**1. Introduction**

Endophytic fungi refer to harmless parasitic fungi that live in the internal organs of plants and animals without causing any adverse reactions. The host provides nutrients for endophytes, and endophytes produce bioactive substances, giving the host an advantage in survival competition [1]. Symbionts coexist with symptomless fish, sponges, algae, and soft corals that grow in a relatively harsh marine environment characterized by high salinity, scarce nutrients, and high osmotic and hydraulic pressures, which provides many environment-specific microorganisms that could coevolve with their hosts by undergoing the rapid and dynamic change of their genomes [2,3]. Thus, endophytic fungi are considered as a treasure trove of unique structural compounds and bioactive metabolites.

Indolyl diketopiperazines (IDKPs), cyclic dipeptides produced by the condensation of l-tryptophan and a second amino acid, were commonly isolated from fungi, especially from the genera *Aspergillus* and *Penicillium* [4,5]. IDKPs had drawn considerable attention from synthetic chemists, natural products researchers, and synthetic biologists for decades due to their significant biological activities, such as antiviral [6,7], anticancer [8–10], immunomodulatory [11,12], antioxidant [13], and α-glucosidase inhibitory activities [14]. Specifically, the vascular disrupting and tubulin-depolymerizing agent plinabulin, a synthetic analog based on the natural diketopiperazine (DKP) product halimide generated by the marine-derived *Aspergillus* sp. CNC-139, had entered the last stage of clinical study for the treatment of non-small-cell lung cancer [15,16]. Since the first IDKP alkaloid chaetomin isolated from the fungus *Chaetomium cochliodes* in the early 1940s, a series of DKPs and their biosynthesis clusters were reported [17–22]. In our continuous investigations of novel bioactive agents from the endophytic fungi [23,24], the endophytic strain *Aspergillus versicolor* DY180635 isolated from the sea crab was selected based on the bio-evaluation results. The ethyl acetate extracts of a rice solid culture of *A. versicolor* DY180635 showed 80% inhibition on the anti-inflammation model of the *Propionibacterium acnes*-induced THP-1 cells at the concentration of 0.1 mg/mL [25]. High-performance liquid chromatography (HPLC) analysis of the ethyl acetate extracts indicated the presence of IDKPs with a diode array detector (DAD) through ultraviolet characteristics at λmax 236, 289, and 336 nm [26]. Thus, to discover structurally complex and/or bioactive DKPs, the spectroscopic-guided isolation was performed in this research.

Spectroscopic-guided isolation resulted in the identification of four new IDPKs, aspamides A–E (**1–4**) and two new DPKs, aspamides F–G (**5–6**), along with 11 known diketopiperazines and intermediates from the ethyl acetate (EtOAc) extracts of the solid culture of *A. versicolor* (Figure 1). The couple of epimers **1–2** were the first samples of brevianamides with an oxygenated aza-acetal structure at the proline motif. All isolated compounds were tested for anti-inflammation in *P. acnes*-induced THP-1 cells. Unfortunately, none showed active effect. With the appearance and spread of SARS-CoV-2 at the end of 2019, compounds **1–17** were selected for the virtual screening on the 3CL hydrolase (Mpro) of SARS-CoV-2, which had been exploited as a potential drug target to fight COVID-19 [27]. The docking scores of compounds **1–2**, **5**, **6**, **8**, and **17** were top among all screened molecules (docking scores: −5.389, −4.772, −5.146, −4.962, −5.158), which may be helpful in fighting COVID-19 after further studies. Herein, we reported the isolation, structural identification, and bio-evaluation of isolated compounds.

**Figure 1.** Structures of compounds **1–17**.
