Cannabinoid receptor type 2 (CB2)-selective N-aryl-oxadiazolyl-propionamides: synthesis, radiolabelling, molecular modelling and biological evaluation

Abstract

Background: The endocannabinoid system is involved in many physiological and pathological processes. Two receptors (cannabinoid receptor type 1 (CB1) and type 2 (CB2)) are known so far. Many unwanted psychotic side effects of inhibitors of this system can be addressed to the interaction with CB1. While CB1 is one of the most abundant neuroreceptors, CB2 is expressed in the brain only at very low levels. Thus, highly potent and selective compounds for CB2 are desired. N-aryl-((hetero)aromatic)-oxadiazolyl-propionamides represent a promising class of such selective ligands for the human CB2. Here, a library of various derivatives is studied for suitable routes for labelling with 18F. Such 18F-labelled compounds can then be employed as CB2-selective radiotracers for molecular imaging studies employing positron emission tomography (PET).

Results: By varying the N-arylamide substructure, we explored the binding pocket of the human CB2 receptor and identified 9-ethyl-9H-carbazole amide as the group with optimal size. Radioligand replacement experiments revealed that the modification of the (hetero)aromatic moiety in 3-position of the 1,2,4-oxadiazoles shows only moderate impact on affinity to CB2 but high impact on selectivity towards CB2 with respect to CB1. Further, we could show by autoradiography studies that the most promising compounds bind selectively on CB2 receptors in mouse spleen tissue. Molecular docking studies based on a novel three-dimensional structural model of the human CB2 receptor in its activated form indicate that the compounds bind with the N-arylamide substructure in the binding pocket. 18F labelling at the (hetero)aromatic moiety at the opposite site of the compounds via radiochemistry was carried out.

Conclusions: The synthesized CB2-selective compounds have high affinity towards CB2 and good selectivity against CB1. The introduction of labelling groups at the (hetero)aromatic moiety shows only moderate impact on CB2 affinity, indicating the introduction of potential labelling groups at this position as a promising approach to develop CB2-selective ligands suitable for molecular imaging with PET. The high affinity for human CB2 and selectivity against human CB1 of the herein presented compounds renders them as suitable candidates for molecular imaging studies.

Scheme 1

N-aryl-((hetero)aromatic)-oxadiazolyl-propionamides synthesized in this study.

Scheme 2

Synthesis of N-aryl-((hetero)aromatic)-oxadiazolyl-propionamides.

Scheme 3

Two strategies to introduce fluorine into the molecule, allowing labelling with¹⁸F.

Figure 1

X-ray crystal structure of 6f (50% thermal ellipsoids). The planar ring systems have been noted by A, B and C and are highlighted in grey.

Figure 2

Radioligand displacement study of 6f and 6h against [³H]CP55,940 on membrane homogenates of hCB2-CHO cells.

Figure 3

Influence of substitution at ring C (cf. Figure1) on affinity towards hCB2. The pK_i values were calculated according to pK_i = −log (K_i). High pK_i values represent high affinity.

Figure 4

Autoradiogram of representative coronal sections of CD-1 mouse spleen tissue. (a) Incubated with 6 nM [³H]CP55,940. HD binding and LD binding represent the white and the red pulp, respectively (cf. text). (b) Incubation with 1 μM selective antagonists for CB2 (SR144528) and CB1 (SR141716A). (c) Compounds 6e, 6a and 6h compete at 1 μM with the binding of [³H]CP55,940 on CB2 in good agreement with the determined binding data: 6e >6a >6h.

Figure 5

Model of the human cannabinoid receptor type 2. (a) Side view with the proposed transmembrane region shaded in grey. The protein is shown with the N-terminus at the top. (b) Top view from the extracellular space. The extracellular loops and the transmembrane helices are labelled to guide the reader. Only the residues forming the binding pocket are shown. The binding pocket identified by the Site Finder module of MOE (Chemical Computing Group Inc., Montreal, Canada) is shown as a surface with colour-coded features: H bonding (magenta), lipophilic (green), mild polar (blue).

Figure 6

Predicted binding modes of 6e, 6a, 6h to human CB2 model after docking with GOLD. All compounds bind with their carbazole moiety (ring A, cf. Figure 1) in the binding pocket indicated by black dots. The figure was created with the software MOE (Chemical Computing Group Inc., Montreal, Canada) using the module PostDock [45]: The visibility of the surfaces is proportional to their binding energies based on Boltzman statistics. The surfaces are colour-coded with respect to the rmsd difference to the ligand with the lowest binding energy (from yellow to blue). As reference, the best-docked ligand is shown with cyan sticks.

Figure 7

Comparison of the predicted binding modes of 7h and 6h to the human CB2 model. 6h (cyan) binds with the carbazole moiety in the binding pocket. For 7h (orange), the preferred binding is with ring A pointing outside of the cavity. This figure was created using MOE (Chemical Computing Group Inc., Montreal, Canada).

Scheme 4

Radiochemistry.