Isolation, Structure Determination, and Semisynthesis of Diphenazine Compounds from a Deep-Sea-Derived Strain of the Fungus <i>Cystobasidium laryngis</i> and Their Biological Activities

Abstract

Phenazostatins E–J (1–6), six new diphenazine derivatives, were isolated from the EtOAc extract of the culture broth of a strain of Cystobasidium laryngis derived from deep-sea sediments of the Indian Ocean Ridge. The structures of 1–6 were elucidated based on the HRESIMS and 1D and 2D NMR spectra. The absolute configurations of 1–6, except for 3 and 6, were determined by modified Mosher’s method, ECD data analysis, and calculations of optical rotation values. The absolute configurations of 3 and 6 were identified by chemical derivatization and comparing the specific rotation values with those of semisynthetic 3 obtained by the oxidation of 1 and saphenic acid (7). Phenazostatin J (6) was semisynthesized using saphenic acid (7) to prepare additional material for biological testing. During the purification of semisynthetic 6, a side product 9 was obtained from the reaction mixture along with 6. Compounds 1–6, along with previously reported 7 and 8, were assessed for anti-neuroinflammatory activity in LPS-induced BV-2 microglia cells. Compound 6 exhibited the highest anti-neuroinflammatory effect with an IC50 value of 0.30 μM, but it showed cytotoxicity at higher concentrations than 1.0 μM. Accordingly, cytotoxicities of 1–9 were evaluated against six human cancer cell lines. Among tested compounds, 6 and 9 showed potent cytotoxicity (IC50 values: 7.7–72 nM). Especially, 6 exhibited the strongest cytotoxicity with an IC50 value of 7.7 nM against the NUGC-3 (stomach) cell line, displaying 19-fold stronger activity than the positive control, adriamycin.