Abstract!

Two new triterpenoids (1, 2) and two new ste-roids (3, 4) along with twelve related known com-pounds (5–16) were isolated from the bark ofMe-lia azedarach. The new structures were elucidatedby means of spectroscopic methods and molecu-lar modeling studies and found to be 21,24-cyclo-eupha-7-ene-3β,16β,21α,25-tetrol (1), 3β-ace-toxy-12β-hydroxy-eupha-7,24-dien-21,16β-olide(2), 29-hydroperoxy-stigmasta-7,24(28)E-dien-3β-ol (3), and 24ξ-hydroperoxy-24-vinyl-latho-sterol (4). All isolated compounds were tested for

their cytotoxic activity against three human can-cer cell lines (A549, H460, HGC27) using the Cell-Titer Glo™ luminescent cell viability assay.Among them, compounds 2–4, 24ξ-hydroperoxy-24-vinyl-cholesterol (6), kulinone (7), meliastatin3 (8), 3-oxo-olean-12-en-28-oic acid (10), and(22E,24S)-5α,8α-epidioxy-24-methyl-cholesta-6,22-dien-3β-ol (12) were found to have cytotoxiceffects, with IC50 values of 5.6–21.2 µg/mL.

Supporting information available online athttp://www.thieme-connect.de/ejournals/toc/plantamedica

Cytotoxic Triterpenoids and Steroidsfrom the Bark ofMelia azedarach

Authors Shi-Biao Wu1, Qiu-Ying Bao1, Wen-Xuan Wang1, Yun Zhao1, Gang Xia1,Zheng Zhao1, Huaqiang Zeng2, Jin-Feng Hu1

Affiliations 1 Department of Natural Products for Chemical Genetic Research, Key Laboratory of Brain Functional Genomics,Ministry of Education and Shanghai Key Laboratory of Brain Functional Genomics, East China Normal University,Shanghai, PR China

2 Department of Chemistry, National University of Singapore, Singapore

Key wordsl" Melia azedarachl" Meliaceael" triterpenoidsl" steroidsl" cytotoxicity

received Sept. 16, 2010revised Nov. 9, 2010accepted Dec. 7, 2010

BibliographyDOI http://dx.doi.org/10.1055/s-0030-1250673Published online January 17,2011Planta Med 2011; 77: 922–928© Georg Thieme Verlag KGStuttgart · New York ·ISSN 0032‑0943

CorrespondenceProf. Dr. Jin-Feng HuDepartment of Natural Productsfor Chemical Genetic ResearchKey Laboratoryof Brain Functional GenomicsMinistry of Education &Shanghai Key Laboratoryof Brain Functional GenomicsEast China Normal University3663 Zhongshan Road NShanghai 200062ChinaPhone: + 862162237510Fax: + 862162606791jfhu@brain.ecnu.edu.cn

922

Wu S-B et al. Cytotoxic Triterpenoids

Original Papers

Introduction!

Cortex meliae Radicis, known as “Ku-Lian-Pi” inChinese, is the dried stem bark or root bark ofMe-lia azedarach L. orM. toosendan Sieb. et Zucc. (Me-liaceae). “Ku-Lian-Pi” has been used as a tradi-tional Chinesemedicine (TCM) to cure dermatosisand repel worms [1]. As a part of our ongoingproject towards the discovery of novel biologi-cally active compounds from plants [2,3], twonew (1, 2) and four known (7–10) triterpenoids,as well as two new (3, 4) and eight known (5, 6,11–16) steroids were obtained from the bark ofM. azedarach. The cytotoxicities of the isolatedcompounds against two human lung cancer celllines (A549, H460) and one human gastric carci-noma cell line (HGC27) were evaluated using theCellTiter Glo™ luminescent cell viability assay.

Materials and Methods!

General experimental proceduresMelting points were measured on a WRS-1A Dig-ital Melting-Point Apparatus (Shanghai YICE Ap-paratus & Equipments CO., Ltd.), and are present-ed herein without being subjected to correction.

and… Planta Med 2011; 77: 922–928

Optical rotations were determined by using a Per-kin-Elmer 341 polarimeter. IR spectra were mea-sured on a Nicolet NEXUS-670 FT‑IR spectropho-tometer. NMR spectra were recorded on a BrukerAvance DRX-500 spectrometer. Chemical shiftsare expressed in δ (ppm), and were referenced tothe residual solvent signals. Electrospray ioniza-tion mass spectra (ESI‑MS) and HR‑ESI‑MS weremeasured on a Bruker Daltonics micrOTOF‑QIImass spectrometer. Semipreparative HPLC wasperformed on a Beckman system consisting of aBeckman Coulter System Gold 508 autosampler,Gold 126 gradient HPLC pumps with a BeckmanSystem Gold 168UV detector, a Sedex 80 (SE-DERE) evaporative light-scattering detector(ELSD), and a YMC-Pack ODS‑A column (250 ×10mm, dp 5 µm). Column chromatography (CC)was performed using silica gel (200–300 mesh;Ji-Yi-Da Silysia Chemical Ltd.), MCI gel CHP20P(75–150 µ; Mitsubishi Chemical Industries), andSephadex LH-20 (GE Healthcare Bio-SciencesAB). Silica gel-precoated plates (GF254, 0.25mm;Kang-Bi-Nuo Silysia Chemical Ltd.) were used forTLC. Spots were visualized using UV light (254and/or 366 nm) and by spraying with 5% (v/v)H2SO4-EtOH followed by heating to 120°C.

923Original Papers

Plant materialThe dried bark ofM. azedarachwas purchased from Shanghai Jiu-Zhou-TongMedicine Co. Ltd. andwas originally collected from Si-chuan, a southwestern province of China. The plant was identi-fied by Prof. Bao-Kang Huang (Department of Pharmacognosy,the Second Military Medical University of China). A voucherspecimen (No. 060318) was deposited at the Herbarium of theShanghai Key Laboratory of Brain Functional Genomics, East Chi-na Normal University.

Extraction and isolationThe dried bark (10.0 kg) was extracted three times with 95%EtOH (3 × 10 L) at room temperature. The solvent was removedunder reduced pressure to give a brown residue (ca. 671.0 g).The entire crude extract was suspended in H2O (3 L), and ex-tracted with EtOAc five times (5 × 3 L) to give an EtOAc extract(ca. 235.0 g), which was then subjected to CC over silica gel(4.2 kg, column: 100 × 9 cm) with a petroleum ether (PE)-acetonegradient (15 :1, 12:1, 10:1, 8 :1, 4 :1, 2 :1, 1 :1, 0 :1, 3 L each) toyield twelve fractions (Fr.1–Fr.12). Fr.3 (between 1.2 L and 3.8 L,1.9 g) was chromatographed on silica gel (250 g, column: 70 ×4 cm) with a CH2Cl2-acetone gradient (10:1, 9 :1, 8 :1, 600mLeach) to furnish compounds 2 (between 550mL and 780mL,13.2mg) and 7 (between 880mL and 1260mL, 9.5mg). Com-pounds 9 (between 450mL and 700mL, 24.2mg), 10 (between980mL and 1200mL, 29.8mg), and 12 (between 820mL and980mL, 18.5mg) were isolated from Fr.4 (between 3.8 L and6.2 L, 3.2 g) by eluting on silica gel CC (220 g, column: 70 × 3 cm)with a PE-EtOAc gradient (8 :1, 6 :1, 4 :1, 500mL each). Com-pound 13 (77.6mg) was obtained from Fr.5 (between 6.2 L and8.5 L, 2.9 g) by crystallization from EtOAc.Fr.6 (between 8.5 L and 14.2 L, 5.8 g) was chromatographed onsilica gel (480 g, column: 70 × 6 cm) using a gradient of CH2Cl2-EtOAc (8:1, 6 :1, 4 :1, 900mL each) to afford six subfractions(Fr.6A–Fr.6F). Compounds 3 (between 0.88 L and 1.2 L, 12.1mg),4 (between 2.0 L and 2.8 L, 13.7mg), 6 (between 2.8 L and 3.3 L,17.4mg), and 8 (between 3.4 L and 3.8 L, 9.0mg) were isolatedfrom Fr.6B (between 1.4 L and 1.6 L, 1.8 g) by silica gel CC (180 g,column: 70 × 3 cm) with PE-EtOAc (5:1, 4.0 L) and were furtherpurified by gel permeation chromatography (GPC) on SephadexLH-20 (column: 110 × 2.5 cm) in MeOH. Fr.6C (between 1.6 Land 1.8 L, 1.1 g) was subjected to CC overMCI‑gel using a stepwisegradientelutionwithMeOH‑H2O(from1:1 toMeOHneat) toyieldcompounds 5 (between 1.5 L and 2.1 L, 9.2mg), 11 (between 2.4 Land 2.8 L, 15.0mg), and 14 (between 3.2 L and 3.4 L, 24.3mg).Compound 1 (23.1mg) was purified from Fr.7 (between 14.2 Land 15.5 L, 4.2 g) by using semipreparative HPLC. The method de-veloped consisted of a linear gradient of CH3CN in H2O from 30%to 70% over 10min, followed by an isocratic gradient of 70%CH3CN for 30min, followed by 95% CH3CN for 5min (flow rate:3mL/min; 1: tR = 29.0min). Compounds 15 (12.0mg) and 16(20.6mg) were purified from Fr.10 (between 19.8 L and 21.2 L1:1, 1.9 g) by using semipreparative HPLC. The method was anisocratic gradient of 30% CH3CN in H2O over 8.0min, followedby a linear gradient of CH3CN from 30% to 50% for 25.0min, andeventually followed by 95% CH3CN for 5.0min (flow rate: 3mL/min; 15: tR = 30.4min; 16: tR = 32.6min).

Compound characterization21,24-Cycloeupha-7-ene-3β,16β,21α,25-tetrol (1): Colorless amor-phous powder, [α]D22: + 3.0 (c 0.145, MeOH); IR (KBr) νmax = 3420(br), 2928, 1634, 1458, 1383, 1169, 1097, 1034, 735, 620 cm−1; 1H

and 13C NMR data seel" Tables 1 and 2; (+) ESI‑MSm/z = 497 [M +Na]+, 971 [2M + Na]+; HR‑ESI‑MS m/z = 497.3600 (calcd. forC30H50O4Na, 497.3601).3β-Acetoxy-12β-hydroxy-eupha-7,24-dien-21,16β-olide (2): Col-orless amorphous powder, [α]D22: − 20.1 (c 0.530, MeOH); IR(KBr) νmax = 3437 (br), 2956, 2923, 1778, 1732, 1624, 1454,1382, 1247, 1028, 743, 628 cm−1; 1H and 13C NMR data seel" Tables 1 and 2; (+) ESI‑MS m/z = 535 [M + Na]+, 1047 [2M +Na]+; HR‑ESI‑MS m/z = 535.3429 (calcd. for C32H48O5Na,535.3394).29-Hydroperoxy-stigmasta-7,24(28)E-dien-3β‑ol (3): Colorlessamorphous powder, [α]D22: + 35.9 (c 0.185, MeOH); IR (KBr)νmax = 3424 (br), 2936, 2871, 1655, 1462, 1381, 1031, 665, 559,467 cm−1; 1H and 13C NMR data see l" Tables 1 and 2; (+) ESI‑MSm/z = 467 [M + Na]+, 911 [2M + Na]+; (−) ESI‑MS m/z = 489 [M +HCOO−]−; HR‑ESI‑MS m/z = 467.3505 [M + Na]+ (calcd. for C29H48

O3Na, 467.3496).24ξ-Hydroperoxy-24-vinyl-lathosterol (4): Colorless amorphouspowder, [α]D22: + 8.6 (c 0.385, MeOH); IR (KBr) νmax = 3424 (br),2939, 2872, 1662, 1461, 1381 cm−1; 1H and 13C NMR data seel" Tables 1 and 2; (+) ESI‑MS m/z = 467 [M + Na]+, 911 [2M +Na]+; HR‑ESI‑MS m/z = 467.3488 [M + Na]+ (calcd. for C29H48

O3Na, 467.3496).2α,3α,20R-Trihydroxy-5α-pregnane 16β-methacrylate (5): Mono-clinic crystals (CHCl3); m.p. 206–207°C; [α]D22: + 38.5 (c 0.50,MeOH); IR (KBr) νmax = 3428 (br), 2929, 2857, 1701, 1633, 1450,1381, 1325, 1299, 1173, 1046, 1006, 873, 619, 530 cm−1; 1H and13C NMR data see l" Tables 1 and 2; (+) ESI‑MS m/z = 421 [M +H]+, 841 [2M + H]+; HR‑EI‑MS m/z = 421.2936 [M + H]+ (calcd. forC25H41O5, 421.2949).

X‑ray crystallographic analysis of 5A colorless crystal (0.35 × 0.28 × 0.17mm3) was obtained from asolution of CHCl3. The X‑ray data was collected on a Bruker-AXSSMART APEX II CCD diffractometer. The crystal was triclinic, withspace group P1 and cell parameters a = 6.0445(4) Å, b = 12.4052(8) Å, c = 16.8601(11) Å, V = 1180.07(13) Å3; Z = 1, d = 1.229mg/m3. The final indices were R1 = 0.0452, wR2 = 0.1045. Crystallo-graphic data for compound 5 has been deposited at the Cam-bridge Crystallographic Data Centre as supplementary publica-tion number CCDC-753300. Copies of the data can be obtained,free of charge, on application to the Director, 12 Union Road,Cambridge CB2 1EZ, UK [fax: + 44-(0)1223336033 or e-mail:deposit@ccdc.cam.ac.uk].

Cytotoxicity assayThe A549 and H460 human lung cancer cell lines as well as theHGC27 human gastric carcinoma cancer cell line were purchasedfrom the cell bank of the Shanghai Institute of Cell Biology. TheA549 and H460 cell lines were cultured in the RPMI-1640 me-dium, while the HGC27 cell line was cultured in the MEM me-dium. All mediawere supplementedwith 10% fetal bovine serum(FBS), 100 units/mL penicillin, and 100 units/mL streptomycin(Invitrogen). The cells were maintained at 37°C in a humidifiedenvironment with 5% CO2. The cell viability was determined byusing the CellTiter Glo™ luminescent cell viability assay (Prome-ga) [5,6]. Briefly, the cancer cells were seeded into 384-wellplates at an initial density of 2000 cells/well in 40 µL of medium.Then the cells were treated with compounds at varying concen-trations. Fluorouracil (5-FU; Sigma-Aldrich, catalog No. F6627)was used as a positive control. After incubation for 72 h, 10% ofCellTiter Glo™ reagent was added, and luminescent signals were

Wu S-B et al. Cytotoxic Triterpenoids and… Planta Med 2011; 77: 922–928

Table 1 1H NMR data of compounds 1–5 (500MHz, J in Hz).

No. 1a 2b 3c 4c 5b

1 1.13, 1H, m 1.24, 1H, m 1.10, 1H, m 1.08, 1H, m 1.25, 1H, m

1.65, 1H, m 1.70, 1H, m 1.78, 1H, m 1.80, 1H, m 1.68, 1H, m

2 1.60, 1H, m 1.64, 1H, m 1.70, 1H, m 1.68, 1H, m 3.71, 1H, brd, 10.0

1.62, 1H, m 1.69, 1H, m 2.08, 1H, m 2.06, 1H, m

3 3.17, 1H, dd, 10.5, 5.2 4.49, 1H, dd, 11.5, 4.0 3.86, 1H, m 3.85, 1H, m 3.91, 1H, m

4 1.58, 1H, m 1.54, 1H, m 1.42, 1H, overlapped

1.98, 1H, m 1.96, 1H, m 1.53, 1H, m

5 1.32, 1H, dd 1.42, 1H, dd, 12.0, 5.8 1.40, 1H, m 1.43, 1H, m 1.58, 1H, m

6 2.00, 1H, m 2.00, 1H, m 1.80, 1H, m 1.78, 1H, m 0.94, 1H, m

2.12, 1H, m 2.17, 1H, m 1.82, 1H, m 1.79, 1H, m 1.73, 1H, m

7 5.28, 1H, brd, 2.9 5.32, 1H, brd, 2.1 5.21, 1H, brs 5.20, 1H, brs 1.48, 1H, m

1.60, 1H, m

8 1.53, 1H, m

9 2.26, 1H, m 2.32, 1H, dd, overlapped 1.64, 1H, m 1.66, 1H, m 0.81, 1H, m

11 1.54, 2H, m 1.50, 1H, m 1.48, 1H, overlapped 1.47, 1H, m 1.12, 1H, m

2.31, 1H, m 1.52, 1H, m 1.56, 1H, m 1.23, 1H, m

12 1.52, 1H, m 4.01, 1H, dd, 9.1, 4.6 1.20, 1H, m 1.20, 1H, m 1.20, 1H, m

1.78, 1H, m 1.99, 1H, m 2.04, 1H, m 2.10, 1H, m

14 1.82, 1H, overlapped 1.81, 1H, dd, overlapped 0.97, 1H, m

15 1.58, 1H, overlapped 1.71, 1H, dd, 13.7, 4.6 1.47, 1H, overlapped 1.41, 1H, m 1.12, 1H, m

2.19, 1H, overlapped 2.24, 1H, dd, 13.7, 10.4 1.58, 1H, overlapped 1.53, 1H, m 2.43, 1H, m

16 4.16, 1H, m 4.18, 1H, m 1.23, 1H, m 1.27, 1H, m 5.09, 1H, m

1.87, 1H, m 1.95, 1H, m

17 1.68, 1H, overlapped 2.51, 1H, dd, 12.5, 11.6 1.20, 1H, m 1.29, 1H, m 1.41, 1H, overlapped

18 0.86, 3H, s 0.79, 3H, s 0.57, 3H, s 0.59, 3H, s 0.95, 3H, s

19 0.78, 3H, s 0.83, 3H, s 0.85, 3H, s 0.85, 3H, s 0.78, 3H, s

20 1.78, 1H, m 2.39, 1H, m 1.42, 1H, m 1.47, 1H, m 4.10, 1H, m

21 3.73, 1H, dd, 8.5, 8.3 1.04, 3H, d, 6.5 1.08, 3H, d, 6.5 1.14, 3H, d, 5.8

22 1.50, 1H, m 1.59, 1H, m 1.22, 1H, m 1.39, 1H, m

1.52, 1H, m 1.99, 1H, m 1.58, 1H, overlapped 1.82, 1H, m

23 1.28, 1H, m 1.65, 1H, m 1.02, 1H, m 1.90, 1H, m

1.80, 1H, m 2.16, 1H, m 1.53, 1H, m 2.13, 1H, m

24 1.89, 1H, m 5.10, 1H, m 5.52, 1H, brs

6.00, 1H, brs

25 2.30, 1H, m 2.39, 1H, m 1.88, 3H, brs

26 1.20, 3H, s 1.68, 3H, brs 1.05, 3H, d, 6.7 1.09, 1.5H, d, 6.5

1.10, 1.5H, d, 6.5

27 1.20, 3H, s 1.62, 3H, s 1.05, 3H, d, 6.7 1.00, 1.5H, d, 6.5

1.02, 1.5H, d, 6.5

28 0.92, 3H, s 0.93, 3H, s 5.68, 1H, t, 7.0 6.03, 0.5H, dd, 17.2, 11.8

6.05, 0.5H, dd, 17.2, 11.8

29 0.85, 3H, s 0.85, 3H, s 4.94, 2H, d, 7.0 5.41, 0.5H, dd, 11.8, 1.2

5.42, 0.5H, dd, 11.8, 1.2

5.35, 0.5H, dd, 17.2, 1.2

5.38, 0.5H, dd, 17.2, 1.2

30 1.21, 3H, s 1.34, 3H, s

OAc 2.05, 3H, s

a Measured in CD3OD; b measured in CDCl3; c measured in C5D5N

924 Original Papers

read on a SpectraMax M5 reader (Molecular Devices Corp.). TheIC50 value was calculated from the curves generated by plottingthe percentage of the viable cells versus test concentrations on alogarithmic scale using SigmaPlot 10.0 software.

Supporting informationThe ab initio density functional theory (DFT) calculations of com-pound 1 at the B3LYP/6-31G* level, the single crystal X‑ray anal-ysis of compound 5 and the MS and NMR spectra of compounds1–6 are available as Supporting Information.

Wu S-B et al. Cytotoxic Triterpenoids and… Planta Med 2011; 77: 922–928

Results and Discussion!

The ethanol extract from the bark of M. azedarach was succes-sively subjected to CC over silica gel, MCI gel, Sephadex LH-20,and semipreparative HPLC to afford sixteen compounds (1–16)(l" Fig. 1 and Fig. 1S). Comparing their MS and NMR data andtheir physical properties with the literature, or by direct compar-ison with authentic samples, the known compounds were ulti-mately identified as 2α,3α,20R-trihydroxy-5α-pregnane 16β-methacrylate (5) [7], 24ξ-hydroperoxy-24-vinyl-cholesterol (6)[4,8], kulinone (7) [9,10], meliastatin 3 (8) [10], cabralenoe (9)[11], 3-oxo-olean-12-en-28-oic acid (10) [12], (24R)-24-ethyl-5α-cholestane- 3β,5,6β-triol (11) [13], (22E,24S)-5α,8α-epidioxy-24-

Table 2 13C NMR data of compounds 1–6 (125MHz, J in Hz).

No. 1a 2b 2ab,d 3c 4c 5b 6c

1 38.4 36.7 36.9 38.1 37.7 40.4 37.5

2 28.4 24.0 27.4 32.9 32.5 69.2 32.3

3 79.8 80.7 78.9 70.8 70.4 69.2 71.0

4 40.0 37.8 38.9 39.5 39.1 34.3 43.2

5 52.3 50.9 50.6 41.1 40.7 38.2 141.6

6 25.1 23.8 23.9 30.6 30.2 31.9 120.9

7 119.6 119.0 119.2 118.5 118.0 20.7 31.9

8 146.8 142.9 142.7 140.1 139.8 34.3 31.8

9 49.8 48.4 48.4 50.2 49.9 54.2 50.2

10 36.1 35.1 35.2 35.0 34.6 37.0 36.6

11 18.3 29.1 29.1 22.3 21.9 27.6 21.1

12 33.7 72.1 72.0 40.2 39.9 40.3 39.7

13 46.7 44.4 44.3 44.1 43.7 42.8 42.2

14 50.5 54.8 54.8 56.4 55.4 53.9 56.6

15 45.6 36.2 36.1 23.8 23.4 35.4 24.2

16 78.4 82.2 82.2 28.7 28.4 75.7 28.2

17 61.5 53.2 53.2 55.7 56.556.7

62.3 56.056.2

18 23.2 13.0 12.9 12.5 12.2 13.1 11.7

19 13.6 19.8 19.8 13.7 13.3 12.5 19.3

20 50.5 45.5 45.5 37.5 37.037.2

66.0 36.136.3

21 81.8 180.5 180.9 19.5 19.319.4

23.1 18.818.9

22 24.6 30.0 30.0 36.9 29.229.3

166.9 28.9

23 28.0 26.0 25.9 27.4 29.829.9

136.5 29.229.3

24 55.6 123.6 123.6 152.9 88.088.1

125.4 87.687.7

25 73.5 132.6 132.6 35.5 32.832.9

18.1 32.432.5

26 27.3 25.7 25.7 22.5 17.317.4

16.917.0

27 28.9 17.9 17.9 22.6 18.1 17.7

28 28.2 27.5 27.3 118.0 139.1139.2

138.7138.8

29 15.3 15.7 14.6 74.1 115.5115.6

115.1115.2

30 28.3 33.6 33.6

OAc 171.021.3

a Measured in CD3OD; b measured in CDCl3; c measured in C5D5N. d The 13C NMR data for 2a are adopted from the literature [29]

Fig. 1 Chemical structures of compounds 1–6.

925

Wu S-B et al. Cytotoxic Triterpenoids and… Planta Med 2011; 77: 922–928

Original Papers

Fig. 2 Key HMBC and selected NOESY correlations of 1.

926 Original Papers

methyl-cholesta-6,22-dien-3β-ol (12) [14,15], β-sitosterol (13),daucosterine (14), 7α-hydroxysitosterol-3-O-β-D-glucoside (15)[16], and 7β-hydroxysitosterol-3-O-β-D-glucoside (16) [16].The molecular weight of compound 1 and its chemical formulaC30H50O4 were determined from the positive mode HR‑ESI‑MS,which gave a pseudomolecular ion peak at m/z 497.3600 [M +Na]+. The 1H NMR spectrum (l" Table 1) of 1 displayed signals ofseven methyl groups at δ 0.86 (3H, s, Me-18), 0.78 (3H, s, Me-19), 1.20 (6H, s, Me-26/Me-27), 0.85 (3H, s, Me-28), 0.92 (3H, s,Me-29), and 1.21 (3H, s, Me-30), three oxymethines at δ 3.17(1H, dd, J = 10.5, 5.2 Hz, H-3α), 4.16 (1H, m, H-16α), and 3.73(1H, dd, J = 8.5, 8.3 Hz, H-21β), and one olefinic proton at δ 5.28(1H, brs, H-7). The 13C NMR and DEPT spectra of 1 exhibited thepresence of thirty carbons classified as seven tertiary methyls,eight methylenes, nine methines, and six non-protonated car-bons (l" Table 2). These data demonstrated that 1 has featuressimilar to those of toosendanone A, a euphane-type triterpenoidrecently isolated from M. toosendan [17], bearing an unusual21,24-cyclopentane ring in the side chain. The most obvious dif-ference between these two compounds was that the ketonic car-bonyl signal at δ 216.8 (C-3) of toosendanone Awas replaced by amethine carbon resonating at δ 79.8 in 1 in its 13C NMR spectrum.The β-orientation of the hydroxyl group at C-3 was ascertaineddue to the characteristic splitting pattern (dd, 10.5 and 5.2 Hz)of H-3 [17].Unlike toosendanone A, compound 1 was insoluble in CHCl3. Theplanar structure of the 21,24-cyclopentanyl moiety with a sec-ondary hydroxyl group at C-21 in 1was confirmed by 2DNMRex-periments. Especially the HMBC correlations of 1 (l" Fig. 2) werefound to be identical to that of toosendanone A [17]. However,the 13C NMR data around the cyclopentane ring of these twocompounds were dissimilar (Fig. 2S), indicating that the com-pounds have a different configuration at C-20, C-21, or C-24. Therelative configuration of the cyclopentane ring in 1 was deter-mined through the NOESY NMR experiment (l" Fig. 2) and corro-borated by ab initio calculations (Table 1S and Fig. 3S). Clear NOEcorrelations were observed between Me-18 at δ 0.86 and H-16 atδ 4.16, between H-16 and H-20 at δ 1.78, between H-20 and Me-26/27 at δ 1.20, between H-17 at δ 1.68 and H-21 at δ 3.73, be-tween H-21 and H-24 at δ 1.89, as well as between H-17 andMe-30 at δ 1.21 (l" Fig. 2).A molecular modeling was incorporated to support the NOE-based relative stereochemistry of the cyclopentane ring in com-pound 1. The diastereoisomer 1 d was the most probable config-uration adopted by compound 1 (Supporting Information). As anNOE correlation could be observed when the Boltzmann-aver-aged interproton distance is generally less than or approximately3 Å through space [18,19], the calculated 1H-1H interproton dis-tances between Me-18 and H-16 (2.31 Å), between H-16 and H-20 (2.39 Å), between H-20 and Me-26/27 (2.75 Å), between H-17and H-21 (2.33 Å), between H-21 and H-24 (2.23 Å), as well as be-tween H-17 and Me-30 (2.10/2.86/3.63 Å) (Table 1S) in 1 d wereapproximately consistent with the aforementioned findings inthe NOESY NMR experiment. Moreover, the optimized geome-tries at the B3LYP/6-31G* level of 1 were carried out to calculatethe possible low-energy configurations (Supporting informa-tion). Among the eight diastereomers (1a–1 h) of the cyclopen-tane ring in 1 (Fig. 3S), 1 d (≤ 1.23 kcal/mol) is one of the potentialstable inequivalent conformers (a conformer within a calculatedenergy of 3 kcal/mol or less is relatively stable at room tempera-ture [20–23]). Thus, compound 1 was deduced as 21,24-cycloeu-pha-7-ene-3β,16β,21α,25-tetrol. Naturally occurring triterpe-

Wu S-B et al. Cytotoxic Triterpenoids and… Planta Med 2011; 77: 922–928

noids possessing a 21,24-cyclopentane ring rather than a tetrahy-drofuran ring in the side chain are quite rare, andwere previouslyonly found inM. toosendan [17], Inonotus obliquus [24,25],Mono-cyclanthus vignei [26], and Thalictrum thunbergii [27,28]. It isworthy to point out that these isolated triterpenoids were foundto adopt the theoretically stable conformers (Table 1S and Fig. 3S,Supporting Information) of 1b (toosendanone A [17] and inonot-sutriol B [24,25]) or 1 d (inonotsutriol A [24,25]).In the HR-EIMS spectrum, compound 2 showed a pseudomolecu-lar ion peak [M + Na]+ at m/z 535.3429 corresponding to its mo-lecular formula C32H48O5. The IR spectrum of 2 exhibited absorp-tion bands attributed to the functional groups of double bond(1624 cm−1), ester carbonyl (1732 cm−1), γ-lactone (1778 cm−1),and hydroxyl (3420 cm−1). The 1H and 13C NMR data (l" Tables 1and 2) showed that 2 has features very similar to those of cina-modiol (2a), a euphane-type triterpenoid possessing a notableβ-hydroxyl at C-12 and a γ-lactone ring between C-16 and C-21(l" Fig. 1) [29]. The significant difference between 2 and 2a is that2 has an acetoxy group at C-3 indicated by the fact that H-3 reso-nating at δ 3.24 in 2a [29] was shifted downfield to δ 4.49 (1H, dd,J = 11.5, 4.0 Hz) in 2. Detailed analyses of 2DNMR spectra (COSY,HSQC, and HMBC) of 2 were performed to corroborate the pro-posed structure. Therefore, 2 was established as 3β-acetoxy-12β-hydroxy-eupha-7,24-dien-21,16β-olide. Compound 2 wasdetected in the crude ethanol extract by TLC and HPLC analyses,suggesting that it was not a purification artifact.Compound 3 exhibited an [M + Na]+ ion peak atm/z 467.3505, in-dicating its molecular formula to be C29H48O3. The 1H NMR spec-trum (l" Table 1) of 3 showed signals of five methyl groups reso-nating at δ 0.57 (3H, s), 0.85 (3H, s), 1.04 (3H, d, J = 6.5 Hz), and1.05 (6H, d, J = 6.7 Hz), one oxymethylene at δ 4.94 (2H, d,J = 7.0 Hz), an oxymethine at δ 3.86 (1H, m), and two olefinic pro-tons at δ 5.21 (1H, brs) and 5.68 (1H, t, J = 7.0 Hz). The 13C NMR(DEPT) spectrum of 3 revealed the presence of twenty-nine car-bonsclassifiedasfivemethyls, elevensp3methylenes (oneoxygen-ated at δ 74.1), seven sp3methines (one oxygenated at δ 70.8), andtwosp3quaternarycarbons, inaddition tofourolefiniccarbonsatδ152.9 (C), 140.1 (C), 118.5 (CH), and 118.0 (CH) (l" Table 2). Theabove data indicated that 3 is a C29 steroid possessing similarspectral properties with those of compound 6, a C-24 epimericmixture that was previously obtained from marine algae and tu-nicates [4,8]. The NMR data of 24ξ-hydroperoxy-24-vinyl-cho-lesterol (6) were recorded in C5D5N for the first time in this study(see Supporting information and l" Table 2). The obvious differ-ences were that the locations of the double bonds were differentin 3 compared to 6. The hydroperoxy group was therefore at-

Table 3 IC50 values of isolated compounds against three human cancer celllines.

Compounda IC50 (µg/mL, mean ± SEM)b

A549 H460 HGC27

1 > 50.0 > 50.0 > 50.0

2 12.7 ± 0.5 14.3 ± 0.8 21.2 ± 0.8

3 13.4 ± 0.5 15.6 ± 1.6 17.8 ± 0.1

4 18.5 ± 1.8 18.7 ± 1.1 14.9 ± 0.6

5 > 50.0 > 50.0 11.3 ± 0.5

6 10.5 ± 1.1 10.7 ± 0.7 10.4 ± 0.3

7 6.2 ± 0.1 7.8 ± 1.2 5.6 ± 0.2

8 12.5 ± 0.4 12.3 ± 0.1 10.9 ± 0.8

9 > 50.0 > 50.0 > 50.0

10 14.7 ± 3.5 17.5 ± 3.3 15.4 ± 2.3

11 > 50.0 > 50.0 > 50.0

12 10.9 ± 0.4 10.9 ± 0.4 11.6 ± 0.6

13 > 50.0 > 50.0 > 50.0

14 > 50.0 > 50.0 > 50.0

15 > 50.0 > 50.0 > 50.0

16 > 50.0 > 50.0 > 50.0

5-FUc 1.3 ± 0.3 2.1 ± 0.3 1.5 ± 0.4

a The purity of all tested compounds and the positive control ranged from 96.2% to

99.5% as determined by analytical HPLC with ELSD detection. b IC50 values refer to the

50% inhibition concentration and were calculated from regression using ten different

concentrations with triplicate determinations. c 5-FU: 5-Fluorouracil was used as the

positive control

Fig. 3 Selected 1H-1H COSY, HMBC, and NOE correlations of 3.

927Original Papers

tached to the terminal C-29 in 3 rather than C-24 in 6 [4,8]. Thelocations of Δ7 and Δ24(28) in 3 were unambiguously confirmedby 2DNMR experiments. Two spin systems of -CH2CH2CH(OH)-CH2CHCH2CH=(H2-1 through H-7) and =CHCH2O-(H-28 at δ 5.68and H2-29 at δ 4.94) were found in the COSY spectrum (shown bybold-faced lines in l" Fig. 3). In its HMBC spectrum, Me-19 at δ0.85 displayed nJ (n = 2, 3) correlations with C-1 (δ 38.1), C-5 (δ41.1), C-9 (δ 50.2), and C-10 (δ 35.0); H-7 at δ 5.21 showed corre-lations with C-5 (δ 41.1), C-6 (δ 30.6), and C-8 (δ 140.1); H-29 at δ4.94 was found to have correlations with C-24 (δ 152.9) and C-28(δ 118.0), and the terminal isopropyl (Me-26/Me-27) at δ 1.05 ex-hibited correlations with C-24 and C-25 (δ 35.5) (l" Fig. 3). The E-geometry of the Δ24(28) double bond in 3 was substantially deter-mined by its NOESY NMR experiment, in which H-28 at δ 5.68was found to have clear NOE correlations with H-25 and H-26/27. Moreover, the chemical shift (δ 2.30) of H-25 was in goodagreement with such an E isomer; in the Z isomer, H-25 wouldbe expected to give a signal shifted downfield to ~ δ 3.0 ppm[30–34]. Consequently, 3 was elucidated to be 29-hydroperoxy-stigmasta-7,24(28)E-dien-3β-ol.The molecular formula C29H48O3 of compound 4was determinedby HR-ESIMS, which gave an [M + Na]+ ion peak at m/z 467.3488.The general features of its 1H and 13C NMR spectra closely re-sembled those of 24ξ-hydroperoxy-24-vinyl-cholesterol (6) [4,8]. The major difference between compounds 4 and 6 is that theΔ5 double bond in ring B of 6was relocated to Δ7 in 4, as substan-tiated by its 2DNMR experiments. A spin system of -CH2CH2CH(OH)-CH2CHCH2CH=(H2-1 through H-7) was found in the COSYspectrum of 4. In the HMBC spectrum, Me-19 at δ 0.85 (3H, s)showed correlations with C-1 (δ 37.7), C-5 (δ 40.7), C-9 (δ 49.9),and C-10 (δ 34.6), and H-7 at δ 5.20 displayed correlations withC-5 (δ 40.7), C-6 (δ 30.2), C-9 (δ 49.9), and C-14 (δ 55.4). Com-pound 4 was isolated as an inseparable mixture (ratio: 1 :1) of24-epimers, as evidenced by the fact that the carbon signalsaround C-24 in the side chain were each split into two pairs(l" Table 2). The Δ28 olefinic protons each also appeared as twopairs of signals [H-28: δ 6.03 and 6.05 (each 0.5H, dd, J = 17.2,11.8 Hz), H-29a: δ 5.41 and 5.42 (each 0.5H, dd, J = 11.8, 1.2 Hz);H-29b: δ 5.35 and 5.38 (each 0.5H, dd, J = 17.2, 1.2 Hz)] (l" Table 1).The hydroperoxy group at C-24 was confirmed by the HMBC cor-relations between C-24 at δ 81.0/88.1 and H-28, H-29, Me-26 [δ1.09 and 1.10 (each 1.5H, d, J = 6.5 Hz)] and Me-27 [δ 1.00 and1.02 (each 1.5H, d, J = 6.5 Hz)]. Thus, compound 4was ascertainedto be 24ξ-hydroperoxy-24-vinyl-lathosterol.

2α,3α,20R-Trihydroxy-5α-pregnane 16β-methacrylate (5) waspreviously reported as a semisynthetic from azedarachol [7].The NMR data of 5 are reported herein for the first time (l" Tables1 and 2) and its stereochemistry was further secured by a singlecrystal X‑ray diffraction analysis (Fig. 4S). Although Melia plantshave been extensively investigated [35], it is still an active area ofresearch for natural product chemists. Triterpenoids such as me-liastatin 3 (8) [10] bearing a hydroperoxy group in the side-chainwere often found from Melia sp. [36,37]; however, steroids (e.g.,3, 4, and 6) bearing a hydroperoxy group in the side-chain havenot been isolated before from the genus Melia. The origin of C-24peroxidized steroids has long been questioned. While some sci-entists suggested that the inseparable 24-hydroperoxy steroidswere probably artifacts arising during sample storage and extrac-tion [38,39], others assumed that they might be biosynthesized[40].Compound 6 was previously found to show significant cytotoxiceffects against P-388, KB, A549 and HT-29 cancer cell lines, withED50 values of 0.3–7.1 µg/mL [8]. Kulinone (7) previously exhib-ited significant inhibitory effects against a small panel of humancancer cell lines (MCF-7, SF268, KM20L2, H460, BXPC-3, and DU-145), with GI50 values of 3.6–5.7 µg/mL [10]. Meanwhile, kuli-none (7) and meliastatin 3 (8) possessed antineoplastic activityagainst the P-388 lymphocytic leukemia cell linewith an ED50 val-ue of 4.6 and 3.1 µg/mL, respectively [10]. Recently, (22E,24S)-5α,8α-epidioxy-24-methyl-cholesta-6,22-dien-3β-ol (12) hasbeen reported to have moderate antiproliferative activity againstthe A2780 human ovarian cancer cell line with an IC50 value of16 µg/mL [41]. In this study, the isolated compounds (1–16) weretested for their in vitro cytotoxicities against three human cancercell lines (A549, H460, and HGC27) by using the CellTiter Glo™luminescent cell viability assay [5,6]. Among them, compounds2–4, 6–8, 10 and 12 were found to show significant cytotoxic ef-fects with IC50 values of 5.6–21.2 µg/mL (l" Table 3). Compound 5,

Wu S-B et al. Cytotoxic Triterpenoids and… Planta Med 2011; 77: 922–928

928 Original Papers

however, showed a significant cytotoxic effect against the HGC27gastric cancer cell line with an IC50 value of 11.3 µg/mL, but nocytotoxic effects (IC50s > 50.0 µg/mL) against either A549 orH460 lung cancer cell lines. The cytotoxicities of 24ξ-hydroper-oxy-24-vinyl-cholesterol (6) against the A549 cell line (IC50 ca.10.5 µg/mL, lit. [8]: ED50 5.9 µg/mL), and kulinone (7) against theH460 cell line (IC50 ca. 7.8 µg/mL, lit. [10]: GI50 3.6–5.7 µg/mL) arecomparable to the previous reported data.

Acknowledgements!

The authors wish to thank Prof. Bao-Kang Huang (The SecondMilitary Medical University of PR China) for the plant identifica-tion. This work was supported by an NSFC grant (No. 90713040)and STCSM grants (No. 06DZ19002, 07DZ22006, 08PJ140410).

References1 China Pharmacopoeia Committee. Pharmacopoeia of the P. R. C. The firstdivision of 2005 edition (English). Beijing: Chinese Chemical and Tech-nologic Press; 2005: 45

2 Wu SB, Ji YP, Zhu JJ, Zhao Y, Xia G, Hu YH, Hu JF. Steroids from the leavesof ChineseMelia azedarach and their cytotoxic effects on human cancercell lines. Steroids 2009; 74: 761–765

3 Li XW, Guo ZT, Zhao Y, Zhao Z, Hu JF. Chemical constituents from Saus-surea cordifolia. Phytochemistry 2010; 71: 682–687

4 Guyot M, Davoust D, Belaud C. Hydroperoxy-24 vinyl-24 cholesterol,nouvel hydropéroxyde naturel isolé de deux tuniciers: Phallusia ma-millata et Ciona intestinalis. Tetrahedron Lett 1982; 23: 1905–1906

5 Węsierska-Gądek J, Schloffer D, Gueorguieva M, Uhl M, Skladanowski A.Increased susceptibility of poly (ADF-Pibose) polymerase-1 knockoutcells to antitumor triazoloacridone C-1305 is associated with perma-nent G2 cell cycle arrest. Cancer Res 2004; 64: 4487–4497

6 Lövborg H, Wojciechowski J, Larsson R, Węsierska-Gądek J. Action of anovel anticancer agent, CHS 828, on mouse fibroblasts: increased sen-sitivity of cells lacking poly (ADF-ribose) polymerase-1. Cancer Res2002; 62: 4206–4211

7 Nakatani M, Takao H, Miura I, Hase T. Azedarachol, a steroid ester anti-feedant from Melia azedarach var. japonica. Phytochemistry 1985; 24:1945–1948

8 Sheu JH, Wang GH, Sung PJ, Chiu YH, Duh CY. Cytotoxic sterols from theFormosan brown alga Turbinaria ornata. Planta Med 1997; 63: 571–572

9 Chiang CK, Chang FC. Tetracyclic triterpenoids from Melia azedarach,L.-III. Tetrahedron 1973; 29: 1911–1929

10 Pettit GR, Numata A, Iwamoto C, Morito H, Yamada T, Goswami A, Clew-low PJ, Cragg GM, Schmidt JM. Antineoplastic agents. 489. Isolation andstructures of meliastatins 1–5 and related euphane triterpenes fromtreeMelia dubia. J Nat Prod 2002; 65: 1886–1891

11 Aalbersberg W, Singh Y. Dammarane triterpenoids from Dysoxylum ri-chii. Phytochemistry 1991; 30: 921–926

12 Shirane N, Hashimoto Y, Ueda K, Takenaka H, Katoh K. Ring-A cleavageof 3-oxo-olean-12-en-28-oic acid by the fungus Chaetomium longi-rostre. Phytochemistry 1996; 43: 99–104

13 Notaro G, Piccialli V, Sica D, Corriero G. 3β,5α,6α-Trihydroxylated sterolswith a saturated nucleus from two populations of the marine spongeCliona copiosa. J Nat Prod 1991; 54: 1570–1575

14 Ioannou E, Abdel-Razik AF, Zervou M, Christofidis D, Alexi X, Vagias C,Alexis MN, Roussis V. 5α,8α-Epidioxysterols from the gorgonian Euni-cella cavolini and the ascidian Trididemnum inarmatum: isolation andevaluation of their antiproliferative activity. Steroids 2009; 74: 73–80

15 Sheikh YM, Djerassi C. Steroids from sponges. Tetrahedron 1974; 30:4095–4103

16 Chaurasia N, Wichtl M. Sterols and steryl glycosides from Urtica dioica.J Nat Prod 1987; 50: 881–885

17 Sang YS, Zhou CY, Lu AJ, Yin XJ, Min ZD, Tan RX. Protolimonoids fromMelia toosendan. J Nat Prod 2009; 72: 917–920

Wu S-B et al. Cytotoxic Triterpenoids and… Planta Med 2011; 77: 922–928

18 Stephens PJ, Pan JJ, Devlin FJ, Cheeseman JR. Determination of the abso-lute configurations of natural products using TDDFT optical rotationcalculations: the iridoid oruwacin. J Nat Prod 2008; 71: 285–288

19 Chen LX, Zhu HJ, Wang R, Zhou KL, Jing YK, Qiu F. ent-Labdane diterpe-noid lactone stereoisomers from Andrographis paniculata. J Nat Prod2008; 71: 852–855

20 Wang B, Dossey AT, Walse SS, Edison AS, Merz Jr KM. Relative configura-tion of natural products using NMR chemical shifts. J Nat Prod 2009;72: 709–713

21 Viano Y, Bonhomme D, Camps M, Briand JF, Ortalo-Magné A, Blache Y,Piovetti L, Culioli G. Diterpenoids from the Mediterranean brown algaDictyota sp. evaluated as antifouling substances against a marine bac-terial biofilm. J Nat Prod 2009; 72: 1299–1304

22 XuWH, Jacob MR, Agarwal AK, Clark AM, Liang ZS, Li XC. VerbesinosidesA–F, 15,27-cyclooleanane saponins from the American native plantVerbesina virginica. J Nat Prod 2009; 72: 1022–1027

23 Jiao WX, Blunt JW, Cole ALJ, Munro MHG. Fumagiringillin, a new fuma-gillinde rivative from a strain of the fungus Aspergillus fumigatus. J NatProd 2004; 67: 1434–1437

24 Taji S, Yamada T, Tanaka R. Three new lanostane triterpenoids, inonot-sutriols A, B, and C, from Inonotus obliquus. Helv Chim Acta 2008; 91:1513–1524

25 Shin Y, Tamai Y, Terazawa M. Chemical constituents of inonotus obli-quus II: a new triterpene, 21,24-cyclopentalanosta-3β,21,25-triol-8-ene from sclerotium. J Wood Sci 2001; 47: 313–316

26 Achenbach H, Frey D. Cycloartanes and other terpenoids and phenyl-propanoids from Monocyclanthus vignei. Phytochemistry 1992; 31:4263–4274

27 Yoshimitsu H, Nishida M, Nohara T. Three new cycloartane glycosidesfrom Thalictrum thunbergii D.C. Tetrahedron 2001; 57: 10247–10252

28 Yoshimitsu H, Nishida M, Yahara S, Nohara T. Two new cycloartanegly-cosides from Thalictrum thunbergii D.C. Tetrahedron Lett 1998; 39:6919–6920

29 Kelecom A, Cabral MMO, Garcia ES. A new euphane triterpene from theBrazilianMelia azedarach. J Braz Chem Soc 1996; 7: 39–41

30 DʼAuria MV, Riccio R, Uriarte E, Minale L, Tanaka J, Higa T. Isolation andstructure elucidation of seven new polyhydroxylated sulfated sterolsfrom the ophiuroid Ophiolepis superba. J Org Chem 1989; 54: 234–239

31 Preus MW, McMorris TC. The configuration at C‑24 in oogoniol(24R‑3β,11α,15β,29-tetrahydroxystigmast-5-en-7-one) and identifi-cation of 24(28)-dehydroogoniols as hormones in Achlya. J Am ChemSoc 1979; 101: 3066–3071

32 Bates RB, Brewer AD, Knights BR, Rowe JW. Double bond configurationsof 24-ethylidine sterols. Tetrahedron Lett 1968; 9: 6163–6167

33 De Marino S, Iorizzi M, Zollo F, Minale L, Amsler CD, Baker BJ, McClintockJB. Isolation, structure elucidation, and biological activity of the steroidoligoglycosides and polyhydroxysteroids from the Antarctic starfishAcodontaster conspicuus. J Nat Prod 1997; 60: 959–966

34 Zollo F, Finamore E, Riccio R, Minale L. Starfish saponins, Part 37. Steroi-dal glycoside sulfates from starfishes of the genus Pisaster. J Nat Prod1989; 52: 693–700

35 Vishnukanta ACR. Melia azedarach: A phytopharmacological review.Pharmacognosy Rev 2008; 2: 173–179

36 Tan QG, Li XN, Chen H, Feng T, Cai XH, Luo XD. Sterols and terpenoidsfrom Melia azedarach. J Nat Prod 2010; 73: 693–697

37 Zhang Y, Tang CP, Ke CQ, Yao S, Ye Y. Limonoids and triterpenoids fromthe stem bark ofMelia toosendan. J Nat Prod 2010; 73: 664–668

38 Kobayashi M, Krishna MM, Ishida K, Anjaneyulu V. Marine sterols. XXII.Occurrence of 3-oxo-4,6,8(14)-triunsaturated steroids in the spongeDysidea herbacea. Chem Pharm Bull 1992; 40: 72–74

39 Sera Y, Adachi K, Shizuri Y. A new epidioxy sterol as an antifouling sub-stance from a Palauan marine sponge, Lendenfeldia chondrodes. J NatProd 1999; 62: 152–154

40 Sheu JH, Wang GH, Sung PJ, Duh CY. New cytotoxic oxygenated fuco-sterols from the brown alga Turbinaria conoides. J Nat Prod 1999; 62:224–227

41 Cao SG, Brodie P, Miller JS, Birkinshaw C, Rakotondrafara A, Andriantsi-ferana R, Rasamison VE, Kingston DG. Antiproliferative compounds ofHelmiopsis sphaerocarpa from the Madagascar rainforest. Nat ProdRes 2009; 23: 638–643