Dibenzocyclooctadiene Lignans and Norlignans ... - ACS Publications

Jan 17, 2013 - Sciences, Kunming 650204, Yunnan, People,s Republic of China. ‡ ... Key Laboratory of Tobacco Chemistry of Yunnan Province, Yunnan ...
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Dibenzocyclooctadiene Lignans and Norlignans from Fruits of Schisandra wilsoniana Guang-Yu Yang,†,§ Rui-Rui Wang,‡ Huai-Xue Mu,†,⊥ Yin-Ke Li,†,⊥ Xiao-Nian Li,† Liu-Meng Yang,‡ Yong-Tang Zheng,‡ Wei-Lie Xiao,*,† and Han-Dong Sun*,† †

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, Yunnan, People’s Republic of China ‡ Key Laboratory of Animal Models and Human Disease Mechanisms and Laboratory of Molecular Immunopharmacology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223, Yunnan, People’s Republic of China § Key Laboratory of Tobacco Chemistry of Yunnan Province, Yunnan Academy of Tobacco Science, Kunming 650106, Yunnan, People’s Republic of China ⊥ School of Chemistry and Biotechnology, Yunnan Nationalities University, Kunming 650031, Yunnan, People’s Republic of China S Supporting Information *

ABSTRACT: Seven new dibenzocyclooctadiene lignans, marlignans M−S (1−7), four new norlignans, marphenols C−F (8−11), and 21 known compounds (12−32) were isolated from the fruits of Schisandra wilsoniana. The structures of 1−11 were elucidated by spectroscopic methods including 1D- and 2D-NMR techniques and CD experiments. Compounds 1−11 were evaluated for their anti-HIV activities and showed EC50 values in the range 2.97−6.18 μg/mL and therapeutic index values of 5.33−29.13.

P

lants of the genus Schisandra are used commonly in Traditional Chinese Medicine for their diverse beneficial bioactivities.1,2 The fruits of Schisandra plants are commonly used in China as sedative and tonic agents.3 Previous studies have shown that this genus is a rich sources of lignans4 and triterpenoids.5 Schisandra wilsoniana A. C. Smith (Schisandraceae) is a climbing plant mainly distributed in Heqing, Lijiang, Dali, and Yulong Prefectures of Yunnan Province in mainland China.6,7 A number of bioactive compounds, such as highly oxygenated nortriterpenoids, carotane sesquiterpenoids, and dibenzocyclooctadiene lignans, have been isolated from this plant, and some of these compounds showed in vitro anti-HIV-1 and antiHBV activities.7−10 Our group has previously studied the leaves and stems of this plant.7,8 Motivated by a search for new bioactive metabolites, our group has investigated chemical constituents of the fruits of S. wilsoniana. We now report the isolation and characterization of seven new dibenzocyclooctadiene lignans, four new norlignans, and 21 known substances. The structures of the new compounds (1−11) were established by means of MS and extensive NMR spectra. The absolute configurations of 1−7 were determined by CD and ROESY experiments. This paper deals with the isolation, structural characterization, and anti-HIV activity of these compounds.

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EtOAc layer was subjected repeatedly to column chromatography on silica gel, Sephadex LH-20, RP-18, and semipreparative RP-HPLC to afford 11 new lignans [marlignans M−S (1−7) and marphenols C−F (8−11)] and 21 known lignans (12−32). Information related to the known compounds can be seen in the Supporting Information.

RESULTS AND DISCUSSION

A 70% aqueous acetone extract prepared from the fruit of S. wilsoniana was partitioned between EtOAc and H2O. The © 2013 American Chemical Society and American Society of Pharmacognosy

Received: October 26, 2012 Published: January 17, 2013 250

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Compound 1 was obtained as a yellow gum and assigned the molecular formula C23H30O7, from its HRESIMS [m/z 419.2074 [M + H]+ (calcd m/z 419.2070)]. The 1H, 13C, and DEPT NMR spectra showed signals for 23 carbon and 30 hydrogen atoms, corresponding to two aromatic rings (δC 107.2−153.7) with two aromatic protons (δH 6.83 and 6.75), one methylene (δC 39.4), two methines (δC 38.3 and 39.8), one oxygenated methine (δC 80.4), two methyls (δC 16.6 and 16.6), five O-methyl groups (δC 55.8, 55.9, 60.5, 60.6, and 60.8), and one phenolic OH proton (δH 10.23). The IR spectrum showed absorption bands of OH (3452 cm−1) and aromatic (1635, 1588, 1542, and 1476 cm−1) groups, and the UV spectrum showed absorption bands at 211 and 242 nm. The 1H−1H COSY correlations of H-6/H-7/H-8/H-9, H-7/H-17, and H-8/ H-18, together with HMBC correlations (Figure 1) of H-11

Figure 2. Key ROESY (↔) correlations of 1.

Compound 2 was obtained as a yellow gum and showed an ion at m/z 405.1918 [M + H]+ in the HRESIMS (calcd m/z 405.1913), corresponding to the molecular formula C22H28O7. The 1H and 13C NMR spectra of 2 were similar to those of 1. The obvious chemical shift differences were the disappearance of an O-methyl group signal and the appearance of a phenolic OH signal (δH 11.22) in 2. This implied that 2 was a dibenzocyclooctadiene lignan possessing two phenolic OH groups and four O-methyl groups. The CD spectrum gave a negative Cotton effect at 250 nm and a positive Cotton effect at 223 nm, indicating that 2 also had an S-biphenyl configuration.11 HMBC correlations of one OH signal (δH 10.52) with C-1 (δC 148.7), C-2 (δC 140.6), and C-16 (δC 120.4) and another OH signal (δH 11.22) with C-13 (δC 141.3), C-14 (δC 148.1), and C-15 (δC 119.9) revealed that the OH groups were located at C-1 and C-14. The four O-methyl groups were located at C-2, C-3, C-12, and C-13, respectively, as supported by HMBC correlations of the four O-methyl proton signals (δH 3.85, 3.91, 3.92, and 3.96) with C-2 (δC 140.6), C-3 (δC 152.3), C-12 (δC 151.9), and C-13 (δC 141.3). Thus, the structure of 2 was established, and it was given the trivial name marlignan N. Comparison of the NMR data of 3 with those of 1 and 2 disclosed that 3 was a dibenzocyclooctadiene lignan possessing one methylenedioxy group (δC 101.6; δH 5.90, 5.99), one phenolic OH group (δH 11.29), and three O-methyl groups (δC 60.7, 59.6, 60.7; δH 3.93, 3.93, 3.98). The CD spectrum indicated that 3 also had an S-biphenyl configuration.11 The methylenedioxy group attached at C-12 and C-13 was supported by HMBC correlations of the methylenedioxy protons (δH 5.90, 5.99) with C-12 (δC 148.4) and C-13 (δC 137.1). HMBC correlations of the phenolic OH (δH 11.29) with C-2 (δC 142.4), C-3 (δC 152.0), and C-4 (δC 111.7) suggested that the OH was attached to C-3. The O-methyl groups at C-1, C-2, and C-14 were supported by HMBC correlations between three O-methyl protons (3.93, 3.93, and 3.98) and C-1 (δC 153.0), C-2 (δC 142.4), and C-14 (δC 139.9), respectively. Thus, the structure of 3 was also established and given the name marlignan O. Compound 4 had the molecular formula C22H24O7, as derived from its HRESIMS at m/z 401.1605 [M + H]+. Its 1H, 13 C, and DEPT NMR spectra and the strong negative Cotton effect at 253 nm in the CD spectrum indicated that 4 was a dibenzocyclooctadiene lignan with an S-biphenyl configuration.11 The 1H and 13C NMR spectra of 4 were very similar to those of methylgomisin R (18).14 The only difference was an OH in 4 rather than an O-methyl group in 18, which was supported by the appearance of a phenolic OH signal (δH 9.19) in 4. HMBC correlation of the O-methyl signal (δH 3.94) with C-1 (δC 141.7) revealed that the O-methyl group should be located at C-1. HMBC correlations of the phenolic OH (δH 9.19) with C-13 (δC 135.8), C-14 (δC 137.3), and C-15 (δC

Figure 1. Selected HMBC (↷) and 1H−1H COSY () correlations of 1.

(δH 6.75 s) with C-9 (δC 39.4), C-10 (δC 136.9 s), and C-15 (δC 122.9 s) and of H-4 (δH 6.83 s) with C-5 (δC 135.0 s), C-6 (δC 80.4), and C-16 (δC 124.1 s), implied that 1 should be a dibenzocyclooctadiene lignan.11 The HMBC correlations of the OH proton (δH 10.23) with C-2 (δC 139.8), C-3 (δC 151.8), and C-4 (δC 111.4) indicated that the OH was at C-2. Attachment of five O-methyl groups at C-1, C-3, C-12, C-13, and C-14 was supported by HMBC correlations of the Omethyl proton signals (δH 3.70, 3.75, 3.83, 3.90, and 3.93) with C-1 (δC 152.7), C-3 (δC 139.8), C-12 (δC 153.7), C-13 (δC 142.1), and C-14 (δC 152.9), respectively. In the cyclooctadiene ring, the oxygenated methine carbon was assigned to C-6 on the basis of its chemical shift (δC 80.4)8 and the HMBC correlations of H-4 (δH 6.83) with C-6 and of an OH signal (δH 4.62) with C-6. Thus, the planar structure of 1 was established. The configuration of the biphenyl groups in all isolated dibenzocyclooctadiene lignans was determined on the basis of their characteristic circular dichroism (CD) spectra. The CD spectra of S-biphenyl-configured lignans show a positive Cotton effect at 215−225 nm and a negative Cotton effect at 240−260 nm. However, R-biphenyl-configured lignans show a negative Cotton effect at 215−230 nm and a positive Cotton effect at 240−260 nm.8,11 The CD spectrum of 1 gave a negative Cotton effect at 252 nm and a positive Cotton effect at 226 nm, indicating that 1 had an S-biphenyl configuration.11 The ROESY correlations (Figure 2) of H-4/CH3-17, H-11/H-9β, and H-11/H-8 in 1 suggested a twist-boat-chair conformation for the cyclooctadiene ring.11 The 6-OH was deduced to be βoriented by the chemical shift of C-6 (δC 80.4), which was similar to that of the 6-β-oriented derivatives of gomisins8,12 and was distinct from that of 6-α-oriented components12,13 in the dibenzocyclooctadiene lignan family. This deduction was supported by the ROESY correlation between H-4/H-6α. Thus, the structure of 1 was established as shown, and it was given the name marlignan M. 251

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and C-8 (δC 48.6), and of the carboxyl proton (δH 11.3) with C-8 (δC 48.6), suggested a OH−CH2−CH(CH2OH)−CH(COOH)−CHOH structural unit in 8 (Figure 3). The 1H

122.4) indicated the OH was located at C-14. Two methylenedioxy units attached to C-2 and C-3, and C-12 and C-13, were supported by HMBC correlations of one pair of methylenedioxy signals (δH 5.95, 5.98) with C-12 (δC 148.7) and C-13 (δC 135.8), and another pair of methylenedioxy signals (δH 6.02, 6.04) with C-2 (δC 136.5) and C-3 (δC 147.5), respectively. The 6-OMe was deduced as β-oriented by the chemical shift of C-6 (δC 90.1), which was similar to that of the 6-β-oriented derivatives of gomisins.15,16 The above deduction was supported by the ROESY correlation of H-4/H-6α. The cyclooctadiene ring of 4 was indicated to be a twist-boat-chair conformation by the ROESY correlations of H-4/CH3-17 and H-9β/H-11. Thus, the structure of 4 was established as shown, and it was named marlignan P. Compounds 5−7 (marlignans Q−S) were all obtained as yellow gums. By comparison of their IR, UV, 1H and 13C NMR, and ROESY spectra with those of 4, compounds 5−7 were also assigned as S-biphenyl-configured dibenzocyclooctadiene lignans with 6-β-oriented O-methyl derivatives. The obvious chemical shift differences resulted from the substituent group variations on the aromatic rings. For compound 5, the HMBC correlations of the O-methyl signal (δH 3.89) with C-14 (δC 141.7) revealed that the O-methyl group should be located at C-14, and correlations of the phenolic OH signal (δH 8.88) with C-1 (δC 138.0), C-2 (δC 136.5), and C-16 (δC 124.0) indicated the OH was located at C-1. This was also supported by the downshift of C-14 from δC 137.3 to δC 141.5 and the upshift of C-1 from δC 141.7 to δC 138.0, when compared with these of 4. When the NMR spectra were compared with those of 4, the disappearance of a methylenedioxy unit and appearance of two O-methyl signals was observed in compound 6. The methylenedioxy unit attached to C-2 and C-3 was supported by HMBC correlations of the methylenedioxy proton (δH 5.96) with C-2 (δC 138.0) and C-3 (δC 148.1). The corralations of three O-methyl signals (δH 3.82, 3.90, and 3.90) with C-1 (δC 142.3), C-12 (δC 138.0), and C-13 (δC 148.1) suggested that the O-methyl groups should be located at C-1, C-12, and C-13, respectively. The phenolic OH at C-14 was determined by correlation of the OH signal (δH 8.99) with C-13 (δC 140.4), C-14 (δC 147.5), and C-15 (δC 122.3). HRESIMS showed that compounds 6 and 7 had the same molecular formula. Comparison of their NMR data suggested that the substituent groups differed at C-1 and C-14. A methylenedioxy unit attached to C-2 and C-3, three O-methyl groups located at C-12, C-13, and C-14, and a phenolic OH located at C-1 were supported by analysis of its HMBC correlations. Thus, the structures of 5−7 were established as shown. Compound 8 had the molecular formula C13H18O7 (by HRESIMS). Its 1H and 13C NMR spectra showed signals of 18 protons and 13 carbons, corresponding to one aromatic ring (δC 131.2 s, 110.8 d, 149.0 s, 148.3 s, 116.5 d, 119.8 d) with three aromatic protons (δH 7.01, 7.18, and 7.23), two oxidized methylene groups (δC 70.4 and 70.4), two methine groups (δC 46.7 and 48.6), one oxidized methine (δC 86.7), one carboxyl group (δC 178.9), and one O-methyl group (δC 55.9). HMBC correlations of aromatic protons H-2 (δH 7.25) and H-6 (δH 7.23) with C-7 (δC 86.7) indicated that the aromatic ring was attached to C-7. The 1H−1H COSY correlations of H-7/H-8/ H-8′/H-9′ and H-8′/H-7′, together with HMBC correlations of H-7 (δH 4.81) with C-8 (δC 48.6), C-9 (δC 178.9), and C-8′ (δC 46.7), of H-7′ (δH 4.32) with C-8′ (δC 46.7), C-9′ (δC 70.42),

Figure 3. Selected HMBC (↷) and 1H−1H COSY () correlations of 8 and 11.

NMR spectrum revealed that the aromatic ring was a 1,2,4trisubstituted phenolic moiety. The O-methyl group at C-3 was supported by HMBC correlations of the O-methyl signal (δH 3.75, s) with C-3 (δC 149.0). HMBC correlations of the aromatic OH signal (δH 10.4) with C-3 (δC 149.0), C-4 (δC 148.3), and C-5 (δC 116.5) indicated that the OH was located at C-4. Thus, the structure of 8 was determined as shown, and it was named marphenol C. Compound 9 had the molecular formula C14H20O8. Its 1H and 13C NMR spectra showed signals of 20 protons and 14 carbons, respectively, corresponding to one aromatic ring with two aromatic protons, two oxidized methylene groups, two methine groups, one oxidized methine, one carboxyl group, and two O-methyl groups. The 1H and 13C NMR spectra of 9 were similar to those of 8. Obvious differences were the substituent groups on aromatic rings. An aromatic proton in 8 was substituted by an O-methyl group (δC 55.7, δH 3.75) in 9. HMBC correlations placed O-methyl groups on C-3 and C-4, and the OH on C-5. Compound 9 was given the name marphenol D. Since the C−C bonds can rotate randomly, the relative configuration of compounds 8 and 9 could not be determined by analysis of their ROESY experiments. Mosher ester analysis could be useful to establish the absolute configuration at C-8. However, after carrying out the bioassays, insufficient amounts of 8 and 9 remained for Mosher experiments. Compound 10 had the molecular formula C20H24O8. Its 1H and 13C NMR spectra showed signals of 24 protons and 20 carbons, corresponding to two aromatic rings (δC 110.9− 148.9) with six aromatic protons (δC 6.84−7.11), two oxidized methylene groups, three methine groups, one carboxyl group, and two O-methyl groups. The HMBC spectrum of 1 showed cross-peaks between H-7 and carbons of both aromatic rings (C-2, C-6, C-2′, and C-6′), which indicated that the two aromatic rings were both linked to the same carbon (C-7). The 1 H−1H COSY correlations (Figure 3) of H-7/H-8/H-8′/H-9′ and H-8′/H-7′, together with HMBC correlations of H-7 with C-8, C-9, and C-8′, of H-7′ with C-8′, C-9′, and C-8, and of the carboxyl proton with C-8, suggested a OH−CH2−CH(OH− CH2)−CH(COOH)−CH structural unit in 10. On the aromatic rings, the HMBC correlations of two O-methyl groups with C-4 and C-4′ indicated that the O-methyl groups should be located at C-4 and C-4′. The relative configuration of 10 was obtained through analysis of coupling constants and the ROESY spectrum.17,18 H-7, H-8, and H-8′ were determined to be α-, β-, and β-oriented, respectively, on the basis of the ROESY correlation of H-9/H-8 and the coupling constant (J = 252

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Journal of Natural Products Table 1.

13

C NMR Data of Compounds 1−7 (δ in ppm) 1a

position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 OMe-1 OMe-2 OMe-3 OMe-12 OMe-13 OMe-14 OMe-6 −OCH2O− a

Article

2a

152.7 139.8 151.8 111.4 135.0 80.4 39.8 38.3 39.4 136.9 107.2 153.7 142.1 152.9 122.9 124.1 16.6 16.6 60.5

s s s d s d d d t s d s s s s s q q q

55.8 55.9 60.5 60.8

q q q q

3a

148.7 140.6 152.3 107.2 133.8 80.5 39.5 37.7 39.0 134.7 104.4 151.9 141.3 148.1 119.9 120.4 17.2 17.3

s s s d s d d d t s d s s s s s q q

60.3 55.7 55.8 60.4

q q q q

4b

153.0 142.4 152.0 111.7 134.7 80.4 39.9 38.2 39.5 137.5 104.6 148.4 137.1 139.9 121.9 123.0 16.9 16.9 60.7 59.6

s s s d s d d d t s d s s s s s q q q q

141.7 136.5 147.5 106.7 132.7 90.1 38.8 36.6 38.0 135.0 103.7 148.6 135.8 137.3 122.4 123.0 17.3 17.3 59.6

s s s d s d d d t s d s s s s s q q q

138.0 136.5 147.4 107.0 133.8 90.0 39.6 36.9 38.1 135.1 102.3 148.5 134.5 141.5 121.3 124.0 17.2 17.2

60.7 q 55.8 q 100.8, 101.1 t

101.6 t

5b

6b

s s s d s d d d t s d s s s s s q q

142.3 138.0 148.1 106.3 133.0 90.2 38.8 36.5 38.3 136.6 107.3 152.8 140.4 147.5 122.3 123.0 17.0 17.0 60.7

59.5 q 55.8 q 100.5, 101.0 t

7b s s s d s d d d t s d s s s s s q q q

139.1 137.7 148.3 107.2 132.3 90.1 38.7 36.4 38.2 136.0 106.3 152.7 141.5 151.7 122.1 122.7 17.1 17.3

s s s d s d d d t s d s s s s s q q

55.8 q 60.5 q 55.9 q 101.1 q

55.7 60.4 60.6 55.7 101.0

q q q q q

10

11

Data were recorded in C5D5N. bData were recorded in CDCl3.

Table 2. position 1 2 3 4 5 6 7 8 9 1′ 2′

13

C NMR Data of Compounds 8−11 (δ in ppm, data were recorded in C5D5N) 8 131.2 110.8 149.0 148.3 116.5 119.8 86.7 48.6 178.9

9 s d s s d d d d s

132.5 105.1 151.2 136.7 148.0 110.2 85.1 47.9 178.8

10 s d s s s d s d d

136.3 110.9 144.9 148.8 114.7 119.7 58.8 49.8 178.6 136.8 111.0

position

11 s d s s d d d d s s d

134.2 112.4 146.0 149.6 116.5 112.4 58.4 49.5 179.7 133.3 108.3

s d s s d d d d s s d

3′ 4′ 5′ 6′ 7′ 8′ 9′ OMe-3 OMe-4 OMe-3′ −OCH2O−

11.9 Hz) between H-7 and H-8. Compound 10 was named marphenol E. Compound 11 was assigned the molecular formula C20H22O8 by HRESIMS. The 1H and 13C NMR spectra of 11 were similar to those of 10. The difference was the disappearance of a signal of an O-methyl group and an OH in 10 and appearance of a methylenedioxy unit in 11. HMBC correlations (Figure 3) of methylenedioxy protons (δH 5.82, 5.87) with C-3′ (δC 147.9) and C-4′ (δC 145.9) and of the O-methyl proton (δC 3.75) with C-3 (δC 149.6) indicated that the methylenedioxy unit should be connected with C-3′ and C-4′, and the O-methyl group should be located at C-3, respectively. Thus, the structure of 11 was determined, and it was given the trivial name marphenol F. Since certain dibenzocyclooctadiene lignans from Schisandra species exhibit potential anti-HIV activities, the new compounds 1−11 were tested for their potencies in preventing the cytopathic effects of HIV-1 in C8166 cells. Cytotoxicity was

8

70.4 46.7 70.4 55.9

9

t d t s

69.8 46.8 69.8 55.6 55.7

t d t q q

145.0 148.9 114.7 119.8 70.0 49.8 70.0 55.9

s s d d t d t s

145.9 147.9 108.8 120.5 70.4 46.7 70.4 55.8

s s d d t d t s

55.9 s 101.6 t

measured in parallel with the determination of antiviral activity, using AZT as a positive control (EC50 = 0.0043 μg/mL and CC50 > 200 μg/mL).19 The results are shown in Table 3. Compounds 1−11 showed anti-HIV-1 activity with EC50 values in the range 2.97−6.18 μg/mL and therapeutic index (TI) values of 5.33−29.13.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a Horiba SEPA-300 polarimeter. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. CD spectra were measured on a JASCO J-810 spectropolarimeter. A Tenor 27 spectrophotometer was used for scanning IR spectrometry. 1D and 2D NMR spectra were recorded on a DRX-500 NMR spectrometer with TMS as internal standard. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. HRESIMS was performed on a VG Autospec-3000 spectrometer. Semipreparative HPLC was performed on a Shimadzu LC-8A preparative liquid chromatograph

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Marlignan N (2): yellow gum; [α]24.8 D −18.6 (c 0.22, MeOH); UV (MeOH) λmax (log ε) 212 (4.86), 240 (3.72), 324 (0.78) nm; CD (c 0.06, MeOH), nm (Δε) 250 (−38.2), 223 (+15.6), 219 (+8.22); IR (KBr) νmax 3458, 2946, 2855, 1640, 1582, 1465, 1398, 1276, 1168, 1054, 974, 826 cm−1; 1H NMR (C5D5N, 500 MHz) δ 6.49 (1H, s, H4), 4.67 (1H, d, J = 8.1 Hz, H-6), 2.10 (1H, m, H-7), 2.01 (1H, m, H8), 2.45 (1H, m, H-9α), 2.71 (1H, m, H-9β), 6.43 (1H, s, H-11), 0.82−0.90 (3H, d, overlap, H-17), 0.82−0.90 (3H, overlap, H-18), 3.85, 3.91, 3.92, 3.96 (3H each, s, 4 × OMe), 10.52, 11.22 (1H each, s, 2 × OH); 13C NMR data, see Table 1; positive ESIMS m/z 405 [M + H]+; positive HRESIMS m/z 405.1918 [M + H]+ (calcd for C22H29O7, 405.1913). Marlignan O (3): yellow gum; [α]24.2 D +38.4 (c 0.22, MeOH); UV (MeOH) λmax (log ε) 212 (4.68), 240 (3.75), 330 (1.74) nm; IR (KBr) νmax 3450, 2945, 2874, 1646, 1574, 1452, 1327, 1262, 1142, 1015, 986, 853 cm−1; 1H NMR (C5D5N, 500 MHz) δ 6.98 (1H, s, H4), 4.54 (d, J = 8.0 Hz, H-6), 2.06 (1H, m, H-7), 1.90 (1H, m, H-8), 2.22 (1H, m, H-9α), 2.49 (1H, m, H-9β), 6.77 (1H, s, H-11), 0.88 (3H, d, J = 7.8 Hz, H-17), 1.12 (3H, d, J = 7.7 Hz, H-18), 3.93, 3.93, 3.98 (3H each, s, 3 × OMe), 5.90, 5.99 (2H, s, −OCH2O−), 11.29 (1H s, Ar-OH); 13C NMR data, see Table 1; positive ESIMS m/z 403 [M + H]+; positive HRESIMS m/z 403.1756 [M + H]+ (calcd C22H27O7 for 403.1757). Marlignan P (4): yellow gum; [α]23.5 D −28.5 (c 0.22, MeOH); UV (MeOH) λmax (log ε) 210 (4.86), 252 (3.81), 325 (2.44), 340 (1.86) nm; CD (c 0.04, MeOH), nm (Δε) 253 (−46.2), 236 (+42.8), 220 (+11.2); IR (KBr) νmax 3480, 2955, 2920, 2875, 2854, 1626, 1478, 1272, 1205, 1082, 1075, 1042, 978, 857 cm−1; 1H NMR (CDCl3, 500 MHz) δ 6.50 (1H, s, H-4), 3.78 (1H, d, J = 4.9 Hz, H-6), 1.67 (1H, m, H-7), 1.85 (1H, m, H-8), 2.02 (1H, m, H-9α), 2.33 (1H, m, H-9β), 6.41 (1H, s, H-11), 0.86−0.92 (3H, overlap, H-17), 0.86−0.92 (3H, overlap, H-18), 3.06 (3H, s, OMe-6), 3.94 (3H, s, OMe-1), 5.95, 5.98 (2H, s, −OCH2O−), 6.02, 6.04 (2H, s, −OCH2O−), 9.19 (1H s, ArOH); 13C NMR data, see Table 1; positive ESIMS m/z 401 [M + H]+; HRESIMS m/z 401.1605 [M + H]+ (calcd for C22H25O7, 401.1600). Marlignan Q (5): yellow gum; [α]23.8 D +38.6 (c 0.25, MeOH); UV (MeOH) λmax (log ε) 210 (4.73), 250 (3.66), 285 (2.54), 325 (2.08), 340 (1.55) nm; CD (c 0.05, MeOH), nm (Δε) 254 (−76.4), 234 (+63.8), 220 (+8.7); IR (KBr) νmax 3482, 2924, 2850, 1625, 1480, 1270, 1208, 1085, 1072, 1047, 676, 852 cm−1; 1H NMR (CDCl3, 500 MHz) δ 6.54 (1H, s, H-4), 3.76 (d, J = 5.2 Hz, H-6), 1.63 (1H, m, H7), 1.81 (1H, m, H-8), 1.98 (1H, m, H-9α), 2.27 (1H, m, H-9β), 6.37 (1H, s, H-11), 0.84−0.89 (3H, overlap, H-17), 0.84−0.89 (3H, overlap, H-18), 3.01 (3H, s, OMe-6), 3.89 (3H, s, OMe-1), 5.90, 5.93 (2H, s, −OCH2O−), 5.98 (2H, s, −OCH2O−), 8.88 (1H s, Ar-OH); 13 C NMR data, see Table 1; positive ESIMS m/z 401 [M + H]+; HRESIMS m/z 401.1594 [M + H]+ (calcd for C22H25O7, 401.1600). Marlignan R (6): yellow gum; [α]23.9 D +43.8; UV (MeOH) λmax (log ε) 210 (4.82), 246 (3.79), 286 (2.64), 329 (1.68) nm; CD (c 0.06, MeOH), nm (Δε) 245 (−45.4), 224 (+39.1), 223 (+10.58),. 219 (−13.5); IR (KBr) νmax 3430, 2962, 2934, 2875, 1625, 1594, 1578, 1462, 1425, 1412, 1329, 1234, 1192, 1148, 1094, 1022, 967, 853 cm−1; 1 H NMR (CDCl3, 500 MHz) δ 6.52 (1H, s, H-4), 3.78 (1H overlap, H-6), 1.62 (1H, m, H-7), 1.47 (1H, m, H-8), 2.08 (1H, m, H-9α), 2.30 (1H, m, H-9β), 6.48 (1H, s, H-11), 0.91−0.93 (3H, overlap, H-17), 0.91−0.93 (3H, overlap, H-18), 3.05 (3H, s, OMe-6), 3.82, 3.90, 3.90 (3H each, s, 3 × OMe), 5.96 (2H, s, −OCH2O−), 8.99 (1H s, ArOH); 13C NMR data, see Table 1; positive ESIMS m/z 417 [M + H]+; HRESIMS m/z 417.1907 [M + H]+ (calcd for C24H30NaO7, 417.1913). Marlignan S (7): yellow gum; [α]24.1 D +45.8; UV (MeOH) λmax (log ε) 210 (4.74), 245 (3.83), 280 (2.54), 325 (1.74) nm; IR (KBr) νmax 3435, 2966, 2930, 2908, 2876, 1628, 1592, 1574, 1460, 1452, 1416, 1325, 1232, 1188, 1146, 1090, 1022. 965, 850 cm−1; CD (c 0.05, MeOH), nm (Δε) 246 (−48.4), 222 (+28.7), 220 (+6.24), 215 (−15.8); 1H NMR (CDCl3, 500 MHz) δ 6.46 (1H, s, H-4), 3.72 (1H, d, J = 6.4 Hz, H-6), 1.83 (1H, m, H-7), 1.66 (1H, m, H-8), 2.03 (1H, m, H-9α), 2.32 (1H, m, H-9β), 6.44 (1H, s, H-11), 0.86−0.88 (3H, overlap, H-17), 0.86 −0.88 (3H, overlap, H-18), 3.00 (3H, s, OMe-6), 3.78, 3.86, 3.86 (3H each, s, 3 × OMe), 5.98 (2H, s, −OCH2O−),

Table 3. Anti-HIV Activities of Compounds 1−11 no.

CC50 (μg/mL)

EC50 (μg/mL)

TI (CC50/EC50)

1 2 3 4 5 6 7 8 9 10 11 AZT

32.1 47.4 70.6 74.6 22.5 36.7 42.8 84.5 93.8 73.5 82.1 >200

3.42 6.18 5.84 3.18 4.22 3.87 4.14 3.14 3.22 3.47 2.97 0.0043

9.39 7.67 12.09 23.46 5.33 9.48 10.34 26.91 29.13 21.18 27.64 >46511.6

with Zorbax PrepHT GF (21.2 mm × 25 cm) or Venusil MP C18 (20 mm × 25 cm) columns. Column chromatography (CC) was performed using silica gel (200−300 mesh, Qing-dao Marine Chemical, Inc., Qingdao, People’s Republic of China), Lichroprep RP-18 gel (40−63 μm, Merck, Darmstadt, Germany), and MCI gel (75−150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan). Fractions were monitored by TLC, and spots were visualized by heating silica gel plates sprayed with 5% H2SO4 in EtOH. Plant Material. The fruits of S. wilsoniana6 were collected on Marer Mountain in Heqing Prefecture, Yunnan Province, People’s Republic of China, in September 2007. Identification of the plant material was verified by Prof. Xi-Wen Li of Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (KIB 077-23) has been deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences. Extraction and Isolation. The air-dried and powdered fruit of S. wilsoniana (4.5 kg) was extracted with 70% aqueous Me2CO (4 × 5 L) at room temperature and filtered. The filtrate was evaporated to smaller volume (4−5 L) under reduced pressure and partitioned with EtOAc (3 × 4 L). The EtOAc-soluble portion (385.0 g) was applied to silica gel (200−300 mesh) CC, eluting with a CHCl3−MeOH gradient system (20:1, 9:1, 8:2, 7:3, 6:4, 5:5), to give five fractions, A−E. Further separation of fraction A (126.0 g) by silica gel CC, eluted with petroleum ether−acetone (20:1−2:1), yielded mixtures A1−A7. Fraction A2 (12.6 g) was subjected to silica gel CC using petroleum ether−acetone and semipreparative HPLC (72% MeOH−H2O) to give 1 (9.4 mg), 4 (8.5 mg), and 7 (18.1 mg). Fraction A3 (8.9 g) was subjected to silica gel CC using petroleum ether−acetone and semipreparative HPLC (68% MeOH−H2O) to afford 5 (11.3 mg) and 6 (15.1 mg). Fraction A4 (5.2 g) was subjected to silica gel CC using petroleum ether−acetone and semipreparative HPLC (62% MeOH− H2O) to give 2 (8.8 mg) and 3 (7.4 mg). Further separation of fraction B (63.0 g) by silica gel CC, eluted with petroleum ether−acetone (8:2 −2:1), yielded mixtures B1−B5. Fraction B3 (11.4 g) was subjected to silica gel CC using petroleum ether−acetone and semipreparative HPLC (35% MeOH−H2O) to give 10 (11.5 mg) and 11 (6.2 mg). Fraction B4 (6.8 g) was subjected to silica gel CC using petroleum ether−acetone and semipreparative HPLC (28% MeOH−H2O) to afford 8 (2.6 mg) and 9 (3.2 mg). Marlignan M (1): yellow gum; [α]23.6 D +22.8 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 211 (4.76), 242 (3.68), 322 (1.82) nm; CD (c 0.02, MeOH), nm (Δε) 252 (−17.8), 226 (+11.8), 220 (−4.76); IR (KBr) νmax 3452, 2942, 2880, 2826, 1635, 1588, 1542, 1476, 1390, 1268, 1235, 1195, 1084, 1037, 975, 842 cm−1; 1H NMR (C5D5N, 500 MHz) δ 6.83 (1H, s, H-4), 4.61 (1H, d, J = 7.8 Hz, H-6), 1.99−2.02 (1H, overlap, H-7), 1.99−2.02 (1H, overlap, H-8), 2.23 (1H, m, H9α), 2.52 (1H, m, H-9β), 6.75 (1H, s, H-11), 0.90−0.96 (3H, overlap, H-17), 0.90−0.96 (3H, overlap, H-18), 3.70, 3.75, 3.83, 3.90, 3.93 (5 H each, s, 5 × OMe), 10.23 (1H s, Ar-OH); 13C NMR data, see Table 1; positive ESIMS m/z 419 [M + H]+; HRESIMS m/z 419.2074 [M + H]+ (calcd for C23H31O7, 419.2070). 254

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Journal of Natural Products

Article

9.17 (1H s, Ar-OH); 13C NMR data, see Table 1; positive ESIMS m/z 417 [M + H]+; HRESIMS m/z 417.1916 [M + H]+ (calcd for C24H30NaO7, 417.1913). +18.6 (c 0.20, Marphenol C (8): white, amorphous solid; [α]25.8 D MeOH); UV (MeOH) λmax (log ε) 205 (4.87), 278 (4.12), 326 (2.26), nm; IR (KBr) νmax 3450, 2962, 2941, 2872, 2830, 1618, 1590, 1462, 1378, 1345, 1272, 1225, 1165, 1140, 970, 820 cm−1; 1H NMR (C5D5N, 500 MHz) δ 7.25 (1H, s, H-2), 7.01 (1H, d, J = 8.1 Hz, H-5), 7.14 (1H, d, J = 8.1 Hz, H-6), 4.80 (1H, d, J = 8.1 Hz, H-7), 3.30 (1H, m, H-8), 4.29−4.45 (2 H, overlap, H-7′), 3.61 (1H, m, H-8′), 4.29− 4.45 (2H, overlap, H-9′), 11.3 (1H, brs, −COOH), 10.43 (1H, brs, ArOH), 3.75 (3H s, −OMe); 13C NMR data, see Table 2; positive ESIMS m/z 309 [M + Na]+; HRESIMS m/z 309.0954 [M + Na]+ (calcd for C13H18NaO7, 309.0950). +22.5 (c 0.21, Marphenol D (9): white, amorphous solid; [α]25.4 D MeOH); UV (MeOH) λmax (log ε) 210 (4.82), 276 (4.15), 325 (2.30), nm; IR (KBr) νmax 3452, 2958, 2939, 2875, 2824, 1622, 1592, 1472, 1375, 1343, 1267, 1229, 1166, 1138, 976, 819 cm−1; 1H NMR (C5D5N, 500 MHz) δ 7.06 (1H, s, H-2), 7.35 (1H, s, H-6), 4.82 (1H, d J = 8.1 Hz, H-7), 3.23 (1H, m, H-8), 4.34−4.38 (2 H, overlap, H-7′), 3.59 (1H, m, H-8′), 4.34−4.38 (2 H, overlap, H-9′), 3.89, 3.92 (3H s, 2 × −OMe); 13C NMR data, see Table 2; positive ESIMS m/z 309 [M + Na]+; HRESIMS m/z 339.1051 [M + Na]+ (calcd for C14H20NaO8, 339.1056). Marlphenol E (10): yellow gum; [α]24.8 D +19.6 (c 0.18, MeOH); UV (MeOH) λmax (log ε) 205 (4.92), 282 (4.18), 326 (2.25) nm; IR (KBr) νmax 3480, 2968, 2947, 2885, 2843, 1622, 1592, 1517, 1458, 1368, 1255, 1262, 1152, 1022, 977, 824 cm−1; 1H NMR (C5D5N, 500 MHz) δ 7.11 (1H, s, H-2), 6.80−6.84 (1H, overlap, H-5), 6.91−6.95 (1H, overlap, H-6), 3.60 (1H, d J = 11.5 Hz, H-7), 3.24 (1H, m, H-8), 7.08 (1H, s, H-2′), 6.80−6.84 (1H, overlap, H-5′), 6.91−6.95 (1H, overlap, H-6′), 4.27−4.34 (2 H, overlap, H-7′), 2.35 (1H, m, H-8′), 4.27−4.34 (2 H, overlap, H-7′), 11.13 (1H, brs, −COOH), 10.54 (2H, brs, Ar-OH),3.77, 3.77 (3H s, 2 × −OMe), 13C NMR data, see Table 2; positive ESIMS m/z 415 [M + Na]+; HRESIMS (positive ion mode) m/z 415.1362 [M + Na]+ (calcd for C20H24NaO8, 415.1369). Marlphenol F (11): yellow gum; [α]24.8 D +26.8 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 208 (4.82), 282 (4.22), 325 (2.18) nm; IR (KBr) νmax 3468, 2977, 2942, 2887, 2849, 2816, 1627, 1593, 1514, 1455, 1363, 1254, 1265, 1153, 1031, 1018, 987, 806 cm−1; 1H NMR (C5D5N, 500 MHz) δ 7.10 (1H, s, H-2), 6.76−6.79 (1H, overlap, H5), 6.99−7.01 (1H, overlap, H-6), 3.61 (1H, d, J = 11.9 Hz, H-7), 3.14 (1H, m, H-8), 7.37 (1H, s, H-2′), 6.76−6.79 (1H, overlap, H-5′), 6.99 −7.01 (1H, overlap, H-6′), 4.36−4.50 (2 H, overlap, H-7′), 2.35 (1H, m, H-8′), 4.36−4.50 (2 H, overlap, H-7′), 11.13 (1H, brs, −COOH), 10.69 (1H, brs, Ar-OH), 5.82, 5.87 (1H, s, −OCH2O−), 3.75 (3H s, −OMe); 13C NMR data, see Table2; positive ESIMS m/z 413 [M + Na]+; HRESIMS (positive ion mode) m/z 413.1217 [M + Na]+ (calcd for C20H22NaO8, 413.1212). Anti-HIV-1 Assay. The cytotoxicity assay against C8166 cells (CC50) was assessed using the MTT method, and anti-HIV-1 activity was evaluated by the inhibition assay for the cytopathic effects of HIV1 (EC50).19

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported financially by CAS grants (KSCX2EW-Q-10 and KSCX1-YW-R-24), the Natural Science Foundation of Yunnan Province (2012FB178), the NSFC (20802082 and 30830115), the 973 programs (2009CB522300 and 2009CB940900), the Young Academic and Technical Leader Rising Foundation of Yunnan Province (2006PY01-47), and the Key Scientific and Technological Projects of China (2009ZX09501-029).

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR, HSQC, HMBC COSY, ROESY, HRESIMS, and CD spectra of 1, 1H and 13C NMR, HSQC, and HMBC of 4 and 8, 1H and 13C NMR spectra of 2, 3, 5−7, and 9−11, detailed isolation and extraction procedures of compounds 1− 32, and the structures, literature references, and other related information of known compounds 12−32. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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Corresponding Author

*Tel: (86) 871-5223251. Fax: (86) 871-5216343. E-mail: xwl@ mail.kib.ac.cn; [email protected]. 255

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