Limonoids from the fruits of Melia azedarach and their cytotoxic activities

Phytochemistry 89 (2013) 59–70

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Limonoids from the fruits of Melia azedarach and their cytotoxic activities Toshihiro Akihisa a,⇑, Xin Pan a, Yasuhiro Nakamura a, Takashi Kikuchi a, Nami Takahashi a, Masahiro Matsumoto a, Eri Ogihara a, Makoto Fukatsu a, Kazuo Koike b, Harukuni Tokuda c a

College of Science and Technology, Nihon University, 1-8-14 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan School of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi-shi, Chiba 274-8510, Japan c Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640, Japan b

a r t i c l e

i n f o

Article history: Received 31 July 2012 Received in revised form 4 January 2013 Accepted 29 January 2013 Available online 27 February 2013 Keywords: Melia azedarach Meliaceae Fruits Limonoids Cytotoxic activities Epstein–Barr virus early antigen (EBV-EA)

a b s t r a c t Thirty-one limonoids and one tirucallane-type triterpenoid were isolated from the fruits of Melia azedarach (Meliaceae). The structures of 14 of these isolated compounds were elucidated on the basis of spectroscopic analyses and comparison with literature. All of these compounds were evaluated for their cytotoxic activities against HL60, A549, AZ521, and SK-BR-3 human cancer cell lines. Meliarachin C (IC50 0.65 lM) and 3-O-deacetyl-40 -demethyl-28-oxosalannin (IC50 2.8 lM) exhibited potent cytotoxic activity against HL60 cells, and this was demonstrated mainly due to the induction of apoptosis by flow cytometry. Western blot analysis suggested that both compounds induced apoptosis via both the mitochondrial and death receptor-mediated pathways. In addition, 25 compounds were evaluated for their inhibitory effects against the Epstein–Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Plants of the Meliaceae family have been well documented for their ability to metabolize structurally diverse and biologically significant limonoids and triterpenoids (Tan and Luo, 2011; Zhao et al., 2010). In the course of a search for potential bioactive compounds from Meliaceae plants, a detailed investigation on the limonoid constituents of Azadirachta indica (neem) seed extracts was undertaken and this showed that some limonoids exhibit potent inhibitory activities against melanogenesis in B16 melanoma cells, against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice, and against TPA-induced Epstein– Barr virus early antigen (EBV-EA) activation (Akihisa et al., 2009, 2011), as well as cytotoxic and apoptosis-inducing activities (Kikuchi et al., 2011). In a continuing study on the limonoid constituents of Meliaceae plants, the fruit extract of Melia azedarach L., was investigated, and thirty-one limonoids (1–31) were isolated, including 14 new compounds, and one tirucallane-type triterpenoid (32). This paper describes the structure elucidation of these compounds and evaluation of their cytotoxic activities and inhibitory effects on the induction of EBV-EA activation induced with TPA in Raji cells. M. azedarach is indigenous to Japan and other countries in the southeastern Asia. Its bark and fruit, which are known as ‘‘Kurenpi’’ and ‘‘Kurenshi’’, respectively, in Japan, have ⇑ Corresponding author. Fax: +81 3 3293 7572. E-mail address: [email protected] (T. Akihisa). 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.01.015

been used for vermicide, anodyne, and skin disease (Namba, 1994; Okada, 2002). Many constituents including limonoids, triterpenoids, and steroids have been isolated from various parts of M. azedarach (Carpinella et al., 2003; Liu et al., 2011; Nakatani et al., 1995; Ntalli et al., 2010; Ochi et al., 1978a; Su et al., 2011; Wu et al., 2011; Zhou et al., 2004, 2005). Several of the limonoids isolated from M. azedarach have been reported to possess antimicrobial (Liu et al., 2011; Su et al., 2011), cytotoxic (Wu et al., 2011; Zhou et al., 2004), antifeedant (Carpinella et al., 2003; Nakatani et al., 1995), and insecticidal (Carpinella et al., 2003) activities.

2. Results and discussion 2.1. Cytotoxic activity of the extracts of M. azedarach fruits Dried and powdered M. azedarach fruits were extracted with nhexane, and the defatted residue was then extracted with MeOH. The MeOH extract was fractionated into EtOAc-, n-BuOH-, and H2O-soluble fractions. The n-hexane and MeOH extracts and the three fractions from the MeOH extract 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 summarized in Table 1. The EtOAc-soluble fraction exhibited potent activity against HL60, A549, and AZ521 cells, and the n-BuOH-soluble

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T. Akihisa et al. / Phytochemistry 89 (2013) 59–70

Table 1 Cytotoxicity of extracts and fractions of Melia azedarach fruits against four human cancer cell lines. Extract and fraction

n-Hexane extract MeOH extract EtOAc-soluble fraction n-BuOH-soluble fraction H2O-soluble fraction

IC50 (ng/ml)a HL 60 (Leukemia)

A549 (Lung)

AZ521 (Stomach)

SK-BR-3 (Breast)

75.5 ± 1.7 2.9 ± 0.4 0.05b ± 0.006 1.9 ± 0.4 >100

>100 64.1 ± 5.4 4.2 ± 0.5 >100 >100

77.0 ± 1.1 2.9 ± 1.3 1.9 ± 0.2 2.0 ± 0.3 >100

>100 21.0 ± 3.3 34.7 ± 2.4 >100 >100

a IC50 Values based on quintuple points. Cells were treated with test samples (1  104–1  106 g/ml) for 48 h, and cell viability was analyzed by the MTT assay. Each value represents the mean ± SD (n = 3). b Range of concentration of test sample assayed: 1  106–1  108 g/ml.

fraction against HL60 and AZ521 cells. The EtOAc-soluble fraction was further investigated for their constituents in this study.

2.2. Isolation, identification, and structure elucidation The EtOAc-soluble fraction was subjected to successive column chromatography (CC) on silica gel (SiO2) and octadecyl silica gel (ODS) columns, and to reversed-phase HPLC which led to the isolation of thirty-one limonoids, 1–31, including 14 new compounds, 5, 6, 13, 15, and 17–26, and one tirucallane-type triterpenoid, 32. Among these compounds, 17 known compounds were identified as meliarachin C (1) (Su et al., 2011), toosendanin (2; C-29 epimeric mixture) (Kim et al., 1999; Ochi et al., 1978a,b), meliarachin K (3) (Su et al., 2011), meliarachin G (4) (Su et al., 2011), trichilinin D (7) (Nakatani et al., 1999), 1-O-cinnamoyltrichilinin (8) (Rajab and Bentley, 1988), salannin (9) (Johnson and Morgan, 1997), 3-O-deacetylsalannin (10) (Madyastha and Venkatakrishnan, 2000), 3-O-deacetyl-3-O-tigloylsalannin (11) (Madyastha and Venkatakrishnan, 2000), ohchinin (12) (Fukuyama et al., 1983), ohchinin acetate (14) (Ochi et al., 1978b), 1-O-decinnamoyl-1-O-benzoylohchinin acetate (16) (Ochi et al., 1978b), ohchinolal (27) (Fukuyama et al., 1983), mesendanin E (28) (Dong et al., 2010), 1-O-detigloyl-1-O-benzoylohchinolal (29) (Zhou et al., 2004), 1-O-detigloyl-1-O-cinnamoylohchinolal (30) (Zhou et al., 2004), nimbolinin D (31) (Nakatani et al., 2000), and meliasenin E (32) (Zhang et al., 2010) by MS, 1H NMR, and 13C NMR spectroscopic comparison with corresponding literature data (Fig. 1). The structures of the 14 new compounds were elucidated on the basis of spectroscopic data and by comparison with literature as described below, and their proposed structures were supported by analysis of the DEPT, 1H–1H COSY, HMQC, HMBC, and NOESY data. The molecular formula of compound 5 was determined as C29H38O9 by HRESIMS. The 1H and 13C NMR spectroscopic data of 5 indicated that it was structurally similar to compound 3 (Su et al., 2011), with the only difference being the absence of an AcO group. The AcO group was located at C-3 of 5 by the HMBC for H-3 (dH 4.92) and the AcO group (dC 169.8). The NOESY correlation (Fig. 2) between H-3 and H-29 supported the S-configuration at C-29 (29-endo). Hence, the structure of compound 5 was assigned as (29S)-19,29-epoxy-29-O-methyl- 1a,3a,7a,29-tetrahydroxymeliacane-11,15-dione 3-acetate which was named 12dehydroneoazedarachin D. Compound 6 had the molecular formula C29H38O9 as determined by HRESIMS. The 1H and 13C NMR spectra (Tables 2 and 5) of 6 resembled those of 5, except for the downfield shift of H-3 signal (dH 5.35) suggesting that 6 is a 29-exo-isomer (Huang et al., 1994) of 5. Thus, 6 was assigned as (29R)-19,29-epoxy-29-O-methyl-1a,3a,7a, 29-tetrahydroxymeliacane-11,15-dione 3-acetate (12-dehydro29-exo-neoazedarachin D). The NOESY correlation (Fig. 2) between H2-19 and H-29 supported the configuration.

The HRESIMS of compound 13 displayed a quasi molecular ion peak at m/z 603.2945 ([M+H]+) consistent with the molecular formula of C36H42O8. The 1H and 13C NMR spectra of 13 were almost superimposable on those of 12 (Fukuyama et al., 1983), except for the coupling constant between the 1H signals of H-20 and H-30 . Compound 12 exhibited the resonances with larger coupling constant between these signals (JH-20 ,30 = 15.9 Hz) (Fukuyama et al., 1983), while compound 13 showed them with smaller coupling constant (JH-20 ,30 = 12.4 Hz) which implied that the latter was the cis-diastereoisomer of the former (Akihisa et al., 2000). Thus, structure 13 was assigned as 1-O-Z-cinnamoylsalannic acid methyl ester (1-O-decinnamoyl-1-O-Z-cinnamoylohchinin). The cis-cinnamoyl group of 13 was supported from the NOE correlation between the 1H signals of H-20 and H-30 (Fig. 2). The HRESIMS of compound 15 displayed a sodiated molecular ion peak at m/z 599.2543 [M+Na]+ corresponding to the molecular formula C34H40O8. The 1H and 13C NMR spectroscopic data (Tables 2 and 5) of 15 were similar to those of 13, except for the presence of a benzoyl group and the absence of a cis-cinnamoyl group. In HMBC experiments, the H-1 signal at dH 5.22 showed a cross-peak with the carbonyl resonance (dC 165.1) of the benzoyl group, indicating that 13 had a benzoyl group at the C-1 position in place of a cis-cinnamoyl group in 13. Thus, structure 15 was elucidated as 1-O-benzoylsalannic acid methyl ester (1-O-decinnamoyl-1-Obenzoylohchinin). Compound 17 was shown to have the molecular formula C36H42O9 by HRESIMS (m/z 641.2724 ([M+Na]+, C36H42O9Na+). The 1H and 13C NMR spectroscopic data (Tables 2 and 5) of 17 were analogous to those of compound 12 (Fukuyama et al., 1983), although the b-furyl ring signals for 12 were lacking for 17. The presence of an a,b-unsaturated-c-lactone ring instead of the b-furyl ring at C-17 was deduced by the 1H [dH 4.27 and 4.50 (each 1H; H2-23), and 7.06 (H-22)] and 13C [dC 135.1 (C-20), 174.3 (C-21), 145.0 (C-22), and 70.0 (C-23)] signals (Mohamad et al., 2009). The HMBC correlations for H-17 and the C-20, C-21, and C-22 indicated the presence of b-substituted-c-lactone ring. Hence, the structure of 17 was assigned as 1-O-cinnamoyl-17-defurano-17(2-buten-4-olide-2-yl)-salannic acid methyl ester which was named ohchininolide. The NOE correlation between H-17 and Me-18 supported that the c-lactone ring at C-17 was a-oriented. Compound 18 was shown to have the molecular formula C34H40O9 by HRESIMS. The 1H and 13C NMR spectroscopic data (Tables 3 and 5) of 18 resembled those of 17, except for the presence of a benzoyl group and the absence of a cinnamoyl group. In HMBC experiments, the H-1 signal at dH 5.18 showed a crosspeak with the carbonyl resonance (dC 164.9) of the benzoyl group, indicating that 18 had a benzoyl group at C-1 position in place of a cinnamoyl group in 17. Hence, structure 18 was elucidated as 1-O-benzoyl-17-defurano-17-(2-buten-4-olide-2-yl)-salannic acid methyl ester (1-O-decinnamoyl-1-O-benzoylohchininolide). Compound 19 has the molecular formula of C37H44O10, as determined by the sodiated ion at m/z 671.2821 ([M+Na]+) in HRESIMS.

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T. Akihisa et al. / Phytochemistry 89 (2013) 59–70 23

AcO HO O 4

AcO

8 10 5 H 7 6

29

20

H HO

O

HO

17 R = Cin 18 R = Bz

O CinO

O

H

O

21 R = Cin 22 R = Bz

OMe O RO

O

O H

OH HO

O

H

HO

O

H O

O 24

23

O

25 R = Bz 26 R = Met O

MeO

OMe O RO

O

H

OH

H

OH

H AcO

O

H OAc 27 R = Tig H O 28 R = Met 29 R = Bz 30 R = Cin

H

MeO

O

H HO

O

O

H

OMe

O

H

O

O HO

O CinO

O H

OMe

O 9 R = Ac, R' = Tig 10 R = H, R' = Tig 11 R = R' = Tig 12 R = H, R' = Cin 13 R = H, R' = Z-Cin 14 R = Ac, R' = Cin 15 R = H, R' = Bz 16 R = Ac, R' = Bz OH

O RO

19 20

O

OMe

HO

O

O

H

H

O

H

7R=H 8 R = Ac

O

O

RO

OH

H

H

O

13 17

15

OMe

O

O

H

RO

O CinO

O 21

11

H

OMe

23

18

12

14 16

H O OH

H

O R'O

H

R R' 3 R = H, R' = OMe, R" = OAc 4 R = OMe, R' = H, R" = OAc 5 R = H, R' = OMe, R" = H 6 R = OMe, R' = H, R" = H

22

O RO

H

AcO

R' 1 R= H, R' = OMe 2 R = H, R' = OH and R = OH, R' = H

OMe

H CinO

O

28

OMe

OAc

O

HO

O OH

H

R

20

13 17 16 1415

11 30 9

19

O

R"

21

H

12

O

1 2 3

18

O

O

22

O

H

O OBz

H

O HO

31

H

32

O O

O 1'

O 3'

4'

1'

2'

E

4' 5'

6'

Z

7'

Ac

Bz

O

O 3'

2'

Cin

1'

2' 4'

Z-Cin

Met

3' 1'

2'

3' 4'

5'

Tig

Fig. 1. Structures of compounds 1–32.

The 1H and 13C NMR spectroscopic data (Tables 3 and 5) of 19 were closely related to those of 17, except for the presence of an additional MeO group. The MeO group was located to be at C-23 by the key HMBC for MeO-23 (dH 3.40) and C-23 (dC 102.6). The relative configuration of 19 was mainly deduced from the NOESY spectrum, and also by comparing the NMR data with those of 17. Thus, 19 was determined to be (23n)-1-O-cinnamoyl-17-defurano-17(4-methoxy-2-buten-4-olide-2-yl)-salannic acid methyl ester which was named 23-methoxyohchininolide A. The stereochemistry at C-23 of 19 remained undetermined. Compound 20 was shown to have the molecular formula C37H44O10 by HRESIMS, same as that of compound 19. The 1H

and 13C NMR spectroscopic data (Tables 3 and 5), and the HMBC and NOESY data of 20 were similar to those of 19, with the differences being the downfield shift [i.e., dH 5.57 (20) vs. 5.24 (19)] of H23 signal and upfield shift [i.e., dH 3.21 (20) vs. 3.40 (19)] of MeO-23 signal in the 1H NMR spectrum. This suggested that 20 was a stereoisomer at C-23 of 19, and, hence, the structure of 20 was assigned as (23n)-1-O-cinnamoyl-17-defurano-17-(4-methoxy-2buten-4-olide-2-yl)-salannic acid methyl ester which was named 23-methoxyohchininolide B. The stereochemistry at C-23 of 20 remained undetermined. The molecular formula of compound 21 was determined as C36H42O10 from its HRESIMS. The 1H and 13C NMR spectra (Tables

62

T. Akihisa et al. / Phytochemistry 89 (2013) 59–70

H

H H

O H O Me H 29 H H 3

19

30

Me H 5

H

H

11 18

H

H

Me

14

H

H

OH Me

17

H H O H Me O H 19 H H O 29 OH H H HH Me H Me H H H H O H Me H H O H OH

H O H O

HH

9

H

Me HO O H H

O

O

Me

O

5 30

H Me

Me

1

HH

3

H 28

O

H H 12

29

6

H

OO O H

H OH H

2'

H

7

Me Me H

HO 17 15

O

H

H

O Me Me H Me O H

18

H

H H

H

HO

H

H

O OH OO H H

Me H

Me

H H H

O

H

H

H

O H

H

H H

H

H H

H H

H

H

O H

H

3'

H

H

6

19

H Me H

O

13

25

Fig. 2. Major NOE correlations (M) for compounds 5, 6, 13, and 25. Drawings correspond to energy-minimized conformation of compounds. Calculation was performed using CAChe CONFLEX with the MM2 force field (CAChe version 5.5; Fujitsu Co., Tokyo, Japan).

Table 2 1 H NMR spectroscopic data of compounds 5, 6, 13, 15, and 17 from Melia azedarach fruits (400 MHz CDCl3, d in ppm, J in Hz). Position

5

1 2

4.14 1.81 2.87 4.92 2.28 1.74 2.46 3.89 3.42

3 5 6 7 9 11 12

6 (br. d, 4.6) (a; br. d, 16.0) (b; dt, 4.6, 16.0) (br. d, 2.8) (t, 7.6) (a; dt, 3.5, 14.9) (b; dd, 2.5, 14.9) (t, 2.5) (s)

4.08 1.81 2.85 5.35 2.45 1.78 2.02 3.94 3.45

13 (m) (a; dt, 1.8, 16.0) (b; dt, 4.6, 16.0) (br. d, 2.8) (br. dd, 2.8, 13.7) (a; dt, 3.2, 13.8) (b; dd, 2.8, 14.2) (m) (s)

2.33 (a; d, 16.8) 2.62 (b; d, 16.8) 3.12(s)

2.33 (a; d, 17.0) 2.65 (b; d, 17.0) 3.15 (s)

2.55 (2H, d, 10.5)

2.58 (2H, d, 10.0)

21 22 23

3.36 0.96 4.04 4.34 7.29 6.28 7.39

3.36 0.99 4.09 4.52 7.32 6.31 7.42

28

0.85 (s)

0.87 (s)

29 30 2’ 3’ 4’ 5’ 6’ 7 AcO-3 MeO-12 MeO-29

4.16(s) 1.04 (s)

4.42 (s) 1.05 (s)

14 15 16 17 18 19

(dd, 4.8, 16.0) (s) (d, 12.3) (d, 12.3) (br. s) (br. s) (t, 1.2)

(t, 10.6) (s) (d, 12.8) (d, 12.8) (br. s) (dd, 1.2, 1.2) (t, 1.2)

15 (t, 3.2) (a; dt, 3.0, 16.4) (b; m) (m) (d, 12.4) (dd, 3.2, 12.4)

5.22 2.13 2.31 3.93 2.90 4.05

(t, 3.2) (a; m) (b; m) (t, 3.4) (d, 12.4) (dd, 3.2, 12.4)

5.06 2.24 2.34 3.91 2.76 4.03

(d, 2.8) (a; m) (b; m) (m) (d, 12.4) (dd, 3.7, 12.4)

4.19 2.68 2.18 2.34

(d, 3.2) (dd, 2.5, 9.8) (m) (dd, 10.0, 15.6)

4.22 2.72 2.19 2.35

(d, 3.2) (dd, 3.2, 10.1) (m) (m)

4.25 2.63 2.11 2.34

(d, 3.7) (dd, 3.7, 8.7) (m) (m)

5.50 2.28 2.16 3.63 1.65 0.93

(tq, 1.4, 6.8) (a; m) (b; m) (br. d, 8.7) (d, 1.8) (s)

5.41 2.25 2.11 3.59 1.64 1.00

(tq, 1.4, 6.8) (a; m) (b; m) (br. d, 9.2) (d, 1.4) (s)

5.36 2.18 2.08 3.54 1.68 1.00

(tq, 1.6, 6.4) (a; m) (b; m) (br. d, 8.0) (d, 1.8) (s)

7.06 4.27 4.50 4.16 3.66 1.18 1.32 6.46 7.66

(dd, 1.6, 2.8) (dt, 1.6, 18.0) (dt, 1.6, 18.0) (a; br. d, 7.8) (b; d, 7.8) (s) (s) (d, 16.0) (d, 16.0)

7.26 (d, 2.4) 6.32 (dd, 0.8, 2.0) 7.31 (t, 1.6)

7.08 (d, 0.9) 6.07 (dd, 0.9, 0.9) 7.17 (t, 1.8)

4.05 3.60 1.11 1.31 6.10 7.05

4.20 3.67 1.19 1.32

(a; d, 7.8) (b; d, 7.8) (s) (s) (d, 12.4) (d, 12.4)

7.62 (dt, 2.0, 7.8) 7.40 (tt, 2.0, 7.8) 7.36 (tt, 2.0, 7.8) 2.09 (s)

2.09 (s)

3.31 (s)

3.40 (s)

17

5.06 1.98 2.22 3.79 2.62 4.00

3.21 (s)

(a; br. d, 7.8) (b; d, 7.8) (s) (s)

8.08 (dt, 1.4, 7.8) 7.50 (dt, 1.4, 7.8) 7.61 (dt, 1.4, 7.8)

2.72 (s)

7.55 (m) 7.42 (m) 7.42 (m) 3.38 (s)

63

T. Akihisa et al. / Phytochemistry 89 (2013) 59–70 Table 3 1 H NMR spectroscopic data of compounds 18–22 from Melia azedarach fruits (400 MHz, CDCI3, d in ppm, J in Hz). Position

18

1 2

5.18 2.21 2.40 3.91 2.88 4.03 4.22 2.67 2.09 2.37 5.28 2.25 2.08 3.50 1.68 1.03 6.93 4.48 4.54 4.17 3.65 1.19 1.32

3 5 6 7 9 11 15 16 17 18 19 22 23 28 29 30 2’ 3’ 4’ 5’ 6’ 7’ MeO-12 MeO-23

19 (d, 2.8) (a; m) (p; m) (t, 3.2) (d, 12.3) (dd, 3.2, 12.3) (d, 3.2) (dd, 4.6, 7.8) (m) (m) (tq, 2.2, 6.4) (a; m) (p; m) (br. d, 8.7) (d, 1.4) (s) (dd, 1.4, 3.0) (dt, 1.4, 18.2) (dt, 1.4, 18.2) (a; d, 7.3) (p; d, 7.3) (s) (s)

8.04 (dt, 1.4, 7.3) 7.47 (t, 7.3) 7.59 (tt, 1.4, 7.3)

3.17 (s)

20

5.06 2.12 2.25 3.91 2.76 4.02 4.23 2.64 2.31 2.39 5.31 2.10

(d, 3.0) (a; m) (p; m) (t, 2.8) (d, 12.4) (dd, 3.2, 12.4) (d, 3.2) (dd, 3.6, 8.7) (m) (m) (br. t, 7.2) (2H, m)

3.54 1.67 0.99 6.83 5.24

21

22

(br. d, 8.3) (d, 1.4) (s) (s) (s)

5.06 2.24 2.36 3.90 2.74 4.03 4.23 2.64 2.11 2.24 5.34 2.25 2.10 3.53 1.70 0.99 6.75 5.57

(d, 3.0) (a; m) (p; m) (t, 2.7) (d, 12.4) (dd, 3.2, 12.4) (d, 3.2) (dd, 3.7, 8.4) (m) (m) (br. t, 6.4) (a; m) (p; m) (br. d, 8.8) (d, 1.3) (s) (t, 1.4) (t, 1.2)

5.14 2.15 2.38 3.93 2.75 3.99 4.24 2.40 2.09 2.33 5.31 2.19 2.08 3.39 1.75 0.93 6.11 5.46

(d, 2.8) (a; m) (p; m) (t, 2.8) (d, 12.4) (dd, 3.2, 12.4) (d, 3.2) (m) (m) (m) (br. t, 7.6) (a; m) (p; m) (m) (d, 1.9) (s) (t, 1.6) (br. d, 9.6)

5.28 2.11 2.38 3.97 2.87 4.03 4.27 2.47 2.24 2.45 5.31 2.29 2.09 3.43 1.75 0.98 6.19 5.66

(t, 3.2) (a; m) (p; m) (t, 2.8) (d, 12.8) (dd, 3.2, 12.4) (d, 3.2) (br. d, 10.6) (m) (m) (br. t, 6.4) (a; m) (p; m) (br. d, 6.4) (d, 1.8) (s) (br. s) (d, 10.3)

4.15 3.66 1.17 1.32 6.46 7.67

(a; d, 7.8) (p; d, 7.8) (s) (s) (d, 16.2) (d, 16.2)

4.15 3.66 1.17 1.32 6.46 7.68

(a; d, 7.8) (p; d, 7.8) (s) (s) (d, 16.0) (d, 16.0)

4.20 3.66 1.15 1.28 6.46 7.67

(a; d, 7.8) (p; d, 7.8) (s) (s) (d, 16.4) (d, 16.4)

4.23 3.68 1.19 1.30

(a; d, 7.3) (p; d, 7.3) (s) (s)

7.56 7.44 7.44 3.44 3.40

(m) (m) (m) (s) (s)

7.56 7.41 7.41 3.38 3.21

(m) (m) (m) (s) (s)

7.63 7.41 7.41 3.39

(m) (m) (m) (s)

8.05 (dt, 1.4, 7.3) 7.54 (t, 7.3) 7.65 (tt, 1.4, 7.3)

3.31 (s)

Table 4 1 H NMR spectroscopic data of compounds 23–26 from Melia azedarach fruits (400 MHz, CDCl3, d in ppm, J in Hz). Position

23

1 2

4.99 2.40 2.28 3.91 2.73 4.03 4.26 2.66 2.12 2.32 5.56 2.15 2.39 3.77 1.68 1.02 5.81 5.90

(m) (a; m) (P; m) (t, 3.2) (d, 12.3) (dd, 3.2, 12.3) (d, 3.2) (m) (m) (m) (t, 6.9) (a; m) (P; m) (br. d, 9.2) (s) (s) (s) (s)

29 30 2’ 3’

4.14 3.66 1.17 1.30 6.44 7.68

(a; br. d, 7.8) (P; d, 7.8) (s) (s) (d, 16.0) (d, 16.0)

3.70 4.19 1.19 1.41 6.47 7.70

(P; d, 7.8) (a; br. d, 7.8) (s) (s) (d, 16.0) (d, 16.0)

4’ 5’ 6’ 7’ MeO-12

7.56 7.42 7.42 3.52

(m) (m) (m) (br. s)

7.54 7.41 7.41 3.25

(dd, 3.6, 7.8) (m) (m) (s)

3 5 6 7 9 11 15 16 17 18 19 21 22 23 28

24 5.19 2.13 2.36 3.95 2.79 4.09 4.17 2.83 2.36 2.44 5.11 2.48 2.85

25 (t, 3.0) (a; dt, 2.7, 16.5) (P; dt, 2.7, 16.5) (dt, 2.7, 8.2) (d, 12.8) (dd, 3.4, 12.8) (d, 3.4) (t, 3.2) (dd, 2.3, 15.6) (dd, 10.5, 15.6) (tt-like, 1.8, 6.2) (a; dd, 3.6, 17.0) (P; dd, 3.6, 17.0)

1.75 (d, 1.8) 1.04 (s)

3 and 5) of 21 were similar to those of 19 and 20, except for the signals indicating an OH group at C-23 rather than a MeO group. The HMBC spectrum of 21 exhibited cross-peaks for H-22 (dH 6.11)

5.08 2.18 2.25 4.17 3.56 4.56 4.27 2.87 2.21 2.32 5.42 2.28 2.13 3.60 1.66 1.07 7.06 6.03 7.16

26 (t, 2.8) (a; m) (P; m) (t, 2.8) (d, 12.3) (dd, 3.2, 12.3) (d, 3.2) (dd, 3.9, 8.9) (m) (m) (br. t, 6.8) (a; m) (P; m) (br. d, 8.4) (d, 1.4) (s) (s) (br. s) (t, 1.6)

4.87 2.14 2.23 4.13 3.39 4.52 4.24 2.84 2.18 2.34 5.46 2.27 2.10 3.65 1.68 1.03 7.23 6.27 7.32

(t, 3.0) (a; m) (P; m) (t, 2.8) (d, 12.4) (dd, 3.6, 12.4) (d, 3.6) (dd, 4.0, 9.0) (m) (m) (br. t, 7.2) (a; m) (P; m) (br. d, 8.7) (d, 1.6) (s) (s) (br. s) (t, 1.6)

1.29 (s) 1.37 (s)

1.25 (s) 1.36 (s)

8.12 (d, 7.8)

5.60 (t, 1.6) 6.22 (br. s) 2.03 (s)

7.46 7.57 7.46 8.12 2.76

(t, 7.8) (d, 7.8) (t, 7.8) (d, 7.8) (s)

3.24 (s)

with C-17, C-20, C-21, and C-23, and H-23 (dH 5.46) with C-21 and C-22, which supported the presence of an OH group at C-23. Hence, 21 was characterized as (23n)-1-O-cinnamoyl-17-defur-

64

T. Akihisa et al. / Phytochemistry 89 (2013) 59–70

ano-17-(4-hydroxy-2-buten-4-olide-2-yl)-salannic acid methyl ester (23-hydroxyohchininolide). The stereochemistry at C-23 of 21 remained undetermined. Compound 22 was established to have the molecular formula C34H40O10 by HRESIMS. The 1H and 13C NMR data (Tables 3 and 5) of 22 resembled those of 21, except for the presence of a benzoyl group and the absence of a cinnamoyl group. In HMBC experiment, 21 exhibited cross peaks for H-1 (dH 5.28) with C-3, C-5, and C-10 , indicating that 22 had a benzoyl group at the C-1 position in place of a cinnamoyl group in 21. Thus, the structure of 22 was elucidated as (23n)-1-O-benzoyl-17-defurano-17-(4-hydroxy-2buten-4-olide-2-yl)-salannic acid methyl ester (1-O-decinnamoyl1-O-benzoyl-23-hydroxyohchininolide). The stereochemistry at C-23 of 22 remained undetermined. Compound 23 was shown to have the molecular formula C36H42O10 by HRESIMS, same as that of compound 21. The 1H and 13C NMR spectra (Tables 4 and 5) of 23 were closely related to those of 21, although the 23-hydroxybut-20(22)-ene-21,23c-lactone ring signals for 21 were lacking for 23. Instead of the c-lactone ring, the presence of an 21-hydroxybut-20(22)ene-21,23-c-lactone ring at C-17 was deduced by the 1H [dH 5.81 (H-21) and 5.90 (H-22)] and 13C [dC 169.0 (C-20), 97.7 (C-21), 119.8 (C-22), and 170.3 (C-23)] signals (Siddiqui et al., 1986a,b). The HMBC cross-correlations for H-17 (dH 3.77) with C-20 and C-21, and H-22 (dH 5.90) with C-17, C-20, C-21, and C-23, and the NOE correlations between the 1H signals of H-7b (dH 4.26) and H-16b (dH 2.39), and H-16b and H-17b of 23 supported that the c-lactone ring was located at C-17 with an a-orientation. Thus, the structure of 23 was assigned as (21n)-1-O-cinnamoyl-

17-defurano-17-(4-hydroxy-2-buten-4-olide-3-yl)-salannic acid methyl ester which was named 21-hydroxyisoohchininolide. The stereochemistry at C-21 of 23 remained undetermined. The molecular formula of compound 24 was determined as C32H38O8 from its HRESIMS. The 1H and 13C NMR spectroscopic data (Tables 4 and 5) of 24 resembled those of 12 (Fukuyama et al., 1983), except for the absence of the signals due to a furanring attached at C-17. Compound 24 exhibited, however, a 13C signal due to a C@O group (dC 206.1) located most probably at C-17. The HMBC cross-correlations for H-16b (dH 2.85) with C-14, C-15, and C-17, and Me-18 (dH 1.75) with C-13, C-14, and C-17, confirmed the presence of a C@O group at C-17. Hence, the structure of 24 was assigned as 1-O-cinnamoyl-17-defurano-17-oxosalannic acid methyl ester (17-defurano-17-oxoohchinin). Compound 25 was shown to have the molecular formula C34H38O9 by HRESIMS. The 1H and 13C NMR spectroscopic data (Tables 4 and 5) of 25 were similar to those of 15, with obvious differences being the lack of oxygenated CH2 group and the presence of C@O group (dC 177.6) at C-28 for 25 when compared with 15. This suggested the presence of a 6,28-c-lactone ring in 25 like in nimbolide (Bokel et al., 1990). In the HMBC experiment, 25 exhibited cross peaks for H-5 (dH 3.56) with C-3, C-4, C-6, C-7, C-10, C-19, and C-28, and Me-29 (dH 1.29) with C-3, C-4, C-5, and C-28, supported the presence of 6,28-c-lactone ring structure in 25, and, hence, the structure of 25 was assigned as 1-O-benzoyl28-oxosalannic acid methyl ester (1-O-decinnamoyl-1-O-benzoyl28-oxoohchinin). Compound 26 showed a molecular formula C31H38O9 as established by HRESIMS. The 1H and 13C NMR spectroscopic data

Table 5 13 C NMR spectroscopic data of 14 compounds from Melia azedarach fruits (100 MHz, CDCI3, d in ppm). Position

5

6

13

15

17

18

19

20

21

22

23

24

25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 28 29 30 1’ 2’ 3’ 4’ 5’ 6’ 7’ AcO-3

70.7 36.2 76.7 40.4 26.0 25.0 69.6 45.0 52.1 43.4 211.0 50.8 43.3 62.3 219.6 42.1 41.8 28.0 58.3 122.0 140.2 110.3 143.4 18.3 102.7 20.4

70.8 35.2 74.4 39.7 28.1 23.1 69.4 44.8 51.8 41.6 210.6 50.5 41.6 62.4 219.7 43.3 41.0 28.0 63.4 121.8 140.1 110.2 143.3 19.5 102.8 20.1

72.6 30.5 70.7 44.1 39.0 72.4 85.7 49.0 39.4 40.6 30.1 172.8 134.8 146.4 87.8 41.0 49.3 13.0 15.5 127.1 138.8 110.8 142.8 77.8 19.6 17.0 164.8 119.5 143.7 134.6 129.7 128.2 129.3

73.3 30.9 70.7 44.2 38.9 72.4 85.9 48.9 39.7 41.2 30.8 172.5 135.4 146.2 88.0 41.1 49.3 13.3 15.2 127.1 138.4 110.6 142.8 77.9 20.0 16.9 165.1 130.2 129.6 128.8 133.3

73.2 30.4 70.8 44.2 39.3 72.3 86.0 49.0 38.7 41.0 30.5 173.1 134.1 148.7 87.4 40.0 48.6 13.1 15.2 135.1 174.3 145.0 70.0 77.9 19.9 16.7 165.2 117.8 145.3 132.7 128.1 129.0 130.7

73.4 30.2 70.7 44.2 38.8 72.3 86.0 49.0 39.5 41.1 30.7 172.9 132.8 148.6 87.4 39.9 48.7 13.2 15.1 135.0 174.2 144.8 70.0 77.9 20.0 16.8 164.9 130.2 129.6 128.5 133.1

73.1 30.5 70.8 44.1 38.7 72.2 86.0 49.0 39.0 40.9 30.4 173.0 132.0 149.3 87.2 39.7 48.3 13.0 15.2 139.3 171.4 142.6 102.6 77.9 19.9 16.8 165.3 117.7 145.3 134.2 128.2 128.9 130.6

73.0 30.6 70.7 44.1 38.8 72.2 85.8 49.1 39.1 40.8 30.5 172.9 132.1 149.1 87.2 39.8 48.6 13.2 15.2 139.4 171.3 142.3 102.1 77.8 19.8 16.8 165.4 117.6 145.5 134.0 128.1 129.0 130.6

72.6 30.2 70.7 44.2 38.5 72.2 86.1 48.1 39.1 41.3 30.8 174.3 132.8 147.3 87.3 40.0 48.7 13.2 15.1 137.0 171.3 141.7 96.7 77.9 19.8 16.1 164.7 117.6 145.3 133.8 128.6 128.9 130.8

72.7 31.1 70.5 44.2 38.8 72.4 86.1 48.2 39.2 41.3 30.1 174.3 132.8 147.3 87.3 40.1 48.7 13.2 15.2 137.1 171.4 141.5 96.6 77.9 19.9 16.2 164.7 129.8 129.4 128.8 133.4

73.3 30.3 70.8 44.2 38.9 72.3 86.0 49.5 39.2 40.9 30.8 174.4 130.6 150.5 88.2 39.0 52.8 13.0 15.2 169.0 97.7 119.8 170.3 78.0 20.0 16.9 165.5 117.4 146.1 133.9 128.4 129.1 130.9

72.5 30.8 70.6 44.2 39.0 72.1 85.0 50.0 40.6 41.1 30.5 172.2 134.1 176.5 78.8 45.8 206.1 8.2 15.4

78.0 19.6 16.3 165.5 117.1 146.2 133.9 128.4 129.1 131.0

71.7 29.7 68.8 47.7 37.3 74.3 83.2 48.7 38.8 40.5 31.2 172.0 136.0 145.0 88.4 40.9 49.3 13.2 15.4 126.6 138.3 110.3 142.7 177.6 15.6 16.5 165.3 130.3 129.8 128.3 133.0

21.4 169.8

21.4 169.3 51.5

51.0

51.5

51.3

51.9 57.1

51.6 55.6

52.6

52.5

52.5

51.6

MeO-12 MeO-23 MeO-29

55.6

56.9

51.2

65

T. Akihisa et al. / Phytochemistry 89 (2013) 59–70

(Tables 4 and 5) of 26 were analogous to those of compound 25, although the benzoyl signals for 25 were lacking for 26. The presence of the 2-methyl-2-propenoyl (methacryl) group instead of the benzoyl group at C-1 of 25 was deduced by the 1H [dH 5.60 and 6.22 (each 1H; H2-30 ), and 2.03 (Me-40 )] and 13C [dC 166.2 (C-10 ), 136.3 (C-20 ), 126.0 (C-30 ), and 18.2 (C-40 )] signals (Dong et al., 2010). This was confirmed by the HMBC correlations for H-1 (dH 4.87) with C3, C-10, and C-10 . Thus, compound 26 was assigned as 1-O-methacryl-28-oxosalannic acid methyl ester (3-O-deacetyl-40 -demethyl28-oxosalannin). 2.3. Cytotoxic activity of compounds Thirty-two compounds, 1–32, isolated from the EtOAc-soluble fraction of the MeOH extract of M. azedarach fruits and two reference anticancer drugs, cisplatin and 5-fluorouracil, were evaluated against four human cancer cell lines by MTT assay, and the results are summarized in Table 6. Twelve compounds, 1, 2, 6, 8, 14, 19, 22, 26, and 29–32, exhibited IC50 values (concentration of compound required to inhibit the growth of the cells by 50%) of 0.005–

9.9 lM against one or more cancer cell lines. Thus, compounds 1, 2, 6, 8, 14, 19, 26, and 29–32 against HL60 cell line, compounds 1, 2, and 26 against AZ521 cell line, and compound 22 against SK-BR-3 cell line showed potent activities. In addition, the cytotoxicities of compounds 5 and 32 against AZ521 cells, and compounds 15 and 32 against SK-BR-3, were observed to be almost comparable with those of the reference cisplatin (AZ521: IC50 9.5 lM; SK-BR-3: IC50 18.8 lM). The lung cancer cells (A549) were less sensitive to the compounds tested, and compounds 1, 14–16, 22, 29, and 32 showed weak cytotoxicities (IC50 42.3–94.2 lM), being less active than cisplatin (IC50 18.4 lM). Among the thirty-two compounds described in this paper, potent cytotoxicity (IC50 0.0054– 0.15 lM) of compound 2 against HL60, PC3 (prostate), BEL7404 (liver), SH-SY5Y (CNS), U251 (CNS), and U937 (histocyte) cells (Zhang et al., 2005), and no cytotoxicity of compounds 29 and 30 against HeLa S3 (epithelial) cells (Zhou et al., 2004) have previously been reported. On the basis of the results in Table 6, the following conclusions can be drawn about the structure–activity relationship of the compounds evaluated: (i) Among the trichilin-type limonoids, those

Table 6 Cytotoxicity of compounds 1–32 against four human cancer cell lines. Compound

IC50 (lM)a HL 60 (Leukemia)

A549 (Lung)

AZ521 (Stomach)

SK-BR-3 (Breast)

Trichilin-type limonoid 1 21 3 4 5 6

0.65b ± 0.12 0.005c ± 0.0002 91.4 ± 4.0 82.0 ± 1.9 11.8 ± 1.5 9.1 ± 1.0

63.6 ± 3.5 >100 >100 >100 >100 >100

1.5 ± 0.5 0.009c)±0.001 >100 >100 11.8 ± 7.8 18.8 ± 8.8

90.4 ± 1.3 >100 >100 >100 >100 >100

Vilasinin-type limonoid 7 8

61.2 ± 7.8 5.8 ± 0.8

>100 >100

>100 16.2 ± 1.7

41.5 ± 3.2 >100

Salannin-type limoni 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

67.1 ± 4.1 23.5 ± 8.5 26.6 ± 4.7 56.3 ± 5.1 32.9 ± 6.7 9.9 ± 0.6 54.8 ± 2.2 12.4 ± 2.6 31.7 ± 4.1 14.1 ± 0.3 4.9 ± 0.5 15.2 ± 2.2 25.1 ± 3.2 12.6 ± 4.1 22.7 ± 1.6 50.4 ± 8.9 22.7 ± 2.4 2.8 ± 0.6

>100 >100 >100 >100 >100 94.3 ± 1.1 82.3 ± 0.3 54.5 ± 1.4 >100 >100 >100 >100 >100 90.1 ± 0.2 >100 >100 >100 >100

55.4 ± 6.1 42.1 ± 4.2 85.0 ± 2.6 >100 >100 >100 35.1 ± 3.9 59.8 ± 3.5 82.9 ± 1.2 34.7 ± 5.8 >100 30.0 ± 6.3 78.5 ± 2.9 55.7 ± 7.1 >100 >100 61.7 ± 7.1 3.2 ± 0.6

88.1 ± 1.2 30.6 ± 5.7 31.6 ± 0.7 >100 >100 35.2 ± 3.6 14.9 ± 2.1 82.7 ± 0.5 >100 54.5 ± 4.1 >100 >100 >100 4.3 ± 1.5 91.5 ± 2.2 >100 >100 >100

Nimbin-type limonoid 27 28 29 30

39.2 ± 4.4 32.1 ± 3.7 5.1 ± 1.3 8.5 ± 2.8

>100 >100 83.5 ± 2.6 >100

>100 80.0 ± 0.8 81.6 ± 1.3 >100

94.4 ± 1.0 70.5 ± 3.8 85.0 ± 0.8 94.8 ± 1.5

Nimbolinin-type limonoid 31

5.4 ± 2.8

>100

96.8 ± 0.6

60.8 ± 9.1

Tirucallane-type triterpenoid 32

6.7 ± 0.6

42.3 ± 9.6

11.9 ± 0.8

18.6 ± 2.3

Reference compounds Cisplatin 5-Fluorouracil

4.2 ± 1.1 6.3 ± 2.3

18.4 ± 1.9 >100

9.5 ± 0.5 28.7 ± 0.5

18.8 ± 0.6 >100

a IC50 values based on quintuple points. Cells were treated with compounds (1  104–1  106 M) for 48 h, and cell viability was analyzed by the MTT assay. Each value represents the mean ± SD (n = 3). b Range of concentration of test compound assayed: 3  106–3  108 M. c Range of concentration of test compound assayed: 3  107–3  109 M.

T. Akihisa et al. / Phytochemistry 89 (2013) 59–70

compounds (1 and 2) with a b-oriented epoxy-ring at C-14/C-15 are far more active than those of their 15-oxo analogues (3–6) against HL60 and AZ521 cells. Furthermore, deacetoxylation at C12 increased the activity against HL60 and AZ521 cells among the 15-oxo trichilins (5 vs. 3 and 6 vs. 4). Potent cytotoxicity against HL60 cells of the compounds possessing a C-14/C-15 b-epoxy-ring was observed also for some azadiradione- and gedunin-type limonoids (Kikuchi et al., 2011). (ii) Acetylation of the hydroxy group at C-3 of vilasanin- (8 vs. 7) and salannin-type limonoids (14 vs. 12/13; 16 vs. 15) with a benzoyl or a cinnamoyl group at C-1 increased the activity against HL60 cells. On the contrary to this, acetylation at C-3 reduced activity against HL60, AZ521, and SK-BR-3 cells for salannins possessing a tigloyl group at C-1 (9 vs. 10), and 1-O-detigloyl-1-O-benzoylsalannins (16 vs. 15) against AZ521 and SK-BR-3 cells. (iii) Substitution of a methacrylic group at C-1 with a benzoyl group reduced the activity against HL60 and AZ521 cells for 28-oxo-salannin-type limonoids (26 vs. 25). (iv) Substitution of a benzoyl or cinnamoyl group at C-1 by a methacrylic or a tigloyl group for nimbin-type limonoids reduced the activity against HL60 cells (29 and 30 vs. 27 and 28) is consistent with our recent observation (Kikuchi et al., 2011). 2.4. Apoptosis-inducing activity of compounds 1 and 26

1

(A) 24

48 (h)

5.9 %

12.2 %

5.1 %

31.6 %

67.4 %

14.9 %

11.7 %

52.1 %

26

(B) 24

48 (h)

4.1 %

9.7 %

8.4 %

38.7 %

71.9 %

14.4 %

21.4 %

31.6 %

PI

66

(C)

Negative control 24

Compounds 1 and 26, which exhibited potent cytotoxic activities against HL60 cells (IC50 0.65 and 2.8 lM, respectively), were evaluated for their apoptosis-inducing activity using HL60 cells. HL60 cells were incubated with these test compounds for 24 and 48 h, and then the cells were analyzed by means of flow cytometry with annexin V–propidium 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 (Martin et al., 1995). Annexin V is a calcium-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, PI double positive), or necrotic (annexin V negative, PI positive) cell death. The ratio of early apoptotic cells (lower right) was increased after treatment with 1 in HL60 cells for 24 h (14.9% vs. 1.8% of negative control) and 48 h (52.1% vs. 2.1% of negative control), and that of late apoptotic cells (upper right) was increased after 48 h (31.6% vs. 1.2% of negative control) [Fig. 3(A)]. In the case of compound 26, the ratio of early apoptotic cells was increased after treatment for 48 h (31.6% vs. 2.1% of negative control), and that of late apoptotic cells (upper right) was increased after 24 h (38.7% vs. 1.2% of negative control) [Fig. 3(B)]. These results demonstrated that most of the cytotoxic activities of compounds 1 and 26 against HL60 cells are due to inducing apoptotic cell death. Caspases are known to mediate the apoptotic pathway (Salvesen and Dixit, 1997; Thornberry and Lazebnik, 1998). Caspases-8 and -9, which are initiator caspases, appear to be the apical caspases activated in death receptor- and mitochondrial stress-induced apoptotic cell death, respectively. Initiator caspases are responsible for either directly or indirectly activating various effector caspases, including caspases-3, -6, and -7, which contain short prodomains. Effector caspases cleave a number of structural and regulatory proteins and are directly responsible for many of the apoptotic features, which are nuclear and cytoplasmic condensation, DNA fragmentation, cell membrane composition, and others (Bratton et al., 2000). In order to clarify the mechanisms by which compounds 1 and 26 induce apoptotic cell death, activation of caspases-3, -8, and -9 was evaluated by Western blot analysis. The levels of procaspases-8, -9, and -3 diminished, almost in a time-dependent manner, after treatment with compounds 1 and 26 (Fig. 4). These results suggest that compounds 1 and 26 induced apoptotic

48 (h)

2.9 %

2.6 %

2.5 %

1.2 %

92.5 %

1.8 %

94.3 %

2.1 %

Annexin V-FITC Fig. 3. Compounds 1 and 26 induced apoptosis against HL60 cells. (A) HL60 cells were cultured with compound 1 (5 lM) for 24 and 48 h. (B) HL60 cells were cultured with compound 26 (10 lM) for 24 and 48 h. (C) Negative control. Each value is the mean of three experiments.

1 0

24

26 48 (h)

0

8

24 (h)

Procaspase-8 (57 kDa) Procaspase-9 (47 kDa) Procaspase-3 (32 kDa) β-Actin (45 kDa) Fig. 4. Western blot analysis of HL60 cells treated with compounds 1 (5 lM) and 26 (10 lM). The results are from one representative experiment among three runs, which showed similar patterns to one another.

cell death via both the mitochondrial- and the death receptor mediated pathways. 2.5. Cytotoxicity of compounds 1 and 26 against normal lymphocyte cells Compounds 1 and 26 and cisplatin were then tested for their cytotoxicity against a normal lymphocyte cell line, RPMI 1788, and their selectivity index (SI) value (Pati et al., 2008), which

T. Akihisa et al. / Phytochemistry 89 (2013) 59–70 Table 7 Cytotoxic activities of compounds 1 and 26 and cisplatin against leukemia (HL60) and normal lymphocyte (RPMI 1788) cell lines. Compound

1 26 Cisplatinc

IC50 (lM)a

67

4. Experimental 4.1. General

SIb

HL60 (Leukemia)

RPMI 1788 (Normal lymphocyte)

0.65 ± 0.12 2.8 ± 0.6 4.2 ± 1.1

10.6 ± 3.1 15.0 ± 8.9 1.4 ± 0.03

16.31 5.36 0.33

a IC50 Values based on quintuple points. Cells were treated with compounds (1  104–1  108 M) for 48 h, and cell viability was analyzed by the MTT assay. Each value represents the mean ± SD (n = 3). b SI refers to the selectivity index, which was obtained by dividing the IC50 value for the normal lymphocyte cells by the IC50 value for HL60 cells. c Reference compound.

was determined by dividing the IC50 value for the normal cell line (RPMI 1788) by the IC50 value for the cancer cell line (HL60). As shown in Table 7, compounds 1 and 26 exhibited SI values of 16.31 and 5.36, respectively, greater than that of cisplatin (SI 0.33). From the results of the cytotoxicity evaluation of the limonoids from M. azedarach fruit extracts, it appears that some of these compounds may be valuable anticancer lead compounds. Furthermore, compounds 1 and 26 which induced apoptotic cell death in leukemia and displayed high selective toxicity against leukemia, may be especially promising in this regard. The cytotoxicity of compound 2 against U937 (histocyte) cells has previously been revealed to be associated with the induction of apoptosis (Zhang et al., 2005). 2.6. Inhibitory effects on EBV-EA induction The inhibitory effects on the induction of EBV-EA induced by TPA were further examined as a preliminary evaluation of the potential anti-tumor-promoting effects (Akihisa et al., 2003) for 25 compounds, 1–8, 12, 13, 15–25, 27, 28, 31, and 32. All of the compounds tested exhibited moderate inhibitory effects with IC50 values in the range of 347–530 mol ratio/32 pmol TPA with preservation of high viability (60%) of Raji cells (Supplementary data). Among the compounds tested, three compounds, 1, 6, and 28, exhibited more potent than or almost equivalent inhibitory effects (IC50 347–401 mol ratio/32 pmol TPA) with the reference compound, b-carotene (IC50 397 mol ratio/32 pmol TPA), a vitamin A precursor studied widely in cancer chemoprevention animal models. Since the inhibitory effects against EBV-EA activation have been demonstrated to closely parallel to those against tumor promotion in vivo (Akihisa et al., 2003), compounds 1, 6, and 28 could be valuable anti-tumor-promoters.

3. Conclusions Thirty-one limonoids, including 14 new compounds and one tirucallane-type triterpenoid, were isolated from the fruits of M. azedarach (Meliaceae). Evaluation of cytotoxic activity against four human cancer cell lines for these compounds established that meliarachin C and 3-O-deacetyl-40 -demethyl-28-oxosalannin exhibited potent cytotoxic activity against HL60 cells, and this was demonstrated mainly due to the induction of apoptosis by flow cytometry. Western blot analysis suggested that both compounds induced apoptosis via both the mitochondrial and death receptor-mediated pathways. In addition, 25 compounds were evaluated for their inhibitory effects against the EBV-EA activation induced by TPA in Raji cells. It appears that meliarachin C and 3-Odeacetyl-40 -demethyl-28-oxosalannin may hold promise as effective anticancer agents, especially against HL60 cells. This work has thus provided a further example of the importance of limonoids as potential anticancer agents.

Crystallizations were performed in MeOH, and melting points were determined on a Yanagimoto micro melting point apparatus and are uncorrected. Optical rotations were measured on a JASCO P-2010 polarimeter in EtOH or MeOH at 25 °C. UV spectra, on a JASCO V-630Bio spectrophotometer, and IR spectra, using a JASCO FTIR-300 E spectrometer, were recorded in EtOH and KBr disks, respectively. NMR spectra were acquired with a JEOL ECX-400 (1H, 400 MHz; 13C, 100 MHz) spectrometer in CDCl3. Chemical shifts (d) values are given in ppm with TMS as internal standard, and coupling constants (J) in Hz. HRESIMS were obtained on an Agilent 1100 LC/MSD TOF (time-of-flight) system [ionization mode: positive; nebulizing gas (N2) pressure: 35 psig; drying gas (N2): flow, 12 l min1; temp: 325 °C; capillary voltage: 3000 V; fragmentor voltage: 225 V]. SiO2 (silica gel 60, 230–400 mesh; Merck) and ODS (Chromatorex-ODS, 100–200 mesh; Fuji Silysia Chemical, Ltd., Aichi, Japan) were used for column chromatography (CC). Reversed-phase preparative HPLC was carried out on ODS columns (25 cm  10 mm i.d.) at 25 °C; on a Pegasil ODS-II 5 lm column (Senshu Scientific Co., Ltd., Tokyo, Japan) with MeCN–H2O– AcOH [50:50:0.1 (HPLC system I) or 40:60:0.1 (HPLC system II); flow rate 3.0 ml min1]; on a TSK-ODS-120A 5 lm column (Toso Co., Tokyo, Japan) with MeOH–H2O–AcOH [60:40:0.1, flow rate 3.0 ml min1 (HPLC system III)]; or on a Capcell pak AQ 5 lm column (Shiseido Co., Ltd., Tokyo, Japan) with MeOH–H2O–AcOH [68:32:1, flow rate 2.0 ml min1 (HPLC system IV)] or with MeCN–H2O–AcOH [50:50:0.1, flow rate 2.0 ml min1 (HPLC system V); or 48:52:1, flow rate 3.0 ml min1 (HPLC system VI)]. 4.2. Chemicals and materials Mature fruits of M. azedarach L. (Meliaceae) were collected from plants cultivated at the Toho University herbal garden (Funabashishi, Chiba, Japan) on 31st January, 2008. The plant material was authenticated by one (K.K.) of the authors, and a voucher specimen (TA-TH-MA-0801) was deposited in the herbarium of the School of Pharmaceutical Sciences, Toho University. Chemicals and reagents were purchased as follows: RPMI-1640 medium, fetal bovine serum (FBS; for RPMI-1640 medium), antibiotics (100 units ml1 penicillin and 100 lg ml1 streptomycin), and non essential amino acid (NEAA) from Invitrogen Co. (Carlsbad, CA, USA), Dulbecco’s modified Eagle’s medium (D-MEM), Eagle’s minimal essential medium (MEM), and MTT from Sigma–Aldrich Japan Co. (Tokyo, Japan), 5-fluorouracil, the EBV cell-culture reagents, and butanoic acid from Nacalai Tesque, Inc. (Kyoto, Japan), cisplatin and b-carotene from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and recombinant human (rh) Annexin V/FITC kit (Bender MedSystems) from Cosmo Bio Co. Ltd. (Tokyo, Japan). All other chemicals and reagents were of anal. grade. 4.3. Extraction and isolation Air-dried fruits of M. azedarach (19.7 kg) were pulverized and extracted with hexane (2 h, under conditions of reflux, 3), which gave a hexane extract (588 g). The defatted residue was then extracted with MeOH (room temp., 3 days, 3) to give the extract (1036 g) which was partitioned in an EtOAc–H2O mixture. The aq. layer was extracted with n-BuOH, and removal of the solvent under reduced pressure from the EtOAc-, n-BuOH-, and H2Osoluble portions yielded 103 g, 260 g, and 658 g of residue, respectively. A portion of the EtOAc fraction (91 g) was subjected to SiO2 CC column (800 g). Step gradient elution was conducted with

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n-hexane–EtOAc (1:0 ? 0:1) to give eleven fractions, A–K. Fraction F (3.07 g), from the eluate of n-hexane–EtOAc (3:2 ? 1:1), was subjected to SiO2 CC (120 g) using n-hexane–EtOAc (1:0 ? 0:1) to give eight fractions, F1–F8. Preparative HPLC (system I) of fraction F5 (439 mg) yielded compounds 28 (12.5 mg, tR 22.4 min), 27 (64.0 mg, tR 24.8 min), and 29 (2.2 mg, tR 29.7 min). Fraction F6 (1.43 g) was subjected to an ODS CC [25 g; eluent: MeOH–H2O (11:9 ? 1:0)] to give four fractions, F6a–F6d. Prep. HPLC (system I) of fraction F6b (497 mg) yielded compounds 30 (1.9 mg, tR 29.3 min), 31 (1.5 mg, tR 38.5 min), and 11 (1.2 mg, tR 43.5 min), respectively. Fraction G (5.06 g), from the eluate of n-hexane– EtOAc (1:1 ? 9:11), was subjected to SiO2 CC (160 g) using CHCl3– EtOAc (1:0 ? 0:1) to give seven fractions, G1–G7. Upon ODS CC [70 g; MeOH–H2O (1:1 ? 0:1)] and subsequent SiO2 CC [60 g; nhexane–EtOAc (4:1 ? 7:3)], fraction G3 yielded a fraction (74 mg) from which compounds 9 (2.6 mg, tR 30.0 min) and 14 (7.4 mg, tR 40.8 min) were isolated by HPLC (system III). Fraction G4 was passed through an ODS [50 g; MeCN–H2O (2:3 ? 1:0)] to give a fraction (206 mg) from which was isolated compound 32 (17.0 mg, tR 42.0 min) by HPLC (system V). Fraction H (3.81 g), from the eluate of n-hexane–EtOAc (2:3), was subjected to SiO2 CC [100 g; n-hexane–EtOAc (4:1 ? 0:1)] to afford eleven fractions, Frs. H1–H11. Further ODS CC [30 g; MeOH–H2O (9:1 ? 3:2)] of fraction H7 (1.32 g) yielded six fractions, H7a–H7f. HPLC of fractions H7d (298 mg; system I) and H7f (146 mg; system IV) yielded compounds 1 (2.6 mg, tR 30.0 min) and 25 (9.1 mg, tR 42.2 min), and compounds 26 (5.8 mg, tR 32.8 min) and 16 (2.0 mg, tR 37.2 min), respectively. Fraction I (15.54 g), from the eluate of nhexane–EtOAc (3:7 ? 1:4), was subjected to SiO2 CC [500 g; n-hexane–EtOAc (1:0 ? 0:1)] to give ten fractions, I1–I10. ODS CC [40 g; MeCN–H2O (3:7 ? 1:0)] of fraction I4 (4.27 g) yielded a fraction (1.17 g). HPLC (system I) of a portion (250 mg) of the fraction gave compounds 10 (15.0 mg, tR 26.4 min) and 15 (23.0 mg, tR 30.0 min). Further ODS CC [80 g; MeOH–H2O (9:11 ? 1:0)] of fraction I5 (4.96 g) yielded eight fractions, I5a–I5 h. HPLC of fractions I5c (1.43 g; system II), I5d (707 mg; system VI), I5f (716 mg; system VI), and I5 g (141 mg; system V) yielded compound 2 (303.1 mg, tR 14.2 min), compounds 4 (12.0 mg, tR 20.0 min), 3 (2.2 mg, tR 24.0 min), and 6 (3.9 mg, tR 17.2 min), compounds 7 (15.3 mg, tR 32.0 min), 12 (78.7 mg, tR 36.8 min), and 13 (4.0 mg, tR 48.0 min), and compound 8 (4.8 mg, tR 53.6 min), respectively. Upon SiO2 CC [135 g; n-hexane–EtOAc (4:1 ? 0:1)] and subsequent HPLC (system VI), fraction I5e (1.78 g) yielded compounds 21 (7.2 mg, tR 20.0 min), 18 (4.4 mg, tR 21.2 min), 22 (7.4 mg, tR 23.2 min), and 5 (15.3 mg, tR 25.0 min). Fraction J (2.88 g), from the eluate of n-hexane–EtOAc (1:9), was subjected to ODS CC [80 g; MeOH–H2O (3:7 ? 1:0)] to give six fractions, J1–J6. HPLC of fractions J3 (497 mg; system V) and J4 (150 mg; system III) yielded compounds 17 (12.7 mg, tR 27.6 min) and 24 (3.4 mg, tR 35.2 min), and compounds 19 (1.3 mg, tR 32.0 min) and 20 (2.8 mg, tR 36.0 min), respectively. Fraction K (2.83 g), from the eluate of n-hexane–EtOAc (0:1), was passed through an ODS column [100 g; MeCN–H2O (1:4 ? 1:0)] and a fraction (166 mg) obtained was subjected to HPLC (system II) which yielded compound 23 (1.6 mg, tR 36.8 min). 4.3.1. 12-Dehydroneoazedarachin D (5) Fine needles; mp 140–143 °C; [a]D25 + 18.6 (c 0.51, MeOH); IR (KBr) mmax cm1: 3350 (OH), 1732, 1715 (C@O), 874 (furan); For 1 H NMR and 13C NMR spectroscopic data, see Tables 2 and 5; HRESIMS m/z: 553.2451 [M+Na]+ (calcd for C29H38O9Na, 553.2413). 4.3.2. 12-Dehydro-29-exo-neoazedarachin D (6) Fine needles; mp 193–196 °C; [a]D25 71.0 (c 0.48, EtOH); IR (KBr) mmax cm1: 3368 (OH), 1730, 1710 (C@O), 874 (furan); For

1

H NMR and 13C NMR spectroscopic data, see Tables 2 and 5; HRESIMS m/z: 553.2437 [M+Na]+ (calcd for C29H38O9Na, 553.2413). 4.3.3. 1-O-Decinnamoyl-1-O-Z-cinnamoylohchinin (13) Fine needles; mp 131–134 °C; [a]D24 + 50.7 (c 0.85, MeOH); UV (EtOH) kmax (log e) nm: 217 (3.61), 280 (3.41); IR (KBr) mmax cm1: 3367 (OH), 1730, 1710 (C@O), 1640, 1453 (cinnnamoyl), 873 (furan); For 1H NMR and 13C NMR spectroscopic data, see Tables 2 and 5; HRESIMS m/z: 603.2945 [M+H]+ (calcd for C36H43O8, 603.2957). 4.3.4. 1-O-Decinnamoyl-1-O-benzoylohchinin (15) Fine needles; mp 155–158 °C; [a]D24 + 62.2 (c 1.22, MeOH); UV (EtOH) kmax (log e) nm: 245 (3.78); IR (KBr) mmax cm1: 3338 (OH), 1735, 1710 (C@O), 1657 (benzoyl), 872 (furan); For 1H NMR and 13 C NMR spectroscopic data, see Tables 2 and 5; HRESIMS m/z: 599.2543 [M+Na]+ (calcd for C34H40O8Na, 599.2620). 4.3.5. Ohchininolide (17) Fine needles; mp 134–137 °C; [a]D24 + 103.5 (c 1.12, MeOH); UV (EtOH) kmax (log e) nm: 217 (4.36), 276 (4.13); IR (KBr) mmax cm1: 3400 (OH), 1765 (c-lactone), 1740, 1717 (C@O), 1638 (cinnamoyl); For 1H NMR and 13C NMR spectroscopic data, see Tables 2 and 5; HRESIMS m/z: 641.2724 [M+Na]+ (calcd for C36H42O9Na, 641.2726). 4.3.6. 1-O-Decinnamoyl-1-O-benzoylohchininolide (18) Amorphous solid; [a]D25 + 63.7 (c 0.88, EtOH); UV (EtOH) kmax (log e) nm: 226 (4.11); IR (KBr) mmax cm1: 3338 (OH), 1750 (c-lactone), 1732, 1715 (C@O), 1657 (benzoyl); For 1H NMR and 13C NMR spectroscopic data, see Tables 3 and 5; HRESIMS m/z: 615.2560 [M+Na]+ (calcd for C34H40O9Na, 615.2570). 4.3.7. 23-Methoxyohchininolide A (19) Fine needles; mp 136–139 °C; [a]D25 + 160.2 (c 0.55, EtOH); UV (EtOH) kmax (log e) nm: 225 (4.20), 275 (4.06); IR (KBr) mmax cm1: 3361 (OH), 1766 (c-lactone), 1732, 1712 (C@O), 1639 (cinnamoyl); For 1H NMR and 13C NMR spectroscopic data, see Tables 3 and 5; HRESIMS m/z: 671.2821 [M+Na]+ (calcd for C37H44O10Na, 671.2832). 4.3.8. 23-Methoxyohchininolide B (20) Fine needles; mp 132–135 °C; [a]D25 + 141.0 (c 0.80, EtOH); UV (EtOH) kmax (log e) nm: 220 (4.08), 277 (3.96); IR (KBr) mmax cm1: 3350 (OH), 1765 (c-lactone), 1735, 1713 (C@O), 1638 (cinnamoyl); For 1H NMR and 13C NMR spectroscopic data, see Tables 3 and 5; HRESIMS m/z: 671.2835 [M+Na]+ (calcd for C37H44O10Na, 671.2832). 4.3.9. 23-Hydroxyohchininolide (21) Fine needles; mp 148–151 °C; [a]D24 + 29.2 (c 1.30, EtOH); UV (EtOH) kmax (log e) nm: 217 (4.20), 276 (3.81); IR (KBr) mmax cm1: 3368 (OH), 1750 (c-lactone), 1735, 1710 (C@O), 1637 (cinnamoyl); For 1H NMR and 13C NMR spectroscopic data, see Tables 3 and 5; HRESIMS m/z: 657.2666 [M+Na]+ (calcd for C36H42O10Na, 657.2675). 4.3.10. 1-O-Decinnamoyl-1-O-benzoyl-23-hydroxyohchininolide (22) Amorphous solid; [a]D25 + 61.1 (c 1.48, EtOH); UV (EtOH) kmax (log e) nm: 226 (4.06), 274 (3.99); IR (KBr) mmax cm1: 3368 (OH), 1750 (c-lactone), 1730, 1717 (C@O), 1655 (benzoyl); For 1H NMR and 13C NMR spectroscopic data, see Tables 3 and 5; HRESIMS m/z: 631.2508 [M+Na]+ (calcd for C34H40O10Na, 631.2519). 4.3.11. 21-Hydroxyisoohchininolide (23) Fine needles; mp 150–153 °C; [a]D25 + 327.1 (c 0.30, EtOH); UV (EtOH) kmax (log e) nm: 220 (4.11), 276 (3.90); IR (KBr) mmax cm1:

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3350 (OH), 1760 (c-lactone), 1733, 1710 (C@O), 1638 (cinnamoyl); For 1H NMR and 13C NMR spectroscopic data, see Tables 4 and 5; HRESIMS m/z: 657.2645 [M+Na]+ (calcd for C36H42O10Na, 657.2675). 4.3.12. 17-Defurano-17-oxoohchinin (24) Fine needles; mp 125–128 °C; [a]D25 + 92.5 (c 0.54, EtOH); UV (EtOH) kmax (log e) nm: 217 (4.34), 240 (4.10), 279 (4.41); IR (KBr) mmax cm1: 3350 (OH), 1733, 1710 (C@O), 1638 (cinnamoyl); For 1H NMR and 13C NMR spectroscopic data, see Tables 4 and 5; HRESIMS m/z: 573.2523 [M+Na]+ (calcd for C32H38O8Na, 573.2464). 4.3.13. 1-O-Decinnamoyl-1-O-benzoyl-28-oxoohchinin (25) Fine needles; mp 204–207 °C; [a]D25 + 123.8 (c 0.76, EtOH); UV (EtOH) kmax (log e) nm: 230 (3.91); IR (KBr) mmax cm1: 3368 (OH), 1778 (c-lactone) 1730, 1714 (C@O), 1656 (benzoyl), 873 (furan); For 1H NMR and 13C NMR spectroscopic data, see Tables 4 and 5; HRESIMS m/z: 613.2412 [M+Na]+ (calcd for C34H38O9Na, 613.2413). 4.3.14. 3-O-Deacetyl-40 -demethyl-28-oxosalannin (26) Fine needles; mp 228–231 °C; [a]D25 + 87.4 (c 0.19, EtOH); IR (KBr) mmax cm1: 3338 (OH), 1772 (c-lactone), 1732, 1714 (C@O), 872 (furan); For 1H NMR and 13C NMR spectroscopic data, see Tables 4 and 5; HRESIMS m/z: 577.2424 [M+Na]+, (calcd for C31H38O9Na, 577.2413). 4.4. Cell line and culture condition Cell lines HL60 (human leukemia), AZ521 (stomach), A549 (lung), and SK-BR-3 (breast) were obtained from Riken Cell Bank (Tsukuba, Ibaraki, Japan). A normal cell line, RPMI 1788 (lymphocytic), was obtained from Health Science Research Resources Bank (Osaka, Japan). Three cell lines, HL60, SK-BR-3, and RPMI 1788, were grown in RPMI 1640 medium, while A549 and AZ521 cell lines were grown in D-MEM and in 90% D-MEM + 10% MEM + 0.1 mM NEAA, respectively. The medium was supplemented with 10% FBS and antibiotics. Cells were incubated at 37 °C in a 5% CO2 humidified incubator. 4.5. Biological evaluation The protocols of cytotoxicity assay, Annexin V–propidium iodide (PI) double staining, western blot analysis, and in vitro EBVEA activation experiment (Akihisa et al., 2006; Kikuchi et al., 2010, 2011; Takaishi et al., 1992) were described in Supplementary data. Acknowledgements This work was supported, in part, by a Grant-in-Aid from the Japan Society for the Promotion of Science (No. 23590017), 2011–2013.

Appendix A. Supplementary data Supplementary data Protocols of biological evaluation (S1), inhibitory effects on the induction of Epstein–Barr virus early antigen (EBV-EA) of compounds from M. azedarach fruit extract (S2), and 1H and 13C NMR spectra of the new compounds (S3–S30). Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2013. 01.015.

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