DNal1, DCpa2, DPal3, Aph5 (For), DAph6 (For), ILys8, DAla10] GnRH,[Ac-DNal1, DCpa2, DPal3Aph5 (Ac), DAph6 (Ac),-ILys8, DAla10] GnRH (acyline),[Ac-DNal1, DCpa2, DPal3, Aph5 (Pio), DAph6 (Pio), ILys8, DAla10] GnRH,[Ac-DNal1, DCpa2, DPal3Aph5 (Atz), DAph6 (Ac), ILys8, DAla10] GnRH,[Ac-DNalDCpa2, DPal3Aph5 (Atz-/3Ala), DAph6 (Atz-/3Ala), ILys8, DAla10] GnRH,[Ac-DNal1, DCpa2, DPal3, Aph5 (Atz-Gab), DAph6 (Atz-Gab), ILys8, DAla10] 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. Na-Methylated backbone substitutions at position 5 yielded analogues that were significantly more hydrophilic presumably because of the breakage of the NHa-Tyr5 to Arg8-CO hydrogenbond reported to stabilize a/3-tum encompassing residues 5-8 and which favored/?-sheet formation as shown earlier by Haviv et al. 2 This substitution resulted, however, in an increased potency in the histamine release assay and insignificantly 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 inthe 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-D2Qal1, DCpa2, DPal3, Aph5 (Atz), DAph6 (Atz), ILys8, DAla10]-GnRH). Introductionof 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, Na-methylation of amino acids on the resin, and elimination of the undesired N, u-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 CH2CI2.