Cytotoxic prenylated bibenzyls and flavonoids from Macaranga kurzii

Fitoterapia 99 (2014) 261–266

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Cytotoxic prenylated bibenzyls and flavonoids from Macaranga kurzii Da-Song Yang a,1, Jian-Guo Wei a,1, Wei-Bing Peng b, Shuang-Mei Wang a, Chen Sun b, Yong-Ping Yang a, Ke-Chun Liu b, Xiao-Li Li a,⁎ a Key Laboratory of Economic Plants and Biotechnology; Germplasm Bank of Wild Species in Southwest China; Institute of Tibetan Plateau Research at Kunming, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China b Biology Institute of Shandong Academy of Sciences, Jinan 250014, PR China

a r t i c l e

i n f o

Article history: Received 14 September 2014 Accepted in revised form 30 September 2014 Available online xxxx Keywords: Macaranga kurzii Euphorbiaceae Prenylated flavonoids Prenylated bibenzyls Cytotoxicity

a b s t r a c t One unique prenylated bibenzyl, kurzphenol A (1), two new prenylated flavonoids, kurzphenols B and C (2 and 3), as well as fourteen known compounds (4–17) were isolated from the twigs of Macaranga kurzii. Compound 1 was the first example of prenylated bibenzyl which possesses a benzofuran ring. All the known compounds were isolated from M. kurzii for the first time. Their structures were elucidated on the basis of extensive spectroscopic interpretation. Compounds 1– 17 were tested for their cytotoxicity against A-549 and Hep G2 cancer cell lines and showed IC50 values in the range of 9.76–30.14 μg/mL. © 2014 Published by Elsevier B.V.

1. Introduction The genus Macaranga is one of the largest genera of the Euphorbiaceae, and many of the plants in this genus were characteristics of secreting red juice after injury. An overview of the literature indicated that prenylated stibenes [1–4] and flavonoids [5–9] are their typical secondary metabolites, and the biological activities of those metabolites encompass virtually all fields of pharmacological sciences [10,11]. Due to the differences in the prenylation position on the aromatic rings, various lengths of prenyl chain, and further modifications of the prenyl moiety such as cyclization and hydroxylation, prenylation contributes strongly to the structural diversity of aromatic products [12]. Studies have showed that the presence of isoprenoid chains is a major determinant of the bioactivity of

⁎ Corresponding author. Tel./fax: +86 871 65223231. E-mail address: [email protected] (X.-L. Li). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.fitote.2014.10.003 0367-326X/© 2014 Published by Elsevier B.V.

prenylated aromatic compounds and made them exhibit much higher biological activities than their mother compounds without derivatization or decoration [13]. Macaranga kurzii was a small arbor distributed in tropical rainforests. Previous phytochemical investigations on M. kurzii showed that it contained flavonoids and stibenes, together with their prenylated derivatives [14]. In the course of an investigation to identify anticancer bioactive compounds from the genus Macaranga, a phytochemical investigation on M. kurzii led to the isolation and characterization of one unique prenylated bibenzyl (1) and two new prenylated flavonoids (2 and 3), coupled with fourteen known compounds (4–17) including one prenylated bibenzyl (4), eight flavonoids (5–12, seven prenylated ones), two polyacetylenes (13 and 14), one ionone (15), one coumarin (16) and one phenolic compound (17) (Fig. 1) . Although numbers of prenylated stilbenes have been reported in genus Macaranga, bibenzyls which were the reduction products of the stilbene were reported in this genus for the first time, and compound 1 was the first example of prenylated bibenzyl which possesses a benzofuran ring. Herein, we report the isolation and structure elucidation of those

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compounds, as well as their cytotoxicity against A-549 and Hep G2 cancer cell lines. 2. Experimental 2.1. General ORD spectra were recorded on a Horiba SEPA-300 polarimeter. CD spectra were obtained on an Automated Circular Dichroism spectrometer (Applied Photophysics). UV data were obtained on a Shimadzu UV-2401PC spectrophotometer. IR spectra were recorded on a Bio-Rad FTS-135 spectrometer with KBr pellets. 1D and 2D NMR experiments were recorded on Bruker AM-400, DRX-500 or Avance III 600 spectrometers with TMS as internal standard. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. ESI-MS was recorded using a Finnigan MAT 90 instrument and HR-ESI-MS was performed on an API QSTAR time-of-flight spectrometer. HREI-MS was performed on a Waters AutoSpec Premier P776. Column chromatography (CC) was performed on Sephadex LH-20, silica gel (200–300 mesh, Qingdao Marine Chemical inc., Qingdao, PR China), RP-18 gel (LiChroprep, 40–63 μm, Merck, Darmstadt, Germany), and MCI gel CHP20P (75–150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan). Semi-preparative HPLC was performed on a Hewlett-Packard instrument (column: Zorbax SB-C18, 250 × 9.4 mm; DAD detector). Fractions were monitored by TLC and visualized by heating plates sprayed with 15% H2SO4 in EtOH. 2.2. Plant material The twigs of M. kurzii were collected from Xishuangbanna of Yunnan province, PR China, in August 2012. A voucher specimen (Yangyp—20120806) was deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences, which was identified by one of the authors (Prof. Yong-Ping Yang).

and 9 (4.3 mg). Fr.5 was subject to Sephadex LH-20 CC (CHCl3–MeOH, 1:1), followed by semipreparative HPLC (MeOH–H2O, 85:15) to afford 1 (15.2 mg) and 4 (9.3 mg). Fr.6 was subjected to Sephadex LH-20 CC (CHCl3–MeOH, 1:1), followed by preparative TLC with petroleum ether–EtOAc (4:1), to give 13 (6.6 mg) and 14 (5.1 mg). 2.4. Spectroscopic data 2.4.1. Kurzphenol A (1) Colorless oil; [α]25 D −2.2 (c 0.22, MeOH); UV (MeOH) λmax (log ε) 208 (4.62), 251 (4.06), 294 (3.81) nm; IR (KBr) νmax 3442, 2965, 2925, 2858, 1616, 1537, 1495, 1452, 1376, 1309, 1254, 1209, 1135, 1070, 1045, 830, 770, 751, 734, 699 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table 1; negative ESIMS m/z 305 [M – H]−, 611 [2M – H]−; HR-EI-MS m/z 306.1613 [M]+ (calcd for C21H22O2, 306.1620). 2.4.2. Kurzphenol B (2) Yellow oil; [α]26 D −1.77 (c 0.28, MeOH); UV (MeOH) λmax (log ε) 204 (4.68), 300 (4.27), 346 (3.74) nm; IR (KBr) νmax 3421, 2968, 2916, 1631, 1452, 1374, 1276, 1230, 1182, 1112, 1003, 766, 698 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table 1; negative ESI-MS m/z 407 [M – H]−; HR-EI-MS m/z 408.1913 [M]+ (calcd for C25H28O5, 408.1937). 2.4.3. Kurzphenol C (3) Yellow oil; [α]24 D −1.31 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 202 (4.77), 274 (4.36) nm; IR (KBr) νmax 3424, 2924, 2854, 1657, 1611, 1553, 1506, 1452, 1434, 1359, 1300, 1268, 1230, 1178, 1159, 1115, 1067, 1009, 946, 835, 635, 578 cm−1; 1 H NMR (acetone-d6, 600 MHz) and 13C NMR (acetone-d6, 100 MHz) data, see Table 1; negative ESI-MS m/z 367 [M – H]−, 735 [2M – H]−; HR-ESI-MS m/z 367.1187 [M – H]− (calcd for C21H19O6, 367.1182). 2.5. Cytotoxicity assay

2.3. Extraction and isolation The air-dried and powdered twigs of M. kurzii (10 kg) were extracted with 90% EtOH (4 × 25 L) for 24 h at room temperature and concentrated in vacuo to give a crude extract. The extract was suspended in H2O and then extracted with EtOAc. The EtOAc fraction (337 g) was subjected to MCI gel CC (MeOH–H2O, 10:90 to 100:0) in a gradient to afford six fractions (Fr.1–6). Fr.1 was subjected to silica gel CC eluting with CHCl3–MeOH (20:1 to 4:1) and then purified by Sephadex LH-20 CC (CHCl3–MeOH, 1:1) to afford 15 (12.3 mg) and 17 (20.6 mg). Fr.2 was subjected to silica gel CC eluting with CHCl3–acetone (10:1 to 3:2) in a gradient and then purified by Sephadex LH-20 CC (CHCl3–MeOH, 1:1) to afford 10 (2.4 mg) and 16 (17.0 mg). Fr.3 was subjected to silica gel CC eluting with CHCl3–EtOAc (9:1 to 3:2) to yield three major subfractions (Fr.3a–3c). Fr.3a was purified by semipreparative HPLC (MeOH–H2O, 90:10) to afford 3 (12.7 mg), 7 (3.4 mg) and 8 (5.1 mg). Fr.3b was repeatedly subjected to Sephadex LH-20 CC (CHCl3–MeOH, 1:1) to obtain 6 (7.9 mg), 11 (13.4 mg) and 12 (14.0 mg). Fr.4 was subject to silica gel CC eluting with CHCl3–EtOAc (10:1 to 3:1) and then purified by Sephadex LH-20 CC (CHCl3–MeOH, 1:1) to afford 2 (16.8 mg), 5 (20.1 mg)

Compounds 1–17 were tested for their cytotoxicity against human lung carcinoma (A-549) and human hepatocellular (Hep G2) by the MTT method, 5-FU was used as a positive control. Briefly, 100 μL of cell suspension (1 × 105 cells/mL) was seeded into 96-well microtiter plates and cultured for 24 h before the compound was added. Then, different concentrations of the compounds were added to the plates, the cells were cultivated for 48 h, and 10 μL of MTT (5 mg/mL) was added to each well. After 4 h, the culture medium was removed and the formazan crystals were completely dissolved with 150 μL DMSO in each well by vigorously shaking the plate. Finally, formazan absorbance was assessed by a BioRad microplate reader at 570 nm. 3. Results and discussion Kurzphenol A (1) was obtained as colorless oil. The molecular formula of 1 was determined as C21H22O2 by HR-EI-MS, requiring 11 degrees of unsaturation. The IR spectrum showed absorption bands for OH (3432 cm−1) and aromatic ring (1616, 1537 cm−1) moieties. The UV spectrum of 1 showed absorption maxima at 208 nm (4.62) and 294 nm (3.81), which

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263

Table 1 1 H and 13C NMR data of compounds 1–3 (δ in ppm, J in Hz). No.

1 (in CDCl3) δHa

1 2 3 4 5 6 7 8 9 10 α β 1′ 2′, 6′ 3′, 5′ 4′ 1″ 2″ 3″ 4″ 5″ 1‴ 2‴ 3‴ 4‴ 5‴ OMe 5-OH 7-OH

6.92, s

6.67, d (2.1) 7.49, d (2.1)

3.15, dd (9.5, 6.8) 2.90, dd (9.5, 6.8) 7.20, d (7.3) 7.31, t (7.3) 7.24, t (7.3) 3.44, d (6.6) 5.11, t (6.6) 1.75, s 1.85, s

2 (in CDCl3) δC

b

132.5 s 121.2 s 152.8 s 97.0 d 153.6 s 120.8 s 105.1 d 143.3 d

32.7 t 37.0 t 141.6 s 128.3 d 128.4 d 126.0 d 25.5 t 122.6 d 134.2 s 25.7 q 18.0 q

3 (in acetone-d6)

δHa

b

δC

5.02, d (11.9) 4.49, d (11.9)

83.1 d 72.5 d 196.2 s 158.8 s 107.9 s 163.2 s 107.0 s 157.6 s 100.3 s

7.54, d (7.1) 7.44, t (7.1) 7.41, t (7.1) 3.34, d (7.0) 5.22, t (7.0) 1.74, s 1.81, s 3.27, d (7.1) 5.17, t (7.1) 1.70, s 1.66, s 11.49, s 6.49, s

136.6 s 127.3 d 128.5 d 129.1 d 21.2 t 121.4 d 134.9 s 25.8 q 17.9 q 21.7 t 121.5 d 134.4 s 25.8 q 17.8 q

δHc

δCb

6.33, s

156.7 s 138.9 s 179.7 s 160.8 s 99.0 d 162.0 s 107.1 s 155.0 s 105.9 s

8.04, d (8.9) 7.02, d (8.9) 3.51, d (6.9) 5.23, t (6.9) 1.64, s 1.76, s

3.86, s 12.74, s

122.9 s 131.1 d 116.4 d 160.7 s 22.2 t 123.5 d 132.0 s 25.8 q 18.1 q

60.1 q

a 1

H NMR data measured at 500 MHz. C NMR data measured at 100 MHz. 1 H NMR data measured at 600 MHz.

b 13 c

indicated the presence of an unconjugated aromatic system [15]. Analysis of the 1H and 13C NMR (Table 1) data of 1 aided by HSQC revealed resonances for one prenyl [δC 25.5 (t), 122.6 (d), 134.2 (s), 25.7 (q), 18.0 (q); δH 3.44 (2H, d, J = 6.6 Hz), 5.11 (1H, d, J = 6.6 Hz), 1.75 (3H, s), 1.85 (3H, s)], one monosubstituted benzene ring [δH 7.20 (2H, d, J = 7.3 Hz), 7.31 (2H, t, J = 7.3 Hz), 7.24 (1H, t, J = 7.3 Hz)], one 1,3-dihydroxy substituted benzene ring [δC 97.0 (d); δH 6.92 (1H, s)], two adjacently benzylic methylenes [δH 3.15 (2H, dd, J = 9.5, 6.8 Hz), 2.90 (2H, t, J = 9.5, 6.8 Hz)] and one benzofuran ring [δC 105.1 (d), 143.3 (d); δH 6.67 (1H, d, J = 2.1 Hz), 7.49 (1H, d, J = 2.1 Hz)]. These signals showed similarities to those of 3,5dihydroxy-2-(3-methyl-2-butenyl)bibenzyl and 6-hydroxy-4(2-phenylethyl)benzofuran [16] and revealed that 1 may be a prenylated bibenzyl which possesses a benzofuran ring. This deduction was confirmed by the 1H–1H COSY correlations of H-7/H-8; H-1″/H-2″ and the HMBC correlations of H-7/C-5, C-6; H-1″/C-1, C-2, C-3; Me-4″/C-2″, C-3″, C-5″; Me-5″/C-2″ (Fig. 2). Therefore, the structure of 1 was determined as 3-hydroxy-2-(3-methyl-2-butenyl)-1-(2-phenylethyl) benzofuran, and it is the first example of prenylated bibenzyl which possess a benzofuran ring. Kurzphenol B (2) was obtained as optically active yellow oil ([α]26 D −1.77, c 0.28, MeOH) and the molecular formula was deduced to be C25H28O5 based on its HR-EI-MS (m/z 408.1913), suggesting 12 degrees of unsaturation. The IR spectrum of 2 suggested characteristic bands of hydroxyl (3421 cm−1) and carbonyl (1631 cm−1) groups. The UV spectrum showed the

characteristic absorbances for a dihydroflavonol [λmax (log ε) 300 (4.27) and 346 (3.74, sh) nm] [17]. An analysis of the NMR data of 2 (Table 1) suggested the presence of two prenyls [δC 21.2 (t), 121.4 (d), 134.9 (s), 25.8 (q), 17.9 (q); δH 3.34 (2H, d, J = 7.0 Hz), 5.22 (1H, d, J = 7.0 Hz), 1.74 (3H, s), 1.81 (3H, s); δC 21.7 (t), 121.5 (d), 134.4 (s), 25.8 (q), 17.8 (q); δH 3.27 (2H, d, J = 7.1 Hz), 5.17 (1H, d, J = 7.1 Hz), 1.70 (3H, s), 1.66 (3H, s)], one monosubstituted benzene ring [δH 7.54 (2H, d, J = 7.1 Hz), 7.44 (2H, t, J = 7.1 Hz), 7.41 (1H, t, J = 7.1 Hz)], two adjacently oxygenated methines [δC 83.1 (d), 72.5 (d); δH 5.02 (1H, d, J = 11.9 Hz), 4.49 (1H, d, J = 11.9 Hz)] and one ketone [δC 196.2]. A comparison of the 1D NMR data (Table 1) of 2 with those of 6,8-diprenylaromadendrin [17] revealed that 2 may be a diprenyldihydroflavonol. The differences between them could be rationalized to the carbon signals corresponding to an oxygenated quaternary carbon (C-4′, δC 156.4) in 6,8diprenylaromadendrin, which were replaced by a nonoxygenated methine [δC 129.1 (d), δH 7.41 (1H, d, J = 7.1 Hz)] in 2. This deduction was confirmed by the 1H–1H COSY correlations of H-3′/H-4′/H-5′ (Fig. 2). The observed HMBC (Fig. 2) correlations of H-1″/C-5, C-6, C-7 and H-1‴/C-7, C-8, C-9 demonstrated that two prenyls were located at C-6 and C-8, respectively. The absolute configurations of 2 were elucidated by the comparison of the coupling constant patterns with those reported data and CD spectra. Two AB systems at δH 5.02 (1H, d, J = 11.9 Hz) and 4.49 (1H, d, J = 11.9 Hz) are characteristic of H-2 and H-3 in the axial conformation of a 2,3-trans-

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Fig. 1. Structures of compounds 1–17.

8 7

O 5

6 1

1''' 3 2

OH

HO

8 9

7 6

1'' 1''

O

HO

8 9

7

3

OH O

2 Fig. 2. Selected HMBC (

O

OH

5

OH O 1

OH

1''

) and 1H–1H COSY (▬) correlations of compounds 1–3.

3

O

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Conflict of interest

Table 2 Cytotoxicity of compounds from Macaranga kurzii. compounds

1 3 4 11 12 14 15 17 5-FU

265

The authors declare that there is no conflict of interest.

IC50 (μg/mL) A-549

Hep G2

N100 17.11 16.01 9.76 15.32 18.22 18.23 12.01 13.26

30.14 N100 N100 N100 N100 N100 N100 N100 13.60

dihydroflavonol [17]. The stereochemistry at C-2 and C-3 was determined to be both R by analysis of the CD spectrum, in which a positive Cotton effect (+1.24) was observed at 319 nm along with a negative Cotton effect (−7.18) at 294 nm [18]. Therefore, the structure of 2 was determined as (2R, 3R)6,8-diprenylpinobanksin. Kurzphenol C (3) possessed a molecular formula of C21H20O6 as deduced from its HR-ESI-MS ([M – H]−, m/z 367.1187), suggesting 12 degrees of unsaturation. The IR spectra of 3 indicated the characteristic bands of hydroxyl (3424 cm− 1) and conjugated carbonyl (1657 cm− 1) groups, which was further supported by its NMR data. The 1D NMR data (Table 1) exhibited one 8-substituted kaempferol [δC 138.9 (s), 179.7 (s), 99.0 (d); δH 6.33 (1H, s); 7.02 (2H, d, J = 8.9 Hz), 8.04 (2H, d, J = 8.9 Hz)], one prenyl [δC 22.2 (t), 123.5 (d), 132.0 (s), 25.8 (q), 18.1 (q); δH 3.51 (2H, d, J = 6.9 Hz), 5.23 (1H, d, J = 6.9 Hz), 1.64 (3H, s), 1.76 (3H, s)], and one methoxy group (δC 60.1). A comparison of the NMR data of 3 with those of icaritin [19] indicated that they were very similar except for the presence of a methoxy group (δC 60.1) in 3. The location of methoxy group and prenyl were confirmed by HMBC correlations of H-OMe (δH 3.86)/C-3; H1″/C-7; C-8, C-9; H-2″/C-8 (Fig. 2). As a result, the structure of 3 was determined as 3-O-methyl-8-(3-methyl-2-butenyl) kaempferol. The known compounds (Fig. 1) were identified as 3,5dihydroxy-4-(3-methyl-2-butenyl)bibenzyl (4) [20], 6,8diprenylgalangin (5) [21], licoflavonol (6) [22], icaritin (7) [19], 3,4′-di-O-methyl-8-(3-methyl-2-butenyl)kaempferol (8) [23], 5,7-dihydroxy-6,8-diprenylflavanone (9) [24], isosakuranetin (10) [25], 8-prenylnaringenin (11) [26], glepidotin B (12) [27], atractylodin (13) [28], acetylatractylodinol (14) [29], blumenol A (15) [30], scopoletin (16) [31], and salicylic acid (17) [32], by comparison of their experimental and reported spectroscopic data. All the compounds were isolated from M. kurzii for the first time. Since prenylated stilbenes and flavonoids are reported to have modest or strong anticancer activities [10,11], the cytotoxicity of compounds 1–17 were evaluated against human lung carcinoma (A-549) and human hepatocellular (Hep G2) cell lines by MTT method, with 5-FU as a positive control [33]. The results are shown in Table 2. Compounds 3, 4, 11, 12, 14, 15 and 17 inhibited the proliferation of A-549 cell line with IC50 values of 17.11, 16.01, 9.76, 15.32, 18.22, 18.23 and 12.01 μg/mL, respectively. Compound 1 exhibited modest cytotoxicity against Hep G2 cell line with IC50 value of 30.14 μg/mL. The other compounds were inactive in current tests.

Acknowledgments This study was financially supported by National Natural Science Foundation of China (31300293 and 81202584), General Project of Applied Foundation Research, Yunnan Province (2013FB067), Basic Research Project of Ministry of Science and Technology of China (2012FY110300), Major State Basic Research Development Program (2010CB951704).

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.10.003.

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