Nitric Oxide Inhibitory Dimeric Sesquiterpenoids ... - ACS Publications

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Nitric Oxide Inhibitory Dimeric Sesquiterpenoids from Artemisia rupestris Chen Zhang,† Shu Wang,‡ Ke-Wu Zeng,† Jun Li,§ Daneel Ferreira,⊥ Jordan K. Zjawiony,⊥ Bing-Yu Liu,† Xiao-Yu Guo,† Hong-Wei Jin,† Yong Jiang,*,† and Peng-Fei Tu*,† †

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People’s Republic of China ‡ Department of Medicinal Chemistry and Pharmaceutical Analysis, Logistics College of Chinese People’s Armed Police Forces, Tianjin 300162, People’s Republic of China § Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China ⊥ Department of BioMolecular Sciences, Division of Pharmacognosy, and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677-1848, United States S Supporting Information *

ABSTRACT: Twelve new dimeric sesquiterpenoids (1−12) were isolated from the dried whole plants of Artemisia rupestris. Their structures were determined using MS and NMR data, and the absolute configurations were elucidated on the basis of experimental and calculated ECD spectra. Compounds 1−9 are presumably formed via biocatalyzed [2+2] or [4+2] cycloaddition reactions. Stereoselectivity of the [4+2] Diels−Alder reaction dictated the formation of endo-products. The dimeric sesquiterpenoids exhibited moderate inhibition on NO production stimulated by lipopolysaccharide in BV-2 microglial cells, with IC50 values in the range 17.0−71.8 μM. MS/MS-guided fractionation and dereplication, and 12 new dimeric sesquiterpenoids (1−12) were obtained. Compounds 1−3 were presumably formed by [2+2] cycloaddition of two sesquiterpenoid moieties; compounds 4−9 were formed via [4+2] cycloadditions, and compounds 10−12 were linked through an ester bond. Herein, the isolation and structural elucidation of the new compounds and their inhibitory effects on lipopolysaccharide (LPS)-induced NO production in BV-2 microglial cells are described.

Artemisia rupestris L. (Asteraceae), as a traditional ethnic medicine, is widely distributed in the Xinjiang Uygur Autonomous Region, People’s Republic of China. It has been commonly used by local people to cure influenza and rheumatism and to reduce fever.1 Previous phytochemical investigations of A. rupestris resulted in the isolation of flavonoids and sesquiterpenoids,2−11 with the latter compounds showing significant anti-inflammatory and antiviral effects.12,13 The guaianes and eudesmanes are the main structural types of sesquiterpenoids isolated from A. rupestris, most of which are Δ11,13-didehydro derivatives and, hence, biologically active Michael acceptors.14 Recently, a series of trace dimeric sesquiterpenoids with remarkable cytotoxicity and antineuroinflammatory activities have been isolated from Artemisia species. Their structures comprise two sesquiterpenoid moieties linked through [4+2] Diels−Alder cycloaddition reactions.15−17 As part of an ongoing search for bioactive dimeric sesquiterpenoids from the Artemisia genus, the 95% aqueous EtOH extract of the whole plants of A. rupestris was investigated by LC-DAD© XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The whole powdered plants of A. rupestris were extracted with 95% aqueous EtOH and partitioned with petroleum ether and CHCl3, successively. LC-DAD-MS/MS analysis suggested that the CHCl3 extract is rich in sesquiterpenoid dimers based upon Received: October 8, 2015

A

DOI: 10.1021/acs.jnatprod.5b00894 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

Figure 1. Key HMBC, 1H−1H COSY, and NOESY correlations of 1, 4, and 7.

characteristic UV absorptions at 210 nm (vinylic group) or 254 nm (α,β-unsaturated carbonyl), molecular weights ranging from m/z 400 to 600, and the retro-Diels−Alder fragment ions such as m/z 247 or 233 (Figure S1, Supporting Information). Thus, the CHCl3 extract was subjected to silica gel, Sephadex LH-20, and ODS column chromatography, followed by semipreparative HPLC, to afford 12 new dimeric sesquiterpenoids (1−12). Artepestrin A (1) was obtained as a white, amorphous powder. A deprotonated molecular ion [M − H]− at m/z 479.2799 (calcd for C30H39O5, 479.2803) observed in the negative-ion HRESIMS in conjunction with 13C NMR spectroscopic data indicated a molecular formula of C30H40O5

with 11 indices of hydrogen deficiency. The IR spectrum showed absorption bands for hydroxy (3401 cm−1) and α,βunsaturated carbonyl (1691 cm−1) functionalities. The 30 wellresolved resonances in the 13C NMR spectrum were classified as sp3 carbons (four CH3, eight CH2, six CH, three C) and sp2 carbons (one CH2, two CH, six C) using the HSQC data. The nine sp2 resonances were assigned to three carbonyls and three pairs of olefinic carbons, which account for six indices of hydrogen deficiency and, thus, requires the presence of five rings in the structure. The presence of characteristic signals of α,β-unsaturated carboxylic acid (δC 123.2, 147.4, 170.6) and α,β-unsaturated cyclopentenone (δC 47.0, 42.4, 211.1, 138.6, B



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absolute configuration was assessed by the quantum chemical calculation of the electronic circular dichroism (ECD) spectra using TDDFT at the B3LYP/6-31G level in MeOH solution with the CPCM model. Four possibilities with the absolute conformations of (1S,7R,10S,11R,3′R,4′S,5′R,7′R,10′S)-1, (1S,7R,10S,11S,3′R,4′S,5′R,7′R,10′S)-1, and their enantiomers were considered. During the conformational optimization with the Chem3D model, less steric hindrance in the 11R compound was evident compared to the 11S epimer. The calculated ECD spectrum of (1S,7R,10S,11R,3′R,4′S,5′R,7′R,10′S)-1 conformed well with the experimental spectrum (Figure 2). Therefore, the structure of artepestrin A (1) was elucidated as shown. Artepestrin B (2) has the same molecular formula of C30H40O5 as 1 as determined by the negative HRESIMS ion at m/z 479.2795 [M − H]− (calcd for C30H39O5, 479.2803) and 13 C NMR data. The 1H and 13C NMR data of 2 are comparable to those of 1, implying that 2 is also a disesquiterpenoid. The two monomeric units in 2 are connected via the C-11 to C-2′ and C-13 to C-1′ bonds (Scheme 1) through a symmetryallowed [π2s+π2a] cycloaddition reaction based upon the HMBC correlations between H3-14′ and C-1′/C-5′/C-9′; H213 and C-11/C-1′; and H-2′ and C-11, as well as 1H−1H COSY correlations of H2-13/H-1′/H-2′/H-3′. The Δ3′,4′

180.5) functionalities suggested that 1 is a dimeric sesquiterpenoid.18 The structure of 1 was constructed on the basis of 1H−1H COSY and HMBC data. Three proton-bearing structural fragments as shown in bold in Figure 1, corresponding to H2-2/H-1/H-10/H2-9/H2-8/H-7/H2-6, H-1′/H-2′/H-3′, and H-5′/H2-6′/H-7′/H2-8′/H2-9′, were observed in the 1H−1H COSY spectrum. These fragments were connected by the HMBC correlations of H3-15/C-3, C-4, C-5; H3-14/C-1, C-9, C-10; H2-2/C-3, C-5; H-7/C-8, C-11, C-12; H3-14′/C-1′, C-5′, C-9′, C-10′; H3-15′/C-3′, C-4′, C-5′; H-2′/C-3′, C-4′; and H213′/C-11′, C-7′, C-12′. On the basis of the above data, the monomeric A and B units were deduced as a guaiane-type moiety similar to pechueloic acid and a eudesmane-type moiety similar to illicic acid, respectively, which have both been isolated from this herb.11 The HMBC correlations of H3-15′/ C-13; H-3′/C-4′, C-7, C-11, C-12; and H2-13/C-11, C-3′, C-4′, C-5′ indicated that units A and B are linked via C-11/C-3′ and C-13/C-4′, with the four-membered ring presumably formed through a [2+2] cycloaddition reaction (Scheme 1). Scheme 1. Cycloaddition Types of 1, 2, 4, and 7

olefinic bond and, hence, participation of the Δ1′,2′ double bond (suprafacial fashion) in the symmetry-allowed [π2s+π2a] cycloaddition formation of the cyclobutane moiety were shown by the HMBC correlations of H-1′/C-3′ and H3-15′/C-3′, C4′. Thus, compound 2 was shown to be a regioisomer of 1. The relative configurations of C-1′ and C-2′ were assigned by NOE correlations of H3-14′/H-1′/H-2′, suggesting that H-1′ and H2′ are proximal and β-oriented. The small coupling constant between H-2′ (br s) and H-1′ (br s) indicated a dihedral angle of ca. 90° and supported their cis-relationship. Differention between the 11S and 11R absolute configurations was achieved by comparison of the experimental and calculated ECD spectra (Figure 2). The absolute configuration of 2 was subsequently elucidated as (1S,7R,10S,11S,1′S,2′S,5′S,7′R,10′S)-2, and the structure of artepestrin B (2) was defined as shown. The positive-ion HRESIMS data of artepestrin C (3) showed a protonated molecular ion at m/z 497.2906 [M + H]+ (calcd for C30H41O6, 497.2909). Taken in conjunction with the 13C NMR spectroscopic data, this indicated a molecular formula of C30H40O6, with one more oxygen atom than those of 1 and 2. Comparison of the NMR data of 3 with those of 2 suggested that C-3′ is oxygenated (δC 72.3) in 3. A Δ4′,5′ olefinic bond in 3 was substantiated by the HMBC correlations of H-3′ and C1′/C-2′/C-4′/C-5′/C-15′ and of H3-15′ and C-3′ (δC 72.3)/C4′ (130.4)/C-5′ (139.7). The 2.8 Hz coupling constant between H-2′ and H-3′ implies that OH-3′ is β-axially oriented. Comparison of the ECD spectra of 1 and 3 showed similar Cotton effects at ca. 248 and 310 nm, but opposite Cotton effects at ca. 202 nm. The Cotton effects at the longer wavelengths resulted from the π−π* and n−π* transitions of the α,β-unsaturated cyclopentenone and α,β-unsaturated carboxylic functionalities, while the short-wavelength Cotton effects are due to the olefinic moiety of unit B.23 Different helicities of 1 and 3, i.e., P for 3, while M for 1 (Figure 3), manifested in their opposite Cotton effects near 202 nm. Compound 3, with an olefinic moiety of P-helicity, suggested the (1′S,2′S,3′S,7′R,10′S) configuration of unit B. Therefore, the absolute configuration of artepestrin C (3) was deduced as (1S,7R,10S,11S,1′S,2′S,3′S,7′R,10′S) and substantiated by the

In natural guaiane- and eudesmane-type sesquiterpenoids,20,21 H-7 adopts an α-orientation, thus permitting assignment of the relative configuration of 1 via its NOESY data. In unit A (guaiane moiety), the NOE correlations of H314/H-6β/H-8β and H-7/H-1 indicated a preferred chairlike conformation of the seven-membered ring (Figure 1). When comparing with the NMR data of guaia-4,11-dien-3-one analogues,22 the chemical shifts of C-1 (δC 47.0)/C-10 (36.7)/C-14 (12.6) and H-1 (δH 3.19)/H-10 (2.12)/H3-14 (0.59) of 1 were in good agreement with those of H-1α, H-7α, and H-10α of guaiane derivatives. In turn, NOE correlations of H3-14′/H-6′β/H-8′β and H-7′/H-5′ implied the usual transfused eudesmane unit B. In a 1D-NOE experiment, the intensity of the H-3′ and H3-15′ resonances was enhanced when irradiating the H3-14′ signal (Figure S11, Supporting Information), indicating their cofacial relationship. Such orientations are in line with the stereochemical course of the symmetry-allowed [π2a+π2s] cycloaddition reaction: the Δ11,13 double bond of unit A (the π2a moiety used in an antarafacial fashion) approaching the Δ3′,4′ double bond of the diene moiety in unit B (the π2s participant used in a suprafacial fashion) from the less-hindered α-face (Figure 1). The relative configuration of the newly formed C-11 stereogenic center was hard to establish by NOE correlations. Thus, the 11R or 11S C



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Figure 2. Comparison of the calculated ECD spectra for (1S,7R,10S,11R,3′R,4′S,5′R,7′R,10′S)-1, (1S,7R,10S,11S,3′R,4′S,5′R,7′R,10′S)-1; (1S,7R,10S,11S,1′S,2′S,5′S,7′R,10′S)-2, (1S,7R,10S,11R,1′S,2′S,5′S,7′R,10′S)-2; (1S,7R,10S,11R,2′S,4′S,7′R,10′S)-4, (1S,7R,10S,11S,2′S,4′S,7′R,10′S)-4; and their enantiomers with the experimental ECD spectra for 1, 2, and 4 in MeOH (blue line).

calculated ECD spectrum (Figure S35, Supporting Information). Artepestrin D (4) has the same molecular formula, C30H40O5, as 1. Analysis of the 1D- and 2D-NMR data suggested that 4 possesses two pechueloic acid-type constituent units, one of which is the same as unit A of 1−3, and the other is a guaia-1(5),11(13)-dien-12-olic acid-like moiety.24 The HMBC correlations between H3-15′/H-2′ and C-11 and between H2-13 and C-1′/C-2′/C-3′, as well as 1H−1H COSY correlations of H2-3′/H-2′/H2-13, indicated that units A and B are linked via C-13/C-2′ and C-11/C-4′. The formation of the bicyclic [2.2.1]hept-2-ene structural moiety is explicable in terms of a symmetry-allowed [π4s+π2s] Diels−Alder cycloaddition reaction of the two guaiane units depicted in Scheme 1. The 2′,4′-cis relative configuration is necessitated by the orbital symmetry rules that require the diene π-system to be utilized in a suprafacial fashion and is supported by the NOESY experiment that showed cross-peaks of H3-14/H-6β/H-8β and H3-14′/H-6′β/8′β, indicative of chairlike conformations of the two seven-membered rings of 4. In the NOE spectrum, enhancement of the H-3′α resonance (δH 1.82, d, J = 8.0 Hz) upon irradiation of H-7′ suggested they are cofacially αoriented (Figure 1), indicating that the dienophile approached

Figure 3. Comparison of the experimental ECD spectra between 1 and 3 and the assignment of the Cotton effects at around 202, 248, and 310 nm.

D



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

the diene from the β-face. The β-orientation of the C-11 carboxylic group stems from the preferential formation of the endo-adducts in Diels−Alder reactions.25−27 The calculated ECD spectra indicated that the spectrum of the 11R-epimer showed a better fit with the experimental spectrum than the 11S-epimer (Figure 2). Thus, the absolute configuration of artepestrin D (4) was defined as (1S,7R,10S,11R,2′S,4′S,7′R,10′S). 6α-Hydroxyartepestrin D (5) has a molecular formula of C30H40O6 based on the [M + H]+ ion at m/z 497.2907 (calcd for C30H41O6, 497.2909) in the positive-ion HRESIMS and 13C NMR data, thus having one more oxygen atom than 4. The 1H and 13C NMR data of 5 were found to be similar to those of 4, except for the deshielded signals at δH 4.94 (1H, d, J = 11.1 Hz) and δC 82.7, suggesting that one of the carbons of 4 is oxygenated in 5. In the HMBC spectrum, the proton at δH 4.94 showed correlations with C-4, C-5, C-7, and C-8 and was assigned to H-6. The NOE association between H3-14 and H-6 suggested an α-orientation for OH-6. The similar NOE correlations and ECD spectrum (Figure S58, Supporting Information) to those of 4 defined the structure and absolute configuration of 5 as shown. Artepestrin E (6) has the same molecular formula, C30H40O5, as 4, as assigned via a [M + H]+ ion at m/z 481.2955 (calcd for C30H41O5, 481.2949) and the 13C NMR spectroscopic data. Analysis of the 1D- and 2D-NMR data of 6 indicated that the difference between 6 and 4 involved the linkage mode between the two guaiane-type units. In the HMBC spectrum, H3-15′ showed long-range correlations with C-5′/C-4′/C-13, and H-2′ showed correlations with C-12/C-5′/C-13. However, the H213 protons resonated as two mutually coupled but isolated doublets at δH 2.21 (d, J = 12.1 Hz) and 1.01 (d, J = 12.1 Hz). These data indicated that the dimerization between the diene and dienophile involved C-11/C-2′ and C-13/C-4′ rather than C-11/C-4′ and C-13/C-2′ for the formation of 5. The bridgehead C-3′ in 6 occupied the same plane of the 5,7bicyclic system as Me-14′ based on the NOESY cross-peak of H3-14′/H-3′β (Figure S67, Supporting Information), which indicated that the terminal double bond Δ11,13 of unit A approached the diene moiety of unit B from the α-face. The C11 carboxylic group was thus α-axially oriented based upon the preference for endo-product fromation.25−27 In this case, the

absolute configuration of 6 was deduced to be (1S,7R,10S,11R,2′S,4′S,7′R,10′S), as confirmed by its experimental and calculated ECD spectra (Figures S69 and S71, Supporting Information). Artepestrin F (7) was obtained as an amorphous, white powder and gave a deprotonated molecular ion at m/z 481.2955 [M − H]− indicative of a molecular formula of C30H42O5 when taken in conjunction with the 13C NMR spectroscopic data. The NMR data of 7 showed resonances reminiscent of two guaiane-type units. In unit A, two oxygenated carbon signals at δC 84.2 (C-4) and 99.0 (C-5) in the 13C NMR spectrum were similar to those of rupestonic acid C.11 However, the deshielded C-5 (ΔδC +14.6) resonance indicated the presence of a lactone moiety bridging C-5 and C11 similar to the same functionality in pulicazine.28 HMBC correlations from H3-15′ to C-3′, C-4′, and C-5′ and from H22′ to C-4′ and C-5′ showed that unit B possesses a Δ4′,5′ olefinic bond [C-4′ (δC 137.4), C-5′ (145.6)], with the remaining correlations similar to those reported for pechueloic acid.14 The HMBC correlations of the two CH2-13 doublets with C-1′/C-5′/C-11/C-3′ and the correlation of H-3′ with C12 indicated that these two units are connected through C-13/ C-1′ and C-11/C-3′ (Scheme 1). In the NOESY spectrum, the cross-peaks of H-1/H-6α, H-1/H-9α, and H3-15/H-6α suggested that OH-4 is β-oriented with the lactone moiety extended above the plane of the 5/7-bicyclic unit A, consistent with the relative configuration of pulicazine.28 The Me-14′ protons showed a NOE correlation with H-2′β, indicating that CH3-14′ is cofacial with the bridge of the norbornene moiety (Figure 1). Thus, in the symmetry-allowed [π4s+π2s] cycloaddition reaction, the terminal double bond of unit A approached from the α-side of the diene moiety of unit B, as shown in Scheme 1. The ECD spectrum of 7 showed a positive Cotton effect at 214 nm (Figure 4), which suggested the (1S,4S,5R,7R,10S) configuration of unit A based on the δlactone ECD rule29 and the relative configuration as defined by the NOE data. Considering the similarity of the B-units in compounds 4−6 and compound 7, the absolute configuration of the latter was defined as (1S,4S,5R,7R,10S,11R,1′R,3′R,7′R,10′S), while the calculated ECD spectrum of its enantiomer showed Cotton effects dissimilar to the experimental spectrum (Figure 4). E



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Table 1. 1H and 13C NMR Data of Compounds 1−3 (δ in ppm, J in Hz, 500 MHz for 1H and 125 MHz for 13C, in Methanol-d4) 1

2

δC

δH

δC

1 2

47.0, CH 42.4, CH2

3.19, br s 2.59, dd (18.9, 6.5) 2.01, d (18.9)

47.2, CH 42.3, CH2

3 4 5 6

211.1, C 138.6, C 180.5, C 34.5, CH2

7 8

42.3, CH 30.3, CH2

9

36.9, CH2

10 11 12 13

36.7, CH 50.9, C 179.3, C 47.0, CH2

14 15 1′ 2′ 3′ 4′ 5′ 6′

12.6, CH3 7.9, CH3 143.9, CH 123.8, CH 49.0, CH 34.4, C 54.2, CH 29.2, CH2

7′ 8′

41.9, CH 28.5, CH2

9′

42.7, CH2

10′ 11′ 12′ 13′

35.4, C 147.4, C 170.6, C 123.2, CH2

14′ 15′

20.7, CH3 25.2, CH3

position

2.88, 2.42, 2.21, 1.79, 1.25, 1.77, 1.77, 2.12,

d (19.8) dd (19.8, 10.5) t (10.5) m m m m tq (6.8, 3.5)

2.16, 1.92, 0.59, 1.63, 5.84, 5.70, 3.15,

d (12.5) d (12.5) d (7.0) s dd (10.0, 2.7) dd (10.0, 2.7) t (2.7)

1.59, 1.54, 1.31, 2.44, 1.62, 1.45, 1.56, 1.40,

m m m m m m m m

6.14, 5.59, 0.98, 1.11,

s s s s

3 δH

3.09, br s 2.57, dd (18.9, 6.4) 2.00, d (18.9)

211.0, C 138.3, C 178.8, C 36.4, CH2 43.4, CH 30.8, CH2 38.2, CH2 40.4, CH 56.9, C 180.0, C 31.8, CH2 12.3, CH3 7.9, CH3 40.4, CH 41.5, CH 120.8, CH 140.2, C 42.5, CH 27.3, CH2 42.5, CH 28.0, CH2 35.5, CH2 34.1, C 147.5, C 170.6, C 123.3, CH2

2.83, 2.30, 1.87, 1.97, 1.32, 1.82, 1.64, 2.11,

d (19.8) dd (19.8, 10.5) t (10.5) m m dd (14.1, 2.5) d (14.1) m

2.33, 1.71, 0.60, 1.64, 2.10, 3.20, 5.36,

m m d (7.0) s t (8.9) br s br s

2.20, 1.70, 1.34, 2.49, 1.60, 1.53, 1.32, 1.22,

d (12.6) m m dq (12.0, 6.1, 3.6) m m d (13.0) dt (13.0, 3.4)

6.16, 5.61, 0.72, 1.67,

18.1, CH3 21.8, CH3

s s s s

δC

δH

47.1, CH 42.6, CH2

3.27, br s 2.56, dd (18.7, 6.7) 2.02, d (18.7)

211.6, C 138.1, C 181.0, C 39.1, CH2 37.8, CH 29.1, CH2 38.4, CH2 37.0, CH 58.2, C 180.4, C 32.5, CH2 12.7, CH3 8.1, CH3 37.6, CH 43.8, CH 72.3, CH 130.4, C 139.7, C 33.1, CH2 41.7, CH 28.8, CH2 41.4, CH2 37.6, C 147.4, C 170.6, C 123.4, CH2 18.4, CH3 18.4, CH3

3.15, 2.23, 2.71, 1.93, 1.41, 1.88, 1.80, 2.15,

d (18.4) dd (18.4, 10.5) t (10.5) m m m m m

2.18, 2.12, 0.62, 1.65, 2.26, 2.33, 3.91,

m m d (7.0) s m dd (13.8, 2.8) d (2.8)

2.68, 1.77, 2.41, 1.65, 1.60, 1.76, 1.38,

d (12.9) dd (12.9, 11.1) t (11.1) m m m m

6.18, 5.62, 1.05, 1.72,

s s s s

Artepestrin G (9) has the same molecular formula of C30H42O6 as 8 on the basis of HRESIMS and 13C NMR data. These two compounds possess two similar guaiane-type units, and their differences included the linkage and oxygenation positions via analysis of 2D-NMR data. The HMBC correlations from the two CH2-13 doublet protons to C-7/C12/C-1′/C-5′ and from H-3′ to C-11/C-13/C-5′ suggested that the linkages of the two units are C-11/C-3′ and C-13/C-5′. The deshielded oxygenated tertiary carbon (δC 93.4) showed an HMBC correlation with Me-15′ and was hence assigned to C-4′. The NOE cross-peak of H3-15′/H-7′ (Figure S103, Supporting Information) suggested they are cofacial and that the bridgehead C-4′ occupies the plane of the 5,7-bicyclic system opposite Me-14′. The Δ11,13 double bond of unit A thus approached the diene functionality of unit B from the β-face. Therefore, the absolute configuration of 9 was deduced to be (1S,4S,5R,7R,10S,11S,3′R,4′S,5′R,7′R,10′S) and was supported by the experimental and calculated ECD spectra (Figure S105, Supporting Information).

2′α-Hydroxyartepestrin F (8) has a molecular formula of C30H42O6, one more oxygen atom than that of 7, as deduced from the [M − H]− ion at m/z 497.2911 (calcd for C30H41O6, 497.2909) in the negative HRESIMS and 13C NMR data. The NMR spectra of 8 were comparable to those of 7 except that one more oxygenated methine signal [δH 3.33 (1H, d, J = 2.0 Hz); δC 85.0] was observed in 8, indicating that it is an oxygenated derivative of 7. In the HMBC spectrum, the oxymethine proton (δH 3.33) showed correlations with C-11, C-13, C-4′, and C-5′, which were used to locate the hydroxy group at C-2′. Enhancement of the signal intensity of H-2′ when irradiating H3-14′ (Figure S92, Supporting Information) suggested the α-orientation of the OH-2′ group. Its similar NOE correlations and ECD spectrum (Figure S94, Supporting Information) with those of 7 suggested the absolute configuration of C-2′ to be S and, hence, a (1S,4S,5R,7R,10S,11R,1′S,2′S,3′R,7′R,10′S) absolute configuration for compound 8. F



Article

active, with IC50 values of 27.3 ± 0.7, 23.0 ± 1.5, and 17.0 ± 0.6 μM, respectively. Their different NO inhibitory effects might be related to the molecular geometry and the presence of hydroxy groups. Comparison of the effects of 7 and 8 showed that the presence of the 2′-hydroxy group reduced the inhibitory effect of 8. The above results could partly account for the traditional usage of A. rupestris for the treatment of inflammation-related diseases.

Rupestrinate A (10) exhibited a deprotonated molecular ion at m/z 495.2736 [M − H]− in the negative-ion HRESIMS, which together with the 13C NMR spectroscopic data was consistent with the molecular formula of C30H40O6 with 11 indices of hydrogen deficiency. Its IR spectrum revealed the presence of hydroxy group (3452 cm−1) and α,β-unsaturated ester (1694 cm−1) functionalities. Compound 10 is also a dimeric sesquiterpenoid, and the two monosesquiterpenoid moieties were deduced to be an esterified pechueloic acid (unit A) and a eudesmane moiety similar to 1-hydroxyeudesma3(4),11(13)-dieno-12-oic acid (unit B).30 In unit B, an additional oxymethine (δH 5.14, δC 74.5) group was observed when compared with 1-hydroxyeudesma-3(4),11(13)-dieno12-oic acid,30 and its proton showed HMBC correlations with C-1′/C-3′/C-4′/C-10′/C-12, indicating C-2′ is oxygenated, and the two units are connected through a C-2′-O-C-12 ester bond. In the NOESY spectrum, the NOE correlations of H314′/H-1′ and H-2′/H-5′ (Figure S114, Supporting Information) suggested H-2′ is α-oriented, and the dihedral angle between H-1′ and H-2′ was ca. 90° based on the broadened singlet of H-1′ in the 1H NMR spectrum. The Cotton effects at 208, 245, and 310 nm in the ECD spectrum of 10 suggested the (1S,7R,10S,1′R,2′R,5′S,7′R,10′R) absolute configuration on the basis of the M-helicity rule and the π−π* and n−π* transitions of the α,β-unsaturated cyclopentenone and α,β-unsaturated carboxyl groups. This is also supported by the experimental and calculated ECD spectra (Figure S116, Supporting Information). Rupestrinate B (11) has a molecular formula of C30H42O6, as deduced from the sodium adduct ion at m/z 521.2851 (calcd for C30H42O6Na, 521.2874) in the HRESIMS and the 13C NMR data. The differences between 11 and 10 involve unit B, which was deduced to be a C-2 oxygenated ilicic acid-like moiety due to the deshielded C-2′ resonance (δC 65.5).19 The HMBC correlation between H-2′ and C-12 suggested that these two units are linked via a C-2′−O−C-12 ester bond. The α-orientation of the OH-2′ and OH-4′ space groups was deduced from the NOESY cross-peaks of H3-14′/H3-15′/H-2′ (Figure S124, Supporting Information). The absence of the Cotton effect at around 210 nm in the ECD spectrum of 11

■

EXPERIMENTAL SECTION

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 Bruker APEX IV FT-MS and 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 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 (100−200 or 200−300 mesh, Qingdao Marine Chemical Inc., People’s Republic of China), ODS C18 (50 μm, Merck, Germany), and Sephadex LH-20 (Amersham Biosciences, Sweden). Semipreparative HPLC was performed on an Agilent 1200 liquid chromatograph with a Waters XBridge Prep Shield RP 18 column (250 mm × 10 mm, 5 μm). Analytical HPLC was performed on an Aglient 1100 system with an Aglient Extend-C18 column (4.6 × 150 mm, 5 μm). All purified compounds submitted for bioassay were at least 95% pure as judged by HPLC analysis. Plant Material. The whole dried plants of Artemisia rupestris were collected in the Xinjiang Uygur Autonomous Region, People’s Republic of China, in February 2010. The plant was identified by one of us (P.F.T.). A voucher specimen (No. 30973630) is deposited at the Herbarium of the Peking University Modern Research Center for Traditional Chinese Medicine, Beijing, People’s Republic of China. LC-MS Dereplication Procedure. HPLC conditions: The mobile phase consisted of a MeCN (A)/0.1% HCOOH aqueous solution (B) (v/v) using a linear gradient elution from 40% A to 100% A in 30 min. The eluent flow at 1.0 mL/min was roughly split as 5:1 (v/v) before entering the ESI source. The DAD data were collected from 200 to 400 nm, and the representative chromatograms were recorded at 210 and 254 nm. MS conditions: negative-ion mode; electrospray voltage, −3.5 kV; detector voltage, 1.7 kV; curved desolvation line temperature, 200 °C; nebulizing gas (N2), 1.5 L/min; drying gas (N2) pressure, 100 kPa; scan range, m/z 400−800 for MS1, 100−600 for MS2. Ultrapure argon was used as the collision gas for the collision-induced dissociation experiment, and the collision energy was set at 50% for MS2; TOF region pressure, 1.4 × 10−4 Pa; ion trap pressure, 1.8 × 10−2 Pa; ion accumulated time, 30 ms. The data acquisition and analysis were performed by LCMS Solution version 3 software (Shimadzu, Kyoto, Japan). Extraction and Isolation. The plant material (50 kg) was powdered and extracted with 95% aqueous EtOH (400 L × 2.5 h × 3, 80 °C). After evaporation in vacuo, the concentrated extract (5 kg) was suspended in H2O and partitioned with petroleum ether and CHCl3, successively. The CHCl3 extract (0.9 kg) was chromatographed over silica gel (100−200 mesh) using petroleum ether/EtOAc (1:0, 9:1, 3:1, 1:1, and 0:1, v/v) as eluents to produce 20 fractions (Frs. A−T) based on TLC analysis. Frs. N (45 g), O (32 g), and P (25 g) were subjected to silica gel (200−300 mesh) CC eluting with petroleum ether/EtOAc (1:0, 3:1, 1:1, and 0:1) to produce Frs. NA− NL, OA−OJ, and PA−PJ, respectively. Frs. NC (5.8 g), OA (4.2 g),

compared to 10 resulted from the absence of the Δ3′,4′ double bond. The absolute configuration of 11 was defined as (1S,7R,10S,2′R,4′R,5′R,7′R,10′S) and was confirmed by the experimental and calculated ECD spectra (Figure S126, Supporting Information). Rupestrinate C (12) showed a sodium adduct ion at m/z 533.2510 [M + Na]+ (calcd for C30H38O7Na, 533.2512), consistent with its molecular formula of C30H38O7 and 13C NMR data. The NMR data implied that unit B is a 5β-hydroxy4-oxo-2(3),11(13)-dienoiphionan-12-oic acid-like moiety.31 The deshielded C-1′ resonance (δC 84.5) and HMBC correlation of H-1′ (δH 5.23) and C-2′/C-3′/C-10′/C-14′/C5′/C-12 suggested that C-1′ is oxygenated and that the two units are connected through a C-1′−O−C-12 ester bond. The NOESY cross-peak of H3-14′/H-1′ (Figure S135, Supporting Information) indicated that OH-1′ is α-oriented, the other correlations being consistent with literature data.31 The experimental and calculated ECD spectra (Figure S137, Supporting Information) defined the absolute configuration of 12 as (1S,7R,10S,1′S,5′R,7′R,10′S). The dimeric sesquiterpenoids were evaluated for their inhibitory effects on LPS-stimulated NO production in BV-2 microglial cells (Table 4). Compounds 1, 5, and 7 were most G


H

36.3, CH 63.2, C 179.2, C 43.2, CH2

12.4, CH3 8.3, CH3 152.6, C 45.2, CH

56.5, CH2

61.5, C 138.1, C 32.8, CH2

40.5, CH 33.7, CH2

10 11 12 13

14 15 1′ 2′

3′

4′ 5′ 6′

7′ 8′

18.8, CH3

38.4, CH2

9

14′

49.3, CH 30.2, CH2

7 8

34.7, CH 148.4, C 169.8, C 121.9, CH2

138.1, C 180.0, C 37.3, CH2

4 5 6

10′ 11′ 12′ 13′

211.3, C

3

35.8, CH2

47.7, CH 42.1, CH2

1 2

9′

δC

position

δH

dd (11.5, 4.1) dd (11.5, 4.1) d (7.0) s

d (15.5) d (15.5) t (10.9) m m m m m

6.00, br s 5.43, s 1.07, d (7.2)

2.36, 2.12, 2.28, 1.44, 1.80, 1.72, 1.62, 2.51,

1.82, d (8.0) 1.25, d (8.0)

2.45, br s

2.84, 1.13, 0.60, 1.53,

3.12, d (20.3) 2.95, dd (20.3, 11.5) 1.61, m 1.71, m 1.47, m 1.79, m 1.61, m 2.05, tq (6.5, 3.3)

3.10, br s 2.64, dd (18.7, 6.3) 1.89, d (18.7)

4

12.9, CH3

34.2, CH 148.7, C 170.4, C 124.0, CH2

36.1, CH2

41.1, CH 31.3, CH2

56.2, C 138.0, C 35.7, CH2

53.3, CH2

12.9, CH3 9.4, CH3 151.8, C 45.9, CH

36.8, CH 61.7, C 183.4, C 35.0, CH2

36.7, CH2

45.1, CH 26.5, CH2

142.9, C 168.9, C 82.7, CH

211.1, C

45.6, CH 42.0, CH2

δC

δH

td (8.6, 3.6) m d (7.2) s

t (11.1) m m m m m

d (8.7) dd (8.7, 1.9) m m m m m m

6.19, d (1.2) 5.64, s 1.12, d (7.2)

2.30, 2.27, 2.73, 1.79, 1.74, 1.75, 1.75, 2.57,

2.40, d (8.1) 1.20, d (8.1)

2.69, t (1.8)

1.87, 1.80, 0.66, 1.85,

2.39, 1.82, 1.43, 1.82, 1.82, 2.14,

4.94, d (11.1)

3.36, m 2.67, dd (19.4, 6.5) 2.06, d (19.4)

5

21.2, CH3

35.2, CH 148.6, C 170.5, C 123.1, CH2

35.8, CH2

42.5, CH 33.4, CH2

53.4, C 145.9, C 32.3, CH2

55.1, CH2

12.6, CH3 8.0, CH3 147.4, C 53.1, CH

36.8, CH 65.1, C 180.2, C 45.1, CH2

37.5, CH2

45.9, CH 30.4, CH2

138.3, C 179.4, C 37.6, CH2

211.4, C

47.3, CH 42.4, CH2

δC

δH

d (12.1) d (12.1) d (7.1) s

m m t (10.3) m m m m m

6.14, d (1.2) 5.58, s 1.15, d (7.0)

2.22, 2.10, 2.66, 1.72, 1.85, 1.82, 1.76, 2.53,

1.31, d (8.3) 1.23, d (8.3)

3.21, t (1.6)

2.21, 1.01, 0.60, 1.62,

2.92, d (20.1) 2.22, dd (20.1, 11.3) 1.64, m 2.12, d (13.7) 1.43, dd (13.7, 3.0) 1.85, m 1.74, m 2.12, m

3.17, br s 2.58, dd (18.9, 6.4) 1.99, d (18.9)

6

CH3 CH3 C CH2

17.5, CH3

37.4, CH 148.3, C 170.6, C 123.1, CH2

33.5, CH2

39.2, CH 28.1, CH2

137.4, C 145.6, C 33.9, CH2

56.6, CH

15.5, 21.9, 61.7, 47.8,

32.8, CH 60.2, C 180.0, C 43.0, CH2

24.0, CH2

36.4, CH 26.6, CH2

84.2, C 99.0, C 31.7, CH2

38.4, CH2

51.8, CH 28.1, CH2

δC

δH

d (13.8) d (13.8) d (7.3) s

m m m m m tq (6.5, 3.2)

1.78, 1.71, 2.74, 2.29, 1.91, 1.88, 1.72, 1.92,

m m t (8.7) d (11.3) m m m m

1.73, m 1.37, d (8.1) 2.82, br s

2.32, 1.37, 0.92. 1.25,

1.91, 1.85, 1.50, 1.82, 1.64, 2.14,

1.81, m 1.53, m

2.47, dt (10.6, 7.9) 2.28, dd (12.7, 3.5) 1.91, m 1.98, m 1.72, m

7

CH3 CH3 C CH

16.3, CH3

33.3, CH 149.1, C 170.3, C 122.7, CH2

33.2, CH2

40.1, CH 33.6, CH2

135.0, C 144.4, C 36.4, CH2

59.0, CH

15.2, 21.9, 64.1, 85.0,

32.6, CH 60.5, C 183.0, C 41.1, CH2

31.6, CH2

36.6, CH 25.9, CH2

84.1, C 99.9, C 28.0, CH2

38.3, CH2

51.7, CH 24.0, CH2

δC

8 δH

d (12.8) d (12.8) d (7.4) s

t (4.5) m m m m tq (6.8, 3.2)

6.15, s 5.60, s 1.06, d (7.2)

2.66, d (14.1) 2.23, dd (14.1, 2.9) 2.53, t (9.0) 1.80, m 1.73, m 1.84, m 1.84, m 2.09, m

2.69, d (1.7)

3.33, d (2.0)

2.27, 1.85, 0.92, 1.24,

2.02, 1.93, 1.51, 1.86, 1.57, 2.17,

2.21, d (14.1) 1.99, d (14.1)

2.48, dt (11.5, 8.0) 1.83,m 1.68, m 2.01, m 1.76, m

Table 2. 1H and 13C NMR Data of Compounds 4−9 (δ in ppm, J in Hz, 500 MHz for 1H and 125 MHz for 13C, in Methanol-d4)

18.6, CH3

38.8, CH 149.7, C 170.7, C 122.7, CH2

35.5, CH2

40.1, CH 35.6, CH2

93.4, C 60.8, C 33.4, CH2

60.0, CH

15.1, CH3 22.0, CH3 157.9, C 127.2, CH

32.6, CH 59.4, C 182.5, C 42.9, CH2

31.4, CH2

36.6, CH 24.6, CH2

84.0, C 100.4, C 27.2, CH2

38.4, CH2

52.2, CH 23.9, CH2

δC

δH dt (11.1, 8.5) m m m m

d (13.0) d (13.0) d (7.5) s

m m t (10.8) m m m m m

6.12, s 5.60, s 1.14, d (7.2)

1.79, 1.72, 2.79, 1.99, 1.75, 1.83, 1.70, 2.84,

2.60, d (3.6)

5.88, d (3.6)

2.37, 1.39, 0.91, 1.23,

2.14, m 1.81, m 1.38, m 1.81, d (14.2) 1.56, dd (14.2, 6.9) 2.16, m

2.09, dd (14.8, 3.4) 1.94, dd (14.8, 4.0)

2.45, 1.81, 1.65, 1.99, 1.75,

9

Journal of Natural Products Article



Article

21.5, CH3 15.2, CH3 15.1, CH3 18.4, CH3 20.1, CH3

δC

18.7, CH3

position

15′

Table 2. continued

4

1.40, s

δH

δC

5

1.14, s

δH

δC

6

1.13, s

δH

δC

7

δH

δC

8

1.76, s

δH

δC

9

1.31, s

δH

and PB (3.8 g) were subjected to CC over Sephadex LH-20 by eluting with MeOH to afford Frs. NCA−NCG, OAA−OAH, and PBA−PBG, respectively. Frs. NCC (856 mg), OAA (1.1 g), and PBA (783 mg) were chromatographed over an ODS-column eluting with MeOH/ H2O (4:6−8:2, v/v) to obtain Frs. NCCA−NCCJ, OAAA−OAAL, and PBAA−PBAK, respectively. Fr. NCCA (93 mg) was purified repeatedly by semipreparative HPLC [MeCN/0.1% THF (60:40, v/ v)] to give 1 (22 mg, tR 7.0 min), 4 (5 mg, tR 8.8 min), 5 (8 mg, tR 4.7 min), and 6 (11 mg, tR 10.3 min). Fr. OAAE (167 mg) was purified repeatedly by semipreparative HPLC [MeCN/0.1% THF (50:50, v/ v)] to give 2 (4 mg, tR 16.6 min), 3 (5 mg, tR 6.4 min), 8 (3 mg, tR 12.8 min), 9 (4 mg, tR 20.9 min), and 10 (11 mg, tR 14.2 min). Fr. PBAE (68 mg) was separated repeatedly by semipreparative HPLC [MeCN/ 0.1% THF (50:50, v/v)] to give 7 (4 mg, tR 13.3 min), 11 (3 mg, tR 13.7 min), and 12 (2 mg, tR 17.2 min). Artepestrin A (1): white, amorphous powder; [α]21 D −36 (c 0.1, MeOH); IR (KBr) νmax 3401, 2961, 2928, 2542, 2235, 2072, 1691, 1622, 1383, 978 cm−1; UV (MeOH) λmax (log ε) 243 (2.21) nm; ECD (MeOH) λmax (Δε) 202 (−17.5), 245 (+6.8), 310 (−1.6) nm; 1H and 13 C NMR data, see Table 1; negative-ion HRESIMS m/z 479.2799 [M − H]− (calcd for C30H39O5, 479.2803). Artepestrin B (2): white, amorphous powder; [α]21 D +286 (c 0.1, MeOH); IR (KBr) νmax 3400, 2932, 2236, 2072, 1692, 1623, 1383, 982 cm−1; UV (MeOH) λmax (log ε) 242 (1.83) nm; ECD (MeOH) λmax (Δε) 239 (+71.1), 310 (−1.4) nm; 1H and 13C NMR data, see Table 1; negative-ion HRESIMS m/z 479.2795 [M − H]− (calcd for C30H39O5, 479.2803). Artepestrin C (3): white, amorphous powder; [α]21 D +274 (c 0.1, MeOH); IR (KBr) νmax 3399, 2929, 2103, 1683, 1207, 1140, 840, 801 cm−1; UV (MeOH) λmax (log ε) 242 (1.83) nm; ECD (MeOH) λmax (Δε) 202 (+11.0), 244 (+11.2), 308 (−1.2) nm; 1H and 13C NMR data, see Table 1; positive-ion HRESIMS m/z 497.2906 [M + H]+ (calcd for C30H41O6, 497.2909). Artepestrin D (4): white, amorphous powder; [α]21 D +211 (c 0.1, MeOH); IR (KBr) νmax 3428, 2958, 2932, 2873, 1693, 1620, 1453, 1384, 1221, 1161, 916 cm−1; UV (MeOH) λmax (log ε) 241 (2.01) nm; ECD (MeOH) λmax (Δε) 211 (−5.0), 242 (+19.8), 308 (−2.2) nm; 1 H and 13C NMR data, see Table 2; positive-ion HRESIMS m/z 481.2926 [M + H]+ (calcd for C30H41O5, 481.2949). 6α-Hydroxyartepestrin D (5): white, amorphous powder; [α]21 D +277 (c 0.1, MeOH); IR (KBr) νmax 3394, 2956, 2929, 2871, 1694, 1513, 1206, 1006, 837, 799 cm−1; UV (MeOH) λmax (log ε) 237 (2.20); ECD (MeOH) λmax (Δε) 238 (+13.4), 322 (−1.5) nm; 1H and 13 C NMR data, see Table 2; positive-ion HRESIMS m/z 497.2907 [M + H]+ (calcd for C30H41O6, 497.2909). Artepestrin E (6): white, amorphous powder; [α]21 D +123 (c 0.1, MeOH); IR (KBr) νmax 3250, 2961, 2928, 2866, 1684, 1204, 1140, 840, 800 cm−1; UV (MeOH) λmax (log ε) 243 (2.14) nm; ECD (MeOH) λmax (Δε) 208 (+7.5), 247 (+16.3), 307 (−3.1) nm; 1H and 13 C NMR data, see Table 2; positive-ion HRESIMS m/z 481.2955 [M + H]+ (calcd for C30H41O5, 481.2949). Artepestrin F (7): white, amorphous powder; [α]21 D +160 (c 0.1, MeOH); IR (KBr) νmax 3400, 2930, 2872, 2525, 2239, 2073, 1679, 1202, 1135, 978, 802 cm−1; UV (MeOH) λmax (log ε) 203 (1.92) nm; ECD (MeOH) λmax (Δε) 214 (+10.8) nm; 1H and 13C NMR data, see Table 2; negative-ion HRESIMS m/z 481.2964 [M − H]− (calcd for C30H41O5, 481.2949). 2′α-Hydroxyartepestrin F (8): white, amorphous powder; [α]21 D −41 (c 0.1, MeOH); IR (KBr) νmax 3359, 2929, 2873, 1676, 1318, −1 1273, 1071, 975 cm ; UV (MeOH) λmax (log ε) 204 (1.83) nm; ECD (MeOH) λmax (Δε) 210 (+18.1), 245 (+3.9) nm; 1H and 13C NMR data, see Table 2; negative-ion HRESIMS m/z 497.2911 [M − H]− (calcd for C30H41O6, 497.2909). Artepestrin G (9): white, amorphous powder; [α]21 D +182 (c 0.1, MeOH); IR (KBr) νmax 3395, 2931, 2873, 1679, 1192, 1150, 940 cm−1; UV (MeOH) λmax (log ε) 205 (1.98) nm; ECD (MeOH) λmax (Δε) 211 (+38.9) nm; 1H and 13C NMR data, see Table 2; positiveion HRESIMS m/z 521.2881 [M + Na]+ (calcd for C30H42O6Na, 521.2874), 537.2621 [M + K]+ (calcd for C30H42O6K, 537.2608). I



Article

Table 3. 1H and 13C NMR Data of Compounds 10−12 (δ in ppm, J in Hz, 500 MHz for 1H and 125 MHz for 13C, in Methanold4) 10 position

11

δC

1 2

47.3, CH 42.4, CH2

3 4 5 6

210.5, C 138.4, C 178.2, C 39.4, CH2

7 8

39.3, CH 32.9, CH2

9

37.7, CH2

10 11 12 13

36.7, CH 148.1, C 167.5, C 124.1, CH2

14 15 1′

δH 3.25, br s 2.60, d (19.1) 2.04, d (19.1)

2.87, 2.58, 2.95, 1.76, 1.70, 1.85, 1.85, 2.14,

d (20.0) dd (20.0, 11.1) t (11.1) m m m m m

12.4, CH3 7.9, CH3 76.7, CH

5.70, 6.17, 0.67, 1.62, 3.36,

s s d (7.1) s br s

2′ 3′

74.5, CH 118.1, CH

5.14, d (4.0) 5.40, d (4.0)

4′ 5′ 6′

143.2, C 40.6, CH 30.1, CH2

7′ 8′

41.6, CH 27.6, CH2

9′

35.0, CH2

10′ 11′ 12′ 13′

37.0, C 147.4, C 170.5, C 123.4, CH2

14′ 15′

16.7, CH3 21.4, CH3

2.38, 1.97, 1.31, 2.49, 1.66, 1.61, 1.90, 1.23,

6.16, 5.62, 0.90, 1.71,

d (13.1) m d (13.1) ddd (16.0, 10.1, 4.4) m m dd (13.1, 4.5) dd (13.1, 4.5)

s s s s

12

δC

δH

δC

δH

47.4, CH 42.5, CH2

3.22, br s 2.60, dd (18.8, 6.8) 2.00, d (18.8)

47.4, CH 42.5, CH2

3.29, br s 2.63, dd (19.0, 7.3) 2.05, d (19.0)

211.1, C 138.5, C 178.3, C 39.6, CH2 39.2, CH 33.0, CH2 37.9, CH2 36.8, CH 149.5, C 167.1, C 123.4, CH2 12.5, CH3 8.0, CH3 50.7, CH2 65.5, CH 48.2, CH2 87.2, C 53.8, CH 28.2, CH2 41.7, CH 28.8, CH2 46.2, CH2 35.0, C 147.4, C 170.5, C 123.5, CH2 20.5, CH3 20.3, CH3

Rupestrinate A (10): white, amorphous powder; [α]21 D +89 (c 0.1, MeOH); IR (KBr) νmax 3452, 2933, 1694, 1625, 1383, 1149, 951 cm−1; UV (MeOH) λmax (log ε) 242 (1.63) nm; ECD (MeOH) λmax (Δε) 208 (−10.9), 248 (+8.3), 310 (−1.7) nm; 1H and 13C NMR data, see Table 3; negative-ion HRESIMS m/z 495.2736 [M − H]− (calcd for C30H39O6, 495.2752). Rupestrinate B (11): white, amorphous powder; [α]21 D +56 (c 0.1, MeOH); IR (KBr) νmax 2930, 1678, 1626, 1441, 1204, 1140, 842, 802, 724 cm−1; UV (MeOH) λmax (log ε) 245 (1.54) nm; ECD (MeOH) λmax (Δε) 246 (+10.5), 310 (−2.2) nm; 1H and 13C NMR data, see Table 3; positive-ion HRESIMS m/z 521.2851 [M + Na]+ (calcd for C30H42O6Na, 521.2874). Rupestrinate C (12): white, amorphous powder; [α]21 D +112 (c 0.1, MeOH); IR (KBr) νmax 3400, 2931, 1688, 1626, 1440, 1203, 1184, 1140, 832 cm−1; UV (MeOH) λmax (log ε) 250 (2.02) nm; ECD (MeOH) λmax (Δε) 220 (−8.9), 249 (+14.0), 318 (−1.1) nm; 1H and 13 C NMR data, see Table 3; positive-ion HRESIMS m/z 533.2510 [M + Na]+ (calcd for C30H38O7Na, 533.2512).

2.82, 2.50, 2.95, 1.85, 1.76, 1.85, 1.57, 2.14,

d (20.0) d (20.0) t (11.4) m m m m m

6.15, 5.61, 0.66, 1.62, 1.80, 1.15, 3.84, 3.08, 1.56,

s s d (7.1) s m d (11.5) m d (12.0) m

1.77, 1.86, 1.40, 2.55, 1.67, 1.48, 1.57, 1.49,

m m m m m m m m

6.13, 5.58, 1.04, 1.52,

s s s s

211.2, C 138.5, C 178.6, C 39.7, CH2 39.4, CH 32.8, CH2 37.7, CH2 36.9, CH 147.9, C 168.0, C 124.9, CH2 12.5, CH3 8.0, CH3 84.5, CH 143.1, CH 153.2, C 200.0, C 85.4, C 37.3, CH2 37.6, CH 29.3, CH2 38.2, CH2 48.4, C 146.6, C 170.3, C 123.4, CH2 14.7, CH3 28.7, CH3

2.90, 2.59, 3.03, 1.79, 1.77, 1.88, 1.86, 2.16,

d (18.4) m t (11.0) m m m m m

6.31, 5.74, 0.69, 1.63, 5.23,

s s d (7.0) s d (3.1)

6.94, d (3.1)

2.71, 1.52, 2.21, 1.67, 1.37, 1.76, 1.34,

d (12.4) t (12.4) m m m m m

6.16, 5.58, 1.10, 2.41,

s s s s

ECD Calculations. 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 and using the Sybyl-X 1.1 software package. The conformers were further optimized by using the TDDFT method at the B3LYP/631G(d) level, and 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/631+G(d) level in MeOH as solvent. ECD spectra of different conformers were simulated using SpecDis with a half-bandwidth of 0.16−0.3 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. Inhibition of NO Production. BV-2 microglial cells were purchased from Peking Union Medical College Cell Bank (Beijing, People’s Republic of China). Cell maintenance, experimental J



Article

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Table 4. Inhibitory Effects of Isolates against LPS-Induced NO Production in BV-2 Cells

a b

compound

IC50 (μM)

cell viability (%)a

1 2 3 4 5 6 7 8 9 10 11 12 quercetinb

27.3 39.8 29.8 32.7 23.0 38.6 17.0 71.8 43.6 33.0 40.6 30.1 8.7

± ± ± ± ± ± ± ± ± ± ± ± ±

95.7 91.1 94.6 90.7 91.1 90.7 89.1 91.8 77.3 96.7 97.2 95.4 99.5

0.7 2.7 1.4 0.8 1.5 1.7 0.6 5.0 2.3 1.3 0.9 0.5 0.3

Cell viability was expressed as a percentage (%) of control group. Positive control.

procedures, and data presentation for the inhibition of NO production and viability assay were the same as previously described.11 Quercetin (IC50 value of 8.7 μM) 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).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00894. ECD spectra calculation details of compounds 1−12; copies of 1D- and 2D-NMR, IR, HRESIMS, and ECD spectra for compounds 1−12 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y. Jiang). Tel/Fax: +86-1082802719. *E-mail: [email protected] (P.-F. Tu). Tel/Fax: +86-1082802750. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by 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 (Nos. 2012ZX09301002-002-002 and 2012ZX09304-005).

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REFERENCES

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