Meroterpenoids from a Medicinal Fungus Antrodia cinnamomea

Article pubs.acs.org/jnp

Meroterpenoids from a Medicinal Fungus Antrodia cinnamomea Mei-Chuan Chen,†,‡ Ting-Yu Cho,† Yueh-Hsiung Kuo,§,# and Tzong-Huei Lee*,⊥ †

Graduate Institute of Pharmacognosy and ‡The Ph.D. program for the Clinical Drug Discovery from Botanical Herbs, Taipei Medical University, Taipei 11031, Taiwan § Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 40447, Taiwan # Department of Biotechnology, Asia University, Taichung 41354, Taiwan ⊥ Institute of Fisheries Science, National Taiwan University, Taipei 10617, Taiwan S Supporting Information *

ABSTRACT: Antrodia cinnamomea, a medicinal fungus indigenous to Taiwan, has been shown to exhibit a broad spectrum of bioactivities for the treatments of alcoholic intoxication, diarrhea, abdominal pain, and fatigue, and a number of active principles have been identified. Among the bioactive entities, clinical trials of antroquinonol and 4-acetyl antroquinonol B are being carried out for curing cancer, hypercholesterolemia, and hyperlipidemia. The total synthesis of antroquinonol has been achieved; however, investigating the structure−activity relationship of this class of compounds remained difficult due to the lack of available analogues. Twenty antroquinonols isolated from A. cinnamomea IFS006 are reported herein. Their structures were elucidated using spectral analysis and by comparison with literature values. Of these, 11 antroquinonol analogues, namely, antroquinonols N−X (1−11), were previously unreported. The growth inhibitory activity of all the antroquinonol analogues was evaluated against human A549 and PC-3 cancer cell lines, and antroquinonol A exhibited the most potent activity, with GI50 values of 5.7 ± 0.2 and 13.5 ± 0.2 μM, respectively. Antroquinonols V (9) and W (10) also showed growth inhibitory activity against A549 cells with GI50 values of 8.2 ± 0.8 and 7.1 ± 2.1 μM, respectively, compared to 5-fluorouracil (GI50 = 4.2 ± 0.2 μM). (SAR) was not possible because only a few analogues were available. By adopting in vitro cell line assays and molecular docking analysis, it was demonstrated that inhibition of Ras and Rho enzyme activation through suppression of farnesyl transferase activity was the key mechanism for cytotoxicity.16 Thus, an in vitro cytotoxicity assay using the Ras mutant cell lines, such as human non-small-cell lung cancer A549 cells and human prostate cancer PC-3 cells that are sensitive to farnesyl transferase inhibitors, could be a promising strategy for studying the SAR of antroquinonol analogues. In continuing our investigations on the bioactive constituents of A. cinnamomea, 20 antroquinonol analogues were isolated and identified from the solid-state fermented products of A. cinnamomea IFS006. Their structures were elucidated by spectral analysis and comparison with literature values. Among these, 11 antroquinonol analogues, namely, antroquinonols N−X (1−11), were previously unreported. Herein, we reported the isolation and structural elucidation of 1−11 as well as their cytotoxicities on A549 and PC3 cancer cell lines.

Antrodia cinnamomea Chang & Chou [synonyms Antrodia camphorata (Zang & Su) Wu, Ryvarden & Chang and Taiwanof ungus camphoratus (Zang & Su) Wu, Yu, Dai & Su] is an endemic fungal species of Taiwan.1,2 Its bitter-tasting reddish fruiting body growing in the decayed inner heartwood wall of Cinnamomum kanehirae Hayata has been used as folk remedies for over one hundred years for the treatments of alcoholic intoxication,3 diarrhea,4 abdominal pain,4,5 fatigue,5 and many other complaints.6 In addition to folk medicinal uses, a broad spectrum of biological activities indicating hepatoprotective,7 cytotoxicity,8 and anti-inflammatory9 qualities have been published within the past decade. The potential active principles in A. cinnamomea were shown to be antroquinonols, triterpenoids, and benzenoids.6 Of these, the antroquinonols, classified as merotertenoids, were speculated to originate from the combination of an orsellinic acid-derived C7 cyclohexenone and a C15 sesquiterpene.10 Among the antroquinonols, antroquinonol and 4-acetyl antroquinonol B have been subjected to clinical trials as anticancer11,12 and antihypercholesterolemia and antihyperlipidemia agents.13 In the recent past, the total synthesis of antroquinonol has been achieved;14,15 however, the study of its structure and activity relationship © 2017 American Chemical Society and American Society of Pharmacognosy

Received: March 14, 2017 Published: September 12, 2017 2439

DOI: 10.1021/acs.jnatprod.7b00223 J. Nat. Prod. 2017, 80, 2439−2446

Journal of Natural Products

Article

Chart 1

Table 1. 13C NMR Spectroscopic Data for Compounds 1−11 (δ in ppm, mult.) no.

1a,c

2b,c

3b,c

4b,c

5b,c

6b,c

7b,c

8b,c

9b,c

10b,c

11a,c

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 −OAc

184.8 s 144.4d 144.3d 183.9 s 141.5 s 138.9 s 25.3 t 119.3 d 137.3 s 39.5 t 26.4 t 128.1 d 131.9 s 48.2 t 65.7 d 127.5 d 134.7 s 25.7 q 18.2 q 16.1 q 16.2 q 12.0 q 61.1 q 61.1 q

198.8 s 152.1 s 116.7 d 65.1 d 47.4 d 43.4 d 28.2 t 123.3 d 138.1 s 40.8 t 27.6 t 128.4 d 132.6 s 49.0 t 67.7 d 129.0 d 137.8 s 68.2 t 14.1 q 16.7 q 16.2 q 13.1 q 55.4 q

199.6 s 137.2 s 164.8 s 67.4 d 45.7 d 41.5 d 28.2 t 122.9 d 138.4 s 40.8 t 27.6 t 128.4 d 132.7 s 49.0 t 67.6 d 129.1 d 137.7 s 68.2 t 14.1 q 16.7 q 16.3 q 12.9 q 60.8 q 58.9 q

199.1 s 138.8 s 160.6 s 70.3 d 44.2 d 42.5 d 28.0 t 122.1 d 138.7 s 40.7 t 27.5 t 138.4 d 132.7 s 49.0 t 67.7 d 129.1 d 137.7 s 68.2 t 14.1 q 16.7 q 16.3 q 13.1 q 61.1 q 60.2 q 20.8 q; 171.5 s

184.3 s 134.0 s 184.3 s 70.6 d 47.1 d 40.0 d 26.2 t 123.9 d 138.3 s 40.8 t 27.7 t 128.5 d 132.6 s 49.0 t 67.7 d 129.1 d 137.8 s 68.2 t 14.1 q 16.7 q 16.3 q 17.1 q 60.0 q

198.8 s 152.1 s 116.7 d 65.1 d 47.4 d 43.4 d 28.2 t 123.5 d 137.9 s 40.6 t 27.4 t 129.3 d 131.7 s 45.9 t 78.9 d 35.7 t 35.1 d 182.7 s 16.0 q 16.5 q 16.2 q 13.1 q 55.4 q

199.1 s 138.8 s 160.6 s 70.3 d 44.3 d 42.5 d 28.0 t 122.1 d 138.6 s 40.7 t 27.4 t 127.6 d 133.5 s 48.9 t 78.8 d 37.6 t 41.8 d 105.3 d 17.2 q 16.7 q 16.3 q 13.1 q 61.1 q 60.2 q 20.8 q; 171.4 s

199.6 s 137.2 s 164.9 s 67.4 d 45.7 d 41.5 d 28.1 t 123.0 d 138.3 s 40.7 t 27.6 t 129.0 d 132.0 s 48.1 t 68.1 d 143.0 d 131.2 s 173.3 s 13.5 q 16.7 q 16.2 q 12.8 q 60.8 q 58.9 q

199.6 s 137.1 s 164.8 s 67.4 d 45.8 d 41.5 d 28.2 t 122.9 d 138.4 s 40.9 t 27.4 t 125.3 d 135.9 s 49.5 t 27.5 t 126.5 d 135.9 s 69.0 t 13.7 q 16.1 q 16.2 q 12.9 q 60.8 q 58.9 q

199.6 s 137.2 s 164.8 s 67.4 d 45.8 d 41.5 d 28.2 t 122.9 d 138.4 s 41.0 t 27.5 t 125.3 d 136.2 s 41.0 t 26.5 t 34.0 t 36.8 d 68.5 t 17.1 q 16.0 q 16.2 q 12.9 q 60.8 q 58.9 q

137.5 s 146.3 s 97.7 d 146.4 s 113.2 s 119.2 s 119.8 d 127.4 d 77.3 s 40.6 t 22.6 t 124.1 d 135.2 s 39.7 t 26.7 t 124.3 d 131.3 s 25.7 q 17.7 q 16.0 q 25.6 q 10.6 q 55.9 q

a

Measured in chloroform-d (125 MHz). bMeasured in methanol-d4 (125 MHz). cMultiplicties were obtained from phase-sensitive HSQC experiments. dSignals are exchangeable.

■

RESULTS AND DISCUSSION In an attempt to increase the chemical diversity of secondary metabolites from A. cinnamomea, an OSMAC (one strain, many compounds) strategy was applied,17 and culture media varied to induce the expression of a cryptic gene cluster and thus afford new products. In this study, A. cinnamomea IFS006 was cultured initially in different combinations of authentic nutrients and cereal (data not shown), and 20 antroquinonol analogues including 11 new compounds (1−11) were obtained

from the fermented products cultured in potato dextrose agar plus barley. Of the known antroquinonols isolated, antroquinonols A, B, and D, 4-acetyl antroquinonol B, and antrocamols LT1, LT2, and LT3 were all identified by comparison of experimental data with published information.18−21 The ubiquinone Q3 was found previously in 11 fungal species several decades ago;22 however, this is the first report to describe the isolation of this compound from A. cinnamomea. Antroquinonol L was isolated initially by supplementing 2440


1.99, 2.09 1.99, 2.09 5.15 br t (6.9)

2.08

4.36 m

10 11 12

14

15

2441

a

1.66 1.61 1.72 2.00 3.97 3.96

d (1.3) d (0.6) d (0.8) s s s

1.69 d (1.3)

1.67 1.65 1.63 1.17 3.61

d d d d s

3.92 s

(1.3) (0.6) (0.6) (7.0)

5.39 dq (8.6, 1.3)

1.67 1.66 1.67 1.15 3.59 4.06

br s br s br s d (6.9) s s

3.93 s

5.39 dq (8.6, 1.4)

4.48 ddd (8.6, 6.8, 6.8)

2.23

2.24

4.48 ddd (8.6, 6.8, 6.8)

2.09

2.06, 2.13 2.07, 2.14 5.19 br t (6.9)

2.10

2.04, 2.12 2.05, 2.12 5.18 br t (6.9)

5.25 br t (7.3)

2.24

2.49 dq (11.4, 6.9)

1.68 m

4.35 d (3.2)

3b,c

1.67 1.66 1.58 1.18 3.62 4.00 2.09

3.92

br s br s br s d (6.9) s s s

5.39 br d (8.7)

4.48 ddd (8.7, 6.8, 6.8)

2.24

2.11

2.03, 2.12 2.03, 2.12 5.20 br t (7.2)

5.16 br t (6.7)

2.02, 2.28

2.52 dq (10.8, 6.9)

1.95 m

5.77 d (3.2)

4b,c

1.67 1.65 1.57 1.26 3.56

d (1.2) br s br s d (7.4) s

3.92 s

5.39 dq (8.7, 1.3)

4.48 ddd (8.7, 6.8, 6.8)

2.24

2.10

2.03, 2.11 2.04, 2.11 5.18 br t (6.6)

5.12 br t (6.8)

1.98, 2.25

2.49 dq (3.4, 7.4)

1.93 m

4.39 d (4.5)

5b,c

d (7.2) br s br s d (7.0) s

0.99 1.65 1.58 1.18 3.62 4.00 2.09

d (7.0) d (0.9) br s d (6.9) s s s

1.91 m 2.13 m 4.93 d (2.0)

2.20m 2.74 m

1.23 1.68 1.64 1.17 3.61

1.62 m

2.38 dd (13.0, 6.5) 4.21 m

2.18 m

2.03, 2.12 2.03, 2.12 5.21 br t (7.1)

5.16 br t (6.7)

2.53 dq (10.8, 6.9) 2.04, 2.27

1.94 m

5.77 d (3.1)

7a,c

1.99 m

2.08 dd (13.8, 6.3) 2.39 dd (13.8, 6.3) 4.69 m

2.08, 2.16 2.08, 2.16 5.26 br t (7.0)

5.23 br t (7.3)

2.68 dq (9.7, 7.0) 2.18, 2.26

5.93 d (5.6) 4.50 dd (5.6, 3.7) 1.83 m

6a,c

1.82 1.67 1.66 1.15 3.58 4.06

d (1.4) br s br s d (6.9) s s

6.52 dq (8.7, 1.4)

2.28 dd (13.4, 6.9) 4.52 m

2.15

2.06, 2.13 2.06, 2.14 5.20 br t (6.9)

5.24 br t (7.4)

2.48 dq (11.4, 6.9) 2.24

1.68 m

4.35 d (3.2)

8a,c

1.64 1.62 1.67 1.15 3.58 4.05

br s br s br s d (6.9) s s

1.37 1.55 3.30 3.41 dd (10.4, 5.9) 0.89 d (6.5) 1.60 br s 1.67 br s 1.15 d (7.0) 3.59 s 4.06 s

1.46 1.05

2.07 5.39 br t (7.3)

1.97

2.06 2.13 5.13 br t (7.0)

5.24 br t (7.4)

2.49 dq (11.4, 6.9) 2.24 t (7.4)

1.67 m

1.38

3.91 s

10a,c 4.34 d (3.3)

2.03

2.13

2.02

2.06 2.13 5.15 br t (6.9)

5.25 br t (7.5)

2.49 dq (11.4, 6.9) 2.24 t (7.5)

1.68 m

4.34 d (3.3)

9a,c

Measured in chloroform-d (500 MHz). bMeasured in methanol-d4 (500 MHz). cSignals without multiplicity were overlapped and picked from COSY or HMBC spectra.

19 20 21 22 23 24 −OAc

17 18

16

5.11 dqq (8.3, 1.3, 1.3)

4.92 br t (7.0)

8

5.23 br t (7.5)

2.18, 2.26

3.16 d (7.0)

7

6

1.82 dddd (9.9, 9.7, 5.7, 3.5) 2.68 dq (9.9, 7.0)

5

2b,c

5.94 d (5.6) 4.50 dd (5.6, 3.5)

1a,c

3 4

no.

Table 2. 1H NMR Spectroscopic Data for Compounds 1−11 [δ in ppm, mult. (J in Hz)]

1.56 1.56 1.33 2.18 3.81

br s d (1.0) s s s

1.65 d (1.3)

2.03 5.06 br t (7.0)

1.94

2.02

5.48 d (10.0) 6.47 d (10.0) 1.66 m 2.09 5.09 br t (7.0) 1.94

6.26 s

11b,c

Journal of Natural Products Article



Article

methyl attached to a tertiary carbon, three methyls located on double bonds, and one methoxyl functionality, respectively. Furthermore, two oxygenated methines at δH 4.50 (1H, dd, J = 5.6, 3.5 Hz, H-4) and 4.48 (1H, ddd, J = 8.6, 6.8, 6.8 Hz, H-15) and one oxymethylene at δH 3.92 (2H, s, H2-18); four methylenes at δH 2.10−2.26 (8H, H2-7, -10, -11, and -14); two methines at δH 1.82 (1H, dddd, J = 9.9, 9.7, 5.7, 3.5 Hz, H-5) and 2.68 (1H, dq, J = 9.9, 7.0 Hz, H-6); and four olefinic methines at δH 5.18 (1H, br t, J = 6.9 Hz, H-12), 5.23 (1H, br t, J = 7.5 Hz, H-8), 5.39 (1H, dq, J = 8.6, 1.3 Hz, H-16), and 5.94 (1H, d, J = 5.6 Hz, H-3) were observed (Table 2). The 13C NMR data (Table 1) coupled with the HSQC spectrum of 2 showed 23 carbon signals corresponding to five methyls at δC 13.1 (C-22), 14.1 (C-19), 16.2 (C-21), 16.7 (C-20), and 55.4 (2-OMe); five methylenes at δC 27.6 (C-11), 28.2 (C-7), 40.8 (C-10), 49.0 (C-14), and 68.2 (C-18); seven methines at δC 43.4 (C-6), 47.4 (C-5), 65.1 (C-4), 116.7 (C-3), 123.3 (C-8), 128.4 (C-12), and 129.0 (C-16); and five other carbons at δC 132.6 (C-13), 137.8 (C-17), 138.1 (C-9), 152.1 (C-2), and 198.8 (C-1). The NMR data for 2 were consistent with those of antroquinonol D,20 except for the distinctive downfield shifts at δH 3.92 (H2-18)/δC 68.2 (C-18) and δH 4.48 (H-15)/δC 67.7 (C-15), indicating that 2 was the 15,18-dihydroxy analogue of antroquinonol D. These assignments were further supported by key cross-peaks of H2-14/H-15 and H-15/H-16 in the COSY spectrum and key cross-peaks of H-15/C-13 and -17 and H218/C-16, -17, and -19 (Figure 1). The relative configurations of

orsellinic acid, the putative intermediate polyketide product, in the cultural medium of a Δpks63787 mutant A. cinnnamomea strain,23 and the same compound was also obtained in this study. Compound 1, a yellowish oil, was determined to have a molecular formula of C24H34O5, as evidenced by its 13C NMR spectrum (Table 1) and HRESIMS analysis. The IR absorptions at 3518 and 1652 cm−1 revealed the presence of a hydroxy and conjugated carbonyl group, respectively. The 1H NMR data (CDCl 3 , 500 MHz) (Table 2) contained conspicuous resonances corresponding to four three-proton doublets at δH 1.61 (3H, d, J = 0.6 Hz, H3-20), 1.66 (3H, d, J = 1.3 Hz, H3-19), 1.69 (3H, d, J = 1.3 Hz, H3-18), and 1.72 (3H, d, J = 0.8 Hz, H3-21), one three-proton singlet at δH 2.00 (3H, s, H3-22), and two methoxyl singlets at δH 3.96 (3H, s, H3-24) and 3.97 (3H, s, H3-23), all attached to olefinic functionalities, as evidenced from their downfield shifts and small J values. Additionally, four methylenes were observed at δH 1.99−2.08 (6H, H2-10, -11, and -14) and 3.16 (2H, d, J = 7.0 Hz, H2-7). Four methine signals at δH 4.36 (1H, m, H-15), 4.92 (1H, br t, J = 7.0 Hz, H-8), 5.11 (1H, dqq, J = 8.3, 1.3, 1.3 Hz, H-16), and 5.15 (1H, br t, J = 6.9 Hz, H-12) were characteristic of one carbinol and three double bonds, respectively. The 13C NMR (CDCl3, 125 MHz) (Table 1) coupled with the HSQC spectrum of 1 indicated 24 carbon signals corresponding to seven methyls at δC 12.0 (C-22), 16.1 (C-20), 16.2 (C-21), 18.2 (C-19), 25.7 (C-18), and 61.1 (C-23 and -24); four methylenes at δC 25.3 (C-7), 26.4 (C-11), 39.5 (C-10), and 48.2 (C-14); four methines at δC 65.7 (C-15), 119.3 (C-8), 127.5 (C-16), and 128.1 (C-12); and nine other carbons at δC 131.9 (C-13), 134.7 (C-17), 137.3 (C-9), 138.9 (C-6), 141.5 (C-5), 144.3 (C-3), 144.4 (C-2), 183.9 (C-4), and 184.8 (C-1). The distinctive downfield shifted signals at δC 183.9 and 184.8 in the 13C NMR spectrum, an IR adsorption signal at 1652 cm−1, and a UV absorption λmax at 272 nm suggested the presence of a p-benzoquinone moiety.24 When comparing the spectral data of 1 with those of ubiquinone Q3, 1 was almost identical with ubiquinone Q3 except that the molecular weight of 1 was 16 Da higher than that of ubiquinone Q3 and the C-15 of 1 downfield shifted to δC 65.7, indicating an additional hydroxy group attached at C-15. The key COSY spectrum of 1 displayed two sets of mutual-coupled protons of H2-14/H-15 and H-15/H-16, which were further supported by key crosspeaks of H-15/C-13 and -17 in the HMBC spectrum. Compound 1 was thus deduced to be the 15-hydroxy analogue of ubiquinone Q3. The configurations of both Δ8 and Δ12 were deduced to be the E form as corroborated by the chemical shifts of C-20 and C-21 at δC 16.2 and 16.1, respectively, in contrast to those of the Z configurations at around δC 25.0.19 Based on the biogenetic relationship between 1 and antrocamols LT1 and LT2, possessing a 15S-hydroxy group,21 all isolated in this study, the configuration of C-15 of 1 was rationally assigned to be the S form. Accordingly, the structure of 1 was determined as shown, and this compound was named antroquinonol N. Compound 2 was isolated as a colorless oil, and its molecular formula, C23H36O5, was determined by its 13C NMR (Table 1) and HRESIMS data. The IR spectrum indicated the presence of hydroxy and α,β-unsaturated ketone groups based on the absorption bands at 3376 and 1685 cm−1, respectively. In the 1 H NMR spectrum of 2 (Table 2), five three-proton signals at δH 1.17 (3H, d, J = 7.0 Hz, H3-22), 1.63 (3H, d, J = 0.6 Hz, H321), 1.65 (3H, d, J = 0.6 Hz, H3-20), 1.67 (3H, d, J = 1.3 Hz, H3-19), and 3.61 (3H, s, H3-23) revealed the presence of one

Figure 1. Key COSY and HMBC correlations of 2.

H-4−H-6 were deduced to be qusai-equatorial, quasi-axial, and quasi-axial, respectively, the same as those of antroquinonol D,20 on the basis of 3JH‑4/H‑5 = 3.5 Hz and 3JH‑5/H‑6 = 9.9 Hz. The absolute configurations of H-4−H-6 of antroquinonol D have been established to be all R form by total synthesis.25 Compound 2 has the same optical rotational sign ([α]26D +33.4) as those of antrocamols LT1 ([α]26D +27.6) and LT2 ([α]26D +83.3). Consequently, the absolute configurations of C4−C-6 and C-15 of 2 were determined to be 4R, 5R, 6R, and 15S, respectively, as shown. The physical and NMR data of 3−5 and 8 were in accordance with those of 2, except for some different substituents borne by their C-3, -4, and -18. Comprehensive spectroscopic data analysis allowed the complete assignments of 3−5 and 8 to be 3-methoxyl, 4-acetyl-3-methoxyl, 3-hydroxy, and 4-acetyl-3-methoxyl-18-oic acid analogues of 2, respectively. Of these, the cyclohexenone moiety of compound 5 with an enolized carbonyl of C-1−C-3 was prone to form an enol− enol tautomerism rapidly (Figure 2), which was in conformity with the published data.26 The chemical shift of both C-1 and C-3 of 5 was suspected to be the average of δ values of interchangeable carbonyl and enol groups. The 3JH‑5/H‑6 (3.4 Hz) of 5, much smaller than that of other compounds, was resulted from the mutual coupled Hquasi‑equatorial -5 and Hquasi‑equatorial-6, as evidenced from key cross-peaks of H-4/H5, H-4/H3-22, H-5/H3-22, and H-6/H2-7 in the ROESY spectrum (Figure 2). 2442



Article

molecular weight of 10 was 2 Da higher than that of 9 by HRESIMS analysis, indicating the lack of a double bond. When comparing the NMR of 10 with that of 9 (Tables 1 and 2), distinctive upfield shifts of δH 5.39 (H-16)/δC 126.5 (C-16) and δC 135.9 (C-17) in 9 to respective δH 1.05 and 1.37 (H216)/δC 34.0 (C-16) and δH 1.55 (H-17)/δC 36.8 (C-17) in 10 as confirmed by key cross-peaks of H2-15/H2-16, H2-16/H-17, and H-17/H2-18 and H3-19 in the COSY spectrum and H2-16/ C-18 and H3-19/C-16, -17, and -18 in the HMBC spectrum also indicated 10 was a Δ16-reducing analogue of 9. Hence, the structure of 10 is as shown, and it was named antroquinonol W. Compound 11, a colorless oil, was assigned a molecular formula of C23H32O3 by HRESIMS accompanied by 13C NMR (Table 1), indicating eight degrees of unsaturation. The 13C NMR coupled with DEPT spectra showed six methyls, four methylenes, five methines, and eight other carbons (Table 1). The distinguishing feature of 2H-chromene was revealed from the aromatic proton at δH 6.26 (1H, s, H-3) and two ciscoupled olefinic protons at δH 6.47 (1H, d, J = 10.0 Hz, H-7) and 5.48 (1H, J = 10.0 Hz, H-8) in the 1H NMR data (Table 2). The NMR data of 11 were compatible with those of albatrelin C,27 except that one additional methoxyl group at δH 3.81 (3H, s, H3-23)/δC 55.9 (C-23) in 11 and one aromatic carbon signal at δC 110.2 in albatrelin C substituted by one alcohol-bearing aromatic carbon signal at δC 137.5 (C-1) were observed (Tables 1 and 2). In the HMBC spectrum of 11, key cross-peaks of H3-22/C-1 and -5, H-3/C-1 and -5, H-7/C-6, H8/C-5, and H3-23/C-2 established the positions of H3-22, OH1, and OMe-2 (Figure 4). The only chiral center (C-9) was determined as S form by comparing the optical rotational value of 11 ([α]26D = +17.6) with that of albatrelin C ([α]16D = −18.5).27

Figure 2. Key ROESY correlations (left) and rapid enol−enol tautomerism (right) of 5.

Compound 6 was obtained as a colorless oil, and its molecular formula, C23H34O5, determined by its 13C NMR (Table 1) and HRESIMS data, was 30 Da less than that of antroquininol B.19 The spectral data of 6 were almost compatible with those of antroquinonol B except that a 3methoxyl resonance disappeared and an additional proton was found at δH 5.93 (1H, d, J = 5.6 Hz, H-3) in the 1H NMR of 6 (Table 2), which were also reflected in its 13C NMR, a δC 59.1 signal replaced by a δC 116.7 signal (Table 1). The probable biogenetic relationship between 6 and 2 and a key cross-peak of H-15/H3-19 in the NOESY spectrum of 6 allowed the determination of the absolute configurations of C-4, -5, -6, -15, and -17 to be 4R, 5R, 6R, 15S, and 17R, respectively. Taken together, the structure of 6 was therefore deduced to be as shown. Compound 7, a colorless oil, has a molecular formula of C26H40O7, on the basis of 13C NMR (Table 1) and HRESIMS. It exhibited IR absorption bands at 3438, 1745, and 1672 cm−1, indicating the presence of hydroxy, ester carbonyl, and conjugated carbonyl functionalities. When comparing the 1H NMR data of 7 with those of 4-acetyl antroquinonol B (Table 2), a distinctive resonance at δH 4.93 (1H, d, J = 2.0 Hz, H-18) was observed in 7, and its corresponding resonance in 13C NMR at δC 105.3 was assigned using the HSQC spectrum of 7, suggesting a geminal diol-bearing analogue of 4-acetyl antroquinonol B. Further assignments of all the NMR data of 7 including key cross-peaks such as H2-14/H-15, H-15/H2-16, H2-16/H-17, H-17/H-18, and H-17/H3-19 in the COSY spectrum and H-15/C-13, H-18/C-17, and H3-19/C-16 and -18 in the HMBC spectrum (Figure 3) corroborated the gross


In an attempt to evaluate the growth inhibitory activity of the isolated compounds against cancer cells, their effects on cell proliferation were assessed using A549 and PC3 cells by sulforhodamine B (SRB) assay. As shown in Table 3, PC-3 prostate cancer cells were resistant to tested compounds except antroquinonol A and the reference compound 5-fluorouracil. Antroquinonol A exhibits the most potent growth inhibitory activity, with GI50 values of 13.5 ± 0.2 and 5.7 ± 0.2 μM against malignant solid tumors A549 and PC-3 cells, respectively. Addition of a hydroxy group to the C-15 and/or C-18 of the farnesyl moiety resulted in slightly reduced potency against A549 cells as shown for compounds 3, 9, and antrocamol LT1 when compared with antroquinonol A (Table 3). Attachment of a γ-lactone ring at the C-15−C-19 of the farnesyl moiety (antroquinonol B) also decreased the cytotoxic activity compared to antroquinonol A, suggesting a bulky substitution attached to the end of the farnesyl moiety could possibly reduce the antitumor activity. The carbonyl group located at C-4 of the cyclohexene ring (1, antroquinonol L, and ubiquinone Q3) greatly diminished cytotoxicity (Table 3). However, a methoxyl group located at C-3 of the cyclohexene ring seemed to be


structure of 7. 4-Acetyl antroquinonol B was speculated to be biosynthesized from 7 via sequential dehydration and oxidation. Thus, the absolute configurations of all the chiral centers of 7 were deduced to be the same as 4-acetyl antroquininol B. This compound was stable through all the experiments in this study. The NMR data of 9 were almost identical with those of antroquinonol A except that a terminal methyl at δH 1.65 (H318)/δC 25.7 (C-18) in antroquinonol A was substituted by an oxymethylene at δH 3.91 (H2-18)/δC 69.0 (C-18) in 9 (Tables 1 and 2). The molecular formula of 9, C24H38O5, deduced by its 13 C NMR (Table 1) coupled with HRESIMS, also supported an additional oxygen in 9 when compared to antroquinonol A. Unambiguously, 9 was identified as the 18-hydroxy analogue of antroquinonol A and was named antroquinonol V. The 2443



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dextrose agar (Becton, Dickinson and Company, Sparks, USA), 20 g of barley, and 15 mL of deionized water. The fermentation was conducted at 25−30 °C for 30 days. Extraction and Isolation of Secondary Metabolites. Fermented products were lyophilized, ground into a powder, and extracted three times with equal volumes of methanol. Extracts were first partitioned with n-hexane, and the methanol layers redissolved in deionized H2O, then partitioned with ethyl acetate and concentrated to obtain a dried n-hexane extract (5.6 g) and ethyl acetate extract (3.8 g). For compound separation, the n-hexane extract was subjected to silica gravity column chromatography (5.0 i.d. × 15 cm), using nhexane−EtOAc as the eluent with a stepwise gradient mode to give 36 fractions. All the fractions were combined into 32 portions as I−XXXII based on the results of TLC analysis. Portion V eluted by n-hexane− EtOAc (94:6, v/v) was further purified on PTLC using the same eluent to afford ubiquinone Q3 (9.7 mg). Portions VI−IX eluted by nhexane−EtOAc (90:10, v/v) were rechromatographed on a Sephadex LH-20 column (1.5 i.d. × 34.5 cm) with CHCl3−MeOH (1:1, v/v) as eluent to afford 4 subportions (I−IV). Subportion II was further purified by PTLC using CHCl3 as eluent to obtain 1 (1.6 mg) and 12 (0.9 mg). Portion XV eluted by n-hexane−EtOAc (70:30, v/v) was further purified by a Phenomenex Luna 5 μ PFP semipreparative column, 10 i.d. × 250 mm, using 90% MeOHaq as eluent to give 2 (2.3 mg). Portion XVI eluted by n-hexane−EtOAc (70:30, v/v) was further purified by the same HPLC column using 85% MeOHaq as eluent to afford antroquinonol A (3.0 mg). Portion XVII eluted by n-hexane− EtOAc (70:30, v/v) was further purified by the same HPLC column using 85% MeOHaq as eluent to obtain 4-acetyl antroquinonol B (21.7 mg) and antrocamol LT2 (2.0 mg). Portion XIX eluted by n-hexane− EtOAc (60:40, v/v) was further purified by the same HPLC column using 85% MeOHaq as eluent to obtain 8 (1.7 mg). Portion XXI eluted by n-hexane−EtOAc (50:50, v/v) was further purified by the same HPLC column using 85% MeOHaq as eluent to give 10 (2.0 mg), 11 (2.2 mg), antroquinonol B (6.5 mg), antrocamol LT1 (2.0 mg), and antrocamol LT3 (1.5 mg). Portion XXVIII eluted by n-hexane−EtOAc (90:10, v/v) was further purified by PTLC using CHCl3 as eluent to obtain 5 (4.7 mg). The ethyl acetate extract was chromatographed on a Sephadex LH-20 column (2.5 i.d. × 69.0 cm) using MeOH as the eluent with a flow rate of 2.2 mL/min. Each fraction (20 mL) collected was checked for its compositions by TLC, and the fractions were combined into 10 portions (I−X). Portion III was rechromatographed on a Diaion HP-20 column (5.0 i.d. × 10 cm) with MeOH−H2O (30:70; 50:50; 70:30; 100:0, v/v) as eluent to afford 4 subportions (I− IV). Subportion III eluted by MeOH−H2O (70:30, v/v) was further purified with a Phenomenex Luna 5 μ PFP semipreparative column, 10 i.d. × 250 mm, using 70% MeOHaq as eluent to give 3 (13.9 mg), 4 (3.8 mg), 6 (2.3 mg), and 9 (2.3 mg). Subportion IV eluted by MeOH was further purified by the same HPLC column using 85% MeOHaq as eluent to obtain 7 (2.7 mg) and antroquinonol D (1.7 mg). Antroquinonol N (1): yellowish oil; [α]26D +14.3 (c 0.4, MeOH); UV (MeOH) λmax (log ε) = 272 (4.07) nm; IR (ZnSe) νmax 3518, 2920, 2848, 1652, 1610, 1445, 1382, and 1264 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS [M + Na]+ at m/z 425.2298 (calcd 425.2304 for C24H34O5Na). Antroquinonol O (2): colorless oil; [α]26D +33.4 (c 0.7, MeOH); UV (MeOH) λmax (log ε) = 258 (3.69) nm; IR (ZnSe) νmax 3376, 2921, 2862, 1685, 1635, 1448, 1382, 1248, 1077, and 1036 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS [M + Na]+ at m/z 415.2459 (calcd 415.2460 for C23H36O5Na). Antroquinonol P (3): colorless oil; [α]26D +45.3 (c 0.7, MeOH); UV (MeOH) λmax (log ε) = 268 (3.86) nm; IR (ZnSe) νmax 3382, 2923, 2855, 1658, 1619, 1451, 1239, 1138, and 1015 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS [M + Na]+ at m/z 445.2554 (calcd 445.2566 for C24H38O6Na). Antroquinonol Q (4): colorless oil; [α]26D +76.8 (c 0.9, MeOH); UV (MeOH) λmax (log ε) = 256 (4.04) nm; IR (ZnSe) νmax 3416, 2925, 2858, 1744, 1670, 1627, 1452, 1370, 1232, 1144, and 1014 cm−1; 1 H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS [M + Na]+ at m/z 487.2661 (calcd 487.2672 for C26H40O7Na).

Table 3. Growth Inhibitory Activities of 1−11 and Other Analogues Isolated in This Study against A549 and PC-3 Cancer Cell Lines GI50a (μM) compound

A549b

PC-3c

1 2 3 4 5 6 7 8 9 10 11 Antroquinonol A Antroquinonol B 4-Acetyl antroquinonol B Antroquinonol D Antroquinonol L Antrocamol LT1 Antrocamel LT2 Antrocamel LT3 Ubiquinone Q3 5-Fluorouracild

>30 >30 12.0 ± 2.5 29.0 ± 1.5 >30 >30 15.4 ± 1.0 >30 8.2 ± 0.8 7.1 ± 2.1 >30 5.7 ± 0.2 14.1 ± 1.5 11.3 ± 0.7 16.4 ± 3.1 >30 9.6 ± 0.6 20.7 ± 2.4 23.8 ± 2.1 >30 4.2 ± 0.2

>30 >30 >30 >30 >30 >30 >30 >30 >30 >30 >30 13.5 ± 0.2 >30 >30 >30 >30 >30 >30 >30 >30 11.7 ± 1.3

a Cells were treated with test compounds for 48 h. bA549, human nonsmall-cell lung cancer cell line. cPC-3, human prostate cancer cell line. d 5-Fluorouracil was used as a positive control.

essential for cell growth suppression by comparing the GI50 values of 2 and 6 with those of 3 and antroquinonol B (Table 3), respectively. Moreover, an acetoxy group substituted at C-4 of the cyclohexene ring decreased cytotoxicity. These data supported partially the reported SAR as characterized with the aid of a docking model of antroquinonol A and farnesyl transferase.16 This is the first report to discuss the SAR of antroquinonol analogues on the basis of growth inhibitory activities against two Ras mutant cancer cell lines.

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EXPERIMENTAL SECTION

General Experimental Procedures. 1H and 13C NMR were acquired on a Bruker Avance DRX-500 and AVIII-500 spectrometer (Ettlingen, Germany). Low- and high-resolution mass spectra were obtained using an API4000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, USA) and Synapt High Definition Mass Spectrometry system with an ESI interface and a TOF analyzer (Waters Corp., Manchester, UK), respectively. IR spectra were recorded on a JASCO FT/IR 4100 spectrometer (Tokyo, Japan). UV spectra were measured on a Thermo UV−vis Heλios α spectrophotometer (Thermo Scientific, Waltham, MA, USA). Sephadex LH-20 (Amersham Biosciences, Filial Sverige, Sweden) and Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan) were used for open column chromatography. Preparative TLC (PTLC) was performed for compound separation using silica gel 60 F254 plates (0.25 mm, Merck). An HPLC pump L-7100 (Hitachi, Japan) equipped with a refractive index detector (Bischoff, Leonberg, Germany) was used for compound purification. All the chemicals were purchased from Sigma-Aldrich (USA). Fungal Strain and Culture. Antrodia cinnamomea IFS006 was isolated and identified by one of us (T.-H.L.). ITS sequences of nuclear rDNA of this fungal strain were submitted to GenBank, and the accession number was KX216402. The mycelium of this strain was inoculated into 250 mL flasks, each containing 0.2 g of Difco potato 2444



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Antroquinonol R (5): colorless oil; [α]26D +25.6 (c 0.6, MeOH); UV (MeOH) λmax (log ε) = 294 (3.78) nm; IR (ZnSe) νmax 3368, 2924, 2858, 1750, 1602, 1497, 1448, 1375, 1073, and 1011 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS [M + Na]+ at m/z 431.2401 (calcd 431.2410 for C23H36O6Na). Antroquinonol S (6): colorless oil; [α]26D +29.7 (c 0.5, MeOH); UV (MeOH) λmax (log ε) = 257 (3.76) nm; IR (ZnSe) νmax 3489, 2924, 2858, 1764, 1687, 1634, 1454, 1371, 1250, 1200, and 1148 cm−1; 1 H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS [M + Na]+ at m/z 413.2300 (calcd 413.2304 for C23H34O5Na). Antroquinonol T (7): colorless oil; [α]26D +67.2 (c 0.7, MeOH); UV (MeOH) λmax (log ε) = 265 (3.86) nm; IR (ZnSe) νmax 3438, 2926, 2862, 1745, 1672, 1628, 1455, 1367, 1232, 1144, and 1014 cm−1; 1 H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS [M − H2O + Na] + at m/z 487.2660 (calcd 487.2672 for C26H40O7Na). Antroquinonol U (8): colorless oil; [α]26D +89.2 (c 0.4, MeOH); UV (MeOH) λmax (log ε) = 267 (4.10) nm; IR (ZnSe) νmax 3393, 2925, 2858, 1658, 1619, 1451, 1383, 1241, 1137, and 1017 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS [M + Na]+ at m/z 459.2346 (calcd 459.2359 for C24H36O7Na). Antroquinonol V (9): colorless oil; [α]26D +56.7 (c 0.01, MeOH); UV (MeOH) λmax (log ε) = 267 (3.94) nm; IR (ZnSe) νmax 3410, 2923, 2855, 1658, 1618, 1452, 1361, 1140, and 1015 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS [M + Na]+ at m/z 429.2614 (calcd 429.2617 for C24H38O5Na). Antroquinonol W (10): colorless oil; [α]26D +27.2 (c 0.6, MeOH); UV (MeOH) λmax (log ε) = 266 (3.41) nm; IR (ZnSe) νmax 3400, 2925, 2862, 1662, 1620, 1454, 1367, 1240, 1136, and 1024 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS [M + Na]+ at m/z 431.2756 (calcd. 431.2773 for C24H40O5Na). Antroquinonol X (11): colorless oil; [α]26D +17.6 (c 0.3, MeOH); UV (MeOH) λmax (log ε) = 223 (4.18), 281 (3.45), and 325 (3.71) nm; IR (ZnSe) νmax 3557, 2922, 2855, 1614, 1478, 1448, 1358, 1305, 1247, 1201, 1133, and 1057 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS [M + Na]+ at m/z 379.2239 (calcd 379.2249 for C23H32O3Na). Cell Culture. The culture of the A549 and PC-3 cell lines was performed as described previously.28 A549 (non-small-cell lung cancer) and PC-3 (prostate cancer) cells were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 containing 10% fetal bovine serum and penicillin (100 units/mL)/streptomycin (100 μg/mL)/amphotericin B (0.25 μg/mL). All cells were maintained in humidified air containing 5% CO2 at 37 °C. Sulforhodamine B Assay. The cellular proliferation was measured using the SRB assay, as described previously.28 Cells were seeded in 96-well plates and cultured overnight followed by the exposure to various concentrations of compounds for 48 h. Cells were fixed with 10% trichloroacetic acid, representing cell population at the time of drug treatment. After incubation with vehicle or compounds for 48 h, cells were fixed with 10% trichloroacetic acid and then stained with SRB at 0.4% (w/v) in 1% acetic acid. Excess SRB was washed away by 1% acetic acid, and cells were lysed with 10 mM Trizma base. The absorbance was measured at a wavelength of 515 nm. Growth inhibition of 50% (GI50) is determined as the drug concentration that results in 50% reduction of total protein increase in control cells during the 48 h incubation time.

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Article

AUTHOR INFORMATION

Corresponding Author

*Tel (T.-H. Lee): 886-2-33881828. E-mail: [email protected]. ORCID

Tzong-Huei Lee: 0000-0001-8036-7563 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology, Taiwan. We thank Ms. Shou-Ling Huang, Instrumentation Center of the College of Science, National Taiwan University, for the NMR data acquisition.

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REFERENCES

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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.7b00223. 1 H and 13C NMR spectra of the new compounds 1−11, chemical structures and spectroscopic data of the known isolates, and a picture of A. cinnamomea IFS006 (DOCX) 2445



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(25) Sulake, R. S.; Chen, C. Org. Lett. 2015, 17, 1138−1141. (26) Martínez-Richa, A.; Mendoza-Díaz, G.; Joseph-Nathan, P. Appl. Spectrosc. 1996, 50, 1408−1412. (27) Liu, L.-Y.; Li, Z.-H.; Ding, Z.-H.; Dong, Z.-J.; Li, G.-T.; Li, Y.; Liu, J.-K. J. Nat. Prod. 2013, 76, 79−84. (28) Hsiao, C.-J.; Hsiao, G.; Chen, W.-L.; Wang, S.-W.; Chiang, C.P.; Liu, L.-Y.; Guh, J.-H.; Lee, T.-H.; Chung, C.-L. J. Nat. Prod. 2014, 77, 758−765.

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Meroterpenoids from a Medicinal Fungus Antrodia cinnamomea

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