Lycodine-Type Lycopodium Alkaloids from the Whole Plants of Huperzia serrata

Abstract Three new lycodine-type Lycopodium alkaloids, namely 1-methyllycodine (1), 8α-hydroxy-15,16-dehydro-des-N-methyl-α-obscurine (2), N-methyl-16-hydroxyhuperzine B (3), and one new natural lycodine-type Lycopodium alkaloid, N-methylhuperzine A (4), along with 11 known analogues (5–15), were isolated from the whole plants of club moss Huperzia serrata. The structures of 1–4 were elucidated on the basis of NMR spectroscopic and mass spectrometry data. Among them, compound 1 was the first lycodine-type alkaloid possessing a methyl group at C-1. In addition, the structure of 5 was confirmed by the single-crystal X-ray crystallography data and its 13C NMR was reported for the first time in current study. Compounds 1–5 were tested their BACE1 inhibitory activity. Graphical Abstract Electronic supplementary material The online version of this article (doi:10.1007/s13659-017-0140-z) contains supplementary material, which is available to authorized users.

have attracted broad interest from chemists and pharmacologists worldwide due to their intriguing carbon skeletons and biological activities [1][2][3][4][5][6]. Such alkaloids were divided into four major classes (lycopodine, lycodine, fawcettimine, and phlegmarine) by Canadian famous chemists Ayer et al. [7]. Particularly, lycodine-type alkaloids, which generally characterized by four connected sixmembered rings, including a pyridone or pyridine ring, a piperidine ring, and a bicyclo [3.3.1]nonane core [8], are a unique class of compounds and have attracted great interest for their biological activities especially the extraordinary acetylcholinesterase (AChE) inhibition by huperzine A that has a potential of becoming a therapeutic agent for the treatment of Alzheimer's disease [2,9].

Results and Discussion
The air-dried and powdered whole plants of H. serrata were extracted with 60% EtOH for three times. The extract was partitioned between EtOAc and 1.0% HCl/H 2 O. Water-soluble materials, which were adjusted to pH 9 with 17% ammonia solution, were then extracted with CHCl 3 to afford an alkaloidal extract. Further column chromatography (CC) over MCI gel, normal-phase silica gel, and Sephadex LH-20 led to the isolation of three new lycodinetype alkaloids (1-3), one new natural lycodine-type Lycopodium alkaloid (4), together with 11 known ones (5-15).   (Table 1), respectively, in the 1 H NMR spectrum. The above information suggested that 1 should be a lycodine-related Lycopodium alkaloid. Comparison of the 1 H and 13 C NMR spectroscopic data of 1 with those of the known alkaloid lycodine (10) [19] revealed that they were structural analogues. A major difference was the presence of one more methyl group [d H 2.55 (3H, s), d C 24.2] at C-1 in 1, which was confirmed by the significant HMBC correlations from H-17 (d H 2.55) to C-1 (d C 157.8) and C-2 (d C 122.0) (Fig. 3), as well as the obvious triplet signal of H-1 in 10 was disappeared in 1. It is worth noting that 1 was the first lycodine-type Lycopodium alkaloid possessing a methyl group at C-1.
The relative configuration of 1 was deduced by the ROESY experiment (Fig. 3) and the coupling constant. Biogenetially, H-12 and H-7 of the lycodine-type Lycopodium alkaloids were band a-orientated, respectively, which were also supported by the diagnostic ROESY correlations of H-6a with H-11b and H-7 with H-11a. Irradiation of H-12 enhanced the signal of H-14a, indicating that these protons were on the same facial plane. Furthermore, the methyl group at C-15 was located at an equatorial position by the large coupling constant (11.9 Hz) between H-14a and H-15. Thus, the structure of 1 was determined and named as 1-methyllycodine. , requiring seven degrees of unsaturation. The IR spectrum was indicative of the presence of a hydroxy (3426 cm -1 ) and an amide carbonyl (1660 cm -1 ) groups. Its 1D NMR spectroscopic data (Tables 1, 2) showed general features analogous to those of des-N-methyl-a-obscurine [23,27]. Differing from the latter, compound 2 has one more hydroxy group and a D 15 double bond in its structure. The carbon resonance at d C 78.8 was assigned to C-8 bearing a hydroxy group, based on the HMBC correlations of H-8 (d H 3.93) with C-12 (d C 38.1), C-14 (d C 42.4), and C-16 (d C 115.1), and of H 2 -6 (d H 1.56 and 2.33) and H-7 (d H 1.94) with C-8, together with the 1 H-1 H COSY correlations of H-7/H-8 (Fig. 4) In the ROESY spectrum (Fig. 4), the band a-orientation of H-12 and H-7, respectively, were revealed by the observed correlations of H-6a/H-11b, H-7/H-11a, and H-12/H-14b. Furthermore, the obvious ROESY correlation of H-8/H-6b allowed the assignment of OH-8 as a-orientated. On the basis of the above evidence, the structure of 2 was assigned as 8a-hydroxy-15,16-dehydro-des-N-methyla-obscurine.  (Tables 1, 2). Analysis of the 1D NMR spectra of 3 revealed that its spectroscopic data closely resembled those of 16-hydroxyhuperzine B (15) [25], a known lycodine-type alkaloid previously isolated from Lycopodium casuarinoides. The only difference between those two compounds was that 3 possessed an additional Nmethyl group. Consequently, compound 3 was assumed to be the N-methylated derivative of 16-hydroxyhuperzine B, which was confirmed by observed key HMBC correlations from N-methyl (H-17) at d H 2.69 to C-9 (d C 51.8) and C-13   114.3, 118.1, 125.8, and 142.3), two sp 3 methylenes (d C 35.2 and 50.7), and three methyls (d C 12.5, 22.7, and 29.7) with corresponding protons as double signal at d H 1.71 and singlet signals at d H 1.53 and 2.09, respectively, in the 1 H NMR spectrum (Table 1). The above data allowed 4 to be a N-methyl derivative of huperzine A, which was confirmed by the observed key HMBC correlation between the proton at d H 2.09 with the sp 3 quaternary carbon C-13 at d C 61.1 (Fig. 5). The observed ROESY correlations (Fig. 5) of H-6b/H-8 and of H-7/H-10 (d H 1.71) indicated that H-7 was a-orientated. The R * configuration of C-13 was assigned by the clear ROESY correlations of H-3 with H-14a and H-11 with H-17. Accordingly, the structure of 4 was characterized as N-methylhuperzine A, which reported previously as a synthetic product from the methylation of huperzine A [2,9]. To our knowledge, compound 4 was isolated for the first time from natural resources.
Compounds 1-5 were evaluated for their b-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) inhibitory activity. Unfortunately, the results showed that all the compounds were inactive (IC 50 values [100 lM).

Gernal Experimental Procedures
UV spectra were recorded with a Shimadzu UV-2401A spectrophotometer. IR spectra were recorded on Bruker Tensor 27 spectrometer with KBr pellets. 1D and 2D NMR spectra were carried out on Bruker AM-400, DRX-500, or AVANCE III-600 spectrometers. Chemical shifts were reported using TMS as the internal standard. ESI-MS were run on Shimadzu UPLC-IT-TOF-MS instrument. HR-ESI-MS spectra were measured using Agilent G 6230 TOF MS (Agilent). EI-MS and HR-EI-MS spectra were measured with a Waters AutoSpec Premier P776 mass spectrometer (Waters, Milford, MA, USA).Crystal analysis were performed on a Bruker APEX DUO diffractometer equipped with an APEX II CCD, using Cu Ka radiation (k = 1.54178 Å ). Cell refinement and data reduction were performed with Bruker SAINT. Column chromatography (CC) was performed on silica gel (100-200 or 200-300 mesh; Qingdao Marine Chemical Co. Ltd., Qingdao, China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sala, Sweden) and MPLC was performed on a Lisui EZ Purify III System packed with MCI gel (CHP20P, 75-150 mm; Mitsubishi Chemical Corporation, Tokyo, Japan). Precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd.) were used for thinlayer chromatography (TLC). Fractions were monitored by TLC and spots were visualized by Dragendorff's reagent.

Plant Material
The club moss H. serrata was collected from Taijiang

Extraction and Isolation
The aerial parts of club moss H. serrata (40 kg) were chopped into sections and extracted with 60% EtOH/H 2 O under reflux for three times (24 h 9 3). The resultant extract was partitioned between EtOAc and 1% HCl/H 2 O solution to afford ethyl acetate and water soluble fractions, respectively. The water-soluble fractions were adjusted to pH 9 with 17% ammonia solution, and then extracted with CHCl 3 to give an alkaloidal extract (106 g). The alkaloidal extract was subjected to a MCI gel CC (MeOH/H 2 O, 5 to 100%) to afford fractions I-V. Fraction I (18.0 g) was further chromatographed over a silica gel CC (CHCl 3 /MeOH,

X-ray Crystal Structure Analysis
Crystal analysis were performed on a Bruker APEX DUO diffractometer equipped with an APEX II CCD, using Cu Ka radiation (k = 1.54178 Å ). Cell refinement and data reduction were performed with Bruker SAINT.
The structure of 5 was solved by direct methods using SHELXS-97. Refinements were performed with SHELXL-97 using full-matrix least-squares, with anisotropic displacement parameters for all the non-hydrogen atoms. The H-atoms were placed in calculated positions and refined using a riding model. Crystallographic data for 5 in this paper have been deposited with the Cambridge Crystallographic Data Centre (CCDC 1518517). Copies of the data can be obtained free of charge from the CCDC via www. ccdc.cam.ac.uk.

BACE1 Inhibitory Activity Assay
Compounds 1-5 were assessed for b-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) inhibitory activity. BACE1 inhibitory evaluation was tested using a fluorescence resonance energy transfer (FRET) assay kit supplied by PanVera (Kit P2985, Madison, WI, USA). The kit was using purified baculovirus expression BACE1 and substrates of a new red FRET peptide substrates, which were based on the ''Swedish'' mutation. The BACE1 FRET assay was carried out according to the principle described in Ref. [26]. The first orally available non-peptidic b-secretase inhibitor LY2811376, [28] which had an IC 50 value of 401.21 nM, was using as a positive control.