Lyconadins G and H, Two Rare Lyconadin-Type Lycopodium Alkaloids from Lycopodium complanatum

Abstract Two rare lyconadin-type Lycopodium alkaloids, lyconadins G (1) and H (2), together with four known ones (3–6), were isolated from Lycopodium complanatum. The structures were determined on the basis of their spectroscopic analyses, and the absolute configuration of 1 was established by an X-ray crystallographic analysis. It is the first time to establish the absolute configuration of lyconadin-type Lycopodium alkaloid by an X-ray diffraction experiment. In addition, these findings may provide more information for the biosynthesis of lyconadins. Graphical Abstract Electronic supplementary material The online version of this article (doi:10.1007/s13659-016-0111-9) contains supplementary material, which is available to authorized users.


Introduction
The Lycopodium alkaloids are a unique family of complex natural products that have garnered long-standing interest from chemists as challenging targets for total synthesis [1][2][3][4][5], due to their fascinating structure complexity and wide-ranging biological activities [6][7][8]. Of these, huper-zine A (Hup A), isolated from the Chinese folk medicinal herb Qian Ceng Ta [whole plant of Huperzia serrata (Thunb.ex Murray) Trev.], is a promising agent to treat Alzheimer's disease with highly specific and potent inhibitory activity against acetylcholinesterase (AChE) [9]. As a consequence, these structurally complex Lycopodium alkaloids have inspired many research groups to study the chemical constituents in the Lycopodium plants [10][11][12][13]. Lycopodium complanatum (L.) Holub, mainly distributed in the temperate and subtropical regions of the world [14], was used as a traditional Chinese herbal medicine for the treatment of arthritic pain, quadriplegia, and contusion [15]. Previously, lycospidine A, a unique C 15 N Lycopodium alkaloid possibly derived from proline instead of the lysine biosynthetically, was isolated from this species [16]. As a part of an ongoing research program to discover more Lycopodium alkaloids with fascinating structures and bioactivities serving as lead compounds for drug discovery [17,18], two new rare lyconadins, termed lyconadins G and H (1 and 2), were isolated from L. complanatum (Fig. 1), together with four known ones, lyconadin A (3) [19] and lyconadins C-E (4-6) [20,21]. To the best of our knowledge, although more than 300 Lycopodium alkaloids have been reported so far [22][23][24][25], only six ones belong to the lyconadin-type [19][20][21]26]. In this paper, we describe the isolation and structure elucidation of lyconadins G (1) and H (2).

Results and Discussion
The air-dried and powdered whole plant of L. complanatum was extracted with MeOH three times. The extract was partitioned between EtOAc and 1 % HCl/H 2 O. The pH of the water-soluble portion was adjusted to pH 9 with saturated Na 2 CO 3 aq. Then, it was extracted with CHCl 3 to afford an alkaloidal extract. Further column chromatography over MCI gel, silica gel, and semipreparative HPLC led to the isolation of compounds 1 (7 mg), 2 (2 mg), 3 (11 mg), 4 (8 mg), 5 (4 mg) and 6 (6 mg).
Lyconadin G (1), obtained as colorless blocks, was assigned to have a molecular formula of C 16 H 20 N 2 O by HR-EI-MS (m/z 256.1576, calcd 256.1576) and 13 C NMR spectroscopic data ( Table 1). The IR absorption implied the presence of amide carbonyl (1647 cm -1 ) functionality. The 13 C NMR and DEPT spectra (Table 1) exhibited 16 carbon resonances, including four quaternary carbons (one carbonyl and three olefinic), seven methines [including three olefinic (d C 120.7, 116.8, and 146.6) with corresponding protons as singlet signal at d H 6.28 and two mutually coupled signals at d H 6.34 and 7.30, respectively, in the 1 H NMR spectrum], four methylenes, and one methyl. Based on the above evidence, the structure of 1 was determined to be similar to that of lyconadin C (4) [20]. Comparison of the NMR data of 1 and 4 suggested that the most obvious differences were the loss of two sp 3 carbons (one sp 3 methine carbon and one sp 3 methylene carbon) and the presence of one more trisubstituted double bond in 1. The 1 H-1 H COSY spectrum of 1 (Fig. 2) showed two partial structures a (C-2/C-3), b (C-9 to C-16 and C-8/ C-15), implying that the additional double bond in 1 was located between C-6 and C-7. The HMBC correlations from H-15 and H 2 -8 to C-7 and from H-6 to C-4, C-5, and C-8 ( Fig. 2) further supported the planar structure of 1 as shown in Fig. 2.  In the ROESY spectrum of 1, the ROESY correlations of H-12/H-8b, H-12/H-14b suggested that they were in the same orientations. The fact that C-16 was in an equatorial position was deduced from the ROESY cross-peak of H-8b/H 3 -16. However, there were no solid proof which can be used to establish the relative configurations of C-10 and C-13. Fortunately, crystals suitable for single-crystal X-ray diffraction of 1 was obtained from CHCl 3 -MeOH. The structure of 1 was confirmed by single-crystal X-ray diffraction using the anomalous scattering of CuK radiation with a Flack parameter of 0.2(2) (Fig. 3 (Table 1) spectrum of 2 gave signals due to four quaternary carbons (one carbonyl, three olefinic), five methines (including one olefinic (d C 123.1) with corresponding proton as a singlet signal at d H 5.85 in the 1 H NMR spectrum), six methylenes, and one methyl, implying that the structure of 2 was similar to that of lyconadin G (1). The sole difference was the loss of a 1,2-disubstituted double bond between C-2 and C-3, which was confirmed by the HMBC correlations of H 2 -2 and H 2 -3 with C-4, and of H 2 -3 with C-5, as well as the 1 H-1 H COSY correlation between H 2 -2 and H 2 -3.
Among Lycopodium alkaloids, lyconadins are a unique family which contain intriguing tetra-or pentacyclic ring systems fused by the unique C-4-C-10 linkage and C-6-N-9 bond, and thus offer a new horizon for organic chemists [27][28][29]. Biogenetically, the discovery of two new compounds (1 and 2), together with four known lyconadins (3-6) from the title plant can provide some new insight into the biosynthesis of lyconadins [19,20]. Possible biogenetic pathway for lyconadins G (1), H (2), and A (3) is proposed in Scheme 1. As illustrated in Scheme 1, the key intermediate A might arise from phlegmarane skeleton by C-4 and C-10 carbon bond formation and subsequent loss of H 2 O and oxidation to yield compounds 1 and 2. In addition, the double bond located between C-6 and C-7 in 1  could serve as a suitable electrophile in the process of the desired C-6-N-9 bond formation to produce lyconadin A (3). The potent biological activities of 1 and 2 against AChE using the improved Ellman method [30,31] were also evaluated. However, neither of them showed obvious activity (IC 50 [ 100 lM).

General
Optical

Extraction and Isolation
The club moss L. complanatum (L.) Holub (100 kg) was chopped into sections and extracted with methanol under reflux for three times (4, 3, 3 h, respectively,). The

Acetylcholinesterase Inhibitory Activity
AChE inhibitory activity of lyconadins G (1) and H (2) was assayed by the spectrophotometric method developed by Ellman with slightly modification. S-Acetylthiocholine iodide, S-butyrylthiocholine iodide, 5,5 0 -dithio-bis-(2-nitrobenzoic) acid (DTNB, Ellman's reagent), AChE derived from human erythrocytes were purchased from Sigma Chemical. Compounds were dissolved in DMSO. The reaction mixture (totally 200 lL) containing phosphate buffer (pH 8.0), test compound (50 lM), and AChE (0.02 U/mL), was incubated for 20 min (30°C). Then, the reaction was initiated by the addition of 40 lL of solution containing DTNB (0.625 mM) and acetylthiocholine iodide (0.625 mM) for AChE inhibitory activity assay, respectively. The hydrolysis of acetylthiocholine was monitored at 405 nm every 3 min for 1 h. Tacrine was used as positive control with final concentration of 0.333 lM. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition = (E -S)/E 9 100 (E is the activity of the enzyme without test compound and S is the activity of enzyme with test compound).