CA2238570A1 - New lh-rh antagonists with improved effectiveness - Google Patents
New lh-rh antagonists with improved effectiveness Download PDFInfo
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- CA2238570A1 CA2238570A1 CA002238570A CA2238570A CA2238570A1 CA 2238570 A1 CA2238570 A1 CA 2238570A1 CA 002238570 A CA002238570 A CA 002238570A CA 2238570 A CA2238570 A CA 2238570A CA 2238570 A1 CA2238570 A1 CA 2238570A1
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/02—Drugs for disorders of the endocrine system of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin
- A61P5/04—Drugs for disorders of the endocrine system of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin for decreasing, blocking or antagonising the activity of the hypothalamic hormones
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- C07C257/10—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines with replacement of the other oxygen atom of the carboxyl group by nitrogen atoms, e.g. amidines
- C07C257/18—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines with replacement of the other oxygen atom of the carboxyl group by nitrogen atoms, e.g. amidines having carbon atoms of amidino groups bound to carbon atoms of six-membered aromatic rings
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- C07D207/18—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
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- C07D207/24—Oxygen or sulfur atoms
- C07D207/26—2-Pyrrolidones
- C07D207/263—2-Pyrrolidones with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms
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- C07D209/04—Indoles; Hydrogenated indoles
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Abstract
New LH-RH antagonists are disclosed, in particular peptidomimetics and peptides modified in a side chain, their salts with pharmaceutically acceptable acids and a process for preparing these LH-RH antagonists and their salts. The disclosed peptides represent analogues of the luteinising hormone releasing hormone (LH-RH). The disclosed compounds have a high antagonistic power and are free of undesirable side effects, in particular edematogenic effects.
Description
.. ..
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 .- - .... - . . . . .
- 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 ....''.'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
-:- .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, .
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
~) 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-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 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 , ~ 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.
.
, 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, _ (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 -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.
,~ ' 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.
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 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.
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 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~.
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
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 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 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) -.
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:
_ 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 \ R3 (CH2)n NH
(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 Example R'-CO R2lR3 R~
T~;nuO.ua~ 3 ~~ - H/H ~ ~, 6 c~_CH, NH 4~3CN
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 .- - .... - . . . . .
- 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 ....''.'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
-:- .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, .
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
~) 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-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 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 , ~ 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.
.
, 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, _ (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 -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.
,~ ' 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.
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 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.
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 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~.
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
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 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 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) -.
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:
_ 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 \ R3 (CH2)n NH
(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 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 O OCH~
14 .cH~ Nff~
1 5 ~0~
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~
-Table 1 ( Contd . ) Example R'-CO R2lR3 R4 ~ HIH
~~~
~ 1~0 27 ~a~ a 28 ~
cooet 29 /~
CH~ t H--~NH
C~ J~,N
31 'CH,~NH~(~
32 \_J "3 ~ ~ --N
O
34 a~ 3 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 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) ) .
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 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:
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 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 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.
, ~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
36 CH2~3 C~2 CHa ..
Table 3 (Contd. ) Example R~ - CO R2 R3 R4 CH~ CH2 ~ ~ NH
o CH, NH2 CH, 41 ~J
42CH2 ~
CH2~
CHl N
44 N ~
~ .
45~'~'c~
CH, 46C~2 ~ CH, 47 ~N ~N
CH
4~8 NH~
_ 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
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 (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.
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]
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 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.
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,~
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 ¦ \ R3 (CH2)n NH
Co-R4 (~ormula I) _ ,~ , 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
Ta~le 5 ( Contd . ) Example R1 CO R2tR3 R4 l'~X ~f ~\O H/H /~NH
62 ~~
W lo 64 ~
65 ~0 66 ~3 O
~~
_ 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 o 74 ~1 0~0 ~o~O
~0~0 O
77 - ~
~ .
o~o CA 02238570 l998-05-26 - ~3 -Table 5 (Contd.) Example ~ - CO R2~3 R4 H/H ~ ~ NH
--~~~~ NH~,H2 /~0~0 ,~
\~ ~~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]
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 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, ..
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 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 -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 , 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 -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 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
CH~ N~
NH~
12 'CH~NH
OBzl=8ereyloxy O OCH~
14 .cH~ Nff~
1 5 ~0~
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~
-Table 1 ( Contd . ) Example R'-CO R2lR3 R4 ~ HIH
~~~
~ 1~0 27 ~a~ a 28 ~
cooet 29 /~
CH~ t H--~NH
C~ J~,N
31 'CH,~NH~(~
32 \_J "3 ~ ~ --N
O
34 a~ 3 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 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) ) .
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 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:
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 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 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.
, ~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
36 CH2~3 C~2 CHa ..
Table 3 (Contd. ) Example R~ - CO R2 R3 R4 CH~ CH2 ~ ~ NH
o CH, NH2 CH, 41 ~J
42CH2 ~
CH2~
CHl N
44 N ~
~ .
45~'~'c~
CH, 46C~2 ~ CH, 47 ~N ~N
CH
4~8 NH~
_ 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
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 (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.
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]
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 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.
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,~
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 ¦ \ R3 (CH2)n NH
Co-R4 (~ormula I) _ ,~ , 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
Ta~le 5 ( Contd . ) Example R1 CO R2tR3 R4 l'~X ~f ~\O H/H /~NH
62 ~~
W lo 64 ~
65 ~0 66 ~3 O
~~
_ 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 o 74 ~1 0~0 ~o~O
~0~0 O
77 - ~
~ .
o~o CA 02238570 l998-05-26 - ~3 -Table 5 (Contd.) Example ~ - CO R2~3 R4 H/H ~ ~ NH
--~~~~ NH~,H2 /~0~0 ,~
\~ ~~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]
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 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, ..
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 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 -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 , 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 -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 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
Claims (18)
1. Compound of the general formula I
in which n is the number 3 or 4, R1 is 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, where the substitution can in turn consist of an aryl group or heteroaryl group, or -NR2R3 is an amino acid group, and R4 is a group having the formula (II) ~(CH2)p~CO~NR5R6 (II) in which p is an integer from 1 to 4, R5 is hydrogen or an alkyl group and R6 is an unsubstituted or substituted aryl or heteroararyl group, or R4 is a ring of the general formula (III) in which q is the number 1 or 2, R7 is a hydrogen atom or an alkyl group, R8 is a hydrogen atom or an alkyl group and X is 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-configuration, and its salts with pharmaceutically acceptable acids.
in which n is the number 3 or 4, R1 is 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, where the substitution can in turn consist of an aryl group or heteroaryl group, or -NR2R3 is an amino acid group, and R4 is a group having the formula (II) ~(CH2)p~CO~NR5R6 (II) in which p is an integer from 1 to 4, R5 is hydrogen or an alkyl group and R6 is an unsubstituted or substituted aryl or heteroararyl group, or R4 is a ring of the general formula (III) in which q is the number 1 or 2, R7 is a hydrogen atom or an alkyl group, R8 is a hydrogen atom or an alkyl group and X is 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-configuration, and its salts with pharmaceutically acceptable acids.
2. .alpha.-N-Z-[.epsilon.-N'-4-(4-Amidinophenyl)amino-1,4-dioxo-butyl]lysinamide and its salts with pharmaceutically acceptable acids.
3. .alpha.-N-Z-[.epsilon.-N'-4-(4-Amidinophenyl)amino-1,5-dioxo-pentyl]lysinamide and its salts with pharmaceutically acceptable acids.
4. .alpha.-N-Z-[.epsilon.-N'-(Imidazolidin-2-on-4-yl)-formyl]lysin-amide and its salts with pharmaceutically acceptable acids.
5. Compound according to one of Claims 1 to 4, in which the salt is an embonate.
6. Compound of the general formula V
Ac-D-Nal(2)1-D-(pCl)Phe2-D-Pal(3)3-Ser4-Tyr5-D-Xxx6-Leu7-Arg8-Pro9-D-Ala10-NH2 (V) in which D-Xxx is an amino acid group of the general formula (VI) in which n is the number 3 or 4, R4 is a group of the formula (II) in which p is an integer from 1 to 4, R5 is hydrogen or an alkyl group and R6 is an unsubstituted or substituted aryl group or heteroaryl group, or R4 is a ring of the general formula (III) in which q is the number 1 or 2, R7 is a hydrogen atom or an alkyl group, R8 is a hydrogen atom or an alkyl group and X is an oxygen or sulphur atom, and its salts with pharmaceutically acceptable acids.
Ac-D-Nal(2)1-D-(pCl)Phe2-D-Pal(3)3-Ser4-Tyr5-D-Xxx6-Leu7-Arg8-Pro9-D-Ala10-NH2 (V) in which D-Xxx is an amino acid group of the general formula (VI) in which n is the number 3 or 4, R4 is a group of the formula (II) in which p is an integer from 1 to 4, R5 is hydrogen or an alkyl group and R6 is an unsubstituted or substituted aryl group or heteroaryl group, or R4 is a ring of the general formula (III) in which q is the number 1 or 2, R7 is a hydrogen atom or an alkyl group, R8 is a hydrogen atom or an alkyl group and X is an oxygen or sulphur atom, and its salts with pharmaceutically acceptable acids.
7. Compound according to Claim 6, in which xxx is a [.epsilon.-N-4-(4-amidinophenyl)amino-1,4-dioxobutyl]lysyl group.
8. Compound according to Claim 6, in which Xxx is a [.epsilon.-N-(imidazolidin-2-on-4-yl)formyl]lysyl group.
9. Compound according to one of Claims 6 to 8, in which the salt is an embonate.
10. Pharmaceutical composition comprising a compound according to one of Claims 1 to 9.
11. Process for the preparation of a compound according to Claim 6, comprising the steps of (a) providing the .alpha.-amino and the carboxylic acid group of D-lysine or D-ornithine with suitable protective groups, (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 R4 is as de~ined in Claim 1, (c) removing the protective group on the .alpha.-carboxylic acid group of the compound obtained in step (b) for the purpose of incorporation in pos. 6 in step (h), (d) coupling of D-alanine provided on the amino group with a protective group to a solid support in the form of a resin, (e) removing the protective group on the amino group of the alanine, (f) reacting the alanine bound to the solid support with proline which is provided with a protective group on the nitrogen atom, (g) removing 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) removing the compound obtained in step (h) from the support and, if appropriate, purifying, in particular by HPhC, (j) if desired, reacting with a pharmaceutically acceptable acid, preferably embonic acid.
12. Process for the preparation of a compound according to Claim 6, comprising the steps of (a) coupling D-alanine provided with a protective group on the amino group to a support suitable for solid-phase synthesis, (b) removing the protective group on the amino group of the alanine, (c) reacting the alanine 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 formula (V), in the sequence from 8 to 1, (f) removing the compound obtained in step (e) from the support, (g) reacting with a carboxylic acid of the formula (VII) R4-COOH (VII) in which R4 is as defined in Claim 1, (h) if desired, reacting with a pharmaceutically acceptable acid, preferably embonic acid
13. Process for the preparation of a compound according to Claim 6, comprising the steps of a) coupling D-alanine provided with a protective group on the amino group to a support suitable for solid-phase synthesis, (b) removing the protective group on the amino group of the alanine, (c) reacting the alanine 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 6 to 8 according to the general formula (V), in the sequence from 8 to 6, (f) removing the .epsilon.-amino protective group from D-lysine or D-ornithine in pos. 6 and reacting with a carboxylic acid of the formula (VII), R4-COOH (VII) in which R4 is as defined in Claim 1, (g) removing the protective group on the .alpha.-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) removing the compound obtained in step (h) from the resin and purifying it, in particular by HPLC, (j) if desired, reacting with a pharmaceutically acceptable acid, preferably embonic acid
14. Process according to one of Claims 11 to 13, in which N-(4-amidinophenyl)amino-4-oxobutyric acid is used as the carboxylic acid of the general formula (VII).
15. Process according to one of Claims 11 to 13, in which imidazolidin-2-one-4-carboxylic acid is used as the carboxylic acid of the general formula (VII).
16. Process according to one of Claims 11 to 15, in which embonic acid is used as the pharamceutically acceptable acid.
17. Use of the substances according to Claims 1 to 9 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.
18. Process for the production of medicaments comprising compounds according to Claims 1 to 9, characterized in that a substance according to Claims 1 to 9 is mixed with the customary excipients and auxiliaries and formulated as a medicament.
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| Application Number | Priority Date | Filing Date | Title |
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| DE19544212.1 | 1995-11-28 | ||
| DE19544212A DE19544212A1 (en) | 1995-11-28 | 1995-11-28 | New LH-RH antagonists with improved effects |
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| AT (1) | ATE289588T1 (en) |
| AU (1) | AU706546B2 (en) |
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| BR (1) | BR9611760A (en) |
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| CZ (1) | CZ290098B6 (en) |
| DE (2) | DE19544212A1 (en) |
| DK (1) | DK0876337T3 (en) |
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| DE19911771B4 (en) * | 1999-03-17 | 2006-03-30 | Zentaris Gmbh | LHRH antagonist, process for its preparation and its use |
| WO2001068676A2 (en) * | 2000-03-14 | 2001-09-20 | Zentaris Ag | Lhrh-antagonists, production and use thereof as medicament |
| US7005418B1 (en) | 1999-09-23 | 2006-02-28 | Zentaris Gmbh | Method for the therapeutic management of extrauterine proliferation of endometrial tissue, chronic pelvic pain and fallopian tube obstruction |
| SE0100567D0 (en) * | 2001-02-20 | 2001-02-20 | Astrazeneca Ab | Compounds |
| CN104086632A (en) * | 2014-08-05 | 2014-10-08 | 杭州诺泰制药技术有限公司 | Method for preparing cetrorelix |
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| US5140009A (en) * | 1988-02-10 | 1992-08-18 | Tap Pharmaceuticals, Inc. | Octapeptide LHRH antagonists |
| US5110904A (en) * | 1989-08-07 | 1992-05-05 | Abbott Laboratories | Lhrh analogs |
| US5300492A (en) * | 1988-02-10 | 1994-04-05 | Tap Pharmaceuticals | LHRH analogs |
| IL101074A (en) * | 1991-03-14 | 1997-09-30 | Salk Inst For Biological Studi | GnRH ANALOGS AND THEIR PREPARATION |
| DE593491T1 (en) * | 1991-04-25 | 1994-11-17 | Romano S.-Cergue Deghenghi | LHRH antagonists. |
| JPH08504209A (en) * | 1992-12-04 | 1996-05-07 | アボツト・ラボラトリーズ | 6-position modified decapeptide LHRH antagonist |
| CA2136079A1 (en) * | 1992-12-18 | 1994-07-07 | Fortuna Haviv | Lhrh antagonists having modified aminoacyl residues at positions 5 and 6 |
| IL108509A0 (en) * | 1993-02-22 | 1994-05-30 | Salk Inst For Biological Studi | GnRH antagonist peptides |
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