GONADOTROPIN-RELEASING-HORMONE ANTAGONISTS - NOVEL MEMBERS OF THE AZALINE-B FAMILY

A series of antagonists of gonadotropin-releasing hormone (GnRH) homol ogous to azaline B 3),Aph(5)(Atz),DAph(6)(Atz),ILys(8),DAla(10)]GnRH) was synthesized, characterized, and tested in a rat antiovulatory assa y (AOA). Selected analogues were also tested in both an in vitro dispe rsed rat pituitary cell culture assay for inhibition of GnRH-stimulate d luteinizing hormone release and an in. vitro histamine release assay . The duration of action of some of the most potent and safest analogu es in those assays was also determined in the castrated male rat in or der to measure the extent (efficacy and duration of action) of inhibit ion of luteinizing hormone release. Structurally, this series of analo gues has novel substitutions (X and Y) in the structure of the azaline B precursor: al(3),-Aph(5)(X),DAph(6)(Y),ILys(8),DAla(10)]GnRH. These substitutions were designed to confer increased hydrophilicity as com pared 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 limit ations of the receptor's cavity. These substitutions include acylated 4-aminophenylalanine at positions 5 and/or 6 (29 analogues), N-alpha-m ethylated backbone substitutions (six analogues), N-omega-isopropylami nophenylalanine 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 test ed for long duration of action in this series, only seven a(2),DPal(3) ,Aph(5),DAph(6),ILys(8),DAla(10)]GnRH, [Ac-DNal(1),DCpa(2),DPal(3), Ap h(5)(For),DAph(6)(For), ILys(8),DAla(10)]GnRH, c-DNal(1),DCpa(2),DPal( 3),Aph(5)(Ac),DAph(6)(Ac),- ILys(8),DAla(10)]GnRH (acyline), 3),Aph(5) (Pio),DAph(6)(Pio),ILys(8,)DAla(10)]GnRh, (3),Aph(5)(Atz),DAph(6)(Ac), ILys(8),DAla(10)]GnRH, [Ac-DNalDCpa(2),DPal(3),Aph(5)(Atz-beta Ala),DA ph(6)(Atz- beta Ala),ILys(8),DAla(10)]GnRH, )(Atz-Gab),DAph(6)(Atz-Gab ),ILys(8),DAla(10)]GnRH) had relative potencies and/or duration of act ion comparable to those of azaline B. The others were one-half to one- tenth as effective as azaline B. N-alpha-Methylated backbone substitut ions at position 5 yielded analogues that were significantly more hydr ophilic presumably because of the breakage of the NH alpha-Tyr(5) to A rg(8)-CO hydrogen bond reported to stabilize a beta-turn encompassing residues 5-8 and which favored beta-sheet formation as shown earlier b y Haviv et al.(2) This substitution resulted, however, in an increased potency in the histamine release assay and in significantly shorter d uration 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. Cha nges in chirality at position 1 or 10 resulted in analogues that were one-tenth and one-half as potent, respectively, as acyline. Introducti on of a relatively hydrophilic acetylated residue in position 1 (Ac-4- aminophenylalanine, Ac-2-quinolylalanine, Ac-3-quinolylalanine) also r esulted in potent analogues in the AOA in the latter two cases (yet ve ry short acting in the case of ,Aph(5)(Atz),DAph(6)(Atz),ILys(8),DAla( 10)]-GnRH). Introduction of either mesityl, (2-chlorophenyl)isourea, o r (3-chlorophenyl)isourea as a substituent on the 4-amino function at residues 5 and 6 of the azaline B precursor was considerably less succ essful. In this article, we describe in details, improved synthetic pr otocols for all novel amino acis, N alpha-methylation of amino acids o n the resin, and elimination of the undesired N omega-methylation of p yridylalanine at position 3 as the result of base treatment (piperidin e or hydrazine) during the deprotection of the Fmoc group or formation of the triazole moiety in the presence of CH2Cl2.