Novel triaryl sulfonamide derivatives as selective cannabinoid receptor 2 inverse agonists and osteoclast inhibitors: discovery, optimization, and biological evaluation

Abstract

Cannabinoid receptors have gained increasing attention as drug targets for developing potential therapeutic ligands. Here, we report the discovery and optimization of triaryl sulfonamides as a novel series possessing significant CB2 receptor affinity and selectivity. Four sets of triaryl ligands were designed and synthesized for further structural modifications and led to the identification of eight compounds as potent and selective CB2 inverse agonists with high binding affinity (CB2K(i) < 10 nM). Especially, compound 57 exhibited the strongest binding affinity on the CB2 receptor (CB2K(i) of 0.5 nM) and the best selectivity over the CB1 receptor (selectivity index of 2594). Importantly, 57 also showed potent inhibitory activity on osteoclast formation, and it was confirmed by a cell viability assay that the inhibition effects were not derived from the cytotoxicity. Finally, 3D QSAR studies confirmed our SAR findings that three bulky groups play an important role for CB2 receptor binding affinity.

Figure 1

Representative cannabinoids with various chemical scaffolds.

Figure 2

A novel CB₂ ligand 10 discovered within an in vitro high-throughput screening research program and further modified for SAR studies. (A) Lead compound 10; (B) 10 was validated by [³H]CP-55040 radiometric binding assays showing high CB₂ receptor binding affinity: Ki = 192 nM and CB₁/CB₂ selectivity (26 fold); (C) Step-by-step medicinal chemistry optimization.

Figure 3

Structures of the lead compound 10 and the modified target compounds 25, 48, 50, 56, and 57.

Figure 4

Comparisons of LANCE signal of different CB₂ receptor ligands in stably transfected CHO cells expressing human CB₂ receptors in a concentration-dependent fashion. EC₅₀ values of compounds 21, 48, 54, 57, and 5 are 268.4 ± 14.5 nM, 16.4 ± 2.84 nM, 608.6 ± 6.06 nM, 42.7 ± 1.35 nM, and 153.8 ± 5.58 nM respectively. EC₅₀ for CP-55,940 and HU308 are 47.1 ± 3.43 nM and 83.8 ± 5.63 nM. Data are mean ± S.E.M. of all experiments of two or more performed in duplicate or triplicate.

Figure 5

Anti-osteoclastogenesis activity of top compounds. (A) Compounds 25, 48 and 57 inhibit RANKL-induced osteoclastogenesis in a dose-dependent manner. RAW 264.7 cells (3 × 10³ cells/well) were treated with or without RANKL (15 ng/mL), followed by addition of the indicated concentrations of 25, 48 and 57 for 5 days and stained for TRAP expression. The data are the mean of three experiments carried out in triplicate. The bar indicates the SD. (B) Photographs of cells in the test of compound 57 (original magnification 100×).

Figure 6

Cytotoxic effect of top compounds 48 (A) and 57 (B) on osteoclast precursor. RAW 264.7 cells (3 × 10³ cells/well) were plated on 96-well plates. Cells were incubated with the indicated doses of compounds 48 and 57 for 3 days. The percentage of cell survival was determined with the MTT assay. The data are the mean ± S.E.M. of all experiments carried out in triplicate.

Figure 7

Overall alignments of training set molecules (A) and test set molecules (B) to the compound 57 as well as CoMFA contour maps of compound 57 showing steric and electrostatic (C) interactions. Sterically (bulk) favored areas are color-coded in green and sterically unfavored areas are in yellow. The red and blue contours reflect whether electropositive or electronegative substituents are favored at a particular position.

Figure 8

Plots of CoMFA-calculated and experimental binding affinity values (pK_i) for the training set.

Figure 9

Plots of CoMFA-calculated and experimental binding affinity values (pK_i) for the test set.

Scheme 1

General Synthesis of Triaryl Sulfonamide Derivativesa ^aReagents and conditions: (i) adamantan-1-amine, methanol, refluxed, 10 h; (ii) heptan-1-amine, methanol, refluxed, 12 h; (iii) p-toluidine or 4-chloroaniline, methanol, refluxed, 12 h; (iv) NaBH₄, methanol, r.t, 12 h; (v) acyl chloride or sulfonyl chloride, anhydrous DCM, TEA, r.t, 12 h.