Uncommon Bis-Amide Matrine-type Alkaloids From Sophora alopecuroides With Anti-inflammatory Effects

Four new alkaloids (1–4) belonging to rare examples of bis-amide matrine-type were isolated from the seeds of sophora alopecuroides. Their structures including absolute configuration were determined by extensive spectroscopic analysis, electronic circular dichroism (ECD) interpretation, and X-ray diffraction crystallography. Chemically, bis-amide matrine-type alkaloids can provide new molecular template for structural modification. Compounds 3–4 displayed obvious anti-inflammatory effects based on the inhibition of two key pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] in a dose-dependent manner, with IC50 values from 35.6 to 45.8 μm.

INTRODUCTION Sophora alopecuroides L. which belongs to the family of Leguminosae, is a salt-tolerant perennial herb plant and distributed in arid desert grassland of northwest China (Wang et al., 2012;Deng et al., 2019). The seeds of S. alopecuroides (Chinese name: Ku-Dou-Zi) has been regarded as a well-known traditional Chinese medicine for the treatment of fever, rheumatism, bacterial infection and inflammatory diseases (Huang et al., 2016). Previous phytochemical investigations on S. alopecuroides revealed alkaloids as one of principal active chemical constituents. Among them, matrine-type alkaloids exhibit diverse bioactivities such as antiviral (Zou et al., 2020), anti-insect (Huang et al., 2017), anti-tumor , and promising anti-inflammatory activities (Huang et al., 2016;Li et al., 2020).
Chemically, matrine-type alkaloids are considered as ideal lead compounds for further structure modifications because of special chemical structure, widespread biological activities, high safety threshold, as well as available commercial sources. In order to improve the activities and amplify their applicants, many matrine-type derivatives have been synthesized and reported in the recent years (Huang et al., 2017;Cai et al., 2018;Cheng et al., 2018;Lv et al., 2018;Cheng et al., 2020;Xu et al., 2020). Interestingly, we have noticed that almost all structural modifications were concentrated in the variations of D ring, such as introducing substituents to C-13 and C-14 sites, opening D ring, fusing D ring and further molecular simplification. However, the amide bond located at D ring is a critical part that can responsible for many biological activities according to the molecular docking analysis (Peng et al., 2020). It is necessary to search more matrine-type template for the development of structural modification strategy.
As a part of continuous systematic search for structurally unique and biologically meaningful natural products from the sophora species (Fan et al., 2019;Luo et al., 2021;Zhang et al., 2018a;Zhang et al., 2018b;Zhang et al., 2016;Zhang et al., 2017), four new matrine-type alkaloids (1-4) were obtained and identified ( Figure 1). It is worth mentioning that compounds 1-4 are uncommon examples of bis-amide matrine with the second amide group at C-2 or C-10. To data, hundreds of matrine-type alkaloids have been reported but only few cases such as 2-oxymatrine, 10-oxy-5,6-dehydromatrine, and 10oxysophoridine that possessed the bis-amide bond at C-2 or C-10 Zhang et al., 2018a). Additionally, all isolates were evaluated their anti-inflammatory activities in vitro based on production of two key pro-inflammatory cytokines (TNF-α and IL-6) in lipopolysaccharides (LPS)-stimulated RAW264.7 cells. Herein, the isolation, strucutre elucidation, and anti-inflammatory activites of those isolates are discussed.

Plant Material
The dry seeds of S. alopecuroides L. were collected from the area (GPS coordinates, 37°98′−38°22′ N, 106°20′−106°66′ E) of Wuzhong City, Ningxia Hui Autonomous Region, People's Republic of China on August 2014. A voucher specimen (accession no. SA-2014-08-28) was authenticated by Prof. Guang-Xiong Zhou (Jinan University) and available for inspection at the Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, P. R. China.

Single X-Ray Diffraction Data Analysis
The single-crystal X-ray diffraction data of one and three were acquired on an Agilent diffractometer with Cu Kα radiation. The structures were solved by the SHELXT structure solution program and refined with full-matrix least-squares minimization on F 2 using SHELXL via OLEX2 software package. Crystallographic data for one and three have been deposited in the Cambridge Crystallographic Data Centre (free of charge at https://www.ccdc.cam.ac.uk/) under deposition numbers CCDC are 2055031 and 2095514, respectively.

Electronic Circular Dichroism Calculations
Conformational analysis was initially performed using sybyl-X-2.1.1 program. Conformers occurring within a 10 kcal/mol energy window from the global minimum were chosen for geometry optimization and energy calculation using DFT with the B3LYP functional and the 6-311++G (d,p) basis set with the Gaussian09 program. Calculated results were completed using TD-DFT with the CPCM model in MeOH based on the optimized geometries. Finally, the Boltzmannaveraged ECD spectra of the two compounds were obtained and visualized with SpecDis 1.61 and drawn using the Origin Pro nine program. The half bandwidth (σ) and UV correction values applied in SpecDis for the final calculated ECD spectra are 0.30 eV and −5 nm, 0.30 eV and −6 nm for compounds 2 and 4, respectively, which are within the reasonable range. Their optimized conformation geometries, thermodynamic parameters, and populations of all conformations (2 and 4) are provided in supporting information.
Compound 4 [α] 25 D −12.2 (c 0.01, CH 3 OH), was obtained simultaneously with three in the same preparation liquid phase condition. Comprehensive analysis of its spectroscopic data indicated that four possessed the same molecular formula and almost identical 1D NMR resonances (Table 1) as that of 3, except for the second amide group exchanged from A ring to B ring. In the 2D NMR spectra of 4, the 1 H-1 H COSY correlations of H 2 -2/H 2 -3/H 2 -4 and H 2 -8/H 2 -9, as well as HMBCs from H 2 -2 to C-10, and from H 2 -8 to C-6/C-7/C-9 suggested the second amide group is located on B ring ( Figure 2). Additionally, good consistency between the experimental CD curve and calculated ECD curve ( Figure 5) allowed the assignment of (5R,11R) absolute configuration. Thus, compound 4 was elucidated and named as (−)-10-oxy-5-hydroxy-6,7-dehydromatrine.
At present, the structural modifications of matrine-type alkaloids have mainly focused on the variations of D ring due to the amide group at C-15 is an active reaction site. (Cai et al., 2018;Cai et al., 2020). Compounds 1-4 possessing rare bis-amide matrine-type motif could provide new molecular template and ideas for synthetic chemists. For example, new derivatives could be designed by the introduction of a protecting group on the D ring and subsequent structural modification via the second amide bond at C-2 or C-10 position on the A or B ring. So that the key part of D ring responsible for biological activity can be retained, and some unexpected good results may emerge.
IL-6 and TNF-α are essential mediators in inflammation processes. Owing to the extracts of S. alopecuroides possessing remarkable clinical effects on various kinds of inflammation, all  isolated alkaloids (1-4) were evaluated for their LPS-stimulated TNF-α and IL-6 production in RAW 264.7 cells using ELISA. Firstly, a MTT cytotoxicity assay was used to examine the cell viability of murine RAW 264.7 cells. The results displayed that all alkaloids at the concentration ranges from 3.125 to 50 μM were nontoxic on RAW 264.7 cells after 24 h treatment. Therefore, the tested alkaloids were applied to those concentration range to perform the next experiment. The production of IL-6 and TNF-α in the culture medium of the LPS-treated group both increased significantly (p < 0.001) in comparison with the control group after 24 h. However, coincubation with compounds 3-4 suppressed the secretion of IL-6 and TNF-α in a dose-dependent manner, suggesting that compounds 3-4 are inhibitor of the initial phase of the inflammatory cascade initiated by LPS stimulation. As shown in Table 2, compounds 3-4 can inhibit the expression of those two proinflammatory mediator secretions (TNF-α and IL-6) with IC 50 values from 35.6 to 45.8 μm, while compounds 1-2 were inactive. In light of the structures and the anti-inflammatory activity, the existence of unsaturated double bond of Δ 6(7) may favor this inhibitory effect.

CONCLUSIONS
The systematic investigation of the seeds of S. alopecuroides led to the isolation of four uncommon bis-amide matrine-type alkaloids. Their special chemical structure can provide a new perspective for developing novel modificatory strategies based on A or B ring. The biological assay revealed two new compounds displayed obvious anti-inflammatory activity. This study not only enriched the structural diversity of matrine-type alkaloids, but also provided an attractive molecular candidate for pharmaceutical chemists.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

AUTHOR CONTRIBUTIONS
DL performed the extract, isolation, and structural elucidation of all chemical structures, and wrote the preliminary manuscript. ZT and WY contributed to the pharmacological assay. CF, NC, and ZW assisted in acquiring the experimental data. YZ, GW, and YL supervised and guided all experiments, rewrote and reviewed the manuscript. All authors have read and approved the published version of manuscript. 45.8 ± 1.9 >50 ± 0.6 Dex b 8.8 ± 1.3 7.2 ± 0.5 a Results were expressed as means ± SD (n 3). b Dex (dexamethasone) was select as positive control.