Steroids represent a special class of biomolecules in human beings and other organisms that play significant roles in membrane structure and signaling [1]. As a remarkable group of natural products, steroids have demonstrated their potential in anti-inflammatory, antitumor, antimycotic and other biological applications. Until now, steroid-based drugs have occupied the second-largest category of drugs in clinical practice for the treatment of various diseases and disorders [2], [3]. In addition to the different transformation and rearrangement patterns within the special tetracyclic core of steroids, Diels–Alder [4 + 2] cycloadduction is another unique approach to generating intriguing scaffolds [4]. Only three natural Diels–Alder [4 + 2] cycloadducts of steroids have been reported thus far [5], [6]. As a result, discovering analogs of Diels–Alder cycloaddition steroids and exploring their biological diversities are still of great significance.

Penicillium herquei, belonging to the genus Penicillium of the Trichocomaceae family, is mainly isolated from Ceriops tagal [7], Melia azedarach (MELIACEAE) [8], seaweeds and marine mangrove [[9], [10], [11]]. Previous studies of P. herquei have led to the discovery of a series of ergosterol peroxides, phenalenones, long-chain polyenes, diazabicyclo [2.2.2]octane derivatives, some of which exhibited potential cytotoxic, anti-hepatitis C virus, anti-inflammatory, and anti-indoleamine 2,3-dioxygenase 1 (IDO1) activities.

In our continuing research on natural bioactive metabolites from fungi P. herquei, the chemical investigation led to the isolation of sixteen ergosteroids, including two novel naturally occurring [4 + 2] Diels–Alder cycloaddition ergosteroids (1 and 2), three undescribed oxidized ergosteroids (3–5), and eleven known analogs (6–16) (See Fig. 1). Herein, the isolation, structure elucidation, and anti-inflammatory activity of these compounds were presented. Additionally, the possible biosynthetic pathways of 1–6 were proposed in this paper.