Ingol-Type Diterpenes from Euphorbia antiquorum with Mouse 11β

Article pubs.acs.org/jnp

Ingol-Type Diterpenes from Euphorbia antiquorum with Mouse 11βHydroxysteroid Dehydrogenase Type 1 Inhibition Activity Wei-Yan Qi,† Wei-Yi Zhang,† Yu Shen,‡ Ying Leng,‡ Kun Gao,*,† and Jian-Min Yue*,†,‡ †

State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, People’s Republic of China ‡ State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China S Supporting Information *

ABSTRACT: Eighteen new ingol-type diterpenes, euphorantins A−R (1−18), along with four known analogues (19− 22), were isolated from the aerial parts of Euphorbia antiquorum. Compounds 1−3 are the first examples of C-17oxygenated ingol-type diterpenes, and compounds 16−18 represent a rare class of 2,3-di-epimers of ingols. Diterpenes 1, 14, and 22 exhibited inhibitory activities against mouse 11βHSD1 with IC50 values of 12.0, 6.4, and 0.41 μM, respectively.

T

1.6 Hz, 1H), 2.00 (dq, J = 7.2, 1.6 Hz, 3H), 1.94 (dq, J = 1.6, 1.6 Hz, 3H)], a methoxy group (δH 3.36, 3H), a carbonyl (δC 207.3), and a trisubstituted double bond (δH 5.73, s; δC 120.0 and 143.1). In addition, the NMR spectra also showed signals for seven oxygenated sp3 carbons (one methylene, four methine, and two quaternary carbons), four methyls (two tertiary and two secondary), and an sp3 quaternary carbon (δC 19.6). The aforementioned data and biosynthetic reasoning suggested that 1 is an ingol-type diterpenoid.3 In the HMBC spectrum (Figure 1), the correlation of H-7 (δH 5.65) with the angelate carbonyl (δC 167.6) was used to locate the angeloyloxy group at C-7. In addition, the correlations from H-3 (δH 5.28) and H-12 (δH 4.92) to each of the acetyl carbonyls at δC 170.4 and 170.7 suggested that the two acetoxy groups are attached at C-3 and C-12, respectively, while the correlations from H-8 to the carbon of OMe helped place the methoxy group at C-8, and the carbonyl could be located at C-14 from the HMBC correlation from H3-20 to C-14 at δC 207.3. In turn, the mutual cross-peaks from H2-17 (δH 4.62) to C-5 and C-6 and from H-5 to C-17 were used to reveal the presence of a hydroxy group at C-17. The relative configuration of 1 was assigned by analysis of its 1 H NMR and ROESY spectra. The coupling constant of J2,3 = 8.5 Hz and the chemical shifts of H2-1 were used to assign the H-2 and H-3 protons in an α-orientation,9 and this was supported by the ROESY correlations of H-1β/Me-16, H-2/H3, and H-3/H-5 (Figure 1). The E-geometry was assigned to the Δ5 double bond from the ROESY correlation between H-5 and H-7. In addition, the ROESY correlations of H2-17/H-7, H2-17/H-8, H-8/H3-19, H-12/H3-19, and H-13/H3-19 revealed that H-7, H-8, H-12, and H-13 are cofacial, and these

he genus Euphorbia (family Euphorbiaceae) has more than 2000 species and is widely distributed over the world in tropical and temperate regions.1 Chemical investigations on this plant genus have led to the isolation of a large array of diterpenoids with diverse structural classes such as the jatrophane, lathyrane, tigliane, ingenane, myrsinol, and ingol types, in which some diterpenoids showed important biological activities including cytotoxic, antimicrobial, anti-inflammatory, and anti-HIV activities.2 The stems and leaves of Euphorbia antiquorum L. have long been used as an herbal medicine in mainland China for the treatment of toothache, dropsy, palsy, and amaurosis.3 Previous chemical studies on this plant growing in Sri Lanka afforded a number of diterpenoids and triterpenoids.3−5 In the continuation of a search for new and bioactive diterpenoids from the family Euphorbiaceae,6−8 18 new ingol-type diterpenes, namely, euphorantins A−R (1−18), as well as four known compounds (19−22) were isolated from the aerial parts of E. antiquorum. Among them, compounds 1− 3 are the first examples of C-17-oxygenated ingol-type diterpenes, while compounds 16−18 are 2,3-di-epimers of ingol analogues, which are very rare and have been found only in E. bungei and E. canariensis hitherto.9,10 Herein are described the isolation, structure elucidation, and biological evaluation of these isolated diterpenoids.

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RESULTS AND DISCUSSION Compound 1 was obtained as a white powder. Its HRESIMS displayed a molecular ion at m/z 585.2671 [M + Na]+ (calcd for 585.2676), consistent with a molecular formula of C30H42O10, with 10 degrees of unsaturation. The IR spectrum showed the presence of hydroxy (3434 cm−1) and carbonyl (1737, 1712 cm−1) groups. The 1D NMR spectra of 1 (Tables 1 and 2) revealed the presence of two methyl group signals (δH 2.07 and 2.13, each 3H), an angeloyl unit [δH 6.12 (qq, J = 7.2, © 2014 American Chemical Society and American Society of Pharmacognosy

Received: March 10, 2014 Published: May 19, 2014 1452

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4.62, δC 61.2) of 1, which resulted in the H-5 signal at δH 6.65 being shifted downfield about Δδ 0.9 as compared to that of 1. This was confirmed by the key HMBC correlations of H-17/C6, H-5/C-17, and H-5/C-6 (Figure S18, Supporting Information). The relative configuration of 3 was proposed as being the same as 1 on the basis of the ROESY spectrum (Figure S19, Supporting Information). Compounds 4−6 were found to share the same molecular formula, C30H42O10, as determined by HRESIMS. The characteristic 1H and 13C NMR spectroscopic data (Tables 1 and 2) indicated that they are ingol-type diterpenoids structurally resembling the known compound 19, with the evident differences being in the oxygenated substituents. Comprehensive analysis of the NMR data revealed 4−6 to contain two acetyls and one epoxyangeloyl group with either a trans- or a cis-configuration, which could be distinguished by the chemical shifts of H-3′, H-4′, and C-5′ (δH 3.35, 1.38, and δC 13.0 for a trans-configuration; δH 3.05, 1.30, and δC 19.0 for a cis-configuration).12,13 For compound 4, two acetoxy groups were located at C-3 and C-12, and a cis-configured epoxyangeloyloxy group (δH 3.06, q, J = 5.4 Hz, H-3′; 1.27, d, J = 5.4 Hz, H-4′; δC 19.1, C-5′) was located at C-7 by the interpretation of the HMBC spectrum (Figure S25, Supporting Information). NMR data analysis showed that compound 5 is closely related to 4 in structure and possesses a trans-configured epoxyangeloyloxy group (δH 3.34, q, J = 5.4 Hz, H-3′; 1.36, d, J = 5.4 Hz, H-4′; δC 13.2, C-5′) at C-7. Compound 6 was identified as the isomer of 5 by NMR analysis, in particular the HMBC spectrum (Figure S39, Supporting Information). A trans-configured epoxyangeloyloxy group was assigned to C-12 in 6 from the correlation from H-12 (δH 4.86) to the epoxyanglate carbonyl (δC 170.7). The molecular formula, C30H42O10, of compound 7 was determined from the molecular ion at m/z 585.2672 [M + Na]+ in the HRESIMS. The 1H and 13C NMR spectroscopic data of 7 showed its structure resembling that of compound 4. The difference was the presence of a 3-hydroxy-2-methylenebutyrate group (δH 6.25 and 5.87, br s, each 1H; 4.67, t, J = 6.4 Hz, 1H; 1.41, d, J = 6.4 Hz, 3H) at C-7 in compound 7 in place of the epoxyangeloyloxy group of 4. This was confirmed by the HMBC correlation from H-7 (δH 5.30) to the carbonyl (δC 165.6) of the 3-hydroxy-2-methylene-butyryl group (Figure S46, Supporting Information). The HRESIMS ion at m/z 549.2458 [M + Na]+ (calcd for 549.2464) gave a molecular formula of C30H38O8 for compound 8. Initial analysis of the 1H and 13C NMR data showed the presence of an acetyl, a benzoyl, and a methoxy group. In the 1H NMR spectrum (Table 1), the H-12 (δH 3.28) signal was shifted upfield (Δδ 1.6) as compared to this same signal in 4. This indicated that a hydroxy group was attached at C-12 of compound 8. Furthermore, the acetoxy and benzoyloxy groups were connected to C-3 and C-7, respectively, from the HMBC cross-peaks from H-3 (δH 5.18) to the carbonyl (δC 170.4) of the acetyl and from H-7 (δH 5.49) to the carbonyl (δC 165.6) of the benzoyl group. The structure of euphorantin H (8) was thus assigned as depicted. Euphorantin I (9) gave the same molecular formula, C30H38O8, as that of 8. The 1H and 13C NMR spectroscopic data analysis (Tables 1 and 2) revealed that compounds 8 and 9 are isomers bearing the same acyl groups. In the 1H NMR spectrum of 9, the H-3 signal resonated upfield at δH 4.25 as compared with compound 8, indicating the presence of a hydroxy group at C-3. In turn, the downfield shifted H-12 at δH

were assigned arbitrarily in a β-orientation. Furthermore, the epoxide ring is β-orientated, which was supported by the ROESY correlations of H3-18/H-9 and H3-18/H-11. Thus, the structure of 1 was elucidated, and this compound has been named euphorantin A. Compound 2, a white, amorphous powder, gave a molecular formula of C32H40O10 as determined by the HRESIMS ion at m/z 607.2521 [M + Na]+ (calcd for 607.2519). The 1H and 13 C NMR spectroscopic data of 2 (Tables 1 and 2) resembled those of compound 1. The only difference was the occurrence of a benzoyloxy group at C-7 of 2 replacing the C-7 angeloyloxy group of 1. The structure of 2 was confirmed from its 2D NMR data (Figures S10 and S11, Supporting Information). In the HMBC spectrum, a key correlation from H-7 (δH 5.84) to the benzoate carbonyl (δC 166.2) was observed. The relative configuration of 2 was corroborated by analysis of the coupling constants and the ROESY correlations of the key protons. Compound 3 was assigned a molecular formula of C30H40O10 by HRESIMS at m/z 583.2525 [M + Na]+ (calcd for 583.2519). The 1H and 13C NMR spectroscopic data of 3 (Table 1 and 2) were closely comparable to those of 1. The main differences indicated the presence of a C-17 aldehyde (δH 10.35, δC 191.6) in 3 instead of the C-17 hydroxymethyl (δH 1453

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Table 1. 1H NMR Spectroscopic Data of Compounds 1−9 [400 MHz, CDCl3, δ (ppm)]a position 1α 1β 2 3 5 7 8 9 11 12 13 16 17

18 19 20 Ac-3 Ac-7 Ac-12 OMe 3′ 4′ 5′

1

2

3

4

5

6

7

8

9

2.81 dd (15.0, 9.0) 1.71 dd (15.0, 1.2) 2.49 m 5.28 d (8.5) 5.73 br s 5.65 br s 3.13 dd (10.2, 2.0) 1.20b 0.89b

2.78 dd (15.0, 9.0) 1.71 d (15.5)

2.92 dd (15.2, 9.0) 1.81b

2.80 dd (15.0, 9.0) 1.69 d (15.0)

2.81 dd (15.0, 9.0) 1.71 d (15.0)

2.79 dd (15.0, 9.0) 1.70 d (15.2)

2.73 dd (15.0, 9.0) 1.71 d (15.0)

2.77 dd (15.0, 9.2) 1.62b

2.47 m 5.18 d (8.5) 5.84 br s 5.84 br s 3.24 dd (10.2, 2.0) 1.35 t (9.6) 1.02b

2.64 m 5.45 d (8.5) 6.65 br s 6.14 br s 2.99 dd (10.2, 2.0) 1.12b 0.92b

2.80 dd (15.0, 9.0) 1.69 dd (15.0, 1.4) 2.55 m 5.20 d (8.8) 5.61 br s 5.38 br s 2.92 dd (10.0, 2.0) 1.16 t (9.6) 1.02b

2.48 m 5.33 d (8.6) 5.58 br s 5.25 br s 2.90 dd (10.0, 2.0) 1.13b 1.00b

2.48 m 5.34 d (8.6) 5.57 br s 5.35 br s 2.92 dd (10.0, 1.6) 1.14b 1.02 d (11.0)

2.46 m 5.30 d (8.5) 5.64 br s 5.30 br s 2.95 dd (10.0, 1.4) 1.18b 0.99b

2.38 m 4.25 d (8.2) 5.77 br s 5.64 br s 3.03 dd (10.2, 2.0) 1.12b 1.03b

4.92 dd (11.0, 4.0) 3.12 m 0.96 d (7.6) 4.62 br s

4.97 dd (10.7, 4.0) 3.18 m 0.93 d (7.4) 4.63 dd (12.0, 3.8) 4.71 dd (12.0, 4.0) 1.18 s 1.02 s 1.06 d (7.2) 2.06 s

4.82 dd (11.1, 4.0) 2.64 m 1.00 d (7.6) 10.35 s

4.87 dd (11.0, 4.0) 2.90 m 0.91 d (7.6) 2.13 s

4.86 dd (11.0, 4.0) 2.90 m 0.96 d (7.6) 2.04 s

4.86 dd (11.0, 4.0) 2.90 m 0.98 d (7.4) 2.05 s

4.88 dd (11.0, 4.0) 2.92 m 0.97 d (7.6) 2.08 s

2.48 m 5.18 d (8.4) 5.71 br s 5.49 br s 2.90 dd (10.0, 1.8) 1.34b 0.74 dd (10.6, 9.1) 3.28 m

1.08 0.96 1.02 2.11

1.09 0.97 1.07 2.06

1.10 0.97 1.05 2.08

1.10 0.97 1.06 2.10 2.13

1.11 0.98 1.07 2.13

2.14 s 3.93 s 8.04 dd (8.4, 1.4) 7.46 t (7.8)

2.11 s 3.43 s 6.14 qq (7.2, 1.6) 2.01 dq (7.2, 1.6) 1.96 dq (1.6, 1.6)

1.10 0.99 1.04 2.07

s s d (7.2) s

2.13 s 3.36 s 6.12 qq (7.2, 1.6) 2.00 dq (7.2, 1.6) 1.94 dq (1.6, 1.6)

7.58 t (7.3)

s s d (7.2) s

s s d (7.2) s

s s d (7.2) s

s s d (7.2) s s

s s d (7.2) s

2.13 s 3.30 s 3.06 q (5.4)

2.12 s 3.29 s 3.34 q (5.4)

3.32 s 3.28 q (5.4)

2.08 s 3.33 s 4.67 t (6.4)

1.27 d (5.4)

1.36 d (5.4)

1.38 d (5.4)

1.59 s

1.54 s

1.55 s

2.74 m 0.93 d (7.2) 2.14 s

1.21 1.14 1.29 2.06

s s d (7.2) s

4.93 dd (11.1, 4.0) 2.97 m 1.03 d (7.6) 2.14 s

1.14 s 1.00 s 1.09 d (7.2)

1.41 d (6.4)

3.26 s 8.04 dd (8.4, 1.2) 7.46 t (7.6)

2.14 s 3.36 s 8.05 dd (8.4, 1.2) 7.46 t (7.6)

6.25 br s

7.59 t (7.6)

7.58 t (7.2)

5.87 br s a

Proton coupling constants (J) in Hz are given in parentheses. Assignments of 1H NMR data are based on HMQC and HMBC experiments. b Overlapping signals.

used to locate the trans-epoxyangeloyloxy group at C-3. In turn, the HMBC correlations from H-7 (δH 5.15), H-8 (δH 4.62), and H-12 (δH 4.86) to the carbonyl carbons δC 169.8, 170.7, and 170.3 of the acetyl groups, respectively, suggested that three acetoxy groups are attached at C-7, C-8, and C-12 (Figure S73, Supporting Information). For compound 12, the H-8 signal (δH 4.61) showed an HMBC cross-peak with the carbonyl carbon (δC 170.0) of the trans-epoxyangeloyloxy group and indicated it was present at C-8, and the three acetoxy groups were assigned to C-3, C-7, and C-12, respectively, by their corresponding HMBC correlations in a similar way (Figure S80, Supporting Information). Hence, the structures of compounds 11 and 12 were elucidated as shown. Compounds 13 and 14 (euphorantins M and N) were assigned the same molecular formula, C29H42O9, as established by HRESIMS. Their NMR data (Tables 2 and 3) indicated the presence of two acetyls and one 2-methylbutanoyl group. In compound 13, the two acetoxy groups and the 2methylbutanoyloxy group were connected to C-3, C-12, and C-8, respectively, from the HMBC correlations from H-3, H12, and H-8 to the corresponding carbonyl in each of the acyl groups (Figure S87, Supporting Information). For compound 14, two acetoxy groups were located at C-7 and C-12 on the basis of the HMBC cross-peaks from H-7 (δH 5.27) and H-12 (δH 4.88) to the corresponding acetyl carbonyl (δC 169.4 and

4.93 showed an HMBC correlation with the acetyl carbonyl at δC 170.8, revealing an acetoxy group to be located at C-12. Finally, the HMBC correlation from H-7 (δH 5.64) to the carbonyl carbon (δC 166.4) of the benzoyl group suggested that a benzoyloxy unit was attached at C-7 (Figure S59, Supporting Information). The structure of 9 was thus elucidated as shown. Euphorantin J (10) gave a molecular formula of C26H38O7 as established by the HRESIMS ion peak at m/z 485.2519 [M + Na]+ (calcd for 485.2515). Its 1H and 13C NMR spectroscopic data (Tables 2 and 3) showed close similarities to those of 19, except that the H-3 (δH 4.29) and H-12 (δH 3.24) signals were shifted upfield (Δδ = 1.0 and 1.6, respectively), indicating the presence of hydroxy groups at C-3 and C-12. The angeloyloxy group was located at C-7 based on the HMBC correlation from H-7 (δH 5.45) to the angeloyl carbonyl (δC 166.6) (Figure S66, Supporting Information). Thus, the structure of compound 10 was established as shown. Euphorantins K (11) and L (12) gave the same molecular formula of C31H42O11. Analysis of the 1H and 13C NMR data of these compounds (Tables 2 and 3) revealed them to be isomers and different from the known compound 20 only in their acylation patterns.11 The NMR data of 11 and 12 showed resonances for both compounds bearing a trans-epoxyangeloyl unit and three acetyl groups. For compound 11, the HMBC correlation from H-3 (δH 5.33) to the carbonyl (δC 170.7) was 1454

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1455

a

170.7 21.1 56.7 166.2 129.6 129.9 128.4 133.3

31.6 29.5 76.9 72.8 120.1 142.8 72.2 79.8 26.9 19.7 30.3 71.7 43.5 207.3 71.6 16.6 61.2 29.5 16.9 13.2 170.3 20.4

2

170.5 20.5 56.9 166.2 127.3 139.5 15.9 20.5

31.7 29.7 76.0 73.2 135.0 141.7 66.5 79.6 26.6 19.2 30.4 70.4 44.4 205.6 70.7 16.8 191.6 29.2 16.4 13.4 170.3 20.5

3

170.7 21.1 56.5 169.1 59.8 60.0 13.4 19.1

31.5 29.4 77.1 73.3 117.3 139.5 74.6 78.5 26.9 19.3 30.5 71.0 43.0 207.4 71.1 16.9 17.2 29.2 16.5 13.3 170.4 20.4

4

170.8 21.1 56.6 170.8 57.4 58.3 13.4 13.2

31.5 29.4 76.4 73.5 117.4 138.8 75.1 78.6 26.9 19.3 29.2 71.0 42.9 207.5 71.2 17.0 17.5 29.2 16.5 13.5 170.4 20.5

5

56.5 170.7 57.5 58.3 13.4 13.3

31.4 29.4 76.2 73.4 117.6 139.1 74.8 78.5 26.9 19.4 30.5 71.0 42.8 207.5 71.2 16.5 17.7 29.2 17.0 13.4 170.4 20.6 170.8 21.1

6

170.8 21.1 56.5 165.6 143.7 67.1 22.01 24.7

31.4 29.3 76.4 73.4 117.4 139.3 74.8 78.5 27.3 19.4 30.6 71.0 42.8 207.5 71.2 16.5 17.6 29.3 17.0 13.4 170.8 20.5

7

Assignments of 13C NMR data are based on HMQC and HMBC experiments.

OMe 1′ 2′ 3′ 4′ 5′

Ac-12

Ac-8

170.7 21.1 56.6 167.6 127.6 139.0 15.9 20.4

31.5 29.5 76.0 72.9 120.0 143.1 71.5 79.5 27.0 19.6 30.4 71.2 43.4 207.3 71.4 16.9 61.2 29.3 16.6 13.2 170.4 20.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Ac-3

Ac-7

1

carbon

56.4 165.6 129.6 130.0 128.5 133.2

31.2 29.2 76.5 74.0 117.2 140.0 75.2 78.7 27.1 18.8 34.1 71.7 43.2 213.1 70.9 16.9 18.0 29.6 16.0 14.7 170.4 20.5

8

170.8 21.1 56.6 166.4 129.7 130.0 128.5 133.3

31.8 32.0 76.1 75.8 117.5 139.4 74.4 79.0 27.2 19.3 30.6 71.0 43.8 207.8 73.0 16.6 17.9 29.4 16.1 13.3

9

Table 2. 13C NMR Spectroscopic Data of Compounds 1−18 [125 MHz, CDCl3, δ (ppm)]a

56.4 166.6 127.5 139.2 15.9 20.7

31.4 31.7 76.1 76.4 117.6 140.8 74.1 78.8 27.2 18.7 34.0 71.6 43.1 213.3 72.6 16.1 18.1 29.5 16.0 14.6

10

170.8 57.4 58.0 13.5 13.2

169.8 21.0 170.7 20.6 170.3 21.1

31.4 29.4 76.4 73.3 117.5 139.1 76.1 72.9 24.7 19.4 30.9 70.5 43.0 207.4 71.2 17.0 17.4 29.1 16.4 13.3

11

170.0 57.4 57.8 13.9 13.2

170.4 21.0

31.4 29.4 76.4 73.4 117.4 139.3 76.6 72.8 24.5 19.6 30.8 70.5 43.0 207.4 71.2 17.0 17.4 29.0 20.6 13.4 170.7 20.6 169.7 21.1

12

176.3 41.0 26.8 16.4 11.6

170.5 21.0

31.7 29.7 77.5 73.7 116.6 141.3 76.5 73.8 23.5 19.1 30.8 70.8 43.2 207.5 71.4 17.0 17.3 29.1 16.5 13.3 170.6 20.6

13

176.1 40.9 26.6 16.4 11.5

170.4 21.0

169.4 21.1

31.8 32.0 76.3 75.7 117.4 139.4 76.5 71.4 24.7 19.2 30.7 70.7 43.1 207.7 72.9 16.1 17.6 29.1 16.2 13.3

14

166.2 129.6 129.8 128.6 133.3

31.3 29.4 77.6 74.2 117.0 141.6 76.6 74.9 26.6 18.8 34.4 71.4 43.4 213.1 71.2 17.0 17.7 29.4 15.8 14.5 170.6 20.7

15

56.4 167.0 127.7 138.9 16.1 20.7

31.8 32.6 76.7 72.9 116.9 138.4 74.6 78.6 27.1 18.7 34.0 71.8 43.2 213.5 73.2 12.0 18.0 29.5 15.9 14.6

16

170.8 21.1 56.5 166.9 127.6 138.9 15.9 20.7

32.1 29.2 76.6 72.6 116.8 138.0 74.1 78.7 27.2 19.3 30.4 71.2 43.1 207.8 73.2 13.4 17.9 29.2 16.6 12.0

17

176.3 40.9 26.8 16.4 11.6

170.5 21.0

32.8 32.4 77.2 71.1 114.7 141.0 76.2 73.6 23.4 19.0 30.7 70.8 43.3 207.4 73.1 12.3 17.4 29.0 16.5 13.2 170.3 20.8

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by comparison of their NMR data with those of 1 (Tables 1, 2, and 3) and/or by analysis of their ROESY spectra. In particular, the coupling constants between H-2 and H-3 (J2,3 = 8−9 Hz) observed for these compounds indicated that the two protons are α-oriented.9 The molecular formula of euphorantin P (16) was determined as C26H38O7 based on its HRESIMS data. An angeloyl group (δH 6.11, qq, J = 7.6, 1.6 Hz, 1H; 1.94, dq, J = 1.6, 1.6 Hz, 3H; 2.02, dq, J = 7.2, 1.6 Hz, 3H) and a methoxy group (δH 3.33) were identified from their 1H NMR and 13C NMR data (Tables 2 and 3). The angeloyloxy and methoxy groups were assigned to C-7 and C-8, respectively, by the HMBC correlations from H-7 (δH 5.40) to the angeloyl carbonyl (δC 167.0) and from H-8 to the OMe (δC 56.4). The NMR data of 16 showed many similarities to those of 10, with the main differences being the coupling constant between H-2 and H-3 (J2,3 = 4.9 Hz) and the chemical shifts of H2-1, which shifted to δH 2.19 and 1.97 in 16 as compared with those of 10 (δH 2.73 and 1.62). These data revealed that H-2 and H-3 in 16 are β-oriented,9 which was supported by the ROESY correlations of H-3/H-1β (δH 1.97) and H3-16/H-1α (δH 2.19) (Figure S109, Supporting Information). Compound 16 was thus assigned as the 2,3-di-epimer of 10.

Figure 1. Key HMBC (→) (a) and ROESY (↔) (b) correlations of euphorantin A (1).

170.4), respectively. Similarly, the 2-methylbutanoyloxy group was thus assigned to C-8 from the 13 by the HMBC spectrum (Figure S94, Supporting Information). Compound 15 gave a molecular formula of C29H36O8. Its NMR spectroscopic data shared many similarities to those of 8. A key HMBC correlation from H-8 (δH 4.70) to the carbonyl carbon (δC 166.2) of the benzoyl group indicated it was attached at C-8. The only acetoxy group was fixed to C-3 by the HMBC cross-peaks from H-3 to the carbonyl of the acetyl group (Figure S101, Supporting Information). Compounds 4−15 (euphorantins D−O) were assigned as having the same relative configuration in the diterpenoid core

Table 3. 1H NMR Spectroscopic Data of Compounds 10−18 [400 MHz, CDCl3, δ (ppm)]a position 1α 1β 2 3 5 7 8 9 11 12 13 16 17 18 19 20 Ac-3 Ac-7 Ac-8 Ac-12 OMe 2′ 3′

4′ 5′ a b

10

11

12

13

14

15

16 b

17

18 2.28 dd (13.7, 10.6) 2.00 dd (13.8, 6.8) 2.12 m 5.33 d (5.5) 5.86 br s 4.19 br s 4.57 dd (10.8, 1.6) 1.38 dd (10.8, 9.2) 1.11b

2.73 dd (15.8, 10.0) 1.62b

2.79 dd (15.0, 9.0) 1.71 d (15.0)

2.79 dd (15.0, 9.0) 1.70 d (15.2)

2.82 dd (15.0, 9.2) 1.69b

2.77 dd (15.0, 9.2) 1.62b

2.78 dd (15.0, 9.2) 1.73b

2.19

1.97b

2.25 dd (13.3, 9.6) 1.95b

2.41 m 4.29 d (8.5) 5.67 br s 5.45 br s 2.82 dd (10.0, 1.8) 1.17 d (9.5)

2.49 m 5.33 d (8.6) 5.62 br s 5.15 br s 4.62 dd (11.0, 2.0) 1.29b

2.49 m 5.33 d (8.6) 5.62 br s 5.11 br s 4.61 dd (10.8, 1.6) 1.30b

2.57 m 5.23 d (8.4) 5.80 br s 4.21 br s 4.58 dd (11.0, 1.5) 1.39b

2.42 m 4.32 t (8.7) 5.69 br s 5.27 br s 4.60 dd (10.8, 2.0) 1.29b

2.59 m 5.25 d (8.4) 5.87 br s 4.36 br s 4.70 dd (10.8, 1.6) 0.96b

2.04 m 4.05 t (4.9) 6.07 br s 5.40 br s 2.83 dd (10.0, 1.8) 1.19b

2.09 m 4.03 t (4.9) 6.03 br s 5.44 br s 2.96 dd (10.0, 1.8) 1.21b

0.68 dd (10.6, 9.2) 3.24 m

1.15b

1.14b

1.10b

1.12b

4.86 dd (11.0, 4.0) 2.91 m 0.97 d (7.4) 2.10 s 1.11 s 0.84 s 1.07 d (7.2) 2.09 s 2.15 s

4.87 dd (11.0, 4.0) 2.92 m 0.92 (7.6) 2.05 s 1.08 s 0.86 s 1.07 d (7.5) 2.10 s

4.88 dd (11.0, 4.0) 2.93 m 1.04 d (7.6) 2.13 s 1.12 s 0.86 s 1.07 d (7.2)

0.72 dd (10.6, 9.2) 3.24 m

1.03b

4.86 dd (11.0, 4.0) 2.93 m 0.97 d (7.4) 2.10 s 1.12 s 0.86 s 1.07 d (7.2)

0.82 dd (10.6, 9.2) 3.29 m 2.74 0.94 2.12 1.21 0.98 1.30 2.13

2.72 1.03 2.09 1.15 1.12 1.26

2.11 s

2.10 s

2.69 1.05 2.09 1.14 1.12 1.27

m d (7.4) s s s d (7.4)

2.15 s 2.10 s 2.11 s

4.88 dd (11.0, 4.0) 2.95 m 1.02 d (7.0) 2.08 s 1.08 s 0.86 s 1.05 d (7.6)

4.87 dd (11.0, 4.0) 2.92 m 0.94 d (7.2) 2.02 s 1.08 s 0.87 s 1.06 d (7.2) 2.15 s

2.13 s 3.34 s

2.11 s

3.33 s 8.07 dd (8.3, 1.4)

6.11 qq (7.6, 1.6)

6.10 qq (7.5, 2.0)

2.02 dq (7.2, 1.6) 1.94 dq (1.6, 1.6)

2.01 dq (7.4, 1.5) 1.93 dq (1.6, 1.6)

m d (7.4) s s s d (7.2) s

m d (6.4) s s s d (7.2)

2.15 s 2.11 s

3.33 s 6.13 qq (7.6, 2.0)

3.29 q (5.2)

3.20 q (5.4)

2.40 q (6.9) 1.50 m

2.33 q (6.9) 1.46 m

2.02 dq (7.2, 1.6) 1.94 dq (1.6, 1.6)

1.36 d (5.4)

1.35 d (5.4)

1.71 m 0.93 t (7.5)

1.67 m 0.90 t (7.5)

7.49 t (7.6)

1.49 s

1.48 s

1.15 d (7.0)

1.11 d (6.8)

7.60 t (7.2)

2.40 q (6.9) 1.50 m 1.70 m 0.93 t (7.2) 1.15 d (6.9)

Proton coupling constants (J) in Hz are given in parentheses. Assignments of 1H NMR data are based on HMQC and HMBC experiments. Overlapping signals. 1456

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(Euan-2012-1Y) has been deposited in the Shanghai Institute of Materia Medica. Extraction and Isolation. The air-dried powder of the plant material (6.0 kg) was extracted three times with 95% EtOH at room temperature (each 10 L) to give an EtOH extract (600 g), which was partitioned between H2O and EtOAc. The EtOAc-soluble part (270 g) was separated on a MCI gel column eluted with MeOH−H2O (3:7 to 9:1, v/v) to produce three fractions, A−C. Fraction C (23 g) was separated on a silica gel column and eluted with gradient mixtures of petroleum ether−acetone (from 20:1 to 1:1) to afford three major fractions, C1−C3. Fraction C1 was separated on a column of reversedphase C18 silica gel (MeOH in H2O, 60−80%) to yield three major components, with each purified by semipreparative HPLC with CH3CN−H2O (65:35, 3 mL/min) as the mobile phase to give 19 (12.6 mg, 28 min), 20 (3.6 mg, 31 min), and 3 (2 mg, 22 min), respectively. Fraction C2 was chromatographed on a reversed-phase C18 silica gel column (MeOH in H2O, 50−90%) to give subfractions C2-1 to C2-3. Fraction C2-1 was subjected to passage over a column of Sephadex LH-20 eluted with MeOH to give two major components, and each of these was purified by semipreparative HPLC with CH3CN−H2O (55:45, 3 mL/min) as the mobile phase to give compounds 4 (4.5 mg, 30 min) and 17 (9.3 mg, 32 min), respectively. Fraction C2-2 was subjected to CC over silica gel eluted with CHCl3− MeOH (150:1) to obtain fractions C2-2a to C2-2d. Fraction C2-2b was separated by semipreparative HPLC (55% CH3CN in H2O, 3 mL/ min) to afford 21 (7.2 mg, 23 min), 7 (1.1 mg, 33 min), 6 (3.3 mg, 38 min), 11 (2.2 mg, 41 min), and 13 (6.5 mg, 42 min), sequentially. In the same way, compounds 12 (3.1 mg, 30 min) and 18 (15 mg, 32 min) were obtained from fraction C2-2c, and compounds 8 (2.5 mg, 36 min) and 16 (0.7 mg, 42 min) were furnished from fraction C2-3. Fraction C3 was chromatographed on a reversed-phase C18 silica gel column eluted with MeOH−H2O mixtures (50−90%) to give fractions C3-1 to C3-2. Fraction C3-2 was fractionated on a silica gel column (CHCl3−MeOH, 150:1), and each major fraction was purified by semipreparative HPLC (45% CH3CN in H2O, 3 mL/min) to afford 2 (2.3 mg, 27 min), 9 (2.6 mg, 30 min), 10 (2.5 mg, 34 min), and 15 (1.4 mg, 51 min), respectively. Fraction C3-2 afforded compound 5 (2.1 mg) by silica gel column chromatography (CHCl3−MeOH, 120:1). Fraction C3-3 was separated by semipreparative HPLC (55% CH3CN in H2O, 3 mL/min) to yield 1 (1.7 mg, 28 min), 14 (0.9 mg, 35 min), and 22 (2.8 mg, 18 min), sequentially. Euphorantin A (1): white, amorphous powder; [α]24D −102 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 219 (3.58) nm; IR (KBr) νmax 3434, 2935, 1737, 1712, 1455, 1375, 1238 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 585.2671 [M + Na]+ (calcd for C30H42O10Na, 585.2676). Euphorantin B (2): white, amorphous powder; [α]24D +18 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (3.61), 272 (2.91) nm nm; IR (KBr) νmax 3438, 2927, 1735, 1454, 1373, 1272 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 607.2521 [M + Na]+ (calcd for C32H40O10Na, 607.2519). Euphorantin C (3): white, amorphous powder; [α]24D −190 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (3.89) nm; IR (KBr) νmax 3434, 2931, 1729, 1679, 1646, 1457, 1375, 1232 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 583.2525 [M + Na]+ (calcd for C30H40O10Na, 583.2519). Euphorantin D (4): white, amorphous powder; [α]24D −172 (c 0.1, MeOH); IR (KBr) νmax 3444, 2927, 2134, 1737, 1637, 1452, 1376, 1234 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 585.2677 [M + Na]+ (calcd for C30H42O10Na, 585.2676). Euphorantin E (5): white, amorphous powder; [α]24D −64 (c 0.1, MeOH); IR (KBr) νmax 3480, 2935, 1735, 1706, 1625, 1450, 1371, 1236 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 585.2668 [M + Na]+ (calcd for C30H42O10Na, 585.2676). Euphorantin F (6): white, amorphous powder; [α]24D −68 (c 0.1, MeOH); IR (KBr) νmax 3446, 2937, 1737, 1635, 1454, 1375, 1240 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 585.2679 [M + Na]+ (calcd for C30H42O10Na, 585.2676). Euphorantin G (7): white, amorphous powder; [α]24D +18 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (3.88) nm; IR (KBr) νmax

The molecular formula of euphorantin Q (17) was determined as C28 H 40 O 8 on the basis of HRESIMS. Spectroscopic data analysis showed that compounds 16 and 17 possess very similar structures, with the only difference occurring at C-12, as reflected by the H-12 chemical shift (δH 4.88) of 17, which was downfield shifted (Δδ = 1.6) as compared with that of 16, indicating the presence of a C-12 acetate in 17 replacing the C-12 hydroxy group of 16. This conclusion was supported by the additional NMR signals observed for an acetyl moiety (δH 2.13; δC 170.8, 21.1) in 17. The structure of euphorantin Q (17) was thus assigned as depicted. Euphorantin R (18) showed the same molecular composition (C29H42O9) as 13, and the NMR spectroscopic data of these two compounds were similar to each other. The major difference was the coupling constants between H-2 and H-3, where J2,3 = 5.5 Hz was observed for 18, indicating that H-2 and H-3 are β-configured. This was corroborated by the key ROESY correlations of H-3/H-1β and H3-16/H-1α (Figure S123, Supporting Information). Hence, compound 18 was assigned as the 2,3-di-epimer of 13. Four known diterpenes isolated from the aerial parts of E. antiquorum were identified, 3,12-diacetyl-7-angeloyl-8-methoxyingol (19),11 3,8,12-triacetyl-7-tigloylingol (20),11 12-acetyl-7angeloyl-8-methoxyingol (21),14 and 3,12-diacetyl-7-benzoyl-8nicotinylingol (22),15 by comparison of their spectroscopic data with values reported in the literature. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is an NADPH-dependent enzyme that is widely expressed in many tissues such as liver, adipose tissue, and brain.16 It can influence factors of metabolic syndrome such as insulin resistance and dyslipidemia. Consequently, it has recently become an attractive therapeutic target for the treatment of a number of diseases such as obesity and metabolic and cardiovascular disease.17 The inhibitory effects of compounds 1, 4, 7, 9, 11− 15, 17−19, and 20−22 on both mouse and human 11β-HSD1 were evaluated using a scintillation proximity assay.18,19 All the test compounds showed no significant inhibitory effects on human 11β-HSD1, while compounds 1, 14, and 22 inhibited mouse 11β-HSD1 with IC50 values of 12.0, 6.4, and 0.41 μM, respectively. These compounds were also tested for their cytotoxic activities against the HL-60 (human premyelocytic leukemia) and A-549 (human lung adenocarcinoma) cell lines and inhibitory activities against the PTP1B enzyme, but none of them were active (IC50 < 10 μM).

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EXPERIMENTAL SECTION

General Expermental Procedures. UV spectra were recorded on a Shimadzu UV-2550 spectrophotometer. IR spectra were made on a PerkinElmer 577 IR spectrometer with KBr disks. NMR spectra were acquired on Varian Mercury-400, Bruker AV-500, and INOVA-600 spectrometers referenced to deuterated solvent peaks. ESIMS and HRESIMS were obtained on a Bruker Daltonics Esquire 3000 Plus and a Waters-Micromass Q-TOF Ultima Global mass spectrometer, respectively. Semipreparative HPLC was carried out on Waters equipment (515 pump and 2487 photodiode array detector) with a YMC-Pack ODS-A column (250 × 10 mm, S-5 μm). Silica gel (300− 400 mesh, Qingdao Marine Chemical Factory, Qingdao, People’s Republic of China), C18 reversed-phase silica gel, and MCI gel were used for column chromatography. Plant Material. The aerial parts of Euphorbia antiquorum were collected from Donglan County of Guangxi Zhuang Autonomous Region of People’s Republic of China and authenticated by Professor Shao-Qing Tang of Guangxi Normal University. A voucher specimen 1457

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3459, 2937, 1735, 1454, 1373, 1243 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 585.2672 [M + Na]+ (calcd for C30H42O10Na, 585.2676). Euphorantin H (8): white, amorphous powder; [α]24D −76 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (3.80), 273 (3.14) nm; IR (KBr) νmax 3430, 2917, 1644, 1430, 1375 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 549.2458 [M + Na]+ (calcd for C30H38O8Na, 549.2464). Euphorantin I (9): white, amorphous powder; [α]24D +21 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (3.77), 274 (3.13) nm; IR (KBr) νmax 3444, 2933, 1727, 1631, 1452, 1375, 1245 cm−1; 1H and 13 C NMR data, see Tables 1 and 2; HRESIMS m/z 549.2449 [M + Na]+ (calcd for C30H38O8Na, 549.2464). Euphorantin J (10): white, amorphous powder; [α]24D −48 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (3.91) nm; IR (KBr) νmax 3438, 2931, 1720, 1646, 1457, 1380, 1232 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 485.2519 [M + Na]+ (calcd for C26H38O7Na, 485.2515). Euphorantin K (11): white, amorphous powder; [α]24D −17 (c 0.1, MeOH); IR (KBr) νmax 3446, 2937, 1739, 1455, 1371, 1238 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 613.2630 [M + Na]+ (calcd for C31H42O11Na, 613.2625). Euphorantin L (12): white, amorphous powder; [α]24D −137 (c 0.1, MeOH); IR (KBr) νmax 3444, 2937, 1739, 1631, 1455, 1371, 1238 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 613.2617 [M + Na]+ (calcd for C31H42O11Na, 613.2625). Euphorantin M (13): white, amorphous powder; [α]24D −80 (c 0.1, MeOH); IR (KBr) νmax 3434, 2937, 1722, 1631, 1459, 1375, 1243 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 557.2726 [M + Na]+ (calcd for C29H42O9Na, 557.2727). Euphorantin N (14): white, amorphous powder; [α]24D +39 (c 0.1, MeOH); IR (KBr) νmax 3450, 2933, 1733, 1459, 1369, 1238 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 569.2509 [M + Na]+ (calcd for C29H42O9Na, 569.2517). Euphorantin O (15): white, amorphous powder; [α]24D +47 (c 0.1, MeOH); IR (KBr) νmax 3475, 2937, 1739, 1635, 1454, 1375, 1249 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 535.2311 [M + Na]+ (calcd for C29H36O8Na, 535.2308). Euphorantin P (16): white, amorphous powder; [α]24D −68 (c 0.1, MeOH); IR (KBr) νmax 3434, 2925, 1637, 1455, 1378 cm−1; 1H and 13 C NMR data, see Tables 2 and 3; HRESIMS m/z 485.2510 [M + Na]+ (calcd for C26H38O7Na, 485.2515). Euphorantin Q (17): white, amorphous powder; [α]24D −161 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 219 (3.96) nm; IR (KBr) νmax 3365, 2937, 1737, 1452, 1371, 1240 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 549.2701 [M + Na]+ (calcd for C29H40O8Na, 549.2700). Euphorantin R (18): white, amorphous powder; [α]24D −56 (c 0.1, MeOH); IR (KBr) νmax 3448, 2937, 1737, 1459, 1373, 1238 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 569.2516 [M + Na]+ (calcd for C29H42O9Na, 569.2517). Bioassays. 11β-HSD1 Inhibitory Activity Assay. Inhibition against human and mouse 11β-HSD1 enzymatic activities was tested via scintillation proximity assay using microsomes containing 11β-HSD1, and glycyrrhetinic acid was used as the positive control (IC50 = 5.7 nM).17 Cytotoxicity Assay. Cytotoxic activities were evaluated against the HL-60 and P-388 cell lines using the MTT method performed according to a previously reported protocol.20 PTP1B Inhibitory Activity Assay. PTP1B inhibitory activity was tested according to the protocols reported previously.21

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Article

AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-0931-8912592. E-mail: [email protected] (K. Gao). *Tel: +86-21-50806718. E-mail: [email protected] (J. M. Yue). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 31270396) and the 111 Project of China.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

IR, HRESIMS, and 1D and 2D NMR spectra of compounds 1− 18 are available free of charge via the Internet at http://pubs. acs.org. 1458

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