New Phenol Derivatives from the Haima Cold Seep-Derived Fungus Aspergillus subversicolor CYH-17

Seven new phenol derivatives, subversins A–E (1–5), subversic acid A (6) and epi-wortmannine G (7); one new natural product, 4-hydroxy-7-methoxyphthalide (8); and five known compounds (9–13) were isolated from the fungus Aspergillus subversicolor CYH-17 collected from the Haima cold seep. The structures and absolute configurations of these compounds were determined via NMR, MS, optical rotation, electronic circular dichroism (ECD) calculation, X-ray diffraction analysis and comparison with the literature. Compounds 2 and 5 were two pairs of enantiomers. All compounds were tested for their α-glucosidase and acetylcholinesterase (AChE) inhibitory activity, antioxidant activity and antibacterial activity, but no obvious activity was observed among these studied compounds.


Introduction
Cold seeps have attracted increasing amounts of research interest since they were first discovered in 1983 [1].In cold seeps, hydrocarbons such as methane, hydrogen sulfide and carbon dioxide are carried to the ocean floor due to geological activity, which leads to abundant chemosynthetic ecosystems [2].The biological resources found in cold seeps are abundant and include archaea, bacteria, fungi, tubeworms, clams and mussels [3].Over the past 40 years, most related research has focused on the taxonomy of species [4,5] and the ecological role of microorganisms [6,7].However, few studies have reported that new secondary metabolites are produced by cold seep-derived creatures [8].
In fact, cold seep organisms possess the potential to produce intriguing natural products as they survive in extreme environments [9].Under extreme conditions, cold seep organisms have evolved unique pathways to produce structurally diverse and biologically active secondary metabolites.According to previous reports, there were a great deal of new compounds that were found in the microorganisms derived from cold seeps, involving alkaloids [10], polyketides [11], terpenoids [12], glycosides [13], macrolides [14] and so forth.The new secondary metabolites displayed significant biological activity, including antimicrobial activity, cytotoxic activity and antioxidant activity.Therefore, cold seeps are a new and significant source for the discovery of active natural products.
Compound 4 was yielded as yellow oil with molecular formula of C 12 H 14 O 2 according to the molecular ion peak at m/z 191.1074 [M + H] + (calculated for C 12 H 15 O 2 , 191.1067), implying six degrees of unsaturation.According to the NMR data, 4 possessed an isobenzofuran skeleton and was similar to riboxylarinol B [24], and the differences were the presence of one double bond between C-9 and C-10 and the absence of the two hydroxyl groups on C-9 and C-10 in 4. The 1 H-1 H COSY correlations between H-3/H-8/H-9/H-10/H-11, the HMBC correlations from H-9 (δ H 5.37-5.52,m) and H-10 (δ H 5.59-5.64,m) to C-8 (δ C 41.85) and C-11 (δ C 18.23) and the mass data indicated the differences.The geometric configuration of the double bond was determined in the same way as 2. Compared with optical rotation data of the (S)-3-deoxyisoochracinic acid [25] [α]     [28] and the changes were the presence of a carboxyl (δ C 175.62) and the absence of one methyl group in 6.This suggested that the carboxyl group might replace the methyl group in 6.The above deduction was supported via the HMBC correlations from H-14 (δ H 6.60, t, J = 6.8 Hz) and H 3 -17 (δ H 1.96, s) to C-16 (δ C 175.62) and the MS data.The NOESY correlations between H 2 -13 (δ H 3.48, d, J = 7.0 Hz) and H 3 -17 indicated that the geometric configuration of the double bond was determined as 14E.Compound 6 was named subversic acid A.
Compound 7 was a yellow oil with the molecular formula of C 12 H 14 O 4 based on the molecular ion peak at m/z 245.0796 [M + Na] + (calculated for C 12 H 14 NaO 4 , 245.0784), implying six degrees of unsaturation.The NMR and the mass data of 7 proved that 7 had the same planar structure as the known compound wortmannine G [29].However, based on the optical rotation data of wortmannine G ([α] 20 D = +4.0(c 4 mM, CHCl 3 )), the absolute structure of 7 ([α] 25 D = −5.9(c 0.1, CHCl 3 )) was defined as 3R.Compound 7 was named epi-wortmannine G.

Biological Test
All compounds were tested for antibacterial activity, antioxidant activity, α-glucosidase inhibitory activity and acetylcholinesterase inhibitory activity.Compound 10 displayed inhibitory activity against five Gram-positive bacteria (B.subtilis, E. profundum, E. faecalis, S. aureus and MRSA) and one Gram-negative bacterium (A.baumannii).Specifically, compound 10 potently inhibited B. subtilis with an MIC value of 0.1 µM.No obvious activity of the compounds was observed in terms of antioxidant activity and enzyme inhibitory activity.The IC 50 and MIC values of the compounds larger than 200 µM were not included in the results of the bioassays (Tables S32 and S33).The structure and activity analysis of 10 and its analogues 6, 9 and 11 indicated that the dibenzofuran skeleton played an essential role in the antibacterial activity, which was consistent with the literature [31,32].

Fungal Materials
The fungus was separated from the sediment (−1363 m) obtained from the Haima cold seep in 2021.The DNA of the fungus was extracted according to the instructions of the DNA extraction kit.Then, a polymerase chain reaction instrument was used to amplify the purified DNA of the fungus with ITS primers (ITS1:5'-CTTGGTCATTTAGAGGAAGTAA-3'; ITS4: 5'-TCCTCCGCTTATTGATATGC-3').According to the ITS region sequence in the NCBI database, the strain was 99.59% identical to A. subversicolor (accession No. NR_135446.1).In terms of the results of the morphological features and the ITS region sequence, the strain was determined to be A. subversicolor and was named A. subversicolor CYH-17.The fungus was stored in the Research Network for Applied Microbiology (RNAM) Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences.

Fermentation, Extraction and Purification
The fungus was fermented on PDA plates and then transferred to 200 flasks containing medium (rice 100.0 g, artificial sea salt 3.0 g, distilled water 0.1 L).The strain was fermented statically at 25 • C for a month.The rice was extracted with ethyl acetate three times to gather 77.0 g of the ethyl acetate extract.
The ethyl acetate extract of the fungus was fractionated by silica gel column chromatography (CC) eluted with petroleum ether and ethyl acetate gradient (100:0 to 0:100) to obtain seven fractions (Frs.1-7).Fr.4 was purified via HPLC with MeOH-H 2 O gradient   13 C NMR data, see Table 3.

X-ray Crystal Structure Analysis
Crystallographic data of compound 1 were yielded on a Rigaku XtaLAB AFC12 single-crystal diffractometer (Rigaku, Japan) via Cu Kα radiation.The crystal was kept at 100.5 (9) K during the data collection.Using Olex2, the structure was solved with the SHELXT structure solution program using Intrinsic Phasing and refined with the SHELXL refinement package using Least Squares minimization.The crystallographic data of compound 1 were stored in the Cambridge Crystallographic Data Centre database (deposition numbers 2324024).Copies of the data are available free of charge from the CCDC at www.ccdc.cam.ac.uk, accessed on 6 March 2022.
Crystal data for compound 1:  The bacteria Vibrio alginolyticus XSBZ14, Enterococcus faecalis ATCC 29212, Acinetobacter baumannii ATCC 19606, Escherichia coli ATCC 25922, Bacillus subtilis BS01, Klebsiella pneumoniae ATCC 13883, Exiguobacterium profundum Staphylococcus aureus ATCC 29213 and MRSA 107352 were used to measure antibacterial activity.The procedures of the antibacterial test were the same as Zhang [39].Ciprofloxacin was used as the positive control.

AChE Inhibitory Assay
The procedures of the AChE inhibitory test were the same as Yang [40].Tacrine was used as the positive control.

α-Glucosidase Inhibitory Assay
The procedures of the α-glucosidase inhibitory test were the same as Ding [41].Acarbose was used as the positive control.

DPPH Radical Scavenging Assay
The procedures of the DPPH radical scavenging test were the same as Zhong [42].Ascorbic acid was used as the positive control.

Conclusions
Chemical exploration of the fungus Aspergillus subversicolor CYH-17 resulted in the isolation and elucidation of seven new phenol derivatives, subversins A-E (1-5), subversic acid A (6) and epi-wortmannine G (7); one new natural product, 4-hydroxy-7-methoxyphthalide (8); and five known secondary metabolites (9-13).The structural frameworks of the compounds included benzoic acid, isobenzofuran-1(3H)-one, isobenzofuran and isochroman-4-one.Compound 10 inhibited six bacteria with MIC values ranging from 0.1 to 50 µM.No obvious activity of the compounds was seen in the enzyme inhibitory activity and antioxidant activity.Future research should focus on exploring the diverse structures of dibenzofuran and isobenzofuran derivatives through OSMAC strategies and elucidating the structure-activity relationship of the compounds in this fungus.

Figure 2 .
Figure 2. The crystal structure for compound 1.

Figure 2 .
Figure 2. The crystal structure for compound 1.Compounds 2-3 were obtained as oils with the molecular formulas of C 13 H 14 O 3 and C 13 H 14 O 4 on the basis of molecular ion peaks at m/z 219.1018 [M + H] + (calculated for C 13 H 15 O 3 , 219.1016) and 235.0966 [M + H] + (calculated for C 13 H 15 O 4 , 235.0965), respectively.NMR data showed that 2-3 shared the same isobenzofuran-1(3H)-one molecular framework.2 was close to the known compound (S)-3-allyl-7-methoxyisobenzofuran-1(3H)-one [22], with the exception of the presence of one methyl (δ C 18.12/δ H 1.61) at the C-10 position in 2. The above change was proven by the 1 H-1 H COSY correlations between H-10/H-11 and the HMBC correlations from H 3 -11 (δ H 1.61) to C-9 (δ C 124.92) and C-10 (δ C 131.17).Compared with 2, there was a hydroxyl group at the C-8 position in 3.This was confirmed by the detailed HMBC and 1 H-1 H COSY correlations (Figure3).Thus, the planar structures of 2 and 3 were determined, and 2-3 were named subversins B-C.

Figure 2 .
Figure 2. The crystal structure for compound 1.
a Spectra were measured in DMSO-d 6 ; b spectra were measured in methanol-d 4 .
a Spectra were measured in methanol-d 4 ; b spectra were measured in chloroform-d.
a Spectra were measured in methanol-d 4 .