Gonadotropin-releasing hormone antagonists: novel members of the azaline B family.

A series of antagonists of gonadotropin-releasing hormone (GnRH) homologous to azaline B ([Ac-DNal(1),DCpa(2),DPal(3),Aph(5)(Atz),DAph(6)(Atz),ILys(8),DAla(10)]GnRH) was synthesized, characterized, and tested in a rat antiovulatory assay (AOA). Selected analogues were also tested in both an in vitro dispersed rat pituitary cell culture assay for inhibition of GnRH-stimulated luteinizing hormone release and an in. vitro histamine release assay. The duration of action of some of the most potent and safest analogues in those assays was also determined in the castrated male rat in order to measure the extent (efficacy and duration of action) of inhibition of luteinizing hormone release. Structurally, this series of analogues has novel substitutions (X and Y) in the structure of the azaline B precursor: [Ac-DNal(1),DCpa(2),DPal(3),-Aph(5)(X),DAph(6)(Y),ILys(8),DAla(10)]GnRH. These substitutions were designed to confer increased hydrophilicity as compared to that of azaline B (determined by relative retention times on a C-18 reverse phase column using a triethylammonium phosphate buffer at pH 7.3) or to make them more easily accessible synthetically. Some bulky substituents were introduced in order to probe the spatial limitations of the receptor's cavity. These substitutions include acylated 4-aminophenylalanine at positions 5 and/or 6 (29 analogues), N-alpha-methylated backbone substitutions (six analogues), N-omega-isopropylaminophenylalanine at position 8, and hydrophilic amino acids at position 1. Out of 20 novel analogues tested for long duration position 8, and hydrophilic amino acids at position 1. Out of 20 novel analogues tested for long duration of action in this series, only seven ([Ac-DNal(1),DCpa(2),DPal(3),Aph(5),DAph(6),ILys(8),DAla(10)]GnRH, [Ac-DNal(1),DCpa(2),DPal(3), Aph(5)(For),DAph(6)(For), ILys(8),DAla(10)]GnRH, [Ac-DNal(1),DCpa(2),DPal(3),Aph(5)(Ac),DAph(6)(Ac),- ILys(8),DAla(10)]GnRH (acyline), [Ac-DNal(1),DCpa(2),DPal(3),Aph(5)(Pio),DAph(6)(Pio),ILys(8,)DAla(10)]GnRh, [Ac-DNal(1),DCpa(2),DPal(3),Aph(5)(Atz),DAph(6)(Ac),ILys(8),DAla(10)]GnRH, [Ac-DNalDCpa(2),DPal(3),Aph(5)(Atz-beta Ala),DAph(6)(Atz- beta Ala),ILys(8),DAla(10)]GnRH, [Ac-DNal(1),DCpa2,DPal(3),Aph(5)(Atz-Gab),DAph(6)(Atz-Gab),ILys(8),DAla(10)]GnRH) had relative potencies and/or duration of action comparable to those of azaline B. The others were one-half to one-tenth as effective as azaline B. N-alpha-Methylated backbone substitutions at position 5 yielded analogues that were significantly more hydrophilic presumably because of the breakage of the NH alpha-Tyr(5) to Arg(8)-CO hydrogen bond reported to stabilize a beta-turn encompassing residues 5-8 and which favored beta-sheet formation as shown earlier by Haviv et al.(2) This substitution resulted, however, in an increased potency in the histamine release assay and in significantly shorter duration of action.(3) Similarly, attempts at replacing isopropyllysine in position 8 by either isopropyl-4-aminophenylalanine or isopropyl-4(aminomethyl)phenylalanine resulted in loss of potency in the AOA. Changes in chirality at position 1 or 10 resulted in analogues that were one-tenth and one-half as potent, respectively, as acyline. Introduction of a relatively hydrophilic acetylated residue in position 1 (Ac-4-aminophenylalanine, Ac-2-quinolylalanine, Ac-3-quinolylalanine) also resulted in potent analogues in the AOA in the latter two cases (yet very short acting in the case of ([Ac-D2Qal(1),DCpa(2),DPal(3),Aph(5)(Atz),DAph(6)(Atz),ILys(8),DAla(10)]-GnRH). Introduction of either mesityl, (2-chlorophenyl)isourea, or (3-chlorophenyl)isourea as a substituent on the 4-amino function at residues 5 and 6 of the azaline B precursor was considerably less successful. In this article, we describe in details, improved synthetic protocols for all novel amino acis, N alpha-methylation of amino acids on the resin, and elimination of the undesired N omega-methylation of pyridylalanine at position 3 as the result of base treatment (piperidine or hydrazine) during the deprotection of the Fmoc group or formation of the triazole moiety in the presence of CH2Cl2.