HETEROCYCLES

Note | Special issue | Vol. 80, No. 1, 2010, pp. 613-621
Received, 19th May, 2009, Accepted, 25th June, 2009, Published online, 26th June, 2009.
DOI: 10.3987/COM-09-S(S)28

■ Biyouxanthones A - D, Prenylated Xanthones from Roots of Hypericum chinense

Naonobu Tanaka, Takuji Mamemura, Shuhei Abe, Kiyoshi Imabayashi, Yoshiki Kashiwada, Yoshihisa Takaishi, Tetsuro Suzuki, Yutaka Takebe, Takaaki Kubota, and Jun'ichi Kobayashi*

Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12 Nishi 6, Kita-ku, Sapporo, Hokkaido 060-0812, Japan

Abstract

Four new prenylated xanthones, biyouxanthones A – D (1 – 4), were isolated from roots of Hypericum chinense. The gross structures of 1 – 4 were elucidated from spectroscopic data, and 2 was assigned as a racemate by chiral HPLC analysis. Biyouxanthones A (1) and B (2) exhibited anti-viral activity against hepatitis C virus (HCV).

The plants, belonging to the genus Hypericum (family Clusiaceae), are known to be a traditional medicine for the treatment of burns, bruises, swelling, inflammation, and anxiety as well as bacterial and viral infections.1-4 In our continuing search for bioactive compounds from Hypericum spp,5 three new prenylated xanthones possessing 2-hydroxycyclohexa-2,4-dienone moiety, biyouxanthones A – C (1 – 3), and one new prenylated xanthone having a 5-methyl-2-(prop-1-en-2-yl)hex-4-enyl group, biyouxanthone D (4), were isolated from roots of H. chinense. In this paper, we describe the isolation, structure elucidation of 1 – 4 and anti-HCV activity of 1 and 2.

The dried roots of H. chinense were extracted with MeOH, and the extracts were partitioned successively with n-hexane, EtOAc, and H2O. n-Hexane-soluble portions were separated by a silica gel column (n-hexane/EtOAc) and then a Sephadex LH-20 column (MeOH) to afford a mixture of xanthone derivatives, which was purified by C18 reversed-phase HPLC (MeOH/H2O) to yield biyouxanthones A (1, 0.00017%), B (2, 0.00016%), and C (3, 0.00087%). EtOAc-soluble portions were purified by a silica gel column (CHCl3/MeOH) and a Sephadex LH-20 (MeOH) column chromatographies, and C18 reversed-phase HPLC (MeOH/H2O, 0.1% TFA) to give biyouxanthone D (4, 0.00012%).

The molecular formula of biyouxanthone A (1), C38H48O6, was revealed by HRESIMS (m/z 599.3366 [M−H]−, Δ −0.6 mmu). IR absorptions at 1671 and 1636 cm-1 implied the presence of carbonyl

functionalities. 1H and 13C NMR data of 1 (Table 1) revealed the presence of three carbonyl groups, seven trisubstituted olefins, two tetrasubstituted olefins, two sp3 quaternary carbons, six sp3 methylenes, nine tertiary methyls, and one hydrogen-bonded hydroxy group. 1H–1H COSY correlations for H2-11 and H-12, H2-16 and H-17, and H2-31 and H-32, and HMBC cross-peaks of H3-14 to C-12, C-13, and C-15, H3-19 to C-17, C-18, and C-20, H3-34 to C-32, C-33, and C-35 suggested the presence of three prenyl groups. The

presence of a geranyl group was implied by 1H–1H COSY correlations for H2-21 and H-22, H2-24 and H2-25, H2-25 and H-26, and HMBC cross-peaks of H3-30 to C-22, C-23, and C-24, and H3-28 to C-26, C-27, and C-29. NOESY correlations for H2-21 to H3-30 and H-22 to H2-24 suggested the 22E configuration for the geranyl group. From these data, 1 was presumed to be a xanthone derivative possessing one geranyl and three prenyl groups. The chemical shift of OH-1 (δH 13.53) revealed the presence of hydrogen-bonded hydroxy group at C-1. The substitution pattern of A-ring (C-1 to C-4, C-4a, and C-9a) was deduced from HMBC cross-peaks of H-2 to C-1, C-3, and C-9a, and H2-16 to C-3, C-4, C-4a, and C-11. HMBC correlations for H2-31 to C-7, C-8, C-8a, and C-21 indicated that C-7, C-8a, a prenyl group, and a geranyl group were attached to an sp3 quaternary carbon (C-8). Connectivities of C-8a to C-10a, C-10a to C-5, and C-5 to C-7 were deduced from HMBC correlations for H-5 to C-6, C-7, C-8a, and C-10a. Thus, the gross structure of biyouxanthone A was elucidated to be 1 (Figure 1).

The molecular formula of biyouxanthone B (2), C34H42O6, was established by HRESIMS (m/z 545.2892 [M−H]−, Δ −1.1 mmu). IR absorptions at 1668 and 1645 cm-1 implied the presence of carbonyl functionalities. 1H and 13C NMR data of 2 (Table 2) suggested that 2 was a xanthone derivative possessing one geranyl, two prenyl, one methoxy, and two hydroxy groups. Carbon chemical shifts of ring B (C-5 to C-8, C-8a, and C-10a) and C-16 to C-30 were similar to corresponding carbon chemical shifts of 1. The substitution pattern of A-ring (C-1 to C-4, C-4a, and C-9a) as shown in Figure 2 were deduced by HMBC cross-peaks of a hydrogen-bonded hydroxy proton (OH-1) to C-1, C-2, and C-9a, H2-11 to C-3, C-4, and C-4a, H-2 and 3-OMe to C-3, and the NOESY correlation between H-2 and 3-OMe. Thus, the gross structure of biyouxanthone B was elucidated to be 2. Biyouxanthone B (2) showed no specific rotation. Chiral HPLC analysis of biyouxanthone B (2) gave two peaks with almost same peak areas due to (−)-biyouxanthone B (tR 19.0 min) and (+)-biyouxanthone B (tR 24.0 min). In

addition, patterns of Cotton effects for respective enantiomers in the CD spectra were symmetrical each other. From these data, biyouxanthone B (2) was assigned as a racemate.

Biyouxanthone C (3) showed the pseudomolecular ion peak at m/z 531 [M−H]− in the ESIMS, and the HRESIMS analysis revealed that the molecular formula of 3 was C33H40O6, whose molecular weight is

smaller by 14 as compared with that of biyouxanthone B (2). 1H and 13C NMR data of biyouxanthone C (3) (Table 2) were similar to those of 2, except for the absence of the signal for a methoxy group at C-3. These data suggested that 3 had a hydroxy group at C-3 in place of a methoxy group of 2. It was supported by detailed analyses of 2D NMR spectra of 3 (Figure 3). Thus, the gross structure of biyouxanthone C was elucidated to be 3.

Biyouxanthone D (4) had a molecular formula of C28H32O6 based on HRESIMS. 1H and 13C NMR data of 4 (Table 3) revealed that 4 was a xanthone derivative possessing one prenyl group, four hydroxy groups, and one monoterpene substituent. The substituent was elucidated to be a 5-methyl-2-(prop-1-en-2-yl)hex-4-enyl group by 1H–1H COSY correlations for H2-16 and H-17, H-17 and H2-18, and H2-18 and H-19, and HMBC cross-peaks of H3-21 to C-19, C-20, and C-22, and H3-25 to C-17, C-23, and C-24. The locations of these substituents were deduced from the HMBC correlations as shown in Figure 4. Thus, the gross structure of biyouxanthone D was elucidated to be 4. Biyouxanthone D (4) showed optical rotation, [α]23D +37.9, while the stereochemistry of C-17 was not assigned.

Biyouxanthones A (1) and B (2) inhibited the HCV core protein level in the culture of HCV-infected human hepatoma Huh7 cells (89% and 61%, respectively) at 10 µM.

EXPERIMENTAL
General Experimental Procedures
Optical rotations were recorded on a JASCO P-1030 digital polarimeter. IR, UV, and CD spectra were recorded on JASCO FT/IR-230, Shimadzu UV-1600PC, and JASCO J-720 spectrophotometers, respectively. NMR spectra were measured by a JEOL ECA 500 spectrometer. The 7.26 and 77.0 ppm resonances of residual CHCl3 were used as internal references for 1H and 13C NMR spectra, respectively. ESIMS spectra were recorded on a JEOL JMS 700-TZ spectrometer.
Plant Material
Hypericum chinense was cultivated at the botanical garden of the University of Tokushima and collected in January 2006. Herbarium specimens were deposited in Experimental Station for Medicinal Plants Studies, Hokkaido University (specimen number: UTP98014).

Extraction and Isolation
Roots of H. chinense (2.52 kg, dry) were extracted with MeOH (10 L x 3), and the extracts were partitioned between n-hexane (1 L x 3) and H2O (1 L). The n-hexane-soluble portions were subjected to a silica gel column (n-hexane / EtOAc), a Sephadex LH-20 column (MeOH), and then C18 reversed-phase HPLC [LUNA 5µ C18(2), Phenomenex, 10 x 250 mm; flow rate 3.0 mL/min; UV detection at 254 nm; eluent MeOH/H2O, 95:5] to afford biyouxanthones A (1, 4.2 mg, 0.00017%), B (2, 3.9 mg, 0.00016%), and C (3, 21.8 mg, 0.00087%). EtOAc-soluble portions were purified by a silica gel column (CHCl3/MeOH) and a Sephadex LH-20 (MeOH) column chromatographies, and C18 reversed-phase HPLC (Mighty sil RP-18, Kanto Chemical Co. Ltd, 10 x 250 mm; flow rate 2.0 mL/min; UV detection at 254 nm; eluent MeOH/H2O, 9:1, 0.1% TFA) to give biyouxanthone D (4, 2.9 mg, 0.00012%).

Biyouxanthone A (1): yellow amorphous solid; [α]23D 0 (c 0.1 CHCl3); UV (MeOH) λmax 306 (ε 3100) and 418 (4400) nm; IR (film) vmax 3347, 1671, and 1636 cm-1; 1H and 13C NMR data (Table 1); ESIMS m/z 599 [M−H]−; HRESIMS: m/z 599.3366 [M−H]− (calcd for C38H47O6, 599.3372).

Biyouxanthone B (2): yellow amorphous solid; [α]23 D 0 (c 0.3 CHCl3); UV (MeOH) λmax 260 (ε 12900), 280 (10100), 314 (13400), and 421 (9200) nm; IR (film) vmax 3367, 1668, and 1645 cm-1; 1H and 13C NMR data (Table 2); ESIMS m/z 545 [M−H]−; HRESIMS: m/z 545.2892 [M−H]− (calcd for C34H41O6, 545.2903).

Biyouxanthone C (3): yellow amorphous solid; [α]23 D 0 (c 0.3 CHCl3); UV (MeOH) λmax 250 (ε 10800), 277 (10900), and 419 (5600) nm; IR (film) vmax 3347, 1670, and 1645 cm-1; 1H and 13C NMR data (Table 2); ESIMS m/z 531 [M−H]−; HRESIMS: m/z 531.2747 [M−H]− (calcd for C33H39O6, 531.2750).

Biyouxanthone D (4): yellow amorphous solid; [α]23D +37.9 (c 0.5 CHCl3); UV (MeOH) λmax 254 (ε 33600), 286 (11300), and 331 (16000) nm; IR (KBr) vmax 3430, 2923, 1617, and 1591 cm-1; 1H and 13C NMR data (Table 3); ESIMS m/z 487 [M+Na]+; HRESIMS: m/z 487.2088 [M+Na]+ (calcd for C28H32O6Na, 487.2097).

Chiral HPLC analysis of biyouxanthone B (2)
Biyouxanthone B (2) was subjected to a chiral HPLC [Chiralpak AD; Daicel Chemical Industry, Ltd., 4.6 x 250 mm; n-hexane / i-PrOH (99:1); flow rate 0.5 mL/min; UV detection at 254 nm] to afford (−)-biyouxanthone B (tR 19.0) and (+)-biyouxanthone B (tR 24.0 min). (−)-Biyouxanthone B: [α]21D −9.6 (c 0.1 CHCl3); CD (MeOH) λext 280 (Δε −0.96), 257 (+0.28), 237 (−0.88), 226 (−0.23), 220 (−0.49), and 211 (+4.52) nm. (+)-Biyouxanthone B: [α]21D +13.7 (c 0.1 CHCl3); CD (MeOH) λext 280 (Δε +0.79), 257 (−0.55), 237 (+0.63), 226 (+0.14), 220 (+0.60), and 211 (−3.67) nm.

Measurement of anti-HCV activity
Huh7 cells infected with HCV JFH-1 isolate6 were treated with biyouxanthones A − D (1 − 4) for 3 days. HCV core protein in cell culture supernatants was quantified using an enzyme immunoassay (Ortho HCV antigen ELISA Kit; Ortho Clinical Diagnostics, Tokyo, Japan), following the manufacturer's instructions.

ACKNOWLEDGMENTS
We thank S. Oka, Center for Instrumental Analysis, Hokkaido University, for measurements of HRESIMS. This work was partly supported by a grant from the Kuribayashi Foundation and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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