Eremophilane-Type Sesquiterpenoids from Fungus Aspergillus aurantiobrunneus

Abstract

Six previously undescribed sesquiterpenoids, aurantiophilanes A–F (1–6), along with six identified analogues (7–12), were isolated from the fungus Aspergillus aurantiobrunneus. Among these, compounds 1 and 3 were identified as highly oxygenated eremophilane sesquiterpenoids, with compound 1 featuring a rare ketone functional group at C-1. The structures of all compounds were unambiguously elucidated using comprehensive spectroscopic analyses, including HRESIMS, NMR, and UV spectroscopy, supplemented by electronic circular dichroism (ECD) analyses and single-crystal X-ray diffraction. All identified compounds were evaluated for immunosuppressive activity; none showed significant effects at concentrations up to 40 µM.

1. Introduction

Sesquiterpenoids, biosynthesized from farnesyl pyrophosphate under the catalysis of sesquiterpene cyclases (STCs), represent one of the most structurally diverse groups of terpenoids, with over 300 distinct carbon skeletons identified to date [1,2,3,4,5,6]. Eremophilane sesquiterpenes, which consist of three isoprenes, share a characteristic decalin framework with eudesmanes and form a small but significant family of sesquiterpenoids [7,8]. Due to their structural variability, eremophilane sesquiterpenes exhibit a range of clinical therapeutic potentials including antitumor, neuroprotective [9], anti-inflammatory [10], antibacterial, cytotoxic [11], and immunosuppressive [12] activities. For example, paraconulones B showed inhibitory effects on lipopolysaccharide-induced NO production in BV2 cells [13]. These attributes highlight the importance of further research into eremophilane-type sesquiterpenoids for their promising biological properties.

The genus Aspergillus represents a highly significant fungal taxon, renowned for its representative secondary metabolites, among which terpenoids are particularly prominent [14]. Notably, between 2019 and 2024, sesquiterpenoids accounted for the largest proportion among the 217 new terpenoids isolated from various Aspergillus species [15]. Motivated by these findings, we turned our attention to less-explored members of the genus and selected A. aurantiobrunneus for further investigation. To date, only a single study has documented the secondary metabolites of this species, identifying sesquiterpenoids as its major chemical constituents [16]. To search for structurally unique and bioactive chemistry, six undescribed sesquiterpenoids and six known analogues were isolated from A. aurantiobrunneus (Figure 1). Accordingly, this study details the processes of extracting, isolating, and determining the molecular structures and assessing their biological properties.

2. Results and Discussion

Aurantiophilane A (1) was initially attained as colorless crystals. The molecular formula of C₁₅H₁₈O₄ was deduced from the HRESIMS peak at m/z 285.1107 [M + Na]⁺ (calcd. for C₁₅H₁₈O₄Na⁺, 285.1103), indicating seven sites of unsaturation. Analysis of the ¹H NMR data (

Table 1. ¹H (400 MHz) and ¹³C (100 MHz) NMR data for compounds 1–3.

	1 a		2 b		3 b
No.	δC, Type	δH, (J in Hz)	δC, Type	δH, (J in Hz)	δC, Type	δH, (J in Hz)
1	204.2, C		128.3, CH	6.30, s	119.0, CH	5.49, dd (3.4, 3.1)
2	41.4, CH2	2.45, m	185.1, C		40.7, CH2	3.03, dd (14.0, 3.1)
		1.74, m				2.71, dd (14.0, 3.4)
3	29.3, CH2	1.83, m	128.0, CH	6.19, s	210.5, C
		1.75, m
4	42.1. CH	2.06, m	157.9, C		54.2, CH	2.55, m
5	47.5, C		42.3, C		42.8, C
6	36.7, CH2	2.86, d (12.5)	31.4, CH2	3.04, d (16.5)	46.4, CH2	1.98, d (14.6)
		2.53, d (12.5)		2.53, d (16.5)		1.52, d (14.6)
7	159.4, C		144.8, C		78.4, C
8	100.8, C		152.1, C		103.9, C
9	127.7. CH	6.29, s	106.9, CH	6.23, s	33.8, CH2	2.78, d (15.2)
						2.59, d (15.2)
10	150.3, C		162.3, C		140.1, C
11	124.7, C		125.7, C		153.5, C
12	173.5, C		169.9, C		67.7, CH2	4.43, m
						4.37, m
13	8.4, CH3	1.83, s	9.1, CH3	2.01, s	104.4, CH2	5.22, d (2.6)
						4.94, d (2.6)
14	18.4, CH3	0.81, s	29.1, CH3	1.37, s	20.1, CH3	1.05, s
15	15.3, CH3	1.07, d (6.9)	19.4, CH3	2.12, s	7.3, CH3	1.03, d (6.6)
O-Me					48.5, CH3	3.30, s

^a Measured in CD₃OD; ^b Measured in CDCl₃.

) and HSQC spectroscopic data of 1, three methyl groups (δ_H 0.81, s; 1.07, d, J = 6.9 Hz; 1.83, s) and one olefinic proton (δ_H 6.29, s) were observed. The ¹³C NMR and DEPT data displayed 15 carbon resonances, comprising three methyls (δ_C 8.4, 15.3, and 18.4), three methylenes (δ_C 29.3, 36.7, and 41.4), one olefinic methine (δ_C 127.7), one quaternary carbon (δ_C 47.5), four nonprotonated carbons (including one oxygenated at δ_C 100.8, and three olefinic δ_C 124.7, 150.3, and 159.4), and one ester carbonyl at δ_C 173.5, along with one ketone at δ_C 204.2. One α, β-conjugated carbonyl, one double bond, and the ester carbonyl occupied four degrees, indicating the presence of the tricyclic ring skeleton. The ¹H−¹H COSY correlations of H₂-2/H₂-3/H-4/H₃-15, along with HMBC correlations from H₂-2 to C-1, C-3, C-4, and C-10, from H₂-6 to C-5, C-7, C-8, C-10, and C-11, from H-9 to C-1, C-5, C-7, C-8, and C-10, and from H₃-14 to C-4, C-5, C-6, and C-10 (Figure 2), constructed an eremophilane-type sesquiterpene framework [17]. The chemical shift of C-8 (δ_C 100.8) revealed the presence of a hemiacetal fragment. Consequently, the HMBC correlations from H₃-13 to C-7, C-11, and C-12 confirmed the existence of γ-lactone. The data of single-crystal X-ray diffraction were acquired by utilizing Cu Kα radiation, which determined the planar structure and absolute configuration of 1 as 4S, 5S, 8R (Figure 3).

Aurantiophilane B (2) was isolated as yellow crystals. Its molecular formula was determined to be C₁₅H₁₄O₃, established by the [M + Na]⁺ ion peak at m/z 265.0835 (calcd. for C₁₅H₁₄O₃Na⁺, m/z 265.0841) in the HRESIMS, corresponding to nine degrees of unsaturation. The ¹H NMR data (Table 1) of 2 revealed three methyl groups (δ_H 1.37, s; 2.01, s; 2.12, s), and three olefinic protons (δ_H 6.19, s; 6.23, s; 6.30, s). The ¹³C NMR data (Table 1) displayed signals for three methyls (δ_C 9.1, 19.4, and 29.1), one methylene (δ_C 31.4), three olefinic methines (δ_C 106.9, 128.0, and 128.3), one quaternary carbon (δ_C 42.3), five olefinic nonprotonated carbons (δ_C 125.7, 144.8, 152.1, 157.9, and 162.3), and two carbonyls (an ester carbonyl at δ_C 169.9 and one ketone at δ_C 185.1). Among the 15 carbons, the existence of two carbonyls and four double bonds accounted for six degrees of unsaturation, illustrating that 2 possessed a tricyclic ring system. Comparison of the aforementioned data with the reported 2-oxo-3-hydroxy-eremophila-1(10),3,7(11),8-tetraen-8,12-olide revealed a major difference: the hydroxyl group at C-3 was absent in 2 [18], as further confirmed by HMBC correlations from H-1 to C-3 and from H₃-15 to C-3, C-4, and C-5 (Figure 2). The absolute configuration of 2 was established as 5R by single-crystal X-ray diffraction (Figure 3).

Aurantiophilane C (3), identified as colorless oil, was determined as C₁₆H₂₂O₄, representing six sites of unsaturation, which was confirmed by an ion peak at m/z 301.1416 [M + Na]⁺ (calcd. for C₁₆H₂₂O₄Na⁺, 301.1416) in the HRESIMS. The ¹H and ¹³C NMR data (Table 1) of 3 were quite comparable to those of septoreremophilane D [10]. The HMBC correlation (Figure 2) from OMe-8 to C-8 indicated that the hydroxyl group at C-8 in septoreremophilane D was replaced by a methoxyl group in 3. The NOESY correlations (Figure 4) of H₃-14/H₃-15, H₃-14/H-9β, H-4/H-6α indicated that H₃-14 and H₃-15 were cofacial and were assigned as β-oriented; H₃-14/H-6β, H-6α/H-12α, and H-12β/OCH₃-8 indicated that OH-7 and OCH₃-8 were on the same side and assigned as β-orientated. The calculated spectrum of 4R, 5R, 7R, 8S-3 showed a close similarity with the experimental ECD spectra of 3. Thus, the absolute configuration of 3 was established to be 4R, 5R, 7R, 8S. (Figure 5).

Aurantiophilane E (4), identified as yellow oil, was confirmed as C₁₅H₂₂O₄ ([M + Na]⁺ m/z 273.1467 (calcd. For C₁₅H₂₂O₃Na, 273.1467) by its HRESIMS spectra, which possessed five sites of unsaturation. The ¹H and ¹³C NMR data (

Table 2. ¹H (400 MHz) and ¹³C (100 MHz) NMR data for compounds 4–6.

	4 a		5 b		6 b
No.	δC, Type	δH, (J in Hz)	δC, Type	δH, (J in Hz)	δC, Type	δH, (J in Hz)
1	73.2, CH	4.51, m	128.0, CH	6.16, d (2.6)	54.4, CH2	2.31, m
2	34.1, CH2	2.21, m	138.8, CH	6.25, m	198.8, C
		1.94, m
3	26.3, CH2	2.08, m	32.6, CH2	2.23, m	127.3, CH	5.89, s
		1.65, m		2.22, m
4	44.0, CH	1.77, m	37.9, CH	1.80, m	161.8, C
5	42.6, C		38.5, C		47.4, CH	2.46, m
6	43.1, CH2	3.29, d (13.6)	38.1, CH2	2.84, d (14.2)	28.1, CH2	1.93, m
		2.40, d (13.6)		2.28, d (14.2)		1.48, m
7	130.9, C		132.0, C		55.4, CH	2.10, m
8	195.4, C		187.4, C		66.4, CH	3.79 dt (10.2, 4.5)
9	129.2, CH		124.3, CH	5.78, s	47.4, CH2	1.96, m
						1.39, m
10	169.6, C		163.1, C		39.0, C
11	146.9, CH	4.53, m	137.2, C		145.7, C
12	63.8, CH2	4.40, m	172.6, C		114.0, CH2	4.97, d (3.1)
						4.93, d (3.1)
13	18.4, CH3	2.35, s	17.0, CH3	2.01, s	19.5, CH3	1.78, s
14	18.7, CH3	1.43, s	17.1, CH3	0.98, s	18.1, CH3	0.94, s
15	15.9, CH3	1.25, d (6.8)	14.7, CH3	1.02, d (6.8)	22.3, CH3	1.90, s
O-Me			52.5, CH3	3.80, s

^a Measured in CD₃OD; ^b Measured in CDCl₃.

) indicated 4 to be an eremophilane derivative [19]. The significant difference between (4aS, 5S, 8R)-5,6,7,8-tetrahydro-8-hydroxy-3-(1-hydroxypropan-2-yl)-4a,5-dimethylnaph-thalen-2(4aH)-one and compound 4 was that the double bond Δ⁶ was replaced by Δ^7,11 in 4, which was supported by the HMBC correlations (Figure 2) from H₂-6 to C-7, C-8, C-10, and C-11, from H₃-13 to C-7, C-11, and C-12, and from H-9 to C-7. NOESY correlations (Figure 4) of H₃-14/H₃-15, H-1/H₃-14 indicated that H-1, H₃-14, and H₃-15 were on the same side and assigned as β-oriented, while OH-1 was α-oriented. The Z geometry for the double bond Δ^7,11 was evident from the NOESY correlations of H-6 and H₃-13. The absolute configuration was assigned as 1S, 4S, 5R, supported by a comparison of the experimental ECD spectrum with the calculated ECD spectrum of 4 (Figure 5).

Aurantiophilane F (5) was isolated as colorless oil. The molecular formula of C₁₆H₂₀O₃, indicating seven sites of unsaturation, was determined by an ion peak at m/z 283.1313 [M + Na]⁺ (calcd. for C₁₆H₂₀O₃Na, 283.1310) in the HRESIMS spectrum. The ¹H NMR spectroscopic data (Table 2), along with the HSQC spectrum, suggested the presence of three methyl groups (δ_H 0.98, s; 1.02, d, J = 6.8 Hz; 2.01, s) and three olefinic protons (δ_H 5.78, s; 6.16, d, J = 2.6 Hz; 6.25, m). The ¹³C NMR data (Table 2) displayed signals for three methyls (δ_C 14.7, 17.0, and 17.1), two methylenes (δ_C 32.6 and 38.1), four methines including three olefinic ones (δ_C 124.3, 128.0, and 138.8), one quaternary carbon (δ_C 38.5), three olefinic nonprotonated carbons (δ_C 132.0, 137.2, and 163.1), one ester carbonyl (δ_C 172.6), and one ketone (δ_C 187.4). One α, β-conjugated carbonyl, two double bonds, and the ester carbonyl occupied five degrees, indicating the existence of the bicyclic ring skeleton in 5. The ¹H−¹H COSY correlations of H-1/H-2/H₂-3/H-4/H₃-15 and HMBC correlations (Figure 2) from H₃-14 to C-4, C-5, C-6, and C-10, from H₃-15 to C-3, C-4, and C-5, from H₂-6 to C-5, C-7, C-8, and C-11, from H-9 to C-1, C-5, and C-7, from H₃-13 to C-7, C-11, and C-12, and from OMe-12 to C-12 confirmed the planar structure of 5. NOESY correlations (Figure 4) of H-4/H₃-14, H₃-15/H-6β, H-6α/H₃-15, H₃-15/H-3β, and H-3β/H-6β indicated the contrary orientation of H₃-14 and H₃-15. The Z geometry for the double bond Δ^7,11 was evident from the NOESY correlations of H-6/H₃-13. The calculated ECD curve for 4S, 5R-5 was in good accordance with the experimental ECD spectrum, assigning the absolute configuration of 5 as 4S, 5R (Figure 5).

Aurantiophilane G (6) was purified as a white powder with the molecular formula of C₁₅H₂₂O₂([M + Na]⁺ m/z 257.1514 (calcd. for C₁₅H₂₂O₂Na, 257.1517) by HRESIMS. The ¹H NMR data (Table 2) of 6 revealed three methyl groups (δ_H 0.94, s; 1.78, s; 1.90, s), two terminal olefinic protons (δ_H 4.97, d, J = 3.1 Hz; 4.93, d, J = 3.1 Hz), and one olefinic proton (δ_H 5.89, s). The ¹³C NMR and DEPT data (Table 2) displayed signals for three methyls (δ_C 18.1, 19.5, and 22.3), four methylenes including one olefinic (δ_C 28.1, 47.4, 55.4, and 114.0), one olefinic methine (δ_C 127.3), one quaternary carbon (δ_C 39.0), two olefinic nonprotonated carbons (δ_C 145.7 and 161.8), and one ketone (δ_C 198.8). The ¹H−¹H COSY correlations of H-5/H₂-6/H-7/H-8/H₂-9 and HMBC correlations (Figure 2) from H-1 to C-2, C-3, C-9, and C-10, from H₂-9 to C-1, C-5, C-7, C-8, and C-10, from H₃-13 to C-7, C-11 and C-12, from H₃-14 to C-1, C-5, C-9, and C-10, and from H₃-15 to C-3, C-4, C-5, and C-6 constructed an eudesmane-type sesquiterpenoid framework. The NOESY correlation (Figure 4) of H₃-14/H-8 suggested that H₃-14 and H-8 were on the same side and assigned as β-oriented; accordingly, OH-8 was α-oriented. The NOESY correlations of H₃-14/H-9β, H-9α/H-5, H-5/H-7 suggested that H-5 and H-7 were α-oriented. Finally, the absolute configurations of 6 were deduced as 5R, 7R, 8S, 10S by comparing the calculated and experimental ECD spectra (Figure 5).

Six new compounds, aurantiophilanes A–H (1–6) and six known ones, neoalantolactone (7) [20], artefreynic acid D (8) [8], dihydrobipolaroxin D (9) [21], septoreremophilane F (10) [10], rel-[(4S, 5R)-9β, 10β-epoxy-8β-hydroxy-eremophil-12,8-olide] (11) [22], tsoongianus (12) [23], were identified as a result of chemical analysis of the fungus Aspergillus aurantiobrunna. Compounds 1–3 may be derived from 4 via nucleophilic addition and subsequent hydroxylation (Figure 6). According to the methods reported in the previous report [24], the inhibitory activities of compounds 1–12 against LPS-induced B lymphocyte proliferation were estimated. However, none of them exhibited any notable activity up to a concentration of 40 µM. Analyzing the experimental results, it is hypothesized that the phenomenon may be attributed to the presence of an unstable hemiacetal moiety in the molecular structure, coupled with the low bioavailability of the compounds.

3. Materials and Methods

3.1. General Experimental Procedures

A PerkinElmer PE-341 polarimeter (PerkinElmer, Waltham, MA, USA) was used to obtain optical rotations. IR spectra were acquired with a Bruker Vertex 70 FTIR spectrophotometer (Bruker, Karlsruhe, Germany). A PerkinElmer Lambda 35 spectrophotometer (PerkinElmer, Inc., Shelton, CT, USA) was employed to measure UV spectra. A JASCO-810 spectrometer was used to obtain experimental ECD spectra. A Bruker NMR spectrometer (Bruker, Germany) was employed to measure the NMR spectra. The chemical shifts for the CD₃OD (δ_H 3.31/δ_C 49.0) and CDCl₃ (δ_H 7.26/δ_C 77.16) signals are provided in ppm. For chromatographic separations, Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and silica gel (Qingdao Marine Chemical, Inc., Qingdao, China) were used. Precoated plates (200–250 µm thickness, silica gel 60 F254, Qingdao Marine Chemical, Inc.) were utilized for thin-layer chromatography analyses. Semi-preparative HPLC purifications were achieved on an H&E HPLC system utilizing a reversed-phase (RP) column (5 µm, 10 × 250 mm, Welch Ultimate XB-C18). A microTOF II instrument (Bruker, Karlsruhe, Germany) was used to obtain HRESIMS data. Graphite-monochromated Cu Kα radiation was applied in single-crystal X-ray diffraction investigations utilizing a Bruker D8 Quest diffractometer. An X-4B microscopic melting point device (Shanghai Shenguang, Shanghai, China) was used to determine the melting points.

3.2. Fungal Material

The fungal strain employed in this investigation was procured from the BeNa Culture Collection (BNCC, accession no. 465.65). According to ITS sequence analysis, its sequence was 99% similar to that of Aspergillus aurantiobrunneus, which was deposited in the culture center of Tongji Medical College, Huazhong University of Science and Technology.

3.3. Fermentation, Extraction, and Isolation

The Aspergillus aurantiobrunneus seed culture was preserved on potato dextrose agar medium at 28 °C for 7 days. Erlenmeyer flasks (1 L) with 250.0 g of rice and 200.0 mL of distilled water that had previously been autoclaved (120 °C, 30 min) were then infected with a piece of mycelium. All flasks were kept in a foster environment at 28 °C for 45 days. Following cultivation, ethyl acetate/H₂O (1:1) was used to extract the fermented rice, which was first extracted using 95% EtOH. The ethyl acetate-soluble fraction (380.0 g) was gained under reduced pressure. This portion was separated using column chromatography on silica gel [100–200 mesh, petroleum ether/ethyl acetate/methanol system (20:1:0 → 0:1:0 → 0:3:1, v:v:v)] to attain eight fractions (Fr. 1–Fr. 8).

Fr. 7 (87.7 g) was split into eight fractions (Fr. 7.1–Fr. 7.8) using an ODS column (MeOH–H₂O 30:70 → 90:10, v:v). Fr7.1 (11.1 g) was separated into 12 fractions (Fr. 7.1.1–Fr. 7.1.12) by using silica gel CC (petroleum ether/ethyl acetate, 10:1–1:1). Sephadex LH-20 (MeOH) was employed to separate Fr. 7.1.5, yielding two fractions (Fr. 7.1.5.1–Fr. 7.1.5.2). Fr. 7.1.5.1 was purified by semi-preparative HPLC (MeOH/H₂O, 48/52, v/v, 2.5 mL/min) separations to yield compound 11 (5.0 mg, t_R = 61.1 min) Five fractions (Fr. 7.1.8.1–Fr. 7.1.8.5) were extracted by subjecting Fr. 7.1.8 to silica gel CC (petroleum ether/dichloromethane/methanol, 2:1:0–0:0:1). Fr. 7.1.8.5 was further separated using Sephadex LH-20 (MeOH) to obtain five fractions (Fr. 7. 1.8.5.1–Fr. 7.1.8.5.5). Purification of Fr. 7.1.8.5.4 was performed by semi-preparative HPLC (MeOH/H₂O, 29/71, v/v, 3.0 mL/min) separations to collect 10 (12.6 mg, t_R = 32.2 min). Fr. 7.2 was further purified using Sephadex LH-20 (MeOH) to obtain two fractions (Fr. 7.2.1 and Fr. 7.2.4), Fr. 7.2.2 was then submitted using silica gel column chromatography (100–200 mesh, petroleum ether/ethyl acetate, 20:1–0:1) to obtain ten fractions (Fr. 7.2.2.1–Fr. 7.2.2.10). Subsequently, Fr. 7.2.2.6 was purified by semi-preparative HPLC (MeCN/H₂O, 26/74, v/v, 3.0 mL/min) separations to yield 1 (22.5 mg, t_R = 47.8 min) and 4 (24.1 mg, t_R = 57.6 min). Fr7.3 (2.3 g) was split into 12 fractions (Fr. 7.3.1–Fr. 7.3.12) by subjecting it to silica gel CC (petroleum ether/ethyl acetate, 15:1–1:1). Fr. 7.3.2 was further purified by using semi-preparative HPLC (MeCN/H₂O, 28/72, v/v, 3.0 mL/min) to yield 3 (12.5 mg, t_R = 30.0 min). Then, 9 (1.5 mg, t_R = 25.4 min) was purified from Fr. 7.3.5 using semi-preparative HPLC (MeCN/H₂O, 32/68, v/v, 3.0 mL/min). Fr. 7.3.7 was purified to afford 2 (11.8 mg, t_R = 85.7 min) and 6 (8.8 mg, t_R =70.8) using semi-preparative HPLC (MeOH/H₂O, 48/52, v/v, 3.0 mL/min). Fr. 7.6 was separated to afford four fractions (Fr. 7.6.1–Fr. 7.6.4) using silica gel column chromatography (100–200 mesh, petroleum ether/dichloromethane, 5:1–0:1). Then, 5 (2.6 mg, t_R = 36.0 min) was purified from Fr. 7.6.1 using semi-preparative HPLC (MeCN/H₂O, 47/53, v/v, 3.0 mL/min). Fr7.5 (11.2 g) was split into four fractions (Fr. 7.5.1–Fr. 7.5.4) by using silica gel CC (petroleum ether/ethyl acetate, 20:1–1:1). Fr. 7.5.3 was separated to obtain three fractions (Fr. 7.5.3.1–Fr. 7.5.3.3) by using silica gel column chromatography (100–200 mesh, petroleum ether/dichloromethane, 5:1–0:1). Then, 8 (12.4 mg, t_R = 37.5 min) was purified from Fr. 7.5.3.3 using semi-preparative HPLC (MeCN/H₂O, 55/45, v/v, 3.0 mL/min). Fr. 7.8 (4.5 g) was separated using silica gel column chromatography (100–200 mesh, petroleum ether/dichloromethane, 10:1–0:1) to obtain three fractions (Fr. 7.8.1–Fr. 7.8.2). Compounds 7 (4.6 mg, t_R = 81.5 min) and 12 (4.1 mg, t_R = 42.8 min) were further purified from Fr. 7.8.1 using semi-preparative HPLC (MeCN/H₂O, 49/51, v/v, 3.5 mL/min).

3.3.1. Aurantiophilane A

Colorless crystal; m.p. 185.1–187.0 °C; [α${]}_{\mathrm{D}}^{20}$ −158 (c 0.1, MeOH); IR v_max = 3396, 2924, and 1767 cm⁻¹; UV (CH₃CN) λ_max (log ε) = 223 (4.13) nm; ECD (CH₃CN) λ_max (Δε) 218 (+11.56), and 242 (−11.64) nm. For ¹H (400 MHz) and ¹³C NMR (100 MHz) data in CD₃OD, see Table 1; (+)-HRESIMS [M + Na]⁺ m/z 285.1107 (calcd. for C₁₅H₁₈O₄Na, 285.1103).

3.3.2. Aurantiophilane B

Yellow crystal; m.p. 193.5–195.0 °C; [α${]}_{\mathrm{D}}^{20}$ −447 (c 0.1, MeOH); IR v_max = 3420, 1775, and 1655 cm⁻¹; UV (CH₃CN) λ_max (log ε) = 213 (4.00), 263 (3.85), and 336 (4.20) nm; ECD (CH₃CN) λ_max (Δε) 219 (+2.54), 245 (−2.97), 321 (+1.71), and 382 (−5.95) nm. For ¹H (400 MHz) and ¹³C NMR (100 MHz) data in CDCl₃, see Table 1; (+)-HRESIMS [M + Na]⁺ m/z 265.0835 (calcd. for C₁₅H₁₄O₃Na, 265.0841).

3.3.3. Aurantiophilane C

Colorless oil; [α${]}_{\mathrm{D}}^{20}$ −135 (c 0.1, MeOH); IR v_max = 3433, 2922, and 1715 cm⁻¹; UV (CH₃CN) λ_max (log ε) = 191 (4.05) nm; ECD (CH₃CN) λ_max (Δε) 216 (−2.1), 254 (+0.3), and 357 (−1.7) nm. For ¹H (400 MHz) and ¹³C NMR (100 MHz) data in CDCl₃, see Table 1; (+)-HRESIMS [M + Na]⁺ m/z 301.1416 (calcd. for C₁₆H₂₂O₄Na, 301.1416).

3.3.4. Aurantiophilane D

Yellow oil; [α${]}_{\mathrm{D}}^{20}$ +279 (c 0.1, MeOH); IR v_max = 3422, 2923 and 1658 cm⁻¹; UV (CH₃CN) λ_max (log ε) = 191 (4.06) and 245 (3.94) nm; ECD (CH₃CN) λ_max (Δε) 199 (+8.8), 246 (+8.3), and 280 (−3.1) nm. For ¹H (400 MHz) and ¹³C NMR (100 MHz) data in CD₃OD, see Table 2; (+)-HRESIMS [M + Na]⁺ m/z 273.1467 (calcd. for C₁₅H₂₂O₃Na, 273.1467).

3.3.5. Aurantiophilane E

Colorless oil; [α${]}_{\mathrm{D}}^{20}$ +360 (c 0.1, MeOH); IR v_max = 3431, 2921,1731,1665 and 1617 cm⁻¹; UV (CH₃CN) λ_max (log ε) = 197 (3.92) and 302 (4.13) nm; ECD (CH₃CN) λ_max (Δε) 223 (+7.9), 265 (+14.3), and 311 (−3.1) nm. For ¹H (400 MHz) and ¹³C NMR (100 MHz) data in CDCl₃, see Table 2; (+)-HRESIMS [M + Na]⁺ m/z 283.1313 (calcd. for C₁₆H₂₀O₃ Na, 283.1310).

3.3.6. Aurantiophilane F

White powder; [α${]}_{\mathrm{D}}^{20}$ +291 (c 0.1, MeOH); IR v_max = 3419, 2920, and 1660 cm⁻¹; UV (CH₃CN) λ_max (log ε) = 190 (4.02) and 236 (4.05) nm; ECD (CH₃CN) λ_max (Δε) 202 (−1.2) and 228 (+4.7) nm. For ¹H (400 MHz) and ¹³C NMR (100 MHz) data in CDCl₃, see Table 2; (+)-HRESIMS [M + Na]⁺ m/z 257.1514 (calcd. for C₁₅H₂₂O₂Na, 257.1517).

3.4. X-Ray Crystallographic Analysis

Yellow crystals of 1 and 2 were obtained from MeOH at room temperature. Crystallographic data have been deposited at the Cambridge Crystallographic Data Center with the CCDC numbers 2484815 and 2484816. The data can be obtained free of charge from the CCDC, 12 Union Road, Cambridge CB1EZ, U.K. [fax: Int. +44-1223-336-033; deposit@ccdc.cam.ac.uk]

Crystal Data of Aurantiophilane (1): C₁₅H₁₈O₄, M = 262.29, a = 9.0657 (16) Å, b = 10.0172 (18) Å, c = 14.548 (3) Å, α = 90°, β = 90°, γ =90°, V = 1321.1 (4) Å, T = 100 (2) K, space group P2₁2₁2₁, Z = 4, μ (Cu Kα) = 0.517 mm⁻¹, 45435 reflections measured, 2698 independent reflections (R_int = 0.0608). The final R₁ values were 0.0290 (I > 2σ (I)). The final wR (F²) values were 0.0734 (I > 2σ (I)). The final R₁ values were 0.0300 (all data). The final wR (F²) values were 0.0740 (all data). The goodness of fit on F² was 1.061. Flack parameter = −0.21 (8).

Crystal Data of Aurantiophilane (2): C₁₅H₁₄O₃, M = 242.26, a= 13.1718 (14) Å, b = 13.6360 (14) Å, c = 13.6360 (14) Å, α = 90°, β = 90°, γ =90°, V = 2449.2 (4) Å, T = 100 (2) K, space group P2₁2₁2₁, Z = 8, μ (Cu Kα) = 0.491 mm⁻¹, 69507 reflections measured, 6129 independent reflections (R_int = 0.0989). The final R₁ values were 0.0754 (I > 2σ (I)). The final wR (F²) values were 0.1780 (I > 2σ (I)). The final R₁ values were 0.1230 (all data). The final wR (F²) values were 0.2113 (all data). The goodness of fit on F² was 0.958. Flack parameter = 0.02 (14).

3.5. Biological Activity

The experimental procedures and methods of immunosuppression used have been described in our previous reports [25].

4. Conclusions

In summary, six undescribed compound aurantiophilanes A–H (1–6) and six known ones (7–12) were first isolated from the fungus A. aurantiobrunneus. These findings indicated the structural diversity of sesquiterpenoids in A. aurantiobrunneus, which covered eremophilane-type sesquiterpenoids, eudesmane-type sesquiterpenoids, and sesquiterpenoid lactones. Despite the bioactivity assessments, none of them displayed any positive results up to the concentration of 40 µM; these novel compounds have significantly enriched the eremophilane-type sesquiterpenoid family with their diverse chemical structures.