Synthesis and biological evaluation of berberine-thiophenyl hybrids as multi-functional agents: Inhibition of acetylcholinesterase, butyrylcholinesterase, and Aβ aggregation and antioxidant activity

Article abstract of DOI:10.1016/j.bmc.2013.07.011

A series of berberine-thiophenyl hybrids were designed, synthesised, and evaluated as inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and β-amyloid (Aβ) aggregation and as antioxidants. Among these hybrids, compounds 4f and 4i, berberine linked with o-methylthiophenyl and o-chlorothiophenyl by a 2-carbon spacer, were observed to be potent inhibitors of AChE, with IC₅₀ values of 0.077 and 0.042 μM, respectively. Of the tested compounds, 4i was also the most potent inhibitor of BuChE, with an IC₅₀ value of 0.662 μM. Kinetic studies and molecular modelling simulations of the AChE-inhibitor complex indicated that a mixed-competitive binding mode existed for these berberine derivatives. The biological studies also demonstrated that these hybrids displayed interesting activities, including Aβ aggregation inhibition and antioxidant properties.

Full text of DOI:10.1016/j.bmc.2013.07.011

Bioorganic & Medicinal Chemistry 21 (2013) 5830–5840
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of berberine–thiophenyl hybrids
as multi-functional agents: Inhibition of acetylcholinesterase,
butyrylcholinesterase, and Ab aggregation and antioxidant activity
a,
Tao Su a, Shishun Xie a, Hui Wei b, Jun Yan a, Ling Huang a, , Xingshu Li
⇑
⇑
a Institute of Drug Synthesis and Pharmaceutical Process, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
b School of Chemistry and Chemical Engineering, Guangdong Pharmaceutical 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–thiophenyl hybrids were designed, synthesised, and evaluated as inhibitors of
acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and b-amyloid (Ab) aggregation and as anti-
oxidants. Among these hybrids, compounds 4f and 4i, berberine linked with o-methylthiophenyl and
o-chlorothiophenyl by a 2-carbon spacer, were observed to be potent inhibitors of AChE, with IC50 values
Received 14 May 2013
Revised 4 July 2013
Accepted 5 July 2013
Available online 24 July 2013
of 0.077 and 0.042
BuChE, with an IC50 value of 0.662
l
M, respectively. Of the tested compounds, 4i was also the most potent inhibitor of
M. Kinetic studies and molecular modelling simulations of the
l
Keywords:
AChE-inhibitor complex indicated that a mixed-competitive binding mode existed for these berberine
derivatives. The biological studies also demonstrated that these hybrids displayed interesting activities,
including Ab aggregation inhibition and antioxidant properties.
Alzheimer’s disease
berberine–thiophenyl hybrids
Inhibitors of AChE
b-Amyloid aggregation
Antioxidants
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
The accumulating evidence suggests that oxidative stress induced
by Ab is implicated in the neurodegeneration of the brains of AD
Alzheimer’s disease (AD) is a neurodegenerative disorder char-
acterised by progressive cognitive decline, memory loss, extensive
neuronal loss, decrease in cholinergic transmission, and the pres-
ence of senile plaques. Although the aetiology of AD is not com-
pletely understood, several diverse hallmarks, such as b-amyloid
patients, and this stress is widely believed to be an important
mechanism of Ab fibril toxicity.9 Therefore, antioxidant protection
is important during aging and especially in AD patients as the
endogenous antioxidant protection system declines rapidly.10 In-
deed, several antioxidants have demonstrated efficacy in a number
of recent studies.11
(Ab) deposits, s-protein aggregation, oxidative stress, and low lev-
els of acetylcholine (ACh) play significant roles in the pathophysi-
ology of the disease.1 The ‘cholinergic hypothesis’ was proposed2
based on the correlation of cholinergic activity in the central ner-
vous system (CNS) of AD patients with their degree of cognitive
function disorder. In recent decades, cholinesterase (ChE) has be-
come one of major targets in current AD3 therapy, and most of
the clinically approved drugs for the treatment of AD are acetyl-
cholinesterase inhibitors (AChEI), such as tacrine, rivastigmine,
galanthamine and donepezil. The progressive deposition of Ab fi-
brils in the brain is another key feature in the pathology of patients
with AD.4 Therefore, based on the ‘amyloid hypothesis’, the pre-
vention of the aggregation of Ab is another possible therapeutic ap-
proach to Alzheimer’s disease.5 A number of Ab aggregation
inhibitors have been developed, including substituted bisphenol,6
coumarin analogs,7 N-arylnaphthylamine derivatives8 and so on.
Because of the complex multifactorial aetiology of AD, the mul-
ti-target-directed ligand (MTDL) strategy was developed, which
has been proven to be a successful strategy to approach this mul-
tifaceted disease according to a large amount of accumulating evi-
dence in recent years.12 In our earlier study, we developed a new
class of 9-O- and 9-N-substituted berberine derivatives that are
dual AChE and BuChE inhibitors with IC50 value ranges from
0.020 to 6.50 lM and 0.029 to 18.20 l
M, respectively.13
In this study, we report a new series of berberine-thiophenyl
hybrids wherein the oxygen or NH group of the berberine deriva-
tives is replaced with a sulfur atom, which may result in additional
antioxidant properties.
2. Results and discussion
2.1. Chemistry
⇑
Corresponding authors. Tel./fax: +86 20 3994 3051 (L.H.); tel./fax: +86 20 3994
3050 (X.L.).
The synthesis of berberine-thiophenyl hybrids is described in
Scheme 1. First, the intermediates 1a–n were synthesised by
E-mail addresses: Huangl72@mail.sysu.edu.cn (L. Huang), lixsh@mail.sysu.
edu.cn (X. Li).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.011

T. Su et al. / Bioorg. Med. Chem. 21 (2013) 5830–5840
5831
Scheme 1. Reagents and conditions: (i) Br(CH2)nBr, K2CO3, KI, acetone, rt, 5 h; (ii)190 °C, 20–30 mm Hg, 15 min; (iii) 1a–n, acetonitrile, reflux, 16 h.
reacting commercially available thiophenol or its analogues with
dibromoalkane in acetone (48–67%). Alternatively, berberrubine
(3) was obtained through selective demethylation of berberine
(2) at 190 °C under vacuum, with a yield of 66%. Finally, interme-
diates 1a–n reacted with berberrubine (3) in acetonitrile to pro-
duce the target compounds 4a–n with yields of 25–39%.
Table 1
In vitro inhibition of AChE and BuChE, and the antioxidant activities (ORAC, Trolox
equivalents) of compounds 4a–n and berberine
Troloxequive
a
Compd
IC50
(
l
M)
Selectivity for
AChEd
AChEb
BuChEc
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
0.177 0.008 1.521 0.046
0.257 0.003 1.494 0.062
0.192 0.030 2.185 0.098 11.38
0.543 0.010 1.661 0.082
0.972 0.036 1.586 0.045
0.077 0.010 1.360 0.018 17.66
0.207 0.026 1.605 0.147
0.299 0.038 1.794 0.102
0.042 0.007 0.662 0046
0.233 0.012 1.253 0.007
0.484 0.031 1.401 0.010
0.083 0.005 1.713 0.007 20.63
0.087 0.006 1.756 0.015 20.18
0.151 0.003 1.092 0.020
8.59
5.81
0.89 0.04
0.94 0.03
0.95 0.04
0.89 0.04
0.77 0.01
1.11 0.08
1.32 0.02
1.86 0.06
0.75 0.08
0.47 0.02
0.82 0.05
1.01 0.07
1.15 0.08
1.94 0.15
0.40 0.01
2.2. In vitro inhibition studies of AChE and BuChE
3.05
1.63
The inhibitory activity of the berberine-thiophenyl hybrids 4a–
n was evaluated toward AChE from electric eel and BuChE from
equine serum with the method of Ellman,14 using berberine as
the reference standard. The activity results are summarised in
Table 1.
As shown in Table 1, most of the hybrids demonstrated potent
AChE and BuChE inhibitory activity with the IC50 values in the
micromolar to sub-micromolar range. Among all the hybrids, com-
pound 4i, with a chlorine substituent in the ortho-position of thio-
phenyl moiety, gave the most potent AChE inhibitory activity with
7.75
6.00
15.76
5.37
2.89
7.23
Berberine 0.374 0.04n 18.21 0.683 48.82
a
The mean SD of at least three independent measurements.
Acetylcholine substrate for the evaluation of the acetylcholinesterase activity.
an IC50 value of 0.042
lM, approximately ninefold and sevenfold
b
higher than that of berberine and tacrine, respectively. From the
IC50 values of 4a–e, it can be concluded that as the alkylene chain
linker is extended, the inhibitory activity of the hybrids become
weak. For example, compound 4a, with two alkylene chains be-
tween the berberine and thiophenyl group, provides an IC50 value
AChE from electric eel.
c
Butyrylcholine substrate for the evaluation of the butyrylcholinesterase
activity. BChE from equine serum.
d
Selectivity for AChE = IC50(BuChE)/IC50(AChE).
The data are expressed as lmol of Trolox equivalent/lmol of tested compound
e
(the means SD of three independent measurements).
of 0.177
of 0.257
l
l
M, whereas 4b, with a three-alkylene chain, gave a value
M. The position of the substituted groups on the phenyl
of BuChE in all of the compounds is not obvious as that of AChE,
and there were moderate activity differences. All the above results
suggest that these hybrids may have interesting potential as dual
AChE and BuChE inhibitors for the treatment of AD.
ring appears also to dramatically influence the inhibitory activities.
These results suggest that substituent groups in the ortho-position
are favourable in terms of the activity compared with substituents
in the meta- and para-positions. For instance, 4f (IC50 = 0.077
with a methyl group in the ortho-position, had approximately
threefold the inhibitory activities of 4g (IC50 = 0.207 M) and 4h
(IC50 = 0.299 M), which have methyl groups in the meta- and
para-position, respectively. The same trend was also observed in
compounds 4i, 4j and 4k. Compounds 4l–n, with different substi-
tuted groups in the ortho-position, also exhibited good AChE inhi-
bition with an IC50 value range from 0.083 to 0.151 lM,
respectively. In addition, all of the target compounds were potent
BuChE inhibitors with an IC50 value range from 0.662 to
lM),
2.3. Kinetics of AChE and BuChE inhibition
l
Using compound 4f and 4i as typical examples, the inhibition
type of AChE and BuChE was investigated by graphical analysis
of the steady state inhibition data (Figs. 1 and 2). The inhibition
constants Ki were estimated from the plots of the slope versus
the concentration of 4f and 4i (Table 2). Reciprocal plots (Linewe-
aver–Burk plots) describing 4f and 4i inhibition showed both
increasing slopes and increasing intercepts with higher inhibitor
concentration, indicating a mixed-type inhibition. In particular,
4f and 4i were able to bind to both the active site and PAS of AChE.
l
2.185
lM, which is more potent than the leading compound, ber-
berine (18.21
lM). However, the structure–activity relationship

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T. Su et al. / Bioorg. Med. Chem. 21 (2013) 5830–5840
Figure 1. Steady state inhibition by 4i and 4f of AChE hydrolysis of ACh. Reciprocal plots of initial velocity and substrate concentration showing mixed-type inhibition for 4i
and 4f on AChE.
Figure 2. Steady state inhibition by 4i and 4f of BuChE hydrolysis of BuCh. Reciprocal plots of initial velocity and substrate concentration showing mixed-type inhibition for
4i and 4f on BuChE.
3 and 4. The most probable conformations of the ligands were cho-
sen based on the docked energy value. The modelling results
showed that 4f and 4i target the catalytic active site (CAS) and
the peripheral anionic site (PAS) of both AChE and BuChE with a
similar manner, which are agreement with the mode of kinetic
data. The Docking model of compounds–TcAChE complexes
(Fig. 3b and c) demonstrated that the berberine moiety of 4f and
4i reached the top of the cavity and interacts with the PAS residue
Table 2
AChE and BuChE inhibition constants of selected compounds (4f and 4i)
Compound
Enzyme
AChE
Km
ki (
l
M)
Type of inhibition
4f
4i
4f
4i
0.154 0.016
0.071
0.018
0.299
0.177
Mixed
Mixed
Mixed
Mixed
BuChE
0.286 0.027
Trp279 (4.97 Å and 4.96 Å, respectively) through p–p stacking. The
conformation of the side-chain of 4f and 4i fits nicely into the
The inhibitory behaviour of 4f and 4i on BuChE, as illustrated in
Figure 2, are similar to those on AChE. Therefore, from the kinetic
profile, we concluded that compound 4f and 4i causes a mixed type
of inhibition and could interact with both CAS and PAS on AChE
and BuChE.
shape of the cavity. At the bottom of the binding site, the phenyl
moieties of 4f and 4i have
p–p stacking interactions with Trp84
(4.36 Å and 4.12 Å, respectively) and Phe330 (3.90 Å and 3.97 Å,
respectively). Meanwhile, the thiophenyl group of 4f forms a direct
hydrogen bond with the backbone OH group of Tyr121, in which
the sulfur atom serves as a hydrogen-bond acceptor (average
Sꢀ ꢀ ꢀH distance = 2.43 Å). As shown in Figure 4, similar with AChE
interaction model, the ligands interacts with Tyr332 located in
2.4. Molecular modelling studies
The possible interaction mode between the berberine-thio-
phenyl hybrids and AChE was studied by molecular docking simu-
lations using the CDOCKER program in DISCOVERY STUDIO 2.1 software.
Compound 4f and 4i were initially docked to the TcAChE and Hu-
BuChE active sites cavity based on the structure of the enzyme
complex (TcAChE: PDB entry 2CMF; HuBuChE: PDB entry 2PM8),
and the resulting structures of the active site are shown in Figures
the PAS, forming a face-to-face
moiety. Besides, the phenyl groups of 4f and 4i interacte by means
of stacking with the indole ring of Trp82.
Hybrids 4g and 4h were also docked into TcAChE using the
same modeling method. Superpositioning of 4g and 4h with 4f
(Fig. 5) showed that the berberine moieties essentially overlap, ex-
cept for the slight differences in the side-chains. The difference in
p–p interaction with the berberine
p–p

T. Su et al. / Bioorg. Med. Chem. 21 (2013) 5830–5840
5833
Figure 3. (a) Stereoviews looking down the gorge of TcAChE binding with 4f (yellow) and 4i (blue). (b) 4f docked into the catalytic gorge of TcAChE (code ID: 2CMF). The
protein residues belonging to CAS and PAS that establish the main interactions are highlighted. (c) 4i docked into the catalytic gorge of TcAChE (code ID: 2CMF). The protein
residues belonging to CAS and PAS that establish the main interactions are highlighted.
the inhibitory activities of these hybrids implied that the steric ef-
fect might played a key role, which have stacking interactions
between the phenyl moiety of the ligands and Trp84 (due to the
different spatial positions of the phenyl moieties, the distance
p–p

5834
T. Su et al. / Bioorg. Med. Chem. 21 (2013) 5830–5840
Figure 4. (a) Stereoviews looking down the gorge of HuBuChE (code ID: 2PM8) binding with 4f (yellow) and 4i (blue). (b) Compound 4f docked into the catalytic gorge of
HuBuChE. The protein residues belonging to CAS and PAS that establish the main interactions are highlighted. (c) Compound 4i docked into the catalytic gorge of HuBuChE.
The protein residues belonging to CAS and PAS that establish the main interactions are highlighted.

T. Su et al. / Bioorg. Med. Chem. 21 (2013) 5830–5840
5835
Figure 5. Docking model of compounds–enzyme complex. berberine–thiophenyl hybrids 4g (green), 4h (yellow) docked into the catalytic gorge of TcAChE(code ID: 2CMF)
and superposition with 4f (red).
between the phenyl moieties of 4f, 4g or 4h and Trp84 were 4.35,
4.43 and 4.45 Å, respectively).
2.5. Antioxidant activity
2.5.1. ORAC assay
The antioxidant activities of the hybrids derived from berberine
were determined using an oxygen radical absorbance capacity as-
say using fluorescein (ORAC-FL), according to the method origi-
nally described by Ou et al.15 and modified by Daevalos.16 The
vitamin analogue Trolox was used as a standard, and the antioxi-
dant activity was expressed as Trolox equivalents (
lmol of Trol-
ox/ mol of the tested compound). As shown in Table 1, all
l
designed compounds exhibited moderate antioxidant capacity,
with ORAC-FL values of 0.47–1.94 Trolox equivalents. These values
are better than that of the leading compound, berberine (0.40 Trol-
ox equivalents). Compounds 4f and 4n (1.11 and 1.94 Trolox equiv-
alents, respectively) yielded better results than did 4a (0.89 Trolox
equivalents), and compounds 4i–g (0.75–0.82 Trolox equivalents)
exhibited poor activities, indicating that the electron donating
groups in the thiophenyl ring are favourable for antioxidant
capacity.
Figure 6. The H2O2 scavenging capacity of compound 4f. Each point represents the
mean values from three sample SD.
the mixture indicated that there was little of compound 4f
([MꢁBr]+ = 472.3) remaining, and a new peak [MꢁBr]+ = 488.2
was observed (see the Fig. 7). Thus, we speculated that the mech-
anism of scavenging H2O2 by berberine-thiophenyl hybrids may be
that they act as reductive agents to produce the oxidative product
sulfoxide.
2.5.2. Hydrogen peroxide scavenging activity
The antioxidant protection provided by removing ROS such as
hydrogen peroxide is important in older individuals, especially
AD patients, because the endogenous antioxidant protection sys-
tem declines rapidly with age. To evaluate the scavenging H2O2
capacity of these berberine–thiophenyl hybrids, a previously re-
ported method17 was chosen for the assay, and the results are
shown in Figure 6. The decreasing curve reveals the proportional
relationship between the concentrations of compound 4f and the
scavenging capacity in a certain time (72 h). When the concentra-
2.6. Ab1–42 aggregation inhibitory activity
The ability of berberine–thiophenyl hybrids to inhibit Ab1–42
self-aggregation was studied through a thioflavin-T based fluori-
metric assay with curcumin (Cur) and resveratrol (Res) as standard
compounds (Fig. 8). The results indicated that all hybrids exhibited
more potent inhibitory activities (65.1–76.8%) than curcumin and
resveratrol (51.3% and 61.1%, respectively). In particular, com-
pounds 4l and 4n, with electron donating groups in thiophenyl
tion of compound 4f was 100 lM, 100 lM H2O2 was scavenged
completely. In addition, to investigate the mechanism of the scav-
enging of H2O2, an LC–MS assay was also used. The LC–-MS assay of

5836
T. Su et al. / Bioorg. Med. Chem. 21 (2013) 5830–5840
Figure 7. The LC–MS spectrum of compound 4f treated with H2O2 after 72 h.
towards AChE and b-amyloid aggregation. Further investigations
into AD candidates based on these results are in progress.
4. Experimental section
4.1. Chemistry
The 1H NMR and 13C NMR spectra were recorded with TMS as
the internal standard on a BrukerBioSpin GmbH 400 MHz spec-
trometer. The coupling constants were given in Hz. High-resolu-
tion mass spectra were obtained with a Shimadazu LCMS-IT-TOF
mass-spectrometer. Flash column chromatography was performed
with 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 chromatog-
raphy on silica gel. Compound 3 was synthesised as previously
reported.18
4.2. General procedure for the preparation of 1a–n
Figure 8. Effects on Ab1–42 peptide aggregation inhibition at 20 lM of selective
compounds. The thioflavin-T fluorescence method was used. The values represent
the mean SD from at least two independent measurements.
To
a stirred suspension of thiophenol or its analogues
(10 mmol) and K2CO3 (15 mmol) in acetone (50 mL), dibromoal-
kane (20 mmol) and KI (0.1 mmol) were added. After stirring at
room temperature for 4 h, the mixture was filtered, and the filtrate
was concentrated under vacuum to give the crude product, which
was chromatographed on silica gel, with EtOAc/petroleum ether as
the eluent to afford the compounds.
(74.8% and 76.8%, respectively), displayed better aggregation inhi-
bition than 4a–c (69.1–70.6%), which lack thiophenyl substituents.
3. Conclusion
In summary, a series of new berberine-thiophenyl hybrids were
synthesised and subjected to pharmacological evaluation. The re-
sults indicated that most of the berberine-thiophenyl hybrids pos-
sessed moderate to high AChE inhibition activity. Assays of these
hybrids evaluating their ability to inhibit self-induced b-amyloid
aggregation indicated that most of the tested compounds exhibited
more potent inhibition activities than that of the reference com-
pound curcumin. The antioxidant activity ORAC assay indicated
that the hybrids were moderate to good antioxidants. The hydro-
gen peroxide scavenging activity assay indicated these compounds
could efficiently eliminate H2O2. It is worth noting that compounds
4f and 4i, berberine linked with o-methylthiophenyl and o-chloro-
thiophenyl by a 2-carbon spacer, displayed high inhibitory activity
4.2.1. (2-Bromoethyl)(phenyl)sulfane (1a)
Thiophenol was treated with 1,2-dibromoethane according to
the general procedure to give the desired product 1a as an oil
(68.9% yield). 1H NMR (400 MHz, CDCl3) d 7.41 (dd, J = 8.3,
1.0 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 7.27 (dd, J = 8.8, 5.6 Hz, 1H),
3.51–3.44 (m, 2H), 3.35–3.26 (m, 2H).
4.2.2. (3-Bromopropyl)(phenyl)sulfane (1b)
Thiophenol was treated with 1,3-dibromopropane according to
the general procedure to give the desired product 1b as an oil
(57.1% yield). 1H NMR (400 MHz, CDCl3) d 7.38–7.32 (m, 2H),
7.29 (t, J = 7.7 Hz, 2H), 7.23–7.12 (m, 1H), 3.52 (t, J = 6.4 Hz, 2H),
3.07 (t, J = 6.9 Hz, 2H), 2.18–2.11 (m, 2H).

T. Su et al. / Bioorg. Med. Chem. 21 (2013) 5830–5840
5837
4.2.3. (4-Bromobutyl)(phenyl)sulfane (1c)
(55%yield). 1H NMR (400 MHz, CDCl3) d 7.39–7.33 (m, 1H), 7.20 (m,
3H), 3.46 (dd, J = 8.8, 7.8 Hz, 2H), 3.32–3.4 (m, 2H), 2.80 (q,
J = 7.5 Hz, 2H), 1.50 (t, J = 7.5 Hz, 3H).
Thiophenol was treated with 1,4-dibromobutane according to
the general procedure to give the desired product 1c as an oil
(58.2% yield). 1H NMR (400 MHz, CDCl3) d 7.30 (ddd, J = 16.0,
10.0, 2.1 Hz, 4H), 7.19 (dd, J = 10.2, 4.2 Hz, 1H), 3.41 (t, J = 6.7 Hz,
2H), 2.94 (t, J = 7.1 Hz, 2H), 2.00 (dd, J = 14.7, 6.8 Hz, 2H), 1.85–
1.75 (m, 2H).
4.2.13. (2-Bromoethyl)(2-fluorophenyl)sulfane (1m)
o-Fluorothiophenol was treated with 1,2-dibromoethane
according to the general procedure to give the desired product
1m as an oil (64%yield). 1H NMR (400 MHz, CDCl3) d 7.43 (td,
J = 7.8, 1.8 Hz, 1H), 7.33–7.25 (m, 1H), 7.18–7.00 (m, 2H), 3.49–
3.41 (m, 2H), 3.31–3.4 (m, 2H).
4.2.4. (5-Bromopentyl)(phenyl)sulfane (1d)
Thiophenol was treated with 1,5-dibromoputane according to
the general procedure to give the desired product 1d as an oil
(48% yield). 1H NMR (400 MHz, CDCl3) d 7.35–7.25 (m, 4H), 7.20–
7.14 (m, 1H), 3.39 (td, J = 6.7, 1.0 Hz, 2H), 2.96–2.89 (m, 2H),
1.92–1.82 (m, 2H), 1.73–1.63 (m, 2H), 1.59 (dd, J = 14.7, 7.5 Hz, 2H).
4.2.14. (2-Bromoethyl)(2-methoxyphenyl)sulfane (1n)
o-Methoxythiophenol was treated with 1,2-dibromoethane
according to the general procedure to give the desired product
1n as an oil (71% yield). 1H NMR (400 MHz, CDCl3) d 7.35 (dd,
J = 7.6, 1.7 Hz, 1H), 7.29–7.4 (m, 1H), 6.93 (td, J = 7.5, 1.2 Hz, 1H),
6.89 (dd, J = 8.2, 1.1 Hz, 1H), 3.90 (s, 3H), 3.48–3.42 (m, 2H),
3.31–3.25 (m, 2H).
4.2.5. (6-Bromohexyl)(phenyl)sulfane (1e)
Thiophenol was treated with 1,6-dibromohexane according to
the general procedure to give the desired product 1e as an oil
(55% yield). 1H NMR (400 MHz, CDCl3) d 7.35–7.23 (m, 4H), 7.17
(t, J = 7.2 Hz, 1H), 3.39 (t, J = 6.8 Hz, 2H), 2.92 (t, J = 7.3 Hz, 2H),
1.88–1.81 (m, 2H), 1.71–1.61 (m, 2H), 1.45 (dt, J = 7.0, 3.4 Hz, 4H).
4.3. General procedure for preparation of 4a–n
Compounds 1a–n (3.0 mmol) were added to the solution of
berberrubine (3) in acetonitrile (10 mL). The mixture was refluxed
for 16 h. After the reaction finished, the solvent was evaporated
under vacuum to give the crude product, which was chromato-
graphed on an Al2O3 column with CH2Cl2/MeOH as the eluent to af-
ford the target compound.
4.2.6. (2-Bromoethyl)(o-tolyl)sulfane (1f)
o-Methylthiophenol was treated with 1,2-dibromoethane
according to the general procedure to give the desired product 1f
as an oil (67% yield). 1H NMR (400 MHz, CDCl3) d 7.37–7.31 (m,
1H), 7.23–7.13 (m, 3H), 3.49–3.42 (m, 2H), 3.27 (dd, J = 9.7,
6.8 Hz, 2H), 2.41 (s, 3H).
4.3.1. 9-o-(2-Thiophenyl)ethyl-berberine bromide(4a)
4.2.7. (2-Bromoethyl)(m-tolyl)sulfane (1g)
Berberrubine was treated with compound 1a according to the
general procedure to afford compound 4a as a yellow solid (33%
yield). Mp 228.4–229.1 °C; 1H NMR (400 MHz, DMSO) d 9.86 (s,
1H), 8.94 (s, 1H), 8.18 (d, J = 9.1 Hz, 1H), 8.00 (d, J = 9.1 Hz, 1H),
7.80 (s, 1H), 7.39 (d, J = 7.5 Hz, 2H), 7.34 (t, J = 7.6 Hz, 2H), 7.5(t,
J = 7.0 Hz, 1H), 7.10 (s, 1H), 6.18 (s, 2H), 4.93 (t, J = 6.2 Hz, 2H),
4.46 (t, J = 6.8 Hz, 2H), 4.00 (s, 3H), 3.53 (t, J = 6.8 Hz, 2H), 3.4–
3.19 (m, 2H). 13C NMR (101 MHz, DMSO) d 150.1, 149.8, 147.6,
145.2, 141.9, 137.4, 135.1, 132.9, 130.6, 129.1, 128.4, 126.5,
126.1, 123.6, 121.5, 120.3, 120.1, 108.3, 105.4, 102.1, 71.7, 56.9,
55.3, 31.9, 26.3. HRMS m/z [MꢁBr]+ calcd for C27H24NO4S
458.1421, found 458.1407.
m-Methylthiophenol was treated with 1,2-dibromoethane
according to the general procedure to give the desired product
1g as an oil (63% yield). 1H NMR (400 MHz, CDCl3) d 7.23–7.16
(m, 3H), 7.06 (dd, J = 6.1, 1.9 Hz, 1H), 3.46 (dd, J = 9.7, 6.7 Hz, 2H),
3.28 (dd, J = 9.8, 6.8 Hz, 2H), 2.34 (s, 3H).
4.2.8. (2-Bromoethyl)(p-tolyl)sulfane (1h)
p-Methylthiophenol was treated with 1,2-dibromoethane
according to the general procedure to give the desired product
1h as an oil (67% yield). 1H NMR (400 MHz, CDCl3) d 7.31 (d,
J = 7.9 Hz, 2H), 7.13 (d, J = 7.8 Hz, 2H), 3.48–3.40 (m, 2H), 3.28–
3.18 (m, 2H), 2.33 (s, 3H).
4.3.2. 9-o-(3-Thiophenyl)propyl-berberine bromide (4b)
Berberrubine was treated with compound 1b according to the
general procedure to afford compound 4b as a yellow solid (35%
yield). Mp 230.8–231.2 °C; 1H NMR (400 MHz, DMSO) d 9.80 (s,
1H), 8.95 (s, 1H), 8.19 (d, J = 9.1 Hz, 1H), 8.00 (d, J = 9.1 Hz, 1H),
7.80 (s, 1H), 7.42–7.29 (m, 4H), 7.20 (t, J = 7.1 Hz, 1H), 7.09 (s,
1H), 6.18 (s, 2H), 4.94 (s, 2H), 4.42 (t, J = 6.1 Hz, 2H), 4.04 (s, 3H),
3.28–3.15 (m, 4H), 2.21–2.13 (m, 2H). 13C NMR (101 MHz, DMSO)
d 150.2, 149.8, 147.6, 145.2, 142.6, 137.4, 135.9, 133.0, 130.6,
129.0, 128.2, 126.6, 125.7, 123.4, 121.5, 120.4, 120.2, 108.4,
105.4, 102.0, 72.8, 57.1, 55.3, 29.2, 28.7, 26.3.HRMS m/z [MꢁBr]+
calcd for C28H26NO4S 472.1577, found 472.1565.
4.2.9. (2-Bromoethyl)(2-chlorophenyl)sulfane (1i)
o-Chlorothiophenol was treated with 1,2-dibromoethane
according to the general procedure to give the desired product 4i
as an oil (54% yield). 1H NMR (400 MHz, CDCl3) d 7.40 (dd,
J = 14.0, 7.8 Hz, 2H), 7.5 (ddd, J = 23.2, 10.0, 4.9 Hz, 2H), 3.52–3.45
(m, 2H), 3.38–3.31 (m, 2H).
4.2.10. (2-Bromoethyl)(3-chlorophenyl)sulfane (1j)
m-Chlorothiophenol was treated with 1,2-dibromoethane
according to the general procedure to give the desired product 1j
as an oil (58% yield). 1H NMR (400 MHz, CDCl3) d 7.36 (s, 1H), 7.4
(s, 3H), 3.50–3.42 (m, 2H), 3.35–3.25 (m, 2H).
4.3.3. 9-o-(4-Thiophenyl)butyl-berberine bromide (4c)
4.2.11. (2-Bromoethyl)(4-chlorophenyl)sulfane (1k)
p-Chlorothiophenol was treated with 1,2-dibromoethane
according to the general procedure to give the desired product
1k as an oil (61% yield). 1H NMR (400 MHz, CDCl3) d 7.31 (q,
J = 8.7 Hz, 4H), 3.44 (dd, J = 9.4, 6.7 Hz, 2H), 3.27 (dd, J = 9.5,
6.8 Hz, 2H).
Berberrubine was treated with compound 1c according to the
general procedure to obtain compound 4c as a yellow solid (29%
yield). Mp 226.8–227.4 °C; 1H NMR (400 MHz, DMSO) d 9.74 (s,
1H), 8.93 (s, 1H), 8.19 (d, J = 9.0 Hz, 1H), 7.98 (d, J = 9.1 Hz, 1H),
7.79 (s, 1H), 7.35–7.26 (m, 4H), 7.16 (t, J = 6.8 Hz, 1H), 7.09 (s,
1H), 6.17 (s, 2H), 4.92 (t, J = 6.0 Hz, 2H), 4.30 (t, J = 6.3 Hz, 2H),
4.03 (s, 3H), 3.5–3.16 (m, 2H), 3.09 (t, J = 7.2 Hz, 2H), 2.04–1.96
(m, 2H), 1.87–1.78 (m, 2H). 13C NMR (101 MHz, DMSO) d 150.3,
149.8, 147.6, 145.2, 142.7, 137.4, 136.4, 132.9, 130.6, 128.9,
127.9, 126.6, 125.5, 123.4, 121.6, 120.4, 120.2, 108.4, 105.4,
4.2.12. (2-Bromoethyl)(2-ethylphenyl)sulfane (1l)
O-Ethylthiophenol was treated with 1,2-dibromoethane accord-
ing to the general procedure to give the desired product 1l as an oil

5838
T. Su et al. / Bioorg. Med. Chem. 21 (2013) 5830–5840
102.0, 73.7, 57.0, 55.3, 31.9, 28.5, 26.3, 25.1. HRMS m/z [MꢁBr]+
1H), 8.92 (s, 1H), 8.16 (d, J = 9.2 Hz, 1H), 7.98 (d, J = 9.1 Hz, 1H),
7.79 (s, 1H), 7.30–7.4 (m, 2H), 7.13 (d, J = 7.9 Hz, 2H), 7.09 (s,
1H), 6.17 (s, 2H), 4.91 (t, J = 6.2 Hz, 2H), 4.43 (t, J = 6.8 Hz, 2H),
3.99 (s, 3H), 3.45 (t, J = 6.8 Hz, 2H), 3.23–3.16 (m, 2H), 2.25 (s,
3H). 13C NMR (101 MHz, DMSO) d 150.1, 149.8, 147.6, 145.3,
142.0, 137.5, 135.8, 132.9, 131.3, 130.6, 129.7, 129.3, 126.5,
123.5, 121.6, 120.4, 120.1, 108.4, 105.4, 102.1, 71.8, 56.9, 55.3,
32.6, 26.3, 20.5. HRMS m/z [MꢁBr]+ calcd for C28H26NO4S
472.1577, found 472.1560.
calcd for C29H28NO4S 486.1734, found 486.1713.
4.3.4. 9-o-(5-Thiophenyl)pentyl-berberine bromide(4d)
Berberrubine was treated with compound 1d according to the
general procedure to obtain compound 4d as a yellow solid (32%
yield). Mp 216.5–218.1 °C; 1H NMR (400 MHz, DMSO) d 9.73 (s,
1H), 8.92 (s, 1H), 8.18 (d, J = 9.2 Hz, 1H), 7.98 (d, J = 9.1 Hz, 1H),
7.79 (s, 1H), 7.30 (d, J = 3.6 Hz, 4H), 7.16 (s, 1H), 7.08 (s, 1H), 6.16
(s, 2H), 4.92 (t, J = 5.9 Hz, 2H), 4.27 (t, J = 6.5 Hz, 2H), 4.03 (s, 3H),
3.5–3.16 (m, 2H), 3.01 (t, J = 6.9 Hz, 2H), 1.92–1.83 (m, 2H), 1.72–
1.57 (m, 4H). 13C NMR (101 MHz, DMSO) d 150.4, 149.8, 147.7,
145.2, 142.8, 137.4, 136.4, 133.0, 130.6, 128.9, 127.9, 126.6,
125.4, 123.3, 121.6, 120.4, 120.2, 108.4, 105.3, 102.0, 57.0, 56.0,
55.2, 31.9, 28.9, 28.3, 26.3, 24.4. HRMS m/z [MꢁBr]+ calcd for C30-
H30NO4S 500.1890, found 500.1877.
4.3.9. 9-o-[2-(o-Chloro)thiophenyl]ethyl-berberine bromide(4i)
Berberrubine was treated with compound 1i according to the
general procedure to obtain compound 4i as a yellow solid (30%
yield). Mp 218.0–218.6 °C; 1H NMR (400 MHz, DMSO) d 9.92 (s,
1H), 8.96 (s, 1H), 8.19 (d, J = 9.2 Hz, 1H), 8.01 (d, J = 9.1 Hz, 1H),
7.80 (s, 1H), 7.56–7.43 (m, 2H), 7.37 (dd, J = 11.0, 4.3 Hz, 1H),
7.26–7.18 (m, 1H), 7.10 (s, 1H), 6.18 (s, 2H), 5.00–4.87 (m, 2H),
4.52 (t, J = 6.5 Hz, 2H), 4.02 (s, 3H), 3.61 (t, J = 6.6 Hz, 2H), 3.26–
4.3.5. 9-o-(6-Thiophenyl)hexyl-berberine bromide(4e)
3.18 (m, 2H).13
C NMR (101 MHz, DMSO) d 150.18, 149.82,
Berberrubine was treated with compound 1e according to the
general procedure to obtain compound 4e as a yellow solid (31%
yield). Mp 211.2–211.6 °C; 1H NMR (400 MHz, DMSO) d 9.73 (s,
1H), 8.92 (s, 1H), 8.18 (d, J = 9.1 Hz, 1H), 7.98 (d, J = 9.0 Hz, 1H),
7.79 (s, 1H), 7.30 (d, J = 4.1 Hz, 4H), 7.16 (dd, J = 8.3, 3.9 Hz, 1H),
7.08 (s, 1H), 6.16 (s, 2H), 4.92 (t, J = 5.7 Hz, 2H), 4.27 (t, J = 6.6 Hz,
2H), 4.03 (s, 3H), 3.19 (t, J = 5.7 Hz, 2H), 2.98 (t, J = 7.1 Hz, 2H),
1.90–1.81 (m, 2H), 1.67–1.57 (m, 2H), 1.49 (m, 4H). 13C NMR
(101 MHz, DMSO) d 150.3, 149.7, 147.6, 145.0, 142.7, 137.3,
136.3, 133.0, 130.6, 129.0, 127.8, 126.5, 125.5, 123.4, 121.6,
120.3, 120.1, 108.4, 105.3, 101.9, 74.1, 56.9, 55.3, 31.8, 29.2, 28.3,
27.7, 26.3, 24.7. HRMS m/z [MꢁBr]+ calcd for C31H32NO4S
514.2047, found 514.2042.
147.67, 145.38, 141.93, 137.47, 134.66, 132.96, 131.49, 130.61,
129.59, 127.84, 127.64, 126.79, 126.51, 123.67, 121.55, 120.38,
120.17, 108.40, 105.41, 102.06, 71.26, 56.97, 55.31, 30.89, 26.31.
HRMS m/z [MꢁBr]+ calcd for C27H23NO4SCl 492.1031, found
492.1011.
4.3.10. 9-o-[2-(m-Chloro)thiophenyl]ethyl-berberine
bromide(4j)
Berberrubine was treated with compound 1j according to the
general procedure to obtain compound 4j as a yellow solid (38%
yield). Mp 215.8–216.4 °C;1H NMR (400 MHz, DMSO) d 9.87 (s,
1H), 8.95 (s, 1H), 8.19 (d, J = 9.2 Hz, 1H), 8.01 (d, J = 9.1 Hz, 1H),
7.80 (s, 1H), 7.43 (s, 1H), 7.39–7.31 (m, 2H), 7.30–7.23 (m, 1H),
7.10 (s, 1H), 6.18 (s, 2H), 4.94 (t, J = 6.1 Hz, 2H), 4.47 (t, J = 6.6 Hz,
4.3.6. 9-o-[2-(o-Methyl)thiophenyl]ethyl-berberine bromide(4f)
Berberrubine was treated with compound 1f according to the
general procedure to obtain compound 4f as a yellow solid (36%
yield). Mp 228.8–229.6 °C; 1H NMR (400 MHz, DMSO) d 9.87 (s,
1H), 8.94 (s, 1H), 8.18 (d, J = 9.2 Hz, 1H), 8.00 (d, J = 9.1 Hz, 1H),
7.79 (s, 1H), 7.39 (d, J = 7.7 Hz, 1H), 7.21 (t, J = 7.6 Hz, 2H), 7.15–
7.05 (m, 2H), 6.17 (s, 2H), 4.92 (t, J = 6.1 Hz, 2H), 4.47 (t,
J = 6.8 Hz, 2H), 3.99 (s, 3H), 3.50 (t, J = 6.8 Hz, 2H), 3.23–3.18 (m,
2H), 2.28 (s, 3H). 13C NMR (101 MHz, DMSO) d 150.1, 149.8,
147.6, 145.3, 142.0, 137.4, 136.6, 134.3, 132.9, 130.6, 130.1,
127.3, 126.6, 126.5, 125.7, 123.6, 121.6, 120.4, 120.1, 108.4,
105.4, 102.1, 71.6, 56.9, 55.3, 31.3, 26.3, 19.8. HRMS m/z [MꢁBr]+
calcd for C28H26NO4S 472.1577, found 472.1558.
2H), 4.01 (s, 3H), 3.59 (t, J = 6.6 Hz, 2H), 3.25–3.17 (m, 2H).13
C
NMR (101 MHz, DMSO) d 150.2, 149.8, 147.7, 145.3, 141.9, 138.0,
137.5, 133.7, 133.0, 130.7, 130.6, 127.0, 126.6, 126.5, 125.8,
123.7, 121.6, 120.4, 120.2, 108.4, 105.4, 102.1, 71.5, 56.9, 55.3,
31.6, 26.3. HRMS m/z [MꢁBr]+ calcd for C27H23NO4SCl 492.1031,
found 492.1015.
4.3.11. 9-o-[2-(p-Chloro)thiophenyl]ethyl-berberine
bromide(4k)
Berberrubine was treated with compound 1k according to the
general procedure to obtain compound 4k as a yellow solid (30%
yield). Mp 213.0–213.6 °C; 1H NMR (400 MHz, DMSO) d 9.83 (s,
1H), 8.93 (s, 1H), 8.17 (d, J = 9.2 Hz, 1H), 7.99 (d, J = 9.1 Hz, 1H),
7.79 (s, 1H), 7.45–7.32 (m, 4H), 7.09 (s, 1H), 6.17 (s, 2H), 4.92 (t,
J = 6.1 Hz, 2H), 4.45 (t, J = 6.6 Hz, 2H), 4.00 (s, 3H), 3.53 (t,
J = 6.6 Hz, 2H), 3.4–3.17 (m, 2H). 13C NMR (101 MHz, DMSO) d
149.9, 149.8, 147.6, 144.9, 141.7, 137.4, 134.2, 132.9, 130.7,
130.5, 130.0, 128.9, 126.4, 123.7, 121.4, 120.5, 120.0, 108.3,
105.3, 101.9, 71.5, 56.8, 55.4, 32.2, 26.3. HRMS m/z [MꢁBr]+ calcd
for C27H23NO4SCl 492.1031, found 492.1011.
4.3.7. 9-o-[2-(m-Methyl)thiophenyl]ethyl-berberine
bromide(4g)
Berberrubine was treated with compound 1g according to the
general procedure to obtain compound 4g as a yellow solid (38%
yield). Mp 217.8–218.4 °C; 1H NMR (400 MHz, CDCl3) d 10.32 (s,
1H), 8.42 (s, 1H), 7.97 (d, J = 9.0 Hz, 1H), 7.68 (d, J = 9.1 Hz, 1H),
7.38 (s, 1H), 7.19 (dd, J = 17.4, 9.9 Hz, 3H), 6.98 (d, J = 7.4 Hz, 1H),
6.76 (s, 1H), 6.02 (s, 2H), 5.21 (t, J = 6.2 Hz, 2H), 4.65 (t, J = 6.5 Hz,
2H), 3.91 (s, 3H), 3.62 (t, J = 6.5 Hz, 2H), 3.29 (t, J = 6.3 Hz, 2H),
2.32 (s, 3H). 13C NMR (101 MHz, DMSO) d 150.1, 149.7, 147.6,
145.3, 141.9, 138.5, 137.4, 134.8, 132.9, 130.6, 128.9, 128.8,
126.8, 126.4, 125.4, 123.6, 121.6, 120.3, 120.1, 108.4, 105.4,
102.0, 71.7, 56.9, 55.3, 31.8, 26.3, 20.8. HRMS m/z [MꢁBr]+ calcd
for C28H26NO4S 472.1577, found 472.1583.
4.3.12. 9-o-[2-(o-Ethyl)thiophenyl]ethyl-berberine bromide (4l)
Berberrubine was treated withcompound1l accordingtothe gen-
eral procedure to obtain compound 4l as a yellow solid (33% yield).
Mp 222.8–223.6 °C; 1H NMR (400 MHz, DMSO) d 9.88 (s, 1H), 8.95
(s, 1H), 8.18 (d, J = 9.2 Hz, 1H), 8.00 (d, J = 9.1 Hz, 1H), 7.80 (s, 1H),
7.43 (d, J = 7.8 Hz, 1H), 7.26–7.14 (m, 3H), 7.10 (s, 1H), 6.18 (s, 2H),
4.93 (t, J = 6.2 Hz, 2H), 4.47 (t, J = 6.8 Hz, 2H), 4.00 (s, 3H), 3.51 (t,
J = 6.8 Hz, 2H), 3.4–3.18 (m, 2H), 2.67 (q, J = 7.5 Hz, 2H), 1.13 (t,
J = 7.5 Hz, 3H).13C NMR (101 MHz, DMSO) d 150.1, 149.8, 147.6,
145.3, 142.7, 142.0, 137.4, 133.6, 132.9, 130.6, 128.5, 128.0, 126.7,
126.5, 126.1, 123.6, 121.5, 120.4, 120.1, 108.4, 105.4, 102.0, 71.7,
4.3.8. 9-o-[2-(p-Methyl)thiophenyl]ethyl-berberine
bromide(4h)
Berberrubine was treated with compound 1h according to the
general procedure to obtain compound 4h as a yellow solid (42%
yield). Mp 222.8–223.6 °C; 1H NMR (400 MHz, DMSO) d 9.83 (s,

T. Su et al. / Bioorg. Med. Chem. 21 (2013) 5830–5840
5839
56.94, 55.3, 31.8, 26.2, 14.5. HRMS m/z [MꢁBr]+ calcd for C29H28NO4-
to be measured at various time points. The plots were assessed by a
weighted least square analysis that assumed the variance of V to be
a constant percentage of V for the entire data set. The 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.
S486.1734, found 486.1729.
4.3.13. 9-o-[2-(o-Fluro)thiophenyl]ethyl-berberine
bromide(4m)
Berberrubine was treated with compound 1m according to the
general procedure to obtain compound 4m as a yellow solid (35%
yield). Mp 221.9–222.4 °C; 1H NMR (400 MHz, DMSO) d 9.88 (s,
1H), 8.94 (s, 1H), 8.18 (d, J = 9.2 Hz, 1H), 8.00 (d, J = 9.1 Hz, 1H),
7.80 (s, 1H), 7.53 (td, J = 7.8, 1.6 Hz, 1H), 7.35–7.28 (m, 1H), 7.28–
7.18 (m, 2H), 7.10 (s, 1H), 6.18 (s, 2H), 4.93 (t, J = 6.3 Hz, 2H),
4.46 (t, J = 6.6 Hz, 2H), 3.99 (s, 3H), 3.53 (t, J = 6.6 Hz, 2H), 3.25–
3.17 (m, 2H). 13CNMR (101 MHz, DMSO) d 150.1, 149.8, 147.6,
145.3, 141.9, 137.4, 132.9, 131.0, 130.6, 128.6, 128.5, 126.5,
125.2, 123.6, 121.5, 120.4, 120.1, 115.7, 115.5, 108.4, 105.4,
102.0, 71.6, 56.9, 55.3, 31.7, 26.3. HRMS m/z [MꢁBr]+ calcd for C27-
H23NO4FS 476.1326, found 476.1312.
4.4.3. Molecular modelling
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, whereas the water molecules present in the PDB file
were maintained in their position. The 3D structures of the 9-
substituted berberine derivatives were generated and optimised
using the DISCOVERY STUDIO 2.1 package (Accelrys Inc., San Diego,
CA). The CDOCKER program in DISCOVERY STUDIO 2.1 was used to per-
form docking simulations. The program allows full flexibility of the
ligand.
4.3.14. 9-o-[2-(o-Methoxyl)thiophenyl]ethyl-berberine
bromide(4n)
4.4.4. The oxygen radical absorbance capacity-fluorescein
(ORAC-FL) assay
Berberrubine was treated with compound 1n according to the
general procedure to obtain compound 4n as a yellow solid (42%
yield). Mp 215.4–216.2 °C; 1H NMR (400 MHz, DMSO) d 9.89 (s,
1H), 8.95 (s, 1H), 8.18 (d, J = 9.2 Hz, 1H), 8.00 (d, J = 9.1 Hz, 1H),
7.80 (s, 1H), 7.36–7.27 (m, 1H), 7.25–7.16 (m, 1H), 7.10 (s, 1H),
7.03–6.91 (m, 2H), 6.18 (s, 2H), 4.97–4.82 (m, 2H), 4.45 (t,
J = 6.8 Hz, 2H), 4.00 (s, 3H), 3.79 (s, 3H), 3.44 (t, J = 6.8 Hz, 2H),
3.25–3.18 (m, 2H). 13C NMR (101 MHz, DMSO) d 156.7, 150.1,
149.8, 147.7, 145.4, 142.0, 137.4, 132.9, 130.6, 128.3, 127.2,
126.5, 123.6, 122.9, 121.6, 121.0, 120.4, 120.1, 111.1, 108.4,
105.4, 102.1, 71.7, 56.9, 55.7, 55.3, 30.4, 26.3. HRMS m/z [MꢁBr]+
calcd for C28H26NO5S 488.1526, found 488.1502.
All the assays were conducted using 75 mM phosphate buffer
(pH 7.4), and the final reaction mixture was 200
lL. The antioxi-
dant (20 L) and fluorescein (120 L, 300 nM final concentration)
l
l
were placed in the wells of a black 96-well plate, and the mixture
was incubated for 10 min at 37 °C. Then, AAPH (Aldrich) solution
(60
lL, 12 mM final concentration) was added rapidly. The plate
was immediately placed into a Spectrafluor Plus plate reader (Te-
can, Crailsheim, Germany), and the fluorescence was measured
every 60 s for 4 h with excitation at 485 nm and emission at
535 nm. Trolox was used as a standard (1–10 mM final concentra-
tion). A blank (FL+AAPH) using phosphate buffer instead of antiox-
idant and Trolox calibration were conducted in each assay. The
samples were measured at different concentrations (0.5–10 mM).
All reaction mixtures were prepared three times, and at least three
independent runs were performed for each sample. The fluores-
cence measurements were normalised to the curve of the blank
(without antioxidant). The ORAC-FL values were calculated as de-
scribed in the reference, and the final results were expressed in
mM of Trolox equivalent/mM of pure compound.
4.4. Biological activity
4.4.1. In vitro inhibition studies on AChE and BuChE
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel),
butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum),
5,5-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), acet-
ylthiocholine chloride (ATC), and butylthiocholine chloride (BTC)
were purchased from Sigma Aldrich. Berberine derivatives were
dissolved in DMSO (10 mM) and then diluted in 0.1 M KH2PO4/
K2HPO4 buffer (pH 8.0) to the final concentration.
4.4.5. Hydrogen peroxide scavenging activity
Briefly, a solution of 4f (100
lL) was added to H2O2 (0.1 mM,
All the in vitro assays were conducted out in 0.1 M KH2PO4/
K2HPO4 buffer, using a Shimadzu UV-2450 Spectrophotometer.
The AChE and BuChE solutions were prepared to give 2.0 units/
mL in 2-mL aliquots. The assay medium (1 mL) consisted of phos-
100 L). Then, 0.1 M phosphate buffer (pH 7.4, 200 l
The solution was incubated for 72 h at 37 °C, and a solution con-
taining red phenol (0.2 mg/mL) and horseradish peroxidase
l
L) was added.
(0.4 mg/mL) in 0.1 M phosphate buffer (500
lL) was added. After
phate buffer (pH 8.0), 50
lL of 0.01 M DTNB, 10
lL of enzyme, and
15 min, a 1 M NaOH solution (100 L) was added, and the
l
50 L of 0.01 M substrate (ACh chloride solution). Briefly, the test
l
absorbance was recorded at 610 nm. The result is expressed as
compounds were added to the assay solution and preincubated
at 37 °C with enzyme for 15 min followed by the addition of sub-
strate. The activity was determined by measuring the increase of
absorbance at 412 nm at 60 s intervals at 37 °C. The calculations
were performed according to the method of the equation of Ellman
et al. Each compound was assayed in triplicate.
the percentage of reduction of H2O2: % reduction of H2O2 ¼
ðAH ꢁ AsampleÞ=AH ꢂ 100. AH
refers to the control solution
2O2
2O2
2O2
with pure acetone and without the compound 4f, and Asample refers
to compound-containing solution.
4.4.6. Inhibition of Ab1–42 peptide aggregation12d
The in vitro BuChE assay (BuCh as the substrate) was performed
similar to the above described method.
Hexafluoroisopropanol (HFIP) pretreated Ab1–42 samples (Ana-
Spec) were resolubilised with 50 mM phosphate buffer (pH 7.4)
to have a stable stock solution ([Ab] = 200
incubated in 50 mM phosphate buffer (pH 7.4) at 37 °Cfor 48 h (fi-
nal Ab concentration 50 M) with and without the tested com-
pound at 20 M. After incubation, the samples were diluted to a
final volume of 200 L with 50 mM glycine–NaOH buffer (pH
8.0) containing thioflavin T. Then, a 300-second time scan of fluo-
rescence intensity was performed (kexc = 450 nm, kem = 485 nm),
and the values at the plateau were averaged after subtracting the
background fluorescence of thioflavin T solution.
lM). The peptide was
4.4.2. Kinetic characterisation of AChE inhibition
The kinetic characterisation of AChE was performed using a pre-
viously reported method. Briefly, the 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. The kinetic
characterisation of the hydrolysis of ATC catalysed by AChE was
performed spectrophotometrically at 412 nm. A parallel control
with no inhibitor in the mixture allowed adjusting of the activities
l
l
l

5840
T. Su et al. / Bioorg. Med. Chem. 21 (2013) 5830–5840
A. I. Biochemistry 1999, 38, 7609; (b) Butterfield, D. A. Curr. Med. Chem. 2003, 10,
Acknowledgments
2651.
10. (a) Di Bona, D.; Scapagnini, G.; Candore, G.; Castiglia, L.; Colonna-Romano, G.;
Duro, G.; Nuzzo, D.; Iemolo, F.; Lio, D.; Pellicano, M.; Scafidi, V.; Caruso, C.;
Vasto, S. Curr. Pharm. Des. 2010, 16, 684; (b) Valko, M.; Leibfritz, D.; Moncol, J.;
Cronin, M. T. D.; Mazur, M.; Telser, J. Int. J. Biochem. Cell B 2007, 39, 44.
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This work was supported by the Foundation for Distinguished
Young Talents in Higher Education of Guangdong (2012
LYM_003), China Postdoctoral Science Foundation (2012M2
51651), Ph.D. Programs Foundation of Ministry of Education of
China (20120171120045), and Natural Science Foundation of China
(20972198).
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