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To whom correspondence should be addressedWhile the clinical significance of gonadotrophin-releasing hormone (GnRH)agonists is well recognized, the potential use of GnRH antagonists in humansawaits the availability of potent analogues with no untoward side-effects. Wehave designed, synthesized and tested several hundred linear and cyclic analogues(agonists and antagonists) of GnRH in different rat models; some have highhistamine releasing activity and others have poor solubility in aqueous bufferswith a pH >6.0. Furthermore, we have identified analogues exhibiting short( 72 h) duration of action in the rat(50 |Xg s.c. dose/rat). We have concluded that the basis for such resistance todegradation and elimination must be specific. In order to gain further informationon the optimal nature and sterical requirements of side-chains, preliminaryexperiments were carried out using betidamino acids. Finally, mono- and dicyclicanalogues of GnRH with potencies comparable with that of the most potentlinear analogues were also obtained. Our approach to the development of suchanalogues included the use of nuclear magnetic resonance and computationaltechniques as well as that of state-of-the-art synthetic approaches. We intend touse the information derived from these structure/activity relationship studies todesign conformationally-similar peptido-mimetics.Key words: acyline/azaline B/betidamino acids/drug design/GnRH antagonists

A series of GnRH antagonists with substitutions at positions 1, 2, 3, 5 and 6 that included the recently reported homoArg-N omega-cyano-N omega'-alkyl- or Lysine-N epsilon-5'-(3-amino-1H-1,2,4-triazole) [Lys(atz)] amino acid derivatives was synthesized, characterized and tested for antiovulatory and anaphylactoid activities and binding affinity. Overall, these analogs were found to be considerably more soluble at neutral pH than their homologs Nal-Glu or Antide. The decapeptides with these substitutions in positions 5 and/or 6 retained high in vivo potency while those with similar substitutions at positions 1, 2 and 3 were significantly less potent than Nal-Glu or Antide. Of the 16 new analogs reported here, Azaline (Ac-DNal1, DCpa2, DPal3, Lys5(atz),DLys6(atz), ILys8,DA1a10]-GnRH) showed the most promising physico-chemical and biological properties [Lys(atz) = N epsilon-5'-(3-amino-1H-1,2,4-triazole) lysine]. Azaline is readily soluble in dilute buffers at pH 7.0, completely inhibits ovulation at 2.0 to 3.0 micrograms per rat, is equipotent to GnRH in releasing histamine in the rat and has a weaker anaphylactoid response in the rat than other analogs such as Nal-Glu or even Antide.

Binding of the GnRH agonist [DAla6,NMe-Leu7,Pro9Net]GnRH to bovine anterior pituitary membranes is inhibited by guanyl nucleotides. The effect of guanyl nucleotides is temperature dependent, in that significant binding inhibition is observed when the receptor-hormone interaction is measured at 37 C, and no inhibition is seen at 4 C. Micromolar concentrations of the nonhydrolyzable GTP analog 5'-guanylylimidodiphosphate [Gpp(NH)p] displace the bound agonist in a dose-dependent manner, with half-maximal displacement occurring in a concentration range of 0.1-0.5 microM, and maximum displacement occurring at a concentration of 50 microM Gpp(NH)p. At a concentration of 50 microM, the other nucleotides GTP and GDP inhibit binding to a lesser extent, while GMP, cGMP, 5'-adenylylimidodiphosphate [App(NH)p], ATP, and cAMP have no effect on the binding. At 37 C, Gpp(NH)p reduces the affinity of the agonist by a factor of 6 and increases its dissociation rate. In the presence of Gpp(NH)p at 37 C, there is also a 2-fold increase in the total number of binding sites. Under the same conditions as those used for the agonist, there is no displacement of the bound antagonist [Ac-D2Nal1,4ClDPhe2,D3Pal3,DLys6,Lys8,D Ala10]-GnRH by doses up to 50 microM Gpp(NH)p. The modulation of the binding of the agonist, but not that of the antagonist, by guanyl nucleotides is characteristic of receptors that are coupled to GTP-binding proteins. Thus, the GnRH receptor appears to be coupled to a GTP-binding protein that may play a role in the mechanism of action of GnRH at the pituitary.

In order to minimize the adverse effect of histamine release in the rat of some gonadotropin releasing hormone (GnRH) antagonists, such as [Ac-D2Nal1,D4FPhe2,DTrp3,DArg6]-GnRH, new structures with modifications at positions 1, 2, 3, 5, 6, 7, and 10 were synthesized and tested in several biological systems. In vitro: the affinity for the pituitary GnRH receptor was measured as was the ability of the analogues to inhibit GnRH-stimulated release of luteinizing hormone (LH) by dispersed anterior pituitary cells in culture and to release histamine from rat mast cells. In vivo: inhibition of ovulation in the cycling rat was determined after subcutaneous (sc) injection of the peptides at noon on the day of proestrus; the duration of action of the peptides was evaluated by measuring LH levels in the castrated male rat after sc injection of some selected analogues. [Ac-D2Nal1,D4ClPhe2,D3Pal3,Arg5,D-4-p-methoxy benzoyl-2-aminobutyric acid6,DAla10]-GnRH was found to be one of the most potent analogues of this series, causing a 100% inhibition of ovulation at 5 micrograms/kg or less. Release of histamine was observed at doses 10-25 times that required for [Ac-D2Nal1,D4FPhe2,DTrp3,DArg6]-GnRH. Thus, introduction of arginine in position 5 with a hydrophobic amino acid in position 6 is compatible with high potency in several biological systems and results in compounds with lowered potency to release histamine compared to homologous peptides with tyrosine in position 5 and D-arginine in position 6.

Experimental protocols have been developed for the synthesis and resolution of numerous ring substituted phenylalanines and tryptophans in half mole quantities. Physical constants on these amino acids are given and their behavior on ion exchange supports (amino acid analyzer and post column ortho-phthalaldehyde derivatization) as well as that of some selected N-methylated amino acids is described. Those amino acids were then derivatized (N alpha-protection with the t-butyloxycarbonyl group) for solid phase peptide synthesis.

Experimental protocols have been developed for the synthesis and resolution of numerous ring substituted phenylalanines and tryptophans in half mole quantities. Physical constants on these amino acids are given and their behavior on ion exchange supports (amino acid analyzer and post column ortho-phthalaldehyde derivatization) as well as that of some selected N-methylated amino acids is described. Those amino acids were then derivatized (N alpha-protection with the t-butyloxycarbonyl group) for solid phase peptide synthesis.

In order to minimize the adverse effect of histamine release in the rat of some gonadotropin releasing hormone (GnRH) antagonists, such as [Ac-D2Nal1,D4FPhe2,DTrp3,DArg6]-GnRH, new structures with modifications at positions 1, 2, 3, 5, 6, 7, and 10 were synthesized and tested in several biological systems. In vitro: the affinity for the pituitary GnRH receptor was measured as was the ability of the analogues to inhibit GnRH-stimulated release of luteinizing hormone (LH) by dispersed anterior pituitary cells in culture and to release histamine from rat mast cells. In vivo: inhibition of ovulation in the cycling rat was determined after subcutaneous (sc) injection of the peptides at noon on the day of proestrus; the duration of action of the peptides was evaluated by measuring LH levels in the castrated male rat after sc injection of some selected analogues. [Ac-D2Nal1,D4ClPhe2,D3Pal3,Arg5,D-4-p-methoxy benzoyl-2-aminobutyric acid6,DAla10]-GnRH was found to be one of the most potent analogues of this series, causing a 100% inhibition of ovulation at 5 micrograms/kg or less. Release of histamine was observed at doses 10-25 times that required for [Ac-D2Nal1,D4FPhe2,DTrp3,DArg6]-GnRH. Thus, introduction of arginine in position 5 with a hydrophobic amino acid in position 6 is compatible with high potency in several biological systems and results in compounds with lowered potency to release histamine compared to homologous peptides with tyrosine in position 5 and D-arginine in position 6.

Three new resins have been developed that allow for the solid phase synthesis of C-terminal peptide N-alkylamides using Boc amino acids, usual side chain protecting groups and hydrogen fluoride cleavage and deprotection. These resins were prepared by reacting the appropriate alkylamine (NH2CH3, NH2CH2CH3, NH2CH2CF3) to Merrifield's 1% divinylbenzene cross-linked chloromethylated polystyrene resin. The application of these resins to the synthesis of C-terminal GnRH N-alkylamides illustrates the versatility of this approach. GnRH analogs were tested for their ability to release LH from cultured rat anterior pituitary cells. [DGlu6, Pro9-NHCH2CH3]-GnRH was synthesized for the first time using the solid phase approach and found to be three times more potent than [DGlu6]-GnRH. Other analogs including [DTrp6, Pro9-NHCH2CH3]-GnRH, [DAla6, Pro9-NHCH2CF3]-GnRH and related peptides were found to be equipotent and to have the same properties (HPLC retention times, amino acid analysis and specific rotation) as the corresponding peptides synthesized using less amenable strategies; yields were equivalent or better than those reported earlier.

The preparation of “Nal—Glu” antagonist (Ac— d -Nal— d -Cpa— d -Pal—Ser—Arg— d -2-amino-5-oxo-5-(4-methoxyphenyl)pentanoic acid—Leu—Arg—Pro— d -Ala—NH 2 ) was accomplished in two steps: (i) preparation of [Ac— d -Nal 1 , d -Cpa 2 , d -Pal 3 , Arg 5 , d -Glu 6 , d -Ala 10 ]-GnRH via standard solid-phase synthetic techniques and (ii) acylation of anisole (Friedel—Crafts) by the glutamic acid residue in position 6. The preparative-scale, reversed-phase high-performance liquid chromatographic (HPLC) purification of the crude “Nal—Glu” antagonist employs first a triethylammonium phosphate (TEAP) (Ph 2.25)—acetonitrile solvent system, followed by an HPLC-based desalting procedure, yielding the acetate salt of the peptide. This repetitive process of purification in TEAP—acetonitrile, followed by counter-ion exchange with 0.5% acetic acid—acetonitrile is highly reproducible and allows large amounts of a given peptide to be purified efficiently in a batchwise fashion. The procedure described for the synthesis, purification and characterization of the “Nal—Glu” antagonist is presented as a model for the multi-gram synthesis and purification of peptides to be used in clinical investigations.