Chemical constituents from fruiting bodies of Basidiomycete Perenniporia subacida

Chemical constituents from fruiting bodies of Basidiomycete Perenniporia subacida

Fitoterapia 109 (2016) 179–184 Contents lists available at ScienceDirect Fitoterapia journal homepage: www.elsevier.com/locate/fitote Chemical cons...

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Fitoterapia 109 (2016) 179–184

Contents lists available at ScienceDirect

Fitoterapia journal homepage: www.elsevier.com/locate/fitote

Chemical constituents from fruiting bodies of Basidiomycete Perenniporia subacida Chun-Nan Wen a,c, Dong-Bao Hu d, Xue Bai a, Fang Wang a, Zheng-Hui Li b, Tao Feng a,b,⁎, Ji-Kai Liu a,b,⁎ a

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China College of Pharmacy, South-Central University for Nationalities, Wuhan 430074, PR China University of Chinese Academy of Sciences, Beijing 100039, PR China d College of Resource and Environment, Yuxi Normal University, Yuxi 653100, PR China b c

a r t i c l e

i n f o

Article history: Received 4 December 2015 Received in revised form 4 January 2016 Accepted 8 January 2016 Available online 11 January 2016 Keywords: Perenniporia subacida Abietane diterpenoids Antifungal activities Cytotoxic activities

a b s t r a c t Four new aromatic abietane diterpenoids and two new benzene derivatives, namely perenacidins A–F (1–6), have been isolated from the fruiting bodies of Basidiomycete Perenniporia subacida. The structures were elucidated by means of extensive spectroscopic methods and computational ECD method. The antifungal activities against Canidia albicans and the cytotoxic activities against four cancer cell lines (including K-562, A-549, SMMC-7721, MCF-7) were evaluated in vitro. © 2016 Elsevier B.V. All rights reserved.

1. Introduction

2. Experimental section

Perenniporia Murrill is a cosmopolitan genus of bracket-forming or encrusting polypores containing about 60 currently recognized species, 29 of which have been reported in China [1,2]. Some species in the genus could attack living or dead hardwood and conifers, playing a key role in the substance circulation of forest ecosystem. Perenniporia subacida have been used as a medicine in treatment for tumor and pruritus in China. Previous phytochemical investigations on this genus revealed the presence of triterpenoids [3,4], naphthalenones [5], and sesquiterpenoids [6]. However, secondary metabolites produced by the fungus P. subacida have not been reported. As part of our efforts to search for bioactive secondary metabolites from higher fungi [7–9], we have carried out a chemical investigation on the EtOH extract of the fruiting bodies of P. subacida, which led to the isolation of four new aromatic abietane diterpenoids and two new benzene derivatives, namely perenacidins A–F (1–6). Herein, we report the isolation, structural elucidation and biological activities of these compounds.

2.1. General experimental procedures

⁎ Corresponding authors at: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China. E-mail addresses: [email protected] (T. Feng), [email protected] (J.-K. Liu).

http://dx.doi.org/10.1016/j.fitote.2016.01.006 0367-326X/© 2016 Elsevier B.V. All rights reserved.

Optical rotations were measured with a P-1020 polarimeter (Jasco, Japan). UV spectra were recorded using a UV-2401A spectrophotometer (Shimadzu, Japan) equipped with a DAD and a 1-cm path-length cell. Samples in methanol solution were scanned from 190 to 400 nm in 1 nm steps. IR spectra were obtained on a Bruker FT-IR Tensor 27 spectrometer using KBr pellets. 1D and 2D NMR spectra were run on a Bruker Avance III-600 MHz spectrometer (Karlsruhe, Germany). Chemical shifts (δ) were expressed in ppm with reference to solvent signals. HR-MS were recorded on a Waters Auto Premier P776 spectrometer (Waters, USA) or an Agilent G6230AA Accurate Mass TOF LC/MS instrument (Agilent, USA). An Agilent 1200 series instrument equipped with Zorbax SB-C18 column (5 μm, 4.6 mm × 150 mm, Agilent, USA; detector: DAD) was used for high performance liquid chromatography (HPLC) analysis with a flow rate of 1.0 mL/min, and an Agilent 1100 series instrument with a reverse-phase preparative Zorbax SB-C18 column (5 μm, 9.4 mm × 150 mm, Agilent, USA) was used for the sample preparation with a flow rate of 10 mL/min. Column chromatography (CC) was performed on silica gel (200–300 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao, China), RP-18 (5 μm, Fuji Silyisa Chemical Ltd., Japan), and Sephadex LH-20 (Amersham Biosciences, Sweden). Fractions were monitored by TLC (GF254, Qingdao Haiyang Chemical

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Co. Ltd., Qingdao, China), and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in ethanol. 2.2. Fungal material Fruiting bodies of P. subacida were collected in a suburb of Helsinki, Finland in September, 2014 and identified by Prof. Yu-Cheng Dai (Beijing Forestry University). A specimen (No. KIB2014-7#) was deposited at Kunming Institute of Botany, Chinese Academy of Sciences. 2.3. Extraction and isolation The dried and powdered fruiting bodies (860 g) of P. subacida were extracted four times (48 h each time) with MeOH at room temperature. The organic layer was concentrated under reduced pressure to afford a crude extract (71 g), and the residue was subjected to silica gel column chromatography separated on a silica gel column (8 × 40 cm) eluted with step-gradient of CHCl3–MeOH (from 1:0 to 0:1) to yield ten fractions (Fr. 1–10). Fr. 3 (13 g) was separated on a reverse-phase (RP) C18 column (5 μm, 4 × 18 cm) using a step-gradient of MeOH–H2O (v/v: 8:2, 4:6, 6:4, 8:2, 0:10) to yield seven sub-fractions (3a–3 g). Fraction 3e (570 mg) was separated by preparative-HPLC (MeCN–H2O, from 20:80 to 40:60 in 20 min) to give compounds 3 (3.3 mg, retention time (tR) = 11.5 min), and 4 (1.5 mg, tR = 9.8 min). Fraction 5 (6.3 g) was subjected on a RP-C18 silica gel with MeOH–H2O (v/v, 0:100, 30:70, 60:40, 100:0) to get five sub-fractions (5a–5e). After preparative-HPLC (MeCN–H2O, from 15:85 to 30:70 in 15 min) and Sephadex LH-20 (acetone) gel column, 1 (35 mg, tR = 7.9 min) and 2 (12 mg, tR = 10.1 min) were obtained from fraction 5b (815 mg). Fraction 6 (1.4 g) was also applied to a RP-C18 silica gel (MeOH–H2O from 20:80 to 100:0) to obtain four fractions (6a–6d). Fr. 6b (550 mg) was further subjected to Sephadex LH-20 (acetone), and subsequently separated over a preparative-HPLC (MeCN–H2O, from 0:100 to 40:60 in 40 min), yielding compounds 5 (1.8 mg, tR = 20.0 min) and 6 (2.3 mg, tR = 26.5 min). Perenacidin A (1): yellowish oil, [α]24 D = −5.5 (c 0.71, MeOH); UV (MeOH) λmax (log ε): 210 (4.19), 254 (3.83), 299 (3.07); IR (KBr) vmax 3438, 2978, 2934, 1704, 1674, 1607, 1386, 1237, 1163, 1126, 1032 cm− 1; 1H and 13C NMR data (see Table 1); HR-EI-MS: m/z 346.1764 [M]+ (calcd for C20H26O5, 346.1780). Table 1 13 C NMR (150 MHz) and 1H NMR (600 MHz) data for compounds 1 and 2 in acetone-d6. No.

1

Perenacidin B (2): yellowish oil, [α]24 D = +11.2 (c 0.22, MeOH); UV (MeOH) λmax (log ε): 210 (4.15), 254 (3.88), 299 (3.02); IR (KBr) vmax 3439, 2970, 2932, 1702, 1683, 1610, 1460, 1385, 1240, 1208, 1122, 1064, 1039 cm−1; 1H and 13C NMR data (see Table 1); HR-EI-MS: m/z 346.1775 [M]+ (calcd for C20H26O5, 346.1780). Perenacidin C (3): yellowish oil, [α]24 D = +11.5 (c 0.37, MeOH); UV (MeOH) λmax (log ε): 194 (3.77), 233 (4.10), 250 (3.87), 296 (2.97); IR (KBr) vmax 3441, 3432, 2956, 2924, 1683, 1631, 1566, 1392, 1360, 1241, 1119, 1065, 1039 cm−1; 1H and 13C NMR data (see Table 2); HR-EI-MS: m/z 330.1469 [M]+ (calcd for C19H22O5, 330.1467). Perenacidin D (4): yellowish oil, [α]24 D = −2.3 (c 0.12, MeOH); UV (MeOH) λmax (log ε): 194 (3.77), 233 (4.10), 250 (3.82), 296 (2.90); IR (KBr) vmax 3439, 3428, 2955, 2923, 2854, 1686, 1637, 1566, 1452, 1385, 1239, 1119, 1042 cm−1; 1H and 13C NMR data (see Table 2); HRTOF-ESI-MS (pos.): m/z 353.1359 [M + Na]+ (calcd for C19H22NaO5, 353.1365). Perenacidin E (5): yellowish oil, [α]24 D = +10.7 (c 0.20, MeOH); UV (MeOH) λmax (log ε): 203 (4.15), 226 (3.72), 280 (3.10); IR (KBr) vmax 3440, 2957, 2923, 2854, 1631, 1446, 1383, 1248, 1162, 1113, 1035 cm− 1; 1H and 13C NMR data (see Table 3); HR-TOF-ESI-MS (pos.): m/z 287.0891 [M + Na]+ (calcd for C14H16NaO5, 287.0895). Perenacidin F (6): yellowish oil, [α]24 D = −8.0 (c 0.23, MeOH); UV (MeOH) λmax (log ε): 195 (4.16), 216 (4.62), 238 (4.02), 354 (2.54) IR (KBr) vmax 3442, 2956,2924, 2854, 1751, 1629, 1450, 1428, 1381, 1314, 1248, 1163, 1112, 1053 cm−1; 1H and 13C NMR data (see Table 3); HR-EI-MS: m/z 190.0631 [M]+ (calcd for C11H10O3, 190.0630). 2.4. Computational methods All DFT and TD-DFT calculations were carried out at 298 K in the gas phase with Gaussian 09 [10]. Conformational searches were carried out at the molecular mechanics level of theory employing MMFF force fields [11–13]. The conformers with relative energy within 10 kcal/mol of the lowest-energy conformer were selected and further geometry optimized at the B3LYP/6–311++G(2d,p) level. All the lowest-energy conformers, which correspond to 99% of the total Boltzmann distribution, were selected for ECD spectra calculation. The Boltzmann factor for each conformer was calculated based on Gibbs free energy. Vibrational analysis at the B3LYP/6–311++G (2d,p) level of theory resulted in no imaginary frequencies, confirming the considered conformers as real minima. TDDFT was employed to calculate excitation energy (in nm) and rotatory strength R in dipole velocity form, at the B3LYP/6– 311++G(2d,p) level.

2

δC

δH (J in Hz)

δC

δH (J in Hz)

1 2

75.8, d 29.1, t

70.0, d 25.6, t

3

35.4, t

4.03 (dd, 9.2, 5.0) 1.92 (overlap) 1.92 (overlap) 1.95 (overlap) 1.70 (m)

4.54 (br s) 2.21 (dddd, 14.3, 14.2, 3.7, 1.9) 1.85 (br d, 14.3) 2.46 (ddd, 14.2, 13.1, 3.8) 1.47 (br d, 13.1)

4 5 6

46.6, s 44.4, d 37.9, t

7 8 9 10 11 12 13 14 15 16 17 18 19 20

198.4, s 131.4, s 154.2, s 44.1, s 127.3, d 131.2, d 149.2, s 123.4, d 71.7, s 32.0, q 32.0, q 179.0, s 16.5, q 17.7, q

2.63 (dd. 14.1, 3.2) 2.34 (dd, 18.1, 3.2) 2.85 (dd, 18.0, 14.1)

8.43 (d, 8.5) 7.69 (dd, 8.5, 2.1) 8.03 (d, 2.1) 1.50 (s) 1.50 (s) 1.32 (s) 1.29 (s)

30.3, t 46.7, s 38.3, d 38.1, t 197.9, s 132.0, s 151.6, s 43.8, s 126.1, d 131.1, d 148.7, s 123.4, d 71.7, s 32.0, q 32.0, q 179.2, s 16.8, q 24.0, q

3.28 (dd, 14.7, 3.1) 2.36 (dd, 17.0, 3.1) 2.81 (dd, 17.0, 14.7)

7.53 (d, 8.3) 7.74 (dd, 8.3, 2.1)

2.5. Antifungal activity Compounds 1–4 were tested for their antimicrobial activities against Canidia albicans in vitro used a turbidimetric method. Amphotericin B was used as a positive control. C. albicans was inoculated in potato dextrose broth (formulated identically to potato dextrose agar (PDA), omitting the agar, prepared in this laboratory) and diluted with medium to 1 × 106 CFU mL− 1. Aliquots of 90 μL were filled in 96-well Ubottomed microplates, and then treated with compounds 1–4 at the maximum concentration of 20 μg/mL. After culturing at 37 °C for 24 h, the absorbance was measured at 620 nm with the microplate reader. The percentage inhibition of cell growth below 50% was regarded as inactive. 2.6. Cytotoxic activity

8.08 (d, 2.1) 1.51 (s) 1.51 (s) 1.35 (s) 1.32 (s)

The assignments were based on 13C, DEPT, and HSQC experiments.

Hepatocellular carcinoma SMMC-7721, lung cancer A-549 cells, breast cancer MCF-7 and human leukemia K-562 cell lines were used in the cytotoxic assay. All the cells were cultured in RPMI-1640 or DMEM medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA) in 5% CO2 at 37 °C. The cytotoxicity assay was performed by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

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Table 2 13 C NMR (150 MHz) and 1H NMR (600 MHz) data for compounds 3 and 4 ameasured in acetone-d6, bmeasured in methanol-d4. 3a

No.

4b

δC

δH (J in Hz)

δC

δH (J in Hz)

1

70.0, d

4.60 (br s)

47.0, t

2

25.8, t

64.8, d

3

30.3, t

1.89 (br d, 14.4) 2.25 (dddd, 14.4, 14.2, 3.7, 1.9) 1.52 (br d, 13.0) 2.46 (ddd, 14.2, 13.0, 3.9)

2.74 (br d, 12.1) 1.53 (dd, 12.1, 11.4) 4.10 (dddd, 11.7, 11.4, 1.8, 1.8)

4 5 6

46.8, s 38.0, d 38.0, t

7 8 9 10 11 12 13 14 15 17 18 19 20

197.1, s 132.6, s 158.5, s 44.6, s 127.4, d 133.0, d 135.8, s 127.8, d 197.2, s 26.6, q 178.8, s 16.8, q 23.6, q

3.30 (dd, 14.6, 2.5) 2.41 (dd, 17.2, 2.5) 2.88 (overlapped)

7.75 (d, 8.3) 8.12 (dd, 8.3, 1.9) 8.49 (d, 1.9) 2.60 (s) 1.37 (s) 1.37 (s)

46.5, t 48.8, s 44.8, d 38.4, t 199.3, s 131.7, s 161.2, s 40.8, s 125.7, d 134.6, d 136.7, s 128.7, d 199.4, s 26.7, q 181.2, s 18.1, q 24.5, q

2.03 (dd, 12.4, 1.8) 1.74 (dd, 12.4, 11.7) 2.68 (dd, 14.4, 2.7) 2.51 (dd, 17.7, 2.7) 2.83 (dd, 17.7, 14.4)

7.66 (d, 8.3) 8.20 (dd, 8.3, 1.7) 8.54 (d, 8.3) 2.62 (s) 1.37 (s) 1.34 (s)

The assignments were based on 13C, DEPT, and HSQC experiments.

sul-fophenyl)-2H tetrazolium] assay according to the manufacturer's instructions. Briefly, 100 μL adherent cells were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition to initial density of 1 × 105 cells/mL. Each tumor cell line was exposed to the tested compounds 1–6 dissolved in DMSO at various concentrations in triplicates for 48 h, with cisplatin and taxol (Sigma, USA) as positive controls. After compound treatment, cell viability was detected by the absorbance at 490 nm, and a cell growth curve was graphed. IC50 values were calculated by Reed and Muench's method [14]. 3. Results and discussion Compound 1 was obtained as a yellowish oil. The molecular formula C20H26O5 was determined on the basis of HR-EI-MS at m/z 346.1764 [M]+ (calcd. for C20H26O5, 346.1780), indicating eight degrees of unsaturation. Its IR spectrum revealed the presence of hydroxyl (3438 cm−1), carboxyl (1704 cm−1), carbonyl (1674 cm−1) and aromatic ring (1607 cm−1). The 1H NMR spectrum (Table 1) exhibited resonances for four methyl groups at δH 1.50 (s, H6-16, 17), 1.32 (s, H3-19), and 1.29 (s, H3-20), one oxygenated methine group at δH 4.03 (dd, J = Table 3 13 C NMR (150 MHz) and 1H NMR (600 MHz) data for compounds 5 and 6 in methanol-d4. No.

5 δC

1 2 3 4 5 6 7 8 9 10 11 12 13 14-OMe

126.0, s 122.4, d 154.6, s 148.9, s 123.2, d 133.2, d 171.1, s 72.8, s 31.9, q 31.9, q 86.3, s 171.1, s 23.6, q 53.9, q

6 δH (J in Hz) 7.98 (d, 1.6)

7.64 (d, 8.2) 7.93 (dd, 8.2, 1.6)

1.56 (s) 1.56 (s)

δC 139.6, s 126.5, d 127.5, s 157.1, s 123.8, d 135.2, d 198.7, s 26.9, q 171.6, s 79.8, d 20.3, q

1.87 (s) 3.74 (s)

The assignments were based on 13C, DEPT, and HSQC experiments.

δH (J in Hz) 8.42 (d, 1.0)

7.73 (d, 8.0) 8.35 (dd, 8.0, 1.0) 2.67 (s) 5.72 (q, 6.8) 1.64 (d, 6.8)

9.2, 5.0 Hz), and three aromatic protons at δH 7.69 (dd, J = 8.5, 2.1 Hz, H-12), 8.03 (d, J = 2.1 Hz, H-14), and 8.43 (d, J = 8.5 Hz, H-11). The 13 C NMR spectrum (Table 1) indicated the presence of a carboxyl carbon (δC 179.0), a carbonyl carbon (δC 198.4), six aromatic carbons (δC 131.4, 154.2, 127.3, 131.2, 149.2, 123.4), an oxygenated methine (δC 75.8), and four methyl carbons (δC 16.5, 17.7, 32.0, 32.0). Analyses of the 1H and 13 C NMR data for compound 1 revealed structural features similar to those of 15-hydroxy-7-oxodehydroabietic acid [15]. The main difference was the presence of one methine bearing a hydroxy group (δC 75.8; δH 4.03) in compound 1 rather than a methylene in 15-hydroxy-7oxodehydroabietic acid. The hydroxy group was located at C-1 deduced from the HMBC correlations (Fig. 2) of H-1(δH 4.03) with C-3 (δC 35.4) and C-20 (δC 17.7). In detail, the characteristic coupling constants of H1 (J = 9.2, 5.0 Hz) indicated the axial position for the hydroxy group. Further HMBC spectrum (Fig. 2) showed correlations of H-3 (δH 1.95, 1.70) and H-5 (δH 2.63) with the carboxyl carbon (C-18, δC 179.0), H2-6 and H-14 with C-7 (δC 198.4), and H6-16, 17 (δH 1.50) with C-13 (δC 149.2) and C-15 (δC 71.7). Cross peaks in the ROESY spectrum (Fig. 3) between H-1/H-5 and between H3-19 (δH 1.32)/H-6β (δH 2.85)/H3-20 (δH 1.29) suggested the β orientation of the hydroxyl group and α orientation of carboxylic group, respectively. In contrast to 15-hydroxy-7oxodehydroabietic acid, the large downfield shift (Δ = 1.0 ppm) of H11 (δH 8.43) in 1 mainly resulted from the interaction with OH-1β due to the close distance in computer-generated 3D drawing (Fig. 3) with minimized energy by MM2 calculation, as evidenced in 11β,15dihydroxyabieta-8,11,13-trien-18-oic acid [16]. Thus, compound 1 was identified as 1β,15-dihydroxy-7-oxoabieta-8,11,13-trien-18-oic acid, namely perenacidin A. Compound 2 was also obtained as a yellowish oil. It exhibited a molecular ion peak at m/z 346.1775 [M] + (calcd. for C20H26O5, 346.1780) in the HR-EI-MS, indicating eight indices of hydrogen deficiency. Its IR spectrum revealed the presence of hydroxy (3439 cm− 1), carboxyl (1702 cm−1), carbonyl (1683 cm−1) and aromatic ring (1610 cm−1). The 1H NMR and 13C NMR spectra (Table 1) were very similar to those of 1. The further 1H–1H COSY and HMBC experiments (Fig. 2) suggested compound 2 did possess the same framework as those of compound 1. However, the change of H-1 [δH 4.54 (br s) in 2; instead of δH 4.03 (dd, J = 9.2, 5.0 Hz) in 1], as well as tiny changes of NMR data of CH2-2 and CH2-3, suggested that the stereoconfiguration of C-1 in 2 was different from that of 1. The broad singlet of H-1 indicated that it was axial and

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Fig. 1. Structures of compounds 1–6 from Perenniporia subacida.

possessed α orientation for the OH, which was supported by the ROESY correlations (Fig. 3) of H-1/H3-20 and H3-19/H-6β/H3-20. Therefore, the structure of compound 2 was assigned as 1α,15-dihydroxy-7-oxoabieta8,11,13-trien-18-oic acid, and it was given the name perenacidin B. Compound 3 was obtained as a yellowish oil. Its molecular formula, C19H22O5, was established based on the molecular ion peak at m/z 330.1469 [M]+ (calcd. for C19H22O5, 330.1467) in HR-EI-MS, corresponding to nine degrees of unsaturation. The IR data at 3441, 1683, 1631, and 1566 cm−1 revealed the presence of hydroxyl, carbonyl groups, and benzene ring, respectively. The 1D NMR (Table 2) show an AMX spin systematic protons [δH 7.75 (1H, d, J = 8.3), 8.12 (dd, J = 8.3, 1.9 Hz), and 8.49 (d, J = 1.9 Hz); δC 132.6 (s), 158.5 (s), 127.4 (d), 133.0 (d), 135.8 (s), 127.8 (d)], indicating a 1,2,4-trisubstituted benzene ring. Apart from the above-mentioned data, the 13C spectral data (Table 2) were also exhibited 13 carbon resonances including three methyls, three methylenes, two methines (one oxygenated), and five quaternary carbons (two carbonyl, one carboxylic). By intensive comparison of the 1H and 13C data with those of 2, the significant difference was the absence of two methyls [δH 1.51 (6H, s); δC 32.0] and an oxygenated carbon (δC 71.7) and the presence of an acetyl group [δC 197.2, 26.6; δH 2.60] in compound 3, which was positioned at C-13 on the base of HMBC correlations (Fig. 2) of H3-17 (δH 2.60) with C-13 (δC 135.8), and H-12 (δH 8.12) with C-15 (δC 197.2). The deuterated solvent of acetone-d6 was changed to chloroform-d for another ROESY experiment due to the same chemical shift of H3-19 and H3-20

and the key proton H-6β overlapped in water residual peaks. The pseudo-axially oriented proton H-6β (δH 2.78, dd, J = 17.2, 14.6 Hz, in chloroform-d) showed ROESY correlations (Fig. 3) with H3-19 (δH 1.37, in chloroform) and H3-20 (δH 1.32, in chloroform), revealing βand axial orientation for H3-20 and H3-19. In addition, cross peaks of ROESY correlations (Fig. 3) between H3-20 and H-1 (δH 4.60) suggested that OH-1 was α configuration. Consequently, compound 3 was elucidated as 1α-hydroxy-7,15-dioxo-16-norabieta-8,11,13-trien-18-oic acid and was given the name perenacidin C. Compound 4 was obtained as a yellowish oil and yielded a pseudomolecular ion peak at m/z 353.1359 ([M + Na]+, calcd. 353.1365) in the positive HR-ESI-MS, indicative of the molecular formula of C19H22O5 with nine degrees of unsaturation. The 1D NMR spectrum (Table 2) revealed 19 carbon resonances, which were ascribed to three methyls, three sp3 methylenes, five methines (including three sp2 carbons), eight quaternary carbons (including two carbonyls, and one carboxyl). The spectral features of NMR and IR data suggested that compounds 3 and 4 had similar structures. However, the hydroxy group in compound 4 was located at C-2, which was proven by the evidence of 1H–1H COSY correlations (Fig. 2) of H2-1 (δH 2.74, 1.53) and H2-3 (δH 2.03, 1.74) with H-2 (δH 4.10). Furthermore, H-2 showing ROESY correlations (Fig. 3) with H3-19 (δH 1.37) and H3-20 (δH 1.34), as well as its coupling constants (dddd, J = 11.7, 11.4, 1.8, 1.8), revealed α and equatorial orientation of the hydroxy group. Hence, the structure of compound 4 was determined as 2α-hydroxy-7,15-dioxo-16-norabieta8,11,13-trien-18-oic acid, which was given the name perenacidin D. Compound 5 was obtained as a yellowish oil, with the molecular formula of C14H16O5, deduced from the pseudo-molecular ion peak at m/z 287.0891 (calcd. for C14H16NaO5, 287.0895) in the positive HRESIMS, requiring seven indices of hydrogen deficiency. Its IR spectrum revealed the presence of hydroxy (3440 cm−1), and carboxyl (1631 cm− 1) groups. The 1D NMR spectrum (Table 3) showed six aromatic carbons [δC 122.4 (d), 123.2 (d), 133.2 (d), 126.0 (s), 154.6 (s), 148.9 (s)] and proton signals with characteristic coupling constants [δH 7.98 (d, 1.6), 7.64 (d, 8.2), 7.93 (dd, 8.2, 1.6)], indicating the presence of a 1,2,4-trisubstituted benzene ring. C-7 (δC 171.1) showed HMBC correlations (Fig. 2) from H-2 (δH 7.98) and H-6 (δH 7.93), and H3-14 (δH 3.74), which linked a methyl carboxyl group to C-1 (δC 126.0). HMBC correlations (Fig. 2) from the two methyls at δH 1.56 (s) to C-3 (δC 154.6) and C8 (δC 72.8), and from H3-13 (δH 1.87) with C-4 (δC 148.9), C-11 (δC 86.3), and C-12 (δC 171.0, C = O), supported the planar structure as shown. Taking into account the degrees of unsaturation and the chemical formula, C-12 was connected to C-8 (δC 72.8) through an ester group. In order to determine the absolute configuration, a TDDFT-ECD calculation protocol was pursued. As can be seen from Fig. 4, ECD curves for the two

Fig. 2. Key HMBC correlations (→) and 1H–1H COSY correlations (\ \) of compounds 1–6.

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183

Fig. 3. Key ROESY correlations (↔) and distance (OH-1 → H-11) in compounds 1–4.

possible stereostructures (11R; 11S) were calculated using the TDDFT theory method. The calculated curves of 11S were in good agreement with the experimental CD spectrum. Thus, the absolute configuration of C-11 in 5 was established as S. Finally, compound 5 was identified, and given the name perenacidin E. Compound 6 was obtained as a yellowish powder. It gave a molecular ion peak at m/z 190.0631 in the HREIMS, indicating the molecular formula of C11H10O3, (calcd., 190.0630), which implying seven indices of hydrogen deficiency. Its IR spectrum showed the absorption of hydroxyl (3442 cm− 1), carbonyl (1751 cm− 1), and carboxylic (1629 cm−1) groups. The 1D NMR (Table 3) showed a 1,2,4-trisubstituted aromatic ring due to the characteristic data [δH 8.42 (d, J = 1.0 Hz), 8.35 (dd, J = 8.0, 1.0 Hz), 7.73 (d, J = 8.0 Hz); δC 139.6 (s), 126.5 (d), 127.5 (s), 157.1 (s), 123.8 (d), 135.2 (d)]. What's more, an acetyl was located at C-1 based on the observation of HMBC correlations (Fig. 2) between H3-8 (δH 2.67) with C-7 (δC 198.7) and C-1 (δC 139.6). HMBC correlations (Fig. 2) between H-2 (δH 8.42) with C-9 (δC 171.6), H-10 (δH 5.72) with C-3 (δC 127.5), C-4 (δC 157.1), and C-11 (δC 20.3),

suggesting the carboxylic and 1-O-substituted ethyl [δH 5.72 (q, J = 6.8 Hz), 1.64 (d, J = 6.8 Hz); δC 79.8 (d), 20.3 (q)] at C-3 and C-4, respectively. HMBC correlation from H-10 to C-9, indicated the presence of the γ-lactone, which also accounted for one unsaturation degree. The absolute configuration of 6 was determined by the same method as compound 5: the calculated ECD spectrum of the 10R conformer matched very well with the experimental one (Fig. 5). As a result, the structure of compound 6 was deduced as shown in Fig. 1, and named perenacidin F. The selected compounds were evaluated in vitro for the antifungal activities against C. albicans and the cytotoxic activities against four cancer cell lines (including K562, A549, SMMC7721, MCF-7). Unfortunately, none of selected compounds showed obviously inhibitory effect against C. albicans (b50%) and four cancer cell lines (IC50 N 40 μM). Abietane diterpenoids were widely distributed in the plant kingdom (mainly in the families of Labiatae, Verbenzaceae, Euhorbiaceae, Pinaceae, Cupressaceae, Taxodiaceae). However, C-1 or C-2 hydroxylated abietane derivatives were rarely found in natural products from plants.

Fig. 4. Experimental and calculated ECD for compound 5.

Fig. 5. Experimental and calculated ECD for compound 6.

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