Sikokianin D, A New C-3/C-3"-Biflavanone from the Roots of Wikstroemia indica

Abstract

A new 3,3′′-biflavanone, sikokianin D (1), was isolated from the roots of Wikstroemia indica, together with two known compounds. Their structures were elucidated by chemical evidence and spectral analyses, including HR-ESI-MS, and 1D- and 2D-NMR techniques.

1. Introduction

Wikstroemia indica (Linn.) C. A. Mey., a shrub of the Thymelaeaceae family, is wildely distributed in the southeast of China. Known as Liaogewang, it has long been used as a folk medicine in southern China for treating arthritis, tuberculosis, syphilis and pertussis [1]. Moreover, W. indica has antifungal, anti-inflammatory, anti-cancer, antiviral and antimalarial effects [2,3,4,5,6,7]. The chemical constituents of the roots have been investigated previously, leading to the identification of groups of flavonoid, coumarin and lignan compounds [2,3,4,5,6,7,8,9]. In previous paper [10], we have reported several C-3/C-3"-biflavanones from the roots of Stellera chamaejasme L. (Thymelaeaceae) collected in Yunnan. C-3/C-3"-Biflavanones have been shown to exhibit a wide range of pharmacological activities, such as antibacterial, anti-inflammatory, antimalarial, and antitumor activities [4,11,12,13,14]. In connection with these interesting biflavanones, we examined the chemical constituents of other Thymelaeaceae plants and one new C-3/C-3"-biflavanone, sikokianin D (1), together with two known compounds, namely sikokianin B (2) and sikokianin A (3) (Figure 1) was isolated from the roots of Wikstroemia indica. This paper describes the isolation and structure elucidation of these compounds.

Figure 1. Chemical Structures of 1–3.

Figure 1. Chemical Structures of 1–3.

2. Results and Discussion

Compound 1 was obtained as a pale yellow amorphous powder with optical activity ([α] : + 231). The HR-ESI-MS of 1 exhibited a quasi-molecular-ion peak ([M+H]⁺) at m/z 557.1442 (calc. 557.1448), corresponding to the molecular formula C₃₁H₂₄O₁₀. Moreover, this compound showed positive reaction with HCl-Mg reagent, indicating that it is a flavonoid. The ¹H-NMR spectrum of 1 (Table 1) displayed signals of one methoxyl group (δ_H 3.79, s, 3H), two H-atoms corresponding to H-2 (δ_H 5.57, 1H, d, J = 5.0 Hz) and H-2" (δ_H 5.19, 1H, d, J = 9.5 Hz), and two H-atoms corresponding to H-3 (δ_H 3.19, 1H, br s) and H-3" (δ_H 3.26, 1H, dd, J = 9.5, 3.0 Hz) at the rings C and C' of the biflavanone. In the ¹H- and ¹³C-NMR established by ¹H-¹H COSY and HMQC experiments (Table 1), the spectra showed its structural fragments to include two sets of typical 5,7-dioxygenated A rings (δ_H 5.74, 5.77, each 1H, d, J = 2.0 Hz; δ_H 5.78, 5.98, each 1H, d, J = 2.0 Hz), and two sets of para-oxygenated B rings (δ_H 7.22, 6.90, each 2H, d, J = 8.5 Hz; δ_H 6.93, 6.63, each 2H, d, J = 8.5 Hz). From the ¹³C-NMR data (Table 1), two carbonyl groups (δ_C 198.5, 196.1) were also observed. These structural fragments were connected to form the given carbon framework of 1 as a dimer of flavanonol derivatives. The partial (-CH-CH-CH-CH-) structure inferred from the ¹H-¹H COSY spectrum (bold line in Figure 2) suggested that the linkage of the two flavanones was possible only at the C-3 and C-3" positions, which was supported by the comparison of the ¹H- and ¹³C-NMR data of 1 with those of known 3,3"-biflavanones [4,6,8,10], and further confirmed by the HMBC correlations of H-2 (δ_H 5.57) with C-3" (δ_C 51.0). The B ring could be located at C-2, based on the observation of the clear cross-peaks of H-2' and H-6' (δ_H 7.22) with C-2 (δ_C 81.2). In the same way, linkage of the B' ring to C-2" of the C' ring was deduced by the correlations of H-2"'and H-6"'(δ_H 6.93) with C-2" (δ_C 83.3). The HMBC cross-peak between the methoxyl group and C-4' on the B ring indicated that the methoxyl group was connected to C-4'.

The stereochemistry at the C-2/C-3 and C-2"/C-3" positions in 1 was determined as cis-trans by comparison of the J values (J_H-2 = 5.0 Hz and J_H-2" = 9.5 Hz) with those of the known 3,3"-biflavanones. The key NOESY correlations between H-2" (δ_H 5.19) with H-2'(H-6') (δ_H 7.22) further confirmed the conclusion above. The relative stereochemistry of compound 1 was confirmed as shown in Figure 1 and the compound named sikokianin D.

Compound 2 was first reported as sikokianin B of which the location of MeO group was unsettled [8], and the exact configuration was elucidated by Nunome [4]. Sikokianin B and sikokianin C were determined by comparing their ¹H- and ¹³C-NMR and MS data with published values.

Table 1. NMR data of sikokianin D (1) in CD₃OD (500 MHz for ¹H, 125 MHz for ¹³C).

No.	δH Mult (J = Hz)	δC
2	5.57 d (5.0)	81.2 d
3	3.19 br s	49.3 d
4	-	198.5 s
5	-	165.0 s
6	5.74 d (2.0)	96.0 d
7	-	168.1 s
8	5.77 d (2.0)	97.0 d
9	-	165.0 s
10	-	103.6 s
1'	-	130.0 s
2'	7.22 d (8.5)	128.4 d
3'	6.90 d (8.5)	114.9 d
4'	-	160.9 s
5'	6.90 d (8.5)	114.9 d
6'	7.22 d (8.5)	128.4 d
2''	5.19 d (9.5)	83.3 d
3''	3.26 dd (9.5, 3.0)	51.0 d
4''	-	196.1 s
5''	-	165.3 s
6''	5.78 d (2.0)	97.0 d
7''	-	167.9 s
8''	5.98 d (2.0)	96.4 d
9''	-	163.9 s
10''	-	105.1 s
1'''	-	128.9 s
2'''	6.93 d (8.5)	130.3 d
3'''	6.63 d (8.5)	116.1 d
4'''	-	158.9 s
5'''	6.63 d (8.5)	116.1 d
6'''	6.93 d (8.5)	130.3 d
4'-OCH3	3.79 s	55.7 q

Table 1. NMR data of sikokianin D (1) in CD₃OD (500 MHz for ¹H, 125 MHz for ¹³C).

No.	δH Mult (J = Hz)	δC
2	5.57 d (5.0)	81.2 d
3	3.19 br s	49.3 d
4	-	198.5 s
5	-	165.0 s
6	5.74 d (2.0)	96.0 d
7	-	168.1 s
8	5.77 d (2.0)	97.0 d
9	-	165.0 s
10	-	103.6 s
1'	-	130.0 s
2'	7.22 d (8.5)	128.4 d
3'	6.90 d (8.5)	114.9 d
4'	-	160.9 s
5'	6.90 d (8.5)	114.9 d
6'	7.22 d (8.5)	128.4 d
2''	5.19 d (9.5)	83.3 d
3''	3.26 dd (9.5, 3.0)	51.0 d
4''	-	196.1 s
5''	-	165.3 s
6''	5.78 d (2.0)	97.0 d
7''	-	167.9 s
8''	5.98 d (2.0)	96.4 d
9''	-	163.9 s
10''	-	105.1 s
1'''	-	128.9 s
2'''	6.93 d (8.5)	130.3 d
3'''	6.63 d (8.5)	116.1 d
4'''	-	158.9 s
5'''	6.63 d (8.5)	116.1 d
6'''	6.93 d (8.5)	130.3 d
4'-OCH3	3.79 s	55.7 q

Figure 2. Key ¹H-¹H COSY and HMBC correlations of 1.

Figure 2. Key ¹H-¹H COSY and HMBC correlations of 1.

3. Experimental

3.1. General

Melting points were measured on a Thermal Values analytical microscope and are uncorrected. Optical rotations were recorded on a Perkin-Elmer 341 polarimeter. IR spectra were recorded on a Nicolet FI-IR 200SXY spectrophotomer. The spectra of high resolution-electrospray ionization-mass spectrometry (HR-ESI-MS) were acquired with a Micromass Q-TOF mass spectrometer (Waters Corporation USA). ¹H- and ¹³C-NMR spectra were measured in CD₃OD with TMS as the internal standard on a Bruker DMX-500 NMR instrument. Silica gel G₂₅₄ and H (Qingdao Sea Chemical Factory, China) were used for TLC and column chromatography, respectively.

3.2. Plant Material

The roots of Wikstroemia indica were purchased from a Chinese medicine pharmacy in Guangzhou, China, in September, 2011. The authentication process was carried out by Le Cai (Yunnan University). A voucher specimen was deposited in the Zhejiang University City College.

3.3. Extraction and Isolation

Air-dried powder roots (2.6 kg) of W. indica were extracted exhaustively with 95% aq. EtOH (9 L × 3) at r. t. After concentration in vacuo, a crude extract (270 g) was obtained, which was suspended in 1 L H₂O, and the suspension was extracted successively with petroleum ether (PE, 1 L × 3), EtOAc (1 L × 3), and BuOH (1 L × 3) to yield 34, 110, 89 g fractions, resp. The EtOAc extract was subjected to CC with PE/EtOAc gradient system of increasing polarity (9/1→5/5, 3600 mL) to give five fractions (Fraction 1–5). Fraction 3 was chromatographed repeatedly over SiO2 column with MeOH/H₂O (7/3→9/1, 1,200 mL) to afford 3 (15 mg). Fraction 4 was subjected to MPLC on octadecyl silica gel (3.5 × 30 cm) eluting by gradient elution with MeOH-H₂O (5 mL/min, linear gradient, 50:50→90:10) to yield compounds 1 (28 mg) and 2 (36 mg).

Sikokianin D (1). Yellow amorphous powder, mp 213–215 °C; ([α] : +231 (c = 0.48, MeOH); IR (KBr, cm⁻¹): 3362, 1643; ¹H-NMR and ¹³C-NMR data, see Table 1; HR-ESI-MS: m/z 557.1442 [M+H]⁺, calcd for C₃₁H₂₅O₁₀, 557.1448.

Sikokianin B (2). Yellow amorphous powder. ¹H-NMR: δ_H 3.23 (1H, t, J = 3.5 Hz, H-3), 3.33 (1H, dd, J = 9.5, 3.0 Hz, H-3"), 3.76 (3H, s, OCH₃), 5.17 (1H, d, J = 9 Hz, H-2"), 5.53 (1H, d, J = 4.5 Hz, H-2), 5.75 (1H, d, J = 2.0 Hz, H-6), 5.84 (1H, d, J = 2.0 Hz, H-8), 5.86 (1H, d, J = 2.0 Hz, H-6"), 5.97 (1H, d, J = 2.0 Hz, H-8"), 6.74~7.16 (8H, m, H-Ar). HR-ESI-MS: m/z 557.1446 [M+H]⁺. Spectral data were in accordance with those reported in the literature [4,8], which confirmed that the isolated compound 2 was sikokianin B.

Sikokianin A (3). Yellow amorphous powder. ¹H-NMR: δ_H 2.91 (1H, d, J = 2.0 Hz, H-3), 2.98 (1H, d, J = 2.0 Hz, H-3"), 3.82 (3H, s, OCH₃), 5.32 (1H, d, J = 2.0 Hz, H-2), 5.37 (1H, d, J = 2.0 Hz, H-2"), 5.75 (2H, d, J = 0.5 Hz, H-6, H-6"), 5.88 (2H, d, J = 0.5 Hz, H-8, H-8"), 6.63~7.04 (8H, m, H-Ar). HR-ESI-MS: m/z 557.1448 [M+H]⁺. Spectral data were in accordance with those reported in the literature [8], which confirmed that the isolated compound 3 was sikokianin A.

4. Conclusions

In conclusion, one new biflavanone, 5,5',7,7'-tetrahydroxy-2-(4-hydroxyphenyl)-2'-(4-methoxy-phenyl)-[3,3'-bichroman]-4,4'-dione (1), together with two known compounds, sikokianin B (2) and sikokianin A (3) was isolated from the EtOH extract of the roots of Wikstroemia indica.