New Lycopodium alkaloids from Lycopodium obscurum

Three new Lycopodium alkaloids, obscurumines C-E (1–3), along with nine known compounds, were isolated from the club moss Lycopodium obscurum L. Structures of the new compounds were determined on the basis of their spectroscopic analysis and the relative configurations of 1 were established by X-ray crystallographic analysis. All the new isolates were tested for the acetylcholinesterase (AChE) inhibitory activity. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s13659-013-0015-x and is accessible for authorized users.


Introduction
The Lycopodium alkaloids represent a large family of plant secondary metabolites obtained from the club moss belonging to Lycopodiaceae. 1,2 Since the impressively diverse skeletons and interesting biological activities, these alkaloids continue to be of interest from a biogenetic and biological point of view and some of them have been challenging targets for total synthesis. [3][4][5] Lycopodium obscurum L., one of the club moss, has been used in China as a traditional folk medicine for the treatment of contusion, dysmenorrhea, quadriplegia, and arthritic pain. 6,7 Previously phytochemical investigation indicated triterpenoids were the main compounds of this plant and only few Lycopodium alkaloids were isolated. [8][9][10] As part of an ongoing program aimed at discovering structurally interesting and bioactive Lycopodium alkaloids, 11,12 three new Lycopodium alkaloids, obscurumines C-E (1-3), along with nine known compounds, were isolated from the whole herb of L. obscurum L. Herein, we report the isolation, structure elucidation, and acetylcholinesterase (AChE) inhibitory activity of the new isolates.

Results and Discussion
The alkaloidal extract of L. obscurum was separated by normal-phase silica gel, RP-18 silica gel, HPLC, and Sephadex LH-20 chromatography to afford three new Lycopodium alkaloids, obscurumines C-E (1-3), along with nine known compounds. The structures of known compounds, compared with literatures data, were identified as lycopodine, 13 anhydrolycodoline, 14 obscurinine, 9 obscurinine B, 8 lycoflex-ine, 15 acetyldihydrolycopodine, 16 (+)-acetylfawcettiine, 17 Ndemethyl-α-obscurine, 18 N-demethyl-β-obscurine. 19 Obscurumine C (1), colorless crystals, has a molecular formula C 16 (Table 1) revealed 16 carbon signals due to five quaternary carbons (three ketone carbons, one olefinic carbon, and one sp 3 quaternary carbon), four methine groups (one olefinic carbon), six methylene groups, and one methyl group ( H 0.71, d, J = 5.9 Hz). The 1 H-1 H COSY correlations revealed the existence of two fragments, a (C-1-C-4) and b (C-6-C-8, C-8/C15/C-14, C-15-C-16), as shown in Figure 2. In the HMBC spectrum, the correlations of the proton ( H 2.42, H-4) at the terminal carbon (C-4) of fragment a and the protons ( H 2.71, 2.39, H-6) at the terminal carbon (C-6) of fragment b with the carbonyl carbon (C-5,  C 205.7) indicated the connection of C-4/C-5/C-6. While, the connection of C-4/C-13/C-14 were derived from HMBC cross-peaks of H-4 and H-14 to C-13. Meanwhile, the HMBC correlations of H-1 with C-9 and C-13 established the connections of C-1, *To whom correspondence should be addressed. E-mail: qinshizhao@mail.kib.ac.cn C-9, and C-13 through a nitrogen atom. The above analysis indicated 1 should be a lycopodine-type alkaloid with many similarities to that of anhydrolycodoline, 14 a known compound also isolated during the isolation. The obvious differences were that 1 possessed two more carbonyl groups one of which was connected to C-9 ( C 155.8) as deduced from the HMBC correlations of H-1 and H-11 with C-9. The other carbonyl group was speculated to attached at C-10 according to the 13 C chemical shift and confirmed by the HMBC correlations of H-11 to C-10. Thus, the gross structure of obscurumine C (1) was elucidated to be 1 possessing a lycopodine-type skeleton with a scarce two adjacent ketones unit.
The relative configuration of 1 was established by ROESY experiment. In the ROESY spectrum, the correlations of H-4 with H-6b and H-6a with H-15 were observed which indicated the α-orientation of H-4 and Me-16. Since the two adjacent ketone unit is scarce in Lycopodium alkaloid and to confirm the structure and the relative configuration, a X-ray crystallographic analysis of 1 ( Figure 2) was carried out which unambiguously certificated the structure and the relative configurations of compound 1.
Compound 2 was obtained as colorless oil. The HREIMS analysis gave an m/z at 261.1735 ([M + ]) (calc. 261.1729) that established the molecular formula C 16 H 23 NO 2 , corresponding to six degrees of unsaturation. The 13 C NMR spectrum displayed 16 carbon signals due to one methyl, eight methylene, four methine, and three quaternary carbons. Among them, one sp 3 quaternary carbon ( C 96.5) was ascribed to the carbon (C-13) which bearing both an oxygen atom and a nitrogen atom and one sp 2 quaternary carbons were attributable to the ketone group ( C 218.2). Partial structures a-c (C-1-C-4, C-6-C-8/C-15, and C-9-C-11) were deduced from 1 H-1 H COSY ( Figure 3) of 2. Further detailed 2D NMR analysis suggested compound 2 was a fawcettimine-type alkaloid which was similar to that of fawcettimine. 20 The obvious difference was that 2 has one oxygened methine group rather than a methylene group in fawcettimine, which was connected to C-10 as deduced from the 1 H-1 H COSY

H and 13 C NMR data for 13 (δ in ppm and J in Hz)
No.  .1885] spectrum, implying six degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl (3439 cm -1 ) and ketone (1640 cm -1 ) groups. The 13 C NMR and DEPT spectra revealed the existence of seventeen carbons due to three quaternary carbons, six methine groups, six methylene groups, and two methyl groups. Detailed analysis of its 1D and 2D NMR data suggested that 3 was similar to that of epilobscurinol. 18 The only difference was that the double bond between C-3/C-4 in epi-lobscurinol was shift to C-2/C-3 as deduced from the 1 H-1 H COSY correlations of H-1/H-2/H-3/H-4 and confirmed by the HMBC correlations of H-2 with C-1 and C-4. In the ROESY spectrum, the correlations of H-4/H-11b and H-5/H-7 were observed which suggested the relative configuration of 3 was the same as epi-lobscurinol. So, the structure of 3 was deduced and shown in Figure 1.
The new compounds obscurumine C-E (1-3) were tested for acetylcholine esterase (AChE) inhibitory activity. However, none of them showed obvious activity.

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s13659-013-0015-x and is accessible for authorized users.