C3-heteroaroyl cannabinoids as photolabeling ligands for the CB2 cannabinoid receptor

Abstract

A series of tricyclic cannabinoids incorporating a heteroaroyl group at C3 were prepared as probes to explore the binding site(s) of the CB1 and CB2 receptors. This relatively unexplored structural motif is shown to be CB2 selective with K(i) values at low nanomolar concentrations when the heteroaromatic group is 3-benzothiophenyl (41) or 3-indolyl (50). When photoactivated, the lead compound 41 was shown to successfully label the CB2 receptor through covalent attachment at the active site while 50 failed to label. The benzothiophenone moiety may be a photoactivatable moiety suitable for selective labeling.

Figure 1

Aroyl cannabinoids.

Figure 2

Accessible conformers within 6 kcal mol⁻¹ of the global energy minimum for 40 (magenta), 41 (orange), and AM755 (blue). Analogues are shown superimposed at their aromatic rings. The global minimum energy conformer for each compound is shown in stick representation.

Figure 3

Compound 41 inhibits the specific binding of [³H]CP-55,940 to mCB2 receptor. HEK293 cell membranes expressing wild type mouse cannabinoid receptor 2 (mCB2) were suspended in TME buffer (25 mM Tris-Base, 5 mM MgCl₂, 1 mM EDTA, pH 7.4) with 0.1% BSA, containing 0.34 µM 41 (i.e. 10-fold K_i of 41 for WT mCB2). A membrane devoid of 41 was used as a parallel control. Incubations of both samples were performed in silanized glass tubes for 30 min in a 37°C water bath. Subsequently, the samples were irradiated for 1 h using Black-Ray long wavelength ultraviolet lamp at 365 nm in ice-cold silanized Petri dishes. The membranes were washed once with 1% BSA TME buffer to remove unbound ligands, and once with TME buffer (no BSA) to remove BSA. Saturation binding assays were carried out after photo-labeling using [³H]CP-55940 as a radioligand. The membrane sample with 41 (34.2 nM; 10 × K_i) exhibited a 67% reduction in its Bmax when compared to control.

Scheme 1

Reagents and conditions: (a) pTsOH, CHCl₃/acetone (4/1), 0 °C, 1 h; rt, 1 h; (b) CH₂Cl₂, cat. DMAP, pyr, Ac₂O, 0 °C to rt, 12 h; (c) KOH, MeOH, 0 °C, 1.5 h; 68% from 4, 5; (d) TMSOTf, MeNO₂, 0 °C, 2.5 h; (e) PhNTf₂, Et₃N, CH₂Cl₂, 0 °C to rt; 57% from 7; (f) NaBH₄, MeOH, rt, 1 h; β/α ca. 95/5, 97%; (g) MeOCH₂Cl, iPr₂NEt, CH₂Cl₂, 0 °C to rt, 2.5 h; 93%.

Scheme 2

Reagents and conditions: (a) DMF, CO, LiCl, BHT, 4Å MS, 110 °C, ArSnBu₃, PdCl₂(dppf)·CH₂Cl₂, 24 h.

Scheme 3

Reagents and conditions: (a) CH₂Cl₂, DIBAL, −78 °C; 96%; (b) ArBr, n-BuLi, THF, −78 °C; (c) MnO₂, CH₂Cl₂; 30, Ar = 3-fluorophenyl, 74%; 31, Ar = 3-(trifluoromethyl)phenyl, 81%; 32, 52% (2 steps); (d) KOH, MeOH, indole; (e) DMF, NaH, MeI; 97%.

Scheme 4

Reagents and conditions: (a) TMSBr, CH₂Cl₂, −40 °C, 1.5 h; 0 °C, 1 h.