HETEROCYCLES

Short Paper | Regular issue | Vol. 89, No. 2, 2014, pp. 465-471
Received, 16th November, 2013, Accepted, 17th December, 2013, Published online, 24th December, 2013.
DOI: 10.3987/COM-13-12887

■ Two New Linear Furanocoumarins from Angelica apaensis

Yong Li, Min Zhang, Min Luo, Xue Wu, Qiuli Xiao, Juan Hua, and Xian Li*

School of Pharmacy, Kunming Medical University, 1168 West Chun Rong Road, Cheng Gong, Kunming, Yunnan 650500, China

Abstract

Two new linear furanocoumarins, together with 13 known analogues, were isolated from the roots of Angelica apaensis. The structures of new compounds were elucidated by spectroscopic methods including extensive 1D and 2D NMR experiments, as well as high-resolution EI mass analysis. All furanocoumarins obtained were assayed for their cytotoxic ability against K562 and SMMC-7721 human tumor cell lines.

The genus Angelica, which belongs to the family Umbelliferae, includes more than 60 species of medicinal herbs. From various phytochemical studies, it has been now established that majority of Angelica species comprise high amounts of biologically active coumarins with antiproliferative, antitumour and anticancer, analgesic and anti-inflammatory, acetylcholinesterase inhibitory, antiatherogenesis, antimicrobial and antiplatelet activities.1,2 Angelica apaensis, which belongs to the family Umbelliferae, is a perennial herb widely distributed in the northeast of Yunnan, China. The roots of this plant have been used extensively as folk medicines to treat cough, asthma, wind colic, tympanites and flatulency.3,4 With an aim to access bioactive constituents from this plant, the present study on A. apaensis, which was collected in Qiaojia County of Yunnan Province, China, afforded two new linear furanocoumarins (1 and 2), along with 13 known analogues, cnidilin (3),5 sen-byakangelicole (4),6 isobyakangelicol (5),4 byakangelicol (6),7 5-(2-hydroxy-3-methoxy-3-methylbutoxy)soralen (7),7 9-(2-hydroxy-3-methoxy-3-methylbutoxy)ergapten (8),7 byakangelicin (9),8 isobyakangelicin (10),9 5,8-di(2,3-dihydroxy-3-methylbutoxy)psoralen (11),10 marmesin (12),11 7-methoxycoumarin (13),12 scopoletin (14),10 hamaudol (15).8 In this study, we describe the isolation and structure determination of two new linear furanocoumarins (1 and 2), and their cytotoxic activity against K562 and SMMC-7721 human tumor cell lines.

Compound (1) was obtained as pale yellow amorphous solid with a quasi-molecular ion peak at m/z 436 [M]+ in its positive-ion EI-MS, corresponding to molecular formula of C22H28O9, which was verified by HR-EIMS spectrum (found: 436.1740; calcd: 436.1733). Its strong yellowish green fluorescence under UV light (268 nm) and the signals of the aromatic region in its 1H and 13C NMR spectra were typical of furocoumarins.13 In the 1H NMR spectrum of 1, two doublet signals (1H, J = 9.8 Hz) at δ 8.33 and 6.24 were assigned as the protons of pyrone ring. Two doublet signals (1H, J = 2.3 Hz) at δ 7.89 and 7.23 were determined the protons of furan ring.10 The 1H NMR showed the presence of 5,8-disubstituted furanocoumarin moiety, a 2″-hydroxy-3″-methoxy-3″-methylbutoxy moiety [δ 4.67 (1H, dd, J = 9.9, 2.2 Hz, H-1″), 4.27 (1H, m, overlap, H-1″), 3.92 (1H, dd, J = 7.6, 1.4 Hz, H-2″), 3.21 (3H, s, H-6″), 1.24 (3H, s, H-5″), 1.18 (3H, s, H-4″)], and a 2″′,3″′-dihydroxy-3″′-methylbutoxy moiety [δ 4.59 (1H, dd, J = 10.1, 2.9 Hz, H-1″′), 4.27 (1H, m, overlap, H-1″′), 3.85 (1H, dd, J = 8.0, 2.9 Hz, H-2″′), 1.23 (3H, s, H-5″′), 1.21 (3H, s, H-4″′)]. The position of the 2″-hydroxy-3″-methoxy-3″-methylbutoxy moiety was established by both a 1H-13C long-range correlation between H-1″ (δ 4.67 and 4.27) with C-5 (δ 144.6) in the HMBC spectrum of 1 (Figure 1) and a positive NOE effect between H-3′ (δ 7.23) and H-1″. The 2″′,3″′-dihydroxy-3″′-methylbutoxy moiety was deduced to be attached to C-8 based on the HMBC correlation between H-1″′ (δ 4.59) to C-8 (δ 128.1). Full assignments were established by 2D NMR including 1H-1H COSY, HSQC, HMBC, ROESY experiments. Therefore, Compound 1 was identified as 5-[2-hydroxy-3-methoxy-3-methylbutoxy]-8-[2,3-dihydroxy-3-methylbutoxy]psoralen.

Compound (2), also obtained as pale yellow amorphous solid, exhibited a molecular ion peak at m/z 450 [M]+ in its positive-ion EI-MS, in combination with the 1H and 13C NMR spectra and HR-EIMS spectrum (found: 450.1895; calcd: 450.1890), established a molecular formula of C23H30O9. A general analysis of the NMR data of 2 showed that it resembled compound 1 very much except for the substituent of C-3″′. The difference can be rationalized as the hydroxy group at C-3″′ in 1 replaced by a methoxy (C-6″′ δ 49.2) in 2. The 13C NMR signal of C-3″′ at δ 77.6 which is 6.1 ppm lower than that of free byakagelicin, suggested that methoxy moiety was linked to C-3″′ position of 2.14 The location of methoxy moiety was established by the HMBC long-range correlation between C-3″′ and H-6″′ (δ 3.31) (Figure 1). Full assignments were established by 2D NMR including 1H-1H COSY, HSQC, HMBC, ROESY experiments. Thus, compound 2 was determined to be 5,8-di-(2-hydroxy-3-methoxy-3-methylbutoxy)psoralen.

Compounds (3–15) were known compounds, whose structures were elucidated by comparisons with the literature.
Compounds (1–15) were tested for cytotoxicity against human-tumor K562 and SMMC-7721 cell lines, with fluorouracil as the positive control. Compound 4 exhibited weak cytotoxic inhibition with the IC50 values of 43.97 μg/mL and 38.06 μg/mL, respectively. Other compounds showed noncytotoxicity with IC50 values of > 100 μg/mL. Although compound 3 has an active center of dimethylallyl group,15 this compound was completely inactive which suggested that the substituents in the molecules also affected its cytotoxicity.

EXPERIMENTAL
General. Column chromatography (CC): silica gel (200–300 mesh). TLC: silica gel GF254; Melting points were obtained on an XRC-1 apparatus, and uncorrected. Optical rotations: SEPA-300 polarimeter. IR spectra: BIO-RAD FTS-135 spectrometer with KBr pellets; νmax in cm-1. UV Spectra: Shimadzu 210A double-beam spectrophotometer; λmax log (ε) in nm. 1H- and 13C-NMR spectra: Bruker AM-400 and DRX-500 spectrometer with acetone-d6 as solvent and TMS as internal standard. MS: VG Auto Spec-3000 magnetic sector instrument; m/z (rel. %).

Plant Material. The roots of Angelica apaensis were collected in Qiaojia, Yunnan Province, P.R. China, in October 2011. The identity of the plant material was verified by Prof. Qi-Tai Zhang. A voucher specimen (KIB-2011-66 Zhang) was deposited in State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, P.R. China.
Extraction and Isolation. The air-dried and powdered roots parts of A. apaensi (6.0 kg) were extracted three times with MeOH at room temperature for 24 h and filtered. The filtrate was evaporated and the resulting residue partitioned between H2O and EtOAc. The EtOAc fraction (165.0 g) was subjected to column chromatography over a silica gel (200–300 mesh, 2.5 kg) column, eluting with CHCl3/Me2CO (1:0–0:1 gradient system) to give 5 fractions L1–L5. Fraction L1 was subjected to silica gel column chromatography, eluting with petroleum ether/EtOAc (from 9:1 to 6:4), Sephadex LH-20 (Me2CO) and recrystallization to yield 1 (6.0 mg), 2 (8.0 mg), 4 (15.0 mg), 5 (6.0 mg), 6 (21.0 mg), 8 (11.0 mg), and 11 (5.0 mg). Fractions L2 were submitted to repeated chromatography over silica gel with petroleum ether/ Me2CO (9:1), Sephadex LH-20 (Me2CO), MCI–gel (60–100% MeOH–H2O, MeOH, Me2CO) and recrystallization to yield 7 (25.0 mg), 10 (6.0 mg) 12 (18.0 mg), and 14 (10.0 mg). Fraction L3 was subjected to chromatography over silica gel with petroleum ether/2-propanol (20:1), MCI–gel (60–100% MeOH–H2O, MeOH, Me2CO) and recrystallization to yield 3 (10.0 mg), 9 (6.0 mg), 13 (12.0 mg), and 15 (9.0 mg).
5-(2-Hydroxy-3-methoxy-3-methylbutoxy)-8-(2,3-dihydroxy-3-methylbutoxy)psoralen (1): pale yellow amorphous solid, mp 124–126 °C; [α]D19.3 -0.59 (c 0.21, MeOH); UV (MeOH) λmax (log ε): 221 (4.44) nm; IR (KBr) νmax cm-1: 3474, 3440, 2973, 1705, 1592, 1480, 1460, 1438, 1364, 1352, 1315, 1152, 1094, 1055; EIMS (70 eV) m/z (rel. int.): 436 [M]+ (18), 334 (13), 219 (12), 218 (82), 217 (24), 73 (100), 59 (52); HR-EIMS m/z: 436.1740 [M]+ (calcd. for C22H28O9, 436.1733); 1H and 13C NMR: see Table 1.
5,8-Di-(2-hydroxy-3-methoxy-3-methylbutoxy)psoralen (2): pale yellow amorphous solid, mp 126–1286 °C; [α]D19.3 +1.72 (c 0.16, MeOH); UV (MeOH) λmax (log ε): 222 (4.42) nm; IR (KBr) νmax cm-1: 3447, 2975, 1728, 1593, 1479, 1437, 1362, 1353, 1324, 1204, 1147, 1089, 1061; EIMS (70 eV) m/z (rel. int.): 450 [M]+ (11), 334 (21), 219 (10), 218 (77), 217 (18), 73 (100); HR-EI-MS m/z: 450.1895 [M]+ (calcd. for C23H30O9, 450.1890); 1H and 13C NMR: see Table 1.
Cytotoxicity Bioassay. Cytotoxicity of compounds against suspended tumor cells was determined by the trypan blue exclusion method, and against adherent cells by sulforhodamine B (SRB) assay. Cells were plated in a 96-well plate 24 h before treatment and continuously exposed to different concentrations (100, 10, 1, and 0.1 μM) of compounds for 72 h. After compound treatments, cells were counted (suspended cells) or fixed and stained with SRB (adherent cells) as described in the literature.16 The assays were performed in triplicate, on separate occasions. The IC50 values were calculated by the Logit method. The percentage of inhibition was calculated according to the inhibiton(%) = [(OD0–OD1)/OD0] × 100 formula, where OD0 is the optical density of control, OD1 is that of the sample.

ACKNOWLEDGEMENTS
Financial support of this research was provided by the Natural Science Foundation of Yunnan Province (No. 2011FB058), and the National Natural Science Foundation of China (No. 21102060).

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