Synthesis of Novel Chloro-Benzo [d]imidazole Regioisomers as Selective CB2 Receptor Agonists: Indirect Functional Evaluation and Molecular Insights

<b>Background/Objectives:</b> The cannabinoid type 2 receptor (CB₂ receptor) has been extensively studied in recent years due to the benefits associated with its modulation, including the regulation of the inflammatory response, neuroimmunomodulatory properties, and antitumor effects, all with the advantage of lacking significant psychoactive effects. Herein, we report the design, synthesis, characterization, biological assays, and molecular modelling analyses of novel (5/6-chloro-2-aryl-1<i>H</i>-benzo [<i>d</i>]imidazol-1-yl)(4-methoxyphenyl)methanone and 5/6-chloro-1-(4-methoxybenzyl)-2-aryl-1<i>H</i>-benzo [<i>d</i>]imidazole regioisomers as potential cannabinoid type 2 receptor ligands. <b>Methods:</b> The compounds were evaluated for their presumed CB₂ agonist activity using an indirect receptor-dependent apoptotic cell death assay exerted by cannabinoids, using the cell lines HEK293 (low CB₁/CB₂ expression), U-87 MG (high CB₁ expression), and HL-60 (exclusive CB₂ expression), and including the known cannabinoid ligands WIN-55,212-2 and AM630 as reference ligands. Flow cytometry was performed to assess apoptosis. Molecular docking and molecular dynamics simulations were used to explore ligand-receptor interactions at the CB₂ active site. <b>Results:</b> Compounds <b>3a</b>, <b>3b'</b>, <b>3c</b>, and <b>4b</b> selectively reduced HL-60 cell viability, similar to WIN-55,212-2, while showing no toxicity toward HEK293 or U-87 MG cells. Flow cytometry indicated that compounds <b>3a</b> and <b>3c</b> induced apoptosis in HL-60 cells comparable to WIN-55,212-2. Computational studies suggested that both compounds bind within the CB₂ receptor active site predominantly through π-π and hydrophobic interactions involving their benzo [<i>d</i>]imidazole cores, 2-aryl moieties, and 4-methoxybenzoyl scaffolds, resembling the binding patterns of established CB₂ ligands. <b>Conclusions:</b> Compounds <b>3a</b> and <b>3c</b> exert selective cytotoxicity against HL-60 cells, likely via a CB₂ agonist-mediated apoptotic mechanism. The applied combined experimental and computational approach provides a rapid, informative strategy for preliminary evaluation of CB₂ ligands and guides subsequent detailed pharmacological studies.