Gonadotropin-releasing hormone antagonists with N.omega.-triazolylornithine, -lysine, or -p-aminophenylalanine residues at positions 5 and 6

Abstract

In order to be used as fertility regulators in humans, gonadotropin releasing hormone (GnRH) antagonists must be extremely potent and long acting and exhibit negligible side effects such as stimulating histamine release. To this aim, we have recently synthesized a series of analogues with the standard Ac-DNal1-DCpa2-DPal3 substitutions, where the N omega-amino function of ornithine, lysine, or p-aminophenylalanine (Aph) was converted to the aminotriazolyl (atz) derivatives at positions 5 and 6 with further modifications at positions 7 and 10. The analogues were tested for their ability to bind to pituitary cell membranes, to release histamine in a mast cell assay, to inhibit luteinizing hormone (LH) secretion by castrated male rats or cultured pituitary cells, and to interfere with the ovulation in intact female rats. While the subcutaneous (sc) injection of 50 micrograms of Azaline A (7, [Ac-DNal1,DCpa2,DPal3,Lys5(atz),DLys6++ +(atz),ILys8,DAla10]GnRH) dissolved in 0.2 mL of an aqueous media significantly inhibited LH release in the castrated male rat for 24 h, the same dose of Azaline B (11), [Ac-DNal1,DCpa2,DPal3,Aph5(atz),DAph6++ +(atz),ILys8,DAla10]GnRH, inhibited LH release for 72 h. A similar long duration of action was observed for Antide ([Ac-DNal1,DCpa2,DPal3,Lys5(Nic),DLys6(Nic ),ILys8,DAla10]GnRH) but not for Nal-Glu ([Ac-DNal1,DCpa2,DPal3,Arg5,4-(pmethoxybenzoy l)-D-2-Abu6,DAla10]GnRH). In the same paradigm, a 5-fold dilution of the peptide (50 micrograms in 1 mL) and the use of three injection sites rather than one resulted in significantly shorter duration of action for most of the peptides tested. This suggested that long duration of action might be the result of slow release from the injection site(s). In order to investigate this possibility, Nal-Glu and Azaline B were injected intravenously (i.v.) at three doses (10, 50, 250 micrograms) to castrated male rats. At all doses, both peptides significantly lowered LH levels for 8 h. By 24 h, Nal-Glu (250 micrograms) and Azaline B (50 and 250 micrograms) still measurably inhibited LH secretion. Finally, only Azaline B (250 micrograms) was still active at 48 h. These findings demonstrate that subtle structural modifications will yield peptides with different half-lives after iv administration. These findings led us to investigate the effects of other structural modifications on duration of action. We observed that systematic substitutions at positions 7 (NMeLeu) and 10 (Pro9-NHEt, and Gly-NH2) were found to be deleterious. Of interest was the observation that only the DAla10-NH2 substitution led to long duration of action and enzymatic stability under the conditions tested.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological Characterization of a Novel, Orally Active Small Molecule Gonadotropin-Releasing Hormone (GnRH) Antagonist Using Castrated and Intact Rats

“…In competition binding assays, GnRH-A bound with comparable affinity to human, rat, and mouse receptors, whereas GnRH and antide exhibited marked species differences but corresponded well with historical values reported in the literature for these peptides at these receptors (Rivier et al, 1992;Chi et al, 1993;Perrin et al, 1993;Flanagan et al, 1994). In contrast, CMPD1 bound with similar low nanomolar affinities to recombinant human and mouse and rat pituitary GnRH receptors.…”

Astressin Analogues (Corticotropin-Releasing Factor Antagonists) with Extended Duration of Action in the Rat

“…We also noted that further deletions (residues 8-14) produced antagonists such as astressin {cyclo (30)(31)(32)(33) were ca. twice and 1/100 as potent as astressin, respectively, suggesting a putative turn that encompasses residues 30-33 (previous paper: Koerber et al J. Med.…”

“…While oCRF and hCRF have been used in the clinic, CRF antagonists | Abbreviations: IUPAC rules are used for nomenclature of peptides including one-letter codes for amino acids. Also: Ac, acetyl; ACTH, adrenocorticotropin hormone; adx, adrenalectomized; astressin, cyclo- (30)(31)(32)(33)[DPhe 12 ,Nle 21,38 ,Glu 30 ,Lys 33 ]hCRF(12-41); Boc, tert-butyloxycarbonyl; BOP, benzotriazolyloxy-tris(dimethylamino)phosphonium hexafluorophosphate; BSA, bovine serum albumin; CD, circular dichroism; CRF, corticotropin-releasing factor (o, ovine; h, human); CSF, cerebrospinal fluid; CZE, capillary zone electrophoresis; DCM, dichloromethane; DIC, diisopropylcarbodiimide; DMF, dimethylformamide; FBS, fetal bovine serum; Fmoc, 9-fluorenylmethoxycarbonyl; HBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; HF, hydrogen fluoride; HOBt, 1-hydroxybenzotriazole; IA, intrinsic activity; ic, intracisternal; LSIMS, liquid secondary ion mass spectrometry; MBHA, 4-methylbenzhydrylamine; NMP, N-methylpyrrolidinone; NMR, nuclear magnetic resonance; OFm, O-fluorenylmethyl; SAR, structure-activity relationships; TBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate; TEAP 2.25, 4.5, and 6.5, triethylammonium phosphate, pH 2.25, 4.5, and 6.5; TFA, trifluoroacetic acid; TFE, trifluoroethanol.…”

Novel Analogues of Degarelix Incorporating Hydroxy-, Methoxy-, and Pegylated-Urea Moieties at Positions 3, 5, 6 and the N-Terminus. Part III

“…We had shown however, that such substitution also increased the propensity of these analogues to release histamine. 28 Since histamine release could be traced to the presence of a hydrophobic N-terminus and basic residues at position 5, 6 and 8, (best illustrated for Nal-Arg 29 and Nal-Glu 25 in Table 2) we investigated whether it was still true after the introduction of neutral functionalities such as a hydroxy-carbamoyl (7), methoxy-carbamoyl (22, 23) and PEG (9, 13 and 14) at positions 5, 6 (on the para-amino function of 4-aminophenylalanine) and at the N-terminus. Interestingly, the expectation that 23 would be more potent than 22 at releasing histamine as a result of the introduction of the N α -methylation of 4Aph 5 did not materialize, suggesting a relatively important contribution of the methoxy-carbamoyl group on the para-amino function of 4Aph 5 to histamine release.…”

“…Compounds 26-28 had the favored methoxy-urea or PEG functionalities at 4Aph 5 and D-4Aph 6 and their N-terminal was either acylated with butanoic acid (26) or octanoic acid (27) and pegylated (28). The short duration of action of 26 and 28 and the inactivity of 27, despite significant antagonist potency, was different from that of the same analogue 22 acetylated at the N-terminus.…”