Immunosuppressive Sesquiterpenoids from the Edible Mushroom Craterellus odoratus

Abstract

The aim of this work was to comprehensively understand the chemical constituents of the edible mushroom Craterellus ordoratus and their bioactivity. A chemical investigation on this mushroom led to the isolation of 23 sesquiterpenoids including eighteen previously undescribed bergamotane sesquiterpenes, craterodoratins A–R (1–18), and one new victoxinine derivative, craterodoratin S (19). The new structures were elucidated by detailed interpretation of spectrometric data, theoretical nuclear magnetic resonance (NMR) and electronic circular dichroism (ECD) calculations, and single-crystal X-ray crystallographic analysis. Compounds 1 and 2 possess a ring-rearranged carbon skeleton. Compounds 3, 10, 12–15, 19, 20 and 23 exhibit potent inhibitory activity against the lipopolysaccharide (LPS)-induced proliferation of B lymphocyte cells with the IC₅₀ values ranging from 0.67 to 22.68 μM. Compounds 17 and 20 inhibit the concanavalin A (ConA)-induced proliferation of T lymphocyte cell with IC₅₀ values of 31.50 and 0.98 μM, respectively. It is suggested that C. ordoratus is a good source for bergamotane sesquiterpenoids, and their immunosuppressive activity was reported for the first time. This research is conducive to the further development and utilization of C. ordoratus.

1. Introduction

Edible mushrooms are a large and fascinating group of fungi. Many species of wild edible mushrooms are valued ingredients due to their unique taste and short growth cycle. Yunnan Province is located in southwest China. Its unique climate and geological diversity make this area very favorable for the growth of wild mushrooms. It is estimated that more than 40% of the world’s and 90% of Chinese edible mushrooms (about 900 species) grow in Yunnan [1,2]. Many wild edible mushrooms are regarded as local delicacies such as Tricholoma matsutake, Collybia albuminosa, Cantharellus cibarius, and several species of the genus Boletus. Studying the chemical constituents of these edible mushrooms, therefore, has become our long-term research project [2,3,4]. The systematic mining of chemical components and evaluation of their biological activity will be beneficial to the scientific development and utilization of these edible fungi.

Species of the genus Craterellus (Cantharellaceae) include well-known edible mushrooms. At present, 142 records of Craterellus have been found and about 74 species are currently recognized as members of the genus. Ten species are originally described from Asia and four of these species have been reported in China [5]. Of them, C. odoratus is an edible mushroom in the family widespread in mainland China and characterized by possessing a bright orange or yellow cap [5,6,7,8]. In China, this wild edible mushroom is especially popular in Yunnan Province, where it is a delicacy on the dinner table from June to August each year. In northern Thailand, C. odoratus is also a wild edible mushroom. Its nutritive value including the content of protein, fat, crude fiber, carbohydrate, and mineral contents was assessed. From the perspective of secondary metabolites, this mushroom has not been systematically studied, only a few polyketides and terpenoids have been reported [9,10,11,12,13]. Therefore, it is necessary to conduct systematic chemical component mining and biological activity evaluation on this mushroom. In this study, a total of 23 sesquiterpenoids (1–23, Figure 1) including eighteen new bergamotane-type ones, namely craterodoratins A–R (1–18), and one new victoxinine derivative, namely craterodoratin S (19), were isolated from C. odoratus. Their structures with absolute configurations were elucidated by extensive spectroscopic methods (including 1D and 2D NMR, MS, UV, and IR technologies), single-crystal X-ray diffraction analysis, as well as NMR and ECD calculations.

Previous pharmacological studies on bergamotane metabolites have demonstrated α-glucosidase inhibitory activity [14], antibacterial activity [9,15,16], inhibition of pancreatic lipase [17], and phytotoxic effects against Johnson grass and Sorghum [18,19,20,21]. In this study, all compounds were evaluated for their cytotoxic activities against five human cancer cell lines, and for their immunosuppressive activity on T-cell and B-cell proliferation. Herein the isolation, structural elucidation, and bioactivities of these isolates are reported.

2. Materials and Methods

2.1. General Experimental Procedures

IR spectra were obtained on a Shimadzu Fourier transform infrared spectrometer using KBr pellets. UV spectra were obtained by using a double beam spectrophotometer UH5300 (Hitachi High-Technologies, Tokyo, Japan). Optical rotations were measured on a Rudolph Autopol IV polarimeter (Hackettstown, NJ, USA). High resolution electrospray ionization mass spectra (HRESIMS) were recorded on an Agilent 6200 Q-TOF MS system or a Thermo Scientific Q Exactive Orbitrap MS system. Circular dichroism (CD) spectra were measured with an Applied Photophysics spectrometer (Chirascan, New Haven, CT, USA). NMR spectra were recorded with a Bruker Avance III 600 MHz spectrometer (Bruker, Karlsruhe, Germany). Sephadex LH-20 (GE Healthcare, Pittsburgh, PA, USA), Silica gel (200–300 mesh), and RP-18 gel (20–45 μM, FuJi) were used for column chromatography (CC). HPLC was performed on an Agilent 1260 liquid chromatography system equipped with Zorbax SB-C18 columns (5 μM, 9.4 mm × 150 mm, or 21.2 mm × 150 mm).

2.2. Fungal Material

The fungus Craterellus odoratus was collected from the Southern part of the Gaoligong Mountains in Yunnan Province, China, in July 2007. The fungus was identified by Prof. Mu Zang at the Kunming Institute of Botany. A voucher specimen (HFC2007-20180714-DQ1) has been deposited in the School of Pharmaceutical Sciences, South-Central University for Nationalities.

2.3. Fermentation, Extraction and Isolation

The rice culture medium was composed of glucose 5%, yeast 5%, pork peptone 0.15%, KH₂PO₄ 0.05%, and MgSO₄ 0.05%. The initial pH was adjusted to 6.0. Cultures were grown in an Erlenmeyer flask (220 rpm, 24 °C) for 6 days until the mycelium biomass reached a maximum. This was then transferred to a rice medium and incubated at 24 °C in the dark for 40 days. The rice medium contained 50 g of rice and 50 mL of water, in a 250 mL Erlenmeyer flask, sterilized at 121 °C for 15 min. A total of 400 flasks were used in this work.

The solid rice culture broth of C. odoratus (20 kg) was extracted six time with MeOH to give a crude extract. The extract was partitioned between water and ethyl acetate (EtOAc). The EtOAc layer was concentrated under reduced pressure to give an organic extract (167 g). It was subjected to CC over silica gel (200–300 mesh) eluted with a solvent system of CHCl₃/MeOH (from 1:0 to 0:1, v/v) to obtain nine fractions A–I. Fraction D (20 g) was fractionated by MPLC over RP-18 silica gel eluted with MeOH/H₂O (from 5:95 to 100:0, v/v) to give 13 subfractions (D₁–D₁₃). Fraction D₆ (5 g) was repeatedly fractionated by CC over silica gel eluted with CHCl₃/MeOH (10:1) to give compounds 9 (7 mg), 10 (4 mg), and 11 (18 mg). Fraction D₄ (3 g) was fractionated by CC over Sephadex LH-20 (MeOH) and then purified by prep-HPLC (CH₃CN/H₂O 35:65 in 30 min) to give compounds 1 (3.0 mg, retention time (t_R) = 16.3 min), 8 (3.2 mg, t_R = 20.1 min), and 4 (3.4 mg, t_R = 24.7 min). Fraction D₃ (5 g) was fractionated by CC over Sephadex LH-20 (MeOH) and then purified by prep-HPLC (CH₃CN/H₂O 32:68 in 25 min) to give compounds 17 (2.3 mg, t_R = 13.2 min), 20 (3.0 mg, t_R = 15.4 min), and 18 (2.6 mg, t_R = 22.0 min). Fraction E (9 g) was fractionated by MPLC over RP-18 eluted with MeOH/H₂O (from 5:95 to 100:0, v/v) to give seven subfractions (E₁–E₇). Fraction E₃ (800 mg) was subjected to CC over silica gel (80–100 mesh), Sephadex LH-20 (MeOH), and then purified by prep-HPLC (CH₃CN/H₂O from 25:75 to 61:39 in 25 min) to give compounds 3 (2.1 mg, t_R = 11.2 min), 6 (3 mg, t_R = 15.3 min), 7 (2.5 mg, t_R = 17.9 min), and 16 (3.8 mg, t_R = 23.1 min). Fraction E₅ (36 mg) was prepared by prep-HPLC (CH₃CN/H₂O 30:70 in 25 min) to give compounds 14 (2.1 mg, t_R = 14.8 min), 13 (2.4 mg, t_R = 18.6 min), and 15 (2.6 mg, t_R = 23.0 min). Fraction F (7 g) was separated by MPLC over RP-18 to give five subfractions F₁–F₅. Fraction F₃ (23 mg) was prepared by prep-HPLC (CH₃CN/H₂O 38:62 in 25 min) to give compounds 2 (2.3 mg, t_R = 15.1 min), 12 (2.0 mg, t_R = 17.6 min), and 5 (3.1 mg, t_R = 20.3 min). Fraction F₄ (30 mg) was prepared by prep-HPLC (CH₃CN/H₂O 40:60 in 25 min) to give compounds 21 (2.3 mg, t_R = 13.0 min), 22 (1.8 mg, t_R = 17.0 min), and 19 (3.1 mg, t_R = 21.3 min). Fraction G (1.2 g) was fractionated by CC over silica gel eluted with CHCl₃/CH₃OH (10:1) to give subfractions G₁−G₆. Compound 23 (3.2 mg) precipitated as acicular crystals from fraction G₃.

Craterodoratin A (1). Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ −1.9 (c 0.11, MeOH); UV (MeOH) λ_max (log ε) 210 (1.04) nm; IR (KBr) ν_max 3365, 2945, 2833, 1660, 1454, 1114, 1031 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see

Table 1. ¹H (600 MHz) and ¹³C (150 MHz) NMR Spectroscopic Data for 1–4 in Methanol-d₄.

Entry	1		2		3		4
	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)
1	49.9, C	−	43.5, CH	2.25, d (4.3)	55.8, C	−	41.5, C	−
2	76.3, CH	4.02, d (9.1)	81.8, C	−	79.7, CH	3.51, s	83.5, CH	3.44, s
3a 3b	37.6, CH2	2.16, m 1.00, dd, (13.3, 3.3)	48.2, CH2	1.98, dd, (14.3, 4.3) 1.28, d, (13.7)	43.2, C	−	48.5, C	−
4	41.9, CH	1.79, m	42.3, CH	1.86, t, (4.3)	47.1, CH	1.83, m	46.5, CH	1.80, s
5a 5b	27.5, CH2	1.69, m 1.27, m	20.3, CH2	1.73, m 1.51, m	26.5, CH2	1.70, m 1.39, m	25.8, CH2	1.68, m 1.41, m
6a 6b	25.8, CH2	1.97, m 1.23, m	26.5, CH2	1.72, m 1.16, m	21.9, CH2	1.62, m 1.10, m	25.0, CH2	1.69, m 0.99, m
7a 7b	50.9, C	−	52.5, C	−	37.2, CH2	1.54, d, (10.1) 1.11, m	40.4, CH2	1.48, m 1.11, d, (10.3)
8a 8b	30.9, CH2	1.46, td, (12.8, 12.3, 5.1) 1.17, td, (12.8, 12.3, 5.1)	33.7, CH2	1.98, dd, (14.3, 4.3) 1.74, m	42.3, CH2	1.36, m	48.9, CH2	1.71, m 1.45, m
9a 9b	24.5, CH2	2.25, m 2.07, m	66.4, CH	4.64, m	24.7, CH2	2.15, m	65.3, CH	4.58, m
10	141.1, CH	6.69, t, (7.4)	141.0, CH	6.48, d, (7.8)	144.1, CH	6.72, t, (7.4)	144.6, CH	6.57, d, (9.28)
11	128.5, C	−	129.4, C	−	128.8, C	−	126.6, C	−
12	171.3, C	−	171.2, C	−	172.0, C	−	170.4, C	−
13	11.2, CH3	1.80, s	11.8, CH3	1.84, s	12.3, CH3	1.78, s	11.4, CH3	1.84, d, (1.5)
14	12.3, CH3	0.85, s	22.4, CH3	1.25, s	17.0, CH3	0.86, s	18.5, CH3	1.07, s
15a 15b	15.5, CH3	0.93, s	63.4, CH2	3.64, d, (11.0) 3.47, d, (11.0)	65.1, CH2	3.59, d, (11.0) 3.51, d, (11.0)	16.1, CH3	0.91, s

; HRESIMS m/z 253.17986 [M + H]⁺ (calcd for C₁₅H₂₅O₃⁺, 253.17982).

Craterodoratin B (2). Colorless crystals (MeOH), mp 138.2–140.3 °C; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ − 11.4 (c 0.09, MeOH); UV (MeOH) λ_max (log ε) 215 (1.88) nm; IR (KBr) ν_max 3442, 2951, 2841, 1647, 1018 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see Table 1; HRESIMS m/z 267.15903 [M + H]⁺ (calcd for C₁₅H₂₃O₄⁺, 267.15909).

Craterodoratin C (3). Colorless crystals (MeOH), mp 180.7–182.4 °C; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ − 14.5 (c 0.11, MeOH); UV (MeOH) λ_max (log ε) 230 (3.12) nm; IR (KBr) ν_max 3464, 2924, 1647, 1280, 1265 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see Table 1; HRESIMS m/z 291.15650 [M + Na]⁺ (calcd for C₁₅H₂₄O₄Na⁺, 291.15668).

Craterodoratin D (4). Colorless crystals (MeOH), mp 159.1–162.5 °C; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ + 32 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 225 (3.02) nm; IR (KBr) ν_max 3468, 2967, 2541, 1647, 1016 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see Table 1; HRESIMS m/z 267.14712 [M − H]⁻ (calcd for C₁₅H₂₃O₄⁻, 267.16018).

Craterodoratin E (5). Colorless crystals (MeOH), mp 140.9–142.4 °C; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ + 6.9 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 210 (1.27) nm; IR (KBr) ν_max 3408, 2951, 2841, 1651, 1018 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see

Table 2. ¹H (600 MHz) and ¹³C (150 MHz) NMR Spectroscopic Data for 5–8 in Methanol-d₄.

Entry	5		6		7		8
	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)
1	49.9, C	−	49.8, C	−	52.0, CH	2.64, t, (5.3, 10.8)	49.1, CH	2.79, t, (5.4)
2	86.0, CH	3.34, d (1.3)	82.2, CH	3.47, d (1.3)	150.0, C	−	151.0, C	−
3a 3b	47.3, C	−	47.8, C	−	23.1, CH2	2.61, m 2.33, m	24.3, CH2	2.65, m 2.40, m
4a 4b	44.0, CH	2.06, d, (3.8)	44.7, CH	2.23, d, (3.5)	23.5, CH2	2.11, m 1.82, m	24.7, CH2	1.96, m
5a 5b	27.0, CH2	1.71, m 1.47, m	27.6, CH2	1.65, m 1.39, m	39.4, CH	2.32, m	40.6, CH	2.27, m
6a 6b	26.7, CH2	1.72, m 0.99, m	26.7, CH2	1.66, m 0.99, m	26.5, CH2	2.20, m 1.47, d, (9.7)	29.1, CH2	2.28, m 1.42, m
7a 7b	41.9, CH2	1.38, d, (10.6) 1.13, d, (10.6)	41.5, CH2	1.47, d, (9.7) 1.07, d, (9.7)	54.7, C	−	56.5, C	−
8a 8b	47.0, CH2	1.70, m 1.53, dd, (2.6, 14.8)	80.5, CH	3.39, d, (6.0)	40.4, CH2	2.50, dd, (12.4, 9.7) 2.02, dd, (12.4, 4.2)	39.4, CH2	2.46, dd, (6.7, 11.9) 1.92, dd, (9.5, 11.9)
9	66.6, CH	4.60, m	70.6, CH	4.38, dd, (9.1, 6.0)	73.7, CH	4.91, m	75.7, CH	4.90, s
10	142.6, CH	6.49, d (8.2)	138.4, CH	6.53, d (8.6)	143.0, CH	6.74, dd, (1.5, 8.0)	144.2, CH	6.65, d, (8.2)
11	131.5, C	−	135.7, C	−	128.1, C	−	130.2, C	−
12	174.9, C	−	176.1, C	−	169.8, C	−	171.3, C	−
13	13.1, CH3	1.80, s	14.3, CH3	1.86, s	11.4, CH3	1.85, d, (1.5)	12.8, CH3	1.85, s
14a 14b	64.0, CH2	3.64, d, (11.7) 3.53, d, (11.7)	19.9, CH3	1.04, s	106.4, CH	4.59, s	106.8, CH	4.57, s
15a 15b	19.4, CH3	1.03, s	12.2, CH3	0.81, s	107.0, CH2	4.74, q, (1.8) 4.68, dt, (3.0, 1.5)	107.8, CH2	4.66, s 4.63, s
OCH3	−	−	−	−	53.3, CH3	3.29, s	55.4, CH3	3.28, s

; HRESIMS m/z 307.15146 [M + Na]⁺ (calcd for C₁₅H₂₄O₅Na⁺, 307.15159).

Craterodoratin F (6). Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ + 18 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 210 (0.84) nm; IR (KBr) ν_max 3400, 2949, 2837, 1653, 1456, 1411, 1112, 1024 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see Table 2; HRESIMS m/z 307.15161 [M + Na]⁺ (calcd for C₁₅H₂₄O₅Na⁺, 307.15159).

Craterodoratin G (7). Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ + 1.10 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 210 (1.74) nm; IR (KBr) ν_max 3444, 2933, 1647, 1091, 1016 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see Table 2; HRESIMS m/z 279.15903 [M + H]⁺ (calcd for C₁₅H₂₃O₄⁺, 279.15909).

Craterodoratin H (8). Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ − 30.6 (c 0.16, MeOH); UV (MeOH) λ_max (log ε) 210 (0.68) nm; IR (KBr) ν_max 3398, 2949, 2835, 1653, 1452, 1112, 1031 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see Table 2; HRESIMS m/z 279.15920 [M + H]⁺ (calcd for C₁₅H₂₃O₄⁺, 279.15909).

Craterodoratin I (9). Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ + 31.6 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 230 (3.01) nm; IR (KBr) ν_max 3379, 2947, 2835, 1653, 1456, 1114, 1031 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see

Table 3. ¹H (600 MHz) and ¹³C (150 MHz) NMR Spectroscopic Data for 9–12 in Methanol-d₄.

Entry	9		10		11		12
	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)
1	51.0, CH	2.10, t, (5.4)	48.0, CH	2.29, t, (5.6)	51.3, CH	2.09, t (5.7)	47.4, CH	2.70, t, (5.3)
2	87.4, C	−	88.2, C	−	87.3, C	−	89.2, C	−
3a 3b	32.0, CH2	1.88, m 1.61, m	27.4, CH2	1.92, m 1.56, td, (10.2, 4.2)	72.2, CH	3.63, d, (6.8)	29.5, CH2	2.06, m 1.83, m
4a 4b	22.4, CH2	1.88, m 1.76, m	22.0, CH2	1.91, m 1.82, m	32.8, CH2	2.25, m 1.85, m	22.4, CH2	1.95, m 1.81, m
5	39.2, CH	2.24, q, (5.4)	39.2, CH	2.32, m	37.8, CH	2.27, m	40.2, CH	2.43, q, (5.3)
6a 6b	22.2, CH2	2.13, m 1.50, d, (10.1)	22.3, CH2	2.14, m 1.51, d, (10.2)	21.7, CH2	2.11, m 2.05, m	22.1, CH2	2.28, m 1.75, d, (10.9)
7	55.3, C	−	53.7, C	−	54.7, C	−	53.6, C	−
8a 8b	32.4, CH2	1.91, m 1.72, m	40.2, CH2	2.01, dd, (14.2, 4.2) 1.84, m	40.1, CH2	2.00, dd, (14.2, 4.0) 1.81, dd, (14.2, 4.0)	43.4, CH2	3.15, q, (3.2)
9a 9b	24.5, CH2	2.19, m 2.10, m	65.7, CH	4.45, td, (8.7, 3.8)	65.8, CH	4.42, td, (9.0, 4.0)	199.4, C	−
10	141.7, CH	6.77, t, (7.5)	143.6, CH	6.67, d, (7.7)	141.7, CH	6.57, d, (9.0)	130.6, CH	7.11, s
11	127.9, C	−	127.3, C	−	129.3, C	−	142.4, C	−
12	170.4, C	−	170.5, C	−	172.2, C	−	169.9, C	−
13	11.0, CH3	1.80, s	11.4, CH3	1.83, s	11.8, CH3	1.83, s	13.4, CH3	2.15, s
14a 14b	70.2, CH2	3.82, d, (8.8) 3.47, d, (8.8)	71.7, CH2	3.94, d, (9.4) 3.63, d, (9.4)	71.5, CH2	3.85, d, (9.4) 3.61, d, (9.4)	179.3, C	−
15a 15b	24.0, CH3	1.24, s	65.9, CH2	3.49, d, (11.4) 3.45, d, (11.4)	21.0, CH3	1.31, s	23.6, CH3	1.51, s

; HRESIMS m/z 251.16409 [M + H]⁺ (calcd for C₁₅H₂₃O₃⁺, 251.16417).

Craterodoratin J (10). Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ + 52 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 210 (1.48) nm; IR (KBr) ν_max 3379, 2947, 2835, 1653, 1456, 1114, 1031 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see Table 3; HRESIMS m/z 305.13602 [M + Na]⁺ (calcd for C₁₅H₂₂O₅Na⁺, 305.13594).

Craterodoratin K (11). Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ + 37 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 210 (1.34) nm; IR (KBr) ν_max 3456, 2924, 2815, 1647, 1396, 1018 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see Table 3; HRESIMS m/z 283.15136 [M + H]⁺ (calcd for C₁₅H₂₃O₅⁺, 283.15400).

Craterodoratin L (12). Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ + 53 (c 0.11, MeOH); UV (MeOH) λ_max (log ε) 235 (1.12) nm; IR (KBr) ν_max 3390, 2947, 2833, 1653, 1456, 1112, 1031 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see Table 3; HR-ESIMS m/z 279.12250 [M + H]⁺ (calcd for C₁₅H₁₉O₅⁺, 279.12270).

Craterodoratin M (13). Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ − 23 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 215 (1.90) nm; IR (KBr) ν_max 3487, 1645 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see

Table 4. ¹H (600 MHz) and ¹³C (150 MHz) NMR Spectroscopic Data for 13–16 in Methanol-d₄.

Entry	13		14		15		16
	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)
1	40.3, CH	2.04, m	73.5, C	−	43.2, CH	2.48, d, (6.7)	47.6, CH	1.87, m
2	39.2, CH	1.96, q, (5.1)	40.3, CH	1.38, m	149.4, C	−	150.2, C	−
3a 3b	37.3, CH2	1.84, m 1.76, m	29.6, CH2	1.86, m 0.93, m	24.7, CH2	2.64, m 2.32, m	23.5, CH2	2.63, m 2.33, m
4a 4b	23.9, CH2	3.16, m	26.7, CH2	1.79, m 1.08, m	30.2, CH2	1.95, m 1.75, m	22.6, CH2	1.95, m 1.88, m
5	37.3, CH	2.13, m	47.3, CH	1.33, m	74.9, C	−	37.6, CH	2.18, q, (5.4)
6a 6b	18.0, CH2	1.63, m 1.27, m	25.9, CH2	1.92, m 1.12, dd, (3.7, 12.5)	34.6, CH2	2.33, m 1.79, d, (9.5)	25.8, CH2	2.33, m 1.47, d, (10.1)
7	42.1, C	−	48.4, C	−	48.6, C	−	46.7, C	−
8a 8b	22.9, CH2	2.01, d, (5.7) 1.37, m	38.1, CH2	1.53, t, (1.5)	32.3, CH2	1.82, m 1.68, m	29.9, CH2	1.87, m
9a 9b	23.7, CH	1.84, m	22.3, CH2	2.21, m	23.8, CH2	2.28, m 2.21, m	23.5, CH2	2.33, m 2.23, q, (8.0)
10	140.7, CH	6.71, dd, (7.4, 14.5)	138.9, CH	6.60, t, (6.6)	142.5, CH	6.81, t, (7.0)	142.8, CH	6.84, t, (8.0)
11	129.3, C	−	130.8, C	−	127.8, C	−	127.5, C	−
12	171.3, C	−	174.5, C	−	171.3, C	−	170.8, C	−
13	11.4, CH3	1.81, s	11.8, CH3	1.81, s	11.1, CH3	1.82, s	11.0, CH3	1.83, s
14a 14b	15.8, CH3	0.89, s	22.0, CH3	1.09, s	15.6, CH3	0.81, s	61.1, CH2	3.43, d, (11.7) 3.33, d, (11.7)
15a 15b	65.3, CH2	3.34, dd, (2.6, 6.0)	67.3, CH2	3.34, d, (3.3)	106.1, CH2	4.63, d, (2.0)	105.9, CH2	4.64, br s 4.60, br s

; HRESIMS m/z 253.17992 [M + H]⁺ (calcd for C₁₅H₂₅O₃⁺, 253.17982).

Craterodoratin N (14). Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ − 11 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 210 (1.04) nm; IR (KBr) ν_max 3487, 1645 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see Table 4; HRESIMS m/z 291.15658 [M + Na]⁺ (calcd for C₁₅H₂₄O₄Na⁺, 291.15668).

Craterodoratin O (15). Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ − 28.6 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 210 (1.48) nm; IR (KBr) ν_max 3460, 2951, 2843, 1645, 1016 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see Table 4; HRESIMS m/z 251.16431 [M + H]⁺ (calcd for C₁₅H₂₃O₃⁺, 251.16417).

Craterodoratin P (16). Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ − 27.9 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 210 (1.77) nm; IR (KBr) ν_max 3367, 2943, 2833, 1654, 1456, 1112, 1031 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see Table 4; HRESIMS m/z 251.16417 [M + H]⁺ (calcd for C₁₅H₂₃O₃H⁺, 251.16417).

Craterodoratin Q (17). Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ + 22 (c 0.07, MeOH), UV (MeOH) λ_max (log ε) 210 (1.37) nm; IR (KBr) ν_max 3468, 1645, 1016 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see

Table 5. ¹H (600 MHz) and ¹³C (150 MHz) NMR Spectroscopic Data for 17–19 in Methanol-d₄.

Entry	17		18		19
	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)
1	48.2, CH	3.00, t, (5.3, 10.6)	45.0, CH	2.03, t, (6.2)	40.8, CH	2.31, br s
2	149.7, C	−	44.2, CH	1.94, q, (6.9)	42.2, CH	2.82, s
3a 3b	23.2, CH2	2.64, m 2.34, m	96.5, C	−	159.1, C	−
4a 4b	22.6, CH2	1.90, m	41.0, CH2	2.24, m 1.87, d, (12.4)	58.7, CH2	3.51, m 3.08, dd, (10.6, 2.1)
5	38.5, CH	2.21, m	37.7, CH3	2.33, q, (6.2)	58.8, CH2	3.51, m
6a 6b	44.1, C	−	35.6, CH2	2.49, m 1.16, d, (9.7)	58.7, CH	1.99, br s
7a 7b	25.5, CH2	2.43, m 1.51, d, (10.0)	39.2, C	−	48.4, C	−
8a 8b	37.9, CH2	2.17, dd, (8.7, 15.0) 1.81, m	39.5, CH2	1.85, m 1.71, dd, (14.8, 4.4)	40.8, CH2	1.41, m 1.33, m
9a 9b	65.7, CH	4.60, m	65.7, CH	4.45, m	24.8, CH2	1.63, m 1.08, m
10	143.1, CH	6.65, d, (8.4)	141.4, CH	6.52, d, (8.2)	44.7, CH	1.08, m
11	127.8, C	−	129.7, C	−	30.9, CH	1.35, m
12	171.6, C	−	172.6, C	−	20.2, CH3	0.87, d, (6.5)
13	11.6, CH3	1.83, s	12.0, CH3	1.85, s	20.0, CH3	0.81, d, (6.5)
14a 14b	64.8, CH2	4.00, d, (11.9) 3.93, d, (11.9)	69.3, CH2	4.01, d, (10.2) 3.94, d, (10.2)	103.4, CH2	5.02, s 4.74, s
15a 15b	106.7, CH2	4.67, br s 4.64, br s	13.8, CH3	1.06, d, (6.9)	20.1, CH3	0.98, s
-OOCCH3	19.3, CH3	2.00, s	45.0, CH	2.03, t, (6.2)	48.2, CH2	3.26, t, (6.0)
-OOCCH3	171.6, C	−	44.2, CH	1.94, q, (6.9)	174.0, C	−

; HRESIMS m/z 331.15140 [M + Na]⁺ (calcd for C₁₇H₂₄O₅Na⁺, 331.15159).

Craterodoratin R (18). Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ − 42 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 215 (1.82) nm; IR (KBr) ν_max 3412, 2924, 1637, 1574, 1435, 1065 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see Table 5; HRESIMS m/z 305.13596 [M +Na]⁺ (calcd for C₁₅H₂₂O₅Na⁺, 305.13594).

Craterodoratin S (19). Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{21}$ − 125 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 210 (1.04) nm; IR (KBr) ν_max 3412, 2924, 1637, 1574, 1435, 1065 cm⁻¹; ¹H and ¹³C NMR spectroscopic data, see Table 5; HRESIMS m/z 278.21140 [M +H]⁺ (calcd for C₁₇H₂₈NO₂⁺, 278.21146).

X-ray Crystallographic Data for Craterodoratin B (2). C₁₅H₂₂O₄·H₂O, M = 284.34, a = 8.6658(3) Å, b = 10.0982(4) Å, c = 17.1595(6) Å, α = 90°, β = 90°, γ = 90°, V = 1501.61(9) ų, T = 100(2) K, space group P212121, Z = 4, μ(Cu Kα) = 0.768 mm⁻¹, 12029 reflections measured, 2967 independent reflections (R_int = 0.0391). The final R₁ values were 0.0314 (I > 2σ(I)). The final wR(F²) values were 0.0802 (I > 2σ(I)). The final R₁ values were 0.0325 (all data). The final wR(F²) values were 0.0813 (all data). The goodness of fit on F² was 1.049. Flack parameter = 0.06(7). CCDC: 2059695 (https://www.ccdc.cam.ac.uk (accessed on 13 November 2021)).

X-ray Crystallographic Data for Craterodoratin C (3). C₁₅H₂₄O₄, M = 268.34, a = 9.9718(3) Å, b = 7.2863(2) Å, c = 10.4844(3) Å, α = 90°, β = 111.5850(10)°, γ = 90°, V = 708.35(4) ų, T = 100(2) K, space group P1211, Z = 2, μ(Cu Kα) = 0.729 mm⁻¹, 13370 reflections measured, 2751 independent reflections (R_int = 0.0334). The final R₁ values were 0.0290 (I > 2σ(I)). The final wR(F²) values were 0.0749 (I > 2σ(I)). The final R₁ values were 0.0291 (all data). The final wR(F²) values were 0.0750 (all data). The goodness of fit on F² was 1.039. Flack parameter = 0.06(6). CCDC: 2059696 (https://www.ccdc.cam.ac.uk (accessed on 13 November 2021)).

X-ray Crystallographic Data for Craterodoratin D (4). C₁₅H₂₄O₄, M = 268.34, a = 6.4802(4) Å, b = 10.8539(7) Å, c = 10.8186(7) Å, α = 90.00°, β = 101.555(2)°, γ = 90.00°, V = 745.51(8) ų, T = 297(2) K, space group P1211, Z = 2, μ(Cu Kα) = 1.54178, 15119 reflections measured, 3109 independent reflections (R_int = 0.0263). The final R₁ values were 0.0293 (all data). The final wR(F²) values were 0.0798 (all data). The goodness of fit on F² was 1.051. Flack parameter = 0.09(4). CCDC: 2059697 (https://www.ccdc.cam.ac.uk (accessed on 13 November 2021)).

X-ray Crystallographic Data for Craterodoratin E (5). C₁₅H₂₄O₅·H₂O, M = 302.36, a = 25.5045(11) Å, b = 6.4589(3) Å, c = 19.4274(8) Å, α = 90°, β = 97.923(2)°, γ = 90°, V = 3169.7(2) ų, T = 100(2) K, space group C121, Z = 8, μ(Cu Kα) = 0.805 mm⁻¹, 50,328 reflections measured, 6168 independent reflections (R^int = 0.0956). The final R₁ values were 0.0680 (I > 2σ(I)). The final wR(F²) values were 0.1739 (I > 2σ(I)). The final R₁ values were 0.0741 (all data). The final wR(F²) values were 0.1821 (all data). The goodness of fit on F² was 1.085. Flack parameter = 0.26(11). CCDC: 2059698 (https://www.ccdc.cam.ac.uk (accessed on 13 November 2021)).

2.4. NMR and ECD Calculations

Details of NMR and ECD calculations for compounds 1, 6‒8, 10, 11 and 18 were given in the Supplementary Materials.

2.5. Cytotoxicity Assay

There are five human cancer cell lines using in this assay including human myeloid leukemia HL-60, human breast cancer MCF-7, human colon cancer SW480, human hepatocellular carcinoma SMMC-7721, and human lung cancer A-549 cells. All selected cells were stored in DMEM medium or RPMI-1640, supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) at 37 °C. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method in 96-well microplates was used for cytotoxicity assay [22]. In brief, 100 µL adherent cells were seeded into each well for 12 h before drug addition, while suspended cells were seeded just before adding the test compound with initial density of 1 × 10⁵ cells/mL. The tumor cell line was exposed to the test compound at concentrations of 0.0625, 0.32, 1.6, 8 and 40 μM in triplicates for 48 h. Taxol (Sigma, St. Louis, MO, USA) was used as a positive control. The cell viability was detected, and the cell growth curve was graphed after compound treatment. The IC₅₀ values were calculated by the Reed and Muench’s method [23].

2.6. Immunosuppressive Activities Assay

2.6.1. Preparation of Spleen Cells from Mice

Female BALB/c mice were sacrificed by cervical dislocation, and their spleens were aseptically removed. After cell debris, mononuclear cell suspensions were prepared, and clumps were removed. Erythrocytes were depleted with ammonium chloride buffer solution. Lymphocytes were washed and resuspended in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 mg/mL).

2.6.2. Cytotoxicity Assay

We used the Cell Counting Kit-8 (CCK-8) assay to test cytotoxicity. In brief, fresh spleen cells were obtained from female BALB/c mice (18–20 g). Spleen cells (1 × 10⁶ cells) were seeded in triplicate in 96-well flat plates and cultured at 37 °C for 48 h, with or without compounds of various concentrations, in a humidified and 5% CO₂-containing incubator. A certain amount of CCK-8 was added to each well during the last 8–10 h of culture. At the end of the culture, we use a microplate reader (Bio-Rad 650) to measure the OD value at 450 nm. Cyclosporin A (CsA) is an immunosuppressant agent which is used as a positive control with definite activity. Only the OD value of culture medium is used as background. The cytotoxicity of compounds was expressed as the concentration of the compounds that reduces cell viability to 50% (CC₅₀).

2.6.3. T and B Cell Function Assay

As mentioned above, fresh spleen cells were obtained from female BALB/c mice (18–20 g). Similarly, the 5 × 10⁵ spleen cells were cultured at the same conditions. The cultures, with or without various concentrations of compounds, were stimulated with 5 µg/mL of concanavalin A (ConA) to induce the T cells’ proliferative response or 10 µg/mL of lipopolysaccharide (LPS) to induce B cells’ proliferative response. Proliferation was assessed in terms of uptake of [³H]-thymidine during 8 h of pulsing with 25 µL/well of [³H]-thymidine, then cells will be harvested onto glass fiber filters. A Beta scintillation counter was used to count the incorporated radioactivity. Cells treated without any stimuli were used as a negative control. The immunosuppressive activity was expressed as the concentration of the compound that inhibited T or B cell proliferation to 50% (IC₅₀) of the control value. Both the cytotoxicity and proliferation assessment were repeated twice.

3. Results and Discussion

Compound 1 was isolated as a colorless oil. Its molecular formula of C₁₅H₂₄O₃ was determined by positive high-resolution electrospray ionization mass spectrometry (HRESIMS) analysis, corresponding to four degrees of unsaturation. In the ¹H NMR data, three singlets for methyl groups were readily observed at δ_H 1.80 (3H, s, Me-13), δ_H 0.93 (3H, s, Me-15), δ_H 0.85 (3H, s, Me-14) (Table 1). The ¹³C NMR and DEPT data revealed 15 carbon resonances including three CH₃, five CH₂, three CH, and four non-protonated carbons (Table 1). Of them, two olefinic carbons at δ_C 141.1 (C-10) and 128.5 (C-11), and one carboxy carbon at δ_C 171.3 (C-12) occupied two degrees of unsaturation, which suggested that 1 should be a bicycle sesquiterpenoid. Preliminary analysis of 1D and 2D NMR data, with respect to those in previous isolated from the same source, suggested that 1 might be a bergamotane-type sesquiterpenoid. The ¹H-¹H COSY spectrum afforded evidence to form a fragment as shown in Figure 2. Through the HMBC correlations from δ_H 0.85 (3H, s, Me-14) to δ_C 49.9 (C-1), 76.3 (C-2), 25.8 (C-6), and 50.9 (C-7), as well as correlations from δ_H 0.93 (3H, s, Me-15) to δ_C 49.9 (C-1), 41.9 (C-4), 50.9 (C-7), and 30.9 (C-8), the positions of two methyl groups including Me-14 at C-1 and Me-15 at C-7 were established (Figure 2). Furthermore, the carboxyl group was identified to be conjugated with the double bond by HMBC correlations from δ_H 1.80 (3H, s, Me-13) to δ_C 141.1 (C-10), 128.5 (C-11), and 171.3 (C-12). In the ROESY spectrum (Figure 3), the cross peaks of H₃-15/H-5a (δ_H 1.69), H₃-15/H-6b (δ_H 1.23), and H-3a (δ_H 2.16)/H-8a (δ_H 1.46) indicated that Me-15 and CH₂-5, CH₂-6 were on the same side. The cross peaks of H₂-9/H₃-13 and the absent correlation between H₃-13 and H-10 confirmed the E-configured double bond. However, the relative configuration of 2-OH could not be determined by the ROESY data. The theoretical NMR calculations and DP4+ probability analyses were employed on two possible structures of (1S*,2R*,4S*,7R*)-1a and (1S*,2S*,4S*,7R*)-1b, and the calculations messages suggested that (1S*,2R*,4S*,7R*)-1a was the correct relative configuration for 1 (see Table S3 in Section S1). Finally, the absolute configuration of 1 was established to be 1S,2R,4S,7R by ECD calculations (Figure 5). Therefore, compound 1 was identified and named as craterodoratin A.

Compound 2 was isolated as colorless crystals. Its molecular formula of C₁₅H₂₂O₄ was determined by HRESIMS analysis, corresponding to five degrees of unsaturation. All the spectroscopic data indicated similar patterns to those of 1, suggesting that 2 has a similar structure to 1. Detailed analysis of 1D and 2D NMR data revealed the differences. Firstly, Me-15 was oxygenated into a hydroxymethylene in 2 as suggested by the HMBC correlations from δ_H 3.64 (1H, d, J = 11.0 Hz, H-15a) and δ_H 3.47 (1H, d, J = 11.0 Hz, H-15b) to δ_C 43.5 (C-1), 42.3 (C-4), 52.5 (C-7) and 33.7 (C-8) (Figure 2). Secondly, Me-14 shifted from C-1 to C-2 in 2 which supported by HMBC correlations from δ_H 1.25 (3H, s, Me-14) to δ_C 43.5 (C-1), 81.8 (C-2), and 48.2 (C-3). Thirdly, analysis of the MS data and carbon shifts at δ_C 81.8 (C-2) and δ_C 66.4 (C-9), as well as the HMBC from δ_H 4.64 (1H, m, H-9) to δ_C 81.8 (C-2) indicated an ether bond between C-2 and C-9. The ROESY (Figure 3) cross peaks of H₃-14/H-5b, and H₃-14/H-6b indicated Me-14, H-5b, and H-6b were on the same side, cross peaks of H₂-15/H-6a, and H₂-15/H-5a indicated CH₂-15, H-5a, and H-6a were on the same side, while correlations between H-9/H₃-13 and the absence of the ROESY correction of between H₃-13/H-10 confirmed the E-configured double bond. Finally, the single-crystal X-ray diffraction established the structure of 2 with the absolute configuration (Flack parameter = 0.06 (7), CCDC: 2059695. Figure 4). Therefore, compound 2 was identified and named as craterodoratin B.

Compound 3 was isolated as colorless crystals. Its molecular formula of C₁₅H₂₄O₄ was determined by HRESIMS data, corresponding to four degrees of unsaturation. The ¹³C NMR and DEPT data (Table 1) displayed 15 carbon resonances including two methyl carbons (δ_C 12.3 and 17.0), one olefinic methine carbons (δ_C 144.1), one oxygenated methine carbon (δ_C 79.7), six methylenes, one methine, two sp³ quaternary carbons (δ_C 55.8 and 43.2), and one carboxyl carbon (δ_C 172.0). Analysis of 2D NMR data including ¹H-¹H COSY and HMBC correlations as shown in Figure 2 suggested that 3 should be a bergamotane-type sesquiterpenoid related to (Z)-2α-hydroxyalbumol [24]. The differences were that C-12 and C-15 in 3 were oxidized into a carboxy group and a hydroxymethylene group, respectively. In the ROESY spectrum, cross peaks of H-7a and H-8 supported that C-7 and C-8 were on the same side. Besides, the double bond between C-10 and C-11 in 3 was assigned as E geometry by the ROESY correlation of H-9/H₃-13. The single-crystal X-ray diffraction established the absolute configuration of 3 (Flack parameter = 0.06(6), CCDC: 2059696, Figure 4). Therefore, compound 3 was identified and named as craterodoratin C.

Compound 4 was isolated as colorless crystals. Its molecular formula of C₁₅H₂₄O₄ was determined by HRESIMS data, the same as that of 3. All the spectroscopic data showed similar patterns to those of 3, except one oxygenated methylene group of C-15 was reduced to a methyl group in 4. This was supported by the key HMBC correlations from δ_H 0.91 (3H, s, Me-15) to δ_C 41.5 (C-1), 83.5 (C-2), and 25.0 (C-6). In addition, C-9 was oxidized into a hydroxymethine group, as demonstrated by the HMBC correlations from δ_H 4.58 (1H, m, H-9) to δ_C 48.9 (C-8), 144.6 (C-10), and 126.6 (C-11). The single-crystal X-ray diffraction not only confirmed the planar structure as elucidated above but also established the absolute configuration (Flack parameter = 0.09(4), CCDC: 2059697, Figure 4). Therefore, compound 4 was identified and named as craterodoratin D.

Compound 5 was isolated as colorless crystals. Its molecular formula of C₁₅H₂₄O₅ was determined by HRESIMS data, corresponding to four degrees of unsaturation. The 1D NMR data (Table 2) indicated that 5 has a closely related structure to that of 4, except that one methyl group of C-14 was oxygenated into a hydroxyl methylene group in 5. It was supported by the loss of one methyl signal in the ¹H NMR spectrum and the HMBC correlations from δ_H 3.64 (1H, d, J = 11.7 Hz, H-14a) and δ_H 3.53 (1H, d, J = 11.7 Hz, H-14b) to δ_C 86.0 (C-2), 47.3 (C-3), 44.0 (C-4), and 47.0 (C-8). The single-crystal X-ray diffraction confirmed the planar structure and established the absolute configuration (Flack parameter = 0.26(11), CCDC: 2059698. Figure 4). Thus, compound 5 was identified and named as craterodoratin E.

Compound 6 was isolated as a colorless oil. Its molecular formula of C₁₅H₂₄O₅ was determined by HRESIMS data, corresponding to four degrees of unsaturation. All the spectroscopic data indicated that 6 had a similar structure to that of 4. Detailed analysis of 1D and 2D NMR data revealed that C-8 was oxygenated into a hydroxymethine group in 6 as established by the shift at δ_C 80.5 (C-8) and MS data. The coupling constant (³J_H-H = 6.0 Hz) of H-8/H-9 of 6 observed in ¹H NMR spectrum and a strong ROESY correlation between H-9 and H-2 suggested anti configuration of H-8 and H-9 [25]. To determine the stereochemistry of C-8, the theoretical NMR calculations and DP4 + probability analyses were employed on two possible relative structures (1R*,2R*,3R*,4S*,8S*,9S*)-6a and (1R*,2R*,3R*,4S*,8R*,9S*)-6b. The results suggested that (1R*,2R*,3R*,4S*,8R*,9S*)-6b was the correct relative configuration for 6 (see Table S6 in Section S2). Based on this, the absolute configuration of 6 was suggested to be 1R,2R,3R,4S,8R,9S by the ECD calculations (Figure 5). Therefore, compound 6 was identified and named as craterodoratin F.

Compounds 7 and 8 were isolated as a pair of epimers. They possessed the same molecular formula of C₁₆H₂₂O₄, on the basis of HRESIMS data. Analysis of 1D (Table 1) and 2D NMR data suggested that the planar structures of 7 and 8 were similar to that of donacinoic acid A (23) [9], except the ether bond between C-2 and C-14 was cut off to give a methoxy group at C-14. In the ROESY spectra, the observed correlations of H-14/H-15a in 7 and H-5/H-14 in 8 suggested that the stereo-configurations of C-14 in 7 and 8 were different. The theoretical NMR calculations and DP4 + probability analyses were employed to elucidate the relative configurations of 7 and 8 (see Tables S9 and S10). Then, the ECD calculations established the absolute configurations to be 7S,9S,14S for 7 and 7S,9S,14R for 8 (Figure S9 in Section S3). Finally, compounds 7 and 8 were identified and named as craterodoratins G and H, respectively.

Compound 9 was isolated as a colorless oil. Its molecular formula of C₁₅H₂₂O₃ was determined on the basis of the HRESIMS data, corresponding to five degrees of unsaturation. Analysis of its NMR data (Table 3) indicated similar patterns to those of massarinolin B [15], except that C-9 in massarinolin B was reduced to a methylene in 9 as determined by the MS data and ¹H–¹H COSY correlations (Figure 2). Therefore, compound 9 was identified and named as craterodoratin I.

Compound 10 was isolated as a colorless oil. Its molecular formula of C₁₅H₂₂O₅ was determined by HRESIMS analysis, corresponding to five degrees of unsaturation. The ¹H and ¹³C NMR (Table 3) spectroscopic characteristics were similar to those of 9, except for two hydroxy groups placed at C-15 and C-9 in 10. These were determined by the HMBC correlations from δ_H 4.45 (1H, td, J = 8.7, 3.8 Hz, H-9) to δ_C 40.2 (C-8), 143.6 (C-10), 127.3 (C-11), and from δ_H 3.49 (1H, d, J = 11.4 Hz, H-15a), 3.45 (1H, d, J = 11.4 Hz, H-15b) to δ_C 88.2 (C-2), 27.4 (C-3), 48.0 (C-1). According to the ROESY correlation between H-9 and H-13, the double bond was established as E form. The absolute configuration was established by ECD calculations as shown in Figure 5. Therefore, compound 10 was identified and named as craterodoratin J.

Compound 11 was isolated as a colorless oil. Its molecular formula of C₁₅H₂₂O₅ was determined by HRESIMS analysis, corresponding to five degrees of unsaturation. The ¹H and ¹³C NMR and DEPT (Table 3) data of 11 displayed signals for structural features similar to 10, except that one hydroxy was transfered from C-15 to C-3. The location was determined by the HMBC correlations from δ_H 1.31 (3H, s, Me-15) to δ_C 87.3 (C-2), 72.2 (C-3), 51.3 (C-1), and from δ_H 3.63 (1H, d, J = 6.8 Hz, H-3) to δ_C 87.3 (C-2), 32.8 (C-4). The stereo-configurations for C-3 and C-9 could not be established according to the ROESY data. Thus, the theoretical NMR calculations and DP4 + probability analyses were employed on four possible structures (see Table S17 in Section S5). Based on these data, the absolute configuration of 11 was suggested to be 1R,2S,3S,5R,7S,9S by the ECD calculations (Figure 5). Therefore, compound 11 was identified and named as craterodoratin K.

Compound 12 was isolated as a colorless oil. Its molecular formula of C₁₅H₁₈O₅ was determined by HRESIMS analysis, corresponding to seven degrees of unsaturation. The ¹H and ¹³C NMR and DEPT (Table 3) data of 12 displayed signals for structural features similar to 9, except that C-14 and C-9 were oxidized into two carbonyl carbons in 12. The locations were determined by the HMBC correlations from δ_H 3.15 (2H, q, J = 3.2 Hz, H-8) to δ_C 40.2 (C-5), 53.6 (C-7), 179.3 (C-14), and 199.4 (C-9). Therefore, compound 12 was identified and named as craterodoratin L.

Compound 13 was isolated as a colorless oil. Its molecular formula of C₁₅H₂₄O₃ was determined by HRESIMS analysis, corresponding to four degrees of unsaturation. The ¹³C NMR and DEPT spectrum (Table 4) displayed 15 carbon resonances including two methyl carbons, one olefinic methine carbon, one quaternary olefinic carbon, five methylenes, three methines, and one quaternary carbon. Analysis of 1D and 2D NMR data suggested that 13 had a similar structure to that of massarinolin C (21) [15]. The locations of 7-Me and 2-CH₂OH were determined by the HMBC correlations from δ_H 0.89 (3H, s, Me-14) to δ_C 40.3 (C-1), 39.2 (C-5), 42.1 (C-7), 37.3 (C-8), and from δ_H 3.34 (2H, dd, J = 6.0, 2.6 Hz, H-15) to δ_C 40.3 (C-1), 37.2 (C-2), 18.0 (C-3). In the ROESY spectrum (Figure 3), the cross peak of H₃-14/H-2 indicated that Me-14 and H-2 were on the same side, while the cross peak of H-9/H-13 indicated the E form of the double bond. Therefore, compound 13 was identified as craterodoratin M.

Compound 14 was isolated as a colorless oil. Its molecular formula of C₁₅H₂₄O₄ was determined by HRESIMS analysis, corresponding to four degrees of unsaturation. All 1D and 2D NMR data suggested that 14 had a structure closely related to that of 13 except that one more hydroxy group at C-1 in 14, which was supported by the HMBC correlations from δ_H 1.09 (3H, s, Me-14) to δ_C 73.5 (C-1), 47.3 (C-5), and 48.4 (C-7). Detailed analysis of 2D NMR data suggested that the other parts of 14 were the same as those of 13. Therefore, compound 14 was identified and named as craterodoratin N.

Compound 15 was isolated as a colorless oil. Its molecular formula of C₁₅H₂₂O₃ was determined by HRESIMS analysis, corresponding to five degrees of unsaturation. The ¹H and ¹³C NMR and DEPT (Table 4) data of 15 displayed a close resemblance to those of 13. One significant difference was that the hydroxy group was substituted at C-5, as evidenced by HMBCs from δ_H 2.33 (1H, m, H-6a) and 1.79 (1H, d, J = 9.5 Hz, H-6b) to δ_C 43.2 (C-1), 74.9 (C-5), and 48.6 (C-7). In addition, one terminal double bond was established between C-2 and C-15, as proved by HMBC correlations from δ_H 4.63 (2H, d, J = 2.0 Hz, H-15) to δ_C 43.2 (C-1), 149.4 (C-2), and 24.7 (C-3). Therefore, compound 15 was identified and named as craterodoratin O.

Compound 16 was isolated as a colorless oil. Its molecular formula of C₁₅H₂₂O₃ was determined to be the same to that of 15 by HRESIMS data. Detailed analysis of 1D and 2D NMR data suggested that 16 had a very similar structure to that of 15 except for the hydroxy group at C-14 in 16 replaced at C-5 in 15. It was supported by the HMBC correlations from δ_H 3.43 (1H, d, J = 11.7 Hz, H-14a) and δ_H 3.33 (1H, d, J = 11.7 Hz, H-14b) to δ_C 47.6 (C-1), 37.6 (C-5), 29.9 (C-8). Therefore, compound 16 was identified and named as craterodoratin P.

Compound 17 was isolated as a colorless oil. Its molecular formula of C₁₇H₂₄O₅ was determined by HRESIMS analysis, corresponding to six degrees of unsaturation. The ¹H and ¹³C NMR and DEPT (Table 5) data of 17 displayed a close resemblance to those of massarinolin C (21) [15]. Analysis of 2D NMR data suggested that one more O-acetyl group was substituted at C-14, as evidenced by HMBC correlations from δ_H 4.00 (1H, d, J = 11.9 Hz, H-14a) and 3.93 (1H, d, J = 11.9 Hz, H-14b) to δ_C 48.2 (C-1), 38.5 (C-5), 37.9 (C-8), 171.6 (-OOCCH₃). The absolute configuration of 17 was established by ECD calculations as shown in Figure S18 in Section S6. Therefore, compound 17 was identified and named craterodoratin Q.

Compound 18 was isolated as a colorless oil. Its molecular formula of C₁₅H₂₂O₅ was determined by HRESIMS data, corresponding to five degrees of unsaturation. The 1D NMR data (Table 5) indicated that 18 should have a closely related structure to that of brasilamide A [26]. One significant difference was that the amide group was replaced by a carboxy group at δ_C 172.6 (C-12), as evidenced by the MS data. In addition, C-9 and C-14 in 18 was reduced into a hydroxymethine group (δ_C 65.7) and a methylene (δ_C 69.3), respectively, as supported by the HMBC correlations from δ_H 4.45 (1H, m, H-9) to δ_C 39.5 (C-8) and 141.4 (C-10), and from δ_H 4.01 (1H, d, J = 10.2 Hz, H-14a) and 3.94 (1H, d, J = 10.2 Hz, H-14b) to δ_C 45.0 (C-1), 39.2 (C-7), 96.5 (C-3), and 37.7 (C-5). Based on the ROESY data, cross peaks of H-9/H-14 supported that H-9 and C-14 were on the same side; cross peaks of H-2/H-6b supported that H-2 and C-14 were on the same side. Finally, the absolute configuration of 18 was suggested to be 1S, 2S, 3R, 5R, 7S, 9S by the ECD calculations (Figure 5). Therefore, compound 18 was identified and named as craterodoratin R.

Compound 19 was isolated as a colorless oil. Its molecular formula of C₁₇H₂₇NO₂ was determined by HRESIMS data, which suggested an N-containing structure. The 1D and 2D NMR data of 19 were similar to those of victoxinine [19,27], except that one oxygenated methylene carbon at C-17 in victoxinine was replaced by a carboxyl group in 19 as proved by HMBC correlation from δ_H 3.26 (2H, t, J = 6.0 Hz, H-16) to δ_C 174.0 (C-17). Detailed analysis of 2D NMR data suggested that the other parts of 19 were the same as that of victoxinine. Therefore, compound 19 was identified and named as craterodoratin S.

In addition to the new compounds as described above, four known sesquiterpenoids obtained in this study were identified as massarinolin B (20) [15,28], massarinolin C (21) [15], massarinolin A (22) [15], and donacinoic acid A (23) [9] by comparison of their spectroscopic data with those reported in the literature. The biosynthesis of the isolated compounds 1−18 and 20−23 was proposed as shown in Scheme 1. Bergamotane sesquiterpenoids, bearing a borneol ring system or a β-pinene ring system, are naturally occurring in plants and fungi (Scheme 1) [9,14,15,16,17,29,30,31,32,33,34]. In this study, compounds 1 and 2 possess a rare carbon skeleton while the methyl group (C-14) in 2 had a further 1,2-migration. The sesquiterpenes 7–18 belong to β-pinene derivatives (Scheme 1), which produced compounds 3–6 by an alkyl migration.

All compounds were evaluated for their cytotoxicity to five human cancer cell lines (HL-60, A-549, SMMC-7721, SW480, MCF-7). However, no compounds are active at the concentration of 40 µM. In addition, all the compounds were investigated for their in vitro immunomodulatory effect on BALB/c mice T and B lymphocyte proliferation. Compounds 3, 10, 12–15, 19, 20, and 23 exhibited potent inhibitory activity against LPS-induced proliferation of B lymphocyte cell with IC₅₀ values ranging from 0.67 to 22.68 μM. Compounds 17 and 20 exhibited inhibitions on ConA-induced proliferation of T lymphocyte cells with IC₅₀ values of 31.50 and 0.98 μM, respectively (

Table 6. Immunosuppressive Tests of the Isolates.

Entry	CC50 (μM)	Con A-Induced T-Cell Proliferation		LPS-Induced B-Cell Proliferation
		IC50 (μM)	SI a	IC50 (μM)	SI a
3	>40	−	−	12.62 ± 1.14	>3.17
10	>40	−	−	19.40 ± 0.48	>2.06
12	>40	−	−	13.71 ± 0.65	>2.92
13	>40	−	−	15.43 ± 1.03	>2.59
14	>40	−	−	13.26 ± 1.29	>3.02
15	>40	−	−	17.12 ± 1.14	>2.34
17	>40	31.50 ± 1.79	>1.27	−	−
19	>40	−	−	22.68 ± 1.67	>1.76
20	>40	0.98 ± 0.01	>40.82	0.67 ± 0.004	>59.26
23	>40	−	−	13.23 ± 0.97	>3.02
CsA	>2.80	0.04	>70.00	0.47	>5.95

^a SI (selectivity index) is determined as the ratio of the concentration of the compounds that reduced cell viability to 50% (CC₅₀) to the concentration of the compounds needed to inhibit the proliferation by 50% relative to the control value (IC₅₀).

).

Structurally, almost all bergamotane sesquiterpenoids in this study contain an α,β-unsaturated carboxylic acid moiety, which might be the key functional group for their immunosuppressive activity. However, it seems that the compounds (10 and 12–15) with a basic core of a β-pinene showed a wider range of biological activities. Immunosuppressants are a kind of drug that can inhibit human immunity. They are mainly used in organ transplantation to combat rejection and autoimmune diseases. At present, there are several immunosuppressive drugs that work by inhibiting T cell proliferation, but new, efficient, and safe immunosuppressive drugs inhibiting B cell proliferation are still unavailable [35,36]. In this study, many bergamotane sesquiterpenoids were found to have potential inhibition of B cell proliferation. To the best of our knowledge, bergamotane sesquiterpenoids were reported for their immunosuppressive activity for the first time.

4. Conclusions

In summary, a total of 23 sesquiterpenoids including 19 new ones were isolated from the edible fungus C. ordoratus. Of them, 22 compounds belong to bergamotane sesquiterpenoids in three carbon skeletons, while compounds 1 and 2 possess a rare ring-rearranged backbone. In addition, many bergamotane sesquiterpenoids exhibited selective inhibitions on LPS-induced B cell proliferation. This study suggests that C. odoratus is rich in bergamotane sesquiterpenoids with promising immunosuppressive activity and provides strong support for the further development and utilization of the edible mushroom C. odoratus.