Tiliabisflavan A, a New Flavan‐3‐ol Dimer from Tilia amurensis with Cytotoxic and Anti‐Inflammatory Effects

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In the search for anticancer compounds from Korean medicinal plants, themethanolic extract from the trunk ofTilia amurensis Rupr. (Tiliaceae) was found to have significant cytotoxicity against A549 (lung carcinoma), SK-OV-3 (ovary malignant ascites), SK-MEL-2 (skinmelanoma), andHCT-15 (colon adenocarcinoma) in our screening test. T. amurensis is commonly known as “bee tree” and widely distributed in the mountainous areas of Korea, China, and Japan. T. amurensis has been used to treat various diseases as a Korean traditional medicine from ancient times. Its flowers have been applied for alleviating a fever, and its leaves have also been traditionally used for analgesia and rheumatoid arthritis. Importantly, an extract of this tree has been used to treat stomach cancer without any side effects. A recent pharmacological study revealed the potential of T. amurensis as an anticancer agent by evaluating the DNA topoisomerase inhibitory activity. However, only a few constituents associated with the anticancer activity from T. amurensis have been reported. Recently, our phytochemical investigations on T. amurensis revealed the presence of bioactive lignan constituents with antitumor and anti-inflammatory activities. Our continuing interest in further research on the bioactive constituents from T. amurensis led us to investigate bioactive metabolites of T. amurensis trunk. A bioassay-guided fractionation and chemical investigation of its MeOH extract resulted in the isolation and identification of four flavan-3-ols, including a new flavan-3-ol dimer, tiliabisflavanA (1) (Figure 1). The isolated compounds were evaluated for their antiproliferative activities against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines, as well as their inhibitory effects on NO production in a lipopolysaccharide (LPS)-activated BV-2 cell line. Compound 1 was obtained as a colorless gum. The molecular formula of 1 [ α 1⁄2 25 D +31.5 (c 0.35, MeOH)] was determined to be C31H28O12 based on positive high-resolution electrospray ionization mass spectrometry (HR-ESIMS) data (m/z 615.1479 [M +Na], calcd for C31H28NaO12, 615.1478). The infrared (IR) spectrum showed absorption bands for hydroxyl group (3358 cm) and aromatic rings (1614 and 1450 cm). The structure of 1 was further elucidated by detailed analysis of H and C NMR chemical shifts and by heteronuclear multiple-quantum correlation (HMQC) and heteronuclear multiple-bond correlation (HMBC) experiments. The H and CNMR spectra of 1were similar to those of2 except for the particular signals assigned tomethylene carbon [δH 3.88 (s); δC 15.5]. The assigned proton and carbon resonances of 1 in CD3OD are summarized in Table 1. The H, C NMR, and HMQC spectra of 1 displayed the characteristic signals for theC-ring of flavan-3-ol [δH4.75 (br s,H-2), 4.10 (br s, H-3), 2.82 (dd, J = 16.5, 4.5, H-4a), and 2.67 (dd, J = 16.5, 3.5, H-4b); δC 79.2 (C-2), 66.0 (C-3), and 27.7 (C-4)]. In addition, the 1,3,4-trisubstituted B ring signals [δH 6.94 (d, J = 1.5,H-20), 6.72 (overlap,H-50, H-60); δC144.7 (C-30, C-40), 130.5 (C-10), 118.5 (C-60), 114.7 (C-50), and 114.2 (C-20)] were observed, together with the A-ring carbon signals [δC 154.6 (C-5), 154.0 (C-7), 152.4 (C-9), 105.5 (C-8), 99.3 (C10), and 95.6 (C-6)]. The NMR data were closely related to those of 2; however, instead of the two A ring aromatic methines (C-6, C-8) in 2, the NMR spectra of 1 showed only one aromatic methine (δC 95.6, CH) for the A ring and an additional quaternary aromatic carbon (δC 105.5, C) as well as particular benzylic methylene signals [δH 3.88 (s); δC 15.5 (CH2)]. This indicated that compound 1 is comprised symmetrically of two epicatechin units connectingC-6 andC-8 positions through amethylene bridge. In theHMBCspectrum (Figure 2), the CH2 proton signal at δH 3.88 showed HMBC correlations with δC 154.0 (C-7), 152.4 (C-9), and 105.5 (C-8), which revealed that the two epicatechin units in 1 were linked between their C-8 position through amethylenebridge.Theother partial structures of 1 were clearly elucidated by the interpretation of the H–H correlation spectroscopy (COSY) and HMBC, and thus, the gross structure of 1 was established as shown in Figure 1 and named tiliabisflavan A. The relative configurations of C-2 and C-3 in 1 were determined to be cis for (2R,3R) or (2S,3S) configuration [δC 79.2 (C-2)/66.0 (C-3) and J3,4a = 4.5 Hz, J3,4b = 3.5 Hz for 1] on the basis of the comparison with the chemical shifts of C-2/ C-3 [δC 79.9/67.5 for (+)-epicatechin (2) and δC 82.6/68.7 for (+)-catechin (3)] and the coupling constants of H-3/H-4 Note DOI: 10.1002/bkcs.10011 K. H. Kim et al. BULLETIN OF THE KOREAN CHEMICAL SOCIETY