11β-HSD1 Inhibitors from Walsura cochinchinensis - Journal of

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11β-HSD1 Inhibitors from Walsura cochinchinensis Mei-Ling Han, Yu Shen, Guo-Cai Wang, Ying Leng, Hua Zhang,* and Jian-Min Yue* State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People’s Republic of China S Supporting Information *

ABSTRACT: A search for inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) from Walsura cochinchinensis yielded 10 new limonoids, cochinchinoids A−J (1−10), and two new triterpenoids, 3-epimesendanin S (11) and cochinchinoid K (12). Their structures were assigned on the basis of spectroscopic data, with the absolute configurations of 1 and 12 being established by X-ray diffraction analysis. Of these compounds, cochinchinoid K (12) displayed inhibitory activity against mouse 11β-HSD1 with an IC50 value of 0.82 μM. spectrum showed absorption bands of hydroxy (3433 cm−1) and carbonyl (1743 and 1712 cm−1) groups. Analysis of the NMR data (Tables 1 and 4) revealed characteristic signals of an α,β-unsaturated carbonyl, a 3-substituted furanyl group, two 2methylbutyryloxy moieties, four oxygenated methines, an oxygenated methylene, and four tertiary methyl carbons. These functionalities accounted for seven indices of hydrogen deficiency requiring five extra rings in the structure of 1. In addition, two exchangeable hydroxy protons were deducible after all other NMR resonances were assigned with the aid of DEPT and HSQC data. The above observations indicated that 1 was likely a vilasinin-type limonoid featuring a 6,28-ether bridge.9 HMBC data (Figure 1) enabled assembly of the aforementioned fragments to corroborate the vilasinin backbone of 1, placing it in the ring-intact series of limonoids.9 More specifically, the two 2-methylbutyryloxy residues were assigned to C-3 and C-7, as supported by the HMBC correlations from H-3 and H-7 to corresponding ester carbonyls, while the presence of 1-OH and 17-OH was indicated by the chemical shifts of C-1 (δC 71.61) and C-17 (δC 80.65), respectively. The relative configuration of 1 was characterized mainly by interpretation of ROESY data (Figure 1). The strong NOE interactions of H3-29 with H-2β, H-6, and H3-19; H3-19 with H-2β, H-6, and H3-30; and H3-30 with H-6 indicated that H2β, H-6, Me-19, Me-29, and Me-30 were β-axially oriented, hence giving the 6,28-ether bridge an α-orientation. In addition, the magnitude of J5,6 (12.5 Hz) suggested that H-5 was α-axially oriented, consequently establishing that H-9 and Me-18 were α-oriented as supported by the ROESY correlations of H-9 with both H-5 and H3-18. Moreover, H-1, H-3, and H-7 were determined to be β-equatorially oriented on the basis of the small values of J1,2β (2,4 Hz), J2β,3 (2.8 Hz), and J6,7 (3.2 Hz), respectively, thus leaving the 1-, 3-, and 7-substituents all α-

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is an enzyme that regulates interconversion of the active/inactive forms of glucocorticoids and acts tissue-specifically depending on the presence/absence of NADPH cofactor.1,2 As 11β-HSD1 is widely expressed in many tissues such as the liver and central nervous system, it has recently become an attractive therapeutic target for the treatment of a number of diseases especially metabolic disorders.2,3 Consequently, the development of 11βHSD1 inhibitors has become a hot research topic.4,5 Walsura cochinchinensis (Baill.), one of only three Chinese species of the Walsura genus (family Meliaceae),6 has proven to be a source of structurally interesting and biologically attractive nortriterpenoids.7 As part of a project supported by the National Natural Science Foundation (NNSF, No. 21272244) that is focusing on the identification of potential 11β-HSD1 inhibitors from this genus, a preliminary fractionation of the EtOH extract of W. cochinchinensis afforded two rearranged limonoids with a new carbon framework from the inactive fraction.8 Analysis of the fractions showing inhibition against 11β-HSD1 resulted in the isolation and identification of 12 new compounds including four vilasinin-class (1−4) and three azadirone-type (8−10) ring-intact limonoids, three ring D-seco limonoids (5−7), and two tirucallane-type triterpenoids (11 and 12), along with a known tirucallane analogue (13). Compounds 12 and 13 were moderate inhibitors of both human and mouse 11β-HSD1, while compound 8 showed selective inhibition against mouse 11β-HSD1. Herein we report the purification, structure characterization, and biological testing of these compounds.

■

RESULTS AND DISCUSSION

Vilasinin-Class Limonoids. Compound 1 was obtained as colorless crystals (mp 205−207 °C) with an [α]D value of −26.7 (in MeOH). HRESI(−)MS analysis revealed a quasi molecular ion at m/z 671.3422 ([M + HCO2]−, calcd 671.3431) that suggested a molecular formula of C36H50O9 incorporating 12 indices of hydrogen deficiency. The IR © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 6, 2013

A

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resemblance and ROESY data. The structure of 2 was thus identified and named cochinchinoid B. The molecular formula of compound 3 was established to be C36H46O8, as indicated by the quasi molecular ion at m/z 651.3184 ([M + HCO2]−, calcd 651.3169) in the HRESI(−)MS analysis. Comparison of the NMR data (Tables 1 and 4) of 3 with those of 1 revealed that they were structural analogues of the vilasinin class. The main differences were signals of two tigloyloxy groups in 3 in place of those of the two 2methylbutyryloxy fragments in 1 and the resonances of an extra methine (δC 60.7 and δH 3.38, CH-17) in 3 replacing the oxygenated quaternary carbon (δC 80.65, C-17) in 1. These structural differences were confirmed by the HMBC correlations from H-3 (δH 5.14) and H-7 (δH 5.78) to the respective tigloyloxy carbonyls (δC 166.7 and 166.5) and from H-22 (δH 6.24) to C-17. The relative configuration of 3 was the same as that of the core structure of 1 via NMR comparison and ROESY data. In particular, the β-oriented H-17 was supported by its NOE interaction with H-12β (δH 2.05). The structure of 3 was thereby established and named cochinchinoid C. Compound 4 displayed a protonated molecular ion peak at m/z 667.3478 in the HRESI(+)MS spectrum, consistent with a molecular formula of C38H50O10 (calcd 667.3482). Analysis of the NMR data (Tables 1 and 4) revealed that 4 was a vilasinintype limonoid.9 Compared with 3, limonoid 4 incorporated a 7O-(2-methylbutyryl) fragment instead of the 7-tigloyloxy unit and a 12-acetoxy group (δH 2.11; δC 170.0 and 21.5), which was confirmed by the HMBC correlations from H-7 (δH 5.74) and H-12 (δH 5.20) to the corresponding ester carbonyls (δC 175.0 and 170.0, respectively). The configurations of C-1, C-3, and C7 were identical with those of 3, as deduced from the coupling patterns of H-1, H-3, and H-7, while the configuration of the C12 stereocenter was assigned on the basis of the strong ROESY correlation between H-12 and H3-18 (δH 1.10). The configuration of the 2-methylbutyryloxy moiety remained unassigned (as with most cases in the literature9) owing to the lack of data from comparable analogues such as the examples of 1/2 and 6/7 (see below). The structure of compound 4 was hence identified and named cochinchinoid D. D-seco Limonoids. Compound 5 exhibited a sodiated molecular ion at m/z 721.2847 (calcd 721.2836) in the HRESI(+)MS analysis, supportive of a molecular formula of C37H46O13. The NMR data (Tables 1 and 4) showed typical signals of a β-furanyl group (δH 7.39, 7.37, and 6.29), four tertiary methyls (δH 1.32, 1.37, 1.23, and 1.18), a tigloyloxy residue (δC 166.5, 137.7, 128.7, 14.4, and 12.1), and three acetoxy units (δH 2.08, 2.08, and 1.91). The NMR data indicated that 5 incorporated a D-seco limonoid scaffold, as evidenced by the lactone carbon at δC 166.7.9 HMBC data (Figure 3) facilitated assembly of the gross structure of 5, displaying identical structural fragments to piscidofuran,13 including the 6,28-ether bridge and the 14,15-oxirane moiety. Compared with piscidofuran,13 compound 5 featured one more acetyl and one more acetoxy group, which were located at C-7 and C-11 via the long-range HMBC correlations from H-7 (δH 5.06) and H-11 (δH 5.10) to the corresponding ester carbonyls at δC 169.0 and 169.7, respectively. The locations of the 1acetoxy and 3-tigloyloxy moieties were confirmed via HMBC data. The relative configuration of 5 was assigned on the basis of the ROESY data (Figure 3) and comparison of the 1H NMR couplings with those of piscidofuran.13 As with piscidofuran, H2β, H-6, Me-19, Me-29, and Me-30 were determined to be β-

oriented. Finally, 17β-OH was assigned via comparison of NMR data with other limonoids possessing the same C-, D-, and E-ring structural fragments.10,11 While a limited amount of sample prevented us from elucidating the configuration of the 2-methylbutyryloxy moieties by chemical means, crystallization of 1 in methanol with a trace of water afforded high-quality crystals suitable for X-ray diffraction analysis. The X-ray data confirmed the relative configuration of 1 assigned by spectroscopic methods and also established its absolute configuration as 1S, 3R, 4R, 5R, 6R, 7S, 8R, 9R, 10R, 13R, 17S, 2′R, 2″R [absolute structure parameter: −0.05(18)].12 The structure of 1 was thus unequivocally characterized and named cochinchinoid A. Compound 2 was assigned the same molecular formula as 1 via the HRESI(−)MS ion at m/z 671.3442 ([M + HCO2]−, calcd 671.3431), indicative of their isomeric nature. Analysis of the NMR data (Tables 1 and 4) confirmed this conclusion, revealing that most resonances of 2 and 1 were nearly superimposable (ΔδH < 0.03 and ΔδC < 0.05 ppm). The major NMR differences were associated with carbon signals (ΔδC > 0.05 ppm) of the two respective 2-methylbutyryloxy moieties and of those of the aglycone in close proximity of the two substituents, i.e., C-2 (ΔδC 0.10), C-4 (ΔδC 0.08), and C-18 (ΔδC 0.06). This indicated that 2 differed from 1 only at the two ester substituents possessing 2′S and 2″S configurations. Such an assignment was supported by the fact that 1 and 2 were separated via an achiral YMC-Pack ODS-A HPLC column. The relative configuration of the core skeleton of 2 was identical with that of 1 based on excellent NMR B

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Table 1. 1H NMR Data for Compounds 1−5 in CDCl3a

a

position

1

2

3

4

1 2α 2β 3 5 6 7 9 11 12α 12β 15 17 18 19 21 22 23 28α 28β 29 30 2′ 3′ 4′ 5′ 2″ 3″ 4″ 5″ 1-OH 1-OAc 7-OAc 11-OAc 12-OAc 17-OH

3.58, ddd (8.7, 3.2, 2.4) 2.34,b m 1.99, ddd (16.3, 2.8, 2.4) 5.11, dd (2.8, 2.8) 2.32,b d (12.5) 4.22, dd (12.5, 3.2) 5.71, d (3.2) 2.88, dd (11.8, 7.3) 1.76, 1.81, m 1.58, ddd (13.2, 9.8, 2.9) 2.46,c m 5.90, s

3.58, ddd (8.8, 3.3, 2.3) 2.35,d m 1.99, ddd (16.2, 2.9, 2.3) 5.12, dd (2.9, 2.9) 2.33,d d (12.5) 4.22, dd (12.5, 3.2) 5.71, d (3.2) 2.89, dd (12.0, 7.5) 1.76, 1.81, m 1.63, s 2.46,e m 5.90, s

3.55, brs 2.03, m 2.35,i ddd (16.4, 3.3, 2.6) 5.15, dd (2.6, 2.6) 2.38,i d (12.6) 4.25, dd (12.6, 2.9) 5.74, d (2.9) 3.04, dd (12.9, 7.1) 1.67, 2.40,i m 5.20, d (8.4)

1.003,k s 0.998,k s 7.54, dd (1.7, 0.8) 6.37, dd (1.7, 0.8) 7.40, dd (1.7, 1.7) 3.38, brd (7.6) 3.54, d (7.6) 1.19, s 1.31, s 2.32,b m 1.45, 1.68, m 0.89, t (7.5) 1.16, d (7.0) 2.23, m 1.38, 1.65, m 0.82, t (7.4) 1.10, d (7.0) 2.46,c d (8.7)

1.01, s 1.01, s 7.55, dd (1.7, 0.8) 6.37, dd (1.7, 0.8) 7.41, dd (1.7, 1.7) 3.36, brd (7.6) 3.54, d (7.6) 1.20, s 1.31, s 2.32,d m 1.49, 1.67, m 0.92, t (7.5) 1.13, d (6.9) 2.24, m 1.38, 1.64, m 0.83, t (7.4) 1.10, d (7.1) 2.48,e d (8.8)

3.60, ddd (8.1, 3.4, 2.2) 2.04,f m 2.37, ddd (16.2, 3.4, 2.8) 5.14, dd (2.8, 2.8) 2.43, d (12.6) 4.24, dd (12.6, 3.1) 5.78, d (3.1) 3.08, dd (11.3, 7.4) 1.79,g m, 2H 1.77,b−j m 2.05,f m 5.98, s 3.38, s 1.03, s 1.02, s 7.44, brs 6.24, brd (1.6) 7.40, dd (1.6, 1.6) 3.30, brd (7.7) 3.52, d (7.7) 1.21, s 1.31, s 6.77,h m 1.78,g dq (7.1, 1.1) 1.80,g brs 6.80,h m 1.71, dq (7.1, 1.1) 1.75, dq (1.1, 1.1) 2.49, d (8.1)

6.07, s 3.73, s 1.10, s 0.98, s 7.40,j m 6.17, brs 7.39,j m 3.31, brd (7.9) 3.53, d (7.9) 1.20, s 1.41, s 6.80, 1.79, 1.82, 2.19, 1.34, 0.80, 1.05,

brq (7.2) brd (7.2) brs m 1.63, m t (7.4) d (7.0)

5 4.82, 2.21, 2.31, 4.97, 2.58, 4.11, 5.06, 2.94, 5.10, 2.12, 1.49, 3.77, 5.56, 1.27, 1.23, 7.37, 6.29, 7.39, 3.55, 3.52, 1.19, 1.32,

dd (3.0, 2.4) ddd (16.8, 3.0, 3.0) ddd (16.8, 2.4, 2.4) dd (3.0, 2.4) d (12.7) dd (12.7, 3.1) d (3.1) d (3.5) ddd (9.6, 3.9, 3.5) dd (15.1, 9.6) dd (15.1, 3.9) s s s s brs brs dd (1.7, 1.7) brd (7.7) d (7.7) s s

6.88, dq (7.1, 1.3) 1.76, brd (7.1) 1.83, brs

1.91, s 2.08, s 2.08, s 2.11, s 2.50, s

2.43, s

Chemical shifts (ppm) referenced to solvent peak (δH 7.26) at 400 MHz.

axially oriented via the NOE interactions of H-6/H3-29, H3-29/ H-2β, H-2β/H3-19, and H3-19/H3-30, while H-5, H-9, and Me18 were determined to be α-axially oriented via J5,6 (12.7 Hz) and ROESY correlations of H-5/H-9 and H-9/H3-18. In addition, the configurations of C-1, C-3, C-7, and C-11 were established on the basis of the small coupling constants of J1,2α/β (3.0/2.4 Hz), J2α/β,3 (3.0/2.4 Hz), J6.7 (3.1 Hz), and J9,11 (3.5 Hz). The assignment of H-12α was supported by its strong ROESY correlation with H-11, and H-17 was determined to be β-oriented as evidenced by the cross-peak of H-12β/H-17. Finally, the 14β,15β-oxirane moiety was corroborated by the strong NOE interaction between H-7 and H-15. The structure of 5 was thereby deduced and named cochinchinoid E. Compounds 6 and 7 were inseparable even on a CHIRALPAK AD-H column, indicative of a pair of highly similar analogues. These compounds were assigned the same molecular formula, C37H48O13, via the HRESI(+)MS ion at m/z 701.3185 ([M + H]+, calcd 701.3173), which was in accord with dihydro derivatives of 5. The NMR data of 6/7 (Tables 2 and 4) were close to those of 5, showing signals supportive of a 2-methylbutyryloxy residue instead of those of the tigloyloxy group at C-3 in the latter. This was supported by an HMBC

b−j

Overlapping signals. kInterchangeable assignments.

correlation from H-3 (δH 4.91/4.92) to an unconjuaged ester carbonyl (δC 175.4). Comparison of the remaining NMR resonances of 6/7 and 5, including key 1H−1H couplings, were supportive of a common configuration for the molecular backbones of these metabolites. Moreover, most of the NMR signals of 6 and 7 were indistinguishable, revealing only one set of signals except for those of the A-ring and the 2methylbutyryloxy moiety. Therefore, the structures of 6 and 7 were identified to possess different substituents at C-3, and by comparing their NMR data with those of 1 and 2, 2′R and 2′S configurations were assigned to the C-3 ester substituents of 6 and 7, respectively. Compounds 6 and 7 were named cochinchinoids F and G, respectively. Azadirone-Type Limonoids. The molecular formula of C33H42O7 for compound 8 was determined via the quasi molecular ion at m/z 595.2910 ([M + HCO2]−, calcd 595.2907) in the HRESI(−)MS spectrum. The NMR data (Tables 2 and 4) revealed diagnostic signals of a limonoid, including a β-substituted furanyl moiety (δH 7.43, 7.42, and 6.20), five tertiary methyls (δH 1.60, 1.51, 1.10, 1.07, and 0.99),9 an acetoxy (δC 170.1 and 21.7), and 2-methylbutyryloxy (δC 175.5, 41.6, 26.5, 17.8, and 12.3) groups. Other readily C

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Table 2. 1H NMR Data for Compounds 6−10 in CDCl3a position

8

9

10 α 1.50, ddd (13.0, 11.1, 6.8) β 1.91, ddd (13.0, 7.3, 3.5) α 2.45, ddd (16.1, 6.8, 3.5) β 2.60, ddd (16,1, 11,1, 7.3)

4.77, brs

7.10, d (10.3)

7.06, d (10.2)

2

2.22, 2.26, m

5.93, d (10.3)

5.89, d (10.2)

3 5 6

4.91,b dd/4.92,b dd 2.44,c d (12.8)/2.45,c d (12.7) 4.08, dd (12.7, 3.2) 5.05, d (3.2) 2.94, d (3.4) 5.12, ddd (9.5, 3.8, 3.4)

12

α 2.12, dd (15.1, 9.5) β 1.48, dd (15.1, 3.8) 3.75, s 5.55, s 1.26, s 1.22, s 7.37, brs 6.29, brs 7.39, dd (1.7, 1.7) α 3.33, brd (7.8) β 3.52, d (7.8) 1.18, s 1.31, s 2.26, m 1.37−1.48, 1.62−1.73, m 0.88, t (7.4) 1.15,d d (7.1)/1.12,d d (6.9) 2.03, s 2.08, s 2.07, s

α 2.81, dd (13.7, 9.3) β 1.68, dd (13.7, 7.2) 5.99, s 3.41, s 0.99, s 1.51, s 7.43,g brs 6.20, brs 7.42,g brs 1.07, s

2.19, brd (12.6) α 1.90, brd (14.8) β 2.05, brdd (14.8, 12.6) 5.38, brs 2.53, dd (12.5, 6.9) α 2.62, ddd (15.6, 8.1, 6.9) β 1.83, dd (15.6, 12.5) 5.25, d (8.1)

1.86,h m 1.72, 2.38, m

7 9 11

2.18, brd (12.6) α 1.91, dd (13.9, 2.9) β 2.11, ddd (13.9, 12.6, 2.0) 5.34, dd (2.9, 2.0) 2.59, d (6.0) 5.82, ddd (9.3, 7.2, 6.0)

5.95, s 3.75, s 1.05,e,j s 1.22, s 7.41,f brs 6.17, brs 7.40,f brs 1.06,e,j s

5.96, s 3.72, s 1.060,i,k s 1.10, s 7.42, m 6.18, m 7.40, dd (1.6, 1.6) 1.01, s

1.10, 1.60, 2.16, 1.34, 0.79, 1.05,

1.09, s 1.46, s 2.21, m 1.35, 1.60, m 0.80, t (7.4) 1.06,e d (7.0)

1.057,i,k s 1.43, s 2.22, m 1.37, 1.64, m 0.82, t (7.4) 1.08, d (7.0)

15 17 18 19 21 22 23 28 28 29 30 2′ 3′ 4′ 5′ 1-OAc 7-OAc 11-OAc 12-OAc a

6/7

1

s s m 1.62, m t (7.4) d (7.1)

5.36, brs 2.35, m α 1.84,h m β 2.01, m 5.21, d (7.8)

2.14, s 2.12, s

Chemical shifts (ppm) referenced to solvent peak (δH 7.26) at 400 MHz.

distinguishable resonances included two α,β-unsaturated carbonyls (δC 203.6, 155.3, and 126.7; 204.0, 190.5, and 124.9) and two sp3 oxymethine carbons (δC 74.3 and 67.2), suggesting that 8 was a ring-intact limonoid of the azadirone class,9 but with a higher degree of oxidation. Analysis of the HMBC data (Figure 4) verified the azadirone skeleton and further confirmed the existence of the 16-oxo-Δ14,15 moiety and the 11-acetoxy group based on the correlations from H-17 (δH 3.41) to C-14, C-15, and C-16 (δC 190.5, 124.9, and 204.0, respectively) and from H-11 (δH 5.82) to the acetyl carbonyl carbon (δC 170.1). The 2-methylbutyryloxy fragment was allocated to C-7 via the HMBC cross-peak between H-7 (δH 5.34) and the corresponding ester carbonyl carbon (δC 175.5). The relative configuration of 8 was established via the ROESY data (Figure 4). The cross-peaks of H-6β/H3-29, H3-29/H3-19, and H3-19/H3-30 indicated that H-6β, Me-19, Me-29, and Me30 were β-axially oriented as in 1. The J5,6 value (12.6 Hz) suggested that H-5 was α-axially oriented, while the correlations of H-5/H-9 and H-9/H3-18 supported α-orientations for both H-9 and Me-18. Furthermore, H-7 was established to be βequatorially oriented on the basis of the magnitudes of J6α/β,7 (2.9/2.0 Hz), while H-11 was determined to be α-oriented via the relatively small J9,11 value (6.0 Hz), in contrast to J9,11α (7.3 Hz) and J9,11β (11.8 Hz) of 1. Finally, H-17 was assigned a βorientation via its ROESY correlation with H-12β (δH 1.68).

b−i

2.12, s

Overlapping signals.

j,k

Interchangeable assignments.

The structure of 8 was thereby elucidated and named cochinchinoid H. Compounds 9 and 10 had molecular formulas of C33H42O7 and C33H44O7, as deduced from the HRESI(−)MS ions at m/z 595.2910 and 597.3068 (both [M + HCO2]−, Δmmu 0.3 and 0.4), suggestive of isomeric and dihydro analogues of 8, respectively. As with 8, the NMR data of 9 (Tables 2 and 4) revealed characteristic resonances of an azadirone-type limonoid, with the only difference being the alteration of 1 H−1H coupling patterns of the C-ring. This supported the shift of the acetoxy group at C-11 in 8 to C-12 in 9, as evidenced by the couplings of H-9 (δH 2.53) with a methylene (δH 2.62 and 1.83, H2-11) and the HMBC correlation from H318 (δH 1.05) to an oxymethine (δC 70.3, C-12). The NMR data (Tables 2 and 4) of 10 exhibited more similarities to 9 than to 8 and revealed most signals that were observed for 9, including the 3-(2-methylbutyryl)oxy and 12-acetoxy functionalities. The major differences were associated with resonances of the A-ring, where the α,β-unsaturated carbonyl carbons (δC 156.1, 126.4, and 203.9) in 9 were replaced by two methylene carbons (δC 38.5 and 33.8; C-1 and C-2) and a nonconjugated carbonyl (δC 215.7, C-3) in 10, supporting that 10 was a 1,2-dihydro derivative of 9. The planar structures of 9 and 10 were further corroborated by HMBC data, with their relative configurations being established via ROESY data. In particular, the 12βD

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Table 3. 1H NMR Data for Compounds 11 and 12 in CDCl3a position

11

1

1.48,b 2H, m

2 3 5 6

1.61,c 1.88,d m 3.47, dd (2.6, 2.6) 1.77, dd (11.9, 5.7) 1.92,e 2.06, m

7 9 11 12 15 16 17 18 19 20 21 22 23 24 26 27 28 29 30 22-OH

5.30, dd (6.2, 2.9) 2.32, m 1.47,b 2.22,f m 3.85, dd (8.4, 7.8) 1.58,c 2H, m 1.95,e 2.25,f m 2.54, ddd (10.0, 10.0, 8.2) 0.83, s 0.80, s 1.89,d m 0.75, d (6.8) 4.21, brs 6.09, 1.98, 2.23, 0.94, 0.92, 1.14, 3.69,

brs s s s s s brs

oxymethine carbon (δC 79.3, C-3) and from H-20 (δH 2.69) to an ester carbonyl (δC 178.2, C-12). Furthermore, formation of this γ-lactone ring was corroborated by the strong IR absorption band at 1765 cm−1, excluding the possibility of six- and seven-membered lactone moieties that would appear at lower wavenumbers below 1750 cm−1.16 The β-orientation of 3-OH was assigned via the splitting pattern of the H-3 signal (dd, J = 11.2, 4.2 Hz). The assignment of the C-24 configuration via NMR data proved challenging, as the couplings of H-24 in both epimers were similar.15 However, crystals suitable for X-ray diffraction studies were obtained in a ternary solvent system of n-hexane/acetone/MeOH (10:1:0.2). The subsequent X-ray analysis permitted assignment of the absolute configuration (Figure 5) of 12 as 3S, 5R, 9R, 10R, 13S, 14S, 17S, 20S, 23R, 24S [absolute structure parameter: 0.07(12)].12 The structure of 12 was thus established and named cochinchinoid K. While the absolute configurations of compounds 1 and 12 were unambiguously established via X-ray data, lack of direct evidence precluded confirmative assignments for those of the other analogues. Nevertheless, given the co-occurrence of these metabolites in W. cochinchinensis, we tentatively assigned the absolute configurations of the core backbones of 2−11 and 13 as shown on the basis of biogenetic considerations. Preliminary testing of the pure isolates at 10 μM in the scintillation proximity assay17,18 revealed that cochinchinoid K (12) and mesendanin S (13) showed >60% inhibition against both human and mouse 11β-HSD1, while cochinchinoid H (8) selectively inhibited human 11β-HSD1 with weaker activity (>50%). In addition, 3-epimesendanin S (11) exhibited ca. 45% inhibition against both types of 11β-HSD1 at 10 μM. Further bioassays (Figure 6) established that 8, 12, and 13 were moderate human 11β-HSD1 inhibitors with IC50 values of 11.44, 3.20, and 3.74 μM, respectively, while 12 and 13 displayed more potent inhibition against mouse 11β-HSD1 with IC50 values of 0.82 and 1.15 μM, respectively.

12 α 1.13, ddd (13.4, 12.2, 4.2) β 1.68,g m 1.60,h 1.65,g m 3.23, dd (11.2, 4.2) 1.31 (m) α 2.14, m β 1.97, (dddd, 17.8, 12.0, 3.3, 2.4) 5.27, dd (6.4, 2.9) 2.21,i m 1.57,h 2H, m 1.70,g 1.78, m 1.55,h 2H, m 1.49, 1.84, m 2.35,j m 0.82, s 0.75, s 2.69, ddd (12.0, 9.0, 5.8) 2.19,i 2.39,j m 4.59, ddd (10.1, 6.1, 1.8) 3.28, d (1.8) 1.29, s 1.34, s 0.96, s 0.86, s 1.02, s

a

Chemical shifts (ppm) referenced to solvent peak (δH 7.26) at 400 MHz. b−jOverlapping signals.

acetoxy groups in 9 and 10 were assigned on the basis of the correlation between H-12 and H3-18. The structures of 9 and 10 were hence identified and named cochinchinoids I and J, respectively. Tirucallane-Type Triterpenoids. HRESI(−)MS analysis of compound 11 revealed a quasi molecular ion at m/z 517.3521 ([M + HCO2]−, calcd 517.3529), indicative of a molecular formula of C30H48O4 and isomeric with mesendanin S (13).14 Analysis of the NMR data (Tables 3 and 4) confirmed this deduction, with diagnostic couplings of H-3/H2-2 (dd, J = 11.2, 4.2 Hz; 3α-H) in 13 being resolved in 11 in a different pattern (dd, J = 2.6, 2.6 Hz; 3β-H), together with minor variations for other NMR resonances of the A-ring. These observations were in agreement with an inversed C-3 configuration in 11. Excellent comparison between the remaining NMR data of the two metabolites supported common structural features, including the configurations at all other stereogenic centers and double bonds, which were also validated by ROESY data. Triterpenoid 11 was thus identified as the C-3 epimer of mesendanin S (13). The molecular formula (C30H48O5) of compound 12 was established via the HRESI(−)MS ion at m/z 533.3486 ([M + HCO2]−, calcd 533.3478), indicative of an isomeric analogue of melianodiol.15 The NMR data (Tables 2 and 4) confirmed that 12 possessed the same tirucallane scaffold as melianodiol,15 with the only difference being the presence of the 3-hydroxy and 21-lactone functionalities versus the 3-oxo and 21hemiketal groups in the latter. This was supported by the HMBC correlations from H3-28/29 (δH 0.96/0.86) to an

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined on a Perkin-Elmer 341 polarimeter. Melting points were measured on an SGM X-4 apparatus (Shanghai Precision & Scientific Instrument Co., Ltd.). UV data were acquired on a Shimadzu UV-2550 spectrophotometer. IR spectra were recorded on a Perkin-Elmer 577 spectrometer using KBr disks. NMR experiments were preformed in CDCl3 on a Bruker AM-400 spectrometer referenced to solvent peaks (δH 7.26; δC 77.16). ESIMS and HRESIMS analyses were carried out on a Bruker Daltonics Esquire3000plus and a Waters-Micromass QTOF Ultima Global mass spectrometer, respectively. Semipreparative HPLC was performed on a Waters 1525 binary pump system with a Waters 2489 detector (210 nm) using a YMC-Pack ODS-A (250 × 10 mm, S-5 μm) or a Daicel CHIRALPAK AD-H (250 × 10 mm, S-5 μm) column. Silica gel (200−300 mesh, Qingdao Haiyang Chemical Co. Ltd.), C18 reversed-phase (RP-18) silica gel (150−200 mesh, Merck), CHP20P MCI gel (75−150 μm, Mitsubishi Chemical Industries, Ltd.), D101-macroporous absorption resin (Shanghai Hualing Resin Co., Ltd.), and Sephadex LH-20 gel (Amersham Biosciences) were used for column chromatography (CC). Precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd.) were used for TLC detection. All solvents used for CC were of analytical grade (Shanghai Chemical Reagents Co., Ltd.), and solvents used for HPLC were of HPLC grade (J & K Scientific Ltd.). Plant Material. The twigs and leaves of W. cochinchinensis were collected in July 2010 from Hainan Province, China, and were identified by Prof. S.-M. Huang, Department of Biology, Hainan University, China. A voucher specimen has been deposited in Shanghai E

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Table 4. 13C NMR Data for Compounds 1−12 in CDCl3a position

1

2

3

4

5

6/7

8

9

10

11

12

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 1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″ 4″ 5″ 1-OAc

71.61 30.28 73.34 42.36 40.71 72.37 72.57 46.51 34.77 40.37 14.23 21.68 50.73 193.09 121.07 205.86 80.65 30.44 15.71 122.85 141.53 109.74 142.90

71.63 30.38 73.32 42.44 40.67 72.40 72.60 46.51 34.76 40.40 14.23 21.68 50.73 193.10 121.05 205.85 80.66 30.50 15.71 122.84 141.55 109.74 142.92

71.6 30.4 73.6 42.6 40.8 72.5 73.0 46.4 34.7 40.4 14.65 30.3 48.6 192.2 124.2 205.4 60.7 26.9 15.6 118.8 141.8 111.4 142.8

71.4 30.3 73.3 42.6 40.8 72.2 73.0 46.0 34.4 40.1 24.4 70.6 51.2 188.1 126.2 204.6 52.7 24.9 15.7 118.1 142.2 111.2 143.0

73.0 27.2 71.1 42.7 42.1 72.3 73.9 44.1 39.6 40.1 66.4 38.4 37.9 68.6 54.4 166.7 78.2 18.5 17.3 120.0 141.3 109.8 143.4

71.07/71.12 27.6 73.1 42.42/42.49 41.9 72.2 73.8 44.1 39.5 40.1 66.4 38.4 37.8 68.5 54.3 166.7 78.2 18.4 17.1 120.0 141.3 109.8 143.4

155.3 126.7 203.6 44.1 47.3 24.0 74.3 44.3 42.2 40.6 67.2 40.1 46.5 190.5 124.9 204.0 60.4 26.1 21.7b 118.1 141.8 111.1 143.1

156.1 126.4 203.9 44.3 46.5 23.6 73.7 44.4 37.9 39.9 25.4 70.3 50.8 188.2 125.5 204.3 52.9 25.1 19.3 117.9 142.1 111.1 143.1

38.5 33.8 215.7 47.0 48.5 25.5 74.3 43.8 42.3 37.1 24.1 70.5 51.0 188.6 125.6 204.5 52.8 24.9 15.3 118.0 142.2 111.1 143.1

77.78 19.25 24.95 175.03 41.41 26.58 11.78 16.85 175.16 41.57 26.58 11.98 17.46

77.74 19.26 24.92 175.10 41.27 26.57 11.74 16.60 175.14 41.63 27.01 11.99 17.44

78.2 19.2 26.0 166.7 128.0 138.7 12.4 14.69 166.5 128.3 137.6 12.3 14.5

78.0 19.1 24.8 166.5 128.0 138.7 14.7 12.3 175.0 41.9 26.8 12.1 17.6

78.3 19.4 20.4 166.5 128.7 137.7 12.1 14.4

78.42/78.36 19.4 20.4 175.4 41.54/41.36 26.89/26.32 11.97/11.83 17.13/16.32

27.3 21.6b 28.2 175.5 41.6 26.5 12.3 17.8

27.1 21.4 25.8 175.5 41.7 26.6 12.2 17.6

26.0 21.1 25.4 175.6 41.8 26.7 12.2 17.7

31.4 25.6 76.4 37.5 44.6 24.1 119.1 145.1 48.0 34.9 29.9 74.9 48.1 51.9 35.2 28.4 40.8 19.9 13.1 39.8 11.7 78.7 201.2 119.2 160.1 28.2 21.6 27.9 21.9 28.3

37.3 27.8 79.3 39.1 50.9 24.1c 118.4 145.3 49.0 35.2 17.6 31.3 43.9 50.6 34.0 24.0c 47.3 23.6 13.2 40.4 178.2 29.7 77.4 76.3 72.7 26.7 26.7 27.7 14.8 27.5

169.1 21.3 169.0 21.7 169.7 20.9

169.23/169.26 21.51/21.54 169.8 20.9 169.1 21.7

170.1 21.7b 169.9 21.3

170.0 21.5

7-OAc 11-OAc 12-OAc a

170.0 21.5

Chemical shifts (ppm) referenced to solvent peak (δC 77.16) at 125 MHz.

Institute of Materia Medica, Chinese Academy of Sciences (WACO2007HN-1Y). Extraction and Isolation. The air-dried powder of leaves and twigs of W. cochinchinensis (11 kg) was extracted with 95% EtOH at room temperature to give a crude extract (280 g), which was partitioned between EtOAc and H2O. The EtOAc-soluble partition (130 g) was fractionated on a column of macroporous resin eluted with 30, 80, and 100% MeOH/H2O, and the 80% MeOH elution (90 g) was separated by an MCI gel column (MeOH/H2O, 4:6 to 9:1) to afford seven fractions (A−G). Fraction C (15.0 g) was subjected to CC eluted with petroleum ether/acetone (50:1 to 1:3) to yield 17

b,c

Interchangeable assignments.

fractions (C1−C17), fraction C11 of which was further separated over a column of RP-18 silica gel (MeOH/H2O, 55% to 75%) to give seven subfractions (C11a−C11g). Subfraction C11g was sequentially purified by silica gel CC (CH3Cl/MeOH, 200:1 to 100:1) and semipreparative HPLC (3.0 mL/min, isocratic elution with 70% MeOH/H2O) to give compounds 1 (5 mg) and 2 (2 mg). Subfraction C11f was first subjected to silica gel CC (CH3Cl/MeOH, 200:1 to 100:1) and further purified by HPLC to yield compound 4 (7 mg). Fraction C12 was fractionated in sequence by RP-18 silica gel (MeOH/H2O, 55% to 70%) CC, silica gel CC (CH3Cl/MeOH, 500:1 to 150:1), and finally semipreparative HPLC to afford compounds 3 (2 F

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Figure 1. Key 2D NMR correlations of 1. (Note: C-3 and C-7 substituents, along with the furanyl moiety, were generalized as R groups in the 3D structure to reduce atom overlapping.)

Figure 3. Key 2D NMR correlations of 5. (Note: C-1, C-3, C-7, and C-11 substituents, along with the furanyl moiety, were generalized as R groups in the 3D structure to reduce atom overlapping.)

mg), 5 (31 mg), 6/7 (25 mg), and 12 (9 mg). Further efforts failed to separate 6/7 even on a Daicel CHIRALPAK AD-H column. Fraction D was separated over columns of silica gel (petroleum ether/acetone, 100:1 to 1:2), RP-18 silica gel (MeOH/H2O, 50% to 80%), and silica gel (CH3Cl/MeOH, 500:1 to 100:1), and was eventually purified by semipreparative HPLC to yield compounds 8 (5 mg), 9 (15 mg), 10 (3 mg), 11 (3 mg), and 13 (3 mg). Cochinchinoid A (1): colorless crystals (MeOH/H2O, 100:1); mp 205−207 °C; [α]22D −26.7 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 246 (4.10) nm; IR (KBr) νmax 3433, 2964, 2931, 1743, 1712, 1599, 1462, 1385, 1277, 1182, 1155, 1084, 1026, 874, 793 cm−1; 1H and 13C NMR (CDCl3) see Tables 1 and 4; ESI(+)MS m/z 627.5 [M + H]+, 649.3 [M + Na]+, 1275.8 [2 M + Na]+; ESI(−)MS m/z 671.6 [M + HCO2]−; HRESI(−)MS m/z 671.3422 [M + HCO2]− (calcd for C37H51O11, 671.3431). Cochinchinoid B (2): white powder; [α]22D +8 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 246 (4.07) nm; IR (KBr) νmax 3477, 2970, 2935, 1734, 1709, 1631, 1597, 1460, 1385, 1263, 1180, 1155, 1082, 1051, 1026, 874 cm−1; 1H and 13C NMR (CDCl3) see Tables 1 and 4; ESI(+)MS m/z 627.5 [M + H]+, 1275.8 [2 M + Na]+; ESI(−)MS m/z 671.6 [M + HCO2]−; HRESI(−)MS m/z 671.3442 [M + HCO2]− (calcd for C37H51O11, 671.3431). Cochinchinoid C (3): white powder; [α]22D −10 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 214 (4.56) nm; IR (KBr) νmax 3437, 2929, 1705, 1649, 1599, 1458, 1390, 1257, 1153, 1076, 1030, 874, 733 cm−1;

Figure 4. Key 2D NMR correlations of 8. (Note: C-7 and C-11 substituents, along with the furanyl moiety, were generalized as R groups in the 3D structure to reduce atom overlapping.) 1

H and 13C NMR (CDCl3) see Tables 1 and 4; ESI(+)MS m/z 607.4 [M + H]+, 629.4 [M + Na]+, 1235.7 [2 M + Na]+; ESI(−)MS m/z 651.6 [M + HCO2]−; HRESI(−)MS m/z 651.3184 [M + HCO2]− (calcd for C37H47O10, 651.3169).

Figure 2. Single-crystal X-ray structure of 1. G

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Figure 5. Single-crystal X-ray structure of 12. Cochinchinoid J (10): white powder; [α]22D −32.5 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 233 (3.75) nm; IR (KBr) νmax 3427, 2960, 2933, 1718, 1705, 1612, 1458, 1385, 1228, 1180, 1153, 1024, 872 cm−1; 1H and 13C NMR (CDCl3) see Tables 2 and 4; ESI(+)MS m/z 553.4 [M + H]+, 575.4 [M + Na]+, 1127.7 [2 M + Na]+; HRESI(−)MS m/z 597.3068 [M + HCO2]− (calcd for C34H45O9, 597.3064). 3-Epimesendanin S (11): white powder; [α]22D +65.0 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 241 (4.15) nm; IR (KBr) νmax 3435, 2922, 1626, 1441, 1383, 1026, 559 cm−1; 1H and 13C NMR (CDCl3) see Tables 3 and 4; ESI(+)MS m/z 455.4 [M − H2O + H]+, 968.0 [2 M + Na]+; HRESI(−)MS m/z 517.3521 [M + HCO2]− (calcd for C31H49O6, 517.3529). Cochinchinoid K (12): colorless crystals (n-hexane/acetone/ MeOH, 10:1:0.2); mp 224−226 °C; [α]22D −54.4 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 207 (3.65) nm; IR (KBr) νmax 3427, 2958, 2881, 1765, 1637, 1468, 1385, 1367, 1165, 1036, 980, 920, 756 cm−1; 1 H and 13C NMR (CDCl3) see Tables 3 and 4; ESI(+)MS m/z 511.3 [M + Na]+, 999.8 [2 M + Na]+; ESI(−)MS m/z 533.6 [M + HCO2]−; HRESI(−)MS m/z 533.3486 [M + HCO2]− (calcd for C31H49O7, 533.3478). X-ray Diffraction Analysis. Cochinchinoids A (1) and K (12) were crystallized from MeOH/H2O (100:1) and n-hexane/acetone/ MeOH (10:1:0.2) at room temperature, respectively. Their X-ray crystallographic data were obtained on a Bruker APEX-II CCD detector employing graphite-monochromated Cu Kα radiation (λ = 1.54178 Å) at 132(2) and 140(2) K, respectively (operated in the ϕ−ω scan mode). The structures were solved by direct method using SHELXS-97 (Sheldrick 2008) and refined with full-matrix leastsquares calculations on F2 using SHELXL-97 (Sheldrick 2008). 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 and 12 (for key parameters see Tables S1 and S2, respectively, in the Supporting Information) have been deposited at the Cambridge Crystallographic Data Centre (deposition nos. CCDC 875036 and 939670, respectively). 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 CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [tel: (+44) 1223-336-408; fax: (+44) 1223336-033; e-mail: [email protected]]. Bioassays. Inhibition against human and mouse 11β-HSD1 enzymatic activities was determined via scintillation proximity assay (SPA)17,18 using microsomes containing 11β-HSD1, and glycyrrhetinic acid (97%, G109797, Aladdin) was used as the positive control. The human and mouse 11β-HSD1 enzymes were expressed in

Figure 6. The inhibitory effects of 8, 12, and 13 on 11β-HSD1 of human (A) and mouse (B).

Cochinchinoid D (4): white powder; [α]22D −88.3 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 215 (4.35) nm; IR (KBr) νmax 3442, 2970, 2937, 1736, 1705, 1651, 1604, 1460, 1373, 1257, 1155, 1080, 1024, 729 cm−1; 1H and 13C NMR (CDCl3) see Tables 1 and 4; ESI(+)MS m/z 667.5 [M + H]+, 689.4 [M + Na]+, 1355.7 [2 M + Na]+; ESI(−)MS m/z 711.7 [M + HCO2]−; HRESI(+)MS m/z 667.3478 [M + H]+ (calcd for C38H51O10, 667.3482). Cochinchinoid E (5): white powder; [α]22D +14.6 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 214 (4.06) nm; IR (KBr) νmax 2966, 1745, 1709, 1651, 1437, 1371, 1261, 1225, 1051, 933, 733 cm−1; 1H and 13C NMR (CDCl3) see Tables 1 and 4; ESI(+)MS m/z 699.4 [M + H]+, 1419.7 [2 M + Na]+; HRESI(+)MS m/z 721.2847 [M + Na]+ (calcd for C37H46O13Na, 721.2836). Cochinchinoids F/G (6/7): white powder; 1H and 13C NMR (CDCl3) see Tables 2 and 4; ESI(+)MS m/z 701.3 [M + H]+, 723.3 [M + Na]+, 1423.7 [2 M + Na]+; HRESI(+)MS m/z 701.3185 [M + H]+ (calcd for C37H49O13, 701.3173). Cochinchinoid I (8): white powder; [α]22D +9.0 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 230 (4.25) nm; IR (KBr) νmax 3435, 2968, 2935, 1736, 1709, 1670, 1603, 1460, 1385, 1240, 1153, 1024, 874, 756 cm−1; 1H and 13C NMR (CDCl3) see Tables 2 and 4; ESI(+)MS m/z 551.4 [M + H]+, 573.4 [M + Na]+; HRESI(−)MS m/z 595.2910 [M + HCO2]− (calcd for C34H43O9, 595.2907). Cochinchinoid H (9): white powder; [α]22D −26.2 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 231 (4.16) nm; IR (KBr) νmax 2968, 2939, 1728, 1699, 1668, 1603, 1460, 1371, 1234, 1188, 1163, 1028, 754 cm−1; 1H and 13C NMR (CDCl3) see Tables 2 and 4; ESI(+)MS m/z 551.5 [M + H]+, 1125.7 [2 M + Na]+; HRESI(−)MS m/z 595.2910 [M + HCO2]− (calcd for C34H43O9, 595.2907). H

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(14) Dong, S. H.; He, X. F.; Dong, L.; Wu, Y.; Yue, J. M. Helv. Chim. Acta 2012, 95, 286−300. (15) Puripattanavong, J.; Weber, S.; Brecht, V.; Frahm, A. W. Planta Med. 2000, 66, 740−745. (16) Pretsch, E.; Bü hlmann, P.; Badertscher, M. Structure Determination of Organic Compounds, 4th ed.; Springer-Verlag: Berlin, 2009; p 317. (17) Mundt, S.; Solly, K.; Thieringer, R.; Hermanowski-Vosatka, A. Assay Drug Dev. Technol. 2005, 3, 367−375. (18) Ye, Y. L.; Zhou, Z.; Zou, H. J.; Shen, Y.; Xu, T. F.; Tang, J.; Yin, H. Z.; Chen, M. L.; Leng, Y.; Shen, J. H. Bioorg. Med. Chem. 2009, 17, 5722−5732.

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 11β-HSD1 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 11βHSD1 and was used as the enzyme source for SPA. 11β-HSD1containing 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 tested compounds’ inhibitory effects on 11β-HSD1 were evaluated by detecting the SPA signal. IC50 values were calculated using Prism 5 (GraphPad software, San Diego, CA, USA).

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ASSOCIATED CONTENT

* Supporting Information S

1

H, 13C, and selected 2D NMR spectra of all compounds, together with the preliminary assay results of all pure chemical entities, are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-21-5080-6718. E-mail: [email protected]; h. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the National Natural Science Foundation (No. 21272244) of the People’s Republic of China. H.Z. would also like to thank the Youth Innovation Promotion Association of the Chinese Academy of Sciences for financial support. We thank Prof. S.-M. Huang of Hainan University for the identification of the plant material and Dr. A. M. Piggott of The University of Queensland for the help with the wording and grammar in this paper.

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REFERENCES

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