Development and evaluation of gonadotropin releasing hormone SPECT radioligands

Introduction: The gonadotropin-releasing hormone (GnRH) is involved in the hypothalamic-pituitary-gonadal axis (HPG axis) and its main function is to regulate reproduction. However, the distribution of GnRH-Receptor (GnRH-R) in the brain is especially interesting, because it density may mirror Alzheimer Disease (AD).1-2 The aim of this study was to synthesize small molecule antagonists for the GnRH-R and evaluate them as possible radiotracers. Materials and Method: Initially, we developed a library of novel GnRH-R antagonists using an approach of late-stage diversification via copper-catalyzed azide-alkyne cycloreaction (CuAAC). Concentration dependent competition studies were performed in vitro in presence of 5 nM GnRH antagonist. The receptor binding inhibition was measured in division arrested cell lines stably expressing functional human GnRH-R.3 Furthermore, in order to characterize these promising GnRH-antagonists as potential SPECT radiotracers, a modification was done for the CuAAC to include iodine. Two novel highly promising candidates were then discovered. Na123I radiolabeling and a competitive binding study were performed.4 Stability of these two candidates was tested in rat and human serum for up to 22 hours and analyzed on a LC-MS. The phase I metabolic profile of both candidates was assessed in human and rat microsomes by LC-MS. In addition, the brain GNRH-R binding of the candidates was evaluated in rat tissue cryosections by autoradiographic 125I-triptorelin competition studies. Result and discussion: A library of GnRH-R antagonists was synthesized by the use of late stage diversification CuAAC strategy; several of these compounds were found to show nM affinity to the GnRH-R compared to the reference compound.3 The use of a modified CuAAC to give 5-iodo 1,2,3-triazoles revealed two highly promising candidates. The competitive binding study for these two candidates gave nM affinity to the GnRH-R. In addition, 123I radiolabeling was performed with 76% analytical radiochemical yield.4 Serum stability studies in rat and human serum revealed the stability of the compounds (>80% remained after 22 hours). The metabolic activity revealed the metabolic profile of the two compounds in rat and human liver microsomes indicating low phase I metabolic activity, which is suitable for SPECT radiotracers. The GnRH-R binding of both compounds was demonstrated in rat brain sections. Conclusions: We have successfully synthesized a library of GnRH-R antagonist