Four new isoflavanones from Tadehagi triquetrum

Introduction

Tadehagi triquetrum (Linn.), belonging to the family of Papilionaceae, is an endemic shrub widely distributed in the southern area of Yunnan Province, China. As a traditional DAI medicine in southwest of China, it has been widely used for the treatments of anthelminthic, stomachic, antimicrobial, and inflammation. In addition, it also has been used as a nutrientand appetitive feedstuff.^{1, 2}

Previous phytochemical studies found a few of isoflavones and phenols from this species.^{3, 4} As a part of our continuing research on medicinal plants of DAI ethnopharmacy, ^6-10 we investigated the chemical constituents of the whole plants of T. triquetrum, which led to the isolation of four new isoflavanones, named triquetrumones E–H (1–4). Isoflavanones possessing isoprenoid unit, can be considered as the characteristic constituents in this plant. This paper describes the isolation and the structural elucidation of these four new compounds.

Results and Discussion

Compound 1 was obtained as yellow amorphous powder which showed an orange fluorescence under UV at 365 nm. The molecular formula was determined as C₂₇H₂₆O₇ by HRFABMS ([M + H]⁺ at m/z 463.1738). The IR spectrum showed bands at 3440 (OH), 1637 (C=O), and 1604 (aromatic ring) cm⁻¹. In the ¹H NMR spectrum (Table 1), one singlet at δ_H 8.03 (s, H-2), an aromatic C-methyl group at δ_H 2.12 (s, H-11), two phenolic hydroxyl at δ_H 12.62 (br. s) and 8.05 (br. s), one methoxyl group at δ_H 3.84 (s) and two 2, 2-dimethylpyran rings at δ_H 5.62 (d, J = 9.9, H-5''), 6.71 (d, J = 9.9, H-4''), 1.48 (s, H-7'', 8'') and 5.63 (d, J = 9.9, H-5'''), 6.82 (d, J = 9.9, H-4'''), 1.50 (s, Me-7''' and Me-8''') were observed. In addition, the ¹³C NMR spectrum (Table 2) displayed 15 skeletal C atoms: one carbonyl at δ_C 181.9, 13 quaternary C-atoms at δ_C 104.7–159.1, and as well one CH group at δC 154.7. Considering the above NMR data and the orange fluorescence under UV at 365nm, the structure was proposed to be an isoflavone, possessing two isoprenoids units.³

The HMBC correlations (Fig. 1) of H-5''/C-8 and H-4'' with C-6'', C-7, and C-9, and of H-5'''/C-3', and H-4''' with C-6''' and C-4' suggested that two dimethylpyrano rings were condensed to C-7/8 and C-4'/3', respectively. A methyl at δ_H 2.12 was assigned to connection with C-6 by its correlations with C-5, 6, 7 in the HMBC spectrum. Moreover, two OH groups were placed at C-5 and C-2', respectively, and a methoxyl group was positioned at C-5' by HMBC experiments. Complete analysis of the ¹H and ¹³C NMR, HSQC, HMBC data established the structure of 1 as (5, 2'-dihydroxy-5'-methoxyl-6-methyl[6'', 6''-dimethylpyrano-(2'', 3'':7, 8)]-[6''', 6'''-dimethylpyrano(2''', 3''':4', 3')]-isoflavanone, and named triquetrumone E.

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Compound 2 was isolated as yellow amorphous powder. It gave a molecular formula C₂₁H₂₀O₇ by HRFABMS ([M + H]⁺ at m/z 385.1274). Bands for OH and carbonyl functional groups were assumed by IR absorption at 3421 and 1647 and UV absorptions at 270 nm were typical for an isoflavanone.¹¹ The NMR spectra of compound 2 (Tables 1 and 2) were similar to those of 1''', 2'''-dehydrocyclokievitone, ¹² except for the appearance of a methyoxyl group (δ_H 3.78, δ_C 60.7) in 2, which was also supported by its molecular formula. The methyoxyl group was paced at C-3', supported by the HMBC correlations (Fig. 1). Thus, compound 2 was established to be 5, 2', 4'-trihydroxy-3'-methoxy[6'', 6''-dimethylpyrano(2'', 3'':7, 8)]-isoflavavone, and named triquetrumone F.

Compound 3 appeared as yellow amorphous powder whose molecular was established as C₂₇H₂₆O₈ by HRFABMS at m/z 479.1723 (calcd. for C₂₇H₂₇O₈, 479.1706), which indicated 15 degrees of unsaturation. T. triquetrum are known to contain three 5-hydroxy-coumaronochromones, and all of which fluoresce on silica gel thin-layer plates under 365 nm of UV light.³ Take UV, MS, and NMR data into account, compound 3 can be most logically formulated as an isoflavone, in which C-2 and C-2' was linked by an ether oxygen to form a pentacyclic ring system.¹³ The ¹H NMR spectrum (Table 1) of 3 revealed two 2, 2-dimethylpyrane rings at δ_H 5.77 (d, J = 9.9, H-5''), 6.46 (d, J = 9.9, H-4''), 1.38 (s, Me-7'') and 1.42 (s, Me-8''), together with 5.68 (d, J = 10.0, H-5'''), 6.57 (d, J = 10.0, H-4'''), 1.41 (s, Me-7''') and 1.45 (s, Me-8'''), associated with an aromatic C-methyl group at δ_H1.92 (s) and two singlets at δ_H 6.32 (s, H-2) and 6.89 (s, H-6'), respectively.

The ¹³C and ¹H NMR spectra of 3 implied that this compound might be an analogue of triquetrumone B.³ Careful comparison of the NMR data of two compounds revealed that one more OH group might be appeared at C-3, suggested by downshift signals at δ_C 79.4 (s, C-3), δ_H 6.08 (br. s, OH) in 3. The proposal was further supported by the HMBC correlations (Fig. 1) between OH-3 with δ_C 193.4 (s, C-4), 111.5 (d, C-2) and 79.4 (s, C-3). Thus, 3 was established as 3, 5-dihydroxy-5'-methoxyl-6-methyl[6'', 6''-dimethylpyrano(2'', 3'':7, 8)]-[6''', 6'''-dimethylpyrano(2''', 3''':2', 3')]coumaronochromone, and named triquetrumone G.

Compound 4 was obtained as yellow amorphous powder. The HRFABMS clearly showed the [M + H]⁺ in agreement with the molecular formula C₂₆H₂₄O₈. The UV, IR, and NMR spectroscopic data also suggested a coumaronochromone structure for 4, ¹⁴ which was very similar to those of 3. Detailed comparison of the NMR data of two compounds revealed that a hydroxyl group was appeared on ring C of 4, instead of the methyoxyl in 3, concurring with its molecular formula. The assignment was also supported by the HMBC correlations (Fig. 1) of δ_H 7.50 (s, OH-5') with δ_C 106.9 (s, C-3'), 142.1 (s, C-4') and 143.0 (s, C-5'). Thus, the structure of 4 was established as 3, 5, 5'-trihydroxy-6-methyl[6'', 6''-dimethylpyrano(2'', 3'':7, 8)]-[6''', 6'''-dimethypyrano(2''', 3''':2', 3')]coumaronochromone, and named triquetrumone H.

Experimental Section

General Experimental Procedures. Column chromatography (CC): Silica gel (200–300 mesh, Qingdao Marine Chemical corporation, China) and Sephadex LH-20 (Amersham Biosciences, Sweden); TLC monitoring, visualization by heating the silica gel plates sprayed with 10% H₂SO₄ in EtOH. Optical rotations: Horiba SEPA-300 polarimeter. UV spectra: Shimadzu 210-A double-beam spectrophotometer; λ_max (logε) in nm. IR Spectra: Brucker Tensor 27 spectrometer; KBr pellets; in cm⁻¹. NMR Spectra: Bruker AM-500 spectrometers; δ in ppm with SiMe₄ as internal standard, J in Hz. MS: VG Autospec-3000 and API QSTAR-Palsar-I spectrometer.

Plant Material. The whole plants of Tadehagi triquetrum were collected from Xishuangbanna, Yunnan Province, China, in April 2007. Its identity was confirmed by Mr. Jing-Yun Cui, and a voucher specimen (NO. 2007042) 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 dried and milled sample (10 kg) were soaked in 95 % EtOH (40 L × 3) under reflux (48 h × 3), and the solvent was evaporated in vacuo. The residue was partitioned between EtOAc and H₂O. The EtOAc extract (80 g) was subjected to CC (CHCl₃/Me₂CO 10:1→1:1): eight fractions (Fr.1–8) by TLC. Fr.3 (15 g) was repeatedly subjected to CC (1. Silica-G petroleum ether/Me₂CO 10:1→2:1; 2. Sephadex LH-20, CHCl₃/MeOH 1:1) to afford 1 (6 mg). Fr.4 (12 g) was purified by repeated CC (SiO₂, petrolrum ether/Me₂CO 3:1) to afford 2 (21 mg). Fr.5 (10 g) was resubmitted to CC (1. SiO₂, CHCl₃/MeOH 70:1→50:1; 2. Sephadex LH-20, MeOH/H₂O 9:1) to provide 3 (5 mg) and 4 (25 mg).

Triquetrumone E (1): yellow amorphous powder, UV (MeOH) λ_max (log ε) 273 (4.49), 212 (4.51) nm. IR (KBr) ν_max 3440, 2924, 1637, 1604 cm⁻¹. ¹H and ¹³C NMR data see Tables 1 and 2. ESIMS (positive): 463 ([M + H]⁺). HRFABMS (positive): 463.1738 ([M + H]⁺, C27H27O7, calcd. 463.1757).

Triquetrumone F (2): yellow amorphous powder, [α]_D²⁰ −1.63 (c 0.002, Me₂CO). UV (MeOH) λ_max (log ε) 270 (4.24), 207 (4.42) nm. IR (KBr) ν_max 3421, 1647, 1598 cm⁻¹. ¹H and ¹³C NMR data see Tables 1 and 2. ESIMS (positive): 385 ([M⁺ H]+). HRFABMS (positive): 385.1274 ([M + H]⁺, C₂₁H₂₁O₇, calcd. 385.1287).

Triquetrumone G (3): yellow amorphous powder, [α]_D²⁰ −44.5 (c 0.001, Me₂CO). UV(MeOH) λ_max (log ε) 273 (4.01), 268 (4.02), 212 (4.39) nm. IR (KBr) ν_max 3441, 1632, 1600 cm⁻¹. ¹H and ¹³C-NMR data see Tables 1 and 2. EIMS: 478 [M]⁺. HRFABMS (positive): 479.1723 ([M + H]⁺, C₂₇H₂₇O₈, calcd. 479.1706).

Triquetrumone H (4): yellow amorphous powder, [α]_D²⁰ −32.3 (c 0.001, Me₂CO). UV (MeOH) λ_max (log ε) 274 (4.34), 268 (4.34), 212 (4.45) nm. IR (KBr) ν_max 3424, 1635, 1599 cm⁻¹. ¹H and ¹³C NMR data see Tables 1 and 2. EIMS: 464 [M]⁺. HRFABMS (positive): 465.1556 ([M + H]⁺, C₂₆H₂₅O₈, calcd. 465.1549).

Electronic Supplementary Material

Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-011-0033-5 and is accessible for authorized users.

Notes

Acknowledgments

The authors are grateful to National Basic Research Program of China (973 Program 2009CB522300), and the Chinese Academy of Science (KSCX2-EW-R-15) for financial support and to the members of the analytical group in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, for the spectral measurements.