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 (Li et al., 2020), 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 10-oxysophoridine that possessed the bis-amide bond at C-2 or C-10 (Zhang et al., 2016; 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.

Compound 1 was isolated as colorless block crystals from CH₃OH, mp 147–148°C [α]₂₅ ^D +84.0 (c 0.01, CH₃OH). The molecular formula was deduced to be C₁₅H₂₂N₂O₃ on the basis of the HRESIMS protonated molecular ion peak [M + H]^₊ at m/z 278.1630, suggesting six indices of hydrogen deficiency. The UV spectrum showed maximum absorption at 206 nm was typical for non-conjugated amide of matrine-type backbone. The IR spectrum displayed a hydroxy group (3,202 cm⁻¹) and two lactam functionalities (1,627 and 1,597 cm⁻¹). The ¹H NMR spectrum (Table 1) exhibited signals for [δ _H 4.70 dd (J = 13.2, 4.6 Hz), 2.69 m] and [δ _H 4.53 days (J = 13.0 Hz), 2.66 m], ascribed to two sets diagnostic methylene protons adjacent to N-atom of amide bond. The ¹³C NMR spectrum (Table 1) revealed 15 carbon resonances categorized by DEPT experiments as two carbonyls (δ _C 172.4 and 170.3), five methines (δ _C 74.5, 54.6, 54.3, 42.0, and 31.5), and eight methylenes (δ _C 46.7, 32.4, 32.1, 28.4, 27.9, 25.3, 20.0, 18.9). The above spectroscopic data as well as biogenetic considerations indicated that compound 1 was a derivative of matrine (Zhang et al., 2018b).

The planar structure and relative configuration of one was ascertained by comprehensive analysis of 2D NMR experiment (including ¹H–¹H COSY, HSQC, HMBC, and NOESY spectra). The characteristic HMBC correlations from H₂-17 to C-4/C-5/C-6/C-11/C-17, from H-6 to C-2/C-5/C-7/C-10/C-11, from H₂-8 to C-6/C-7/C-9/C-10/C-11, and from H-9 to C-7/C-8/C-10, together with ¹H−¹H COSY correlations of H-6/H-7/H-8/H-9 in B ring (Figure 2), implied one more amide bond occurring at C-10 and a hydroxy group locating on C-9 of matrine-based skeleton. Furthermore, the coupling constant between H-9 and H₂-8 (³ J = 5.6 Hz) suggested H-9 occupied the equatorial position (α) in six-member ring, supported by the absence of H-9/H-11 cross-peak signal in the NOESY spectrum. Finally, the absolute configuration of (5S,6S,7,9,11R)-1 was unequivocally established by a single crystal X-ray diffraction (Figure 3) using Cu Kα radiation [Flack parameter: 0.09 (13)]. Thus, the structure of one was deduced as shown in Figure 1, and named as (+)-10-oxy-9β-hydroxymatrine.

Compound 2 [α]₂₅ ^D −29.2 (c 0.01, CH₃OH), possessed a molecular formula of C₁₅H₂₂N₂O₃ (calcd for C₁₅H₂₃N₂O₃, 279.1703) via the HRESIMS ion peak at m/z 279.1704 [M + H]⁺ and ¹³C NMR data. Its spectroscopic values showed that two is structurally similar to sophoridine (Zhang et al., 2018a), while the obviously differences were that an additional amide carbon (δ _C 170.5) and a more O-bearing methine [δ _C 64.3, δ _H 4.01 dd (J = 10.4, 4.2 Hz)] are observed. The key HMBC cross-peaks of H₂-8 to C-6/C-7/C-9/C-10/C-11 and H-6 to C-2/C-4/C-8/C-10, together with sequential COSY correlations of H-6/H-7/H₂-8/H-9 in B ring (Figure 2) lead to the full structural assignment of 2, as shown in Figure 1. The configuration of OH-9 was deduced as equatorial orientation (α), which was determined by the large coupling constant of H-8α/H-9 (J _H-9/H-8α = 10.4 Hz) and NOE cross-peaks (Figure 4) of H-9 (δ _H 4.01)/H-8a (δ _H 2.13)/H-11 (δ _H 3.67). Consequently, the absolute structure of two was corroborated by comparing the experiment and calculated CD curve (Figure 5). Compound 2 was thereby deduced and named as (−)-10-oxy-9α-hydroxysophridine.

Compound 3, colorless block crystals, showed an [M + H]⁺ ion peak at m/z 277.1541 (calcd for C₁₅H₂₁N₂O₃, 401.1571) in the HRESIMS spectrum, consistent with the molecular formula of C₁₅H₂₀N₂O₃. The ¹H and ¹³C NMR data of 3 (Table 1) were similar to those of 5-hydroxy-6,7-dehydro-matrine (Zhang et al., 2018b). The most notable difference was that the methylene at C-10 was replaced by another lactam group (δ _C 172.8), which was verified by the key ¹H–¹H COSY correlation of H-2/H-3, and HMBC cross-peaks from H₂-3 to C-2/C-4, from H₂-10 to C-2/C-6/C-8, and from H₂-17 to C-4/C-5/C-6/C-11/C-17 (Figure 2). Moreover, the planar structure and absolute configuration of three were determined by X-ray crystallography (Figure 3) with an excellent Flack parameter [−0.01 (6)]. Hence, the complete structure of three was established and named as (−)-2-oxy-5-hydroxy-6,7-dehydromatrine.

Compound 4 [α]₂₅ ^D −12.2 (c 0.01, CH₃OH), 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 ¹H–¹H COSY correlations of H₂-2/H₂-3/H₂-4 and H₂-8/H₂-9, as well as HMBCs from H₂-2 to C-10, and from H₂-8 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 non-toxic 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, co-incubation 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 pro-inflammatory mediator secretions (TNF-α and IL-6) with IC₅₀ 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 Δ⁶⁽⁷⁾ may favor this inhibitory effect.

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.