Molecular Networking-Guided Discovery of Hypecoleptopines B–H, Seven Pairs of Racemic Spiro-benzylisoquinoline Alkaloids with Anti-Neuroinflammatory Activity from Hypecoum leptocarpum

Hypecoleptopines B-H (1-5, 7, and 8), seven pairs of racemic spiro-benzylisoquinoline alkaloids with a complicated ring system, together with four key biosynthetic precursors (9-12) were isolated from Hypecoum leptocarpum by molecular networking approach. Hypecoleptopines B-H possess a rarely reported 6/6/5/6/6 pentacyclic system with a spirocyclic amide scaffold or fused spironolactone skeleton. Their structures were established by employing a combination of spectroscopic analysis, chiral separation, computational calculations, and X-ray crystal diffraction. The anti-neuroinflammatory effects of all isolates were assessed in lipopolysaccharide (LPS)-stimulated BV-2 microglial model. Remarkably, compounds (+)-11 and (-)-11 demonstrated the most significant inhibition effects with IC₅₀ values of 9.9 and 8.6 μM, compared with the positive control, minocycline (IC₅₀: 25 μM). A further mechanistic study revealed that the two compounds exert the effects by inhibiting the release of inflammatory factors and downregulating iNOS and COX-2 protein expression.