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Three New and Other Limonoids from the Hexane Extract of Melia azedarach Fruits and Their Cytotoxic Activities by Xin Pan a ), Masahiro Matsumoto a ), Yasuhiro Nakamura a ), Takashi Kikuchi a ), Jie Zhang a ), Motohiko Ukiya a ), Takashi Suzuki b ), Kazuo Koike c ), Rima Akihisa d ), and Toshihiro Akihisa* a ) d ) a
) College of Science and Technology, Nihon University, 1-8-14 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan b ) School of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi-shi, Chiba 274-8510, Japan c ) School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan d ) Akihisa Medical Clinic, 1086-3 Kamo, Sanda-shi, Hyogo 669-1311, Japan (fax: þ 81-79-5671980; e-mail: [email protected])
A defatted fraction obtained from the hexane extract of the fruits of Melia azedarach L. (chinaberry tree; Meliaceae) exhibited cytotoxic activities against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines with IC50 values in the range of 2.9 – 21.9 mg/ml. Three new limonoids, 3-deacetyl-4’-demethylsalannin (5), 3-deacetyl-28-oxosalannin (14), and 1-detigloylohchinolal (17), along with 16 known limonoids, 1 – 4, 6 – 13, 15, 16, 18, and 19, and one known triterpenoid, 20, were isolated from the fraction. The structures of new compounds were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. These compounds were evaluated for their cytotoxic activities against the four cancer cell lines mentioned above. 3-Deacetyl-4’-demethyl-28oxosalannin (16), which exhibited potent cytotoxicity against AZ521 (IC50 3.2 mm) cells, induced typical apoptotic cell death in AZ521 cells upon evaluation of the apoptosis-inducing activity by flow cytometry. This work provided, furthermore, valuable information on the structural features of limonoids of the fruits and/or seeds of Melia azedarach and related Meliaceae plants, M. toosendan and Azadirachta indica.
Introduction. – Plants of the Meliaceae family have been well-documented for their ability to metabolize structurally diverse and biologically significant limonoids and triterpenoids [1 – 3]. Melia azedarach L. (chinaberry tree; Meliaceae) is indigenous to Japan, Taiwan, China, and Southeast Asia. Its bark and fruits have been used for vermicide, anodyne, and skin disease [4] [5]. Many constituents including limonoids, triterpenoids, and steroids have been isolated from various parts of M. azedarach [1 – 3] [6 – 24]. The limonoids of M. azedarach have been reported to exhibit antimicrobial [13] [14], cytotoxic [9 – 12] [15], antifeedant [7] [8] [16] [24], insecticidal [16], and antiviral [22] activities. In the course of a search for potential bioactive compounds from Meliaceae plants, we were interested to investigate the limonoid constituents of M. azedarach fruits. Thus, investigation on the MeOH extract, obtained by MeOH extraction after hexane extraction of M. azedarch fruits, has led to the isolation of 32 limonoids and one triterpenoid, and some of which have been shown to exhibit potent cytotoxic and apoptosis-inducing activities against human cancer cell lines [17] [18]. In continuing a study on the constituents of M. azedarach fruits, the hexane extract of the 2014 Verlag Helvetica Chimica Acta AG, Zrich
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fruits was investigated in this work, resulting in the isolation of 19 limonoids, 1 – 19, including three new compounds, 5, 14, and 17 (see below), and one triterpenoid, 20. Herein, we describe the structure elucidation of these compounds and evaluation of their cytotoxic activities against four human cancer cell lines. The seed oil of M. azedarach, as well as the seed oil of Azadirachta indica A. Juss (neem tree), has been reported to exhibit repellent activity against sandflies (Phlebotomus orientalis and P. bergeroti) in Ethiopia [25].
Results and Discussions. – Cytotoxic Activities of the Hexane Extracts of M. azedarach Fruits. Dried and powdered M. azedarach fruits were extracted with hexane, and the residue was then extracted with MeOH [17] [18]. The hexane extract was passed through a SiO2 column in order to remove bulky less-polar oily substance from
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the extract. The hexane extract and the defatted more-polar fraction were evaluated for their cytotoxic activities against four human cancer cell lines, HL60 (leukemia), A549 (lung), AZ521 (stomach), and SK-BR-3 (breast), by means of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, and the results are compiled in Table 1. While the hexane extract was inactive against all cell lines tested, the defatted fraction exhibited cytotoxic activities against all cell lines with IC50 values in the range of 2.9 – 21.9 mg/ml. The defatted fraction was further investigated in this study. Table 1. Cytotoxic Activities of Melia azedarach Fruit Extracts against Four Human Cancer Cell Lines a ) Extract or fraction
Hexane extract Defatted fraction Cisplatin c )
Cytotoxicity ( IC50 S. D. [mg/ml] b )) HL60 ( Leukemia)
A549 ( Lung)
AZ521 (Stomach)
SK-BR-3 ( Breast)
75.5 1.7 2.9 2.4 1.3 0.3
> 100 12.0 0.7 5.5 0.6
77.0 1.1 12.3 0.9 2.9 0.2
> 100 21.9 0.6 5.6 0.2
a ) Cells were treated with test samples (1 10 4 – 1 10 6 g/ml) for 48 h, and cell viability was analyzed by MTT assay. b ) The IC50 values based on triplicate five points. c ) Reference compound.
Isolation, Identification, and Structure Elucidation of Compounds. The defatted fraction of the hexane extract was subjected to column chromatography (CC) over SiO2 and octadecyl silica gel (ODS), and reversed-phase (RP) preparative high-performance liquid chromatography (HPLC), which led to the isolation of 19 limonoids, 1 – 19, including three new limonoids, 5, 14, and 17, and one known triterpenoid, 20. The 17 known compounds were identified as trichilinin D (1) [26], 1-cinnamoyltrichilinin (2) [27], 3a,7a-dihydroxy-21,23-epoxy-1a,6a,12a-triacetoxy-24,25,26,27-tetranorapotirucalla-14,20,22-trien-28-al (3; 3-deacetyl-1,6-diacetylsendanal) [28], 3-deacetylsalannin (4) [29], ohchinin (6) [21], ohchinin acetate (7) [19], 1-decinnamoyl-1benzoylohchinin (8) [17], ohchininolide (9) [17], 1-decinnamoyl-1-benzoylohchininolide (10) [17], 23-methoxyohchininolide A (11) [17], 23-methoxyohchininolide B (12) [17], 23-hydroxyohchininolide (13) [17], 1-decinnamoyl-1-benzoyl-28-oxoohchinin (15) [17], 3-deacetyl-4’-demethyl-28-oxosalannin (16) [17], mesendanin E (18) [30], ohchinolal (19) [21], and azedarachic acid (20) [31] by comparison of their spectral data with those in the literature. The structures of three new limonoids, 5, 14, and 17, were elucidated on the basis of spectroscopic analysis and comparison with literature data as described below. Compound 5 was designated the molecular formula C31H40O8 by its positive-ion mode HR-ESI mass spectrum (m/z 563.2618 ([M þ Na] þ , C31H40NaO þ8 ; calc. 563.2620)). The IR spectrum of 5 showed the absorption bands of OH (3367 cm 1), ester and conjugated ester C¼O (1730 and 1713 cm 1, resp.), and furyl (873 cm 1) groups. The 13C-NMR and DEPT data of 5 (Table 2) indicated the presence of 31 Catoms including nine quaternary C-atoms (including four olefinic and two C¼O Catoms), eleven CH (including five O-bearing sp3 and three olefinic), five CH2 (including one O-bearing and one sp2 ), and six Me (including one O-bearing) groups. The 1H- and 13C-NMR data (Table 2) of 5 were closely similar to those of 4 [29], except
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for the presence of the 2-methylprop-2-enoyl (methacryl; Met) group (d(H) 2.05 (Me(4’)), and 5.65 (t, J ¼ 3.0) and 6.16 (t, J ¼ 1.4) (CH2(3’)); d(C) 18.4 (C(4’)), 126.2 (C(3’)), 136.2 (C(2’)), and 165.0 (C(1’))) [17] instead of the tigloyl group at C(1) of 4. The HMBCs (Table 2) of HC(1) (d(H) 5.04) with C(1’) supported the presence of the Met group at C(1). In addition, the NOE interactions (Fig. 1) between HC(3), Me(29), Me(19), and HC(1), and between Me(19), HC(6), HC(7), HbC(16), and HC(17) indicated the a-orientations of MetO, OH, and the furyl groups at C(1), C(3), and C(17), respectively. Thus, the structure of 5 was elucidated as 1-methacrylsalannic acid methyl ester (¼ 3-deacetyl-4’-demethylsalannin).
Fig. 1. Major NOE correlations ( $ ) for compounds 5, 14, and 17. Drawings correspond to energyminimized conformation of compounds. Calculation was performed using CAChe Conformation Search with the MM2 force field (CAChe version 6.01; Fujitsu Co., Tokyo, Japan).
Compound 14 had the molecular formula C32H40O9 , as indicated by positive-ionmode HR-ESI-MS (m/z 569.2777 ([M þ H] þ , C32H41O þ9 ; calc. 569.2750)). Its IR spectrum suggested the presence of OH (3338 cm 1), g-lactone (1778 cm 1), ester and conjugated ester C¼O (1731 and 1718 cm 1, resp.), and furyl (860 cm 1) moieties. The 13 C-NMR data of 14 (Table 2), along with the DEPT data, indicated the presence of 32 C-atoms including ten quaternary C-atoms (including three C¼O and two olefinic Catoms), twelve CH (including five O-bearing sp3 and four olefinic), three CH2 , and seven Me groups. The 1H- and 13C-NMR data (Table 2) of 14 were analogous to those of 4, with obvious differences being the lack of O-bearing CH2 group and the presence of a C¼O group (d(C) 177.5) at C(28) in 14 when compared with 4. This suggested the presence of a 6,28-g-lactone ring in 14 like in nimbolide [32]. In the HMBC (Table 2) experiment, 14 exhibited cross-peaks of HC(5) (d(H) 2.39) and Me(29) (d(H) 1.25)
18 19 20 21 22 23
17
12 13 14 15 16
10 11
6 7 8 9
3 4 5
1 2
Position
7.24 (br. s) 6.26 (dd, J ¼ 1.0, 2.0) 7.31 (t, J ¼ 1.8)
1.65 (d, J ¼ 1.4) 0.96 (s)
5.39 (br. t, J ¼ 7.6) 2.23 – 2.27 (m, Ha ), 2.08 – 2.14 (m, Hb ) 3.61 (br. d, J ¼ 7.2)
2.00 – 2.18 (m) 2.24 – 2.30 (m)
2.64 (dd, J ¼ 3.0, 9.4)
4.02 (dd, J ¼ 3.2, 12.4) 4.18 (d, J ¼ 3.7)
2.72 (d, J ¼ 12.8)
13.1 15.3 127.1 138.8 110.7 142.9
49.3
172.7 135.0 146.4 87.9 41.2
40.8 30.5
72.5 85.7 49.0 39.5
70.8 44.2 38.8
5.04 (d, J ¼ 8.0) 2.11 – 2.18 (m, Ha ), 2.30 – 2.37 (m, Hb ) 3.87 – 3.91 (m)
20, 22, 23 20, 21, 23 20, 21, 22
13, 14 15, 17, 20 13, 14, 20 13, 14, 15, 16, 20, 21, 22 13, 14, 17 1, 5, 9, 10
8, 12 8, 12
10, 11, 12, 19, 30
3, 4, 6, 7, 10, 19, 28 5, 7 5, 6, 8, 9
1
7.25 (s) 6.28 (br. s) 7.33 (br. s)
1.68 (s) 1.03 (s)
5.45 (t, J ¼ 7.6) 2.20 – 2.26 (m, Ha ), 2.06 – 2.16 (m, Hb ) 3.65 (d, J ¼ 8.7)
2.13 – 2.22 (m) 2.26 – 2.35 (m)
2.83 (dd, J ¼ 3.9, 8.9)
4.52 (dd, J ¼ 3.4, 12.6) 4.25 (d, J ¼ 3.7)
3.39 (d, J ¼ 12.4)
4.88 (d, J ¼ 3.0) 2.14 – 2.22 (m, Ha ), 2.25 – 2.32 (m, Hb ) 4.13 (dd, J ¼ 4.3, 6.2)
73.1 30.0
d( H ) 3, 5, 1’ 10
14 d(H )
d(C )
5 HMBC
13.0 15.5 126.8 138.8 110.5 143.0
49.4
172.4 135.9 145.5 88.4 41.0
40.3 30.8
74.3 83.1 47.7 38.7
68.9 48.9 37.4
71.3 29.4
d(C )
22, 23 20, 21, 23 20, 21, 22
7, 13, 14 13, 15, 17, 20 13, 14, 15, 17, 20 13, 14, 15, 16, 20, 21, 22 13, 14, 17 1, 5, 9, 10
9, 10, 12 9, 10, 12
8, 10, 11, 12, 14, 19, 30
3, 4, 6, 7, 10, 19, 28, 29 7 5, 6, 9, 30
1
3, 5, 1’ 4, 10
HMBC
7.12 (s) 6.11 (s) 7.31 (t, J ¼ 1.2)
1.71 (d, J ¼ 1.3) 0.98 (s)
5.50 (br. t, J ¼ 7.6) 2.25 (dd, J ¼ 6.4, 1.4, Ha ), 2.00 – 2.08 (m, Hb ) 3.64 (br. d, J ¼ 7.6)
2.35 (dd, J ¼ 4.1, 17.4) 2.46 (dd, J ¼ 5.5, 17.4)
2.68 (br. t, J ¼ 4.6)
5.22 (dd, J ¼ 3.2, 12.3) 4.11 (d, J ¼ 2.7)
12.9 16.3 126.8 138.6 110.9 143.3
49.6
177.2 135.4 146.4 87.5 41.5
42.9 30.9
69.0 86.3 47.4 40.1
76.2 49.4 34.0
3.72 (br. t, J ¼ 8.0) 3.60 (d, J ¼ 12.4)
72.5 29.0
d(C )
3.66 – 3.68 (m) 1.97 – 2.10 (m, 2 H )
d( H )
17
14 14, 15, 20 15, 20 15, 18, 20, 22
20, 22, 23 20, 21, 23 20, 21, 22
13, 14 1, 5, 9, 10
13, 13, 13, 13,
8, 10, 12 8, 10, 12
30
6-MeCO 5, 6, 9, 30
4, 10, 19, 28, 29
1
3 10
HMBC
Table 2. 1H- and 13C-NMR (400 and 100 MHz, resp.), and HMBC (H ! C) Data of Compounds 5, 14, and 17 (recorded in CDCl3 ; d in ppm, J in Hz)
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51.5
12
3.22 (s)
51.5
12.0 14.5
16.6 15.6 166.7 128.8 137.9
12
2’, 3’, 5’ 1’, 2’, 3’
1’, 2’, 4’, 5’
3, 4, 5, 28 7, 8, 9, 14
17
0.93 (s) 1.36 (s)
9.68 (s)
d( H )
3.58 (s)
3.21 (s)
1’, 2’, 3’
6.95 (dd, J ¼ 6.0, 13.6) 1.82 (d, J ¼ 6.9) 1.90 (s)
1.25 (s) 1.36 (s)
HMBC
12-MeO
18.4
1’, 2’, 4’
3, 4, 5, 28 7, 8, 9, 14
177.5
d(C )
1.95 (s)
5.65 (t, J ¼ 3.0), 6.16 (t, J ¼ 1.4) 2.05 (s)
19.8 16.9 165.0 136.2 126.2
77.8
3.64 (d, J ¼ 7.3), 4.13 (d, J ¼ 7.3) 1.16 (s) 1.31 (s) 3, 4, 29
14 d(H )
d( H ) HMBC
d(C )
5
4’ 5’ 6-AcO
29 30 1’ 2’ 3’
28
Position
Table 2 (cont.)
21.0, 170.4 51.7
13.8 17.4
208.0
d(C )
12
6-MeCO
3, 4, 5, 28 7, 8, 9, 14
4, 5, 29
HMBC
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with C(28), indicating the presence of 6,28-g-lactone ring structure. Further, the NOE correlations (Fig. 1) between HC(1), Me(19), Me(29), and HC(3), and between Me(19), HC(6), Me(30), HC(7), HbC(16), and HC(17) indicated the aorientations of TigO and OH groups at C(1) and C(3), respectively, the g-lactone ring at C(6)/C(28), and the furyl ring at C(17). Hence, the structure of 14 was assigned as 3-deacetyl-28-oxosalannic acid methyl ester ( ¼ 3-deacetyl-28-oxosalannin). Compound 17 gave a [M þ H] þ ion peak in the positive-ion-mode HR-ESI mass spectrum at m/z 531.2579 (C29H39O þ9 ; calc. 531.2594), consistent with the molecular formula C29H38O8 . The IR spectrum of 17 indicated the presence of OH (3360 cm 1), ester C¼O (1740 cm 1), and furyl (870 cm 1) moieties. The 13C-NMR and DEPT data of 17 (Table 2) indicated the presence of 29 C-atoms including eight quaternary Catoms (including two C¼O and three olefinic), twelve CH (including five O-bearing sp3, one O-bearing sp2, and three olefinic), three CH2 , and six Me groups. The 1H- and 13 C-NMR data (Table 2) of 17 were analogous to those of 19 [21], although the signals of the tigloyl group of ohchinolal were lacking for 17, suggesting that it possesses the structure of 1-detigloylohchinolal. The NOE interactions (Fig. 1) between HC(3), Me(29), Me(19), and HC(1), between Me(19), HC(6), HC(7), HbC(16), and HC(17), and between Me(19) and Me(30) supported the relative configurations proposed. This study has led to the isolation of 19 limonoids including two of vilasinin-type, 1 and 2, one of havanensin-type, 3, 13 of salannin-type, 4 – 16, and three of nimbin-type, 17 – 19, along with one euphane-type triterpenoid, 20, from the defatted fraction of the hexane extract of M. azedarch fruits (Table 3). Among the 19 limonoids, 15, i.e., 1, 2, 4, 6 – 13, 15, 16, 18, and 19, have recently been isolated also from the MeOH extract of M. azedarch fruits [17], and the euphane-type triterpenoid, 20, also from the fruits of M. azedarach [31]. Limonoid 3 has so far been isolated only from the fruits of M. toosendan [28]. Structural Features of the Limonoids of the Fruits and/or Seeds of Melia azedarach, M. toosendan, and Azadirachta indica. Limonoids are biosynthesized from euphane- or tricallane-type triterpenoids through protolimonoids as biogenetic intermediates [1 – 3]. The Scheme shows a basic structure for one compound from each type of representative limonoids found in Meliaceae, and their possible biogenetic interrelationships [1] [3]. This study has established the occurrence of vilasinin-, havanensin-, salannin-, and nimbin-type limonoids, of which ring C-seco-salannin-type limonoids predominated in the hexane extract of M. azedarch fruits. The structural features of the limonoid constituents of this extract were similar to those of the MeOH extract of M. azedarch fruits [17] [18], although the latter contained, in addition, toosendanin, a ringintact trichilin-type limonoid and one of the widely known limonoids [2], as the most predominant limonoid constituent, along with other trichilin-type and several nimbolinin-type limonoids [17]. Co-occurrence of trichilin- [8] [10] [13] [17] [18] [22] [24], vilasinin- [17], salannin- [10] [13] [17] [19 – 21] [23], nimbin- [9] [17], and nimbolinintype [9] [11] [13] [17] limonoids as constituents seems to be the general feature of M. azedarch fruits and/or seeds. M. toosendan has long been recognized as an insecticidal and medicinal plant in China, and its fruits are used for treatment of malaria and for stomachaches caused by roundworms [33]. Limonoids present in the fruits of M. toosendan are closely related to
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Table 3. Cytotoxic Activities (IC50 S.D. [mm]) of the Compounds Isolated from the Defatted Fration of the Hexane Extracts of Melia azedarach Fruits against Four Human Cancer Cells a ) b ) Compound Vilasinin-Type Limonoid 1 Trichilinin D c ) 2 1-Cinnamoyltrichilinin c ) Havanensin-Type Limonoid 3 3-Deacetyl-1,6-diacetylsendanal
HL 60 A549 (Leukemia) ( Lung) 61.2 7.8 > 100 5.8 0.8 > 100 3.6 1.0 > 100
Salannin-Type Limonoids 23.5 8.5 4 3-Deacetylsalannin c ) 5 3-Deacetyl-4’-demethylsalannin 9.6 1.2 6 Ohchinin c ) 56.3 5.1 7 Ohchinin acetate c ) 9.9 0.6 8 1-Decinnamoyl-1-benzoylohchinin c ) 54.8 2.2 9 Ohchininolide c ) 31.7 4.1 10 1-Decinnamoyl-1-benzoylohchininolide c ) 14.1 0.3 11 23-Methoxyohchininolide A c ) 4.9 0.5 12 23-Methoxyohchininolide B c ) 15.2 2.2 13 23-Hydroxyohchininolide c ) 25.1 3.2 14 3-Deacetyl-28-oxosalannin 39.1 3.6 15 1-Decinnamoyl-1-benzoyl-28-oxoohchinin c ) 22.7 2.4 16 3-Deacetyl-4’-demethyl-28-oxosalannin c ) 2.8 0.6 17 1-Detigloylohchinolal 5.0 0.9 18 Mesendanin E c ) > 100 19 Ohchinolal c ) 39.2 4.4
> 100 > 100 > 100 94.3 1.1 82.3 0.3 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 25.7 2.5 > 100 > 100
Euphane-Type Triterpenoid 20 Azedarachic acid
4.6 0.6 > 100
Cisplatin d )
4.2 1.1
18.4 1.9
AZ521 ( Stomach)
SK-BR-3 ( Breast)
> 100 41.5 3.2 16.2 1.7 > 100 22.0 1.5 > 100 42.1 4.2 47.5 3.6 > 100 > 100 35.1 3.9 82.9 1.2 34.7 5.8 > 100 30.0 6.3 78.5 2.9 > 100 61.7 7.1 3.2 0.6 7.3 1.3 81.2 1.2 > 100
30.6 5.7 > 100 > 100 35.2 3.6 14.9 2.1 > 100 54.5 4.1 > 100 > 100 > 100 > 100 > 100 > 100 76.5 5.8 77.8 7.2 94.4 1.0
21.5 3.8
49.7 4.3
9.5 0.5
18.8 0.6
a ) Cells were treated with compounds (1 10 4 – 1 10 6 m) for 48 h, and cell viability was analyzed by the MTT assay. b ) The IC50 values based on triplicate five points. c ) Data from [17]. d ) Reference compound.
those of M. azedarach fruits. Thus, M. toosendan fruits have been reported to contain toosendanin [34] [35] and other trichilin-type limonoids [28] [34 – 39], along with vilasinin- [35 – 38] [40], havanensin- [28] [40], salannin- [38] [41], nimbin- [38], and nimbolinin-type [28] [35 – 38] [40 – 44] limonoids, which is consistent with our recent [17] and present observation on the M. azedarach fruits. However, M. toosendan fruits can be discriminated from M. azedarach fruits by containing nimbolinin-type limonoids abundantly but salannin-type limonoids with lower abundance. Due to the importance as a valuable source of bioactive limonoids, the limonoid constituents of Azadirachta indica (neem tree) have been the subject of intensive research [1 – 3]. The fruits and/or seeds of A. indica contain azadirone- [45 – 51], vilasinin- [48] [51], azadirachtin- [48] [50] [52 – 55], salannin- [51] [53] [54] [56], nimbin[32] [47] [49 – 51] [53] [54] [57], and gedunin-type [45] [47] [49 – 51] [58] limonoids, among which azadirone- [50] [51] and salannin-type [51] limonoids predominate. Co-
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Scheme. A Possible Biogenetic Sequence and Classification of Meliaceous Limonoids. Reproduced from [3] with a minor modification.
occurrence of azadirone-, azadirachtin-, and gedunin-type limonoids, in addition to vilasinin-, salannin-, and nimbin-type limonoids, which also occur in M. azedarach and M. toosendan fruits and/or seeds (vide supra), and the apparent absence of trichilinand nimbolinin-type limonoids seem to be the characteristic feature of the limonoids of A. indica fruits and/or seeds. Limonoids of azadirone and gedunin types have been detected also in the seeds of A. indica var. siamensis (Siamese neem tree) [59]. Cytotoxic Activity. The cytotoxic activities of three new compounds, 5, 14, and 17, and of two known compounds, 3 and 20, along with the reference chemotherapeutic drug, cisplatin, were evaluated against the human cancer cell lines HL60 (leukemia), A549 (lung), AZ521 (stomach), and SK-BR-3 (breast) by the MTT assay, and the results are compiled in Table 3. The cytotoxicities of 15 known compounds, 1, 2, 4, 6 – 13, 15, 16, 18, and 19, evaluated in our recent study [17], were also included in Table 3 to
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interpret the cytotoxicities (Table 1) of the defatted fraction of the hexane extract. All compounds exhibited cytotoxicities against one or more cancer cell lines tested with IC50 values in the range of 2.8 – 94.4 mm. In particular, the cytotoxicities of 2, 3, 5, 7, 11, 16, 17, and 20 against HL60 (IC50 2.8 – 9.9 mm), of 17 against A549 (IC50 25.7 mm), of 16 and 17 against AZ521 (IC50 3.2 and 7.3 mm, resp.), and of 8 against SK-BR-3 (IC50 14.9 mm) were observed to be superior to, or almost comparable with, those of cisplatin (HL60: IC50 4.2 mm ; A549: IC50 18.4 mm ; AZ521: IC50 9.5 mm ; and SK-BR-3: IC50 18.8 mm). Based on these results, it is highly possible that the compounds exhibiting potent or moderate cytotoxicities are the cytotoxic principles of the defatted fraction of the hexane extract of M. azedarach fruits (Table 1). It might be worthy to note that demethacrylation of 18 and detigloylation of 19 at C(1), to afford compound 17, greatly enhance the cytotoxicities against HL60, A549, and AZ521 cells. The cell death of HL60 cells by compound 16 has recently been established to be, at least in part, due to apoptosis induced via both the mitochondrial and the death receptor-mediated pathways [17]. Apoptosis-Inducing Activity of Compound 16. Compound 16, which exhibited potent cytotoxic activitiy (IC50 3.2 mm) against AZ521 cells, was evaluated for its apoptosis-inducing activity using AZ521 cells. AZ521 Cells were incubated with the test compound for 24 and 48 h, and then they were analyzed by means of flow cytometry with annexin Vpropidium iodide (PI) double staining. Exposure of the membrane phospholipid phosphatidylserine to the external cellular environment is one of the earliest markers of apoptotic cell death [60]. Annexin V is a Ca-dependent phospholipid-binding protein with high affinity for phosphatidylserine expressed on the cell surface. PI does not enter whole cells with intact membranes and was used to differentiate between early apoptotic (annexin V, positive; PI, negative), late apoptotic (annexin V and PI, double positive), or necrotic (annexin V, negative; PI, positive) cell death. The ratio of early apoptotic cells (Fig. 2, lower right) increased after treatment with 16 in AZ521 cells for 24 h (from 2.7 to 28.8%) and 48 h (16.8%), and that of late apoptotic cells (upper right) increased after 48 h (from 1.1 to 41.0%). These results demonstrated that most of the cytotoxicity of compound 16 against AZ521 cells is due to inducing apoptotic cell death.
Fig. 2. Detection of 16-induced early and late apoptotic cells by annexin VPI double staining in AZ521 cells. The cells were cultured with 10 mm 16 for 24 and 48 h.
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Conclusions. – This study has established that the defatted fraction of the hexane extract of Azadirachta indica fruits exhibit cytotoxic activities against HL60, A549, A521, and SK-BR-3 human cancer cell lines. Investigation of the fraction has led to the isolation of 19 limonoids, 1 – 19, and one triterpenoid, 20, of which three, i.e., 3-deacetyl4’-demethylsalannin (5), 3-deacetyl-28-oxosalannin (14), and 1-detigloylohchinolal (17), were new compounds. These compounds exhibited potent cytotoxicities (2, 3, 5, 7, 11, 16, 17, and 20 against HL60; 17 against A549; 16 and 17 against AZ521; and 8 against SK-BR-3), suggesting that they might be, at least in part, responsible for the cytotoxicities of the defatted fraction of A. indica fruit hexane extract. Moreover, 3deacetyl-4’-demethyl-28-oxosalannin (16) was revealed to induce apoptotic cell death in AZ521 cells. Compounds isolated in this study seem to be responsible for the repellent activity of M. azedarach seed oil against sandflies [25], since the seeds are expected to contain limonoids and triterpenoids, similar to those occurring in the fruits, and since meliaceous limonoids exhibit insect antifeedant and insecticidal activities [1] [2]. This work has provided, furthermore, valuable information on the structural features of limonoids of the fruits and/or seeds of Melia azedarach and related Meliaceae plants, M. toosendan and Azadirachta indica. Experimental Part General. Column chromatography (CC): silica gel (SiO2 , 230 – 400 mesh; Merck) and octadecyl silica gel (ODS; Chromatorex-ODS, 100 – 200 mesh; Fuji Silysia Chemical, Ltd., Aichi, Japan). Prep. HPLC (with refractive-index detector): ODS columns (25 cm 10 mm i.d.) at 258 on a Pegasil ODS-II 5 mm column (Senshu Scientific Co., Ltd., Tokyo, Japan) with MeCN/H2O/AcOH (flow rate, 3.0 ml/min) 48 : 52 : 0.1 (system I), 50 : 50 : 0.1 (system II), or 55 : 45 : 0.1 (system III), on a Capcell pak AQ 5 mm column (Shiseido Co., Ltd., Tokyo, Japan) with MeCN/H2O/AcOH (flow rate, 2.0 ml/min) 50 : 50 : 0.1 (system IV), 52 : 48 : 0.1 (system V), or 55 : 45 : 0.1 (system VI), or on a TSK ODS-120A 5 mm column (Toso Co., Tokyo, Japan) with MeOH/H2O/AcOH (flow rate, 3.0 ml/min) 60 : 40 : 0.1 (system VII) or with MeCN/H2O/HCOOH (flow rate, 3.0 ml/min) 70 : 30 : 0.1 (system VIII) as mobile phase. Optical rotations: JASCO P-1020 digital polarimeter. UV Spectra: JASCO V-630Bio spectrophotometer; lmax (log e) in nm. IR Spectra: JASCO FTIR-300E spectrometer; ˜n in cm 1. 1D- and 2D-NMR spectra: JEOL ECX-400 (at 400 (1H) and 100 MHz (13C)) spectrometer at r.t.; CDCl3 soln.; d in ppm rel. to Me4Si as internal standard, J in Hz. HR-ESI-MS: Agilent 1100 LC/MSD TOF (time-of-flight) system (ionization mode, positive; cap. voltage, 3000 V; fragmentor voltage, 225 V); m/z. Microplate reader: Sunrise-Basic (Tecan Japan Co., Ltd., Kawasaki, Japan). Flow cytometer: Cell Lab QuantaTM SC (Beckman Coulter K. K., Tokyo, Japan). Materials and Chemicals. Mature fruits of M. azedarach L. (Meliaceae) were collected from plants cultivated at the Toho University herbal garden (Funabashi-shi, Chiba, Japan) on 31st January, 2008. The plant material was authenticated by one (K. K.) of the authors, and a voucher specimen (TA-TH-MA0801) was deposited with the Herbarium of the School of Pharmaceutical Sciences, Toho University. The chemicals were purchased as follows: RPMI-1640 medium, fetal bovine serum (FBS; for RPMI-1640 medium), antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin), and non-essential amino acid (NEAA) from Invitrogen Co. (Carlsbad, CA, USA); Dulbeccos modified Eagles medium (DMEM), Eagles minimal essential medium (MEM), and MTT from SigmaAldrich Japan Co. (Tokyo, Japan); cisplatin from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); and recombinant human (rh) Annexin V/FITC kit (Bender MedSystems GmbH, A-Vienna) from Cosmo Bio Co., Ltd. (Tokyo, Japan). All other chemicals and reagents were of anal. grade. Extraction and Isolation. Air-dried fruits of M. azedarachi (19.7 kg) were pulverized and extracted with hexane (2 l/kg of dried fruits; 3 h, reflux, 3 ), and the residue was then extracted with MeOH [17]. The MeOH extract (1036 g) has recently been investigated for its limonoid constituents and their
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cytotoxicities [17] [18]. In this study, the hexane extract (588 g) was subjected to CC (SiO2 (500 g/40 g extract)) to give a less-polar oily fraction (437 g; hexane/AcOEt 1 : 0 to 4 : 1) and more-polar defatted fraction (52 g; AcOEt/MeOH 1 : 0 to 0 : 1). The defatted fraction was applied to CC (SiO2 (600 g); hexane/AcOEt 1 : 0 to 0 : 1) to afford nine fractions, Frs. A – I. Fr. D (2.82 g) from the eluate of hexane/ AcOEt 11 : 9 was subjected to CC (SiO2 (150 g); hexane/AcOEt 9 : 1 to 0 : 1) to yield eight fractions, Frs. D1 – D8. Further CC (ODS (13 g); MeOH/H2O 2 : 3 to 1 : 0) and subsequent prep. HPLC (system VIII) of Fr. D2 (242 mg) gave compound 20 (retention time (tR ) 63.0 min; 5.0 mg). Fr. D3 (1031 mg), upon CC (ODS (50 g); MeOH/H2O 1 : 1 to 1 : 0) and subsequent prep. HPLC (system III), yielded compounds 19 (tR 32.8 min; 1.9 mg) and 18 (tR 38.4 min; 2.1 mg). Fr. F (2.23 g) eluted with hexane/ AcOEt 3 : 7 was applied to CC (SiO2 (110 g); hexane/AcOEt 1 : 1 to 0 : 1) to furnish nine fractions, Frs. F1 – F9. CC (ODS (27 g); MeOH/H2O 2 : 3 to 1 : 0) of Fr. F4 (1038 mg) afforded seven fractions, Fr. F4-1 – F4-7. Prep. HPLC (system I) of Frs. F4-4 (318 mg) and F4-6 (188 mg) gave compounds 14 (tR 27.2 min; 60.2 mg), 16 (tR 24.0 min; 8.6 mg), and 15 (tR 30.4 min; 11.9 mg), and compound 7 (tR 52.0 min; 9.6 mg), resp. Prep. HPLC (system II) of Fr. F6 (207 mg) yielded compounds 3 (tR 16.8 min; 7.0 mg) and 17 (tR 20.0 min; 3.5 mg). Fr. G (2.53 g) eluted with hexane/AcOEt 1 : 4 was subjected to CC (ODS (57 g); MeOH/H2O 2 : 3 to 1 : 0) which furnished ten fractions, Fr. G1 – G10. Prep. HPLC of Frs. G3 (50 mg; system IV), G5 (138 mg; system V), G7 (100 mg; system V), and G9 (35 mg; system VI) yielded compound 10 (tR 23.2 min; 1.8 mg), compounds 4 (tR 32.0 min; 27.6 mg) and 8 (tR 40.0 min; 21.1 mg), compounds 5 (tR 44.0 min; 3.0 mg) and 6 (tR 56.0 min; 24.7 mg), and compound 2 (tR 54.4 min; 1.9 mg), resp. Fr. H (2.27 g) from the eluate of hexane/AcOEt 1 : 4 was subjected to CC (SiO2 (156 g); hexane/ AcOEt 1 : 0 to 0 : 1) to yield five fractions, Frs. H1 – H5. Further CC (ODS (57 g); MeOH/H2O 4 : 5 to 1 : 0) and subsequent prep. HPLC (system V) of Fr. H3 (1437 mg) gave compounds 13 (tR 20.8 min; 6.3 mg) and 9 (tR 28.0 min; 6.3 mg). Fr. I (1.33 g) eluted with hexane/AcOEt 1 : 9 was subjected to CC (ODS (40 g); MeOH/H2O 2 : 3 to 1 : 0) to yield nine fractions, Fr. I1 – I9. Prep. HPLC of Frs. I6 (116 mg; system VII) and I7 (99 mg; system II) afforded compounds 11 (tR 36.0 min; 3.0 mg) and 12 (tR 31.2 min; 22.2 mg), and compound 1 (tR 34.4 min; 15.4 mg), resp. Data of the New Compounds. 3-Deacetyl-4’-demethylsalannin ( ¼ (2aR,3R,5S,5aR,6R,6aR,8R,9aR,10aS,10bR,10cR)-8-(Furan-3-yl)-2a,4,5,5a,6,6a,8,9,9a,10a,10b,10c-dodecahydro-3-hydroxy-6-(2methoxy-2-oxoethyl)-2a,5a,6a,7-tetramethyl-2H,3H-cyclopenta[d]naphtho[2,3-b:1,8-b’c’]difuran-5-yl 2Methylprop-2-enoate; 5). Amorphous solid. [a] 25 D ¼ þ 49.1 (c ¼ 0.57, EtOH). UV (EtOH): No absorption maximum above 210 nm. IR (KBr): 3367 (OH), 1730, 1713 (C¼O), 873 (furan). 1H- and 13C-NMR: see Table 2. HR-ESI-MS: 563.2618 ([M þ Na] þ , C31H40NaO þ8 ; calc. 563.2621). 3-Deacetyl-28-oxosalannin ( ¼ (2aS,3R,5S,5aR,6R,6aR,8R,9aR,10aS,10bR,10cR)-8-(Furan-3-yl)2a,4,5,5a,6,6a,8,9,9a,10a,10b,10c-dodecahydro-3-hydroxy-6-(2-methoxy-2-oxoethyl)-2a,5a,6a,7-tetramethyl-2-oxo-2H,3H-cyclopenta[d]naphtho[2,3-b:1,8-b’c’]difuran-5-yl (2E)-2-Methylbut-2-enoate; 14). Fine needles. M.p. 200 – 2038. [a] 24 D ¼ þ 50.7 (c ¼ 0.85, MeOH). UV (EtOH): No absorption maximum above 210 nm. IR (KBr): 3338 (OH), 1778 (g-lactone) 1731, 1718 (C¼O), 860 (furan). 1H- and 13C-NMR: see Table 2. HR-ESI-MS: 569.2777 ([M þ H] þ , C32H41O þ9 ; calc. 569.2750). 1-Detigloylohchinolal ( ¼ Methyl [(2R,3aR,4aS,5R,5aR,6S,7R,9S,9aR,10R,10aR)-5-(Acetyloxy)-6formyl-2-(furan-3-yl)-3,3a,4a,5,5a,6,7,8,9,9a,10,10a-dodecahydro-7,9-dihydroxy-1,6,9a,10a-tetramethyl2H-cyclopenta[b]naphtho[2,3-d]furan-10-yl]acetate; 17). Fine needles. M.p. 129 – 1328. [a] 24 D ¼ þ 84.5 (c ¼ 1.01, MeOH). UV (EtOH): 220 (4.17). IR (KBr): 3360 (OH), 1740 (C¼O), 870 (furan). 1H- and 13 C-NMR: see Table 2. HR-ESI-MS: 531.2579 ([M þ H] þ , C29H39O þ9 ; calc. 531.2594). Cell Lines and Culture Conditions. Cell lines HL60 (leukemia), AZ521 (stomach), A549 (lung), and SK-BR-3 (breast) were obtained from Riken Cell Bank (Tsukuba, Ibaraki, Japan). They were grown in the following media: HL60 and SK-BR-3 in RPMI-1640 medium, AZ521 in DMEM medium, and A549 in 90% DMEM þ 10% MEM þ 0.1 mm NEAA. The medium was supplemented with 10% FBS and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin). Cells were incubated at 378 in a 5% CO2 humidified incubator and cultured as described in [61 – 64]. Determination of Cell Proliferation. Cell proliferation was assessed using the MTT ( ¼ 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)-based colorimetric assay as described in [61 – 64].
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Annexin VPropidium Iodide (PI) Double Staining. Annexin VPI double staining was performed as described in [61] [65] [66] with AZ521 (1.5 105 cells/5 ml) cells.
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Three New and Other Limonoids from the Hexane Extract of Melia azedarach Fruits and Their Cytotoxic Activities by Xin Pan a ), Masahiro Matsumoto a ), Yasuhiro Nakamura a ), Takashi Kikuchi a ), Jie Zhang a ), Motohiko Ukiya a ), Takashi Suzuki b ), Kazuo Koike c ), Rima Akihisa d ), and Toshihiro Akihisa* a ) d ) a
) College of Science and Technology, Nihon University, 1-8-14 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan b ) School of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi-shi, Chiba 274-8510, Japan c ) School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan d ) Akihisa Medical Clinic, 1086-3 Kamo, Sanda-shi, Hyogo 669-1311, Japan (fax: þ 81-79-5671980; e-mail: [email protected])
A defatted fraction obtained from the hexane extract of the fruits of Melia azedarach L. (chinaberry tree; Meliaceae) exhibited cytotoxic activities against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines with IC50 values in the range of 2.9 – 21.9 mg/ml. Three new limonoids, 3-deacetyl-4’-demethylsalannin (5), 3-deacetyl-28-oxosalannin (14), and 1-detigloylohchinolal (17), along with 16 known limonoids, 1 – 4, 6 – 13, 15, 16, 18, and 19, and one known triterpenoid, 20, were isolated from the fraction. The structures of new compounds were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. These compounds were evaluated for their cytotoxic activities against the four cancer cell lines mentioned above. 3-Deacetyl-4’-demethyl-28oxosalannin (16), which exhibited potent cytotoxicity against AZ521 (IC50 3.2 mm) cells, induced typical apoptotic cell death in AZ521 cells upon evaluation of the apoptosis-inducing activity by flow cytometry. This work provided, furthermore, valuable information on the structural features of limonoids of the fruits and/or seeds of Melia azedarach and related Meliaceae plants, M. toosendan and Azadirachta indica.
Introduction. – Plants of the Meliaceae family have been well-documented for their ability to metabolize structurally diverse and biologically significant limonoids and triterpenoids [1 – 3]. Melia azedarach L. (chinaberry tree; Meliaceae) is indigenous to Japan, Taiwan, China, and Southeast Asia. Its bark and fruits have been used for vermicide, anodyne, and skin disease [4] [5]. Many constituents including limonoids, triterpenoids, and steroids have been isolated from various parts of M. azedarach [1 – 3] [6 – 24]. The limonoids of M. azedarach have been reported to exhibit antimicrobial [13] [14], cytotoxic [9 – 12] [15], antifeedant [7] [8] [16] [24], insecticidal [16], and antiviral [22] activities. In the course of a search for potential bioactive compounds from Meliaceae plants, we were interested to investigate the limonoid constituents of M. azedarach fruits. Thus, investigation on the MeOH extract, obtained by MeOH extraction after hexane extraction of M. azedarch fruits, has led to the isolation of 32 limonoids and one triterpenoid, and some of which have been shown to exhibit potent cytotoxic and apoptosis-inducing activities against human cancer cell lines [17] [18]. In continuing a study on the constituents of M. azedarach fruits, the hexane extract of the 2014 Verlag Helvetica Chimica Acta AG, Zrich
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fruits was investigated in this work, resulting in the isolation of 19 limonoids, 1 – 19, including three new compounds, 5, 14, and 17 (see below), and one triterpenoid, 20. Herein, we describe the structure elucidation of these compounds and evaluation of their cytotoxic activities against four human cancer cell lines. The seed oil of M. azedarach, as well as the seed oil of Azadirachta indica A. Juss (neem tree), has been reported to exhibit repellent activity against sandflies (Phlebotomus orientalis and P. bergeroti) in Ethiopia [25].
Results and Discussions. – Cytotoxic Activities of the Hexane Extracts of M. azedarach Fruits. Dried and powdered M. azedarach fruits were extracted with hexane, and the residue was then extracted with MeOH [17] [18]. The hexane extract was passed through a SiO2 column in order to remove bulky less-polar oily substance from
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the extract. The hexane extract and the defatted more-polar fraction were evaluated for their cytotoxic activities against four human cancer cell lines, HL60 (leukemia), A549 (lung), AZ521 (stomach), and SK-BR-3 (breast), by means of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, and the results are compiled in Table 1. While the hexane extract was inactive against all cell lines tested, the defatted fraction exhibited cytotoxic activities against all cell lines with IC50 values in the range of 2.9 – 21.9 mg/ml. The defatted fraction was further investigated in this study. Table 1. Cytotoxic Activities of Melia azedarach Fruit Extracts against Four Human Cancer Cell Lines a ) Extract or fraction
Hexane extract Defatted fraction Cisplatin c )
Cytotoxicity ( IC50 S. D. [mg/ml] b )) HL60 ( Leukemia)
A549 ( Lung)
AZ521 (Stomach)
SK-BR-3 ( Breast)
75.5 1.7 2.9 2.4 1.3 0.3
> 100 12.0 0.7 5.5 0.6
77.0 1.1 12.3 0.9 2.9 0.2
> 100 21.9 0.6 5.6 0.2
a ) Cells were treated with test samples (1 10 4 – 1 10 6 g/ml) for 48 h, and cell viability was analyzed by MTT assay. b ) The IC50 values based on triplicate five points. c ) Reference compound.
Isolation, Identification, and Structure Elucidation of Compounds. The defatted fraction of the hexane extract was subjected to column chromatography (CC) over SiO2 and octadecyl silica gel (ODS), and reversed-phase (RP) preparative high-performance liquid chromatography (HPLC), which led to the isolation of 19 limonoids, 1 – 19, including three new limonoids, 5, 14, and 17, and one known triterpenoid, 20. The 17 known compounds were identified as trichilinin D (1) [26], 1-cinnamoyltrichilinin (2) [27], 3a,7a-dihydroxy-21,23-epoxy-1a,6a,12a-triacetoxy-24,25,26,27-tetranorapotirucalla-14,20,22-trien-28-al (3; 3-deacetyl-1,6-diacetylsendanal) [28], 3-deacetylsalannin (4) [29], ohchinin (6) [21], ohchinin acetate (7) [19], 1-decinnamoyl-1benzoylohchinin (8) [17], ohchininolide (9) [17], 1-decinnamoyl-1-benzoylohchininolide (10) [17], 23-methoxyohchininolide A (11) [17], 23-methoxyohchininolide B (12) [17], 23-hydroxyohchininolide (13) [17], 1-decinnamoyl-1-benzoyl-28-oxoohchinin (15) [17], 3-deacetyl-4’-demethyl-28-oxosalannin (16) [17], mesendanin E (18) [30], ohchinolal (19) [21], and azedarachic acid (20) [31] by comparison of their spectral data with those in the literature. The structures of three new limonoids, 5, 14, and 17, were elucidated on the basis of spectroscopic analysis and comparison with literature data as described below. Compound 5 was designated the molecular formula C31H40O8 by its positive-ion mode HR-ESI mass spectrum (m/z 563.2618 ([M þ Na] þ , C31H40NaO þ8 ; calc. 563.2620)). The IR spectrum of 5 showed the absorption bands of OH (3367 cm 1), ester and conjugated ester C¼O (1730 and 1713 cm 1, resp.), and furyl (873 cm 1) groups. The 13C-NMR and DEPT data of 5 (Table 2) indicated the presence of 31 Catoms including nine quaternary C-atoms (including four olefinic and two C¼O Catoms), eleven CH (including five O-bearing sp3 and three olefinic), five CH2 (including one O-bearing and one sp2 ), and six Me (including one O-bearing) groups. The 1H- and 13C-NMR data (Table 2) of 5 were closely similar to those of 4 [29], except
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for the presence of the 2-methylprop-2-enoyl (methacryl; Met) group (d(H) 2.05 (Me(4’)), and 5.65 (t, J ¼ 3.0) and 6.16 (t, J ¼ 1.4) (CH2(3’)); d(C) 18.4 (C(4’)), 126.2 (C(3’)), 136.2 (C(2’)), and 165.0 (C(1’))) [17] instead of the tigloyl group at C(1) of 4. The HMBCs (Table 2) of HC(1) (d(H) 5.04) with C(1’) supported the presence of the Met group at C(1). In addition, the NOE interactions (Fig. 1) between HC(3), Me(29), Me(19), and HC(1), and between Me(19), HC(6), HC(7), HbC(16), and HC(17) indicated the a-orientations of MetO, OH, and the furyl groups at C(1), C(3), and C(17), respectively. Thus, the structure of 5 was elucidated as 1-methacrylsalannic acid methyl ester (¼ 3-deacetyl-4’-demethylsalannin).
Fig. 1. Major NOE correlations ( $ ) for compounds 5, 14, and 17. Drawings correspond to energyminimized conformation of compounds. Calculation was performed using CAChe Conformation Search with the MM2 force field (CAChe version 6.01; Fujitsu Co., Tokyo, Japan).
Compound 14 had the molecular formula C32H40O9 , as indicated by positive-ionmode HR-ESI-MS (m/z 569.2777 ([M þ H] þ , C32H41O þ9 ; calc. 569.2750)). Its IR spectrum suggested the presence of OH (3338 cm 1), g-lactone (1778 cm 1), ester and conjugated ester C¼O (1731 and 1718 cm 1, resp.), and furyl (860 cm 1) moieties. The 13 C-NMR data of 14 (Table 2), along with the DEPT data, indicated the presence of 32 C-atoms including ten quaternary C-atoms (including three C¼O and two olefinic Catoms), twelve CH (including five O-bearing sp3 and four olefinic), three CH2 , and seven Me groups. The 1H- and 13C-NMR data (Table 2) of 14 were analogous to those of 4, with obvious differences being the lack of O-bearing CH2 group and the presence of a C¼O group (d(C) 177.5) at C(28) in 14 when compared with 4. This suggested the presence of a 6,28-g-lactone ring in 14 like in nimbolide [32]. In the HMBC (Table 2) experiment, 14 exhibited cross-peaks of HC(5) (d(H) 2.39) and Me(29) (d(H) 1.25)
18 19 20 21 22 23
17
12 13 14 15 16
10 11
6 7 8 9
3 4 5
1 2
Position
7.24 (br. s) 6.26 (dd, J ¼ 1.0, 2.0) 7.31 (t, J ¼ 1.8)
1.65 (d, J ¼ 1.4) 0.96 (s)
5.39 (br. t, J ¼ 7.6) 2.23 – 2.27 (m, Ha ), 2.08 – 2.14 (m, Hb ) 3.61 (br. d, J ¼ 7.2)
2.00 – 2.18 (m) 2.24 – 2.30 (m)
2.64 (dd, J ¼ 3.0, 9.4)
4.02 (dd, J ¼ 3.2, 12.4) 4.18 (d, J ¼ 3.7)
2.72 (d, J ¼ 12.8)
13.1 15.3 127.1 138.8 110.7 142.9
49.3
172.7 135.0 146.4 87.9 41.2
40.8 30.5
72.5 85.7 49.0 39.5
70.8 44.2 38.8
5.04 (d, J ¼ 8.0) 2.11 – 2.18 (m, Ha ), 2.30 – 2.37 (m, Hb ) 3.87 – 3.91 (m)
20, 22, 23 20, 21, 23 20, 21, 22
13, 14 15, 17, 20 13, 14, 20 13, 14, 15, 16, 20, 21, 22 13, 14, 17 1, 5, 9, 10
8, 12 8, 12
10, 11, 12, 19, 30
3, 4, 6, 7, 10, 19, 28 5, 7 5, 6, 8, 9
1
7.25 (s) 6.28 (br. s) 7.33 (br. s)
1.68 (s) 1.03 (s)
5.45 (t, J ¼ 7.6) 2.20 – 2.26 (m, Ha ), 2.06 – 2.16 (m, Hb ) 3.65 (d, J ¼ 8.7)
2.13 – 2.22 (m) 2.26 – 2.35 (m)
2.83 (dd, J ¼ 3.9, 8.9)
4.52 (dd, J ¼ 3.4, 12.6) 4.25 (d, J ¼ 3.7)
3.39 (d, J ¼ 12.4)
4.88 (d, J ¼ 3.0) 2.14 – 2.22 (m, Ha ), 2.25 – 2.32 (m, Hb ) 4.13 (dd, J ¼ 4.3, 6.2)
73.1 30.0
d( H ) 3, 5, 1’ 10
14 d(H )
d(C )
5 HMBC
13.0 15.5 126.8 138.8 110.5 143.0
49.4
172.4 135.9 145.5 88.4 41.0
40.3 30.8
74.3 83.1 47.7 38.7
68.9 48.9 37.4
71.3 29.4
d(C )
22, 23 20, 21, 23 20, 21, 22
7, 13, 14 13, 15, 17, 20 13, 14, 15, 17, 20 13, 14, 15, 16, 20, 21, 22 13, 14, 17 1, 5, 9, 10
9, 10, 12 9, 10, 12
8, 10, 11, 12, 14, 19, 30
3, 4, 6, 7, 10, 19, 28, 29 7 5, 6, 9, 30
1
3, 5, 1’ 4, 10
HMBC
7.12 (s) 6.11 (s) 7.31 (t, J ¼ 1.2)
1.71 (d, J ¼ 1.3) 0.98 (s)
5.50 (br. t, J ¼ 7.6) 2.25 (dd, J ¼ 6.4, 1.4, Ha ), 2.00 – 2.08 (m, Hb ) 3.64 (br. d, J ¼ 7.6)
2.35 (dd, J ¼ 4.1, 17.4) 2.46 (dd, J ¼ 5.5, 17.4)
2.68 (br. t, J ¼ 4.6)
5.22 (dd, J ¼ 3.2, 12.3) 4.11 (d, J ¼ 2.7)
12.9 16.3 126.8 138.6 110.9 143.3
49.6
177.2 135.4 146.4 87.5 41.5
42.9 30.9
69.0 86.3 47.4 40.1
76.2 49.4 34.0
3.72 (br. t, J ¼ 8.0) 3.60 (d, J ¼ 12.4)
72.5 29.0
d(C )
3.66 – 3.68 (m) 1.97 – 2.10 (m, 2 H )
d( H )
17
14 14, 15, 20 15, 20 15, 18, 20, 22
20, 22, 23 20, 21, 23 20, 21, 22
13, 14 1, 5, 9, 10
13, 13, 13, 13,
8, 10, 12 8, 10, 12
30
6-MeCO 5, 6, 9, 30
4, 10, 19, 28, 29
1
3 10
HMBC
Table 2. 1H- and 13C-NMR (400 and 100 MHz, resp.), and HMBC (H ! C) Data of Compounds 5, 14, and 17 (recorded in CDCl3 ; d in ppm, J in Hz)
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51.5
12
3.22 (s)
51.5
12.0 14.5
16.6 15.6 166.7 128.8 137.9
12
2’, 3’, 5’ 1’, 2’, 3’
1’, 2’, 4’, 5’
3, 4, 5, 28 7, 8, 9, 14
17
0.93 (s) 1.36 (s)
9.68 (s)
d( H )
3.58 (s)
3.21 (s)
1’, 2’, 3’
6.95 (dd, J ¼ 6.0, 13.6) 1.82 (d, J ¼ 6.9) 1.90 (s)
1.25 (s) 1.36 (s)
HMBC
12-MeO
18.4
1’, 2’, 4’
3, 4, 5, 28 7, 8, 9, 14
177.5
d(C )
1.95 (s)
5.65 (t, J ¼ 3.0), 6.16 (t, J ¼ 1.4) 2.05 (s)
19.8 16.9 165.0 136.2 126.2
77.8
3.64 (d, J ¼ 7.3), 4.13 (d, J ¼ 7.3) 1.16 (s) 1.31 (s) 3, 4, 29
14 d(H )
d( H ) HMBC
d(C )
5
4’ 5’ 6-AcO
29 30 1’ 2’ 3’
28
Position
Table 2 (cont.)
21.0, 170.4 51.7
13.8 17.4
208.0
d(C )
12
6-MeCO
3, 4, 5, 28 7, 8, 9, 14
4, 5, 29
HMBC
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with C(28), indicating the presence of 6,28-g-lactone ring structure. Further, the NOE correlations (Fig. 1) between HC(1), Me(19), Me(29), and HC(3), and between Me(19), HC(6), Me(30), HC(7), HbC(16), and HC(17) indicated the aorientations of TigO and OH groups at C(1) and C(3), respectively, the g-lactone ring at C(6)/C(28), and the furyl ring at C(17). Hence, the structure of 14 was assigned as 3-deacetyl-28-oxosalannic acid methyl ester ( ¼ 3-deacetyl-28-oxosalannin). Compound 17 gave a [M þ H] þ ion peak in the positive-ion-mode HR-ESI mass spectrum at m/z 531.2579 (C29H39O þ9 ; calc. 531.2594), consistent with the molecular formula C29H38O8 . The IR spectrum of 17 indicated the presence of OH (3360 cm 1), ester C¼O (1740 cm 1), and furyl (870 cm 1) moieties. The 13C-NMR and DEPT data of 17 (Table 2) indicated the presence of 29 C-atoms including eight quaternary Catoms (including two C¼O and three olefinic), twelve CH (including five O-bearing sp3, one O-bearing sp2, and three olefinic), three CH2 , and six Me groups. The 1H- and 13 C-NMR data (Table 2) of 17 were analogous to those of 19 [21], although the signals of the tigloyl group of ohchinolal were lacking for 17, suggesting that it possesses the structure of 1-detigloylohchinolal. The NOE interactions (Fig. 1) between HC(3), Me(29), Me(19), and HC(1), between Me(19), HC(6), HC(7), HbC(16), and HC(17), and between Me(19) and Me(30) supported the relative configurations proposed. This study has led to the isolation of 19 limonoids including two of vilasinin-type, 1 and 2, one of havanensin-type, 3, 13 of salannin-type, 4 – 16, and three of nimbin-type, 17 – 19, along with one euphane-type triterpenoid, 20, from the defatted fraction of the hexane extract of M. azedarch fruits (Table 3). Among the 19 limonoids, 15, i.e., 1, 2, 4, 6 – 13, 15, 16, 18, and 19, have recently been isolated also from the MeOH extract of M. azedarch fruits [17], and the euphane-type triterpenoid, 20, also from the fruits of M. azedarach [31]. Limonoid 3 has so far been isolated only from the fruits of M. toosendan [28]. Structural Features of the Limonoids of the Fruits and/or Seeds of Melia azedarach, M. toosendan, and Azadirachta indica. Limonoids are biosynthesized from euphane- or tricallane-type triterpenoids through protolimonoids as biogenetic intermediates [1 – 3]. The Scheme shows a basic structure for one compound from each type of representative limonoids found in Meliaceae, and their possible biogenetic interrelationships [1] [3]. This study has established the occurrence of vilasinin-, havanensin-, salannin-, and nimbin-type limonoids, of which ring C-seco-salannin-type limonoids predominated in the hexane extract of M. azedarch fruits. The structural features of the limonoid constituents of this extract were similar to those of the MeOH extract of M. azedarch fruits [17] [18], although the latter contained, in addition, toosendanin, a ringintact trichilin-type limonoid and one of the widely known limonoids [2], as the most predominant limonoid constituent, along with other trichilin-type and several nimbolinin-type limonoids [17]. Co-occurrence of trichilin- [8] [10] [13] [17] [18] [22] [24], vilasinin- [17], salannin- [10] [13] [17] [19 – 21] [23], nimbin- [9] [17], and nimbolinintype [9] [11] [13] [17] limonoids as constituents seems to be the general feature of M. azedarch fruits and/or seeds. M. toosendan has long been recognized as an insecticidal and medicinal plant in China, and its fruits are used for treatment of malaria and for stomachaches caused by roundworms [33]. Limonoids present in the fruits of M. toosendan are closely related to
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Table 3. Cytotoxic Activities (IC50 S.D. [mm]) of the Compounds Isolated from the Defatted Fration of the Hexane Extracts of Melia azedarach Fruits against Four Human Cancer Cells a ) b ) Compound Vilasinin-Type Limonoid 1 Trichilinin D c ) 2 1-Cinnamoyltrichilinin c ) Havanensin-Type Limonoid 3 3-Deacetyl-1,6-diacetylsendanal
HL 60 A549 (Leukemia) ( Lung) 61.2 7.8 > 100 5.8 0.8 > 100 3.6 1.0 > 100
Salannin-Type Limonoids 23.5 8.5 4 3-Deacetylsalannin c ) 5 3-Deacetyl-4’-demethylsalannin 9.6 1.2 6 Ohchinin c ) 56.3 5.1 7 Ohchinin acetate c ) 9.9 0.6 8 1-Decinnamoyl-1-benzoylohchinin c ) 54.8 2.2 9 Ohchininolide c ) 31.7 4.1 10 1-Decinnamoyl-1-benzoylohchininolide c ) 14.1 0.3 11 23-Methoxyohchininolide A c ) 4.9 0.5 12 23-Methoxyohchininolide B c ) 15.2 2.2 13 23-Hydroxyohchininolide c ) 25.1 3.2 14 3-Deacetyl-28-oxosalannin 39.1 3.6 15 1-Decinnamoyl-1-benzoyl-28-oxoohchinin c ) 22.7 2.4 16 3-Deacetyl-4’-demethyl-28-oxosalannin c ) 2.8 0.6 17 1-Detigloylohchinolal 5.0 0.9 18 Mesendanin E c ) > 100 19 Ohchinolal c ) 39.2 4.4
> 100 > 100 > 100 94.3 1.1 82.3 0.3 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 25.7 2.5 > 100 > 100
Euphane-Type Triterpenoid 20 Azedarachic acid
4.6 0.6 > 100
Cisplatin d )
4.2 1.1
18.4 1.9
AZ521 ( Stomach)
SK-BR-3 ( Breast)
> 100 41.5 3.2 16.2 1.7 > 100 22.0 1.5 > 100 42.1 4.2 47.5 3.6 > 100 > 100 35.1 3.9 82.9 1.2 34.7 5.8 > 100 30.0 6.3 78.5 2.9 > 100 61.7 7.1 3.2 0.6 7.3 1.3 81.2 1.2 > 100
30.6 5.7 > 100 > 100 35.2 3.6 14.9 2.1 > 100 54.5 4.1 > 100 > 100 > 100 > 100 > 100 > 100 76.5 5.8 77.8 7.2 94.4 1.0
21.5 3.8
49.7 4.3
9.5 0.5
18.8 0.6
a ) Cells were treated with compounds (1 10 4 – 1 10 6 m) for 48 h, and cell viability was analyzed by the MTT assay. b ) The IC50 values based on triplicate five points. c ) Data from [17]. d ) Reference compound.
those of M. azedarach fruits. Thus, M. toosendan fruits have been reported to contain toosendanin [34] [35] and other trichilin-type limonoids [28] [34 – 39], along with vilasinin- [35 – 38] [40], havanensin- [28] [40], salannin- [38] [41], nimbin- [38], and nimbolinin-type [28] [35 – 38] [40 – 44] limonoids, which is consistent with our recent [17] and present observation on the M. azedarach fruits. However, M. toosendan fruits can be discriminated from M. azedarach fruits by containing nimbolinin-type limonoids abundantly but salannin-type limonoids with lower abundance. Due to the importance as a valuable source of bioactive limonoids, the limonoid constituents of Azadirachta indica (neem tree) have been the subject of intensive research [1 – 3]. The fruits and/or seeds of A. indica contain azadirone- [45 – 51], vilasinin- [48] [51], azadirachtin- [48] [50] [52 – 55], salannin- [51] [53] [54] [56], nimbin[32] [47] [49 – 51] [53] [54] [57], and gedunin-type [45] [47] [49 – 51] [58] limonoids, among which azadirone- [50] [51] and salannin-type [51] limonoids predominate. Co-
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Scheme. A Possible Biogenetic Sequence and Classification of Meliaceous Limonoids. Reproduced from [3] with a minor modification.
occurrence of azadirone-, azadirachtin-, and gedunin-type limonoids, in addition to vilasinin-, salannin-, and nimbin-type limonoids, which also occur in M. azedarach and M. toosendan fruits and/or seeds (vide supra), and the apparent absence of trichilinand nimbolinin-type limonoids seem to be the characteristic feature of the limonoids of A. indica fruits and/or seeds. Limonoids of azadirone and gedunin types have been detected also in the seeds of A. indica var. siamensis (Siamese neem tree) [59]. Cytotoxic Activity. The cytotoxic activities of three new compounds, 5, 14, and 17, and of two known compounds, 3 and 20, along with the reference chemotherapeutic drug, cisplatin, were evaluated against the human cancer cell lines HL60 (leukemia), A549 (lung), AZ521 (stomach), and SK-BR-3 (breast) by the MTT assay, and the results are compiled in Table 3. The cytotoxicities of 15 known compounds, 1, 2, 4, 6 – 13, 15, 16, 18, and 19, evaluated in our recent study [17], were also included in Table 3 to
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interpret the cytotoxicities (Table 1) of the defatted fraction of the hexane extract. All compounds exhibited cytotoxicities against one or more cancer cell lines tested with IC50 values in the range of 2.8 – 94.4 mm. In particular, the cytotoxicities of 2, 3, 5, 7, 11, 16, 17, and 20 against HL60 (IC50 2.8 – 9.9 mm), of 17 against A549 (IC50 25.7 mm), of 16 and 17 against AZ521 (IC50 3.2 and 7.3 mm, resp.), and of 8 against SK-BR-3 (IC50 14.9 mm) were observed to be superior to, or almost comparable with, those of cisplatin (HL60: IC50 4.2 mm ; A549: IC50 18.4 mm ; AZ521: IC50 9.5 mm ; and SK-BR-3: IC50 18.8 mm). Based on these results, it is highly possible that the compounds exhibiting potent or moderate cytotoxicities are the cytotoxic principles of the defatted fraction of the hexane extract of M. azedarach fruits (Table 1). It might be worthy to note that demethacrylation of 18 and detigloylation of 19 at C(1), to afford compound 17, greatly enhance the cytotoxicities against HL60, A549, and AZ521 cells. The cell death of HL60 cells by compound 16 has recently been established to be, at least in part, due to apoptosis induced via both the mitochondrial and the death receptor-mediated pathways [17]. Apoptosis-Inducing Activity of Compound 16. Compound 16, which exhibited potent cytotoxic activitiy (IC50 3.2 mm) against AZ521 cells, was evaluated for its apoptosis-inducing activity using AZ521 cells. AZ521 Cells were incubated with the test compound for 24 and 48 h, and then they were analyzed by means of flow cytometry with annexin Vpropidium iodide (PI) double staining. Exposure of the membrane phospholipid phosphatidylserine to the external cellular environment is one of the earliest markers of apoptotic cell death [60]. Annexin V is a Ca-dependent phospholipid-binding protein with high affinity for phosphatidylserine expressed on the cell surface. PI does not enter whole cells with intact membranes and was used to differentiate between early apoptotic (annexin V, positive; PI, negative), late apoptotic (annexin V and PI, double positive), or necrotic (annexin V, negative; PI, positive) cell death. The ratio of early apoptotic cells (Fig. 2, lower right) increased after treatment with 16 in AZ521 cells for 24 h (from 2.7 to 28.8%) and 48 h (16.8%), and that of late apoptotic cells (upper right) increased after 48 h (from 1.1 to 41.0%). These results demonstrated that most of the cytotoxicity of compound 16 against AZ521 cells is due to inducing apoptotic cell death.
Fig. 2. Detection of 16-induced early and late apoptotic cells by annexin VPI double staining in AZ521 cells. The cells were cultured with 10 mm 16 for 24 and 48 h.
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Conclusions. – This study has established that the defatted fraction of the hexane extract of Azadirachta indica fruits exhibit cytotoxic activities against HL60, A549, A521, and SK-BR-3 human cancer cell lines. Investigation of the fraction has led to the isolation of 19 limonoids, 1 – 19, and one triterpenoid, 20, of which three, i.e., 3-deacetyl4’-demethylsalannin (5), 3-deacetyl-28-oxosalannin (14), and 1-detigloylohchinolal (17), were new compounds. These compounds exhibited potent cytotoxicities (2, 3, 5, 7, 11, 16, 17, and 20 against HL60; 17 against A549; 16 and 17 against AZ521; and 8 against SK-BR-3), suggesting that they might be, at least in part, responsible for the cytotoxicities of the defatted fraction of A. indica fruit hexane extract. Moreover, 3deacetyl-4’-demethyl-28-oxosalannin (16) was revealed to induce apoptotic cell death in AZ521 cells. Compounds isolated in this study seem to be responsible for the repellent activity of M. azedarach seed oil against sandflies [25], since the seeds are expected to contain limonoids and triterpenoids, similar to those occurring in the fruits, and since meliaceous limonoids exhibit insect antifeedant and insecticidal activities [1] [2]. This work has provided, furthermore, valuable information on the structural features of limonoids of the fruits and/or seeds of Melia azedarach and related Meliaceae plants, M. toosendan and Azadirachta indica. Experimental Part General. Column chromatography (CC): silica gel (SiO2 , 230 – 400 mesh; Merck) and octadecyl silica gel (ODS; Chromatorex-ODS, 100 – 200 mesh; Fuji Silysia Chemical, Ltd., Aichi, Japan). Prep. HPLC (with refractive-index detector): ODS columns (25 cm 10 mm i.d.) at 258 on a Pegasil ODS-II 5 mm column (Senshu Scientific Co., Ltd., Tokyo, Japan) with MeCN/H2O/AcOH (flow rate, 3.0 ml/min) 48 : 52 : 0.1 (system I), 50 : 50 : 0.1 (system II), or 55 : 45 : 0.1 (system III), on a Capcell pak AQ 5 mm column (Shiseido Co., Ltd., Tokyo, Japan) with MeCN/H2O/AcOH (flow rate, 2.0 ml/min) 50 : 50 : 0.1 (system IV), 52 : 48 : 0.1 (system V), or 55 : 45 : 0.1 (system VI), or on a TSK ODS-120A 5 mm column (Toso Co., Tokyo, Japan) with MeOH/H2O/AcOH (flow rate, 3.0 ml/min) 60 : 40 : 0.1 (system VII) or with MeCN/H2O/HCOOH (flow rate, 3.0 ml/min) 70 : 30 : 0.1 (system VIII) as mobile phase. Optical rotations: JASCO P-1020 digital polarimeter. UV Spectra: JASCO V-630Bio spectrophotometer; lmax (log e) in nm. IR Spectra: JASCO FTIR-300E spectrometer; ˜n in cm 1. 1D- and 2D-NMR spectra: JEOL ECX-400 (at 400 (1H) and 100 MHz (13C)) spectrometer at r.t.; CDCl3 soln.; d in ppm rel. to Me4Si as internal standard, J in Hz. HR-ESI-MS: Agilent 1100 LC/MSD TOF (time-of-flight) system (ionization mode, positive; cap. voltage, 3000 V; fragmentor voltage, 225 V); m/z. Microplate reader: Sunrise-Basic (Tecan Japan Co., Ltd., Kawasaki, Japan). Flow cytometer: Cell Lab QuantaTM SC (Beckman Coulter K. K., Tokyo, Japan). Materials and Chemicals. Mature fruits of M. azedarach L. (Meliaceae) were collected from plants cultivated at the Toho University herbal garden (Funabashi-shi, Chiba, Japan) on 31st January, 2008. The plant material was authenticated by one (K. K.) of the authors, and a voucher specimen (TA-TH-MA0801) was deposited with the Herbarium of the School of Pharmaceutical Sciences, Toho University. The chemicals were purchased as follows: RPMI-1640 medium, fetal bovine serum (FBS; for RPMI-1640 medium), antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin), and non-essential amino acid (NEAA) from Invitrogen Co. (Carlsbad, CA, USA); Dulbeccos modified Eagles medium (DMEM), Eagles minimal essential medium (MEM), and MTT from SigmaAldrich Japan Co. (Tokyo, Japan); cisplatin from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); and recombinant human (rh) Annexin V/FITC kit (Bender MedSystems GmbH, A-Vienna) from Cosmo Bio Co., Ltd. (Tokyo, Japan). All other chemicals and reagents were of anal. grade. Extraction and Isolation. Air-dried fruits of M. azedarachi (19.7 kg) were pulverized and extracted with hexane (2 l/kg of dried fruits; 3 h, reflux, 3 ), and the residue was then extracted with MeOH [17]. The MeOH extract (1036 g) has recently been investigated for its limonoid constituents and their
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cytotoxicities [17] [18]. In this study, the hexane extract (588 g) was subjected to CC (SiO2 (500 g/40 g extract)) to give a less-polar oily fraction (437 g; hexane/AcOEt 1 : 0 to 4 : 1) and more-polar defatted fraction (52 g; AcOEt/MeOH 1 : 0 to 0 : 1). The defatted fraction was applied to CC (SiO2 (600 g); hexane/AcOEt 1 : 0 to 0 : 1) to afford nine fractions, Frs. A – I. Fr. D (2.82 g) from the eluate of hexane/ AcOEt 11 : 9 was subjected to CC (SiO2 (150 g); hexane/AcOEt 9 : 1 to 0 : 1) to yield eight fractions, Frs. D1 – D8. Further CC (ODS (13 g); MeOH/H2O 2 : 3 to 1 : 0) and subsequent prep. HPLC (system VIII) of Fr. D2 (242 mg) gave compound 20 (retention time (tR ) 63.0 min; 5.0 mg). Fr. D3 (1031 mg), upon CC (ODS (50 g); MeOH/H2O 1 : 1 to 1 : 0) and subsequent prep. HPLC (system III), yielded compounds 19 (tR 32.8 min; 1.9 mg) and 18 (tR 38.4 min; 2.1 mg). Fr. F (2.23 g) eluted with hexane/ AcOEt 3 : 7 was applied to CC (SiO2 (110 g); hexane/AcOEt 1 : 1 to 0 : 1) to furnish nine fractions, Frs. F1 – F9. CC (ODS (27 g); MeOH/H2O 2 : 3 to 1 : 0) of Fr. F4 (1038 mg) afforded seven fractions, Fr. F4-1 – F4-7. Prep. HPLC (system I) of Frs. F4-4 (318 mg) and F4-6 (188 mg) gave compounds 14 (tR 27.2 min; 60.2 mg), 16 (tR 24.0 min; 8.6 mg), and 15 (tR 30.4 min; 11.9 mg), and compound 7 (tR 52.0 min; 9.6 mg), resp. Prep. HPLC (system II) of Fr. F6 (207 mg) yielded compounds 3 (tR 16.8 min; 7.0 mg) and 17 (tR 20.0 min; 3.5 mg). Fr. G (2.53 g) eluted with hexane/AcOEt 1 : 4 was subjected to CC (ODS (57 g); MeOH/H2O 2 : 3 to 1 : 0) which furnished ten fractions, Fr. G1 – G10. Prep. HPLC of Frs. G3 (50 mg; system IV), G5 (138 mg; system V), G7 (100 mg; system V), and G9 (35 mg; system VI) yielded compound 10 (tR 23.2 min; 1.8 mg), compounds 4 (tR 32.0 min; 27.6 mg) and 8 (tR 40.0 min; 21.1 mg), compounds 5 (tR 44.0 min; 3.0 mg) and 6 (tR 56.0 min; 24.7 mg), and compound 2 (tR 54.4 min; 1.9 mg), resp. Fr. H (2.27 g) from the eluate of hexane/AcOEt 1 : 4 was subjected to CC (SiO2 (156 g); hexane/ AcOEt 1 : 0 to 0 : 1) to yield five fractions, Frs. H1 – H5. Further CC (ODS (57 g); MeOH/H2O 4 : 5 to 1 : 0) and subsequent prep. HPLC (system V) of Fr. H3 (1437 mg) gave compounds 13 (tR 20.8 min; 6.3 mg) and 9 (tR 28.0 min; 6.3 mg). Fr. I (1.33 g) eluted with hexane/AcOEt 1 : 9 was subjected to CC (ODS (40 g); MeOH/H2O 2 : 3 to 1 : 0) to yield nine fractions, Fr. I1 – I9. Prep. HPLC of Frs. I6 (116 mg; system VII) and I7 (99 mg; system II) afforded compounds 11 (tR 36.0 min; 3.0 mg) and 12 (tR 31.2 min; 22.2 mg), and compound 1 (tR 34.4 min; 15.4 mg), resp. Data of the New Compounds. 3-Deacetyl-4’-demethylsalannin ( ¼ (2aR,3R,5S,5aR,6R,6aR,8R,9aR,10aS,10bR,10cR)-8-(Furan-3-yl)-2a,4,5,5a,6,6a,8,9,9a,10a,10b,10c-dodecahydro-3-hydroxy-6-(2methoxy-2-oxoethyl)-2a,5a,6a,7-tetramethyl-2H,3H-cyclopenta[d]naphtho[2,3-b:1,8-b’c’]difuran-5-yl 2Methylprop-2-enoate; 5). Amorphous solid. [a] 25 D ¼ þ 49.1 (c ¼ 0.57, EtOH). UV (EtOH): No absorption maximum above 210 nm. IR (KBr): 3367 (OH), 1730, 1713 (C¼O), 873 (furan). 1H- and 13C-NMR: see Table 2. HR-ESI-MS: 563.2618 ([M þ Na] þ , C31H40NaO þ8 ; calc. 563.2621). 3-Deacetyl-28-oxosalannin ( ¼ (2aS,3R,5S,5aR,6R,6aR,8R,9aR,10aS,10bR,10cR)-8-(Furan-3-yl)2a,4,5,5a,6,6a,8,9,9a,10a,10b,10c-dodecahydro-3-hydroxy-6-(2-methoxy-2-oxoethyl)-2a,5a,6a,7-tetramethyl-2-oxo-2H,3H-cyclopenta[d]naphtho[2,3-b:1,8-b’c’]difuran-5-yl (2E)-2-Methylbut-2-enoate; 14). Fine needles. M.p. 200 – 2038. [a] 24 D ¼ þ 50.7 (c ¼ 0.85, MeOH). UV (EtOH): No absorption maximum above 210 nm. IR (KBr): 3338 (OH), 1778 (g-lactone) 1731, 1718 (C¼O), 860 (furan). 1H- and 13C-NMR: see Table 2. HR-ESI-MS: 569.2777 ([M þ H] þ , C32H41O þ9 ; calc. 569.2750). 1-Detigloylohchinolal ( ¼ Methyl [(2R,3aR,4aS,5R,5aR,6S,7R,9S,9aR,10R,10aR)-5-(Acetyloxy)-6formyl-2-(furan-3-yl)-3,3a,4a,5,5a,6,7,8,9,9a,10,10a-dodecahydro-7,9-dihydroxy-1,6,9a,10a-tetramethyl2H-cyclopenta[b]naphtho[2,3-d]furan-10-yl]acetate; 17). Fine needles. M.p. 129 – 1328. [a] 24 D ¼ þ 84.5 (c ¼ 1.01, MeOH). UV (EtOH): 220 (4.17). IR (KBr): 3360 (OH), 1740 (C¼O), 870 (furan). 1H- and 13 C-NMR: see Table 2. HR-ESI-MS: 531.2579 ([M þ H] þ , C29H39O þ9 ; calc. 531.2594). Cell Lines and Culture Conditions. Cell lines HL60 (leukemia), AZ521 (stomach), A549 (lung), and SK-BR-3 (breast) were obtained from Riken Cell Bank (Tsukuba, Ibaraki, Japan). They were grown in the following media: HL60 and SK-BR-3 in RPMI-1640 medium, AZ521 in DMEM medium, and A549 in 90% DMEM þ 10% MEM þ 0.1 mm NEAA. The medium was supplemented with 10% FBS and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin). Cells were incubated at 378 in a 5% CO2 humidified incubator and cultured as described in [61 – 64]. Determination of Cell Proliferation. Cell proliferation was assessed using the MTT ( ¼ 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)-based colorimetric assay as described in [61 – 64].
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Annexin VPropidium Iodide (PI) Double Staining. Annexin VPI double staining was performed as described in [61] [65] [66] with AZ521 (1.5 105 cells/5 ml) cells.
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