HETEROCYCLES

Short Paper | Regular issue | Vol. 87, No. 3, 2013, pp. 637-643
Received, 19th November, 2012, Accepted, 28th December, 2012, Published online, 7th January, 2013.
DOI: 10.3987/COM-12-12631

■ Isolation and Structure Elucidation of Alkaloids from Pinellia ternata

Zhihao Zhang, Wenjie Li, Ruichao Lin,* Zhong Dai,* and Xiaofeng Li

Faculty of Chinese Medicine, Macau University of Science and Technolog, Avenida Wai Long, Taipa, Macau 999078, China

Abstract

A new compound, 3-(6,7-dimethoxyisoquinolin-1-yl)-4,7-dimethoxy-3-methylisobenzofuran-1(3H)-one (1) named alkterlactone was isolated from Pinellia ternata, together with four known compounds N-trans-feruloyloctopamine (2), 2'-O-methyladenosine (3), 5'-S-methyl-5'-thioadenosine (4) and 2'-deoxy-thymidine (5). Compounds 2–5 were isolated for the first time from Pinellia ternata. The structures of these compounds were elucidated and characterized on the basis of 1D NMR, 2D NMR and MS data.

Pinellia ternata (Thunb.) Berit. called banxia in China is a classic traditional Chinese medicine widely distributed in Sichuan, Guizhou and Anhui Provinces of China. Its rhizome is used in clinic for antiemetic, antitussive, sedative and anti-inflammatory purposes.1,2 However, crude banxia may cause side effects such as tongue numbing, tongue swelling, salivation, slurred speech and hoarseness. Unfortunately, the toxic ingredients have not yet been identified unambiguously. So processed products of banxia are always used in clinic, especially Rhizoma Pinelliae Preparata (qingbanxia) and Rhizoma Pinelliae Preparatum (fabanxia), both of which are recorded in Chinese Pharmacopoeia (2010 Edition). Qingbanxia is the product of raw banxia processed with alum and fabanxia is the product of raw banxia processed with lime solution and Glycyrrhiza uralensis.
Previously, phytochemicals from this plant that have been characterized include alkaloids,3,4 volatile oils,5 polysaccharides,6 amino acids,7 triterpenes, sterols, fatty acids,8 cerebrosides,9 and proteins.10 Here, We report the isolation and characterization of a new alkaloid, 3-(6,7-dimethoxyisoquinolin-1-yl)-4,7- dimethoxy-3-methylisobenzofuran-1(3H)-one (1) and four known alkaloids (Figure 1) N-trans-feruloyloctopamine (2), 2′-O-methyladenosine (3), 5'-S-methyl-5'-thioadenosine (4) and 2'-deoxythymidine (5), which were isolated for the first time from banxia.

The compounds 1-5 were isolated using silica gel, Sephadex LH-20 gel column chromatography and preparative liquid chromatography from a 95% EtOH extract of banxia. The structures of compounds were characterized by examination of their HR ESI-MS, NMR (1D and 2D) data and comparison with literature reports.
Compound 1 was obtained as a colorless solid; [α]25D -0.7° (c 0.003, CH3OH). UV (CH3OH) λmax (nm) (log ε): 239, 315, 326 (4.4, 3.5, 3.5). Its molecular formula was assigned as C22H21NO6, suggesting thirteen degrees of unsaturation, on the basis of the [M+H]+ ion peak at m/z 396.1424 (calcd. for C22H21NO6, 396.1447) in the HR-ESI-MS. 1H-NMR (CD3OD, 500 MHz) showed six downfield proton signals, including an AB-pattern for two heteroaromatic protons at δ 8.23 (1H, d, 5.5 Hz) and 7.56 (1H, d, 5.5 Hz), and another AB-pattern for two aromatic protons at δ 7.37 (1H, d, 8.0 Hz), δ 7.27 (1H, d, 8.5 Hz), and two aromatic protons at δ 7.46 (1H, s), δ 7.21 (1H, s). Fifteen highfield proton signals, including five singlet protons at δ 3.99 (3H, s, OCH3), δ 3.90 (3H, s, OCH3), δ 3.86 (3H, s, OCH3), δ 3.78 (3H, s, OCH3), δ 2.09 (3H, s, CH3) were also observed. The 13C-NMR data (Table 1), DEPT and HSQC spectra of compound 1 allowed the assignment of 22 carbon signals to six tertiary, 11 quaternary carbons, four methoxy and one methyl.

The carbon skeleton of compound 1 is as same as the carbon skeleton of 9-methyldecumbenine C which has been reported in literature.11 The 1H-NMR data was similar to that of 1-[1'-(6',7'-dimethoxyphtha1ide)]-6,7-dimethoxyisoquinoline synthesized by V. Smul.12 The major difference in compound 1 was the two methoxy groups at C-4 and C-7 in contrast to methoxy groups at C-6' and C-7' in that compound. What's more, H-3 was replaced by methyl in compound 1. These differences were confirmed by HMBC spectrum and 1H, 1H-COSY spectrum (Figure 2).
In the HMBC spectrum, the observation of diagnostic correlations from methyl protons to C-9, C-10' and C-3, allowed the methyl group to be attached to C-3. Four methoxy groups (methoxy protons at δH = 3.78, 3.90, 3.86, 3.99) were assigned to C-6', C-7', C-4 and C-7, respectively, based on the correlations of H (δ 3.78, 3H, s) with C-6', H (δ 3.90, 3H, s) with C-7', H (δ 3.86, 3H, s) with C-4 and H (δ 3.99, 3H, s) with C-7. The attribution of the carbons and hydrogen were further established by the HMBC spectrum. These data pooled together would suggest compound 1 to be 3-(6,7-dimethoxyisoquinolin-1-yl)-4,7-dimethoxy-3-methylisobenzofuran-1(3H)-one.

C-3 is a chiral carbon. Since the specific rotation values of compound 1 was -0.7° (c 0.003, CH3OH), which is close to zero and CD spectrum indicated no obvious cotton effects. We deduced the compound 1 to be racemates. These data established the structure of compound 1 as 3-(6,7-dimethoxyisoquinolin-1-yl)-4,7-dimethoxy-3-methylisobenzofuran-1(3H)-one. The novel natural product is tentatively named alkterlactone. Further work on the biological activities of compounds 1-5 and structural determination of other interesting alkaloids are in progress.

EXPERIMENTAL
General. Agilent 6320 Ion TRAP LC/MS and Waters XevoTM UPLC-QTof were employed for MS analysis. 1H- and 13C-NMR spectra were recorded on Bruker Avance DRX 500 instrument or Varian Unity VNS 600 using DMSO-d6 or CD3OD as solvent, with TMS as internal standard. The specific rotation was measured on AUTOPOL IV Automatic Polarimeter (Rudolph, Hackettstown, NJ, USA). UV spectra were recorded on an Agilent 8453 UV/Vis Spectrophotometer (Agilent, Santa Clara, CA, USA). CD spectra were taken on a JASCO J-815 Spectropolarimeter (JASCO, Tokyo, Japan) using a 0.1 cm standard cell and spectrophotometric-grade MeOH. IR spectra were taken on a Nicolet 5700 FTIR Spectrometer (Thermo, Waltham, MA, USA). Compounds were purified by Dalian Elite P230p preparative high performance liquid chromatography [(Dalian, Liaoning Province, China), Phenomenex Luna C18 columns (250 mm × 4.6 mm and 250 mm × 21.2 mm i.d.)]. Sephadex LH-20 was purchased from Amersham Pharmacia Biotech AB (Uppsala, Sweden). AB-8 resin (20–60 mesh) was acquired from Nankai University (Tianjin, China). Silica gel (160–200 mesh, 200–300 mesh) for column chromatography was purchased from Qingdao Marine Chemical Plant (Qingdao, Shandong Province, China). All other chemicals were of analytical reagent grade and used without any further purification.

Plant Material. Crude roots of banxia were collected from Hezhang County, Guizhou Province, China, in June 2011. The species was identified by Professor Zhang. J. (National Institutes for Food and Drug Control, NIFDC for short). The voucher specimens were deposited at the herbarium of NIFDC. The roots were air-dried and ground to a powder using a grinding mill (Tianjin, China).

Extraction and isolation. The powder (30 kg) was extracted three times with hot 95% EtOH (12 L), for 2 h each time. The extract was concentrated to afford a syrup (0.8 kg), which was dissolved in 95% EtOH (1 L). The solution was chromatographed over a AB-8 resin (20–60 mesh) column (100 × 12 cm i.d.) eluting with a gradient of EtOH-H2O (0:100, 30:70, 60:40, 90:10) to give four fractions. The 90% EtOH eluent and 60% EtOH eluent were combined and were concentrated under vacuum to obtain the extract (132 g). The extract was chromatographed over a silica gel (160–200 mesh) column (100 × 12 cm i.d.) with petroleum ether first and then CH2Cl2/MeOH (100% CH2Cl2, CH2Cl2:MeOH = 100:1, CH2Cl2:MeOH = 80:1, … 100% MeOH) to afford 10 fractions (M01–M10). Fraction M02 (19.0 g) was chromatographed over a silica gel (200–300 mesh) column (100 × 6.0 cm i.d.) with CH2Cl2/MeOH (100% CH2Cl2 to 100% MeOH) to afford 105 fractions. Fractions 28-46 (1.2 g) were chromatographed over a silica gel (200–300 mesh) column (45 × 2.5 cm i.d.) with CH2Cl2/MeOH (50:1 to 10:1) to afford 11 fractions. Fractions 4-8 (0.54 g) were subjected to Sephadex LH-20 column chromatography (100 × 2.5 cm i.d.) with MeOH to afford 25 fractions. Fractions 7-10 (260 mg) were subjected to Sephadex LH-20 column chromatography (100 × 2.0 cm i.d.) with MeOH to afford 28 fractions. The fraction 12 was purified by preparative HPLC to yield compound 1 (3.3 mg) using MeOH-H2O (70:30) as mobile phase at a flow rate of 10 mL/min. Fraction M04 (0.53 g) was subjected to Sephadex LH-20 column chromatography (100 × 2.5 cm i.d.) with MeOH to afford 26 fractions. The fractions 14-15 were purified by preparative HPLC to yield compound 2 (6.7 mg) using MeOH-H2O (55:45) as mobile phase at a flow rate of 10 mL/min. The 30% EtOH eluent was concentrated under vacuum to obtain the extract (178 g). The extract was chromatographed over a silica gel (160–200 mesh) column (100 × 12 cm i.d.) with CH2Cl2/MeOH (100% CH2Cl2, CH2Cl2:MeOH = 100:1, CH2Cl2:MeOH = 50:1, … 100% MeOH) to afford 10 fractions (N01-N10). Fraction N04 (5.6 g) was chromatographed over a silica gel (200–300 mesh) column (45 × 4.0 cm i.d.) with CH2Cl2/MeOH (100% CHCl2 to 100% MeOH) to afford 33 fractions. Fractions 19-23 (820 mg) were subjected to Sephadex LH-20 column chromatography (100 × 2.5 cm i.d.) with MeOH to afford 22 fractions. Fractions 21-22 were compound 4 (13.9 mg). Fractions 18-20 were purified by preparative HPLC to yield compound 3 (4.0 mg) using MeOH-H2O (25:75) as mobile phase at a flow rate of 10 mL/min. Fractions 12-14 were purified by preparative HPLC to yield compound 5 (4.0 mg) using MeOH-H2O (10:90) as mobile phase at a flow rate of 10 mL/min.
(±)-Alkterlactone (1): Colorless solid. [α]25D -0.7° (c 0.003, CH3OH). UV (CH3OH) λmax (nm) (log ε): 239, 315, 326 (4.4, 3.5, 3.5). HR-ESI-MS: m/z 396.1424 [M+H]+ (calcd. for C22H21NO6, 396.1447). The 1H- and 13C-NMR spectral data are listed in Table 1.
N-trans-Feruloyloctopamine (2): Colorless oil. ESI-MS: m/z 314 [M+H]+. C18H19NO4. The 1H- and 13C-NMR spectral data are consistent with published data.13,14
2′-O-Methyladenosine (3): Colorless solid. ESI-MS: m/z 282 [M+H]+. C11H15N5O4. The 1H- and 13C-NMR spectral data are consistent with published data.15
5'-S-Methyl-5'-thioadenosine (4): Colorless oil. ESI-MS: m/z 298 [M+H]+. C11H15N5O3S. 13C-NMR (DMSO, 100 MHz): δ 156.1 (C-6), 152.7 (C-2), 149.6 (C-4), 139.9 (C-9), 119.2 (C-7), 87.4 (C-1′), 83.8 (C-4′), 72.7 (C-2′), 72.7 (C-3′), 36.1 (C-5′), 15.6 (-CH3). The 1H-NMR spectral data are consistent with the published data.16,17 The 13C-NMR spectral data has not been reported previously.
2'-Deoxy-thymidine (5): Colorless solid. ESI-MS: m/z 243 [M+H]+. C10H15N2O5. The 1H- and 13C-NMR spectral data are consistent with published data.18

ACKNOWLEDGEMENTS
This project was supported by National “Twelfth Five-Year” Plan for Science and Technology Program of China 2009BAI73B02. We thank Zhang J. from National Institutes for Food and Drug Control, Beijing 100050, for the identification of the investigated medicinal herb.

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