SK14332002A3 - LHRH-antagonists, production and use thereof as medicament - Google Patents
LHRH-antagonists, production and use thereof as medicament Download PDFInfo
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- SK14332002A3 SK14332002A3 SK1433-2002A SK14332002A SK14332002A3 SK 14332002 A3 SK14332002 A3 SK 14332002A3 SK 14332002 A SK14332002 A SK 14332002A SK 14332002 A3 SK14332002 A3 SK 14332002A3
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
Description
Oblasť technikyTechnical field
Vynález sa týka antagonistov LHRH so zlepšeným správaním pri rozpúšťaní, spôsobu výroby týchto zlúčenín, liečiv, v ktorých sa tieto zlúčeniny nachádzajú, a použitia týchto liečiv na liečenie hormonálne závislých nádorov a nemalígnych chorôb ovplyvnených hormónmi, ako je napríklad benígna hyperplázia prostaty (BPH) a endometrióza.The invention relates to LHRH antagonists with improved dissolution behavior, to a process for the production of these compounds, to medicaments containing them, and to the use of these medicaments for the treatment of hormone-dependent tumors and non-malignant hormone-affected diseases such as benign prostatic hyperplasia (BPH); endometriosis.
Doterajší stav technikyBACKGROUND OF THE INVENTION
Nomenklatúra používaná na definovanie peptidov sa zhoduje s nomenklatúrou vysvetlenou komisiou IUPAC-IUB pre biochemickú nomenklatúru (European J. Biochem. 1984, 138, 937), v ktorej sa v súlade s obvyklým opisom aminoskupiny na N-konci uvádzajú vlavo a karboxylová skupina na C-konci vpravo. Antagonisty LH-RH, ako peptidy podlá vynálezu,_ obsahujú v prírode sa vyskytujúce a syntetické aminokyseliny, pričom tie prvé obsahujú Ala, Val, Leu, íle, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gin, Cys, Met, Phe, Tyr, Pro, Trp a His. Tieto skratky pre jednotlivé aminokyseliny sú založené na triviálnych názvoch aminokyselín a sú: Ala = alanín, Arg = arginín, Gly - glycín, Leu = leucín, Lys = lyzín, Pal (3) = (3-pyridyl)alanín, Nal(3) = 3-(2-naftyl)alanín, Phe = fenylalanín, Cpa = 4-chlórfenylalanín, Pro = prolin, Ser = serín, Thr = treonín, Trp = tryptofán, Tyr = tyrozín a Sar = sarkozín. Všetky tu uvedené aminokyseliny pochádzajú z L-radu, ak nie je uvedené inak. Napríklad D-Nal(2) je skratka pre 3-(2-naftyl)-D-alanín a Ser je skratka pre L-serín.The nomenclature used to define peptides is consistent with the nomenclature explained by the IUPAC-IUB Biochemical Nomenclature Commission (European J. Biochem. 1984, 138, 937), where, in accordance with the usual description of the amino group at the N-terminus, the left and carboxyl group are indicated on C -the right. LH-RH antagonists, such as peptides of the invention, contain naturally occurring and synthetic amino acids, the former comprising Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gln, Cys, Met, Phe, Tyr, Pro, Trp, and His. These amino acid abbreviations are based on the trivial amino acid names and are: Ala = alanine, Arg = arginine, Gly-glycine, Leu = leucine, Lys = lysine, Pal (3) = (3-pyridyl) alanine, Nal (3) = 3- (2-naphthyl) alanine, Phe = phenylalanine, Cpa = 4-chlorophenylalanine, Pro = proline, Ser = serine, Thr = threonine, Trp = tryptophan, Tyr = tyrosine and Sar = sarcosine. All amino acids provided herein are of the L-series unless otherwise indicated. For example, D-Nal (2) is an abbreviation for 3- (2-naphthyl) -D-alanine and Ser is an abbreviation for L-serine.
Substitúcie na ε-aminoskupine v bočnom reťazci lyzínu sú uvádzané prostredníctvom výrazu v zátvorkách za Lys, poprípade vo forme skratky.Substitutions on the ε-amino group in the lysine side chain are indicated by the term in brackets after Lys, or abbreviation.
Iné použité skratky:Other abbreviations used:
Ac acetyl·Ac acetyl ·
B 4-(4-amidinofenyl)amino-1,4-dioxobutylB 4- (4-amidinophenyl) amino-1,4-dioxobutyl
Boe terc-butyloxykarbonylBoe tert-butyloxycarbonyl
Bop benzotriazol-l-oxy-tris(dimetylamino)fosfóniumhexafluorofosfátBop benzotriazole-1-oxy-tris (dimethylamino) phosphonium hexafluorophosphate
DCC dicyklohexylkarbodiimidDCC dicyclohexylcarbodiimide
DCM dichlórmetánDCM dichloromethane
Ddz dimetoxyfenyl-dimetylmetylenoxykarbonyl (dimetoxydimetyl-Z)Ddz dimethoxyphenyl-dimethylmethylenoxycarbonyl (dimethoxydimethyl-Z)
DIC diizopropylkarbodiimidDIC diisopropylcarbodiimide
DIPEA N,N-diizopropyletylamínDIPEA N, N-diisopropylethylamine
DMF dimetylformamidDMF dimethylformamide
Fmoc fluorenylmetoxykarbonylFmoc fluorenylmethoxycarbonyl
HF . kyselina fluorovodíkováHF. hydrofluoric acid
Hobt 1-hydroxybenzotriazolHobt 1-hydroxybenzotriazole
HPLC vysokotlaková kvapalinová chromatografiaHPLC high pressure liquid chromatography
Me me tyl__________Me me tyl__________
TFA kyselina trifluóroctováTFA trifluoroacetic acid
Z benzyloxykarbonyl.Z - benzyloxycarbonyl.
Peptidy podľa vynálezu predstavujú analógy hormónu uvoľňujúceho luteinizačný hormón (LH-RH), ktorý má nasledujúcu štruktúru:The peptides of the invention are luteinizing hormone releasing hormone (LH-RH) analogues having the following structure:
p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, [LH-RH, gonadorelín]. p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, [LH-RH, gonadorelin].
Viac ako 20 rokov vyhľadávali výskumníci selektívne účinné antagonisty dekapeptidu LH-RH [M. Karten und E. Riviér, Endocrine Reviews 7, 44-66 (1986)]. Veľký záujem o také antagonisty je založený na ich užitočnosti v oblasti endokrinológie, gynekológie, antikoncepcie a rakoviny. Vyrobil sa veľký počet zlúčenín ako potenciálnych antagonistov LH-RH. Najzaujímavejšími zlúčeninami, ktoré sa doteraz objavili, sú tie zlúčeniny, ktorých štruktúra predstavuje modifikáciu štruktúry LH-RH.For over 20 years, researchers have been looking for selectively effective decapeptide LH-RH antagonists [M. Karten and E. Riviera, Endocrine Reviews 7, 44-66 (1986)]. Great interest in such antagonists is based on their usefulness in the fields of endocrinology, gynecology, contraception and cancer. A large number of compounds have been produced as potential LH-RH antagonists. The most interesting compounds that have been discovered so far are those whose structure represents a modification of the structure of LH-RH.
Prvý rad účinných antagonistov sa získal zavedením zvyškov aromatických aminokyselín do polôh 1, 2, 3 a 6 alebo 2, 3 a 6. Obvyklé písomné vyjadrenie týchto zlúčenín vyzerá nasledovne: Najskôr sa uvádzajú aminokyseliny, ktoré do peptidovéhoho reťazca LH-RH vstúpili na miesto pôvodne sa vyskytujúcich aminokyselín, pričom polohy, v ktorých sa uskutočnila zámena, sa označujú hornými číslicami. Ďalej sa pomocou nasledujúceho označenia „LH-RH vyjadrí, že sa jedná o analógy LH-RH, v ktorých sa uskutočnila zámena.The first series of potent antagonists was obtained by introducing aromatic amino acid residues at positions 1, 2, 3, and 6, or 2, 3, and 6. Typical writing of these compounds is as follows: First, the amino acids that entered the LH-RH peptide chain at the site originally amino acids, the positions at which the swapping was performed are denoted by upper digits. In addition, the following designation "LH-RH" indicates that these are LH-RH analogues in which the substitution has taken place.
Známymi antagonistami sú:Known antagonists are:
[Ac-D-Cpa1,2, D-Trp3'6]LH-RH (D. H. Coy et al. , In: Gross, E. and Meienhofer, J. (Eds) Peptides; Proceedings of the 6th American Peptide Symposium, str. 775-779, Pierce Chem. Co., Rockville III. (1979):[Ac-D-Cpa 1,2 , D-Trp 3 ' 6 ] LH-RH (DH Coy et al., In: Gross, E. and Meienhofer, J. (Eds) Peptides; Proceedings of the 6th American Peptide Symposium) , pp. 775-779, Pierce Chem. Co., Rockville III (1979):
[Ac-Pro1, D-Cpa2, D-Nal (2) 3'6]LH-RH (US patent č. 4.419.347) a[Ac-Pro 1 , D-Cpa 2 , D-NaI (2) 3 ' 6 ] LH-RH (US Patent No. 4,419,347), and
ÍAc-Pro1, D-Cpa2.,_D-Trp3'-6]LH-RH—(-J-.—L·:—Pineda et al~ Tľ Olin.Ac-Pro 1 , D-Cpa 2 ', D-Trp 3 ' - 6 ] 1 H-RH - (-) - Pineda et al.
Endocrinol. Metab. 56, 420, 1983).Endocrinol. Metab. 56, 420 (1983).
Aby sa zlepšil účinok antagonistov, zavádzali sa neskôr zásadité aminokyseliny, napríklad D-Arg, do polohy 6, príkladom je [Ac-D-Cpa1'2, D-Trp3, D-Arg6, D-Ala10]LH-RH (ORG30276) (D. H. Coy et al., Endocrinology 100, 1445, 1982) a [ÄC-D-Nal (2) 1, D-Phe(4-F)2, D-Trp3, D-Arg6]LH-RH (ORF 18260) (J. E. Riviér et in: Vickery B. H. Nestor, Jr. J. J., Hafez, E. S. E. (Eds). LHRH and its Analogs, str. 11-22 MTP Press, Lancaster, UK 1984).In order to improve the effect of the antagonists, basic amino acids, for example D-Arg, were later introduced into the 6-position, for example [Ac-D-Cpa 1 ' 2 , D-Trp 3 , D-Arg 6 , D-Ala 10 ] LH- RH (ORG30276) (DH Coy et al., Endocrinology 100, 1445, 1982) and [α-D-Nal (2) 1, D-Phe (4-F) 2 , D-Trp 3 , D-Arg 6 ] LH-RH (ORF 18260) (JE Rivier et al: Vickery BH Nestor, Jr. JJ, Hafez, ESE (Eds). LHRH and its Analogs, pp. 11-22 MTP Press, Lancaster, UK 1984).
Ďalšie účinné antagonisty LH-RH sú opísané vo WO 92/19651, WO 94/19370, WO 92/17025, WO 94/14841, WO 94/13313Other potent LH-RH antagonists are described in WO 92/19651, WO 94/19370, WO 92/17025, WO 94/14841, WO 94/13313
US-A 5,300,492, US-A 5,140,009, EP 0 413 209 Al a DE 195 44 212 Al.US-A 5,300,492, US-A 5,140,009, EP 0 413 209 A1 and DE 195 44 212 A1.
Posledný dokument zverejňuje zlúčeniny s modifikovanou ornitínovou alebo lyzínovou stavebnou zložkou v polohe 6, ktoré majú nasledujúci vzorec:The latter document discloses compounds with a modified ornithine or lysine building component at the 6-position having the following formula:
Ac-D-Nal (2) 1-D-Cpa2-D-Pal (3) 3-Ser4-Tyr5-D-Xxx6-Leu7-Arg8-Pro9-DAla10-NH2, kde D-Xxx predstavuje aminokyselinovú skupinu všeobecného vzorca VI:Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -Tyr 5 -D-Xxx 6 -Leu 7 -Arg 8 -Pro 9 -DAla 10 -NH 2 where D-Xxx represents the amino acid group of formula VI:
-NH-CH-COI (CH2)„-NH-CH-COI (CH 2 ) n
II
NHNH
II
CO-RCO-R
Ďalšími známymi antagonistami LH-RH sú antarelix, ganirelix a cetrorelix.Other known LH-RH antagonists are antarelix, ganirelix and cetrorelix.
Antarelix® (INN: Teverelix):Antarelix (INN: Teverelix):
Ac-D-Nal (2) 1-D-Cpa2-D-Pal (3) 3-Ser4-Tyr5-D-Hci6-Leu7-Lys (iPr) 8Pro9-D-Ala10-NH2;_____Ac-D-Nal (2) 1 -D-Ca 2 -D-Pal (3) 3 -Ser 4 -Tyr 5 -D-Hci 6 -Leu 7 -Lys (iPr) 8 Dec 9 -D-Ala 10 - NH 2 ; _____
Ganirelix:ganirelix:
Ac-D-Nal (2)1-D-Cpa2-D-Pal(3)3-Ser4-Tyr5-D-hArg (Et) 26-Leu7hArg (Et) 28-Pro9-D-Ala10-NH2;Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -Tyr 5 -D-hArg (Et) 2 6 -Leu 7 hArg (Et) 2 8 -Pro 9 - D-Ala 10 -NH 2 ;
Cetrorelix:cetrorelix:
Ac-D-Nal (2)1-D-Cpa2-D-Pal (3)3-Ser4-Tyr5-D-Cit6-Leu7-Arg8-Pro9-DAla10-NH2.Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -Tyr 5 -D-Cit 6 -Leu 7 -Arg 8 -Pro 9 -Da 10 -NH 2 .
Cieľom predloženého vynálezu je poskytnúť nové antagonisty LH-RH, ktoré majú zvýšenú enzymatickú stabilitu a výrazne zlepšenú rozpustnosť vo vode.It is an object of the present invention to provide novel LH-RH antagonists having enhanced enzymatic stability and markedly improved water solubility.
Podstata vynálezuSUMMARY OF THE INVENTION
Táto úloha sa rieši prostredníctvom zlúčenín nasledujúceho všeobecného vzorca IThis problem is solved by compounds of the following general formula I
Α-Χχχ1-Χχχ2-Χχχ3-Χχχ4-Χχχ5-Χχχ6-Χχχ7-Χχχ8-Χχχ9-Χχχ1θ-ΝΗ2 (I) v ktoromΑ-Χχχ 1 -Χχχ 2 -Χχχ 3 -Χχχ 4 -Χχχ 5 -Χχχ 6 -Χχχ 7 -Χχχ 8 -Χχχ 9 -Χχχ 1θ -ΝΗ 2 (I) in which
amino-1,4-dioxobutyl]-Lys (skratka: D-Lys(B)),amino-1,4-dioxobutyl] -Lys (abbreviation: D-Lys (B)),
Xxx7 znamená Leu alebo Nie,Xxx 7 means Leu or No,
Xxx8 znamená Arg alebo Lys(iPr),Xxx 8 stands for Arg or Lys (iPr)
Xxx9 znamená Pro aXxx 9 stands for Pro and
Xxx10 znamená D-Ala alebo Sar, s tým pravidlom,.----že keď Xxx6 znamená D-Lys (B), potom Xxx7 je Nie, keď Xxx6 znamená D-Cit, potom Xxx7 je Nie a Xxx10 je DAla alebo keď Xxx6 znamená D-Hci, potom Xxx7 je Leu a Xxx10 je D-Ala, a ich solí s farmaceutický prijateľnými kyselinami, najmä acetátov, pamoátov (embonátov, t.j. solí kyseliny l,l'-metylénbis-(2-hydroxy-3-naftoovej))a trifluóracetátov.Xxx 10 stands for D-Ala or Sar, with the rule that - if Xxx 6 stands for D-Lys (B) then Xxx 7 is No, if Xxx 6 stands for D-Cit, then Xxx 7 is No and Xxx 10 is DAla or when Xxx 6 is D-Hci, then Xxx 7 is Leu and Xxx 10 is D-Ala, and salts thereof with pharmaceutically acceptable acids, especially acetates, pamoates (embonates, i.e. 1,1'-methylenebis acid salts) (2-hydroxy-3-naphthoic)) and trifluoroacetates.
Podľa ďalšieho aspektu vynálezu sú obzvlášť prednostné nasledujúce zlúčeniny a taktiež ich soli s farmaceutický prijateľnými kyselinami:According to a further aspect of the invention, the following compounds are particularly preferred as well as their salts with pharmaceutically acceptable acids:
Ac-D-Nal (2)1-D-Cpa2-D-Pal (3) 3-Ser4-N-Me-Tyr5-D-Lys (B) 6-Nle7Arg8-Pro9-Sar10-NH2;Ac-D-Nal (2) 1 -D-Ca 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 Arg 8 -Pro 9 -Sar 10 -NH 2 ;
Ac-D-Nal(2)1-D-Cpa2-D-Pal (3)3-Ser4-N-Me-Tyr5-D-Lys(B)6-Nle7Arg8-Pro9-D-Ala10-NH2;Ac-D-Nal (2) 1 -D-Ca 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 Arg 8 -Pro 9 -D -Ala 10 -NH 2 ;
Ac-D-Nal (2)1-D-Cpa2-D-Pal (3)3-Ser4-N-Me-Tyr5-D-Lys (B) 6-Nle7Lys (iPr) 8-Pro9-Sar10-NH2;Ac-D-Nal (2) 1 -D-Ca 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 Lys (iPr) 8 -Pro 9 -Sar 10 -NH 2 ;
Ac-D-Nal (2) '‘-D-Phe (4-CI) 2-D-Pal (3) 3-Ser4-N-Me-Tyr5-D-Lys (B) 6Nle7-Lys (iPr) 8-Pro9-D-Ala10-NH2;Ac-D-Nal (2) -D-Phe (4-Cl) 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 Nle 7 -Lys (iPr) 8 -Pro 9 -D-Ala 10 -NH 2 ;
Ac-D-Nal (2) X-D-Cpa2-D-Pal (3) 3-Ser4-N-Me-Tyr5-D-Cit6-Nle7-Arg8Pro9-D-Ala10-NH2;Ac-D-Nal (2) X -D-Ca 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Cit 6 -Nle 7 -Arg 8 Dec 9 -D-Ala 10 -NH 2 ;
Ac-D-Nal (2)1-D-Cpa2-D-Pal (3)3-Ser4-N-Me-Tyr5-D-Hci6-Leu7-Arg8Pro9-D-Ala10-NH2;Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Hci 6 -Leu 7 -Arg 8 Dec 9 -D-Ala 10 -NH 2 ;
Ac-D-Nal(2)1-D-Cpa2-D-Pal (3)3-Ser4-N-Me-Tyr5-D-Cit6-Nle7Lys (iPr) 8-Pro9-D-Ala10-NH2;Ac-D-Nal (2) 1 -D-Ca 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Cit 6 -Nle 7 Lys (iPr) 8 -For 9 -D -Ala 10 -NH 2 ;
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-N-Me-Tyr5-D-Hci6-Leu7Lys (iPr) 8-Pro9-D-Ala10-NH2.Ac-D-Nal (2) 1 -D-Ca 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Hci 6 -Leu 7 Lys (iPr) 8 -For 9 -D -Ala 10 -NH 2 .
Podlá ďalšieho aspektu vynálezu sa poskytujú zlúčeniny podlá vynálezu vo forme acetátových, trifluóracetátových alebo pamoátových solí.According to another aspect of the invention, compounds of the invention are provided in the form of acetate, trifluoroacetate or pamoate salts.
Podlá ďalšieho aspektu vynálezu sa zlúčeniny podlá vynálezu môžu používať ako liečivá alebo farmaceutické prípravky.According to a further aspect of the invention, the compounds of the invention may be used as medicaments or pharmaceutical preparations.
Podlá ďalšieho aspektu vynálezu sa poskytujú farmaceutické prípravky obsahujúce aspoň jednu zo zlúčenín podlá vynálezu a obvyklé nosiče a pomocné látky.According to a further aspect of the invention there are provided pharmaceutical compositions comprising at least one of the compounds of the invention and conventional carriers and excipients.
Podľa ďalšieho aspektu vynálezu sa poskytuje spôsob výroby zlúčenín všeobecného vzorca I podľa vynálezu, pri ktorom sa vytvárajú fragmenty zo stavebných zložiek Xxxm s vhodnými chrániacimi skupinami, kde m znamená celé číslo 1 až 10 a Xxx1 je acetylovaný, na tuhom (nerozpustnom) nosiči alebo v roztoku obvyklým spôsobom, potom sa fragmenty na tuhom nosiči zlučujú spájaním segmentov a po ukončení spájania sa zlúčeniny všeobecného vzorca I oddeľujú od nerozpustného nosiča obvyklým spôsobom za amidácie na stavebnej zložke Xxx10.According to a further aspect of the invention there is provided a process for the preparation of compounds of formula I according to the invention, wherein fragments are formed from Xxx m building blocks with suitable protecting groups, wherein m is an integer 1 to 10 and Xxx 1 is acetylated, on a solid (insoluble) carrier. or in solution in a conventional manner, then the fragments on the solid support are pooled by segment joining and upon completion of the bonding, the compounds of formula I are separated from the insoluble support in the usual manner by amidation on component Xxx 10 .
Podľa ďalšieho aspektu vynálezu sa poskytuje použitie zlúčenín podľa vynálezu na výrobu liečiv na liečenie hormonálne závislých nádorov, najmä rakoviny prostaty alebo rakoviny prsníka, a taktiež na liečenie nemalígnych indikácií, ktorých liečenie si vyžaduje hormonálnu supresiu LH-RH.According to a further aspect of the invention there is provided the use of the compounds of the invention for the manufacture of a medicament for the treatment of hormone-dependent tumors, in particular prostate cancer or breast cancer, as well as for the treatment of non-malignant indications whose treatment requires hormone suppression.
Podľa ďalšieho aspektu vynálezu sa poskytuje spôsob výroby farmaceutických prípravkov, pri ktorom sa aspoň jedna zlúčenina podľa jedného z nárokov 1 až 10 zmieša s obvyklými nosičmi a pomocnými látkami a upraví do formy liečiva.According to a further aspect of the invention there is provided a process for the manufacture of pharmaceutical compositions, wherein at least one compound according to any one of claims 1 to 10 is mixed with conventional carriers and excipients and formulated as a medicament.
Podľa ďalšieho aspektu vynálezu sa poskytuje spôsob liečenia hormonálne závislých nádorov, najmä rakoviny prostaty, rakoviny prsníka alebo myómu maternice, a taktiež nemalígnych indikácií, ktorých liečenie si vyžaduje hormonálnu supresiu LH-RH, ako je napríklad endometrióza, benígna hyperplázia prostaty (BPH), a taktiež sa poskytuje spôsob liečenia porúch plodnosti u samíc alebo samcov cicavcov, najmä u ľudí, prostredníctvom podávania účinnej dávky aspoň jednej zlúčeniny podľa vynálezu.According to a further aspect of the invention there is provided a method of treating hormone-dependent tumors, particularly prostate cancer, breast cancer or uterine fibroid, as well as non-malignant indications whose treatment requires hormone suppression of LH-RH such as endometriosis, benign prostatic hyperplasia (BPH), and also provided is a method of treating fertility disorders in female or male mammals, particularly humans, by administering an effective dose of at least one compound of the invention.
Zlúčeniny podlá vynálezu sa môžu použiť na liečenie hormonálne závislých nádorov, najmä rakoviny prostaty, rakoviny prsníka alebo myómu maternice, a taktiež na liečenie nemalígnych indikácií, ktorých liečenie si vyžaduje hormonálnu supresiu LH-RH, ako je napríklad endometrióza, benígna hyperplázia prostaty (BPH). Ďalej sa môžu použiť na liečenie porúch plodnosti žien alebo mužov, napríklad na riadenú ovariálnu superstimuláciu v rámci umelého oplodnenia (in vitro fertilizácia). Na to sa obvykle zmiešajú o sebe známym spôsobom s obvyklými nosičmi a pomocnými látkami a upravia do formy liečiva.The compounds of the invention may be used for the treatment of hormone-dependent tumors, in particular prostate cancer, breast cancer or uterine fibroid, as well as for the treatment of non-malignant indications for which hormonal suppression of LH-RH such as endometriosis, benign prostatic hyperplasia (BPH) . In addition, they can be used to treat fertility disorders in women or men, for example, for controlled ovarian superstimulation in artificial insemination (in vitro fertilization). To this end, they are usually mixed in conventional manner with the usual carriers and auxiliaries and formulated as medicaments.
Syntéza zlúčenín vzorca I sa môže uskutočňovať nielen klasickou kondenzáciou fragmentov alebo syntézou na tuhom nosiči podľa Merrifielda s navzájom za sebou nasledujúcim spájaním použitím D-lyzínu už acylovaného karboxylovou kyselinou všeobecného vzorca R* 1 * * *-COOH v bočnom reťazci, ale aj reakciou stavebnej zložky dekapeptidu s príslušnými karboxylovými kyselinami amidovým zlučovaním v bočnom reťazci D-lyzínu6. Teda zavedenie skupiny R1-CO- sa môže uskutočňovať v troch rôznych bodoch spôsobu; pred kondenzáciou jednotlivých stavebných zložiek na peptid, po zabudovaní lyzínu alebo ornitínu do peptidového reťazca, ale aj pred kondenzáciou nasledujúcej stavebnej zložky alebo po kondenzácii všetkých stavebných zložiek.The synthesis of the compounds of formula I can be carried out not only by classical fragment condensation or solid-phase synthesis according to Merrifield with successive coupling using D-lysine already acylated with a carboxylic acid of the formula R * 1 * * * -COOH in the side chain, the decapeptide component with the corresponding carboxylic acids by amide coupling in the side chain of D-lysine 6 . Thus, the introduction of the group R 1 -CO- can be carried out at three different process points; before condensation of the individual building components to the peptide, after incorporation of lysine or ornithine into the peptide chain, but also before condensation of the next building component or after the condensation of all building components.
Zlúčeniny vzorca I sa syntetizujú známymi metódami, ako je napríklad technika na tuhom nosiči, čiastočná technika na tuhom nosiči (takzvaná kondenzácia fragmentov) alebo klasickým spájaním v roztoku (viď M. Bodanszky, „Principles of Peptide Synthesis, Springer Verlag 1984).Compounds of formula I are synthesized by known methods, such as a solid support technique, a partial solid support technique (the so-called fragment condensation), or classical solution coupling (see M. Bodanszky, "Principles of Peptide Synthesis, Springer Verlag 1984).
Metódy syntézy na tuhom nosiči sú opísané napríklad v učebnici „Solid Phase Peptide Synthesis, J. M. Stewart and J. D. Young, Pierce Chem. Company, Rockford, III, 1984, a v práci G. Barany and R. B. Merrifield „The Peptides, kap.Methods for solid support synthesis are described, for example, in the textbook "Solid Phase Peptide Synthesis" by J. M. Stewart and J. D. Young by Pierce Chem. Company, Rockford, III, 1984, and in G. Barany and R. B. Merrifield, "The Peptides, Chap.
1, str. 1-285, 1979, Academic Press Inc. Klasické syntézy v roztoku sú podrobne opísané v rozprave „Methoden der1, p. 1-285 (1979), Academic Press Inc. Classical solutions in solution are described in detail in the 'Methoden der
Organischen Chemie (Houben-Weyl), Synthese von Peptiden. E.Organischen Chemie (Houben-Weyl), Synthes von Peptiden. E.
Wunsch (vydavateľ) 1974, Georg Thieme Verlag, Stuttgart, SRN.Wunsch (Publisher) 1974, Georg Thieme Verlag, Stuttgart, Germany.
Postupná tvorba sa uskutočňuje napríklad tak, že sa najskôr aminokyselina s karboxy-koncom, ktorej a-aminoskupina je chránená, kovalentne viaže na bežný nerozpustný nosič určený nezávisle tento účel, chrániaca skupina a-aminoskupiny tejto aminokyseliny sa odštiepi, na takto získanú voľnú aminoskupinu sa cez svoju karboxylovú skupinu naviaže ďalšia chránená aminokyselina a týmto spôsobom sa postupne v správnom slede zlučujú ostatné aminokyseliny syntetizovaného peptidu a po zlúčení všetkýchaminokyselín sa vytvorený peptid oddelí od nosiča a poprípade sa oddelia ďalšie existujúce chrániace skupiny vedľajších funkčných skupín. Postupná kondenzácia sa uskutočňuje syntézou príslušných, obvyklým spôsobom chránených aminokyselín bežným postupom.The sequential formation is carried out, for example, by first covalently bonding the carboxy-terminal amino acid of which the .alpha.-amino group is protected to a conventional insoluble carrier designed independently for this purpose, the .alpha.-amino protecting group of this amino acid is cleaved; through its carboxyl group, it binds another protected amino acid, and in this manner the other amino acids of the synthesized peptide are sequentially combined in the correct sequence, and after all amino acids have been combined, the peptide formed is separated from the carrier and optionally further existing protecting groups of side functional groups are separated. The sequential condensation is carried out by synthesis of the corresponding, normally protected amino acids by a conventional procedure.
Vzájomné naviazanie jednotlivých aminokyselín sa uskutočňuje obvyklými metódami určenými na tento účel.The binding of the individual amino acids is carried out by conventional methods designed for this purpose.
Do úvahy prichádzajú najmä:In particular:
• metóda symetrických anhydridov v prítomnosti dicyklohexylkarbodiimidu alebo diizopropylkarbodiimidu (DCC), DIC);The method of symmetrical anhydrides in the presence of dicyclohexylcarbodiimide or diisopropylcarbodiimide (DCC), DIC);
• karbodiimidová metóda všeobecne;The carbodiimide method in general;
-· karbodiimid-hydroxybenzotriazolová metóda (viď TheCarbodiimide - hydroxybenzotriazole method (see
Peptides, zv. 2, Ed. E. Gross and J. Meienhofer).Peptides, Vol. 2, Ed. E. Gross and J. Meienhofer).
Pri spájaní fragmentov sa využíva prednostne azidové zlučovanie prebiehajúce bez racemizácie alebo DCC-l-hydroxybenzotriazolová, poprípade DCC-3-hydroxy-4-oxo-3,4-dihydro1,2,3-benzotriazínová metóda. Môžu sa použiť aj aktivované estery fragmentov.The coupling of the fragments preferably uses the azide coupling without racemization or the DCC-1-hydroxybenzotriazole or DCC-3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine method. Activated fragment esters may also be used.
Na postupnú kondenzáciu aminokyselín sú vhodné najmä aktivované estery N-chránených aminokyselín, ako je napríklad N-hydroxysukcínimidester alebo 2,4,5-trichlórfenylester. Aminolýza sa dá veľmi dobre katalyzovať N-hydroxyzlúčeninami, ktoré majú približne kyslosť kyseliny octovej, ako je napríklad 1-hydroxybenzotriazol.Activated esters of N-protected amino acids, such as the N-hydroxysuccinimide ester or 2,4,5-trichlorophenyl ester, are particularly suitable for the sequential condensation of amino acids. Aminolysis is very well catalyzed by N-hydroxy compounds having approximately acetic acid, such as 1-hydroxybenzotriazole.
Ako intermediárne chrániace skupiny aminoskupiny sú vhodné dehydrogenačné skupiny, ako je napríklad benzyloxykarbonylový zvyšok (= zvyšok Z) alebo slabo kyslé odštiepitelné skupiny. Ako chrániace skupiny pre a-aminoskupiny prichádzajú do úvahy napríklad: terciárne butyloxykarbonylové skupiny, fluorenylmetyloxykarbonylové skupiny, karboxybenzoxyskupiny, poprípade karbobenztioskupiny (poprípade s p-bróm- alebo p-nitrobenzylovým zvyškom), trifluóracetylová skupina, ftalylový zvyšok, o-nitrofenoxyacetylová skupina, tritylová skupina, p-toluénsulfonylskupina, benzylskupina, benzylové zvyšky substituované na benzénovom jadre (p-bróm- alebo p-nitrobenzylový zvyšok) a α-fenyletylový zvyšok. Za týmto účelom sa taktiež odkazuje na práce Jesse P. Greenstein and Milton Winitz, Chemistry of Amino Acids, New York 1961, John Wiley and Sons, Inc., zväzok 2, napríklad strana 883 a ďalšie, „Principles of Peptide Synthesis, Springer Verlag 1984, „Solid Phase Peptide Synthesis, J. M. Stewart and J. D. Young, Pierce Chem. Company, Rockford, III, 1984, G. Barany and R. B. Merrifield „The Peptides, kap. 1, str. 1-285, 1979, Academic Press Inc.Suitable intermediate amino protecting groups are dehydrogenation groups such as, for example, the benzyloxycarbonyl radical (= radical Z) or weakly acid cleavable groups. Suitable protecting groups for .alpha.-amino groups include, for example: tertiary butyloxycarbonyl, fluorenylmethyloxycarbonyl, carboxybenzoxy, optionally carbobenzthio (optionally with p-bromo- or p-nitrobenzyl radical), trifluoroacetyl radical, trifluoroacetyl radical, trifluoroacetyl radical, trifluoroacetyl radical , p-toluenesulfonyl, benzyl, benzyl radicals substituted on the benzene ring (p-bromo- or p-nitrobenzyl) and α-phenylethyl. For this purpose reference is also made to Jesse P. Greenstein and Milton Winitz, Chemistry of Amino Acids, New York 1961, John Wiley and Sons, Inc., Volume 2, for example page 883 et seq., "Principles of Peptide Synthesis, Springer Verlag" 1984, " Solid Phase Peptide Synthesis, " JM Stewart & JD Young, Pierce Chem. Company, Rockford, III, 1984, G. Barany and R. B. Merrifield " The Peptides, Chap. 1, p. 1-285 (1979), Academic Press Inc.
_a taktiež The Peptides. zväzok 2f Ed._EL._Gross and-ÓLMaienhofer, Academic Press, New York. Tieto chrániace skupiny prichádzajú do úvahy principiálne taktiež na ochranu ďalších funkčných vedľajších skupín (skupín OH, skupín NH2) príslušných aminokyselín.and also The Peptides. Volume 2 f Ed._EL._Gross and-ÓLMaienhofer, Academic Press, New York. These protecting groups are suitable in principle also for the protection of further functional side groups (OH groups, NH2 groups) of the relevant amino acids.
Existujúce hydroxyskupiny (serín, treonín) sa chránia prednostne pomocou benzylových a podobných skupín. Ďalšie aminoskupiny, ktoré nie sú v polohe α (napríklad aminoskupiny v polohe ω, guanidínová skupina arginínu) sa prednostne chránia ortogonálne.Existing hydroxy groups (serine, threonine) are preferably protected with benzyl and the like groups. Other amino groups that are not in the α position (e.g. amino groups in the ω position, guanidine group of arginine) are preferably protected orthogonally.
Jednotlivé stavebné zložky aminokyselín sú s výnimkou lyzínu modifikovaného skupinou R1-CO- komerčne dostupné.The individual amino acid builders are commercially available, with the exception of the R 1 -CO- modified lysine.
Možný priebeh spôsobu výroby lyzínu modifikovaného skupinou R1-CO- je nasledujúci:The possible course of a process for the production of lysine modified by the group R 1 -CO- is as follows:
1. α-Karboxylová skupina sa vhodne chráni, napríklad esterifikáciou.1. The α-carboxyl group is suitably protected, for example by esterification.
2. ε-Aminoskupina sa chráni, napríklad skupinou Z.2. The ε-amino group is protected, for example by a Z group.
3. α-Aminoskupina sa chráni tak (napríklad skupina Boe), že sa poskytne selektivita vzhľadom na neskoršie odštiepenie chrániacich skupín aminoskupiny.3. The .alpha.-amino group is protected (e.g., Boe) to provide selectivity with respect to the later cleavage of the amino protecting groups.
4. Skupina Z na ε-aminoskupine sa odštiepi.4. The Z group on the ε-amino group is cleaved off.
5. Na ε-aminoskupinu sa naviaže požadovaná skupina R1-CO-.5. The desired R 1 -CO- group is bound to the ε-amino group.
6. Chrániaca skupina na α-aminoskupine sa odštiepi.6. The α-amino protecting group is cleaved off.
7. α-Aminoskupina sa poprípade reverzibilne derivatizuje, napríklad skupinou Z.7. The α-amino group is optionally reversibly derivatized, for example by the Z group.
Na zavedenie skupiny R1-CO- reakciou aminoskupiny lyzínu s príslušnou karboxylovou kyselinou, poprípade derivátom karboxylovej kyseliny, prichádzajú do úvahy principiálne rovnaké spôsoby, ako sa opisujú vyššie pre zlučovanie aminokyselín. Obzvlášť prednostná je však kondenzácia použitím karbodiimidu, napríklad l-etyl-3-(3-dimetylaminopropyl)karbodiimidu a 1-hydroxybenzotriazolu.For the introduction of the group R 1 -CO- the reaction of the amino group of lysine with appropriate carboxylic acid or carboxylic acid derivative, there are suitable in principle the same method as described above to couple the amino acids. However, condensation using carbodiimide, for example 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 1-hydroxybenzotriazole, is particularly preferred.
Reakcia zlučovania aminokyselín sa uskutočňuje v obvyklom indiferentnom rozpúšťadle alebo suspendačnom prostredí určenom na tento účel (napríklad v dichlórmetáne), pričom sa poprípade na zlepšenie rozpustnosti môže pridať dimetylformamid.The amino acid coupling reaction is carried out in a conventional, indifferent solvent or suspending medium for this purpose (e.g. dichloromethane), whereby dimethylformamide may optionally be added to improve solubility.
Ako syntetické nosné materiály prichádzajú do úvahy nerozpustné polyméry, ako je napríklad v organickom rozpúšťadle napučiavajúca polystyrénová živica v granulovanej forme (napríklad kopolymér polystyrénu a 1% divinylbenzénu). Vytváranie chráneného dekapeptidamidu na metylbenzhydryl12 amínovej živici (živica MBHA, t.j. polystyrénová živica s metylbenzhydrylamínovými skupinami), ktorá poskytuje požadovanú C-koncovú amidovú funkčnú skupinu peptidu po H.Fodštiepení od nosiča, sa môže uskutočňovať podlá nasledujúcej blokovej schémy:Suitable synthetic carrier materials are insoluble polymers, such as an organic solvent swelling polystyrene resin in granular form (for example a copolymer of polystyrene and 1% divinylbenzene). The formation of the protected decapeptidamide on the methylbenzhydryl12 amine resin (MBHA resin, i.e. polystyrene resin with methylbenzhydrylamine groups), which provides the desired C-terminal amide functional group of the peptide after H. Cleavage from the support may be carried out according to the following flow chart:
Bloková schémaBlock scheme
Protokol syntézy peptidov použitím aminokyselín chránených BoePeptide synthesis protocol using Boe protected amino acids
Να-Boc-chránené aminokyseliny sa zlučujú obvyklým spôsobom v trojnásobnom molovom nadbytku v prítomnosti diizopropylkarbodiimidu (DIC) a 1-hydroxybenzotriazolu (HOBt) v CH2CI2/DMF v priebehu 90 minút a chrániaca skupina Boe sa odštiepi polhodinovým pôsobením 50% kyseliny trifluóroctovej (TFA) v CH2CI2. Na kontrolu úplného zreagovania môže slúžiť chloranilový test podľa Christensena a Kaiserov ninhydrínový test. Zvyšky volných funkčných aminoskupín sa blokujú acetyláciou v päťnásobnom nadbytku acetylimidazolu v CH2C12. Sled reakčných stupňov výstavby peptidu na živici vyplýva z blokovej schémy. Za účelom odštiepenia peptidov naviazaných na živici sa daný konečný produkt syntézy na tuhom nosiči vysuší vo vákuu nad P2O5 a spracuje v 500-násobnom nadbytku HF/anizolu (10:1, objem:objem) pri 0 °C v priebehu 60 minút.The .alpha.-Boc-protected amino acids are combined in a conventional manner in triple molar excess in the presence of diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) in CH2Cl2 / DMF over 90 minutes and the Boe protecting group is cleaved for half an hour with 50% trifluoroacetic acid. in CH2Cl2. Christensen's chloranil test and Kaiser's ninhydrin test can be used to control complete reaction. Radicals free of functional amino groups were blocked by acetylation in a five fold excess of acetylimidazole in CH 2 C1 2nd The sequence of the reaction steps of peptide-resin construction follows from the flow chart. In order to cleave the resin-bound peptides, the final solid support synthesis product is dried under vacuum over P2O5 and treated in a 500-fold excess of HF / anisole (10: 1, v / v) at 0 ° C for 60 minutes.
Po oddestilovaní HF a anizolu vo vákuu vzniknú peptidy vo forme bielych tuhých látok zmiešaním s bezvodým etyléterom. Oddelenie od polymérneho nosiča sa uskutočňuje premývaním 50% vodným roztokom kyseliny octovej. Opatrným zahustením roztokov obsahujúcich kyselinu octovú vo vákuu sa môžu získať peptidy vo forme vysoko viskóznych olejov, ktoré sa po pridaní absolútneho éteru za chladu menia na biele tuhé látky.Upon distillation of HF and anisole in vacuo, the peptides are formed as white solids by mixing with anhydrous ethyl ether. Separation from the polymeric support is accomplished by washing with 50% aqueous acetic acid. By carefully concentrating the acetic acid-containing solutions in vacuo, the peptides can be obtained in the form of highly viscous oils, which, upon addition of absolute ether, turn into white solids when cold.
Ďalšie čistenie sa uskutočňuje rutinnými metódami preparatívnej vysokotlakovej kvapalinovej chromatografie (HPLC).Further purification is accomplished by routine preparative high pressure liquid chromatography (HPLC) methods.
Premena peptidov na ich adičné soli s kyselinami sa môže uskutočniť ich reakciou s kyselinami o sebe známym spôsobom. Voľné peptidy sa zasa môžu získať reakciou svojich adičných solí s kyselinami so zásadami. Pamoáty peptidov sa môžu vytvoriť reakciou solí kyseliny trifluóroctovej (TFA-solí) peptidu s voľnou kyselinou pamoovou (embónovou, l,l'-metylénbis-(2-hydroxy-3-naftoovou)) alebo s príslušnou disodnou soľou kyseliny pamoovej. Na to sa TFA-soľ peptidu zmieša vo vodnom roztoku s roztokom pamoátu disodného v polárnom aprotónovom prostredí, prednostne v dimetylacetamide, a vzniknutá svetložltá zrazenina sa izoluje.Conversion of peptides to their acid addition salts can be accomplished by reaction with acids in a manner known per se. The free peptides can in turn be obtained by reacting their acid addition salts with bases. The peptide pamoates can be formed by reacting the trifluoroacetic acid (TFA) salts of the peptide with free pamoic acid (embonic, 1,1'-methylenebis- (2-hydroxy-3-naphthoic)) or the appropriate pamoic acid disodium salt. To this end, the TFA salt of the peptide is mixed in aqueous solution with a solution of pamoate disodium in a polar aprotone medium, preferably in dimethylacetamide, and the resulting pale yellow precipitate is isolated.
Nasledujúce príklady slúžia na objasnenie vynálezu bez toho, aby ho obmedzovali.The following examples serve to illustrate the invention without limiting it.
Príklady uskutočnenia vynálezuDETAILED DESCRIPTION OF THE INVENTION
Príklad 1 (D-68968):Example 1 (D-68968):
Ac-D-Nal (2) 1-D-Cpa2-D-Pal (3) 3-Ser4-N-Me-Tyr5-D-Lys (B) 6-Nle7Arg8-Pro9-Sar10-NH2 Ac-D-Nal (2) 1 -D-Ca 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 Arg 8 -Pro 9 -Sar 10 -NH 2
Syntéza dekapeptidu sa uskutočňovala na polymérnom nosiči s hustotou nasýtenia 0,55 mmol/g (aminometylsubstituovaná živica, chrániaca skupina Fmoc, typ D-1675, firma Bachem). Lyzín sa naviazal vo forme Fmoc-D-Lys(Boe)OH, chrániace skupiny Fmoc sa odštiepili pomocou 20% piperidínu v DMF. Po súčasnom odštiepení všetkých chrániacich skupín v bočnom reťazci a uvoľnení od polymérneho nosiča sa izolovaný surový peptid čistil prostredníctvom preparatívnej HPLC. Po lyofilizácii sa získal 98,5% dekapeptid.The decapeptide synthesis was carried out on a polymeric carrier with a saturation density of 0.55 mmol / g (aminomethyl-substituted Fmoc protecting group, type D-1675, from Bachem). Lysine was bound as Fmoc-D-Lys (Boe) OH, the Fmoc protecting groups were cleaved with 20% piperidine in DMF. After simultaneous cleavage of all side chain protecting groups and release from the polymeric carrier, the isolated crude peptide was purified by preparative HPLC. After lyophilization, 98.5% decapeptide was obtained.
Substitúcia na ε-dusíku D-lyzínu pôsobením kyseliny 4—(4— -aminofenyl)amino-1,4-dioxobutánovej sa uskutočnila pomocou PyBop v DMF za prídavku DIPEA. Čistenie izolovaného surového peptidu sa uskutočňovalo prostredníctvom preparatívnej HPLC. Následná lyofilizácia poskytla približne 99% produkt (trifluóracetát) sumárneho vzorca C82H106C1NÍ9O15 s príslušným FAB-MS 1633 (M+H) (vypoč. 1631,78096).Substitution on the ε-nitrogen of D-lysine with 4- (4-aminophenyl) amino-1,4-dioxobutanoic acid was performed with PyBop in DMF with the addition of DIPEA. Purification of the isolated crude peptide was carried out by preparative HPLC. Subsequent lyophilization gave approximately 99% product (trifluoroacetate) of the general formula C82H106ClN9O15 with the corresponding FAB-MS 1633 (M + H) (calc. 1631.78096).
Príklad 2 (D-68969):Example 2 (D-68969):
Ac-D-Nal (2)1-D-Cpa2-D-Pal(3)3-Ser4-N-Me-Tyr5-D-Lys(B)6-Nle7Arg8-Pro9-D-Ala10-NH2 Ac-D-Nal (2) 1 -D-Ca 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 Arg 8 -Pro 9 -D -Ala 10 -NH 2
Syntéza dekapeptidu sa uskutočňovala na polymérnom nosiči s hustotou nasýtenia 0,55 mmol/g (aminometylsubstituovaná živica, chrániaca skupina Fmoc, typ D-1675, firma Bachem). Lyzín sa naviazal vo forme Fmoc-D-Lys(Boe)-OH, chrániace skupiny Fmoc sa odštiepili pomocou 20% piperidínu vThe decapeptide synthesis was carried out on a polymeric carrier with a saturation density of 0.55 mmol / g (aminomethyl-substituted Fmoc protecting group, type D-1675, from Bachem). Lysine was bound as Fmoc-D-Lys (Boe) -OH, the Fmoc protecting groups were cleaved with 20% piperidine in
DMF. Po súčasnom odštiepení všetkých chrániacich skupín v bočnom reťazci a uvoľnení od polymérneho nosiča izolovaný surový peptid s obsahom približne 71 % (HPLC) bez čistenia ďalej reagoval.DMF. After cleavage of all side-chain protecting groups and release from the polymeric support, the isolated crude peptide of about 71% (HPLC) was further reacted without purification.
Substitúcia v bočnom reťazci D-lyzínu pôsobením kyseliny 4-(4-aminofenyl)amino-1,4-dioxobutánovej sa uskutočnila pomocou PyBop v DMF za prídavku DIPEA. Izolovaný surový peptid sa čistil prostredníctvom preparátívnej HPLC. Po následnej lyofilizácii sa získal 98,8% produkt (trifluóracetát) sumárneho vzorca C82H106CIN19O15 s príslušným FAB-MS 1633 (M+H) (vypoč. 1631,78096).Substitution in the D-lysine side chain with 4- (4-aminophenyl) amino-1,4-dioxobutanoic acid was performed with PyBop in DMF with the addition of DIPEA. The isolated crude peptide was purified by preparative HPLC. Subsequent lyophilization gave 98.8% product (trifluoroacetate) of the general formula C82H106CIN19O15 with the corresponding FAB-MS 1633 (M + H) (calc. 1631.78096).
Príklad 3 (D-68971):Example 3 (D-68971):
Ac-D-Nal (2) 1-D-Cpa2-D-Pal (3)3-Ser4-N-Me-Tyr5-D-Lys(B)6-Nle7Lys (iPr) 8-Pro9-Sar10-NH2 Ac-D-Nal (2) 1 -D-Ca 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 Lys (iPr) 8 -Pro 9 -Sar 10 -NH 2
Syntéza dekapeptidu sa uskutočňovala na polymérnom nosiči s hustotou nasýtenia 0,55 mmol/g (aminometylsubstituovaná živica, chrániaca skupina Fmoc, typ D-1675, firma Bachem). Lyzín sa naviazal vo forme Fmoc-D-Lys(Boe)-OH,_ chrániace skupiny Fmoc sa odštiepili pomocou 20% piperidínu v DMF. Po súčasnom odštiepení všetkých chrániacich skupín v bočnom reťazci a uvoľnení od polymérneho nosiča sa izolovaný surový peptid (obsah približne 59 %, HPLC) čistil prostredníctvom preparatívnej HPLC. Po lyofilizácii sa získal 95% dekapeptid.The decapeptide synthesis was carried out on a polymeric carrier with a saturation density of 0.55 mmol / g (aminomethyl-substituted Fmoc protecting group, type D-1675, from Bachem). Lysine was bound as Fmoc-D-Lys (Boe) -OH, Fmoc protecting groups were cleaved with 20% piperidine in DMF. After concomitant cleavage of all side chain protecting groups and release from the polymeric support, the isolated crude peptide (approximately 59% content, HPLC) was purified by preparative HPLC. After freeze-drying, a decapeptide of 95% was obtained.
Substitúcia D-lyzínu v bočnom reťazci pôsobením kyseliny 4-(4-aminofenyl)amino-1,4-dioxobutánovej sa uskutočnila pomocou PyBop v DMF za prídavku DIPEA. Čistenie izolovaného surového peptidu sa uskutočňovalo prostredníctvom preparatívnej HPLC. Po následnej lyofilizácii sa získal 96,6% produkt (trifluóracetát) sumárneho vzorca C85Hi12ClNi70i5 s príslušným FAB-MS 1648 (M+H) (vypoč. 1645,8218).Substitution of D-lysine in the side chain with 4- (4-aminophenyl) amino-1,4-dioxobutanoic acid was performed with PyBop in DMF with the addition of DIPEA. Purification of the isolated crude peptide was carried out by preparative HPLC. Subsequent lyophilization gave 96.6% of the product (trifluoroacetate) of the general formula C 85 H 12 ClN 17 O 15 with the corresponding FAB-MS 1648 (M + H) (calc. 1645.8218).
Príklad 4 (D-68987)Example 4 (D-68987)
Ac-D-Nal (2)1-D-Phe(4-CI)2-D-Pal(3) 3-Ser4-N-Me-Tyr5-D-Lys (B) 6Nle7-Lys (iPr) 8-Pro9-D-Ala10-NH2 Ac-D-Nal (2) 1 -D-Phe (4-Cl) 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 Nle 7 -Lys ( iPr) 8 -Pro 9 -D-Ala 10 -NH 2
Syntéza D-Lys-6-nesubsitutovaného dekapeptidu sa uskutočňovala na 9,09 g polymérneho nosiča s hustotou nasýtenia 0,55 mmol/g. Lyzín6 sa naviazal vo forme Fmoc-DLys(Boc)-OH. Po odštiepení živice sa izolovalo 8,15 g surového peptidu. Čistenie surového peptidu sa uskutočňovalo prostredníctvom preparatívnej HPLC.The synthesis of D-Lys-6-unsubstituted decapeptide was carried out on 9.09 g of polymer support with a saturation density of 0.55 mmol / g. Lysine 6 was coupled as Fmoc-DLys (Boc) -OH. After cleavage of the resin, 8.15 g of crude peptide was isolated. Purification of the crude peptide was performed by preparative HPLC.
Substitúcia D-lyzínu v bočnom reťazci pôsobením kyseliny 4-(4-aminofenyl)amino-1,4-dioxobutánovej sa uskutočnila pomocou PyBop v DMF za prídavku DIPEA. Izolovaný surový peptid sa čistil prostredníctvom preparatívnej HPLC. Po následnej lyofilizácii sa získal 94,6% produkt (trifluóracetát) sumárneho vzorca C85Hii2ClN170i5 s príslušným FAB-MS 1646,8 (M+H); (vypoč. 1645,82).Substitution of D-lysine in the side chain with 4- (4-aminophenyl) amino-1,4-dioxobutanoic acid was performed with PyBop in DMF with the addition of DIPEA. The isolated crude peptide was purified by preparative HPLC. Subsequent lyophilization gave 94.6% of the product (trifluoroacetate) of the general formula C 85 H 12 ClN 17 O 15 with the corresponding FAB-MS 1646.8 (M + H); (calc. 1645.82).
Príklad 5:Example 5:
Ac-D-Nal (2)1-D-Cpa2-D-Pal(3)3-Ser4-N-Me-Tyr5-D-Cit6-Nle7-Arg8Pro9-D-Ala10-NH2 Ac-D-Nal (2) 1 -D-Ca 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Cit 6 -Nle 7 -Arg 8 Dec 9 -D-Ala 10 -NH 2
Príklad 6:Example 6:
Ac-D-Nal (2)1-D-Cpa2-D-Pal(3) 3-Ser4-N-Me-Tyr5-D-Hci6-Leu7-Arg8Pro9-D-Ala10-NH2 Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Hci 6 -Leu 7 -Arg 8 Dec 9 -D-Ala 10 -NH 2
Príklad 7:Example 7:
Ac-D-Nal (2) ^D-Cpa^D-Pal (3) 3-Ser4-N-Me-Tyr5-D-Cit6-Nle7Lys (iPr) 8-Pro9-D-Ala10-NH2 Ac-D-Nal (2) -D-Ca-D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Cit 6 -Nle 7 Lys (iPr) 8 -For 9 -D-Ala 10 -NH 2
Príklad 8:Example 8:
Ac-D-Nal (2) 1-D-Cpa2-D-Pal (3) 3-Ser4-N-Me-Tyr5-D-Hci6-Leu7Lys (iPr) 8-Pro9-D-Ala10-NH2 Ac-D-Nal (2) 1 -D-Ca 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Hci 6 -Leu 7 Lys (iPr) 8 -For 9 -D -Ala 10 -NH 2
Všeobecné pracovné postupy výroby peptidov podlá príkladov 5 až 8:The general procedures for producing peptides according to Examples 5 to 8:
Dekapeptidy sa môžu vytvárať Merrifieldovou syntézou na tuhom nosiči (SPPS) a taktiež klasickou kondenzáciou fragmentov v roztoku. Vytváranie peptidovej sekvencie na polymérnom nosiči je z ekonomických dôvodov prednostné a môže sa uskutočniť principiálne voliteľne podľa 1) Boe- alebo 2) Fmoc-stratégie; podlá toho sa môže na C-koncové naviazanie D-alanínu použiť buď metylbenzhydrylamínová živica (pri 1) alebo Fmoc-2,4-dimetoxy-4'-(karboxymetyloxy)benzhydrylamínová živica (pri 2).Decapeptides can be formed by Merrifield solid support synthesis (SPPS) as well as by classical condensation of fragments in solution. The formation of the peptide sequence on a polymeric carrier is preferred for economic reasons and can be performed in principle optionally according to 1) Boe- or 2) Fmoc-strategy; accordingly, either the methylbenzhydrylamine resin (at 1) or the Fmoc-2,4-dimethoxy-4 '- (carboxymethyloxy) benzhydrylamine resin (at 2) can be used for the C-terminal binding of D-alanine.
Syntéza na tuhom nosiči, Merrifieldov spôsob:Solid support synthesis, Merrifield method:
Dekapeptidy sa syntetizujú za štandardizovaných reakčných podmienok (bloková schéma, tabulka 1) na syntézu na tuhom nosiči podľa Fmoc-stratégie použitím 5 gramov polymérneho nosiča Fmoc-2,4-dimetoxy-4'-(karboxymetyloxy)benzhydrylamínovej živice od firmy Bachem D1675, hustota nasýtenia približne 0,55 mmol/g, veľkosť zŕn 200 až 400 mesh.Decapeptides are synthesized under standardized reaction conditions (flow chart, Table 1) for solid support synthesis according to Fmoc strategy using 5 grams of polymer support Fmoc-2,4-dimethoxy-4 '- (carboxymethyloxy) benzhydrylamine resin from Bachem D1675, density 0.55 mmol / g, 200 to 400 mesh.
Postupné vytváranie sekvencie na živici sa uskutočňuje pomocou Να-Fmoc-chránených aminokyselín, podlá nasledujúcej blokovej schémy:Sequential sequencing on the resin is accomplished by Να-Fmoc-protected amino acids, according to the following block diagram:
Tabulka 1Table 1
(* podlá T. Christensena, Acta Chem. Scand. B 33, 163-166, 1313)(* according to T. Christensen, Acta Chem. Scand. B 33, 163-166, 1313)
Reakčné parametre typické pre postup (opakovanej) syntézy dekapeptidov na tuhom nosiči podlá vyššie uvedenej schémy:Reaction parameters typical for the (repeated) solid support synthesis process of decapeptides according to the above scheme:
- odštiepenie chrániacej skupiny Fmoc pomocou 20% piperidínu v DMF, 2x5 min pri teplote miestnosti (stupeň 2);cleavage of the Fmoc protecting group with 20% piperidine in DMF, 2x5 min at room temperature (step 2);
- zlučovanie v trojnásobnom molovom nadbytku Fmocaminokyselín pomocou diizopropylkarbodiimidu (DIC) v prítomnosti hydroxybenzotriazolu (HOBt) (stupeň 8);combining in a triple molar excess of Fmocamino acids with diisopropylcarbodiimide (DIC) in the presence of hydroxybenzotriazole (HOBt) (step 8);
- C-koncové odštiepenie polymérneho nosiča vrátane odstránenia chrániacich skupín v bočnom reťazci aminokyselín pomocou kyseliny trifluóroctovej (TFA).C-terminal cleavage of the polymeric carrier including removal of the side chain amino acid protecting groups with trifluoroacetic acid (TFA).
Po odštiepení polymérneho nosiča vzniká pri použití 5 gramov živice približne 5 až 6 gramov surovej zmesi peptidov s obsahom približne 70 až 80 % požadovanej zložky. Táto sa získa následnou preparatívnou chromatografiou HPLC. Prepara.tívne čistenie dekapeptidov pomocou HPLC; podmienky chromatografie :After cleavage of the polymeric carrier, about 5 to 6 grams of crude peptide mixture containing about 70 to 80% of the desired component is formed using 5 grams of resin. This was obtained by subsequent preparative HPLC. Preparative decapeptide purification by HPLC; conditions of chromatography:
Preparatívna HPLC, firma Shimadzu, kolóna Dynamax RP18, 12 μπι, 30 nm, L = 250 mm, ID = 41,4 mmPreparative HPLC, Shimadzu, Dynamax RP18 column, 12 µπ, 30 nm, L = 250 mm, ID = 41.4 mm
Gradientový systém s časovým programom, 40 % B -> 90 % B, 50 minGradient system with time program, 40% B -> 90% B, 50 min
Eluent A: 970 ml H2O + 30 ml CH3CN + 1 ml CF3COOHEluent A: 970 mL H 2 O + 30 mL CH 3 CN + 1 mL CF 3 COOH
Eluent B: 300 ml H2O + 700 ml CH3CN + 1 ml CF3COOHEluent B: 300 mL H 2 O + 700 mL CH 3 CN + 1 mL CF 3 COOH
UV-detekcia, λ = 220 nm, prietoková rýchlosť 60 ml/min.UV detection, λ = 220 nm, flow rate 60 ml / min.
Získané frakcie sa zahustia vo vákuu a lyofilizujú. Dekapeptidy vznikajú vo forme ľahkého bezfarebného materiálu. Premena na acetátovú soľnú formu požadovanú pre farmakologický vývoj sa nslaitočňn jo nánlodno pm'strprinir.tvnni chromatografickej iónovej výmeny.The fractions obtained are concentrated in vacuo and lyophilized. The decapeptides are formed in the form of a light colorless material. The conversion to the acetate salt form required for pharmacological development is practiced neatly. chromatographic ion exchange.
Skúšky biologickej účinnosti:Biological activity tests:
Zlúčeniny vzorca I podľa vynálezu sa skúmali z hľadiska ich väzby k receptoru. Tento spôsob sa úzko opiera o spôsob opísaný v práci Beckers et al., Eur. J. Biochem. 231, 535-543 (1995). Podľa vyššie zverejnenej syntézy získaný cetrorelix sa j ódu j e. pôsobením [125I] (Amersham; špecifická aktivita 80,5 Bq/fmol) použitím činidla IodoGen (Pierce). Reakčná zmes sa čistí vysokoúčinnou kvapalinovou chromatografiou s obrátenými fázami, pričom sa získa monojódovaný cetrorelix bez neoznačeného peptidu. Približne 80 % [125I]-cetrorelixu a neoznačenej zlúčeniny podľa vynálezu je vhodných na . špecifickú väzbu s receptorom.The compounds of the formula I according to the invention have been investigated for their receptor binding. This method relies heavily on the method described by Beckers et al., Eur. J. Biochem. 231: 535-543 (1995). According to the above-published synthesis, the cetrorelix obtained is iodine. with [ 125 I] (Amersham; specific activity 80.5 Bq / fmol) using IodoGen reagent (Pierce). The reaction mixture was purified by reverse phase high performance liquid chromatography to give mono-iodinated cetrorelix without unlabeled peptide. About 80% of [ 125 I] -cetrorelix and the unlabeled compound of the invention are suitable for. specific receptor binding.
Zlúčeniny podlá vynálezu sa môžu testovať nasledujúcimi metódami 1 a 2 na svoju účinnosť in vitro, pričom sa stanovia väzbové afinity pri väzbovej skúške s [125I]-cetrorelixom (metóda 1) a funkčné aktivity s triptorelínom ako agonistickým stimulom (metóda 2) .The compounds of the invention can be tested by the following methods 1 and 2 for their in vitro potency by determining binding affinities in the [ 125 I] -cetrorelix binding assay (method 1) and functional activities with triptorelin as agonist stimulus (method 2).
Metóda 1 (stanovenie KD na príklade cetrorelixu):Method 1 (determination of K D on the example of cetrorelix):
Skúška väzby k receptoru podľa práce Beckers, T., Marheineke, K., Reiländer, H., Hilgard P. (1995) „Selection and characterization of mamalian celí lines with stable overexpression of human pituitary receptors for gonadoliberin (GnRH) Eur. J. Biochem. 231, 535 - 543.Receptor Binding Assay according to Beckers, T., Marheineke, K., Reiländer, H., Hilgard P. (1995) "Selection and characterization of mammalian cell lines with stable overexpression of human pituitary receptors for gonadoliberin (GnRH) Eur. J. Biochem. 231, 535-543.
Pri skúške naviazania k receptoru sa cetrorelix jóduje použitím činidla IodoGen (Pierce) s [125I] (Amersham; špecifická aktivita 80,5 Bq/fmol). Reakčná zmes sa čistí vysokoúčinnou kvapalinovou chromatografiou s obrátenými fázami, pričom sa získa monojódovaný cetrorelix bez neoznačeného ppptidn·—Približne 8Q—%-[125I]-cetrorelixu bolo' schopných špecificky sa viazať na receptor.In the receptor binding assay, cetrorelix is iodinated using IodoGen (Pierce) with [ 125 I] (Amersham; specific activity 80.5 Bq / fmol). The reaction mixture was purified by reverse phase high performance liquid chromatography to yield mono-iodinated cetrorelix without unlabeled ppptid. Approximately 8% - [ 125 I] -cetrorelix was able to specifically bind to the receptor.
Skúška naviazania na receptor sa uskutočňuje s neporušenými bunkami za fyziologických podmienok tak, ako boli opísané (Beckers et al., 1995). Subkonfluentné kultúry stabilne transfekovaných LTK-buniek, ktoré exprimujú humánny receptor LHRH, sa oddelia inkubáciou v NaCl/Pi (137 mM NaCl, 2,7 mM KC1, 8,1 mM Na2HPO4, 11,47 mM KH2PO4)/1 mM EDTA a zhromaždia centrifugovaním. Bunková peleta sa resuspenduje vo väzbovom tlmivom roztoku (DMEM bez Η2003, so 4,5 g/1 glukózy, 10 mM HEPES, pH 7,5, 0,5 % (hmotnosť/objem) BSA, 1 g/1 bacitracínu, 0,1 g/1 SBTI, 0,1 % (hmotnosť/objem) NaN3) . Na skúšku potlačenia sa inkubuje 0,25 x 106 buniek/100 μΐ s približne 225 pM [125I]-cetrorelixu (špecifická aktivita 5 až 10 x 105 dpm/pmol) a rozličnými koncentráciami neoznačenej ' zlúčeniny podľa vynálezu ako konkurentmi. Bunková suspenzia v 100 μΐ väzbového média sa v 400 μΐ skúšobnej rúrke potiahne 200 μΐ 84 % (objem) silikónového oleja (Merck, typ 550)/16 % (objem) parafínového oleja. Po inkubácii počas 1 hodiny pri 37 °C za pomalého, kontinuálneho pretrepávania sa bunky oddelia centrifugovaním v priebehu 2 minút pri 9000 ot./min (Rotortyp HTA13,8; Heraeus Sepatec, Osterode/Nemecko) od inkubačného média. Špičky rúrky, ktoré obsahujú bunkovú peletu, sa odrežú. Bunková peleta a supernatant sa potom analyzujú počítaním žiarenia γ. Množstvo nešpecifických naviazaní sa stanoví za vloženia neoznačeného cetrorelixu pri 1 μΜ konečnej koncentrácii a typicky je > 10 % z celkových naviazaní. Analýza údajov o väzbách sa uskutočňuje pomocou programu pre.analýzu ligandov EBDA (Biosoft V3,0).The receptor binding assay is performed with intact cells under physiological conditions as described (Beckers et al., 1995). Subconfluent cultures of stably transfected LTK cells that express the human LHRH receptor are separated by incubation in NaCl / Pi (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 , 11.47 mM KH 2 PO 4 ) / 1 mM EDTA and collected by centrifugation. The cell pellet is resuspended in binding buffer (DMEM without 00 2 00 3 , with 4.5 g / l glucose, 10 mM HEPES, pH 7.5, 0.5% (w / v) BSA, 1 g / l bacitracin 0.1 g / l SBTI, 0.1% (w / v) NaN 3 ). For the suppression assay, 0.25 x 10 6 cells / 100 μΐ are incubated with approximately 225 µM [ 125 L] -cetrorelix (specific activity 5 to 10 x 10 5 dpm / pmol) and various concentrations of unlabeled compound of the invention as competitors. The cell suspension in 100 μΐ binding medium is coated with 200 μΐ 84% (volume) silicone oil (Merck type 550) / 16% (volume) paraffin oil in a 400 μΐ test tube. After incubation for 1 hour at 37 ° C with slow, continuous shaking, cells are separated by centrifugation for 2 minutes at 9000 rpm (Rotortyp HTA13.8; Heraeus Sepatec, Osterode / Germany) from the incubation medium. The tube tips containing the cell pellet are cut off. The cell pellet and supernatant are then analyzed by counting γ radiation. The amount of non-specific binding is determined by loading unlabeled cetrorelix at 1 μΜ final concentration and is typically> 10% of total binding. Binding data analysis is performed using an EBDA ligand analysis program (Biosoft V3.0).
Cetrorelix má hodnotu KD 170 pikomol na liter (pM) (počet nezávislých uskutočnených pokusov: 21).Cetrorelix has a K D value of 170 picomol per liter (pM) (21 independent experiments performed).
Metóda 2 (funkčná skúška na stanovenie antagonistickej účinnosti—(hodno-ty IC50) ) :Skúška sa uskutočňuje s nižšie uvedenými modifikáciami tak, ako sa opisuje v práci Beckers, T., Reiländer, H.,Method 2 (functional assay to determine antagonist activity— (IC50 values)): The assay is carried out with the modifications described below as described by Beckers, T., Reiländer, H.,
Hilgard P. (1997) „Characterization of gonadotropin-releasing hormone analogs based on a sensitive cellular luciferase reportér gene assay, Analyt. Biochem. 251, 17 - 23 (Beckers et ab 1997). 10 000 buniek na jamku, ktoré exprimujú humánny receptor LHRH a oznamujúci gén pre luciferázu, sa kultivujú 24 hodín na mikrotitračných platniach použitím DMEM s prísadami a 1 % (objem/objem) FCSi. Bunky sa potom 6 hodín stimulujú pomocou 1 nM [D-Trp6]LHRH. Antagonistické zlúčeniny podľa vynálezu sa pridajú pred stimuláciou a bunky sa nakoniec lyžujú za účelom kvantifikácie bunkovej Luc-aktivity. Výpočet hodnôt IC50 z kriviek závislosti účinnosti od dávky sa uskutočňuje nelineárnou regresnou analýzou použitím Hillovho modelu (program EDX 2.0 od C. Grunwalda, Arzneimittelwerk, Drážďany).Hilgard P. (1997) 'Characterization of gonadotropin releasing hormone analogs based on a sensitive cellular luciferase reporter gene assay, Analyt. Biochem. 251, 17-23 (Beckers et al 1997). 10,000 cells per well that express the human LHRH receptor and the reporting luciferase gene are cultured for 24 hours in microtiter plates using DMEM with additives and 1% (v / v) FCSi. Cells are then stimulated with 1 nM [D-Trp 6 ] LHRH for 6 hours. The antagonist compounds of the invention are added prior to stimulation and the cells are finally lysed to quantitate cellular Luc activity. Calculation of IC 50 values from dose-response curves is performed by non-linear regression analysis using the Hill model (EDX 2.0 program from C. Grunwald, Arzneimittelwerk, Dresden).
Kvantifikácia Luc-aktivity sa uskutočňuje v podstate tak, ako sa opisuje (Promega Technical Bulletins #101/161) použitím skúšobného systému pre luciferázu (Promega E4030) dvojito. Pridaním koenzýmu A (CoA) sa uskutoční oxidácia lyciferyl-CoA s výhodnou kinetikou. Po odstránení kultivačného média z mikrotitračnej platne sa bunky lyžujú pridaním 100 μί lyzačného tlmivého roztoku (25 mM Trisfosfátu pH 7,8, 2 mM ditiotreitolu, 2 mM kyseliny 1,2-diaminocyklohexán-N,N,N',N'-tetraoctovej (CDTA), 10 % (objem/objem) glycerolu, 1% (objem/objem) tritónu X-100. Po 15-minútovej inkubácii pri teplote miestnosti sa 10 μΐ bunkové lyzáty prenesú na bielu mikrotitračnú platňu (Dynatech) vhodnú na luminometrickú detekciu. Enzymatická reakcia sa iniciuje pridaním 50 μί skúšobného tlmivého roztoku (20 mM tricínu pH —1,07- mM—(MgCO3)4Mg (OH)2,—2, 67Quantification of Luc activity was performed essentially as described (Promega Technical Bulletins # 101/161) using the luciferase assay system (Promega E4030) in duplicate. Addition of Coenzyme A (CoA) oxidizes lyciferyl-CoA with advantageous kinetics. After removing the culture medium from the microtiter plate, cells are lysed by adding 100 µl of lysis buffer (25 mM Trisphosphate pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N, N, N ', N'-tetraacetic acid ( CDTA), 10% (v / v) glycerol, 1% (v / v) tritone X-100 After incubation for 15 minutes at room temperature, 10 μΐ cell lysates are transferred to a white microtiter plate (Dynatech) suitable for luminometric detection. The enzymatic reaction is initiated by the addition of 50 μί of assay buffer (20 mM Tricine pH mM1,07 mM - (MgCO 3 ) 4 Mg (OH) 2 , 22,67
MgSOí, 0,1 mM kyseliny etylendiamíntetraoctovej (EDTA), 33,3 mM ditiotreitolu, 270 μΜ koenzýmu A, 470 μΜ luciferínu zo svätojánskej mušky (Photinus pyralis), 530 μΜ rATPNa2) . Po 1 minúte sa stanoví pre celkový čas jednej sekundy luminiscencia so signálnou polovičnou hodnotou času 5 minút použitím zariadenia EG&G Berthold MicroLumat LB 96 P.MgSOí, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 33.3 mM dithiothreitol, 270 μΜ of coenzyme A, 470 μ sv of firefly luciferin (Photinus pyralis), 530 μΜ rATPNa 2 ). After 1 minute, a luminescence with a signal half-time of 5 minutes is determined for a total time of one second using an EG&G Berthold MicroLumat LB 96 P.
V tabuľke 2 sú zhrnuté fyzikálno-chemické a in vitro údaje zlúčenín podľa vynálezu. IC50 platí pre funkčnú aktivitu a pM znamená pikomol na liter. Rozpustnosť vo vode sa stanovila podľa spôsobu opísaného pod poznámkou 2):Table 2 summarizes the physicochemical and in vitro data of the compounds of the invention. IC 50 is valid for functional activity and pM means picomol per liter. The water solubility was determined according to the method described in note 2):
Tabulka 2Table 2
Poznámky:notes:
1) Čislo v zátvorke udáva počet navzájom nezávislých pokusov.1) The number in brackets indicates the number of independent attempts.
2) Rozpustnosť vo vode sa stanovila podlá ďalej opísanej metódy:(2) The water solubility was determined according to the following method:
Rozpustnosť podľa metódy uvedenej vo vestníku v Ringerovom roztoku:Solubility according to Ringer's solution method:
Na stanovenie rozpustnosti podľa metódy uvedenej vo vestníku sa testovaná látka v nadbytku zmieša s inertným nosným materiálom, ako je napríklad piesok, a naplní do sklenej kolóny (objem približne 10 ml). Na dno kolóny sa predtým vložilo sito Z vaty a filter zn.Bklňných vlhkí m-Zmes látky a piesku sa nechá 1 hodinu napučiavať v 1,0 ml rozpúšťadla, v ktorom sa má stanoviť rozpustnosť. Potom sa 10 ml rozpúšťadla naplní do sklenej kolóny. Pomocou hadicového čerpadla sa roztok čerpá v okruhu. Toto stanovenie sa uskutočňuje pri teplote miestnosti (približne 20 °C). Rozpustnosť sa stanoví, keď hmotnostné koncentrácia navzájom za sebou odoberaných frakcií je konštantná.. Stanovenie hmotnostnej koncentrácie sa uskutočňuje prostredníctvom nižšie opísanej metódy HPLC.To determine the solubility according to the journal method, the test substance is mixed in excess with an inert support material such as sand and packed into a glass column (approximately 10 ml volume). A cotton sieve was previously applied to the bottom of the column and a filter of total wet moisture was swelled for 1 hour in 1.0 ml of solvent to determine solubility. 10 ml of solvent is then packed into a glass column. The solution is pumped in the circuit using a hose pump. This determination is carried out at room temperature (about 20 ° C). Solubility is determined when the mass concentration of the fractions collected in succession is constant. The mass concentration is determined by the HPLC method described below.
Metóda HPLCHPLC method
Zariadenie:Appliances:
Systém HPLC: Hewlett Packard 1100; detektor: Hewlett Packard DAD Šerieš 110HPLC system: Hewlett Packard 1100; detector: Hewlett Packard DAD Šerieš 110
Kolóna: materiál kolóny: Nucleosil® 120-3 Cis veľkosť častíc: 3 pm rozmery kolóny: 125 x 4 mm výrobca: Macherey & Na-gelColumn: column material: Nucleosil® 120-3 Cis particle size: 3 pm column dimensions: 125 x 4 mm manufacturer: Macherey & Na-gel
Parametre zariadenia: vstrekovací objem: 15 μΐ prietok: 1,0 ml/min teplota pece: 45 °C vlnová dĺžka: 226 nm čas zastavenia: 15 minDevice parameters: injection volume: 15 μΐ flow rate: 1.0 ml / min furnace temperature: 45 ° C wavelength: 226 nm stop time: 15 min
55% mobilná fáza A: Zmieša sa 970 ml MÍ11Í-Q-H2O, 30 ml acetonitrilu a 1 ml kyseliny trifluóroctovej. Výsledné pH je približne 1,9.55% mobile phase A: Mix 970 mL of M11H-Q-H2O, 30 mL of acetonitrile and 1 mL of trifluoroacetic acid. The resulting pH is about 1.9.
45% mobilná fáza B: Zmieša sa 300 ml Milli-Q-H2O, 700 ml acetonitrilu a 1 ml kyseliny trifluóroctovej. Výsledné pH je približne 1,8.45% mobile phase B: 300 ml of Milli-Q-H2O, 700 ml of acetonitrile and 1 ml of trifluoroacetic acid are mixed. The resulting pH is about 1.8.
Podávanie zlúčenín podlá vynálezu sa môže uskutočňovať rôznymi formami vhodnými pre peptidové účinné látky. Vhodné ap1i kác ie sú odborníkom dobre známe. Aplikácia sa môže__ uskutočňovať napríklad injekciou. Aplikácia sa môže uskutočňovať napríklad parenterálne. Prednostné je pri tom subkutánne (s.c.), intramuskulárne (i.m.), intravenózne (i.v.), bukálne (napríklad sublingválne) alebo rektálne podávanie. Podávanie i.s. a i.m. je obzvlášť prednostné.The administration of the compounds of the invention may be carried out in various forms suitable for peptide active substances. Suitable applications are well known to those skilled in the art. Administration can be carried out, for example, by injection. The administration can be carried out, for example, parenterally. Preferred are subcutaneous (s.c.), intramuscular (i.m.), intravenous (i.v.), buccal (e.g., sublingual) or rectal administration. Administration i.s. and i.m. is particularly preferred.
Zlúčeniny podlá vynálezu sú vhodné na výrobu rôznych aplikačných foriem, napríklad lyofilizátov, roztokov alebo suspenzií. Vhodné aplikačné formy a ich výroba sú odborníkom známe. Ako vhodné pomocné látky a látky tvoriace skelet liečivého prípravku sú napríklad hexitoly, ako je napríklad manitol, najmä D-manitol, L-manitol alebo D,L-manitol, sorbitol, ako je napríklad D-sorbitol, D- alebo L-altritol, iditol, glucitol a dulcitol. Výroba sa uskutočňuje o sebe známymi spôsobmi, napríklad zmiešaním, suspendovaním alebo lyofilizáciou.The compounds of the invention are suitable for the preparation of various dosage forms, for example lyophilisates, solutions or suspensions. Suitable dosage forms and their preparation are known to those skilled in the art. Suitable excipients and skeletal formers are, for example, hexitols such as mannitol, especially D-mannitol, L-mannitol or D, L-mannitol, sorbitol such as D-sorbitol, D- or L-altritol, iditol, glucitol and dulcitol. The preparation is carried out in a manner known per se, for example by mixing, suspending or lyophilizing.
Príklad A: Lyofilizát na výrobu s.c. injekčného roztoku mg zlúčeniny z príkladu 1 (zodpovedajúc 0,26 až 0,27 mg acetátovej soli); 0 až 16,9 hmotnostných dielov D-manitolu, prednostne 0,1 až 7 hmotnostných dielov vzhladom na príklad 1 a voda na injekčné účely (na výrobu injekčného roztoku z lyofilizátu).Example A: Lyophilisate for production s.c. solution of injection of the compound of Example 1 (corresponding to 0.26 to 0.27 mg of the acetate salt); 0 to 16.9 parts by weight of D-mannitol, preferably 0.1 to 7 parts by weight with respect to Example 1, and water for injection (for the preparation of a solution for injection from a lyophilisate).
Príprava: 1,62 g z príkladu 1 sa rozpustí v 30% kyseline octovej (približne 1,5 1 vody na injekčné účely a 91,17 g kyseliny octovej). Roztok sa zriedi 1,5 1 vody, pridá sa 82,2 g manitolu, uskutoční sa sterilná filtrácia, zmes sa plní do ml injekčných flaštičiek za aseptických podmienok a lyofilizuje. Získa sa 1 mg lyofilizátu zlúčeniny podlá príkladu 1.Preparation: 1.62 g of Example 1 are dissolved in 30% acetic acid (approximately 1.5 L of water for injection and 91.17 g of acetic acid). The solution is diluted with 1.5 L of water, 82.2 g of mannitol is added, sterile filtration is performed, the mixture is filled into ml vials under aseptic conditions and lyophilized. 1 mg of the lyophilisate of the compound of Example 1 is obtained.
Zlúčeniny podľa vynálezu sú vhodné napríklad na liečenie hormonálne závislých malígnych alebo nemalígnych chorôb, napríklad na liečenie rakoviny prsníka, rakoviny prostaty, endometriózy, myómu maternice, benígnej hyperplázie prostaty (BPH), a taktiež na liečenie porúch plodnosti žien alebo_ mužov, napríklad na zabránenie predčasnej ovulácii u pacientiek, ktoré sa podrobujú riadenej ovariálnej stimulácii nasledovanej odňatím vaječnej bunky a technikami asistovanej reprodukcie. Uvedené liečenia sa môžu uskutočňovať u cicavcov, najmä u ľudí.The compounds of the invention are suitable, for example, for the treatment of hormone-dependent malignant or non-malignant diseases, for example for the treatment of breast cancer, prostate cancer, endometriosis, uterine myoma, benign prostate hyperplasia (BPH), and also for the treatment of fertility disorders in women or men. ovulation in patients undergoing controlled ovarian stimulation followed by egg cell withdrawal and assisted reproduction techniques. Said treatments may be carried out in mammals, especially humans.
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| CN101037472B (en) * | 2006-03-14 | 2013-03-27 | 中国人民解放军军事医学科学院毒物药物研究所 | LHRH antagonist with low-histamine releasing function |
| DK2252627T3 (en) | 2008-01-24 | 2017-08-14 | Esperance Pharmaceuticals | MERGER CONSTRUCTION WITH LYTIC DOMAIN AND METHOD FOR PRODUCING AND USING SAME. |
| CN101597321B (en) * | 2008-06-03 | 2013-04-24 | 中国人民解放军军事医学科学院毒物药物研究所 | LHRH antagonist with long-acting low-histamine release side effect |
| WO2010142060A1 (en) * | 2009-06-11 | 2010-12-16 | 中国人民解放军军事医学科学院毒物药物研究所 | Lhrh antagonist with long potency and low histamine releasing effect |
| CA2889475A1 (en) | 2012-10-30 | 2014-05-08 | Esperance Pharmaceuticals, Inc. | Antibody/drug conjugates and methods of use |
| CN104418936B (en) * | 2013-08-20 | 2018-06-05 | 中国人民解放军军事医学科学院毒物药物研究所 | Lhrh antagonist derivative and its medicinal usage |
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| US5110904A (en) * | 1989-08-07 | 1992-05-05 | Abbott Laboratories | Lhrh analogs |
| IL101074A (en) | 1991-03-14 | 1997-09-30 | Salk Inst For Biological Studi | GnRH ANALOGS AND THEIR PREPARATION |
| DE69214818T2 (en) | 1991-04-25 | 1997-02-27 | Romano Deghenghi | LHRH antagonists |
| US5698522A (en) | 1992-12-04 | 1997-12-16 | Abbott Laboratories | 6-position modified decapeptide LHRH antagonists |
| ATE206136T1 (en) | 1992-12-18 | 2001-10-15 | Abbott Lab | LHRH ANTAGONISTS WITH MODIFIED AMINOACYL RESIDUALS 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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| DE19911771B4 (en) * | 1999-03-17 | 2006-03-30 | Zentaris Gmbh | LHRH antagonist, process for its preparation and its use |
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