Spiro Meroterpenoids from Ganoderma applanatum - Journal of

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Spiro Meroterpenoids from Ganoderma applanatum Qi Luo,†,‡ Xiao-Yi Wei,§ Jing Yang,† Jin-Feng Luo,# Rui Liang,# Zheng-Chao Tu,*,# and Yong-Xian Cheng*,† †

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China § Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, People’s Republic of China # Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, People’s Republic of China S Supporting Information *

ABSTRACT: Spiroapplanatumines A−Q (1−12, 14−16, 18, and 20), new spiro meroterpenoids respectively bearing a 6/5/7 or 6/5/5 ring system, along with three known compounds, spirolingzhines A, B, and D, were isolated from the fruiting bodies of the fungus Ganoderma applanatum. Their structures including absolute configurations were assigned by using spectroscopic methods, ECD and 13C NMR calculations, and single-crystal X-ray diffraction analysis. Biological evaluation of all the compounds disclosed that compounds 7 and 8 inhibited JAK3 kinase with IC50 values of 7.0 ± 3.2 and 34.8 ± 21.1 μM, respectively.

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T

RESULTS AND DISCUSSION The EtOH extract of G. applanatum was suspended in H2O and partitioned with EtOAc. A combination of chromatography of an EtOAc-soluble fraction afforded 21 spiro meroterpenoids, including new spiroapplanatumines A−Q (1−12, 14−16, 18, 20). Spiroapplanatumine A (1) has the molecular formula C16H14O7 deduced from analysis of its HRESIMS, 13C NMR, and DEPT data, indicating 10 degrees of unsaturation. The 1H NMR spectrum (Table 1) contains three typical aromatic signals at δH 6.97 (1H, d, J = 2.7 Hz, H-3), 7.12 (1H, dd, J = 8.8, 2.7 Hz, H-5), and 6.92 (1H, d, J = 8.8 Hz, H-6), suggesting the presence of a 1,2,4-trisubstituted benzene substructure. The 13 C NMR and DEPT spectra of 1 (Table 3) contain 16 resonances attributable to three methylenes, five methines (four sp2), a ketone, two carboxylic carbonyls, four sp2 carbons (two oxygenated), and an oxygenated aliphatic carbon. Some of these signals resemble those of spirolingzhine D (13), a meroterpenoid previously isolated from G. lingzhi,13 indicating that they are analogues differing in ring C. The 1H−1H COSY correlations of H-3′/H-4′/H-5′/H-6′ and HMBC correlations of H-3′, H-8′/C-1′ (δC 205.9), C-2′ (δC 89.3), H-8′/C-6′, C-7′, H-5′, H-6′/C-7′, and H-8′/C-3′ disclose the presence of a seven-membered ring C, incorporated with ring B via C-2′. Two carboxylic acid groups are respectively connected to C-3′ and C-7′, gaining support from HMBC correlations of H-3′, H-

he genus Ganoderma (Ganodermataceae) is distributed in the tropical and subtropical regions.1 Many Ganoderma species are of great value and have been used as folk medicines especially in some Asian countries to treat a wide range of diseases.2 For instance, G. lucidum and G. atrum have been used to treat cancer, asthma, chronic hepatitis, nephritis, arthritis, and insomnia.3 So far, more than 400 secondary metabolites have been characterized from Ganoderma species. Among these, triterpenoids and polysaccharides are considered as the major constituents of this genus.4,5 In 2013, we reported a pair of structurally novel and reno-protective meroterpenoid enantiomers from G. lucidum;6 thereafter, several similar compounds were described by us,7−13 adding new facets for the genus Ganoderma and providing new targets for synthesis.14,15 As part of our ongoing study of structurally diverse meroterpenoids from Ganoderma species, the fruiting bodies of G. applanatum were investigated, and novel meroterpenoids from this species have been reported by us.16,17 Further chemical investigations on this species resulted in the isolation of 21 spiro meroterpenoids characteristic of a 6/5/7 or 6/5/5 ring system. To explore the biological potentials of these isolated compounds, ethnopharmacological knowledge was utilized. Considering that G. applanatum is used for treating cancer and tuberculous toxic-allergic arthritis in Chinese folk medicine, and overexpression of JAK3 is implicated in several disorders such as cancer, organ transplantation, diabetic nephropathy, and autoimmune diseases,18−21 the inhibitory effects of these meroterpenoids against JAK3 were therefore evaluated. © 2016 American Chemical Society and American Society of Pharmacognosy

Received: May 12, 2016 Published: December 20, 2016 61

DOI: 10.1021/acs.jnatprod.6b00431 J. Nat. Prod. 2017, 80, 61−70

Journal of Natural Products

Article

Chart 1

Table 1. 1H NMR Data of Compounds 1−6 (δ in ppm) in Methanol-d4 no. 3 5 6 3′ 4′ 5′ 6′ 8′

1a 6.97, d (2.7) 7.12, dd (8.8, 2.7) 6.92, d (8.8) 3.31, overlap Ha: 2.26, m Hb: 2.01, m Ha: 2.68, m Hb: 2.48, m 7.34, t (5.8) Ha: 2.99, d (15.5) Hb: 2.62, d (15.5)

9′-OCH3 10′-OCH3 a

2b 6.91, 7.13, 6.96, 3.27, 2.29, 2.01, 2.65, 2.45, 7.42, 2.96, 2.91,

d (2.7) dd (8.8, 2.7) d (8.8) dd (12.2, 3.0) m m m m t (6.3) d (14.7) d (14.7)

3b 6.97, 7.14, 6.93, 3.35, 2.22, 2.03, 2.67, 2.49, 7.31, 2.99, 2.66,

4b

d (2.7) dd (8.8, 2.7) d (8.8) dd (10.7, 3.6) m m m m t (5.7) d (15.6) d (15.6)

3.31, s

6.91, 7.13, 6.93, 3.30, 2.27, 1.95, 2.65, 2.45, 7.40, 2.98, 2.91,

d (2.7) dd (8.8, 2.7) d (8.8) dd (12.0, 3.3) m m m m t (6.3) d (14.9) d (14.9)

5b 6.96, 7.08, 6.86, 3.18, 2.25, 1.93, 2.67, 2.43, 7.34, 2.96, 2.57, 3.64,

6b

d (2.7) dd (8.8, 2.7) d (8.8) dd (10.7, 3.1) m m m m t (6.3) d (15.3) d (15.3) s

6.89, 7.11, 6.93, 3.23, 2.27, 2.01, 2.65, 2.45, 7.39, 2.92,

d (2.5) dd (8.8, 2.5) d (8.8) dd (10.3, 3.0) m m m m dd (8.3, 5.5) s

3.65, s

3.46, s

400 MHz. b600 MHz.

stereochemistry at C-2′. Using the same computational methods as those of 1, the absolute configuration of 2 was finally determined as 2′R,3′R (Figure 1), which is actually a diastereoisomer of 1. This conclusion could be confirmed by comparison of their CD profiles [CD (MeOH) Δε218 +10.54, Δε317 −4.59, Δε360 +5.20 for 1; CD (MeOH) Δε214 −6.58, Δε237 +8.97, Δε324 +11.26, Δε366 −10.73 for 2]. Spiroapplanatumines C (3) and D (4) were assigned to have the same planar structure by analysis of their HRESIMS and 1D and 2D NMR data. A detailed comparison of 1H and 13C NMR data (Tables 1 and 3) between 3 and 1 discloses that 3 is a methyl ester derivative of 1. The methoxy group is connected to C-10′ by the observed HMBC correlation of OCH3 (δH 3.31)/C-10′ (δC 172.6) in 3 and HMBC correlation of OCH3 (δH 3.46)/C-10′ (δC 173.7) in 4. The absolute configurations of 3 and 4 were respectively determined as 2′S,3′R and 2′R,3′R by comparing their CD spectra with those of 1 and 2 (Figure 2).

4′/C-10′ and H-6′, H-8′/C-9′. There are two chiral centers in 1, but the limited ROESY interactions make it impossible to clarify the relative configuration of 1 using this technique. Therefore, the absolute configuration of 1 was assigned by electronic circular dichroism (ECD) calculations22 (Computational methods, Supporting Information). We found that calculated weighted ECD spectra of (2′S,3′R)-1 and (2′S,3′S)-1 have obvious differences at 230−260 nm (positive Cotton effect for 2′S,3′R at 250 nm, but negative Cotton effective for 2′S,3′R at 250 nm). Therefore, the ECD spectrum of (2′S,3′R)-1 agrees well with the experimental spectrum of 1, leading to the assignment of the absolute configuration at the stereogenic centers (Figure 1). Spiroapplanatumine B (2) has the molecular formula C16H14O7 deduced from its HRESIMS, 13C NMR, and DEPT data. The planar structure of 2 was found to be the same as that of 1 by detailed interpretation of its 1D and 2D NMR data (Tables 1 and 3). Compound 2 differs from 1 only in the 62



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Table 2. 1H NMR Data of Compounds 7−11 (δ in ppm) in Methanol-d4 7b

no. 3 5 6 3′ 4′ 5′ 6′ 7′ 8′ 9′ 9′-OCH3 10′-OCH3 a

8b

9b

6.97, d (2.7) 7.12, overlap 6.87, d (8.8) 3.34, dd (9.5, 3.6) Ha: 2.31, m Hb: 2.10, m Ha: 2.86, m Hb: 2.66, m 7.12, overlap

6.87, 7.13, 6.94, 3.27, 2.36, 2.05, 2.83, 2.63, 7.22,

d (2.7) dd (8.8, 2.7) d (8.8) dd (9.5, 3.0) m m m m t (6.1)

Ha: 2.91, d (15.5) Hb: 2.49, d (15.5) 9.34, s

2.92, d (14.6) 2.74, d (14.6) 9.34, s

6.97, 7.14, 6.88, 3.40, 2.27, 2.15, 2.87, 2.67, 7.10,

10a

d (2.7) dd (8.8, 2.7) d (8.8) dd (8.9, 6.7) m m m m t (5.7)

11b

6.96, d (2.1) 7.13, dd (8.8, 2.1) 6.92, d (8.8) 3.09, dd (10.2, 3.0) 2.28, m 1.92, m 2.09, m 1.54, m Ha: 2.16, m Hb: 1.67, m 2.87, m 2.13, m

2.90, d (15.8) 2.54, d (15.8) 9.34, s

6.88, 7.18, 6.97, 3.35, 2.15,

d (2.7) dd (8.8, 2.7) d (8.8) dd (8.7, 1.8) m

2.10, m 2.94, dd (11.1, 7.4)

5.07, s 4.95, s 3.79, s

3.59, s 3.31, s

3.52, s

400 MHz. b600 MHz.

Table 3. 13C NMR (150 MHz) Data of Compounds 1−12 (δ in ppm) in Methanol-d4 no.

1

2

3

4

5

6

7

8

9

10

11

12

1 2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ OCH3

166.8 121.9 108.7 153.3 127.5 114.6 205.9 89.3 52.6 24.3 27.8 146.0 130.7 31.9 170.6 174.3

165.6 123.8 108.3 153.6 127.3 114.6 203.2 86.6 57.5 24.7 28.0 146.6 128.9 35.1 170.1 175.2

166.7 121.7 108.5 153.9 128.0 114.8 204.8 89.2 52.2 23.9 27.7 145.8 130.0 31.5 170.4 172.6 52.0

165.6 123.6 108.3 153.8 127.5 114.6 202.9 86.6 57.0 24.6 27.8 146.2 129.2 35.0 170.3 173.7 52.3

166.9 122.6 108.7 153.4 127.1 114.7 206.3 89.3 55.4 24.7 28.2 146.6 130.3 32.0 169.2 177.3 52.4

165.6 124.0 108.3 153.7 127.2 114.6 203.4 86.6 58.2 24.8 28.1 146.8 128.7 35.2 168.8 175.8 52.5

166.7 121.9 108.7 153.9 127.8 114.7 204.9 89.3 52.2 24.3 28.9 158.1 140.7 28.5 194.9 173.8

165.7 123.5 108.3 153.7 127.5 114.7 202.9 86.6 57.2 24.6 28.9 158.3 139.4 31.4 194.5 175.0

166.5 121.6 108.6 154.1 128.1 114.7 204.4 89.5 51.7 24.2 28.8 157.9 140.3 28.3 194.9 172.5 52.0

166.7 122.0 108.7 153.8 127.6 114.7 205.9 91.2 53.1 27.1 29.3 34.4 39.9 37.4 177.2 175.1 52.4

167.2 122.8 107.7 154.0 128.7 114.7 204.1 98.4 55.6 27.4 30.5 53.7 145.4 113.7 65.4 173.1 52.3

167.1 123.3 107.9 153.7 128.2 114.6 205.2 98.8 58.1 28.4 30.7 53.9 146.2 113.1 65.4 174.3

Figure 1. Comparison of CAM-B3LYP/TZVP-calculated ECD spectra for 2′S,3′R and 2′S,3′S with the experimental spectra of 1and 2 in MeOH.

Interpretation of HRESIMS and 1D and 2D NMR data of spiroapplanatumines E (5) and F (6) reveals that they have the same planar structure. The only difference between 5 and 1 is that the methoxy group in 5 is connected to C-9′ instead of C10′ in 1, evidenced from the HMBC correlation of OCH3 (δH 3.64)/C-9′ (δC 169.2). The methoxy group in 6 is connected to

C-9′, supported by HMBC correlation of OCH3 (δH 3.65)/C9′ (δC 168.8) (Figure 3). Likely, comparison of CD curves indicates 2′S,3′R for 5 and 2′R,3′R for 6 (Figure 2). Spiroapplanatumines G (7) and H (8) were also found to be a pair of diastereoisomers. The structure difference between 7 and 1 is that 9′-COOH of 1 was reduced to 9′-CHO, evident 63



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Figure 2. Experimental CD spectra of 1−9 observed in MeOH.

with the exception that a methoxy group is connected to C-10′ (δC 172.5). This change is supported by an HMBC correlation of OCH3 (δH 3.31)/C-10′ (Figure 3). Likewise, the CD curve similarities between 9 and 1 suggest that they bear the same configurations (Figure 2). The molecular formula of spiroapplanatumine J (10) was determined to be C17H18O7 on the basis of HRESIMS, 13C NMR, and DEPT data. Comparison of the NMR data for 10 and 5 indicates the absence of a Δ6′(7′) double bond in 10. This conclusion is further confirmed by the 1H−1H COSY correlations of H-3′/H-4′/H-5′/H-6′/H-7′/H-8′. The ROESY correlation between H-3′/H-7′ (Figure 4) suggests that H-3′ and H-7′ are at the same side of ring C, whereas the lack of effective correlation makes it impossible to assign the relative configuration at C-2′ by using ROESY observations. As a result, computational methods were used to clarify the absolute configuration of 10. The conformers of (2′S,3′R,7′S)10 and (2′R,3′R,7′S)-10 were calculated by using the Gaussian09 software. It was found that the calculated weighted ECD spectra of (2′S,3′R,7′S)-10 are in good accordance with the experimental CD spectrum of 10 (Figure 5). Consequently, the absolute configuration of 10 was unambiguously assigned. Spiroapplanatumine K (11) was obtained as a yellow crystal from MeOH−H2O (100:1). Its molecular formula of C17H18O7 was deduced on the basis of its HRESIMS, 13C NMR, and DEPT data (Tables 2 and 3). The NMR data of 11 are similar to those of 13,13 indicating that they are analogues. The only

Figure 3. Key 1H−1H COSY (bold blue lines) and HMBC (↷) correlations for the compounds 1−12, 14−16, 18, and 20.

from HMBC correlations of H-6′, H-8′/C-9′ (δC 194.9). Finally, the absolute configurations of 7 and 8 were respectively established as 2′S,3′R and 2′R,3′R based on their CD similarities with those of 1 and 2 (Figure 2). Spiroapplanatumine I (9) possesses the molecular formula C17H16O6 deduced from its HRESIMS, 13C NMR, and DEPT data. The NMR data of 9 (Tables 2 and 3) resemble those of 7

Figure 4. Key ROESY correlations for compounds 10, 12, 14, 15, 18, and 20. 64



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Figure 5. Comparison of B3LYP/6-311G(d,p)-calculated ECD spectra for 2′S,3′R,7′S; 2′R,3′S,7′R; 2′R,3′R,7′S; and 2′S,3′S,7′R with the experimental spectrum of 10 in MeOH.

Figure 7. Experimental CD spectra of 11, 12, and 13 observed in MeOH.

difference between 11 and 13 is that a methoxy group in 11 is connected to C-10′ (δC 173.1), supported by an HMBC correlation of OCH3/C-10′. In the ROESY spectrum, the correlation of H-3′/H-6′ is not detected, suggesting that H-3′ and H-6′ might be on the opposite side of ring C. This assumption is further confirmed by single-crystal X-ray diffraction using Cu Kα radiation (Figure 6), which meanwhile assigned the stereochemistry at C-2′. Thus, the absolute configuration of 11 was unambiguously assigned as 2′R,3′R,6′S.

DEPT data. Interpretation of 1D and 2D NMR data discloses that 14 and 12 possess the same backbone. The ROESY correlation of H-3′/H-6′ suggests these two protons are on the same side of the ring C. Of note, the scarcity of effective correlations makes it impossible to assign the relative configuration at C-2′ by using ROESY observations. To clarify the absolute configuration of 14, ECD calculations were performed using four low-energy conformers as in the abovementioned methods (Figure 9). It was found that the calculated ECD spectrum of (2′S,3′R,6′R)-14 has a positive Cotton effect at 241 nm, while that of (2′S,3′S,6′S)-14 has a weak Cotton effect at 236 nm, indicating that the calculated data of (2′S,3′S,6′S)-14 match well with the experimental ones, clarifying the absolute configuration of 14. Spiroapplanatumine N (15) has the molecular formula C16H14O6 on the basis of its HRESIMS, 13C NMR, and DEPT data. A detailed analysis of NMR data (Tables 4 and 5) between 15 and 12 indicates that an oxygenated methylene in 12 is replaced by an aldehyde group (δC 195.2) in 15. This conclusion is supported by the observed HMBC correlations of H-6′/C-9′ and H-9′/C-8′. Two protons of H-3′ and H-6′ are on opposite sides of the ring C, supported by ROESY correlations of H-3′/Hb-4′, Hb-5′/H-8′, and H-6′/Ha-4′, Ha5′. Compound 15 was isolated as a racemate, which was separated by chiral HPLC to afford enantiomers 15a and 15b. To clarify the absolute configuration of 15, ECD calculations were conducted as in the above-mentioned methods (Figure 10). The results show that both ECD spectra of the (2′R,3′S,6′R)-configuration and (2′R,3′R,6′S)-configuration agree well with the experimental CD spectrum of 15a, indicating the limitation of ECD calculations in configuration assignment of 15a. Thus, 13C NMR chemical shift calculations were utilized. The results show that the R2 (0.99900) and CMAE (1.18) for (2′R,3′S,6′R)-15a are less than those of (2′R,3′R,6′S)-15a [R2 (0.99971), CMAE (0.63)] (Figure 11, Supporting Information). Hence, the absolute configuration for 15a was determined to be 2′R,3′R,6′S. Spiroapplanatumine O (16) was determined to have the molecular formula C17H16O6 on the basis of its HREIMS, 13C NMR, and DEPT data. The resemblance of NMR data between 15 and 16 suggests that they are analogues. Compound 16 differs from 15 in that an additional methoxy group is present in 16. The HMBC correlation of OCH3 (δH 3.48)/C-10′ (δC 172.8) indicates the position of this moiety. Likewise, the CD curve of 16 exhibits similar Cotton effects to those of 15a, indicative of the same 2′R,3′R,6′S configuration.

Figure 6. Plot of the X-ray crystallographic data of 11.

The molecular formula of spiroapplanatumine L (12) was assigned as C16H16O6 by means of HRESIMS, 13C NMR, and DEPT data. Close comparison of NMR data between compounds 12 and 11 reveals that they are analogues. The ROESY correlations of H-3′/Hb-5′, Hb-5′/H-9′, and H-6′/Ha4′, Ha-5′ are observed, suggesting that H-3′ and H-6′ are on the opposite sides of ring C. In addition, it is also impossible to assign the relative configuration at C-2′ by using ROESY data. To clarify the absolute configuration of 12, comparison of CD curves between those of (2′R,3′R,6′S)-11, (2′R,3′S,6′R)-13, and 12 was undertaken (Figure 7), suggesting that compound 12 is actually a diastereoisomer of 13 with 2′R,3′R,6′Sconfiguration. This conclusion was further supported by the results of 13C NMR chemical shift calculations (Figure 8, Supporting Information), which show that (2′R,3′R,6′S)-12 rather than (2′S,3′R,6′S)-12 is more reasonable by comparison of the correlation coefficient (R2) and CMAE (the corrected mean absolute error) [0.99829 and 1.66 ppm for (2′R,3′R,6′S)12; 0.99662 and 2.36 ppm for (2′S,3′R,6′S)-12]. Thus, the absolute configuration of 12 was assigned as 2′R,3′R,6′S. Spiroapplanatumine M (14) has the molecular formula C16H16O6 derived by analysis of its HREIMS, 13C NMR, and 65



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Figure 8. Regression analysis of experimental versus calculated 13C NMR chemical shifts of (2′R,3′R,6′S)-12 and (2′S,3′R,6′S)-12 at the MPW1PW91-SCRF/6-311+G(2d,p) level. Linear fitting is shown as a line.

Table 5. 13C NMR (150 MHz) Data of 14−16, 18, and 20 (δ in ppm) in Methanol-d4

Figure 9. Comparison of B3LYP/6-311G(d,p)-calculated ECD spectra for 2′S,3′S,6′S; 2′R,3′R,6′R; 2′S,3′R,6′R; and 2′R,3′S,6′S with the experimental spectrum of 14 in MeOH.

Analysis of HRESIMS, 13C NMR, and DEPT data of spiroapplanatumine P (18) indicates that 18 is a methyl ester derivative of 17, previously characterized from G. lingzhi.13 The HMBC correlation from OCH3 to C-10′ (δC 173.3) indicates that the methoxy moiety is at C-10′ in 18. There are four chiral centers in the structure of 18. The ROESY correlations of H3′/Ha-5′ and Ha-5′/H-7′, Ha-9′ reveal the relative configurations at C-3′ and C-6′. In addition, analysis of 1H NMR of 17 (δH‑7′ 1.75, δH‑8′ 0.62, δHa‑9′ 3.58, δHb‑9′ 3.39), 18 (δH‑7′ 1.70, δH‑8′ 0.59, δHa‑9′ 3.54, δHb‑9′ 3.37), and 19 (δH‑7′ 1.62, δH‑8′ 0.96, δHa‑9′ 3.15, δHb‑9′ 3.03) suggest that the configurations of 17 and 18 at C-7′ are the same. ECD calculations were conducted to

no.

14

15

16

18

20

1 2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ OCH3

167.8 124.0 107.9 153.6 128.2 114.6 204.4 99.1 55.3 25.5 30.5 53.5 145.5 113.5 65.4 174.9

166.6 123.2 108.1 153.7 127.9 114.3 204.6 96.2 56.1 28.0 30.0 47.3 147.9 138.1 195.2 174.3

166.6 123.0 108.0 154.1 128.2 114.4 203.9 96.4 55.7 27.7 30.2 47.2 147.6 138.2 195.0 172.8 52.2

167.1 123.0 107.9 154.1 128.4 114.7 205.0 99.2 57.4 27.8 30.5 53.5 36.7 17.4 66.9 173.3 52.1

168.0 123.8 107.8 153.8 128.2 114.7 203.5 97.5 50.0 22.2 32.1 79.2 171.8

52.0

clarify the absolute configuration of 18. We found that the calculated weighted ECD spectra of (2′R,3′S,6′R,7′S)-18 and (2′S,3′S,6′R,7′S)-18 have obvious differences in the ranges of 195−260 and 300−400 nm, revealing that the calculated ECD spectrum of (2′R,3′S,6′R,7′S)-18 agrees well with the experimental one of 18. Therefore, the absolute configuration of 18 was assigned as 2′R,3′S,6′R,7′S (Figure 12).

Table 4. 1H NMR Data of Compounds 12, 14−16, 18, and 20 (δ in ppm) in Methanol-d4 no. 3 5 6 3′ 4′ 5′ 6′ 7′ 8′ 9′

12a

14b

15a

16a

6.89, d (2.7) 7.13, dd (8.8, 2.7) 6.93, d (8.8) 3.31, overlap Ha: 2.13, m Hb: 2.11, m Ha: 2.08, m Hb: 2.07, m 3.00, t (8.4)

6.91, d (2.7) 7.11, dd (8.8, 2.7) 6.92, d (8.8) 3.49, t-like (8.6) Ha: 2.42, m Hb: 2.08, overlap Ha: 2.23, m Hb: 2.08, overlap 3.00, dd (12.5, 6.8)

6.88, d (2.7) 7.09, dd (8.8, 2.7) 6.84, d (8.8) 3.43, m Ha: 2.21, m Hb: 2.15, m Ha: 2.11, m Hb: 2.08, m 3.40, m

6.87, d (2.7) 7.11, dd (8.8, 2.7) 6.85, d (8.8) 3.43, m Ha: 2.21, m Hb: 2.15, m Ha: 2.11, m Hb: 2.08, m 3.40, m

Ha: 5.07, s Hb: 4.93, s 3.80, s

5.02, s

6.51, s 6.22, s 9.27, s

6.52, s 6.20, s 9.25, s

3.76, s

OCH3 a

3.48, s

18a 6.92, 7.21, 7.02, 3.20, 2.09,

d (2.1) dd (8.8, 2.1) d (8.8) t (8.4) m

2.20, 1.74, 2.29, 1.70, 0.59,

m m m m d (6.7)

20b 6.96, 7.18, 7.04, 3.45, 2.39, 1.98, 2.16, 1.98, 4.31,

Ha: 3.54, dd (10.7, 3.5) Hb: 3.37, dd (10.7, 6.5) 3.49, s

d (2.7) dd (8.8, 2.7) d (8.8) dd (9.5, 2.1) m m m m dd (10.4, 7.5)

3.33, s

400 MHz. b600 MHz. 66



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Figure 10. Comparison of B3LYP/6-311G(d,p)-calculated ECD spectra for 2′R,3′S,6′R; 2′S,3′R,6′S; 2′S,3′S,6′R; and 2′R,3′R,6′S with the experimental spectra of 15a, 15b, and 16 in MeOH.

Figure 11. Regression analysis of experimental versus calculated 13C NMR chemical shifts of (2′R,3′S,6′R)-15a and (2′R,3′R,6′S)-15a at the MPW1PW91-SCRF/6-311+G(2d,p) level. Linear fitting is shown as a line.

Figure 13. Comparison of B3LYP/6-311G(d,p)-calculated ECD spectra for 2′R,3′R,6′S; 2′S,3′S,6′R; 2′R,3′S,6′R; and 2′S,3′R,6′S with the experimental spectrum of 20 in MeOH.

Figure 12. Comparison of B3LYP/6-311G(d,p)-calculated ECD spectra for 2′R,3′S,6′R,7′S; 2′S,3′R,6′S,7′R; 2′R,3′R,6′S,7′R; and 2′S,3′S,6′R,7′S with the experimental spectrum of 18 in MeOH.

configuration, which is further supported by the comparison of R2 and CMAE [0.99927 and 1.17 ppm for (2′R,3′S,6′R)-20; 0.99938, and 0.92 ppm for (2′R,3′R,6′S)-20] (Figure 14, Supporting Information). Thus, the absolute configuration of 20 was assigned as 2′R,3′R,6′S. It is notable that compounds 3−6, 9−11, 16, 18, and 20 are all methylated substances, either at C-10′ or at C-9′, thus prompting the speculation that they may be artificial products formed during the isolation procedure. Further efforts to clarify this were not carried out by LC-MS due to low abundance of these substances (ca. 0.1−1 ppm). We consider them as natural products because ethylation rather than methylation should be expected since extraction was conducted under reflux of EtOH. Also, methylation is commonly seen in biosynthetic processes.

Spiroapplanatumine Q (20) has the molecular formula C14H14O6 derived from its HRESIMS, 13C NMR, and DEPT data. A detailed analysis of 1D and 2D NMR spectra of 20 reveals the presence of a 6/5/5 ring system similar to that of 19. Intriguingly, compound 20 is a trinormeroterpenoid relative to 19, which is supported by the 1H−1H COSY correlations of H-3′/H-4′/H-5′/H-6′ and HMBC correlations of H-6′/C-1′, C-2′, C-3′, and C-4′. The ROESY correlation of H-3′/H-6′ indicates that they are on the same side of ring C. In the same manner as that of 18, ECD calculations were performed for four low-energy conformers (Figure 13). The results show that the calculated ECD data of the (2′R,3′R,6′S)-configuration are more similar to those of 20 than the (2′R,3′S,6′R)67



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Figure 14. Regression analysis of experimental versus calculated 13C NMR chemical shifts of (2′R,3′S,6′R)-20 and (2′R,3′R,6′S)-20 at the MPW1PW91-SCRF/6-311+G(2d,p) level. Linear fitting is shown as a line. Fr.3.4 (8.0 g) was submitted to RP-18 CC (MeOH−H2O, 10−55%) to afford Fr.3.4.1−Fr.3.4.8. Among them, Fr.3.4.4 (2.6 g) was gel filtrated via Sephadex LH-20 (MeOH) and purified by semipreparative HPLC (MeOH−H2O, 36:64) to give 7 (70.0 mg), 8 (210.9 mg), 12 (1.7 mg), 14 (1.5 mg), and 15 (82.4 mg). Fr.3.4.7 (1.5 g) was purified by Sephadex LH-20 CC (MeOH) and semipreparative HPLC (MeOH−H2O, 35:65) to yield 3 (4.0 mg), 9 (4.9 mg), 13 (43.1 mg), 16 (1.5 mg), and 19 (53.8 mg). Compound 15 was isolated as a racemate, which was further purified by chiral phase on Daicel Chiralpak IC (n-hexane−EtOH, 80:20, flow rate 2.5 mL/min) to afford enantiomers 15a (26.2 mg) and 15b (24.3 mg). Fr.3.6 (7.6 g) was separated by using Sephadex LH-20 (MeOH) followed by an RP18 column (MeOH−H2O, 40:60−55:45) to afford Fr.3.6.1−Fr.3.6.6. Further purification of Fr.3.6.5 (3.3 g) by semipreparative HPLC (MeOH−H2O, 35:65) gave 4 (11.1 mg), 5 (4.0 mg), 6 (9.0 mg), 9 (4.0 mg), 10 (4.8 mg), 11 (44.1 mg), and 18 (9.2 mg). Spiroapplanatumine A (1): yellow gum, [α]23D +93.5 (c 0.79, MeOH); UV (MeOH) λmax (log ε) 363 (3.56), 250 (3.88), 219 (4.31) nm; CD (MeOH) Δε218 +10.54, Δε317 −4.59, Δε360 +5.20; ESIMS m/ z 317 [M − H]−; HRESIMS m/z 317.0662 [M − H]− (calcd for C16H13O7, 317.0661); 1H and 13C NMR data, see Tables 1 and 3. Spiroapplanatumine B (2): yellow gum, [α]23D +83.6 (c 0.82, MeOH); UV (MeOH) λmax (log ε) 363 (3.54), 246 (3.88), 219 (4.19) nm; CD (MeOH) Δε214 −6.58, Δε237 +8.97, Δε324 +11.26, Δε366 −10.73; ESIMS m/z 317 [M − H]−; HRESIMS m/z 317.0663 [M − H]− (calcd for C16H13O7, 317.0661); 1H and 13C NMR data, see Tables 1 and 3. Spiroapplanatumine C (3): yellow gum, [α]23D +51.1 (c 0.42, MeOH); UV (MeOH) λmax (log ε) 364 (3.57), 250 (3.95), 215 (4.34) nm; CD (MeOH) Δε218 +7.13, Δε317 −3.09, Δε360 +4.02; ESIMS m/z 331 [M − H]−; HRESIMS m/z 331.0826 [M − H]− (calcd for C17H15O7, 331.0818); 1H and 13C NMR data see Table 1 and Table 3. Spiroapplanatumine D (4): yellow gum, [α]23D +57.8 (c 0.39, MeOH); UV (MeOH) λmax (log ε) 366 (3.61), 252 (3.89), 218 (4.25) nm; CD (MeOH) Δε214 −3.68, Δε237 +7.57, Δε324 +9.57, Δε366 −9.36; ESIMS m/z 331 [M − H]−; HRESIMS m/z 331.0822 [M − H]− (calcd for C17H15O7, 331.0818); 1H and 13C NMR data, see Tables 1 and 3. Spiroapplanatumine E (5): yellow gum, [α]23D +74.2 (c 0.24, MeOH); UV (MeOH) λmax (log ε) 363 (3.58), 248 (3.87), 221 (4.27) nm; CD (MeOH) Δε218 +9.97, Δε317 −4.68, Δε360 +4.86; ESIMS m/z 331 [M − H]−; HRESIMS m/z 331.0824 [M − H]− (calcd for C17H15O7, 331.0818); 1H and 13C NMR data, see Tables 1 and 3. Spiroapplanatumine F (6): yellow gum, [α]23D +74.1 (c 0.58, MeOH); UV (MeOH) λmax (log ε) 363 (3.54), 248 (3.87), 221 (4.21) nm; CD (MeOH) Δε214 −5.14, Δε237 +6.25, Δε324 +9.02, Δε366 −8.17; ESIMS m/z 331 [M − H]−; HRESIMS m/z 331.0820 [M − H]− (calcd for C17H15O7, 331.0818); 1H and 13C NMR data, see Tables 1 and 3. Spiroapplanatumine G (7): yellow gum, [α]23D +79.1 (c 0.58, MeOH); UV (MeOH) λmax (log ε) 363 (4.04), 248 (4.42), 222 (4.74) nm; CD (MeOH) Δε218 +5.98, Δε317 −6.42, Δε360 +6.51; ESIMS m/z 301 [M − H]−; HRESIMS m/z 301.0710 [M − H]− (calcd for C16H13O6, 301.0712); 1H and 13C NMR data, see Tables 2 and 3.

Finally, similar methylated meroterpenoids to natural products have also been detected in natural origins of Ganoderma fungus.7,9 Three known compounds were identified as spirolingzhine D (13),13 spirolingzhine A (17),13 and spirolingzhine B (19),13 respectively, by comparison of their NMR data with those in the literature. All the isolated compounds were evaluated for their potentials against JAK3. The results show that compounds 7 and 8 display inhibitory properties on JAK3 kinase with IC50 values of 7.0 ± 3.2 and 34.8 ± 21.1 μM, respectively, suggesting that they could be beneficial as lead compounds for JAK3overexpressed disorders.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a Horiba SEPA-300 polarimeter. UV spectra were collected on a Shimadzu double-beam 210A spectrometer. CD spectra were measured on a Chirascan instrument. NMR spectra were determined on a Bruker AV-400 and an Avance III 600 spectrometer. ESIMS and HRESIMS were measured on an API QSTAR Pulsar 1 spectrometer. Column chromatography (CC) was performed on silica gel (200−300 mesh, Qingdao Marine Chemical Inc., People’s Republic of China), RP-18 (40−60 μm, Daiso Co., Japan), MCI gel CHP 20P (75−150 μm, Tokyo, Japan), and Sephadex LH-20 (Amersham Biosciences, Sweden). Semipreparative HPLC was carried out using an Agilent 1200 liquid chromatograph; the columns used were a 250 mm × 9.4 mm i.d., 5 μm, Zorbax SB-C18 and a 250 mm × 10 mm, i.d., 5 μm, Daicel Chiralpak (IC), with a flow rate of 2.5 mL/min. Fungal Material. The fruiting bodies of G. applanatum were purchased from Tongkang Pharmaceutical Co. Ltd. in Guangzhou Province, People’s Republic of China, in September 2013. The material was identified by Prof. Zhu-Liang Yang at Kunming Institute of Botany, Chinese Academy of Sciences, and a voucher specimen (CHYX-0590) was deposited at the State Key Laboratory of Photochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, People’s Republic of China. Extraction and Isolation. Powdered fruiting bodies of G. applanatum (30.0 kg) were extracted by refluxing with 80% EtOH (3 × 120 L × 2 h) and concentrated under reduced pressure to give a crude extract, which was suspended in water followed by extraction with EtOAc to afford an EtOAc-soluble extract. The EtOAc extract (0.9 kg) was divided into seven parts (Fr.1−Fr.7), by using an MCI gel CHP 20P column eluted with aqueous MeOH (MeOH−H2O, 20:80− 100:0). Fr.3 (73.0 g) was separated by using MCI gel CHP 20P eluted with gradient aqueous MeOH (MeOH−H2O, 30:70−60:40) to yield nine fractions (Fr.3.1−Fr.3.9). Fr.3.1 (9.0 g) was further divided into three portions (Fr.3.1.1−Fr.3.1.3) by RP-18 CC with aqueous MeOH (10−50%). Fr.3.1.1 (2.1 g) was separated by Sephadex LH-20 (MeOH) followed by semipreparative HPLC (MeOH−H2O, 35:65) to yield 1 (140.0 mg), 2 (69.4 mg), 17 (7.8 mg), and 20 (7.4 mg). 68



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Computational Studies. Molecular Merck force field (MMFF) and DFT/TDDFT calculations were performed with the Spartan’14 software package (Wavefunction Inc., Irvine, CA, USA) and the Gaussian09 program package.22 The conformational search generated low-energy conformers within a 10 kcal/mol energy and was finished with the software Conflex 7. The predominant conformers were optimized by DFT calculation at the B3LYP/6-311G(d,p) level. Under the circumstances, the calculation of 13C NMR chemical shifts at the MPW1PW91-SCRF/6-311+G(2d,p) level with the PCM in MeOH was carried out. ECD calculations were conducted at the B3LYP/6311G(d,p) level with the PCM in MeOH solution. For comparisons of the calculated curves and experimental CD spectra, the program SpecDis23 was used. In Vitro JAK3 Kinase Inhibition Activity Assay. Inhibition activity of compounds against recombinant human kinase JAK3 (Life Technologies, Carlsbad, CA, USA) was determined using the FRETbased Z′-Lyte assay system according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The reactions were carried out in 384-well plates in a 10 μL reaction volume with the appropriate amount of kinases in 50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, and 0.01% Brij-35. The reactions were incubated 1 h at room temperature in the presence of 2 μM substrate with 10 μM ATP and in the presence of various concentrations of the compounds. Then 5 μL of development reagent was added for a further 2 h room-temperature incubation followed by the addition of 5 μL of stop solution. A fluorescence signal ratio of 445 nm (coumarin)/ 520 nm (fluorescin) was examined with an EnVision multilabel reader (PerkinElmer, Inc.)]. Staurosporine was used as the positive control with an IC50 value of 0.31 nM. The data were analyzed using Graphpad Prism5 (Graphpad Software, Inc.).

Spiroapplanatumine H (8): yellow gum, [α]23D +17.2 (c 0.99, MeOH); UV (MeOH) λmax (log ε) 363 (3.60), 221 (4.34) nm; CD (MeOH) Δε214 −8.14, Δε237 +2.93, Δε324 +7.96, Δε366 −7.56; ESIMS m/z 301 [M − H]−; HRESIMS m/z 301.0710 [M − H]− (calcd for C16H13O6, 301.0712); 1H and 13C NMR data, see Tables 2 and 3. Spiroapplanatumine I (9): yellow gum, [α]23D +19.7 (c 0.17, MeOH); UV (MeOH) λmax (log ε) 367 (3.56), 250 (3.95), 223 (4.35) nm; CD (MeOH) Δε218 +0.80, Δε317 −3.99, Δε360 +5.15; ESIMS m/z 315 [M − H]−; HRESIMS m/z 315.0878 [M − H]− (calcd for C17H15O6, 315.0869); 1H and 13C NMR data, see Tables 2 and 3. Spiroapplanatumine J (10): yellow gum, [α]23D +3.86 (c 0.23, MeOH); UV (MeOH) λmax (log ε) 364 (3.80), 252 (4.03), 220 (4.40) nm; CD (MeOH) Δε207 +3.58, Δε254 −5.42, Δε317 −7.33, Δε360 +7.38; ESIMS m/z 333 [M − H]−; HRESIMS m/z 333.0979 [M − H]− (calcd for C17H17O7, 333.0974); 1H and 13C NMR data, see Tables 2 and 3. Spiroapplanatumine K (11): yellow crystal, [α]23D −186.1 (c 1.81, MeOH); UV (MeOH) λmax (log ε) 374 (3.59), 256 (3.89), 217 (4.18) nm; CD (MeOH) Δε198 +9.75, Δε235 −13.61, Δε320 +2.12, Δε368 −4.90; ESIMS m/z 317 [M − H]−; HRESIMS m/z 317.1032 [M − H]− (calcd for C17H17O6, 317.1025); 1H and 13C NMR data, see Tables 2 and 3. Spiroapplanatumine L (12): yellow gum, [α]23D −142.0 (c 0.23, MeOH); UV (MeOH) λmax (log ε) 372 (3.51), 254 (3.81), 217 (4.12) nm; CD (MeOH) Δε196 +8.64, Δε235 −6.11, Δε322 + 2.72, Δε368 −3.86; ESIMS m/z 303 [M − H]−; HRESIMS m/z 303.0874 [M − H]− (calcd for C16H15O6, 303.0947); 1H and 13C NMR data, see Tables 3 and 4. Spiroapplanatumine M (14): yellow gum, [α]23D +40.4 (c 0.25, MeOH); UV (MeOH) λmax (log ε) 372 (3.55), 255 (3.82), 215 (4.14) nm; CD (MeOH) Δε200 −22.02, Δε232 +1.76, Δε320 −10.53, Δε365 +13.36; ESIMS m/z 303 [M − H]−; ESIMS m/z 303 [M − H]−; HRESIMS m/z 303.0874 [M − H]− (calcd for C16H15O6, 303.0947); 1 H and 13C NMR data, see Tables 4 and 5. Spiroapplanatumine N (15): yellow gum, UV (MeOH) λmax (log ε) 371 (3.59), 254 (3.86), 218 (4.28) nm; {[α]23D −199.6 (c 0.22, MeOH); CD (MeOH) Δε198 +11.75, Δε235 −13.71, Δε320 +2.10, Δε368 −4.94; (−)-spiroapplanatumine N (15a)}; {[α]23D +166.9 (c 0.67, MeOH); CD (MeOH) Δε198 −10.36, Δε235 + 10.63, Δε322 −2.13, Δε368 +3.02; (+)-spiroapplanatumine N (15b)}; ESIMS m/z 301 [M − H]−; HRESIMS m/z 301.0726 [M − H]− (calcd for C16H13O6, 301.0790); 1H and 13C NMR data, see Tables 4 and 5. Spiroapplanatumine O (16): yellow gum, [α]23D −240.0 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 373 (3.69), 255 (4.07), 215 (4.47) nm; CD (MeOH) Δε198 +11.81, Δε235 −14.83, Δε320 +1.62, Δε368 −4.26; ESIMS m/z 315 [M − H]−; HRESIMS m/z 315.0868 [M − H]− (calcd for C17H15O6, 315.0947); 1H and 13C NMR data, see Tables 4 and 5. Spiroapplanatumine P (18): yellow gum, [α]23D −42.1 (c 0.42, MeOH); UV (MeOH) λmax (log ε) 373 (3.57), 256 (3.87), 219 (4.17) nm; CD (MeOH) Δε235 −6.02, Δε320 −1.41, Δε368 +2.56; ESIMS m/z 319 [M − H]−; HRESIMS m/z 319.1189 [M − H]− (calcd for C17H19O6, 319.1182); 1H and 13C NMR data, see Tables 4 and 5. Spiroapplanatumine Q (20): yellow gum, [α]23D +14.3 (c 0.31, MeOH); UV (MeOH) λmax (log ε) 370 (3.47), 254 (3.79), 217 (4.09) nm; CD (MeOH) Δε229 −12.08, Δε320 −10.47, Δε365 +13.84; ESIMS m/z 277 [M − H]−; HRESIMS m/z 277.0722 [M − H]− (calcd for C14H13O6, 277.0790); 1H and 13C NMR data, see Tables 4 and 5. X-ray crystallographic data for 11: C17H18O6, M = 318.31, orthorhombic, a = 6.10060(10) Å, b = 15.0443(2) Å, c = 16.0425(2) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 1472.37(4) Å3, T = 100(2) K, space group P212121, Z = 4, μ(CuKα) = 0.914 mm−1, 9165 reflections measured, 2682 independent reflections (Rint = 0.0286). The final R1 values were 0.0274 (I > 2σ(I)). The final wR(F2) values were 0.0711 (I > 2σ(I)). The final R1 values were 0.0274 (all data). The final wR(F2) values were 0.0711 (all data). The goodness of fit on F2 was 1.056. Flack parameter = 0.08(13). The Hooft parameter is 0.07(4) for 1067 Bijvoet pairs. The deposition number CCDC 1447625 can be obtained free of charge from the Cambridge Crystallographic Data Centre.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00431. HRESIMS, CD, and 1D and 2D NMR spectra of new compounds (PDF) Crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Z.-C. Tu). *Tel/Fax: 86-871-65223048. E-mail: [email protected] (Y.-X. Cheng). ORCID

Yong-Xian Cheng: 0000-0002-1343-0806 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Science Fund for Distinguished Young Scholars (81525026), NSFCJoint Foundation of Yunnan Province (U1202222), and National Natural Science Foundation of China (21472199). We gratefully acknowledge support from the Guangzhou Branch of the Supercomputing Center of Chinese Academy of Sciences.

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REFERENCES

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Spiro Meroterpenoids from Ganoderma applanatum - Journal of

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