Isolation of a new lycodine alkaloid from Lycopodium japonicum

A new lycodine alkaloid, N-methylhydroxypropyllycodine (1), was isolated from the club moss Lycopodium japonicum Thunb, together with five known compounds, N-methyllycodine (2), huperzinine (3), β-obscurine (4), α-obscurine (5) and des-N-methyl-α-obscurine (6). Their structures were elucidated by spectroscopic analyses, including 2D NMR techniques.


Introduction
Lycopodium japonicum Thunb (Lycopodiaceae) is a traditional Chinese medicinal plant for the treatment of sprains, strains and myasthenia (Zhang & Zhang 2004;Chinese Pharmacopoeia Commission 2010). Previous investigations have shown that this plant is a rich source of lycodine alkaloids possessing diverse structures of unprecedented skeletons (Sun et al. 2008;. As a part of our research of structurally unique and biologically active compounds from medicinal plants of Yunnan, China, we have isolated and identified a new lycodine alkaloid, N-methylhydroxypropyllycodine (1), as well as five known compounds, N-methyllycodine (2), huperzinine (3), b-obscurine (4), a-obscurine (5) and des-N-methyl-a-obscurine (6) from L. japonicum. Our procedures and findings are reported in this article ( Figure 1).

Results and discussion
Compound 1 was obtained as a colourless gum. The molecular formula of 1 was determined to be C 20 H 30 N 2 O by HR-EI-MS ([M] þ 314.2364), implying seven degrees of unsaturation. The IR spectrum showed the absorption of hydroxyl group at 3441 cm 21 . The 1 H and 13 C NMR and DEPT spectra of 1 displayed three methyls, seven sp 3 CH 2 , four sp 3 and two sp 2 CH, three sp 2 and one sp 3 quaternary C-atoms. The 13 C NMR spectra [d 157.2 (s), 157.1 (s), 135.7 (s), 135.1 (d), 121.5 (d)] showed that 1 had a trisubstituted pyridine ring. 1 H-1 H COSY and HSQC analyses indicated the existence of four fragments, including an isolated 2-hydroxy-propyl group. Careful inspection in the 1 H and 13 C NMR spectra revealed that 1 was similar to hydroxypropyllycodine (7) previously isolated from Lycopodium obscurum (Ayer & Kasitu 1989). Compound 1 differs from 7 only in the additional N-Me group instead of an NZH bond, which was further confirmed by HMBC correlations from H-17 (d 2.65) to C-9 (d 50.5) and C-13 (d 59.4). Therefore, the structure of 1 was elucidated to be N-methylhydroxypropyllycodine.

Apparatus and reagents
Optical rotations were determined on a Horiba SEAP-300 spectropolarimeter (Horiba International Corporation, Kyoto, Japan). NMR spectra were recorded on a Bruker DRX-AV-500 spectrometer (Bruker BioSpin Group, Rheinstetten, Germany) at 500 MHz for 1 H and 125 MHz for 13 C using standard pulse sequence programs. All chemical shifts were recorded with respect to TMS as an internal standard. MS was obtained on a VG Auto Spec-3000 spectrometer (VG PRIMA, Birmingham, England). IR was measured on a Perkin-Elmer 241 polarimeter (PerkinElmer, Boston, MA, USA). Column chromatography was carried out on silica gel H (10-40 mm, Qingdao Haiyang Chemical Factory, Qingdao, China) and RP-18 (40-75 mm, Fuji Chemical Industrial Co., Ltd, Tochigi, Japan). TLC was performed on silica gel GF 254 (Yantai Jiangyou Silica Gel Co., Ltd, Yantai, China). Solvents were of industrial purity and distilled prior to use.

Plant material
The whole plants of L. japonicum were collected from Pingbian County of Yunnan Province, China in August, 2011 and identified by Prof. Shugang Lu, School of Life Science, Yunnan University, Kunming, China, where a voucher specimen (No. 1108018) has been deposited.

Conclusion
In our study, six lycodine alkaloids were isolated from the club moss L. japonicum Thunb. N-methylhydroxypropyllycodine (1) was a new alkaloid with 2-hydroxy-propyl and N-Me groups.

Supplementary material
Supplementary material relating to this article is available online.