Synthesis and evaluation of candidate PET radioligands for corticotropin-releasing factor type-1 receptors

Abstract

Introduction A radioligand for measuring the density of corticotrophin-releasing factor subtype-1 receptors (CRF1 receptors) in living animal and human brain with positron emission tomography (PET) would be a useful tool for neuropsychiatric investigations and the development of drugs intended to interact with this target. This study was aimed at discovery of such a radioligand from a group of CRF1 receptor ligands based on a core 3-(phenylamino)pyrazin-2(1H)-one scaffold. Methods CRF1 receptor ligands were selected for development as possible PET radioligands based on their binding potency at CRF receptors (displacement of [125I]CRF from rat cortical membranes), measured lipophilicity, autoradiographic binding profile in rat and rhesus monkey brain sections, rat biodistribution, and suitability for radiolabeling with carbon-11 or fluorine-18. Two identified candidates (BMS-721313 and BMS-732098) were labeled with fluorine-18. A third candidate (BMS-709460) was labeled with carbon-11 and all three radioligands were evaluated in PET experiments in rhesus monkey. CRF1 receptor density (Bmax) was assessed in rhesus brain cortical and cerebellum membranes with the CRF receptor ligand, [3H]BMS-728300. Results The three ligands selected for development showed high binding affinity (IC50 values, 0.3–8 nM) at CRF1 receptors and moderate lipophilicity (LogD, 2.8–4.4). [3H]BMS-728300 and the two 18F-labeled ligands showed region-specific binding in rat and rhesus monkey brain autoradiography, namely higher binding density in the frontal and limbic cortex, and cerebellum than in thalamus and brainstem. CRF1 receptor Bmax in rhesus brain was found to be 50–120 fmol/mg protein across cortical regions and cerebellum. PET experiments in rhesus monkey showed that the radioligands [18F]BMS-721313, [18F]BMS-732098 and [11C]BMS-709460 gave acceptably high brain radioactivity uptake but no indication of the specific binding as seen in vitro. Conclusions Candidate CRF1 receptor PET radioligands were identified but none proved to be effective for imaging monkey brain CRF1 receptors. Higher affinity radioligands are likely required for successful PET imaging of CRF1 receptors.

Synthesis, F-18 radiolabeling, and microPET evaluation of 3-(2,4-dichlorophenyl)-N-alkyl-N-fluoroalkyl-2,5-dimethylpyrazolo[1,5-a]pyrimidin-7-amines as ligands of the corticotropin-releasing factor type-1 (CRF1) receptor

“…The behavior of [ 18 F] 27 , [ 18 F] 28 , [ 18 F] 28- d 8 , and [ 18 F] 29 in the brain of a cynomolgus monkey is similar to what was reported for [ 11 C] 7 and [ 11 C] 8 in the brain of a baboon, 85 and [ 18 F] 10 and [ 11 C] 12 in the brain of a rhesus monkey 88 with regards to the shapes of the TACs, relatively even distribution throughout the brain, and an apparent lack of specific binding. All of these compounds displayed high binding affinities to the CRF 1 receptor when evaluated in in vitro binding assays and compounds [ 18 F] 10 and [ 18 F] 11 displayed specific binding in autoradiography studies with rhesus brain slices.…”

“…The TACs of [ 18 F] 27 in the brain (Figure 8) were also similar to those of [ 18 F] 28 , [ 18 F] 28- d 8 , and [ 18 F] 29 with peak brain uptake achieved between ~5 – 15 min followed by a steady washout and with no discernable difference between regions with high CRF 1 receptor density (cerebellum and cortex) 85, 88, 107, 108 and regions of low CRF 1 receptor density (striatum and thalamus) which suggests little or no specific binding. The TACs of [ 18 F] 26 in the brain (Figure 9) show that it entered the brain more rapidly and washed out more rapidly than [ 18 F] 27 , [ 18 F] 28 , [ 18 F] 28- d 8 , and [ 18 F] 29 .…”

“…All of these compounds displayed high binding affinities to the CRF 1 receptor when evaluated in in vitro binding assays and compounds [ 18 F] 10 and [ 18 F] 11 displayed specific binding in autoradiography studies with rhesus brain slices. 88 Furthermore, 8 blocked the binding of [ 125 I]sauvagine, [ 125 I]oCRF, and [ 3 H] 6 in rat autoradiography studies, 130, 131 and numerous other studies have used autoradiography to confirm the presence of the CRF 1 receptor and to quantify its density. 85, 88, 107, 108, 132-134 Thus, it is not clear why specific binding is not being observed during PET studies.…”

“…Based on this preliminary data, compound [ 18 F] 28 (prepared by the one-step radiolabeling method) was evaluated by microPET for brain uptake and specific binding in an anesthetized male cynomolgus monkey. The microPET images in Figure S2 (Supplementary Material) show that brain penetrance was achieved but the TACs in Figure 3 show that uptake of [ 18 F] 28 was equal across all brain regions including regions with high CRF 1 receptor density (cerebellum and cortex) 85, 88, 107, 108 and regions of low CRF 1 receptor density (striatum and thalamus), thus suggesting little or no specific binding. This uniform brain distribution is similar to what was reported for [ 11 C] 6 , [ 11 C] 7 , [ 11 C] 8 , [ 18 F] 10 , [ 18 F] 11 , and [ 11 C] 12 .…”

“…84, 85 Compounds [ 18 F] 9 – [ 18 F] 11 and [ 11 C] 12 have recently been reported, and these compounds were able to penetrate the BBB of a monkey but they did not display specific binding. 87, 88 Thus, attempts to develop a viable CRF 1 receptor radiotracer have been hampered by low brain entry, little or no in vivo specific binding when brain entry is achieved, and rapid metabolism. A successful CRF 1 receptor radiotracer will, therefore, require improvements in binding affinity that will enable specific binding to be detected in vivo , a reduction in lipophilicity to increase brain penetrance and reduce non-specific binding, and increased metabolic stability.…”