Lycodine- Type alkaloids from lycopodiastrum casuarinoides and their acetylcholinesterase inhibitory activity

Article abstract of DOI:10.3390/molecules19079999

Four new lycodine- Type alkaloids, namely 16-hydroxyhuperzine B (1), N-methyl-11- Acetoxyhuperzine B (2), 8,15-dihydrolycoparin A (3) and (7S,12S,13R)- huperzine D-16-O-β-D-glucopyranoside (4), along with ten known analogues 5-14, were isolated from the whole plant of Lycopodiastrum casuarinoides. The structures of the new compounds were elucidated by means of spectroscopic techniques (IR, MS, NMR, and CD) and chemical methods. Compounds 1 and 2 possessed four connected six-membered rings, while compounds 3 and 4 were piperidine ring cleavage products. In particular, compound 4 was a lycopodium alkaloidal glycoside which is reported for the first time. Among the isolated compounds N-demethylhuperzinine (7), huperzine C (8), huperzine B (9) and lycoparin C (13) possessed significant inhibitory activity against acetylcholinesterase, and the new compound 1 showed moderate inhibitory activity. The structure activity relationships were discussed.

Full text of DOI:10.3390/molecules19079999

Molecules 2014, 19, 9999-10010; doi:10.3390/molecules19079999
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Lycodine-Type Alkaloids from Lycopodiastrum casuarinoides
and Their Acetylcholinesterase Inhibitory Activity
Dong-Bo Zhang 1, Jian-Jun Chen 1,*, Qiu-Yan Song 1, Li Zhang 1 and Kun Gao 1,2,
*
1
State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical
Engineering, Lanzhou University, Lanzhou 730000, China
2
School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology,
Zhejiang University; Ningbo 315100, China
* Authors to whom correspondence should be addressed; E-Mails: chenjj@lzu.edu.cn (J.-J.C.);
npchem@lzu.edu.cn (K.G.); Tel.: +86-931-891-2592 (K.G.); Fax: +86-931-891-2582 (K.G.).
Received: 23 May 2014; in revised form: 18 June 2014 / Accepted: 27 June 2014 /
Published: 10 July 2014
Abstract: Four new lycodine-type alkaloids, namely 16-hydroxyhuperzine B (1),
N-methyl-11-acetoxyhuperzine B (2), 8,15-dihydrolycoparin A (3) and (7S,12S,13R)-
huperzine D-16-O-β-D-glucopyranoside (4), along with ten known analogues 5−14, were
isolated from the whole plant of Lycopodiastrum casuarinoides. The structures of the new
compounds were elucidated by means of spectroscopic techniques (IR, MS, NMR, and
CD) and chemical methods. Compounds 1 and 2 possessed four connected six-membered
rings, while compounds 3 and 4 were piperidine ring cleavage products. In particular,
compound 4 was a lycopodium alkaloidal glycoside which is reported for the first time.
Among the isolated compounds N-demethylhuperzinine (7), huperzine C (8), huperzine B
(9) and lycoparin C (13) possessed significant inhibitory activity against acetylcholinesterase,
and the new compound 1 showed moderate inhibitory activity. The structure activity
relationships were discussed.
Keywords: Lycopodiastrum casuarinoides; Lycopodiaceae; lycodine alkaloid;
acetylcholinesterase; 16-hydroxyhuperzine B; N-methyl-11-acetoxyhuperzine B;
8,15-dihydrolycoparin A; (7S,12S,13R)-huperzine D-16-O-β-D-glucopyranoside

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1. Introduction
Alzheimer’s Disease (AD) is a chronic neurological disorder characterized by memory impairment,
cognitive dysfunction, behavioral disturbances and deficits in activities of daily living [1–3].
According to the cholinergic hypothesis, memory impairment in patients suffering from AD is a result
of decreased levels of the neurotransmitter acethylcholine (ACh) in the cortex [4]. Acetylcholinesterase
(AChE) inhibitors that can block the cholinergic degradation of ACh are therefore considered to be a
promising approach for the treatment of AD. Additionally, recent studies have shown that AChE
inhibitors also prevent the assembly of β-amyloid peptide into amyloid plaque which is the first step of
AD [5,6]. This discovery further stimulated a great interest in searching for useful leads which could
become new candidates for the development of rational drug design against AD.
Lycopodiastrum casuarinoides (Spring) Holub (Lycopodiaceae), the only species of the genus
Lycopodiastrum, has been used as a folk medicine for relaxing tendons and stimulating blood
circulation [7]. Previous phytochemical research on this plant indicated that lycodine-type alkaloids
were its main chemical and bioactive ingredients [8–13], in particular, a type of alkaloids that
possessed extraordinary AChE inhibition, such as the well-known huperzine A [12]. According to
previous reports and our research, it was found that the total alkaloidal extract of the plant exhibited
significant AChE inhibitory activity [8,10–13]. Further bioactivity-guided chromatographic
fractionation led to four new (compounds 1−4) and ten known lycodine-type alkaloids 5–14 (Figure 1).
Herein, we reported the isolation, structural elucidation, AChE inhibitory activity of these compounds.
Furthermore, the preliminary structure-activity relationships are discussed.
Figure 1. Structures of compounds 1–14 and huperzine A.
2. Results and Discussion
Compound 1, obtained as a colorless gum, showed an [M+H]+ ion peak at m/z 273.1600 in its
HRESIMS, corresponding to the molecular formula of C16H20N2O2 (calcd. for C16H21N2O2, 273.1598),

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implying eight degrees of unsaturation. The IR absorption band (1,654 cm−1) of compound 1 indicated
the presence of an α,β-unsaturated carbonyl group. The existence of an α-pyridone moiety was
revealed by the absorption bands (227 and 306 nm) in its UV spectrum and two characteristic proton
1
signals [δH 7.79, 6.48 (each 1H, d, J = 9.0 Hz)] in the low field region of the H-NMR spectrum [13].
Additionally, a hydroxymethyl group [δH 3.87, 3.83 (2H, ABq), δC 65.9] and an olefinic proton
1
[δH 5.81 (br d, J = 4.8 Hz)] were also displayed in the H-NMR spectrum (Table 1). The 13C- and
DEPT NMR spectra showed 16 carbon signals, including six methylenes (one oxygenated at δC 65.9
and one nitrided at δC 42.0), five methines and five quaternary carbons (two nitrogenated at δC 57.6 and
145.6, and one amide carbonyl carbon at δC 165.5) (Table 2). These spectroscopic data of compound 1
were similar to those of the known huperzine B (9) [12] which co-occurs in this species [9], indicating
that compound 1 was a lycodine alkaloid possessing four connected six-membered rings.
Table 1. 1H-NMR data of compounds 1–4, δ in ppm and J in Hz.
No.
2
δH (1) a
6.48, d (9.0)
7.79, d (9.0)
2.90, dd (18.6, 6)
2.39, br d (18.6)
2.59, m
δH (2) b
6.41, d (9.2)
δH (3) a
6.46, d (9.6)
δH (4) b
6.48, d (9.6)
3
7.79, d (9.2)
7.66, d (9.6)
7.87, d (9.6)
6a
6b
7
2.96, dd (17.6, 4.8)
2.39, br d (17.6)
2.68, m
3.01, dd (18.6, 6.6)
2.36, br d (18.6)
2.16, m
2.99, dd (17.6, 4.3)
2.38, br d (17.6)
2.60, m
8a
8b
9a
5.81, br d (4.8)
5.42, br d (5.2)
1.91, br d (13.8)
1.78, ddd (13.8, 13.2, 3.6)
5.82, br d (4.1)
3.06, br d (12.0)
2.71, ddd (14.4,
14.2, 2.0)
9b
2.55, overlapped
1.80, overlapped
1.80, overlapped
1.39, dddd (13.2,
12.6, 12.3, 4.6)
1.69, m
2.66, overlapped
1.75, m
10a
5.33, dd (16.8, 1.8)
5.10, dd (10.2, 1.8)
5.42, dd (17.0, 2.0)
5.27, dd (10.2, 2.0)
10b
1.64, m
11a
4.76, ddd (11.2,
10.8, 5.2)
6.02, ddd (16.8, 10.2, 10.2) 6.09, ddd (17.0, 10.2, 10.1)
11b
12
2.07, br d (12.6)
2.51, d (16.2)
2.25, d (16.2)
2.07, dd (10.8, 3.6)
2.65, d (16.7)
2.95, dd (10.8, 3.6)
2.19, dd (12.6, 12.6)
1.66, dd (12.6, 3)
2.05, m
3.06, dd (10.3, 4.1)
3.11, d (18.4)
14a
14b
15
1.76, d (16.7)
2.16, d (18.4)
16
3.87, 3.83; ABq
(13.8)
1.56, br s
4.11, 4.02; ABq (12.2)
Glu
1'
2'
4.22, d (7.8)
3.17, dd (9.5, 7.8)
3.23, t (9.5)
3'
4'
3.25, t (9.5)
5'
3.34, m
6'a
3.64, dd (11.8, 5.4)
3.85, dd (11.8, 1.3)
2.70, s
6'b
N-Me A
N-Me B
11-OAc
2.63, s
2.04, s
2.58, s
2.58, s
2.70, s
Note: 2 in CDCl3, 1, 3 and 4 in CD3OD. a Data were measured at 600 MHz (1H); b Data were measured at 400 MHz (1H).
Assignments were based on DEPT, HSQC, 1H-1H COSY, and HMBC experiments.

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Table 2. 13C-NMR data of compounds 1−4.
No.
δC (1) a
165.5
119.6
140.6
115.3
145.6
29.8
δC (2) b
165.0
118.1
140.8
119.8
142.6
29.3
δC (3) a
165.0
118.8
142.4
117.7
146.4
30.1
δC (4) b
165.5
118.9
143.1
117.5
144.9
29.4
1
2
3
4
5
6
7
34.3
29.7
38.7
39.7
8
127.1
42.0
124.0
48.6
37.5
128.8
9
10
24.8
25.3
117.3
141.4
48.1
119.1
139.8
46.2
11
25.0
71.2
12
39.3
37.3
13
57.6
58.1
64.2
64.3
14
41.6
43.2
41.9
40.6
15
136.6
65.9
132.6
23.0
41.4
135.6
72.8
16
180.8
Glu
1'
2'
102.5
75.0
77.9
71.7
78.1
62.8
40.2
40.2
3'
4'
5'
6'
N-Me A
N-Me B
11-OAc
11-OAc
37.2
40.2
40.2
170.5
21.1
Note: 2 in CDCl3, 1, 3 and 4 in CD3OD. Data were measured at 150 MHz (13C); Data were measured at
a
b
100 MHz (13C). Assignments were based on DEPT, HSQC, 1H-1H COSY, and HMBC experiments.
The most significant difference between two compounds was that the presence of an additional
hydroxyl group in compound 1. The hydroxyl group might be localized to C-16, which was confirmed
by HMBC correlations between the C-16 (δC 65.9) with H-14, H-8 (Figure 2).
Figure 2. Key HMBC (H→C) correlations and 1H-1H COSY ( ) of compounds 1–4.

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The relative configuration of compound 1 was provided by the NOE difference spectra. In the
biogenetic consideration of lycodine-type alkaloids derivatives isolated from Lycopodiaceae species,
H-12 was assigned as the β-orientation. Irradiation of H-12 enhanced the signals of H-14a, thus, the H-7
was α-oriented. The specific rotation of compound 1 was determined to be [α]2D5 −70 (c 0.1, MeOH),
which was similar to the value of [α]2D5 −54.2 (c 0.2, MeOH) observed for huperzine B (9) [12]. Also, the
CD spectrum of compound 1 (Figure 3) showed a positive Cotton effect around 230 nm and a negative
one near 307 nm, which was in agreement with that of huperzine B (9) [12], indicating a (13R)
configuration [13]. Taken together, the absolute configuration of compound 1 was assigned to be
7S/12R/13R. Finally, the structure of compound 1 was elucidated and named as 16-hydroxyhuperzine B.
Figure 3. CD spectra of compounds 1–3 and 9.
30
1
2
3
25
9
20
15
10
5
0
-5
-10
220 240 260 280 300 320 340 360 380 400
Wavelenth [nm]
Compound 2, obtained as a yellow powder, has an [M+H]+ ion peak at m/z 329.1863 in its
HRESIMS, corresponding to the molecular formula of C19H24N2O3 (calcd. for C19H25N2O3, 329.1860),
implying nine degrees of unsaturation. The NMR data of compound 2 were similar to those of
huperzine B (9) [12], except for the presence of an additional acetoxy group [δH 2.04 (3H, s), δC 21.1,
170.5] and one methyl group [δH 2.63 (3H, s), δC 37.2]. The acetoxy group might be localized at C-11
in compound 2, which was supported by the shift of the C-11 signal in compound 2 (δC 71.2) to lower
field region relative to huperzine B (9) (δC 28.1), and was further confirmed by observed key HMBC
correlations from H-11 (δH 4.76) to the acetoxy carbonyl carbon at δC 170.5 (Figure 2). The methyl
could be attached to the N-atom, which was confirmed by HMBC correlations between the proton at δH
2.63 with C-13 (δC 58.1) and C-9 (δC 48.6) (Figure 2). The relative configuration of compound 2 was
deduced by the NOE difference spectra experiment and the coupling constants. Like compound 1, the
H-12 was assigned a β-orientation and the H-7 was α-oriented. Furthermore, the large coupling
constant between H-11 and H-12 (J11, 12 = 10.8 Hz) indicated the vicinal protons H-11 (δH 4.76) and
H-12 (δ 2.07) both took axial orientations. The CD spectrum of compound 2 showed a positive Cotton
effect at 229 nm and a negative one at 307 nm (Figure 3), which matched well with that of huperzine B
(9) [12]. Thus, the absolute configuration of compound 2 was assigned as 7R/11R/12R/13R.

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Compound 3, a white powder, showed an [M+H]+ ion peak at m/z 303.1709 in its HRESIMS,
corresponding to the molecular formula of C17H22N2O3 (calcd. for C17H23N2O3, 303.1703), implying
1
eight degrees of unsaturation. In the H-NMR spectrum of compound 3, an ABX spin system for the
exomethylene moiety (CH2 = CH) resonating at δH 6.02 (1H, ddd, J = 16.8, 10.2, 10.2 Hz), 5.33 (1H, dd,
J = 16.8, 1.8 Hz) and 5.10 (1H, dd, J = 10.2, 1.8 Hz) suggested that compound 3 was a piperidine ring
(C ring) cleavage product. The NMR data of compound 3 were highly similar to those of lycoparin A
(14) [8], except for the fact the Δ8(15) double bond was saturated, which implied the structure of
compound 3 to be 8,15-dihydrolycoparin A, which was supported by its 2D NMR experiments (Figure 2).
In particular, a long spin system [−CH2−CH(X)−CH2−CH−CH2−(H2-6/H-7(X)/H2-8/H-15/H2-14),
X = −CH−CH−CH2−(H-12/H-11/H2-10)] was displayed in the 1H-1H COSY spectrum of compound 3.
The relative configuration of compound 3 was deduced by the NOE difference spectra experiment and
the coupling constants. In the consideration of the biogenesis of the lycodine-type alkaloid derivatives
isolated from Lycopodiaceae species, the H-12 was assigned as the β-orientation. Irradiation of H-12
enhanced the signals of H-8b and H-14a, indicating that these protons were on the same facial plane,
the H-7 was therefore assigned as the α-orientation. The large coupling constant between H-15 and
H-14a (J14a, 15 = 12.6 Hz) indicated that these two protons both took axial orientations, thus, H-15 was
on the other side. Similarly, the CD curve of compound 3 showed a positive Cotton effect around 230
nm and a negative value near 310 nm (Figure 3). Consequently, the absolute configuration of
compound 3 was established as 7S/12R/13R/15R.
Compound 4, obtained as a colorless powder, showed a molecular ion peak at m/z 449.2288
[M+H]+ in HRESIMS, consistent with the molecular formula of C23H32N2O7 (calcd. for 449.2282,
C23H33N2O7) requiring nine degrees of unsaturation. The strong IR absorptions at 3,385 and 1,658 cm−1
indicated the presence of hydroxyl and carbonyl groups, respectively. The NMR data of compound 4
were analogous to those of huperzine D (12) [11] with a C-15 hydroxymethyl group except for a set of
signals of one hexose group including the anomeric proton signal at δH 4.22 (1H, d, J = 7.8 Hz), the
other 6 proton signals at δH 3.17–3.85, the anomeric carbon signal at δC 102.5 (C-1) and another 5
carbon signals at δC 62–78. The hexose was suggested to be a D-glucose by the comparison of
13C-NMR data with those reported in the literature, and further confirmed by TLC comparison with an
authentic sample and by its specific rotation value after acidic hydrolysis of compound 4 in
HCl-methanol (9%) yielded huperzine D (12) and glucose [14]. The coupling constant of H-1' (δH
4.22, d, J = 7.8 Hz) indicated the D-glucose had a β-linkage. The C-16 of compound 4 should be
glycosylated, which was confirmed by the observed key HMBC correlation from the anomeric proton
at H-1' (δH 4.22) to C-16 (δC 72.8) (Figure 2). Accordingly, the structure of compound 4 was assigned
as (7S,12S,13R)-huperzine D-16-O-β-D-glucopyranoside.
By comparison of our spectroscopic data with those reported in the literature, the remaining known
compounds were identified as huperzinine (5) [11], casuarinine A (6) [13], N-demethylhuperzinine (7) [10],
huperzine C (8) [11], huperzine B (9) [12], casuarinine E (10) [13], carinatumin B (11) [15], huperzine
D (12) [11], lycoparin C (13) [8], and lycoparin A ( 14) [8], respectively.
Lycodine-type alkaloids are a group of structurally unique secondary metabolites, characterized by
four or three connected six-membered rings, including a pyridone or pyridine ring (ring A), a
piperidine ring (ring C), and a bicyclo[3.3.1]nonane core formed by rings B and D. Many of them
continued to be the hot spots of research interest because of their promising bioactivity [16] and

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challenging total synthesis [17,18]. In our study, four new lycodine-type alkaloids and ten known
analogues were isolated from L. casuarinoides. As far as we know, compound 4 is the first reported
example of a lycopodium alkaloidal glycoside. It may be worthwhile to point out that we initially
doubted the origin of compound 2 because it could be an artifact produced from the present extraction
and isolation procedure, which employed EtOAc. In order to rule out that possibility, the MeOH
extract of the plant was re-examined by the HPLC with pure compound 2 as reference (Figure 4). The
unequivocal detection of the same compound in the original extract with an identical HPLC tR value as
that of the reference compound proved that compound 2 was a natural product and not an artifact.
Figure 4. (A) HPLC analysis of compound 2 (retention time: 7.848 min); (B): HPLC
analysis of the MeOH extracts of L. casuarinoides (retention time: 7.821 min). HPLC
analyses were performed on Waters 1525-2998 series HPLC system (C-18 column, Sun
Fire, 5 μm, 4.6 mm × 150 mm); mobile phase, CH3CN/H2O = 7/3; flow rate, 1.0 mL/min;
injection volume, 10 μL).
The isolated lycodine-type alkaloids 1–14 were evaluated for their AChE inhibiting activity by
Ellman’s method in 96-well microplates [19,20]. The results are listed in Table 3. Huperzine A with an
IC50 value of 74.3 nM was used as a reference compound, and it showed good agreement with
literature data (IC50 = 72.4 nM) [21]. Huperzine C (8) possessed the most potent inhibition against
AChE, with an IC50 value of 0.6 μM. Also, N-demethylhuperzinine (7), huperzine B (9) and lycoparin
C (13) showed significant AChE inhibitory activity with IC50 values of 1.9, 20.2 and 23.9 μM,
respectively, and the new compound 1 exhibited moderate inhibitory activity with IC50 value of 87.3 µM.
Interestingly, N-demethylhuperzinine (7), huperzine C (8), huperzine B (9) and lycoparin C (13) have
been previously reported to show inhibition of AChE activity with IC50 values of 15.0, 0.489, 19.3 and
25.0 µM, repectively [8,13,21]. Our reports were found to be close to the literature data. In fact, the
inhibiton of huperzine C (8) with an amino group at C-13 was 3-fold higher than that of N-demethyl-
huperzinine (7), and huperzinine (5) show very weak activity, suggested that the N-methyl group on
the position 13 might cause the sharp decrease in AChE inhibition. In other words, the amino group on
the positions 13 may be a structural requirement for the anti-AChE activity of lycodine-type alkaloids,
which was also supported by comparing the structure-activity relationships between casuarinine E (10)
(IC50 > 250 μM) and lycoparin C (13). Morever, the activity of huperzine C (8) with a methyl at C-15

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was 40-fold higher than those of lycoparin C (13) with a hydroxymethyl at C-15, indicated the methyl
of the three-carbon bridge ring was important for AChE inhibition in this kind of alkaloids.
Interestingly, huperzine A is very similar to huperzine C (8), except for the position of double bond,
but the activity of the former was 8-fold higher than that of the latter, implying the exocyclic double
bond was required for high anti-AChE activity. The findings mentioned above were consistent with
previous observations [22].
Table 3. AChE inhibiting activity of compounds 1, 7, 8, 9 and 13.
Compounds
Compound 1
IC50 (μM)
87.3 ± 1.9
N-Demethylhuperzinine (7)
Huperzine C (8)
Huperzine B (9)
Lycoparin C (13)
Huperzine A a
1.9 ± 0.2
0.6 ± 0.1
20.2 ± 1.3
23.9 ± 2.2
(74.3 ± 2.8) ×10−3
Values are expressed as mean ± SD (n = 3). a Positive control.
3. Experimental
3.1. General
Optical rotations were measured on a Perkin-Elmer 341 polarimeter. IR spectra were recorded on a
170SX FT-IR instrument using KBr discs over the range of 400–4,000 cm−1. UV spectra were
measured using a Shimadzu UV-260 spectrophotometer. CD spectra were obtained on an Olis DSM
1000 spectrometer. NMR spectra were recorded on a Bruker AM-400 and a Varian Mercury-600BB
NMR (600 MHz) spectrometer using TMS as an internal standard. High-resolution electrospray
ionization mass spectra (HRESIMS) were measured on a Bruker Daltonics APEX II 47e spectrometer.
Column chromatography was performed on silica gel (200–300 mesh, Qingdao Marine Chemical Inc.,
Qingdao, People’s Republic of China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala,
Sweden) and RP-C18 (100–200 µm, Waters). Semipreparative HPLC was performed on a Waters 1525
binary pump system with a Waters 2489 detector (210 nm) using a YMC-Pack ODS-A (250 × 10 mm,
S-5 μm) column. Fractions were monitored by TLC, which were visualized by heating the silica gel
plates after being sprayed with 5% H2SO4 in EtOH.
3.2. Plant Material
The whole L. casuarinoides were collected in Tunchang County of Hainan Province, China
(19°36′N; 110°12′E; elevation 328 m), in July 2009, and identified by Qiongxin Zhong of Hainan
Normal University. The voucher specimen (No. 2009020) was deposited in the State Key Laboratory
of Applied Organic Chemistry, Lanzhou University, China.
3.3. Extraction and Isolation
An air-dried and powdered sample (4.7 kg) was extracted with 95% MeOH three times (each time
30 L for 7 days) at room temperature. Evaporation of the solvent gave a residue (430 g), which was

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partitioned between EtOAc (3 × 2 L) and 2% HCl solution (2 L). The acidic water-soluble materials,
adjusted to pH 9–10 with 10% ammonia solution, were extracted with CHCl3 (4 × 2.5 L) and BuOH
(4 × 2.5 L). The CHCl3 extract (4.3 g) was subjected to silica gel (Φ 4 × 40, 200–300 mesh, 400 g)
column eluting with a CHCl3/MeOH (80:0, 80:1, 40:1, 20:1, 10:1, 5:1, 2:1, 1:1, 0:1, each 1.5 L)
gradient system to give fractions 1–9. Fraction 3 (0.15 g) was chromatographed on silica gel column
(Φ 1 × 10, 200–300 mesh, 8 g) eluting with PE/EtOAc/Et2NH (1:1:0.002, 0.1 L) to give compound 2
(6.0 mg). Huperzinine (5, 900 mg) was recrystallized in MeOH from fraction 4 (1.4 g). Fraction 5 (0.3 g)
was subjected to silica gel column chromatography (Φ 1.5 × 15, 200–300 mesh, 30 g) eluting with
CHCl3/EtOAc/MeOH/Et2NH (7:7:1:0.015, 0.15 L) to yield casuarinine A (6) (20.6 mg), N-demethyl-
huperzinine (7) (15.6 mg) and huperzine C (8) (10.3 mg). Fraction 6 (0.3 g) was rechromatographed on
silica gel (Φ 1.5 × 15, 200–300 mesh, 30 g) eluting with EtOAc/MeOH/Et2NH (10:1:0.01, 0.13 L) to
give huperzine B (9) (15.7 mg). Fraction 7 (0.5 g) was chromatographed on a silica gel column (Φ
2 × 10, 200–300 mesh, 35 g) eluted with CHCl3/EtOAc/MeOH, (5:5:1) to afford three fractions (7A–C).
Then fraction 7B was purified by reversed-phase preparative HPLC (MeOH/H2O, 28:72, v/v; flow rate,
2.0 mL/min) to yield compound 3 (1.2 mg, tR = 16.9 min), casuarinine E (10) (2.8 mg, tR = 20.9 min),
carinatumin B (11) (18 mg, tR = 18.6 min) and huperzine D (12) (19.2 mg, tR = 35.3 min). The BuOH
extract (2.3 g) was subjected to an HP-20 column (Φ 5 × 20, 0.8 kg) eluting with a H2O/MeOH (10:0,
9:1, 8:2, 7:3, 6:4, 5:5, each 2 L) gradient system to give fractions 1–6. Fraction 2 (0.15 g) was
chromatographed on a reversed-phase column (Φ 1 × 15, 30 g) using ODS and was eluted with 20%
MeOH (fractions 2A–B). Then fraction 2B was purified by reversed-phase preparative HPLC
(MeOH/H2O, 16:84, v/v; flow rate, 2.0 mL/min) to yield lycoparin C (13) (3.1 mg, tR = 8.9 min),
lycoparin A (14) (3.8 mg, tR = 11.2 min), compound 1 (1.8 mg, tR=19.3 min) and compound 4 (6.3 mg,
tR = 12.6 min).
3.4. Spectral Data
16-Hydroxyhuperzine B (1). Colorless gum; [α]2D5 = −70 (c 0.1, MeOH); IR (KBr) νmax: 3340, 2919,
2850, 1654, 1611, 1464, 1115, 752 cm−1; UV (MeOH) λmax (log ε): 227 (3.90), 306 (3.81) nm;
1H-NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD, 150 MHz) data: see Tables 1 and 2; HRESIMS
m/z 273.1600 [M+H]+ (273.1598 calcd. for C16H21N2O2).
N-Methyl-11-acetoxyhuperzine B (2). Yellow powder; [α]2D5 = −50 (c 0.1, MeOH); IR (KBr) νmax: 3373,
2919, 2850, 1658, 1598, 1456, 1095, 761 cm−1; UV (MeOH) λmax (log ε): 226 (3.86), 306 (3.79) nm;
13
1H-NMR CDCl3, 400 MHz) and C-NMR (CDCl3, 100 MHz) data: see Tables 1 and 2; HRESIMS
m/z 329.1863 [M+H]+ (329.1860 calcd. for C19H25N2O3).
8, 15-Dihydrolycoparin A (3). White powder; [α]2D5 = −120 (c 0.1, MeOH); IR (KBr) νmax: 3384, 2919,
2851, 1658, 1609, 1462, 1119, 796 cm−1; UV (MeOH) λmax (log ε): 227 (3.85), 306 (3.75) nm;
1H-NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD, 150 MHz) data: see Tables 1 and 2; HRESIMS
m/z 303.1709 [M+H]+ (303.1703 calcd. for C17H23N2O3).
(7S,12S,13R)-Huperzine D-16-O-β-D-glucopyranoside (4). Colorless powder; [α]2D5 = −70 (c 0.2,
MeOH); IR (KBr) νmax: 3385, 2919, 2851, 1658, 1602, 1459, 1093, 761 cm−1; UV (MeOH) λmax (log ε):

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227 (3.81), 305 (3.69) nm; 1H-NMR (CD3OD, 400 MHz) and 13C-NMR (CD3OD, 100 MHz) data: see
Tables 1 and 2; HRESIMS m/z 449.2288 [M+H]+ (449.2282 calcd. for C23H33N2O7).
3.5. Hydrolysis of Compound 4
Compound 4 (5 mg) was dissolved in 9% dry HCl-Methanol (2 mL) at 80 ºC for 3 h. After
neutralization with NaHCO3, the mixture was evaporated. The residue was resuspended in H2O and
then filtered to yield compound 1 (1.2 mg). The sugar components in the filtrate were identified by
TLC (on silica gel, developed with CHCl3/MeOH = 5:1) as D-(+)-glucose (Rf 0.40) by comparison
with an authentic sample.
3.6. Biological Material
AChE (EC3.1.1.7, Sigma product no. C2888), acetylthiocholine iodide (ATCI), 5,5'-dithiobis
[2-nitrobenzoic acid] (DTNB) and huperzine A (purity 98%) were purchased from Sigma (St. Louis,
MO, USA).
3.7. Assay of AChE Inhibition
The fourteen compounds were tested for AChE inhibitory activities by the modified Ellman’s
method in 96-well microplates [19,20]. Briefly, 0.1 M sodium phosphate buffer (140 µL, pH = 8.0),
sample solution (20 µL) and enzyme solution (15 µL) were mixed and incubated at 4 °C for 20 min.
Ten µL of 0.01 M DTNB was added and the reaction was then started by adding 0.075 M ATCI (10 µL).
After incubating the reaction solution at 37 °C for 20 min, the optical densities were measured in a
96-well plate reader at 405 nm immediately. A blank positive control was set up by adding 20 µL
huperinze A (0.100 µg/mL in phosphate buffered saline) instead of 20 µL sample solution. Blanks
were set up by adding 20 µL buffer solutions instead of 20 µL sample solution. Experiment control
was set up by adding 15 µL buffer solutions instead of 15 µL enzyme solution in order to deduct the
sample background. All reactions were carried out thrice. The inhibition rate (%) was calculated by the
following equation:
Inhibition% = [(Blank − Blank positive control) − (Experiment − Experiment
control)]/(Blank − Blank positive control) × 100%
The concentration of test samples that inhibited the hydrolysis of acetylthiocholine by 50% (IC50)
was determined by monitoring the effect of increasing concentrations of these samples in assays on the
inhibition values. Huperzine A was chosen as the positive control, with an IC50 value of 74 nmol.
4. Conclusions
A bioactivity-guided separation of the alkaloidal extracts of L. casuarinoides led to four new
(compounds 1–4) and ten known (compounds 5–14) lycodine-type alkaloids. Their isolation,
purification, and structural determination are reported. Within the series of lycodine-type alkaloids
tested for AChE inhibiting activity, N-demethylhuperzinine (7), huperzine C (8), huperzine B (9) and
lycoparin C (13) showed significant AChE inhibitory activity, with IC50 values of 1.9, 0.6, 20.2 and

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23.9 μM, respectively, and the new compound 1 exhibited moderate inhibitory activity with an IC50
value of 87.3 μM. The structure-activity relationships disclosed that the amino group at C-13, methyl
of the three-carbon bridge ring and the exocyclic double bond were all required for the anti-AChE
activity of these lycodine-type alkaloids. These findings indicated that the promising AChE inhibitory
activity of N-demethylhuperzinine (7), huperzine C (8), huperzine B (9) and lycoparin C (13) could
stimulate future development of new anti-AD agents.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/7/9999/s1.
Acknowledgments
The research work was financially supported by National Natural Science Foundation of China
(No. 31270396, No. 21002046).
Author Contributions
KG, JJC and DBZ designed research; DBZ, QYS and LZ performed research and analyzed the data;
DBZ, KG, and JJC wrote the paper. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Jann, M.W. Preclinical pharmacology of metriphonate. Pharmacotherapy 1998, 18, 55–67.
2. Adams, R.L.; Crai, P.L.; Parsons, O.A. Neuropsychology of dementia. Neurol. Clin. 1984, 4,
387–405.
3. Aisen, P.S.; Davis, K.L. The search for disease-modifying treatment for Alzheimer’s Disease.
Neurology 1997, 48, 35–41.
4. Lahiri, D.K.; Farlow, M.R.; Greig, N.H.; Sambamurti, K. Current drug targets for alzheimer’s
disease treatment. Drug Dev. Res. 2002, 56, 267–281.
5. Munoz-Torrero, D.; Camps, P. Dimeric and hybrid antialzheimer drug candidates. Curr. Med.
Chem. 2006, 13, 399–422.
6. Pang, Y.P.; Quiram, P.; Jelacic, T.; Hong, F.; Brimijoin, S. Highly potent, selective, and low cost
bis-tetrahydroaminacrine inhibitors of acetylcholinesterase-steps toward novel drugs for treating
alzheimer’s disease. J. Biol. Chem. 1996, 271, 23646–23649.
7. Jiangsu New Medical College. Dictionary of the Traditional Chinese Medicine; Shanghai Science
and Technology Press: Shanghai, China, 1986; p. 2406.
8. Hirasawa, Y.; Kato, E.; Kobayashi, J.; Kawahara, N.; Goda, Y.; Shiro, M.; Morita, H. Lycoparins
A–C, new alkaloids from Lycopodium casuarinoides inhibiting acetylcholinesterase. Bioorg. Med.
Chem. 2008, 16, 6167−6171.

Molecules 2014, 19
10010
9. Yin, S.; Fan, C.Q.; Wang, X.N.; Yue, J.M. Lycodine-type alkaloids from Lycopodium
casuarinoides. Helv. Chim. Acta 2006, 89, 138−143.
10. Shen, Y.C.; Chen, C.H. Alkaloids from Lycopodium casuarinoides. J. Nat. Prod. 1994, 57,
824−826.
11. Liu, J.S.; Huang, M.F. The alkaloids huperzines C and D and huperzinine from Lycopodiastrum
casuarinoides. Phytochemistry 1994, 37, 1759−1761.
12. Liu, J.S.; Zhu, Y.L.; Yu, C.M.; Zhou, Y.Z.; Han, Y.Y.; Wu, F.W.; Qi, B.F. The structures of
huperzine A and B,two new alkaloids exhibiting marked anticholinesterase activity. J. Can. Chem.
1986, 64, 837−839.
13. Tang, Y.; Fu, Y.; Xiong, J.; Li, M.; Ma, G.L.; Yang, G.X.; Wei, B.G.; Zhao, Y.; Zhang, H.Y.;
Hu, J.F. Casuarinines A−J, lycodine-type alkaloids from Lycopodiastrum casuarinoides. J. Nat. Prod.
2013, 76, 1475–1484.
14. Matsuda, H.; Kageura, T.; Inoue, Y.; Morikawa, T.; Yoshikawa, M. Absolute stereostructures
and syntheses of saussureamines A, B, C, D and E, amino acid-sesquiterpene conjugates with
gastroprotective effect, from the roots of Saussurea lappa. Tetrahedron 2000, 56, 7763–7777.
15. Choo, C.Y.; Hirasawa, Y.; Karimata, C.; Koyama, K.; Sekiguchi, M.; Kobayashi, J.; Morita, H.
Carinatumins A-C, new alkaloids from Lycopodium carinatum inhibiting acetylcholinesterase.
Bioorg. Med. Chem. 2007, 15, 1703−1707.
16. Ma, X.Q.; Gang, D.R. The lycopodium alkaloids. Nat. Prod. Rep. 2004, 21, 752–772.
17. Sha, C.K.; Lee, F.K.; Chang, C.J. Tandem radical cyclizations initiated with α-carbonyl radicals:
First total synthesis of (+)-paniculatine. J. Am. Chem. Soc. 1999, 121, 9875–9876.
18. Williams, J.P.; Friedrich, D.; Pinard, E.; Roden, B.A.; Paquette, L.A.; Laurent, D.R.S. Total
synthesis of the lycopodium alkaloids magellanine and magellaninone by three-fold annulation of
2-cyclopentenone. J. Am. Chem. Soc. 1994, 116, 4689–4696.
19. Ellman, G.L.; Courtney, K.D.; Andres, V.; Featherstone, R.M. A new and rapid colorimetric
determination of acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 88–95.
20. Orhan, I.; Sener, B.; Choudhary, M.I.; Khalid, A. Acetylcholinesterase and butyrylcholinesterase
inhibitory activity of some Turkish medicinal plants. J. Ethnopharmacol. 2004, 91, 57–60.
21. Feng, S.; Wang, Z.F.; He, X.C.; Zheng, S.X.; Xia, Y.; Jiang, H.L.; Tang, X.C.; Bai, D.L.
Bis-huperzine B: Highly Potent and Selective Acetylcholinesterase Inhibitors. J. Med. Chem.
2005, 48, 655–657.
22. Olafsdόttir, E.S.; Halldorsdottir, E.S.; Pich, N.M.; Omarsdottir, S. Lycopodium Alkaloids:
Pharmacology. In Natural Products; Ramawat, K.G., Merillon, J.M.; Eds.; Springer: Berlin,
Heidelberg, Germany, 2013; pp. 1239–1262.
Sample Availability: Samples of all compounds are available from corresponding author.
© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
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