Structurally diverse triterpenoid alkaloids from Buxus rugulosa

Abstract Four new nortriterpenoid alkaloids, namely buxrugulines E–H (1–4), along with five known ones (5–9), were isolated from the twigs and leaves of Buxus rugulosa. Their structures were identified based on extensive NMR data and MS spectroscopic analyses. Our bioassays revealed that compounds 5, 6 and 8 exhibited potent cytotoxicity in vitro against MCF-7 cell lines, with IC50 values ranging from 6.70 to 11.00 μM, respectively.


Introduction
The genus Buxus, as the largest genus of the Buxaceae family, are consisted of about 70 species, mainly distributing in the Middle East, Tropical and South Africa, East Asia, and North and Central America [1-3].Besides being widely used for landscaping purposes, plants of the genus Buxus have also been employed in traditional medicine for the treatment of malaria, skin infections, rheumatism, and cardio dysfunction [4].The nortriterpenoid alkaloids are the dominant constituents in this genus containing a unique 5a-pregnane-type steroidal alkaloid structure with C-4 methyls, 9b, 10b-cycloartenol system and a degraded C-20 side chain [4,5].Phytochemical studies on Buxus plants have resulted in the identification of over 200 alkaloids, which have shown widely biological activities, including cardio-protective, anticholinesterase, antitumor, activitiesantimalaria, antituberculosis, anti-HIV, antibacterial [1-6].Especially for its cardioprotective effect, one Buxus alkaloid, named cyclovirobuxinum D (CVBD) has been used in China to treat myocardial ischemia, myocardial infarction, arrhythmias and coronary heart disease [6,7].
Buxus rugulosa, a dwarf evergreen shrub, grow widely in the rocky areas of Sichuan and Yunnan Province [4].Previous phytochemical studies on the title plant have resulted the isolation of eight new nortriterpenoidal alkaloids with cytotoxic activities [4,8].In our continuing efforts to discover structurally diverse and biologically significant nortriterpenoid alkaloids from plants [1], four new triterpenoidal alkaloids, named buxrugulines E-H (1-4, Figure 1), together with five known triterpenoidal bases (5-9), were obtained from B. rugulosa.Moreover, all compounds were screened for cytotoxic activities against MCF-7 and MDA-MB-231 tumor cell lines.Herein, we report the isolation, structural elucidation and cytotoxic activities of the compounds.

Results and discussion
The ethanol extracts of the twigs and leaves of B. rugulosa were partitioned into ethyl acetate and dichlormethane fractions.The dichlormethane fraction was repeatedly subjected to silica gel, MCI, RP-18 column, preparative HPLC to obtain four new triterpenoid alkaloids (1-4), and five known analogues (5-9).
Compound 1 was isolated as a white amorphous powder.The HRESIMS exhibited a quasi-molecular ion peak at m/z 579.3796 2), respectively.A one-proton signal at d H 6.31 (d, J ¼ 9.0 Hz) owing to an exchangeable amidic NH showed the 1 H-1 H COSY correlation (Figure 2) with H-3 (d H 3.97), and the latter showed the HMBC cross-peak with the carbonyl carbon (d C 169.4) of the benzamide group, indicating that the benzamide group was located C-3.The HMBC correlations of H-6 (d H 4.92) with the carbonyl carbon (d C 170.3) of the acetoxy group demonstrated that the acetoxy group was assigned to C-6 (d C 77.0).Combining the molecular mass and the above NMR data analysis, we could infer that there was an additional N, N-dimethyl amino group   Compound 2 was also obtained as a white amorphous powder.The HRESIMS exhibited a quasi-molecular ion peak at m/z 521.3740 ([M þ H] þ ), indicating the molecular formula C 33 H 48 N 2 O 3 .The NMR data (Table 1) of 2 were similar to those of 1, except that a hydrogen at C-7 was replaced by a hydroxyl group in 2, accompanied with the absence of a hydroxyl group at C-2 and of an acetoxyl group at C-6.These differences could be confirmed by the HMBC correlations (Figure 3   Compound 4 was obtained as a white amorphous powder.The HRESIMS exhibited a quasi-molecular ion peak at m/z 397.3580 ([M þ H] þ ), indicating the molecular formula C 27 H 44 N 2 .Its NMR data (Table 1) exhibited four methyl singlets at d H 0.78, 0.69, 1.03 and 0.72 corresponding to C-18, C-30, C-31 and C-32, respectively, and a doublet methyl at d H 1.14 (d, J ¼ 6.1 Hz) due to C-21.The N(CH 3 ) 2 protons appeared as a 6H singlet at d H 2.35, while a 3H singlet at d H 2.45 was assigned to the NHCH 3 protons.Four olefinic signals at d H 6.11 (dd, J ¼ 10.1, 2.2 Hz), 5.65, 5.67 and 6.01 were ascribed to C-1, C-2, C-11 and C-19, respectively.This deduction could be confirmed by the HMBC correlations (Figure 5)   In addition, five known compounds were identified to be (-)-Buxanoldine (5) [11], N-Benzoylbuxidienine-F (6) [12], (-)-31-Acetoxyl-N a -benzoylbuxidienine (7) [13], Buxidin(8) [9], and Buxaustroine B (9) [6] by comparing their spectroscopic data with those in the literature.
In summary, four new triterpenoid alkaloids, buxrugulines E-H, together with five known triterpenoidal basesanglues (5-9), were isolated from the twigs and leaves of B. rugulosa.Compounds 5, 6 and 8 exhibited strong cytotoxic activities against MCF-7 cells and moderate MDA reversal activities against MDA-MB-231 cells.Our present research fully reflected the structural diversity of triterpenoid alkaloids and provided new chemotypes for the development of anti-tumor lead compounds.

General experimental procedures
Optical rotations were recorded on a JASCO P-1020 polarimeter (Jasco, Tokyo, Japan).UV spectra were acquired using Shimadzu UV-2450 spectrophotometer (Shimadzu, Tokyo, Japan).Infrared spectra were performed on a Bruker Tensor-27 instrument using KBr pellets (Bruker, Karlsruhe, Germany).The CD spectra in MeOH were recorded on a JASCO J-810 spectrometer (Jasco, Tokyo, Japan).1D and 2D NMR spectra were measured on a Bruker AVANCE III 500 MHz or Bruker AVIIIHD 600 MHz spectrometer with tetramethylsilane (TMS) as internal standard for chemical shifts.HR-ESI-MS data were obtained on an Agilent 6520B Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA).Silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), RP-C 18 (40-63 mm, FuJi Silysia Chemical Ltd., Aichi, Japan) were used for column chromatography.An Agilent 1200 Series instrument with a DAD detector using a shim-pack VP-ODS column (2504.6 mm, i.d.) was used for HPLC analysis.Preparative HPLC was conducted on a Shimadzu LC-6A instrument with an SPD-20A detector using a shim-packRP-C 18 column (20 � 200 mm, i.d. 10 lm, Shimadzu, Tokyo, Japan).TLC was performed on precoated TLC plates (F 254 Si gel 60, Qingdao Marine Chemical, Inc.) with compounds visualized by spraying modified bismuth potassium iodide solution.

Plant material
The twigs and leaves of Buxus rugulosa were collected from Kunming, Yunnan province of China in March 2019, and identified by Prof. Mian Zhang of the Research Department of Pharmacognosy, China Pharmaceutical University.A voucher specimen (No.BR201903) was stored at the Department of Natural Medicinal Chemistry, China Pharmaceutical University (Nanjing, China).

Cytotoxic activity assay
The cytotoxicity of all isolates against two human cancer cell lines MCF-7 and MDA-MB-231 was tested by the MTT method as reported previously [14].Doxorubicin was used as the positive control.The experiments were performed in triplicate.
) from H-7 (d H 4.22) to C-9 (d C 137.0) and from the proton [d H 6.18 (d, J ¼ 9.6 Hz)] of the benzamide NH to C-2 (d C 30.3).Similar to 1, the N, N-dimethyl amino signals were also missing.The a-orientation of H-7 was assigned by the ROESY correlations (Figure3) of H-7 with H-5 and H 3 -32.Furthermore, The ROESY correlations displayed that the rest of chiral centers (C-3, C-5, C-8, C-13, C-14, C-16, C-17, C-20) had the same stereochemistry as those of 1. Thereby, the structure of 2 was elucidated and named buxruguline F.Compound 3 was isolated as a white amorphous powder.The HRESIMS exhibited a quasi-molecular ion peak at m/z 429.3475([M þ H] þ ),indicating the molecular formula C 27 H 44 N 2 O 2 .The NMR data (Table 1) of 3 showed the presence of two methyl singlets at d H 1.27 and 0.94 for the C-18 and C-32, a doublet methyl at d H 1.21 (d, J ¼ 6.8 Hz) for C-21, two N-methyl amino singlets at d H 2.88 and 2.55, together with an alcyclopropyl methylene group [d H 0.09 (d, J ¼ 4.4 Hz) and 0.34 (d, J ¼ 4.2 Hz)] and an acetylamide group (d H 2.11; d C 22.7 and 172.2).The above data of 3 implied a 9b, 10b cycloartane-type nortriterpenoid skeleton of typical Buxus alkaloids [3].A terminal
from H-19 to C-11 (d C 132.0), C-1 (d C 136.4), C-5 (d C 51.1) and C-8 (d C 49.0) and by the 1 H-1 H COSY correlations (Figure 5) of H-2 with H-1 and H-3 (d H 2.92).Above data confirmed the presence of the D 1,2 9(10!19) abeo triene system in 4 [5].The HMBC correlations of N(CH 3 ) 2 protons with C-3 (d C 70.7) and of NHCH 3 protons with C-20 (d C 58.7) indicated that the N(CH 3 ) 2 group was located at C-3 and that the NHCH 3 group was bound to C-20.The ROESY correlations (Figure5) of 4 suggested that the chiral centers had the same relative configurations as those of previously discussed compound 1.Thereby, the structure of 4 was elucidated and named buxruguline H.
indicating the molecular formula C 35 H 50 N 2 O 5 .The NMR data of 1 (Table 1) indicated the presence of four methyl singlets at d H 0.71, 0.98, 1.16 and 0.93 for C-18, C-30, C-31, and C-32, a doublet methyl at d H 0.94 (d, J ¼ 6.2 Hz) for C-21, an acetoxy group (d H 2.08; d