Bousmekines A-E, New Alkaloids from Two Bousigonia Species: B.angustifolia and B. mekongensis

Abstract Four new monoterpenoid indole alkaloids, bousmekines A-D (1–4), and one new pyranopyridine alkaloid, bousmekine E (5), were isolated from the twigs and leaves of Bousigonia angustifolia and Bousigonia mekongensis. Their structures including absolute configurations were elucidated by a combination of MS, NMR, ECD calculation, and single-crystal X-ray diffraction analysis. Compound 2 was an eburnea-type MIAs characterized by a rare chlorine atom while 5 possessed a novel pyranopyridine moiety. Their cytotoxicities against several human cancer cell lines were evaluated and compound 1 exhibited significant cytotoxicity with IC50 values of 0.8–7.4 μM. Graphic Abstract Electronic supplementary material The online version of this article (doi:10.1007/s13659-020-00278-6) contains supplementary material, which is available to authorized users.


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of them are distributed in southwestern China [7]. Previous chemical studies on this genus have resulted in the identification of more than 100 alkaloids, primarily consisted of aspidosperma, eburnea, and aspidosperma-eburnea type MIAs [8][9][10][11][12]. In order to further investigation on novel and bioactive MIAs and proffer new vision into the constitutions of the two Bousigonia species, their alkaloidal extracts were investigated and four new MIAs (1)(2)(3)(4) and one new pyranopyridine alkaloid (5) was isolated. This paper herein describes the isolation, structural elucidation and the cytotoxicities of the isolates (Fig. 1). Detailed analysis of its NMR data (Tables 1 and 2) indicated that compound 1 had a high similarity with that of 3α-acetonyltabersonine [13], except for the presence of an additional methoxy (δ H 3.81; δ C 55.5) and a nonprotonated quaternary carbon (δ C 160.0). The key HMBC correlations of OMe (δ H 3.81), d,dd,J = 8.4,2.4 Hz) to C-11 (δ C 160.0) indicated that the methoxy group was located at C-11 (Fig. 2), and thus established the planar structure of 1 as 11-methoxy derivative of 3α-acetonyltabersonine. 2D NMR spectra (HSQC, HMBC, and 1 H-1 H COSY) confirmed the other parts of 1 were the same as that of 3α-acetonyltabersonine (Fig. 3). The ROESY correlation of H-3/H-21, and of H-21/H-19a indicated that these protons were co-facial and arbitrarily assigned as α-oriented. Therefore, the relative configurations of 1 was assigned as (3S*, 7R*, 20R*, 21S*)-1. The ECD calculation results for (3S, 7R, 20R, 21S)-1 matched well with its experimental ECD spectrum finally established the absolute configuration of 1 (Fig. 4).

Structure Elucidation of the Compounds
Bousmekine B (2) 13 C and DEPT spectra suggested that 2 possessed 19 carbons including one methyl, five methylenes, eight methines, and five nonprotonated carbons. The NMR data demonstrated that 2 was an eburneantype alkaloid similar as meloyunine [14], with exception of an additional methine (δ H 3.92; δ C 54.9). The abundance ration of 329.1414/331.1388 (3:1) in HRESIMS spectrum indicated the presence of a chlorine atom in 2 (Figure S16, Supporting Information). HMBC correlation of H-14 (δ H 3.92) to C-20 (δ C 44.3) established that the chlorine atom was located at C-14. Finally, the planar structure and relative configuration of 2 was established by 2D NMR spectra (Fig. 2), and its absolute configuration was determined by X-ray single crystal diffraction analysis with Flack parameter = 0.104 (12) (Fig. 5).
Bousmekine D (4) had the same molecular formula with 3, as established by HRESIMS analysis at m/z 313.1908 [M + H] + (calcd 313.1911). Compared its NMR data with 3 indicated that both compounds had the same skeleton. The coupling constant of H-16 (br d, J = 4.8 Hz) demonstrated that H-16 took β orientation and thus compound 4 was assigned as the C-16 epimer of 3. Further 2D NMR spectra  .1019), corresponding to five degrees of unsaturation. The 13 C and DEPT spectra showed that 5 comprises 10 carbons, including two methyls, one methylene, five methines and two nonprotonated carbons.
The ROESY correlation of H-4 and H-6 indicated that both protons took the same orientation and were arbitrarily designated as α-orientation, thus its relative configuration was determined as (4S*,6S*)-5 (Fig. 3). To allocate the absolute configuration of 5, the optical rotation (OR) value of (4S,6S)-5 was calculated by using density functional theory (DFT) method. The calculated [α] value for (4S,6S)-5 was -67, which is close to its experimental value (-66), and thus the absolute configuration of 5 was assigned as (4S,6S).

Plant Material
The twigs and leaves of B. angustifolia and B. mekongensis were collected in Xishuangbanna, Yunnan Province, People's Republic of China, in July 2018. The samples were identified by Mr. Yu Chen, Kunming Botanical Garden. A specimen (no. ZY20180623 and no. ZY20180624) was deposited at State Key Laboratory of Phytochemistry and Plant Resource in West China, Kunming Institute of Botany, Chinese Academy of Science (CAS).

Extraction and Isolation
The dried twigs and leaves of B. mekongensis (30.5 kg) were powdered and extracted three times with methanol. The extract was adjusted to 2-3 with hydrochloric acid (5%) and then extracted three times with petroleum ether. The water fraction was basified to pH 9-10 with sodium hydroxide (10%), then extracted with chloroform to get the crude alkaloids. The crude alkaloids (530 g) were separated on a silica gel column (100-200 mesh), and eluted with a gradient of CHCl 3 -MeOH

Cytotoxicity Assays
Cytotoxicity evaluations were performed according to the previously described protocol [10,17].

Concluding Remarks
In this investigation, four new MIAs (1)(2)(3)(4) and one pyranopyridine alkaloid (5) were isolated from the twigs and leaves of two Bousigonia species: B. angustifolia and B. mekongensis. Among them, compound 2 was an eburnea-type MIAs and featured by a rare chlorine atom at C-14, while 5 was characterized by a novel pyranopyridine moiety. Moreover, compound 1 exhibited significant cytotoxicities against several human cancer cell lines with IC 50 values of 0.8-7.4 μM. The findings enriched the diversity of secondary metabolites of the Bousigonia genus.