Cytotoxic Deoxybenzoins and Diphenylethylenes from Arundina

Article pubs.acs.org/jnp

Cytotoxic Deoxybenzoins and Diphenylethylenes from Arundina graminifolia Qiu-Fen Hu,†,‡ Bin Zhou,†,‡ Yan-Qing Ye,† Zhi-Yong Jiang,† Xiang-Zhong Huang,† Yin-Ke Li,† Gang Du,† Guang-Yu Yang,*,† and Xue-Mei Gao*,† †

Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission and Ministry of Education, Yunnan University of Nationalities, Kunming 650031, Yunnan, People’s Republic of China ‡ Key Laboratory of Tobacco Chemistry of Yunnan Province, Yunnan Academy of Tobacco Science, Kunming 650106, Yunnan, People’s Republic of China S Supporting Information *

ABSTRACT: Eight new C-4-alkylated deoxybenzoins (1−8), three new diphenylethylenes (9−11), and five known diphenylethylenes were isolated from Arundina graminifolia. The structures of 1−11 were elucidated by spectroscopic methods including extensive 1D and 2D NMR techniques. Compounds 9−11 are the first naturally occurring diphenylethylenes possessing a hydroxyethyl unit. Compounds 1−11 were evaluated for cytotoxicity against five human tumor cell lines. Compounds 4, 5, and 9−11 showed significant cytotoxicity against five cancer cell lines, with IC50 values ranging from 1.8 to 8.7 μM.

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RESULTS AND DISCUSSION Whole plants of A. graminifolia were extracted with 70% aqueous acetone. The extract was subjected repeatedly to column chromatography on silica gel, RP-18, and semipreparative RP-HPLC separation to afford compounds 1−11 and the known compounds dihydropinosylvin,9 4′-methylpinosylvin, 9 3-(γ,γ-dimethylallyl)resveratrol, 1 0 5-(γ,γdimethylallyl)oxyresveratrol,10 and 3-hydroxy-4,3′,5′-trimethoxy-trans-stilbene.11 The 1H and 13C NMR data of the compounds 1−11 are listed in Tables 1 and 2. Compound 1 was obtained as a yellow gum, and its HRESIMS revealed a peak at m/z 363.1208 [M + Na]+ indicative of the molecular formula C20H20O5, corresponding to 11 degrees of unsaturation. The IR spectrum indicated the presence of OH (3438 cm−1), carbonyl (1674 cm−1), and aromatic (1612, 1540, 1439 cm−1) groups. 1H, 13C, and DEPT NMR spectra showed signals for 20 carbons and 20 hydrogen atoms, a typical pattern of a deoxybenzoin system12 [δC 144.9 s, 110.8 d, 156.3 s, 112.2 s, 162.8 s, 113.1 d, 133.5 s, 130.9 d (2C), 115.8 d (2C), 161.8 s, 197.5 s, 45.6 t] with six aromatic protons [δH 6.26 s, 1H, 6.40 s, 1H, 7.91 d, J = 8.6 (2H), and 7.01 d, J = 8.6 (2H)], one methoxy group (δC 55.9 q; δH 3.80 s), and an isoamyl ketone group13 [δC 191.9 s, 50.3 t, 80.2 s, 25.8 q (2C); δH 2.55 s, 1.57 s, (6H)]. Long-range HMBC correlations (Figure 1) of H-8 (δH 2.55 s) to C-4 (δC 112.2 s), C-9 (δC 80.2

Arundina graminifolia D. Don (Orchidaceae), known as bamboo orchid, is a terrestrial multiperennial with reedy stems that forms clumps and grows to a height between 70 cm and 2 m. This tropical genus extends from India, Nepal, Thailand, Malaysia, Singapore, and South China to Indonesia and across the Pacific Islands.1 It has been widely used for sore throat, detoxification, and dissipating blood stasis by Dai people living in Xishuangbanna, Yunnan Province.2 Previous phytochemical studies of A. graminifolia revealed the presence of stilbenoids,3 bibenzyls,4 phenanthrenes,5,6 and other phenolic compounds.7,8 Phenolic compounds possessing antitobacco mosaic virus (anti-TMV) and anti-HIV-1 properties have been isolated from A. gramnifolia grown in Xishuangbanna and Honghe Prefecture.7,8 Motivated by a search for new bioactive metabolites from local plants, our group investigated the chemical constituents of the whole plant of A. graminifolia growing in Dehong Prefecture, which led to the isolation and characterization of eight new deoxybenzoins (1−8), three new diphenylethylenes (9−11), and five known diphenylethylenes. Compounds 1−8 are C-4-alkylated deoxybenzoins reported from nature for the first time. Compounds 9−11 are the first naturally occurring diphenylethylenes possessing a hydroxyethyl moiety. This paper deals with the isolation and structural characterization of these compounds and their cytotoxicity against five human tumor cell lines. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 11, 2013

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dx.doi.org/10.1021/np400379u | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

Table 1. 1H and 13C NMR Data of Compounds 1−6 (δ in ppm, in CDCl3) 1 no. 1 2 3 4 5 6 7 8 9 10 11

δC (mult) 144.9 s 110.8 d 156.3 s 112.2 s 162.8 s 113.1 d 191.9 s 50.3 t 80.2 s 25.8 q 25.8 q

2 δH (J in Hz) 6.26 s

6.40 s 2.55 s 1.57 s 1.57 s

δC (mult) 143.9 s 110.9 d 156.1 s 111.9 s 164.9 s 113.8 d 191.4 s 50.0 t 80.6 s 26.0 q 26.0 q

3 δH (J in Hz) 6.28 s

6.33 s 2.50 s 1.57 s 1.57 s

δC (mult) 146.9 s 111.0 d 160.3 s 112.9 s 164.9 s 114.7 d 193.1 s 132.3 d 154.1 s 21.9 q 71.8 t

4 δH (J in Hz)

δC (mult) 146.9 s 111.4 d 160.1 s 112.6 s 164.6 s 114.3 d 192.6 s 132.8 d 154.6 s 22.0 q 71.6 t

6.46 s

6.63 s 6.31 s 1.99 s 4.57 s

5 δH (J in Hz) 6.50 s

6.67 s 6.33 s 1.98 s 4.60 s

12 1′ 2′, 6′

133.5 s 130.9 d

3′, 5′

115.8 d

4′ 7′ 8′ OMe-5 OMe-4′ Ar-OH-4′ Ar-OH-5

161.8 s 197.5 s 45.6 t

4.05 s

55.9 q

3.80 s

7.91 d (8.6) 7.01 d (8.6)

9.47 brs

133.6 s 131.1 d 115.9 d 161.6 s 197.8 s 45.4 t 55.8 q 55.9 q

7.92 d (8.6) 7.00 d (8.6)

4.04 s 3.83 s 3.80 s

133.8 s 130.9 d 115.4 d

7.93 d (8.6) 7.02 d (8.6)

161.5 s 197.1 s 45.0 t

4.07 s

55.9 q

3.81 s

134.0 s 131.1 d 116.5 d 158.9 s 197.1 s 45.5 t

9.34 brs

7.90 d (8.6) 6.99 d (8.6)

4.02 s

9.11 brs 9.32 brs

δC (mult)

6

δH (J in Hz)

δC (mult)

145.1 s 108.4 d 162.4 s 111.6 s 162.4 s 108.4 d 204.1 s 40.8 t 31.9 t 28.0 d 21.3 q

2.97 1.68 1.87 0.92

21.3 q

0.92 d (6.2)

21.8 q

137.9 s 130.0 d

7.46 d (7.6)

133.3 s 115.7 d

128.6 d 132.0 d 198.5 s 44.9 t

6.70 s

6.70 s t (7.1) m m d (6.2)

7.35 dd (7.0, 7.6) 7.41 d (7.0) 4.04 s

9.20 brs

145.4 s 108.9 d 161.9 s 111.3 s 161.9 s 108.9 d 204.2 s 41.0 t 32.2 t 27.0 d 21.8 q

129.9 d

δH (J in Hz) 6.66 s

6.66 s 2.88 t (7.1) 1.65 m 1.84 m 0.92 d (6.2) 0.92 d (6.2) 7.81 d (8.6) 7.02 dd (8.6)

160.0 s 198.0 s 45.3 t

3.99 s

55.9 q

3.80 s 9.11 brs

formulas C21H22O5, C20H18O5, and C19H15O5, respectively. The 1H and 13C NMR spectra of compounds 2−4 were similar to those of 1, with differences that resulted from altered substituents at C-3, C-4, C-4′, and C-5. Detailed comparison of NMR spectra showed the major difference between 1 and 2 to be the disappearance of a phenolic OH (δH 9.47 brs) and appearance of an OCH3 signal (δC 55.8 and δH 3.83) in 2. HMBC correlation between the methoxy protons and C-5 (δC 164.9) confirmed a methoxy group at C-5. The main structural differences between compounds 3 and 1 were indicated by the appearance of an O-methylene ether signal (δC 71.8, δH 4.57) and the lack of a methyl signal (δC 26.0, δH 1.57) in 3. This indicated that the methyl group in 1 was an O-methylene ether group in compound 3. Olefinic carbons (δC 132.3 d and 154.1 s) suggested a double bond between C-8 and C-9. The HMBC spectrum exhibited correlations of the O-methylene ether signal H2-11 (δH 4.57 s) with C-3 (δC 160.3 s), C-8 (δC 132.3 d), C-9

s), and C-10, 11 (δC 25.8 q) were observed in 1. This indicated that the isoamyl ketone moiety was attached to the aromatic ring at positions C-3 and C-4, forming a dimethyl-2,3dihydropyran-4-one moiety. The HMBC correlation of methoxy protons (δH 3.80 s) with C-4′ (δC 161.8 s) revealed that this OCH3 group was located at C-4′. The chelated OH located at C-5 was supported by HMBC correlations of the OH proton (δH 9.47) with C-4 (δC 112.2 s), C-5 (δC 162.8 s), and C-6 (δC 113.1 d). Two doublets (δH 7.91 d, J = 8.6, 2H, and 7.01 d, J = 8.6, 2H) and two singlets (δH 6.26 s, 1H, and 6.40 s, 1H) in the 1H NMR spectrum also supported the substituent positions in compound 1. Thus, the structure of 1 was established as shown, and it was given the name gramideoxybenzoin A. Compounds 2−4, obtained as yellow gums, showed quasimolecular ions at m/z 377.1358, 361.1059, and 323.0917 [M + Na]+ in the HRESIMS, corresponding to the molecular B



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Table 2. 1H and 13C NMR Data of Compounds 7−11a (δ in ppm) 7 no. 1 2 3 4 5 6 7 8 9 10 11 12 1′ 2′, 6′ 3′, 5′ 4′ 7′ 8′ OMe-3 OMe-3′, 5′ Ar-OH-3 Ar-OH-5 Ar-OH-3′, 5′ −OAc a

δC (mult) 145.8 s 111.7 d 159.5 s 108.6 s 164.1 s 113.4 d 182.1 s 106.3 d 168.2 s 32.4 d 20.5 q 20.5 q 137.7 s 129.3 d 128.1 d 132.2 d 198.3 s 44.9 t


6.63 s 6.38 s 2.60 m 1.02 d (6.8) 1.02 d (6.8) 7.51 d (7.4) 7.39 dd (7.0, 7.4) 7.44 d (7.0) 4.09 s

8 δC (mult) 145.0 s 112.8 d 158.3 s 111.1 s 160.7 s 114.5 d 182.3 s 106.3 d 168.4 s 33.2 d 20.4 q 20.4 q 137.9 s 129.6 d 128.2 d 132.0 d 198.4 s 45.0 t

9 δC (mult)

δH (J in Hz)

138.3 s 105.1 d 157.0 s 112.4 s 157.0 s 105.1 d 19.6 t 62.9 t

6.52 s

6.79 s 6.36 s


6.55 s 2.81 t (7.1) 4.15 t (7.1)

10 δC (mult) 137.0 s 103.5 d 158.3 s 112.4 s 154.6 s 105.0 d 20.2 t 62.4 t

11


6.50 s 2.88 t (7.1) 4.11 t (7.1)

δC (mult) 137.7 s 105.5 d 156.5 s 112.5 s 156.5 s 105.5 d 20.4 t 63.0 t

6.54 s

6.54 s 2.83 t (7.1) 4.15 t (7.1)

2.54 m 1.05 d (6.8) 1.05 d (6.8) 139.5 105.7 159.6 102.6 126.4 125.9

7.53 d (7.4) 7.41 dd (7.0, 7.4) 7.49 d (7.0) 4.05 s

s d s d d d

6.45 d (1.9) 6.15 t (1.9) 7.24 d (16.0) 6.92 d (16.0)

139.2 s 105.8 d 159.2 s 102.9 d 126.4 d 125.9 d 55.9 q

6.44 d (1.9) 6.26 7.28 7.06 3.79

t (1.9) d (16.0) d (16.0) s

138.3 103.2 162.6 101.0 126.8 125.8

s d s d d d

55.7 q 10.90 brs 10.90 brs 11.13 brs

9.49 brs 169.8 s 21.2 q

δH (J in Hz)

11.01 brs 11.27 brs

6.47 d (1.9) 6.25 t (1.9) 7.29 d (16.1) 7.03 d (16.1) 3.80 s 11.16 brs 11.16 brs

2.02 s

Compounds 7 and 8 were measured in CDCl3; compounds 9−11 were measured in C5D5N.

(δH 2.97) with C-4, together with the 1H−1H COSY correlations between H-8/H-9/H-10/H-11(H-12), confirmed the isohexyl ketone group at C-4 in 5. The HMBC correlations of two hydroxy protons (δH 9.20) to C-3 (δC 162.4 s) and C-5 (δC 162.4 s) placed phenolic OH groups at C-3 and C-5, respectively. A two-proton singlet at δH 6.70 was assigned to H2 and H-6. Doublets at δH 7.46 (J = 7.6 Hz) and δH 7.41 (J = 7.0 Hz) as well as a doublet of doublets at δH 7.35 (J = 7.0, 7.6 Hz) indicated no substituent at C-4′. Thus, the structure of 5 was established as shown, and it was given the name gramideoxybenzoin E. Compound 6 was assigned the molecular formula C21H24O5 by HRESIMS. Comparison of the 1H and 13C NMR spectra of 6 to those of 5 revealed that the only difference between them was due to the appearance of a methoxy group (δC 55.9 q, δH 3.80 s) in 6. The HMBC correlation of the methoxy proton signal (δH 3.80) with C-4′ (δC 160.0) placed the methoxy group at C-4′. Thus, the structure of compound 6 was determined as shown, and this compound was given the name gramideoxybenzoin F. Compound 7 was obtained as a yellow gum; C20H17O4 by HRESIMS. The NMR spectra, together with the UV and IR experiments, suggested that the structure of 7 was similar to that of compound 5. Apart from the deoxybenzoin moiety, an isohexenyl ketone [δC 182.1 s, 106.3 d, 168.2 s, 32.4 d, 20.5 q (2C); δH 6.38 s, 2.58−2.61 m, 1.02 d, J = 6.8] was observed in the NMR spectra. HMBC correlations of H-8 (δH 6.38) to C-4 (δC 108.6), C-7 (δC 180.1), C-9 (δC 168.2), and C-10 (δC 32.4) indicated that the isohexenyl ketone moiety was fused to the aromatic ring at C-3 and C-4, forming an isopropylpyran-4-one ring. The phenolic OH at C-5 in 7 was established by HMBC

Figure 1. Selected HMBC (↷) correlation of 1 and 9.

(δC 154.1 s), and C-10 (δC 21.9 q) and those of H-8 (δH 6.31 s) with C-4 (δC 112.9 s), C-7 (δC 193.1 s), C-9, C-10, and C-11 (δC 71.8 t). These correlations suggested a seven-membered ring fused to the aromatic ring at C-3 and C-4. The chemical shift differences between compounds 3 and 4 indicated that a methoxy group (δC 55.9 and δH 3.81) at C-4′ in 3 was replaced by a phenolic hydroxy group (δH 9.11) in compound 4. The HMBC spectrum also supported OH groups at C-4′ and C-5 in compound 4. Accordingly, the structures of gramideoxybenzoins C and D (2−4) were determined as shown. Compound 5 gave a parent ion by HRESIMS at m/z 325.1448 [M − H]− corresponding to the molecular formula C20H22O4. The NMR, UV, and IR spectra suggested that 5 was also a deoxybenzoin analogue. The 1H and 13C NMR signals of 5 (Table 1) were assigned by the DEPT, HSQC, and HMBC spectra. Comparison of the 13C NMR data of 5 with those of 1 indicated that the major differences between them were the substituents at C-4 and C-4′. The 1H NMR spectrum (Table 1) revealed a triplet at δH 2.97 (J = 7.1 Hz, 2H), a multiplet at δH 1.67−1.71 (2H), a multiplet at δH 1.85−1.89 (1H), and a doublet at δH 0.92 (J = 6.2 Hz, 6H), which established the presence of an isohexyl ketone.14 HMBC correlation of H-8 C



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considered to be inactive (IC50 values >10 μM) in all five tumor cell lines. Compound 4 showed cytotoxicity against A549, SHSY5Y, and PC3 cells with IC50 values of 2.2, 1.8, and 3.4 μM, respectively; 5 showed cytotoxicity against NB4 and SHSY5Y cells with IC50 values of 2.1 and 3.2 μM; 10 was cytotoxic against NB4 and PC3 cells with IC50 values of 3.3 and 2.2 μM, respectively.

correlations of the hydroxy proton (δH 9.49) to C-4 (δC 108.6), C-5 (δC 164.1), and C-6 (δC 113.4). Accordingly, the structure of 7 was established, and it was named gramideoxybenzoin G. The appearance of an acetoxy group (δH 2.02; δC 21.2, 169.8) and the disappearance of a phenolic OH (δH 9.49) in the NMR spectra of 8 suggested that the OH at C-5 in 7 was replaced by an acetoxy group in 8. This compound was named gramideoxybenzoin H. The HRESIMS of 9 displayed a quasi-molecular ion [M − H]− at m/z 287.0914, consistent with the molecular formula C16H16O5. The 1H NMR spectrum of 9 (Table 2) showed resonances for two meta-aromatic protons from a 1,3,4,5substituted phenyl ring at δH 6.55 (s, 2H), three meta-coupled aromatic protons at δH 6.15 (t, J = 1.9 Hz, 1H) and 6.45 (d, J = 1.9 Hz, 2H) from a 1,3,5-trisubstituted phenyl ring, and transolefinic protons, at δH 6.92 and 7.24 (d, J = 16.0 Hz, 1H each), typical of a trans-stilbene.15 Two two-proton triplets at δH 2.81 and 4.15 with the same coupling constant (J = 7.1 Hz) suggested the existence of a hydroxyethyl group. The hydroxyethyl group was placed at C-4 on the basis of HMBC correlations of H-7 (δH 2.81) with C-3 (δC 157.0), C-4 (δC 112.4), and C-5 (δC 157.0) and of H-8 (δH 4.15) with C-4 (δC 112.4). Long-range correlations between Ar-OH (δH 10.90, 2H) and the signals of C-2, C-3, C-4, C-5, and C-6 confirmed OH groups at C-3 and C-5, respectively. Correlations of Ar-OH (δH 11.13, 2H) with the signals of C-2′, C-3′, C-4′, C-5′, and C-6′ indicated additional OH groups at C-3′ and C-5′, respectively. Compound 9 was thus elucidated as (E)-5-(3,5dihydroxystyryl)-2-(2-hydroxyethyl)benzene-1,3-diol and was given the name gramistilbenoid A. Gramistilbenoids B (10) and C (11) had NMR spectra similar to those of 9. The molecular formula C17H18O5 was assigned to compound 10 on the basis of its HRESIMS data. It differed structurally from 9 only at C-3, with the replacement of one hydroxy group in 9 by a methoxy group in 10, as determined by comparison of their NMR data. HMBC correlation of the methoxy protons (δH 3.79) to C-3 also implied that the methoxy group was attached to C-3. The molecular formula of 11 was C18H20O5. HMBC correlations of methoxy protons with C-3′ and C-5′ in 11 supported the positions of methoxy groups at C-3′ and C-5′, respectively. Thus, the structures of compounds 10 and 11 were established as shown. Since some previously reported stilbenes from Orchidaceae plants exhibited cytotoxicity,16−18 we tested compounds 1−11 for cytotoxicity against five human tumor cell lines (NB4, A549, SHSY5Y, PC3, and MCF7) using the MTT method as reported previously.19 Paclitaxel was used as the positive control. The results are depicted in Table 3. Compounds 1, 3, and 6−8 were

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General Experimental Procedures. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. A Tenor 27 spectrophotometer was used for scanning IR spectroscopy with KBr pellets. 1D and 2D NMR spectra were recorded on a DRX-500 NMR spectrometer with TMS as internal standard. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. HRESIMS was performed on a VG Autospec-3000 spectrometer. Semipreparative HPLC was performed on a Shimadzu LC-8A preparative liquid chromatograph with Zorbax PrepHT GF (21.2 mm × 25 cm) or Venusil MP C18 (20 mm × 25 cm) columns. Column chromatography (CC) was performed using silica gel (200−300 mesh, Qing-dao Marine Chemical, Inc., Qingdao, People’s Republic of China), Lichroprep RP-18 gel (40−63 μm, Merck, Darmstadt, Germany), and MCI gel (75−150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan). Fractions were monitored by TLC, and spots were visualized by heating silica gel plates sprayed with 5% H2SO4 in EtOH. Plant Material. The whole plant of A. graminifolia was collected in Zhaifang village, Dehong Prefecture, Yunnan Province, People’s Republic of China, in September 2011. The identification of the plant material was verified by Dr. N. Yuan of Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (YNNU 2011-9-31) has been deposited in our laboratory. Extraction and Isolation. The air-dried and powdered A. graminifolia (6.0 kg) plant material was extracted four times with 70% aqueous acetone (4 × 10 L) at room temperature and filtered. The filtrate was evaporated under reduced pressure, and the crude extract (520 g) was decolorized by MCI. The portion of the extract soluble in 90% methanol (457 g) was chromatographed on a silica gel column eluting with a CHCl3−MeOH gradient system (20:1, 9:1, 8:2, 7:3, 6:4, 5:5), to give six fractions (A−F). Separation of fraction B (9:1, 35.5 g) by silica gel CC, eluted with petroleum ether−acetone (9:1−1:2), yielded fractions B1−B7. Fraction B2 (8:2, 6.27 g) was subjected to silica gel CC using petroleum ether−acetone and semipreparative HPLC (68% MeOH−H2O) to give 1 (6.5 mg), 2 (8.9 mg), 3 (8.7 mg), 7 (12.1 mg), and 8 (8.4 mg). Fraction B3 (7:3 3.51 g) was subjected to silica gel CC using petroleum ether−acetone and semipreparative HPLC (64% MeOH−H2O) to give 4 (8.6 mg), 5 (9.2 mg), 6 (10.2 mg), dihydropinosylvin (6.7 mg), 3-(γ,γ-dimethylallyl)resveratrol (12.1 mg), and 5-(γ,γ-dimethylallyl)oxyresveratrol (15.3 mg). Separation of fraction C (8:2, 25.3 g) by silica gel CC, eluted with petroleum ether−acetone (9:1−1:2), yielded fractions C1−C7. Fraction C2 (8:2, 2.64 g) was subjected to silica gel CC using petroleum ether−acetone and semipreparative HPLC (50% MeOH− H2O) to give 3-hydroxy-4,3′,5′-trimethoxy-trans-stilbene (17.8 mg). Fraction C3 (7:3, 3.45 g) was subjected to silica gel CC using petroleum ether−acetone and semipreparative HPLC (55% MeOH− H2O) to give 9 (8.1 mg), 10 (14.8 mg), 11 (9.5 mg), 12 (21.5 mg), and 4′-methylpinosylvin (10.4 mg). Gramideoxybenzoin A (1): yellow gum; UV (MeOH) λmax (log ε) 210 (4.47), 265 (4.08), 358 (3.22) nm; IR (KBr) νmax 3438, 2922, 2876, 1715, 1674, 1612, 1540, 1482, 1439, 1352, 1134, 948, 862 cm−1; 1 H and 13C NMR data (CDCl3, 500 and 125 MHz), see Table 1; positive ESIMS m/z 363 [M + Na]+; positive HRESIMS m/z 363.1208 [M + Na]+ (calcd for C20H20O5Na, 363.1208). Gramideoxybenzoin B (2): yellow gum; UV (MeOH) λmax (log ε) 210 (4.38), 268 (4.11), 356 (3.27) nm; IR (KBr) νmax 3441, 2919, 2879, 1712, 1677, 1615, 1537, 1480, 1442, 1350, 1136, 942, 865 cm−1; 1 H and 13C NMR data (CDCl3, 500 and 125 MHz), see Table 1;

Table 3. Cytotoxicity Data (IC50, μM) for Compounds 1−11 from A. graminifoliaa

a

compound

NB4

A549

SHSY5Y

PC3

MCF7

2 4 5 9 10 11 paclitaxel

>10 7.6 2.1 5.5 3.3 4.8 0.03

6.4 2.2 5.2 8.1 6.5 5.5 0.02

>10 1.8 3.2 7.8 4.5 6.2 0.2

>10 3.4 6.8 5.6 2.2 8.7 0.2

8.5 5.4 5.5 6.4 5.3 5.9 0.1

EXPERIMENTAL SECTION

The values for compounds 1, 3, 6, 7, and 8 were all greater than 10. D



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positive ESIMS m/z 377 [M + Na]+; positive HRESIMS m/z 377.1358 [M + Na]+ (calcd for C21H22O5Na, 377.1365). Gramideoxybenzoin C (3): yellow gum; UV (MeOH) λmax (log ε) 210 (4.42), 262 (3.81), 305 (3.97) nm; IR (KBr) νmax 3448, 2926, 2862, 1708, 1638, 1602 1574, 1467, 1386, 1204, 1146, 972, 874 cm−1; 1 H and 13C NMR data (CDCl3, 500 and 125 MHz), see Table 1; positive ESIMS m/z 361 [M + Na]+; positive HRESIMS m/z 361.1059 [M + Na]+ (calcd for C20H18O5Na, 361.1052). Gramideoxybenzoin D (4): yellow gum; UV (MeOH) λmax (log ε) 210 (4.46), 258 (3.77), 302 (3.89) nm; IR (KBr) νmax 3445, 2927, 2860, 1705, 1636, 1605, 1576, 1462, 1389, 1203, 1141, 968, 870 cm−1; 1 H and 13C NMR data (CDCl3, 500 and 125 MHz), see Table 1; negative ESIMS m/z 323 [M − H]−; negative HRESIMS m/z 323.0917 [M − H]− (calcd for C19H15O5, 323.0919). Gramideoxybenzoin E (5): yellow gum; UV (MeOH) λmax (log ε) 215 (4.28), 256 (3.82), 278 (3.93), 336 (3.46) nm; IR (KBr) νmax 3462, 2931, 2839, 1710, 1668, 1611, 1584, 1468, 1372, 1196, 1135, 965, 862 cm−1; 1H and 13C NMR data (CDCl3, 500 and 125 MHz), see Table 1; negative ESIMS m/z 325 [M − H]−; negative HRESIMS m/z 325.1448 [M − H]− (calcd for C20H21O4, 325.1440). Gramideoxybenzoin F (6): yellow gum; UV (MeOH) λmax (log ε) 218 (4.22), 263 (3.91), 282 (3.86), 338 (3.26) nm; IR (KBr) νmax 3465, 2928, 2831, 1705, 1672, 1615, 1580, 1460, 1365, 1197, 1142, 979, 866 cm−1; 1H and 13C NMR data (CDCl3, 500 and 125 MHz), see Table 1; positive ESIMS m/z 379 [M + Na]+; positive HRESIMS m/z 379.1525 [M + Na]+ (calcd for C21H24O5Na, 379.1521). Gramideoxybenzoin G (7): yellow gum; UV (MeOH) λmax (log ε) 215 (4.36), 258 (3.80), 305(3.62) 346 (2.87) nm; IR (KBr) νmax 3350, 2917, 2875, 1696, 1635, 1608, 1562, 1458, 1439, 1368, 1279, 1157, 1109, 1064, 894, 753 cm−1; 1H and 13C NMR data (CDCl3, 500 and 125 MHz), see Table 2; negative ESIMS m/z 321 [M − H]−; negative HRESIMS m/z 321.1120 [M − H]− (calcd for C20H17O4, 321.1127). Gramideoxybenzoin H (8): yellow gum; UV (MeOH) λmax (log ε) 218 (4.26), 265 (3.80), 308 (3.57) 348 (2.96) nm; IR (KBr) νmax 3354, 2922, 2877, 1736, 1698, 1638, 1605, 1567, 1454, 1435, 1364, 1274, 1150, 1114, 1067, 890, 759 cm−1; 1H and 13C NMR data (CDCl3, 500 and 125 MHz), see Table 2; positive ESIMS m/z 387 [M + Na]+; positive HRESIMS m/z 387.1208 [M + Na]+ (calcd for C22H20O5Na, 387.1208). Gramistilbenoid A (9): yellow gum; UV (MeOH) λmax (log ε) 210 (4.26), 238 (2.87), 285 (3.86) nm; IR (KBr) νmax 3422, 2918, 2886, 1622, 1536, 1454, 1360, 1262, 1030, 861, 774 cm−1; 1H and 13C NMR data (C5D5N, 500 and 125 MHz), see Table 2; negative ESIMS m/z 287 [M − H]−; negative HRESIMS m/z 287.0914 [M − H]− (calcd for C16H15O5, 287.0919). Gramistilbenoid B (10): yellow gum; UV (MeOH) λmax (log ε) 210 (4.36), 241 (2.76), 288 (3.91) nm; IR (KBr) νmax 3418, 2921, 2879, 1618, 1534, 1450, 1367, 1257, 1132, 1035, 864, 770 cm−1; 1H and 13C NMR data (C5D5N, 500 and 125 MHz), see Table 2; positive ESIMS m/z 325 [M + Na]+; positive HRESIMS m/z 325.1057 [M + Na]+ (calcd for C17H18O5Na, 325.1052). Gramistilbenoid C (11): yellow gum; UV (MeOH) λmax (log ε) 210 (4.32), 240 (2.80), 288 (3.96) nm; IR (KBr) νmax 3415, 2927, 2874, 1613, 1530, 1454, 1362, 1262, 1135, 1038, 869, 762 cm−1; 1H and 13C NMR data (C5D5N, 500 and 125 MHz), see Table 2; positive ESIMS m/z 339 [M + Na]+; positive HRESIMS m/z 339.1202 [M + Na]+ (calcd for C18H20O5Na, 339.1208). Cytotoxicity Assay. Colorimetric assays were performed to evaluate cytotoxicity. NB4 (human acute promyelocytic leukemia cells), A549 (human lung adenocarcinoma), SHSY5Y (human neuroblastoma), PC3 (human prostate), and MCF7 (human breast adenocarcinoma) tumor cell lines were purchased from the American Type Culture Collection (ATCC). All cells were cultured in RPMI1640 or DMEM medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere with 5% CO2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA). Briefly, 100 μL of suspended adherent cells was seeded into

each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition. In addition, suspended cells were seeded just before drug addition, with an initial density of 1 × 105 cells/mL in 100 μL of medium. Each tumor cell line was exposed to each test compound at various concentrations in triplicate for 48 h; paclitaxel (Sigma, purity >95%) was used as a positive control. After the incubation, MTT (100 μg) was added to each well, and the incubation was continued for 4 h at 37 °C. The cells were lysed with 100 μL of 20% SDS−50% DMF after removal of 100 μL of the medium. The optical density of the lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by Reed and Muench’s method.20

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ASSOCIATED CONTENT

S Supporting Information *

1

H, and 13C NMR, HSQC, HMBC, and HRESIMS spectra of 1; 1H and 13C NMR spectra of 2−11; these materials are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: 86-871-5913043. E-mail: [email protected] (X. Gao). *E-mail: [email protected] (G. Yang). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported financially by the Excellent Scientific and Technological Team of Yunnan High School (2010CI08), the Yunnan University of Nationalities Green Chemistry and Functional Materials Research for Provincial Innovation Team (2011HC008), and Open Research Fund Program of Key Laboratory of Ethnic Medicine Resource Chemistry (Yunnan University of Nationalities) (2010XY08).

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REFERENCES

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