New butanolide derivatives from the marine derived fungus Aspergillus terreus GZU-31-1 by chemical epigenetic manipulation

Abstract Chemical epigenetic manipulation of Aspergillus terreus GZU-31-1 led to the discovery of five butanolide derivatives (1–5), including two new ones (1 and 2), and four known diphenyl ether derivatives (6−9). Compound 1 featured a Z-configuration double bond in the isoprenyl group was a potential anti-inflammatory bioactive group. Compound 2 was a new natural product. Moreover, compound 3 with a deacetylated group at C-4 was rarely reported as a butanolide analogue, which was isolated from the liquid culture treated with polyketide pathway inhibitor sodium citrate dihydrate. All of the isolates (1−9) were tested for their anti-inflammatory effects on the production of nitric oxide in lipopolysaccharide-induced microglial cells (RAW 264.7 cells). Compounds 1, 7, 8 and 9 exhibited more potent anti-inflammatory activity with IC50 values of 16.31, 20.16, 9.53 and 21.64 μM than the positive control (indomethacin, IC50, 24.0 μM).


Introduction
Aspergillus were widely used for fermentation in food processing industries and were prolific producers of secondary metabolites with intriguing pharmaceutical activities.Especially, A. monascus and A. terreus were famous fungi that belong to the Aspergillus, which metabolize the famous anti-hyperlipidemia drug lovastatin and monacolin K (Endo et al. 1986).Previous chemical investigation of A. terreus showed that the metabolites including butyrolactones, meroterpenoids and alkaloids exhibited various biological activities, for example, butyrolactone derivative aspernolide A with antiviral and antifouling activities (Nong et al. 2014), C 7 -alkylated salicylaldehyde derivatives with anti-inflammatory and antioxidative activities (Lin et al. 2021), and meroterpenoid furanasperterpene A with lipid-lowing effects (Tang et al. 2021).Consequently, the bioactive metabolites of Aspergillus may have the potential to be lead compounds for drug discovery.
As part of our ongoing investigation to find more bioactive compounds from marine-derived fungi, a chemical investigation of the fungus A. terreus GZU-31-1, isolated from the intestinal tract of Onchidium struma, was carried out (Tang, Li, et al. 2020;Tang, Liu et al. 2020;Tang et al. 2021).The EtOAc extract of a fermentation broth of the fungus led to the isolation and identification of five butanolide derivatives (1-5) and four known diphenyl ether derivatives (6À9) (Figure 1).They were evaluated for their anti-inflammatory activities on nitric oxide (NO) production in RAW 264.7 cells.Among them, compounds 1, 7, 8 and 9 showed good activity in reducing NO production.Herein, details of the isolation, structure elucidation, and bioactivity of these compounds are described.
Further detailed analysis of the NMR data, found that 1 might be a dehydration product of 5 at C-1 000 and C-2 000 to form a new double bond (Z-configuration, J ¼ 9.8 Hz), which supported by the 1 HÀ 1 H COSY relationships (Figure S3) of H-1 000 /H-2 000 and the HMBC correlations from H-1 000 to C-2 0 , C-3 0 and C-4 0 , from H-2 000 to C-3 000 .Meanwhile, the HMBC correlations from H-4 000 and H-5 000 (d H 1.34) to C-2 000 and C-3 000 confirmed the existence of the double bond between C-1 000 and C-2 000 .At last, the specific structure of 1 was determined by the 2 D NMR, as shown in Figure S3.The absolute configuration at C-4 in 1 were determined by comparison of the experimental and simulated electronic circular dichroism (ECD) spectra.The experimental ECD spectrum of 1 matched well with the theoretical ECD spectrum for 4 R-1 (Figure S17).Finally, compound 1 was named as asperbutyrolactone A.
Asperbutyrolactone B (2) that previously isolated as a synthetic product (Pal and Banik 2020) is a new natural product for the first time to be reported.The 1 H NMR spectra of 2 (Table S1) displayed the signals for a 1, 4-disubstituted benzene ring at d H 7.63 (2H, d, J ¼ 8.9 Hz, H-2 0 and H-6 0 ) and d H 6.93 (2H, d, J ¼ 8.9 Hz, H-3 0 and H-5 0 ), a monosubstituted benzene moiety at d H 6.85 (2H, d, J ¼ 6.5 Hz, H-2 00 and H-6 00 ) and d H 7.14 (3H, m, H-3 00 , H-4 00 and H-5 00 ) and a methoxy group at d H 3.79 (3H, s, 5-OMe).Detailed analyses of the 2 D NMR data of 2, the benzyl group connected to C-4 of the c-butenolide nucleus was established by the HMBC correlations (Figure S11) from H 2 -5 to C-1 000 (d C 132.8) and C-6 000 (d C 130.6).Meanwhile, the HMBC correlations from H-2 00 and H-6 00 to C-3 confirmed that the 1,4-disubstituted benzene ring was connected to C-3 in the c-butenolide nucleus.Moreover, the methoxy group was linked to C-5, which was further established by the HMBC correlation from the 5-OMe to C-5.Thus, the planar structure of 2 was successfully established.
Besides, the absolute configuration of stereogenic centers for 2 was determined as 4 R by comparing the calculated ECD curves with the experimental curves as shown in Figure S18.Thus, compound 2 could be identified and named as asperbutyrolactone B.
All of the isolated metabolites were evaluated for their anti-inflammatory activities on the lipopolysaccharide-induced microglial cells (RAW 264.7 cells).Compounds 1, 7, 8 and 9 were found to reveal notable inhibitory effects against nitric oxide production with IC 50 values of 16.31, 20.16, 9.53 and 21.64 lM, respectively (Table S2), while the remained compounds exhibited weak activity (IC 50 >50 mM).From the structural viewpoint, the Z-configuration double bond in 1 might play the key role in the anti-inflammation by comparing with 5. Besides, by comparison of the compounds 6À9, the carboxyl group at C-7 0 rather than the methyl ester in 6 might decrease the antiinflammatory activity.This finding provides new viewpoint to discover new antiinflammatory agents.

Fungal material
The fungal strain GZU-31-1 was isolated from the intestinal tract of the Onchidium sp.collected from Xuwen in Guangdong province, China.The fungus was identified by our team as Aspergillus terreus GZU-31-1, according to a molecular biological protocol by DNA amplification and sequencing of the ITS region (deposited in GenBank, accession No. MN860009).The strain was stored in School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, China, with the access code, GZU-31-1.

Biological assay
RAW 264.7 cells were cultured at 37 C in DMEM supplemented with 10% FBS with penicillin G (60 U/mL) and streptomycin (100 mg/mL) in a 5% CO 2 air atmosphere.The cells were seeded in 96-well plates (2 Â 10 4 cells/well) and brooded for 12 h, which were co-brooded with the tested compounds in suitable concentration for 2 h.Then the LPS (1 mg/mL) was added and cultured for another 48 h.The NO concentration was detected by the Griess reagent.Slightly, 100 mL of cell-free supernatant was mixed with 100 mL of Griess reagent (Griess reagent I and Griess reagent II, Beyotime).After breeding at room temperature for 5 min, the OD values were measured at 540 nm using a microplate reader and repeated in parallel three times.The cytotoxicity was detected by MTT method, the cells were seeded in 96-well plates (2 Â 10 4 cells/well) and brooded to adhere for 12 h, then the cells were co-brooded with fresh medium (200 mL/well) and treated with the tested compounds at various final concentrations (0 À 100 mM) for 2 h, added LPS (1 mg/mL), after 48 h, MTT (5 mg/mL) was subsequently added and brooded for 4 h, and the cells of the positive control group were cobrooded with indomethacin.The OD values at 570 and 490 nm were measured with a microplate reader after the culture medium was removed and the crystals were dissolved synchronously in DMSO.All the compounds were dissolved in DMSO and diluted with DMEM medium until the final concentration < 0.1%.