New Adducts of Iriflophene and Flavonoids Isolated from Sedum aizoon L. with Potential Antitumor Activity

Four new special compounds with character of an iriflophene unit and a flavonoid unit connecting via a furan ring were isolated from the roots of Sedum aizoon L. Their corresponding structures were elucidated on the basis of spectroscopic analysis. The in vitro anti-proliferative activities against BXPC-3, A549, and MCF-7 tumor cell lines were evaluated. Compounds 3 and 4 exhibited moderate cytotoxic activities with IC50 ranging from 24.84 to 37.22 μmol L−1, which was capable for further drug exploration.


Introduction
Sedum aizoon L., is an endemic plant, named 'jingtiansanqi' in folk medicine. It is distributed in Japan, North Korea, Mongolia, and China. The whole plant is used as a traditional medicine to treat traumatism, hemorrhage, palpitation, and neurasthenia [1][2][3]. Previously, the phytochemical constituents of Sedum aizoon L. have been extensively reported but only restricted to the aerial part [4][5][6][7][8][9]. So far, no investigation has been reported regarding the chemical constituents and biological activities of the underground part. In order to find new biologically active compounds, we extracted the roots of Sedum aizoon L. and four new special flavonoids were obtained and identified with character of an iriflophene unit and a flavonoid unit connecting via a furan ring (Figure 1). These rare dimers were discovered for the first time. The anti-proliferative activities in vitro against BXPC-3, A549, and MCF-7 tumor cell lines were evaluated by MTT assay.

Results and Discussion
The ethanol extract of Sedum aizoon L. was concentrated and stored at room temperature to yield a crude extract with sediment separated out at the bottom. The sediment was presumed to have low polarity because it dissolved in ethanol but separated out during concentration. Spectroscopic analysis of purified compounds led to the structures of Compounds 1-4. The 1 H-NMR and 13 C-NMR, IR, UV, HRESIMS, DEPT, HSQC, HMBC, and CD of Compounds 1-4 are available as supporting data.
The linkage between carbons and hydrogen was characterized by the HSQC while the HMQC data effectively positioned the hydroxyl groups and all non-protonated carbons. The hydrogen at δH 5.86 (H-6) showed correlations to the carbons at δC 168.7 (C-7), 163.3 (C-5), 98.1 (C-10), 95.2 (C-8), while the one at δH 5.77 (H-8) exhibited correlations to the carbons at δC 191.3 (C-4), 168.5 (C-7), 160.5 (C-9), 98.1 (C-10), and 96.9 (C-6) which confirmed the structure of ring A as a common disubstituted ring of a flavonoid. The structure of ring C was confirmed as a trisubstituted phenyl connected to the C-2 by a series correlations of the hydrogen at

Results and Discussion
The ethanol extract of Sedum aizoon L. was concentrated and stored at room temperature to yield a crude extract with sediment separated out at the bottom. The sediment was presumed to have low polarity because it dissolved in ethanol but separated out during concentration. Spectroscopic analysis of purified compounds led to the structures of Compounds 1-4.  [10]. A saturated quaternary carbon with an oxygen atom at δ C 79.89 (C-3) was readily characterized, while the dioxygenated carbon at δ C 117.01(C-2) was identified by comparison with the chemical shifts of dihydroflavonol moieties from the daphnodorins isolated from Daphne odora Thunb [11][12][13].
The determination of the absolute configuration of C-2 and C-3 in Compounds 1-4 was established by circular dichroic (CD) spectra (Figure 3). The CD spectra showed a negative cotton effect similar to that of daphnodorin F and H at 275 and 321 nm. Therefore, the absolute configuration of C-2 and C-3 was assigned as 2S, 3R [23,24]. group at C-7. Therefore, the structure of Compound 4 was elucidated as 1,8,10,10b-tetrahydroxy-5a-(4-hydroxy-3-methoxyphenyl)-9-(4-hydroxybenzoyl)-3-methoxy-5a,10bdihydro-11H-benzofuro[2,3-b]chromen-11-one, an iriflophene unit and a rhamnazin unit connecting via a furan ring [22]. The determination of the absolute configuration of C-2 and C-3 in Compounds 1-4 was established by circular dichroic (CD) spectra (Figure 3). The CD spectra showed a negative cotton effect similar to that of daphnodorin F and H at 275 and 321 nm. Therefore, the absolute configuration of C-2 and C-3 was assigned as 2S, 3R [23,24].  Table 1. With those distinct frameworks and promising activities, Compounds 3 and 4 can be considered as potential lead compounds for the further structural modification and biological evaluation.

General Procedures
Optical rotations were determined on an Anton Paar MCP-200 polarimeter (Anton Paar, Graz, Austria) in MeOH at 20 °C. UV spectra were obtained on a UV-1700 visible apectrophotometer (Shimadzu, Kyōto, Japan). IR spectra were recorded using a Bruker IFS-55 IR spectrometer with KBr disks. NMR experiments were performed on a Bruker 400 MHz 600 MHz AV Ш HD spectrometers (Bruker Biospin, Rheinstetten, Germany). HR-ESI-MS carried out on an Agilent Technologies 6540 UHD accurate mass Q-TOF MS apparatus (Agilent, Santa Clara, CA, USA). ECD spectra were Subsequently, the isolated Compounds 1-4 were evaluated for in vitro cytotoxicity against BXCP-3, MCF-7, and A549 tumor cell lines. The result revealed that Compounds 3 and 4 exhibited moderate cytotoxic activities to all three cell lines with IC 50 ranging from 24.84 to 37.22 µmol L −1 , as shown in Table 1. With those distinct frameworks and promising activities, Compounds 3 and 4 can be considered as potential lead compounds for the further structural modification and biological evaluation.

Plant Material
The roots of S. aizoon L. were collected at Heze, Shandong Province, China, in August 2016 and identified by Prof. Jincai Lu (School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, China). A voucher specimen (No.20160930) was deposited in the Herbarium of Shenyang Pharmaceutical University.