Pepluane and Paraliane Diterpenoids from Euphorbia peplus with

Article pubs.acs.org/jnp

Pepluane and Paraliane Diterpenoids from Euphorbia peplus with Potential Anti-inflammatory Activity Luo-Sheng Wan,† Rui Chu,†,§ Xing-Rong Peng,† Guo-Lei Zhu,†,§ Mu-Yuan Yu,†,§ Lei Li,†,§ Lin Zhou,† Shuang-Yang Lu,†,§ Jin-Run Dong,†,§ Zhi-Run Zhang,† Yan Li,† and Ming-Hua Qiu*,†,§ †

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China § University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Twelve new diterpenoids based on two rare skeletal types, namely, paralianones A−D (1−4) and pepluanols A−H (5−12), along with five known compounds, were isolated from an acetone extract of Euphorbia peplus. Their structures were proposed based on 1D and 2D NMR spectroscopic data analysis. These diterpenoids were evaluated for potential anti-inflammatory activity in a lipopolysaccharidestimulated mouse macrophage cellular model. Compounds 3, 4, 11, 13, and 16 displayed moderate inhibitory effects on NO inhibition, with IC50 values ranging from 29.9 to 38.3 μM.

Euphorbia peplus L. (family Euphorbiaceae) is a well-known herbal medicine, and the whole plant has been used commonly in folkloric medicine to treat various skin diseases, such as sunspots, warts, and corns.1,2 Ingenol mebutate, a drug that was recently approved by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) to treat actinic keratosis, is an ingenane-type diterpenoid isolated from this plant.3 Previous studies on the chemical constituents of E. peplus resulted in the identification of numerous bioactive diterpenoids, mainly based on the ingenane, jatrophane, pepluane, and segetane skeletons.4−12 Of these compounds, pepluanin A displayed multidrug resistance-reversing activity by inhibiting P-glycoprotein,9 whereas pepluanone (17) inhibited the production of NO, PGE2, and TNF-α by reducing the expression of iNOS, COX-2, and TNF-α mRNA to exert an anti-inflammatory effect in vitro.10 Following a preliminary chemical investigation of the biologically active components of this plant,12,13 12 new compounds based on rare skeletons, representing the paraliane (paralianones A−D, 1−4) and pepluane (pepluanols A−H, 5−12) diterpenoid types, were obtained, along with five known analogues. The close structural analogy of these compounds (1−16) with pepluanone (17) suggested that they should be examined for potential antiinflammatory activity. The potencies of compounds 3, 4, 11, 13, and 16 were found to be greater than that of pepluanone (17) in the in vitro bioassay used.

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was subjected to repeated macroporous resin, silica gel, Sephadex LH20, and ODS column chromatography (CC), followed by preparative TLC and semipreparative HPLC, to afford four new paraliane (1−4) and eight new pepluane (5− 12) diterpenoids. Compound 1 was assigned a molecular formula of C31H40O9 (with 12 degrees of unsaturation) from the (+)-HRESIMS peak at m/z 579.2568 [M + Na]+ (calcd 579.2570), which was consistent with the 13C NMR data. Its NMR spectra (Table 1) showed typical signals for two acetoxy groups, a benzoyloxy group, five methyls (one secondary and four tertiary), three methylenes, six methines (three oxygenated at δH 5.83, 5.92, and 4.93), six quaternary carbons (two oxygenated at δC 85.3 and 84.4), and a carbonyl group at δC 221.9. These functionalities accounted for eight degrees of unsaturation, and thus, four additional rings must exist in 1. Analyzing the NMR data revealed that the structure of 1 is similar to that of 5,8,14-triacetoxy-3-benzoyloxy-15-hydroxy-9-oxoparaliane (13),14 except for the presence of a hydroxy group (δH 3.39) replacing the acetoxy group at C-8 in the latter. This deduction was supported by the downfield shifts of C-7, C-9, and C-12 from δC 41.2, 216.3, and 40.4 to δC 44.1, 221.9, and 51.2, respectively; the upfield shift of C-8 from δC 90.9 to 85.3; and the diagnostic HMBC correlations from OH-8 to C-7, C-8, C9, and C-12 (Figure 1). The relative configuration of 1 was established from its ROESY spectrum (Figure 1), in which the cross-peaks of H-AA′ (of OBz)/H-5/OH-8/H-12/OH-15/H3-

RESULTS AND DISCUSSION

The whole powdered plants of E. peplus were extracted with acetone and partitioned with petroleum ether. The latter extract © XXXX American Chemical Society and American Society of Pharmacognosy

Received: March 8, 2016

A

DOI: 10.1021/acs.jnatprod.6b00206 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(C-14) and the absence of an oxygenated methine. Thus, the oxygenated methine in 3 was inferred as being replaced by a carbonyl group at C-14 in 4, which was supported by the HMBC correlations from OH-15 and H3-20 to C-14. Since its ROESY correlations were very similar to those of 3, the relative configuration of 4 could be assigned. Therefore, the structure of 4 (paralianone D) was delineated as shown. The (+)-HRESIMS of 5 exhibited a peak at m/z 639.2774 [M + Na]+ (calcd for C33H44O11Na, 639.2781) and yielded a molecular formula of C33H44O11, requiring 12 degrees of unsaturation. Its 1D NMR spectra revealed the presence of three acetoxy groups, a benzoyloxy unit, four methyls, four methylenes, seven methines (four oxygenated at δC 76.3, 70.0, 65.2, and 73.6), and five quaternary carbons (three oxygenated at δC 90.7, 68.8, and 84.1). Three acetoxy groups and one benzoyloxy group accounted for eight of the 12 degrees of unsaturation, and the remaining four required 5 to be tetracyclic. The above-mentioned analysis suggested that 5 is a typical diterpenoid derivative based on a pepluane skeleton.5 Further analysis of the NMR data (Table 2) revealed that the structure of 5 is identical to that of 5,8,9,15-tetraacetoxy-3benzoyloxy-11,16-dihydroxypepluane (14),6 except for the absence of an acetoxy group, which is replaced by a hydroxy group at C-9 in 5, accounting for the 42-mass-units difference relative to 14. This conclusion was further supported by the downfield shifts of C-8 and C-10 from δC 88.6 and 41.6 to 90.7 and 44.4 and the upfield shifts of H-9 and C-9 from δH 5.79 to 4.66 and δ C 68.3 to 65.2, respectively. The relative configuration of 5 was elucidated by ROESY correlations, which were also identical to those of 14. Thus, the structure of 5 (pepluanol A) was established as shown. Similarly, compound 6 (pepluanol B), which has the same molecular formula as 5 according to its (+)-HRESIMS, was directly determined by comparing the chemical shifts of C-7, C8, C-9, and C-13 with those of 14.6 The downfield shifts of C-7, C-9, and C-13 from δC 39.8, 68.3, and 47.0 to δC 44.2, 72.5, and 49.1, respectively, and the upfield shift of C-8 from δC 88.6 to 77.8 suggested that a hydroxy group was located at C-8 in 6 rather than the acetoxy group in 14. Compound 7 was determined to have a molecular formula of C28H42O11 based on the (+)-HRESIMS peak at m/z 577.2617 [M + Na]+ (calcd for C28H42O11Na, 577.2625), which is 104 mass units less than that of 14,6 suggesting the absence of the benzoyloxy group at C-3 in 7. This difference is consistent with the fact that the signals corresponding to this group disappeared. Additionally, in the HMBC spectrum, HMBC correlations from OH-3 to C-2 (δC 36.0) and C-3 (δC 74.4) confirmed this deduction. The OH-3 substituent was determined as β-oriented by the ROESY correlations of OH3/OH-16 and OH-3/H-5. Therefore, the structure of 7 (pepluanol C) was elucidated as shown. Compound 8 gave a molecular formula of C37H48O14 as assigned by the (+)-HRESIMS peak at m/z 739.2937 [M + Na]+ (calcd 739.2942). Its NMR spectra (Table 2) revealed that the structure of 8 is similar to that of 14,6 except for the presence of an additional acetoxy group (δH 2.12; δC 169.7 and 20.6), an additional oxygenated methine (δH 5.03; δC 74.6), and the concomitant absence of a methylene group in 8. The additional oxygenated methine and acetoxy group were assigned based on the 1H−1H COSY correlations of H-1 (δH 5.03)/H-2 (δH 2.87)/H-3 (δH 5.76)/H-4 (δH 2.39)/H-5 (δH 5.87) and the HMBC correlations from H-1 to the carbonyl carbon (δC 169.7) of this acetoxy group. The relative

16, H-12/H-14, and H-12/H3-19 indicated that OBz, H-5, OH8, H-12, H-14, OH-15, H3-16, and H3-19 are cofacial, and these were assigned randomly as β-oriented. In turn, the ROESY correlations of H-4/H3-17/H3-20 revealed that these groups are α-oriented. Therefore, the structure of compound 1 was elucidated as shown and named paralianone A. Compound 2 was isolated as a white, amorphous powder. Its (+)-HRESIMS peak at m/z 679.2722 [M + Na]+ (calcd 679.2731) established a molecular formula of C35H44O12, which is 58 mass units more than that of 13,14 suggesting that it is an acetylated derivative of 13. The additional acetoxy group (δH 2.06; δC 170.5 and 21.2) and oxygenated methylene (H2-17 at δH 4.32 and C-17 at δC 63.4) allowed the location of an acetoxy group at C-17, as confirmed by the HMBC correlations of H217 to the carbonyl carbon of the acetoxy group, C-5, and C-7. The α-orientation of H2-17 was assigned as that of 1 by ROESY correlations of H-4/H2-17/H3-20. Thus, the structure of 2 (paralianone B) was depicted as shown. Compound 3 yielded a molecular formula of C31H40O8 based on its 13C NMR and the (+)-HRESIMS peak at m/z 563.2619 [M + Na]+ (calcd 563.2621). Analyzing its NMR data (Table 1) revealed that its structure resembles that of 1, except that the oxygenated quaternary carbon C-8 was replaced by a methine (δH 3.14 and δC 46.4) in the former. This difference was supported by the correlations of H2-7 (δH 1.45 and 1.78)/H-8 (δH 3.14)/H-12 (δH 4.26)/H2-11 (δH 1.77 and 1.92) in the 1 H−1H COSY spectrum. The H-8 proton was assigned as βoriented by the ROESY correlations from H-8 to H-5, H-12, and H3-19. The structure of 3 (paralianone C) was therefore defined as shown. Compound 4, a white powder, was determined to have a molecular formula of C29H36O7 by its (+)-HRESIMS peak at m/z 519.2351 [M + Na]+ (calcd 519.2359). On comparing its NMR data with those of 3 (Table 1), the 13C NMR of 4 indicated the presence of a second carbonyl group at δC 207.0 B



Article

Table 1. 1H and 13C NMR Data for Compounds 1−4a 1 δH 1α 1β 2 3 4 5 6 7α 7β 8 9 10 11α 11β 12 13 14 15 16 17 18 19 20 8-OH 15-OH 3-OBz

2.14 1.51 2.56 5.83 2.45 5.92

m dd (14.1, 4.9) m m dd (11.9, 4.5) d (11.9)

1.69 d (15.7) 1.83 d (15.7)

1.72 dd (14.4,3.9) 2.06 dd (14.4, 11.0) 4.22 dd (11.0, 3.9) 4.93 s 1.07 1.07 1.09 1.29 0.64 3.39 3.11

d (5.6) s s s s s s

7.92 AA′ 7.59 C 7.46 BB′ 5-OAc

2.01 s

2 δC, type 44.3, CH2 36.0, 76.4, 47.7, 70.2, 51.8, 44.1,

CH CH CH CH C CH2

85.3, 221.9, 46.3, 33.7,

C C C CH2

51.2, 53.2, 73.5, 84.4, 16.5, 16.3, 23.9, 29.3, 16.4,

CH C CH C CH3 CH3 CH3 CH3 CH3

165.8, 133.4, 129.3, 129.4, 128.7, 172.0, 20.9,

C CH C CH CH C CH3

17-OAc a

2.16 1.50 2.47 5.78 2.56 6.07

m dd (14.4, 5.6) m m dd (12.2, 3.8) d (12.2)

2.04 m 2.14 m

1.79 m 2.15 m 4.44 brd 4.96 s 1.05 4.32 1.12 1.26 0.78

d (7.1) m s s s

7.92 AA′ 7.58 C 7.45 BB′ 1.98 s 1.81 s

2.11 s

3 δC, type

δH

44.5, CH2 36.3, 76.3, 48.1, 68.5, 54.6, 36.0,

CH CH CH CH C CH2

90.1, 216.0, 45.9, 33.3,

C C C CH2

49.7, 53.0, 73.0, 84.3, 16.1, 63.4, 25.6, 28.6, 17.4,


165.5, 133.3, 129.8, 129.3, 128.5, 169.7, 20.8, 169.9, 20.6, 169.6, 20.9, 170.5, 21.2,

C CH C CH CH C CH3 C CH3 C CH3 C CH3

3.23 s

8-OAc 14-OAc

δH

169.7, C 21.0, CH3

2.12 s 2.06 s

2.13 1.47 2.51 5.81 2.39 5.59

m m m m dd (11.9, 4.3) d (11.9)

1.45 m 1.78 m 3.14 m

1.77 m 1.92 m 4.26 m 4.94 s 1.06 1.09 1.05 1.11 0.60

d (7.5) s s s s

4 δC, type 44.4, CH2 36.1, 76.5, 48.1, 69.0, 52.9, 35.4,

CH CH CH CH C CH2

46.4, 225.1, 47.0, 35.5,

CH C C CH2

40.7, 52.3, 73.4, 84.4, 16.3, 16.6, 22.7, 29.4, 15.7,


165.7, 133.3, 129.6, 129.4, 128.7, 170.8, 20.9,

C CH C CH CH C CH3

3.18 s 7.93 AA′ 7.59 C 7.48 BB′ 2.02 s

2.11 s

δH 2.61 1.64 2.54 5.87 2.30 5.96

m dd (14.5, 4.4) m m dd (11.9, 4.3) d (11.9)

1.54 dd (14.0, 7.4) 1.92 dd (14.0, 10.0) 3.35 m

1.69 dd (14.4, 5.1) 1.82 dd (14.4, 10.0) 4.42 m

1.10 1.00 1.06 1.10 0.82

d (7.5) s s s s

δC, type 42.1, CH2 35.8, 77.0, 52.6, 67.9, 57.5, 35.7,

CH CH CH CH C CH2

48.2, 223.7, 47.3, 35.8,

CH C C CH2

43.9, 61.6, 207.0, 86.7, 16.3, 16.6, 22.7, 28.7, 14.5,

CH C C C CH3 CH3 CH3 CH3 CH3

165.7, 133.5, 129.6, 129.5, 128.8, 170.8, 21.0,

C CH C CH CH C CH3

3.38 s 7.97 AA′ 7.60 C 7.49 BB′ 2.05 s

169.8, C 21.0, CH3

δ in ppm, J in Hz, 600 MHz for 1H and 150 MHz for 13C, in CDCl3.

formula, C33H42O11, which is 42 mass units less than that of pepluanone (17),10 revealing the absence of an acetoxy group in each case. Their 1D NMR spectroscopic data (Table 3) were also comparable with those of 17, except for differences in the chemical shifts in ring D. Specifically, the downfield shifts of C10, C-12, and C-19 and the upfield shift of C-11 suggested that the acetoxy group of 17 is replaced by a hydroxy group at C-11 in 9 and 10. The similar ROESY correlations between 9 and 17 suggested that H3-19 has an α-orientation (Figure 2). In turn, the β-orientation of H3-19 in 10 was established by correlations from H3-19 to H-10β and H-12 in its ROESY spectrum (Figure 2). Compound 11 was isolated as a white, amorphous powder. Its molecular formula, C33H40O10, was determined from the (+)-HRESIMS data (m/z 619.2509 [M + Na]+, calcd 619.2519), which indicated 14 degrees of unsaturation. The 1D NMR spectroscopic data (Table 3) of 11 exhibited signals attributable to three acetoxy groups, a benzoyloxy group, four methyls (one secondary and three tertiary by 1H NMR), three

Figure 1. (a) Three-bond HMBC (red arrows) and 1H−1H COSY correlations (thick blue lines) of 1. (b) Key ROESY correlations (dashed red lines) of 1.

configuration of H-1 was assigned as α-oriented by the ROESY correlations of H-1/H-4. Therefore, the structure of 8 was delineated and named pepluanol D. According to their (+)-HRESIMS data, compounds 9 (pepluanol E) and 10 (pepluanol F) have the same molecular C



Article

Table 2. 1H and 13C NMR Data for Compounds 5−8a 5 δH 1α 1β 2 3 4 5 6 7α 7β 8 9 10α 10β 11 12α 12β 13 14 15 16 17 18 19 20 3-OH 16-OH 1-OAc

2.15 1.49 2.52 5.79 2.40 5.81

m dd (14.2, 4.9) m m m d (12.0)

1.94 d (15.8) 2.40 m 4.66 d (3.5) 1.80 m 1.94 d (15.8) 1.67 m 1.82 m 4.22 dd (12.6, 6.5) 5.03 s 1.04 1.08 1.31 0.91

d (7.3) s s s

6 δC, type 44.6, CH2 35.8, CH 76.3, CH 48.3,CH 70.0, CH 49.0, C 39.4, CH2 90.7, C 65.2, CH 44.4, CH2 68.8, C 33.6, CH2 47.1, 51.4, 73.6, 84.1, 16.3, 16.4, 31.5, 16.1,

CH C CH C CH3 CH3 CH3 CH3

3.16 s

3-OBz 7.92 AA′ 7.53 C 7.40 BB′ 5-OAc

1.96 s

8-OAc

1.69 s

a

2.16 1.49 2.58 5.82 2.49 5.95

m dd (14.2, 4.5) m m dd (11.9, 4.6) d (11.9)

1.55 m 1.66 m 4.93 d (6.0) 1.70 m 2.35 dd (16.4 5.9) 1.59 m 1.73 m 4.21 dd (13.0, 6.4) 5.03 s 1.05 1.08 1.28 0.90

d (7.3) s s s

165.6, 133.1, 129.9, 129.4, 128.4, 170.0, 20.9, 171.1, 22.2,

C CH C CH CH C CH3 C CH3

7.91 AA′ 7.56 C 7.42 BB′ 2.00 s

δH

44.6, CH2 36.0, 76.2, 47.8, 71.4, 49.4, 44.2,

CH CH CH CH C CH2

77.8, C 72.5, CH 42.1, CH2 68.7, C 34.0, CH2 49.1, 52.7, 73.8, 84.2, 16.5, 16.3, 30.9, 15.5,


169.8, C 21.0, CH3

165.8, 133.4, 129.3, 129.4, 128.6, 172.8, 21.0,

C CH C CH CH C CH3

2.05 1.48 2.20 3.97 1.98 5.65

m dd (13.9, 5.9) m brs m d (12.0)

1.66 d (16.0) 2.63 d (16.0) 5.74 d (4.8) 1.86 d (16.8) 1.96 m 1.68 m 1.71 m 4.32 dd (12.7, 6.7) 4.96 s 1.14 1.12 1.27 0.89 4.12 5.24

d (7.2) s s s s s

2.12 s

169.6, 21.5, 169.7, 20.9,

C CH3 C CH3

8 δC, type

δH

2.13 s

2.05 s 2.09 s

δC, type

44.2, CH2

5.03 d (9.9)

74.6, CH

36.0, 74.4, 48.5, 73.4, 48.3, 39.5,

2.87 5.76 2.39 5.87

37.7, 72.4, 43.4, 69.3, 49.2, 39.5,

CH CH CH CH C CH2

89.1, C 67.6, CH 40.8, CH2 68.2, C 33.9, CH2 46.7, 51.0, 73.0, 84.3, 15.7, 16.2, 32.0, 15.9,


m m dd (11.8, 5.2) d (11.8)

1.59 m 2.50 d (15.9) 5.81 d (5.3) 1.87 d (16.9) 2.00 m 1.70 m 1.79 m 4.22 dd (13.0, 6.4) 4.91 s 0.82 1.06 1.31 0.92

d (7.5) s s s

2.93 s 2.12 s

8.03 AA′ 7.51 C 7.37 BB′

2.01 s 2.04 s

2.11 s

7 δC, type

2.88 s

9-OAc 15-OAc

δH

174.1, 21.4, 170.6, 22.5, 169.1, 21.1, 169.7, 20.9,

C CH3 C CH3 C CH3 C CH3

1.91 s 1.75 s 2.03 s 2.14 s

CH CH CH CH C CH2

88.3, C 67.4, CH 41.4, CH2 68.5, C 33.6, CH2 47.1, 50.8, 72.0, 82.0, 10.2, 16.3, 31.2, 15.8,


169.7, 20.6, 165.9, 133.0, 130.0, 129.7, 128.2, 169.7, 20.8, 170.2, 22.1, 168.9, 21.4, 169.8, 20.8,

C CH3 C CH C CH CH C CH3 C CH3 C CH3 C CH3


methylenes, seven methines (one olefinic at δH 5.99 and δC 125.1; three oxygenated at δH 5.76 and δC 76.5, δH 6.00 and δC 68.3, and δH 5.02 and δC 72.4, respectively), and six quaternary carbons (one carbonyl at δC 195.2, one olefinic at δC 164.7, and two oxygenated at δC 83.4 and 84.4, respectively). These data were similar to those of compound 5, except for the chemical shifts in ring D, specifically, a double bond rather than the methylene and hydroxy groups at C-10 and C-11 in 11, which could be supported by the upfield shift of C-9 from δC 206.0 to 195.2. Moreover, in the HMBC spectrum, the correlations of H3-19 (δH 2.03) to C-10 (δC 125.1), C-11 (δC 164.7), and C-12 (δC 30.8) further supported this deduction. Thus, the structure of 11 (pepluanol G) was delineated as shown. Compound 12, a white, amorphous powder, yielded a molecular formula of C35H42O12 from the (+)-HRESIMS peak at 677.2569 [M + Na]+ (calcd 677.2574), which is 42 mass

units more than that of 11, suggesting the presence of an additional acetoxy group. On comparing its NMR data (Table 3) to those of 11, the 13C NMR spectrum of 12 revealed the presence of resonances corresponding to the additional acetoxy group at δH 2.04 and δC 170.5 and 21.2, an oxygenated methylene at δH 4.41 and δC 63.0, and the absence of a methyl group at C-18. These findings suggested that the methyl group of 11 is substituted by an acetoxy group at C-18 in 12, as supported by the HMBC correlations of H2-18 (δH 4.41) to the carbonyl carbon (δC 170.5) of the acetoxy group. The relative configuration of H2-18 was assigned as α-oriented based on the correlations from H2-18 to H-4 and H3-20 in the ROESY spectrum. The structure of 12 (pepluanol H) was thus elucidated as shown. Five known diterpenoids, 5,8,14-triacetoxy-3-benzoyloxy-15hydroxy-9-oxoparapliane (13),14 5,8,9,15-tetraacetoxy-3-benD



Article

Table 3. 1H and 13C NMR Data for Compounds 9−12a 9 δH 1α 1β 2 3 4 5 6 7α 7β 8 9 10α 10β 11 12α 12β 13 14 15 16 17 18 19 20 16-OH 3-OBz

2.12 1.49 2.56 5.81 2.41 5.84

m dd (14.2, 4.7) m m dd (12.0, 4.4) d (12.0)

1.77 d (16.1) 2.56 m

2.49 d (16.8) 2.73 d (16.8) 1.31 m 1.85 dd (12.4, 6.4) 4.68 dd (13.0, 6.4) 4.96 s 1.07 1.12 1.36 0.67 3.17

d (7.3) s s s s

7.95 AA′ 7.58 C 7.45 BB′ 5-OAc

1.96 s

8-OAc

1.92 s

15-OAc

2.12 s

10 δC, type 44.3, CH2 35.7, 76.3, 48.2, 69.5, 49.3, 37.7,

CH CH CH CH C CH2

88.8, C 206.0, C 52.1, CH2 72.9, C 34.5, CH2 49.3, 51.9, 72.7, 84.2, 16.4, 16.2, 31.0, 15.9,


165.6, C 133.3, CH 129.8, C 129.4, CH 128.5, CH 170.2, C 20.9, CH3 170.7, C 20.9, CH3 169.7, C 20.8, CH3

11

δH 2.13 1.50 2.57 5.79 2.41 5.86

m dd (14.6, 5.1) m m dd (12.0, 4.3) d (12.0)

1.73 m 2.54 m

2.44 d (15.1) 2.80 d (15.1) 1.45 m 1.79 m 4.39 dd (12.6, 6.4) 4.98 s 1.07 1.11 1.63 0.69 3.33

d (7.3) s s s s

7.96 AA′ 7.59 C 7.46 BB′ 1.95 s 1.88 s 2.11 s

δC, type

δH

44.3, CH2 35.7, 76.5, 48.1, 69.5, 49.4, 38.1,

2.17 1.46 2.47 5.76 2.38 6.00

CH CH CH CH C CH2

2.22 d (18.8) 2.93 dd (18.8, 8.1) 4.18 d (8.1)


5.02 s 1.02 1.11 2.03 0.75 3.40


44.5, CH2 36.2, 76.5, 48.4, 68.3, 48.3, 46.5,

CH CH CH CH C CH2

83.4, C 195.2, C 125.1, CH

5.99 s

72.7, C 36.2, CH2 48.2, 52.1, 72.7, 84.3, 16.2, 16.2, 30.2, 16.3,

m dd (14.5, 5.7) m m dd (11.9, 3.9) d (9.4)

1.64 d (15.4) 2.26 d (15.4)

88.4, C 204.7, C 51.5, CH2

12 δC, type

d (7.2) s s s s

164.7, C 30.8, CH2 48.7, 56.3, 72.4, 84.4, 16.0, 16.4, 24.5, 15.4,



7.90 AA′ 7.57 C 7.44 BB′ 2.01 s 1.89 s 2.14 s

18-OAc a

δH 2.16 1.47 2.45 5.76 2.48 6.09

δC, type

m dd (14.4, 5.7) m m dd (12.2, 4.1) d (12.2)

2.00 m 2.20 m

36.2, 76.1, 48.4, 67.7, 51.1, 40.7,

CH CH CH CH C CH2

82.5, C 194.9, C 125.0, CH

6.01 s

2.21 m 2.95 dd (18.7, 8.5) 4.24 d (8.5) 5.03 s 1.02 4.41 2.04 0.76 3.39

44.4, CH2

d (7.2) m s s s

164.7, C 30.6, CH2 48.7, 56.7, 71.9, 84.4, 16.0, 63.0, 24.5, 15.7,


165.4, C 133.2, CH 129.8, C 129.2, CH 128.5, CH 168.9, C 20.8, CH3 170.8, C 20.4, CH3 169.8, C 20.9, CH3 170.5, C 21.2, CH3

7.89 AA′ 7.57 C 7.44 BB′ 2.01 s 1.88 s 2.12 s 2.04 s


All compounds were tested for inhibitory activity against NO production in the lipopolysaccharide (LPS)-stimulated mouse macrophage cellular model (Table 4). Compounds 3, 4, 11, 13, and 16 displayed inhibitory effects on NO inhibition, with IC50 values of 33.7, 38.3, 36.6, 29.9, and 37.1 μM, respectively. These values were comparable with that of pepluanone (17) Table 4. Inhibitory Effects on LPS-Stimulated NO Production in RAW264.7 Cells Figure 2. Key ROESY correlations (dashed red lines) of 9 and 10.

zoyloxy-11,16-dihydroxypepluane (14),6 5,8,11,15-tetraacetoxy3-benzoyloxy-9,16-dihydroxypepluane (15),8 5,8,9,11,15-pentaacetoxy-3-benzoyloxy-16-hydroxypepluane (16),7 and pepluanone (17),10 were also isolated from this plant. These compounds were identified by comparing their spectroscopic data with reported values.

compound

IC50 (μM)

compound

IC50 (μM)

compound

IC50 (μM)

1 2 3 4 5 6

43.2 >50 33.7 38.3 >50 >50

7 8 9 10 11 12

>50 >50 >50 >50 36.6 >50

13 14 15 16 17 MG-132a

29.9 >50 >50 37.1 47.5 0.18

a

E

Positive control. DOI: 10.1021/acs.jnatprod.6b00206 J. Nat. Prod. XXXX, XXX, XXX−XXX


Article

Paralianone A (1): white, amorphous powder; [α]25D −17.2 (c 0.36, MeOH); UV (MeOH) λmax (log ε) 200 (3.86), 229 (4.04), 272 (2.84) nm; IR (KBr) νmax 3452, 2931, 1727, 1453, 1381, 1280, 1112, 1026, 712 cm−1; 1H and 13C NMR data, see Table 1; (+)-HRESIMS m/z [M + Na]+ 579.2568 (calcd for C31H40O9Na, 579.2570). Paralianone B (2): white, amorphous powder; [α]25D −12.2 (c 0.48, MeOH); UV (MeOH) λmax (log ε) 199 (3.81), 229 (4.01), 272 (2.78) nm; IR (KBr) νmax 3441, 2934, 1740, 1452, 1370, 1208, 1106, 1026, 713 cm−1; 1H and 13C NMR data, see Table 1; (+)-HRESIMS m/z [M + Na]+ 679.2722 (calcd for C35H44O12Na, 679.2731). Paralianone C (3): white, amorphous powder; [α]25D −43.1 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 200 (3.86), 229 (4.03), 271 (2.95) nm; IR (KBr) νmax 3458, 2968, 1741, 1452, 1370, 1230, 1108, 1025, 712 cm−1; 1H and 13C NMR data, see Table 1; (+)-HRESIMS m/z [M + Na]+ 563.2619 (calcd for C31H40O8Na, 563.2621). Paralianone D (4): white, amorphous powder; [α]25D −16.1 (c 0.37, MeOH); UV (MeOH) λmax (log ε) 199 (3.86), 229 (4.09), 272 (2.85) nm; IR (KBr) νmax 3457, 2966, 1715, 1453, 1381, 1277, 1109, 1027, 713 cm−1; 1H and 13C NMR data, see Table 1; (+)-HRESIMS m/z [M + Na]+ 519.2351 (calcd for C29H36O7Na, 519.2359). Pepluanol A (5): white, amorphous powder; [α]25D +35.4 (c 0.33, MeOH); UV (MeOH) λmax (log ε) 199 (3.74), 229 (4.06), 272 (2.80) nm; IR (KBr) νmax 3433, 2964, 1739, 1369, 1284, 1241, 1024, 712 cm−1; 1H and 13C NMR data, see Table 2; (+)-HRESIMS m/z [M + Na]+ 639.2774 (calcd for C33H44O11Na, 639.2781). Pepluanol B (6): white, amorphous powder; [α]25D −5.0 (c 0.16, MeOH); UV (MeOH) λmax (log ε) 200 (3.75), 229 (4.01), 272 (2.76) nm; IR (KBr) νmax 3472, 2926, 1720, 1628, 1373, 1243, 1027, 715 cm−1; 1H and 13C NMR data, see Table 2; (+)-HRESIMS m/z [M + Na]+ 639.2776 (calcd for C33H44O11Na, 639.2781). Pepluanol C (7): white, amorphous powder; [α]25D +2.1 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 202 (3.40) nm; IR (KBr) νmax 3435, 2927, 1740, 1631, 1373, 1245, 1023 cm−1; 1H and 13C NMR data, see Table 2; (+)-HRESIMS m/z [M + Na]+ 577.2617 (calcd for C28H42O11Na, 577.2625). Pepluanol D (8): white, amorphous powder; [α]25D +30.3 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 199 (3.70), 229 (4.02), 273 (2.74) nm; IR (KBr) νmax 3440, 2931, 1746, 1633, 1370, 1239, 1029, 715 cm−1; 1H and 13C NMR data, see Table 2; (+)-HRESIMS m/z [M + Na]+ 739.2937 (calcd for C37H48O14Na, 739.2942). Pepluanol E (9): white, amorphous powder; [α]25D +34.8 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 200 (3.77), 229 (4.03), 272 (2.86) nm; IR (KBr) νmax 3434, 2926, 1740, 1371, 1236, 1115, 1025, 712 cm−1; 1H and 13C NMR data, see Table 3; (+)-HRESIMS m/z [M + Na]+ 637.2616 (calcd for C33H42O11Na, 637.2625). Pepluanol F (10): white, amorphous powder; [α]25D +25.9 (c 0.27, MeOH); UV (MeOH) λmax (log ε) 199 (3.75), 229 (4.05), 271 (2.88) nm; IR (KBr) νmax 3460, 2928, 1736, 1631, 1373, 1242, 1123, 1024, 713 cm−1; 1H and 13C NMR data, see Table 3; (+)-HRESIMS m/z [M + Na]+ 637.2622 (calcd for C33H42O11Na, 637.2625). Pepluanol G (11): white, amorphous powder; [α]25D −64.3 (c 0.22, MeOH); UV (MeOH) λmax (log ε) 198 (3.94), 232 (4.32) nm; IR (KBr) νmax 3444, 2928, 1740, 1671, 1372, 1227, 1025, 713 cm−1; 1H and 13C NMR data, see Table 3; (+)-HRESIMS m/z [M + Na]+ 619.2509 (calcd for C33H40O10Na, 619.2519). Pepluanol H (12): white, amorphous powder; [α]25D −61.2 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 198 (3.89), 232 (4.22) nm; IR (KBr) νmax 3443, 2925, 1740, 1672, 1371, 1226, 1027, 713 cm−1; 1H and 13C NMR data, see Table 3; (+)-HRESIMS m/z [M + Na]+ 677.2569 (calcd for C35H42O12Na, 677.2574). Nitric Oxide Production in RAW264.7 Macrophages.16 RAW264.7 cells were seeded in 96-well cell culture plates (2 × 105 cells/well) containing RPMI-1640 (Hyclone). After a 24 h preincubation, the cells were treated with gradient dilutions of the compounds with a maxium concentration of 100 μM, followed by stimulation with LPS (1 μg/mL) for 18 h. NO production in the supernatant was assessed by the Griess reagent (Sigma). After a 5 min incubation, the absorbance at 550 nm was measured with a 2104 Envision multilabel plate reader (PerkinElmer Life Sciences, Inc., Boston, MA, USA). MG-132, an inhibitor of proteasome, was used as

(IC50 value of 47.5 μM), which has been extensively studied by previous researchers.10,15 In addition, none of the test compounds displayed any obvious cytotoxicity to RAW264.7 cells.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded with a JASCO P-1020 polarimeter. UV spectra were obtained using a Shimadzu UV2401PC instrument. Infrared spectra were recorded on a Bruker Tensor-27 instrument by using KBr pellets. 1 H and 13C NMR spectra were measured on Bruker AV-400 and DRX500 NMR spectrometers. Chemical shifts (δ) are expressed in ppm with reference to the solvent peak (δH 7.26 and δC 77.0). EIMS and HRTOFEIMS data were collected on a Waters Auto Spec Premier 776 spectrometer. HRESIMS and HRTOFESIMS were performed with an API QSTAR Pulsar spectrometer. An Agilent 1100 series instrument equipped with an Agilent Zorbax SB-C18 column (5 μm, 10 mm × 250 mm) was used for high-performance liquid chromatography (HPLC) analysis. Preparative thin-layer chromatography (TLC) and analytical TLC were performed on precoated TLC plates (200−250 μm thickness, F254 Si gel 60, Qingdao Marine Chemical, Inc.), and compounds were visualized by spraying the dried plates with 5% aqueous H2SO4 followed by heating until dryness. Silica gel (200− 300) mesh (Qingdao Marine Chemical, Inc.), Lichroprep RP-18 (40− 63 μm, Fuji), and Sephadex LH-20 (20−150 μm, Pharmacia) were used for CC. Methanol, chloroform, ethyl acetate, acetone, petroleum ether, and 2-propanol were purchased from Tianjing Chemical Reagents Co. (Tianjing, People’s Republic of China). Plant Material. The whole plants of E. peplus were collected in September of 2013 in Kunming, People’s Republic of China, and were identified by M.-H.Q. A voucher specimen has been deposited in the herbarium of the Kunming Institute of Botany, Chinese Academy of Science (no. kep-09-13). Extraction and Isolation. The air-dried powder of the plant material (25 kg) was extracted twice with acetone (100 L) and heated under reflux for 4 h to yield 6 kg of a crude extract, which was suspended in water (20 L) and partitioned with petroleum ether (5 × 20 L). The petroleum ether-soluble fraction (3 kg) was fractioned by CC on a macroporous resin and eluted with a MeOH/H2O mixture (50−100%) to give four fractions, F1−F4. Fraction F2 (430 g) was subjected to silica gel CC and eluted with petroleum ether/EtOAc (100:0−0:100) to give eight subfractions, subF1−subF8. Of these, subF1 (40 g) was subjected to RP-18 CC and eluted with MeOH/ H2O (20:80 to 100:0) to obtain 36 subfractions, subF1-1−subF1-36. SubF1-18 (50 mg) was further purified by preparative TLC (petroleum ether/2-propanol, 80:1) to yield 3 (1.8 mg), 4 (32 mg), and 17 (4.0 mg). SubF6 (120 g) was separated by RP-18 CC and eluted with MeOH/H2O (20:80 to 100:0) to afford 50 subfractions, subF6-1−subF6-50. SubF6-6 (12 mg) was passed over Sephadex LH20 (100% MeOH) to yield 15 (2.0 mg). SubF6-25 (30 mg) was successively subjected to Sephadex LH-20 (100% MeOH) and preparative HPLC (75% CH3CN) purification to afford 1 (3.1 mg), 2 (2.2 mg), and 13 (1.7 mg). SubF6-41 (28 mg) was sequentially separated with Sephadex LH-20 (100% MeOH) and by preparative TLC (petroleum ether/2-propanol, 50:1) to yield 6 (4.1 mg). SubF7 (110 g) was subjected to RP-18 CC and eluted with MeOH/H2O (20:80 to 100:0) to give 45 subfractions, subF7-1−subF7-45. Next, subF7-1 (20 mg) was successively separated over Sephadex LH-20 (100% MeOH) and preparative TLC (petroleum ether/2-propanol, 50:1) to yield 16 (3.0 mg). SubF7-23 (45 g) was subjected to silica gel CC (petroleum ether/2-propanol, 200:1 to 20:80) to afford seven fractions (subF7-23a−subF7-23g). SubF7-23a (11 g) was separated by Sephadex LH-20 CC (100% MeOH) and preparative TLC (petroleum ether/2-propanol, 20:1) to yield 5 (2.8 mg), 7 (10 mg), 10 (1.1 mg), and 12 (2.0 mg). Finally, subF7-23d (8 g) was sequentially subjected to purification by Sephadex LH-20 (100% MeOH) and by preparative HPLC (75% CH3CN) to yield 8 (15 mg), 9 (8.0 mg), 11 (3.3 mg), and 14 (4.2 mg). F



Article

positive control. The viability of RAW264.7 cells was evaluated by the MTT assay simultaneously to exclude the interference of the cytotoxicity of the test compounds. The absorbance was read at 595 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.6b00206. 1D, 2D, and HRESIMS spectra of compounds 1−12 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-871-65223327. Fax: +86-871-65223255. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (NSFC, Grant 81403050) and NSFCJoint Foundation of Yunnan Province (Grant U1132604).

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REFERENCES

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Pepluane and Paraliane Diterpenoids from Euphorbia peplus with

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