Bioactive Diterpenoids from the Stems of Euphorbia royleana - Journal

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Bioactive Diterpenoids from the Stems of Euphorbia royleana Peixia Wang,†,¶ Chunfeng Xie,†,¶ Lijun An,† Xueyuan Yang,† Yaru Xi,† Shuo Yuan,† Chenyue Zhang,† Muhetaer Tuerhong,‡ Da-Qing Jin,⊥ Dongho Lee,§ Jie Zhang,# Yasushi Ohizumi,∥ Jing Xu,*,† and Yuanqiang Guo*,†

J. Nat. Prod. Downloaded from pubs.acs.org by UNIV DE BARCELONA on 02/07/19. For personal use only.

†

State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300350, People’s Republic of China ‡ College of Chemistry and Environmental Sciences, Laboratory of Xinjiang Native Medicinal and Edible Plant Resources Chemistry, Kashgar University, Kashgar 844000, People’s Republic of China ⊥ School of Medicine, Nankai University, Tianjin 300071, People’s Republic of China § Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea # Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi 832003, People’s Republic of China ∥ Kansei Fukushi Research Institute, Tohoku Fukushi University, Sendai 989-3201, Japan S Supporting Information *

ABSTRACT: Two ingenane- (1 and 2), two ent-atisane- (3 and 4), two ent-kaurane- (5 and 6), two ent-abietane- (7 and 8), and one ent-isopimarane-type (9) diterpenoid and 12 known analogues have been isolated from the methanolic extract of the stems of Euphorbia royleana. Their structures, including absolute configurations, were determined by extensive spectroscopic methods and ECD data analysis. The nitric oxide inhibitory activities of those diterpenoids were examined biologically in lipopolysaccharide-stimulated BV-2 cells, with compounds 1, 2, 5−7, 10, and 12 having IC50 values lower than 40 μM. Molecular docking was used to investigated the possible mechanism of compounds 1, 2, 5−7, 10, and 12.

T

species.8−13 In our search for anti-inflammatory agents from traditional herbal medicines,14,15 the chemical constituents of E. royleana were investigated. Nine previously undescribed diterpenoids (1−9), as well as 12 known diterpenoids (10− 21), were obtained from the stems of E. royleana. The structures of compounds 1−9, including absolute stereochemistry, were established by a combination of spectroscopic and electronic circular dichroism data analysis. The isolated diterpenoids were also examined biologically for their nitric oxide (NO) inhibitory effects in BV-2 cells.

he genus Euphorbia contains about 2000 species around the world.1 Some Euphorbia plants, including E. pekinensis, E. f ischeriana, E. kansui, and E. hirta, have been used for multiple pathologic symptoms.2 Previous phytochemical investigations have revealed the genus Euphorbia to be a rich source of diterpenoids, triterpenoids, and aromatic compounds showing antitumor, anti-inflammatory, antimicrobial, and multidrug-resistance-reversing activities.3,4 The Euphorbia plants have been widely investigated phytochemically for the presence of structurally diverse diterpenoids with multiple potential biological effects.3,4 Recently, several diterpenoids with novel skeletons have been reported from some species of Euphorbia, such as pepluanols and heliojatrones.5,6 The structural diversity and potential biological significance of diterpenoids from Euphorbia species have prompted further investigation on this genus. Euphorbia royleana Boiss. is a succulent shrub growing widely in Guangxi, Sichuan, and Yunnan provinces of mainland China. E. royleana is used to remove poisonous or harmful substances and relieve inflammation and rheumatic pains.7 Previous investigations revealed that diterpenoids and triterpenoids are the main constituents in this plant © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Compound 1, isolated as a colorless oil, gave the molecular formula C30H40O7 by the 13C NMR data (Table 1) and from the HRESIMS pseudomolecular ion peak [M + H]+ at m/z 513.2851 (calcd for C30H41O7, 513.2852). The IR spectrum displayed characteristic absorption bands at 3475, 1706, and 1650 cm−1, consistent with hydroxy, carbonyl, and olefinic groups, respectively. The 1H NMR spectroscopic data of 1 Received: June 19, 2018

A

DOI: 10.1021/acs.jnatprod.8b00493 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

In order to establish the relative configuration of 1, its NOESY spectrum was measured. The cross-peaks of H-8/H11, H-8/H2-17, and H2-17/H-12β indicated that they adopted a β-orientation, while the NOESY correlations of H3-18/H12α, H-13/H-12α, and H-13/H-14 revealed that they adopted an α-orientation (Figure 2). The relative configurations of C-3, C-4, C-5, and C-10 stereocenters were found to be identical to those of known analogues18,19 through comparing their chemical shifts and coupling constants and from the key NOESY correlation of H-3/H-5. Based on comparison of the calculated and experimental electronic circular dichroism (ECD) spectra (Figure 3A),20−22 the absolute configurations of C-3, C-4, C-5, C-8, C-10, C-11, C-13, C-14, and C-15 in compound 1 were determined as 3S, 4S, 5R, 8S, 10S, 11R, 13R, 14R, and 15R, respectively. The structure of 1 was finally elucidated as (3S,4S,5R,8S,10S,11R,13R,14R,15R)-3β-O-angeloyl-17-tigloyloxy-20-deoxyingenol. Compound 2 gave the molecular formula C 32 H 38 O 7 according to HRESIMS. The 1D NMR spectroscopic data of compound 2 were comparable to those of 1, suggesting that their structures resembled each other. The only difference found was that compound 2 possesses a benzoyloxy group instead of a tigloyloxy group as in 1, which was confirmed by the long-range correlation of H2-17 to the carbon at δC 166.8 of the benzoyloxy group. NOESY correlations and Chem3D modeling were used to disclose that the skeletal conformation of 2 is the same as that of compound 1. In turn, the C-3 angeloyloxy group, the C-4 and C-5 hydroxy groups, and the C-17 benzoyloxy group all were defined as being β-oriented. The calculated ECD spectrum displayed similar Cotton effects to the experimental ECD curve (Figure 3B), allowing the assignment of a (3S,4S,5R,8S,10S,11R,13R,14R,15R) configuration for compound 2. Conclusively, the structure of 2 was constructed as (3S,4S,5R,8S,10S,11R,13R,14R,15R)-3β-O-angeloyl-17-benzoyloxy-20-deoxyingenol.

showed the presence of six olefinic methyl groups [δH 1.77 (s), 1.79 (d, J = 1.5 Hz), 1.84 (dq, J = 7.0, 1.1 Hz), 1.81 (t, J = 1.1 Hz), 1.92 (t, J = 1.5 Hz), and 2.00 (dq, J = 7.2, 1.5 Hz)], two aliphatic methyl groups [δH 0.98 (d, J = 7.1 Hz) and 1.14 (s)], four olefinic protons [δH 5.72 (1H, m), 6.05 (1H, d, J = 1.5 Hz), 6.16 (1H, qd, J = 7.2, 1.5 Hz), and 6.88 (1H, qd, J = 7.0, 1.1 Hz)], two oxymethine protons [δH 3.68 (1H, brs) and 5.49 (1H, brs)], and a pair of oxymethylene protons [δH 4.21 and 4.33 (each 1H, d, J = 12.2 Hz)] (Table 2). Analysis of the carbon resonances at δC 168.3, 127.1, 140.0, 20.7, 15.9, 168.4, 128.7, 137.1, 14.4, and 12.1 in the 13C NMR spectrum, combined with 1H NMR data (Table 2), was suggestive of the existence of an angeloyloxy and a tigloyloxy group.16 The DEPT data, associated with HMQC experiment, exhibited an additional 20 carbons comprising four methyls, two methylenes [one oxymethylene (δC 65.5)], eight methines [two oxymethines (δC 77.4 and 82.9) and two olefinic methines (δC 123.2 and 132.1)], and six quaternary carbons [including one ketone (δC 206.2), two olefinic (δC 135.9 and 137.8), and one oxygenated (δC 84.9)]. The aforementioned spectroscopic evidence revealed compound 1 to be an ingenane-type diterpenoid bearing both an angeloyloxy and a tigloyloxy residue.16,17 The ketone carbonyl, olefinic, oxygenated carbons at δC 206.2 (C-9), 132.1 (C-1), 135.9 (C-2), 137.8 (C-6), 123.2 (C-7), 82.9 (C-3), 84.9 (C-4), 77.4 (C-5), and 65.5 (C17) as well as the other skeletal carbons were assigned based on the comprehensive interpretation of the 2D NMR spectra. The HMBC correlation of H-3 with an ester carbonyl carbon (δC 168.3) demonstrated that the angeloyloxy group was linked to C-3, while the long-range correlation from H2-17 to an ester carbonyl carbon (δC 168.4) suggested that the tigloyloxy group was connected to C-17, respectively. The planar structure of 1 was thus constructed according to the above spectroscopic data analysis. B

DOI: 10.1021/acs.jnatprod.8b00493 J. Nat. Prod. XXXX, XXX, XXX−XXX

C

a

1 2 3 4 5 1 2 3/7 4/6 5

132.1 135.9 82.9 84.9 77.4 137.8 123.2 43.0 206.2 72.1 38.7 30.9 24.1 23.8 27.7 24.5 65.5 16.9 15.6 21.9 168.3 127.1 140.0 20.7 15.9 168.4 128.7 137.1 14.4 12.1

1

CH C CH C CH C CH CH C C CH CH2 CH CH C CH3 CH2 CH3 CH3 CH3 C C CH CH3 CH3 C C CH CH3 CH3

132.1 135.9 82.9 84.9 77.3 137.9 123.2 43.1 206.1 72.1 38.8 31.0 24.1 23.9 27.8 24.6 66.2 16.8 15.6 21.9 168.3 127.0 140.1 20.7 15.9 166.8 130.4 129.6 128.4 132.9

2

C CH CH CH

CH C CH C CH C CH CH C C CH CH2 CH CH C CH3 CH2 CH3 CH3 CH3 C C CH CH3 CH3

position

OR-19a

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

3

171.0 20.8

39.1 34.7 213.2 52.1 57.6 19.7 38.7 32.8 51.1 37.3 23.2 32.0 23.3 27.3 52.3 74.0 68.9 20.5 66.2 14.1

C CH3

CH2 CH2 C C CH CH2 CH2 C CH C CH2 CH CH2 CH2 CH2 C CH2 CH3 CH2 CH3

CH2 CH2 C CH CH CH2 CH2 C CH C CH2 CH CH2 CH2 CH2 C CH2 CH3 CH3

12.0

4 39.2 37.3 213.6 45.1 53.7 22.3 38.1 32.7 49.1 37.2 23.3 32.2 23.6 27.3 52.6 74.2 69.0 11.6

A number with a superscript indicates the location of the substituent group on the parent skeleton.

OR-17a

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

position

Table 1. 13C NMR Data for Compounds 1−9 (δ in ppm, 100 MHz, in CDCl3)

176.6 41.0 26.6 16.5 11.6

39.9 34.8 213.2 51.9 56.9 21.1 41.5 43.2 55.9 38.7 19.2 26.5 40.7 38.0 52.2 79.5 69.8 21.0 65.6 17.6

5

C CH CH2 CH3 CH3

CH2 CH2 C C CH CH2 CH2 C CH C CH2 CH2 CH CH2 CH2 C CH2 CH3 CH2 CH3

168.0 128.2 137.9 14.5 12.0

40.1 34.9 213.5 52.1 56.9 21.2 41.5 43.2 56.0 38.8 19.1 26.6 40.6 38.0 52.1 79.6 69.7 20.8 66.0 17.7

6

C C CH CH3 CH3

CH2 CH2 C C CH CH2 CH2 C CH C CH2 CH2 CH CH2 CH2 C CH2 CH3 CH2 CH3

80.4 25.4 39.0 33.3 55.1 23.6 36.9 151.6 52.0 45.6 30.7 75.6 156.3 114.5 116.8 175.3 8.3 33.3 21.4 12.5 170.5 21.9

7 CH CH2 CH2 C CH CH2 CH2 C CH C CH2 CH C CH C C CH3 CH3 CH3 CH3 C CH3

77.5 29.6 33.1 47.1 47.4 23.5 36.9 152.5 52.7 37.7 30.8 76.5 157.2 114.2 116.1 175.9 8.2 71.3 17.4 11.7

8 CH CH2 CH2 C CH CH2 CH2 C CH C CH2 CH C CH C C CH3 CH2 CH3 CH3

CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH C CH CH CH2 CH3 CH3 CH3

17.3 14.2

9 36.6 26.3 79.5 75.9 54.3 20.9 34.3 136.1 51.3 38.6 27.5 73.2 43.3 128.4 146.0 113.7 17.5

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a

0.98 d (7.1) 1.79 d (1.5)

1.77 s

6.16 qd (7.2, 1.5) 6.15 qd (7.3, 1.4)

2.00 dq (7.2, 1.5) 1.92 t (1.5) 6.88 qd (7.0, 1.1) 1.84 dq (7.0, 1.1)

1.81 t (1.1)

3

4 5 3/7 4/6

5

18 19

20

OR-3a

D

7.56 t (7.3)

1.99 dq (7.3, 1.4) 1.91 t (1.4) 8.06 d (7.3) 7.46 t (7.3)

1.73 s

0.99 d (7.0) 1.80 d (1.4)

20 OAc

19b

18 19a

17b

14b 15a 15b 17a

14a

13b

12 13a

9 11α 11β

6β 7α 7β

2α 2β 4 5 6α

1α 1β

position 3

4

1.85 m 1.11 m 1.23 m 3.43 d (11.2) 3.58 d (11.2) 1.15 s 4.63 d (11.7) 3.95 d (11.7) 1.22 s 2.00 s

0.85 m

1.52 m

1.83 m 1.66 m

1.16 s

1.90 m 1.13 m 1.23 m 3.44 d (11.0) 3.59 d (11.0) 0.99 d (6.5)

0.85 m

1.48 m

1.83 m 1.66 m

1.32 mb 2.07 m 1.31 m

1.32 mb 2.06 m 1.26 m

m m mb m m

1.50 m 1.40 m, 1.14 m

2.31 2.48 2.29 1.08 1.60

1.30 m 1.92 m

1.55 m 1.13 m 1.45 m

1.35 mb 1.55 m

2.33 m 2.75 m

1.33 m 1.93 m

position

m m mb m m

mb mb d (10.9) d (10.9)

4 5

2.34 m 1.34 m, 1.58 m 0.87 t (7.5) 1.10 d (6.8)

OR-19a

4.60 d (11.4) 3.94 d (11.4)

1.14 s

1.44 1.42 3.40 3.48

1.97 brd (12.4) 1.03 m

1.63 m 2.12 m

2.10 m 1.62 m 1.86 m

1.46 m 1.46 m 1.17 brd (9.0)

2.38 2.76 1.38 1.63 1.45

1.27 s 2 3

5 1.35 m 2.20 m

20

19a 19b

18

15a 15b 17a 17b

14β

14α

12β 13

11α 11β 12α

7α 7β 9

2α 2β 5 6α 6β

1α 1β

6

m m mb m m

mb mb d (10.9) d (10.9)

1.78 d (6.8) 1.79 s

6.80 q (6.8)

1.29 s

4.68 d (11.4) 3.99 d (11.4)

1.18 s

1.45 1.41 3.40 3.49

1.98 brd (12.3) 1.06 m

1.60 m 2.13 m

2.04 m 1.63 m 1.86 m

1.48 m 1.48 m 1.18 brd (9.1)

2.38 2.78 1.37 1.65 1.48

1.37 m 2.22 m

OAc

19 20

18b

16a 16b 17 18a

15

14

11β 12

7β 9 11α

6α 6β 7α

2α 2β 3α 3β 5

1α 1β

position

2.12 s

0.87 s 1.10 s

1.84 s 0.91 s

2.50 m 2.40 brd (7.5) 2.62 dd (13.2, 5.3) 1.31 mb 5.00 dd (13.2, 5.3) 6.30 brs

4.72 dd (10.9, 4.6) 0.88 m 1.85 m 1.39 m 1.48 m 1.25 dd (12.1, 3.6) 1.34 m 1.90 m 1.45 m

7

0.78 s 1.02 s

3.12 d (10.9)

1.83 s 3.42 d (10.9)

2.48 m 2.43 brd (7.5) 3.75 dd (13.2, 6.1) 1.45 mb 5.00 dd (13.2, 6.1) 6.29 brs

1.25 m 1.74 m 1.30 m 1.72 m 1.58 dd (12.4, 1.8) 1.43 m 1.77 m 2.19 m

3.57 dd (8.8, 5.3)

8

9

1.14 s 0.79 s

5.76 dd (17.4, 10.9) 5.08 d (17.4 5.12 d (10.9) 1.06 s

5.16 brs

1.75 m 3.58 dd (12.2, 4.2)

2.34 m 1.99 mb 1.82 m

1.76 m 2.02 m 2.29 m

3.48 dd (12.4, 4.7) 1.32 mb

1.77 m 1.50 m

1.29 m 1.70 m

A number with a superscript indicates the location of the substituent group on the parent skeleton. bSignals were in overlapped regions of the spectrum, and the multiplicities could not be discerned.

OR-17a

16 17a 17b

2.49 m 0.97 m 1.19 dd (12.2, 8.2) 1.22 s 4.54 d (11.9) 4.40 d (11.9)

brs m brd (12.2) m m

2.36 m 0.93 m 1.12 dd (11.8, 8.6) 1.14 s 4.33 d (12.2) 4.21 d (12.2)

3.68 5.74 4.22 2.46 1.81

12β 13 14

brs m brd (11.8) m m

3.68 5.72 4.15 2.46 1.83

2

6.06 d (1.4) 5.48 brs

5 7 8 11 12α

1

6.05 d (1.5) 5.49 brs

1 3

position

Table 2. 1H NMR Data for Compounds 1−9 (δ in ppm, 400 MHz, in CDCl3)

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Figure 1. 1H−1H COSY and key HMBC correlations of compounds 1−9.

spectrum (Figure 1). The analysis of the above NMR data pointed toward an ent-atisane-type diterpenoid skeleton for 3. The NOE correlations of H3-18/H-5, H3-18/H-6β, H2-19/ H3-20, H2-19/H-2α, H2-19/H-6α, H3-20/H-2α, H3-20/H-6α, H3-20/H-11α, H3-20/H-13α (14α), H-5/H-9, H-7β/H-9, H9/H-15b, H-15a/H-14β, H2-17/H-15a, and H2-17/H-13β (Figure 2) gave information on the spatial conformation of 3. According to this conformation of 3, rings A and B exist in a twist chair and a chair conformation and are trans-fused with H-5 adopting a β-axial orientation and C-20 being αorientated. In turn, ring B and the six-membered ring consisting of C-8/C-9/C-11/C-12/C-15/C-16 share C-8 and C-9 and are also trans-fused, with H-9 β-oriented and the C13−C-14 bridge connection unit on the α-face, with C-19 occupying an α-axial position. According to the coincidentally calculated and experimental ECD data depicted in Figure 3C, the absolute structure of compound 3 was defined as (4R,5S,8S,9R,10S,12S,16S)-ent-19-acetoyloxy-16α,17-dihydroxyatisan-3-one. Compound 4 exhibited a positive pseudomolecular ion peak [M + H]+ at m/z 307.2273 (calcd 307.2273) in the HRESIMS, consistent with the molecular formula C19H30O3. The 1D NMR spectra of compound 4 resembled those of 3, with the major differences being the disappearance of any signals for the acetyloxy group and C-19 oxymethylene. The NMR spectroscopic evidence, together with its HRESIMS data, suggested compound 4 to be a C-19 norditerpenoid of the entatisane type. The only skeletal difference found between compounds 3 and 4 was that the C-19 oxymethlyene in 3 was replaced by a proton in 4, and the two isolates were considered to have the same skeletal conformation from a biosynthetic

Compound 3 was assigned the molecular formula C22H34O5 deduced from the HRESIMS data. Its 13C NMR data (Table 1) established the presence of 22 carbon resonances. Signals at δC 171.0 and 20.8 for an acetyloxy group were evident. The remaining 20 carbons were sorted into two methyls, 10 methylenes (including two oxygenated), three methines, and five quaternary carbons (including one oxygen-bearing and one ester carbonyl) on the basis of DEPT and HMQC spectra, indicating a diterpenoid framework. This diterpenoid scaffold was established by performing 2D NMR experiments. The HMBC cross-peaks of H2-2 to C-1/C-3/C-4/C-10, H-5 to C1/C-3/C-4/C-6/C-7/C-9/C-10/C-18/C-19/C-20, H-9 to C1/C-5/C-7/C-8/C-10/C-20, H3-18 to C-3/C-4/C-5/C-19, H2-19 to C-3/C-4/C-5/C-18, and H3-20 to C-1/C-5/C-9/C10, combined with the 1H−1H COSY correlations (Figure 1), established the existence of fused six-membered rings A and B. Additionally, one more six-membered ring consisting of C-8/ C-9/C-11/C-12/C-15/C-16 was also deduced according to the 1H−1H COSY and HMBC correlations, as depicted in Figure 1. The tricyclic 6,6,6-fused ring skeleton was thus established based on the above analysis. Subsequently, the ketone carbonyl and oxygenated carbon resonances at δC 213.2 (C-3), 66.2 (C-19), 74.0 (C-16), and 68.9 (C-17) and the other carbon signals of the three six-membered rings were assigned, respectively, based on the 2D NMR experiments. The only remaining methylene carbons at δC 23.3 and 27.3, attributable to C-13 and C-14, were shown to connect the bridgehead carbons C-8 and C-12 and constituted a bridge ring, as supported by the HMBC correlations and the spin system of H-12/H2-13/H2-14 from the 1H−1H COSY E

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Figure 2. Conformations and key NOESY correlations of compounds 1, 3, 5, 7, and 9.

determined the structural conformation as depicted in Figure 2, with trans-fused A/B rings with H-5 adopting a β-axial position and C-20 adapting an α-axial position, while rings B and C share a C-8−C-9 unit and are cis-fused and H-9 and C15 are both β-oriented. Accordingly, C-19 was assigned as being α-axially oriented by the NOESY cross-peak of H2-19/ H3-20, and the configuration of C-16 bearing a hydroxymethyl group (CH2OH-17) was assigned as rel-16S, as supported by the NOESY couplings of H2-17/H-14β and H2-17/H-15a. However, it was not feasible to assign the configuration of the chiral carbon of the 2-methylbutyryloxy group because of its flexibility. The calculated ECD spectrum displayed similar Cotton effects to the experimental ECD curve (Figure 3E), allowing the definition of an absolute structure for compound 5 as (4R,5S,8S,9R,10S,13R,16S)-ent-16α,17-dihydroxy-19-(2ξmethylbutanoyloxy)kauran-3-one. Analysis of its 1D spectra indicated that compound 6 is a diterpenoid containing a tigloyloxy group [δC 168.0, 137.9, 128.2, 14.5, and 12.0; δH 6.80 (1H, q, J = 6.8 Hz), 1.78 (3H, d, J = 6.8 Hz), and 1.79 (3H, s)]. The NMR data of compound 6 exhibited similarities to those of compound 5, suggesting these two compounds share the same skeleton. The location at C-19 of the tigloylxoy group on the ent-kaurane scaffold was inferred from the HMBC long-range correlation of H2-19 to the carbon at δC 168.0. NOESY data analysis showed compound 6 to have the same relative configuration as 5. The structure of compound 6 was assigned as (4R,5S,8S,9R,10S,13R,16S)-ent16α,17-dihydroxy-19-tigloyloxykauran-3-one via comparison of the experimental and calculated ECD spectra (Figure 3F). Compound 7 showed a positive pseudomolecular ion peak [M + H]+ at m/z 359.2218 (calcd 359.2222) in the HRESIMS,

standpoint, as confirmed by analysis of their NOESY spectra. The calculated ECD spectrum of (4R,5R,8S,9R,10R,12S,16S)4 matched the experimental data closely (Figure 3D). Compound 4 was consequently elucidated as (4R,5R,8S,9R,10R,12S,16S)-ent-16α,17-dihydroxy-19-noratisan-3-one. Compound 5 was assigned the molecular formula C25H40O5 from its HRESIMS data. Its 13C NMR spectrum exhibited 25 carbons including a ketone carbonyl (δC 213.2) and an ester carbonyl (δC 176.6). This ester carbonyl (δC 176.6) and the carbon signals at δC 41.0, 26.6, 16.5, and 11.6, together with the proton signals at δH 2.34 (m), 1.34 (m), 1.58 (m), 0.87 (t, J = 7.5), and 1.10 (d, J = 6.8), revealed the presence of a 2methylbutyryloxy group. The remaining 20 carbons indicated a diterpenoid skeleton. Considering only one ketone carbonyl was observed and no olefinic carbons occurred in the 13C NMR spectrum, compound 5 was inferred as having four rings, according to the total index of hydrogen deficiency. On detailed comparison of the 13C NMR data with those of compound 3 (a tetracyclic ent-atisane diterpenoid) and tetracyclic ent-kaurane diterpenoids,23 an ent-kaurane-type diterpenoid scaffold was proposed for compound 5. Comprehensive analysis of 2D NMR spectroscopic results led to the establishment of the A/B/C/D rings, with the proton and carbon signals fully assigned. The HMBC crosspeak of H2-19 to the carbon δC 176.6 suggested the 2methylbutyryloxy group was linked to C-19. NOESY correlations of H3-18/H-5, H3-18/H-6β, H-5/H-9, H-9/H15b, H-9/H-1β, H2-17/H-14β, H2-17/H-15a, H-15a/H-14β, H2-19/H3-20, H2-19/H-2α, H2-19/H-6α, H3-20/H-1α, H320/H-2α, H3-20/H-12α, H3-20/H-14α, H-12α/H-14α, and H-7α/H-15a (Figure 2), together with Chem3D simulations, F

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Figure 3. Calculated and experimental ECD spectra of compounds 1−9 (A−I) in acetonitrile.

consistent with the molecular formula of C22H31O4. From the 1D NMR spectra, one acetyloxy group was evident (δH 2.12; δC 170.5, 21.9). The additional 20 resonances included four olefinic, two oxygenated carbons, and one ester carbonyl (Table 1). The ester carbonyl carbon signal at δC 175.3 and the two olefinic carbon signals at δC 156.3 and 116.8, together with the oxygenated carbon (δC 75.6), were extrapolated to constitute a five-membered unsaturated lactone ring bearing a methyl group at C-15, which was substantiated by the supportive 2D NMR experiments. In addition to this lactone ring, the 6/6/6 fused tricyclic system was established by examination of the 1H−1H COSY and HMBC data, where the carbon signals at δC 151.6, 114.5, and 80.4 were assigned as C8, C-14, and C-1, respectively. It was found that ring C and the lactone ring are fused in sharing C-12 and C-13, and an entabietane-type diterpenoid skeleton for compound 7 was thus elucidated. The HMBC long-range correlation from H-1 to the carbon δC 170.5 demonstrated that the aectyloxy group was linked to C-1. After defining the planar structure of compound 7, NOESY cross-peaks of H-12/H3-20, H3-19/H3-20, H3-20/ H-2α, H-2α/H3-19, H3-19/H-6α, H-3β/H3-18, H3-18/H-5, H1/H-5, H-1/H-9, H-5/H-9, H-9/H-7β, H-5/H-7β, and H-9/ H-11β disclosed the structural conformation as shown in Figure 2. Rings A and B are trans-fused and both exist in a normal chair conformation according to the molecular arrangement of compound 7, while H-1, H-5, and H-9 are βaxially oriented and Me-19, Me-20, and H-12 α-axially

oriented. The calculated ECD spectrum displayed similar Cotton effects with the experimental ECD curve (Figure 3G), allowing the definition of an absolute structure for compound 7 as (1S,5R,9R,10R,12R)-1α-acetoyloxy-ent-abieta-8(14),13(15)-dien-12α,l6-olide. Compound 8 gave the molecular formula C 20 H 28 O 4 according to the HRESIMS result. The NMR spectra of compound 8 were very similar to those of 7, except for the absence of signals for an acetyloxy group, suggesting both compounds to be close analogues. Comparison of 13C NMR data revealed an additional oxymethylene signal (δC 71.3) in 8, implying that Me-18 in compound 7 is replaced by this moiety. A NOESY experiment revealed compound 8 to have the same molecular conformation as that of 7, with the C-18 oxymethylene being β-equatorially oriented. The absolute configuration of 8 was assigned as 1S, 4S, 5R, 9R, 10S, and 12R by comparison of calculated and experimental ECD data (Figure 3H). Compound 9 gave a molecular formula of C19H30O3, as shown by the HRESIMS (m/z 307.2273 [M + H]+, calcd for C19H31O3, 307.2273). The 13C NMR spectrum exhibited 19 carbon signals, which included two oxymethines (δC 79.5 and 73.2), one oxygen-bearing tertiary carbon (δC 75.9), and four olefinic carbons [two olefinic methines (δC 128.4 and 146.0), one olefinic methylene (δC 113.7), and one olefinic quaternary carbon (δC 136.1)], associated with the DEPT and HMQC spectra. The additional 12 carbons comprised three methyls, G

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five methylenes, two methines, and two quaternary carbons (Table 1). These spectroscopic features were comparable to those of 3α,12α-dihydroxy-ent-8(14),15-isopimaradien-18-al (12),24 implying compound 9 to be an ent-isopimarane-type diterpenoid derivative. The main differences between 9 and 12 were that the formyl group present in 12 was not observed and one more oxygenated tertiary carbon (δC 75.9) appeared in compound 9, suggesting that the formyl group in 12 is replaced by a hydroxy group in 9. The above deductions were substantiated by combinational examination of the NMR and HRESIMS data, and compound 9 was determined as an entisopimarane-type 18-norditerpenoid. NOESY correlations of H3-19/H3-20, H3-19/H-6α, H3-20/H-1α, H3-20/H-2α, H320/H-6α, H3-20/H-11α, H3-20/H3-17, H-1β/H-3, H-3/H-5, H-5/H-9, H-5/H-7β, H-7β/H-9, H-9/H-12, and H-12/H-16a (Figure 3) revealed the trans-fusion of rings A and B, the βaxial orientations of H-3, H-5, and H-9, and the α-axial orientations of Me-17, Me-19, and Me-20. The calculated ECD spectrum displayed similar Cotton effects to the experimental ECD curve (Figure 3I), allowing the definition of an absolute structure for compound 9 as (3R,4R,5S,9R,10S,12S,13S)-ent18-nor-8(14),15-isopimaradiene-3β,12β,4α-triol. Other compounds obtained were identified as antiquorine A (10),25 sandaracopimaradienolal (11),26 3α,12α-dihydroxyent-8(14),15-isopimaradien-18-al (12),24 ent-13S-hydroxyatis16-ene-3,14-dione (13),27 ent-3β,13S-dihydroxyatis-16-en-14one (14),27 ent-3α,13S-dihydroxyatis-16-en-14-one (15),25 ent3β,19-dihydroxykaur-16-ene (16),28 eurifoloid D (17),16 eurifoloid E (18),16 7-angeloyl-12-acetyl-8-methoxyingol (19),29 3,7,12-triacetyl-8-benzoylingol (20),29 and 3,12diacetyl-7-hydroxy-8-methoxyingol (21).29 Studies on inflammation have suggested that compounds inhibiting NO production might be of use in treating neurodegenerative diseases.30 The effects of these isolates 1− 21 on LPS-stimulated NO release were thus measured through the Griess reaction in BV-2 cells.31,32 The isolates 1, 2, 5−7, 10, and 12 exhibited more potent nitric oxide inhibitory effects in BV-2 cells (Table 3).

free binding energies and the binding residues. The possible inhibitory mechanism on NO production is to bind to the active site of the iNOS protein as implied by the results of molecular docking.

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Table 3. IC50 Values of Compounds 1−21 Inhibiting NO Production in BV-2 Cells compound

IC50 (μM)a

compound

IC50 (μM)a

1 2 3 4 5 6 7 8 9 10 11

28.6 ± 0.5 14.5 ± 0.9 >50 >50 32.6 ± 2.1 19.3 ± 2.3 12.0 ± 0.4 >50 >50 38.4 ± 4.6 >50

12 13 14 15 16 17 18 19 20 21 SMTa

32.3 ± 0.8 >50 >50 >50 50.1 ± 3.7 >50 >50 >50 >50 >50 3.7 ± 0.0

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations and infrared (IR) spectra were determined on an IP120 automatic polarimeter (InsMark) and a Tensor 27 FT-IR instrument (Bruker), respectively. ECD spectra were taken with a J-715 CD spectropolarimeter (JASCO). 1D and 2D NMR spectra were recorded on a AV 400 spectrometer (Bruker) using tetramethylsilane as internal references. HRESIMS data were recorded on an IonSpec 7.0 T FTICR MS (IonSpec Co., Ltd., Lake Forest, CA, USA), while ESIMS data were acquired on a Thermo Finnigan LCQ-Advantage mass spectrometer. Preparative HPLC separations were conducted using a CXTH LC3000 system (Shodex RI-102 detector) equipped with a YMC-pack ODS-AM column (5 μm, 250 mm × 20 mm). Biological reagents and chemical reagents were purchased from Sigma Co. and Tianjin Chemical Reagent Co., respectively. Column chromatography was performed using silica gel (100−200 mesh) as stationary phases. The BV-2 cell line was provided by Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Plant Material. The stems of E. royleana (7.1 kg) were collected in Yunnan Province, People’s Republic of China, in March 2016. The plant sample was authenticated by Y.G., and the deposition of a voucher specimen (No. 20160301) was performed in the Laboratory of Bioactive Substances and Functions of Natural Medicines, College of Pharmacy, Nankai University. Extraction and Isolation. The powdered stems of E. royleana (7.1 kg) were extracted with methanol (3 × 35 L) by refluxing. The methanol extract was concentrated to form a residue (700 g) and then was suspended with H2O (1 L), and the resulting H2O layer was partitioned with ethyl acetate three times (each 1 L). The ethyl acetate-partitioned section (318 g) was separated through column chromatography over silica gel and eluted with a gradient solvent system of petroleum ether−acetone. The eluent was combined based on the TLC analysis to give fractions F1−F9. Fraction F4 was chromatographed by MPLC on octadecylsilyl silica gel (ODS) eluted with gradient mixtures of MeOH−H2O (65:35−93:7) to afford subfractions F4−1−F4−9. Subfraction F4−7 was purified by preparative HPLC using MeOH−H2O (84:16) to yield compounds 1 (tR = 46 min, 5.7 mg), 2 (tR = 48 min, 8.1 mg), and 11 (tR = 57 min, 15.4 mg). Compounds 7 (tR = 38 min, 9.6 mg) and 20 (tR = 31 min, 21.8 mg) were purified from subfraction F4−6 by preparative HPLC using MeOH−H2O (81:19). The purification of F4−3 (MeOH−H2O, 72:28) and F4−8 (MeOH−H2O, 90:10) gave compounds 16 (tR = 42 min, 10.7 mg) and 19 (tR = 44 min, 5.7 mg), respectively. Fraction F8 was purified by MPLC, eluted with 63−92% MeOH in H2O, to afford five subfractions, F8−1−F8−5, and compounds 3 (tR = 33 min, 15.4 mg), 8 (tR = 29 min, 34.0 mg), and 9 (tR = 40 min, 10.4 mg) were purified from subfraction F8−2 by preparative HPLC using 67% MeOH in H2O as eluent. Using the same method, F7 produced nine subfractions, F7−1−F7−9. Subfraction F7−2 was further purified using MeOH−H2O (68:32) as mobile phase to give compounds 4 (tR = 41 min, 8.0 mg), 12 (tR = 51 min, 6.9 mg), and 18 (tR = 38 min, 9.5 mg), while the purification of F7−4 (MeOH−H2O, 74:26) led to the isolation of compound 6 (tR = 48 min, 13.1 mg). Fractions F5 and F6 yielded subfractions F5−1−F5−6 and subfractions F6−1−F6−11, respectively. Based on the above-mentioned HPLC strategy, compound 5 (tR = 54 min, 8.3 mg) was further purified from F6−5 (MeOH−H2O, 74.5:25.5), compounds 10 (tR = 44 min, 8.1 mg) and 17 (tR = 40 min, 19.4 mg) were further purified from F5−2 (MeOH− H2O, 78:22), compounds 13 (tR = 29 min, 87.1 mg) and 15 (tR = 22 min, 25.4 mg) were separated from F5−1 (MeOH−H2O, 71:29), and compounds 14 (tR = 22 min, 45.7 mg) and 21 (tR = 32 min, 10.2 mg) were further purified from F6−2 (MeOH−H2O, 73:27). (3S,4S,5R,8S,10S,11R,13R,14R,15R)-3β-O-Angeloyl-17-tigloyloxy20-deoxyingenol (1): colorless oil; [α]20 D +19.5 (c 0.3, CH2Cl2); ECD

a

SMT (2-methyl-2-thiopseudourea, sulfate) was used as positive control. Data are presented based on three experiments.

iNOS is a key enzyme and regulates NO production in the inflammatory process.33,34 Compounds 1, 2, 5−7, 10, and 12 were subsequently subjected to molecular docking with the iNOS protein.33,35 The results obtained revealed that the iNOS protein had strong affinities with all of these diterpenoids (Figure 4). Table 4 gave the information on the logarithm of H

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Figure 4. Molecular docking simulations obtained at the lowest energy conformation, highlighting potential hydrogen contacts of compounds 1 (A), 2 (B), 5−7 (C−E), 10 (F), and 12 (G), respectively (colored by atom: carbon is cyan; nitrogen is blue; oxygen is red; hydrogen is gray; sulfur is orange). For clarity, only interacting residues are labeled. Hydrogen-bonding interactions are shown by dashes. These figures were created by PyMOL. (CH3CN) λmax (Δε) 204 (+2.22), 218 (+4.50) nm; IR (film) νmax 3475, 2955, 1706, 1650, 1305, 1262, 971, 780 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 513 [M + H]+; HRESIMS m/z 513.2851 [M + H]+ (calcd for C30H41O7, 513.2852). (3S,4S,5R,8S,10S,11R,13R,14R,15R)-3β-O-Angeloyl-17-benzoyloxy-20-deoxyingenol (2): colorless oil; [α]D20 +152.3 (c 0.1, CH2Cl2); ECD (CH3CN) λmax (Δε) 193 (+0.74), 202 (+2.29),

204 (+2.16), 220 (3.72) nm; IR (film) νmax 3484, 2925, 1715, 1229, 960, 712, 689 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 535 [M + H]+; HRESIMS m/z 535.2703 [M + H]+ (calcd for C32H39O7, 535.2696). (4R,5S,8S,9R,10S,12S,16S)-ent-19-Acetoyloxy-16α,17-dihydroxyatisan-3-one (3): colorless oil; [α]20 D −6.5 (c 0.3, CH2Cl2); ECD (CH3CN) λmax (Δε) 199 (−0.29), 212 (−0.65), 278 (+0.68) nm; IR I

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Table 4. Logarithms of Free Binding Energies (FBE, kcal/ mol) of NO Inhibitors to the Active Cavities of iNOS (PDB code: 3E6T) and Targeting Residues of the Binding Site Located on the Mobile Flap compound

− log(FBE)

1 2 5 6

−9.3 −9.8 −8.9 −8.5

7 10 12

−8.8 −9.1 −9.2

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00493. NMR, HRESIMS, and IR spectra of compounds 1−9 (PDF)

targeting residues ARG-260 ARG-382 GLN-257 H4B-902 TYR-341 TYR-367 ARG-260 GLN-257 TYR-341 TYR-367 GLN-257 GLN-381 ARG-375 ARG-375 ARG-382 TYR-341 TYR-367 GLN-257 GLN-381 TYR-341 TYR-341 TYR-367 ARG-382 ARG-260

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax (J. Xu): 86-22-23507760. E-mail: xujing611@nankai. edu.cn. *Tel/Fax (Y. Guo): 86-22-23502595. E-mail: victgyq@nankai. edu.cn. ORCID

−1

Dongho Lee: 0000-0003-4379-814X Jing Xu: 0000-0003-0847-4510 Yuanqiang Guo: 0000-0002-5297-0223

(film) νmax 3392, 2927, 1704, 1386, 1174, 1080, 734 cm ; for H and 13 C NMR data, see Tables 1 and 2; ESIMS m/z 379 [M + H]+; HRESIMS m/z 379.2492 [M + H]+ (calcd for C22H35O5, 379.2484). (4R,5R,8S,9R,10R,12S,16S)-ent-16α,17-Dihydroxy-19-noratisan3-one (4): colorless oil; [α]20 D +98.0 (c 0.2, CH2Cl2); ECD (CH3CN) λmax (Δε) 218 (−0.04), 296 (−0.54) nm; IR (film) νmax 3399, 2925, 1702, 1451, 1138, 734 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 307 [M + H]+; HRESIMS m/z 307.2273 [M + H]+ (calcd for C19H31O3, 307.2273). (4R,5S,8S,9R,10S,13R,16S)-ent-16α,17-Dihydroxy-19-(2ξmethylbutanoyloxy)kauran-3-one (5): white powder; [α]20 D −5.8 (c 0.2, CH2Cl2); ECD (CH3CN) λmax (Δε) 224 (−0.31), 253 (−0.01), 294 (−0.18) nm; IR (film) νmax 3435, 2928, 1730, 1459, 1147, 1013, 734 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 421 [M + H]+; HRESIMS m/z 421.2965 [M + H]+ (calcd for C25H41O5, 421.2954). (4R,5S,8S,9R,10S,13R,16S)-ent-16α,17-Dihydroxy-19-tigloyloxykauran-3-one (6): white needles (MeOH); [α]20 D +10.0 (c 0.3, CH2Cl2); ECD (CH3CN) λmax (Δε) 211 (−0.35), 237 (+0.29), 298 (−0.32) nm; IR (film) νmax 3410, 2926, 1706, 1264, 701 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 419 [M + H]+; HRESIMS m/z 419.2816 [M + H]+ (calcd for C25H39O5, 419.2797). (1S,5R,9R,10R,12R)-1α-Acetoyloxy-ent-abieta-8(14),13(15)-dien12α,l6-olide (7): colorless oil; [α]20 D +71.7 (c 0.3, CH2Cl2); ECD (CH3CN) λmax (Δε) 209 (−4.42), 238 (−4.07), 278 (+10.16) nm; IR (film) νmax 2927, 1666, 1239, 1018, 970, 765, 647 cm−1; for 1H and 13 C NMR data, see Tables 1 and 2; ESIMS m/z 359 [M + H]+; HRESIMS m/z 359.2218 [M + H]+ (calcd for C22H31O4, 359.2222). (1S,4S,5R,9R,10S,12R)-1α,18-Dihydroxy-ent-abieta-8(14),13(15)dien-12α,l6-olide (8): colorless oil; [α]20 D +160.0 (c 0.3, CH2Cl2); ECD (CH3CN) λmax (Δε) 213 (−6.03), 238 (−6.99), 278 (+19.63) nm; IR (film) νmax 3411, 2926, 1720, 1660, 1264, 1019, 733 cm−1; for 1 H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 333 [M + H]+; HRESIMS m/z 333.2079 [M + H]+ (calcd for C20H29O4, 333.2066). (3R,4R,5S,9R,10S,12S,13S)-ent-18-nor-8(14),15-Isopimaradiene3β,12β,4α-triol (9): colorless oil; [α]20 D +108.9 (c 0.2, CH2Cl2); ECD (CH3CN) λmax (Δε) 197 (+1.64), 231 (−0.23), 286 (+0.15) nm; IR (film) νmax 3392, 2925, 1706, 1450, 1377, 1243, 1000, 735 cm−1; for 1 H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 307 [M + H]+; HRESIMS m/z 307.2273 [M + H]+ (calcd for C19H31O3, 307.2273). Computational Methods. The calculations for ECD spectra were conducted as published previously.15 Bioassay for Nitric Oxide Inhibitory Effects. The bioassay experiment was carried out according to the previous method.15 Molecular Docking Research. The molecular docking was conducted as published previously.15 1

Author Contributions ¶

P. Wang and C. Xie contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (Nos. U1703107, 21642016, and 21372125), the National Key Research and Development Program of China (No. 2018YFA0507204), the Hundred Young Academic Leaders Program of Nankai University, the Natural Science Foundation of Tianjin, China (No. 16JCYBJC27700), and the Fundamental Research Funds for the Central Universities.

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Journal of Natural Products

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DOI: 10.1021/acs.jnatprod.8b00493 J. Nat. Prod. XXXX, XXX, XXX−XXX