Cytotoxic Lanostanoids from Poria cocos - Journal of Natural Products

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Cytotoxic Lanostanoids from Poria cocos Kuei-Hung Lai,†,‡,◆ Mei-Chin Lu,§,⊥,◆ Ying-Chi Du,† Mohamed El-Shazly,†,∥ Tung-Ying Wu,† Yu-Ming Hsu,† Astrid Henz,‡ Juan-Cheng Yang,∇,○ Anders Backlund,*,‡ Fang-Rong Chang,*,†,□,# and Yang-Chang Wu*,†,∇,○,¶ †

Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden § Graduate Institute of Marine Biology, National Dong Hwa University, Pingtung 944, Taiwan ⊥ National Museum of Marine Biology & Aquarium, Pingtung 944, Taiwan ∥ Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, Ain-Shams University, Organization of African Unity Street, Abassia, Cairo 11566, Egypt ∇ School of Pharmacy, College of Pharmacy, China Medical University, Taichung 40402, Taiwan ○ Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung 40447, Taiwan □ Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan # Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan ¶ Center for Molecular Medicine, China Medical University Hospital, Taichung 40447, Taiwan ‡

S Supporting Information *

ABSTRACT: Six new and 16 known lanostanoids were isolated from the sclerotia of Poria cocos. The structures of the new isolates were elucidated to be 16α-hydroxy-3-oxo-24methyllanosta-5,7,9(11),24(31)-tetraen-21-oic acid (1), 3β,16α,29-trihydroxy-24-methyllanosta-7,9(11),24(31)-trien21-oic acid (2), 3β,16α,30-trihydroxy-24-methyllanosta7,9(11),24(31)-trien-21-oic acid (3), 3β-acetoxy-16α,24βdihydroxylanosta-7,9(11),25-trien-21-oic acid (4), 3β,16αdihydroxy-7-oxo-24-methyllanosta-8,24(31)-dien-21-oic acid (5), and 3α,16α-dihydroxy-7-oxo-24-methyllanosta-8,24(31)-dien-21-oic acid (6), based on extensive spectroscopic analyses. The absolute configuration of 4 was determined using Mosher’s method. The antiproliferative activity of the isolated compounds (except 3 and 4) was evaluated against four leukemic cell lines (Molt 4, CCRF-CEM, HL 60, and K562). Dehydropachymic acid (9), dehydroeburicoic acid (12), pachymic acid (14), and lanosta-7,9(11),24-trien-21-oic acid (20) exhibited an antiproliferative effect on the CCRF-CEM cancer cell line with IC50 values of 2.7, 6.3, 4.9, and 13.1 μM, respectively. Both dehydropachymic acid (9) and dehydroeburicoic acid (12) showed antiproliferative effects against Molt 4 (IC50 13.8 and 14.3 μM) and HL 60 (IC50 7.3 and 6.0 μM) leukemic cell lines. Primary computational analysis using a chemical global positioning system for natural products (ChemGPS-NP) on the active lanostanoids from P. cocos suggested that targets other than topoisomerases may be involved in the antiproliferative activity.

Lanostanoids from P. cocos exhibited potent cytotoxic activity against several cancer cell lines including DU145 and LNCaP prostate carcinoma cell lines,4 human non-small-cell lung cancer A549 cells,5 H-ras transformed cells,6 CRL1579 cells, human leukemia (HL60) and melanoma (CRL1579) cells,7 human stomach cancer cells (NUGC-3), and human acute lymphoblastoid leukemia cells (Ball-1).8 They were also active against ovarian (NIH:OVCAR-3), breast (SK-BR-3), stomach (AZ521), and pancreatic (PANC-1) cancer cell lines.9 Lanostanoids target certain proteins and enzymes that are particularly important for inflammation and cancer develop-

Poria cocos (Schw.) Wolf (Polyporaceae) is a saprophytic fungus that parasitizes on the roots of Pinus densif lora or Pinus massoniana trees.1 It is widely distributed in China, Japan, and Korea.2 The sclerotia of this fungus, well known as f u-ling or hoelen, are used in traditional Chinese and Japanese medicine for their diuretic, sedative, and tonic effects.1 Several chemical constituents such as choline, ergosterol, histidine, β-pachyman, and lanostane-type triterpenoids were identified from P. cocos. Both polysaccharide and lanostanoids were considered to be the two major classes of secondary metabolites isolated from this fungus. Recent reports indicated that the polysaccharides are responsible for the immune-stimulating properties of the mushroom, while the lanostanoids are responsible for the antiinflammatory and cytotoxic activities.3 © 2016 American Chemical Society and American Society of Pharmacognosy

Received: June 22, 2016 Published: November 3, 2016 2805

DOI: 10.1021/acs.jnatprod.6b00575 J. Nat. Prod. 2016, 79, 2805−2813

Journal of Natural Products

Article

Chart 1. Lanostanoids from Poria cocos (Schw.) Wolf

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RESULTS AND DISCUSSION The dried sclerotia of P. cocos were refluxed four times in 95% ethanol at 75 °C, for 2 h each time. The ethanolic extract was fractionated into ethyl acetate- and water-soluble fractions. The fractionation of the EtOAc-soluble fraction yielded six new (1− 6) and 16 known lanostanoids including 3-(2-hydroxyacetoxy)5α,8α-peroxydehydrotumulosic acid (7),21 polyporenic acid C (8),22 dehydropachymic acid (9),23 dehydrotumulosic acid (10),23 3-epi-dehydrotumulosic acid (11),23 dehydroeburicoic acid (12),16 5α,8α-peroxydehydrotumulosic acid (13),10 pachymic acid (14),23 tumulosic acid (15),23 poriacosone B (16),24 poriacosone A (17),24 daedaleanic acid B (18),25 3β,16α-dihydroxylanosta-7,9(11),24-trien-21-oic acid (19),26 lanosta-7,9(11),24-trien-21-oic acid (20),27 16α-hydroxytrametenolic acid (21),26 and poricoic acid A (22).28 Compound 1 was isolated as a white, amorphous powder. Its molecular formula was deduced as C31H44O4 by the analysis of its 13C NMR and HRESIMS data, which suggested 10 degrees

ment. They inhibit TPA (12-O-tetradecanoylphorbol-13acetate)-induced inflammation,10−12 DNA polymerases,8,13 and DNA topoisomerases I and II.8,14 In the past decade our group has focused on studying the cytotoxic effect of triterpenoids isolated from medicinal mushrooms.15−17 Among the compounds identified, dehydroeburicoic acid was the most potent. It is a lanostanoid derivative18 that was isolated from Antrodia cinnamomea19 and P. cocos.20 Dehydroeburicoic acid exhibited potent antileukemic effects in both in vitro and in vivo assays,16 and this study was the first report on the in vivo antitumor activity of a lanostanoid derivative. The promising bioassay results along with the previous reports indicating that more than 50 lanostanoids were isolated from P. cocos3 motivated us to conduct further investigations on this fungus to identify other dehydroeburicoic acid-like compounds possessing antileukemic activity. 2806



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Table 1. 1H NMR Spectroscopic Data of 1−6 (Measured at 400 and 600 MHz in C5D5N, δ in ppm, J in Hz) 1a

position 1 2

2b

2.01, m 1.81, dt (13.8, 5.4) 2.63, dd (9.9, 5.7)

1.96, m 1.46, m 1.92, m

dd (10.8, 4.8) m m m d (6.0) d (6.0)

3.37, 1.32, 2.22, 2.16, 5.76, 5.42,

t (7.5) dd (12.0, 3.6) dd (6.6, 3.6) d (12.0) d (6.6) d (5.4)

d (18.0) m m d (13.2) t (6.0) dd (11.2, 6.0) s s m m m m m

2.84, 2.48, 2.59, 2.42, 4.61, 3.07, 1.18, 1.08, 3.00, 2.70, 2.43, 2.56, 2.42,

d (17.4) m d (13.2) m t (6.9) dd (11.4, 6.0) s s m m m m m

6.03, d (6.6)

7 11

5.76, d (6.6) 5.52, d (4.8)

4.24, 1.99, 2.30, 2.20, 5.58, 5.42,

12

2.74, 2.55, 2.47, 1.99, 4.56, 2.90, 1.09, 1.06, 2.96, 2.66, 2.46, 2.56, 2.41,

2.72, 2.44, 2.46, 1.90, 4.53, 2.89, 1.09, 1.15, 2.96, 2.66, 2.49, 2.55, 2.42,

15 16 17 18 19 20 22 23

4a

2.00, m 1.56, m 2.01, m

3 5 6

d (18.0) m dd (12.6, 9.0) d (12.6) t (6.9) dd (11.1, 5.7) s s td (10.5, 2.4) m m m m

3a

24 25 26

2.29, quin (6.6) 1.01, d (6.6)

2.29, quin (6.8) 1.00, d (6.8)

2.29, quin (6.6) 1.01, d (6.6)

27 28 29

1.00, d (6.6) 1.25, s 1.43, s

1.00, d (6.6) 1.11, s 1.11, s

30

1.49, s

0.99, 1.16, 4.17, 3.71, 1.46,

31

4.99, s 4.86, s

4.98, s 4.85, s

d (6.8) s d (10.8) d (10.8) s

4.48, 3.86, 4.98, 4.86,

d (10.2) d (9.6) s s

3-OAc a

1.90, 1.42, 1.78, 1.72, 4.71, 1.27, 2.07,

m m m m dd (12.0, 4.2) dd (9.6, 5.4) m

5.56, br s 5.31, br s 2.65, 2.41, 2.41, 1.90, 4.57, 2.89, 0.99, 1.01, 2.97, 2.38, 1.24, 2.25, 2.17, 4.52,

m m m d (12.6) br t m s s m m m m m br t

4.87, 5.19, 1.88, 0.90, 0.99,

s s s s s

1.48, s

5b

6b

1.67, m 1.29, m 1.88, m

1.44, m

3.42, t (7.6) 1.74, dd (11.6, 5.6) 2.63, m

2.18, m 2.12, 2.07, 2.91, 2.80, 4.56, 2.78, 1.10, 1.13, 2.93, 2.66, 2.50, 2.56, 2.41,

m m d (14.4) m t (7.0) m s s m m m m m

2.20, 1.82, 3.60, 2.57, 2.58,

m m br s m m

2.33, 2.18, 2.14, 2.05, 2.88, 2.80, 4.54, 2.78, 1.11, 1.14, 2.92, 2.67, 2.49, 2.55, 2.40,

m m m m d (14.0) m t (6.8) m s s m m m m m

2.29, quin (6.8) 1.00, d (6.8)

2.30, quin (6.8) 1.00, d (6.8)

1.00, d (6.8) 1.12, s 1.06, s

0.99, d (6.8) 1.09, s 0.91, s

1.60, s

1.49, s

5.00, s 4.86, s

4.99, s 4.85, s

2.05 s b

Measured at 600 MHz. Measured at 400 MHz.

was confirmed by the HMBC correlations (Figure 1) from H-6 to C-4, C-5, C-7, and C-8; from H-7 to C-4, C-5, C-6, C-9, and C-14; and from H-11 to C-8 and C-10. The IR absorptions of OH (3410 cm−1) and CO (1704 cm−1), together with the carbon signals at δC 214.5 and 76.4 and the proton signals at δH 4.56 (t, J = 6.9 Hz), indicated the presence of a carbonyl and hydroxy groups. The HMBC cross-peaks (Figure 1) from H-1, H-2 to C-3 (δC 214.5) and from H-15, H-17 to C-16 (δC 76.4) indicated the attachment of the carbonyl group to C-3 and the hydroxy group to C-16, respectively. The lanostanoid sidechain was identified as a 24(31)-ene-21-oic side-chain by the comparison of its 1D and 2D NMR data to those of compound 8. The ROESY cross-correlations of H-16/H3-18, H-16/H-20, and H-17/H3-30 supported the α-, α-, and β-orientation assignments of OH-16, H-17, and H-20, respectively. Thus, compound 1 was elucidated as 16α-hydroxy-3-oxo-24-methyllanosta-5,7,9(11),24(31)-tetraen-21-oic acid. Compound 2, a white, amorphous powder, showed the molecular formula C31H48O5 determined by HRESIMS and 13C NMR data. The IR spectra indicated the presence of OH (3406 cm−1) and CC (1638 cm−1) functionalities. The 1H (Table

of unsaturation. The IR spectrum showed absorptions for OH (3410 cm−1), CO (1704 cm−1), and CC (1638 cm−1) functionalities. The 1H NMR data (Table 1) showed five tertiary methyls at δH 1.06 (s), 1.09 (s), 1.25 (s), 1.43 (s), and 1.49 (s), two secondary methyls at δH 1.00 (d, J = 6.6 Hz) and 1.01 (d, J = 6.6 Hz), an oxymethine proton at δH 4.56 (t, J = 6.9 Hz), three olefinic protons at δH 6.03 (d, J = 6.6 Hz), 5.76 (d, J = 6.6 Hz), and 5.52 (br d, J = 4.8 Hz), and an olefinic methylene at δH 4.86 (s) and 4.99 (s). The 13C NMR data (Table 2) revealed 31 carbon signals sorted by HSQC and DEPT. They included a carbonyl carbon at δC 214.5, a carboxylic carbon at δC 178.8, and eight olefinic carbons at δC 156.1, 150.2, 143.0, 140.9, 122.0, 117.5, 116.1, and 107.0, and an oxymethine carbon at δC 76.4. Detailed analysis of these NMR data suggested that compounds 1 and 8 are closely related, with the only difference being an Δ5,7,9(11)-triene system in the B/C ring [1: δC 150.2 (C-5), δH 6.03 (H-6)/δC 117.5 (C-6), δH 5.76 (H-7)/δC 116.1 (C-7), δC 140.9 (C-8), δC 143.0 (C-9), δH 5.52 (H-11)/δC 122.0 (C-11); 8: δH 1.62 (H-5)/δC 51.0 (C-5), δC 23.8 (C-6), δC 120.6 (C-7), δC 142.8 (C-8), δC 144.7 (C-9), δC 117.6 (C-11)]. This double conjugated system 2807



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Table 2. 13C NMR Spectroscopic Data of 1−6 (Measured at 100 and 150 MHz in C5D5N, δ in ppm)

a

position

1a

2b

3a

4a

5b

6b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 3-OAc

33.6 34.2 214.5 49.0 150.2 117.5 116.1 140.9 143.0 40.3 122.0 37.0 45.1 49.0 44.1 76.4 57.4 17.7 30.4 48.5 178.8 31.4 33.2 156.1 34.1 21.9 22.0 29.5 27.5 25.8 107.0

36.1 28.2 73.0 43.1 42.6 23.3 121.2 142.6 146.3 37.7 116.5 36.3 45.1 49.4 44.4 76.4 57.6 17.6 23.6 48.6 178.4 31.5 33.2 156.1 34.1 21.8 22.0 13.3 67.3 26.5 107.0

36.2 28.6 77.9 39.3 49.5 23.6 124.8 138.0 146.8 38.0 117.0 35.9 45.5 55.6 39.2 77.1 58.4 18.8 22.9 48.7 178.9 31.5 33.2 156.1 34.1 21.9 22.0 28.7 16.6 66.2 107.0

35.6 24.5 80.6 37.8 49.6 23.1 120.5 142.9 145.6 37.6 117.3 36.2 45.1 49.3 44.3 76.0 57.4 17.8 22.8 48.9 178.5 29.2 34.4 75.6 150.1 110.3 17.7 28.2 17.1 26.6

35.1 28.3 77.0 39.5 50.5 37.1 198.7 139.2 164.9 40.1 23.5 29.0 46.6 46.7 45.8 76.9 55.9 17.9 18.3 48.8 179.8 31.6 33.3 156.2 34.1 21.9 22.0 27.8 16.0 26.2 106.9

29.7 26.4 74.0 38.0 44.7 36.9 198.8 139.2 165.6 40.2 23.3 28.9 46.6 46.8 45.8 76.9 55.9 17.9 18.2 48.9 179.4 31.6 33.3 156.2 34.1 21.9 22.0 28.2 22.0 26.2 106.9

disappearance of a methyl group signal. A substitution with CH2OH at position 29 can be deduced by HMBC cross-peaks from H-5 and H3-28 to C-29 and from H2-29 to C-3, C-4, and C-28 (Figure 1). The ROESY correlations of H2-29/H3-19, H229/Hβ-6, H3-28/H-3, and H3-28/H-5 confirmed that the additional hydroxy group is connected to the β-oriented C-29 rather than to the α-oriented C-28. Thus, compound 2 was identified as 3β,16α,29-trihydroxy-24-methyllanosta7,9(11),24(31)-trien-21-oic acid. The same structure has been named as 29-hydroxydehydrotumulosic acid by the Cai group in 2011.29 Differences in the NMR assignments between 2 and 29-hydroxydehydrotumulosic acid were observed, especially at positions 3, 4, 5, 15, and 28 [2 (C5D5N, at 400 MHz): δH 4.24 (H-3)/δC 73.0 (C-3), δC 43.1 (C-4), δH 1.99 (H-5)/δC 42.6 (C-5), δ H 1.90, 2.46 (H 2 -15), δ C 13.3 (C-28); 29hydroxydehydrotumulosic acid29 (C5D5N, at 500 MHz): δH 3.52 (H-3)/δC 78.3, δC 34.7 (C-4), δH 1.60 (H-5)/δC 51.4 (C5), δH 4.59 (H2-15), δC 27.2 (C-28)]. The NMR data of 2 and 29-hydroxydehydrotumulosic acid were compared to those of compound 10 [10 (C5D5N, at 400 MHz): δH 3.44 (H-3)/δC 78.0 (C-3), δC 39.3 (C-4), δH 1.34 (H-5)/δC 49.8 (C-5), δH 1.93, 2.42 (H2-15), δC 28.8 (C-28)]. An electronegative substituent (−OH) can cause α- and β-effects30 on the downfield-shifting signals of C-29 and C-4 as well as a γeffect30 on the upfield-shifting signals of C-3, C-5, and C-28. Detailed analysis of the NMR data of 2, 10, and 29hydroxydehydrotumulosic acid focusing on these positions (Figure 2) revealed the consistency of the corresponding

170.5 21.1

Figure 2. Inspections for α-, β-, and γ-effects with an additional 29hydroxy substitution. Label with red: downfield shifting compared to 10; label with blue: upfield shifting compared to 10.

Measured at 150 MHz. bMeasured at 100 MHz.

1), 13C (Table 2), DEPT, and HSQC NMR spectra displayed six methyls, one olefinic methylene, one oxymethylene, two olefinic methines, two oxymethines, three olefinic tertiary carbons, and one carboxylic carbon. The NMR data of 2 resembled those of the known compound 10 with the exception of an additional oxymethylene signal at δC 67.3 [δH 3.71 (d, J = 10.8 Hz), δH 4.17 (d, J = 10.8 Hz)] and the

signals in 2 and the prediction that showed the downfield αand β-effects at C-29 (ΔδC = +50.7) and C-4 (ΔδC = +3.8) and the upfield γ-effect at C-3 (ΔδC = −5.0), C-5 (ΔδC = −7.2), and C-28 (ΔδC = −15.5). In contrast, 29-hydroxydehydrotumulosic acid violated the rule at C-4 (ΔδC = −4.6), C-3 (ΔδC =

Figure 1. Key HMBC correlations (from H to C) for compounds 1−6. 2808



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+0.3), and C-5 (ΔδC = +1.6). Furthermore, the peak at δH 4.59 in the 29-hydroxydehydrotumulosic acid was assigned to H-15, which is an abnormal downfield shift for a sp3 methylene signal without any attached electronegative substituent. Accordingly, the proposed assignment of compound 2 was in line with the accepted rules of assignments for this class of compounds and with the previously reported data for similar structures. Compound 3 was obtained as a white, amorphous powder. The molecular formula was determined as C31H48O5, based on the [M + Na]+ pseudomolecular HRESIMS ion peak and 13C NMR data. The IR spectrum showed an absorption of a hydroxy group and another absorption for a CC functionality at 3388 and 1652 cm−1, respectively. The 13C (Table 2) and DEPT data suggested the presence of 31 carbons that were identical to those of 2, including a carboxylic carbon at δC 178.9, six olefinic carbons at δC 156.1, 146.8, 138.0, 124.8, 117.0, and 107.0, two oxymethine carbons at δC 77.9 and 77.1, and an oxymethylene carbon at δC 66.2. A pair of characteristic doublet oxymethylene protons at δH 3.86 (d, J = 9.6 Hz) and 4.48 (d, J = 10.2 Hz), as well as a carbon signal at δC 66.2 (Table 1), indicated the presence of an OH substitution at a methyl group. The HMBC correlations (Figure 1) from the oxymethylene protons (δH 3.86 and 4.48) to C-8, C-13, and C15 and from H-15 to δC 66.2 suggested that OH is connected to C-30. Since a hydroxy group was attached to C-30, carbons 8, 14, and 15 followed the β- and γ-effects.30 They displayed a downfield effect at C-14 (ΔδC = +6.2) and an upfield effect at C-8 (ΔδC = −4.7) and C-15 (ΔδC = −5.2). According to the ROESY correlation data of Hα-12/H2-30 and H-17/H2-30, the orientation of CH2OH substitution at C-30 was assigned as α. Consequently, compound 3 was established as 3β,16α,30trihydroxy-24-methyllanosta-7,9(11),24(31)-trien-21-oic acid. The molecular formula of compound 4 was shown to be C32H48O6, from the HRESIMS and 13C NMR data, suggesting nine degrees of unsaturation. The IR spectrum suggested the presence of OH (3397 cm−1), aliphatic ester carbonyl (1733 cm−1), and CC (1652 cm−1) functionalities. Detailed analysis of 1H (Table 1) and 13C (Table 2) NMR data suggested that the structure of 4 was similar to that of 9 with the exception of a hydroxy group bonded to C-24 instead of a methylene group. The 7,9(11)-diene system in the B/C ring and the 3β-acetoxy and 16α-hydroxy groups can be supported by the HMBC (Figure 1) (from H-7 and H3-19 to C-9; from H-11 and H3-30 to C-8; from H-1, H-2, and H3-29 to C-3; and from H-15 and H-17 to C-16) and ROESY (H-3/H-5 and H-3/H3-28; H-16/ H3-18 and H-16/H-20) cross-peaks. The presence of a 24βhydroxy-25-en-21-oic side-chain was suggested based on the HMBC (Figure 1) correlations from a pair of terminal olefinic methylenes at δH 4.87 and 5.19 (H-26) to the oxymethine carbon at δC 75.6 (C-24) and the methyl carbon at δC 17.7 (C27), as well as the 2J and 3J HMBC cross-correlations from a deshielded methyl group at δH 1.88 (H3-27) to δC 75.6 (C-24) and δC 150.1 (C-25), respectively. In order to determine the configuration at C-24, the (S)- and (R)-MTPA ester derivatives, 4s and 4r, were synthesized with (−)-(R)-MTPA-Cl and (+)-(S)-MTPA-Cl, respectively. The Δδ-values indicated a 24(R) configuration (Figure S74). On the basis of the above information, the structure of compound 4 was established as 3β-acetoxy-16α,24β-dihydroxylanosta-7,9(11),25-trien-21-oic acid. Compound 5, a white, amorphous powder, was found to possess a molecular formula of C31H48O5 based on the quasimolecular ion peak in the HRESIMS and 13C NMR data,

suggesting eight degrees of unsaturation. The absorptions for OH (3397 cm−1) and CC (1652 cm−1) functionalities were detected from the IR spectrum. The 1D NMR data (Tables 1 and 2) combined with the HSQC spectra revealed the presence of five tertiary methyls at δH 1.06, 1.10, 1.12, 1.13, and 1.60, an isopropyl group with two methyls at δH 1.00 (d, J = 6.8 Hz) and 1.00 (d, J = 6.8 Hz), two oxymethines at δH 3.42 (δC 77.0) and 4.56 (δC 76.9), one terminal olefinic methylene at δH 4.86 and 5.00 (δC 106.9), two tertiary olefinic carbons at δC 139.2 and 164.9, a carboxylic carbon at δC 179.8, and a carbonyl carbon at δC 198.7. The 1H NMR spectrum of 5 was very similar to the spectrum of compound 15 with a slight difference in 13C NMR: an additional carbonyl carbon at δC 198.7 in the 13C NMR spectrum of 5 along with downfield shifts of two tertiary olefinic carbons (from δC 134.8 and 134.9 to δC 139.2 and 164.9). According to the NMR data, the attachment of the carbonyl group to the B/C ring system was suggested based on the HMBC cross-correlations (Figure 1) from δH 1.74 (H-5) and 2.63 (H-6) to the carbonyl carbon at δC 198.7. Also, the carbonyl carbon was assigned at position 7. The ROESY crosspeaks of H-3/H-5, H-3/H3-28, H-16/H-18, and H-16/H-20 suggested the β- and α-orientation assignments of OH-3 and OH-16, respectively. The 24(31)-en-21-oic side-chain from C17 was suggested by comparison of the 1D and 2D NMR data of 5 to that of compound 15. Hence, the structure of compound 5 was established as 3β,16α-dihydroxy-7-oxo-24methyllanosta-8,24(31)-dien-21-oic acid. Compound 6, a white, amorphous powder, gave a pseudomolecular ion peak, and the analysis of its HRESIMS and 13C data suggested a molecular formula of C31H48O5, with eight degrees of unsaturation. The IR spectrum indicated the presence of OH and CC functional groups based on the absorptions at 3407 and 1652 cm−1, respectively. The 1H (Table 1), 13C (Table 2), and HSQC NMR spectra revealed the presence of five tertiary methyls at δH 0.91, 1.09, 1.11, 1.14, and 1.49, an isopropyl group with two methyls at δH 0.99 (d, J = 6.8 Hz) and 1.00 (d, J = 6.8 Hz), two oxymethines at δH 3.60 (δC 74.0) and 4.54 (δC 76.9), one terminal olefinic methylene at δH 4.85 and 4.99 (δC 106.9), two tertiary olefinic carbons at δC 139.2 and 165.6, a carboxylic carbon at δC 179.4, and a carbonyl carbon at δC 198.8. Detailed analysis of the 1D and 2D NMR data of both compounds 5 and 6 suggested that they were diastereomers. The orientation of the hydroxy group at C3 was different based on the coupling patterns at H-3 and the ROESY correlations [5: δH 3.42 (t, J = 7.6 Hz) (H-3)/δC 77.0 (C-3); ROESY: H-3/H-5, H-3/H3-28; 6: δH 3.60 (br s) (H-3)/ δC 74.0 (C-3); ROESY: H-3/H3-29]. Moreover, the β-oriented assignment of OH-3 in compound 5 placed H-3 at an axial position in the chair form of the A ring, resulting in a larger coupling constant (t, J = 7.6 Hz) with H2-2.31 On the other hand, the orientation of H-3 in 6 was equatorial, as suggested by the small coupling constant with H2-2, resulting in a broad singlet. Accordingly, compound 6 was elucidated as 3α,16αdihydroxy-7-oxo-24-methyllanosta-8,24(31)-dien-21-oic acid. In this study, we isolated six novel lanostane-type triterpenoids (1−6) from the sclerotia of P. cocos. Compound 1 was found to possess a unique 5,7,9(11)-triene at the B/C ring, whereby this double conjugated system in lanostanoids from P. cocos has never previously been reported. Chung et al. reported the isolation of a lanostanoid with a triene at the B/C ring from hulls of Oryza sativa.32 However, no lanostanoid has ever been isolated from the hulls of O. sativa except in this 2809



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Scheme 1. Plausible Biogenetic Pathway

Table 3. Antiproliferative Activity (IC50, μM)a compound 8 9 12 14 15 20 doxorubicinc a

Molt 4 27.6 13.8 14.3 8.6 30.1 16.2 0.01

± ± ± ± ± ± ±

0.5 2.1 2.9 4.3 9.8 3.0 0.001

CCRF-CEM 25.5 2.7 6.3 4.9 32.2 13.1 0.01

± ± ± ± ± ± ±

2.3 2.1 2.1 1.2 4.4 2.7 0.001

HL 60 31.8 7.3 6.0 15.4 20.0 20.0 0.01

± ± ± ± ± ± ±

5.2 2.5 1.6 8.1 2.5 1.3 0.001

K562 26.3 18.6 25.6 25.5 NAb 14.8 0.5

± ± ± ±

1.8 5.1 12.1 9.2

± 5.3 ± 0.2

The other tested compounds were inactive, with IC50 > 40 μM. bNA (nonactive) = IC50 > 40 μM. cPositive control.

report. Even the hulls of O. sativa are not known as a source of triterpenoids. Compound 1 might also act as an intermediate in the biosynthetic pathway of 6α-hydroxypolyporenic acid C to compounds 8 and 13 (Scheme 1).33,34 Compounds 2 and 3 possess characteristic hydroxy substitutions on the terminal methyl groups, and the CH2OH group on position 30 was identified for the first time in lanostanoids isolated from P. cocos.1 The 24α-hydroxy-25-en-21-oic side-chain in lanostanoids of P. cocos was observed only in compound 4. Compounds 5 and 6 showed novel 7-oxo B rings that have never been found in other lanostanoids of P. cocos. The characteristic 3-O-hydroxyacetoxy group in compound 7 suggested that this compound might be formed from compound 13, which carries the same functional group (Scheme 1).34 Four of the new compounds (except 3 and 4 because they were isolated in small quantities), along with the other 16 known lanostane-type triterpenoids (7−22), were evaluated for their antiproliferative activity against Molt 4, CCRF-CEM, HL 60, and K562 leukemic cell lines. Compounds 9, 12, 14, and 20 showed potent antiproliferative effects against CCRF-CEM human T cell lymphoblastic leukemia cells with IC50 values of 2.7, 6.3, 4.9, and 13.1 μM, respectively. Both 9 and 12 demonstrated antiproliferative effects against both Molt 4 human acute lymphoblastic leukemia (IC50 13.8 and 14.3 μM) and HL 60 human promyelocytic leukemia (IC50 7.3 and 6.0 μM). Compound 12 also showed antiproliferative activity on NUGC-3 and Ball-1 cancer cell lines8 through its inhibitory effect on topoisomerases in a previous study. Both compounds 12 and 22 inhibited HL 60 cell growth, while 12 showed

further antitumor effect in an HL 60 xenograft animal model.16,28 Compound 14 exhibited potent cytotoxic activity against A549 cells5 and induced apoptotic effects on DU145 and LNCaP prostate cancer cells.4 Moreover, preliminary studies revealed that compounds 9, 14, and 15 inhibited cell growth of human colon carcinoma, and it was suggested that the antiproliferative effect of this class of compounds was mediated through the inhibition of DNA topoisomerases I and II.14 ChemGPS-NP (chemical global positioning system for natural products) is a computational model based on the principal component analysis (PCA) of physical−chemical properties. These properties can be calculated from simplified molecular input line entry specification (SMILES) structure data and by performing score prediction in the ChemGPS-NP model. This system provides a bioinformatics tool for charting and navigating the biologically relevant chemical space.35 Smallscale ChemGPS-NP applied analysis plays a role in discussing structure−activity relationships, while large-scale analysis can reveal the trends and predict the possible bioactivities or targets for specific groups of compounds. As shown in Figure 3, lanostanoids isolated from P. cocos were plotted in ChemGPSNP using the calculated prediction scores of the online tool ChemGPS-NPWeb (http://chemgps.bmc.uu.se) together with the previously studied topoisomerase I and II inhibitors from our ChemGPS-NP database. The active lanostanoids explored particularly in the third dimension (PC3, which describes lipophilicity, polarity, and H-bond capacity) formed a cluster in the analyzing graphic. Since none of the active compounds appear in the clusters of the topoisomerase I and II inhibitors groups, and the previous studies did not demonstrate solid evidence for the inhibitory effect on topoisomerases I and II, it 2810



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plate. Silica gel 60 (Merck, 40−63 μm and 63−200 μm) were used for column chromatography. A Shimadzu LC-20AT pump and a Shimadzu SPD-20A UV−vis detector (Shimadzu Inc., Kyoto, Japan) along with a COSMOSIL 5C18-MS-II Waters (20 × 250 mm, C18) column were used for HPLC. Fungal Material. The dried sclerotia of P. cocos were provided by Sun-Ten Pharmaceutical Co., Ltd. and the nonprofit Organization Brian Research Institute of Taiwan. A voucher specimen (SPC110307) was deposited in the Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan. Extraction and Isolation. The dried sclerotia of P. cocos (4.7 kg) were extracted by reflux using 95% ethanol (w/v = 1:10) at 75 °C for 2 h, and the process was repeated four times. An ethanolic extract weighing 76.7 g was obtained (extraction rate: 1.63%). The ethanolic extract was partitioned between EtOAc and H2O (1:1, v/v), yielding the EtOAc- (24.7 g) and H2O-soluble fractions. The EtOAc-soluble fraction (24.7 g) was subjected to normal-phase column chromatography on silica gel 60 (Merck, 63−200 μm, column size: 15 × 50 cm), using gradients of CHCl3−MeOH (100:0, 30:1, 20:1, 10:1, 5:1) providing 11 subfractions (SPC1−11). Subfraction SPC5 (4.3 g) was further fractionated into six fractions by normalphase column chromatography on silica gel 60 (Merck, 40−63 μm, column size: 5 × 25 cm, mobile phase: CHCl3−MeOH, 25:1). Subfraction SPC5-3 (775.1 mg) was subjected to reversed-phase HPLC (C18, column size: 20 × 250 mm) eluted with the mobile phase MeOH−H2O, 92:8 (0.1% acetic acid in H2O), resulting in the separation of 1 (2.4 mg), 8 (39.8 mg), 9 (67.2 mg), 12 (3.9 mg), 14 (111.0 mg), and 20 (15.8 mg). Subfraction SPC5-6 (475.3 mg) was chromatographed over reversed-phase HPLC (C18, column size: 20 × 250 mm, mobile phase: MeOH−H2O, 92:8, 0.1% acetic acid in H2O) to furnish 7 (2.6 mg), 10 (76.1 mg), 11 (9.8 mg), 13 (8.7 mg), 15 (125.2 mg), 19 (4.7 mg), and 21 (3.3 mg). Subfraction SPC8 (2.0 g) was subjected to normal-phase fractionation on silica gel 60 (Merck, 40−63 μm, column size: 5 × 25 cm, mobile phase: CHCl3−MeOH, 15:1), which yielded seven subfractions. Subfraction SPC8-5 (356.0 mg) was purified over reversed-phase HPLC (C18, column size: 20 × 250 mm, mobile phase: MeOH−H2O, 85:15, 0.1% acetic acid in H2O) to obtain 5 (5.7 mg), 6 (8.3 mg), 13 (15.0 mg), 16 (12.5 mg), 17 (6.4 mg), and 18 (17.2 mg). Subfraction SPC8-6 (287.5 mg) was further purified by reversed-phase HPLC (C18, column size: 20 × 250 mm, mobile phase: MeOH−H2O, 80:20, 0.1% acetic acid in H2O) to obtain 3 (3.0 mg) and 4 (3.3 mg). The purification of subfraction SPC8-7 (87.5 mg) using reversed-phase HPLC (C18, column size: 20 × 250 mm, mobile phase: MeOH−H2O, 85:15, 0.1% acetic acid in H2O) led to the separation of 2 (2.2 mg) and 22 (4.3 mg). 16α-Hydroxy-3-oxo-24-methyllanosta-5,7,9(11),24(31)-tetraen-21-oic acid (1): white, amorphous powder; [α]25 D +102.5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 242 (3.46), 252 (3.34), 309 (3.83), 323 (3.89), 340 (3.70) nm; IR (neat) νmax 3410, 2947, 1704, 1638, 1562, 1457, 1404, 1376, 1019 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 503.3135 [M + Na]+ (calcd for C31H44O4Na, 503.3137). 3β,16α,29-Trihydroxy-24-methyllanosta-7,9(11),24(31)trien-21-oic acid (2): white, amorphous powder; [α]25 D +38.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (3.79), 235 (3.98), 243 (4.03), 252 (3.86) nm; IR (neat) νmax 3406, 2929, 1638, 1590, 1405, 1381, 1038 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 523.3397 [M + Na]+ (calcd for C31H48O5Na, 523.3399). 3β,16α,30-Trihydroxy-24-methyllanosta-7,9(11),24(31)trien-21-oic acid (3): white, amorphous powder; [α]25 D +61.5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (3.64), 236 (3.94), 243 (3.99), 252 (3.83) nm; IR (neat) νmax 3388, 2929, 1652, 1564, 1405, 1371, 1029 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 523.3392 [M + Na]+ (calcd for C31H48O5Na, 523.3399). 3β-Acetoxy-16α,24β-dihydroxy-lanosta-7,9(11),25-trien-21oic acid (4): white, amorphous powder; [α]25 D +25.3 (c 0.1, MeOH);

Figure 3. ChemGPS-NP analysis of lanostanoids from P. cocos. Score plot of the three dimensions (principal components 1−3) consisting of PC1 (red; represents size, shape, and polarizability), PC2 (orange; aromatic- and conjugation-related properties), and PC3 (green; lipophilicity, polarity, and H-bond capacity) and from analysis of novel (purple; compounds 1−6), known (dark blue; compounds 7− 22), and antileukemic (transparent yellow; compounds 8, 9, 12, 14, 15, and 20) lanostanoids from P. cocos in the ChemGPS-NP model addressed by Rosén et al. in 2009 together with previously studied topoisomerase I (light blue) and II (light green) inhibitors from our ChemGPS-NP database.

was suggested that targets other than topoisomerases may be involved in the antiproliferative effect of lanostanoids from P. cocos.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation spectra were recorded on a JASCO-P-1020 polarimeter (cell length 10 mm) (JASCO, Tokyo, Japan). UV spectra were measured using JASCO UV530 ultraviolet spectrophotometers (JASCO, Tokyo, Japan). IR spectra were obtained on a PerkinElmer FTIR system and Spectrum 2000 spectrophotometer. NMR spectra were obtained on a Mercury Plus 400 MHz FT-NMR and a Varian Unity 600 MHz FT-NMR (Varian Inc., Palo Alto, CA, USA). ESIMS data were collected on a VG Biotech Quattro 5022 mass spectrometer. HRESIMS data were obtained on a Bruker APEX II spectrometer (FT-ICR/MS, FTMS) (Bruker Daltonics Inc., Billerica, MA, USA). TLC was performed on Kieselgel 60 F254 (0.25 mm, Merck, Darmstadt, Germany) and/or RP18 F254S (0.25 mm, Merck, Darmstadt, Germany) coated plates and then visualized by spraying with 50% H2SO4 and heating on a hot 2811



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UV (MeOH) λmax (log ε) 206 (3.67), 236 (3.85), 243 (3.89), 252 (3.74) nm; IR (neat) νmax 3397, 2919, 1733, 1652, 1571, 1410, 1371, 1248, 1024 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 551.3342 [M + Na]+ (calcd for C32H48O6Na, 551.3349). 3β,16α-Dihydroxy-7-oxo-24-methyllanosta-8,24(31)-dien21-oic acid (5): white, amorphous powder; [α]25 D +28.5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (3.46), 253 (3.72) nm; IR (neat) νmax 3397, 2957, 1652, 1580, 1410, 1376, 1090, 1023 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 523.3396 [M + Na]+ (calcd for C31H48O5Na, 523.3399). 3α,16α-Dihydroxy-7-oxo-24-methyllanosta-8,24(31)-dien21-oic acid (6): white, amorphous powder; [α]25 D −1.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (3.49), 253 (3.69) nm; IR (neat) νmax 3407, 2919, 1652, 1578, 1410, 1371, 1019 cm−1; 1H and 13 C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 523.3397 [M + Na]+ (calcd for C31H48O5Na, 523.3399). Preparation of (R)- and (S)-MTPA Esters (4r, 4s). Compound 4 (3.3 mg) was divided and stored in two NMR tubes and dried under vacuum. Deuterated pyridine (0.75 mL) and (R)-MTPA-Cl (10 μL) were added to one of the NMR tubes. The reaction NMR tubes were permitted to stand at room temperature and monitored by 400 MHz NMR every hour. After 3 h, the reaction was found to be completed, and the 1H NMR data was obtained (400 MHz, in C5D5N). The (S)MTPA ester of 4 (4s) was obtained, and the 1H NMR data were analyzed. Similar to 4s, (S)-MTPA-Cl (10 μL) and deuterated pyridine (0.75 mL) were reacted at room temperature for 3 h, to afford the (R)MTPA ester derivative (4r), in a separate experiment, and the 1H NMR spectrum was measured with 400 MHz NMR in C5D5N. (S)MTPA ester 4 (4s): 1H NMR δH (400 MHz, C5D5N) 5.369 (1H, d, J = 6.4 Hz, H-7), 5.211 (1H, d, J = 6.8 Hz, H-11), 5.168 and 5.036 (both 1H and s, H-31), 4.716 (1H, t, J = 4.8 Hz, H-3), 4.670−4.680 (1H, m, H-16), 4.670−4.680 (1H, m, H-24), 2.117 (3H, s, H-27), 2.107 (3H, s, H-30), 2.054 (3H, s, OAc), 0.976 (3H, s, H-18), 0.966 (3H, s, H-29), 0.964 (3H, s, H-19), 0.863 (3H, s, H-28). (R)-MTPA ester 4 (4r): 1H NMR δH (400 MHz, C5D5N): 5.356 (1H, d, J = 6.8 Hz, H-7), 5.230 (1H, d, J = 6.0 Hz, H-11), 5.168 and 5.058 (both 1H and s, H-31), 4.715 (1H, t, J = 4.6 Hz, H-3), 4.670−4.680 (1H, m, H16), 4.670−4.680 (1H, m, H-24), 2.121 (3H, s, H-27), 2.107 (3H, s, H-30), 2.054 (3H, s, OAc), 0.984 (3H, s, H-18), 0.967 (3H, s, H-29), 0.964 (3H, s, H-19), 0.862 (3H, s, H-28). MTT Cell Proliferative Assay. Molt 4 (human acute lymphoblastic leukemia), CCRF-CEM (human T cell lymphoblastic leukemia), HL-60 (human acute promyelocytic leukemia), and K562 (human chronic myelogenous leukemia) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Rat PBMCs were generated as described previously.36 Cells were maintained in RPMI-1640 medium supplemented with 10% FCS, 2 mM glutamine, and antibiotics (100 units/mL penicillin and 100 μg/ mL streptomycin) at 37 °C in a humidified atmosphere of 5% CO2. Cells were seeded at 4 × 104 per well in 96-well culture plates before treating with different concentrations of the test compounds. The compounds were dissolved in DMSO (less than 0.02%) and made immediately to 1.25, 2.5, 5, 10, and 20 μg/mL prior to the experiments. After treatment for 72 h, the cytotoxicity of the tested compounds was determined using the MTT cell proliferation assay (thiazolyl blue tetrazolium bromide, Sigma-M2128). The MTT is reduced by the mitochondrial dehydrogenases of the viable cells to a purple formazan product. The MTT-formazan product was dissolved in DMSO. Light absorbance values (OD = OD570 − OD620) were recorded at wavelengths of 570 and 620 nm using an ELISA reader (Anthos Labtec Instrument, Salzburg, Austria) to calculate the concentration that caused 50% inhibition (IC50), i.e., the cell concentration at which the light absorbance value of the experimental group was half that of the control group. These results were expressed as a percentage of the control ± SD established from n = 4 wells per one experiment from four separate experiments. ChemGPS-NP Analysis. The PCA-based model ChemGPS-NP (http://chemgps.bmc.uu.se) is a tool for navigation in biologically relevant chemical space. It has eight principal components (PCs)

based on 35 carefully selected chemical descriptors describing physical−chemical properties such as size, shape, polarizability, lipophilicity, polarity, flexibility, rigidity, and hydrogen bond capacity. The ChemGPS-NP prediction scores were calculated for all compounds isolated from P. cocos using the online tool ChemGPSNPWeb37 (http://chemgps.bmc.uu.se) on the basis of their structural information as SMILES derived via ChemBioDraw version 13.0. All compounds were then mapped into ChemGPS-NP chemical property space using the software Grapher 2.0 (Mac OS) together with previously studied topoisomerase I and II inhibitors from our ChemGPS-NP database.

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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.6b00575. 1D and 2D NMR data of 1−6 and 1D NMR data of 8− 10 and 15 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +46-18-471-4498. E-mail: [email protected] (A. Backlund). *Tel: +886-7-3121-101-2162. Fax: +886-7-3114-773. E-mail: [email protected] (F.-R. Chang). *Tel: +886-4-22057153. Fax: +886-4-22060248. E-mail: [email protected] (Y.-C. Wu). Author Contributions ◆

K.-H. Lai and M.-C. Lu contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research project on Poria cocos (Schw.) Wolf was supported by grants from the Ministry of Science and Technology of Taiwan (MOST 103-2320-B-037-005-MY2 and 102-2628-B-037-003-MY3 awarded to F.-R.C.), the Excellence for Cancer Research Center Grant, the Ministry of Health and Welfare, Executive Yuan, Taipei, Taiwan (MOHW104-TDU-B-212-124-003), the grant for Health and Welfare Surcharge of Tobacco Products, and Kaohsiung Medical University “Aim for the Top Universities Grant”, Grant No. KMU-TP103. This work was also supported by National Science Council (NSC 103-2911-I-002-303) (NSC101-2632-B039-001-MY3), National Health Research Institutes (NHRI-EX102-10241BI), and CMU under the Aim for Top University Plan of the Ministry of Education, Taiwan, awarded to Y.-C.W.

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REFERENCES

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