Design, synthesis and anti-inflammatory evaluation of tricyclic diterpenoid carbamates in LPS-stimulated macrophages

A focused medicinal-chemistry campaign was undertaken to optimize the phenotypic hit QW60 against lipopolysaccharide (LPS)-induced macrophage inflammation. A total of 42 tricyclic diterpenoid carbamates were synthesized and evaluated by measuring tumor necrosis factor-alpha (TNF-α) production in RAW264.7 cells. Replacing the hydrolytically labile C-3 ester of QW60 with carbamates identified the N-propargyl analogue 1–18 , which reduced TNF-α to 71.4 ± 8.6% of the LPS control at 10 μM. Removal of the C-13 acetyl group afforded 1–22 (58.5 ± 5.7%), and subsequent optimization of the C-12 alkoxy substituent delivered the n-propoxy derivative 1–30 (36.3 ± 4.0%). C-7 oxidation and most C-13 acyl, bromo, or amide substitutions were unfavorable. Compound 1–30 caused little loss of RAW264.7 cell viability at concentrations up to 20 μM in complementary MTT and LDH-release assays and dose-dependently reduced TNF-α protein and interleukin-1 beta (IL-1β) mRNA in LPS-stimulated cells. These data define a coherent preliminary structure-activity relationship for this scaffold and identify 1–30 as a lead for further mechanistic, pharmacokinetic, and in vivo evaluation.