Antcamphins AâL, Ergostanoids from Antrodia camphorata - Journal of

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Antcamphins A−L, Ergostanoids from Antrodia camphorata Yun Huang,† Xionghao Lin,† Xue Qiao,† Shuai Ji,† Kedi Liu,† Chi-tai Yeh,‡ Yew-min Tzeng,*,§ Dean Guo,† and Min Ye*,† †

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People’s Republic of China ‡ Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei 11031, Taiwan § Institute of Biochemical Sciences and Technology, Chaoyang University of Technology, Taichung 41349, Taiwan S Supporting Information *

ABSTRACT: Twelve ergostanoids, named antcamphins A−L (1−12), together with 20 known triterpenoids, were isolated from fruiting bodies of the medicinal fungus Antrodia camphorata. Compounds 1 and 2 represent the first examples of norergostanes isolated from A. camphorata, and compounds 3 and 4 are the first pair of cis−trans isomers of ergostane-type triterpenoids containing an aldehyde group. Compounds 5− 12 are four pairs of C-25 epimers. The structures of 1−12 were elucidated on the basis of extensive spectroscopic data analysis including NMR and HRESIMS. Particularly, the absolute configurations at C-25 for 5−12 were determined by the modified Mosher’s method. These triterpenoids exhibited weak cytotoxic activities against MDA-MB-231 breast cancer cells and A549 lung cancer cells, but did not inhibit the growth of normal cells in the sulforhodamine B assay. antcin K,9 camphoratin A, camphoratin C,2 antcin F,10 3β,15αdihydroxylanosta-7,9(11),24-trien-21-oic acid, dehydrosulphurenic acid, sulphurenic acid,9 3-oxodehydrosulphurenic acid,11 15α-acetyl dehydrosulphurenic acid,12 24-methylenelanost-8ene-3β,15α,21-triol,13 dehydroeburicoic acid, and eburicoic acid,9 were isolated from an ethyl acetate extract of fruiting bodies of A. camphorata. Moreover, the absolute configurations of C-25 for the ergostanoids were determined by the modified Mosher’s method with an esterification reagent, 1-(9-anthryl)2,2,2-trifluoroethanol. The cytotoxicities of 16 compounds on the MDA-MB-231 breast cancer cell line, A549 lung cancer cell line, and HS68 normal foreskin fibroblast cell line were also evaluated.

Antrodia camphorata (synonyms: Taiwanof ungus camphoratus; Ganoderma camphoratum; Antrodia cinnamomea) (Polyporaceae family, Aphyllophorales) is a rare and precious parasitic fungus growing restrictedly on the inner heartwood wall of Cinnamomum kanehirae Hay (Lauraceae), an endangered tree endemic to Taiwan.1 The fruiting body of A. camphorata has long been used in Taiwan folk medicine for the treatment of food, alcohol, and drug intoxication, diarrhea, abdominal pain, hypertension, skin inflammation, and liver cancer.2 Previous studies on the chemical constituents of A. camphorata revealed it was rich in ergostane and lanostane triterpenoids, which showed anti-inflammatory, immunomodulatory, hepatoprotective, and antitumor activities.3,4 Interestingly, ergostanoids in A. camphorata are usually present as C-25 epimeric pairs. These epimers were extremely difficult to separate due to their similar properties. In addition, determining the configuration of C-25 was challenging due to flexibility of the side chain. Although 28 ergostanoids have been reported from A. camphorata, so far, only four epimeric pairs (antcin B, antcin C, antcin H, and antcin K) were obtained as pure compounds. Their absolute configurations were determined by the modified Mosher’s method.5,6 This information encouraged us to further explore structural diversity of ergostanoids from A. camphorata and to investigate their bioactivities. In the present study, 12 new ergostanoids, antcamphins A−L (1−12), together with 20 known triterpenoids, 25R-antcin H, 25S-antcin H, 25S-antcin B, 25R-antcin B, antcin A, 25S-antcin C, 25R-antcin C,7,8 25S-antcin K, 25R© 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

Antcamphin A (1) was obtained as a white, amorphous powder. The HRESIMS spectrum gave an [M + H]+ ion at m/z 405.2631 (calcd 405.2636), indicating a molecular formula of C24H37O5, five carbon atoms fewer than previously reported ergostanoids. The 13C NMR spectra displayed 24 signals, also indicating that 1 should have a pentanorergostane skeleton. The IR spectrum showed the presence of hydroxyl (3422 cm−1) and conjugated carbonyl (1674 cm−1) groups. The UV absorption maximum at 270 nm revealed the presence of a 8(9)-ene-7,11-dione moiety, which was corroborated by the Received: September 23, 2013 Published: January 3, 2014 118

dx.doi.org/10.1021/np400741s | J. Nat. Prod. 2014, 77, 118−124

Journal of Natural Products

Article

Figure 1. Key HMBC and 1H−1H COSY correlations for compounds 1 and 2.

was supported by the 13C NMR spectrum, which displayed 26 signals. The IR spectrum revealed the presence of a cyclohexanone group (1711 cm−1) and a conjugated carbonyl group (1676 cm−1), but no band for a hydroxyl group. The UV spectrum showed similar absorption maximum (ca. 270 nm) to compound 1, indicating the presence of a 8(9)-ene-7,11-dione moiety. This was corroborated by the carbon resonances at δC 202.6 (C-11), 200.8 (C-7), 152.0 (C-9), and 145.5 (C-8). The carbonyl group at δC 209.9 was assigned to C-3 due to the HMBC correlations of H-5 (1.85, m) and CH3-29 (1.01, d, J = 5.2 Hz) with C-3. The above evidence, together with the methyl signals for CH3-18 (0.67, s) and CH3-19 (1.58, s), suggested the structure of 2 was very similar to that of antcin B.8 Comparison of the 13C NMR data of 2 with those of antcin B indicated their structures differed in the side chain moiety. The HMBC correlations of CH3-21 (δH 0.79, brs)/C-22 (δC 18.3), H2-22 (δH 1.75, m; 1.25, m)/C-24 (δC 208.2), and CH325 (δH 2.09, s)/C-23 (δC 40.3) and C-24, together with the 1 H−1H COSY correlation of H2-22/H2-23 (Figure 1), indicated 2 had an aliphatic side chain containing a carbonyl group at C-24 and a terminal methyl at C-25. The NOE enhancement between CH3-19 (δH 1.58, s) and H-4 (δH 2.43, m) indicated the α-configuration of CH3-29, which was the same as antcin B. Therefore, compound 2 was identified as a derivative of antcin B with a 26,27,28-trinorergostane skeleton. Antcamphin C (3) was isolated as a white powder. The HRESIMS spectrum ([M + H]+ m/z 439.3206, calcd 439.3207) established the molecular formula C29H42O3. Its UV absorption maximum at 251 nm and IR absorption bands at 1638 and 1710 cm−1 suggested the presence of an 8(9)-en11-one moiety, which was confirmed by the carbon resonances at δC 199.3 (C-11), 156.9 (C-8), and 138.8 (C-9). The other quaternary carbonyl signal at δC 211.8 was assigned to C-3 due to its HMBC correlation with CH3-29 (δH 1.13, d, J = 6.4 Hz). The evidence above indicated the A, B, C, and D rings in 3 were the same as those in antcin A.8 The conjugated aldehyde signal at δC 191.2, along with the absence of a carboxyl signal, indicated that 3 had a different side chain from known ergostanoids of A. camphorata. The HMBC correlations of H26 (δH 10.29, s)/C-24 (δC 158.6), C-25 (δC 131.9), and C-27 (δC 10.6); CH3-27 (δH 1.84, s)/C-24, C-25, and C-26 (δC 191.2); and CH3-28 (δH 2.05, s)/C-23 (δC 33.9), C-24, and C25 suggested that the terminal carboxyl group at C-26 in antcin A was replaced by an aldehyde group in 3 and that the double bond at Δ24,28 moved to Δ24,25. The NOE enhancement between H-26 and CH3-28 indicated the trans-relationship of C-26 and C-23. Therefore, the structure of compound 3 was identified as 4α-methyl-(24E)-ergosta- 8(9),24(25)-diene-3,11dione-26-aldehyde.

carbon signals at δC 204.0 (C-11), 202.0 (C-7), 153.1 (C-9), and 144.4 (C-8). The 1H NMR, 13C NMR, and HSQC spectra of 1 showed the presence of four methyl groups at δH 1.53 (3H, s), 1.11 (3H, d, J = 6.4 Hz), 1.03 (3H, d, J = 6.4 Hz), and 0.80 (3H, s), suggesting that compound 1 contained a 4methylergosta-8(9)-ene-7,11-dione skeleton similar to that of antcin H.7 The hydroxyl groups at C-3 and C-12 were established by the HMBC correlations of CH3-29 (δH 1.03, d, J = 6.4 Hz)/C-3 (δC 69.2), H-3 (δH 3.85, brs)/C-1 (δC 28.6) and C-5 (δC 41.6), CH3-18 (δH 0.80, s)/C-12 (δC 81.0), and H-12 (δH 4.52, s)/C-11 (δC 204.0) and C-13 (δC 50.3). When compared with the 13C NMR data of antcin H, the side chain signals, including C-24(28) olefinic carbons, C-25 methine, C26 carboxyl, and C-27 methyl groups, in antcin H were replaced by a new secondary hydroxylated carbon at δC 59.7 in 1. Thus, compound 1 had a 24,25,26,27,28-pentanorergostane skeleton. The new carbon was assigned to C-23 based on the 1H−1H COSY correlation between H2-22 and H2-23 as well as the HMBC correlation of CH3-21 (δH 1.03, d, J = 6.4 Hz)/C-22 (δC 39.7, CH2) (Figure 1). The relative configuration of 1 was determined by 1H NMR and NOE experiments. H-3 (δH 3.85) showed a broad peak with a half-height width of 8.5 Hz, indicating that H-3 had an equatorial-axial coupling with H-4.14 Thus, 3-OH was determined to have an α-orientation. The αorientation of 12-OH was established by the NOE enhancements of H-12/CH3-18 and CH3-21. On the basis of the above evidence, compound 1 was identified as a derivative of antcin H with a 24,25,26,27,28-pentanorergostane skeleton. Antcamphin B (2) was obtained as a pale yellow syrup. The [M + H]+ ion at m/z 413.2692 (calcd 413.2686) in the HRESIMS spectrum corresponded to the molecular formula C26H37O4, indicating that 2 should be a trinorergostane. This 119



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Antcamphin D (4) has the same molecular formula as 3, according to HRESIMS analysis ([M + H]+ m/z 439.3209, calcd 439.3207). Their 1H and 13C NMR spectra were almost identical, indicating the structures were very similar. The only difference was chemical shifts for positions 26 (δH 10.32, s; δC 190.3), 27 (δH 1.78, s; δC 11.0), and 28 (δH 1.82, s; δC 22.1). We also observed that compounds 3 and 4 were unstable and could be interconverted at room temperature. They were assumed to be a pair of cis−trans-isomers. The cis-isomer, compound 4, produced a mixture of cis- and trans-forms on standing after the 1H and 13C NMR spectra were recorded. However, we were able to assign its NMR signals based on 2D NMR data of the mixture. The cis-configuration of 4 was confirmed by NOE experiment of the mixture, which showed enhancements of H-27/CH3-28 and H2-23/H-26 (Figure 2). Therefore, the structure of compound 4 was established as 4αmethyl-(24Z)-ergosta-8(9),24(25)-diene-3,11-dione-26-aldehyde.

Figure 3. Determination of the absolute configuration of C-25 by 1H NMR analysis of (R)- and (S)-1-(9-anthryl)-2,2,2-trifluoroethanonyl (AT) esters of compound 5.

pair of ergostane C-25 epimers. The NMR data for 7 and 8 were very similar to those of 5 and 6 except for an additional methyl signal at δH 3.67 (s) and δC 51.7, which suggested that 7 and 8 were methyl esters of 5 and 6. This was confirmed by the HMBC correlation from this new methyl group to C-26 (δC 176.8). Moreover, 7 and 8 could be converted into compounds 5 and 6 during long time storage, respectively. Therefore, the absolute configuration of C-25 for compounds 7 and 8 was determined to be 25S and 25R, respectively. HRESIMS analysis ([M + H]+ m/z 485.2892 and 485.2889, respectively) of antcamphins I (9) and J (10) established the molecular formula C29H40O6. Their UV maximum at 270 nm and IR absorptions at 1671, 1710, and 3499 cm−1 indicated the presence of an 8(9)-ene-7,11-dione moiety, a carboxyl group, and a hydroxyl group. Comparison of their 13C NMR data with those of antcin B suggested that compounds 9 and 10 should be hydroxylated products of antcin B.8 The additional hydroxyl group was assigned to C-12 due to the HMBC correlations of CH3-18 (δH 0.78, s)/C-12 (δC 80.6) and H-12 (δH 4.48, s)/C11 (δC 203.7). The NOE enhancements of H-12 with CH3-18 suggested that 12-OH was α-oriented. To determine the absolute configuration of C-25, we conducted the modified Mosher’s reaction of 10. After reaction with (R)- and (S)-1-(9anthryl)-2,2,2-trifluoroethanonyl, the 1H NMR spectra showed that CH3-27 of the (R)-AT-ester resonated at δH 1.32, while that of the (S)-AT-ester appeared at δH 1.29. Therefore, the absolute configuration of compound 10 was determined to be 25S. The configuration of C-25 in 9 was thus deduced to be 25R. HRESIMS analysis ([M + H]+ m/z 473.3262 and 473.3265, respectively) established the molecular formula of antcamphins K (11) and L (12) as C29H44O5. They showed identical UV, IR, and NMR spectra but different HPLC retention and, therefore, could be a pair of C-25 epimers. Their NMR spectra were similar to those of antcin C.8 The only difference was the presence of 3-OH in 11 and 12 rather than a carbonyl group, which was confirmed by the HMBC correlation of CH3-29 (δH 1.29, d, J = 6.4 Hz) and C-3 (δC 75.4). H-3 (δH 3.34) appeared as a broad multiplet with a half-height width of 33.5 Hz, indicating that H-3 had an axial−axial coupling with H-4.14 Therefore, 3-OH was β-oriented, which was confirmed by the NOE enhancement of H-3 with H-5. The β-configuration of 7OH was deduced by the NOE enhancements of H-7 with H-5 and H-14.5 The low amounts of compounds 11 and 12 prevented us from preparing Mosher’s esters for absolute

Figure 2. Key NOE enhancements for compounds 3 and 4.

Antcamphins E (5) and F (6) were obtained respectively as a white powder. HRESIMS analysis established their molecular formula as C29H42O6 ([M + H]+ m/z 487.3046 and 487.3049, respectively). Their UV, IR, and NMR spectra were identical, although they showed different high-performance liquid chromatography (HPLC) retention. These data suggested they could be ergostane C-25 epimers. The IR absorption bands at 1635, 1715, and 3433 cm−1 suggested the presence of an 8(9)-en-11-one moiety, a carboxyl group, and a hydroxyl group, respectively. Their 1H and 13C NMR spectra were similar to those of antcin C except for a new quaternary carbon signal at δC 76.3.8 Moreover, CH3-29 at δH 1.90 appeared as a singlet instead of a doublet. The HMBC correlation from CH329 to C-4 (δC 76.3) indicated that a hydroxyl group was substituted at C-4. The NOE enhancements of CH3-29/H-5 (δH 1.79, dd, J = 13.2, 0.2 Hz) and H-7 (δH 4.66, t, J = 8.4 Hz)/ H-5 and H-14 (δH 2.78, dd, J = 12.0, 7.2 Hz) demonstrated that compounds 5 and 6 had the same stereoconfigurations as antcin C for the tetracyclic core. To determine the spatial configuration of C-25, we conducted a modified Mosher’s reaction using (R)- and (S)-1-(9-anthryl)-2,2,2-trifluoroethanonyl (AT) to obtain (R)- and (S)-AT-esters of 5, respectively (Figure 3).5 The chemical shifts for CH3-18 and CH3-21 of the (S)-AT-ester were 0.72 and 0.79, respectively, which were significantly downfield compared to 0.62 and 0.72 for the (R)AT-ester. Therefore, the absolute configuration of C-25 for compound 5 was established as 25S, and thus the configuration for compound 6 was deduced to be 25R. Antcamphins G (7) and H (8) share the same molecular formula C30H44O6, as deduced from HRESIMS spectra ([M + H]+ m/z 501.3201 and 501.3202, respectively). They showed identical UV (λmax 254 nm), IR (νmax 1639, 1735, and 3350 cm−1), and NMR spectra, indicating that 7 and 8 were another 120



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Table 1. NMR Spectroscopic Data (400 MHz, pyridine-d5) for Compounds 1−4 1

2

position

δC, type

1

28.6, CH2

2

30.2, CH2

3 4 5 6

69.2, 35.3, 41.6, 38.6,

7

202.0, C

200.8, C

8 9 10 11 12

144.4, 153.1, 39.0, 204.0, 81.0,

145.5, 152.0, 38.6, 202.6, 57.4,

CH CH CH CH2

C C C C CH

13 14 15

50.3, C 42.8, CH 24.6, CH2

16

27.5, CH2

17 18 19 20 21 22

46.5, 11.7, 16.4, 33.2, 18.3, 39.7,

23

59.6, CH2

24 25 26 27 28 29

CH CH3 CH3 CH CH3 CH2

δH (J in Hz) 2.71, 1.90, 1.83, 1.29, 3.85, 1.66, 2.57, 2.60, 2.43,

m m m m brs m m m m

4.52, s

3.56, 2.84, 1.66, 1.98, 1.36, 2.31, 0.80, 1.53, 1.77, 1.11, 1.99, 1.40, 3.93, 3.87,

dd (12.4, 7.6) m m m m m s s m d (6.4) m m m m

1.03, d (6.4)

4

δH (J in Hz)

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

34.9, CH2

3.15, 1.39, 2.56, 1.91,

m m m m

35.4, CH2

3.40, 1.37, 2.56, 2.43,

m m m m

35.8, CH2

3.40, 1.37, 2.56, 2.43,

m m m m

2.43, 1.85, 2.39, 2.36,

m m m m

2.36, 1.31, 1.65, 1.31, 2.24, 2.06,

m m m m m m

2.36, 1.31, 1.65, 1.31, 2.24, 2.06,

m m m m m m

39.2, CH2 209.9, 43.9, 48.8, 37.7,

C CH CH CH2

C C C C CH2

47.2, C 49.4, CH 25.3, CH2 27.9, CH2 53.9, 12.1, 16.2, 35.4, 18.3, 29.6,


40.3, CH2 208.2, C 29.7, CH3

16.4, CH3

3

δC, type

11.5, CH3

38.1, CH2 211.8, 44.4, 50.7, 21.1,

C CH CH CH2

30.2, CH2

2.98, d (13.6) 2.49, d (13.6) 2.74, 2.72, 1.53, 1.84, 1.25, 1.30, 0.67, 1.58, 1.32, 0.79, 1.75, 1.25, 2.39, 2.29,

156.9, 138.8, 36.9, 199.3, 57.9,

C C C C CH2

47.3, C 53.0, CH 23.8, CH2

m m m m m m s s m brs m m m m

27.7, CH2 54.8, 12.0, 17.6, 36.4, 18.4, 33.1,


33.9, CH2

2.09, s

1.01, d (5.2)

158.6, 131.9, 191.2, 10.6, 17.2, 12.1,

C C CH CH3 CH3 CH3

2.93, d (14.0) 2.44, d (14.0) 2.56, 1.72, 1.42, 1.87, 1.25, 1.38, 0.71, 1.46, 2.47, 0.88, 1.61, 1.08, 2.14, 2.00,

m m m m m m s s m d (5.6) m m m m

10.29, s 1.84, s 2.05, s 1.13, d (6.4)

38.0, CH2 211.8, 44.4, 50.6, 21.4,

C CH CH CH2

30.8, CH2 156.9, 138.7, 37.3, 199.3, 57.9,

C C C C CH2

47.2, C 52.9, CH 23.8, CH2 27.6, CH2 54.7, 12.3, 18.0, 36.9, 18.7, 33.4,


35.4, CH2 159.3, 132.3, 190.3, 11.0, 22.1, 12.5,

C C CH CH3 CH3 CH3

2.93, d (14.0) 2.44, d (14.0) 2.56, 1.72, 1.42, 1.87, 1.25, 1.38, 0.71, 1.46, 2.47, 0.88, 1.61, 1.00, 2.17, 2.03,

m m m m m m s s m d (5.6) m m m m

10.32, s 1.78, s 1.82, s 1.13, d (6.4)

To our best knowledge, antcamphins A (1) and B (2) are the first examples of norergostanoids isolated from Antrodia camphorata, and compounds 3 and 4 are the first pair of cis− trans-isomers of ergostane-type triterpenoids containing an aldehyde group. Compounds 5/6, 7/8, 9/10, and 11/12 are four pairs of C-25 epimers, and their absolute configurations were determined by the modified Mosher’s method. In addition, the β-configuration of 3-OH in antcamphins K (11) and L (12) was first discovered in ergostanoids, although it is well known to occur in lanostanoids. From a biosynthetic point of view, compounds 1 and 2 could be derived from antcins H and B, respectively, through elimination of five or three carbon atoms. Compounds 3 and 4 could be biosynthetic precursors for antcin A, a major constituent of A. camphorata, considering that the aldehyde group could be oxidized into a carboxyl group. Compounds 5/6 and 11/12 are 4β- and 3βhydroxylated derivatives of antcin C, respectively. Compounds 9/10 are 12α-hydroxylated derivatives of antcin B. Antcamphins G (7) and H (8) are probably isolation artifacts because

configuration determination. However, we conducted reduction reactions of their analogue antcin B and found that 11 and 12 were the reduction product of 25S- and 25R-antcin B, respectively, by LC/MS analysis.15 Furthermore, 11 and 12 were purified from the reduction products of antcin B (epimeric mixture). Therefore, the configuration of compounds 11 and 12 was identified as 25S and 25R, respectively. To investigate the cytotoxic potential of the triterpenoids from Antrodia camphorata, breast cancer (MDA-MB-231), lung cancer (A549), and normal foreskin fibroblast cell lines (HS68) were incubated with each compound for 48 h. The effects on cell proliferation were determined by the sulforhodamine B assay.16 As a result, most of the ergostane-type triterpenoids showed IC50 values ranging from 22.0 to 93.5 μM in both breast and lung cancer cell lines (Table S5 in the Supporting Information). All the compounds were nontoxic to normal cells (HS68). We also noted that 25R ergostane stereoisomers showed higher activities than the corresponding 25S form for antcin B, antcin C, and antcin H. 121



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Table 2. NMR Spectroscopic Data (400 MHz, pyridine-d5) for Compounds 5−12a 5/6 position

δC, type

1

36.7, CH2

2

34.5, CH2

3 4 5 6

214.2, 76.3, 50.8, 30.5,

C C CH CH2

7 8 9 10 11 12

70.3, 155.4, 141.8, 37.7, 201.1, 58.4,

CH C C C C CH2

7/8

δH (J in Hz) 3.29, 1.60, 3.06, 2.50,

1.79, 2.66, 2.51, 4.66,

m m m m

dd (13.2, 0.2) m m t (8.4)

3.00, d (14.0) 2.48, d (14.0)

δC, type 36.7, CH2 34.4, CH2 214.2, 76.3, 50.6, 30.5,

C C CH CH2

70.3, 155.4, 141.9, 37.7, 201.1, 58.5,

CH C C C C CH2

9/10

δH (J in Hz) 3.30, 1.60, 3.07, 2.51,

1.79, 2.66, 2.51, 4.68,

m m m m

δH (J in Hz)

δC, type

34.8, CH2

3.01, 1.45, 2.52, 2.28,

34.4, CH2

37.7, CH2

dd (10.0, 0.2) m m t (8.0)

3.02, d (13.6)

11/12

δC, type

209.8, 43.8, 48.6, 39.3,

C CH CH CH2

200.4, 144.9, 151.1, 38.2, 203.7, 80.6,

C C C C C CH

m m m m

32.2, CH2

2.41, m 1.81, m 2.55, m

75.4, 39.1, 46.8, 32.7,

CH CH CH CH2

4.48, s

69.9, 155.1, 142.3, 37.5, 201.5, 58.7,

CH C C C C CH2

2.51, d (13.6) 13 14 15

47.8, C 53.8, CH 25.6, CH2

16

28.3, CH2

17 18 19 20 21 22

55.0, 12.5, 20.7, 36.2, 18.6, 34.5,

23

31.7, CH2

24 25 26 27 28

150.3, 46.8, 176.9, 17.2, 110.4,

29 OCH3


C CH C CH3 CH2

24.3, CH3

2.78, 2.56, 2.16, 1.93, 1.37,

dd (12.0, 7.2) m m m m

1.43, 0.88, 1.91, 1.42, 0.90, 1.75, 1.29, 2.41, 2.23,

m s s m d (5.6) m m m m

3.46, m 1.50, 5.24, 5.07, 1.55,

d (7.2) s s d (6.4)

47.7, C 53.6, CH 25.6, CH2 28.3, CH2

54.7, 12.5, 20.7, 36.1, 18.6, 34.3,


31.7, CH2 149.4, 45.8, 176.8, 16.7, 111.0,

C CH C CH3 CH2

24.3, CH3 51.7, CH3

m m m m

1.31, 1.40, 0.89, 1.91, 1.35, 0.88, 1.63, 1.24, 2.23, 2.05,

m m s s m d (5.2) m m m m

27.3, CH2

3.29, d (6.4) 1.35, 5.07, 5.01, 1.54, 3.66,

d (7.2) s s d (6.4) s

3.07, 1.76, 2.09, 1.91, 3.34, 1.66, 1.10, 2.49, 1.72, 4.53,

m m m m m m m m m t (8.0)

2.98, d (13.6) 2.49, d (13.6)

50.1, C 42.7, CH 24.5, CH2

2.80, 2.60, 2.19, 1.93,

δH (J in Hz)

46.0, 11.7, 16.4, 35.8, 18.1, 34.5,


31.9, CH2 150.3, 46.4, 177.6, 17.0, 110.5,

C CH C CH3 CH2

11.5, CH3

3.52, 2.84, 1.63, 1.97,

m m m m

1.31, 2.27, 0.78, 1.64, 1.46, 1.06, 1.74, 1.41, 2.22, 2.45,


3.44, d (6.8) 1.49, 5.21, 5.07, 0.99,

d (6.8) s s d (6.8)

48.0, C 53.7, CH 25.5, CH2 28.2, CH2

54.8, 12.5, 18.7, 36.2, 18.7, 34.5,


31.7, CH2 150.5, 46.7, 176.9, 17.2, 110.5,

C CH C CH3 CH2

15.8, CH3

2.77, 2.54, 2.12, 1.92,

dd (12.0, 7.4) m m m

1.34, 1.41, 0.87, 1.50, 1.41, 0.90, 1.29, 1.13, 2.40, 2.21,


3.46, d (7.2) 1.51, 5.24, 5.07, 1.29,

d (6.8) s s d (6.4)

a

Note: Compounds 5−12 are four pairs of 25-S/R epimers isolated in pure form from A. camphorata. The epimers showed identical NMR spectral data. Inc., China). Spots were visualized under UV light (365 nm) by spraying with 10% alcoholic sulfuric acid and heating at 105 °C. Column chromatography (CC) was performed using ODS C18 (DAISO Company, Japan), silica gel (200−300 mesh, Qingdao Marine Chemical Inc., China), or Sephadex LH-20 (GE Healthcare Bio-Science AB, USA). Semipreparative HPLC was performed on an Agilent 1200 instrument using a YMC Pack ODS-A column (250 mm × 10 mm, i.d., 5 μm, YMC Co. Ltd., Japan) eluted with 0.03% trifluoroacetic acid (TFA) in CH3CN−H2O or CH3OH−H2O at a flow rate of 2 mL/min. Preparative HPLC was performed on a Waters 2545 purification system using an Alltima ODS column (250 mm × 22 mm, i.d., 5 μm, Alltech Co. Ltd., USA) eluted with 0.05% TFA in CH3OH−H2O at a flow rate of 15 mL/min. Fungal Material. The fruiting bodies of Antrodia camphorata (BCRC 35396) were cultivated on wood sections of Cinnamomum kanehirae (Bull camphor tree) by one of the coauthors (Y.-M.T.). The cultivation continued for 12 months before harvesting. The rDNA ITS

they could not be detected in the EtOAc extract of A. camphorata by LC/MS analysis.

■

EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra (400 MHz for 1 H and 100 MHz for 13C) were obtained on a Bruker 400 MHz spectrometer in pyridine-d5 with TMS as reference, unless otherwise stated. NOE experiments were conducted using an Inova 600 MHz spectrometer. Optical rotations were measured on a Rudolph Autopol III automatic polarimeter. IR spectra were recorded as KBr disks on a Nicolet NEXUS-470 FT-IR spectrometer. UV spectra were measured on a Cary 300 Bio UV−visible spectrophotometer. HRESIMS spectra were recorded on a Bruker APEX IV FT-MS spectrometer. LC/MS analysis was conducted on an LCQ Advantage ion-trap mass spectrometer (ThermoFisher, San Jose, CA, USA). TLC was carried out on precoated silica gel GF254 plates (Qingdao Marine Chemical 122



Article

Antcamphin J (10): white powder (MeOH); [α]25D +100 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 270 (3.59) nm; IR (KBr) νmax 3499, 2963, 2933, 1710, 1671, 1458, 1238, 1063 cm−1; 1H NMR and 13 C NMR data, see Table 2; HRESIMS m/z 485.2889 [M + H]+ (calcd for C29H41O6 485.2898). Antcamphin K (11): white powder (MeOH); [α]25D +41 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 254 (3.58) nm; IR (KBr) νmax 3422, 2964, 1734, 1635, 1262, 1078 cm−1; 1H NMR and 13C NMR data, see Table 2; HRESIMS m/z 473.3262 [M + H]+ (calcd for C29H45O5 473.3262). Antcamphin L (12): white powder (MeOH); [α]25D +63 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 254 (3.36) nm; IR (KBr) νmax 3432, 2925, 1714, 1644, 1163, 1064 cm−1; 1H NMR and 13C NMR data, see Table 2; HRESIMS m/z 473.3265 [M + H]+ (calcd for C29H45O5 473.3262). Synthesis of (R)- and (S)-AT Esters. The absolute configurations for C-25 of ergostane epimers were determined by the modified Mosher’s method following the procedure reported by Du et al after minor modifications.5 The esterification reagent was 1-(9-anthryl)2,2,2-trifluoroethanol (AT). For instance, the (R)-AT-ester of 10 was prepared as follows. 10 (2.85 mg, 0.006 mmol), (R)-1-(9-anthryl)2,2,2-trifluoroethanol (1.63 mg, 0.006 mmol), 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (3.39 mg, 0.018 mmol), triethylamine (1.5 μL, 0.012 mmol), and 4-(dimethylamino)pyridine (1.07 mg, 0.009 mmol) were dissolved in 1 mL of deuterochloroform. The mixture was blended with ultrasound for 20 min and stood for 1 d. The reaction mixture was concentrated under reduced pressure and then partitioned between CHCl3 and H2O. The CHCl3 layer was evaporated to obtain the (R)-AT-ester of 10. The (S)-AT-ester of 10, together with (R)-/(S)-AT-esters of 5, 25R/S-antcin H, 25R/S-antcin B, 25R/S-antcin C, and 25R/S-antcin K, were prepared in similar procedures. 1H NMR spectra of these esters were determined directly without further purification. Reduction of 25R- and 25S-Antcin B. 25R-Antcin B (3 mg, 0.006 mmol) and 25S-antcin B (3 mg), each in pure form, were reacted with NaBH4 (0.35 mg, 0.010 mmol) in an ice bath for 10 min. The reaction products, together with pure compounds 11 and 12, were analyzed by LC/MS. Likewise, a mixture of 25R- and 25S-antcin B (100 mg) was reacted with NaBH4 (11.5 mg, 0.321 mmol), and compounds 11 (1.2 mg) and 12 (1.4 mg) were separated from the reaction products by repeated semipreparative HPLC. Sulforhodamine B Assay. Sulforhodamine B (SRB) (SigmaAldrich Chemie GmbH, Munich, Germany) was used to test the effects of 16 triterpenoids on cell growth and viability. Cisplatin was used as the positive control. Based on our previous method described by Yeh et al.,16 the compounds were dissolved in dimethylsulfoxide (DMSO) before diluting with the growth medium to a final DMSO concentration of

Antcamphins AâL, Ergostanoids from Antrodia camphorata - Journal of

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