Two aromatic acid derivatives and a xanthone from Hypericum hengshanense

Abstract Three previously undescribed compounds including two aromatic acid derivatives (1–2), and one xanthone (3), together with ten known compounds (4–13) were isolated from the aerial part of Hypericum hengshanense. The planar structures of three new compounds were established by 1 D and 2 D NMR and MS data. And the absolute configurations of compounds 1–2 were determined by the quantum chemical ECD calculations. Compounds 1–2 showed weak cytotoxicity against Hep-2 human cancer cell lines with IC50 values of 65.1 ± 2.7 and 78.0 ± 1.0 μg/mL, respectively. Graphical Abstract

Hypericum hengshanense, a traditional medicinal herb in China, is distributed in eastern Hunan Province and western Jiangxi Province, China.It is used as anti-inflammation, anti-depression, liver disease, perennial herb grown as a rare endemic species in Southwest and East China (Wang and Xu 2013).However, there are limited studies on the material basis and pharmacodynamics of the plant.In our search for bioactive metabolites from H. hengshanense, the systematically chemical isolation of the petroleum ether and ethyl acetate extracts of H. hengshanense led to three previously undescribed compounds, including two aromatic acid derivatives (1-2) and one xanthone (3) (Figure 1).All compounds were evaluated for their cytotoxic activity against Hep-2 (laryngeal cancer cells), Mcf-7 (breast cancer cells) and Eca-109 (esophageal cancer cells) human cancerous cells.Further, the key HMBC correlation from H-2 at d H 5.58 to C-7 0 at d C 171.5 revealed the linkage of the feruloyl and 1-methyl malic acid moieties was located between C-2 and C-7 0 via an ester bond (Figure S1).The absolute configuration of 1 was determined by comparing its specific rotation with the reported data for similar derivatives to 1. (S)-benzoyl malic acid has a negative specific rotation value [a] 21 D ¼ À7 (c ¼ 1.0, CHCl 3 ), whereas that of (R)-galloyl malic acid dimethyl ester is positive [a] 20 D ¼ 21 (c ¼ 0.1, MeOH) (Ma et al. 2012).Thus, the absolute configuration of compound 1 was suggested to assign as 2 R configuration based on its specific rotation being a positive value i.e. [a] 25 D ¼ þ20.2.ECD calculations were used to further verify the absolute configuration of compound 1.The ECD profile of R-1 was in good agreement with the experimental data by comparison of the experimental and calculated ECD spectra (Figure S2).Thus, the structure of 1 was confirmed as shown and named (R)-2-O-(4 0 -hydroxy-3 0 -methoxy-benzoyl)-1methoxy-malic acid.

Results and discussion
Compound 2 was obtained as a colorless amorphous solid, and its molecular formula of C 12 H 12 O 8 was determined by the HRESIMS peak at m/z 285.0607 Comparison of its 1 H and 13 C NMR data with those of 1 (Table S1, S2) revealed structural similarity with compound 1, the only difference being that the C-1 in compound 2 is attached to a hydroxy group instead of a methoxy group, which was further confirmed by the ROESY and HMBC data.Subsequently, comparison of the calculated ECD with the experimental ECD (Figure S2), the absolute configuration of 2 was assigned as 2 R. Thus, the structure of 2 was therefore elucidated as (R)-2-O-(4 0 -hydroxy-3 0 -methoxy-benzoyl)-malic acid.

General experimental procedures
UV and IR spectra were determined on a Shimadzu UV-250 spectrometer and a Shimadzu FTIR-8400S spectrometer, respectively.Optical rotations were measured using an Autopol IV-T automatic polarimeter (Rudolph Research Analytical, USA).Circular dichroism (CD) spectra were recorded with Chirascan Plus circular dichroism (Applied Photophysics Ltd., London, UK).LC-PDA-ESIMS data were recorded on a Waters ACQUITY SQD MS system (Waters, Milford, MA, USA) connected to a Waters 1525 HPLC with a 2998 Photodiode Array Detector (Waters, Milford, MA, USA) and a Waters Sunfire TM C 18 column (5 lm, 4.6 Â 150 mm).NMR (MeOH-d 4 ) spectra were acquired on a Bruker Ascend IIITM 600 MHz NMR spectrometer (Bruker Corporation, Fallanden, Switzerland).The chemical shifts (d) were reported in ppm, and coupling constants (J) were given in Hz.The HRESIMS data were recorded on a UHPLC System and the Q Exactive HF Mass Spectrometer (Thermo Fisher Scientific, USA).Separations were performed on Thermo C 18 columns (5 lm, 10 Â 150 mm) (Waters, Ireland) and COSMOSIL C 18 columns (10 Â 250 mm) using MeCN-H 2 O as eluent with a flow rate of 4 mL/min.All the solvents used for chromatography were of high-performance liquid chromatography (HPLC) grade and all the other chemicals were of analytical-reagent grade.HPLC-grade acetonitrile (MeCN) and methanol (MeOH) were purchased from Merck Chemical Company (Darmstadt, Germany).For column chromatography (CC), HP20 macroporous resin (Mitsubishi, Japan), silica gel (300-400 mesh, Qingdao Marine Chemical, Inc. Qingdao, China), Sephadex LH-20 (GE Health Care, Uppsala, Sweden) were used.Ethanol, petroleum ether, ethyl acetate, chloroform and methanol were purchased from Wuhan Tong Guang Technology Biology Company.

Plant material
The aerial parts of H. hengshanense were collected from Hengyang City, Hunan Province, China, in August 2019.Plant material was identified by Professor Dingrong Wan, South-Central Minzu University (SCUEC), Wuhan, China.A voucher specimen (reference number: No. SC0866) was deposited at the Plant specimen Bank, School of Pharmaceutical Sciences, SCUEC, Wuhan, China.

Computational calculations
In general, the conformational analyses were carried out via random searching in the Sybyl-X 2.1.1 software using the MMFF force field.Subsequently, the generated conformers for all conformers of compounds 1-2 were subjected to optimization with density functional theory (DFT) at the B3LYP/6-31G(d,p) level by the Gaussian 09 software.The conformers with a Boltzmann population of over 1% were chosen for ECD calculations and then the optimized conformers were calculated using the timedependent density functional theory (TD-DFT) method at the B3LYP/6-311G(d,p) level, solvent effects of the MeOH solution were evaluated at the same DFT level using the SCRF/PCM method.Finally, The Boltzmann-averaged ECD spectra were generated by the program SpecDis 1.62 (Bruhn et al. 2013) using a Gaussian band shape with a 0.30 eV exponential halfwidth from dipole-length dipolar and rotational strengths.By comparing the experimental spectra with the calculated ECD spectra, the absolute configurations of 1-2 were resolved.

Cytotoxic assay
Cytotoxicities of the compounds 1-13 were assessed using the MTT assay against human cell line Hep-2, Mcf-7 and Eca-109 cells according to the method described in previous literature (Karakas ¸et al. 2017).The experiments were repeated three times, and the cytotoxicity was expressed as the IC 50 value, which reduces the number of viable cells by 50%.

Conclusions
In conclusion, three new compounds (1-3), together with ten known compounds (4-13) were isolated from the aerial parts of H. hengshanense.Based on their spectroscopic data (IR, UV, HRESIMS, 1 D and 2 D NMR) and by comparison with related literatures, the structures of compounds 1-13 were elucidated.In addition, the absolute conformational structures of compounds 1-2 were elucidated based on the comparison of experimental and calculated ECD spectra.This paper enriches the research of H.
hengshanense to a great extent and lays the foundation for the research and development of new drugs.
Compound 1 was obtained as a colorless amorphous solid with molecular formula C 13 H 14 O 8 determined by the HRESIMS spectrum at m/z 297.0619 ([M -H] -, calcd for C 13 H 13 O 8 C 153.3, 148.8 and 121.6, three carbonyl quaternary carbons at d C 167.0, 171.5 and 173.2, three aromatic tertiary carbons at d C 113.7, 116.0 and 125.5, one oxygenated tertiary carbon at d C 70.5, one methylene carbon at d C 37.4, and two methoxy carbons at d C 56.4 and d C 53.0.The key HMBC correlations of the aromatic proton at d H 7.55 to the carbons at d C 148.8, 153.3, 125.5 and 167.0, the aromatic proton at d H 6.85 to the carbons at d C 153.3, 148.8, and 121.6, and the aromatic proton at d H 7.57 to the carbons at d C 153.3, 113.7 and 167.0, as well as the methoxy proton at d H 3.89 (3H, s) to carbon at d C 148.8, confirmed the substitution pattern of 3 0 -methoxy-4 0hydroxy and constructed a feruloyl moiety.In addition, the key 1 H-1 H COSY correlation of the methine at d H 5.58 to the methylene at d H 2.96, and the key HMBC correlations of the methine at d H 5.58 to the carbons at d C 171.5, 173.2 and 37.4, the methylene atFigure 1. Experimental and calculated ECD spectra of 1.
-297.0610).The IR absorption bands indicated the presence of hydroxy (2955 cm À1 ), ester carbonyl (1717 cm À1 ) and aromatic ring (1597, 1516 and 1431 cm À1 ) moieties.The UV spectrum of 1 displayed two characteristic absorption bands at 220 and 265 nm.The 1 H NMR spectrum (TableS1) showed the presence of a typical ABX system [d H 7.57 (1H, dd, J ¼ 8.2, 2.0 Hz), 7.55 (1H, d, J ¼ 2.0 Hz), 6.85 (1H, d, J ¼ 8.2 Hz)], two methoxy groups at d H 3.89 (3H, s) and d H 3.76 (3H, s), a methylene at d H 2.96 (2H, d, J ¼ 6.1 Hz) and an oxygenated methine at d H 5.58 (1H, t, J ¼ 6.1 Hz).The 13 C NMR and DEPT spectra (TableS2) exhibited 13 carbons, including three aromatic quaternary carbons at d These data suggested that 3 was a xanthone derivative having one methoxy group and three hydroxy groups.The specific position and plane structure of substituents are determined by HMBC and ROESY spectrums.The key HMBC correlations of the low-field aromatic proton at d H 8.10 to the carbons at d C115.1, 177.8, 114.9, 159.6 and 165.2, the aromatic proton at d H 6.88 to the carbons at d C 128.9 , 165.2, 103.2 and 115.1, and the aromatic proton at d H 6.94 to the carbons at d C 159.6 and 165.2 confirmed a hydroxy group at C-6 of A ring.Additionally, the key HMBC correlations of the aromatic proton at d H 7.24 to the carbons at d C114.3, 177.8,  142.7, 144.1 and 147.3, and the methoxy proton at d H 3.96 (3H, s)to the carbons at d C 97.2 and 144.1, as well as the key ROESY correlation of the proton at d H 7.24 to the methoxy proton at d H 3.96 confirmed the substitution pattern of 2-methoxy-3,4-dihydroxy in B ring.Therefore, compound 3 was determined as 3,4,6-trihydroxy-2methoxyxanthone.