*Article* **Polyketide Derivatives from the Endophytic Fungus** *Phaeosphaeria* **sp. LF5 Isolated from** *Huperzia serrata* **and Their Acetylcholinesterase Inhibitory Activities**

**Yiwen Xiao 1,2,†, Weizhong Liang 2,†, Zhibin Zhang <sup>1</sup> , Ya Wang <sup>2</sup> , Shanshan Zhang <sup>2</sup> , Jiantao Liu <sup>2</sup> , Jun Chang <sup>2</sup> , Changjiu Ji <sup>3</sup> and Du Zhu 1,2,\***


**Abstract:** The secondary metabolites of *Phaeosphaeria* sp. LF5, an endophytic fungus with acetylcholinesterase (AChE) inhibitory activity isolated from *Huperzia serrata*, were investigated. Their structures and absolute configurations were elucidated by means of extensive spectroscopic data, including one- and two-dimensional nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) analyses, and calculations of electronic circular dichroism (ECD). A chemical study on the solid-cultured fungus LF5 resulted in 11 polyketide derivatives, which included three previously undescribed derivatives: aspilactonol I (**4**), 2-(1-hydroxyethyl)-6 methylisonicotinic acid (**7**), and 6,8-dihydroxy-3-(10*R*, 20*R*-dihydroxypropyl)-isocoumarin (**9**), and two new natural-source-derived aspilactonols (G, H) (**2**, **3**). Moreover, the absolute configuration of de-*O*-methyldiaporthin (**11**) was identified for the first time. Compounds **4** and **11** exhibited inhibitory activity against AChE with half maximal inhibitory concentration (IC50) values of 6.26 and 21.18 µM, respectively. Aspilactonol I (**4**) is the first reported furanone AChE inhibitor (AChEI). The results indicated that *Phaeosphaeria* is a good source of polyketide derivatives. This study identified intriguing lead compounds for further research and development of new AChEIs.

**Keywords:** *Phaeosphaeria* sp.; secondary metabolite; polyketide; AChE inhibitor; biosynthetic pathways
