Synthesis, biological evaluation of 9-N-substituted berberine derivatives as multi-functional agents of antioxidant, inhibitors of acetylcholinesterase, butyrylcholinesterase and amyloid-β aggregation

Article abstract of DOI:10.1016/j.ejmech.2011.09.051

A series of 9-N-substituted berberine derivatives were synthesized and biologically evaluated as antioxidant and inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase and amyloid-β aggregation. Most of these compounds exhibited very good antioxidant activities, inhibitive activities of AChE and amyloid-β aggregation. Among them, compound 8d, (o-methylphenethyl)amino linked at the 9-position of berberine, was found to be a good antioxidant (with 4.05 μM of Trolox equivalents), potent inhibitor of AChE (an IC₅₀ value of 0.027 μM), and high active inhibitor of amyloid-β aggregation (an IC₅₀ value of 2.73 μM).

Full text of DOI:10.1016/j.ejmech.2011.09.051

European Journal of Medicinal Chemistry 46 (2011) 5885e5893
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological evaluation of 9-N-substituted berberine derivatives as
multi-functional agents of antioxidant, inhibitors of acetylcholinesterase,
butyrylcholinesterase and amyloid-b aggregation
*
Wen-Jun Shan, Ling Huang, Qi Zhou, Fan-Chao Meng, Xing-Shu Li
Institute of Drug Synthesis and Pharmaceutical Processing, 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:
Received 4 May 2011
Received in revised form
28 September 2011
Accepted 28 September 2011
Available online 5 October 2011
A series of 9-N-substituted berberine derivatives were synthesized and biologically evaluated as anti-
oxidant and inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase and amyloid-
Most of these compounds exhibited very good antioxidant activities, inhibitive activities of AChE and
amyloid- aggregation. Among them, compound 8d, (o-methylphenethyl)amino linked at the 9-position
of berberine, was found to be a good antioxidant (with 4.05 M of Trolox equivalents), potent inhibitor of
AChE (an IC50 value of 0.027 M), and high active inhibitor of amyloid- aggregation (an IC50 value of
2.73 M).
b aggregation.
b
m
m
b
m
Keywords:
9-N-substituted berberine derivative
Acetylcholinesterase inhibitor
Ó 2011 Elsevier Masson SAS. All rights reserved.
Amyloid-b aggregation inhibitory
Antioxidant properties
Alzheimer’s disease
1. Introduction
At present, only AChE inhibitors, such as tacrine [6] (A in Fig. 1),
galanthamine [7] (B in Fig. 1), huperzine A [8] (C in Fig. 1) and done-
pezil [9] (D in Fig.1), have been used mainly for the clinical treatment
of AD. However, clinical experience shows that inhibition of ChEs is
a palliative treatment, which does not address AD’s etiology [10]. The
patients are beneficial in improving cognitive and behavioral symp-
toms, but the neurodegeneration has not been prevented [11]. Due to
the multi-pathogenesis of AD, the approach is gradually moving from
the development of single target drugs to the “multi-target-directed
ligands”, and the design strategy is usually to combine distinct
pharmacophores of different drugs in one hybrid molecule [12,13]. In
recent years, a significant number of studies of multivalent ligand
strategies to design hybrid compounds for simultaneously inhibiting
Alzheimer’s disease (AD), a neurodegenerative disorder char-
acterized by a progressive deterioration of memory and cognition,
occurs more frequently in elderly people. Several diverse hall-
marks, such as deposits of aberrant proteins (b-amyloid (Ab) and
s
-protein), oxidative stress, dyshomeostasis of biometals, and low
levels of acetylcholine (ACh) appear to play significant roles in the
pathophysiology of the disease [1]. Among them, the deposition of
Ab in the brain is the pathological hallmark of AD and one of the
potential reasons of the neuronal damage [2]. According to the
“amyloid hypothesis”, the production and accumulation of oligo-
meric aggregates of Ab are a central event in the pathogenesis of
AD, as they are thought to be able to initiate the pathogenic
cascade, ultimately leading to neuronal loss and dementia [3].
acetylcholine hydrolysis, AChE-induced Ab aggregation and other
pathogeneses have been designed and synthesized [14e20].
However, from both fundamental and practical standpoints, it is
desirable to develop new “multi-target-directed ligands” candidate
drugs with natural medicines as the lead compounds.
Berberine, an isoquinoline alkaloid isolated from the roots and
stem-bark of berberis species, is widely used as a traditional
medicine for treating diarrhea [21] and gastrointestinal disorders
[22]. In recent years, berberine has been demonstrated to show
a wide range of pharmacological activities including antihyperten-
sive, anti-inflammatory, antioxidant, antidepressant, anticancer,
anti-diarrheal, cholagogue, hepatoprotective and antimicrobial [23].
Therefore, to prevent A
b self-aggregation is an attractive thera-
peutic strategy for the treatment of AD [4]. At the same time, AD is
probably associated with multifaceted etiologies and pathogenic
phenomena, and all mechanisms seem to share oxidative stress as
a unifying factor, which is thought to have a causative role in the
pathogenesis of AD recently [5].
* Corresponding author. Tel./fax: þ86 20 39943050.
E-mail address: lixsh@mail.sysu.edu.cn (X.-S. Li).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.051

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W.-J. Shan et al. / European Journal of Medicinal Chemistry 46 (2011) 5885e5893
Fig. 1. Structure of reported AChE inhibitors.
In a study examining the treatment of AD, berberine was also
found to inhibit acetylcholinesterase [24] and significantly reverse
the amyloid peptide (Ab)-induced memory damage when assessed
in the Morris water maze [25]. In our previous work on the design,
synthesis and biological evaluation of dual inhibitors of AChE/
BuChE using berberine as the lead structure, a series of novel 9-O
substituted berberine derivatives were synthesized. Biological
evaluation showed that berberine linked with phenol by 4-carbon
spacers (E in Fig. 1) [26], berberine linked with 3-methylpyridinium
by a 2-carbon spacer (F in Fig. 1) [27], and a cyclohexylamino group
linked to berberine by a three carbon spacer (G in Fig. 1) [28],
exhibited highly potent AChE inhibition with IC50 values of 0.097,
Scheme 1. Reagents and condition: (a) Br(CH2)nBr, K2CO3, TEBAC, acetone, 24 h; (b)
phenol, K2CO3, DMF; (c) Hydrazine Hydrate, EtOH, reflux, 4 h; (d) Amines, EtOH, reflux,
16e24 h.
Finally, treatment of berberine with primary amines produced the
target products amino-isoquinolinium derivatives. Other
9-N-substituted berberine derivatives, such as 6ae6c, 7ae7c,
8ae8h and 10ae10c, were prepared by the direct reaction of
berberine with corresponding amines that are commercially
available. The reaction of berberine with amines was carried out
according to the precedure in the literature [35].
0.048 and 0.020 mM, respectively. Molecular modeling showed that
these 9-O substituted berberine derivatives could interact with the
catalytic active site (CAS) and the peripheral cationic site (PAS) of
AChE simultaneously.
It is well-known that aromatic amines have antioxidant prop-
erties as which may be potential sites for the attack of both OH_and
2.2. Biological activities
ꢀ
e radicals [29]. Therefore, the replacement of an oxygen atom of the
2.2.1. In vitro inhibition studies of AChE and BuChE
9-O substituted berberine derivatives by a NH may result in some
different properties. On the other hand, as many literatures re-
ported some dual binding site AChE inhibitors exhibited a signifi-
cant inhibitory activity on A
speculated that berberine derivatives might be able to inhibit the
self-induced A aggregation. With these concepts in mind, in this
paper, we design, synthesize, and evaluate a new series of 9-N
substituted berberine derivatives which were found to show
interesting biological activities including AChE and BuChE inhibi-
tion, inhibition of self-induced A
properties.
The inhibitory activity of the 9-N substituted berberine deriva-
tives 5e10 were evaluated toward AChE from electric eel and
BuChE from equine serum, using berberine as the reference stan-
dard and the method of Ellman et al. [36]. The IC50 values for AChE
and BuChE inhibition are summarized in Table 1. In contrast to
berberine, the results revealed that most of the berberine deriva-
tives were potent inhibitors of BuChE with IC50 values ranging from
micromolar to sub-micromolar concentrations. However, some of
the 9-N substituted berberine derivatives exhibited slightly poorer
AChE inhibitory activities compared with their 9-O substituted
relatives. Nonetheless, a similar trend between the inhibitory
potency and the length of the alkylene chain was observed using
a simple structureeactivity relationship analysis. For instance,
series 5, with 2, 3, 4, 5 and 6 methylene groups between the
berberine and phenoxyl group, compound 5c was the best inhibitor
b self-aggregation [18,30e34], we
b
b aggregation and antioxidant
2. Results and discussion
2.1. Chemistry
(0.349
lent 9-O substituted berberine derivative (0.097
interesting that compound 6b, with a phenyl linked with berberine
by two methylene groups, provided the highest potency (0.209 M)
in series 6. The results indicated that the two methylene groups
used as the linker were favorable for the inhibition observed for this
series. Inspired by these results, a series of berberine derivatives
m
M) in this series, which was much lower than the equiva-
The synthetic pathway of 9-N-substituted berberine derivatives
is shown in Scheme 1. 5ae5e, 9a and 9b are synthesized via four
steps. First, the reaction of phthalimide with dibromoalkanes in the
presence of K2CO3 provided the intermediate 2. Compound 2 was
then reacted with phenol to give 3, which was reduced by hydra-
zine to afford the primary amines 4 in almost quantitative yields.
m
M) [26]. It was
m

W.-J. Shan et al. / European Journal of Medicinal Chemistry 46 (2011) 5885e5893
5887
Table 1
In vitro inhibition IC50
(
mM) and selectivity of compounds 5e10 for AChE and BuChE and antioxidant activities.
Entry
Compounds
n
IC50
(
mM)
Selectivity for AChEc
Trolox equivd
AChE ꢁ SEMa
BuChE ꢁ SEMb
1
2
3
4
5
6
7
8
5a
5b
5c
5d
5e
6a
6b
6c
7a
7b
7c
8a
8b
8c
8d
8e
2
3
4
5
6
1
2
3
2
2
2
2
2
2
2
2
2
2
2
4
4
0
1
3
e
e
e
0.688 ꢁ 0.016
0.588 ꢁ 0.012
0.349 ꢁ 0.020
1.094 ꢁ 0.091
1.419 ꢁ 0.037
0.936 ꢁ 0.036
0.209 ꢁ 0.011
0.680 ꢁ 0.017
1.261 ꢁ 0.106
0.608 ꢁ 0.029
1.160 ꢁ 0.098
0.990 ꢁ 0.030
0.238 ꢁ 0.021
0.693 ꢁ 0.021
0.027 ꢁ 0.002
0.091 ꢁ 0.005
0.449 ꢁ 0.027
0.160 ꢁ 0.010
0.130 ꢁ 0.014
0.400 ꢁ 0.030
1.167 ꢁ 0.032
1.914 ꢁ 0.037
1.143 ꢁ 0.094
0.576 ꢁ 0.036
0.374 ꢁ 0.036
0.097 ꢁ 0.005
0.311 ꢁ 0.009
0.727 ꢁ 0.038
0.216 ꢁ 0.007
1.201 ꢁ 0.028
1.556 ꢁ 0.126
1.300 ꢁ 0.044
2.282 ꢁ 0.061
0.406 ꢁ 0.039
0.785 ꢁ 0.013
1.391 ꢁ 0.109
0.555 ꢁ 0.032
2.495 ꢁ 0.063
2.885 ꢁ 0.089
0.445 ꢁ 0.017
0.841 ꢁ 0.052
0.713 ꢁ 0.016
0.674 ꢁ 0.011
0.821 ꢁ 0.011
5.818 ꢁ 0.052
0.424 ꢁ 0.015
0.511 ꢁ 0.032
2.936 ꢁ 0.089
3.280 ꢁ 0.357
2.016 ꢁ 0.099
6.323 ꢁ 0.046
18.2 ꢁ 0.683
4.89 ꢁ 0.035
0.041 ꢁ 0.003
1.06
0.37
3.44
1.42
0.92
2.43
1.94
1.15
1.10
0.91
2.15
2.91
1.86
1.21
26.4
7.4
1.83
36.4
3.26
1.28
2.52
1.71
1.76
11.0
48.6
50.4
0.13
5.15 ꢁ 0.06
5.40 ꢁ 0.34
3.68 ꢁ 0.23
3.64 ꢁ 0.17
2.80 ꢁ 0.22
4.32 ꢁ 0.18
4.80 ꢁ 0.11
3.51 ꢁ 0.32
6.06 ꢁ 0.18
4.18 ꢁ 0.14
6.40 ꢁ 0.51
4.68 ꢁ 0.14
4.48 ꢁ 0.31
2.83 ꢁ 0.01
4.05 ꢁ 0.09
4.73 ꢁ 0.35
4.56 ꢁ 0.16
5.30 ꢁ 0.05
3.36 ꢁ 0.18
2.49 ꢁ 0.04
4.63 ꢁ 0.37
2.17 ꢁ 0.06
5.12 ꢁ 0.09
5.70 ꢁ 0.39
0.40
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
8f
8g
8h
9a
9b
10a
10b
10c
Berberine
E
1.0
e
Tacrine
SEM: Standard Error of the Mean.
a
50% inhibitory concentration (means ꢁ SEM of three experiments) of AChE from electric eel.
50% inhibitory concentration (means ꢁ SEM of three experiments) of BuChE from equine serum.
Selectivity for AChE ¼ IC50 (BuChE)/IC50 (AChE).
b
c
d
Data are expressed as
mmol of trolox equivalent/
m
mol of tested compound (means ꢁ SEM of three experiments).
with two methylene groups between the berberine and various
aromatic ring units were synthesized and evaluated. Compounds
7ae7c, with nitrogen or sulfur-containing groups linked with
berberine, gave IC50 values from 0.680 to 1.261 mM. Most berberine
derivatives linked with a substituted phenyl exhibited potent
simulations using the CDOCKER program in the Discovery studio
2.1 software. Compound 8d was initially docked to the TcAChE
active site cavity based on the structure of the T. californica enzyme
complex (TcAChE; PDB entry 2CMF) and the resulting structure of
the active site was shown in Fig. 3. The most likely conformations of
the ligand were selected based on the calculated docking score for
the bonding strength. The modeling demonstrated that the
inhibitory activities. Among them, compound 8d, the most potent
for AChE inhibition, gave an IC50 value of 0.027 mM, about 13-fold
higher than that of berberine. Additionally, the position of the
substituted groups on the phenyl ring also influenced the inhibitory
activities. Compounds 8e and compound 8f, with m-methyl or
p-methyl, both gave relatively higher IC50 values than those of their
o-substituted homologs (Table 1, entries 16ꢀ17). The results also
indicated that bi-substituted compounds such as 8g and 8h, gave
moderate inhibitory activities. The bicyclic derivative 9a showed
slightly lower activities than its monocyclic homolog 5c, which was
in accord with our previous studies.
2.2.2. Kinetic study of ACHE
It is important to determinate the type of inhibition in an effort
to understand the mechanism of inhibition and the binding sites of
the inhibitor sites. To investigate this, the 9-N-berberine derivative
8d, which was the most potent AChE inhibitor, was used in a kinetic
study of AChE inhibition. The results (Fig. 2) showed that the
inhibitory behavior of 8d on EeAChE is similar to that of bis-
tetrahydroaminoacridine [37] inhibitors of EeAChE. Therefore,
from the kinetic profile, we concluded that compound 8d was
a mixed-type of inhibitor, which binds at both the catalytic and the
peripheral sites of AChE.
2.2.3. Molecular modeling studies
The possible interaction mode between the 9-N-berberine
derivatives and AChE was studied by molecular docking
Fig. 2. Steady-state inhibition by 8d of AChE hydrolysis of ACh. Reciprocal plots of
initial velocity and substrate concentration showing mixed-type inhibition for 8d on
AChE.

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W.-J. Shan et al. / European Journal of Medicinal Chemistry 46 (2011) 5885e5893
Fig. 4. Docking model of compoundeenzyme complex. 9-N-berberine derivatives 8e
(blue), 8f (yellow) docked into the catalytic gorge of TcAChE(code ID: 2CMF) and
superposition with 8d (amaranthine). (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
Fig. 3. 9-N-berberine derivatives 8d (gray) docked into the catalytic gorge of TcAChE
(code ID: 2CMF), highlighting the protein residues belonging to CAS and PAS that
establish the main interactions.
aromatic ring B in 8d reached the top of the cavity and interacted
equivalents. Other compounds derived from 9-N-(2-phenylethyl)
berberine also showed good antioxidant activities, which exceeded
4-fold the value of trolox. On the other hand, berberine and
compound E, which berberine linked by 9-O with phenol via
4-carbon spacers, provided only 0.40 and 1.0 Trolox equivalents.
The results indicated that the 9-N structure of berberine derivatives
was indeed necessary for high potent antioxidant activities.
with PAS residue Trp279 (4.28 Å) through
pep stacking. The
conformation of the side-chain of 8d fits nicely into the shape of the
cavity. At the bottom of the binding site, the phenyl moiety made
pep stacking interactions with Trp84 and Phe330, with distances
of 4.227 Å and 4.533 Å, respectively. The 9-amino-group of 8d
formed a direct hydrogen bond with the backbone OH group of
Tyr121, in which the oxygen atom served as hydrogen-bond
acceptor (average O.H distance ¼ 2.925 Å).
2.2.5. Effects on the Ab1e42 peptide aggregation
On the basis of the results of in vitro inhibition studies of AChE
and BuChE, the ability of 9-N-berberine derivatives to reduce
For estimating the ability to penetrate bloodebrain barrier of 9-
N-substituted berberine derivatives, the logBBB value of compound
8d was predicted with the ADMET program in Discovery studio 2.1.
The result (0.885 of logBBB value) indicated that 8d could penetrate
bloodebrain barrier easily.
Ab1e42 self-aggregation was studied through a thioflavin T-based
fluorimetric assay with curcumin (Cur) as standard compound. The
results summarized in Table 2 (the IC50 values were also exhibited
in the table) indicated that all tested 9-N-berberine derivatives
showed higher potencies compared to those of the curcumin
(52.1%) and berberine (36.3%) at 20 mM. Among them, compound
8d, which expressed the most potent ability for AChE inhibition,
9-N-Berberine derivatives 8e and 8f were also docked into
TcAChE using the same modeling method. Superpositioning of 8e
and 8f with 8d (Fig. 4) showed that the berberine moieties essen-
tially overlap, except for the slight differences in the side-chains.
The difference in the inhibitory activities implied that the steric
effect most likely played a key role for these 9-N-Berberine deriv-
Table 2
atives, which might have
pep stacking interactions between the
Inhibition of self-induced Ab1e42 aggregation by some 9-N-berberine derivatives
with curcumin as a reference.
phenyl moiety of the ligands and Trp84 (due to the different spatial
positions of the phenyl moieties, the distance between the phenyl
moieties of 8d, 8e or 8f and Trp84 were 4.533, 4.623 and 4.776 Å,
respectively).
Compound
Inhibition ratio (%)a
20 10 mM
IC50 (mM)
m
M
Curcumin
Berberine
52.1
36.3
92.2
87.8
91.1
90.4
82.6
95.0
93.6
94.0
70.1
93.6
30.7
8.9
n.t.
n.t.
n.t.
n.t.
2.56
2.58
2.94
2.73
1.80
3.75
>5
2.2.4. Antioxidant activity assay
6b
7b
8a
8b
8c
8d
8e
8f
79.2
72.8
79.0
76.3
73.7
80.0
79.0
80.0
54.1
81.3
The antioxidant activities of 9-N-berberine derivatives were
determined by the method of oxygen radical absorbance capacity of
fluorescein (ORAC-FL), which were described by Ou et al. [38], and
then optimized by Dávalos et al. [39]. Vitamin E analog, trolox was
used as a standard (the activities were expressed as trolox equiv-
alents, mmol of trolox equivalents/mmol of tested compound). The
results in Table 1 showed that all 9-N-berberine derivatives
exhibited high potent peroxyl radical absorbance capacities ranging
from 2 to 6-fold the value of trolox. Among them, compounds 7a
and 7c, which bearing an indole or a pyridine group on 9-position
of berberrine, gave the best results with 6.40 and 6.06 Trolox
8g
8h
2.22
n.t.: not tested.
a
Inhibition of self-induced Ab1e42 aggregation (50
M and 10 M concentration.
mM) produced by the tested
compound at 20
m
m

W.-J. Shan et al. / European Journal of Medicinal Chemistry 46 (2011) 5885e5893
5889
showed the highest potency of A
b
aggregation inhibition (95.0%).
eluent) to give the products. The preparation of compounds 3ae3e
Similarly, compounds 8e and 8f, the substitution isomerisms of 8d,
gave 93.6% and 94.0% inhibition potency, respectively. On the other
hand, compounds 8b and 8h, with halogen substituents on the
phenyl, gave 90.4% and 93.6% inhibition potency. However,
compound 8g, with 3,4-dimethoxyphenyl substituent, was found
less favorable for the inhibition potency (70.1%).
was according to the method previously described [42].
4.3.1. 2-(2-Phenoxyethyl)isoindoline-1,3-dione (3a)
Compound 3a was synthesized according to the general proce-
dure to give the desired product in 90% yield. 1H NMR (400 MHz,
CDCl3)
d 7.88e7.74 (m, 2H), 7.74e7.69 (m, 2H), 7.27e7.21 (m, 2H),
6.94e6.86 (m, 3H), 4.23 (t, J ¼ 6.0 Hz, 2H), 4.11 (t, J ¼ 6.0 Hz, 2H); 13C
NMR (101 MHz, CDCl3)
d 168.17, 158.29, 134.02, 132.05, 129.43,
3. Conclusions
123.35, 121.11, 114.62, 64.61, 37.35.
In summary, a series of new 9-N-substituted berberine deriva-
tives were synthesized and subjected to pharmacological evalua-
tion. The results showed that most of 9-N-substituted berberine
derivatives possessed moderate to high AChE inhibition activity.
Kinetic studies and molecular modeling simulations indicated that
a mixed-competitive binding mode for these berberine derivatives.
Antioxidant activity assay indicated that most of these berberine
4.3.2. 2-(3-Phenoxypropyl)isoindoline-1,3-dione (3b)
Compound 3b was synthesized according to the general
procedure to give the desired product in 92% yield. 1H NMR
(400 MHz, CDCl3) d 7.86e7.82 (m, 2H), 7.73e7.69 (m, 2H), 7.26e7.21
(m, 2H), 6.94e6.89 (m, 1H), 6.82e6.79 (m, 2H), 4.03 (t, J ¼ 6.0 Hz,
2H), 3.91 (t, J ¼ 7.2 Hz, 2H), 2.22e2.15 (m, 2H); 13C NMR (101 MHz,
CDCl3) d 168.37, 158.69, 133.91, 132.17, 129.37, 123.23, 120.76, 114.47,
derivatives were good antioxidants with a range of 2.2e6.4
Trolox equivalents. These berberine derivatives also showed high
potencies to inhibit self-induced amyloid- aggregation, all the ten
mM
65.55, 35.52, 28.34.
b
4.3.3. 2-(4-Phenoxybutyl)isoindoline-1,3-dione (3c)
tested compounds exhibited more potent inhibitive activities than
that of the reference compound curcumin. It is worthy to note that
compound 8d, with (o-methylphenethyl)amino linked at the
9-position of berberine, showed high inhibitory activity toward
Compound 3c was synthesized according to the general proce-
dure to give the desired product in 87% yield. 1H NMR (400 MHz,
CDCl3)
6.94e6.86 (m, 3H), 3.99 (t, J ¼ 6.0 Hz, 2H), 3.77 (t, J ¼ 7.2 Hz, 2H),
1.93e1.80 (m, 4H); 13C NMR (101 MHz, CDCl3)
168.41, 158.91,
d 7.86e7.82 (m, 2H), 7.73e7.69 (m, 2H), 7.27e7.23 (m, 2H),
AChE (with an IC50 value 0.027
aggregation (an IC50 value of 2.73
m
M for AChE) and amyloid-
d
b
mM). Further investigations into
133.86, 132.15, 129.38, 123.19, 120.62, 114.53, 67.05, 37.67, 26.66,
25.34.
AD candidates based on these results are in progress.
4. Experimental section
4.3.4. 2-(5-Phenoxypentyl)isoindoline-1,3-dione (3d)
Compound 3d was synthesized according to the general
4.1. Chemistry
procedure to give the desired product in 91% yield. 1H NMR
(400 MHz, CDCl3) d 7.86e7.82 (m, 2H), 7.73e7.69 (m, 2H), 7.28e7.24
The NMR spectra were recorded with TMS as the internal
standard on a Bruker 400 MHz spectrometer. Coupling constants
were given in Hz. MS spectra were recorded on a Agilent LCeMS
6120 instrument with an ESI mass selective detector. Flash column
chromatography was performed with silica gel (200e300 mesh)
purchased from Qingdao Haiyang Chemical Co. Ltd or alumina from
Sinopharm Chemical Reagent Co. Ltd. All the reactions were
monitored by thin layer chromatography on silica gel. Berberine
chloride was isolated from Chinese herbal medicine Coptis chi-
nensis Franch and recrystallized from hot water. Compounds 5e10
were synthesized according to the reported procedures [40,41].
(m, 2H), 6.94e6.85 (m, 3H), 3.95 (t, J ¼ 6.4 Hz, 2H), 3.72 (t, J ¼ 7.2 Hz,
2H), 1.87e1.73 (m, 4H), 1.57e1.49 (m, 2H); 13C NMR (101 MHz,
CDCl3) d 168.45,158.97,133.88,132.14,129.39,123.19,120.52,114.47,
67.45, 37.86, 28.84, 28.35, 23.43.
4.3.5. 2-(6-Phenoxyhexyl)isoindoline-1,3-dione (3e)
Compound 3e was synthesized according to the general proce-
dure to give the desired product in 95% yield. 1H NMR (400 MHz,
CDCl3)
d 7.85e7.82 (m, 2H), 7.72e7.69 (m, 2H), 7.28e7.23 (m, 2H),
6.94e6.86 (m, 3H), 3.94 (t, J ¼ 6.4 Hz, 2H), 3.70 (t, J ¼ 7.2 Hz, 2H),
1.81e1.68 (m, 4H), 1.55e1.39 (m, 4H); 13C NMR (101 MHz, CDCl3)
d
168.48, 159.03, 133.87, 132.15, 129.38, 123.18, 120.47, 114.48, 67.63,
4.2. General procedures for the preparation of 2ae2e
37.94, 29.13, 28.53, 26.62, 25.69.
Phthalimide (40 mmol), K2CO3 (120 mmol) and benzyl-
triethylammonium chloride (4.4 mmol) were suspended in acetone
(100 mL). Dibromoalkanes (100 mmol) was added to the suspen-
sion and then the reaction mixture was stirred at room temperature
for 24 h. The mixture was filtered and then evaporated under
vacuum to afford the crude product, which was purified by chro-
matography on a silica gel column with EtOAc/petroleum ether as
eluent to give the products.
4.3.6. 2-(4-(Pyridine-3-yloxy)butyl)isoindoline-1,3-dione (3f)
Compound 3f was synthesized according to the general proce-
dure to give the desired product in 55% yield. 1H NMR (400 MHz,
CDCl3)
d
8.28 (d, J ¼ 1.8 Hz, 1H), 8.19 (dd, J ¼ 4.1, 1.7 Hz, 1H), 7.85 (m,
2H), 7.76e7.68 (m, 2H), 7.23e7.13 (m, 2H), 4.04 (t, J ¼ 5.8 Hz, 2H),
3.78 (t, J ¼ 6.7 Hz, 2H), 1.97e1.81 (m, 4H); 13C NMR (101 MHz,
CDCl3)
d 168.41, 155.02, 142.04, 137.97, 133.96, 132.05, 123.77,
123.22, 121.02, 67.46, 37.51, 26.49, 25.20.
4.3. General procedures for the preparation of 3ae3g
4.3.7. 2-(4-(Naphthalen-1-yloxy)butyl)isoindoline-1,3-dione (3g)
Compound 3g was synthesized according to the general
procedure to give the desired product in 90% yield. 1H NMR
To a stirred suspension of phenol (12 mmol) and K2CO3
(20 mmol) in DMF (20 mL), intermediates 2ae2e (10 mmol) were
added. After stirred at room temperature for 10e12 h, the mixture
was quenched with water and extracted with EtOAc for three times.
The combined organic phase was dried over MgSO4, filtered and
concentrated to give the crude product, which was purified by
chromatography on an silica gel column (EtOAc/petroleum ether as
(400 MHz, CDCl3)
d 8.25 (m, 1H), 7.84 (m, 1H), 7.81 (m, 1H),
7.71e7.69 (m, 2H), 7.49e7.40 (m, 2H), 7.38e7.32 (m, 2H), 6.80e6.78
(m, 1H) 4.18 (t, J ¼ 5.8 Hz, 2H), 3.83 (t, J ¼ 6.7 Hz, 2H), 2.01e1.99 (m,
4H); 13C NMR (101 MHz, CDCl3)
d 168.46, 154.62, 134.48, 133.89,
132.10, 127.41, 126.34, 125.76, 125.14, 123.19, 122.05, 120.16, 104.61,
67.29, 37.73, 26.73, 25.53.

5890
W.-J. Shan et al. / European Journal of Medicinal Chemistry 46 (2011) 5885e5893
4.4. General procedures for the preparation of 4ae4g
CDCl3)
d
11.52 (s,1H), 7.87 (s,1H), 7.58 (s,1H), 7.52 (d, J ¼ 8.5 Hz,1H),
7.33e7.20 (m, 3H), 7.12 (d, J ¼ 8.5 Hz, 1H), 6.88 (m, 3H), 6.80 (s, 1H),
6.08 (s, 2H), 5.06 (t, J ¼ 6.1 Hz, 2H), 3.96 (t, J ¼ 6.5 Hz, 2H), 3.91 (s,
3H), 3.83 (d, J ¼ 6.3 Hz, 2H), 3.15 (t, J ¼ 6.0 Hz, 2H), 1.95e1.80 (m,
An solution of 3ae3g (11 mmol) and hydrazine hydrate(80%)
(9 mL, 142 mmol) in ethanol (60 mL) was refluxed for 4 h, and then
cooled to room temperature. The mixture was filtered and the solid
was washed with 95% EtOH. The whole filtrate was concentrated
and the solid was dissolved in CH2Cl2. The mixture was dried over
MgSO4, filtered and concentrated to give the crude product, which
was used in the next step without further purification.
4H), 1.56e64 (m 2H); 13C NMR (101 MHz, CDCl3)
d 159.00, 150.04,
148.19, 147.29, 146.12, 139.27, 135.30, 133.11, 129.58, 129.29, 124.91,
120.37, 118.60, 117.04, 114.42, 113.94, 108.46, 104.76, 101.99, 67.70,
56.89, 54.22, 46.68, 30.89, 28.98, 27.82, 23.38; ESI-MS m/z: 483.2
[M ꢀ Cl]þ.
4.5. General procedures for the preparation of 5e10
4.5.5. 9-N-6-Phenoxyhexyl berberine (5e)
Berberine was treated with 6-phenoxyhexylamine according to
general procedure to give the desired product 5e as a red solid,
yield 42%; decomposition point: 200 ꢂC, IR (KBr): 3415, 2935, 2854,
To a solution of berberine (0.3718 g, 1 mmol) in anhydrous
ethanol, the substituted amine (4e10 mmol) were added. The
mixture was stirred and heated for 10e24 h at 78 ꢂC. After the
reaction completed (monitored by TLC), the mixture was concen-
trated in vacuo and the residual oil was purified on Al2O3 column
with CH2Cl2/CH3OH as eluent to give the proposed compound.
2049, 2025, 1632, 1508, 1393, 1068, 953, 863 cmꢀ1 1H NMR
;
(400 MHz, CDCl3) d 11.43 (s,1H), 7.88 (s,1H), 7.48e7.53 (m, 2H), 7.29
(s,1H), 7.27e7.21 (m, 2H), 7.13 (d, J ¼ 8.5 Hz,1H), 6.95e6.83 (m, 3H),
6.78 (s, 1H), 6.06 (s, 2H), 5.04 (t, J ¼ 6.2 Hz, 2H), 3.94 (t, J ¼ 6.6 Hz,
2H), 3.91 (s, 3H), 3.79 (dd, J ¼ 13.5, 6.4 Hz, 2H), 3.14 (t, J ¼ 6.0 Hz,
2H), 1.86e1.73 (m, 4H), 1.59e1.41 (m,4H); 13C NMR (101 MHz,
4.5.1. 9-N-2-Phenoxyethyl berberine (5a)
Berberine was treated with 2-phenoxyethanamine according to
general procedure to give the desired product 5a as a red solid,
yield 43%; decomposition point: 242 ꢂC, IR (KBr): 3483, 2052, 2025,
CDCl3) d 159.13,150.16,148.32, 147.72,146.15,139.93,135.44,133.26,
129.77, 129.36, 125.25, 120.47, 118.46, 117.12, 114.51, 113.39, 108.62,
104.71, 102.05, 67.85, 57.09, 54.15, 46.77, 31.23, 29.26, 27.97, 26.75,
25.87; ESI-MS m/z: 497.3 [M ꢀ Cl]þ.
1637, 1385, 1120, 862 cmꢀ1 1H NMR (400 MHz, CDCl3)
; d 11.51
(s, 1H), 7.91 (s, 1H), 7.57 (d, J ¼ 8.6 Hz, 2H), 7.30 (s, 1H), 7.25e7.14 (m,
3H), 6.87e6.77 (m, 4H), 6.08 (s, 2H), 5.10e4.99 (t, J ¼ 5.8 Hz, 2H),
4.39 (t, J ¼ 5.8 Hz, 2H), 4.25 (q, J ¼ 6.0 Hz, 2H), 3.91 (s, 3H), 3.18e3.10
4.5.6. 9-N-Benzylberberine (6a)
Berberine was treated with benzylamine according to general
procedure to give the desired product 6a as a red solid, yield 45%;
decomposition point: 237 ꢂC, IR (KBr): 3374, 2049, 2025,1633, 1507,
(t, J ¼ 5.8 Hz, 2H); 13C NMR (101 MHz, CDCl3)
d 158.66, 150.16,
148.29, 147.68, 147.15, 138.94, 135.40, 133.03, 129.59, 129.24, 124.69,
120.42, 118.79, 117.92, 115.48, 114.62, 108.50, 104.87, 102.05, 68.23,
56.72, 54.38, 45.67, 27.81; ESI-MS m/z: 441.2 [M ꢀ Cl]þ.
1390, 864, 746, 701 cmꢀ1; 1H NMR (400 MHz, MeOD)
d 9.61 (s, 1H),
8.46 (s, 1H), 7.83 (d, J ¼ 8.8 Hz, 1H), 7.64e7.55 (m, 2H), 7.38 (d,
J ¼ 7.2 Hz, 2H), 7.32 (t, J ¼ 7.4 Hz, 2H), 7.25 (t, J ¼ 7.2 Hz, 1H), 6.92 (s,
1H), 6.08 (s, 2H), 4.80 (s, 2H), 4.72 (t, J ¼ 6.0 Hz, 2H), 3.96 (s, 3H),
4.5.2. 9-N-3-Phenoxypropyl berberine (5b)
Berberine was treated with 3-phenoxypropylamine according to
general procedure to give the desired product 5b as a red solid,
yield 45%; decomposition point: 222 ꢂC, IR (KBr): 3413, 2049, 2025,
3.25e3.17 (m, 2H); 13C NMR (101 MHz, MeOD)
d 151.81, 149.79,
147.21, 141.09, 138.09, 134.75, 131.38, 129.73, 128.62, 124.68, 121.88,
121.30, 119.29, 109.35, 106.23, 103.57, 57.40, 56.96, 53.26, 28.39;
HRMS (ESI): calcd for (M ꢀ Cl)þ (C26H23N2O3) requires m/z 411.1709,
found 411.1703.
1638, 1506, 1215, 1067, 952, 864, 756 cmꢀ1 1H NMR (400 MHz,
;
CDCl3)
d
11.52 (s, 1H), 7.89 (s, 1H), 7.61 (t, J ¼ 6.0 Hz, 1H), 7.52 (d,
J ¼ 8.6 Hz,1H), 7.30 (s,1H), 7.26e7.19 (m, 2H), 7.15 (d, J ¼ 8.5 Hz,1H),
6.87 (m, 3H), 6.79 (s, 1H), 6.07 (s, 2H), 5.02 (t, J ¼ 6.2 Hz, 2H), 4.18 (t,
J ¼ 6.0 Hz, 2H), 4.02 (q, J ¼ 6.7 Hz, 2H), 3.89 (s, 3H), 3.12 (t, J ¼ 6.0 Hz,
4.5.7. 9-N-(2-Phenylethyl)berberine (6b)
Berberine was treated with 2-phenylethanamine according to
general procedure to give the desired product 6b as a red solid [40],
yield 60%; decomposition point: 247 ꢂC, IR (KBr): 3414, 2048, 2026,
2H), 2.32 (p, J ¼ 6.4 Hz, 2H); 13C NMR (101 MHz, CDCl3)
d 158.99,
150.11, 148.28, 147.59, 146.32, 139.41, 135.28, 133.07, 129.55, 129.32,
124.96, 120.38, 118.59, 117.19, 114.44, 114.06, 108.51, 104.77, 102.04,
65.90, 56.85, 54.11, 44.06, 30.75, 27.86. ESI-MS m/z: 455.2 [M ꢀ Cl]þ.
1635, 1512, 1391, 1146, 861, 750, 700 cmꢀ1 1H NMR (400 MHz,
;
CDCl3)
d
11.53 (s, 1H), 7.89 (s, 1H), 7.75 (t, J ¼ 6.2 Hz, 1H), 7.52 (d,
J ¼ 8.6 Hz, 1H), 7.30 (t, J ¼ 3.4 Hz, 3H), 7.25 (dd, J ¼ 10.2, 4.8 Hz, 2H),
7.15 (dt, J ¼ 7.2, 2.9 Hz, 2H), 6.80 (s, 1H), 6.07 (s, 2H), 5.12e5.01 (t,
J ¼ 6.4 Hz, 2H), 4.10e3.98 (m, 2H), 3.90 (s, 3H), 3.20e3.06 (m, 4H);
4.5.3. 9-N-4-Phenoxybutyl berberine (5c)
Berberine was treated with 4-phenoxybutylamine according to
general procedure to give the desired product 5c as a red solid, yield
46%; decomposition point: 220 ꢂC, IR (KBr): 3415, 2938, 2049, 2025,
13C NMR (101 MHz, CDCl3)
d 150.17, 148.32, 147.76, 146.41, 139.84,
139.40, 135.38, 133.19, 129.66, 129.15, 128.19, 125.90, 125.15, 120.50,
118.46, 117.37, 114.00, 108.58, 104.75, 102.05, 56.93, 54.17, 48.02,
37.81, 27.92; HRMS (ESI): calcd for (M ꢀ Cl)þ (C27H25N2O3) requires
m/z 425.1865, found 425.1860.
1632, 1507, 1215, 1068, 953, 863 cmꢀ1 1H NMR (400 MHz, CDCl3)
;
d
11.41 (s, 1H), 7.92 (s, 1H), 7.51 (d, J ¼ 8.3 Hz, 2H), 7.31 (d, J ¼ 6.1 Hz,
2H), 7.17e7.23 (m, 3H), 6.83e6.88 (m, 3H), 6.76 (s, 1H), 6.05 (s, 2H),
5.02 (t, J ¼ 6.0 Hz, 2H), 4.02 (d, J ¼ 5.8 Hz, 2H), 3.87 (s, 3H), 3.84 (d,
J ¼ 6.1 Hz, 2H), 3.12 (t, J ¼ 6.0 Hz, 2H), 2.02e1.86 (m, 4H); 13C NMR
4.5.8. 9-N-(3-Phenylpropyl)berberine (6c)
(101 MHz, CDCl3)
d
158.98, 150.10, 148.27, 147.48, 146.14, 139.40,
Berberine was treated with 3-phenyl-1-propylamine according
to general procedure to give the desired product 6c as a red solid,
yield 35%; decomposition point: 232 ꢂC, IR (KBr): 3400, 2921, 2851,
135.31, 133.16, 129.58, 129.29, 125.06, 120.38, 118.57, 117.11, 114.45,
113.82, 108.52, 104.77, 102.04, 67.56, 56.93, 54.12, 46.34, 27.78,
26.79; ESI-MS m/z: 469.2 [M ꢀ Cl]þ.
2049, 2025, 1633, 1507, 1393, 862, 750, 700 cmꢀ1 1H NMR
;
(400 MHz, CDCl3)
d
11.61 (s, 1H), 7.85 (s, 1H), 7.78 (t, J ¼ 6.1 Hz, 1H),
4.5.4. 9-N-5-Phenoxypentyl berberine (5d)
7.49 (d, J ¼ 8.5 Hz,1H), 7.28e7.21 (m, 5H), 7.16e7.05 (m, 2H), 6.80 (s,
1H), 6.08 (s, 2H), 5.13e5.00 (t, J ¼ 6.0 Hz, 2H), 3.85 (dd, J ¼ 13.4,
5.9 Hz, 2H), 3.78 (s, 3H), 3.15 (t, J ¼ 5.8 Hz, 2H), 2.77 (t, J ¼ 7.6 Hz,
Berberine was treated with 5-phenoxypentylamine according to
general procedure to give the desired product 5d as a red solid,
yield 40%; decomposition point: 208 ꢂC, IR (KBr): 3444, 2938, 2856,
2H), 2.20e2.07 (m, 2H); 13C NMR (101 MHz, CDCl3)
d 150.10, 148.28,
2052, 2025, 1632, 1598, 1507, 1479, 860 cmꢀ1
;
1H NMR (400 MHz,
147.55, 146.04, 142.33, 139.49, 135.27, 133.14, 129.57, 128.55, 128.11,

W.-J. Shan et al. / European Journal of Medicinal Chemistry 46 (2011) 5885e5893
5891
125.45, 125.09, 120.50, 118.49, 117.04, 113.59, 108.52, 104.77, 102.03,
56.78, 54.08, 46.26, 33.38, 32.80, 27.91; HRMS (ESI): calcd for
(M ꢀ Cl)þ (C28H27N2O3) requires m/z 439.2022, found 439.2003.
2917, 2025, 1633, 1508, 1479, 860 cmꢀ1; 1H NMR (400 MHz, CDCl3)
d
11.56 (s, 1H), 7.89 (s, 1H), 7.75 (t, J ¼ 6.2 Hz, 1H), 7.53 (d, J ¼ 8.6 Hz,
1H), 7.30 (s, 1H), 7.29e7.23 (m, 3H), 7.15 (d, J ¼ 8.5 Hz, 1H), 6.92 (t,
J ¼ 8.8 Hz, 2H), 6.81 (s, 1H), 6.08 (s, 2H), 5.11e5.02 (t, J ¼ 6.0 Hz, 1H),
4.02 (dd, J ¼ 15.5, 6.3 Hz, 2H), 3.90 (s, 3H), 3.22e3.14 (t, J ¼ 6.0 Hz,
2H), 3.14e3.06 (t, J ¼ 7.2 Hz, 2H); 13C NMR (101 MHz, CDCl3)
4.5.9. 9-N-[2-(5-Methoxy-1H-indol-3-yl)]ethylberberine (7a)
Berberine was treated with 2-(5-methoxy-1H-indol-3-yl)
ethanamine according to general procedure to give the desired
product 7a as a red solid, yield 45%; decomposition point: 153 ꢂC, IR
(KBr): 3373, 2903, 2834, 2049, 2025, 1632, 1507, 1215, 1068, 953,
d
150.26, 148.38, 147.86, 146.39, 139.56, 135.55, 133.31, 130.57,
129.77, 125.40, 120.49, 118.42, 117.47, 115.00, 114.79, 113.92, 108.67,
104.71, 102.09, 57.04, 54.17, 48.00, 36.93, 27.96; HRMS (ESI): calcd
for (M ꢀ Cl)þ (C27H24N2O3F) requires m/z 443.1771, found 443.1767.
862 cmꢀ1 1H NMR (400 MHz, MeOD)
; d 9.00 (s, 1H), 8.24 (s, 1H),
7.66 (d, J ¼ 8.8 Hz, 1H), 7.49 (s, 1H), 7.41 (d, J ¼ 8.7 Hz, 1H), 7.13 (d,
J ¼ 8.8 Hz, 1H), 6.99 (s, 1H), 6.89 (s, 1H), 6.76 (d, J ¼ 2.3 Hz, 1H), 6.62
(dd, J ¼ 8.8, 2.4 Hz, 1H), 6.08 (s, 2H), 4.32 (t, J ¼ 6.2 Hz, 2H),
4.00e3.91 (m, 5H), 3.62 (s, 3H), 3.07 (t, J ¼ 6.0 Hz, 2H), 3.03 (t,
4.5.14. 9-N-[2-(4-Nitrophenyl)]ethylberberine (8c)
Berberine was treated with 2-(4-nitrophenyl)ethanamine
according to general procedure to give the desired product 8c as
a red solid, yield 25%; decomposition point: 226 ꢂC, IR (KBr): 3444,
J ¼ 6.0 Hz, 2H); 13C NMR (101 MHz, MeOD)
d 154.91, 151.75, 149.83,
146.46, 137.36, 134.55, 131.28, 128.83, 125.55, 123.92, 121.97, 121.09,
118.98, 112.99, 111.92, 109.27, 106.18, 103.53, 101.71, 57.25, 56.47,
56.20, 50.44, 28.40, 28.06; ESI-MS m/z: 494.3 [M ꢀ Cl]þ.
2921, 2841, 2049, 2025, 1633, 1512, 1343, 860 cmꢀ1 1H NMR
;
(400 MHz, CDCl3)
d
11.61 (s, 1H), 8.10 (d, J ¼ 8.6 Hz, 2H), 7.91 (s, 1H),
7.76 (t, J ¼ 5.9 Hz, 1H), 7.50e7.55 (m, 3H), 7.30 (s, 1H), 7.18 (d,
J ¼ 8.6 Hz, 1H), 6.82 (s, 1H), 6.09 (s, 2H), 5.07 (t, J ¼ 6.2 Hz, 2H), 4.09
(dd, J ¼ 14.6, 6.6 Hz, 2H), 3.90 (s, 3H), 3.29 (t, J ¼ 7.6 Hz, 2H), 3.17 (t,
4.5.10. 9-N-[2-(Thiophen-2-yl)]ethylberberine (7b)
Berberine was treated with 2-(thiophen-2-yl) ethanamine
according to general procedure to give the desired product 7b as
a red solid, yield 45%; decomposition point: 232 ꢂC, 1H NMR
J ¼ 6.0 Hz, 2H); 13C NMR (101 MHz, CDCl3)
d 150.38, 148.36, 147.90,
146.74, 146.44, 138.12, 136.06, 133.42, 129.93, 125.16, 123.35, 120.27,
118.84, 117.78, 115.36, 108.58, 104.71, 102.06, 56.86, 54.73, 47.40,
37.40, 27.67; HRMS (ESI): calcd for (M ꢀ Cl)þ (C27H25N2O3) requires
m/z 470.1716, found 470.1697.
(400 MHz, CDCl3)
d 11.58 (s, 1H), 7.90 (s, 1H), 7.86 (s, 1H), 7.54 (d,
J ¼ 8.6 Hz, 1H), 7.30 (s, 1H), 7.16 (d, J ¼ 8.5 Hz, 1H), 7.08 (dd, J ¼ 5.0,
1.1 Hz,1H), 6.91 (dt, J ¼ 4.9, 3.0 Hz, 2H), 6.80 (s,1H), 6.07 (s, 2H), 5.05
(t, J ¼ 5.9 Hz, 1H), 4.08 (m, 2H), 3.93 (s, 3H), 3.39e3.28 (t, J ¼ 7.2 Hz,
4.5.15. 9-N-[2-(o-Tolyl)]ethylberberine (8d)
2H), 3.21e3.09 (t, J ¼ 6.0 Hz, 2H); 13C NMR (101 MHz, CDCl3)
d
150.21,
Berberine was treated with 2-(o-tolyl)ethanamine according to
general procedure to give the desired product 8d as a red solid,
yield 65%; decomposition point: 239 ꢂC, IR (KBr): 3409, 2905, 2836,
148.36, 147.76, 146.35, 142.16, 139.09, 135.43, 133.21, 129.64, 126.79,
125.26, 123.23, 120.50, 118.52, 117.37, 114.14, 108.60, 104.78, 102.08,
56.96, 54.19, 48.09, 31.92, 27.93; ESI-MS m/z: 431.1 [M ꢀ Cl]þ.
2051, 2025, 1633, 1508, 1425, 1170, 941, 860, 763 cmꢀ1 1H NMR
;
(400 MHz, CDCl3)
d
11.59 (s, 1H), 7.89 (s, 1H), 7.82 (t, J ¼ 6.0 Hz, 1H),
4.5.11. 9-N-[2-(Pyridin-2-yl)]ethylberberine (7c)
7.52 (d, J ¼ 8.6 Hz, 1H), 7.30 (s, 1H), 7.26e7.22 (m, 1H), 7.20e7.03 (m,
4H), 6.80 (s, 1H), 6.08 (s, 2H), 5.07 (t, J ¼ 6.2 Hz, 2H), 4.01 (dd,
J ¼ 15.8, 6.2 Hz, 2H), 3.90 (s, 3H), 3.25e3.03 (m, 4H), 2.41 (s, 3H); 13C
Berberine was treated with 2-(pyridin-2-yl) ethanamine
according to general procedure to give the desired product 7c as
a red solid, yield 44%; decomposition point: 203 ꢂC, IR (KBr): 3445,
NMR (101 MHz, CDCl3)
d 150.21, 148.35, 147.86, 146.44, 139.55,
2924, 2050, 2025, 1633, 1506, 1068, 953, 862, 773 cmꢀ1
;
1H NMR
137.93, 136.61, 135.47, 133.27, 130.22, 129.57, 126.08, 125.71, 125.23,
120.53, 118.40, 117.47, 113.85, 108.60, 104.71, 102.03, 56.93, 54.21,
46.71, 35.31, 27.96, 19.47; HRMS (ESI): calcd for (M ꢀ Cl)þ
(C28H26N2O3) requires m/z 439.2022, found 439.2003.
(400 MHz, MeOD)
d
9.66 (s,1H), 8.40 (t, J ¼ 5.2 Hz, 2H), 7.80e7.67 (m,
2H), 7.60e7.49 (m, 2H), 7.33 (d, J ¼ 7.8 Hz,1H), 7.22 (dd, J ¼ 6.8, 5.2 Hz,
1H), 6.91 (s, 1H), 6.07 (s, 2H), 4.83 (2H), 4.01e3.93 (m, 5H), 3.24 (t,
J ¼ 6.0 Hz, 1H), 3.13 (t, J ¼ 6.8 Hz, 2H); 13C NMR (101 MHz, MeOD)
d
160.42,151.80,149.86,147.14,138.61,138.03,137.73,134.78,131.44,
4.5.16. 9-N-[2-(m-Tolyl)]ethylberberine (8e)
125.50, 124.40, 123.21, 121.89, 121.27, 119.57, 109.36, 106.22, 103.58,
Berberine was treated with 2-(m-tolyl)ethanamine according to
general procedure to give the desired product 8e as a red solid,
yield 60%; decomposition point: 238 ꢂC, IR (KBr): 3382, 2927, 2837,
57.29, 56.95, 49.86, 39.95, 28.40; ESI-MS m/z: 426.3 [M ꢀ Cl]þ.
4.5.12. 9-N-[2-(4-Methoxyphenyl)]ethylberberine (8a)
2049, 2025, 1633, 1512, 1450, 1149, 942, 861, 701 cmꢀ1 1H NMR
;
Berberine was treated with 2-(4-methoxyphenyl)ethanamine
according to general procedure to give the desired product 8a as
a red solid, yield 45%; decomposition point: 242 ꢂC, IR (KBr): 3400,
(400 MHz, CDCl3)
d
11.45 (s, 1H), 7.90 (s, 1H), 7.69 (t, J ¼ 6.2 Hz, 1H),
7.52 (d, J ¼ 8.6 Hz, 1H), 7.30 (s, 1H), 7.19e7.06 (m, 4H), 6.97 (d,
J ¼ 7.3 Hz, 1H), 6.79 (s, 1H), 6.07 (s, 2H), 5.04 (t, J ¼ 6.2 Hz, 2H), 4.02
(dd, J ¼ 15.8, 6.2 Hz, 2H), 3.90 (s, 3H), 3.15 (t, J ¼ 6.0 Hz, 2H), 3.06 (t,
2925, 2836, 2051, 2025, 1633, 1509, 1036, 867 cmꢀ1 1H NMR
;
(400 MHz, CDCl3)
d
11.48 (s, 1H), 7.88 (s, 1H), 7.70 (t, J ¼ 6.2 Hz, 1H),
J ¼ 7.6 Hz, 2H), 2.29 (s, 3H); 13C NMR (101 MHz, CDCl3)
d 150.20,
7.52 (d, J ¼ 8.6 Hz, 1H), 7.30 (s, 1H), 7.21 (d, J ¼ 8.5 Hz, 2H), 7.14 (d,
J ¼ 8.5 Hz, 1H), 6.79 (d, J ¼ 8.5 Hz, 3H), 6.08 (s, 2H), 5.06 (t,
J ¼ 6.2 Hz, 2H), 4.01 (dd, J ¼ 15.6, 6.3 Hz, 2H), 3.91 (s, 3H), 3.76 (s,
3H), 3.16 (t, J ¼ 6.2 Hz, 2H), 3.05 (t, J ¼ 7.6 Hz, 2H); 13C NMR
148.35, 147.76, 146.47, 139.70, 137.77, 135.42, 133.20, 130.02, 129.70,
128.12, 126.69, 126.14, 125.08, 120.53, 118.49, 117.40, 114.02, 108.61,
104.77, 102.07, 56.94, 54.23, 48.11, 37.79, 27.94, 21.36; HRMS (ESI):
calcd for (M ꢀ Cl)þ (C28H26N2O3) requires m/z 439.2022, found
439.2009.
(101 MHz, CDCl3)
d 157.89, 150.19, 148.35, 147.84, 146.44, 139.59,
135.42, 133.23, 131.91, 130.11, 129.73, 125.20, 120.53, 118.42, 117.40,
113.88, 113.66, 108.63, 104.73, 102.07, 57.01, 55.25, 54.16, 48.22,
36.86, 27.95; HRMS (ESI): calcd for (M ꢀ Cl)þ (C28H27N2O4) requires
m/z 455.1971, found 455.1953.
4.5.17. 9-N-[2-(p-Tolyl)]ethylberberine (8f)
Berberine was treated with 2-(p-tolyl)ethanamine according to
general procedure to give the desired product 8f as a red solid, yield
60%; decomposition point: 243 ꢂC, IR (KBr): 3445, 2049, 2025,1632,
4.5.13. 9-N-[2-(4-Fluorophenyl)]ethylberberine (8b)
1511, 1428, 1069, 946, 862 cmꢀ1; 1H NMR (400 MHz, CDCl3)
d 11.42
Berberine was treated with 2-(4-fluorophenyl)ethanamine
according to general procedure to give the desired product 8b as
a red solid, yield 63%; decomposition point: 234 ꢂC, IR (KBr): 3422,
(s, 1H), 7.90 (s, 1H), 7.64 (t, J ¼ 5.6 Hz, 1H), 7.51 (d, J ¼ 8.6 Hz, 1H),
7.30 (s, 1H), 7.17 (t, J ¼ 7.6 Hz, 3H), 7.06 (d, J ¼ 7.8 Hz, 2H), 6.79 (s,
1H), 6.06 (s, 2H), 5.04 (t, J ¼ 6.2 Hz, 2H), 4.00 (dd, J ¼ 15.3, 6.1 Hz,

5892
W.-J. Shan et al. / European Journal of Medicinal Chemistry 46 (2011) 5885e5893
2H), 3.89 (s, 3H), 3.15 (t, J ¼ 6.0 Hz, 2H), 3.06 (t, J ¼ 7.6 Hz, 2H), 2.29
(s, 3H); 13C NMR (101 MHz, CDCl3)
150.18, 148.26, 146.88, 146.54,
138.07, 136.26, 135.55, 133.05, 129.69, 128.90, 124.27, 120.35, 118.96,
117.42, 115.47, 108.45, 104.80, 102.03, 56.64, 54.81, 48.49, 37.01,
27.67, 20.89; HRMS (ESI): calcd for (M ꢀ Cl)þ (C28H26N2O3) requires
m/z 439.2022, found 439.2024.
118.45, 117.23, 113.56, 108.63, 104.67, 102.05, 68.11, 57.08, 54.13,
d
46.19, 27.94, 27.48, 26.65. ESI-MS m/z: 470.3 [M ꢀ Cl]þ.
4.5.22. 9-N-(1-Phenylethyl)berberine (10a)
Berberine was treated with 1-phenylethanamine according to
general procedure to give the desired product 10a as a red solid,
yield 35%; decomposition point: 234 ꢂC, IR (KBr): 3381, 2959, 2925,
4.5.18. 9-N-[2-(3,4-Dimethoxyphenyl)]ethylberberine (8g)
2852, 2051, 2025, 1634, 1508, 1385, 1039, 762, 702 cmꢀ1 1H NMR
;
Berberine was treated with 2-(3,4-dimethoxyphenyl)ethan-
amine according to general procedure to give the desired product
8g as a red solid, yield 50%; decomposition point: 247 ꢂC, IR (KBr):
(400 MHz, MeOD)
d
9.51 (s, 1H), 8.49 (s, 1H), 7.85 (d, J ¼ 8.8 Hz, 1H),
7.65 (d, J ¼ 8.8 Hz, 1H), 7.59 (s, 1H), 7.35 (d, J ¼ 7.2 Hz, 2H), 7.27 (t,
J ¼ 7.5 Hz, 2H), 7.18 (t, J ¼ 7.3 Hz, 1H), 6.93 (s, 1H), 6.09 (s, 2H), 5.02
(q, J ¼ 6.7 Hz,1H), 4.77e4.65 (m, 2H), 3.96 (s, 3H), 3.20 (d, J ¼ 6.8 Hz,
3386, 2913, 2834, 2049, 2025, 1633, 1513, 942, 855, 809 cmꢀ1 1H
;
NMR (400 MHz, CDCl3)
d
11.27 (s, 1H), 7.89 (s, 1H), 7.54e7.49 (m,
2H); 13C NMR (101 MHz, MeOD)
d 150.48, 149.42, 148.42, 145.67,
2H), 7.29 (s, 1H), 7.17 (d, J ¼ 9.0 Hz, 1H), 6.88 (s, 1H), 6.80e6.72 (m,
3H), 6.08 (s, 2H), 5.04 (t, J ¼ 5.2 Hz, 2H), 4.04 (t, J ¼ 7.2 Hz, 2H),
3.92 (s, 3H), 3.86 (s, 3H), 3.81 (s, 3H), 3.18 (t, J ¼ 5.8 Hz, 2H), 3.05
144.67, 136.53, 135.64, 133.47, 130.01, 128.44, 128.24, 127.00, 125.93,
123.52, 120.46, 120.03, 119.09, 119.04, 107.91, 104.88, 102.16, 58.52,
55.98, 55.55, 26.92, 23.34; ESI-MS m/z: 425.3 [M ꢀ Cl]þ.
(t, J ¼ 7.2 Hz, 2H); 13C NMR (101 MHz, CDCl3)
d 150.21, 148.78,
148.32, 147.23, 146.90, 138.67, 135.66, 133.14, 132.25, 129.90,
124.60, 121.01, 120.38, 118.68, 117.56, 114.92, 112.56, 111.16, 108.57,
104.70, 102.03, 56.83, 55.93, 54.67, 48.21, 36.99, 27.73; HRMS (ESI):
calcd for (M ꢀ Cl)þ (C29H28N2O5) requires m/z 485.2076, found
485.2062.
4.5.23. 9-N-(2-Methylpropyl)berberine (10b)
Berberine was treated with 2-methylpropan-1-amine according
to general procedure to give the desired product 10b as a red solid,
yield 45%; decomposition point: 251 ꢂC, IR (KBr): 3485, 2958, 2868,
2052, 2026, 1633, 1508, 1397, 1163, 1099 cmꢀ1; 1H NMR (400 MHz,
CDCl3)
d
11.49 (s, 1H), 7.89 (s, 1H), 7.52 (d, J ¼ 8.5 Hz, 2H), 7.30 (s,
4.5.19. 9-N-[2-(2,4-Dichlorophenyl)]ethylberberine (8h)
1H), 7.13 (d, J ¼ 8.5 Hz, 1H), 6.79 (s, 1H), 6.07 (s, 2H), 5.08 (t,
J ¼ 6.2 Hz, 2H), 3.92 (s, 3H), 3.62 (t, J ¼ 6.4 Hz, 2H), 3.21e3.11 (m,
2H), 2.20e2.06 (m, 1H), 1.01 (d, J ¼ 6.7 Hz, 6H); 13C NMR (101 MHz,
Berberine was treated with 2-(2,4-dichlorophenyl)ethanamine
according to general procedure to give the desired product 8h as
a red solid, yield 55%; decomposition point: 242 ꢂC, IR (KBr): 3391,
CDCl3) d 150.13,148.29,147.65,146.07,140.15,135.43, 133.28,129.81,
2923, 2850, 2048, 2025, 1633, 1507, 1392, 859, 819 cmꢀ1
;
1H NMR
125.28, 120.55, 118.41, 116.95, 113.15, 108.62, 104.67, 102.02, 57.03,
(400 MHz, CDCl3)
d
11.61 (s, 1H), 8.10 (d, J ¼ 8.6 Hz, 2H), 7.91 (s, 1H),
54.18, 30.02, 27.96, 20.43; ESI-MS m/z: 377.5 [M ꢀ Cl]þ.
7.76 (t, J ¼ 6.2 Hz, 1H), 7.52 (dd, J ¼ 12.0, 8.6 Hz, 3H), 7.30 (s,1H), 7.18
(d, J ¼ 8.6 Hz, 1H), 6.82 (s, 1H), 6.09 (s, 2H), 5.07 (t, J ¼ 6.2 Hz, 2H),
4.09 (dd, J ¼ 14.6, 6.6 Hz, 2H), 3.90 (s, 3H), 3.37e3.23 (t, J ¼ 6.8 Hz,
4.5.24. 9-N-[3-(N,N-Dimethylamino)propyl]berberine (10c)
Berberine was treated with N0,N0-dimethylpropane-1,3-amine
according to general procedure to give the desired product 10c as
a red solid, yield 40%; decomposition point: 214 ꢂC, IR (KBr): 3418,
2H), 3.23e3.11 (m, 2H); 13C NMR (101 MHz, CDCl3)
d 150.27, 148.40,
147.79, 146.64, 139.19, 136.32, 135.47, 134.69, 133.00, 132.28, 129.72,
128.75, 126.91, 124.87, 120.50, 118.53, 117.54, 114.26, 108.68, 104.72,
102.11, 56.68, 54.24, 45.82, 34.84, 27.97; HRMS (ESI): calcd for
(M ꢀ Cl)þ (C27H24Cl2N2O3) requires m/z 493.1086, found 493.1089.
2971, 2848, 2048, 2025, 1633, 1507, 1395, 1068 cmꢀ1 1H NMR
;
(400 MHz, MeOD)
d
9.73 (s, 1H), 8.52 (s, 1H), 7.89 (d, J ¼ 8.8 Hz, 1H),
7.63 (t, J ¼ 4.3 Hz, 2H), 6.95 (s, 1H), 6.10 (s, 2H), 4.83 (2H), 4.07 (s,
3H), 3.67 (t, J ¼ 6.9 Hz, 2H), 3.25 (t, J ¼ 6.0 Hz, 2H), 2.52 (t, J ¼ 7.2 Hz,
2H), 2.31 (s, 6H), 1.96e1.84 (m, 2H); 13C NMR (101 MHz, MeOD)
4.5.20. 9-N-[4-(Naphthalen-1-yloxy) butyl] berberine (9a)
Berberine was treated with 4-(naphthalen-1-yloxy) butan-1-
amine (4g) according to general procedure to give the desired
product 9a as a red solid, yield 39%; decomposition point: 201 ꢂC, IR
(KBr): 3445, 2921, 2850, 2026,1650,1633,1507,1067 cmꢀ1; 1H NMR
d 151.81,149.82,149.56, 147.45, 138.77, 137.75,134.88,131.47,124.50,
122.00, 121.32, 119.19, 118.87, 109.35, 106.25, 103.56, 58.04, 57.44,
56.91, 45.40, 29.27, 28.45; ESI-MS m/z: 406.55 [M ꢀ Cl]þ.
(400 MHz, CDCl3)
d
11.54 (s, 1H), 8.09 (d, J ¼ 8.5 Hz, 1H), 7.82e7.67
4.6. Biological activity and molecular modeling
(m, 3H), 7.51e7.40 (m, 2H), 7.38e7.28 (m, 3H), 7.25 (s, 1H), 7.03 (d,
J ¼ 8.6 Hz,1H), 6.80 (d, J ¼ 7.5 Hz, 2H), 6.08 (s, 2H), 5.00 (t, J ¼ 6.3 Hz,
2H), 4.24 (d, J ¼ 5.4 Hz, 2H), 3.94 (d, J ¼ 5.9 Hz, 2H), 3.84 (s, 3H), 3.11
(t, J ¼ 5.6 Hz, 2H), 2.07e2.11 (m, 4H); 13C NMR (101 MHz, CDCl3)
4.6.1. In vitro inhibition studies on AChE and BuChE
The method of Ellman et al. was followed for the in vitro inhi-
bition studies on AChE and BuChE. All the assays were under 0.1 M
KH2PO4/K2HPO4 buffer, pH 8.0, using a Shimadzu 2450 Spectro-
photometer. Enzyme solutions were prepared to give 2.0 units/mL
in 2 mL aliquots. The assay medium contained phosphate buffer, pH
d
154.75, 150.15, 148.32, 147.69, 146.12, 139.78, 135.31, 134.35,
133.22,129.73,127.24,126.04,125.60,125.32,124.89,122.04,120.57,
119.63, 118.26, 117.21, 113.32, 108.65, 104.64, 102.06, 67.74, 57.04,
53.98, 46.24, 27.95, 27.59, 27.00; ESI-MS m/z: 519.3 [M ꢀ Cl]þ.
8.0 (1 mL), 50
mL of 0.01 M DTNB, 20 mL of enzyme, 10 mL of inhibitor
and 50 L of 0.01 M substrate (acetylthiocholine chloride or
m
4.5.21. 9-N-[4-(Pyridin-3-yloxy) butyl] berberine (9b)
butylthiocholine chloride). Test compounds were added to the
assay solution and pre-incubated with the enzyme for 15 min fol-
lowed by the addition of substrate. Assays were done with a blank
containing all components except AChE or BChE in order to account
for nonenzymatic reaction. The activity was determined by
measuring the increase in absorbance at 412 nm at 1 min intervals
at 37 ꢂC. The reaction rates were compared, and the percent inhi-
bition due to the presence of test compounds was calculated. Each
concentration was analyzed in triplicate, and IC50 values were
determined graphically from log concentration-inhibition curves.
Kinetic characterization of AChE was performed using the re-
ported method [43]. Six different concentrations of the substrate
Berberine was treated with 4-(pyridin-3-yloxy) butan-1-amine
(4f) according to general procedure to give the desired product
9b as a red solid, yield 43%; decomposition point: 115 ꢂC, IR (KBr):
3415, 2921, 2850, 2051, 2025, 1633, 1508, 1149, 800 cmꢀ1; 1H NMR
(400 MHz, CDCl3)
d
11.62 (s, 1H), 8.23 (d, J ¼ 2.1 Hz, 1H), 8.14 (dd,
J ¼ 4.2, 1.7 Hz, 1H), 7.87 (s, 1H), 7.69 (t, J ¼ 6.0 Hz, 1H), 7.54 (d,
J ¼ 8.6 Hz, 1H), 7.29 (s, 1H), 7.18 (m, 2H), 7.13 (d, J ¼ 8.5 Hz, 1H), 6.80
(s, 0H), 6.08 (s, 2H), 5.13e5.02 (t, J ¼ 6.0 Hz, 2H), 4.11 (t, J ¼ 5.8 Hz,
2H), 3.90 (5H), 3.15 (t, J ¼ 6.0 Hz, 2H), 2.07e1.92 (m, 4H); 13C NMR
(101 MHz, CDCl3)
d 155.23, 150.21, 148.35, 147.68, 146.14, 141.61,
139.72,138.26,135.52,133.32,129.76,125.43,123.74,120.90,120.49,

W.-J. Shan et al. / European Journal of Medicinal Chemistry 46 (2011) 5885e5893
5893
were mixed in the 1 mL 0.1 M KH2PO4/K2HPO4 buffer (pH 8.0),
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fourfold and at least four independent runs were performed for
each sample. Fluorescence measurements were normalized to the
curve of the blank (without antioxidant). The ORAC-FL values were
calculated as described in the reference [44] and the final results
were in mM of Trolox equivalent/mM of pure compound.
4.6.4. Inhibition of Ab1e42 peptide aggregation
As reported in a previously published protocol [45], HFIP pre-
treated Ab1e42 samples (AnaSpec) were resolubilized with a 50 mM
phosphate buffer (pH ¼ 7.4) in order to have a stable stock solution
([A
b
] ¼ 200
mM). The peptide was incubated in 50 mM phosphate
buffer (pH ¼ 7.4) at 37 ꢂC for 48 h (final A
b
concentration 50
M. After incubation, the
L with 50 mM glyci-
neeNaOHbuffer(pH8.0)containingthioflavinT.Then, a300-seconds-
time scan of fluorescence intensity was performed (
exc ¼ 450 nm;
mM) with
and without the tested compound at 20
m
samples were diluted to a final volume of 200
m
l
l
em ¼ 485 nm). Each measurement was run in triplicate. The fluo-
rescence intensities wererecorded and the percentageof inhibitionon
aggregationwas calculatedby using thefollowingequation: (1 e IFi/IFc)
*100%, in which IFi and IFc were the fluorescence intensities obtained
for absorbance in the presence and absence of inhibitors, respectively,
after subtracting the fluorescence of respective blanks.
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Acknowledgments
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We thank the Natural Science Foundation of China (20972198)
and the Ministry of Science and Technology of China (No.
2009ZX09501-017) for financial support of this study.