HETEROCYCLES

Paper | Special issue | Vol. 79, No. 1, 2009, pp. 917-924
Received, 5th October, 2008, Accepted, 20th November, 2008, Published online, 27th November, 2008.
DOI: 10.3987/COM-08-S(D)64

■ Petiolins D and E, Phloroglucinol Derivatives from Hypericum pseudopetiolatum var. kiusianum

Naonobu Tanaka, Takaaki Kubota, Haruaki Ishiyama, Yoshiki Kashiwada, Yoshihisa Takaishi, Junji Ito, Yuzuru Mikami, Motoo Shiro, and Jun'ichi Kobayashi*

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

Abstract

A new phloroglucinol derivative possessing citran skeleton, petiolin D (1), and a new chromone glucoside, petiolin E (2), were isolated from aerial parts of Hypericum pseudopetiolatum var. kiusianum. The structures of 1 and 2 were elucidated by spectroscopic data, and a single-crystal X-ray diffraction analysis of 1 revealed that 1 was a racemic mixture.

INTRODUCTION
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,5-10 we previously isolated new phloroglucinol derivatives, petiolins A – C, from the aerial parts of Hypericum pseudopetiolatum var. kiusianum.11 Further investigation of extracts from this plant resulted in the isolation of a new phloroglucinol derivative possessing citran skeleton, petiolin D (1), and a new chromone glucoside, petiolin E (2), were isolated from aerial parts of H. pseudopetiolatum var. kiusianum. In this paper, we describe the isolation and structure elucidation of 1 and 2.

RESULTS AND DISCUSSION
The aerial parts of H. pseudopetiolatum var. kiusianum were extracted with MeOH, and the extracts were partitioned successively with n-hexane, EtOAc, and H2O. n-Hexane-soluble portions were subjected to a silica gel column (n-hexane/EtOAc) and then a Sephadex LH-20 column (EtOH) chromatographies to afford a mixture of phloroglucinol derivatives, which was purified by a C18 column (MeOH/H2O) and C18 HPLC (MeOH/H2O) to yield petiolin D (1, 0.0008%). EtOAc-soluble portions were applied to a Sephadex LH-20 column (MeOH/H2O), a C18 column (MeOH/H2O), a silica gel column (CHCl3/MeOH) chromatographies, and C18 HPLC (MeOH/H2O) to give petiolin E (2, 0.0008%).

The molecular formula of petiolin D (1), C30H42O4, was established by HRESIMS [m/z 489.2973 (M+Na)+, Δ -0.8 mmu]. 1H and 13C NMR data (Table 1) of 1 revealed the presence of one hydrogen-bonded hydroxyl (δH 14.07), one fully substituted benzene ring (δC 163.1, 160.4, 156.0, 110.8, 106.0, and 104.9), two trisubstituted olefins [δH 5.20 and 5.08 (each 1H, t, J = 7.0 Hz); δC 134.2, 131.0, 124.5, and 122.9], one 2-methylpropanoyl group [δH 3.79 (1H, sept, J = 6.5 Hz), 1.19 and 1.18 (each 3H, d, J = 6.5 Hz); δC 209.8, 38.9, 19.6, and 19.1], two sp3 quaternary carbons attached to an oxygen atom [δC 84.5 and 75.9], two methines [δH 2.82 (1H, brs) and 2.20 (1H, m); δC 45.9 and 27.6], three methylenes [δH 2.18 (1H, ddd, J = 13.2, 4.4, 3.0 Hz), 1.86 (1H, dd, J = 13.2, 1.5 Hz), 1.82, 1.47, 1.32, 0.90 (each 1H, m); δC 37.4, 34.8, and 21.9], and three tertiary methyl groups [δH 1.55, 1.43, and 1.06 (each 3H, s); δC 29.6, 28.7, and 24.2]. The presence of a geranyl group was implied by 1H–1H COSY correlations of H2-11 to H-12 and H2-15 to H-17, HMBC correlations of H3-14 to C-12, C-13, and C-15, H3-19 to C-17, C-18, and C-20, and NOESY correlations of H2-11 to H3-14 and H-12 to H2-15. The 1H–1H COSY spectrum suggested the connectivity of C-24 to C-26. HMBC correlations of H3-27 to C-24, C-25, and C-26 indicated that an oxygenated sp3 quaternary carbon (C-25) was attached to C-24, C-26, and C-27. Connectivities of C-28 to C-22, C-29, and C-30 were deduced from HMBC correlations of H3-30 to C-22, C-28, and C-29. 13C NMR chemical shifts of a benzene ring suggested the 1,3,5-trihydroxy substitution. HMBC correlations of H2-11 to C-1, C-2, and C-3, and H-21 to C-4 indicated that C-11 and C-21 were attached to C-2 and C-4, respectively (Figure 1). The molecular formula and the unsaturation degree of 1 implied that C-3 and C-5 were connected to C-25 or C-28 through an ether linkage, respectively.
Petiolin D (1) was crystallized from methanol/water as colorless platelets. A single-crystal X-ray diffraction analysis of 1 revealed the structure and relative stereochemistry. This crystal consisted of a pair of enantiomers, suggesting that 1 was a racemate. The ORTEP drawing of one enantiomer of 1 was shown in Figure. 2. Thus, the structure of petiolin D was elucidated to be 1, a new phloroglucinol derivative possessing citran skeleton.

Petiolin E (2) showed the pseudomolecular ion peak at m/z 431 (M+Cl)- in the ESIMS, and the HRESIMS analysis revealed the molecular formula to be C19H2435ClO9 [m/z 431.11126 (M+35Cl)-, Δ +1.2 mmu]. IR absorptions (1662, 1621, and 1579 cm-1) suggested the presence of chromone functionality.12 The 1H NMR spectrum showed signals of a pair of meta-coupled aromatic protons [δH 6.69 and 6.48 (each 1H, d, J = 1.8 Hz)], an olefin proton [δH 6.11 (1H, s)], an isobutyl group [δH 2.67 (1H, tq, J = 7.0, 7.0 Hz), 1.76 and 1.64 (each 1H, dq, J = 7.0, 7.0 Hz), 1.30 and 0.94 (each 3H, d, J = 7.0 Hz)], and an anomeric proton [δH 5.03 (1H, d, J = 6.9 Hz)]. The 13C NMR spectrum exhibited signals due to a conjugated carbonyl carbon, six aromatic carbons, a trisubstituted olefin, an isobutyl group, and a sugar moiety (Table 2). From these data, 2 was elucidated to be a isobutylchromone glycoside. The aglycone of 2 was deduced to be 5,7-dihydroxy-2-isobutylchromone from the analysis of the 1H-1H COSY and HMBC spectra (Figure 3). 13C NMR chemical shifts of the sugar moiety in 2 were coincident with those of quercetin-3-O-β-D-glucoside.13 The HMBC correlation of H-1’ to C-7 indicated that the glucosyl moiety was connected to a hydroxyl group at C-7, and its β-glycoside linkage was derived from the J-value (6.9 Hz) of the anomeric proton signal.

The sugar moiety was assigned as D-glucopyranose by chiral HPLC analysis of O-benzoyl derivatives of the methanolysis products of 2.14 Thus, the structure of 2 was elucidated to be 5,7-dihydroxy-2-isobutylchromone-7-O-β-D-glucopyranoside.

Petiolin D (1) is a new phloroglucinol derivative consisting of a citran skeleton, a geranyl group, and a 2-methylpropanoyl group, while petiolin E (2) is a new chromone-7-O-β-D-glucopyranoside having an isobutyl group at C-2. Though various phloroglucinol derivatives consisted of a citran skeleton were found in natural sources,15,16 a derivative having a citran skeleton with a geranyl group attached to its benzene ring like petiolin D (1) has not been reported so far. Petiolins D (1) and E (2) showed no cytotoxicity against murine lymphoma L1210 cells and human epidermoid carcinoma KB cells (both IC50 >10 μg/mL), while 1 and 2 exhibited a weak antifungal activity against Aspergillus niger (both MIC, 33.3 μg/mL).

EXPERIMENTAL
General Experimental Procedures
Optical rotations were recorded on a JASCO P-1030 digital polarimeter. IR and UV spectra were recorded on JASCO FT/IR-230 and Shimadzu UV-1600PC spectrophotometers, respectively. NMR spectra were measured by a JEOL ECA 500 spectrometer. The 7.27 and 76.9 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-T100LP.

Plant Material
Hypericum pseudopetiolatum var. kiusianum was collected in Kochi Prefecture, Japan in August 2005. Herbarium specimens were deposited in the botanical garden of the University of Tokushima (specimen number: UTP98013).

Extraction and Isolation
The aerial parts of H. pseudopetiolatum var. kiusianum (320 g) were extracted with MeOH (3L x 3), and the extracts were partitioned successively with n-hexane (300 mL x 3), EtOAc (300 mL x 3), and H2O (300 mL). The n-hexane-soluble portions were subjected to a silica gel column (n-hexane / EtOAc), a Sephadex LH-20 column (EtOH), a C18 column (MeOH/H2O, 85: 15) chromatographies, and then C18 reversed-phase HPLC (Mightysil RP-18, Kanto Chemical Co., Ltd, 10 x 250 mm; flow rate 3.0 mL/min; UV detection at 254 nm; eluent MeOH/H2O, 95:5) to afford petiolin D (1, 2.5 mg, 0.0008%). The EtOAc-soluble portions were applied to a Sephadex LH-20 column (H2O → MeOH), a C18 column (MeOH/H2O), a silica gel column (CHCl3/MeOH), and then C18 reversed-phase HPLC (Mighty sil RP-18, Kanto Chemical Co. Ltd, 10 x 250 mm; flow rate 3.0 mL/min; UV detection at 254 nm; eluent MeOH/H2O, 1:1, 0.1 % TFA) to give petiolin E (2, 2.4 mg, 0.0008%).

Petiolin D (1): Colorless crystal; mp 110 – 112 oC; UV (MeOH) λmax 240 (ε 15100), 299 (14200), 364 (2470) nm; IR (KBr) vmax 3411 and 1613 cm-1; 1H and 13C NMR data (Table 1); ESIMS m/z 489 (M+Na)+; HRESIMS: m/z 489.2973 (M+Na)+ (calcd for C30H42O4Na, 489.2981).

Petiolin E (2): Colorless amorphous; [α]23D -34.4 (c 0.48 MeOH); UV (MeOH) λmax 257 (ε 9200), 286 (3695), and 320 (2175) nm; IR (KBr) vmax 3408, 1662, 1621, and 1579 cm-1; 1H and 13C NMR data (Table 2); ESIMS m/z 431 (M+35Cl)-; HRESIMS: m/z 431.1113 (M+35Cl)- (calcd for C19H2435ClO9, 431.1101).

X-Ray Analysis of Petiolin D (1)
Petiolin D (1) was crystallized as colorless platelets from MeOH/water. The crystal having approximate dimensions of 0.20x0.20x0.02 mm was mounted in a roop. All measurements were made on a Rigaku RAXIS PAPID imaging plate area detector with graphite monochromated Cu-Kα radiation (1.54187Å) at -180oC. Crystal data: Formula C30H42O4, Formula weight 466.66, Space group P-1(#2), a=9.56002(17)Å, b=9.74718(18), c=28.0157(5), α=88.8543(7)o, β=86.5058(7), γ=85.7846(7), V=2598.38(8)Å3, Z=4, Dcalcd=1.193 g/cm3, 39766 reflections measured, 9338 reflections unique, 2θmax=136.5o, Rint=0.062, R1= Σ ||Fo|-|Fc|| / Σ |Fo| = 0.0572 for 6648 reflections with I>2σ(I), wR2=[Σ (w(Fo2-Fc2)2 / Σ w(Fo2)2)1/2 = 0.1581 for all reflections, goodness of fit 1.043. The structure was solved by direct methods (SIR2002)17 and expanded using Fourier thechniques.18 The non-hydrogen atoms were refined anisotropically. Hydrogen atoms were refined using the riding model. All calculations were performed using CrystalStructure19 except for refinement, which was performed using SHELXL-97.20 Crystallographic data for petiolin D (1) have been deposited at the Cambridge Crystallographic Data Center (deposition number CCDC 692371).

Stereochemical Assignment of the Sugar Moiety in Petiolin E (2).
Petiolin E (2, 0.3 mg) was treated with 3% HCl/MeOH (300 μL) at 110 oC for 1 h. After the solvent was removed by nitrogen stream, to the residue was added EtOAc (100 μL), and the EtOAc solution was extracted with H2O (100 μL x 3). The aqueous fraction evaporated in vacuo was treated pyridine (100 μL), triethylamine (15 μL), and benzoyl chloride (15 μL), at rt for 21 h. After addition of MeOH (100 μL), the reaction mixture was extracted with n-hexane (100 μL x 3). The n-hexane-soluble fraction was evaporated in vacuo to afford O-benzoyl/methyl derivative of the sugar units of 2. Authentic D- and L-glucose were treated with benzoyl chloride as described above to afford O-benzoyl/methyl derivatives of D- and L-glucose, respectively. The O-benzoyl/methyl derivatives were subjected to chiral HPLC analyses using Chiralpak OP(+) (Daicel Chemical Industry, Ltd., 4.6 x 250 mm; MeOH; flow rate 0.5 mL/min; UV detection at 254 nm). The retention time of O-benzoyl/methyl derivative of methanolysis product of 2 was found to be 13.7 min, while the retention times of O-benzoyl/methyl derivatives of authentic D- and L-glucose were found to be 13.7 and 14.5 min, respectively.

ACKNOWLEDGMENTS
We thank T. Akiyama and M. Inagaki, the Kochi Prefectural Makino Botanical Garden, for collection and botanical identification of the plant, S. OKa, A. Tokumitsu, and A. Miyao, Center for Instrumental Analysis, Hokkaido University, for measurements of HRESIMS. This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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