Ganocochlearic acid A, a rearranged hexanorlanostane triterpenoid, and cytotoxic triterpenoids from the fruiting bodies of Ganoderma cochlear

Abstract

Ganocochlearic acid A (1), a rearranged hexanorlanostane triterpenoid featuring a γ-lactone ring and a five-membered carbon ring, and eleven new lanostane triterpenoids (2–12), along with six known analogues (13–18) were isolated from the fruiting bodies of Ganoderma cochlear. Their structures, including absolute configurations, were established on the basis of the MS, NMR, X-ray crystallographic, and ECD analysis. A plausible biosynthetic pathway for 1 was proposed. Compounds 7–9, 11–13 showed moderate cytotoxic activities against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW480) with IC₅₀ values ranging from 8 to 30 μM. Compound 4 exhibited relatively potent cytotoxic activity against MCF-7 cells (IC₅₀: 9.15 μM), compared to the positive control (cisplatin, IC₅₀: 12.7 μM).

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Introduction

Ganoderma (Ganodermataceae) is a popular dietary supplement in East Asia, where it is taken to enhance health and longevity. Its use in traditional Chinese and other Asian medicines in the treatment of different diseases, especial various types of cancers, and is backed by two thousand years of anecdotal evidence and a growing body of scientific data.^1–5 Lanostane triterpenoids are one of the main groups, which are known to inhibit cancer growth and metastasis by modulating the immune system, inducing cell-cycle arrest, and triggering apoptosis.^6–9 Ganoderma lucidum is probably the most well-studied species of all the lanostane-containing fungi, nevertheless, other species of the same genus have also been studied and identified as good sources of potential anticancer agents.^10–12

G. cochlear is medicinally consumed in China. Previous phytochemical investigations, including our recent work, have resulted in the identification of triterpenoids and aromatic meroterpenoids.^13–17 In our continuing effort aimed at the isolation of bioactive triterpenoids, we investigated the fruiting bodies of G. cochlear and eighteen triterpenoids (Fig. 1), including ganocochlearic acid A (1), a hexanorlanostane triterpenoid with a γ-lactone ring and a five-membered carbon ring, three 3,4-seco-lanostane triterpenoids (2, 3 and 6), and eight new lanostane triterpenoids (7–12), as well as six known analogues (13–18) were isolated. In addition, compounds 1–13 were evaluated for their cytotoxic activities against five human tumor cell lines by MTS method.

Fig. 1 Chemical structure of compounds 1–18.

Results and discussion

Ganocochlearic acid A (1) was obtained as white powder. Its molecular formula C₂₄H₃₄O₅ was deduced from the HRESIMS [M + Na]⁺ ion at m/z 425.2307 (calcd 425.2304), corresponding to eight degrees of unsaturation. The IR spectrum showed the presence of hydroxyl (3439 cm⁻¹) and α,β-unsaturated carbonyl (1709 cm⁻¹) groups. The ¹H NMR spectrum showed three singlet methyl signals at δ 1.48, δ 1.09 and δ 0.78, as well as a doublet methyl signal at δ 0.89 (d, J = 6.6 Hz). The ¹³C NMR spectrum displayed twenty-four carbon resonances, including four methyls, nine methylenes, three methines and eight quaternary carbons (one ketone carbonyl, one carboxyl, two sp² quaternary and one oxyquaternary carbons), suggesting that compound 1 was a lanostane-type nortriterpenoid and had the same rings C, D and side chain as fornicatin B,¹⁸ which were supported by the 2D NMR spectral analysis (Fig. 2).

Fig. 2 Key HMBC (), ¹H–¹H COSY () and ROESY () correlations of ganocochlearic acid A (1).

The comparative analysis of its ¹³C NMR (C₅D₅N) data with those of fornicatin B showed the strong downfield shift of C-8 (δ 164.1 for fornicatin B; δ 179.3 for 1) and the highfield shift of C-9 (δ 135.4 for fornicatin B; δ 133.0 for 1) and C-11 (δ 199.8 for fornicatin B; δ 198.1 for 1), indicating that ring B of 1 should be different from that of fornicatin B. These differences were proved by the HMBC correlations of H-10 (δ 3.11, m), H₂-6 (δ 2.31, m; δ 1.97, m) and H₂-7 (δ 2.86, m; δ 2.32, m) to C-8 and C-9; of H-10 to C-11, together with the ¹H–¹H COSY correlations of H-10/H₂-6/H₂-7 (Fig. 2). Thus, we unambiguously deduced that ring B of 1 was a five-membered carbon ring.

Apart from seven degrees of unsaturation occupied by three rings, a carboxyl group, a carbonyl group, a double bond and a ester carbonyl group, the remaining one degree of unsaturation was assumed to be representative of another ring (ring A). The HMBC correlations (Fig. 2) of H-10 and H₂-6 to C-5 (δ 90.7), of H₃-19 (δ 1.50, s), H₂-1 (δ 2.70, m; δ 1.86, m) and H₂-2 (δ 1.74, m; δ 1.45, m) to C-5 and C-3 (δ 177.4), coupled with the ¹H–¹H COSY correlation of H₂-1/H₂-2, deduced ring A of 1 to be a γ-lactone ring. Thus, the planar structure of 1 was established.

The relative orientation of H-10 was determined to be β based on the ROESY correlation of H-10/H₃-18. Thus, only two diastereoisomers (1a, 5S,10S; 1b, 5R,10S) remained. The absolute configuration of 1 was established by comparison of the experimental CD spectrum and the calculated ECD data (Fig. 3). The optimized conformations of 1a and 1b were obtained using the time-dependent density functional theory (TD-DFT) method at the B3LYP/6-31+G(d,p) level in the gas phase by using the Gaussian 09 (see ESI†). Further ECD calculations were performed at the PCM-B3LYP/6-31+G(d,p) level in MeOH solution. The result showed that the calculated weighted ECD spectrum of 1a is accordance with the experimental spectrum (Fig. 3). Consequently, the absolute configuration of compound 1 was assigned as 5S,10S.

Fig. 3 Calculated and experimental CD spectra of different diastereomers. (— Calcd.1 for 1a, — Calcd.2 for 1b, — Expl. experimental in MeOH).

Cochlate C (2) was obtained as white powder, and its molecular formula was established as C₂₈H₃₈O₇ by HRESIMS ion at m/z 509.2509 [M + Na]⁺ (calcd 509.2510), indicating 10 degrees of unsaturation. The ¹H NMR spectrum showed three singlet methyl signals at δ 1.66, 1.39, 0.89; one doublet methyl signal at δ 0.75 (d, J = 6.0 Hz); one methoxyl signal at δ 3.60 (s); three oxymethine signals at δ 5.40 (d, J = 5.3 Hz), δ 5.21 (s) and δ 4.68 (t, J = 2.4 Hz); as well as one terminal double bond proton signals at δ 4.99 (s), δ 4.87 (s). The ¹³C-DEPT data (Table 2) displayed the presence of the characteristic signals for the terminal double bond (δ 143.6, C; δ 115.1, CH₂), α,β-unsaturated carbonyl group (δ 168.0, C; δ 125.2, C; δ 196.9, C), and two ester carbonyl groups (δ 170.8, C; δ 174.2, C). Its 1D NMR data were similar to those of cochlate A¹³ except that two oxymethines in 2 replaced two methylenes in cochlate A. The ¹H–¹H COSY correlations (Fig. 4) between the oxymethine proton (δ 4.68, m) and H-16, between H-16 and H-17, between H-17 and H-20, and between H-20 and H₃-21, conjugated with the HMBC correlations of H₃-30 to C-13, C-14 and the oxymethine, and of the oxymethine proton to C-13, C-14, C-16, and C-17 indicated that C-15 was bonded to hydroxy group.

Fig. 4 Key HMBC () and ¹H–¹H COSY () correlations of compounds 2, 3, 4 and 6.

Additionally, another oxymethine was assigned to be C-19 on the basis of the HMBC correlations of H-19 (δ 5.21, s) to C-10, C-5, C-8, C-9 and C-11. Moreover, H-19 also showed the HMBC correlation with C-3 (δ 170.8), which deduced that a δ-lactone was between C-3 and C-19. The ROESY correlations (Fig. 5) of H-15/H₃-18 and of H-19/H-5/H₃-30 proved that the relative orientation of H-15 and H-19 were β and α, respectively. Therefore, the structure of 2 was determined, named cochlate C.

Fig. 5 Key ROESY correlations of compound 2.

Compound 3 was isolated as colorless needle crystals. The molecular formula of 3 was established as C₂₇H₃₈O₆ by HRESIMS ion at m/z 481.2560 [M + Na]⁺ (calcd 481.2566). Analysis of ¹H ¹³C, and DEPT spectra (Tables 1 and 2) revealed the presence of a double bond (δ 115.6, CH₂; δ 146.4, C), an α,β-unsaturated carbonyl moiety (δ 164.9, C; δ 137.2, C; δ 199.8, C), a carboxyl group (δ 176.4, C), a ester carbonyl group (δ 177.1, C), an oxyquaternary carbon (δ 87.7, C) and an oxymethine (δ 67.7, C), suggesting that compound 3 had the same skeleton as fornicatin B.¹⁸ However, comparison of 1D NMR data between 3 and fornicatin B showed five singlet methyls in 3 but four singlet methyls and one doublet methyl in fornicatin B. Meanwhile, a methine in fornicatin B was replaced by the oxyquaternary carbon (δ 87.7) in 3, indicating that C-20 could be linked to an oxygen. Furthermore, the HMBC correlations (Fig. 4) of H₃-21 to C-20, C-17, and C-22, of H₂-22 to C-20, C-17, C-23, and C-24, and of H₂-23 to C-20, C-22, and C-24, along with the analysis of the molecular weight illustrated that a 20 → 24 γ-lactone was present in 3. The planar structure of 3 was finally determined.

Table 1 The ¹H NMR spectroscopic data of compounds 1–12

Position	1^a	2^b	3^b	4^a	5^b	6^a	7^b	8^a	9^a	10^a	11^a	12^a
	^1H (J)	^1H (J)	^1H (J)	^1H (J)	^1H (J)	^1H (J)	^1H (J)	^1H (J)	^1H (J)	^1H (J)	^1H (J)	^1H (J)
a Measured in CDCl3.b Measured in C5D5N. ^1H and ^13C NMR spectra (δ) were measured at 600 (150) MHz for 1, 4, 6, 8–12; at 500 (125) MHz for 5 and 7; at 400 (100) MHz for 2 and 3. The assignments were based on COSY, HSQC, and HMBC experiments.
1	2.70, m; 1.86, m	2.71, m	3.24, m	1.98, m; 1.43, m	2.02, m; 1.51, m	1.83, m; 1.55, m	2.05, m; 1.52, m	2.04, m; 1.54, m	1.97, m; 1.51, m	2.31, m; 1.78, m	2.27, m; 1.76, m	1.72, m; 1.22, m
2	1.74, m; 1.45, m	2.11, m; 1.91, m	2.67, m	1.67, m	1.95, m	1.26, m; 2.20, m	1.95, m	1.78, m; 1.66, m	1.70, m	2.34, m; 2.03, m	2.76, m; 2.35, m	1.69, m; 1.58, m
3				3.25, dd (11.5, 4.0)	3.46, d (7.7)		3.46, d (7.8)	3.27, dd (11.6, 4.2)	4.50, dd (11.7, 4.3)			3.24, dd (11.5, 4.0)
4
5		2.67, m	2.50, m	1.08, m	1.29, m	2.23, m	1.27, m	1.16, m		1.54, m	1.27, m
6	2.13, m; 1.97, m	2.31, m	2.24, m	2.08, m	2.18, m	2.56, m	2.17, m	2.09, m	2.14, m	2.21, m; 2.07, m	2.17, m; 2.10, m	1.67, m; 1.53, m
7	2.86, m; 2.32, m	5.40, d (5.3)	4.69, t (7.9)	5.48, d (5.5)	5.57, d (4.7)	5.39, br s	5.57, d (5.0)	5.58, d (6.5)	5.85, d (6.5)	5.56, d (6.6)	5.90, d (6.1)	1.72, m; 1.61, m
8
9
10	3.11, d (9.2)
11				5.31, d (5.7)	5.41, d (4.7)	5.33 br s	5.41, d (5.0)	5.67, d (5.5)	5.31 d (6.0)	5.23 br s	5.39, d (5.4)	2.04, m
12	2.71, d (16.0); 2.54, d (16.0)	2.83, d (18.3); 2.60, d (18.3)	2.89, d (16.3); 2.78, d (16.3)	2.08, m; 2.25, m	2.35, d (16.0); 2.20, d (16.0)	2.19, m; 2.26, m	2.18, d (18.0); 2.42, d (18.0)	3.48, br s	2.35, m; 2.04, m	4.29, s	2.35, m; 2.07, m	1.63, m
13
14
15	2.91, m; 1.73, m	4.68, m	3.08, m; 1.70, m	1.64, m; 1.43, m	1.75, m; 1.45, m	1.64, m; 1.33, m	1.70, m; 1.49 m	1.65, m; 1.46, m	4.27, s	1.75, m; 1.38, m	4.29, t (6.9)	1.75, m
16	2.10, m; 1.48, m	2.07, m	1.82, m; 1.70, m	1.42, m; 2.10, m	2.28, m; 1.51, m	2.04, m	2.22, m; 1.51, m	2.06, m; 1.36, m	2.01, m; 1.77, m	2.08, m; 1.59, m	2.01, m; 1.78, m	2.02, m
17	1.80, m	1.92, m	2.20, m	1.39, m	2.40, m	1.97, m	2.45, m	2.56, m	2.07, m	2.15, m	2.08, m	1.89, m
18	0.81, s	0.90, s	1.32, s	0.57, s	0.74, s	0.64, s	0.73, s	0.56, s	0.60, s	0.57, s	0.64, s	0.71, s
19	1.50, s	5.21, s	1.43, s	0.97, s	1.10, s	0.97, s	1.09, s	0.97, s	1.00, s	1.23, s	1.19, s	0.98, s
20	1.48, m	1.27, m		1.94, m	1.68, m	1.44, dd (6.8, 3.4)	1.69, m	1.44, m	1.38, m	1.57, m	1.40, m	1.49, m
21	0.92, d (6.0)	0.75, d (6.0)	1.34, s	0.91, d (6.0)	1.21, d (6.0)	0.89, d (6.8)	1.24, d (6.5)	0.93, d (6.8)	0.87, d (6.3)	1.09, d (6.3)	0.88, d (6.6)	0.87, d (6.8)
22	1.35, m; 1.86, m	1.31 m; 1.82, m	2.65, m; 2.45, m	3.99, d (9.9)	4.43, d (5.7)	3.68, m	4.11, m	3.70, m	3.59, m	3.76, m	3.60, m	4.30, m
23	2.44, m; 2.30, m	2.38, m; 2.24, m	2.06, m; 1.79, m	1.48, m; 1.78, m	2.05, m; 1.45, m	2.00, m; 2.29, m	1.75, m; 1.46, m	2.29, m	2.28, m; 1.99, m	2.03, m	2.28, m; 1.99, m	5.61, br s
24				4.29, d (9.7)	4.10, d (9.5)	5.14, t (7.0)	6.05, t (6.7)	5.16, t (7.2)	5.13, t (7.2)	5.14, t (7.3)	5.13, t (6.7)	5.16 br s
25
26				4.83, s; 5.00, s	1.53, s	1.64, s	4.32, s	1.64, s	1.63, m	1.64, s	1.64, s	1.28, s
27				1.74, s	1.56, s	1.72, s	1.87, s	1.73, s	1.73, s	1.73, s	1.73, s	1.28, s (overlap)
28		4.99, s; 4.87, s	4.96, s; 5.03, s	0.88, s	1.13, s	4.64, s; 4.67, s	1.20, s	1.01, s	0.88, s	1.08, s	1.08, s	0.80, s
29		1.66, s	1.80, s	1.00, s	1.20, s	1.66, s	1.12, s	0.89, s	0.94, s	1.12, s	1.12, s	1.00, s
30	1.11, s	1.40, s	1.28, s	0.91, s	0.96, s	0.85, s	0.97, s	1.07, s	0.96, s	0.95, s	0.96, s	0.92, s
OCH3		3.60, s						3.33, s				3.16, s
C̲H̲3̲CO									2.05, s

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Table 2 The ¹³C-DEPT data of compounds 1–12

Position	1^a	1a^a	2^b	3^b	4^a	5^b	6^a	7^b	8^a	9^a	10^a	11^a	12^a
	^13C	^13C	^13C	^13C	^13C	^13C	^13C	^13C	^13C	^13C	^13C	^13C	^13C
a Measured in CDCl3.b Measured in C5D5N. ^1H and ^13C NMR spectra (δ) were measured at 600 (150) MHz for 1, 4, 6, 8–12; at 500 (125) MHz for 5 and 7; at 400 (100) MHz for 2 and 3. The assignments were based on COSY, HSQC, and HMBC experiments.
1	32.0 CH2	32.0 CH2	28.0 CH2	32.6 CH2	35.4 CH2	36.3 CH2	34.2 CH2	36.3 CH2	35.6 CH2	35.6 CH2	36.5 CH2	36.5 CH2	35.5 CH2
2	29.9 CH2	29.9 CH2	26.8 CH2	30.8 CH2	27.7 CH2	28.7 CH2	28.3 CH2	28.7 CH2	27.5 CH2	24.5 CH2	34.6 CH2	34.7 CH2	27.8 CH2
3	177.4 C	177.4 C	170.8 C	176.4 C	78.9 CH	78.1 CH	178.3 C	78.0 CH	78.7 CH	80.5 CH	216.9 C	216.7 C	78.9 CH
4			143.6 C	146.4 C	38.6 C	39.3 C	149.1 C	39.3 C	38.6 C	35.3 C	47.6 C	47.4 C	38.8 C
5	90.7 C	90.7 C	48.3 CH	44.9 CH	49.0 CH	49.8 CH	50.6 CH	49.7 CH	48.8 CH	49.0 CH	50.6 CH	50.4 CH	50.3 CH
6	27.0 CH2	27.0 CH2	38.6 CH2	35.3 CH2	22.9 CH2	23.5 CH2	28.3 CH2	23.5 CH2	22.9 CH2	22.7 CH2	23.6 CH2	23.5 CH2	18.2 CH2
7	33.9 CH2	33.9 CH2	74.2 CH	67.7 CH	120.3 CH	120.9 CH	117.5 CH	120.9 CH	123.3 CH	121.0 CH	121.3 CH	121.0 CH	27.6 CH
8	179.3 C	179.3 C	168.0 C	164.9 C	142.5 C	143.0 C	141.6 C	143.0 C	142.6 C	140.8 C	141.8 C	140.9 C	134.2 C
9	133.0 C	133.0 C	125.2 C	137.2 C	145.8 C	146.5 C	136.9 C	146.5 C	148.6 C	145.7 C	145.5 C	144.6 C	134.4 C
10	51.8 CH	51.8 CH	80.8 C	41.2 C	37.3 C	37.8 C	38.3 C	37.8 C	37.6 C	37.2 C	36.9 C	37.2 C	36.9 C
11	198.1 C	198.1 C	196.9 C	199.8 C	116.2 CH	116.6 CH	120.2 C	116.6 CH	113.8 CH	116.2 CH	122.0 C	117.0 CH	26.4 CH2
12	49.8 CH2	49.8 CH2	49.6 CH2	51.9 CH2	37.8 CH2	38.3 CH2	38.8 CH2	38.2 CH2	82.6 CH	38.5 CH2	73.9 CH	38.5 CH2	30.9 CH2
13	50.0 C	49.9 C	46.6 C	47.3 C	43.7 C	44.0 C	44.1 C	44.0 C	47.3 C	44.1 C	49.1 C	44.1 C	44.4 C
14	50.1 C	50.1 C	52.4 C	51.9 C	50.2 C	50.7 C	50.1 C	50.7 C	49.7 C	51.9 C	51.5 C	51.9 C	39.8 C
15	29.7 CH2	29.7 CH2	70.5 CH	31.9 CH2	31.4 CH2	31.9 CH2	30.8 CH2	31.9 CH2	32.5 CH2	74.7 CH	31.5 CH2	74.6 CH	30.8 CH2
16	27.5 CH2	27.5 CH2	38.6 CH2	22.2 CH2	27.2 CH2	27.7 CH2	27.2 CH2	27.8 CH2	26.8 CH2	39.5 CH2	27.2 CH2	39.4 CH2	20.9 CH2
17	49.5 CH	49.5 CH	49.0 CH	53.2 CH	42.2 CH	47.6 CH	47.5 CH	47.8 CH	40.1 CH	45.2 CH	49.6 CH	45.2 CH	46.7 CH
18	17.3 CH3	17.2 CH3	17.7 CH3	19.2 CH3	15.3 CH3	16.0 CH3	16.3 CH3	16.0 CH3	16.7 CH3	15.8 CH3	10.3 CH3	15.9 CH3	15.7 CH3
19	27.8 CH3	27.8 CH3	74.5 CH	22.2 CH3	22.6 CH3	23.1 CH3	21.9 CH3	23.0 CH3	22.5 CH3	22.8 CH3	21.8 CH3	22.1 CH3	19.4 CH3
20	35.7 CH	35.7 CH	35.9 CH	87.7 C	46.9 CH	42.4 CH	40.2 CH	41.7 CH	40.4 CH	40.1 CH	38.8 CH	40.1 CH	41.9 CH
21	18.4 CH3	18.2 CH3	17.7 CH3	25.9 CH3	12.1 CH3	12.7 CH3	11.6 CH3	12.4 CH3	10.6 CH3	11.7 CH3	11.9 CH3	11.7 CH3	11.9 CH3
22	31.0 CH2	31.1 CH2	31.1 CH2	28.0 CH2	74.4 CH	73.6 CH	73.2 CH	72.8 CH	73.6 CH	73.2 CH	74.5 CH	73.2 CH	73.7 CH
23	31.2 CH2	31.3 CH2	31.1 CH2	31.9 CH2	40.2 CH2	36.8 CH2	34.2 CH2	34.9 CH2	34.1 CH2	34.2 CH2	35.0 CH2	34.3 CH2	132.6 CH
24	179.0 C	174.6 C	174.2 C	177.1 C	76.8 CH	79.6 CH	120.8 CH	123.2 CH	120.9 CH	120.7 CH	120.7 CH	120.6 CH	134.7 CH
25					147.6 C	72.4 CH	134.5 C	137.4 C	134.0 C	134.8 C	134.9 C	134.9 C	74.7 C
26					110.5 CH2	25.8 CH3	18.2 CH3	68.0 CH2	17.9 CH3	18.0 CH3	18.1 CH3	18.1 CH3	25.8 CH3
27					17.9 CH3	25.9 CH3	26.0 CH3	14.3 CH3	25.7 CH3	25.9 CH3	26.0 CH3	25.8 CH3	26.0 CH3
28			115.1 CH2	115.6 CH2	15.7 CH3	16.6 CH3	111.8 CH2	16.6 CH3	28.1 CH3	27.9 CH3	25.3 CH3	25.3 CH3	15.4 CH3
29			22.4 CH3	22.2 CH3	28.0 CH3	28.8 CH3	21.9 CH3	28.8 CH3	15.7 CH3	16.9 CH3	22.6 CH3	22.4 CH3	27.9 CH3
30	23.8 CH3	23.9 CH3	19.1 CH3	27.8 CH3	25.5 CH3	26.3 CH3	23.9 CH3	25.9 CH3	28.5 CH3	17.1 CH3	24.9 CH3	17.0 CH3	24.3 CH3
OCH3		51.8 CH3	51.3 CH3						55.2 CH3				50.4 CH3
C̲H̲3̲CO										21.3 CH3
										170.9 C

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The ROESY correlation of H-7/H₃-30 deduced 7-OH to be β. The absolute configuration of C-20 was determined to be S on the basis of the X-ray crystal diffraction analysis (Fig. 6). Accordingly, the structure of 3 was established to be (20S)-7β-hydroxy-11-oxo-3,4-seco-25,26,27-trinorlanosta-4(28),8-dien-3-oic-20 → 24 lactone and named as cochlearic acid A (3).

Fig. 6 X-ray structures of compounds 3 and 4.

Compound 4 was isolated as colorless needle crystals with a molecular formula of C₃₀H₄₈O₃ based on HREIMS ion at m/z 456.3612 [M]⁺ (calcd 456.3603). The 1D NMR spectroscopic data (Tables 1 and 2) showed the following signals: six singlet methyls (δ 1.74, δ 1.00, δ 0.97, δ 0.91, δ 0.87 and δ 0.56); one doublet methyl (δ 0.91, d, J = 6.0 Hz); eight methylenes, including one sp² methylene (δ 4.83, s; δ 5.00, s); eight methines, including three oxymethines (δ 3.25, dd, J = 11.5 and 4.0 Hz; δ 3.99, d, J = 9.9 Hz; δ 4.29, d, J = 9.7 Hz) and two sp² methines [δ 5.48 (d, J = 5.5 Hz), δ 5.31 (d, J = 5.7 Hz)]; as well as seven quaternary carbons (three sp² quaternary carbons). These data indicated that the structural skeleton of compound 4 was a lanosta-7,9(11)-dien-3-ol, which was supported by the 2D NMR spectra (Fig. 4).

The ¹H–¹H COSY correlations between H₃-21 and H-20, between H-22 (δ 3.99, d, J = 9.9 Hz) and H₂-23, between H₂-23 and H-24 (δ 4.29, d, J = 9.7 Hz), together with the HMBC correlations of H₃-21 to C-20, C-17 and C-22 (δ 74.4), of H-22 to C-20, C-24 (δ 76.8) and C-25 (δ 147.6), of H-23 to C-22, C-24, C-25, and C-26 (δ 110.5), of H₃-27 to C-24, C-25 and C-26, indicated that compound 4 had the same side chain moiety (22,24-dihydroy-25-ene) as inontsutriol D.¹⁹ The stereocenters (C-22 and C-24) of the side chain moiety in 4 were confirmed by the X-ray crystal diffraction analysis. A single X-ray crystallographic analysis (Cu Kα radiation, Fig. 6) of 4 assigned C-22 and C-24 as S and R, respectively. Thus, the structure of 4 was determined to be lanosta-7,9(11),25-trien-3β,22S,24R-triol and named as ganodercochlearin D (4).

Compound 5 was found to possess the molecular formula C₃₀H₅₀NaO₃, as evidenced by its positive HRESIMS. Its 1D NMR data (Tables 1 and 2) closely resembled those of 4. The main difference observed in the ¹³C NMR spectra was that the signals corresponding to the terminal double bond (δ 147.6, δ 110.5) at C-25 and C-26 were replaced by an oxymethine (δ 72.4, CH) and a methyl (δ 25.8 CH₃), respectively. This difference was confirmed by the HMBC correlations of H-24 (δ 4.10, d, J = 9.5 Hz) with C-25 (δ 72.4), C-26 (δ 25.8) and C-27 (δ 25.9), of H₃-26 (δ 1.53, s) and H₃-27 with C-24 (δ 79.6) and C-25. Therefore, the structure of 5 was established to be lanosta-7,9(11)-3β,22S,24R,25-tetraol, named ganodercochlearin E (5).

Based on the positive HREIMS of 6, its molecular formula C₃₀H₄₆O₃ was deduced, containing eight degrees of unsaturation. Its ¹³C NMR spectrum showed thirty carbon resonances. Among them, the signals at δ 117.5, δ 141.6, δ 136.9 and δ 120.2 were characterized for the conjugated double bond (Δ⁷ and Δ^9,11). Meanwhile, the signals at δ 149.1, C; δ 111.8, CH₂ and δ 178.3, C were assigned as the terminal double bond (C-4, C-28) and the carboxyl group (C-3) on the basis of the DEPT, HSQC and HMBC spectra. These informations indicated that compound 6 had the same cyclic structure as 16-deoxyporicoic acid B²⁰ with a 3,4-seco-7,9(11)-dien-lanostane skeleton. Additionally, one double bond signals (δ 120.8, CH; δ 134.5, C) and one oxymethine signal (δ 73.2, CH) were also observed in the ¹³C NMR spectrum of 6. Moreover, the HMBC correlations of H₃-21 (δ 0.89, d, J = 6.8 Hz) with C-17 (δ 47.5), C-20 (δ 40.2), and C-22 (δ 73.2), of H-22 (δ 3.68, m) with C-17, C-20, C-21, C-23, and C-24, together with the ¹H–¹H COSY correlations of H₃-21/H-20/H-22/H-23/H-24, confirmed that the side chain of compound 6 resembled that of compound 5, except for the replacement of one oxymethine (C-24) and one oxyquaternary carbon (C-25) in 5 by two sp² olefinic carbons in 6. Accordingly, the structure of 6 was assigned as (22S)-hydroxy-3,4-seco-lanosta-4(28),7,9(11),24-tetraen-3-oic acid and named as cochlearic acid B (6).

The molecular formula of ganodercochlearin F (7) was deduced from the HRESIMS ion at m/z 479.3493 [M + Na]⁺ (calcd 479.3501). Its 1D NMR spectroscopic data (Tables 1 and 2) and the DEPT analysis of 7 showed that this compound bore a close resemblance to compound 5. However, the presence of the signals at δ 72.8 (CH), δ 123.2 (CH), and δ 137.4 (C), conjugated with a set of 2D NMR correlations indicated that compound 7 had the same side chain moiety as compound 6. Further comparison of the ¹³C-DEPT data between 6 and 7 displayed that an oxymethylene in 7 replaced a methyl in 6. The HMBC correlations (Fig. 4) of the oxymethylene protons (δ 4.32, s) with C-24, C-25 and C-27 were observed, which indicated that the hydroxyl group was linked to C-26.

The geometry of Δ²⁴ were determined to be Z on the basis of the ROESY correlation of H-24/H₂-26. Thus, the structure of 7 was established to be (22S)-lanosta-7,9(11),24Z-triene-3β,22,26-triol and named as ganodercochlearin F (7).

Ganodercochlearin G (8) possessed a molecular formula of C₃₁H₅₀O₃ as determined by HREIMS (m/z 470.3755 [M]⁺, calcd 470.3760). The signals in the ¹H and ¹³C NMR spectroscopic data (Tables 1 and 2) of 8 strikingly matched those of 7, indicating that they shared the same backbone. The major difference were the absence of the oxymethylene and the presence of an oxymethine and a methoxyl group in 8. The key HMBC correlations of H₃-18 with the C-13, C-14, C-17, and the oxymethine (δ 82.6); of the oxymethine proton (δ 3.48, br s) with C-9, C-11, C-13 and C-14, as well as of the methoxyl protons (δ 3.33, s) with C-12, which indicated that the methoxyl group was linked to C-12.

12β-OMe was deduced by the ROESY correlation of H-12/H₃-30. Therefore, the structure of 8 was assigned as (22S)-12β-methoxyl-lanosta-7,9(11),24-triene-3β,22-diol, named ganodercochlearin G (8).

The molecular formula of ganodercochlearin H (9) was determined to be C₃₂H₅₀O₄ from the HREIMS ion at m/z 498.3715 [M]⁺, calcd 498.3709. The 1D NMR data (Tables 1 and 2) of 9 obviously showed the presence of an acetyl (δ 2.05, s; δ 21.3, CH₃; δ 170.9, C). On the basis of the HMBC correlation of H-3 with the acetyl carbonyl carbon (δ 170.9), this acetoxyl group was located at C-3. In addition, by comparison of the 1D NMR spectra of 9 and 7, an oxymethine (δ 74.7) was observed in 9 instead of the oxymethylene in 7. The obvious HMBC correlations of H₃-30 with C-13, C-14, C-8, and the oxymethine, of the oxymethine proton (δ 4.27, s) with C-13, C-14, C-30, C-16, and C-17 indicated that the oxymethine was located at C-15. Moreover, the ROESY correlation of H-15/H₃-18 assigned 15-OH as α-orientation. Accordingly, the structure of 9 was established to be (22S)-3β-acetoxyl-lanosta-7,9(11),24-trien-15α,22-diol, named ganodercochlearin H (9).

Ganodercochlearin I (10) and ganodercochlearin J (11) gave the same molecular formula, C₃₀H₄₆O₃ (HREIMS). Both of them had thirty carbon resonances, including eight methyls, six methylenes, eight methines (three sp² methines and two oxymethines), and eight quaternary carbons (one carbonyl and three sp² quaternary carbons). These signals showed that their backbone was 22-hydroxy-lanosta-7,9(11),24-triene-3-one. This was supported by the 2D NMR analysis. Thus, the only significant difference between 10 and 11 could be the position of another oxymethine (δ 73.9 for 10; δ 74.6 for 11). The HMBC correlations of the oxymethine proton (δ 4.29, s) in 10 with C-11, C-13, and C-18, as well as of the oxymethine (δ 4.29, t, J = 6.9 Hz) in 11 with C-13, C-14, C-30, C-16, and C-17 confirmed that C-12 in 10 and C-15 in 11 were connected to the hydroxyl group, respectively. Similarly, on the basis of the ROESY correlations of H-12/H₃-30 and of H-15/H₃-18, 12-OH and 15-OH were assigned as β-orientation and α-orientation, respectively. Therefore, the structures of 10 and 11 were determined to be (22S)-12β,22-dihydroxy-lanosta-7,9(11),24-triene-3-one and (22S)-15α,22-dihydroxy-lanosta-7,9(11),24-triene-3-one, named ganodercochlearins I and J (10 and 11).

Ganodercochlearin K (12) possessed a molecular formula of C₃₁H₅₂O₃ as determined by HREIMS ion at m/z 472.3924 [M]⁺ (calcd 472.3916). The 1D NMR data (Tables 1 and 2) of 12 were similar to those of ganodercochlearin C (13), except for the absence of the conjugated double bonds (Δ⁷ and Δ^9,11) and the presence of a double bond between C-8 and C-9, which was confirmed by the HMBC correlations of H₂-6 and H₂-7 with C-8 (δ 134.2), of H₂-11 with C-8 (δ 134.2, C), C-9 (δ 134.4, C) and C-10. The structure of 12 was finally assigned as (22S)-25-methoxyl-lanosta-8,23-dien-3β,22-diol and named as ganodercochlearin K (12).

The six known lanostane triterpenoids were identified by comparison of experimental and literature spectroscopic data as ganodercochlearin C (13),¹³ ganodermadiol (14),²¹ polycarpol (15),²² ganoderic acid C2 (16),²³ 3β,15α,22β-trihydroxylanosta-7,9(11),24-trien-26-oic acid (17),²⁴ and ganoderiol F (18).²⁵

Ganocochlearic acid A (1) is a hexnorlanostane triterpenoid possessing a γ-lactone ring (A ring) and a five-membered carbon ring (B ring). A ring is connected with B ring through a single bond (C-5–C-10). Detailed comparison of the structures between fornicatin B and 1, it is found that they have the same C, D rings and the side chain moiety. Thus, we deduce that compound 1 could derive from fornicatin B via the degradation of C-4, C-28 and C-29, Baeyer–Villiger reaction and Wagner–Meerwein rearrangement. A possible biosynthetic pathway is proposed and shown in Scheme 1.

Scheme 1 The plausible biosynthetic pathway for 1.

Cytotoxic effects of compounds 1–13 were tested against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW480) by MTS method. Compounds 7–9, 11–13 had moderate cytotoxic activities against five cell lines with IC₅₀ values ranging from 8 to 30 μM (Table 3). Compound 4 exhibited cytotoxic activity against MCF-7 and SW480 cancer cells with respective IC₅₀ value of 9.15 and 15.9 μM, while the positive control, cisplatin (DDP), showed the activity toward these cancer cells with IC₅₀ values of 12.7 and 12.5 μM. Compound 10 displayed weak inhibition against HL-60, SMMC-7721, A-549 and MCF-7 cells (IC₅₀: 32.9, 28.1, 38.5 and 25.1 μM). In the other hand, 3,4-seco-lanostane triterpenoids (1–3, 6) exhibited no inhibitory effects against all the test human tumor cells. Ganodercochlearin D (4) and ganodercochlearin E (5) had the same structure, nevertheless, compound 5 show weak cytotoxicity with IC₅₀ values of >40 μM, suggesting that the terminal double bond in the side chain of 4 could be the key structural feature. The results are shown in Table 3.

Table 3 The cytotoxicity data of compounds 4, 7–13^a. (IC₅₀: μM)

Compounds	HL-60	SMMC-7721	A-549	MCF-7	SW480
a DDP = cis-dichlorodiamineplatinum(II) or cisplatin, a positive control.
4	6.91 ± 1.02	10.5 ± 2.11	12.1 ± 2.72	9.15 ± 1.23	15.9 ± 3.11
7	16.3 ± 3.34	17.2 ± 3.63	16.5 ± 3.15	16.8 ± 3.43	17.8 ± 4.01
8	13.9 ± 2.84	17.0 ± 3.36	15.8 ± 3.24	17.4 ± 3.77	17.1 ± 3.72
9	16.2 ± 3.32	16.8 ± 3.21	14.7 ± 2.83	15.8 ± 3.45	15.9 ± 3.45
10	32.9 ± 4.32	28.1 ± 4.25	38.5 ± 4.83	25.1 ± 4.26	>40
11	14.7 ± 2.32	17.6 ± 3.22	26.8 ± 4.53	19.3 ± 3.91	22.3 ± 4.10
12	13.6 ± 2.71	16.3 ± 3.02	16.9 ± 3.03	21.6 ± 3.81	19.2 ± 3.72
13	19.3 ± 3.81	10.1 ± 2.12	8.84 ± 1.47	13.4 ± 2.22	15.9 ± 2.83
DDP	1.14 ± 0.23	4.63 ± 0.55	4.68 ± 0.56	12.7 ± 2.31	12.5 ± 2.34

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3,4-seco-Norlanostane and 7,9(11),24-trien-lanostane triterpenoids were widely distributed in the fruiting bodies of G. cochlear. Indeed, these kinds of triterpenoids were also found in other Ganoderma species and exhibited various bioactivities. 3,4-seco-Nortriterpenoids, fornicatins A, D, and F, lowered the ALT and AST levels in HepG2 cells treated with H₂O₂.¹³ Meanwhile, 7,9(11),24-trien-lanostane triterpenoids showed antitumor, antiviral, antifungal (Fitoterapia, 2003, 74, 375), and antimycobacterial activities.^26–30 Previous and present researches indicated that lanostane triterpenoids play an important role in explaining the wide application of Ganoderma, and deserve further research.

Conclusions

In conclusion, twelve new lanostane triterpenoids, including a hexanorlanostane triterpenoid (1), three 3,4-seco-lanostane triterpenoids (2, 3, 6), and nine 7,9(11),24-trien-lanostane triterpenoids (4, 5, and 7–12), along with six known analogues (13–18) were isolated from the fruiting bodies of G. cochlear. Among of them, compounds 4, 7–13 showed cytotoxic activities.

Experimental section

General experimental procedures

Optical rotations were obtained with a Jasco P-1020 polarimeter. UV spectra was recorded using Shimadzu UV2401PC spectrophotometer. ¹H and ¹³C NMR spectra were measured on Bruker AV-400 and DRX-500 instruments (Bruker, Zurich, Switzerland) using TMS as internal standard for chemical shifts. Chemical shifts (δ) are expressed in ppm with reference to the TMS resonance. ESIMS and HRTOF-ESIMS data were recorded on an API QSTAR Pulsar spectrometer. EIMS and HRTOF-EIMS data were taken on an Waters Auto Spec Premierp 776 spectrometer (America, Waters). Infrared spectra were recorded on a Bruker Tensor-27 instrument by using KBr pellets. An Agilent 1100 series instrument equipped with an Agilent ZORBAX SB-C18 column (5 μm, 9.4 mm × 250 mm) was used for high-performance liquid chromatography (HPLC) analysis.

Fungal material

The fruiting bodies of G. cochlear were purchased in July 2013 from Traditional Chinese Medicine Market in Kunming. The mushroom was identified by Prof. Liu Peigui, Kunming Institute of Botany, Chinese Academy of Science. A specimen (no. 13071501) was deposited in the Herbarium of the Department of Taxonomy, Kunming Institute of Botany, Chinese Academy of Sciences.

Extraction and isolation

G. cochlear (68 kg) mushrooms were chipped and extracted with 95% EtOH under reflux three times (three hours per time). The combined ethanol extracts were evaporated under reduced pressure. The residue was suspended in H₂O and extracted with EtOAc. The volume of the combined EtOAc extracts was reduced to one-third under reduced pressure. The residue was fractionated by macroporous resin (D-101; MeOH–H₂O, 50 : 50, 70 : 30 and 90 : 10, v/v): fractions I–III.

We taken out 100 g of fraction II and used the reversed-phase (RP-18) silica gel chromatography to treat it and obtain eight subfractions (MeOH–H₂O: 50 : 50 → 80 : 20, Fr. II-1 → Fr. II-8). Every fractions was treated by Sephadex G-25 (MeOH) to three parts. Fr. II-4-2 was purified by preparative TLC (P-TLC, CHCl₃–MeOH–acetic acid: 80 : 1 : 1) to give compounds 1 (10 mg) and 2 (12 mg). Compound 3 (22 mg) was obtained from Fr. II-4-3 by recrystallization (MeOH). Fr. II-6-2 was absorbed to column chromatography (CC, silica gel) and eluted with CHCl₃–acetone: 80 : 1, 60 : 1, 40 : 1 to give four subfractions (Fr. II-6-2a → Fr. II-6-2d). Fr. II-6-2b was purified by P-TLC (CHCl₃–MeOH: 50 : 1) to gain ganodermadiol (14, 13 mg) and polycarpol (15, 19 mg). Four subfractions (Fr. II-7-2a → Fr. II-7-2d) were obtained from Fr. II-7-2 by CC (silica gel, CHCl₃–acetone, step gradient). Fr. II-7-2a was treated by the recrystallization to obtain compound ganodermatriol (18, 21 mg). Similarly, compound 5 (12 mg) was isolated from the Fr. II-7-2b by CC (silica gel). Compound 7 (7 mg) was obtained from the Fr. II-7-2c by P-TLC. Fr. II-8-2 was treated by the P-TLC to gain ganoderic acid C2 (16, 12 mg) and 3β,15α,22β-trihydroxylanosta-7,9(11),24-trien-26-oic acid (17, 23 mg).

Fraction III (1.5 kg) was treated by column chromatography (CC, silica gel; CHCl₃–MeOH step gradients) to give four subfractions (80 : 1, 50 : 1, 20 : 1 and 5 : 1, III-1–III-4). Fraction III-2 (50 g) was further fractionated by RP-18 to obtain ten subfractions (MeOH–H₂O: 50 : 50 → 80 : 20). On the basis of the TLC results, Fr. III-2-3 and Fr. III-2-6 were chosen for the further separation.

Fr. III-2-3 was separated by the RP-18 (MeOH–H₂O, 50 : 50 → 80 : 20, v/v) to obtain seven subfractions (Fr. III-2-3a → Fr. III-2-3g). Compound 6 (23 mg) was isolated from the Fr. III-2-3b, which was treated by the Sephadex G-25 (MeOH) and recrystallization (MeOH). Fr. III-2-3c was absorbed to Sephadex G-25 and eluted with MeOH to give four fractions. Fr. III-2-3c-2 was further treated by the preparative TLC (P-TLC, CHCl₃–MeOH: 50 : 1) to give compounds 4 (25 mg) and 10 (18 mg). Compound 8 (35 mg) was obtained from Fr. III-2-3 by CC (silica gel; CHCl₃–MeOH) and P-TLC. Fr. III-2-3f was further separated by reversed-phase HPLC (CH₃CN–H₂O, 85%) to afford compound 12 (24 mg) and ganodercochlearin C (13, 30 mg). By repeated CC, HPLC separation, and recrystallization (MeOH/H₂O), from fraction III-2-3g, compounds 9 (12 mg) and 11 (21 mg) were isolated. The spectroscopic data of the new compounds (1–12) are listed herein.

Ganocochlearic acid A (1). White powder, [α]²⁰_D +45.2 (c 0.04, MeOH); UV (MeOH) λ_max (log ε) nm: 258 (3.69), 202 (3.72) nm; IR (KBr) ν_max: 3439, 2969, 2934, 2881, 1709, 1671 cm⁻¹; ¹H and ¹³C-DEPT data: see Tables 1 and 2; ESIMS m/z 425 [M + Na]⁺, HREIMS m/z 402.2401 [M]⁺ (calcd for C₂₄H₃₄O₅, 402.2406).

Cochlate C (2). White powder, [α]²¹_D +91.0 (c 0.19, MeOH); UV (MeOH) λ_max (log ε) nm: 257 (3.92) nm; IR (KBr) ν_max: 3430, 2975, 1711, 1676 cm⁻¹; ¹H and ¹³C-DEPT data: see Tables 1 and 2; ESIMS m/z 509 [M + Na]⁺, HRESIMS m/z 509.2509 [M + Na]⁺ (calcd for C₂₈H₃₈NaO₇, 509.2515).

Cochlearic acid A (3). Colorless needles, [α]²¹_D +144.4 (c 0.19, MeOH); UV (MeOH) λ_max (log ε) nm: 257 (3.87), 227 (3.66) nm; IR (KBr) ν_max: 3443, 2987, 1706, 1656 cm⁻¹; ¹H and ¹³C-DEPT data: see Tables 1 and 2; ESIMS m/z 457 [M − H]⁻, HRESIMS m/z 481.2560 [M + Na]⁺ (calcd for C₂₇H₃₈NaO₆, 481.2561).

Crystal data for 3. C₂₇H₃₈O₆·H₂O, M = 476.59, triclinic, a = 8.1696(8) Å, b = 11.5686(9) Å, c = 13.0762(8) Å, α = 89.962(4)°, β = 89.807(2)°, γ = 87.127(4)°, V = 1234.28(17) ų, T = 100(2) K, space group P1, Z = 2, μ(CuKα) = 0.742 mm⁻¹, 9504 reflections measured, 4501 independent reflections (R_int = 0.0409). The final R₁ values were 0.0697 (I > 2σ(I)). The final wR(F²) values were 0.1913 (I > 2σ(I)). The final R₁ values were 0.0736 (all data). The final wR(F²) values were 0.1976 (all data). The goodness of fit on F² was 1.087. Flack parameter = −0.2(3).

Ganodecochlearin D (4). Colorless needles, [α]²⁵_D +46.3 (c 0.23, MeOH); UV (MeOH) λ_max (log ε) nm: 242 (4.18), 199 (3.80) nm; IR (KBr) ν_max: 3443, 2987, 1706, 1656 cm⁻¹; ¹H and ¹³C-DEPT data: see Tables 1 and 2; EIMS m/z 456 [M]⁺, HREIMS m/z 456.3612 [M]⁺ (calcd for C₃₀H₄₈O₃, 456.3603).

Crystal data for 4. C₃₀H₄₈O₃·CH₄O, M = 488.73, monoclinic, a = 6.2648(3) Å, b = 33.5254(16) Å, c = 6.9559(3) Å, α = 90.00°, β = 107.704(2)°, γ = 90.00°, V = 1391.76(11) ų, T = 100(2) K, space group P21, Z = 2, μ(CuKα) = 0.580 mm⁻¹, 11 475 reflections measured, 4453 independent reflections (R_int = 0.0469). The final R₁ values were 0.0597 (I > 2σ(I)). The final wR(F²) values were 0.1645 (I > 2σ(I)). The final R₁ values were 0.0598 (all data). The final wR(F²) values were 0.1646 (all data). The goodness of fit on F² was 1.089. Flack parameter = 0.0(3). The Hooft parameter is −0.04(7) for 2035 Bijvoet pairs.

Ganodercochlearin E (5). White powder; [α]²¹_D +102.5 (c 0.12, MeOH); UV (MeOH) λ_max (log ε) nm: 245 (4.12) nm; IR (KBr) ν_max: 3385, 2973, 2961, 2843, 1465 cm⁻¹; ¹H and ¹³C-DEPT data: see Tables 1 and 2; ESIMS m/z 497 [M + Na]⁺, HRESIMS m/z 497.3603 [M + Na]⁺ (calcd for C₃₀H₅₀NaO₄, 497.3607).

Cochlearic acid B (6). White powder; [α]²⁶_D +19.6 (c 0.12, MeOH); UV (MeOH) λ_max (log ε) nm: 240 (4.06) and 202 (4.12) nm; IR (KBr) ν_max: 3385, 2973, 2961, 2843, 1465 cm⁻¹; ¹H and ¹³C-DEPT data: see Tables 1 and 2; EIMS m/z 454 [M]⁺, HREIMS m/z 454.3441 [M]⁺ (calcd for C₃₀H₄₆O₃, 454.3447).

Ganodercochlearin F (7). White powder; [α]²¹_D +34.8 (c 0.14, MeOH); UV (MeOH) λ_max (log ε) nm: 245 (3.98) nm; IR (KBr) ν_max: 3385, 2973, 2961, 2843, 1465 cm⁻¹; ¹H and ¹³C-DEPT data: see Tables 1 and 2; ESIMS m/z 497 [M + Na]⁺, HRESIMS m/z 479.3493 [M + Na]⁺ (calcd for C₃₀H₄₈NaO₃, 479.3501).

Ganodercochlearin G (8). White powder; [α]²⁵_D +65.2 (c 0.12, MeOH); UV (MeOH) λ_max (log ε) nm: 246 (3.03) and 201 (3.89) nm; IR (KBr) ν_max: 3385, 2973, 2961, 2843, 1465 cm⁻¹; ¹H and ¹³C-DEPT data: see Tables 1 and 2; EIMS m/z 470 [M]⁺, HREIMS m/z 470.3755 [M]⁺ (calcd for C₃₁H₅₀O₃, 470.3760).

Ganodercochlearin H (9). White powder; [α]²⁵_D +56.0 (c 0.21, MeOH); UV (MeOH) λ_max (log ε) nm: 242 (4.05) and 201 (3.88) nm; IR (KBr) ν_max: 3385, 2973, 2961, 2843, 1465 cm⁻¹; ¹H and ¹³C-DEPT data: see Tables 1 and 2; EIMS m/z 498 [M]⁺, HREIMS m/z 498.3715 [M]⁺ (calcd for C₃₂H₅₀O₄, 498.3709).

Ganodercochlearin I (10). White powder; [α]²⁵_D −5.9 (c 0.08, MeOH); UV (MeOH) λ_max (log ε) nm: 245 (4.11) and 201 (3.94) nm; IR (KBr) ν_max: 3385, 2973, 2961, 2843, 1465 cm⁻¹; ¹H and ¹³C-DEPT data: see Tables 1 and 2; EIMS m/z 454 [M]⁺, HREIMS m/z 454.3440 [M]⁺ (calcd for C₃₀H₄₆O₃, 454.3447).

Ganodercochlearin J (11). White powder; [α]²⁵_D +90.3 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) nm: 242 (4.24) and 201 (4.17) nm; IR (KBr) ν_max: 3385, 2973, 2961, 2843, 1465 cm⁻¹; ¹H and ¹³C-DEPT data: see Tables 1 and 2; EIMS m/z 454 [M]⁺, HREIMS m/z 454.3441 [M]⁺ (calcd for C₃₀H₄₆O₃, 454.3447).

Ganodercochlearin K (12). White powder; [α]²⁵_D +24.9 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) nm: 222 (3.41) and 200 (4.12) nm; IR (KBr) ν_max: 3385, 2973, 2961, 2843, 1465 cm⁻¹; ¹H and ¹³C-DEPT data: see Tables 1 and 2; EIMS m/z 472 [M]⁺, HREIMS m/z 472.3924 [M]⁺ (calcd for C₃₁H₅₂O₃, 472.3916).

X-ray diffraction analysis of 3 and 4

The crystal structure of 3 and 4 were solved by direct method SHELXS-97 (Sheldrich, G. M. University of Gottingen; Gottingen, Germany, 1997) and the full-matrix least-squares deposited in the Cambridge Crystallographic Data Centre (deposition number: 963836 for 3; 1405439 for 4).†

Computational methods

Quantum chemical method was used to assign the absolute configuration of compound 1 by comparing the experimental and calculated electronic circular dichroism (ECD) spectra at time-dependent density functional theory (TDDFT). Conformational analysis was initially carried out by using Discovery Studio 4.1 Client conformational searching and molecular mechanics methods (MMFF94). The selected conformers were then optimized at the B3LYP/6-31+G(d,p) level in the gas phase by using the Gaussian09.³¹ Further ECD calculations were performed at the PCM-B3LYP/6-31+G(d,p) level in MeOH solution.

Cytotoxicity assay

The cytotoxicity of the tested compounds was evaluated using the MTS assay.³² Briefly, 1 × 10⁵ cells per mL from five human cancer cell lines, breast cancer MCF-7, hepatocellular carcinoma SMMC-7721, human myeloid leukemia HL-60, lung cancer A-549 cells, and colon cancer SW480 were seeded in 96-well plates. After 24 h of incubation, the cells were treated with or without test compounds or cisplatin (DDP, positive control) at a given concentrations for 48 h. MTS was then added to each well, and the plates were stored for 4 h. Absorbance was read at 490 nm. Cytotoxicity is reported as percent of control, where 100% is the viability of cells without test compounds.

Acknowledgements

The project was financially supported by the General Program of NSFC (No. 81172940) and Knowledge Innovation Program of the CAS (Grant No. KSCX2-YW-G-038, KSCX2-YW-R-194), Top Talents Program from the Department of Science and Technology in Yunnan Province (20080A007), as well as Foundation of State Key Laboratory of Phytochemistry and Plant Resources in West China (P2010-ZZ14).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. CCDC 963836 and 1405439. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra16796e

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