Lupane Triterpenoids from the Stems of - American Chemical Society

Jan 27, 2014 - ABSTRACT: Fifteen new lupane-type triterpenoids (1−15) and 10 known triterpenoids (16−25) were isolated from the stems of Euonymus ...
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Lupane Triterpenoids from the Stems of Euonymus carnosus Jian Zhou, Chuang-Jun Li, Jing-Zhi Yang, Jie Ma, Yan Li, Xiu-Qi Bao, Xiao-Guang Chen, Dan Zhang, and Dong-Ming Zhang* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Fifteen new lupane-type triterpenoids (1−15) and 10 known triterpenoids (16−25) were isolated from the stems of Euonymus carnosus. The structures of the new compounds were elucidated on the basis of spectroscopic analyses, and the absolute configuration of compound 1 was confirmed by X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation. In addition, the compounds were tested for their cytotoxic activity against five human cancer cell lines and their ability to inhibit LPS-induced nitric oxide production in the murine microglia BV2 cell line. Compound 11 exhibited moderate cytotoxicity against several human cancer cell lines, and compounds 1, 2, 4, 5, 20, and 25 showed neuritis inhibitory activity against microglial inflammation factor, with IC50 values of 7.39, 7.48, 7.80, 3.48, 2.54, and 6.09 μM, respectively. Euonymus carnosus Hemsl (Celastraceae), a deciduous shrub or small tree, is widely distributed in mainland China and Japan1 and has been extensively used as a folk medicine to treat rheumatic arthralgia, dysmenorrhea, and traumatic injuries.2 Previous phytochemical investigations of plants in the genus Euonymus have led to the identification of triterpenoids, sesquiterpenoids, alkaloids, flavonoids, steroids, and lignanoids, which showed diverse biological activities, such as antitumor, anti-inflammatory, anti-HIV, and insecticidal effects.3 As part of our ongoing research to identify bioactive substances from the genus Euonymus, we investigated a 95% EtOH extract of the stems of E. carnosus and identified 10 new lupane triterpenoids (1−4, 6−11), five new 30-norlupan triterpenoids (5, 12−15), and 10 known compounds (16−25). Compounds 1, 2, 4, 5, 10−15, 18−20, and 24 are among the relatively few naturally occurring lupane triterpenoids that incorporate a 3α-hydroxy or a 3α-acetoxy group. In this paper, the isolation and structural elucidation of the new compounds as well as an evaluation of their biological activities are reported.

graphic steps, using silica gel, macroporous resin PRP-512, Sephadex LH-20, ODS, and preparative HPLC to afford compounds 1−25. Compound 1 was crystallized from CDCl3 as colorless needles. The molecular formula, C30H50O3, was determined from HREIMS (m/z 458.3778 [M]+, calcd for 458.3760) and 13 C NMR spectroscopic data, requiring six indices of hydrogen deficiency. The IR spectrum showed the presence of carbonyl (1727 cm−1) and hydroxy (3260 cm−1) functionalities. The 1H NMR data indicated the presence of six tertiary methyl groups [δH 0.78, 0.83, 0.86, 0.94, 0.95, and 1.04 (each 3H, s)], a methyl doublet [δH 1.05 (3H, d, J = 7.0 Hz)], an oxymethine proton [δH 3.40 (1H, brt, J = 2.5 Hz)], and the typical lupane Hβ-19 resonance at δH 2.31 (1H, m). The 13C NMR spectrum revealed 30 carbon resonances (Table 2) and displayed a resonance at δC 181.0, suggesting the presence of a carboxylic group. This assignment was supported by the MS fragment at m/z 385 formed by the loss of a CH3−CH−COOH group from the molecular ion. The aforementioned spectroscopic analysis suggested that 1 was a lupane triterpenoid with a hydroxy group at C-3 and a C-29 carboxylic group. The location of the C-3 hydroxy group was determined via an

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RESULTS AND DISCUSSION A 95% EtOH extract of the stems of E. carnosus was suspended in H2O and extracted successively with petroleum ether, CHCl3, EtOAc, and n-BuOH. The petroleum ether- and CHCl3-soluble fractions were subjected to multiple chromato© 2014 American Chemical Society and American Society of Pharmacognosy

Received: October 10, 2013 Published: January 27, 2014 276

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structure of 1 was determined as (20S)-3α-hydroxylupan-29-oic acid. The molecular formula of compound 2 was assigned as C32H52O4 according to its HRESIMS (m/z 523.3754 [M + Na]+, calcd for 523.3758) and 13C NMR data. The 1H and 13C NMR data for compounds 2 and 1 (Tables 1 and 2) were similar, except that the hydroxy group at C-3 in 1 was replaced by an acetoxy group [δH 2.08 (3H, s), δC 21.4, 170.8] in 2. This assignment was deduced from the correlations of H-3 (δH 4.62, brt, J = 2.5 Hz) with C-1 (δC 33.9), C-5 (δC 50.1), and the acetoxy carbonyl carbon (δC 170.8) in the HMBC experiment. The relative and absolute configurations of both 2 and 1 were identical according to the observed NOE correlations and the chemical shift values of the Me-28 (δH 0.78) and Me-30 (δH 1.05) protons.5 Therefore, 2 was defined as (20S)-3αacetoxylupan-29-oic acid. Compound 3 was isolated as a white, amorphous solid. Its molecular formula was determined to be C30H48O3 from the 13 C NMR data and molecular ion [M]+ at m/z 456.3611 (calcd for 456.3603) in the HREIMS spectrum, which indicated that the molecular mass of 3 is two mass units less than that of 1. The 1H and 13C NMR data for 3 were similar to those for 1, implying that these compounds were based on the same carbon skeleton. Analysis of its 1H−1H COSY, HSQC, and HMBC data revealed that the C-3 hydroxy group in 1 was replaced by a carbonyl group (δC 218.2) in 3. The absolute configuration at C-20 of 3 was identical to that of 1 on the basis of the chemical shift values for the Me-28 (δH 0.79) and Me-30 (δH 1.05) protons.5 Therefore, the structure of compound 3 was determined to be (20S)-3-oxolupan-29-oic acid. The structure of compound 4 has the same molecular formula as 1 based on its HREIMS and 13C NMR data. Analysis of the 1D and 2D NMR data revealed that compound 4 had the same molecular structure as 1, except for the C-20 absolute configuration. By comparison of the chemical shift values of Me-28 [δH 0.75 (3H, s)] and Me-30 [δH 1.15 (3H, d, J = 6.5 Hz)] in 4 with those of the known (20R)- and (20S)-3βhydroxylupan-29-oic acids [(20R): δH 0.74 (3H, s, Me-28), 1.15 (3H, d, J = 6.5 Hz, Me-30); (20S): δH 0.77 (3H, s, Me-28), 1.05 (3H, d, J = 6.5 Hz, Me-30)],5 the absolute configuration of C20 in 4 was determined to be R. This result was confirmed by the negative sign of the specific rotation for 4 ([α]20D = −60.7) compared to that of the two known compounds (20R, [α]20D = −52.6; 20S, [α]20D = +8.7).5 Therefore, compound 4 was determined as (20R)-3α-hydroxylupan-29-oic acid. Compound 5 exhibited the molecular formula C29H50O2, on the basis of its 13C NMR and HREIMS data. The NMR data for 5 (Tables 1 and 2) were similar to those for 24, suggesting that they were homologues. The major difference was that the C-20 carbonyl group in the latter was replaced by a hydroxy group in 5. The HMBC correlations from H-20 [δH 3.99 (1H, m)] to C18 (δC 47.0), C-21 (δC 21.3), and C-29 (δC 23.0) indicated that the hydroxy group was located at C-20. This was further confirmed by a separated spin−spin system (H-19/H-20/H29) in the 1H−1H COSY spectrum. On the basis of the NOE correlations and 1H NMR data, the relative and absolute configurations of 5 were similar to those of 4. Therefore, the structure of 5 was assigned as (20R)-30-norlupane-3α,20-diol. Comparison of the HRESIMS and NMR data for compound 6 with those for foliasalacin B26 indicated that their structures were closely related. On the basis of the HMBC correlations of H-7 [δH 3.88 (1H, dd, J = 6.0, 9.5 Hz)] with C-6 (δC 30.3), C-8 (δC 46.7), C-14 (δC 44.6), and C-26 (δC 10.0), compound 6

HMBC experiment in which H-3 exhibited 2J correlations with C-2 (δC 25.3) and C-4 (δC 37.5) and 3J correlations with C-1 (δC 33.2), C-5 (δC 49.0), C-23 (δC 22.1), and C-24 (δC 28.2). Similarly, the carboxylic group was assigned at C-20 on the basis of the HMBC correlations of H-19/C-29, H-20/C-29, and Me-30/C-29. In addition, the cross-peaks between H-1/H-2, H-2/H-3, H-18/H-19, H-19/H-20, H-19/H-21, and H-20/Me30 in the 1H−1H COSY spectrum confirmed key connections among the specific protons (Figure 1). Collectively, the NMR data indicated that the molecular structure of 1 was similar to wallichianic acid,4 except for the orientation of the C-3 hydroxy group. The α-orientation of the C-3 hydroxy group was assigned on the basis of the small J values (δH 3.40, brt, J = 2.5 Hz) of H-3. This was confirmed by the NOE correlations from H-3 to Me-23 and Me-24 (δH 0.83 and 0.94). The 20S absolute configuration was assigned on the basis of the method of Corbett and co-workers.5 A single-crystal X-ray diffraction pattern using the anomalous scattering of Cu Kα radiation confirmed the 3R, 5R, 8R, 9S, 10R, 13R, 14R, 17R, 18S, 19R, and 20S absolute configurations (Figure 2). Therefore, the 277

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278

2.80 1.71 1.40 1.37

1.04 0.83 0.94 0.86 1.04 0.95 0.78

20 21a 21b 22a

22b 23 24 25 26 27 28a

29b

29a

28b

1.00 1.48 1.32 1.18 2.31

15a

15b 16a 16b 18 19

1.68 m

7b 9 11a 11b 12a 12b 13

m s s s s s s

m m m m

m m m m m

m m m m m m m

1.36 1.41 1.51 1.31 1.66 1.24 1.76

5 6a 6b 7a

1a

1.42 m 1.23 m 1.94 m 1.54 m 3.40 brt (2.5) 1.20 m 1.40 me 1.40 me 1.43 m

1a 1b 2a 2b 3

no.

m m m m m m m

1.06 0.84 0.88 0.86 1.05 0.99 0.78

2.80 1.72 1.40 1.37

1.01 1.49 1.35 1.19 2.32

m s s s s s s

m m m m

m m m m m

1.70 m

1.38 1.40 1.50 1.31 1.68 1.25 1.77

1.43 m 1.11 m 1.87 m 1.58 m 4.62 brt (2.5) 1.18 m 1.40 me 1.40 me 1.44 m

2a

me m m m m m m

1.05 1.03 1.08 0.95 1.08 0.94 0.79

2.78 1.73 1.40 1.38

1.03 1.49 1.34 1.20 2.32

m s s s s s s

m m m m

m m m m m

1.12 0.83 0.94 0.86 1.04 0.94 0.75

2.80 1.77 1.77 1.32

0.96 1.44 1.34 1.40 1.78

m s s s s s s

m me me m

m m m m m

1.68 m

m m m m m m m

1.35 1.41 1.53 1.30 1.52 1.30 1.68

m me me me

4a 1.42 m 1.25 m 1.93 m 1.54 m 3.40 brt (2.5) 1.20 m 1.39 me 1.39 me 1.42 m

m m m m

1.70 m

1.44 1.39 1.49 1.39 1.69 1.25 1.79

1.32 1.47 1.47 1.44

1.92 1.40 2.49 2.41

3a

m m m m m m m

m s s s s s s

m me me m

m m m m m

1.12 d (6.0)

1.11 0.84 0.94 0.86 1.05 0.96 0.73

3.99 1.66 1.66 1.37

1.00 1.47 1.38 1.30 1.68

1.71 m

1.39 1.43 1.52 1.30 1.57 1.30 1.73

1.42 m 1.25 m 1.94 m 1.55 m 3.39 t (2.5) 1.22 m 1.41 me 1.41 me 1.46 m

5a m m m m

6a

m m m m m m

m s s s s s s

m m m m

m m m m m

3.81dd (4.0, 10.0) 3.41 t (9.5)

1.02 1.03 1.09 0.94 1.12 1.03 0.76

1.92 1.67 1.38 1.36

1.38 1.53 1.37 1.30 1.76

1.91 m

1.32 1.52 1.46 1.65 1.30 1.69

1.42 m 1.62 me 1.62 me 3.88 dd (6.0, 9.5)

1.91 1.33 2.50 2.42

Table 1. 1H NMR Spectroscopic Data of Compounds 1−15

m m m m m m m

m me me m

m m m m

m m m m

m m m m

3.81 dd (4.5, 10.5) 3.43 dd (8.5, 10.5)

1.01m 1.03 s 1.07 s 0.95 s 1.17 s 0.95 s 0.80 s

1.96 1.71 1.42 1.38

1.79 1.28 1.34 1.74

4.16 dd (5.0, 11.0)

1.62 1.32 1.52 1.33 1.67 1.39 1.71

1.35 1.49 1.49 1.86

1.93 1.41 2.48 2.44

7a m m m m

m m m m m m

m s s s s s s

4.90 brs

4.94 d (1.0)

1.25 1.03 1.08 0.92 1.10 1.05 0.80

2.07 m 1.35 m 1.41 m

1.43 m 1.55 m 1.43 m 1.47 m 2.29 dt (5.0, 11.0)

1.92 m

1.29 1.45 1.37 1.46 1.12 1.65

1.40 m 1.60 me 1.60 me 3.87 dd (6.5, 9.5)

1.88 1.32 2.48 2.40

8a

m m m m m m m

m m m m

m m me me

5.91 brs

m s s s s s s

4.87f

1.38 0.82 0.93 0.82 1.02 0.94 0.82

2.15 m 1.20 m 1.44 m

1.03 m 1.50 m 1.42 m 1.64 m 2.75 dt (6.0, 11.2)

6.29 brs

m s s s s s s

me m m m m m m

m me me me

m m m m t (2.8)

1.68 m

1.39 1.35 1.37 1.19 1.68 1.05 1.65

1.19 1.40 1.40 1.39

1.40 1.20 1.91 1.51 3.38

10c

4.96 d (1.0)

1.29 1.02 1.06 0.96 1.19 1.01 0.87

2.11 m 1.39 m 1.40 m

1.71 m 1.45 m 1.57 m 2.27 dt (5.0, 11.0)

4.13 dd (5.0, 11.5)

1.60 1.38 1.47 1.29 1.50 1.23 1.68

1.42 1.54 1.45 2.01

1.92 1.46 2.46 2.46

9b

me m m m m m m

4.90 brs

1.24 m 0.87 s 0.83 s 0.84 s 1.04 s 1.03 s 3.79 d (10.5) 3.32 d (10.5) 4.95 brs

2.11 m 1.41 m 1.91 m

1.08 m 1.94 m 1.24 m 1.71 m 2.29 dt (5.5, 11.0)

1.74 m

1.39 1.38 1.42 1.20 1.37 1.08 1.63

1.39 m 1.10 m 1.86 m 1.56 m 4.61 brt (2.5) 1.17 m 1.43 me 1.43 me 1.39 me

11a

m m m m m m m

m m m m

m m m m t (2.5)

1.19 m 0.86 s 0.83 s 0.84 s 1.01 s 1.05 s 3.78 d (10.5) 3.24 d (11.0) 2.15 s

2.08 m 1.52 m 1.96 m

1.09 m 1.93 m 1.29 m 2.07 m 2.61 dt (6.0, 11.5)

1.69 m

1.39 1.39 1.43 1.21 1.09 1.01 1.57

1.16 1.43 1.35 1.46

1.39 1.08 1.85 1.56 4.61

12a

m m m m m m m

1.19 m 0.82 s 0.93 s 0.83 s 1.01 s 1.02 s 3.78 d (11.0) 3.23 d (11.0) 2.15 s

2.08 m 1.53 m 1.96 m

1.08 m 1.93 m 1.29 m 2.05 m 2.60 dt (6.0, 11.5)

1.68 m

1.36 1.42 1.46 1.21 1.12 1.01 1.55

1.37 m 1.21 m 1.92 m 1.53 m 3.38 brt (2.5) 1.21 m 1.39 me 1.39 me 1.46 m

13a

m m m m m m m

2.16 s

1.20 m 0.82 s 0.93 s 0.83 s 1.013 s 1.012 s 4.20 dd (1.5, 9.0) 3.78 d (9.0)

2.09 m 1.50 m 1.80 m

1.06 m 1.85 m 1.32 m 2.08 m 2.66 dt (5.0, 11.5)

1.67 m

1.38 1.40 1.48 1.21 1.07 0.98 1.57

1.37 m 1.20 m 1.93 m 1.53 m 3.38 brt (2.5) 1.20 m 1.40 me 1.40 me 1.47 m

14d

s s s s s s

2.37 s

0.83 0.94 0.89 1.14 0.96 1.28

2.28 d (2H, 8.0)

1.25 m 1.81 m 1.61 m

1.39 m 1.43 m 1.59 m 1.52 m 1.60 m 1.37 m 2.79 dd (2.5, 5.5) 1.89 m

1.28 m 1.23 m 1.95 m 1.56 m 3.41 brt (2.5) 1.24 m 1.45 m 1.38 m 1.45 m

15d

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2.07 s 2.07 s 4.13 d (14.5) 4.09 d (14.0) 2.07 s 4.02 d (14.5)

9.51 s 4.06 d (14.5)

4.14 d (14.5) 4.10 d (14.5) 0.96 0.97 d (6.5) 1.15 d (7.0) 1.05 d (7.0)

2.08 s

1.05 d (7.0)

OAc-3 OAc-28

30b

1.05 d (7.0) 30a

had one more hydroxy group compared to foliasalacin B2, and this additional group was located at C-7. The β-orientation of the C-7 hydroxy group was deduced from the NOESY correlation of H-7 with Me-27 (δH 1.03). Therefore, compound 6 was defined as (20R)-7β,29-dihydroxylupan-3-one. Compound 7 exhibited the same molecular formula, C30H50O3, as 6, based on the HRESIMS ion at m/z 481.3665 [M + Na]+ and the 13C NMR data. The 1H NMR and 13C NMR data for 7 revealed that it possessed the same skeleton as compound 6, except for a hydroxy group at C-15 in 7 instead of at C-7 in compound 6. This assignment was confirmed by the HMBC correlations from H-15 [δH 4.16 (1H, dd, J = 5.0, 11.0 Hz)] to C-8 (δC 42.3), C-14 (δC 48.1), C-16 (δC 46.5), and C27 (δC 8.1). The α-orientation of the C-15 hydroxy group was deduced from the NOESY correlations of H-15 with Me-26 (δH 1.17) and Me-28 (δH 0.80). Therefore, compound 7 was determined to be (20R)-15α,29- dihydroxylupan-3-one. Compounds 8 and 9 possessed the same molecular formula, C30H48O3, and their NMR spectroscopic data (Tables 1 and 2) were similar. Their IR spectra revealed the presence of hydroxy and carbonyl functionalities. The 1H NMR spectra indicated the presence of an oxymethine proton, a hydroxymethyl group, two vinyl protons, and six tertiary methyl groups. These data suggested that compounds 8 [H-29a, δH 4.94 (1H, d, J = 1.0 Hz), H-29b, 4.90 (1H, brs)] and 9 [H-29a, δH 4.96 (1H, d, J = 1.0 Hz), H-29b, 4.87 (1H, overlapped)] were lupane triterpenoids with a Δ20,29 double bond. Their 13C NMR spectra also confirmed the presence of a terminal double bond, a ketocarbonyl, a hydroxymethyl, and an oxymethine carbon. The NMR data of 8 and 9 resembled those for 16,8 except for the addition of a hydroxy group. The position and orientation of the C-7 hydroxy group in 8 [H-7, δH 3.87 (1H, dd, J = 6.5, 9.5 Hz)] and the C-15 hydroxy group in 9 [H-15, δH 4.13 (1H, dd, J = 5.0, 11.5 Hz)] were determined to be the same as those in 6 and 7, respectively, on the basis of their 1H−1H COSY, HSQC, HMBC, NOE, and NOESY spectra. Therefore, compounds 8 and 9 were determined to be 7β,30dihydroxylup-20(29)-en-3-one and 15α,30-dihydroxylup20(29)-en-3-one, respectively. Compound 10, obtained as a white, amorphous solid, possessed a molecular formula of C30H48O2 on the basis of its HRESIMS and 13C NMR data. The UV absorption band at 225 nm indicated the presence of an α,β-unsaturated formyl group. Analysis of the NMR data indicated that compound 10 and the known 3β-hydroxylup-20(29)-en-30-al7 possessed the same molecular structure. The difference between these compounds was the orientation of the C-3 hydroxy group. The small coupling constant (J = 2.8 Hz) between H-3 and H-2 and the NOE correlations of H-3 with Me-23 (δH 0.82) and Me-24 (δH 0.93) indicated that the C-3 hydroxy group was α-oriented. Interestingly, the 13C NMR spectrum of 10 (Supporting Information S78) displayed signals for only 29 carbon atoms. Using HSQC and HMBC experiments, we assigned the δC 36.9 resonance in the 13C NMR spectrum to C-19 and the δH 2.75 proton resonance (1H, dt, J = 6.0, 11.2 Hz) to H-19. The absence of a resonance for C-19 was most likely due to angularly dependent through-space effects, as reported by Reynolds and co-workers.7 Therefore, compound 10 was confirmed to be 3α-hydroxylup-20(29)-en-30-al. Compound 11 was obtained as a white, amorphous solid, and its molecular formula was determined to be C32H50O4 by 13 C NMR and HRESIMS data. It is 42 mass units higher than that of 3β,28,30-lup-20(29)-ene-triol.8 A comparison of the

Recorded in CDCl3 at 500 MHz. bRecorded in methanol-d4 at 500 MHz. cRecorded in CDCl3 at 400 MHz. dRecorded in CDCl3 at 600 MHz. eAverage chemical shift for unresolved CH2 protons. f Signals overlapped. Proton coupling constants (J) in Hz are given in parentheses. The assignments were based on 1H−1H COSY, HSQC, and HMBC experiments.

13a 12a 11a 10c 9b 8a 7a f

6a 5a 4a 3a 2a 1a no.

Table 1. continued

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a

14d

15d

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Table 2. 13C NMR Spectroscopic Data of Compounds 1−15 no.

1a

2a

3a

4a

5a

6a

7a

8a

9b

10c

11a

12a

13a

14d

15d

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 OCOCH3-3 OCOCH3-3 OCOCH3-28 OCOCH3-28

33.2 25.3 76.3 37.5 49.0 18.3 34.2 41.0 49.8 37.2 20.6 26.4 37.7 43.1 27.2 35.4 43.1 47.2 40.1 40.9 23.6 40.4 22.1 28.2 15.9 16.0 14.4 17.9 181.0 9.6

33.9 22.9 78.4 36.7 50.1 18.1 34.1 41.00 49.8 37.1 20.6 26.3 37.7 43.07 27.2 35.5 43.10 47.2 40.1 40.97 23.6 40.3 21.7 28.0 15.8 16.0 14.5 17.9 181.7 9.5 21.4 170.8

39.6 34.1 218.2 47.3 55.9 19.6 33.6 40.8 49.4 36.8 21.3 26.3 37.8 43.0 27.2 35.4 43.1 47.2 40.1 41.0 23.6 40.3 21.1 26.6 15.9 15.8 14.3 17.9 181.9 9.6

33.2 25.4 76.3 37.5 48.9 18.3 34.2 41.1 49.8 37.2 20.8 27.0 37.7 43.2 27.3 35.4 43.2 48.6 43.5 42.0 23.8 39.7 22.2 28.2 15.9 16.0 14.5 17.8 181.3 17.1

33.3 25.4 76.2 37.5 49.0 18.3 34.2 41.1 49.8 37.3 20.7 27.1 37.6 43.0 27.2 35.2 43.2 47.0 46.1 68.7 21.3 40.2 22.2 28.2 15.9 16.0 14.6 18.3 23.0

39.3 34.1 217.3 47.0 52.1 30.3 74.0 46.7 49.3 36.8 21.4 27.2 38.4 44.6 31.2 35.8 42.8 47.3 43.7 38.1 23.1 40.3 21.0 26.4 15.5 10.0 14.8 17.5 64.4 18.1

39.9 34.2 218.2 47.2 54.5 19.8 37.0 42.3 49.8 37.0 21.6 27.2 37.72 48.1 69.6 46.5 43.0 47.2 43.0 37.75 23.4 39.8 21.0 26.7 16.0 16.3 8.1 18.8 64.4 18.0

39.3 34.1 217.2 47.0 52.1 30.4 74.0 46.7 49.6 36.8 21.5 26.7 38.5 44.3 31.3 35.8 42.7 48.7 43.9 154.7 31.7 39.9 21.0 26.4 15.6 10.0 14.8 17.6 106.9 65.0

41.1 35.1 221.2 48.3 55.8 21.0 37.9 43.5 51.8 38.3 23.0 28.1 39.4 49.5 70.3 47.2 44.0 49.6 44.4 155.7 33.0 40.6 21.4 27.3 16.3 16.6 8.5 19.2 107.3 65.1

33.4 25.5 76.3 37.6 49.1 18.4 34.3 41.1 50.2 37.4 20.9 27.8 37.9 42.9 27.4 35.5 43.4 51.3 36.9 157.4 32.7 40.0 22.2 28.2 15.9 16.0 14.6 17.9 132.8 194.5

33.9 22.9 78.1 36.7 50.2 18.1 34.0 41.1 50.1 37.2 20.8 26.8 37.1 42.8 27.0 29.2 47.8 49.4 43.4 154.7 31.8 33.8 21.7 27.8 16.0 15.0 15.9 60.3 106.9 65.1 21.4 170.5

33.8 22.8 78.2 36.7 50.2 18.1 33.9 41.0 50.0 37.2 20.7 27.2 36.2 42.7 27.0 28.9 47.8 49.6 52.1 212.1 27.7 33.9 21.7 27.8 16.0 15.85 15.93 60.5 29.3

33.2 25.3 76.0 37.3 49.0 18.2 34.0 41.2 50.0 37.6 20.7 27.2 36.2 42.6 26.9 28.8 47.8 49.8 52.2 212.2 27.6 33.9 22.1 28.1 15.87 15.91 14.8 60.4 29.3

33.2 25.4 76.2 37.5 48.9 18.2 33.9 40.9 50.0 37.3 20.6 27.3 36.4 42.6 26.9 29.4 46.4 49.5 51.7 211.8 27.5 34.4 22.1 28.2 15.8 16.0 14.8 62.5 29.7

33.4 25.4 76.0 37.5 49.0 18.2 34.4 41.5 50.5 37.4 20.5 26.6 41.0 46.7 27.3 36.8 43.0 184.2 142.2 203.8 204.3 51.8 22.1 28.2 16.3 16.4 15.7 25.0 32.5

21.4 170.8 21.0 171.6

a Recorded in CDCl3 at 125 MHz. bRecorded in methanol-d4 at 125 MHz. cRecorded in CDCl3 at 100 MHz. dRecorded in CDCl3 at 150 MHz. The assignments were based on DEPT, 1H−1H COSY, HSQC, and HMBC experiments.

the presence of an acetoxy group in 11. The acetoxy group was located at C-3 on the basis of the HMBC correlations from H-3 [δH 4.61 (1H, t, J = 2.5 Hz)] to C-1 (δC 33.9), C-5 (δC 50.2), C-23 (δC 21.7), C-24 (δC 27.8), and an acetoxy carbonyl carbon (δC 170.5). In the NOE experiment, the NOE correlations of H-3 with Me-23 and Me-24 as well as the small coupling constant (J = 2.5 Hz) between H-3 and H-2 were used to assign an α-orientation to the C-3 acetoxy group. Therefore, compound 11 was determined as 3α-acetoxy-28,30dihydroxylup-20(29)-ene. Compound 12 possessed a molecular formula of C31H50O4 according to its HRESIMS (m/z 509.3604 [M + Na]+, calcd for 509.3601) and 13C NMR data. The 1H and 13C NMR data for 12 (Tables 1 and 2) were similar to those for 24,15 with the main difference being that the C-28 methyl group in 24 was changed into a hydroxymethyl group [δH 3.78 (1H, d, J = 10.5 Hz), 3.24 (1H, d, J = 11.0 Hz)]. This assignment was based on the HMBC correlations from H-28 to C-16 (δC 28.9), C-17 (δC 47.8), and C-22 (δC 33.9). The other difference between these two compounds was that the C-3 hydroxy group in 24 was replaced by an acetoxy group in 12, which was based on the HMBC correlations of H-3 (δH 4.61, t, J = 2.5 Hz) with C-1 (δC 33.8), C-4 (δC 36.7), C-5 (δC 50.2), C-23 (δC 21.7), C-24 (δC 27.8), and an acetoxy carbonyl carbon (δC 170.8). In

Figure 1. Key 1H−1H COSY, HMBC, and NOE correlations of compound 1.

NMR data of 11 (Tables 1 and 2) with those of the latter compound indicated that the two compounds differed due to 280

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Figure 2. X-ray crystal structure of compound 1.

Compound 15 was isolated as a white, amorphous solid. Its molecular formula was determined as C29H44O3 on the basis of the 13C NMR data and [M + H]+ peak at m/z 441.3375 (calcd for C29H45O3, 441.3363) in its HRESIMS spectrum, which indicated eight indices of hydrogen deficiency. The IR spectrum indicated the presence of hydroxy (3433 cm−1) and α,βunsaturated carbonyl groups (1690 cm−1). The 1H and 13C NMR data (Tables 1 and 2) indicated seven methyl [δH 0.83, 0.89, 0.94, 0.96, 1.14, 1.28, and 2.37 (each 3H, s)] and nine methylene groups, four methines (one oxygenated), a carbonyl group (δC 203.8), and an α,β-unsaturated carbonyl [δC 142.2 (C-18), 184.2 (C-19), and 204.3 (C-21)]. The molecular structure of 15 was determined from the combined 1D and 2D NMR data in comparison with the spectroscopic data for 24,15 which indicated that 15 had a C-30 norlupan skeleton bearing an α-oriented C-3 hydroxy group and an α,β-unsaturated carbonyl moiety at C-18−19(21). Thus, the structure of 15 was assigned as 3α-hydroxy-30-norlupan-18(19)-ene-20,21-dione. The 10 known compounds, 30-hydroxylup-20(29)-en-3-one (16),9 28-hydroxylup-20(29)-en-3-one (17),9 3-epi-lupeol (18),10 lup-20(30)-ene-3α,29-diol (19),11 3α,28-dihydroxylup20(29)-ene (20),12 lupeol (21),13 lup-20(29)-ene-3β,30-diol (22),14 betulin (23),9 obibanmol I (24),15 and 30-norlupan-3βol-20-one (25),16 were identified by analysis of their spectroscopic data and through comparison of these data with the literature values. Compounds 1−25 were tested for their cytotoxic activity against five human cancer cell lines (HCT-8, Bel-7402, BGC823, A549, and A2780). Only compound 11 exhibited moderate cytotoxicity for the HCT-8 (IC50 3.21 ± 0.36 μM), Bel-7402 (IC50 2.77 ± 0.45 μM), BGC-823 (IC50 4.09 ± 0.54 μM), and A549 (IC50 3.35 ± 0.26 μM) cell lines (Table 3). The

addition, the relative configuration of compound 12 was identical to that of 24 on the basis of their NOE correlations and proton coupling constants. This evidence permits definition of the structure of 12 as 3α-acetoxy-28-hydroxy-30norlupan-20-one. The molecular formula of compound 13 was assigned as C29H48O3 on the basis of the 13C NMR and HRESIMS data. The molecular mass is thus 42 mass units less than that of 12. Analysis of the spectroscopic data indicated that 13 shared the same skeleton as 12. Comparison of the chemical shift of H-3 [δH 3.38 (1H, brt, J = 2.5 Hz)] for 13 with that of 12 (δH 4.61) suggested that the C-3 acetoxy group in 12 was replaced by a hydroxy group in 13. This assignment was verified by the HMBC correlations of H-3 with C-1 (δC 33.2), C-4 (δC 37.3), C-5 (δC 49.0), C-23 (δC 22.1), and C-24 (δC 28.1) and was further confirmed by the deshielded shift of C-3 from δC 76.0 in 13 to δC 78.2 in 12. The NOE correlations from H-3 to Me-23 and Me-24 as well as the small coupling constant (J = 2.5 Hz) between H-3 and H-2 were used to assign an α-orientation to the C-3 hydroxy group. Therefore, the structure of 13 was determined to be 3α,28-dihydroxy-30-norlupan-20-one. The HRESIMS and 13C NMR data of compound 14 revealed a molecular formula of C31H50O4. The 2D NMR data for 14 exhibited a pattern similar to that of 13, except for the presence of an additional acetyl group. The acetyl group was linked to C28, as determined from the key HMBC correlations of H-28 [δH 4.20 (1H, dd, J = 1.5, 9.0), 3.78 (1H, d, J = 9.0 Hz)] with the ester carbonyl carbon (δC171.6). The relative configurations of both 14 and 13 were identical according to the NOE correlations and proton coupling constants. Therefore, the structure of compound 14 was determined to be 3αhydroxy-28-acetoxy-30-norlupan-20-one. 281

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Table 3. Cytotoxic Activities of Compound 11 against Five Human Cancer Linesa IC50 (μM) compound

HCT-8

Bel-7402

BGC-823

A549

A2780

11 Taxolb

3.21 ± 0.36 0.020 ± 0.006

2.77 ± 0.45 0.011 ± 0.002

4.09 ± 0.54 0.026 ± 0.006

3.35 ± 0.26 0.032 ± 0.003

>10 μM 0.59 ± 0.11

Results are reported as the mean ± SD based on three independent experiments. The other compounds were inactive (IC50 > 10 μM). bPositive control. a

Table 4. Inhibitory Effects of Compounds 1−25 against LPS-Induced NO Production in Murine Microglia BV2 Cellsa compound 1 2 4 5 a b

IC50 (μM) 7.39 7.48 7.80 3.48

± ± ± ±

0.62 0.83 0.56 0.43

cell viabilityb

compound

IC50 (μM)

cell viabilityb

± ± ± ±

20 25 curcuminc

2.54 ± 0.42 6.09 ± 0.75 0.53 ± 0.08

89.3 ± 2.3 93.5 ± 3.8 93.3 ± 2.9

87.3 98.5 96.8 94.2

1.6 4.3 3.5 1.8

Results are reported as the mean ± SD based on three independent experiments. Compounds 3, 6−19, and 21−24 were inactive (IC50 > 10 μM). Cell viability is expressed as a percentage (%) of the LPS-only treatment group. cPositive control.

other compounds were inactive (IC50 > 10 μM). These compounds were also evaluated for their inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine microglia BV2 cells. As shown in Table 4, compounds 1, 2, 4, 5, 20, and 25 showed moderate abilities to inhibit NO production and had no influence on cell viability, as determined by the MTT method. The remaining compounds exhibited weak effects (IC50 > 10 μM). The data indicated that a hydroxy or acetoxy group at C-3 may play an important role in modulating inhibition of NO production. The presence of a C-3 carbonyl group (compounds 3 and 17) may decrease the inhibitory activities against NO production in murine microglia BV2 cells.

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The petroleum ether extract (180 g) was subjected to column chromatography on silica gel (200−300 mesh, 3.6 kg) using a stepwise gradient elution of petroleum ether−EtOAc (50:1, 20:1, 10:1, 6:1, 4:1, 2:1, 1:2, and 0:1, v/v) to afford eight subfractions (P1−P8). Fraction P2 (6.1 g) was chromatographed over a Sephadex LH-20 column using petroleum ether−CHCl3−MeOH (5:5:1) as a mobile phase to afford five fractions (P2a−P2e). Separation of fraction P2b (2.6 g) on an RP-18 column (ODS, 50 μm, YMC) with MeOH−H2O (65−100%) followed by preparative HPLC using CH3CN−H2O (95:5, v/v) as the mobile phase yielded 2 (8 mg), 14 (7 mg), 18 (17 mg), and 21 (14 mg). Fraction P5 (17.1 g) was purified by macroporous resin PRP512 column chromatography by eluting with 95% EtOH and subsequently chromatographed on a silica gel column (200−300 mesh, 340 g) eluted with n-hexane−EtOAc gradients (from 20:1 to 5:1, v/v) to obtain five subfractions (P5a−P5e). Fraction P5a (2.5 g) was purified using an RP-18 column (ODS, 50 μm, YMC) with MeOH− H2O (60−100%) followed by preparative HPLC using CH3CN−H2O (80:20, v/v) to yield 16 (21 mg), 17 (25 mg), 20 (16 mg), 23 (20 mg), 24 (40 mg), and 25 (20 mg). Fraction P7 (14.5 g) was also passed through a macroporous resin PRP-512 column with 95% EtOH and subjected to an RP-18 column (ODS, 50 μm, YMC) with MeOH−H2O (60−100%) to yield 13 subfractions (P7a−P7l). Compound 8 (5.4 mg) was obtained from fraction P7d (120 mg) by preparative HPLC using CH3CN−H2O (65:35, v/v) as the mobile phase. Fraction P7f (150 mg) was subjected to preparative HPLC using CH3CN−H2O (65:35, v/v) to afford compounds 11 (15 mg), 12 (16 mg), and 15 (1.1 mg). Fraction P7h (510 mg) was purified by preparative HPLC using MeOH−H2O (90:10, v/v) to yield compounds 1 (37 mg), 3 (26 mg), 19 (105 mg), and 22 (70 mg). Fraction P7j (460 mg) was further separated by preparative HPLC using CH3CN−H2O (90:10, v/v) to yield compounds 4 (20 mg), 5 (14 mg), and 10 (9.3 mg). The CHCl3 extract (102 g) was fractionated via silica gel CC (200− 300 mesh, 2.5 kg) and was eluted with CHCl3−MeOH (20:1) to afford seven fractions (C1−C7) based on TLC analysis. Fraction C7 (10.0 g) was subjected to column chromatography over silica gel (200−300 mesh, 200 g) eluted with petroleum ether−acetone (from 9:1 to 1:1, v/v) to afford subfractions C7a−C7h. Fraction C7b (1.0 g) was separated by column chromatography on silica gel (200−300 mesh, 25 g) eluted with a petroleum ether−EtOAc system (6:1) and was further purified via preparative HPLC using CH3CN−H2O (60:40, v/v) to afford 6 (2.0 mg), 7 (2.5 mg), 9 (1.3 mg), and 13 (5.3 mg). (20S)-3α-Hydroxylupan-29-oic acid (1): colorless needles (CDCl3); mp 215− 218 °C; [α]20D +13 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 203 (3.19) nm; IR (microscope) νmax 3427, 3260, 2956, 2607, 1727, 1694, 1457, 1385, 1243, 1038, 990 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on an XT5B microscopic melting point apparatus and are uncorrected. Optical rotations were measured on a JASCO P2000 automatic digital polarimeter. The UV spectra were obtained on a JASCO V-650 spectrophotometer, and the IR data were recorded on a Nicolet 5700 spectrometer using an FT-IR microscope transmission method. The NMR spectra were acquired on Mercury-400, INOVA500, Bruker AV500-III, and Bruker AV600-III spectrometers. HREIMS data were collected with an AutoSpec Ultima-TOF MS spectrometer. HRESIMS spectra were obtained on an Agilent 1100 series LC/MSD ion trap mass spectrometer. Preparative HPLC was performed on a Shimadzu LC-6AD instrument with an SPD-20A detector and a YMCPack ODS-A column (250 × 20 mm, 5 μm). Silica gel (200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China), macroporous resin PRP-512 (Beijing Jufu Resin Inc., Beijing, People’s Republic of China), and ODS (50 μm, YMC, Japan) were used for column chromatography (CC). TLC was performed using glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with 10% H2SO4 in 95% EtOH followed by heating. Plant Material. The stems of E. carnosus were collected in Jiujiang, Jiangxi Province, China, in April 2011 and were identified by Professor Ce-ming Tan from the Jiujiang Botany Institute of Forest. A voucher specimen (No. ID-22158) was deposited at the Herbarium of the Institute of Material Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, China. Extraction and Isolation. The air-dried stems of E. carnosus (15 kg) were crushed to a coarse solid and refluxed with 95% EtOH (120 L × 2 h × 3). After evaporation of the EtOH in vacuo, the aqueous residue (700 g) was diluted with H2O and successively partitioned with petroleum ether (3 L × 4), CHCl3 (3 L × 4), EtOAc (3 L × 4), and n-BuOH (3 L × 4). 282

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Tables 1 and 2; HREIMS m/z 458.3778 [M]+ (calcd for C30H50O3, 458.3760). (20S)-3α-Acetoxylupan-29-oic acid (2): white, amorphous solid; [α]20D +5 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 203 (3.41) nm; IR (microscope) νmax 2942, 2860, 1739, 1709, 1458, 1374, 1245, 1180, 1035, 984 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Tables 1 and 2; HRESIMS m/z 523.3754 [M + Na]+ (calcd for C32H52NaO4, 523.3758). (20S)-3-Oxolupan-29-oic acid (3): white, amorphous solid; [α]20D +32 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 203 (3.36) nm; IR (microscope) νmax 2943, 2627, 1710, 1453, 1387, 1207, 1141, 1114, 1073,987, 919 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Tables 1 and 2; HREIMS m/z 456.3611 [M]+ (calcd for C30H48O3, 456.3603). (20R)-3α-Hydroxylupan-29-oic acid (4): white, amorphous solid; [α]20D −61 (c 0.25, CHCl3); UV (MeOH) λmax (log ε) 203 (2.55) nm; IR (microscope) νmax 3395, 2936, 2863, 1703, 1460, 1382, 1235, 1210, 1068, 990 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Tables 1 and 2; HREIMS m/z 458.3758 [M]+ (calcd for C30H50O3, 456.3760). (20R)-30-Norlupane-3α,20-diol (5): white, amorphous solid; [α]20D −40 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 203 (3.39) nm; IR (microscope) νmax 3431, 2949, 2866, 2852, 1739, 1456, 1382, 1127, 1068, 1043, 988 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Tables 1 and 2; HREIMS m/z 430.3801 [M]+ (calcd for C29H50O2, 430.3811). (20R)-7β,29-Dihydroxylupan-3-one (6): white, amorphous solid; [α]20D −45 (c 0.09, CHCl3); UV (MeOH) λmax (log ε) 204 (3.31) nm; IR (microscope) νmax 3420, 2947, 2870, 1701, 1595, 1459, 1383, 1250, 1203, 1121, 1024, 965 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Tables 1 and 2; HRESIMS m/z 481.3665 [M + Na]+ (calcd for C30H50NaO3, 481.3652). (20R)-15α,29-Dihydroxylupan-3-one (7): white, amorphous solid; [α]20D −22 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 203 (3.43) nm; IR (microscope) νmax 3508, 3433, 2944, 2847, 1702, 1598, 1450, 1385, 1039, 1019, 968 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Tables 1 and 2; HRESIMS m/z 481.3674 [M + Na]+ (calcd for C30H50NaO3, 481.3652). 7β,30-Dihydroxylup-20(29)-en-3-one (8): white, amorphous solid; [α]20D −19 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 204 (3.95) nm; IR (microscope) νmax 3511, 2943, 2869, 1721, 1696, 1458, 1382, 1266, 1113, 1100, 1037, 1027 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Tables 1 and 2; HRESIMS m/z 479.3496 [M + Na]+ (calcd for C30H48NaO3, 479.3496). 15α,30-Dihydroxylup-20(29)-en-3-one (9): white, amorphous solid; [α]20D −10 (c 0.3, CHCl3); UV (MeOH) λmax (log ε) 204 (3.79) nm; IR (microscope) νmax 3403, 2947, 2869, 1701, 1649, 1460, 1384, 1111, 1058, 1012, 967 cm−1; 1H NMR (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125 MHz), see Tables 1 and 2; HRESIMS m/z 479.3501 [M + Na]+ (calcd for C30H48NaO3, 479.3496). 3α-Hydroxylup-20(29)-en-30-al (10): white, amorphous solid; [α]20D −13 (c 0.3, CHCl3); UV (MeOH) λmax (log ε) 225 (3.67) nm; IR (microscope) νmax 3505, 2942, 2866, 2697, 1690, 1617, 1458, 1382, 1232, 1068, 990, 938 cm−1; 1H NMR (CDCl3, 400 MHz) and 13 C NMR (CDCl3, 100 MHz), see Tables 1 and 2; HRESIMS m/z 463.3555 [M + Na]+ (calcd for C30H48NaO2, 463.3547). 3α-Acetoxy-28,30-dihyroxylup-20(29)-ene (11): white, amorphous solid; [α]20D −27 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 204 (3.67) nm; IR (microscope) νmax 3407, 2946, 2869, 1714, 1648, 1456, 1376, 1248, 1181, 1031, 984 cm−1; 1H NMR (CDCl3, 500 MHz) and 13 C NMR (CDCl3, 125 MHz), see Tables 1 and 2; HRESIMS m/z 523.3775 [M + Na]+ (calcd for C32H50NaO4, 523.3758). 3α-Acetoxy-28-hydroxy-30-norlupan-20-one (12): white, amorphous solid; [α]20D −50 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 203 (3.08) nm; IR (microscope) νmax 3470, 2945, 2868, 1714, 1458, 1375, 1249, 1180, 1033, 984 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Tables 1 and 2; HRESIMS m/z 509.3604 [M + Na]+ (calcd for C31H50NaO4, 509.3601).

3α,28-Dihydroxy-30-norlupan-20-one (13): white, amorphous solid; [α]20D −38 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 203 (2.70) nm; IR (microscope) νmax 3575, 3463, 2941, 2867, 1702, 1458, 1378, 1354, 1167, 1034, 991 cm−1; 1H NMR (CDCl3, 500 MHz) and 13 C NMR (CDCl3, 125 MHz), see Tables 1 and 2; HREIMS m/z 444.3622 [M]+ (calcd for C29H48O3, 444.3603). 3α-Hydroxy-28-acetoxy-30-norlupan-20-one (14): white, amorphous solid; [α]20D −15 (c 0.05, CHCl3); UV (MeOH) λmax (log ε) 204 (3.51) nm; IR (microscope) νmax 3431, 2942, 2864, 1737, 1717, 1460, 1378, 1237, 1071, 1033, 991 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Tables 1 and 2; HRESIMS m/z 509.3614 [M + Na]+ (calcd for C31H50NaO4, 509.3601). 3α-Hydroxy-30-norlupan-18(19)-ene-20,21-dione (15): white, amorphous solid; [α]20D −49 (c 0.02, CHCl3); UV (MeOH) λmax (log ε) 202 (3.94), 237 (4.01) nm; IR (microscope) νmax 3433, 2937, 2867, 1708, 1690, 1620, 1453, 1383, 1352, 1171, 1070, 994 cm−1; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Tables 1 and 2; HRESIMS m/z 441.3375 [M + H]+ (calcd for C29H45O3, 441.3363). X-ray Crystallographic Analysis of Compound 1. C30H50O3· 2.5CDCl3, M = 757.12, orthorhombic, space group, P212121, a = 10.7324(5) Å, b = 23.7833(14) Å, c = 28.8248(12) Å; α = β = γ = 90.00°, V = 7357.6(6) Å3, Z = 8, μ(Cu Kα) = 5.509 mm−1, ρcalc = 1.367 g·cm−3, F(000) = 3192, 37 856 reflections independent and 14 223 reflections observed (|F|2 ≥ 2σ|F|2). R1 = 0.0636, wR2 = 0.1512, Flack parameter = −0.008(14). Colorless crystals of 1 were crystallized from CDCl3. The intensity data were collected on an Agilent Xcalibur Eos Gemini diffractometer equipped with a Cu Kα radiation source. The crystal structure of 1 was solved by direct methods (SHELXS-97), expanded using a difference Fourier technique, and refined by the SHELXS-97 program and the full-matrix least-squares calculations. All non-hydrogen atoms were refined anisotropically, and all of the hydrogen atoms were fixed at calculated positions. The crystallographic data for compound 1 have been deposited at the Cambridge Crystallographic Data Centre (deposition number: CCDC 961233). Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk (or e-mail: deposit@ ccdc.cam.ac.uk). Cytotoxicity Assay. The cytotoxicity of compounds 1−25 against a set of human cancer cell lines that included Bel-7402 (human liver carcinoma), BGC-823 (human stomach carcinoma), A549 (human lung carcinoma), and A2780 (human ovarian carcinoma) was assessed using the MTT assay.17 The cells were maintained in RPMI 1640 medium, supplemented with 10% fetal bovine serum, penicillin (100 UI/mL), and streptomycin (100 μg/mL) in a 37 °C incubator under an atmosphere of 5% CO2. The cells were plated at a final concentration of 2 × 104 cells per/well in a 96-well cell culture plate for 24 h. Each tumor cell line was subsequently treated with 100 μL of a DMSO solution of the samples at various concentrations in three parallel wells. After treatment for 96 h, cell growth was evaluated using an MTT assay procedure. A 100 μL MTT solution (500 μg/mL) was added to each well, and the plate was incubated for 4 h at 37 °C. The absorbance at 570 nm was measured using a microplate reader after the formazan crystals had been dissolved with 150 μL of DMSO. Taxol was used as a positive control. The IC50 value was defined as the concentration of a sample that caused 50% inhibition of in vitro cell growth. Inhibitory Effects on NO Production in LPS-Activated Microglia. Compounds 1−25 were tested for their ability to inhibit LPS-activated NO production in the BV2 cell line. The murine microglial BV2 cells were obtained from the Cell Culture Centre at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and LPS (from Escherichia coli 055:B5) was purchased from Sigma-Aldrich. The BV2 cells were plated into a 96-well plate. After being preincubated for 24 h, the cells were pretreated with various concentrations of isolated compounds and stimulated with 300 ng/mL LPS for an additional 24 h. Nitrite, which is a soluble oxidation product of NO, was determined in the culture supernatant using the Griess reaction. NaNO2 was used as a standard to assay the NO2− 283

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concentration. The OD value of the samples at 550 nm was measured. Cell viability was assessed using an MTT assay. Curcumin was used as the positive control.18

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ASSOCIATED CONTENT

* Supporting Information S

Copies of spectra of compounds 1−15 and X-ray crystallographic data of compound 1 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86-10-63165227. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the Department of Instrumental Analysis, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, for the measurement of the UV, IR, NMR, MS, HREIMS, and HRESIMS spectra. This research program is financially supported by the National Natural Science Foundation of China (No. 21132009), National Science and Technology Project of China (2011ZX09307-002-01), and the Program for Changjiang Scholars and Innovative Research Team in University of the Ministry of Education of China (IRT1007).

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REFERENCES

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