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

Abstract

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.

1. 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.

With the aim of uncovering new secondary metabolites from cold seep-derived fungi, our team carried out a series of works. As a result, we isolated some fungi from the Haima cold seep and discovered several new diketopiperazine alkaloids from the fungi Aspergillus fumigatus CYH-5 [15] and Toxicocladosporium sp. CYH-18 [16]. Recently, the fungus Aspergillus subversicolor CYH-17, isolated from the sediment of the Haima cold seep at a depth of 1363 m in 2021, attracted our attention. 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 products, diorcinol (9) [17], 3,7-dihydroxy-1,9-dimethyldibenzofuran (10) [18], 2-methoxyl cordyol C (11) [19], farnesylemefuranone E (12) [12] and citreorosein (13) [20] (Figure 1), were separated from the fungus A. subversicolor CYH-17. NMR, MS, optical rotation, electronic circular dichroism calculation and X-ray diffraction analysis were used to confirm the planar structures and absolute configurations of these compounds. In this study, the separation, structural elucidation and biological activity of those secondary metabolites are reported.

2. Results and Discussion

2.1. Structural Elucidation

Compound 1 was obtained as a colorless crystal with the molecular formula of C₁₉H₂₄O₅ based on the molecular ion peak at m/z 333.1696 [M + H]⁺ (calculated for C₁₉H₂₅O₅, 333.1697), suggesting eight degrees of unsaturation. After analyzing detailed NMR data, 1 was found to have a benzoic acid skeleton similar to that of 2,4-dihydroxy-6-(5,7-dimethyl-2-oxo-trans-3-trans-5-nonadienyl)-3-methylbenzaldehyde [21], except for the aldehyde group being a carboxyl group in 1. The downshift of C-1 (δ_C 173.23) in 1 and the mass data revealed the difference. The coupling constant (J_10,11 = 15.9 Hz) and the NOESY correlations between H-10 (δ_H 6.12, d, J = 15.9 Hz) and H₃-18 (δ_H 1.75, s) and between H-11 (δ_H 7.22, d, J = 15.9 Hz) and H-13 (δ_H 5.81, d, J = 9.7 Hz) suggested that the geometric configurations of the double bonds were determined to be 10E and 12E. Compared with the optical rotation data of 2,4-dihydroxy-6-(5,7-dimethyl-2-oxo-trans-3-trans-5-nonadienyl)-3-methylbenzaldehyde (${[\alpha ]}_{\mathrm{D}}^{23}$ = +51.0 (c 1.0, CHCl₃)), the absolute configuration of 1 (${[\alpha ]}_{\mathrm{D}}^{25}$ = +31.5 (c 0.02, CHCl₃)) was determined as 14S. After repeated crystallization, the crystal of compound 1 was obtained, and the absolute configuration of compound 1 was unambiguously confirmed as 14S based on the Cu Kα radiation data with a good flack parameter (−0.01 (8)) (Figure 2). Compound 1 was named subversin A.

Compounds 2–3 were obtained as oils with the molecular formulas of C₁₃H₁₄O₃ and C₁₃H₁₄O₄ on the basis of molecular ion peaks at m/z 219.1018 [M + H]⁺ (calculated for C₁₃H₁₅O₃, 219.1016) and 235.0966 [M + H]⁺ (calculated for C₁₃H₁₅O₄, 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 ¹H-¹H COSY correlations between H-10/H-11 and the HMBC correlations from H₃-11 (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 ¹H-¹H COSY correlations (Figure 3). Thus, the planar structures of 2 and 3 were determined, and 2-3 were named subversins B–C.

The geometric configuration of the double bond of 2–3 was confirmed to be 9E via the NOESY correlations between H-9 and H₃-11. Compound 2 was a racemic mixture (${[\alpha ]}_{\mathrm{D}}^{25}$ = 0 (c 0.1, CHCl₃)), which was separated via chiral HPLC into two optically pure enantiomers: (+)-2 and (−)-2 (Figure S72). Based on optical rotation data of the (S)-3-allyl-7-methoxyisobenzofuran-1(3H)-one (${[\alpha ]}_{\mathrm{D}}^{34}$ = −43.8 (c 1.0, CHCl₃)) [22], the absolute structures of (+)-2 (${[\alpha ]}_{\mathrm{D}}^{25}$ = +18.5 (c 0.04, CHCl₃)) and (−)-2 (${[\alpha ]}_{\mathrm{D}}^{25}$ = −26.2 (c 0.05, CHCl₃)) were defined as 3R and 3S, respectively. Additionally, the absolute configurations of (+)-2 and (−)-2 were confirmed further by ECD calculations, and the calculated ECD spectra for (3R)-2 and (3S)-2 agreed with the experimental curves (Figure S73). The relative configuration of 3 was confirmed as 3R*, 8S* (3b) by DP4+ probability analysis using GIAO NMR chemical shift calculations [23] (Table S31). The absolute configuration of 3 was determined through ECD calculation. The calculated ECD spectrum for (3R,8S)-3 agreed with the experimental curve (Figure S81).

Compound 4 was yielded as yellow oil with molecular formula of C₁₂H₁₄O₂ according to the molecular ion peak at m/z 191.1074 [M + H]⁺ (calculated for C₁₂H₁₅O₂, 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 ¹H-¹H 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] $({[\alpha ]}_{\mathrm{D}}^{22}$ = −13.0 (c 0.08, MeOH)), the configuration of 4 (${[\alpha ]}_{\mathrm{D}}^{25}$ = −24.7 (c 0.08, MeOH)) was defined as 3S. Compound 4 was named subversin D.

Table 1. ¹H NMR and ¹³C NMR data of compounds 1, 5 and 6 (700, 176 MHz, δ in ppm, J in Hz).

Position	1 a		5 b		6 b
	δC	δH	δC	δH	δC	δH
1	173.23, C		173.84, C		159.53, C
2	104.89, C				103.91, CH	6.17 (t, 2.1)
3	162.93, C		69.19, CH2	5.22 (s)	160, C
3a			129.65, C
4	108.61, C		146.91, C		111.46, CH	6.26 (s)
5	159.10, C		142.04, C		141.57, C
6	110.94, CH	6.15 (s)	156.63, C		111.71, CH	6.33 (s)
7	136.70, C		99.61, CH	6.94 (s)	157.12, C
7a			121.58, C
8	47.47, CH2	4.09–4.16 (m)			104.77, CH	6.30 (s)
9	197.23, C		72.84, CH2	4.12 (m) 4.03–4.09 (m)	157.35, C
10	124.45, CH	6.12 (d, 15.9)	35.75, CH2	2.00–2.08 (m) 1.81–1.89 (m)	120.99, C
11	146.61, CH	7.22 (d, 15.9)	39.92, CH	2.68 (m)	139.9, C
12	131.76, C		184.64, C		113.02, CH	6.30 (s)
13	148.35, CH	5.81 (d, 9.7)	18.46, CH3	1.19 (d, 6.8)	26.46, CH2	3.48 (d, 7.0)
14	34.37, CH	2.44–2.48 (m)	56.66, CH3	3.88 (s)	140.17, CH	6.60 (t, 6.8)
15	29.48, CH2	1.36–1.42 (m) 1.24–1.30 (m)			130.37, C
16	11.84, CH3	0.81 (t, 7.4)			175.62, C
17	20.09, CH3	0.96 (d, 6.6)			13.09, CH3	1.96 (s)
18	12.29, CH3	1.75 (s)			19.99, CH3	2.20 (s)
19	8.14, CH3	1.94 (s)			21.55, CH3	2.21 (s)

^a Spectra were measured in DMSO-d₆; ^b spectra were measured in methanol-d₄.

Table 1. ¹H NMR and ¹³C NMR data of compounds 1, 5 and 6 (700, 176 MHz, δ in ppm, J in Hz).

Position	1 a		5 b		6 b
	δC	δH	δC	δH	δC	δH
1	173.23, C		173.84, C		159.53, C
2	104.89, C				103.91, CH	6.17 (t, 2.1)
3	162.93, C		69.19, CH2	5.22 (s)	160, C
3a			129.65, C
4	108.61, C		146.91, C		111.46, CH	6.26 (s)
5	159.10, C		142.04, C		141.57, C
6	110.94, CH	6.15 (s)	156.63, C		111.71, CH	6.33 (s)
7	136.70, C		99.61, CH	6.94 (s)	157.12, C
7a			121.58, C
8	47.47, CH2	4.09–4.16 (m)			104.77, CH	6.30 (s)
9	197.23, C		72.84, CH2	4.12 (m) 4.03–4.09 (m)	157.35, C
10	124.45, CH	6.12 (d, 15.9)	35.75, CH2	2.00–2.08 (m) 1.81–1.89 (m)	120.99, C
11	146.61, CH	7.22 (d, 15.9)	39.92, CH	2.68 (m)	139.9, C
12	131.76, C		184.64, C		113.02, CH	6.30 (s)
13	148.35, CH	5.81 (d, 9.7)	18.46, CH3	1.19 (d, 6.8)	26.46, CH2	3.48 (d, 7.0)
14	34.37, CH	2.44–2.48 (m)	56.66, CH3	3.88 (s)	140.17, CH	6.60 (t, 6.8)
15	29.48, CH2	1.36–1.42 (m) 1.24–1.30 (m)			130.37, C
16	11.84, CH3	0.81 (t, 7.4)			175.62, C
17	20.09, CH3	0.96 (d, 6.6)			13.09, CH3	1.96 (s)
18	12.29, CH3	1.75 (s)			19.99, CH3	2.20 (s)
19	8.14, CH3	1.94 (s)			21.55, CH3	2.21 (s)

^a Spectra were measured in DMSO-d₆; ^b spectra were measured in methanol-d₄.

Table 2. ¹H NMR and ¹³C NMR data of compounds 2–4 (700, 176 MHz, δ in ppm, J in Hz).

Position	2 a		3 a		4 b
	δC	δH	δC	δH	δC	δH
1	171.03, C		171.06, C		58.97, CH2	4.95 (s)
3	81.58, CH	5.46–5.53 (m)	84.49, CH	5.42 (d, 4.0)	70.89, CH	4.82 (dd, 7.9, 5.3)
3a	154.00, C		151.26, C		142.03, C
4	115.06, CH	7.10 (d, 7.3)	116.16, CH	7.15 (d, 7.6)	118.13, CH	6.98 (d, 7.7)
5	137.92, CH	7.69 (dd, 8.1, 7.7)	137.57, CH	7.67 (t, 8.0)	129.32, CH	7.20 (t, 7.9)
6	112.03, CH	7.08 (d, 8.0)	112.25, CH	7.09 (d, 8.3)	116.16, CH	6.82 (d, 8.0)
7	159.92, C		159.90, C		156.63, C
7a	114.35, C		114.97, C		122.95, C
8	38.31, CH2	2.68–2.75 (m) 2.46–2.59 (m)	74.23, CH	4.42–4.45 (m)	41.85, CH2	2.38–2.45 (m)
9	124.92, CH	5.25–5.35 (m)	129.15, CH	5.51 (m)	126.79, CH	5.37–5.52 (m)
10	131.17, CH	5.58 (m)	130.49, CH	5.73 (m)	130.04, CH	5.59–5.64 (m)
11	18.12, CH3	1.61 (dd, 6.5, 1.4)	17.94, CH3	1.68 (m)	18.23, CH3	1.70 (d, 6.3)
12	56.33, CH3	3.96 (s, 3H)	56.30, CH3	3.96 (s)

^a Spectra were measured in methanol-d₄; ^b spectra were measured in chloroform-d.

Table 2. ¹H NMR and ¹³C NMR data of compounds 2–4 (700, 176 MHz, δ in ppm, J in Hz).

Position	2 a		3 a		4 b
	δC	δH	δC	δH	δC	δH
1	171.03, C		171.06, C		58.97, CH2	4.95 (s)
3	81.58, CH	5.46–5.53 (m)	84.49, CH	5.42 (d, 4.0)	70.89, CH	4.82 (dd, 7.9, 5.3)
3a	154.00, C		151.26, C		142.03, C
4	115.06, CH	7.10 (d, 7.3)	116.16, CH	7.15 (d, 7.6)	118.13, CH	6.98 (d, 7.7)
5	137.92, CH	7.69 (dd, 8.1, 7.7)	137.57, CH	7.67 (t, 8.0)	129.32, CH	7.20 (t, 7.9)
6	112.03, CH	7.08 (d, 8.0)	112.25, CH	7.09 (d, 8.3)	116.16, CH	6.82 (d, 8.0)
7	159.92, C		159.90, C		156.63, C
7a	114.35, C		114.97, C		122.95, C
8	38.31, CH2	2.68–2.75 (m) 2.46–2.59 (m)	74.23, CH	4.42–4.45 (m)	41.85, CH2	2.38–2.45 (m)
9	124.92, CH	5.25–5.35 (m)	129.15, CH	5.51 (m)	126.79, CH	5.37–5.52 (m)
10	131.17, CH	5.58 (m)	130.49, CH	5.73 (m)	130.04, CH	5.59–5.64 (m)
11	18.12, CH3	1.61 (dd, 6.5, 1.4)	17.94, CH3	1.68 (m)	18.23, CH3	1.70 (d, 6.3)
12	56.33, CH3	3.96 (s, 3H)	56.30, CH3	3.96 (s)

^a Spectra were measured in methanol-d₄; ^b spectra were measured in chloroform-d.

Compound 5 was obtained as a yellow oil with the molecular formula of C₁₄H₁₆O₇ on the basis of the molecular ion peak at m/z 295.0829 [M − H]⁻ (calculated for C₁₄H₁₅O₇, 295.0823), suggesting seven degrees of unsaturation. The ¹H and ¹³C NMR data indicated that 5 also had a isobenzofuran-1(3H)-one unit, similar to the known compound (+)-5-(3-carboxy-butoxy)-7-hydroxy-4,6-dimethylphthalide [26,27], with the exception of the different substituents on C-4, C-6 and C-7 of the benzene ring in 5. The differences were proven by the HMBC correlations from H₂-3 (δ_H 5.22, s) to C-4 (δ_C 146.91, s), from H₃-14 (δ_H 3.88, s) to C-6 (δ_C 156.63) and from H-7 (δ_H 6.94, s) to C-1 (δ_C 173.84) and C-7a (δ_C 121.58). Compound 5 was also a racemic mixture (${[\alpha ]}_{\mathrm{D}}^{25}$ = 0 (c 0.1, MeOH)), which was separated via chiral HPLC into two optically pure enantiomers: (+)-5 and (−)-5 (Figure S74). Based on optical rotation data of the (+)-5-(3-carboxy-butoxy)-7-hydroxy-4,6-dimethylphthalide (${[\alpha ]}_{\mathrm{D}}^{25}$ = +6.2 (c 0.36, MeOH)), the absolute structures of (+)-5 (${[\alpha ]}_{\mathrm{D}}^{25}$ = +7.1 (c 0.1, MeOH)) and (−)-5 (${[\alpha ]}_{\mathrm{D}}^{25}$ = −7.7 (c 0.08, MeOH)) were defined as 11S and 11R, respectively. Compound 5 was named subversin E.

Compound 6 was purified as a yellow oil with the molecular formula of C₁₉H₂₀O₅ according to the molecular ion peak at m/z 329.1393 [M + H]⁺ (calculated for C₁₉H₂₁O₅, 329.1384), indicating 10 degrees of unsaturation. The NMR data of 6 were close to those of the known compound verticilatin [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 H₃-17 (δ_H 1.96, s) to C-16 (δ_C 175.62) and the MS data. The NOESY correlations between H₂-13 (δ_H 3.48, d, J = 7.0 Hz) and H₃-17 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 C₁₂H₁₄O₄ based on the molecular ion peak at m/z 245.0796 [M + Na]⁺ (calculated for C₁₂H₁₄NaO₄, 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 (${[\alpha ]}_{\mathrm{D}}^{20}$ = +4.0 (c 4 mM, CHCl₃)), the absolute structure of 7 (${[\alpha ]}_{\mathrm{D}}^{25}$ = −5.9 (c 0.1, CHCl₃)) was defined as 3R. Compound 7 was named epi-wortmannine G.

Compound 8 was obtained as a white powder with the molecular formula of C₉H₈O₄ on the basis of the molecular ion peak at m/z 181.0498 [M + H]⁺ (calculated for C₉H₉O₄, 181.0495), suggesting six degrees of unsaturation. The ¹H-¹H COSY correlations between H-5/H-6 and the HMBC correlations from H₂-3 (δ_H 5.22, s) to C-1 (δ_C 172.00), C-3a (δ_C 136.44), C-4 (δ_C 147.22) and C-7a (δ_C 114.58), from H-5 (δ_H 7.05, d, J = 8.7 Hz) to C-3a (δ_C 136.44) and C-7 (δ_C 152.87), and from H-6 (δ_H 6.92, d, J = 8.7 Hz) to C-4 (δ_C 147.22) and C-7a (δ_C 114.58), confirmed the planar structure of compound 8. Compound 8 was first synthesized by Keay [30] in 1984 and this was the first time that compound 8 had been reported from nature.

Table 3. ¹H NMR and ¹³C NMR data of compounds 7–8 (700, 176 MHz, δ in ppm, J in Hz).

Position	7 a		8 a
	δC	δH	δC	δH
1	58.42, CH2	5.03 (d, 16.2) 4.84 (d, 16.2)	172.00, C
3	97.69, C		68.88, CH2	5.22 (s)
3a			136.44, C
4	193.27, C		147.22, C
4a	130.64, C
5	118.59, CH	7.46 (d, 7.6)	123.37, CH	7.05 (d, 8.7)
6	128.87, CH	7.23 (t, 7.9)	113.43, CH	6.92 (d, 8.7)
7	120.56, CH	7.00 (dd, 8.0, 0.7)	152.87, C
7a			114.58, C
8	154.19, C		56.53, CH3	3.87 (s)
8a	130.29, C
9	39.75, CH2	2.02 (ddd, 13.5, 11.7, 4.8) 1.77 (ddd, 13.6, 11.7, 4.8)
10	17.61, CH2	1.51 (m) 1.31–1.39 (m)
11	14.76, CH3	0.93 (t, 7.4)

^a Spectra were measured in methanol-d₄.

Table 3. ¹H NMR and ¹³C NMR data of compounds 7–8 (700, 176 MHz, δ in ppm, J in Hz).

Position	7 a		8 a
	δC	δH	δC	δH
1	58.42, CH2	5.03 (d, 16.2) 4.84 (d, 16.2)	172.00, C
3	97.69, C		68.88, CH2	5.22 (s)
3a			136.44, C
4	193.27, C		147.22, C
4a	130.64, C
5	118.59, CH	7.46 (d, 7.6)	123.37, CH	7.05 (d, 8.7)
6	128.87, CH	7.23 (t, 7.9)	113.43, CH	6.92 (d, 8.7)
7	120.56, CH	7.00 (dd, 8.0, 0.7)	152.87, C
7a			114.58, C
8	154.19, C		56.53, CH3	3.87 (s)
8a	130.29, C
9	39.75, CH2	2.02 (ddd, 13.5, 11.7, 4.8) 1.77 (ddd, 13.6, 11.7, 4.8)
10	17.61, CH2	1.51 (m) 1.31–1.39 (m)
11	14.76, CH3	0.93 (t, 7.4)

^a Spectra were measured in methanol-d₄.

2.2. 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 IC₅₀ 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].

By the end of 2023, over 575 isobenzofuran derivatives had been reported, mainly in Umbelliferae plants and fungi [33,34,35,36,37,38]. Among the isobenzofuran derivatives, 97 originate from marine fungi. Based on the literature [36,37], it was reported that isobenzofuran derivatives exhibited effects on neuroprotective, anti-inflammatory, hepatoprotective and cytotoxicity assays.

3. Materials and Methods

3.1. 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.

3.2. General

Sephadex LH-20 (GE Healthcare, Stockholm, Sweden) and 100–200 and 200–300 mesh Silica gel (Qingdao Marine Chemical Group Co., Qingdao, China) were used for column chromatography (CC), while an Agilent 1260 HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with an ODS C-18 column (5 μm, 10 × 250 mm) was used for HPLC separation. In addition, all the data for structural elucidation were collected from an MCP500 automatic polarimeter (AntonPaar, Graz, Austria), UV-2600 spectrometer (Shimadzu, Kyoto, Japan), IR Affinity-1 spectrometer (Shimadzu, Kyoto, Japan), AVANCE IIIHD 700 MHz Digital NMR Spectrometer (Bruker, Billerica, MA, USA), MaXis quadrupole time-of-flight mass spectrometer (Bruker, Mannheim, Germany), Rigaku XtaLAB AFC12 single-crystal diffractometer (Rigaku, Tokyo, Japan) and chirascan circular dichroism spectrometer (Applied Photophysics, Leatherhead, UK).

3.3. 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-H₂O gradient (5:95 to 100:0) to obtain six fractions (Frs.4.1–Fr.4.6). Fr.4.3 was fractionated via silica gel CC eluted with CH₂Cl₂-MeOH (100:0 to 90:10) to yield two parts (Frs.4.3.1–Fr.4.3.2). Fr.4.3.1 was purified by HPLC (65% MeOH/H₂O) to obtain 7 (3.1 mg). Fr.4.3.2 was subjected to Sephadex LH-20 (CH₂Cl₂/MeOH = 1:1) to give 4 (1.5 mg). Fr.4.4 was fractionated via silica gel CC eluted with CH₂Cl₂-MeOH (100:0 to 90:10) to yield three subfractions Frs.4.4.1–Fr.4.4.3. Fr.4.4.2 was subjected to Sephadex LH-20 (CH₂Cl₂/MeOH = 1:1) to give 9 (46.7 mg) and three fractions (Frs.4.4.2.1–4.4.2.3). Fr.4.4.2.1 was purified by HPLC (60% ACN/H2O) to obtain 2 (1.1 mg). Fr.4.4.2.2 was separated by HPLC (60% ACN/H₂O) to yield 11 (1.8 mg). Fr.4.4.2.3 was fractionated by HPLC (60% ACN/H₂O) to give 10 (3.9 mg). Fr.4.4.3 was subjected to Sephadex LH-20 (CH₂Cl₂/MeOH = 1:1) to obtain Fr.4.4.3.1. Fr.4.4.3.1 was separated via silica gel CC eluted with CH₂Cl₂-MeOH (100:0 to 90:10) to yield 13 (2.2 mg) and 6 (3.1 mg). Fr.4.5 was fractionated via silica gel CC eluted with CH₂Cl₂-MeOH (100:0 to 95:5) to give the two parts Fr.4.5.1–Fr.4.5.2. Fr.4.5.1 was purified by HPLC (70% ACN/H₂O) to obtain 12 (3.0 mg). Fr.4.5.2 was fractionated via Sephadex LH-20 (CH₂Cl₂/MeOH = 1:1) to yield 1 (6.0 mg). Fr.5 was separated via HPLC with MeOH-H₂O gradient (5:95 to 100:0) to obtain two fractions (Frs.5.1–Fr.5.2). Fr.5.2 was subjected to Sephadex LH-20 (CH₂Cl₂/MeOH = 1:1) to give Fr.5.2.1–Fr.5.2.2. Fr.5.2.1 was purified by HPLC (43% MeOH/H₂O) to obtain 3 (2.2 mg). Fr.5.2.2 was fractionated by HPLC (55% MeOH/H₂O) to obtain 8 (1.2 mg). Fr.6 was separated via HPLC with MeOH-H₂O gradient (5:95 to 100:0) to yield three fractions (Frs.6.1–Fr.6.3). Fr.6.2 was subjected to Sephadex LH-20 (MeOH) to give Fr.6.2.1. Fr.6.2.1 was purified by HPLC (37% ACN/H₂O) to obtain 5 (1.5 mg).

3.3.1. Subversin A (1)

Colorless crystal; ${[\alpha ]}_{\mathrm{D}}^{25}$ = +41.1 (c 0.1, MeOH), ${[\alpha ]}_{\mathrm{D}}^{25}$ = +31.5 (c 0.02, CHCl₃); HR-ESI-MS at m/z 333.1696 [M + H]⁺ (calculated for C₁₉H₂₅O₅, 333.1697); UV (MeOH) λ_max (log ε) 219 (4.38), 279 (4.33) nm; IR (film) ν_max 3340, 2949, 1647, 1018, 671 cm⁻¹; ¹H NMR and ¹³C NMR data, see Table 1.

3.3.2. Subversin B (2)

Colorless oil; ${[\alpha ]}_{\mathrm{D}}^{25}$ = 0 (c 0.1, CHCl₃); HR-ESI-MS at m/z 219.1018 [M + H]⁺ (calculated for C₁₃H₁₅O₃, 219.1016); UV (MeOH) λ_max (log ε) 215 (4.12), 235 (3.94) 298 (3.74) nm; IR (film) ν_max 2926, 1759, 1607,1298,1038,787 cm⁻¹; ¹H NMR and ¹³C NMR data, see Table 2.

(+)-2. Colorless oil; ${[\alpha ]}_{\mathrm{D}}^{25}$ = +18.5 (c 0.04, CHCl₃); CD (MeOH): 209 nm (Δε = 2.94), 240 nm (Δε = −1.11), 296 nm (Δε = −0.65).

(−)-2. Colorless oil; ${[\alpha ]}_{\mathrm{D}}^{25}$ = −26.2 (c 0.05, CHCl₃); CD (MeOH): 210 nm (Δε = −2.33), 241 nm (Δε = 1.06), 296 nm (Δε = 0.66).

3.3.3. Subversin C (3)

Colorless oil; ${[\alpha ]}_{\mathrm{D}}^{25}$ = +6.4 (c 0.1, MeOH); HR-ESI-MS at m/z 235.0966 [M + H]⁺ (calculated for C₁₃H₁₅O₄, 235.0965); UV (MeOH) λ_max (log ε) 213 (4.13), 236 (3.84) 299 (3.64) nm; IR (film) ν_max 3385, 2954, 1744, 1026, 689 cm⁻¹; CD (MeOH): 209 nm (Δε = −9.85), 243 nm (Δε = 2.31), 298 nm (Δε = 1.25); ¹H NMR and ¹³C NMR data, see Table 2.

3.3.4. Subversin D (4)

Yellow oil; ${[\alpha ]}_{\mathrm{D}}^{25}$ = −17 (c 0.1, CHCl₃), ${[\alpha ]}_{\mathrm{D}}^{25}$ = −24.7 (c 0.08, MeOH); HR-ESI-MS at m/z 191.1074 [M + H]⁺ (calculated for C₁₂H₁₅O₂, 191.1067); UV (MeOH) λ_max (log ε) 220 (3.72), 281 (3.31) nm; IR (film) ν_max 3327, 2945,1018, 671 cm⁻¹; ¹H NMR and ¹³C NMR data, see Table 2.

3.3.5. Subversin E (5)

Yellow oil; ${[\alpha ]}_{\mathrm{D}}^{25}$ = 0 (c 0.1, MeOH); HR-ESI-MS at m/z 295.0829 [M − H]⁻ (calculated for C₁₄H₁₅O₇, 295.0823); UV (MeOH) λ_max (log ε) 216 (4.21), 266 (3.65) nm; IR (film) ν_max 3319, 2947, 1651, 1018, 675 cm⁻¹; ¹H NMR and ¹³C NMR data, see Table 1.

(+)-5. Yellow oil; ${[\alpha ]}_{\mathrm{D}}^{25}$ = +7.1 (c 0.1, MeOH); CD (MeOH): 223 nm (Δε = 0.30).

(−)-5. Yellow oil; ${[\alpha ]}_{\mathrm{D}}^{25}$ = −7.7 (c 0.08, MeOH); CD (MeOH): 224 nm (Δε = −0.39).

3.3.6. Subversic Acid A (6)

Yellow oil; HR-ESI-MS at m/z 329.1393 [M + H]⁺ (calculated for C₁₉H₂₁O₅, 329.1384); UV (MeOH) λ_max (log ε) 219 (4.61), 282 (3.73) nm; IR (film) ν_max 3364, 1595, 1522, 1153, 837, 677 cm⁻¹; ¹H NMR and ¹³C NMR data, see Table 1.

3.3.7. epi-Wortmannine G (7)

Yellow oil; ${[\alpha ]}_{\mathrm{D}}^{25}$ = −5.9 (c 0.1, CHCl₃); HR-ESI-MS at m/z 245.0796 [M + Na]⁺ (calculated for C₁₂H₁₄NaO₄, 245.0784); UV (MeOH) λ_max (log ε) 223 (4.18), 260 (3.86) 318 (3.41) nm; IR (film) ν_max 3334, 2954, 1690,1020, 754, 677 cm⁻¹; ¹H NMR and ¹³C NMR data, see Table 3.

3.3.8. 4-Hydroxy-7-methoxyphthalide (8)

White powder; HR-ESI-MS at m/z 181.0498 [M + H]⁺ (calculated for C₉H₉O₄, 181.0495); UV (MeOH) λ_max (log ε) 217 (3.96), 237 (3.57) 324 (3.37) nm; IR (film) ν_max 3343, 2947, 1649, 1018, 669 cm⁻¹; ¹H NMR and ¹³C NMR data, see Table 3.

3.4. 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: C₃₈H₄₈O₁₀, M = 664.76, triclinic, space group P1 (no.1), a = 6.32630 (10) Å, b = 10.8354 (2) Å, c = 13.0039 (2) Å, α = 91.6160(10)°, β = 103.8220 (10)°, γ = 92.5990 (10)°, V = 863.99 (3) ų, Z = 1, T = 100.5 (9) K, μ (Cu Kα) = 0.751 mm⁻¹, Dcalc = 1.278 g/cm³, 17397 reflections measured (7.006° ≤ 2Θ ≤ 148.54°), 6466 unique (R_int = 0.0298, R_sigma = 0.0318) which were used in all calculations. The final R₁ was 0.0340 (I > 2σ (I)) and wR₂ was 0.0928 (all data). The goodness of fit on F² was 1.079. Flack parameter = −0.01 (8), melting point: 184.0–185.0 °C.

3.5. Bioassays

3.5.1. Antibacterial Assay

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.

3.5.2. AChE Inhibitory Assay

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

3.5.3. 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.

3.5.4. 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.

3.6. Chiral HPLC Separation of Compounds 2 and 5

Chiral HPLC separations of the compounds were recorded on the HPLC (Agilent 1260) equipped with CHIRALPAK^® IA (250 × 4.6 mm, 5 μm). A phase: hexane with 0.1% formic acid; B phase: isopropanol.

Compound 2: gradient program: 0 min (93%A–7%B) to 25 min (93%A–7%B); flow rate: 1 mL/min; detection: UV 215 nm.

Compound 5: gradient program: 0 min (80%A–20%B) to 12 min (80%A–20%B); flow rate: 1 mL/min; detection: UV 215 nm.

4. 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.