HETEROCYCLES

Short Paper | Regular issue | Vol. 85, No. 3, 2012, pp. 689-696
Received, 6th January, 2012, Accepted, 6th February, 2012, Published online, 15th February, 2012.
DOI: 10.3987/COM-12-12420

■ New Dihydroagarofuranoid Sesquiterpenes from Celastrus angulatus

Jiang-yun Liu, Bei-bei Tan, Xue-long Gong, Qiong-ming Xu,* Xiao-fei Sun, Pei-lie Cai, and Shi-lin Yang

School of Pharmacy, Soochow University, Suzhou 215123, China

Abstract

Three new highly esterified β-dihydroagarofuran sesquiterpenes (1-3) were isolated from the root barks of Celastrus angulatus Max. together with eight (4–11) known ones. The new compounds were identified as 1β-picolinoyloxy-2β,6α-diacetoxy-8β,13-diisobutanoyloxy-9α-benzoyloxy-4α-hydroxy-β-dihydroagarofuran (1), 1β-picolinoyloxy-2β,6α-diacetoxy-8β,13-diisobutanoyloxy-9α-furanoyloxy-4α-hydroxy-β-dihydroagarofuran (2), and 1β,6α-diacetoxy-2β-nicotinoyloxy-8β,13-diisobutanoyloxy-9α-benzoyloxy-4α-hydroxy-β-dihydroagarofuran (3) on the basis of spectroscopic methods. Compounds 1-11 were all possessed cytotoxic activities against three human tumor cell lines by MTT assay.

There are about 50 species in the genus Celastus, family Celastraceae, and some of them have been applied as important folk medicines to treat fever, chill, joint pain, edema, rheumatoid arthritis, and bacterial infection or as natural insecticides in China and South America for a long history.1 The family Celastraceae is well-known to provide various highly oxygenated dihydroagarofuran derivatives. As for the same skeleton of Celastrus angulatus, these compounds usually posses six different polyester substitutes in carbon positions C-1, 2, 6, 8, 9 and 13, such as acetoxy (Ac), isobutanoyloxy (iBu), (α-methyl)-butanoyloxy (iPet), benzoyloxy (Bz), furanoyloxy (Fu), nicotinoyloxy (Nic), picolinoyloxy (Pic) and cinnamoyloxy esters etc. Different species of genus Celastus may take different oxygenated level in C-2, 4, 6, 8 or 13 of the skeleton, such as Celastrus Hindsii, Celastrus orbiculatus, Celastrus paniculatus, Celastrus Stephanotiifolius and Celastrus Wulcanicola, etc. Considering the amounts of polyester substitutes and connection positions variations, these compounds have abundant molecular diversities, with more than 100 of them reported to date.2 These ingredients were responsible in major for traditional functions of Celastus plants such as insecticidal, antitumor, multidrug-resistance reversing, antituberculosis, and immunosuppressive activities. In the present study, three new highly esterified sesquiterpenes (1–3, Scheme 1) were isolated from the root barks of Celastrus angulatus Max., along with eight known ones (4–11), 1α,2α,6β-triacetoxy-8α,13-di(α-methyl)-butanoyloxy-9β-benzoyloxy-4β-hydroxy-β-dihydroagarofuran (4),3 1α,2α,6β-triacetoxy-8α-isobutanoyloxy-9β-benzoyloxy-13-(α-methyl)-butanoyloxy-4β-hydroxy-β-dihydro-agarofuran (5),4 1α,2α,6β-triacetoxy-8α,13-diisobutanoyloxy-9β-benzoyloxy-4β-hydroxy-β-dihydroagarofuran (6),4 1α,6β,8α-triacetoxy-2α,13-diisobutanoyloxy-9β-benzoyloxy-4β-hydroxy-β-dihydroagarofuran (7),5 1α,2α,6β-triacetoxy-8α-isobutanoyloxy-9β-furanoyloxy-13-(α-methyl)-butanoyloxy-4β-hydroxy-β-dihydroagarofuran (8),6 1α,6β,8β-triacetoxy-2α,13-diisobutanoyloxy-9β-furanoyloxy-4β-hydroxy-β-dihydroagarofuran (9),7 1α,2α,6β-triacetoxy-8α,13-diisobutanoyloxy-9β-furanoyloxy-4β-hydroxy-β-dihydroagarofuran (10),8 and 1α,2α-diacetoxy-8α,13-diisobutanoyloxy-9β-benzoyloxy-4β,6β-dihydroxy-β-dihydroagarofuran (11).9 In this paper we describe the isolation and structural elucidation of the three new compounds, as well as the evaluation of their cytotoxic activities against human HeLa, SMMC-7721, and HL-60 tumor cell lines.

Compound 1 was obtained as a white amorphous powder (CHCl3) and gave positive Vanillin reaction. Its moleucular formula was analyzed for C40H49O14N at m/z 768.3222 ([M+H]+, calcd 768.3226) by HR-ESIMS. The 13C-NMR spectrum of 1 revealed nine methyls, two methylenes, nine oxygenated carbon signals (between δC 65.9–91.5), and six ester C=O groups (δC 163.9-177.2). Two signals at δC 91.5 (5-C) and 54.4 (10-C) are common characteristics of β-dihydroagarofuran skeleton. The 1H-NMR spectrum of 1 indicated the proton signals of a β-dihydroagarofuran skeleton, such as three tertiary methyls and seven oxygenated methylenes (between δH 4.92–6.33), and the proton signals of six ester groups to be two methyls of two acetate esters, four secondary methyls of two isobutanoate esters, five aromatic proton signals of one benzoate ester, and four aromatic proton signals of one picolinoylate ester, by comparisons with the corresponding signals of angulatusine A (1α,2α,8β-triacetoxy-9α-benzoyloxy-13-picolinoyloxy-β-dihydroagarofuran)10 and other known compounds. Assignments of the H- and C-atom signals of 1 were obtained by extensive 2D NMR (HSQC, 1H-1H COSY, HMBC and NOESY) analyses as shown in Table 1 and Figure 1. The linkage of the six esters to the skeleton locations were confirmed by the HMBC correlations, as between δH 5.30 (H-8) and δC 175.8 (8-C=O of iBu), δH 4.92/5.02 (H-13) and δC 177.2 (13-C=O of iBu), δH 5.88 (H-1) and δC 163.9 (1-C=O of Pic), δH 5.72 (H-9) and δC 164.3 (9-C=O of Bz), δH 6.33 (H-6) and δC 169.5 (6-C=O of Ac). The HMBC correlation of remaining one acetyl group could not be observed, and it should be connected to the remaining position at C-2. Assignments of the relative configurations of parent skeleton of compound 1 were based on the splitting patterns and the coupling constants of proton signals, and by comparisons with the corresponding signals of known compounds.10,11 The singlet peak of H-6 could be observed as the dihedral angels of H-6 and H-7 were near 90°. Similarly, the singlet peak of H-9 suggested H-8 and H-9 both take equatorial orientations. The coupling constant between H-1 (J = 3.6 Hz) and H-2 suggested they take same orientations. Usually, H-1 and H-6 should have same axial orientations by comparing with other compounds of this class. In the NOESY spectrum of compound 1, cross-peaks between H-1 (δH 5.88)/H-2 (δH 5.71) /H-3β (δH 2.34), Me-12 (δH 1.52)/H-6 (δH 6.33)/ H-7 (δH 2.40), H-6 (δH 6.33)/CH2-13 (δH 4.92 and 5.02) /H-1 (δH 5.88) further confirmed their stereochemistry configurations. Accordingly, structure of 1 was elucidated as 1α-picolinoyloxy-2α,6β-diacetoxy-8α,13-diisobutanoyl-oxy-9β-benzoyloxy-4β-hydroxy-β-dihydroagarofuran.

Compound 2 was obtained as a white amorphous powder (CHCl3). Its moleucular formula was determined to be C38H47O15N at m/z 758.3016 ([M+H]+, calcd 758.3018) by HR-ESIMS. By comparisons with 1α-nicolinoyloxy-2α,6β,8α-triacetoxy-13-isobutanoyloxy-9β-furanoyloxy-4β-hydroxy-β-dihydroagarofuran11 and 1, the 1H and 13C-NMR data of 2 were similar to those of 1, with difference of a furanoyloxy ester (δC 109.4, 117.6, 143.7, 148.7, 160.7) substituted at C-9 instead of a benzoyloxy group

of 1. Assignments of the H- and C-atom signals of 2 were obtained by 2D-NMR analyses as shown in Table 1. The linkage of the five esters to the skeleton locations were confirmed by the HMBC corelations, as between δH 5.25 (H-8) and δC 175.7 (8-C=O of iBu), δH 4.92/4.98 (H-13) and δC 177.1 (13-C=O of iBu), δH 5.84 (H-1) and δC 164.2 (1-C=O of Pic), δH 5.61 (H-9) and δC 160.7 (9-C=O of Fu), δH 6.29 (H-6) and δC 169.5 (6-C=O of Ac). The left one acetyl group was then assigned to the remaining position at C-2. Compound 2 was comfirmed as 1α-picolinoyloxy-2α,6β-diacetoxy-8α,13-diisobutanoyloxy-9β-furanoyloxy-4β-hydroxy-β-dihydroagarofuran.
Compound 3 was obtained as a white amorphous powder (CHCl3). Its moleucular formula was analyzed for C40H49O14N at m/z 768.3233 ([M+H]+, calcd 768.3226) by HR-ESIMS, same as that of 1. By comparison with 1α-nicolinoyloxy-2α,6β,8α-triacetoxy-13-isobutanoyloxy-9β-furanoyloxy-4β-hydroxy-β-dihydroagarofuran11 and 1, the 1H and 13C-NMR data of 3 were similar to those of 1, with a different substitute ester of a nicotinoyloxy ester in stead of a picotiloxy group of 1. Assignments of the H- and C-atom signals of 3 were obtained by comparing with the corresponding signals of 1 and by 2D-NMR analyses as shown in Table 1. In HMBC spectrum of compound 3, correlations between δH 5.89 (H-2), 5.66 (H-9) and δC 164.4×2 (both C=O of Nic and 9-Bz) confirmed the linkage of the nicotinoyloxy ester at C-2. Other HMBC correlations could be observed between δH 5.31 (H-8) and δC 175.9 (8-C=O of iBu), δH 4.73/5.12 (H-13) and δC 176.9 (13-C=O of iBu), δH 5.75 (H-1), 6.34 (H-6) and δC 169.5 (1- and 6- C=O of Ac). Compound 3 was thus elucidated as 1α,6β-diacetoxy-2α-nicolinoyloxy-8α,13-diisobutanoyloxy-9β-benzoyloxy-4β-hydroxy-β-dihydroagarofuran.

Cytotoxic activities of isolated compounds 1-11 were tested by MTT assay in vitro and expressed as IC50 values. They all exhibited moderate cytotoxic activities against human HeLa, SMMC-7721, and HL-60 tumor cell lines, with IC50 values ranging from 14.77 to 24.38 μM (Table 2). Compounds 1-11 with a hydroxy group at C-4 showed similar cytotoxic activity results with literatures,12 which suggested that each of these derivatives might display a unique mode of antitumor action. The present results suggested all these compounds might be, at least in part, responsible for the traditional therapeutic effect of root barks of Celastrus angulatus.

EXPERIMENTAL
General
Melting points were determined by the XT5 micro-melting-point apparatus (XT5, Beijing families instrument light instrument plant, China) and are uncorrected. Optical rotations were obtained on a Perkin-Elmer model 241 polarimeter. IR spectra were taken on a Perkin-Elmer 983 G spectrometer. 1H-, 13C-NMR and 2D NMR spectra were recorded on a Varian Inova 400 spectrometer in CDCl3 using tetramethylsilane (TMS) as internal standard. EI-MS spectra were determined on a Micromass Zabspec spectrometer. HRESI-MS spectra were determined on a Micromass Q-TOF2 spectrometer. Preparative HPLC was carried out on the columns of ODS (3.5 × 400 cm, Cosmosil 75C18-Prep, 250 × 20 mm i.d., 5 µm PRC-ODS column; Shimadzu Co. Ltd.), the flow rate was 2 mL/min and the wavelength for detection was 254 nm. Silica gel (200-300 mesh) for column chromatography was obtained from Qingdao Marine Chemical Factory, Qingdao, China. Precoated plates of silica gel used for TLC were obtained from Qingdao Marine Chemical Factory, Qingdao, China. Compounds on the TLC were colored by 10% sulfuric acid alcohol solution. HeLa uterocervical carcinoma cell, SMMC-7721 hepato carcinoma cell and HL-60 leukocythemia carcinoma cell were purchased from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
Plant material
The root barks of Celastrus angulatus Max. were collected from Liu’an city, Anhui Province, China, in March 2008, and the botanical origin of material was identified by Prof. Chun-yu Liu, College of Pharmaceutical Science, Soochow university, and the voucher specimen (No. CA080601) was deposited at department of natural medicinal chemistry, College of Pharmaceutical Science, Soochow University.
Extraction and isolation
The air-dried plants (15 kg) were extracted with 95% EtOH (60 L × 3) under reflux for 2 h. After evaporation, the residue (1.3 kg) was suspended in H2O (15 L) and partitioned with petroleum ether (PE, 60–90 °C; 15 L × 4). The PE layer was concentracted to give a residue (169 g), which was subjected to vacuum liquid column on silica gel (30 × 25 cm, 200-300 mesh; Qingdao Haiyang Chemical Co. Ltd.) using a stepwise gradient elution of PE - EtOAc (9 : 1; 8 : 2; 7 : 3; 6 : 4; 0 : 10; each 3.0 L) to afford 14 subfractions (CA-1~14). CA-3 (6.2 g) was subjected to silica gel columns (4 × 60 cm), eluted with PE - EtOAc (90:10; 80:20; 75:25) repeatedly, and purified by Sephadex LH-20 (CHCl3 : MeOH = 1:1) to yield 4 (14.2 mg). CA-4 (5.1 g) was chromatographed on silica gel (4 × 60 cm) with gradient elution of PE - EtOAc (90:10; 80:20; 75:25; 70:30) to afford 11 subfractions (CA-4.1 to CA-4.11); CA-4.7 (1.4 g) was then chromatographed by MPLC over ODS-C18 column (3.5 × 40 cm, Cosmosil 75 C18-Prep), eluted with MeOH-H2O (70:30; 75:25; 80:20; 85:15, 100:0; each 800 mL) to afford compounds 5 (18 mg), 6 (15 mg) and 7 (12 mg) from MeOH-H2O (80:20 and 85:15) fraction. CA-5 (5.4 g) was chromatographed on silica gel (4 × 60 cm), eluted with PE - EtOAc (80:20; 75:25; 70:30; 60:40) to afford 8 subfractions (CA-5.1 to CA-5.8); CA-5.5 (1.8 g) was then chromatographed by MPLC over ODS-C18 column (3.5 × 40 cm, Cosmosil 75 C18-Prep), eluted with MeOH-H2O (60:40; 70:30; 75:25; 85:15, 100:0; each 800 mL) to afford compounds 8 (11 mg), 9 (10 mg) and 10 (13 mg) from MeOH-H2O (75:25) fraction. CA-6 (4.5g) was chromatographed on silica gel (4 × 60 cm) with gradient elution of PE - EtOAc (80:20; 75:25; 70:30; 60:40; each 800 mL) to afford 6 subfractions (CA-6.1 to CA-6.6); CA-6.3 (2.3 g) was then chromatographed by MPLC over ODS-C18 column (3.5 × 40 cm, Cosmosil 75 C18-Prep; Nacalai Tesque Inc.), eluted with MeOH-H2O (65:35; 70:30; 75:25; 100:0; each 800 mL) to afford 11 subfractions (6.3.1 to CA-6.3.11); CA-6.3.3 (0.9 g) was further purified by HPLC (250 × 20 mm i.d., 5 µm PRC-ODS column; Shimadzu Co. Ltd.), eluted with MeOH-H2O (70:30 – 75:25, 6 mL/min) to yield compounds 1 (11 mg) , 2 (10 mg) and 3 (13 mg). CA-7 (4.5 g) was subjected to silica gel columns (4 × 60 cm), eluted with PE - EtOAc (75:25; 70:30) repeatedly, and purified by Sephadex LH-20 (CHCl3 : MeOH = 1:1) to yield 11 (14 mg).
1α-Picolinoyloxy-2α,6β-diacetoxy-8α,13-diisobutanoyloxy-9β-benzoyloxy-4β-hydroxy-β-dihydroagarofuran (1): White amorphous powder, [α]20 D -9.0 (c 0.21, CHCl3). UV λmax (CHCl3) nm: 226, 230 (sh), 274. IR (KBr) cm-1: 3485, 2978, 1746, 1725, 1601, 1588, 1280, 1240, 745, 715. HR-ESIMS m/z 768.3222 [M+H] + (calcd for C38H47O15N: 768.3226). 1H- and 13C- data are given in Table 1.
1α-Picolinoyloxy-2α,6β-diacetoxy-8α,13-diisobutanoyloxy-9β-furanoyloxy-4β-hydroxy-β-dihydroagarofuran (2): White amorphous powder, [α]20 D -17.0 (c 0.22, CHCl3). UV λmax (CHCl3) nm: 226, 245. IR (KBr) cm-1: 3488, 2982, 1742, 1730, 1600, 1590, 1280, 1218, 750, 717. HR-ESIMS m/z 758.3016 [M+H]+ (calcd for C40H49O14N: 758.3018). 1H- and 13C- data are given in Table 1.
1α,6β-Diacetoxy-2α-nicolinoyloxy-8α,13-diisobutanoyloxy-9β-benzoyloxy-4β-hydroxy-β-dihydroagarofuran (3): White amorphous powder, [α]20 D -11.0 (c 0.18, CHCl3). UV λmax (CHCl3) nm: 216, 232 (sh), 273. IR (KBr) cm-1: 3480, 2980, 1745, 1718, 1600, 1585, 1310, 1235, 741, 712. HRESI-MS m/z 768.3233 [M+H]+ (calcd for C40H49O14N: 768.3226). 1H- and 13C- data are given in Table 1.
Cytotoxic activity
MTT colorimetric assay in vitro was performed to evaluate the cytotoxic activities of compounds 1-11 (Purity higher than 90% as determined by HPLC) against human HeLa, SMMC-7721, and HL-60 tumor cell lines (Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) as reported,13 with norcantharidin (Purity higher than 99.0% as determined by HPLC; Nanjing Zelang Medical Technology Co. Ltd.) as positive control.

ACKNOWLEDGEMENT
We are grateful to Prof. Xue-dong Yang of the Department of Pharmaceutical Science and Technology, Tianjin University, for the HRESI-MS measurement and Mrs. Li-Ping Shi of the Instrumentation Center of Shanghai Institute of Organic Chemisty, Chinese Academy of Science for the NMR measurements. This paper was funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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