Meroterpenoids from the Fungus Ganoderma sinensis and First Absolute Configuration Clarification of Zizhine H.

Five new meroterpenoids, zizhines P-S and U (1-4,7), together with two known meroterpenoids (5 and 6) were isolated from Ganoderma sinensis. Their structures including absolute configurations were assigned by using spectroscopic, computational, and chemical methods. Racemics zizhines P and Q were purified by HPLC on chiral phase. Biological evaluation found that 4, 5 and 6 are cytotoxic toward human cancer cells (A549, BGC-823, Kyse30) with IC50 values in the range of 63.43-80.83 μM towards A549, 59.2 ± 2.73 μM and 64.25 ± 0.37 μM towards BGC-823, 76.28 ± 1.93 μM and 85.42 ± 2.82 μM towards Kyse30.

1. Introduction

Ganoderma fungi, worldwide known mushrooms, are mainly distributed in the tropical and subtropics regions [1]. Due to the medicinal significance of this genus, plenty of studies have been conducted in the last decades which reveal the presence of triterpenoids, polysaccharides, alkaloids, fatty acids, nucleotides, proteins, peptides, trace elements and sterols thereof [2,3,4,5]. A recent search by SciFinder according to “Ganoderma” found 25,917 related papers, indicating the importance of Ganoderma in the scientific community. Ganoderma is known in China as a mythic name “immortal herbs” [6], which has been used for the treatment of a wide range of diseases such as trachitis, chronic hepatitis, neurasthenia, dyspepsia, hypertension, and tumor [7]. Despite that Ganoderma embraces more than 30 fungal species, so far only G. lucidum and G. sinensis are recorded in Pharmacopoeia of People’s Republic of China (2015 edition). We have conducted an extensive and oriented study on meroterpenoids from G. lucidum and found structurally and biologically intriguing meroterpenoids. [8,9,10] Inspired by these previous findings, we have embarked an investigation on G. sinensis, leading to the isolation of (±)-sinensilactam A and biologically important meroterpenoids with a para-hydroxycinnamyl group in the structure [11,12,13]. As a continuous study on G. sinensis, the current investigation resulted in the characterization of five new meroterpenoids, zizhines P−S and U (1−4,7) along with two previously reported meroterpenoids (5 and 6) (Figure 1). To reveal their biological importance, all the isolates were evaluated for their cytotoxic properties against several human cancer cells.

Figure 1

The structures of compounds 1–7.

2. Results and Discussion

2.1. Structure Elucidation of the Compounds

Compound 1 was isolated as a yellowish gum. Its molecular formula was deduced as C21H26O7 by analysis of its positive HRESIMS, 13C-NMR, and DEPT spectra. The 1H-NMR spectrum of 1 (Table 1) gives a typical ABX spin system [δH 7.26 (1H, d, J = 3.0 Hz, H-3), 7.01 (1H, dd, J = 8.9, 3.0 Hz, H-5), 6.78 (1H, d, J = 8.9 Hz, H-6)]. The 13C-NMR and DEPT spectra (Table 1) show one methyl, five sp3 methylenes, two sp3 oxygenated methylenes, five sp2 methines, eight nonprotonated carbons (including a ketone group at δC 204.1 and a carboxyl group at δC 177.4). Within the context of meroterpenoids isolated from Ganoderma species, these data prompted us to associate 1 with meroterpenoid. Inspection of 2D NMR data of 1 reveals 1H−1H COSY correlations (Figure 2) of H2-4′/H2-5′/H-6′ (δH 5.47) and H2-8′/H2-9′/H-10′ (δH 5.38) and HMBC correlations (Figure 2) of H2-12′ (δH 3.91), H3-13′/C-10′, C-11′ (δC 136.4), H2-12′/C-13′, H2-14′ (δH 4.26, 4.21), H2-8′/C-6′, C-7′, H2-14′/C-8′, and Ha-5′/C-7′, indicating the presence of two isoprenyl moieties in the side chain of 1. Besides, the observation of HMBC correlations of H2-2′/C-1′ (δC 204.1), C-4′, C-15′ (δC 177.4), Hb-4′/C-3′ (δC 82.3), C-15′, H2-14′/C-3′ in 1 suggest the presence of another isoprenyl residue and a seven-membered ring via the formation of C-formed by C-3′-O-C-14′. The terpenoidal group is connected with the, benzene ring via C-2-C-1′ based on the HMBC observation of H-3, H2-2′/C-1′. As a result, the planar structure of 1 was assigned (Figure 1).

Table 1

1H and 13C-NMR data of 1 and 2.

No.	1 a		No.	2 b
	δH (J in Hz)	δC		δH (J in Hz)	δC
1		156.7	1		156.7
2		121.0	2		121.0
3	7.26, d (3.0)	115.9	3	7.26, d (2.9)	115.9
4		150.6	4		150.6
5	7.01, dd (8.9, 3.0)	126.1c	5	7.01, dd (8.9, 2.9)	126.1
6	6.78, d (8.9)	119.7	6	6.78, d (8.9)	119.7
1′		204.1	1′		204.0
2′	3.78, d (16.7)	45.4	2′	3.79, d (17.3)	45.4
	3.38, d (16.7)			3.34, d (17.3)
3′		82.3	3′		82.2
4′	2.35, overlap	36.2	4′	2.33, overlap	36.2
	2.21, m			2.20, m
5′	2.35, overlap	24.6	5′	2.33, overlap	24.6
	2.26, m			2.25, m
6′	5.47, br s	125.8d	6′	5.47, overlap	126.1
7′		141.1	7′		140.8
8′	1.97, m	36.7	8′	1.99, m	36.5
9′	2.11, m	27.5	9′	2.14, m	27.5
10′	5.38, t (7.1)	126.0 c,d	10′	5.47, overlap	129.5
11′		136.4	11′		132.0
12′	3.91, s	68.9	12′	4.56, s	70.8
13′	1.63, s	13.7	13′	1.68, s	14.1
14′	4.26, d (16.1)	66.9	14′	4.27, d (16.1)	66.8
	4.21, d (16.1)			4.18, d (16.1)
15′		177.4	15′		177.2
			1′′		161.3
			2′′, 6′′	6.80, d (8.6)	116.8
			3′′, 5′′	7.45, d (8.6)	131.2
			4′′		127.1
			7′′	7.61, d (15.9)	146.5
			8′′	6.33, d (15.9)	115.2
			9′′		169.1

Recorded at 500 MHz for 1H and 150 MHz for 13C-NMR in methanol-d4. Recorded at 500 MHz for 1H and 125 MHz for 13C-NMR in methanol-d4. Signals with the same symbol might be interchangeable.

Figure 2

The 1H−1H COSY, Key HMBC and ROESY correlations for 1–4 and 7.

As for the geometry of 1, significant ROESY correlation (Figure 2) of H-10′/H2-12′ shows that ∆10′(11′) double bond is E configuration. It was noted that 1 was isolated as a racemic mixture indicated by its chiral HPLC analysis. Racemic 1 was further separated by HPLC on chiral phase to afford (+)-1 and (−)-1, respectively. There is only one chiral center in the structure of 1. To clarify the absolute configurations of (+)-1 and (−)-1, ECD calculations were carried out at B3LYP/6-311+g(2d,p) level. It was found that the experiment ECD spectrum of (+)-1 matches well with the calculated ECD spectrum of (S)-1 (Figure 3). Thus, the absolute configurations of (−)-1 and (+)-1 were respectively assigned as 3′R and 3′S. Finally, the (−)-1 and (+)-1 was determined to be (R,E)-2-(2-(2,5-dihydroxyphenyl)-2-oxoethyl)-6-(5-hydroxy-4-methylpent-3-en-1-yl)-2,3,4,7-tetrahydrooxepine-2-carboxylic acid and (S,E)-2-(2-(2,5-dihydroxyphenyl)-2-oxoethyl)-6-(5-hydroxy-4-methylpent-3-en-1-yl)-2,3,4,7-tetrahydrooxepine-2-carboxylic acid respectively. Hence, the structure of 1, named zizhine P, was deduced.

Figure 3

The calculated and experimental ECD spectra of 1 and 2.

Compound 2 has a molecular formula of C30H32O9 deduced from its positive HRESIMS, 13C-NMR, and DEPT spectra. After careful analysis of the data of 1 and 2 (Table 1), it was found that the only difference between 2 and 1 is that a 4-hydroxycinnamic acid group is connected to C-12′ via an oxygen atom, this conclusion is supported by the HMBC correlation (Figure 2) of H2-12′ (δH 4.56)/C-9′′ (δC 169.1). The stereochemistry of 2 was assigned using ROSEY evidences. The ROESY correlation (Figure 2) of H2-9′ (δH 2.14)/H3-13′ (δH 1.68) indicate that the configuration of Δ10′(11′) double bond is E-form. The large coupling constants (nearly 16.0 Hz) of the one pair of olefinic protons suggest the trans form for the Δ7′’(8′′) double bond. Compound 2 was also isolated as a racemic mixture. Further separation by chiral phase HPLC afforded (−)-2 and (+)-2. To clarify their absolute configurations, ECD curve comparison with 1 was used. It is obvious that the experiment CD spectrum of (+)-2 agrees well with the experiment CD spectrum of (+)-1 (Figure 3). The absolute configurations of (−)-2 and (+)-2 were thus assigned as 3′R and 3′S, respectively. Ultimately, the (−)-2 and (+)-2 was determined to be (R)-2-(2-(2,5-dihydroxyphenyl)-2-oxoethyl)-6-((E)-5-(((E)-3-(4-hydroxyphenyl)acryloyl)oxy)-4-methylpent-3-en-1-yl)-2,3,4,7-tetrahydrooxepine-2-carboxylic acid and (S)-2-(2-(2,5-dihydroxyphenyl)-2-oxoethyl)-6-((E)-5-(((E)-3-(4-hydroxyphenyl)acryloyl)oxy)-4-methylpent-3-en-1-yl)-2,3,4,7-tetrahydrooxepine-2-carboxylic acid respectively. In this way, the structure of 2 was deduced and named zizhine Q.

The molecular formula of compound 3 was was determined to be C30H34O8 by analysis of its HRESIMS, 13C-NMR, and DEPT spectra. The 1H-NMR spectrum of 3 (Table 2) shows five aromatic signals including an ABX spin system at δH 6.66 (2H, H-3 and H-6), 6.54 (1H, dd, J = 8.5, 3.0 Hz, H-5), and an AA′BB′ system [δH 6.89 (2H, d, J = 8.6 Hz, H-2′′ and H-6′′), δH 7.55 (2H, d, J = 8.6 Hz, H-3′′ and H-5′′)]. The 13C-NMR and DEPT spectra (Table 2) display one methyl (δC 13.3), seven sp3 methylenes (including two oxygenated), twelve sp2 methines, and ten nonprotonated carbons (three sp2 oxygenated, one carbonyl at δC 169.5 and one ester carbonyl at δC 166.5). These data are similar to those of zizhine K [13]. The only difference is that the ketone group in zizhine K is reduced to a sp3 methylene in 3. This alteration is supported by the HMBC correlations (Figure 2) of H2-1′ (δH 3.70)/C-1, C-2, C-3, C-2′, C-3′, and C-15′ (δC 169.5). As for the stereochemistry of 3, ROESY correlations (Figure 2) of H-2′ (δH 6.03)/H2-4′ (δH 2.35), H2-5′ (δH 2.25)/H2-14′ (δH 4.10), H-10′ (δH 5.51)/H2-12′ (δH 4.54), and the coupling constant of H-7′′ (δH 7.62, 1H, d, J = 16.0 Hz) indicate theΔ2′(3′) and Δ6′(7′) double bonds are Z form, and the Δ10′(11′) and Δ7′′(8′′) double bonds are E form. As a result, compound 3 was identified as (2Z,5Z,9E)-2-(2-(2,5-dihydroxyphenyl)ethylidene)-6-(hydroxymethyl)-11-(((E)-3-(4-hydroxyphenyl)acryloyl)oxy)-10-methylundeca-5,9-dienoic acid and named zizhine R.

Table 2

1H- and 13C-NMR data of 3 and 4.

No.	3 a		No.	4 b
	δH (J in Hz)	δC		δH (J in Hz)	δC
1		148.1	1		157.0
2		126.1 c	2		120.6
3	6.66, overlap	116.7	3	7.11, d (2.9)	116.2
4		150.4	4		150.7
5	6.54, dd (8.5, 3.0)	113.8	5	7.01, dd (8.9, 2.9)	126.2
6	6.66, overlap	114.7	6	6.81, d (8.9)	119.7
1′	3.70, d (7.9)	30.5	1′		197.6
2′	6.03, t (7.9)	139.9	2′	6.89, s	130.4
3′		131.5	3′		146.0
4′	2.35, m	34.8	4′	2.52, t (7.3)	35.2
5′	2.25, m	27.0	5′	2.39, q (7.3)	26.9
6′	5.26, t (7.4)	125.8	6′	5.35, t (7.3)	127.3
7′		139.7	7′		140.9
8′	2.16, overlap	34.2	8′	2.24, overlap	35.4
9′	2.16, overlap	26.4	9′	2.24, overlap	27.4
10′	5.51, t (6.1)	129.0	10′	5.52, br s	130.1
11′		130.4	11′		131.8
12′	4.54, s	69.4	12′	4.54, s	71.0
13′	1.67, s	13.3	13′	1.67, s	14.1
14′	4.10, s	58.9	14′	4.13, s	60.0
15′		169.5	15′		170.1
1′′		159.7	1′′		161.3
2′′, 6′′	6.89, d (8.6)	116.7	2′′, 6′′	6.78, d (8.6)	116.8
3′′, 5′′	7.55, d (8.6)	130.1	3′′, 5′′	7.45, d (8.7)	131.2
4′′		126.2 c	4′′		127.1
7′′	7.62, d (16.0)	144.5	7′′	7.60, d (15.9)	146.6
8′′	6.38, d (16.0)	114.7	8′′	6.32, d (15.9)	115.2
9′′		166.5	9′′		169.1
			-OCH3	3.66, s	52.7

Recorded at 500 MHz for 1H and 125 MHz for 13C-NMR in acetone-d6. Recorded at 500 MHz for 1H and 125 MHz for13C-NMR in methanol-d4. Signals with the same symbol might be interchangeable.

Compound 4 has a molecular formula of C31H34O9 deduced from its HRESIMS, 13C-NMR, and DEPT spectra. It bears the same carbon skeleton and geometry as those of zizhine K [13] by inspection of their NMR spectra (Table 2). The only difference between them is that an oxygenated methyl (δC 52.7) in 4 instead of a hydroxyl in zizhine K connected with C-15′ (δC 170.1) is observed, which is supported by the obvious HMBC correlation (Figure 2) of -OCH3 (δH 3.66)/C-15′. Moreover, the ROESY correlations (Figure 3) of H-2′ (δH 6.89)/H2-4′ (δH 2.52), H2-5′ (δH 2.39)/H2-14′ (δH 4.13), H-10′ (δH 5.52)/H2-12′ (δH 4.54), and the coupling constant of H-7′′ (δH 7.60, 1H, d, J = 15.9 Hz) indicate that theΔ2′(3′) and Δ6′(7′) double bonds are Z forms, and the Δ10′(11′) and Δ7′′(8′′) double bonds are E forms. As a result, compound 4 was identified as (2Z,5Z,9E)-methyl 2-(2-(2,5-dihydroxyphenyl)-2-oxoethylidene)-6-(hydroxymethyl)-11-(((E)-3-(4-hydroxyphenyl)acryloyl)oxy)-10-methylundeca-5,9-dienoate and named zizhine S.

Compounds 5 and 6 was isolated by chiral HPLC. Their planar structures were identified as that of zizhine H. There are two chiral centers in 5 and 6, the absolute configuration at C-8′ of 5 and 6 were both assigned as S form according to the Mosher’s method. Briefly, treatment of 5 with (R)- or (S)-a-methoxy-atrifluoromethyl phenylacetic acyl chloride (MTPA-Cl) in deuterated pyridine was carried out to acquire the (S)-MTPA ester (5a) and (R)-MTPA ester (5b) (Figure 4), respectively. Analysis of the 1H-NMR signals of 5a and 5b indicates a 8′S configuration judged from theΔδH values of 5a and 5b. In the same manner as that of 5, the absolute configuration of C-8′ of 6 was identified as S. To clarify the stereochemistry at C-1′ in 5 and 6, ECD calculations of two model compounds 5c and 6c (Figure S62) were performed. It was found that the configuration at C-8′ has no influence on CD curves of 5 or 6 which means that CD comparison of 5 or 6 with similar compounds makes sense to assign the absolute configuration at C-1′. With this idea, ECD comparisons between 5 or 6 with those of (+)-ganocapenoid A [14] and (−)-ganocapenoid A [14] were conducted. The results show that the CD spectra of 5 and 6 agree well with those of (+)-ganocapenoid A and (−)-ganocapenoid A, respectively, indicating the absolute configurations of 5 and 6 are 1′R,8′S and 1′S,8′S, respectively. In fact, this conclusion is also identical with that of ECD calculations of 5c and 6c (Figure 5).

Figure 4

Δδ(S-R) values for the the Mosher’s esters of 5 and 6 in pyridine-d5.

Figure 5

The calculated and the experimental ECD spectra of 5 and 6.

It was noted that zizhine H has been incorrectly reported by us [13] as enantiomers. Our present results show that 5 and 6 are epimers rather than enantiomers. In this case, a careful analysis of the NMR spectra of zizhine H by Luo found that pairs of peaks are present, supporting our current conclusion. After the revision and clarification of the structures 5 and 6, we renamed them as zizhine T for 5 ((E)-(S,2E,6E)-9-((R)-5-(2,5-dihydroxyphenyl)-2-oxo-2,5-dihydrofuran-3-yl)-5-hydroxy-6-(hydroxymethyl)-2-methylnona-2,6-dien-1-yl 3-(4-hydroxyphenyl)acrylate) and 1′-epimer of zizhine T for 6 ((E)-(S,2E,6E)-9-((S)-5-(2,5-dihydroxyphenyl)-2-oxo-2,5-dihydrofuran-3-yl)-5-hydroxy-6-(hydroxymethyl)-2-methylnona-2,6-dien-1-yl 3-(4-hydroxyphenyl)acrylate).

Compound 7 has the molecular formula of C16H18O6 (eight degrees of unsaturation) on the basis of its HRESIMS, 13C-NMR, and DEPT spectra. The 1H-NMR spectrum of 7 (Table 3) shows an ABX spin system at δH 7.15 (1H, d, J = 3.0 Hz, H-3), 7.04 (1H, dd, J = 9.0, 3.0 Hz, H-5), 6.84 (1H, d, J = 9.0 Hz, H-6). The 13C-NMR and DEPT spectra (Table 3) display one methyl (δC 21.3), two sp3 methylenes, one sp3 oxygenated methylene, five sp2 methines, and seven quaternary carbons (one carbonyl at δC 199.0 and one carboxyl at δC 171.5). These data resemble those of fornicin D [15]. The only difference between them is that one more hydroxyl group attached to the terminal methyl in 7 is observed, gainning support by the obvious HMBC correlaations (Figure 3) of H2-8′ (δH 4.03)/C-6′ (δC 127.4), C-7′ (δC 137.2), C-9′ (δC 21.3). For the stereochemistry of 9, the ROESY correlations (Figure 3) of H-2′ (δH 7.63)/H2-4′ (δH 2.66), H2-5′ (δH 2.28)/H2-8′ (δH 4.03), H-6′ (δH 5.22)/H3-9′ (δH 1.62), indicate that the double bonds Δ2′(3′) and Δ6′(7′) are both Z forms. Thus, the compound 7 was deduced as (2Z,5Z)-2-(2-(2,5-dihydroxyphenyl)-2-oxoethylidene)-7-hydroxy-6-methylhept-5-enoic acid and named zizhine U.

Table 3

1H- and 13C-NMR data of 5–7.

No.	5 a		No.	6 a		No.	7 b
	δH (J in Hz)	δC		δH (J in Hz)	δC		δH (J in Hz)	δC
1		149.0	1		149.0	1		157.2
2		123.3	2		123.3	2		121.4
3	6.46, d (2.9)	113.4	3	6.47, d (2.9)	113.4	3	7.15, d (3.0)	115.8
4		151.4	4		151.4	4		150.8
5	6.61, dd (8.7, 2.9)	117.3	5	6.61, dd (8.7, 2.9)	117.3	5	7.04, dd (9.0, 3.0)	126.4
6	6.68, d (8.7)	117.3	6	6.67, d (8.7)	117.3	6	6.84, d (9.0)	119.8
1′	6.23, d (1.5)	79.8	1′	6.23, d (1.5)	79.9	1′		199.0
2′	7.37, d (1.5)	151.4	2′	7.37, d (1.5)	151.4	2′	7.63, s	131.3
3′		132.8	3′		132.9 c	3′		148.5
4′	2.41, m	26.1	4′	2.39, m	26.2 d	4′	2.66, m	29.8
5′	2.46, m	26.4	5′	2.45, m	26.4 d	5′	2.28, m	28.2
6′	5.58, t (6.9)	129.2	6′	5.58, t (7.0)	129.3	6′	5.22, t (7.6)	127.4
7′		142.6	7′		142.5	7′		137.2
8′	4.18, t (6.5)	75.2	8′	4.18, t (6.5)	75.3	8′	4.03, s	61.2
9′	2.32, m	35.4	9′	2.33, m	35.4	9′	1.62, s	21.3
10′	5.53, t (6.5)	126.6	10′	5.54, t (6.5)	126.6	10′		171.5
11′		133.1	11′		133.1c
12′	4.56, s	70.8	12′	4.56, s	70.8
13′	1.69, s	14.4	13′	1.69, s	14.4
14′	4.19, d (12.2)	58.1	14′	4.19, d (12.2)	58.1
	4.11, d (12.2)			4.13, d (12.2)
15′		176.7	15′		176.7
1′′		161.2	1′′		161.3
2′′, 6′′	6.80, d (8.6)	116.8	2′′, 6′′	6.80, d (8.6)	116.8
3′′, 5′′	7.45, d (8.6)	131.2	3′′, 5′′	7.45, d (8.6)	131.2
4′′		127.1	4′′		127.1
7′′	7.61, d (15.9)	146.6	7′′	7.61, d (15.9)	146.6
8′′	6.33, d (15.9)	115.2	8′′	6.32, d (15.9)	115.2
9′′		169.1	9′′		169.1

Recorded at 500 MHz for 1H and 125 MHz for 13C-NMR in methanol-d4. Recorded at 500 MHz for 1H and 150 MHz for 13C-NMR in methanol-d4. , Signals with the same symbol might be interchangeable.

2.2. Biological Evaluation

Ganoderma fungi have been reported to be beneficial for cancer patients [16]. The responsible compounds for cancer might be triterpenoids [17,18], polysacchride [19,20,21]. Meroterpenoids are widely present in the genus Ganoderma [22]. However, such compounds were largely ignored before 2013. Thereafter, increasing numbers of meroterpenoids were characterized by us. Our previous study disclosed that meroterpenoids are also in vitro active toward cancer cells [23,24,25]. Whether meroterpenoids resulting from the present study are also potent against cancer cells needs examination. Herein, all the isolated compounds were evaluated for their cytotoxic activity toward three human cancer cell lines and HDF (normal human embryonic lung fibroblasts). It was found that only BGC-823 and KYSE30 cells are sensitive to compounds 5 and 6, and A549 cells are sensitive to compound 4, all the other compounds are not active (Table 4). Although the cytotoxic potential of the active compounds are not so strong indicated by their larger IC50 values, they appear to be not harmful to human normal cells (HDF) with IC50 values larger than 160 μM. Cytotoxicity assay is just one approach to evaluate the role of the compounds against cancer, the derivatives of such meroterpenoids was found to be active toward Protein Tyrosine Phosphatase 1B (PTP1B) [26], indicating the usefulness of such meroterpenoids in cancer from an alternative aspect. Therefore, it is necessary to explore new screening methods to gain a deep insight into the biological role of the isolated meroterpenoids.

Table 4

IC50 values for 1–7 and 5-FU on different cell lines.

Compound	Cell Lines (IC50, μM)
	BGC-823	KYSE30	A549	HDF
(−)-1	>160	>160	>160	>160
(+)-1	>160	>160	>160	>160
(−)-2	>160	>160	>160	>160
(+)-2	>160	>160	>160	>160
3	>160	>160	>160	>160
4	>160	>160	72.13 ± 8.7 *	>160
5	59.2 ± 2.73 *	76.28 ± 1.93 *	>160	>160
6	64.25 ± 0.37 *	85.42 ± 2.82 *	>160	>160
7	>160	>160	>160	>160
5-FU	37.72 ± 1.87	8.43 ± 0.19	35.02 ± 2.42	51.07 ± 2.43

BGC-823: human gastric cancer cells. KYSE30: human esophageal cancer cells. A549: human lung cancer cells. HDF: normal human embryonic lung fibroblasts. * p < 0.05 (vs. 5-FU group).

3. Experimental Section

3.1. General Procedures

Optical rotations were measured on a Bellingham + Stanley ADP 440 + digital 9 polarimeter (Bellingham & Stanley, Kent, UK). UV spectra were obtained on a Shimadzu UV-2600 spectrometer (Shimadzu Corporation, Tokyo, Japan). CD spectra were measured on a Chirascan instrument (Agilent Technologies, Santa Clara, CA, USA). NMR spectra were recorded on a Bruker AV-500 and AV-600 spectrometer (Bruker, Karlsruhe, Germany) with TMS as an internal standard. HRESIMS of was collected by a Shimazu LC-20AD AB SCIEX triple TOF 5600+ MS spectrometer (Shimadzu Corporation, Tokyo, Japan) or a Waters Xevo G2-XS QTOF. MCI gel CHP 20P (75–150 μm, Tokyo, Japan), C-18 silica gel (40–60 μm; Daiso Co., Tokyo, Japan), YMC gel ODS-A-HG (40–60 μm; YMC Co., Tokyo, Japan), silica gel (200–300 mesh; Qingdao Marine Chemical Inc., Qingdao, China), silica gel GF254 (80–100 mesh, Qingdao Marine Chemical Inc., China) and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden) were used for column chromatography (CC). Semi-preparative HPLC was taken on a saipuruisi chromatograph with a Phenomenex Kinetex (250 mm × 10 mm, i.d., 5 μm) or a YMC-Pack ODS-A column (250 mm × 10 mm, i.d., 5 μm). Preparative HPLC was taken on a Chuangxin-Tongheng chromatograph equipped with a Thermo Hypersil GOLD-C18 column (250 × 21.2 mm, i.d., 5 μm). Racemic compounds and epimers were purified by chiral HPLC on a Daicel Chiralpak column (IC, 250 mm × 10 mm, i.d., 5 μm) and a Daicel Chiralpak column (IC, 250 mm × 4.6 mm, i.d., 5 μm).

3.2. Fungal Material

The fruiting bodies of G. sinensis were purchased from Tongkang Pharmaceutical Co. Ltd. in Guangdong Province, China, in September 2018. The material was authenticated by Prof. Xiang-Hua Wang at Kunming Institute of Botany, Chinese Academy of Sciences, China, and a voucher specimen (CHYX-0621) is deposited at School of Pharmaceutical Sciences, Shenzhen University Health Science Center, China.

3.3. Extraction and Isolation

The powdered fruiting bodies of G. sinensis (93.0 kg) were extracted by reflux with 80% EtOH (3 × 300 L × 3 h) to give a crude extract. The extract was suspended in water and partitioned with EtOAc to obtain an EtOAc-soluble extract (1.6 kg). The EtOAc extract was divided into ten parts (Fr.1-Fr.10) by using a MCI gel CHP 20P column eluted with aqueous MeOH (40–100%).

Fr.7 (40.0 g) was subjected to Sephadex LH-20 CC (MeOH) to obtain six parts (Fr.7.1–Fr.7.6). Among them, Fr.7.3 (14.0 g) was cut by a C-18 column (MeOH/H2O, 30–100%) to afford eight portions (Fr.7.3.1–Fr.7.3.8). Of which, Fr.7.3.5 (4.7 g) was divided by Sephadex LH-20 (MeOH) to provide Fr.7.3.5.1 and Fr.7.3.5.2. The first part (2.6 g) was further divided by silica gel CC eluted with increasing MeOH in CH2Cl2 (200:1–1:1) to afford Fr.7.3.5.1.1–Fr.7.3.5.1.8. Fr.7.3.5.1.6 (731.0 mg) was cut into three parts (Fr.7.3.5.1.6.1–Fr.7.3.5.1.6.3) by preparative thin layer chromatography (PTLC) (EtOAc:EtOH:H2O = 15:2:1). One portion from PTLC (220.0 mg) (Rf = 0.5) was first submitted to Sephadex LH-20 (MeOH) to get two parts (Fr.7.3.5.1.6.3.1 and Fr.7.3.5.1.6.3.2). Fr.7.3.5.1.6.3.2 (121.0 mg) was separated by preparative HPLC [MeOH/H2O (0.05% TFA), 35%-100%] to obtain five portions (Fr.7.3.5.1.6.3.2.1–Fr.7.3.5.1.6.3.2.5). Fr.7.3.5.1.6.3.2.3 (21.0 mg) was purified by semi-preparative HPLC (acetonitrile/H2O containing 0.05% TFA, 40%, flow rate: 3 mL/min) to afford compound 9 (1.1 mg, tR = 32.7 min). Fr.7.3.6 (4.5 g) was further divided by silica gel CC eluted with gradient CH2Cl2/MeOH (100:1–1:1) to obtain Fr.7.3.6.1–Fr.7.3.6.8. Among them, Fr.7.3.6.5 (251.0 mg) was submitted to preparative HPLC [MeOH/H2O (0.05% TFA), 30%-100%] to provide four portions (Fr.7.3.6.5.1–Fr.7.3.6.5.4). Fr.7.3.6.5.1 (51.0 mg) was first submitted to semi-preparative HPLC [MeOH/H2O (0.05% TFA), 57%, flow rate: 3 mL/min] to obtain 3.0 mg, and then purified by semi-preparative HPLC (acetonitrile/H2O containing 0.05% TFA, 31%, flow rate: 3 mL/min) to yeild compound 1 (1.8 mg, tR = 26.0 min). Fr.7.3.6.6 (782.0 mg) was separated by preparative HPLC [MeOH/H2O (0.05% TFA), 35–100%] to obtain five portions (Fr.7.3.6.6.1–Fr.7.3.6.6.5). Among them, Fr.7.3.6.6.2 (166.0 mg) was cut by preparative HPLC [MeOH/H2O (0.05% TFA), 35%-100%] to obtain eight parts (Fr.7.3.6.6.2.1–Fr.7.3.6.6.2.8). Of which, Fr.7.3.6.6.2.5 (70.0 mg) was fractionated by semi-preparative HPLC (acetonitrile/H2O containing 0.05% TFA, 40%, flow rate: 3 mL/min) to obtain four parts (Fr.7.3.6.6.2.5.1–Fr.7.3.6.6.2.5.4). Fr.7.3.6.6.2.5.3 (43.0 mg) was further purified by chiral HPLC (n-hexane/ethanol, 82:18, flow rate: 3 mL/min) to yeild compounds 5 (12.4 mg, tR = 18.2 min) and 6 (12.2 mg, tR = 22.3 min).

Fr.8 (68.0 g) was gel filtrated over Sephadex LH-20 (MeOH) to provide six parts (Fr.8.1–Fr.8.6). Of which, Fr.8.3 (34.0 g) was separated by a C-18 silica gel column eluted with aqueous MeOH (40–100%) to afford eight portions (Fr.8.3.1–Fr.8.3.8). Among them, Fr.8.3.4 (28.2 g) was divided by silica gel CC eluted with CH2Cl2:MeOH (50:1–1:1) to afford five parts (Fr.8.3.4.1–Fr.8.3.4.5). 100.0 mg was taken out of Fr.8.3.4.5 (13.0 g) and purified by semi-preparative HPLC (acetonitrile/H2O containing 0.05% TFA, 38%, flow rate: 3 mL/min) to yeild compound 3 (31.3 mg, tR = 33.1 min). Fr.8.3.6 (1.2 g) was submitted to Sephadex LH-20 (MeOH) to afford four parts (Fr.8.3.6.1–Fr.8.3.6.4). Among them, Fr.8.3.6.2 (369.0 mg) was separated by a C-18 silica gel column eluted with aqueous MeOH (40–100%) to provide Fr.8.3.6.2.1–Fr.8.3.6.2.11. Of which, Fr.8.3.6.2.6 (145.0 mg) was cut into eight portions (Fr.8.3.6.2.6.1–Fr.8.3.6.2.6.8) by PTLC (CH2Cl2:MeOH = 8:1). Fr.8.3.6.2.6.3 (33.0 mg) (Rf = 0.8) was gel filtrated over Sephadex LH-20 (MeOH) to afford 13.0 mg followed by purification by semi-preparative HPLC (acetonitrile/H2O containing 0.05% TFA, 50%, flow rate: 3 mL/min) to afford compound 4 (5.88 mg, tR = 29.0 min). Fr.8.3.6.3 (377.0 mg) was separated by PTLC (CH2Cl2:MeOH = 8:1) to obtain five portions (Fr.8.3.6.3.1–Fr.8.3.6.3.5). Among them, Fr.8.3.6.3.4 (55.0 mg) (Rf = 0.3) was gel filtrated over Sephadex LH-20 (MeOH) to obtain 38.0 mg, then was purified by semi-preparative HPLC (acetonitrile/H2O containing 0.05% TFA, 45%, flow rate: 3 mL/min) to yeild compound 2 (10.2 mg, tR = 20.1 min).

3.4. Compound Characterization Data

(±)-Compound 1: yellowish gum; UV (MeOH) λmax (logε) 386 (3.56), 259 (3.87), 227 (4.16) nm; {[α]20 D −8.2 (c 0.06, MeOH); CD (MeOH) Δε214 +1.33, Δε249 +0.39, Δε256 +0.54, Δε276 +0.11, Δε292 +0.14, Δε361 −0.24; (−)-1}; {[α]D20 +8.0 (c 0.05, MeOH); CD (MeOH) Δε217 −1.38, Δε247 −0.12, Δε257 −0.35, Δε277 −0.02, Δε290 −0.17, Δε360 +0.42; (+)-1}; HRESIMS m/z 391.1762 [M + H]+ (calcd for C21H27O7, 391.1757); 1H and 13C-NMR data see Table 1.

(±)-Compound 2: yellow gum; UV (MeOH) λmax (logε) 363 (3.40), 314 (4.24), 263 (3.88), 228 (4.28) nm; {{[α]20 D −10.6 (c 0.39, MeOH); CD (MeOH) Δε209 +0.57, Δε217 +1.21, Δε227 +0.53, Δε235 +1.09, Δε251 +0.37, Δε264 +0.64, Δε273 +0.14, Δε309 +0.50, Δε364 −0.35; (−)-2}; {[α]D20 +14.0 (c 0.28, MeOH); CD (MeOH) Δε208 −1.82, Δε219 −2.04, Δε229 −1.88, Δε234 −1.66, Δε250 −0.48, Δε262 −0.85, Δε275 +0.29, Δε312 −0.12, Δε361 +1.01; (+)-2}; HRESIMS m/z 559.1947 [M + Na]+ (calcd for C30H32O9Na, 559.1944); 1H and 13C-NMR data see Table 1.

Compound 3: yellow gum; UV (MeOH) λmax (logε) 309 (4.17), 202 (4.46) nm; HRESIMS m/z 523.2324 [M + H]+ (calcd for C30H35O8, 523.2332); 1H and 13C-NMR data see Table 2.

Compound 4: yellow gum; UV (MeOH) λmax (logε) 312 (3.83), 202 (4.08) nm; HRESIMS m/z 573.2101 [M + Na]+ (calcd for C31H34O9Na, 573.2101); 1H and 13C-NMR data see Table 2.

Compound 5: yellow gum; UV (MeOH) λmax (logε) 310 (4.20), 220 (4.20) nm; {[α]20 D +42.2 (c 0.32, MeOH); CD (MeOH) Δε210 +16.74, Δε255 −0.42, Δε302 +1.14; (+)-5; HRESIMS m/z 559.1945 [M + Na]+ (calcd for C30H32O9Na, 559.1944); 1H and 13C-NMR data see Table 3.

Compound 6: yellow gum; UV (MeOH) λmax (logε) 310 (4.19), 220 (4.19) nm; {[α]20 D −32.4 (c 0.28, MeOH); CD (MeOH) Δε210 −13.58, Δε256 +0.54, Δε306 −0.35; (−)-6; HRESIMS m/z 554.2371 [M + NH4]+; (calcd for C30H36O9N, 554.2385); 1H and 13C-NMR data see Table 3.

Compound 7: yellow gum; UV (MeOH) λmax (logε) 377 (3.17), 260 (3.63), 221 (3.79), 203 (3.94) nm; HRESIMS m/z 307.1188 [M + H]+ (calcd for C16H19O6, 307.1182); 1H and 13C-NMR data see Table 4.

3.5. MTPA Esterification of 5 and 6

The absolute stereostructure of 5 and 6 was confirmed by Mosher’s method [27]. Compound 5 (1.0 mg) was dissolved in 1 mL of anhydrous deuteration pyridine, which was divided into two equal portions in NMR sample tube. To each portion was added 2 μL of either R-MTPA-Cl or S-MTPA-Cl to give S-MTPA ester (5a) or R-MTPA ester (5b) derivatives, and then the mixtures was kept at room temperature for 2 h. Finally, without purification, the 1H-NMR of the mixtures was tested. Preparation of the MTPA derivatives of 6 is same as that of 5.

3.6. Cell Viability Assay

All cell lines were purchased from the Cell Bank of China Science Academy (Shanghai, China) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 100 U/mL penicillin-streptomycin, and incubated at 37 °C in an atmosphere of 5% CO2. Cell viability was evaluated by the CCK8 assay kit (Dojindo Laboratories, Tokyo, Japan) according to the manufacturer’s instructions. Exponentially growing cells were seeded at 2–8 × 103 cells per well in 96-well culture plates for 24 h. Cells were exposed to increasing concentrations (0–80 μM) of 4, 5, 6, or 5-FU for 48 h. The equal volume of DMSO was used as the solvent control. CCK8 solution (10 μL) was added to each well and incubated for another 1–4 h. Light absorbance of the solution was measured at 450 nm (Epoch 2; BioTek Instruments, Inc. Winooski, VT, USA). The IC50 values were calculated using the GraphPad prism 7 and analyzed by fitting a curve using nonlinear regression [28,29].

4. Conclusions

To conclude, the present investigation on G. sinensis led to the characterization of five new meroterpenoids. With the aid of ECD calculations and chemical methods, the ambiguous structure of zizhine H was firstly clarified as a pair of epimers. Biological evaluation of these meroterpenoids disclosed that such type of compounds might be beneficial for the preventive or treatment of cancer. The current study will add new facets for chemical profiling of Ganoderma fungal species.