Chemical Constituents with Anti-Proliferative Activity on Pulmonary Arterial Smooth Muscle Cells from the Roots of Anthriscus sylvestris (L.) Hoffm.

A chemical investigation of Anthriscus sylvestris roots led to the isolation and characterization of two new nitrogen-containing phenylpropanoids (1–2) and two new phenol glycosides (8–9), along with fifteen known analogues. Structure elucidation was based on HRESIMS, 1D and 2D NMR spectroscopy, and electronic circular dichroism (ECD). In addition, compounds 3, 6, 9–10, 12, and 17 exhibited inhibitory effects against the abnormal proliferation of pulmonary arterial smooth muscle cells with IC50 values ranging from 10.7 ± 0.6 to 57.1 ± 1.1 μM.

Pulmonary arterial hypertension (PAH) is a chronic, progressive disease of the pulmonary circulation characterized by vascular remodeling [9].Pulmonary vascular remodeling involves a variety of cells, including vascular endothelial cells, pulmonary artery smooth muscle cells (PASMCs), and multiple inflammatory cells [10,11].The previous study demonstrated that diarylpentanoids and phenylpropanoids isolated from the roots of A. sylvestris could inhibit the abnormal proliferation of PASMCs induced by hypoxia [12], which attracted our interest to search for more natural products with anti-proliferative effects from this plant.In this study, two new nitrogen-containing phenylpropanoids (1-2), and two new phenol glycosides (8)(9), along with fifteen known analogues, were isolated from the roots of A. sylvestris, and their anti-proliferative effects against the abnormal proliferation of pulmonary arterial smooth muscle cells were evaluated.

Structure Characterization
The chemical investigations on the extract of the roots of A. sylvestris resulted in the characterization of compounds (1-19) (Figure 1).

Structure Characterization
The chemical investigations on the extract of the roots of A. sylvestris resulted in the characterization of compounds (1-19) (Figure 1).Major discrepancies were concentrated on the absence of the methoxy group at C-16 and the presence of the methoxy group at C-5, and the ∆ 8 (9) double bond linked with a methylene carbon, which was confirmed by the 1 H-1 H COSY correlations of H-9 with H-8 and H-10 and the HMBC crosspeaks from H-8 to C-2 and C-6, from H-10 to C-11, and from the hydrogens of the methoxy group (δ H 3.82) to C-5 (Figure 2).Based on these data, the structure of compound 1 was identified and named as anthriscusin O.  [13].Major discrepancies were concentrated on the absence of the methoxy group at C-16 and the presence of the methoxy group at C-5, and the Δ 8 (9) double bond linked with a methylene carbon, which was confirmed by the 1 H-1 H COSY correlations of H-9 with H-8 and H-10 and the HMBC crosspeaks from H-8 to C-2 and C-6, from H-10 to C-11, and from the hydrogens of the methoxy group (δH 3.82) to C-5 (Figure 2).Based on these data, the structure of compound 1 was identified and named as anthriscusin O. Compound 2 was obtained as a colorless solid.Its molecular formula, C21H19NO4, was established by the HRESIMS ion at m/z 372.1205 [M + Na] + (calcd.for 372.1206).The 1 H and 13 C NMR data (Table 1) of 2 revealed the presence of a tetrasubstituted aromatic ring [δH 6.52 (1H, s, H-2), 6.47 (1H, s, H-6); δC 150.6 (C-3), 144.9 (C-5), 135.9 (C-4), 133.9 (C-1), 108.0 (C-6), 100.Compound 2 was obtained as a colorless solid.Its molecular formula, C 21 H 19 NO 4 , was established by the HRESIMS ion at m/z 372.1205 [M + Na] + (calcd.for 372.1206).The 1 H and 13 C NMR data (Table 1) of 2 revealed the presence of a tetrasubstituted aromatic ring [δ H 6.52 (1H, s, H-2), 6.47 (1H, s, H-6); δ C 150.6 (C-3), 144.9 (C-5), 135.9 (C-4), 133.9 were similar to those of (2E)-3-(1,3-benzodioxol-5-yl)-2-propen-1-yl]benzenemethanol [14], except for the absence of the aromatic group and the presence of a quinoline unit at C-11 and an additional methoxy group at C-5, which was deduced by the HMBC correlations from H-11 to C-13 and C-15, and from the hydrogens of the methoxy group (δ H 3.82) to C-5 (Figure 2).To define the absolute configuration, the ECD calculation was performed at the B3LYP/6-311G(d) level using the TDDFT method.Furthermore, the agreement of the Cotton effects of the calculated ECD spectrum of (R)-2 with the experimental ECD spectrum of 2 allowed the absolute configuration of 2 to be assigned as (R) (Figure 3).Thus, the structure of 2 was elucidated as shown and named as anthriscusin P. and an additional methoxy group at C-5, which was deduced by the HMBC correlations from H-11 to C-13 and C-15, and from the hydrogens of the methoxy group (δH 3.82) to C-5 (Figure 2).To define the absolute configuration, the ECD calculation was performed at the B3LYP/6-311G(d) level using the TDDFT method.Furthermore, the agreement of the Cotton effects of the calculated ECD spectrum of (R)-2 with the experimental ECD spectrum of 2 allowed the absolute configuration of 2 to be assigned as (R) (Figure 3).Thus, the structure of 2 was elucidated as shown and named as anthriscusin P.  Compound 8 was acquired as a white amorphous powder with a molecular formula of C 17 H 24 O 9 , as determined by its HRESIMS and 13 C NMR data.The 1 H and 13 C NMR data (Table 2) of 8 were similar to those of 12 [15], except for the replacement of the methoxy group at C-8 by the ethyl group in 8, which was corroborated by the HMBC correlations (Figure S31, Supplementary Materials) of H-8 with δ C 201.9 (C-7) and 8.7 (C-9).Additionally, the hexose moiety was determined as D-glucose through the chiral-phase HPLC analysis of a monosaccharide produced by the hydrolysis of compound 8 (Figure S63, Supplementary Materials).The anomeric proton (δ H 4.95) of hexose moiety had a large coupling constant (J = 7.4 Hz), indicating a β-configuration.Thus, the structure of 8 was defined as shown and named as 1-(3-β-D-glucopyranosyloxy)-4,5-dimethoxyphenyl)propan-1-one.S64, Supplementary Materials).Moreover, the anomeric proton (δ H 4.25) of D-glucose had a large coupling constant (J = 7.8 Hz), and the anomeric proton (δ H 4.79) of L-rhamnose had a small coupling constant (J = 1.2 Hz), which suggested the configurations of the anomeric carbons of D-glucose and L-rhamnose were β and α, respectively.In the HMBC spectrum, the anomeric proton of the D-glucose unit showed a correlation with C-7, and the anomeric proton of the L-rhamnose unit was correlated with the C-6 ′ of D-glucose unit.These NMR data mentioned above were similar to those of compound 14 [16], except for the presence of three methyl groups.The three methyl groups were respectively located at C-2, C-3, and C-4, which was determined by the key HMBC correlations from the methyl protons at δ H 2.28 to C-1, C2, and C-3, from the methyl protons at δ H 2.18 to C-3 and C-4, and from the methyl protons at δ H 2.25 to C-4 and C-5 (Figure 2).Based on these data, the structure of compound 9 was identified and named as 2,3,4-trimethylbenzylalcohol-α-L-rhamnopyranosyl-(6→1)-β-D-glucopyranoside.

Biological Activity
The isolates 1-19 were screened for their inhibitory effects on PASMCs' abnormal proliferation induced by hypoxia in vitro.As shown in Figure 4 trum, the anomeric proton of the D-glucose unit showed a correlation with C-7, and the anomeric proton of the L-rhamnose unit was correlated with the C-6′ of D-glucose unit.These NMR data mentioned above were similar to those of compound 14 [16], except for the presence of three methyl groups.The three methyl groups were respectively located at C-2, C-3, and C-4, which was determined by the key HMBC correlations from the methyl protons at δH 2.28 to C-1, C2, and C-3, from the methyl protons at δH 2.18 to C-3 and C-4, and from the methyl protons at δH 2.25 to C-4 and C-5 (Figure 2).Based on these data, the structure of compound 9 was identified and named as 2,3,4-trimethylbenzylalcoholα-L-rhamnopyranosyl-(6→1)-β-D-glucopyranoside.

Biological Activity
The isolates 1-19 were screened for their inhibitory effects on PASMCs' abnormal proliferation induced by hypoxia in vitro.As shown in Figure 4, compared with the normal (NC) group, the proliferation rate of PASMCs in the model (M) group was significantly increased (p < 0.01).Compared with the M group, the proliferation rates of the compounds 3, 6, 9-10, 12, and 17 groups were significantly decreased (p < 0.01), which indicated that compounds 3, 6, 9-10, 12, and 17 can significantly inhibit the abnormal proliferation of PASMCs at 5 µM.Then, these cells were treated with the compounds (3, 6, 9-10, 12, and 17) with different concentrations (1, 2.5, 5, 10, 20, 50, and 100 µM).Compounds 3, 6, 9-10, 12, and 17 suppressed the abnormal proliferation of PASMCs, with IC50 values of 56.3 ± 0.5, 10.7 ± 0.65, 57.1 ± 1.1, 44.0 ± 0.75, 41.5 ± 0.87, and 35.3 ± 0.42 µM, respectively.Notably, the comparison of compounds 8 and 12 showed that the methoxy fragment connected to C-8 may be responsible for the inhibition.Additionally, compounds 9 and 10 exhibited an inhibitory effect in contrast to compounds 14 and 15, which might be attributed to the presence of a 2,3,4-trimethylphenyl group.Pharmacological studies have shown that deoxypodophyllotoxin isolated from A. sylvestris possesses antitumor, antibacterial, and antiviral activities [7,8,27,28], which has led to the pharmacological effects of A. sylvestris being mainly focused on antitumor effects, with less research and attention paid to other pharmacological effects.In this study, the inhibitory effects of compounds isolated from A. sylvestris on the hypoxia-induced cell proliferation of PASMCs were investigated for the first time.The preliminary results of this experiment have an important potential value for future development and research on A. sylvestris.

Plant Material
The dried roots of A. sylvestris were collected in November 2021 from Leshan city, Sichuan province, China, and identified by Professor Chengming Dong of Henan University of Chinese Medicine.A voucher specimen (No. 20211116A) was deposited at the Department of Natural Medicinal Chemistry, Henan University of Chinese Medicine, Zhengzhou, China.

Extraction and Isolation
The chopped dried roots (48.0 kg) were extracted with 70% aqueous acetone (smashed tissue extraction).The extract (15 kg) was suspended in water and sequentially partitioned with petroleum ether, EtOAc, and n-BuOH for fifteen times, respectively.

Computational Analysis
The conformations of 2 were analyzed by GMMX software (6.0) using the MMFF94 force field.The conformers were optimized with density functional theory (DFT) at the B3LYP/6-31G using the Gaussian 2016 package.The ECD calculations of conformers with Boltzmann distributions over 1% were further calculated by the TDDFT method at the B3LYP/6-311G (d) level in CH 3 OH.The ECD spectra were simulated by the SpecDis 1.71 software [29].

MTT Assay
Briefly, the PASMCs were seeded in 96-well plates at 2 × 10 4 cells/well at 37 • C in an atmosphere of 5% CO 2 .The cells were divided into the normal group (NC), model group (M), and treatment groups (isolated compounds).Then, the MTT assay was performed as previously described [12].All data were analyzed by SPSS software version 26.0 (IBM, New York, NY, USA) and presented as the mean ± standard deviation.Concentration-response analysis was performed to determine the compound concentrations required to inhibit the growth of cells by 50% (IC 50 ) using GraphPad Prism 8.02 software.

Conclusions
Four new compounds (1-2, 8, and 9), together with fifteen known analogs were isolated from the roots of A. sylvestris.All compounds were isolated from the plant for the first time, which greatly enriches the chemical content of this plant.In preliminary in vitro bioassays, the results showed that 3, 6, 9-10, 12, and 17 exhibited anti-proliferation effects on hypoxia-induced PASMCs' cell proliferation, suggesting that they may further act as potential lead molecules for the development of therapeutic agents for PAH.Then, we will discover more bioactive compounds from A. sylvestris and carry out further research on the mechanism with potential compounds for the treatment of PAH.

Figure 3 .
Figure 3. Experimental and calculated ECD spectra of compound 2.