Nantenine as an acetylcholinesterase inhibitor: SAR, enzyme kinetics and molecular modeling investigations

Article abstract of DOI:10.3109/14756361003671078

Nantenine, as well as a number of flexible analogs, were evaluated for acetylcholinesterase (AChE) inhibitory activity in microplate spectrophotometric assays based on Ellman'a€s method. It was found that the rigid aporphine core of nantenine is an import

Full text of DOI:10.3109/14756361003671078

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(1): 46–55
Copyright © 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756361003671078
RESEARCH ARTICLE
Nantenine as an acetylcholinesterase inhibitor: SAR, enzyme
kinetics and molecular modeling investigations
Stevan Pecic1, Marie A. McAnuff2, and Wayne W. Harding1
2
1City University of New York Hunter College, Department of Chemistry, 695 Park Avenue, New York, USA, and City
University of New York, Hunter College, Department of Biology, 695 Park Avenue, New York, USA
Abstract
Nantenine, as well as a number of flexible analogs, were evaluated for acetylcholinesterase (AChE) inhibitory activity
in microplate spectrophotometric assays based on Ellman’s method. It was found that the rigid aporphine core
of nantenine is an important structural requirement for its anticholinesterase activity. Nantenine showed mixed
inhibition kinetics in enzyme assays. Molecular docking experiments suggest that nantenine binds preferentially to
the catalytic site of AChE but is also capable of interacting with the peripheral anionic site (PAS) of the enzyme, thus
accounting for its mixed inhibition profile. The aporphine core of nantenine may thus be a useful template for the
design of novel PAS or dual-site AChE inhibitors. Inhibiting the PAS is desirable for prevention of aggregation of the
amyloid peptide Aβ, a major causative factor in the progression of Alzheimer’s disease (AD).
Keywords: Aporphine, AChE, alzheimer’s, peripheral anionic site, ICM
Introduction
deficits. Four compounds are commonly prescribed
for treatment of AD based on this mechanism of action
namely galanthamine (1), tacrine (2), donepezil (3) and
rivastigmine (4) (Figure 1) [13,14]. ese therapeutics
offer short-term improvement in treating symptoms of
the disease with modest benefits in slowing the decline
in behavior, function and cognition associated with the
disease [15–17]. All of the approved AChE inhibitors have
potentially serious side-effects especially with long-term
use, and more efficacious and safer alternatives are desir-
able [18–21].
Alzheimer’s disease (AD) is an incapacitating condition
which afflicts millions of people worldwide. e disease
is especially prevalent among the elderly and is associ-
ated with severe deficits in cognition and memory [1–3].
In the advanced stages of AD, dramatic changes in the
emotional state, psychological well-being and personal-
ity are observed [4,5]. e onset and progression of AD is
thought to be a consequence of the formation of neuritic
plaques via aggregation of the amyloid peptide Aβ (the
amyloid hypothesis of AD) [6–10].
AChE has two main binding domains, the catalytic
site and a peripheral anionic site (PAS) [22,23]. ere is
evidence that AChE facilitates the formation of amyloid
fibrils via a set of amino acids located in the vicinity of the
PAS and that molecules which bind to the PAS prevent
aggregation of the amyloid peptide Aβ [24,25]. Based on
these developments, there has recently emerged a new
therapeutic strategy which involves the use of molecules
Research over the years has supported early hypoth-
eses that a deficit in cholinergic neurotransmission plays
a major role in the neurodegeneration associated with
the disease [11,12]. Acetylcholinesterase (AChE) is an
enzyme which is responsible for the degradation of the
neurotransmitter acetylcholine. Most of the current treat-
ments for AD employ compounds which act as inhibitors
of acetylcholinesterase and thereby improve cholinergic
Address for Correspondence: Wayne Harding, Department of Chemistry, Hunter College, CUNY, 695 Park Avenue, New York, NY 10065, USA.
(Received 04 November 2009; revised 21 December 2009; accepted 12 January 2010)
46

Nantenine as an acetylcholinesterase inhibitor
47
endowed with the ability to bind to the catalytic site and
PAS of AChE simultaneously [26–29]. Such dual-acting
molecules should be more efficacious in decelerat-
ing the progression of the disease via prevention of Aβ
accumulation via PAS inhibition as well as providing the
usual palliative cognitive and memory improvements
associated with the inhibition of the catalytic site of the
enzyme.
Materials and Methods
Chemistry
General Methods and Instrumentation
High resolution electron impact mass spectra (HREIMS)
were obtained using an Agilent 6520 Q-TOF (Santa
Clara, CA)instrument. NMR data were collected on
a Bruker 500 MHz machine (Billerica, MA) with TMS
as internal standard and CDCl3 (Sigma-Aldrinc Inc.,
St. Louis, MO) as solvent unless stated otherwise.
Chemical shift (δ) values are reported in ppm and
coupling constants in Hertz (Hz). Melting points were
obtained on a Mel-Temp capillary electrothermal melt-
ing point apparatus. Reactions were monitored by TLC
(Newark, Delaware, NJ) with Analtech Uniplate silica gel
G/UV 254 (1101D, Dubuque, IA) precoated plates
(0.2 mm). TLC plates were visualised by UV (254 nm)
and by staining with phosphomolybdic acid reagent fol-
lowed by heating. Flash column chromatography was
performed with Silicagel 60 (EMD Chemicals Darmstadt,
Germany, 230-400 mesh, 0.04-0.063 µm particle size).
Aporphines are a family of compounds that are
structurally and biogenetically related to isoquinoline
alkaloids [30]. A number of aporphines have shown
biological activity as dopamine receptor and serotonin
receptor ligands and there are reports of some mem-
bers which have cytotoxic and antimalarial activities
[31–39]. Relatively few aporphines have been evaluated
as AChE inhibitors. In that regard, a recent report indi-
cated that aporphinoids 5 and 6 (Figure 2) ex Corydalis
turtschaninovii Besser (Papaveraceae) show moderate
activity with half maximal inhibitory concentration
(IC50) values of 27.1 and 48.7 μM respectively [40].
Structurally-related synthetic oxoaporphines and iso-
oxoaporphines have been recently reported to pos-
sess anticholinesterase activity, inhibiting the enzyme
non-competitively [41]. e aporphine 7 has also been
reported to have moderate AChE inhibitory activity
(34.5% inhibition at 10−4 M). e structural similarity
between nantenine (8), (an aporphine which we are
currently investigating as a 5-HT2A receptor ligand)
and the aforementioned aporphines prompted us to
investigate the anticholinesterase activity of 8. We
initially compared 8 with the known AChE inhibitor
galanthamine (1) in a TLC anticholinesterase bioau-
tographic assay [42] and found that 8 showed activity.
To understand the mode of inhibition, enzyme kinetics
studies were undertaken. In order to explore the influ-
ence of molecular rigidity on AChE inhibitory activity
of 8, we have synthesised and evaluated a small library
of nantenine congeners. We also performed molecular
docking studies on nantenine in order to better under-
stand its mode(s) of binding to the enzyme.
Synthesis
3-(benzo[d][1,3]dioxol-5-yl)-4,5-dimethoxybenzaldehyde
(11) A mixture of Pd(PPh3)4 (2.35 g, 2.04 mmol) and
commercially available 5-bromoveratraldehyde 10 (5 g,
20.4 mmol) in DME (250 mL) was stirred for 15 min at
20°C under argon. 2M aqueous K2CO3 (71.5 mL, 142.8
mmol) was added to the mixture, followed by boronic acid
9 (6.77 g, 40.8 mmol) in DME. e mixture was refluxed
for 18 h and then cooled to RT. e reaction mixture was
treated with water and ethyl acetate and the layers were
separated. e organic extract was washed sequentially
with 1M NaOH and water then dried over Na2SO4. e
solvent was evaporated to give crude 10, which was puri-
fied by column chromatography (hexanes:EtOAc, 4:1).
Compound 11 (5.66 g, 96%) was obtained as a pale yel-
low oil: 1H NMR (500 MHz, CDCl3): δ 9.92 (s, 1H), 7.44 (d,
1H, J= 2 Hz), 7.42 (d, 1H, J= 2 Hz), 7.06 (d, 1H, J= 1.7 Hz),
7 (dd, 1H, J= 8, 1.7 Hz), 6.89 (d, 1H, J= 8 Hz), 6.02 (s, 2H),
3.97 (s, 3H); 3.71 (s, 3H); 13C NMR (125 MHz, CDCl3): δ
191.2, 153.7, 151.9, 147.5, 147.2, 135.7, 132.3, 130.8, 127.2,
122.7, 109.7, 109.3, 108.3, 101.2, 60.7, 56.1. HREIMS calcd.
for C16H14O5 [M]+ 286.0841; found 286.0841.
N
NH2
O
O
N
H
(E)-5-(2,3-dimethoxy-5-(2-nitrovinyl)phenyl)benzo[d][1,3]
dioxole (12) A mixture of aldehyde 11 (5.31 g, 18.56
mmol), ammonium acetate (1.43 g, 18.56 mmol),
nitromethane (4.98 mL, 92.82 mmol), and glacial AcOH
was refluxed for 4 h. After cooling to RT, the product
was filtered and recrystallised from EtOH to afford 12,
as a yellow solid (5.15 g, 84%): mp 124–127°C. 1H NMR
(500 MHz, CDCl3): δ 7.97 (d, 1H, J= 13.6 Hz), 7.55 (d, 1H,
J= 13.6 Hz), 7.14 (d, 1H, J= 2.1 Hz), 7.02 (d, 1H, J= 1.7 Hz),
7.01 (d, 1H, J= 2.1 Hz), 6.96 (dd, 1H, J= 8, 1.7 Hz), 6.88 (d,
1H, J= 8 Hz), 6.02 (s, 2H), 3.95 (s, 3H); 3.68 (s, 3H); 13C
NMR (125 MHz, CDCl3): δ 153, 150.3, 147.8, 147.5, 139.2,
HO
1
2
O
O
O
O
N
O
N
N
3
4
Figure 1. Clinically available AChE inhibitors.
© 2011 Informa UK, Ltd.

48
Stevan Pecic et al.
13C NMR (125 MHz, CDCl3): δ 161.4, 153.1, 147.4, 146.8,
135.4, 134.4, 133.6, 131.9, 122.7, 122.6, 111.7, 109.9, 108.2,
101.2, 60.6, 56.1, 39.3, 35.5. HREIMS calcd. for C18H20NO5
[M+H]+ 330.1263; found 330.1333.
OH
O
O
O
O
O
N+
O
N
O
N
O
O
O
O
N-(3-(benzo[d][1,3]dioxol-5-yl)-4,5-dimethoxyphenethyl)
acetamide (14b) e amine 13 (0.32 g, 1.06 mmol),
acetyl chloride (0.12 mL, 5.5 mmol) and triethylamine
(0.45 mL, 10 mmol) were mixed with dichloromethane
(10 mL) and stirred for 6 hours at 0°C. e reaction was
treated with saturated sodium bicarbonate, extracted
with DCM (3 × 35 mL), dried over Na2SO4 and concen-
trated under reduced pressure to give a yellow oil, that
was subsequently purified by column chromatogra-
phy (MeOH:DCM, 1:99). Compound 14b (0.33 g, 91%)
O
O
O
5
6
7
O 2
A
D
B
N
O
1
C
O
9
O
1
was obtained as an orange-red oil: H NMR (500 MHz,
8
CDCl3): δ 8.12 (s, 1H), 7.06 (br. s, 1H), 6.98 (d, 1H, J = 8
Hz), 6.86 (d, 1H, J = 8 Hz), 6.73 (br. s, 1H), 5.98 (s, 2H), 5.71
(br. s, 1H), 3.88 (s, 3H), 3.58 (s, 3H), 3.51 (dd, 2H, J = 13.2,
6.5 Hz), 2.78 (t, 2H, J = 7 Hz), 1.95 (s, 3H); 13C NMR
(125 MHz, CDCl3): δ 170.1, 153.1, 147.4, 146.8, 145,
135.4, 134.7, 131.9, 122.6, 122.5, 111.7, 109.8, 108.1, 101,
60.5, 56, 40.7, 35.6, 23.4. HREIMS calcd. for C19H22NO5
[M+H]+ 344.1415; found 344.1488.
Figure 2. Structures of known aporphinoid AChE inhibitors (5, 6 and
7) and nantenine (8).
136.6, 136.6. 130.9, 125.9, 125.1, 122.9, 111, 109.9, 108.5,
101.4, 61, 56.3. HREIMS calcd. for C17H16NO6 [M+H]+
330.0899; found 330.0971.
2-(3-(benzo[d][1,3]dioxol-5-yl)-4,5-dimethoxyphenyl)
ethanamine (13) trimethylsilyl chloride (TMSCl) (5 mL,
24.2 mmol) was added to a vigorously stirred suspension
of LiBH4 (0.35 g, 7.62 mmol) in anhydrous tetrahydro-
furan (THF) (15 mL) over a period of 2 min. After the gas
evolution had ceased, the trimethylsilane was removed
by purging the solution with argon. en, over a period of
5 min, a solution of 12 (1 g, 3.03 mmol) in anhydrous THF
(15 mL) was added and the mixture was heated for 18 h
at reflux. After cooling to RT, the mixture was quenched
carefully with methanol (25 mL) at 0°C. e solvent was
removed with a rotatory evaporator, the resulting residue
dissolved in 20% aq KOH (50 mL), and extracted with
DCM (3 × 50 mL). e combined extracts were dried
(Na2SO4) and then concentrated under reduced pressure.
Synthesis of compounds 15a and 15b To a stirred solu-
tion of amide 14a (0.46 g, 1.4 mmol) in acetonitrile (5 mL)
was added POCl3 (0.65 mL, 7 mmol) at RT, and the result-
ing mixture was heated at 50°C for 12 h. e reaction
mixture was concentrated, and the quaternary salt was
dissolved in DCM (25 mL). After the mixture was cooled
to 0°C, it was diluted with water, made basic with 5%
aqueous NH4OH, and extracted with DCM (3 × 25 mL).
e organic solution was washed with water (30 mL),
dried with anhydrous Na2SO4 and concentrated to give
a yellow crude dihydroisoquinoline. Sodium borohy-
dride (1.04 g, 27.6 mmol) was added portion-wise to
a stirred solution of the crude dihydroisoquinoline in
methanol (20 mL) and the mixture was stirred at 0°C for
3 h. e reaction mixture was concentrated, and excess
NaBH4 was destroyed by adding water and glacial ace-
tic acid until gas evolution ceased. e mixture was
extracted with DCM (3 × 25 mL), dried over Na2SO4 and
concentrated to give orange oil. is crude oily tetrahy-
droisoquinoline was treated with aqueous formaldehyde
solution, (37%, 2.59 mL, 2.55 mmol) in anhydrous DCM
(10 mL) and then with NaBH(OAc)3 (1.35 g, 6.38 mmol).
e mixture was allowed to stir at RT for 24h. e reac-
tion was quenched with 5% aqueous sodium bicarbon-
ate (25 mL), and extracted with ethyl acetate (2 × 25 mL),
dried over Na2SO4 and concentrated to dryness. e resi-
due was purified via silica flash column chromatography
(MeOH:DCM, 2:98) to provide 15a. Similar procedures
were used to prepare 15b from 14b.
1
is gave 13 as an oil (0.90 g, 99%). H NMR (500 MHz,
CDCl3): δ 7.1 (d, 1H, J= 1.3 Hz), 7.04 (d, 1H, J= 1.5, 8 Hz),
6.88 (d, 1H, J= 8 Hz), 6.77 (s, 1H), 6.76 (s, 1H), 6.02 (s, 2H),
3.92 (s, 3H), 3.62 (s, 3H), 3.01 (t, 2H, J= 7 Hz), 2.75 (t, 2H,
J= 7 Hz). 13C NMR (125 MHz, CDCl3): δ 153, 147.3, 146.7,
144.8, 135.7, 135.2, 132.2, 122.6, 122.6, 111.9, 109.9, 108.1,
101, 60.5, 56, 43.5, 40.
N-(3-(benzo[d][1,3]dioxol-5-yl)-4,5-dimethoxyphenethyl)
formamide (14a) Amine 13 (0.46 g, 1.53 mmol), ethyl
formate (0.31 mL, 3.9 mmol) and triethylamine (0.43 mL,
3.10 mmol) were heated to reflux for 48 h. Removal of
excess ethyl formate and triethylamine under reduced
pressure gave a dark-brown oil, that was purified by col-
umn chromatography (MeOH:DCM, 1:99). Compound
14a (0.46 g, 90.8%) was obtained as an orange-red oil: 1H
NMR (500 MHz, CDCl3): δ 8.12 (s, 1H), 7.05 (s, 1H), 6.98
(d, 1H, J= 8 Hz), 6.85 (d, 1H, J= 8 Hz), 6.73 (br. s, 2H), 5.98
(s, 2H), 3.88 (s, 3H), 3.56 (m, 6H), 2.82 (t, 2H, J= 6.9 Hz);
8-(benzo[d][1,3]dioxol-5-yl)-6,7-dimethoxy-2-methyl-1,2,3,
4-tetrahydroisoquinoline (15a) 15a was prepared from
14a in 88% overall yield as bright yellow crystals: mp
Journal of Enzyme Inhibition and Medicinal Chemistry

Nantenine as an acetylcholinesterase inhibitor
49
82–84°C. 1H NMR (500 MHz, CDCl3): δ 6.87 (d, 1H, J= 7.9
Hz), 6.70–6.65 (m, 3H), 6.01 (d, 2H, J= 8.4 Hz), 3.88 (s,
3H), 3.54 (s, 3H), 3.15 (br. s, 2H), 2.94 (br. s, 2H), 2.65 (br.
s, 2H), 2.35 (s, 3H); 13C NMR (125 MHz, CDCl3): δ 151,
147.3, 146.5, 144.8, 134, 129.8, 129.5, 125.8, 122.6, 111.6,
110, 108.2, 101, 60.8, 56.5, 55.8, 52.6, 46.3, 29.8; HREIMS
calcd. for C19H21NO4 [M]+ 327.1478; found 327.1471.
Preparation of Homology Model
Since a high-resolution crystal structure of E. electricus
AChE is not available, the homology model for molecular
modeling purposes was built based on the X-ray crystal
structure of Torpedo californica AChE in complex with
bis-(5)-tacrine (PDB code 2CMF) which was obtained
from the Protein Data Bank. e tacrine ligand and water
molecules present in the pdb file were removed prior to
building the model. e sequence of E. electricus AChE
was aligned with the sequence of Torpedo californica
AChE, and showed 70% identity. For the sequence align-
ment step, the following parameters were used:
Alignment Algorithm: ZEGA
8-(benzo[d][1,3]dioxol-5-yl)-6,7-dimethoxy-1,2-dimethyl-
1,2,3,4-tetrahydroisoquinoline (15b)
Prepared from 14b, as a mixture of isomers, in 90% over-
all yield as bright yellow crystals: mp 101–104°C. 1H NMR
(500 MHz, CDCl3): δ 6.87–6.84 (m, 1H), 6.76–6.67 (m, 1H),
6.66–6.65 (m, 2H), 6.03–6 (m, 2H), 3.87 (s, 3H), 3.78–3.71
(m, 1H), 3.53–3.49 (m, 3H), 3.08–3.07 (m, 2H), 2.77–2.71
(m, 2H), 2.38–2.46 (m, 3H), 0.97–0.94 (m, 3H); 13C NMR
(125 MHz, CDCl3) (doubling of signals observed; average
values for isomeric shifts reported): δ 151, 147.3, 146.5,
145.1, 134.6, 130.8, 129.9, 128.6, 123.3, 112, 110.6, 108.1,
101, 60.8, 55.7, 55, 44.1, 42, 26.3, 16.2; HREIMS calcd. for
C20H23NO4 [M]+ 341.1627; found 341.1628.
Gap Open: 2.4
Gap Extension: 0.15
maxPenalizedGap: 99
is alignment, along with a file containing the atomic
coordinates of Torpedo californica AChE, was used as an
input to the ICM PRO software package to generate the
homology model. For the homology model building step
the following parameters were used:
3D template: 2CMF
AChE Inhibition Assays
Max loop length: 100
Nterm extension: 1
AChE inhibition was determined using the method of
Adsersen et al [43] with ATCI as substrate. In brief, to a
96-well microplate, 25 μL substrate, 15mM ATCI in water,
125 μL 3 mM DTNB in buffer C (50 mM Tris–HCl, pH 8,
0.1M NaCl, 0.02 M MgCl2·6H2O), 72.5 μL buffer B (50 mM
Tris–HCl, pH 8, 0.1% bovine serum albumin) and 2.5 μL
test compound solution dissolved in DMSO were added
and the absorbance was measured seven times at 405 nm
every 19 s in a PowerWave 200 Microplate Scanning
Spectrophotometer (Winooski, VT). en 25 μL AChE
(0.22 U/mL in buffer B) was added to each of the wells
and the absorbance was measured again seven times at
405 nm every 19 s. e reaction rate was calculated by
the GraphPad Prism software version 5.0 (LaJolla, CA)
and Microsoft Excel. Any increase in absorbance due to
the spontaneous hydrolysis of substrate was corrected
by subtracting the rate of the reaction before adding
the enzyme. Percentage inhibition was calculated by
comparing the rates for the samples to the blank (2.5 μL
DMSO instead of test compound solution). IC50 values
were calculated by nonlinear regression analysis using
GraphPad Prism. Each experiment was performed in
triplicate [43].
Cterm extension: 1
Expand gaps by: 1
After energy-minimising using ICM-PRO, the receptor
model was saved and used for docking studies.
Generation of ligands
e 2D structure of nantenine (or analogs 15a, 15b,
16 and 17) was drawn with ChemDraw Ultra version
9.0 (Cambridge, MA) and energy minimised through
Chem3D Ultra version 9.0/MOPAC (Cambridge, MA), Job
Type: Minimum RMS Gradient of 0.01 kcal/mol and RMS
distance of 0.1 Å, and saved as MDL MolFile (*.mol).
Docking using the Molsoft ICM 3.6-1 program [45]
To perform the ICM small molecule docking the fol-
lowing steps were executed according to the program
guidelines:
(1) Setup docking project:
(i) e project name was set.
(ii) e enzyme (receptor) was setup: (e receptor
molecule was entered in the receptor molecule
data entry box, the binding sites were identified
by clicking on the “identify binding sites” but-
ton. In this way potential ligand binding pock-
ets were identified. After the receptor setup was
complete, the program displayed the receptor
with selected binding site residues highlighted
in yellow x-7stick presentation).
(iii) e binding site was reviewed and adjusted:
ICM made a box around the ligand binding
site based on the information entered in the
receptor setup section. e position of the
box encompassed the residues expected to be
involved in ligand binding.
Docking studies with Nantenine
e binding of the nantenine and Electrophorus elec-
tricus AChE was examined in silico. All docking studies
were performed using Internal Coordinate Mechanics
(Molsoft ICM version 3.6-1, LaJolla, CA) package. e
ICM score considers several free energy terms such as
van der Waals, hydrogen bonding, Poisson electrostatic
desolvation and entropy. ICM sequence-structure align-
ment is based on zero end gap global alignment (ZEGA)
sequence alignment [44] (Needleman and Wunsch algo-
rithm with zero gap and penalties).
© 2011 Informa UK, Ltd.

50
Stevan Pecic et al.
(iv) Receptor maps were made: Energy maps of
the environment within the docking box were
constructed. Flexibility of the receptor residues
was set to 4.0.
To determine the mode of inhibition exhibited by
nantenine, we carried out an evaluation of the steady-
state kinetics of the enzyme in the presence of nantenine.
A double reciprocal (1/V vs 1/[S], Lineweaver-Burk)
plot was performed and this indicated mixed inhibi-
tion since lines of increasing gradient showed increas-
ing x-intercepts (Figure 4). Figure 5 shows a Dixon plot
which allowed for calculation of the Ki of nantenine in
this assay (18.7 μM).
(2) Docking simulation was executed:
Interactive docking was used to dock nantenine
and the other analogs. e molecules were docked
in non-chiral mode. e thoroughness level was
set to the maximum value of 10. ICM scores were
obtained after this procedure.
Docking experiments
To further understand the structural basis for the anti-
AChE activity seen in 8, molecular docking experiments
were performed with the program ICM Pro® [54]. e
crystal structure of Torpedo californica AChE (TcAChE)
bound to bis-(5)-tacrine was obtained from the Protein
Data Bank (PDB) – PDB code 2CMF. Bis-(5)-tacrine
was removed and a homology model was generated
for Electrophorus electricus AChE using the homology
modeling features of the program. Manipulations were
performed to allow for identification of the binding
sites in the homology modelled enzyme. After energy-
minimisation, nantenine was docked into the identified
binding regions of the enzyme. Scoring functions from
this docking experiment are reported in Table 2. e
known catalytic site inhibitor galanthamine (1) was also
docked for comparison. e docking scores suggest that
nantenine may bind preferentially to the catalytic site
since the three most favorable conformations are located
in this site.
Results
Chemistry
Compounds 15a and 15b were prepared as outlined in
Scheme 1. Commercially available boronic acid 9 and
bromoaldehyde 10 were coupled under Suzuki condi-
tions [46,47] affording the biaryl intermediate (11). A
nitro-aldol reaction [48,49] on 11 followed by reduction
of the nitrostyrene (12), gave amine 13. Compound
13 was condensed with ethyl formate or with acetyl
chloride to give amides 14a and 14b. These amides
were then subjected to Bischler-Napieralski [50,51]
cyclisation to afford intermediate dihydroisoquino-
lines which were immediately reduced with NaBH4 to
give intermediate tetrahydroisoquinoline products
(due to instability of the imines). Methylation of the
tetrahydroisoquinoline intermediates under reduc-
tive-amination conditions then furnished the target
seco-ring C derivatives 15a and 15b. Compounds 16
and 17 (Figure 3) were prepared as reported by us
elsewhere [52].
Catalytic site interactions of nantenine
Inspection of the top binding pose in the active site indi-
cates that nantenine interacts with the enzyme via π-π
stacking interactions between the Trp84 and rings A and
D as well as similar interactions between the Tyr330 and
ring D. Both of these interactions occur in the anionic
sub-site [55] region of the enzyme. Of the three amino
acid residues in the catalytic triad [56] (His440, Ser200
and Glu327), nantenine interacts only with Ser 200 (via
the hydrophobic contacts with ring B). Nantenine also
shows interactions with Gly118 in the oxyanion hole [57]
region of AChE. Other hydrophobic interactions help
to stabilise the molecule in the active site. Interactions
of nantenine in the catalytic site of the enzyme have
been displayed with LigPlot [58] (Figure 6) for ease of
viewing.
Enzyme inhibition and kinetics assays
IC50 values for compounds 8, 15a, 15b, 16 and 17
determined with a microplate assay [53] using lyophi-
lised Electrophorus electricus AChE (Type VI, Sigma-
Aldrich, MO) and based on Ellman’s method, are
summarised in Table 1. The lead compound nantenine,
showed the highest inhibitory activity with an IC50 of
1.09 μM. The seco-ring C analogs 15a and 15b were pre-
pared to evaluate whether the rigid aporphine frame-
work was required for activity. We were also interested
in evaluating these compounds since they are more
accessible synthetically, so that any follow-up SAR
studies would be facilitated. Both compounds were
approximately two-fold less active than nantenine.
Compound 16, in which the biaryl bond of nantenine is
absent, was 17 times less active than the lead molecule.
The flexible analog 17 had the lowest inhibitory activ-
ity among the ring-truncated analogs, being inactive
(IC50 > 100 μM). Taking together the results obtained
from the SAR analysis of this set of compounds, it is
apparent that ring C and ring B are required to be
intact for anticholinesterase activity. Therefore it
appears that the rigid aporphine core of nantenine is
an important structural feature for AChE inhibitory
activity.
O
O
O
O
N
N
O
O
O
O
16
17
Figure 3. Other seco-ring C nantenine analogs.
Journal of Enzyme Inhibition and Medicinal Chemistry

Nantenine as an acetylcholinesterase inhibitor
51
B(OH)2
O
O
O
O
NO2
c
O
O
CHO
O
O
CHO
+
a
b
NH2
O
O
Br
10
9
O
O
O
O
O
O
11
12
13
d or e
O
O
f, g, h
HN
R
N
O
O
O
R
O
O
O
O
15a R = H
15b R = CH3
14a R = H
14b R = CH3
Scheme 1. Synthesis of target analogs 15a-15b. Reagents and conditions: (a) Pd(PPh3)4, DME, K2CO3, reflux, 24h; (b) CH3NO2, NH4OAc, reflux, 4h; (c)
LiBH4, TMSCI, THF, reflux; (d) HCOOEt, Et3N, DCM for 14a; (e) acetyl chloride, Et3N, DCM for 14b; (f) POCl3, MeCN, 50°C, 12h; (g) NaBH4, MeOH,
0°C, 4h; (h) HCNO, NaBH(OAc)3, DCM, rt, 24h.
Table 1. AChE inhibitory activity of analogues.
Compound
120
100
80
60
40
20
0
No Inhibitor
10 uM
IC50 AChE (μM)a
25 uM
Nantenine (8)
15a
15b
16
1.09 0.17
2.63 0.38
2.38 0.21
18.06 1.92
> 100b
50 uM
100 uM
17
Galanthaminec
0.53 0.11
aResults are the mean of three replications ( SEM of three
experiments).
bConsidered inactive
cReference compound.
−0.2 −0.15 −0.1 −0.05
0
0.05
0.1
0.15
0.2
[S]−1 mM−1
PAS interactions of nantenine
e ICM docking experiments also predicted bind-
ing poses for nantenine in the PAS. Figure 7 shows a
LigPlot model for the key interactions of pose number
four which indicates that nantenine is stabilised via a
hydrogen-bonding interaction between the C2 oxygen
atom and the phenolic group of Tyr70 in the PAS. π-π
stacking interactions between the aromatic rings of
nantenine and Trp279 as well as between Y334 and ring
A also seems to be important for binding to the PAS,
based on inspection of our ICM model.
Figure 4. Lineweaver-Burk steady-state inhibition plot showing mixed
inhibition for nantenine.
However, in the case of compound 16, among the top
three binding poses, one was in the PAS, one in the
catalytic site and the third in both the catalytic site and
PAS. Compound 17 did not dock into the catalytic site
or the PAS of the enzyme which is consistent with the
low activity observed with this compound (Table 1).
Visual inspection showed that compound 17 was
docked in a region on the outer surface of the enzyme.
erefore, the relatively good ICM score obtained for
compound 17 is clearly not due to the active site or PAS
inhibition.
e ICM models may be used to explain the mode of
inhibition exhibited by nantenine; e mixed mode of
inhibition is most likely to be due to nantenine binding
to both the catalytic site and PAS.
Docking of 15a, 16 and 17
In order to begin to rationalise the trends in anticho-
linesterase activity seen in our analogs, ICM docking
simulations were then performed with compounds
15a, 16 and 17. Table 3 summarises results from these
docking experiments. e docked poses were evaluated
by visual inspection. e three lowest energy poses for
compound 15a were all located in the catalytic site.
Discussion
Alzheimer’s disease (AD) is a neurodegenerative con-
dition, the onset and progression of which is associ-
ated with deficits in cholinergic neurotransmission as
well as aggregation of the amyloid peptide Aβ [6–10]. A
major biological target for the development of anti-AD
© 2011 Informa UK, Ltd.

52
Stevan Pecic et al.
drugs is the enzyme acetylcholinesterase. Inhibitors of
acetylcholinesterase have proved to be of therapeutic
value in the treatment of Alzheimer’s disease and have
been the front-line approach in this regard [13,14]. is
enzyme is known to possess two binding domains: a
catalytic site which binds the endogenous neurotrans-
mitter acetylcholine and catalyses its degradation and
a peripheral anionic site [22,23]. Clinically available
inhibitors have the inhibition of the catalytic site of
the enzyme as an integral part of their mode of action.
is mode of action of anti-AD drugs is in line with the
cholinergic hypothesis of Alzheimer’s disease [59,60].
However, current interest is in the identification and
development of molecules which potently inhibit
both the catalytic site and peripheral anionic site of
the enzyme [27]. Dual-site inhibition of the enzyme is
beneficial, since inhibition of the catalytic site results
in improvements in cholinergic deficits, while inhibi-
tion of the peripheral anionic site prevents aggregation
of toxic amyloid peptide Aβ [24,25,61]. A number of
chemical scaffolds have been identified which fit this
role, some of which utilise a dimeric design for dual
AChE inhibition [62–64]. Our work here indicates that
the aporphine scaffold may represent another oppor-
tunity to develop novel dual-site AChE inhibitors.
Table 2. ICM docking scores for nantenine (8) and galanthamine
(1) in AChE.
100
Pose numbera
ICM docking scores
−59.2
Binding site
Catalytic
Catalytic
Catalytic
PAS
7.5 mM ATCI
15 mM ATCI
30 mM ATCI
45 mM ATCI
60 mM ATCI
75 mM ATCI
1
80
2
−55.09
3
−53.9
60
40
20
0
4
−53.75
5
−53.56
Catalytic
Catalytic
PAS
6
−53.4
7
−52.61
8
−52.56
PAS
9
−52.31
PAS
−40
−20
0
20
40
60
80
100
120
10
−51.43
−59.52b
Catalytic
Catalytic
−20
Galanthamine
[Nantenine] µM
aranked in order of increasing energy
btop score which corresponds to the lowest energy in ICM
Figure 5. Dixon plot. (Intercept on the x- axis is -Ki).
A
B
Figure 6. A is an ICM model of nantenine in the catalytic site of AChE. Pose shown is for lowest energy conformation (pose number 1 in Table 2) and
B the LigPlot model of nantenine in the catalytic site of AChE.
Journal of Enzyme Inhibition and Medicinal Chemistry

Nantenine as an acetylcholinesterase inhibitor
53
A
B
Figure 7. A is an ICM model of nantenine in the PAS of AChE. Pose shown is for the lowest energy conformation in the PAS (pose number 4 in Table
2) and B is the LigPlot model of Nantenine in the PAS.
A number of analogues of our lead molecule, nan-
tenine were synthesised and evaluated in an Ellman
assay for AChE inhibition. ese analogs were designed
to probe the requirements for structural rigidity of the
aporphine core of the lead compound in inhibiting the
enzyme. We found that the rigidity of nantenine was
an important structural feature for AChE inhibition.
Others have found that the less rigid tetrahydroisoqui-
noline precursors of some aporphines show higher lev-
els of AChE inhibition than the aporphines [65]. ese
results together suggest that the structural rigidity of
aporphines as a group per se is not the only determi-
nant of enzyme activity, although in the case of nant-
enine this rigidity does appear to play a significant role.
Nantenine showed mixed inhibition kinetics in our
enzyme assays. Interestingly, nantenine was previously
shown to be inactive in an AChE assay with rat syn-
aptosomal membrane as the enzyme source [66]. e
assay conditions or source of the enzyme may account
for the discrepancy in both the previous and currently
reported results.
Table 3. ICM predicted binding sites for 15a, 16 and 17.
Compound Pose number
ICM scores
−60.84
−57.93
−56.9
Binding sitea
Catalytic
Catalytic
Catalytic
PAS
15ab
1
2
3
1
2
3
16c
−69.7
−68.14
−67.75
Catalytic
PAS/
Catalytic
17
1
2
3
−85.27
−84.49
−84.34
–
–
−
aCatalytic site or PAS
bTop nine binding poses were in catalytic site, tenth in PAS
cBinding poses four to ten are in PAS
of inhibition. is study is the first to examine the dock-
ing of aporphines and the flexible analogs, although
there is one recent study where the docking of structur-
ally related oxoaporphines and oxoisoaporphines was
reported [67]. Like nantenine, the molecular model-
ling experiments suggest that the oxoaporphines and
oxoisoaporphines interact with the PAS. In the case of
the flexible analogs 15a, 16 and 17, we found that visual
Our docking experiments suggest that nantenine
is capable of binding to both the catalytic site and the
PAS of AChE, which could account for its mixed mode
© 2011 Informa UK, Ltd.

54
Stevan Pecic et al.
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inspection of the docked ICM poses seems to be a good
method for predicting activity or lack thereof in this
series of compounds. However, the top three ICM scores
for nantenine (8), 15a and 16 did not show good qualita-
tive correlation with the observed inhibitory activities,
suggesting that the ICM score is not a good predictor
of relative AChE inhibitory activity for this series. is
may be due in part to the occurrence of multiple binding
modes for these compounds.
In considering the design of novel anti-AD drugs,
dual inhibition of AChE is a significant strategy to
explore. Nantenine is an interesting lead for such
studies. Apart from the dual inhibitory profile, phar-
macokinetic factors such as blood-brain barrier pen-
etrability will also be important in the future design
of potential anti-AD nantenine analogs. e optimal
clogP for blood-brain barrier penetrability is 2–5 [68].
In this regard, nantenine is perhaps a better lead than
some other anti-AChE aporphinoids previously identi-
fied such as compound 5 [40], which predictably will
not cross the blood-brain barrier due to its quaternised
nitrogen functionality (clogP of nantenine and 5 = 3.6
and -1.4 respectively as calculated with ChemBioDraw
Ultra version 11).
Conclusions
SAR explorations have revealed that the rigid structure
of nantenine is important for anticholinesterase activ-
ity; increasing molecular flexibility was associated with a
decrease in AChE inhibition. Since the inhibition was of
the mixed type, it is perceptible that nantenine binds to
both the PAS and the catalytic site. is is corroborated
by our molecular docking studies which indicated key
H-bond and π-π stacking interactions of nantenine in
the PAS. e aporphine scaffold of nantenine may be
useful for the synthesis of novel PAS or dual-site AChE
inhibitors.
Declaration of interest
e authors report no conflict of interest.
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