**1. Introduction**

Immunosuppressants are the main clinical drugs for the treatment of undesirable or abnormal activations in the body, such as immune system activation, which is associated with a variety of autoimmune diseases and allergic reactions, including rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, and glomerulonephritis, and responses in organ transplantation recipients [1]. Most of the representative immunosuppressive drugs used in the clinic were developed from microbial secondary metabolites, such as cyclosporin A (CsA), tacrolimus (FK506), and rapamycin [2]. These drugs form complexes with intracellular immunophilin receptors. Among them, the CsA/cyclophilin and FK506/FKBP complexes share the same pharmacodynamic property of suppressing activated T cells by inhibiting the activity of calcineurin (CN) phosphatase; thus, these complexes prevent the dephosphorylation and nuclear translocation of activated T cell nuclear factor (NFAT) and NFAT-mediated transcription of a large number of cytokine genes, such as interleukin 2 (IL-2) [3,4]. However, despite their undeniable satisfactory therapeutic effects, the aforementioned immunosuppressive drugs have been found to cause dramatic side effects, such as nephrotoxicity, hepatotoxicity, neurotoxicity, malignancy, and other adverse effects [5,6]. Thus, the discovery of new clinically applied immunosuppressants with a high efficacy but no major cytotoxicity is urgently needed.

Endophytic fungi isolated from mangrove trees are one of the most pivotal and promising sources of bioactive natural products, presumably owing to their intriguing structural skeleton and the promising pharmacological effect of their secondary metabolites, making them attractive repositories for therapeutic agents and lead compounds [7,8]. Over

**Citation:** Feng, Z.; Zhang, X.; Wu, J.; Wei, C.; Feng, T.; Zhou, D.; Wen, Z.; Xu, J. Immunosuppressive Cytochalasins from the Mangrove Endophytic Fungus *Phomopsis asparagi* DHS-48. *Mar. Drugs* **2022**, *20*, 526. https://doi.org/10.3390/ md20080526

Academic Editor: Ipek Kurtboke

Received: 9 July 2022 Accepted: 16 August 2022 Published: 18 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

School of Chemical Engineering and Technology, Hainan University, Haikou 570228, China

**<sup>\*</sup>** Correspondence: happyjing3@hainanu.edu.cn; Tel.: +86-898-6627-9226

50% of mangrove-derived endophytic fungal bioactive secondary metabolites are produced by the genera *Aspergillus* and *Penicillium*, while *Pestalotiopsis*, *Alternaria,* and *Phomopsis* are considered the predominant producers [9]. The *Phomopsis* (teleomorph *Diaporthe*) fungi, which contains more than 900 species named from a wide range of hosts, have attracted considerable attention from natural product researchers in recent years [10,11]. Versatile bioactive metabolites, such as cytotoxic phomopchalasins B and C [12]; cytochalasins J1–J3, H1, and H2 [13]; dicerandrols [14]; antibiotic isopentylated diphenyl ethers [15], phomoxanthone A [16] and phompsichalasin [17]; *β*-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitory protoilludane-type sesquiterpenoids [18]; and anti-Tobacco mosaic virus (TMV) cytosporone U [19] and arylbenzofurans [20] have been isolated from *Phomopsis* strains. Among them, cytochalasins are a diverse group of polyketide synthase nonribosomal peptide synthetase (PKS-NRPS)-derived fungal metabolites characterized by a perhydroisoindolone moiety, which is typically fused to a macrocyclic ring (ring size 9–14) [21]. Since the first representatives, cytochalasin A and B, were isolated in 1966 [22], the number of natural products belonging to this family has increased to over 200 [12]. As part of our research on mangrove-derived fungi, a series of structurally novel and biologically active metabolites have been discovered [23–27]. Our primary application of in vitro immunosuppressive activity screening indicated that the MeOH extracts from the endophytic fungus strain *Phomopsis asparagi* DHS-48, which were obtained from a fresh root of the mangrove plant *Rhizophora mangle*, strongly inhibited ConA/LPS-induced spleen lymphocyte proliferation and CN enzyme activity, with IC50 values of 6.20 ± 0.20 μg/mL, 10.28 ± 0.24 μg/mL, and 78.03 ± 0.45 μg/mL, respectively. Bioassay-guided investigation of the immunosuppressive constituents obtained from the large-scale fermentation of the abovementioned *P. asparagi* DHS-48 resulted in the isolation of three new cytochalasins, namely, phomoparagins A-C (**1**–**3**), along with five known analogs, phomopchalasin A and B (**4**,**5**) [12], cytochalasin H (**6**) [28], and J (**7**) [29] and fragiformin B (**8**) [30] (Figure 1). Herein, we report the isolation, structure elucidation, and immunosuppressive activity, as well as the plausible biosynthetic pathway, of the isolated compounds.

**Figure 1.** Structures of the isolated Compounds **1**–**8**.
