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Cembranoids from the Gum Resin of Boswellia carterii as Potential Antiulcerative Colitis Agents Jin Ren, Yan-Gai Wang, Ai-Guo Wang, Lian-Qiu Wu, Hai-Jing Zhang, Wen-Jie Wang, Ya-Lun Su, and Hai-Lin Qin* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Eight new cembranoids, boscartins A−H (1, 2, and 4−9), and the known incensole oxide were isolated from the gum resin of Boswellia carterii. The absolute configurations of 1, 2, 4, and incensole oxide were unequivocally resolved using single-crystal X-ray diffraction analysis with Cu Kα radiation, and the absolute configuration of 5 was resolved via electronic circular dichroism data. The antiulcerative colitis activities of the compounds were evaluated in an in vitro x-box-binding protein 1 (XBP 1) transcriptional activity assay using dual luciferase reporter detection. At 10 μM, compounds 1, 5, 6, and 7 significantly activated XBP 1 transcription with EC50 values of 0.34, 1.14, 0.88, and 0.42 μM, respectively, compared with the pGL3-basic vector control.

T

he gum resin of Boswellia carterii Birdw., also known as frankincense, is a famous crude drug in traditional Chinese medicine (TCM), and it has been used to treat rheumatic arthralgia, chest obstruction, dysmenorrhea, and amenorrhea.1 Frankincense also possesses analgesic, anti-inflammatory, sedative, antihyperlipidemic, and antibacterial activities and has been used to treat rheumatoid arthritis, osteoarthritis, and ulcers.2 Previous phytochemical investigations of frankincense have indicated that various diterpenoids, such as cembranoids and prenylaromadendranoids, are the main constituents of this natural medicine.2b,3 Cembrane-type diterpenoids share the 4isopropyl-1,7,11-trimethylcyclotetradecane basic structural feature on the basis of systematic nomenclature.4 Hundreds of cembranoids have been isolated and identified from marine organisms, especially from soft corals and gorgonians.5 In terrestrial plants, cembranoids have been found only in tobacco, pinus, myrrh, and frankincense. In recent years, cembranoids have attracted increasing interest in the community of natural product research because many of these compounds exhibit a wide range of biological functions, such as ichthyotoxic, anticancer, anti-inflammatory, antihuman immunodeficiency virus (HIV), and antimicrobial activities.6 In our ongoing search for natural compounds with bioactivity against ulcerative colitis (UC), the activity of a 95% EtOH extract of frankincense was investigated, and eight new cembranoids (1, 2, and 4−9), in addition to the known compound 3, were isolated and identified. The isolated compounds were tested for anti-UC efficacy in the x-box-binding protein 1 (XBP 1) transcriptional activity dual luciferase reporter assay.7 This study describes the isolation and structure elucidation of related compounds, and it also reports the anti-UC activity of these compounds. © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The pulverized gum resin of B. carterii was extracted with 95% EtOH. The extract was purified by multiple chromatographic procedures and/or crystallizations, yielding eight new (1, 2, and 4−9) and one known (3) compound. The compounds were identified as diterpenoids sharing 20 carbon atoms based on their molecular formulas obtained from the HRESIMS measurements in the positive ion mode. The nine compounds were further determined to belong to the cembranoid class of diterpenoids Received: February 2, 2015

A

DOI: 10.1021/acs.jnatprod.5b00104 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H NMR Spectroscopic Data for Compounds 1−5 δH (type, J) position

1

2a 2b 3 5a 5b 6a 6b 7 9a 9b 10a 10b 11 13a,b 14a,b 15 16 17 18 19 20

2.03, br d (14.4)a 1.68, dd (14.4, 7.6)a 3.92, d (7.6) 2.19, dd (12.6, 9.0)a 1.68, ddd (12.6, 12.6, 7.8)a 2.10, m 1.86, m 4.17, dd (11.2, 4.4) 5.52, ddd (10.8, 6.8, 1.2)

a

2.95, ddd (12.0, 10.8, 2.0) 1.79, ddd (12.0, 11.2, 6.8) 3.81, dd (11.2, 2.0) 2.15, m; 1.70, m 1.99, m; 1.46, m 2.10, m 0.90, d (6.8) 0.93, d (6.8) 1.15, s 1.74, br s 1.17, s

2 1.95, br d (15.0)a 1.56, dd (15.0, 7.2)a 3.40, d (7.2) 3.37, dd (10.8, 4.2) 2.24, ddd (13.2, 11.4, 4.2)a 1.63, ddd (13.2, 10.8, 4.8)a 3.58, dd (11.4, 4.8) 1.92, dd (10.8, 10.8)a 1.58, dd (10.8, 10.8)a 2.03, ddd (17.4, 10.8, 9.0)a 1.51, ddd (17.4, 10.8, 6.6)a 4.01, dd (9.0, 6.6) 1.94, m; 1.57, m 1.94, m; 1.36, m 2.13, sept (6.6) 0.93, d (6.6) 0.94, d (6.6) 1.30, s 1.17, s 1.10, s

3

4

1.81, dd (15.6, 2.4)a 1.51, dd (15.6, 5.4)a 2.99, dd (5.4, 2.4) 2.01, m 1.27, ddd (12.6, 12.6, 4.2)a 2.17, m 2.07, m 5.15, dd (6.6, 6.6) 2.15, m 2.03, dd (12.6, 12.6)a 1.99, dd (13.2, 12.6)a 1.39, m 3.37, d (10.8) 2.01, m; 1.82, m 1.92, m; 1.44, m 2.15, m 0.90, d (6.6) 0.94, d (6.6) 1.17, s 1.66, br s 1.08, s

1.79, dd (15.6, 1.8)a 1.46, dd (15.6, 5.4)a 2.99, dd (5.4, 1.8) 2.42, dd (12.0, 3.6)a 1.28, dd (12.0, 12.0)a 4.50, ddd (12.0, 9.0, 3.6) 5.29, d (9.0) 2.33, br d (13.8)a 2.03, ddd (13.8, 13.8, 1.8)a 2.09, ddd (13.8, 13.8, 2.4)a 1.49, ma 3.26, d (10.8) 1.97, m; 1.83, m 1.97, m; 1.43, m 2.16, sept (6.6)a 0.88, d (6.6) 0.95, d (6.6) 1.19, s 1.85, s 1.07, s

5 1.77, d (15.6)a 1.38, dd (15.6, 6.0)a 2.86, d (6.0) 2.34, dd (13.8, 4.2)a 1.28, ma 3.14, ddd (12.6, 12.6, 12.0)a 2.44, ma 6.54, dd (12.0, 4.8) 2.67, ddd (13.8, 13.8, 4.2)a 2.03, ddd (13.8, 4.8, 4.2)a 1.80, ddd (13.8, 13.8, 4.8)a 1.66, dddd (13.8, 10.8, 4.2, 4.2)a 2.90, d (10.8) 2.00, m; 1.81, m 1.90, m; 1.37, m 2.07, sept (6.6)a 0.86, d (6.6) 0.92, d (6.6) 1.22, s 10.21, s 1.08, s

Calculated from the 1D TOCSY spectrum (600 MHz).

coupling correlations in the 1H−1H COSY spectrum from CH2-2 to CH-3, from CH2-5 to CH2-6 and CH2-6 to CH-7, from  CH-9 to CH2-10 and CH2-10 to CH-11, and from CH2-13 to CH2-14 unambiguously assigned the four structural fragments CH 2 (2)−CH(3), CH 2 (5)−CH 2 (6)−CH(7), CH(9)− CH2(10)−CH(11), and CH2(13)−CH2(14) in the cembranoid core with CH-3, CH-7, and CH-11 being categorized as oxymethine groups and CH-9 as an olefinic methine according to their chemical shifts in the 1H and 13C NMR spectra. The HMBC experiment, coupled with the molecular formula showing four indices of hydrogen deficiency, established the 2D structure of compound 1 as 1:12,4:7-diepoxycembr-8-ene3,11-diol or 1:4,7:12-diepoxycembr-8-ene-3,11-diol using the following correlations: from Me-18 to C-3, 4, and 5; from Me-19 to C-7, 8, and 9; from Me-20 to C-11, 12, and 13; from Me-16 and Me-17 to C-1 and 15; from H-3 to C-1, 4, and 5; from H-7 to C-8 and 9; from H-11 to C-12 and 13; and from H2-14 to C-2. The former was established as the 2D structure of 1 via the NOE correlation between H-3 and H-10a at δH 2.95 (1H, ddd, J = 12.0, 10.8, 2.0 Hz) in the NOESY experiment. The NOESY experiment, along with the coupling constants, also established the relative configuration of 1 (Figure 1). The NOE correlations of H-3/H-10a, H-11/H-10a, and H-10b/Me-20, along with the absence of NOE correlations of H-11/H-10b and H-11/Me-20, showed that OH-3 was in an α-orientation, OH-11 was in an exo and α-orientation, and Me-20 was in a β-orientation. The torsional angle of approximately 180° between the intersecting H(11)C(11)C(10) and H(10b)C(10)C(11) planes was ascertained by the data including not only the above-mentioned NOE correlations but also the large coupling constant of 11.2 Hz between H-11 and H-10b, which also confirmed the above assigned orientations of OH-3, OH-11, and Me-20. The NOE correlations of H-3/H-6b, H-7/H-6a, and H-7/Me-18 suggested that both Me-18 and H-7 were in α-orientations. On the basis of the NOE interactions of H-9/Me-19, the olefinic geometry was assigned as 8Z. In addition, the NOE correlation of H-3/H-15

according to the common features in their NMR spectroscopic data (Tables 1−3). One isopropyl group was detected in the higher field region. With the exception of compound 5, which showed singlets of a formyl group and two methyl groups as the manifestation of the three methyl groups of the cembranoid core, each of the remaining eight compounds had three methyl singlets in their 1H NMR spectra, which is characteristic of the majority of cembranoids. These features were confirmed by HSQC and DEPT NMR experiments. The three methyl groups in compounds 1−4 and 6−9 or the formyl group and the two methyl groups in compound 5 are connected to C-8, C-4, and C12, respectively, and the isopropyl group is connected to C-1 of the cembranoid skeleton. Compound 1 was obtained as colorless prisms from acetone. The molecular formula was determined as C20H34O4 by 13C NMR data and by an HRESIMS experiment in the positive ion mode with the protonated ion at m/z 339.2530 and the sodium adduct ion at m/z 361.2357, which is indicative of four indices of hydrogen deficiency. The IR spectrum displayed absorption bands of hydroxy (3355 and 3264 cm−1) and olefinic (1678 cm−1) functionalities. Except for the aforementioned diagnostic signals of cembranoids in the 1H NMR spectrum that resonated at δH 0.90 (3H, d, J = 6.8 Hz, Me-16), 0.93 (3H, d, J = 6.8 Hz, Me17), 1.15 (3H, s, Me-18), 1.74 (3H, br s, Me-19), 1.17 (3H, s, Me-20), and 2.10 (1H, m, H-15), well-resolved signals of three oxymethine protons at δH 3.92 (1H, d, J = 7.6 Hz, H-3), 4.17 (1H, dd, J = 11.2, 4.4 Hz, H-7), and 3.81 (1H, dd, J = 11.2, 2.0 Hz, H-11) and an olefinic proton at δH 5.52 (1H, ddd, J = 10.8, 6.8, 1.2 Hz, H-9) were also evident (Table 1). The DEPT NMR experiment classified the 20 carbons into five methyls, six methylenes, five methines, including the tertiary C-15 (δC 32.9), an olefinic methine at δC 124.6 (C-9), and three oxygenated methines at δC 73.3 (C-3), 81.3 (C-7), and 79.8 (C-11), three oxygenated tertiary carbons at δC 89.2 (C-1), 84.3 (C-4), and 85.3 (C-12), and a quaternary olefinic carbon at δC 136.5 (C-8) (Table 3). Along with the HSQC experiment, the homonuclear B

DOI: 10.1021/acs.jnatprod.5b00104 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 1H NMR Spectroscopic Data for Compounds 6−9 δH (type, J) position 2a 2b 3 5a 5b

6

7

8

9

1.75, dd (15.6, 1.8)a 1.61, dd (15.6, 5.4)a 3.16, dd (5.4, 1.8) 2.59, dd (12.6, 4.8)a

1.82, dd (15.6, 2.4) 1.61, dd (15.6, 4.8) 3.14, dd (4.8, 2.4) 2.27, ddd (13.6, 3.6, 3.6) 1.35, m

2.00, dd (15.6, 5.2)a 1.50, dd (15.6, 2.4)a 2.77, dd (5.2, 2.4)

2.45, dd (14.0, 3.2) 2.34, dd (14.0, 11.2) 8.16, dd (11.2, 3.2)

1.44, dd (12.6, 11.4)a 4.65, ddd (11.4, 8.4, 4.8)

6a

9 10a 10b 11 13a,b 14a,b 15 16 17 18 19 20 a

1.12, ddd (13.8, 13.8, 3.0)a

Figure 1. NOE (H↔H) correlations of 1.

6b 7

2.31, ddd (13.8, 4.8, 3.0)a

2.46, m

2.58, dddd (13.8, 11.4, 4.8, 3.0)a

3.99, dd (13.2, 2.0)

2.46, m

2.25, br dd (13.8, 13.8)a 6.94 br d (11.4)

2.12, dd (13.2, 5.6) 3.82, dd (5.6, 2.0) 1.87, m 2.24, dd (13.2, 9.2) 1.70, m

6.69, br d (8.4)

6.84, dd (6.0, 6.0)

3.89, d (12.6)a 2.10, dd (12.6, 10.2)a 3.45, d (10.2)

3.86, d (12.4)

3.81, d (12.6)a

2.06, dd (12.4, 10.4)

2.06, dd (12.6, 9.6)a

3.53, d (10.4)

3.50, d (9.6)a

2.13, m; 1.79, m 2.02, m; 1.47, m 2.18, sept (6.6) 0.91, d (6.6) 0.97, d (6.6) 1.14, s 1.94 br s 1.16, s

2.13, m; 1.82, m 2.03, m; 1.49, m 2.21, sept (6.8) 0.92, d (6.8) 0.98, d (6.8) 1.13, s 1.79, br s 1.14, s

2.29, m; 1.87, m

1.89, m

4.32, dd (8.8, 4.0) 1.85, m; 1.44, dd (11.6, 8.0) 1.95, dd (11.6, 8.0); 1.74, m 1.82, m

0.87, d (6.8) 0.98, d (6.8) 1.35, s 1.77, br s 1.23, s

0.93, d (6.8) 1.07, d (6.8) 1.76, br s 1.21, s 1.10, s

2.03, m; 2.03, m

confirmed that the isopropyl group was in the same β-orientation as H-3. Finally, the structure of 1 was unequivocally assigned by single-crystal X-ray diffraction analysis using the anomalous scattering of Cu Kα radiation with a Flack parameter of 0.04 (10), which not only affirmed that the relative configuration of 1 was consistent with the data from the NOESY experiment but also assigned the absolute configuration of (1S, 3R, 4S, 7R, 8Z, 11S, 12R) according to the numbering system of cembranoids. On the other hand, no intramolecular H-bond was found, but the intermolecular H-bonds between 1 and two H2O molecules were observed. The ORTEP diagram showing the absolute configuration of 1 is shown in Figure 2. Thus, the structure of 1 was established as (1S,3R,4S,7R,11S,12R,8Z,)-1:12,4:7-diepoxycembr-8-ene-3,11-diol. This series of compounds was given the general name of boscartins, and boscartin A was recommended as the trivial name of compound 1. Compound 2 was obtained as colorless prisms from acetone. The molecular formula was determined as C20H34O5 by 13C NMR data and by an HRESIMS experiment in the positive ion mode with the protonated ion at m/z 355.2480 and the sodium adduct ion at m/z 377.2311, indicating four indices of hydrogen deficiency. The IR spectrum displayed absorption bands of hydroxy functionalities (3338 and 3188 cm−1). The diagnostic

Calculated from the 1D TOCSY spectrum (600 MHz).

Table 3. 13C NMR Spectroscopic Data for Compounds 1−9 position

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

89.2 40.9 73.3 84.3 37.0 32.0 81.3 136.5 124.6 30.2 79.8 85.3 37.6 31.0 32.9 18.8 16.9 20.1 25.0 19.7

88.8 41.2 74.3 64.2 60.1 33.0 76.1 84.1 37.4 26.4 80.1 84.9 29.7 29.2 31.3 18.9 16.6 10.7 15.6 26.1

88.3 36.0 60.0 59.0 38.1 23.9 123.4 135.6 32.8 30.3 77.8 84.4 36.7 29.7 32.7 18.5 16.9 16.8 18.8 20.0

88.4 36.8 59.9 56.9 47.6 65.7 127.2 140.9 32.1 31.2 77.9 84.6 37.0 29.5 32.3 18.6 16.6 17.6 20.5 19.7

88.0 36.7 59.4 57.8 38.8 23.5 152.2 141.0 23.2 32.7 76.8 84.1 37.0 29.6 32.5 18.5 16.5 16.8 191.9 20.2

88.9 38.2 60.6 56.6 46.7 65.7 142.7 139.5 204.5 40.8 77.0 84.8 36.5 30.6 33.0 18.6 16.9 17.4 11.9 19.6

88.9 37.8 60.7 58.6 37.3 25.4 144.3 137.1 203.3 40.8 78.1 84.8 36.6 30.3 32.8 18.7 16.9 16.1 11.2 19.7

88.5 29.0 60.5 59.5 38.6 25.3 144.3 135.8 203.2 41.1 75.5 84.4 35.4 31.4 36.2 19.1 16.4 17.0 10.6 21.7

88.6 33.8 147.6 135.1 203.1 44.5 77.0 87.11 36.2 28.6 83.6 87.15 31.2 36.2 42.2 18.6 17.8 10.8 19.2 25.3

C

DOI: 10.1021/acs.jnatprod.5b00104 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 2. ORTEP drawings of 1 (left) and 2 (right).

Figure 3. NOE (H↔H) correlations of 2−9.

signals of cembranoids in the 1H NMR spectrum resonated at δH 0.93 (3H, d, J = 6.6 Hz, Me-16), 0.94 (3H, d, J = 6.6 Hz, Me-17), 1.30 (3H, s, Me-18), 1.17 (3H, s, Me-19), 1.10 (3H, s, Me-20), and 2.13 (1H, sept, J = 6.6 Hz, H-15). Well-resolved signals of four oxymethine protons at δH 3.40 (1H, d, J = 7.2 Hz, H-3), 3.37 (1H, dd, J = 10.8, 4.2 Hz, H-5), 3.58 (1H, dd, J = 11.4, 4.8 Hz, H7), and 4.01 (1H, dd, J = 9.0, 6.6 Hz, H-11) were also evident (Table 1). The DEPT NMR experiment classified the 20 carbons into five methyls, six methylenes, five methines, including the tertiary C-15 (δC 31.3) and four oxygenated methines at δC 74.3 (C-3), 60.1 (C-5), 76.1 (C-7), and 80.1 (C-11), and four oxygenated tertiary carbons at δC 88.8 (C-1), 64.2 (C-4), 84.1

(C-8), and 84.9 (C-12). Along with the HSQC experiment and considering the chemical shifts and the molecular formula with four indices of hydrogen deficiency, the HMBC spectrum revealed that an oxirane ring was present at C-4 (δc 64.2) and C5 (δC 60.1) based on the correlations from H-3 to C-4 and 5; from H-5 to C-6; and from Me-18 to C-3, 4, and 5. In addition, the homonuclear coupling correlations from CH2-2 to CH-3, from CH-5 to CH2-6, and from CH2-6 to CH-7 in the 1H−1H COSY spectrum of compound 2 supported this linkage. The NOE correlations of Me-19/H-11 and Me-17/Me-20 established that the two epoxy groups, which were also deduced from the molecular formula and chemical shifts, bridged C-8 and C-11 and D

DOI: 10.1021/acs.jnatprod.5b00104 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 4. ORTEP drawings of 3 (left) and 4 (right).

C-1 and C-12, respectively. The 1H−1H COSY correlations (from CH2-9 to CH2-10; from CH2-10 to CH-11; from CH2-13 to CH2-14; and from CH-15 to Me-16 and Me-17), in addition to the HMBC correlations (from H-7 to C-8, 9, and 19; from H-11 to C-10, 12, 13, and 20; and from H-15 to C-1, 2, and 14), established the 2D structure of compound 2 as 1:12,4:5,8:11triepoxycembrane-3,7-diol. The relative configuration of compound 2 was determined using NOE spectra. Irradiation of the Me-20 signal resulted in the NOE enhancements of the Me-17 and H-11 signals, and irradiation of H-11 led to the NOE enhancements of Me-19 and Me-20, suggesting that H-11, Me19, Me-20, and the isopropyl group were all in β-orientations. Irradiation of the H-3 signal caused the NOE enhancement of the H-15 signal, but no NOE enhancement was observed for Me-18. Irradiation of Me-18 caused the NOE enhancement of H-7. Irradiation of H-7 caused the NOE enhancement of Me-18, but no NOE enhancement was observed for Me-19. Irradiation of H5 caused the NOE enhancement of Me-19. These results suggested that both OH-3 and Me-18 were in α-orientations, and both H-5 and OH-7 were in β-orientations (Figure 3). The structure of 2, including the absolute configuration, was also assigned by single-crystal X-ray diffraction analysis using the anomalous scattering of Cu Kα radiation with a Flack parameter of 0.0(2). The ORTEP diagram showing the absolute configuration of 2 is shown in Figure 2. The relative configurations of the stereogenic centers were confirmed to be consistent with the findings of the NOE data. In addition, no intramolecular H-bond was found, but the intermolecular Hbond between 2 and a H2O molecule was observed. Thus, the structure of compound 2 was established as (1S,3R,4S,5S,7S,8R,11S,12R)-1:12,4:5,8:11-triepoxycembrane3,7-diol and named boscartin B. Compound 3 was obtained as colorless prisms from acetone. The molecular formula was determined as C20H34O3 by 13C NMR data and by an HRESIMS experiment in the positive ion mode with the protonated ion at m/z 323.2587 and the sodium adduct ion at m/z 345.2407, indicating four indices of hydrogen deficiency. The IR spectrum displayed absorption bands of hydroxy (3449 cm−1) and olefinic (1667 cm−1) functionalities. The diagnostic signals of cembranoids in the 1H NMR spectrum resonated at δH 0.90 (3H, d, J = 6.6 Hz, Me-16), 0.94 (3H, d, J =

6.6 Hz, Me-17), 1.17 (3H, s, Me-18), 1.66 (3H, br s, Me-19), 1.08 (3H, s, Me-20), and 2.15 (1H, m, H-15). Well-resolved signals of two oxymethine protons at δH 2.99 (1H, dd, J = 5.4, 2.4 Hz, H-3) and 3.37 (1H, d, J = 10.8 Hz, H-11), as well as an olefinic proton at δH 5.15 (1H, dd, J = 6.6, 6.6 Hz, H-7), were also evident (Table 1). The DEPT NMR experiment classified the 20 carbons into five methyls, seven methylenes, four methines, including the tertiary C-15 (δC 32.7), an olefinic methine at δC 123.4 (C-7), and two oxygenated methines at δC 60.0 (C-3) and 77.8 (C-11), three oxygenated tertiary carbons at δC 88.3 (C-1), 59.0 (C-4), and 84.4 (C-12), and a quaternary olefinic carbon at δC 135.6 (C8). Considering the chemical shifts and the molecular formula with four indices of hydrogen deficiency, along with the HSQC experiments, the HMBC spectrum revealed that an oxirane ring was present at the C-3−C-4 bond and an epoxy group bridged C1 and C-12 based on the following correlations: from H-3 to C-1 and 2; from H2-2 to C-4; from H-11 to C-12; from Me-18 to C-3, 4, and 5; from Me-20 to C-11, 12, and 13; and from Me-16 and 17 to C-1. After the four structural fragments of CH2(2)− CH(3), CH2(5)−CH2(6)−CH(7), CH2(9)−CH2(10)−CH(11), and CH2(13)−CH2(14) in the cembranoid core were deduced via the 1D TOCSY spectra (data not shown), the 2D structure of compound 3 was confirmed as 1:12,3:4-diepoxycembr-7-en-11-ol using the HMBC experiment, which showed the following additional correlations: from H2-5 to C-4 and 7; from H2-6 to C-7 and 8; and from H2-10 to C-8 and 12. This constitution was the same as the known cembranoid incensole oxide, which was also isolated from frankincense in 1968.8 However, there has been no report on the complete NMR data and configuration of incensole oxide. Therefore, the structure and configurations of compound 3 were assessed by NOESY and single-crystal X-ray diffraction analysis. The NOE correlations of H-3/H-15, H-3/H-5b, Me-18/H-2a, Me-18/H2b, H-11/H-2a, H-10a/H-7, and Me-19/6a, along with the absence of the NOE correlation of H-11/Me-20, suggested that H-3, Me-20, and the isopropyl group were all in β-orientations, Me-18 was in an α-orientation, OH-11 was in an exo and αorientation, and the geometry of the double bond was 7E (Figure 3). The X-ray crystallographic analysis (Figure 4) revealed that the absolute configuration was (1S, 3R, 4R, 7E, 11S, 12R). Thus, E

DOI: 10.1021/acs.jnatprod.5b00104 J. Nat. Prod. XXXX, XXX, XXX−XXX

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an HRESIMS experiment in the positive ion mode with the sodium adduct ion at m/z 375.2142, indicating five indices of hydrogen deficiency. The IR spectrum showed absorption bands of hydroxy (3427 cm−1) and α,β-unsaturated carbonyl (1712 and 1667 cm−1) functionalities. The 1H, 13C, and DEPT NMR data of compound 6 closely resembled those of compound 4. The primary difference was that the resonances at δH 2.33 (1H, br d, J = 13.8 Hz) and 2.03 (1H, ddd, J = 13.8, 13.8, 1.8 Hz) and δC 32.1 for the C-9 methylene in compound 4 were replaced by that of a ketocarbonyl moiety at δC 204.5 in compound 6 (Tables 1−3). Compared with the data of compound 4, deshieldings of C-7 and C-10 (Δδ +15.5 and +9.6, respectively) were observed in the 13C NMR spectrum of compound 6. These differences and the assignment were also supported by the correlations from H-7, H2-10, H-11, and Me-19 to C-9 in the HMBC spectrum. The NOE correlations of H-3/H-15, H-3/H-5b, Me-18/H-5a, Me18/H-6, H-11/H-2a, H-11/H-10a, H-10a/H-7, Me-19/H-6, and Me-20/H-10b, along with the absence of the NOE correlation of H-11/Me-20, suggested that H-3, OH-6, Me-20, and the isopropyl group were all in β-orientations, Me-18 was in an αorientation, OH-11 was in an exo and α-orientation, and the geometry of the double bond was 7E (Figure 3). Thus, the structure of compound 6 was established as (rel)(1S,3R,4R,6S,11S,12R,7E)-1:12,3:4-diepoxy-6,11-dihydroxycembr-7-en-9-one and named boscartin E. Compound 7 was obtained as a colorless oil. The molecular formula was determined as C20H32O4 by 13C NMR data and by an HRESIMS experiment in the positive ion mode with the protonated ion at m/z 337.2375 and the sodium adduct ion at m/ z 359.2199, indicating five indices of hydrogen deficiency. Analysis of the IR spectrum of compound 7 suggested the presence of hydroxy (3456 cm−1 ) and α,β-unsaturated ketocarbonyl (1727 and 1663 cm−1) functionalities. The 1H, 13 C, and DEPT NMR data of compound 7 closely resembled those of compound 6. The primary difference was that the resonances at δH 4.65 (1H, ddd, J = 11.4, 8.4, 4.8 Hz) and δC 65.7 for the C-6 oxymethine in compound 6 were replaced by those of a methylene at δH 2.46 (2H, m) and δC 25.4 in compound 7 (Tables 2 and 3). Compared with compound 6, a shielding of C6 (Δδ −40.3) was observed in compound 7. Homonuclear correlations from CH2-5 to CH2-6 and CH2-6 to CH-7 were observed in the 1H−1H COSY spectrum, and the correlations from H2-6 to C-4 and 8 and from H-7 to C-6 were observed in the HMBC spectrum, which confirmed this assignment. The NOE correlations of H-3/H-15, H-3/H-5b, Me-18/H-5a, Me-18/H2a, Me-18/H-2b, H-11/H-2a, H-11/H-10a, Me-20/H-10b, H10a/H-7, and Me-19/6a, along with the absence of the NOE correlations of H-3/Me-18 and H-11/Me-20, suggested that H3, Me-20, and the isopropyl group were all in β-orientations, Me18 was in an α-orientation, OH-11 was in an exo and αorientation, and the geometry of the double bond was 7E (Figure 3). Thus, the structure of compound 7 was established as (rel)(1S,3R,4R,11S,12R,7E)-1:12,3:4-diepoxy-11-hydroxycembr-7en-9-one and named boscartin F. Compound 8 was obtained as a colorless oil. The molecular formula was assigned as C20H32O4 by 13C NMR data and by an HRESIMS experiment in the positive ion mode with the protonated ion at m/z 337.2374 and the sodium adduct ion at m/ z 359.2197. This molecular formula was the same as that for compound 7. The IR spectrum suggested the presence of hydroxy (3455 cm−1) and α,β-unsaturated ketocarbonyl (1728 and 1662 cm−1) functionalities. A detailed comparison of NMR data between compounds 8 and 7 indicated that these two

the structure of compound 3 was established as (1S,3R,4R,11S,12R,7E,)-1:12,3:4-diepoxycembr-7-en-11-ol. Compound 4 was obtained as colorless prisms from acetone. The molecular formula was established as C20H34O4 by 13C NMR data and by an HRESIMS experiment in the positive ion mode with the sodium adduct ion at m/z 361.2350, indicating four indices of hydrogen deficiency. The IR spectrum displayed absorption bands of hydroxy (3526 and 3299 cm−1) and olefinic (1642 cm−1) functionalities. The 1H, 13C, and DEPT NMR data of compound 4 closely resembled those of compound 3, except that the resonances at δH 2.17 and 2.07 and δC 23.9 for the C-6 methylene group in compound 3 were replaced by those at δH 4.50 (1H, ddd, J = 12.0, 9.0, 3.6 Hz) and δC 65.7 of the oxygenbearing methine of C-6 in compound 4 (Tables 1 and 3). Along with the HSQC spectrum, the correlations from H2-5 and H-7 to C-6 and from H-6 to C-8 in the HMBC spectrum confirmed this assignment. The structure and configurations of compound 4 were assessed by the different NOE spectra and single-crystal Xray diffraction analysis. The NOE correlations of H-3/H-15, H3/H-5b, Me-18/H-6, H-6/H-5a, H-3/H-7, H-3/H-10a, H-11/ H-2a, H-10a/H-7, Me-19/H-6, and Me-19/H-11, along with the absence of the NOE correlation of H-11/Me-20, suggested that H-3, OH-6, Me-20, and the isopropyl group were all in βorientations, Me-18 was in an α-orientation, OH-11 was in an exo and α-orientation, and the geometry of the double bond was 7E (Figure 3). The X-ray crystallographic analysis (Figure 4) revealed that the absolute configuration was (1S, 3R, 4R, 6S, 7E, 11S, 12R). Thus, the structure of compound 4 was established as (1S,3R,4R,6S,11S,12R,7E,)-1:12,3:4-diepoxycembr-7-ene-6,11diol, and it was given the trivial name boscartin C. Compound 5 was isolated as a colorless oil. The molecular formula was determined as C20H32O4 by 13C NMR data and by an HRESIMS experiment in the positive ion mode with the protonated ion at m/z 337.2384 and the sodium adduct ion at m/ z 359.2200, indicating five indices of hydrogen deficiency. The IR spectrum displayed absorption bands of hydroxy (3475 cm−1) and α,β-unsaturated aldehyde (1713 and 1672 cm−1) functionalities. The 1H, 13C, and DEPT NMR data of compound 5 closely resembled those of 3, except that the resonances of Me-19 at δH 1.66 (3H, br s, Me-19) and δC 18.8 in compound 3 were replaced by those of a formyl group at δH 10.21 (1H, s, H-19) and δC 191.9 (C-19) in compound 5. Moreover, a deshielding (Δδ +28.8) of C-7 was observed in the 13C NMR spectrum of compound 5 compared with compound 3 (Table 3). These differences and the assignment were supported by the correlations from H-19 of the formyl group to C-7 and 8 and from H-7 and H2-9 to C-19 in the HMBC spectrum. The NOE correlations of H-3/H-15, H-3/H5b, Me-18/H-2a, Me-18/H-2b, Me-18/H-5a, H-11/H-10a, H10a/H-7, H-19/6a, and Me-20/H-10b, along with the absence of the NOE correlation of H-11/Me-20, suggested that H-3, Me-20, and the isopropyl group were all in β-orientations, Me-18 was in an α-orientation, OH-11 was in an exo and α-orientation, and the geometry of the double bond was 7Z (Figure 3). The assignment of the absolute configuration of C-11 was simplified by the electronic circular dichroism (ECD) data of the in situ-formed [Rh2(OCOCF3)] complex. The ECD spectrum of the Rh complex of compound 5 showed a positive Cotton effect at ca. 349 nm, indicating an 11S absolute configuration based on the bulkiness rule.9 Thus, the structure of compound 5 was established as (1S,3R,4R,11S,12R,7Z,)-1:12,3:4-diepoxy-11-hydroxycembr-7-ene-19-carbaldehyde and named boscartin D. Compound 6 was isolated as a colorless oil. The molecular formula was determined as C20H32O5 by 13C NMR data and by F

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using the NOE difference spectra. The NOE correlations of H-3/ H-6a, Me-19/H-6a, Me-19/H-11, and Me-20/H-11, along with the absence of the NOE correlations of H-3/Me-18 and H-7/ Me-19, suggested that H-11, OH-7, Me-19, Me-20, and the isopropyl group were all in β-orientations and the geometry of the double bond was 3E (Figure 3). Thus, the structure of compound 9 was established as (rel)-(1S,7S,8R,11S,12R,3E,)1:12,8:11-diepoxy-7-hydroxycembr-3-en-5-one and named boscartin H. To evaluate the potential anti-UC activity, the cytotoxicity toward intestinal epithelial cell-6 (IEC-6) was first examined at 10 μM using the MTT assay. Compounds 1−9 showed no obvious cytotoxicity when incubated with IEC-6 cells for 72 h; survival ranged from 87% to 102% (Figure 5), suggesting that all

compounds shared identical functional groups, but they had noticeable differences in terms of 1H and 13C NMR chemical shifts (Tables 2 and 3). Along with the HSQC experiment, the identical 2D structures were confirmed using the HMBC experiment, which showed the following correlations in compound 8: from H-15 to C-1 and C-2; from Me-16 and 17 to C-1; from H2-2 to C-1, 3, 4, and 14; from Me-18 to C-3, 4, and 5; from H-7 to C-5 and 9; from Me-19 to C-7, 8, and 9; from H210 to C-9, 11, and 12; from H-11 to C-12 and 13; and from Me20 to C-11, 12, and 13. This information showed that the configuration of compound 8 differed from that of compound 7. As in the case of compound 7, the relative configuration of compound 8 was assessed by a NOESY experiment. The correlations of H-3/H-5b, H-3/H-11, H-7/H-10a, H-11/H-10a, and Me-19/H-6a, along with the absence of the NOE correlations of H-3/Me-18, H-3/H-15, and H-11/Me-20, suggested that Me-18, Me-20, and the isopropyl group were all in β-orientations, H-3 was in an α-orientation, OH-11 was in an exo and α-orientation, and the geometry of the double bond was 7E (Figure 3). Accordingly, the structure of compound 8 was established as (rel)-(1S,3S,4S,11S,12R,7E)-1:12,3:4-diepoxy-11hydroxycembr-7-en-9-one and named boscartin G. Compound 9 was obtained as a colorless oil. The molecular formula was determined as C20H32O4 by 13C NMR data and by an HRESIMS experiment in the positive ion mode with the protonated ion at m/z 337.2376 and the sodium adduct ion at m/ z 359.2193, indicating five indices of hydrogen deficiency. The IR spectrum displayed absorption bands of hydroxy (3501 cm−1) and α,β-unsaturated ketocarbonyl (1731 and 1648 cm−1) functionalities. The diagnostic signals of cembranoids in the 1H NMR spectrum resonated at δH 0.93 (3H, d, J = 6.8 Hz, Me-16), 1.07 (3H, d, J = 6.8 Hz, Me-17), 1.76 (3H, br s, Me-18), 1.21 (3H, s, Me-19), 1.10 (3H, s, Me-20), and 1.82 (1H, m, H-15). Well-resolved signals of two oxymethine protons at δH 3.82 (1H, dd, J = 5.6, 2.0 Hz, H-7) and 4.32 (1H, dd, J = 8.8, 4.0 Hz, H-11) and an olefinic methine proton at δH 8.16 (1H, dd, J = 11.2, 3.2 Hz, H-3) were also evident (Table 2). The DEPT NMR experiment classified the 20 carbons into five methyls, six methylenes, four methines, including the tertiary C-15 (δC 42.2), an olefinic methine at δC 147.6 (C-3), and two oxygenated methines at δC 77.0 (C-7) and 83.6 (C-11), three oxygenated tertiary carbons at δC 88.6 (C-1), 87.11 (C-8), and 87.15 (C-12), a ketocarbonyl carbon at δC 203.1 (C-5), and a quaternary olefinic carbon at δC 135.1 (C-4). Along with the HSQC experiment and considering the chemical shifts and the molecular formula showing five indices of hydrogen deficiency, the correlations (from H2-2 to C-3 and 4; from H-3, H2-6, and H7 to C-5; and from Me-18 to C-3, 4, and 5) observed in the HMBC spectrum revealed the Δ3,4 trisubstituted double bond and the C-5 carbonyl group. Two epoxy groups were confirmed to be between C-1 and C-12 and between C-8 and C-11 using HMBC correlations (from H2-13 to C-1; from H2-14 to C-12; from Me-19 to C-7, 8, and 9; from Me-20 to C-11, 12, and 13; and from Me-16 and 17 to C-1) and the NOE correlation of Me19/H-11 in the NOE difference experiment. After the four structural fragments of CH2(2)−CH(3), CH2(6)−CH(7), CH2(9)−CH2(10)−CH(11), and CH2(13)−CH2(14) were established by the 1H−1H COSY spectrum, the 2D structure of compound 9 was defined as 1:12,8:11-diepoxy-7-hydroxycembr-3-en-5-one using the HMBC experiment, which showed the following additional correlations: from H2-6 to C-8; from H7 to C-8 and 9; from H-11 to C-10, 12, and 13; and from H2-14 to C-15. The relative configuration of compound 9 was assessed

Figure 5. Toxicity results of compounds 1−9 on IEC-6 cells determined by the MTT assay.

the compounds are suitable for studying additional bioactivities, such as anti-UC activity. Compounds 1−9 were then evaluated at 10 μM for the activation of the XBP 1 target gene using a dual luciferase reporter assay. This transcription factor (TF) is associated with the occurrence, exacerbation, and potential treatment of UC.7 The results are shown in Figure 6, in which

Figure 6. Effects of compounds 1−9 on XBP 1 transcriptional activity.

con 1 was used as background contrast and con 2 was the pGL3basic vector control. As a new potential target for anti-UC, there is no other agonist of XBP 1, except 8-oxodihyrocoptisine (8OC), which was selected as the positive control in this study. 8OC was synthesized from the natural quaternary coptisine and has been shown to activate XBP 1 transcription in our laboratory.10 At 10 μM, all the compounds activated XBP 1 transcription. Compound 6 exhibited more significant activity than the positive control, and compounds 1, 5, and 7 were more effective than the other tested compounds but had weaker G

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activity than 8-OC. Compounds 1, 5, 6, and 7 and 8-OC had relative activating ratios of 1.5-, 1.6-, 2.5-, 1.5-, and 2.2-fold, respectively, compared with con 2. In addition, the significant activity of compounds 1, 5, 6, and 7 was demonstrated by the significant dose−effect relationship observed for these compounds, with EC50 values of 0.34, 1.14, 0.88, and 0.42 μM, respectively (Figures 7−10). Although the activity was slightly or

Figure 10. EC50 value of 7 (EC50: 0.42 μM). spectrophotometer, and IR spectra were recorded on a Thermo Nicolet 5700 FT-IR microscopic spectrophotometer. NMR spectra were acquired on either a Varian Inova 600 MHz or a Bruker Avance III 400 MHz NMR spectrometer. CDCl3 was used both as a solvent and as an internal reference at δH 7.26 and δC 77.0. Positive ion mode HRESIMS experiments were conducted on an Agilent 1100 series LC/ MSD Trap SL mass spectrometer. Analytical HPLC was performed on an Agilent series 1200 HPLC instrument with a DIKMA-C18 column (250 × 4.6 mm; 5 μm). Preparative HPLC was performed on a Shimadzu LC-6AD instrument with a SPD-20A detector and a YMCPack ODS-A column (250 × 20 mm; 5 μm). Silica gel (160−200 mesh and 200−300 mesh; Qingdao Marine Chemical Inc., Qingdao, China) and silica gel H (Qingdao Marine Chemical Inc., Qingdao, China) were used for column chromatography (CC), and precoated GF254 silica gel plates (Qingdao Marine Chemical Inc., Qingdao, China) was used for TLC analyses. The spots were visualized by spraying with 10% H2SO4 in EtOH and then with 5% anisaldehyde in EtOH followed by heating under infrared radiation. Plant Material. Gum resin of Boswellia carterii was kindly furnished by Beijing Tongrentang Co., Ltd., and identified by Professor Wan-Zhi Song (Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China) according to the identification procedure for the gum resin of B. carterii in the State Pharmacopoeia of China.1a A voucher specimen (No. ID-S-2385) was deposited in the Herbarium of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College. Extraction and Isolation. The gum resin of B. carterii (11 kg) was powdered and extracted with 95% EtOH several times under reflux conditions until the extract solvent was transparent. The EtOH extract was filtered and concentrated under reduced pressure to yield a dark brown residue (8.0 kg). The residue was separated by flash silica gel CC (160−200 mesh and 20 × 200 cm), eluting sequentially with petroleum ether, CH2Cl2, and EtOAc, to yield three fractions. The CH2Cl2 fraction (500 g) was chromatographed on a column of silica gel, using gradient elution of petroleum ether with increasing proportions of EtOAc, to give 19 subfractions, Fr. 1−19. Fr. 6 (70 g) was separated by RP-C18 vacuum CC, eluting with a gradient MeOH in H2O (60−100%), to give 17 subfractions, Fr. 6-A to Fr. 6-Q. Fr. 6-H (80% MeOH) was recrystallized from acetone to afford compound 3 (99 mg) as colorless prisms. Fr. 10 (40 g) was separated by RP-C18 vacuum CC, eluting with a gradient of MeOH in H2O (70−100%), to give 10 subfractions, Fr. 10-A to 10-J. Fr. 10-A (2 g; 70% MeOH) was subjected to CC of silica gel H, eluting with isocratic CH2Cl2−Et2O (8:1), to afford four subfractions, Fr. 10-A-1 to 10-A-4. Fr. 10-A-2 (600 mg) was separated by CC on silica gel H, using isocratic petroleum ether−EtOAc (3:1) as the eluent, and the eluate was then separated through preparative RP-HPLC [mobile phase of MeOH−H2O (65%, v/v); flow rate of 6 mL min−1; and UV detection at 230 nm] to yield compounds 5 (9.5 mg; tR = 11.61 min), 7 (2.0 mg; tR = 15.03 min), 8 (2.1 mg; tR = 21.15 min), and 9 (6.2 mg; tR = 25.50 min). Fr. 10-A-3 (800 mg) was separated by preparative RP-HPLC [mobile phase of MeOH−H2O (70%, v/v); flow rate of 6 mL min−1; and UV detection at 210 nm] to afford compound 1 (tR = 6.43 min), which was

Figure 7. EC50 value of 1 (EC50: 0.34 μM).

Figure 8. EC50 value of 5 (EC50: 1.14 μM).

Figure 9. EC50 value of 6 (EC50: 0.88 μM).

significantly weaker than the activity of the positive control (EC50 0.08 μM), the findings that all nine compounds can activate XBP 1 transcription provided support for the application of frankincense in some traditional medicine systems to treat ulcers.1b The total syntheses of active boscartins is ongoing in our laboratory to further evaluate their activities in experimental animal models.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on an XT5B microscopic melting point apparatus. Optical rotations were measured on a Rudolph Research Autopol III automatic polarimeter. ECD spectra were measured using a JASCO J-815 CD spectrometer. UV spectra were recorded on a JASCO V-650 H

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337.2379), m/z 359.2197 [M + Na]+ (calcd for C20H32NaO4, 359.2198). Boscartin H (9): colorless oil; [α]25 D −9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (3.31), 238 (3.53) nm; IR (neat) νmax 3501, 2963, 2931, 2877, 1731, 1648, 1457, 1381, 1283, 1238, 1173, 1124, 1061, 1032 cm−1; 1 H NMR (CDCl3, 400 MHZ), see Table 2; 13C NMR (CDCl3, 100 MHZ), see Table 3; HRESIMS m/z 337.2376 [M + H]+ (calcd for C20H33O4, 337.2379), m/z 359.2193 [M + Na]+ (calcd for C20H32NaO4, 359.2198). Single-Crystal X-ray Structure Determination of Boscartins A−C (1, 2, and 4) and Incensole Oxide (3). Colorless crystals of boscartins A−C (1, 2, and 4) and incensole oxide (3) were obtained from acetone. The crystal data of compounds 1, 3, and 4 were obtained on an Agilent Gemini E diffractometer with monochromatic Cu Kα radiation (λ = 1.541 84 Å), and the crystal data of compound 2 were obtained on a Rigaku MicroMax 002+ diffractometer with monochromatic Cu Kα radiation (λ = 1.541 87 Å). The structures were solved by direct methods (SHELXS-97) and refined using full-matrix leastsquares difference Fourier techniques. All non-hydrogen atoms were refined anisotropically, and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters. Crystallographic data for structures 1−4 were deposited at the Cambridge Crystallographic Data Centre (CCDC). Copies of the data can be obtained free of charge by application to the Director, CCDC, 12 Union Road, Cambridge CB21EZ, UK (fax: + 44(0)1223 336033 or e-mail: [email protected]). The CCDC reference numbers are as follows: 1035970 (1), 1036082 (2), 1035968 (3), and 1035969 (4). Crystal data of boscartin A (1): C20H38O6, M = 374.5, triclinic, a = 7.5468(4) Å, b = 8.5723(4) Å, c = 10.2666(6) Å, α = 99.326(4)°, β = 107.867(5)°, γ = 114.786(5)°, V = 540.47(5) Å3, T = 100.4 K, space group P1 (no. 1), Z = 1, μ(Cu Kα) = 0.675, 6380 reflections measured, and 3611 unique (Rint = 0.0196). These values were used in all calculations. The final wR(F2) was 0.0981 (all data). Crystal data of boscartin B (2): C20H36O6, M = 372.5, monoclinic, a = 6.0890(12) Å, b = 16.766(3) Å, c = 9.846(2) Å, β = 95.88(3)°, V = 999.9(3) Å3, T = 293 K, space group P21 (no. 4), Z = 2, μ(Cu Kα) = 0.729, 13 102 reflections measured and 3769 unique (Rint = 0.0513). These values were used in all calculations. The final wR(F2) was 0.0984 (all data). Crystal data of incensole oxide (3): C20H34O3, M = 322.47, orthorhombic, a = 6.38937(15) Å, b = 15.4884(4) Å, c = 18.7967(4) Å, V = 1860.16(7) Å3, T = 101.8 K, space group P212121 (no. 19), Z = 4, μ(Cu Kα) = 0.588, 6027 reflections measured and 3500 unique (Rint = 0.0195). These values were used in all calculations. The final wR(F2) was 0.0953 (all data). Crystal data of boscartin C (4): C20H34O4, M = 338.47, monoclinic, a = 6.9899(4) Å, b = 15.3898(8) Å, c = 8.8510(7) Å, β = 98.479 (7)°, V = 941.72(5) Å3, T = 100.2 K, space group P21 (no. 4), Z = 2, μ(Cu Kα) = 0.645, 5962 reflections measured and 3558 unique (Rint = 0.0188). These values were used in all calculations. The final wR(F2) was 0.0878 (all data). Assays of Cytotoxicity and XBP1-Activating Activity in Vitro. Cytotoxicity assay toward IEC-6 cells, the dual luciferase reporter assay in vitro for all isolated compounds, and the EC50 value assay for compounds 1, 5, 6, and 7 were carried out as described previously by our laboratory.11

recrystallized from acetone to afford colorless prisms (27 mg). The EtOAc fraction (300 g) was separated by vacuum CC on silica gel H, using gradient elution of petroleum ether−EtOAc (10:1 to 2:1), to give 14 subfractions, Fr. E1 to E14. Fr. E13 (petroleum ether−EtOAc, 2:1) was separated by RP-C18 vacuum CC, eluting with a gradient of MeOH in H2O (60−100%), to give 12 subfractions, Fr. E13-1 to Fr. E13-12. Fr. E13-3 (10.3 g; 60% MeOH) was recrystallized from acetone to give compound 2 as colorless prisms (100 mg). Fr. E13-4 (17.6 g; 70% MeOH) was fractionated by CC on silica gel H, using an isocratic elution system of petroleum ether−EtOAc (2:1), to give six subfractions, Fr. E13-4-1 to Fr. E13-4-6. Fr. E13-4-5 (8.3 g) was chromatographed on a column of silica gel H, using an isocratic elution system of CH2Cl2−acetone (8:1), to afford 15 subfractions, Fr. E13-4-51 to Fr. E13-4-5-15. Fr. E13-4-5-8 (200 mg) was separated by preparative RP-HPLC [mobile phase of MeOH−H2O (60%, v/v); flow rate of 6 mL min−1; and UV detection at 210 nm] to afford compounds 4 (tR = 8.67 min) and 6 (3.2 mg; tR = 5.80 min). Compound 4 was obtained as colorless prisms (29 mg) by recrystallization from acetone. Boscartin A (1): colorless prisms (acetone); mp 194.7−196.0 °C; [α]25 D −18 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 205 (3.77) nm; IR (neat) νmax 3355, 3264, 2958, 2878, 1678, 1474, 1449, 1380, 1092, 1067, 934 cm−1; 1H NMR (CDCl3, 400 MHZ), see Table 1; 13C NMR (CDCl3, 100 MHZ), see Table 3; positive ion mode HRESIMS m/z 339.2530 [M + H]+ (calcd for C20H35O4, 339.2535), m/z 361.2357 [M + Na]+ (calcd for C20H34NaO4, 361.2355). Boscartin B (2): colorless prisms (acetone); mp 215.5−215.9 °C; [α]25 D +62 (c 0.5, MeOH); IR (neat) νmax 3338, 3188, 2974, 2925, 2881, 1466, 1378, 1324, 1099, 1071, 1051, 880 cm−1; 1H NMR (CDCl3, 600 MHZ), see Table 1; 13C NMR (CDCl3, 100 MHZ), see Table 3; HRESIMS m/z 355.2480 [M + H]+ (calcd for C20H35O5, 355.2484), m/ z 377.2311 [M + Na]+ (calcd for C20H34NaO5, 377.2304). Incensole oxide (3): colorless prisms (acetone); mp 146.0−148.0 °C; [α]25 D −25 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 204 (3.62) nm; IR (neat) νmax 3449, 2956, 2923, 2862, 1667, 1462, 1449, 1386, 1293, 1085, 1043, 949, 860, 825 cm−1; 1H NMR (CDCl3, 600 MHZ), see Table 1; 13 C NMR (CDCl3, 150 MHZ), see Table 3; HRESIMS m/z 323.2587 [M + H]+ (calcd for C20H35O3, 323.2586), m/z 345.2407 [M + Na]+ (calcd for C20H34NaO3, 345.2406). Boscartin C (4): colorless prisms (acetone); mp 171.1−179.1 °C; [α]25 D −6 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 203 (3.69) nm; IR (neat) νmax 3526, 3299, 2962, 2928, 2872, 1642, 1466, 1440, 1385, 1365, 1100, 1053, 1022, 1005, 977 cm−1; 1H NMR (CDCl3, 600 MHZ), see Table 1; 13C NMR (CDCl3, 100 MHZ), see Table 3; HRESIMS m/z 361.2350 [M + Na]+ (calcd for C20H34NaO4, 361.2355). Boscartin D (5): colorless oil; [α]25 D −40 (c 0.8, MeOH); UV (MeOH) λmax (log ε) 206 (3.20), 240 (3.49) nm; Rh2(OCOCF3)4induced ECD (CHCl3) λmax (log Δε) 349 (1.34) nm; IR (neat) νmax 3475, 2960, 2925, 2875, 1713, 1672, 1451, 1384, 1370, 1095, 1080, 1034, 975 cm−1; 1H NMR (CDCl3, 600 MHZ), see Table 1; 13C NMR (CDCl3, 100 MHZ), see Table 3; HRESIMS m/z 337.2384 [M + H]+ (calcd for C20H33O4, 337.2379), m/z 359.2200 [M + Na]+ (calcd for C20H32NaO4, 359.2198). Boscartin E (6): colorless oil; [α]25 D +5 (c 0.3, MeOH); UV(MeOH) λmax (log ε) 204 (3.21), 223 (3.21) nm; IR (neat) νmax 3427, 2967, 2935, 2878, 1712, 1667, 1457, 1382, 1252, 1082, 1045, 978, 914, 733 cm−1; 1H NMR (CDCl3, 600 MHZ), see Table 2; 13C NMR (CDCl3, 150 MHZ), see Table 3; HRESIMS m/z 375.2142 [M + Na]+ (calcd for C20H32NaO5, 375.2147). Boscartin F (7): colorless oil; [α]25 D −13 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (3.54), 231 (3.72) nm; IR (neat) νmax 3456, 2964, 2939, 2876, 1727, 1663, 1460, 1384, 1285, 1098, 1080, 1044, 920, 877, 733 cm−1; 1H NMR (CDCl3, 400 MHZ), see Table 2; 13C NMR (CDCl3, 100 MHZ), see Table 3; HRESIMS m/z 337.2375 [M + H]+ (calcd for C20H33O4, 337.2379), m/z 359.2199 [M + Na]+ (calcd for C20H32NaO4, 359.2198). Boscartin G (8): colorless oil; [α]25 D +4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (3.34), 231 (3.53) nm; IR (neat) νmax 3455, 2963, 2939, 2878, 1728, 1662, 1455, 1384, 1289, 1076, 916, 886, 733 cm−1; 1H NMR (CDCl3, 400 MHZ), see Table 2; 13C NMR (CDCl3, 100 MHZ), see Table 3; HRESIMS m/z 337.2374 [M + H]+ (calcd for C20H33O4,

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00104. 1 H, 13C, and DEPT NMR spectra; 1H−1H COSY, HSQC, HMBC, and 2D NOESY/1D difference NOE spectra; and HRESIMS spectra for compounds 1−9 (DOCX) I

DOI: 10.1021/acs.jnatprod.5b00104 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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AUTHOR INFORMATION

Corresponding Author

*Tel (H. L. Qin): +86-010-83172503. Fax: +86-010-63017757. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Science and Technology Project of China (No. 2012ZX09301002-002; 2012ZX09301002-006; 2011ZX09307-002-01) and by PCSIRT (No. IRT 1007).

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REFERENCES

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DOI: 10.1021/acs.jnatprod.5b00104 J. Nat. Prod. XXXX, XXX, XXX−XXX