Limonoids and Triterpenoids as 11β-HSD1 Inhibitors from Walsura

Mar 3, 2016 - *E-mail for J.-M.Y.: [email protected]. Tel: 86-21-50806718. ... Their structures and absolute configurations were determined by spectros...
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Limonoids and Triterpenoids as 11β-HSD1 Inhibitors from Walsura robusta Guo-Cai Wang, Jin-Hai Yu, Yu Shen, Ying Leng, Hua Zhang,* and Jian-Min Yue* State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 ZuChongZhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People’s Republic of China S Supporting Information *

ABSTRACT: Nine new cedrelone-type limonoid derivatives, walsunoids A−I (1−9), and 11 known compounds were isolated from the leaves of Walsura robusta. Walsunoid A (1) is a new degradation product of cedrelone-type limonoids, and walsunoid I (9) is a rare cedrelone-type limonoid amide. Their structures and absolute configurations were determined by spectroscopic data, single-crystal X-ray diffraction, and ECD data analyses. Five compounds showed moderate inhibitory activities against human and/or mouse 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) with IC50 values ranging from 0.69 to 9.9 μM.

T

he genus Walsura (Meliaceae), which is widely distributed in the tropical zone of Asia,1 is known for its structurally diverse secondary metabolites, particularly limonoids and triterpenoids, with insecticidal, cytotoxic, and 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitory activities.2−10 Walsura robusta, one of the three Chinese endemic species, mainly occurs in the Guangdong and Yunnan provinces of China.1 Previous chemical studies on W. robusta led to the isolation of a series of sesquiterpenoids, limonoids, and phenolic glucosides, some of which exhibited antioxidant, antibacterial, and antigiardial activities.11−13 The 11β-HSD1 enzymes have been demonstrated as potential therapeutic targets associated

with a number of metabolic diseases.14−16 As part of a continuing search for 11β-HSD1 inhibitors from Meliaceae plants,8,9,17 nine new cedrelone-type limonoid derivatives, walsunoids A−I (1− 9), five known tirucallane-type triterpenoids (11−15), and six known cedrelone-type limonoids (10, 16−20) were isolated from the leaves of W. robusta. Walsunoid A (1) is a new degradation product of cedrelone-type limonoids and walsunoid I (9) is a rare cedrelone-type limonoid amide. Biological tests showed that five compounds exhibited moderate inhibitory activities against human and/or mouse 11β-HSD1. Herein, the isolation, structure elucidation, and biological evaluation of these compounds are presented.

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Received: October 24, 2015

RESULTS AND DISCUSSION Compound 1 possessed a molecular formula of C25H32O7 derived from (−)-HRESIMS data showing a deprotonated © XXXX American Chemical Society and American Society of Pharmacognosy

A

DOI: 10.1021/acs.jnatprod.5b00952 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H NMR Spectroscopic Data of Compounds 1−5 1a,c position

3b,c

(J in Hz)

(J in Hz)

1

6.97, d (9.9)

7.29, d (9.9)

7.29, d (9.9)/7.25, d (9.9)

2

6.15, d (9.9)

6.31, d (9.9)

6.30, d (9.9)/6.27, d (9.9)

9 11 12

2.48, s 4.61, dd (9.8, 6.1) α 1.99, dd (15.2, 6.1) β 1.91, d (15.2) 3.94, s α 1.51, m β 2.30, m 2.16, m 0.85, s 1.55, s a 2.40, dd (16.0, 5.7) b 2.32, m

2.84, s 5.05, d (6.2) α 2.25, dd (15.2, 6.2) β 2.89, d (15.2) 4.06, s 2.11, m

2.86, s/2.82, s 5.03, d (6.2) α 2.28, dd (15.2, 6.2)/2.19, dd (15.2, 6.2) β 2.93, d (15.2) 4.09, s/4.06, s α 2.08, m β 2.19, m 3.05, m 0.99, s/0.92, s 1.82, s/1.81, s

15 16 17 18 19 20 22 23 28 29 30 OMe 6-OH 11-OH 23-OH a

(J in Hz)

2b,d

4.25, m 1.57, s 1.51, s 1.35, s

3.03, dd (10.6, 6.7) 0.95, s 1.81, s

7.05, brs 5.91, brs 1.89, s 1.89, s 1.77, s 3.45, s

7.20, brs/7.17, brs 6.48, brs/6.46, brs 1.89, s/1.88, s 1.87, s/1.84, s 1.77, s

6.37, s 2.49, d (9.8) 3.05, t (4.8)

Measured in CDCl3. bMeasured in pyridine-d5.

4a,e

5a,d

(J in Hz)

(J in Hz)

α 1.68, m β 2.29, m α 2.71, m β 2.63, m 2.22, s 4.49, dd (9.0, 6.4) α 2.06, dd (15.8, 6.4) β 2.38, d (15.8) 3.98, s α 2.02, m β 2.29, m 2.73, m 0.76, s 1.34, s

α 1.70, m β 2.29, m α 2.72, m β 2.63, m 2.23, s 4.49, dd (9.0, 6.4) α 2.08, dd (15.8, 6.4) β 2.41, d (15.8) 3.98, s α 2.05, m β 2.31, m 2.73, m 0.73, s 1.34, s

6.77, brs 5.73, brs 1.52, s 1.42, s 1.34, s 3.56, s 6.25, s 2.29, d (9.0)

7.12, m 4.83, m 1.52, s 1.43, s 1.34, s 6.25, s 2.28, d (9.0)

c,d, and e

Data were measured at 500, 600, and 400 MHz, respectively.

molecule at m/z 443.2068 [M − H]− (calcd 443.2075) and 13C NMR data, indicating 10 indices of hydrogen deficiency. The IR spectrum showed the presence of hydroxy (3408 cm−1) and carbonyl (1675 cm−1) functionalities. Analysis of the NMR data (Tables 1 and 2), including DEPT and HSQC spectra (Figure S3, Supporting Information), revealed the presence of 25 carbon resonances corresponding to three ketocarbonyls (δC 208.6, 203.5, and 197.3, two as enones), two double bonds, four sp3 quaternary carbons, one oxygenated sp3 tertiary carbon, four sp3 methines (two oxygenated), four sp3 methylenes (one oxygenated), and five methyls. The two double bonds and three carbonyl groups accounted for five out of the 10 indices of hydrogen deficiency, requiring five rings in the structure. The 2D structure of 1 was constructed by 2D NMR analysis as a degradation product of cedrelone-type limonoids. Analysis of the 1 H−1H COSY spectrum revealed the presence of three structural fragments as drawn with bold bonds (Figure 1A). The connection of three structural fragments, quaternary carbons, and the other functional groups were then completed by analysis of the HMBC correlations (Figure 1A). Particularly, the presence of two conjugated carbonyl groups and the 6-OH group in rings A and B were confirmed by the HMBC correlations of H-1/C-3; H3-28 (29)/C-3, C-4, and C-5; H3-19/C-1, C-5, C-9, and C-10; H3-30/C-7, C-8, C-9, and C-14; and 6-OH/C-5, C-6, and C-7. The chemical shifts of C-14 (δC 69.3) and C-15 (δC 59.0) indicated the presence of an oxirane ring. The chemical shifts and HMBC correlations from H2-20 and H2-23 (δH 4.25) to C-22 (δC 208.6) indicated the presence of a carbonyl and a hydroxy group at C-22 and C-23, respectively. Finally, the last hydroxy group was positioned at C-11 by the chemical shifts of H-11 (δH 4.61) and C-11 (δC 67.2). Therefore, the 2D structure of 1 was established as a degradation product of cedrelone-type limonoids

possessing the identical rings A−D compared to 11βhydroxycedrelone.10 The relative configuration of 1 was defined by interpretation of ROESY data (Figure 1B) and comparison of NMR data with those of 11β-hydroxycedrelone.10 The ROESY correlations of H3-30 with both H3-19 and 11-OH showed that they were in pseudo 1,3-diaxial relationships and were randomly assigned βorientations. The ROESY cross-peaks of H-11/H-9, H-9/H3-18, and H3-18/H2-20 indicated that H-9, Me-18, and the C-17 side chain were α-oriented. The 14,15-epoxy ring was fixed as βoriented based on the matching NMR data of the related protons and carbons with those of 11β-hydroxycedrelone.10 Compound 1 (walsunoid A) was thus characterized as shown. Compounds 2 and 10 exhibited the same molecular formula of C27H32O8 as deduced from the (−)-HRESIMS ions at m/z 483.2029 and 483.2011 [M − H]− (calcd 483.2024) and 13C NMR data. Comparison of the 1D (Tables 1−3) and 2D NMR data (Figures S15, S16, S89, and S90, Supporting Information) of 2 and 10 showed similarity except for the slight variations of the chemical shifts of C-21 (ΔδC 0.3), C-22 (ΔδC 0.2), and C-23 (ΔδC 0.4) in the α,β-unsaturated-γ-lactone moiety, suggesting they are C-23 epimers. Compound 10 was identified as the same as the recently reported 11β-hydroxy-23-O-methylwalsuranolide3 via their identical NMR data in pyridine-d5. Because the configuration of C-23 in 10 was not previously assigned, it was not possible to distinguish between 10 and 2 on the basis of the available spectroscopic data. However, compound 10 yielded high-quality crystals in MeOH, which permitted the utilization of X-ray diffraction analysis (Figure 2) to establish its absolute configuration as 8R, 9R, 10R, 11S, 13S, 14R, 15R, 17R, 23S [absolute structure parameter: − 0.01 (7)].18 Compound 2 (walsunoid B) was thus characterized to be the 23α-epimer of 10. B

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Table 2. 13C NMR Spectroscopic Data of Compounds 1−10

a

position

1a,c

2b,d

3b,c

4a,e

5a,d

6b,c

7b,d

8a,c

9a,d

10b,e

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 OAc

151.7 127.7 203.5 48.7 134.8 140.9 197.3 45.8 45.5 40.5 67.2 46.0 41.0 69.3 59.0 32.8 41.5 21.5 25.6 37.6

154.0 127.6 204.4 49.5 134.2 144.0 199.1 47.5 46.0 41.9 66.5 47.2 41.7 69.6 56.4 31.7 43.4 23.0 26.8 138.2 171.9 146.7 103.6 27.7 22.1 23.6 56.9

154.1/150.0 127.59/127.56 204.4 49.51/49.48 134.2/134.1 144.02/143.99 199.08/199.06 47.6/47.5 46.01/45.98 41.68/41.66 66.6 47.2/47.1 41.9/41.7 69.69/69.66 56.5 32.0/31.9 43.3/43.2 23.0/22.8 26.8 136.9/136.8 172.7/172.4 149.4/149.1 98.8/98.6 27.7 22.1 23.6

35.4 32.8 214.3 48.0 140.3 140.1 198.0 45.5 48.3 39.6 66.9 46.7 41.3 69.7 58.3 31.1 42.7 22.5 16.8 137.8 170.7 145.0 102.5 24.5 20.5 23.1 57.2

35.4 32.8 214.3 48.0 140.4 140.1 198.2 45.6 48.3 39.6 66.9 46.9 41.2 69.8 58.4 31.1 43.1 22.5 16.8 133.6 173.7 147.3 70.5 24.5 20.5 23.1

152.6/152.8 128.0 204.1 49.6 133.5 144.3 198.1 47.5 44.5 41.2 68.2 42.8/44.0 41.8 69.3 56.4 31.1 45.4/46.8 22.7 25.5 170.1 100.0/101.1 120.3/121.3 171.6 27.6 22.0 23.5

36.1 33.4 214.5 48.9 139.2 143.2 198.6 47.0 47.0 39.8 68.8 42.9 41.8 69.2 56.4 31.0 45.3/46.7 22.9 17.1 170.2 100.0/101.1 120.2/121.0 171.6 24.8 21.5 23.9

150.8 127.9 203.3 48.6 133.7 141.1 197.4 46.1 43.9 40.62 68.2 42.6 40.56 68.6 56.0 31.5 42.1 22.3 24.9 122.8 139.6 110.6 143.3 27.0 21.3 23.1

151.5 127.8 203.4 48.7 135.1 140.9 197.4 45.9 45.4 41.0 67.1 46.6 42.1 69.5 58.1 30.8 43.4 22.7 25.7 149.8 170.6 130.4 169.5 27.0 21.36 22.85

154.0 127.6 204.4 49.5 134.2 144.0 199.0 47.5 46.0 41.7 66.5 47.1 41.7 69.7 56.5 31.8 43.4 23.0 26.8 138.2 171.6 146.5 103.2 27.7 22.1 23.6 56.4

171.0 21.8

171.0 21.8

170.6 21.6

208.6 68.5 27.0 21.3 22.8

Measured in CDCl3. bMeasured in pyridine-d5.

c,d, and e

Data were measured at 125, 150, and 100 MHz, respectively.

It should be noted that the reported specific rotation for 10 ([α]27D − 2.1, c 0.1, MeOH)3 significantly differed from that ([α]27D + 22, c 0.8, MeOH) obtained in this study, which could be attributed to the impurities in the sample reported in reference3. Compound 3 was obtained as inseparable mixture of epimers in a ca. 1:1 as inferred from the 1H NMR data (pyridine-d5) (Figure S22, Supporting Information). The molecular formula of C26H30O8 was assigned by the (−)-HRESIMS ion at m/z 469.1867 [M − H]− (calcd 469.1868) and 13C NMR data. Comparison of the NMR data (Tables 1 and 2) of 3 to those of 10 (Tables 2 and 3) showed that they were closely related analogues featuring identical carbon frameworks. The main distinction was attributable to the α,β-unsaturated-γ-lactone motif. The absence of the methoxy signals and the shielded chemical shifts of C-23 as compared with those of 2 and 10 suggested that compound 3 is a mixture of C-23 hemiacetal epimers. Further analysis of 2D NMR data (Figures S24−S26, Supporting Information) confirmed this conclusion, and the structures of the C-23 epimers of 3 (walsunoid C) were thus assigned as shown. The molecular formula of C27H34O8 of compound 4 was determined by the (+)-HRESIMS ion at m/z 995.4435 [2 M + Na]+ (calcd 995.4400) and 13C NMR data, with two mass units more than that of 10 suggestive of a dihydro analogue. Analysis of the NMR data (Tables 1 and 2) of 4 confirmed this deduction by the presence of diagnostic resonances of two more sp 3 methylenes (δC, 32.8 and 35.4) and a carbonyl group (δC, 214.3) replacing those of the α,β-conjugated carbonyl group (δC,

Figure 1. 1H−1H COSY and selected HMBC (A) and key ROESY (B) correlations of 1.

C

DOI: 10.1021/acs.jnatprod.5b00952 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 3. 1H NMR Spectroscopic Data of Compounds 6−10 6a,c position

(J in Hz)

8b,c

(J in Hz)

1

7.29, d (9.8)

2

6.27, d (9.8)/6.29, d (9.8)

9 11 12

3.05, s 6.05, d (6.1) α 2.47, dd (15.3, 6.1) β 2.71, d (15.3) 4.10, s α 2.10, m β 2.24, dd (13.5, 6.3) 3.17, m 1.01, s/1.14, s 1.40, s 6.42, brs/6.35, brs 6.11, brs/6.14, brs

α 1.86, m β 2.24, m α 2.80, dd (18.8, 9.1) β 2.61, dd (18.8, 9.7) 2.72, s 5.84, d (6.6) α 2.35, dd (15.4, 6.6) β 2.66, d (15.4) 4.08, s α 2.10, m β 2.22, m 3.16, dd (10.1, 6.3) 1.01, s/1.15, s 1.19, s 6.44, brs/6.36, brs 6.11, brs/6.15, brs

1.83, s 1.86, s 1.59, s

1.73, s 1.82, s 1.55, s

2.17, s

2.11, s

15 16 17 18 19 21 22 23 28 29 30 OMe OAc 6-OH 11-OH a

7a,d

Measured in pyridine-d5. bMeasured in CDCl3.

(J in Hz)

9b,d (J in Hz)

10a,e (J in Hz)

6.96, d (10.0)

6.96, d (9.0)

7.30, d (9.9)

6.14, d (10.0)

6.15, d (9.0)

6.30, d (9.9)

2.71, s 5.71, m 2.06, m

2.45, s 4.66, dd (8.8, 6.2) α 2.07, dd (15.8, 6.2) β 2.42, d (15.8) 4.04, s α 2.09, m β 2.36, m 2.87, dd (10.6, 6.3) 0.77, s 1.56, s

2.82, s 5.04, m 2.18, m 2.90, d (15.2) 4.08, s 2.05, dd (13.5, 11.0) 2.16, dd (13.5, 6.4) 2.98, dd (11.0, 6.4) 0.90, s 1.81, s

6.30, brs

7.00, brs 5.93, brs 1.89, s 1.88, s 1.77, s 3.42, s

3.89, s α 1.94, dd (13.6, 11.0) β 2.30, dd (13.6, 6.4) 2.72, dd (11.0, 6.4) 0.70, s 1.33, s 7.14, brs 6.15, brs 7.35, brs 1.56, s 1.50, s 1.40, s 2.13, s 6.47, s

1.57, s 1.51, s 1.39, s

6.35, s 2.33, d (8.8)

c,d, and e

Data were measured at 500, 600, and 400 MHz, respectively.

Figure 2. ORTEP drawing of 10. Figure 3. ORTEP drawing of 4.

absence of NMR signals for the methoxy and dioxymethine groups, and the presence of those for an oxymethylene (CH2− 23: δH 4.83, 2H; δC 70.5), established 5 to be the 23-demethoxy derivative of the former, which was confirmed by the HMBC correlations of H2-23/C-20 and C-22 (Figure S43, Supporting Information). The relative configuration of 5 was assigned via NMR data comparison of corresponding stereogenic centers in 4. Thus, the structure of compound 5 (walsunoid E) was established as shown. Both compounds 6 (walsunoid F) and 7 (walsunoid G) were isolated as ca. 4:1 mixtures of epimers based on the proton integrals in their 1H NMR spectra (Figures S48 and S57, Supporting Information). The molecular formulas of C28H32O9 and C28H34O9 of 6 and 7 were determined by 13C NMR data and the deprotonated (−)-HRESIMS ions at m/z 511.1970 [M − H]− (calcd 511.1974) and 513.2130 [M − H]− (calcd 513.2130), respectively. Analysis of the NMR data (Tables 2 and 3) of 6

151.7, 127.6, and 203.5) of 10 (Table S1, Supporting Information) in the A-ring. Further examination of 2D NMR data (Figures S33−S35, Supporting Information) corroborated the structural difference between the two compounds defining compound 4 as the 1,2-dihydro congener of 10. Particularly, 23OMe in 4 was assigned to be α-oriented by comparing the relevant NMR data with those of the epimeric compounds 2 and 10 (Table S1, Supporting Information). The absolute configuration of 4 (walsunoid D) was unequivocally established as shown (Figure 3) by single-crystal X-ray diffraction analysis [absolute structure parameter: −0.04(9)].18 Compound 5 displayed a sodiated molecular ion at m/z 479.2032 [M + Na]+ (cacld 479.2040) in the (+)-HRESIMS spectrum, which is consistent with a molecular formula of C26H32O7. Analysis of the NMR data (Tables 1 and 2) of 5 implied that the structure of 5 resembled that of 4 with the major differences occurring at the lactone ring. Compared to 4, the D

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Compound 10 and compounds 1−3, 6, 8, and 9 possessed the same chromophores in the limonoid core and had similar ECD curves (Figure 5), indicating that they shared identical absolute

revealed that it was an 11-O-acetyl derivative of 11βhydroxyisowalsuranolide3 as supported by the extra proton and carbon signals for an acetyl group (δH 2.17; δC 171.0 and 21.8), which was further confirmed by the deshielded H-11 resonance (ΔδH 0.93) and the HMBC correlation (Figure S51, Supporting Information) from H-11 to the acetyl carbonyl. The relative configuration of 6 was shown to be identical to that of 11βhydroxyisowalsuranolide3 based on their similar coupling constants and supported by the NOESY data (Figure S52, Supporting Information). As for compounds 4 and 10, compound 7 (Tables 2 and 3) was the 1,2-dihydro derivative of 6 by analysis of their NMR differences involving the signals for two sp3 methylenes and a carbonyl group instead of those for an α,β-unsaturated carbonyl moiety. The structure of 7 was also confirmed by inspection of 2D NMR data (Figures S59−S61, Supporting Information). Compound 8 was assigned a molecular formula of C28H32O7 via the (−)-HRESIMS ion at m/z 479.2081 [M − H]− (calcd 479.2075) and 13C NMR data, indicative of an acetylated analogue of 11β-hydroxycedrelone.10 Analysis of the NMR data (Tables 2 and 3) of 8 supported this hypothesis with additional resonances for an acetyl group (δH 2.13; δC 170.6 and 21.6), which was located at C-11 based on the deshielded H-11 resonance (ΔδH 1.09) and the HMBC correlation (Figure S69, Supporting Information) from H-11 to the acetyl carbonyl. The relative configuration of compound 8 was established as the same as that of 11β-hydroxycedrelone via NMR data comparison and NOESY data (Figure S70, Supporting Information). Therefore, the structure of compound 8 (walsunoid H) was elucidated as shown. Compound 9 gave a molecular formula of C26H29NO7 as determined by the (−)-HRESIMS ion at m/z 466.1864 [M − H]− (calcd 466.1871) and 13C NMR data. Comparison of the NMR data of 9 (Tables 2 and 3) with those of 10 (Table S1, Supporting Information) in CDCl3 suggested that compounds 9 and 10 had identical A−D rings but had different C-17 substituents. A maleimide residue at C-17 was established on the basis of the molecular composition of 9 and the remaining NMR data (δH 6.30; δC 130.4, 149.8, 169.5, and 170.6), which was confirmed by the HMBC correlations (Figure 4) of H-17/C20, C-21, and C-22; and H-22/C-20, C-21, and C-23. The relative configuration of 9 was assigned the same as that of 10 based upon NMR comparison and the NOESY data (Figure S80, Supporting Information). Therefore, the structure of compound 9 (walsunoid I) was assigned as shown.

Figure 5. ECD spectra of compounds 1−3, 6, and 8−10.

configurations in the A−D rings. The absolute configurations of compounds 1−3, 6, 8, and 9 were thus assigned as shown. Similarly, the absolute configurations of compounds 5 and 7 were established as depicted by comparing their ECD curves with that of 4 (Figure 6).

Figure 6. ECD spectra of compounds 4, 5, and 7.

Compounds 2 and 10 with a methyl acetal group at C-23 were verified as true natural products by the LC-ESIMS analysis of the ethanolic crude extract, in which the ESIMS molecular ion peaks at m/z 483 [M − H]− and m/z 485 [M + H]+ for 2 and 10 were observed (Figures S92−S95, Supporting Information). Although the molecular ion peaks of compound 4 was not found in the LCESIMS analysis due likely to the lower abundance in the crude extract, it is believed as a true natural product. In addition, the known compounds were characterized as niloticin (11),19,20 dihydroniloticin (12),20,21 piscidinol A (13),22,23 22S,23R-epoxytirucalla-7-ene-3α,24,25-triol (14),24 3-epimesendanin S (15),8 11β-hydroxyisowalsuranolide (16),3 isowalsuranolide (17),10 11β-hydroxy-1,2-dihydroisowalsuranolide (18),3 11β-hydroxydihydrocedrelone (19),10 11β-acetoxydihydrocedrelone (20)10 by comparing their observed and reported spectroscopic data. All the isolates were evaluated in vitro for their inhibitory effects against both human and mouse 11β-HSD1 using the scintillation proximity assay (SPA).25,26 The preliminary testing of these compounds at 10 μM demonstrated that compound 14

Figure 4. 1H−1H COSY (bold bond) and selected HMBC correlations of 9. E

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EtOAc (3 × 1.5 L) to obtain the EtOAc fraction (430 g), which was separated into fractions A, B, and C by D101 macroporous absorption resin eluted with EtOH-H2O (50:50, 80:20, and 95:5, v/v). Fraction B (200 g) was applied to an MCI gel column eluted with MeOH-H2O (50−100%) to give six fractions (B1− B6). Fraction B2 (12 g) was subjected to a silica gel column using petroleum ether-acetone (20:1 to 1:3) to give nine fractions (B2a−B2i). Fractions B2g and B2h were separated by columns of silica gel with CHCl3-MeOH (100:1 to 10:1) into subfractions B2g1−B2g5 and B2h1−B2h3, respectively. Compound 9 (5 mg) was purified by semipreparative HPLC (3.0 mL/min, 42% MeCN-H2O isocratic elution) from fraction B2g4. Fraction B2g3 was purified by semipreparative HPLC (3.0 mL/min, 47% MeCN-H2O) to afford compound 5 (3 mg). Fraction B2h3 was separated by a column of C18 reversed-phase silica gel with MeOH-H2O (50−100%) to afford three subfractions (B2h3a− B2h3c). Compound 3 (13 mg) was isolated from B2h3b by semipreparative HPLC (3.0 mL/min, 37% MeCN-H2O isocratic elution). Fraction B2h2 was applied to a silica gel column eluted with petroleum ether-acetone (20:1 to 1:3) to get four subfractions (B2h2a−B2h2d). Subfraction B2h2c was further separated by semipreparative HPLC (3.0 mL/min, 40% MeCNH2O) to afford compounds 6 (36 mg) and 7 (15 mg). Compound 1 (5 mg) was purified by semipreparative HPLC (3.0 mL/min, 37% MeCN-H2O) from B2h2d. Fraction B4 (10 g) was subjected to a silica gel column eluted with petroleum ether-acetone (20:1 to 1:3) to yield eight fractions (B4a−B4h). Fraction B4b was separated over a column of silica gel eluted with CHCl3-MeOH (100:1 to 10:1), and the major part was purified by semipreparative HPLC (3.0 mL/min, 60% MeCN-H2O) to give compound 8 (16 mg). Fraction B 4f was subjected to a silica gel column eluted with CHCl3-MeOH (100:1 to 10:1) to give fractions B 4f1−B 4f4. Fraction B 4f4 was applied to a column of silica gel eluted with petroleum ether-EtOAc (8:1 to 1:2) to obtain three major components, each of which was purified by semipreparative HPLC (3.0 mL/min, 52% CH3CN-H2O) to afford compounds 2 (25 mg), 4 (4 mg), and 10 (40 mg), respectively. 11β-Hydroxy-23-O-methylwalsuranolide (10). Colorless crystals (MeOH); mp 204−205 °C; [α]27D + 22 (c 0.8, MeOH); UV (MeOH) λmax(log ε) 216 (4.10), 279 (3.98) nm; ECD (MeOH) λ (Δε) 195 (+27.0), 279 (+19.3), 319 (−15.7) nm; 1H and 13C NMR data (pyridine-d5) see Tables 2 and 3, 1H and 13C NMR data (CDCl3) see Table S1, Supporting Information; (−)-HRESIMS m/z 483.2011 [M − H]− (calcd for C27H31O8, 483.2024). Walsunoid A (1). White powder; [α]27D − 18 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 216 (3.96), 278 (3.64) nm; ECD (MeOH) λ (Δε) 198 (+12.2), 278 (+20.3), 321 (−14.8) nm; IR (KBr) νmax 3408, 2936, 1675, 1354, 1251, 1032, 733 cm−1; 1H and 13C NMR data see Tables 1 and 2; (+)-ESIMS m/z 445 [M + H]+, (−)-ESIMS m/z 443 [M − H]−; (−)-HRESIMS m/z 443.2068 [M − H]− (calcd for C25H31O7, 443.2075). Walsunoid B (2). White powder; [α]27D − 66 (c 0.9, MeOH); UV (MeOH) λmax (log ε) 216 (4.11), 279 (3.96) nm; ECD (MeOH) λ (Δε) 194 (+34), 228 (+6.0), 281 (+21.6), 319 (−18.0) nm; IR (KBr) νmax 3410, 2978, 1767, 1678, 1365, 1265, 1091, 1033, 732 cm−1; 1H and 13C NMR data (pyridine-d5) see Tables 1 and 2, 1H and 13C NMR data (CDCl3) see Table S1, Supporting Information; (+)-ESIMS m/z 485 [M + H]+, (−)-ESIMS m/z 483 [M − H]− ; (−)-HRESIMS m/z 483.2029 [M − H]− (calcd for C27H31O8, 483.2024).

showed >50% inhibition against both enzymes, while compound 8 selectively inhibited the human 11β-HSD1 and compounds 11−13 displayed better inhibitory activity against the mouse subtype than against the human subtype (Tables S2 and S3, Supporting Information). The IC50 values of those compounds exhibiting >50% inhibition rate at the tested single concentration was further acquired and the results are shown in Table 4. Five compounds showed moderate inhibitory activities against human and/or mouse 11β-HSD1 with IC50 values ranging from 0.69 to 9.9 μM. Table 4. Inhibitory Activities of Compounds 8 and 11−14 against Human and Mouse 11β-HSD1 (IC50 ± SD in μM)

a

compound

human

mouse

8 11 12 13 14 glycyrrhetinic acid

9.9 ± 1.4 NTa NT NT 1.9 ± 0.16 8.8 ± 1.6 nM

NAa 0.69 ± 0.03 3.8 ± 0.67 0.88 ± 0.29 1.2 ± 0.15 9.4 ± 1.0 nM

“NA” − Not active; “NT” − IC50 not tested

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EXPERIMENTAL SECTION General Experimental Procedures. Melting points were measured using an SGM X-4 apparatus (Shanghai Precision & Scientific Instrument Co., Ltd., P.R. China). Optical rotations were acquired on a PerkinElmer 341 polarimeter. UV spectra were recorded on a Shimadzu UV-2550 UV−visible spectrophotometer. IR spectra were obtained on a PerkinElmer 577 spectrometer with KBr disks. 1D and 2D NMR spectra were performed on Bruker AM-400, AM-500, and AM-600 NMR spectrometers with TMS as internal standard. (±)-ESIMS and (±)-HRESIMS data were collected on an Esquire 3000plus LCMS and a Waters Q-TOF Ultima Global mass spectrometer. ECD data were acquired on a JASCO 810 Spectrophotometer. The X-ray crystallographic data were obtained on a Bruker SMART CCD detector employing graphite monochromated Cu−Kα radiation. D101-macroporous absorption resin (Sinopharm Chemical Reagent Co., Ltd., Shanghai, P. R. China), MCI gel (CHP20P, 75−150 μm, Mitsubishi Chemical Industries Ltd., Japan), Silica gel (Silica gel H, 200−300 mesh, 300−400 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, P. R. China), Sephadex LH-20 gel (Amersham Biosciences, Sweden) and C18 reversed-phase silica gel (150−200 mesh, Merck, Germany) were used for column chromatography. Precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao, P. R. China) were used for TLC analyses. Semipreparative HPLC was performed on a Waters 1525 pump equipped with a Waters 2489 detector and YMC-Pack ODS-A column (250 × 10 mm, S5 μm, 12 nm). Plant Material. The leaves of W. robusta were collected in August 2013 from Jianfengling on the Hainan Island, People’s Republic of China, and were authenticated by Prof. S.-M. Huang from Department of Biology, Hainan University. A voucher specimen (deposition no.: WR-2013-Y1) has been deposited at Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Extraction and Isolation. The air-dried powder of the leaves of W. robusta (12 kg) was extracted with 95% EtOH (3 × 20 L) at room temperature to afford a crude extract (1.2 kg). The crude extract was suspended in H2O (1.5 L) and partitioned with F

DOI: 10.1021/acs.jnatprod.5b00952 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Walsunoid C (3). White powder; [α]27D − 21 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 216 (4.08), 276 (3.94) nm; ECD (MeOH) λ (Δε) 194 (+20.3), 234 (+4.2), 278 (+16.0), 319 (−12.5) nm; IR (KBr) νmax 3409, 2927, 1760, 1675, 1355, 1251, 1092, 1032, 734 cm−1; 1H and 13C NMR data see Tables 1 and 2; (+)-ESIMS m/z 471 [M + H] + , 963 [2 M + Na] + ; (−)-HRESIMS m/z 469.1867 [M − H]− (calcd for C26H29O8, 469.1868). Walsunoid D (4). Colorless crystals (MeOH); mp 255−256 °C; [α]27D + 13 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 204 (3.79), 278 (3.80) nm; ECD (MeOH) λ (Δε) 220 (−9.1), 274 (+6.2), 319 (−3.1) nm; IR (KBr) νmax 3410, 2927, 1766, 1707, 1674, 1359, 1249, 1092, 1031, 734 cm−1; 1H and 13C NMR data see Tables 1 and 2; (+)-ESIMS m/z 487 [M + H]+, (−)-ESIMS m/z 485 [M − H]−; (+)-HRESIMS m/z 995.4435 [2 M + Na]+ (calcd for C54H68O16Na, 995.4400). Walsunoid E (5). White powder; [α]27D − 19 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (3.99), 278 (3.94) nm; ECD (MeOH) λ (Δε) 223 (−7.5), 275 (+8.2), 319 (−4.2) nm; IR (KBr) νmax 3403, 2929, 1751, 1708, 1674, 1348, 1249, 1087, 1033, 733 cm−1; 1H and 13C NMR data see Tables 1 and 2; (+)-ESIMS m/z 457 [M + H]+, (−)-ESIMS m/z 455 [M − H]−; (+)-HRESIMS m/z 479.2032 [M + Na] + (calcd for C26H32O7Na, 479.2040). Walsunoid F (6). White powder; [α]27D − 49 (c 1.5, MeOH); UV (MeOH) λmax (log ε) 217 (4.19), 278 (3.87) nm; ECD (MeOH) λ (Δε) 198 (+8.3), 275 (+17.9), 322 (−17.2) nm; IR (KBr) νmax 3409, 2974, 2935, 1766, 1739, 1680, 1364, 1252, 1034, 957 cm−1; 1H and 13C NMR data see Tables 2 and 3; (+)-ESIMS m/z 513 [M + H]+, (−)-ESIMS m/z 511 [M − H]−; (−)-HRESIMS m/z 511.1970 [M − H]− (calcd for C28H31O9, 511.1974). Walsunoid G (7). White powder; [α]27D − 39 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 208 (4.19), 278 (4.03) nm; ECD (MeOH) λ (Δε) 218 (−19.0), 257 (+6.2), 317 (−3.9) nm; IR (KBr) νmax 3411, 2972, 2936, 1762, 1736, 1712, 1360, 1250, 1034, 955 cm−1; 1H and 13C NMR data see Tables 2 and 3; (+)-ESIMS m/z 515 [M + H]+, (−)-ESIMS m/z 513 [M − H]−; (−)-HRESIMS m/z 513.2130 [M − H]− (calcd for C28H33O9, 513.2130). Walsunoid H (8). White powder; [α]27D − 47 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 217 (4.06), 278 (3.93) nm; ECD (MeOH) λ (Δε) 197 (+10.5), 278 (+16.0), 320 (−13.9) nm; IR (KBr) νmax 3400, 2978, 2940, 1729, 1686, 1380, 1356, 1252, 1035 cm−1; 1H and 13C NMR data see Tables 2 and 3; (+)-ESIMS m/z 481 [M + H]+, (−)-ESIMS m/z 479 [M − H]−; (−)-HRESIMS m/z 479.2081 [M − H]− (calcd for C28H31O7, 479.2075). Walsunoid I (9). White powder; [α]27D + 7 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 225 (4.13), 280 (3.77) nm; ECD (MeOH) λ (Δε) 193 (+14.5), 280 (+15.6), 322 (−10.6) nm; IR (KBr) νmax 3436, 2926, 1715, 1675, 1344, 1093, 1034, 735 cm−1; 1 H and 13C NMR data see Tables 2 and 3; (+)-ESIMS m/z 468 [M + H]+, (−)-ESIMS m/z 466 [M − H]−; (−)-HRESIMS m/z 466.1864 [M − H]− (calcd for C26H28NO7, 466.1871). X-ray Crystallographic Analysis. Compounds 4 and 10 were crystallized from methanol at room temperature. The X-ray crystallographic data were obtained on a Bruker SMART CCD detector using graphite monochromated Cu Kα radiation (λ = 1.54178 Å) at 140(2) (operated in the ϕ−ω scan mode). The structures were established by direct method using SHELXS-97 (Sheldrick 2008) and improved with full-matrix least-squares calculations on F2 using SHELXL-97 (Sheldrick 2008). Crystallographic data for 4 (Table S4, Supporting Information)

and 10 (Table S5, Supporting Information) have been deposited at the Cambridge Crystallographic Data Center (deposition numbers: CCDC 1432398 for 4 and CCDC 1418722 for 10). The copy of the data can be obtained free of charge on application to the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [tel: (+44) 1223−336−408; fax: (+44) 1223−336−033; email: [email protected]]. 11β-HSD1 Inhibitory Activity Assay. The 11β-HSD1 inhibitory assay was performed as previously reported, with glycyrrhetinic acid being used as positive control.8

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00952. IR, ESIMS/HRESIMS, 1D and 2D NMR spectrum of compounds 1−10 (PDF) X-ray crystallographic data of compound 4 (CIF) X-ray crystallographic data of compound 10 (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail for J.-M.Y.: [email protected]. Tel: 86-21-50806718. Fax: 86-21-50806718. *E-mail for H.Z.: [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 (Nos. 21532007, 81273398) of the People’s Republic of China. We thank Prof. S. M. Huang from Department of Biology, Hainan University, for the identification of the plant material.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.5b00952 J. Nat. Prod. XXXX, XXX, XXX−XXX