Inhibition of cholinesterase activity and amyloid aggregation by berberine-phenyl-benzoheterocyclic and tacrine-phenyl-benzoheterocyclic hybrids

Article abstract of DOI:10.1016/j.bmc.2012.02.059

A series of berberine-phenyl-benzoheterocyclic (26-29) and tacrine-phenyl-benzoheterocyclic hybrids (44-46) were synthesised and evaluated as multifunctional anti-Alzheimer's disease agents. Compound 44b, tacrine linked with phenyl-benzothiazole by 3-carbon spacers, was the most potent AChE inhibitor with an IC₅₀ value of 0.017 μM. This compound demonstrated similar Aβ aggregation inhibitory activity with cucurmin (51.8% vs 52.1% at 20 μM, respectively), indicating that this hybrid is an excellent multifunctional drug candidate for AD.

Full text of DOI:10.1016/j.bmc.2012.02.059

Bioorganic & Medicinal Chemistry 20 (2012) 3038–3048
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of cholinesterase activity and amyloid aggregation by
berberine-phenyl-benzoheterocyclic and tacrine-phenyl-
benzoheterocyclic hybrids
⇑
⇑
Ling Huang, Tao Su, Wenjun Shan, Zonghua Luo, Yang Sun, Feng He , Xingshu Li
Institute of Drug Synthesis and Pharmaceutical Process, 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:
A series of berberine-phenyl-benzoheterocyclic (26–29) and tacrine-phenyl-benzoheterocyclic hybrids
(44–46) were synthesised and evaluated as multifunctional anti-Alzheimer’s disease agents. Compound
44b, tacrine linked with phenyl-benzothiazole by 3-carbon spacers, was the most potent AChE inhibitor
Received 14 January 2012
Revised 27 February 2012
Accepted 27 February 2012
Available online 10 March 2012
with an IC50 value of 0.017 lM. This compound demonstrated similar Ab aggregation inhibitory activity
with cucurmin (51.8% vs 52.1% at 20
lM, respectively), indicating that this hybrid is an excellent multi-
functional drug candidate for AD.
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Alzheimer’s disease
Cholinesterase
Amyloid
Multifunctional drug
1. Introduction
been reported, including curcumin and its analogs (1; Fig. 1) as
well as bisphenol A derivatives (2; Fig. 1).7
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder that, as of 2010, has affected approximately 36 million
people.1 The etiology of AD is still elusive, and multiple factors,
Because AD is a complex neurodegenerative disorder resulting
from multiple factors, molecules that modulate the activity of a
single protein target are unable to significantly alter the progres-
sion of the disease. Recently, a strategy of developing drugs that
simultaneously affect multiple targets,8 which is also called the
‘multitarget-directed ligands’ (MTDLs) design strategy, has been
proposed instead of the ‘one protein, one target, one drug’ strategy.
Following the MTDLs design strategy, we have developed a novel
series of benzenediol-berberine hybrids (3; Fig. 1), which simulta-
neously target three molecular abnormalities of AD.9 Herein, we
describe the design, synthesis, and evaluation of berberine-
phenyl-benzoheterocyclic and tacrine-phenyl-benzoheterocyclic
hybrids as multifunctional anti-AD agents that target cholinester-
ase activity and amyloid aggregation.
such as amyloid b (Ab) deposits, s-protein aggregation, oxidative
stress, and low levels of acetylcholine (ACh), have been suggested
to contribute to the development of AD.2 In the past decade, treat-
ment strategies for AD have mainly been aimed at improving cho-
linergic neurotransmission in brain, which was mostly based on
the ‘cholinergic hypothesis’.3 Many approaches have been investi-
gated based on this hypothesis, but cholinesterase inhibitors
(ChEIs) were the first and, to date, the only species that showed
some promise in the treatment of AD.4 The following four cholin-
esterase inhibitors have been approved by the US Food and Drug
Administration: tacrine, donepezil, rivastigmine and galantamine.
These inhibitors have beneficial effects on cognitive, functional
and behavioural symptoms of AD. The amyloid hypothesis, which
is another hypothesis regarding the etiology of AD, states that
the accumulation of Ab in the brain is the primary influence driving
AD pathogenesis.5 According to this hypothesis, the development
of anti-AD drugs to reduce the concentration of Ab oligomers by
inhibiting aggregation could possibly be another therapeutic strat-
egy.6 Recently, a number of Ab fibril formation inhibitors have
2. Results and discussion
2.1. Design and chemistry
Howlett and coworkers described a series of benzofuran ana-
logues (4; Fig. 2) as inhibitors of Ab fibril formation via a process
involving the binding of benzofuran to a peptide.10 Recently, a
new series of hybrid molecules based on benzofuran analogues
and N-methyl-N-benzylamine as inhibitors of acetylcholinesterase
(AChE)/butylcholinesterase (BuChE) and Ab aggregation were
reported.11 The results of this study suggest that MTDLs could be
⇑
Corresponding authors.
E-mail address: lixsh@mail.sysu.edu.cn (X. Li).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.02.059

L. Huang et al. / Bioorg. Med. Chem. 20 (2012) 3038–3048
3039
using carbon spacers of varying lengths (26a–b, 46b–c; Fig. 2) to
investigate their activity when simultaneously affecting multiple
targets. To evaluate the effect on AChE on the phenyl-benzohetero-
cyclic moiety, we substituted the benzofuran moiety with indole
(27a–b; Fig. 2), benzothiazole (28a–b, 44b–e; Fig. 2) and benzoxaz-
ole (29a–b, 45b–c; Fig. 2) for comparison.
The synthetic route of berberine-phenyl-benzoheterocyclic hy-
brids is shown in Scheme 1. Firstly, 4-methoxy-phenyl-benzofuran
(10) was synthesised from the commercially available salicylic
aldehyde (7) as previously reported.13 Then, the O-methyl group
of 10 was removed via a reaction with BBr3 to yield intermediate
11. Compound 14, 4-hydroxybenzoindole, was synthesised by
reacting aniline and 2-bromoacetophenone (13) under microwave
irradiation without any catalysts. Compound 17, 4-hydroxy-phe-
nyl-benzothiazole, was prepared from 2-aminobenzenethiol and
p-hydroxybenzaldehyde with good yields (80%). Compound 20,
4-hydroxy-phenyl-benzooxazole, was synthesised via the conden-
sation of 2-aminophenol (18) with 4-hydroxybenzoic acid (19) in
the presence of p-toluenesulfonic acid. Alkylations of intermedi-
ates 11, 14, 17 and 20 with 1,2-dibromoethane or 1,3-dibromopro-
pane in acetone afforded compounds 21–24, respectively. Target
molecules 26–29 were obtained via reactions of 21–24 with berb-
errubine (25) in 31–69% yields.
Figure 1. Structures of a curcumin analog (1), a bisphenol A derivative (2) and
benzenediol-berberine hybrids (3).
The preparation of hybrids 34–35 is outlined in Scheme 2. The
reaction of commercially available indole-2-carboxylic acid (30)
and benzofuran-2-carboxylic acid (31) with 3-bromopropylmine
produced amides 32–33, which reacted with berberrubine to yield
target compounds 34 and 35.
more therapeutically effective if their structural elements or phar-
macophores were combined rationally to a single molecule. In pre-
vious work, we determined that berberine (5; Fig. 2) could inhibit
AChE in the sub-micromolar range (IC50 = 0.374 lM), which sug-
Tacrine-phenyl-benzoheterocyclic hybrids were synthesised
with phthalimide 36 as the starting material (Scheme 3). Firstly,
gested that berberine was an appropriate leading compound for
development of anti-AD drugs.12 With this new paradigm, we com-
bined the phenyl-benzofuran moiety (anti-Ab aggregation activity)
with berberine and tacrine (6; Fig. 2) (anti-cholinesterase moiety)
the reaction of phthalimide with
a,x-dibromoalkanes in the
presence of K2CO3 provided intermediates 37a–d, which were
Figure 2. Design strategy for compounds 26–29, 44–46.

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L. Huang et al. / Bioorg. Med. Chem. 20 (2012) 3038–3048
Scheme 1. Reagents and conditions: (a) EtOH, NaBH4; (b) acetonitrile, PPh3 HBr; (c) toluene, 4-methoxybenzoyl chloride, NEt3; (d) CH2Cl2, BBr3 (e) CuBr2, EtOH; (f) aniline,
C2H5OC2H4OH, microwave; (g) toluene, reflux, overnight; (h) p-TsOH, xylene, reflux, 12 h; (i) Br(CH2)nBr, K2CO3, actone, reflux; (j) K2CO3, DMF, 80 °C.
Scheme 2. Reagents and conditions: (a) 3-bromopropylamine, EDCI, THF/DMF; (b) K2CO3, DMF, 80 °C, 24 h.
subsequently reacted with compounds 11, 17 and 20 to give com-
pounds 38–40, respectively. Reduced with hydrazine, compounds
38–40 were converted to amines 41–42 with almost quantitative
yields. Finally, the reaction of 9-chloro-1,2,3,4-tetrahydroacridine
with amines 41–42 provided hybrids 44–46 in good yields.
2.2. In vitro inhibition studies of AChE and BuChE
The AChE and BuChE inhibitory effects of the hybrids were
determined by the spectroscopic method described by Ellman
et al. using tacrine as the standard.14 AChE (E.C.3.1.1.7) and BuChE

L. Huang et al. / Bioorg. Med. Chem. 20 (2012) 3038–3048
3041
Scheme 3. Reagents and conditions: (a) Br(CH2)nBr, K2CO3, TEBAC, acetone, 24 h; (b) 11, 17, 20, K2CO3, DMF; (c) hydrazine hydrate, EtOH, reflux, 4 h; (d) 9-chloro-1,2,3,4-
tetrahydroacridine, pentanol, reflux, 24 h.
(E.C. 3.1.1.8) were obtained from electric eel and equine serum,
respectively.
2.3. Kinetics of AChE inhibition
The IC50 values for AChE and BuChE inhibition are summarised
in Table 1. Most of the hybrids demonstrated potent inhibitory
activity against AChE and BuChE with IC50 values in the micromo-
lar to sub-micromolar range. In particular, compound 44b, tacrine
and phenyl-benzofuran linked with a 3-carbon spacer, exhibited
The inhibition type of AChE was investigated by graphical anal-
ysis of steady state inhibition data (Fig. 3) using compound 44b as
a
typical example. Reciprocal plots (Lineweaver–Burk plots)
describing 44b inhibition showed both increasing slopes and
increasing intercepts with higher inhibitor concentration, indicat-
ing a mixed-type inhibition. These results revealed that compound
44b was able to bind to both the active site and PAS of AChE, which
is also in agreement with the results of our molecular modeling
studies.
the most potent inhibition of AChE (IC50 = 0.017 lM), which is
18-fold more potent than tacrine. A simple structure–activity
relationship analysis showed that the AChE inhibitory potency
was closely related to the length of the alkylene chain. From
the IC50 values of the tested compounds, it appears that a 3-car-
bon spacer seems to be the proper length for a linker between the
two pharmacophore groups in both series. For example, com-
pounds 26b, 27b, 28b, 29b and 44b, in which berberine or tacrine
was linked with phenyl-benzofuran via a 3-carbon spacer, exhib-
ited greater inhibition (IC50 = 1.92, 0.774, 1.48, 2.12, and
0.017 lM, respectively) than their relatives, 26a, 27a, 28a, 29a
and 44c–e, which contained linkers that were either 2-, 4-, or
5-carbon spacers (IC50 = 3.59, 1.07, 1.64, 3.30, 0.105, 0.040, and
0.093 lM, respectively). The replacement of the alkylene chain
2.4. Molecular modeling studies
To explain the interaction modes of the hybrids to AChE, molec-
ular docking simulations for derivative 44b to TcAChE were per-
formed using the CDOCKER program in the Discovery studio 2.1
software based on the X-ray crystal structure of the TcAChE-bis-
(7)-tacine complex (PDB entry 2CMF). The docking results showed
that hybrid 44b could simultaneously bind to both the central
pocket and peripheral sites (Fig. 4a, b). The tacrine moiety of the
hybrid was observed to enter the AChE gorge, adopting parallel
with an acyl alkyl chain resulted in only minor changes in AChE
inhibitory activity (27b vs 34, with IC50 values of 0.774 and
p–p interactions with Trp84 (3.67 Å) and Phe330 (4.32 Å) in a
0.976
l
M; 26b vs 35, with IC50 values 1.92 and 1.94
l
M, respec-
‘sandwich’ form. As shown in Figure 4b, the benzothiazole moiety
stacks with Trp279 of PAS with a distance of 4.65 Å of ring-to-ring.
In addition, the benzyl moiety, linked at the rim of the gorge, has a
tively). Most tacrine-phenyl-benzoheterocyclic hybrids were
more potent inhibitors of AChE than the berberine-phenyl-benzo-
heterocyclic hybrids, indicating that the stereospecificity of the
functional groups is very important for inhibitory activity. The re-
sults also indicated that the structure of the benzoheterocyclic
side chain also affects the inhibitory activity of these compounds
(Table 1). The order of activity for the four benzoheterocyclic
groups is as follows: indole > benzothiazole > benzofuran > benz-
oxazole. However, the same trend was not observed in BuChE
inhibition. Almost all the compounds showed potent activity
against BuChE. Additionally, some compounds demonstrated
higher selectivity (i.e., 26–29, 45c) for BuChE than AChE. Com-
pound 44e, the most potent for BuChE inhibition
face-to-edge
p–p interaction with Phe331 (4.71 Å), which could
also fold in a proper conformation into the gorge to interact with
Tyr70, Asp72, Trp271, Phe330 and Tyr334 through hydrophobic
interactions.
2.5. Inhibition of Ab1–42 aggregation
Ab1–40 and Ab1–42 are the main forms of Ab peptides found in
the amyloid plaques, which are released from an amyloid precur-
sor protein through sequential cleavages by b- and
c-secretases.
Although Ab1–40 is the predominant product of this proteolytic
pathway, Ab1–42 is far more fibrillogenic.15 Therefore, we chose
Ab1–42 to study the inhibition of the hybrid compounds. The ability
of novel hybrids to inhibit Ab1–42 aggregation was assessed by the
(IC50 = 0.082 lM), exhibited almost equal inhibitory activity with
AChE. Based on these results, these hybrids should be good dual
AChE/BChE inhibitors to treat AD.

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L. Huang et al. / Bioorg. Med. Chem. 20 (2012) 3038–3048
Table 1
In vitro inhibition of AChE and BuChE and Ab self-aggregation of berberine, cucurmin, and compounds 27–29, 34–35, and 44–46
a
Compound
X
Y
n
IC50
(lM)
Selectivity for AChEd
Ab self-aggregation inhibitorye (IC50
, lM)
AChE/AChb
BuChE/BuChc
Berberine
26a
26b
27a
27b
28a
28b
29a
29b
34
35
44b
44c
44d
44e
45b
45c
—
C
C
C
C
—
O
O
NH
NH
S
—
2
3
2
3
2
3
2
3
—
—
3
4
5
6
3
4
3
4
—
—
0.374 0.024
3.59 0.270
1.92 0.058
1.07 0.005
0.774 0.013
1.64 0.015
1.48 0.091
3.30 0.018
2.12 0.021
0.976 0.060
1.94 0.094
0.017 0.002
0.105 0.014
0.040 0.004
0.093 0.006
0.475 0.002
0.947 0.023
0.058 0.001
0.201 0.014
0.311 0.009
n.t.
18.2 0.683
0.688 0.062
0.990 0.028
0.421 0.001
0.711 0.048
0.175 0.013
0.587 0.031
0.346 0.007
0.639 0.011
2.19 0.127
1.54 0.137
0.122 0.006
0.186 0.010
0.171 0.015
0.082 0.007
0.352 0.010
0.207 0.018
0.156 0.002
0.238 0.008
0.041 0.003
n.t.
48.6
0.2
0.5
0.4
0.9
0.1
0.4
0.1
0.3
1.1
1.7
7.2
1.8
4.2
0.9
0.7
0.2
2.7
1.2
0.13
—
36.3%f
5.78 0.37
84.6%
4.03 0.14
4.69 0.50
6.45 0.44
79.2%
3.61 0.09
3.75 0.10
73.5%
69.1%
51.8%
37.8%
40.7%
41.9%
57.6%
33.2%
f
N
N
N
N
—
—
N
N
N
N
N
N
C
f
S
O
O
—
—
S
S
S
S
O
O
O
O
—
—
f
f
f
f
f
f
f
f
f
46b
46c
Tacrine
Curcumin
62.8%
f
C
—
—
46.3%
n.t.g
18.7 0.9952.1%f
a
b
c
d
e
f
Mean SD of at least three independent measurements.
Acetylcholine substrate for evaluation of antiacetylcholinesterase activity.
Butyrylcholine substrate for evaluation of antibutyrylcholinesterase activity.
Selectivity for AChE = IC50 (BuChE)/IC50 (AChE).
The thioflavin-T fluorescence method was used. Value represents as the mean SD from at least two independent measurements.
All values were measured at the inhibitors’ concentration of 20
n.t. = not tested.
lM.
g
thioflavin T fluorescence assay using curcumin as a standard (Table
1). Significantly, all the berberine-phenyl-benzoheterocyclic
hybrids (IC50 = 3.61–6.45
greater inhibitory activities than curcumin (IC50 = 18.7
at 20 M) and berberine (36.3% at 20 M). Among these, com-
pound 29a, the berberine-phenyl-benzoxazole hybrid, was the
most potent inhibitor of Ab aggregation (IC50 = 3.61 M), which is
lM, 69.1–84.6% at 20
lM) exhibited
lM, 52.1%
l
l
l
5.2-fold more potent than curcumin. Conversely, most of the ber-
berine-phenyl-benzoheterocyclic hybrids exhibited higher or sim-
ilar Ab aggregation inhibitory activity with cucurmin. Among
them, compound 46b exhibits the highest inhibitory activity of
62.8% at 20
AChE inhibitor, demonstrated similar Ab aggregation inhibitory
activity with cucurmin (51.8% vs 52.1% at 20 M, respectively),
lM. In particular, compound 44b, the most potent
l
which indicated that compound 44b is a good multifunctional can-
Figure 3. Steady state inhibition by 44b of AChE hydrolysis of Ach. The plots show
mixed-type inhibition for 44b on AChE.
didate drug for AD.

L. Huang et al. / Bioorg. Med. Chem. 20 (2012) 3038–3048
3043
4.1.1. 2-(Hydroxymethyl)phenol (8)
Sodium borohydride (6 mmol) was added to a stirring solution
of 2-hydroxybenzaldehyde (12 mmol) in ethanol (20 mL) at 0 °C.
The reaction mixture was stirred at room temperature for 1 h. After
the solvent was removed, 40 mL of 1 N HCl solution was added to
the residue and extracted with diethyl ether (40 mL). The organic
phase was dried over Na2SO4 and filtered. The filtrate was concen-
trated to give compound 8 (90%). 1H NMR (400 MHz, CDCl3) d 7.48
(s, 1H), 7.24 (d, J = 6.9 Hz, 1H), 7.22–7.12 (m, 1H), 7.02 (d, J = 6.9 Hz,
1H), 6.92–6.78 (m, 2H), 4.78 (s, 2H), 2.91 (s, 1H); 13C NMR
(101 MHz, CDCl3)
116.40, 64.27.
d 155.80, 129.48, 128.04, 124.87, 120.19,
4.1.2. 2-Hydroxybenzyltriphenylphosphonium bromide (9)
A solution of 8 (10 mmol) and triphenylphosphine hydrobro-
mide (10 mmol) in acetonitrile (40 mL) was stirred under reflux
for 1 h. The solid that formed was filtered and washed with aceto-
nitrile to give compound
9
as white solid. (85%). 1H NMR
(400 MHz, CDCl3) d 9.00 (s, 1H), 7.79–7.68 (m, 3H), 7.63–7.44 (m,
12H), 7.27 (m, 1H), 7.02–6.95 (m, 1H), 6.95–6.90 (m, 1H), 6.59
(t, J = 7.4 Hz, 1H), 4.55 (d, J = 13.5 Hz, 2H).
4.1.3. 2-(4-Methoxyphenyl)benzofuran (10)
A mixture of 9 (5 mmol) and 4-methoxybenzoyl chloride
(5 mmol) in a mixed solvent (toluene 20 mL and triethylamine
0.5 mL) was stirred under reflux for 2 h. The precipitate was re-
moved by filtration. The filtrate was concentrated, and the residue
was recrystalized with ethyl acetate to give 10 (30%). 1H NMR
(400 MHz, CDCl3) d 7.76–7.62 (m, 2H), 7.43 (ddd, J = 26.7, 9.3,
4.8 Hz, 2H), 7.19–7.07 (m, 2H), 6.91–6.82 (m, 2H), 6.77 (d,
J = 0.7 Hz, 1H), 3.74 (s, 3H); 13C NMR (101 MHz, CDCl3) d 158.94,
155.02, 153.66, 128.46, 125.37, 122.69, 122.30, 121.79, 119.53,
113.21, 109.94, 98.64, 54.29.
Figure 4. Docking models of the compound–enzyme complex. (a) Stereoviews
looking down the gorge of TcAChE binding with 44b. (b) Representation of
compound 44b docked into the binding site of AChE, highlighting the protein
residues that form the main interactions with the inhibitor.
3. Conclusion
4.1.4. 4-(Benzofuran-2-yl)phenol (11)
BBr3 (12 mL,1 M solution in CH2Cl2) was added to a solution of
10 (2 mmol) in CH2Cl2 (30 mL) dropwise in an ice bath. The mix-
ture was allowed to warm to room temperature and stirred for
30 min. Water (40 mL) was added while the reaction mixture
was cooled in an ice bath. The mixture was extracted with ethyl
acetate, and the organic phase was dried over Na2SO4 and filtered.
The filtrate was concentrated, and the residue was purified by
chromatography on a silica gel column with EtOAc/petroleum
ether as eluent to give 11 (63%). 1H NMR (400 MHz, CDCl3) d
9.35 (s, 1H), 7.61 (d, J = 8.2 Hz, 2H), 7.45 (d, J = 6.9 Hz, 1H), 7.39
(d, J = 7.5 Hz, 1H), 7.18–7.07 (m, 2H), 6.87–6.77 (m, 3H); 13C
NMR (101 MHz, CDCl3) d 163.10, 161.18, 159.15, 134.26, 131.18,
128.33, 127.63, 126.30, 125.26, 120.74, 115.58, 103.72.
In conclusion, we have synthesised and evaluated a series of
berberine-phenyl-benzoheterocyclic (26–29) and tacrine-phenyl-
benzoheterocyclic hybrids (44–46) as multifunctional anti-AD
agents. Our results demonstrate that these compounds possess po-
tent AChE and Ab aggregation inhibitory activities. Compound 44b,
tacrine linked with phenyl-benzothiazole by 3-carbon spacers, was
the most potent AChE inhibitor with an IC50 of 0.017
ally, compound 44b demonstrated similar Ab aggregation inhibi-
tory activity with cucurmin (51.8% vs 52.1% at 20 M,
lM. Addition-
l
respectively), indicating that this compound is an excellent multi-
functional drug candidate for AD. Kinetic analyses and molecular
docking stimulations revealed that the inhibitors could simulta-
neously interact with the central pocket, gorge, and peripheral
pocket of AChE. Finally, our results indicate that these new
compounds represent useful templates for the development of
new multifunctional anti-AD agents.
4.1.5. 2-Bromo-1-(4-hydroxyphenyl)ethanone (13)
CuBr2 (17.92 g, 80 mmol) was added to a stirring solution of 12
(5.44 g, 40 mmol) in ethanol (120 mL) in room temperature. The
mixture was stirred under reflux for 6 h. The precipitate was re-
moved by filtration. The filtrate was concentrated, and the residue
was dissolved in ethyl acetate. The solution was washed with
water, and the organic phase was dried over Na2SO4 and filtered.
The filtrate was concentrated, and the residue was purified by
recrystalization with ethanol to give 6.25 g of 13 (75.8%). 1H
NMR (400 MHz, DMSO) d 10.44 (d, J = 64.4 Hz, 1H), 7.89 (d,
J = 6.6 Hz, 2H), 6.87 (d, J = 8.0 Hz, 2H), 4.99–4.56 (m, 2H); 13C
NMR (101 MHz, DMSO) d 189.81, 162.61, 131.40, 130.01, 125.35,
115.38, 40.45, 33.43.
4. Experimental section
4.1. Chemistry
The 1H NMR and 13C NMR spectra were recorded using TMS as
the internal standard on a Bruker BioSpin GmbH spectrometer at
400.132 MHz and 100.614 MHz, respectively. Coupling constants
are given in Hz. High-resolution mass spectra were obtained using
a
Shimadzu LCMS-IT-TOF mass spectrometer. Flash column
chromatography was performed using Silica gel (200–300 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 using silica gel.
4.1.6. 4-(1H-Indol-2-yl)phenol (14)
A solution of 13 (0.56 g, 2.5 mmol) and aniline (0.7 g, 7.5 mmol)
in 2-ethoxyethanol (5 mL) was placed in a microwave tube. The

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L. Huang et al. / Bioorg. Med. Chem. 20 (2012) 3038–3048
mixture was heated in a microwave reactor at 140 °C for 10–12 min.
The mixture was filtered and then evaporated under vacuum .The
residue was purified by recrystalizaiton with ethanol and water,and
then was purified again by chromatography on a silica gel column to
give a yellow solid 0.2 g of 14 (38.3%). 1H NMR (400 MHz, CDCl3) d
10.68 (s, 1H), 9.08 (s, 1H), 7.54 (d, J = 8.3 Hz, 2H), 7.42 (d,
J = 7.7 Hz, 1H), 7.30 (d, J = 7.9 Hz, 1H), 6.97 (dd, J = 14.7, 6.8 Hz,
4.2.3. 2-(4-(3-Bromoethoxy)phenyl)-1H-indole (22a)
Compound 14 was treated with 1,2-dibromethane according to
general procedure to give the desired product 22a (38% yield). 1H
NMR (400 MHz, CDCl3) d 8.27 (s, 1H), 7.59 (t, J = 8.7 Hz, 3H), 7.37
(d, J = 8.0 Hz, 1H), 7.17 (dd, J = 11.1, 4.0 Hz, 1H), 7.11 (t, J = 7.4 Hz,
1H), 6.97 (d, J = 8.7 Hz, 2H), 6.72 (d, J = 1.3 Hz, 1H), 4.32 (t,
J = 6.2 Hz, 2H), 3.65 (t, J = 6.2 Hz, 2H); 13C NMR (101 MHz, CDCl3)
d 158.48, 137.87, 136.68, 129.43, 126.51, 125.49, 121.96, 120.38,
120.20, 115.10, 110.72, 98.94, 65.48, 32.34, 29.90.
1H), 6.91 (t, J = 7.1 Hz, 1H), 6.80 (d, J = 8.2 Hz, 2H), 6.52 (s, 1H); 13
C
NMR (101 MHz, CDCl3) d 155.99, 137.61, 135.80, 128.43, 125.69,
123.22, 120.38, 119.03, 118.76, 115.04, 109.88, 96.86.
4.2.4. 2-(4-(3-Bromopropoxy)phenyl)-1H-indole (22b)
4.1.7. 4-(Benzothiazole-2-yl)phenol (17)
Compound 14 was treated with 1,3-dibrompropane according
to general procedure to give the desired product 22b (54% yield).
1H NMR (400 MHz, CDCl3) d 8.25 (s, 1H), 7.59 (t, J = 8.3 Hz, 3H),
7.38 (d, J = 7.9 Hz, 1H), 7.17 (t, J = 7.4 Hz, 1H), 7.11 (t, J = 7.3 Hz,
1H), 6.98 (d, J = 8.6 Hz, 2H), 6.71 (s, 1H), 4.15 (t, J = 5.8 Hz, 2H),
3.62 (t, J = 6.4 Hz, 2H), 2.42–2.25 (m, 2H); 13C NMR (101 MHz,
CDCl3) d 158.48, 137.87, 136.68, 129.43, 126.51, 125.49, 121.96,
120.38, 120.20, 115.10, 110.72, 98.94, 76.41, 65.48, 32.34, 29.90.
A mixture of 15 (1.88 g, 15 mmol) and 16 (1.83 g, 15 mmol) in
toluene (50 mL) was heated to reflux for overnight. After cooling
to room temperature, the precipitate was removed by filtration.
The filtrate was concentrated, and the residue was purified by
recrystalization with toluene to give 2.1 g of 13(61.7%). 1H NMR
(400 MHz, DMSO) d 10.20 (s, 1H), 8.07 (d, J = 7.9 Hz, 1H), 7.97 (d,
J = 7.9 Hz, 1H), 7.95–7.90 (m, 2H), 7.53–7.46 (m, 1H), 7.42–7.36
(m, 1H), 6.96–6.89 (m, 2H).
4.2.5. 2-(4-(3-Bromoethoxy)phenyl)benzo[d]thiazole (23a)
Compound 17 was treated with 1,2-dibromethane according to
general procedure to give the desired product 23a (22% yield). 1H
NMR (400 MHz, CDCl3) d 8.03 (dd, J = 9.2, 2.4 Hz, 3H), 7.87 (d,
J = 7.9 Hz, 1H), 7.50–7.44 (m, 1H), 7.39–7.32 (m, 1H), 7.03–6.97
(m, 2H), 4.34 (t, J = 6.2 Hz, 2H), 3.66 (t, J = 6.2 Hz, 2H); 13C NMR
4.1.8. 4-(Benzoxazole-2-yl)phenol (20)
A mixture of 18 (1.09 g, 10 mmol) and 19(1.66 g, 12 mmol) and
p-toluenesulfonic(9.51 g, 50 mmol) in xylene (40 mL) was heated
to reflux for 12 h. Ethyl acetate(120 ml) was added to the mixture,
and then washed with saturated sodium bicarbonate (20 ꢀ 50 ml)
and brine (50 ml).The organic phase was dried over Na2SO4 and fil-
tered. The filtrate was concentrated to get the crude product 1.26 g,
which was used in the next step without further purification. 1H
NMR (400 MHz, CDCl3) d 14.67 (s, 1H), 12.74 (d, J = 8.1 Hz, 2H),
12.35 (d, J = 5.9 Hz, 1H), 12.29–12.23 (m, 1H), 12.01 (d, J = 2.2 Hz,
(101 MHz, CDCl3)
d 166.55, 159.32, 153.16, 133.85, 128.16,
126.08, 125.23, 123.87, 121.86, 120.51, 114.01, 66.87, 27.72.
4.2.6. 2-(4-(3-Bromopropoxy)phenyl)benzo[d]thiazole (23b)
Compound 17 was treated with 1,3-dibrompropane according
to general procedure to give the desired product 23b (35% yield).
1H NMR (400 MHz, CDCl3) d 7.93 (t, J = 7.3 Hz, 3H), 7.77 (d,
J = 7.9 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 7.26 (t, J = 7.5 Hz, 1H), 6.89
(d, J = 8.7 Hz, 2H), 4.09 (dt, J = 11.5, 5.9 Hz, 2H), 3.52 (t, J = 6.4 Hz,
2H), 2.31–2.18 (m, 2H); 13C NMR (101 MHz, CDCl3) d 166.79,
159.96, 153.10, 133.77, 128.09, 125.52, 125.20, 123.80, 121.76,
120.48, 113.82, 64.43, 31.15, 28.79.
2H), 11.64 (d, J = 8.1 Hz, 2H); 13C NMR (101 MHz, CDCl3)
168.08, 165.94, 155.26, 147.03, 134.19, 129.33, 129.24, 124.09,
122.55.
d
4.2. General procedures for the preparation of 21a–b, 22a–b,
23a–b, 24a–b
To a stirred suspension of selective compounds (11, 14, 17, 20)
(5 mmol) and K2CO3 (15 mmol) in acetone (35 mL), dibromoalk-
anes (20 mmol) were added. The mixture was heated to reflux
for 12–24 h, and then was filtered. The filtrate evaporated under
vacuum to afford the crude product, which was purified by chro-
matography on an silica gel column with EtOAc/petroleum ether
as eluent to give the target products.
4.2.7. 2-(4-(3-Bromoethoxy)phenyl)benzo[d]oxazole (24a)
Compound 20 was treated with 1,2-dibromethane according to
general procedure to give the desired product 24a (40% yield). 1H
NMR (400 MHz, CDCl3) d 8.13 (d, J = 8.7 Hz, 2H), 7.67 (dd, J = 8.4,
4.4 Hz, 1H), 7.48 (dd, J = 8.4, 4.4 Hz, 1H), 7.29–7.23 (m, 2H), 6.97
(d, J = 8.7 Hz, 2H), 4.30 (t, J = 6.2 Hz, 2H), 3.60 (t, J = 6.2 Hz, 2H);
13C NMR (101 MHz, CDCl3) d 161.92, 159.75, 149.67, 141.20,
128.46, 123.71, 123.47, 119.40, 118.67, 113.99, 109.40, 66.87,
27.66, 21.11.
4.2.1. 2-(4-(2-Bromoethoxy)phenyl)benzofuran (21a)
Compound 10 was treated with 1,2-dibromethane according to
general procedure to give the desired product 21a (47% yield). 1H
NMR (400 MHz, CDCl3) d 7.80 (dd, J = 12.6, 5.7 Hz, 2H), 7.55 (dd,
J = 7.8, 6.6 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.30–7.27 (m, 1H),
7.25–7.18 (m, 1H), 6.99 (d, J = 8.9 Hz, 2H), 6.93–6.88 (m, 1H),
4.35 (t, J = 6.3 Hz, 2H), 3.67 (t, J = 6.3 Hz, 2H); 13C NMR (101 MHz,
CDCl3) d 157.41, 154.76, 153.70, 128.39, 125.48, 123.07, 122.85,
121.85, 119.62, 114.02, 109.99, 98.96, 66.89, 27.90.
4.2.8. 2-(4-(3-Bromopropoxy)phenyl)benzo[d]oxazole (24b)
Compound 20 was treated with 1,3-dibrompropane according
to general procedure to give the desired product 24b (32% yield).
1H NMR (400 MHz, CDCl3) d 8.13 (d, J = 8.7 Hz, 2H), 7.67 (dd,
J = 7.8, 5.1 Hz, 1H), 7.51–7.44 (m, 1H), 7.26 (p, J = 7.0 Hz, 2H),
6.96 (d, J = 8.8 Hz, 2H), 4.21–4.06 (m, 2H), 3.56 (t, J = 6.4 Hz, 2H),
2.35–2.22 (m, 2H); 13C NMR (101 MHz, CDCl3) d 162.07, 160.39,
149.65, 141.22, 128.40, 123.62, 123.42, 118.90, 118.61, 113.83,
109.37, 64.47, 31.17, 28.73.
4.2.2. 2-(4-(3-Bromopropoxy)phenyl)benzofuran (21b)
Compound 10 was treated with 1,3-dibrompropane according
to general procedure to give the desired product 21b (62% yield).
1H NMR (400 MHz, CDCl3) d 7.72 (d, J = 8.8 Hz, 2H), 7.48 (d,
J = 7.2 Hz, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.20 (d, J = 1.2 Hz, 1H),
7.14 (dd, J = 12.5, 4.6 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 6.82 (s, 1H),
4.13–4.01 (m, 2H), 3.59–3.49 (m, 2H), 2.34–2.21 (m, 2H); 13C
NMR (101 MHz, CDCl3) d 158.05, 154.93, 153.68, 128.44, 125.42,
122.76, 122.57, 121.82, 119.57, 113.80, 109.97, 98.75, 64.40,
31.29, 28.88.
4.3. General procedures for the preparation of 26a–b, 27a–b,
28a–b, 29a–b
Compounds 21a–b, 22a–b, 23a–b, 24a–b (2 mmol) was added
to a magnetically stirred suspension of 25 (2 mmol), K2CO3
(6 mmol) in DMF(15 mL) under an argon atmosphere. The mixture
was allowed to heat in reflux for 24 h, and monitored by TLC. When

L. Huang et al. / Bioorg. Med. Chem. 20 (2012) 3038–3048
3045
the mixture was cooled to room temperature, filtered, and then
evaporated under vacuum. The crude product was chromato-
graphed on an Al2O3 column, eluted with CHCl3/MeOH as eluent
to afford the proposed compound.
142.69, 137.63, 137.47, 136.88, 133.00, 130.62, 128.75, 126.68,
126.34, 124.98, 123.40, 121.55, 121.00, 120.39, 120.21, 119.60,
119.18, 114.88, 111.04, 108.36, 105.39, 102.04, 97.30, 71.24,
64.44, 59.71, 57.01, 55.22, 29.43. HRMS m/z [M-Br]+ Calcd for
C36H30N2O5 571.2233, found 571.2249.
4.3.1. 9-O-2-(4-(2-Ethoxy)phenyl)benzofuran-berberine
bromide (26a)
4.3.5. 9-O-2-(4-(3-Ethoxy)phenyl)benzo[d]thiazole-berberine
bromide (28a)
Compound 21a was treated with berberrubine 25 according to
general procedure to give the desired product 26a as a yellow solid
(39% yield). Mp 216.3–218.3 °C; 1H NMR (400 MHz, DMSO) d 9.79
(s, 1H), 8.93 (s, 1H), 8.23 (d, J = 9.1 Hz, 1H), 8.03 (d, J = 9.1 Hz, 1H),
7.80 (d, J = 8.7 Hz, 2H), 7.76 (s, 1H), 7.61 (d, J = 7.1 Hz, 1H), 7.57 (d,
J = 7.8 Hz, 1H), 7.31–7.22 (m, 3H), 7.02 (s, 1H), 6.98 (d, J = 8.7 Hz,
2H), 6.14 (s, 2H), 4.83 (s, 2H), 4.71 (s, 2H), 4.53 (s, 2H), 4.08 (s,
3H), 3.13 (s, 2H). 13C NMR (101 MHz, DMSO) d 154.17, 150.94,
148.91, 148.05, 144.33, 141.99, 139.38, 138.14, 135.22, 133.07,
131.27, 128.46, 127.02, 126.47, 125.59, 125.98, 123.02, 121.94,
120.36, 120.11, 119.23, 119.09, 118.20, 113.54, 110.71, 109.13,
106.47, 100.13, 96.41, 74.29, 63.12, 57.88, 52.07. HRMS m/z [M-
Br]+ Calcd for C35H27NO6 558.1917, found 558.1929.
Compound 23a was treated with berberrubine 25 according to
general procedure to give the desired product 28a as a yellow solid
(35% yield). Mp 219.3–220.8 °C; 1H NMR (400 MHz, DMSO) d 9.79
(s, 1H), 8.92 (s, 1H), 8.23 (d, J = 9.2 Hz, 1H), 8.10 (d, J = 7.8 Hz, 1H),
8.04–7.94 (m, 4H), 7.74 (s, 1H), 7.52 (dd, J = 11.2, 4.1 Hz, 1H), 7.45–
7.41 (m, 1H), 7.09–6.98 (m, 3H), 6.13 (s, 2H), 4.89–4.80 (m, 2H),
4.73 (s, 2H), 4.57 (s, 2H), 4.11 (d, J = 21.9 Hz, 3H), 3.16–3.10 (m,
2H); 13C NMR (101 MHz, DMSO)
d 166.75, 160.52, 153.55,
150.21, 149.75, 147.60, 145.16, 142.40, 137.42, 134.17, 132.99,
130.44, 128.79, 126.62, 126.49, 125.65, 125.12, 123.62, 122.42,
122.12, 121.71, 120.27, 120.21, 115.23, 108.28, 105.32, 102.00,
72.08, 67.52, 57.02, 55.26, 30.63. HRMS m/z [M-Br]+ Calcd for
C34H26N2O5S 575.1641, found 575.1663.
4.3.2. 9-O-2-(4-(3-Propoxy)phenyl)benzofuran-berberine
bromide (26b)
4.3.6. 9-O-2-(4-(3-Opropoxy)phenyl)benzo[d]thiazole-berberine
bromide (28b)
Compound 21b was treated with berberrubine 25 according to
general procedure to give the desired product 26b as a yellow solid
(42% yield). Mp 210.3–213.0 °C; 1H NMR (400 MHz, DMSO) 9.82 (s,
1H), 8.57 (s, 1H), 8.30 (d, J = 9.1 Hz, 1H), 8.01 (d, J = 9.1 Hz, 1H),
7.80–7.67 (m, 3H), 7.50 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H),
7.17–7.01 (m, 4H), 6.90–6.81 (m, 1H), 6.75 (s, 1H), 6.16 (s, 2H),
4.91 (s, 2H), 4.49 (s, 2H), 4.37 (s, 2H), 4.07 (s, 3H), 3.22 (s, 2H),
2.41 (s, 2H). 13C NMR (101 MHz, DMSO) d 159.41, 153.66, 151.71,
149.37, 146.13, 144.50, 139.21, 138.86, 138.78, 134.10, 133.75,
126.79, 126.38, 125.21, 124.89, 124.10, 122.41, 121.57, 120.87,
120.01, 119.94, 118.32, 116.35, 110.59, 107.48, 105.29, 103.90,
96.09, 72.14, 65.37, 57.71, 56.91, 54.05, 28.62. HRMS m/z [M-Br]+
Calcd for C36H29NO6 572.2073, found 572.2094.
Compound 23b was treated with berberrubine 25 according to
general procedure to give the desired product 28b as a yellow solid
(25% yield). Mp 204.3–206.6 °C; 1H NMR (400 MHz, DMSO) d 9.82
(s, 1H), 8.93 (s, 1H), 8.19 (d, J = 9.1 Hz, 1H), 8.13–8.06 (m, 1H), 8.01
(dd, J = 15.0, 8.9 Hz, 4H), 7.77 (s, 1H), 7.52 (t, J = 7.3 Hz, 1H), 7.43 (t,
J = 7.2 Hz, 1H), 7.14 (d, J = 8.7 Hz, 2H), 7.06 (s, 1H), 6.15 (s, 2H), 4.92
(s, 2H), 4.52 (t, J = 6.0 Hz, 2H), 4.39 (t, J = 6.0 Hz, 2H), 4.02 (s, 3H),
3.20 (s, 2H), 2.42–2.36 (m, 2H); 13C NMR (101 MHz, DMSO) d
166.88, 161.04, 153.63, 150.23, 149.83, 147.67, 145.18, 142.73,
137.48, 134.19, 133.05, 130.54, 128.83, 126.71, 126.43, 125.59,
125.05, 123.38, 122.40, 122.08, 121.54, 120.35, 120.22, 115.21,
108.33, 105.38, 102.02, 71.14, 64.79, 57.02, 55.24, 29.36, 26.33.
HRMS m/z [M-Br]+ Calcd for C35H28N2O5S 589.1797, found
589.1826.
4.3.3. 9-O-2-(4-(3-Ethoxy)phenyl)-1H-indole-berberine bromide
(27a)
Compound 22a was treated with berberrubine 25 according to
general procedure to give the desired product 27a as a yellow solid
(30% yield). Mp 211.4–213.7 °C; 1H NMR (400 MHz, DMSO) d 11.36
(s, 1H), 9.79 (s, 1H), 8.93 (s, 1H), 8.23 (d, J = 9.2 Hz, 1H), 8.03 (d,
J = 9.1 Hz, 1H), 7.76 (d, J = 3.5 Hz, 2H), 7.74 (s, 1H), 7.48 (d,
J = 7.7 Hz, 1H), 7.36 (d, J = 7.9 Hz, 1H), 7.10–7.02 (m, 2H), 6.97 (t,
J = 8.0 Hz, 3H), 6.72 (d, J = 1.5 Hz, 1H), 6.07 (d, J = 59.8 Hz, 2H),
4.92–4.75 (m, 2H), 4.70 (dd, J = 5.3, 2.7 Hz, 2H), 4.60–4.44 (m,
2H), 4.19–3.96 (m, 3H), 3.17–3.06 (m, 2H); 13C NMR (101 MHz,
DMSO) d 157.47, 150.29, 149.79, 147.65, 145.21, 142.48, 137.48,
137.44, 136.87, 132.97, 130.47, 128.72, 126.62, 126.29, 125.15,
124.31, 123.61, 121.76, 121.06, 120.31, 120.23, 119.61, 119.20,
114.83, 111.01, 108.32, 105.37, 102.03, 97.42, 72.21, 67.12, 57.02,
54.85. HRMS m/z [M-Br]+ Calcd for C35H28N2O5 557.2100, found
557.2076.
4.3.7. 9-O-2-(4-(3-Ethoxy)phenyl)benzo[d]oxazole-berberine
bromide (29a)
Compound 24a was treated with berberrubine 25 according to
general procedure to give the desired product 29a as a yellow solid
(33% yield). Mp 221.4–224.1 °C; 1H NMR (400 MHz, DMSO) d 9.80
(s, 1H), 8.92 (s, 1H), 8.23 (d, J = 9.2 Hz, 1H), 8.09 (d, J = 8.8 Hz, 2H),
8.03 (d, J = 9.1 Hz, 1H), 7.74 (td, J = 5.2, 1.7 Hz, 3H), 7.42–7.38 (m,
2H), 7.08 (d, J = 8.8 Hz, 2H), 7.02 (s, 1H), 6.13 (s, 2H), 4.88–4.79
(m, 2H), 4.73 (s, 2H), 4.58 (s, 2H), 4.07 (s, 3H), 3.17–3.19 (m, 2H);
13C NMR (101 MHz, DMSO) d 163.45, 163.01, 151.43, 151.09,
150.37, 146.21, 144.79, 141.97, 141.29, 138.62, 135.34, 132.77,
130.25, 129.07, 127.04, 125.33, 124.86, 122.42, 121.55, 121.10,
120.17, 117.46, 116.36, 111.11, 106.24, 104.98, 100.86, 78.88,
70.57, 67.75, 55.67, 54.12. HRMS m/z [M-Br]+ Calcd for
C34H26N2O6 559.1869, found 559.1883.
4.3.4. 9-O-2-(4-(3-Propoxy)phenyl)-1H-indole-berberine
bromide (27b)
4.3.8. 9-O-2-(4-(3-Propoxy)phenyl)benzo[d]oxazole-berberine
bromide (29b)
Compound 22b was treated with berberrubine 25 according to
general procedure to give the desired product 27b as a yellow solid
(38% yield). Mp 192.3–194.9 °C; 1H NMR (400 MHz, DMSO) d 11.44
(s, 1H), 9.81 (s, 1H), 8.94 (s, 1H), 8.20 (d, J = 9.1 Hz, 1H), 8.00 (d,
J = 9.1 Hz, 1H), 7.80 (d, J = 9.0 Hz, 3H), 7.49 (d, J = 7.7 Hz, 1H),
7.37 (d, J = 8.0 Hz, 1H), 7.04 (dd, J = 19.3, 14.0 Hz, 4H), 6.97 (t,
J = 7.4 Hz, 1H), 6.75 (s, 1H), 6.16 (s, 2H), 4.90 (s, 2H), 4.51 (s, 2H),
4.33 (s, 2H), 4.03 (s, 3H), 3.19 (s, 2H), 2.37 (s, 2H); 13C NMR
Compound 24b was treated with berberrubine 25 according to
general procedure to give the desired product 29b as a yellow solid
(42% yield). Mp 210.5–212.4 °C; 1H NMR (400 MHz, DMSO) d 9.81
(s, 1H), 8.92 (s, 1H), 8.19 (d, J = 9.2 Hz, 1H), 8.15 (d, J = 8.9 Hz, 2H),
8.00 (d, J = 9.1 Hz, 1H), 7.78–7.73 (m, 3H), 7.42–7.38 (m, 2H), 7.19
(d, J = 8.9 Hz, 2H), 7.07 (s, 1H), 6.16 (s, 2H), 4.94–4.88 (m, 2H), 4.51
(t, J = 6.2 Hz, 2H), 4.40 (t, J = 6.1 Hz, 2H), 4.02 (s, 3H), 3.22–3.17 (m,
2H), 2.42–2.36 (m, 2H); 13C NMR (101 MHz, DMSO) d 162.28,
161.44, 150.23, 150.06, 149.78, 147.64, 145.20, 142.67, 141.60,
(101 MHz, DMSO)
d 158.01, 150.28, 149.80, 147.65, 145.23,

3046
L. Huang et al. / Bioorg. Med. Chem. 20 (2012) 3038–3048
137.45, 132.99, 130.60, 129.13, 126.66, 125.00, 124.71, 123.41,
121.52, 120.36, 120.19, 119.38, 118.76, 115.25, 110.66, 108.36,
105.35, 102.03, 79.12, 71.11, 64.80, 56.99, 55.21, 30.64, 28.27.
HRMS m/z [M-Br]+ Calcd for C35H28N2O6 573.2026.1869, found
573.2048.
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.
4.7. General procedures for the preparation of 38a–d, 39a–b,
40a–b
4.4. General procedures for the preparation of 32, 33
To a stirred mixture of 11, 17, 20(12 mmol) and K2CO3(20 m-
mol) in DMF (20 mL), 37a–d (10 mmol) were added. After stirred
at room temperature for 10–12 h, the mixture was quenched with
water and extracted with EtOAc for three times.The combined or-
ganic phase was dried over Na2SO4, filtered and concentrated to
give the crude product, which was purified by chromatography
on an silica gel column with EtOAc/petroleum ether aseluent to
give the products.
To 30, 31 (10 mmol) solution in THF/DMF (50 ml), 3-bromopro-
pan-1-amine (11 mmol) was slowly added at 0 °C and then EDCI
(11 mmol). After stirred for 1 h at room temperature, the reaction
mixture was washed with NaHCO3 solution (2ꢀ50 ml), water
(2ꢀ50 ml), brine (1ꢀ50 ml), and then dried with Na2SO4, concen-
trated in vacuo to afford the resulting compound (32, 33) which
was used for the next step without purification.
4.5. General procedures for the preparation of 34, 35
4.8. General procedures for the preparation of 41a–d, 42a–b,
43a–b
Compounds 32, 33 (2 mmol) was added to a magnetically stir-
red suspension of berberrubine 25 (2 mmol), K2CO3 (6 mmol) in
DMF (15 mL) under an argon atmosphere. The mixture was al-
lowed to heated in reflux for 24 h, and monitored by TLC. When
the mixture was cooled to room temperature,filtered, and then
evaporated under vacuum. The crude product was chromato-
graphed on an Al2O3 column, eluted with CHCl3/MeOH as eluent
to afford the proposed compound.
An solution of 38a–d, 39a–b, 40a–b (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 fil-
tered and the solid was washed with 95% EtOH. The whole filtrate
was concentrated and the solid was dissolved in CH2Cl2. The mix-
ture was dried over Na2SO4, filtered and concentrated to give the
crude product, which was used in the next step without further
purification.
4.5.1. 9-O-N-(3-Bromopropyl)-1H-indole-2-carboxamide-
berberine bromide (34)
4.9. General procedures for the preparation of 44a-d,45a-b,46a-
b
Compound 32 was treated with berberrubine 25 according to
general procedure to give the desired product 34 as a yellow solid
(34% yield). Mp 173.3–176.1 °C; 1H NMR (400 MHz, DMSO) d 11.59
(s, 1H), 9.99 (s, 1H), 8.92 (s, 1H), 8.78 (t, J = 5.6 Hz, 1H), 8.19 (d,
J = 9.2 Hz, 1H), 7.98 (d, J = 9.1 Hz, 1H), 7.78 (s, 1H), 7.59 (d,
J = 8.0 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.22–7.14 (m, 2H), 7.11 (s,
1H), 7.02 (t, J = 7.4 Hz, 1H), 6.18 (s, 2H), 5.10–4.96 (m, 2H), 4.39
(t, J = 6.0 Hz, 2H), 3.61 (dd, J = 12.3, 6.3 Hz, 2H), 3.28–3.17 (m,
2H), 2.21–2.09 (m, 2H); 13C NMR (101 MHz, DMSO) d 161.32,
150.26, 145.36, 137.42, 136.31, 132.95, 131.61, 130.67, 126.98,
123.35, 121.39, 119.72, 112.22, 108.38, 102.55, 102.02, 71.80,
56.99, 56.00, 29.93, 26.33, 18.44. HRMS m/z [M-Br]+ Calcd for
To a solution of 9-chloro-1,2,3,4-tetrahydroacridine (0.217 g,
1 mmol) in pentanol, 41a–d, 42a–b, 43a–b (4–10 mmol) were
added. The mixture was stirred in reflux for 10–24 h. After the
reaction completed (monitored by TLC), the mixture was concen-
trated in vacuo and the residual was purified purified by chroma-
tography on an silica gel column with EtOAc/petroleum ether
aseluent to give the products.
4.9.1. N-(3-(4-(benzo[d]thiazol-2-yl)phenoxy)propyl)-1,2,3,4-
tetrahydroacridin-9-amine (44a)
C
31H27N3O5 522.2029, found 522.2031.
Compound 41a was treated with 9-chloro-1,2,3,4-tetrahydroac-
ridine according to general procedure to give the desired product
44a as a white solids (39% yield). Mp 152.7–154.0 °C; 1H NMR
(400 MHz, CDCl3) d 8.09–8.00 (m, 3H), 7.97 (d, J = 7.8 Hz, 1H),
7.94–7.85 (m, 2H), 7.56 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.51–7.44
(m, 1H), 7.40–7.30 (m, 2H), 7.05–6.96 (m, 2H), 4.22 (t, J = 5.7 Hz,
2H), 3.74 (t, J = 6.2 Hz, 2H), 2.76 (t, J = 5.5 Hz, 2H), 1.92–1.75 (m,
8H); 13C NMR (101 MHz, CDCl3) d 167.68, 160.91, 158.64, 154.21,
150.46, 147.48, 134.99, 129.21, 128.86, 128.37, 126.83, 126.27,
124.90, 123.88, 122.88, 122.60, 121.54, 120.43, 114.82, 66.42,
46.84, 34.05, 30.74, 25.01, 23.06, 22.80. HRMS m/z [M-Br]+ Calcd
for C29H27N3OS 466.1953, found 466.1959.
4.5.2. 9-O-N-(3-Propyl)benzofuran-2-carboxamide-berberine
bromide (35)
Compound 33 was treated with berberrubine 25 according to
general procedure to give the desired product 36 as a yellow solid
(29% yield). Mp 168.4–169.9 °C; 1H NMR (400 MHz, DMSO) d 9.77
(s, 1H), 8.89 (s, 1H), 8.69 (t, J = 5.7 Hz, 1H), 8.22 (d, J = 9.2 Hz, 1H),
8.00 (d, J = 9.1 Hz, 1H), 7.74 (s, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.40 (d,
J = 8.0 Hz, 1H), 7.29–7.20 (m, 2H), 7.06 (s, 1H), 6.94 (t, J = 7.3 Hz,
1H), 6.20 (s, 2H), 5.17–5.99 (m, 2H), 4.47 (t, J = 6.1 Hz, 2H), 3.67–
3.74 (m, 2H), 3.22–3.15 (m, 2H), 2.17–2.05 (m, 2H); 13C NMR
(101 MHz, DMSO)
d 163.01, 154.44, 147.91, 138.55, 135.62,
133.85, 131.92, 130.07, 127.68, 125.47, 124.74, 118.45, 110.57,
107.68, 103.27, 103.00, 72.7, 57.18, 56.37, 28.83, 25.47, 20.07.
HRMS m/z [M-Br]+ Calcd for C31H26N2O6 523.1869, found
523.2048.
4.9.2. N-(4-(4-(Benzo[d]thiazol-2-yl)phenoxy)butyl)-1,2,3,4-
tetrahydroacridin-9-amine (44b)
Compound 41b was treated with 9-chloro-1,2,3,4-tetrahydroac-
ridine according to general procedure to give the desired product
44b as a white solids (21% yield). Mp 151.5–152.3 °C;1H NMR
(400 MHz, CDCl3) d 8.06–7.98 (m, 3H), 7.96 (d, J = 8.5 Hz, 1H),
7.92 (d, J = 7.9 Hz, 1H), 7.87 (d, J = 7.5 Hz, 1H), 7.55 (ddd, J = 8.3,
6.8, 1.2 Hz, 1H), 7.49–7.42 (m, 1H), 7.38–7.30 (m, 2H), 6.99–6.92
(m, 2H), 4.05 (t, J = 5.7 Hz, 2H), 3.57 (t, J = 6.6 Hz, 2H), 3.06 (d,
J = 6.1 Hz, 2H), 2.73 (d, J = 5.7 Hz, 2H), 1.96–1.82 (m, 8H); 13C
NMR (101 MHz, CDCl3) d 167.77, 161.16, 158.57, 154.22, 150.51,
147.50, 134.85, 129.13, 128.83, 128.31, 126.53, 126.22, 124.82,
4.6. General procedures for the preparation of 37a–d
Phthalimide (36) (40 mmol), K2CO3 (120 mmol) and benzyltri-
ethylammonium chloride(4.4 mmol) were suspended in acetone
(100 mL). Dibromoalkanes (100 mmol) was added to the mixture
and then the reaction mixture was stirred at room temperature
for 24 h. The mixture was filtered and then evaporated under

L. Huang et al. / Bioorg. Med. Chem. 20 (2012) 3038–3048
3047
123.74, 122.74, 121.50, 120.33, 116.23, 114.84, 67.66, 49.07, 34.04,
28.51, 26.65, 24.88, 23.06, 22.78. HRMS m/z [M-Br]+ Calcd for
30H29N3OS 480.0210, found 480.2133.
4.9.7. N-(3-(4-(benzofuran-2-yl)phenoxy)propyl)-1,2,3,4-
tetrahydroacridin-9-amine (46a)
C
Compound 43a was treated with 9-chloro-1,2,3,4-tetrahydroac-
ridine according to general procedure to give the desired product
46a as a white solids (38% yield). Mp 147.9–149.3 °C; 1H NMR
(400 MHz, DMSO) d 8.15 (d, J = 8.5 Hz, 1H), 7.83 (d, J = 8.0 Hz,
2H), 7.71 (d, J = 8.4 Hz, 1H), 7.65–7.55 (m, 2H), 7.53 (t, J = 7.6 Hz,
1H), 7.32 (dd, J = 14.4, 6.6 Hz, 1H), 7.26 (m, 3H), 7.01 (d,
J = 8.1 Hz, 2H), 4.13 (t, J = 5.7 Hz, 2H), 3.66–3.53 (m, 2H), 2.89 (d,
J = 6.0 Hz, 2H), 2.75 (d, J = 5.4 Hz, 2H), 2.13–2.00 (m, 2H), 1.80 (m,
4H); 13C NMR (101 MHz, CDCl3) d 159.20, 158.51, 155.97, 154.70,
150.59, 147.43, 129.46, 128.77, 128.35, 126.44, 123.76, 123.45,
122.79, 120.59, 120.27, 116.14, 114.77, 110.98, 99.74, 67.53,
49.11, 34.02, 28.55, 26.70, 24.87, 23.05, 22.77. HRMS m/z [M-Br]+
Calcd for C30H28N2O2 449.3129, found 449.3140.
4.9.3. N-(5-(4-(Benzo[d]thiazol-2-yl)phenoxy)pentyl)-1,2,3,4-
tetrahydroacridin-9-amine (44c)
Compound 41c was treated with 9-chloro-1,2,3,4-tetrahydroac-
ridine according to general procedure to give the desired product
44c as a glassy solids (27% yield). Mp 145.8–147.8 °C; 1H NMR
(400 MHz, CDCl3) d 8.07–8.00 (m, 3H), 7.98 (d, J = 8.5 Hz, 1H),
7.94 (d, J = 8.4 Hz, 1H), 7.91–7.86 (m, 1H), 7.57 (ddd, J = 8.3, 6.9,
1.2 Hz, 1H), 7.51–7.45 (m, 1H), 7.40–7.33 (m, 2H), 7.01- 6.94 (m,
2H), 4.04 (t, J = 6.2 Hz, 2H), 3.55 (t, J = 7.1 Hz, 2H), 3.08 (s, 2H),
2.73 (s, 2H), 1.93 (t, J = 3.2 Hz, 4H), 1.89–1.83 (m, 2H), 1.80–1.74
(m, 2H), 1.65–1.60 (m, 2H); 13C NMR (101 MHz, CDCl3) d 167.85,
161.31, 158.38, 154.21, 150.74, 147.30, 134.83, 129.31, 129.11,
128.53, 126.30, 124.81, 123.74, 122.80, 120.86, 116.02, 114.81,
67.76, 49.33, 33.93, 31.47, 28.91, 24.81, 23.54, 23.04, 22.75. HRMS
m/z [M-Br]+ Calcd for C31H31N3OS 494.2266, found 494.2257.
4.9.8. N-(4-(4-(Benzofuran-2-yl)phenoxy)butyl)-1,2,3,4-
tetrahydroacridin-9-amine (46b)
Compound 43b was treated with 9-chloro-1,2,3,4-tetrahydroac-
ridine according to general procedure to give the desired product
46b as a glassy solids (41% yield). Mp 146.6–148.4 °C; 1H NMR
(400 MHz, DMSO) d 8.15 (d, J = 8.5 Hz, 1H), 7.83 (d, J = 8.0 Hz,
2H), 7.71 (d, J = 8.4 Hz, 1H), 7.65–7.55 (m, 2H), 7.53 (t, J = 7.6 Hz,
1H), 7.32 (dd, J = 14.4, 6.6 Hz, 1H), 7.26 (m, 3H), 7.01 (d,
J = 8.1 Hz, 2H), 4.13 (t, J = 5.7 Hz, 2H), 3.66–3.53 (m, 2H), 2.89 (d,
J = 6.0 Hz, 2H), 2.75 (d, J = 5.4 Hz, 2H), 2.13–2.00 (m, 2H), 1.80 (m,
4H); 13C NMR (101 MHz, CDCl3) d 159.20, 158.51, 155.97, 154.70,
150.59, 147.43, 129.46, 128.77, 128.35, 126.44, 123.76, 123.45,
122.79, 120.59, 120.27, 116.14, 114.77, 110.98, 99.74, 67.53,
49.11, 34.02, 28.55, 26.70, 24.87, 23.05, 22.77. HRMS m/z [MꢁBr]+
Calcd for C31H30N2O2 463.2386, found 463.2392.
4.9.4. N-(6-(4-(Benzo[d]thiazol-2-yl)phenoxy)hexyl)-1,2,3,4-
tetrahydroacridin-9-amine (44d)
Compound 41d was treated with 9-chloro-1,2,3,4-tetrahydroac-
ridine according to general procedure to give the desired product
44d as a glassy solids (24% yield). 1H NMR (400 MHz, CDCl3) d
8.04–8.00 (m, 3H), 7.96 (d, J = 7.8 Hz, 1H), 7.92 (d, J = 8.2 Hz, 1H),
7.87 (dd, J = 7.9, 0.5 Hz, 1H), 7.59–7.51 (m, 1H), 7.49–7.44 (m,
1H), 7.39–7.30 (m, 2H), 7.02–6.93 (m, 2H), 4.02 (t, J = 6.4 Hz, 2H),
3.51 (t, J = 7.2 Hz, 2H), 3.07 (s, 2H), 2.71 (s, 2H), 1.92 (dd, J = 6.5,
3.3 Hz, 4H), 1.81 (dd, J = 14.0, 6.5 Hz, 2H), 1.70 (dd, J = 14.2,
7.0 Hz, 2H), 1.50 (dt, J = 11.9, 6.3 Hz, 4H); 13C NMR (101 MHz,
CDCl3) d 167.88, 161.40, 158.37, 154.21, 150.84, 134.84, 129.10,
128.65, 128.38, 126.26-, 124.79, 123.68, 122.80, 121.51, 120.20,
117.66, 115.92, 114.83, 67.92, 49.41, 33.95, 31.72, 29.06, 26.72,
25.87, 24.80, 23.04, 22.76. HRMS m/z [M-Br]+ Calcd for C32H33N3OS
508.2423, found 508.2427.
4.10. Biological activity
4.10.1. In vitro inhibition studies on AChE and BuChE
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel), buty-
lcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum), 5,50-dithi-
4.9.5. N-(3-(4-(Benzo[d]oxazol-2-yl)phenoxy)propyl)-1,2,3,4-
tetrahydroacridin-9-amine (45a)
obis-(2-nitrobenzoic
acid)
(Ellman’s
reagent,
DTNB),
acetylthiocholine chloride (ATC), butylthiocholine chloride (BTC),
and tarcine hydrochloride were purchased from Sigma–Aldrich.
Berberine derivatives were dissolved in DMSO and diluted in
0.1 M KH2PO4/K2HPO4 buffer (pH 8.0) to provide a final concentra-
tion range. DMSO was diluted to a concentration in excess of 1 in
10000, and no inhibitory action on either AChE or BuChE was de-
tected in separate prior experiments.
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 Spec-
trophotometer. Enzyme solutions were prepared to give
2.0 units/mL in 2 mL aliquots. The assay medium (1 mL) consisted
Compound 42a was treated with 9-chloro-1,2,3,4-tetrahydroac-
ridine according to general procedure to give the desired product
45a as a white solids (26% yield). Mp 148.9–150.1 °C; 1H NMR
(400 MHz, DMSO) d 8.13 (t, J = 9.7 Hz, 3H), 7.84–7.67 (m, 3H),
7.52 (t, J = 7.5 Hz, 1H), 7.43–7.36 (m, 2H), 7.33 (t, J = 7.6 Hz, 1H),
7.16–7.05 (m, 2H), 4.17 (d, J = 5.9 Hz, 2H), 3.60 (d, J = 6.3 Hz, 2H),
2.89 (d, J = 5.9 Hz, 2H), 2.74 (s, 2H), 2.12–2.03 (m, 2H), 1.79 (m,
4H). 13C NMR (101 MHz, DMSO) d 159.00, 155.37, 153.98, 129.07,
128.19, 127.89, 126.21, 123.96, 123.30, 123.02 (d, J = 9.7 Hz),
122.38, 120.72, 120.31, 114.94, 110.86, 65.46, 52.02, 33.40, 30.03,
25.06, 22.67. HRMS m/z [M-Br]+ Calcd for C29H27N3O2 450.2281,
found 450.2293.
of phosphate buffer (pH 8.0), 50
ll of 0.01 M DTNB, 10 ll of en-
zyme, and 50 l of 0.01 M substrate (ACh chloride solution). Test
l
compounds were added to the assay solution and preincubated
at 37 °C with the enzyme 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.15 Each concentration was assayed in triplicate.
In vitro BuChE assay was similar with the method described
above.
4.9.6. N-(4-(4-(Benzo[d]oxazol-2-yl)phenoxy)butyl)-1,2,3,4-
tetrahydroacridin-9-amine (45b)
Compound 42b was treated with 9-chloro-1,2,3,4-tetrahydroac-
ridine according to general procedure to give the desired product
45b as a white solids (30% yield). Mp 145.8–147.8 °C; 1H NMR
(400 MHz, CDCl3) d 8.19 (d, J = 8.9 Hz, 2H), 7.97 (d, J = 8.5 Hz, 1H),
7.92 (d, J = 8.5 Hz, 1H), 7.76–7.70 (m, 1H), 7.59–7.52 (m, 2H),
7.37–7.30 (m, 3H), 7.00 (d, J = 8.9 Hz, 2H), 4.08 (t, J = 5.7 Hz, 2H),
3.59 (t, J = 6.5 Hz, 2H), 3.07 (s, 2H), 2.74 (s, 2H), 1.96–1.87 (m,
9H); 13C NMR (101 MHz, CDCl3) d 161.56, 158.59, 150.51, 142.25,
129.41, 128.86, 128.33, 124.55 (d, J = 19.5 Hz), 123.75, 122.68,
120.32, 119.63, 116.25, 114.80, 110.39, 67.65, 49.10, 34.07, 28.53,
26.64, 24.89, 23.06, 22.79. HRMS m/z [M-Br]+ Calcd for
4.10.2. Kinetic characterization of AChE inhibition
Kinetic characterization of AChE was performed using a reported
method.16 Test compound was added into the assay solution and
pre-incubated with the enzyme at 37 °C for 15 min, followed by
the addition of substrate. Kinetic characterization of the hydrolysis
of ATC catalyzed by AChE was done spectrometrically at 412 nm. A
C30H29N3O2 464.2338, found 464.2349.

3048
L. Huang et al. / Bioorg. Med. Chem. 20 (2012) 3038–3048
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 squareanalysis 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 plotted against the concentration
of the inhibitors in a weighted analysis and Ki was determined as
the ratio of the replot intercept to the replot slope.
References and notes
1. Alzheimer’s Society: http://www.alzheimers.org.uk/.
2. Mayer, S. C.; Kreft, A. F.; Harrison, B.; Abou-Gharbia, M.; Antane, M.; Aschmies,
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J.; Fan, K.; Galante, R.; Gonzales, C.; Ho, D. M.; Hoke, M. E.; Hu, Y.; Huryn, D.;
Jain, U.; Jin, M.; Kremer, K.; Kubrak, D.; Lin, M.; Lu, P.; Magolda, R.; Martone, R.;
Moore, W.; Oganesian, A.; Pangalos, M. N.; Porte, A.; Reinhart, P.; Resnick, L.;
Riddell, D. R.; Sonnenberg-Reines, J.; Stock, J. R.; Sun, S.-C.; Wagner, E.; Wang,
T.; Woller, K.; Xu, Z.; Zaleska, M. M.; Zeldis, J.; Zhang, M.; Zhou, H.; Jacobsen, J.
S. J. Med. Chem. 2008, 51, 7348.
3. Bartus, R. T.; Dean, R. L.; Pontecorvo, M. J.; Flicker, C. Ann. N. Y. Acad. Sci. 1985,
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4. (a) Rogers, S. L.; Farlow, M. R.; Doody, R. S.; Mohs, R.; Friedhoff, L. T.; Group, D.
S. Neurology 1998, 50, 136; (b) Cummings, J. L. Am. J. Psychiat. 2000, 157, 4.
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Huang, Z.-S. Bioorg. Med. Chem. 2011, 19, 5596; (b) Zhou, Y.; Jiang, C.; Zhang, Y.;
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4.10.3. Inhibition of Ab1–42 peptide aggregation17
HFIP pretreated Ab1–42 samples (AnaSpec) were resolubilized
with a 50 mM phosphate buffer (pH = 7.4) in order to have a stable
stock solution ([Ab] = 200
50 mM phosphate buffer (pH = 7.4) at 37 °C for 48 h (final Ab
concentration 50 M) with and without the tested compound at
at different concentrations. After incubation, the samples were di-
luted to a final volume of 200 L with 50 mM glycine–NaOH buffer
lM). The peptide was incubated in
l
l
(pH 8.0) containing thioflavin T. Then, a 300-s-time scan of fluores-
cence intensity was performed (kexc = 450 nm; kem = 485 nm), and
values at plateau were averaged after subtracting the background
fluorescence of thioflavin T solution.
4.11. Molecular modeling
9. Jiang, H.; Wang, X.; Huang, L.; Luo, Z.; Su, T.; Ding, K.; Li, X. Bioorg. Med. Chem.
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24.
The simulation system 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 optimized by Discovery
studio 2.1 package (Accelrys Inc., San Diego, CA). The CDOCKER
pro- gramin Discovery studio 2.1 software was used to perform-
docking simulations, which allows full flexibility of the ligand.
Acknowledgment
We thank the Natural Science Foundation of China (20972198,
81173577) and Guangdong Province Natural Science Foundation
(8451008901000380) for financial support of this study.
17. Rosini, M.; Simoni, E.; Bartolini, M.; Cavalli, A.; Ceccarini, L.; Pascu, N.;
McClymont, D. W.; Tarozzi, A.; Bolognesi, M. L.; Minarini, A.; Tumiatti, V.;
Andrisano, V.; Mellor, I. R.; Melchiorre, C. J. Med. Chem. 2008, 51, 4381.