ent-Abietane and Tigliane Diterpenoids from the Roots of Euphorbia

Apr 6, 2017 - Nesreen A. Safwat , Mona T. Kashef , Ramy K. Aziz , Khadiga F. Amer , Mohammed A. Ramadan. Tuberculosis 2018 108, 106-113 ...
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ent-Abietane and Tigliane Diterpenoids from the Roots of Euphorbia fischeriana and Their Inhibitory Effects against Mycobacterium smegmatis Chun-Jie Wang,⊥ Qiu-Long Yan,⊥ Yu-Fang Ma, Cheng-Peng Sun, Chang-Ming Chen, Xiang-Ge Tian, Xiu-Yan Han, Chao Wang,* Sa Deng, and Xiao-Chi Ma* College of Pharmacy, Academy of Integrative Medicine, and Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, People’s Republic of China S Supporting Information *

ABSTRACT: An investigation on the bioactive chemical constituents of the roots of Euphorbia fischeriana has been conducted, with 21 diterpenoids obtained using various chromatographic techniques. On the basis of spectroscopic data analysis, the new compounds were elucidated as four ent-abietane-type diterpenoids (1−4) and four tigliane-type diterpenoids (13−16). Also obtained were eight known ent-abietane (5−12) and five known tigliane (17−21) diterpenoids. The potential antituberculosis effects of these diterpenoids were evaluated using a Mycobacterium smegmatis model. The most potent compound according to the in vitro bioassay used was 17hydroxyjolkinolide B (12) (MIC 1.5 μg/mL).

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The herbaceous plant Euphorbia fischeriana Steud is a perennial of the family Euphorbiaceae, which is distributed mainly in northeastern mainland China.1 The roots of E. fischeriana are named “Lang-du” and are used traditionally for the treatment of edema, ascites, and cancer. An extract of E. fischeriana is also used as a component of the drug “Jie-He-Ling”, which is employed clinically for the treatment of lymphoid and pulmonary tuberculosis.2,3 This plant is also a well-known traditional folk medicine in Korea, utilized as a remedy for skin diseases, intestinal parasites, psoriasis, and cancer.4 Chemical investigations of E. fischeriana have revealed the presence of polysaccharides,5−7 diterpenoids,8 triterpenoids,9 steroids,10 and acetophenone derivatives.11 Among these, the major chemical constituents of this plant are diterpenoids, including compounds of the abietane, tigliane, tigliane glycoside, and pimarane types, as well as dimeric diterpenoids, and norditerpenoids.12−17 Some of these substance have displayed biological activities, including antibacterial, antituberculosis, anti-inflammatory, and cytotoxic effects.18−21 On considering the use of E. fischeriana to treat tuberculosis, a phytochemical investigation of the roots of this plant was conducted. On the basis of chromatographic separation and spectroscopic data interpretation, four new ent-abietane-type diterpenoids (1−4), four new tigliane-type diterpenoids (13− 16), and 13 known diterpenoids were identified. The inhibitory effects of the isolated diterpenoids were evaluated against Mycobacterium smegmatis. © 2017 American Chemical Society and American Society of Pharmacognosy

RESULTS AND DISCUSSION Compound 1 was obtained as a white amorphous powder and showed a molecular formula of C20H26O5 as established by HRESIMS m/z 369.1663 (calcd 369.1678, [M + Na]+), which indicated eight degrees of unsaturation. The UV spectrum suggested the presence of a diene carbonyl group from the absorption observed at λ 281 nm. The 1H NMR spectrum displayed the signals of an oxymethylene unit at δH 4.17 (2H, s); an olefinic moiety at δH 6.62 (br s), two oxymethine groups at δH 4.31 (d, J = 3.0 Hz) and 4.89 (d, J = 3.0 Hz); and three tertiary methyl groups at δH 1.06 (3H, s), 0.96 (3H, s), and 0.96 (3H, s) (Table 1). A combination of 13C NMR and HSQC experiments indicated a total of 20 carbons for 1, confirming the presence of the above-mentioned groups and revealing other moieties, including a carbonyl group (δC 215.0) and an α,β-unsaturated γ-lactone (δC 173.4, 154.7, 121.0, and 79.0) (Table 1). On the basis of spectroscopic data analysis, compound 1 was deduced as being an ent-abietane diterpenoid, similar to helioscopinolides D and E, which have a carbonyl moiety in ring A.22 The 1H−1H COSY spectrum displayed three spin−spin systems: H-1 (δH 2.18)/H-2 (δH 2.38), H-6 (δH 2.52)/H-7 (δH 1.43), and H-9 (δH 2.19)/H-11 (δH 4.31)/ H-12 (δH 4.89) (Figure 1). A carbonyl group at C-3 was determined by the long-range correlations of H-18 (δH 0.96)/ C-3 (δC 215.0), H-19 (δH 1.06)/C-3 (δC 215.0), H-2 (δH 2.61, 2.38)/C-3 (δC 215.0) observed in the HMBC spectrum (Figure Received: August 28, 2016 Published: April 6, 2017 1248

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by the experimental ECD method (Figure 3). Therefore, compound 1 was elucidated as 11α,17-dihydroxyhelioscopinolide E.4,22 Compound 2 gave the molecular formula C20H26O6, as determined by HRESIMS. This compound displayed a UV absorption band at 275 nm, similar to that of compound 1. Analysis of the spectroscopic data suggested that the planar structure of compound 2 is similar to that of 1, except for the presence of an extra hydroxy group (Table 1). This additional hydroxy group was assigned at C-7 by the HMBC correlation of H-14 (δH 6.82)/C-7 (δC 72.2) (Figure 1). On the basis of the correlations between H-7 (δH 1.77)/H-18, H-11/H-9, and H12/H-20 observed in the NOESY spectrum (Figure 2), the orientations of OH-7, OH-11, and H-12 were established as β, α, and α, respectively. Compound 2 showed a π−π* negative Cotton effect at 234.5 nm of the α,β-unsaturated γ-lactone in its ECD spectrum and hence was assigned the same configuration at C-12 as that of 1 (Figure S55, Supporting Information). Thus, compound 2 was established as 6β,11α,17-trihydroxyhelioscopinolide E.4,22 Compound 3, a white amorphous powder, showed a molecular formula of C20H28O5, as determined by its 13C

1). The HMBC correlations of H-9 (δH 2.19)/C-11 (δC 63.3), C-12 (δC 79.0), H-11/C-12, C-13 (δC 154.7) supported the locations of oxygenated methine carbons at C-11 and C-12. A 17-oxymethine carbon was determined by the long-range correlations of H-17/C-16 (δC 173.4), C-15 (δC 121.0). Thus, the planar structure of 1 was established as an ent-abietane diene-lactone with a carbonyl group at C-3 and two hydroxy groups at C-11 and C-12. The relative configuration of 1 was assigned by a NOESY experiment, in which H-11 and H-12 were determined to have a β-orientation and an α-orientation, respectively, on the basis of NOESY correlations of H-11/H-9 and H-12/H-20 (Figure 2). The absolute configuration of compound 1 was established by an ECD experiment. The π−π* negative Cotton effect at 236.5 nm of the α,βunsaturated γ-lactone suggested the S configuration of C12.23,24 The calculation of ECD spectra using time-dependent density functional theory (TDDFT) was applied to confirm the absolute configuration.25−27 The calculated ECD spectrum of (5S, 9R, 10R, 11S, 12S)-1 matched very well with the experimental spectrum, while the calculated ECD curve of its enantiomer was opposite to the experimental data, thus confirming the absolute structure of compound 1 as assigned 1249

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Table 1. NMR Spectroscopic Data of ent-Abietane Diterpenoids 1−4 (δH in ppm, J in Hz) 1a number 1 2

δH 2.18 1.77 2.61 2.38

δC m m m m

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

2b 36.2 34.0

δH 2.29 1.91 2.70 2.51

δC m m m m

215.0

1.77 2.52 2.18 1.73 1.43

m m m m m

2.19 s 4.31 d (3.0) 4.89 d (3.0) 6.62 br s

4.17 0.96 1.06 0.96

s s s s

46.8 53.7 35.9 24.1 149.1 60.7 39.1 63.3 79.0 154.7 114.3 121.0 173.4 54.0 21.3 26.4 16.2

3a 38.1 35.3 218.4

2.33 dd (13.0, 10.0) 1.89 m 1.77 m 4.44 t (2.5)

2.77 br s 4.50 d (3.5) 5.06 d (3.0) 6.82 br s

4.37 d (1.0) 1.06 s 1.12 s 1.08 s

48.2 48.4 32.5 72.2 151.0 56.9 40.9 65.3 81.4 156.6 116.9 124.4 175.9 55.6 22.1 26.9 16.5

δH

4b δC

1.88 1.06 1.47 1.39 1.38 1.14

m m m m m m

1.60 1.67 1.26 1.52 0.99

m m m m m

1.87 s

5.71 d (2.5) 3.94 d (3.0)

2.10 d (2.5) 0.81 s 0.82 s 0.57 s

42.1 18.1 41.9 33.0 44.2 15.4 21.6 72.6 64.8 35.5 193.5 86.2 145.7 74.0 134.5 173.1 9.8 32.6 21.4 15.8

δH

δC

1.83 1.48 1.55 1.47 1.43 1.25

m m m m m m

1.31 1.82 1.49 2.57 2.28

m m m m m

3.76 d (6.5)

6.47 br s

4.33 m 0.87 s 0.93 s 0.78 s

41.3 20.0 43.1 34.5 55.6 24.5 37.5 157.2 60.8 42.0 70.9 104.6 158.0 114.7 122.1 173.9 55.4 22.4 34.0 15.7

The NMR data were recorded in DMSO-d6 at 500 MHz for δH and at 125 MHz for δC. bThe NMR data were recorded in methanol-d4 at 500 MHz for δH and at 125 MHz for δC. a

Figure 1. Key HMBC (H → C) and 1H−1H COSY correlations of ent-abietane diterpenoids (1 and 2).

Figure 3. Measured ECD spectra of 1 (black line) and the calculated ECD spectra of (5S, 9R, 10R, 11S, 12S)-1 (red line) and the enantiomer of (5R, 9S, 10S, 11R, 12R)-1 (blue line).

unsaturated five-membered lactone ring (δC 173.1, 145.7, 134.5, and 86.2) (Table 1). Analysis of the spectroscopic data indicated that compound 3 is also based on an ent-abietane diterpenoid lactone skeleton. This compound was found to be similar to ebracteolatanolide B28 and yuexiandajisu E,29 isolated from Euphorbia ebracteolata. On the basis of an HMBC experiment, the oxygenated C-8 and C-14 could be determined from the long-range correlations of H-14 (δH 3.94)/C-8 (δC 72.6), H-9 (δH 1.87)/C-8 (δC 72.6), H-7b (δH 1.52)/C-8 (δC

Figure 2. Key NOESY correlations of ent-abietane diterpenoids (1 and 2).

NMR and HRESIMS data (m/z 349.2001 [M + H]+; calcd 349.2015). The NMR spectroscopic data revealed signals for a carbonyl (δC 193.5), two oxymethines (δH 3.94, δC 74.0; δH 5.71, δC 86.2), an oxygenated quaternary carbon (δC 72.6), four tertiary methyls (δH 2.10, 0.81, 0.82, and 0.57), and an α,β1250

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Table 2. NMR Spectroscopic Data of Tigliane Diterpenoids 13−16 (δC in ppm, J in Hz)a 13b

14b

15b

16b

number

δH

δC

δH

δC

δH

δC

δH

δC

1 2 3 4 5

7.48 s

159.2 132.4 208.2 73.0 37.2

7.53 s

160.4 135.2 209.1 74.3 37.6

7.50 s

159.2 132.6 208.1 72.9 37.7

7.49 s

159.2 132.6 208.1 72.9 37.7

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

2.35 d (18.5) 2.24 d (18.5) 5.48 s 2.83 br s 1.86 m 2.99 s 2.02 m 1.46 m

1.07 s 0.96 s 0.79 br s 1.65 br s 3.77 s

140.7 127.9 38.1 35.8 56.1 74.9 32.4 63.6 31.3 22.7 22.8 15.5 18.6 9.9 66.1

2.93 d (20.0) 2.59 d (20.0) 6.94 s 3.23 m 3.05 m 2.05 m 2.14 dd (14.5, 7.0) 1.59 dd (14.5, 10.5) 1.00 d (5.5) 1.20 s 1.08 s 0.92 d (6.5) 1.75 s

141.0 146.5 41.3 77.8 57.2 37.6 32.9 65.1 33.0 24.4 23.3 15.8 19.0 10.2 186.6

Glc-1′ Glc-2′ Glc-3′ Glc-4′ Glc-5′ Glc-6′

2.42 d (19.0) 2.34 d (19.0) 5.63 d (3.5) 2.91 m 3.02 m 1.85 m 2.03 m 1.47 dd (14.5, 11.0) 0.83 m 1.08 s 0.98 s 0.81 d (6.5) 1.67 d (1.5) 4.18 d (11.5) 3.94 d (11.5) 4.27 d (8.0) 3.09 m 3.45 m 4.69 t (9.5) 3.40 m 3.37 m 3.32 m

136.9 131.2 38.3 75.0 56.1 35.8 32.4 63.6 31.1 22.79 22.76 15.6 18.7 10.0 74.2 102.2 73.7 74.1 71.4 74.8 60.8

2.41 d (19.5) 2.32 d (19.5) 5.63 d (4.0) 2.90 m 3.02 m 1.85 m 2.03 dd (15.0, 7.5) 1.47 dd (15.0, 11.0) 0.84 m 1.07 s 0.97 s 0.81 d (6.5) 1.66 d (1.5) 4.16 d (12.0) 3.95 d (12.0) 4.29 d (7.5) 3.20 m 4.93 t (9.5) 3.35 m 3.22 m 3.66 m 3.48 m

136.8 131.3 38.3 75.0 56.1 35.8 32.4 63.6 31.1 22.79 22.76 15.6 18.7 10.0 74.0 102.0 71.6 77.9 68.0 76.7 60.7

For the signals of the other ester groups, see the Experimental Section. bThe NMR data were recorded in DMSO-d6 at 500 MHz for δH and at 125 MHz for δC. a

correlated with H-20, which confirmed the OH-11 as β. Similar to the ECD data of compound 1 and 2, the π−π* positive Cotton effect of the α,β-unsaturated γ-lactone of 4 suggested the S configuration at C-12 (Figure S57, Supporting Information).23,24 Therefore, compound 4 was elucidated as 7-deoxylangduin B. Compound 13 was obtained as an amorphous powder. HRESIMS was used to establish the molecular formula, C22H30O6, from the ion peak at m/z 435.2024 (calcd 435.2019, [M + COOH]−). The UV spectrum showed an absorption maximum at 239 nm, which indicated the presence of an α,β-unsaturated carbonyl group. In the 1H NMR spectrum, the signals of five tertiary methyls (δH 0.79, 0.96, 1.07, 1.65, 1.99, each 3H, s), an oxymethine (δH 3.77, 2H, s), and two trisubstituted olefinic bonds (δH 7.48 s, 5.48 s) were observed (Table 2). The 13C NMR spectrum displayed 22 carbon signals, which confirmed the above-mentioned moieties and suggested the presence of an α,β-unsaturated carbonyl group (δC 159.2, 132.4, and 208.2), an oxygenated quaternary carbon (δC 74.9, 73.0, and 63.6) (Table 2), and an acetyl group (δC 172.0, 20.8). Comparison of the NMR data of 13 and the tigliane-type diterpenoid, prostratin (19), indicated these two compounds to be closely comparable to one another.30 The difference between 13 and prostratin was found to be in the position of a hydroxy group. The 1H NMR spectrum of 13 showed a singlet for CH3-18 instead of a doublet as in prostratin, which suggested a tetrasubstituted C-11 in 13.

72.6), H-14 (δH 3.94)/C-13 (δC 145.7), and H-9 (δH 1.87)/C14 (δC 74.0). The HMBC spectrum aided in the assignment of hydroxy groups at C-8 (δH 5.38, s) and C-14 (δH 6.52, d, J = 3.0 Hz) while the carbonyl moiety could be located at C-11 from the HMBC correlation of H-9/C-11 (δC 193.5). In the NOESY spectrum, the correlations of OH-8/H-9, H-12/H-9, and H-14/ H-9 supported the orientations proposed for OH-8, H-12 and OH-14 as β, β, and α, respectively. In its ECD spectrum, compound 3 showed a π−π* positive Cotton effect at 219 nm of the α,β-unsaturated γ-lactone, which indicated the R configuration of C-12 (Figure S56, Supporting Information). Therefore, the structure of 3 (11-oxo-ebracteolatanolide B) was elucidated as shown. The spectroscopic data (UV, NMR, and HRESIMS) obtained suggested that compound 4 is also an ent-abietane diene diterpenoid lactone, with an oxymethine (δH 3.76, δC 70.9) and an oxymethine group (δH 4.33, δC 55.4) (Table 1). Additionally, the carbon signal at δC 104.6 suggested the presence of a hemiketal-lactol moiety. On the basis of spectroscopic data analysis, compound 4 was deduced to be similar to the known diterpenoid langduin B, except for the absence of one of its hydroxy groups.15 The HMBC experiment showed long-range correlations of H-11 (δH 3.76)/C-9 (δC 60.8), C-12 (δC 104.6), H-14 (δH 6.47)/C-12, H-11 (δH 3.76)/ C-12, H-17 (δH 4.33)/C-16 (δC 173.9), C-15 (δC 122.1), and C-13 (δC 158.0), which supported the presence of hydroxy groups at C-11, C-12, and C-17. In a NOESY experiment, H-11 1251

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16.32 It was deduced that there were differences in the position of the galloyl group in the structures of 15, 16, and fischeroside B. On the basis of HSQC, HMBC, and 1H−1H COSY spectra, the proton and carbon signals were assigned for the glucopyranosyl groups of 15 and 16, respectively (Table 2). A HMBC experiment on 15 showed correlations from glc-4-H (δH 4.69) to the galloyl carbonyl carbon (δC 165.1), leading to the location of the galloyl group at glc-C-4. In the same way, the galloyl group was located at the glc-C-3 position for the structure of 16 by the HMBC correlation of glc-H-3 (δH 4.93)/ gall-C-1 (δC 165.4). The experimental ECD data of compounds 15 and 16 were similar to that of compound 13 (Figure S58, Supporting Information), which suggested the same configuration for their tigliane skeleton.31 On the basis of the above data analysis and comparison, compounds 15 and 16 were elucidated as prostratin 20-O-(4′-galloyl)-β-D-glucopyranoside and prostratin 20-O-(3′-galloyl)-β-D-glucopyranoside, respectively. On comparison of their physical and spectroscopic data with published values, the known compounds were identified as the ent-abietane-type and tigliane-type diterpenoids 7β,11β,12βtrihydroxy-ent-abieta-8(14),13(15)-dien-16,12-olide (5),10 langduin B (6),15 (4R,4aR)-dihydroxy-3-hydroxymethyl-7,7,10atrimethyl- 2,4,4a,5,6,6a,7,8,9,10,10a,l0bdodecahydrophenanthro[3,2-b]furan-2-one (7),33 yuexiandajisu E (8),29 jolkinolide A (9),15 17-hydroxyjolkinolide A (10),15 jolkinolide B (11),15 17-hydroxyjolkinolide B (12),15 fischeroside A (17),22 phorbol-13-acetate (18),17 12-deoxyphorbol-13acetate (prostratin) (19),34 12-deoxyphorbaldehyde-13-acetate (20),12 and langduin A (21).8 The MIC values of the isolated compounds against M. smegmatis were evaluated, and the results are shown in Table 3.

Additionally, a proton signal was observed at C-9 from the 1Dand 2D-NMR data. The occurrence of a C-11 hydroxy group rather than one at C-9 was supported by the HMBC correlations from the hydroxy proton at δH 4.67 (OH-11) to C-11. The oxygenated C-11 was also confirmed by the HMBC correlations of H-10 (δH 2.99)/C-11 (δC 74.9), H-12a (δH 2.02)/C-11 (δC 74.9), and H-18 (δH 0.79)/C-11 (δC 74.9). Tigliane-type diterpenoids hydroxylated at C-11 have been reported occasionally. Additionally, the OH-4 and OH-20 were established by the HMBC correlations from the hydroxy proton at δH 5.60 (OH-4) to C-4, and from δH 4.70 (OH-20) to C-20. By comparison with the C-13 signal (δC 63.8) of prostratin,30 the acetyl group was located at C-13 of compound 13. Although no obvious HMBC correlations were observed between the acetyl group and diterpenoid skeleton, the NOESY correlations were obtained between the acetyl (δH 1.99) and CH3-16 (δH 1.07), CH3-17 (δH 0.96), which confirmed the position of the acetyl group. As shown in Figure S58 (Supporting Information), the ECD spectrum of 13 exhibited a n−π* positive Cotton effect of an α,β-unsaturated ketone at 229.5 nm, which suggested the S configuration at C10.31 Thus, compound 13 was established as 9-deoxy-11βhydroxyprostratin. Analysis of the spectroscopic data of compound 14 also suggested it to be a tigliane-type diterpenoid. The molecular formula, C22H28O7, indicated one more degree of unsaturation in comparison with compound 13 and prostratin. The NMR spectra showed similar functionalities to those of prostratin,30 except for the oxygenated substituent at C-20 (Table 2). For most tigliane-type diterpenoids isolated from the genus Euphorbia, the C-20 functional group occurs as a methyl, oxymethine, or aldehyde group. However, the 13C NMR spectrum of 14 indicated the absence of a primary alcohol at C20 and the presence of a carboxyl group. The chemical shift of H-6 was shifted by δH 0.46, and C-6 was shifted by ΔδC 18.6, with C-20 established as a carboxylic acid unit (δC 186.6). In the HMBC experiment, no obvious correlations confirmed the position of a 13-acetyl group. As a prostratin analogue, the NOESY correlations between the acetyl group (δH 2.06) and H-12, H-16, H-17, H-18 were used to locate the acetyl group at C-13. Therefore, the structure of compound 14 as 20-oxoprostratin was established as shown. Compounds 15 and 16 both showed the same molecular formula, C35H44O15, as established by HRESIMS at the same ion peak m/z 727.2551 (calcd 727.2578 [M + Na]+). On the basis of spectroscopic data analysis (Table 2), a prostratin diterpenoid was established, together with an additional saccharide and galloyl group in each case. Accordingly, both compounds were deduced as tigliane-type diterpenoid glycosides with galloyl moieties, similar to fischeroside B.11 The NMR spectroscopic data of 15 and 16 were compared with analogous values for fischeroside B, especially the 13C NMR data (Table 2). Differences were observed for the carbon signals in the saccharide groups of compounds 15 and 16 and fischeroside B. Hence, hydrolytic reactions were conducted to identify the sugar units. Alkali hydrolysis was carried out using potassium carbonate to hydrolyze the galloyl group. Then, the glycoside bonds were removed by acid hydrolysis with trifluoroacetic acid under heating. Each saccharide residue was prepared as a 5-methyl-2-phenyl-1,2-dihydropyrazol-3-one (PMP) derivative, which was then analyzed by HPLC. Finally, by comparison with the reference materials (D-glucose, Dgalactose), glucopyranosyl groups were determined for 15 and

Table 3. MIC Values for Compounds 1−21 against M. smegmatis

a

compound

MIC (μg/mL)

compound

MIC (μg/mL)

1 2 3 4 5 6 7 8 9 10 11

>400 >400 300 200 400 >400 >400 >400 >400 100 25

12 13 14 15 16 17 18 19 20 21 kanamycina

1.5 >400 >400 >400 >400 >400 >400 >400 >400 >400 10

Positive control.

In general, the ent-abietane diterpenoids were more potent than the tigliane diterpenoids. The most active compound, 17hydroxyjolkinolide B (12), was found to exhibit an MIC value of 1.5 μg/mL.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained on a JASCO P2000 automatic digital polarimeter. UV spectra were recorded on a JASCO V-650 spectrophotometer. NMR spectra were acquired with a Bruker-500 NMR spectrometer in various solvents with tetramethylsilane (TMS) as an internal standard. ESIMS data were determined on API 3200 mass spectrometer (AB Sciex, Framingham, MA, U.S.A.). HRESIMS were collected on an Agilent 1100 series LC/MSD ion trap mass spectrometer. Analytical HPLC

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20-Oxo-prostratin (14). Compound 14 was produced as a white amorphous solid; [α]25 D − 48.6 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 204 (3.60) nm; 1H NMR (DMSO-d6, 500 MHz), see Table 2 and δH 2.06 (3H, s, Ac-13); 13C NMR (DMSO-d6, 125 MHz), see Table 2 and δC 21.1, 175.0 (Ac-13); HRESIMS m/z 403.1765 (calcd for C22H27O7, 403.1757). Prostratin 20-O-(4′-galloyl)-β-D-glucopyranoside (15). Compound 15 was produced as a white amorphous solid; [α]25 D − 13.5 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 210 (3.90), 274 (3.65) nm; ECD (CH3OH) Δε230 + 3.34, Δε275 − 0.22; 1H NMR (DMSOd6, 500 MHz), see Table 2 and δH 1.99 (3H, s, Ac-13), 6.95 (2H, s, glc4′-Gall); 13C NMR (DMSO-d6, 125 MHz), see Table 2 and δC 20.8, 171.9 (Ac-13), 165.1 (C-1″), 119.4 (C-2″), 108.8 (C-3″), 145.5 (C4″), 138.4 (C-5″), 145.5 (C-6″), 108.8 (C-7″); HRESIMS m/z 727.2551 (calcd for C35H44O15Na, 727.2578). Prostratin 20-O-(3′-galloyl)-β-D-glucopyranoside (16). Compound 16 was produced as a white amorphous solid; [α]25 D − 10.4 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 210 (3.90), 274 (3.65) nm; ECD (CH3OH) Δε231 + 3.98, Δε333 − 0.24; 1H NMR (DMSOd6, 500 MHz), see Table 2 and δH 1.99 (3H, s, Ac-13), 6.97 (2H, s, glc4′-Gall); 13C NMR (DMSO-d6, 125 MHz), see Table 2 and δC 20.8, 171.9 (Ac-13), 165.4 (C-1″), 120.1 (C-2″), 108.8 (C-3″), 145.4 (C4″), 138.0 (C-5″), 145.4 (C-6″), 108.8 (C-7″); HRESIMS m/z 727.2551 (calcd for C35H44O15Na, 727.2578). Hydrolysis of Tigliane Diterpenoid Glycosides 15 and 16.32 Each diterpenoid glycoside (1 mg) was hydrolyzed using 5 mg of potassium carbonate in 2 mL of water at room temperature with stirring for 12 h. Then, the mixture was neutralized with 10% aqueous HCl. The reaction mixture was evaporated in vacuo, and the residue was dissolved in methanol, which contained the diterpenoid glycoside with the absence of the galloyl group. The hydrolysis products were fully hydrolyzed using 0.25 mL of trifluoroacetic acid aqueous solvent (4 mol/L), at 110 °C, for 4 h. After evaporation of the aqueous solvent, each residue was dissolved in water, which afforded the saccharide products from the diterpenoid glycoside. The resultant sugar was prepared as a 5-methyl-2-phenyl-1,2-dihydropyrazol-3-one (PMP) derivative by treatment with 0.15 mol/L aqueous NaOH at 75 °C for 30 min. After being neutralized with 0.3 mL of 0.15 mol/L aqueous HCl, the saccharide-PMP derivatives were analyzed using an HPLC instrument, using reference compounds (D-glucopyranose- and D-galactopyranose-PMP derivatives). The HPLC analytical method was carried out on a Waters Xbridge C18 column (100 × 10 mm, 2.5 μm) with a mobile phase of acetonitrile (A) and 10 mM ammonium acetate aqueous solution (B) with the gradient program as 0/21/60 min, 16%/16%/24% (A) at a flow rate of 0.45 mL/min. The column temperature was maintained at 30 °C. The retention time of reference standards D-glucopyranose- and D-galactopyranose-PMP derivatives were 41.6 and 44.5 min, respectively, which were used for monosaccharide identification. ECD Calculations. Conformational analysis was carried out via Monte Carlo searching with the MMFF94 molecular mechanics force field using the Spartan 10 software.25 The conformers were reoptimized using DFT at the B3LYP/6-31+G (d, p) level in vacuo with the Gaussian 09 program.26 The B3LYP/6-31G+(d, p) harmonic vibrational frequencies were further calculated to confirm their stability. The energies, oscillator strengths, and rotational strengths of the first 20 electronic excitations were calculated using the TDDFT methodology at the B3LYP/6-311++G (2d, 2p) level in vacuum. The ECD spectra were simulated by the Gausssum 2.25 program27 (σ = 0.3 eV). All quantum computations were performed using Gaussian 09 program package, on an IBM cluster machine located at the High Performance Computing Center of Peking Union Medical College. Inhibitory Effects on Mycobacterium smegmatis. The inhibitory effects of different extracts and isolated diterpenoids against M. smegmatis were conducted using an Alamar blue cell viability assay (Thermo Fisher Scientific Inc.) in 96-well microplates, as described previously.35 The test materials were dissolved in DMSO at an initial concentration at 0.4 mg/mL. The positive control antimicrobial agent, kanamycin, was used at a concentration of 0.1 mg/mL. The M. smegmatis strain was preincubated for 48 h and incubated for an

was conducted on an UltiMate 3000 instrument (Thermo Scientific Dionex) equipped with a diode array detector (DAD) and a Waters Xbridge RP C18 column (250 × 4.6 mm, 5 μm). Preparative HPLC was performed on an Agel instrument with an UV detector and a YMC C18 column (250 × 10 mm, 5 μm). Column chromatography was performed using silica gel (200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China). TLC was carried out on glass precoated silica gel GF254 plates (5 × 10 cm, 2.5 × 7.5 cm, Qingdao Marine Chemical Inc.). Spots were visualized under UV light or by spraying with 10% sulfuric acid in EtOH followed by heating at 105 °C. Plant Material. The fresh roots of Euphorbia fischeriana were collected in Donggang, Liaoning Province, People’s Republic of China, in September 2014. A voucher specimen (No. P-220) was identified to be E. fischeriana by Professor Jingming Jia and has been deposited at the College of Pharmacy, Dalian Medical University. Extraction and Isolation. Air-dried roots of Euphorbia fischeriana (20 kg) were extracted with 80% ethanol (1.5 h × 3). After evaporation of EtOH in vacuo, the aqueous residue was diluted with water and then partitioned with EtOAc. The EtOAc extract (1959 g) was subjected to silica gel column chromatography by elution with petroleum ether−acetone mixtures (30:1−1:2) to give 40 fractions. Fraction A31 (23 g) was subjected to column chromatography over silica gel with CHCl3−MeOH (1:0−2:1) to afford 12 fractions (B1− B12). Subfractions B1−B7 were purified by preparative-HPLC (RP C18 column; detected at 250 nm, 280 nm) eluted by CH3CN−water (0.01% TFA v/v) (20−60%) to yield compounds 1 (5 mg), 2 (11 mg), 3 (6 mg), 4 (13 mg), 5 (10 mg), 6 (21 mg), 7 (20 mg), and 8 (14 mg). Subfractions B9−B12 were separated by preparative-HPLC with a RP C18 column (detected at 210 and 254 nm; at 8 mL/min; 10−30% CH3CN−H2O, 0.01% TFA v/v) to give compounds 15 (8 mg), 16 (6 mg), and 17 (14 mg). Fraction A28 (13 g) was separated by MPLC equipped with an RP C18 column and detected at 210 and 280 nm after elution with CH3OH−water (20 mL/min), to give subfractions C1−C23. Preparative HPLC with an RP C18 column and CH3CN−water (0.01% TFA v/v) (20−60%) elution (detected at 210, 250, and 280 nm, 8 mL/min) was applied to purify subfractions (C1− C15), and the purified diterpenoids were obtained as 9 (10 mg), 10 (5 mg), 11 (7 mg), 12 (14 mg), 13 (11 mg), 14 (3 mg), 18 (10 mg), 19 (12 mg), 20 (6 mg), and 21 (2 mg). 11α,17-Dihydroxyhelioscopinolide E (1). Compound 1 was produced as a white amorphous solid; [α]D25 − 15.0 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 281 (3.80) nm; ECD (CH3OH) Δε236.5 − 4.30, Δε277 + 14.1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz), see Table 1; HRESIMS m/z 369.1663 (calcd for C20H26O5Na, 369.1678). 6β,11α,17-Trihydroxyhelioscopinolide E (2). Compound 2 was produced as a white amorphous solid; [α]25 D − 21.0 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 275 (4.15) nm; ECD (CH3OH) Δε234.5 − 1.43, Δε275 + 7.2; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz), see Table 1; HRESIMS m/z 385.1162 (calcd for C20H26O6Na, 385.1627). 11-Oxo-ebracteolatanolide B (3). Compound 3 was produced as a white amorphous solid; [α]25 D − 8.5 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 248 (3.80) nm; ECD (CH3OH) Δε219 + 0.45; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz), see Table 1; HRESIMS m/z 349.2001 (calcd for C20H29O5, 349.2015). 7-Deoxylangduin B (4). Compound 4 was produced as a white amorphous solid; [α]25 D − 31.2 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 272 (3.75) nm; ECD (CH3OH) Δε248.5 + 1.83, Δε287.5 − 3.13; 1 H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz), see Table 1; HRESIMS m/z 371.1825 (calcd for C20H28O5Na, 371.1834). 9-Deoxy-11β-hydroxyprostratin (13). Compound 13 was produced as a white amorphous solid; [α]25 D − 54.7 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 239 (3.43) nm; ECD (CH3OH) Δε229.5 + 10.32, Δε272 − 0.90; 1H NMR (DMSO-d6, 500 MHz), see Table 2 and δH 1.99 (3H, s) (Ac-13); 13C NMR (DMSO-d6, 125 MHz), see Table 2 and δC 20.8, 172.0 (Ac-13); HRESIMS m/z 435.2024 (calcd for C23H31O8, 435.2019). 1253

DOI: 10.1021/acs.jnatprod.6b00786 J. Nat. Prod. 2017, 80, 1248−1254

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additional 24 h at 37 °C after the addition of the samples. Then, cell viability was determined by absorbance measurement at 590 nm.

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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.6b00786. Copies of spectra of new compounds (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail for X.C.M.: [email protected]. Tel.: +86-41186110419. Fax: +86-411-86110408. *E-mail for C.W.: [email protected]. ORCID

Xiao-Chi Ma: 0000-0003-4397-537X Author Contributions ⊥

C.-J.W. and Q.-L.Y. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research program was supported financially by the National Natural Science Foundation of China (Nos. 81503201 and 81473334), and Dalian Outstanding Youth Science and Technology Talent awards (2014J11JH132 and 2015J12JH201), and the Distinguished Professor of Liaoning Province, Liaoning Bai Qian Wan Talents Program, and by the Innovation Team of Dalian Medical University. The calculated ECD experiment was performed by Ming-Hua Chen (Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China).

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REFERENCES

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DOI: 10.1021/acs.jnatprod.6b00786 J. Nat. Prod. 2017, 80, 1248−1254