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Sesquiterpenoids from Artemisia vestita
Phytochemistry 147 (2018) 194e202
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Sesquiterpenoids from Artemisia vestita Shuai-Hua Tian 1, Chen Zhang 1, Ke-Wu Zeng, Ming-Bo Zhao, Yong Jiang**, Peng-Fei Tu* State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 September 2017 Received in revised form 14 December 2017 Accepted 5 January 2018
Eleven previously undescribed sesquiterpenoids, arvestolides DeJ, arvestonates AeC, and arvestonol were isolated from the aerial parts of Artemisia vestita W. Their structures were elucidated by extensive analysis of HRMS, UV, IR, and NMR spectroscopic data, and the absolute configurations were determined by single crystal X-ray diffraction, empirical rules, and comparison of calculated and experimental ECD data. Arvestolides H and I showed inhibitory effects on nitric oxide production in BV-2 cells induced by lipopolysaccharide with IC50 values of 43.2 and 39.9 mM, respectively. © 2018 Elsevier Ltd. All rights reserved.
Keywords: Artemisia vestita W. Compositae Sesquiterpenoid NO inhibition
1. Introduction Artemisia vestita W., belonging to the Composite family, is a wild bush endemic to the western high-altitude area of China, and its aerial parts have been traditionally used as a folk medicine for the treatment of inflammatory diseases (Wu and Peter, 1992). Sesquiterpenoids, as a type of characteristic metabolites of Artemisia species, have been reported for their diverse structures and antiinflammatory, cytotoxic, and anti-virus bioactivities (Braulio, 2011; Kawazoe et al., 2003; Ma et al., 2000; Wang et al., 2013). A number of anti-inflammatory and cytotoxic sesquiterpenoids and their dimers formed via Diels-Alder [4þ2] cycloaddition have been isolated from the Artemisia species in our previous studies (Tian et al., 2013a, 2013b; Turak et al., 2014; Zan et al., 2012; Zhang et al., 2014, 2016). As an ongoing search for active sesquiterpenoids and their derivatives from the Artemisia genus, the 95% aqueous EtOH extract of A. vestita was phytochemically investigated and afforded 11 previously undescribed sesquiterpenoids, including eight eudesmanes (1e5 and 7e9), one germacranolide (6), one nor-dilactone (10), and one seco-sesquiterpenoid (11). Their structures were elucidated by HRESIMS, IR, UV, and NMR spectroscopic data. The absolute configurations of previously undescribed
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Jiang), [email protected] (P.-F. Tu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.phytochem.2018.01.004 0031-9422/© 2018 Elsevier Ltd. All rights reserved.
compounds were established based on single-crystal X-ray diffraction, empirical rules, and comparison of the calculated and experimental ECD spectra. Compounds 1e11 were tested for their cytotoxic and nitric oxide (NO) inhibitory effects on BV-2 cells induced by lipopolysaccharide (LPS). Only compounds 5 and 6 exhibited weak NO inhibitory effects with IC50 values of 43.2 and 39.9 mM, respectively.
2. Results and discussion The powdered aerial parts of A. vestita (45 kg) were extracted with 95% aq. EtOH and partitioned with petroleum ether and CHCl3, successively. The CHCl3-soluble portion, which was analyzed by LC/ MS to be rich of sesquiterpenoids, was subjected repeatedly to silica gel, Sephadex LH-20, and ODS column chromatography (CC), followed by semipreparative HPLC to afford 11 previously undescribed sesquiterpenoids (1e11) (see Fig. 1). Arvestolide D (1) was obtained as a white amorphous powder. A sodium adduct ion [M þ Na]þ at m/z 345.1317 (calcd. 345.1314) in the HRESIMS indicated a molecular formula of C17H22O6, requiring seven indices of hydrogen deficiency. The IR spectrum showed absorption bands for hydroxy (3381 cm1) and lactone (1738, 1056 cm1) functionalities. The resonances for olefinic and oxygenated protons at dH 5.96 (1H, br, s), 5.68 (1H, s), and 4.39 (1H, d, J ¼ 11.6 Hz) were observed along with four methyl signals at dH 2.21 (3H, s), 2.05 (3H, s), 1.30 (3H, s), and 1.26 (3H, d, J ¼ 6.8 Hz) in the 1H NMR spectrum. A total of 17 carbon resonances in the 13C NMR spectrum were classified into four methyl, two methylene,
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five methine, and six quaternary carbons by the multi-edited HSQC spectrum. Among them, signals for carbonyl (dC 191.1, 177.2, and 170.3), double bond (dC 128.5 and 155.4), and oxygenated (dC 83.8, 77.4, and 75.7) carbons were assigned via their chemical shifts. These 1D NMR data were similar to those of 1-acetyleudesma-2oxo-3-en-6,12-olide (Anake et al., 1992), and their difference was one more oxygenated tertiary carbon resonance at dC 75.7 in 1. The HMBC correlations from H3-15 to C-3, C-4, and C-5 and from H-3 to C-1, C-2, and C-5 assigned this carbon to be C-5 at which a hydroxy group was attached. Thus, 1 was deduced as 1-O-acetyleudesma-5hydroxy-2-oxo-3-en-6,12-olide. The relative configuration of 1 was established by the coupling constants and NOE correlations. The NOE cross-peaks of H3-14/H-6, H-6/H-11, and H3-14/H-1 indicated that they were cofacial and arbitrarily assigned as b-orientation. The large coupling constants of H-6/H-7 (J ¼ 11.6 Hz) and H-7/H-11 (J ¼ 13.6 Hz) suggested H-7 to be a-oriented, which was also supported by the NOE cross-peaks of H-7/H3-13 and H-7/H-9a (Fig. 2).
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Based on the NOE correlations of 5-OH/H-1, H3-14, and H-6 determined in DMSO-d6, the relative configuration of 5-OH was deduced as b-oriented. The positive Cotton effect at 245 nm in the ECD spectrum, arising from the p-p* orbital transition of a,b-unsaturated ketene, indicated a positive helicity as shown in Fig. 3 according to the a,b-unsaturated ketone helicity rule (Kirk, 1986). While a negative Cotton effect at 323 nm (Dε 3.2) mainly ascribed to the n-p* orbital transition of the conjugated ketene was determined in terms of the Octant rule (Pei, 2015) (Fig. 3). Both of these two Cotton effects assigned the absolute configuration of 1 to be (1R,5S,6S,7S,10S,11S), which was also corroborated by comparison of the experimental and calculated ECD data (Fig. 4). The calculated ECD spectrum of (1R,5S,6S,7S,10S,11S)-1 agreed well with the experimental one. Therefore, the structure of arvestolide D (1) was determined as (1R,5S,6S,7S,10S,11S)-1-O-acetyleudesma-5hydroxy-2-oxo-3-en-6,12-olide. Arvestolide E (2) was obtained as a white gum, and its molecular formula was determined as C17H22O6 by the HRESIMS deprotonated ion [M e H]e at m/z 321.1328 (calcd. 321.1338), indicating seven indices of hydrogen deficiency. The IR spectrum showed the presence of carbonyl (1774 cm1) and lactone (1739 and 1068 cm1) functionalities. The 13C NMR spectrum presented signals for an a,bunsaturated lactone group (dC 126.3, 154.4, and 170.9), an acetyl (dC 170.5 and 21.1), four oxygenated carbons (dC 70.1, 72.9, 75.4, and 89.6), four methylene, three methyl, and an aliphatic quaternary carbons with the aid of HSQC experiment. These data indicated that 2 is a sesquiterpenoid similar to 13-acetoxyeudesma-4-hydroxy7,11-en-6,12-olide (Irwin and Geissman, 1973). Their differences are the acetoxyl position and one more oxygenated tertiary carbon resonance in the 13C NMR data of 2. The existence of 1-O-acetyl was
Fig. 1. Structures of compounds 1e11.
Fig. 2. Key 1H-1H COSY, HMBC, and NOESY correlations of arvestolide D (1).
Fig. 4. Comparison of the calculated ECD spectra of (1R,5S,6S,7S,10S,11S)-1 and its enantiomer with the experimental ECD spectrum of 1.
Fig. 3. Helicity and Octant rules applied for the Cotton effects at 245 nm (p/p*) and 323 nm (n/p*) of ECD spectrum of 1.
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deduced from the HMBC correlation from H-1 to the acetyl carbon (dC 170.5). Based on the HMBC correlations of H3-15/C-5 and H3-14/ C-5, an epoxy between C-5 and C-6 was deduced from the remarkable deshielded chemical shifts of C-5 (dC 70.1) and C-6 (dC 89.6) and the indices of hydrogen deficiency of 2. Thus, the planar structure of 2 was elucidated as 1-acetoxyeudesma-4-hydroxy-5,6epoxy-7,11-en-6,12-olide. The NOE correlations of H3-14/H-1, H314/H-2b, and H3-15/H-3a indicated that H-1, H3-14, and 4-OH were b-oriented. It is difficult to assign the a- or b-orientation of epoxy (C-5-O-C-6) through NOEs, whereas, C-5 and C-6 configurations were found to have profound influence on the Cotton effect at around 250 nm in the ECD spectrum. By comparison of the calculated ECD spectra of the four stereoisomers of 2 (Fig. S25, Supporting Information) with the experimental one, the absolute configurations of C-5 and C-6 were determined as 5S and 6R, while the other stereocenters could not be determined since their minor attributions to the Cotton effects. The structure of arvestolide E (2) was thus elucidated as shown. Arvestolide F (3) was obtained as colorless needles with a molecular formula of C15H18O5 determined by a protonated ion [M þ H]þ at m/z 279.1227 (calcd. 279.1232) in the HRESIMS. The IR spectrum showed absorption bands at 1772, 1677, and 1054 cm1 ascribable to the carbonyl and lactone functionalities. The 1H and 13 C NMR data of 3 were similar to those of a-santonin (Fujimoto et al., 1979), indicating a similar eudesmane-type structure of 3. Comparison of the NMR data with those of a-santonin, an oxymethylene [dH 4.65 (1H, d, J ¼ 12.0 Hz) and 4.06 (1H, d, J ¼ 12.0 Hz); dC 58.1] and a pair of oxygenated quaternary carbons (dC 65.0 and 69.8) in 3 substituted the signals of a methyl and a pair of olefinic carbons of a-santonin. These differences were further supported by 2D NMR spectra, especially by HMBC correlations. The oxymethylene was assigned as the hydroxymethyl of CH2-15 based on its HMBC correlations with C-3, C-4, and C-5. Two oxygenated carbons, C-4 and C-5, were elucidated to form an epoxy moiety by the HMBC correlations of H3-14/C-5, H2-15/C-3/C-4/C-5, and H-7/C5, together with the indices of hydrogen deficiency. Namely, the 4,5-double bond of a-santonin was oxygenated into an epoxy group in 3. In the NOESY spectrum, the cross-peaks of H-11/H-6, H-6/H8b/H3-14, H-7/H-9a, and H-7/H3-13, together with the coupling constants of JH-6/H-7 (11.5 Hz), JH-7/H-11 (12.5 Hz), and JH-7/H-8b (12.8 Hz) indicated the same relative configurations of H-6b, H-7a, and H3-14b as those of a-santonin. The NOEs of H2-15/H-7 indicated the epoxy has a b-orientation and the two rings of A and B were cis-fused. The relative configuration of 3 was further defined by the X-ray diffraction analysis. Due to the large Flack parameter [0.2(2)], the calculated ECD spectra (Fig. S35, Supporting Information) were adopted to determine the absolute configuration of arvestolide F (3) as (4R,5S,6S,7S,10S,11S)-eudesma-15-hydroxy-3keto-1,2-en-4,5-epoxy-6,12-olide. Arvestolide G (4) was obtained as a white amorphous powder. A sodium adduct ion [M þ Na]þ at m/z 331.1524 (calcd. 331.1521) in the HRESIMS indicated a molecular formula of C17H24O5. In the 1H NMR spectrum, two methyl (dH 1.26, 3H, d, J ¼ 7.0 Hz; 1.22, 3H, s), an acetoxy methyl (dH 2.08, 3H, s), two oxygenated methine (dH 4.79, 1H, dd, J ¼ 5.1, 2.5 Hz; 4.62, 1H, d, J ¼ 11.3 Hz), and one oxymethylene (dH 4.40, 1H, d, J ¼ 12.1 Hz; 4.12, 1H, d, J ¼ 12.1 Hz) protons were observed. Seventeen carbon resonances were assigned as three methyl, five methylene, four methine, and five quaternary carbons with the help of multi-edited HSQC spectrum, including a pair of vinyl carbons (dC 130.1 and 131.6), a lactone carbon (dC 178.0), and an acetyl group (dC 170.7 and 21.2). Collectively, these data were similar to those of 1,15-dihydroxyeudesma4,5-11,13-dien-6,12-olide (Krautmann et al., 2007), except that the 1-OH was acetylated, and the exocyclic double bond D11(13) was hydrogenated to a methyl in 4. These deductions were supported
by the HMBC correlations from H-1 to the acetyl carbon (dC 170.7) and H3-13 to C-11/C-7/C-12. The NOEs of H3-14/H-6/H-8b, H3-14/H1, H-6/H-11, H-7/H-9a, and H-7/H3-13 indicated the same relative configuration of 4 as that of 1,15-dihydroxyeudesma-4,5-11,13dien-6,12-olide (Krautmann et al., 2007) or 1. The absolute configuration of 4 was determined as (1S,6S,7S,10R,11S) by comparison of the calculated and experimental ECD data (Fig. S45, Supporting Information). Thus, the structure of arvestolide G (4) was determined as (1S,6S,7S,10R,11S)-1-O-acetyl-eudesma- 15hydroxyl-4,5-en-6,12-olide. Arvestolide H (5) was obtained as a white amorphous powder. The HRESIMS spectrum showed a sodium adduct ion [M þ Na]þ at m/z 371.1821 (calcd. 371.1834) corresponding to a molecular formula of C20H28O5. The 1H NMR spectrum presented an olefinic proton at dH 6.16 (1H, q, J ¼ 7.0 Hz), two terminal vinyl protons at dH 5.22 (1H, s) and 5.06 (1H, s), three oxygenated methine at dH 4.85 (1H, t, J ¼ 3.0 Hz), 4.30 (1H, t, J ¼ 3.0 Hz), and 4.05 (1H, t, J ¼ 10.9 Hz), and four methyl signals at dH 0.94 (3H, s), 1.25 (3H, d, J ¼ 7.0 Hz), 1.97 (3H, s), and 2.06 (3H, d, J ¼ 7.0 Hz). The corresponding carbon signals for oxymethine (dC 72.3, 75.5, and 79.2), vinyl (dC 113.3, 127.4, 139.7, and 145.3), methyl (dC 12.5, 15.9, 17.8, and 20.8), and carbonyl groups (dC 166.7 and 179.1) were observed in the 13C NMR spectrum. These 1D NMR data indicated that 5 is a sesquiterpenoid similar to 1-acetylerivanin (Mengi et al., 1991) except that the acetyl group is replaced by an angelica acyl group in 5. This deduction was supported by the HMBC correlation from H-1 to the angelica acyl carbon (dC 166.7). The NOE cross-peaks of H314/H-1, H-3, H-6, H-6/H-11, H-5/H-7, and H-7/H3-13, together with the coupling constants of H-6 (1H, t, J ¼ 10.9 Hz) and H-3 (1H, t, J ¼ 3.0 Hz) indicated 5 has the same relative configuration as that of 1-acetylerivanin. The ECD spectrum (Fig. S55, Supporting Information) of 5 showed the Cotton effects at 206 nm (Dε þ2.0) and 300 nm (Dε 2.7), deriving from a p/p* electron transition of the terminal vinyl of D4,15 and a n/p* electron transition of the angelica acyl. According to the Olefin octant rule (Scott and Wrixon, 1970), the positive Cotton effect reveals an S configuration of C-5 (Fig. S55, Supporting Information). The calculated ECD spectra indicated that the spectrum of (1S,3R,5S,6S,7S,10R,11S)-5 agreed well with the experimental one (Fig. S55, Supporting Information). Thus, the structure of arvestolide H (5) was deduced as (1S,3R,5S,6S,7S,10R,11S)-1-O-angelica acyl-eudesma-3-hydroxy4,15-en-6,12-olide. Arvestolide I (6) was obtained as colorless needles from tetrahydrofuran-water system. Its molecular formula was determined as C17H24O5 by a sodium adduct ion [M þ Na]þ at m/z 331.1517 (calcd. 331.1521) in the HRESIMS. The 1H and 13C NMR data revealed the presence of vinyl (dH 5.51; dC 126.2, and 143.5), carbonyl (dC 202.4), lactone (dC 177.3), and acetyl (dH 2.01; dC 21.6 and 170.3) groups. The proton spin systems from H-1 to H-3, H-6 to H-10, and H-1 and H-10 were connected through 1H-1H COSY correlations. By comparison of the 13C NMR data with those of asantonin and its analogues in literature (Shimizu et al., 1979), 6 was deduced as a 5-keto germacranolide. 1-O-acetyl was assigned via HMBC correlation from H-1 to the acetyl group (dC 170.3). The vinyl proton (dH 5.51) showed HMBC correlations with C-1, C-5, and C-15, suggesting a double bond existed between C-3 and C-4. Thus, 6 was elucidated as 1-O-acetyl-3-en-5-keto-germacranolide. The relative configuration of 6 was established by NOESY spectrum. The crosspeaks of H-1/H-3, H-6/H-10, and H-6/H3-15 indicated that the 10membered ring moiety has a boat-chair conformation (Pui-Hay et al., 2009) and these protons were assigned as b-oriented. The large coupling constant (J ¼ 9.7 Hz) between H-6 and H-7 suggested that H-7 is a-oriented. The a-orientation of H3-13 was elucidated by the NOE correlation of H-7/H3-13 and the large coupling constant (J ¼ 12.2 Hz) between H-7 and H-11. The absolute
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configuration of 6 was finally determined as (1S,6S,7S,10R,11S) by the X-ray crystallography (Fig. 5). This result was further confirmed by comparison of the experimental and calculated ECD spectra (Fig. S65, Supporting Information). Therefore, the structure of arvestolide I (6) was defined as (1S,6S,7S,10R,11S)-1-O-acetyl-3-en5-keto-germacranolide. Arvestonate A (7), obtained as a colorless gum, has a molecular formula of C16H26O5 determined by the HRESIMS (m/z 321.1682 [M þ Na]þ, calcd. 321.1678) and 13C NMR data. The NMR data of 7 were almost the same with those of crossostephin (Wu et al., 2009), a eudesmane sesquiterpenoid. Their difference was the terminal D11(13) double bond of crossostephin was hydrogenated into a methyl group in 7, which was supported from the HMBC correlations from H3-13 to C-7, C-11, and C-12. The NOE correlations of H314/H-6 and the coupling constant (J ¼ 10.4 Hz) between H-6 and H7 both indicated the same configurations of H3-14b, H-6b, and H-7a as those of crossostephin. The coupling constants of 11.5 and 5.2 Hz between H-1 and H2-2 suggested that H-1 was axial and a-oriented, which was corroborated by the NOE correlation between H1 and H-9a. The configuration of 5-OH determined the trans- or cisfused pattern of the eudesmane, which was found to be closely related with the ECD Cotton effect of 7. The positive Cotton effect at 205 nm of 7 (Fig. 6) revealed an S configuration based on the Olefin octant rule (Scott and Wrixon, 1970). By comparison of the experimental and calculated ECD spectra (Fig. S75, Supporting Information), the absolute configuration of 7 was finally established as (1R,5R,6S,7S,10S). However, the configuration of H-11 is hard to determine from the present data. Therefore, the structure of 7 was determined as Methyl (1R,5R,6S,7S,10S)-1,5,6-trihydroxy-eudesma4,15-en-12-oate. Arvestonate B (8) has a molecular formula of C18H28O6 determined from a sodium adduct ion [M þ Na]þ at m/z 363.1777 (calcd. 363.1784) in the HRESIMS and the 13C NMR data. The similar IR and NMR data with those of 7 indicated that they shared the same
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eudesmane scaffold. A group of additional acetyl signals were observed in 8 in comparison with 7. The 1-O-acetyl was assigned from the HMBC correlation from H-1 to the acetyl group. The NOE correlations of H3-14/H-1 and H3-14/H-6 and the coupling constant between H-6 and H-7 (J ¼ 11.5 Hz) defined a relative configuration of H-1b, H3-14b, H-6b, and H-7a. However, a negative Cotton effect at 205 nm in the ECD spectrum of 8 was inconsistent with that of 7. After changing solvent to DMSO-d6, the NOE correlations of 5-OH (dH 4.55) with H-1, H-6, and H3-14 indicated that they are cofacial and the two rings are cis-fused. By comparison of the structures between 7 and 8, the configuration of C-5 was found to have a determined effect on the conformation of methylenecyclohexane moiety and thus these two compounds have a mirror-like Cotton effect at around 205 nm in the ECD spectra (Fig. 6). The quantum chemical calculation of ECD spectra and Olefin octant rule indicated that the absolute configuration of 8 is (1S,5S,6S,7S,10S) (Fig. S85, Supporting Information). The configuration of C-11 is also hard to determine exactly since its flexible residue conformation. Arvestonol (9) was obtained as a colorless gum. Its molecular formula was determined as C15H24O3 based on the 13C NMR data and a protonated ion at m/z 253.1816 (calcd. 253.1804) in the HRESIMS. The IR spectrum showed absorption bands for hydroxy (3351 cm1) and conjugated carbonyl (1741 cm1) groups. The 1H and 13C NMR data indicated that 9 is an eudesmane sesquiterpenoid similar to 3-oxo-eudesma-4,5-en-9,12-diol (Liu et al., 1998). Their difference was the position of the hydroxy group, which shifted from C-9 to C-1 in 9, as proved by the HMBC correlations from H-1 (dH 3.81) to C-3 (dC 197.2) and C-5. Thus, the structure of 9 was defined as 3-oxo-eudesma-4-en-1,12-diol. The relative configuration was established by NOESY spectrum. The cross-peaks of H3-14/H-6b/H-8b/H-7 indicated that H-7 was b-oriented. The coupling constants between H-1 and H2-2 (J ¼ 12.8 and 5.3 Hz) indicated H-1 was axial and a-oriented, which was supported by NOE correlation between H-1 and H-9a. The positive Cotton effect
Fig. 5. X-ray ORTEP drawings of compounds 3, 6, and 10.
Fig. 6. The comparison of experimental ECD spectra and Olefin octant rule applied for 7 and 8.
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at 240 nm in the ECD spectrum of 9 (Fig. S100, Supporting Information), arising from the p-p* electron transition, indicated a clockwise helicity of a,b-unsaturated carbonyl according to the empirical helicity rule (Kirk, 1986). Correspondingly, the absolute configuration of 9 was determined as (1R,7S,10R), in combination of the aforementioned established NOE correlations. Comparison of the calculated and experimental ECD spectra of 9 (Fig. S100, Supporting Information) corroborated the above deduction. The configuration of C-11 is hard to determine as that of 7 and 8. Therefore, the structure of arvestonol (9) was determined as (1R,7S,10R)-3-oxo-eudesma-4,5-en-1,12-diol. Arvestolide J (10) was obtained as colorless needles from acetone-water (5:1) solvent. Its molecular formula was determined as C14H18O5 based on a sodium-adduct ion [M þ Na]þ at m/z 289.1059 (calcd. for 289.1052) in the HRESIMS and the 13C NMR data. IR spectrum showed hydroxy (3347 cm1) and lactone (1757, 1013 cm1) absorption bands. In the 1H NMR spectrum, an olefinic proton (dH 5.82), an oxymethine (dH 3.59) and three methyl signals were distinctively observed. Fourteen carbon resonances were classified into three methyl, two methene, four methine, and five quaternary carbons by multi-edited HSQC spectrum. Among them, two carbonyls (dC 181.1 and 170.1), a double band (dC 120.4 and 167.6), and two oxy-carbon signals (dC 93.1 and 110.9) were detected. These 1D NMR data were similar to those of norsantolinidilactone A (Jakupovic et al., 1991), a nor-dilactone sesquiterpenoid isolated from A. santolinifolia. In comparison of their 13C NMR data, an obvious deshielded chemical shift of C-5 (DdC þ23.6) assigned by the HMBC correlations of H3-14/C-5 and H3-15/C-5 indicated a hydroxy group was attached, which was consistent with its molecular formula. Therefore, the planar structure of 10 was determined as 5-hydoxy-norsantolinidilactone A. The relative configuration of 10 was elucidated from the NOESY spectrum in which the cross-peaks of H3-14/H-6, H-6/H-8b, and H-6/H-11 were present, indicating these b-oriented protons were cofacial. The large coupling constant (J ¼ 12.0 Hz) of the doublet H-6 indicated an a-orientation of H-7. However, the 5-OH configuration is hard to establish. A relative configuration of (5R,6S,7S,10R,11S)-10 was achieved by the X-ray crystallographic analysis (Fig. 5) due to the large Flack parameter [0.27(17)]. Finally, by comparison of calculated and experimental ECD data (Fig. S110, Supporting Information), the absolute configuration of arvestolide J (10) was determined as 5R,6S,7S,10R,11S. Arvestonate C (11) was obtained as a colorless gum, and its molecular formula of C17H26O6 was determined by a sodium adduct ion [M þ Na]þ at m/z 349.1624 (calcd. 349.1627) and its 13C NMR data. IR spectrum showed similar absorption bands for hydroxy (3511 cm1) and lactone (1735 and 1054 cm1) functionalities as those of 10. Seventeen carbon resonances were detected in the 13C NMR spectrum and further classified into four methyl, five methylene, three methine, and five quaternary carbons by the multiedited HSQC spectrum. The 1H-1H COSY spectrum gave the mutual correlations of H2-2/H2-3, H-6/H-7/H2-8/H2-9, and H-7/H11/H3-13. The HMBC correlations of H2-15/C-5, C-3, H3-16/C-1, H23/C-1, H3-13/C-7, C-12, and H3-17/C-12 demonstrated the presence of two residues of 1-methoxy-4-methylene-1,5-dioxopentan-5-yl (C-1 to C-5) and 1-methoxy-1-oxopropan-2-yl (C-11 to C-12). These two residues were connected to a cyclopentane moiety at the C-7 and C-10 positions, which was proved by the HMBC correlations of H3-14/C-5 and C-9, and H3-13/C-7 and C-12. 6-OH Substituent was deduced from the chemical shifts (dH 3.61 and dC 85.4) and the HMBC correlations of H3-14/C-6 and H-6/C-8 and C-9. This terpene framework of 11 was similar to that of santolinifolide A (Jakupovic et al., 1991), a seco-sesquiterpenoid, and the structure of 11 was determined as methyl 4-(2-hydroxy-3-(1-methoxy-1oxopropan-2-yl)-1-methylcyclopentate-1-carbonyl)pent-4-enoate.
The relative configuration of 11 was established by NOESY spectrum in which the correlations of H3-14/H-6, H-8a, and H-11, H-6/ H-11, and H-7/H3-13 were observed, indicating H3-14 and H-6 are a-oriented while H-7 is b-oriented in combination with the coupling constant (J ¼ 7.0 Hz) of H-6/H-7. The positive ECD Cotton effect at 225 nm (Fig. S120, Supporting Information) is arisen from the conjugated ketene at C-10 (Marina et al., 2004; Mori, 1974) and its absolute configuration was deduced as 6R,7R,10S by comparison of experimental and calculated ECD spectra (Fig. S120, Supporting Information). Therefore, the structure of arvestonate C (11) was established as depicted. A. vestita, as a folk medicine in Tibet area, has been traditionally used to treat various inflammatory diseases (Tian et al., 2013b). Some sesquiterpenoids and their dimers isolated from Artemisia species in previous studies (Turak et al., 2014; Zan et al., 2012; Zhang et al., 2014, 2016) showed remarkable cytotoxic and NO inhibitory activities. Thus, the isolates were screened on the same models. The pre-screening for cytotoxic activities on HepG2 cell line showed that compounds 1e11 had no effect with IC50 values larger than 100 mM. Compounds 5 and 6 showed inhibition against LPS-induced NO production in BV-2 cells with IC50 values of 43.2 and 39.9 mM, respectively, while the other compounds were all larger than 100 mM. 3. Conclusions In this study, 11 previously undescribed sesquiterpenoids including eight eudesmane sesquiterpenoids (1e5 and 7e9), a germacrene sesquiterpenoid (6), and two seco-sesquiterpenoids (10 and 11) were isolated from A. vestita. Their structures were elucidated from HRESIMS, IR, and NMR spectroscopic data. Their absolute configurations were determined by empirical rules, X-ray diffraction analysis, and comparison of experimental and calculated ECD data. The configuration of C-5 was disclosed to be responsible for the mirror-like ECD Cotton effect at around 205 nm for 7 and 8, which was corroborated by the Olefin octant rule and quantum calculation of ECD spectra. Compounds 5 and 6 showed NO inhibitory effects with IC50 values of 43.2 and 39.9 mM, respectively. The results obtained herein give a proof for the traditional use of A. vestita for the treatment of inflammation-related diseases. 4. Experimental 4.1. General experimental procedures Optical rotations were measured using a Rudolph Autopol III automatic polarimeter. ECD spectra were recorded on a JASCO J-810 CD spectrometer. IR spectra were recorded (KBr disks) on a Thermo Nicolet Nexus 470 FT-IR spectrometer. NMR spectra were obtained at 500 MHz for 1H and 125 MHz for 13C on a Varian INOVA-500 NMR spectrometer in methanol-d4. The HSQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HRMS data were performed on a Bruker APEX IV FT-MS or a Waters Xevo G2 Q-TOF spectrometers fitted with an ESI source. LC-MS analysis was performed on a Shimadzu LCMS-IT-TOF instrument equipped with a Shimadzu Prominence HPLC system (Shimadzu, Kyoto, Japan). TLC analysis was carried out on the precoated silica gel GF254 plates (Qingdao Marine Chemical Inc., 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. Column chromatography (CC) was performed using silica gel (100e200 mesh or 200e300 mesh, Qingdao Marine Chemical Inc., People's Republic of China), ODS C18 (50 mm, Merck, Germany), and Sephadex LH-20 (Amersham Biosciences, Sweden). Semipreparative HPLC was performed on an Agilent 1200 liquid chromatography
S.-H. Tian et al. / Phytochemistry 147 (2018) 194e202
with a Waters XBridge™ Prep shield RP 18 column (250 mm 10 mm, 5 mm). Analytical HPLC was performed on an Aglient 1100 system with an Aglient Extend-C18 column (4.6 150 mm, 5 mm). All purified compounds submitted for bioassay were at least 95% pure as judged by HPLC analysis. 4.2. Plant materials The aerial parts of Artemisia vestita W. were collected in Ninger town, Puer city, Southwest of Yunnan Province, People's Republic of China, in September 2010. The plant material was authenticated by one of the authors (P.-F. Tu). A voucher specimen (No. 20100905) has been deposited at the herbarium of the Peking University Modern Research Center for Traditional Chinese Medicine. 4.3. Extraction and isolation The dried aerial parts of A. vestita (45 kg) were extracted with 95% aqueous EtOH (360 L 3 h 3) at room temperature. The concentrated extracts (3.8 kg) was suspended in H2O (15 L) and extracted with petroleum ether (PE) and chloroform (CHCl3) successively. The CHCl3 extract (450 g) were subjected to a silica gel CC eluting with a step-wise gradient of PE-EtOAc (1:0, 8:1, 3:1, 1:1, and 0:1, v/v) to produce 10 fractions (Frs.1e10), monitored by TLC analysis. Frs. 5e7 were combined and subjected to silica gel CC (200e300 mesh) eluting with gradient n-hexane-acetone (30:1, 10:1, 5:1, 2:1, and 0:1, v/v) to yield five fractions (Frs. 5ae5e). Fr. 5a was further subjected to Sephadex LH-20 CC eluting with methanol to afford fractions 5a1e5a5. Fr. 5a2 was repeatedly purified over silica gel CC (200e300 mesh) using CHCl3-EtOAc (20:1 / 10:1 / 5:1) to yield 1 (2 mg). Fr. 5a3 was separated on Sephadex LH-20 CC to obtain sub-fractions 5a3ae5a3f and the subfraction 5a3c was purified by silica gel (200e300 mesh) CC repeatedly to yield 11 (3 mg). Fr. 5a4 was subjected to silica gel CC eluted with CHCl3-acetone (30:1 / 10:1 / 4:1) to obtain sub-
fractions 5a4ae5a4g. Sub-fraction 5a4c was purified repeatedly by reversed-phase C18 CC eluting with MeOH-H2O (2:8 / 3:7 / 5:5 / 7:3) to yield 2 (2 mg) and 3 (4 mg). Subfraction 5a4e was purified repeatedly by reversed-phase C18 CC eluting with (2:8 / 3:7 / 5:5 / 7:3) to yield 5 (4 mg) and 10 (3 mg). Fr. 5a5 was separated by silica gel CC using CHCl3-acetone (20:1 / 10:1 / 5:1) to obtain sub-fractions 5a5ae5a5e and subfraction 5a5b were purified repeatedly by semipreparative HPLC (MeCN-H2O 40:60) to yield 6 (4 mg, tR ¼ 15 min). Fr. 5b was subjected to Sephadex LH-20 CC using MeOH as eluting solvent to yield fractions 5b1e5b5. Fr. 5b2 was separated by silica-gel CC using CHCl3-EtOAc (20:1 / 10:1) to obtain sub-fractions 5b2ae5b2e. Sub-fraction 5b2b was purified by reversed-phase C18 CC eluting with MeOH-H2O (20:80 / 40:60 / 60:40 / 80:20) to yield 4 (2 mg) and 7 (3 mg). Sub-fraction 5b2d was purified by reversedphase C18 CC eluting with MeOH-H2O (20:80 / 40:60 / 60:40 / 80:20) to yield 8 (3 mg) and 9 (2 mg).
4.3.1. Arvestolide D (1) White powder; [a]20 D e4.6 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 227 (3.5) nm; ECD (MeOH) lmax (D3 ) 323 (3.2), 245 (þ3.7) nm; IR (KBr) nmax 3381, 2957, 2870, 1738, 1455, 1369, 1056, 1010, 788, 750 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 1; HRESIMS m/z 345.1317 [M þ Na]þ (calcd for C17H22O6Na, 345.1314).
4.3.2. Arvestolide E (2) White gum; [a]20 D þ69.0 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 228 (3.5) nm; ECD (MeOH) lmax (D3 ) 252 (þ37.2) nm; IR (KBr) nmax 3742, 3710, 3652, 2939, 2865, 1774, 1739, 1541, 1257, 1068, 786 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 1; HRESIMS m/z 321.1328 [M e H]e (calcd for C17H21O6, 321.1338).
Table 1 1 H and 13C NMR Data of Compounds 1e5 (d in ppm, J in Hz, 500 MHz for 1H and 125 MHz for Position
1
dH 1 2a 2b 3a 3b 4 5 6 7 8a 8b 9a 9b 10 11 12 13 14 15
5.68, s
acetyl
2.21, s
2
dC
dH
77.4 4.86, br s 75.4 191.1 2.28, m 22.3 1.73, dd (14.7, 3.9) 5.96, s 128.5 2.05, dd (14.3, 3.9) 33.8 1.69, d (14.3) 155.4 72.9 75.7 70.1 4.39, d (11.6) 83.8 89.6 1.62, ddd (13.6, 11.6, 3.4) 47.4 154.4 1.89, d (13.0) 23.8 2.57, m 18.8 1.49, m 2.23, m 1.87, d (13.0) 30.4 2.25, m 26.9 1.41, m 1.08, dd (13.3, 5.5) 47.1 39.0 2.38, dq (13.6, 6.8) 41.6 126.3 177.2 170.9 1.26, d (6.8) 12.7 1.89, s 8.6 1.30, s 16.9 1.29, s 21.4 2.05, s 22.6 1.47, s 27.9 21.6 2.10, s 170.3
13
C, in CDCl3).
3
dC
199
dH 6.31, d (10.5) 5.94, d (10.5)
4
dC
dH
5
dC
154.1 4.79, dd (5.1, 2.5) 76.1 123.1 1.88, m 22.7 1.88, m 195.2 2.32, m 25.9 2.22, m 65.0 130.1 69.8 131.6 4.55, d (11.5) 77.1 4.62, d (11.3) 82.4 2.21, m 50.3 1.75, m 52.8 1.99, m 23.2 1.97, ddd (12.9, 5.2, 3.2) 23.7 1.64, qd (13.6, 12.8, 4.4) 1.58, td (12.9, 3.2) 1.97, m 37.9 1.75, m 33.0 1.53, dd (13.7, 4.4) 1.46. dt (13.5, 3.2) 41.6 40.4 2.41, dq (12.5, 6.8) 41.3 2.34, m 41.1 176.9 178.0 1.27, d (7.0) 12.6 1.26, d (7.0) 12.3 1.42, s 21.5 1.22, s 26.1 4.65, d (12.0) 58.1 4.40, d (12.1) 62.9 4.06, d (12.0) 4.12, d (12.1) 21.1 2.08, s 21.2 170.5 170.7
dH
dC
4.85, 1.54, 2.13, 4.30,
t (3.0) m m t (3.0)
3.07, 4.05, 1.64, 1.89, 1.54, 1.71, 1.42,
d (10.9) t (10.9) m dd (13.0, 3.5) m m dt (13.5, 3.5)
75.5 33.3 72.3 145.3 43.2 79.2 52.3 22.8 33.0
41.7 2.35, dq (13.5, 6.8) 41.1 179.1 1.25, d (7.0) 12.5 0.94, s 17.8 5.22, s 113.3 5.06, s
angelica acyl 6.16, q (7.0) 2.06, d (7.0) 1.97, s
166.7 127.4 139.7 15.9 20.8
200
S.-H. Tian et al. / Phytochemistry 147 (2018) 194e202
4.3.3. Arvestolide F (3) Colorless needles; mp 160e162 C; [a]20 D e18.3 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 229 (3.5) nm; ECD (MeOH) lmax (D3 ) 340 (þ18.5) nm; IR (KBr) nmax 3707, 2945, 1772, 1677, 1054, 1031, 670 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 1; HRESIMS m/z 279.1227 [M þ H]þ (calcd for C15H19O5, 279.1232).
4.3.8. Arvestonate B (8) Colorless gum; [a]20 D e29.1 (c 0.2, MeOH); UV (MeOH) lmax (log ε) 207 (3.1) nm; ECD (MeOH) lmax (D3 ) 205 (e37.1) nm; IR (KBr) nmax 3451, 2948, 1732, 1245, 1026, 966 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 2; HRESIMS m/z 363.1777 [M þ Na]þ (calcd for C18H28O6Na, 363.1784).
4.3.4. Arvestolide G (4) White powder; [a]20 D þ4.4 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 210 (3.3) nm; ECD (MeOH) lmax (D3 ) 210 (e18.2) nm; IR (KBr) nmax 3706, 3354, 2972, 2943, 1788, 1057, 1032 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 1; HRESIMS m/z 331.1524 [M þ Na]þ (calcd for C17H24O5Na, 331.1521).
4.3.9. Arvestonol (9) Colorless gum; [a]20 D þ52.6 (c 0.2, MeOH); UV (MeOH) lmax (log ε) 248 (3.4) nm; ECD (MeOH) lmax (D3 ) 248 (þ67.3) nm; IR (KBr) nmax 3351, 2950, 2868, 1741, 1443, 1194, 1023, 749 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 2; HRESIMS m/z 253.1816 [M þ Na]þ (calcd for C15H24O3Na, 253.1804).
4.3.5. Arvestolide H (5) White amorphous powder; [a]20 D þ32.5 (c 0.3, MeOH); UV (MeOH) lmax (log ε) 218 (3.5) nm; ECD (MeOH) lmax (D3 ) 210 (þ2.1), 300 (e2.7) nm; IR (KBr) nmax 3755, 3383, 2949, 2863, 1732, 1594, 1444. 1055, 1014 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 1; HRESIMS m/z 371.1821 [M þ Na]þ (calcd for C20H28O5Na, 371.1834).
4.3.10. Arvestolide J (10) Colorless needles; mp 132e134 C; [a]20 D þ26.3 (c 0.6, MeOH); UV (MeOH) lmax (log ε) 215 (3.4) nm; ECD (MeOH) lmax (D3 ) 222 (e17.8) nm; IR (KBr) nmax 3347, 2970, 1757, 1252, 1013, 925 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 3; HRESIMS m/z 289.1059 [M þ Na]þ (calcd for C14H18O5Na, 289.1052).
4.3.6. Arvestolide I (6) Colorless needles; mp 157e159 C; [a]20 D e11.4 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 246 (3.2) nm; ECD (MeOH) lmax (D3 ) 252 (þ59.8) nm, 320 (þ21.5) nm; IR (KBr) nmax 3723, 3383, 2936, 1782, 1735, 1058, 754 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 2; HRESIMS m/z 331.1517 [M þ Na]þ (calcd for C17H24O5Na, 331.1521).
4.3.11. Arvestonate C (11) Colorless gum; [a]20 D þ2.5 (c 0.9, MeOH); UV (MeOH) lmax (log ε) 222 (3.4) nm; ECD (MeOH) lmax (D3 ) 225 (þ4.8), 327 (e5.1) nm; IR (KBr) nmax 3511, 2953, 2873, 1735, 1453, 1260, 1054, 667 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 3; HRESIMS m/z 349.1624 [M þ Na]þ (calcd for C17H26O6Na, 349.1627).
4.3.7. Arvestonate A (7) Colorless gum; [a]20 D þ54.6 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 202 (3.1) nm; ECD (MeOH) lmax (D3 ) 205 (þ6.3) nm; IR (KBr) nmax 3331, 2945, 2868, 1708, 1502, 1375, 1292, 1192, 1024, 754, 693 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 2; HRESIMS m/z 321.1682 [M þ Na]þ (calcd for C16H26O5Na, 321.1678).
4.4. X-ray crystallographic analysis Crystal analysis were performed on a Bruker APEX diffractometer equipped with an APEX Ⅱ CCD, using Cu Ka radiation (l ¼ 1.54178 Å). Cell refinement and data reduction were performed with Bruker SAINT. The structures of 3, 6, and 10 were solved by direct methods using SHELXS-97. Refinements were performed
Table 2 1 H and 13C NMR Data of Compounds 6e9 (d in ppm, J in Hz, 500 MHz for 1H and 125 MHz for Position
6
7
dH 1 2a 2b 3a 3b 4 5 6 7 8a 8b 9a 9b 10 11 12
4.67, 2.50, 2.18, 5.51,
4.59, 2.54, 2.08, 1.14, 1.28,
ddd (12.0, 4.0, 1.6) m m ddd (11.3, 5.8, 1.6)
d (9.7) m dt (9.2, 3.5) m m
dC
dH
76.5 29.6
4.07, 1.88, 1.52, 2.73, 2.09,
126.2 143.5 202.4 80.6 48.6 27.5 26.6
1.45, m 2.24, dq (12.2, 6.8)
32.8 41.0 177.3
13 14 15
1.31, d (6.8) 0.92, d (6.7) 2.03, s
12.7 16.9 22.6
16 acetyl
2.01, s
21.6 170.3
C, in CDCl3). 8
dd (11.5, 5.2) dtd (12.6, 5.2, 1.8) m td (13.5, 6.1) ddd (13.5, 5.4, 1.8)
4.13, d (10.4) 2.16, m 1.61, m 1.40 qd (13.3, 4.3) 1.80, td (13.3, 4.3) 1.53, m 2.82, dq (2.4, 7.3)
1.23, 0.77, 5.00, 4.76, 3.70,
13
d (7.3) s br s br s s
dC
dH
72.8 31.1
5.09, 1.84, 1.63, 2.68, 2.15,
30.2 148.1 78.4 68.4 41.7 22.5 29.0 43.2 40.4 177.4 13.2 12.6 123.1 51.7
3.80, 2.59, 1.59, 1.34, 1.64, 1.48,
9
dd (12.1, 4.8) dt (12.8, 3.5) m m dt (13.9, 3.5)
d (11.5) dq (11.5, 5.6) m td (13.3, 4.4) m dt (14.4, 3.5)
2.61, m
1.16, 1.02, 5.37, 5.15, 3.69, 2.03,
d (6.9) s s s s s
dC
dH
dC
76.7 27.0
3.81, dd (12.8, 5.3) 2.64, dd (16.4, 5.3) 2.55, dd (16.4, 12.8)
74.5 42.3
31.6 144.1 78.3 76.3 41.8 22.5 25.9 43.7 42.3 177.7 13.2 16.8 115.5 51.7 21.2 170.6
197.2
2.75, 1.93, 1.51, 1.69, 1.52, 1.33, 2.13,
d (15.3) t (12.8) m m m t (13.3) dt (13.3, 3.2)
1.66, 3.67, 3.58, 1.00, 1.17, 1.77,
m dd (10.6, 5.8) dd (10.6, 6.3) d (7.0) s s
129.4 161.9 30.4 39.6 25.2 37.8 41.5 40.4 65.8 13.5 16.2 10.9
S.-H. Tian et al. / Phytochemistry 147 (2018) 194e202 Table 3 1 H and 13C NMR Data of Compounds 10 and 11 (d in ppm, J in Hz, 500 MHz for 1H and 125 MHz for 13C, in CDCl3). Position
10
11
dH 1 2a 2b 3a 3b 4 5 6 7 8a 8b 9a 9b 10 11 12 13 14 15 16 17
5.82, s
3.59, 2.64, 1.87, 1.33, 2.64, 1.74,
d (12.0) br s m m m m
2.28, m 1.27, d (6.5) 1.21, s 2.17, s
dC
dH
170.1
2.46, 2.44, 2.58, 2.45,
120.4 167.6 119.7 93.1 44.2 21.6 38.9 51.9 43.0 181.5 13.2 25.4 15.2
3.61, 2.19, 1.83, 1.32, 2.34, 1.61,
dC m m m m
d (7.0) p (8.7) ddd (12.5, 8.7, 3.8) m dt (13.4, 8.7) ddd (13.4, 8.7, 3.8)
2.46, m 1.17, 1.36, 5.80, 5.72, 3.66, 3.69,
d (7.0) s s s s s
173.2 32.8 28.5 145.4 208.7 85.4 43.4 25.1 34.5 56.7 49.7 176.9 15.1 24.4 123.1 51.5 51.7
with SHELXL-97 using full-matrix least-squares, with anisotropic displacement parameters for all the non-hydrogen atoms. The Hatoms were placed in calculated positions and refined using a riding model. Crystallographic data have been deposited with the Cambridge Crystallographic Data Center (CCDC 1535635 for 3; CCDC 918234 for 6; CCDC 930778 for 10). Crystal data for 3: C15H18O5, M ¼ 278.29 g/mol, size ¼ 0.26 0.18 0.15 mm3, orthorhombic, space group P21, a ¼ 9.4913(19) Å, b ¼ 10.299(2) Å, c ¼ 14.739(3) Å, b ¼ 90.0 , V ¼ 1440.8 (5) Å, T ¼ 296(2) K, Z ¼ 4, d ¼ 1.283 g/m3, m(CuKa) ¼ 1.54178 Å, F(000) ¼ 328, completeness qmax ¼ 97.5%, 2486 independent reflections, Rint ¼ 0.0252, 2416 reflections with jFj2 2sjFj2, 0 restraint, R1 ¼ 0.0354, wR2 ¼ 0.09716, S ¼ 1.044, GOF ¼ 1.044. The Flack parameter is 0.2(2). Crystal data for 6: C17H24O5, M ¼ 308.36 g/mol, size ¼ 0.22 0.15 0.10 mm3, monoclinic, space group P21, a ¼ 10.323(2) Å, b ¼ 7.6545(15) Å, c ¼ 11.528(2) Å, b ¼ 115.16(3) , V ¼ 824.4 (3) Å, T ¼ 296(2) K, Z ¼ 2, d ¼ 1.242 g/m3, m(CuKa) ¼ 1.54178 Å, F(000) ¼ 332, completeness qmax ¼ 97.4%, 2741 independent reflections, Rint ¼ 0.0191, 2579 reflections with jFj2 2sjFj2, 23 restraint, R1 ¼ 0.0326, wR2 ¼ 0.0896, S ¼ 1.0484, GOF ¼ 1.048. The Flack parameter is 0.1(2). Crystal data for 10: C14H18O5 þ H2O, M ¼ 284.30 g/mol, size ¼ 0.12 0.10 0.10 mm3, triclinic, space group P1, a ¼ 6.8239(14) Å, b ¼ 6.8327(14) Å, c ¼ 9.0715(18) Å, b ¼ 74.68(3) , V ¼ 359.21 (13) Å, T ¼ 296(2) K, Z ¼ 1, d ¼ 1.314 g/m3, m(CuKa) ¼ 1.54178 Å, F(000) ¼ 152, completeness qmax ¼ 91.9%, 2096 independent reflections, Rint ¼ 0.0121, 2091 reflections with jFj2 2sjFj2, 3 restraint, R1 ¼ 0.0315, wR2 ¼ 0.0836, S ¼ 1.078, GOF ¼ 1.078. The Flack parameter is 0.27(17). 4.5. Conformational energy calculation of the main conformers of 1‒10 The relative configurations were initially established according to 1D-gradient NOE or NOESY spectra, and submitted to random conformational analysis with the MMFF94s force field using the Sybyl-X 1.1 software package. The conformers were further optimized by using the TDDFT method at the B3LYP/6-31G(d) level and
201
the frequency was calculated at the same level of theory. The stable conformers without imaginary frequencies were subjected to ECD calculation by the TDDFT method at the B3LYP/6-31þG(d) level in MeOH as solvent. ECD spectra of different conformers were simulated using SpecDis with a half-band width of 0.3 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. 4.6. Cytotoxic assay Cell proliferation inhibition was determined using the MTT method according to the established protocols (Zan et al., 2012). Taxol was used as the positive control. 4.7. NO inhibitory effect BV-2 microglial cells were purchased from Peking Union Medical College (PUMC) Cell Bank (Beijing, People's Republic of China). Cell maintenance, experimental procedures, and data presentation for the inhibition of NO production and viability assay were conducted as previously described (Zhang et al., 2014). Quercetin (IC50 value of 10.0 mM) was used as a positive control. All the compounds were prepared as stock solutions in DMSO (final solvent concentration less than 0.5% in all assays). Acknowledgements This work was financially supported by the grants from the National Natural Science Foundation of China (Nos. 81373294, 81222051, and 81303253) and the National Key Technology R&D Program “New Drug Innovation” of China (No. 2017ZX09101003008-003). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.phytochem.2018.01.004. References Anake, K., Luis, M.M.V., Jose, M.C., Werner, H., 1992. An eudesmanolide from Picris spinifera. Phytochemistry 31, 3635e3636. Braulio, M.F., 2011. Natural sesquiterpenoids. Nat. Prod. Rep. 28, 1580e1610. Fujimoto, Y., Shimizu, T., Ohmori, M., Tatsuno, T., 1979. Modification of a-santonin. Ⅲ. Synthesis of dihdrocostunolide. Chem. Pharm. Bull. 27, 923e933. Irwin, M.A., Geissman, T.A., 1973. Arbusculin-D from Artemisia arbuscula ssp. Arbuscula. Phytochemistry 12, 853e855. Jakupovic, J., Tan, R.X., Bohlmann, F., Jia, Z.J., Huneck, S., 1991. Seco- and norsesquiterpene lacones with a new carbon skeleton from Artemisia santolinifolia. Phytochemistry 30, 1941e1946. Kawazoe, K., Tsubouchi, Y., Abdullah, N., Takaishi, Y., Shibata, H., Higuti, T., Hori, H., Ogawa, M., 2003. Sesquiterpenoids from Artemisia gilvescens and an anti-MRSA compound. J. Nat. Prod. 66, 538e539. Kirk, D.N., 1986. The chiroptical properties of carbonyl compounds. Tetrahedron 42, 810e818. rez, Y., Catala n, C.A.N., Krautmann, M., Riscala, E.C., Burgueno-Tapia, E., Mora-Pe Joseph-Nathlan, P., 2007. C-15-Functionalized eudesmanolides from Mikania campanulata. J. Nat. Prod. 70, 1173e1179. Liu, L.J., Chen, Y.G., Xiong, Z.M., Li, J., Zhou, G., Li, Y.L., 1998. A facile synthesis of 3oxo-7aH-eudesma-4-ene-9b,12-diol. Chin. Chem. Lett. 9, 625e627. Ma, C.M., Nakamura, N., Hattori, M., Zhu, S., Komatsu, K., 2000. Guaiane dimers and germacranolide from Artemisia caruifolia. J. Nat. Prod. 63, 1626e1629. Marina, D., Cinzia, D.M., Armando, Z., Brigida, D., 2004. Isolation and phytotoxicity of Apocarotenoids from Chenopodium album. J. Nat. Prod. 67, 1492e1495. Mengi, N., Taneja, S.C., Mahajan, V.P., Mathela, C.S., 1991. Eudesmanolides from Senecio chrysanthemoides. Phytochemistry 30, 2329e2330. Mori, K., 1974. Synthesis of optically active grasshopper ketone and dehydrovomifoliol as a synthetic support for the revised absolute configuration of (þ)-abscisic acid. Tetrahedron 30, 1065e1072. Pei, Y.H., 2015. Absolute determination of stereochemical configuration of some
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marine natural products. J. Int. Pharm. Res. 42, 786. Pui-Hay, B.P., He, Z.D., Ma, S.C., Chan, Y.M., Shaw, P.C., Ye, W.C., Jiang, R.W., 2009. Antiviral constituents against respiratory viruses from Mikania micrantha. J. Nat. Prod. 72, 925e928. Scott, A.I., Wrixon, A.D., 1970. A symmetry rule for chiral olefins. Tetrahedron 26, 3695e3715. Shimizu, T., Fujimoto, Y., Tatsuno, T., 1979. Modification of a-Santonin. Ⅳ. Epoxidation of germacranolde-type sesquiterpenes. Chem. Pharm. Bull. 27, 934e945. Tian, S.H., Chai, X.Y., Zan, K., Zeng, K.W., Guo, X.Y., Jiang, Y., Tu, P.F., 2013a. Arvestolides A-C, new rare sesquiterpenes from the aerial parts of Artemisia vestita. Tetrahedron Lett. 54, 5035e5038. Tian, S.H., Chai, X.Y., Zan, K., Zeng, K.W., Tu, P.F., 2013b. Three new eudesmane sesquiterpenes from Artemisia vestita. Chin. Chem. Lett. 24, 797e800. Turak, A., Shi, S.P., Jiang, Y., Tu, P.F., 2014. Dimeric guaianolides from Artemisia absinthium. Phytochemistry 105, 109e114. Wang, S., Li, J., Sun, J., Zeng, K.W., Cui, F.X., Jiang, Y., Tu, P.F., 2013. NO inhibitory
guaianolide-derived terpenoids from Artemisia argyi. Fitoterapia 85, 169e175. Wu, Z.Y., Peter, H.R., 1992. Flora of China. Editorial Board of Flora of China, vol. 76. Science Press, Beijing, p. 49. Wu, Q., Zou, L., Yang, X.W., Fu, D.X., 2009. Novel sesquiterpene and coumarin constituents from the whole herbs of Crossostephium chinense. J. Asian Nat. Prod. Res. 11, 85e90. Zan, K., Chai, X.Y., Chen, X.Q., Wu, Q., Fu, Q., Zhou, S.X., Tu, P.F., 2012. Artanomadimers A-F: six new dimeric guaianolides from Artemisia anomala. Tetrahedron 68, 5060e5065. Zhang, C., Wang, S., Zeng, K.W., Cui, F.X., Jin, H.W., Guo, X.Y., Jiang, Y., Tu, P.F., 2014. Rupestonic acids B-G, NO inhibitory sesquiterpenoids from Artemisia rupestris. Bioorg. Med. Chem. Lett. 24, 4318e4322. Zhang, C., Wang, S., Zeng, K.W., Li, J., Ferreria, D., Zjawiony, J.K., Liu, B.Y., Guo, X.Y., Jin, H.W., Jiang, Y., Tu, P.F., 2016. Nitric oxide inhibitory dimeric sesquiterpenoids from Artemisia rupestris. J. Nat. Prod. 79, 213e223.
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Sesquiterpenoids from Artemisia vestita Shuai-Hua Tian 1, Chen Zhang 1, Ke-Wu Zeng, Ming-Bo Zhao, Yong Jiang**, Peng-Fei Tu* State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 September 2017 Received in revised form 14 December 2017 Accepted 5 January 2018
Eleven previously undescribed sesquiterpenoids, arvestolides DeJ, arvestonates AeC, and arvestonol were isolated from the aerial parts of Artemisia vestita W. Their structures were elucidated by extensive analysis of HRMS, UV, IR, and NMR spectroscopic data, and the absolute configurations were determined by single crystal X-ray diffraction, empirical rules, and comparison of calculated and experimental ECD data. Arvestolides H and I showed inhibitory effects on nitric oxide production in BV-2 cells induced by lipopolysaccharide with IC50 values of 43.2 and 39.9 mM, respectively. © 2018 Elsevier Ltd. All rights reserved.
Keywords: Artemisia vestita W. Compositae Sesquiterpenoid NO inhibition
1. Introduction Artemisia vestita W., belonging to the Composite family, is a wild bush endemic to the western high-altitude area of China, and its aerial parts have been traditionally used as a folk medicine for the treatment of inflammatory diseases (Wu and Peter, 1992). Sesquiterpenoids, as a type of characteristic metabolites of Artemisia species, have been reported for their diverse structures and antiinflammatory, cytotoxic, and anti-virus bioactivities (Braulio, 2011; Kawazoe et al., 2003; Ma et al., 2000; Wang et al., 2013). A number of anti-inflammatory and cytotoxic sesquiterpenoids and their dimers formed via Diels-Alder [4þ2] cycloaddition have been isolated from the Artemisia species in our previous studies (Tian et al., 2013a, 2013b; Turak et al., 2014; Zan et al., 2012; Zhang et al., 2014, 2016). As an ongoing search for active sesquiterpenoids and their derivatives from the Artemisia genus, the 95% aqueous EtOH extract of A. vestita was phytochemically investigated and afforded 11 previously undescribed sesquiterpenoids, including eight eudesmanes (1e5 and 7e9), one germacranolide (6), one nor-dilactone (10), and one seco-sesquiterpenoid (11). Their structures were elucidated by HRESIMS, IR, UV, and NMR spectroscopic data. The absolute configurations of previously undescribed
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Jiang), [email protected] (P.-F. Tu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.phytochem.2018.01.004 0031-9422/© 2018 Elsevier Ltd. All rights reserved.
compounds were established based on single-crystal X-ray diffraction, empirical rules, and comparison of the calculated and experimental ECD spectra. Compounds 1e11 were tested for their cytotoxic and nitric oxide (NO) inhibitory effects on BV-2 cells induced by lipopolysaccharide (LPS). Only compounds 5 and 6 exhibited weak NO inhibitory effects with IC50 values of 43.2 and 39.9 mM, respectively.
2. Results and discussion The powdered aerial parts of A. vestita (45 kg) were extracted with 95% aq. EtOH and partitioned with petroleum ether and CHCl3, successively. The CHCl3-soluble portion, which was analyzed by LC/ MS to be rich of sesquiterpenoids, was subjected repeatedly to silica gel, Sephadex LH-20, and ODS column chromatography (CC), followed by semipreparative HPLC to afford 11 previously undescribed sesquiterpenoids (1e11) (see Fig. 1). Arvestolide D (1) was obtained as a white amorphous powder. A sodium adduct ion [M þ Na]þ at m/z 345.1317 (calcd. 345.1314) in the HRESIMS indicated a molecular formula of C17H22O6, requiring seven indices of hydrogen deficiency. The IR spectrum showed absorption bands for hydroxy (3381 cm1) and lactone (1738, 1056 cm1) functionalities. The resonances for olefinic and oxygenated protons at dH 5.96 (1H, br, s), 5.68 (1H, s), and 4.39 (1H, d, J ¼ 11.6 Hz) were observed along with four methyl signals at dH 2.21 (3H, s), 2.05 (3H, s), 1.30 (3H, s), and 1.26 (3H, d, J ¼ 6.8 Hz) in the 1H NMR spectrum. A total of 17 carbon resonances in the 13C NMR spectrum were classified into four methyl, two methylene,
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five methine, and six quaternary carbons by the multi-edited HSQC spectrum. Among them, signals for carbonyl (dC 191.1, 177.2, and 170.3), double bond (dC 128.5 and 155.4), and oxygenated (dC 83.8, 77.4, and 75.7) carbons were assigned via their chemical shifts. These 1D NMR data were similar to those of 1-acetyleudesma-2oxo-3-en-6,12-olide (Anake et al., 1992), and their difference was one more oxygenated tertiary carbon resonance at dC 75.7 in 1. The HMBC correlations from H3-15 to C-3, C-4, and C-5 and from H-3 to C-1, C-2, and C-5 assigned this carbon to be C-5 at which a hydroxy group was attached. Thus, 1 was deduced as 1-O-acetyleudesma-5hydroxy-2-oxo-3-en-6,12-olide. The relative configuration of 1 was established by the coupling constants and NOE correlations. The NOE cross-peaks of H3-14/H-6, H-6/H-11, and H3-14/H-1 indicated that they were cofacial and arbitrarily assigned as b-orientation. The large coupling constants of H-6/H-7 (J ¼ 11.6 Hz) and H-7/H-11 (J ¼ 13.6 Hz) suggested H-7 to be a-oriented, which was also supported by the NOE cross-peaks of H-7/H3-13 and H-7/H-9a (Fig. 2).
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Based on the NOE correlations of 5-OH/H-1, H3-14, and H-6 determined in DMSO-d6, the relative configuration of 5-OH was deduced as b-oriented. The positive Cotton effect at 245 nm in the ECD spectrum, arising from the p-p* orbital transition of a,b-unsaturated ketene, indicated a positive helicity as shown in Fig. 3 according to the a,b-unsaturated ketone helicity rule (Kirk, 1986). While a negative Cotton effect at 323 nm (Dε 3.2) mainly ascribed to the n-p* orbital transition of the conjugated ketene was determined in terms of the Octant rule (Pei, 2015) (Fig. 3). Both of these two Cotton effects assigned the absolute configuration of 1 to be (1R,5S,6S,7S,10S,11S), which was also corroborated by comparison of the experimental and calculated ECD data (Fig. 4). The calculated ECD spectrum of (1R,5S,6S,7S,10S,11S)-1 agreed well with the experimental one. Therefore, the structure of arvestolide D (1) was determined as (1R,5S,6S,7S,10S,11S)-1-O-acetyleudesma-5hydroxy-2-oxo-3-en-6,12-olide. Arvestolide E (2) was obtained as a white gum, and its molecular formula was determined as C17H22O6 by the HRESIMS deprotonated ion [M e H]e at m/z 321.1328 (calcd. 321.1338), indicating seven indices of hydrogen deficiency. The IR spectrum showed the presence of carbonyl (1774 cm1) and lactone (1739 and 1068 cm1) functionalities. The 13C NMR spectrum presented signals for an a,bunsaturated lactone group (dC 126.3, 154.4, and 170.9), an acetyl (dC 170.5 and 21.1), four oxygenated carbons (dC 70.1, 72.9, 75.4, and 89.6), four methylene, three methyl, and an aliphatic quaternary carbons with the aid of HSQC experiment. These data indicated that 2 is a sesquiterpenoid similar to 13-acetoxyeudesma-4-hydroxy7,11-en-6,12-olide (Irwin and Geissman, 1973). Their differences are the acetoxyl position and one more oxygenated tertiary carbon resonance in the 13C NMR data of 2. The existence of 1-O-acetyl was
Fig. 1. Structures of compounds 1e11.
Fig. 2. Key 1H-1H COSY, HMBC, and NOESY correlations of arvestolide D (1).
Fig. 4. Comparison of the calculated ECD spectra of (1R,5S,6S,7S,10S,11S)-1 and its enantiomer with the experimental ECD spectrum of 1.
Fig. 3. Helicity and Octant rules applied for the Cotton effects at 245 nm (p/p*) and 323 nm (n/p*) of ECD spectrum of 1.
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deduced from the HMBC correlation from H-1 to the acetyl carbon (dC 170.5). Based on the HMBC correlations of H3-15/C-5 and H3-14/ C-5, an epoxy between C-5 and C-6 was deduced from the remarkable deshielded chemical shifts of C-5 (dC 70.1) and C-6 (dC 89.6) and the indices of hydrogen deficiency of 2. Thus, the planar structure of 2 was elucidated as 1-acetoxyeudesma-4-hydroxy-5,6epoxy-7,11-en-6,12-olide. The NOE correlations of H3-14/H-1, H314/H-2b, and H3-15/H-3a indicated that H-1, H3-14, and 4-OH were b-oriented. It is difficult to assign the a- or b-orientation of epoxy (C-5-O-C-6) through NOEs, whereas, C-5 and C-6 configurations were found to have profound influence on the Cotton effect at around 250 nm in the ECD spectrum. By comparison of the calculated ECD spectra of the four stereoisomers of 2 (Fig. S25, Supporting Information) with the experimental one, the absolute configurations of C-5 and C-6 were determined as 5S and 6R, while the other stereocenters could not be determined since their minor attributions to the Cotton effects. The structure of arvestolide E (2) was thus elucidated as shown. Arvestolide F (3) was obtained as colorless needles with a molecular formula of C15H18O5 determined by a protonated ion [M þ H]þ at m/z 279.1227 (calcd. 279.1232) in the HRESIMS. The IR spectrum showed absorption bands at 1772, 1677, and 1054 cm1 ascribable to the carbonyl and lactone functionalities. The 1H and 13 C NMR data of 3 were similar to those of a-santonin (Fujimoto et al., 1979), indicating a similar eudesmane-type structure of 3. Comparison of the NMR data with those of a-santonin, an oxymethylene [dH 4.65 (1H, d, J ¼ 12.0 Hz) and 4.06 (1H, d, J ¼ 12.0 Hz); dC 58.1] and a pair of oxygenated quaternary carbons (dC 65.0 and 69.8) in 3 substituted the signals of a methyl and a pair of olefinic carbons of a-santonin. These differences were further supported by 2D NMR spectra, especially by HMBC correlations. The oxymethylene was assigned as the hydroxymethyl of CH2-15 based on its HMBC correlations with C-3, C-4, and C-5. Two oxygenated carbons, C-4 and C-5, were elucidated to form an epoxy moiety by the HMBC correlations of H3-14/C-5, H2-15/C-3/C-4/C-5, and H-7/C5, together with the indices of hydrogen deficiency. Namely, the 4,5-double bond of a-santonin was oxygenated into an epoxy group in 3. In the NOESY spectrum, the cross-peaks of H-11/H-6, H-6/H8b/H3-14, H-7/H-9a, and H-7/H3-13, together with the coupling constants of JH-6/H-7 (11.5 Hz), JH-7/H-11 (12.5 Hz), and JH-7/H-8b (12.8 Hz) indicated the same relative configurations of H-6b, H-7a, and H3-14b as those of a-santonin. The NOEs of H2-15/H-7 indicated the epoxy has a b-orientation and the two rings of A and B were cis-fused. The relative configuration of 3 was further defined by the X-ray diffraction analysis. Due to the large Flack parameter [0.2(2)], the calculated ECD spectra (Fig. S35, Supporting Information) were adopted to determine the absolute configuration of arvestolide F (3) as (4R,5S,6S,7S,10S,11S)-eudesma-15-hydroxy-3keto-1,2-en-4,5-epoxy-6,12-olide. Arvestolide G (4) was obtained as a white amorphous powder. A sodium adduct ion [M þ Na]þ at m/z 331.1524 (calcd. 331.1521) in the HRESIMS indicated a molecular formula of C17H24O5. In the 1H NMR spectrum, two methyl (dH 1.26, 3H, d, J ¼ 7.0 Hz; 1.22, 3H, s), an acetoxy methyl (dH 2.08, 3H, s), two oxygenated methine (dH 4.79, 1H, dd, J ¼ 5.1, 2.5 Hz; 4.62, 1H, d, J ¼ 11.3 Hz), and one oxymethylene (dH 4.40, 1H, d, J ¼ 12.1 Hz; 4.12, 1H, d, J ¼ 12.1 Hz) protons were observed. Seventeen carbon resonances were assigned as three methyl, five methylene, four methine, and five quaternary carbons with the help of multi-edited HSQC spectrum, including a pair of vinyl carbons (dC 130.1 and 131.6), a lactone carbon (dC 178.0), and an acetyl group (dC 170.7 and 21.2). Collectively, these data were similar to those of 1,15-dihydroxyeudesma4,5-11,13-dien-6,12-olide (Krautmann et al., 2007), except that the 1-OH was acetylated, and the exocyclic double bond D11(13) was hydrogenated to a methyl in 4. These deductions were supported
by the HMBC correlations from H-1 to the acetyl carbon (dC 170.7) and H3-13 to C-11/C-7/C-12. The NOEs of H3-14/H-6/H-8b, H3-14/H1, H-6/H-11, H-7/H-9a, and H-7/H3-13 indicated the same relative configuration of 4 as that of 1,15-dihydroxyeudesma-4,5-11,13dien-6,12-olide (Krautmann et al., 2007) or 1. The absolute configuration of 4 was determined as (1S,6S,7S,10R,11S) by comparison of the calculated and experimental ECD data (Fig. S45, Supporting Information). Thus, the structure of arvestolide G (4) was determined as (1S,6S,7S,10R,11S)-1-O-acetyl-eudesma- 15hydroxyl-4,5-en-6,12-olide. Arvestolide H (5) was obtained as a white amorphous powder. The HRESIMS spectrum showed a sodium adduct ion [M þ Na]þ at m/z 371.1821 (calcd. 371.1834) corresponding to a molecular formula of C20H28O5. The 1H NMR spectrum presented an olefinic proton at dH 6.16 (1H, q, J ¼ 7.0 Hz), two terminal vinyl protons at dH 5.22 (1H, s) and 5.06 (1H, s), three oxygenated methine at dH 4.85 (1H, t, J ¼ 3.0 Hz), 4.30 (1H, t, J ¼ 3.0 Hz), and 4.05 (1H, t, J ¼ 10.9 Hz), and four methyl signals at dH 0.94 (3H, s), 1.25 (3H, d, J ¼ 7.0 Hz), 1.97 (3H, s), and 2.06 (3H, d, J ¼ 7.0 Hz). The corresponding carbon signals for oxymethine (dC 72.3, 75.5, and 79.2), vinyl (dC 113.3, 127.4, 139.7, and 145.3), methyl (dC 12.5, 15.9, 17.8, and 20.8), and carbonyl groups (dC 166.7 and 179.1) were observed in the 13C NMR spectrum. These 1D NMR data indicated that 5 is a sesquiterpenoid similar to 1-acetylerivanin (Mengi et al., 1991) except that the acetyl group is replaced by an angelica acyl group in 5. This deduction was supported by the HMBC correlation from H-1 to the angelica acyl carbon (dC 166.7). The NOE cross-peaks of H314/H-1, H-3, H-6, H-6/H-11, H-5/H-7, and H-7/H3-13, together with the coupling constants of H-6 (1H, t, J ¼ 10.9 Hz) and H-3 (1H, t, J ¼ 3.0 Hz) indicated 5 has the same relative configuration as that of 1-acetylerivanin. The ECD spectrum (Fig. S55, Supporting Information) of 5 showed the Cotton effects at 206 nm (Dε þ2.0) and 300 nm (Dε 2.7), deriving from a p/p* electron transition of the terminal vinyl of D4,15 and a n/p* electron transition of the angelica acyl. According to the Olefin octant rule (Scott and Wrixon, 1970), the positive Cotton effect reveals an S configuration of C-5 (Fig. S55, Supporting Information). The calculated ECD spectra indicated that the spectrum of (1S,3R,5S,6S,7S,10R,11S)-5 agreed well with the experimental one (Fig. S55, Supporting Information). Thus, the structure of arvestolide H (5) was deduced as (1S,3R,5S,6S,7S,10R,11S)-1-O-angelica acyl-eudesma-3-hydroxy4,15-en-6,12-olide. Arvestolide I (6) was obtained as colorless needles from tetrahydrofuran-water system. Its molecular formula was determined as C17H24O5 by a sodium adduct ion [M þ Na]þ at m/z 331.1517 (calcd. 331.1521) in the HRESIMS. The 1H and 13C NMR data revealed the presence of vinyl (dH 5.51; dC 126.2, and 143.5), carbonyl (dC 202.4), lactone (dC 177.3), and acetyl (dH 2.01; dC 21.6 and 170.3) groups. The proton spin systems from H-1 to H-3, H-6 to H-10, and H-1 and H-10 were connected through 1H-1H COSY correlations. By comparison of the 13C NMR data with those of asantonin and its analogues in literature (Shimizu et al., 1979), 6 was deduced as a 5-keto germacranolide. 1-O-acetyl was assigned via HMBC correlation from H-1 to the acetyl group (dC 170.3). The vinyl proton (dH 5.51) showed HMBC correlations with C-1, C-5, and C-15, suggesting a double bond existed between C-3 and C-4. Thus, 6 was elucidated as 1-O-acetyl-3-en-5-keto-germacranolide. The relative configuration of 6 was established by NOESY spectrum. The crosspeaks of H-1/H-3, H-6/H-10, and H-6/H3-15 indicated that the 10membered ring moiety has a boat-chair conformation (Pui-Hay et al., 2009) and these protons were assigned as b-oriented. The large coupling constant (J ¼ 9.7 Hz) between H-6 and H-7 suggested that H-7 is a-oriented. The a-orientation of H3-13 was elucidated by the NOE correlation of H-7/H3-13 and the large coupling constant (J ¼ 12.2 Hz) between H-7 and H-11. The absolute
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configuration of 6 was finally determined as (1S,6S,7S,10R,11S) by the X-ray crystallography (Fig. 5). This result was further confirmed by comparison of the experimental and calculated ECD spectra (Fig. S65, Supporting Information). Therefore, the structure of arvestolide I (6) was defined as (1S,6S,7S,10R,11S)-1-O-acetyl-3-en5-keto-germacranolide. Arvestonate A (7), obtained as a colorless gum, has a molecular formula of C16H26O5 determined by the HRESIMS (m/z 321.1682 [M þ Na]þ, calcd. 321.1678) and 13C NMR data. The NMR data of 7 were almost the same with those of crossostephin (Wu et al., 2009), a eudesmane sesquiterpenoid. Their difference was the terminal D11(13) double bond of crossostephin was hydrogenated into a methyl group in 7, which was supported from the HMBC correlations from H3-13 to C-7, C-11, and C-12. The NOE correlations of H314/H-6 and the coupling constant (J ¼ 10.4 Hz) between H-6 and H7 both indicated the same configurations of H3-14b, H-6b, and H-7a as those of crossostephin. The coupling constants of 11.5 and 5.2 Hz between H-1 and H2-2 suggested that H-1 was axial and a-oriented, which was corroborated by the NOE correlation between H1 and H-9a. The configuration of 5-OH determined the trans- or cisfused pattern of the eudesmane, which was found to be closely related with the ECD Cotton effect of 7. The positive Cotton effect at 205 nm of 7 (Fig. 6) revealed an S configuration based on the Olefin octant rule (Scott and Wrixon, 1970). By comparison of the experimental and calculated ECD spectra (Fig. S75, Supporting Information), the absolute configuration of 7 was finally established as (1R,5R,6S,7S,10S). However, the configuration of H-11 is hard to determine from the present data. Therefore, the structure of 7 was determined as Methyl (1R,5R,6S,7S,10S)-1,5,6-trihydroxy-eudesma4,15-en-12-oate. Arvestonate B (8) has a molecular formula of C18H28O6 determined from a sodium adduct ion [M þ Na]þ at m/z 363.1777 (calcd. 363.1784) in the HRESIMS and the 13C NMR data. The similar IR and NMR data with those of 7 indicated that they shared the same
197
eudesmane scaffold. A group of additional acetyl signals were observed in 8 in comparison with 7. The 1-O-acetyl was assigned from the HMBC correlation from H-1 to the acetyl group. The NOE correlations of H3-14/H-1 and H3-14/H-6 and the coupling constant between H-6 and H-7 (J ¼ 11.5 Hz) defined a relative configuration of H-1b, H3-14b, H-6b, and H-7a. However, a negative Cotton effect at 205 nm in the ECD spectrum of 8 was inconsistent with that of 7. After changing solvent to DMSO-d6, the NOE correlations of 5-OH (dH 4.55) with H-1, H-6, and H3-14 indicated that they are cofacial and the two rings are cis-fused. By comparison of the structures between 7 and 8, the configuration of C-5 was found to have a determined effect on the conformation of methylenecyclohexane moiety and thus these two compounds have a mirror-like Cotton effect at around 205 nm in the ECD spectra (Fig. 6). The quantum chemical calculation of ECD spectra and Olefin octant rule indicated that the absolute configuration of 8 is (1S,5S,6S,7S,10S) (Fig. S85, Supporting Information). The configuration of C-11 is also hard to determine exactly since its flexible residue conformation. Arvestonol (9) was obtained as a colorless gum. Its molecular formula was determined as C15H24O3 based on the 13C NMR data and a protonated ion at m/z 253.1816 (calcd. 253.1804) in the HRESIMS. The IR spectrum showed absorption bands for hydroxy (3351 cm1) and conjugated carbonyl (1741 cm1) groups. The 1H and 13C NMR data indicated that 9 is an eudesmane sesquiterpenoid similar to 3-oxo-eudesma-4,5-en-9,12-diol (Liu et al., 1998). Their difference was the position of the hydroxy group, which shifted from C-9 to C-1 in 9, as proved by the HMBC correlations from H-1 (dH 3.81) to C-3 (dC 197.2) and C-5. Thus, the structure of 9 was defined as 3-oxo-eudesma-4-en-1,12-diol. The relative configuration was established by NOESY spectrum. The cross-peaks of H3-14/H-6b/H-8b/H-7 indicated that H-7 was b-oriented. The coupling constants between H-1 and H2-2 (J ¼ 12.8 and 5.3 Hz) indicated H-1 was axial and a-oriented, which was supported by NOE correlation between H-1 and H-9a. The positive Cotton effect
Fig. 5. X-ray ORTEP drawings of compounds 3, 6, and 10.
Fig. 6. The comparison of experimental ECD spectra and Olefin octant rule applied for 7 and 8.
198
S.-H. Tian et al. / Phytochemistry 147 (2018) 194e202
at 240 nm in the ECD spectrum of 9 (Fig. S100, Supporting Information), arising from the p-p* electron transition, indicated a clockwise helicity of a,b-unsaturated carbonyl according to the empirical helicity rule (Kirk, 1986). Correspondingly, the absolute configuration of 9 was determined as (1R,7S,10R), in combination of the aforementioned established NOE correlations. Comparison of the calculated and experimental ECD spectra of 9 (Fig. S100, Supporting Information) corroborated the above deduction. The configuration of C-11 is hard to determine as that of 7 and 8. Therefore, the structure of arvestonol (9) was determined as (1R,7S,10R)-3-oxo-eudesma-4,5-en-1,12-diol. Arvestolide J (10) was obtained as colorless needles from acetone-water (5:1) solvent. Its molecular formula was determined as C14H18O5 based on a sodium-adduct ion [M þ Na]þ at m/z 289.1059 (calcd. for 289.1052) in the HRESIMS and the 13C NMR data. IR spectrum showed hydroxy (3347 cm1) and lactone (1757, 1013 cm1) absorption bands. In the 1H NMR spectrum, an olefinic proton (dH 5.82), an oxymethine (dH 3.59) and three methyl signals were distinctively observed. Fourteen carbon resonances were classified into three methyl, two methene, four methine, and five quaternary carbons by multi-edited HSQC spectrum. Among them, two carbonyls (dC 181.1 and 170.1), a double band (dC 120.4 and 167.6), and two oxy-carbon signals (dC 93.1 and 110.9) were detected. These 1D NMR data were similar to those of norsantolinidilactone A (Jakupovic et al., 1991), a nor-dilactone sesquiterpenoid isolated from A. santolinifolia. In comparison of their 13C NMR data, an obvious deshielded chemical shift of C-5 (DdC þ23.6) assigned by the HMBC correlations of H3-14/C-5 and H3-15/C-5 indicated a hydroxy group was attached, which was consistent with its molecular formula. Therefore, the planar structure of 10 was determined as 5-hydoxy-norsantolinidilactone A. The relative configuration of 10 was elucidated from the NOESY spectrum in which the cross-peaks of H3-14/H-6, H-6/H-8b, and H-6/H-11 were present, indicating these b-oriented protons were cofacial. The large coupling constant (J ¼ 12.0 Hz) of the doublet H-6 indicated an a-orientation of H-7. However, the 5-OH configuration is hard to establish. A relative configuration of (5R,6S,7S,10R,11S)-10 was achieved by the X-ray crystallographic analysis (Fig. 5) due to the large Flack parameter [0.27(17)]. Finally, by comparison of calculated and experimental ECD data (Fig. S110, Supporting Information), the absolute configuration of arvestolide J (10) was determined as 5R,6S,7S,10R,11S. Arvestonate C (11) was obtained as a colorless gum, and its molecular formula of C17H26O6 was determined by a sodium adduct ion [M þ Na]þ at m/z 349.1624 (calcd. 349.1627) and its 13C NMR data. IR spectrum showed similar absorption bands for hydroxy (3511 cm1) and lactone (1735 and 1054 cm1) functionalities as those of 10. Seventeen carbon resonances were detected in the 13C NMR spectrum and further classified into four methyl, five methylene, three methine, and five quaternary carbons by the multiedited HSQC spectrum. The 1H-1H COSY spectrum gave the mutual correlations of H2-2/H2-3, H-6/H-7/H2-8/H2-9, and H-7/H11/H3-13. The HMBC correlations of H2-15/C-5, C-3, H3-16/C-1, H23/C-1, H3-13/C-7, C-12, and H3-17/C-12 demonstrated the presence of two residues of 1-methoxy-4-methylene-1,5-dioxopentan-5-yl (C-1 to C-5) and 1-methoxy-1-oxopropan-2-yl (C-11 to C-12). These two residues were connected to a cyclopentane moiety at the C-7 and C-10 positions, which was proved by the HMBC correlations of H3-14/C-5 and C-9, and H3-13/C-7 and C-12. 6-OH Substituent was deduced from the chemical shifts (dH 3.61 and dC 85.4) and the HMBC correlations of H3-14/C-6 and H-6/C-8 and C-9. This terpene framework of 11 was similar to that of santolinifolide A (Jakupovic et al., 1991), a seco-sesquiterpenoid, and the structure of 11 was determined as methyl 4-(2-hydroxy-3-(1-methoxy-1oxopropan-2-yl)-1-methylcyclopentate-1-carbonyl)pent-4-enoate.
The relative configuration of 11 was established by NOESY spectrum in which the correlations of H3-14/H-6, H-8a, and H-11, H-6/ H-11, and H-7/H3-13 were observed, indicating H3-14 and H-6 are a-oriented while H-7 is b-oriented in combination with the coupling constant (J ¼ 7.0 Hz) of H-6/H-7. The positive ECD Cotton effect at 225 nm (Fig. S120, Supporting Information) is arisen from the conjugated ketene at C-10 (Marina et al., 2004; Mori, 1974) and its absolute configuration was deduced as 6R,7R,10S by comparison of experimental and calculated ECD spectra (Fig. S120, Supporting Information). Therefore, the structure of arvestonate C (11) was established as depicted. A. vestita, as a folk medicine in Tibet area, has been traditionally used to treat various inflammatory diseases (Tian et al., 2013b). Some sesquiterpenoids and their dimers isolated from Artemisia species in previous studies (Turak et al., 2014; Zan et al., 2012; Zhang et al., 2014, 2016) showed remarkable cytotoxic and NO inhibitory activities. Thus, the isolates were screened on the same models. The pre-screening for cytotoxic activities on HepG2 cell line showed that compounds 1e11 had no effect with IC50 values larger than 100 mM. Compounds 5 and 6 showed inhibition against LPS-induced NO production in BV-2 cells with IC50 values of 43.2 and 39.9 mM, respectively, while the other compounds were all larger than 100 mM. 3. Conclusions In this study, 11 previously undescribed sesquiterpenoids including eight eudesmane sesquiterpenoids (1e5 and 7e9), a germacrene sesquiterpenoid (6), and two seco-sesquiterpenoids (10 and 11) were isolated from A. vestita. Their structures were elucidated from HRESIMS, IR, and NMR spectroscopic data. Their absolute configurations were determined by empirical rules, X-ray diffraction analysis, and comparison of experimental and calculated ECD data. The configuration of C-5 was disclosed to be responsible for the mirror-like ECD Cotton effect at around 205 nm for 7 and 8, which was corroborated by the Olefin octant rule and quantum calculation of ECD spectra. Compounds 5 and 6 showed NO inhibitory effects with IC50 values of 43.2 and 39.9 mM, respectively. The results obtained herein give a proof for the traditional use of A. vestita for the treatment of inflammation-related diseases. 4. Experimental 4.1. General experimental procedures Optical rotations were measured using a Rudolph Autopol III automatic polarimeter. ECD spectra were recorded on a JASCO J-810 CD spectrometer. IR spectra were recorded (KBr disks) on a Thermo Nicolet Nexus 470 FT-IR spectrometer. NMR spectra were obtained at 500 MHz for 1H and 125 MHz for 13C on a Varian INOVA-500 NMR spectrometer in methanol-d4. The HSQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HRMS data were performed on a Bruker APEX IV FT-MS or a Waters Xevo G2 Q-TOF spectrometers fitted with an ESI source. LC-MS analysis was performed on a Shimadzu LCMS-IT-TOF instrument equipped with a Shimadzu Prominence HPLC system (Shimadzu, Kyoto, Japan). TLC analysis was carried out on the precoated silica gel GF254 plates (Qingdao Marine Chemical Inc., 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. Column chromatography (CC) was performed using silica gel (100e200 mesh or 200e300 mesh, Qingdao Marine Chemical Inc., People's Republic of China), ODS C18 (50 mm, Merck, Germany), and Sephadex LH-20 (Amersham Biosciences, Sweden). Semipreparative HPLC was performed on an Agilent 1200 liquid chromatography
S.-H. Tian et al. / Phytochemistry 147 (2018) 194e202
with a Waters XBridge™ Prep shield RP 18 column (250 mm 10 mm, 5 mm). Analytical HPLC was performed on an Aglient 1100 system with an Aglient Extend-C18 column (4.6 150 mm, 5 mm). All purified compounds submitted for bioassay were at least 95% pure as judged by HPLC analysis. 4.2. Plant materials The aerial parts of Artemisia vestita W. were collected in Ninger town, Puer city, Southwest of Yunnan Province, People's Republic of China, in September 2010. The plant material was authenticated by one of the authors (P.-F. Tu). A voucher specimen (No. 20100905) has been deposited at the herbarium of the Peking University Modern Research Center for Traditional Chinese Medicine. 4.3. Extraction and isolation The dried aerial parts of A. vestita (45 kg) were extracted with 95% aqueous EtOH (360 L 3 h 3) at room temperature. The concentrated extracts (3.8 kg) was suspended in H2O (15 L) and extracted with petroleum ether (PE) and chloroform (CHCl3) successively. The CHCl3 extract (450 g) were subjected to a silica gel CC eluting with a step-wise gradient of PE-EtOAc (1:0, 8:1, 3:1, 1:1, and 0:1, v/v) to produce 10 fractions (Frs.1e10), monitored by TLC analysis. Frs. 5e7 were combined and subjected to silica gel CC (200e300 mesh) eluting with gradient n-hexane-acetone (30:1, 10:1, 5:1, 2:1, and 0:1, v/v) to yield five fractions (Frs. 5ae5e). Fr. 5a was further subjected to Sephadex LH-20 CC eluting with methanol to afford fractions 5a1e5a5. Fr. 5a2 was repeatedly purified over silica gel CC (200e300 mesh) using CHCl3-EtOAc (20:1 / 10:1 / 5:1) to yield 1 (2 mg). Fr. 5a3 was separated on Sephadex LH-20 CC to obtain sub-fractions 5a3ae5a3f and the subfraction 5a3c was purified by silica gel (200e300 mesh) CC repeatedly to yield 11 (3 mg). Fr. 5a4 was subjected to silica gel CC eluted with CHCl3-acetone (30:1 / 10:1 / 4:1) to obtain sub-
fractions 5a4ae5a4g. Sub-fraction 5a4c was purified repeatedly by reversed-phase C18 CC eluting with MeOH-H2O (2:8 / 3:7 / 5:5 / 7:3) to yield 2 (2 mg) and 3 (4 mg). Subfraction 5a4e was purified repeatedly by reversed-phase C18 CC eluting with (2:8 / 3:7 / 5:5 / 7:3) to yield 5 (4 mg) and 10 (3 mg). Fr. 5a5 was separated by silica gel CC using CHCl3-acetone (20:1 / 10:1 / 5:1) to obtain sub-fractions 5a5ae5a5e and subfraction 5a5b were purified repeatedly by semipreparative HPLC (MeCN-H2O 40:60) to yield 6 (4 mg, tR ¼ 15 min). Fr. 5b was subjected to Sephadex LH-20 CC using MeOH as eluting solvent to yield fractions 5b1e5b5. Fr. 5b2 was separated by silica-gel CC using CHCl3-EtOAc (20:1 / 10:1) to obtain sub-fractions 5b2ae5b2e. Sub-fraction 5b2b was purified by reversed-phase C18 CC eluting with MeOH-H2O (20:80 / 40:60 / 60:40 / 80:20) to yield 4 (2 mg) and 7 (3 mg). Sub-fraction 5b2d was purified by reversedphase C18 CC eluting with MeOH-H2O (20:80 / 40:60 / 60:40 / 80:20) to yield 8 (3 mg) and 9 (2 mg).
4.3.1. Arvestolide D (1) White powder; [a]20 D e4.6 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 227 (3.5) nm; ECD (MeOH) lmax (D3 ) 323 (3.2), 245 (þ3.7) nm; IR (KBr) nmax 3381, 2957, 2870, 1738, 1455, 1369, 1056, 1010, 788, 750 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 1; HRESIMS m/z 345.1317 [M þ Na]þ (calcd for C17H22O6Na, 345.1314).
4.3.2. Arvestolide E (2) White gum; [a]20 D þ69.0 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 228 (3.5) nm; ECD (MeOH) lmax (D3 ) 252 (þ37.2) nm; IR (KBr) nmax 3742, 3710, 3652, 2939, 2865, 1774, 1739, 1541, 1257, 1068, 786 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 1; HRESIMS m/z 321.1328 [M e H]e (calcd for C17H21O6, 321.1338).
Table 1 1 H and 13C NMR Data of Compounds 1e5 (d in ppm, J in Hz, 500 MHz for 1H and 125 MHz for Position
1
dH 1 2a 2b 3a 3b 4 5 6 7 8a 8b 9a 9b 10 11 12 13 14 15
5.68, s
acetyl
2.21, s
2
dC
dH
77.4 4.86, br s 75.4 191.1 2.28, m 22.3 1.73, dd (14.7, 3.9) 5.96, s 128.5 2.05, dd (14.3, 3.9) 33.8 1.69, d (14.3) 155.4 72.9 75.7 70.1 4.39, d (11.6) 83.8 89.6 1.62, ddd (13.6, 11.6, 3.4) 47.4 154.4 1.89, d (13.0) 23.8 2.57, m 18.8 1.49, m 2.23, m 1.87, d (13.0) 30.4 2.25, m 26.9 1.41, m 1.08, dd (13.3, 5.5) 47.1 39.0 2.38, dq (13.6, 6.8) 41.6 126.3 177.2 170.9 1.26, d (6.8) 12.7 1.89, s 8.6 1.30, s 16.9 1.29, s 21.4 2.05, s 22.6 1.47, s 27.9 21.6 2.10, s 170.3
13
C, in CDCl3).
3
dC
199
dH 6.31, d (10.5) 5.94, d (10.5)
4
dC
dH
5
dC
154.1 4.79, dd (5.1, 2.5) 76.1 123.1 1.88, m 22.7 1.88, m 195.2 2.32, m 25.9 2.22, m 65.0 130.1 69.8 131.6 4.55, d (11.5) 77.1 4.62, d (11.3) 82.4 2.21, m 50.3 1.75, m 52.8 1.99, m 23.2 1.97, ddd (12.9, 5.2, 3.2) 23.7 1.64, qd (13.6, 12.8, 4.4) 1.58, td (12.9, 3.2) 1.97, m 37.9 1.75, m 33.0 1.53, dd (13.7, 4.4) 1.46. dt (13.5, 3.2) 41.6 40.4 2.41, dq (12.5, 6.8) 41.3 2.34, m 41.1 176.9 178.0 1.27, d (7.0) 12.6 1.26, d (7.0) 12.3 1.42, s 21.5 1.22, s 26.1 4.65, d (12.0) 58.1 4.40, d (12.1) 62.9 4.06, d (12.0) 4.12, d (12.1) 21.1 2.08, s 21.2 170.5 170.7
dH
dC
4.85, 1.54, 2.13, 4.30,
t (3.0) m m t (3.0)
3.07, 4.05, 1.64, 1.89, 1.54, 1.71, 1.42,
d (10.9) t (10.9) m dd (13.0, 3.5) m m dt (13.5, 3.5)
75.5 33.3 72.3 145.3 43.2 79.2 52.3 22.8 33.0
41.7 2.35, dq (13.5, 6.8) 41.1 179.1 1.25, d (7.0) 12.5 0.94, s 17.8 5.22, s 113.3 5.06, s
angelica acyl 6.16, q (7.0) 2.06, d (7.0) 1.97, s
166.7 127.4 139.7 15.9 20.8
200
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4.3.3. Arvestolide F (3) Colorless needles; mp 160e162 C; [a]20 D e18.3 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 229 (3.5) nm; ECD (MeOH) lmax (D3 ) 340 (þ18.5) nm; IR (KBr) nmax 3707, 2945, 1772, 1677, 1054, 1031, 670 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 1; HRESIMS m/z 279.1227 [M þ H]þ (calcd for C15H19O5, 279.1232).
4.3.8. Arvestonate B (8) Colorless gum; [a]20 D e29.1 (c 0.2, MeOH); UV (MeOH) lmax (log ε) 207 (3.1) nm; ECD (MeOH) lmax (D3 ) 205 (e37.1) nm; IR (KBr) nmax 3451, 2948, 1732, 1245, 1026, 966 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 2; HRESIMS m/z 363.1777 [M þ Na]þ (calcd for C18H28O6Na, 363.1784).
4.3.4. Arvestolide G (4) White powder; [a]20 D þ4.4 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 210 (3.3) nm; ECD (MeOH) lmax (D3 ) 210 (e18.2) nm; IR (KBr) nmax 3706, 3354, 2972, 2943, 1788, 1057, 1032 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 1; HRESIMS m/z 331.1524 [M þ Na]þ (calcd for C17H24O5Na, 331.1521).
4.3.9. Arvestonol (9) Colorless gum; [a]20 D þ52.6 (c 0.2, MeOH); UV (MeOH) lmax (log ε) 248 (3.4) nm; ECD (MeOH) lmax (D3 ) 248 (þ67.3) nm; IR (KBr) nmax 3351, 2950, 2868, 1741, 1443, 1194, 1023, 749 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 2; HRESIMS m/z 253.1816 [M þ Na]þ (calcd for C15H24O3Na, 253.1804).
4.3.5. Arvestolide H (5) White amorphous powder; [a]20 D þ32.5 (c 0.3, MeOH); UV (MeOH) lmax (log ε) 218 (3.5) nm; ECD (MeOH) lmax (D3 ) 210 (þ2.1), 300 (e2.7) nm; IR (KBr) nmax 3755, 3383, 2949, 2863, 1732, 1594, 1444. 1055, 1014 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 1; HRESIMS m/z 371.1821 [M þ Na]þ (calcd for C20H28O5Na, 371.1834).
4.3.10. Arvestolide J (10) Colorless needles; mp 132e134 C; [a]20 D þ26.3 (c 0.6, MeOH); UV (MeOH) lmax (log ε) 215 (3.4) nm; ECD (MeOH) lmax (D3 ) 222 (e17.8) nm; IR (KBr) nmax 3347, 2970, 1757, 1252, 1013, 925 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 3; HRESIMS m/z 289.1059 [M þ Na]þ (calcd for C14H18O5Na, 289.1052).
4.3.6. Arvestolide I (6) Colorless needles; mp 157e159 C; [a]20 D e11.4 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 246 (3.2) nm; ECD (MeOH) lmax (D3 ) 252 (þ59.8) nm, 320 (þ21.5) nm; IR (KBr) nmax 3723, 3383, 2936, 1782, 1735, 1058, 754 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 2; HRESIMS m/z 331.1517 [M þ Na]þ (calcd for C17H24O5Na, 331.1521).
4.3.11. Arvestonate C (11) Colorless gum; [a]20 D þ2.5 (c 0.9, MeOH); UV (MeOH) lmax (log ε) 222 (3.4) nm; ECD (MeOH) lmax (D3 ) 225 (þ4.8), 327 (e5.1) nm; IR (KBr) nmax 3511, 2953, 2873, 1735, 1453, 1260, 1054, 667 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 3; HRESIMS m/z 349.1624 [M þ Na]þ (calcd for C17H26O6Na, 349.1627).
4.3.7. Arvestonate A (7) Colorless gum; [a]20 D þ54.6 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 202 (3.1) nm; ECD (MeOH) lmax (D3 ) 205 (þ6.3) nm; IR (KBr) nmax 3331, 2945, 2868, 1708, 1502, 1375, 1292, 1192, 1024, 754, 693 cm1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 2; HRESIMS m/z 321.1682 [M þ Na]þ (calcd for C16H26O5Na, 321.1678).
4.4. X-ray crystallographic analysis Crystal analysis were performed on a Bruker APEX diffractometer equipped with an APEX Ⅱ CCD, using Cu Ka radiation (l ¼ 1.54178 Å). Cell refinement and data reduction were performed with Bruker SAINT. The structures of 3, 6, and 10 were solved by direct methods using SHELXS-97. Refinements were performed
Table 2 1 H and 13C NMR Data of Compounds 6e9 (d in ppm, J in Hz, 500 MHz for 1H and 125 MHz for Position
6
7
dH 1 2a 2b 3a 3b 4 5 6 7 8a 8b 9a 9b 10 11 12
4.67, 2.50, 2.18, 5.51,
4.59, 2.54, 2.08, 1.14, 1.28,
ddd (12.0, 4.0, 1.6) m m ddd (11.3, 5.8, 1.6)
d (9.7) m dt (9.2, 3.5) m m
dC
dH
76.5 29.6
4.07, 1.88, 1.52, 2.73, 2.09,
126.2 143.5 202.4 80.6 48.6 27.5 26.6
1.45, m 2.24, dq (12.2, 6.8)
32.8 41.0 177.3
13 14 15
1.31, d (6.8) 0.92, d (6.7) 2.03, s
12.7 16.9 22.6
16 acetyl
2.01, s
21.6 170.3
C, in CDCl3). 8
dd (11.5, 5.2) dtd (12.6, 5.2, 1.8) m td (13.5, 6.1) ddd (13.5, 5.4, 1.8)
4.13, d (10.4) 2.16, m 1.61, m 1.40 qd (13.3, 4.3) 1.80, td (13.3, 4.3) 1.53, m 2.82, dq (2.4, 7.3)
1.23, 0.77, 5.00, 4.76, 3.70,
13
d (7.3) s br s br s s
dC
dH
72.8 31.1
5.09, 1.84, 1.63, 2.68, 2.15,
30.2 148.1 78.4 68.4 41.7 22.5 29.0 43.2 40.4 177.4 13.2 12.6 123.1 51.7
3.80, 2.59, 1.59, 1.34, 1.64, 1.48,
9
dd (12.1, 4.8) dt (12.8, 3.5) m m dt (13.9, 3.5)
d (11.5) dq (11.5, 5.6) m td (13.3, 4.4) m dt (14.4, 3.5)
2.61, m
1.16, 1.02, 5.37, 5.15, 3.69, 2.03,
d (6.9) s s s s s
dC
dH
dC
76.7 27.0
3.81, dd (12.8, 5.3) 2.64, dd (16.4, 5.3) 2.55, dd (16.4, 12.8)
74.5 42.3
31.6 144.1 78.3 76.3 41.8 22.5 25.9 43.7 42.3 177.7 13.2 16.8 115.5 51.7 21.2 170.6
197.2
2.75, 1.93, 1.51, 1.69, 1.52, 1.33, 2.13,
d (15.3) t (12.8) m m m t (13.3) dt (13.3, 3.2)
1.66, 3.67, 3.58, 1.00, 1.17, 1.77,
m dd (10.6, 5.8) dd (10.6, 6.3) d (7.0) s s
129.4 161.9 30.4 39.6 25.2 37.8 41.5 40.4 65.8 13.5 16.2 10.9
S.-H. Tian et al. / Phytochemistry 147 (2018) 194e202 Table 3 1 H and 13C NMR Data of Compounds 10 and 11 (d in ppm, J in Hz, 500 MHz for 1H and 125 MHz for 13C, in CDCl3). Position
10
11
dH 1 2a 2b 3a 3b 4 5 6 7 8a 8b 9a 9b 10 11 12 13 14 15 16 17
5.82, s
3.59, 2.64, 1.87, 1.33, 2.64, 1.74,
d (12.0) br s m m m m
2.28, m 1.27, d (6.5) 1.21, s 2.17, s
dC
dH
170.1
2.46, 2.44, 2.58, 2.45,
120.4 167.6 119.7 93.1 44.2 21.6 38.9 51.9 43.0 181.5 13.2 25.4 15.2
3.61, 2.19, 1.83, 1.32, 2.34, 1.61,
dC m m m m
d (7.0) p (8.7) ddd (12.5, 8.7, 3.8) m dt (13.4, 8.7) ddd (13.4, 8.7, 3.8)
2.46, m 1.17, 1.36, 5.80, 5.72, 3.66, 3.69,
d (7.0) s s s s s
173.2 32.8 28.5 145.4 208.7 85.4 43.4 25.1 34.5 56.7 49.7 176.9 15.1 24.4 123.1 51.5 51.7
with SHELXL-97 using full-matrix least-squares, with anisotropic displacement parameters for all the non-hydrogen atoms. The Hatoms were placed in calculated positions and refined using a riding model. Crystallographic data have been deposited with the Cambridge Crystallographic Data Center (CCDC 1535635 for 3; CCDC 918234 for 6; CCDC 930778 for 10). Crystal data for 3: C15H18O5, M ¼ 278.29 g/mol, size ¼ 0.26 0.18 0.15 mm3, orthorhombic, space group P21, a ¼ 9.4913(19) Å, b ¼ 10.299(2) Å, c ¼ 14.739(3) Å, b ¼ 90.0 , V ¼ 1440.8 (5) Å, T ¼ 296(2) K, Z ¼ 4, d ¼ 1.283 g/m3, m(CuKa) ¼ 1.54178 Å, F(000) ¼ 328, completeness qmax ¼ 97.5%, 2486 independent reflections, Rint ¼ 0.0252, 2416 reflections with jFj2 2sjFj2, 0 restraint, R1 ¼ 0.0354, wR2 ¼ 0.09716, S ¼ 1.044, GOF ¼ 1.044. The Flack parameter is 0.2(2). Crystal data for 6: C17H24O5, M ¼ 308.36 g/mol, size ¼ 0.22 0.15 0.10 mm3, monoclinic, space group P21, a ¼ 10.323(2) Å, b ¼ 7.6545(15) Å, c ¼ 11.528(2) Å, b ¼ 115.16(3) , V ¼ 824.4 (3) Å, T ¼ 296(2) K, Z ¼ 2, d ¼ 1.242 g/m3, m(CuKa) ¼ 1.54178 Å, F(000) ¼ 332, completeness qmax ¼ 97.4%, 2741 independent reflections, Rint ¼ 0.0191, 2579 reflections with jFj2 2sjFj2, 23 restraint, R1 ¼ 0.0326, wR2 ¼ 0.0896, S ¼ 1.0484, GOF ¼ 1.048. The Flack parameter is 0.1(2). Crystal data for 10: C14H18O5 þ H2O, M ¼ 284.30 g/mol, size ¼ 0.12 0.10 0.10 mm3, triclinic, space group P1, a ¼ 6.8239(14) Å, b ¼ 6.8327(14) Å, c ¼ 9.0715(18) Å, b ¼ 74.68(3) , V ¼ 359.21 (13) Å, T ¼ 296(2) K, Z ¼ 1, d ¼ 1.314 g/m3, m(CuKa) ¼ 1.54178 Å, F(000) ¼ 152, completeness qmax ¼ 91.9%, 2096 independent reflections, Rint ¼ 0.0121, 2091 reflections with jFj2 2sjFj2, 3 restraint, R1 ¼ 0.0315, wR2 ¼ 0.0836, S ¼ 1.078, GOF ¼ 1.078. The Flack parameter is 0.27(17). 4.5. Conformational energy calculation of the main conformers of 1‒10 The relative configurations were initially established according to 1D-gradient NOE or NOESY spectra, and submitted to random conformational analysis with the MMFF94s force field using the Sybyl-X 1.1 software package. The conformers were further optimized by using the TDDFT method at the B3LYP/6-31G(d) level and
201
the frequency was calculated at the same level of theory. The stable conformers without imaginary frequencies were subjected to ECD calculation by the TDDFT method at the B3LYP/6-31þG(d) level in MeOH as solvent. ECD spectra of different conformers were simulated using SpecDis with a half-band width of 0.3 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. 4.6. Cytotoxic assay Cell proliferation inhibition was determined using the MTT method according to the established protocols (Zan et al., 2012). Taxol was used as the positive control. 4.7. NO inhibitory effect BV-2 microglial cells were purchased from Peking Union Medical College (PUMC) Cell Bank (Beijing, People's Republic of China). Cell maintenance, experimental procedures, and data presentation for the inhibition of NO production and viability assay were conducted as previously described (Zhang et al., 2014). Quercetin (IC50 value of 10.0 mM) was used as a positive control. All the compounds were prepared as stock solutions in DMSO (final solvent concentration less than 0.5% in all assays). Acknowledgements This work was financially supported by the grants from the National Natural Science Foundation of China (Nos. 81373294, 81222051, and 81303253) and the National Key Technology R&D Program “New Drug Innovation” of China (No. 2017ZX09101003008-003). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.phytochem.2018.01.004. References Anake, K., Luis, M.M.V., Jose, M.C., Werner, H., 1992. An eudesmanolide from Picris spinifera. Phytochemistry 31, 3635e3636. Braulio, M.F., 2011. Natural sesquiterpenoids. Nat. Prod. Rep. 28, 1580e1610. Fujimoto, Y., Shimizu, T., Ohmori, M., Tatsuno, T., 1979. Modification of a-santonin. Ⅲ. Synthesis of dihdrocostunolide. Chem. Pharm. Bull. 27, 923e933. Irwin, M.A., Geissman, T.A., 1973. Arbusculin-D from Artemisia arbuscula ssp. Arbuscula. Phytochemistry 12, 853e855. Jakupovic, J., Tan, R.X., Bohlmann, F., Jia, Z.J., Huneck, S., 1991. Seco- and norsesquiterpene lacones with a new carbon skeleton from Artemisia santolinifolia. Phytochemistry 30, 1941e1946. Kawazoe, K., Tsubouchi, Y., Abdullah, N., Takaishi, Y., Shibata, H., Higuti, T., Hori, H., Ogawa, M., 2003. Sesquiterpenoids from Artemisia gilvescens and an anti-MRSA compound. J. Nat. Prod. 66, 538e539. Kirk, D.N., 1986. The chiroptical properties of carbonyl compounds. Tetrahedron 42, 810e818. rez, Y., Catala n, C.A.N., Krautmann, M., Riscala, E.C., Burgueno-Tapia, E., Mora-Pe Joseph-Nathlan, P., 2007. C-15-Functionalized eudesmanolides from Mikania campanulata. J. Nat. Prod. 70, 1173e1179. Liu, L.J., Chen, Y.G., Xiong, Z.M., Li, J., Zhou, G., Li, Y.L., 1998. A facile synthesis of 3oxo-7aH-eudesma-4-ene-9b,12-diol. Chin. Chem. Lett. 9, 625e627. Ma, C.M., Nakamura, N., Hattori, M., Zhu, S., Komatsu, K., 2000. Guaiane dimers and germacranolide from Artemisia caruifolia. J. Nat. Prod. 63, 1626e1629. Marina, D., Cinzia, D.M., Armando, Z., Brigida, D., 2004. Isolation and phytotoxicity of Apocarotenoids from Chenopodium album. J. Nat. Prod. 67, 1492e1495. Mengi, N., Taneja, S.C., Mahajan, V.P., Mathela, C.S., 1991. Eudesmanolides from Senecio chrysanthemoides. Phytochemistry 30, 2329e2330. Mori, K., 1974. Synthesis of optically active grasshopper ketone and dehydrovomifoliol as a synthetic support for the revised absolute configuration of (þ)-abscisic acid. Tetrahedron 30, 1065e1072. Pei, Y.H., 2015. Absolute determination of stereochemical configuration of some
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