Synthesis and structure-Activity relationship of nuciferine derivatives as potential acetylcholinesterase inhibitors

Article abstract of DOI:10.1007/s00044-013-0905-9

Acetylcholinesterase inhibitors (AChEIs) are currently the best available pharmacotherapy for Alzheimer patients, but because of bioavailability issues, there is still great interest in discovering better AChEIs. The aporphine alkaloid is an important class of natural products, which shows diverse biological activity, such as acetylcholinesterase inhibitory activity. To find new lead AChEIs compounds, eight aporphine alkaloids were synthesized by O-dealkylation, N-dealkylation, and ring aromatization reactions using nuciferine as raw material. The anti-Acetylcholinesterase activity of synthesized compounds was measured using modified Ellman's method. The results showed that some synthesized compounds exhibited higher affinity to AChE than the parent compound nuciferine. Among these compounds, 1,2-dihydroxyaporphine (2) and dehydronuciferine (5) were the most active compounds (IC₅₀ = 28 and 25 μg/mL, respectively). Preliminary analysis of structure-Activity relationships suggested that aromatization of the C ring, the presence of the alkoxyl group at C1 and the hydroxy group at C2 position as well as the alkyl substituent at the N atom were favorable to the acetylcholinesterase inhibition. Molecular docking was also applied to predict the binding modes of compounds 1, 2, and 9 into the huperzine A binding site of AChE.

Full text of DOI:10.1007/s00044-013-0905-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0905-9
ORIGINAL RESEARCH
Synthesis and structure–activity relationship of nuciferine
derivatives as potential acetylcholinesterase inhibitors
•
Zhongduo Yang Zhuwen Song Weiwei Xue
•
•
•
Jie Sheng Zongmei Shu Yin Shi
•
•
•
Jibei Liang Xiaojun Yao
Received: 8 July 2013 / Accepted: 21 December 2013
Ó Springer Science+Business Media New York 2013
Abstract Acetylcholinesterase inhibitors (AChEIs) are
currently the best available pharmacotherapy for Alzhei-
mer patients, but because of bioavailability issues, there is
still great interest in discovering better AChEIs. The
aporphine alkaloid is an important class of natural pro-
ducts, which shows diverse biological activity, such as
acetylcholinesterase inhibitory activity. To find new lead
AChEIs compounds, eight aporphine alkaloids were syn-
thesized by O-dealkylation, N-dealkylation, and ring aro-
matization reactions using nuciferine as raw material. The
anti-acetylcholinesterase activity of synthesized com-
pounds was measured using modified Ellman’s method.
The results showed that some synthesized compounds
exhibited higher affinity to AChE than the parent com-
pound nuciferine. Among these compounds, 1,2-dihydr-
oxyaporphine (2) and dehydronuciferine (5) were the most
active compounds (IC50 = 28 and 25 lg/mL, respec-
tively). Preliminary analysis of structure–activity relation-
ships suggested that aromatization of the C ring, the
presence of the alkoxyl group at C1 and the hydroxy group
at C2 position as well as the alkyl substituent at the N atom
were favorable to the acetylcholinesterase inhibition.
Molecular docking was also applied to predict the binding
modes of compounds 1, 2, and 9 into the huperzine A
binding site of AChE.
Keywords Aporphine alkaloids Á
Acetylcholinesterase inhibitors Á
Structure–activity relationships
Introduction
Alzheimer’s disease (AD), the most common form of
dementia in the elderly, is a progressive neurodegenerative
disorder characterized by cognitive impairments and
memory loss. It has been reported that approximately 10 %
of the population over the age of 65 years is affected by
AD (Racchi et al., 2004). To date, although the etiology of
AD is not completely known, two diverse hypotheses, the
amyloid hypothesis and the cholinergic hypothesis, play
significant roles in the pathophysiology of the disease
(Scarpini et al., 2003). The former indicates that acetyl-
cholinesterase (AChE) can accelerate formation of
b-amyloid fibrils which can lead to AD (Hardy and Selkoe,
2002). The latter suggests that decreased levels of acetyl-
choline in the hippocampus and cortex is one of the most
important causes of AD. Therefore, some acetylcholines-
terase inhibitors (AChEIs) are effective agents for the
treatment of AD symptoms (Lahiri et al., 2002). Recent
research demonstrated that AChEIs not only alleviate the
cognitive defects of AD patients by elevating acetylcholine
(ACh) levels, but also act as disease modifying agents by
preventing the first step of AD, the assembly of b-amyloid
peptides (Ab) into amyloid plaques (Munoz and Camps,
Z. Yang (&) Á Z. Song Á J. Sheng Á Z. Shu Á J. Liang
School of Life Science and Engineering, Lanzhou University
of Technology, Lanzhou 730050, Gansu,
People’s Republic of China
e-mail: yangzhongduo@126.com
W. Xue Á X. Yao
Department of Chemistry, Lanzhou University,
Lanzhou 730000, People’s Republic of China
Y. Shi
Key Lab of New Animal Drug Project, Gansu Province, Key Lab
of Veterinary Pharmaceutical Development, Ministry of
Agriculture, Lanzhou Institute of Husbandry and Pharmaceutical
Sciences of CAAS, Lanzhou 730050,
People’s Republic of China
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Med Chem Res
nuciferine derivative, isolated from Nelumbo nucifera, as a
potent AChEI, was reported in our previous paper (Yang
et al., 2012). To explore the structure–activity relationships
of this type of compounds against AChE, we synthesized
a series of nuciferine derivatives 1–8 (Scheme 1) by
O-dealkylation, N-dealkylation, as well as aromatization
and evaluated their activity against AChE.
Results and discussion
Chemistry
For preparing the target derivatives 1–8 from nuciferine,
we followed the synthetic route outlined in Scheme 1.
First, the regioselective O-demethylation of nuciferine at
140 °C with dibenzyl diselenide in the presence of sodium
borohydride (NaBH4) in dry N,N-dimethylformamide
(DMF) gave 1-hydroxy-2-methoxyaporphine (1) in 76 %
yield (Ahmad et al., 1977). Next, O-demethylation
of 1 with AlBr3 in methyl cyanide at 60 °C gave
Fig. 1 The structures of compounds 9–12
2006). This discovery stimulated great interest in the dis-
covery of new AchEIs.
The aporphine alkaloid is an important and typical class
of natural products. N-methylasimilobine (10) (Fig. 1), a
Scheme 1 Synthesis of
derivatives 1–8
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Med Chem Res
Table 1 The inhibitory ratio and IC50 values of all compounds
Name
Inhibitory ratio % (final
concentration, 0.103 mg/mL)
IC50 (lg/mL)
Nuciferine
25.0 1.7
18.5 1.2
72.9 2.1
61.7 1.5
37.3 2.2
79.7 2.0
19.9 1.6
2.0 1.8
–
1
–
Fig. 2 The mechanism of forming 7-hydroxy-dehydronuciferine 3
2
28 0.4
3
73 0.4
4
–
1,2-dihydroxyaporphine (2) in 63 % yield (Horie et al.,
1995). 7-Hydroxy-dehydronuciferine (3) was obtained in
61 % yield by reacting nuciferine with I2 and CaO in the
mixed tetrahydrofuran (THF) and methanol solution. It is
worth mentioning that the obtainment of 3 was accidental.
Originally, we planned to remove the methyl at the N-atom
to obtain compound 6 using CaO and I2 reported by Kirk
(Acosta et al., 1994). Unfortunately, 6 was not successfully
obtained but resulted in a new product 3 identified by MS
and NMR. The proposed mechanism (Fig. 2) was that the
initial step was the formation of a benzyl radical (II) in the
presence of I2 and CaO, instead of a vinyl amine 5 through
an iminium cation. Further oxidation would transform II
into a ketone III which isomerizes to form the enol IV.
Treatment of nuciferine with 30 % H2O2 in methanol at
room temperature furnished nuciferine N-oxide (4) in 90 %
yield. Reaction of nuciferine with I2 and NaOAc in THF
gave dehydronuciferine (5) in 50 % yield (Cava et al.,
1972). Reduction of 4 with hydrated ferrous sulfate (FeS-
O4Á7H2O) in methanol at room temperature afforded
nornuciferine (6) in 40 % yield (Huang et al., 2002) which
was executed via O-demethylation using the same method
of synthesizing 1 to yield 1-hydroxy-2-methoxynor-
aporphine (7) (73 % yield). However, synthesis of 1,2-di-
hydroxynoraporphine (8) was unsuccessful using many
O-demethylation methods such as the use of dimethyl
sulfide (Me2S), methanesulfonic acid (MeSO3H) (Couture
et al., 1996), HBr–HOAc (Zou et al., 2008), AlBr3 (Horie
et al., 1992), etc. as catalysts. Finally, compound 8 was
obtained in 70 % yield using 40 % HBr as catalyst in the
presence of sodium iodide in CF3COOH solution at 140 °C
high temperature.
5
25 0.2
6
–
7
–
8
28.1 2.3
59.0 1.2
94.0 2.1
98.1 0.5
–
9
86 0.3
1.5 0.2
0.017 0.002
10
Huperzine A
- The IC50 values were not measured for the corresponding
compounds
N-methylasimilobine (10) reported in our group (Yang
et al., 2012), the activities obviously increased in com-
parison to the parent compound nuciferine. The pre-
liminary structure–activity relationships could be drawn
from the results as follows:
(1) The O-dealkylation of C1 decreased the AChE
inhibition. For example, the inhibition ratio of
O-demethylated product 1 (18.5 %) was lower than
that of the parent compound nuciferine (25.0 %). This
influence was also reflected between the compounds 2
and 10, the IC50 values of them were 28 and 1.5 lg/
mL, respectively. Moreover, this conclusion could
also be summarized by comparing the inhibition ratio
of 6 (19.9 %) and 7 (2.0 %). This indicated that the
presence of the methoxy group at C1 is favorable to
increasing the AChE inhibition.
(2) The hydroxyl group at the C2 position plays an
important role for AChE inhibition. For example,
compound 2, sharing a hydroxyl at C2 position, was
obtained from 1 by an O-dealkylation reaction. The
activity of the former was approximately fourfold
higher than the latter (the inhibition ratio was 72.9
and 18.5 %, respectively). This influence was also
reflected between compounds 7 and 8. This result was
consistent with the previous molecular docking
results reported by our group, which showed that
the hydroxyl at C2 position of N-methylasimilobine
(10) formed a H-bond with the carbonyl of Ser 293 in
AchE (Yang et al., 2012).
Biological activities and structure–activity relationship
The AChE inhibitory activity of nuciferine derivatives was
assayed according our previously described method (Yang
et al., 2012) using huperzine A as reference compound.
Table 1 shows the activity of all compounds expressed
either as inhibition ratio or IC50 values. As shown in
Table 1, most of the synthesized compounds exhibited
moderate activity against acetylcholinesterase. Compounds
2 and 5 had higher inhibition than others with IC50 values
of 28 and 25 lg/mL, respectively. Although their activities
were much less than Huperzine A and weaker than
(3) The presence of the N–CH3 is also important. Com-
pound 8, with an unsubstituted NH, failed to inhibit
acetylcholinesterase, compared with compound 2.
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Med Chem Res
Compound 7, a N-demethylated product of 1, had
relatively lower activity than 1. This result was also
consistent with our previous molecular docking results
which demonstrated that the N-atom of compound 10
formed a H-bond with the hydroxyl of Tyr 124 in
AChE. The fact that the inhibitory activity decreased
after N-demethylation reaction could be explained by
the reduction of electron-cloud density on the N-atom.
The cleavage of the methyl group (a relatively stronger
electron-donating group than the H-atom) could
decrease the electron-cloud density on the N-atom
causing reduction of H-bond forming ability and a
decreased affinity between the enzyme and inhibitors.
(4) The aromatization of the C-ring significantly
increases the inhibitory activity. For example, after
the C-ring of nuciferine was aromatized to form
compounds 3 and 5, the inhibition ratio of the latter
two were increased to 61.7 and 79.7 %, respectively.
The aromatization of the C-ring could lead to an
increase of p electron-cloud density and the aroma-
ticity of the molecule, which reinforces the p–p
interaction of inhibitor molecules with the aromatic
amino acid residues of AChE.
Fig. 3 The predicted binding model of compound 3 to AChE
The above analysis suggests that the aromatization of
the C-ring and the alkoxyl of C1, the hydroxyl at the C2
position and the alkyl substituent on the N-atom were very
important to maintain the AChE inhibition of nuciferine
derivatives. In our future research, we will retain these
pharmacophores to insure no loss of inhibitory activity.
The docking program Glide (Glide, version 5.5) was
applied to predict the binding modes of the representative
compounds (nuciferine, huperzine A, and compounds 1, 2,
and 9) into the huperzine A binding site of AChE. As
shown in Figs. 3, 4, 5, 6, 7, and 8, huperzine A, nuciferine,
compounds 1, 2, and 9 were docked into the active site of
AChE around the residue Trp 84 (Trp 86 residue in the pdd
code: 1C2B). In addition, the calculated binding free
energy of compounds in the molecular docking study was
found to correlate well with its inhibition activity, as shown
in Table 2 and the RMSD for the re-dock study of huper-
Fig. 4 The predicted binding model of compound 1 to AChE
design and synthesis of a series of aporphine alkaloids, and
measurement of their anti-AChE activity. The structure–
activity relationship of these compounds was preliminarily
analyzed and determined. Variations of activity between
the different compounds tested suggest promising future
structural modifications for activity improvement.
˚
zine A is 0.79 A. These results could explain the observed
differences in inhibitory activity. The aporphine alkaloids
is an important class of natural products, which shows
diverse biological activity, such as antiplatelet aggregation
(Kuo et al., 2001), anti-poliovirus (Boustie et al., 1998),
etc. However, except for a strong AChEI 10 reported by
our group (Yang et al., 2012) and two weak AChEIs 11, 12
(Fig. 1) reported by Mankee et al. (2006), aporphine
alkaloids with anti-AChE activity have been rarely
obtained by isolation or synthesis. Moreover, this is the
first report of the structure–activity relationships of this
type of alkaloid against AChE. This paper describes the
Experimental
Materials and measurements
Acetylcholinesterase (EC 3.1.1.7, Sigma product NO.
C2888), huperzine A were purchased from Sigma (St. Louis,
123

Med Chem Res
Fig. 5 The predicted binding model of compound 2 to AChE
Fig. 7 The predicted binding model of nuciferine to AChE
Fig. 8 Superposition of the crystal structure (pink) and the confor-
mation (cyan) from docking result for huperzine A at the active site of
AChE (Color figure online)
Fig. 6 The predicted binding model of compound 9 to AChE
MO, USA). Sodium borohydride, N,N-dimethylformamide,
dimethyl sulfide, methanesulfonic acid, aluminum bromide,
and sodium iodide were purchased from Aladdin-Reagent
Co., Ltd. (Shanghai, China). Silica gel G plates and silica gel
(200–300 mesh) were obtained from Qingdao Haiyang
Chemical Co., Ltd. (Qingdao, China). The leaves of N. nu-
cifera were purchased from Huanghe Herb Market in
Lanzhou. The herb was identified by Associate Professor Lin
Yang who majored in plant classification, School of Life
Science and Engineering, Lanzhou University of Technol-
ogy, Lanzhou. China. Other reagents were of analytical
grade. Melting points were recorded on a Mel-Temp II
Table 2 The docking score of the studied compounds by Glide
program
Inhibitors
Binding free energy (Kcal/mol)
Nuciferine
-7.58
-7.54
-8.39
-8.07
-8.62
1
2
9
Huperzine A
1
melting-point apparatus. H NMR and 13C NMR spectra
shifts are expressed in parts per million (ppm) and coupling
constants in Hz. Mass spectra (ESI–MS, positive) was
recorded on a Bruker Daltonics esquire 6000 spectrometer.
were recorded on a Brucker Advance-AM-400 MHz spec-
trometer with TMS as the internal standard. Proton Chemical
123

Med Chem Res
Extraction and isolation of (6aR)-1,2-dimethoxy-6-methyl-
5,6,6a,7-tetrahydro-4H-dibenzo[de, g]quinoline
(nuciferine)
NMR (125 MHz, CDCl3) d: 147.8 (C-1), 120.5 (C-1a),
129.9 (C-1b), 143.4 (C-2), 111.2 (C-3), 127.4 (C-3a), 28.8
(C-4), 54.6 (C-5), 64.3 (C-6a), 36.4 (C-7), 137.5 (C-7a),
129.2 (C-8), 128.9 (C-9), 127.5 (C-10), 125.0 (C-11), 133.7
(C-11a), 56.6 (C-2, OMe), 44.7 (N–CH3). ESI MS: 282.3
[M?H]? (Barolo et al., 2006).
The leaves of N. nucifera (10 kg) were crushed into a
powder and extracted with 95 % ethanol (30 L 9 2, 80 °C,
2 h). The supernatant was separated from the solid residue
by paper filtration and evaporated to dryness under reduced
pressure. The residue was suspended in water (1 L). The
suspension solution was adjusted to pH = 2 by the addition
of HCl (6 M), which would be filtered after one night. The
filtrate solution was basified to pH = 11 with NaOH (1 M)
and then extracted with chloroform (l L 9 4) and evapo-
rated to dryness to obtain total alkaloid (11.94 g). The total
alkaloid extract was repeatedly isolated and purified using
MCI–GEL and recrystallization to obtained nuciferine
(5.153 g). M.p. 165–166 °C. 1H NMR (400 MHz, CDCl3),
d: 2.55 (s, 3H, N–CH3), 2.50 (m, 1H), 2.59–2.71 (m, 2H),
3.02–3.20 (m, 4H), 3.65 (s, 3H, O–CH3), 3.88 (s, 3H, O–
CH3), 6.63 (s, 1H), 7.23-7.33 (m, 3H), 8.36 (d, J = 8.0 Hz,
1H). 13C NMR (125 MHz, CDCl3) d:144.4 (C-1), 125.9
(C-1a), 129.0 (C-1b), 151.7 (C-2), 112.0 (C-3), 127.9 (C-
3a), 28.4 (C-4), 52.3 (C-5), 71.0 (C-6a), 34.6 (C-7),136.0
(C-7a), 128.1 (C-8), 127.3 (C-9), 126.9 (C-10), 126.7 (C-
11), 131.4 (C-11a), 43.9 (N–CH3), 60.0 (C-1, OMe), 55.7
(C-2, OMe) (Guinaudeau et al., 1979).
(6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-
dibenzo[de,g]quinolin-1,2-diol (2, 1,2-dihydroxyapor
phine)
Compound 2 was prepared by the following method: To a
solution of anhydrous AlBr3 (1.0171 g, 3.8 mmol) in
10 mL dry MeCN was added compound 1 (0.182 g,
0.6 mmol) under N2. The mixture was heated 70 °C for
48 h. The solvent was evaporated under reduced pressure.
The residue was dissolved by acetone and collected the
filtrate by filter. Purification by recrystallization from acr-
tone/chloroform gave the desired 2 (0.104 g, 65 %) as
white amorphous powder. M.p. 162–164 °C. 1H NMR
(400 MHz, CD3OD), d: 2.83–2.96 (m, 3H), 3.18 (s, 3H, N–
CH3), 3.40–3.47 (m, 2H), 3.76 (dd, J = 12.4, 5.6 Hz 1H),
4.20 (br d, J = 13.6 Hz, 1H), 6.64 (s, 1H), 7.20–7.35 (m,
3H), 7.91 (s, 1H), 8.44 (d, J = 8.0 Hz 1H). 13C NMR
(125 MHz, CD3OD), d: 143.7 (C-1), 121.1 (C-1a), 122.8
(C-1b), 147.0 (C-2), 114.3 (C-3), 122.4 (C-3a), 27.1 (C-4),
54.1 (C-5), 63.4 (C-6a), 34.0 (C-7), 134.4 (C-7a), 129.8 (C-
8), 128.9 (C-9), 128.1 (C-10), 128.1 (C-11),133.7 (C-11a),
41.9 (N–CH3). ESI MS: 268.2 [M?H]? (Kelly et al.,
1976).
Synthesis
(6aR)-2-methoxy-6-methyl-5,6,6a,7-tetrahydro-4H-
dibenzo[de,g]quinolin-1-ol (1, 1-hydroxy-2-
methoxyaporphine)
1,2-dimethoxy-7-hydroxy-6-methyl-5,6-dihydro-4H-
dibenzo[de,g]quinoline (3)
Compound 1 was prepared by the following method: To a
stirred solution of dibenzyl diselenide (0.22 g, 0.7 mmol)
in 10 mL of anhydrous N,N-dimethylformamide (DMF)
was added excess NaBH4 (0.3 g, 7.9 mmol) under N2.
After 20 min, nuciferine (0.28 g, 0.95 mmol) in 15 mL of
DMF was introduced. The mixture was refluxed under
nitrogen until all the alkaloid was consumed (monitored by
TLC). The mixture was cooled and solvent was evaporated
under reduced pressure. The residue was taken up in 5 %
H2SO4 and the nonalkaloidal components were extracted
with benzene. The acid solution was basified with 10 %
NH4OH (pH = 9) and exhaustively extracted with chlo-
roform. The combined chloroform extracts were dried
(anhydrous Na2SO4) and evaporated. The product was
further purified by column chromatography (chloroform–
methanol–triethylamine, 40:1:0.001, v/v/v) to give 1
(0.204 g, 76 %) as colorless needle crystals. M.p.
161–163 °C. 1H NMR (400 MHz, CDCl3), d: 2.58 (s, 3H),
2.48–2.68 (m, 3H), 2.88–3.11 (m, 4H), 3.82 (s, 3H), 6.54
(s, 1H), 7.18–7.31 (m, 3H), 8.35 (d, J = 8.0 Hz, 1H). 13C
Compound 3 was prepared by the following method: A
mixture of nuciferine (0.095 g, 0.3 mmol) and calcium
oxide (0.146 g, 2.6 mmol) in THF (2 mL) and methanol
(2 mL) was chilled in an ice-bath. Iodine (0.164 g,
0.6 mmol) in THF (1 mL) was added. The mixture was
stirred at 0 °C for 2.5 h. The mixture was filtered and the
filtrate was evaporated under reduced pressure. Purification
by flash chromatography on a silica–gel column (chloro-
form–methanol–triethylamine, 30:1:0.001, v/v/v) afforded
3 (0.057 g, 61 %) as colorless needle crystals. M.p.
258–260 °C. 1H NMR (400 MHz, CDCl3), d: 2.26 (s, 3H),
2.90 (m, 1H), 3.13 (m, 1H), 3.40 (m, 2H), 3.99 (s, 3H), 4.07
(s, 3H), 7.15 (s, 1H), 7.23 (t, J = 8.0 Hz, 1H), 7.34 (d,
J = 8 Hz, 1H), 7.42 (t, J = 8 Hz, 1H), 9.70 (d,
J = 8.0 Hz, 1H). 13C NMR (125 MHz, CDCl3) d: 145.7
(C-1), 126.2 (C-1a), 121.5 (C-1b), 151.0 (C-2), 112.3 (C-
3), 131.1 (C-3a), 27.0 (C-4), 50.2 (C-5), 143.4 (C-6a),
126.1 (C-7), 126.9 (C-7a), 127.9 (C-8), 123.7 (C-9), 126.2
(C-10), 126.2 (C-11),134.8 (C-11a), 41.6 (N–CH3), 60.0
123

Med Chem Res
(C-1, OMe), 58.5 (C-2, OMe). ESI MS: 310.2 [M?H]?
(Wafo et al., 1999).
(6aR)-1,2-dimethoxy-5,6,6a,7-tetrahydro-4H-
dibenzo[de,g]quinoline (6, nornuciferine)
Compound 6 was prepared by the following method: To a
stirred solution of 4 (0.31 g, 1 mmol) in 2 mL methanol
was added FeSO4Á7H2O (0.304 g, 2 mmol) at 0 °C. The
reaction mixture was allowed to warm in the room tem-
perature and stir for 12 h. A solution of saturated aqueous
NaHCO3 (10 mL) was added to the reaction mixture and
the suspension was stirred at room temperature for 20 min
and then extracted with chloroform (100 mL 9 3). The
combined chloroform layer was washed with water
(50 mL 9 3), brine and dried over Na2SO4. After removal
of the organic solvent under reduced pressure, the residue
was purified with flash chromatograph (eluting with chlo-
roform–methanol–triethylamine, 10:1:0.001, v/v/v) to gave
6 (0.112 g, 40 %) as colorless needle crystals. M.p.
(6S,6aR)-1,2-dimethoxy-6-methyl-6-oxo-5,6,6a,7-
tetrahydro-4H-dibenzo[de,g]quinoline (4, nuciferine
N-oxide)
Compound 4 was prepared by the following method: To a
solution of nuciferine (0.295 g, 1 mmol) in 15 mL MeOH
was added 7.5 mL 30 % H2O2 at room temperature. After
the starting material had been consumed (ca 24 h), as
monitored by TLC, the reaction mixture was extracted with
CHCl3 (200 mL 9 3). The CHCl3 layer after being washed
with water (50 mL 9 3) and dried over Na2SO4 was con-
densed under reduced pressure to give the residue, which
was purified on a silica–gel column (chloroform–metha-
nol–triethylamine, 30:1:0.001, v/v/v) to give 4 (0.279 g,
90 %) as green oil. 1H NMR (400 MHz, CDCl3), d:
2.82–2.86 (m, 1H), 3.15–3.18 (m, 1H), 3.37–3.44 (m, 1H),
3.59–3.89 (m, 2H), 4.31–4.41 (m, 2H), 3.64 (s, 3H), 3.74
(s, 3H), 3.84 (s, 3H), 6.69 (s, 1H), 7.26–7.33 (m, 3H), 8.35
(d, J = 8.0 Hz, 1H). 13C NMR (125 MHz, CDCl3) d:
146.1 (C-1), 120.2 (C-1a), 125.6 (C-1b), 153.4 (C-2), 110.9
(C-3), 127.9 (C-3a), 24.5 (C-4), 63.6 (C-5), 71.9 (C-6a),
56.3 (N–CH3), 30.0 (C-7), 132.8 (C-7a), 127.9 (C-8), 128.2
(C-9), 128.4 (C-10), 128.3 (C-11), 128.2 (C-11a), 60.3 (C-
1, OCH3), 55.9 (C-2, OCH3). ESI MS: 312.2 [M?H]? (Lu
et al., 1987).
1
128–129 °C. H NMR (400 MHz, CDCl3), d: 2.69–2.78
(m, 3H), 2.83–3.02 (m, 2H), 3.37–3.39 (m, 1H), 3.81–3.88
(m, 1H), 3.67 (s, 3H), 3.89 (s, 3H), 6.65 (s, 1H), 7.20–7.32
(m, 3H), 8.39 (d, J = 8.0 Hz, 1H). 13C NMR (400 MHz,
CDCl3), d: 145.5 (C-1), 126.7 (C-1a), 129.3 (C-1b), 152.6
(C-2), 111.7 (C-3), 129.3 (C-3a),29.6 (C-4), 42.6 (C-5),
53.3 (C-6a), 36.4 (C-7), 135.2 (C-7a), 128.4 (C-8), 127.9
(C-9), 127.6 (C-10), 127.2 (C-11), 132.0 (C-11a), 55.9 60.2
(C-1, OMe), (C-2, OMe). ESI MS: 282.2 [M?H]? (Gu-
inaudeau et al., 1983).
(6aR)-2-methoxy-5,6,6a,7-tetrahydro-4H-
Dibenzo[de,g]quinolin-1-ol (7, 1-hydroxy-2-methoxyn
oraporphine)
1,2-dimethoxy-6-methyl-5,6-dihydro-4H-
dibenzo[de,g]quinoline (5, dehydronuciferine)
Compound 7 was prepared from 0.4 mmol 6 and
0.35 mmol dibenzyl diselenide by the same way of syn-
thesis of 1. Compound 7 as colorless needle crystals,
Compound 5 was prepared by the following method: A
solution of iodine (0.25 g, 1 mmol) in 20 mL THF was
added dropwise during 30 min to a refluxing solution of
nuciferine (0.3 g, 1 mmol) in 10 mL THF containing sus-
pended anhydrous NaOAc (0.3 g, 4 mmol). The stirred
mixture was refluxed gently for an addition 2 h and then
evaporated to dryness. Dissolution of the residue with
CHCl3 (100 mL 9 3) and evaporation of the extract gave
an oil which crystallized from absolute EtOH to give 5
(0.146, 50 %) as colorless needle crystals. M.p.
130–131 °C. 1H NMR (400 MHz, CDCl3), d: 3.09 (s, 3H),
3.28 (t, J = 6.0 Hz, 2H,), 3.38 (t, J = 6.0 Hz, 2H), 3.90 (s,
3H), 4.02 (s, 3H), 6.63 (s,1H), 7.02 (s, 1H), 7.34 (t,
J = 8.0 Hz, 1H), 7.44 (t, J = 8.0 Hz, 1H), 7.65 (d,
J = 8.0 Hz, 1H), 9.46 (d, J = 8.0 Hz, 1H). 13C NMR
(125 MHz, CDCl3) d:145.3 (C-1), 118.4 (C-1a), 125.3 (C-
1b), 151.4 (C-2), 111.2 (C-3), 127.1 (C-3a), 30.5 (C-4),
59.3 (C-5), 123.5 (C-6a), 40.4 (N–CH3), 126.3 (C-7), 134.2
(C-7a), 126.1 (C-8), 124.5 (C-9), 124.4 (C-10), 126.1 (C-
11), 128.5 (C-11a), 56.1 (C-1, OCH3), 50.0 (C-2, OCH3).
ESI MS: 294.1 [M?H]? (Wang et al., 2009).
1
0.068 g was obtained, yield 73 %. M.p. 208–210 °C. H
NMR (400 MHz, CD3OD), d: 2.61–2.68 (m, 2H),
2.79–2.95 (m, 3H), 3.25–3.30 (m, 1H), 3.69–3.74 (m, 1H),
3.86 (s, 3H), 6.66 (s, 1H), 7.12–7.24 (m, 3H), 8.41 (d,
J = 8.0 Hz, 1H). 13C NMR (400 MHz, CD3OD), d: 141.8
(C-1), 119.9 (C-1a), 123.3 (C-1b), 146.5 (C-2), 110.7 (C-
3), 127.6 (C-3a), 28.4 (C-4), 42.8 (C-5), 53.1 (C-6a), 36.6
(C-7), 135.8 (C-7a), 128.4 (C-8), 128.4 (C-9), 126.2 (C-
10), 126.0 (C-11), 132.4 (C-11a), 55.9 (C-2, OMe). ESI
MS: 268.1 [M?H]?(Han et al., 1989; Guinaudeau et al.,
1979).
(6aR)-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-1,2-
diol (8, 1,2-dihydroxynoraporphine)
Compound 8 was prepared by the following method: The
NaI (0.392 g, 2 mmol) was added to 8 mL 40 % HBr.
After 30 min, then, a solution of 7 (0.134 g, 0.5 mmol) in
123

Med Chem Res
5 mL CF3COOH was added to the reaction mixture. The
reaction mixture was heated to 140 °C and refluxed for 5 h.
The reaction mixture was cooled down to 100 °C and
evaporated under reduced pressure. The flash chromatog-
raphy (chloroform–methanol–triethylamine, 10:1:0.001,
v/v/v) was performed to give 8 (0.088 g, 70 %) as colorless
obtained from the Protein Data Bank (PDB code: 1GPK).
Before docking the ligands into the protein active site, the
protein structures were prepared including adding hydro-
gen atoms, assigning partial charges using the OPLS-2005
force field and assigning protonation states. The grid box
was defined by centering on the huperzine A in the AChE
active site. The docking of huperzine A, nuciferine and
compound 1, 2, and 9 into the prepared grid were carried
1
oil. H NMR (400 MHz, CDCl3), d: 2.72–2.76 (m, 2H),
2.88–2.91 (m, 1H), 3.05–3.08 (m, 2H), 3.48–3.52 (m, 1H),
4.05–4.08 (m, 1H), 6.50 (s, 1H), 7.08–7.14 (m, 3H), 8.33
(d, J = 8.0 Hz, 1H). 13C NMR (400 MHz, CDCl3), d:
141.5 (C-1), 122.9 (C-1a), 126.5 (C-1b), 146.6 (C-2), 112.7
(C-3), 127.6 (C-3a), 30.4 (C-4), 42.6 (C-5), 55.1 (C-6a),
39.2 (C-7), 136.8 (C-7a), 128.2 (C-8), 127.6 (C-9), 126.5
(C-10), 127.8 (C-11), 132.3 (C-11a). ESI MS: [M?H]?
(Wang et al., 2009).
¨
out using Glide program (Glide, version 5.5, Schrodinger,
LLC, New York, NY, 2009) with the default parameter, for
which standard precision (SP) mode was selected. The best
scoring poses from the largest cluster were considered for
further structural and interaction analysis.
Conclusion
(6aR)-1-methoxy-5,6,6a,7-tetrahydro-4H-
dibenzo[de,g]quinolin-2-ol (9, asimilobine)
Nuciferine was modified by O-dealkylation, N-dealkyla-
tion, and ring aromatization reactions and eight derivatives
were obtained. The anti-acetylcholinesterase ability of
these compounds was detected at the final concentration of
0.1 mg/mL. The results showed that 1,2-dihydroxy apor-
phine (2) and dehydronuciferine (5) had moderate anti-
cholinesterase activity with IC50 values of 28 and 25 lg/
mL. Analysis of the structure–activity relationship sug-
gested that the alkoxyl of the C1, hydroxyl at the C2
position and the alkyl substituent on the N-atom are the
basic pharmacophore for maintaining the AChE inhibition.
Compound 9 was purchased from Southwest Jiaotong
University and identified by NMR spectroscopy. M.p.
177–179 °C, 1H NMR (400 MHz, CDCl3), d: 3.58 (s, 3H),
6.66 (s, 1H), 7.22–7.30 (m, 3H), 8.29 (d, J = 7.5 Hz, 1H).
13C NMR (400 MHz, CDCl3), d: 143.0 (C-1), 135.0 (C-
1a), 125.5 (C-1b), 148.6 (C-2), 114.7 (C-3), 128.8 (C-3a),
27.2 (C-4), 42.5 (C-5), 53.5 (C-6a), 36.0 (C-7), 134.8 (C-
7a), 128.4 (C-8), 127.9 (C-9), 126.3 (C-10), 127.1 (C-11),
131.3 (C-11a). (Rollinger et al., 2006).
Acknowledgments The authors are grateful to the National Natural
Science Foundation of China (No. 21262022), the Elitist Program of
Lanzhou University of Technology (No. J201303) and Zhejiang
Provincial Natural Science Foundation of China (No. LY12B02005).
The authors also thank Miss Karen Tenney, a researcher of University
of California, Santa Cruz, for revising the grammar errors
Bioassay procedures for AChE inhibition
The procedures of testing AChE inhibiting activity were
same with those described in our previous paper (Yang
et al., 2012).
Conflict of interest The authors declare that there are no conflicts
of interest.
Molecular modeling
In order to further study the structure activity relationship,
we docked five compounds (compounds 1, 2, 9, nuciferine,
and huperzine A) into the active site of AChE. We selected
these five compounds due to their difference in inhibition
activity. The structure of huperzine A was extracted from
the co-crystal structure of AChE/huperzine A (PDB code:
1GPK). The structures of nuciferine and compound 1, 2,
and 9 were constructed using Maestro (Maestro, version
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