
PicLys5, D-PicLys6 and IOrn8;
MNicLys5, D-MNicLys6 and IOrn8;
PzcLysS, D-PzcLys6 and IOrn8;
Tyr5, D-NicLys and ILys ;
TyrS, D-NicLys and IOrn ;
N-Ac-D-2-Nall, D-pClPhe2, D-3-Pal3, Ser4, NicLysS, D-
NicLys6, Leu7, ILys8, Pro9 and D-Alal0NH2; and
N-Ac-D-2-Nall, D-pClPhe2, D-3-Pal3, Ser4, PicLys5, cis D-
PzACAla6, Leu7, ILys8, Pro9 and D-Alal0NH2.
The present invention further involves use of the
above decapeptides in a process for inhibiting ovulation
in an animal. This process comprises administering to
said animal a decapeptide preferably having the structure:
N-Ac-D-2-Nall, D-pClPhe2, D-3-Pal3, Ser4, NicLys5, D-
NicLys6, Leu7. ILys8, Pro9 and D-Alal0NH2. Likewise, the
inventive process may be used to inhibit ovulation in an
~339659
-
animal; to inhibit the onset of puberty in an animal; to
inhibit the sexual impetus of an animal; to alter the
gonadal function of an animal; to inhibit the growth of
hormone-dependent tumors in an animal; and to lower LH and
FSH levels in serum of post-menopausal women. These and
other related uses will be apparent to those skilled in
the art upon examination of this specification.
Abbreviations and formulas used herein include the
following:
a = alpha
BOC = t-butoxycarbonyl
Br-Z = o-bromobenzyloxycarbonyl
nBuOAc = n-butylacetate
n-BuOH = n-butanol
c = cis
CDC13 = deuterochloroform
CHC13 = chloroform
CH2C12 = dichloromethane
CH3CN = acetonitril
Cl-Z = o-chlorobenzyloxycarbonyl
d = delta
DCC = dicyclohexylcarbodiimide
DIEA = diisopropylethylamine
DMF = dimethylformamide
E = eta
Et = ethyl
EtOAc = ethyl acetate
EtOH = ethanol
Et2O = diethyl ether
HF = hydrogen fluoride
HOAc = acetic acid
KH2PO4 = potassium dihydrogen phosphate
MeOH = methanol
MgSO4 = magnesium sulfate
1339659
'., g
NH40Ac = ammonium acetate
iPrOH = 2-propanol
py = pyridine
t = trans
TFA = trifluoroacetic acid
THF = tetrahydrofuran
TOS = p-toluensulfonyl
m = micro
Z = benzyloxycarbonyl
Abu = 2-aminobutyric acid
Aile = alloisoleucine
AnGlu = 4-(4-methoxyphenylcarbamoyl)-2-
aminobutyric acid
BzLys = NE-benzoyllysine
Cit = citrulline
C12Phe = 3,4-dichlorophenylalanine
CypLys = NE-cyclopentyllysine
DMGLys = NE-N,N-dimethylglycyl)lysine
Dpo = Nd-(4,6-dimethyl-2-pyrimidyl)
ornithine
Et2hArg = NG,NG-diethylhomoarginine
FPhe = A-fluorophenylalarine
HOBLys = NE-(4-hydroxybenzoyl)lysine
Ilys = NE-isopropyllysine
INicLys = NE-isonicotinoyllysine
IOrn = Nd-isopropylornithine
Me3Arg = NG,NG,NGl-trimethylarginine
Me2Lys = NE,NE-dimethyllysine
MNicLys = N -(6-methylnicotinoyl)lysine
MPicLys = NE-~6-methylpicolinoyl)lysine
NACAla = 3(4-nicotinoylaminocyclohexyl)alanine
2-Nal = 3-(2-naphthyl)alanine
NicLys = NE-nicotinoyllysine
NicOrn = Nd-nicotinoylornithine
Nle = norleucine, 2-aminohexanoic acid
'' -lO- 1~39659
NMeLeu = N-methylleucine
Nval = norvaline, 2-aminopentanoic acid
3-Pal = 3-(3-pyridyl)alanine
pClPhe = 3-(4-chloro)phenylalanine
PicLys = N -picoloyllysine
Pip = piperidine-2-carboxylic acid
PmcLys = NE-(4-pyrimidinylcarbonyl)lysine
PmACAla = 3[4(4-
pyrimidinylcarbonyl)aminocyclohexyl]alanine
PzACAla = 3(4-
pyrazinylcarbonylaminocyclohexyl)alanine
3-PzAla = 3-pyrazinylalanine
PzcLys = NE-pyrazinylcarbonyllysine
Sar = N-methylglycine
TinGly = 3-thienylglycine
Most natural amino acids were obtained from Peninsula
Laboratories, San Carlos, CA. The hydroxyl group of Ser
was protected as the benzyl ether, the phenolic hydroxyl
group of Tyr as the Br-Z derivative, and E-amino group of
Lys as the Cl-Z derivative, the guanidino group of Arg and
the imidazole group of His as the TOS derivatives. The
a-amino function was protected as the BOC derivative.
BOC-Orn(Z) was obtained from Sigma Chemical Co., St.
Louis, Mo. BOC-D-2-Nal, BOC-D-3-Pal, BOC-D-C12Phe, BOC-
pClPhe and BOC-ILys(Z) dicyclohexylamine salt were
provided by the Southwest Foundation for Biomedical
Research, San Antonio, TX. The benzhydrylamine
hydrochloride resin was obtained from Beckman Bioproducts,
Palo Alto, CA. The nitrogen content was about 0.65
mmoles/g. The CH2C12 was distilled before use.
The present invention involves the design, synthesis
and use of LHRH antagonists with high antiovulatory
potency and diminished activity to release histamine (1).
13~9G59
- ' --11--
These new antagonists feature, for example, D-N -
nicotinoyllsine (D-NicLys) in position 6 and N -
isopropyllysine (ILys) in position 8. The solution of D-
Arg6, particularly in combination with Arg8 and a cluster
of hydrophobic aromatic amino acid residues at the N-
terminal, have been implicated in the release of histamine
(2-4).
Other reductions of anaphylactoid activity were
obtained by increasing the distance between the positive
charges in positions 6 and 8 by Arg5 and by inclusion of a
neutral residue in position 6 as in [N-Ac-D-2-Nall,D-
pClPhe2,D-3-Pal3,Arg5,D-4(p-methoxybenzoyl)-2-amino-
butyric acid6,D-Alal0]-LHRH (2-Nal represents 3-(2-
naphthyl) alanine; PClPhe represents 3(4-
chlorophenyl)alanine; 3-Pal represents 3(3-
pyridyl)alanine) by Rivier et al. (5) and [N-Ac-D-2-
Nal ,D-aMepClPhe2~D-Trp3,Arg5~D-Tyr6~D-Alal0]-LHRH
(aMepClPhe represents 2 methyl-3(4-chlorophenyl)alanine)
by Roeske et al. (6). Further modifications in position 6
are reductive alkylation of D-Lys6 by Hocart et al. (7),
incorporation of N,N-diethylhomoarginine by Nestor et al.
(9). The cyclic analogs recently synthesized by Rivier et
1 did not show any lowering in histamine release
compared to the linear counterparts (10).
From the peptides of the present invention, two were
initially selected as models for further design. The
peptide [N-Ac-D-2-Nal , D-pClPhe2, D-3-Pal3, NicLys5, D-
NicLys6, ILys8, D-Alal0]-LHRH (named Antide) had an
impressive combination of potency and low histamine
release, antiovulatory activity (AOA) was 100~ at lug and
36% at 0.5ug; ED50 for histamine release, in vitro, was
consistently above 300ug/ul as compared to about 0.17 for
the standard analog [N-Ac-D-2-Nall,D-pFPhe2,D-Trp3,D-
Arg6]-LHRH (pFPhe represents 3(4-fluorophenyl)alanine)
133965~
-12-
(5). Another analog was identical to Antide except for
PicLys5 and D-PicLys6 (PicLys represents N-
picoloyllysine); 100~ AOA at 0.5ug and 40~ at 0.25ug;
ED50, 93+11.
Included herein are results from LHRH analogs with
acylated aminocyclohexylalanine residues in position 6,
from analogs in which Leu7 has been substituted with other
neutral residues, from a comparison of ILys8 vs. IOrn8,
and from tests on oral activity and duration of
antagonists activity when administered orally or
parenterally (s.c.)
Melting points are uncorrected. NMR data are
reported as d-values downfield from TMS.
Before acylation, the Z and Cl-Z groups of Lys and
Orn were cleaved by hydrogenolysis in MeOH in the presence
of 10% Pd/C.
BOC-D-BzLys was synthesized by acylation of BOC-D-Lys
with benzoyl chloride as described for the L- isomer by
Bernardi et al. (17).
BOC-DMG-Lys was prepared by acylation of BOC-Lys with
chloracetyl chloride using the same method and the
reacting the crude product from 10 mmoles BOC-Lys in 10 ul
THF with 10 ul 40~ aq. dimethylamine. The reaction
mixture was stirred 15 minutes in ice bath and then 2.5
hours at room temperature. After evaporation in vacuo the
crude product was dissolved in 10 ul H2O and applied on a
~io-Rad AGl-X8 column, acetate form, 1 x 25 cm. The
column was first washed with 200 ul water and then the
product was eluted with 6% HOAc and lyophilized several
times to remove the HOAc. Yield 60-70%. Amorphous mass.
Rf (n-BuOH:py:HOAc:H2O = 30:10:3:12) = 0.27. Purity ~
-13- 1~9659
9S~. NMR (CDC13):1.45,s,9H,t-butoxy group; 1.85-
1.48,m,6H,B,y,d,CH2 groups; 2.6,s,6H,N(CH3)2; 3.25,m,2H,
E-CH2; 3.37,s,2H,N-CH2-CO; 4.15,m,1H,a-CH.
The other acylated Lys derivatives in the tables were
prepared from BOC-D or L-Lys and the corresponding p-
nitrophenyl ester.
p-Nitrophenyl nicotinate. To 9.85 g, 80 mmoles,
nicotinic acid and 13.35 g, 96 mmoles p-nitrophenol in 250
ul DMF was added 16.5 g, 80 mmoles DCC with stirring in
ice-bath. After 1 hour at O'C and 3 hours at room
temperature the urea was filtered off and the product was
- precipitated by the addition of an equal volume of water.
Filtration, drying in vacuo and recrystallization from i-
PrOH gave 11.22 g, 57~ of white needles, m.p. 172.5-173 C
(24)
p-nitrophenyl isonicotinate was prepared, in the same
manner 12 g, 61%, m.p. 139-141'C, m.p. 137-139'C. (18)
Also p-nitrophenyl 6-methylnicotinate was prepared in
the same way. Yield from 70 mmoles 6-methylnicotinic
acid: 6.0 g, 33% after recrystallization from MeOH. M.p.
156-157 C. Rf (2% MeOH in CHC13) = 0.57 NMR (CDC13):
2.7,s,3H,CH3; 7.36,d,1H,py H5;7.45,m,2H,H adjacent to the
oxygen in the phenyl ring; 8.34,m,3H,H adjacent to the NO2
group in the phenyl ring overlapping with py H4;
9.27,d,lH,py H2.
P-nitrophenyl picolinate. 4.92 g, 40 mmoles,
picolinic acid and 5.84 g, 42 mmoles p-nitrophenol were
suspended/dissolved in 200 ul CH2C12. Then 8.24 g 40
mmoles, DCC was added in 20 ul CH2C12 with vigorous
stirring. Stirring was continued in room temperature for
17 hours. Then the mixture was filtered and the filter
1~396S9
- ~ -14-
cake washed with 30-40 ul CH2C12. The raw product was
first treated with 100 ul Et20 with stirring in ice-bath
and filtered. Recrystallization from 250 ul iPrOH gave
6.24 g, 63~ product. M.p. 154-6 C (dec.). M.p. 145-7 C
(18).
Pyrazinecarboxylic acid p-nitrophenylester. This
compound was prepared using the same method as the
previous compound. From 40 mmoles pyrazinecarboxylic acid
and 44 mmoles p-nitrophenol was obtained 35.2 mmoles, 88%,
ester. M.p. 180-182 C (dec.). Rf (CHC13:MeOH = 49:1) =
0.72. NMR (CDC13): 7.5,m and 8.37m,2H each, hydrogens
adjacent to the oxygen and nitro group respectively in the
phenol ring; 8.84,m,1H,pyrazine H5; 8.9,d,1H,pyrazine H ;
9.48,d,1H,pyrazine H3.
BOC-NicLys. 2.5 g BOC-Lys (L or D) was suspended in
200 ul DMF with stirring. Then 1.1 equivalent of p-
nitrophenyl nicotinate was added and the mixture stirred
at room temperature for 36 hours. The mixture was then
filtered and the filtrate evaporated to dryness at reduced
pressure to yield a yellow oil. The residue was stirred
with 2x50 ul Et20 in ice-bath. The first Et20 phase was
decanted, the second was filtered off. Recrystallization
from EtOAc/hexanes gave 2.05 g product, 58% (L-form).
M.p. 138 C, lit. (17) 138-141 C. L-form [a] 20D= -2.91
(MeOH), D-form [a]20D= 3.35 (MeOH).
L- and D-BOC-INicLys were prepared similarly by
acylating 10 mmoles L or D BOC-Lys with p-nitrophenyl
isonicotinate in 100 ul DMF, 40 hours, room temperature.
The crude product was partitioned between 120 ul EtOAc and
50 ul H2O. The EtOAc phase was extracted with 2 x 50 ul
H2O and 50 ul brine. The original aqueous phase was
back-extracted with 30 ul EtOAc. The combined EtOAc
phases were then dried (MgSO4) and evaporated and the
-15- 13396S9
residue was treated with Et2O and recrystallized as above
to give 1.07 g, BOC- L-INicLys, 30.5%. The yield for the
D compound was 1.26 g, 36~. NMR (Acetone d6):
1.4,s,9H,t-butoxy group; 1.8-1.48,m,6H,B,y,d,-CH2-;
3.44,t,2H,E-CH~; 4.13,m,1H,a-CH; 7.77,m,2H,py H5 and H ;
8.70,m,2H,py H and H .
L- and D-BOC-PicLys. 1.23 g, 5 mmoles, of L- or D-
BOC-Lys was stirred with 1.34 g, 5.5 mmoles, p-nitrophenyl
picolinate in 60 ul DMF for 16 hours. After filtration
and evaporation and product was purified by column
chromatography on silica gel on a 4.5 x 32 cm column and
the solvent system n-BuOH:py:HOAc:H2O = 30:10:3:12. The
product after chromatography was dissolved in EtOAc and
washed with H2O, brine, dried and evaporated in vacuo.
The yields were usually 60-70%. NMR (CDC13):
1.43,s,9H,t-butoxy group; 1.73-1.45,m,6H,B,y,d-CH2;
3.47,m,2H,E-CH ; 4.32,m,lH,a-CH; 7.43,m,lH,py H5;
7.85,m,1H,py H~; 8.2,m,1H,py H3; 8.55,m,1H,py H6.
L- and D-BOC-MNicLys. 10 mmoles BOC-Lys and 10.5
mmoles p-nitrophenyl 6-methylnicotinate were allowed to
react in 150 ul DMF in the usual manner. After 27 hours
filtration and evaporation yielded a yellow oil. Et2O
treatment (2 x 50 ul) gave 3.3 g product which was
recrystallized from 50 ul 20% MeOH in EtOAc/hexane. Yield
2.87 g, 78.6% (L-form). Rf(n-BuOH:py:HOAc:H2O =
32:10:3:12) = 0.61. NMR(CDC13): 1.46,s,9H,t-butoxy group;
1.9-1.5,m,6H,B,y,d-CH2; 2.57,s,3H,py CH3; 3.36,m,2H,E-CH2;
4.11,m,lH,a-CH; 7.22,d,lH,py H5; 8.08,m,lH,py H4;
8.95,broad s,lH,py H2.
L- and D-BOC-PzcLys. Using the method above was
obtained from 7.7 mmoles pyrazine carboxylic acid p-
nitrophenyl ester and 7 mmoles BOC-Lys, L or D, in 100 ul
DMF about 6 mmoles product after recrystallization from
~ -16- 1339659
_.
iPrOH. Rf(n-BuOH:py:HOAc:H2O = 30:10:3:12) = 0.47. NMR
(CDC13): 1.45,s,9H,t-butoxy group; 1.9-1.48,m,6H,B,y,d-
CH2-; 3.51,m,2H,E-CH2; 4.29,m,1H,a-CH; 8.52,q,1H,pyrazine
H5; 8.77,d,1H,pyrazine H6; 9.41,d,1H,pyrazine H3.
BOC-L-NicOrn. This compound was prepared the usual
way by reacting 7 mmoles p-nitrophenyl nicotinate with 5
mmoles BOC-Orn in 75 ul DMF for 36 hours. Evaporation and
recrystallization from EtOAc gave 3.5 mmoles, 70%, NicOrn,
m.p. 143-144 C. Rf(n-BuOH:HOAc:H2O = 4:1:2) = 0.70.
NMR(CDC13): 1.45,s,9H,t-butoxy group; 7.46,m,1H,py H5;
8.27,m,1H,py H4; 8.69,m,1H,py H6; 9.05,m,1H,py H .
BOC-D-trans-NACAla. 1.43 g, 5 mmoles, BOC-D-trans-
3(4-aminocyclohexyl) alanine (provided by the Southwest
Foundation for Biomedical Research) was stirred with 1.35
g, 5.5 mmoles, p-nitrophenyl nicotinate in 60 ul DMF for
120 hours in room temperature. The mixture was then
filtered, evaporated, treated with Et2O in ice bath and
filtered again. Recrystallization was done by heating in
12 ul EtOH and adding 18 ul hot H2O. This produced a
clear solution from which crystals separated on cooling.
This procedure was repeated twice. Yield: 0.98 g, 50%.
Purity >95%. M.p. >220 C. NMR(DMSO d6): 1.46,s,9H,t-
butoxy group; 1.9-1.48,m,11H,ring CH2, ring CH in position
1 and B-CH2; 3.72,m,lH,ring CH in position 4; 3.95,m,lH,
a-CH; 7.48,m,lH,py H5; 8.16,m,lH,py H ; 8.67,m,lH,py H6;
8.96,m,lH,py H .
BOC-D-cis-NACAla. 5 mmoles BOC-D-cis-3(4-
aminocyclohexyl)alanine (source: as above) and 5.5 mmoles
p-nitrophenyl nicotinate were allowed to react in DMF as
above. Reaction time: 25 hours. Purification was
achieved by Et2O treatment as above and silica gel
chromatography on a 4.5 x 32 cm column using the solvent
system CHC13:MeOH:py:HOAc = 75:10:10:5. Yield 1.3 g, 61%,
-17- 1~39559
_
amorphous powder. Rf (column system) = 0.58. NMR
(CDC13): 1.44,s,9H,t-butoxy group; 1.95-1.45,m,11H,ring
CH2, ring CH in position 1 and B-CH2; 4.22,m,1H,a-CH;
4.35,m,1H,ring CH in position 4: 7.35, 8.24, 8.63 and
8.98, lH each, assignments as previous compound.
BOC-IOrn(Z). This compound was prepared from BOC-
Orn(Z) by reductive alkylation with acetone and H2/Pd as
described by Prasad et al. (23) followed by conversion to
the Nd- Z derivative with benzyl chloroformate in aqueous
alkali (Schotten-Baumann conditions). Purification was
achieved by chromatography on silica gel with CHC13/MeOH
85:15. Rf (CHC13;MeOH:HOAc = 85:15:3) = 0.8. NMR(CHC13):
1.10,d,6H, isopropyl CH3; 1.40,s,9H,t-butoxy group; 1.7-
1.5,m,4H,B,y-CH2; 3.09,m,2H, d-CH2; 4.2,m,lH,a-CH;
5.10,s,2H,benzyl CH2; 7.3,m,5H,aromatics.
BOC-CypLys(Z). 2.04 g BOC-Lys(Z) was dissolved in 8
ul of cyclopentanone and 32 ul H2O containing 0.22 g NaOH.
Hydrogenation was performed in the presence of 0.4 g 10%
Pd/C at 50-60 psi in a Parr apparatus. After 4 hours the
hydrogenation was interrupted and 2 ul 0.5 M NaOH and 10
ul MeOH were added. The hydrogenation was then continued
for 16 hours at 50-60 psi. Then filtration and
evaporation. The residue was dissolved in 75 ul H2O and
the aqueous phase extracted with three times with Et2O and
once with hexane. The pH was then brought to 6-7 with HCl
and the solution evaporated in rotary evaporator, bath
temperature 40'C. The resulting product was then
converted to the Z-derivative using benzyl chloroformate
in aqueous NaOH (Schotten-Baumann conditions). Yield:
1.3 g, 58% overall. Rf (n-BuOH:py:HOAc:H2O - 30:10:3:12)
= 0.69. Purity >95~. NMR (CDC13): 1.45,s,9H,t-butoxy
group; 1.95-1.35,m,14H,ring CH2 + B,y,d-CH2; 3.13,broad
t,2H,E-CH2; 4.34-4.05,m,2H,a-CH + ring CH;
5.13,s,2H,benzyl CH2; 7.35,m,5H,aromatic protons.
-18- 1339~59
BOC-Me2Lys, D- and L-. These compounds were prepared
by hydrogenolysis of the corresponding Z- or Cl-Z-
derivatives in the presence of 37~ formaldehyde
essentially as described by L. Benoiton (22) for the Na _
acetyl analog. Purification was achieved by
chromatography on silica gel with the solvent system n-
BuOH:py:H2O = 2:2:1. The yields are 40-65% and the
products are amorphous. NMR (CDC13): 1.41,s,9H,t-butoxy
group; 1.9-1.5,m,6H,B,y,d-CH2; 2.6,s,6H,N(CH3)2;
2.8,m,2H,E-CH2; 4.03,m,lH,a-CH.
BOC-D-AnGlu. 0.62 g, 3 mmoles, DCC was added to the
ice-cooled solution of 1.10 g, 3 mmoles, BOC-D-glutamic
acid a-benzylester and 0.39 g, 3 mmoles, p-anisidine in 25
ul CH2C12. The reaction mixture was stirred while warming
up to room temperature and then another 17 hours. The
dicyclohexylurea was then filtered off and CHC13 added to
a total volume of 125 ul. This solution was extracted
with 2 x lN H2SO4, H2O, saturated NaHCO3, 2 x H2O and
dried (MgSO4). Evaporation and recrystallization from
EtOH gave 0.99 g, 74% product, m.p. 129.5-131 C. Rf (4%
MeOH in CHC13) = 0.53. This product was dissolved in 30
ul MeOH and 10 ul EtOH and hydrogenated in the presence of
0.3 g Pd/C at 50 psi for 2.5 hours. Filtration and
evaporation gave a quantitative yield of BOC-D-AnGlu. Not
crystalline. Purity >98~. NMR (CDC13): 1.45,s,9H,t-
butoxy group; 2.35-1.95,m,2H,B-CH2; 2.6-2.4,m,2H,y-CH2;
3.76,s,3H,OCH3; 4.3,m,1H,a-CH; 6.82 and 7.42, broad d, 2H
each, aromatic protons.
BOC-Me3Ara. First, N,N,N',S-tetramethylisothiourea
was prepared by the procedure of Lecher and Hardy (19).
B.p. (15 mm) = 74 C, lit(above) 68 C at 11 mm. BOC-Orn,9
mmoles, and teramethylisothiourea, 10 mmoles, were
dissolved in 15 ul DMF and 2 ul triethylamine and
incubated at 100'C for 2 hours and at room temperature for
-lg- 1339659
10 hours. Then the reaction mixture was evaporated to
dryness and passed through a silica gel column eluted by
iPrOH:triethylamine:H2O = 42:6:13. The white solid so
obtained was dissolved in H2O and the solution was
acidified with 6N HCl and lyophilized to give 5.5 mmoles
product. Rf (column eluant) = 0.50. NMR (D2O):
1.42,s,9H,t-butoxy group, 2.80,m,1H,a-CH; 2.89,s,3H, CH3
on guanidino group; 2.96,s,6H, (CH3)2N; 3.25,t,2H,d-CH2;
1.50,m,4H,B,y-CH2.
BOC-Dpo. From 10 mmoles arginine hydrochloride and
1.72 g sodium hydrogen carbonate dissolved in 17 ul H2O,
28.6 ul acetylacetone and 28.6 ul EtOH was obtained 7.5
mmoles Dpo following the procedure of F.-S. (20). The
product was then converted to the corresponding BOC-
derivative using di-t-butyl dicarbonate in 50~ aqueous
dioxane in the presence of sodium hydroxide. This
reaction proceeds in essentially quantitative yield.
Rf(nBuOH:HOAc:H2O = 4:1:2) = 0.63. NMR (CDC13):
1.45,s,9H,t-butoxy group; 1.9-1.5,4H,B,y-CH2;
2.33,s,6H,CH3; 3.46,m,2H,d-CH2; 4.24,m,1H,a-CH; 6.35,s,1H,
aromatic H. L- and D- forms react similarly.
BOC-D-Et2hArg. This compound was prepared by the
method of Nestor and Vickery, U.S. Pat. 4,530,920, July
23, 1985. Rf(nBuOH:HOAc:H2O = 4:1:2) = 0.52.
The peptides of the present invention were
synthesized by the solid phase method using a Beckman
Model 990 Peptide Synthesizer. (1, 11) The
benzhydrylamine hydrochloride resin (BHA-resin) was used
as a solid support. The program of the synthesizer was
divided into subprograms.
1. Deprotection: 1. CH2C12 (2 x wash, 1 or 2
min); 2. 50% TFA in CH2C12 containing 0.1~ indole (1 x
'. -20- 13~9G59
wash, 1 or 2 min); 3. 50% TFA in CH2C12 containing 0.1%
indole (deprotection, 20 min); 4. CH2C12 (2 x wash).
2. Neutralization: 1. CH2C12 (2 x wash, 1 or 2
min); 2. DIEA (10% in CH2C12) (2 x wash, 1 or 2 min); 3.
DIEA (10% in CH2C12) (neutralization, 5 min); 4. CH2C12
(2 x wash, 1 or 2 min).
3. DCC Coupling: 1. CH2C12 (2 x wash, 1 or 2
min); 2. amino acid solution in CH2C12 (delivery,
transfer, mix, 5 min); 3. DCC (10% in CH2C12, (delivery
and mix, 180 min); 4. CH2C12 (2 x wash, 1 or 2 min).
4. Active Ester Coupling: not used.
5. Final Wash: 1. CH2C12 (2 x wash, 1 or 2 min);
2. i-PrOH (3 x wash, 1 or 2 min); 3. DMF (3 x wash, 1 or
2 min); 4. CH2C12 (3 x wash, 1 or 2 min).
6. Wash after TFA Treatment: 1. CH2C12 (2 x wash,
1 or 2 min); 2. i-PrOH (2 x wash, 1 or 2 min); CH2C12 (3
x wash, 1 or 2 min).
7. Acetylation: 1. CH2C12 (2 x wash, 1 or 2 min);
2. 25% Ac2O and Py in CH2C12 (1 x wash, 1 or 2 min); 3.
25% Ac2O and Py in CH2C12 (acetylation, 20 min); 4.
CH2C12 (2 x wash, 1 or 2 min).
The first amino acid was attached to the resin by the
program sequence 2-3-5. Before placing the resin into the
reaction vessel, the resin was washed in a separatory
funnel with 25 ul CH2C12/g resin to remove the fine
particles. In all couplings, usually a 3-4 fold excess of
the Boc-amino acid over the nitrogen content of the resin
was used. This procedure generally resulted in a complete
coupling reaction. If a positive ninhydrin color reaction
-21- 1339659
. ~
was observed, a second coupling was performed (program
sequence 3-5). Then, the resin was acetylated (program
sequence 7-5).
The next amino acid was attached by the program
sequence 1-6-2-3-5. For DCC coupling, all amino acids
were dissolved in CH2C12. Acetylation of the amino acid
residue in position 1 was performed using the program
sequence 1-6-2-7-5. The volume of the solvents and the
reagents used for the washing and the performing of the
chemical reactions was about 10 ul/g resin.
After all of the amino acids had been coupled, the
peptide resin was dried overnight, in vacuo. The resin
was then treated with double-distilled liquid hydrogen
fluoride (10 ul/g resin) containing 10-25~ distilled
anisole or p-cresol for 1 hour at 0 C. Then, the HF was
evaporated under reduced pressure and the residue was
dried overnight, in vacuo, by an oil pump. The mixture
was then extracted several times with Et2O (25 ul/g
resin), then with aqueous. HOAc, 30%, 50%, 10%, and once
with 25 ul distilled, deionized water. The combined
aqueous solution was lyophilized to yield the crude
peptide.
Most peptides were purified by silica gel
chromatography (1 x 60 cm column) using one of the solvent
systems nBuOH:HOAc:H2O = 4:1:2 or 4:1:5 upper phase or
nBuOAc:nBuOH:HOAc:H2O = 2:8:2:3 followed by gel filtration
over Sephadex*G 25 with 6% HOAc as the eluant. In the
case of unsatisfactory purity after this procedure the
peptides were further purified by semipreparative HPLC
using a Waters*liquid chromatograph equipped with a 660
solvent programmer. A 1.2 x 25 cm m-Bondapak*C18 column
was used with the solvent system A = 0.1 M NH40Ac pH 5.0
and B = 20% A + 80% CH3CN. Different gradients of
* a trade-mark
~ B-~
-22- . ~339659
increasing amounts of B in 15 - 25 minutes were employed
to effect purification.
An alternate purification scheme has been gel
5 filtration over Sephadex*G-25 with 6% HOAc followed by --
chromatography over Sephadex*LH 20 (2.5 x 100 cm) with the
solvent system H2O:nBuOH:HOAc:MeOH = 90:10:10:8. If
necessary, the latter procedure was repeated 1 - 2 times.
The purity of the peptides was assessed by thin layer
chromatography on Merck silica gel plates in at least four
different solvent systems as shown in Table II. The spots
were developed with the chlorine/o-tolidine reagent. In
Table II are also shown the conditions and results of
analytical HPLC. The equipment was the one described
above except that an analytical m-Bondapak*C18 column (3.9
mm x 30 cm) was used.
Amino acid analyses were performed on a Beckman model
118 CL amino acid analyzer. Samples of about 0.5 ug were
hydrolyzed in 6N hydrochloric acid in sealed glass tubes
for 24 hours at 110 C. The residue was then evaporated
and dissolved in citrate buffer, pH 2.2 and applied to the
analyzer. The results are in Tables I and II.
The antiovulatory activity, AOA, in rats was
determined as described by Humphries et al. (12). The
wheal test was performed by intradermally injecting 10 ug
of peptide in 100 ul of saline into anaesthesized rats,
measuring the ideally circular wheal response and
calculating the area. The in vitro histamine release test
was done as described by Karten et al. (4).
The results of these bioassays are presented in Table
I and other Tables appended hereto.
* a trade-mark
r
1339659
-23-
Of the 57 peptides in Table I, 21 had an AOA of about
90% or more at a dosage of 1 ug in the present assay. Of
the 37 peptides of Table 1 tested for histamine release in
the rat mast cell assay, 10 had ED50 values of 300 or more
as compared to 0.17 for the standard compound [N-Ac-D-2-
Nall,D-4-F-Phe2,D-Trp3,D-Arg6]-LHRH. Nine additional
analogs had ED50 values ranging from 86 to 288, i.e. they
do not release more histamine than clinically used
"superagonists".
Of the thirty-seven peptides of Table 1 tested in the
rat mast cell assay, seven (numbers 4, 23, 24, 43
(Antide), 44, 53, 55) had both an AOA of about 90% or more
at 1 ug and an ED50 value of about > 86 ug/ul. This
included the potent analog, No. 53, which had 100% AOA at
0.5 ug and 40% AOA at 0.25 ug. The ED50 value for this
analog was 93+28. It was thus demonstrated that high AOA
with low histamine release could be found in the analogs
of the present invention.
Structural features in common for these seven
peptides are: 1) A D-Lys residue in position 6 which was
acylated by the weakly basic nicotinic acid or analogs
like picolinic and 6-methylnicotinic acid. 2) The
corresponding acylated L-Lys residue or the natural Tyr in
position 5. 3) The alkylated derivatives ILys or IOrn in
position 8. 4) Arg is absent from the sequence.
Two examples of the influence of Arg on histamine
release are the pairs 43,10 and 4,1. No. 43 (Antide) has
the sequence N-Ac-D-2-Na ,D-pClPhe sub,D-3-
Pal3,Ser ,NicLys5,D-NicLys6,Leu7,ILys3,Pro9,D-Ala -NH2.
Its ED50 value is >300. No. 10 is identical in sequence
except that NicLys5 is replaced by Arg5. This caused the
ED50 value to decrease to 4.3+0.52. No. 4 has identical
sequence as No. 43 except for Tyr in position 5. Its ED50
1~39659
-24-
value is 133+22. In No. 1, ILys8 in this sequence is
replaced by Arg8 which caused the ED50 va1ue to decrease
to 39.2+7. It thus seems that position 5 is more
sensitive than position 8 for Arg substitution.
In position 8, the alkylated ILys and IOrn residues
are superior to Lys and Orn, respectively, both with
respect to AOA and histamine release (pairs 3,4 and 6,7).
Whether ILYs8 or IOrn8 is best seems to be sequence
dependent.
For the determination of duration of action, the
antagonist was administered s.c. or orally to 26 days old
female rats at a specific time before administration of
the agonist, [D-Qal6]-LHRH. The serum levels of rat
luteinizing hormone (LH) and rat follicle stimulating
hormone (FSH) were then measured 2 hours after the agonist
administration by RIA. The oral administration was done
through force-feeding with feeding tubes.
Table IV shows data on AOA and histamine release for
analogs containing acylated aminocyclohexylalanine
residues. For the analogs with NicLys5, D-NACAla6, IV-1
and IV-2, (NACAla represents 3(4-nicotinoyl-
aminocyclohexyl)alanine), analog 2 with cis-D-NACAla6 is
somewhat more active, 100% vs. 70% AOA at lug. Analogs
IV-7 and IV-8 with NicLys5, D-PzACAla6 (PzACAla represents
3(4-pyrazinylcarbonylaminocyclohexyl)alanine) show the
opposite order of activity. The trans residue has the
higher AOA, 88% vs. 25% at lug.
Analogs IV-3 and IV-4 with PicLys5, trans and cis
PACAla6 (PACAla represents 3(4-
picolinoylaminocyclohexyl)alanine) are equipotent, 50 and
54% AOA at 0.5ug, respectively, whereas in the case of
PicLys5, trans and cis PzACAla6 the cis compound is more
1339659
- -25-
than twice as active. The former, analog IV-5 is about as
potent as analogs IV-3 and IV-4 (44% at 0.5ug) while the
latter, analog 6, has 100%, 73%, and 29% AOA at 0.5, 0.25,
and 0.125ug, respectively. The high potency analog IV-6
is unique in comparison with the low activity of the
structurally similar analog IV-8.
Analog IV-9 has cis-PzACAla5, D-PicLys6 and, although
residues 5 and 6 are reversed, retained the high potency
of analog IV-6, 90~ and 67% at 0.5 and 0.25ug,
respectively.
As for histamine release, all analogs tested, in
vitro, have lower ED50 values than the parent compounds.
The ED50 values range from about 30 to about 60 compared
to >300 and 93+11 for Antide and analog V-10. The tests
for wheal response show a range from 99.5 to 129.6, which
is similar to Antide (132.7) and analog V-10 (123.0). The
lack of correlation between the two tests may primarily
reflect assay variation.
In summary, for the analogs with NicLys5,
incorporation of aminocyclohexylalanine derivatives in
position 6 resulted in substantial increase in, in vitro,
histamine release and unchanged or lowered AOA. For the
PicLys5 analogs with the same substitutions there was
lowering of AOA potency in all cases except one, where a
considerable increase was observed. The combination
PicLys5 and cis-D-PzACAla6 evidently possesses some
beneficial structure. Histamine release for the PicLys5
analogs was increased by 50-100%.
In Table V, are the results from substitutions in
position 7 of analog V-10. This position allows some
structural freedom although none of the peptides show
higher AOA than analog V-10. Analogs V-12, V-14, and V-16
~9659
-26-
having Aile7 (alloisoleucine), Val7 and Abu7 (2-
aminobutyric acid), are equipotent with analog V-10.
Analog V-16 with the straight chain Abu7 is slightly more
potent than analogs V-13 and V-15 with Nle7 (norleucine)
and Nval7 (norvaline), respectively, which should more
closely resemble the natural Leu7.
For compound V-17 with the small Ala7, the AOA
decreased to 60% at 0.5ug. Incorporation of Trp7 which is
the natural residue in chicken II, salmon and lamprey
LHRH's (13-15), gave analog 18 with only 10% AOA at 0.5ug.
Trp7 may be too large considering the size of the adjacent
D-PicLys6 and Ilys8.
The most interesting feature of Table V is the, in
vitro, histamine release data. The three analogs with
similar AOA potency as analog V-10 show markedly
diminished histamine release. The ED50 values for analogs
V-12, V-14, and V-16 are >300, 213+30 and 273+27,
respectively; i.e., a 2-3 fold decrease in histamine
release is achieved by small changes in side chain
structure. Also, the wheal response is diminished for all
analogs compared to V-10.
It was noted earlier (1) that whether ILys or IOrn is
the best substituent in position 8 is sequence dependant.
To further investigate this aspect, the IOrn8 analogs
corresponding to some of the best peptides were
synthesized and tested. The results in Table VI indicate
that ILys8 may be better. For two of the pairs, analogs
VI-10, VI-l9 and VI-12, VI-21, ILys8 and IOrn8 were about
equivalent. For the other three pairs, the analogs with
ILys were more active, but the differences were not
large. The largest difference was for the pair with Val7,
where the ILys8-analog VI-14 showed 90% AOA at 0.5ug vs.
57% for the IOrn8-analog VI-20.
~ -27- 1c~39659
Analog VI-l9 was tested, in vitro, for histamine
release. The ED50 value is 42+3.1; i.e., the histamine
release is 2-fold that of the analog with one more CH2
unit. The wheal response did not change conspicuously
except for the Aile7 and IOrn8 analog 21 which had the low
value of 78.6+4.5 compared to the ILys analog 12 which had
97.9+2.9.
Table VII shows the duration of action of Antide and
two analogs. When Antide was injected 44 hours before 50
ng of [D-Qal6]-LHRH (Qal represents 3(3-quinolyl)alanine),
a superagonist, at doses of 3, 10, and 30ug, significant
reductions in serum LH were observed at the two higher
doses. The LH decreased from 113+11 to 46+12 and 5+0.7
ng/ul. Serum FSH was also decreased, most significantly
from about 300 to about 300 ng/ul at 30ug.
Analog VII-24, [Tyr5]-Antide, and analog IV-6 were
similarly injected 24 hours before the agonist. Analog
VII-24 showed high activity, reducing the LH level to
19+4, 3+0.4 and 0.3+0.03 ng/ul at doses of 3, 10, and
30ug, respectively. The corresponding figures for analog
IV-6 are 42+7, 15+3, and 3.4+2 ng/ul. This is interesting
since in the antiovulatory assay analog IV-6 is
considerably more potent, 73% at 0.25 ug vs. 45% at 0.5
ug. Perhaps, analog IV-6 is enzymatically degraded faster
than analog VII-24. The long duration of action of these
analogs s.c. may also be due to "depot" effects at the
site of injection.
Table VIII shows the duration of action of Antide
after oral administration. Forty-eight hours after
administration of 100 or 300ug dose levels of Antide,
there were significantly reduced levels of LH which had
been released by 5 ng of [D-Qal6]-LHRH s.c. Reductions
from 21+3 to 4+0.8 and 8+2 ng/ul, respectively, were
-28- 1~396S9
observed. The results are about the same in the -24 hour
experiment (9+2 and 6+0.3 ng/ul). Antide appears to
possess considerable resistance towards degrading en2ymes.
When Antide was given 2 hours before the agonist, a strong
decrease in LH levels was observed. At a dose of 30ug, a
significant lowering of the LH level to 6+1 ng/ul was
seen. At 100 and 300ug, the levels were 1+0.3 and 0.4+.4
ng/ul, i.e., very low levels. When 10 ng of agonist was
used, the results are qualitatively very similar.
For comparison, the last three entries in Table VIII
are from experiments with [N-Ac-D-pClPhe ' ,D-Trp ,D-
Arg6,D-Alal0]-LHRH, VIII-25, an analog that has been
reported to have oral activity, (16). These data show
that Antide is more active than VIII-25, since a dose of
30ug given 2 hours before the agonist reduced the LH level
from 44+4 to 22+4 ng/ul (p<0.01}. The value for analog
VIII-25 is 39+6 (NS). At 100 ug, the corresponding
numbers are 7+3 (p<0.001) and 26+7 (p<0.05). The FSH
levels were, in general, lowered when Antide was
administered at -2 hours at 100 or 300ug dose levels.
The results in Table IX show that there is no
significant difference between administration of Antide in
water or in corn oil.
Antide has also been tested orally in the
antiovulatory assay (Table X). The AOA values at 300,
600, and 1200ug dose levels are 18, 73, and 100%
respectively. Expressed as rats ovulated/total rats, the
numbers are 9/11, 3/11, and 0/11. For analog VIII-25, the
numbers 9/11, 4/11, and 0/11 have been reported at dose
levels of 500, 1000, and 2000ug, respectively, (16).
Antide was about twice as active as analog VIII-25.
~ 29- i 3~9659
Table XI shows a comparison of the oral activities of
Antide and four analogs. One was as active as Antide, one
was considerably less active and two were less active at
low doses (30 and 100ug) and about as active at 300ug.
After a 15 ng s.c. dose of [D-Qal6]-LHRH, the LH
level rose to 91+4.6 ng/ul. At oral dose levels of 30,
100, and 300ug of Antide, reduced levels of LH of 75+3,
20+4, and 5+1 ng/ul, respectively, were recorded. Analog
4 with PicLys5, and D-PACAla6 showed no significant
reduction of LH at 30 and 100ug levels, but there was a
reduction to 51+6 ng/ul at a 300ug dose.
Analog V-12 with PicLys5, D-PicLys6, and Aile7 and
analog IV-6 with PicLys5, cis-D-PzACAla6 are less active
than Antide at 30 and 100ug, but were equally active at
300 ug. Both of these peptides were substantially more
active than Antide in the s.c. antiovulatory assay.
Analog 26 was equipotent with Antide. This is not
suprising since the only structural difference between
these analogs is a pyrazine instead of a pyridine moiety
in the NE-acyl group of the D-Lys6 residue.
Table XI and XII also shows results with Antide, for
example, when 50 ng of the agonist was used. Comparison
of these results with the data from the experiments using
15 ng of agonist shows a dose-response relationship which
is expected from competitive antagonism. Using 15 ng of
agonist, 100 and 300ug of Antide reduced the LH level from
115+15 ng/ul to 20+4 and 5+1 ng/ul respectively, while in
the experiments using 50 ng of agonist, 300 and 900ug of
Antide reduced the LH to the same level (19+3 and 5.3+1.2
ng/ul).
_30_ i3~9G59
Table XIII shows the biological effects of Antide in
a dispersed pituitary cell culture system.
The structures and biological activites of certain
preferred LHRH analogs inhibiting more than 50% of
ovulatory activity at a dose of 0.25 ug are shown in Table
XIV.
It is proposed that Antide and other antagonists of
the present invention may be utilized to induce a state of
reversible medical castration that will be of value in the
treatment of a rather large number of diseased states such
as endometriosis, uterine fibroids and hormonal dependent
cancers (prostate, breast). In some patients temporary
inhibition of the function of the gonads with Antide, for
example, while the patient is receiving chemotherapeutic
agents and/or irradiation may prevent or minimize adverse
effects of these agents on the gonads and thus help to
preserve future fertility. Therapeutic examples would be
irradiation during bone marrow transplantation, cervical
carcinoma, metastatic thyroid and uterine carcinoma,
possibly thyrotoxicosis, etc. during chemotherapy for
disseminated lupus erythematosus and certain stages of
organ transplantation. More physiological usages of the
antagonists of the present invention such as Antide would
be to inhibit fertility in both females and males.
More unique possible usages of Antide or other
decapeptides of the present invention would be to modify
sexual behavior during select disease states. Such
disease states could involve patients with AIDS, the
aggressive behavior of sex offenders in prisons or
aggressive adolescents confined to corrective
institutions. It is also possible is that high serum
gonadotrophin levels of post-menopausal women may induce
functional abnormalities in fat cells that cause weight
'- -31- i339659
gain or in bone cells that play a role in accelerated
osteoporosis. These functional abnormalities could
potentially be reduced with administration of Antide by
inhibiting the high LH and/or FSH level in serum of post
menopausal women.
Selective LH-RH antagonists mainly with charged amino
acid substitutions in position 6 and/or 8 of the
decapeptides probably stimulate histamine release by a
direct effect on mast cells to release histamine while
other LH-RH antagonists like Antide do not. It is thus
proposed that the mast cell-stimulating antagonists
applied locally to wounds of the skin may accelerate
healing while non-histamine stimulating antagonists may
prevent some of the allergic reactions which occur in
humans.
To delay the onset of puberty in short stature
children by administration of Antide with and without
concommitant administration of GH or GH-releasing peptides
is proposed as a unique method to enhance body height.
The presence of gonadal hormones fuse the epiphysis of
long bone and prevent their further elongation. This
approach should extend and augment the use and
effectiveness of GH and GH-releasing peptides.
The administration of LH-RH antagonists of the
present invention acutely inhibits the function of the
gonads within 24 hours. Continuous administration of LH-
RH superagonists also inhibits the function of the gonadsbut this is only after several days of stimulating the
gonads to hyperfunction. Such superagonist administration
introduces a number of potential undesirable clinical
problems in patients with prostate cancer, endometriosis,
uterine fibroids as well as with sex offenders and those
subjected to a temporary induction of medical castration.
13~9659
-32-
For these reasons it is proposed that LH-RH antagonists
will be more desirable agents than LH-RH agonists for
introducing a reversible state of medical castration. At
the diagnostic level, such as differentiating the anatomic
source of steroid secretion from the adrenal versus the
ovary or to reveal the degree of calcium excretion
dependency on gonadal steroid hormones, the rapid onset of
inhibiting gonadal function with LH-RH antagonists makes
them an unequivocally superior agent over LH-RH agonists.
It is proposed that, in every clinical situation where
LH-RH superagonists have been utilized to inhibit gonadal
function, the LH-RH antagonists will be the agents of
choice.
References:
1. Ljungqvist, A., Feng, D.-M., Tang, P.-F.L., Kubota,
M., Okamoto, T., Zhang, Y., Bowers, C.Y., Hook, W.A. &
Folkers K. (1987) Biochem. BioDhvs. Res. Commun. 148 (2),
849-586.
2. Karten, M.D. & Rivier, J.E. (1986) Endocr. Rev. 7,
44-56.
3. Hook, W.A., Karten, M. & Siraganian, R. P. (1985)
Fed. Proc. Fed. Am. Soc. Exptl. Biol. 44, 1323.
4. Karten, M.D., Hook, W.A., Siraganian7 R.P., Coy,
D.H., Folkers, K., Rivier, J.E. & Roeske, R.W. (1987) in
LHRH and its Analoqs; ContraceDtive and TheraDeutic
ADplications ~L~ 2, eds. Vickery, B.H. & Nestor, J.J.,
Jr., (MTP Press Ltd., Lancaster, England) pp. 179-190.
1~39659
-33-
5. Rivier, J.E., Porter, J., Rivier, C.L., Perrin, M.,
Corrigan, A., Hook, W.A., Siraganian, R.P. & Vale, W.W.
(1986) J. Med. Chem. 29, 1846-1851.
6. Roeske, R.W., Chaturvedi, N.C., Hrinyo-Pavlina, T., &
Kowalczuk, M. (1987) in LHRH and its Analoqs;
Contraceptive and Therapeutic Applications Part 2, eds.
Vickery, B.H. & Nestor, J.J., Jr., (MTP Press Ltd.,
Lancaster, England) pp. 17-24.
7. Hocart, S.J., Nekola, M.V. & Coy, D.H. (1987) J. Med.
Chem. 30, 739-743.
8. Nestor, J.J., Tahilramani, R., Ho, T.L., McRae, G.I.
& Vickery, B.H. (1988) J. Med. Chem. 31, 65-72.
9. Bajusz, S., Kovacs, M., Gazdag, M., Bokser, L.,
Karashima, T., Csernus, V.J., Janaky, T., Guoth, J. &
Schally, A.V. (1988) Proc. Natl. Acad. Sci. USA 85, 1637-
1641.
10. Rivier, J., Kupryszewski, G., Varga, J., Porter, J.,
Rivier, C., Perrin, M., Hagler, A., Struthers, S.,
corrigan, A. & Vale, W. (1988) J. Med. Chem. 31, 677-682.
11. Folkers, K., Bowers, C.Y., Shieh, H.-M., Liu, Y.-Z.,
Xiao, S.-B., Tang, P.-F.L. & Chu, J.-Y. (1984) Biochem.
Biophys. Res. Commun. 123 (3) 1221-1226.
12. Humphries, J., Wan, Y.-P., Folkers, K. & Bowers, C.Y.
(1978) J. Med. Chem. 21(1), 120-123.
13. Miyamoto, K., Hasegawa, Y., Nomura, M., Igarashi, M.,
Kanagawa, K. & Matsuo, H. (1984) Proc. Natl. Acad. Sci.
USA 81, 3874-3878.
~ ~34~ i3~96S9
14. Sherwood, N., Eiden, L., Brownstein, M., Spiess, J.,
Rivier, J., & Vale, W. (1983) Proc. Natl. Acad. Sci. USA
80, 2794-2798.
15. Sherwood, N.M., Sower, S.A., Marshak, D.R., Fraser,
B.A. & Brownstein, M.J. (1986) J. Biol. Chem. 261, 4812-
4819.
16. Nekola, M.V., Horvath, A., Ge, L.-J., Coy, D.H. &
Schally, A.V. (1982) Science 218, 160-161.
17. Bernardi, et al., J. Pharm. Pharmacol. 19, 95 (1967).
18. Fife, T.H. and Przystas, T.J., J. Am. Chem. Soc. 107,
1041 (1985).
19. Lecher et al., U.S. 2,872,484, Feb. 3, 1959, Chem.
Abstr. 53, 11238c.
20. Tjoeng et al., Chem. Ber. 108, 862 (1975).
21. Humphries et al., J. Med. Chem. 21(1), 120 (1978).
22. Benoiton, L., Can. J., Chem. 42, 2043 (1969).
23. Prasad et al., J. Med. Chem. 19, 492 (1976).
24. Zinner, H. and Fiedler, H., Arch. Pharm. 291(63), 330
(1958).
Changes may be made in the particular amino acid or
derivatives and their assembly described herein or in the
steps or the sequence of steps of the method described
herein without departing from the concept and scope of the
invention as defined in the following claims.
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