A series of antagonists of gonadotropin-releasing hormone (GnRH) of the general formula Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph/4Amf(P)-D4Aph/D4Amf(Q)-Leu-ILys-Pro-DAla-NH2 was synthesized, characterized, and screened for duration of inhibition of luteinizing hormone release in a castrated male rat assay. Selected analogues were tested in a reporter gene assay (IC50 and pA2) and an in vitro histamine release assay. P and Q contain urea/carbamoyl functionalities designed to increase potential intra- and intermolecular hydrogen bonding opportunities for structural stabilization and peptide/receptor interactions, respectively. These substitutions resulted in analogues with increased hydrophilicity and a lesser propensity to form gels in aqueous solution than azaline B [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Atz)-D4Aph(Atz)-Leu-ILys-Pro-DAla-NH2 with Atz = 3'-amino-1H-1',2',4'-triazol-5'-yl, 5], and in some cases they resulted in a significant increase in duration of action after subcutaneous (s.c.) administration. Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(L-hydroorotyl)-D4Aph(carbamoyl)-Leu-ILys-Pro-DAla-NH2 (acetate salt is FE200486) (31) and eight other congeners (20, 35, 37, 39, 41, 45-47) were identified that exhibited significantly longer duration of action than acyline [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Ac)-D4Aph(Ac)-Leu-ILys-Pro-DAla-NH2] (6) when administered subcutaneously in castrated male rats at a dose of 50 microg in 100 microL of phosphate buffer. No correlation was found between retention times on a C18 reverse phase column using a triethylammonium phosphate buffer at pH 7.0 (a measure of hydrophilicity) or affinity in an in vitro human GnRH report gene assay (pA2) and duration of action. FE200486 was selected for preclinical studies, and some of its properties were compared to those of other clinical candidates. In the intact rat, ganirelix, abarelix, azaline B, and FE200486 inhibited plasma testosterone for 1, 1, 14, and 57 days, respectively, at 2 mg/kg s.c. in 5% mannitol (injection volume = 20 microL). Based on the information that 31, 33, 35 and 37 were significantly shorter acting than acyline or azaline B after intravenous administration (100 microg/rat), we surmised that the very long duration of action of the related FE200486 (for example) was likely due to unique physicochemical properties such as solubility in aqueous milieu, comparatively low propensity to form gels, and ability to diffuse at high concentrations in a manner similar to that described for slow release formulations of peptides. Indeed, in rats injected s.c. with FE200486 (2 mg/kg), plasmatic concentrations of FE200486 remained above 5 ng/mL until day 41, and the time after which they dropped below 3 ng/mL and plasma LH levels started rising until full recovery was reached at day 84 with levels of FE200486 hovering around 1 ng/mL. Additionally, FE200486 was less potent at releasing histamine from isolated rat mast cells than any of the GnRH antagonists presently described in preclinical reports.

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Reactions of carbanions of aryl chloromethyl and dichloromethyl sulfones 3 and 4 and tert‐butyl chloro‐ and dichloroacetates (5 and 6) with 1‐halo‐2,4‐dinitrobenzenes 1 and 2 proceed by nucleophilic replacement of a hydrogen atom. The SNAr of the halogen atom occurs to a negligible extent.

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ABSTRACT:Degarelix is a novel competitive gonadotropin-releasing hormone receptor blocker (antagonist). In this study, the nonclinical metabolism and excretion of degarelix was investigated in SpragueDawley rat, beagle dog, and cynomolgus monkey. Degarelix was found to be stable when incubated in microsomes and cryopreserved hepatocytes from animal liver tissue. Absorption, distribution, metabolism, and excretion studies in male rat, dog, and monkey showed that after a subcutaneous dose of tritium-labeled degarelix, the peptide was rapidly absorbed with C max in plasma of 1 to 2 h. The predominant route of excretion was via the kidneys and the bile. In rat and dog, most of the degarelix dose was eliminated within 48 h via urine and feces in equal amounts (40-50% in each matrix), whereas in monkey the major route of excretion was fecal (50%) and renal (22%). In plasma and urine samples from all three species, mainly intact degarelix was detected. In bile and feces samples from rats and dogs, the same truncated peptides of the parent decapeptide were detected. The major metabolites identified represented the N-terminal tetrapeptide, the pentapeptide, and the heptapeptide. From the animal studies, it could be concluded that degarelix is subject to common peptidic degradation in the liver and bile and is fully excreted via metabolic and biliary (as metabolites and parent compound) and urinary (mainly as parent compound) pathways. Systemic exposure to metabolic products seems to be low.