Synthesis, biological evaluation, and molecular modeling of berberine derivatives as potent acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.12.035

By targeting the dual active sites of acetylcholinesterase (AChE), a new series of berberine derivatives was designed, synthesized, and evaluated as AChE inhibitors. Most of the derivatives inhibited AChE in the sub-micromolar range. Compound 8c, berberine linked with phenol by a 4-carbon spacer, showed the most potent inhibition of AChE. A kinetic study of AChE and BuChE indicated that a mix-competitive binding mode existed for these berberine derivatives. Molecular modeling studies confirmed that these hybrids target both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. This is the first report where AChE inhibitory activity has been associated with berberine as a lead molecule.

Full text of DOI:10.1016/j.bmc.2009.12.035

Bioorganic & Medicinal Chemistry 18 (2010) 1244–1251
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation, and molecular modeling of berberine
derivatives as potent acetylcholinesterase inhibitors
*
*
Ling Huang, Anding Shi, Feng He , Xingshu Li
The Industrial Institute of Fine Chemicals and Synthetic Drugs, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
By targeting the dual active sites of acetylcholinesterase (AChE), a new series of berberine derivatives was
designed, synthesized, and evaluated as AChE inhibitors. Most of the derivatives inhibited AChE in the
sub-micromolar range. Compound 8c, berberine linked with phenol by a 4-carbon spacer, showed the
most potent inhibition of AChE. A kinetic study of AChE and BuChE indicated that a mix-competitive
binding mode existed for these berberine derivatives. Molecular modeling studies confirmed that these
hybrids target both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. This is
the first report where AChE inhibitory activity has been associated with berberine as a lead molecule.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 22 October 2009
Revised 9 December 2009
Accepted 10 December 2009
Available online 16 December 2009
Keywords:
Berberine derivatives
AChE inhibitors
Mix-competitive binding mode
1. Introduction
tion of amyloid-b fibrils that are associated with plaque deposition
in AD patients. AChE inhibitors simultaneously blocking both the
Alzheimer’s disease (AD), the most common dementia in the
elderly, is a progressive neurodegenerative disorder characterized
by memory loss and other cognitive impairments. Although the
etiology of AD is not completely known, several diverse hallmarks
catalytic and peripheral sites might not only alleviate the cognitive
defect of AD patients by elevating ACh levels, but also act as dis-
ease-modifying agents delaying amyloid plaque formation.7,8 This
discovery stimulated a great interest toward a bivalent ligand
strategy to design hybrid compounds for simultaneously inhibiting
acetylcholine hydrolysis and AChE-induced Ab aggregation. Gener-
ally, bivalent hybrid ligands are obtained by connecting two iden-
tical or distinct moieties through a linker of suitable length to
make contact with both the CAS and PAS, including tacrine-related
homo- and heterobivalent hybrids,9,10 bis-galanthamine hybrids,11
and huperzine A dimeric inhibitors,12 with IC50 values from sub-
nanomolar to picomolar.
such as b-amyloid (Ab) deposits, s-protein aggregation, oxidative
stress, or low levels of acetylcholine (ACh) play significant roles
in the pathophysiology of the disease.1 The cholinergic hypothesis
of AD suggests that low levels of ACh in specific regions of the brain
result in learning and memory dysfunction.2 This hypothesis indi-
cates the therapeutic potential of increasing the levels of ACh
through inhibition of the acetylcholinesterase (AChE). Acetylcho-
linesterase inhibitors (AChEIs) represent a well-established class
of drugs for the symptomatic treatment of AD, which include
tacrine (CognexÒ), 1a, Fig. 1), donepezil (AriceptÒ, 1b, Fig. 1), galan-
thamine (ReminylÒ, 1c, Fig. 1), and huperzine A (1d, Fig. 1). Among
them, galanthamine and huperzine A are naturally-occurring alka-
loids from the genus Galanthus (Amaryllidaceae) and the club
moss Huperzia serrata (Lycopodiaceae), respectively.
In the past decade, a series of crystal structures of AChE/inhib-
itor complexes have been reported,3–6 which indicate that AChE
has two binding sites: a catalytic active site (CAS) and a peripheral
anionic site (PAS). The CAS of AChE is located at the bottom of a
narrow gorge, which consists of two sub sites, an anionic and
esteratic site. The second ‘anionic’ site, known as the PAS, lies
around 14 Å from the active site. The PAS is involved in the forma-
* Corresponding authors.
E-mail address: lixsh@mail.sysu.edu.cn (X. Li).
Figure 1. Stucture of tacrine (1a), donepezil (1b), galanthamine (1c), huperzine A
(1d) and berberine (1e).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.035

L. Huang et al. / Bioorg. Med. Chem. 18 (2010) 1244–1251
1245
Berberine (1e, Fig. 1) could be isolated as the principal qua-
2.2. In vitro inhibition studies of AChE and BuChE
ternary base from a traditional Chinese herb, Coptis chinensis
Franch, the roots of which are widely used as traditional medi-
cines for treating diarrhea13 and gastrointestinal disorders.14 It
can inhibit acetylcholinesterase15 and play an important role in
metabolic syndrome.16 Chemically, berberine possesses a quater-
nary nitrogen and three aromatic rings that might bind to CAS or
The AChE inhibitory effects of all target compounds were exam-
ined by the method of Ellman et al.18 on AChE from electric eel
using commercial galanthamine as the reference standard. The
BuChE inhibitory on equine serum BuChE were also examined by
the same method. The IC50 values for AChE and BuChE inhibition
are summarized in Table 1.
PAS of AChE by
p–p stacking and electronic interaction. Enlight-
ened by the interesting structure of berberine, we designed sev-
eral series of berberine derivatives by combining it with diverse
aromatic rings that have two binding dual sites. The berberine
moiety was expected to bind the PAS of AChE, and the aromatic
rings of non-berberine moiety would interact with the catalytic
All berberine derivatives were potent inhibitors of AChE, with
IC50 values ranging from micromolar to sub-micromolar. A simple
structure–activity relationship analysis showed that the AChE
inhibitory potency closely related to the length of the alkylene
chain. Compounds 6c, 7c, and 8c, with four methylene groups be-
tween the berberine and aromatic ring units, were the best inhib-
itors in their series. Especially, compound 8c showed the best AChE
inhibitory activity of all the berberine derivatives, with an IC50 va-
center of AChE through a cation–p interaction. In this paper,
we disclose the synthesis of berberine derivatives and their high
AChE inhibitory activity, with IC50 values in the sub-micromolar
range.
lue of 0.097 lM. However, the same trend was not shown for
BuChE inhibition.
It is surprising that series 6, which possesses berberine–carba-
zole hybrids, showed a remarkably low inhibitory activity com-
pared to the bicyclic heterodimeric hybrids in series 7 and
monocyclic heterodimeric hybrids in series 8. Monocyclic deriva-
tives (8a–e) showed most potent inhibitory activity of 9- to 20-fold
more potent activity than 4-hydroxy carbazole derivatives. The rel-
ative rigidity of the 4-hydroxy carbazole scaffold could hinder pen-
etration into the AChE gorge to reach the binding site.19
From the IC50 values of the compounds, it appeared that the 4-
hydroxy carbazole derivatives (6a–e), showing a better activity to-
ward BuChE than that of AChE. Compound 6a, the most potent for
2. Results and discussion
2.1. Chemistry
The synthetic pathway of 9-substituted berberine derivatives
6a–e, 7a–e, 8a–e are shown in Scheme 1. First, the reaction of 4-
hydroxycarbazole, 1-hydroxybenzotriazole and phenol in buta-
none with
formed to provide responding
a
,
x
-dibromoalkanes in the presence of K2CO3 was per-
-bromoalkyl ether derivatives (3a–
x
e, 4a–e, and 5a–e). The selective demethylation of berberine 1 at
190 °C under vacuum gave berberrubine 217 in 68% yield. Finally,
the target compounds 6a–e, 7a–e, 8a–e were obtained by reaction
of berberrubine 2 with 3a–e, 4a–e, and 5a–e, respectively, in
CH3CN for 12–24 h.
BuChE inhibition, had an IC50 value of 0.029 lM, about 50-fold
higher than for AChE. Recent studies indicated that inhibition of
brain BuChE may represent an important therapeutic target for
AD. It plays a key role and can partly compensate the function of
Table 1
In vitro inhibition and selectivity of compounds 1, 6a–e, 7a–e, 8a–e, and galantha-
mine on AChE and BChE activities
Compd
n
IC50
(l
M) SEM
BuChEb
Selectivity for AChEc
AChEa
1
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
0.374 0.024
1.23 0.161
1.12 0.08
18.2 0.683
48.6
0.024
0.128
0.116
0.064
0.04
6.6
8.0
18.6
8.9
2
3
4
5
6
2
3
4
5
6
2
3
4
5
6
0.029 0.004
0.143 0.009
0.099 0.006
0.107 0.011
0.086 0.007
2.48 0.220
2.89 0.430
4.17 0.391
2.49 0.139
2.83 0.142
3.00 0.080
2.22 0.213
4.89 0.035
2.69 0.166
4.18 0.147
15.7 0.787
0.856 0.096
1.68 0.059
2.10 0.131
0.375 0.022
0.359 0.034
0.224 0.019
0.280 0.039
0.515 0.016
0.224 0.017
0.123 0.007
0.097 0.005
0.398 0.024
0.520 0.083
0.623 0.099
5.5
13.4
18.0
50.4
6.8
8.0
25.3
Galanthamine
a
50% inhibitory concentration (means SEM of three experiments) of AChE from
electric eel.
b
50% inhibitory concentration (means SEM of three experiments) of BuChE
Scheme 1. 9-Substituted berberine derivatives 6a–e, 7a–e, 8a–e. Reagents and
conditions: (i) Br(CH2)nBr, K2CO3, KI butanone, 1–4 h; (ii) 190 °C, 20–30 mmHg,
15 min; (iii) 3a–e, 4a–e, 5a–e, K2CO3, CH3CN, reflux.
from equine serum.
c
Selectivity for AChE = IC50 (BuChE)/IC50 (AChE).

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L. Huang et al. / Bioorg. Med. Chem. 18 (2010) 1244–1251
AChE.20,21 Consequently, the berberine derivatives could be inter-
esting dual AChE/BChE inhibitors for Alzheimer’s disease.
Table 2
AChE and BuChE inhibition constants of some selected derivatives
Compound
Enzyme
ki SE (lM)
Type of inhibition
2.3. Kinetic study of AChE and BuChE
Berberine
8c
Berberine
8c
AChE
AChE
BuChE
BuChE
0.289 0.075
0.045 0.004
9.20 0.791
1.02 0.076
Non-competitive
Mixed
Non-competitive
Mixed
Determination of the inhibition type is important in under-
standing the mechanism of inhibition and the inhibitor binding
sites. Graphical analysis of the steady-state inhibition data could
give information about the binding mode of the selected com-
pounds. Based on the in vitro inhibition experiments, berberine
and its derivative 8c, one of the most potent AChE inhibitors, were
used to investigate the mechanisms of inhibition of AChE and
BuChE (Fig. 2). The estimates of inhibition constants Ki (Table 2)
were estimated from the plots of the slope versus the concentra-
tion of berberine and 8c.
genesis.24–26 For example, xanthostigmine derivatives,27 a class of
long-lasting AChE inhibitors, and bis-(ꢀ)-nor-meptazinols,28 de-
creased Ab aggregation and may be disease-modifying agents for
AD. Because the berberine derivatives have a binding mode similar
to xanthostigmine derivatives and bis-(ꢀ)-nor-meptazinols, ber-
berine could be a lead compound for AD treatment.
In the Lineweaver–Burk plots of Figure 2A and B, lines crossing
the x axis in the same point revealed unchanged Km and decreased
mmax with increasing inhibitor concentrations. This is a typical
trend for non-competitive inhibition, which was similar to that
of propidium.22 The inhibitory behavior of 8c on AChE and BuChE,
as illustrated in Figure 2C and D, is similar to that of bis-tetrahyd-
roaminoacridine23 inhibitors of AChE. Therefore, from the kinetic
profile, we concluded that compound 8c causes a mixed type of
inhibition and could interact with both CAS and PAS.
Based on the kinetic analysis, we assumed that the rather wide
shape of berberine binds to the PAS, and this binding might change
the conformation of the enzyme and sterically block the gorge,
making it harder for bulky berberine derivatives to enter the active
site in the base of a deep, narrow gorge. Therefore, steric factors
contribute to AChE inhibitory activity in the berberine hybrids, as
the monocyclic benzene berberine hybrids have stronger activity
than the tricyclic berberine carbazole hybrids.
Recent studies for non-catalytic functions of AChE suggested
that the PAS, besides its role in allosteric regulation of AChE-cata-
lyzed hydrolysis of released acetylcholine, also mediates heterolo-
gous protein associations, which contribute to cell recognition and
adhesion processes during synaptogenesis, and the nucleation of
amyloid peptides during Alzheimer’s disease onset. AChEIs interact
with both CAS and the PAS and inhibit AChE-induced Ab fibrillo-
2.4. Molecular modeling studies
To explore the interaction mode and the structure–activity rela-
tionships of 9-substituted berberine derivatives with AChE, molec-
ular docking simulations were performed with the software
AUTODOCK based on the structure of the complex of T. californica en-
zyme (TcAChE) (PDB entry 2CMF). Based on the in vitro inhibition
Figure 2. Steady state inhibition by berberine (A), 8c (C) of AChE hydrolysis of ATCh and berberine (B), 8c (D) of BuChE hydrolysis of BuTCh; the plots show non-competitive
inhibition for berberine on AChE (A) and BuChE (B), and mixed-type inhibition for 8c on AChE (C) and BuChE (D).

L. Huang et al. / Bioorg. Med. Chem. 18 (2010) 1244–1251
1247
results, we selected compound 8c, the highest AChE inhibitor,
respectively, as ligand example.
potent inhibitor, 8c, berberine linked with phenol by 4-carbon
spacers, inhibited AChE with IC50 of 0.097 M. The preliminary
l
As shown in Figure 3, the bivalent phenyl–berberine hybrid 8c
could favorably interact with the central catalytic pocket and the
PAS along the active-site gorge of the enzyme with its three major
functional groups: the berberine moiety, alkyl chain, and phenyl
structure–activity relationship showed that the potency of AChE
inhibition was mainly influenced by the function at the end of
the chain, as well as the length of the connecting tether. A kinetic
study indicated that the berberine derivatives cause a mixed type
of inhibition and could interact with both CAS and PAS, consistent
with the results of molecular molding. Thus, berberine and its
derivatives bind and inhibit the peripheral anionic site of the AChE
and may allow for rational design of novel AChE inhibitors.
moiety. The phenyl ring of 8c displays a classic parallel p–p stacking
with the electron-rich indole ring of the Trp86 side chain near the
bottom of the gorge. Its coplanar berberine moiety could stack above
the PAS residue Trp279. Residue Phe330, which forms the ‘bottle
neck’ of the active site gorge, adopts an ‘open’ conformation, allow-
ing largegroupsto reach thebaseof thegorge. Thephenylmoietybe-
4. Experimental section
4.1. Chemistry
tween Trp84 and Phe330 form face-to-face
p–p stacking
interactions in a ‘sandwich’ form. Indeed, the four-methylene ali-
phatic spacer (alkyl tether) links them, snaking along the gorge,
andislongenoughtoallowaproperinteractionbetween8candboth
sites of the AChE. The conformation of the side chain fits the shape of
the gorge. Compound 8c, the highest AChE inhibitor, respectively, as
a typical example to understand the ligand–protein interactions.
In addition, the berberine moiety, linked at the rim of the gorge,
The NMR spectra were recorded with TMS as the internal stan-
dard on a Varian 400 MHz spectrometer. Coupling constants were
given in Hz. MS spectra were recorded on a Agilent LC–MS 6120
instrument with an ESI mass selective detector. Flash column chro-
matography was performed with silica gel (200–300 mesh) pur-
chased from Qingdao Haiyang Chemical Co. Ltd or alumina from
Sinopharm Chemical Reagent Co. Ltd. All the reactions were mon-
itored by thin layer chromatography on silica gel.
Berberine chloride was isolated from Chinese herbal medicine
C. chinensis Franch and recrystallized from hot water. Compound
2 was prepared according to the reported procedure.17 Compounds
3a–e, 4a–e, 5a–e, 6a–e, 7a–e, and 8a–e were synthesized followed
the reported procedures.
gives a face-to-edge p–p interaction with Phe278. The long tether
could fold with a proper conformation in the gorge that might favor-
ably interact with Tyr70, Asp72, Tyr121, Trp279, Ile287, Phe330, and
Tyr334 by hydrophobic interactions. Therefore, the simultaneous
interactions of phenyl–berberine hybrid 8c in the central pocket,
gorge, and peripheral pocket of TcAChE produce inhibitory potency.
3. Conclusion
In summary, three series of berberine derivatives were de-
signed, synthesized, and evaluated for their inhibitory activity
against AChE. Most of them are potent inhibitors of AChE, with
IC50 values in the micromolar and sub-micromolar range. The most
4.2. General procedures for the preparation of 3a–e, 4a–e, 5a–e
To a stirred suspension of selective compounds (3–5) (10 mmol)
and K2CO3 (15 mmol) in butanone (50 mL), KI (1 mmol) and dib-
Figure 3. Predicted binding orientation of the 8C on the binding domain of AChE. (A) Predicted positions of 8C in the active site of AChE. (B) Schematic representation of the
8C (purple) interactions with AChE (red). Hydrogen bonds are indicated with dashed lines. Residues involved in hydrophobic interactions with the ligand are shown with red
rays.

1248
L. Huang et al. / Bioorg. Med. Chem. 18 (2010) 1244–1251
romoalkanes (12 mmol) were added. The mixture was stirred at
room temperature for 1–4 h, filtered, and then evaporated under
vacuum. The crude product was chromatographed on a silica gel
column, eluted with EtOAc/petroleum ether as eluent to afford
the proposed compound.
4c as a white solid, yield 79%; 1H NMR (400 MHz, CDCl3) d: 8.01
(d, J = 8.4 Hz, 1H), 7.51–7.59 (m, 2H), 7.40 (t, J = 7.6 Hz, 1H), 4.60
(t, J = 6.0 Hz, 2H), 3.53 (t, J = 6.4 Hz, 2H), 2.16–2.23 (m, 2H), 2.02–
2.09 (m, 2H).
4.2.9. 1-(5-Bromopentyloxy)-benzotriazole (4d)
4.2.1. 4-(2-Bromoethoxy)-9H-carbazole (3a)
1-Hydroxybenzotriazole (4) was treated with 1,5-dibromopen-
tane according to general procedure to give the desired product
4d as a white solid, yield 77%; 1H NMR (400 MHz, CDCl3) d: 8.00
(d, J = 8.4 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.52 (t, J = 8.4 Hz, 1H),
7.39 (t, J = 8.0 Hz, 1H), 4.57 (t, J = 6.4 Hz, 2H), 3.46 (t, J = 6.4 Hz,
2H), 1.87–2.00 (m, 4H), 1.71–1.78 (m, 2H).
4-Hydroxycarbazole (3) was treated with 1,2-dibromoethane
according to general procedure to give the desired product 3a as
white solid, yield 71%; 1H NMR (400 MHz, CDCl3) d: 8.37 (d,
J = 7.6 Hz, 1H), 8.00 (br s, exchangeable with D2O, 1H), 7.30–7.41
(m, 2H), 7.24–7.27 (m, 2H), 7.02 (d, J = 8.4 Hz, 1H), 6.60 (t,
J = 8.0 Hz, 1H), 4.52 (t, J = 6.0 Hz, 2H), 3.82 (t, J = 6.0 Hz, 2H).
4.2.10. 1-(6-Bromohexyloxy)-benzotriazole (4e)
4.2.2. 4-(3-Bromopropoxy)-9H-carbazole (3b)
1-Hydroxybenzotriazole (4) was treated with 1,6-dibromohex-
ane according to general procedure to give the desired product
4e as a white solid, yield 79%; 1H NMR (400 MHz, CDCl3) d: 8.00
(d, J = 8.4 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H),
7.39 (t, J = 7.6 Hz, 1H), 4.56 (t, J = 6.4 Hz, 2H), 3.44 (t, J = 6.4 Hz,
2H), 1.85–1.95 (m, 4H), 1.51–1.63 (m, 4H).
4-Hydroxycarbazole (3) was treated with 1,3-dibromopropane
according to general procedure to give the desired product 3b as
a white solid, yield 76%; 1H NMR (400 MHz, CDCl3) d: 8.22 (d,
J = 7.6 Hz, 1H), 7.92 (br s, exchangeable with D2O, 1H), 7.32–7.39
(m, 2H), 7.21–7.28 (m, 2H), 6.96 (d, J = 8.4 Hz, 1H), 6.64 (t,
J = 8.0 Hz, 1H), 4.33 (t, J = 6.4 Hz, 2H), 3.72 (t, J = 6.4 Hz, 2H),
2.44–2.50 (m, 2H).
4.2.11. (2-Bromoethoxy)benzene (5a)
Phenol (5) was treated with 1,2-dibromoethane according to
general procedure to give the desired product 5a as oil, yield
87%; 1H NMR (400 MHz, CDCl3) d: 7.17–7.23 (m, 2H), 6.83–6.90
(m, 3H), 4.39 (t, J = 6.0 Hz, 2H), 3.81 (t, J = 6.4 Hz, 2H).
4.2.3. 4-(4-Bromobutoxy)-9H-carbazole (3c)
4-Hydroxycarbazole (3) was treated with 1,4-dibromobutane
according to general procedure to give the desired product 3c as
a white solid, yield 70%; 1H NMR (400 MHz, CDCl3) d: 8.27 (d,
J = 7.6 Hz, 1H), 7.92 (br s, exchangeable with D2O, 1H), 7.21–7.37
(m, 4H), 6.96 (d, J = 8.0 Hz, 1H), 6.61 (t, J = 8.0 Hz, 1H), 4.21 (t,
J = 6.4 Hz, 2H), 3.52 (t, J = 6.4 Hz, 2H), 2.09–2.20 (m, 4H).
4.2.12. (3-Bromopropoxy)benzene (3b)
Phenol (5) was treated with 1,3-dibromopropane according to
general procedure to give the desired product 5b as oil, yield
90%; 1H NMR (400 MHz, CDCl3) d: 7.16–7.22 (m, 2H), 6.81–6.89
(m, 3H), 4.02 (t, J = 6.0 Hz, 2H), 3.52 (t, J = 6.4 Hz, 2H), 2.20–2.26
(m, 2H).
4.2.4. 4-(5-Bromopentyloxy)-9H-carbazole (3d)
4-Hydroxycarbazole (3) was treated with 1,5-dibromopentane
according to general procedure to give the desired product 3d as a
white solid, yield 68%; 1H NMR (400 MHz, CDCl3) d: 8.30 (d,
J = 7.6 Hz, 1H), 7.99 (br s, exchangeable with D2O, 1H), 7.23–7.38 (m,
4H), 7.00 (d, J = 8.0 Hz, 1H), 6.63 (t, J = 8.0 Hz, 1H), 4.22 (t, J = 6.4 Hz,
2H), 3.46 (t, J = 6.8 Hz, 2H), 1.97–2.04 (m, 4H), 1.54–1.80 (m, 2H).
4.2.13. (4-Bromobutoxy)benzene (5c)
Phenol (5) was treated with 1,4-dibromobutane according to
general procedure to give the desired product 5c as oil, yield
86%; 1H NMR (400 MHz, CDCl3) d: 7.25–7.30 (m, 2H), 6.87–6.96
(m, 3H), 3.99 (t, J = 6.0 Hz, 2H), 3.48 (t, J = 6.4 Hz, 2H), 2.03–2.10
(m, 2H), 1.90–1.97 (m, 2H).
4.2.5. 4-(6-Bromohexyloxy)-9H-carbazole (3e)
4-Hydroxycarbazole (3) was treated with 1,6-dibromohexane
according to general procedure to give the desired product 3e as
a white solid, yield 67%; 1H NMR (400 MHz, CDCl3) d: 8.20 (d,
J = 7.6 Hz, 1H), 7.78 (br s, exchangeable with D2O, 1H), 7.12–7.28
(m, 4H), 6.84 (d, J = 8.0 Hz, 1H), 6.53 (t, J = 8.0 Hz, 1H), 4.08 (t,
J = 6.4 Hz, 2H), 3.31 (t, J = 6.8 Hz, 2H), 1.77–1.88 (m, 4H), 1.44–
1.51 (m, 4H).
4.2.14. (5-Bromopentyloxy)benzene (5d)
Phenol (5) was treated with 1,5-dibromopentane according to
general procedure to give the desired product 5d as oil, yield
81%; 1H NMR (400 MHz, CDCl3) d: 7.26–7.31 (m, 2H), 6.89–6.98
(m, 3H), 3.87 (t, J = 6.4 Hz, 2H), 3.40 (t, J = 6.4 Hz, 2H), 1.76–1.90
(m, 4H), 1.58–1.67 (m, 2H).
4.2.6. 1-(2-Bromoethoxy)-benzotriazole (4a)
4.2.15. (6-Bromohexyloxy)benzene (5e)
1-Hydroxybenzotriazole (4) was treated with 1,2-dibromoeth-
ane according to general procedure to give the desired product
4a as a white solid, yield 80%; 1H NMR (400 MHz, CDCl3) d: 8.01
(d, 1H, J = 8.8 Hz), 7.70 (d, 1H, J = 8.4 Hz), 7.55 (t, 1H, J = 8.0 Hz),
7.42 (t, 1H, J = 7.6 Hz), 4.86 (t, J = 6.4 Hz, 2H), 3.69 (t, J = 6.4 Hz, 2H).
Phenol (5) was treated with 1,6-dibromohexane according to
general procedure to give the desired product 5e as oil, yield
79%; 1H NMR (400 MHz, CDCl3) d: 7.28–7.34 (m, 2H), 6.90–6.99
(m, 3H), 3.73 (t, J = 6.0 Hz, 2H), 3.38 (t, J = 6.4 Hz, 2H), 1.73–1.82
(m, 4H), 1.52–1.69 (m, 4H).
4.2.7. 1-(3-Bromopropoxy)-benzotriazole (4b)
4.3. General procedures for the preparation of 6a–e, 7a–e, 8a–e
1-Hydroxybenzotriazole (4) was treated with 1,3-dibromopro-
pane according to general procedure to give the desired product
4b as a white solid, yield 78%; 1H NMR (400 MHz, CDCl3) d: 7.94
(d, J = 8.4 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H),
7.33 (t, J = 8.0 Hz, 1H), 4.64 (t, J = 6.0 Hz, 2H), 3.66 (t, J = 6.4 Hz,
2H), 2.32–2.38 (m, 2H).
Compounds 3a–e, 4a–e, 5a–e (2.4 mmol) was added to a mag-
netically stirred suspension of 2 (2 mmol), K2CO3 (6 mmol) in
CH3CN (15 mL). The mixture was heated in reflux for 12–24 h,
and monitored by TLC. When the mixture was cooled to room tem-
perature, filtered, and then evaporated under vacuum. The crude
product was chromatographed on an Al2O3 column, eluted with
CHCl3/MeOH (100:1–50:1) as eluent to afford the proposed
compound.
4.2.8. 1-(4-Bromobutoxy)-benzotriazole (4c)
1-Hydroxybenzotriazole (4) was treated with 1,4-dibromobu-
tane according to general procedure to give the desired product

L. Huang et al. / Bioorg. Med. Chem. 18 (2010) 1244–1251
1249
4.3.1. 9-O-[2-(9H-Carbazole-4-yloxy)ethyl] berberine bromide
(6a)
LC/MS (ESI) m/z: [MꢀBr]+ 571.7. Anal. Calcd for C37H35BrN2O5: C,
66.57; H, 5.28; N, 4.20. Found: C, 66.49; H, 5.25; N, 4.27.
Berberrubine (2) was treated with 4-(2-bromoethoxy)-9H-car-
bazole (3a) according to general procedure to give the desired
product 6a as a yellow solid, yield 61%; 1H NMR (400 MHz,
DMSO-d6) d: 11.20 (br s, exchangeable with D2O, 1H), 9.64 (s,
1H), 8.71 (s, 1H), 8.24 (d, J = 9.2, 1H), 7.94 (d, J = 9.0, 1H), 7.61 (s,
1H), 7.31–7.33 (m, 3H), 7.18 (d, J = 6.9, 1H), 7.08–7.03 (m, 1H),
6.95 (s, 1H), 6.80–6.71 (m, 2H), 6.16 (s, 2H), 4.88 (t, J = 6.7, 2H),
4.71 (t, J = 6.2, 2H), 4.55 (t, J = 6.5, 2H), 4.13 (s, 3H), 2.68 (t,
J = 6.5, 2H); LC/MS (ESI) m/z: [MꢀBr]+ 531.6. Anal. Calcd for
C33H27BrN2O5: C, 64.82; H, 4.45; N, 4.58. Found: C, 64.77; H,
4.40; N, 4.45.
4.3.6. 9-O-[2-(Benzotriazole-1-yloxy)ethyl] berberine bromide
(7a)
Berberrubine (2) was treated with 1-(2-bromoethoxy)-benzo-
triazole (4a) according to general procedure to give the desired
product 7b as a yellow solid, yield 71%; 1H NMR (400 MHz,
DMSO-d6) d: 10.03 (s, 1H), 9.03 (s, 1H), 8.29 (d, J = 9.2, 1H), 8.15–
8.09 (m, 2H), 7.94 (d, J = 8.3, 1H), 7.88 (s, 1H), 7.73–7.68 (m, 1H),
7.56–7.52 (m, 1H), 7.17 (s, 1H), 6.24 (s, 2H), 5.14 (t, J = 6.4, 2H),
5.02 (t, J = 6.1, 2H), 4.79 (t, J = 6.0, 2H), 4.11.(s, 3H), 3.31 (t,
J = 6.1, 2H); LC/MS (ESI) m/z: [MꢀBr]+ 483.4. Anal. Calcd for
C27H23BrN4O5: C, 57.56; H, 4.11; N, 9.94. Found: C, 57.39; H,
4.10; N, 9.88.
4.3.2. 9-O-[3-(9H-Carbazole-4-yloxy)propyl] berberine bromide
(6b)
Berberrubine (2) was treated with 4-(3-bromopropoxy)-9H-car-
bazole (3b) according to general procedure to give the desired
product 6b as a yellow solid, yield 55%; 1H NMR (400 MHz,
DMSO-d6) d: 11.35 (br s, exchangeable with D2O, 1H), 9.80 (s,
1H), 8.88 (s, 1H), 8.13 (d, J = 9.2, 1H), 8.07 (d, J = 7.7, 1H), 7.97–
7.90 (m, 1H), 7.76 (s, 1H), 7.44 (d, J = 8.1, 1H), 7.31 (t, J = 7.9, 3H),
7.06 (dd, J = 9.1, 4.4, 2H), 6.77 (d, J = 7.9, 1H), 6.17 (s, 2H), 4.78 (t,
J = 6.2, 2H), 4.67 (t, J = 6.2, 2H), 4.51 (t, J = 5.9, 2H), 3.92 (s, 3H),
3.11 (t, J = 6.2, 2H), 2.54 (t, J = 6.1, 2H); LC/MS (ESI) m/z: [MꢀBr]+
545.6. Anal. Calcd for C34H29BrN2O5: C, 65.29; H, 4.67; N, 4.48.
Found: C, 65.21; H, 4.55; N, 4.51.
4.3.7. 9-O-[3-(Benzotriazole-1-yloxy)propyl] berberine bromide
(7b)
Berberrubine (2) was treated with 1-(3-bromopropoxy)-benzo-
triazole (4b) according to general procedure to give the desired
product 7b as a yellow solid, yield 66%;1H NMR (400 MHz,
DMSO-d6) d: 9.83 (s, 1H), 8.94 (s, 1H), 8.22 (d, J = 9.2, 1H), 8.09
(d, J = 8.4, 1H), 8.01 (d, J = 9.1, 1H), 7.87 (d, J = 8.4, 1H), 7.81 (s,
1H), 7.64 (t, J = 7.4, 1H), 7.51–7.45 (m, 1H), 7.10 (s, 1H), 6.18 (s,
2H), 4.91 (dd, J = 14.5, 6.3, 4H), 4.56 (t, J = 6.2, 2H), 4.07 (s, 3H),
3.21 (t, J = 6.4, 2H), 2.43 (m, 2H); LC/MS (ESI) m/z: [MꢀBr]+
497.4. Anal. Calcd for C28H25BrN4O5: C, 58.24; H, 4.36; Br, 13.84;
N, 9.70. Found: C, 58.20; H, 4.25; Br, 13.84; N, 9.51.
4.3.3. 9-O-[4-(9H-Carbazole-4-yloxy)butyl] berberine bromide
(6c)
Berberrubine (2) was treated with 4-(2-bromobutoxy)-9H-car-
bazole (3c) according to general procedure to give the desired
product 6c as a yellow solid, yield 50%; 1H NMR (400 MHz, MeOD)
4.3.8. 9-O-[4-(Benzotriazole-1-yloxy)butyl] berberine bromide
(7c)
Berberrubine (2) was treated with 1-(4-bromobutoxy)-benzo-
triazole (4c) according to general procedure to give the desired
product 7c as a yellow solid, yield 61%; 1H NMR (400 MHz,
DMSO-d6) d: 9.79 (s, 1H), 8.94 (s, 1H), 8.21 (d, J = 9.2, 1H), 8.08
(d, J = 8.5, 1H), 8.00 (d, J = 9.1, 1H), 7.86 (d, J = 8.4, 1H), 7.81 (s,
1H), 7.68–7.62 (m, 1H), 7.52–7.46 (m, 1H), 7.10 (s, 1H), 6.18 (s,
2H), 4.95 (t, J = 6.1, 2H), 4.70 (t, J = 6.3, 2H), 4.38 (t, J = 6.2, 2H),
4.04 (s, 3H), 3.22 (t, J = 6.1, 2H), 2.20–2.11 (m, 2H), 2.11–2.02 (m,
2H); LC/MS (ESI) m/z: [MꢀBr]+ 511.5. Anal. Calcd for C29H27BrN4O5:
C, 58.89; H, 4.60; N, 9.47. Found: C, 58.77; H, 4.55; N, 9.50.
d: 9.22 (s, 1H), 8.18 (s, 1H), 7.96 (d, J = 7.8, 1H), 7.67 (s, 1H), 7.57 (d,
J = 9.1, 1H), 7.50–7.43 (m, 2H), 7.37 (d, J = 8.1, 1H), 7.29–7.24 (m,
1H), 7.15 (t, J = 8.0, 1H), 6.96 (t, J = 7.4, 1H), 6.82 (t, J = 4.0, 2H),
6.57 (d, J = 7.9, 1H), 6.12 (s, 2H), 4.68 (d, J = 5.9, 2H), 4.41 (dt,
J = 10.6, 5.7, 4H), 3.94 (s, 3H), 2.99 (d, J = 6.2, 2H), 2.27–2.19 (m,
4H); LC/MS (ESI) m/z: [MꢀBr]+ 559.6. Anal. Calcd for C35H31BrN2O5:
C, 65.73; H, 4.89; N, 4.30. Found: C, 65.59; H, 4.81; N, 4.22.
4.3.4. 9-O-[5-(9H-Carbazole-4-yloxy)butyl] berberine bromide
(6d)
4.3.9. 9-O-[5-(Benzotriazole-1-yloxy)pentyl] berberine bromide
(7d)
Berberrubine (2) was treated with 4-(2-bromopentyloxy)-9H-
carbazole (3d) according to general procedure to give the desired
product 6d as a yellow solid, yield 48%; 1H NMR (400 MHz,
DMSO-d6) d: 11.32 (br s, exchangeable with D2O, 1H), 9.76 (s,
1H), 8.91 (s, 1H), 8.14 (dd, J = 13.4, 8.5, 2H), 7.96 (d, J = 9.1, 1H),
7.78 (s, 1H), 7.43 (d, J = 8.1, 1H), 7.29 (t, J = 7.9, 2H), 7.11–6.99
(m, 3H), 6.70 (d, J = 7.9, 1H), 6.18 (s, 2H), 4.88 (t, J = 6.0, 2H), 4.36
(t, J = 6.6, 2H), 4.27 (t, J = 6.1, 2H), 4.02 (s, 3H), 3.00 (t, J = 6.1,
2H), 2.10–2.00 (m, 4H), 1.82 (dt, J = 14.7, 7.4, 2H); LC/MS (ESI) m/
z: [MꢀBr]+ 571.7. Anal. Calcd for C36H33BrN2O5: C, 66.16; H, 5.09;
N, 4.29. Found: C, 66.11; H, 5.15; N, 4.13.
Berberrubine (2) was treated with 1-(5-bromopentyloxy)-ben-
zotriazole (4d) according to general procedure to give the desired
product 7d as a yellow solid, yield 60%; 1H NMR (400 MHz,
DMSO-d6) d: 9.77 (s, 1H), 8.93 (s, 1H), 8.20 (d, J = 9.2, 1H), 8.07
(d, J = 8.4, 1H), 8.00 (d, J = 9.1, 1H), 7.84 (d, J = 8.3, 1H), 7.79 (s,
1H), 7.63 (t, J = 7.6, 1H), 7.51–7.44 (m, 1H), 7.09 (s, 1H), 6.18 (s,
2H), 4.94 (t, J = 6.2, 2H), 4.63 (t, J = 6.4, 2H), 4.34 (t, J = 6.5, 2H),
4.05 (s, 3H), 3.18 (t, J = 6.4, 2H), 1.97 (dd, J = 14.2, 6.8, 2H), 1.90
(dd, J = 14.4, 6.7, 2H), 1.80–1.72 (m, 2H); LC/MS (ESI) m/z: [MꢀBr]+
525.5. Anal. Calcd for C30H29BrN4O5: C, 59.51; H, 4.83; N, 9.25.
Found: C, 59.44; H, 4.91; N, 9.43.
4.3.5. 9-O-[6-(9H-Carbazole-4-yloxy)butyl] berberine bromide
(6e)
4.3.10. 9-O-[6-(Benzotriazole-1-yloxy)hexyl] berberine
bromide (7e)
Berberrubine (2) was treated with 4-(2-bromohexyloxy)-9H-
carbazole (3e) according to general procedure to give the desired
product 6e as a yellow solid, yield 49%; 1H NMR (400 MHz,
DMSO-d6) d: 11.23 (br s, exchangeable with D2O, 1H), 9.72 (s,
1H), 8.87 (d, J = 7.4, 1H), 8.32 (s, 1H), 8.13 (dd, J = 12.4, 8.3, 1H),
7.78 (d, J = 6.4, 1H), 7.79 (s, 1H),7.43 (d, J = 8.0, 1H), 7.27 (t,
J = 7.9, 2H), 7.11–6.99 (m, 3H), 6.75 (d, J = 7.9, 1H), 6.18 (s, 2H),
4.88 (d, J = 6.2, 2H), 4.33 (t, J = 6.7, 2H), 4.23 (t, J = 5.9, 2H), 4.01
(s, 3H), 3.16 (t, J = 6.1, 2H), 1.97–2.05 (m, 4H), 1.68–1.74 (m, 4H);
Berberrubine (2) was treated with 1-(6-bromohexyloxy)-ben-
zotriazole (4e) according to general procedure to give the desired
product 7e as a yellow solid, yield 57%; 1H NMR (400 MHz,
DMSO-d6) d: 9.76 (s, 1H), 8.93 (s, 1H), 8.20 (d, J = 9.2, 1H), 8.07
(d, J = 8.5, 1H), 7.99 (d, J = 9.1, 1H), 7.83 (d, J = 8.3, 1H), 7.80 (s,
1H), 7.63 (t, J = 7.6, 1H), 7.51–7.45 (m, 1H), 7.10 (s, 1H), 6.18 (s,
2H), 4.94 (t, J = 6.2, 2H), 4.59 (t, J = 6.4, 2H), 4.32 (t, J = 6.7, 2H),
4.05 (s, 3H), 3.20 (t, J = 6.2, 2H), 1.97–1.89 (m, 2H), 1.88–1.80 (m,

1250
L. Huang et al. / Bioorg. Med. Chem. 18 (2010) 1244–1251
2H), 1.67–1.56 (m, 4H); LC/MS (ESI) m/z: [MꢀBr]+ 539.7. Anal.
Calcd for C31H31BrN4O5: C, 60.10; H, 5.04; N, 9.04. Found: C,
60.15; H, 5.08; N, 9.11.
4.4. Biological activity
4.4.1. In vitro inhibition studies on AChE and BuChE
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel),
butylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum), 5,50-
dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), acetylthi-
ocholine chloride (ATC), butylthiocholine chloride (BTC), and
tarcine hydrochloride were purchased from Sigma–Aldrich. Ber-
berine derivatives were dissolved in DMSO and diluted in 0.1 M
KH2PO4/K2HPO4 buffer (pH 8.0) to provide a final concentration
range. DMSO was diluted to a concentration in excess of 1 in
10,000, and no inhibitory action on either AChE or BuChE was
detected in separate prior experiments.
4.3.11. 9-O-[2-(Phenylol-1-yloxy)ethyl] berberine bromide (8a)
Berberrubine (2) was treated with (2-bromoethoxy)benzene
(5a) according to general procedure to give the desired product
8a as a yellow solid, yield 71%; 1H NMR (400 MHz, DMSO-d6) d:
9.76 (s, 1H), 8.93 (s, 1H), 8.20 (d, J = 9.2 Hz, 1H), 8.02 (d,
J = 9.2 Hz, 1H), 7.79 (s, 1H), 7.28 (t, J = 8.0 Hz, 2H), 7.07 (s, 1H),
6.93 (t, J = 7.6 Hz, 1H), 6.86 (d, J = 7.6 Hz, 2H), 6.17 (s, 2H), 4.78
(t, J = 6.0 Hz, 2H), 4.67 (t, J = 6.1 Hz, 2H), 4.43 (t, J = 6.5 Hz, 2H),
4.06 (s, 3H), 3.12 (t, J = 6.4 Hz, 2H); LC/MS (ESI) m/z: [MꢀBr]+
442.4. Anal. Calcd for C27H24BrNO5: C, 62.08; H, 4.63; N, 2.68.
Found: C, 62.01; H, 4.60; N, 2.78.
4.4.2. In vitro AChE assay
All the assays were carried out under 0.1 M KH2PO4/K2HPO4
buffer, pH 8.0, using a Shimadzu UV-2450 Spectrophotometer. En-
zyme solutions were prepared to give 2.0 units/mL in 2 mL ali-
quots. The assay medium (1 mL) consisted of phosphate buffer
4.3.12. 9-O-[3-(Phenylol-1-yloxy)propyl] berberine bromide
(8b)
Berberrubine (2) was treated with (3-bromopropoxy)benzene
(5b) according to general procedure to give the desired product
8b as a yellow solid, yield 75%; 1H NMR (400 MHz, DMSO-d6) d:
9.78 (s, 1H), 8.94 (s, 1H), 8.18 (d, J = 9.2 Hz, 1H), 7.98 (d,
J = 9.2 Hz, 1H), 7.80 (s, 1H), 7.31 (t, J = 8.0 Hz, 2H), 7.09 (s, 1H),
6.93–6.99 (m, 3H), 6.18 (s, 2H), 4.87 (t, J = 6.0 Hz, 2H), 4.48 (t,
J = 6.0 Hz, 2H), 4.26 (t, J = 6.4 Hz, 2H), 4.00 (s, 3H), 3.19 (t,
J = 6.0 Hz, 2H), 2.34 (t, J = 6.4 Hz, 2H); LC/MS (ESI) m/z: [MꢀBr]+
456.5. Anal. Calcd for C28H26BrNO5: C, 62.69; H, 4.89; N, 2.61.
Found: C, 62.60; H, 4.77; N, 2.78.
(pH 8.0), 50 lL of 0.01 M DTNB, 10 lL of enzyme, and 50 lL of
0.01 M substrate (ACh chloride solution). Test compounds were
added to the assay solution and preincubated at 37 °C with the en-
zyme for 15 min followed by the addition of substrate. The activity
was determined by measuring the increase in absorbance at
412 nm at 1 min intervals at 37 °C. Calculations were performed
according to the method of the equation in Ellman et al.18 Each
concentration was assayed in triplicate.
In vitro BuChE assay was similar with the method described
above.
4.3.13. 9-O-[4-(Phenylol-1-yloxy)butyl] berberine bromide (8c)
Berberrubine (2) was treated with (4-bromobutoxy)benzene
(5c) according to general procedure to give the desired product
8c as a yellow solid, yield 76%; 1H NMR (400 MHz, DMSO-d6) d:
9.81 (s, 1H), 8.97 (s, 1H), 8.23 (d, J = 9.2 Hz, 1H), 8.04 (d,
J = 9.2 Hz, 1H), 7.84 (s, 1H), 7.34 (t, J = 7.6 Hz, 2H), 7.14 (s, 1H),
6.95–7.00 (m, 3H), 6.22 (s, 2H), 4.99 (t, J = 6.0 Hz, 2H), 4.42 (t,
J = 6.0 Hz, 2H), 4.13 (t, J = 5.2 Hz, 2H), 4.09 (s, 3H), 3.25 (t,
J = 6.0 Hz, 2H), 2.02–2.10 (m, 4H); LC/MS (ESI) m/z: [MꢀBr]+
470.5. Anal. Calcd for C29H28BrNO5: C, 63.28; H, 5.13; N, 2.54.
Found: C, 63.33; H, 5.14; N, 2.49.
4.5. Kinetic characterization of ChE inhibition
Kinetic characterization of AChE and BuChE was performed
using a reported method.29 Test compound was added into the as-
say solution and pre-incubated with the enzyme at 37 °C for
15 min, followed by the addition of substrate. Kinetic characteriza-
tion of the hydrolysis of ATC catalyzed by AChE was done spectro-
metrically at 412 nm. A parallel control with no inhibitor in the
mixture, allowed adjusting activities to be measured at various
times. The plots were assessed by a weighted least square analysis
that assumed the variance of V to be a constant percentage of V for
the entire data set. Slopes of these reciprocal plots were then plot-
ted against the concentration of the inhibitors in a weighted anal-
ysis and Ki was determined as the ratio of the replot intercept to
the replot slope.
4.3.14. 9-O-[5-(Phenylol-1-yloxy)pentyl] berberine bromide
(8d)
Berberrubine (2) was treated with (5-bromopentyloxy)benzene
(5d) according to general procedure to give the desired product 8d
as a yellow solid, yield 66%; 1H NMR (400 MHz, DMSO-d6) d: 9.75
(s, 1H), 8.92 (s, 1H), 8.19 (d, J = 9.1, 1H), 8.00 (d, J = 9.2, 1H), 7.79
(s, 1H), 7.33–7.23 (m, 2H), 7.09 (s, 1H), 6.95–6.89 (m, 3H), 6.17
(s, 2H), 4.95 (t, J = 6.0, 2H), 4.31 (t, J = 6.0, 2H), 4.05 (s, 3H), 4.02
(t, J = 6.4, 2H), 3.21 (t, J = 6.4, 2H), 2.01–1.79 (m, 4H), 1.64 (tdd,
J = 24.2, 11.8, 6.8, 2H); LC/MS (ESI) m/z: [MꢀBr]+ 484.5. Anal. Calcd
for C30H30BrNO5: C, 63.83; H, 5.36; N, 2.48. Found: C, 63.81; H,
5.30; N, 2.56.
Kinetic characterization of BuChE assay use the similar method
described above.
4.6. Molecular modeling
The initial model of AChE for docking studies was built based on
the X-ray crystal structure of the bistacrine–AChE complex which
was obtained from the Protein Data Bank (PDB entry 2CMF). The
original ligand was removed while water molecules present in
the PDB file were maintained in their position. 3D structures of
the 9-substituted berberine derivatives were generated and opti-
mized by DISCOVERY STUDIO 1.7 package (Accelrys Inc., San Diego,
CA). AUTODOCK 3.0.5 package30 was used to perform docking simula-
tions, which adopts the hybrid Lamarckian Genetic Algorithm as
searching algorithm and allows full flexibility of the ligand. Solva-
tion parameters and Kollman charges for all atoms in AChE were
assigned by using AUTODOCK Tools. The grid for energy evaluation
was centered at residue Trp84 in AChE with grid points in the x-,
y-, z-axes set to 50 ꢁ 50 ꢁ 50 and separated by 0.375 Å. The initial
population size and maximum number of energy evaluations were
set to 100 and 1.0 ꢁ 107 respectively. The docked results within an
4.3.15. 9-O-[2-(Phenylol-1-yloxy)hexyl] berberine bromide (8e)
Berberrubine (2) was treated with (6-bromohexyloxy)benzene
(5e) according to general procedure to give the desired product
8e as a yellow solid, yield 61%; 1H NMR (400 MHz, DMSO-d6) d:
9.74 (s, 1H), 8.92 (s, 1H), 8.19 (d, J = 9.1, 1H), 8.00 (d, J = 9.2, 1H),
7.79 (s, 1H), 7.32–7.24 (m, 2H), 7.09 (s, 1H), 6.91 (dd, J = 7.5, 4.6,
3H), 6.17 (s, 2H), 5.95 (t, J = 6.4, 2H), 4.30 (dd, J = 11.9, 6.6, 2H),
4.05 (s, 3H), 3.98 (dd, J = 13.0, 6.6, 2H), 3.21 (dd, J = 10.8, 5.4, 2H),
1.95–1.72 (m, 4H), 1.54 (dd, J = 8.6, 5.6, 4H). LC/MS (ESI) m/z:
[MꢀBr]+ 498.6. Anal. Calcd for C31H32BrNO5: C, 64.36; H, 5.58; N,
2.42. Found: C, 64.32; H, 5.51; N, 2.53.

L. Huang et al. / Bioorg. Med. Chem. 18 (2010) 1244–1251
1251
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53, 753.
RMSD of 1.5 Å were clustered, and the final results of each ligand
were selected considering both the embedded empirical binding
free energy evaluation and the clustering analysis.
13. Zhang, M. F.; Shen, Y. Q. Zhongguo Yao Li Xue Bao 1989, 10, 174.
14. Zhou, H.; Mineshita, S. J. Pharmacol. Exp. Ther. 2000, 294, 822.
15. Cho, K. M.; Yoo, I. D.; Kim, W. G. Biol. Pharm. Bull. 2006, 29, 2317.
16. Li, Y. H.; Peng, Y.; Kong, W. J.; Wang, Y. X.; Hu, C. Q.; Zuo, Z. Y.; Wang, Y. M.;
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We thank the Natural Science Foundation of China (20472116),
the Natural Science Foundation of Guangdong (84510089010
00380) for financial support of this study.
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