Phytochemistry 104 (2014) 79–88

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Lathyrane-type diterpenoids from the seeds of Euphorbia lathyris Jin Lu a, Guoyu Li b, Jian Huang a,c,⇑, Cui Zhang a, Lan Zhang a, Ke Zhang b, Pingya Li c, Ruichao Lin d, Jinhui Wang a,b,⇑ a

School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China College of Pharmacy, Xinjiang Medical University, Urumqi 830011, People’s Republic of China c Department of New Drug, Institute of Frontier Medical Sciences, Jilin University, Changchun 130021, People’s Republic of China d School of Traditional Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China b

a r t i c l e

i n f o

Article history: Received 18 April 2013 Received in revised form 10 January 2014 Available online 23 May 2014 Keywords: Euphorbia lathyris Euphorbiaceae Lathyrane-type diterpenoid Euphorbia Factor L12–L21 Cytotoxicity

a b s t r a c t Ten lathyrane-type diterpenoids named Euphorbia Factor L12–L21 (1–10) and twelve known diterpenoids (11–22) were isolated from seeds of Euphorbia lathyris. The structures of these compounds were determined by extensive spectroscopic (UV, IR, HRESIMS, 1D and 2D NMR) analyses. In addition, the configuration of Euphorbia Factor L12 (1) was further confirmed by X-ray crystallographic and circular dichroism (CD) analyses. A putative biogenetic relationship to these compounds was proposed. Cytotoxicity of the isolated compounds against C6 and MCF-7 cell lines were evaluated. Compounds 1, 5, 7, 12 and 17 exhibited considerable cytotoxic activities (IC50 12.4–36.2 lM). Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The seeds of Euphorbia lathyris, which is a common traditional Chinese medicine, have been used to treat hydropsy, ascites, terminal schistosomiasis, and snakebites (Chinese Materia Medica, 1998). Up to this study, previous chemical and biological research on this plant resulted in series of diterpenoids named Euphorbia Factors L1–L11 (Adolf and Hecker, 1971, 1975; Appendino et al., 2003; Liao et al., 2005) and two rearranged lathyrane skeletons called lathyranoic acid A (Liao et al., 2005) and lathyranone A (Gao et al., 2007), respectively. Among them, the lathyrane-type factors were demonstrated to be powerful P-glycoprotein inhibitors; these compounds are also well-known as modulators of multidrug resistance (MDR) (Appendino et al., 2003; Engi et al., 2007; Duarte et al., 2007; Reis et al., 2013). Though the genus Euphorbia is a rich source of lathyrane-type diterpenoids (Shi et al., 2008), few reports detailing their cytotoxic activity are available (Gao et al., 2007; Itokawa et al., 1989). Herein, described are the isolation and structural elucidation of the lathyrane-type diterpenoids (1–22, ten new and twelve known) from the seeds of E. lathyris,

⇑ Corresponding authors. Address: School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China. Tel./fax: +86 24 23986479 (J. Huang), +86 993 2055001 (J. Wang). E-mail addresses: [email protected] (J. Huang), wjh.1972@yahoo. com.cn (J. Wang). http://dx.doi.org/10.1016/j.phytochem.2014.04.020 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

as well as the cytotoxic activities against cancer cell lines (C6, MCF-7, Hela and HepG2). 2. Results and discussion Compound 1 has a molecular formula of C29H36O6 as determined by HRESIMS [m/z 503.2411 [M+Na]+ (calcd for C29H36O6Na, 503.2410)] and was obtained in colorless crystal form. The presence of an ester carbonyl and conjugated ketone groups (1744, 1657 and 1628 cm1) were evident from analysis of its IR spectrum. The 1H and 13C NMR spectra of 1 indicated the occurrence of one acetate group [dH 2.16 (3H, s); dC 169.5], and one ketone group (dC 194.9) (Tables 1 and 2). Additionally, the 1H NMR spectrum contained resonances attributed to five methyl groups [dH 1.01 (3H, d, J = 6.6 Hz), 1.09 (3H, s), 1.16 (3H, s), 1.21 (3H, s), 1.90 (3H, s)] and one benzoate group [dH 8.11 (2H, dd, J = 7.8, 1.0 Hz), 7.45 (2H, t, J = 7.8 Hz), 7.60 (1H, dt, J = 7.8, 1.0 Hz)]. The 1H–1H COSY spectrum demonstrated the presence of three structural fragments (a, b and c) with correlated protons (Fig. 2), whose connectivities were determined using the HMBC spectrum. The existence of correlations from dH 1.76 (H-4) to dC 57.3 (C-5), dH 1.98 (Ha-7) to dC 57.3 (C-5) and dC 63.9 (C-6) implied a 5,6-epoxy group was present at C5, C6. Correlations between dH 1.16 (H-17), dC 38.4 (C-7) and dC 57.3 (C-5) were indication of a CH3 group at C-17. The HMBC correlations of H-1 with C-14 and C-15, H-4 with C-14, H-18 and H-19 with C-9, C-10 and C-11, H-20 with C-12, C-13 and C-14 (Fig. 2), respectively, supported the presence of a

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J. Lu et al. / Phytochemistry 104 (2014) 79–88

Table 1 H NMR spectroscopic data for the diterpene moiety of compounds 1–5 in CDCl3.

1

a b

No.

1a

1a 1b 2 3 4 5 7a 7b 8a 8b 9 11 12 16 17a 17b 18 19 20

3.64 1.71 2.21 5.68 1.76 3.28 1.98 1.48 2.02 1.47 1.17 1.54 6.98 1.01 1.16

2a dd (13.8, 7.6) m m t (3.3) dd (9.3, 3.3) d (9.3) dd (13.8, 1.8) d (1.8) m m d (6.7) dd (11.0, 6.7) d (11.0) d (6.6) s

3.45 1.41 2.03 5.38 1.56 2.79 1.31 1.92 1.47 1.26 1.13 1.98 6.97 0.87 1.07

1.21 s 1.09 s 1.90 s

3b dd (13.8, 7.6) d (13.8) m t (3.8) dd (9.0, 3.8) d (9.0) m d (14.1) m m d (6.7) dd (10.8, 6.7) d (10.8) d (6.6) s

1.20 s 1.06 s 1.83 s

4b

3.40 1.78 2.35 5.78 2.92 6.36 5.54

dd (14.1, 8.4) dd (14.1, 11.5) m t (3.6) dd (7.8, 3.6) d (7.8) dd (9.0, 3.3)

2.24 2.36 1.33 1.52 6.51 0.95 5.25 5.51 1.20 1.27 1.82

m m m dd (11.0, 8.4) d (11.0) d (6.6) s s s s s

3.44 1.60 2.26 5.55 2.76 6.08 2.07 2.20 1.96 1.75 1.15 1.39 6.48 0.89 4.99 4.72 1.17 1.15 1.69

5a dd (14.1, dd (14.1, m t (3.3) dd (10.2, d (10.2) m m m m m dd (11.4, d (11.4) d (5.1) s s s s s

8.7) 12.0)

3.55 1.67 2.39 5.86 2.91 6.20 2.06 2.18 2.27 2.19 1.17 1.41 6.53 0.96 5.02 4.77 1.17 1.18 1.72

3.3)

8.4)

dd (14.4, 8.4) dd (14.4, 12.0) m t (3.5) dd (9.3, 3.5) d (9.3) m m m m m dd (11.0, 8.4) d (11.0) d (7.2) s s s s s

Recorded at 600 MHz in CDCl3. Recorded at 300 MHz in CDCl3.

Table 2 C NMR spectroscopic data for the diterpene moiety of compounds 1–10 in CDCl3 (150 MHz).

13

No.

1

2

3

4

5

6

7

8

9

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

46.4 38.4 80.8 51.6 57.3 63.9 38.4 23.4 34.0 26.4 29.9 144.4 134.6 194.9 91.9 13.8 20.0 29.1 16.5 12.6

45.7 37.7 80.4 51.1 56.8 63.3 38.3 29.7 33.8 26.3 23.1 144.2 134.2 194.7 91.4 13.4 19.9 29.1 16.4 12.3

47.9 37.7 79.5 52.9 64.2 141.7 79.0 28.7 31.3 24.7 27.8 142.4 135.7 197.6 91.9 14.2 119.1 28.7 16.6 12.8

48.3 37.2 80.2 52.2 65.8 144.4 35.0 21.2 35.4 25.3 28.5 146.7 134.2 196.9 92.4 14.1 115.5 29.0 16.8 12.5

48.5 37.7 81.6 52.1 65.4 144.4 34.9 21.7 35.4 25.4 28.6 146.6 134.2 196.5 92.4 14.2 115.5 29.0 16.0 12.5

45.0 38.9 81.6 50.8 124.9 140.6 32.2 28.5 34.1 24.9 29.3 147.1 132.6 194.3 94.8 14.0 64.0 29.1 16.4 12.4

44.6 38.3 81.2 50.6 124.7 140.2 32.1 28.4 34.1 24.7 29.3 147.0 132.3 194.6 92.5 13.7 63.9 29.1 16.1 12.3

44.6 38.2 81.3 50.3 122.0 144.7 31.4 28.2 34.3 24.9 29.8 147.2 132.1 194.6 94.6 13.7 62.8 29.1 16.1 12.3

48.9 37.2 81.2 54.9 63.6 134.5 133.2 23.8 30.3 25.0 27.7 142.2 134.6 196.7 93.0 14.2 65.4 28.6 17.0 11.9

45.7 38.5 76.8 55.1 63.9 144.7 79.7 29.9 25.6 16.2 29.7 81.8 75.2 219.3 85.8 13.8 124.0 29.1 19.1 21.6

3,15-di-O-acyljolkinol-5b,6b-oxide skeleton for 1 (Duarte et al., 2007; Tian et al., 2011; Uemura et al., 1976; Adolf et al., 1984a; Valente et al., 2004; Vasas et al., 2004). Additionally, the correlation of dH 5.68 (H-3) with dC 165.4 (C-10 ) indicated a benzoate moiety was located at C-3. Therefore, compound 1 was deduced to be 15-O-acetyl-3-O-benzoyljolkinol-5b,6b-oxide, which was previously reported from Euphorbia characias (Seip and Hecker, 1983), but without supporting spectroscopic data or a proposed configuration. In the NOESY spectrum, the key NOE correlations for H-4/H-2, H3/H-2/H-4 and H3-17/H-4 supported that H-2, H-3 and H3-17 were all in an a-orientation (Xu et al., 2012). Meanwhile, the H-5/Hb-7/ Hb-8 correlation indicated that H-5 was in a b-orientation. The NOE effect observed between H-20 and H-11 was consistent with a (E)-geometry for the C-12/C-13 double bond; this assignment was confirmed by the upfield-shifted carbon signal of C-20 (dC 12.3) (Liao et al., 2005). The correlations for H3-18 with H-9 and H-11 established a cis orientation for H-9 and H-11, similar to other lathyrane diterpenoids (Evans and Taylor, 1983). Based on the above, the proposed relative structure of 1 was elucidated as shown

(Fig. 2) and confirmed by single-crystal X-ray crystallographic analysis; the perspective ORTEP plot was shown (Fig. 3). This structure was similar to that of (12E,2S,3S,4R,5R,6R,9S,11S,15R)-3,15-diacetoxy-5,6-epoxylathyr-12-en-14-one (Tian et al., 2011), as well as jolkinols A and B (Adolf et al., 1984a), which also had an assemblable 3b substituent. Because the absolute structure of the former was validated by single-crystal X-ray diffraction after isolation from the same genus E. micractina Boiss, the characteristic Cotton effects (223 nm, ester group, p?p⁄, De 2.4699; 265 nm, a,b-unsaturated ketone, p?p⁄, De +2.8274 in CH3OH) in the circular dichroism (CD) spectrum of 1 (Fig. 4) were consistent with the model compound. Therefore, compound 1 was identified as 15-O-acetyl-3-O-benzoyljolkinol-5b,6b-oxide. Compound 2 had a molecular formula of C30H38O6 as determined by HRESIMS and showed similar UV and IR data to those of 1. A comparison of the NMR spectroscopic data for 2 and 1 (Tables 1 and 2) indicated that the benzoyloxy group in 1 was replaced with a phenylactyloxy group (dH 3.71, 7.35, 7.30 and 7.25 and dC 170.0, 42.2, 134.7, 129.5, 128.4 and 127.0) in 2. In the HMBC spectrum, the correlation between dH 5.38 (H-3) and

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J. Lu et al. / Phytochemistry 104 (2014) 79–88

2 16

O

20

1 O 14 13

3

R O

H

10

R1 O

17

H

O C

R O

H O

O R 2

O C

3 R 1 = benzoyl R 2 = salicyl

O C

H

O 6 R 1 = benzoyl

R2

5 R = salicyl

16 R = benzoyl 17 R = phenylacetyl 18 R = nicotinoyl

O

O

H

O

C

O

H

O

H R1 O

H

H O

4 R = acetyl

15 R 1 = R2 = benzoyl

H R1 O

H

O

H

H

2 R = phenylacetyl

O

O

O

C

H

18

1 R = benzoyl

O

O

C

H 19

15 12 119 4 8 5 6 7

O

O C

O

O

O C

R3

H O

O

H

R2

R O O C

H O

O

R 2 = acetyl

9 R 1 = benzoyl R2 = acetyl R3 = H

7 R 1 = phenylacetyl R 2 = acetyl

13 R1 = benzoyl R 2 = H R 3 = acetyl

14 R = benzoyl

8 R 1 = phenylacteyl R2 = H

20 R1 = benzoyl R 2 = R 3 = acetyl

21 R = phenylacetyl

11 R1 = cinnamyl

R2 = H

12 R1 = hexanoyl

R2 = acetyl

19 R1 = cinnamyl

R2 = acetyl

O

H

HO HO HO R1 O O C

O C

H O

H O

O

C O O

O R 2

HH

10 R1 = R 2 = benzoyl

O C

OH

22 Fig. 1. Compounds 1–22.

O O C

O

b

O C

O

a O

c

A

B

Fig. 2. 1H–1H COSY (bold), key HMBC (A) NOESY (B) correlations of 1.

dC 170.0 (C-10 ) was used to locate the phenylactyloxy group at C-3. Compound 2 was identified as 15-O-acetyl-3-O-phenylacteyljolkinol-5b,6b-oxide. For compound 3, its HRESIMS indicated a molecular formula of C38H42O10 through the presence of a peak at m/z 681.2679 [M+Na]+. The 1H and 13C NMR spectra of 3 (Tables 1 and 2) suggested that it was similar to compound 15 (Seip and Hecker, 1983). The correlations of the signals at dH 5.78 (H-3), 6.36 (H-5),

5.54 (H-7) and dC 166.0(C-10 ), 169.7 (C-100 ), 169.1 (C-1000 ) demonstrated the presence of acyloxy groups at C-3, C-5 and C-7, respectively. Moreover, the only difference between 3 and 15 was that C-7 was connected to a salicylate moiety (Section 4) in 3 other than a benzoate group in 15. Consequently, the structure 3 was established. The same relative configuration of 3 and 15 was established using the NOESY plot (Supplementary Information, Fig. S28). The absolute configuration of 3 was established

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J. Lu et al. / Phytochemistry 104 (2014) 79–88

Fig. 3. X-ray ORTEP drawing of compound 1.

compound 1 compound 3 compound 4 compound 6 compound 9 compound 10

30

Δε(M-1cm-1)

through the exciton chirality method (Narayanan et al., 1971; Harada et al., 1975, 1981). In the CD spectrum of 3 (Fig. 4), a positive maximum (246 nm, De +1.4691) and a negative maximum (223 nm, De 2.6846) due to two chromophoric groups (benzoate and salicylate) at C-3 and C-7 were observed indicating that the benzoate and salicylate groups have a clockwise relationship (Supplementary Information, Fig. S29). Therefore, the absolute configuration of 3 was elucidated and named as 5,15-di-O-acetyl3-O-benzoyl-7-O-salicyllathyol. The HRESIMS of 4 and 5 exhibited [M+Na]+ at m/z 483.2358 and 561.2462, respectively, where corresponded to molecular formulae of C26H36O7 and C31H38O8. Compounds 4 and 5, as well as 16, 17, 18, were found to differ only in the type of ester group attached to C-3. The occurrence of signals typical for an acetyloxy group in 4 and a salicyloxy group in 5 (Section 4), instead of the benzoyloxy, phenylactyloxy and nicotinoxy groups in 16, 17, 18, together with the HMBC correlation in 4 and 5 for H-3 (dH 5.55 and 5.86), with the corresponding ester carbonyls (dC 170.7 and 169.4) confirmed the attachment of the acetyloxy and salicyloxy groups to C-3 in 4 and 5. Therefore, the gross structures of 4 and 5 were established. The same configuration was inferred for 4 and 5 based on the NOESY and CD spectra of 4, 5, 16, 17 and 18 (Fig. 4) (Jiao et al., 2008). The structures of 4 and 5 were elucidated as 3,5, 15-tri-O-acetyllathyrol and 5,15-di-O-acetyl-3-O-salicyllathyrol, respectively. Compound 6 was obtained as colorless, blocky, crystals. The HRESIMS indicated the molecular formula as C31H38O7. Analysis of the 1H and 13C NMR spectra (Tables 2 and 3) suggested that it was similar to 17-hydroxyjolkinol (Tian et al., 2011; Adolf et al., 1984b; Jiao et al., 2009); that lathyrane derivative has, however, a 5,6-double bond, as confirmed by the 2D NMR measurements. Two acyl groups (benzoyloxy and acetoxy) (Section 4) were located at C-3 and C-17, respectively, using the correlations between H-3 and H-17, as well as and the carbon signals for the ester carbonyls. The NOE and the similarity of the CD data between 6 (Fig. 4) and (5E,12E,2S,3S,4S,9S,11S,15R)-3,15-diacetoxylathyra-5,12-dien-14one (Tian et al., 2011) demonstrated that compound 6 was 15,17di-O-acetyl-3-O-benzoyl-17-hydroxyjolkinol. The structure of compound 6 had been depicted by Appendino and coworkers

20

10

0

-10 190

240

290

340

390

wavelength(nm) Fig. 4. CD spectra for compounds 1, 3, 4, 6, 9, 10 (in MeOH).

(Appendino et al., 2003), without a structural interpretation or reference. Compound 7 was determined to have a molecular formula of C32H40O7 via HRESIMS. Using the NMR spectroscopic data for 7 and 6 (Tables 2 and 3), the only difference was the occurrence of signals typical for an additional phenylacetyloxy group in 7 (Section 4) instead of those for a benzoyloxy group bonded to C-3 in 6 (similar to 2 and 1). Additionally, compound 8 was a 17-deacetyl derivative of 7, because one acetoxymethyl in 7 was replaced with a CH2OH (IR, 3430 cm1) in 8; the CH2OH group was located at C-17 because C-17 was shifted upfield to dC 62.8 from 63.9 (Table 2). Accordingly, compound 7 was identified as 15,17-diO-acetyl-3-O-phenylacetyl-17-hydroxyjolkinol, and 8 was 15-Oacetyl-3-O-phenylacetyl-17-hydroxyjolkinol. Compound 9 was a 5-deacetyl derivative of the known compound 20 (Adolf et al., 1984b), as indicated by its spectroscopic data (Tables 2 and 3). Meanwhile, the chemical shift for C-5 moved from 65.3 to 63.6, indicating replacement of the 5-OAc group in 20 with a 5-OH in 9. Therefore, compound 9 was 15,17-di-O-acetyl-3O-benzoyl-5,17-dihydroxyisolathyol.

83

J. Lu et al. / Phytochemistry 104 (2014) 79–88 Table 3 H NMR spectroscopic data for the diterpene moiety of compounds 6–10 in CDCl3 (600 MHz).

1

No.

6

1a 1b 2 3 4 5 7a 7b 8a 8b 9 11 12 16 17a 17b 18 19 20

3.67 1.62 2.32 5.54 2.65 5.68 2.34 1.99 2.21 1.42 1.09 1.43 6.58 0.89 4.39 4.11 1.18 1.04 1.87

7 dd (14.2, 8.2) dd (14.2, 12.4) m t (3.3) dd (11.0, 3.3) d (11.0) m m m m m m d (11.6) d (6.6) d (12.2) d (12.2) s s

8

3.50 1.38 2.15 5.26 2.65 5.19 2.13 1.91 2.15 1.23 1.05 1.37 6.41 0.89 4.27 4.05 1.17 1.03 1.80

dd (14.0, 8.0) m m t (3.2) dd (10.9, 3.2) d (10.9) m m m m m m d (11.4) d (6.6) d (12.2) d (12.2) s s s

3.50 1.40 2.15 5.24 2.61 5.08 2.10 2.09 2.15 1.26 1.07 1.26 6.42 0.90 3.86 3.62 1.16 1.03 1.80

Compound 10 had a molecular formula of C36H42O10 according to the HRESIMS data (m/z 657.2718 [M+Na]+, calcd for C36H42O10 Na, 657.2676). The presence of a hydroxyl group (3426 cm1) was apparent in its IR spectrum. The 1H and 13C NMR spectroscopic data resembled closely those of 15 (Tables 2 and 3), except for the absence of signals for the 12,13-double bond and the presence of resonance for two sp3 hybrid C-12 and C-13 (dC 81.8, 75.2). Therefore, 10 was a 12,13-dihydroxy analogue of 15. In addition, an acetyl moiety was absent, although it is located at C-15, according to the literature (Gao et al., 2007; Hohmann et al., 1999). Analysis of the NOESY spectrum of 10, H-12/H3-19/Hb-8 and H3-20/H-3 established the presence of OHa-12 and OHb-13 moieties. Similar coupling constants and Cotton effects for compound 10, 3 and 15 identified compound 10 as 5-O-acetyl-3,7-di-O-benzoyl-12a,13bdihydroxylathyol. Compounds 11–14 were reported as natural products for the first time. Their 1H and 13C NMR spectroscopic data were assigned

Table 4 H (600 MHz) and

1

No.

dd (14.0, 8.0) m m t (3.4) dd (11.2, 3.4) d (11.2) m m m m m m d (11.4) d (6.6) d (12.6) d (12.6) s s s

10

3.51 1.64 2.36 5.90 2.61 5.12 5.61

dd (14.4, 8.6) m m t (3.3) dd (8.3, 3.3) d (8.3) dd (10.9, 5.6)

2.27 1.88 2.25 5.58 3.23 6.05 5.63

dd (11.0, 7.5) t (11.0) t (7.5) t (3.6) dd (11.0, 3.6) d (11.0) d (11.0)

2.38 2.35 1.26 1.47 6.50 0.98 4.51 4.51 1.17 1.26 1.76

m m m dd (11.5, 8.6) d (11.5) d (6.6) s s s s s

2.14 2.57 0.99 1.25 4.49 0.95 5.48 5.22 1.17 1.36 1.39

m m m m s d (6.6) s s s s s

via 2D NMR experiments including 1H–1H COSY, HSQC, HMBC and NOESY (Table 4). The known compounds were identified by comparing their spectroscopic data with that reported in the literature as Euphorbia Factor L2 (15) (Appendino et al., 1999a), Euphorbia Factor L3 (16) (Appendino et al., 1999a), deoxy Euphorbia Factor L1 (17) (Appendino et al., 1999b), Euphorbia Factor L8 (18) (Itokawa et al., 1990), Euphorbia Factor L7a (19) (Adolf et al., 1984b), Euphorbia Factor L7b (20) (Adolf et al., 1984b), Euphorbia Factor L1 (21) (Appendino et al., 1999a) and Lathyranoic acid A (22) (Gao et al., 2007), respectively (Fig. 1). The isolated compounds include different patterns of lathyrane skeletons, including the jolkinols (1 and 2), 7-hydroxylathyrols (3, 10, 15 and 22), lathyrols (4, 5, 16, 17 and 18), 17-hydroxyjolkinols (6, 7, 8, 11, 12 and 19), 17-hydroxyisolathyrols (9, 13 and 20) and epoxylathyrols (14 and 21). The different types of lathyrane skeletons may be obtained via oxidation, hydrolyzation, dehydration

13

C NMR (150 MHz) spectroscopic data for the diterpene moiety of compounds 11–14 in CDCl3.

11

12

H 1a 1b 2 3 4 5 6 7a 7b 8a 8b 9 10 11 12 13 14 15 16 17a 17b 18 19 20

9

3.58 1.56 2.23 5.41 2.77 5.58

dd (14.0, 8.2) dd (14.0, 12.2) m t (3.6) dd (11.0, 3.6) d (11.0)

2.38 2.25 2.20 1.58 1.10

m m m m m

1.39 dd (11.5, 8.0) 6.59 d (11.5)

1.01 3.97 3.69 1.17 1.05 1.85

d (6.5) d (12.4) d (12.4) s s s

13

C

H

44.8

3.54 1.43 2.18 5.30 2.72 5.63

dd (14.0, 8.0) m m t (3.5) dd (10.9, 3.5) d (10.9)

2.40 2.04 1.47 2.26 1.46

m m m m m

38.6 81.1 50.4 145.0 122.2 31.6 29.1 34.4 24.9 28.4 147.2 132.3 194.7 94.7 13.9 62.9 29.3 16.2 12.4

1.37 m 6.55 d (11.2)

0.96 4.35 4.12 1.18 1.10 1.84

d (6.6) d (12.5) d (12.5) s s s

C

H

44.7

3.49 1.67 2.37 5.91 2.99 6.45

38.5 80.6 50.5 125.0 140.3 32.4 28.4 34.0 24.8 29.3 146.9 132.5 194.4 94.5 13.8 64.0 29.1 16.2 12.4

14

dd (14.4, 8.4) dd (14.4, 12.0) m t (3.3) dd (8.4, 3.3) d (8.4)

5.56 dd (12.4, 3.8) 2.71 m 2.36 m 1.26 m 1.27 dd (11.5, 8.7) 6.53 d (11.5)

0.96 4.06 4.06 1.19 1.35 1.78

d (6.6) s s s s s

C

H

48.4

3.49 1.63 2.26 5.73 1.99 6.08

dd (14.1, 8.1) dd (14.1, 12.4) m t (3.1) dd (8.8, 3.1) d (8.8)

2.01 0.93 2.08 1.76 1.09

m m m m m

37.5 80.9 52.5 65.5 135.1 132.1 24.3 29.7 25.5 31.0 143.2 134.3 196.8 92.7 14.3 64.4 28.7 17.1 12.1

C

1.51 dd (11.4, 8.0) 6.48 d (11.4)

0.91 2.51 2.36 1.21 1.23 1.89

d (6.6) d (3.5) d (3.5) s s s

48.1 38.2 80.9 50.0 65.1 59.0 33.5 20.1 34.9 25.7 29.2 143.8 136.0 196.8 92.1 13.5 55.3 29.0 16.9 12.5

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O

H

HO

n

tio

O HO

a xid

H HO H

o

O

ep

H jolkinodiol

ox

6, 7, 8 11, 12, 19

OH 17-hydroxyjolkinol

H

H HO

various esterfication

H

HO

ida

tio

various esterification H

n HO

1, 2

H

O jolkinol +H2 O

O

O

H

HO

HO

O H

-H 2 O

-H 2 O H

HO H HO isolathyrol

O

lathyrol

on

ti ida

x

o ep

ox

ida

tio

n O

O

H

H

HO

HO H

H

HO H HO

4, 5, 16 17, 18

HO H HO

H HO HO

oxidation HO

various esterfication

H

H

H HO

HO

HO H HO O

OH 17-hydroxyisolathyrol

epoxylathyrol

various esterification

various esterification

9, 13, 20

14, 21

HO H HO

H

OH

H

7-hydroxylathyrol various esterfication (cleavege)

3, 10, 15, 22

Fig. 5. Proposed biosynthetic pathway to compounds 1–22.

and esterification from the same jolkinodiol precursor (Dewick, 2002). Therefore, a plausible biogenetic pathway for compounds 1–22 is proposed (Fig. 5). All of the isolated compounds were evaluated for their cytotoxic activities against four cancer cell lines MCF-7, C6, Hela and HepG2 (Table 5). Compounds 1, 7, 12 and 17 exhibited moderate cytotoxic activities against MCF-7 (IC50 12.4–23.1 lM). However, these same compounds showed only weak cytotoxicity against C6 (IC50 > 20 lM) due to their selective activity. Only compound 5 exhibited considerable antitumor potency against the C6 cell line (IC50 15.3 lM). Unfortunately, all tested compounds were inactive against the Hela and HepG2 cell lines at 50 lM. The preliminary structure–activity relationships for the lathyrane diterpenoids with a 5b,6b-epoxy type exhibits the highest Table 5 Cytotoxicity of compounds 1–22.a Compound

1 5 7 8 12 17 Taxolc

IC50 ± SD (lM) C6

MCF-7

23.4 ± 1.8 15.3 ± 0.8 –b 47.3 ± 1.9 –b 36.2 ± 1.9 8.4 ± 0.8

12.4 ± 0.3 –b 13.1 ± 0.7 33.3 ± 2.1 23.1 ± 1.4 15.1 ± 0.6 6.8 ± 1.7

inhibitory activity against C6 and MCF-7 (e.g., 1). In contrast, 6,17-epoxy (e.g., 14 and 21), 6,7-double bond type (e.g., 9, 13 and 20) have no cytotoxicity. However, when C-7 is substituted, no cell lines show inhibitory activity (e.g., 3, 10 and 15). Comparing compounds 7 and 6, 8 and 6, 17 and 16 indicated that the phenylacetyl group dramatically increased the anticancer activity relative to the benzoyl group at C-3. From these results, it can be proposed that the cytotoxic activity of the lathyrane diterpenoids is related to the structure of the parent-skeleton, the substituent pattern and position, as well as the cell line type. 3. Conclusions Ten new lathyrane-type diterpenoids named Euphorbia Factor L12–L21 (1–10) and twelve known derivatives (11–22) were isolated from the seeds of E. lathyris. The structures and relative configurations of 1–14 were determined based on spectroscopic interpretations. The absolute configurations were established through CD analysis. Among the isolated diterpenoids, compounds 1, 5, 7, 12 and 17 showed considerable cytotoxicity against C6 or MCF-7 cell lines. 4. Experimental 4.1. General procedures

a

Compounds 2–4, 6, 9–11, 13–16, 18–22 were all inactive against C6 and MCF-7 cell lines. b IC50 value above 50 lM. c Positive control.

Optical rotations were measured with a Perkin–Elmer 241 MC polarimeter, whereas UV spectra were recorded on a Shimadzu UV-2201 spectrophotometer. CD spectra were obtained with a

J. Lu et al. / Phytochemistry 104 (2014) 79–88

Biologic MOS-450 spectrometer. The IR spectra were determined on a Bruker IFS-55 infrared spectrophotometer with KBr disks. The 1H and 13C NMR and 2D NMR spectra were recorded on Bruker Avance III 600 spectrometers using TMS as an internal standard. The HRESIMS data were obtained using a Waters LCT Premier XE time-of-flight mass spectrometer (Waters, America). Column chromatography (CC) was performed on silica gel (90–150 lm, Qingdao Marine Chemical Company, Qingdao, China), Sephadex LH-20 (40–70 lm, Amersham Pharmacia Biotech AB, Uppsala, Sweden) and ODS columns (40–63 lm, Merck, Germany); RP-HPLC was performed on an HITACHI unit equipped with a UV–VIS detector and a YMC ODS-A column (250  10 mm, 5 lm). 4.2. Plant material Seeds of E. lathyris were purchased from Anguo, Hebei Province, People’s Republic of China, in April 2010. The plant material was identified by Professor Jimin Xu (National Institutes for Food and Drug Control). A voucher specimen (No. 20100420) has been deposited at the Research Department of Natural Medicine, Shenyang Pharmaceutical University. 4.3. Extraction and isolation Crushed air-dried seed material (8.0 kg) was extracted with EtOH–H2O (95:5, 3  80 L) under conditions of refluxing for 3 h. The combined EtOH extracts were concentrated in vacuo to generate a crude residue (667 g) that was suspended in H2O (1.5 L). The suspension was partitioned successively with petroleum ether (60–90 °C), CHCl3, EtOAc and n-BuOH (each three times). The petroleum ether (60–90 °C) portion (200 g) was separated by a silica gel CC eluted with petroleum ether (60–90 °C)–acetone (from 100:0 to 0:100) to yield 10 fractions (A1–A10). Fraction A2 (3.2 g) was separated via preparative RP-HPLC (MeOH–H2O, 82:18, v/v) to yield compounds 21 (3.0 g) and 5 (14.2 mg). Fraction A3 (6.7 g) was purified by crystallizing from petroleum ether-acetone to obtain compounds 15 (1.5 g), 16 (2.5 g) and 17 (3.4 g). Fraction A4 (6.0 g) was applied to a Sephadex LH-20 column (MeOH–CHCl3, 1:1, v/v) to give sub-fractions A4B1–A4B4 that were further purified by preparative RP-HPLC (MeOH–H2O, 82:18, v/v) to obtain compounds 22 (8.7 mg), 3 (27.8 mg) and 4 (13 mg). Fraction A5 (6.5 g) was separated over an ODS column (MeOH–H2O, 20:80– 100:0, v/v) to obtain eight sub-fractions (A5B1–A5B8). Sub-fractions A5B3–A5B8 were further purified by RP-HPLC using different mobile phrases. Sub-fraction A5B3 (MeOH–H2O, 72:28, v/v) yielded compounds 13 (10.1 mg), 14 (8.7 mg), 1 (23.6 mg), 2 (6.2 mg) and 20 (1.8 g). Compounds 6 (2.7 mg) and 12 (50.4 mg) were isolated from sub-fraction A5B6 (MeCN–H2O, 77:23, v/v). Fraction A6 (0.91 g) was separated over the same ODS column and eluted with a gradient of MeOH–H2O (from 30:70 to 80:20, v/v), to obtain five fractions; sub-fraction A6B3 was further subjected to RP-HPLC (MeCN–H2O, 69:31, v/v) to obtain compounds 7 (29 mg), 8 (2.6 mg) and 11 (4.5 mg), respectively. Fraction A7 (2.7 g) underwent by preparative RP-HPLC (MeOH–H2O, 80:20, v/ v) to yield compounds 10 (6.3 mg) and 19 (106 mg). Fraction A8 (2.0 g) was fractionated using the same ODS column and eluted with MeOH–H2O (from 40:60 to 80:20, v/v) to obtain fractions A8B1–A8B6; sub-fraction A8B2 was further chromatographed by RP-HPLC (MeOH–H2O, 76:24, v/v) to give compounds 9 (4.1 mg) and 18 (65.2 mg). 4.3.1. Euphorbia Factor L12 (1) Colorless crystals; [a]D20 +2.0 (c 1.0, CH3OH); UV (CH3OH) kmax (log e) nm 265 (1.00); IR (KBr) mmax 3452, 2929, 1744, 1725, 1657, 1628, 1453, 1383, 1271, 1230, 1149, 1115, 1067, 1042, 939, 711, 526 cm1; For 1H NMR (CDCl3, 600 MHz) spectroscopic data for

85

the diterpene moiety, see Table 1, for the 3-O-benzoyl unit, [d 8.11 (2H, dd, J = 7.8, 1.0 Hz, H-30 /70 ), 7.45 (2H, t, J = 7.8 Hz, H-40 / 60 ), 7.60 (1H, dt, J = 7.8, 1.0 Hz, H-50 )]; 15-O-acetyl unit, [d 2.16 (3H, s, H-200 )]; For 13C NMR (CDCl3, 150 MHz) spectroscopic data for the diterpene moiety, see Table 2, for the 3-O-benzoyl unit, [d 165.4 (C-10 ), 130.4 (C-20 ), 129.8 (C-30 /C-70 ), 128.6 (C-40 /C-60 ), 133.1 (C-50 )]; 15-O-acetyl unit, [d 169.5 (C-100 ), 21.5 (C-200 )]; HRESIMS m/z 503.2411 [M+Na]+ (calcd for C29H36O6Na, 503.2410). 4.3.2. Euphorbia Factor L13 (2) Colorless crystals; [a]D20 +2.0 (c 1.0, CH3OH); UV (CH3OH) kmax (log e) nm 263 (0.75); IR (KBr) mmax 3433, 2925, 1739, 1658, 1626, 1454, 1380, 1270, 1231, 1150, 1061, 1042, 1003, 909, 857, 713, 526 cm1; For 1H NMR (CDCl3, 600 MHz) spectroscopic data for the diterpene moiety, see Table 1, for the 3-O-phenylacetyl unit, [d 3.71 (2H, s, H-20 ), 7.35 (2H, d, J = 7.6 Hz, H-40 /80 ), 7.30 (2H, t, J = 7.6 Hz, H-50 /70 ), 7.25(1H, d, J = 7.6 Hz, H-60 )]; 15-O-acetyl unit, [d 2.04 (3H, s, H-200 )]; For 13C NMR (CDCl3, 150 MHz) spectroscopic data for the diterpene moiety, see Table 2, for the 3-O-phenylacetyl unit, [d 170.0 (C-10 ), 42.2 (C-20 ), 134.7 (C-30 ), 129.5 (C-40 /C-80 ), 128.4 (C-50 /C-70 ), 127.0 (C-60 )]; 15-O-acetyl unit, [d 169.3 (C-100 ), 21.4 (C-200 )]; HRESIMS m/z 517.2568 [M+Na]+ (calcd for C30H38O6Na, 517.2566). 4.3.3. Euphorbia Factor L14 (3) Amorphous solid; [a]D20 +6.9 (c 0.70, CH3OH); UV (CH3OH) kmax (log e) nm 234 (1.31); IR (KBr) mmax 3427, 2924, 2853, 1744, 1720, 1630, 1486, 1454, 1384, 1275, 1241, 1218, 1158, 1114, 866, 761, 713, 617, 526 cm1; For 1H NMR (CDCl3, 300 MHz) spectroscopic data for the diterpene moiety, see Table 1, for the 3-O-benzoyl unit, [d 8.06 (2H, dd, J = 6.9, 1.0 Hz, H-30 /70 ), 7.46 (2H, d, J = 6.9 Hz, H-40 / 60 ), 7.58 (1H, dt, J = 6.9, 1.0 Hz, H-50 )]; 5-O-acetyl unit, [d 1.37 (3H, s, H-200 )]; 7-O-salicyl unit, [d 10.61 (1H, s, OH-3000 ), 6.93 (1H, d, J = 7.8 Hz, H-4000 ), 6.76 (1H, dd, J = 7.8, .1.8 Hz, H-5000 ), 7.40 (1H, t, J = 7.8 Hz, H-6000 ), 7.70 (1H, dd, J = 7.8, 1.8 Hz, H-7000 )]; 15-O-acetyl unit, [d 2.21 (3H, s, H-20000 )]; For 13C NMR (CDCl3, 150 MHz) spectroscopic data for the diterpene moiety, see Table 2, for the 3-O-benzoyl unit, [d 166.0 (C-10 ), 130.3 (C-20 ), 129.7 (C-30 /C-70 ), 128.4 (C-40 / C-60 ), 133.1 (C-50 )]; 5-O-acetyl unit, [d 169.4 (C-100 ), 21.0 (C-200 )]; 7-O-salicyl unit, [d 169.1 (C-1000 ), 112.2 (C-2000 ), 161.7 (C-3000 ), 117.6 (C-4000 ), 135.9 (C-5000 ), 119.1 (C-6000 ), 129.9 (C-7000 )]; 15-O-acetyl unit, [d 169.7 (C-10000 ), 21.9 (C-20000 )]; HRESIMS m/z 681.2679 [M+Na]+ (calcd for C38H42O10Na, 681.2676). 4.3.4. Euphorbia Factor L15 (4) White powder; [a]D20 +11.2 (c 0.58, CH3OH); UV (CH3OH) kmax (log e) nm 275 (1.39); IR (KBr) mmax 3439, 2924, 2856, 1740, 1627, 1453, 1383, 1273, 1254, 1236, 1115, 1059, 1032, 1018, 904, 713, 620, 450 cm1; For 1H NMR (CDCl3, 300 MHz) spectroscopic data for the diterpene moiety, see Table 1, for the 3-O-acetyl unit, [d 2.04 (3H, s, H-20 )]; 5-O-acetyl unit, [d 1.98 (3H, s, H-200 )]; 15-O-acetyl unit, [d 2.09 (3H, s, H-2000 )]; For 13C NMR (CDCl3, 150 MHz) spectroscopic data for the diterpene moiety, see Table 2, for the 3-O-acetyl unit, [d 170.7 (C-10 ), 20.9 (C-20 )]; 5-O-acetyl unit, [d 170.6 (C-100 ), 21.2 (C-200 )]; 15-O-acetyl unit, [d 169.9 (C-1000 ), 22.0 (C-2000 )]; HRESIMS m/z 483.2358 [M+Na]+ (calcd for C26H36O7Na, 483.2359). 4.3.5. Euphorbia Factor L16 (5) White powder; [a]D20 +17.0 (c 1.0, CH3OH); UV (CH3OH) kmax (log e) nm 266 (0.99); IR (KBr) mmax 3429, 2926, 2855, 1743, 1673, 1650, 1616, 1486, 1456, 1383, 1371, 1302, 1250, 1226, 1159, 1091, 1035, 1023, 906, 759, 701 cm1; For 1H NMR (CDCl3, 600 MHz) spectroscopic data for the diterpene moiety, see Table 1, for the 3-O-salicyl unit, [d 10.50(1H, s, OH-30 ), 7.00 (1H, d, J = 7.4 Hz, H-40 ), 7.48 (1H, dt, J = 7.4, 1.2 Hz, H-50 ), 6.89 (1H, t, J

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= 7.8 Hz, H-60 ), 7.84 (1H, dd, J = 7.8, 1.2 Hz, H-70 )]; 5-O-acetyl unit, [d 1.83 (3H, s, H-200 )]; 15-O-acetyl unit, [d 2.23 (3H, s, H-2000 )]; For 13 C NMR (CDCl3, 150 MHz) spectroscopic data for the diterpene moiety, see Table 2, for the 3-O-salicyl unit, [d 169.4 (C-10 ), 112.2 (C-20 ), 161.5 (C-30 ), 117.7 (C-40 ), 136.0 (C-50 ), 119.2 (C-60 ), 130.2 (C-70 )]; 5-O-acetyl unit, [d 170.0 (C-100 ), 20.8 (C-200 )]; 15-O-acetyl unit, [d 169.7 (C-1000 ), 22.0 (C-2000 )]; HRESIMS m/z 561.2462 [M+Na]+ (calcd for C31H38O8Na, 561.2464). 4.3.6. Euphorbia Factor L17 (6) Colorless block crystals; [a]D20 +3.8 (c 1.0, CH3OH); UV (CH3OH) kmax (log e) nm 229 (1.21); IR (KBr) mmax 3429, 2928, 1741, 1723, 1650, 1619, 1453, 1371, 1272, 1233, 1150, 1114, 1067, 1028, 939, 908, 864, 711 cm1; For 1H NMR (CDCl3, 600 MHz) spectroscopic data for the diterpene moiety, see Table 3, for the 3-O-benzoyl unit, [d 8.11 (2H, dd, J = 7.6, 1.2 Hz, H-30 /70 ), 7.49 (2H, dt, J = 7.6, 1.2 Hz, H-40 /60 ), 7.62 (1H, t, J = 7.6 Hz, H-50 )]; 15-O-acetyl unit, [d 2.09 (3H, s, H-200 )]; 17-O-acetyl unit, [d 2.02 (3H, s, H2000 )]; For 13C NMR (CDCl3, 150 MHz) spectroscopic data for the diterpene moiety, see Table 2, for the 3-O-benzoyl unit, [d 165.9 (C-10 ), 130.3 (C-20 ), 129.6 (C-30 /C-70 ), 128.5 (C-40 /C-60 ), 133.1 (C50 )]; 15-O-acetyl unit, [d 169.4 (C-100 ), 21.4 (C-200 )]; 17-O-acetyl unit, [d 170.7 (C-1000 ), 20.9 (C-2000 )]; HRESIMS m/z 545.2519 [M+Na]+ (calcd for C31H38O7Na, 545.2515). 4.3.7. Euphorbia Factor L18 (7) Colorless oil; [a]D20 +7.5 (c 1.0, CH3OH); UV (CH3OH) kmax (log e) nm 273 (1.21); IR (KBr) mmax 3447, 2931, 1737, 1649, 1615, 1455, 1370, 1233, 1150, 1125, 1030, 1006, 969, 857, 723, 632 cm1; For 1 H NMR (CDCl3, 600 MHz) spectroscopic data for the diterpene moiety, see Table 3, for the 3-O-phenylacetyl unit, [d 3.68 (2H, s, H-20 ), 7.29–7.35 (5H, m)]; 15-O-acetyl unit, [d 2.01 (3H, s, H-200 )]; 17-O-acetyl unit, [d 1.99 (3H, s, H-2000 )]; For 13C NMR (CDCl3, 150 MHz) spectroscopic data for the diterpene moiety, see Table 2, for the 3-O-phenylacetyl unit, [d 170.7 (C-10 ), 42.2 (C-20 ), 134.6 (C30 ), 129.4 (C-40 /C-80 ), 128.5 (C-50 /C-70 ), 127.2 (C-60 )]; 15-O-acetyl unit, [d 169.5 (C-100 ), 21.4 (C-200 )]; 17-O-acetyl unit, [d 170.6 (C-1000 ), 20.9 (C-2000 )]; HRESIMS m/z 559.2670 [M+Na]+ (calcd for C32H40O7Na, 559.2672). 4.3.8. Euphorbia Factor L19 (8) Colorless oil; [a]D20 +1.8 (c 1.0, CH3OH); UV (CH3OH) kmax (log e) nm 265 (0.88); IR (KBr) mmax 3430, 2926, 2855, 1736, 1649, 1614, 1455, 1382, 1271, 1237, 1149, 1125, 1058, 1033, 1006, 860, 718, 632 cm1; For 1H NMR (CDCl3, 600 MHz) spectroscopic data for the diterpene moiety, see Table 3, for the 3-O-phenylacetyl unit, [d 3.69 (2H, s, H-20 ), 7.34–7.36 (5H, m)]; 15-O-acetyl unit, [d 2.01 (3H, s, H-200 )]; For 13C NMR (CDCl3, 150 MHz) spectroscopic data for the diterpene moiety, see Table 2, for the 3-O-phenylacetyl unit, [d 170.9 (C-10 ), 42.2 (C-20 ), 134.5 (C-30 ), 129.4 (C-40 /C-80 ), 128.6 (C-50 / C-70 ), 127.2 (C-60 )]; 15-O-acetyl unit, [d 169.5 (C-100 ), 21.5 (C-200 )]; HRESIMS m/z 517.2536 [M+Na]+ (calcd for C30H38O6Na, 517.2566). 4.3.9. Euphorbia Factor L20 (9) White powder; [a]D20 +1.0 (c 0.42, CH3OH); UV (CH3OH) kmax (log e) nm 224 (1.85); IR (KBr) mmax 3750, 3442, 2926, 2855, 1738, 1633, 1559, 1456, 1384, 1277, 1238, 1118, 1043, 713, 620 cm1; For 1H NMR (CDCl3, 600 MHz) spectroscopic data for the diterpene moiety, see Table 3, for the 3-O-benzoyl unit, [d 8.12 (2H, dd, J = 7.6, 1.0 Hz, H-30 /70 ), 7.46 (2H, dt, J = 7.6, 1.0 Hz, H-40 /60 ), 7.59 (1H, t, J = 7.6 Hz, H-50 )]; 15-O-acetyl unit, [d 2.19 (3H, s, H-200 )]; 17-O-acetyl unit, [d 2.05 (3H, s, H-2000 )]; For 13C NMR (CDCl3, 150 MHz) spectroscopic data for the diterpene moiety, see Table 2, for the 3-O-benzoyl unit, [d 166.1 (C-10 ), 130.7 (C-20 ), 129.7 (C-30 /C-70 ), 128.4 (C-40 /C-60 ), 133.0 (C-50 )]; 15-O-acetyl unit, [d 169.8 (C-100 ), 22.2 (C-200 )]; 17-O-acetyl unit, [d

170.9 (C-1000 ), 21.2 (C-2000 )]; HRESIMS m/z 561.2466 [M+Na]+ (calcd for C31H38O8Na, 561.2464). 4.3.10. Euphorbia Factor L21 (10) White powder; [a]D20 +3.2 (c 1.0, CH3OH); UV (CH3OH) kmax (log e) nm 226 (1.04); IR (KBr) mmax 3426, 2923, 2853, 1741, 1723, 1646, 1453, 1383, 1272, 1134, 1069, 1027, 935, 712, 623 cm1; For 1H NMR (CDCl3, 600 MHz) spectroscopic data for the diterpene moiety, see Table 3, for the 3-O-benzoyl unit, [d 8.11 (2H, dd, J = 7.6, 1.0 Hz, H-30 /70 ), 7.46 (2H, t, J = 7.6 Hz, H-40 / 60 ), 7.58 (1H, dt, J = 7.6, 1.0 Hz, H-50 )]; 5-O-acetyl unit, [d 1.66 (3H, s, H-200 )]; 7-O-benzoyl unit, [d 7.87 (2H, dd, J = 7.6, 1.0 Hz, H-3000 /7000 ), 7.30 (2H, t, J = 7.6 Hz, H-4000 /6000 ), 7.44 (1H, dt, J = 7.6, 1.0 Hz, H-5000 )]; For 13C NMR (CDCl3, 150 MHz) spectroscopic data for the diterpene moiety, see Table 2, for the 3-O-benzoyl unit, [d 165.9 (C-10 ), 130.4 (C-20 ), 129.7 (C-30 /C-70 ), 128.4 (C-40 /C-60 ), 132.9 (C-50 )]; 5-O-acetyl unit, [d 168.7 (C-100 ), 20.8 (C-200 )]; 7-Obenzoyl unit, [d 165.5 (C-1000 ), 130.2 (C-2000 ), 129.7 (C-3000 /C-7000 ), 128.2 (C-4000 /C-6000 ), 132.8 (C-5000 )]; HRESIMS m/z 657.2718 [M+Na]+ (calcd for C36H42O10Na, 657.2676). 4.3.11. Euphorbia Factor L22 (11) Colorless oil; [a]D20 +14.0 (c 1.0, CH3OH); UV (CH3OH) kmax (log e) nm 277 (1.27); IR (KBr) mmax 3428, 2927, 2856, 1739, 1714, 1637, 1452, 1382, 1271, 1238, 1170, 1150, 1124, 1059, 1009, 907, 863, 768, 710, 632 cm1; For 1H NMR (CDCl3, 600 MHz) spectroscopic data for the diterpene moiety, see Table 4, for the 3-O-cinnamoyl unit, [d 6.49 (1H, d, J = 16.0 Hz, H-20 ), 7.73 (1H, d, J = 16.0 Hz, H-30 ), 7.56 (2H, m, H-50 /90 ), 7.42 (2H, m, H-60 / 80 ), 7.41 (1H, m, H-70 )]; 15-O-acetyl unit, [d 2.08 (3H, s, H-200 )]; For 13C NMR (CDCl3, 150 MHz) spectroscopic data for the diterpene moiety, see Table 4, for the 3-O-cinnamoyl unit, [d 166.4 (C-10 ), 118.0 (C-20 ), 124.8 (C-30 ), 132.3 (C-40 ), 128.1 (C-50 /90 ), 129.0(C-60 / 80 ), 130.5 (C-70 )]; 15-O-acetyl unit, [d 169.5 (C-100 ), 21.5 (C-200 )]; HRESIMS m/z 529.2576 [M+Na]+ (calcd for C31H38O6Na, 529.2566). 4.3.12. Euphorbia Factor L23 (12) Colorless oil; [a]D20 +0.5 (c 1.0, CH3OH); UV (CH3OH) kmax (log e) nm 270 (0.97); IR (KBr) mmax 3449, 2931, 2862, 1739, 1650, 1616, 1455, 1370, 1270, 1228, 1171, 1114, 1059, 1030, 1007, 969, 937, 908, 857, 778, 734, 631 cm1; For 1H NMR (CDCl3, 600 MHz) spectroscopic data for the diterpene moiety, see Table 4, for the 3-Ohexanoyl unit, [d 2.40 (2H, t, J = 7.2 Hz, H-20 ), 1.70 (2H, t, J = 7.2 Hz, H-30 ), 1.37 (4H, m, H-40 /50 ), 0.93 (3H, t, J = 6.9 Hz, H-60 )]; 15-O-acetyl unit, [d 2.02 (3H, s, H-200 )]; 17-O-acetyl unit, [d 2.01 (3H, s, H-2000 )]; For 13C NMR (CDCl3, 150 MHz) spectroscopic data for the diterpene moiety, see Table 4, for the 3-O-hexanoyl unit, [d 173.0 (C-10 ), 34.5 (C-20 ), 24.8 (C-30 ), 31.4 (C-40 ), 22.4 (C-50 ), 13.9 (C-60 )]; 15-O-acetyl unit, [d 169.5 (C-100 ), 21.4 (C-200 )]; 17-Oacetyl unit, [d 170.7 (C-1000 ), 20.9 (C-2000 )]; HRESIMS m/z 539.2988 [M+Na]+ (calcd for C30H44O7Na, 539.2985). 4.3.13. Euphorbia Factor L24 (13) White powder; [a]D20 +9.1 (c 0.42, CH3OH), UV (CH3OH) kmax (log e) nm 268 (0.90); IR (KBr) mmax 3445, 2927, 1742, 1721, 1629, 1454, 1370, 1277, 1234, 1149, 1114, 1028, 945, 713 cm1; For 1H NMR (CDCl3, 600 MHz) spectroscopic data for the diterpene moiety, see Table 4, for the 3-O-benzoyl unit, [d 8.02 (2H, dd, J = 7.4, 1.0 Hz, H-30 /70 ), 7.45 (2H, t, J = 7.4 Hz, H-40 /60 ), 7.58 (1H, dt, J = 7.4, 1.0 Hz, H-50 )]; 5-O-acetyl unit, [d 1.68 (3H, s, H-200 )]; 15O-acetyl unit, [d 2.24 (3H, s, H-2000 )]; For 13C NMR (CDCl3, 150 MHz) spectroscopic data for the diterpene moiety, see Table 4, for the 3-O-benzoyl unit, [d 166.0 (C-10 ), 130.1 (C-20 ), 129.6 (C-30 /C70 ), 128.5 (C-40 /C-60 ), 133.3 (C-50 )]; 5-O-acetyl unit, [d 169.1 (C-100 ), 21.0 (C-200 )]; 15-O-acetyl unit, [d 169.8 (C-1000 ), 22.2 (C-2000 )]; HRESIMS m/z 561.2460 [M+Na]+ (calcd for C31H38O8Na, 561.2464).

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4.3.14. Euphorbia Factor L25 (14) Colorless block crystals; [a]D20 +9.9 (c 0.76, CH3OH); UV (CH3OH) kmax (log e) nm 272 (1.04); IR (KBr) mmax 3428, 2927, 1743, 1719, 1652, 1624, 1454, 1383, 1274, 1233, 1210, 1114, 1066, 1040, 902, 865, 714, 560, 420 cm1; For 1H NMR (CDCl3, 600 MHz) spectroscopic data for the diterpene moiety, see Table 4, for the 3-O-benzoyl unit, [d 8.00 (2H, dd, J = 7.8, 1.2 Hz, H-30 /70 ), 7.45 (2H, t, J = 7.8 Hz, H-40 /60 ), 7.58 (1H, dt, J = 7.8, 1.2 Hz, H-50 )]; 5-O-acetyl unit, [d 1.82 (3H, s, H-200 )]; 15-O-acetyl unit, [d 2.23 (3H, s, H-2000 )]; For 13C NMR (CDCl3, 150 MHz) spectroscopic data for the diterpene moiety, see Table 4, for the 3-O-benzoyl unit, [d 165.8 (C-10 ), 129.9 (C-20 ), 129.7 (C-30 /C-70 ), 128.4 (C-40 /C-60 ), 133.2 (C-50 )]; 5-O-acetyl unit, [d 170.4 (C-100 ), 21.0 (C-200 )]; 15-Oacetyl unit, [d 169.5 (C-1000 ), 21.0 (C-2000 )]; HRESIMS m/z 561.2462 [M+Na]+ (calcd for C31H38O8Na, 561.2464). 4.3.15. X-ray crystallographic data for 1 C29H36O6, Mr = 480.25, crystal size 0.27  0.23  0.22 mm3, space group Monoclinic, P2 (1), unit cell dimensions: a = 10.369 (2) Å, b = 10.020 (2) Å, c = 14.508 (3) Å, a = 90.00°, b = 108.76°, c = 90.00°, V = 1427.2 (5) Å3, Z = 2, Dc = 1.193 mg/m3, l = 0.084 mm1, F(0 0 0) = 552. 14109 unique reflections were collected in range 3.02° < h < 27.42°, in which 6399 reflections were observed (|F|2 P 2r|F|2), R1 = 0.0570, wR2 = 0.1607, S = 1.096. CCDC number: 928708. 4.4. Cytotoxic activity against HepG2, MCF-7, Hela and C6 cells The HepG2, MCF-7, Hela and C6 cells were obtained from Peking Union Medical College, Beijing, China. The cells were cultured in Dulbecco’s Modified Eagle’s Medium–High glucose medium (GIBCO, NY, U.S.A.) supplemented with 10% Fetal calf serum (FCS) (Shengma Yuanheng, Beijing, China), 100 IU/ml streptomycin, 100 IU/ml penicillin and 2 mM L-glutamine and maintained at 37 °C with 5% CO2 in a humidified atmosphere. Compounds 1–22 were dissolved in dimethyl sulfoxide (DMSO), generating stock solutions. The DMSO concentration was maintained below 0.05% throughout the cell culture period; DMSO did not exert any detectable effect on cell growth or cell death. The HepG2, MCF-7, Hela and C6 cells were incubated at 5  104 cells/ ml in 96-well plates, respectively. After incubation for 24 h, the cells were treated with the 22 compounds at various concentrations. After 24 h, 10 lL MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5diphenyltetrazolium bromide; 5 mg/ml) was added to each well. The cells were incubated at 37 °C for 4 h to allow the incorporation and conversion of MTT to the formazan derivative. The formazan derivative was solubilized by adding 100 lL DMSO; the absorbance was measured at 570 nm with an enzyme immunoassay instrument (Bio-Rad Model 680). Supplementary data The crystallographic data for 1 has been deposited with the Cambridge Crystallographic Data Centre. These data can be obtained free of charge via the internet at http://www.ccdc.cam. ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK. Fax: +44 1223 336033; e-mail: deposit@ ccdc.cam.ac.uk). Acknowledgments This work was supported by the Key Projects of the National Science and Technology Pillar Program (2012BAI30B02), National Natural Science Foundation (81260628) and Shenyang Science &

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Technology Program (F12-157-9-00). We are grateful to Professor Jimin Xu for identifying the plant material.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem. 2014.04.020.

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