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Jun 10, 2014 - Eremophilane-Type Sesquiterpenoids with Diverse Skeletons from Ligularia sagitta ... were obtained from the aerial parts of Ligularia s...
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Eremophilane-Type Sesquiterpenoids with Diverse Skeletons from Ligularia sagitta Jian-Jun Chen, Chao-Jun Chen, Xiao-Jun Yao, Xiao-Jie Jin, and Kun Gao* State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Tian-shui Road 222, Lanzhou 730000, People’s Republic of China S Supporting Information *

ABSTRACT: Five new highly oxygenated eremophilane-type sesquiterpenoids, possessing C19 (1 and 2), C15 (3 and 4), and C14 (8) skeletons, along with eight known eremophilenolides were obtained from the aerial parts of Ligularia sagitta. The absolute configuration of 1 was assigned by X-ray diffraction analysis and that of 3 by ECD spectroscopy. Compounds 1−10 were evaluated for their antibacterial activities against Staphyloccocus aureus, Bacillus subtilis, Escherichia coli, Bacillus cereus, and Erwinia carotovora. Compounds 4 and 5 displayed broad-spectrum inhibitory activity against these bacteria with MIC values of approximately 7.25 μg/mL, followed by 3 and 6 with MIC values in the range of 23.0−125.0 μg/mL. Compounds 3 and 8 showed mild activity against three human tumor cell lines (IC50 ≈ 13 μM). Preliminary structure−activity relationships for these eremophilenolides are reported.

T

isolation, structural elucidation, as well as biological evaluation of these compounds are described in this report.

he genus Ligularia is a medicinally important member of the family Compositae, which consists of approximately 150 species. More than 100 species of this genus are distributed in China, of which 27 species have long been used in Chinese folk medicine. It is recorded in the Chinese pharmacopoeia that Ligularia has been used to treat hemoptysis, rheumatism, pulmonary tuberculosis, urinary tract blockages, asthma, hepatitis, and bronchitis for hundreds of years.1,2 Biological and phytochemical investigations showed that Ligularia species produce a variety of metabolites which have interesting structures and unique biological activities. More importantly, sesquiterpenoids such as eremophilanes, oplopanes, bisabolanes, germacrenes, benzofurans, and eudesmanes possess a variety of core backbones and display a series of bioactivities, including cytotoxic, antibacterial, insecticidal, anti-inflammatory, antiproliferative, and plant growth regulatory activities. Therefore, studies of this genus have been of considerable interest to natural products researchers.3,4 Ligularia sagitta (Maxim) Maettf (Compositae) is mainly distributed in western China, and its rhizomes are used to treat inflammation, eliminate phlegm, suppress coughs, relieve pain, and stimulate blood flow.2 Previous studies of L. sagitta have resulted in the isolation of several bioactive eremophilenolides and triterpenoids.5 In searching for new bioactive sesquiterpenoids from Ligularia,6 we reinvestigated the aerial parts of L. sagitta and isolated five new (1−4, 8) and eight known (5−7, 9−13) highly oxygenated eremophilane derivatives. The © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 14, 2014

A

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RESULTS AND DISCUSSION The aerial parts of L. sagitta were extracted with MeOH, and the extract was partitioned with EtOAc and n-BuOH. The EtOAc extract was fractionated by column chromatography (CC) over silica gel, Sephadex LH-20, and semipreparative HPLC to afford five new highly oxygenated eremophilane-type sesquiterpenoids, sagittacins A−E (1−4, 8), which feature three types of carbon skeletons. Eight known compounds (5−7, 9− 13) were also isolated. By comparing their observed and reported spectroscopic data, the structures of the known compounds were assigned as 6β-(2′-hydroxymethylacryloyloxy)-1β,10β-epoxy-8β-hydroxyeremophil-7(11)-en-8α(12)olide (5),7 1β,10β-epoxy-6β,8α-dihydroxyeremophil-7(11)-en8β(12)-olide (6),8 1β,10β-dihydroxy-8β-methoxyeremophil7(11)-en-8α(12)-olide (7),9 1β-hydroxy-8,11-dioxoeremophil12-nor-6,9-diene (9),10 1β-hydroxy-8-oxoeremophil-6,9-diene11-trinor-7-ol (10),5g (11S)-1β-hydroxy-8-oxoeremophil-6,9dien-12-al (11), (11R)-1β-hydroxy-8-oxoeremophil-6,9-dien12-al (12),11 and (11S)-1β-hydroxy-8-oxoeremophil-6,9-diene12-nor-11-ol (13).12 Sagittacin A (1) was crystallized as colorless needles from CHCl3, [α]20 D +9 (c 0.6, MeOH). Its molecular formula was assigned as C19H26O6 by the HRESIMS (m/z: [M + Na]+ calcd 373.1622; found, 373.1630) and 13C NMR spectroscopic data (Table 1), indicating seven indices of hydrogen deficiency. The IR spectrum displayed absorptions for hydroxy (3389 cm−1) and carbonyl (1719 cm−1) groups. The 13C NMR spectrum (CDCl3) displayed an acyl carbon resonance at δ 175.7 and two vinylic carbon resonances at δ 149.5 and 138.2, accounting for two indices of hydrogen deficiency. The five remaining indices

of hydrogen deficiency indicated that 1 has a pentacyclic skeleton. The 1H NMR spectrum displayed resonances for three methyl groups at δH 1.03 (d, J = 7.2 Hz, H3-14), 1.15 (s, H3-15), and 1.90 (d, J = 1.8 Hz, H3-13), an oxygenated methylene at δH 3.77 (d, J = 10.8 Hz, H-19a) and 3.68 (d, J = 10.8 Hz, H-19b), and three oxygenated methines at δH 3.93 (br s, H-1), 3.95 (br s, H-6), and 4.62 (br d, J = 4.2 Hz, H-12). The 13 C NMR spectrum of 1 displayed resonances for 19 carbons, comprising three methyl, five methylene (one oxygenated), four methine (three oxygenated), and seven quaternary (one acyl, two sp2 hybridized, and two oxygenated) carbons. These observations indicated that 1 was an eremophilane-type sesquiterpenoid derivative with a C19 skeleton.5e In the 1 H−1H COSY spectrum, correlations from the oxygenated methine (H-1) to the methylene (H2-2), together with the spin system from H2-2 through H2-3 to H-4, continuing to H3-14, established the segment, −OCH(1)−CH2(2)−CH2(3)−CH(4)−CH3(14) (Figure 1). The HMBC experiment showed

Figure 1. 1H−1H COSY (bold) and selected HMBC (arrows) correlations of 1, 3, and 8.

cross-peaks of H2-9/C-1, C-5, C-7, C-8, C-10; Me-13/C-7, C11, C-12; Me−15/C-4, C-5, C-6, C-10; and Me-14/C-3, C-4, C-5. These observations suggested that 1 possessed the normal eremophilane-type sesquiterpenoid carbon skeleton previously observed from L. sagitta.5b Furthermore, the HMBC crosspeaks of H2-16/C-17, C-18, C-19; and H2-19/C-16, C-18 revealed that an additional carbon chain (C-16−C-19) existed in 1. The correlation from H-12 to H2-16 in the 1H−1H COSY spectrum indicated C-12 was connected to C-16. The coupling patterns of H2-9 (δH 2.68, d, J = 13.2 Hz, 2.80 d, J = 13.2 Hz) and H2-16 (δH 3.68, d, J = 10.8 Hz, 3.77 d, J = 10.8 Hz) combined with the HMBC cross-peaks of H2-9/C-17 and H219/C-8, C-16, C-17 indicated that C-8 was directly connected to C-17. Comparison with the 13C NMR data of the known compounds, the deshielded C-10 resonance at δ 94.6, suggested that the ester moiety was connected to C-10, although no direct HMBC cross-peak was evident (Figure 1).13 In addition, the HMBC cross-peaks of H-12/C-7, C-8, C-17 revealed the presence of an oxygen bridge between C-8 and C-12. Thus, the molecular structure with a C19 skeleton was deduced as shown in 1. The 1H NMR coupling constants and NOE experiments indicated that 1 and ligulasagitin B possessed the same relative configuration.5e The proposed structure and relative configuration were confirmed by single-crystal X-ray diffraction analysis using Cu Kα radiation at 292.11 (10) K (Figure 2). Thus, the absolute configuration of 1 was assigned as (1R, 4S, 5S, 6S, 8R, 10R, 12R, 17S) based on a Flack absolute structure parameter of −0.05(16). Compound 2 was an optically active, [α]20 D +30 (c 0.1, MeOH), colorless gum. The molecular formula of 2 was assigned as C23H30O8 by the HRESIMS (m/z: [M + Na]+ calcd 457.1833; found, 457.1842) and 13C NMR spectroscopic data

13

Table 1. C NMR Data of Compounds 1−4, and 8 (δ in ppm) position

1a,c

2a,d

3b,d

4a,d

8b,d

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 OCH3 1′ 2′ 3′ 4′ 5′

74.4 30.5 27.1 38.8 47.6 74.9 138.2 87.2 28.0 94.6 149.5 84.1 10.8 18.1 10.7 37.7 56.4 175.7 67.5

74.3 29.6 25.8 36.7 46.4 73.4 132.7 86.3 27.1 91.7 149.4 83.5 10.6 16.8 11.3 36.4 54.6 172.0 66.4

62.5 19.7 23.7 31.8 43.1 73.4 153.3 104.1 43.1 60.7 130.6 170.7 8.1 15.4 16.1

62.5 19.7 23.6 31.7 43.3 73.6 154.4 101.2 43.3 60.8 130.4 170.6 7.9 15.4 15.6

73.6 33.0 24.5 37.8 40.9 66.7 63.6 191.3 123.2 163.5 200.6

50.7 165.9 126.6 143.6 15.6 64.7

163.2 124.6 144.1 16.3 65.2

165.4 139.4 126.5 62.4

28.0 15.7 19.3

a

Recorded at 150 MHz. bRecorded at 100 MHz. cRecorded in methanol-d4. dRecorded in CDCl3. B

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characteristic carbon resonance for a quaternary carbon at δ 104.1 indicated an eremophil-7(11)-en-8(12)-olide skeleton with seven indices of hydrogen deficiency for 3.16 Consistent with the 13C NMR resonances at δ 62.5 (C-1) and 60.7 (C-10), the remaining hydrogen deficiency could be attributed to the presence of an epoxy moiety. The HMBC data revealed correlations of H-1/C-2, C-3, C-5, C-9, and C-10, and H-2, H315/C-10, suggesting that the epoxy moiety involved C-1 and C10.8,16 The (Z)-2′-hydroxymethylbut-2′-enoyloxy group was placed at C-6, as shown by the HMBC cross-peak between H-6 and C-1′. Furthermore, the methoxy group was located at C-8 via the HMBC correlation of OCH3/C-8, thus permitting assignment of the molecular structure of 3. The relative configuration of 3 was defined via NOE data and 3 J coupling constants. Considering the biosynthesis relationship of eremophilane-type sesquiterpenoids isolated from Compositae species, both CH3-14 and CH3-15 were assigned to be βoriented.17 The characteristic resonance at δH 3.17 (d, J = 4.8, H-1) suggested that the 1,10- epoxy moiety was in the βorientation and that the A/B ring system was cis-fused.16 The observed NOE correlations between H-6 (δ 5.81) and H-4 (δ 1.78) (3%), and OCH3 (δ 3.28) (2%) indicated that H-6 and the C-8 OCH3 group were α-oriented (Figure 3). According to the Naya rules for eremophil-7(11)-en-8(12)-olide derivatives with oxygenation at C-8, a homoallylic coupling (J = 1.5−2.0 Hz) between H-6α and CH3-13 should be observed in the 8β(12)-olide isomer due to a dihedral angle approximating 90° between C(6)-α-H and C(11)−CH3(13). In the 8α(12)-olide isomer, the dihedral angle was approximately 20°, which indicated that the coupling should be negligible.18 Indeed, homoallylic coupling (J6,13 = 1.6 Hz) between H-6 and Me-13 was observed for 3, which further confirmed the above conclusion. To determine the absolute configuration of 3, the electronic circular dichroism (ECD) spectra for (1R,4S,5S,6S,8R,10S)-3 and its enantiomer were calculated using TDDFT theory at the B3LYP/6-311++G(d,p) level and were compared with the experimental data for 3 (Supporting Information).19 This method is a highly effective method for determining the absolute configuration of chiral natural products.6e,20 The calculated ECD curve for (1R,4S,5S,6S,8R,10S)-3 agreed well with the experimental spectrum for 3 (Figure 4). Correspondingly, the calculated Cotton effects for the electronic transitions of the α,βunsaturated γ-lactone group around 207 (negative, π−π* transition), 230 nm (positive, π−π* transition), and 253 (negative, n−π* transition) were in accord with those Cotton effects observed around 208, 229, and 250 nm in the experimental spectrum.21 Therefore, the absolute configuration of sagittacin C was determined to be 1R,4S,5S,6S,8R,10S.

Figure 2. ORTEP drawing of 1.

(Table 1), indicating nine indices of hydrogen deficiency. The 1 H and 13C NMR spectra showed resonances similar to those of 1. Significant differences were that a 2′-(hydroxymethyl)acryloyloxy moiety was present [δH 4.34 (2H, s), 5.87 (1H, br s), 6.30 (1H, br s); δC 165.4, 139.4, 126.5, 62.4] and that H-6 was deshielded from δ 3.95 to 5.57 in 2. These results indicated that in compound 2 the C-6 hydroxy group was replaced by a 2′-(hydroxymethyl)acryloyloxy group. This conclusion was confirmed by deacylation of 2 to afford 1.15 Thus, the structure of sagittacin B was assigned as shown in 2. The molecular formula of sagittacin C (3), a colorless gum, was assigned as C21H28O7 by the HRESIMS (m/z: [M + Na]+ calcd 415.1727; found, 415.1720) and 13C NMR spectroscopic data (Table 1), indicating eight indices of hydrogen deficiency. A (Z)-2′-hydroxymethylbut-2′-enoyloxy and a methoxy moiety were observed in 3 from the 1H NMR resonances at δ 6.60 (1H, q, J = 7.6 Hz), 4.36 (1H, d, J = 12.8 Hz), 4.32 (1H, d, J = 12.8 Hz), 2.15 (3H, d, J = 7.6 Hz), and 3.28 (3H, s), together with the 13C NMR resonances at δ 165.9, 143.6, 126.6, 64.7, 15.6, and 50.7.5f In addition to the carbons belonging to the aforementioned groups, the 13C NMR spectrum of 3 also displayed 15 carbon resonances classified as three methyl, three methylene, three methine (two oxygenated), six quaternary (one acyl, two sp2 hybridized, and two oxygenated) carbons (Table 1), suggesting that 3 had a sesquiterpenoid skeleton. The IR spectrum showed an absorption of an α,β-unsaturatedγ-lactone moiety at 1772 cm−1.5b The above data as well as the three methyl proton resonances at δ 1.86 (d, J = 1.6 Hz, H-13), 1.16 (s, H-15), and 1.06 (d, J = 8.0 Hz, H-14) and the

Figure 3. Key NOE correlations of 3, 4, and 8. C

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1.39) (4%) suggested that the 6,7-epoxy moiety was in the αorientation (Figure 3). Therefore, the structure of 8 was established as 1β-hydroxy-6α,7α-epoxy-8,11-dioxoeremophil12-nor-9-ene. Compounds 1 and 2 are rare examples of C19 eremophilanes possessing a 6/6/6 fused-ring skeleton. Thus far, only a few C19 eremophilanes containing a 1(18)-olide moiety have been found in nature.5f,13 Moreover, the 10(18)-olide moiety of the C19 eremophilanes 1 and 2 was only found in ligulasagitin B, which had been previously isolated from L. sagitta collected in the Gannan Tibet Autonomous Region, China.5e A plausible biosynthetic pathway for 1 and 2 might involve a sequential Diels−Alder reaction and esterification of a common C15 eremophilane sesquiterpene and 2-formylacrylic acid.5e,13 The establishment of the absolute configurations of 1−3 was based on X-ray crystallography, chemical methods, ECD data, and computational approaches, but there is no direct evidence to confirm the assignments for 4 and 8. The absolute configurations of the parent skeletons of 4 and 8, as shown, were tentatively assigned using biosynthesis correlations based on the co-occurrence of these eremophilanes in L. sagitta. Considering the folkloric use of L. sagitta, the antibacterial activities of 1−10 against Staphyloccocus aureus, Bacillus subtilis, Escherichia coli, Bacillus cereus, and Erwinia carotovora were tested using a microbroth dilution method.22 Ampicillin served as a positive control (Table 2). Compounds 4 and 5 were the

Figure 4. Experimental ECD spectrum of 3 overlaid with calculated spectra for (1R,4S,5S,6S,8R,10S)-3 and (1S,4R,5R,6R,8S,10R)-3.

According to the HRESIMS and 13C NMR spectroscopic data, the molecular formula of sagittacin D (4) was established as C20H26O7. When comparing the 1D NMR spectroscopic data, a structural similarity was observed between 4 and known compound 5 (Tables 1 and 3), and the main difference between 4 and 5 lies in the fact that the 2′-(hydroxymethyl)acryloyloxy group at C-6 in 5 was replaced by a (Z)-2′hydroxymethylbut-2′-enoyloxy group in 4. The structure of 4 was further confirmed through a series of 2D NMR experiments. The characteristic resonance at δH 3.20 (d, J = 5.4, H-1) and the NOEs between H-6 (δ 6.01) and H-4 (δ 1.80) (3%), and CH3-13 (δ 1.79) (3%) suggested that the 1,10epoxy moiety was in the β-orientation and that H-6 was in the α-orientation (Figure 3). Compared with 3, no homoallylic coupling between H-6 and CH3-13 was observed in the 1H NMR spectra of either 4 or 5, which suggested that the dihedral angle between C(6)-α-H and C(11)−CH3(13) was approximately 20°. In this case, the hydroxy group at C-8 was unambiguously assigned to be in the β-orientation.18 Thus, the structure of sagittacin D (4) was defined as 6β-[(Z)-2′hydroxymethylbut-2′-enoyloxy]-1β,10β-epoxy-8β-hydroxyeremophil-7(11)-en-8α(12)-olide. Sagittacin E (8) was shown to have the molecular formula C14H18O4 by HRESIMS (m/z: [M + Na]+ calcd 273.1097; found, 273.1092) and 13C NMR spectroscopic data (Table 1), indicating six indices of hydrogen deficiency in the molecule. The IR spectrum showed absorptions at 3438, 1769, 1723, and 1673 cm−1, suggesting that hydroxy, carbonyl, and α,βunsaturated carbonyl groups were present in 8. In the 13C NMR spectrum, 14 carbon resonances were displayed, and they were classified as three methyl, two methylene, four methine (one sp2 hybridized, and two oxygenated), and five quaternary (one sp2 hybridized, one oxygenated, and two carbonyls) carbons. These data implied that 8 was a 1β-hydroxy-8,11dioxoeremophil-12-nor-9-ene derivative, similar to 9.10 Comparison of the 1H NMR data of 8 and 9 showed that the olefinic H-6 resonance was absent in 9, whereas a singlet appeared at δ 3.49 (1H, s) in 8, which indicated oxygenation at C-6. The 13C NMR chemical shifts of C-6 (δ 66.7) and C-7 (δ 63.6) further demonstrated that the 6,7-double bond in 9 was replaced with a 6,7-epoxy moiety in 8 (Table 1). The 2D NMR spectra further confirmed the structure of 8 (Figure 1). The NOE correlations between H-6 (δ 3.49) and H-14 (δ 1.11) (3%), and H-15 (δ

Table 2. Antibacterial Activity of Compounds 3−6, 8, and 9 MIC (μg/mL) compound

B. cereus

S. aureus

B. subtilis

E. coli

E. carotovora

3 4 5 6 8 9 ampicillin

62.5 15.50 7.25 62.5 >250.0 >250.0 25.0

125.0 7.25 7.25 125.0 >250.0 >250.0 25.0

125.0 7.25 15.50 125.0 >250.0 >250.0 12.5

62.5 7.25 7.25 125.0 31.25 31.25 100.0

125.0 7.25 7.25 125.0 62.5 >250.0 100.0

most active metabolites and displayed broad-spectrum inhibitory activity against all the tested bacteria with MIC values of approximately 7.25 μg/mL. Compounds 3 and 6 displayed moderate activity against these bacteria with MIC values ranging from 23.0−125.0 μg/mL; however, compound 8 exhibited only moderate activity against E. coli and E. carotovora with MIC values of 31.25 μg/mL and 62.5 μg/mL, respectively, and compound 9 exhibited only moderate activity against E. coli with an MIC value of 31.25 μg/mL. The remaining compounds showed no activity against any of the tested bacteria. When comparing the antibacterial activities of 3−7, the presence of a 1β,10β-epoxy group seemed to be crucial. Furthermore, the fact that compounds 4 and 5 showed stronger activity than 3 indicated that the C-8 methoxy group might be responsible for the decrease in antibacterial activities for the 1β,10β-epoxy-8hydroxyeremophil-7(11)-en-8β(12)-olides. Using the previously reported sulforhodamine B method, the cytotoxicity of compounds 1−10 was also evaluated against the HL-60 (acute leukemia), SMMC-7721 (hepatic cancer), and HeLa (cervical carcinoma) human tumor cell lines. 23 Mitomycin was employed as a positive control and showed an IC50 ≈ 3 μM. Only compounds 3 and 8 exhibited mild activity against the three cell lines (IC50 ≈ 13 μM). Although compounds 1 and 2 showed no bioactivity, their discovery D

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Table 3. 1H NMR Data of Compounds 1−4, and 8 (δ in ppm, J in Hz)

a

position

1a,c

2a,d

3b,d

4a,d

8b,d

1 2a 2b 3a 3b 4 6 9a 9b 12 13 14 15 16a 16b 19a 19b −OCHH3 3′a 3′b 4′ 5′a 5′b

3.93, br s 2.09, m 1.65, m 2.08, m 1.28, m 2.05, m 3.95, br s 2.80, d (13.2) 2.68, d (13.2) 4.62, br d (4.2) 1.90, d (1.8) 1.03, d (7.2) 1.15, s 1.75, dd (12.0, 4.2) 1.70, br d (12.0) 3.77, d (10.8) 3.68, d (10.8)

4.03, br s 1.95, m 1.63, m 2.09, m 1.26, m 2.20, m 5.57, br s 3.03, d (13.2) 2.65, d (13.2) 4.65, br s 1.64, d (2.4) 0.77, d (6.6) 1.32, s 1.95, dd (12.0, 4.2) 1.83, d (12.0) 3.90, d (10.8) 3.85, d (10.8)

3.17, d (4.8) 2.05, m 1.97, m 1.69, m 1.48, m 1.78, m 5.81, d (1.6) 2.30, d (13.2) 1.78, d (13.2)

3.20, d (5.4) 2.04, m 1.97, m 1.69, m 1.45, m 1.80, m 6.01, br s 2.30, d (13.2) 1.80, d (13.2)

4.40, br s 1.94, m 1.70, m 1.76, m 1.52, m 1.98, m 3.49, s 5.90, s

1.86, d (1.6) 1.06, d (8.0) 1.16, s

1.79, s 1.06, d (7.2) 1.16, s

2.34, s 1.11, d (5.6) 1.39, s

3.28, s 6.60, q (7.6)

6.60, q (7.6)

2.15, d (7.6) 4.36, d (12.8) 4.32, d (12.8)

2.17, d (7.6) 4.42, dd (12.0, 4.8) 4.14, dd (12.0, 6.0)

6.30, br s 5.87, br s 4.34, s

Recorded at 600 MHz. bRecorded at 400 MHz. cRecorded in methanol-d4. dRecorded in CDCl3.

MeOH (3 × 5 L, 7 days each time). After evaporation under reduced pressure, an extract (630 g) was obtained and partitioned with EtOAc and n-BuOH. The EtOAc-soluble partion (97.6 g) was subjected to silica gel CC (1200 g) eluting with petroleum ether/acetone (1:0−0:1, gradient system). On the basis of TLC analysis, six fractions A−F were obtained. Four fractions, C.1−C.4, were obtained after fraction C (3.3 g) was separated by silica gel CC (90 g) eluting with petroleum ether/EtOAc (8:1−1:1, gradient system). Next, C.3 (1.5 g) was subjected to silica gel CC eluting with CHCl3/EtOAc (20:1− 5:1, gradient system) to afford three fractions, C.3.1−C.3.3. With CHCl3/EtOAc (10:1) as the eluent, C.3.2 (0.4 g) was separated by silica gel CC, affording 2 (3 mg) and a mixture of 3 and 4. This mixture was separated by semipreparative HPLC (eluted with 55% MeOH/H2O, YMC-Pack ODS-A, 5 μm, 250 × 10 mm column, flow rate 2.0 mL/min, 70 min) to yield 3 (8 mg) and 4 (5 mg). Similarly, four fractions, D.1−D.4, were obtained after separating fraction D (3.9 g) on silica gel CC (120 g) eluting with CHCl3/EtOAc (8:1−2:1, gradient system). Among these fractions, D.2 (1.3 g) was subjected to silica gel CC eluting with CHCl3/EtOAc (4:1, 2:1) to afford four fractions, D.2.1− D.2.4. Compounds 1 (12 mg) and 7 (6 mg), 5 (4 mg) and 9 (15 mg), and a mixture (2 mg) of 11 and 12 were obtained by separating D.2.2 (0.25 g), D.2.3 (0.29 g), and D.2.4 (0.12 g) on a silica gel CC eluting with CHCl3/ EtOAc (5:1) and then Sephadex LH-20 CC (MeOH/CHCl3, 1:1), respectively. Fraction E (5.1 g) was subjected to silica gel CC (130 g) eluting with CHCl3/EtOAc (10:1−2:1, gradient system) to yield E.1−E.4. Using CHCl3/EtOAc (5:1, 3:1) as eluent, four fractions (E.2.1− E.2.4) were obtained by separating E.2 (1.2 g) on silica gel CC (30 g). Likewise, 6 (13 mg) and 8 (5 mg) were afforded by further separating E.2.2 (0.15 g) on silica gel CC (CHCl3/EtOAc, 6:1), followed by Sephadex LH-20 CC (MeOH/CHCl3, 1:1). Using the same

contributes to the diversity and complexity of C19 eremophilanes.

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EXPERIMENTAL SECTION General Experimental Procedures. Optical rotations were acquired with a Perkin−Elmer 341 polarimeter. Infrared spectra were obtained on a Nicolet NEXUS 670 FT-IR spectrometer. Melting points were measured using an X-4 digital display micro melting point apparatus and are uncorrected. 1H, 13C, and 2D NMR spectra were obtained on a Varian Mercury-600BB or Bruker Avance III-400 instrument. ECD spectra were acquired with a JASCO J-720 spectropolarimeter. X-ray diffraction data were collected on a SuperNova, Dual, Eos diffractometer; the structure was solved with Superflip program using charge flipping and refined with the ShelXL program (using graphite-monochromated Cu Kα radiation). HRESIMS data were acquired on a Bruker APEXII mass spectrometer. Column chromatography (CC) was performed with silica gel (200−300 mesh, Qingdao Marine Chemical Factory, China) and Sephadex LH-20 (Amersham Pharmacia Biotech). A Waters 1525 series instrument equipped with a YMC-Pack ODS-A column (250 × 10 mm, 5 μm) was used for semipreparative HPLC. TLC was conducted on GF254 plates. Ampicillin and mitomycin were purchased from Sigma (St. Louis, MO). S. aureus, E. coli, B. subtilis, B. cereus, and E. carotovora were obtained from Northwest A&F University, China. Plant Material. The aerial parts of L. sagitta, collected in Zhang County, Gansu Province, China, in August 2010 and identified by Zhang Guoliang, a professor at Lanzhou University. A voucher specimen (no. 20100816-2) was stored at the Natural Product Laboratory of State Key Laboratory of Applied Organic Chemistry, Lanzhou University. Extraction and Isolation. At room temperature, the aerial parts of L. sagitta (8.2 kg) were chipped and extracted with E

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nated sheep blood (5%), and samples (0.1 mL) were transferred to 96-well microtiter plates and inoculated using a Dynatech MIC-2000 instrument. In each well, the final concentration of inoculum was controlled in the range of 2 × 105−7 × 105 CFU/mL. After incubation for 18 h at 35 °C, the lowest concentration of antibiotic with no bacterial growth due to the absence of hemolysis of erythrocytes was defined as the MIC end point. The experiments were carried out in triplicate. Cytotoxicity Assay. For the cytotoxic assay protocol, refer to ref 6e.

method, 10 (16 mg) and 13 (1 mg) were obtained by further separating E.2.3 (0.18 g). Sagittacin A (1). Colorless needles (CHCl3); mp 233−234 °C; [α]20 D +9 (c 0.6, MeOH); IR (KBr) νmax 3389, 1719, 1654, 1453 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z: [M + Na]+ calcd for C19H26O6Na, 373.1622; found, 373.1630. Sagittacin B (2). Colorless gum; [α]20 D +30 (c 0.1, MeOH); IR (KBr) νmax 3393, 1740, 1655, 1596, 1462 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z: [M + Na]+ calcd for C23H30O8Na, 457.1833; found, 457.1842. Sagittacin C (3). Colorless gum; [α]20 D −158 (c 0.4, MeOH); IR (KBr) νmax 3441, 1772, 1723, 1649 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z: [M + Na]+ calcd for C21H28O7Na, 415.1727; found, 415.1720. Sagittacin D (4). Colorless gum; [α]20 D −8 (c 0.4, MeOH); IR (KBr) νmax 3372, 1764, 1738, 1651 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z: [M + NH4]+ calcd for C20H30O7N, 396.2017; found, 396.2027. Sagittacin E (8). Colorless gum; [α]20 D +90 (c 0.5, MeOH); IR (KBr) νmax 3438, 1769, 1723, 1673 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z: [M + Na]+ calcd for C14H18O4Na, 273.1097; found, 273.1092. X-ray Diffraction Analysis of Sagittacin A (1). A colorless block crystal with dimensions 0.39 × 0.35 × 0.28 mm of 1 was obtained from CHCl3 and used for X-ray analysis (CCDC980785). Charge flipping was applied to the structure analysis of 1, which was refined with the ShelXL program. Combined with three sets of exposures, data were collected over a hemisphere of reciprocal space. At T = 292.11 (10) K the crystal was observed to belong to the orthorhombic space group P212121, with a = 9.1403(2) Å, b = 12.3986(3) Å, c = 15.0582(4) Å, V = 1706.50(7) Å3, Z = 4, Dcalc = 1.364 g/cm3, λ = 1.5418 Å, μ (Cu Kα) = 0.832 mm−1, and F (000) = 752. A total of 10 415 reflections, collected in the range of 5.66° ≤ θ ≤ 72.54°, yielded 3358 unique reflections. The structure was determined by direct methods and was refined using full-matrix least-squares on F2 values for 3288 I > 2σ(I). Hydrogen atoms were fixed at calculated positions and non-hydrogen atoms were refined anisotropically. The final indices gave the following values: R = 0.0344, Rw = 0.0962, goodness of fit = 1.045. The scattering factors were referred to the International Tables for X-ray Crystallography.24 Hydrolysis of Sagittacin B (2). Sagittacin B (2, 1.0 mg, 0.0023 mmol) was added to 3 mL of K2CO3−MeOH (5%), and the reaction mixture was stirred for 10 h at room temperature. The MeOH was removed under vacuum, and the residue was diluted with EtOAc. The organic phase was washed with NaHCO3 and brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuum. The crude product was purified via silica gel CC (CHCl3/EtOAc, 5:1) to give 1 (0.6 mg). Antibacterial Assay. The preparation of the inoculum was conducted as follows: First, several colonies from an overnight culture of S. aureus, E. coli. , B. subtilis, B. cereus, and E. carotovora on sheep blood agar media (5%) were suspended in Mueller−Hinton broth. Second, they were adjusted to a turbidity of a 0.5 McFarland standard (about 1.5 × 108 CFU/mL). Next, a further 10-fold dilution was carried out in Mueller−Hinton broth. With sterile water, stock solutions of compounds 1−10 and ampicillin were adjusted to 1 g/L, and they were used at once. The samples were doubly diluted (250−0.125 μg/mL) in Mueller−Hinton broth with defibri-

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ASSOCIATED CONTENT

S Supporting Information *

Supplemetary data associated with this article (NMR, IR, and HRESIMS spectra for compounds 1−4, and 8; computational details for 3). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86-931-8912582. Tel.: +86-931-8912592. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported financially by the National Basic Research Program of China (no. 2014CB138703), the NSFC (no. 31270396), and the Fundamental Research Funds for the Central Universities (lzujbky-2013-51).

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REFERENCES

(1) How, K. C. A Dictionary of the Famiiies and Genera of Chinese Seed Plants; Science Press: Beijing, 1982; pp 276. (2) Jiangsu College of New Medicine. A Dictionary of Traditional Chinese Medicines; Shanghai Science and Technology Press: Shanghai, 1977; p 7, 154, 549, 1152, and 2349. (3) Wu, Q. X.; Shi, Y. P.; Jia, Z. J. Nat. Prod. Rep. 2006, 23, 699−734. (4) Chen, K.; Baran, P. Nature 2009, 459, 824−828. (5) (a) Yang, L.; Peng, H. R.; Jia, Z. J. Chin. Chem. Lett. 1995, 6, 875−876. (b) Li, X. Q.; Gao, K.; Jia, Z. J. Planta Med. 2003, 69, 356− 360. (c) Li, L.; Xu, L. W.; Wang, H. Q. Helv. Chim. Acta 2004, 87, 1125−1129. (d) Liu, S. J.; Liu, J. Q.; Zhang, M.; Zhang, C. F.; Wang, Z. T. Biochem. Syst. Ecol. 2007, 35, 245−247. (e) Li, P. L.; Wang, C. M.; Zhang, Z. X.; Jia, Z. J. Tetrahedron 2007, 63, 12665−12670. (f) Li, P. L.; Zhang, Z. X.; Jia, Z. J. Chem. Lett. 2008, 37, 308−309. (g) Li, P. L.; Jia, Z. J. Helv. Chim. Acta 2008, 91, 1717−1727. (6) (a) Wu, Q. H.; Wang, C. M.; Chen, S. G.; Gao, K. Tetrahedron Lett. 2004, 45, 8855−8858. (b) Liu, C. M.; Fei, D. Q.; Wu, Q. H.; Gao, K. J. Nat. Prod. 2006, 69, 695−699. (c) Fei, D. Q.; Li, S. G.; Liu, C. M.; Wu, G.; Gao, K. J. Nat. Prod. 2007, 70, 241−245. (d) Li, W. X.; Lei, M.; Fei, D. Q.; Gao, K. Planta Med. 2009, 75, 635−640. (e) Chen, J. J.; Li, W. X.; Gao, K.; Jin, X. J.; Yao, X. J. J. Nat. Prod. 2012, 75, 1184− 1188. (7) Jia, Z. J.; Zhao, Y.; Tan, R. X. Planta Med. 1992, 58, 365−367. (8) Zhao, Y.; Jia, Z. J.; Tan, R. X.; Yang, L. Phytochemistry 1992, 31, 2785−2787. (9) Reina, M.; Santanab, O.; Domíngueza, D. M.; Villarroeld, L.; Fajardoe, V.; Rodríguezf, M. L.; González-Coloma, A. Chem. Biodiversity 2012, 9, 625−643. (10) Zhao, Y.; Peng, H. R.; Jia, Z. J. J. Nat. Prod. 1994, 57, 1626− 1630. (11) Wang, Q.; Mu, Q.; Shibano, M.; Morris-Natschke, S. L.; Lee, K. H.; Chen, D. F. J. Nat. Prod. 2007, 70, 1259−1262. (12) Zhao, Y.; Jia, Z. J. Chin. Chem. Lett. 1993, 4, 323−326. F

dx.doi.org/10.1021/np5003302 | J. Nat. Prod. XXXX, XXX, XXX−XXX

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(13) Zhao, Y.; Parsonsz, S.; Smart, B. A.; Tan, R. X.; Jia, Z. J.; Sun, H. D.; Rankin, D. W. H. Tetrahedron 1997, 53, 6195−6208. (14) Flack, H. D. Acta Crystallogr. 1983, A39, 876−881. (15) Plattner, J.; Gless, R.; Rapoport, H. J. Am. Chem. Soc. 1972, 94, 8613−8615. (16) Wang, W. S.; Gao, K.; Jia, Z. J. J. Nat. Prod. 2002, 65, 714−717. (17) Moriyama, Y.; Takahashi, T. Bull. Chem. Soc. Jpn. 1976, 49, 3196−3199. (18) Naya, K.; Nogi, N.; Makiyama, Y.; Takashina, H.; Imagawa, T. Bull. Chem. Soc. Jpn. 1977, 50, 3002−3006. (19) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, revision A.1; Gaussian, Inc.: Wallingford, CT, 2009. (20) (a) Berova, N.; Bari, L. D.; Pescitelli, G. Chem. Soc. Rev. 2007, 36, 914−931. (b) Diedrich, C.; Grimme, S. J. Phys. Chem. A 2003, 107, 2524−2539. (c) Ding, Y. Q.; Li, X. C.; Ferreira, D. J. Nat. Prod. 2010, 73, 435−440. (d) Yuan, T.; Zhu, R. X.; Zhang, H.; Odeku, O. A.; Yang, S. P.; Liao, S. G.; Yue, J. M. Org. Lett. 2010, 12, 252−255. (21) Edwards, J. A.; Carpio, H.; Cross, A. D. Tetrahedron Lett. 1964, 5, 3299−3304. (22) Ericsson, H. M.; Sherris, J. C. Acta Pathol. Micro. Sc. B 1971, 217 (Suppl), 1−90. (23) Skehan, P.; Storeng, R.; Scudiero, D. J. Natl. Cancer Inst. 1990, 82, 1107−1112. (24) Ibers, J. A.; Hamilton, W. C. International Tables for X-Ray Crystallography; The Kynoch Press: Birmingham, U.K., 1974; Vol. IV.

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