Bioassay- and Chemistry-Guided Isolation of Scalemic Caged

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Bioassay- and Chemistry-Guided Isolation of Scalemic Caged Prenylxanthones from the Leaves of Garcinia bracteata Sheng-Li Niu,†,‡ Da-Hong Li,‡ Xin-Yu Li,‡ Yue-Tong Wang,§ Sheng-Ge Li,‡ Jiao Bai,‡ Yue-Hu Pei,‡ Yong-Kui Jing,§ Zhan-Lin Li,*,‡ and Hui-Ming Hua*,‡ †

Key Laboratory of Zoonosis of Liaoning Province, School of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang 110866, People’s Republic of China ‡ Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education; School of Traditional Chinese Materia Medica, and §School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China S Supporting Information *

ABSTRACT: With bioassay- and chemistry-guided fractionation, seven new caged prenylxanthones including two scalemic mixtures, epiisobractatin (1), 13-hydroxyisobractatin (2), 13hydroxyepiisobractatin (3), 8-methoxy-8,8a-dihydrobractatin (4), 8-ethoxy-8,8a-dihydrobractatin (5), garcibracteatone (6), and 8-methoxy-8,8a-dihydroneobractiatin (7), and the eight known compounds 8−15 were isolated from the leaves of Garcinia bracteata. The structures were unambiguously elucidated through analysis of spectroscopic data. The 2D structures and relative configurations of 1 and 5 were confirmed by X-ray crystallographic analysis. The separation of the enantiomers of 1−5 was accomplished by chiral-phase HPLC. The absolute configurations of the enantiomers of 1 and 5 were assigned by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. The absolute configurations of the related compounds were determined via comparisons of their ECD data with those of the enantiomers of 1 and 5, respectively. Notably, compound 7, with a neo caged skeleton, is the first representative of a novel type of caged xanthone lacking a Δ8(8a) double bond. The isolated compounds exhibited significant cell growth inhibitory activities in vitro against human leukemic HL-60 and K562 cell lines, with GI50 values ranging from 0.2 to 8.8 μM. A preliminary structure−activity relationship is discussed. carbonyl type, represented by gambogic acid. To date, only five C-5 carbonyl-containing caged xanthones, such as neobractatin, have been reported.13−15 G. bracteata C. Y. Wu ex Y. H. is commonly encountered in the southern regions of the Guangxi and Yunnan Provinces of People’s Republic of China. The main active compounds isolated from G. bracteata are caged xanthones,13−15 classic xanthones,14 and benzophenones.14 Previously, we reported five new classic xanthones and two rearranged caged xanthones from G. bracteata with significant cytotoxic activities.17,18 The intriguing structures and diverse bioactivities prompted further investigation of the bioactive compounds from G. bracteata. A CHCl3-soluble fraction from the leaves of G. bracteata exhibited strong antiproliferative activity against the HL-60 cell line (GI50 < 3.13 μg/mL). HPLC-DAD analysis and the 1H and 13C NMR data of the CHCl3 extract suggested the presence of caged prenylxanthones. Therefore, a systematic study to explore the chemical components of the CHCl3 extract of the leaves of G.

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he genus Garcinia has been widely studied from the perspective of phytochemical constituents and biological activity. Caged xanthones, with “privileged structures” characterized by an unusual 4-oxatricyclo[4.3.1.03,7]dec-2-one skeleton, are a special class of bioactive ingredients mainly derived from several Garcinia plants,1−3 namely, G. hanburyi,4,5 G. morella,6,7 G. gaudichaudii,8,9 G. scortechinii,10,11 G. laterif lora,12 and G. bracteata.13−15 During the past few years, caged xanthones have increasingly attracted the attention of both synthetic and natural-product chemists due to the fascinating and unique structures, biosynthesis, and notable biological activities of these compounds.16 More than 120 caged xanthones from Garcinia have been reported, and most of these caged xanthones possess a broad array of pharmacological and biological properties, such as cytotoxic, antibacterial, and anti-inflammatory activities.1−3 Caged xanthones may be classified into two types: a normal type with a C-6 carbonyl group and a novel type possessing a rearranged caged backbone with a C-5 carbonyl moiety. Each type includes the following subtypes: caged xanthones with or without a Δ8(8a) double bond. Most caged xanthones from Garcinia belong to the C-6 © 2018 American Chemical Society and American Society of Pharmacognosy

Received: May 26, 2017 Published: March 22, 2018 749

DOI: 10.1021/acs.jnatprod.7b00454 J. Nat. Prod. 2018, 81, 749−757

Journal of Natural Products

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bracteata was launched. In the present study, seven new (1−7) and eight known (8−15) caged xanthones were isolated from the leaves of G. bracteata. In addition, the antiproliferative activity of these caged xanthones was measured in vitro against the human leukemic HL-60 and K562 cell lines. Herein, the isolation of the new caged xanthones, the elucidation of their structures, and the growth inhibitory activity of the isolated compounds against two human leukemic cell lines are discussed.

molecular formula of C28H32O6. The UV absorption maxima at 218 and 355 nm were indicative of a caged xanthone derivative with a Δ8(8a) double bond.13 The IR spectrum showed absorption bands of a hydrogen-bonded hydroxy group (3445 cm−1), an unconjugated carbonyl group (1739 cm−1), and a hydrogen-bonded carbonyl group (1637 cm−1). The 1H NMR data of 1 (Table 1) exhibited signals due to a hydrogen-bonded hydroxy group at δH 13.14 (1H, brs, 1-OH), an olefinic proton of an α,β-unsaturated carbonyl unit at δ 7.47 (1H, d, J = 7.0 Hz, H-8), an aromatic proton at δ 6.01 (1H, s), a typical olefinic proton of a 3-methylbut-2-enyl moiety at δ 4.35 (1H, t, J = 7.9 Hz, H-22), a methyl doublet at δ 1.29 (3H, d, J = 6.6 Hz, H13), and six methyl singlets at δH 1.06, 1.27, 1.37, 1.42, 1.50, and 1.73. The 13C NMR data (Table 2) showed 28 carbon resonances, which were classified by the HSQC spectrum as five quaternary carbons, six oxygenated tertiary carbons, seven methyls, two methylenes, six methines, and two carbonyl carbons at δc 203.9 and 179.0. In addition, the typical signals of a bicyclo[2.2.2]octane unit at δ 3.49 (dd, J = 4.8, 7.0 Hz, H-7), 2.34 (dd, J = 4.8, 13.4 Hz, Ha-16), and 2.53 (d, J = 9.3 Hz, H17) were shown in the 1H NMR spectrum. In conjunction with the presence of three oxygenated tertiary carbon signals at δC 91.1 (C-10a), 84.9 (C-5), and 83.1 (C-18), 1 was determined to possess a caged xanthone scaffold.12,20 Comparison of the 1H and 13C NMR spectroscopic data of 1 (Tables 1 and 2) with those of isobractatin (8)13 showed their close structural relationship (Figure S17, Supporting Information). Differences were observed for the C-13 and C-14 signals of the dihydrofuran unit in the 13C NMR spectrum and the H-12, Me-14, and Me-15 signals in the 1H NMR spectrum, suggesting that the two products were a pair of C-12 epimers, which was consistent with the HMBC correlations shown in Figure 1a. The key NOESY correlations (Figure 1b) of H3-13/H3-15, H3-15/Ha-21, H-12/H3-14, and H3-14/H3-19 indicated the same orientation of the C-5 prenyl and the C-12 methyl groups (Me-13). Thus, H-12 was trans to the C-5 prenyl and was arbitrarily assigned an α-orientation This was confirmed by the similar δH values but the distinctly different δC values (ΔδC ca. +8 ppm) of the gem-dimethyl moiety (Me-14, 15) reported previously.10,11,19 The assignment of the relative configuration of 1 was confirmed by analysis of the single-crystal X-ray diffraction data (Figure 2). Compound 1 showed a specific rotation, [α]25 D −15 (c 0.1, CHCl3), implying that this compound may be a scalemic mixture. It was reported that natural caged xanthones are scalemic mixtures with enantiomeric ratios as high as 1.9:1.13,21,22 Thus, compound 1 was resolved using HPLC on a chiral-phase IC column, and the corresponding enantiomers 25 (+)-1 ([α]25 D +145.3, c 0.1, CHCl3) and (−)-1 ([α]D −161.8, c 0.1, CHCl3), with opposite electronic circular dichroism (ECD) curves, were isolated in a ratio of ca. 1:1.1 (Figure S67, Supporting Information). The computation of ECD data using time-dependent density functional theory (TDDFT) was used to determine the absolute configuration of (−)-1 and (+)-1.23−25 Since compound 1 must be one of the two enantiomers (5R,7S,10aS,12R,17S)-1 (1a) or (5S,7R,10aR,12S,17R)-1 (1b) based on the relative configuration that was confirmed by X-ray crystallographic analysis, the two enantiomers were differentiated by the ECD method. The experimental ECD curve of (−)-1 matched well with the calculated curve of 1a, whereas the observed ECD spectrum for (+)-1 was similar to that calculated for 1b (Figure 1c). The absolute configurations of (−)-1 and

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RESULTS AND DISCUSSION The HPLC-DAD analysis (Figure S1, Supporting Information) of the CHCl3-soluble fraction of the 95% EtOH extract of the leaves of G. bracteata showed that several peaks exhibited the characteristic UV absorption of caged prenylxanthones at approximately 230 and 350 nm.10,19 The 1H NMR spectrum (Figure S2, Supporting Information) showed the presence of methyl signals of prenyl groups, aromatic protons, and hydrogen-bonded phenolic protons. The conjugated carbonyl groups of the xanthone skeleton (δ 175−185), unconjugated carbonyls (δ 200−210), and oxygenated carbons (δ 82−92 ppm) characteristic of caged moieties were observed in the 13C NMR spectrum (Figure S3, Supporting Information).13,20 These data indicated the likely presence of caged prenylxanthones. The CHCl3 extract was purified by successive column chromatography using silica gel, Sephadex LH-20, and semipreparative HPLC, which permitted the isolation of seven new (1−7) and eight known (8−15) caged xanthones. Compounds 1−7 were obtained as scalemic mixtures, of which 1−5 were resolved by chiral-phase HPLC. Compound 1 was isolated as yellow needles. The HRESIMS data of this compound exhibited a sodium adduct ion [M + Na]+ at m/z 487.2092 (calcd for 487.2091) corresponding to a 750

DOI: 10.1021/acs.jnatprod.7b00454 J. Nat. Prod. 2018, 81, 749−757

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Table 1. 1H NMR Data (600 MHz, CDCl3) of Compounds 1−7 (δH, mult., J in Hz) no.

1

2

3

4

5

6

7

2 7 8 8a 12

6.01, s 3.49, dd (7.0, 4.8) 7.47, d (7.0)

6.09, s 3.51, dd (7.0, 4.8) 7.49, d (7.0)

6.07, s 3.50, dd (7.0, 4.8) 7.49, d (7.0)

4.53, q (6.6)

4.49, dd (8.0, 3.1)

4.40, dd (8.1, 3.3)

13a 13b 14 15 16a 16b 17 19 20 21a 21b

1.29, d (6.6) 1.42, s 1.50, s 2.34, dd (13.4, 4.8) 1.37, m 2.53, d (9.3) 1.27, s 1.73, s 2.64, d (13.9) 2.55,dd (13.9, 7.9)

3.83, dd (11.8, 3.1) 3.75, dd (11.8, 8.0) 1.60, s 1.48, s 2.34, dd (13.4, 4.8) 1.38, m 2.55, d (8.0) 1.27, s 1.73, s 2.64, d (13.9) 2.53, dd (13.9, 8.0)

6.08, s 2.82, t (5.4) 4.45, d (3.5) 3.32, brs 6.25, dd (17.8, 10.6) 5.34, d (17.8) 5.30, d (10.6) 1.67, s 1.62, s 1.96, dd (14.6, 4.8) 1.38, dd (14.6, 8.6) 2.53, d (8.6) 1.13, s 1.39, s 2.87, dd (14.8, 5.0) 2.71, dd (14.8, 9.1)

6.07, s 2.91, t (4.8) 4.41, d (4.1) 3.16, brs 6.22, dd (17.8, 10.6) 5.35, d (17.8) 5.30, d (10.6) 1.58, s 1.55, s 1.97, dd (14.4, 5.9) 1.42, m 2.57, d (8.4) 1.17, s 1.44, s 6.18, d (16.0)

5.95, s 2.95, t (4.1) 4.28, brs 3.06, brs 6.46, dd (17.8, 10.6) 5.45, d (17.8) 5.35, d (10.6) 1.61, s 1.54, s 2.43, dd (15.2, 6.4) 2.00, dd (14.6, 8.6) 2.60, m 1.28, s 1.26, s 2.34, d (7.9)

4.35, t (7.9) 1.06, s 1.37, s

4.35, d (9.7) 1.05, s 1.37, s

4.01, dd (10.9, 8.1) 3.92, dd (10.9, 3.3) 1.70, s 1.27, s 2.34, dd (13.4, 4.8) 1.37, m 2.52, d (9.2) 1.25, s 1.72, s 2.66, d (13.9) 2.50, dd (13.9, 10.5) 4.37, d (10.5) 1.08, s 1.37, s

6.09, s 2.82, t (5.7) 4.35, d (4.3) 3.32, brs 6.25, dd (17.8, 10.6) 5.36, d (17.8) 5.31, d (10.6) 1.67, s 1.62, s 1.97, dd (14.6, 6.2) 1.38, dd (14.6, 8.6) 2.54, d (8.6) 1.13, s 1.39, s 2.88, dd (14.6, 5.7) 2.70, dd (14.6, 8.9) 5.31, m 1.62, s 1.67, s 3.30, s

5.71, d (16.0) 1.33, s 1.34, s 3.34, s

5.02, t (7.9) 1.63, s 1.73, s 3.44, s

13.14, s

13.13, s

13.08, s

5.35, m 1.63, s 1.67, s 3.59, m; 3.46, m 1.16, t (14.0) 12.10, s 6.93, brs

11.94, s 6.94, brs

11.86, s 7.39, brs

22 24 25 26 27 1-OH 3-OH

12.08, s 6.92, brs

Table 2. 13C NMR Data (150 MHz) of Compounds 1−7 (δC, CDCl3) no.

1

2

3

4

5

6

7

1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

166.5 92.8 168.5 112.6 156.7 85.0 203.9 47.3 134.3 135.3 179.0 101.5 91.1 43.6 91.8 16.5 28.4 20.2 26.1 49.7 83.1 29.2 31.1 29.1 117.7 133.9 17.0 25.7

166.6 92.9 167.8 112.5 156.3 84.9 203.7 47.3 134.5 135.3 179.2 101.6 91.1 42.9 95.1 62.4 29.7 19.4 26.1 49.6 83.1 29.2 31.0 29.1 117.8 133.7 16.9 25.6

166.4 93.0 167.9 113.1 156.1 84.8 203.7 47.2 134.5 135.4 179.2 101.7 91.1 42.9 94.7 61.4 25.0 21.3 26.1 49.5 83.1 29.2 30.9 29.1 117.7 133.7 17.0 25.7

162.7 99.5 165.6 111.6 158.2 86.9 209.3 44.9 74.8 47.4 194.8 103.3 88.9 41.6 149.4 113.6 30.5 27.5 20.4 44.1 81.2 27.6 30.7 28.3 118.0 134.1 18.2 26.2 55.9

162.6 99.5 165.7 111.5 158.2 86.9 209.4 44.9 73.2 48.2 194.8 103.2 88.9 41.6 149.4 113.5 30.5 27.5 20.5 44.1 81.2 27.6 30.7 28.3 118.0 133.8 18.2 26.1 63.9 15.3

162.5 99.4 165.7 111.4 158.1 88.2 208.6 44.4 74.1 47.6 194.3 102.4 89.6 41.2 149.5 113.6 29.8 27.0 20.1 43.4 82.2 27.5 30.2 28.3 144.8 117.5 29.5 29.5 56.1

163.4 99.3 165.8 111.5 159.9 206.3 83.2 45.0 75.6 54.9 192.8 102.5 81.7 41.4 150.0 113.4 28.3 26.2 28.3 41.8 84.0 25.8 29.9 30.4 116.7 136.6 18.4 26.1 56.9

(+)-1 were assigned as (5R,7S,10aS,12R,17S) (1a) and (5S,7R,10aR,12S,17R) (1b), respectively. The absolute configuration of the caged skeleton of (−)-1 assigned by ECD analysis is also consistent with that of oliganthone,25 the absolute configuration of which was unambiguously determined based on the experimental and computed ECD spectra. Consequently, the structures of compounds (−)-1 and (+)-1 were defined as shown, and the compounds were named (−)-epiisobractatin and (+)-epiisobractatin, respectively. Compound 2 was obtained as a yellow, amorphous powder and had a molecular formula of C28H32O7, as established by HRESIMS ([M + H]+ at m/z 481.2210), which is 16 mass units higher than that of 1, indicating the presence of an additional oxygen atom in 2. The UV and IR spectra of this compound resembled those of 1. The 1H and 13C NMR spectra of 2 (Tables 1 and 2) were also similar to those of 1; however, one methyl resonance (Me-13) in the spectrum of 1 was replaced by hydroxymethyl signals (δH 3.83 and 3.75; δC 62.4) in the spectrum of 2, which was consistent with the result of the HRESIMS analysis. The C-12 location of the hydroxymethyl group was based on the HMBC correlations of H2-13 with C11 and C-12. The attachment of other substituents was further confirmed via a detailed HMBC analysis (Figure S25, Supporting Information) and was identical to those of 1. The relative configuration of 2 was assigned to be similar to that of 1 by analyzing the NOESY data (Figure S26, Supporting Information). The key NOESY correlations of H2-13/H3-15, H3-15/Ha-21, H-12/H3-14, and H3-14/H3-19 showed that the C-5 prenyl group and the C-13 hydroxymethyl group were cofacial. The α-orientation of H-12 of 2 was ascertained by the similar δH values but distinctly different δC values (ΔδC ca. +10 ppm) of the gem-dimethyl moiety in the dihydrofuran unit.10,11 The enantiomers of compound 2 were resolved by chiral-phase HPLC on an IC column to yield the corresponding enantiomers (−)-2 ([α]25 D −124.3, c 0.1, CHCl3) and (+)-2 751

DOI: 10.1021/acs.jnatprod.7b00454 J. Nat. Prod. 2018, 81, 749−757

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Figure 1. (a) Key HMBC correlations, (b) key NOESY correlations, and (c) experimental ECD spectra of compounds (−)-1 and (+)-1 and the calculated ECD spectra of 1a and 1b using the TDDFT method at the B3LYP/6-31+G(d,p) level.

Compound 3 was assigned the same molecular formula as 2, i.e., C28H32O7, by HRESIMS data. The IR and UV spectra resembled those of 2. In addition, the 1H and 13C NMR data (Tables 1 and 2) of these compounds were also similar except in the chemical shifts of the gem-dimethyl substituents of the dihydrofuran moiety. Differences were observed in the C-14 and C-15 signals of the dihydrofuran units in the 13C NMR spectrum and in the H-12, Me-14, and Me-15 proton signals in the 1H NMR spectrum, suggesting that the two products are a pair of C-12 epimers (Figure S37, Supporting Information). The linkages of the other substituents were identical to those in 2, as determined by HMBC analysis (Figures S34 and S35, Supporting Information). The key NOESY correlations (Figure S36, Supporting Information) of H-12/H3-15, H3-15/Ha-21, H2-13/H3-14, and H3-14/H3-19 indicated that H-12 was βoriented, which was further confirmed by the similar δC values (ΔδC ca. +3 ppm) of the C-18 gem-dimethyl groups.10,11 The scalemic mixture of 3 was further analyzed and resolved by chiral-phase HPLC on an IC column to yield the corresponding enantiomers (−)-3 ([α]25 D −202.5, c 0.1, CHCl3) and (+)-3 +198.7, c 0.1, CHCl ([α]25 D 3) in a ratio of ca. 1.3:1 (Figure S69, Supporting Information). The absolute configurations of (−)-3 (5R,7S,10aS,12S,17S) and (+)-3 (5S,7R,10aR,12R,17R) were unambiguously assigned by comparing the ECD spectra of these compounds with those of compounds (−)-2 and (+)-2, respectively. Consequently, the structures of compounds (−)-3 and (+)-3 were determined to be (−)- and (+)-13hydroxyepiisobractatin, respectively.

Figure 2. ORTEP drawing of compound 1 based on single X-ray crystallographic analysis.

([α]25 D +118.8, c 0.1, CHCl3) in a ratio of ca. 1.2:1 (Figure S68, Supporting Information). The absolute configurations of (−)-2 and (+)-2 were established by comparing their ECD spectra to those of compounds (−)-1 and (+)-1. The similarity of the ECD spectrum of compound (+)-2 with that of (+)-1 and of compound (−)-2 with that of (−)-1 indicated an absolute configuration of (5S,7R,10aR,12S,17R) for (+)-2 and (5R,7S,10aS,12R,17S) for (−)-2. Therefore, compounds (−)-2 and (+)-2 were assigned as (−)- and (+)-13hydroxyisobractatin, respectively. 752

DOI: 10.1021/acs.jnatprod.7b00454 J. Nat. Prod. 2018, 81, 749−757

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Figure 3. (a) Key HMBC correlations, (b) key NOESY correlations, and (c) experimental ECD spectra of compounds (−)-4 and (+)-4 and the calculated ECD spectra of 4a and 4b using the TDDFT method at the B3LYP/6-31+G(d,p) level. 25 ([α]25 D +79.7, c 0.1, CHCl3) and (−)-4 ([α]D −95.5, c 0.1, CHCl3) were isolated in a ratio of ca. 1:1.1 (Figure S70, Supporting Information). The absolute configurations of the caged units of (−)-4 and (+)-4 were defined as (5R,7S,8S,8aR,10aS,17S) (4a) and (5S,7R,8R,8aS,10aR,17R) (4b), respectively, by comparing the experimental and calculated ECD spectra using the Gaussian 09 program (Figure 3c). Compounds (−)-4 and (+)-4 also exhibited similar ECD spectra to (−)- and (+)-doitunggarcinone,21 respectively, revealing that these compounds have the same absolute configuration. Therefore, compounds (−)-4 and (+)-4 were defined as (−)- and (+)-8-methoxy-8,8a-dihydrobractatin, respectively. Based on the HRESIMS data, compound 5 was shown to have a molecular formula of C30H38O7, which is 14 mass units higher than that of 4, suggesting the presence of an additional methylene group in 5. Compound 5 showed similar IR and UV data to 4, indicating that it is also a caged xanthone derivative. The 1H and 13C NMR data (Tables 1 and 2) of these compounds were also similar except for the fact that a methoxy group at C-8 in 4 was replaced by an ethoxy group (δH 3.46, 3.59) in 5, which was consistent with the molecular formula of 5. The relative configuration of 5 was assigned to be the same as that of 4 by comparing their NMR data and NOESY correlations. Finally, X-ray crystallographic analysis confirmed the unambiguous assignment of the 2D structure and relative configuration of 5 (Figure 4). The scalemic mixture was resolved via chiral-phase HPLC, affording two enantiomers, 25 (+)-5 ([α]25 D +76.1, c 0.1, CHCl3) and (−)-5 ([α]D −98.8, c 0.1, CHCl3), in a ratio of ca. 1:1.4 (Figure S71, Supporting Information). The absolute configurations of (−)-5 and (+)-5 were established as (5R,7S,8S,8aR,10aS,17S) (5a) and

Compound 4 was obtained as colorless needles, and the molecular formula was determined to be C29H36O7 based on a positive HRESIMS ion peak. The UV spectrum showed absorption maxima at 221 and 298 nm that were indicative of a caged xanthone derivative lacking a Δ8(8a) double bond.10,13 The IR spectrum showed absorption bands of a hydrogenbonded hydroxy (3376 cm−1), an unconjugated carbonyl (1736 cm−1), and a hydrogen-bonded carbonyl group (1633 cm−1). The 1H−1H COSY spectrum displayed a continuous spin system comprising H-8a, H-8, H-7, H2-16, and H-17, which indicated a CHCHCHCH2CH structure unit (Figure 3a). The NMR data of 4 (Tables 1 and 2) were similar to those of 1-Omethyl-8-methoxy-8,8a-dihydrobractatin (9)13 except for the presence of a hydrogen-bonded hydroxyl resonance at δH 12.09 in 4 instead of the methoxy singlet observed in the spectrum of 9. The HMBC cross-peaks (Figure 3a) of the hydroxy group at δH 12.09 with the carbon resonances at δC 126.7 (C-1), 103.3 (C-9a), and 99.5 (C-2) confirmed that the methoxy group in 9 replaced the hydrogen-bonded hydroxy substituent in 4. The methoxy group at δH 3.30 was assigned to C-8 due to the HMBC cross-peak between the methoxy protons and C-8 (δC 74.8). The C ring of 4 adopted a chair conformation. Thus, due to the absence of the anisotropic shielding zone of the A ring, the olefinic proton signal of H-22 was deshielded from δH 4.39 to δH 5.31, which is characteristic of a caged xanthone lacking the Δ8(8a) double bond.13 The trans relationship between H-8 and H-8a was identified by the cross-peaks of H-8/Ha-16 and H-8a/H2-21 in the NOESY spectrum (Figure 3b). The 8,8atrans configuration was assigned based on the appearance of H8a as a broad singlet.12,13 The scalemic mixture of 4 was resolved by HPLC on a chiralphase IC column, and the corresponding enantiomers (+)-4 753

DOI: 10.1021/acs.jnatprod.7b00454 J. Nat. Prod. 2018, 81, 749−757

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bond was assigned as E based on the coupling constant (JH‑21/H‑22 = 16.0 Hz). The 8,8a-trans configuration was based on the appearance of H-8a as a broad singlet12,13 and the key NOESY correlations of H-8/Ha-16, H-8a/H2-21, and H-8a/ CH3O-8, which were identical to those of 4. The closely comparable NOESY profiles of 4 and 6 indicated the same relative configurations of these compounds. Compound 6, with a specific rotation of [α]25 D −27 (c 0.1, MeOH), was analyzed by chiral-phase HPLC, showing the presence of two well-resolved peaks of the two enantiomers in a ratio of ca. 1.1:1 (Figure S72, Supporting Information). However, because of an insufficient amount of material, the enantiomers of compound 6 could not be further resolved. Thus, the structure of garcibracteatone was defined as shown. Compound 7, with a molecular formula of C29H36O7 deduced by the HRESIMS data, displayed IR and UV absorption bands similar to those of 4. The 1H and 13C NMR data indicated significant difference in the H2-16 protons (δH 2.43 and 2.00) and three oxygenated tertiary carbons (δC 84.0, 83.2, and 81.7) in the caged scaffold compared with those of 4, suggesting that 7 might belong to the type of isomerized caged xanthones with a carbonyl group at C-5.13−15 Comparison of the 1H NMR spectroscopic data with those of neobractiatin (10)10,15 indicated that these compounds are of similar structure except that a deshielded olefinic proton signal (δH 7.15) in 10 replaced two methine proton signals (δH 4.28, 3.06) and a methoxy signal (δH 3.44). The 1H−1H COSY spectrum showed the presence of a spin system comprising H8a, H-8, H-7, H-17, and H2-16. These features further suggested that 7 had the same type of caged skeleton as neobractiatin (10) but lacked the Δ8(8a) double bond. The locations of other substituents were also identical to 10, as determined by HMBC data analysis. The relative configuration of 7 was determined by NOESY data analysis. The observed key NOESY correlations of H-8/ Ha-16, H-8a/H2-21, and H-8a/CH3O-8 as well as the broad singlet of H-8a12,13 revealed that H-8 and H-8a adopted β- and α-orientations, respectively. Compound 7 was shown to be a 1:1.1 mixture of enantiomers by chiral-phase HPLC analysis but was not resolved due to the small amount of compound remaining (Figure S73, Supporting Information). Hence, the structure of 8-methoxy-8,8a-dihydroneobractiatin (7) was established as shown. By comparing the physicochemical and spectroscopic properties with reported data, the structures of the eight known caged xanthones were elucidated as isobractatin (8),13 1-O-methyl-8-methoxy-8,8a-dihydrobractatin (9),13 neobractatin (10),15 3-O-methyoneobractatin (11),15 bractatin (12),13 3O-methyobractatin (13),15 neoisobractatin A (14),14 and neoisobractatin B (15).14 The 2D structure and relative configuration of 13 was further verified by X-ray diffraction analysis (Figure S5, Supporting Information). To date, the novel type of naturally occurring caged xanthones, possessing an isomerized caged skeleton with a C5 carbonyl group, has exclusively been found in G. bracteata. Compounds 7, 10, 11, 14, and 15 belong to this type of caged xanthones, of which compound 7 is a new member of rearranged caged xanthones and the first representative lacking the Δ8(8a) double bond. The mechamisms that account for the formation of the regular and novel caged motifs have been evaluated.26 Notably, xanthones with the regular caged motif are the major constituents, while those with the novel scaffold are observed as minor products when the A ring is electron rich

Figure 4. ORTEP drawing of compound 5 based on single X-ray crystallographic analysis.

(5S,7R,8R,8aS,10aR,17R) (5b), respectively, by comparing the experimental and calculated ECD spectra (Figure 5).

Figure 5. Experimental ECD spectra of compounds (−)-5 and (+)-5 and the calculated ECD spectra of 5a and 5b using the TDDFT method at the B3LYP/6-31+G(d,p) level.

Compounds (−)-5 and (+)-5 also exhibited similar ECD spectra to compounds (−)-4 and (+)-4, respectively, implying that these compounds have the same absolute configurations (Figure S86, Supporting Information). Therefore, the structures of compounds (−)-5 and (+)-5 were defined and named (−)- and (+)-8-ethoxy-8,8a-dihydrobractatin, respectively. Compound 6 had a molecular formula of C29H36O8, as deduced by HRESIMS, which is 16 mass units higher than that of 4, indicating the presence of an additional oxygen atom in 6. The UV spectrum resembled that of 4.10 The 1H and 13C NMR data (Tables 1 and 2) were also similar to those of 4 except that signals for a 3-hydroxy-3-methylbut-1-enyl moiety [δH 6.18 (1H, d, J = 16.0 Hz, H-21), 5.71 (1H, d, J = 16.0 Hz, H-22), 1.34 (3H, s, Me-24), and 1.33 (3H, s, Me-25) ] in 6 replaced those of the 3-methylbut-2-enyl group at C-5 in 4, which was consistent with the additional oxygen atom in the molecular formula of 6. The linkage of a 3-hydroxy-3-methybut-1-enyl group at C-5 was further ascertained by the HMBC cross-peaks of H-21 (δH 6.18) with C-5 (δC 88.2), C-6 (δC 208.4), and C10a (δC 89.6) and of H-22 (δH 5.71) with C-5 (δC 88.2). Additionally, the key HMBC correlation between the methoxy protons at δH 3.34 and C-8 (δC 74.1) established the location of the methoxy group at C-8. The geometry of the Δ21(22) double 754

DOI: 10.1021/acs.jnatprod.7b00454 J. Nat. Prod. 2018, 81, 749−757

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Table 3. GI50 Values of Compounds 1−15, Which Inhibited Growth of HL-60 and K562 Cellsa GI50 ± SD (μM)

GI50 ± SD (μM)

compound

HL-60

K562

compound

HL-60

K562

1 2 3 4 5 6 7 8 9

1.2 ± 0.1 1.1 ± 0.2 1.1 ± 0.3 7.3 ± 1.0 4.5 ± 0.3 8.8 ± 0.1 6.5 ± 0.03 1.9 ± 0.2 3.4 ± 0.04

2.1 ± 0.3 2.1 ± 0.1 2.1 ± 0.2 3.4 ± 0.5 2.1 ± 0.1 3.8 ± 0.2 3.5 ± 0.1 3.6 ± 0.2 1.6 ± 0.02

10 11 12 13 14 15 gambogic acid doxorubicin hydrochloride

0.8 ± 0.2 1.0 ± 0.04 0.4 ± 0.02 0.2 ± 0.02 0.9 ± 0.04 0.7 ± 0.1 0.3 ± 0.03 0.02 ± 0.002

1.3 ± 0.1 1.5 ± 0.2 1.0 ± 0.03 0.8 ± 0.02 1.6 ± 0.1 1.7 ± 0.1 0.6 ± 0.03 0.02 ± 0.001

a HL-60 and K562 cells were treated for 3 days; GI50 is the concentration that inhibited 50% of cell growth. Total cell numbers were counted. The cell growth inhibition in the treated cells was compared with control cells. The data shown are means ± SD of three independent experiments.

recorded on a Shimadzu UV-2201 spectrophotometer. The IR spectra were obtained on a Bruker IFS-55 spectrometer with KBr discs. HRESIMS data were acquired on a Bruker microTOFQ-Q mass spectrometer. NMR (1H NMR, 13C NMR, COSY, HSQC, HMBC, and NOESY) spectra were acquired on a Bruker-AV-600 NMR spectrometer. Silica gel was purchased from Qingdao Ocean Chemical Factory (Qingdao, China) and Sephadex LH-20 from GE Healthcare (Sweden). Analytical and semipreparative HPLC was conducted on a Shimadzu SPD-20A with a DAD detector and an LC-6AD series pump equipped with a C18 column (4.6 × 250 mm, 5 μm; Eclipse Plus C18, Agilent Co. Ltd., or 20 mm × 250 mm, 5 μm; YMC Co. Ltd.). LC-MS was performed on an Agilent 1260−6210 single-quadrupole spectrometer equipped with a C18 column (4.6 × 100 mm, 1.8 μm; Eclipse Plus C18, Agilent Co. Ltd.) by using ESIMS and a DAD detector. Chiral-phase analytical HPLC was performed with a CHIRALPAK IC column (4.6 × 250 mm, 5 μm, Daicel Co. Ltd., P. R. China), equipped with a Shimadzu SPD-M20A DAD monitor and an LC-20AB series pump. Plant Material. Dried leaves of G. bracteata were collected in October 2010 from Xishuangbanna Tropical Botanical Garden of Yunnan Province, People’s Republic of China. The plant was authenticated by Mr. Jingyun Cui (Xishuangbanna Tropical Botanical Garden). A voucher specimen (GB-20101008) was deposited at Shenyang Pharmaceutical University. Extraction and Isolation. Air-dried leaves of G. bracteata (15.5 kg) were extracted with 95% EtOH (3 × 30 L) three times (2 h, 2 h, 1 h) under reflux. The crude EtOH extract was evaporated under vacuum to yield a brown-yellow crude gum (1.2 kg). The crude extract was then suspended in water (5 L) and extracted successively with petroleum ether (3 × 5 L), CHCl3 (3 × 5 L), and n-BuOH (3 × 5 L). The CHCl3 extract (300 g) was purified by CC over silica gel using solvent mixtures of increasing polarity: petroleum ether (60−90 °C)− acetone (100:0−0:100, v/v). The fractions were detected by TLC under UV light at 254 nm and combined to afford 10 major fractions (Fr. A−Fr. J). Fr. A (petroleum ether−acetone, 100:3, 2.5 g) was separated via semipreparative HPLC using MeCN−H2O (55:45) as the mobile phase to afford 2 (4.8 mg, tR = 18 min), 3 (6.7 mg, tR = 20 min), 14 (5.2 mg, tR = 31 min), and 15 (4.1 mg, tR = 33 min). Fr. B (petroleum ether−acetone, 100:3, 1.2 g) was repeatedly recrystallized using MeOH to afford 11 (30.5 mg). Fr. C (petroleum ether−acetone, 100:3, 1.5 g) was fractionated by preparative TLC using petroleum ether−acetone (2:1) and further subjected to Sephadex LH-20 CC (MeOH) to obtain 13 (20.2 mg). Fr. D (petroleum ether−acetone, 100:4, 2.2 g) was isolated by semipreparative HPLC (MeCN− H2O,75:25) to give 1 (10.3 mg, tR = 17 min) and 8 (30.5 mg, tR = 16 min). Fr. E (petroleum ether−acetone, 100:5, 1.1 g) was separated by semipreparative HPLC (MeCN−H2O, 60:40) to yield 7 (4.3 mg, tR = 12 min). Fr. F (petroleum ether−acetone, 100:7, 1.2 g) was purified by semipreparative HPLC (MeOH−H2O, 70:30) to afford 4 (8.1 mg, tR = 21 min), 5 (8.6 mg, tR = 24 min), and 6 (4.5 mg, tR = 15 min). Fr. G (petroleum ether−acetone, 100:5, 1.8 g) was separated by preparative TLC using petroleum ether−acetone (2:1) and then repeatedly

owing to the site-selectivity of the tandem Claisen/Diels−Alder rearrangement. The quantitative ratio of the regular caged scaffolds to the corresponding novel caged scaffolds that have been isolated from G. bracteata is approximately 2:1 (e.g., 1 versus 15, 12 versus 10, and 4 versus 7), which is consistent with reported results.26 Notably, compounds 4, 5, 6, 7, and 9 may be MeOH- or EtOH-addition products of enone precursor, e.g., 12. To determine whether these compounds were artifacts, a simple and rapid determination of the chemical composition of the CHCl3 extract of G. bracteata was successfully accomplished via LC-MS analysis. Acetonitrile and CHCl3 were used as solvents or mobile phases instead of MeOH or EtOH. The results indicated that the 8-O-methyl or 8-O-ethoxy products are indeed naturally occurring (Figures S80−S84, Supporting Information). The compounds were tested for cell-growth inhibition of the human leukemic HL-60 and K562 cell lines with doxorubicin hydrochloride and gambogic acid as positive controls (GI50 values are listed in Table 3). All compounds displayed antiproliferative activity against the two cancer cell lines, with GI50 values ranging from 0.2 to 8.8 μM. Of these compounds, 13 was the most effective in inhibiting the two cancer cell lines, with GI50 values of 0.2 and 0.8 μM. Compound 13 exhibited more potent inhibition of the HL-60 cell line than gambogic acid, a potential antitumor molecule. A preliminary structure−cytotoxicity study of compounds 1− 15 showed that the Δ8,8a double bond plays a significant role in the mediation of the cytotoxicity of these caged xanthones,27,28 and the novel type of caged xanthones, possessing an isomerized caged skeleton with a C-5 carbonyl group and comprising a Δ8,8a double bond, such as compounds 10, 11, 14, and 15, showed more potent activity than other caged xanthone compounds. In addition, when the prenyl group at C-4 was cyclized to a dihydrofuran moiety, the inhibitory activity decreased, revealing that the hydrophobic unsaturated acyclic prenyl group at C-4 may be crucial for antitumor activity. The C-12 epimers in compounds with dihydrofuran rings (2 versus 3, 1 versus 8, and 14 versus 15) did not influence the growthinhibitory activity.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points (°C) were determined on an electrothermal melting point apparatus without correction (ShenGuang WRS-1B). Optical rotations were measured on a JASCO DIP-370 polarimeter. ECD spectra were collected by a Biologic MOS 450 spectrometer. The UV absorption spectra were 755

DOI: 10.1021/acs.jnatprod.7b00454 J. Nat. Prod. 2018, 81, 749−757

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(+)-8-Ethoxy-8,8a-dihydrobractatin (5b): tR 52 min, 2.4 mg, [α]25 D +76.1 (c 0.17, CHCl3); ECD (MeOH) λmax (Δε) 228 (−19.2), 312 (+11.9), 351 (−0.7) nm. Garcibracteatone (6): white, amorphous powder (MeOH); [α]25 D −27 (c 0.11, CHCl3); UV (MeOH) λmax (log ε) 218 (4.26), 296 (4.13), 338 (3.62) nm; IR (KBr) νmax 3378, 1739, 1635, 1583 cm−1; 1 H and 13C NMR, see Tables 1 and 2; ESIMS m/z 535 [M + Na]+, 511 [M − H]−; HRESIMS m/z 511.2340 [M − H]− (calcd for C29H35O8, 511.2337). 8-Methoxy-8,8a-dihydroneobractatin (7): white, amorphous powder (MeOH); [α]25 D −16 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 222 (4.25), 288 (4.12), 330 (3.65) nm; IR (KBr) νmax 3423, 1752, 1635, 1589 cm−1; 1H and 13C NMR, see Tables 1 and 2; ESIMS m/z 519 [M + Na]+, 495 [M − H]−; HRESIMS m/z 495.2390 [M − H]− (calcd for C29H35O7, 495.2388). X-ray Crystallographic Analysis of Compound 1. Yellow, needle-shaped crystals of 1 were obtained from a MeOH solution subjected to slow evaporation in a refrigerator for 1 week. Singlecrystal X-ray diffraction data were collected using a Bruker SMART APEX-II CCD diffractometer equipped with a multilayer monochromator and Mo Kα radiation; C28H32O6, Mw = 464.54; monoclinic, space group P21/c, a = 10.105(5) Å, b = 11.810(5) Å, c = 20.030(5) Å, α = 90.00(5)°, β = 90.00(5)°, γ = 90.00(5)°; V = 2390.4(17) Å3, Z = 4, dc = 1.291 g/m3, F(000) = 992.0, λ(Mo Kα) = 0.71 mm−1, μ = 0.090 mm−1, and T = 293(2) K. The total number of reflections measured were 12 115 (−11 ≤ h ≤ 11, −12 ≤ k ≤ 14, −23 ≤ l ≤ 22), leading to 4222 unique reflections (I ≥ 2σ(I) Rint = 0.054) (all data). Final indices: R1 = 0.046 and wR2 = 0.0975, goodness-of-fit S = 0.999. X-ray Crystallographic Analysis of Compound 5. Colorless, needle-shaped crystals of 5 were obtained from a MeOH solution subjected to slow evaporation in a refrigerator for 1 week. Singlecrystal X-ray diffraction data were collected by a Bruker SMART APEX-II CCD diffractometer equipped with a multilayer-monochromator and Mo Kα radiation. C30H38O7, Mw = 510.60; monoclinic, space group P21/c, a = 14.317(5) Å, b = 8.341(5) Å, c = 22.569(5) Å, α = 90.00(5)°, β = 98.28(5)°, γ = 90.00(5)°; V = 2667.1(19) Å3, Z = 4, dc = 1.272 g/m3, F(000) = 1096.0, λ(Mo Kα) = 0.71 Å mm−1, μ = 0.089 mm−1, and T = 293(2) K. The total number of reflections measured were 9918 (−15 ≤ h ≤ 14, −8 ≤ k ≤ 8, −23 ≤ l ≤ 20), leading to 3310 unique reflections (I ≥ 2σ(I), Rint = 0.1127) (all data). Final indices: R1 = 0.0594 and wR2 = 0.1269, goodness-of-fit S = 0.962. Crystallographic data for compounds 1 and 5 have been submitted to the Cambridge Crystallographic Data Center as supplementary publication no. CCDC 1545287 and 1545286. Copies of the data are available online free of charge at http://www.ccdc.cam.ac.uk (or from CCDC, 12 Union Road, Cambridge CB2 1EZ, e-mail: deposit@ccdc. cam.ac.uk, fax: +44-1223-336033). Computational Methods. The geometries of 1 and 5 determined by X-ray data analysis were subjected to geometry optimization by TDDFT theory at the B3LYP/6-31G** level in the Gaussian 09 program package.29 TDDFT ECD calculations for optimized conformers were carried out at the B3LYP/6-31+G** level with a CPCM model in MeOH solvent, and the calculated ECD spectra were generated using SpecDis 1.51.30 The calculated ECD curve of 4 was generated by Boltzmann weighting of the selected low-energy conformers using SpecDis 1.51.30 Cell Growth Inhibition Assays. The cell growth inhibitory activities of the caged xanthones were evaluated by the trypan blue method using the human leukemia HL-60 and K-562 cell lines. Detailed methodology for the cell growth inhibition test was described in a previous paper,31 with doxorubicin hydrochloride used as a positive control. Three independent repeat experiments were performed.

recrystallized from MeOH to obtain 9 (4.2 mg) and 10 (60.8 mg). Fr. H (petroleum ether−acetone, 100:7, 1.7 g) was fractionated in the same way as Fr. G to afford 12 (120.7 mg). Chiral-Phase HPLC Separation of Scalemic 1−5 and ChiralPhase HPLC Analysis of 6 and 7. Scalemic mixtures of 1 (8.5 mg), 2 (3.9 mg), 3 (4.1 mg), 4 (6.5 mg), and 5 (6.6 mg) were subjected to HPLC on a chiral-phase column (CHIRALPAK IC column, 4.6 × 250 mm, 5 μm) to yield pure enantiomers. The mobile phase was nhexane−2-propanol in the ratio 75:25 for 1, 92:8 for 2 and 3, 85:15 for 4, and 90:10 for 5, with a flow rate of 0.5 mL/min. Compounds 6 and 7 were analyzed by analytical chiral-phase HPLC using the conditions described for 1 to yield two peaks for the enantiomers at retention times of tR 35 and 40 min in an approximate ratio of 1.1:1 for 6 (nhexane−2-propanol, 98:2) and at retention times of tR 19 and 20 min in an approximate ratio of 1.1:1 for 7 (n-hexane−2-propanol, 75:25). Owing to the small amount of material remaining, compounds 6 and 7 could not be chirally resolved. Epiisobractatin (1): yellow needles (CHCl3), mp 180−185 °C; UV (MeOH) λmax (log ε) 218 (4.58), 355 (4.23) nm; IR (KBr) νmax 3445, 2969, 2925, 1739, 1637, 1587, 1464, cm−1; 1H and 13C NMR, see Tables 1 and 2; ESIMS m/z 465 [M + H]+, 487 [M + Na]+; HRESIMS m/z 487.2092 [M + Na]+ (calcd for C28H32O6Na, 487.2091). (−)-Epiisobractatin (1a): tR 15 min, 3.5 mg, [α]25 D −161.8 (c 0.15, CHCl3); ECD (MeOH) λmax (Δε) 210 (+11.1), 281 (+7.0), 307 (+6.5), 352 (−18.4) nm. (+)-Epiisobractatin (1b): tR 18 min, 3.2 mg, [α]25 D +145.3 (c 0.16, CHCl3); ECD (MeOH) λmax (Δε) 225 (+9.6), 283 (−8.1), 306 (−6.9), 352 (+18.7) nm. 13-Hydroxyisobractatin (2): yellow, amorphous powder (CHCl3); UV (MeOH) λmax (log ε) 212 (4.62), 354 (4.16) nm; IR (KBr) νmax 3443, 2966, 2925, 1739, 1635, 1590, 1469 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 481.2210 [M + H]+ (calcd for C28H33O7, 481.2221). (−)-13-Hydroxyisobractatin (2a): tR 12 min, 1.8 mg, [α]25 D −124.3 (c 0.1, CHCl3); ECD (MeOH) λmax (Δε) 210 (+10.8), 222 (−6.4), 285 (+8.5), 310 (+8.3), 354 (−19.8) nm. (+)-13-Hydroxyisobractatin (2b): tR 15 min, 1.3 mg, [α]25 D +118.8 (c 0.1, CHCl3); ECD (MeOH) λmax (Δε) 225 (+13.9), 283 (−7.7), 306 (−6.9), 351 (+19.5) nm. 13-Hydroxyepiisobractatin (3): yellow, amorphous powder (CHCl3); UV (MeOH) λmax (log ε) 214 (4.59), 354 (4.15) nm; IR (KBr) νmax 3494, 2968, 2929, 1742, 1633, 1592, 1475 cm−1; 1H and 13 C NMR, see Tables 1 and 2; HRESIMS m/z 481.2222 [M + H]+ (calcd for C28H33O7, 481.2221). (−)-13-Hydroxyepiisobractatin (3a): tR 15 min, 2.1 mg, [α]D25 −202.5 (c 0.18, CHCl3); ECD (MeOH) λmax (Δε) 224 (−7.9), 282 (+7.7), 308 (+6.5), 354 (−19.2) nm. (+)-13-Hydroxyepiisobractatin (3b): tR 17 min, 1.6 mg, [α]25 D +198.7 (c 0.12, CHCl3); ECD (MeOH) λmax (Δε) 209 (−14.7), 282 (−6.0), 313 (−5.6), 354 (+18.2) nm. 8-Methoxy-8,8a-dihydrobractatin (4): colorless needles (MeOH), mp 212−216 °C; UV (MeOH) λmax (log ε) 221 (4.18), 298 (4.10) nm; IR (KBr) νmax 3376, 2933, 1736, 1633, 1413 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 497.2514 [M + H]+ (calcd for C29H37O7, 497.2534). (−)-8-Methoxy-8,8a-dihydrobractatin (4a): tR 13 min, 2.9 mg, [α]25 D −95.5 (c 0.14, CHCl3); ECD (MeOH) λmax (Δε) 239 (+4.98), 305 (−9.6) nm. (+)-8-Methoxy-8,8a-dihydrobractatin (4b): tR 11 min, 2.6 mg, [α]25 D +79.7 (c 0.15, MeOH); ECD (MeOH) λmax (Δε) 223 (−9.1), 307 (+9.3) nm. 8-Ethoxy-8,8a-dihydrobractatin (5): colorless needles (MeOH), mp 215−219 °C; UV (MeOH) λmax (log ε) 220 (4.24), 297 (4.15) nm; IR (KBr) νmax 3381, 2974, 1733, 1633, 1583, 1412 cm−1; 1H and 13 C NMR, see Tables 1 and 2; HRESIMS m/z 511.2692 [M + H]+ (calcd for C30H39O7, 511.2690). (−)-8-Ethoxy-8,8a-dihydrobractatin (5a): tR 57 min, 3.3 mg, [α]25 D −98.8 (c 0.19, CHCl3); ECD (MeOH) λmax (Δε) 226 (+18.5), 311 (−12.2), 351 (+0.8) nm.

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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.7b00454. 756

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Experimental detail; details of the quantum chemical ECD calculations for compounds 1, 4, and 5; HRESIMS, UV, IR, NMR, and ECD spectra of compounds 1−7 (PDF) X-ray data of compounds 1, (CIF) 5, and (CIF) 13 (CIF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Tel: +86-24-23986465. Fax: +8624-23986465. ORCID

Zhan-Lin Li: 0000-0001-5306-3396 Hui-Ming Hua: 0000-0002-0258-3647 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support for this investigation from the National Natural Science Foundation of China (Grant Nos. 31570350 and 31602112) and Scientific Research Foundation of Shenyang Agricultural University (Grant No. 880415026) is gratefully acknowledged.

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DOI: 10.1021/acs.jnatprod.7b00454 J. Nat. Prod. 2018, 81, 749−757