Benzenediol-berberine hybrids: Multifunctional agents for Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2011.09.040

We designed and synthesized a series of hybrid molecules, in an effort to identify novel multifunctional drug candidates for Alzheimer's disease (AD), by reacting berberine with benzenediol, melatonin, and ferulic acid. The products were evaluated for: (i

Full text of DOI:10.1016/j.bmc.2011.09.040

Bioorganic & Medicinal Chemistry 19 (2011) 7228–7235
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Bioorganic & Medicinal Chemistry
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Benzenediol-berberine hybrids: Multifunctional agents for Alzheimer’s disease
Huailei Jiang a,b, Xu Wang b, Ling Huang b, Zonghua Luo b, Tao Su b, Ke Ding a, , Xingshu Li b,
⇑
⇑
a Institute of Chemical Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510663, People’s Republic of China
b The Industrial Institute of Fine Chemicals and Synthetic Drugs, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed and synthesized a series of hybrid molecules, in an effort to identify novel multifunctional
drug candidates for Alzheimer’s disease (AD), by reacting berberine with benzenediol, melatonin, and
ferulic acid. The products were evaluated for: (i) the ability to inhibit multiple cholinesterases (ChEs);
(ii) the capacity to prevent amyloid b (Ab) aggregation; and (iii) antioxidant activity. All of the derivatives
were better antioxidants, and inhibited Ab aggregation to a greater extent, than the lead compound, ber-
berine. Two of the hybrids, in particular, have the potential to be excellent candidates for AD therapy: the
berberine-pyrocatechol hybrid (compound 8) was a much better inhibitor of acetylcholinesterase (AChE)
Received 5 August 2011
Revised 21 September 2011
Accepted 22 September 2011
Available online 8 October 2011
Keywords:
Benzenediol-berberine hybrids
Cholinesterases
Amyloid b
than unconjugated berberine (IC50: 0.123 vs 0.374 lM); and the berberine-hydroquinone hybrid (com-
pound 12) displayed high antioxidant activity, could inhibit AChE (IC50 of 0.460
lM), and had the greatest
ability to inhibit Ab aggregation.
Antioxidant
Ó 2011 Elsevier Ltd. All rights reserved.
Alzheimer’s disease
1. Introduction
Because of the complex multifactorial etiology of AD, research-
ers have developed the multi-target-directed ligand (MTDL) strategy
for drug discovery. It has become a valuable tool for developing
drugs that target multiple factors implicated in AD pathogenesis.13
In the MTDL strategy, distinct pharmacophores from different
drugs are combined in a single molecule to produce multifunc-
tional hybrids.14,15 One of the most widely adopted approaches
has been to modify the structure of AChE inhibitors to confer addi-
tional biological properties that are likely to be useful for treating
AD.
In our previous work using berberine (1, Fig. 1) as the lead
structure, we designed and synthesized a series of novel deriva-
tives and found that most of them were capable of inhibiting both
AChE and BuChE.16 The most potent inhibitor, berberine linked
with phenol by 4-carbon spacers (2, Fig. 1), strongly inhibited AChE
AD is a neurodegenerative disorder characterized by progres-
sive cognitive decline, decreased cholinergic transmission, and
the presence of senile plaques. Although the etiology of AD is not
fully known, the presence of Ab deposits, increased oxidative
stress, and reduced levels of acetylcholine (ACh) are thought to
play significant roles in the pathophysiology of the disease.1
There are two types of ChEs in the central nervous system
(CNS), both of which can hydrolyze ACh: acetylcholinesterase
(AChE) and butyrylcholinesterase (BuChE). Recent studies indicate
that as AChE activity steadily decreases from mild to severe stages
of AD (eventually reaching 10–15% of normal values), BuChE levels
remain unchanged or may rise. In fact, evidence suggests that
inhibiting BuChE raises ACh levels and improves cognition.2,3
Dual-acting ChE inhibitors affect the activities of both AChE and
BuChE.4,5
(IC50 of 0.097 lM). We continued to pursue this promising strategy
with the design, synthesis, and evaluation of numerous other com-
The formation of Ab fibrils and their deposition into neurotoxic
amyloid plaques appear to be the primary cause of AD.6–8 Ab
deposits inflict oxidative injury to surrounding neurons and elicit
an acute inflammatory response that contributes to the neurode-
generation.9,10 Senile plaques release free radicals which are extre-
mely toxic11, and the accumulation of these reactive oxygen
species (ROS) damages major cell components. Thus, oxidative
stress is thought to play a major role in the pathogenesis of AD. In-
deed, several antioxidant compounds have demonstrated efficacy
in a number of recent studies.12
pounds with therapeutic potential for treating AD.
Certain phenol and diphenol derivatives have previously been
shown to possess both antioxidant activity17,18 and the ability to
inhibit Ab aggregation.19,20 For example, curcumin, which is pro-
duced by the rhizomes of Curcuma longa, is an effective scavenger
of ROS. Ferulic acid (4, Fig. 1), one of the predominant phenolic
acids in wheat (Triticum aestivum) and eucalyptus (Eucalyptus
globulus), has been reported to impart resistance to Ab toxicity in
the brains of mice receiving long-term administration of this
compound.21,22 Melatonin (3, Fig. 1), a hormone produced by the
pineal gland, is also a potent antioxidant, and is able to scavenge
a variety of ROS.23,24 We generated a series of hybrids of berberine,
diphenol, ferulic acid, and melatonin; in effect combining two
⇑
Corresponding authors.
E-mail address: lixsh@mail.sysu.edu.cn (X. Li).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.040

H. Jiang et al. / Bioorg. Med. Chem. 19 (2011) 7228–7235
7229
Figure 1. Structure of berberine (1), the phenol-berberine derivative (2), melatonin (3), and ferulic acid (4).
distinct pharmacophores to form hybrid compounds. In this report,
2.2. In vitro inhibition studies of AChE and BuChE
we describe the synthesis of these hybrids and evaluate their po-
tential, in vitro, as candidates for treating AD. We demonstrate that
these compounds display antioxidant activity, and that some of
them also have the ability to strongly inhibit cholinesterases and
reduce Ab aggregation.
The ability of the derivatives to inhibit AChE activity was as-
sessed by the method of Ellman,27 using AChE from the electric
eel and commercial galanthamine as the reference standard. The
ability of these compounds to inhibit horse serum BuChE was
determined using a similar method. The IC50 values for AChE and
BuChE inhibition are summarized in Table 1. The berberine-
2. Results and discussion
2.1. Chemistry
benzenediol hybrids (7–13, IC50 values of 0.628–0.123
better inhibitors of AChE than the berberine-melatonin hybrids
(17–19, IC50 values of 1.11–1.44 M) or the berberine-ferulic acid
hybrid (21, IC50 of 3.21 M). However, the berberine-phenol
derivative (2, IC50 of 0.097 M) was the most potent inhibitor of
AChE. The berberine-pyrocatechol hybrid (8) was also a strong
AChE inhibitor (IC50 of 0.123 M).
All of the berberine-benzenediol derivatives were potent inhib-
itors of BuChE with IC50 values of 1.04–2.72 M, which was better
than that of the leading compound berberine (Table 1, entry 1,
18.02 M). As BuChE could also hydrolyze ACh, BuChE inhibition
lM) were
l
l
l
The synthesis of the berberine-benzenediol derivatives (7–13)
is presented in Scheme 1. First, berberrubine (5) was obtained with
a yield of 66% through selective demethylation of berberine (1) at
190 °C under vacuum,25 and was then alkylated with dibromoalk-
anes in dimethylformamide (DMF)26 to produce compounds 6a–c
with yields of 60–75%. Finally, 6a–c were reacted with benzenediol
in DMF for 4 h to produce 7–13 at high yields.
Berberine-melatonin hybrids (17–19) were synthesized as
shown in Scheme 2. First, different b-bromo-N-arylpropiontrypta-
mides (14–16) were obtained by reacting b-bromopropionyl chlo-
ride with the appropriate tryptamine in the presence of pyridine.
Compounds 14–16 were then coupled with berberrubine (5) in
DMF to produce the berberine-melatonin hybrids (17–19).
The synthesis of the berberine-ferulic acid hybrid (21) is shown
in Scheme 3. The reaction of ferulic acid with 1,3-dibromopropane
in the presence of Et3N at 50 °C gave compound 20 with a yield of
67%. The reaction of 20 with berberrubine (5) in DMF produced the
berberine-ferulic acid hybrid (21) with a yield of 43%.
l
l
l
was also evaluated in the presence of ACh as the enzyme substrate.
The results showed that most of these hybrids exhibited similar
selectivity ratios of AChE/BuChE (Table 1), which indicated they
could effectively inhibit both AChE and BuChE, and may be inter-
esting as dual AChE/BuChE inhibitors for AD.
2.3. Kinetics of AChE inhibition
To investigate the kinetics of AChE inhibition, the most potent
inhibitor, compound 8, was chosen for further kinetic studies
Scheme 1. 9-Substituted berberine derivatives 7–13. Reagents and conditions: (i) 190 °C, 20–30 mm Hg, 15 min; (ii) Br(CH2)nBr, DMF, 1–4 h; (iii) Benzenediol, DMF, ꢀ80 °C.

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H. Jiang et al. / Bioorg. Med. Chem. 19 (2011) 7228–7235
Scheme 2. Synthesis of the berberine-melatonin hybrids 17–19.
Scheme 3. Synthesis of the berberine-ferulic acid hybrid 21.
(Fig. 2.). The intersection point of Lineweaver–Burk reciprocal plot
was in the third quadrant, which was considered as a mixed-type
inhibition of uncompetitive inhibition and non-competitive inhibi-
tion.29 Noncompetitive inhibition takes place when an inhibitor
can bind to the enzyme at a site that is distinct from the active site.
Uncompetitive inhibition, also known as anti-competitive inhibi-
tion, takes place when an enzyme inhibitor binds only to the com-
plex formed between the enzyme and the substrate (the E–S
complex). Both of them could only bind to the allosteric sites of en-
zyme. This phenomena may be explained as the hydroxyl group of
phenol moiety in compound 8 hindered the penetration of inhibi-
tor into the AChE gorge, which was consisted of hydrophobic ami-
hybrid, because of the existence of the phenolic hydroxyl. Com-
pound 12, which possessed a hydroxyl at the para-position on the
benzene ring, was the strongest antioxidant (ORAC-FL value of
9.53). Comparing the ORAC-FL values of the berberine-melatonin
hybrids (17–19) with that of melatonin (3), it is apparent that the
berberine moiety does not significantly contribute to the radical
scavenging ability of the hybrids. This is also evident when compar-
ing the antioxidant activities of ferulic acid and the berberine-ferulic
acid hybrid.
2.5. Inhibition of Ab1–42 aggregation
no acids, to reach the catalytic active site33
.
The ability of the novel berberine derivatives to inhibit Ab1–42
aggregation was assessed using the thioflavin T fluorescence assay,
with a curcumin standard (Fig. 3). Significantly, all the berberine
hybrids exhibited greater inhibitory activity than curcumin
(52.1%), berberine-phenol hybrid 2 (57.4%), and berberine (36.3%)
2.4. Antioxidant activity
The antioxidant activity of the hybrids derived from berberine
were determined using the oxygen radical absorbance capacity as-
say using fluorescein (ORAC-FL), according to the method originally
described by Ou et al.30 and modified by Daevalos31 The vitamin E
analog, Trolox, was used as a standard, and the antioxidant activity
at a concentration of 20 lM. Among these, compound 12, the
hydroquinone-berberine hybrid, was the most potent inhibitor of
Ab aggregation (92.0%). Compound 12 also possessed the greatest
antioxidant activity among the berberine derivatives (9.54 Trolox
equivalents). The position of the phenolic hydroxyl on the benzene
ring appears to influence the compound’s ability to inhibit Ab
aggregation, paralleling its crucial role in the ability to inhibit AChE
and BuChE. For example, compound 7, with the phenolic hydroxyl
at the ortho position, gave a lower inhibitory activity (83.5%) than
the other isomers (compound 10, resorcinol-berberine hybrid,
85.9%, and compound 12, hydroquinone-berberine hybrid, 92%).
Other hybrids, berberine-melatonin hybrids (17–19, 75.8–82.8%)
and the berberine-ferulic acid hybrid (21, 75.8%) also exhibited
was expressed as Trolox equivalents (lmol of Trolox/lmol of tested
compound). As shown in Table 1, berberine (1) and the berberine-
phenol derivative (2) exhibited only mild antioxidant activity. All
of the hybrids, including berberine-benzenediol (7–13), berberine-
melatonin (17–19), and berberine-ferulic acid (21) demonstrated
excellent antioxidant activity with ORAC-FL values of 3.1–9.53
Trolox equivalents. Moreover, most of the berberine-benzenediol
derivatives (9–13) exhibited much better antioxidant activity than
the berberine-melatonin hybrids or the berberine-ferulic acid

H. Jiang et al. / Bioorg. Med. Chem. 19 (2011) 7228–7235
7231
Table 1
In vitro inhibition of AChE and BuChE and Oxygen Radical Absorbance Capacity (ORAC, Trolox equivalents) by berberine (1), melatonin (3), ferulic acid (4), galanthamine and the
berberine derivatives 2, 7–13, 17–19, 21
R
O
O
Br
Br
N
O
N
R
O
O
(CH2)nO
O
O
NH
O
O
7-13
17-19
N
H
O
O
Br
N
OH
O
O
O
OCH3
21
O
Selectivity for AChEd
IC50 (l
M) BuChE/AChe
Trolox equivf
a
Compound
R
n
IC50
(lM)
AChEb
BuChEc
1
2
–
–
0.374 0.024
0.097 0.005
18.2 0.683
4.89 0.035
48.6
50.4
n.t.g
n.t.
0.4 0.01
1.0 0.02
7
8
OH
3
4
0.490 0.032
0.123 0.003
1.74 0.028
2.09 0.14
3.55
17.0
1.30 0.021
1.74 0.007
3.41 0.25
3.54 0.27
9
10
11
OH
OH
2
3
4
0.422 0.034
0.628 0.008
0.547 0.002
2.64 0.063
1.13 0.014
2.40 0.021
6.26
1.80
4.39
2.26 0.007
0.898 0.071
1.92 0.059
4.63 0.01
5.31 0.33
5.30 0.41
12
13
3
4
0.460 0.013
0.304 0.005
2.06 0.035
1.04 0.077
4.48
3.42
1.66 0.106
0.95 0.098
9.54 0.62
6.45 0.39
17
18
19
21
3
4
H
CH3
OCH3
–
–
–
–
1.11 0.021
1.42 0.042
1.44 0.054
3.21 0.155
n.t.
>100
>100
0.623 0.099
1.64 0.206
1.96 0.014
2.72 0.035
2.40 0.042
n.t.
n.t.
n.t.
15.7 0.787
7.96
1.39
1.88
0.75
–
–
–
25.3
1.46 0.015
1.79 0.212
2.49 0.024
2.25 0.113
n.t.
n.t.
n.t.
n.t.
3.78 0.01
3.11 0.21
3.61 0.30
3.43 0.11
2.3 0.1h
3.13 0.12
n.t.
–
–
–
Curcumin
Galanthamine
n.t.
a
b
c
Mean SD of at least three independent measurements.
Acetylcholine substrate for evaluation of antiacetylcholinesterase activity. AChE from electric eel.
Butyrylcholine substrate for evaluation of antibutyrylcholinesterase activity. BuChE from equine serum.
Selectivity for AChE = IC50 (BuChE)/IC50 (AChE).
d
e
f
Acetylcholine substrate for evaluation of antibutyrylcholinesterase activity.
Data are expressed as
n.t. = not tested.
Data from Ref.
l mol of trolox equivalent/l mol of tested compound and are the mean (n = 3) SD.
g
h
28
better inhibitory activity than that of curcumin, berberine and
compound 2 which without the hydroxyl group on the phenyl ring.
inhibition. Combining antioxidant activity with the ability to inhi-
bit ChEs and impede Ab aggregation, these hybrid compounds are
excellent multifunctional drug candidates for AD. Further investi-
gation into these potential therapeutic agents is in progress.
3. Conclusion
In summary, we designed and synthesized a series of novel
berberine conjugates, including berberine-benzenediol derivatives
(7–13), berberine-melatonin hybrids (17–19), and a berberine-
ferulic acid hybrid (21). We evaluated their potential as multifunc-
tional drug candidates for AD. All of these compounds exhibited
antioxidant activity and inhibited Ab aggregation better than
unconjugated berberine, although some of them had a reduced
ability to inhibit AChE. The hydroquinone-berberine hybrid, com-
pound 12, had the greatest ability to suppress Ab aggregation. It
was also an excellent antioxidant and a reasonable AChE and
BuChE inhibitor. Compound 8 was a potent AChE inhibitor and a
strong antioxidant. It also inhibited Ab aggregation. A study of en-
zyme kinetics revealed that the inhibition of 8 was a mixed-type
inhibition of uncompetitive inhibition and non-competitive
4. Material and methods
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
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
wasperformedusing silicagel(200–300 mesh)purchasedfromQing-
dao Haiyang Chemical Co. Ltd or alumina from Sinopharm Chemical
Reagent Co. Ltd All the reactions were monitored by thin layer chro-
matography using silica gel. Berberine chloride was isolated from
Chinese herbal medicine C. chinensis Franch and recrystallized from

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H. Jiang et al. / Bioorg. Med. Chem. 19 (2011) 7228–7235
(s, 1H), 8.88 (s, 1H), 8.19 (d, J = 9.1 Hz, 1H), 8.00 (d, J = 9.0 Hz, 1H),
7.80 (s, 1H), 7.09 (s, 1H), 6.98 (d, J = 7.2 Hz, 1H), 6.78 (d,
J = 15.1 Hz, 3H), 6.18 (s, 2H), 4.92–4.79 (m, 2H), 4.51 (t, J = 6.2 Hz,
2H), 4.25 (t, J = 6.1 Hz, 2H), 4.02 (s, 3H), 3.18 (s, 2H), 2.38–2.23 (m,
2H). 13C NMR (101 MHz, DMSO) d 150.37, 149.84, 147.69, 146.91,
146.77, 145.20, 142.82, 137.46, 133.06, 130.61, 126.79, 123.36,
121.59, 121.24, 120.39, 120.22, 119.23, 115.74, 114.11, 108.38,
105.41, 102.05, 71.45, 65.28, 57.01, 55.29, 29.52, 26.33.HRMS m/z
[MꢀBr]+ Calcd for C28H25NO6 472.1760, found 472.1781.
4.2.2. 9-O-[4-(Phenylol-2-yloxy)butyl] berberine bromide (8)
Compound 6c was treated with 1,2-benzenediol according to a
general protocol to obtain compound 8 as a yellow solid (31%
yield). mp 197.2–198.9 °C; 1H NMR (400 MHz, MeOD) d 9.70 (s,
1H), 8.70 (s, 1H), 8.12 (d, J = 9.1 Hz, 1H), 8.02 (s, 1H), 7.68 (s, 1H),
6.98 (s, 1H), 6.93 (dd, J = 10.6, 6.8 Hz, 1H), 6.77 (t, J = 2.8 Hz, 2H),
6.13 (s, 2H), 4.90 (d, J = 6.6 Hz, 2H), 4.55 (s, 2H), 4.20 (d,
J = 5.9 Hz, 2H), 4.11 (s, 3H), 3.24 (s, 2H), 2.14 (dd, J = 11.3, 6.0 Hz,
4H). 13C NMR (101 MHz, MeOD) d 161.50, 159.24, 155.67, 150.61,
147.82, 145.90, 144.98, 144.42, 135.45, 130.6, 126.9, 125.20,
124.93, 123.29, 120.70, 120.23, 118.8, 112.6, 110.5, 106.8, 103.9,
102.1, 71.62, 65.37, 56.93, 53.13, 30.175, 28.46, 26.38. HRMS m/z
[MꢀBr]+ Calcd for C29H27NO6 486.1917, found 486.1899.
Figure 2. Steady state inhibition by compound 8 of AChE hydrolysis of ACh; the
plots show mixed type inhibition for compound 8 on AChE.
hot water. Compound 5 and intermediates6a, 6band 6cwere synthe-
sized as previously reported.26
4.2.3. 9-O-[2-(Phenylol-3-yloxy)ethyl] berberine bromide (9)
Compound 6a was treated with 1,3-benzenediol according to
general protocol to obtain compound 9 as a yellow solid (33%
yield). mp 217.4–218.8 °C; 1H NMR (400 MHz, MeOD) d 9.63 (s,
1H), 8.68 (s, 1H), 8.09 (dd, J = 41.2, 9.1 Hz, 2H), 7.60 (d,
J = 27.5 Hz, 1H), 7.05 (t, J = 8.2 Hz, 1H), 6.94 (s, 1H), 6.38 (dd,
J = 8.0, 1.9 Hz, 1H), 6.29 (dd, J = 8.1, 2.1 Hz, 1H), 6.18 (d,
J = 2.1 Hz, 1H), 6.12 (s, 2H), 4.81–4.73 (m, 2H), 4.72–4.61 (m, 2H),
4.40–4.27 (m, 2H), 4.14 (s, 3H), 3.19–3.05 (m, 2H). 13C NMR
4.2. General procedure for the preparation of 7–13
To a solution of compounds 6a, 6b or 6c (1 mmol) in DMF
(15 ml), benzenediol (2 mmol) was added at room temperature.
After stirring at 80 °C for 4 h, the mixture was cooled to room tem-
perature and evaporated under vacuum. The crude product was
chromatographed on an Al2O3 column and eluted with CHCl3/
MeOH (100:1–50:1) to obtain purified compound.
(101 MHz, MeOD)
d 160.93, 159.95, 152.17, 152.13, 149.90,
146.31, 144.53, 139.48, 135.02, 131.71, 131.27, 127.73, 124.92,
123.94, 121.73, 121.51, 109.39, 109.34, 106.54, 106.24, 103.68,
102.88, 73.82, 68.29, 57.60, 57.32, 28.13. HRMS m/z [MꢀBr]+ Calcd
for C27H23NO6 458.1604, found 458.1624.
4.2.1. 9-O-[3-(Phenylol-2-yloxy) propyl] berberine bromide (7)
Compound 6b was treated with 1,2-benzenediol according to a
general protocol to obtain compound 7 as a yellow solid (37% yield).
mp 213.1–215.7 °C; 1H NMR (400 MHz, DMSO) d 9.77 (s, 1H), 8.94
Figure 3. Effects on the Ab1–42 peptide aggregation inhibition at 20 lM of berberine derivatives.

H. Jiang et al. / Bioorg. Med. Chem. 19 (2011) 7228–7235
7233
4.2.4. 9-O-[3-(Phenylol-3-yloxy)propyl] berberine bromide (10)
Compound 6b was treated with 1,3-benzenediol according to
general protocol to obtain compound 10 as a yellow solid (40%
yield). mp 211.5–214.0 °C; 1H NMR (400 MHz, MeOD) d 9.43 (s,
1H), 8.60 (s, 1H), 8.03 (d, J = 9.1 Hz, 1H), 7.93 (d, J = 9.1 Hz, 1H),
7.56 (s, 1H), 7.02 (t, J = 8.1 Hz, 1H), 6.87 (s, 1H), 6.42–6.28 (m,
3H), 6.04 (s, 2H), 4.53 (dd, J = 12.6, 6.6 Hz, 4H), 4.25 (s, 2H), 3.99
(s, 3H), 3.12–3.03 (m, 2H), 2.30 (p, J = 5.9 Hz, 2H). 13C NMR
and concentrated under vacuum to generate compounds 14–16,
which were used for the next step without purification.
4.4. General protocol for the preparation of 17–19
Compounds 14–16 (2.4 mmol) were added to a magnetically-
stirred suspension of berberrubine (5; 2 mmol) in CH3CN (15 ml)
and the mixture was stirred in a reflux apparatus for 12–24 h.
The mixture was cooled to room temperature, filtered, and then
evaporated under vacuum. The crude product was chromato-
graphed on an Al2O3 column and eluted with CHCl3/MeOH
(100:1–50:1) to obtain the purified compound.
(101 MHz, MeOD)
d 159.43, 158.74, 150.72, 150.20, 147.28,
146.59, 143.12, 139.90, 134.04, 133.16, 132.01, 126.17, 124.95,
124.05, 122.35, 122.07, 109.21, 107.34, 105.94, 104.25, 102.67,
101.03, 72.53, 69.29, 58.47, 56.88, 29.91, 26.73. HRMS m/z [MꢀBr]+
Calcd for C28H25NO6 472.1760, found 472.1779.
4.4.1. 9-O-[(3-Oxo-tryptamino)propyl]-berberine bromide (17)
Berberrubine was treated with compound 14 according to a
general protocol to obtain compound 17 as a yellow solid (27%
yield). mp 156.4–158.7 °C; 1H NMR (400 MHz, MeOD) d 9.44 (s,
1H), 8.62 (s, 1H), 8.09 (s, 1H), 8.02 (s, 1H), 7.63 (s, 1H), 7.55 (dt,
J = 6.7, 2.7 Hz, 1H), 7.08 (d, J = 1.8 Hz, 2H), 6.98–6.87 (m, 3H),
6.15 (s, 2H), 4.64–4.54 (m, 4H), 4.12 (s, 3H), 3.64 (t, J = 6.8 Hz,
2H), 3.18–3.09 (m, 2H), 2.99 (t, J = 6.8 Hz, 2H), 2.81 (t, J = 5.6 Hz,
2H). 13C NMR (101 MHz, MeOD) d 169.55, 160.75, 158.57, 152.44,
149.35, 146.73, 146.11, 141.56, 136.44, 132.59, 131.56, 127.30,
124.01, 122.27, 122.18, 121.46, 119.56, 118.69, 117.09, 112.80,
111.30, 109.22, 104.22, 100.87,100.57, 73.26, 59.80, 57.94, 57.20,
39.91, 39.76, 27.53. HRMS m/z [MꢀBr]+ Calcd for C32H29N3O5
536.2185, found 536.2171.
4.2.5. 9-O-[4-(Phenylol-3-yloxy)butyl] berberine bromide (11)
Compound 6c was treated with 1,3-benzenediol according to
general protocol to obtain compound 11 as a yellow solid (42%
yield). mp 205.1–207.3 °C; 1H NMR (400 MHz, MeOD) d 9.53 (s,
1H), 8.56 (s, 1H), 7.98 (d, J = 9.1 Hz, 1H), 7.88 (s, 1H), 7.53 (s, 1H),
6.83 (s, 1H), 6.62 (d, J = 9.1 Hz, 2H), 6.56 (d, J = 9.1 Hz, 2H), 5.99
(s, 2H), 4.79–4.74 (m,2H), 4.39 (s, 2H), 3.97 (s, 3H), 3.93 (t,
J = 6.0 Hz, 2H), 3.14–3.08 (m, 2H), 2.03–1.95 (m, 2H), 1.91 (dd,
J = 7.5, 5.8 Hz, 2H). 13C NMR (101 MHz, MeOD) d 160.83, 156,57,
151.92, 148.73, 147.17, 146.50, 146.45, 144.73, 141.28, 131.64,
130.88, 130.75, 123.84, 120.64, 111.75, 116.79, 110.06, 106.87,
106.72, 103.56, 101.25, 101.06, 72.37, 69.46, 57.70, 56.04, 29.11,
27.79, 27.65 HRMS m/z [MꢀBr]+ Calcd for C29H27NO6 486.1917,
found 486.1928.
4.4.2. 9-O-[(3-Oxo-5-methyltryptamino)propyl]-berberine
bromide (18)
4.2.6. 9-O-[3-(Phenylol-4-yloxy)propyl] berberine bromide (12)
Compound 6b was treated with 1,4-benzenediol according to a
general protocol to obtain compound 12 as a yellow solid (55%
yield). mp 218.5–220.8 °C; 1H NMR (400 MHz, MeOD) d 9.52 (s,
1H), 8.68 (s, 1H), 8.10 (d, J = 9.0 Hz, 1H), 8.00 (d, J = 9.0 Hz, 1H),
7.63 (s, 1H), 6.94 (s, 1H), 6.81 (d, J = 8.9 Hz, 2H), 6.73 (d,
J = 8.9 Hz, 2H), 6.61 (s, 1H), 6.11 (s, 2H), 4.60 (dt, J = 11.6, 5.9 Hz,
4H), 4.26 (t, J = 5.7 Hz, 2H), 4.06 (s, 3H), 3.19–3.11 (m, 2H), 2.41–
2.30 (m, 2H). 13C NMR (101 MHz, MeOD) d 160.27, 158.30,
151.27, 151.03, 147.94, 144.08, 143.37, 139.55, 134.52, 130.73,
130.26, 126.50, 123.71, 122.67, 120.55, 120.36, 108.39, 108.27,
105.68, 105.50, 101.90, 101.07, 73.10, 64.95, 57.47, 56.75, 29.14,
28.08. HRMS m/z [MꢀBr]+ Calcd for C28H25NO6 472.1760, found
472.1751.
Berberrubine was treated with compound 15 according to a
general protocol to obtain compound 18 as a yellow solid (23%
yield). mp 150.9–153.2 °C; 1H NMR (400 MHz, MeOD) d 9.33 (s,
1H), 8.50 (s, 1H), 8.02 (s, 1H), 7.95 (s, 1H), 7.54 (s, 1H), 7.27 (s,
1H), 6.99 (s, 1H), 6.96–6.83 (m, 2H), 6.73–6.63 (m, 1H), 6.12 (s,
2H), 4.61–4.47 (m, 4H), 4.09 (s, 3H), 3.60 (t, J = 6.7 Hz, 2H), 3.08
(s, 2H), 2.92 (t, J = 6.6 Hz, 2H), 2.80 (t, J = 5.3 Hz, 2H), 2.26 (s, 3H).
13C NMR (101 MHz, MeOD) d 168.99, 161.27, 157.24, 150.89,
150.03, 147.13, 146.56, 139.34, 135.72, 133.87, 130.15, 128.37,
123.93, 122.35, 121.98, 121.69, 120.27, 119.36, 118.36, 113.57,
110.97, 109.72, 106.48, 103.81,101.04, 72.45, 59.07, 56.98, 40.93,
39.88, 27.52, 25.28, 22.41. HRMS m/z [MꢀBr]+ Calcd for
C33H31N3O5 550.2342, found 550.2338.
4.2.7. 9-O-[4-(Phenylol-4-yloxy)butyl] berberine bromide (13)
Compound 6c was treated with 1,4-benzenediol according to
general protocol to obtain compound 13 as a yellow solid (55%
yield). mp 200.3–202.1 °C; 1H NMR (400 MHz, MeOD) d 9.40 (s,
1H), 8.57 (s, 1H), 8.00 (d, J = 9.1 Hz, 1H), 7.90 (d, J = 9.1 Hz, 1H),
7.53 (s, 1H), 6.99 (t, J = 8.1 Hz, 1H), 6.84 (s, 1H), 6.38–6.27 (m,
3H), 6.01 (s, 2H), 4.50 (dd, J = 13.3, 7.2 Hz, 5H), 4.20 (t, J = 5.8 Hz,
2H), 3.97 (s, 3H), 3.11–3.01 (m, 2H), 2.28 (p, J = 5.9 Hz, 2H). 13C
NMR (101 MHz, MeOD) d 161.68, 159.96, 152.31, 152.18, 149.91,
145.92, 144.68, 139.60, 135.18, 131.80, 131.20, 127.86, 124.69,
123.55, 121.79, 121.61, 109.35, 109.16, 106.71, 106.53, 103.68,
103.00, 72.30, 65.25, 57.58, 57.18, 30.89, 28.22.HRMS m/z [MꢀBr]+
Calcd for C29H27NO6 486.1917, found 486.1932.
4.4.3. 9-O-[(3-Oxo-5-methoxytryptamino)propyl]-berberine
bromide (19)
Berberrubine was treated with compound 16 according to gen-
eral protocol to obtain compound 19 as a yellow solid (33% yield).
mp 189.1–191.3 °C; 1H NMR (400 MHz, DMSO) d 10.60 (s, 1H), 9.80
(s, 1H), 8.91 (s, 1H), 8.29 (t, J = 5.7 Hz, 1H), 8.19 (d, J = 9.2 Hz, 1H),
8.00 (d, J = 9.1 Hz, 1H), 7.78 (s, 1H), 7.24 (d, J = 7.4 Hz, 1H), 7.16 (t,
J = 8.5 Hz, 2H), 7.12–7.06 (m, 2H), 6.99 (d, J = 2.4 Hz, 1H), 6.66 (dd,
J = 8.7, 2.4 Hz, 1H), 6.18 (s, 2H), 4.90–4.78 (m, 2H), 4.48 (t,
J = 6.0 Hz, 2H), 4.06 (s, 3H), 3.72 (s, 3H), 3.25–3.13 (m, 3H), 2.78
(t, J = 7.3 Hz, 2H), 2.71 (t, J = 6.0 Hz, 2H). 13C NMR (101 MHz,
DMSO) d 165.53, 154.06, 151.17, 148.33, 147.15, 146.99, 146.30,
145.74, 141.48, 131.60, 131.72, 131.63, 127.55, 123.21, 123.0
121.54, 117.82, 116.20, 112.44, 112.19, 112.00, 109.47, 103.59,
101.26, 100.45, 71.59, 59.23, 57.83, 55.98, 40.32, 38.87, 29.30,
28.59. HRMS m/z [MꢀBr]+ Calcd for C33H31N3O6 566.2291, found
566.2319.
4.3. General protocol for the preparation of compounds 14–16
To a solution of substituted tryptamine (10 mmol) in chloro-
form (re-distilled, 50 ml), b-bromopropionyl chloride (11 mmol)
was slowly added at 0 °C, followed by pyridine (11 mmol). After
stirring 1 h at room temperature, the reaction mixture was washed
with NaHCO3 solution (2 ꢁ 50 ml), water (2 ꢁ 50 ml), and brine
(1 ꢁ 50 ml), consecutively, then dried with anhydrous Na2SO4
4.4.4. (E)-3-Bromopropyl 3-(4-hydroxy-3-methoxyphenyl)
acrylate (20)
1,3-Dibromopropane (4.0 mmol) was added to a magnetically-
stirred suspension of ferulic acid (2 mmol) in acetone (30 ml) in

7234
H. Jiang et al. / Bioorg. Med. Chem. 19 (2011) 7228–7235
the presence of Et3N and the mixture was stirred in a reflux appa-
ratus for 4 h. Then the mixture was cooled to room temperature
and evaporated under vacuum. The crude product was chromato-
graphed on a silica gel column to obtain compound 20 as an oil
(82% yield). 1H NMR (400 MHz, CDCl3) d 7.62 (d, J = 15.9 Hz, 1H),
7.08 (dd, J = 8.2, 1.9 Hz, 1H), 7.03 (d, J = 1.8 Hz, 1H), 6.92 (d,
J = 8.2 Hz, 1H), 6.28 (d, J = 15.9 Hz, 1H), 5.90 (s, 1H), 4.34 (t,
J = 6.0 Hz, 2H), 3.93 (s, 3H), 3.52 (t, J = 6.6 Hz, 2H), 2.33–2.18 (m,
2H).
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.
4.5.3. Inhibition of Ab1–42 peptide aggregation
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 con-
centration 50 M) with and without the tested compound at
20 M. After incubation, the samples were diluted to a final vol-
ume of 200 L with 50 mM glycine-NaOH buffer (pH 8.0) contain-
lM). The peptide was incubated in
4.4.5. 9-O-[3-(Ferulic acid)propyl] berberine bromide (21)
Compound 20 (2.4 mmol) was added to a magnetically-stirred
suspension of berberrubine (5; 2 mmol) in CH3CN (15 ml) and the
mixture was stirred in a reflux apparatus for 12 h. The mixture
was cooled to room temperature, filtered, and then evaporated un-
der vacuum. The crude product was chromatographed on an Al2O3
column and eluted with CHCl3/MeOH (100:1–50:1) to generate
compound 21 as a yellow solid (43% yield). mp 202.4–203.7 °C; 1H
NMR (400 MHz, DMSO) d 9.82 (s, 1H), 8.93 (s, 1H), 8.20 (d,
J = 9.2 Hz, 1H), 8.01 (d, J = 9.1 Hz, 1H), 7.78 (s, 1H), 7.56
(d, J = 15.9 Hz, 1H), 7.30 (d, J = 1.5 Hz, 1H), 7.09 (s, 2H), 6.81 (d,
J = 8.1 Hz, 1H), 6.48 (d, J = 15.9 Hz, 1H), 6.18 (s, 2H), 5.02–4.89 (m,
2H), 4.43 (dd, J = 11.5, 6.0 Hz, 4H), 4.05 (s, 3H), 3.81 (s, 3H), 3.21 (s,
2H), 2.27 (s, 2H). 13C NMR (101 MHz, DMSO) d 166.71, 150.27,
149.83, 149.27, 147.87, 147.68, 145.25, 145.13, 142.63, 137.50,
133.05, 130.63, 126.74, 125.49, 123.44, 123.05, 121.56, 120.39,
120.19, 115.45, 114.29, 111.33, 108.37, 105.40, 102.04, 71.08,
60.76, 57.07, 55.71, 55.30, 29.13, 26.32. HRMS m/z [MꢀBr]+ Calcd
for C32H29NO8 556.1971, found 556.1977.
l
l
l
ing thioflavin T. Then, a 300-seconds-time scan of fluorescence
intensity was performed (kexc = 450 nm; kem = 485 nm), and values
at plateau were averaged after subtracting the background fluores-
cence of thioflavin T solution.
4.5.4. The antioxidant activity assay
The antioxidant activity was determined by the oxygen radical
absorbance capacity-fluorescein (ORAC-FL) assay.30,31,34 All the as-
says were under 75 mM phosphate buffer (pH 7.4) and the final
reaction mixture was 200
(120 L, 300 nM final concentration) 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 L; 12 mM final con-
lL. Antioxidant (20 lL) and fluorescein
l
l
centration) was added rapidly. The plate was immediately placed
into a Spectrafluor Plus plate reader (Tecan, Crailsheim, Germany)
and the fluorescence was measured every 60 s for 4 h with exita-
tion at 485 nm and emission at 535 nm. Trolox was used as stan-
4.5. Biological activity
dard (1–10 lM, final concentration). A blank (FL+AAPH) using
phosphate buffer instead of antioxidant and Trolox calibration
were carried out in each assay. The samples were measured at dif-
ferent concentrations (0.5–10 lM). All reaction mixtures were pre-
pared fourfold and at least four independent runs were performed
for each sample. Fluorescence measurements were normalized to
the curve of the blank (without antioxidant). The ORAC-FL values
were calculated as described in the reference34 and the final results
4.5.1. In vitro inhibition of AChE and BuChE
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from the electric eel),
butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum),
5,50-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 and then diluted in 0.1 M KH2PO4/K2HPO4 buf-
fer (pH 8.0) to the final concentration.
were in
lM of Trolox equivalent/lM of pure compound.
All the in vitro AChE assays were carried out in 0.1 M KH2PO4/
K2HPO4 buffer, pH 8.0, using a Shimadzu UV-2450 Spectrophotom-
eter. AChE and BuChE solutions were prepared to give 2.0 units/ml
in 2 ml aliquots. The assay medium (1 ml) consisted of phosphate
Acknowledgment
We thank the Natural Science Foundation of China (20972198)
for financial support of this study.
buffer (pH 8.0), 50 ll of 0.01 M DTNB, 10 ll of enzyme, and 50 ll
of 0.01 M substrate (ACh chloride solution). Test compounds were
added to the assay solution and preincubated at 37 °C with the en-
zyme for 15 min, followed by the addition of substrate. 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 Ellman et al.27 Each concentration
was assayed in triplicate.
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