Communications ChemMedChem doi.org/10.1002/cmdc.202000256 1 2 3 Discovery of a Lead Brain-Penetrating Gonadotropin- 4 5 Releasing Hormone Receptor Antagonist with Saturable 6 Binding in Brain 7 8 Roberto B. W. Bekker,[a] Richard Fjellaksel,[b, c, d] Trine Hjornevik,[e] Syed Nuruddin,[f] 9 Waqas Rafique,[a] Jørn H. Hansen,[d] Rune Sundset,[b, c] Ira H. Haraldsen,[g] and 10 [a, f, g] 11 Patrick J. Riss* 12 13 We report the synthesis, radiosynthesis and biological charac- conditions in comparison to pretreatment with a receptor- 14 saturating dose of GnRH antagonist revealed saturable uptake 15 terisation of two gonadotropin-releasing hormone receptor À (0.1%ID/mL) into the brain. 16 (GnRH R) antagonists with nanomolar binding affinity. A small À 17 library of GnRH R antagonists was synthesised in 20–67% 18 overall yield with the aim of identifying a high-affinity Our attention has been drawn towards the gonadotropin 19 antagonist capable of crossing the blood–brain barrier. Binding À releasing hormone receptor (GnRHÀ R) because of its role in 20 affinity to rat GnRH R was determined by autoradiography in 125 hormone related behaviour and in the pathophysiology of 21 competitive-binding studies against [ I]buserelin, and inhib- several diseases. Gonadotropin releasing hormone is a decapep- 22 ition constants were calculated by using the Cheng–Prusoff tide hormone (pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ) 23 equation. The radioligands were obtained in 46–79% radio- 2 > and a key neurotransmitter in the hypothalamus-pituitary- 24 chemical yield and 95% purity and with a molar activity of gonadal axis. Apart from the classical role in reproduction, 25 19–38 MBq/nmol by direct nucleophilic radiofluorination. Posi- GnRH plays a role in cell proliferation,[1,2] tumour progression,[3–5] 26 tron emission tomography imaging in rat under baseline metastasis[4] and angiogenesis,[5] and has been linked to neuro- 27 degenerative cascades, for example, in dementia through 28 [a] R. B. W. Bekker, Dr. W. Rafique, Prof. P. J. Riss nonclassical mechanisms.[6–8] In circulation, the peptide acts as 29 Department of Chemistry, University of Oslo an endocrine signalling hormone that mediates the release of 30 Sem Sælands vei, 260371 Oslo (Norway) E-mail: [email protected] follicle stimulating hormone and luteinising hormone by 31 [b] Dr. R. Fjellaksel, Prof. R. Sundset GnRHÀ R activation in the pituitary gland and other regions.[2,5–8] 32 Department of Clinical Medicine As such, hormonal signalling cascades following GnRHÀ R 33 UiT The Arctic University of Norway Hansine Hansens veg 18 activation might catalyse the multiple actions of GnRH and 34 9019 Tromsø (Norway) mediate hormone signalling in the central nervous system 35 [c] Dr. R. Fjellaksel, Prof. R. Sundset (CNS). The GnRHÀ R is widely expressed in CNS; highester levels 36 PET Imaging Center University Hospital of North Norway are found in the olfactory bulb, hypothalamus, and 37 Sykehusvegen 38, 9019 Tromsø (Norway) hippocampus.[5,7–9] Furthermore, GnRHÀ R plays an established 38 [d] Dr. R. Fjellaksel, Prof. J. H. Hansen role in clinical care, for example, in the treatment of hormone- 39 Department of Chemistry UiT – The Arctic University of Norway responsive cancers, reproductive disorders and for behavioural 40 Hansine Hansens veg 18 modification of sexual offenders through these mechanisms. 41 9019 Tromsø (Norway) Recent observations also demonstrate crosstalk between 42 [e] Dr. T. Hjornevik Department of Diagnostic Physics GnRHÀ R and growth factor receptors.[1–6] 43 Oslo University Hospital Few publications have investigated GnRHÀ R antagonists for 44 Sognsvannsveien 20, 0372 Oslo (Norway) PET. However, the role GnRH plays in pathophysiology of 45 [f] Dr. S. Nuruddin, Prof. P. J. Riss Norwegian Medical Cyclotron AS, Rikshospitalet diseases emphasises the potential of GnRHÀ R PET imaging. 46 Sognsvannsveien 20, Oslo, (Norway) In this study our main goal was to develop a PET imaging 47 [g] Dr. I. H. Haraldsen, Prof. P. J. Riss probe for GnRHÀ R in brain.[16–18] In more detail, we were 48 Clinical Neurosciences Oslo University Hospital-Ulleval interested in the development of a moderate-strength GnRHÀ R 49 Kirkeveien 166, post code? Oslo (Norway) antagonist to target brain imaging of GnRHÀ R as means to 50 Supporting information for this article is available on the WWW under detect and characterise brain cancer, both primary tumours and 51 https://doi.org/10.1002/cmdc.202000256 metastases, as well as to elucidate CNS mechanisms of 52 This article belongs to the Special Collection “Nordic Medicinal Chemistry 2019–2020” behaviour, brain damage and cognition involving GnRHÀ R.[9–11] 53 © 2020 The Authors. Published by Wiley-VCH GmbH. In our reasoning, a reversibly binding antagonist of GnRHÀ R 54 This is an open access article under the terms of the Creative Commons would be particularly interesting because of its lack of receptor 55 Attribution Non-Commercial NoDerivs License, which permits use and dis- stimulatory side effects and superior passive tissue permeability 56 tribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. to peptide agonists.[12] 57 ChemMedChem 2020, 15, 1624–1628 1624 © 2020 The Authors. Published by Wiley-VCH GmbH Wiley VCH Freitag, 21.08.2020 2017 / 173464 [S. 1624/1628] 1 Communications ChemMedChem doi.org/10.1002/cmdc.202000256 Several classes of small-molecular antagonist have been lipophilicity (log D ) of 4 and 4.6, respectively, held promise of 1 7.4 investigated for imaging GnRHÀ R, and tested for positron decent brain uptake. Based on these considerations and 2 emission tomography (PET) imaging in the brain, however, previous work wherein structure–activity relationships, affinity 3 without success.[13–14] The authors point out several reasons for and off target binding were assessed, we selected benzimida- 4 failure such as affinity, molecular size and association kinetics. zol-derivative 3 and elagolix analogue 4, for further study.[12,15–18] 5 We have pursued a library of GnRHÀ R antagonists that The primary objective in developing GnRHÀ R radiotracers for 6 addresses the moderate to high-affinity range allowing com- brain imaging is achieving brain uptake and saturable binding 7 petition of the ligand in circulation with endogenous GnRH at in brain, which has been elusive so far. 8 GnRHÀ R binding sites. While the use of moderate affinity Reference 3 and the corresponding labelling precursor 5 9 antagonists may permit assessment of the receptor availability, were obtained by copper-mediated azide-alkyne cycloaddition 10 a measure reflecting both ligand concentration and the as described previously; however, 1.05 equiv. Cu was required 11 concentration of the receptor in tissues, high-affinity ligands to achieve useful conversion of the starting material.[13] 12 may be required to image low concentration targets. Elagolix Compound 6 was prepared by cyclisation of elagolix using bis 13 (1) and relugolix (2) are potent small-molecule GnRH antago- 2-methylprop-2-yl pyrocarbonate in MeCN in a yield of 92% (a). 14 nists from a novel class drugs unrelated to the earlier peptide- Demethylation was achieved using boron tribromide in di- 15 based ligands (Scheme 1). Given the pharmacokinetic profiles of chloromethane furnish compound 7 in 64% yield (b). Com- 16 these approved drugs, we were certain that 1 and 2 would not pound 7 was converted into the reference compound 4 ((R)-5- 17 suffer from metabolic instability. At the same time, the high (2-fluoro-3-(2-fluoroeth-1-oxy)phenyl)-1-(2-fluoro-6- 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 Scheme 1. Competitive binding affinities (K ) for compounds 1–4 and synthetic route for radiolabelling compounds [18F]3 and [18F]4: a) BOC O, DMAP, MeCN, 56 i 2 92%; b) BBr3, CH2Cl2, 64%; c) Cs2CO3, ethylene ditosylate, MeCN, 64%; d) crypt-222, K2CO3, MeCN, 79%; i) crypt-222, K2CO3, MeCN, 46 %. 57 ChemMedChem 2020, 15, 1624–1628 www.chemmedchem.org 1625 © 2020 The Authors. Published by Wiley-VCH GmbH Wiley VCH Freitag, 21.08.2020 2017 / 173464 [S. 1625/1628] 1 Communications ChemMedChem doi.org/10.1002/cmdc.202000256 (trifluoromethyl)benzyl)-6-methyl-3-(2-(2-oxopyrrolidin-1-yl)-2- Ammonium formate (0.1 M, pH 7); 1:1 as mobile phase at a 1 phenylethyl)pyrimidine-2,4(1H,3H)-dione) by using 2-fluoro eth- flow rate of 8.5 mL/min. The HPLC fraction containing the 2 1-yl 4-methylbenzenesulfonate dissolved in anhydrous MeCN product was collected 7–9 min after the injection. The product 3 with cesium carbonate as base in up to 40% yield (c). The fraction was diluted and [18F]3 was extracted using a waters 4 labelling precursor (R)-2-(2-fluoro-3-(1-(2-fluoro-6- SepPak plus C cartridge pre-rinsed with EtOH and water. The 5 18 (trifluoromethyl)benzyl)-6-methyl-2,4-dioxo-3-(2-(2-oxopyrroli- cartridge was washed with H O (10 mL) afterwards and the 6 2 din-1-yl)-2-phenylethyl)-1,2,3,4-tetrahydropyrimidin-5-yl)- retained product was eluted into a clean glass vial with 7 phenoxy)ethyl 4-methylbenzene sulfonate (8) was obtained in a endotoxin free physiological saline (0.9% NaCl) using EtOH (< 8 yield of 64% by treating 7 with an excess of ethylene ditosylate 10%). The tracer was obtained in a nondecay corrected activity 9 in MeCN (d). The purity of each compound was determined by yield of 11% (380 MBq) with a molar activity of 18.5 MBq/nmol 10 reversed-phase HPLC prior to testing of the compounds’ bind- at delivery to the scanner.