Iterative Approach to the Discovery of Novel Degarelix Analogues:  Substitutions at Positions 3, 7, and 8. Part II

Degarelix (FE200486, Ac-<SCP>d</SCP>-2Nal¹-<SCP>d</SCP>-4Cpa²-<SCP>d</SCP>-3Pal³-Ser⁴-4Aph(<SCP>l</SCP>-Hor)⁵-<SCP>d</SCP>-4Aph(Cbm)⁶-Leu⁷-ILys⁸-Pro⁹-<SCP>d</SCP>-Ala¹⁰-NH<INF>2</INF>) is a potent and very long acting antagonist of gonadotropin-releasing hormone (GnRH) after subcutaneous administration in mammals including humans. Analogues of degarelix were synthesized, characterized, and screened for the antagonism of GnRH-induced response in a reporter gene assay in HEK-293 cells expressing the human GnRH receptor. The duration of action was also determined in the castrated male rat assay to measure the extent (efficacy and duration of action) of inhibition of luteinizing hormone (LH) release. Structurally, this series of analogues has novel substitutions at positions 3, 7, and 8 and N^α-methylation at positions 6, 7, and 8 in the structure of degarelix. These substitutions were designed to probe the spatial limitations of the receptor's cavity and to map the steric and ionic boundaries. Some functional groups were introduced that were hypothesized to influence the phamacokinetic properties of the analogues such as bioavailability, solubility, intra- or intermolecular hydrogen bond forming capacity, and ability to bind carrier proteins. Substitutions at positions 3 ([N^β-(2-pyridyl-methyl)<SCP>d</SCP>-Dap³]degarelix, IC<INF>50</INF> = 2.71 nM) (<BO>5</BO>), 7 ([Pra⁷]degarelix, IC<INF>50</INF> = 2.11 nM) (<BO>16</BO>), and 8 ([N^δ-(IGly)Orn⁸]degarelix, IC<INF>50</INF> = 1.38 nM) (<BO>20</BO>) and N-methylation ([N^α-methyl-Leu⁷]degarelix, IC<INF>50</INF> = 1.47 nM) (<BO>32</BO>) yielded analogues that were equipotent to degarelix (<BO>2</BO>) in vitro (IC<INF>50</INF> = 1.64 nM) but shorter acting in vivo. Out of the 33 novel analogues tested for the duration of action in this series, two analogues ([N^ε-cyclohexyl-Lys⁸]degarelix, IC<INF>50</INF> = 1.50 nM) (<BO>23</BO>) and ([N^β-(IβAla)Dap⁸]degarelix, IC<INF>50</INF> = 1.98 nM) (<BO>26</BO>) had antagonist potencies and duration of action similar to that of azaline B {inhibited LH (>80%) release for >72 h after sc injection to castrated male rats at a standard dose of 50 μg/rat in 5% mannitol}. Under similar conditions analogues ([N^γ-(IGly)Dab⁸]degarelix, IC<INF>50</INF> = 1.56 nM) (<BO>21</BO>) and ([IOrn⁸]degarelix, IC<INF>50</INF> = 1.72 nM) (<BO>18</BO>) had a longer duration of action {inhibited LH (>96 h) release} than azaline B; however they were shorter acting than degarelix. Hydrophilicity of these analogues, a potential measure of their ability to be formulated for sustained release, was determined using RP-HPLC at neutral pH yielding analogues with shorter as well as longer retention times. No correlation was found between retention times and antagonist potency or duration of action.