Lycoplanines B-D, Three Lycopodium Alkaloids from Lycopodium complanatum

Abstract A novel C17N Lycopodium alkaloid (LA), lycoplanine B (1), containing an unusual formyl group, along with two new LAs, lycoplanines C (2) and D (3), were isolated from the whole plant of Lycopodium complanatum. Their structures were elucidated by extensive NMR techniques, including 1D- and 2D-NMR experiments, as well as comparing their spectral data with those of the known analogues. A possible biogenetic pathway for 1 was also proposed. Graphical Abstract Electronic supplementary material The online version of this article (10.1007/s13659-018-0161-2) contains supplementary material, which is available to authorized users.


Introduction
Lycopodium complanatum belonging to the family Lycopodiaceae, is mainly distributed in temperate and subtropical areas. This plant has been used as a folk medicine for the treatment of arthritic pain, quadriplegia, contusion, and blood stasis [1,2]. Previous phytochemical investigations of this plant led to the isolation of a number of Lycopodium alkaloids (LAs) [3][4][5][6], which have attracted great interest from biogenetic [7,8], synthetic [9][10][11], and biological [12,13] points of view.
In our continuing efforts to find biogenetically interesting and structurally unique LAs, we reported a LA, lycoplanine A, posessing an intriguing tricyclic (6/9/5) ring skeleton from L. complanatum [14]. Further chemical investigation of the extracts of this plant resulted in the isolation of three new LAs, lycoplanines B-D (1-3), along with three known compounds lycopodine (4), lycopodine N-oxide (5), and des-N-methyl-a-obscurine (6). In current study, we describe the isolation, structure elucidation, and anti-AChE activity of 1-3 ( Fig. 1), as well as a plausible biogenetic path for lycoplanine B (1). ) in the HRESIMS, which corresponded to the molecular formula C 17 H 23 NO 2 , and was further confirmed by its 13 C NMR and DEPT analysis. The IR absorption bands indicated the existence of carbonyl (1703 cm -1 ), and olefinic functional (1637 cm -1 ) groups. The 13 C NMR spectroscopic data of 1 (Table 1), with the aid of an HSQC NMR experiment, showed 17 carbons due to one methyl (d C 22.8), seven sp 3 methylenes, one sp 2 (d C 158.6) methine, four sp 3 methines, one sp 3 (d C 62.2) quaternary carbon, one sp 2 (d C 113.2) quaternary carbon, one carbonyl group (d C 212.1), and one aldehyde group (d C 189.4), of which one sp 3 quaternary carbon (d C 62.2) and one sp 3 methylene carbon (d C 49.1) were attributed to those attached to a nitrogen atom.
The relative configuration of 1 was elucidated by the ROESY spectrum ( Lycoplanine C (2), obtained as colorless oil, had a molecular formula of C 16 3 , 278.1751) with six degrees of unsaturation. The IR absorption at 3414 cm -1 suggested the presence of hydroxy group. In agreement with its molecular formula, all the sixteen carbon signals were observed in the 13 C NMR spectrum (Table 1), and were further classified by DEPT experiments into one methyl (d C 22.5), six methylenes, six methines (one olefinic at d C 123.2), and three quaternary carbons (one olefinic at d C 143.5, and one carbonyl at d C 210.1). The 1 H NMR (Table 1) spectrum of 2 displayed one methyl (d H 0.84, d, J = 6.1 Hz) and one olefinic proton (d H 5.82, d, J = 4.5 Hz). Careful comparison of the NMR spectroscopic data of 2 with those of lycoposerramine K [15] suggested that they possessed the same carbon skeleton. The fragments of a C-1/C-2/C-3/C-4, b C-9/C-10/C-11, and c C-6/C-7/C-8/C-15(C-16)/C-14 ( Fig. 3) determined by the 1 H-1 H COSY correlations (Fig. 3), in combination with a series of HMBC correlations (Fig. 3) of H-9 with C-1 and C-13, H-1 with C-9 and C-13, H-4 with C-5, C-12 and C-13, H-6 with C-4, C-5 and C-12, and H-7 with C-11 and C-12, further confirmed the above deduction. The only difference between 2 and lycoposerramine K was that 2 possessed an additional hydroxy group connected to C-10, as inferred from the HMBC correlations of H-9 and H-11 with C-10 (d C 64.3).
The relative configuration of 2 was assigned on the basis of ROESY experiment. The ROESY correlations (Fig. 3   methylenes, two methines, and five quaternary carbons (one amide, two olefinic carbons, and two sp3 quaternary carbons). Further 2D NMR analysis indicated that 3 should be a lycodine-type alkaloid with many similarities to that of des-N-methyl-a-obscurine [16], which was also isolated in present study. Differing from des-N-methyl-a-obscurine, the molecular formula of 3 contains one more oxygen atom. Meanwhile, the methine group (d C 44.5) at C-12 in des-N-methyl-a-obscurine was replaced by a sp3 quaternary carbon (d Compounds 1-3 were also tested for AChE inhibitory activities using the Ellman method [18] reported previously (huperzine A as positive control, IC 50 = 0.03 lM). Unfortunately, they showed no inhibitory activity (IC 50-[ 100 lM) after two repeated experiments.

Extraction and Isolation
The air-dried and powdered sample (30 kg) was extracted with 60% EtOH (24 h 9 3), the extract was partitioned between EtOAc and 1.0% HCl/H 2 O. Water-soluble materials, which were adjusted to pH 10 with saturated Na 2 CO 3 aq, were extracted with CHCl 3 to give an alkaloidal extract (60 g). The alkaloidal extract was subjected to medium pressure liquid chromatography (MPLC) over RP-