J. Med. Chem. 1994,37, 701-705
70 1
In Vitro and in Vivo Activities of Reduced-Size Antagonists of Luteinizing Hormone-Releasing Hormonettl Fortuna Haviv,' Timothy D. Fitzpatrick, Charles J. Nichols, Eugene N. Bush, Gilbert Diaz, Gary Bammert, A. T. Nguyen, Edwin S. Johnson, Judith Knittle,t and Jonathan Greer TAP Pharmaceuticals, Inc., and the Pharmaceutical Products Division of Abbott Laboratories, Abbott Laboratories, Abbott Park, Illinois 60064 Received October 27, 1993"
A novel series of octapeptide LHRH antagonists was designed on the basis of the structure of the (2-9) fragment of a LHRH agonist. By adopting a systematic SAR study, we were able to improve first the in vitro activity and then the in vivo LH suppression, raising them up to the range of the decapeptide antagonists NalGlu (51) and A-75998 (50), resulting in A-76154 (49). The octapeptide antagonist A-76154 is the most potent reduced-size LHRH antagonist reported. It suppresses LH in the castrated rat by over 80% for a period of 4 h following sc bolus administration of 30 pg/kg.
Introduction Antagonists of luteinizing hormone-releasinghormone (LHRH, pGlu-His-Trp-Ser-~-Gly-Leu-Arg-Pro-GlyNH2) bind to the LHRH receptor in the pituitary gonadotrophs causing inhibition of gonadotropin release, which subsequently causes the suppression of sex steroids in mammals.lI2 This property of suppressing sex hormones renders the LHRH antagonists potentially useful in the treatment of a large variety of endocrine-based conditions such as prostate cancer, endometriosis,and precocious puberty.'s2 SeveralLHRH agonists are currently used in the treatment of the above indication^.^^^ Because of their peculiar mechanism of action, the LHRH agonists suppress gonadotropins and sex hormones only following chronic administration for 2-4 weeks. In the first days of administration agonists increase plasma gonadotropins and sex steroids, occasionally causing an initial clinical flare.lp2 Antagonists, on the other hand, are devoid of this initial hormone surge. Over the last 10 years a large number of LHRH antagonistshave been synthesized and tested? All of them are either nona- or decapeptides containing four or five D-aminoacids. We previouslyreported a series of reducedsize hexapeptide analogues of LHRH, both agonists and antagonists,B which contain only one D-aminO acid and were designed based on the (3-9) fragment of the agonist In that [ D L ~ ~ ~ , P ~ O ~ N H E ~known I L H RasHleuprolide. , series, the most potent antagonist, [N-(1-naphthylpropionyl)-Ser4,~LeuG,ProgNHEtl (3-9)LHRH, had a ~ KofI 9.55 for receptor binding and a pA2 of 9.28 for in vitro LH inhibition. However, in vivo in the castrated rat model the compound was active only by infusion, presumably because of a short duration of action.6 In an attempt to increase both the in vitro potency and the in vivo duration TAP Pharmaceuticals, Inc. Partofthisworkwaspresentedasaposter attheThirteenth American PeptideSymposium,Edmonton,Alberta, Canada,June 20-25,1993. Abstr. P-070. #Abbreviations: The abbreviations for the amino acids are in accordance with the recommendations of the IUPAC-IUB Joint Commissionon BiochemicalNomenclature (Eur.J . Biochem. 1984,138,9-37). The symbols represent the L-isomer except when indicated otherwise. Additional abbreviations: ~ l N a l ~-3-(l-naphthyl)alanine; , NMeTyr, N-methyltyrosine;DLys(Nic), D-lysine(N-e-nicotiny1);Lys(Isp1, lysine(N-e-iaopfopyl);~Glu(i+)!4-@-methoxybenzoyl)-~2-aminobutyric acid; HPLC, high-pressureliquid chromatography; LH, luteinizing hormone; BC, subcutaneous;FABMS,fast atom bombardment mass spectrum;AAA, amino acid analysis. * Abstract published in Aduance ACS Abstracts, February 1, 1994. t
t
of action, we decided to evaluate other fragments of LHRH analogues as the departure point for the design of a novel series of reduced-sizeLHRH antagonists, which would be active in the range of standard decapeptide antagonists following sc bolus injection in the rat. As our starting point we selected the longer (2-9) fragment (2, Table 1) of the agonist [Phe2,~Trp6,ProgNHEtlLHRH (1): which had a pK1 of 7.08 and a pD2 of 7.64 (for definitions of pK1 and pD2, see footnotes of Table 1). The compounds were tested in vitro for LHRH rat pituitary receptor binding, for suppression of LH release from rat pituitary cells and release of histamine from rat peritoneal mast cells, and in vivo for LH suppression in the castrated rat.
Chemical Synthesis All the peptides were synthesized by solid-phase synthesis techniques (SPPS)? Peptides containing Pro-ethyl amide at the C-terminus were synthesized using Boc-Pro attached to Merrifield resin, and the peptide was cleaved from the resin withethylamine. Peptides containing DAla amide at the C-terminus were synthesized using Boc-DAla attached to 4-methylbenzhydrylamine resin, and the peptide was cleaved with anhydrous HF.&1° The synthesis protocol, cleavage of the peptide from the resin, removal of the protecting groups, workup, and HPLC purification were analogous to those described in our recent publications.819 All the peptides were characterized by analytical HPLC, FABMS, and AAA.
Biological Testing Antagonists were tested in vitro for LHRH receptor bindin$ and for LH release from cultured rat pituitary cells.6 The binding affinities are reported as ~ K I The . in vitro LH release activities of agonists are reported as pD2. The LH inhibition potencies for antagonists are reported as pA2 (for definition of pA2, see footnote of Table 1).For initial characterizationof the safety profile, the antagonists were tested for histamine release from rat peritoneal mast cells.'1J2 All antagonists were tested for LH suppression in castrated male rats following sc administration of 30 pgl kg dose, dissolved in 1:4 propylene glycollsaline solution, to groups of three animals. Serial blood samples were collected for 24 h after dosing. Plasmas were separated by centrifugation and frozen at -20 OC until assayed. LH
0022-2623/94/1837-0701$04.50/00 1994 American Chemical Society
702 Journal of Medicinal Chemistry, 1994, Vol. 37, No. 5
Haviv et al.
Table 1. In Vitro FunctionalPropertiesof Reduced-SizeLHRH Antagonists
H1-Phe2-Trp3-Sefi-Tyr5-~Trp6-Leu7-Ar~-Pro~-NHEt -~
compd 1
substitution
tRa
MH+*
pGlul
2
as above
15.83
1181
3
NAcPhe2
20.00
1223
4
phenylacetyl2
44.46
1152
5
(4-chlorophenyl)acety12
47.15
1186
6
(4-hydroxyphenyl)acety12
40.03
1168
7
indolyl-2-carbonyl2
37.12
1177
8
(4-chlorophenyl)propionylz
49.50
1200
9
(4-fluorophenyl)propiony12
43.87
1184
10
(4-methoxyphenyl)propionylZ
46.01
1196
11
[4-(trifluoromethyl)phenyl]propiony12
45.65
1234
12
(2-fluorophenyl)propionylz
44.89
1184
13
(4hydroxyphenyl)propiony12
39.67
1182
14
(3-fluorophenyl)propiony12
46.29
1184
15
[4-(trifluoromethyl)phenyl]acety12,~Trp3
35.66
1220
16
indolyl-2-carbony12,~Trp3
44.29
1205
17
5-fluoroindolyl-2-carb0nyl~,~Trp~
47.20
1195
18
(4-fluorophenyl)propiony12,~Trp3
47.14
1184
19
(4-chlorophenyl)propiony12,~Trp3
44.60
1200
20
(4-chlorophenyl)propionylz,~Tyr3
44.16
1177
21
(4-chlorophenyl)propiony12,Tyr3
40.18
1177
22
(2,4-difluorophenyl)propiony12,~Trp3
46.75
1202
23
(3,4-difluorophenyl)propiony12,~Trp3
47.51
1200
24
(4-fluorophenyl)propiony12,~4c1Phe3
49.80
1179
25
(4-fluorophenyl)propiony12,~Tyr3
41.41
1161
26
(4-fluorophenyl)propiony12,~lNa13
50.23
1195
27
(4-fluorophenyl)propiony12,1Na13
51.24
1195
28
(4-fluorophenyl)propiony12,~Pro3
41.89
1095
29
(4-fluorophenyl)propiony12,~Lys(Nic)6
37.21
1231
30
(4-fluorophenyl)propiony12,~Trps,~Lys(Nic)6
35.88
1231
31
(4-fluorophenyl)propiony12,~T~
41.34
1161
32
(4-fluorophenyl)propiony12,~4ClPhe6
50.04
1179
33
(4-fl~orophenyl)propiony1~,D~p~,DT~
41.24
1161
34
(4-fluorophenyl)propiony12,~Trp3,NMSer4
46.35
1199
35
(4-fluorophenyl)propiony12,~Trp3,Pro4
49.89
1194
36
(4-fluorophenyl)propiony12,~Trp3,Pro(40H)4
46.55
1210
37
(4-fluorophenyl)propiony12,~Trp3,NMeTyr5
35.79
1198
38
(4-fluorophenyl)propiony12,~Trp3,NMeT~,~Lys( Nic)6
34.63
1245
pKf 10.61 (k0.05) 7.08 (k0.28) 9.78 (k0.13) 9.08 (k0.33) 9.08 (10.33) 9.09 (10.28) 7.77 (k0.13) 10.28 (f0.1) 10.48 (k0.14) 10.11 (kO.19) 9.56 (k0.14) 10.24 (k0.05) 10.07 (k0.23) 8.87 (k0.16) 9.19 (10.12) 10.12 (k0.23) 7.91 (k0.15) 10.54 (10.01) 10.12 (k0.14) 10.75 (k0.25) 10.12 (f0.23) 10.40 (10.08) 10.28 (h0.27) 10.05 (10.0) 10.65 (10.06) 10.77 (fO.18) 10.77 (f0.17) 10.93 (10.21) 9.46 (k0.22) 10.63 (A0.03) 10.42 (A0.24) 10.74 (k0.21) 10.99
EDJ-
pDzd 10.81 (k0.13) 7.64 (A0.33) 8.02 (k0.16) 8.26 (A0.26) 8.26 (10.26) 7.35 (f0.31) 7.75 (k0.225) 9.90 (h0.4) 9.23 (k0.27) 8.62 (k0.22) 9.02 (10.30) 9.35 (kO.09) 7.81 (10.28) 9.05 (k0.05) 8.58 (fO.09) 9.07 (k0.05) 7.38 (10.04) 10.41 (k0.31) 9.78 (h0.06) 10.08 (k0.125) 9.07 (10.05) 10.85 (h0.05) 10.45 (i0.05) 10.75 (10.15) 9.66 (f0.13) 11.15 (k0.15) 11.30 (kO.0)
9.87 (10.15) 9.08 (4~0.18) 8.89
8.62 (A0.29) 7.68 (10.25) 10.02 (k0.28) 7.90 (k0.24) 11.05 (10.25) 10.50 (fO.10) 9.94 (10.02) 7.67 (AO.01) 7.68
(kO.0)
(hO.0)
10.48 (k0.08) 10.78 (A0.24)
11.15 (h0.05) 10.46 (AO.10)
(kO.0)
