Hepatoprotective Prenylaromadendrane-Type Diterpenes from theGum Resin of Boswellia carteriiYan-gai Wang, Jin Ren, Ai-guo Wang, Jian-bo Yang, Teng-fei Ji, Qin-Ge Ma, Jin Tian, and Ya-lun Su*

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy ofMedical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China

*S Supporting Information

ABSTRACT: Chemical examination of the exuded gum resinof Boswellia carterii resulted in the isolation of nine newprenylaromadendrane-type diterpenes, boscartols A−I (1−9).The structures of these compounds were established byextensive 1D and 2D NMR spectroscopic analyses, massspectrometric data, and circular dichroism spectra. Compounds1−3, 5, 6, 8, and 9 (10 μM) showed moderate hepatoprotectiveactivity against D-galactosamine-induced HL-7702 cell damage.

Olibanum, a gum resin also known as frankincense, is theexudate produced by secretory tissue in the bark of the

Boswellia species that are native to Ethiopia, Somalia, India, andthe Arabic peninsula.1 Olibanum has been important in Unani(an Islamic system of healing) and Chinese medicine for thetreatment of rheumatoid arthritis, osteoarthritis, dysmenorrhea,and ulcers, as well as the swelling and pain from injuries.2

Previous chemical and pharmacological studies of olibanumhave shown that the extract and its constituents possess manytypes of biological activities, including anti-inflammatory,cytotoxic, antifungal, immunomodulatory, and antibacterialactivities, and these activities are due to diverse secondarymetabolites including triterpenes (such as boswellic acids) andcembrane-type diterpenes.3

To elucidate the structural diversity of olibanum and findnovel and effective lead compounds, a detailed chemicalanalysis of the gum resin of Boswellia carterii Birdw.(Burseraceae) was conducted. Chromatographic separation ofthe CH2Cl2 fraction of a 95% alcoholic extract led to theisolation of nine new prenylaromadendrane-type diterpenes(1−9). This type of diterpene has rarely been reported.2e,4 Tothe best of our knowledge, only one prenylaromadendrane-typediterpene has been isolated from olibanum, and its absoluteconfiguration has not yet been reported.2e We report herein theisolation, structural elucidation, and hepatoprotective activitiesof compounds 1−9.

■ RESULTS AND DISCUSSION

Boscartol A (1) was isolated as a colorless oil; its molecularformula was determined from the HREIMS data (m/z302.2233, calcd for C20H30O2, 302.2246) and was consistentwith six degrees of unsaturation. IR absorptions (3393 and1634 cm−1) suggested that it contained hydroxy and olefinicfunctionalities. The 13C NMR and DEPT spectra exhibited 20carbon resonances, which were attributed to three methyls, sixmethylenes, seven methines, and four tertiary carbons; six

olefinic carbons in three double bonds and two oxygenatedcarbons were indicated. Thus, a tricyclic skeleton for 1 wouldaccount for the remaining degrees of unsaturation. The subunitspin systems were established by the 1H−1H gCOSYrelationships from C-1 to C-3, from C-5 to C-9, from C-5 to

Received: June 30, 2013Published: November 6, 2013

Article

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© 2013 American Chemical Society andAmerican Society of Pharmacognosy 2074 dx.doi.org/10.1021/np400526b | J. Nat. Prod. 2013, 76, 2074−2079

C-1, and from C-16 to C-15 and C-17. Their connectivity wasdetermined through HMBC correlations. The HMBC relation-ships from H3-11 (δH 1.23, s) to C-3 (δC 41.7), C-4 (δC 80.8),and C-5 (δC 53.6) indicated the connection of the subunits toform a cyclopentane ring. HMBC relationships from the 1,1-disubstituted double-bond hydrogens H-12a (δH 4.72, s) andH-12b (δH 4.70, s) to C-1 (δC 52.9), C-9 (δC 38.5), and C-10(δC 152.9) confirmed the connection of the subunits to form acycloheptane ring. The two rings were fused at C-1 and C-5. Athree-membered ring was formed by C-6, C-7, and C-13, andthe methyl group (C-14) was deduced from the HMBCinteractions from H3-14 (δH 1.19, s) to C-6 (δC 31.5), C-7 (δC29.0), and C-13 (δC 26.55). Thus, the skeleton ofaromadendrane was established for compound 1. The prenylgroup was established by additional 1H−1H gCOSY cross-peaksbetween H-16 and H-15 and H-17 and HMBC relationshipsfrom H3-20 (δH 1.84, s) to C-17 (δC 128.4), C-18 (δC 133.5),and C-19 (δC 61.9) and from H2-19 (δH 4.26, s) to C-17, C-18,and C-20 (δC 21.5). The connection of the aromadendrane andprenyl subunits via C-13 was determined by the HMBCinteractions from H-15 to C-6, C-7, C-13, and C-14. Finally,the presence of OH groups at C-4 and C-19 was evident fromthe chemical shifts of C-4 (δC 80.8) and C-19 (δC 61.9).Therefore, a planar structure similar to that of olibanumol D2e

was established for 1.The relative configuration of 1 was established from the

coupling constants and NOESY interactions. The double bondswere determined to be 15E and 17Z according to the NOESYcross-peaks between H-15 and H-17 and between H-16 and H-19 and the coupling constants, JH‑15,H‑16 = 14.8 Hz and JH‑16,H‑17= 10.8 Hz. The NOESY relationship between H-6 and H-7suggested a cis-fusion between the cycloheptane and cyclo-

propane rings, and H-6 and H-7 were arbitrarily assigned as α-oriented. The α-orientations for H-1, H-15, and H3-11 wereevident from the NOESY interactions from H-6 to H-1, H-15,and H3-11. Lack of NOESY correlation between H-1 and H-5indicated that H-5 was β-oriented. Additional NOESYinteractions from H3-14 to H-5 indicated that H3-14 was alsoβ-oriented.The absolute configuration of the C-4 tertiary alcohol was

deduced by the CD data of the in s i tu- formed[Rh2(OCOCF3)4] complex,5 and the inherent contributionwas subtracted. The Rh complex of 1 showed a positive Cottoneffect at ca. 350 nm, correlating to a 4S absolute configurationon the basis of the bulkiness rule.5,6 Thus, the structure ofc o m p o u n d 1 w a s d e t e r m i n e d t o b e(1R,4S,5S,6R,7R,13S,15E,17Z)-4β-hydroxy-15-(3-methyl-4-hy-droxy-2-butenyl)aromadendr-10(12),15-diene, named boscar-tol A.Boscartol B (2) had the molecular formula C20H32O2, as

determined by the HRESIMS data (m/z 327.2292, calcd forC20H30NaO2, 327.2295). Its

1H and 13C NMR data (Tables 1and 3) featured a prenylaromadendrane-based skeleton closelyrelated to compound 1. The 1D and 2D NMR data revealedthat 2 shared the same aromadendrane structure as 1, but 2 hadonly one double bond in its prenyl group. Comparison of theNMR data disclosed that the double bond was between C-17and C-18. This assignment was supported by the chemicalshifts of H-15 (δH 1.35 and 1.18; each 1H, m)/C-15 (δC 43.5)and H-16 (δH 2.18 and 2.13; each 1H, m)/C-16 (δC 24.9) andthe HMBC cross-peaks from H-17 to C-15 and C-16, from H-16 to C-15, C-17, and C-18, and from H-15 to C-16 and C-17.Similar NOESY relationships and CD data indicated that 2possessed the same relative and absolute configurations as 1.

Table 1. 1HNMR Spectroscopic Data for Compounds 1−5a

no. 1 2 3 4b 5

1 2.23, td (10.8, 6.0) 2.22, td (10.0, 6.0) 2.22, td (10.0, 6.0) 2.26, td (10.8, 6.4) 2.24, td (10.8, 6.8)2a 1.91, m 1.88, m 1.90, m 1.91, m 1.69, m2b 1.65, m 1.64, m 1.66, m 1.63, m 1.58, m3a 1.78, m 1.74, m 1.78, m 1.74, m 1.85, m3b 1.57, m 1.60, m 1.62, m 1.61, m 1.22, m4 2.08, m5 1.45, t (10.8) 1.32, t (10.0) 1.36, t (10.0) 1.35, t (10.8) 1.40, q (10.8)6 0.83, dd (10.8, 10.0) 0.51, dd (10.8, 10.0) 0.56, dd (11.2, 10.0) 0.61, dd (10.8, 10.0) 0.62, dd (10.8, 9.6)7 0.76, m 0.72, ddd (10.8, 10.0, 6.4) 0.77, ddd (11.2, 10.0, 6.0) 0.99, m 0.87, ddd (10.8, 9.6, 6.0)8a 2.00, m 1.95, m 2.03, m 2.07, m 2.00, m8b 1.11, m 0.98, m 1.07, m 1.05, m 1.00, m9a 2.45, dd (13.2, 6.0) 2.43, dd (12.8, 6.4) 2.43, dd (13.2, 6.0) 2.45, dd (13.2, 6.4) 2.43,d (13.2, 6.0)9b 2.06, m 2.03, t (12.8) 2.07, m 2.04, m 2.07, m11 1.23, s 1.29, s 1.29, s 1.26, s 0.96, d (7.2)12a 4.72, s 4.70, s 4.70, s 4.72, s 4.63, s12b 4.70, s 4.67, s 4.67, s 4.70, s 4.63, s14 1.19, s 1.02, s 1.01, s 1.02, s 1.04, s15a 5.30, d (14.8) 1.35, m 2.00, m 2.85, d (7.6) 1.78, dd (14.4, 4.0)15b 1.18, ddd (16.0, 10.0, 6.0) 1.93, m 1.13, dd (14.4, 10.4)16a 6.25, dd (14.8, 10.8) 2.18, m 5.65, m 5.70, dd (16.0, 7.6) 4.70, ddd (10.4, 8.0, 4.0)16b 2.13, m17 5.89, d (10.8) 5.29, m 5.61, d (15.6) 5.80, d (16.0) 5.34, d (8.0)19a 4.26, s 4.16, d (12.0) 1.31, s 1.34, s 4.23, d (12.4)19b 4.12, d (12.0) 4.03, d (12.4)20 1.84, s 1.78, t (1.2) 1.31, s 1.33, s 1.81, d (1.2)

a1H NMR spectra (δH) were measured in CDCl3 at 400 MHz for 1−5. Coupling constants (J) in Hz are given in parentheses. bData for OMe-12 in4: δH 3.27 (s). The assignments were based on NOESY, HSQC, and HMBC experiments.

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Boscartol C (3) was assigned the same molecular formula,C20H32O2 (five degrees of unsaturation), as 2 by HRESIMS. 1Hand 13C NMR data (Tables 1 and 3) suggested that 3 was also aprenylaromadendrane-type diterpene. The principal differencesbetween 3 and 2 were the presence of a double bond betweenC-16 and C-17 and an OH at C-18 (in the prenyl group) in 3.These differences were supported by the HMBC correlationsfrom H-16 (δH 5.65, m) to C-15 (δC 45.3), C-17 (δC 139.7),and C-18 (δC 70.6), from H-17 [δH 5.61, d (15.6 Hz)] to C-15,C-16 (δC 124.7), C-18, C-19 (δC 29.9), and C-20 (δC 29.8),and from H3-19 and H3-20 to C17 and C-18. The relative andabsolute configurations of 3 were the same as those of 1, asdeduced from similar NOESY correlations and the CD data ofthe in situ-formed [Rh2(OCOCF3)4] complex.The NMR data of boscartol D (4, C21H32O2) (Tables 1 and

3) were comparable to those of 3, except for the presence of anadditional methoxy group. The methoxy and oxygenatedmethine hydrogens were at δH 3.27 (3H, s) and δH 2.85(1H, d, J = 7.6 Hz), respectively, and their correspondingcarbons were at δC 56.3 (CH3) and 90.3 (CH). Because of thefree rotation of the C-13 and C-15 single bond, the absolute

configuration at C-15 was not determined. Biogeneticconsiderations and the similar NOE interactions shown by 4and 3 suggest that these compounds have the sameconfigurations at the other stereogenic centers.Boscartol E (5) had the molecular formula C20H32O2. The

NMR spectra of 5 (Tables 1 and 3) were similar to those of 2,with the exception that the resonance of C-4 was shifted upfield(δC 35.5) compared to that of 2. This suggested the absence ofan OH at C-4 in 5, which was supported by the characteristic1H NMR signal of H3-11 [δH 0.96, d (7.2 Hz)]. Anotherexception was in the prenyl group, which showed the presenceof two OH groups. The OH groups were at C-16 and C-19, asshown by the HMBC correlations from H-16 [δH 4.70, ddd(10.4, 8.0, 4.0 Hz)] to C-15 (δC 50.4), C-17 (δC 131.0), and C-18 (δC 138.2) and from H2-19 [δH 4.29 and 4.03, each 1H, d(12.4 Hz)] to C-17, C-18, and C-20 (δC 21.8). The double-bond geometry was determined to be 17Z according to theNOESY correlations of H-16/H2-19 and H-17/H3-20. Therelative configurations of 5 were the same as those of 2, asindicated by the NOESY spectrum. The 16S configuration at C-16 was determined on the basis of CD data of the in situ-formed [Rh2(OCOCF3)4] complex.The HRESIMS data of boscartol F (6) agreed with a

molecular formula of C20H28O2 and the presence of sevendegrees of unsaturation. The IR spectrum indicated theexistence of OH (3429 cm−1), aldehyde (2736, 1685 cm−1),and double bond (1654, 1613 cm−1) groups. The 1H NMRspectrum of 6 indicated the presence of three methyls [δH 1.82(3H, s, H3-20), 1.25 (3H, s, H3-14), 1.20 (3H, s, H3-11)] and aconjugated diene aldehyde system [δH 9.34 (1H, s, CHO), 6.79(1H, d, J = 11.4 Hz, H-17), 6.43 (1H, dd, J = 15.0, 11.4 Hz, H-16), 5.80 (1H, d, J = 15.0 Hz, H-15)], which was confirmed bythe λmax (304 nm) in the UV spectrum. HMBC correlationsindicated that the planar structure of compound 6 was the sameas hemerocallal A.4f The 15E and 17Z geometries were deducedfrom the JH‑15/H‑16 (15.0 Hz) and JH‑16/H‑17 (11.4 Hz) values.The singlet peak of H3-20 proved that H3-20 and H-17 were ina cis-relationship, further supporting the 17Z assignment.4f

Compound 7 had the molecular formula C20H28O3 byHRESIMS. The NMR data (Tables 2 and 3) indicated a partialaromadendrane structure similar to that of 1. IR absorptionsrevealed OH (3434, 3358 cm−1) and α,β-unsaturated ketone(1689, 1632 cm−1) functionalities. The NMR spectra revealedan α,β-unsaturated ketone fraction [(δH 7.15, m)/(δC 156.2,143.7, 208.0)] in the prenyl subunit, which was supported bythe HMBC correlations from H-16 [δH 4.87, dd (4.0, 2.0)] toC-15 (δC 67.7), C-17 (δC 156.2), and C-18 (δC 143.7); fromH3-20 [δH 1.80, t (1.5)] to C-17, C-18, and C-19 (δC 208.0);from H-17 (δH7.15, m) to C-15, C-16, C-18, and C-19; andfrom H-15 [δH 1.63, d (2.0)] to C-14, C-16, and C-19. Theassignments were supported by the molecular weight anddegrees of unsaturation. Biogenetic considerations and thesimilar ROESY interactions of 7 and 1 resulted in theassignment of the same configurations as those of thearomadendrane skeleton for the corresponding stereogeniccenters of these compounds.In addition, the ROESY correlations of H-6/H-7/H-15 and

H3-14/H-16 demonstrated that H-15 and H-16 were in a trans-relationship. From the MM2 energy-minimized conformation,two conformations agreed with the ROESY correlations. TheCD spectrum displayed positive and negative Cotton effects at230 nm (Δε +4.49) and 350 nm (Δε −0.51), respectively,which predicted the 15S, 16R conformation, according to the

Table 2. 1HNMR Spectroscopic Data of Compounds 6−9a

no. 6 7 8 9

1 2.23, td (10.8,6.0)

2.33, td (10.0,6.5)

2.23, td (10.0,6.5)

2.25, td (10.5,6.5)

2a 1.91, dq(12.0, 6.0)

1.95, m 1.93, m 1.95, m

2b 1.64, m 1.69, m 1.64, m 1.67, m3a 1.77, ddd

(12.6, 6.0,1.8)

1.85, dd (12.0,9.5)

1.78, m 1.79, m

3b 1.57, td (12.6,6.0)

1.70, m 1.63, m 1.64, m

45 1.50, t (10.8) 1.43, t (10.0) 1.37, t (10.0) 1.39, t (10.5)6 1.00, t (10.8) 0.84, t (10.0) 0.55, dd

(11.0, 10.0)0.67, dd(10.5,10.0)

7 1.23, dd(10.8, 4.2)

1.13, ddd (10.5,10.0, 6.5)

0.93, ddd(11.0, 10.0,6.0)

0.76, ddd (11.0,10.5, 6.0)

8a 2.04, ddd(12.6, 10.8,6.0)

2.12, dddd (12.5,10.5, 6.5, 1.0)

2.06, m 2.06, m

8b 1.17, m 0.98, m 1.01, m 1.05, m9a 2.45, dd

(12.6, 6.0)2.45, dd (12.5,6.5)

2.43, dd(13.0, 6.0)

2.45, dd (13.0,5.5)

9b 2.07, t (12.6) 2.01, t (12.5) 2.02, m 2.03, t (13.0)11 1.22, s 1.40, s 1.29, s 1.35, s12a 4.71, s 4.75, s 4.71, s 4.73, s12b 4.68, s 4.71, s 4.69, s 4.70, s14 1.27, s 0.80, s 1.14, s 1.16, s15a 6.79, d (11.4) 1.63, d (2.0) 1.73, dd

(14.5, 4.5)1.56, m

15b 1.40, dd(14.5, 9.0)

1.56, m

16 6.43, dd(15.0, 11.4)

4.87, dd (4.0,2.0)

5.05, m 5.08, m

17 5.79, d (15.0) 7.15, m 7.17, m 7.06, m19a 9.36, s19b20 1.82, s 1.80, t (1.50) 1.91, t (2.0) 1.91, t (2.0)

a1H NMR spectra (δ) were measured in CDCl3 at 600 MHz for 6 andat 500 MHz for 7−9. Coupling constants (J) in Hz are given inparentheses. The assignments were based on NOESY, HSQC, andHMBC experiments.

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literature.7 Therefore, the absolute configuration of compound7 was assigned as 1R, 4S, 5S, 6R, 7R, 13S, 15S, 16R.The molecular formula of boscartol H (8) was the same as 7,

and IR absorptions (3449, 1746, and 1634 cm−1) suggestedthat it contained OH, carbonyl, and olefinic groups. Analysis ofits NMR data (Tables 2 and 3) revealed the same partialaromadendrane structure as 7. Additional NMR resonancescorresponding to a methylene group (δH 1.73, 1.40)/(δC 46.7)and an α,β-unsaturated lactone (δH 7.17, m)/δC (129.5, 149.5,174.3) were observed in the spectrum of 8. The assignmentswere confirmed by the HMBC correlations from H-15 (δH1.73,1.40) to C-17 (δC 149.5); from H-16 (δH 5.05, m) to C-15 (δC46.7), C-17 (δC 149.5), and C-18 (δC 129.5); from H-17 (δH7.17, m) to C-16 (δC 80.0), C-18, C-19, and C-20 (δC 10.6);and from H3-20 to C-17, C-18, and C-19. Similar ROESYcorrelations indicated that 7 and 8 had the same relativeconfigurations. The absolute configuration of C-16 wasdetermined from the circular dichroism (CD) spectrum. TheCD spectrum of 8 showed negative Cotton effects at 230.5 nm(Δε −2.49), and the 16S configuration was consistent with theCD spectrum.8 Hence, the absolute configuration of compound8 was assigned as 1R, 4S, 5S, 6R, 7R, 13S, 16S.Boscartol I (9) has the same molecular formula as 8, and the

NMR spectra of 9 strongly resembled those of 8. Examinationof the ROESY interactions of 9 revealed that the relativeconfigurations of the chiral centers at C-1, C-4, C-5, C-6, C-7,and C-14 were identical to those of 8. However, a slightdifference of chemical shift (in CDCl3) was observed for H-15(δH 1.56, 2H, m), as the H-15 of the corresponding resonancesof 8 was split into two peaks [(δH 1.73, dd (14.5) and 1.40, dd(14.5, 9.0)]. These findings suggested that 9 was the C-16epimer of 8. The positive Cotton effect at 231.5 nm (Δε +2.41)in the CD spectrum of 9 confirmed this assignment.Compounds 1−9 were tested for cytotoxicity against five

human tumor cell lines, HCT-8 (human ileocecal adenocarci-noma), Bel-7402 (human hepatoma), BGC-823 (human gastriccancer), A549 (human lung epithelial), and A2780 (human

ovarian cancer), and for neuroprotective activity against MPP+-induced neural injury. However, the compounds showedneither cytotoxicity (IC50 >10 μM, all cell lines) norneuroprotective activity. They were also bioassayed forhepatoprotective activity against D-galactosamine-induced tox-icity in HL-7702 cells, and the hepatoprotective activity drugbicyclol was used as the positive control.9 As shown in Table 4,compounds 1−3, 5, 6, 8, and 9 exhibited moderatehepatoprotective activity.

■ EXPERIMENTAL SECTIONGeneral Experimental Procedures. Optical rotations were

measured using a Rudolph Research Autopol III automatic polar-imeter. UV, CD, and IR spectra were recorded using a Cary 300spectrometer, a JASCO J-815 CD spectrometer, and a Nicolet 5700FT-IR spectrometer (FT-IR microscope transmission), respectively.1H and 13C NMR data were acquired with Varian Mercury-400 andInova-500 spectrometers using a solvent signal (CDCl3) as a reference.

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

no. 1 2 3 4b 5 6 7 8 9

1 52.9 52.7 52.8 51.8 53.2 52.8 48.2 52.2 51.32 26.6 26.2 26.3 25.6 29.2 26.7 23.7 26.0 25.53 41.7 41.7 41.6 41.3 35.0 41.9 38.9 41.9 40.94 80.8 81.0 81.1 81.0 35.5 80.8 79.9 80.8 80.65 53.6 53.8 53.8 52.8 43.3 53.4 52.8 53.6 53.46 31.5 28.9 28.2 23.7 27.3 32.9 24.4 28.5 28.17 29.0 26.8 26.3 27.1 26.6 30.5 23.4 26.9 26.68 24.3 24.8 24.8 24.6 24.5 24.3 25.2 24.6 24.89 38.5 38.8 38.8 38.6 38.8 38.2 38.8 38.6 38.710 152.9 153.5 153.4 153.3 154.2 152.4 154.1 153.2 153.411 26.6 24.2 24.3 27.4 21.3 26.1 22.2 22.0 21.712 106.8 106.3 114.5 106.7 105.8 107.3 106.8 106.6 106.713 21.6 25.6 26.1 25.9 17.0 28.2 23.6 25.4 25.114 12.7 13.7 14.0 9.5 13.2 12.2 10.7 14.2 14.015 144.4 43.5 45.3 90.3 50.4 155.8 67.7 46.7 46.916 119.7 24.9 124.7 125.4 66.3 120.1 72.4 80.0 79.817 128.4 123.9 139.7 141.0 131.0 149.9 156.2 149.5 148.918 133.5 134.0 70.6 70.5 138.2 135.0 143.7 129.5 129.519 61.9 61.5 29.9 29.8 62.2 194.9 208.0 174.3 173.720 21.5 21.3 29.8 29.7 21.8 9.4 10.0 10.6 10.7

a13C NMR spectra (δ) were measured in CDCl3 at 100 MHz for 1−6 and at 125 MHz for 7−9. bData for OMe-12 in 4: δC 56.3. The assignmentswere based on NOESY(ROESY), HSQC, and HMBC experiments.

Table 4. Hepatoprotective Effects of Compounds 1−3, 5, 6,8, and 9 (10 μM) against D-Galactosamine-Induced Toxicityin HL-7702 Cellsa

compound cell survival rate (% of normal) inhibition (% of control)

normal 100 ± 1.1control 58 ± 1.5bicyclol 76 ± 5.1b 42.51 69 ± 1.5c 26.12 73 ± 1.5c 33.63 70 ± 0.6d 27.05 73 ± 2.0c 36.36 88 ± 2.4d 69.68 68 ± 1.5b 24.39 73 ± 2.0c 33.6

aResults are expressed as means ± SD (n = 3; for normal and control,n = 6); bicyclol was used as positive control (10 μM). bp < 0.05. cp <0.01. dp < 0.001.

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EIMS and HREIMS data were measured using a Micromass Autospec-Ultima ETOF spectrometer. ESIMS data were obtained using a Q-Trap LC/MS/MS (turbo ionspray source) spectrometer. Columnchromatography (CC) was performed using silica gel H, 160−200mesh and 200−300 mesh (Qingdao Marine Chemical Inc., China).HPLC separation was performed using an Agilent 1200 series with a4.6 × 250 mm DIKMA-analyzed column packed with C18 (5 μm).Preparative HPLC was performed using a Shimadazu LC-6ADinstrument with an SPD-20A detector and a YMC-Pack ODS-Acolumn (250 × 20 mm, 5 μm). TLC utilized precoated silica gelGF254 plates. The spots were visualized under UV light (254 or 356nm) or by spraying with 10% H2SO4 in 95% EtOH and 5%anisaldehyde followed by heating.Plant Material. Gum resin of B. carterii was obtained from the

Beijing Tongrentang Pharmacy, People’s Republic of China, which wasverified by Mr. Wanzhi Song (Institute of Materia Medica, Beijing100050, China). A voucher specimen (No. ID-S-2385) was depositedat the herbarium of the Department of Medicinal Plants, Institute ofMateria Medica, Chinese Academy of Medical Sciences and PekingUnion Medical College, Beijing 100050, China.Extraction and Isolation. The gum resin of B. carterii (11.8 kg)

was extracted three times with 95% EtOH. The extracts werecombined and evaporated under reduced pressure to yield a darkbrown residue (8.2 kg). The residue was separated by silica gel columnchromatography (160−200 mesh, 20 × 200 cm) and elutedsuccessively with petroleum ether (20 L), CH2Cl2 (20 L), EtOAc(20 L), and MeOH (10 L) to yield four fractions (A−D). Fraction B(500 g) was separated by silica gel CC into bands B1 to B19. FractionB14 (64.4 g) was separated on a RPC18 column (60% to 100% CH3OHin H2O) to provide subfractions B14‑1−B14‑19. Separation of B14‑3 (3.46g) repeatedly by silica gel CC, followed by RP HPLC (70% MeOH inH2O), gave 6 (15 mg) and 7 (119 mg). Fraction B14‑4 (2.31 g) waschromatographed over silica gel and further fractionated using RPHPLC (60% MeOH in H2O) to afford 8 (12 mg) and 9 (4 mg).Fraction B14‑7 (6.70 g) was separated by silica gel CC to give B14‑7‑1−B14‑7‑6. Fraction B14‑7‑5 (1.76 g) was separated by silica gel H CCeluting with 3:1 petroleum ether/acetone and then further fractionatedby RP HPLC (80% MeOH in H2O) to afford 1 (16 mg), 2 (19 mg), 3(41 mg), and 5 (118 mg). B14‑7‑6 (0.92 g) was fractionated via silica gel(7:1 CH2Cl2/acetone) followed by RP HPLC (75% MeOH in H2O)to yield 4 (8 mg).Boscarterol A (1): colorless oil; [α]20D −14.6 (c 1.0, CHCl3); UV

(CHCl3) λmax (log ε) 257 (3.23) nm; Rh2(OCOCF3)4-induced CD (c3.4 × 10−3 M, CHCl3) λmax (Δε) 350 (+0.0082) nm; IR νmax 3393(br), 2938, 2869, 1634, 1451, 1377, 1261, 1095 cm−1; 1H NMR(CDCl3, 400 MHz) data, see Table 1; 13C NMR (CDCl3, 100 MHz)data, see Table 3; HREIMS m/z 302.2233 (calcd for C20H30O2,302.2246).Absolute Configuration of the Tertiary Alcohol in 1. A sample

of 1 (2.59 mg) was dissolved in a dry solution of CHCl3 (2.59 mL).The first CD spectrum was recorded. Then, the CHCl3 solution of 1was mixed with [Rh2(OCOCF3)4] complex (1.8 mg), and its timeevolution was monitored until stationary (ca. 5 min after mixing). Theinherent CD was subtracted. The observed sign of the E band at ca.350 nm in the induced CD spectrum was correlated to the absoluteconfiguration of the C-4 tertiary moiety.Boscarterol B (2): colorless oil; [α]20D −6.25 (c 1.6, CHCl3); UV

(CHCl3) λmax (log ε) 250 (2.68) nm; Rh2(OCOCF3)4-induced CD (c3.3 × 10−3 M, CHCl3) λmax (Δε) 353.5 (+0.017) nm; IR νmax 3374(br), 3081, 2924, 2863, 1667, 1634, 1451, 1376, 1096 cm−1; 1H NMR(CDCl3, 400 MHz) data, see Table 1; 13C NMR (CDCl3, 100 MHz)data, see Table 3; (−)-ESIMS m/z 339.4 [M + Cl]−; (−)-ESIMS m/z339.4 [M + Cl]−; (+)-HRESIMS m/z 327.2292 (calcd forC20H32O2Na, 327.2295).Boscarterol C (3): colorless oil; [α]20D −14.5 (c 1.0, CHCl3); UV

(CHCl3) λmax (log ε) 245 (2.57) nm; Rh2(OCOCF3)4-induced CD (c3.3 × 10−3 M, CHCl3) λmax (Δε) 346 (+0.015) nm; IR νmax 3419 (br),2975, 2893, 1637, 1454, 1379, 1139, 1095 cm−1; 1H NMR (CDCl3,400 MHz) data, see Table 1; 13C NMR (CDCl3, 100 MHz) data, seeTable 3; (+)-ESIMS m/z 327.2 [M + Na]+, (−)-ESIMS m/z 339.5 [M

+ Cl]−; (+)-HRESIMS m/z 327.2293 (calcd for C20H32O2Na,327.2295).

Boscarterol D (4): colorless oil; [α]20D −7.7 (c 0.2, CHCl3); UV(CHCl3) λmax (log ε) 242 (2.88) nm; IR νmax 3404 (br), 2964, 2853,1635, 1456, 1376, 1238, 1091 cm−1; 1H NMR (CDCl3, 400 MHz)data, see Table 1; 13C NMR (CDCl3, 100 MHz) data, see Table 3;(+)-HRESIMS m/z 317.2475 (calcd for C21H33O2, 317.2475).

Boscarterol E (5): colorless oil; [α]20D −0.47 (c 2.38, CHCl3); UV(CHCl3) λmax (log ε) 246 (2.12) nm; Rh2(OCOCF3)4-induced CD (c3.3 × 10−3 M, CHCl3) λmax (Δε) 350 (+0.46) nm; IR νmax 3421 (br),2965, 1467, 1372, 1235, 1028 cm−1; 1H NMR (CDCl3, 400 MHz)data, see Table 1; 13C NMR (CDCl3, 100 MHz) data, see Table 3;HREIMS m/z 304.2384 (calcd for C20H32O2 304.2402).

Boscarterol F (6): colorless oil; [α]28D −28.6 (c 0.36, CHCl3); UV(CHCl3) λmax (log ε) 304 (4.62) nm; IR νmax 3429 (br), 2962, 2738,2919, 2835, 1654, 1613, 1447, 1388, 1206, 1011 cm−1; 1H NMR(CDCl3, 600 MHz) data, see Table 2; 13C NMR (CDCl3, 100 MHz)data, see Table 3; HREIMS [M]+ m/z 300.2085 (calcd for C20H28O2,300.2089).

Boscarterol G (7): colorless oil; [α]20D +63.2 (c 3.6, CHCl3); UV(CHCl3) λmax (log ε) 243 (2.97) nm; CD (c 3.2 × 10−3 M, MeOH)λmax (Δε) 230 (+4.49), 330.5 (−0.51) nm; IR νmax 3434 (br), 3358,3093, 2930, 2870, 1689, 1632, 1448, 1377, 1289, 1049 cm−1; 1H NMR(CDCl3, 500 MHz) data, see Table 2; 13C NMR (CDCl3, 125 MHz)data, see Table 3; (+)-HRESIMS m/z 339.1938 (calcd forC20H28NaO3, 339.1931).

Boscarterol H (8): colorless oil; [α]20D −41.0 (c 0.53, CHCl3); UV(CHCl3) λmax (log ε) 243 (2.89) nm; CD (c 3.2 × 10−3 M, MeOH)λmax (Δε) 230 (−2.50) nm; IR νmax 3449 (br), 3079, 2927, 2867, 1746,1634, 1471, 1449, 1375, 1204, 1097 cm−1; 1H NMR (CDCl3, 500MHz) data, see Table 2; 13C NMR (CDCl3, 125 MHz) data, see Table3; (+)-ESIMS m/z 339.3 [M + Na]+; (−)-ESIMS m/z 351.5 [M +Cl]−; (+)-HRESIMS m/z 339.1938 (calcd for C20H28O3Na,339.1931).

Boscarterol I (9): colorless oil; [α]20D −7.7 (c 0.2, CHCl3);UV(CHCl3) λmax (log ε) 242 (2.88) nm; CD (c 3.2 × 10−3 M,MeOH) λmax (Δε) 230.5 (2.41) nm; IR νmax 3467 (br), 2927, 2866,1754, 1634, 1450, 1377, 1099 cm−1; 1H NMR (CDCl3, 500 MHz)data, see Table 2; 13C NMR (CDCl3, 125 MHz) data, see Table 3;(+)-ESIMS m/z 339.3 [M + Na]+; (−)-ESIMS m/z 351.3 [M + Cl]−;(+)-HRESIMS m/z 339.1936 (calcd for C20H28O3Na, 339.1931).

Cytotoxicity Assay. Compounds 1−9 were tested for cytotoxicityagainst HCT-8 (human colon carcinoma), Bel-7402 (human livercarcinoma), BGC-823 (human stomach carcinoma), A549 (humanlung carcinoma), and A2780 (human ovarian carcinoma) by means ofan MTT method described in the literature.10

Protective Effect on Cytotoxicity Induced by D-Galactos-amine in HL-7702 Cells. The hepatoprotective effects of compounds1−9 were determined by a (MTT) colorimetric assay9,10 in HL-7702cells. Each cell suspension of 2 × 104 cells in 200 μL of RPMI 1640containing fetal calf serum (10%), penicillin (100 U/mL), andstreptomycin (100 μg/mL) was placed in a 96-well microplate andprecultured for 24 h at 37 °C under a 5% CO2 atmosphere. Freshmedium (100 μL) containing bicyclol and test samples was added, andthe cells were cultured for 1 h. The cultured cells were exposed to 25mM D-galactosamine for 24 h. Then, 100 μL of 0.5 mg/mL MTT wasadded to each well after the withdrawal of the culture medium andincubated for an additional 4 h. The resulting formazan was dissolvedin 150 μL of DMSO after aspiration of the culture medium. Theoptical density (OD) of the formazan solution was measured on amicroplate reader at 492 nm. Inhibition (%) was obtained by thefollowing formula:

= −

− ×

inhibition (%) [(OD OD )

/(OD OD )] 100

(sample) (control)

(normal) (control)

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■ ASSOCIATED CONTENT*S Supporting InformationThis material (1H, 13C NMR, DEPT, HSQC, HMBC, COSY,NOESY, ESI, HRESIMS, IR, ORD, and UV spectroscopic datafor compounds 1−9) is available free of charge via the Internetat http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Tel: 86-10-63165327. Fax: 86-10-63017757. E-mail: suyalun@imm.ac.cn.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSFinancial support from the National Science and TechnologyProject of China (No. 2012ZX09301002-002) and PCSIRT(No. IRT1007) and from the National Science and TechnologyProject of China (2011ZX09307-002-01) is acknowledged..

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