Artemisia freyniana

Article Cite This: J. Nat. Prod. 2018, 81, 866−878

pubs.acs.org/jnp

Nitric Oxide Inhibitory Sesquiterpenoids and Its Dimers from Artemisia f reyniana Chen Zhang,† Ran Wen,† Xiao-Li Ma,† Ke-Wu Zeng,† Yang Xue,† Pu-Ming Zhang,† Ming-Bo Zhao,† Yong Jiang,† Guo-Qing Liu,*,‡ and Peng-Fei Tu*,† †

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People’s Republic of China ‡ Institute of Cardiovascular Sciences and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Peking University, Beijing 100191, People’s Republic of China S Supporting Information *

ABSTRACT: Two new disesquiterpenoids (1 and 2) and 11 new (3−13) and 10 known (14−23) sesquiterpenoids were isolated from the whole plants of Artemisia f reyniana. Their structures were elucidated by spectroscopic data analysis and comparison with published NMR data. The absolute configurations of the new isolates (1−13) were assigned based on singlecrystal X-ray diffraction data and comparison of the experimental and calculated ECD data. The eremophilane derivatives 8 and 9 possess an unprecedented 2-isopropyl-3,7,7a-trimethyl-2,4,5,6,7,7a-hexahydro-1H-indene scaffold, and a putative biosynthetic pathway for these compounds is proposed. Compounds 4, 5, and 9 exhibited inhibitory effects against LPS-stimulated nitric oxide (NO) production in RAW 264.7 macrophage cells with IC50 values of 10.8, 12.6, and 11.7 μM, respectively.

T

assignments of the new compounds and their nitric oxide (NO) inhibitory effects are described.

he eremophilane class of sesquiterpenoids has been found from numerous medicinal plants, endophytes, and marinederived fungi.1 They share the same decalin core with the eudesmanes, except for the presence of a methyl group at C-5 rather than at C-10.2 The genera Ligularia and Senecio contain a rich array of eremophilanes with structural diversity and significant biological properties, such as cytotoxic and antibacterial properties.3 Recently, the number of eremophilanes derived from endophytic and marine fungi has increased significantly, with some displaying cytotoxic,4 anti-inflammatory,5 and antifungal activities.6 Our research into the natural products from Artemisia species led to the identification of new sesquiterpenoids and their dimers.7 Only a few eremophilane sesquiterpenoids have been reported from Artemisia species, and the dimeric compounds were speculated to be formed by free-radical formation of the C−C bond linkage.8 Guided by LC-MS/MS screening, a phytochemical examination of a 95% aq. EtOH extract of A. f reyniana afforded 13 new isolates (1− 13) including two disesquiterpenoids (1 and 2), seven eremophilanes (3−9), and four eudesmanes (10−13). Herein, the isolation, structural elucidation, and absolute configuration © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The 95% aq. EtOH extract of the whole plants of A. f reyniana was dissolved in H2O and partitioned sequentially with petroleum ether and CHCl3. LC-DAD-MS/MS analyses indicated that the CHCl3 portion is rich in sesquiterpenoid content and is accompanied by the dimeric analogues in trace amounts. Subsequent separation and purification guided by LCMS were carried out. The CHCl3 portion was repeatedly subjected to silica gel, ODS, and Sephadex LH-20 column chromatography (CC) and semipreparative C18-HPLC to yield two new dimers, 1 and 2, and 11 new sesquiterpenoids, 3−13, together with 10 known compounds (14−23), including 8αhydroxyeudesma-4,11(13)-dien-12-oic acid (14),9 8α-acetoxyeudesma-4,11(13)-dien-12-oic acid (15),9 debneyol (16),10 eremophil-8-oxo-9,11(7)-dien-12-oic acid (17),11 eremophilReceived: November 9, 2017 Published: March 8, 2018 866

DOI: 10.1021/acs.jnatprod.7b00947 J. Nat. Prod. 2018, 81, 866−878

Journal of Natural Products

Article

Chart 1

9,11(13)-dien-12,8-olide (18),9 eremophil-3-hydroxy-9,11(13)dien-12-oic acid (19),12 eremophil-9,11(13)-dien-12-oic acid (20),9 eudesma-4-en-12,8-olide (21),13 3-oxocostic acid (22),14 and 5-oxojasoniolide (23)15 (see Chart 1). Artefreynisin A (1) was obtained as a white amorphous powder, and its molecular formula was assigned as C30H40O5 based on the protonated ion [M + H]+ detected at m/z 481.2955 (calcd 481.2954) in the HRESIMS and its 13C NMR data. The IR spectrum indicated the presence of hydroxy (3444 cm−1) and lactone (1745, 1018 cm−1) groups. A total of 30 carbon resonances were detected in the 13C NMR spectrum and five methyls, eight methylenes, an sp2 and seven sp3 methines, an oxygenated tertiary carbon, three sp2 and three sp3 quaternary carbons, and two carbonyl carbons were classified by the HSQC spectrum. On the basis of their chemical shifts, two carbonyls (δC 180.9 and 183.6), four olefinic carbons (δC 114.2, 141.2, 156.8, and 158.2), and three oxygenated carbons (δC 72.6, 76.5, and 84.7) are present. The corresponding proton signals were observed in the 1H NMR spectrum including an olefinic proton at δH 5.55 (1H, d, J = 5.2 Hz), oxygenated methines at δH 4.97 (1H, t, J = 5.2 Hz), and 5.12 (1H, d, J = 9.7 Hz) and five methyls at δH 0.84 (3H, d, J = 6.4 Hz), 0.94 (3H, s), 1.17 (3H, d, J = 6.9 Hz), 1.42 (3H, s), and 1.52 (3H, s). Overlapping signals at around δH 1.71−2.48 were ascribed to methylene resonances (Table 1). These 1D NMR data and the 11 indices of hydrogen deficiency indicated that 1 is a disesquiterpenoid.16 Further analyses were conducted via 2D NMR data. The two methyls [δH 0.84 (3H, d, J = 6.4 Hz) and δH 0.94 (3H, s)] both show HMBC correlations with C-4 and C-5, indicating that they are located at adjacent carbons. HMBC correlations of H3-

15/C-3, H3-14/C-6 and C10, H-8/C-6, C-10, and C-12, and H9/C-1 and C-5 and COSY correlations of H2-1/H2-2/H2-3/H4/H3-15 and H2-6/H-7/H-8/H-9 (Figure 1) suggested the presence of an eremophil-9-en-12,8-olide moiety. 9 The remaining NMR signals indicated the presence of a 10hydroxyguaia-1(5)-en-12,6-olide moiety,17 which was also supported by the HMBC correlations of H3-14′/C-1′, C-9′, and C-10′; H3-15′/C-3′, C-4′, and C-5′; H3-13′/C-7′, C-11′, and C-12′; and H-6′/C-1′, C-8′, and C-12′; and the COSY correlations of H-6′/H-7′/H2-8′/H2-9′ and H-7′/H-11′/H313′ (Figure 1). The remaining indices of hydrogen deficiency indicated the presence of an additional ring. The HMBC correlations of H3-15′/C-11, H-2′/C-13, and H2-3′/C-11 and C-13 and the H2-13/H-2′/H2-3′ proton spin system suggested that the two sesquiterpenoid units were connected via C-11−C4′ and C-13−C-2′, probably formed by a symmetry-allowed [π4s + π2s] Diels−Alder cycloaddition. The A-ring of the eremophilane unit adopted a chair conformation with a β-axial Me-14 and β-equatorial Me-15, as shown by the NOEs of H3-14/H-1β, H-3β, H-6β, and H3-15 and H3-15/H-6α (Figure 2). A large coupling constant between H-7 and H-6β (J = 14.5 Hz) revealed that H-7 was α-oriented. H-8 coupled with H-7 and H-9 in a triplet pattern (J = 5.2 Hz) suggested its syn-orientation with H-7. In the guaiane unit, the NOEs of H-6′/H-8′β/H-11′ indicated that they are cofacial and that H-7′ is α-oriented based on its large coupling constants with H-6′ (J = 9.7 Hz) and H-11′ (J = 12.3 Hz). The key NOEs of H-7/H-6′ and H-8/H3-15′ revealed that the dienophile (eremophilane unit) approached the diene (guaiane unit) from the β-face (Figure 2). Thus, the α-orientation of H2′ and H3-15′ were necessitated according to the orbital 867



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Table 1. 1H and 13C NMR Data of Compounds 1 and 2 (δ in ppm, J in Hz, 600 MHz for 1H and 150 MHz for 13C, in Methanold4) artefreynisin A (1) position 1α 1β 2α 2β 3α 3β 4 5 6α 6β 7 8α 8β 9 10 11 12 13

δC, type 32.9, CH2 30.9, CH2 31.8, CH2 40.0, CH 39.9, C 35.4, CH2 38.8, CH 76.5. CH 114.2, CH 158.2, C 61.6, C 183.6, C 37.7, CH2

14 15 1′α 1′β 2′α 2′β 3′a/3′α 3′b/3′β 4′ 5′ 6′α 6′β 7′ 8′α 8′β 9′α 9′β 10′ 11′ 12′ 13′

20.2, CH3 15.9, CH3 156.8, C

72.6, C 42.9, CH 180.9, C 12.9, CH3

14′ 15′

28.5, CH3 18.7, CH3

artefreynisin B (2)

δH (J in Hz)

δC, type

2.04, 2.36, 1.32, 1.91, 1.50, 1.50, 1.56,

d (12.7) dd (12.7, 4.6) d (4.9) m m m m

1.89, 0.82, 2.26, 4.97,

dd (14.3, 5.2) dd (14.5, 14.3) dt (14.5, 5.2) t (5.2)

5.55, d (5.2)

2.17, 1.90, 0.94, 0.84,

dd (12.0, 3.6) m s d (6.4)

35.5, CH2 23.3, CH2 30.3, CH2 32.6, CH 42.5, C 39.5, CH2 29.5, CH 33.5. CH2 80.6, CH 73.4, C 147.1, C 170.2, C 122.9, CH2 16.4, CH3 16.2, CH3 34.1, CH2

43.8, CH

3.01, br s

27.3, CH2

53.5, CH2

1.94, m 1.18, m

31.4, CH2

65.4, C 141.2, C 84.7, CH 51.8, CH 26.0, CH2 40.6, CH2

44.8, CH 41.2, C 44.1, CH2

5.12, d (9.7) 1.98, 1.97, 1.78, 1.79, 1.79,

m m m m m

48.3, CH 200.3, C 124.1, CH

1.17, d (6.9)

174.1, 142.1, 167.6, 128.8,

1.42, s 1.52, s

16.4, CH3 15.5, CH3

2.40, dq (12.3, 6.9)

C C C CH2

δH (J in Hz) 1.64, 1.78, 1.63, 1.46, 1.33, 1.42, 2.40,

m m m m m m m

1.77, 1.25, 2.88, 1.57, 1.95, 5.16,

m m t (12.8) dd (13.5, 3.0) ddd (13.5, 12.8, 3.0) t (3.0)

6.15, 5.51, 0.87, 0.90, 2.29, 2.41, 1.87, 1.55, 1.55, 1.55, 1.54,

br s br s s d (6.8) dd (13.3) m m m m m m

2.22, t (13.7) 2.07, dd (13.7, 4.7) 3.64, dd (13.7, 4.7)

5.68, d (1.1)

6.29, 5.73, 1.23, 0.93,

br s br s s d (6.0)

Figure 1. Key 1H−1H COSY and HMBC correlations of 1.

shielding of C-8′ (ΔδC −7.7) compared to artepestrin D7e due to the γ-gauche effect18,19 derived from the β-oriented 10-OH and the 13C NMR shift of Me-14′ (δC 28.5) in comparison with

symmetry rules and C-3′ extends below the plane of the cyclohexene moiety. An α-equatorial Me-14′ was deduced based on its NOE correlation with H-2′ and supported by the 868



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Figure 2. Key NOESY correlations of 1.

those of anabsinthin (δC 29.3) and absinthin E (δC 22.5).7c Furthermore, by comparison of the experimental and calculated ECD data, the absolute configuration of 1 was determined. The calculated ECD spectrum of (4S,5R,7R,8R,11S,2′S,4′S,6′S,7′S,10′S,11′S)-1 agreed well with the experimental curve, while its enantiomer showed mirror-like Cotton effects (Figure 3). Therefore, the structure of artefreynisin A (1) was defined as shown.

NMR data,20,21 the two sesquiterpenoid units were deduced to be 9,10-dihydroxyeremophil-11(13)-en-12-oic acid and eremophil-8-oxo-9,11(13)-dien-12-oic acid. This deduction was supported by the HMBC correlations of H3-14/C-4, C-5, C6, and C-10; H3-15/C-3, C-4, and C-5; H-9/C-1, C-5, and C-7; H2-13/C-7, C-11, and C-12; H3-14′/C-4′, C-5′, C-6′, and C10′; H3-15′/C-3′, C-4′, and C-5′; H-9′/C-1′, C-5′, and C-7′; and H2-13′/C-7′, C-11′, and C-12′ and the COSY correlations as shown in bold in Figure S1 (Supporting Information). The shielding of C-12′ (δC 167.6, Δδ = −2.6) and the HMBC correlation of H-9/C-12′ implied that the eremophilane units were connected via the C-9−C-12′ ester linkage. The NOESY correlations of H3-14/H-1β, H-3β, H-6β, and H3-15 and H-6β/ H-8β indicated chair conformations for both rings A and B (Figure S2, Supporting Information). Their cis-fused pattern was deduced by the NOEs of H-4/H-2α and H-7, and thus, 10OH was assigned as β-oriented. The H-9β-equatorial orientation was assigned based on its coupling constants (J = 3.0 Hz) with H2-8. The β-orientations of H3-14′ and H3-15′ were deduced by the NOEs of H3-14′/H-1′β, H-3′β, and H315′. H-7′ was axial and β-oriented, as deduced from its NOE with H3-14′ and large coupling constant with H-6′α (J = 13.7 Hz). The positive Cotton effect at 242 nm arising from the π → π* electronic transition of the trans-enone unit defined the (4′S,5′R,7′S) absolute configurations (Figure S3, Supporting Information) based on the Kirk helicity rule.22 The absolute configuration of 2 was defined as (4S,5R,7S,9R,10R,4′S,5′R,7′S) by comparison of the experimental and calculated ECD data (Figure S3, Supporting Information). Therefore, the structure of artefreynisin B (2) was defined as shown. Artefreynic acid A (3) was obtained as a yellowish oil. Its molecular formula was determined as C15H20O3 by the deprotonated ion [M − H]− detected at m/z 247.1338 (calcd for 247.1334) in the HRESIMS and its 13C NMR data. The IR spectrum showed absorption bands for conjugated carboxylic functionalities (3737−3444, 1681, and 1623 cm−1).23 Both the 1H and 13C NMR data of 3 were similar to those of dehydroflourensic acid,23 an eremophil-9-oxo1(10)-11(13)-dien-12-oic acid sesquiterpenoid. The structural difference involved the position of the cis-enone unit by analyses of the HMBC data. HMBC correlations of H-7/C-9 and H3-14/C-10 assigned the Δ9(10) double bond and the carbonyl group was assigned to be at C-1 (δC 208.5) based on the HMBC correlation between H-9 and C-1. The 2D structure of 3 was thus defined as eremophil-1-oxo-9,11(13)-dien-12-oic

Figure 3. Comparison between calculated ECD spectra (4S,5R,7R,8R,11S,2′S,4′S,6′S,7′S,10′S,11′S)-1 (red line) and its enantiomer (blue line) with the experimental spectrum (black line) of 1.

Artefreynisin B (2) was assigned a molecular formula of C30H42O6 based on the deprotonated ion [M − H]− detected at m/z 497.2897 (calcd 497.2903) in the HRESIMS and its 13C NMR data. Two sets of characteristic eremophilane signals were observed in the 1H and 13C NMR data (Table 1), showing the presence of two α-substituted acrylic acid moieties [δH 5.51 (1H, br s), 6.15 (1H, br s), δC 122.9, 147.1, and 170.2; δH 5.73 (1H, br s), 6.29 (1H, br s), δC 128.8, 142.1, and 167.6)] and four methyl groups [δH 0.87 (3H, s), 0.90 (3H, d, J = 6.8 Hz), 1.23 (3H, s), and 0.93 (3H, d, J = 6.0 Hz); δC 16.2, 16.4, 15.5, and 16.4], indicating that 2 is a disesquiterpenoid. Two oxygenated carbons (δC 73.4 and 80.6) and an α,β-unsaturated carbonyl group (δC 124.1, 174.1, and 200.3) were assigned based on their chemical shifts. By comparison with published 869



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Figure 4. Kirk helicity rule applied for the cis-enone of 3 accounting for the positive Cotton effect at 234 nm and negative one at 310 nm.

Table 2. 13C NMR Data of Compounds 3−13 (δ in ppm, J in Hz, 125 MHz, in Methanol-d4 and DMSO-d6) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ethoxy

3

4

5

6

7

8a

9

10

11

12

13

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

208.5, C 41.1, CH2 30.1, CH2 38.3, CH 42.1, C 41.4, CH2 33.4, CH 31.8. CH2 129.8, CH 149.3, C 146.5, C 170.6, C 123.3, CH2 21.1, CH3 15.1, CH3

36.6, CH2 24.1, CH2 30.4, CH2 41.7, CH 41.9, C 43.8, CH2 37.4, CH 38.5. CH2 75.5, CH 90.1, C 145.9, C 170.4, C 127.2, CH2 18.3, CH3 16.4, CH3

39.5, CH2 24.9, CH2 30.0, CH2 39.1, CH 54.0, C 41.5, CH2 36.5, CH 42.4. CH2 174.0, C 218.2, C 145.2, C 170.1, C 126.8, CH2 20.3, CH3 16.1, CH3 61.4, CH2 14.5, CH3

76.4, CH 36.1, CH2 27.6, CH2 40.5, CH 40.1, C 42.8, CH2 33.4, CH 32.7. CH2 123.9, CH 147.6, C 146.5, C 170.6, C 122.6, CH2 23.4, CH3 16.1, CH3

124.2, CH 26.2, CH2 28.1, CH2 35.8, CH 39.1, C 38.8, CH2 43.5, CH 73.2, CH 39.9, CH2 141.7, C 145.1, C 170.9, C 124.6, CH2 21.1, CH3 16.5, CH3

23.0, CH2 26.5, CH2 29.4, CH2 44.3, CH 51.9, C 44.2, CH2 41.8, CH 133.6, C 188.2, CH 173.9, C 144.1, C 167.9, C 122.3, CH2 18.5, CH3 16.2, CH3

25.8, CH2 27.9, CH2 31.0, CH2 46.2, CH 53.1, C 45.9, CH2 46.0, CH 125.7, C 169.5, C 167.9, C 144.1, C 170.5, C 123.1, CH2 19.5, CH3 16.6, CH3

35.4, CH2 18.5, CH2 36.8, CH2 75.5, C 76.3, C 33.3, CH2 42.9, CH 73.4, CH 43.5, CH2 39.9, C 143.8, C 170.2, C 126.6, CH2 23.3, CH3 25.9, CH3




21.1, CH3 172.5, C

20.9, CH3 172.4, C

21.0, CH3 172.5, C

acetyl methoxy a

52.2, CH3

Recorded in DMSO-d6. 1

H and 13C NMR data (Tables 2 and 3) suggested that the structure of 4 is similar to that of eremophil-9(10)-epoxy-12-oic acid. 21 The difference is that the −CH(CO 2 H)−CH 3 functionality at C-7 is replaced by an acrylic acid moiety in 4. This deduction was supported by the HMBC correlations from H2-13 to C-7, C-11, and C-12. Thus, the 2D structure of 4 was determined to be eremophil-9(10)-epoxy-11(13)-en-12-oic acid. The cis-fused pattern of 4 was deduced from the close proximity of H-4 and H-7 (Figure S2, Supporting Information). The H-9α-equatorial orientation was deduced from its coupling constant (J = 3.5 Hz) with H-8β. The ECD spectrum of 4 showed a negative Cotton effect at 232 nm (Δε −3.5), which suggested a (7S) absolute configuration.24 The remaining stereocenters were thus deduced to be (4S,5R,9S,10R)configured based on the established relative configuration. This result was corroborated by comparison of the calculated and experimental ECD spectra (Figure S5, Supporting

acid. The NOE correlations of H3-14/H3-15, H-3β, and H-6β, and H-6β/H-8β indicated that A-ring adopts a chair conformation, and Me-14 and Me-15 are β-oriented. On the basis of the NOE association of H-7/H-4 and the large coupling constants of H-7/H-6β and H-8β (J = 11.1 Hz), H-7 was assigned an α-axial orientation. The ECD Cotton effects at 234 nm (Δε + 6.8) and 310 nm (Δε −1.7), mainly arising from the π → π* electronic transition of the cis-enone unit, defined the (4S,5R,7R) absolute configuration according to Kirk helicity rule22 (Figure 4). This deduction was confirmed by comparison of the experimental and calculated ECD data (Figure S4, Supporting Information). Therefore, the structure of artefreynic acid A (3) was defined as (4S,5R,7R)-eremophil-1-oxo9,11(13)-dien-12-oic acid. Artefreynic acid B (4) was obtained as a colorless oil with a molecular formula of C15H22O3, as determined from the protonated ion [M + H]+ detected at m/z 251.1648 (calcd 251.1647) in the HRESIMS and from the 13C NMR data. The 870



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Table 3. 1H NMR Data of Compounds 3−8 (δ in ppm, J in Hz, 500 MHz, in Methanol-d4 and DMSO-d6) 3 δH (J in Hz)

position 1α 1β 2α 2β 3α 3β 4 6α 6β 7 8α 8β 9α 9β 13 14 15 ethoxy a

4 δH (J in Hz)

1.89, 1.77, 2.15, 2.11, 1.25, 2.79, 2.32, 2.04, 6.15,

m m m m dd (13.6, 11.1) t (11.1) m ddd (17.6, 11.1, 2.4) dd (6.3, 2.4)

1.95, m 1.97, m 2.06, tt (11.9, 5.5) 1.58, m 1.37, m 1.31, td (12.7, 4.3) 2.23, m 1.66, dt (13.5, 3.5) 1.99, m 2.89, m 2.21, m 1.86, dd (14.7, 5.9, 3.5) 4.12, dd (9.0, 3.5)

6.18, 5.55, 0.90, 0.96,

br s br s s d (6.7)

6.12, 5.59, 0.92, 0.87,

2.43, m

5

br s br s s d (6.8)

6

δH (J in Hz)

8a

7

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

2.36, dd (7.7, 6.0)

4.23, t (2.9)

5.43, p (1.9)

1.71, m 1.90, m 1.83, m 1.57, ddd (13.3, 9.0, 3.8) 2.02, m 1.97, dd (14.8, 7.5) 1.86, dd (14.8, 4.6) 3.03, dd (12.9, 7.5) 2.53, m 2.55, m

1.56, 1.87, 1.38, 1.83, 1.71, 1.98, 1.14, 2.65, 2.08, 1.95, 5.65,

m m d (12.9) m m m dd (12.7, 11.8) t (11.8) d (13.6) dd (13.6, 11.8) dd (6.5, 1.8)

2.11, m 2.00, ddt (7.5, 4.6, 2.9) 1.51, m 1.51, m 1.71, m 1.61, dd (13.8, 2.9) 1.26, dd (13.8, 12.1) 2.34, ddd (12.1, 7.0, 2.9)

6.17, 5.62, 0.98, 0.88, 4.04, 1.19,

6.11, 5.49, 1.11, 0.85,

br s br s s d (6.6)

br s br s s d (6.8) q (7.1) t (7.1)

4.03, 1.93, 2.70, 6.21, 5.64, 0.88, 0.86,

ddd (8.1, 7.0, 1.1) dd (14.3, 1.1) m br s br s s d (6.7)

3.25, 2.18, 1.34, 1.89, 1.41, 1.34, 1.39, 1.98, 1.48, 3.76,

d (14.2) td (14.2, 5.4) m m m m m dd (13.4, 10.6) dd (13.4, 2.5) dd (10.6, 2.5)

9.96, br s 5.96, 5.15, 0.94, 0.81,

s s s d (5.9)

Recorded in DMSO-d6.

Figure 5. Octant rule applied for 5 accounting for the positive Cotton effect at around 300 nm.

carbonyl groups at C-9 and C-10, respectively, and 5 was thus elucidated as a 9,10-seco-eremophilane. This deduction was further corroborated by the HMBC correlations of H3-15/C-3, C-4, and C-5; H3-14/C-4, C-5, C-6, and C-10; H2-1/C-10; H28/C-9; and H2-6/C-8 and COSY correlations of H2-1/H2-2/ H2-3/H-4/H3-15 and H2-6/H-7/H2-8, as shown in Figure S1 (Supporting Information). An ethyl group was connected to C9 via an ester linkage, as proved by the HMBC correlation from the ethoxy protons to the carbonyl carbon. Thus, the 2D structure of 5 was defined as 9-ethoxy-9,10-seco-eremophil-9,10dioxo-11(13)-en-12-oic acid. The chair conformation of ring A was deduced from the NOE correlations of H3-14/H-1β, H-3β, and H3-15 and H-4/H-2α (Figure S2, Supporting Information). The positive Cotton effect at 300 nm in the ECD spectrum was ascribed to the n → π* electronic transition of the cyclohexanone unit, which permitted assignment of the (5R) absolute configuration based on the octant rule25 (Figure 5),

Information). Therefore, the structure of artefreynic acid B (4) was determined as (4S,5R,7S,9S,10R)-eremophil-9(10)-epoxy11(13)-en-12-oic acid. Artefreynic acid C (5), a colorless gum, had a molecular formula of C17H26O5, as determined from the deprotonated ion [M − H]− detected at m/z 309.1704 (calcd 309.1702) in the HRESIMS and its 13C NMR data. The 1H and 13C NMR data (Tables 2 and 3) exhibited signals for an α-substituted acrylic acid moiety [δH 5.62 (1H, br s), 6.17 (1H, br s); δC 126.8, 145.2, and 170.1], an ethoxy group [δH 4.04 (2H, q, J = 7.1 Hz), 1.19 (3H, t, J = 7.1 Hz); δC 61.4 and 14.5], a carbonyl group (δC 218.2), and an ester (δC 174.0) moiety. Two methyl groups [δH 0.98 (3H, s) and 0.88 (3H, d, J = 6.8 Hz)] with eremophilane-type features were evident in the 1H NMR data. H-7 was assigned as the proton at δH 3.03 based on the HMBC correlations from H2-13 to C-7, C-11, and C-12. The HMBC correlations of H-7/C-9 and H3-14/C-10 assigned the ester and 871



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Figure 6. Comparison of the experimental ECD spectra between 8 (black line) and 9 (red line), and the X-ray single crystal structure of 8 drawing in ORTEP.

The large coupling constant of H-7/H-6β (J = 12.1 Hz) suggested that H-7 was α-oriented, and thus, H-4 was αoriented based on its NOEs with H-7 and H-2α. These NOE correlations indicated a twist-boat conformation of ring B as shown in Figure S2 (Supporting Information). In contrast to the ECD spectrum of 6, a positive Cotton effect at 205 nm was observed in the spectrum of 7. The absolute configuration of 7 was defined as (4S,5R,7R,8S) by comparison of the experimental and calculated ECD data (Figure S8, Supporting Information). The structure of artefreynic acid E (7) was thus defined as (4S,5R,7R,8S)-8-hydroxyeremophil-1(10)-11(13)dien-12-oic acid. Artefreynic acid F (8) was obtained as colorless crystals and was deduced to have a molecular formula of C15H20O3 based on a deprotonated ion [M − H]− at m/z 247.1333 (calcd 247.1334) in the HRESIMS and its 13C NMR data. The IR spectrum indicated the presence of hydroxy (3289 cm−1) and conjugated carbonyl (1689, 1649, 1621 cm−1) groups. The 13C NMR spectrum coupled with the HSQC correlations showed the presence of two methyls, an sp2 and four sp3 methylenes, two methines, an sp3 and three sp2 quaternary carbons, and two carbonyl carbons. Combining these with the 1H NMR data, the resonances for an α-substituted acrylic acid [δH 5.15 (1H, s), 5.96 (1H, s); δC 122.3, 144.1, and 167.9], a double bond (δC 133.6 and 173.9), and a formyl group [δH 9.96 (br s); δC 188.2] were assigned. These 1D NMR data were similar to those of eremophilane derivatives, differing only in the formyl signals. HMBC correlations from H-7 to C-8, C-9, and C-10; H2-1 to C-8; and H3-14 to C-10 and the COSY correlations (Figure S1, Supporting Information) suggested that the B-ring of 8 is a fivemembered ring carrying a formyl residue at C-8 and a Δ8(10) double bond. Thus, the 2D structure of 8 was defined as 2-(3formyl-7,7a-dimethyl-2,4,5,6,7,7a-hexahydro-1H-inden-2-yl)acrylic acid. The NOE correlations of H3-14/H-1β, H-3β, H315, and H-6β, and H-4/H-2α and H-6α revealed a chair conformation for ring A (Figure S2, Supporting Information). The coupling constant (J = 2.5 Hz) between H-7 and H-6β indicated that the dihedral angle between these protons was near 90°, thus indicating an α-orientation of H-7. A crystal of 8 was obtained from a MeOH−H2O (9:1) solution, and the absolute configuration of 8 (Figure 6) was unambiguously defined as (4S,5R,7S) by single-crystal X-ray diffraction analysis. The HRESIMS data of artefreynic acid G (9) showed a deprotonated ion [M − H]− at m/z 263.1282 (calcd 263.1283), corresponding to a molecular formula of C15H20O4, which is 16 mass units more than that of 8. Comparison of the 1H and 13C NMR data with those of 8 revealed that the formyl group of 8 was replaced by a hydroxycarbonyl group (δC 169.5) in 9. The

and thus, the (4S) absolute configuration was determined based on the NOE correlations. By comparison of the calculated and experimental ECD data (Figure S6, Supporting Information), the positive Cotton effect at 218 nm, arising from the π → π* electronic transition of the α,β-unsaturated carboxylic acid group, defined the (7S) absolute configuration. Therefore, the structure of artefreynic acid C (5) was defined as (4S,5R,7S)-9ethoxy-9(10)-seco-eremophil-9(10)-dioxo-11(13)-en-12-oic acid. Artefreynic acid D (6) was isolated as a colorless gum. Its HRESIMS spectrum showed a deprotonated ion [M − H]− at m/z 249.1489 (calcd 249.1491), corresponding to a molecular formula of C15H22O3. By comparison with published NMR data,26 the structure of 6 was shown to be similar to that of eremophil-9,11(13)-dien-12-oic acid. A set of oxymethine resonances [δH 4.23 (1H, t, J = 2.9 Hz); δC 76.4] of 6 was assigned to H-1 and C-1 based on the HMBC correlations of H-9/C-1, H-1/C-3, C-5, and C-10 and the COSY correlations as shown in Figure S1 (Supporting Information). The shielding of C-3 (ΔδC −4.9) compared to compound 20 supported the presence of 1-OH in terms of the γ-gauche effect.18,19 Thus, the 2D structure of 6 was elucidated as 1-hydroxyeremophil9,11(13)-dien-12-oic acid. The NOE correlations of H3-14/H3β, H3-15, and H-6β and H-4/H-2α indicated a chair conformation for ring A. On the basis of the NOE correlation of H-7/H-4 and the coupling constants of H-7/H-6β and H-8β (J = 11.8 Hz) an α-orientation was assigned to H-7. The triplet of H-1 (JH‑1/H2‑2 = 2.9 Hz) and its NOE with H-9 suggested that H-1 is equatorial and α-oriented. A negative Cotton effect at 205 nm and a low-amplitude positive Cotton effect at 238 nm in the ECD spectrum of 6 may be the result of the π → π* electronic transitions of the allylic alcohol and α,β-unsaturated carboxylic acid groups. By comparison of the calculated and experimental ECD data (Figure S7, Supporting Information), the absolute configuration of 6 was defined as (1R,4S,5R,7R), and the structure of artefreynic acid D (6) was thus defined as (1R,4S,5R,7R)-1-hydroxyeremophil-9,11(13)-dien-12-oic acid. The molecular formula of artefreynic acid E (7) is C15H22O3, which is the same as that of 6, as determined by negative-ion HRESIMS and 13C NMR data. Their similar 1H and 13C NMR data implied that 7 is an isomer of 6. The HMBC correlations of H-8/C-6, C-10, and C-11; H-1/C-9; and H3-14/C-10 and the COSY correlations (Figure S1, Supporting Information) assigned the hydroxy group to C-8 and indicated a Δ1(10) double bond. The 2D structure of 7 was thus defined as 8hydroxyeremophil-1(10)-11(13)-dien-12-oic acid. The NOE correlations of H3-14/H-3β, H-6β, and H-9β and H-8/H-6β indicated their cofacial relationship, and H-8 was β-oriented. 872



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Table 4. 1H NMR Data of Compounds 9−13 (δ in ppm, J in Hz, 500 MHz, in Methanol-d4) position 1α 1β 2α 2β 3α 3β 4 6α 6β 7 8 9α 9β 13 14 15 acetyl methoxy

9

10

11

12

13

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

3.46, 2.06, 1.32, 1.86, 1.48, 1.39, 1.43, 2.12, 1.53, 3.96,

dd (14.5, 5.6) td (14.5, 5.4) m m m dd (12.1, 3.3) m dd (13.4, 10.7) dd (13.4, 2.8) dd (10.7, 2.8)

6.12, 5.36, 0.96, 0.86,

s s s d (6.3)

1.70, 0.99, 1.33, 1.91, 1.91, 1.36,

dd (13.3, 5.0) d (13.3) m m m m

0.99, 1.58, 1.65, 1.37, 1.85, 1.85,

d (12.2) m m m m m

1.03, 1.96, 1.58, 1.58, 2.66, 2.07,

d (13.3) dd (13.3, 4.8) m m td (13.4, 6.1) dd (13.4, 4.1)

1.37, 1.55, 1.55, 1.60, 1.99, 1.89,

td (12.5, 3.4) m m m d (17.3) d (17.3)

1.67, 2.27, 3.09, 5.45, 1.28, 1.92, 6.19, 5.68, 1.37, 1.13,

dd (13.5, 4.9) t (13.5) ddd (13.5, 11.3, 4.9) td (11.3, 5.1) t (11.3) dd (11.3, 5.1) br s br s s s

1.55, 2.38, 2.90, 5.48, 1.48, 1.79, 6.15, 5.60, 1.24, 1.27,

m t (13.5) ddd (13.5, 11.2, 4.4) td (11.2, 4.8) dd (12.0, 11.2) dd (12.0, 4.8) br s br s s s

1.69, 2.23, 3.23, 5.39, 1.86, 1.50, 6.19, 5.66, 1.00, 4.79, 4.63, 1.93,

dd (13.3, 4.4) t (13.3) ddd (13.3, 11.3, 4.4) td (11.3, 5.1) t (11.3) dd (11.3, 5.1) br s br s s br s br s s

2.59, 2.02, 2.32, 4.04, 1.21, 1.82, 6.25, 5.71, 1.60, 1.11,

dd (14.1, 3.9) d (14.1) ddd (14.1, 10.9, 3.9) td (10.9, 4.3) dd (12.5, 10.9) dd (12.5, 4.3) br s br s s s

1.91, s

1.95, s

3.74, s

(Figure S9, Supporting Information). Therefore, the structure of artefreynic acid H (10) was defined as (4S,5S,7R,8S,10R)-8O-acetyl-5-hydroxyilicic acid. Artefreynic acid I (11) has a molecular formula of C17H24O5 based on a deprotonated ion [M − H]− at m/z 307.1548 (calcd 307.1545) in the HRESIMS, 18 mass units less than that of 10. The NMR data were comparable with those of 10, and differences included the shielding of C-4 (ΔδC −10.0) and C-5 (ΔδC −6.1) in 11. A 4,5-epoxy moiety was thus identified in conjunction with the indices of hydrogen deficiency. The 2D structure of 11 was defined as 8-O-acetyleudesma-4(5)-epoxy11(13)-en-12-oic acid. The NOE correlation of H-8/H3-14 and the large coupling constants of H-8/H-7 and H-9α (J = 11.2 Hz) revealed an H-8β-axial orientation. On the basis of the NOE correlation of H3-14/H-2β and the 3D simulation (Figure S2, Supporting Information), the epoxy group was deduced to be α-oriented. The same Cotton effect at 228 nm (Δε −1.1) (Figure S10, Supporting Information) as observed for 10 indicated a (7R) absolute configuration that facilitated assignment of (4R,5S,8S,10R) absolute configuration based on the NOE correlations. Therefore, the structure of artefreynic acid I (11) was defined as (4R,5S,7R,8S,10R)-8-O-acetyleudesma-4(5)-epoxy-11(13)-en-12-oic acid. Artefreynic acid J (12) was obtained as an amorphous powder. A deprotonated ion [M − H]− at m/z 307.1552 (calcd 307.1545) in the HRESIMS together with 13C NMR data revealed a molecular formula of C17H24O5. In both the 1H and 13 C NMR spectra, the distinct signals for an acetyl group (δH 1.93, 3H, s; δC 21.0 and 172.5), an α-substituted acrylic acid (δH 5.66, 1H, s and 6.19, 1H, s; δC 126.6, 143.5, and 170.0), a terminal double bond (δH 4.63, 1H, s and 4.79, 1H, s; δC 107.9 and 152.7), and two oxygenated carbons (δH 5.39, td, J = 11.3 and 5.1 Hz; δC 73.1, δC 75.0) were observed. These 1D NMR data were similar to those of 5-hydroxycostic acid,29 differing only in the acetyl residue. HMBC correlations from H3-14 and H2-15 to C-5 and from H-8 to the acetyl carbonyl carbon revealed that the 2D structure of 12 is 8-O-acetyl-5hydroxycostic acid. The NOE correlations of H3-14/H-6β, H-

2D structure of 9 was elucidated as 2-(1-carboxyvinyl)-7,7adimethyl-2,4,5,6,7,7a-hexahydro-1H-indene-3-carboxylic acid. Because the NOE correlations and Cotton effects in the ECD spectrum (Figure 6) were the same as those of 8, the absolute configuration of 9 was defined as (4S,5R,7R). Artefreynic acid H (10) was obtained as a white, amorphous powder. Its molecular formula was determined as C17H26O6 based on the deprotonated ion [M − H]− at m/z 325.1654 (calcd 325.1651) in the HRESIMS and its 13C NMR data. The IR spectrum showed absorption bands for hydroxy (3444 cm−1) and conjugated hydroxycarbonyl (1712, 1646 cm−1) functional groups. The presence of an acetyl group [δH 1.91 (3H, s); δC 21.1 and 172.5] was deduced from the 1D NMR data, and the remaining 15 carbon resonances were classified as two methyls, six methylenes, two methines, and five nonprotonated carbons based on the HSQC spectrum. An oxymethine [δH 5.45 (1H, td, J = 11.3, 4.9 Hz); δC 73.4], two oxygenated tertiary carbons (δC 75.5 and 76.3), and an αsubstituted acrylic acid moiety [δH 5.68 (1H, br s) and 6.19 (1H, br s); δC 126.6, 143.8, and 170.2] were assigned based on their proton and carbon chemical shifts. These 1D NMR data (Tables 2 and 4) indicated that 10 is an eudesmane sesquiterpenoid of 8-O-acetyl-5-hydroxyilicic acid.27,28 An 8O-acetyl group was assigned based on the HMBC correlation from H-8 to the acetyl carbonyl group. The relative configuration of 10 was determined from the NOESY spectrum. The cross-correlation peaks of H3-14/H-2β, H-6β and H-8, H-7/H-9α, and H3-15/H-6α suggested that the two rings were trans-fused and had chair conformations (Figure S2, Supporting Information). The large coupling constants of H-7/ H-6β (J = 13.5 Hz) and H-7/H-8 (J = 11.3 Hz) confirmed that H-7 was axial and α-oriented. The negative Cotton effect at 228 nm in the ECD spectrum, arising from the π → π* electronic transition of α,β-unsaturated hydroxycarbonyl moiety, indicated the (7R) absolute configuration,24 and the remaining stereocenters were assigned as (4S,5S,8S,10R) based on the established relative configuration. This result was confirmed by comparison of the calculated and experimental ECD data 873



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Figure 7. Comparison of the experimental ECD spectra between 10 and 12 and olefin octant rule applied for 12 viewed from C-15 to C-4.

and the aromatic ring, as is evident in the 3D model of the RMPA ester (Figure S12, Supporting Information). The C-7 and C-10 stereocenters were assigned (7R,10R) absolute configuration based on the relative configurations. This result was confirmed by comparison of the calculated and experimental ECD data (Figure S13, Supporting Information). Therefore, the structure of 13 was defined as methyl (7R,8S,10R)-8hydroxyeudesma-4,11(13)-dien-12-oate. In conclusion, 13 new compounds (1−13), including 11 sesquiterpenoids (3−13) and two sesquiterpenoid dimers (1 and 2), together with 10 known compounds (14−23) were isolated from the whole plants of A. f reyniana. The cytotoxicities of these compounds were tested against the HCT116 and DU145 cell lines, and the results showed that compounds 1−13 had weak effects with IC50 values greater than 50 μM. NO inhibitory effects on LPS-stimulated RAW 264.7 cells were tested for the new compounds. Compounds 4, 5, and 9 showed IC50 values of 10.8, 12.6, and 11.7 μM, respectively, which were comparable to the value of the positive control, dexamethasone (10.7 μM) (Table 5). Artefreynisin A (1), the dimer of two sesquiterpenoids formed via a Diels−

8, H-6β/H-8, and H-7/H-9α indicated a chair conformation of ring B. The large coupling constants (J = 11.3 Hz) of H-8/H-7 and H-8/H-9α suggested a β-axial orientation of H-8. The NOEs between H-7 and H2-15 suggested that the two rings are cis-fused, and that 5-OH is β-oriented (Figure S2, Supporting Information). By comparing the ECD spectra of 10 and 11, an additional negative Cotton effect at 215 nm was observed for 12, which was derived from the π → π* electronic transition of the allylic alcohol moiety. This CE indicated a (5S) absolute configuration according to the olefin octant rule (Figure 7).30 Correspondingly, the other stereogenic carbons were defined as (7R,8S,10R) based on the relative configuration. This result was corroborated by comparison of the calculated and experimental ECD spectra (Figure S11, Supporting Information). Therefore, the structure of artefreynic acid J (12) was defined as (5S,7R,8S,10R)-8-O-acetyl-5-hydroxycostic acid. Compound 13 was isolated as a colorless gum. Its molecular formula was determined as C16H24O3 based on a sodium adduct ion [M + Na]+ at m/z 287.1624 (calcd 287.1623) in the HRESIMS and its 13C NMR data. The 1H and 13C NMR data (Tables 2 and 4) suggested that 13 is a sesquiterpenoid similar to 8-hydroxyeudesma-4,11(13)-dien-12-oic acid,9 differing only in the additional methoxy group with resonances at δH 3.74 and δC 52.2. The presence of a methyl ester group was evidenced by the HMBC cross-peak from the methoxy protons to the carbonyl carbon (δC 169.2). The 2D structure of 13 was thus defined as methyl 8-hydroxyeudesma-4,11(13)-dien-12-oate. On the basis of the NOE correlations of H3-14/H-1β, H-2β, H6β, and H-8β, H-7/H-9α, and H3-15/H-6α, a relative configuration of (H-7α, H-8β, H3-14β) was established. The large coupling constants of H-8/H-7 (J = 10.9 Hz), H-7/H-6β (J = 14.1 Hz), and H-8/H-9α (J = 10.9 Hz) suggested axial positions for H-8 and H-7. The absolute configuration of C-8 was determined as S by the chemical shifts of MPA (methoxyphenylacetic acid) ester derivatives. Distinct deshielded shifts (ΔδH = δR − δS) of the H-7 (+0.12), H2-13 (+0.39 and +0.37) and methyl ester (+0.11) signals along with the shielded signals of H2-9 (−0.01 and −0.02) and H3-14 (−0.03) were observed. The minor shifts of H2-9 and H3-14 resulted from the considerable distance between these protons

Table 5. Inhibitory Effects of Isolates Against LPS-Induced NO Production in RAW 264.7 Cells (n = 3)a compound 1 3 4 5 6 7 8 9 10 13 dexamethasoneb

IC50 (μM) 38.3 21.6 10.8 12.6 18.9 41.1 21.9 11.7 15.0 30.6 10.7

± ± ± ± ± ± ± ± ± ± ±

0.6 0.3 0.8 1.1 0.5 0.6 0.8 0.3 0.4 0.7 0.3

Only compounds with observable inhibitory effects (IC50 < 50 μM) were listed. bPositive control. a

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Scheme 1. Putative Biosyntheic Pathway to 8 and 9

AF201609) has been deposited at the Herbarium of the Peking University Modern Research Center for Traditional Chinese Medicine. Extraction and Isolation. The dried whole plants of A. f reyniana (15 kg) were extracted with 95% aq. EtOH (3 × 90 L, each 2 h) at 80 °C to yield the extract (1120 g). The extract was suspended in H2O and partitioned sequentially with petroleum ether (2 × 2.5 L) and CHCl3 (3 × 2.5 L). The CHCl3 extract (486 g) was subjected to silica gel (100−200 mesh) CC and eluted with a petroleum ether−EtOAc gradient (10:1, 5:1, 3:1, and 1:1, v/v) to yield nine fractions (A−I). Fraction E (65 g) was separated into three fractions (E1−E3) via RPC18 CC, eluting with a gradient of MeOH−H2O (4:6 → 6:4 → 8:2). Fraction E2 was subjected to RP-C18 CC, eluting with MeOH−H2O (4:6 → 5:5), and 20 subfractions (E2a−E2t) were obtained. Fraction E2f was separated by semipreparative HPLC, eluting with MeCN− H2O (3:7, 3 mL/min) to obtain subfractions E2f1−E2f5. From Fraction E2f3, compounds 3 (1.5 mg, tR = 13 min), 11 (2.2 mg, tR = 18 min), 22 (2 mg, tR = 14 min), and 23 (3 mg, tR = 21 min) were purified via semipreparative HPLC, repeatedly eluting with MeCN− H2O (2:8 → 3:7, 30 min, 3 mL/min). From Fraction E2f4, compounds 4 (2 mg, tR = 18 min), 5 (3 mg, tR = 19 min), 10 (2.4 mg, tR = 22 min), and 12 (3 mg, tR = 24 min) were purified via semipreparative HPLC, eluting with MeCN−H2O (3:7, 3 mL/min). Compound 8 (21 mg, tR = 10 min) was purified from Fraction E2h by semipreparative HPLC, eluting with MeCN−H2O (3:7, 3 mL/min) and crystallized from a MeOH−H2O (9:1) solution. Fraction E3 was subjected to RP-C18 CC and eluted with MeOH−H2O (5:5 → 6:4) to yield 12 subfractions, E3a−E3q. Compound 6 (1.2 mg, tR = 9 min) was purified from Fraction E3c via semipreparative HPLC, eluting with MeCN−H2O (5:5, 3 mL/min). Compound 19 (2.0 mg, tR = 7 min) was purified from Fraction E3e via semipreparative HPLC, eluting with MeCN−H2O (4:6, 3 mL/min). Fraction F (85 g) was separated using a similar protocol as that used for Fraction E. Subfractions F1− F3 were obtained via RP-C18 CC eluting with a MeOH−H2O gradient (4:6 → 6:4 → 8:2). Fraction F2 was separated into 40 subfractions (F2-1−F2-40) via RP-C18 CC, eluting with MeOH−H2O (5:5). Fractions F2-4, F2-24, and F2-31 were further purified by semipreparative HPLC, eluting with MeCN−H2O (4:6, 5:5, and 6:4, each at 3 mL/min), to yield 17 (1.8 mg, tR = 9 min), 18 (3.2 mg, tR = 14 min), and 21 (2.2 mg, tR = 9 min). Fraction F2-26 was separated into three subfractions (F2-26a−F2-26c) by semipreparative HPLC, eluting with MeCN−H2O (5:5, 3 mL/min). Compound 16 (3.1 mg, tR = 6 min) was obtained from Fraction F2-26b via semipreparative HPLC, eluting with MeCN/H2O (5:5, 3 mL/min). Compound 13 (2.1 mg, tR = 18 min) was separated from Fraction F226c via semipreparative HPLC under the same conditions as those used for F2-26b. Fraction F3 was subjected to RP-18 CC, eluting with MeOH−H2O (6:4 → 8:2) to yield subfractions F3-1−F3-35. Fraction F3-7 was separated into three subfractions, F3-7a−F3-7c, via semipreparative HPLC, eluting with MeCN−H2O (4:6, 3 mL/min). Compounds 9 (2.4 mg, tR = 7 min), 7 (2.0 mg, tR = 8 min), and 14 (1.5 mg, tR = 9 min) were purified from Fraction F3-7b via semipreparative HPLC, eluting with MeCN−H2O (4:6, 3 mL/min). Fractions F3-24 and F3-27 were separated via semipreparative HPLC, repeatedly eluting with MeCN−H2O (6:4, 3 mL/min) to yield 1 (1.5 mg, tR = 10 min) and 2 (1.2 mg, tR = 17 min).

Alder [4 + 2] cycloaddition, was an exo-type product, based on the “Endo-rule”,31 suggesting that the reaction could be catalyzed by a Diels−Alderase.32 Compounds 8 and 9 had 2isopropyl-3,7,7a-trimethyl-2,4,5,6,7,7a-hexahydro-1H-indene cores, which is unprecedented in eremophilane-type derivatives. Their putative biosynthetic pathways are depicted in Scheme 1. The known compound 20 was assumed to be a precursor, and the biosynthetic pathway involves oxidative cleavage, an enolate ion intermediate, condensation between C-8 and C-10, and a dehydration sequence, which lead to the formation of 8, which is further oxidized to 9. In an effort to prove that compounds 2, 5, and 13 are derived from a natural source, an LC-MS/MS analysis was performed on the MeOH percolate of A. f reyniana. Compounds 2 and 5 were detected by LC-MS/MS, suggesting that these compounds occur naturally (Figure S132, Supporting Information), while the natural form of 13 could be a free carboxylic acid.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an SGW X-4 melting point apparatus (Shanghai Precision & Scientific Instrument Co. Ltd.). Optical rotations were measured using a Rudolph Autopol III automatic polarimeter (NJ). UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer (Tokyo, Japan). ECD spectra were recorded on a J-810 CD spectrophotometer (JASCO, Japan). IR spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrometer (MA). NMR spectra were recorded on a Varian INOVA-500 spectrometer (CA) and Bruker Ultrashield 600 Plus NMR spectrometers (Bruker Biospin Rheinstettern, Germany) by using methanol-d4 or DMSO-d6 as a solvent. HRESIMS data were collected on a Waters Xevo G2 Q-TOF mass spectrometer. LC-MS/MS analysis was performed on a Shimadzu UFLC system (Kyoto, Japan) coupled with a Sciex 4500 Qtrap mass spectrometer (Foster City, CA) via an ESI interface. X-ray crystallographic data were collected using a Rigaku-dTREK diffractometer with CrysAlisPro and a goniometer with Cu Kα radiation (Agilent Technologies, Yarnton, Oxfordshire, U.K.). Column chromatography (CC) was performed on silica gel (100−200 mesh or 200−300 mesh, Qingdao Marine Chemical Co. Ltd., People’s Republic of China), ODS (50 μm, Merck, Germany), and Sephadex LH-20 (Amersham Biosciences, Sweden). Preparative HPLC was carried out using a Waters XBridge Prep Shield RP C18 column (10 mm × 250 mm, 5 μm) on an Agilent 1200 series LC instrument with a DAD. TLC analyses were carried out on precoated silica gel GF254 plates (Qingdao Marine Chemical Co. Ltd., People’s Republic of China). Spots were visualized under UV light (254 and 365 nm) or by heating after spraying with 2% vanillin−H2SO4 solution. All the solvents used for isolation were of analytical grade, and the solvents used for HPLC were of HPLC grade. Plant Material. Whole dried plants of Artemisia f reyniana were collected from Inner Mongolia Province, China, in September 2016 and were identified by Prof. Peng-Fei Tu. A voucher specimen (no. 875



Article

Artefreynisin A (1): white, amorphous powder; [α]25 D +102 (c 0.2, MeOH); UV (MeOH) νmax (log ε) 205 (2.11) nm; IR (KBr) νmax 3444, 1745, 1729, 1713, 1163, 1018, 902, 677, 576 cm−1; ECD (MeOH) νmax (Δε) 214 (−31.5) nm; 1H and 13C NMR data, see Table 1; positive-ion HRESIMS m/z 481.2955 [M + H]+ (calcd for C30H41O5, 481.2954). Artefreynisin B (2): white, amorphous powder; [α]25 D +65 (c 0.1, MeOH); UV (MeOH) νmax (log ε) 205 (3.21), 240 (3.03) nm; IR (KBr) νmax 3421, 2918, 2851, 1678, 1207, 1139, 843, 724 cm−1; ECD (MeOH) νmax (Δε) 242 (+3.9), 300 (−1.2) nm; 1H and 13C NMR data, see Table 1; negative-ion HRESIMS m/z 497.2897 [M − H]− (calcd for C30H41O6, 497.2903). Artefreynic Acid A (3): yellowish oil; [α]25 D +85 (c 0.2, MeOH); UV (MeOH) νmax (log ε) 240 (3.96) nm; IR (KBr) νmax 3444, 2917, 1712, 1681, 1222, 1017, 576 cm−1; ECD (MeOH) νmax (Δε) 234 (+6.8), 310 (−1.7) nm; 1H and 13C NMR data, see Tables 2 and 3; negativeion HRESIMS m/z 247.1338 [M − H]− (calcd for C15H19O3, 247.1334). Artefreynic Acid B (4): colorless oil; [α]25 D +124 (c 0.2, MeOH); UV (MeOH) νmax (log ε) 225 (4.07) nm; IR (KBr) νmax 3419, 2917, 2849, 1681, 1381, 1019, 576 cm−1; ECD (MeOH) νmax (Δε) 232 (−3.5) nm; 1H and 13C NMR data, see Tables 2 and 3; positive-ion HRESIMS m/z 251.1648 [M + H]+ (calcd for C15H23O3, 251.1647). Artefreynic Acid C (5): colorless gum; [α]25 D +23 (c 0.2, MeOH); UV (MeOH) νmax (log ε) 210 (2.05) nm; IR (KBr) νmax 3445, 2920, 1697, 1438, 1250, 1148, 1024, 829 cm−1; ECD (MeOH) νmax (Δε) 218 (+9.7), 300 (+3.1) nm; 1H and 13C NMR data, see Tables 2 and 3; negative-ion HRESIMS m/z 309.1704 [M − H]− (calcd for C17H25O5, 309.1702). Artefreynic Acid D (6): colorless gum; [α]25 D +90 (c 0.2, MeOH); UV (MeOH) νmax (log ε) 202 (4.23) nm; IR (KBr) νmax 3444, 2918, 1712, 1681, 1142, 1018, 522 cm−1; ECD (MeOH) νmax (Δε) 205 (−2.1), 238 (+0.3) nm; 1H and 13C NMR data, see Tables 2 and 3; negative-ion HRESIMS m/z 249.1489 [M − H]− (calcd for C15H21O3, 249.1491). Artefreynic Acid E (7): colorless gum; [α]25 D +88 (c 0.2, MeOH); UV (MeOH) νmax (log ε) 210 (4.04) nm; IR (KBr) νmax 3364, 2922, 1693, 1622, 1461, 1265, 1021, 841, 575 cm−1; ECD (MeOH) νmax (Δε) 205 (+23.1), 245 (+4.2) nm; 1H and 13C NMR data, see Tables 2 and 3; negative-ion HRESIMS m/z 249.1491 [M − H]− (calcd for C15H21O3, 249.1491). Artefreynic Acid F (8): colorless crystals; mp 162−164 °C; [α]25 D − 67 (c 0.3, MeOH); UV (MeOH) νmax (log ε) 210 (3.68), 254 (3.45) nm; IR (KBr) νmax 3289, 2936, 2863, 1689, 1649, 1432, 1029, 831 cm−1; ECD (MeOH) νmax (Δε) 250 (+8.5) nm; 1H and 13C NMR data, see Tables 2 and 3; negative-ion HRESIMS m/z 247.1333 [M − H]− (calcd for C15H19O3, 247.1334). Artefreynic Acid G (9): colorless gum; [α]25 D −75 (c 0.1, MeOH); UV (MeOH) νmax (log ε) 205 (4.07), 233 (3.89) nm; IR (KBr) νmax 3445, 2919, 2851, 1712, 1646, 1362, 1020, 529 cm−1; ECD (MeOH) νmax (Δε) 243 (+8.2) nm; 1H and 13C NMR data, see Tables 2 and 4; negative-ion HRESIMS m/z 263.1282 [M − H]− (calcd for C15H19O4, 263.1283). Artefreynic Acid H (10): white, amorphous powder; [α]25 D −14 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 (3.57) nm; IR (KBr) νmax 3444, 2918, 2850, 1712, 1646, 1202, 1023, 574 cm−1; ECD (MeOH) λmax (Δε) 228 (−1.3) nm; 1H and 13C NMR data, see Tables 2 and 4; negative-ion HRESIMS m/z 325.1654 [M − H]− (calcd for C17H25O6, 325.1651). Artefreynic Acid I (11): colorless oil; [α]25 D +33 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 (3.38) nm; IR (KBr) νmax 3420, 2919, 2851, 1734, 1435, 1027, 827 cm−1; ECD (MeOH) λmax (Δε) 228 (−1.1) nm; 1H and 13C NMR data, see Tables 2 and 4; negative-ion HRESIMS m/z 307.1548 [M − H]− (calcd for C17H23O5, 307.1545). Artefreynic Acid J (12): white, amorphous powder; [α]25 D +48 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 217 (3.49) nm; IR (KBr) νmax 3448, 2937, 2867, 1713, 1258, 1024, 951, 899 cm−1; ECD (MeOH) λmax (Δε) 215 (−15.1) nm; 1H and 13C NMR data, see Tables 2 and 4; negative-ion HRESIMS m/z 307.1552 [M − H]− (calcd for C17H23O5, 307.1545).

Methyl (7R,8S,10R)-8-hydroxyeudesma-4,11(13)-dien-12-oate (13): colorless gum; [α]25 D +62 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 (4.04) nm; IR (KBr) νmax 3420, 2918, 2850, 1713, 1437, 1362, 1025, 579 cm−1; ECD (MeOH) λmax (Δε) 228 (−8.6) nm; 1H and 13C NMR data, see Tables 2 and 4; positive-ion HRESIMS m/z 287.1624 [M + Na]+ (calcd for C16H24O3Na, 287.1623). X-ray Crystallographic Analysis of 8. Upon crystallization from MeOH−H2O (9:1) using the vapor diffusion method, crystals of 8 were obtained at room temperature. A suitable crystal was selected and mounted on a Rigaku-dTREK diffractometer with CrysAlisPro and a goniometer with Cu Kα radiation. The crystal was kept at 293 K during data collection. Using Olex2, the structure was solved with the ShelXT structure solution program by using Direct Methods and refined with the ShelXL refinement package using Least Squares minimization. Crystallographic data for 8 [Flack parameter 0.01(9), Table S1, Supporting Information] have been deposited at the Cambridge Crystallographic Data Center (Deposition No. CCDC 1560531). These data can be obtained free of charge from CCDC via the Internet at www.ccdc.cam.ac.uk/conts/retrieving.html. ECD Calculations. The relative configurations of the compounds were initially established on the basis of their NOESY spectra, optimized by the MM2 force field using ChemOffice 2014 software, and then submitted to random conformational analysis with the MMFF94s force field using the Sybyl-X 2.0 software package. The conformers were further optimized by using the TDDFT method at the B3LYP/6-31G(d) level, and the frequency was calculated at the same theoretical level. Stable conformers without imaginary frequencies were subjected to ECD calculation by the TDDFT method at the B3LYP/6-31+G(d) level with the CPCM model in MeOH. ECD spectra of different conformers were simulated using SpecDis v1.51 with a half-bandwidth of 0.3−0.5 ev, and the final ECD spectra were obtained according to the Boltzmann-calculated contribution of each conformer. The calculated ECD spectra were compared with the experimental data. All calculations were performed with the Gaussian 09 program package. Preparation of MPA Esters Derived from 13 (13a/13b). Samples of 13 (0.7 mg each) were treated with (R)- or (S)-MPA (0.9 mg) with dicyclohexylcarbodiimide (1 mg) and 4-dimethylaminopyridine (0.6 mg) in dry CH2Cl2 (2 mL). The reaction was stirred for 4 h at room temperature. The solvent was removed in vacuo, and the residue was purified by TLC (n-hexane−EtOAc, 7:1) to give a white, amorphous powder. The powders were dissolved in MeOH and further purified by semipreparative HPLC, eluting with MeCN−H2O (7:3) to obtain the (R)-MPA ester 13a or the (S)-MPA ester 13b. (R)-MPA Ester (13a): colorless oil; 1H NMR (500 MHz, methanold4): δ 1.37 (m, 1H, H-1α), 1.50 (m, 1H, H-1β), 1.55 (m, 1H, H-2α), 1.60 (m, 1H, H-2β), 1.99 (m, 1H, H-3α), 1.89 (d, J = 15.2 Hz, 1H, H3β), 2.66 (d, J = 14.7 Hz, 1H, H-6α), 2.14 (d, J = 14.7 Hz, 1H, H-6β), 2.56 (t, J = 11.6 Hz, 1H, H-7), 5.43 (td, J = 11.6, 4.6 Hz, 1H, H-8), 1.30 (m, 1H, H-9α), 1.88 (m, 1H, H-9β), 6.12 (br s, 1H, H-13a), 5.62 (br s, 1H, H-13b), 1.12 (s, 3H, H3-14), 1.60 (s, 3H, H3-15), 3.75 (s, 3H, OCH3). (S)-MPA Ester (13b): colorless oil; 1H NMR (500 MHz, methanold4): δ 1.37 (m, 1H, H-1α), 1.50 (m, 1H, H-1β), 1.55 (m, 1H, H-2α), 1.60 (m, 1H, H-2β), 1.99 (m, 1H, H-3α), 1.89 (m, 1H, H-3β), 2.57 (d, J = 13.8 Hz, 1H, H-6α), 2.07 (d, J = 13.8 Hz, 1H, H-6β), 2.44 (t, J = 11.8 Hz, 1H, H-7), 5.41 (td, J = 11.8, 4.3 Hz, 1H, H-8), 1.32 (m, 1H, H-9α), 1.89 (m, 1H, H-9β), 5.73 (br s, 1H, H-13a), 5.25 (br s, 1H, H13b), 1.15 (s, 3H, H3-14), 1.58 (s, 3H, H3-15), 3.64 (s, 3H, OCH3). LC-MS/MS Analysis. LC-MS/MS analysis was performed on a Shimadzu UFLC system (CBM-20A controller, two LC-20AD binary pumps, a SPD-M20A diode array detector, a SIL-20AC autosampler, a CTO-20A column oven, and a DGU-20A5 degasser) coupled with a Sciex 4500 Qtrap mass spectrometer (Foster City, CA) via an ESI interface. Data acquisition and processing were performed by Sciex Analyst 1.6.2 software. Both negative and positive scan modes of MRM surveys (m/z 497.2 and 267.0 for 2; m/z 309.4 and 245.0 for 5; m/z 265.4 and 247.0 for 13) were utilized for detecting compounds 2, 5, and 13, based on their different fragmentation patterns. For 2 and 5, the ion source parameters were maintained as follows: polarity, 876



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negative; ion spray voltage, −4500 V; source temperature, 500 °C; curtain gas (CUR), 35 psi; ion source gas 1 (GS1), 50 psi; and ion source gas 2 (GS2), 50 psi. For 13, the ion source parameters were maintained as follows: polarity, positive; ion spray voltage, 5500 V; source temperature, 500 °C; curtain gas (CUR), 35 psi; ion source gas 1 (GS1), 50 psi; and ion source gas 2 (GS2), 50 psi. Cytotoxic Assay. Cell growth inhibition was determined using the MTT method according to the established protocols.33 Taxol was used as the positive control. NO Inhibitory Assay. The RAW 264.7 mouse macrophage cell line was purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cell maintenance, experimental procedures, and data presentation for the inhibition of NO production and viability assay were conducted as previously described.34 The effects of the compounds on cell growth were measured using the MTT method. None of the compounds showed cytotoxicity at a dosage of 50 μM. The experiments were performed three times in parallel, and the results are presented as the mean ± SD (n = 3). Dexamethasone (IC50 value of 10.7 μM) was used as a positive control. All the compounds were prepared as stock solutions in DMSO.

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Gao, K. J. Nat. Prod. 2007, 70, 241−245. (d) Wang, Q.; Mu, Q.; Shibano, M.; Morris-Natschke, S. L.; Lee, K. H.; Chen, D. F. J. Nat. Prod. 2007, 70, 1259−1262. (e) Mohamed, A. E.-H. H.; Ahmed, A. A. J. Nat. Prod. 2005, 68, 439−442. (4) (a) McDonald, L. A.; Barbieri, L. R.; Bernan, V. S.; Janso, J.; Lassota, P.; Carter, G. T. J. Nat. Prod. 2004, 67, 1565−1567. (b) Oh, H.; Jensen, P. R.; Murphy, B. T.; Fiorilla, C.; Sullivan, J. F.; Ramsey, T.; Fenical, W. J. Nat. Prod. 2010, 73, 998−1001. (5) (a) Liu, J. M.; Zhang, D. W.; Zhang, M.; Zhao, J. L.; Chen, R. D.; Wang, N.; Zhang, D.; Dai, J. G. J. Nat. Prod. 2016, 79, 2229−2235. (b) Cheng, Z. B.; Zhao, J. J.; Liu, D.; Proksch, P.; Zhao, Z. M.; Lin, W. H. J. Nat. Prod. 2016, 79, 1035−1047. (6) Zhao, Y. X.; Schenk, D. J.; Takahashi, S.; Chappell, J.; Coates, R. M. J. Org. Chem. 2004, 69, 7428−7435. (7) (a) Wang, S.; Li, J.; Sun, J.; Zeng, K. W.; Cui, J. R.; Jiang, Y.; Tu, P. F. Fitoterapia 2013, 85, 169−175. (b) Chen, Z.; Wang, S.; Zeng, K. W.; Cui, F. X.; Jin, H. W.; Guo, X. Y.; Jiang, Y.; Tu, P. F. Bioorg. Med. Chem. Lett. 2014, 24, 4318−4322. (c) Turak, A.; Shi, S. P.; Jiang, Y.; Tu, P. F. Phytochemistry 2014, 105, 109−114. (d) Zan, K.; Chai, X. Y.; Chen, X. Q.; Wu, Q.; Fu, Q.; Zhou, S. X.; Tu, P. F. Tetrahedron 2012, 68, 5060−5065. (e) Zhang, C.; Wang, S.; Zeng, K. W.; Li, J.; Ferreira, D.; Zjawiony, J. K.; Liu, B. Y.; Guo, X. Y.; Jin, H. W.; Jiang, Y.; Tu, P. F. J. Nat. Prod. 2016, 79, 213−223. (8) (a) Xie, W. D.; Liu, Y. H.; Weng, C. W.; Zhao, H.; Row, K. H. J. Chin. Chem. Soc. 2011, 58, 412−414. (b) Xie, W. D.; Weng, C. W.; Li, X.; Row, K. H. Helv. Chim. Acta 2010, 93, 1983−1989. (c) Zhan, Z. J.; Ying, Y. M.; Ma, L. F.; Shan, W. G. Nat. Prod. Rep. 2011, 28, 594−605. (9) Bohlmann, F.; Trinks, C.; Jakupovic, J.; Huneck, S. Phytochemistry 1985, 24, 995−997. (10) Whitehead, I. M.; Ewing, D. F.; Threlfall, D. R. Phytochemistry 1988, 27, 1365−1370. (11) Bohlmann, F.; Le-Van, N. Phytochemistry 1978, 17, 1173−1178. (12) Bohlmann, F.; Zdero, C.; King, R. M.; Robinson, H. Liebigs Ann. Chem. 1986, 1986, 799−813. (13) Marco, J. A.; Carda, M. Magn. Reson. Chem. 1987, 25, 1087− 1091. (14) Garcez, F. R.; Garcez, W. S.; Hamerski, L.; Miranda, A. C. d. M. Quim. Nova 2010, 33, 1739−1742. (15) Jakupovic, J.; Ganzer, U.; Pritschow, P.; Lehmann, L.; Bohlmann, F.; King, R. M. Phytochemistry 1992, 31, 863−880. (16) Yang, Y. X.; Shan, L.; Liu, Q. X.; Shen, Y. H.; Zhang, J. P.; Ye, J.; Xu, X. K.; Li, H. L.; Zhang, W. D. Org. Lett. 2014, 16, 4216−4219. (17) El-Masry, S.; Ghazy, N. M.; Zdero, C.; Bohlmann, F. Phytochemistry 1984, 23, 183−185. (18) Lei, C.; Huang, S. X.; Chen, J. J.; Yang, L. B.; Xiao, W. L.; Chang, Y.; Lu, Y.; Huang, H.; Pu, J. X.; Sun, H. D. J. Nat. Prod. 2008, 71, 1228−1232. (19) Huang, H. C.; Ahmed, A. F.; Su, J. H.; Chao, C. H.; Wu, Y. C.; Chiang, M. Y.; Sheu, J. H. J. Nat. Prod. 2006, 69, 1554−1559. (20) Zheng, Q. X.; Xu, Z. J.; Sun, X. F.; Guéritte, F.; Cesario, M.; Cheng, C. H. K.; Sun, H. D.; Zhao, Y. Chin. Chem. Lett. 2003, 14, 393−396. (21) Camargo, M. J. de; Miranda, M. L. D.; Kagamida, C. M.; Rodrigues, E. D.; Garcez, F. R.; Garcez, W. S. Quim. Nova 2013, 36, 1008−1013. (22) Kirk, D. N. Tetrahedron 1986, 42, 777−818. (23) Mata, R.; Bye, R.; Linares, E.; Macías, M.; Rivero-Cruz, I.; Pérez, O.; Timmermann, B. N. Phytochemistry 2003, 64, 285−291. (24) Mori, K. Tetrahedron 1974, 30, 1065−1072. (25) Cerda-García-Rojas, C. M.; Coronel, A. C.; de Lampasona, M. E. P.; Catalán, C. A. N.; Joseph-Nathan, P. J. Nat. Prod. 2005, 68, 659− 665. (26) Bohlmann, F.; Zdero, C. Phytochemistry 1978, 17, 2032−2033. (27) Wang, G. C.; Li, G. Q.; Geng, H. W.; Li, T.; Xu, J. J.; Ma, F.; Wu, X.; Ye, W. C.; Li, Y. L. Phytochemistry 2013, 96, 201−207. (28) Zheng, Q. X.; Xu, Z. J.; Sun, X. F.; Guéritte, F.; Cesario, M.; Sun, H. D.; Cheng, C. H. K.; Hao, X. J.; Zhao, Y. J. Nat. Prod. 2003, 66, 1078−1081.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00947. Crystallographic data of 8 (CIF) NMR, MS, and ECD spectra of 1−13, key HMBC and COSY correlations for 2−13, key NOESY correlations for 2−13, and detailed quantum calculations of ECD spectra of 1−7 and 10−13 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +86-10-82802769. Fax: +86-10-82802769. E-mail: [email protected]. *Phone: +86-10-82802750. Fax: +86-10-82802750. E-mail: [email protected]. ORCID

Yong Jiang: 0000-0002-8450-7786 Peng-Fei Tu: 0000-0003-3553-1840 Notes

The authors declare no competing financial interest. Crystallographic data for 8 have been deposited at the Cambridge Crystallographic Data Center (Deposition No. CCDC 1560531). These data can be obtained free of charge from CCDC via the Internet at www.ccdc.cam.ac.uk/conts/ retrieving.html.

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ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (NSFC; Nos. 81373294), and National Key Technology R&D Program “New Drug Innovation” of China (No. 2018ZX09711001-008-003).

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REFERENCES

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Artemisia freyniana

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