Isolation of Ten New Sesquiterpenes and New Abietane-Type Diterpenoid with Immunosuppressive Activity from Marine Fungus Eutypella sp.

Abstract

Background: Ten new sesquiterpenes, including eight eremophilane-type sesquiterpenes (1–8) and two compounds (9–10) with a cyclopentane ring, representing an undescribed subtype of sesquiterpene, along with a new abietane-type diterpenoid (11), were isolated and identified from a deep-sea-derived fungus: Eutypella sp. Methods: Their structures were elucidated on the basis of various spectroscopic analyses, mainly including nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HRESIMS) data, ¹³C NMR calculations with DP4+ probability analyses, electronic circular dichroism (ECD) calculations, and single-crystal X-ray diffraction experiments. Results: Furthermore, compound 11 exhibited potent immunosuppressive activity with IC₅₀ values of 8.99 ± 1.08 μM in a lipopolysaccharide (LPS) model and 5.39 ± 0.20 μM in a concanavalin A (ConA) model.

1. Introduction

The dynamic environmental conditions of the ocean, characterized by significant fluctuations in pressure, salinity, temperature, pH, nutrient availability, and light intensity, exert selective pressures that drive marine microorganisms to evolve unique metabolic pathways for enhanced adaptation. This evolutionary process endows marine-derived secondary metabolites with remarkable bioactivities such as antitumor, antibacterial, and antiviral properties, while their distinct chemical scaffolds provide invaluable molecular resources for innovative drug discovery [1]. Marine fungus-derived bioactive compounds are potently applied for treatments of diseases [2,3], such as the antibiotics cephalosporin C, griseofulvin, and fusidic acid, used for the treatment of various pathogenic infections [4]. Research on fungi of the genus Eutypella over the past two decades has yielded diverse compounds, including terpenoids, cytochalasins, steroids, lactones, and diketopiperazines, which demonstrate notable cytotoxic, antibacterial, and anti-inflammatory activities [5,6,7,8,9,10]. The researchers isolated γ-lactones, sesquiterpenoids, and diterpenoids with moderate cytotoxic activity from the fermentation broth of Eutypella sp. BCC-13199, an endophyte of Zingiber species native to Thailand [6]. Epigenetic manipulation activated the sesquiterpene pathway in the fungus Eutypella sp., and twenty-six new eremophilane-type sesquiterpenoids were isolated, and two of their compounds exerted significant inhibition on NO production [11]. Jiao Binghua et al. isolated seven pimarane-type diterpenoid derivatives from Eutypella sp. D-1, a fungal strain obtained from high-latitude Arctic soils [8]. These compounds exhibited cytotoxic activity against human cancer cell lines and inhibited lipopolysaccharide (LPS)-induced NO production in lymphocytes. Terpenoids, with their unique chemical structures and broad spectrum of biological activities, demonstrate significant potential in the field of immunosuppression. Representative terpenoid immunosuppressive agents include triptolide [12], oridonin [13], and pseudopterosins [14]. With the aim of discovering bioactive natural products from deep sea organisms, we examined the metabolites of an unidentified fungus species, Eutypella sp. MCCC 3A00281, that was obtained from the deep sea sediment at a depth of 5610 m in the South Atlantic Ocean. Chromatographic separation of the EtOAc extract from the fermented fungus yielded a series of terpenes, including ten new sesquiterpenes (1–10) and one novel abietane-type diterpene (11) (Figure 1). Herein, we report their isolation and structural elucidation, and an evaluation of immunosuppressive activities.

2. Results and Discussion

2.1. Structural Elucidation

Compound 1 was isolated as a colorless oil. The molecular formula was established as C₁₅H₂₄O₂ on the basis of an HREIMS peak at m/z 259.1663 [M + Na⁺] (calcd for C₁₅H₂₄O₂Na⁺, 259.1674), consistent with 4 degrees of unsaturation. The ¹H NMR data (

Table 1. ¹H NMR data (δ in ppm, J in Hz) for compounds 1–5.

No.	1 a,d	2 a,c	3 a,c	4 b,d	5 a,c
1	5.38,1H, d (4.9)	1.99, 1H, ddt (13.1, 11.2, 2.0), ax 2.19, 1H, ddd (13.1, 4.9, 2.2), eq	1.04, 1H, dd (12.8, 11.1), ax 1.40, 1H, m, eq	5.36, 1H, m	3.53, 1H, dt (9.4, 4.6), ax
2	3.84, 1H, d (5.2), eq	3.34, 1H, d (4.9), ax	3.39, 1H, dp (10.1, 5.1), ax	1.96, 2H, m, ax	1.33 *, 1H, eq 1.63 *, 1H, ax
3	1.35, 1H, d (13.6), eq 1.49, 1H, td (13.4, 4.4), ax	1.25, 1H, td (12.3, 10.6), ax 1.60, 1H, ddt (12.2, 5.0, 2.4), eq	1.52, 1H, ddd (9.5, 5.0, 1.9), eq 1.13, 1H, m, ax	1.40, 2H, dp (9.0, 4.2)	1.30 *, 1H, ax 1.43, 1H, m, eq
4	1.64, 1H, m, ax	1.30, 1H, m, ax	1.09 *, 1H, ax	1.33, 1H, m, ax	1.69 *, 1H
5
6	1.06, 1H, t (13.1), ax 1.61, 1H, dd (13.1, 3.5), eq	1.45 *, 1H, ax 1.47 *, 1H, eq	1.44 *, 1H, eq 0.91, 1H, t (12.9), ax	1.83, 1H, dd (13.0, 3.2) 0.78, 1H, t (13.0)	2.37, 1H, d (14.8) 2.44, 1H, d (14.8)
7	2.18 *, 1H, ax	2.10, 1H, dt (9.2, 4.3), ax	2.05, 1H, ddd (13.5, 10.3, 3.7), ax	1.90, 1H, m, ax
8	3.26, 1H, m	3.92, 1H, m	3.29, 1H, dt (10.5, 5.2)	3.73 *, 1H
9	2.21 *, 2H	5.45, 1H, dd (5.5, 1.8)	1.45 *, 1H, eq 1.24, 1H, td (12.3, 10.6), ax	2.31, 2H, m	2.31, 1H, d (16.4) 2.66, 1H, d (16.4)
10			1.10 *, 1H, ax
11
12	4.72, 2H, brs	4.66, 1H, brs 4.76, 1H, brs	4.67, 2H, brs	3.75 *, 2H	1.81, 3H, brs
13	1.65, 3H, s	1.75, 3H, s	1.63, 3H, s	1.19, 3H, s	1.75, 3H, brs
14	0.84, 3H, s	0.85, 3H, s	0.68, 3H, s	0.95, 3H, s	0.83, 3H, s
15	0.80, 3H, d (6.6)	0.86, 3H, brs	0.72, 3H, d (5.9)	0.88, 3H, d (6.4)	0.77, 3H, d (6.6)
1-OH					4.35, 3H, d (4.3)
2-OH	4.43, 1H, d (5.3)	4.65, 1H, brs	4.43, 1H, d (4.7)
8-OH	4.45, 1H, d (5.6)	4.06, 1H, d (6.6)	4.19, 1H, d (5.6)
10-OH					4.45, 3H, s

* overlapped; ^a DMSO-d₆; ^b CDCl₃; ^c 600 MHz; ^d 400 MHz.

) and heteronuclear single quantum coherence (HSQC) spectrum of compound 1 (Figure S4) reveal the following signals: three methyls (δ_H 1.65, 0.84, and 0.80), two terminal olefinic protons (δ_H 4.72), three methylenes (δ_H 1.35/1.49, 1.06/1.61, 2.21), two oxygenated methines (δ_H 3.84, 3.26), two methines (δ_H 1.64, 2.18), one olefinic methine (δ_H 5.38), and two exchangeable proton signals (δ_H 4.43, 4.45). The ¹³C NMR data (

Table 2. ¹³C NMR data (δ in ppm) for compounds 1–5.

No.	1 a,c	2 a,c	3 a,c	4 b,d	5 a,c
1	124.2, CH	41.7, CH2	40.0, CH2	122.6, CH	71.3, CH
2	62.3, CH	68.9, CH	68.7, CH	30.0, CH2	30.9, CH2
3	36.2, CH2	39.9, CH2	40.1, CH2	27.2, CH2	28.4, CH2
4	33.9, CH	40.4, CH	41.8, CH	40.7, CH	32.4, CH
5	37.2, C	37.5, C	35.4, C	37.5, C	42.2, C
6	42.7, CH2	34.7, CH2	42.9, CH2	39.2, CH2	35.9, CH2
7	48.5, CH	42.1, CH	48.6, CH	47.7, CH	131.1, C
8	71.2, CH	63.9, CH	70.9, CH	73.9, CH	203.4, C
9	41.6, CH2	124.4, CH	37.8, CH2	42.6, CH2	45.0, CH2
10	143.9, C	144.2, C	40.6, CH	140.2, C	78.3, C
11	147.4, C	147.1, C	148.0, C	76.4, C	137.7, C
12	111.2, CH2	110.0, CH2	110.9, CH2	70.0, CH2	22.2, CH3
13	19.5, CH3	22.2, CH3	19.6, CH3	24.4, CH3	21.2, CH3
14	16.5, CH3	16.4, CH3	11.2, CH3	18.3, CH3	14.2, CH3
15	15.5, CH3	15.4, CH3	15.3, CH3	16.1, CH3	16.0, CH3

^a DMSO-d₆; ^b CDCl₃; ^c 150 MHz; ^d 100 MHz.

) and DEPT-135 spectrum (Figure S3) confirm the presence of 15 carbons, including the following: three methyls (δ_C 15.5, 16.5, 19.5), three methylenes (δ_C 36.2, 41.6, 42.7), one olefinic methylene (δ_C 111.2), two oxygenated methines (δ_C 62.3, 71.2), one olefinic methine (δ_C 124.2), two methines (δ_C 33.9, 48.5), one quaternary carbon (δ_C 37.2), and two olefinic quaternary carbons (δ_C 143.9, 147.4). The ¹³C NMR data of compound 1 closely align with those of the known eremophilane-type sesquiterpene 7βH-eremophil-1(10),11(12)-dien-2β,8β-diol [15]. However, distinct differences are observed in the chemical shifts and coupling constants of H-1, H-2, and H₂-3 and their corresponding carbons (C-1, C-2, C-3), suggesting potential structural modifications in these regions. The planar structure of Compound 1 was determined to be identical to that of the known compound based on heteronuclear multiple bond correlation (HMBC) and ¹H-¹H correlation spectroscopy (¹H-¹H COSY) correlations (Figure 2).

The relative configurations were established on the basis of nuclear Overhauser effect spectroscopy (NOESY) cross peaks. As a general tendency, biosynthesis results in eremophilane structures with CH₃-14 in β-orientation; consequently, this position was assigned accordingly. The key NOESY cross-peaks (Figure 3) between H-3ax (δ_H 1.49)/CH₃-15 (δ_H 0.80)/CH₃-14 (δ_H 0.84), CH₃-14/H-6eq (δ_H 1.61)/H-7 (δ_H 2.18), OH-2 (δ_H 4.43)/H-4 (δ_H 1.64), H-4/H-6ax (δ_H 1.06), and H-6ax/H-8 (δ_H 3.26) collectively support the β-orientations of CH₃-14, CH₃-15, and H-7, while H-2, H-4, and H-8 adopt α-orientations. Therefore, the relative configuration of compound 1 was deduced as 2S*,4S*,5R*,7S*,8R*. Finally, the absolute configuration of compound 1 was assigned as 2S,4S,5R,7S,8R by comparison of its experimental and computed ECD curves (Figure 4).

Compound 2 was isolated as a colorless oil. The molecular formula was established as C₁₅H₂₄O₂ on the basis of a HREIMS peak at m/z 259.1668 [M + Na⁺] (calcd for C₁₅H₂₄O₂Na⁺, 259.1674). The HMBC and ¹H-¹H COSY correlations revealed that the double bond position in compound 2 was altered compared to compound 1, shifting from C-1–C-10 to C-9–C-10 (Figure 2). The key NOESY cross-peaks of CH₃-15 (δ_H 0.80)/CH₃-14 (δ_H 0.84) and H-3ax (δ_H 1.49), CH₃-14 and H-7 (δ_H 2.18), and OH-2 (δ_H 4.43)/H-4 (δ_H 1.64)/H-6ax (δ_H 1.06)/H-8 (δ_H 3.26) suggested that H-7, CH₃-14, and CH₃-15 were on the same side with β-orientations, while H-2, H-4, and H-8 was on the opposite side (α-orientation). The absolute configuration of compound 2 (2R,4S,5R,7S,8S) was determined by comparative analysis of ECD calculations and experimental data (Figure 4).

Compound 3 was determined to have the molecular formula C₁₅H₂₆O₂, as deduced from the HRESIMS data (m/z 261.1826 [M + Na⁺], calcd for C₁₅H₂₆O₂Na⁺, 261.1830). The ¹H NMR data (Table 1) displayed three methyls (δ_H 1.63, 0.72, and 0.68), five methylenes (δ_H 4.67, 1.52/1.13, 1.44/0.91, 1.45/1.24, and 1.40/1.04), five methines (δ_H 3.39, 3.29, 2.05, 1.10, and 1.09), and two hydroxyls (δ_H 4.43 and 4.19). The ¹³C NMR spectrum (Table 2) showed 15 carbon signals attributed to three methyls (δ_C 19.6, 15.3, and 11.2), five methylenes (δ_C 110.9, 42.9, 40.1, 40.0, and 37.8), five methines (δ_C 70.9, 68.7, 48.6, 41.8, and 40.6), and two non-protonated carbons (δ_C 148.0 and 35.4). The ¹D-NMR data of 3 combined with the sequential ¹H-¹H COSY correlations of Me-15/H-4/CH₂-3/H-2/CH₂-1/H-10/CH₂-9/H-8/H-7/CH₂-6, as well as the key HMBC from Me-15 to C-3/C-4/C-5, Me-14 to C-4/C-5/C-6/C-10, C-6 to C-4, and Me-13 to C-12/C-7/C-11 suggested an eremophilane sesquiterpene with a 6/6-fused ring system skeleton.

Key NOESY correlations (Figure 3) revealed spatial proximities: CH₃-14 (δ_H 0.68) to H-7 (δ_H 2.05) and H-3ax (δ_H 1.13), and Me-15 (δ_H 0.72) to H-3ax, supporting β-orientations for CH₃-14, CH₃-15, and H-7; H-2 (δ_H 3.39)/H-4 (δ_H 1.09)/H-10 (δ_H 1.10)/H-8 (δ_H 3.29)/H-6ax (δ_H 0.91), indicating α-orientations for these protons. The absolute configuration of compound 3 (2R,4S,5R,7S,8S,10S) was determined by comparative analysis of ECD calculations and experimental data.

Compound 4 gave a molecular formula of C₁₅H₂₆O₃, as evidenced via its HRESIMS data. The ¹H and ¹³C NMR signals (Table 1 and Table 2), along with the ¹H−¹H COSY and HMBC spectrum, showed that the planar structure of compound 4 is similar to valenc-1(10)-ene-8,11-diol [16]. Unlike known compounds, the hydroxymethyl group was assigned to C-12 based on the HMBC long-range correlations between the proton signal at δ_H 3.75 (H-12) and C-7, C-11, and C-13. The NOESY correlations (Figure 3) from CH₃-14 (δ_H 0.95) to H-6β (δ_H 1.83)/H-7 (δ_H 1.90), and cross-peaks of H-4 (δ_H 1.33)/H-6ax (δ_H 0.78)/CH₃-12 (δ_H 3.75) determined that CH₃-14, CH₃-15, and H-7 were on the same side with β-orientations. Considering the biosynthetic pathway and single-crystal X-ray diffraction experiment, the absolute configuration of compound 4 was confirmed as 4S,5R,7R,8R,11S by the single-crystal X-ray diffraction analysis using Cu Kα radiation (Figure 5) [Flack parameter = 0.03(12), CCDC 2431789].

Compound 5 was isolated as a colorless oil and provided a molecular formula of C₁₅H₂₄O₃, according to its HRESIMS data (m/z 275.1631 [M + Na⁺]), which suggested four degrees of unsaturation. The ¹H NMR data (Table 1) displayed four methyls (δ_H 1.81, 1.75, 0.83, and 0.77), four methylenes (δ_H 2.66/2.31, 2.44/2.37, 1.63/1.33, and 1.43/1.30), two methines (δ_H 3.53 and 1.69), and two hydroxyls (δ_H 4.45 and 4.35). The ¹³C NMR spectrum (Table 2) of compound 5 showed 15 carbon signals attributed to four methyls (δ_C 22.2, 21.2, 16.0, and 14.2), four methylenes (δ_C 45.0, 35.9, 30.9, and 28.4), two methines (δ_C 71.3 and 32.4), and five non-protonated carbons (δ_C 203.4, 137.7, 131.1, 78.3, and 42.2). The planar structure of compound 5 was inferred to be identical to eutyperemophilane R by ¹H-¹H COSY and HMBC data (Figure 2) [11]. The difference between compound 5 and eutyperemophilane R was that C-10 was hydroxylated in compound 5.

The NOE correlations of Me-14 (δ_H 0.83) with OH-10 (δ_H 4.45)/ H-1 (δ_H 3.53), H-3ax (δ_H 1.30) with Me-14 (δ_H 0.83)/ Me-15 (δ_H 0.77), and H-4 (δ_H 1.69) with H-2ax (δ_H 1.32) revealed relative configuration. The absolute configuration of compound 5 (1S,4S,5R,10S) was determined by comparative analysis of ECD calculations and experimental data (Figure 4).

Compound 6 gave a molecular formula of C₁₅H₂₄O₃, as evidenced via its HRESIMS data. Although compound 6 shares the same molecular formula as compound 5, its planar structure was deduced to have a 1,3-dihydroxy substitution based on ¹H-¹H COSY and HMBCs (Figure 2). Analysis of the NOESY correlations (Figure 3), the spatial proximities between CH₃-14 and H-1, H-3, and CH₃-15 indicate β-configurations for H-1, H-3, and CH₃-15. The correlation between H-10 and H-4 suggests an α-configuration for H-10. The relative configuration of compound 6 was determined as 1S*,3R*,4R*,5R*,10S*, while absolute configuration was confirmed by comparative analysis of ECD calculations and experimental data.

Compound 7 was isolated as a colorless needle crystal. The molecular formula was established as C₁₈H₂₇NO₄ on the basis of a HREIMS peak at m/z 344.1835 [M + Na⁺] (calcd for C₁₈H₂₇NO₄Na⁺, 344.1838). The ¹H and ¹³C NMR data (

Table 3. ¹H NMR data (δ in ppm, J in Hz) for compounds 6–10.

No.	6 b,c	7 b,d	8 b,d	9 b,c	10 a,c
1	3.49 *, 1H, ax			3.55, 1H, m 3.68, 1H, td (8.4, 7.3, 4.3)	3.25, 1H, m 3.46, 1H, ddd (11.3, 5.2, 2.7)
2	1.39, 1H, q (11.5), eq 2.39, 3H, dt (11.8, 4.6), ax	2.38 *, 1H 2.42 *, 1H	2.37 *, 1H 2.41 *, 1H	3.81, 1H, ddd (10.0, 6.8, 2.9), ax	3.41, 1H, m, ax
3	3.49 *, 3H, ax	1.66, 1H, td (13.2, 6.0), ax 1.92 *, 1H, eq	1.63 *, 1H, ax 1.92, 1H, m, eq
4	1.25, 1H, dd (10.3, 6.7), ax	2.04, 1H, ddd (12.6, 6.6, 3.9), ax	2.02, 1H, ddd (12.6, 6.8, 4.0), eq	5.28, 1H, s	5.25 *, 1H
5				1.92, 1H, t (12.9) 2.04, 1H, m	1.86 *, 1H, ax 2.12, 1H, m, eq
6	1.96, 1H, m, ax 2.74, 1H, d (15.0), eq	1.94 *, 1H, ax 2.62, 1H, d (13.0), eq	2.17, 1H, d (13.1), ax 2.54, 1H, d (13.1), eq	1.71, 1H, m, ax	2.27, 1H, td (10.0, 8.3), ax
7
8				1.32, 1H, t (11.9) 1.54 *	3.31, 1H, m
9	2.19, 1H, dd (17.3, 12.7) 2.80, 1H, dd (17.3, 5.5	1.48, 1H, dd (14.0, 12.5), ax 2.27, 1H, dd (14.0, 3.4, eq	1.35, 1H, m, ax 2.26, 1H, dd (14.0, 3.4), eq	4.54, 1H, ddd (11.3, 8.1, 2.9), ax	1.84 *, 1H 1.92, 1H, m
10	1.65, 1H, ddd (12.6, 10.2, 5.5), ax	2.75, 1H, dd (12.5, 3.4), ax	2.73, 1H, dd (12.5, 3.3), ax	5.19, 1H, d (8.0)	5.25 *, 1H
11
12	2.03, 3H, d (2.2)			1.73, 3H, s	1.65, 3H, s
13	1.79, 3H, d (1.4)	1.84, 3H, d (1.6)	1.77, 3H, d (1.5)	1.69, 3H, s	1.53, 3H, s
14	0.75, 3H, s	0.51, 3H, s	0.47, 3H, s	0.95, 3H, s	0.96, 3H, s
15	1.08, 3H, d (6.7)	1.02, 3H, d (6.7)	1.00, 3H, d (6.7)	1.65, 3H, s	1.51, 3H, s
16		2.89, 3H, s	3.72, 1H, dddd (14.2, 8.9, 5.2, 1.3) 3.45, 1H, ddd (13.9, 8.8, 7.2)
17		3.40, 1H, ddd (14.8, 6.0, 4.0) 3.51, 1H, m	2.91, 1H, ddd (13.7, 8.8, 5.3) 3.11, 1H, ddd (13.2, 8.9, 7.1)
18		3.79, 2H, brs
19, 23			7.29 *, 2H
20, 22			7.22 *, 2H
21			7.21 *, 1H

* overlapped; ^a DMSO-d₆; ^b CDCl₃; ^c 600 MHz; ^d 400 MHz.

and

Table 4. ¹³C NMR data (δ in ppm) for compounds 6–10.

No.	6 b,c	7 b,d	8 b,c	9 b,c	10 a,c
1	69.5, CH	210.6, C	210.8, C	65.4, CH2	65.0, CH2
2	45.2, CH2	41.3, CH2	41.4, CH2	75.8, CH	71.1, CH
3	70.5, CH	31.3, CH2	31.3, CH2	150.9, C	145.7, C
4	49.6, CH	42.3, CH	42.2, CH	122.5, CH	123.1, CH
5	37.1, C	44.9, C	44.9, C	29.4, CH2	32.2, CH2
6	43.5, CH2	36.9, CH2	36.1, CH2	50.7, CH	44.6, CH
7	129.8, C	148.2, C	150.3, C	45.6, C	55.4, C
8	202.9, C	91.6, C	87.6, C	41.7, CH2	74.7, CH
9	40.9, CH2	32.8, CH2	32.5, CH2	70.3, CH	31.8, CH2
10	50.0, CH	53.6, CH	53.7, CH	126.6, CH	123.4, CH
11	145.6, C	130.6, C	128.1, C	135.3, C	130.8, C
12	23.5, CH3	172.6, C	170.6, C	25.9, CH3	25.7, CH3
13	22.6, CH3	8.3, CH3	8.2, CH3	18.5, CH3	17.8, CH3
14	12.8, CH3	11.7, CH3	11.4, CH3	15.5, CH3	16.9, CH3
15	10.9, CH3	14.8, CH3	14.8, CH3	12.3, CH3	13.0, CH3
16		49.8, OCH3	40.8, OCH2
17		42.7, CH2	35.2, CH2
18		63.0, CH2	139.7, C
19, 23			128.7, CH
20, 22			129.1, CH
21			126.6, CH

^a DMSO-d₆; ^b CDCl₃; ^c 150 MHz; ^d 100 MHz.

) of compound 7 were very similar to those of the known compound eremophilane lactam, which indicated that both compounds shared the same core skeleton [11]. The difference between compound 7 and eremophilane lactam was the substitution of the N-H hydrogen with a hydroxyethyl group, as supported by the key HMBCs from H₂-17 to C-8 and C-12. Considering the NOE correlations and single-crystal X-ray diffraction experiment (Figure 5), the absolute configuration of compound 7 was confirmed as 4S,5R, 8S,10S by the single-crystal X-ray diffraction analysis using Cu Kα radiation [Flack parameter = 0.01(6), CCDC 2431799].

Compound 8 possessed a molecular formula of C₂₃H₂₉NO₃, as determined by the HRESIMS and NMR data. Comprehensive analysis of the NMR data of compound 7 and compound 8 suggested that both compounds were structural analogs; the difference lies in the substitution of a methyl group in compound 7 with a phenethyl group in compound 8, accompanied by N-reduction. This structural modification was corroborated by HMBCs between H₂-16 and C-18, as well as between H₂-17 and C-18, C-19, and C-20 (Figure 2).

By analysis of the NOESY data, Me-14 (δ_H 0.47) showed strong NOESY correlations to Me-15 (δ_H 1.00) and H-9ax (δ_H 1.35), indicating that Me-15, Me-14, and H-9ax were on the same face with β-orientations, while cross-peaks of H-4 (δ_H 2.02)/H-10 (δ_H 2.73) suggested that H-10 was on the opposite side (α-orientation). The correlation of H-16 (δ_H 3.45)/H-9eq (δ_H 2.26) suggested that hyphenated oxygen groups were α-oriented. The absolute configuration was determined by a comparative analysis of ECD calculations and experimental data.

Compound 9 was isolated as a colorless oil. The molecular formula was established as C₁₅H₂₄O₂ on the basis of a HREIMS peak at m/z 259.1668 [M + Na⁺] (calcd for C₁₅H₂₄O₂Na⁺, 259.1674), bearing four degrees of unsaturation. The ¹³C NMR spectrum exhibited 15 carbon resonances, including four olefinic carbons for two double bonds, a sp³ quaternary carbon, three methylenes, three methines, and four methyl groups (Table 3 and Table 4). The COSY correlations of H-5 (δ_H 1.92, 2.04) with H-4 (δ_H 5.28) and H-6 (δ_H 1.71), together with the HMBCs from CH₃-15 (δ_H 1.65) to C-3 (δ_C 150.9), C-4 (δ_C 122.5), and C-7 (δ_C 45.6), and from CH₃-14 (δ_H 0.95) to C-3, C-6 (δ_C 50.7), and C-7, established a dimethylcyclopentene unit. The COSY correlations from H-6 to H-2 (δ_H 3.81) and CH₂-1 (δ_H 3.68), and from H-9 (δ_H 4.54) to CH₂-8 (δ_H 1.32, 1.54) and H-10 (δ_H 5.19), in association with the HMBC from CH₂-1 to C-2 (δ_C 75.8) and C-6, and from H-9 to C-7 and C-2, fused a pyran ring to the cyclopentene nucleus. In addition, a 2-methylpropenyl side chain was deduced based on the HMBCs of CH₃-12 (δ_H 1.73)/CH₃-13 (δ_H 1.69) with C-10 (δ_C 126.6) and C-11 (δ_C 135.3). This side chain was connected to C-9 via the HMBCs between CH₂-8 and C-10 (Figure 2).

The NOE correlations from CH₃-14 and H-2/H-9, as well as between H-1 and H-6, indicate that H-2, CH₃-14, and H-9 are positioned on the opposite face relative to H-6 (Figure 3). The experimental ECD data of compound 9 were similar to the calculated data for (2R,6R,7S,9S)-9 (Figure 6), determining the absolute configuration.

Comparative analysis of 1D NMR data combined with COSY and HMBCs revealed that compound 10 shares identical partial structures (cyclopentene unit and side chain) with compound 9. The key structural divergence lies in the formation of a pyran ring in compound 9 via dehydration–condensation between OH-9 and OH-2, whereas in compound 10, C-8 is oxidized to a hydroxyl group without oxygen ring formation.

In the NOESY spectrum, H-6 showed correlations with H-9, and CH₃-14 exhibited mutual correlations with H-2 and H-5ax. A weak correlation between H-6 and H-5ax, contrasted with a strong correlation between H-6 and H-5eq, indicated that CH₃-14, H-5ax, and H-2 are positioned on the opposite face of the ring relative to H-6. The relative configuration was deduced as 2R*,6R*,7S*. Based on Snatzke’s method [17], the treatment of 10 with Mo₂(OAc)₄ derived an Mo₂(OAc)₄ complex, whose ECD data showed a positive cotton effect at 311 nm and 400 nm in DMSO (Figure 7), reflecting the 2R configuration. To identify the final structure for compound 10, the theoretical ¹³C NMR calculation with DP4+ probability analyses were employed. The ¹³C NMR chemical shifts in two possible isomers, 10A (2R,6R,7S,8R*) and 10B (2R,6R,7S,8S*), were calculated at the B3LYP-D3(BJ)/6-31G* level, and the calculated results for 10B (R² = 0.9973) showed a better match to the experimental data than 10A (R² = 0.995). Furthermore, according to the DP4+ probability analyses, 10B was assigned with a 100% probability (Figure 8). The above messages demonstrated 10B to be the right relative structure for compound 10. The absolute configuration of compound 10 was finally determined as 2R,6R,7S,8S by comparison of the experimental and computed ECD curves (Figure 6).

The molecular formula of compound 11 was established as C₂₀H₂₈O₃ by the HRESIMS data. The ¹H and ¹³C NMR data of compound 11 were comparable to the known compound hydroxyldecandrin G [18], but exhibited an additional proton signal δ_H 2.84 and distinct chemical shift differences in the side chain (C-11–C-17): C-11 (Δδ_C +0.6), C-12 (Δδ_C +1.7), C-13 (Δδ_C +1.7), C-14 (Δδ_C +1.1), C-15 (Δδ_C −37.8), and C-16/17 (Δδ_C −7.5) (

Table 5. ¹H and ¹³C NMR data (δ in ppm, J in Hz) for compound 11 (CD₃OD).

No.	11		Hydroxyldecandrin G
	δHa	δCb	δH	δC
1	1.48, 1H, t (12.1)	45.6, CH2	1.48 t (12.1)	45.6
	2.57, 1H, dd (12.1, 4.6)		2.57 dd (12.1, 4.6)
2	3.75, 1H, ddd (12.1, 9.7, 4.6), ax	69.2, CH	3.75 ddd (12.1, 9.7, 4.6)	69.2
3	2.94 1H, d (9.7), ax	83.4, CH	2.95 d (9.7)	83.4
4		40.4, C		40.4
5	1.83 1H, dd (13.7, 4.1), ax	50.0, CH	1.83 dd (13.9, 4.0)	50.1
6	2.59 1H, dd (18.2, 4.1), eq	36.8, CH2	2.59 dd (18.1, 4.0);	36.9
	2.68 1H, dd (18.2, 13.7), ax		2.68 dd (18.1,13.9)
7		201.3, C		201.2
8		131.3, C		131.1
9		154.4, C		154.9
10		39.8, C		39.9
11	7.29, 1H, d (8.2)	125.6, CH	7.33 d (8.3)	125.0
12	7.40, 1H, dd (8.2, 2.2)	134.1, CH	7.64 dd (8.3, 2.2)	132.4
13		148.4, C		149.4
14	7.72, 1H, d (2.2)	125.2, CH	7.98 d (2.2)	124.1
15	2.84, 1H, m	34.8, CH		72.6
16	1.15, 3H, d (6.9)	24.2, CH3	1.42 s	31.7
17	1.15, 3H, d (6.9)	24.2, CH3	1.42 s	31.7
18	0.98, 3H, s	28.5, CH3	0.98 s	28.5
19	0.90, 3H, s	16.7, CH3	0.88 s	16.7
20	1.20, 3H, s	24.6, CH3	1.20 s	24.6

^a 600 MHz; ^b 150MHz.

). These discrepancies could only be rationalized by proposing the reduction of the OH-15 group. This hypothesis was further corroborated by ¹H–¹H COSY and HMBC analyses (Figure 2), which confirmed the planar structure. The NOESY correlations from CH₃-18 to H-5 and H-3 and from H-2 to CH₃-19 and CH₃-20, along with the coupling constant between H-2 and H-3 (J = 9.7 Hz), revealed the relative configuration of compound 11. The absolute configuration of compound 11 (2S,3S,5S,10R) was determined by comparing its 1D NMR data with the known compound hydroxyldecandrin G, as well as by analyzing the agreement between the calculated and experimentally measured ECD spectra (Figure 9).

2.2. Immunosuppressive Activity

In the concanavalin A (ConA)-induced lymphocyte proliferation assay, compounds 1 and 9 exhibited moderate inhibitory activity with half-maximal inhibitory concentration (IC₅₀) values of 13.55 ± 1.40 μM and 29.25 ± 1.56 μM, respectively. Notably, compound 11 demonstrated more potent suppression against both lipopolysaccharide (LPS)- and ConA-induced lymphocyte proliferation, showing IC₅₀ values of 8.99 ± 1.08 μM (LPS model) and 5.39 ± 0.20 μM (ConA model). Compound 11 exhibited potent activity, presumably attributed to its diterpenoid nature, while the enhanced activity of compound 1 among sesquiterpenoid analogs may be due to the presence of a double bond between C-1 and C-10.

2.3. Spectroscopic Data of Compounds

2.3.1. Compound 1

Colorless oil; ${\left[\alpha \right]}_D^{24}$ −191 (c 0.1, MeOH), UV (MeCN) λ_max (log ε) = 194 (4.16), 245 (2.50), 279 (2.86), 335(0.23) nm; ECD (MeCN) λ_max (Δε) = 199 (−7.87), 222 (+0.26) nm; for ¹H and ¹³C NMR data, see Table 1 and Table 2; HRESIMS m/z 259.1663 [M + Na⁺] (calcd. for C₁₅H₂₄O₂Na⁺, 259.1674).

2.3.2. Compound 2

Colorless oil; ${\left[\alpha \right]}_D^{24}$ +238 (c 0.1, MeOH), UV (MeCN) λ_max (log ε) = 195 (4.02), 226 (2.27) nm; ECD (MeCN) λ_max (Δε) = 198 (+15.25), 218 (+0.01) nm; IR (KBr) ν_max 3360, 2964, 2931, 2856, 1685, 1461, 1443, 1034, 1019 and 888cm⁻¹ cm⁻¹; for ¹H and ¹³C NMR data, see Table 1 and Table 2; HRESIMS m/z 259.1668 [M + Na⁺] (calcd. for C₁₅H₂₄O₂Na⁺, 259.1674).

2.3.3. Compound 3

Colorless oil; ${\left[\alpha \right]}_D^{24}$ −61 (c 0.1, MeOH), UV (MeCN) λ_max (log ε) = 200 (4.20), 220 (2.93) nm; ECD (MeCN) λ_max (Δε) = 209 (−0.13), 245 (+1.64), 258 (−0.08) nm; IR (KBr) ν_max 3325, 2969, 2926, 2857, 1648, 1466, 1444, 1032 and 883 cm⁻¹; for ¹H and ¹³C NMR data, see Table 1 and Table 2; HRESIMS m/z 261.1826 [M + Na⁺] (calcd. for C₁₅H₂₆O₂Na⁺, 261.1830).

2.3.4. Compound 4

Colorless needle crystals; ${\left[\alpha \right]}_D^{24}$ −391 (c 0.1, MeOH), UV (MeCN) λ_max (log ε) = 192 (4.48), 373 (1.00) nm; ECD (MeCN) λ_max (Δε) = 204 (−0.70) nm; IR (KBr) ν_max 3379, 2963, 2923, 2855, 1663, 1459, 1381 and 1039 cm⁻¹; for ¹H and ¹³C NMR data, see Table 1 and Table 2; HRESIMS m/z 277.1771 [M + Na⁺] (calcd. for C₁₅H₂₆O₃Na⁺, 277.1780).

2.3.5. Compound 5

Colorless oil; ${\left[\alpha \right]}_D^{24}$ −339 (c 0.1, MeOH), UV (MeCN) λ_max (log ε) = 200 (3.74), 215 (3.32), 249 (3.88), 383 (1.05) nm; ECD (MeCN) λ_max (Δε) = 202 (−3.98), 246 (+11.87) nm; for ¹H and ¹³C NMR data, see Table 1 and Table 2; HRESIMS m/z 275.1631 [M + Na⁺] (calcd. for C₁₅H₂₄O₃Na⁺, 275.1623).

2.3.6. Compound 6

White amorphous powders; ${\left[\alpha \right]}_D^{24}$ +126 (c 0.1, MeOH), UV (MeCN) λ_max (log ε) = 191 (3.89), 215 (3.36), 249 (3.84), 393 (1.46) nm; ECD (MeCN) λ_max (Δε) = 192 (−2.48), 209 (+0.15), 226 (−0.37), 228 (−0.31), 245 (−1.52) nm; IR (KBr) ν_max 3393, 2968, 2923, 2854, 1671, 1453 and 1043 cm⁻¹; for ¹H and ¹³C NMR data, see Table 3 and Table 4; HRESIMS m/z 275.1626 [M + Na⁺] (calcd. for C₁₅H₂₄O₃Na⁺, 275.1623).

2.3.7. Compound 7

Colorless needle crystals; ${\left[\alpha \right]}_D^{24}$ −286 (c 0.1, MeOH), UV (MeCN) λ_max (log ε) = 214 (3.85), 375 (−0.54) nm; ECD (MeCN) λ_max (Δε) = 192 (+1.20), 203 (+2.29), 223 (−2.01), 237 (−0.77), 253 (−1.26) nm; IR (KBr) ν_max 3429, 2966, 2921, 2850, 1717, 1657 and 1407 cm⁻¹; ¹H and ¹³C NMR data, see Table 3 and Table 4; for HRESIMS m/z 344.1835 [M + Na⁺] (calcd. for C₁₈H₂₇NO₄Na⁺, 344.1838).

2.3.8. Compound 8

White amorphous powders; ${\left[\alpha \right]}_D^{24}$ −341 (c 0.1, MeOH), UV (MeCN) λ_max (log ε) = 210 (4.21), 371 (−0.18) nm; ECD (MeCN) λ_max (Δε) = 205 (+5.16), 252 (−7.58) nm; IR (KBr) ν_max 3337, 2962, 2929, 1709, 1692, 1670, 1454, 1434, 1416, 701 cm⁻¹; for ¹H and ¹³C NMR data, see Table 3 and Table 4; HRESIMS m/z 390.2045 [M + Na⁺] (calcd. for C₂₃H₂₉NO₃Na⁺, 390.2045).

2.3.9. Compound 9

Colorless oil; ${\left[\alpha \right]}_D^{24}$ +76 (c 0.1, MeOH), UV (MeCN) λ_max (log ε) = 196 (4.03) nm; ECD (MeCN) λ_max (Δε) = 199 (+4.53), 215 (−0.25) nm; for ¹H and ¹³C NMR data, see Table 3 and Table 4; HRESIMS m/z 259.1668 [M + Na⁺] (calcd. for C₁₅H₂₄O₂Na⁺, 259.1674).

2.3.10. Compound 10

White amorphous powders; ${\left[\alpha \right]}_D^{24}$ −10 (c 0.1, MeOH), UV (MeCN) λ_max (log ε) = 200 (3.96) nm; ECD (MeCN) λ_max (Δε) = 193 (−16), 200 (−0.35), 208 (−2.54), 222 (+7.60), 236 (+3.35), 250 (−3.18) nm; for ¹H and ¹³C NMR data, see Table 3 and Table 4; HRESIMS m/z 277.1784 [M + Na⁺] (calcd. for C₁₅H₂₆O₃Na⁺, 277.1780).

2.3.11. Compound 11

Pale yellow oil; ${\left[\alpha \right]}_D^{24}$ +7 (c 0.1, MeOH), UV (MeCN) λ_max (log ε) = 210 (4.22), 230 (3.37) nm; ECD (MeCN)λ_max (Δε) = 192 (+2.48), 213 (−0.83), 218 (+0.43), 251 (+0.51), 264 (0.00), 294 (+0.98), 319 (−0.67) nm; for ¹H and ¹³C NMR data, see Table 5; HRESIMS m/z 339.1938 [M + Na⁺] (calcd. for C₂₀H₂₈O₃Na⁺, 339.1936).

3. Materials and Methods

3.1. General Experimental Procedures

Thin-layer chromatography (TLC) was performed with silica gel GF254 glass plates (200−250 μm thickness, Qingdao Marine Chemical Inc., Qingdao, China). Compounds were observed by TLC, and spots were visualized by dipping heated silica gel plates with 10% H₂SO₄ in EtOH. Column chromatography (CC) was implemented with Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden), octadecylsilyl (ODS) (50 μm, YMC Co., Ltd., Kyoto, Japan), and silica gel for column chromatography (100−200 mesh and 200−300 mesh; Qingdao Marine Chemical Inc., China). Compounds were purified using a Waters HPLC system using a reversed-phase (RP) C-18 column (5 µm, 10 × 250 mm, QuikSep SP ODS-A, H&E Co., Ltd., Beijing, China). Optical rotations were obtained using a PerkinElmer 341 polarimeter (PerkinElmer Inc., Fremont, CA, USA). ECD spectra were recorded with a JASCO-810 ECD spectrometer (JASCO Corporation, Tokyo, Japan). Ultraviolet–visible spectroscopy (UV) spectra were measured on a Lambda 35 instrument (PerkinElmer Inc., Waltham, MA, USA) in MeOH. Infrared spectroscopy (IR) spectra were recorded with a Vertex 70 FT-IR spectrophotometer (Bruker, Karlsruhe, Germany). The NMR experiments were conducted on Bruker AM-400 or AM-600 spectrometers (Bruker Corporation, Karlsruhe, Germany), and chemical shifts are reported in parts per million (δ) using the DMSO-d₆ signal (δ_H 2.50; δ_C 39.5), methanol-d4 signal (δ_H 3.31; δ_C 49.0), and chloroform-d signal (δ_H 7.26; δ_C 77.2) as internal standards for ¹H and ¹³C NMR, respectively. HRESIMS data were acquired using SolariX 7.0T Bruker Daltonics (Bruker Corporation, Karlsruhe, Germany).

3.2. Fungus Material

Fungus Eutypella sp. MCCC 3A00281 (family: Diatrypaceae) was isolated from the South Atlantic Ocean deep-sea sediment (5610 m) (GPS 6°25′48″ S, 27°54′00″ W, accession number KT366012). The fungal identification was described in a previous publication [11].

3.3. Fermentation, Extraction and Isolation

Based on the previously established experimental protocols [19], to obtain the seed culture, Eutypella sp. was incubated on potato dextrose agar (PDA) medium at 25 °C for 7 days. Then, the agar was cut into pieces, and the mycelia of the strains grown on PDA were inoculated in autoclaved rice medium (250 g of rice and 250 mL of tap water were placed in 1000 mL Erlenmeyer flasks, with 50 kg of rice in total) and cultured at 25 °C for 35 days. Thereafter, fermented rice was extracted with ethyl alcohol ten times. After the solvent had been evaporated, a nut-brown pasty fluid was obtained, which was evenly dispersed in water and extracted with ethyl acetate eight times. Ultimately, 431 g of crude extract was obtained, which was separated by silica gel column chromatography (100−200 mesh, 800 g) and eluted with a system of petroleum ether-ethyl acetate-methanol (10:1:0–10:10:0–10:10:1, v/v/v) to afford six primary fractions (Fr.1–Fr.6).

Fr.4 (86.3 g) was subjected to ODS column chromatography (CC, MeOH–H₂O, 40–90%) to obtain 7 fractions (Fr.4.1–Fr.4.7). Fr.4.4 was submitted to silica gel CC (petroleum ether–ethyl acetate) to obtain three fractions (Fr.4.4.1–Fr.4.4.3). Compound 1 (1.0 mg; MeOH-H₂O, 64%, v/v, t_R = 33 min, 3.0 mL/min), compound 4 (9.2 mg; MeCN-H₂O, 48%, v/v, t_R = 25 min, 3.0 mL/min), compound 8 (2.4 mg; MeOH-H₂O, 70%, v/v, t_R = 30 min, 3.0 mL/min), and compound 9 (1.0 mg; MeOH-H₂O, 80%, v/v, t_R = 35 min, 2.0 mL/min) were purified by semipreparative high-performance liquid chromatography (HPLC) from Fr.4.4.1. Fr.4.4.2 was subjected to Sephadex LH-20, silica gel CC, and HPLC purification, isolating compound 2 (5.0 mg; MeOH-H₂O, 57%, v/v, t_R = 67 min, 3.0 mL/min), compound 3 (4.1 mg; MeOH-H₂O, 59%, v/v, t_R = 40 min, 3.0 mL/min), compound 5 (1.0 mg; MeOH-H₂O, 61%, v/v, t_R = 40 min, 3.0 mL/min), and compound 11 (1.0 mg; MeOH-H₂O, 60%, v/v, t_R = 76 min, 3.0 mL/min). Fr.5 (24.0 g) was subjected to ODS column chromatography (CC, MeOH−H₂O, 20–100%) to obtain 9 fractions (Fr.5.1–Fr.5.9). Compound 6 (6.0 mg; MeCN-H₂O, 24%, v/v, t_R = 38 min, 3.0 mL/min), compound 7 (10.0 mg; MeCN-H₂O, 33%, v/v, t_R = 43 min, 3.0 mL/min), and compound 10 (0.8 mg; MeOH-H₂O, 65%, v/v, t_R = 38 min, 3.0 mL/min) were purified by semipreparative HPLC from Fr.5.3.

The compounds isolated in this study are terpenoids, exhibiting Rf values of 0.4–0.7 (developed in dichloromethane/methanol, 20:1, v/v), with bluish-purple spots observed after spraying with the detection reagent.

3.4. X-Ray Crystal Structure Analysis

Suitable crystals of compounds 4 and 7 were obtained by slow evaporation from MeOH–THF (10:1) at 20 °C. The intensity data for these compounds were collected with a Bruker APEX DUO diffractometer (Bruker Corporation, Karlsruhe, Germany) equipped with an APEX II CCD using graphite-monochromated Cu Kα radiation. Crystallographic data for the reported structures were deposited in the Cambridge Crystallographic Data Center (CCDC) with deposition number CCDC 2431789 for compound 4 and CCDC 2431799 for compound 7.

Crystal Data for Compound 4. C₁₅H₂₆O₃, M = 254.36 g/mol, a = 11.79120 (10) Å, b = 8.47850 (10) Å, c = 15.1542(2) Å, α = 90°, β = 106.3940 (10)°, γ = 90°, V = 1453.40 (3) ų, T = 99.99 (10) K, space group C₂, Z = 4, μ (Cu Kα) = 0.627 mm⁻¹, 7208 reflections measured, 2842 independent reflections (Rint = 0.0409). The final R₁ value was 0.0365 (I > 2σ(I)). The final wR(F²) value was 0.0978 (I > 2σ(I)). The final R₁ value was 0.0372 (all data). The final wR(F²) value was 0.0983 (all data). The goodness of fit on F² was 1.063. The flack parameter was 0.03 (12).

Crystal Data for Compound 7. C₁₈H₂₇NO₄, M = 321.19 g/mol, a = 7.07560 (10) Å, b = 12.18480 (10) Å, c = 23.0375 (2) Å, α = 90°, β = 90°, γ = 90°, V = 1986.17 (4) ų, T = 99.99 (10) K, space group P2₁2₁2₁, Z = 4, μ (Cu Kα) = 0.794 mm⁻¹, 20,759 reflections measured, 4009 independent reflections (R_int = 0.0427). The final R₁ value was 0.0277 (I > 2σ(I)). The final wR(F²) value was 0.0698 (I > 2σ(I)). The final R₁ value was 0.0285 (all data). The final wR(F²) value was 0.0702 (all data). The goodness of fit on F² was 1.048. The flack parameter was 0.01 (6).

3.5. ECD Calculations

In general, conformational analyses were carried out via random searching in the Sybyl-X 2.0 using the MMFF94S force field with an energy cutoff of 5 kcal/mol. The results showed the nine lowest energy conformers for compound 1, the seven lowest energy conformers for compound 2, the five lowest energy conformers for compound 3, the ten lowest energy conformers for compound 5, the two lowest energy conformers for compound 6, the thirty lowest energy conformers for compound 8, the nine lowest energy conformers for compound 9, the twenty lowest energy conformers for compound 10, and the nine lowest energy conformers for compound 11. Subsequently, geometry optimizations and frequency analyses were implemented at the B3LYP-D3(BJ)/6-31G* level in conductor-like polarizable continuum model (CPCM) methanol using ORCA5.0.1 [20]. All conformers used for property calculations in this work were characterized to be stable on a potential energy surface (PES) with no imaginary frequencies. The excitation energies, oscillator strengths, and rotational strengths (velocity) of the first 60 excited states were calculated using the TD-DFT methodology at the PBE0/def2-TZVP level in CPCM methanol using ORCA5.0.1 [20]. The ECD spectra were simulated by the overlapping Gaussian function (half the bandwidth at 1/e peak height, sigma = 0.30 for all) [21]. Gibbs free energies for conformers were determined by using thermal correction at B3LYP-D3(BJ)/6-31G* level and electronic energies evaluated at the wB97M-V/def2-TZVP level in CPCM methanol using ORCA5.0.1 [20]. To obtain the final spectra, the simulated spectra of the conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (ΔG). By comparing the experiment spectra with the calculated model molecules, the absolute configuration of the chiral center was determined.

3.6. ¹³C NMR Calculations

The details of NMR calculations with DP4+ probability analyses for compound 10 are included in the Supporting Information.

3.7. Biological Activity

Animals: Male BALB/c mice (6–8 weeks old) were obtained from Hubei Biont Biotechnology (Wuhan, China) and were fed in the Tongji Experimental Animal Center. The mice were raised under conditions of a 12 h light/12 h dark cycle, a temperature of 22 ± 1 °C, and a relative humidity of 55 ± 5%. All the mice were acclimatized for 1 week before use. All experiments were performed in accordance with the guidelines of the Ethical Committee for the Experimental Use of Animals at Huazhong University of Science and Technology (Wuhan, China).

Lymphocyte preparation: Briefly, the mice were sacrificed by cervical dislocation, and the spleen was dissected out in a sterile environment. The spleen was ground in a 70 μm filter to obtain a single-cell suspension. After centrifugation at 300× g for 5 min, an appropriate amount of lysis buffer was added to lyse the red blood cells for 5 min. The cells were collected after centrifugation at 300× g for 5 min and washed twice with PBS. Lymphocytes were lysed in RPMI1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS and penicillin–streptomycin solution culture medium. The cells were counted on an automatic counter, and the cell concentration was adjusted to 5 × 10⁶ mL⁻¹. Cell viability was determined by trypan-blue dye exclusion, and cell viability reached up to 95%. The culture media were maintained in a humidified atmosphere of 5% CO₂ at 37 °C.

Lymphocyte proliferation assay: The splenocytes (5 × 10⁵ cells per well) were cultured in 96-well plates in triplicate in the presence of ConA (5 μg mL⁻¹), or LPS (10 μg mL⁻¹) medium in a humidified atmosphere of 5% CO₂ at 37 °C. Next, the corresponding final concentration of the compound was added to the lymphocytes. After 48 h, 10 μL of CCK-8 was added to each well and the plates were incubated for 1 h. The absorbance was measured on an ELISA microplate reader at a wavelength of 450 nm [22].

4. Conclusions

In summary, a total of ten undescribed sesquiterpenes with two types of skeletons, including eight undescribed eremophilane-type sesquiterpenes (1–8), two undescribed sub-type of sesquiterpenes (9–10), and an abietane-type diterpenoid (11), were isolated and identified from the deep-sea fungus Eutypella sp. MCCC 3A00281. Their structures were elucidated on the basis of various spectroscopic analyses, mainly including NMR and HRESIMS data, ¹³C NMR calculations with DP4+ probability analyses, ECD calculations, and single-crystal X-ray diffraction experiments. Immunosuppressive activity assays were performed on all isolated compounds. The results showed that compound 11 exhibited potent immunosuppressive activity with IC₅₀ values of 8.99 ± 1.08 μM (LPS model) and 5.39 ± 0.20 μM (ConA model). Compounds 1 and 9 exhibited moderate inhibitory activity in the ConA model with IC₅₀ values of 13.55 ± 1.40 μM and 29.25 ± 1.56 μM, respectively. Regrettably, there are usually low contents of novel compounds in fungi, and sufficient quantities have not been obtained for more comprehensive and in-depth research on interesting biological activities.