Three new hasubanan-type alkaloids from the Stephania longa

Abstract Three new hasubanan-type alkaloids, stephalonine Q (1), stephalonine R (2) and stephalonine S (3), together with four known alkaloids, isolonganone (4), eletefine (5), aurantiamide (6), N-cinnamoyltyramine (7), were isolated from the whole plant of Stephania longa. Their structures were identified by NMR, HR-ESI-MS, CD methods and x-ray crystallography, as well as by comparison with the literature data. All isolated compounds were evaluated for their antimicrobial activities against five bacteria in vitro. Compound 5 displayed inhibitory activity against only S. aureus, with an MIC value of 50 μg/mL.


Introduction
Stephania genus (Menispermaceae family) contained almost 60 species, which are widely distributed in tropical and subtropical regions of Asia and Africa (Semwal et al. 2010). Many plants of the Stephania genus are used in the traditional Chinese medicine (TCM) or as folklore herbs (Achike andKwan 2002, Joshi et al. 2008).
Previous phytochemical investigation into the Stephania genus had revealed the existence of protoberberine-type (Tanahashi et al. 2000), aporphine-type (Deng et al. 1993), morphine-type (Nakaoji et al. 1997), hasubanan-type (Wang et al. 1994) and bisbenzylisoquinoline-type alkaloids (Ogino et al. 1988). More importantly, these alkaloids exhibit a variety of biological activities, including sedative (Carroll et al. 2010), antitumour (Nakaoji et al. 1997), anti-inflammatory (Kang et al. 1996) and anti-microbial activities (Nawawi et al. 2001, Semwal andRawat 2009) Stephania longa, a herbaceous liana, widely distributed in the southern regions of China, has long been used in folk medicine to treat fever, inflammation and dysentery ( Zhang et al. 2006). Our previous studies on the title plant had resulted in the discovery of five alkaloids with promising anti-inflammatory activity (Liu et al. 2021). In our continuous effort to search bioactive constituents from S. longa, three new hasubanan-type alkaloids (stephalonine Q-S) has been isolated from S. longa together with four known ones ( Figure 1). Their structures were identified by NMR, HR-ESI-MS, CD methods and X-ray crystallography, as well as by comparison with the literature data. In addition, all isolated compounds were evaluated for their antimicrobial activities in vitro. Compound 5 displayed inhibitory effect on only S. aureus, with an MIC value of 50 lg/mL.

Result and discussion
Compound 1 was obtained as a yellow amorphous powder. The UV spectrum of 1 ( Figure S1) showed the absorption maxima at 204 and 279 nm. The IR spectrum of 1 ( Figure S2) exhibited absorption bands of hydroxyl at 3549 cm À1 , carbonyl at 1743 and 1679 cm À1 and aromatic ring at 1617 and 1495 cm À1 . The molecular formula was determined to be C 20 H 23 NO 7 on the basis of the HR-ESI-MS data at m/z 390.1537 [M þ H] þ (calculated for C 20 H 24 NO 7 þ , 390.1547), suggesting ten indices of hydrogen deficiency ( Figure S3). The 1 H-NMR data ( Figure S4)  The 1 H-1 H COSY spectrum of 1 revealed the existence of two spin-coupling system ( Figure S31). These data suggested that compound 1 was a hasubanan-type alkaloid. Comparison of the NMR data of 1 with those of the known longanone (Lao et al. 1981) revealed they were similar, except the presence of an additional carbonyl at d C 176.3 (C-16) in 1, which was supported by the HMBC correlations ( Figure S31) Figure S33) of 1 showed positive cotton effects at 208-237 nm and a negative cotton effect at 283 nm, which confirmed the absolute configuration to be 6S, 7S, 8 R, 10S, 13S, 14S. (Xiao et al. 2019). Therefore, the structure of compound 1 was determined and named as stephalonine Q.
Compound 2 was isolated as a yellow amorphous powder. The UV spectrum of 2 ( Figure S11) displayed the absorption peaks at 205 and 274 nm. The IR spectrum ( Figure S12) showed the characteristic absorption for hydroxy group at 3442 cm À1 , carbonyl at 1724 and 1685 cm À1 and aromatic ring at 1490 and 1456 cm À1 . Its molecular formula was deduced as C 21 H 25 NO 6 by its HR-ESI-MS data at m/z 388.1754 [M þ H] þ (calculated for C 21 H 26 NO 6 þ , 388.1760), accounting for the presence of ten degrees of unsaturation ( Figure S13). The 13 C and DEPT NMR data (Figures S15 and S16) revealed the existence of two carbonyls, seven quaternary carbons, four methines, four methylenes and four methyls in 2. The NMR data of 2 (Table S1) were shown to be quite similar to those of the known alkaloid, prostephabyssine (Zhang and Yue 2005)  ( Figure S33). Therefore, the structure of compound 2 was identified and named as stephalonine R.
Compound 3 was isolated as a yellow amorphous powder. The UV spectrum of 3 ( Figure S21) showed the absorption maxima at 205, 264 and 317 nm. The IR spectrum of 3 ( Figure S22) showed the characteristic absorption bands of hydroxyl group at 3206 cm À1 , carbonyl at 1681 and 1636 cm À1 and aromatic ring at 1575 and 1489 cm À1 . A molecular formula of C 19 H 19 NO 5 was assigned to 3 on the basis of HR-ESI-MS at m/z 342.1336 [M þ H] þ (calculated for C 19 H 20 NO 5 þ , 342.1341), which indicated the presence of eleven degrees of unsaturation ( Figure S23). The 1 D and 2 D NMR spectrum of compound 3 ( Figures S24-S30) revealed the presence of 19 carbon signals, including two carbonyls, seven quaternary carbons, five methylenes, two methines and three methyls. The NMR data of 3 were closely resembled to those prostephabyssine (Zhang and Yue 2005), the obvious differences were the disappearance of an oxygenated tertiary carbon and the presence of an additional double bond between C-9 (d C 116.7) and C-10 (d C 137.0). This was confirmed by the 1 H-1 H COSY correlations between H-9 (d H 5.55) and H-10 (d H 6.82) ( Figure S31). The experimental CD curve of 3 ( Figure S33) was highly similar with 2, suggested that 3 possessed the same absolute configurations as 2, Therefore, the structure of compound 3 was identified and named as stephalonine S. The known compounds were identified as isolonganone (4) (Zhang and Yue 2005), eletefine (5) (da-Cunha et al. 1998), aurantiamide (6) (Xu et al. 2009) and N-cinnamoyltyramine (7) (Chen et al. 2017) by comparing their NMR and spectroscopic data with those reported values in published literature. Compound 4 was further confirmed by the single crystal X-ray diffraction method for the first time.
All isolated compounds were screened for their antimicrobial activities against five bacteria, Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 53820), Pseudomonas aeruginosa (ATCC 5753-735), Klebsiella pneumonia (ATCC 538148) and Acinetobacter baumannii (ATCC 38717) with the positive control of penicillin G for Gram-positive bacteria and ceftazidime for Gram-negative bacteria (Table S2). However, compound 5 displayed inhibitory activity on only S. aureus, with MIC value of 50 lg/mL.

General experimental procedures
UV spectra were recorded on a Shimadzu UV-2700 UV-VIS spectrophotometer (Shimadzu, Japan). IR data were measured on an IRTracer-100 spectrometer (Shimadzu, Japan). Optical rotations were determined using an AUTOPOL IV automatic polarimeter (Rudolph Research Analytical, USA). ECD spectra were obtained on a Chirascan-plus CD spectrometer (Applied Photophysics Ltd., UK). 1 D and 2 D NMR spectra were collected on a Bruker Av 400 (Bruker, Germany) with TMS as the internal standard. HR-ESI-MS data were acquired on a Thermo Scientific Q Exactive mass spectrometer (Thermo Electron, Bremen, Germany). Shimadzu LC-20AP liquid chromatography system (Shimadzu, Japan) with YMC-Pack ODS-A C18 column (250 Â 10 mm, 5 mm, YMC Co. Ltd., Japan) were used for preparative HPLC separation. Silica gel Qingdao,China), octadecylsilyl silica gel (ODS, 50 mm, YMC, Japan) were used for column chromatography (CC).

Plant material
Stephania longa were bought from Wuzhou, Guangxi, China and were identified by Prof. An-ping Yang (Foshan University). A voucher specimen (FJD-201907) was stored in the Department of Pharmacy, School of Medicine, Foshan University.

Extraction and isolation
The general extraction and partition procedures of the S. longa were described in our previous report (Liu et al. 2021). In this work, the separation of Fr. 2-4 (3.0 g) using a preparative HPLC column (MeOH-H 2 O, 80: 20, v/v) obtained compound 4 (10.2 mg).

Antimicrobial assays
The in vitro antimicrobial assay was performed according to the reference (Qin et al. 2016) with some modification. Five pathogenic bacteria used were: S. aureus, E. coli, P. aeruginosa, K. pneumonia and A. baumannii. The MICs values of the isolated compounds were determined by the broth microdilution method in 96-well plate . The test samples were first of all dissolved in DMSO, and the solutions obtained were then added to Mueller Hinton Broth (MHB). The compounds and the reference drugs were initially prepared in 2% DMSO in broth medium at 400 lg/mL. A volume of 200 lL of each test sample was added into the first wells of the 96-well plate. The first concentration was serially diluted two folds and 100 lL of inoculum was standardized at 1.5 Â 105 CFU/mL for bacteria (The optical density from 0.08 to 0.1 at 625 nm Shimadzu UV-2700 spectrophotometer). This gave final concentration ranges from 200 to 0.39 lg/mL for each sample, and the final concentration of DMSO in each well was less than 1% (preliminary analysis that 1% (v/v) DMSO do not interfere the growth of test organism). The positive control of S. aureus was Penicillin G and Ceftazidime for E. coli, p. aeruginosa, K. pneumonia, A. baumannii and the negative control was 200 lL MHB. The plates were covered with the sterile lid, and then agitated to mix the contents of the wells using a plate shaker and incubated at 37 C for 24 h. All tests were performed in triplicate. The MICs here were defined as the lowest concentration of compound that results in inhibition of visible bacterial growth (no turbidity) compared with the positive control drug.

Conclusion
In this study, three new hasubanan-type alkaloids, stephalonine Q-S (1-3) and four known ones, isolonganone (4), eletefine (5), aurantiamide (6) and N-cinnamoyltyramine (7) were isolated from S. longa and heir structures were identified. Additionally, all isolated compounds were evaluated for their antimicrobial activities in vitro. However, compound 5 displayed inhibitory activity against only S. aureus with an MIC value of 50 lg/mL.