BETIDAMINO ACID SCAN OF THE GNRH ANTAGONIST ACYLINE

Strong clinical evidence suggests that GnRH antagonists will replace G nRH agonists in a number of indications because of their ability to in hibit gonadotropin secretion as long as an adequate concentration of t he analogue is present in the circulation whereas superagonists will t ake approximately 2 weeks to desensitize the gonadotrophs. Until recen tly, antagonists were either too weak and/or would release histamine. Azaline B([Ac-D2Nal(1),D4Cpa(2),D3Pal(3), 4Aph(5)(atz),D4Aph(6)(atz),I Lys(8),DAla(10)]GnRH) and long-acting members of the azaline family D3 Pal-Ser-4Aph(X)-D4Aph(Y)-Leu-ILys-Pro-DAla-NH2), however, appear to be promising drug candidates. Because these antagonists tend to form gel s (due to the formation of beta-sheet structures) and, as a result, ar e not readily amenable to formulation for long-term delivery, we have investigated ways of increasing hydrophilicity while retaining high po tency and lack of histamine releasing activity. Betidamino acids (a co ntraction of ''beta'' position and ''amide'') are N'-monoacylated (opt ionally, N'-monoacylated and N-mono- or N,N'-dialkylated) aminoglycine derivatives in which each N'-acyl/alkyl group may mimic naturally occ urring amino acid side chains or introduce novel functionalities. We h ave used unresolved N-alpha-Boc,N'(alpha)-Fmoc-aminoglycine, and N-alp ha-Boc,N'(alpha)(CH3)Fmoc-aminoglycine as templates for the introducti on of betidamino acids in acyline Pal-Ser-4Aph(Ac)-D4Aph(Ac)-Leu-Ilys- Pro-DAla-NH2), a long acting member of the azaline B family, to test b iocompatibility of these betide derivatives. Diastereomeric peptides c ould be separated using RP-HPLC in most cases. Biological results obta ined in vitro (binding affinity to rat pituitary gland membranes) and in vivo (rat antiovulatory assay, AOA) indicate in most cases small di fferences in relative potencies (< 5-fold) between the D-and L-nonalky lated betidamino acid-containing acylines. Importantly, most betide di astereomers have high affinity for the GnRH receptor and were equipote nt with acyline in the AOA. Greater differences in affinity and potenc y between diastereomers were observed after introduction of a methyl g roup on the side chain nitrogen (''beta'' position) of the same analog ues, with one of the diastereomer having an affinity and a potency in the AOA equivalent to that of acyline. These results suggest that chir ality at the cc-carbon coupled to side chain orientation is important for receptor recognition. The duration of action of some of the most p otent analogues 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. Data suggest that introduction of a b etidamino acid results in reduction of duration of action. Also, intro duction of betidamino acids results in peptides with increased hydroph ilicity (as determined by elution times on C-18 silicas at pH 7.3) com pared to that of the parent Compound. N'-Methyl substitution results i n parallel increase in retention times on C-18 silicas as expected.