ent-Strobane and ent-Pimarane Diterpenoids from Siegesbeckia

Article pubs.acs.org/jnp

ent-Strobane and ent-Pimarane Diterpenoids from Siegesbeckia pubescens Jianbin Wang,† Hongquan Duan,‡ Yi Wang,† Bowen Pan,† Chun Gao,† Chunyan Gai,† Qiong Wu,† and Hongzheng Fu*,† †

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People’s Republic of China ‡ Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, People’s Republic of China S Supporting Information *

ABSTRACT: Two strobane diterpenoids, strobols A (1) and B (2), 15 new pimarane diterpenoids (3−6 and 8−18), and the known compounds kirenol (19), darutigenol (20), and ent-2β,15,16,19-tetrahydroxypimar-8(14)-ene (7) were isolated from the aerial parts of Siegesbeckia pubescens Makino. The structures of the new compounds were established based on the interpretation of HRESIMS and NMR analysis. The configurations of 1, 6, and 17 were confirmed by X-ray crystallographic data. Compounds 3, 5, and 11 inhibited the migration of MB-MDA-231 breast cancer cells induced by the chemokine epithelial growth factor, with IC50 values of 4.26, 3.45, and 9.70 μM, respectively.

P

genol (20) [ent-3β,15S,16-trihydroxypimar-8(14)-ene],15 and ent-2β,15,16,19-tetrahydroxypimar-8(14)-ene (7).16 The structural elucidation of the new compounds and their effects on the migration of MB-MDA-231 breast cancer cells are presented in this paper. A putative biosynthesis pathway for these compounds is proposed.

lants of the genus Siegesbeckia (Compositae) are annual herbs, widely distributed in China. Their aerial parts have long been used as a traditional Chinese medicine to treat hypertension, rheumatic arthritis, malaria, and snakebites.1,2 Literature reports indicated that extracts and certain chemical constituents of Siegesbeckia exhibit anti-inflammatory, immunosuppressive, antiallergic, antithrombotic, antihistamine release, and antiobesity capabilities, as well as pathogenic microorganism resistance.3−5 Previous pharmacological studies have indicated that diterpenoids are the primary antirheumatic constituents of Siegesbeckia species.6,7 Phytochemical studies of Siegesbeckia species have been conducted for several decades, and more than 100 compounds have been isolated and characterized, including ent-kaurane and ent-pimarane diterpenoids, sesquiterpenoids, and flavonoids.4 Our team has studied Siegesbeckia pubescens in detail and found that the diterpenoid kirenol (19) is a promising lead compound for rheumatic arthritis.8−10 In order to obtain a sufficient quantity of kirenol (19) for immunomodulation activity studies and to identify new bioactive diterpenoids, a crude extract of S. pubescens was investigated, leading to the isolation and identification of two new ent-strobane diterpenoids (1 and 2),11−13 15 new entpimarane diterpenoids (3−6 and 8−18), and the known kirenol (19) [ent-2α,15S,16,19-tetrahydroxypimar-8(14)-ene],14 daruti© 2016 American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION Strobol A (1) was obtained as colorless crystals, mp 251−252 °C. Its molecular formula was determined as C20H34O4, with four indices of hydrogen deficiency, based on the HRESIMS ion at m/ z 373.2154 [M + Cl]− (calcd 373.2151). The IR spectrum showed absorption bands for hydroxy (3400 cm−1) and olefinic (1671 cm−1) groups. The 1H NMR spectrum of 1 (Figure S1, Supporting Information) exhibited signals for an olefinic proton at δH 5.08 (d, J = 5.2 Hz, H-15) and three methyl groups at δH 1.05 (s, Me-20), 1.26 (s, Me-18), and 1.39 (s, Me-17). 13C NMR and DEPT data (Table 1) showed three methyl, eight methylene (including two oxygenated at δC 64.8 and 64.6), five methine (including one olefinic carbon at δC 121.7 and one oxygenated at δC 64.1), and three quaternary carbons (including one olefinic Received: February 20, 2016 Published: December 23, 2016 19

DOI: 10.1021/acs.jnatprod.6b00150 J. Nat. Prod. 2017, 80, 19−29

Journal of Natural Products

Article

Chart 1

Table 1. 13C NMR Spectroscopic Data (100 MHz, Pyridine-d5, Unless Otherwise Noted, δ in ppm) of Compounds 1−9

a

pos.

1

2

3

4a

5

6

7

8

9

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

48.7, CH2 64.1, CH 42.2, CH2 41.0, C 56.5, CH 24.8, CH2 40.0, CH2 142.0, C 60.1, CH 41.9, C 19.0, CH2 41.3, CH2 76.6, C 45.5, CH 121.7, CH 64.6, CH2 25.8, CH3 28.3, CH3 64.8, CH2 16.4, CH3

36.7, CH2 28.6, CH2 78.1, CH 39.6, C 55.4, CH 24.8, CH2 39.6, CH2 142.3, C 59.6, CH 40.4, C 18.7, CH2 41.3, CH2 76.8, C 45.3, CH 121.1, CH 64.6, CH2 25.8, CH3 28.8, CH3 16.3, CH3 14.6, CH3

54.5, CH2 210.0, C 51.9, CH2 39.1, C 55.3, CH 37.8, CH2 198.7, C 131.6, C 152.7, C 43.5, C 125.4, CH 135.9, CH 137.4, C 128.7, CH − − 21.5, CH3 27.8, CH3 66.1, CH2 26.0, CH3

48.9, CH2 65.5, CH 45.1, CH2 41.2, C 52.3, CH 20.0, CH2 31.8, CH2 135.4, C 147.1, C 40.1, C 125.1, CH 127.6, CH 135.8, C 130.5, CH − − 20.9, CH3 27.8, CH3 65.6, CH2 27.1, CH3

37.6, CH2 28.5, CH2 78.1, CH 39.5, C 54.6, CH 22.7, CH2 36.6, CH2 136.2, C 50.3, CH 37.9, C 19.2, CH2 31.8, CH2 38.7, C 130.9, CH 79.0, CH 63.9, CH2 23.9, CH3 29.1, CH3 16.6, CH3 15.3, CH3




46.2, CH2 68.4, CH 85.5, CH 43.9, C 55.5, CH 22.9, CH2 36.6, CH2 137.6, C 51.0, CH 38.9, C 19.1, CH2 32.8, CH2 38.0, C 130.1, CH 76.7, CH 64.0, CH2 23.2, CH3 24.3, CH3 65.4, CH2 16.8, CH3

Data in methanol-d4.

carbon at δC 142.0) and one oxygenated tertiary carbon at δC 76.6. These spectroscopic features accounted for one out of the

four indices of hydrogen deficiency, suggesting the presence of three rings in the structure of 1. 20



Article

The HMBC cross-peaks from Me-17 (s) to C-13 (δC 76.6), C14, and C-12 and from H-15 (δH 5.08) to C-7, C-9, C-13, C-14, and C-16 (δC 64.6), along with two proton spin systems of H-9/ H-11/H-12 and H-15/H-14/H-16 (δH 4.22, 4.04) in the COSY spectrum, demonstrated a unique seven-membered ring with a hydroxy group at C-13 and a hydroxymethyl group at C-14 (fragment A), as depicted in Figure 1 (red line). In addition, the

Figure 3. X-ray ORTEP drawing of compound 1.

Strobol B (2) was isolated as a white solid. Its molecular formula was established as C20H34O3 from the HRESIMS ion at m/z 367.2487 [M + HCOO]− (calcd 367.2484). Absorption bands at νmax 3357 and 1673 cm−1 in its IR spectrum indicated the presence of hydroxy and olefinic groups. On the basis of 1H and 13C NMR data (Tables 1 and 3), compound 2 possessed an ent-strobane skeleton similar to that of 1. A deshielded oxymethine (δC 78.1 vs 64.1 for C-2 in 1) indicated a 3-OH group in 2 rather than the 2-OH group present in 1, and an hydroxymethyl group present in 1 was replaced by a methyl group (δC16.3, C-19) in 2. This assignment was confirmed by the HMBC cross-peaks from Me-18 (δH 1.17, s, 3H) to C-3 (δC 78.1), C-4 (δC 39.6), C-5 (δC 55.4), and C-19 (δC 16.3). In the NOESY spectrum, the correlations of H-3/Me-18/H-5 revealed the α-orientation of 3-OH. Accordingly, the structure of compound 2 was established as ent-3β,13β,16-trihydroxystrob8(15)-ene. Compound 3 was assigned a molecular formula of C18H22O3 based on the HRESIMS ion at m/z 309.1455 [M + Na]+ (calcd 309.1461). The IR spectrum showed absorption bands at νmax 3397, 1708, and 1679 cm−1, consistent with the presence of hydroxy and α,β-unsaturated carbonyl groups. The 1H NMR spectrum exhibited three aromatic protons at δH 7.33 (1H, dd, J = 8.0, 1.2 Hz), 7.22 (1H, d, J = 8.0 Hz), and 8.07 (1H, d, J = 1.2 Hz) and three methyl signals at δH 2.24 (3H, s), 1.36 (3H, s), and 1.24 (3H, s). The 1H and 13C NMR data of 3 resembled those of 7oxo-16-devinyl-ent-pimar-8,11,13-trien-17-oic acid (3a)17 except that an additional carbonyl group (C-2) was present in 3 and a hydroxycarbonyl group (C-19) in 3a was replaced by a hydroxymethyl group in 3. This assignment was confirmed by the HMBC cross-peaks from Me-18 (δH 1.24, 3H, s) to C-3, C-4, C-5, and C-19 (δC 66.1); from Me-20 to C-1, C-5, C-9, and C-10; and from H-1 and H-3 to C-2 (δC 210.0). The absolute configuration of 3 was defined by the comparison of its experimental electronic circular dichroism (ECD) spectrum with that of 11,14-dihydroxy-7-oxo-16-devinyl-ent-pimar8,11,13-trien-17-oic acid (3b).17 The experimental ECD spectrum of 3 exhibited negative Cotton effects at 210, 282, and 326 nm and positive Cotton effects at 252 and 304 nm (Figure S62, Supporting Information), which were similar to those of 3b. Thus, the structure of compound 3 was characterized as 19-hydroxy-15-devinyl-ent-pimar-8,11,13-triene-2,7-dione. Compound 4 was obtained as an amorphous powder. Its molecular formula was determined as C18H26O2 on the basis of the positive HRESIMS ion at m/z 549.3938 [2 M + H]+. A

Figure 1. Key HMBC and COSY correlations of compound 1.

HMBC cross-peaks from Me-20 (s) to C-1, C-5, C-9, and C-10 and from Me-18 (s) to C-3, C-4, C-5, and C-19 (δC 64.8), together with the COSY correlations of H-1/H-2 (δH 4.03, 3.65)/H-3 and H-5/H-6/H-7, indicated the structural fragment B (blue line) with a hydroxy group at C-2 and a hydroxymethyl group at C-4. Fragments A and B shared carbons 7 and 9. Thus, the structure of 1 was elucidated. A specific rotation value of −168 and the biosynthesis pathway allowed for the identification of 1 as an ent-strobane diterpenoid.11−13 The relative configuration of 1 was established on the basis of a NOESY experiment (Figure 2). The correlations of Me-18/H-5/

Figure 2. Key NOESY correlations of compound 1.

H-9 and H-17/H-16 indicated that the hydroxymethyl group at C-14 and Me-18 adopted β-orientations, and the cross-peak of H-2/Me-20 confirmed the β-orientation of 2-OH. Thus, the structure of compound 1 was assigned as ent-2α,13β,16,19tetrahydroxystrob-8(15)-ene, and this identification was confirmed by single-crystal X-ray diffraction analysis of crystals obtained by recrystallization from MeOH (Figure 3). 21



Article

Table 2. 13C NMR Spectroscopic Data (100 MHz, Pyridine-d5, δ in ppm) of Compounds 10−18 pos.

10

11

12

13

14

15

16

17

18

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

49.6, CH2 64.1, CH 46.1, CH2 40.7, C 47.9, CH 30.5, CH2 72.6, CH 141.4, C 46.9, CH 40.3, C 18.7, CH2 32.5, CH2 38.0, C 133.0, CH 76.6, CH 63.9, CH2 22.9, CH3 28.2, CH3 65.1, CH3 16.5, CH3

48.6, CH2 63.7, CH 45.0, CH2 40.3, C 52.3, CH 32.2, CH2 72.2, CH 140.5, C 49.1, CH 39.5, C 18.7, CH2 32.2, CH2 37.3, C 126.3, CH 76.3, CH 63.7, CH2 22.9, CH3 27.7, CH3 64.5, CH3 16.6, CH3

38.1, CH2 38.6, CH2 211.7, C 45.2, CH 53.5, CH 26.7, CH2 35.4, CH2 137.3, C 48.4, CH 37.9, C 19.3, CH2 32.7, CH2 38.1, C 130.3, CH 76.6, CH 64.0, CH2 23.2, CH3 11.9, CH3

76.5, CH 39.8, CH2 75.5, CH 39.8, C 53.3, CH 22.4, CH2 37.1, CH2 138.9, C 52.3, CH 44.7, C 22.4, CH2 33.6, CH2 37.8, C 130.3, CH 76.9, CH 64.1, CH2 23.4, CH3 28.9, CH3 16.4, CH3 9.5, CH3

35.6, CH2 28.5, CH2 78.0, CH 39.1, C 55.3, CH 128.1, CH 131.4, CH 137.1, C 50.4, CH 37.3, C 18.7, CH2 32.8, CH2 38.8, C 133.0, CH 77.3, CH 63.8, CH2 23.5, CH3 28.5, CH3 16.8, CH3 13.5, CH3

47.5, CH2 64.1, CH 45.8, CH2 40.1, C 56.1, CH 127.8, CH 130.6, CH 136.9, C 51.0, CH 38.7, C 19.1, CH2 41.3, CH2 38.8, C 133.3, CH 77.3, CH 63.8, CH2 23.6, CH3 27.3, CH3 65.0, CH2 15.2, CH3

155.8, CH 126.2, CH 203.8, C 44.5, C 52.2, CH 22.2, CH2 35.9, CH2 136.3, C 46.9, CH 40.4, C 18.7, CH2 32.4, CH2 38.1, C 131.9, CH 76.7, CH 64.0, CH2 23.2, CH3 26.4, CH3 22.3, CH3 16.0, CH3

37.7, CH2 28.1, CH2 78.2, CH 39.0, C 49.9, CH 23.6, CH2 123.6, CH 135.6, C 51.0, CH 35.2, C 19.7, CH2 33.0, CH2 44.0, C 85.5, CH 76.6, CH 72.0, CH2 21.6, CH3 28.5, CH3 15.9, CH3 14.4, CH3

32.0, CH2 26.4, CH2 75.1, CH 37.6, C 44.1, CH 23.7, CH2 124.3, CH 135.9, C 51.5, CH 35.3, C 19.8, CH2 33.3, CH2 44.1, C 85.7, CH 77.1, CH 72.3, CH2 21.8, CH3 28.9, CH3 22.6, CH3 14.6, CH3

13.4, CH3

comparison of the 1H and 13C NMR data of 4 with those of 3 (Tables 1 and 3) indicated that 4 was an analogue of 3. The major difference was the absence of two carbonyl groups [one was replaced by an oxygenated methine (C-2) and the other by a methylene (C-7)]. This conclusion was verified by the HMBC cross-peaks from Me-20 to C-1, C-5, C-9, and C-10 and from Me-18 to C-3, C-4, C-5, and C-19, together with the correlations in the COSY spectrum of H-1/H-2(δH 3.96)/H-3 and H-5/H-6/ H-7. In the NOESY spectrum, the cross-peak of Me-20 with H-2 defined the structure of compound 4 as 2β,19-dihydroxy-15devinyl-ent-pimar-8,11,13-triene. Compound 5 and darutigenol (20) were obtained as a pair of epimers.15 The molecular formula of 5 was C20H34O3 according to the HRESIMS and 13C NMR data. The analysis of their 1D and 2D NMR data indicated two similar structures (Figure 4). The 13C NMR spectrum of 5 showed a diagnostic resonance at δC 79.0 (C-15) deshielded by 2.2 ppm compared to δC 76.8 of darutigenol (20). Thus, compound 5 was concluded to be a 15S epimer of darutigenol (20), a conclusion consistent with a previous study of the C-15 configuration of naturally occurring pimarene-15,16-diols.18,19 The absolute configuration of C-15 was confirmed by the induced ECD spectra of its in situ complex with Mo2(OAc)4 in DMSO solution (Snatzke’s method).20−22 According to the helicity rule relating the sign of the Cotton effect to the configuration of the substrate,23 the negative Cotton effect at 310 nm in the ECD spectrum of darutigenol (20) indicated a 15R configuration. In contrast, the positive Cotton effect in the ECD spectrum of 5 indicated a 15S configuration (Figure 5). Thus, the structure of compound 5 was established as ent-3β,15R,16- trihydroxypimar-8(14)-ene. Compounds 6−8 had the same molecular formula of C20H34O4, as indicated by their 13C NMR data and negative HRESIMS ions at m/z 383.2436, 383.2421, and 383.2431 [M + HCOO]−. The 1H and 13C NMR data of these compounds (Tables 1 and 3) were similar to those of kirenol (19) except for some chemical shift differences corresponding to regions near the C-15, C-2, and C-4 stereogenic centers, respectively. The characteristic carbon signal at δC 79.0 (C-15) defined 6 as the 15S epimer of kirenol (19), which was confirmed by the induced

ECD spectrum (Figure 5). Thus, the structure of compound 6 was defined as ent-2α,15R,16,19- tetrahydroxypimar-8(14)-ene, and this identification was confirmed by single-crystal X-ray diffraction analysis (Figure 6). The 13C NMR data of 7 resembled those of kirenol (19), except for small differences in the chemical shifts of C-1, C-2, C3, C-4, and C-10. These differences could be attributed to the effects of 2-OH, suggesting that 7 was the C-2 epimer of kirenol (19). The 1D and 2D NMR data analysis of 7 indicated that it is the known compound ent-2β,15,16,19-tetrahydroxypimar-8 (14)-ene.16 Compared to kirenol (19), a characteristic carbon resonance of a downshifted oxymethylene at δC 71.3 [deshielded by 6.5 ppm compared to 64.8 (C-19) in 19], together with two proton resonances at δH 3.66 (d, J = 10.3 Hz) and δH 3.38 (d, J = 10.3 Hz), was present in the NMR data of 8. The resonance at δC 71.3 was close to the chemical shift of 18-CH2OH but different from that of 19-CH2OH of naturally occurring pimarene diterpenoids.17 Thus, compound 8 was most likely the C-4 epimer of kirenol (19), which was further confirmed by the cross-peaks in the NOESY spectrum of H-2/Me-20/Me-19 and H-18 (δH 3.66 and 3.38)/H-5/H-9. Thus, the structure of compound 8 was assigned as ent-2α,15,16,18-tetrahydroxypimar-8(14)-ene. Compound 9 had a molecular formula of C20H34O5, as determined by the HRESIMS ion at [M + HCOO]− m/z 399.2383 (calcd 399.2383). A comparison of the NMR data of 9 with those of kirenol (19) (Table 1 and Table S1, Supporting Information) revealed a considerable degree of similarity except for some resonances corresponding to ring A. The large chemical shift difference of C-3 (from δC 45.7 in 19 to 85.5) indicated the presence of a 3-OH group, which was supported by the HMBC cross-peaks from Me-18 to C-3, C-4, C-5, and C-19. The crosspeaks in the NOESY spectrum of H-3 with Me-18 and H-5, and of H-2 with Me-20, suggested the α-orientation of 3-OH and the β-orientation of 2-OH. Therefore, the structure of compound 9 was defined as ent-2α,3β,15,16,19-pentahydroxypimar-8(14)ene. Compounds 10 and 11 were obtained as a pair of epimers. The molecular formula C20H34O5 was deduced from their HRESIMS 22


a

1.78, m

2.13, m 1.52, m 2.07, m 1.65, m

1.33, m 1.83, m 1.45, m 2.23, m 1.95, m

1.93, m

2.16, m 1.67, m 2.05, m 1.62, m

3.40, m 5.08, d (5.2) 4.22, t (9.8) 4.04, m 1.39, 3H, s 1.26, 3H, s

4.03, d (10.5) 3.65, d (10.5) 1.05, 3H, s

19a 19b 20

23

Data in methanol-d4.

2

0.97, 3H, s

1.00, 3H, s

3.42, m 5.07, d (4.8) 4.22, t (9.6) 4.06, dd (9.6, 6.5) 1.40, 3H, s 1.17, 3H, s

1.08, m 1.68, m 1.40, m 2.24, m 2.00, m

2.27, m 1.34, m 4.27, m

1a 1b 2a 2b 3a 3b 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12a 12b 13 14 15 16a 16b 17 18

2.86, m 1.31, m

1.57, m 1.11, m 1.81, m 1.81, m 3.44, m

1

pos.

3.72, d (11.2) 3.42, d (11.2) 1.16, 3H, s

2.22, 3H, s 1.07, 3H, s

2.24, 3H, s 1.24, 3H, s 3.84, brs 2.84, brs 1.36, 3H, s

6.81, s

7.11, d (8.1)

7.33, dd (8.0, 1.2)

8.07, d (1.2)

6.88, d (8.1)

1.41, dd (12.8, 1.6) 1.98, m 1.70, m 2.78, m 2.78, m

2.23, m 0.93, m

2.59, brd (11.7) 1.25, t (11.7) 3.96, m

4a

7.22, d (8.0)

2.52, dd (14.0, 3.8) 3.23, m 3.12, dd (14.0, 3.8)

2.92, dd (13.3, 2.4) 2.35, d, (13.3)

3.17, m 2.73, d (13.3)

3

0.73, 3H, s

1.00, 3H, s

5.8, s 4.06, brd (9.8) 4.13, brd (10.5 3.98, dd (10.5, 9.8) 1.19, 3H, s 1.19, 3H, s

1.54, m 1.54, m 1.92, m 1.29, m

1.64, m

1.05, m 1.58, m 1.32, m 2.30, m 2.10, m

1.60, m 1.10, m 1.80, m 1.80, m 3.45, m

5

4.02, m 4.02, m 0.83, 3H, s

5.75, s 3.61, d (10.4) 4.10, dd (10.4, 2.4) 3.95, m 1.17, 3H, s 1.26, 3H, s

1.64, m 1.64, m 1.89, m 1.24, m

1.80, m

1.30, m 1.71, m 1.28, m 2.30, m 2.10, m

2.88, m 1.33, m

2.32, m 1.36, m 4.23, m

6

4.49, d (10.0) 3.80, d (10.0) 1.26, 3H, s

5.45, s 4.10, d (9.6) 4.18, d (9.6) 3.95, dd (9.6, 9.6) 1.17, 3H, s 1.20, 3H, s

1.77, m 1.60, m 2.43, m 1.10, m

1.77, m

1.37, m 1.80, m 1.54, m 2.32, m 2.15, m

2.48, m 1.43, m

2.04, m 1.46, m 4.38, m

7

Table 3. 1H NMR Spectroscopic Data (400 MHz, Pyridine-d5, Unless Otherwise Noted, δ in ppm, J in Hz) of Compounds 1−9

0.84, 3H, s

5.42, s 4.10, d (10.2) 4.18, m 4.01, dd (10.2, 10.2) 1.14, 3H, s 3.66, d (10.3) 3.38, d (10.3) 0.92, 3H, s

1.72, m 1.57, m 2.40, m 1.46, m

1.90, t (9.8)

1.82, m 1.68, m 1.26, m 2.30, m 2.17, m

2.13, m 2.13, m

2.25, m 1.36, m 4.18, m

8

4.43, d (10 0.8) 3.66, d (10.8) 0.76, 3H, s

5.42, s 4.05, m 4.16, m 4.02, m 1.15, 3H, s 1.52, 3H, s

1.57, m 1.57, m 2.40, d (12.8) 1.04, dd (12.8, 4.0)

1.76,t (8.0)

1.30, m 1.66, m 1.30, m 2.27, d (13.6) 2.06, m

2.18, m 1.41, m 4.19, m 3.56, m

9

Journal of Natural Products Article



Article

Figure 4. Key HMBC, COSY, and NOESY correlations of compound 5 and darutigenol (20).

Figure 5. ECD curves of Mo2(OAc)4 complexes of compounds 5, 6, and darutigenol (20).

ions at [M + HCOO]− m/z 399.2384 and 399.2378 (calcd 399.2383). The 1H and 13C NMR data (Tables 2 and 4) revealed structural features similar to those of kirenol (19), except for a hydroxy group at C-7. This assertion was confirmed by the HMBC cross-peaks from H-7 (δH 4.50 or 4.18) to C-6, C-8, and C-14. The β-orientation of 7-OH in 10 was deduced from the small coupling constant of H-7, a broad singlet with a width at half-peak height of 7.2 Hz, while the α-orientation of 7-OH in 11 was derived from the large coupling constant of H-7 (δH 4.18, dd, J = 11.5, 4.8 Hz). This assumption was confirmed by the NOESY cross-peaks of H-7/Me-20 in 10, indicating the β-orientation of 7-OH, and H-5/H-7/H-9 in 11, indicating the α-orientation of 7OH. Therefore, the structures of compounds 10 and 11 were defined as ent-2α,7α,15,16,19-pentahydroxypimar-8(14)-ene

Figure 6. X-ray ORTEP drawing of compound 6.

24


1.30, m 2.29, m 1.66, m 4.18, dd (11.5, 4.8)

1.78, t (8.0)

1.60, m 1.60, m 2.32, m 1.03, dd (12.8, 3.6)

2.33, m 2.25, d (13.6) 1.73, m 4.50, brs

2.68, t (7.8)

1.73, m 1.73, m 2.40, d (12.8) 1.13, m

6.17, s 3.96, d (10.4) 4.07, d (1.6) 3.88, m 1.09, 3H, s 1.18, 3H, s 3.56, d (10.8) 2.90, m 0.79, 3H, s

2.68, d (11.6) 1.21, m

2.93, d (10.8) 1.45, m

5.74, s 4.08, m 4.16, m 4.01, m 1.10, 3H, s 1.41, 3H, s 4.11, m 3.75, d (10.6) 0.90, 3H, s

2.14, d (11.6) 1.24, m 4.10, m

2.33, m 1.50, m 4.29, brs

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

11

10

pos.

25

5.52, s 4.27, m 4.22, m 4.05, dd (10.2, 9.6) 1.19, 3H, s 1.21, 3H, s 1.07, 3H, s 1.06, 3H, s

0.81, 3H, s

2.46, m 2.23, m 2.51, m 1.16, m

2.06, t (8.0)

1.09, m 1.69, m 1.51, dd (12.8, 4.3) 2.36, m 2.15, m

2.33, m 2.21, m 3.61,dd (12.1, 2.9)

3.81, brd (11.6)

13

5.46, s 4.10, m 4.18, dd (10.3, 1.8) 4.04, m 1.12, 3H, s 1.01, d (6.4)

1.62, m 1.47, m 2.50, m 1.05, m

1.66, m

2.14, m 1.18, m 1.54, m 1.19, m 2.22, m 2.20, m

1.75, m 1.75, m 1.28, m 1.38, m

12

0.77, 3H, s

5.56, s 4.03, m 4.17, d (8.8) 3.98, m 1.22, 3H, s 1.27, 3H, s 1.12, 3H, s

1.50, m 1.50, m 2.55, brd (13.4) 1.14,m 5.56, s 4.01, m 4.14, d (8.6) 3.98, m 1.21, 3H, s 1.36, 3H, s 4.05, d (10 0.8) 3.76, d (10.8) 0.82, 3H, s

1.57, m 1.57, m 2.83, brd (12.8) 1.39, m

2.11, t (8.4)

6.01, dd (9.6, 3.2)

6.17, dd (9.8, 2.9)

1.96, m

1.33, m 6.13, dd (9.6, 1.6)

2.83, m 1.39, m

2.24, m 1.34, m 4.35, m

15

2.0, m 5.77, dd (9.8, 1.9)

1.56, m 1.17, m 1.86, m 1.86, m 3.50, m

14

Table 4. 1H NMR Spectroscopic Data (400 MHz, Pyridine-d5, δ in ppm, J in Hz) of Compounds 10−18

0.79, 3H, s

5.52, s 4.03, m 4.17, m 4.03, m 1.16, 3H, s 1.21, 3H, s 0.98, 3H, s

1.62, m 1.62, m 2.43, m 1.11, m

1.92, t (8.0)

1.77, m 1.47, m 1.28, m 2.28, m 2.10, m

6.00, d (10.2)

6.83, d (10.2)

16

0.81, 3H, s

4.29, s 4.33, brs 4.20, dd (8.6, 6.2) 4.03, dd (8.6, 4.7) 1.33, 3H, s 1.15, 3H, s 1.15, 3H, s

1.42, m 1.42, m 1.72, m 1.40, s

1.70, m

1.22, dd (11.2, 2.2) 2.02, m 2.02, m 5.99, d (2.2)

1.72, d (5.6) 1.10, m 1.83, m 1.83, m 3.45, m

17

0.88, 3H, s

4.29, s 4.33, brs 4.22, dd (8.6, 6.1) 4.02, dd (8.6, 4.4) 1.31, 3H, s 1.14, 3H, s 0.92, 3H, s

1.49, m 1.49, m 1.72, dt (13.5, 3.8) 1.38, m

1.92, m

2.05, m 2.01, m 2.01, m 6.03, d (2.4)

1.97, m 1.51, m 1.94, m 1.80, m 3.64, brs

18

Journal of Natural Products Article



Article

and ent-2α,7β,15,16,19-pentahydroxypimar-8(14)-ene, respectively. Compound 12 was assigned a molecular formula of C19H30O3 based on the HRESIMS ion at m/z 351.2170 [M + HCOO]−. The NMR signals of 12 were comparable to those of ent-15,16dihydroxypimar-8(14)-en-3-one, which was isolated from Acacia pennatula,24 with prominent differences being the absence of a methyl signal. Thus, compound 12 was most likely an entnorpimarane. A diagnostic methyl doublet at δH 1.01 (3H, d, J = 6.4 Hz) indicated this methyl group was located at C-4. In the NOESY spectrum, the correlation of the methyl signal at δH 1.01 with Me-20 indicated the absence of Me-18. Thus, the structure of compound 12 was identified as ent-15,16-dihydroxy-18norpimar-8(14)-en-3-one. Compound 13 was assigned a molecular formula of C20H34O4 based on the negative HRESIMS ion at m/z 383.2424 [M + HCOO]−. 1D and 2D NMR data indicated a carbon skeleton similar to that of darutigenol (20).15 The only difference between the compounds was that a shielded methylene resonance (C-1) in darutigenol (20) was replaced by an oxygenated methine at δC 76.5. This assignment was confirmed by the HMBC cross-peaks from Me-20 (δH 1.06, s) to δC 76.5 (C-1), 53.3 (C-5), 52.3 (C-9), and 44.7 (C-10). The NOESY correlations of H-1/H-5/H-9 indicated the α-orientation of the hydroxy group at C-1. Thus, the structure of compound 13 was defined as ent-1β,3β,15,16tetrahydroxypimar-8(14)-ene. Compound 14 was obtained as an amorphous powder. The molecular formula was determined as C20H32O3 based on the HRESIMS ion at m/z 663.4591 [2 M + Na]+ (calcd 663.4595). The IR spectrum showed absorption bands of hydroxy (3449 cm−1) and olefinic (1633 cm−1) groups. The NMR spectroscopic data showed characteristic signals of the carbons of two double bonds (δC 137.1, 133.0, 131.4, and 128.1), three oxygen-bearing carbons (δC 78.0, 77.3, and 63.8), and four methyl singlets (δC 28.5, 23.5, 16.8, and 13.5). A comparison of the 13C NMR data of compounds 14 and darutigenol (20)15 indicated that both compounds had similar skeletons and substituent patterns. The only difference was that an extra double bond was present in 14. The HMBC cross-peaks from Me-17 to C-12 (δC 32.8), C-13 (δC 38.8), C-14 (δC 133.0), and C-15 (δC 77.3) and from H-14 (δH 5.56) to C-7 (δC 131.4), C-9 (δC 50.4), and C-13 (δC 38.8) defined the Δ6 (7) and Δ8 (14) double bonds. The correlations in the NOESY spectrum of H-3/Me-18/H-5 indicated the αorientation of 3-OH. Therefore, the structure of compound 14 was defined as ent-3β,15,16-trihydroxypimar-6,8(14)-diene. Compound 15 was obtained as a white powder. The molecular formula was determined as C20H32O4, based on the HRESIMS ion at m/z 371.2012 [M + Cl]−. Its NMR spectroscopic data are similar to those of 14, except for an extra hydroxymethyl (δC 65.0) and a shielded oxymethine at δC 64.1 (vs δC 78.0 in 14). The HMBC cross-peaks from Me-18 to C-3, C-4, C-5, and C-19 (δC 65.0), together with the COSY correlations of H-1/H-2 (δH 4.35)/H-3, indicated the locations of 2-OH and 19-CH2OH. The NOESY cross-peaks between H-2 and Me-20 indicated the βorientation of 2-OH. Thus, the structure of compound 15 was defined as ent-2α,15,16, 19-tetrahydroxypimar-6,8(14)-diene. Compound 16 had a molecular formula of C20H30O3, as determined by the HRESIMS ion at m/z 363.2174 [M + HCOO]− (calcd 363.2171). The 1H and 13C NMR data of 16 (Tables 2 and 4) were indicative of the presence of four methyl groups (δH 0.79, 0.98, 1.16, and 1.17) and three olefinic protons (δH 6.83, 6.00, and 5.52). A comparison of the NMR data of 16 with those of ent-15,16-dihydroxypimar-8(14)-en-3-one24 re-

vealed that these compounds were closely related. The position of the extra disubstituted Δ1(2) double bond was confirmed by the HMBC cross-peaks from Me-20 to C-1 (δC 155.8), C-5, C-9, and C-10 and from H-1 to C-3 (δC 203.8). Thus, the structure of compound 16 was defined as ent-15,16-dihydroxypimar-1,8(14)dien-3-one. Compound 17, obtained as colorless crystals, mp 183−185 °C, had a molecular formula of C20H32O3 as determined by the HRESIMS ion at m/z 319.2284 [M − H]−. The IR spectrum showed absorption bands of hydroxy (3356 cm−1) and olefinic (1664 cm−1) groups. The 13C NMR spectrum showed 20 carbon signals, including four methyl, six methylene (one oxygenated at δC 72.0), six methine (one olefinic at δC 123.6 and three oxygenated at δC 85.5, 78.2, and 76.6), and four quaternary carbons (one olefinic at δC 135.6). In addition, the 1H and 13C NMR resonances (Tables 2 and 4) of 17 were similar to those of 14β,16-epoxy-ent-3β,15α,19-trihydroxypimar-8-ene,16,25 except for an extra olefinic proton signal at δH 5.99 (d, J = 2.2 Hz), which suggested that the tetrasubstituted double bond in the known compound was replaced by a trisubstituted double bond in 17. The position of the Δ7(8) double bond was confirmed by the HMBC cross-peaks from the olefinic proton signal at δH 5.99 to C-6 (δC 23.6), C-9 (δC 49.1), and C-14 (δC 51.0). The relative configuration of 17 was established by the NOESY correlations of Me-17 with H-14, rather than H-15, and of Me-18 with H-3 and H-5, suggesting that H-14 was β-oriented and that 3-OH was α-oriented. Therefore, the structure of compound 17 was proposed to be 14β,16-epoxy-ent-3β,15α,19-trihydroxypimar-7ene. X-ray diffraction analysis of crystals obtained by recrystallization from MeOH was used to confirm the structure (Figure 7).

Figure 7. X-ray ORTEP drawing of compound 17·H2O.

Compound 18 was defined as an epimer of 17, as indicated by the HRESIMS and 13C NMR data. The 13C NMR data of 18 were very similar to those of 17 except for the chemical shifts of some carbons near the C-3 stereogenic center. Thus, 18 was concluded to be a C-3 isomer of 17. In the 1H NMR spectrum of 18, the appearance of H-3 as a broad singlet with a width at halfpeak height of 5.8 Hz, revealing small JH‑2a/H‑3 and JH‑2b/H‑3 values, indicated the β-orientation of 3-OH, which was confirmed by the NOESY correlations between H-3 and Me-19, rather than H-5. Thus, the structure of compound 18 was established as 14β,16epoxy-ent-3α,15α,19-trihydroxypimar-7-ene. The known compounds were identified as kirenol (19),14 darutigenol (20),15 and ent-2β,15,16,19-tetrahydroxypimar8(14)-ene (7) by analysis of their 1D and 2D NMR data, as well as comparison with reported data. Compounds 3−7 and 8− 16 were determined as an “ent-pimarane” skeleton on the basis of 26



Article

Figure 8. Putative biosynthesis pathway for compounds 1−2026

their biosynthesis pathway (Figure 8)26 and the comparison of their ECD curves with those of 19 and 20 (Figures S63−65, Supporting Information). Compounds 1−11, 13−15, and 17−20 were tested for their inhibitory effects on nitric oxide production because of the clinical application of Siegesbeckia species and because of the reported anti-inflammatory effects of pimarane diterpenoids.8−10,22 Lipopolysaccharide-stimulated Raw 264.7 macrophage cells were used in the NO inhibitory assay, but no significant activity was observed (IC50 > 100 μM). The inhibitory effects of these compounds against the invasion of MB-MDA231 cells were analyzed. In a screening experiment, compounds 3, 5, and 11 inhibited the EGF-induced invasion of MB-MDA231 cells with IC50 values of 4.26, 3.45, and 9.70 μM, respectively (Table 5). LY294002 was used as a positive control, with an IC50 value of 0.38 μM, and the other compounds showed weak or no effects in the same assay (IC50 > 10 μM). The results of the current study suggest that the number, location, and orientation of the hydroxy groups may be essential for the inhibitory effects of the diterpenoids against the invasion of MB-MDA-231 cells.

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Table 5. Inhibitory Effects of Compounds 1−11, 13−15, and 17−20 on the Invasion of MDA-MB-231 Cells

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using a JASCO P-2000 automatic digital polarimeter. Melting points were measured on an X-5 (Gongyi City Yuhua Instrument Co., Ltd. Gongyi, People’s Republic of China). IR spectra were obtained using a Nicolet Nexus 470 FT-IR spectrometer (Nicolet, Madison, WI, USA) with KBr pellets. TOF-MS data were measured on an XEVOG2QTOF#YCA166 spectrometer. ECD spectra were recorded on a JASCO J-810 spectropolarimeter (Jasco, Hachioji, Tokyo, Japan). NMR spectra were recorded on a Bruker Avance III 400 spectrometer (Bruker, Karlsruhe, Baden−Wuerttemberg, Germany). Spectra were analyzed using Bruker TopSpin 2.1 software. Pyridine-d5 for NMR experiments was purchased from Cambridge Isotope Laboratories, Inc. (Xenia, OH, USA). Crystal data were obtained on a Rigaku MicroMax 002+ single-crystal X-ray diffractometer with the wavelength for Cu Kα (λ = 1.5418 Å). A Laballiance HPLC system consisting of a binary pump, a model 2000 detector, and a YMC-pack C18 column (10 × 250 mm) was used. Silica gel (200−300 mesh, Qingdao Marine Chemical Factory,

a

compound

IC50 (μM)

1 2 3 4 5 6 7 8 9 10 11 13 14 15 17 18 19 20 LY294002b

NA NA 4.29 NA 3.45 NA NA NA NA NA 9.70 NA NA NA NA NA NA NA 0.38

Not active at a concentration of 10 μM. bPositive control.

Qingdao, China), Sephadex LH-20 (Taizhou Luqiao Sijia Chemical Reagents Factory, Taizhou, People’s Republic of China), and ODS (50 μm, YMC, Kyoto, Japan) were used for column chromatography. TLC was conducted with glass precoated with silica gel GF254 (Qingdao Marine Chemical Factory, Qingdao, China). Solvents of analytical grade were purchased from Beijing Chemical Factory, and human EGF was obtained from Peprotech (Rocky Hill, NJ, USA). Plant Material. S. pubescens was purchased from An-Guo medicinal materials market, Hebei Province, People’s Republic of China, in September 2011 and was identified by one of the authors (H.-Z.F.). A voucher specimen (No. 2011009) was deposited at the State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center. 27



Article

ent-2α,15,16,18-Tetrahydroxypimar-8(14)-ene (8): white powder; [α]25 D −52 (c 0.1, MeOH); IR (KBr) νmax 3411, 2927, 1663, 1611, 1521, 1382, 1319, 1261, 1167, and 1014 cm−1; 1H and 13C NMR data (Tables 1 and 3); HRESIMS m/z 383.2431, [M + HCOO]− (calcd for C21H35O6, 383.2434). ent-2α,3β,15,16,19-Pentahydroxypimar-8(14)-ene (9): amorphous powder; [α]25 D −50 (c 0.1, MeOH); IR (KBr) νmax 3374, 2938, 2873, 1684, 1455, 1385, 1080, 1054, and 1033 cm−1; 1H and 13C NMR data (Tables 1 and 3); HRESIMS m/z 399.2383 [M + HCOO]− (calcd for C21H35O7, 399.2383). ent-2α,7α,15,16,19-Pentahydroxypimar-8(14)-ene (10): amorphous powder; [α]25 D −10 (c 0.1, MeOH); IR (KBr) νmax 3369, 2940, 1668, 1614, 1455, 1385,1260, and 1033 cm−1; 1H and 13C NMR data (Tables 2 and 4); HRESIMS m/z 399.2384 [M + HCOO]− (calcd for C21H35O7, 399.2383). ent-2α,7β,15,16,19-Pentahydroxypimar-8(14)-ene (11): white powder; [α]25 D −16 (c 0.1, MeOH); IR (KBr) νmax 3356, 2943, 2871, 2850, 1675, 1465, 1370, 1274, 1139, 1064, and 1032 cm−1; 1H and 13C NMR data (Tables 2 and 4); HRESIMS m/z 399.2378 [M + HCOO]− (calcd for C21H35O7, 399.2383). ent-15,16-Dihydroxy-18-norpimar-8(14)-en-3-one (12): white powder; [α]25 D −40 (c 0.1, MeOH); IR (KBr) νmax 3399, 2935, 2873, 1762, 1708, 1604, 1450, 1378, 1243, 1178, and 1047 cm−1; 1H and 13C NMR data (Tables 2 and 4); HRESIMS m/z 351.2170 [M + HCOO]− (calcd for C20H31O5, 351.2171). ent-1β,3β,15,16-Tetrahydroxypimar-8(14)-ene (13): white powder; [α]25 D −68 (c 0.1, MeOH); IR (KBr) νmax 3368, 2942, 2873, 1691, 1562, 1414, 1204, 1138, 1075, and 1020 cm−1; 1H and 13C NMR data (Tables 2 and 4); HRESIMS m/z 383.2424 [M + HCOO]− (calcd for C21H35O6, 383.2434). ent-3β,15,16-Trihydroxypimar-6,8(14)-diene (14): amorphous powder; [α]25 D −42 (c 0.1, MeOH); IR (KBr) νmax 3368, 2942, 2873, 1691, 1562, 1414, 1204, 1138, 1075, and 1020 cm−1; 1H and 13C NMR data (Tables 2 and 4); HRESIMS m/z 663.4591 [2 M + Na]+ (calcd for C40H64O6Na, 663.4595). ent-2α,15,16,19-Tetrahydroxypimar-6,8(14)-diene (15): white powder; [α]25 D −44 (c 0.1, MeOH); IR (KBr) νmax 3450, 2920, 2852, 1634, 1565, 1466, 1087, 1061, and 1031 cm−1; 1H and 13C NMR data (Tables 2 and 4); HRESIMS m/z 371.2012 [M + Cl]− (calcd for C20H32O4Cl, 371.1995). ent-15,16-Dihydroxypimar-1,8(14)-dien-3-one (16): amorphous powder; [α]25 D −62 (c 0.1, MeOH); IR (KBr) νmax 3434, 2936, 2873, 1725, 1671, 1461, 1373, 1243, 1202, and 1047 cm−1; 1H and 13C NMR data (Tables 2 and 4); HRESIMS m/z 363.2174 [M + HCOO]− (calcd for C21H31O5, 363.2171). 14β,16-Epoxy-ent-3β,15α,19-trihydroxypimar-7-ene (17): colorless crystals; mp 183−185 °C; [α]25 D +90 (c 0.1, MeOH); IR (KBr) νmax 3357, 2932, 2888, 2849, 1723, 1664, 1457, 1364, 1204, 1088, 1045, and 1011 cm−1; 1H and 13C NMR data (Tables 2 and 4); HRESIMS m/z 319.2284 [M − H]− (calcd for C20H31O3, 319.2273). 14β,16-Epoxy-ent-3α,15α,19-trihydroxypimar-7-ene (18): white powder; [α]25 D −10 (c 0.1, MeOH); IR (KBr) νmax 3398, 2937, 2872, 1671, 1453, 1385, 1365, 1210, 1094, 1067, and 1038 cm−1; 1H and 13C NMR data (Tables 2 and 4); HRESIMS m/z 641.4778 [2 M + H]+ (calcd for C40H65O6, 641.4794). X-ray Crystallographic Data of Compounds 1, 6, and 17. All three colorless crystal were grown by slow evaporation in MeOH solution. Diffraction intensity data were acquired with a CCD area detector using graphite-monochromated Cu Kα radiation (λ = 1.541 87 Å). Crystal data of 1: C20H34O4 (M = 338.47); monoclinic crystal (0.30 × 0.25 × 0.20 mm); space group P21; unit cell dimensions a = 7.342(4) Å, b = 15.348(12) Å, c = 8.002(4) Å, V = 891.5 (10) Å3; Z = 2; α = 90°; β = 98.622(8)°; γ = 90°, ρcalcd = 1.261 g/cm3, F(000) = 372.0. The final refinement gave R1 = 0.0420 and wR2 = 0.1153 [I ≥ 2σ(I)], Flack parameter = −0.01(18). Crystal data of 6: C20H34O4 (M = 338.49); orthorhombic crystal (0.40 × 0.40 × 0.30 mm); space group P212121; unit cell dimensions a = 6.776(2) Å, b = 14.138(5) Å, c = 19.569(10) Å, V = 1874.7(13) Å3; Z = 4; α = β = γ = 90°, ρcalcd = 1.199 g/cm3, F(000) = 746.3. The final

Extraction and Isolation. Dried and ground aerial parts of S. pubescens (50 kg), divided into five batches (each 10 kg), were extracted with 75% EtOH (3 × 30 L). After the evaporation of the solvent under reduced pressure, the residue was suspended in H2O and extracted with EtOAc and n-BuOH successively. The residue of the EtOAc layer (350 g) was fractionated by silica gel column chromatography with a glass column (10 × 250 mm) and a stepwise gradient of CHCl3 and MeOH (30:1, 20:1, 10:1, and 5:1, each five column volumes) to give five fractions [A (120.4 g), B (58.6 g), C (41.9 g), D (32.1 g), and E (42.3 g)]. Fraction A (120.4 g) was subjected to silica gel column chromatography (CHCl3−MeOH, 50:1−1:1) to afford subfractions A1, A2, A3, and A4. Subfraction A3 (39.2 g) was subjected to silica gel column chromatography (petroleum ether−EtOAc, 50:1−1:1) to afford 20 (4.1 g). The residue of subfraction A3 was purified by Sephadex LH20 chromatography (MeOH−H2O, 50%) and further separated by preparative HPLC (CH3CN−H2O, 30%) to afford compounds 2 (14.6 mg), 3 (6.7 mg), 5 (16.5 mg), 12 (4.2 mg), 14 (6.1 mg), and 16 (3.9 mg). Fraction A2 (625.4 mg) was chromatographed on C18 reversedphase silica gel eluting with MeOH−H2O (from 50% to 80%) and further purified by preparative HPLC (MeOH−H2O, 80%) to give compounds 4 (5.1 mg), 17 (108.5 mg), and 18 (8.3 mg). Fraction B (58.6 g) was subjected to silica gel column chromatography (CC) (CHCl3−MeOH, 30:1 to 0:1) to afford subfractions B1, B2, and B3. Compound 19 (31.6 g) was obtained from B2 (42.1 g) by the recrystallization method, and the residue of subfraction B2 was purified by CC over silica eluting with a gradient of CHCl3 and MeOH (20:1 to 5:1) to afford three fractions (B2‑1, B2‑2, and B2‑3). Fraction B2‑2 (2.5 g) was purified over a Sephadex LH-20 (MeOH−H2O, 30%) to afford compound 19 (1.5 g). The residue of B2‑2 was purified by ODS gel (MeOH−H2O, 30−70%) and preparative HPLC (CH3CN−H2O, 25%) to afford compounds 1 (19.6 mg), 6 (150.3 mg), 7 (6.0 mg), 8 (5.2 mg), 9 (7.9 mg), 13 (34.8 mg), and 15 (6.2 mg). Fraction B2‑3 (185.6 mg) was purified over an ODS column eluting with a gradient of MeOH and H2O (30−50%) and then further purified by preparative HPLC (MeOH−H2O, 52%) to afford compounds 10 (18.4 mg) and 11 (33.2 mg). Strobol A (1): colorless crystals (MeOH); mp 251−252 °C; [α]25 D −168 (c 0.1, MeOH); IR (KBr) νmax 3400, 2940, 1671, 1466, 1439, 1410, 1103, and 1032 cm−1; 1H and 13C NMR data (Tables 1 and 3); HRESIMS m/z 373.2154 [M + Cl]− (calcd for C20H34O4Cl, 373.2151). Strobol B (2): white solid; [α]25 D −196 (c 0.1, MeOH); IR (KBr) νmax 3357, 2939, 1673, 1465, 1370, 1103, 1072, and 1032 cm−1; 1H and 13C NMR data (Tables 1 and 3); HRESIMS m/z 367.2487 [M + HCOO]− (calcd for C21H35O5, 367.2484). 19-Hydroxy-15-devinyl-ent-pimar-8,11,13-triene-2,7-dione (3): amorphous powder; [α]25 D −38 (c 0.1, MeOH); IR (KBr) νmax 3397, 2923, 2856, 1708, 1679, 1433, 1204, 1184, 1138, and 1033 cm−1; 1H and 13 C NMR data (Tables 1 and 3); HRESIMS m/z 309.1455 [M + Na]+ (calcd for C18H22O3Na, 309.1461). 2β,19-Dihydroxy-15-devinyl-ent-pimar-8,11,13-triene (4): amorphous powder; [α]25 D −82 (c 0.1, MeOH); IR (KBr) νmax 3349, 2934, 1681, 1612, 1454, 1433, 1204, 1184, 1138, and 1033 cm−1; 1H and 13C NMR data (Tables 1 and 3); HRESIMS m/z 549.3938 [2 M + H]+ (calcd for C36H53O4, 549.3938). ent-3β,15R,16-Trihydroxypimar-8(14)-ene (5): white powder; [α]25 D −48 (c 0.1, MeOH); IR (KBr) νmax 3374, 2939, 2871, 1660, 1455, 1383, 1366, 1069, and 1032 cm−1; 1H and 13C NMR data (Tables 1 and 3); HRESIMS m/z 367.2473, [M + HCOO]− (calcd for C21H35O5, 367.2484). ent-2α,15R,16,19-Tetrahydroxypimar-8(14)-ene (6): colorless crystals (MeOH); mp 191−193 °C; [α]25 D −58 (c 0.1, MeOH); IR (KBr) νmax 3260, 2940, 2871, 1637, 1472, 1454, 1368, 1116, 1073, and 1033 cm−1; 1H and 13C NMR data (Tables 1 and 3); HRESIMS m/z 383.2436, [M + HCOO]− (calcd for C21H35O6, 383.2434). ent-2β,15,16,19-Tetrahydroxypimar-8 (14)-ene (7): white powder; [α]25 D −48 (c 0.1, MeOH); IR (KBr) νmax 3367, 2937, 2874, 1677, 1454, 1366, 1116, 1073, and 1031 cm−1; 1H and 13C NMR data (Tables 1 and 3); HRESIMS m/z 383.2421, [M + HCOO]− (calcd for C21H35O6, 383.2434). 28



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refinement gave R1 = 0.0293 and wR2 = 0.0785 [I ≥ 2σ(I)], Flack parameter = −0.09(18). Crystal data of 17: C20H32O3·H2O (M = 338.49 without crystal water); monoclinic crystal (0.11 × 0.39 × 0.60 mm); space group P21; unit cell dimensions a = 7.098(5) Å, b = 11.005(5) Å, c = 12.116(6) Å, V = 935.0(9) Å3; Z = 2; α = 90°; β = 98.904(8)°; γ = 90°, ρcalcd = 1.202 g/ cm3, F(000) = 372. The final refinement gave R1 = 0.0433 and wR2 = 0.1048 [I ≥ 2σ(I)], Flack parameter = −0.0(2). The X-ray crystallographic data of compounds 1, 6, and 17 are presented in the Supporting Information and were deposited in the Cambridge Crystallographic Data Center with deposition numbers CCDC 1057213 (17), CCDC 1057214 (1), and CCDC 1057215 (6). Copies of the data can be obtained free of charge from the CCDC via www.ccdc.cam.ac.uk. Viability Measurements of MDA-MB-231 Cells. The cell viability was tested using the MTT assay. Briefly, cells were seeded at 1 × 104 cells/180 μL/well in 96-well culture plates. The cells were treated with various amounts of tested compound solutions or with the DMSO vehicle (control) and incubated (37 °C, 5% CO2) for 48 h. The supernatants were removed using a micropipet, and 20 μL of the MTT was added to each well and incubated for 4 h at 37 °C. The optical density (OD) was read at a wavelength of 570 nm with a microplate reader. The inhibitory ratio (IR) was calculated as follows: IR % = 1 − (OD with drug/OD without drug) × 100. Invasion Assay. MDA-MB-231 cells were used to perform the chemotaxis invasion assay described by Zhang et al.27 using 8 μm filter membranes (Neuroprobe) that were previously pretreated with 0.001% fibronectin in serum-free medium at 4 °C overnight and air-dried. The lower surface of the filters was coated with a chemoattractant (EGF; 1 ng/mL, 30 μL/well). The control cells and cells pretreated with the test compounds were resuspended in binding medium (RPMI 1640 containing 0.1% BSA and 25 mM HEPES) at a density of 0.5 × 106 cells/mL, placed into the upper compartment of the chamber (50 μL/ well), and incubated at 37 °C in 5% CO2 for 3.5 h. The filter membrane was rinsed, fixed, and stained. The cells were counted, and the inhibitory ratio was calculated as follows: IR % = (1 − number of migrated cells in sample/number of migrated cells in control) × 100%. The potencies of the products were expressed as median inhibitory concentration (IC50) values. LY294002 (Camarillo, CA, USA) was used as a positive control substance for this assay.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Grant No. 81172943) and the National Major Scientific and Technological Special Projects for “Significant New Drugs Development” during the Twelfth Fiveyear Plan Period (No.2012ZX09103201-022).

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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.6b00150. 1D and 2D NMR, HRESIMS, and IR spectra of compounds 1 and 2; 1H and 13C spectra of compounds 3−20 (including 2D for compounds 4, 7, and 18); ECD curves of compounds 3−15, 19, and 20; 1H and 13C NMR data of kirenol (19) and darutigenol (20) (PDF) X-ray crystallographic data of compounds 1, 6, and 17 (CIF) (CIF) (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel (H. Fu): +86-10-82805212. Fax: +86-10-82805212. E-mail: [email protected]. ORCID

Jianbin Wang: 0000-0001-9605-0085 Notes

The authors declare no competing financial interest. 29


ent-Strobane and ent-Pimarane Diterpenoids from Siegesbeckia

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