A new piperidine alkaloid from the leaves of Microcos paniculata L.

Abstract A new piperidine alkaloid, microcosamine C (1), and one known compound, microcosamine A (2) were isolated from the leaves of Microcos paniculata. Structure elucidation was carried out using HR-ESI-MS, 1D and 2D NMR spectroscopic methods and by comparison with data reported in the literature. The absolute configuration at the C-3 hydroxy group of 1 was established by a Mosher esterification procedure. Both the isolates (1–2) were evaluated for cytotoxicity against four selected tumour cell lines and showed only weak activity against RAW 264.7 cell line.


Introduction
Microcos paniculata L. belongs to the family Malvaceae and is distributed in tropical and subtropical areas of South and South-East Asia (Wu et al. 2007). In China, the leaves are used as a folk medicine and as an important herbal tea for the treatment of fever, heatstroke, indigestion and diarrhoea (Luo et al. 2009). Previous phytochemical studies on M. paniculata L. led to the isolation of triterpenoids and flavonoids (Feng et al. 2008). Several studies reported that this species contains 2, 3, 6-trisubstituted piperidine alkaloids as minor components with insecticidal (Bandara et al. 2000), larvicidal (Feng et al. 2008), cytotoxic and nicotinic acetylcholine receptor antagonistic (Still et al. 2013) activities. In this study, one new piperidine alkaloid, microcosamine C (1) (Figure 1), together with one known compound, microcosamine A (2) were isolated. Herein, we report the isolation and structural elucidation of the new compound, as well as in vitro cytotoxicity data against selected tumour cell lines.
The geometry at Δ 1′, 2′ and Δ 7′, 8′ double bonds was assigned as E based on the large coupling constants (J = 15.1 Hz). For the Δ 3′, 4′ and Δ 5′, 6′ double bonds, the configuration could not be determined by coupling constants due to signal overlap. However, the 1 H NMR spectrum of MTPA ester derivatives of 1, taken in pyridine-d 5 solution, clearly showed that each of the eight olefinic proton signals possessed large coupling constants (from 14.5 to 15.0 Hz, Supplementary material Figure S12), supporting a trans configuration for the Δ 3′, 4′ and Δ 5′, 6′ double-bonds.
The relative stereochemistry of the piperidinol ring was determined by analysis of coupling constants and by NOESY experiments. The large proton coupling constant (J = 11.0 Hz) between H-2 and H-3 in the 1 H NMR spectrum (CDCl 3 as solution, Supplementary material Figure S10) indicated that H-2 and H-3 are in axial configuration. NOE correlations (Supplementary material Figure S1) between H-6 and H-2 (δ H 1.99, 1H, m) observed in the NOESY spectrum, indicated that H-2 and H-6 were on the same face of the piperidinol ring. The absolute configuration at the C-3 hydroxyl group position was accomplished using a modified Mosher's ester procedure as described by Still and coworkers. (Still et al. 2013). The final assignment was based on proton resonances of the S-MTPA and R-MTPA ester derivatives. A consistent distribution of positive and negative Δδ H values (Δδ H = δ S -δ R ) around the C-3 esterification site (Supplementary material Figure S14) allowed the assignment of a S-configuration for the C-3 hydroxy position (Freire et al. 2005). Accordingly, 1 was assigned structurally as (2R, 3S, 6R)-6-(deca-1E, 3E, 5E, 7E-tetraenyl)-3-hydroxy-1, 2-dimethylpiperidine and named microcosamine C.
The known compound 2 was identified as microcosamine A by comparison of the spectral data with reported values (Feng et al. 2008).
Both isolates (1-2) were tested for cytotoxic activities against four tumour (HeLa, HepG2, A549 and RAW 264.7) cell lines using the MTT method. Both 1 and 2 were found to be inactive (<50% inhibition of cell proliferation at the dose of 100 μM) against HeLa, HepG2 and A549 tumour cells. However, they showed weak cytotoxicity against RAW 264.7 cell line with IC 50 values of 31.5 and 39.8 μM, respectively.

General
Optical rotations were measured on an Anton Paar MCP-500 polarimeter. UV spectra were determined in methanol on a Shimadzu UV-1800 spectrophotometer. IR spectra were recorded on a Bruker Tensor 27 ATR instrument. NMR spectroscopic data were obtained on a Bruker Avance-600 MHz spectrometer with TMS as the internal standard. HR-ESI-MS data were recorded by a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer. Column chromatography was performed using silica gel (200-300 mesh, Qingdao Marine Chemical Inc China), and Diaion HP-20 resin (Mitsubishi Kasei Kogyo Co Ltd). HPLC isolation was achieved on an Agilent 1260 instrument using a semi-preparative HPLC column (Reprosil-Gold 120 C 18 , 5 μM, 250 × 10 mm). Spots were visualised by spraying with Dragendorff's reagent.

Plant Material
The leaves of M. paniculata L. were purchased from Guangzhou Qingping Professional Market for Traditional Chinese Medicine, Guangdong, PR China, in August 2014, and taxonomically identified by Prof. Yaying Wang, Ximen Medical College, China. An authenticated voucher specimen (No. 20140901) has been deposited at Central Laboratory, Xiamen Medical College, PR China.

Preparation of MTPA esters of microcosamine C (1)
The preparation of MTPA Esters of microcosamine C (1) was carried out directly in NMR tubes according to a previously reported method (Still et al. 2013). After drying compound 1 (each 0.8 mg, 0.0031 mmol) in two NMR tubes overnight in a desiccator, approximately 450 μL of pyridine-d 5 was added under argon, respectively. Then two tubes were treated with 20 μL (S)-MTPA-Cl and (R)-MTPA-Cl, respectively. After 12 h, the reaction was completed and the 1 H NMR (600 MHz, Pyridine-d 5 ) and 1 H− 1 H COSY spectra were recorded. Analysis of the Δδ H (δ S -δ R ) chemical shift values was obtained by 1 H NMR for each of the MTPA diastereomers and allowed the assignment of a S-configuration for the C-3 hydroxy position of 1 (Freire et al. 2005).

Conclusions
A new piperidine alkaloid together with one known compound was obtained from the leaves of M. paniculata. The cytotoxic activity of the isolates against four cancer cell lines was evaluated in vitro. The result showed that both 1 and 2 showed weak cytotoxicities against RAW 264.7 cell line with IC 50 values of 31.5 and 39.8 μM, respectively. The new findings enriched the 2, 3, 6-trisubstituted piperidine alkaloid structural diversity.