Lupane, friedelane, oleanane, and ursane triterpenes from the stem of Siphonodon celastrineus Griff

Phytochemistry xxx (2013) xxx–xxx

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Lupane, friedelane, oleanane, and ursane triterpenes from the stem of Siphonodon celastrineus Griff Wirongrong Kaweetripob a, Chulabhorn Mahidol a,b, Hunsa Prawat a,⇑, Somsak Ruchirawat a,b a b

Chulabhorn Research Institute, Kamphaeng Phet 6 Road, Bangkok 10210, Thailand Chulabhorn Graduate Institute and Center for Environmental Health and Toxicology (EHT), Kamphaeng Phet 6 Road, Bangkok 10210, Thailand

a r t i c l e

i n f o

Article history: Received 23 March 2013 Received in revised form 7 September 2013 Available online xxxx Keywords: Celastraceae Siphonodon celastrineus Lupanes Friedelanes Oleananes Ursanes Triterpenes NMR spectroscopy Cytotoxic activities

a b s t r a c t Twenty-one triterpenes consisting of a lupane derivative, two friedelanes, an oleanane derivative, and 17 ursane-type triterpenoids, together with three known triterpenes, three sterols, a fatty acid, a sesquiterpene alkaloid, and a glycerol derivative, were isolated from the stem of Siphonodon celastrineus. Their structures were characterized by various spectroscopic techniques, as well as comparison with literature data. Twenty-seven metabolites of these were evaluated for cytotoxic activity against six human cancer cell lines. The biosynthetic formation of a 1,4-dioxane bridge is also discussed. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Siphonodon celastrineus Griff. (Celastraceae) is a tree that grows up to 25 m. in height and is found in the northern and central parts of Thailand. In Thai traditional medicine, its roots are used for the treatment of inflammation, abscesses, skin diseases, and as a bone tonic (Chayamarit, 1985). Recently, an ethanolic extract of the leaves of S. celastrineus was shown to be cytotoxic to the breast cancer cell line, MCF-7, with an IC50 value of 17.1 lg/ml (Itharat et al., 2004). Subsequent chemical investigations of the root bark of this plant resulted in isolation of the oleanane triterpene, 3bacetoxy-11a-benzoyloxy-13b-hydroxyolean-12-one, and the quinone methide triterpene, pristimerin, from the methanolic extract (Niampoka et al., 2005). Preliminary screening of dichloromethane extract of the stem of this plant in this laboratory showed cytotoxic activity against the MOLT-3 cancer cell line (86% cytotoxicity at 10 lg/ml). The chemical and biological properties of this species were thus further investigated. In this report, the isolation and structure elucidation of 21 new triterpenoids, (1, 4–23 in Fig. 1), together with nine known compounds isolated from the

⇑ Corresponding author. Tel.: +66 2 553 8555x8983; fax: +66 2 553 8572. E-mail address: [email protected] (H. Prawat).

stem of S. celastrineus, are described. The known compounds were identified as: friedelane-3,21-dione (2) (Gunatilaka et al., 1983), 21b-hydroxyfriedelan-3-one (3) (Nguyen and Tran, 2009), 24-ethylcholesta-1,4-dien-3-one (24) (Lin et al., 2003), 24-ethyl6b-hydroxycholest-4-en-3-one (25) (Arai et al., 1998), 24-ethyl3b-hydroxycholest-5-en-7-one (26) (Guerriero et al., 1993; Notaro et al., 1992), (9S,10E,12Z)-9-hydroxyoctadeca-10,12-dienoic acid (27) (Kato et al., 1984; Tsuboi et al., 1986; Suemune et al., 1985), 11a-methoxyolean-12-en-3b-ol (28) (Mathias et al., 2000; Fujita et al., 2000), mayteine (29) (Itokawa et al., 1993; Han et al., 1990), and (2S)-1-O-palmitoyl glycerol (30) (Qi et al., 2004), based on comparison of their spectroscopic data with those previously described in the indicated literature. Compounds 1–12 and 15– 29 were evaluated for cytotoxicity against six human cancer cell lines (MOLT-3, HuCCA-1, A549, HeLa, HepG2, and MDA-MB-231). 2. Results and discussion Repeated chromatographic separation of the CH2Cl2 extract of S. celastrineus stem gave a total of 30 compounds, including 21 new triterpenoids. These compounds were shown to be a lupane derivative 1, two friedelane derivatives 4 and 5, an oleanane derivative 6, and 17 ursane-type triterpenes 7–23. Compound 1 was obtained as a colorless solid. Its molecular formula, C31H50O2, was determined by HRAPCIMS (m/z 455.3867

0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.09.027

Please cite this article in press as: Kaweetripob, W., et al. Lupane, friedelane, oleanane, and ursane triterpenes from the stem of Siphonodon celastrineus Griff. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.027

2

W. Kaweetripob et al. / Phytochemistry xxx (2013) xxx–xxx

MeO

1H 25

2

10

3

O

4

20 19 H 18 17

5 H 6

28

15 27

12

H

H

1

2 3

O

1

23

RO 25

H

H

24

21

1126 13 8

29

30

29

30

7

4 23

27 18

26

15

16

25

28

30

25 2 3

HO

11

20

26

17

H

5 H

4

27

H

OH

R

6

23

R5

H R 6 23 24

7

24 23

28

12

2

15

1

R3

15

16

20 21 E R 17 25 11 26 13 28 C HD 15 1 2 A 5 H 3 27 R 4 B 4 1

28 21

12 O

29

H

H

24

30 29

26

27

HO

4 R=H 5 R = COPh

24

29

OH O 11 12

17

H

10

30

O 20 21

R1 β-OH, α-H β-OH, α-H β-OH, α-H O β-OH, α-H β-OH, α-H β-OH, α-H β-OH, α-H β-OH, α-H β-OH, α-H β-OH, α-H O β-OH, α-H

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

R2 O β-H, α-OMe β-H, α-OH O β-H, α-OMe O β-H, α-OH β-H, α-OMe β-H, α-OH β-H, α-OH β-H, α-OH O β-H, α-OH

R3 OH OH H OH H OH H OH OH OH OH OH OH

R4 OH OH OH OH OH H OCOPh H OH OH OH OH OCOPh

R5 H H H OH H H H H H H H H H

R6 CH3 CH3 CH3 CH3 CH3 CH2OH CH3 CH3 CHO COOCH3 CH3 CH2OH CHO

OH MeO 3' 4' 5'

30 29

HO

2

O

O

26

25

1 3

19 21

H

4

H

OH

2'

OH H

9' 28

8'

O

25

21

30

H 29 O

7'

MeO 3'

11

H

24 23

H

HO

27 OH

22

11

H

28

15

9' OH

30

H 29 8' O 7' H 12 O

1' H

25

H 12

H HO

4'

6'

1'

27

H

24 23

HO

H

28

15

27 OH

H

24 23

23

Fig. 1. Isolated compounds 1, 4–23 from the stem of Siphonodon celastrineus.

[M+H]+, calcd for C31H51O2, 455.3884). The IR spectrum showed the presence of terminal methylene (1640 cm1) and gem-dimethyl (1380 cm1) groups. The 1H NMR spectrum of 1 (Table 1) displayed signals corresponding to six tertiary methyl groups at dH 0.79, 0.94, 0.95, 0.98, 1.04, and 1.67, one methoxy group at dH 3.38, two olefinic protons at dH 4.56 (s) and 4.69 (d, J = 2.0 Hz), one oxygenated

methine proton at dH 3.23 (t, J = 2.6 Hz), and one acetal proton at dH 5.04 (s). The 13C NMR and DEPT spectra of 1 (Table 1) suggested the presence of a 1,1-disubstituted double bond at dC 109.3 and 150.9, an oxygenated methine carbon at dC 76.0, an acetal carbon at dC 101.4, and a methoxy carbon at dC 54.6. Based on the molecular formula of C31H50O2, the degrees of unsaturation of 1 were deter-

Please cite this article in press as: Kaweetripob, W., et al. Lupane, friedelane, oleanane, and ursane triterpenes from the stem of Siphonodon celastrineus Griff. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.027

3

W. Kaweetripob et al. / Phytochemistry xxx (2013) xxx–xxx Table 1 H (600 MHz) and

1

Position

a

13 a

C NMR (150 MHz) spectroscopic data of compounds 1, 4, and 5 (CDCl3). 1

5

4

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

1

26.3

24.8

23.1 76.0 36.4 52.4 20.9

212.3 58.6 42.3 41.6

7

33.0

1.35–1.46

18.2

2.07, 2.12, 2.23, 2.37, – 2.25, – 1.82, 1.88, 1.48,

m m m ddd (13.3, 4.0, 1.8)

3 4 5 6

8 9 10 11

43.1 45.7 39.8 23.6

54.0 41.1 60.3 30.1

25.8

13 14 15

38.3 40.3 27.8

16

35.6

17

43.0

1.56, – 1.65, 1.24, 2.07, 1.40, 1.53, – – 1.37, 1.60, 1.42, 1.78, –

m

12

– 1.34, – 1.38, 1.65, 0.97, 1.65, 1.62, – 1.06, 1.68, 1.37, 1.48, –

2.13,qd (12.7, 4.4) 2.23, m 2.27, m 2.41, dd (12.7, 3.7) – 2.29, q (6.5) – 1.42, m 1.91, dt (13.0, 2.9) 1.57, m 1.68, m 1.68, m – 1.77, m 1.32, td (13.7, 3.1) 2.05, brd (13.7) 1.42, m 1.59, m – – –

24.6

2

0.72, m 2.16, td (12.1, 2.0) 1.79, m 1.88, m 3.23, t (2.6) – 1.03, m 1.37–1.50

18 19

48.3 47.9

1.37, m 2.38, td (11.0, 5.9)

41.8 36.9

20

150.9

–

42.7

21

29.9

218.7

22

40.0

1.33, m 1.92, m 1.19, q (10.5)

23 24 25

29.6 24.0 101.4

0.95, s 0.98, s 5.04, s

7.0 14.8 65.1

26 27 28 29

16.2 14.7 18.0 109.3

21.5 18.6 33.8 28.8

30 OMe-25 OCOPh ipso ortho metha para

19.3 54.6

1.04, 0.94, 0.79, 4.56, 4.69, 1.67, 3.38,

m m m m m m m m m m

42.6

30.7 39.9 37.9 33.1 34.9 33.0

s s s s d (2.0) s s

55.0

25.0 166.8 130.2 129.5 128.6 133.1

1.39, 1.67, 1.46, 1.80, – 1.86,

m m m m m

1.62, m 1.87, m – 1.83, 2.62, 0.93, 0.87, 4.48, 4.93, 1.18, 1.21, 1.17, 1.07,

m d (13.0) d (6.7) s d (12.3) d (12.3) s s s s

1.16, s –

42.5 212.8 58.5 42.4 41.7 18.0 53.9 42.0 60.7 30.0 31.4 39.9 37.9 33.0 35.0 33.1 41.9 37.0 42.7

m m m m

m m m m m

m m m m

1.82, m 1.62, m 1.86, m –

218.7 55.0 7.0 14.7 62.7 20.9 18.4 33.6 28.8 25.0

1.82, m 2.61, d (12.8) 0.91, d (6.6) 0.83, s 3.90, d (11.7) 3.98, d (11.7) 1.078, s 1.18, s 1.15, s 1.08, s 1.17, s –

7.98, brd (7.5) 7.46, t (7.5) 7.58, t (7.5)

Assignments were based on DEPT, HSQC, and HMBC experiments.

mined to be seven, including one double bond. Thus, 1 should possess a hexacyclic structure. The COSY spectrum of 1 showed that the acetal proton at dH 5.04 had 1H–1H correlation with Ha-1 (dH 0.72). The HMBC spectrum of 1 exhibited long-range correlations between the acetal proton (dH 5.04) and C-1 (dC 26.3), C-3 (dC 76.0), C-5 (dC 52.4), and C-9 (dC 45.7). The methoxy protons (dH 3.38) correlated with C-25 (dC 101.4). The evidence described thus suggested that 1 was closely related to a lupane-type triterpene (pentacyclic, one double bond) with one additional ring system, which was formed via an ether linkage between C-3 and C-25. The NOESY spectrum of 1 showed a nOe effect between H-25 and H3-26, indicating that H-25 and H3-26 were cis to each other and had a b-orientation. Compound 1 was thus concluded to be 3b,25-epoxy-25-methoxylup-20(29)-ene. Compound 4 was obtained as a colorless solid. Its HRAPCIMS exhibited a pseudo-molecular ion peak at m/z 457.3685 [M+H]+ (calcd for C30H49O3, 457.3676) corresponding to the molecular for-

mula, C30H48O3, and indicating seven degrees of unsaturation. The NMR spectra of 4 (Table 1) had resonances attributable to two cyclic hexanones, an oxymethylene, six tertiary methyls, and a secondary methyl. This NMR spectroscopic data, coupled with the molecular formula, indicated that compound 4 was a friedelanetype triterpenoid, with two ketone functionalities, and an oxymethylene group. The location of the hydroxy group at C-25, and two carbonyl groups, at C-3 and C-21, were confirmed by the HMBC correlations of H2-25 (dH 3.90 and 3.98) to C-8 (dC 53.9) and C-10 (dC 60.7), the correlations of H3-23 (dH 0.91, d, J = 6.6 Hz) with C-3 (d 212.8), and between H3-29 (dH 1.08) and H3-30 (dH 1.17) and C-21 (dC 218.7), respectively. Therefore, compound 4 was designated as 25-hydroxyfriedelane-3,21-dione. Compound 5 was obtained as a colorless solid. Its HRAPCIMS exhibited a pseudo-molecular ion peak at m/z 561.3943 [M+H]+ (calcd for C37H53O4, 561.3997) corresponding to the molecular formula, C37H52O4, indicating twelve degrees of unsaturation. The 1H

Please cite this article in press as: Kaweetripob, W., et al. Lupane, friedelane, oleanane, and ursane triterpenes from the stem of Siphonodon celastrineus Griff. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.027

4

W. Kaweetripob et al. / Phytochemistry xxx (2013) xxx–xxx

and 13C NMR spectroscopic data of 5 (Table 1) were similar to those of 4, with few variations. The extra five proton aromatic signals at dH 7.98 (2H, brd, J = 7.5 Hz), 7.58 (1H, t, J = 7.5 Hz), 7.46 (2H, t, J = 7.5 Hz) were attributed to a benzoyl group, and the downfield shift of the oxymethylene protons (H2-25) at dH 4.48 and 4.93 in the 1H NMR spectra of 5 indicated that the hydroxy group at C25 was esterified by benzoic acid. The location of the benzoyl group at C-25 was confirmed by the HMBC correlations of H2-25 (dH 4.48 and 4.93) and the benzoyl protons at dH 7.98, to the ester carbonyl carbon (dC 166.8). Thus, compound 5 was identified as 25benzoyloxyfriedelane-3,21-dione. Compound 6 was isolated as a colorless solid. Its molecular formula was determined to be C30H48O3 from the pseudo-molecular ion peak at m/z 457.3688 [M+H]+ (calcd for C30H49O3, 457.3676) in the HRAPCIMS. The IR spectrum showed absorption bands corresponding to hydroxy (3422 cm1) and a,b-unsaturated ketone (1710 cm1) groups. The 13C NMR and DEPT spectra (Table 2) showed 30 carbon resonances, including eight tertiary methyls (dC 15.5, 16.5, 18.7, 23.2, 23.4, 28.0, 29.2, and 33.1), nine methylenes (dC 17.5, 26.5, 26.7, 27.3, 32.9, 34.7, 36.9, 39.1, and 41.3), four methines (dC 38.7, 55.0, 60.4, and 78.7), and nine quaternary carbons (dC 30.9, 31.3, 37.4, 39.2, 41.5, 46.3, 138.3, 142.8, and 195.3), respectively. These data, together with those obtained from the 1H NMR spectrum [(Table 2) eight tertiary methyls (dC 0.81,

Table 2 H (600 MHz) and

1

Position

a

0.87, 0.90, 0.95, 1.01, 1.13, 1.14, and 1.36), an oxymethine proton at dC 3.23, and a hydroxy proton on a sp2 carbon at dH 6.21], indicated that compound 6 should be an oleanane-type triterpene containing an a,b-unsaturated ketone and two hydroxy groups. The HMBC (Fig. 2) correlations of H3-23 (dH 1.01) and H3-24 (dH 0.81) to C-3 (dC 78.7), C-4 (dC 39.2), and C-5 (dC 55.0) were used to locate one of the hydroxy groups at C-3. Further analysis of the HMBC data showed that the second hydroxy group (dC 6.21) was attached to C-12 (dC 142.8) as indicated by the correlations of OH-12 (dH 6.21) with C-11 (dC 195.3), C-12 (dC 142.8), and C-13 (dC 138.3). In addition, the HMBC correlations of H-9 (dH 2.46) to C-11 (dC 195.3); of H-18 (dH 2.82) to C-12 (dC 142.8) and C-13 (dC 138.3); and of H3-27 to C-13 (dC 138.3) confirmed the placement of a carbonyl and a tetra-substituted double bond at C-11 and C-12/13, respectively. The hydroxy group at C-3 was assigned a b-orientation due to the trans-diaxial coupling constant of H-3 (J = 11.5 Hz). In addition, the NOESY correlation between H-3 and H-5 confirmed the a-orientation of H-3. Thus, compound 6 was determined to be 3b,12-dihydroxyolean-12-en-11-one. Compound 7 was obtained as a colorless solid. Its molecular formula was deduced as C30H48O3 from the HRAPCIMS. The IR spectrum showed absorption bands for hydroxy (3410 cm1), olefinic (1661 cm1), and gem-dimethyl (1381 cm1) groups. In the 1H NMR spectrum of 7 (Table 2), the presence of five tertiary methyl

13 a

C NMR (150 MHz) spectroscopic data of compounds 6–8 (CDCl3). 6

7

8

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

1

39.1

38.3

27.3 78.7 39.2 55.0 17.5

78.9 38.8 54.5 17.9

7

32.9

8 9 10 11 12 13 14 15

46.3 60.4 37.4 195.3 142.8 138.3 41.5 26.5

16

26.7 31.3 38.7 41.3

1.52, 1.90, – 1.14, 1.69,

m m

17 18 19 20 21

30.9 34.7

22

36.9

23 24 25 26 27 28

28.0 15.5 16.5 18.7 23.2 29.2

3.23, – 0.72, 1.43, 1.61, 1.44, 1.64, – 2.46, – – – – – 1.13, 1.83, 0.93, 2.05, – 2.82, 1.24, 1.40, – 1.19, 1.38, 1.29, 1.54, 1.01, 0.81, 1.13, 1.14, 1.36, 0.87,

m m m m dd (11.5, 4.7)

1.04, m 2.75, dt (10.0, 3.4) 1.66, m

3 4 5 6

0.98, 1.85, 1.61, 1.67, 3.22, – 0.74, 1.58, 1.68, 1.53, 1.66, – 1.89, – 5.77, 5.51, – – 4.31,

39.3

2

1.02, m 2.79, dt (13.6, 3.4) 1.65, m

29 30 OH-12

33.1 23.4

0.89, 1.27, 1.49, 1.28, 1.60, 0.98, 0.78, 0.90, 1.16, 1.05, 3.19, 3.61, 1.01, 0.94, –

m m m m m s s s s s dd (6.8, 2.0) d (6.8) d (6.2) d (6.2)

dd (10.9, 5.9) brd (11.0) m m m m s

m td (13.6, 4.3) m td (13.6, 4.3) brdd (13.5, 3.5) m m m m m td (13.7, 4.3) s s s s s s

0.90, s 0.95, s 6.21, s

27.1

35.1 43.0 52.7 36.5 129.4 132.5 85.8 48.9 68.4 37.8 42.7 61.0 37.6 40.6 31.2 34.0 27.7 14.9 17.7 19.9 12.8 76.8 18.1 19.4

dd (11.2, 3.4) m m m m m brd (10.4) dd (10.4, 3.1)

dd (10.3, 6.2)

m m

27.2 78.7 39.0 54.6 17.7 36.2 46.7 59.8 37.3 195.1 144.9 134.0 47.2 68.4 38.5 34.2 48.7 40.4 39.1 30.9 40.7 28.0 15.6 16.6 19.1 15.3 29.4 16.5 20.9

3.25, – 0.73, 1.46, 1.62, 1.69, 1.90, – 2.47, – – – – – 4.28,

dd (10.9, 5.7) d (11.0) m m m td (13.2, 4.1) s

dd (11.4, 5.4)

1.27, 2.00, – 2.48, 1.39,

m t (12.2)

1.08, 1.25, 1.46, 1.35, 1.49, 1.01, 0.81, 1.16, 1.22, 1.37, 0.87,

m m m m m s s s s s s

d (8.5) m

0.82, d (6.2) 0.94, d (6.4) 6.37, s

Assignments were based on DEPT, HSQC, and HMBC experiments.

Please cite this article in press as: Kaweetripob, W., et al. Lupane, friedelane, oleanane, and ursane triterpenes from the stem of Siphonodon celastrineus Griff. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.027

W. Kaweetripob et al. / Phytochemistry xxx (2013) xxx–xxx

O 2

24

17

15

1 5

4

HO

OH 19 12

7 27

23

Fig. 2. HMBC (

) correlations of 6.

groups (dH 0.78, 0.90, 0.98, 1.05, and 1.16), two secondary methyl groups [dH 0.94 (d, J = 6.2 Hz) and 1.01 (d, J = 6.2 Hz)] and an oxymethylene group [dH 3.19 (dd, J = 6.8, 2.0 Hz) and 3.61 (d, J = 6.8 Hz)] of an ursane-triterpene skeleton (Chen et al., 2006; Mencherini et al., 2007) was observed, in which one of the tertiary methyl groups was oxidised. In addition, the two methine protons attached to the oxymethine carbons C-3 (dC 78.9) and C-15 (dC 68.4) appeared as two doublets of doublets at dH 3.22 (J = 11.5, 4.7 Hz) and 4.31 (J = 10.3, 6.2 Hz), respectively. Two cis-olefinic protons at dH 5.77 (brd, J = 10.4 Hz) and 5.51 (dd, J = 10.4, 3.1 Hz), connected to C-11 (dC 129.4) and C-12 (dC 132.5), respectively, were also observed. The HMBC correlations (Fig. 3) between the proton signals of H3-23 (dH 0.98) and H3-24 (dH 0.78) and C-3 (dC 78.9) confirmed the attachment of the first hydroxy group at C-3. The correlations between H-15 (dH 4.31) and C-8 (dC 43.0), C-17 (dC 42.7) and C-27 (dC 12.8), and also between H2-16 (dH 1.52/ 1.90) and H3-27 (dH 1.05) and C-15 (dC 68.4) indicated that the second hydroxy group was located at C-15. The configurations of the hydroxy groups at C-3 and C-15 were assigned as b- and a-orientations, respectively, due to the trans-diaxial coupling constants of H-3 (J = 11.5 Hz) and H-15 (J = 10.3 Hz). Furthermore, in the NOESY experiments of 7 (Fig. 3), correlations between signals at dH 3.22 (H-3), 0.74 (H-5) and 0.98 (H3-23) and those at dH 4.31 (H-15) and 1.16 (H3-26) confirmed the a- and b-orientations of H-3 and H-15, respectively. The HMBC correlations from H2-28 (dH 3.19/3.61) to C-13 (dC 85.8), C-16 (dC 37.8), C-17 (dC 42.7), and C-18 (dC 61.0), as well as H-18 (dH 1.14) to C-13 (dC 85.8), and C-14 (dC 48.9) indicated that compound 7 has an ether linkage between C-13 and C-28. On the basis of the above evidence, compound 7 was identified as 13b,28-epoxyurs-11-ene-3b,15a-diol. Compound 8 was obtained as a colorless solid. Its molecular formula was determined as C30H48O4 on the basis of the HRAPCIMS, indicating seven degrees of unsaturation. Its IR spectrum showed absorption bands for hydroxy (3408 cm1), a,b-unsaturated carbonyl (1704 cm1) and gem-dimethyl (1380 cm1) groups. The 1H and 13C NMR spectra (Table 2) displayed 30 carbon resonances comprising eight methyl groups (including six tertiary and two secondary), seven methylenes, seven methines (two oxygenated at dC 78.7 and 68.4), a carbonyl carbon (dC 195.1), a pair of quaternary olefinic carbons [dC 144.9 (oxygenated carbon) and dC 134.0],

29

H

O

25 1

OH

HO

Fig. 3. HMBC (

HO H

) and NOESY (

3 24

28

12 O

H

26 5H

13 H 17 14 15 16

H OH

H

) correlations of 7.

5

and five quaternary carbons. These data coupled with the molecular formula, indicated that compound 8 was a triterpenoid with five rings, three hydroxy groups, and a carbonyl group. In the HMBC data, the proton signals at dH 1.01 (H3-23) and 0.81 (H324) correlated with the carbon signal at dC 78.7 (C-3), indicating that one hydroxy group was attached to C-3. In addition, the proton signals at dH 1.27/2.00 (H2-16) and 1.37 (H3-27) correlated with the carbon resonance at dC 68.4 (C-15), and the proton resonances at dH 4.28 (H-15) correlated with the carbon signals at dC 46.7 (C-8), 38.5 (C-16), 34.2 (C-17), and 15.3 (C-27). These correlations indicated that a second hydroxy group was positioned at C15. The OH groups at positions C-3 and C-15 must be equatorial based on the large coupling constants of H-3 (J = 10.9 Hz) and H15 (J = 11.4 Hz) consistent with the axial configurations for both methine protons. In addition, the correlations between the signals at dC 3.25 (H-3), 0.73 (H-5), and 1.01 (H3-23), and the resonances at dH 4.28 (H-15), 1.22 (H3-26), and 0.87 (H3-28) in the NOESY experiment confirmed the a- and b-orientations of H-3 and H-15, respectively. The remaining hydroxy group existed as part of an enol system at C-12 (oxygenated carbon) and C-13, which could be deduced from the HMBC correlations from H-9 (dH 2.47), OH12 (dC 6.37) and H-18 (dC 2.48) to C-12 (dC 144.9), and of H3-27 (dH 1.37) and H-18 (dC 2.48) to C-13 (dC 134.0). The carbonyl carbon was assigned at C-11 by the HMBC correlations from OH-12 (dH 6.37) and H-9 (dH 2.47) to C-11 (dC 195.1). On the basis of the above evidence, compound 8 was determined to be 3b,12,15a-trihydroxyurs-12-en-11-one. Compound 9 was isolated as a colorless solid. Its molecular formula was deduced as C31H52O4 from the HRAPCIMS. The 1H and 13C NMR spectroscopic data of 9 (Table 3) closely resembled those of 8, except for the signals corresponding to ring C. Compound 9 was found to differ from 8 due to the presence of a methoxy group (dC 3.18; dC 51.5) and an oxygenated methine group (dC 4.26, d, J = 10.4 Hz; dC 76.4), as well as the absence of a carbonyl group. The NMR resonances at dH 4.26 (H-11), dH 4.67 (OH-12), dC 143.1 (C-12) and 119.2 (C-13) indicated the presence of a 12-hydroxy11-methoxy-12-ene unit. The doublet at dH 1.87 and 4.26 for H-9 and H-11 showed vicinal coupling constants of 10.4 Hz, indicating that H-9 and H-11 were trans-diaxial, and that the methoxy group (11-OMe) should be equatorial (a-oriented). Cross-peaks between resonances at dH 4.26 (H-11), dH 1.12 (H3-25), and 1.17 (H3-26) in the NOESY spectrum confirmed the b-orientation of H-11. Therefore, compound 9 was proposed to be 11a-methoxyurs-12-ene3b,12,15a-triol. Compound 10 was obtained as a colorless solid. Its molecular formula was deduced as C30H50O3 from the HRAPCIMS (m/z 459.3828 [M+H]+, calcd for C30H51O3, 459.3833), indicating six degrees of unsaturation. The 1H and 13C NMR spectroscopic data of 10 (Table 3) were similar to those of 9, except for the signals corresponding to ring C. Compound 10 was found to differ from compound 9 due to the presence of an olefinic proton (dH 5.28) and the absence of a methoxy and an enolic hydroxy group. In addition, it was found that compounds 12 and 14 were similar to 10, with the difference of the presence of a methoxy at C-11 in 12 instead of a hydroxy groups in 10, and a benzoyloxy group at C-15 in 14, instead of a hydroxy group in 10. Therefore, compounds 10, 12, and 14 were identified as urs-12-ene-3b,11a,15a-triol, 11a-methoxyurs-12-ene-3b,15a-diol, and 15a-benzoyloxyurs-12-ene3b,11a-diol, respectively. Compound 11 was isolated as a colorless solid. Its molecular formula was determined to be C30H46O5 on the basis of HRAPCIMS, indicating eight degrees of unsaturation. The NMR spectra of 11 (Table 3) were similar to those of 8, except for the signals corresponding to rings A and E. Compound 11 was found to differ from compound 8 due to the presence of a carbonyl group (dC 216.7) instead of an oxymethine group in ring A, and an additional hydroxy

Please cite this article in press as: Kaweetripob, W., et al. Lupane, friedelane, oleanane, and ursane triterpenes from the stem of Siphonodon celastrineus Griff. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.027

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W. Kaweetripob et al. / Phytochemistry xxx (2013) xxx–xxx

Table 3 H (600 MHz) and

1

Position

a

13 a

C NMR (150 MHz) spectroscopic data of compounds 9–11 (CDCl3). 9

10

11

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

1

39.0

40.8

27.5

1.24, m 2.19, dt (13.6, 3.4) 1.65, m

39.9

2

1.21, m 2.25, dt (13.4, 3.4) 1.62, m

3 4 5 6

78.6 39.1 55.1 18.4

216.7 47.7 55.1 19.0

7

37.4

8 9 10 11 12 13 14 15 16

44.2 46.3 38.5 76.4 143.1 119.2 46.4 68.1 38.7

17 18 19 20 21

34.1 47.5 40.5 39.3 31.0

22

41.2

23 24 25 26 27 28 29 30 OMe-11 OH-12

28.4 15.8 16.2 18.7 17.8 29.0 17.0 21.2 51.5

3.25, – 0.78, 1.44, 1.63, 1.54, 1.75, – 1.52, – 4.26, 5.28, – – 4.19, 1.22, 1.95, – 1.36, 1.33, 0.91, 1.26, 1.43, 1.24, 1.46, 1.01, 0.82, 1.09, 1.11, 1.19, 0.85, 0.89, 0.93,

1.48, m 2.92, ddd (13.5, 7.1, 4.1) 2.40, ddd (15.9, 6.5, 4.1) 2.66, ddd (15.9, 11.3, 7.1) – – 1.32, m 1.53–1.62, m

3.26, – 0.79, 1.40, 1.60, 1.62, 1.72, – 1.87, – 4.26, – – – 4.18, 1.19, 1.92, – 2.29, 1.33, 1.04, 1.24, 1.43, 1.46,

dd (8.5, 7.9)

brd (11.1) m m m m m

1.01, 0.82, 1.12, 1.17, 1.23, 0.86, 0.93, 0.94, 3.18, 4.67,

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

brd (11.4) m m m td (12.7, 4.1) d (10.4) d (10.4)

dd (11.4, 5.8) m t (12.1)

27.4 78.7 39.0 54.9 18.5 36.7 44.6 55.7 38.2 68.1 129.8 143.5 47.7 68.0 38.8 34.0 58.3 39.1 39.2 30.9 40.9 28.2 15.6 16.8 18.6 17.2 29.3 17.5 21.3

dd (11.2, 5.2) dd (12.0, 1.2) m m m td (12.8, 4.0) d (8.6) dd (8.8, 3.3) d (3.3)

dd (11.7, 5.8) m t (11.7) m m m m m m m s s s s s s d (6.0) brs

34.1

35.5 46.6 59.1 36.9 194.4 145.1 133.9 47.4 68.3 38.0 33.7 43.6 41.4 70.9 35.4 35.6 26.4 21.4 15.8 18.9 15.1 29.2 11.6 29.7

1.78, m 1.92, m – 2.57,s – – – – – 4.32, dd (11.7, 5.5) 1.28, m 2.01, t (11.7) – 2.88, dd (11.9, 2.0) 1.72, m – 1.33, m 1.56, m 1.64, m 1.73, m 1.11, s 1.08, s 1.29, s 1.26, s 1.37, s 0.92, s 0.87, d (6.8) 1.22, s 6.34, s

Assignments were based on DEPT, HSQC, and HMBC experiments.

group at C-20 in ring E. In the HMBC spectrum of 11, the proton resonances of the gem-dimethyl groups at dH 1.11 (H3-23) and 1.08 (H3-24) correlated with the carbon signal at dC 216.7, locating a carbonyl group at C-3. The location of the tertiary hydroxy group at C-20 (dC 70.9) was supported by HMBC correlations of H-18 (dH 2.88), H-19 (dH 1.72), H3-29 (dH 0.87), and H3-30 (dH 1.22) with C20. Observation of NOESY correlations between both H3-28 (dH 0.92) and H3-29 (dH 0.87) with H-18 (dH 2.88), but not with H330 (dH 1.22), indicated the b-configuration of the hydroxy group at C-20. On the basis of the above evidence, the structure of 11 was designated as 12,15a,20b-trihydroxyurs-12-ene-3,11-dione. Compound 13 was obtained as a colorless solid, with the same molecular formula as 8 (C30H48O4). The UV, IR, and MS spectra of 13 were identical to those of 8, suggesting the presence of an a,b-unsaturated carbonyl group in 13. The NMR spectroscopic data of 13 (Table 4) were also similar to those of 8, except for the signals corresponding to ring D and the gem-dimethyl groups on ring A. In addition, 13 was found to differ from 8 due to the presence of resonances indicating a methylene group [dH 1.17/1.89, dC 27.5 (C15)], instead of an oxymethine group, in ring D and an oxymethylene [dH 3.35/4.21, dC 64.3 (C-24)], instead of a methyl group on ring A. The location of the primary hydroxy group at C-24 (dC 64.3) was confirmed by HMBC correlations between H-3 (dH 3.48), H-5 (dH 0.83), and H3-23 (dH 1.26) and C-24 (dC 64.3). The cross-peak correlations between Hb-24 (dH 4.21) and H3-25 (dH 1.10) were ob-

served in the NOESY spectrum of 13, indicating the bconfiguration of the oxymethylene group at C-4. On the basis of the evidence described, compound 13 was identified as 3b,12,24trihydroxyurs-12-en-11-one. Compound 15 had the molecular formula C31H52O3, one oxygen atom less than 9. The 1H and 13C NMR spectroscopic data of 15 (Table 5) closely resembled those of 9, except for the signals corresponding to ring D. Thus, 15 was found to differ from 9 due to the presence of a methylene group [dH 1.01/1.79, dC 27.2 (C-15)] instead of an oxymethine group on ring D. Thus, compound 15 was assigned to be 11a-methoxyurs-12-ene-3b,12-diol. Compound 16 was obtained as a colorless solid. Its molecular formula was deduced as C30H48O3 from the HRAPCIMS. The NMR spectra of 16 (Table 5) were identical to those of 9, except for the signals corresponding to a methoxy group on ring C and absence of gem-dimethyl groups on ring A. Thus, compound 16 differed from 9 by the absence of a methoxy group at C-11 on ring C and the appearance of an aldehyde group, instead of a methyl group on ring A. The location of the aldehyde group at C-4 was determined by HMBC correlations between H-3 (dH 3.22), H-5 (dH 1.06), and H3-23 (dH 1.31) and C-24 (dC 207.9). The aldehyde group at C-4 was b-oriented, as deduced from the NOESY correlations between H-24 (dH 9.79) and H3-25 (dH 1.05). On the basis of the evidence described, compound 16 was identified as 3b,11a,12,15a-tetrahydroxyurs-12-en-24-al.

Please cite this article in press as: Kaweetripob, W., et al. Lupane, friedelane, oleanane, and ursane triterpenes from the stem of Siphonodon celastrineus Griff. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.027

7

W. Kaweetripob et al. / Phytochemistry xxx (2013) xxx–xxx Table 4 H (600 MHz) and

1

Position

a

13 a

C NMR (150 MHz) spectroscopic data of compounds 12–14 (CDCl3). 12

13

14

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz; CDCl3 + D2O)

1

39.9

38.9

27.5

27.4

1.24, m 2.18, dt (13.7, 3.4) 1.63, m

3 4 5 6

78.7 39.0 54.9 18.6

80.6 43.1 55.5 17.6

78.7 39.0 54.7 18.5

3.21, dd (11.3, 4.6) – 0.75, m 1.38, m

7

36.5

35.0

8 9 10 11 12 13 14 15

44.2 52.7 38.1 76.5 125.2 144.1 47.7 68.2

3.23, – 0.79, 1,43, 1.61, 1.53, 1.73, – 1.65, – 3.75, 5.46, – – 4.18,

1.04, 2.77, 1.72, 1.92, 3.48, – 0.83, 1.36, 1.67, 1.45, 1.65, – 2.46, –

40.9

2

1.27, m 1.89, dt (13.9, 3.4) 1.62, m

1.13, 1.63, – 1.56, – 4.28, 5.33, – – 5.63,

16

38.8

17 18 19 20 21

33.9 58.7 39.3 39.1 30.9

22

41.0

23 24 25 26 27 28 29 30 OMe-11 OH-12 OCOPh ipso ortho meta para

dd (11.1, 5.1) brd (12.6) m m dt (12.9, 3.0) td (12.9, 4.0) d (8.8) dd (8.8, 3.2) d (3.2)

dd (11.3, 5.6)

28.2 15.6

1.22, 1.95, – 1.36, 0.92, 1.33, 1.26, 1.42, 1.26, 1.46, 1.00, 0.81,

m t (11.3) d (11.1) m m m m m m s s

17.1 18.9 16.6 29.3 17.3 21.3 54.7

1.06, 1.10, 1.17, 0.85, 0.92, 0.93, 3.28,

s s s s d (6.2) brs s

27.6

33.2 45.5 59.7 36.9 195.1 144.5 134.4 41.7 27.5 27.3 33.4 48.9 40.8 39.3 31.2 41.1 22.4 64.3 17.1 18.4 20.9 28.8 16.5 21.0

– – – 1.17, 1.89, 0.96, 2.09, – 2.45, 1.42, 1.07, 1.27, 1.44, 1.39, 1.47, 1.26, 3.35, 4.21, 1.10, 1.14, 1.34, 0.83, 0.79, 0.93, – 6.26,

m dt (13.6,3.6) m m dd (11.8, 4.2) d (11.2) m m m m s

m m m td (13.6, 4.8) d 11.4) m m m m m m s m d (11.2) s s s s d (6.6) d (6.6)

44.7 55.7 38.2 67.8 130.2 142.7 47.0 72.1 34.5 33.9 58.3 39.1 39.2 30.8 40.7 28.2 15.6 16.8 18.6 18.7 28.9 17.5 21.2

s 165.6 131.0 129.5 128.4 132.8

1.43, 2.05, – 1.43, 1.43, 0.94, 0.97, 1.42, 1.28, 1.47, 0.93, 0.76, 1.07, 1.18, 1.48, 0.98, 0.94, 0.95, – – – – 8.01, 7.47, 7.57,

dt (13.3, 3.3) m d (8.7) dd (8.7, 3.4) d (3.4)

dd (11.0, 5.8) m t (11.0) m m m m m m m s s s s s s d (6.0) brs

d (7.5) t (7.5) t (7.5)

Assignments were based on DEPT, HSQC, and HMBC experiments.

NMR spectroscopic data of compounds 17, 18, and 20 were similar to those of 16, but different in substitution pattern. Instead of the aldehyde group on ring A in 16, compound 17 had a carboxylic acid methyl ester, and compound 18 had a methyl group. Compound 20 was a 15-benzoyl derivative of 16. Therefore, compounds 17, 18, and 20 could be assigned as 3b,11a,12,15a-tetrahydroxyurs-12-en24-oic acid methyl ester, urs-12-ene-3b,11a,12,15a-tetrol, and 15a-benzoyloxy-3b,11a,12-trihydroxyurs-12-en-24-al, respectively. Compound 19 had the molecular formula C30H46O5 (HRAPCIMS). The 1H and 13C NMR spectroscopic data of 19 (Table 6) closely resembled those of 13 (Table 4), except for the signals corresponding to rings A and D. Compound 19 was found to differ from compound 13 by the appearance of a carbonyl group at C-3 (dC 219.4) and an oxymethine group [dH 4.29, dd, J = 11.6, 5.7 Hz; dC 68.3] at C-15 instead of oxymethine and methylene groups, respectively. The OH group at C-15 was assigned to be equatorial, based on the large coupling constant of H-15 (J = 11.6 Hz), as well as the NOESY correlations of H-15 (dH 4.29) and H3-26 (dH 1.23)

and H3-28 (dH 0.88). Compound 19 was thus concluded to be 12,15a,24-trihydroxyurs-12-ene-3,11-dione. Compound 21 was isolated as a colorless solid. Its molecular formula was determined as C30H48O4 on the basis of the HRAPCIMS, indicating seven degrees of unsaturation. The IR spectrum suggested the presence of hydroxy (3400 cm1) and carbonyl (1714 cm1) groups. In the 1H NMR spectrum (Table 7), signals were observed for six tertiary methyl groups at dH 0.86, 1.01, 1.02, 1.03, 1.11, and 1.17, two secondary methyl groups at dH 0.83 and 1.05, an aldehyde proton at dH 9.77, a methylene group at dH 2.30 (d, J = 16.4 Hz) and 2.50 (d, J = 16.4 Hz), a trisubstituted olefinic proton at dH 5.20 (brt, J = 4.3 Hz), and an oxygenated methine proton at dH 3.38 (td, J = 11.3, 4.2 Hz). The 13C NMR and DEPT spectra showed 30 carbon resonances including six tertiary methyls (dC 16.9, 19.4, 19.5, 22.9, 23.9, and 28.4), two secondary methyls (dC 15.8 and 17.4), seven methylenes (dC 20.4, 23.8, 26.5, 29.2, 43.0, 47.7, and 50.3), eight methines (dC 38.4, 40.4, 47.1, 47.7, 58.6, 71.8, 125.1, and 207.7), and seven quaternary carbons (dC 35.2,

Please cite this article in press as: Kaweetripob, W., et al. Lupane, friedelane, oleanane, and ursane triterpenes from the stem of Siphonodon celastrineus Griff. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.027

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W. Kaweetripob et al. / Phytochemistry xxx (2013) xxx–xxx

Table 5 H (600 MHz) and

1

Position

a

13 a

C NMR (150 MHz) spectroscopic data of compounds 15–17 (CDCl3). 15

16

17

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

1

39.0

40.7

27.5

1.23, m 2.39, brd, (13.7) 1.88, m

41.7

2

1.22, m 2.24, brd (10.4) 1.62, m

3 4 5 6

78.7 39.2 55.5 18.2

76.9 52.8 56.2 18.5

34.2

37.3

8 9 10 11 12 13 14 15

40.6 46.4 38.4 76.7 142.1 118.2 42.9 27.2

3.22, – 1.06, 1.67, 1.92, 1.72, 1.76, – 1.63, – 4.15, – – – 4.18,

77.9 49.3 56.3 20.2

7

43.8 53.3 38.4 70.2 145.7 116.6 46.7 68.1

– 1.60, d (9.7) – 4.14, d (9.0) – – – 4.17, m

16

27.6

38.8

17 18 19 20 21

33.3 47.7 40.8 39.5 31.3

22

41.6

23 24 25 26 27 28 29 30 OMe OH-12

28.4 15.8 16.2 18.1 23.9 28.5 17.0 21.2 51.4

3.25, – 0.79, 1.37, 1.55, 1.38, 1.51, – 1.87, – 4.26, – – – 1.01, 1.79, 0.83, 2.01, – 2.24, 1.37, 1.03, 1.26, 1.41, 1.36, 1.42, 1.01, 0.82, 1.11, 1.11, 1.20, 0.81, 0.92, 0.93, 3.17, 4.57,

1.22, 2.39, 1.78, 2.04, 3.11, – 0.97, 1.70, 1.87, 1.68,

1.18, 1.92, – 2.16, 1.36, 1.04, 1.23, 1.42, 1.31, 1,48, 1.42, – 0.97, 1.15, 1.24, 0.86, 0.90, 0.92, 3.70, 4.77,

dd (8.9, 7.5) brd (12.2) m m m td (13.6, 4.1) d (10.4) d (10.4)

m td (13.6, 5.1) m td (13.6, 4.7) m m m m m m m s s s s s s d (6.6) d (6.6) s s

28.2

37.2 43.7 52.9 38.3 70.1 145.7 116.7 46.6 68.1 38.9 34.2 47.4 40.5 39.3 31.0 41.0 19.3 207.9 15.8 18.6 18.0 29.3 16.8 21.1

1.18, 1.93, – 2.13, 1.36, 1.02, 1.23, 1.44, 1.32, 1.48, 1.31, 9.79, 1.05, 1.16, 1.23, 0.86, 0.91, 0.93, – 4.71,

t (7.3) m m m m m d (9.2) d (9.3)

dd (12.0, 4.8) m t (12.0) d (11.3) m m m m m m s s s s s s d (6.5) d (6.3) s; 3.14, brs

28.3

34.1 47.3 40.5 39.3 31.0 41.0 23.8 178.4 14.4 18.5 17.8 29.3 16.7 21.0 51.3 –

m dt (13.2, 4.2) dq (13.2, 4.2) qd (13.2, 4.2) td (12.1, 4.4) m m m m

m t (12.2) d (11.1) m m m m m m s s s s s d (6.6) d (6.6) s s; 3.40, d (12.1)

Assignments were based on DEPT, HSQC, and HMBC experiments.

40.0, 42.3, 42.7, 50.7, 139.1, and 175.5), indicated that compound 21 was a tetracyclic triterpene with two carbonyls and one double bond. This conclusion was supported by comparison of the NMR spectroscopic data of 21 (Table 7) with those of the similar compound 3-oxo-2,3-secours-12-en-2-oic acid (Toriumi et al., 2003). The difference between these two compounds is the presence of an oxymethine group [dH 3.38 (td, J = 11.3, 4.7 Hz); dC 71.8] in 21. The position of an additional hydroxy group in 21 at C-21 was determined by the HMBC correlations between the proton signal at dH 1.05 (H3-30) and the carbon signal at dC 71.8 (C-21). Owing to the two large coupling constants (J = 11.3 Hz) of H-21 with H20 and Hb-22, the hydroxy group at C-21 should have an equatorial configuration (b-orientation). Therefore, compound 21 was identified as 21b-hydroxy-3-oxo-2,3-secours-12-en-2-oic acid. Compound 22 was isolated as a colorless solid and its molecular formula, C40H60O7 was determined by HRAPCIMS (m/z 687.4017 [M+Cl], calcd for C40H6035ClO7, 687.4033), indicating 11 degrees of unsaturation. The similarity of the 1H and 13C NMR chemical shifts of 22 with those of 18 (Tables 6 and 7) indicated a common urs-12-ene-3b,15a-diol nucleus for the two compounds. However, in the 1H NMR of 22, the proton signal for H-11 appeared at lower field (dH 4.94) and the resonance for OH-12 was absent. Moreover, the NMR spectra of 22 (Table 7) showed signals suggesting an additional arylpropane unit in the molecule. The 1H NMR spectrum of

22 displayed resonances for three aromatic protons at dH 6.87 (d, J = 1.4 Hz, H-20 ), 6.88 (d, J = 8.1 Hz, H-50 ), 6.82 (dd, J = 8.1, 1.4 Hz, H-60 ), a phenolic hydroxy at dH 5.62 (s, OH-40 ), a methoxy group at dH 3.88 (s, OMe-30 ), a benzylic oxymethine at dH 4.75 (d, J = 10.3 Hz, H-70 ), an oxymethine at dH 4.42 (dt, J = 10.3, 3.6 Hz, H-80 ), and two oxymethylene protons at dH 3.39 (ddd, J = 12.0, 7.2, 4.6 Hz, Ha-90 ) and 3.58 (m, Hb-90 ). These data were consistent with the 13C NMR data, which indicated the presence of: three aromatic methine carbons at dC 109.7 (C-20 ), 114.4 (C-50 ) and 120.2 (C60 ), two oxyquaternary aromatic carbons at dC 145.8 (C-40 ) and 146.6 (C-30 ), a quaternary aromatic carbon at dC 131.1 (C-10 ), a methoxy carbon at dC 55.9 (OMe-30 ), two oxymethine carbons at dC 77.2 (C-70 ) and 75.8 (C-80 ), and one oxymethylene carbon at dC 62.2 (C-90 ). The downfield doublet at dH 4.75 (H-70 ), typical of a benzylic oxymethine, and the doublet of triplet at dH 4.42 (H-80 ), suggested the presence of a 1,4-dioxane bridge between the C-11 and C-12 of the triterpene and C-80 and C-70 of the arylpropane moiety. The positions of the phenolic hydroxy group at C-40 and the methoxy group at C-30 were established by the HMBC correlations (Fig. 4) of OH-40 (dH 5.62) to C-40 , C-50 and C-30 and the correlation of OMe-30 (dH 3.88) to C-30 , respectively. The NOESY correlations (Fig. 4) between H-80 (dH 4.42) and H-11 (dH 4.94) and between H-70 (dH 4.75) and H-29 (dH 1.01) indicated the linkage of triterpene and arylpropane units through a 1,4-dioxane bridge. The linkages

Please cite this article in press as: Kaweetripob, W., et al. Lupane, friedelane, oleanane, and ursane triterpenes from the stem of Siphonodon celastrineus Griff. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.027

9

W. Kaweetripob et al. / Phytochemistry xxx (2013) xxx–xxx Table 6 H (600 MHz) and

1

Position

a

13 a

C NMR (150 MHz) spectroscopic data of compounds 18 and 19 (CDCl3). 18

19

20

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

1

40.9

39.4

27.3 78.5 39.0 54.8 18.5

7

37.0

8 9 10 11 12 13 14 15 16

44.1 54.4 38.2 69.9 145.7 117.0 46.5 68.2 38.7

17 18 19 20 21

34.1 47.3 40.5 39.3 31.0

22

41.1

23 24

28.2 15.6

d (11.2) m m m m m m s s

1.22, 2.39, 1.87, 3.16, – 1.03, 1.57, 1.69, 1.28, 1.64, – 1.68, – 4.18, – – – 5.63, 1.40, 2.04, – 2.19, 1.45, 1.04, 1.22, 1.43, 1.34, 1.49, 1.23, 9.71,

25 26 27 28 29 30 OH OCOPh ipso ortho meta para

16.7 18.6 18.2 29.3 16.8 21.1

1.12, 1.13, 1.22, 0.86, 0.90, 0.92, 4.74,

s s s s d (6.5) d (6.5) brs

1.60, 2.89, 2.55, – – 1.60, 1.52, 1.60, 1.81, 1.88, – 2.59, – – – – – 4.29, 1.28, 2.01, – 2.51, 1.39, 1.08, 1.25, 1.48, 1.35, 1.50, 1.27, 3.50, 3.98, 1.22, 1.23, 1.37, 0.88, 0.82, 0.94, 6.34,

40.9

2 3 4 5 6

1.23, 2.20, 1.62, 3.24, – 0.78, 1.43, 1.62, 1.62, 1.73, – 1.60, – 4.18, – – – 4.19, 1.18, 1.93, – 2.15, 1.35, 1.02, 1.24, 1.43, 1.31, 1.48, 1.01, 0.82,

m brd, (13.4) m dd (10.8, 5.4) d (11.4) m m m td (12.9, 3.4) m d (10.3)

brd (11.1) m t (12.1)

34.4 219.4 51.5 55.7 18.7 35.6 46.3 58.7 36.5 194.2 145.0 134.7 47.4 68.3 38.6 34.2 48.7 40.6 39.1 30.9 40.7 22.0 65.6 16.7 18.6 15.1 29.5 16.6 20.9

m ddd (13.2, 7.7, 5.1) m

m m m dt (13.7, 3.3) td (13.7, 3.7) s

dd (11.6, 5.7) m t (11.6) d (10.1) m m m m m m s d (11.3) d (11.3) s s s s d (6.6) d (6.4) s

28.2 76.9 52.8 55.8 18.6 35.6 43.8 52.7 38.3 70.0 146.1 116.1 45.9 72.2 34.2 34.0 47.4 40.6 39.3 30.9 40.7 18.7 207.6 15.7 19.2 19.5 28.9 16.8 21.0 165.5 131.0 129.5 128.8 132.9

m dt (13.7, 3.3) m m [+D2O; 3.16, dd (8.7, 7.3)] m td (12.7, 2.7) m m m d (9.5) d (9.3)

dd (11.5, 5.8) m t (11.5) d (11.2) m m m m m m s brd (1.6)

1.03, s 1.23, s 1.52, s 0.99, s 0.95, d (6.2) 0.94, d (6.2) 3.15 and 4.77, each s

8.01, d (7.6) 7.47, t (7.6) 7.58, t (7.6)

Assignments were based on DEPT, HSQC, and HMBC experiments.

between the two units were C-11–O–C-80 and C-12–O–C-70 (Fig. 1). The relative trans stereochemistry of the dioxane moiety was determined by the JH-70 /H-80 value of 10.3 Hz (Nunes et al., 2011). The NOESY correlations between (Fig. 4) H-80 , H-11, H-25 and H26, indicated the relative configuration of H-80 as b-orientation. Therefore, the structure of 22 was established as 11a,12-[2(hydroxymethyl)-3-(4-hydroxy-3-methoxyphenyl)ethane-1,2-dioxy]-urs-12-ene-3b,15a-diol. Compound 23 was shown to have the same molecular formula, C40H60O7, as 22 based on HRAPCIMS results. The 1H and 13C NMR spectra of 23 (Table 7) indicated that the triterpene moiety and arylpropane unit of 23 were identical with those of 22. The difference between 22 and 23 was in the positions of the linkage between the two units. The HMBC correlations of H-70 (dH 4.75) to C-11 (dC 75.1) and the NOESY correlations between H-70 (dH 4.75) and H-11 (dH 4.18) and between H-80 (dH 3.62) and H-29 (dH 0.93) indicated that the 1,4-dioxane linkage between the triterpene moiety and the arylpropane unit were C-11–O–C-70 and C-12–O–C80 (Fig. 1). The relative trans stereochemistry of the dioxane moiety was determined by the JH-70 /H-80 value of 9.5 Hz (Nunez et al., 2011). The NOESY correlations between H-70 , H-11, H3-25 and H3-26 indi-

cated the relative configuration of H-70 as b-orientation. Compound 23 was thus determined to be 11a,12-[3-(hydroxymethyl)-2-(4hydroxy-3-methoxyphenyl)ethane-1,2-dioxy]-urs-12-ene-3b,15adiol. The biosynthesis of triterpenes containing a 1,4-dioxane bridge, which is a chemotaxanomic marker of the Celastraceae species, has been hypothesized to occur via Diels–Alder reactions (Shirota et al., 1995; Nunez et al., 2011). All these Diels–Alder adducts contain as a part of triterpene aromatic units. Interestingly, this aromatic unit in compounds 22 and 23 was not observed. Consequently, a possible biosynthetic pathway for the formation of the 1,4-dioxane rings in compounds 22 and 23 (Scheme 1) is proposed to involve a radical mechanism. Phenolic oxidation of coniferyl alcohol generated radical A which could couple with an enoxy radical (C@C–O) 18a resulting in a C–O coupled product B, which then underwent a conjugate addition to generate compound 23. Compound 22 was envisioned to be biosynthesized in a similar manner from oxyradical (C–O) of compound 18. In an effort to identify natural products with potentially useful anticancer properties, the cytotoxic effects of the new compounds 1, 4, 6–12, and 15–23, as well as the known compounds 2, 3 and

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W. Kaweetripob et al. / Phytochemistry xxx (2013) xxx–xxx

Table 7 H (600 MHz) and

1

Position

13 a

C NMR (150 MHz) spectroscopic data in CDCl3 for compounds 21, 22, and 23. 21

22

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

1

43.0

d (16.4) d (16.4)

40.8

175.5 207.7 50.7 47.7 20.4

7

32.1

m m

8 9 10 11

40.0 40.4 42.3 23.8

43.7 51.6 38.4 68.0

43.8 50.5 38.3 75.1

1.12, 2.27, 1.43, 3.20, – 0.75, 1.39, 1.59, 1.68, 1.78, – 1.84, – 4.18,

12 13 14 15

125.1 139.1 42.7 26.5

146.2 116.5 46.6 68.2

– – – 4.19, m

145.0 123.1 47.2 68.1

– – – 4.22, dd (11.7, 5.8)

16

29.2

38.7

35.2 58.6 38.4 47.1 71.8

22

50.3

23 24 25 26 27 28 29 30 10 20 30 40 50 60 70 80 90

23.9 19.5* 19.4* 16.9 22.9 28.4 17.4 15.8

1.20, 1.95, – 2.32, 1.40, 1.02, 1.28, 1.44, 1.31, 1.48, 0.99, 0.81, 1.19, 1.20, 1.25, 0.85, 1.01, 0.94,

38.8

17 18 19 20 21

1.38, 1.56, – 2.16, – 1.97, 2.07, 5.20, – – 1.03, 1.82, 1.06, 1.86, – 1.38, 1.43, 0.85, 3.38,

1.11, 2.23, 1.47, 3.17, – 0.76, 1.42, 1.62, 1.61, 1.75, – 1.77, – 4.94,

40.8

2 3 4 5 6

2.50, 2.30, – 9.77, – 2.29, 1.52,

1.20, 1.95, – 2.42, 1.36, 0.94, 1.28, 1.43, 1.32, 1.48, 1.00, 0.78, 1.08, 1.22, 1.29, 0.85, 0.93, 0.93, – 6.87, – – 6.86, 6.85, 4.75, 3.62, 3.50, 3.57, 3.89, 5.61,

1.23, 1.78, 1.17, 1.11, 1.01, 1.02, 1.03, 0.86, 0.83, 1.05,

s m m

dd (11.6, 4.3) brdd (17.6, 11.6) dt (17.6, 4.3) brt (4.3)

m m m td (11.3, 4.0) m m m td (11.3, 4.2) m dd (11.3, 4.2) s s s s s s d (6.2) d (6.3)

OMe-30 OH-40 a *

23

27.4 78.4 39.0 54.8 18.4 37.2

34.1 46.1 40.3 39.6 31.0 41.1 28.3 15.8 16.7 18.7 18.0 29.3 17.6 21.0 131.1 109.7 146.6 145.8 114.4 120.2 77.2 75.8 62.2 55.9 –

m dt (14.1, 3.3) m t (8.3)

27.7 78.6 39.1 54.9 18.4

brd (11.7) m m m m

37.8

brd (9.2) d (9.1)

m d (12.0)

34.1 46.7 38.9 39.5 31.0

d (11.2) m m m m m m s s s s s s d (6.4) d (6.4)

41.1 28.4 15.8 16.4 19.2 17.6 29.2 18.3 21.1 130.0 109.6 146.4 145.5 114.1 120.1 78.1 81.3 62.0

6.87, d (1.4)

6.88, 6.82, 4.75, 4.42, 3.39, 3.58, 3.88, 5.62,

d (8.1) dd (8.1, 1.4) d (10.3) dt (10.3, 3.6) ddd (12.0, 7.2, 4.6) m s s

56.0 –

m dt (14.1, 3.4) m dd (7.8, 7.0) m m m m td (13.0, 3.8) d (10.4) d (10.4)

m dd (11.7) d (10.7) m m m m m m s s s s s s d (6.4) brs brs

d (8.0) brd (8.0) d (9.5) ddd (9.5, 4.0, 2.8) dd (12.4, 4.0) dd (12.4, 2.8) s brs

Assignments were based on DEPT, HSQC, and HMBC experiments. Exchangeable in the column.

24–29, were evaluated for cytotoxicity against MOLT-3 (acute T lymphoblastic leukemia), HuCCA-1 (cholangiocarcinoma), A549 (lung cancer), HeLa (cervical carcinoma), HepG2 (hepatocarcinoma), and MDA-MB-231 (breast cancer) cancer cell lines. The results showed that compound 21 had good activity against MOLT-3 cells, with an IC50 value of 4.5 lM. It should be noted that this compound exhibited no cytotoxicity against MRC-5 (normal embryonic lung) normal cell line (2.90% cytotoxicity at 106 lM). Compound 3 showed weak activity against HepG2 cells, with an IC50 value of 10.2 lM, whereas 29 showed weak cytotoxic activity with an IC50 value of 13.8 lM against MOLT-3 cells. The triterpenes (1, 2, 4, 6, 7, 11, 16, 17 and 28), the three sterol compounds (24–26) and the fatty acid (27) were not cytotoxic to any of the cell lines tested.

OH 2'

1'

OH 9'

OH

4'

MeO

MeO 6'

30 29

OH H

20 21

8' 7' O

O

O

25

HO

H

3 24

H

H

23

Fig. 4. HMBC (

OH

28

HO H

) and NOESY (

H O H 12 18 11 9 8

5 H

H

15 27

28

H OH

H ) correlations of 22.

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W. Kaweetripob et al. / Phytochemistry xxx (2013) xxx–xxx

OH

OH MeO

MeO

HO

O

OH MeO

OH

HO O

HO

HO

OH

HO

O

O

A

conif eryl alcohol

Compound 18

OH

HO

B

OH

HO

18a

Compound 23

Scheme 1. The proposed mechanism for the formation of the 1,4-dioxane ring in compound 23.

3. Conclusions Twenty-one new triterpenes, consisting of a lupane derivative 1, two friedelane derivatives 4 and 5, an oleanane derivative 6, and 17 ursane-type triterpenoids 7–23, together with three known triterpenes 2, 3, and 28, three sterols 24, 25, and 26, a fatty acid 27, a sesquiterpene alkaloid 29, and a glycerol derivative 30, were isolated from the stem of S. celastrineus. The structures of the isolated compounds were determined mainly using 1D and 2D NMR spectroscopic data (1H, 13C, COSY, NOESY, HSQC, HMBC) and high resolution mass spectrometry, as well as comparison with literature data. Compound 21 was found to have good activity against MOLT-3 cells with IC50 values of 4.5 lM, while compound 3 was weakly active against HepG2 cells with an IC50 value of 10.2 lM. Only one known compound 29 showed cytotoxic activity against MOLT-3 cells, yielding a weak IC50 value of 13.8 lM. The possible pathway for the biosynthesis of 1,4-dioxane bridge in compounds 22 and 23 was also proposed.

4. Experimental section 4.1. General experimental procedures Melting points were determined on a Büchi 535 apparatus and are uncorrected. Optical rotation values were measured on a JASCO P-1020 polarimeter in CHCl3. UV spectra were recorded using a Shimadzu UV-1700 PharmaSpect UVVis spectrophotometer in MeOH. Infrared spectra were obtained on a Perkin Elmer Spectrum One spectrophotometer using the UATR technique. HRMS were performed on a Bruker (Micro ToF) spectrometer. All commercial grade solvents were distilled prior to use, and spectral grade solvents were used for spectroscopic measurements. 1H and 13C NMR spectra were recorded on a Bruker Advance 600 instrument using TMS as the internal standard. Chemical shifts are given in parts per million downfield from TMS, and coupling constants are measured in Hz. DEPT, HSQC, HMBC COSY and NOESY experiments were conducted using standard Bruker Sofware. Medium pressure liquid chromatography (MPLC) was performed using a Buchi Pump Module C-605 and a Buchi UV Monitor C-630. High performance liquid chromatography (HPLC) was performed using a Waters Delta 600 with a Waters 2996 photodiode array detector. Silica gel 60 (Merck, 0.063–0.200 mm.) was used for column chromatography (CC) at normal pressure, while silica gel 60 (Merck, less than 0.063 mm.) was used for CC under reduced pressure. Silica gel 60 PF254 (Merck) was used for preparative thin layer chromatography (TLC). TLC was performed on a precoated aluminium plate (Merck, silica gel 60 F254). The results were visualized by UV absorption at 254 nm and followed by Godin’s reagent stain.

Sephadex LH-20 (GE Helth care Bio-Sciences AB) was used for column gel filtration. 4.2. Plant material The stem material of S. celastrineus was collected from the forest at Sakaerat, Nakon Ratchasima province (Thailand) in October 2008. The plant material was identified by Dr. Wichan Eiadthong, Department of Forest Biology, Faculty of Forestry, Kasetsart University, Bangkok, 10900, Thailand. A voucher specimen (CRI508) was deposited at the Laboratory of Natural Products, Chulabhorn Research Institute, Bangkok, Thailand. 4.3. Extraction and isolation Air-dried and powdered stem of S. celastrineus (6.9 kg) was extracted with CH2Cl2 (10 L  3) at room temperature for 3 days. The solvent was removed by rotary evaporator to give a dark brown residue (89.3 g), which showed cytotoxic activity against the MOLT-3 cancer cell line (86% cytotoxicity at 10 lg/ml). Subsequently, the crude residue was separated on a silica gel column via vacuum liquid chromatography. Elution was carried out using a gradient of acetone in CH2Cl2–hexane (1:1) to obtain four fractions (A–D). Fraction A (63.34 g) was subjected to silica gel CC using a MeOH–CH2Cl2 mobile phase to yield six separated fractions (A1– A6). Fraction A2 (10.64 g, 1.5% MeOH–CH2Cl2) was first filtered through a Sephadex LH-20 column that was washed with MeOH–CH2Cl2 (1:1). Fractions were further separated on a silica gel column using EtOAc–hexane as the eluent to afford seven fractions (A2-1 to A2-7). Fraction A2-1 (117 mg) was applied to a silica gel MPLC column (230  15 mm, Merck, less than 0.063 mm, 4% EtOAc–hexane, flow rate 10 ml/min, k 220 nm), to give compound 1 (13.0 mg). Fraction A2-3 (153.7 mg) was purified by preparative TLC with hexane–CH2Cl2–MeOH (3:1.5:0.2) to give compounds 2 (16.9 mg) and 24 (3.9 mg). Fraction A2-4 (700.0 mg) was purified by recrystallization from a minimal amount of MeOH in CH2Cl2 to give compound 3 (44.0 mg). The mother liquor was evaporated under reduced pressure (656.0 mg). The remaining solid was then filtered through a Sephadex LH-20 column washing with MeOH (16.4 mg) and further purified using on a C18-MS-II HPLC column [250  20 mm, Cosmosil column, MeOH–H2O (97:3), flow rate 8 ml/min, k 275 nm] to give compound 6 (2.9 mg, at 23.5 min). Fraction A2-5 (1.2 g) was filtered twice through a Sephadex LH20 column, first eluting with MeOH–CH2Cl2 (30:70, v/v), and then refiltering using MeOH as eluent to give a mixture compounds (411 mg) that was separated using on a C18-MS-II HPLC column [250  20 mm, Cosmosil column, MeOH–H2O (97:3 to 100:0 for

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W. Kaweetripob et al. / Phytochemistry xxx (2013) xxx–xxx

10 min; 100:0 for 30 min), flow rate 8 ml/min, k 236 nm], to give compounds 5 (9.3 mg, at 15.5 min), and 25 (3.6 mg, at 24.5 min), respectively. Fraction A2-6 (816.5 mg) was recrystallized using a minimal amount of MeOH in CH2Cl2, the precipitate thus formed was filtered to give compound 4 (14.2 mg). The mother liquor was evaporated (728.5 mg), and the remaining solid residue was filtered twice using a Sephadex LH-20 column, first eluting with MeOH–CH2Cl2 (30:70, v/v), and then refiltering using MeOH as eluent to yield a mixture of compounds (202.4 mg). The mixture was separated on an EXODS HPLC column [250  21.2 mm, Hichrom column, MeOH–H2O (80:20 for 30 min; 80:20 to 100:0 for 20 min and 100:0 for 30 min), flow rate 8 ml/min, k 280 nm] to give compounds 7 (14.4 mg, at 31.0 min) and 8 (28.8 mg, at 33.5 min). Fraction A5 (1.6 g) was filtered twice through a Sephadex LH-20 column, first eluting with MeOH–CH2Cl2 (50:50, v/v), and then refiltering using MeOH as the eluent to give a residue (18.7 mg), which was further purified on a C18 HPLC column [250  10 mm, SunFire column, MeOH–H2O (88:12), flow rate 2.5 ml/min, k 284 nm] to yield compound 21 (6.7 mg, at 18.4 min). Fraction B (3.96 g) was reapplied to a Sephadex LH-20 column and eluted with MeOH to provide two fractions (B1–B2). Fraction B1 (938.0 mg) was applied to an EXODS HPLC column [250  21.2 mm, Hichrom column, MeOH–H2O (75:25–100:0 for 25 min), flow rate 8 ml/min, k 236 nm] to yield compound 29 (21.0 mg, at 7.4 min) and a mixture of compounds (46.9 mg). The mixture was further separated on a C18 HPLC column [250  10 mm, SunFire column, MeOH–H2O (72:28), flow rate 2.5 ml/min, k 216 nm], which yielded compounds 9 (3.7 mg, at 34.6 min), 10 (3.4 mg, at 41.1 min), and 27 (8.4 mg, at 45.9 min). Fraction B2 (2.5 g) was subjected to Sephadex LH-20 CC using MeOH–CH2Cl2 (1:1) as eluent to give two fractions (B2-1 and B22). Fraction B2-1 (952.8 mg) was further separated on a C18-MS-II HPLC column [250  20 mm, Cosmosil column, MeOH–H2O (85:15 for 30 min; 85:15–100:0 for 35 min), flow rate 8 ml/min, k 224, 236,280 nm] to give compounds 11 (6.0 mg. at 9.9 min, k 280 nm), 12 (13.5 mg, at 12.8 min, k 236 nm), 26 (4.4 mg, at 24.4 min, k 236 nm), 13 (3.6 mg, at 38.4 min, k 280 nm), 14 (1.6 mg, at 39.5 min, k 224 nm), 15 (4.7 mg, at 48.9 min, k 236 nm), and 28 (8.5 mg, at 56.7 min, k 236 nm). Fraction B2-2 (707.0 mg) was further separated on a C18-MS-II HPLC column [250  20 mm, Cosmosil column, MeOH–H2O (85:15 for 30 min; 85:15 to 100:0 for 5 min), flow rate 8 ml/min, k 236, 280 nm] to give 3 peaks (B2-2-1, B2-2-2, and B2-2-3). Peak B2-2-1 (47.4 mg) was further separated on an EXODS HPLC column [250  21.2 mm, Hichrom column, MeOH–H2O (72:28 for 30 min; 72:28 to 100:0 for 15 min), flow rate 8 ml/min, k 236 nm] to yield compounds 16 (7.9 mg, at 16.7 min), 17 (8.4 mg, at 23.3 min), and 22 (2.3 mg, at 34.2 min). Peak B2-2-2 (76.1 mg) was further separated on an EXODS HPLC column [250  21.2 mm, Hichrom column, MeOH–H2O (77:23), flow rate 8 ml/min, k 236, 280 nm] to give compounds 18 (28 mg, at 17.8 min, k 236 nm), 23 (2.7 mg, at 21.7 min, k 236 nm), and 19 (22.6 mg, at 23.9 min, k 280 nm). Peak B2-2-3 (38.5 mg) was further separated on an EXODS HPLC column [250  21.2 mm, Hichrom column, MeOH–H2O (87:13), flow rate 8 ml/min, k 236 nm] to yield compounds 20 (6.3 mg, at 20.1 min) and 30 (1.9 mg, at 30.9 min).

4.3.1. 3b,25-Epoxy-25-methoxylup-20(29)-ene (1) Colorless solid; mp 192–196 °C; [a]D26 +78.0 (c 1.28, CHCl3); UV kmax (MeOH) nm (log e) 206 (3.2), 220 (sh), 297 (2.1); IR (UATR) mmax 2946, 2867, 1640, 1461, 1380, 1098, 1072, 880, and 739 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 1; HRAPCIMS m/z 455.3867 [M+H]+ (calcd for C31H51O2, 455.3884).

4.3.2. 25-Hydroxyfriedelane-3,21-dione (4) Colorless solid; mp 276–278 °C; [a]D25 +108.8 (c 1.08, CHCl3); UV kmax (MeOH) nm (log e) 205 (3.0), 288 (1.9); IR (UATR) mmax 3539, 2949, 2866, 1706, 1634, 1604, 1513, 1466, 1253, and 1172 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 1; HRAPCIMS m/z 457.3685 [M+H]+ (calcd for C30H49O3, 457.3676). 4.3.3. 25-Benzoyloxyfriedelane-3,21-dione (5) Colorless solid; mp 156–157 °C; [a]D26 +65.1 (c 0.31, CHCl3); UV kmax (MeOH) nm (log e) 230 (4.3), 273 (3.1); IR (UATR) mmax 2940, 2873, 1712, 1466, 1450, 1380, 1272, 1111, and 712 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 1; HRAPCIMS m/z 561.3943 [M+H]+ (calcd for C37H53O4, 561.3997). 2.3.4. 3b,12-Dihydroxyolean-12-en-11-one (6) Colorless solid; mp 177–180 °C; [a]D25 +99.6 (c 0.25, CHCl3); UV kmax (MeOH) nm (log e) 206 (3.8), 224 (sh), 287 (3.6); IR (UATR) mmax 3422, 3280, 2947, 2923, 2854, 1710, 1661, 1631, 1462, 1384, 1365, and 1037 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 2; HRAPCIMS m/z 457.3688 [M+H]+ (calcd for C30H49O3, 457.3676). 2.3.5. 13b,28-Epoxyurs-11-ene-3b,15a-diol (7) Colorless solid; mp 254–256 °C; [a]D27 +75.6 (c 1.64, CHCl3); UV kmax (MeOH) nm (log e) 206 (3.6), 288 (3.0); IR (UATR) mmax 3410 (br), 2926, 2857, 1704, 1661, 1454, 1381, 1264, 1020, 999, 917, and 736 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 2; HRAPCIMS m/z 457.3630 [M+H]+ (calcd for C30H49O3, 457.3676). 4.3.6. 3b,12,15a-Trihydroxyurs-12-en-11-one (8) Colorless solid; mp 195–198 °C; [a]D26 +105.1 (c 3.0, CHCl3); UV kmax (MeOH) nm (log e) 289 (3.7); IR (UATR) mmax 3408 (br), 2946, 2926, 2866, 1704, 1662, 1633, 1455, 1380, 1264, 1015, and 737 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 2; HRAPCIMS m/z 473.363 [M+H]+ (calcd for C30H49O4, 473.3625). 4.3.7. 11a-Methoxyurs-12-ene-3b,12,15a-triol (9) Colorless solid; mp 165–168 °C; [a]D25 +40.6 (c 0.35, CHCl3); UV kmax (MeOH) nm (log e) 220 (sh), 281 (3.4); IR (UATR) mmax 3420 (br), 2926, 2860, 1705, 1456, 1379, 1263, 991, 736, and 703 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 3; HRAPCIMS m/z: 523.3548 [M+35Cl] (calcd for C31H5235ClO4, 523.3560) and 525.3538 [M+37Cl] (calcd for C31H5237ClO4, 525.3546). 4.3.8. Urs-12-ene-3b,11a,15a-triol (10) Colorless solid; mp 202–205 °C; [a]D26 +13.6 (c 0.33, CHCl3); UV kmax (MeOH) nm (log e) 204 (3.5), 230 (sh), 274 (2.5); IR (UATR) mmax 3392 (br), 2924, 2853, 1707, 1455, 1379, 1264, 1036, 998, 974, and 736 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 3; HRAPCIMS m/z 459.3828 [M+H]+ (calcd for C30H51O3, 459.3833). 4.3.9. 12,15a,20b-Trihydroxyurs-12-ene-3,11-dione (11) Colorless solid; mp 256–258 °C; [a]D30 +116.7 (c 0.48, CHCl3); UV kmax (MeOH) nm (log e) 289 (3.0); IR (UATR) mmax 3415 (br), 2928, 2866, 1699, 1660, 1630, 1459, 1370, 1269, 1009, 898, 735, and 702 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 3; HRAPCIMS m/z 487.3415 [M+H]+ (calcd for C30H47O5, 487.3418).

Please cite this article in press as: Kaweetripob, W., et al. Lupane, friedelane, oleanane, and ursane triterpenes from the stem of Siphonodon celastrineus Griff. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.027

W. Kaweetripob et al. / Phytochemistry xxx (2013) xxx–xxx

4.3.10. 11a-Methoxyurs-12-ene-3b,15a-diol (12) Colorless solid; mp 158–162 °C; [a]D26 13.4 (c 0.38, CHCl3); UV kmax (MeOH) nm (log e) 206 (3.9); IR (UATR) mmax 3390 (br), 2925, 2853, 1690, 1456, 1264, 1081, 997, 734, and 704 cm1; for 1 H and 13C NMR (CDCl3) spectroscopic data, see Table 4; HRAPCIMS m/z 472.3891 [M]+ (calcd for C31H52O3, 472.3911). 4.3.11. 3b,12,24-Trihydroxyurs-12-en-11-one (13) Colorless solid; mp 228–232 °C; [a]D26 +137.0 (c 0.42, CHCl3); UV kmax (MeOH) nm (log e) 288 (3.8); IR (UATR) mmax 3408 (br), 2973, 2925, 2866, 1662, 1631, 1456, 1370, 1270, 1039, and 737 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 4; HRAPCIMS m/z 473.3647 [M+H]+ (calcd for C30H49O4, 473.3625). 4.3.12. 15a-Benzoyloxyurs-12-ene-3b,11a-diol (14) Semi solid; [a]D26 +11.8 (c 0.24, CHCl3); UV kmax (MeOH) nm (log e) 229 (3.6), 273 (2.6), 280 (2.7); IR (UATR) mmax 3419 (br), 2925, 2853, 1712, 1451, 1379, 1274, 1175, 1115, 972, 737, and 712 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 4; HRAPCIMS m/z: 597.3705 [M+35Cl] (calcd for C37H5435ClO4, 597.3716) and 599.3708 [M+37Cl] (calcd for C37H5437ClO4, 599.3707). 4.3.13. 11a-Methoxyurs-12-ene-3b,12-diol (15) Colorless solid; mp 117–120 °C; [a]D26 +45.3 (c 0.32, CHCl3); UV kmax (MeOH) nm (log e) 208 (3.8); IR (UATR) mmax 3426 (br), 2924, 2853, 1709, 1673, 1456, 1378, 1263, 1178, 1034, 995, 737, and 704 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 5; HRAPCIMS m/z 472.3902 [M]+ (calcd for C31H52O3, 472.3911). 4.3.14. 3b,11a,12,15a-Tetrahydroxyurs-12-en-24-al (16) Colorless solid; mp 175–178 °C; [a]D30 +38.6 (c 2.06, CHCl3); UV kmax (MeOH) nm (log e) 208 (3.2); IR (UATR) mmax 3422 (br), 2924, 2866, 1704, 1456, 1379, 1253, 1040, and 985 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 5; HRAPCIMS m/z 489.3582 [M+H]+ (calcd for C30H49O5, 489.3575). 4.3.15. 3b,11a,12,15a-Tetrahydroxyurs-12-en-24-oic acid methyl ester (17) Colorless solid; mp 258–261 °C; [a]D26 +55.4 (c 0.84, CHCl3); UV kmax (MeOH) nm (log e) 211 (3.4); IR (UATR) mmax 3406 (br), 2947, 2921, 2866, 1701, 1456, 1433, 1377, 1245, 1192, 1149, 1040, 983, 736, and 704 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 5; HRAPCIMS m/z 519.3692 [M+H]+ (calcd for C31H51O6, 519.3680). 4.3.16. Urs-12-ene-3b,11a,12,15a-tetrol (18) Colorless solid; mp 211–214 °C; [a]D30 +35.6 (c 6.95, CHCl3); UV kmax (MeOH) nm (log e) 209 (3.7); IR (UATR) mmax 3422 (br), 2925, 2866, 1689, 1456, 1379, 1265, 1037, 994, 735, and 704 cm1; for 1 H and 13C NMR (CDCl3) spectroscopic data, see Table 6; HRAPCIMS m/z: 509.3404 [M+35Cl] (calcd for C30H5035ClO4, 509.3403) and 511.3378 [M+37Cl] (calcd for C30H5037ClO4, 511.3389). 4.3.17. 12,15a,24-Trihydroxyurs-12-ene-3,11-dione (19) Colorless gum; [a]D27 +154.3 (c 1.99, CHCl3); UV kmax (MeOH) nm (log e) 288 (4.2); IR (UATR) mmax 3413 (br), 2946, 2923, 2866, 1699, 1661, 1630, 1456, 1372, 1263, 1081, 1040, 1013, and 706 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 6; HRAPCIMS m/z 487.3422 [M+H]+ (calcd for C30H47O5, 487.3418).

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4.3.18. 15a-Benzoyloxy-3b,11a,12-trihydroxyurs-12-en-24-al (20) Colorless solid; mp 188–191 °C; [a]D27 +43.2 (c 0.29, CHCl3); UV kmax (MeOH) nm (log e) 228 (4.2), 273 (3.1), 280 (3.0); IR (UATR) mmax 3456 (br), 2924, 2866, 1708, 1451, 1275, 1117, 737, and 712 cm1; for 1H and 13C NMR (CDCl3): spectroscopic data, see Table 6; HRAPCIMS m/z 593.3837 [M+H]+ (calcd for C37H53O6, 593.3837). 4.3.19. 21b-Hydroxy-3-oxo-2,3-secours-12-en-2-oic acid (21) Colorless solid; mp 227–230 °C; [a]D26 +62.9 (c 0.73, CHCl3); UV kmax (MeOH) nm (log e) 204 (3.7), 225 (sh), 274 (2.9); IR (UATR) mmax 3400 (br), 2924, 2860, 1714, 1457, 1380, 1003, and 737 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 7; HRAPCIMS m/z 473.3644 [M+H]+ (calcd for C30H49O4, 473.3625). 4.3.20. 11a,12-[2-(Hydroxymethyl)-3-(4-hydroxy-3methoxyphenyl)ethane-1,2-dioxy]-urs-12-ene-3b,15a-diol (22) Colorless solid; mp 142–145 °C; [a]D30 +22.2 (c 0.80, CHCl3); UV kmax (MeOH) nm (log e) 206 (4.4), 235 (sh), 280 (3.3); IR (UATR) mmax 3358 (br), 2951, 2841, 1644, 1450, 1409, 1112, and 1014 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 7; HRAPCIMS m/z: 687.4017 [M+35Cl] (calcd for C40H6035ClO7, 687.4033) and 689.3992 [M+37Cl] (calcd for C40H6037ClO7, 689.4028) . 4.3.21. 11a,12-[3-(Hydroxymethyl)-2-(4-hydroxy-3methoxyphenyl)ethane-1,2-dioxy]-urs-12-ene-3b,15a-diol (23) Semi solid; [a]D30 2.2 (c 0.68, CHCl3); UV kmax (MeOH) nm (log e) 212 (4.1), 280 (3.3); IR (UATR) mmax 3380 (br), 2927, 2866, 1697, 1636, 1603, 1518, 1456, 1378, 1265, 1112, 1034, and 735 cm1; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 7; HRAPCIMS m/z: 687.4027 [M+35Cl] (calcd for C40H6035ClO7, 687.4033) and 689.4023 [M+37Cl] (calcd for C40H6037ClO7, 689.4028). 4.4. Cytotoxicity assay Cytotoxic activity for adhesive cell lines, including human hepatocarcinoma (HepG2), human lung cancer (A549), human cholangiocarcinoma (Thai; HuCCA-1), human cervical carcinoma (HeLa), and human breast cancer (MDA-MB-231) cancer cell lines, was evaluated using the MTT assay (Carmichael et al., 1987; Mosmann, 1983). For the non-adhesive T-lymphoblast (MOLT-3) cell line, the cytotoxicity was assessed using the XTT assay (Doyle and Griffiths, 1997). Etoposide was used as the positive control for tested MOLT3 cells with an IC50 value of 0.03 ± 0.009 lM. Doxorubicin was used as the positive control for the HuCCA-1, A549, HeLa, HepG2, and MDA-MB-231 cell lines with IC50 values of 0.78 ± 0.12, 0.59 ± 0.01, 0.36 ± 0.10, 0.72 ± 0.43, and 0.38 ± 0.19 lM, respectively. Acknowledgements We thank CRI colleagues from the Laboratory of Immunology, the Laboratory of Biochemistry, and the Integrated Research Unit for cytotoxicity tests. Partial financial support from the Center of Excellence on Environmental Health and Toxicology (EHT), PERDO, Ministry of Education and Chulabhorn Research Center, Institute of Molecular Biosciences, Mahidol University are gratefully acknowledged. We also thank Prof. Pittaya Tuntiwachwuttikul for valuable discussions and we are grateful to Prof. Geoffrey A. Cordell for English editing and helpful suggestions.

Please cite this article in press as: Kaweetripob, W., et al. Lupane, friedelane, oleanane, and ursane triterpenes from the stem of Siphonodon celastrineus Griff. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.027

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Please cite this article in press as: Kaweetripob, W., et al. Lupane, friedelane, oleanane, and ursane triterpenes from the stem of Siphonodon celastrineus Griff. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.027