Sesquiterpenoids with Various Carbocyclic Skeletons from the

Article pubs.acs.org/jnp

Sesquiterpenoids with Various Carbocyclic Skeletons from the Flowers of Chrysanthemum indicum Lei-Lei Liu,† Thi Kim Quy Ha,‡ Wei Ha,† Won Keun Oh,‡ Jun-Li Yang,*,† and Yan-Ping Shi*,† †

CAS Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Lanzhou 730000, People’s Republic of China ‡ Korea Bioactive Natural Material Bank, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 08826, Republic of Korea S Supporting Information *

ABSTRACT: A phytochemical investigation of the flowers of Chrysanthemum indicum yielded sesquiterpenoids 1−25 with various carbocyclic skeletons, including 10 new (1−10) and 15 known (11−25) analogues. The structures were elucidated via their physical data, while the absolute configuration of compounds 6, 8, and 10 was assessed via electronic circular dichroism analysis. The evaluation of the effect of sesquiterpenoids on porcine epidemic diarrhea virus (PEDV) replication showed that compounds 1−5, 12, 14, 16, 17, 19, and 21 increased cell viability against cell death in PEDV-injected cells. Compounds 2, 12, and 17 were selected and investigated for their inhibition of proteins required for PEDV replication. Compounds 2 and 17 significantly reduced PEDV nucleocapsid and spike protein synthesis compared with azauridin as a positive control.

T

herbs led to the identification of several bioactive terpenoids.8−13 As part of this program, an investigation of an aqueous EtOH extract of the flowers of C. indicum afforded 10 new sesquiterpenoids, i.e., chrysanthemumins A−J (1−10), which included six eudesmane (1−6), an iphionane (7), two germacrane (8 and 9), a guaiane (10), and 15 known sesquiterpenoids (11−25). Herein, the isolation and structure determination of these isolates, as well as their inhibitory activities against porcine epidemic diarrhea virus (PEDV) replication, are described.

he large Asteraceae family, the plants of which mainly produce sesquiterpenoids, contains more than 1000 genera and 25 000−30 000 species.1 The Chrysanthemum genus plants that belong to the Asteraceae family are annual herbs with a distribution from Europe to Asia, Africa, and North America. Different types of chemical constituents have been reported for this genus, with sesquiterpenoids and flavonoids being the most common.2−4 In recent years, increased attention has been focused on Traditional Chinese Medicine (TCM), as it shows potential in the prevention and treatment of widespread epidemic diseases, such as severe acute respiratory syndrome (SARS) and swineorigin influenza A (H1N1). Several studies indicated that TCM preparations for the treatment of these epidemic diseases were mainly based on heat-clearing and detoxifying herbs, an important type of TCM.5,6 The flowers of Chrysanthemum indicum, which are recorded in the Chinese Pharmacopoeia as Ye-Ju-Hua, are well known in TCM. This plant is an important heat-clearing and detoxifying herb used to treat inflammation, headache, and vertigo and for the preparation of a bitter tea used for antibacterial, antioxidant, and anti-inflammatory purposes.7 Over the past years, studies on selected TCM © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Using fractionation with diverse chromatographic materials (e.g., silica gel, RP-C18, and Sephadex LH-20), 10 new (1−10) and 15 known (11−25) sesquiterpenoids were isolated from a 95% ethanol extract of the flowers of C. indicum. Chrysanthemumin A (1) was isolated as a colorless oil with [α]D20 +40 (c 0.3, acetone). Its molecular formula was determined as C15H26O3 based on an HRESIMS ion at m/z Received: July 26, 2016 Published: February 3, 2017 298

DOI: 10.1021/acs.jnatprod.6b00694 J. Nat. Prod. 2017, 80, 298−307

Journal of Natural Products

Article

Chart 1

272.2214 [M + NH4]+ (calcd 272.2220). Its 1H and 13C NMR data (Table 1) showed three methyl singlets (δH 1.10, 1.24, and 1.37), an oxymethine [δH 4.30; δC 74.0 (CH)], two oxygenated tertiary carbons [δC 73.7 (C), 76.2 (C)], and one olefinic bond [δH 4.94, 5.22; δC 109.1 (CH2), 149.4 (C)]. These observations suggested compound 1 to be a eudesmane-type sesquiterpenoid possessing exomethylene and three hydroxy functionalities, which were assigned based on the 1H−1H COSY, HMQC, and HMBC experiments, as shown in Figure 1. The 1D NOE experiment indicated correlations from H-6 to H-7 and H3-12 and from H-8β to H3-13 and H3-14, which suggested that H-6 and H-7 were α-oriented and Me-14 was β-oriented. The αorientation of OH-5 was deduced from the pyridine-induced solvent shifts (Δδ = δCDCl3 − δpyridine‑d5) of H-1α (−0.67), H-3α (−0.55), H-7α (−0.56), and H-9α (−0.61).14 This conclusion was supported by the similarity of the coupling pattern of H-6 and the chemical shifts of C-6/7 in compound 1 (H-6: br s; δC 74.0/43.7 in CDCl3) compared with chrysanthemumin J (H-6: br s; δC 74.4/43.8 in CDCl3).15 Chrysanthemumin B (2), a colorless oil with [α]20 D −13 (c 0.2, acetone), had a molecular formula of C15H26O3 based on

the HRESIMS ion at m/z 277.1778 [M + Na]+ (calcd 277.1774). Its NMR data (Table 1) were similar to those of 1, suggesting an epimeric relationship. This was supported by the HSQC and HMBC data. The 1D NOE experiment supported the correlations from H-6 to H-7, H3-12, and H3-14, which indicated that H-6, H-7, and Me-14 were α-oriented. Following the same procedure as for 1, OH-5 was determined to have an α-orientation via measuring the pyridine-induced solvent shifts of H-7α (−0.31) and H-9α (−0.38) (Table 1).14 Chrysanthemumin C (3), a white solid with [α]20 D +15 (c 0.2, acetone), had a molecular formula of C15H24O2 based on the HRESIMS data. Its 1H and 13C NMR data (Tables 2 and 3) showed the presence of an angular methyl (δH 0.68; δC 9.3), a vinylic methyl (δH 1.77; δC 21.0), two oxymethines (δH 3.86, 4.37; δC 73.7, 74.6), and two exo methylenes [δH 4.68, 4.73 (2H), 4.99; δC 108.4, 110.4, 150.4, 150.5]. The above observations suggested 3 to be a eudesmane-type sesquiterpenoid with two hydroxy groups and two terminal double bonds. Its 2D structure was constructed based on the HMBC crosspeaks from H3-14 (δH 0.68) to C-1 (δC 74.6), C-5 (δC 41.8), C9 (δC 36.7), and C-10 (δC 40.4); from H-1 (δH 3.86) to C-2 (δC 299



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Table 1. 1H and 13C NMR Data for Compounds 1 and 2 1 no.

δ Ca

1

37.0, t

2

21.5, t

3

32.0, t

4 5 6 7 8 9 10 11 12 13 14 15

149.4, 76.2, 74.0, 43.7, 16.4,

s s d d t

34.1, t 36.7, 73.7, 29.0, 28.6, 20.9, 109.1,

2

δ Ha

s s q q q t

1.84, 0.99, 1.73, 1.64, 2.55, 2.16,

4.30, 1.92, 1.92, 1.54, 1.66, 1.21,

1.24, 1.37, 1.10, 5.22, 4.94,

dt (13.2, 4.0) d (13.2) m m dt (13.6, 6.0) dd (13.6, 4.4)

br s m m m m m

s s s s s

δHb 2.51, 1.12, 1.91, 1.66, 3.10, 2.22,

4.86, 2.48, 2.48, 1.85, 2.27, 1.39,

1.51, 1.51, 1.42, 5.92, 5.13,

δ Ca

m d (12.4) m m dt (13.6, 6.0) m

38.4, t 22.1, t 34.1, t 149.1, 75.4, 73.4, 46.0, 17.3,

br s m m m m m

δ Ha

s s d d t

32.8, t

s s s d (2.0) d (2.0)

38.2, 72.6, 28.8, 28.5, 23.3, 111.3,

s s q q q t

1.82, m 1.17, m 1.55, m

δ Hb

2.45, m 2.16, m

2.12, 1.18, 1.63, 1.56, 2.83, 2.16,

m m m m dt (13.2, 4.8) m

4.51, 1.73, 1.92, 1.61, 1.63, 1.14,

d (2.8) m m m m m

4.91, 2.04, 2.30, 1.82, 2.20, 1.18,

d (2.8) dt (12.4, 2.8) m m m m

1.41, 1.23, 1.17, 5.00, 4.78,

s s s d (1.6) s

1.59, 1.51, 1.45, 5.06, 4.99,

s s s s s

a

Data (δ) were measured in CDCl3 at 400 MHz for protons and at 100 MHz for carbons. bData (δ) were measured in pyridine-d5 at 400 MHz. Proton coupling constants (J) in Hz are given in parentheses.

supported by HMBC cross-peaks from H2-15 (δH 2.70, 3.95) to C-3 (δC 31.6), C-4 (δC 61.9), and C-5 (δC 75.4). The 1D NOE experiment supported the correlations from H3-14 to H-15a (0.59%) and H-15b (0.36%), which indicated that H3-14 and H2-15 were β-oriented. The pyridine-induced solvent shifts of H-1α (−0.21), H-7α (−0.25), and H-9α (−0.30) were used to determine that OH-5 was α-oriented. The coupling pattern of H-6 and chemical shifts of C-7/8 of compound 4 (H-6: br s; δC 43.9/16.3 in CDCl3), which were similar to those of chrysanthemumin J (H-6: br s; δC 43.8/17.4 in CDCl3)15 and compound 1 (H-6: br s; δC 43.7/16.4 in CDCl3), were used to determine that H-6 and H-7 were α-oriented. Chrysanthemumin E (5), a colorless oil with [α]20 D +40 (c 0.3, acetone), had the same molecular formula of C15H26O4 as 4 based on the HRESIMS ion at m/z 293.1731 [M + Na]+ (calcd 293.1723). Analysis of its NMR data (Tables 2 and 3) suggested that compound 5 may be a eudesmane-type sesquiterpenoid possessing the same functional groups as 4. The 2D structure was constructed using the HMBC analysis: from H2-15 (δH 3.62, 3.76) to C-3 (δC 24.5), C-4 (δC 65.9), and C-5 (δC 68.2); from H-6 (δH 4.08) to C-4 (δC 65.9), C-5 (δC 68.2), C-7 (δC 47.1), C-8 (δC 16.6), C-10 (δC 31.9), and C11 (δC 73.8); and from H3-12/13 (δH 1.37/1.28) to C-7 (δC 47.1) and C-11 (δC 73.8). The 1D NOE experiment provided the correlations from H3-14 to H2-15, which suggested that Me14 and H2-15 were β-oriented, while the epoxy group involving C-4 and C-5 was on the α-face. An NOE experiment showed that irradiation of H-6 enhanced H-15a (5.08%), H-7 (3.13%), and H3-12 (4.28%). Taken in conjunction with the coupling pattern of H-6 (d, J = 2.8 Hz), this suggested that both H-6 and H-7 were α-oriented. The molecular formula of chrysanthemumin F (6), a yellow gum with [α]20 D +4 (c 0.3, acetone), was deduced to be C15H24O2 based on the HRESIMS ion at m/z 259.1675 [M + Na]+ (calcd 259.1669). The NMR data of 6 (Tables 2 and 3)

Figure 1. Key 1H−1H COSY (H → H) and HMBC (H → C) correlations for sesquiterpenoids 1, 7, and 9.

37.8), C-3 (δC 73.7), and C-10 (δC 40.4); from H-3 (δH 4.37) to C-1 (δC 74.6), C-4 (δC 150.4), and C-15 (δC 110.4); from H2-15 (δH 4.68, 4.99) to C-3 (δC 73.7), C-4 (δC 150.4), and C5 (δC 41.8); and from H3-12 (δH 1.77) to C-11 (δC 150.5), C13 (δC 108.4), and C-7 (δC 45.2). The 1D NOE experiment showed that the irradiation of H-1 enhanced H-5, indicating the α-orientations of H-1 and H-5. The coupling patterns of H-1/3 and chemical shifts of C-1/9/10 in compound 3 (H-1: dd, J = 12.0, 4.8 Hz; H-3: t, J = 3.2 Hz; δC 74.6/36.7/40.4 in CDCl3) were similar to those of 1β,3a-dihydroxy-6β-acetoxyeudesm4(15)-ene (H-1: dd, J = 11.0, 5.0 Hz; H-3: t, J = 2.5 Hz; δC 75.0/37.2/40.6 in CDCl3), which suggested that H-3 and Me14 were β-oriented. The similar chemical shifts of C-6/7/8 in 3 (δC 26.5/45.2/28.6 in CDCl3), 1β,4β-dihydroxyeudesman-11ene (δC 26.4/46.1/26.8 in CDCl3),16 and β-dictyopterol (δC 26.5/45.3/28.9 in CDCl3)17 were used to assign H-7 as αoriented. Chrysanthemumin D (4) was obtained as a colorless oil with [α]20 D +7 (c 0.3, acetone), the molecular formula of which was deduced as C15H26O4 from its HRESIMS ion at m/z 288.2162 [M + NH4]+ (calcd 288.2169). Its 1H and 13C NMR data (Tables 2 and 3) were similar to those of compound 1, except for the signals of an epoxy group (δH 2.70, 3.95; δC 56.7, 61.9) involving C-4 and C-15 instead of the terminal double bond [δH 4.94, 5.22; δC 109.1 (CH2), 149.4 (C)] in 1, which was also 300



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Table 2. 1H (400 MHz) NMR Data for Compounds 3−8 3a

no.

4a

1

3.86, dd (12.0, 4.8)

2 3

2.05, m 1.75, m 4.37, t (3.2)

5

2.34, br d (12.4)

6

12 13

1.62, 1.36, 2.03, 1.68, 1.46, 2.01, 1.25, 1.77, 4.73,

14

0.68, s

15

4.99, s 4.68, s

7 8 9

1.75, 0.98, 1.82, 1.63, 2.49, 1.05,

m m m m, m m m s br s (2H)

5a

m m m m ddd (13.2, 5.6, 2.0) m

1.53, 0.89, 1.69, 1.41, 2.18, 1.93,

m dt (10.8, 2.8) m m dd (14.4, 8.8) dd (14.4, 6.0)

m m m m br s

1.87, 1.49, 2.13, 1.85, 3.16,

5.80, br s

4.12, d (2.4)

1.86, 1.85, 1.52, 1.74, 1.20, 1.35, 1.27,

1.50, 2.05, 1.66, 1.46,

1.37, s 1.28, s

2.30, 1.72, 1.51, 1.44, 1.39, 1.26, 1.74,

1.37, 1.84, 1.55, 1.44, 1.34, 1.32, 1.22,

1.22, s

1.28, s

0.98, s

1.11, s

3.95, dd (5.6, 2.0) 2.70, d (5.6)

3.76, d (11.2) 3.62, d (11.2)

4.35, d (12.0) 4.19, d (12.0)

2.19, s

m m m m m s s

m m m m

m m m m m s s

8b

m m m m dd (10.4, 8.4)

4.08, d (2.8)

m m dq (13.6, 3.2) m m s s

4.51, m 2.78, dd (12.8, 4.8) 1.62, m

2.52, 2.31, 1.88, 1.49, 2.37, 1.98, 1.33, 4.34,

m m m m m dd (14.4, 7.2) dd (14.4, 5.2) dd (12.8, 5.2)

1.67, 4.63, 4.56, 5.10, 4.88, 5.29, 5.07,

s br s br s t (2.0) s s s

4.73, br s

Spectra recorded in CDCl3. bSpectra recorded in acetone-d6.

Table 3. 13C (100 MHz) NMR Data for Compounds 3−8 a

no.

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

74.6, 37.8, 73.7, 150.4, 41.8, 26.5, 45.2, 28.6, 36.7, 40.4, 150.5, 21.0, 108.4, 9.3, 110.4,

a

1.51, 1.41, 2.31, 2.16, 5.82,

7a

4.07, br s

5-OH a

6a

a

a

4 d t d s d t d t t s s q t q t

36.4, 20.0, 31.6, 61.9, 75.4, 70.8, 43.9, 16.3, 34.8, 37.7, 74.6, 28.4, 29.6, 20.2, 56.7,

a

5 t t t s s d d t t s s q q q t

34.4, 15.3, 24.5, 65.9, 68.2, 71.9, 47.1, 16.6, 37.3, 31.9, 73.8, 28.7, 28.8, 21.3, 66.1,

a

6 t t t s s d d t t s s q q q t

36.8, 20.5, 126.5, 135.1, 140.8, 120.7, 48.2, 22.7, 37.8, 32.1, 73.2, 28.2, 25.7, 23.1, 64.7,

7 t t d s s d d t t s s q q q t

38.5, 25.1, 53.8, 214.5, 83.4, 71.7, 46.5, 16.7, 35.8, 45.2, 72.1, 28.9, 28.4, 19.6, 31.3,

experimental ECD spectrum of 6 and the calculated ECD spectrum of 6b confirmed the absolute configuration of compound 6 as (7S,10S). Chrysanthemumin G (7), a colorless oil with [α]20 D +5 (c 0.2, acetone), was assigned a molecular formula of C15H26O4 based on an HRESIMS sodium adduct ion at m/z 293.1728 [M + Na]+ (calcd 293.1723). Its 1H and 13C NMR data (Tables 2 and 3) indicated the presence of four methyl singlets (δH 1.11, 1.22, 1.32, and 2.19), one oxygenated methine (δH 4.12; δC

b

8 t t d s s d d t t s s q q q q

65.8, d 45.8, t 97.8, s 155.6 s 31.0, t 40.7, t 46.2, d 45.8, t 80.4, d 151.8, s 153.4, s 20.8, q 108.9, t 106.5, t 112.0, t

Spectra recorded in CDCl3. bSpectra recorded in acetone-d6.

were similar to those of 7αH,10β-methyeudesma-3,5-dien-11ol,18 except for one more oxymethylene signal (δH 4.19, 4.35; δC 64.7). These observations suggested the presence of one more hydroxy group in 6, which was assigned to C-15 based on the HMBC cross-peaks from H2-15 (δH 4.19, 4.35) to C-3 (δC 126.5), C-4 (δC 135.1), and C-5 (δC 140.8). The similar chemical shifts of C-1, C-7, C-8, C-10, and C-11 of 6 (δC 36.8, 48.2, 22.7, 32.1, and 73.2, in CDCl3) and 7αH,10βmethyleudesma-3,5-dien-11-ol (δC 37.3, 48.5, 23.0, 32.3, and 73.3, in CDCl3)18 were used to determine the relative configuration of (7S*,10S*). The absolute configuration was studied based on electronic circular dichroism (ECD) calculations, which were conducted using the Gaussian 09 program.19 The caculated ECD spectra of 6a and its enantiomer 6b are shown in Figure 2. Comparison of the

Figure 2. Experimental ECD spectrum of compound 6 and calculated ECD spectra for 6a and 6b. 301



Article

71.7), two oxygenated tertiary carbons (δC 72.1, 83.4), and a carbonyl group (δC 214.5). These suggested compound 7 to be a rare iphionane-type sesquiterpenoid possessing a carbonyl and three hydroxy groups. The functional groups were assigned according to the 1H−1H COSY and HMBC data, as shown in Figure 1. The 1D NOE experiment showed that irradiation of H-3 (δH 3.16) enhanced the H-6 and H-2α resonances by 0.53% and 1.17%, respectively. Irradiation of H-6 (δH 4.12) enhanced the signals for OH-5 (0.55%), H-3 (0.73%), and H-7 (0.73%). Taken in conjunction with the coupling constant between H-6 and H-7 (J = 2.4 Hz), these observations suggested the α-orientations of H-3, OH-5, H-6, and H-7. If Me-14 were α-oriented, based on a molecular modeling study on CambridgeSoft Chem3D Ultra 10.0, H3-14 would show NOE correlations to H-3, OH-5, and H2-9 and no correlations to any proton of H2-8. However, the irradiation of H3-14 enhanced one of the H2-8 protons. These observations indicated that Me-14 should be β-oriented. Chrysanthemumin H (8) was obtained as colorless crystals with [α]20 D +5 (c 0.2, acetone). It had a molecular formula of C15H22O3, as deduced from an HRESIMS sodium adduct ion at m/z 273.1466 [M + Na]+ (calcd 273.1461). Its 1H and 13C NMR data (Tables 2 and 3) revealed the presence of a vinylic methyl (δH 1.67; δC 108.9, 153.4) and two oxymethine (δH 4.34, 4.51; δC 65.8, 80.4) groups, as well as one oxygenated tertiary carbon (δC 97.8) and three terminal olefinic bonds (δH 4.56, 4.63, 4.88, 5.07, 5.10, 5.29; δC 106.5, 108.9, 112.0, 151.8, 153.4, 155.6). The 1H−1H COSY, HSQC, and HMBC experiments, together with the above information, were used to determine the 2D structure of 8. The 1D NOE experiment showed that the irradiation of H-9 enhanced the signal for H-7, which indicated that H-7 and H-9 were cofacial. Finally, the structure of 8 was confirmed via single-crystal X-ray diffraction analysis (Figure 3A). This is the second naturally occurring 3,9epoxygermacrene-type compound. To determine the absolute configuration of this rare 3,9-epoxygermacrene sesquiterpenoid, ECD calculations were used.19 As shown in Figure 3B, the experimental ECD spectrum of 8 had a similar pattern to the calculated ECD spectrum of 8b, which confirmed the absolute configuration of compound 8 as (1S,3R,7S,9R). Chrysanthemumin I (9), a colorless oil with [α]20 D +10 (c 0.1, acetone), had a molecular formula of C20H28O5, as deduced from the HRESIMS [M + Na]+ ion at m/z 371.1835 (calcd 371.1829). The 1H and 13C NMR data (Table 4) showed signals for three methyls (δH 1.18, 1.42, and 1.97), one trisubstituted olefinic bond (δH 5.47; δC 125.2, 138.6), one epoxy group (δH 2.88; δC 60.0, 60.4), one lactone moiety (δH 5.47; δC 80.2, 179.0), and one angeloyloxy group (δH 1.97, 1.99, 5.28, 6.10; δC 15.7, 20.6, 72.3, 126.9, 139.9, 166.5). The 1H−1H COSY, HSQC, and HMBC experiments (Figure 1), together with the above information, were used to determine the 2D structure of 9. The relative configuration was analyzed via NMR data comparison with reported analogues, coupling constants, and the 1D NOE experiment. Because the H-5 and H-6 signals overlapped at δH 5.47 in CDCl3, the 1H NMR data were measured in acetone-d6 and assigned using the 1H−1H COSY and HMBC experiments (Table 4). The similarity between the coupling patterns of H-3/5/6 of compound 9 (H-3: dd, J = 5.2, 2.0 Hz; H-5: dd, J = 11.2, 1.2 Hz; H-6: br d, J = 11.2 Hz) and those of leptocarpin (H-3: dd, J = 5.0, 2.0 Hz; H-5: dd, J = 11.0, 2.0 Hz; H-6: dd, J = 11.0, 2.0 Hz)20 suggested the αorientations of H-3 and H-7, the β-orientation of H-6, and a Z Δ4(5) double bond. The 1D NOE experiment indicated

Figure 3. (A) ORTEP diagram of the crystal structure of sesquiterpenoid 8. (B) Experimental ECD spectrum of compound 8 and calculated ECD spectra for 8a and 8b.

correlations from H-3 to H-1 and H3-15, from H-6 to H3-14, and from H3-13 to H-7, indicating that H-1, H-7, and H3-13 were α-oriented and H-6 and H3-14 were β-oriented. Chrysanthemumin J (10), a yellow gum with [α]20 D +30 (c 0.1, acetone), was assigned a molecular formula of C17H22O5 based on the HRESIMS [M + Na]+ ion at m/z 329.1366 (calcd 329.1359). Analyses of the 1H and 13C NMR data (Table 4) indicated that 10 had a 2D structure similar to that of the monoacetate of eupahakonin-A,21 with a major difference in the presence of a methyl doublet (δH 1.28, d, J = 6.6 Hz) in 10 instead of an exocyclic methylene. This was confirmed using the 1H−1H COSY, HSQC, and HMBC data. The relative configuration of 10 was assigned via 1D NOE analysis and by comparing chemical shifts with those of analogues. The 1D NOE experiment showed that irradiation of H-8 enhanced H-6 and H-11, which suggested that H-6, H-8, and H-11 were αoriented and Me-13 was β-oriented. Further NOE correlations 302



Article

Table 4. 1H and 13C NMR Data for Compounds 9 and 10 9 no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ CH3CO

δ Ca 60.0, d 31.2, t 72.3, 138.6, 125.2, 80.2, 45.2, 24.4,

d s d d d t

40.7, t 60.4, 36.2, 179.0, 10.4, 16.4, 23.4, 166.5, 126.9, 139.9, 15.7, 20.6,

10

δ Ha

s d s q q q s s d q q

δHb

2.88, 2.62, 1.82, 5.28,

dd (10.0, 4.8) dt (15.6, 4.8) ddd (15.6, 10.0, 2.0) dd (5.6, 2.0)

2.88, 2.69, 1.78, 5.26,

dd (10.4, 4.4) dt (15.6, 4.8) m dd (5.2, 2.0)

5.47, 5.47, 2.12, 1.75, 1.49, 2.41, 1.04,

m m t (10.8) m m m m

5.59, 5.47, 2.25, 1.74, 1.48, 2.32, 1.02,

dd (11.2, 1.2) br d (11.2) br dd (10.8, 9.6) m m m m

2.92, dq (9.2, 7.2)

3.04, dq (9.6, 7.2)

1.18, d (7.2) 1.42, s 1.97, s

1.05, d (6.8) 1.46, s 1.94, d (1.2)

6.10, dq (7.2, 1.2) 1.97, d (1.2) 1.99, dd (7.2, 1.2)

6.11, m 1.94, m 1.94, m

δ Ca 83.0, s 46.6, t 123.2, 142.7, 64.8, 79.0, 51.3, 73.9,

d s d d d d

123.3, d 140.1, 41.0, 177.7, 15.4, 24.0, 17.9,

s d s q q q

21.1, q and 170.3, s

a

δ Hc 2.61, m 2.59, d (10.8, 1.8) 5.46, br s 2.67, 3.94, 2.50, 5.34,

d (10.8) dd (12.0, 10.8) m dd (9.0, 1.8)

5.33, d (1.2)

2.50, m 1.28, d (6.6) 1.94, d (1.2) 1.94, br s

2.12, s

b

Data (δ) were measured in CDCl3 at 400 MHz for protons and at 100 MHz for carbons. Data (δ) were measured in acetone-d6 at 400 MHz. cData (δ) were measured in CDCl3 at 600 MHz. Proton coupling constants (J) in Hz are given in parentheses.

from H3-13 to H-7 and from H-5 to H-7 indicated the βorientations of H-5 and H-7. The chemical shifts of C-1 in 10 (δC 83.3 in acetone-d6) and eupahakonin-A (δC 83.4 in acetone-d6)21 were used to determine that OH-1 and H-5 were cofacial. ECD calculations19 was used to determine the absolute configuration of compound 10. As shown in Figure 4, the experimental ECD spectrum of 10 shared a similar pattern to the calculated ECD spectrum of 10b. This observation determined the absolute configuration of 10 as (1R,5S,6R,7S,8R,11R). On the basis of the literature and spectroscopic data, the known sesquiterpenoids were defined as 11(7→6)abeo-14norcarbrane-4,7-dione (11),11 6,8-cycloeudesm-4(15)-en-1-ol (12),22 (4R,5R)-4,5-dihydroxycaryophyll-8(13)-ene (13),23 11hydroxy-1-oxo-4α,5α,7β,10β-eremophilane (14),24 spathulenol (15),25 chrysanthediol A (16),17 1β-hydroxy-4(15),5E,10(14)germacratriene (17),26 5α-hydroxy-β-eudesmol (18),27 ligucyperonol (19),28 intermedeol (20),29 eudesm-4(15)-ene-1β,6αdiol (21),11 angeloylcumambrin B (22),30 cumambrin A (23),30 11,13-dehydrodesacetylmatricarin (24),31 and matricarin (25).32 PEDV is a type of single-stranded RNA virus that belongs to the Coronaviridae family.33 PEDV infections have severely affected the swine industry in Asian, American, and European countries due to the lack of effective prevention and control approaches.34 Among the three key steps (viral entry, viral replication, and viral assembly and budding) within the life cycle of the virus, viral replication has been considered a promising target for antiviral research because of its core role in the life cycle and the fact that most current antiviral agents target this stage.35 In this study, the sesquiterpenoids from C. indicum flowers were screened for their inhibition against

Figure 4. Experimental ECD spectrum of compound 10 and calculated ECD spectra for 10a and 10b.

PEDV replication using azauridin as the positive control. The cytotoxicity of the compounds was measured in Vero cells. As a result, no cytotoxicity was observed for compounds 1−5, 11, 12, 14−17, 19, and 21 but was observed for compounds 6, 8, 10, 24, and 25 (Supporting Information). Accordingly, the cytopathic effect (CPE) inhibition assay was performed at 40 or 303



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Figure 5. Effects of C. indicum sesquiterpenoids on PEDV replication. Vero cells were injected with PEDV for 2 h, and then the cells were treated with azauridin as the positive control: test compounds (A); compounds 2, 12, and 17 at different concentrations (B). The antiviral activity was determined using a CPE inhibition assay after 3 days of incubation. The data are presented as the mean ± SD (n = 3); *p < 0.05 and **p < 0.01 compared with the virus-injected control group.

50 μM for compounds 1−5, 11, 12, 14−17, 19, and 21 and at 20 μM for cytotoxic compounds. After inoculation with PEDV for 2 h, Vero cells were treated with test compounds and incubated for 3 days. As shown in Figure 5A, compounds 1−5, 12, 14, 16, 17, 19, and 21 protected cells against viral infection. In addition, compounds 2, 12, and 17 also dose-dependently inhibited PEDV replication at concentrations ranging from 20 to 90 μM (Figure 5B). Furthermore, three structurally representative sesquiterpenoids, 2, 12, and 17, were selected and investigated for their potential effects on proteins required for PEDV replication. The virus-infected cells were incubated with or without test compounds at 40 or 45 μM. After 24 h of incubation, the proteins were lysed from cells and analyzed using Western blot, which was performed using nucleocapsid and spike antibodies. As shown in Figure 6A, compounds 2 and 17 were found to significantly reduce PEDV nucleocapsid and spike protein synthesis compared with azauridin as the positive control. However, nucleocapsid and spike protein levels were slightly decreased when the virus-infected cells were treated with compound 12. Compound 2 was measured in detail for its

inhibition of PEDV protein expression, which demonstrated that compound 2 dose-dependently inhibited nucleocapsid and spike protein synthesis at concentrations of 80, 40, 20, and 10 μM (Figure 6B). On the basis of these observations, an immunofluorescence assay was performed to identify the inhibitory effects of compound 2 on PEDV replication. As shown in Figure 7, the virus-injected cells showed a significant green fluorescence compared with the noninjected cells. However, when the virus-injected cells were co-incubated with compound 2 at various concentrations (80, 40, and 20 μM) or azauridin (20 μM) as the positive control, the cells expressing virus antigens were effectively reduced. The results indicated that compound 2 exhibited potential inhibition of the viral protein synthesis in a dose-dependent manner. Herein, a total of nine different sesquiterpenoid frameworks have been discovered in the flowers of C. indicum. Except for eudesmane and germacrane, the remaining seven skeletons were reported from this species for the first time. The C. indicum sesquiterpenoids showed potential antiviral activity on PEDV replication. This study provides a promising natural source, C. indicum, for finding more novel sesquiterpenoids 304



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Article

EXPERIMENTAL SECTION

General Experimental Procedures. The melting points are uncorrected and were determined using an X-4 digital display micromelting point apparatus. A PerkinElmer model 341 polarimeter with a 1 dm cell and a Nicolet NEXUS 670 FT-IR spectrometer were used to measure optical rotation values and IR spectra, respectively. Varian INOVA-300, Varian INOVA-600, and Bruker AVANCE III-400 spectrometers were used to record NMR spectra, with chemical shifts as δ (ppm), using tetramethylsilane as the internal standard. ESIMS data were acquired on an Esquire 6 000 mass spectrometer. HRESIMS data were collected on a Bruker APEX II mass spectrometer. Sephadex LH-20 (GE Healthcare Bioscience AB, Sweden), C18 reverse-phase silica gel (YMC, ODS-A, AA120S50), and silica gel (200−300 mesh, Qingdao Marine Chemical Factory, Qingdao, People’s Republic of China) were used for column chromatography (CC). TLC using silica GF254 (10−40 μm) was detected at 254 nm, and the spots were visualized by spraying with 5% H2SO4 in EtOH (v/v) followed by heating. Plant Material. The flowers of C. indicum were purchased at the Huanghe Medicinal Material Market in Gansu in 2009 and authenticated by Associate Prof. Qi of Lanzhou Institute of Chemical Physics, where a voucher specimen (No. ZY2009C001) was deposited. Extraction and Isolation. The flowers of C. indicum (9.0 kg) were air-dried, pulverized, and extracted with 95% ethanol (3 × 10 L, 3 h each) at 50 °C. After being concentrated in vacuo, the residue was suspended in H2O (2.5 L) and successively partitioned with petroleum ether, EtOAc, and n-BuOH. The petroleum ether fraction (300 g) was subjected to silica gel CC (2.5 kg) with a gradient system of petroleum ether−EtOAc (60:1, 30:1, 15:1, 10:1, 7:1, 5:1, 3:1, and 1:1) to afford eight fractions (A−H) based on TLC analysis. Fraction B was subjected to silica gel CC with petroleum ether−EtOAc (20:1) to yield three subfractions (B1−B3). Further purification of subfraction B3 via silica gel CC elution with petroleum ether−CHCl3 (10:1) yielded a mixture (65 mg), which was further loaded onto preparative TLC (petroleum ether−acetone, 15:1) to yield compounds 15 (10.1 mg) and 20 (25.6 mg). Fraction D was subjected to silica gel CC elution with petroleum ether−EtOAc (15:1) to yield two subfractions (D1 and D2). From subfraction D1, compounds 12 (31 mg) and 17 (45.9 mg) were obtained via repeated silica gel CC elution with petroleum ether−CHCl3 (7:1) and then separated over C18 reversed-phase silica gel using MeOH−H2O (70:30). Subfraction D2 yielded compound 22 (8.9 mg) after fractionation via repeated silica gel CC with petroleum ether−EtOAc (15:1). Fractions E−G were fractionated over Sephadex LH-20 using CHCl3−MeOH (2:1) to yield three subfractions (E1, F1, and G1) without pigments. The fractionation of subfraction E1 via repeated silica gel CC with petroleum ether−CHCl3−EtOAc (10:2:1) and then with petroleum ether−EtOAc (10:1) yielded compound 1 (67.2 mg), compound 11 (10.1 mg), and a mixture of compounds 19 and 21 (35.1 mg). This mixture was further separated via C18 reversedphase silica gel using MeOH−H2O (70:30, 60:40) to yield purified 19 (5.4 mg) and 21 (25.2 mg). Subfraction F1 was separated via silica gel CC and eluted repeatedly with CHCl3−EtOAc (25:1, 15:1, and 10:1) to yield compound 2 (14.3 mg), compound 4 (79.8 mg), and one subfraction, F1A. F1A was further fractionated via silica gel CC and was eluted successively with petroleum ether−EtOAc (10:1) and CHCl3−acetone (15:1) to yield compounds 7 (23.2 mg), 9 (17.2 mg), 10 (4.5 mg), and 13 (6.7 mg) and one more subfraction, F1B. F1B was purified via C18 reversed-phase silica gel (MeOH−H2O, 70:30) to yield compounds 14 (25.3 mg), 16 (34.5 mg), and 18 (98.5 mg) and a mixture (15.7 mg). This mixture was repeatedly purified via C18 reversed-phase silica gel (MeOH−H2O, 60:40) to yield compounds 24 (3.6 mg) and 25 (6.1 mg). Subfraction G1 was fractionated over silica gel and eluted successively with CHCl3−acetone (10:1) and petroleum ether−acetone (8:1.5) to yield compounds 3 (81 mg), 5 (55.3 g), and 6 (16.7 mg) and a mixture that was further purified via C18 reversed-phase silica gel (MeOH−H2O, 65:35) to afford compounds 8 (24 mg) and 23 (45.5 mg). Chrysanthemumin A (1): colorless oil; [α]20 D +40 (c 0.3, acetone); UV (MeOH) λmax (log ε) 205 (2.10) nm; IR (KBr) νmax 3420, 2973,

Figure 6. Western blot analysis demonstrated the inhibition by sesquiterpenoids of nucleocapsid and spike protein synthesis. The virus-injected cells were treated with compounds 2, 12, and 17 at a concentration of 40 or 45 μM (A) and compound 2 at different concentrations of 80, 40, 20, and 10 μM (B). After 24 h of incubation, the target proteins were evaluated via Western blot. The data are expressed as the mean ± SD (n = 3); *p < 0.05 and **p < 0.01 compared with the virus-injected nucleocapsid protein control group, while #p < 0.05 and ##p < 0.01 compared with the virus-injected spike protein control group.

with potential antiviral activity on PEDV replication by inhibiting the expression of viral proteins. 305



Article

Figure 7. (A) Morphology of Vero cells showing the effects of compounds 2, 12, and 17 on the PEDV-induced CPE inhibition assay. (B, C) Immunofluorescence assay of PEDV-infected cells with nucleocapsid (B) and spike (C) antibodies. The virus-injected cells were incubated with compound 2 at different concentrations for 24 h. The cells were fixed, permeabilized, and stained with monoclonal antibody directed against nucleocapsid or spike protein. 2943, 2928, 2871, 1641, 1567, 1458, 1374, 1168, 962, 733 cm−1; 1H and 13C NMR data, Table 1; ESIMS m/z 277.2 [M + Na]+; HRESIMS m/z 272.2214 [M + NH4]+ (calcd for C15H30O3N, 272.2220). Chrysanthemumin B (2): colorless oil; [α]20 D −13 (c 0.2, acetone); UV (MeOH) λmax (log ε) 206 (2.08) nm; IR (KBr) νmax 3369, 2928, 2866, 1641, 1454, 1377, 1170, 1047, 952, 907 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 277.1778 [M + Na]+ (calcd for C15H26O3Na, 277.1774). Chrysanthemumin C (3): white solid; [α]20 D +15 (c 0.2, acetone); UV (MeOH) λmax (log ε) 206 (2.92) nm; IR (KBr) νmax 3357, 2928, 2859, 1646, 1442, 1381, 1152, 1040, 1016, 892 cm−1; 1H NMR data, Table 2; 13C NMR data, Table 3; HRESIMS m/z 259.1677 [M + Na]+ (calcd for C15H24O2Na, 259.1669). Chrysanthemumin D (4): colorless oil; [α]20 D +7 (c 0.3, acetone); IR (KBr) νmax 3422, 2943, 2869, 1706, 1458, 1376, 1171, 1129, 1043, 993, 910 cm−1; 1H NMR data, Table 2; 13C NMR data, Table 3; HRESIMS m/z 288.2162 [M + NH4]+ (calcd for C15H30O4N, 288.2169). Chrysanthemumin E (5): colorless oil; [α]20 D +40 (c 0.3, acetone); IR (KBr) νmax 3365, 2944, 1654, 1459, 1379, 1277, 1152, 1051, 1023, 927 cm−1; 1H NMR data, Table 2; 13C NMR data, Table 3; HRESIMS m/z 293.1731 [M + Na]+ (calcd for C15H26O4Na, 293.1723). Chrysanthemumin F (6): yellow gum; [α]20 D +4 (c 0.3, acetone); UV (MeOH) λmax (log ε) 205 (2.48), 219 (2.05) nm; IR (KBr) νmax 3525, 3414, 3005, 2922, 1713, 1648, 1423, 1363, 1223, 1093, 531 cm−1; 1H NMR data, Table 2; 13C NMR data, Table 3; HRESIMS m/z 259.1675 [M + Na]+ (calcd for C15H24O2Na, 259.1669). Chrysanthemumin G (7): colorless oil; [α]20 D +5 (c 0.2, acetone); IR (KBr) νmax 3402, 2940, 2872, 1690, 1446, 1362, 1167, 1093, 960 cm−1; 1 H NMR data, Table 2; 13C NMR data, Table 3; HRESIMS m/z 293.1728 [M + Na]+ (calcd for C15H26O4Na, 293.1723). Chrysanthemumin H (8): colorless crystal; mp 111−113 °C; [α]20 D +5 (c 0.2, acetone); UV (MeOH) λmax (log ε) 204 (3.04), 220 (2.76) nm; IR (KBr) νmax 3371, 2923, 2856, 1664, 1645, 1446, 1094, 1077, 1027, 908, 889 cm−1; 1H NMR data, Table 2; 13C NMR data, Table 3; HRESIMS m/z 273.1466 [M + Na]+ (calcd for C15H22O3Na, 273.1461). Chrysanthemumin I (9): colorless oil; [α]20 D +10 (c 0.1, acetone); UV (MeOH) λmax (log ε) 205 (3.29), 223 (2.57) nm; IR (KBr) νmax 3405, 2931, 1766, 1716, 1450, 1382, 1233, 1151, 1038, 966 cm−1; 1H and 13C NMR data, Table 4; HRESIMS m/z 371.1835 [M + Na]+ (calcd for C20H28O5Na, 371.1829). Chrysanthemumin J (10): yellow gum; [α]20 D +30 (c 0.1, acetone); UV (MeOH) λmax (log ε) 206 (3.30) nm; IR (KBr) νmax 3405, 2924, 1772, 1742, 1660, 1377, 1239, 1157, 1023, 960, 906 cm−1; 1H and 13C

NMR data, Table 1; HRESIMS m/z 329.1366 [M + Na]+ (calcd for C17H22O5Na, 329.1359). X-ray crystallographic data of 8 (CCDC 797080): colorless crystal from petroleum ether−acetone (3:1), C15H22O3, orthorhombic, space group C2221, M = 250.33, a = 6.6634(17) Å, b = 13.334(4) Å, c = 31.049(8) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 2758.8(12) Å3, Z = 8, Dx = 1.205 mg/m3. Graphite-monochromated Mo Kα radiation (λ = 0.710 73 Å) was used to analyze a crystal with dimensions of 0.22 × 0.19 × 0.10 mm on a Bruker Axs Smart APEX II imaging plate area detector. Among the 2548 independent reflections measured, 2108 were considered to be observed (|F|2 > 2σ|F|2). Final indices: R(gt) = 0.0455, Rw = 0.1193 [w = 1/[σ2(Fo2) + (0.1000P)2 + 1.0443P], where P = (Fo2 + 2Fc2)/3]. The structure was solved, expanded, and refined using direct methods (SHELXL-97), Fourier techniques, and the NOMCSDP program with full-matrix least-squares calculations. Computational Methods and Details for the ECD Spectra of 8. To define the absolute configurations, the Gaussian 09 suite of programs was used for quantum mechanical calculations on the basis of the B3LYP/6-311++G(d,p) level of theory.19 The B3LYP functional was employed.36 On the basis of the optimized geometries at the above level, the B3LYP/6-311++G(d,p) method was used for the ECD calculations by TDDFT. The IEFPCM solvation model was used to simulate the experimental conditions, and MeOH was used as the solvent for the optimizations and ECD calculations. Cell Culture and Virus Stock. This part is the same as the reported method.37 Cytotoxicity Assay and Cytopathic Effect Inhibition Assay. These assays were conducted according to published protocols37 with modifications (Supporting Information). Western Blot Analysis. This assay was conducted according to a published protocol37 with modifications (Supporting Information). Immunofluorescence Assay. This assay was conducted according to a published protocol37 with modifications (Supporting Information). Statistical Analysis. This part is the same as the reported method.37

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00694. 1D and 2D NMR spectra of the new compounds 1−10; cytotoxicity assessment of the isolated compounds as 306



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Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision, A. 02; Gaussian, Inc.: Wallingford, CT, 2009. (20) Martínez, J. R.; Ayamante, B. I. S.; Núñez-Alarcón; Juana, A.; de Vivar, A. R. Phytochemistry 1979, 18, 1527−1528. (21) Ito, K.; Sakaibara, Y.; Haruna, M. Phytochemistry 1982, 21, 715− 720. (22) Yamamura, S.; Niwa, M.; Ito, M.; Saito, Y. Chem. Lett. 1982, 11, 1681−1684. (23) Collado, I. G.; Hanson, J. R.; Hitchcock, P. B.; Macías-Sánchez, A. J. J. Org. Chem. 1997, 62, 1965−1969. (24) Arantes, S. F.; Hanson, J. R.; Hitchcock, P. B. Phytochemistry 1999, 52, 1063−1067. (25) Wang, X. R.; Wu, Q. X.; Shi, Y. P. Chem. Biodiversity 2008, 5, 279−289. (26) Brown, G. D.; Liang, G. Y.; Sy, L. K. Phytochemistry 2003, 64, 303−323. (27) El-Askary, H. I.; Meselhy, M. R.; Galal, A. M. Molecules 2003, 8, 670−677. (28) Naya, K.; Okayama, T.; Fujiwara, M.; Nakata, M.; Ohtsuka, T.; Kurio, S. Bull. Chem. Soc. Jpn. 1990, 63, 2239−2245. (29) Salgueiro, L. R.; Vila, R.; Tomi, F.; Tomas, X.; Caňigueral, S.; Casanova, J.; Proenca da Cunha, A.; Adzet, T. Phytochemistry 1997, 45, 1177−1183. (30) Jang, D. S.; Yang, M. S.; Ha, T. J.; Park, K. H. Arch. Pharmacal Res. 1998, 21, 591−594. (31) Ohno, N.; Gershenzon, J.; Roane, C.; Mabry, T. J. Phytochemistry 1980, 19, 103−106. (32) Ahmed, A. A.; Gati, T.; Hussein, T. A.; Ali, A. T.; Tzakou, O. A.; Couladis, M. A.; Mabry, T. J.; Toth, G. Tetrahedron 2003, 59, 3729− 3735. (33) Wang, Y.; Li, J.; Sun, M.; Ni, B.; Huan, C.; Huang, L.; Fan, H.; Ren, X.; Mao, X. Antiviral Res. 2014, 106, 33−41. (34) Mole, B. Nature 2013, 499, 388. (35) (a) Belema, M.; Meanwell, N. A. J. Med. Chem. 2014, 57, 5057− 5071. (b) Gigante, A.; Canela, M.-D.; Delang, L.; Priego, E.-M.; Camarasa, M.-J.; Querat, G.; Neyts, J.; Leyssen, P.; Pérez-Pérez, M.-J. J. Med. Chem. 2014, 57, 4000−4008. (36) Berova, N.; Di Bari, L.; Pescitelli, G. Chem. Soc. Rev. 2007, 36, 914. (37) Yang, J. L.; Ha, T. K. Q.; Dhodary, B.; Pyo, E.; Nguyen, N. H.; Cho, H.; Kim, E.; Oh, W. K. J. Med. Chem. 2015, 58 (3), 1268−1280.

analyzed by an MTT assay, and the procedures for the cytotoxicity assay, CPE inhibition assay, Western blot analysis, and immunofluorescence assay (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail (J.-L. Yang): [email protected]. *Tel (Y.-P. Shi): +86-931-4968208. Fax: +86-931-4968088. Email: [email protected]. ORCID

Jun-Li Yang: 0000-0001-7199-0214 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Prof. P.-P. Zhou at Lanzhou University for calculating the ECD spectra. This work was financially supported by the International Partnership Program of Chinese Academy of Sciences (No. 153631KYSB20160004), National Natural Science Foundation of China (No. 81673325), CAS Pioneer Hundred Talents Program, and scientific research project of the Central Asia Drug Discovery and Development Centre of the Chinese Academy of Sciences (CAM 201404).

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REFERENCES

(1) Wu, Q. X.; Shi, Y. P.; Jia, Z. J. Nat. Prod. Rep. 2006, 23, 699−734. (2) Yoshikawa, M.; Morikawa, T.; Murakami, T.; Toguchida, I.; Harima, S.; Matsuda, H. Chem. Pharm. Bull. 1999, 47, 340−345. (3) Matsuda, H.; Morikawa, T.; Toguchida, I.; Harima, S.; Yoshikawa, M. Chem. Pharm. Bull. 2000, 48, 651−656. (4) Matsuda, H.; Morikawa, T.; Toguchida, I.; Harima, S.; Yoshikawa, M. Chem. Pharm. Bull. 2002, 50, 972−975. (5) Xiao, X. H. Chin. Tradit. Herb. Drugs 2003, 34, 669−671. (6) Zheng, H. P.; Yang, Z.; Zhang, F. C.; Zhang, A. M.; Wang, J.; Hong, W. X.; Chen, J. F.; Tan, X. H. China J. Tradit. Chin. Med. Pharm. 2010, 25, 780−782. (7) Feng, Z. M.; Song, S.; Xia, P. F.; Jiang, J. S.; Zhang, P. C. Helv. Chim. Acta 2009, 92, 1823−1828. (8) Chen, W. H.; Qi, H. Y.; Shi, Y. P. J. Nat. Prod. 2009, 72, 1410− 1413. (9) Chen, W. H.; Wu, Q. X.; Wang, R.; Shi, Y. P. Phytochemistry 2010, 71, 1002−1006. (10) Liu, L. L.; Wang, R.; Yang, J. L.; Shi, Y. P. Helv. Chim. Acta 2010, 93, 595−601. (11) Wang, R.; Liu, L. L.; Shi, Y. P. Helv. Chim. Acta 2010, 93, 2081− 2085. (12) Wang, R.; Chen, W. H.; Shi, Y. P. J. Nat. Prod. 2010, 73, 17−21. (13) Yang, J. L.; Liu, L. L.; Shi, Y. P. Planta Med. 2011, 77, 271−276. (14) (a) Demarco, P. V.; Farkas, E.; Doddrell, D.; Mylari, B. L.; Wenkert, E. J. Am. Chem. Soc. 1968, 90, 5480−5486. (b) Su, B. N.; Misico, R.; Park, E. J.; Santarsiero, B. D.; Mesecar, A. D.; Fong, H. H. S.; Pezzuto, J. M.; Kinghorn, A. D. Tetrahedron 2002, 58, 3453−3466. (15) Liu, L. L.; Wang, R.; Yang, J. L.; Shi, Y. P. Chin. J. Chem. 2012, 30, 1255−1260. (16) Li, X.; Yang, M.; Han, Y.-F.; Gao, K. Planta Med. 2005, 71, 268− 272. (17) Hu, L. H.; Chen, Z. L. Phytochemistry 1997, 44, 1287−1290. (18) Barrero, A. F.; Mar Herrador, M.; Quilez, J. F.; AlvarezManzaneda, R.; Portal, D.; Gavin, J. A.; Gravalos, D. G.; Simmonds, M. S. J.; Blaney, W. M. Phytochemistry 1999, 51, 529−541. (19) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; 307


Sesquiterpenoids with Various Carbocyclic Skeletons from the

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