Cytotoxic Phorbol Esters of Croton tiglium - Journal of Natural

Article pubs.acs.org/jnp

Cytotoxic Phorbol Esters of Croton tiglium Xiao-Long Zhang,†,§ Lun Wang,†,§ Fu Li,† Kai Yu,‡ and Ming-Kui Wang*,† †

Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization & Ecological Restoration Biodiversity Conservation, Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, People’s Republic of China ‡ School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610013, People’s Republic of China § Graduate University of the Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Chemical investigation of the seeds of Croton tiglium afforded eight new phorbol diesters (three phorbol diesters, 1−3, and five 4-deoxy-4α-phorbol diesters, 4−8), together with 11 known phorbol diesters (nine phorbol diesters, 9−17, and two 4-deoxy-4α-phorbol diesters, 18 and 19). The structures of compounds 1−8 were determined by spectroscopic data information and chemical degradation experiments. The cytotoxic activities of the phorbol diesters were evaluated against the SNU387 hepatic tumor cell line, and compound 3 exhibited the most potent activity (IC50 1.2 μM).

Croton tiglium L. (family Euphorbiaceae) is distributed in tropical and subtropical zones.1 The seeds of C. tiglium are well known as “Badou” in mainland China. The use of this species was first described in the Chinese medical literature 2200 years ago. C. tiglium has been utilized widely to treat gastrointestinal disorders, intestinal inflammation, rheumatism, headache, peptic ulcer, and visceral pain.2 In 1963, Van Duuren and his colleagues reported the tumor-promoting principles of C. tiglium seeds.3 After that, many bioactive phorbol esters were isolated and evaluated from this species.4−9 The major constituent, 12-O-tetradecanoylphorbol-13-acetate (TPA), has been used widely in biochemical experiments as a standard tumor-promoting agent.6−9 In addition, phorbol ester antiHIV-1 agents have been separated from this species,10 and TPA was reported to be a potent inhibitor of HIV-1-induced cytopathic effects (CPEs), with an IC100 value of 0.48 ng·mL−1.11 Phorbol esters with cytotoxic, antileukemic, and antimycobacterial activities were also purified and identified.12−15 Previous studies have shown the cytotoxic effects of extracts and compounds from C. tiglium.16,17 In the present study, the seeds of C. tiglium were collected in Sichuan Province, and the phytochemical study was carried out using a bioguided isolation strategy. Eight new phorbol diesters (three phorbol diesters, 1− 3, and five 4-deoxy-4α-phorbol diesters, 4−8), together with 11 known phorbol diesters (nine phorbol diesters, 9−17, and two 4-deoxy-4α-phorbol diesters, 18 and 19), were isolated and characterized. Herein, the isolation and identification of these compounds and their in vitro cytotoxic activities against the SNU387 hepatic tumor cell line are reported. © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Compound 1 was assigned the molecular formula C30H42O8 on the basis of the HRESIMS (m/z 553.2751 [M + Na]+, calcd 553.2772) and NMR data, indicating 10 degrees of unsaturation. The IR spectrum showed the absorptions of hydroxy (3415 cm−1) and carbonyl (1699 cm−1) groups. The 1D-NMR and HSQC spectroscopic data (Table 1) of 1 displayed signals for an α,β-unsaturated carbonyl (δH 7.61, s, H1; δC 160.9, C-1; δC 132.8, C-2; δC 209.0, C-3), a trisubstituted double bond (δH 5.70, d, J = 3.8 Hz, H-7; δC 129.4, C-7; δC 140.3, C-6), an oxymethylene (δH 4.00, d, J = 12.7 Hz and 4.04, d, J = 12.7 Hz, H2-20; δC 68.1, C-20), an oxymethine (δH 5.47, d, J = 10.4 Hz, H-12; δC 76.5, C-12), four methyls (δH 1.23, s, Received: November 29, 2012

A

dx.doi.org/10.1021/np300832n | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data for Compounds 1−3 in CDCl3 (δ in ppm) 1 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 R1 1′ 2′ 3′ 4′ 5′ R2 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 11″ 12″ 13″ 14″ 15″ 16″ 17″ 18″ a−e

δH (J in Hz) 7.61, s

2.55, d (18.3) 2.49, d (18.3) 5.70, d (3.8) 3.27, brm 3.26, brm 2.15, m 5.47, d (10.4) 1.11, d (5.0) 1.23, 1.28, 0.91, 1.79, 4.04, 4.00,

2.38, 1.67, 1.48, 0.93, 1.16,

s s d (7.0) s d (12.7) d (12.7)

m m m t (7.4) d (7.0)

6.89, m 1.77, d (2.4) 1.80, s

2 δC, type 160.9, 132.8, 209.0, 73.7, 38.7,

CH C C C CH2

140.3, 129.4, 39.2, 78.2, 56.2, 43.0, 76.5, 65.3, 36.5, 26.7, 23.9, 16.9, 14.4, 10.1, 68.1,

C CH CH C CH CH CH C CH C CH3 CH3 CH3 CH3 CH2

176.1 41.9 26.0 11.6 17.1 169.7 128.3 139.6 14.4 11.7

3

δH (J in Hz)

δC

7.60, s

2.57, d (19.0) 2.49, d (19.0) 5.69, d (4.6) 3.26, brm 3.26, brm 2.17, m 5.46, d (10.3) 1.08, d (5.1) 1.21, 1.28, 0.89, 1.76, 4.04, 3.99,

s s d (6.4) s d (12.8) d (12.8)

6.84, m

position

160.8 132.8 209.0 73.7 38.6

1 2 3 4 5

140.4 129.3 39.1 78.3 56.2 43.3 73.7 65.5 36.5 27.7 23.8 16.9 14.4 10.1 68.1

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

167.8 128.5 137.5

R2 1″ 2″ 3″

1.76, d (7.0) 1.84, s

14.4 12.2

2.37, m 1.15, t (7.6)

177.0 27.7 8.7

δH (J in Hz) 7.57, s

2.52, d (19.0) 2.37, d (19.0) 5.66, d (4.5) 3.19, brm 3.12, brm 2.01, m 3.98, d (9.8) 1.01a 1.22, s 1.24, s 1.01a 1.77, s 4.48, d (12.4) 4.43, d (12.4)

4″ 5″ R3 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴ 10‴ 11‴ 12‴ 13‴ 14‴ 15‴ 16‴ 17‴ 18‴

δC 160.3 132.3 208.7 73.3 38.9 136.2 129.7 39.2 78.2 56.7 44.9 69.1 65.1 35.3 27.1 23.6 16.9 14.1 10.1 77.3

2.11, s

173.6 21.0

2.29, t (7.6) 1.59, m 1.27−1.33, mb 1.27−1.33, mb 1.27−1.33, mb 1.27−1.33, mb 2.01, md 5.34, mc 5.34, mc 2.01, md 1.27−1.33, mb 1.27−1.33, mb 1.27−1.33, mb 1.27−1.33, mb 1.27−1.33, mb 1.27−1.33, mb 0.88, t (7.0)

174.0 34.2 24.9 29.1−29.8e 29.1−29.8e 29.1−29.8e 29.1−29.8e 27.2 130.0 133.0 27.2 29.1−29.8e 29.1−29.8e 29.1−29.8e 29.1−29.8e 31.9 22.6 15.0

Overlapping signals within the same column.

δC 23.9, CH3-16; δH 1.28, s, δC 16.9, CH3-17; δH 0.91, d, J = 7.0 Hz, δC 14.4, CH3-18; δH 1.79, s, δC 10.1, CH3-19), a methylene (δH 2.55, d, J = 18.3 Hz and 2.49, d, J = 18.3 Hz, H2-5; δC 38.7, C-5), and four methines (δH 3.27, m, H-8; δC 39.2, C-8; δH 3.26, m, H-10; δC 56.2, C-10; δH 2.15, m, H-11; δC 43.0, C-11; δH 1.11, d, J = 5.0 Hz, H-14; δC 36.5, C-14).11,18 The 1H−1H COSY correlations (Figure 1) indicated the presence of C-1− C-10, C-7−C-8−C-14, and C-12−C-11−C-18 moieties. NOESY correlations (Figure 2) were observed among H-8,

H-11, and H-17. These signals suggested that compound 1 possesses the tigliane (phorbol) backbone similar to those of the known compounds 9−15 and 17.11,18−21 Other characteristic resonances included signals for a tiglyl group and a 2methylbutyryl moiety.11,19 In the HMBC spectrum, the H-12 proton showed a 3J correlation with the carbonyl carbon of the 2-methylbutyryl group (δC 176.1), confirming the location of the 2-methylbutyrate group at C-12. Thus, the structure of 1 B



Article

same 4-deoxy-4α-phorbol unit as the known compound 18.19 The main 1H and 13C NMR signals indicated the presence of an α,β-unsaturated carbonyl, a trisubstituted double bond, an oxymethylene, an oxymethine, four methyls, a methylene, and five methines, which were confirmed by HSQC and HMBC correlations (Figure 1).19,22 The 1H−1H COSY spectrum indicated the presence of C-1−C-10−C-4−C-5, C-7−C-8−C14, and C-12−C-11−C-18 moieties (Figure 1). In addition, the signals at δH 4.04 (H-12), δC 75.1 (C-12), δH 4.34, 4.47 (H-20), and δC 70.5 (C-20) suggested acylation at C-20, rather than at C-12. In the HMBC spectrum, long-range correlations between H3-2″ (δH 2.09, s) and C-13, as well as H2-2‴ (δH 2.37, t, J = 7.3 Hz) and C-20, confirmed acylations at C-13 with a acetyl group and C-20 with a linoleoyl group. Hydrolysis of 4 with 0.1 M NaOMe gave methyl linoleate, which was analyzed by GCMS (tR 23.1 min, m/z 294 [M]+).11 Furthermore, the NOESY correlation (Figure 2) between H-4 and H-10 revealed that H-4 is α-oriented. Therefore, the structure of 4 was determined to be 13-O-acetyl-4-deoxy-4α-phorbol-20-linoleate. Compound 5 exhibited a [M − H]− peak at m/z 653.4405 in the HRESIMS (calcd 653.4417), consistent with a molecular formula of C40H62O7, requiring 10 degrees of unsaturation. The 1 H and 13C NMR data (Tables 2 and 3) of 5 were quite similar to those of 4, except that the signals of a linoleoyl group in 4 were replaced by signals of an oleoyl group in 5. After hydrolysis with 0.1 M NaOMe, methyl oleate was identified by GC-MS (tR 21.7 min, m/z 296 [M]+). In the HMBC spectrum, the signal at δH 2.09 (H-2″) was coupled to the signal at δC 67.8 (C-13), and the signal at δH 2.38 (H-2‴) was coupled to the signal at δC 70.5 (C-20). Hence, the structure of 5 was assigned as 13-O-acetyl-4-deoxy-4α-phorbol-20-oleate. Compound 6 gave a molecular ion peak at m/z 607.3596 [M + Na]+ in the HRESIMS (calcd 607.3611), corresponding to the molecular formula C35H52O7 (10 degrees of unsaturation). The 1H and 13C NMR spectroscopic data (Tables 2 and 3) of 6 were similar to those of the known compound 18.19 In addition, the signals of a decanoyl group in 6 were observed to replace those of an acetyl group in 18. Hydrolysis with 0.1 M NaOMe gave methyl decanoate, which was identified by GCMS (tR 5.5 min, m/z 186 [M]+). The H-12 signal (δH 5.52, d, J = 10.4 Hz) showed an HMBC correlation with the signal of carbonyl carbon (δC 167.6) of the tiglyl moiety. Therefore, the structure of 6 was determined to be 12-O-tiglyl-4-deoxy-4αphorbol-13-decanoate. Compound 7 was determined to have the molecular formula C33H40O7, from the HRESIMS (m/z 571.2653 [M + Na]+, calcd 571.2666) and NMR spectra, implying 14 degrees of unsaturation. Comparison of the NMR data (Tables 2 and 3) revealed the backbone of 7 to be the same as that of 18.19 A distinction was recognized by the presence of the phenylacetyl group in 7 instead of the acetyl group in 18. On the basis of the HMBC spectrum, a tiglate group was assigned to C-12. Compound 7 was therefore established as 12-O-tiglyl-4-deoxy4α-phorbol-13-phenylacetate. Compound 8 showed the molecular formula C30H42O7 (10 degrees of unsaturation), on the basis of the HRESIMS (m/z 537.2804 [M + Na]+, calcd 537.2823). The 1H and 13C NMR spectroscopic data (Tables 2 and 3) indicated that the structure of 8 was similar to 18 with the exception of the 2methylbutyrate at C-13.19 The carbonyl carbon of a tiglyl moiety (δC 167.4) exhibited an HMBC correlation with H-12 (δH 5.53, d, J = 10.4 Hz). Thus, the structure of 8 was assigned as 12-O-tiglyl-4-deoxy-4α-phorbol-13-(2-methyl)butyrate.

Figure 1. Key 1H−1H COSY () and HMBC (→) correlations of 1 and 4.

Figure 2. Key NOESY (↔) correlations of 1 and 4.

was determined to be 12-O-(2-methyl)butyrylphorbol-13tiglate. Compound 2 gave a molecular formula of C28H38O8 on the basis of the HRESIMS (m/z 525.2436 [M + Na]+, calcd 525.2459) and NMR data, indicating 10 degrees of unsaturation. The 1H and 13C NMR data (Table 1) of 2 were similar to those of compound 12. However, signals for a 2-methylbutyryl group in 12 were replaced by a propionyl moiety (δC 177.0, δH 2.37, δC 27.7, δH 1.15, and δC 8.7) in 2. The HMBC spectrum showed long-range correlations between H-12 (δH 5.46, d, J = 10.3 Hz) and the carbonyl carbon of the tiglyl group (δC 167.8). Compound 2 was assigned therefore as 12-O-tiglylphorbol-13-propionate. Compound 3 showed a sodiated molecular ion peak (m/z 693.4309 [M + Na]+, calcd 693.4337) in the HRESIMS, consistent with a molecular formula of C40H62O8 (10 degrees of unsaturation). The 1H and 13C NMR data (Table 1) revealed 3 to have similar features to those of 16, with signals at δH 3.98 (H-12), δC 69.1 (C-12), δH 4.43, 4.48 (H2-20), and δC 77.3 (C20).11 In addition, signals for an oleoyl group in 3 were observed to replace those for a linoleoyl moiety in 16. Comparison of the chemical shifts of H-20, C-20, H-12, and C12 in 3 with those in 1 indicated that it was C-20 rather than C12 attached to an electron-withdrawing group. After hydrolysis with 0.1 M NaOMe, methyl oleate was assigned by GC-MS (tR 21.7 min, m/z 296 [M]+). The H2-20 (δH 4.43, d, J = 12.4 Hz, δH 4.48, d, J = 12.4 Hz) showed HMBC correlations to the carbonyl carbon of the oleoyl moiety (δC 174.0). On the basis of HMBC and GC-MS, an acetyl group was assigned to C-13, and an oleoyl group to C-20. Compound 3 was identified as 13O-acetylphorbol-20-oleate. Compound 4 gave a molecular formula of C40H60O7 on the basis of the HRESIMS (m/z 651.4246 [M − H]−, calcd 651.4266) and NMR data, indicating 11 degrees of unsaturation. Comparison of the 1H and 13C NMR data (Tables 2 and 3) indicated that compound 4 possesses the C



Article

Table 2. 1H NMR Data for Compounds 4−8 in CDCl3 (600 MHz, δ in ppm, J in Hz) position 1 4 5 7 8 10 11 12 14 16 17 18 19 20 R2 2″ 3″ 4″ 5″ R3 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴ 10‴ 11‴ 12‴ 13‴ 14‴ 15‴ 16‴ 17‴ 18‴ f−n

4 7.02, 2.71, 3.27, 2.46, 5.17, 1.96, 3.47, 1.59, 4.04, 0.78, 1.20, 1.24, 1.26, 1.76, 4.47, 4.34,

s m dd (9.26, 2.1) dd (9.26, 2.2) s brs m m d (9.6) d (5.0) s s d (6.2) s d (12.4) d (12.4)

2.09, s

5 7.02, 2.71, 3.27, 2.46, 5.17, 1.96, 3.46, 1.60, 4.04, 0.78, 1.20, 1.24, 1.24, 1.76, 4.47, 4.34,


2.09, s

2.37, t (7.3)

2.38, t (6.1)

1.64, m 1.29−1.35, mf 1.29−1.35, mf 1.29−1.35, mf 1.29−1.35, mf 2.05, mg 5.35, mh 5.35, mh 2.77, m 5.35, mh 5.35, mh 2.05, mg 1.29−1.35, mf 1.29−1.35, mf 1.29−1.35, mf 0.89, t (6.0)

1.64, m 1.29−1.35, mi 1.29−1.35, mi 1.29−1.35, mi 1.29−1.35, mi 2.01, mj 5.33, mk 5.33, mk 2.01, mj 1.29−1.35, mi 1.29−1.35, mi 1.29−1.35, mi 1.29−1.35, mi 1.29−1.35, mi 1.29−1.35, mi 0.89, t (6.7)

position

6

1 4 5

7.06, 2.79, 3.46, 2.49, 5.12, 1.97, 3.51, 1.72, 5.52, 0.78, 1.20, 1.24, 1.08, 1.78, 4.12, 3.89,

7 8 10 11 12 14 16 17 18 19 20


7 7.04, 2.77, 3.39, 2.45, 5.07, 1.95, 3.50, 1.72, 5.57, 0.72, 1.02, 1.21, 1.08, 1.77, 3.97, 3.86,


8 7.05, 2.79, 3.57, 2.48, 5.12, 1.98, 3.51, 1.72, 5.53, 0.75, 1.18, 1.25, 1.08, 1.78, 4.02, 3.90,


R1 2′ 3′ 4′ 5′ R2 2″

6.87, m 1.83, d (7.9) 1.88, s

6.88, m 1.84, d (7.1) 1.89, s

6.87, m 1.83, d (7.1) 1.87, s

2.30, m

3.67, d (15.7) 3.60, d (15.7)

2.24, m

3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″

1.60, m 1.23−1.30l 1.23−1.30l 1.23−1.30l 1.23−1.30l 1.23−1.30l 1.23−1.30l 0.87, t (6.90)

7.23, 7.30, 7.24, 7.30, 7.23,

mm mn m mn mm

1.70, 1.43, m 0.92, t (7.5) 1.01, d (7.0)


enhances cytotoxicity against the tumor cell line used. However, phorbol-12,13-diesters with an acetate group at C13 (compounds 10, 11, and 18) did not have remarkable cytotoxicity against these cells.

Eleven known phorbol esters, 12-O-tiglylphorbol-13-isobutyrate (9),19 12-O-tetradecanoylphorbol-13-acetate (10),11 12O-hexadecanoylphorbol-13-acetate (11),23 12-O-tiglylphorbol13-(2-methyl)butyrate (12),11 12-O-acetylphorbol-13-decanoate (13),11 12-O-(2-methyl)butyrylphorbol-13-isobutyrate (14),19 12-O-acetylphorbol-13-dodecanoate (15),15 13-O-acet y l p h o r b o l - 2 0 - l i n o l e a t e (1 6 ), 1 1 1 2-O - ( 2 - m e t h y l ) butyrylphorbol-13-dodecanoate (17),11 12-O-tiglyl-4-deoxy4α-phorbol-13-acetate (18),19 and 12-O-tiglyl-4-deoxy-4αphorbol-13-isobutyrate (19),19 were also isolated and identified by comparison of their spectroscopic data with literature values. The isolated compounds were tested for their cytotoxicity against the SNU387 hepatic tumor cell line using the MTT method, with paclitaxel as the positive control (IC50 0.27 μM). Among these phorbol diesters, compounds 3 and 16 were the most potently cytotoxic, with IC50 values of 1.2 and 3.4 μM, respectively. These data (Table 4) suggest that the presence of an acetate group at the C-13 position of phorbol-12,20-diesters

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a Horiba SEPA-300 polarimeter. UV spectra were run on a Shimadzu UV-2550 spectrophotometer. IR spectra were obtained on a Perkin-Elmer 983 G spectrometer. NMR spectra were recorded on a Bruker Avance 600 spectrometer (600 MHz for 1H and 150 MHz for 13C) with TMS as the internal standard. High-resolution experiments were performed on a Bruker Bio TOF-Q mass spectrometer equipped with an ESI source. GC-MS analysis was performed on Thermo Trace-PolarisQ GC-MS with an Agilent DB-1 column (60 m × 0.25 mm × 0.5 μm). Analytical HPLC was carried out on a LabAlliance Series III with a model 201 (SSI) detector and Ultimate C18 column (250 mm × 4.60 mm, 5 μm). Preparative HPLC was carried out on P3000 with a UV3000 detector (Beijing D



Article

Table 3. 13C NMR Data for Compounds 4−8 in CDCl3 (150 MHz, δ in ppm, J in Hz) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 R2 1″ 2″ 3″ 4″ 5″ R3 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴ 10‴ 11‴ 12‴ 13‴ 14‴ 15‴ 16‴ 17‴ 18‴ o−s

4 155.6, 143.1, 210.9, 45.1, 34.2, 132.2, 128.7, 40.9, 77.7, 48.7, 47.1, 75.1, 67.8, 35.6, 25.8, 24.0, 16.4, 14.0, 10.4, 70.5,

CH C C CH CH2 C CH CH C CH CH CH2 C CH C CH3 CH3 CH3 CH3 CH

5 155.6 143.2 210.9 45.1 34.2 132.2 128.7 40.9 77.7 48.7 47.1 75.2 67.8 35.6 25.9 24.0 16.3 14.1 10.4 70.5

173.6 21.0

173.6 21.0

173.8 31.4 24.8 29.0 29.1−29.6o 29.1−29.6o 29.1−29.6o 27.1 130.1 128.0 25.6 127.9 130.0 26.3 29.1−29.6o 29.1−29.6o 22.5 12.3

173.8 34.3 24.9 29.1−29.7p 29.1−29.7p 29.1−29.7p 29.1−29.7p 27.2 129.7 129.9 27.1 29.1−29.7p 29.1−29.7p 29.1−29.7p 29.1−29.7p 31.8 22.6 12.4

position

6

7

8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 R1 1′ 2′ 3′ 4′ 5′ R2 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″

156.1 143.3 213.2 40.7 25.2 137.1 126.5 43.5 78.1 49.6 47.4 75.5 65.0 37.2 24.2 24.5 16.5 12.3 10.4 69.3

156.1 143.4 213.1 40.7 25.2 137.1 126.3 43.4 78.1 49.6 47.4 75.4 65.6 37.0 24.0 25.1 16.4 12.3 10.4 69.3

156.2 143.3 213.2 40.7 25.2 137.1 126.4 43.7 78.1 49.6 47.4 75.6 64.9 37.3 24.1 25.4 16.2 12.3 10.4 69.3

167.6 128.6 137.6 14.4 11.8

167.6 128.6 137.7 14.5 11.8

167.4 128.6 137.5 14.4 11.9

176.2 34.4 29.0 29.1 29.2q 29.3 29.2q 31.8 22.6 14.1

173.8 41.3 133.0 129.6s 128.6r 126.3 128.6r 129.6s

178.9 41.3 26.1 11.6 16.6


ChuangXinTongHeng Science and Technology Co., Ltd.) and Ultimate C18 column (250 mm × 21.2 mm, 5 μm). Column chromatography was performed on silica gel (200−300 mesh, Qingdao Marine Chemical Co.). TLC was detected with 254 nm UV light and visualized by spraying with a solution of 5% H2SO4 in C2H5OH (v/v) followed by heating. Plant Material. The seeds of C. tiglium were collected from Silie Town, Yibin County, Sichuan Province, People’s Republic of China, in November 2011. The plant was identified by Prof. Wu Feng-e, Chengdu Institute of Biology, Chinese Academy of Sciences. A voucher specimen (CIB-4-304-307) has been deposited at the Center for Natural Products, Chengdu Institute of Biology, Chinese Academy of Sciences. Extraction and Isolation. The air-dried seeds of C. tiglium (7.1 kg) were powdered and percolated with MeOH at room temperature

to exhaustion. The combined extracts were concentrated to yield 957.9 g of residue, which was suspended in water and extracted successively with petroleum ether (60−90 °C), CHCl3, and n-BuOH. The CHCl3 extract (740.4 g) was subjected to column chromatography (CC) over silica gel (100−200 mesh), eluting with EtOAc−petroleum ether (0:10 to 10:0, stepwise), to yield nine fractions: Fr.1 (17.3 g), Fr.2 (27.1 g), Fr.3 (200.5 g), Fr.4 (156.4 g), Fr.5 (64.5 g), Fr.6 (37.1 g), Fr.7 (33.7 g), Fr.8 (7.5 g), and Fr.9 (40.5 g). Fr.7 (33.7 g) was further separated by CC on silica gel (200−300 mesh) eluted with acetone−CH2Cl2 (0:10 to 10:0, stepwise) and monitored by HPLC to obtain 10 further fractions: Fr.7a (1.8 g), Fr.7b (0.9 g), Fr.7c (1.5 g), Fr.7d (2.8 g), Fr.7e (2.5 g), Fr.7f (2.9 g), Fr.7g (3.5 g), Fr.7h (2.7 g), Fr.7i (2.7 g), and Fr.7j (3.1 g). Fr.7c (1.5 g) was separated on a reversed-phase C18 column (MeOH−H2O, 60:40) to obtain five subfractions (Fr.7c1−Fr.7c5). E



Article

Table 4. Cytotoxic Activity Evaluation of Compounds 1−19a compound

SNU387b

compound

SNU387b

1 2 3 4 5 6 7 8 9 10

>10 >10.2 1.2 ± 0.1 8.1 ± 1.3 6.6 ± 0.9 >10 >10 >10 >10 >10

11 12 13 14 15 16 17 18 19 paclitaxel

>10 8.7 ± 1.1 >10 >10 >10 3.4 ± 0.5 >10 >10 >10 0.27 ± 0.04

1122, 1069, 886, 804 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 3; HRESIMS m/z 607.3596 [M + Na]+ (calcd for C35H52O7Na, 607.3605). 12-O-Tiglyl-4-deoxy-4α-phorbol-13-phenylacetate (7): pale yellow gum; [α]25D −30.8 (c 0.11, CHCl3); UV (MeOH) λmax (log ε) 236 (4.11), 341 (3.48) nm; IR (KBr) νmax 3427, 2925, 1710, 1651, 1455, 1379, 1345, 1258, 1134, 1071, 887, 734 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 3; HRESIMS m/z 571.2653 [M + Na]+ (calcd for C33H40O7Na, 571.2666). 12-O-Tiglyl-4-deoxy-4α-phorbol-13-(2-methyl)butyrate (8): colorless gum; [α]25D −17.7 (c 0.19, CHCl3); UV (MeOH) λmax (log ε) 234 (4.01) nm; IR (KBr) νmax 3414, 2927, 1714, 1651, 1458, 1381, 1257, 1153, 1029, 887, 731 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 3; HRESIMS m/z 537.2804 [M + Na]+ (calcd for C30H42O7Na, 537.2823). Cytotoxicity Assays. The cytotoxicity of all isolated compounds against the SNU387 hepatic tumor cell line (from the Shanghai Cell Bank, Chinese Academy of Sciences) was measured using the microculture tetrazolium (MTT) assay.24 The cells were maintained in RPMI1640 medium with 10% fetal bovine serum, harvested, and seeded in 96-well plates. Then, they were treated with the test compounds at various concentrations and incubated for 48 h followed by an MTT assay. Absorbance of the solution was measured using a microplate reader spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA), at a wavelength of 570 nm. Half-maximal inhibitory concentration (IC50) values were calculated using GraphPad Prism 4 (San Diego, CA, USA). Paclitaxel was used as the positive control. All samples were assayed in triplicate.

a Data are expressed as IC50 values (μM). bCompounds with IC50 >10 μM were considered inactive.

Fr.7c3 (100 mg) was purified by semipreparative HPLC (12 mL/min, 50 min 45:55 to 75:25 CH3CN−H2O gradient elution, 210 nm) to yield compounds 6 (10 mg) and 8 (2 mg). Fr.7e (100 mg) was subjected to semipreparative HPLC (12 mL/min, 45 min, 60:40 CH3CN−H2O, 210 nm) to obtain compound 17 (15 mg). Fr.7f (200 mg) was subjected to semipreparative HPLC (12 mL/min, 65 min 60:40 to 85:15 CH3CN−H2O gradient elution, 210 nm) to yield compounds 5 (21 mg), 16 (17 mg), 3 (19 mg), and 1 (3 mg). Fr.7g (3.5 g) was separated on a silica gel column eluted with CH2Cl2− MeOH (10:1 to 1:1) to give five subfractions (Fr.7g1−Fr.7g5). Fr.7g3 (140 mg) was purified by semipreparative HPLC (10 mL/min, 65 min, 55:45 to 80:20 CH3CN−H2O gradient elution, 210 nm) to yield compounds 7 (3 mg) and 19 (14 mg). Fr.7h (250 mg) was purified by semipreparative HPLC (12 mL/min, 40 min 65:35 to 80:20 CH3CN− H2O gradient elution, 210 nm) to obtain compounds 15 (30 mg), 18 (28 mg), and 4 (31 mg). Fr.7i (2.7 g) was separated on a C18 column (MeOH−H2O, 55:45 to 80:20, stepwise) to afford four subfractions (Fr.7i1−Fr.7i4). Fr.7i2 (540 mg) was purified by semipreparative HPLC (12 mL/min, 50 min, 65:35 to 85:15 MeCN−H2O gradient elution, 210 nm) to give compounds 9 (100 mg), 10 (97 mg), 11 (25 mg), and 2 (15 mg). Fr.7i4 was subjected to semipreparative HPLC (12 mL/min, 60 min 80:20 to 95:5 CH3CN−H2O gradient elution, 210 nm), to yield compounds 12 (15 mg), 13 (21 mg), and 14 (14 mg). 12-O-(2-Methyl)butyrylphorbol-13-tiglate (1): colorless gum; [α]25D +21.1 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 229 (3.88) nm; IR (KBr) νmax 3415, 2963, 2036, 1699, 1382, 1262, 1083, 803 cm−1; 1H and 13C NMR (CDCl3) data, see Table 1; HRESIMS m/z 553.2751 [M + Na]+ (calcd for C30H42O8Na, 553.2772). 12-O-Tiglylporbol-13-propionate (2): colorless gum; [α]25D +31.4 (c 0.27, CHCl3); UV (MeOH) λmax (log ε) 233 (3.92) nm; IR (KBr) νmax 3419, 2925, 2124, 2037, 1712, 1651, 1378, 1261, 1079, 1030, 804 cm−1; 1H and 13C NMR (CDCl3) data, see Table 1; HRESIMS m/z 525.2436 [M + Na]+ (calcd for C28H38O8Na, 525.2459). 13-O-Acetylphorbol-20-oleate (3): colorless oil; [α]25D +19.1 (c 0.35, CHCl3); UV (MeOH) λmax (log ε) 232 (3.55) nm; IR (KBr) νmax 3426, 2926, 2855, 1723, 1629, 1457, 1377, 1261, 1172, 1053 cm−1; 1H and 13C NMR (CDCl3) data, see Table 1; HRESIMS m/z 693.4309 [M + Na]+ (calcd for C40H62O8Na, 693.4337). 13-O-Acetyl-4-deoxy-4α-phorbol-20-linoleate (4): colorless oil; [α]25D −11.6 (c 0.68, CHCl3); UV (MeOH) λmax (log ε) 242 (4.08) nm; IR (KBr) νmax 3427, 2927, 2857, 1723, 1377, 1250, 1158, 1052, 992 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 3; HRESIMS m/z 651.4246 [M − H]− (calcd for C40H59O7, 651.4266). 13-O-Acetyl-4-deoxy-4α-phorbol-20-oleate (5): yellow oil; [α]25D −20.6 (c 0.53, CHCl3); UV (MeOH) λmax (log ε) 234 (3.87) nm; IR (KBr) νmax 3419, 2927, 1723, 1639, 1456, 1377, 1250, 1169, 1051, 992, 886 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 3; HRESIMS m/z 653.4405 [M − H]− (calcd for C40H61O7, 653.4423). 12-O-Tiglyl-4-deoxy-4α-phorbol-13-decanoate (6): colorless gum; [α]25D −13.2 (c 0.25, CHCl3); UV (MeOH) λmax (log ε) 237 (3.95) nm; IR (KBr) νmax 3418, 2926, 2857, 1713, 1652, 1456, 1379, 1259,

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ASSOCIATED CONTENT

S Supporting Information *

Spectroscopic data of compounds 1−8. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-28-82890821. Fax: +86-28-82890288. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 Program, No. 2009CB522804) and the National New Drug Innovation Major Project of China (2011ZX09307-002-02).

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REFERENCES

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