Note: Descriptions are shown in the official language in which they were submitted.
CA 02238570 1998-05-26
.. ..
Titie: -~ - -~ - - - --
Novel LR-R~ antaqonists ha~inq imProved action
The invention relates to novel LH-RH antagonists, in
particular peptidomimetics and peptides modified in a
side ch~; n, salts thereof with pharmaceutically
acceptable acids and processes for the preparation of
the LH-RH antagonists and their salts. The peptides
according to the in~ention are analogues o~ the
luteinizing hormone-releasing hormone (LH-RH), which
has the following structure-
p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, rLH-RH,
gonadorelin].
For more than 20 years, research scientists have sought
antagonists of LH-RH decapeptide with selective potency
tM. Karten and J. E. Rivier, ~ndocrine Reviews 7, 4*-66
(1986)]. Thé great interest in such antagonists is
accounted for by 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 f ound to date are those
compounds whose structure is a modification of the LH-
RH structure.
The first series of potent antagoni~ts was obt~;neA by
the introduction of aromatic amino acid esters in
positions 1, 2, 3 and 6 or 2, 3 and 6. The customary
manner of writing the compounds is as follows: first
the amino acids are indicated which are entered in the
peptide ch~;~ of LH-RH in place of the amino acids
originall~- present, the positions in which replacement
took place being marked by superscript figures.
Furthermore, it is expressed by the description ~LH-RHn
placed afterwards that they are LH-RH analogueues in
which replacement took place.
CA 02238570 l998-05-26
-- 2
.- - .... - . . . . .
- Rnow~ antagonists are: ; -
- - ~ c-D-Phé(4-C1~ ~2,. -T ~3~'~ H-~-(D,-H. Coy e al.,-In.
Gross, E. and Meienhofer, J. (Eds) Peptides;
Proceedings of the 6th American Pep~ide Symposium, pp.
775-779, Pierce Chem. Co., Rockville III. (1979):
{Ac-Pro~, D-Phe(4-Cl) 2, D-Nal(2) 3'~] LH-RH (US-Patent No.
4,419,347) and ~AC-Pro1, D-Phe(4-Cl)2, D-Trp36] LH-RH
(J. L. Pineda, et al., J. Clin. Endocrinol. Metab. 56,
420, 1983).
In order to increase the water solubility of
antagonists, basic amino acids, for example D-Arg, were
later introduced in the 6-position. For example tAc-D-
Phe(4-Cl~ 1~2, D-Trp3, D-Arg6, D-Ala10] LH-RH (ORG-30276)
15 (D. H. Coy, et. al., Endocrinology 100, 1445, 1982);
and
tAc-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).
Such analogues not only had the expected improved water
solubility, but also showed an improved antagonistic
activity. Nevertheless, these extremely potent,
hydrophilic analogues with D-Arg' and other basic side
ch~; n~ in the 6-position cause te~-.~o~ary oe~e~-~ on the
~ace and the extremities when they were A~ n; ~tered
subcutaneously to rats in doses o~ 1.25 or 1.5 mg/kg
(F. Schmidt, et al., Contraception 29, 283, 1984: J. E.
Morgan, et al., Int. Archs. Allergy Appl. Immun. 80, 70
(198~). 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 and
EP 0 413 209 A1.
The occurrence of oedematogenic effects in rats after
the a~m; n; ~tration of some of these antagonists have
allowed doubts to arise about their safety when used in
man, and thus the introduction of these medicaments
CA 02238570 1998-05-26
....''.'i'n'~o..c.l.In'i~al us$'has bëen'delaye~ hëre'.is therefore
- .~ ~a' g~eat need for~antagoni6tic pept~es which are -f~ee
of side ef~ect~. -
. 5 ~ccording to the invention, the aforementioned objectis achieved ~y compounds o~ the general ~ormula (I)
~ ' ~ R2
R~--CO--NH--CH--CO--N
\ R3
(l H2)n
NH (l)
CO--~
in which n is the number 3 or 4, Rl i~ an alkyl group,
an alkyloxy group, an aryl group, a heteroaryl group,
an aralkyl group, a heteroaralkyl group, an aralkyloxy
group or a heteroaralkyloxy group, in each case
unsubstituted or substituted, R2 and R3 independently of
one another are each a hydrogen atom, an alkyl group,
an aralkyl group or a heteroaralkyl group, in each case
unsubstituted or substituted, or -NR2R3 is an amino acid
group, and R~ is a group having the ~ormula (II)
--(~ H2)F ~ R6
R5 (~1)
in which p is an integer from 1 to 4, Rs iQ hydrogen or
an alkyl group and R' is an unsubstituted or substituted
aryl group or heteroaryl group, it being possible for
the substitution, in turn, to consist of an aryl group
or heteroaryl group, or ~ is a ring of the general
formula (III) /R7
~X
N
3 5 R8
-
CA 02238570 1998-OS-26
:- .in which .q .18 tl~e.. ;numbe.r 1 or 2, R7.is a.hydro~en atom
_ . _, , _ .
~or an alkyl g;o~, R~ is a hydrogen 'atom or an ~l~yl
group and X i~ an oxygen or sulphur atom, where the
aromatic or heteroaromatic radicals can be partially or
completely hydrogenated, and chiral carbon atoms can
have the R- or S-con~iguration, and their salts with
pharmaceutically acceptable acids.
Preferred combinations of radicals Rl to R~ are:
a) R1 i8 benzyloxy, RZ is hydrogen and R3 is hydrogen,
b) Rl i8 benzyloxy, R2 is hydrogen and R~ is 4-
amidinophenyl, and
c) R2 is hydrogen, R3 is hydrogen and R~ is 4-
amidinophenyl.
Preferred alkyl groups are the methl, ethyl, n-propyl,
i-propyl, n-butyl, i-butyl, t-butyl, 2-ethylhexyl,
dodecyl and hexadecyl groups.
Pre~erred aryl groups are phenyl, n~ph~hyl,
phen~nthrenyl and fluorenyl groups.
Preferred heteroaryl groups are the pyridyl, pyrimidyl,
imidazolyl, imidazopyridyl, indolyl, ; n~701yl~
triazolyl, tetrazolyl, benzimidazolyl, quinolyl, 2,5-
dichlo ~y ~d-3-yl and furyl groups.
Preferred hydrogenated heteroaryl group~ are the
piperidino, piperazinyl, morpholino and pyrrolidinyl
groups.
Aralkyl groups and heteroaralkyl groups are those
groups which are bonded to the correspo~; ng b; n~; ~g
~- 35 sites ~ia an alkylene group, preferably a ~ethylene,
ethylene, n-propylene or n-butylene group.
Pre~erred substituents are halogen atoms such as
fluorine, chlorine, bromine and iodine, and the methyl,
CA 02238570 1998-OS-26
.
ethyl, i'-propyl, tert-bu~yl, :cyano, nitro, car~oxylic
~aci~, carboxamide,- 'ca~boxylic acid methyl'' e~tër~ '' '''
carboxylic acid ethyl e~ter, crotonic acid ethyl ester,
trifluoromethyl, benzoyl, methoxy, benzyloxy,
pyridyloxy, amino, dimethylamino, isopropylamino,
amidino and quinolylmethoxy groups
Furthermore, according to the invention, compounds of
the general formula (V)
Ac-D-Nal(2)l-D(pCl)Phe2-D-Pal(3)3-Ser~-Tyrs-D-Xxx6-Leu7-
Arg~-Pro'-D-Ala10 -NH2 (V)
where D-Xxx is an amino acid group of the general
15 formula (VI) -HN-CH-C0-0-
I
(l H2)n
NH
~)
CO - R4
and n, p, q, R~, Rs, R~, R7, R~ and X are as defined
above, and their salts with pharmaceutically acceptable
acids also achieve the abovementioned object.
The co.,.~o~ds according to the invention have a high
antagonistic potency and are free of undesirable side
effects, in particular free of oedematogenic effects.
If they are not present as salts with poorly water-
soluble, pharm~c~l~tically acceptable acids, they
additionally have an improved water solubility.
Further~more, the compounds have high affinity for the
h-lm~n LH-RH receptor, i.e. are highly potent in
inhibiting the release of gonadotropins from the
pituitary gland in r~mm-l~, including man, exhibit
long-lasting suppression of testosterone in rats, and
35 cause m;n;~m~l hist~m;ne release in vitro.
Pre~erred compounds o~ the general formula (I) are:
a-N-Z-t~-N'-4-(4-amidinophenyl)amino-1,4-dioxo-
CA 02238570 1998-05-26
butyl]lys;n?m;~e and a-N-Z-~~-N~ (imidazolidin-2-on-4J
~yl)~ormyl]lysinamide. Preferred peptides according to
formula (V) are those in which Xxx is the t~-N'-4-(4-
amidinophenyl)amino-1,4-dioxobutyl]lysyl group or the
t~-N'-(imidazolidin-2-on-4-yl)formyl]lysyl group. The
salts with pharmaceutically acceptable acids are
preferably poorly soluble in water. Particularly
pre~erred salts are those of 4,4'-methylene-bis(3-
hydroxy-2-naphthoic acid), also known as embonic acid
or pamoic acid.
The nomenclature used for the definition o~ the
peptides agrees with that no~nclature explained by the
IUPAC-IUB Commission on Biochemical Nomenclature
(European J. Biochem. 1984, 138, 9-37?~ in which in
agree~ent with the conventional representation the
amino groups in the N terminus appear to the le~t and
the carboxyl group in the C terminus appears to the
right. The LH-RH antagonists such as the peptides and
peptidomimetics according to the invention include
20 ~m; no acids occurring in nature 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 abble~iations for the individual
amino acid radicals are based on the trivial names of
the ~m;no acids and are Ala ~l~n;n~, Arg arginine, Gly
glycine, Leu l~l~c;~e, Lys lysine, Pal(3) 3-(3-
pyridyl)~l ~n; n~, Nal(2) 3-(2-naphthyl)~l ~n; n~, Phe
phenyl~l ~n; n~, (pCl)Phe 4-chlorophenyl~l ~n; ne, Pro
proline, Ser serine, Thr thr~on;ne~ Trp tryptophan and
Tyr tyrosine. All amino acids described here originate
~rom the L-series, if not otherwise mentioned. For
example, D-Nal(2) is the abbreviation for 3-(2-
naphthyl)-D-alanine and Ser is the abbre~iation ~or
L-serine. Other abbre~iations used are:
Boc tert-Butyloxycarbonyl
Bop Benzotriazol-1-oxytris-
dimethylamino)phosphonium
- hexa~luorophosphate
DCC Dicyclohexylcarbodiimide
CA 02238570 1998-OS-26
-- 7
DCM - Dichloromethane
~dz Dimethoxyphenyldi~ethylmethylenOxycarbonyl
(dimethoxydimethyl-z)
DIC Diisopropylcarbodiimide
DIPEA N,N-diisopropylethylamine
DMF Dimethylformamide
Fmoc Fluorenylmethyloxycarbonyl
HF Liquid anhydrous hydrofluoric acid
HOBt l-Hydroxybenzotriazole
HPLC High-pressure liquid chromatography
TFA Trifluoroacetic acid
Z senzyloxycarbonyl
According to the invention, compounds of the general
~ormula (I) are prepared by first providing two of the
three ~unctionalities (a-amino, ~-amino and a-
carboxylic acid group) with protective groups and thenreacting the free third functionality in a suitable
manner. If appropriate, it is also possible, where this
leads to better results, to introduce in the first step
intermediate protective groups which are then replaced
a~ter the second step by the desired functionality.
Suitable protecti~e groups and methods ~or attaching
the same are known in the field. Examples of protecti~e
groups are described in ~Principles of Peptide
Synthesis~, Springer Verlag 1984), in the t~thook
~Solid Phase Peptide Synthesisn ~. M. Stewart and
J. D. Young, Pierce Chem. ~o~ny, Rockford, III, 1984,
and in G. Barany and R. B. Merri~ield ~The Peptides~,
Ch. 1, pp. 1-285, 1979, Ac~em;c Press Inc.
The synthesis of compounds according to formula (IV)
can be carried out both either by classical ~ragment
condensation or by solid-phase synthesis according to
Merri~ield with buildir.~-up one on the other in
sequence using D-lysine already acylated in the side
chain by the carboxylic acid o~ the general
~ormuia (VII) and by reaction o~ a decapeptide unit
with the appropriate carboxylic acids by amide linkage
CA 02238570 1998-05-26
,
-- 8
~ in the side chain of D-lysine6. Accordingly., there are
~according to the invention three alternati~es available
for the process for the preparation o~ a compound o~
the general formula (V).
The first possibility comprises the steps of
(a) providing the a-amino and the carboxylic acid
group of D-lysine or D-ornithine with suitable
protective groups,
lo (b) reacting the D-lysine or D-ornithine provided with
.protective groups with a carboxylic acid of the general
formula (VII)
R4-COOH (VII)
in which R~ iS as defined above,
(c) Le~oving the protective group on the a-carboxylic
acid group of the compound obt~; n~ in step (b) for the
purpose of incorporation in pos. 6 in step (h),
(d) coupling of D-AlAn;ne provided on the amino group
with a protective group to a solid support in the form
of a resin (Merrifield synthesis),
(e) re~l.oving the protective group on the amino group
of the alanine,
(f) reacting the AlAn;ne. bound to the solid support
with proline which is provided with a protective group
on the nitrogen atom,
(g) le~ ving the protective group on the nitrogen atom
of the proline,
(h) repeating steps f) and g) with the amino acids 1
to 8 according to the general formula.(V), in the
. sequence from 8 to 1, using modified D-lysine or D-
- ornithine described in step (c) for pos. 6,
(i) le...o~ing the compound obtained in step (h) from
the support and, if appropriate, purifyin~ (e.g. HP~C),
(j) if desired, reacting with a pharmaceutically
acceptable acid, preferably embonic acid.
.
CA 02238570 1998-05-26
,
g
According to the second alternative, the process for
~~he preparation of a compound of the general
formula (V) comprizes the steps of
(a) coupling D~ n;ne provided with a protective
group on the amino group to a support suitable for
solid-phase synthesis,
(b) leu-~ving the protective group on the amino group
of the alanine,
(c) reacting the ~l~n;ne bound to the resin with
proline which is provided with a protective group on
the nitrogen atom,
(d) removing the protective group on the nitrogen atom
of the proline,
(e) repeating steps c) and d) with the amino acids 1
to 8 according to the general ~ormula (v), in the
sequence from 8 to 1,
(f) .e...o~ing the compound obtained in step (e) from
the ~upport,
(g) reacting with a carboxylic acid of the
formula (VII)
R~-COOH (VII)
in which R~ is as defined above,
(h) if desired, reacting with a pharmaceutically
acceptable acid, preferably em~o~;c acid.
The third variant of the proces~ for the preparation of
a compound of the general formula (V) comprizes the
steps of
(a) coupling D-alanine provided with a protective
group on the amino group to a support suita~le for
solid-pha~e synthesis,
(b) ~e...oving the protective group on the amino group
of the alanine,
(c) reacting the alanine bound to the resin with
proline which i8 provided with a protective group on
the -nitrogen atom,
_
CA 02238570 1998-OS-26
- 10 -
(d) removing the protective group on the nitrogen atom
~~f the proline,
(e) repeating steps c) and d) with the amino acids 6
to 8 according to the general formula (v), in the
sequence from 8 to 6,
(f) le.l.~ing the ~-amino protective group from D-
lysine 'or D-ornithine in pO8. 6 and reacting with a
carboxylic acid of the formula (VII),
R~-COOH (VII)
in which R~ is as de~ined above,
(g) Le...o~ing the protective group on the a-amino group
of the D-lysine or D-ornithine,
(h) repeating steps c) and d) with the amino acids 1
to 5 according to the general formula (IV), in the
sequence from 5 to 1,
(i) el..oving the compound obtained in step (h) from
the resin and purifying it (e.g. HPLC),
(j) if desired, reacting with a pharmaceutically
acceptable acid, preferably embonic acid.
Preferred carboxylic acids of the general formula (VII)
are imidazolidin-2-one-4-carboxylic acid and N-(4-
amidinophenyl)amino-4-oxobutyric acid.
The co...~o~Lds of the formula (V) are synthesized
according to the known methods, such as, for example,
by pure solid-phaRe technique, partial solid-phase
technique or by the classical solution couplings (see
M. Bodanszky, ~Principles of Peptide Synthesisn,
Springer Verlag 1984). For example, the methods of
solid-phase synthesis are described in the textbook
~Solid Phase Peptide Synthesisn J. M. Stewart and
- 35 J. D. Young, Pierce Chem. Company, Rockford, III, 1984,
and in G. Barany and R. B. Merrifield "The Peptide~,
Ch. 1, pp. 1-285, 1979, Academic Press Inc. Classical
solu-tion syntheses are described in detail in the
treatment ~Methoden der Organischen Chemie [Methods of
-
CA 02238570 1998-05-26
-- 11 --
Organic Chemistry] (Houben-Weyl), Synthese von Peptiden
~~Peptide Synthesis]" E. Wunsch (Editor) 1974, Georg
Thieme Verlag, Stuttgart, FRG.
s The stepwise synthesis is carried out, for example, by
first covalently binding the car~oxy-terminal amino
acid, whose a-amino group is protected, to an insoluble
support which is customary for this purpose, L~-,-o~ing
the a-amino protective group of this amino acid,
lo bonding the next protected.amino acid to the free amino
group thus obtained via its carboxyl gro~p, and in thi~
manner linking the other amino acids of the peptide to
be synthesized step by ~tep in the correct sequence,
and a~ter linkage of all amino acids removing the
finished peptide from the support and, if appropriate,
removing further side-function protective groups
present. Stepwise condensation is carried out in a
conventional manner by a synthesis from the appropriate
amino acids protected in a customary manner. Likewise,
the use of automatic peptide synthesizers, for example
~abortec SP 650 type from Bachem, Switzerland, is
possible using the co~m~cially av~ hle protected
amino acids.
me 1 ;nk~e of the indi~idual amino acids to one another is
carried out by the m~t~ cu~tomary for this purpose, the
following in par~;~l~ being ~uitable:
~ Symmetric anhydrides method in the presence of
dicyclohexylcarbodiimide or diiso~Lo~ylcarbo~
(DCC, DIC)
~ Carbodiimide method generally
~ Carbodiimide-hydroxybenzotriazole method
(see The Peptides, Volume 2, Ed. E. Gross and
J. Meienhofer). For the linkage of arginine, the
carbodiimide method is preferably used. For the other
amino acids, the symmetric or mixed anhydrides method
is in general used.
CA 02238570 1998-05-26
,~ '
- 12 -
In the fragment coupling, acid coupling, which proceeds
-without racemization, or the Dcc-l-hydroxybenzotriazole
or DCC-3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine
method i8 preferably used. Activated esters of
5 fragments can also be employed.
For the stepwise condensation of amino acids,
particularly highly suitable activated esters are those
of N-protected amino acid~, such as, for example,
lo N-hydroxysuccinimide esters or 2,4,5-trichlorophenyl
esters. The aminolysis can be catalysed very readily by
N-hydroxy compounds which approximately have the
acidity of acetic acid, such as, for example,
l-hydroxybenzotriazole.
1~
Intermediate amino protective groups which are
available are groups which can be removed by
dehydrogenation, such as, for example, the
benzyloxycarbonyl radical (= Z radical) or groups which
can be ~eulv~ed by weak acid. Protective groups for the
a-amino groups are, for example:
tertiary butyloxyc~h~nyl groups, carbobenzoxy groups
or carbobenzothio groups (if appropriate in each case
having a p-bromo or p-nitrobenzyl radical), the
trifluoroacetyl group, the phthalyl radical, the
o-nitrorh~n~yacetyl group, the trityl group, the
p-toluenes~ honyl 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 the book by
Jesse P. Greenstein and Milton Winitz, ~h~m; ~try of
Amino Acids, New York 1961, John Wiley and Sons, Inc.,
Volume 2, for example page 883 et seq. and The
Peptides, Volume 2; Ed. E. Gross and J. Meienhofer,
Academic Press, New Yor~. These protective groups are
fundamentally al80 suitable for the protection of
further functional side groups (OH groups, NH2 groups)
of the corresponding amino acids.
CA 02238570 1998-05-26
Hydroxyl groups present (serine, threonine) are prefer-
-~bly protected by benzyl groups and-- similar groups.
Further amino groups not in the a-position (for example
amino groups in the ~-position, the guanidino group of
arginine) are preferably protected orthogonally.
The reaction for the linkage of amino acids takes place
in a customary indifferent solvent or susp~n~;ng agent
therefor (for example dichloromethane), it being
possible to add dimethylformamide, if necessary, to
improve the solubility.
For introduction of the R~-Co group by reaction o~ the
amino group of the lysine with the carboxylic acid of
the general ~ormula (VII), fundamentally the same pro-
cesses as described above are ~uitable ~or lin~age of
the amino acids. Particularly preferred, however, is
condensation using carho~;;m;de, for example 1-ethyl-3-
(3-dimethyl~m;nopropyl)carbodiimide, and 1-hydroxy-
benzotriazole.
Suitable synthetic supports are insoluble polymers, forexample polystyrene resin in bead form, which can be
~wollen in organic ~olvents (for example a copolymer of
polystyrene and 1~ divinylbenzene). The synth~is of a
protected decapeptide amide on a methylh~n7hydrylamide
resin (MBHA resin, i.e. polystyrene resin provided with
methyl~n7hydrylamide groups) which affords the desired
C-ter~;n~l amide function of the peptide after an HF
cleavage from the support can be carried out according
to the following flow diagram:
CA 02238570 l998-05-26
- 14 --
Flow diaqram
- ~Peptide synthesis protocol
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 ~in
4 Washing Isopropanol 2 x 2 min
W~h;ng Methanol . 2 x 2 min
6 Washing DCM 2 x 3 min
7 Neutralization DCM/DIPEA (9:1) 3 x 5 min
8 Washing MethanoI 2 x 2 min
9 Washing DCM 3 x 3 min
lo STOP Addition of the Boc-As
in DCM + DIC + HOBt
11 Coupling - about 90 min
12 Washing Methanol 3 x 2 min
13 W~h;ng DCM 2 x 3 min
The Na-Boc-protected amino acids are coupled in a
three-fold molar excess in the presence of diisopropyl-
carbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) in
CH2Cl2/DMF in the course of 90 min, and the BOC protec-
tive group i8 le~l~oved by action of 50~ trifluoroacetic
acid (T~A) in CH2Cl2 for half an hour. To check for
complete conversion, the chloranil test according to
Christensen and the Kaiser's ninhydrin test can be
used. Radicals of free amino function are blocked by
acetylation in a five-fold excess of acetylimidazole in
CH2Cl2. The sequence of the reaction steps of peptide
synthesis on the resin follows from the flow diagram.
For the removal of the resin-bound peptides, the
respective final product- of solid-phase synthesis i8
dried in vacuo over P205 and t-eated at 0~C for 60 min
in a ~OO-fold excess of HF/anisole 10:1 (v:v).
After- distilling off HF and anisole in vacuo, the
peptide amides are obtained by stirring with anhydrous
CA 02238~70 1998-0~-26
ethyl ether as white solids; the removal of polymeric
~~upport 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 in
the cold after addition of abs ether.
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 by reaction thereof with acids in
a manner known per se Conversely, free peptides can be
obtained by reaction o~ 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 correspond-
ing disodium salt of ~mho~; C acid. To do this, thepeptide TFA salt is treated in aqueous solution with
the solution of disodium embonate in polar aprotic
medium, preferably dimethylacetamide, and the pale
yellow precipitate formed i8 isolated.
The following examples illustrate the invention without
limiting it.
CA 02238570 1998-05-26
ExamPle
_
Ac-D-Nal(2)-D(pCl)Phe-D-Pal(3)-Ser-Tyr-D-[~-N'-(; m; ~olidin-
2-on-4-yl)formyl3-Ly~-Leu-Arg-Pro-D-Ala-N~
The synthesis was carried out according to the flow
diagram on 5 g of mBHA resin (loading density
1.08 mmol/g). Lysine wa~ coupled as Fmoc-D-Lys(Boc)-OH
and acylated with imidazolidin-2-one-4-carboxylic acid
in a 3-~old exce~s after removal o~ the Boc group in
the side chain. A~ter removal o~ the Fmoc protective
group with 20~ piperidine/DMF, exte~sion was carried
out at the N terminus according to the ~low diagram.
After removal of the polymeric support, ~.2 g o~ crude
peptide were obtained, which were puri~ied by st~n~rd
processes o~ preparative HPLC. After subsequent freeze
drying, 2.1 g of HPLC-homogeneous product of the
empirical formula C~Hg7Nl~OlsCl having the correct FAB-MS
1514 (M+H') (calc. 1512 7) and corresponding lH-NMR
spectrum were obtained.
H-NMR (500 MHz, DMSO-d6, ~ in ppm):
8.56, m, 2H, arom. H; 8.08, m, lH, arom. H; 7.81, m,
lH, arom. H; 7.73 m, 2H, arom. H; 7.66, m, lH, arom. H;
7.60, s, lH, arom. H; 7.44, m, 2H, arom. H; 7.30, d,
lH, arom. H; 7.25, and 7.18, 2d, 2x2H, arom. H p-Cl-
Phe; 6.97 and 6.60, 2d, 2x2H, arom. H Tyr; 9.2-6.3,
several signals, amide NH; 4.8-4.0, several m, Ca-H and
aliph. H; 2.1-1.1, several m, residual aliphat. H;
1.70, s, 3H, acetyl; 1.22, d, 3H, C~-H Ala; 0.85, dd,
6H, C~-H ~eu
Example 2
- 35 Ac-D-Nal~2)-D(pCl)Phe-D-Pal(3)-Ser-Tyr-D-t~-N'-4-(4-
amidinophenyl)amino-1,4-dioxobutyl3-Lys-Leu-Arg-Pro-D-
Ala-NH2
CA 02238570 l998-05-26
-- 17
0.7 mmol (1.03 g) of decapeptide Ac-D-Nal-D-(pCl)Phe-D-
Pal-Ser-Tyr-D-Lys-Leu-Arg-Pro-D-Ala-NH2 was reacted with
l o mmol (0.27 g) of (4-amidinophenyl)amino-4-
oxobutyric acid in the presence of 1.0 mmol (0.16 g) of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and
1.O mmol (0.16 g) of l-hydroxybenzotriazole in freshly
distilled DMF. The solvent was removed after 24 h in
vacuo, the residue obtained was dissolved in water and
the solution was freeze dried. The crude reaction
lo product obtained (1.63 g) .was purified by preparative
reverse-phase HPLC; altogether 0.61 g of HPhC-
homogeneous product of empirical formula C8lH1o~NlgOlsCl
having the correct FAB-MS: 1618.7 (M+H') (calc. 1617.7)
and corresponding lH-NMR spectrum were obtained
H-NMR (500 MHz, DMS0-d~, ~ in ppm):
10.4, s, lH and 9.15, s, 2E, and 8.8, s, lH, NH's of 4-
amidinoaniline; 8.60, m, 2H, arom. H; 8.20, m, lH,
arom. H; 7.80, m, lH, arom. H; 7.73, m, arom. H; 7.61,
s, lH, arom. H; 7.44, m, 2H, arom. H; 7.30, d, lH,
arom. H; 7.25 and 7.20, 2d, 4H, arom. H (pCl)Phe; 7.0
and 6.6, 2D, 4H, arom. H Tyr; 8.3 - 7.2, several
signals, amide-NH; 4.73 - 4.2, several multiplets, Ca-
H; 4.13, m, lH, Ca-H; Ala; 3.78 - 2.4, several
multiplets, C~-H and aliphat. H; 1.72, 8, 3H, acetyl;
1.22, d, 3H, C~ Ala; 0.85, dd, 6H, C~ Leu
ExamPle 3
0.5 g (O.3 mmol) of peptide LH-RH antagonist according
to ~m~le 1, dissolved in 50 ml of HzO, wa~ converted
by reaction with 0.130 g (0.3 mmol) of disodium pamoate
in 2 ml of aqueous solution to peptide embonate, which
rapidly depo~ited from the solution as a yellow
precipitate. 0.281 g of finely crystalline yellow-green
powder were obt~;ne~, em~onic acid content 33~.
CA 02238570 1998-05-26
- 18 -
Example 4
O.3 g (0.17 mmol) of peptide LH-RH antagonist according
to Example 2, dissolved in 5 ml of dimethylacetamide,
was converted by reaction with 0.195 g (0.45 mmol) of
disodium pamoate in 2 ml of aqueous solution to peptide
embonate, which after addition of 50 ml of H20 was
o~tained as a yellow precipitate. 0.330 g o~ ~inely
crystalline yellow product were obtained, embonic acid
content 20~.
Compounds of the general formula I are obt~in~le
according to the following Schem~ 1, 3, ~ and 5, the
three functionalities R1, R3 and R~. being varied
systematically. Scheme 1 shows the synthesis of the
compound o~ Example 1:
Scheme 1
l3~0~NH-CH-CO--NHz ~ HJ~NH2
~ ~I~ xHCI HOOC l'N xHCI
NH2
1 yloxy~is-
th,' ' ~o)~l.osl~l,On;um
h ~ .~ os~l. t(BOP)
2) tl ~l~L fl~
3) 2n NaOH
~ ~) CF3COOH
(3~o~.~NH -CH-CO -NH
x CF3COOH
NH~ NH~
O O ~f NHz
NH
CA 02238~70 1998-0~-26
- 19 -
General ~rocedure for the Preparation of the comPounds
of the qeneral formula I accordinq to Scheme 1
The carboxylic acid R~-COOH substituted by the radical
R~, on which the general formula I and the Synthesis
Scheme 1 are based, which in the case of a basic
radical ~or R4 can also be present as a ~alt, for
example as a hydrochloride, hydrosulphate or acetate,
is dissolved or suspended with exclusion of moisture
and with stirring in a non-polar or dipolar aprotic
organic solvent, such as, for example, tetrahydrofuran,
dioxane, methyl tert-butyl ether, tolulene,
dimethylformamide, dimethylacetamide, N-methyl-
pyrrolidone, dimethyl sulphoxide or methylene chloride
and treated with stirring with a base serving as an
acid trap, such as, for example, with diisopropyl~m;ne,
triethylamine, N-methylmorpholine, dimethyl~m;no-
pyridine or pyridine. A mixture of Z-(L~-lysinamide
hydrochloride in a diduent is then added, a suitable
diduent being that employed above for dissolving the
carboxylic acid R~-COOH substituted by the radical R~.
The pH of the reaction mixture is then adjusted using
one of the bases employed as an acid trap, for example,
to pH 6.5 - 9.0, preferably to 7.0 - 8.5, particularly
to 7.0 - 7.5. Finally, the solution of a coupling
reagent, e.g. benzotriazol-l-yloxy-tris(dimethylAm;no)-
phosphonium hexafluorophosphate (BOP), or benzotriazol-
1-yloxy-tripyrrolidinophosphonium h~Y~ f luorophosphate
(PyBOP) or dicyclohexylcarbodiimide (DCC) is ~e~ to
the reaction mixture with further stirring and the pH
of the solution i8 adjusted again to the abovementioned
pH range after a short time. The suspension is stirred,
for example, at O - 80~C, preferably at 10 - 50~C,
particularly at 20 - 30~C, for 1-15 hours, then
filtered off with suction, the solid is washed and the
filtrate is concentrated to dryness in vacuo. The
residue is crystallized by rubbing with an organic
solvent, for example with toluene, tetrahydro~uran,
acetone, methyl ethyl ketone or isopropyl alcohol or it
_
CA 02238~70 1998-0~-26
- 20 -
is purified by recrystallization, distillation or by
column or ~lash chromatography on silica gel or
alumina. The eluent used, is, for example, a mixture of
methylene chloride, methanol, ammonia (25~) in the
ratio 85:15:1 (vol/vol) or a mixture of methylene
chloride, methanol, ammonia (25~) in the ratio 80:25:5
(vol/vol)
Trifluoroacetate Synthesis:
The compound purified according to the procedure
described above is dissol~ed in protic or aprotic
solvents, e.g in alcohols, such as methanol, EtOH,
isopropanol, or in cyclic ethers, such as, for example,
tetrahydrofuran or dioxane, and adjusted to a pH of
10-11 using 2N sodium hydroxide solution. The ~olid
precipitated is filtered off with suction, washed,
dried in ~acuo and treated in ethanolic solution at a
temperature of 10-80~C, preferably 20-40~C, with a
molar e~uivalent or 2-4 fold molar excess of
tri~luoroacetic acid. After st~n~;ng o~ the solution at
0-4~C for 24 hours the desired trifluoroacetate
crystallizes, which is filtered off with suction and
dried in vacuo.
According to this general procedure, on which Synthesis
Scheme 1 is based, compounds were synthesized which
follow below from the description of Example 5 and the
following Table 1:
Example 5
a-N-rBenzyloxycarbonyll-~-N- rs- [ (4-amidino-phenYl) -
aminol-5-oxo-pentanoyl~-L-lysinamide trifluoroacetate
g (17.5 mmol) of 5-t[4-(aminoiminomethyl)phenyl]-
amino]-5-oxopentanoic acid hydrochloride are suspended
with- stirring and exclusion of moisture in 200 ml of
dimethylformamide and treated with 3 85 ml (35.0 mmol)
-
CA 02238570 1998-05-26
.
- 21 -
of N-methylmorpholine. A mix~ure of 5.53 g (17.5 mmol)
of Z-(L)-lysinamide hydrochloride in 100 ml of
dimethylformamide is added and the pH i~ adjusted to
7.0-7.5 using N-methylmorpholine. Finally, a solution
o~ 9.73 g (21.9 mmol) of benzotriazol-1-yloxy-
tris(dimethylamino)phosphonium hexafluorophosphate
(BOP) is added and after 10-15 minutes the pH is again
adjusted to 7.0-7.5. The yellow-coloured suspension is
stirred with continuous checking of the pH, which
lo should be 7.0-7.5, for 3-4 hours at room temperature,
the colourless precipitate is ~iltered o~ with
suction, washed twice with dimethylformamide and the
yellow-coloured ~iltrate is evaporated to dryness. The
oily residue is digested with a total o~ Sx40 ml of
methyl ethyl ketone in such a way that after each of
the 5 solvent treatments the methyl ethyl ketone phase
is poured of~ and discarded. The residual crude
product, which is obtained in crystalline form, is
~iltered o~ with suction, washed with 30 ml of methyl
ethyl ketone and dried at room temperature in vacuo.
The solid is then dissolved in about 50 ml of ethanol
and adjusted to pH 10-11 using 2N sodium hydroxide
solution. The precipitated base is ~iltered off with
suction, washed with water and ethanol and dried at
35~C in vacuo.
Yield: 5.5 g (62~ of theory)
Trifluoroacetate: 5.5 g of base are treated at 60~C in
ethanolic suspension with a 5-fold molar amount of
trifluoroacetic acid. The solution is stored overnight
at 4~C, and the trifluoroace~ate obtained i~ filtered
off with suction and dried at 35~C in vacuo.
Yield: 5.9 g (87.7~ theory)
Meltinq point: 185~C
Elemental analYsis:
_
CA 02238570 1998-05-26
calc. C 53.84 H s.65 N 13.45
found C 54.11 H 5.74 N 13.33
~H-NMR (500 Mhz DMS0-d6, ~ in pPm):
10.47, s, lH, anilide, 9.14 and 8.8 2s, NH amidine,
7.82, m, lH, lys-~-NH, 7.79 and 7.46, ~s, aromat. H,
7.27 and 6.93 2s, 2H, CONH2, 7.20, d, lH, urethane NH,
5.0, s, 2H, benzyl H, 3.89, m, lH, C~-H, 3.0 and 2.58
and 2.40, 3 m, altogether 6H, aliphat. H, 1.60 - 1.20,
4 m, altogether 6H, rem. Aliphat. H
/ ~2
R1- CO -NH-CH-CO-N
\ R3
(CH2)n
NH
CO - R4
(Formula I)
According to the above procedure, further compounds
shown in Table I below were prepared, n being equal to
4 throughout.
Table 1: a,~-N-substituted L-lysinamide derivatives
according to Synthesis Scheme 1 and to the
general formula I (for all Examples n is
e~ual to 4)~5
CA 02238570 1998-05-26
- 23 -
Example R'-CO R2lR3 R~
T~;nuO.ua~ 3 ~~ - H/H ~ ~,
6 c~_CH, NH 4~3CN
7 c~c~ Nff4,3_F
8 c~--C~,J~ Nff 43 Cl
~ NH ~
CH~ N~
NH~
12 'CH~NH
OBzl=8ereyloxy
13
O OCH~
14 .cH~ Nff~
1 5 ~0~
CA 02238570 1998-05-26
- 24 -
Table 1 (Contd. )
Example R'-CO R2lR3 R~
16 [~ c~J~NH~3C~
17 c~ NH ~3
18 ,C~ NH~J~
1 9 ~ C~f NH ~
~
~COOCh~,
21 C~ch~NH J~ J
N~l
22 b'
23 ~C~ NH ~~1~ NH~
24
-
CA 02238570 1998-05-26
- 25 -
Table 1 ( Contd . )
Example R'-CO R2lR3 R4
~ HIH
~~~
~ 1~0
26
27 ~a~ a
28 ~
cooet
29 /~
CH~ t H--~NH
C~ J~,N
31 'CH,~NH~(~
32 \_J "3
~ ~ --N
O
34 a~ 3
CA 02238570 1998-05-26
- 26 -
The melting points of the compounds according to the
above examples can be seen from Table 2 below:
Table 2: Melting points o~ the compounds according to
Examples 5 to 34
Example m.p.~~Cl Example m.p.~~C~ Example m.p.[~C]
185 15 225 25syrupy
residue
6 185 16211-214 26205-210
7 216-220 17183-186 27172-177
8 22S 18 (oil) 28227-230
9 217-220 19syrupy 29225-229
residue
10 218-222 20 (oil) 30233-235
11 208-212 21 (oil) 31215-218
12 (oil) 22 (oil) 32 155
13 232-236 23syrupy 33 (oil)
residue
14 194-198 24 (oil) 34 (oil)
Precursors for the comPounds of the qeneral formula I
prepared accordinq to Synt~esis Scheme 1, which follow
from Table 1
The Z-(L)lysinamide employed as a starting compound for
the synthesis final stage of ~x~mples 5-34 is
15 ~o~me~cially available. The substituted ~aryln- or
"heteroarlyamino-oxo-alkanoic acids~ used as further
starting materials and following from synthesis Scheme
1 can be prepared by procedures known from the
literature analogueously to Synthesis Scheme 2 (P.R.
Bovy, ~-. Organ. Chem. 58, 7948 (1993) ) .
CA 02238570 1998-05-26
- 27 -
Scheme 2
A~H~ ~ (CH,~, ~ o (CH2 ~ NH
\o
A= Aryl p= 2~ A= Aryl, Heteroaryl
I I.,l~.-.aryl p= 2-5
The aromatic or heteroaromatic ~m; nefi A-NH2 used, which
~ollow from Synthesis Scheme 2, are commercially
available; the aminoimidazotl,2-a]pyridine on which the
compound of ~mple 28 is based can be synthesized
analogueously to procedures known from the literature
(R. Westwood, ~. Med. Chem 31, 1098 (1988)).
The "aryl~- or ~heteroarylamino-oxo-alkanoic acids"
already predesignated as precursors can furthermore be
prepared by, starting from a monomethyl ~lk~ne-
dicarboxylate, e.g. monomethyl suberate and monomethyl
azelate, reacting with an aromatic or heteroaromatic
amine by m~n~ of an aminolysis reaction in a boiling
alcohol, for example in boiling ethanol or butanol, or
optionally in an aromatic solvent, such as, for
example, in toluene or xylene, at boiling heat,
optionally in an autoclave-at the boiling point of the
solvent using a pressure of up to 50 bar, concentrating
the reaction solution in vacuo and purifying the
residue by crystallization from methanol or ethanol or
by column chromatography. The eluent used is, for
example, a mixture of methylene chloride, methanol,
ammonia (25~) in the ratio 85:15:1 (vol/vol) or a
mixture of methylene chloride, methanol, ~mmo~; a (25~)
in the ratio 80:25:5 (~ol/vol).
An alternative course of the process for the
preparation o~ compounds o~ the general ~ormula (I), in
CA 02238570 1998-05-26
- 28 -
which R~ is the benzyloxycarbonyl group and R2 and R3
are a hydrogen atom, is as follows:
1. The a-carboxylic acid group is amidated.
2. The ~-amino group is protected with the Z group.
3. The a-amino group is protected with the Boc group
such that a selectivity with respect to the later
removal of the amino protective groups results.
4. The Z group on the ~-amino group is removed.
5. The desired group R~-CO- is introduced on to the
~-amino group.
6. The Boc group on the a-amino group is removed.
7. The a-amino group is provided with the Z group.
Further compounds of the general Formula I are
obtainable according to the ~ollowing Scheme 3,
representing the synthesis of the compound of Example
35:
CA 02238570 1998-05-26
-- 29
Scheme 3
1.StLlfe
~ Q ~ ~OH ~ t~ ~t~l~td
~HF I t~N
t*l~O~f , '
2. St~tfe
o~c K --~N ~ ~o~C~
o~f~ ~IH ~,C O
o ¦ DMF I NMM
o O ,~NU ~ ~ , K [~N
NMM I 60P Nt t
DMF
--~N NH
UN~
CA 02238S70 1998-0~-26
- 30 -
General ~rocedure for the pre~aration of the compounds
of the qeneral formula I accordinq to Scheme 3:
1st Staqe
Z-Lys(BOC)-OH and a base, for example triethylamine,
diisopropylamine, N-methylmorpholine, N-ethyl-
piperidine, and an aliphatic or aromatic carbonyl
cXloride, for example acetyl chloride, isobutyroyl
chloride, isovaleroyl chloride, pivaloyl chloride,
benzoyl chloride or 4-methoxybenzoyl chloride are added
at a temperature within a range from -30~C to 30~C,
preferably between -20~C to 200C, particularly between
-15~C and 5~C, to a dipolar aprotic or non-polar
organic solvent, such as, for example, tetrahydrofuran,
dimethyl sulphoxide, dimethyl-formamide, acetonitrile,
ethyl acetate, dimethyl-acetamide, N-methylpyrrolidone,
dioxane, toluene, ether, methylene chloride or
chloroform. After ~ome time, for example 30 minutes to
3 hours, a solution or suspension, cooled to -10~C, of
an amine in a dipolar aprotic or non-polar organic
solvent, for example tetrahydrofuran, dimethyl
sulphoxide, dimethylformamide, acetonitrile, ethyl
acetate, dimethylacetamide, N-methylpyrrolidone,
dioxane, toluene, ether, methylene chloride or
chloroform, is added with vigorous stirring. The
su~pension is stirred at a temperature within a range
from -30~C to 30~C, preferably between -20~C and 20~C,
particularly between -15~C and 5~C, ~or 1 to 2 hours.
After ~n~; ng of the reaction, the base is filtered o~f
with suction a~ the hydrochloride and the solvent is
concentrated. The oily residue is treated with an
aprotic or non-polar organic solvent, for example
ether, diisopropyl ether, methyl tert-butyl ether,
- petroleum ether, toluene, xylene, pentane, hexane. The
solution is stirred for some time, for example 30
minutes to 3 hours, until a white powder is
precipitated. The precipitate is filtered off with
suction and dried.
CA 02238~70 1998-0~-26
- 31 -
2nd Staqe
The Z-Lys(Boc)amide obtained according to the above
procedure of the 1st Stage is dissolved in
trifluoroacetic acid at a temperture between -20~C and
30~C, preferably between -10~C and 20~C, particularly
between -S~C and 5~C and stirred for a period of 15
minutes to 1 hour. The excess trifluoroacetic acid is
concentrated and the oily residue is treated wi~h a
dipolar aprotic or non-polar organic solvent, such as,
for example, dimethylformamide, methylene chloride,
tetrahydro~uran, acetonitrile, N-methylpyrrolidone,
ethyl acetate. The desired acid, a base, ~uch as, ~or
example, diisopropylethylamine, N-methylmorpholine and
the suitable coupling reagent such a~, for example,
BOP, PyBOP, DCC are then added in a dipolar aprotic or
non-polar organic solvent, such as, for example,
dimethylformamide, methylene chloride, tetrahydrofuran,
acetonitrile, N-methylpyrrolidone, ethyl acetate. The
reaction takes place at a temperature from -10~C to
100~C, preferably at 0~C to 80~C, particularly between
10~~ and 35~C. After a reaction time of 1 to 5 hours
and st~n~;ng at room temperature for 2* hours, the
solvent is concentrated. The residue is precipitated
using an organic solvent, ~uch as, for example, water,
2S isopropanol, methylene chloride or ether. The crude
product is purified by chromatography on a silica gel
column.
According to this general procedure for Stages 1 and 2,
on which the Synthesis Scheme 3 is based, compounds
were~ synthesized which follow from Table- 3 below, n
being equal to 4 throughout.
CA 02238570 1998-05-26
,
-- 32
~R2
Rt _ CO--NH--CH--CO--N
\ R3
(I ~2)n
NH
o CO--R4
(Formula I)
Ta~le 3: a,~-N-substituted L-lysinamide derivatives
according to synthesis Sche~e 3 and the
general formula I (for all Examples n is
equal to 4)
Example R~ -CO R2 R3 R4
[~ CH2~N ~ ,NH
NH2
36 CH2~3
C~2
37
38
CHa
CA 02238570 1998-05-26
..
Table 3 (Contd. )
Example R~ - CO R2 R3 R4
CH~ CH2 ~ ~ NH
o CH, NH2
CH,
41 ~J
NH2
42CH2 ~
CH2~
CHl N
44 N ~
~ .
45~'~'c~
CH,
46C~2 ~ CH,
47 ~N ~N
CH
4~8 NH~
_
CA 02238570 1998-05-26
-- 34
Table 3 (Contd.)
Example ~ - CO R2 R3 R4
~' CJt2
C~2~
N~
~0
C~2~
~1 ~F
52 N ~
CH~ CH,
~3 ~
CH,
CH2~ N
54
C~ N~
Exam~le 35:
s
N-(a-N-Z-r~-N-4-(4-Amidinophenyl)-amino-1,4-dioxo-
butyl]lysine-N-(3-p~yridylmethyl))amide
1st Staqe
Z-Lys(Boc)-N-(3-pyridylmethyl)amide
N-(a-N-Z-r~-N-tert-butYloxyca~-bonyl~lysine-N-(3-
pyridylmethyl)amide
lS 4 g (10 mmol) of Z-Lys(Boc)-OH, which is commercially
available, 1 g (10 mmol) of triethylamine and 1.26 g
CA 02238570 1998-05-26
- 35 -
(10 mmol) of pivaloyl chloride are added at -15~C to
60 ml of tetrahydrofuran. After 30 minutes, a solution,
precooled to -10~C, of 1.08 g (10 mmol) of
3-(aminomethyl)pyridine in 20 ml of tetrahydrofuran is
added with vigorous stirring. The suspension is stirred
at -15~C for 1 to 2 hours. The triethylamine hydro-
chloride is filtered o~f with suction at low
temperature and the tetrahydrofuran is then evaporated.
The oily residue is treated with 100 ml of diethyl
ether. The solution is stirred until a white powder
precipitates. The precipitate is filtered off with
suction and dried Yield: 4 g (85~ of theory).
~nd Staqe
N-(a-N-Z-r~-N-4-(4-AmidinoPhenYl)-amino-1 4-dioxo-
butYlllYsine-N-(3-p~ridYlmethYl)amide
2 g (4.25 mmol) of Z-Lys(Boc)-N-(3-pyridylmethyl~amide
are dissol~ed in 20 ml of TFA at 0~C and the solution
is stirred for 20 min. The excess TFA is concentrated
and the oily residue is treated with lo ml of DMF. 4.6
ml (42.5 mmol) of N-methylmorpholine, 1.15 g
(4.25 mmol) of 4-~[4-aminoiminomethyl)phenyl3amino]-4-
oxobutyric acid hydrochloride, 2.35 g (5.3 mmol) of BOPand 20 ml of DMF are then added. The mixture is stirred
at room temperature for 24 hours. The DMF i~
concentrated, and the residue is digested twice with 40
ml of water, then filtered off with suction and dried.
The crude product is purified by chromatography on a
silica gel column using the eluent 89b (70~ HCCl3, 40
MeOH, 10~ CH3COO~Na~ in 1 Mol per litre NH40H 25~).
Yield: 340 mg (14~ of theory).
~x~mples 36 to 5S were obtained analogueously to
~mple 35.
CA 02238S70 1998-05-26
- 36 -
Table 4: Melting points of the compounds according to
~xamples 35 to 55
Example m.p [~C] Example m.p.t~C] Example m.p.t~C]
31190-198 38 190 45 189
32218-220 39 198 46 197
33 209 40 213 47
34 195 41 48
35189-191 42 175 49
36215-220 43 196 50 194
37 183 44 217 51
Further compounds of the general Formula I were
prepared according to the ~ollowing Schemes 4 and 5.
Scheme 4: Reaction with carboxylic acids
J,~ H2N~N~ NH~NH2
R~ OH ~ N 1,~ ,)~' O
NH-Boc NH-80c
O ~ TFA - 80c
Il C '-" 'oderBOP o
O HN NH2 ~H2
CA 02238570 1998-05-26
- 37 -
Scheme 5: Reaction with chloroformic acid esters
o o o
R~ O a ~2N~ NH2 Sd~e~Baumann- _o~NH~NH2
~ gungen O ~ ~
NH-Boc NH-Boc
~ lFA - 60c
~ G.L ~ ' ' oder BOP O
,Ob,NH~ ein~un~(HPLC) ~~~~
NH~U'NHJ~NHz H~
1. Acylation with carboxylic acids or chloroformic acid
esters according to S~hemefi 4 and 5:
H-Lys(Boc)-NH2 is reacted at room temperature in an
aprotic solvent (DMF, DMSO) in the presence of a base
(DIPEA, NMM) and of a coupling reagent (DCC, DIC, ED~I) -
25 with a carboxylic acid to give the resulting amide.After le,l~o~dl of the solvent, the residue is treated
with water and the poorly soluble crude product is
filtered off with suction. The product can be purified
by crystallization from alcohol (MeOH, EtOH< 2-PrOH) or
esters (MEK, EA).
The reaction of H-Lys(Boc) -NH2 with carbonyl chlorides
in aqueous-alkaline solution (Schotten-Baumann
conditions) leads to the desired derivatives in 90-95~
yields. The crude product is is~lated by filtering off
with suction and purified by recrystallization from
alcohol (MeOH/EtOH/isopropanol) or ethyl acetate or
met~yl ethyl ketone.
CA 02238570 1998-05-26
- 38 -
2. Removal of the Boc protective group using TFA:
The removal of the Boc protective group at room
temperature in a mixture of dichloromethane and
trifluoroacetic acid (2:1) is quantitative after
approximately 60 min. The isolated, usually oily crude
product Rl-Lys-NH2 is rapidly further reacted without
further purification steps.
3. Acylation where R~ . 4-((4-(aminoiminomethyl)-
phenyl)amino)-4-oxobutyric acid hydrochloride:
The reaction with a further carboxylic acid (R~) is
carried out in aprotic solvents (DMF, DMSO) at room
temperature in the presence of a base (NMM, DIPEA)
using coupling reagents such as EDCI, Bop or PyBop.
After removing the solvents, the product precipitates
on addition of water. Purification is carried out by
means of preparative HPLC on an RPl8 column using
eluent mixtures of water, acetonitrile and
tri~luoroacetic acid. The product is obt~;ne~ as the
TFA salt.
According to this general procedure, on which the
Synthesis Schemes 4 and 5 are based, compounds were
synthesized which follow below from the description of
Example 56 and the following Table 5:
Example 56
32 ~mol of Z-lysinamide hydrochloride and 32 mmol of
4-((4-(aminoiminomethyl)phenyl)amino)-4-oxybutyric acid
hydrochloride are added at room temperature to 120 ml
of dry, degassed N,N-dimethylformamide (DMF).
The starting materials dissolve rapidly with stirring;
after addition of 104 mmol of diisopropylethylamine and
40 mmol of BOP the mixture is stirred at RT for 16 h.
CA 02238570 1998-05-26,~
- 39 -
Solvent and excess DIPEA are stripped of on a rotary
evaporator at a bath temperature of 50-~5~C and about
10 mbar. The oily residue is treated with 250 ml of
water, homogenized in an ultrasonic bath and cooled.
Precipitated crude product is filtered off with suction
and washed with water on the suction filter.
A~ter drying in vacuo over calcium chloride, about 16 g
of beige powder having a purity of about 90~ (HPLC) are
obtained as the HCl salt.
To prepare the corresponding tri~luoroacetate, the
product is suspended in 100 ml of water and treated
with 32 mmol (2.45 ml) of trifluoroacetic acid (9g~).
In order to remove excess acid again, the mixture i~
evacuated briefly on a rotary evaporator, then the
aqueous suspension is lyophilized.
After recrystallization from alcohol (EtOH/MeOH), the
product thus obtained can be lyophilized again for
better solubility.
Yield: 5.26 g
M.p.:210-213~C
~R2
R1- CO -NH-CH-CO-N
¦ \ R3
(CH2)n
NH
Co-R4
(~ormula I)
_
CA 02238570 1998-05-26
,~ ,
- 40 -
Table 5: ~,~-N-substituted L-lysinamide derivatives
according to Schemes 4 and 5 and of the general formula
I (for al~ Examples n is equal to 4)
Example R1 CO ~2~R3 R4
~6 ~3~~--il /~NH~NH2
~7
58 F ~
59 ~ O
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- 41 -
Ta~le 5 ( Contd . )
Example R1 CO R2tR3 R4
l'~X ~f ~\O H/H /~NH
61
62 ~~
63
W lo
64 ~
65 ~0
66 ~3
67
O
68
~~
CA 02238570 1998-05-26
_ 42
Table 5 (Contd. )
Examp~e R' - CO R2~3R4
69 ~ ~ ~NH ~NH
~ O H2
13~
0~0
71 o~N~
o~o
~ - o~o
72
73
o
74 ~1
0~0
~o~O
~0~0
76 J
O
77 - ~
~ .
o~o
78
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- ~3 -
Table 5 (Contd.)
Example ~ - CO R2~3 R4
H/H ~ ~ NH
--~~~~ NH~,H2
81
/~0~0
,~
82
\~ ~~0~0
Table 6: Melting points of the compounds according to
5 Examples 56 to 82
Example m.p t~C] Example m.p.t~C~ Example m.p.t~c]
S6210-213 65218-222 74191-193
57220-223 66216-219 75186-188
58213-21S 67235-238 76220-222
59223-226 68up to 218 77210-215
60up to 233 69205-208 78up to 223
61up to 237 70168-170 79up to 226
62up to 221 71197-202 80194-197
63up to 220 72221-226 81215-222
64230-236 73225-228 82219-222
Note: The statement "up to..." indicates that the
substance formed an amorphous foam having corresponding
physical properties after freeze-drying. A melting
point in the strict sense did not exist, but rather a
slow sintering together until liquefaction.
Salts of the com~ounds of the qeneral Formula I
The compounds according to the invention can also be
present as acid addition salts, for example as salts of
mineral acids, such as, for example, hydrochloric acid,
CA 02238570 1998-05-26
..
- 44 -
sulphuric acid, phosphoric acid, ~alts of organic
acids, such as, for example, acetic acid,
trifluoroacetic acid, lactic acid, malonic acid, maleic
acid, fumaric acid, gluconic acid, glucuronic acid,
citric acid, embonic acid, methanesulphonic acid,
hydroxyethanesulphonic acid, pyruvic acid and succinic
acid.
Both the compounds of the general formula I and their
salts are biologically ac~ive. The compounds o~ the
general formula I can be administered in free form or
as salts with a physiologically tolerable acid.
A~m; n;stration can be carried out orally, parenterally,
intravenously, transdermally or by inhalation.
1~ .
The invention ~urthermore relates to pharmaceutical
preparations cont~in;ng at least one compound of the
formula I or its salt with physiologically tolerable
inorganic or organic acids and, if appropriate,
pharmaceutically utilizable excipients and/or diluents
or auxilaries.
~xamPle 83 B;n~;ng affinities of Cetrorelix,
~x~ple 1, Example 2 and Example 56 to the
human LH-RH receptor
(Cetrorelix: Ac-D-Nal(2)-D-p-Cl-Phe-D-Pal(3)-Ser-Tyr-D-
Cit-Leu-Arg-Pro-D-Ala-NH2)
Method for the determination of the b;n~;ng af f inity
(dissociation constant Kd): -
The b;n~;ng af~inity was determined by a competitiveb;n~;ng test ("displacement binding experiment";
Beckers et al. Eur. J. Bio~hem. 231, 535-5*3, 1995).
The radiolabelled ligand used i~ [12sI] Cetrorelix
(speci~ic activity 5-lOxlOs dpm/pmol; dissolved in 20
v:v acetonitrile, 0.2~ w:v albumin, 0.1~ w:v TFA, ~80~
v:v aqua). The binding ability of the iodinated peptide
is between 60~ and 85~. The non-labelled test compounds
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- 45 -
used are Cetrorelix, Example 1, Example 2 and Example 5
in solution. The substances are employed in concentra-
tions of 0.01 nM - 1000 nM (Cetrorelix, Example 1,
Example 2) or o.01 ~M - lO ~LM (Exa~nple 56)
The cells of the individual cell clone L3.5/78
overexpressing the human hH-RH receptor which are used
for the binding test are removed with PBS/EDTA (PBS
without Ca2~/Mg2~/1 mM EDTA) from a cell culture dish
grown under non-confluent conditions, the cell count is
determined and the cells are resuspended in incubation
medium (Dulbecco's modified Eagle Medium with 4.5 g/l
glucose, 10 mM Hepes pH 7.5, 0.5~ w:v BSA, l g/l
bacitracin, 0.1 g/l SBTI, 0.1~ w:v NaN3) at a corres-
ponding cell density. 200 ~l o~ silicone/para~in oilmixture (84/16~ by volume) are initially introduced
into special 400 ~l reaction vessels (Renner, Beckman
type) and 50 ~l of the cell suspension (2.5xlOs cells)
are pipetted onto it. 50 ~l of binding medium contain-
ing [l25I3 Cetrorelix and the compound to be tested atthe appropriate concentration are added to the cell
suspension on the silicone/paraffin oil layer. The
mixture is then incubated with rotation for 60 min at
37~C in a warm cabinet. After this step, it is centri-
fuged at 9000 rpm (room temperature) for 2 min in theHeraeus Biofuge 15 in the HTA 13.8 rotor. In the course
of this, the cells pelletize through the
silicone/paraffin oil layer and are thus separated from
the b; n~; ng medium. After centrifugation, the reaction
vessels are shock-frozen in liquid N2 and the tip of the
reaction vessel (cell pellet) i8 cut off with a pair of
pincers and the tip containing the cell pellet (bound
ligand [l2sI3 Cetrorelix) and the supernatant (unbound,
~ree ligand ~l2sI] Cetrorelix) are transferred to
counting tubes. To determine the ~x;mnm b;n~;ng (Bo),
no competitor is added. For the determination of non-
specific binding, 1 ~M unlabelled Cetrorelix is added
for -competition. At < 10~ of the total binding Bo, the
non-specific binding is low. Quantification is carried
-
CA 02238570 1998-05-26
- 46 -
out in a r-counter; analysis is carried out using the
EBDA/ligand V3.0 programme (McPherson, J. Pharmacol.
Methods 14, 213-228, 1985). Plotting in the dose-
response graph makes possible the estimation o~ the ICso
tconcentration which causes 50~ inhibition o~ the
reaction at the receptor) and the EBDA/ligand programme
calculates the dissociation constant Kd tnM3 from this.
.
Result: from the competition curve~ (see Fig. 1) it is
evident that all compounds tested compete with the
radiolabelled ligand [l2sI] Cetrorelix) for binding to
human LH-RH receptor. In each case, the binding (in
of the total binding Bo) is plotted again~t the concen-
tration of the competitor. For the compounds shown in
Fig. 1, it was possible to calculate the following
binding affinities as the dissociation constant Kd
~nM]: Cetrorelix (SB-75) - O.214 nM, Example 1
o.305 nM, Example 2 - O.104 nM and Example 56 - 986 nM.
The b; n~; ng affinities as the mean value of various
determinations can be taken from Table 7.
Example 84 Antagonistic action o~ F~mrle 2 and Example 56
in the funct;o~l assay on the human LH-RH
receptor
Method for the determination of IP3 (D-myo-1,3,5-
triphosphate): a subconfluent culture of the cell clone
(L 3.5/78) o~erexpressing the hll~~n ~H-RH receptor i8
washed lx with PBS, the cells are ~e,.,o~ed with PBS/EDTA
and the cell suspension is pelletted. The cells are
resuspended in incubation medium (Dulbecco' 8 modified
Eagle Medium with 4.5 g/l of glucose, 10 mM Hepes
pH 7.5, 0.5~ w:v BSA, S mM of LiCl, 1 g/l of
bacitracin, o.~ g/l of SBTI), aliquoted into 1.5 ml
reaction ~essels and preincubated at 37~C for 30 min.
4x10C cells in a 500 ~l volume are needed per measuring
point. A~ter the preincubation step, LH-RH (stock
solution o.5 ~M in lo mM tris pH 7.5, 1 mM
dithiothreitol, 0.1~ w:v BSA/Bachem Art # H4005) are
CA 02238570 1998-05-26
,
- 47 -
added to the cell suspension at a ~inal concentration
of 10 nM. The action of an antagonist is tested ~y
simultaneous addition at the corresponding concentra-
tion (for example 0.0316, 0.1, 0.316 etc. up to loo nM
~or Example 2). As a negative control, cells without
added LH-RH are incubated. A~ter incubation at 37~C for
15 min, IP3 formed is isolated from the cells by means
o~ trichloroacetic acid (TCA) extraction. To this end,
500 ~l of ice-cold 15~ (w:v) TCA solution are added to
the cell suspension. The resulting precipitate is
pelletted by centrifugation at 4~C in the Heraeus
Biofuge l~R centrifuge at 2000xg for 15 min. The super-
natant of 950 ~l is extracted 3x with 10 vol of cold,
water-saturated diethyl ether in a 15 ml vessel stand-
ing on ice. After the last extraction step, thesolution is adjusted to a pH of 7.5 with 0.5 M NaHC03
solution.
The determination of the IP3 concentration in the cell
extracts is carried out by means of a sensitive
competitive binding test using an IP3 binding protein,
labelled ~3H]-IP3 and unlabelled IP3. To this end, an
assay kit from Amersham (TRK 1000) is used; the deter-
mination is carried out as described in the assay
protocol. A~ter carrying out the various steps, 2 ml of
scintillator for aqueous samples (Rotiszint Ecoplus) is
finally added, the resuspended pellet con~;n;ng the
bound [3H]-IP3 is carefully mixed with it, and measured
in a ~-scintillation counter. The amount of cellular IP3
is calculated using a st~n~rd curve and a dose-
response curve is set up. The IC50 can be estimated from
the inflection point of this curve.
Result: Fig. 1 shows appropriate do~e-respon~e curves
for the peptide antagonists Ex~,nple 2 (Fig. 2), as well
as for the pepti~o~;m~tic Example 56 (Fig. 3).
Stimulation was carried out with 10 nM LH-RH and the
inhibition o~ formation of IP3 determined as a function
of the substance concentration For Example 2 and
-
CA 02238570 1998-05-26
- 48 -
Example 56, it was not possible to determine any
agonistic activity, i.e. the substances by themselves
do not lead to any stimulation of IP3 synthesis. In
control experiments not presented here, it was shown
that non-trans~ected cells cannot be stimulated by
LH-RH to IP3 synthesis. The IP3 concentrations still
measurable at the highest concentrations correspond to
those of unstimulated cells. In Example 2 and
Example 56 we are thus dealing with functional
antagonists of LH-RH. The substances differ, however,
in their potency. Under the experimental conditions
selected, the IC50 o~ Example 2 is approximately 0.4 nM,
the ICso ~or Example 56, however, is approximately 4 ~M.
These activities correlate very well with the in vitro
binding af~inities, determined in the competitive bind-
ing test using [125I]-Cetrorelix, of Kd = O.109 nM ~or
Example 2 and Kd = 1.08 ~M for Example 56.
Exam~le 85 Hormone-suppressive action o~ Example 1,
Example 2 and Example 56 in the healthy male rat
To determine the suppression of testosterone in the
blood of healthy male rats, the substance was injected
subcutaneously into the right flank of the ~n;~l S . The
dosage was 1.5 mg/kg in the case of Example 1 and
Example 2 and 10 mg/kg in the case of Example 56. To
check the testosterone ~alues, about 300 ~l o~ blood
were taken from the ~n; m~l ~ from the sublingual vein at
the times 0, 2, 4, 8 (only Example 56), 24, 48, 72 and
96 hours, and then every 3 days until the end of
supp~ession. Suppression with 1 ng/ml of testosterone
after the administration of Example 1 lasted up to
264 hours in one ~n;m~l, up to 336 hours in two ~n;m~l S
and up to 384 hours in one ~ni m~ ig. 4). After
administration of Example 2, the testosterone evel in
one animal was suppressed for up to 408 hours, and in
four animals for up to 648 hours (Fig. 5). Example 56
(10 mg/kg s.c.) suppressed the testosterone level in
all 5 animals even after 2 hours and maintained this
CA 02238570 1998-OS-26
- 49 -
action for up to 8 hours At the next measuring point
(24 h), the testosterone values rose again (Fig. 6).
Table 7: Biological data
Binding affinities to human LH-~H receptor (expressed
as the dissociation constant Kd [nM]; evaluation using
the EBDA/Ligand Analysis Programme. Mean values from
various experiments are indicated, number of
experiments in brackets ) as well as testosterone
suppression in vivo, histamine release in vitro and
water solubility in comparison to SB-75:
Substance ~finity (1.5 mg/kg, (ICso) H2o
h~m ~n LH-RH single dose) Histamine Solubility
receptor testosterone relea~e [mg/ml]
tnmol/L] suppression [~g/ml]
rats
th]
Cetrorelix 0.202 (10) 144 9.7 9
SB-7s
Example 1 0.306 (2) 336 31.9 27
Example 2 0.109 (2) 648 17.1 23
Example 3 0.170 (2) 864 n.d. n.d.
Example 4 0.206 (2) 696 n.d. n.d.
Example 56 1082 (2)
~~Not determinable because o~ poor solubility
