Organic synthesis, radiofluorination and first biological evaluation of N-aryl-oxadiazolyl-propionamides as potential radioligands for PET imaging of cannabinoid CB₂ receptors

Objectives: The cannabinoid receptor type 1 (CB₁) has been intensively investigated but much less is known about the role and distribution of the cannabinoid receptor type 2 (CB₂) in the central nervous system. Recently, we reported the synthesis and radiofluorination of a novel N-aryl-oxadiazolyl-propionamide for non-invasive imaging of CB₂ with PET. However, the radiolabelling yields were unsatisfactory [1]. Thus, medicinal chemistry approaches were taken to develop fluorinated CB₂ derivatives of 1 without compromising biological activity or selectivity. Radiofluorination and first biological evaluation were performed for the most promising CB₂-selective ligands.

Methods: The reference compounds 2 (X² = -¹⁹F) and 3 (Y² = -¹⁹F) were achieved via a modified synthetic procedure [2] from the respective benzoic acid, succinic anhydride and N-alkyl-3-amino-carbazole. SAR studies were performed by varying both N-carbazole and amide-oxadiazole chain lengths. In addition to this, the substitution pattern of the aromatic region has been investigated. Distribution coefficients (LogD, pH = 7.2) were experimentally determined for 2 and 3 using a RP-HPLC method. Aliphatic [X¹ = LG, Y¹ = -H (LG = leaving group)] and aromatic (X¹ = -H, Y¹ = -LG) radiofluorinations of 1 were performed in AcCN using Kryptofix® 222 (K₂₂₂) as phase transfer catalyst (PTC). The reaction conditions were optimized by varying the amount of PTC and precursor 1 (X¹ or Y¹ = -LG). For in vitro and in vivo studies, ¹⁸F-2 in isotonic saline solution containing 5% EtOH, was used. Rat spleen slices were incubated with ¹⁸F-2 in absence and presence of CB_1/2-specific ligands. Ex vivo autoradiographs were obtained from brain and spleen of CD-1 mice after i.v. injection of 30 MBq of ¹⁸F-2.

Figure 1. Radiofluorination of N-aryl-oxadiazolyl-propionamide precursors 1; Ki = inhibition constant

Results: The N-ethyltosylate-carbazole 1 (X¹ = -OTs, Y¹ = -F) was the best choice for aliphatic radiofluorination. Although several LG were implemented (compound 1, X¹ = H, Y¹ = -LG), aromatic radiofluorination was only successfully achieved using the ammonium salt 1 (Y¹ = Me₃N⁺I^–) as precursor. Under optimized conditions, similar radiolabelling efficiencies (>50%), high radiochemical purities (>97%) and specific activities >250 GBq/µmol were obtained for ¹⁸F-2 and ¹⁸F-3. Increasing amounts of K₂₂₂ lead to a decrease of the labelling efficiencies for ¹⁸F-2. LogD values of 3.8 and 4.4 for 2 and 3, respectively, suggest a good blood-brain barrier penetration. Autoradiography of spleen slices showed high inhibition of ¹⁸F-2 binding (≥–74%) by the CB₁/CB₂-specific CP55,940 and by CB₂-specific ligands (JTE 907, GP1a, SR144528). Notably, no inhibition by the CB₁-specific SR141716A was observed indicating highly selective CB₂ binding of ¹⁸F-2. Ex vivo autoradiographs at 30 min p.i. showed heterogeneous radiotracer distribution in spleen (%ID/g = 6.96), comparable to the in vitro distribution of [³H]CP55,940, and low uptake and homogenous radiotracer distribution in brain (%ID/g = 1.49).

Conclusions: Fluorinated derivatives of 1 have been synthesized and the compounds with the best pharmacological properties were further investigated. Direct nucleophilic ¹⁸F-substitutions successfully delivered highly selective CB₂ radioligands ¹⁸F-2 and ¹⁸F-3. Biological evaluation of ¹⁸F-2 suggests specific CB₂ binding in vivo, which makes the compound a promising candidate for further evaluation.
Acknowledgements: This work has been supported by the DFG (project BR 1360/12-1).
References: [1] Rühl T, et al. (2012), Org Med Chem Lett, 2:32 [2] Cheng Y, et al. (2008), J Med Chem, 51, 5019-5034.