Bioorganic & Medicinal Chemistry Letters 23 (2013) 6682–6687
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Cytotoxic compounds from invasive giant salvinia (Salvinia molesta) against human tumor cells Shiyou Li ⇑, Ping Wang, Guangrui Deng, Wei Yuan, Zushang Su National Center for Pharmaceutical Crops, Arthur Temple College of Forestry and Agriculture, Stephen F. Austin State University, Nacogdoches, TX 75962-6109, USA
a r t i c l e
i n f o
Article history: Received 23 September 2013 Revised 17 October 2013 Accepted 21 October 2013 Available online 29 October 2013 Keywords: Salvinia molesta Giant salvinia Abietane diterpenes Salviniol Salviniside Invasive species Cytotoxicity
a b s t r a c t Giant salvinia (Salvinia molesta) is one of the most noxious invasive species in the world. Our bioactivityguided fractionation of ethanol extract of giant salvinia led to the isolation of 50 compounds. Of the six new compounds (1–6), salviniol (1) is a rare abietane diterpene with a new ferruginol-menthol coupled skeleton and both salviniside I (2) and salviniside II (3) are novel benzofuran glucose conjugates with unique 10-membered macrodiolide structures. Sixteen abietane diterpenes (1, 7–17, and 19–22) demonstrated in vitro activities against human tumor cells, and 7 and 8 showed selective cytotoxicity to tumor cells over normal cells. Ó 2013 Elsevier Ltd. All rights reserved.
Salvinia Ség. is a genus of floating ferns belonging to the family Salviniaceae and has 10–14 species in the world, particularly in the tropics. Salvinia molesta D. S. Mitchell, known as giant salvinia, water fern, or kariba weed, is native to Brazil. The plant could double its biomass in 2–3 days under favorable conditions and may be still alive after drying for days.1a–c Since 1939, it has invaded lake and river systems in warm climates in the world.2 At present, giant salvinia is one of the most widespread and environmentally, economically and socially destructive invasive plant species.3 In addition, giant salvinia provides habitat for snails that are intermediate hosts for Schistosoma sp. which parasitize the human intestinal and urinary tracts. The parasitic disease Schistosomiasis is also known as snail fever, bilharzia, or bilharziosis, is the second most socioeconomically devastating parasitic disease after malaria. Current chemical, biological, and mechanical control measures are very expensive and have failed to achieve their purpose.3,4 Researchers had analyzed the chemical composition of giant salvinia to evaluate its suitability as a source of forage for ruminants but the high content of crude ash, lignin and tannins restrict the use.5 Recently, it was found that salvinia is able to accumulate certain metals makes it potentially useful for waste management and effluent treatment.6a Positive control by harvesting and utilizing giant salvinia has not been developed, and the medical or pharmaceutical potential of giant salvinia remains underexplored. ⇑ Corresponding author. Tel.: +1 936 468 2071; fax: +1 936 468 7058. E-mail address: [email protected] (S. Li). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.10.040
Previous phytochemical investigations reported six phenolics, one iridiod, and one terpenoid in the Salvinia genus and some compounds showed antioxidant activity.6a–f We found that a crude ethanol extract of S. molesta showed moderate cytotoxic activity again human non-small cell lung cell (A549), human prostate cancer cell (PC-3), and human pancreatic cancer cell (PANC-1) but not toxic to normal human lung fibroblasts cells (MRC-5). As a part of our continuing research on the discovery of novel anticancer agents from native Texas plants and important invasive plants, we carried out a bioactivity-guided phytochemical investigation on this plant and obtained 50 compounds (Fig. 1), including 17 abietane diterpenes (1 and 7–22), nine phenolics (2–4, 29–32, 49 and 50), five fatty acids (24–28), five triterpene (35–39), four apocarotenoids (42–45), two acyclic sesquiterpenoids (6 and 23), two monoterpenes (5 and 46), two jasmonates (33 and 34), two steroids (40 and 41), and two coumarin (47 and 48). All abietane diterpenes were isolated from Salvinia for the first time. Among the 50 compounds, six are new compounds (1–6). Of them, salviniol (7(menth-1-en-4-ol)-ferruginol) (1) is a rare abietane diterpene with a new ferruginol-menthol coupled skeleton and both salviniside I (2) and salviniside II (3) are novel benzofuran glucose conjugates with unique 10-membered macrodiolide structure through acylation at C-4 and C-6 of glucopyranosyl. The EtOH extract of S. molesta was found to exhibit moderate selective cytotoxicity against several human cancer cell lines (A549: GI50 84.34 lg/mL; PC-3: GI50 100 lg/mL; PANC-1: GI50 69.92 lg/mL), but no activity for normal lung cell line (MRC-5:
6683
S. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6682–6687 HO
20
1 2
14
5 6 7
4
10′ 1′
H
19
18
3′
3' 7′
4′
6′ 5′
8
8′
HO
9′
OH
HO
2′
3
7 8
4
5a
OH
1 2 10
9
6'
5''' 2''' 3'''
O 1''' R 2= β -H
6'' 5''
O HO
4''
O
3'' 2'' 1''
4 5
9
7
O
3
O
6
11
O
7
CH 3
3 R 1=OH, R 2= β -H or R 1= α -H, R2 =OH 3a R 1=OCH 3, R 2= β -H
R1 ,R 2
1
6
4
5
3
2
14
15
OR2
1
R1
COOH
7 R1 =OH; R2 =H; R 3 =OH 8 R1 =H; R 2=H; R 3=OH 9 R1 =H; R 2=H; R 3=H 10 R1 =H; R 2 =CH3 ; R 3 =H 11 R1 =H; R 2 =CH3 ; R 3 =OH
13
6 R=β -D-glucopyranosyl 23 R=H
10
5b 11
4
8
9
10
12 2
8
OR
OH 4'''
2 R1 =
O
OH
12
5
O
1
HO 6
5'
11
4 3 10
2
1'
O
5
O
2'
4'
6
9
1
13 15 17 12
9
10
3
11
7
8
16
OH
O R3
5
OH
OH
R
R
OH
O O
OH
O
O O
O
R 14 R=H 15 R=OH
12 R=OH 13 R=H
17 R=OH 18 R=H
16
19
20 OH
OH
OH
OH
O
COOCH 3
24 OH
CHO CHO 21
27 COOH
R1
28
OH R2
R
R1
35 R1 =H, R 2 =CH2 , Δ22,29 36 R1 =OAc, R2 =CH 2, Δ22,29 37 R1 =OAc, R2 =CH 3 38 R1 =OAc, R2 =CH 2OH 39 R1 =OAc, R2 =CH 3, Δ16,17
O
R 3=OH
29 3; 30 R 1=COOH; R 2=OH; R 3 =OH 1 31 R =COOH; R 2=H; R 3=OH 32 R 1=CHO; R 2=H; R3 =OH
33 R=OH 34 R=ile
46 O
O R
3
O
47 R 1 =H; R 2=H; R 3=OCH3 48 R 1 =CH 2CH3 ; R 2=CH3 ; R 3=H
O
HO
45
44
R2
O
40 41 Δ 22,23
HO
O
43
42
HO
O
OH
O HO
HO
O
O
O
O
H
R2
R3 R 2 =H;
COOH
25
22
R 1=COOCH
26
OH
O
R1
COOH
HOOC
O
O
O
O
49
50
Figure 1. Structures of the compounds isolated from Salvinia molesta.
GI50 >100 lg/mL). This prompted us to phytochemically examine the chemical constituents responsible for the cytotoxic activity using bioactivity-guided fractionation. This Letter reports the isolation, structural elucidation, and cytotoxic activity of six new compounds, salviniol (7-(menth-1-en-4-ol)-ferruginol) (1), salviniside I (2), salviniside II (3), salvinin A (4), salvinin B (5), and 10-hydroxy-2,6, 10-trimethyl-2E,6E,11-dodecatrienoic acid 10-O-b-D-glucopyranoside (6), together with 16 abietane diterpenes: 14-deoxycoleon U (7),6e,f montbretol (8),7a 5,6-dehydrosugiol (9),7b 7-methoxyrosmanol
(10),7c montbretyl 12-methyl ether (11),7d 11-hydroxysugiol (12),7e sugiol (13),7f ferruginol (14),7g 7-hydroxyferruginol (15),8a 6,7-dehydroferruginol (16),8b 12-hydroxy simonellite (17),8c simonellite (18),8c 1-oxomiltirone (19),8d royleanone (20),7g 6,7-dehydroroyleanone (21),6f 12-hydroxy-6,7-secoabieta-8,11,13-triene-6, 7-dial (22),8e and 28 other known compounds: 10-hydroxy-2,6,10trimethyl-2E,6E,11-dodecatrienoic acid (23),8f 6-hydroxy-7E,9Z-pentadecadienoic acid (24),8g 11-hydroxy-7Z,9E,13E-hexadecatrienoic acid (25),8h 13-hydroxy-9Z,11E,15E-octadecatrienoic acid (26),8i
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S. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6682–6687
13-hydroxy-tetradecanoic acid methyl ester (27), decanoic acid (28),8j 4-hydroxy methyl benzoate (29), 3,4-dihydroxy benzoic acid (30), 4-hydroxy benzoic acid (31), p-hydroxybenzaldehyde (32),9a cucurbic acid (33),9b cucurbinoyl-isoleucine (34),9c 22(29)hopene (35),9d 3b-acetoxy-20(29)-hopene (36), 3b-acetoxy-hopane (37), 3b-acetoxy-29-hydroxy-hopane (38), 3b-acetoxy-16-hopene (39), b-sitosterol (40), b-stigmasterol (41), (3R,6R,7E)-3-hydroxy4,7-megastigmadien-9-one (42),9e annuionone D (43),9f dehydrovomifoliol (44),9g (+)-3-hydroxy-b-ionone (45),9e pubinernoid A (46),9h 3-methoxycoumarin (47),9i 4-methyl-7-ethyl-coumarin (48),9j asiaticin (49),9k 2-(benzoyloxy)-1,1-dimethyl-ethyl (50)9l (Fig. 1). The identities of these known compounds were determined by analyzing their spectroscopic data and confirmed by comparing their values with those in the literature. Compound 1 was isolated as a colorless powder. The molecular formula C30H46O2 was deduced from the [M-H]+ peak in the HRESI-MS, suggesting the presence of eight degrees of unsaturation. The 13C NMR spectrum of 1 (Table 1) indicated the presence of four double bonds [dC 150.9 (s), 148.8 (s), 136.4 (s), 131.6 (s), 127.1 (d), 120.9 (d), 110.8 (d)] and an oxygenated carbon [dC 72.1 (s)]. Its 1H NMR spectrum had signals for three methyl groups (dH 0.87, 0.89, 1.12), two isopropyl groups [dH 0.93, 0.95, 1.23, 1.24 (3H each, d, J = 6.9 Hz)], an olefinic proton (dH 5.35, br s), and two aromatic protons [dH 6.60, 6.87 (1H each, s)] (Table 1). Those data suggested that 1 was a derivative of ferruginol (14)7g exhibiting typical signals of ferruginol: an isopropyl group attached to a phenyl group; two para aromatic protons and a typical Hb-1 proton; three singlet methyl groups.10 The other moiety of 1 was a cyclic monoterpene having a tri-substituted double bond (dC 136.4, 120.9), an isopropyl group, and an oxygenated carbon, suggesting it to be a menth-
Table 1 C and 1H NMR data of compound 1 (d in ppm)
13
No.
1 dC
1
38.8
2
19.4
3
41.8
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 10 20 30
33.4 44.9 21.9 35.3 131.6 148.8 38.0 110.8 150.9 131.6 127.1 27.0 22.8 22.6 33.8 21.7 25.1 136.4 120.9 34.8
40 50
72.1 31.2
0
25.0 36.6 17.0 16.9 46.3
6 70 80 90 100
dH 2.15 1.33 1.72 1.60 1.48 1.19
(1H, (1H, (1H, (1H, (1H, (1H,
m) m) m) m) m) m)
1.39 (1H, m) 1.65 (2H, m) 2.93 (1H, br q, J = 7.5 Hz)
6.60 (1H, s)
6.87 3.08 1.24 1.23 0.89 0.87 1.12
(1H, (1H, (3H, (3H, (3H, (3H, (3H,
s) sept. J = 6.9 Hz) d, J = 6.9 Hz) d, J = 6.9 Hz) s) s) s)
5.35 (br s) 2.23 (1H, m) 1.95 (1H, m) 1.74 1.56 2.19 2.92 0.93 0.95 2.21
(1H, (1H, (2H, (1H, (3H, (3H, (2H,
m) m) m) m) d, J = 6.9 Hz) d, J = 6.9 Hz) m)
1-en-4-ol. An HMBC experiment revealed long-range couplings from H-100 at dC 46.3 to C-6, C-7, C-20 and C-60 , establishing the linkage between ferruginol and menth-1-en-4-ol. The stereochemistry of 1 established by NOESY experiment.10,11 Thus the structure of 1 was established as 7-(menth-1-en-4-ol)-ferruginol. Recently, abietane-type diterpenes with novel skeletons, such as diterpenes attached to sesquiterpenes,10–12 dimeric diterpenes,13 and monoterpenes14 were reported from the bark and heartwood of Cryptomeria japonica, the fruits of C. fortunei, and the bark of Calocedrus macrolepis var. formosana. Compound 1 has a new ferruginol-menthol skeleton and is named as salviniol because of the plant source Salvinia. Compound 2 was obtained as colorless powder and was assigned a molecular formula of C27H26O13, as deduced from the [M-H]+ peak at m/z 557.1296 (calcd for 557.1295) in the HR-ESIMS. In the 13C and 1H NMR spectrum 2 (Table 2) displayed a set of glycoside signals (dC 103.7, 77.7, 75.6, 74.5, 68.7, 65.7). The anomeric proton at dH 4.39 with coupling constant JH1,2 value (d, J = 7.8 Hz) demonstrated the presence of a b-glucopyranosyl moiety.6c The monosaccharide was further determined to be D configuration after chemical degradation of 215,16 and HPLC analysis with authentic sugar. The 1H NMR spectrum of 2 contained signals assigned to an olefinic proton at dH 5.65 (1H, dd, H-3000 ), a vinyl methyl group at dH 1.70 (3H, s, H-5000 ), and two oxygenated methylenes at dH 4.11 (2H, d, J = 6.4 Hz, H-4000 ), 4.06 and 4.21 (1H each, d, J = 12.2 Hz, H-1000 ), respectively. These data determined that 2 possessed a hemiterpene with a trisubstituted double bond.15,16 Further, five aromatic protons were also found in the 1H NMR spectrum (Table 2), of which three signals at dH 7.39 (1H, dd, J = 1.8, 8.2 Hz, H-20 ), 6.83 (1H, d, J = 8.2 Hz, H-30 ), and 7.45 (1H, d, J = 1.8 Hz, H-60 ) were characteristic of a 1,2,4-trisubstituted phenyl moiety, the remaining two at dH 7.67 (1H, d, J = 8.2 Hz, H-6) and 6.79 (1H, d, J = 8.2 Hz, H-7) were assigned to a 1,2,3,4-tetrasubstituted phenyl group.6c A close inspection of the 13C NMR spectrum of 2, together with the MS data, clearly revealed that the 1,2,3, 4-tetrasubstituted phenyl moiety should be a part of a 8-carbon benzofuran skeleton.17,18 In additional, the 13C NMR spectra of 2 displayed also two ester carbonyl carbons at dC 168.0 and 168.5. As observed in the HMBC spectrum of 2, three key HMBC correlation among dH 4.39 (glc H-100 ) and dC 75.8 (hemiterpene C-1000 ) established that the glucose was attached to the assigned position; H-6 at dH 7.67 and Glc H-600 at dH 3.93 with a carbonyl carbon at dC 168.0 (C-11) established the linkage between the benzofuran unit and H-600 of Glc. Similarly, the other ester carbonyl carbon (dC 168.5, C-10) was attached to H-400 of Glc position by the HMBC spectrum analysis. These long-range HMBC correlations of H-20 to C-10 , C-60 and C-2, and of H-60 to C-10 , C-20 and C-2 established the connection of the benzofuran skeleton with 1,2,4-trisubstituted phenyl moiety. The 1,1-ADEQUATE spectrum also confirmed the assignment. The relatively downfield shifted of C-3 at dC 110.2 and the remaining one degree of unsaturation established the linkage of the C-3 and C-10. The complete assignment of the protons and carbons was achieved by a combination of 1H, 13C, HSQC, 1 H-1H COSY, HMBC and 1,1-ADEQUATE spectral analyses. Moreover, NOEs were observed between the methylene protons at C-1000 and C-4000 , indicating the E-configuration of the hemiterpene moiety. The structure of 2 was identified as 100 -O-[(E)-2000 -methyl-but2000 -en-4 000 -ol]-400 ,6 00 -O-[3,5-dicarbonyl-8-hydroxy-2-(40 ,50 -dihydroxy-phenyl)-1-benzofuran-2-yl]-b-D-glucopyranose and was named salviniside I. Compound 3 was obtained as colorless powders and showed an [M-1]+ at m/z 473.0714 (calcd for 473.0720) in the HR-ESI-MS, suggesting the molecular formula to be C22H18O12. The NMR spectroscopic data of 3 (Table 2), was different from those of 2 mainly in the absence of the signals for the hemiterpene moiety, which was attached at the C-1 position of the glucose in 2. Instead,
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S. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6682–6687 Table 2 C and 1H NMR data of compounds 2 and 3 (d in ppm)
13
No.
2
3
a-Anomer
b-Anomer dC 2 3 4 5 6 7 8 9 10 11 10 20 30 40 50 60 Glc-100 Glc-200 Glc-300 Glc-400 Glc-500 Glc-600
dH
158.4 110.2 129.7 114.8 130.0 112.1 149.0 143.5 168.5 168.0 121.6 121.4 116.7 149.4 146.8 115.7 103.7 75.6 74.5 77.7 68.7 65.7
1000
75.8
2000 3000 4000 5000
135.8 128.5 59.2 14.2
dC
7.67 (1H, d, J = 8.2 Hz) 6.79 (1H, d, J = 8.2 Hz)
7.39 (1H, dd, J = 8.2, 1.8 Hz) 6.83 (1H, d, J = 8.2 Hz)
7.45 4.39 3.32 3.59 5.27 3.77 3.93 5.02 4.06 4.21
(1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, J = 1.8 Hz) d, J = 7.8 Hz) dd, J = 8.2, 8.7 Hz) dd, J = 9.6, 9.2 Hz) t, J = 9.6 Hz) td, J = 10.1, 4.1 Hz) t, J = 10.5 Hz) dd, J = 10.5, 4.1 Hz) d, J = 12.2 Hz) d, J = 12.2 Hz)
158.3 110.3 129.7 114.9 130.0 112.1 148.8 143.5 168.7 168.1 121.6 121.5 116.8 149.4 146.8 115.7 98.6 76.7 74.5 77.8 68.9 65.8
dH
dC 158.4 110.2 129.7 114.8 130.0 112.1 148.8 143.5 168.7 168.1 121.6 121.4 116.8 149.4 146.8 115.7 94.4 74.2 71.6 78.4 64.4 66.4
7.70 (1H, d, J = 8.2 Hz) 6.82 (1H, d, J = 8.2 Hz)
7.39 (1H, dd, J = 8.5, 1.8 Hz) 6.84 (1H, d, J = 8.5 Hz)
7.45 4.57 3.27 3.57 5.30 3.78 3.94 5.04
(1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, J = 1.8 Hz) d, J = 7.8 Hz) dd, J = 7.8, 9.2 Hz) dd, J = 9.6, 9.2 Hz) t, J = 9.6 Hz) td, J = 10.5, 4.1 Hz) t, J = 11.0 Hz) dd, J = 10.3, 4.1 Hz)
dH
7.70 (1H, d, J = 8.2 Hz) 6.82 (1H, d, J = 8.2 Hz)
7.39 (1H, dd, J = 8.5, 1.8 Hz) 6.84 (1H, d, J = 8.5 Hz)
7.45 (1H, d, J = 1.8 Hz) 5.14 (1H, d, J = 3.7 Hz) 3.51(1H, dd, J = 3.7, 9.2 Hz) 3.83 (1H, t, J = 10.1 Hz) 5.24 (1H, t, J = 9.6 Hz) 4.24 (1H, td, J = 10.5, 4.1 Hz) 3.91 (1H, t, J = 11.0 Hz) 4.97(1H, dd, J = 10.3, 4.1 Hz)
5.65 (1H, dd, J = 5.9, 6.4 Hz) 4.11 (2H, d, J = 6.4 Hz) 1.70 (3H, s)
compound 3 displayed similarly two set of glycoside signals (Table 2) with two anomeric proton at dH 4.57 (1H, d, J = 7.8 Hz, b-H-100 ) and 5.14 (1H, d, J = 3.7 Hz, a-H-100 ), as well as two protonated carbon at dC-b 98.6 and dC-a 94.4 observed in the HSQC spectrum. Acid hydrolysis of 3 liberated D-glucose identified by coHPLC with authentic sugar.15,16 The analysis of the 1H NMR, COSY, HSQC spectra indicated 3 should be an isomeric mixture of a and b-D-glucopyranosides.6a,c,19 This conclusion was confirmed by production of 3a with b-anomeric methyl glucose upon methylation of 3 with MeOH in 10% H2SO4. Owing to the formation of a mixture of a and b-anomers, each signals of the aglycon part of 3 appeared essentially in duplicate. Further, the complete assignment of the protons and carbons was achieved by a combination of 1H, 13C, HSQC, 1H–1H COSY and HMBC spectral analyses. Thus, compound 3 was identified as 400 ,600 -O-[3,5-dicarbonyl-8-hydroxy-2-(40 ,50 dihydroxy-phenyl)-1-benzofuran-2-yl]-a/b-D-glucopyranose and named salviniside II.
Compound 4 was obtained as a pale yellow powder. The molecular formula C10H8O2 was established by HR-ESI-MS and 1D NMR spectrum (Table 3). The 1H NMR spectrum of compound 4 displayed the signals of five sp2 protons and two sp3 protons. The 13 C NMR spectrum with DEPT experiments displayed 10 carbon resonances comprising one oxygenated methylene, five sp2 methines, and four quaternary carbons (one keto group). The 1H NMR spectrum (dH 8.19, dd, J = 8.4, 2.4 Hz, H-5; dH 7.20, m, H-6 and H-7; dH 7.42, dd, J = 7.6, 2.2 Hz, H-8) revealed an o-disubstituted benzene ring substructure.8j HMBC correlations of H2-10 to C-1, C-2 and C-3, indicated that the only oxygenated methylene (C-10) was placed at C-2. The whole structure was completed by 2D NMR spectrum. Thus, compound 4 was elucidated as 2-hydroxymethyl-indone, namely as salvinin A. Compound 5 was obtained as an amorphous powder. The molecular formula was established as C11H16O2 by HR-ESI-MS and 1D NMR spectrum. The 1H NMR spectrum (Table 4) showed Table 4 C and 1H NMR data of compound 5 (d in ppm)
13
No.
Table 3 C and 1H NMR data of compound 4 (d in ppm)
13
No.
1 2 3 4 5 6 7 8 9 10
dC 4
dC 195.6 126.1 132.6 136.5 121.3 121.9 123.0 110.6 113.6 64.9
5
dH
1 2 3 4 5
172.6 112.4 182.6 87.2 47.6
6 7
63.9 47.9
8 10 11 12
34.8 29.0 24.0 24.4
8.17 (1H, s) 8.19 7.20 7.20 7.42
(1H, dd, J = 8.4, 2.4 Hz) (1H, m) (1H, m) (1H dd, J = 7.6, 2.2 Hz)
4.73 (2H, s)
dH 5.75 (1H, s)
2.45 1.41 4.07 1.97 1.36
(1H, (1H, (1H, (1H, (1H,
dt, J = 12.4, 5.2 Hz) m) m) dt, J = 12.4, 2.8 Hz) m)
1.25 (3H, s) 1.29 (3H, s) 1.57 (3H, s)
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S. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6682–6687
Table 5 Cytotoxic activity of 17 abietane diterpenes against various human tumor cell lines (GI50) (lM) Compd
A549
PC-3
HL-60
PANC-1
BxPC-3
MDA-MB-231
MRC-5
1 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
62.91 ± 9.63 9.10 ± 2.47 11.07 ± 3.16 84.52 ± 18.73 86.50 ± 9.54 21.21 ± 6.21 35.04 ± 6.20 79.8 ± 2.18 30.67 ± 5.22 75.11 ± 7.04 53.79 ± 9.65 42.22 ± 3.89 () 24.89 ± 5.94 30.14 ± 3.64 47.96 ± 7.75 93.75 ± 21.39
54.19 ± 5.90 8.39 ± 0.86 13.17 ± 2.03 >100 N/A N/A 41.03 ± 7.90 >100 56.45 ± 16.51 85.61 ± 5.30 58.12 ± 3.28 43.60 ± 11.88 () 18.97 ± 5.34 27.07 ± 11.76 46.32 ± 6.60 N/A
48.05 ± 10.86 13.33 ± 5.62 11.45 ± 3.13 92.78 ± 9.98 N/A N/A 35.26 ± 0.63 68.64 ± 13.46 27.33 ± 8.95 75.19 ± 1.02 66.52 ± 0.04 50.01 ± 15.23 () 23.27 ± 6.66 36.33 ± 7.14 30.03 ± 8.30 N/A
60.44 ± 16.17 22.59 ± 4.18 23.08 ± 9.16 >100 N/A N/A 43.14 ± 8.32 87.94 ± 11.18 41.13 ± 14.11 78.24 ± 13.46 46.71 ± 10.98 37.53 ± 5.72 () 32.15 ± 2.75 52.55 ± 18.84 51.34 ± 10.04 N/A
87.28 ± 24.51 37.28 ± 7.44 25.0 ± 13.19 >100 N/A N/A 45.01 ± 8.33 36.08 ± 1.50 21.14 ± 0.51 74.73 ± 11.09 37.50 ± 4.01 36.42 ± 8.08 () 20.54 ± 2.00 51.04 ± 10.44 41.74 ± 2.92 N/A
75.0 ± 1.37 >100 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
58.41 ± 6.83 24.75 ± 4.73 20.01 ± 5.14 >100 N/A N/A 40.19 ± 0.91 >100 40.01 ± 7.74 70.49 ± 7.21 59.25 ± 7.56 55.36 ± 4.94 () 20.39 ± 3.36 45.70 ± 2.32 40.19 ± 2.06 N/A
Note: GI50 (mean ± SD) refers to the concentration required to have 50% cell-growth inhibition; () indicates that the isolated is inactive (negative at 100 or GI50 >100 lM); N/A: no data available.
three methyls at dH 1.25, 1.29, and 1.57 as singlets, and a proton of a trisubstituted double bond at dH 5.75. The 13C NMR spectrum (Table 4) showed 11 carbon signals, identified by a DEPT experiment as three methyls, two methylenes, one oxymethine, two double bond carbons, two quaternary carbons and one carbonyl carbon. The 1H–1H COSY spectrum together with the HSQC data revealed a –CH2–CH–CH2– unit in bold (5a) (Fig. 1). HMBC correlations of H3-12/C-3 and C-4, H-2/C-1, C-3, C-4 and C-8, H3-11/C-1, C-8 and C-10 suggested the partial structure 5b (Fig. 1). The two substructures were linked via C-4 and C-5, C-7 and C-8 by the HMBC corre-
lations of H3-11/C-7 and H3-12/C-5, respectively. The only leftover uncertainty for the planar structure of 5 was the remaining one degree of unsaturation, which required the presence of an additional ring. The relatively upfield shifted of the 13C NMR data at C-1 at dC 172.6 and the relatively downfield shifted of C-6 at dC 63.9, suggesting that an ether bridge was present between C-1 and C-6 to form an oxygen ring. The relative stereochemistry of 5 was established on the basis of the ROESY experiment. The strong ROESY correlations of H-5b/H3-12 and H-6b indicated that H-6, and Me12 were b-oriented. The other configurations were established as
50
60
45
Salviniol
Montbretol
50
35
0 uM
30
40 uM 60 uM
25
80 uM
20
100 uM
15 10
Viable BxPC-3 Cells (x1,000)
Viable BxPC-3 Cells (x1,000)
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0 0
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Salviniol
Montbretol 25 0 uM
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80 uM 100 uM
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Viable Panc-1 Cells (x1,000)
25 Viable Panc-1 Cells (x1,000)
30
Incubation Time (hr)
Incubation Time (hr)
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10 5
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Figure 2. Effects of salviniol (1) and montbretol (8) on the proliferation of human exocrine pancreatic cancer cells (PANC-1 and BxPC-3).
S. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6682–6687
the same as compound pubinernoid A by comparing their spectroscopic data.9h The gross structure of 5 was thus established as (4S⁄,6S⁄)-4-hydroxy-4,8,8-trimethyl-9-oxabicyclo[4.2.1]non-1-en3-one, namely as salvinin B. Compound 6 was isolated as a pale, amorphous powder. The molecular formula C21H33O8 was deduced from the [M-H]+ peak at m/z 413.2174 (calcd for 413.2175) in the HR-ESI-MS. The 13C NMR spectrum of 6 (Table S1) showed 21 carbon resonances, including one resonated at dC 98.2 assignable to an anomeric carbon, and five resonated in the region dC 61.4–76.6, which suggest the presence of a hexose residue. In the 1H NMR spectrum, the coupling constant of the anomeric proton at dH 4.31 (H-10 , d, J = 7.8 Hz) indicated that the sugar moiety was a b-glucopyranose,20 which was further determined to be b-D-glucopyranose by co-HPLC analysis with authentic sugar after acid hydrolysis of 6.21 Apart from the sugar moiety, the 1H and 13C NMR data of the aglycone of 6 (Table S1) were very similar to those of 23,8f indicating that 6 was likely a glycosidic derivative of 23. The HMBC correlation between H-10 (dH 4.31) and C-10 (dC 80.5) showed that the sugar moiety was attached to C-10. The E- and E-geometry of the two double bonds at C-2 and C-6 were identified by the analysis of the NOESY spectrum of 6 between H-13/H-4 and H-5, H3-14 and H-8. Thus, the structure of 6 was assigned as 10-hydroxy-2,6,10-trimethyl2E,6E,11-dodecatrienoic acid 10-O-b-D-glucopyranoside. All compounds (1–50) were tested for their cytotoxicity against human exocrine pancreatic cancer (PANC-1 and BxPC-3), nonsmall cell lung cancer (A549), prostate cancer (PC-3), breast cancer (MDA-MB-231), leukemia (HL-60), and normal lung cells (MRC5).22 Sixteen abietane diterpenes (1, 7–17, and 19–22) demonstrated activities against the tumor cells (Table 5). 14-deoxycoleon U (7) and montbretol (8) have high selectivity over normal cells. The suppressive effects of salviniol (1) and montbretol (8) on human PANC-1 and BxPC-3 pancreatic cancer cell proliferation were investigated, and both could inhibit human pancreatic cancer cell proliferation in a dose-dependent manner (Fig. 2). The cytotoxicity of compounds 1, 11, 15, 16, 17, and 22 were reported for the first time. The bioactivities of compounds 7, 8, 12, 20, and 21 against human exocrine pancreatic cancer have never been reported before. It is also the first report of the selective activities of compounds 7 and 8 to human non-small cell lung cancer (A549) but less toxic normal human lung fibroblasts MRC-5. The cytotoxicity of compounds 12 and 21 against human non-small cell lung cancer (A549), prostate cancer (PC-3), and leukemia (HL-60) are reported for the first time. Based on the structure–activity relationship analysis of bioactive abietane diterpenes, the abietane diterpenes with group ‘OH’, ‘OCH3’, or ‘@O’ at C-12 will have cytotoxicity against human tumors, and abietane diterpenes also have one of these groups at C-6 will enhance the bioactivity. 6,7-seco abietane derivatives still possess cytotoxicity, but show less activity than the regular abietanes according to the bioassay results The bioactive compounds from salvinia, particularly salviniol have promising potential in the drug development for cancer. Pharmaceutical production of bioactive agents from salvinia will provide the best way to ‘control’ the most noxious invasive species in the world. Acknowledgments This work was partially supported by the US Department of Agriculture grant 2008-03460 and Stephen F. Austin State University. The authors would like to thank Chris Moret of Texas Parks and Wildlife Department for plant collection; Dr. Vanessa Santiago of Texas A&M University for HR-ESI-MS analysis; and Dr. Youlin Xia of University of Minnesota for NMR analysis.
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Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013. 10.040. References and notes 1. (a) Finlayson, C. M. Australia Aquat. Bot. 1984, 18, 257; (b) Room, P. M.; Thomas, P. A. J. Appl. Ecol. 1986, 23, 1013; (c) Barrett, S. C. H. Sci. Amer. 1989, 260, 90. 2. Room, P. M. Trends Ecol. Evol. 1990, 5, 74. 3. Schooler, S. S.; Salau, B.; Julien, M. H.; Ives, A. R. Nature 2011, 470, 86. 4. Abbasi, S. A.; Nipaney, P. C. Environ. Conserv. 1986, 13, 235. 5. Moozhiyil, M.; Pallauf, J. J. Econ. Bot. 1986, 40, 375. 6. (a) Choudhary, M. I.; Naheed, N.; Abbaskhan, A.; Musharraf, S. G.; Siddiqui, H.; Siddiqui, H.; Atta-ur-Rahman Phytochemistry 2009, 69, 1018; (b) Chantiratikul, P.; Meechai, P.; Nakbanpotec, W. Res. J. Biol. Sci. 2009, 4, 1113; (c) Narasimhulua, M.; Ashalathaa, K.; Sri Laxmib, P.; Sarmaa, A. V. S.; Rama Raoa, B.; Kavi Kishorb, P. B.; David Krupadanamc, G. L.; Zehra Alid, A.; Tiwarid, A. K.; Panneer Selvame, A.; Venkateswarlua, Y. Nat. Prod. Res. 2010, 24, 1390; (d) Srilaxmi, P.; Sareddy, G. R.; Kavikishor, P. B.; Setty, O. H.; Phanithi, P. P. BMC Pharm. 2010, 10, 13; (e) Fraga, B. M.; Diaz, C. E.; Guadano, A.; Gonzalez-Coloma, A. J. Agric. Food Chem. 2005, 53, 5200; (f) Kusumoto, N.; Ashitani, T.; Hayasaka, Y.; Murayama, T.; Ogiyama, K.; Takahashi, K. J. Chem. Ecol. 2009, 35, 635. 7. (a) Ayhan, U.; Gulacti, T. J. Nat. Prod. 1992, 55, 441; (b) Xu, M. F.; Shen, L. Q.; Wang, K. W.; Du, Q. Z.; Wang, N. Nat. Prod. Commun. 2011, 6, 3; (c) Guerrero, I. C.; Andres, L. S.; León, L. G.; Machín, R. P.; Padrón, J. M.; Luis, J. G.; Delgadillo, J. J. Nat. Prod. 1803, 2006, 69; (d) Ulubelen, A.; Topcu, G. J. Nat. Prod. 1992, 55, 441; (e) Xiang, W.; Li, S. H.; Zhao, Q. S.; Na, Z.; Jiang, B.; Zhang, H. J.; Lin, Z. W.; Sun, H. D. Chinese Chem. Lett. 2002, 13, 141; (f) Benjamin, R. Magn. Reson. Chem. 2003, 41, 741; (g) Tezuka, Y.; Kasimu, R.; Li, J. X.; Basnet, P.; Tanaka, K.; Namba, T.; Kadota, S. Chem. Pharm. Bull. 1998, 46, 107. 8. (a) Kuo, Y. H.; Chen, C. H.; Wein, Y. S. Helv. Chim. Acta. 2002, 85, 2657; (b) Kuo, Y. H.; Yeh, M. H. Phytochemistry 1998, 49, 2453; (c) Elisa, G.; Laura, P.; Annamaria, D.; Giuseppe, G. Ann. Chim. 1969, 59, 510; (d) Sairafianpour, M.; Christensen, J.; Staerk, D.; Budnik, B. A.; Arsalan Kharazmi, A.; Bagherzadeh, K.; Jaroszewski, J. W. J. Nat. Prod. 2001, 64, 1398; (e) Fang, J.; Jan, S.; Cheng, Y. J. Chem. Res 1986, 5, 350; (f) Miyazawa, M.; Nankai, H.; Kameoka, H. J. Agric. Food Chem. 1996, 44, 1543; (g) Tamura, S.; Kaneko, M.; Shiomi, A.; Yang, G. M.; Yamaura, T.; Murakami, N. Bioorg. Med. Chem. Lett. 1837, 2010, 20; (h) Xiang, W. S.; Wang, J. D.; Wang, X. J.; Zhang, J. J. Asian Nat. Prod. Res. 2008, 10, 1081; (i) Bang, M. H.; Han, J. T.; Kim, H. Y.; Park, Y. D.; Park, C. H.; Lee, K. R.; Baek, N. I. Arch. Pharm. Res. 2002, 25, 438; (j) Yu, D. Q.; Yang, J. S. Handbook of Analytical Chemistry; Chemical Industry Press: Beijing, 1999. 9. (a) Avupati, V. R.; Yejella, R. P.; Guntuku, G.; Gunta, P. Bioorg. Med. Chem. Lett. 2012, 22, 1031; (b) Fujita, T.; Terato, K.; Nakayama, M. Biosci. Biotechnol. Biochem. 1996, 60, 732; (c) Hans-Dieter, K.; Guenther, S. Phytochemistry 1995, 38, 569; (d) Mahato, S. B.; Kundu, A. P. Phytochemistry 1994, 37, 1517; (e) DellaGreca, M.; Marino, C. D.; Zarrelli, A.; D’Abrosca, B. J. Nat. Prod. 2004, 67, 1492; (f) Zhao, J.; Zhou, L. L.; Li, X.; Xiao, H. B.; Hou, F. F.; Cheng, Y. X. J. Nat. Prod. 2011, 74, 1392; (g) Tan, M. T.; Nonato, M. G.; Kogure, N.; Kitajima, M.; Takayama, H. Biochem. Syst. Ecol. 2012, 40, 4; (h) Huang, S. X.; Yang, J.; Xiao, W. L.; Zhu, Y. L.; Li, R. T.; Li, L. M.; Pu, J. X.; Li, X.; Li, S. H.; Sun, H. D. Helv. Chim. Acta 2006, 89, 1169; (i) Mustafa, A.; Hsihmat, O. H.; Zayed, S. M. A. D.; Ahmed Nawar, A. Tetrahedron 1831, 1963, 19; (j) Brubaker, A. N.; Ruiter, J. D.; Whitmer, W. L. J. Med. Chem. 1986, 29, 1094; (k) Siddiuui, B. S.; Aslam, H.; Ali, S. T.; Khan, S.; Begum, S. J. Asian Nat. Prod. Res. 2007, 9, 407; (l) Fielding, A. J.; Franchi, P.; Roberts, B. P.; Smits, T. M. J. Chem. Soc. 2002, 2, 151. 10. Kazuko, Y.; Toshinori, T.; Akemi, U.; Shigenobu, A. Chem. Pharm. Bull. 2006, 54, 315. 11. Hsieh, C. L.; Tseng, M. H.; Shao, Y. Y.; Chang, J. Y.; Kuo, C. C.; Chang, C. Y.; Kuo, Y. H. J. Nat. Prod. 2006, 69, 1611. 12. Kazuko, Y.; Kentaro, S.; Akemi, U.; Shigenobu, A. Chem. Pharm. Bull. 2006, 54, 574. 13. Shigenobu, A.; Akemi, U.; Shinya, B.; Shinji, I.; Mikako, O.; Mayo, T.; Kazuko, Y. Chem. Pharm. Bull. 2004, 52, 354. 14. Wu, J.; Zhao, W. M. J. Asian Nat. Prod. Res. 2010, 12, 382. 15. Oiso, Y.; Toyota, M.; Asakawa, Y. Chem. Pharm. Bull. 2001, 49, 126. 16. Toyota, M.; Oiso, Y.; Asakawa, Y. Chem. Pharm. Bull. 2002, 50, 508. 17. Maurya, R.; Singh, R.; Deepak, M.; Handa, S. S.; Yadav, P. P.; Mishra, P. K. Phytochemistry 2004, 65, 915. 18. Wu, S. F.; Chang, F. R.; Wang, S. Y.; Hwang, T. L.; Lee, C. L.; Chen, S. L.; Wu, C. C.; Wu, Y. C. J. Nat. Prod. 2011, 989. 19. Ding, H. Y.; Wu, Y. C.; Lin, H. C.; Chan, Y. Y.; Wu, P. L.; Wu, T. S. Chem. Pharm. Bull. 1999, 47, 652. 20. Zhang, Z. Z.; Li, S. Y.; Ownby, S.; Wang, P.; Yuan, W.; Zhang, W. L.; Beasley, R. S. Phytochemistry 2008, 69, 2070. 21. Tanaka, T.; Nakashima, T.; Ueda, T.; Tomii, K.; Kouno, I. Chem. Pharm. Bull. 2007, 55, 899. 22. Wang, P.; Ownby, S.; Zhang, Z. Z.; Yuan, W.; Li, S. Y. Bioorg. Med. Chem. Lett. 2010, 20, 2790.
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Cytotoxic compounds from invasive giant salvinia (Salvinia molesta) against human tumor cells Shiyou Li ⇑, Ping Wang, Guangrui Deng, Wei Yuan, Zushang Su National Center for Pharmaceutical Crops, Arthur Temple College of Forestry and Agriculture, Stephen F. Austin State University, Nacogdoches, TX 75962-6109, USA
a r t i c l e
i n f o
Article history: Received 23 September 2013 Revised 17 October 2013 Accepted 21 October 2013 Available online 29 October 2013 Keywords: Salvinia molesta Giant salvinia Abietane diterpenes Salviniol Salviniside Invasive species Cytotoxicity
a b s t r a c t Giant salvinia (Salvinia molesta) is one of the most noxious invasive species in the world. Our bioactivityguided fractionation of ethanol extract of giant salvinia led to the isolation of 50 compounds. Of the six new compounds (1–6), salviniol (1) is a rare abietane diterpene with a new ferruginol-menthol coupled skeleton and both salviniside I (2) and salviniside II (3) are novel benzofuran glucose conjugates with unique 10-membered macrodiolide structures. Sixteen abietane diterpenes (1, 7–17, and 19–22) demonstrated in vitro activities against human tumor cells, and 7 and 8 showed selective cytotoxicity to tumor cells over normal cells. Ó 2013 Elsevier Ltd. All rights reserved.
Salvinia Ség. is a genus of floating ferns belonging to the family Salviniaceae and has 10–14 species in the world, particularly in the tropics. Salvinia molesta D. S. Mitchell, known as giant salvinia, water fern, or kariba weed, is native to Brazil. The plant could double its biomass in 2–3 days under favorable conditions and may be still alive after drying for days.1a–c Since 1939, it has invaded lake and river systems in warm climates in the world.2 At present, giant salvinia is one of the most widespread and environmentally, economically and socially destructive invasive plant species.3 In addition, giant salvinia provides habitat for snails that are intermediate hosts for Schistosoma sp. which parasitize the human intestinal and urinary tracts. The parasitic disease Schistosomiasis is also known as snail fever, bilharzia, or bilharziosis, is the second most socioeconomically devastating parasitic disease after malaria. Current chemical, biological, and mechanical control measures are very expensive and have failed to achieve their purpose.3,4 Researchers had analyzed the chemical composition of giant salvinia to evaluate its suitability as a source of forage for ruminants but the high content of crude ash, lignin and tannins restrict the use.5 Recently, it was found that salvinia is able to accumulate certain metals makes it potentially useful for waste management and effluent treatment.6a Positive control by harvesting and utilizing giant salvinia has not been developed, and the medical or pharmaceutical potential of giant salvinia remains underexplored. ⇑ Corresponding author. Tel.: +1 936 468 2071; fax: +1 936 468 7058. E-mail address: [email protected] (S. Li). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.10.040
Previous phytochemical investigations reported six phenolics, one iridiod, and one terpenoid in the Salvinia genus and some compounds showed antioxidant activity.6a–f We found that a crude ethanol extract of S. molesta showed moderate cytotoxic activity again human non-small cell lung cell (A549), human prostate cancer cell (PC-3), and human pancreatic cancer cell (PANC-1) but not toxic to normal human lung fibroblasts cells (MRC-5). As a part of our continuing research on the discovery of novel anticancer agents from native Texas plants and important invasive plants, we carried out a bioactivity-guided phytochemical investigation on this plant and obtained 50 compounds (Fig. 1), including 17 abietane diterpenes (1 and 7–22), nine phenolics (2–4, 29–32, 49 and 50), five fatty acids (24–28), five triterpene (35–39), four apocarotenoids (42–45), two acyclic sesquiterpenoids (6 and 23), two monoterpenes (5 and 46), two jasmonates (33 and 34), two steroids (40 and 41), and two coumarin (47 and 48). All abietane diterpenes were isolated from Salvinia for the first time. Among the 50 compounds, six are new compounds (1–6). Of them, salviniol (7(menth-1-en-4-ol)-ferruginol) (1) is a rare abietane diterpene with a new ferruginol-menthol coupled skeleton and both salviniside I (2) and salviniside II (3) are novel benzofuran glucose conjugates with unique 10-membered macrodiolide structure through acylation at C-4 and C-6 of glucopyranosyl. The EtOH extract of S. molesta was found to exhibit moderate selective cytotoxicity against several human cancer cell lines (A549: GI50 84.34 lg/mL; PC-3: GI50 100 lg/mL; PANC-1: GI50 69.92 lg/mL), but no activity for normal lung cell line (MRC-5:
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S. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6682–6687 HO
20
1 2
14
5 6 7
4
10′ 1′
H
19
18
3′
3' 7′
4′
6′ 5′
8
8′
HO
9′
OH
HO
2′
3
7 8
4
5a
OH
1 2 10
9
6'
5''' 2''' 3'''
O 1''' R 2= β -H
6'' 5''
O HO
4''
O
3'' 2'' 1''
4 5
9
7
O
3
O
6
11
O
7
CH 3
3 R 1=OH, R 2= β -H or R 1= α -H, R2 =OH 3a R 1=OCH 3, R 2= β -H
R1 ,R 2
1
6
4
5
3
2
14
15
OR2
1
R1
COOH
7 R1 =OH; R2 =H; R 3 =OH 8 R1 =H; R 2=H; R 3=OH 9 R1 =H; R 2=H; R 3=H 10 R1 =H; R 2 =CH3 ; R 3 =H 11 R1 =H; R 2 =CH3 ; R 3 =OH
13
6 R=β -D-glucopyranosyl 23 R=H
10
5b 11
4
8
9
10
12 2
8
OR
OH 4'''
2 R1 =
O
OH
12
5
O
1
HO 6
5'
11
4 3 10
2
1'
O
5
O
2'
4'
6
9
1
13 15 17 12
9
10
3
11
7
8
16
OH
O R3
5
OH
OH
R
R
OH
O O
OH
O
O O
O
R 14 R=H 15 R=OH
12 R=OH 13 R=H
17 R=OH 18 R=H
16
19
20 OH
OH
OH
OH
O
COOCH 3
24 OH
CHO CHO 21
27 COOH
R1
28
OH R2
R
R1
35 R1 =H, R 2 =CH2 , Δ22,29 36 R1 =OAc, R2 =CH 2, Δ22,29 37 R1 =OAc, R2 =CH 3 38 R1 =OAc, R2 =CH 2OH 39 R1 =OAc, R2 =CH 3, Δ16,17
O
R 3=OH
29 3; 30 R 1=COOH; R 2=OH; R 3 =OH 1 31 R =COOH; R 2=H; R 3=OH 32 R 1=CHO; R 2=H; R3 =OH
33 R=OH 34 R=ile
46 O
O R
3
O
47 R 1 =H; R 2=H; R 3=OCH3 48 R 1 =CH 2CH3 ; R 2=CH3 ; R 3=H
O
HO
45
44
R2
O
40 41 Δ 22,23
HO
O
43
42
HO
O
OH
O HO
HO
O
O
O
O
H
R2
R3 R 2 =H;
COOH
25
22
R 1=COOCH
26
OH
O
R1
COOH
HOOC
O
O
O
O
49
50
Figure 1. Structures of the compounds isolated from Salvinia molesta.
GI50 >100 lg/mL). This prompted us to phytochemically examine the chemical constituents responsible for the cytotoxic activity using bioactivity-guided fractionation. This Letter reports the isolation, structural elucidation, and cytotoxic activity of six new compounds, salviniol (7-(menth-1-en-4-ol)-ferruginol) (1), salviniside I (2), salviniside II (3), salvinin A (4), salvinin B (5), and 10-hydroxy-2,6, 10-trimethyl-2E,6E,11-dodecatrienoic acid 10-O-b-D-glucopyranoside (6), together with 16 abietane diterpenes: 14-deoxycoleon U (7),6e,f montbretol (8),7a 5,6-dehydrosugiol (9),7b 7-methoxyrosmanol
(10),7c montbretyl 12-methyl ether (11),7d 11-hydroxysugiol (12),7e sugiol (13),7f ferruginol (14),7g 7-hydroxyferruginol (15),8a 6,7-dehydroferruginol (16),8b 12-hydroxy simonellite (17),8c simonellite (18),8c 1-oxomiltirone (19),8d royleanone (20),7g 6,7-dehydroroyleanone (21),6f 12-hydroxy-6,7-secoabieta-8,11,13-triene-6, 7-dial (22),8e and 28 other known compounds: 10-hydroxy-2,6,10trimethyl-2E,6E,11-dodecatrienoic acid (23),8f 6-hydroxy-7E,9Z-pentadecadienoic acid (24),8g 11-hydroxy-7Z,9E,13E-hexadecatrienoic acid (25),8h 13-hydroxy-9Z,11E,15E-octadecatrienoic acid (26),8i
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13-hydroxy-tetradecanoic acid methyl ester (27), decanoic acid (28),8j 4-hydroxy methyl benzoate (29), 3,4-dihydroxy benzoic acid (30), 4-hydroxy benzoic acid (31), p-hydroxybenzaldehyde (32),9a cucurbic acid (33),9b cucurbinoyl-isoleucine (34),9c 22(29)hopene (35),9d 3b-acetoxy-20(29)-hopene (36), 3b-acetoxy-hopane (37), 3b-acetoxy-29-hydroxy-hopane (38), 3b-acetoxy-16-hopene (39), b-sitosterol (40), b-stigmasterol (41), (3R,6R,7E)-3-hydroxy4,7-megastigmadien-9-one (42),9e annuionone D (43),9f dehydrovomifoliol (44),9g (+)-3-hydroxy-b-ionone (45),9e pubinernoid A (46),9h 3-methoxycoumarin (47),9i 4-methyl-7-ethyl-coumarin (48),9j asiaticin (49),9k 2-(benzoyloxy)-1,1-dimethyl-ethyl (50)9l (Fig. 1). The identities of these known compounds were determined by analyzing their spectroscopic data and confirmed by comparing their values with those in the literature. Compound 1 was isolated as a colorless powder. The molecular formula C30H46O2 was deduced from the [M-H]+ peak in the HRESI-MS, suggesting the presence of eight degrees of unsaturation. The 13C NMR spectrum of 1 (Table 1) indicated the presence of four double bonds [dC 150.9 (s), 148.8 (s), 136.4 (s), 131.6 (s), 127.1 (d), 120.9 (d), 110.8 (d)] and an oxygenated carbon [dC 72.1 (s)]. Its 1H NMR spectrum had signals for three methyl groups (dH 0.87, 0.89, 1.12), two isopropyl groups [dH 0.93, 0.95, 1.23, 1.24 (3H each, d, J = 6.9 Hz)], an olefinic proton (dH 5.35, br s), and two aromatic protons [dH 6.60, 6.87 (1H each, s)] (Table 1). Those data suggested that 1 was a derivative of ferruginol (14)7g exhibiting typical signals of ferruginol: an isopropyl group attached to a phenyl group; two para aromatic protons and a typical Hb-1 proton; three singlet methyl groups.10 The other moiety of 1 was a cyclic monoterpene having a tri-substituted double bond (dC 136.4, 120.9), an isopropyl group, and an oxygenated carbon, suggesting it to be a menth-
Table 1 C and 1H NMR data of compound 1 (d in ppm)
13
No.
1 dC
1
38.8
2
19.4
3
41.8
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 10 20 30
33.4 44.9 21.9 35.3 131.6 148.8 38.0 110.8 150.9 131.6 127.1 27.0 22.8 22.6 33.8 21.7 25.1 136.4 120.9 34.8
40 50
72.1 31.2
0
25.0 36.6 17.0 16.9 46.3
6 70 80 90 100
dH 2.15 1.33 1.72 1.60 1.48 1.19
(1H, (1H, (1H, (1H, (1H, (1H,
m) m) m) m) m) m)
1.39 (1H, m) 1.65 (2H, m) 2.93 (1H, br q, J = 7.5 Hz)
6.60 (1H, s)
6.87 3.08 1.24 1.23 0.89 0.87 1.12
(1H, (1H, (3H, (3H, (3H, (3H, (3H,
s) sept. J = 6.9 Hz) d, J = 6.9 Hz) d, J = 6.9 Hz) s) s) s)
5.35 (br s) 2.23 (1H, m) 1.95 (1H, m) 1.74 1.56 2.19 2.92 0.93 0.95 2.21
(1H, (1H, (2H, (1H, (3H, (3H, (2H,
m) m) m) m) d, J = 6.9 Hz) d, J = 6.9 Hz) m)
1-en-4-ol. An HMBC experiment revealed long-range couplings from H-100 at dC 46.3 to C-6, C-7, C-20 and C-60 , establishing the linkage between ferruginol and menth-1-en-4-ol. The stereochemistry of 1 established by NOESY experiment.10,11 Thus the structure of 1 was established as 7-(menth-1-en-4-ol)-ferruginol. Recently, abietane-type diterpenes with novel skeletons, such as diterpenes attached to sesquiterpenes,10–12 dimeric diterpenes,13 and monoterpenes14 were reported from the bark and heartwood of Cryptomeria japonica, the fruits of C. fortunei, and the bark of Calocedrus macrolepis var. formosana. Compound 1 has a new ferruginol-menthol skeleton and is named as salviniol because of the plant source Salvinia. Compound 2 was obtained as colorless powder and was assigned a molecular formula of C27H26O13, as deduced from the [M-H]+ peak at m/z 557.1296 (calcd for 557.1295) in the HR-ESIMS. In the 13C and 1H NMR spectrum 2 (Table 2) displayed a set of glycoside signals (dC 103.7, 77.7, 75.6, 74.5, 68.7, 65.7). The anomeric proton at dH 4.39 with coupling constant JH1,2 value (d, J = 7.8 Hz) demonstrated the presence of a b-glucopyranosyl moiety.6c The monosaccharide was further determined to be D configuration after chemical degradation of 215,16 and HPLC analysis with authentic sugar. The 1H NMR spectrum of 2 contained signals assigned to an olefinic proton at dH 5.65 (1H, dd, H-3000 ), a vinyl methyl group at dH 1.70 (3H, s, H-5000 ), and two oxygenated methylenes at dH 4.11 (2H, d, J = 6.4 Hz, H-4000 ), 4.06 and 4.21 (1H each, d, J = 12.2 Hz, H-1000 ), respectively. These data determined that 2 possessed a hemiterpene with a trisubstituted double bond.15,16 Further, five aromatic protons were also found in the 1H NMR spectrum (Table 2), of which three signals at dH 7.39 (1H, dd, J = 1.8, 8.2 Hz, H-20 ), 6.83 (1H, d, J = 8.2 Hz, H-30 ), and 7.45 (1H, d, J = 1.8 Hz, H-60 ) were characteristic of a 1,2,4-trisubstituted phenyl moiety, the remaining two at dH 7.67 (1H, d, J = 8.2 Hz, H-6) and 6.79 (1H, d, J = 8.2 Hz, H-7) were assigned to a 1,2,3,4-tetrasubstituted phenyl group.6c A close inspection of the 13C NMR spectrum of 2, together with the MS data, clearly revealed that the 1,2,3, 4-tetrasubstituted phenyl moiety should be a part of a 8-carbon benzofuran skeleton.17,18 In additional, the 13C NMR spectra of 2 displayed also two ester carbonyl carbons at dC 168.0 and 168.5. As observed in the HMBC spectrum of 2, three key HMBC correlation among dH 4.39 (glc H-100 ) and dC 75.8 (hemiterpene C-1000 ) established that the glucose was attached to the assigned position; H-6 at dH 7.67 and Glc H-600 at dH 3.93 with a carbonyl carbon at dC 168.0 (C-11) established the linkage between the benzofuran unit and H-600 of Glc. Similarly, the other ester carbonyl carbon (dC 168.5, C-10) was attached to H-400 of Glc position by the HMBC spectrum analysis. These long-range HMBC correlations of H-20 to C-10 , C-60 and C-2, and of H-60 to C-10 , C-20 and C-2 established the connection of the benzofuran skeleton with 1,2,4-trisubstituted phenyl moiety. The 1,1-ADEQUATE spectrum also confirmed the assignment. The relatively downfield shifted of C-3 at dC 110.2 and the remaining one degree of unsaturation established the linkage of the C-3 and C-10. The complete assignment of the protons and carbons was achieved by a combination of 1H, 13C, HSQC, 1 H-1H COSY, HMBC and 1,1-ADEQUATE spectral analyses. Moreover, NOEs were observed between the methylene protons at C-1000 and C-4000 , indicating the E-configuration of the hemiterpene moiety. The structure of 2 was identified as 100 -O-[(E)-2000 -methyl-but2000 -en-4 000 -ol]-400 ,6 00 -O-[3,5-dicarbonyl-8-hydroxy-2-(40 ,50 -dihydroxy-phenyl)-1-benzofuran-2-yl]-b-D-glucopyranose and was named salviniside I. Compound 3 was obtained as colorless powders and showed an [M-1]+ at m/z 473.0714 (calcd for 473.0720) in the HR-ESI-MS, suggesting the molecular formula to be C22H18O12. The NMR spectroscopic data of 3 (Table 2), was different from those of 2 mainly in the absence of the signals for the hemiterpene moiety, which was attached at the C-1 position of the glucose in 2. Instead,
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S. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6682–6687 Table 2 C and 1H NMR data of compounds 2 and 3 (d in ppm)
13
No.
2
3
a-Anomer
b-Anomer dC 2 3 4 5 6 7 8 9 10 11 10 20 30 40 50 60 Glc-100 Glc-200 Glc-300 Glc-400 Glc-500 Glc-600
dH
158.4 110.2 129.7 114.8 130.0 112.1 149.0 143.5 168.5 168.0 121.6 121.4 116.7 149.4 146.8 115.7 103.7 75.6 74.5 77.7 68.7 65.7
1000
75.8
2000 3000 4000 5000
135.8 128.5 59.2 14.2
dC
7.67 (1H, d, J = 8.2 Hz) 6.79 (1H, d, J = 8.2 Hz)
7.39 (1H, dd, J = 8.2, 1.8 Hz) 6.83 (1H, d, J = 8.2 Hz)
7.45 4.39 3.32 3.59 5.27 3.77 3.93 5.02 4.06 4.21
(1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, J = 1.8 Hz) d, J = 7.8 Hz) dd, J = 8.2, 8.7 Hz) dd, J = 9.6, 9.2 Hz) t, J = 9.6 Hz) td, J = 10.1, 4.1 Hz) t, J = 10.5 Hz) dd, J = 10.5, 4.1 Hz) d, J = 12.2 Hz) d, J = 12.2 Hz)
158.3 110.3 129.7 114.9 130.0 112.1 148.8 143.5 168.7 168.1 121.6 121.5 116.8 149.4 146.8 115.7 98.6 76.7 74.5 77.8 68.9 65.8
dH
dC 158.4 110.2 129.7 114.8 130.0 112.1 148.8 143.5 168.7 168.1 121.6 121.4 116.8 149.4 146.8 115.7 94.4 74.2 71.6 78.4 64.4 66.4
7.70 (1H, d, J = 8.2 Hz) 6.82 (1H, d, J = 8.2 Hz)
7.39 (1H, dd, J = 8.5, 1.8 Hz) 6.84 (1H, d, J = 8.5 Hz)
7.45 4.57 3.27 3.57 5.30 3.78 3.94 5.04
(1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, J = 1.8 Hz) d, J = 7.8 Hz) dd, J = 7.8, 9.2 Hz) dd, J = 9.6, 9.2 Hz) t, J = 9.6 Hz) td, J = 10.5, 4.1 Hz) t, J = 11.0 Hz) dd, J = 10.3, 4.1 Hz)
dH
7.70 (1H, d, J = 8.2 Hz) 6.82 (1H, d, J = 8.2 Hz)
7.39 (1H, dd, J = 8.5, 1.8 Hz) 6.84 (1H, d, J = 8.5 Hz)
7.45 (1H, d, J = 1.8 Hz) 5.14 (1H, d, J = 3.7 Hz) 3.51(1H, dd, J = 3.7, 9.2 Hz) 3.83 (1H, t, J = 10.1 Hz) 5.24 (1H, t, J = 9.6 Hz) 4.24 (1H, td, J = 10.5, 4.1 Hz) 3.91 (1H, t, J = 11.0 Hz) 4.97(1H, dd, J = 10.3, 4.1 Hz)
5.65 (1H, dd, J = 5.9, 6.4 Hz) 4.11 (2H, d, J = 6.4 Hz) 1.70 (3H, s)
compound 3 displayed similarly two set of glycoside signals (Table 2) with two anomeric proton at dH 4.57 (1H, d, J = 7.8 Hz, b-H-100 ) and 5.14 (1H, d, J = 3.7 Hz, a-H-100 ), as well as two protonated carbon at dC-b 98.6 and dC-a 94.4 observed in the HSQC spectrum. Acid hydrolysis of 3 liberated D-glucose identified by coHPLC with authentic sugar.15,16 The analysis of the 1H NMR, COSY, HSQC spectra indicated 3 should be an isomeric mixture of a and b-D-glucopyranosides.6a,c,19 This conclusion was confirmed by production of 3a with b-anomeric methyl glucose upon methylation of 3 with MeOH in 10% H2SO4. Owing to the formation of a mixture of a and b-anomers, each signals of the aglycon part of 3 appeared essentially in duplicate. Further, the complete assignment of the protons and carbons was achieved by a combination of 1H, 13C, HSQC, 1H–1H COSY and HMBC spectral analyses. Thus, compound 3 was identified as 400 ,600 -O-[3,5-dicarbonyl-8-hydroxy-2-(40 ,50 dihydroxy-phenyl)-1-benzofuran-2-yl]-a/b-D-glucopyranose and named salviniside II.
Compound 4 was obtained as a pale yellow powder. The molecular formula C10H8O2 was established by HR-ESI-MS and 1D NMR spectrum (Table 3). The 1H NMR spectrum of compound 4 displayed the signals of five sp2 protons and two sp3 protons. The 13 C NMR spectrum with DEPT experiments displayed 10 carbon resonances comprising one oxygenated methylene, five sp2 methines, and four quaternary carbons (one keto group). The 1H NMR spectrum (dH 8.19, dd, J = 8.4, 2.4 Hz, H-5; dH 7.20, m, H-6 and H-7; dH 7.42, dd, J = 7.6, 2.2 Hz, H-8) revealed an o-disubstituted benzene ring substructure.8j HMBC correlations of H2-10 to C-1, C-2 and C-3, indicated that the only oxygenated methylene (C-10) was placed at C-2. The whole structure was completed by 2D NMR spectrum. Thus, compound 4 was elucidated as 2-hydroxymethyl-indone, namely as salvinin A. Compound 5 was obtained as an amorphous powder. The molecular formula was established as C11H16O2 by HR-ESI-MS and 1D NMR spectrum. The 1H NMR spectrum (Table 4) showed Table 4 C and 1H NMR data of compound 5 (d in ppm)
13
No.
Table 3 C and 1H NMR data of compound 4 (d in ppm)
13
No.
1 2 3 4 5 6 7 8 9 10
dC 4
dC 195.6 126.1 132.6 136.5 121.3 121.9 123.0 110.6 113.6 64.9
5
dH
1 2 3 4 5
172.6 112.4 182.6 87.2 47.6
6 7
63.9 47.9
8 10 11 12
34.8 29.0 24.0 24.4
8.17 (1H, s) 8.19 7.20 7.20 7.42
(1H, dd, J = 8.4, 2.4 Hz) (1H, m) (1H, m) (1H dd, J = 7.6, 2.2 Hz)
4.73 (2H, s)
dH 5.75 (1H, s)
2.45 1.41 4.07 1.97 1.36
(1H, (1H, (1H, (1H, (1H,
dt, J = 12.4, 5.2 Hz) m) m) dt, J = 12.4, 2.8 Hz) m)
1.25 (3H, s) 1.29 (3H, s) 1.57 (3H, s)
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S. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6682–6687
Table 5 Cytotoxic activity of 17 abietane diterpenes against various human tumor cell lines (GI50) (lM) Compd
A549
PC-3
HL-60
PANC-1
BxPC-3
MDA-MB-231
MRC-5
1 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
62.91 ± 9.63 9.10 ± 2.47 11.07 ± 3.16 84.52 ± 18.73 86.50 ± 9.54 21.21 ± 6.21 35.04 ± 6.20 79.8 ± 2.18 30.67 ± 5.22 75.11 ± 7.04 53.79 ± 9.65 42.22 ± 3.89 () 24.89 ± 5.94 30.14 ± 3.64 47.96 ± 7.75 93.75 ± 21.39
54.19 ± 5.90 8.39 ± 0.86 13.17 ± 2.03 >100 N/A N/A 41.03 ± 7.90 >100 56.45 ± 16.51 85.61 ± 5.30 58.12 ± 3.28 43.60 ± 11.88 () 18.97 ± 5.34 27.07 ± 11.76 46.32 ± 6.60 N/A
48.05 ± 10.86 13.33 ± 5.62 11.45 ± 3.13 92.78 ± 9.98 N/A N/A 35.26 ± 0.63 68.64 ± 13.46 27.33 ± 8.95 75.19 ± 1.02 66.52 ± 0.04 50.01 ± 15.23 () 23.27 ± 6.66 36.33 ± 7.14 30.03 ± 8.30 N/A
60.44 ± 16.17 22.59 ± 4.18 23.08 ± 9.16 >100 N/A N/A 43.14 ± 8.32 87.94 ± 11.18 41.13 ± 14.11 78.24 ± 13.46 46.71 ± 10.98 37.53 ± 5.72 () 32.15 ± 2.75 52.55 ± 18.84 51.34 ± 10.04 N/A
87.28 ± 24.51 37.28 ± 7.44 25.0 ± 13.19 >100 N/A N/A 45.01 ± 8.33 36.08 ± 1.50 21.14 ± 0.51 74.73 ± 11.09 37.50 ± 4.01 36.42 ± 8.08 () 20.54 ± 2.00 51.04 ± 10.44 41.74 ± 2.92 N/A
75.0 ± 1.37 >100 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
58.41 ± 6.83 24.75 ± 4.73 20.01 ± 5.14 >100 N/A N/A 40.19 ± 0.91 >100 40.01 ± 7.74 70.49 ± 7.21 59.25 ± 7.56 55.36 ± 4.94 () 20.39 ± 3.36 45.70 ± 2.32 40.19 ± 2.06 N/A
Note: GI50 (mean ± SD) refers to the concentration required to have 50% cell-growth inhibition; () indicates that the isolated is inactive (negative at 100 or GI50 >100 lM); N/A: no data available.
three methyls at dH 1.25, 1.29, and 1.57 as singlets, and a proton of a trisubstituted double bond at dH 5.75. The 13C NMR spectrum (Table 4) showed 11 carbon signals, identified by a DEPT experiment as three methyls, two methylenes, one oxymethine, two double bond carbons, two quaternary carbons and one carbonyl carbon. The 1H–1H COSY spectrum together with the HSQC data revealed a –CH2–CH–CH2– unit in bold (5a) (Fig. 1). HMBC correlations of H3-12/C-3 and C-4, H-2/C-1, C-3, C-4 and C-8, H3-11/C-1, C-8 and C-10 suggested the partial structure 5b (Fig. 1). The two substructures were linked via C-4 and C-5, C-7 and C-8 by the HMBC corre-
lations of H3-11/C-7 and H3-12/C-5, respectively. The only leftover uncertainty for the planar structure of 5 was the remaining one degree of unsaturation, which required the presence of an additional ring. The relatively upfield shifted of the 13C NMR data at C-1 at dC 172.6 and the relatively downfield shifted of C-6 at dC 63.9, suggesting that an ether bridge was present between C-1 and C-6 to form an oxygen ring. The relative stereochemistry of 5 was established on the basis of the ROESY experiment. The strong ROESY correlations of H-5b/H3-12 and H-6b indicated that H-6, and Me12 were b-oriented. The other configurations were established as
50
60
45
Salviniol
Montbretol
50
35
0 uM
30
40 uM 60 uM
25
80 uM
20
100 uM
15 10
Viable BxPC-3 Cells (x1,000)
Viable BxPC-3 Cells (x1,000)
40
40 0 uM 5 uM
30
10 uM 20 uM
20
40 uM
10
5
0
0 0
10
20
30
40
50
60
70
0
80
10
20
40
50
60
70
80
30
30
Salviniol
Montbretol 25 0 uM
20
40 uM 60 uM
15
80 uM 100 uM
10
5
Viable Panc-1 Cells (x1,000)
25 Viable Panc-1 Cells (x1,000)
30
Incubation Time (hr)
Incubation Time (hr)
20
0 uM 5 uM
15
10 uM 20 uM 40 uM
10 5
0
0 0
10
20
30
40
50
Incubation Time (hr)
60
70
80
0
10
20
30
40
50
60
70
80
Incubation Time (hr)
Figure 2. Effects of salviniol (1) and montbretol (8) on the proliferation of human exocrine pancreatic cancer cells (PANC-1 and BxPC-3).
S. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6682–6687
the same as compound pubinernoid A by comparing their spectroscopic data.9h The gross structure of 5 was thus established as (4S⁄,6S⁄)-4-hydroxy-4,8,8-trimethyl-9-oxabicyclo[4.2.1]non-1-en3-one, namely as salvinin B. Compound 6 was isolated as a pale, amorphous powder. The molecular formula C21H33O8 was deduced from the [M-H]+ peak at m/z 413.2174 (calcd for 413.2175) in the HR-ESI-MS. The 13C NMR spectrum of 6 (Table S1) showed 21 carbon resonances, including one resonated at dC 98.2 assignable to an anomeric carbon, and five resonated in the region dC 61.4–76.6, which suggest the presence of a hexose residue. In the 1H NMR spectrum, the coupling constant of the anomeric proton at dH 4.31 (H-10 , d, J = 7.8 Hz) indicated that the sugar moiety was a b-glucopyranose,20 which was further determined to be b-D-glucopyranose by co-HPLC analysis with authentic sugar after acid hydrolysis of 6.21 Apart from the sugar moiety, the 1H and 13C NMR data of the aglycone of 6 (Table S1) were very similar to those of 23,8f indicating that 6 was likely a glycosidic derivative of 23. The HMBC correlation between H-10 (dH 4.31) and C-10 (dC 80.5) showed that the sugar moiety was attached to C-10. The E- and E-geometry of the two double bonds at C-2 and C-6 were identified by the analysis of the NOESY spectrum of 6 between H-13/H-4 and H-5, H3-14 and H-8. Thus, the structure of 6 was assigned as 10-hydroxy-2,6,10-trimethyl2E,6E,11-dodecatrienoic acid 10-O-b-D-glucopyranoside. All compounds (1–50) were tested for their cytotoxicity against human exocrine pancreatic cancer (PANC-1 and BxPC-3), nonsmall cell lung cancer (A549), prostate cancer (PC-3), breast cancer (MDA-MB-231), leukemia (HL-60), and normal lung cells (MRC5).22 Sixteen abietane diterpenes (1, 7–17, and 19–22) demonstrated activities against the tumor cells (Table 5). 14-deoxycoleon U (7) and montbretol (8) have high selectivity over normal cells. The suppressive effects of salviniol (1) and montbretol (8) on human PANC-1 and BxPC-3 pancreatic cancer cell proliferation were investigated, and both could inhibit human pancreatic cancer cell proliferation in a dose-dependent manner (Fig. 2). The cytotoxicity of compounds 1, 11, 15, 16, 17, and 22 were reported for the first time. The bioactivities of compounds 7, 8, 12, 20, and 21 against human exocrine pancreatic cancer have never been reported before. It is also the first report of the selective activities of compounds 7 and 8 to human non-small cell lung cancer (A549) but less toxic normal human lung fibroblasts MRC-5. The cytotoxicity of compounds 12 and 21 against human non-small cell lung cancer (A549), prostate cancer (PC-3), and leukemia (HL-60) are reported for the first time. Based on the structure–activity relationship analysis of bioactive abietane diterpenes, the abietane diterpenes with group ‘OH’, ‘OCH3’, or ‘@O’ at C-12 will have cytotoxicity against human tumors, and abietane diterpenes also have one of these groups at C-6 will enhance the bioactivity. 6,7-seco abietane derivatives still possess cytotoxicity, but show less activity than the regular abietanes according to the bioassay results The bioactive compounds from salvinia, particularly salviniol have promising potential in the drug development for cancer. Pharmaceutical production of bioactive agents from salvinia will provide the best way to ‘control’ the most noxious invasive species in the world. Acknowledgments This work was partially supported by the US Department of Agriculture grant 2008-03460 and Stephen F. Austin State University. The authors would like to thank Chris Moret of Texas Parks and Wildlife Department for plant collection; Dr. Vanessa Santiago of Texas A&M University for HR-ESI-MS analysis; and Dr. Youlin Xia of University of Minnesota for NMR analysis.
6687
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013. 10.040. References and notes 1. (a) Finlayson, C. M. Australia Aquat. Bot. 1984, 18, 257; (b) Room, P. M.; Thomas, P. A. J. Appl. Ecol. 1986, 23, 1013; (c) Barrett, S. C. H. Sci. Amer. 1989, 260, 90. 2. Room, P. M. Trends Ecol. Evol. 1990, 5, 74. 3. Schooler, S. S.; Salau, B.; Julien, M. H.; Ives, A. R. Nature 2011, 470, 86. 4. Abbasi, S. A.; Nipaney, P. C. Environ. Conserv. 1986, 13, 235. 5. Moozhiyil, M.; Pallauf, J. J. Econ. Bot. 1986, 40, 375. 6. (a) Choudhary, M. I.; Naheed, N.; Abbaskhan, A.; Musharraf, S. G.; Siddiqui, H.; Siddiqui, H.; Atta-ur-Rahman Phytochemistry 2009, 69, 1018; (b) Chantiratikul, P.; Meechai, P.; Nakbanpotec, W. Res. J. Biol. Sci. 2009, 4, 1113; (c) Narasimhulua, M.; Ashalathaa, K.; Sri Laxmib, P.; Sarmaa, A. V. S.; Rama Raoa, B.; Kavi Kishorb, P. B.; David Krupadanamc, G. L.; Zehra Alid, A.; Tiwarid, A. K.; Panneer Selvame, A.; Venkateswarlua, Y. Nat. Prod. Res. 2010, 24, 1390; (d) Srilaxmi, P.; Sareddy, G. R.; Kavikishor, P. B.; Setty, O. H.; Phanithi, P. P. BMC Pharm. 2010, 10, 13; (e) Fraga, B. M.; Diaz, C. E.; Guadano, A.; Gonzalez-Coloma, A. J. Agric. Food Chem. 2005, 53, 5200; (f) Kusumoto, N.; Ashitani, T.; Hayasaka, Y.; Murayama, T.; Ogiyama, K.; Takahashi, K. J. Chem. Ecol. 2009, 35, 635. 7. (a) Ayhan, U.; Gulacti, T. J. Nat. Prod. 1992, 55, 441; (b) Xu, M. F.; Shen, L. Q.; Wang, K. W.; Du, Q. Z.; Wang, N. Nat. Prod. Commun. 2011, 6, 3; (c) Guerrero, I. C.; Andres, L. S.; León, L. G.; Machín, R. P.; Padrón, J. M.; Luis, J. G.; Delgadillo, J. J. Nat. Prod. 1803, 2006, 69; (d) Ulubelen, A.; Topcu, G. J. Nat. Prod. 1992, 55, 441; (e) Xiang, W.; Li, S. H.; Zhao, Q. S.; Na, Z.; Jiang, B.; Zhang, H. J.; Lin, Z. W.; Sun, H. D. Chinese Chem. Lett. 2002, 13, 141; (f) Benjamin, R. Magn. Reson. Chem. 2003, 41, 741; (g) Tezuka, Y.; Kasimu, R.; Li, J. X.; Basnet, P.; Tanaka, K.; Namba, T.; Kadota, S. Chem. Pharm. Bull. 1998, 46, 107. 8. (a) Kuo, Y. H.; Chen, C. H.; Wein, Y. S. Helv. Chim. Acta. 2002, 85, 2657; (b) Kuo, Y. H.; Yeh, M. H. Phytochemistry 1998, 49, 2453; (c) Elisa, G.; Laura, P.; Annamaria, D.; Giuseppe, G. Ann. Chim. 1969, 59, 510; (d) Sairafianpour, M.; Christensen, J.; Staerk, D.; Budnik, B. A.; Arsalan Kharazmi, A.; Bagherzadeh, K.; Jaroszewski, J. W. J. Nat. Prod. 2001, 64, 1398; (e) Fang, J.; Jan, S.; Cheng, Y. J. Chem. Res 1986, 5, 350; (f) Miyazawa, M.; Nankai, H.; Kameoka, H. J. Agric. Food Chem. 1996, 44, 1543; (g) Tamura, S.; Kaneko, M.; Shiomi, A.; Yang, G. M.; Yamaura, T.; Murakami, N. Bioorg. Med. Chem. Lett. 1837, 2010, 20; (h) Xiang, W. S.; Wang, J. D.; Wang, X. J.; Zhang, J. J. Asian Nat. Prod. Res. 2008, 10, 1081; (i) Bang, M. H.; Han, J. T.; Kim, H. Y.; Park, Y. D.; Park, C. H.; Lee, K. R.; Baek, N. I. Arch. Pharm. Res. 2002, 25, 438; (j) Yu, D. Q.; Yang, J. S. Handbook of Analytical Chemistry; Chemical Industry Press: Beijing, 1999. 9. (a) Avupati, V. R.; Yejella, R. P.; Guntuku, G.; Gunta, P. Bioorg. Med. Chem. Lett. 2012, 22, 1031; (b) Fujita, T.; Terato, K.; Nakayama, M. Biosci. Biotechnol. Biochem. 1996, 60, 732; (c) Hans-Dieter, K.; Guenther, S. Phytochemistry 1995, 38, 569; (d) Mahato, S. B.; Kundu, A. P. Phytochemistry 1994, 37, 1517; (e) DellaGreca, M.; Marino, C. D.; Zarrelli, A.; D’Abrosca, B. J. Nat. Prod. 2004, 67, 1492; (f) Zhao, J.; Zhou, L. L.; Li, X.; Xiao, H. B.; Hou, F. F.; Cheng, Y. X. J. Nat. Prod. 2011, 74, 1392; (g) Tan, M. T.; Nonato, M. G.; Kogure, N.; Kitajima, M.; Takayama, H. Biochem. Syst. Ecol. 2012, 40, 4; (h) Huang, S. X.; Yang, J.; Xiao, W. L.; Zhu, Y. L.; Li, R. T.; Li, L. M.; Pu, J. X.; Li, X.; Li, S. H.; Sun, H. D. Helv. Chim. Acta 2006, 89, 1169; (i) Mustafa, A.; Hsihmat, O. H.; Zayed, S. M. A. D.; Ahmed Nawar, A. Tetrahedron 1831, 1963, 19; (j) Brubaker, A. N.; Ruiter, J. D.; Whitmer, W. L. J. Med. Chem. 1986, 29, 1094; (k) Siddiuui, B. S.; Aslam, H.; Ali, S. T.; Khan, S.; Begum, S. J. Asian Nat. Prod. Res. 2007, 9, 407; (l) Fielding, A. J.; Franchi, P.; Roberts, B. P.; Smits, T. M. J. Chem. Soc. 2002, 2, 151. 10. Kazuko, Y.; Toshinori, T.; Akemi, U.; Shigenobu, A. Chem. Pharm. Bull. 2006, 54, 315. 11. Hsieh, C. L.; Tseng, M. H.; Shao, Y. Y.; Chang, J. Y.; Kuo, C. C.; Chang, C. Y.; Kuo, Y. H. J. Nat. Prod. 2006, 69, 1611. 12. Kazuko, Y.; Kentaro, S.; Akemi, U.; Shigenobu, A. Chem. Pharm. Bull. 2006, 54, 574. 13. Shigenobu, A.; Akemi, U.; Shinya, B.; Shinji, I.; Mikako, O.; Mayo, T.; Kazuko, Y. Chem. Pharm. Bull. 2004, 52, 354. 14. Wu, J.; Zhao, W. M. J. Asian Nat. Prod. Res. 2010, 12, 382. 15. Oiso, Y.; Toyota, M.; Asakawa, Y. Chem. Pharm. Bull. 2001, 49, 126. 16. Toyota, M.; Oiso, Y.; Asakawa, Y. Chem. Pharm. Bull. 2002, 50, 508. 17. Maurya, R.; Singh, R.; Deepak, M.; Handa, S. S.; Yadav, P. P.; Mishra, P. K. Phytochemistry 2004, 65, 915. 18. Wu, S. F.; Chang, F. R.; Wang, S. Y.; Hwang, T. L.; Lee, C. L.; Chen, S. L.; Wu, C. C.; Wu, Y. C. J. Nat. Prod. 2011, 989. 19. Ding, H. Y.; Wu, Y. C.; Lin, H. C.; Chan, Y. Y.; Wu, P. L.; Wu, T. S. Chem. Pharm. Bull. 1999, 47, 652. 20. Zhang, Z. Z.; Li, S. Y.; Ownby, S.; Wang, P.; Yuan, W.; Zhang, W. L.; Beasley, R. S. Phytochemistry 2008, 69, 2070. 21. Tanaka, T.; Nakashima, T.; Ueda, T.; Tomii, K.; Kouno, I. Chem. Pharm. Bull. 2007, 55, 899. 22. Wang, P.; Ownby, S.; Zhang, Z. Z.; Yuan, W.; Li, S. Y. Bioorg. Med. Chem. Lett. 2010, 20, 2790.
