Lithocarpic Acids AâN, 3,4-seco-Cycloartane ... - ACS Publications

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Lithocarpic Acids A−N, 3,4-seco-Cycloartane Derivatives from the Cupules of Lithocarpus polystachyus Hongmin Wang,†,‡ Ruonan Ning,†,‡ Yu Shen,§ Zhenhua Chen,† Jinlong Li,† Rujun Zhang,† Ying Leng,*,§ and Weimin Zhao*,† †

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China

§

S Supporting Information *

ABSTRACT: Fourteen new 3,4-seco-cycloartane-type triterpenes, lithocarpic acids A−N (1−14), together with one known compound, coccinetane E (15), were identified from the cupules of Lithocarpus polystachyus. The structures of 1−14 were determined by spectroscopic data analysis and chemical methods, and the absolute configurations of 1 and 4 were defined unequivocally by X-ray crystallography using Cu Kα radiation. Compounds 1−15 are the first examples of 3,4-seco-cycloartane derivatives isolated from the genus Lithocarpus. Among them, compounds 1 and 2, 9 and 10, and 11 and 12 were found to be three pairs of C-24 epimers, while compounds 7 and 8 represent the first examples of 3,4-seco-norcycloartane-type triterpenes. Compound 1, as the major component of the plant extract, showed potent antibacterial activity against Micrococcus luteus and Bacillus subtilis, with MIC values of 3.1 and 6.3 μg/mL, respectively, as well as inhibitory activity against human and mouse 11β-hydroxysteroid dehydrogenase type 1, with IC50 values of 1.9 and 0.24 μM, respectively.

T

weights not yet reported from this genus. Phytochemical investigation on the nonaromatic components of the EtOH extract of this plant part resulted in the identification of 14 new 3,4-seco-cycloartane derivatives (1−14), named lithocarpic acids A−N, along with one known compound, coccinetane E (15). This is the first report of 3,4-seco-cycloartane derivatives isolated from the genus Lithocarpus. Bioassay of selected isolates obtained from this work indicated that the major component of the extract, compound 1, showed potent antibacterial activity against Micrococcus luteus and Bacillus subtilis, with MIC values of 3.1 and 6.3 μg/mL, respectively, as well as inhibitory activity against human and mouse 11βhydroxysteroid dehydrogenase type 1 (11β-HSD1), with IC50 values of 1.9 and 0.24 μM, respectively.

he genus Lithocarpus, belonging to the beech family, Fagaceae, consists of more than 300 species distributed primarily in east and southeast Asia,1 and approximately 130 species are grown in the mountainous regions of southern mainland China.2 Plants of the genus Lithocarpus have an extensive history of use in medicine and as foods.3 In particular, the leaves of Lithocarpus polystachyus Rehder, known as a sweet tea, have been used popularly as a beverage and a traditional medicine to prevent or treat a variety of diseases.4 The sweettasting tea from this plant is claimed to possess multiple bioactivities, including antihypertensive, antiatherosclerotic, vasodilatory, cerebrovascular, hypoglycemic, and antiobesity effects.5−8 Previous phytochemical studies on the leaves of L. polystachyus have resulted in the identification of triterpenoids9−12 and dihydrochalcones such as phloridzin, trilobatin, and 3-hydroxyphloridzin.13 Phloridzin, a well-known sodiumglucose cotransporter (SGLT) inhibitor,14 occurs in the dried leaves of L. polystachyus with up to 7% of the content.15 On the basis of the structure of phloridzin and its pharmacological target, canagliflozin16 and dapaliflozin,17 two selective SGLT-2 inhibitors, have recently been approved by the FDA and EMA as members of a new generation of antidiabetic drugs, while empagliflozin has been granted approval by the EMA,18 and ipragliflozin received approval in Japan in January 2014.19 In a search for natural products with new structures and interesting biological effects, tandem LC-DAD-MS analysis of different parts of L. polystachyus revealed the presence of nonaromatic components in the cupules with molecular © 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The air-dried cupules of L. polystachyus (500 g) were powdered and percolated with 95% EtOH (3 L × 3) at room temperature. After evaporation of the solvent in vacuo, the residue was separated into six fractions by silica gel column chromatography. Repeated column chromatography of fractions 2 and 3 over silica gel (300−400 mesh), MCI gel, and semipreparative HPLC afforded 14 new 3,4-seco-cycloartane derivatives, named lithocarpic acids A−N (1−14), along with one known compound, coccinetane E (15). Received: May 4, 2014 Published: August 6, 2014 1910

dx.doi.org/10.1021/np500379f | J. Nat. Prod. 2014, 77, 1910−1920

Journal of Natural Products

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H-21β and H-21α/H-24 (Figure 2). Crystallization of 1 in CH3CN with a trace of water afforded a high-quality crystal suitable for X-ray diffraction analysis. The X-ray data confirmed the structure of 1 assigned by spectroscopic methods and also established its absolute configuration (Figure 3). Accordingly, 1 was characterized as (5S,8R,9S,10R,13R,14S,17R,20R,24R)21,24-epoxy-25-hydroxy-3,4-seco-cycloart-4(28)-en-3-oic acid and has been named lithocarpic acid A. Lithocarpic acid B (2) was found to share the same molecular formula as that of 1. The 1H and 13C NMR spectra of these two compounds were closely comparable (Tables 1 and 2), but their major differences were associated with signals of the side chain linked at C-17. Detailed analysis of the 1H−1H COSY, HSQC, and HMBC spectra of 2 also revealed the presence of a C-21, C-24 ether bond and a C-25 hydroxy group, suggesting 2 to be the C-24 isomer of 1. The coupling constants of H-21α (dd, J = 11.4, 2.0 Hz), H-21β (d, J = 11.4 Hz), and H-24 (dd, J = 11.0, 2.5 Hz) along with NOE correlations between H-21β and H-20/H-24 indicated that H20 and H-24 are oriented equatorially and axially, respectively (Figure 2). Further analysis of its ROESY spectrum revealed the configurations of C-5, C-8, C-9, C-10, C-13, C-14, and C17 in 2 to be consistent with those of the cycloartane skeleton, according to NOE correlations between H3-30 and H-5 and H17 and between H-8 and H3-18 and H-19β. Accordingly, 2 (lithocarpic acid B) was identified as (5S,8R,9S,10R,13R,14S,17R,20R,24S)-21,24-epoxy-25-hydroxy-3,4-seco-cycloart-4(28)en-3-oic acid. Lithocarpic acid C (3) was obtained as colorless orthorhombic crystals. Its molecular formula was determined to be C30H48O5 based on the quasimolecular ion at m/z 523.3187 [M + Cl]− (calcd for C30H48O5Cl, 523.3190) in the HRESIMS, indicating seven degrees of unsaturation. The 1H NMR spectrum of 3 again exhibited signals characteristic of a 3,4-seco-cycloartane derivative (Tables 1 and 2). Analysis of its 1 H−1H COSY spectrum revealed a C-21/C-20/C-22/C-23/C24 spin coupling system, in which C-21, C-23, and C-24 are oxygenated. In its HMBC spectrum, 1H−13C long-range correlation signals between H-21 and C-17, C-22, and C-23 and between H-23 and C-20, C-21, and C-24 indicated the presence of a C-21, C-23 ether bridge in 3 (Figure 4). The presence of a hydroxy group at C-25 was demonstrated by the chemical shift at δC 73.8 along with 1H−13C long-range correlations between C-25 and H-24, H-26, and H-27. In its ROESY spectrum, NOE correlations from H-8 to H3-18 and H19β, from H3-30 to H-5 and H-17, from H3-18 to H-20 and H21β, and from H-17 to H-21α indicated the same relative configuration of 3 as 1 at C-5, C-8, C-9, C-10, C-13, C-14, C17, and C-20. The assignment of the configuration of C-23 using NOE correlations was challenging, as the 1H NMR signals of H-20 (δH 2.19 m) and H-22β (δH 2.19 m), and of H21β (δH 3.97 m) and H-23 (δH 3.98 m), were overlapped. However, crystals suitable for X-ray diffraction studies were obtained in acetone. The subsequent X-ray analysis permitted assignment of the structure and relative configuration of 3 (lithocarpic acid C) as (5S*,8R*,9S*,10R*,13R*,14S*,17R*,20R*,23S*,24S*)-24,25dihydroxy-21,23-epoxy-3,4-seco-cycloart-4(28)-en-3-oic acid (Figure 5).20 Lithocarpic acid D (4) was isolated as colorless orthorhombic crystals. Its HRESIMS gave a [M + Na] + ion at m/z 497.3599, indicating a molecular formula of C30H50O4, with six degrees of unsaturation. The 1H and 13C NMR data (Tables 1

Lithocarpic acid A (1) was obtained as colorless monoclinic crystals with a molecular formula of C30H48O4 based on the [M + Na]+ ion peak at m/z 495.3445 (calcd 495.3450) in the HRESIMS, suggesting seven degrees of unsaturation. In its 1H NMR spectrum, the signals of a pair of terminal olefinic protons [δH 4.81, 4.73 (each 1H, br s)], a vinyl methyl [δH 1.68 (3H, s)], four tertiary methyls [δH 1.17, 1.13, 1.00, 0.91 (each 3H, s)], and a pair of cyclopropyl methylene protons [δH 0.73, 0.43 (each 1H, J = 4.4 Hz)] were observed (Table 1). The 13C NMR spectrum of 1 exhibited 30 carbon signals, consisting of a terminal alkene (δC 149.5 s, 111.8 t), a carboxylic acid group (δC 179.1 s), an oxygenated methylene (δC 72.8 t), an oxygenated methine (δC 84.2 d), and an oxygenated quaternary carbon (δC 72.1 s), together with five methyls, 11 methylenes, four methines, and four quaternary carbons (Table 2). Interpretation of the 1H−1H COSY, TOCSY, and HSQC spectra of 1 led to the assignment of five isolated spin systems [S1: C-1/C-2; S2: C-5/C-6/C-7/C-8; S3: C-11-C-12; S4: C15/C-16/C-17/C-20/(C-21)/C-22/C-23/C-24; S5: C-28/C4/C-29] in its structure (Figure 1). In the HMBC spectrum of 1, 1H−13C long-range correlation signals between H2-19 and C1, C-5, C-8, and C-11 revealed linkages of S1, S2, and S3, while connections of S2, S3, and S4 were deduced from correlation signals between H3-18 and C-12, C-14, and C-17 and between H3-30 and C-8, C-13, and C-15. The 1H−13C long-range correlation signal between H-1 and C-3 supported the attachment of the carboxylic acid group to C-2. In turn, the correlation signal between H-28 and C-5 indicated the location of the isopropenyl moiety S5 at C-5, and that between H2-21 and C-20, C-22, and C-24 revealed that C-21 and C-24 are connected through an ether bridge to form a 2,5-disubstituted tetrahydropyran ring. In addition, the correlation signals between H-24 and C-25, C-26, and C-27 confirmed the linkage of a 2-propanol moiety at C-24. This evidence suggested that 1 is a 3,4-seco-cycloartane derivative featuring a C-21, C-24 ether bond and a C-25 hydroxy group. The relative configurations of H-20 and H-24 were defined from the coupling constants of H-21α (t, J = 10.9 Hz), H-21β (d, J = 10.9 Hz), and H-24 (d, J = 11.1 Hz) obtained in C5D5N and subsequently confirmed by the NOESY correlations of H-20/ 1911



Article

Table 1. 1H NMR Data of Compounds 1−6 (500 MHz) 1a

no. 1

2.04 m; 1.30 m

2

15

2.53 ddd (16.0, 11.2, 5.4); 2.27 ddd (15.8, 11.6, 4.0) 2.41 dd (11.6, 5.0) α: 1.07 m; β: 1.53 m α: 1.30 m; β: 1.07 m 1.55 m α: 2.09 m; β: 1.31 m 1.53 m; 1.53 m 1.32 m; 1.32 m

16

1.89 m; 1.33 m

17 18 19

3b

4b

2.05 m; 1.25 m 2.54 m;

2.04 m; 1.35 m 2.48 m;

2.05 m; 1.34 m 2.49 m;

2.29 m

2.22 m

2.24 m

1.50 m 1.00 s α: 0.43 d (4.4);

2.42 dd (11.8, 5.0) α: 1.04 m; β: 1.52 m α: 1.30 m; β: 1.12 m 1.54 m α: 2.05 m; β: 1.37 m 1.62 m; 1.62 m 1.32 m; 1.32 m 1.87 m; 1.25 m 1.55 m 0.94 s α: 0.43 d (4.0);

2.50 1.45 1.13 1.32 1.16 1.60 2.19 1.25 1.71 1.71 1.35 1.35 1.97 1.55 1.67 1.03 0.77

β: 0.73 d (4.4)

β: 0.71 d (4.0)

20 21

1.51 m α: 3.03 m;

22

β: 4.17 d (11.5) 1.90 m; 1.10 m α: 1.32 m; β: 1.60 m 3.00 m

1.48 m α: 3.45 dd (11.4, 2.0); β: 4.06 d (11.4) 1.86 m; 1.08 m α: 1.37 m; β: 1.55 m 3.12 dd (11.0, 2.5)

2.51 m α: 1.15 m; β: 1.49 m α: 1.35 m; β: 1.18 m 1.66 m α: 2.13 m; β: 1.36 m 1.60 m; 1.60 m 1.43 m; 1.43 m 1.97 m; 1.35 m 1.81 m 1.04 s α: 0.40 d (4.3); β: 0.80 d (4.3) 2.19 m α: 3.37 m;

1.13 1.17 4.81 4.73 1.68 0.91

1.17 1.16 4.81 4.73 1.68 0.97

5 6 7 8 11 12

23 24 26 27 28 29 30 OAc a

2a

s s br s; br s s s

s s br s; br s s s

β: 3.97 m α: 1.55 m; β: 2.19 m 3.98 m 3.32 m 1.19 1.18 4.94 4.75 1.70 1.01

s s br s; br s s s

5a

6a

7a

2.09 m; 1.25 m

2.05 m; 1.27 m

2.07 m; 1.33 m

2.53 ddd (16.9, 11.3, 5.4); 2.30 ddd (16.9, 12.0, 4.2) 2.43 dd (12.0, 5.2) α: 1.26 m; β: 1.40 m α: 1.33 m; β: 1.11 m 1.57 m α: 2.05 m; β: 1.33 m 1.63 m

2.52 ddd (16.9, 11.8, 5.4); 2.26 m 2.40 m α: 1.04 m; β: 1.56 m α: 1.29 m; β: 1.10 m 1.56 m α: 2.08 m; β: 1.20 m 1.60 m

2.53 ddd (16.0, 11.0, 5.4); 2.28 ddd (16.0, 11.6, 4.2) 2.41 m α: 1.53 m; β: 1.07 m α: 1.30 m; β: 1.08 m 1.58 m α: 2.10 m; β: 1.25 m 1.60 m

1.34 m

1.33 m

1.32 m

m m; m m; m m m; m m; m m; m m; m m s d (4.3);

1.94 m; 1.39 m

1.92 m; 1.33 m

1.88 m; 1.32 m

1.92 m 0.99 s α: 0.43 d (4.4);

1.90 m 0.99 s α: 0.40 d (4.3);

1.88 m 0.97 s α: 0.41 d (4.6);

0.44 d (4.3)

β: 0.73 d (4.4)

β: 0.71 d (4.3)

β: 0.73 d (4.3)

1.43 m 0.94 d (6.5)

1.73 m 4.22 dd (11.4, 2.7);

1.72 m 4.19 dd (11.2, 3.0);

1.54 m 3.76 dd (11.3, 3.4);

1.85 m; 0.97 m 1.31 m; 1.12 m

3.90 1.82 1.57 2.71

3.87 dd (11.2, 6.3) 2.24 m; 2.00 m

3.59 dd (11.3, 6.3) 1.48 m; 1.25 m

5.60 m

1.57 m; 1.57 m

5.60 m

2.46 t (6.8)

1.29 1.29 4.80 4.72 1.67 0.92 2.05

2.15 s

3.17 dd (10.2, 1.4) 1.17 s 1.13 s 4.85 br s; 4.75 br s 1.71 s 1.01 s

5.96 1.87 4.81 4.74 1.68 0.95 2.06

dd (11.7, 5.5) m; m m

br s; 5.77 br s s br s; br s s s s

s s br s; br s s s s

4.81 4.73 1.68 0.93

br s; br s s s

Tested in CDCl3. bTested in CD3OD.

spectra indicated that all these compounds share an identical tetracyclic 3,4-seco-cycloart-4(28)-en-3-oic acid skeleton with structural differences occurring only in the C-17 side chain (Table 2). The molecular formula of lithocarpic acid E (5) was deduced as C32H48O5 by the [M − H]− ion peak at m/z 511.3416 in the HRESIMS, implying three degrees of unsaturation in the C-17 side chain. Analysis of its 1H and 13C NMR data (Tables 1 and 2) revealed characteristic signals for a terminal olefinic bond, a vinyl methyl, a ketone, and an acetoxy group in the C-17 side chain. Detailed analysis of its 1H−1H COSY and HSQC spectra revealed the occurrence of C-21/C-20/C-22/C-23 and C-26/ C-27 spin coupling systems. In its HMBC spectrum, correlations between C-24 and H-22b (δH 1.57) and H2-23 (δH 2.71), and between C-24 and H2-26 and H3-27, and the ester carbonyl at δC 171.4 and H2-21 (δH 4.22 and 3.90) were used to propose the structure of the C-17 side chain. In

and 2) of 4 were similar to those of 3 except for the signals associated with the side chain at C-17. Its 1H and 13C NMR and HSQC spectra revealed the presence of an oxygenated quaternary carbon (δC 73.9 s), an oxygenated methine [δH/δC 3.17 (1H, dd, J = 10.2, 1.4 Hz)/80.6 d], two singlet methyls [δH/δC 1.17, 1.13 (each 3H, s)/25.9 q, 24.7 q], and a doublet methyl [δH/δC 0.94 (3H, d, J = 6.5 Hz)/19.0 q] in the C-17 side chain. The HMBC correlations between H3-21 and C-17, C-20, and C-21; between H-24 and C-23, C-25, C-26, and C27; and between H3-26 and C-24, C-25 and C-27 suggested a 24,25-diol function in the molecule of 4. The structure of 4 (lithocarpic acid D) was characterized as (5S,8R,9S,10R,13R,14S,17R,20R,24S)-24,25-dihydroxy-3,4-secocycloart-4(28)-en-3-oic acid by single-crystal X-ray diffraction analysis (Figure 6).20 Comparison of 13C NMR data of lithocarpic acids E−N (5− 14) with those of 1−4 and also interpretation of their 2D NMR 1912


a

1913

28.9 31.4 179.1 149.5 46.0 27.8 25.2 47.8 21.4 27.1 26.9 32.5 45.1 49.0 35.6 27.0 49.4 18.6 30.3 39.3 72.8 30.2 26.2 84.2 72.1 24.1 26.3 111.8 19.9 19.5

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 OAc

t t s s d t t d s s t t s s t t d q t d t t t d s q q t q q

28.9 31.5 179.2 149.5 46.0 27.9 25.3 48.2 21.4 27.1 26.9 32.6 44.9 49.3 35.7 27.4 42.5 18.9 30.3 35.8 70.6 27.3 21.4 84.4 72.5 23.6 25.8 111.7 19.9 19.7

2a

t t s s d t t d s s t t s s t t d q t d t t t d s q q t q q

Tested in CDCl3. bTested in CD3OD.

1a

no. 30.4 t 32.5 t 177.9 s 150.9 s 47.2 d 29.2e t 26.2 t 49.4 d 22.3 s 28.4 s 28.1 t 34.3 t 46.3 s 50.2 s 36.8 t 29.0e t 53.7 d 18.7 q 31.1 t 37.8 d 19.0 q 34.9 t 29.1e t 80.6 d 73.9 s 25.9 q 24.7 q 112.1 t 20.1 q 19.9 q

4b 28.9 31.5 179.6 149.4 46.0 27.8 25.2 47.9 21.3 27.1 26.9 32.1 45.1 49.2 35.6 27.7 46.8 18.5 30.2 39.5 64.6 25.2 34.3 202.3 144.6 124.6 17.9 111.8 19.9 19.6 171.4 21.2

5a

Interchangeable assignments.

c−e

30.3 t 32.5c t 177.7 s 150.8 s 47.0 d 28.7d t 26.1 t 48.7 d 22.5 s 28.8 s 27.9 t 32.4c t 46.5 s 49.7 s 36.9 t 28.8d t 52.0 d 18.2 q 31.1 t 44.4 d 72.9 t 37.7 t 80.5 d 80.2 d 73.8 s 25.4 q 26.7 q 112.2 t 20.0 q 19.7 q

3b

Table 2. 13C NMR Data of Compounds 1−14 (125 MHz) t t s s d t t d s s t t s s t t d q t d t t t s s t q t q q s s

28.9 31.5 179.4 149.4 45.9 27.9 25.1 47.9 21.3 27.1 26.9 32.0 45.0 49.1 35.6 27.7 46.4 18.5 30.1 40.2 64.5 33.0 124.1 140.4 70.9 29.9 29.9 111.8 19.9 19.5 171.5 21.2

6a t t s s d t t d s s t t s s t t d q t d t t d d s q q t q q s q

28.9 31.5 180.0 149.4 45.9 27.8 25.1 47.9 21.3 27.1 26.9 32.1 45.1 49.2 35.6 27.7 46.6 18.4 30.2 39.4 64.6 25.6 30.5 179.8

111.8 19.9 19.5 171.4 21.2

111.8 t 19.9 q 19.5 q

8a t t s s d t t d s s t t s s t t d q t d t t t t s q

28.9 31.4 179.1 149.5 45.9 27.8 25.1 47.7 21.4 27.2 27.0 32.4 45.0 49.2 35.6 27.6 46.1 18.4 30.0 42.6 62.5 28.8 19.5 43.9 210.0 30.2

7a

t q q s q

t t s s d t t d s s t t s s t t d q t d t t t s

28.9 31.4 179.5 149.4 46.0 27.9 25.2 47.9 21.3 27.1 26.9 32.1 45.0 49.2 35.6 27.7 46.6 18.4 30.2 39.7 64.8 31.1 25.9 76.3 147.5 111.3 17.7 111.7 19.9 19.5 171.5 21.1

9a t t s s d t t d s s t t s s t t d q t d t t t d s t q t q q s q

28.9 31.5 179.6 149.4 46.0 27.8 25.2 47.9 21.3 27.1 26.9 32.1 45.0 49.1 35.6 27.7 46.7 18.4 30.2 39.8 64.9 31.2 26.0 76.6 147.3 111.6 17.4 111.8 19.9 19.5 171.5 21.2

10a t t s s d t t d s s t t s s t t d q t d t t t d s t q t q q s q

29.0 31.3 179.6 149.5 45.9 27.8 25.1 47.8 21.4 27.3 27.0 32.3 45.1 49.2 35.6 27.5 46.4 18.4 29.9 42.7 62.2 30.0 25.2 76.3 147.7 110.8 18.0 111.7 19.9 19.6

11a t t s s d t t d s s t t s s t t d q t d t t t d s t q t q q

29.0 31.6 179.0 149.5 45.9 27.8 25.1 47.8 21.4 27.3 27.0 32.5 45.0 49.2 35.6 27.5 46.4 18.4 30.0 42.4 62.5 30.6 25.0 76.3 147.6 111.0 17.9 111.7 19.9 19.6

12a t t s s d t t d s s t t s s t t d q t d t t t d s t q t q q

28.9 31.3 178.6 149.5 46.0 27.9 25.2 47.9 21.3 27.1 27.0 32.0 45.1 49.1 35.6 27.7 46.7 18.4 30.2 39.5 64.9 30.4 24.9 124.7 131.7 17.8 25.9 111.8 19.8 19.6 171.5 21.2

13a t t s s d t t d s s t t s s t t d q t d t t d d s q q t q q s q

29.0 31.3 178.6 149.6 46.0 27.9 25.2 47.8 21.5 27.2 27.2 33.2 45.3 49.1 36.1 28.2 52.4 18.2 30.1 36.1 18.4 35.8 24.7 127.3 134.4 69.3 13.8 111.7 19.9 19.5

14a t t s s d t t d s s t t s s t t d q t d q t t d s t q t q q

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HRESIMS (m/z 513.3585 [M − H]−), indicating two degrees of unsaturation in the C-17 side chain. According to 1H and 13C NMR data (Tables 1 and 2) of this compound, the presence of an acetoxy group and a 1,2-disubstituted olefinic function in the C-17 side chain could be deduced. The olefinic function was located at C-23, C-24 according to HMBC correlations of C-23 with H-20 and H2-22 and of C-24 with H3-26 and H3-27. The coupling constant between H-23 and H-24 (15.9 Hz, measured in C5D5N) supported the E configuration of the olefinic bond. The HMBC correlations of H2-21 with the acetoxy carbonyl carbon (δC 171.5 s), C-17 (δC 46.4 d), and C-20 (δC 40.2 d) were used to assign the acetoxy group to C-21. Accordingly, compound 6 (lithocarpic acid F) was determined as 21-acetoxy25-hydroxy-3,4-seco-cycloart-4(28),23E-dien-3-oic acid. The HRESIMS of lithocarpic acid G (7) exhibited an [M + Cl]− ion at m/z 493.3071, corresponding to the molecular formula C29H46O4, indicating one degree of unsaturation in the C-17 side chain. Its 13C NMR spectrum displayed seven carbon signals belonging to a C-17 side chain, including a ketone carbon (δC 210.0), a methyl (δC 30.2), four methylenes (δC 62.5, 43.9, 28.8, 19.5), and a methine (δC 42.6) (Table 2), indicating 7 to be a 3,4-seco-norcycloartane-type triterpene. The HMBC correlations of H2-21 with C-17, C-20, and C-22, of H224 with C-22, C-23, C-25, and C-26, and of H3-26 with C-24 and C-25 supported the assignment of a hydroxy group at C21, a ketone group at C-25, and a terminal methyl at C-26. Therefore, compound 7 (lithocarpic acid G) was identified as 21-hydroxy-25-oxo-3,4-seco-27-norcycloart-4(28)-en-3-oic acid. The molecular formula of lithocarpic acid H (8) was deduced as C29H44O6, according to the [M − H]− ion peak at m/z 487.3054 in the HRESIMS, implying two degrees of unsaturation in the C-17 side chain. The 13C NMR spectrum of 8 exhibited seven carbon signals in the C-17 side chain, belonging to one carboxyl group (δC 179.8), one acetoxy group (δC 171.4 s, 21.1 q), three methylenes (δC 64.6, 30.5, and 25.6), and one methine (δC 39.4) (Table 3). The position of the carboxyl group was assigned as C-24 based on the HMBC correlations of H2-23 (δH 2.44, 2.32) with C-20 (δC 39.4 s), C22 (δC 25.6 t), and the carbonyl signal at δC 179.8. The assignment of the acetoxy unit at C-21 was established by the HMBC correlations between H2-21 (δH 4.24, 3.89) and C-17

Figure 1. 1H−1H COSY and key HMBC correlations of 1 and 2.

Figure 2. Relative configuration of C-20 and C-24 in 1 and 2 determined by JH−H analyses and ROESY correlations.

addition, compound 5 exhibited key NOE signals consistent with those of the cycloartane skeleton, including correlations of H-8 with H3-18 and H-19β and of H3-30 with H-5 and H-17. Therefore, compound 5 (lithocarpic acid E) was characterized as 21-acetoxy-24-oxo-3,4-seco-cycloart-4(28),25-dien-3-oic acid. Lithocarpic acid F (6), obtained as a white, amorphous powder, gave the molecular formula C32H50O5 from its

Figure 3. Single-crystal X-ray structure of 1. 1914



Article

Figure 4. Key 2D NMR correlations of 3.

Figure 5. Single-crystal X-ray structure of 3.

Figure 6. Single-crystal X-ray structure of 4.

(δC 46.6 d), C-20, C-22, and the carbonyl signal at δC 171.4. In addition, compound 8 exhibited key NOE correlations consistent with those of the cycloartane core, including correlations from H-8 to H3-18 and H-19β and from H-17 to H3-21 and H3-30. On the basis of the above evidence, the structure of 8 (lithocarpic acid H) could be proposed as 21acetoxy-3,4-seco-25,26,27-trinorcycloart-4(28)-en-3,24-dioic acid.

HRESIMS analysis revealed an identical molecular formula of lithocarpic acids I (9) and J (10) to C32H50O5 (m/z 513.3583 and 513.3588, respectively, [M − H]−), implying two unsaturation degrees in the C-17 side chain. The NMR data of 9 and 10 were also superimposable (ΔδH ≤ 0.01 ppm and ΔδC < 0.3 ppm), suggesting that the two compounds may be stereoisomers. Both the 1D NMR spectra of 9 and 10 displayed characteristic signals of an acetoxy group, a terminal olefinic 1915



Article

Table 3. 1H NMR Data of Compounds 7−14 (CDCl3, 500 MHz) no. 1 2

8 2.04 m; 1.30 m 2.53 ddd (16.0, 11.0, 5.4); 2.28 m

5

2.42 m

6

1.52 1.07 1.28 1.08 1.55 2.10 1.25 1.59 1.33 1.93 1.30 1.88 0.99 0.72 0.42 1.71 4.24 3.89 1.87 1.63 2.44 2.32

m; m m; m m m; m m m m; m m s d (4.3); d (4.3) m dd (11.5, 2.6); dd (11.5, 6.5) m; m m; m

4.81 4.73 1.68 0.93 2.06

br s; br s s s s

7 8 11 12 15 16 17 18 19 20 21 22 23 24 26 27 28 29 30 OAc

11

12

2.09 m; 1.36 m 2.54 ddd (16.0, 11.0, 5.4); 2.28 ddd (16.0, 11.6, 4.2) 2.41 dd (11.6, 4.8)

9

2.09 m; 1.36 m 2.53 ddd (16.0, 11.0, 5.4); 2.27 ddd (16.0, 11.6, 4.2) 2.41 dd (11.6, 4.8)

10

2.04 m; 1.34 m 2.52 m;

2.04 m; 1.35 m 2.50 m;

2.28 m

1.52 1.10 1.30 1.10 1.56 2.10 1.25 1.60 1.32 1.90 1.32 1.90 0.99 0.72 0.41 1.68 4.23 3.91 1.64 1.45 1.55 1.09 4.02 4.94

m; m m; m m m; m m m m; m m s d (4.3); d (4.3) m dd (11.4, 3.3); dd (11.4, 6.2) m; m m; m t (6.2) br s; 4.84 br s

1.52 1.10 1.30 1.10 1.56 2.10 1.25 1.60 1.32 1.90 1.32 1.90 0.99 0.72 0.41 1.68 4.23 3.91 1.64 1.45 1.55 1.09 4.02 4.93

m; m m; m m m; m m m m; m m s d (4.3); d (4.3) m dd (11.4, 3.3); dd (11.4, 6.2) m; m m; m t (6.2) br s; 4.84 br s

1.71 4.81 4.73 1.67 0.93 2.05

s br s; br s s s s

1.71 4.80 4.73 1.67 0.93 2.05

s br s; br s s s s

13

14 2.20 m; 1.36 m 2.58 m;

2.26 m

2.06 m; 1.32 m 2.54 ddd (17.0, 12.1, 5.7); 2.27 m

2.43 m

2.41 m

2.43 m

1.51 m; 1.08 m 1.30 m; 1.10 m 1.56 m 2.10 m; 1.25 m 1.60 m 1.31 m 1.90 m; 1.30 m 1.90 m 0.97 s 0.73 d (4.3); 0.40 d (4.3) 1.50 m 3.76 m; 3.59 m 1.56 m; 1.48 m 1.47 m; 1.10 m 4.07 m 4.95 br s; 4.84 br s 1.73 s 4.81 br s; 4.73 br s 1.68 s 0.94 s

1.52 m; 1.07 m 1.29 m; 1.13 m 1.56 m 2.07 m; 1.24 m 1.65 m 1.31 m 1.87 m; 1.35 m 1.83 m 0.97 s 0.72 d (4.3); 0.40 d (4.3) 1.58 m 3.74 m; 3.49 m 1.61 m; 1.50 m 1.61 m; 1.10 m 4.04 m 4.94 br s; 4.82 br s 1.72 s 4.80 br s; 4.72 br s 1.69 s 0.92 s

1.52 1.08 1.30 1.08 1.56 2.10 1.24 1.61 1.32 1.92 1.33 1.92 0.99 0.73 0.42 1.64 4.22 3.94 1.44 1.25 2.02 1.91 5.09 1.60

m; m m; m m m; m m m m; m m s d (4.3); d (4.3) m dd (11.2, 3.0); dd (11.2, 5.7) m; m m; m t (7.1) s

2.43 dd (11.9, 5.7) 1.50 m; 1.26 m 1.30 m; 1.10 m 1.57 m 2.11 m; 1.09 m 1.63 m 1.40 m 1.90 m; 1.28 m 1.59 m 0.96 s 0.73 d (4.4); 0.43 d (4.4) 1.47 m 0.89 d (6.7)

1.68 4.81 4.74 1.68 0.94 2.07

s br s; br s s s s

2.28 m

1.29 1.08 1.93 1.30 5.40 4.00

m; m m; m t (6.9) s

1.67 4.81 4.73 1.68 0.93

s br s; br s s s

Figure 7. Δδ (δS − δR) values (in ppm) for the MTPA esters of 9−12.

1916



Article

derivatives containing a C-21, C-24 ether bridge, while lithocarpic acid C (3) is the first example of a 3,4-secocycloartane derivative with a C-21, C-23 ether bridge. In turn, lithocarpic acids G (7) and H (8) are the first examples of 3,4seco-norcycloartanes. The isolated compounds were evaluated for antibacterial activities against four bacterial strains and for their effects on 11β-HSD1. The most abundant substance obtained, lithocarpic acid A (1), showed potent antibacterial activity against Micrococcus luteus and Bacillus subtilis, with MIC values of 3.1 and 6.3 μg/mL, respectively, whereas its C-24 isomer, lithocarpic acid B (2), did not show antibacterial activity against the four bacterial strains tested. Lithocarpic acid F (6) and the known compound 15 were also active against Micrococcus luteus and Bacillus subtilis (Table 4).

bond, and an oxygenated methine in their respective C-17 side chain. Analysis of the 1H−1H COSY, HSQC, and HMBC spectra of these compounds was used to locate the acetoxy group at C-21, the hydroxy group at C-24, and the terminal olefinic bond at C-25, C-26. The slight differences in the 13C NMR signals of C-24, C-25, C-26, and C-27 indicated that 9 and 10 are C-24 epimers. To determine the spatial configurations of C-24 in 9 and 10, (S)- and (R)-MTPA esters of 9 and 10 were synthesized for both substances. The Δδ (δS − δR) values observed for signals of the protons close to C-24 revealed a 24R configuration for 9 and a 24S configuration for 10 (Figure 7). Therefore, 9 (lithocarpic acid I) was characterized as (24R)-21-acetoxy-24-hydroxy-3,4-seco-cycloart-4(28),25-dien-3-oic acid, while 10 (lithocarpic acid J) was proposed as (24S)-21-acetoxy-24-hydroxy-3,4-seco-cycloart-4(28),25-dien-3-oic acid. Lithocarpic acids K (11) and L (12) were found to share the same molecular formula, C30H48O4, as deduced by HRESIMS (m/z 471.3480 and 471.3475, respectively, [M − H]−) and exhibited almost identical 13C NMR spectra (Table 3), indicating these compounds to be another pair of epimers. The NMR data of compounds 11 and 12 showed the absence of an acetyl group and upfield shifts of C-21, suggesting them to be deacetylated derivatives of 9 and 10. The absolute configurations of C-24 in 11 and 12 were determined by the modified Mosher’s method, and the Δδ (δS − δR) values observed for signals of the protons close to C-24 revealed the 24R configuration for 11 and 24S for 12 (Figure 7). Therefore, 11 (lithocarpic acid K) was characterized as (24R)-21,24dihydroxy-3,4-seco-cycloart-4(28),25-dien-3-oic acid, while 12 (lithocarpic acid L) was proposed as (24S)-21,24-dihydroxy3,4-seco-cycloart-4(28),25-dien-3-oic acid. Lithocarpic acid M (13) was shown to possess a molecular formula of C32H50O5 according to the HRESIMS at m/z 513.3594 [M − H]−, indicating two degrees of unsaturation in the C-17 side chain. Its 1H and 13C NMR data (Tables 2 and 3) showed typical signals of a trisubstituted olefinic bond, an acetoxy moiety, and two vinyl methyl groups in the C-17 side chain. Detailed analysis of its 1H−1H COSY, HSQC, and HMBC spectra revealed the location of the acetoxy moiety at C-21 and the olefinic double bond at C-24, C-25. Thus, 13 (lithocarpic acid M) was assigned as 21-acetoxy-3,4-secocycloart-4(28),24-dien-3-oic acid. Lithocarpic acid N (14) exhibited an [M + Cl]− ion peak at m/z 491.3306 in the HRESIMS, and its molecular formula was determined as C30H48O3 (calcd 491.3292), implying one degree of unsaturation in the C-17 side chain. Analysis of its 1H and 13 C NMR spectra revealed the presence of a trisubstituted olefinic bond, a vinyl methyl group, and a hydroxymethyl group in the C-17 side chain. The observed HMBC correlations between H-24 and C-25, C-26, and C-27 and between H3-27 and C-24, C-25, and C-26 supported the assignment of the hydroxymethyl group at C-26 and the olefinic bond at C-24, C25. The configuration of the olefinic bond was determined as E by comparison of the chemical shift of C-26 with literature data (E configuration: δC 69.0−70.0; Z configuration: δC 60.0− 62.0).21−24 Therefore, the structure of 14 (lithocarpic acid N) was identified as (24E)-26-hydroxy-3,4-seco-cycloart-4(28),24dien-3-oic acid. Lithocarpic acids A−N (1−14) and coccinetane B (15) are the first examples of 3,4-seco-cycloartane derivatives reported from the genus Lithocarpus. Lithocarpic acids A (1) and B (2) are the first pair of C-24 isomers of 3,4-seco-cycloartane

Table 4. Antibacterial Activity of Selected Compounds Isolated from L. polystachyus MIC (μg/mL)a compound

Sa

Se

MI

Bs

1 2 4 5 6 9 10 11 15 ofloxacin

25.0 >50 >50 >50 25.0 >50 >50 25.0 >50 0.20

12.5 >50 >50 >50 12.5 25.0 25.0 25.0 >50 0.20

3.1 >50 50.0 >50 6.3 12.5 12.5 12.5 6.3 1.6

6.3 >50 50.0 25.0 6.3 12.5 12.5 6.3 3.1 0.013

a Sa: Staphylococcus aureus; Se: Staphylococcus epidermidis; Ml: Micrococcus luteus; Bs: Bacillus subtilis.

11β-Hydroxysteroid dehydrogenase type 1 is an enzyme that regulates interconversion of the active/inactive forms of glucocorticoids and acts tissue-specifically depending on the presence/absence of the NADPH cofactor.25,26 11β-HSD1 inhibitors may have use in the treatment of diabetes and obesity, among other conditions.26 The four most abundant compounds (1, 6, 8, and 11) were selected to evaluate their inhibitory activities against human and mouse 11β-HSD1. Preliminary testing of the pure isolates at 10 μM in a scintillation proximity assay revealed that all of them showed >60% inhibition against both human and mouse 11β-HSD1. Further bioassays showed that compounds 1, 6, 8, and 11 are human 11β-HSD1 inhibitors with IC50 values of 1.9, 2.3, 1.9, and 1.4 μM, respectively, while compounds 1, 6, and 11 displayed more potent inhibition against mouse 11β-HSD1 with IC50 values of 0.24, 0.74, and 0.99 μM, respectively (Table S1, Supporting Information).

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured using a Büchi 510 melting point apparatus (Büchi, Flawil, Switzerland). Optical rotations were determined on a PerkinElmer 341 polarimeter (PerkinElmer, Waltham, MA, USA). IR spectra were recorded on a PerkinElmer 577 spectrometer using KBr disks. NMR experiments were performed in CDCl3, CD3OD, or C5D5N on Bruker AM-400 (Bruker, Ettlingen, Germany), Varian Mercury Plus-400 (Varian, Palo Alto, CA, USA), Bruker Advance III 500, and Bruker Advance III 600 NMR spectrometers, with chemical shifts referenced to solvent peaks (CDCl3: δH/δC 7.26/77.16; CD3OD: δH/δC 3.33/ 1917



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49.00; C5D5N: δH/δC 7.22/123.87). ESIMS analyses were carried out on a Shimadzu LC-MS-2020 (Shimadzu, Kyoto, Japan) with a Shimadzu SPD-M20 diode array detector using a CNW C18 (2.1 × 50 mm, 3.5 μm; Anpel Scientific Instrument Co., Ltd., Shanghai, People’s Republic of China) or a CNW C18 (2.1 × 100 mm, 3.5 μm; Anpel Scientific Instrument Co., Ltd.) column. HRESIMS was carried out on an LCT Premier XE mass spectrometer (Waters, Milford, MA, USA). Semipreparative HPLC was performed on a Unimicro EasySep-1010 binary pump system (Unimicro, Shanghai, People’s Republic of China) with a Unimicro EasySep-1010 detector using YMC-Pack ODS-A (250 × 20 mm, 5 μm; YMC Co., Ltd., Kyoto, Japan), YMCPack ODS-A (150 × 10 mm, 5 μm), or YMC-Pack Diol (250 × 20 mm, 5 μm) columns. Silica gel (300−400 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, People’s Republic of China), C18 reversed-phase (RP-18) silica gel (150−200 mesh; Merck, Whitehouse Station, NJ, USA), polyamide (30−100 mesh, Sinopharm Chemical Reagent Co., Ltd., Shanghai, People’s Republic of China), and CHP20P MCI gel (75−150 μm, Mitsubishi Chemical Industries, Ltd., Tokyo, Japan) were used for column chromatography (CC). Precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd.) were used for TLC detection. Plant Material. The cupules of L. polystachyus were collected in September 2012 from Bama Yao Autonomous County, Guangxi Zhuang Autonomous Region, People’s Republic of China, and identified by Mr. Wenhui Liang of Guangxi Zhuang Autonomous Region Forestry Research Institute. A voucher specimen has been deposited at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (WMZ-20121019-TC). Extraction and Isolation. The air-dried cupules of L. polystachyus (500 g) were powdered and percolated with 95% EtOH at room temperature (3 L × 3) to give a crude extract (74 g), which was fractionated on silica gel (300−400 mesh) eluted with a petroleum ether/acetone gradient (from 10:1 to 0:1, v/v) to afford seven fractions (Fr.1−Fr.7). Fr.2 (15 g) was subjected to separation on an MCI column eluted with MeOH/H2O (1:1, 3:1, 10:1, 1:0, v/v) to yield four subfractions (Fr.21−Fr.24). Fr.22 (4.6 g) was further separated over silica gel eluted with a petroleum ether/acetone (10:1 to 4:1, v/v) gradient to give four subfractions (Fr.221−Fr.224). Fr.223 (2.4 g) was subsequently purified by RP-18 gel CC (50% to 90% MeOH) and repeated semipreparative HPLC (5.0 mL/min, 95% CH3CN) to give compounds 4 (16 mg) and 6 (43 mg). Fr.23 was subjected to silica gel CC (petroleum ether/EtOAc, 10:1 to 5:1, v/v) and further purified by repeated semipreparative HPLC to yield compounds 1 (125 mg), 2 (15 mg), 5 (5 mg), 6 (72 mg), 13 (2 mg), 14 (4 mg), and a mixture of 9 and 10 (25 mg), which was further purified by semipreparative HPLC (5.0 mL/min, 85% CH3CN) to give 9 (5 mg) and 10 (5 mg). Fraction 3 was separated over a polyamide column (50% to 100% MeOH) and silica gel (CH2Cl2/ MeOH, 25:1 to 5:1) and was eventually purified by semipreparative reversed-phase HPLC (3.0 mL/min, 65% CH3CN) and normal-phase HPLC (5 mL/min, 100% to 80% n-hexane/2-propanol) to afford compounds 3 (8 mg), 7 (3 mg), 8 (11 mg), 11 (84 mg), and 12 (32 mg). Lithocarpic acid A (1): colorless, monoclinic-like crystals (CH3CN:H2O, 20:1), mp 235−237 °C; [α]D26 +63.8 (c 0.10, CHCl3); IR (KBr) νmax 3444, 2937, 2871, 1708, 1455, 1376, 1164, 1091, 894 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 1 and 2; 1 H NMR (400 MHz, C5D5N) δH 5.03 (1H, br s, H-28a), 4.87 (1H, br s, H-28b), 4.33 (1H, d, J = 10.9 Hz, H-21β), 3.28 (1H, d, J = 11.1 Hz, H-24), 3.12 (1H, t, J = 10.9 Hz, H-21α), 2.88 (1H, m), 2.60 (2H, m), 2.42 (1H, m), 2.21 (1H, m), 1.94 (2H, t, J = 11.9 Hz), 1.75 (3H, s), 1.48 (3H, s), 1.45 (3H, s), 0.99 (3H, s), 0.92 (3H, s), 0.68 (1H, d, J = 4.2 Hz, H-19a), 0.38 (1H, d, J = 4.3 Hz, H-19b); HRESIMS m/z 495.3445 [M + Na]+ (calcd for C30H48O4Na, 495.3450). Lithocarpic acid B (2): white, amorphous powder; [α]25 D +33.6 (c 0.07, CHCl3); IR (KBr) νmax 3419, 2944, 2863, 1708, 1641, 1454, 1378, 1162, 1081, 890 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 1 and 2; HRESIMS m/z 507.3265 [M + Cl]− (calcd for C30H48O4Cl, 507.3241).

Lithocarpic acid C (3): colorless orthorhombic crystals (acetone); mp 220−223 °C; [α]27 D +51.2 (c 0.05, CHCl3); IR (KBr) νmax 3375, 2939, 2872, 1707, 1640, 1198, 736 cm−1; 1H and 13C NMR (CD3OD) data, see Tables 1 and 2; HRESIMS m/z 523.3187 [M + Cl]− (calcd for C30H48O5Cl, 523.3190). Lithocarpic acid D (4): colorless orthorhombic crystals (acetone); mp 250−253 °C; [α]27 D +53.8 (c 0.08, CHCl3); IR (KBr) νmax 3411, 2937, 2872, 1735, 1709, 1641, 1456, 1379, 1205, 1078, 893 cm−1; 1H and 13C NMR (CD3OD) data, see Tables 1 and 2; HRESIMS m/z 497.3599 [M + Na]+ (calcd for C30H50O4Na, 497.3607). Lithocarpic acid E (5): white, amorphous powder; [α]27 D +53.8 (c 0.08, CHCl3); IR (KBr) νmax 3428, 2929, 2873, 1737, 1708, 1454, 1378, 1238, 1033 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 1 and 2; HRESIMS m/z 511.3416 [M − H]− (calcd for C32H47O5, 511.3424). Lithocarpic acid F (6): white, amorphous powder; [α]27 D +53.8 (c 0.22, CHCl3); IR (KBr) νmax 3433, 2941, 2871, 1739, 1711, 1641, 1456, 1379, 1240, 1153, 1033, 893 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 1 and 2; 1H NMR (400 MHz, C5D5N) δH 6.01 (1H, ddd, J = 15.6, 7.3, 6.0 Hz, H-23), 5.99 (1H, d, J = 15.6 Hz, H-24), 4.99 (1H, br s, H-28a), 4.84 (1H, br s, H-28b), 4.43 (1H, dd, J = 11.2, 3.3 Hz, H-21a), 4.05 (1H, dd, J = 11.2, 7.3 Hz, H-21b), 2.83 (1H, ddd, J = 14.9, 11.6, 5.4 Hz), 2.09 (3H, s, COCH3), 1.71 (3H, s, H3-29), 1.54 (3H, s), 1.53 (3H, s), 1.00 (3H, s), 0.86 (3H, s), 0.70 (1H, d, J = 4.3 Hz, H-19a), 0.43 (1H, d, J = 4.3 Hz, H-19b); HRESIMS m/z 513.3585 [M − H]− (calcd for C32H49O5, 513.3580). Lithocarpic acid G (7): white, amorphous powder; [α]27 D +53.5 (c 0.04, CHCl3); IR (KBr) νmax 3429, 2926, 2871, 1709, 1641, 1457, 1377, 1263, 1033, 890, 732 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 1 and 2; HRESIMS m/z 493.3071 [M + Cl]− (calcd for C29H46O4Cl, 493.3085). Lithocarpic acid H (8): white, amorphous powder; [α]27 D +63.3 (c 0.12, CHCl3); IR (KBr) νmax 3431, 2922, 2877, 1735, 1721, 1645, 1453, 1420, 1379, 1246, 1039, 895 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 3; HRESIMS m/z 487.3054 [M − H]− (calcd for C29H43O6, 487.3060). Lithocarpic acid I (9): white, amorphous powder; [α]27 D +41.4 (c 0.04, CHCl3); IR (KBr) νmax 3432, 2927, 2871, 1739, 1710, 1641, 1456, 1378, 1242, 1033, 893 cm−1; 1H NMR (500 MHz, CDCl3) and 13 C NMR (125 MHz, CDCl3) data, see Tables 2 and 3; HRESIMS m/ z 513.3583 [M − H]− (calcd for C32H49O5, 513.3580). Lithocarpic acid J (10): white, amorphous powder; [α]27 D +53.1 (c 0.08, CHCl3); IR (KBr) νmax 3435, 2930, 2872, 1739, 1710, 1640, 1456, 1378, 1241, 1033, 893 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 3; HRESIMS m/z 513.3588 [M − H]− (calcd for C32H49O5, 513.3580). Lithocarpic acid K (11): white, amorphous powder; [α]27 D +57.9 (c 0.13, CHCl3); IR (KBr) νmax 3415, 2927, 2871, 1709, 1641, 1454, 1377, 1284, 1032, 893 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 3; HRESIMS m/z 471.3480 [M − H]− (calcd for C30H47O4, 471.3474). Lithocarpic acid L (12): white, amorphous powder; [α]27 D +68.9 (c 0.13, CHCl3); IR (KBr) νmax 3417, 2943, 2871, 1709, 1640, 1455, 1378, 1031, 893, 733 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 3; HRESIMS m/z 471.3475 [M − H]− (calcd for C30H47O4, 471.3474). Lithocarpic acid M (13): colorless gum; [α]27 D +68.2 (c 0.04, CHCl3); IR (KBr) νmax 3423, 2926, 2871, 1742, 1708, 1460, 1378, 1240, 1033, 892 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 3; HRESIMS m/z 513.3594 [M − H]− (calcd for C32H49O5, 513.3580). Lithocarpic acid N (14): white, amorphous powder; [α]27 D +48.1 (c 0.02, CHCl3); IR (KBr) νmax 3391, 2959, 2924, 2852, 1647, 1461, 1377, 1261 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 3; HRESIMS m/z 491.3306 [M + Cl]− (calcd for C30H48O3Cl, 491.3292). Preparation of (S)- and (R)-MTPA Esters of 9−12. (R)-(−) and (S)-(+)-MTPA-Cl (10 μL) and a catalytic amount of DMAP were separately added to two different aliquots of 9 (each 2 mg) in anhydrous CDCl3. The resulting mixtures were allowed to stand at 1918



Article

μ(Cu Kα) = 0.577 mm−1, and F(000) = 1004. Crystal dimensions: 0.25 × 0.05 × 0.05 mm3. Independent reflections: 2147 (Rint = 0.0718). The final R1 values were 0.1279, wR2 = 0.3386 (I > 2σ(I)). Flack parameter: 0.1(11). CCDC number: 982762. Crystal Data of 4: C30H50O4, Mr = 474.70, orthorhombic, a = 8.5098(2) Å, b = 12.7020(3) Å, c = 25.9391(4) Å, α = 90°, β = 90°, γ = 90°, V = 2803.80(10) Å3, space group P212121, Z = 4, Dx = 1.125 mg/m3, μ(Cu Kα) = 0.563 mm−1, and F(000) = 1048. Crystal dimensions: 0.26 × 0.16 × 0.06 mm3. Independent reflections: 5098 (Rint = 0.0589). The final R1 values were 0.0453, wR2 = 0.1196 (I > 2σ(I)). Flack parameter: 0.10(14). CCDC number: 982760. Antibacterial Activity Assay. The in vitro antibacterial bioassay against Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Micrococcus luteus ATCC 9341, and Bacillus subtilis ATCC 6633 was conducted using a microdilution method27 and with ofloxacin as the positive control. The microbial cells were suspended in Mueller-Hinton broth to form a final density of 5 × 105−106 cfu/mL and incubated at 37 °C for 18 h under aerobic conditions with the test compounds, which were dissolved in DMSO. The blank controls of microbial culture were incubated with a limited amount of DMSO under the same conditions. DMSO was determined to be nontoxic under the experimental conditions used. 11β-Hydroxysteroid Dehydrogenase Type 1 Assay. Inhibition against human and mouse 11β-HSD1 enzymatic activities was determined via a scintillation proximity assay (SPA) using microsomes containing 11β-HSD1, and glycyrrhetinic acid (97%, G109797, Aladdin) was used as the positive control.28,29 The human and mouse 11β-HSD1 enzymes were expressed in HEK293 cells. Briefly, the sequences of human and murine 11β-HSD1 were obtained from the clones provided by the NIH Mammalian Gene Collection. The expression plasmids were constructed by inserting the murine 11βHSD1 sequence into the multiple clone sites of pcDNA3. HEK293 cells were transfected with the expression plasmid and selected by cultivation in the presence of 700 μg/mL of G418. The microsomal fraction overexpressing 11β-HSD1 was prepared from the HEK293 cells stably transfected with either human or murine 11β-HSD1 and was used as the enzyme source for SPA. 11β-HSD1-containing microsomes were first incubated with NADPH and [3H]-cortisone (Amersham); then the product, [3H]-cortisol, was specifically captured by a monoclonal antibody coupled to protein A-coated SPA beads (GE). The inhibitory effects of the test compounds on 11β-HSD1 were evaluated by detecting the SPA signal. IC50 values were calculated using Prism 5 (GraphPad software, San Diego, CA, USA).

room temperature for 48 h, and then 1H NMR spectra were run without further purification. The related proton signals were assigned by analyzing 1D TOCSY and 1H−1H COSY spectra. By the same procedure, the (S)- and (R)-MTPA esters of 10−12 were prepared, and the related proton signals were also assigned by analyzing the 1DTOCSY and 1H−1H COSY spectra. (S)-MTPA ester of 9 (9a): selected 1H NMR values (400 MHz, CDCl3) δH 5.31 (t, J = 6.4 Hz, H-24), 4.92 (br s, H-26a), 4.89 (br s, H-26b), 4.17 (m, H-21a), 3.85 (m, H-21b), 1.99 (s, OAc), 1.78 (m, H23a), 1.64 (m, H-20), 1.58 (s, H3-27), 1.58 (m, H-23b), 1.41 (m, H22a), 1.33 (m, H-22b). (R)-MTPA ester of 9 (9b): selected 1H NMR values (400 MHz, CDCl3) δH 5.37 (t, J = 6.4 Hz, H-24), 5.02 (br s, H-26a), 4.95 (br s, H-26b), 4.14 (m, H-21a), 3.81 (m, H-21b), 2.00 (s, OAc), 1.74 (m, H23a), 1.70 (s, H3-27), 1.60 (m, H-20), 1.54 (m, H-23b), 1.34 (m, H22a), 1.23 (m, H-22b). (S)-MTPA ester of 10 (10a): selected 1H NMR values (400 MHz, CDCl3) δH 5.33 (t, J = 6.4 Hz, H-24), 5.00 (br s, H-26a), 4.97 (br s, H-26b), 4.12 (m, H-21a), 3.80 (m, H-21b), 1.95 (s, OAc), 1.77 (m, H23a), 1.64 (m, H-20), 1.64 (s, H3-27), 1.57 (m, H-23b), 1.36 (m, H22a), 1.15 (m, H-22b). (R)-MTPA ester of 10 (10b): selected 1H NMR values (400 MHz, CDCl3) δH 5.27 (t, J = 6.4 Hz, H-24), 4.92 (br s, H-26a), 4.88 (br s, H-26b), 4.12 (m, H-21a), 3.80 (m, H-21b), 1.95 (s, OAc), 1.81 (m, H23a), 1.60 (s, H3-27), 1.69 (m, H-20), 1.62 (m, H-23b), 1.44 (m, H22a), 1.25 (m, H-22b). (S)-MTPA ester of 11 (11a): selected 1H NMR values (400 MHz, CDCl3) δH 5.27 (t, J = 6.6 Hz, H-24), 4.91 (br s, H2-26), 4.35 (m, H21a), 4.15 (m, H-21b), 1.78 (m, H-23a), 1.71 (m, H-20), 1.57 (s, H327), 1.57 (m, H-23b), 1.36 (m, H2-22). (R)-MTPA ester of 11 (11b): selected 1H NMR values (400 MHz, CDCl3) δH 5.29 (t, J = 6.6 Hz, H-24), 4.98 (br s, H-26a), 4.94 (br s, H-26b), 4.48 (m, H-21a), 4.00 (m, H-21b), 1.70 (m, H-23a), 1.66 (s, H3-27), 1.64 (m, H-20), 1.49 (m, H-23b), 1.23 (m, H-22a), 1.12 (m, H-22b). (S)-MTPA ester of 12 (12a): selected 1H NMR values (400 MHz, CDCl3) δH 5.29 (m, H-24), 5.02 (br s, H-26a), 4.96 (br s, H-26b), 4.35 (m, H-21a), 4.17 (m, H-21b), 1.74 (m, H-20), 1.67 (s, H3-27), 1.62 (m, H-22a and H-23a), 1.34 (m, H-23b), 1.16 (m, H-22b). (R)-MTPA ester of 12 (12b): selected 1H NMR values (400 MHz, CDCl3) δH 5.21 (t, J = 6.8 Hz, H-24), 4.92 (br s, H-26a), 4.85 (br s, H-26b), 4.49 (m, H-21a), 4.09 (m, H-21b), 1.78 (m, H-20), 1.67 (m, H-23a), 1.58 (m, H-22a), 1.53 (s, H3-27), 1.41 (m, H-23b), 1.15 (m, H2-22b). X-ray Crystallographic Analyses of Compounds 1, 3, and 4. The single-crystal X-ray diffraction data of compounds 1, 3, and 4 were collected on a Bruker APEX-II CCD detector employing graphitemonochromated Cu Kα radiation (λ = 1.54178 Å) at 140(2) K (1 and 4) or 296(2) K (3). The structures were solved by direct methods using SHELXL-97 and refined using full-matrix least-squares calculation on F2 using SHELXL-97. All non-hydrogen atoms were refined anisotropically. The hydrogen atom positions were geometrically idealized and allowed to ride on their parent atoms. Crystallographic data for 1, 3, and 4 have been deposited at the Cambridge Crystallographic Data Centre. Copies of these data can be obtained free of charge via the Internet at www.ccdc.cam.ac.uk/conts/retrieving. html or on application to the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [tel: (+44) 1223-336-408; fax: (+44) 1223-336-033; e-mail: [email protected]]. Crystal Data of 1: C60H96O8·CH3CN·H2O, Mr = 1004.43, monoclinic, a = 16.5653(2) Å, b = 7.60490(10) Å, c = 23.1068(3) Å, α = 90°, β = 98.4080(10)°, γ = 90°, V = 2879.65(6) Å3, space group P21, Z = 2, Dx = 1.158 mg/m3, μ(Cu Kα) = 0.594 mm−1, and F(000) = 1104. Crystal dimensions: 0.30 × 0.25 × 0.20 mm3. Independent reflections: 8927 (Rint = 0.0306). The final R1 values were 0.0545, wR2 = 0.1537 (I > 2σ(I)). Flack parameter: 0.08(11). CCDC number: 982761. Crystal Data of 3: C30H48O5, Mr = 504.68, orthorhombic, a = 7.6982(10) Å, b = 19.440(3) Å, c = 21.085(3) Å, α = 90°, β = 90°, γ = 90°, V = 3155.4(8) Å3, space group P212121, Z = 4, Dx = 1.062 mg/m3,

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ASSOCIATED CONTENT

S Supporting Information *

NMR spectra of all new compounds, CIF files of 1, 3, and 4, and bioassay results of tested compounds are provided. These materials are available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax (W. Zhao): 86-21-50806052. E-mail wmzhao@simm. ac.cn. *Tel/Fax (Y. Leng): 86-21-50806059. E-mail: [email protected]. cn. Author Contributions ‡

H. Wnag and R. Ning contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The investigation was financially supported by Forestry Industry Research Special Fund for Public Welfare Project (No. 201204612). 1919



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REFERENCES

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