HETEROCYCLES

Short Paper | Regular issue | Vol. 89, No. 3, 2014, pp. 731-738
Received, 16th December, 2013, Accepted, 20th January, 2014, Published online, 29th January, 2014.
DOI: 10.3987/COM-13-12916

■ New Compounds from Tabebuia avellanedae

Li Zhang, Isao Hasegawa, Takanori Tatsuno, Tetsuro Kawabata, Tomihisa Ohta, and Takeshi Tadano*

School of Pharmacy and Pharmaceutical Sciences, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan

Abstract

Two new compounds, a compound firstly isolated from plants, a known iridoid and four known lignan derivatives were isolated from water extract of Tabebuia avellanedae. The chemical structures and relative configurations of the new compounds were determined by 1D, 2D NMR and MS spectroscopic analyses.

Tabebuia avellanedae Lorentz ex Griseb, which belongs to the family Bignoniaceae, is a popular tree distributed throughout the tropical rain forests of Central and South America. It is called “divine tree” by indigenous peoples of South America, because it is considered to be one of the most effective, economical and versatile remedies against a multitude of acute and chronic diseases.1 Its inner bark is commonly known as “taheebo”, “lapacho”, “pau d’arco”, and “ipe roxo”, which has been used for various ethnopharmacological prupose for over 1000 years.2 Following its popular use, the chemistry of this plant was extensively studied, and a variety of biologically active constituents have been isolated, such as furanonaphthoquinones, naphthoquinones, quinones, lignans, benzoic acid, cyclopentene dialdehyde, flavonoids, iridoids, phenolic glycosides, saponins, and coumarins.3-11
In this research, we separated the water extract of inner bark of Tabebuia avellanedae and obtained two new compounds (1-2), a compound firstly isolated from plants (3), together with five known compounds (4-8). The structures of 1-8 were listed in Figure 1, and 1H and 13C-NMR spectroscopic data of 1-3 were listed in Table 1 to Table 3.

Compound 1 was obtained as yellow powder. Its molecular formula was determined as C14H14O5 by HRFABMS m/z 261.0759 [M-H]-. The 1H NMR spectrum of two singlet δH 6.55 (s) and 6.35 (s), and HMBC correlations from δH 6.55 (H-5) to δC 126.1 (C-8a) and δC 144.7 (C-7), from δH 6.35 (H-8) to δC 126.3 (C-4a) and δC 145.8 (C-6) suggested the presence of a aromatic ring. The 1H NMR spectrum of four multiplet at δH 2.68 (m), 2.72 (m), 3.96 (m) and 3.80(m), together with 1H-1H COSY correlations between δH 3.96 (H-3α) and δH 2.68(H-4α), from δH 3.80 (H-3β) to δH 2.72 (H-4β), suggested the presence of one oxidized ethyl group, and the HMBC correlations from δH 6.55 (H-5) to δC 28.7 (C-4), from δH 2.72 (H-4β) to 126.1 (C-8a), and from δH 3.80 (H-3β) to δC 126.3 (C-4a) indicated that the oxidized methylene group is located at C-4a of the aromatic ring. The 1H NMR spectrum of a singlet at δH 5.66 (s) suggested the presence of one oxidized methine group, and the HMBC correlations from δH 5.66 (H-1) to δC 114.0 (C-8) and 126.3 (C-4a), indicated that the oxidized methine group is located at C-8a of the aromatic ring. Furthermore, the HMBC correlations from δH 5.66 (C-1) to δC 63.0 (C-3) confirmed the presence of one isochroman group. The 1H NMR spectrum of one singlet at δH 4.47 (2H, s) revealed the presence of one hydroxymethyl group. The remaining signals of two doublet at δH 6.08 (d, J = 3.4) and 6.23 (d, J = 3.4) in the 1H NMR spectrum, the 1H-1H COSY correlations between δH 6.08 and δH 6.23 and the HMBC correlations from δH 6.08 (H-10) to δC 156.3 (C-12), from δH 6.23 (H-11) to δC 156.1 (C-9) were noted, furthermore, its molecular formula was determined as C14H14O5, indicated the presence of one furyl group. In addition, the HMBC correlations from δH 4.47 (H-13) to δC 108.9 (C-11) proved that the hydroxymethyl group is located at the C-12 of the furyl group. The HMBC correlations from δH 5.66 (H-1) to δC 111.7 (C-10) and 156.1 (C-9), indicted the furyl group is located at C-1 of the isochroman group. Thus, compound 1 was determined as shown in Figure 1.

Compound 2 was obtained as colorless oil. Its molecular formula was determined as C20H20O7 by HRFABMS m/z 371.1129 [M-H]-. The 1D NMR spectra and HMQC spectrum showed the presence of two ABX system aromatic rings [δH 6.95 (d, J = 1.7), 6.79 (d, J = 8.5), 6.90 (dd, J = 1.7, 8.5) and 7.03 (d, J = 1.7), 6.77 (d, J = 8.5), 6.84 (dd, J = 1.7,8.5)], one methylenedioxy group [δH 5.93 (2H, s) and δC 102.4] and one methoxy group [δH 3.86 (3H, s) and δC 56.4]; the presence of two doublets [δH 4.04 (d, J = 9.3), 3.84 (d, J = 9.3) and δC 76.1], and an apparent triplet and a double doublets [δH 4.45 (t, J = 8.9), 3.77 (dd, J = 5.8,8.9) and δC 72.0] for nonequivalent geminal methylene protons at C-9' and C-9, respectively, together with one double triplet [δH 3.00 (dt, J = 5.8, 8.9) and δC 62.6], one doublet [δH 4.84 (d, J = 5.8) and δC 87.6], and one singlet [δH 4.66 (s) and δC 89.2] suggested a structure of furanofuran lignan with two ABX aromatic rings. It has the similar spectra with (1S*,2R*,5R*,6S*)-6-(4-hydroxy-3- methoxyphenyl)-2-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]-oactan-1-ol reported by S. Yamauchi et al.,12 expect for the positions of the two ABX system aromatic rings. In Figure 2, HMBC correlations observed from δH 5.93(H-10) to δC 149.4 (C-3), δC 148.7 (C-4), from δH 6.90 (H-6) to δC 107.8 (C-2), δC 148.7 (C-4) and δC 87.6 (C-7), from δH 6.95 (H-2) to δC 148.7 (C-4), δC 121.0 (C-6) and δC 87.6 (C-7), together with 1H-1H COSY correlations between δH 4.84 (H-7) and δH 3.00 (H-8), and among δH 4.45(H-9α), δH 3.77 (H-9β) and δH 3.00 (H-8), supported the methylenedioxy-substituted phenyl group is located at C-7 (not C-7') of furanofuran group. In addition, the NOE correlations (measured in DMSO) were observed between H-8 and H-8'-OH, from H-8 to H-9β, from H-7 to H-7'α, and from H-7 to H-9α. Thus, the structure was established as in Figure 1.
Compound 3 was obtained as yellow amorphous powder. It was synthesized by K. Mori and K. Okada in 198413 but NMR data was not showed. The 1D NMR spectra of seven protons [δH 7.49 (m), 7.44 (dd, J = 7.6,7.9) , 7.86 (d, J = 7.9) , 7.80 (d, J = 8.3) , 7.55 (m), 7.51 (m), 8.07 (d, J = 8.6) ] and carbon signals [δC 137.7, 126.7, 125.2, 128.9, 133.2, 128.6, 126.0, 122.6, 123.2, 130.9], suggested the existence of more than one aromatic rings. Correlations observed in HMBC spectrum, from δH 8.07 (H-9') to δC 126.0 (C-7'), δC 133.9 (C-5'a) and δC 137.7(C-2'), from δH 7.51 (H-8') to δC 130.9 (C-9'a) and δC 128.6 (C-6'), from δH 7.80 (H-6') to δC 122.6 (C-8'), δC 130.9 (C-9'a) and δC 128.9 (C-5'), from δH 7.86 (H-5') to δC 128.6 (C-6'), δC 130.9 (C-9'a) and δC 126.7 (C-3'), from δH 7.44 (H-4') to δC 133.9 (C-5'a) and δC 137.7 (C-2'), from δH 7.49 (H-3') to δC 128.9 (C-5') and δC 130.9 (C-9'a) revealed presence of one naphthyl group. The 1D NMR spectra and HMQC spectrum revealed the presence of two methyl groups [δH 1.27 (3H, s), δC 26.9 and 1.68 (3H, d, J = 6.5), δC 20.7], two methylene groups [δH 2.46 (m), 2.20 (m), δC 46.5, and 1.77 (m), 1.64 (m), δC 42.1], one hydroxymethyl group [δH 3.86 (m), 3.76 (m) and δC 59.6], one oxidized quaternary carbon [δC 72.5], one lower field-shifted methine group and one carbonyl group [δC 171.2]. Furthermore, the HMBC correlations from δH 1.68 (H-10') to δC 137.7 (C-2') and δC 44.6 (C-1') and from δH 7.49 (H-3') to δC 44.6 (C-1') revealed C-1' is located at C-2' of naphthyl group. The HMBC correlations from δH 2.46 (H-2α) to δC 171.2 (C-1), δC 72.5 (C-3) and δC 26.9 (C-6), from δH 1.27 (H-6) to δC 46.5 (C-2), δC 72.5 (C-3) and δC 42.1 (C-4) and from δH 3.86 (H-5α), δH 3.76 (H-5β) to δC 72.5 (C-3) indicated the presence of 3,5-dihydroxy-3-methylpentanyl group. In addition, its molecular formula was determined as C18H23NO3 by HRFABMS m/z 300.1606 [M-H]-, confirmed the presence of nitrogen. The NOE correlations from δH 5.95 (H-1') to δH 8.07 (H-9'), δH 1.68 (H-10') and δH 6.17 (-NH-), from 2.46 (H-2α), 2.20 (H-2β) to 6.17 (-NH-) and δH 1.27 (H-6) revealed the imino group is located to C-1' and C-1with amido bond. Thus, the structure was established as in Figure 1.
The five known compounds epipinoresinol(4),14 pinoresinol(5),14 (+)-balanoponin(6),15 salicifoliol(7),16 and 3-deoxy-artselaenin(8),17 were identified by comparing their spectroscopic data with those in the literature.

EXPERIMENTAL
General. Optical rotations were measured using a Horiba SEPA-3000 high-sensitivity polarimeter. UV spectra were measured using a Shimadzu UV-1600 UV-visible spectrometer. IR spectra were recorded on a Shimadzu IR-460 IR spectrophotometer, whereas NMR spectra were obtained using the ECA600 spectrometer in CD3OD, CDCl3 or DMSO. Chemical shifts were referenced to the residual solvent peaks (δH 3.30 and δC 49.8 for CD3OD, δH 7.24 and δC 77.0 for CDCl3, δH 2.49 and δC 39.5 for DMSO-d6). Mass spectra were measured on the JMS-T100TD and JMS-700 mass spectrometer. Reversed-phase HPLC was carried out on C30-UG-5 (5 µm, Nomura. Chemical, Seto, Japan) and C18-AR-Ⅱ(5 µm, Nacalai Tesque., Japan). Silica gel (63-210 μm, Kanto Kagaku, Japan) and ODS (63-212 μm, Wako Pure Chemical, Japan) were used for open-column chromatography. Thin-layer chromatography (TLC) was carried out on silica gel 60 F254 (Merck) and RP-18 F254S (Merck).
Plant material. Water extract of Tabebuia avellanedae for the present investigation was taxonomically identified and extracted by Taheebo Japan Corporation. In accordance with their method, dried bark of Tabebuia avellanedae (10 kg) were extracted with boiling water (30 L) three times, and the water solutions were combined and concentrated in vacuo to get the crude extract. The plant sample was deposited in a database in our laboratory under registration number T-340.

Extraction and isolation. The water extract (100 g) was suspended in H2O (1 L) and partitioned successively with n-hexane, EtOAc and n-BuOH (each 1 L, 3 times) to yield n-hexane fraction (0.6 g), EtOAc fraction (14.1 g), n-BuOH fraction (31.5 g) and H2O fraction (65.1 g), respectively. The EtOAc fraction (14.0 g) was chromatographed on silica gel with a gradient solvent system (n-hexane/EtOAc/MeOH) to give 15 fractions (A1-A15). A3 and A4 (n-hexane/EtOAc =1/1, 2.3 g) was rechromatographed on ODS with gradient solvent (MeOH/H2O) to afford 14 fractions (B1-B14). Fraction B1 (MeOH/H2O= 0/1, 280mg) was subjected on ODS with gradient solvent (MeOH/ H2O) to afford 12 fractions (C1-C12). Fraction C7 (MeOH/H2O= 1/4, 6.2 mg) was further purified by ODS HPLC (C18-AR-Ⅱ) with 33% MeOH to afford compound 1 (6.0 mg). Fraction B4 (MeOH/H2O= 1/4, 69.0 mg) was subjected on Sephadex LH-20 with MeOH to get 9 fractions (D1-D9). Fraction D6 was separated using ODS HPLC (C18-AR-Ⅱ) with 20% MeOH to afford compound 8 (6.0 mg). Fraction B5 (MeOH/H2O= 1/4, 119.2 mg) was applied to Sephadex LH-20 with MeOH to get 3 fractions (E1-E3), and fraction E3 was further purified by C30 HPLC (C30-UG-5) with 40% MeOH to obtain compound 7 (2.9 mg). Fractions B11 and B12 (MeOH/H2O= 3/4, 79.8 mg) was subjected on Sephadex LH-20 with MeOH to get 6 fractions (F1-F6), and fraction F4 (40.0 mg) was loaded to silica gel with a gradient solvent system (CHCl3/MeOH) to give 11 fractions (G1-G11). Fraction G4 (6.9 mg) was purified by ODS HPLC (C18-AR-Ⅱ) with 50% MeOH and 55% MeOH to afford compound 5 (0.9 mg) and 2 (1.6 mg) and fraction H. Fraction H (1.8 mg) was further purified by C30 HPLC (C30-UG-5) with 52% MeOH to afford compound 4 (1.0 mg). Fractions G6 and G7 (3.0 mg) was purified by C30 HPLC (C30-UG-5) with 50% MeOH to afford compound 6 (1.2 mg). Fraction B13 (MeOH/H2O= 3/4, 100.9 mg) was separated using Sephadex LH-20 with MeOH to get 6 fractions (I1-I6), and fractions I3 and I4 (85.0 mg) was subjected on silica gel with a gradient solvent system (n-hexane/EtOAc) to give 10 fractions (J1-J10). Fraction J9 (21.2 mg) was further purified by ODS HPLC (C18-AR-Ⅱ) with 60% MeOH to afford compound 3 (1.0 mg).

Compound 1.
Yellow powder; [α]D26.4 –0.51 (MeOH, c1.00); UV (MeOH) λmax (log ε) 286 (1.27), 225 (1.71) nm; IR υmax (KBr) 3271, 2926, 2852, 1720, 1651, 1556, 1506, 1456, 1286, 999 cm–1; 1H NMR spectroscopic data (600 MHz, CDCl3) and 13C NMR spectroscopic data (125 MHz, CDCl3), are shown in Table 1. HRFABMS m/z 261.0759 [M-H]- (calcd. for C14H13O5, 261.0763).

Compound 2.
Colorless oil; [α]D26.8 +0.71 (MeOH, c1.90); UV (MeOH) λmax (log ε) 284.5 (1.10), 232 (1.39) nm; IR υmax (KBr) 3334, 2877, 1699, 1606, 1515, 1446, 1242, 1037, 931, 799, 756 cm–1; 1H NMR spectroscopic data (600 MHz, CDCl3) and 13C NMR spectroscopic data (125 MHz, CDCl3), are shown in Table 2. HRFABMS m/z 371.1129 [M-H]- (calcd. for C20H19O7, 371.1131).

Compound 3.
Yellow amorphous powder; [α]D22.6 +5.82 (MeOH, c1.00); UV (MeOH) λmax (log ε) 292 (2.23), 281(2.38), 271 (2.34), 226 (2.97), 212 (2.95) nm; IR υmax (KBr) 3308, 2972, 2932, 1716, 1634, 1539, 1516, 1456, 1396, 1375, 1238, 1121, 1060, 1024, 800, 779, 756 cm–1; 1H NMR spectroscopic data (600 MHz, CDCl3) and 13C NMR spectroscopic data (125 MHz, CDCl3), are shown in Table 3. HRFABMS m/z 300.1606 [M-H]- (calcd. for C18H22NO3, 300.1600).

ACKNOWLEDGEMENTS
The authors thank Taheebo Japan Co., Ltd for generously providing the powdered inner bark of Tabebuia avellanedae.

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