Limonoids with 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitory

Article pubs.acs.org/jnp

Limonoids with 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitory Activities from Dysoxylum mollissimum Bin Zhou, Yu Shen, Yan Wu, Ying Leng,* and Jian-Min Yue* State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, PR China S Supporting Information *

ABSTRACT: Thirteen new limonoids, dysoxylumosins A−M (1−13), along with six known analogues (14−19) were isolated from the twigs of Dysoxylum mollissimum. Their structures were established on the basis of spectroscopic data analysis. Compounds 1−6, 8, and 12 exhibited significant inhibitory activities against human and/or mouse 11βhydroxysteroid dehydrogenase type 1 (11β-HSD1). Dysoxylumosin F (6), the most potent substance isolated, showed an IC50 value of 9.6 ± 0.90 nM against human 11β-HSD1.

T

he genus Dysoxylum of the family Meliaceae is a rich source of limonoids.1−5 There are about 75 species in this genus worldwide, of which 15 species grow in the south of mainland China.6 Several plants in this genus have been used medicinally to treat leprosy, ulcers, and sexually transmitted diseases.7,8 Chemical investigations on the plants of this genus have led to the isolation of a large array of structurally diverse compounds with significant biological activities including cytotoxic,9−11 antimicrobial,12 anti-inflammatory,9,10,13 and vasodilatory effects.14 Our previous work on Dysoxylum mollissimum Blume collected from Xishuangbanna in Yunnan Province afforded six limonoids.15 Because environmental changes may affect the secondary metabolite profiles of plants, we thus re-examined the chemical components of this plant growing in Hainan Province, which resulted in the isolation of 13 new limonoids, named dysoxylumosins A−M (1−13), as well as six known analogues

(14−19) from the twigs. As part of our continuing efforts to identify 11β-HSD1 inhibitors from Meliaceae species,16 the major new limonoids isolated in the current study were tested against both human and mouse 11β-HSD1, which are the potential therapeutic targets associated with a number of metabolic diseases. Presented herein are the isolation, structural characterization, and biological evaluation of these compounds.

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RESULTS AND DICUSSION Compound 1, named dysoxylumosin A, was obtained as a white amorphous powder. The 13C NMR data as well as the (+)-HRESIMS ion peak at m/z 423.2161 [M + H]+ (calcd 423.2166) suggested a molecular formula of C26H30O5 for 1, incorporating 12 indices of hydrogen deficiency. The IR absorption bands at 3444 and 1725 cm−1 showed the presence of hydroxy and carbonyl groups. The diagnostic NMR data (Tables 1 and 2) suggested the presence of a β-substituted furan ring (δH 6.83, 7.27, and 7.36), two α,β-unsaturated keto groups (δC 202.5 and 200.9), and three double bonds. These functionalities accounted for 8 out of the 12 indices of hydrogen deficiency, requiring the presence of 4 additional rings in the structure of 1. The aforementioned data suggested that 1 is a limonoid similar to walsuronoid B,17 which has a rare 18(13→14)abeo-limonoid framework. The planar structure of 1 was assigned from its 2D NMR spectra (Figure 1). In particular, the structural fragments (C1−C2, C9−C11−C12, and C15−C17) were established readily by the 1H−1H COSY correlations; two α,β-unsaturated ketone moieties in rings A and B were placed by the HMBC correlations from H1 and H329 to C3 and from OH6 to C5, C6, and C7, respectively. The presence of a Δ12 double bond and a hydroxy group at C15 were assigned by the HMBC cross peaks of H318/C13 (δC 149.1) and C15 (δC 82.8), and the Received: May 18, 2015

© XXXX American Chemical Society and American Society of Pharmacognosy

A

DOI: 10.1021/acs.jnatprod.5b00442 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H NMR Spectroscopic Data of Compounds 1−5 in (500 MHz, CDCl3) position

1

2

3

4

5

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

1 2

6.94 (d, 10.1) 6.08 (d, 10.1)

9 11

2.75 (dd, 11.2, 5.6) α 2.43 (m) β 2.30 (m) 5.58 (m)

12 15 16 17 18 19 21 22 23 28 29 30 OMe OAc OH6 OH7 OH15

4.52 (dd, 5.6, 3.1) α 2.23 (m) β 2.07 (ddd, 13.8, 8.6, 3.2) 2.75 (dd, 11.2, 5.6) 0.98 (3H, s) 1.38 (3H, s) 7.27 (br s) 6.24 (br s) 7.36 (br s) 1.58 (3H, s) 1.56 (3H, s) 1.48 (3H, s)

6.83 (s)

3.76 (d, 5.6) α 2.83 (d, 19.4) β 2.66 (dd, 19.4, 5.6) 2.96 (d, 2.2) 4.45 (m)

7.05 (d, 10.0) 6.16 (d, 10.0) 3.24 (s) 5.84 (d, 10.1)

3.65(d, 5.7) α 2.85 (d, 19.3) β 2.74 (dd, 19.3, 5.7) 3.77 (s) 5.70 (d, 10.1)

3.70 (d, 5.7) α 2.82 (d, 19.5) β 2.59 (dd, 19.5, 5.7) 3.16 (d, 2.0) 5.44 (m)

α 2.42 (dd, 13.9, 2.8) β 2.92 (dd, 13.9, 3.1) 4.57 (t, 8.6) α 2.90 (m) β 2.76 (m)

6.88 (dd, 10.1, 3.1)

6.76 (dd, 10.1, 3.2)

4.54 (m) α 3.30 (dd, 18.2, 7.2) β 2.70 (d, 18.2)

4.57 (m) α 3.30 (dd, 18.0, 7.2) β 2.68 (d, 18.0)

2.25 (dd, 14.3, 2.8) 3.08 (dd, 14.3, 3.1) 4.58 (t, 8.6) α 2.88 (ddd, 15.4, 8.6, 2.5) β 2.74 (ddd, 15.4, 8.6, 2.5)

1.31 (3H, s) 1.29 (3H, s) 7.49 (br s) 6.54 (br s) 7.42 (br s) 1.51 (3H, s) 1.47 (3H, s) 1.80 (3H, s) 3.35 (3H, s)

0.91 (3H, s) 1.42 (3H, s) 7.53 (br s) 6.63 (br s) 7.45 (br s) 1.58 (3H, s) 1.56 (3H, s) 1.48 (3H, s)

0.95 (3H, s) 1.10 (3H, s) 7.52 (br s) 6.61 (br s) 7.44 (br s) 1.56 (3H, s) 1.48 (3H, s) 1.46 (3H, s) 3.36 (3H, s)

6.72 (s)

6.77 (s)

6.68 (s)

3.63 (br s)

3.62 (br s)

3.29 (br s)

β-furan ring was located at C17 by the HMBC correlations from H17 to C20, C21, and C22. The relative configuration of 1 was assigned by analysis of its NOESY data (Figure 1), in which the correlations of H329/H319, H319/H330, H330/H17, H330/ H11β, H319/H11β, and H17/H16β indicated that they are spatially close, so these were assigned arbitrarily as β-oriented. Consequently, the NOESY cross peaks of H318/H9, H318/H15, and H318/H16α suggested that they are cofacial and adopt an α-orientation. Thus, the structure of 1 (dysoxylumosin A) was elucidated as shown. Compound 2 was obtained as a white amorphous powder, and it was found to possess a molecular formula of C27H34O7 on the basis of the 13C NMR data and the (+)-HRESIMS protonated molecular ion at m/z 471.2376 [M + H]+ (calcd 471.2377), requiring 11 double-bond equivalents (DBEs). Comparison of the NMR data (Tables 1 and 2) of 2 with those of walsuronoid B showed many similarities except for the absence of the Δ1 double bond in ring A and the presence of a methoxy group at C1, as assigned by the HMBC correlation of CH3O (δH 3.35)/C1. The methoxy group was assigned an α orientation by the key NOESY cross peak between H1 and H319 (Figures S14 and S15, Supporting Information). The NOESY correlations of H9/H11 and H9/H318 suggested H11 was α-configured. Thus, the structure of compound 2 (dysoxylumosin B) was established as shown. Compound 3 was obtained as a white amorphous powder. Its molecular formula was determined as C26H28O5 on the basis of the 13C NMR data and the (+)-HRESIMS ion at m/z 421.1997 [M + H]+ (calcd 421.2010), requiring 13 double-bond equivalents. Comprehensive analysis of the NMR data (Tables 1 and 2) of 3 revealed that it is also a close structural analogue of walsuronoid B, except for the presence of a Δ11 double bond,

1.34 (3H, s) 1.04 (3H, s) 7.41 (br s) 6.46 (br s) 7.41 (br s) 1.48 (3H, s) 1.51 (3H, s) 1.80 (3H, s) 3.39 (3H, s) 1.77 (3H, s) 6.71 (s)

which was confirmed by the observed HMBC cross peaks from H11 (δH 5.84) to C13 and from H12 (δH 6.88) to C9 and C17 (Figure S23, Supporting Information). The relative configuration of 3 was assigned by analysis of its NOESY data (Figure S24, Supporting Information). The structure of 3 (dysoxylumosin C) was thereby determined as shown. Compound 4, named dysoxylumosin D, was obtained as a white amorphous powder. It gave a molecular formula of C27H32O6, as determined by the 13C NMR data and from the (+)-HRESIMS peak at m/z 453.2265 [M + H]+ (calcd 453.2272). The 1H NMR and 13C NMR spectroscopic data (Tables 1 and 2) of 4 showed many similarities to those of 2. The main differences were the proton signals assignable to an extra Δ11 double bond in 4 in place of an oxygenated methine (CH11) and a methylene (CH2−12) in 2, which were supported by the HMBC correlations of H9/C11 (δC 125.4) and C12 (δC 124.5) and H11/C13 (Figure S32, Supporting Information). The methoxy group at C1 of 4 was also assigned as α-oriented by the coupling constant (J1,2α = 5.7 Hz), which is similar to that of 2. The structure of 4 was thus deduced as shown. The molecular formula of compound 5, C29H36O8, was determined by 13C NMR and the (+)-HRESIMS ion peak at m/z 513.2483 [M + H]+ (calcd 513.2488). Detailed analysis of the NMR data (Tables 1 and 2) of 5 revealed its structure to be closely related with that of compound 2, with the only difference being the presence of an extra acetyl group. An acetoxy group was located at C11 by the HMBC correlations from H11 (δH 5.44, brs) to the acetyl carbonyl (δ C 170.7; Figure S40, Supporting Information). The configurations of H1 and H11 in 5 were assigned as being identical to those of 2 by the coupling patterns of H1 and H11, respectively. The structure of 5 (dysoxylumosin E) was hence deduced as shown. B



Article

Table 2. 13C NMR Spectroscopic Data of Compounds 1−13 (125 MHz, CDCl3) position

1

2

3

4

5

6

7

8

9

10

11

12

13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 28 29 30 OMe

152.2 126.5 202.5 47.8 140.2 143.0 200.9 49.7 40.3 39.9 23.7 118.2 149.1 53.6 82.8 40.2 38.4 27.9 27.7 128.4 138.9 110.0 143.4 26.4 21.4 18.6

79.9 37.2 213.1 48.1 140.5 142.5 201.9 55.2 40.9 44.7 68.5 34.3 134.8 58.3 81.4 43.6 127.0 24.4 19.6 121.5 140.8 109.7 143.4 23.9 21.4 20.7 57.1

152.5 127.3 202.8 48.2 138.3 142.0 201.0 49.6 45.9 39.7 125.9 124.4 135.4 56.8 79.9 43.4 126.7 23.4 28.4 121.8 141.0 109.6 143.5 26.4 21.1 19.2

79.8 37.9 201.7 48.3 138.4 142.8 201.7 49.5 40.7 43.1 125.4 124.5 135.9 56.7 80.1 43.5 125.7 23.3 18.4 121.9 140.9 109.6 143.4 24.0 21.2 19.8 57.5

79.8 36.9 212.6 48.1 139.4 142.6 201.6 55.5 39.9 44.3 69.9 30.2 134.0 58.0 81.4 43.3 126.4 24.3 18.7 121.6 140.7 109.0 143.5 23.9 21.5 20.3 57.1 170.7 21.5

35.4 32.5 213.5 48.1 142.3 140.6 201.8 55.7 47.8 39.2 69.6 30.1 133.8 57.7 81.4 43.3 126.6 24.3 18.9 121.6 140.8 109.0 143.5 24.1 20.8 18.9

80.3 37.6 213.2 47.8 135.5 142.3 197.8 45.4 38.1 44.1 68.2 42.7 40.7 69.0 56.1 31.6 42.0 22.0 16.2 123.0 139.6 110.8 143.2 24.1 21.0 23.4 57.0 170.9 21.7

155.8 125.8 203.5 48.9 134.9 140.8 196.7 46.7 41.1 40.8 70.1 76.7 46.1 68.1 55.4 31.7 41.1 16.2 24.7 121.9 139.9 110.9 143.2 27.3 21.3 21.2

155.8 125.8 203.5 48.9 134.9 140.8 196.7 46.7 41.1 40.8 70.2 76.6 46.1 68.1 55.4 31.7 41.1 16.3 24.7 121.9 139.9 110.9 143.2 27.3 21.3 21.2

157.7 126.3 205.1 45.1 45.8 71.2 72.5 42.3 44.5 40.8 18.1 34.5 42.3 61.1 220.9 43.5 38.1 27.8 21.3 122.9 140.3 110.9 143.1 31.8 20.8 18.1

157.4 126.6 206.2 45.7 48.8 67.8 77.6 41.6 45.9 40.4 18.2 34.5 42.2 61.7 218.9 43.3 38.1 28.2 21.4 122.7 140.3 110.9 143.1 32.1 20.2 18.7

154.0 125.4 203.2 43.0 55.8 209.1 78.8 45.1 39.3 44.7 23.5 71.6 46.1 59.0 218.9 43.0 38.0 21.3 18.8 122.2 140.5 110.6 143.7 22.3 24.6 17.0

81.8 37.9 213.3 48.2 136.1 144.4 197.4 54.3 49.9 43.5 77.4 51.0 56.3 100.1 69.2 40.9 44.5 19.0 15.5 123.4 139.5 110.3 143.1 24.0 20.7 23.3 56.7

169.9 21.9

172.3 21.3

OAc

170.5 21.4

1′ 2′ 3′ 4′ 5′

178.3 41.8 26.7 17.0 11.7

178.2 41.9 26.8 17.0 11.7

175.9 41.5 26.8 16.9 11.9

the (+)-HRESIMS, consistent with a molecular formula of C29H36O8 and supported by the 13C NMR data. The characteristic NMR signals for a β-furan ring, an α,β-unsaturated carbonyl, and a 14,15-epoxy group were observed in the 1H and 13C NMR spectra (Tables 2 and 3). Comparison of the NMR data of 7 with those of 5 showed great similarities except for changes in rings C and D, which indicated the presence of a methyl at C13 in 7 in place of the methyl at C14 in 5 that is biosynthetically a Wagner− Meerwein rearranged product of 7.17,18 This was confirmed by the HMBC correlations from H318 to C12, C13, C14, and C17. The 14,15-epoxy group was located by the mutual HMBC correlations from H15, H216, H318, and H330 to C14. The planar structure of 7 was confirmed by the HMBC spectrum (Figure 2). The relative configuration of 7 was established by analysis of the NOESY spectrum (Figure 2). In particular, the NOESY correlations of H319/H330, H319/H329, H319/H1, H17/ H12β, and H17/H16β suggested these to be cofacial, and they were assigned randomly as β-oriented. As a consequence, the NOESY correlations of H11/H9, H9/H318, and H318/H16α indicated these protons were α-configured. Accordingly, the structure of 7 was determined as shown. Compound 8 (dysoxylumosin H) gave a molecular formula of C31H38O8, as determined by the 13C NMR data and the (+)-HRESIMS ion at m/z 539.2629 [M + H]+ (calcd 539.2639). Comparison of its NMR data (Tables 2 and 3) with those of

Figure 1. (A) Selected COSY (bold bond), HMBC, and (B) NOESY correlations of 1.

Dysoxylumosin F (6) gave a molecular formula of C28H34O7 according to the 13C NMR data and the pseudomolecular ion peak at m/z 483.2369 [M + H]+ (calcd 483.2377) in (+)-HRESIMS data, 30 mass units less than that of compound 5. Comparison of its NMR spectroscopic data (Tables 2 and 3) with those of 5 revealed the absence of a methoxy group at C1 on 6, consistent with its molecular formula. The structure of 6 was determined as shown with the relative configuration confirmed by HMBC and NOESY spectra (Figures S48 and S49, Supporting Information). Compound 7, named dysoxylumosin G, exhibited a sodiated molecular ion at m/z 535.2290 [M + Na]+ (calcd 535.2302) in C



Article

Table 3. 1H NMR Spectroscopic Data of Compounds 6−10 (500 MHz, CDCl3) position 1 2 5 6 7 9 11 12 14 15 16 17 18 19 21 22 23 28 29 30 OMe OAc OH6 OH7 OH15 2′ 3′ 4′ 5′

6

7

8

9

10

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

α 1.83 (m) β 2.35 (dd, 13.5, 8.8) α 2.73 (m) β 2.53 (m)

3.65 (d, 6.0)

7.47 (d, 9.9)

7.47 (d, 9.9)

7.08 (d, 10.0)

α 2.80 (d, 19.1) β 2.66 (dd, 9.1, 6.0)

5.98 (d, 9.9)

5.98 (d, 9.9)

5.92 (d, 10.0)

2.27 (s) 5.53 (m) α 2.25 (m) β 3.08 (dd, 14.7, 3.1)

3.33 (s) 5.52 (m) 1.98 (m)

3.04 (d, 11.1) 4.54 (ddd, 11.1, 4.6, 2.7) 5.46 (d, 4.6)

3.04 (d, 11.1) 4.54 (ddd, 11.1, 4.6, 2.7) 5.46 (d, 4.6)

4.58 (t, 8.6) α 2.89 (dd, 15.4, 8.6) β 2.73 (m)

3.90 (s) α 1.95 (dd, 13.7, 11.1) β 2.30 (dd, 13.7, 6.5) 2.73 (dd, 11.1, 6.5) 0.74 (3H, s) 1.07 (3H, s) 7.14 (br s) 6.16 (br s) 7.36 (br s) 1.52 (3H, s) 1.43 (3H, s) 1.36 (3H, s) 3.32 (3H, s) 2.13 (3H, s) 6.39 (s)

3.82 (s) α 1.99 (dd, 13.7, 11.1) β 2.31 (dd, 13.7, 6.6) 2.90 (dd, 11.1, 6.6) 0.81 (3H, s) 1.52 (3H, s) 7.14 (br s) 6.09 (br s) 7.32 (br s) 1.58 (3H, s) 1.50 (3H, s) 1.81 (3H, s)

3.82 (s) α 1.99 (dd, 13.7, 11.1) β 2.31 (dd, 13.7, 6.6) 2.90 (dd, 11.1, 6.6) 0.81 (3H, s) 1.52 (3H, s) 7.14 (br s) 6.09 (br s) 7.32 (br s) 1.58 (3H, s) 1.50 (3H, s) 1.81 (3H, s)

6.36 (s)

6.36 (s)

2.36 (m) 1.63 (m) 1.42 (m) 1.07 (3H, d, 7.0) 0.87 (3H, t, 7.4)

2.36 (m) 1.63 (m) 1.40 (m) 1.08 (3H, d, 7.0) 0.84 (3H, t, 7.4)

1.31 (3H, s) 1.08 (3H, s) 7.41 (br s) 6.46 (br s) 7.41 (br s) 1.50 (3H, s) 1.47 (3H, s) 1.81 (3H, s) 1.76 (3H, s) 6.68 (s)

2.68 (d, 12.3) 5.45 (dd, 12.3, 2.2) 3.89 (d, 2.2) 1.47 (m) 1.69 (m) α 1.28 (m) β 2.02 (dt, 14.4, 3.4, 3.4) 2.87 (s) 2.53 (d, 10.2) 3.48 (t, 10.2) 0.80 (3H, s) 1.10 (3H, s) 7.29 (br s) 6.29 (br s) 7.41 (br s) 1.27 (3H, s) 1.17 (3H, s) 1.20 (3H, s) 2.15 (3H, s)

5.60 (s)

Compound 9, named dysoxylumosin L, was found to share the same molecular formula of C31H38O8 as 8, as determined by its (+)-HRESIMS and 13C NMR data. Their NMR spectra were almost identical except for differences in a triplet methyl group (δH 0.87 for 8 and δH 0.84 for 7), which was assigned to the 2-methylbutanoyl group by the characteristic signals in the 1H NMR spectra. These small differences in the triplet methyl group suggested that compounds 8 and 9 are a pair of C2′ epimers. To distinguish them, an empirical rule was applied, in which the proton chemical shift of CH34′ was more deshielded in the case of 2′R than 2′S, which was the opposite in CH35′ (δCH3−4′ (8 − 9) = −6.48 Hz and δCH3−5′ (8 − 9) = 9.97 Hz; Figure S1, Supporting Information).16a,20 Compounds 8 and 9 were thus proposed to have a 2′S and a 2′R configuration, respectively, as shown. The molecular formula of compound 10 was established as C28H36O6, as indicated by the 13C NMR data and the (+)-HRESIMS ion peak at m/z 491.2423 [M + Na]+ (calcd 491.2404). Comparison of the NMR data (Tables 2 and 3) of 10 with those of 6α-O-acetyl-7-deacetylnimocinol21 revealed these compounds to be structural analogues. The main difference was the presence of a keto group at C15 in 10 instead of the Δ14 double bond in the latter limonoid, which was confirmed by the HMBC correlations from H318 to C14 (δC 61.1) and from H14 and H 2 16 to C15 (δ C 220.9; Figure S81, Supporting Information). The H5 and H14 protons were assigned as

Figure 2. (A) Key HMBC correlations and (B) selected NOESY correlations of 7.

11β-acetoxydihydrocedrelone19 revealed that 8 is a close analogue bearing an extra 2-methylbutanoyl group, which was located at C12 by the key HMBC correlation from H12 to C1′ (Figure S66, Supporting Information). The NOESY correlations of H319/H329, H319/H330, H319/H11, H11/H12, H17/H12, and H17/H16β suggested that H11 and H12 are β-oriented. The structure of 8 in the limonoid core was confirmed by its HMBC and NOESY spectra (Figure S67, Supporting Information), as shown. D



Article

α-oriented as judged from the ROESY correlations of H9/H5 and of H9 and H318/H14. The H6 and H7 protons were determined as β-oriented by the ROESY cross peaks of H6/ H329, H6/H7, H6/H330, and H7/H330 (Figure S82, Supporting Information). The structure of 10 (dysoxylumosin J) was thus established as shown. Compound 11, named dysoxylumosin K, was found to share the same molecular formula of C28H36O6 as 10 on the basis of the 13 C NMR data and the (+)-HRESIMS ion peak at m/z 491.2405 [M + Na]+ (calcd 491.2404). Analysis of the NMR data revealed the only difference between two compounds to be in the acetylation patterns at C6 and C7. An acetoxy group was assigned to C7 in compound 11 by the downfield chemical shift of H7 (5.22, d, J = 2.4 Hz) instead of an OAc-6 as in 10, which was confirmed by the HMBC correlation from H7 to the corresponding acetyl carbonyl group (δC 172.3; Figure S90, Supporting Information). The proposed structure of 11 as shown was verified by HMBC and ROESY spectra (Figure S91, Supporting Information). Dysoxylumosin L (12) gave a molecular formula of C31H40O7 as determined by the (+)-HRESIMS ion peak at m/z 525.2845 [M + H]+ (calcd 525.2847) and the 13C NMR data. Analysis of the NMR data (Tables 2 and 4) of 12 showed that its structure is closely related to those of compounds 10 and 11, with the major difference being the presence a 2-methylbutanoyl group, distinguished by its characteristic NMR signal located at C12 from the key HMBC correlation from H12 to C1′. The presence of a keto group was assigned to C6 (δC 209.1) by the HMBC correlations from H5, H7, and OH7 to C6 (Figure S99, Supporting Information). A β configuration was assigned to H7 and H12 by the NOESY correlations of H7/H330, H330/H12, and H12/H17 (Figure S100, Supporting Information). The structure of compound 12 was thus assigned as shown. Compound 13 (dysoxylumosin M) displayed a molecular ion peak at m/z 469.2229 [M − H]− (calcd 469.2232) in the (−)-HRESIMS corresponding to a molecular formula of C27H34O7, which was supported by the 13C NMR data. Comparison of its NMR data (Tables 2 and 4) with those of 7 showed several similarities. However, the major differences were the absence of the diagnostic NMR signals for the 14,15-epoxy moiety and an acetyl group and the presence of a characteristic quaternary carbon signal at δC 100.1. The chemical shifts and the HMBC correlations (Figure 3) of H318/C12, C13, C14, and C17; H11 and H330 (δH 4.60)/C14; and H15 (δH 4.67)/C8 and C17 enabled the proposal of an ether bridge between C11 and C14 and the location of Me18 and OH15 to C13 and C15, respectively. The relative configuration of 13 was deduced by analysis of the ROESY spectrum (Figure 3). The ROESY correlations of H329/H319, H319/H330, H1/H319, H17/H12β, and H17/H16β indicated that they are cofacial, and these were assigned arbitrarily as β-oriented. In turn, H9, H15, H318, and H328 were assigned as α-directed from the ROESY cross peaks of H328/H2α, H9/H318, H9/H12α, H15/H16α, and H318/H16α. The H11 proton was assigned as α-oriented by the key ROESY cross peak between H9 and H318. The structure of 13 was thus characterized unequivocally as shown. Six known limonoids, isocedrelone (14),22 cedrelone (15),22 walsuronoid B (16),16 11β,12α-diacetoxycedrelone (17),23 deacetylanthothecol (18),24 and neotrichileone (19),25 were also isolated. Their structures were identified by NMR and MS analysis as well as by comparison with reported physical and spectroscopic values.

Table 4. 1H NMR Spectroscopic Data of Compounds 11−13 (500 MHz, CDCl3) position

11

12

13

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz) 3.30 (d, 6.1) α 2.79 (d, 18.9) β 2.71 (dd, 18.9, 6.1)

1 2

7.06 (d, 10.0) 5.92 (d, 10.0)

6.86 (d, 10.3) 5.80 (d, 10.3)

5 6 7 9

2.16 (d, 11.8) 4.40 (dd, 11.8, 2.4) 5.22 (d, 2.4) 1.39 (m)

3.83 (s)

11 12

1.70 (m) α 1.30 (m) β 2.05 (dt, 14.4, 3.4, 3.4) 2.32 (s)

14 15 16

3.78 (d, 5.6) 2.24 (dd, 11.6, 2.9) 2.08 (m) 5.20 (t, 3.0)

4.60 (dd, 4.0, 2.4) α 1.42 (m) β 2.19 (m)

3.04 (s)

2.52 (d, 10.1)

2.57 (d, 9.9)

17

3.46 (t, 10.1)

3.47 (t, 9.9)

18 19 21 22 23 28 29 30 OMe OAc OH6 OH7 OH15 2′ 3′



4′ 5′

3.22 (br s)

4.67 (dd, 7.8, 7.8) α 2.46 (dt, 13.9, 7.7) β 2.11 (dd, 13.9, 6.2) 3.33 (dd, 13.9, 6.2) 0.89 (3H, s) 1.14 (3H, s) 7.21 (br s) 6.24 (br s) 7.38 (br s) 1.50 (3H, s) 1.41 (3H, s) 1.54 (3H, s) 3.30 (3H, s)

2.18 (3H, s) 6.36 (s) 3.22 (d, 5.6) 2.54 (d, 8.2) 2.38 (m) 1.66 (m) 1.44 (m) 1.10 (3H, d, 7.0) 0.87 (3H, t, 7.4)

Figure 3. (A) Key HMBC correlations and (B) selected NOESY correlations of 13.

The most abundant new isolates 1−6 as well as 8−13 were evaluated for inhibitory activity against both human and mouse 11β-HSD1, which are NADPH-dependent enzymes acting as regulators of the active/inactive forms of glucocorticoids.26 The test results showed that compounds 1−3 and 6 exhibited strong inhibition against both human and mouse 11β-HSD1, and E



Article

to give six major components (B5b1−B5b6), and each of these was purified by semipreparative HPLC (63% CH3CN in H2O, 3 mL/min) to yield compounds 5 (2.3 mg), 6 (4.2 mg), 7 (6.5 mg), 8 (3.2 mg), and 9 (3.2 mg) and 11β,12α-diacetoxycedrelone (2.2 mg), respectively. Fraction C (30 g) was passaged over a silica-gel column (CHCl3/ MeOH, 100:1 to 50:1) to yield subfractions C1 and C2. Fraction C1 (3.1 g) was then purified by semipreparative HPLC (55% CH3CN in H2O, 3 mL/min) to give compound 13 (10 mg) and deacetylanthothecol (10 mg). Fraction C2 (1.5 g) was separated on a column of Sephadex LH20 to obtain three major parts, and each of these was purified by semipreparative HPLC (55% CH3CN in H2O, 3 mL/min) to yield compounds 10 (3.0 mg) and 11 (7.0 mg) and neotrichileone (5.2 mg), respectively. Dysoxylumosin A (1): white, amorphous powder; [α]24D +1 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 215 (3.95) nm; IR (KBr) νmax 3444, 2970, 2936, 1725, 1675, 1460, 1384, 1185, 1058, 873, 602 cm−1; 1H NMR (CDCl3) see Table 1; 13C NMR (CDCl3) see Table 2; (+)-ESIMS m/z 423.2 [M + H]+, 445.2 [M + Na]+; (+)-HRESIMS m/z 423.2161 [M + H]+ (calcd for C26H31O5, 423.2166). Dysoxylumosin B (2): white, amorphous powder; [α]24D −103 (c 0.7, MeOH); UV (MeOH) λmax (log ε) 288 (3.95), 221 (4.15) nm; IR (KBr) νmax 3393, 2920, 1691, 1609, 1350, 1256, 1028, 873 cm−1; 1H NMR (CDCl3) see Table 1; 13C NMR (CDCl3) see Table 2; (+)-ESIMS m/z 471.4 [M + H]+; (+)-HRESIMS m/z 471.2376 [M + H]+ (calcd for C27H35O7, 471.2377). Dysoxylumosin C (3): white, amorphous powder; [α]24D −95 (c 0.47, MeOH); UV (MeOH) λmax (log ε) 283 (4.23), 223 (3.97) nm; IR (KBr) νmax 3406, 2935, 1680, 1622, 1387, 1363, 1253, 1094, 1063 cm−1; 1 H NMR (CDCl3), see Table 1; 13C NMR (CDCl3), see Table 2; (+)-ESIMS m/z 421.3 [M + H]+, 443.2 [M + Na]+; (+)-HRESIMS m/z 421.1997 [M + H]+ (calcd for C26H29O5, 421.2010). Dysoxylumosin D (4): white, amorphous powder; [α]24D −42 (c 0.50, MeOH); UV (MeOH) λmax (log ε) 274 (4.40) nm; IR (KBr) νmax 3340, 2971, 2918, 1707, 1655, 1624, 1387, 1239, 1091, 1063, 875 cm−1; 1H NMR (CDCl3), see Table 1; 13C NMR (CDCl3), see Table 2; (+)-ESIMS m/z 453.4 [M + H]+, 475.2 [M + Na]+; (+)-HRESIMS m/z 453.2265 [M + H]+ (calcd for C27H33O6, 453.2272). Dysoxylumosin E (5): white, amorphous powder; [α]24D −67 (c 0.23, MeOH); UV (MeOH) λmax (log ε) 287 (3.85), 222 (3.78) nm; IR (KBr) νmax 3418, 2920, 2850, 1739, 1709, 1616, 1371, 1245, 1087, 1063, 1024 cm−1; 1H NMR (CDCl3), see Table 1 and 13C NMR (CDCl3), see Table 2; (+)-ESIMS m/z 513.4 [M + H]+; (+)-HRESIMS m/z 513.2483 [M + H]+ (calcd for C29H37O8, 513.2488). Dysoxylumosin F (6): white, amorphous powder; [α]24D −76 (c 0.30, MeOH); UV (MeOH) λmax (log ε) 287 (4.30), 222 (3.88) nm; IR (KBr) νmax 33423, 2922, 2850, 1734, 1702, 1620, 1351, 1240, 1063 cm−1; 1 H NMR (CDCl3), see Table 3; 13C NMR (CDCl3), see Table 2; (+)-ESIMS m/z 483.4 [M + H]+, 4505.2 [M + Na]+; (+)-HRESIMS m/z 483.2369 [M + H]+ (calcd for C28H35O7, 483.2377). Dysoxylumosin G (7): white, amorphous powder; [α]24D −24 (c 0.65, MeOH); UV (MeOH) λmax (log ε) 278 (3.97) nm; IR (KBr) νmax 3407, 2924, 1733, 1708, 1676, 1383, 1248, 1234, 1089, 1031, 789 cm−1; 1 H NMR (CDCl3), see Table 3; 13C NMR (CDCl3), see Table 2; (+)-ESIMS m/z 513.3 [M + H]+, 535.3 [M + Na]+; (+)-HRESIMS m/z 535.22290 [M + Na]+ (calcd for C29H36O8Na, 535.2302). Dysoxylumosin H (8): white, amorphous powder; [α]24D −81 (c 0.32, MeOH); UV (MeOH) λmax (log ε) 280 (3.80), 217 (3.91) nm; IR (KBr) νmax 3407, 2924, 1733, 1708, 1676, 1383, 1248, 1234, 1089, 1031, 789 cm−1; 1H NMR (CDCl3), see Table 3; 13C NMR (CDCl3), see Table 2; (+)-ESIMS m/z 539.1 [M + H]+, 1099.2 [2 M + Na]+; (+)-HRESIMS m/z 539.2639 [M + H]+ (calcd for C31H39O8, 539.2629). Dysoxylumosin I (9): white, amorphous powder; [α]24D −72 (c 0.32, MeOH); UV (MeOH) λmax (log ε) 281 (3.86), 215 (4.00) nm; IR (KBr) νmax 3420, 2971, 2935, 1730, 1678, 1624, 1461, 1384, 1360, 1185, 1034, 789 cm−1; 1H NMR (CDCl3), see Table 3; 13C NMR (CDCl3), see Table 2; (+)-ESIMS m/z 539.2 [M + H]+, 1099.3 [2 M + Na]+; (−)-HRESIMS m/z 537.2494 [M − H]− (calcd for C31H37O8 537.2488). Dysoxylumosin J (10): white, amorphous powder; [α]24D −72 (c 0.32, MeOH); UV (MeOH) λmax (log ε) 219 (3.90) nm; IR (KBr)

compounds 4, 5, 8, and 12 displayed selective inhibition against human 11β-HSD1 (Table 5). Table 5. Inhibitory Activities of 1−6, 8, and 12 against Human and Mouse 11β-HSD1 (IC50 ± SD, nM)

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compound

human 11β-HSD1

mouse 11β-HSD1

1 2 3 4 5 6 8 12 glycyrrhetinic acid

61 ± 12.1 54 ± 10.2 >100 >100 >100 9.6 ± 0.89 >100 >100 8.8 ± 1.56

>100 >100 >100 >100 >100 >100 >100 >100 9.4 ± 1.03

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined on an Autopol VI polarimeter at room temperature. UV data were obtained by using a Shimadzu UV-2550 spectrophotometer. IR spectra were acquired on a Thermo IS5 spectrometer with KBr disks. NMR spectra were run on a Bruker AM-500 NMR spectrometer with TMS as internal standard. ESIMS was carried out on a Bruker Daltonics Esquire 3000 plus mass spectrometer, and HRESIMS was carried out on a Waters-Micromass Q-TQF Ultima Global or a Thermo Scientific LTQ Velos Pro Orbitrap Elite mass spectrometer. Semipreparative HPLC was carried out on a Waters 1525 binary pump system with a Waters 2489 detector (210 nm) and equipped with a YMCPack ODS-A (250 mm × 10 mm, S-5 μm). Silica gel (200−300 mesh, Qingdao Haiyang Chemical Co., Ltd.), C18 reversed-phase (RP-18) silica gel (20−45 μm, Fuji Silysia Chemical Ltd.), CHP20P MCI gel (75−150 μm, Mitsubishi Chemical Corporation), 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 of D. mollissimum were collected in June 2008 from Ledong County of Hainan Province, People’s Republic of China, and were authenticated by Prof. S.-M. Huang, Department of Biology, Hainan University, People’s Republic of China. A voucher specimen has been deposited in Shanghai Institute of Materia Medica, Chinese Academy of Sciences (accession number: Dysom-2010-1Y). Extraction and Isolation. The air-dried, powdered twigs of D. mollissimum (6 kg) was extracted three times with 95% EtOH at room temperature to give a crude extract (500 g), which was partitioned between EtOAc and H2O. The EtOAc-soluble fraction (350 g) was subjected to passage over an MCI gel column (MeOH/H2O, 3:7 to 9:1) to afford four major fractions (A−D). Fraction B (40 g) was separated over a silica-gel column eluted with gradient mixtures of petroleum ether−acetone (from 50:1 to 1:5) to afford six fractions (B1−B6). Fraction B2 (5.1 g) was purified by semipreparative HPLC (70% CH3CN in H2O, 3 mL/min) to yield compounds 1 (6.3 mg), 12 (4.5 mg), iocedrelone (8.2 mg), and cedrelone (5.1 mg). Fraction B4 (6.2 g) was separated on a reversed-phase column containing C18 silica gel (MeOH/H2O, 30−80%) to yield three major portions (B4a−B4c). Part B4a (2.1 g) was separated over a silica-gel column eluted with petroleum ether-chloroform (from 2:1 to 1:3) to give two components (B4a1 and B4a2), and each of these was purified by semipreparative HPLC (50% CH3CN in H2O, 3 mL/min) to afford compounds 3 (9.3 mg) and 4 (10 mg), respectively. Part B4b (500 mg) was purified by semipreparative HPLC (47% CH3CN in H2O, 3 mL/min) to yield compound 2 (13 mg) and walsuronoid B (15 mg). Fraction B5 (5.9 g) was separated on a column containing reversed-phase C18 silica gel (MeOH/ H2O, 30−80%) to yield four major subfractions. Fraction B5b (2.2 g) was fractionated on a silica-gel column (CHCl3-MeOH, 500:1 to 50:1) F



Article

νmax 3427, 2925, 1724, 1671, 1384, 1251, 1162, 1108, 1029 cm−1; 1H NMR (CDCl3), see Table 3; 13C NMR (CDCl3), see Table 2; (+)-ESIMS m/z 469.2 [M + H]+, 959.5 [2 M + Na]+; (+)-HRESIMS m/z 491.2423 [M + Na]+ (calcd for C28H36O6Na, 491.2404). Dysoxylumosin K (11): white, amorphous powder; [α]24D −1 (c 0.70, MeOH); UV (MeOH) λmax (log ε) 218 (3.94) nm; IR (KBr) νmax 3446, 2954, 1739, 1670, 1383, 1240, 1065, 873 cm−1; 1H NMR (CDCl3), see Table 4; 13C NMR (CDCl3), see Table 2; (+)-ESIMS m/z 469.3 [M + H]+, 959.7 [2 M + Na]+; (+)-HRESIMS m/z 491.2405 [M + Na]+ (calcd for C28H36O6Na, 491.2404). Dysoxylumosin L (12): white, amorphous powder; [α]24D −37 (c 0.45, MeOH); UV (MeOH) λmax (log ε) 280 (4.09), 248 (3.76) nm; IR (KBr) νmax 3397, 2969, 1680, 1388, 1365, 1275, 1059, 1029, 873 cm−1; 1 H NMR (CDCl3), see Table 4; 13C NMR (CDCl3), see Table 2; (+)-ESIMS m/z 525.4 [M + H]+, 547.3 [M + Na]+; (+)-HRESIMS m/z 525.2845 [M + H]+ (calcd for C31H41O7, 525.2847). Dysoxylumosin M (13): white, amorphous powder; [α]24D −51 (c 0.50, MeOH); UV (MeOH) λmax (log ε) 280 (3.87) nm; IR (KBr) νmax 3415, 2970, 1709, 1682, 1459, 1386, 1344, 1247, 1088, 1027, 874, 786 cm−1; 1H NMR (CDCl3), see Table 4; 13C NMR (CDCl3), see Table 2; (−)-ESIMS m/z 469.2 [M − H]−; (−)-HRESIMS m/z 469.2229 [M − H]− (calcd for C27H33O7, 469.2232). 11β-HSD1 Inhibitory Activity Assay. Inhibition against human and mouse 11β-HSD1 enzymatic activities was tested via a scintillation proximity assay (SPA),27 with glycyrrhetinic acid used as the positive control.

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ASSOCIATED CONTENT

* Supporting Information S

IR, ESIMS, HRESIMS, 1D and 2D NMR spectra of compounds 1−13. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00442.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Tel.: +86-21-50806718. Fax: +86-21-50806718. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by the National Natural Science Foundation (nos. 81273398 and 81321092), and the Foundation (2012CB721105) from the Ministry of Science and Technology of the People’s Republic of China. We thank Prof. S.-M. Huang of Hainan University for identification of the plant material.

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REFERENCES

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