ANTAGONISTS OF LHRH

Abstract: LHRH analogs and congeners with high water solubility have been synthesized. These new analogs had 0 %-100 % antiovulatory activity at a 0.5 mu g dosage and 0 %-80 % at 0.25 mu g. The ED50 for histamine release was 30.5 mu g/ml- > 300 mu g/ml.

Description: DESCRIPTION ANTAGONISTS OF LHRH This is a continuation-in-part of application serial number 07/946,056 filed September 5, 1992 which is incorporated by reference herein. This invention was made with government support under contract no. N01-HD-1-3101 awarded by the National Institutes of Health. The government, therefore, has certain rights in the invention. BACKGROUND OF THE INVENTION Many potent analogs of LHRH (the luteinizing hormone releasing hormone, y7Glu 1 ,His 2 ,Trp 3 ,Ser 4 ,Tyr 5 ,Gly 6 ,Leu 7 ,Arg 8 ,Pro 9 ,Gly 10 -NH 2 )have structural features which cause release of histamine from mast cells (Karten et al., 1986). These features include a group of hydrophobic amino acids at the N terminal and strongly basic residues in positions 6 and 8, notably D-Arg , Arg . A prime example of this class of analogs is (N- Ac-D-2-Na1 1 ,D-4F-Phe 2 D-Trp 3 ,D Arg 6 )- LHRH (Schmidt et al.). Some analogs bind to rat peritoneal mast cells and membrane preparations and that the binding was related to the release of histamine (Sundaram et al.). For a recent review of LHRH analogs, see Janecka, et al. (1994). Abbreviations for the unnatural amino acids mentioned herein are included in the following list: AABLys N e -(4-acetylaminobenzoyl)lysine AALys N e -anisinoyl-lysine AAP e 3-(4-acetylaminophenyl)lysine Abu 2-amiπobutyric acid ACLys N f (6-aminocaproyl)lysine ACyh 1 -aminocyclohexanecarboxylic acid ACyp 1-aminocyclopentaπecarboxylic acid Aiie alloisoleucine Ala alanine AnGlu 4-(4-methoxy-phenylcarbamoyl)-2-aminobutyric acid 2ANic 2-aminonicotinic acid 6ANic 6-aminonicotinic acid APic 6-aminopicolinic acid APh 4-aminobenzoic acid APhe 4-aminophenylalanine APz 3-amino-2-pyrazinecarboxylic acid Aze azetidine-2-carboxylic acid Bim 5-benzimidazolecarboxylic acid BzLys N e -benzoyllysine Cit citrulline CI 2 Phe 3-(3,4-dichlorophenyl)alanine Cpa 3 (4-chlorophenyl)alanine cPzACAIa cis-3-(4 pyrazinγlcarbonylaminocyclohexyl)alanine cPmACAIa cis-3-(4-(4-pyrimidylcarbonyl)aminocyclohexyl)alanine Obf 3-(2-dibenzofuranyl)alanine DMGLys N ε -(N,N-dimethylglycyl)lysine Dpo N e (4,6 dimethyl-1 pyrimidyl) omithine F 2 Ala 3,3 difluoroalaniπe hNal 4 (2-naphthyl)-2 aminobutyric acid HOBLys N e - - (4-hydroxybenzoyl)lysine hpCIPhe 4 (4 chlorophenyl)-2 amino-butyric acid Hse omoserine (2 amino-4 hydroxybutanoic acid) ICapLys N e -(6 isopropylamiπocaproyl)lysine ILys N e isopropyllysine Ind indoline 2 carboxylic acid INicLys N e isonicotinoyllysine lOrn N isopropylornithine Me 3 Arg N ϋ ,N b ,N -trimethylarginine Mβ2Lys N e ,N e -dimethyllysine MNal 3-((6-methyl)2-πaphtyl)alanine MNicLys N e -(6-methylpicolinoyl)lysine MOB 4-methoxybenzoyl MpCIPhe N-methyl-3 (4 chlorophenyl)lysine MPZGIu glutamic acid, y-4-methylpiperazine NAcDNal N-acetyl-3-12-naphthyl)alaπiπe NacDQal N-acetyl-3 (3-quinolyl)alanine Nal 3 (2-naphthyl)alanine Nap 2-naphthoic acid NapDThr N-naphthoyl-D threonine NicLys N^-nicotinoyllysine N0 2 B 4-nitrobenzoyl N0 2 Phe 3 (4-nitrophenyl)alanine oCIPhe 3-(2-chlorophenyl)alanine Opt O-phenyl-tyrosiπe Orn ornithine Pal 3-(3-pyridyl)alaπine 21 Pal 3-(2 pyridyl)alanine 2PALys N e -(3 pyridylacetyl)lysine pCapLys N e (5-picolinoylaminocaproyl)lysine pCIPhe 3-(4-chlorophenyl)alanine pFPhe 3-(4 fluorophenyl) alanine Pic picolinic acid PicLys N^-picolinoyllysine PicSar N-picolinoylsarcosinyl Pip piperidine 2-carboxylic acid PmcLys N e -(4-pyrimidylcarbonyl}lysine Ptf 3 (4 trifluoromethyl) phenylalanine Pz pγrazinecarboxylic acid PzAla 3-pyrazinylalanine PzAPhe 3-(4 pyrazinylcarbonylaminophenyl)alanine Qal 3 (3-quiπolyl)alanine Qnd-Lys N ε -quinaldoyllysine Qui 3-quinolinecarboxylic acid Qux 2-quinoxalinecarboxylic acid Tic 1 ,2,3,4-tetrahγdroisoquinoline-3-carboxγlic acid TinGly 2-thienylglycine tNACAIa trans-3 (4-nicotinoylaminocyclohexyl)-alanine tPACAIa trans-3-(4-picolinoylaminocyclohexyl)alanine SUMMARY OF THE INVENTION LHRH analogs having the following formulas have been prepared and tested. (NAcDQal ,DPtf 2 ,DPal 3 ,cisPzACAIa 5 ,DPicLys 6 ,DAIa 10 )LHRH (NAcDNal ,DpCIPhe 2 ,DPal 3 ,cisPzACAIa 5 ,DNicLys 6 ,ILys 8 ,DAIa 10 )LHRH (NAcDNal ,DpCIPhe 2 ,DPal 3 ,Thr 4 ,PicLys 5 ,DPicLys 6 ,ILys 8 ,DAIa 1 0 )LHRH (NAcDNal r DpCIPhe 2 ,DPal 3 ,PicLys 5 ,DPicLys 6 ,Thr 7 ,ILys 8 ,DAIa 10 )LHRH (NapDThr , DpCIPhe 2 , DPal 3 , PicLys 5 ,DPicLys 6 ,ILys 8 ,DAIa 10 )LHRH (NAcDNal , DpCIPhe 2 ,DPal 3 ,NicLys 5 ,DNicLys 6 ,Thr 7 ,ILys 8 ,DAIa 1 0 )LHRH (NAcDNal ,DpCIPhe 2 ,DPal 3 ,Thr 4 NicLys 5 ,DNicLys 6 ,Thr 7 ,ILys 8 ,DAIa 1 0 )LHRH (NAcDNal 1 ,DpCIPhe 2 ,DPal 3 ,PicLys 5 ,D(PicSar)Lys 6 ,ILys 8 ,DAIa 10 )LHRH (NAcDNal 1 , DpCIPhe 2 ,DPal 3 ,D(PicSar)Lys 6 ,ILys 8 ,DAIa 10 )LHRH (NAcDNal 1 ,DpCIPhe 2 ,DPal 3 ,PicLys 5 ,D(6ANic)Lys 6 ,ILys 8 ,DAIa 10 )LHRH (NAcDNal 1 ,DpCIPhe 2 ,DPal 3 ,PicLys 5 ,D(6ANic)0rn 6 ,ILys 8 ,DAIa 1 0 )LHRH (NAcDQal 1 ,DCpa 2 ,DPal 3 ,cisPzACAIa 5 ,DPicLys 6 ,NLeu 7 ,ILys 8 ,DAIa 10 )LHRH (NAcDNal 1 , DCpa 2 ,DPal 3 ,DPicLys 5 ,DAPhe(PicSar) 6 ,ILys 8 ,DAIa 1 0 )LHRH (NAcDQal 1 ,DCpa 2 ,DPal 3 ,PicLys 5 ,DPal 6 ,ILys 8 ,DAIa 10 )LHRH (NAcDNal 1 , DCpa 2 ,DPal 3 ,PicLys 5 ,DOrn(ACyp) 6 ,ILys 8 ,DAIa 10 )LHRH DESCRIPTION OF THE PREFERRED EMBODIMENTS LHRH analogs and congeners with high water solubility have been synthesized. These new analogs had 0%-100% antiovuiatory activity at a 0.5 yg dosage and 0%-80% at 0.25 μg. The ED 50 for histamine release was 30.5 μg/ml- > 300 μg/ml. The compounds of the present invention are water soluble and some of them, as listed below, are also soluble in physiological salt solution without gelling. The preferred analogs show favorable potency and histamine values. These are important for clinical use and are not easily accomplished. An important final point is that many LHRH analogs of the present invention do not contain the PzACAIa substitution and still have favorable potency, histamine releasing effects and water solubility as well as solubility in physiological salt solution without gelling, except as listed below. The reason this is important is because the PzACAIa is difficult to synthesize because of the stereochemical requirements, thus adding substantially to the expense of synthesis in large amounts. A concerted effort was made to eliminate the PzACAIa for this very reason. As you know emphasis on the cost of drugs is a major issue these days. Analogs 27316, 27345, 27234, 27459, and 27490 were soluble at 1 mg/ml in the physiological buffer solution PIPES-AC. Analog 27249 (1 mg/ml) and analog 27355 (0.3 mg/ml were slightly turbid. Analog 27274 gelled slightly at 1 mg/ml and analog 27163 gelled slightly at 1 mg/ml. For comparison, Antide gelled completely at 1 mg/ml while Lystide did not. The following Examples are included to show the best mode of the present invention and not to limit the invention unless specifically claimed. EXAMPLE 1 DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF Analogs OF LHRH BY CRITERIA OF POTENCY, SAFETY AND SOLUBILITY Experimental Materials BOC Ser(OBzl ), BOC-Pro, BOC Leu and BOC D AIa were obtained from Peninsula Laboratories, Belmont, CA. Abu was obtained from Sigma Chemical Company, St. Louis, MO and it was converted to the BOC-derivative using standard procedures (Moroder et al.). BOC-D-2-Nal, BOC-D-/>CIPhe, BOC-D- and L-3-Pal, BOC-ILys.Z) dicyclohexylamine salt, BOC-D- and L-NicLys and BOC-D-3-Qal were all provided by Dr. Narasimha Rao of the Southwest Foundation for Biomedical Research, San Antonio, TX. BOC-D- and L- PzAla were synthesized by literature methods (Petermaπ et al.). BOC-AOPP was kindly provided by Dr. R.M. Freidinger, Merck, Sharp & Dohme, West Point, PA (Freidinger et al., 1980 and 1982). α-BOC cis D- and L-4-amino- cyclohexylalanine were provided by Dr. Narasimha Rao, and were converted to the corresponding BOC-c s-D- and L-PzACAIa derivatives by acylation with the p- nitrophenylester (Folkers et al., 1990) of pyrazinecarboxylic acid from the Aldrich Chemical Company, Milwaukee, Wl, in DMF. BOC-D- and L-PicLys were similarly prepared from σ-BOC-D- and L-Lys and picolinic acid p nitrophenylester (Ljungqvist et al.) in DMF. σ-BOC-D- and L-(DSer)Lys, protected at the D-Ser moiety by the Z group at the amino group and by the benzyl group at the hydroxyl function, were synthesized by acylation of BOC-D- and L-Lys in DMF by Z-D-Ser(0Bzl)-0Np in the presence of 1 -hydroxybenzotriazole. The BOC-D (AcDSer)Lys, protected as the benzyl derivative at the D-Ser hydroxy, was similarly prepared by the reaction between Ac-D-Ser(0Bzl)0Np and BOC-D-Lys. All synthesized amino acids and intermediates were characterized by NMR and were homogenous on TLC. The benzhydrylamine hydrochloride resin was purchased from Beckman Bioproducts, Palo Alto, CA. The dicyclohexylcarbodiimide was from Aldrich and was distilled in vacuo before use. The dichloromethane was distilled from sodium carbonate. All other solvents and reagents were reagent grade. Synthesis The peptides were synthesized by the solid-phase method using a Beckman automated 990 peptide synthesizer. The protocol details used were essentially as described (Folkers et al., 1984). The peptide was cleaved from the resin with concomitant removal of all protecting groups by treatment with doubly distilled HF at 0°C for 1 h in the presence of about 10% anisole in μ-cresol. The HF was then evaporated, in vacua, first by a water aspirator and then by pump vacuum overnight. The residue was then extracted 2-3 times with ether in order to remove non-peptidic material. The crude peptide was subsequently extracted with aqueous acetic acid and the extract was lyophilized. Purification and Characterization Purification was achieved by chromatography on Si0 2 (EM, 230-400 mesh) with the solvent system /.-butanol.acetic acid:water 4:1 :2 or 4:1 :5 (upper phase) followed by gel filtration on Sephadex G 25 with 6% aqueous acetic acid as the eluant. An alternative purification method was gel filtration as above followed by chromatography on Sephadex LH 20 with the solvent system waterrj&_/fø/7σ/:acetic acid:methanol 90:10:10:8. The purity was checked by TLC, amino acid analysis and HPLC. Amino acid analyses were carried out on a Beckman 1 18 CL amino acid analyzer after hydrolysis in constant boiling HCI for 24 h using standard procedures (Folkers et al., 1984). The unnatural amino acids were qualitatively determined with the exception of 3 Pal which was quantified. The purity was further checked by HPLC using a Waters instrument with a 660 solvent programmer and a Vydac C 18 column. The flow rate was 1.5 ml/min and the absorbance was recorded at 210 nm. Different gradients of increasing concentration of acetonitrile in 0.01 M KH 2 P0 4 , adjusted to pH 3 with H 3 P0 , were employed. All peptides were estimated to be 97-99% pure in this system (data not shown). Biological assays AOA in rats was determined as reported (Humphries et al.). The histamine release was assayed in rat mast cells as reported (Hook et al., Karten et al., 1987). The EDg value reported is the concentration in μg/ml that releases 50% of total releasable histamine. The biological data are in the following table. These are more completely explained in United States patent number 4,935,491 that is incorporated, in pertinent part, by reference herein. Unspecified positions are from LHRH ( 7Glu 1 ,His 2 ,Trp 3 ,Ser 4 ,Tyr 5 ,Gly 6 , Leu 7 ,Arg 8 ,Pro 9 ,Gly 10 -NH 2 ) The most preferred LHRH analogs of the table have high antiovuiatory activity and also a high effective dose level for histamine release The following citations are incorporated in pertinent part by reference herein for the reasons cited in the above text. References Folkers et al., (June 19, 1990) U.S. Patent 4,935,491. Folkers et al., Biochem, Biophys. Res. Commun. 123, 1221 (1984). Freidinger et al., Since 210, 656 (1980). Freidinger et al., J. Org. Chem. 47 104 (1982). Humphries et al., Biochem. Biophys. Res. Comm. (1976) 72, 939-944. Humphries et al., (1987) J. Med. Chem. 21, 120-123. Janecka et al. (1994) Amino Acids 6,1 1 1-130. Karten et al., (1986) Endocr. Rev. 7: 44-66. Karten et al., in: LHRH and its Analogs: Contraceptive and Therapeutic Applications II, pp. 179-190, MTP Press LTD, Lancaster, England (1987). Ljungqvist et al., (1988) Acta Chem. Scand. 842: 408-410. Ljungqvist et al., (1987) Biochem. Biophys Res. Comm. 148: 849-856. Ljungqvist et al., Proc. Natl. Acad. Sci. USA 85, 8236 (1988). Moroder et al., Hoppe Seyler's Z. Physiol. Chem. 357, 1651 (1976). Petermaπ et al., Helv. Chim. Acta 66, 1513 (1983). Rao et al., (1987) Int. J. Peptide Protein Res. 29: 1 18-125. Schmidt et al., (1984) Contraception 29: 283-289. Sundaram et al., Agents and Actions 25, 307 (1988).

Claims: CLAIMS: 1. An LHRH analog having the formula (NAcDQal 1 ,DPtf 2 ,DPAI 3 ,cjsPzACAIa 5 ,DPicLys 6 ,DAIa 10 )LHRH. 2. An LHRH analog having the formula (NAcDNal 1 , DpCIPhe 2 ,DPal 3 ,cjsPzACAIa 5 ,DNicLys 6 ,ILys 8 ,DAIa 1 0 )LHRH. 3. An LHRH analog having the formula (NAcDNal 1 ,DpCIPhe 2 ,DPal 3 ,Thr 4 ,PicLys 5 ,DPicLys 6 ,ILys 8 ,DAIa 10 )LHRH. 4. An LHRH analog having the formula (NAcDNal 1 ,DpCIPhe 2 ,DPal 3 ,PicLys 5 ,DPicLys 6 ,Thr 7 ,ILys 8 ,DAIa 10 )LHRH. An LHRH analog having the formula (NapDThr 1 , DpCIPhe 2 , DPal 3 , PicLys 5 ,DPicLys 6 ,ILys 8 ,DAIa 10 )LHRH. 6. An LHRH analog having the formula (NAcDNal 1 , DpCIPhe 2 ,DPal 3 ,NicLys 5 ,DNicLys 6 ,Thr 7 ,ILys 8 ,DAIa 10 )LHRH. 7. An LHRH analog having the formula (NAcDNal 1 , DpCIPhe 2 ,DPal 3 ,Thr 4 NicLys 5 ,DNicLys 6 ,Thr 7 ,ILys 8 ,DAIa 10 )LHRH. 8. An LHRH analog having the formula (NAcDNal 1 ,DpCIPhe 2 ,DPal 3 ,PicLys 5 ,D(PicSar)Lys 6 ,ILys 8 ,DAIa 10 )LHRH. 9. An LHRH analog having the formula (NAcDNal 1 ,DpCIPhe 2 ,DPal 3 ,D(PicSar)Lys 6 ,ILys 8 ,DAIa 10 )LHRH. 10. An LHRH analog having the formula (NAcDNal 1 ,DpCIPhe 2 ,DPal 3 ,PicLys 5 ,D(6ANic)Lys 6 ,ILys 8 ,DAIa 10 )LHRH. 11. An LHRH analog having the formula (NAcDNal 1 ,DpCIPhe 2 ,DPal 3 ,PicLys 5 ,D(6ANic)0rn 6 ,ILys 8 ,DAIa 1 0 )LHRH. 12. An LHRH analog having the formula (NAcDQal 1 ,DCpa 2 ,DPal 3 ,cisPzACAIa 5 ,DPicLys 6 ,NLeu 7 ,ILys 8 ,DAIa 10 )LHRH. 13. An LHRH analog having the formula (NAcDNal 1 ,DCpa 2 ,DPal 3 . DPicLys 5 ,DAPhe(PicSar) β ,ILys 8 ,DAIa l 0 )LHRH. 14. An LHRH analog having the formula (NAcDQal 1 ,DCpa 2 ,DPal 3 ,PicLys 5 ,DPal 6 ,ILys 8 ,DAIa 1 0 )LHRH. 15. An LHRH analog having the formula (NAcDNal 1 , DCpa 2 ,DPal 3 ,PicLys 5 ,DOrn(ACyp) 6 ,ILys 8 ,DAIa 1 0 )LHRH.

Inventors: BOWERS CYRIL Y, FOLKERS KARL A, JANECKA ANNA, BOWERS, CYRIL, Y., FOLKERS, KARL, A., JANECKA, ANNA
Applicants: UNIV TEXAS, UNIV TULANE, BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM, THE ADMINISTRATORS OF THE TULANE EDUCATIONAL FUND
Application year: 1996
Main classification: A61K38
IPC codes: A61K38, C07K7