Note: Descriptions are shown in the official language in which they were submitted.
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Novel LHRH-Antagonists, Production And Use Thereof As
Medicament
The invention relates to LHRH antagonists having
improved solubility properties, processes for the
preparation of these compounds, medicaments in which
these compounds are contained, and the use of the
medicaments for the treatment of hormone-dependent
tumours and hormone-influenced non-malignant disorders
such as benign prostate hyperplasia (BPH) and
endometriosis.
The nomenclature used for the definition of the
peptides agrees with that nomenclature explained by the
IUPAC-IUB Commission on Biochemical Nomenclature
(European J. Biochem. 1984, 138, 9-37), in which, in
agreement with the conventional representation, the
amino groups at the N terminus appear to the left and
the carboxyl group at the C terminus appears to the
right. The LH-RH antagonists such as the peptides
according to the invention include naturally occurring
and synthetic amino acids, the former including Ala,
Val, Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gln,
Cys, Met, Phe, Tyr, Pro, Trp and His. The abbreviations
for the individual amino acid residues are based on the
trivial names of the amino acids and are Ala=alanine,
Arg=arginine, Gly=glycine, Leu=leucine, Lys=lysine,
Pal(3)=3-(3-pyridyl)alanine, Na1(2)=3-(2-naphthyl)-
alanine, Phe=phenylalanine, Cpa=4-chlorophenylalanine,
Pro=proline, Ser=serine, Thr=threonine, Trp=tryptophan,
Try=tyrosine and Sar=sarcosine. All amino acids
described here originate from the L series, if not
mentioned otherwise. For example, D-Nal(2) is the
abbreviation for 3-(2-naphthyl)-D-alanine and Ser is
the abbreviation for L-serine. Substitutions on the E
amino group in the side chain of lysine are represented
by a term placed in brackets behind Lys, if appropriate
in the form of an abbreviation.
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Other abbreviations used are:
Ac Acetyl
B 4-(4-Amidinophenyl)amino-1,4-dioxobutyl
Boc tert-Butyloxycarbonyl
Bop Benzotriazol-l-oxy-tris(dimethylamino)-
phosphonium hexafluorophosphate
DCC Dicyclohexylcarbodiimide
DCM Dichloromethane
Ddz Dimethoxyphenyl-dimethylmethylenoxy-carbonyl
(Dimethoxy-dimethyl-Z)
DIC Diisopropylcarbodiimide
DIPEA N,N-Diisopropylethylamine
DMF Dimethylformamide
Fmoc Fluorenylmethyloxycarbonyl
HF Hydrofluoric acid
HOBt 1-Hydroxybenzotriazole
HPLC High-pressure liquid chromatography
Me Methyl
TFA Trifluoroacetic acid
Z Benzyloxycarbonyl
The peptides according to the invention are analogues
of the luteinizing-hormone-releasing hormone (LH-RH),
which has the following structure:
p-Glu-Hi s -Trp- Ser- Tyr-Gly-Leu-Arg- Pro -Gly-NH2, [LH-RH,
gonadorelin].
For more than 20 years, researchers have sought
selective potent antagonists of the LH-RH decapeptide
[M. Karten. and J.E. Rivier, Endocrine Reviews 7, 44-66
(1986)]. The high interest in such antagonists is based
on their usefulness in the field of endocrinology,
gynaecology, contraception and cancer. A large number
of compounds have been prepared as potential LH-RH
antagonists. The most interesting compounds which have
been found to date are those compounds whose structures
are a modification of the LH-RH structure.
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The first series of potent antagonists was obtained by
the introduction of aromatic amino acid residues into
the positions 1, 2, 3 and 6 or 2, 3 and 6. The
customary way of writing the compounds is as follows:
the amino acids are first indicated which have taken
the place of the amino acids originally present in the
peptide chain of LH-RH, the positions in which the
exchange took place being marked by superscripted
figures. Furthermore, by the notation "LH-RH" placed
afterwards it is expressed that these are LH-RH
analogues in which the exchange has taken place.
Known antagonists are:
[Ac-D-Cpal'2, D-Trp3'6] LH-RH (D.H. Coy et al., In:
Gross, E. and Meienhofer, J. (Eds) Peptides;
Proceedings of the 6th American Peptide Symposium,
pp. 775-779, Pierce Chem. Co., Rockville III. (1979):
[Ac-Prol, D-Cpa2, D-Nal(2)3'6] LH-RH (US Patent
No. 4,419,347) and [Ac-Prol, D-Cpa2, D-Trp3'6] LH-RH
(J.L. Pineda, et al., J. Clin. Endocrinol. Metab. 56,
420, 1983).
In order to improve the action of antagonists, basic
amino acids, for example D-Arg, were later introduced
into the 6 position. For example [Ac-D-Cpa1'2, D-Trp3,
D-Arg6, D-Ala10] LH-RH (ORG-30276) (D.H. Coy, et al.,
Endocrinology 100, 1445, 1982); and
[Ac-D-Nal(2)1, D-Phe(4-F)2, D-Trp3, D-Arg6] LH-RH (ORF
18260) (J.E. Rivier et al., in: Vickery B.H. Nestor,
Jr. J.J., Hafez, E.S.E (Eds). LHRH and its Analogs,
pp. 11-22 MTP Press, Lancaster, UK 1984).
Further potent LH-RH antagonists are described in
WO 92/19651, WO 94/19370, WO 92/17025, WO 94/14841,
WO 94/13313, US-A 5,300,492, US-A 5,140,009,
EP 0 413 209 Al and DE 195 44 212 Al.
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The latter discloses compounds having a modified
ornithine or lysine unit in position 6 and which
correspond to the following formula:
Ac-D-Nal ( 2 )'-D-Cpa2-D-Pal ( 3 ) 3-Ser4-Tyrs-D-Xxx6-Leu'-Arga-
Pro9-D-Alalo-NH2,
in which D-Xxx is an amino acid group of the general
formula (VI)
-HN-CH-CO-
I
(CH2)n
I
NH
l
CO-R
Further known LH-RH antagonists are antarelix,
ganirelix and cetrorelix.
Antarelix (INN: teverelix):
Ac-D-Nal ( 2) '-D-Cpa2-D-Pal ( 3) 3-Ser4-Tyr5-D-Hci6-Leu7-
Lys (iPr) 8-Pro9-D-Alalo-NH2
Ganirelix:
Ac-D-Nal ( 2)1-D-Cpa2-D--Pal ( 3) 3-Ser4-Tyr5-D-hArg ( Et ) 26-Leu'-
hArg (Et) z8-Pro9-D-Ala10-NH2
Cetrorelix:
Ac-D-Nal ( 2 )'-D-Cpa2--D-Pal ( 3 ) 3-Ser4-Tyr5-D-Cit6-Leu7 -Arg8-
Pro9-D-Ala10-NH2
The aim of the invention is to create novel LH-RH
antagonists which have an increased enzymatic stability
and significantly improved water solubility.
This object is achieved by compounds of the following
general formula (I)
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A-X]CX1-XXXZ -XxX3 -x.XX4 -Xxx5 -Xxx6 -Xxx7-XxXa -XXX9 -XXX10 -NH2
(I)
in which
A is an acetyl group,
Xxxl is D-Nal ( 2 ) ,
Xxx2 is D-Cpa,
Xxx3 is D-Pal(3),
Xxx4 is Ser,
Xxx5 is N-Me-Tyr,
xxx 6 is D-Cit, D-Hci or D-[--N'-4-(4-amidinophenyl)-
amino-1,4-dioxobutyl]Lys (abbreviation: D-Lys(B)),
Xxx' is Leu or Nle,
xxx 8 is Arg or Lys(iPr),
Xxx9 is Pro and
Xxxlo is D-Ala or Sar,
with the proviso
that if Xxx6 is D-Lys(B), then Xxx' is Nle,
if Xxx6 is D-Cit, then Xxx7 is Nle and Xxxlo is
D-Ala, or
if Xxx6 is D-Hci, then Xxx' is Leu and Xxxlo is
D-Ala,
and their salts with pharmaceutically acceptable acids,
in particular the acetates, embonates and
trifluoroacetates.
According to a further aspect of the invention, the
following compounds and their salts with
pharmaceutically acceptable acids are particularly
preferred:
Ac-D-Nal ( 2) 1-D-Cpaz-D-Pal ( 3) 3-Ser4-N-Me-Tyr5-D-Lys (B ) 6-
Nle7 -Arge-Pro9-Sarlo-NH2
Ac-D-Nal ( 2) 1-D-Cpa2-D-Pal ( 3) 3-Ser4-N-Me-Tyr5-D-Lys (B ) 6-
Nle7 -Arga-Pro9-D-Ala10-NH2 ,
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-N-Me-Tyr5-D-Lys(B)6-
Nle7 -Lys ( iPr ) 8-Pro9-Sarlo-NHz
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Ac-D-Nal(2)1-D-Phe(4-C1)2 -D-Pal(3)3-Ser4-N-Me-Tyr5-
D-Lys (B) 6-Nle7 -Lys (iPr) $-Pro9-D-Alalo-NH2
Ac-D-Nal ( 2) '-D-Cpa2--D-Pal ( 3) 3-Ser4-N-Me-Tyr5-D-Cit6-Nle7-
Arg8-Pro9-D-Alalo-NH2
Ac-D-Nal ( 2) 1-D-Cpa2-D-Pal ( 3) 3-Ser4-N-Me-Tyr5-D-Hci6-Leu7-
Arg8- Pro9-D-Alalo-NH2
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-N-Me-Tyr5-D-Cit6-Nle7-
Lys (iPr) 8-Pro9-D-Alalo-NH2
Ac-D-Nal ( 2 )'-D-Cpa2-D-Pal ( 3 ) 3-Ser4-N-Me-Tyr5-D-Hci6-Leu7-
Lys (iPr) e-Pro9-D-Alalo-NH2
According to a further aspect of the invention, the
compounds according to the invention are present as an
acetate, trifluoroacetate or embonate salt.
According to a further aspect of the invention, the
compounds according to the invention can be used as
medicaments or pharmaceutical preparations.
According to a further aspect of the invention,
pharmaceutical preparations are provided, comprising at
least one of the compounds according to the invention
and customary vehicles and excipients.
According to a further aspect of the invention, a
process for the preparation of the compounds of the
general formula I according to the invention is
provided, in which fragments from units Xx.-e" provided
with suitable protective groups, in which m is an
integer from 1 to 10 and Xxxl is acetylated, are
synthesized on a solid phase or in solution according
to customary processes, then the fragments are bound to
a solid phase by segment coupling and after conclusion
of the coupling the compounds of the general formula I
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are removed from the solid phase using customary
processes with amidation on the unit Xxxlo
According to a further aspect of the invention, the use
of the compounds according to the invention for the
production of medicaments for the treatment of hormone-
dependent tumours, in particular prostate carcinoma or
breast cancer, and for non-malignant indications whose
treatment necessitates LH-RH hormone suppression, is
provided.
According to a further aspect of the invention, a
process for the production of pharmaceutical
preparations is provided, where at least one compound
according to one of Claims 1 to 10 is mixed with the
customary vehicles and excipients and formulated as a
pharmaceutical.
According to a further aspect of the invention, a
process is provided for the treatment of hormone-
dependent tumours, in particular prostate carcinoma,
breast cancer or uterine myoma, and for non-malignant
indications whose treatment necessitates LH-RH hormone
suppression, such as endometriosis, benign prostate
hyperplasia (BPH), and in the treatment of fertility
disorders of women or of men, in mammals, in particular
in humans, by administration of an efficacious dose of
at least one compound according to the invention.
The compounds according to the invention can be used
for the treatment of hormone-dependent tumours, in
particular prostate carcinoma, breast cancer or uterine
myoma, and also for non-malignant indications whose
treatment necessitates LH-RH hormone suppression, such
as endometriosis or benign prostate hyperplasia (BPH).
Furthermore, they can be employed for the treatment of
fertility disorders in men or in women, for example for
controlled ovarian superstimulation in the course of
in-vitro fertilization. To this end, they are
customarily mixed with conventional vehicles and
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excipients and formulated as medicaments according to
processes known per se.
The synthesis of compounds according to formula (I) can
both be carried out either by classical fragment
condensation or by solid-phase synthesis according to
Merrifield with synthesis following one another using
D-lysine already acylated in the side chain with the
carboxylic acid of the general formula R1-COOH or by
reaction of a decapeptide unit with the appropriate
carboxylic acids by amide linkage in the side chain of
D-lysine6. Accordingly, the introduction of the R1-CO-
group can be performed in three different positions in
the process: before the condensation of the individual
units to give the peptide, after the incorporation of
lysine or ornithine in the peptide chain, but before
the condensation of the next unit or after condensation
of all units.
The compounds of the formula (I) are synthesized
according to the known methods, such as, for example,
by pure solid-phase technique, partly solid-phase
technique (so-called fragment condensation) or by the
classical solution couplings (see M. Bodanszky,
"Principles of Peptide Synthesis", Springer Verlag
1984).
For example, the methods of solid-phase synthesis are
described in the textbook "Solid Phase Peptide
Synthesis", J.M. Stewart and J.D. Young, Pierce Chem.
Company, Rockford, III, 1984, and in G. Barany and
R.B. Merrifield "The Peptides", Ch. 1, pp. 1-285, 1979,
Academic Press Inc. Classical solution syntheses are
described in detail in the treatment "Methoden der
Organischen Chemie [Methods of Organic Chemistry]
(Houben-Weyl), Synthese von Peptiden" [Synthesis of
Peptides] E. Wunsch (Editor) 1974, Georg Thieme Verlag,
Stuttgart, FRG.
The stepwise synthesis is carried out, for example, by
first covalently bonding the carboxy-terminal amino
acid whose a-amino group is protected to an insoluble
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support which is customary for this, removing the
a-amino protective group of this amino acid, bonding
the free amino group thus obtained to the next
protected amino acid via its carboxyl group, and in
this manner linking the customary amino acids of the
peptide to be synthesized in the correct sequence step
for step, and after linkage of all amino acids removing
the finished peptide from the support and removing any
further side function protective groups which may be
present. The stepwise condensation is carried out in a
conventional manner by synthesis from the
corresponding, customarily protected amino acids.
The linkage of the individual amino acids to one
another is carried out according to the methods
customary for this; those particularly suitable are:
= Symmetrical anhydride method in the presence of
dicyclohexylcarbodiimide or diisopropylcarbodiimide
(DCC, DIC)
= Carbodiimide method generally
= Carbodiimide/hydroxybenzotriazole method
(see The Peptides, Volume 2, Ed. E. Gross and
J. Meienhofer).
In the fragment coupling, the azide coupling, which
proceeds without racemization, or the DCC-1-
hydroxybenzotriazole or DCC-3-hydroxy-4-oxo-3,4-dihyro-
1,2,3-benzotriazine method is preferably used.
Activated esters of fragments can also be employed.
Esters of N-protected amino acids, such as, for
example, N-hydroxysuccinimide esters or 2,4,5-
trichlorophenyl esters, are particularly highly
suitable for the stepwise condensation of amino acids.
The aminolysis can be very well catalysed by N-hydroxy
compounds which have approximately the acidity of
acetic acid, such as, for example,
1-hydroxybenzotriazole.
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Intermediate amino protective groups which present
themselves are groups which are removed by
hydrogenation, such as, for example, the
benzyloxycarbonyl radical (= Z radical) or groups which
can be removed by weak acid. Suitable protective groups
for the a-amino groups are, for example:
tertiary butyloxycarbonyl groups, fluorenylmethyl-
oxycarbonyl groups, carbobenzoxy groups or
carbobenzothio groups (if appropriate in each case
having a p-bromo- [sic] or p-nitrobenzyl radical), the
trifluoroacetyl group, the phthalyl radical, the o-
nitrophenoxyacetyl group, the trityl group, the p-
toluenesulphonyl group, the benzyl group, benzyl
radicals substituted in the benzene nucleus (p-bromo-
or p-nitrobenzyl radical) and the a-phenylethyl
radical. Reference is also made here to P. Greenstein
and Milton Winitz, Chemistry of Amino Acids, New York
1961, John Wiley and Sons, Inc., Volume 2, for example
page 883 et seq., "Principles of Peptide Synthesis",
Springer Verlag 1984, "Solid Phase Peptide Synthesis",
J.M. Stewart and J.D. Young, Pierce Chem. Company,
Rockford, III, 1984, G. Barany and R.B. Merrifield "The
Peptides", Ch. 1, pp. 1-285, 1979, Academic Press Inc.,
and also The Peptides, Volume 2, Ed. E. Gross and
J. Maienhofer, Academic Press, New York. These
protective groups are fundamentally also suitable for
the protection of further functional side groups (OH
groups, NH2 groups) of the corresponding amino acids.
Hydroxyl groups present (serine, threonine) are
preferably protected by benzyl groups and similar
groups. Further amino groups not in the a-position (for
example amino groups in the (9-position, guanidino group
of arginine) are preferably orthogonally protected.
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The individual amino acid units, excluding lysine
[lacuna] modified by the R1-CO-group, are commercially
obtainable.
A possible course of the process for the preparation of
lysine modified by R1-CO-group is as follows:
1. The a-carboxylic acid group is suitably protected,
for example by esterification.
2. The E-amino group is protected, for example by the Z
group.
3. The a-amino group is protected (e.g. Boc group) in
such a way that a selectivity with respect to the
later removal of the amino protective groups
results.
4. The Z group on the E-amino group is removed.
5. The desired group R1-CO- is introduced on the 6-amino
group.
6. The protective group on the a-amino group is
removed.
7. The a-amino group is optionally reversibly
derivatized, e.g. with the Z group.
For the introduction of the R1-CO-group by reaction of
the amino group of the lysine with the appropriate
carboxylic acid or carboxylic acid derivative, suitable
processes are fundamentally the same processes as
described above for the linkage of the amino acids.
However, condensation using carbodiimide, for example
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 1-
hydroxybenzotriazole is particularly preferred.
The reaction for the linkage of the amino acids takes
place in an inert solvent or suspending agent which is
customary for this (for example dichloromethane), it
being possible to add dime thyl f ormami de, if necessary,
to improve the solubility.
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Suitable synthetic supports are insoluble polymers, for
example polystyrene resin in bead form, which can be
swollen in organic solvents (for example a copolymer of
polystyrene and 1% divinylbenzene). The synthesis of a
protected decapeptide amide on a methylbenzhydrylamine
resin (MBHA resin, i.e. polystyrene resin provided with
methylbenzhydrylamine groups), which affords the
desired C-terminal amide function of the peptide after
HF cleavage from the support, can be carried out
according to the following flow diagram:
Flow diagram
Peptide synthesis protocol using Boc-protected amino
acids
Stage Function Solvent/Reagent (v/v) Time
1 Washing Methanol 2 x 2 min
2 Washing DCM 3 x 3 min
3 Removal DCM/TFA (1:1) 1 x 30 min
4 Washing Isopropanol 2 x 2 min
5 Washing Methanol 2 x 2 min
6 Washing DCM 2 x 3 min
7 Neutralization DCM/DIPEA (9:1) 3 x 5 min
8 Washing Methanol 2 x 2 min
9 Washing DCM 3 x 3 min
10 STOP Addition of the Boc-As
in DCM + DIC + HOBt
11 Coupling DCM, optionally DCM/DCF approx.
90 min
12 Washing Methanol 3 x 2 min
13 Washing DCM 2 x 3 min
The Na-Boc-protected amino acids are customarily
coupled in a three fold molar excess in the presence of
diisopropylcarbodiimide (DIC) and 1-hydroxybenzo-
triazole (HOBt) in CH2C12/DMF in the course of 90 min,
and the Boc-protected group is removed by action of 50%
trifluoroacetic acid (TFA) in CH2C12 for half an hour.
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To check for complete conversion, the chloranil test
according to Christensen and the Kaiser's ninhydrin
test can be used. Radicals of free amino functions are
blocked by acetylation in a five fold excess of
acetylimidazole in CH2C12. The sequence of the reaction
steps of the peptide synthesis on the resin follows
from the flow diagram. For the removal of the resin-
bound peptides, the respective final product of the
solid phase synthesis is dried in vacuo over P205 and
treated at 0 C for 60 min in a 500-fold excess of
HF/anisole 10:1/v:v.
After distilling of HF and anisole in vacuo, the
peptide amides are obtained as white solids by washing
with anhydrous ethyl ether with stirring, and the
removal of polymeric support additionally obtained is
carried out by washing with 50% strength aqueous acetic
acid. By careful concentration of the acetic acid
solutions in vacuo, the respective peptides can be
obtained as highly viscous oils, which are converted
into white solids after addition of abs. ether in the
cold.
Further purification is carried out by routine methods
of preparative high-pressure liquid chromatography
(HPLC).
The conversion of the peptides into their acid addition
salts can be effected in a manner known per se by
reaction thereof with acids. Conversely, free peptides
can be obtained by reaction of their acid addition
salts with bases. Peptide embonates can be prepared by
reaction of trifluoroacetic acid salts (TFA salts) of
the peptide with free embonic acid (pamoic acid) or the
corresponding disodium salt of embonic acid. For this,
the peptide TFA salt is treated in aqueous solution
with the solution of disodium embonate in polar aprotic
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medium, preferably dimethylacetamide, and the pale
yellow precipitate formed is isolated.
The following examples serve to illustrate the
invention without restricting it.
Example 1 (D-68968):
Ac-D-Nal ( 2) '-D-Cpa2-D-Pal ( 3) 3-Ser4-N-Me-Tyr5-D-Lys (B) 6-
Nle7 -Arg8-Pro9-Sar10-NHZ
The synthesis of the decapeptide was carried out on a
polymeric support having a loading density of
0.55 mmol/g (aminomethyl-substituted resin, Fmoc
protection, type D-1675, Bachem). Lysine was coupled as
Fmoc-D-Lys(Boc) -OH and the Fmoc protective groups were
removed using 20% piperidine/DMF. After simultaneous
removal of all side-chain protective groups and
detachment from the polymeric support, the isolated
crude peptide was purified by means of preparative
HPLC. After freeze-drying, 98.5% strength decapeptide
was obtained.
The substitution on the =-nitrogen of D-lysine with
4-(4-aminophenyl)amino-1,4-dioxobutyric acid was
carried out using PyBop in DMF with the addition of
DIPEA. The purification of the isolated crude peptide
was carried out by means of preparative HPLC. The
subsequent freeze drying afforded about 99% strength
product (trifluoroacetate) of the empirical formula
C82H106C1N19015 with correct FAB-MS 1633 (M+H) (calc.
1631.78096)
Example 2 (D-68969):
Ac-D-Nal ( 2) '-D-Cpa2-D-Pal ( 3) 3-Ser4-N-Me-Tyr5-D-Lys ( B) 6-
N1 e7 -Arg8- Pro9-D-Ala10 -NH2
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The synthesis of the decapeptide was carried out on a
polymeric support having a loading density of
0.55 mmol/g (aminomethyl-substituted resin, Fmoc
protection, type D-1675, Bachem). Lysine was coupled as
Fmoc-D-Lys(Boc)-OH and the Fmoc protective groups were
removed using 20% piperidine/DMF. After simultaneous
removal of all side-chain protective groups and
detachment from the polymeric support, the isolated
crude peptide having a content of about 71% (HPLC) was
reacted further without purification.
The side-chain substitution of D-lysine with
4-(4-aminophenyl)amino-1,4-dioxobutyric acid was
carried out using PyBop in DMF with the addition of
DIPEA. The isolated crude peptide was purified by means
of preparative HPLC. After subsequent freeze drying, a
98.8% strength product (trifluoroacetate) of the
empirical formula C82H106C1N19O15 was obtained with
correct FAB-MS 1633 (M+H) (calc. 1631.78096)
Example 3 (D-68971):
Ac-D-Nal (2 ) '-D-Cpa2-D-Pal ( 3) 3-Ser4-N-Me-Tyr5-D-Lys (B) 6-
Nle7 -Lys ( iPr ) a-Pro9-Sarlo-NH2
The synthesis of the decapeptide was carried out on a
polymeric support having a loading density of
0.55 mmol/g (aminomethyl-substituted resin, Fmoc
protection, type D-1675, Bachem). Lysine was coupled as
Fmoc-D-Lys(Boc) -OH and the Fmoc protective groups were
removed using 20% piperidine/DMF. After simultaneous
removal of all side-chain protective groups and
detachment from the polymeric support, the isolated
crude peptide (content about 59%, HPLC) was purified by
means of preparative HPLC. After freeze drying, 95%
strength decapeptide was obtained.
The side-chain substitution of D-lysine with
4-(4-aminophenyl)amino-1,4-dioxobutyric acid was
CA 02402193 2002-09-09
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carried out using PyBop in DMF with the addition of
DIPEA. The isolated crude peptide was purified by means
of preparative HPLC. After subsequent freeze drying, a
96.6% strength product (trifluoroacetate) of the
empirical formula C85H112C1N17015 ws obtained with correct
FAB-MS 1648 (M+H) (calc. 1645.8218)
Example 4 (D-68987)
Ac-D-Nal(2)1-D-Phe(4-C1 )2-D-Pal(3)3-Ser4-N-Me-Tyr5-
D-Lys (B) 6-Nle7 -Lys (iPr) a-Pro9-D-Ala10-NH2
The synthesis of the D-Lys-6-unsubstituted decapeptide
was carried out on 9.09 g of polymeric support having a
loading density of 0.55 mmol/g, lysine6 was coupled as
Fmoc-D-Lys(Boc)-OH.
After removal from the resin, 8.15 g of crude peptide
were isolated. The purification of the crude peptide
was carried out by means of preparative HPLC.
The side-chain substitution of D-lysine with
4-(4-aminophenyl)amino-1,4-dioxobutyric acid was
carried out using PyBop in DMF, with addition of DIPEA.
The isolated crude peptide was purified by means of
preparative HPLC. After subsequent freeze drying, a
94.6% strength product (trifluoroacetate) of the
empirical formula C85H112C1N17015 was obtained with
corresponding FAB-MS 1646.8 (M+H; calculated 1645.82)
Example 5:
Ac-D-Nal ( 2) '-D-Cpa2-D-Pal ( 3) 3-Ser4-N-Me-Tyr5-D-Cit6-Nle'-
Arg8-Pro9-D-A1a10-NH2
Example 6:
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Ac-D-Nal ( 2) 1-D-Cpa2-D-Pal ( 3) 3-Ser4-N-Me-Tyr5-D-Hci6-Leu7-
Arg8 -Pro9-D-Ala10-NH2
Example 7:
Ac-D-Nal (2 )1-D-Cpa2-D-Pal (3 ) 3-Ser4-N-Me-Tyr5-D-Cit6-Nle7-
Lys (iPr) 8-Pro9-D-Alalo-NHZ
Example 8:
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-N-Me-Tyr5-D-Hci6-Leu7-
Lys (iPr) 8-Pro9-D-Alalo-NH2
General working procedures for the preparation of the
peptides according to Examples 5 to 8:
The decapeptides can be prepared both by the Merrifield
solid-phase synthesis (SPPS) [sic] and by classical
fragment condensation in solution. The synthesis of the
peptide sequence on the polymeric support is to be
preferred for economic reasons and can in principle be
carried out alternatively according to (1)Boc or (2)
Fmoc strategy; correspondingly, in each case either a
methylbenzhydrylamine resin (for 1) or an Fmoc-2,4-di-
methoxy-4'-(carboxymethyloxy)benzhydrylamine resin (for
2) can be employed for the C-terminal bonding of
D-alanine.
Solid-phase synthesis, Merrifield process:
The decapeptides are synthesized according to Fmoc
strategy under standardized reaction conditions (flow
scheme, Table I) for a solid-phase synthesis, using
5 grams of the polymeric support Fmoc-2,4-dimethoxy-
4'-(carboxymethyloxy)benzhydrylamine resin, Bachem
D1675, loading density about 0.55 mmol/gram, grain size
200-400 mesh.
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The stepwise synthesis of the sequence on the resin is
carried out with the aid of N=-Fmoc-protected amino
acids, according to the following flow scheme:
Table I:
Step Function Solvent Time Repetitions
1 Washing DMF 2 min 2 x
2 Removal 20% piperidine in 5 min 2 x
DMF
3 Washing DMF 2 min 2 x
4 Washing Isopropanol 2 min 1 x
5 Washing DMF 2 min 2 x
6 Washing Isopropanol 2 min 1 x
7 Washing DMF 2 min 2 x
8 Coupling Boc-AS-OH, HOBT, 90 1 x
DIC in DMF min
9 Washing DMF 2 min 1 x
Washing Isopropanol 2 min 1 x
11 Washing DMF 2 min 1 x
12 Washing Isopropanol 2 min 1 x
13 Washing DMF 2 min 1 x
14 Washing Isopropanol 2 min 1 x
Checking Chloranil colour
,
test
(*according to T. Christensen, Acta Chem. Scand. B 33,
763-766, 1979)
Process-typical (repetitive) reaction parameters of the
solid-phase synthesis of the decapeptides according to
the above scheme:
-Removal of the Fmoc protective group using 20%
piperidine in DMF, 2 x 5 min at RT (Step 2).
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-Couplings each in a threefold molar excess of Fmoc-
amino acids with diisopropylcarbodiimide (DIC) in the
presence of hydroxybenzotriazole (HOBt) (Step 8).
-C-terminal removal from the polymeric support
including removal of the amino acid side-chain
protective groups using trifluoroacetic acid (TFA).
After removal from the polymeric support, when using
5 grams of resin about 5-6 grams of crude peptide
mixture having a content of about 70-80% of desired
component are formed; this is recovered by subsequent
preparative HPLC chromatography.
Preparative HPLC purification of the decapeptides;
chromatography conditions:
Prep. HPLC, Shimadzu, Dynamax column RP18, 12 m,
300 A, L= 250 mm, ID = 41.4 mm
Gradient system with time programme, 40% B 90% B,
50 min
Eluent A: 970 ml of H20 + 30 ml of CH3CN + 1 ml of
CF3COOH
Eluent B: 300 ml of H20 + 700 ml of CH3CN + 1 ml of
CF3COOH
UV detection, == 220 nm, flow rate 60 ml/min
The fractions obtained are concentrated in vacuo and
lyophilized. The decapeptides are formed as a light
colourless material.
A double decomposition into the acetate salt form
desired for pharmacological development is then carried
out by chromatographic ion-exchange.
Investigations of the biological action:
The compounds according to formula I according to the
invention are investigated for their receptor binding.
The process closely follows the process described in
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Beckers et al., Eur. J. Biochem. 231, 535-543 (1995).
Cetrorelix obtained according to the synthesis
disclosed above is iodinated with [125I] (Amersham;
specific activity 80.5 Bq/fmol) using the IodoGen
reagent (Pierce). The reaction mixture is purified by
reverse-phase high-performance liquid chromatography,
monoiodinated cetrorelix being obtained without
unlabelled peptide. In each case, about 80% of the
[125I]-cetrorelix and the unlabelled compound according
to the invention are suitable for the specific receptor
association.
The compounds according to the invention can be tested
for their in-vitro action using the following Methods 1
and 2, the binding affinities in the binding assay
being determined with [125I]-cetrorelix (Method 1) and
the functional activities being determined with
triptorelin as an agonist stimulus (Method 2).
Method 1 (determination of KD using the example of
cetrorelix):
Receptor binding assay according to Beckers, T.,
Marheineke, K., Reilander, H., ' Hilgard P. (1995)
"Selection and characterization of mammalian cell lines
with stable overexpression of human pituitary receptors
for gonadoliberin (GnRH)" Eur. J. Biochem. 231,
535-543.
For investigation of the receptor binding, cetrorelix
is iodinated using the IodoGen reagent (Pierce) with
[125I] (Amersham; 80.5 Bq/fmol specific activity) . The
reaction mixture is purified by high-performance liquid
chromatography with exchanged phases, monoiodinated
cetrorelix being obtained without unlabelled peptide.
About 80% of the [125I] cetrorelix was capable of
specific receptor association.
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The receptor binding assay is carried out under
physiological conditions as described (Beckers et al.,
1995) using intact cells. Subconfluent cultures of
stably transfected LTK" cells, which express the human
LHRH receptor, are separated off by incubation in
NaCl/Pi (137 mM NaCl, 2.7 mM KC1, 8.1 mM Na2HP04,
11.47 mM KH2PO4) /1 mM EDTA and collected by
centrifugation. The cell pellet is resuspended in
binding buffer (DMEM without H2C03, with 4.5 g/1 of
glucose, 10 mM Hepes pH 7.5, 0.5% (mass/volume) BSA,
1 g/1 bacitracin, 0.1 g/1 SBTI, 0.1% (mass/volume)
NaN3). For displacement assays, 0.25 x 106 cells/100 l
are incubated with approximately 225 pM of the [125I] -
cetrorelix (specific activity 5-10 x 105 dpm/pmol) and
various concentrations of unlabelled compound according
to the invention as competitor. The cell suspension in
100 l of binding medium is layered in 400 l assay
tubes over 200 l of 84% by volume silicone oil (Merck
Type 550)/16% by volume paraffin oil. After incubation
for 1 h at 37 C with slow, continuous shaking, the
cells are separated from the incubation medium by
centrifugation for 2 min at 9000 rpm (rotor type
HTA13.8; Heraeus Sepatec, Osterode/Germany). The tips
of the tubes which contained the cell pellet are cut
off. Cell pellet and supernatants are then analysed by
counting the y radiation. The amount of non-
specifically bound material is determined at a final
concentration of 1 M with inclusion of unlabelled
cetrorelix and is typically _< 10% of the total bound
material. The analysis of the binding data is carried
out using. the EBDA/ligand analysis programme (Biosoft
V3.0).
Cetrorelix has a KD value of 170 picomol per litre (pM)
(number of experiments carried out independently: 21).
Method 2 (functional assay for the determination of the
antagonistic activity (IC50 value)):
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The assay is carried out, provided with the
modifications mentioned below, as described in Beckers,
T., Reilander, H., Hilgard, P. (1997) "Characterization
of gonadotropin-releasing hormone analogs based on a
sensitive cellular luciferase reporter gene assay",
Analyt. Biochem. 251, 17-23 (Beckers et al., 1997).
10,000 cells per well, which express the human LHRH
receptor and a luciferase reporter gene, are cultured
for 24 h in microtitre plates using DMEM with additives
and 1% (v:v) FCS;. The cells are then stimulated with
1'nM [D-Trp6] LHRH for 6 h. Antagonistic compounds
according to the invention are added before the
stimulation and the cells are lysed at the end for the
quantification of the cellular Luc activity. The
calculation of the IC50 values from dose-effect curves
is carried out by non-linear regression analysis using
the Hill model (Programme EDX 2.0 from C. Grunwald,
Arzneimittelwerk Dresden).
The quantification of the Luc activity is carried out
in duplicate essentially as described (Promega
Technical Bulletins #101/161) using the respective
luciferase assay system (Promega E4030). Owing to
addition of coenzyme A (CoA), an oxidation of
luciferyl-CoA takes place with advantageous kinetics.
After the removal of the culture medium from the
microtitre plate, the cells are lysed by addition of
100 l of lysis buffer (25 mM tris-phosphate pH 7.8,
2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-
N,N,N',N'-tetraacetic acid (CDTA), 10% (v:v) glycerol,
1% (v:v) TritoriM X-100). After incubation at room
temperature for 15 min, 10 N.1 of cell lysate are
transferred into a white microtitre plate suitable for
luminometric detection (Dynatech). The enzymatic
reaction is initiated by addition of 50 l of assay
buffer (20 mM tricine pH 7.8, 1.07 mM (MgCO3)9Mg(OH)2,
2.67 mM MgSO4, 0.1 mM ethylenediaminetetraacetic acid
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(EDTA), 33.3 mM dithiothreitol, 270 M coenzyme A,
470 M glow-worm (Photinus pyralis) luciferin, 530 pM
rATPNa2). After one minute, the luminescence is
determined for a total time of one second with a signal
half-life of five minutes using the EG&G Berthold
MicroLumat LB 96 P.
Physicochemical and in-vitro data of the compounds
according to the invention are summarized in Table 2.
IC50 stands for the functional activity and pM denotes
picomoles per litre. The water solubility was
determined according to the process described under
Note 2):
Table 2:
Compound Water solubility2 IC50 [pM]
[mg/ml]
Cetrorelix 0.002 198(5)1
Example 1 (D-68968) 1.03 1300(1)1
Example 2 (D-68969) 1.11 1400(1)1
Example 3 (D-68971) 1.36 4700(1)1
Example 4 (D-68987) 1.18 700(1)1
Notes:
1) the number in brackets indicates the number of
experiments independent of one another
2) the water solubility was determined according to the
method described below:
Solubility according to the official gazette method in
Ringer's solution:
For the determination of the solubility according to
the official gazette method, the test substance is
mixed in an excess with an inert carrier material such
as, for example, sand and packed into a glass column
(volume size about 10 ml). A sieve of cotton wool and a
glass fibre filter had been incorporated into the
column bottom beforehand. The substance-sand mixture is
allowed to swell for 1 hour in 1.0 ml of solvent in
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which the solubility is to be determined. 10 ml of the
solvent are then poured into the glass column. The
solution is recycled by means of a peristaltic pump.
This determination is carried out at room temperature
(about 20 C). The solubility is determined when the
mass concentration of successive fractions withdrawn is
constant.
The determination of the mass concentration is
determined [sic] by means of the HPLC method described
below.
HPLC method:
Equipment
HPLC system: Hewlett Packard 1100; detector: Hewlett
Packard DAD series 1100
Column: column material: Nucleosil 120-3 C18
particle size: 3 m
column size: 125 x 4 mm
manufacturer: Macherey & Nagel
Equipment parameters: injection volume: 15 l
flow: 1.0 ml/min
oven temperature: 45 C
wavelength: 226 nm
stop time: 15 min
TM
55% mobile phase A: 970 ml of Milli-Q-H20, 30 ml of
acetonitrile and 1 ml of trifluoroacetic acid are
mixed. The resulting pH is about 1.9..
45% mobile phase B: 300 ml of Milli-Q-H20, 700 ml of
acetonitrile and 1 ml of trifluoroacetic acid are
mixed. The resulting pH is about 1.8.
The administration of the compounds according to the
invention can be carried out in different forms
suitable for peptide active compounds. Suitable
administrations are well known to the person skilled in
the art. Administration can be carried out, for
example, by injection. Administration can be carried
out, for example, parenterally. Subcutaneous (s.c.),
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intra-muscular (i.m.), intravenous (i.v.), buccal (e.g.
sublingual) or rectal administration are preferred
here. I.s. and i.m. Administration are particularly
preferred.
The compounds according to the invention are suitable
for the preparation of different administration forms,
for example for lyophilizates, solutions or
suspensions. Suitable administration forms and their
preparation are known to the person skilled in the art.
Suitable excipients and bulking agents are, for
example, hexitols, such as mannitol, in particular
D-mannitol, L-mannitol or D,L-mannitol, sorbitol, such
as D-sorbitol, D- or L-altritol, iditol, glucitol and
dulcitol. Preparation is carried out according to
procedures known per se, for example by mixing,
suspending or lyophilizing.
Example A: Lyophilizate for the preparation of an s.c.
injection solution
1 mg of compound according to Example 1 (corresponding
to 0.26-0.27 mg of acetate salt); 0-16.9 parts by
weight of D-mannitol, preferably 0.1-7 parts by weight,
in each case based on Example 1, and water for
injection (for the preparation of the injection
solution from the lyophilizate).
Preparation: Dissolve 1.62 g of Example 1 in 30%
strength acetic acid (about 1.5 litres of water for
injection and 91.17 g of acetic acid). Dilute the
solution with 1.5 litres of water. Add 82.2 g of
mannitol, sterile filter, dispense into sterile 2 ml
injection vials under aseptic conditions and freeze
dry. 1 mg of lyophilizate of the compound according to
Example 1 is obtained.
The compounds according to the invention are suitable,
for example, for the treatment of malignant or non-
malignant hormone-dependent disorders, such as, for
example, for the treatment of breast carcinoma, of
prostate carcinoma, of endometriosis, of uterine myoma,
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benign prostate hyperplasia (BPH) and in the treatment
of female or male fertility disorders, for example for
the prevention of premature ovulation in patients who
are subjected to controlled ovarian stimulation
followed by egg cell removal and techniques of assisted
reproduction. The treatments mentioned can be carried
out in mammals, in particular in humans.
