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

Hypecoleptopines B-H (<b>1</b>-<b>5</b>, <b>7</b>, and <b>8</b>), seven pairs of racemic spiro-benzylisoquinoline alkaloids with a complicated ring system, together with four key biosynthetic precursors (<b>9</b>-<b>12</b>) were isolated from <i>Hypecoum leptocarpum</i> 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 (+)-<b>11</b> and (-)-<b>11</b> 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.