Design, synthesis and evaluation of novel tacrine-rhein hybrids as multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1039/c3ob42010h

A series of tacrine-rhein hybrid compounds have been designed and synthesized as novel multifunctional potent ChE inhibitors. Most of the compounds inhibited ChEs in the nanomolar range in vitro effectively. Compound 10b was one of the most potent inhibitors and was 5-fold more active than tacrine toward AChE, and it also showed a moderate BuChE inhibition with an IC₅₀ value of 200 nM. Kinetic and molecular modeling studies of 10b also indicated that it was a mixed-type inhibitor binding simultaneously to the active and peripheral sites of AChE. In inhibition of the AChE-induced Aβ aggregation assay, compound 10b (70.2% at 100 μM) showed the greatest inhibitory activity. In addition, 10b showed metal-chelating property and low hepatotoxicity. These results suggested that 10b might be an excellent multifunctional agent for AD treatment.

Full text of DOI:10.1039/c3ob42010h

Organic &
Biomolecular Chemistry
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PAPER
Design, synthesis and evaluation of novel
tacrine–rhein hybrids as multifunctional agents for
the treatment of Alzheimer’s disease†
Cite this: Org. Biomol. Chem., 2014,
2, 801
1
Su-Yi Li, Neng Jiang, Sai-Sai Xie, Kelvin D. G. Wang, Xiao-Bing Wang* and
Ling-Yi Kong*
A series of tacrine–rhein hybrid compounds have been designed and synthesized as novel multifunctional
potent ChE inhibitors. Most of the compounds inhibited ChEs in the nanomolar range in vitro effectively.
Compound 10b was one of the most potent inhibitors and was 5-fold more active than tacrine toward
AChE, and it also showed a moderate BuChE inhibition with an IC50 value of 200 nM. Kinetic and mole-
cular modeling studies of 10b also indicated that it was a mixed-type inhibitor binding simultaneously to
the active and peripheral sites of AChE. In inhibition of the AChE-induced Aβ aggregation assay,
compound 10b (70.2% at 100 μM) showed the greatest inhibitory activity. In addition, 10b showed metal-
chelating property and low hepatotoxicity. These results suggested that 10b might be an excellent
multifunctional agent for AD treatment.
Received 6th October 2013,
Accepted 16th November 2013
DOI: 10.1039/c3ob42010h
www.rsc.org/obc
(
CAS) at the bottom and the peripheral anionic site (PAS) near
1
. Introduction
1
0,11
the entrance of the gorge.
Biochemical studies have indi-
Alzheimer’s disease (AD), the fourth leading cause of death in cated that AChE promotes amyloid fibril formation by inter-
people over 65 years old, affects more than 24 million people action through the PAS of the enzyme, giving stable AChE-Aβ
1
12
worldwide. Although 100 years have passed since its discov- complexes, which are more toxic than single Aβ peptides.
ery, treating AD remains a challenge for the pharmaceutical Therefore, dual-site inhibitors that interact with both CAS and
2
community. Over the past few decades, a plethora of targets PAS appear to be a very promising therapeutic strategy, since
have been suggested in the attempt to identify the causative they can simultaneously improve cognition and slow the rate
1
3
factors of AD. These included deposits of aberrant proteins, of Aβ-elicited neurodegeneration. In healthy brains, AChE
namely, β-amyloid (Aβ) and τ-protein, oxidative stress, dys- hydrolyzes about 80% of acetylcholine while BuChE plays a
homeostasis of biometals, and low levels of acetylcholine secondary role. However, as AD progresses, the activity of AChE
3
–5
(ACh).
is greatly reduced in specific brain regions while BuChE
The current therapeutic options for the treatment of AD are activity increases, likely as a compensation for the AChE
1
4
acetylcholinesterase inhibitors (AChEIs) which are based on decrease. Consequently, both enzymes are useful therapeutic
6
,7
the cholinergic hypothesis.
The cholinergic hypothesis of targets for AD.
Many studies have found that overproduction of Aβ also
AD asserts that a decline of ACh levels leads to cognitive and
memory deficits, so sustaining or recovering cholinergic func- plays an important role in the pathogenesis and development
8
tion is supposed to be clinically beneficial. ACh can be of AD, as it leads to synaptic dysfunction, formation of intra-
1
5
degraded by two types of cholinesterases, namely acetylcholin- neuronal fibrillary tangles, and eventually neuron loss. The
9
esterase (AChE) and butyrylcholinesterase (BuChE). The crys- deposited Aβ can cause the shrinkage of neuritis and denatur-
tallographic structure of AChE reveals that it has a narrow 20 Å ing of neurons, and it can also disrupt the calcium channels
2
+
gorge, containing two binding sites: the catalytic active site in the cell membrane, enhancing Ca influx and leading to
1
6
the disequilibrium of calcium. Therefore, inhibitors of the
Aβ aggregation may be effective in blocking the progression of
the pathology. Recent studies have indicated that the level of
1
7
State Key Laboratory of Natural Medicines, Department of Natural Medicinal
Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009,
People’s Republic of China. E-mail: cpu_lykong@126.com, xbwang@cpu.edu.cn;
Tel: +86-25-83271405
metal ions, such as iron, zinc, and copper, in the brain of AD
18
patients is 3–7 fold higher than that of healthy individuals.
In in vitro experiments the elevated concentrations of metals
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c3ob42010h
1
9
1
are able to bind to Aβ, thus promoting its aggregation. It has
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Fig. 1 Structures of some known dual-site inhibitors.
2
+
2+
also been found that redox-active ions like Cu and Fe con- continued with our research on various natural products with
tribute to the production of reactive oxygen species and oxi- potential application in the AD field. In this paper, rhein was
2
0,21
dative stress, which are critical for Aβ neurotoxicity.
selected to hybridize with tacrine to design a series of novel
Therefore, modulation of such biometals in the brain has hybrids exhibiting multifunctional activities. We planned to
been proposed as a potential therapeutic strategy for the treat- use tacrine for its inhibition of cholinesterases (ChEs) through
ment of AD.
the CAS and a rhein scaffold for its metal-chelating, hepatopro-
Tacrine, the first drug approved for the treatment of AD, is tective effects, as well as for its potential interaction with the
a potent inhibitor of both AChE and BuChE that suffers from PAS due to its aromatic character. Regarding the possible
2
2
therapy limiting side effects, mainly liver toxicity. Because of structural modification on the tacrine fragment, we focused on
the clinical effectiveness of AChEIs in general and the high the 6-position substitution and changes in carbocyclic ring
potency of tacrine in particular, this structure has been widely size of the tacrine nucleus to study possible effects on ChE
used for application in hybrid or multitarget compounds in inhibition. Based on the structure of AChE, we considered con-
order to obtain potent AChEIs with other pharmacological pro- necting tacrine and rhein fragments by alkylene linkers of
2
3,24
perties.
For example, tacrine–melatonin hybrids and different lengths. In order to find the optimal linker efficiently,
tacrine–ferulic acid hybrids have been designed as potent hybrids with linkers of even-numbered carbon atoms were
2
5
ChEIs with antioxidant properties, tacrine–4-oxo-4H-chro- synthesized.
mene hybrids have been designed as multifunctional agents In this paper, we describe the synthesis, pharmacological
capable of inhibiting ChE and β-secretase and NO-donor– evaluation, and molecular modeling of a series of novel
2
6
9
,27
tacrine hybrids showed hepatoprotective properties
(Fig. 1).
tacrine–rhein hybrids. The pharmacological evaluation of
Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is these novel compounds include AChE and BuChE inhibition,
one of the most important active components of rhubarb the kinetics of enzyme inhibition, AChE-induced Aβ aggrega-
(
Rheum officinale), a traditional Chinese herb to treat chronic tion, metal chelation, and the hepatoprotective effect. Finally,
liver disease, and shows broad-gauged pharmacological molecular modeling studies were performed to gain insight
effects, such as antibacterial, anti-inflammatory and anti- into the binding mode and structure–activity relationships of
2
8,29
tumor activities.
Numerous reports have demonstrated new hybrid compounds.
that rhein has a hepatoprotective effect. Rhein can ameliorate
fatty liver disease, protect hepatocytes from injury and prevent
the progress of hepatic fibrosis in rats, which may be associ-
ated with the role that rhein plays in antioxidation and anti-
inflammation, inhibiting the expression of TGF-beta1 and sup-
2
. Results and discussion
2.1. Chemistry
3
0–33
pressing the activation of hepatic stellate cells.
Recently, it Total synthesis of hybrids of tacrine–rhein congeners was
has been reported that rhein lysinate could decrease the involved in two kinds of key intermediate compounds 3 and
generation of Aβ in the brain tissues of AD model mice by inhi- 8a–n.
3
4
biting the inflammatory response and oxidative stress. These
Preparation of the key intermediate compounds 8a–n is
results suggest that rhein might reduce the liver toxicity shown in Scheme 2. The POCl -mediated cyclodehydration
3
caused by tacrine and be beneficial for AD treatment.
reaction between 4a–b and cycloketones 5a–c was adapted to
Recently, our group has reported the synthesis of tacrine– the corresponding chlorides 6a–f with moderate yields
3
5
flavonoid hybrids as multifunctional ChEIs against AD. We (62–91%). The amination of the chlorides 8a–n was carried out
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2 2 4 2 2
Scheme 1 Synthesis of intermediates 2 and 3. Reagents and conditions: (a) Ac O, H SO , 3 h, reflux; (b) oxalyl chloride, DMF, CH Cl , 1 h, 30 °C.
Scheme 2 Synthesis of intermediates 8a–n. Reagents and conditions: (c) POCl
–12 h.
3 2 2 n 2
, reflux 3 h. (d) 4–5 equiv. NH (CH ) NH , 1-pentanol, reflux
6
3 2 2 3 2
Scheme 3 Synthesis of tacrine–rhein hybrids 10a–n. Reagents and conditions: (e) Et N, CH Cl , 1 h, r.t.; (f) Et N, acetone, H O, 6 h, 50 °C.
3
8
in reaction with 4 equiv. of α,ω-diamine 7a–d in refluxing spectrophotometric method of Ellman et al. For comparison
3
6
1
-pentanol for 8–16 h, followed by removal of the solvent. purposes, tacrine and galanthamine were used as reference
The resulting mixture was diluted with dichloromethane and compounds. The IC50 values of all tested compounds and their
washed with a large amount of water. Finally the organic selectivity index for AChE over BuChE are summarized in
phase was dried with anhydrous Na
and purified by column chromatography to give products 8a–n pounds showed good inhibitory activities to both ChEs with
as a pale-brown oil. IC50 values ranging from sub-micromolar to nanomolar.
2 4
SO , concentrated in vacuo, Table 1. It could be seen from Table 1 that all novel target com-
On the other hand, rhein 1 as the starting material was Among the target compounds, 10f (IC50 = 22.0 nM) showed the
acetylated in the presence of Ac O and H SO to afford diacer- most potent inhibitory activity for AChE, which was 6 and 121
2
2
4
3
7
ein 2, which was chloridated with oxalyl chloride to give 3
Scheme 1). The obtained compound 3 was finally reacted with (IC50 = 135 nM) and galantamine (IC50 = 2670 nM), respect-
a–n in dichloromethane to furnish desired compounds 10a–n ively. In contrast, 10l exhibited the strongest inhibition to
times stronger than those of the reference compounds tacrine
(
8
in moderate yields (Scheme 3).
BuChE with an IC50 value of 11.0 nM, which was 4- and 1154-
fold more potent than those of tacrine (IC50 = 45.0 nM) and
galanthamine (IC50 = 12 700 nM). From the IC50 values of com-
2
.2. Cholinesterase inhibitory activity
The inhibitory activity of hybrids 10a–n and the related com- pounds 10a–d, it appeared that a suitable tether for the linker
pound 1 against AChE (from the electric eel) and BuChE between the two anchoring groups, tacrine and rhein, seemed
(
from equine serum) was measured according to the to be 6 carbons. It also showed that the variation of chain
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Table 1 Inhibition of AChE and BuChE activities, selectivity index, AChE-induced Aβ aggregation by the synthesized compounds
IC50 (nM)
for AChE
IC50 (nM)
for BuChE
Selectivity
index
% Inhibition of
Aβ(1–40) aggregation
a
b
c
e
Compound
m
n
R
1
>100 000
58.7 ± 4.3
27.3 ± 2.9
148 ± 7
220 ± 12
40.6 ± 3.7
22.0 ± 1.5
966 ± 24
543 ± 16
453 ± 21
513 ± 14
173 ± 16
130 ± 8
>100 000
286 ± 9
200 ± 7
—
10.5 ± 0.8
66.5 ± 1.2
70.2 ± 0.8
63.3 ± 2.4
64.1 ± 1.9
54.7 ± 0.7
56.5 ± 1.6
46.8 ± 1.9
48.1 ± 2.0
41.9 ± 0.8
42.0 ± 1.7
61.2 ± 2.4
64.8 ± 1.8
58.4 ± 1.6
55.9 ± 1.4
6.5 ± 0.8
nd
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
0g
0h
0i
0j
0k
0l
0m
0n
2
2
2
2
2
2
1
1
1
1
3
3
3
3
4
6
8
H
H
H
H
Cl
Cl
H
H
Cl
Cl
H
H
Cl
Cl
4.9
7.3
2.4
1.4
30.4
35.1
0.4
0.4
19.4
1.5
0.5
0.1
5.2
6.6
0.3
4.8
360 ± 14
320 ± 16
1234 ± 45
773 ± 28
432 ± 19
219 ± 16
8816 ± 86
769 ± 23
89.5 ± 6.5
11.0 ± 0.7
1112 ± 56
419 ± 22
45 ± 3
10
4
6
4
6
4
6
4
6
4
6
114 ± 4
63.8 ± 7.5
135 ± 8
Tacrine
Galantamine
Propidium
2670 ± 150
12 700 ± 205
nd
d
nd
82 ± 2
a
b
Inhibitor concentration (mean ± SEM of three experiments) required for 50% inactivation of AChE. Inhibitor concentration (mean ± SEM of
c
d
e
three experiments) required for 50% inactivation of BuChE. Selectivity index = IC50 (BuChE)/IC50 (AChE). Not determined. Inhibition of
AChE-induced Aβ(1–40) aggregation, the thioflavin-T fluorescence method was used, the mean ± SEM of three independent experiments and the
measurements were carried out in the presence of 100 μM compounds.
length for the inhibitors had more influence on their inhibition resulting from the substrate-velocity curves for AChE. For
of AChE than BuChE. This may be due to the conformational AChE, the plot showed both increased slopes (decreased Vmax
,
difference between these two enzymes. By contrast, BuChE does from 0.40 to 0.15) and intercepts (decreased K , from 0.28 to
m
not have a functional peripheral site, and the active site of 0.15) with increasing concentration of the inhibitor (Fig. 2).
BuChE is wider than that of AChE. Therefore, BuChE has less This pattern indicated a mixed-type inhibition and therefore
restriction to inhibitors with varying linker length.
revealed that compound 10b might be able to bind to CAS as
Since the IC50 values of 10a (n = 4, IC50 = 58.7 nM) and 10b well as PAS of AChE. In contrast, a different plot for BuChE
n = 6, IC50 = 27.3 nM) for AChE inhibition were in the nano-
(
molar range, we selected n = 4 and 6 for further study. It is
already known that the chlorine atom at the 6-position
3
9
increases the inhibitory potency of tacrine. A chlorine atom
was introduced to the 6-position of the tacrine moiety producing
1
0e–f that resulted in a slight increase in AChE inhibition, but
a significant decrease in BuChE inhibition. In comparison
with 10a–b, carbocyclic-shrinked congeners 10g–h resulted in
almost 20-fold less potency at AChE and a slight decrease in
BuChE inhibition, respectively. In contrast, ring-expanded
1
0k–l increased the potency against BuChE activity signifi-
cantly (almost 3-fold for 10k and 20-fold for 10l), although
there was a slight decrease against AChE activity. These results
disclosed that BuChE seems to be better able than AChE to
4
0,41
accommodate steric bulk around the catalytic site.
The
presence of a chlorine atom in the ring-expanded 10k–l and
ring-shrinked 10g–h resulted in a slight increase in AChE inhi-
bition and a significant decrease in BuChE inhibition.
2
.3. Kinetic study of ChE inhibition
To gain information on the mechanism of inhibition, the
potent inhibitor 10b was selected for kinetic studies. The type
of inhibition was elucidated from the analysis of Lineweaver–
Burk plots, which were reciprocal rates versus reciprocal sub-
strate concentrations for the different inhibitor concentrations
Fig. 2 Lineweaver–Burk plots resulting from the subvelocity curve of
AChE activity with different substrate concentrations (0.05–0.50 mM) in
the absence and presence of 13.6, 27.3, and 54.6 nM 10b. The Vmax and
K
m
values shown in this table are the mean ± SD of three experiments.
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designed compounds could interact with the PAS of AChE. To
further explore the dual action of these compounds, AChE-
induced Aβ(1–40) aggregation inhibitory activity was examined
4
1,42
using the ThT-based fluorometric assay.
Table 1 summar-
izes the anti-Aβ aggregation activity of the novel hybrids and
reference compounds. From the results, it could be seen that
all the synthesized compounds presented a good inhibitory
activity on AChE-induced Aβ aggregation (from 41.9 to 70.2%
at 100 μM). The results indicated that 10b (70.2% at 100 μM)
was the most potent inhibitor of AChE-induced Aβ aggregation,
which was 11-fold more potent than tacrine and relatively
weaker than the positive control propidium. From the inhi-
bition values of compounds 10a–d, it appeared that the linker
length did not play a role in determining the inhibition of
AChE-induced Aβ aggregation, since no significant change of
the percentages of inhibition was observed along with the
lengthening of the spacer length. Hybrids which consist of a
unit of tacrine 10a–d are clearly more potent than other
hybrids 10g–h and 10k–l. The presence of a chlorine atom at
position 6 of the tacrine unit leads to a slightly decreased effect.
In order to study the correlation between the AChE-induced
Aβ aggregation inhibitory activity and AChE inhibitory potency,
a scatter plot of in vitro inhibitory percent of AChE-induced Aβ
aggregation versus IC50 value for AChE inhibition was obtained
and is shown in Fig. 5. Utilizing a linear fitting procedure, a
statistically significant fit was obtained and it was clearly
shown that the inhibitory effects for Aβ aggregation and AChE
were positively correlated. These results appear to validate that
the compound which binds both CAS and PAS of AChE simul-
taneously could strongly inhibit Aβ aggregation mediated by
the enzyme.
Fig. 3 Lineweaver–Burk plots resulting from the subvelocity curve of
BuChE activity with different substrate concentrations (0.05–0.50 mM)
in the absence and presence of 100.0, 200.0, and 400.0 nM 10b. The
V
max and K
m
values shown in this table are the mean ± SD of three
experiments.
was obtained, showing different Km (from 0.14 to 0.49) and
constant Vmax in different inhibitor concentrations (Fig. 3).
This suggested a competitive inhibition, revealing that these
compounds compete for the same binding site as the substrate
acetylcholine.
It may be observed that the inhibitor concentration in the
aggregation assay (100 μM) is much higher than those necess-
ary to inhibit AChE (in the sub-micromolar and nanomolar).
2
.4. Molecular modeling studies
To further study the interaction mode of compound 10b for
AChE, a molecular docking study was performed using the
software package MOE 2008.10. The X-ray crystal structure of
the TcAChE complex with bis(7)-tacrine (PDB ID 2CKM) was
applied to build the starting model of AChE. As shown in
Fig. 4, the tacrine moiety of 10b was bound to the CAS of
AChE, it being stacked against the phenyl ring of Phe 330 and
the indole ring of Trp 84 with the ring-to-ring distance of 3.55
and 4.03 Å, respectively. The rhein moiety interacted with the
indole ring of Trp 279 and Tyr70 of PAS via π–π stacking inter-
actions with the distance of 3.92 and 3.72 Å, respectively. All
these results clearly indicated that compound 10b could simul-
taneously bind to CAS and PAS binding sites of AChE, thereby
demonstrating the rationality of our molecular design.
4
2
However, as other researchers have pointed out, higher con-
centrations of AChE and Aβ are necessary, since they may
speed up the process of aggregation for us to achieve the
purpose of analysis. So, the inhibitor/AChE concentration
values are in the same magnitude, if the concentration ratio in
both Ellman’s assay for the determination of the AChE inhibi-
tory activity and the thioflavin T-based fluorometric assay for
the determination of the Aβ antiaggregating effect is taken into
4
3
account. In this respect, it seems reasonable that similar
amounts of an inhibitor can carry out simultaneously both the
anticholinesterasic and the antiaggregating actions.
2.6. Metal chelating effect
To further study, the chelating effect of 10b for metals such as
2
.5. Inhibition of AChE-induced Aβ(1–40) aggregation
2+
2+
Cu and Fe in methanol was studied by UV-vis spectrometry
4
4
As is known, AChE directly promotes in vitro the assembly of with wavelength ranging from 200 to 500 nm. In Fig. 6a, UV-
2
+
Aβ peptide into amyloid fibrils, forming a stable AChE–Aβ vis spectra of 10b at increasing Cu concentrations are shown
complex, which could cause neurotoxicity. The concomitant as an example. The increase in absorbance, which could be
inhibition of the PAS of AChE, which is supposedly associated better estimated by an inspection of the differential spectra
2
+
with the aggregation of Aβ, may turn AChEIs into potential (Fig. 6b), indicated that there was an interaction between Cu
disease modifying agents. The kinetic study of AChE inhibition and 10b. A similar behavior was also observed when using
2
+
and molecular modeling studies have demonstrated that the Fe . These observations indicated that 10b could effectively
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Fig. 4 (a) 3D docking model of compound 10b with TcAChE. Atom colors: yellow-carbon atoms of 10b, gray-carbon atoms of residues of TcAChE,
dark blue-nitrogen atoms, red-oxygen atoms. The dashed lines represent the interactions between the protein and the ligand. (b) 2D schematic
diagram of docking model of compound 10b with TcAChE. The figure was prepared using the ligand interactions application in MOE.
2
+
2+
chelate Cu and Fe , and thereby could serve as a metal che- 2.7. Hepatotoxicity studies
lator in treating AD. The ratio of the ligand/metal ion in the
complex was investigated by mixing a fixed amount of the
metal ion with increasing ligand; it was possible to observe
In order to determine whether the designed tacrine–rhein
hybrids had hepatotoxicity in comparison to tacrine, 10b was
selected for the assay with adult mice. After being treated with
that the maximum intensity of difference spectra was reached tacrine and 10b, the heparinized serum of mice was obtained
at a 1 : 1 ratio, which was taken as an indication of the stoichio- at different times, and the levels of aspartate aminotransferase
metry of the complex.
In order to measure the stoichiometry of the complex 10b– (Fig. 8). In comparison to the control, tacrine caused signifi-
(ASAT) and alanine aminotransferase (ALT) were determined
2
+
45
Cu , Job’s method was used by preparing solutions of com- cant hepatotoxicity, as indicated by the increased activity of
pound 10b and CuCl2 so that the sum of concentrations of ASAT and ALT. From the results, it appeared that 10b pos-
both species was constant in all samples, but the proportions sessed higher safety than tacrine.
of both components varied between 0 and 100%. The UV
For morphological studies the liver tissue was stained with
absorbance differences at 250 nm were plotted vs. the mole hematoxylin and eosin (HE). Complete pericentral necrosis
fraction, showing a maximum at 0.53 that revealed a stoichio- and distinct fatty degeneration of the hepatocytes of the sur-
2
+
metry of 1 : 1 for complex 10b–Cu (Fig. 7).
rounding intermediate and periportal zones were seen 30 h
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Fig. 5 Scatter plots representing in vitro inhibitory percent of AChE-
induced Aβ aggregation versus IC50 value for AChE inhibition for a series
of tacrine–rhein hybrids.
2+
Fig. 7 Determination of the stoichiometry of complex 10b–Cu by
Job’s method.
Fig. 8 ALT and ASAT activity after the administration of tacrine and 10b.
Values are expressed as mean ± SEM (n = 8–9; t test, compared to the
control of the same time after administration, *p ≤ 0.05, **p ≤ 0.01).
after administration of tacrine (Fig. 9b). In contrast, only
minor morphological changes were observed after the treat-
ment with compound 10b (Fig. 9c). All these findings
suggested that compound 10b could be safer than tacrine for
the treatment of AD.
Fig. 6 (a) UV-vis (200–500 nm) absorption spectra of 10b (25 μM) in
(2–50 μmol L− ).
1
methanol after addition of ascending amounts of CuCl
2
2+
(
b) The differential spectra due to 10b–Cu
complex formation
obtained by numerical subtraction from the above spectra of those of
2+
Cu and 10b at the corresponding concentrations.
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4.1.1. General procedures for the preparation of inter-
mediates 6a–f
-Chloro-2,3-dihydro-1H-cyclopenta[b]quinoline (6a). General
9
cyclization procedure: o-aminobenzoic acid 4a (3 g, 22 mmol)
and cyclopentanone 5a (1.70 g, 22 mmol) were carefully added
to POCl (15 mL) in an ice bath. The mixture was heated under
3
Fig. 9 Histomorphological appearance of livers of male mice after
treatment with the solvent only (control) (A) or 30 h after administration
of tacrine (B) or 10b (C). HE, original magnification ×200.
reflux for 4 h, then cooled at room temperature, and concen-
trated to give a slurry. The residue was diluted with EtOAc,
neutralized with aqueous K CO , and washed with brine. The
2
3
2 3
organic layer was dried over anhydrous Na CO and concen-
trated in vacuo to furnish a yellow solid. The crude product was
3
. Conclusion
purified by silica gel chromatography with ethyl acetate and
petroleum ether (1 : 10) to afford 6a as a light yellow solid in
In conclusion, a series of tacrine–rhein hybrid compounds
have been designed and synthesized as novel multifunctional
potent ChE inhibitors. Most of the compounds inhibited ChEs
in the nanomolar range in vitro effectively. Compound 10b was
one of the most potent inhibitors and was 5-fold more active
than tacrine toward AChE, and it also showed a moderate
BuChE inhibition with an IC50 value of 200 nM. Kinetic and
molecular modeling studies of 10b indicated that it was a
mixed-type inhibitor binding simultaneously to active and per-
ipheral sites of AChE. In the inhibition of the AChE-induced
Aβ aggregation assay, compound 10b (70.2% at 100 μM)
showed the greatest inhibitory activity. In addition, 10b also
showed metal-chelating property and low hepatotoxicity.
Altogether, the multifunctional effects of the new hybrids
qualified them as potential anti-AD drug candidates, and 10b
might be considered as a promising lead compound for
further research.
41
78.0% yield.
6,9-Dichloro-2,3-dihydro-1H-cyclopenta[b]quinoline (6b). Com-
pound 4b was treated with cyclopentanone 5a according to the
general cyclization procedure to give 6b as a pale solid in
46
73.2% yield.
9-Chloro-1,2,3,4-tetrahydroacridine (6c). Compound 4a was
treated with cyclohexanone 5b according to the general cycliza-
41
tion procedure to give 6c as a pale solid in 81.0% yield.
6,9-Dichloro-1,2,3,4-tetrahydroacridine (6d). Compound 4b
was treated with cyclohexanone 5b according to the general
cyclization procedure to give 6d as a pale solid in 91.0%
4
7
yield.
1-Chloro-7,8,9,10-tetrahydro-6H-cyclohepta[b]quinoline (6e).
1
Compound 4a was treated with cycloheptanone 5c according
to the general cyclization procedure to give 6e as a pale solid
4
1
in 62.0% yield.
3,11-Dichloro-7,8,9,10-tetrahydro-6H-cyclohepta[b]quinoline (6f).
Compound 4b was treated with cycloheptanone 5c according
to the general cyclization procedure to give 6f as a pale solid in
4
8
62.0% yield.
4
. Experimental section
4.1.2. General procedures for the preparation of intermedi-
4
.1. Chemistry
ates 8a–n
1
All chemicals (reagent grade) used were purchased from
N -(1,2,3,4-Tetrahydroacridin-9-yl)butane-1,4-diamine (8a).
Sinopharm Chemical Reagent Co., Ltd (China). The reaction Intermediate 6c (2.0 g, 9.3 mmol), butanediamine 7a (3.0 g,
progress was monitored using analytical thin layer chromato- 37.1 mmol), catalytic amount of KI (0.02 g) and 1-pentanol
graphy (TLC) on precoated silica gel GF254 (Qingdao Haiyang (4 mL) were combined and heated to reflux (160 °C) for 12 h.
Chemical Plant, Qingdao, China) plates and the spots were After cooling to room temperature, the mixture was diluted
detected under UV light (254 nm). The melting point was with CH Cl (30 mL) and then washed with 10% NaOH (30 mL
2
2
measured on an XT-4 micromelting point instrument and × 3) and water (30 mL × 3). The organic layer was dried over
uncorrected. IR (KBr-disc) spectra were recorded using a anhydrous K CO , filtered, concentrated in vacuo, and purified
2
3
1
13
Bruker Tensor 27 spectrometer. H NMR and C NMR spectra by silica gel chromatography with CH Cl –MeOH–Et N
2
2
3
were measured on a Bruker ACF-500 spectrometer at 25 °C and (100 : 20 : 0.5) as an eluent to afford compound 8a as a yellow
9
referenced to TMS. Chemical shifts are reported in ppm (δ) oil in 52.0% yield.
using the residual solvent line as an internal standard. The
1
N -(1,2,3,4-Tetrahydroacridin-9-yl)hexane-1,6-diamine (8b).
splitting patterns are designed as s, singlet; d, doublet; t, Intermediate 6c was treated with hexanediamine 7b according
triplet; m, multiplet. The purity of all compounds was con- to the general procedure like 8a to give the desired product 8b
9
firmed to be higher than 95% through analytical HPLC as a yellow oil in 57.6% yield.
performed with an Agilent 1200 HPLC System. Mass spectra
1
N -(1,2,3,4-Tetrahydroacridin-9-yl)octane-1,8-diamine (8c).
were obtained on a MS Agilent 1100 Series LC/MSD Trap Intermediate 6c was treated with diaminooctane 7c according
mass spectrometer (ESI-MS) and a Mariner ESI-TOF spectro- to the general procedure to give the desired product 8c as a
9
meter (HRESIMS), respectively. Column chromatography yellow oil in 42.3% yield.
was performed on silica gel (90–150 μm; Qingdao Marine
Chemical Inc.).
1
N -(1,2,3,4-Tetrahydroacridin-9-yl)decane-1,10-diamine (8d).
Intermediate 6c was treated with diaminodecane 7d according
808 | Org. Biomol. Chem., 2014, 12, 801–814
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Paper
to the general procedure to give the desired product 8d as a (m, 2H), 2.66–2.57 (m, 2H), 1.92 (dq, J = 11.8, 5.8 Hz, 2H), 1.76
4
7
yellow oil in 54.5% yield.
(dp, J = 16.5, 5.8 Hz, 4H), 1.64 (p, J = 7.8, 7.3 Hz, 2H), 1.50–1.42
1
+
N -(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)butane-1,4-diamine (m, 2H), 1.40–1.30 (m, 4H); ESI-MS m/z: 312.2 [M + H] .
8e). Intermediate 6d was treated with butanediamine 7a
1
(
N -(3-Chloro-7,8,9,10-tetrahydro-6H-cyclohepta[b]quinolin-11-yl)-
according to the general procedure to give the desired product hexane-1,6-diamine (8n). Intermediate 6f was treated with hexane-
4
7
8
e as a yellow oil in 48.5% yield.
diamine 7b according to the general procedure to give the
1
1
N -(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)hexane-1,6-diamine desired product 8n as a yellow oil in 57.6% yield; H NMR
8f). Intermediate 6d was treated with hexanediamine 7b (500 MHz, CD OD) δ 8.08 (d, J = 9.0 Hz, 1H), 7.80 (d, J = 2.2
according to the general procedure to give the desired product Hz, 1H), 7.39 (dd, J = 9.0, 2.2 Hz, 1H), 3.37–3.32 (m, 2H),
(
3
4
7
8
f as a yellow oil in 55.2% yield.
3.15–3.07 (m, 2H), 3.00–2.92 (m, 2H), 2.63–2.54 (m, 2H),
N -(2,3-Dihydro-1H-cyclopenta[b]quinolin-9-yl)butane-1,4-diamine 1.93–1.89 (m, 2H), 1.79–1.71 (m, 4H), 1.68–1.60 (m, 2H), 1.44
8g). Intermediate 6a was treated with butanediamine 7a (p, J = 7.3 Hz, 2H), 1.39–1.28 (m, 4H); ESI-MS m/z: 346.2
1
(
+
according to the general procedure to give the desired product [M + H] .
4
1
8
g as a yellow oil in 53.0% yield.
4.1.3. General procedures for the preparation of com-
1
N -(6-Chloro-2,3-dihydro-1H-cyclopenta[b]quinolin-9-yl)butane- pounds 10a–n. General procedure: to anhydrous dichloro-
,4-diamine (8h). Intermediate 6b was treated with butanedi- methane (25 mL) were added oxalyl chloride (0.42 mL,
1
amine 7a according to the general procedure to give the desired 4.83 mmol) and DMF (0.25 mL). The solution was stirred for
1
product 8h as a yellow oil in 62.4% yield; H NMR (500 MHz, 0.5 h at room temperature and diacerein 2 (1 g, 2.65 mmol)
CD OD) δ 8.03 (d, J = 9.1 Hz, 1H), 7.68 (d, J = 2.2 Hz, 1H), 7.30 was added. The mixture was heated to 30 °C for 1 h. All sol-
3
(
7
(
dd, J = 9.0, 2.2 Hz, 1H), 3.62 (t, J = 7.1 Hz, 2H), 3.23 (t, J = vents were removed under reduced pressure to get acyl chlor-
.2 Hz, 2H), 2.94 (t, J = 7.8 Hz, 2H), 2.67 (t, J = 7.1 Hz, 2H), 2.12 ide 3, which was dissolved in dichloromethane (60 mL). This
p, J = 7.6 Hz, 2H), 1.71–1.64 (m, 2H), 1.61–1.52 (m, 2H); solution was added to 8 (2.65 mmol) and triethylamine
+
ESI-MS m/z: 290.1 [M + H] .
(0.65 mL, 4.62 mmol) in dichloromethane (10 mL) by dripping
1
N -(2,3-Dihydro-1H-cyclopenta[b]quinolin-9-yl)hexane-1,6-diamine at room temperature. When the reaction was complete, it was
8i). Intermediate 6a was treated with hexanediamine 7b diluted with dichloromethane, washed with water, followed by
(
according to the general procedure to give the desired product brine solution. The organic layer was dried over anhydrous
4
9
8
i as a yellow oil in 64.2% yield.
Na SO and concentrated under reduced pressure. The crude
2 4
1
N -(6-Chloro-2,3-dihydro-1H-cyclopenta[b]quinolin-9-yl)hexane- product 9 was dissolved in acetone (30 mL) without purifi-
,6-diamine (8j). Intermediate 6b was treated with hexanedi- cation. Triethylamine (3 mL, 21.35 mmol) and water (2 mL)
1
amine 7b according to the general procedure to give the desired were added. The solution was heated to 50 °C for 6 h, the
1
product 8j as a yellow oil, in 48.5% yield; H NMR (500 MHz, solvent was removed under reduced pressure and the residue
CD
dd, J = 9.1, 2.2 Hz, 1H), 3.61 (t, J = 7.2 Hz, 2H), 3.23 (t, J = 7.2 HCl. The water layer was extracted using dichloromethane and
Hz, 2H), 2.95 (t, J = 7.8 Hz, 2H), 2.66–2.58 (m, 2H), 2.13 (p, J = the organic layer was separated, washed with brine, dried over
3
OD) δ 8.04 (d, J = 9.1 Hz, 1H), 7.69 (d, J = 2.2 Hz, 1H), 7.31 was poured into water, and the pH was adjusted to 6 with 10%
(
7
.5 Hz, 2H), 1.66 (h, J = 6.7, 6.2 Hz, 2H), 1.51–1.44 (m, 2H), Na SO , and concentrated under reduced pressure to give the
2
4
+
1
.45–1.33 (m, 4H); ESI-MS m/z: 318.2 [M + H] .
crude product, which was purified by chromatography
N -(7,8,9,10-Tetrahydro-6H-cyclohepta[b]quinolin-11-yl)butane- (CH Cl –MeOH = 10 : 1) on silica gel to afford 10 as a solid.
1
2
2
1
,4-diamine (8k). Intermediate 6e was treated with butanedi-
4,5-Dihydroxy-9,10-dioxo-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)-
amine 7a according to the general procedure to give the desired amino)butyl)-9,10-dihydroanthracene-2-carboxamide (10a). Inter-
4
1
product 8k as a yellow oil in 60.4% yield.
mediate 9 was treated with 8a according to the general pro-
1
N -(3-Chloro-7,8,9,10-tetrahydro-6H-cyclohepta[b]quinolin-11-yl)- cedure to give the desired product 10a as a red solid in 59.5%
butane-1,4-diamine (8l). Intermediate 6f was treated with butane- yield, m.p. 168–170 °C; IR (KBr) ν 3450.3, 2923.7, 1635.3,
diamine 7a according to the general procedure to give the 1562.7, 1453.4, 1401.5, 1271.1, 1197.5, 1080.2, cm− ; H NMR
1 1
1
desired product 8l as a yellow oil in 68.5% yield; H NMR (500 MHz, CDCl ) δ 8.02 (d, J = 1.7 Hz, 1H), 7.96–7.91 (m, 1H),
3
(
500 MHz, CD OD) δ 8.14 (d, J = 8.8, 1H), 7.86 (d, J = 2.1 Hz, 7.88 (dd, J = 8.3, 1.3 Hz, 1H), 7.83 (dd, J = 7.6, 1.1 Hz, 1H),
3
1
(
2
H), 7.49 (dd, J = 8.8, 2.1 Hz, 1H), 3.40–3.36 (m, 2H), 3.20–3.12 7.76–7.68 (m, 2H), 7.52 (ddd, J = 8.2, 6.7, 1.4 Hz, 1H), 7.33 (td,
m, 2H), 3.07–2.96 (m, 2H), 2.74–2.63 (m, 2H), 1.98–1.95 (m, J = 8.3, 1.3 Hz, 2H), 6.68 (s, 1H), 3.56–3.52 (m, 4H), 3.08–2.99
H), 1.85–1.78 (m, 4H), 1.75–1.66 (m, 2H), 1.61–1.52 (m, 2H); (m, 2H), 2.72 (q, J = 6.0, 4.2 Hz, 2H), 1.92–1.89 (m, 4H),
+
13
ESI-MS m/z: 318.2 [M + H] .
1.82–1.75 (m, 4H). C NMR (125 MHz, CDCl ) δ 192.60,
3
1
N -(7,8,9,10-Tetrahydro-6H-cyclohepta[b]quinolin-11-yl)hexane- 181.11, 165.07, 162.86, 162.76, 150.60, 142.40, 137.63, 134.00,
1
,6-diamine (8m). Intermediate 6e was treated with hexanedi-
1
1
2
33.45, 128.51, 125.06, 123.96, 123.42, 122.61, 120.37, 120.23,
18.97, 117.39, 117.07, 115.82, 48.86, 40.07, 29.71, 29.07,
amine 7b according to the general procedure to give the desired
product 8m as a yellow oil in 57.6% yield; H NMR (500 MHz,
CD OD) δ 8.09 (d, J = 8.5, 1.3 Hz, 1H), 7.83 (dd, J = 8.4, 1.2 Hz,
1
+
7.16, 24.90, 23.03, 22.70; ESI-MS m/z: 536.2 [M + H] ; HRMS:
+
3
calcd for C H N O [M + H] , 536.2180, found 536.2179.
3
2
30 3 5
1
H), 7.58 (ddd, J = 8.3, 6.9, 1.4 Hz, 1H), 7.43 (ddd, J = 8.3, 6.8,
4,5-Dihydroxy-9,10-dioxo-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)-
1
.3 Hz, 1H), 3.36–3.32 (m, 2H), 3.16–3.09 (m, 2H), 3.03–2.94 hexyl)-9,10-dihydroanthracene-2-carboxamide (10b). Intermediate 9
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was treated with 8b according to the general procedure general procedure to give the desired product 10e as a red
to give the desired product 10b as a dark yellow solid in solid in 69.4% yield, m.p. 115–117 °C; IR (KBr) ν 3457.0,
6
1
1
5.0% yield, m.p. 183–185 °C; IR (KBr) ν 3450.3, 2933.8, 2935.6, 1629.2, 1554.6, 1477.3, 1450.4, 1401.1, 1269.6, 1200.7,
625.7, 1546.1, 1474.3, 1384.6, 1272.4, 1210.0, 1162.7, 1157.2, 1082.3 cm− ; H NMR (500 MHz, CDCl
1
1
) δ 7.98 (d, J =
084.2 cm− ; H NMR (500 MHz, CDCl ) δ 8.01 (s, 1H), 7.91 1.7 Hz, 1H), 7.83 (d, J = 9.0 Hz, 1H), 7.81–7.77 (m, 2H),
3
1
1
3
(
(
dd, J = 15.9, 8.5 Hz, 2H), 7.82 (d, J = 7.5 Hz, 1H), 7.78–7.65 7.72–7.65 (m, 2H), 7.31 (dd, J = 8.3, 1.1 Hz, 1H), 7.22 (dd, J =
m, 2H), 7.52 (t, J = 7.7 Hz, 1H), 7.32 (dd, J = 8.5, 5.4 Hz, 2H), 9.0, 2.2 Hz, 1H), 6.78 (t, J = 5.9 Hz, 1H), 5.30 (s, 1H), 4.03 (s,
6
.59–6.48 (m, 1H), 4.08 (s, 1H), 3.52–3.47 (m, 4H), 3.07–3.05 1H), 3.56–3.43 (m, 4H), 3.03–2.94 (m, 2H), 2.70–2.62 (m, 2H),
1
3
(m, 2H), 2.70–2.68 (m, 2H), 1.93–1.90 (m, 4H), 1.72–1.64 (m, 1.92–1.87 (m, 4H), 1.80–1.73 (m, 4H). C NMR (125 MHz,
1
3
4
1
1
1
2
3 3
H), 1.51–1.41 (m, 4H). C NMR (125 MHz, CDCl ) δ 192.73, CDCl ) δ 192.61, 181.11, 165.30, 162.93, 162.78, 159.66, 150.66,
81.26, 165.17, 162.99, 162.88, 154.13, 142.78, 137.76, 134.10, 142.47, 137.74, 134.28, 134.01, 133.48, 130.12, 127.53, 125.16,
33.59, 125.16, 124.22, 124.10, 123.57, 123.18, 120.49, 118.48, 124.64, 124.45, 123.46, 120.45, 118.60, 117.42, 117.23, 116.43,
17.43, 117.30, 115.97, 100.14, 49.17, 40.26, 31.58, 29.86, 115.86, 49.07, 40.15, 34.01, 29.19, 27.20, 24.85, 23.03, 22.71;
+
9.55, 26.69, 26.57, 24.69, 22.97, 22.46; ESI-MS m/z: 562.2 ESI-MS m/z: 570.2 [M + H] ; HRMS: calcd for C H ClN O
3
2
29
3
5
−
−
+
[
M − H] ; HRMS: calcd for C34
found 562.2349.
,5-Dihydroxy-9,10-dioxo-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)- 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide
H
32
N
3
O
5
[M − H] , 562.2347, [M + H] , 570.1790, found 570.1786.
N-(6-((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-
4
amino)octyl)-9,10-dihydroanthracene-2-carboxamide (10c). Inter- (10f). Intermediate 9 was treated with 8f according to the
mediate 9 was treated with 8c according to the general pro- general procedure to give the desired product 10f as a red solid
cedure to give the desired product 10c as a red solid in 56.2% in 58.0% yield, m.p. 153–154 °C; IR (KBr) ν 3448.5, 2931.6,
yield, m.p. 132–133 °C; IR (KBr) ν 3445.8, 2925.6, 1634.2, 1629.3, 1510.1, 1475.5, 1401.3, 1271.3, 1199.5, 1160.0,
474.7, 1399.8, 1272.1, 1201.1, 1080.5 cm−
500 MHz, CDCl
1
;
1
H
NMR 1086.2 cm
) δ 8.01 (d, J = 1.7 Hz, 1H), 7.90 (dd, J = 17.1, 5.5 Hz, 1H), 8.18–8.08 (m, 2H), 7.84 (t, J = 7.9 Hz, 1H),
.5 Hz, 2H), 7.78 (d, J = 7.4 Hz, 1H), 7.73–7.65 (m, 2H), 7.51 7.78–7.72 (m, 2H), 7.65 (d, J = 2.3 Hz, 1H), 7.42 (d, J = 8.3 Hz,
−1
1
1
(
8
6
; H NMR (500 MHz, DMSO-d ) δ 8.84 (t, J =
3
(
(
t, J = 7.5 Hz, 1H), 7.38–7.22 (m, 2H), 6.83–6.68 (m, 1H), 4.11 1H), 7.31 (dd, J = 9.0, 2.3 Hz, 1H), 3.44–3.39 (m, 4H), 2.86 (t,
s, 1H), 3.51–3.44 (m, 4H), 3.06–3.04 (m, 2H), 2.68–2.63 (m, J = 6.1 Hz, 2H), 2.67 (t, J = 5.9 Hz, 2H), 1.82–1.76 (m, 4H),
2
8
1
1
1
2
H), 1.90 (p, J = 3.3 Hz, 4H), 1.69–1.60 (m, 4H), 1.44–1.28 (m, 1.61–1.65 (m, 2H), 1.65–1.60 (m, 2H), 1.36–1.30 (m, 4H).
1
3
13
H). C NMR (125 MHz, CDCl
3
) δ 192.65, 181.20, 165.14,
6
C NMR (125 MHz, DMSO-d ) δ 192.71, 181.25, 165.07,
62.91, 162.83, 151.50, 142.92, 137.69, 134.01, 133.54, 128.93, 162.99, 162.86, 154.18, 142.84, 137.79, 134.15, 133.61, 125.19,
27.88, 125.10, 123.93, 123.54, 123.10, 120.42, 119.75, 117.36, 124.28, 124.17, 123.51, 123.13, 120.36, 118.45, 117.39, 117.28,
15.91, 115.33, 100.12, 49.44, 40.47, 33.43, 31.76, 29.84, 29.52, 115.95, 100.10, 49.19, 40.29, 31.66, 29.94, 29.57, 26.76, 26.65,
−
+
9.18, 26.84, 24.78, 23.07, 22.68; ESI-MS m/z: 590.3 [M − H] ; 24.69, 22.98, 22.47; ESI-MS m/z: 598.2 [M + H] ; HRMS: calcd
−
+
HRMS: calcd for C H N O [M − H] , 590.2674, found for C H ClN O [M + H] , 598.2103, found 598.2101.
36
36
3
5
34 33
3 5
5
90.2677.
N-(4-((2,3-Dihydro-1H-cyclopenta[b]quinolin-9-yl)amino)butyl)-
4
,5-Dihydroxy-9,10-dioxo-N-(10-((1,2,3,4-tetrahydroacridin-9-yl)- 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide
amino)decyl)-9,10-dihydroanthracene-2-carboxamide (10d). Inter- (10g). Intermediate 9 was treated with 8g according to the
mediate 9 was treated with 8d according to the general pro- general procedure to give the desired product 10g as a red
cedure to give the desired product 10d as a dark yellow solid in solid in 59.5% yield, m.p. 228–230 °C; IR (KBr) ν 3452.6,
6
1
1
1
7
7
8.5% yield, m.p. 141–143 °C; IR (KBr) ν 3455.2, 2924.1, 2956.3, 1642.9, 1588.2, 1571.1, 1535.8, 1400.2, 1301.9, 1291.9,
626.2, 1541.5, 1473.3, 1401.1, 1272.1, 1205.4, 1162.6, 1266.5, 1201.2 cm− ; H NMR (500 MHz, DMSO-d ) δ 8.88 (brs,
1
1
6
082.0 cm− ; H NMR (500 MHz, CDCl
1
1
) δ 8.04 (d, J = 1.7 Hz, 1H), 8.23 (d, J = 8.5 Hz, 1H), 8.10 (brs, 1H), 7.78–7.68 (m, 2H),
3
H), 7.96–7.91 (m, 1H), 7.89 (d, J = 8.5 Hz, 1H), 7.81 (dd, J = 7.68–7.66 (m, 1H), 7.57–7.54 (m, 1H), 7.39–7.36 (m, 2H), 6.99
.4, 1.2 Hz, 1H), 7.73 (d, J = 1.7 Hz, 1H), 7.68 (t, J = 7.9 Hz, 1H), (s, 1H), 3.59 (q, J = 6.5 Hz, 2H), 3.19 (t, J = 7.3 Hz, 4H), 2.89 (t,
.57–7.48 (m, 1H), 7.37–7.27 (m, 2H), 6.69–6.59 (m, 1H), 4.01 J = 7.8 Hz, 2H), 2.02 (p, J = 7.6 Hz, 2H), 1.69–1.64 (m, 4H).
1
3
(s, 1H), 3.54–3.41 (m, 4H), 3.04 (t, J = 5.8 Hz, 2H), 2.73–2.64
6
C NMR (125 MHz, DMSO-d ) δ 191.43, 180.24, 169.44,
(
m, 2H), 1.95–1.82 (m, 4H), 1.68–1.60 (m, 4H), 1.41–1.35 (m, 169.31, 164.68, 150.70, 150.37, 147.02, 140.14, 134.96, 134.61,
H), 1.34–1.23 (m, 8H). C NMR (125 MHz, CDCl ) δ 192.74, 134.47, 133.36, 130.74, 129.51, 127.35, 125.68, 124.84, 122.63,
3
1
3
4
1
1
1
2
81.29, 165.07, 162.94, 162.89, 142.92, 137.71, 134.09, 133.59, 121.95, 117.32, 114.43, 45.23, 40.14, 34.83, 29.90, 27.46, 23.23,
−
28.68, 125.13, 123.82, 123.57, 123.04, 120.46, 117.41, 117.33, 21.48; ESI-MS m/z: 556.2 [M − H] ; HRMS: calcd for
−
15.95, 49.58, 40.57, 33.83, 31.87, 29.61, 29.46, 29.39, 29.35,
C
31
H
26
N
3
O
5
[M − H] , 556.1878, found 556.1877.
9.27, 27.02, 26.97, 24.88, 23.17, 22.84; ESI-MS m/z: 618.3
N-(6-((2,3-Dihydro-1H-cyclopenta[b]quinolin-9-yl)amino)hexyl)-
−
−
[
M − H] ; HRMS: calcd for C H N O [M − H] , 618.2987, 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide
3
8
40 3 5
(
10h). Intermediate 9 was treated with 8i according to the
found 618.2984.
N-(4-((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)- general procedure to give the desired product 10h as a red
4
,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide solid in 64.3% yield, m.p. 116–118 °C; IR (KBr) ν 3453.7,
(
10e). Intermediate 9 was treated with 8e according to the 2925.0, 1631.9, 1474.6, 1400.7, 1271.6, 1201.1, 1157.9,
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082.9 cm−
1
;
1
H NMR (500 MHz, DMSO-d
) δ 8.84 (t, J = 10k as a red solid in 57.5% yield, m.p. 209–211 °C; IR (KBr)
1
5
1
6
.6 Hz, 1H), 8.17 (dd, J = 8.5, 1.4 Hz, 1H), 8.09 (d, J = 1.7 Hz, ν 3449.8, 2924.5, 1631.8, 1558.7, 1479.5, 1452.7, 1400.9,
H), 7.80 (t, J = 7.9 Hz, 1H), 7.74–7.69 (m, 2H), 7.66–7.32 1270.8, 1198.2, 1162.8, 1080.7 cm−1
;
1
H NMR (500 MHz,
(
(
(
m, 1H), 7.51–7.48 (m, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.34–7.30 DMSO-d
m, 1H), 6.64 (t, J = 6.1 Hz, 1H), 3.51 (q, J = 6.7 Hz, 2H), 3.29 8.09 (s, 1H), 7.83 (t, J = 7.9 Hz, 1H), 7.77–7.68 (m, 3H),
q, J = 6.6 Hz, 2H), 3.15 (t, J = 7.2 Hz, 2H), 2.85 (t, J = 7.8 Hz, 7.45–7.34 (m, 2H), 5.53 (s, 1H), 3.30–3.26 (m, 4H), 3.03–3.01
6
) δ 8.85 (t, J = 5.6 Hz, 1H), 8.18 (d, J = 9.0 Hz, 1H),
2
H), 2.01 (p, J = 7.6 Hz, 2H), 1.62–1.53 (m, 4H), 1.42–1.33 (m, 2H), 2.92–2.85 (m, 2H), 1.82–1.77 (m, 2H), 1.67–1.55 (m,
1
3
13
(m, 4H). C NMR (125 MHz, DMSO-d ) δ 191.19, 181.08, 8H). C NMR (125 MHz, DMSO-d ) δ 191.41, 181.00, 165.97,
6 6
1
1
1
3
71.83, 167.13, 163.85, 161.50, 161.42, 147.04, 146.74, 141.79, 163.76, 161.34, 161.12, 150.20, 146.54, 141.80, 137.47, 133.47,
37.32, 133.50, 133.28, 128.01, 127.45, 124.49, 123.13, 122.48, 133.27, 132.50, 126.60, 125.10, 124.43, 124.31, 122.96, 122.28,
21.61, 119.21, 118.60, 117.30, 117.27, 116.05, 111.82, 44.02, 120.33, 119.36, 117.49, 117.38, 116.01, 49.35, 36.39, 36.36,
−
3.87, 30.61, 30.56, 28.71, 26.15, 25.78, 22.62, 20.95; ESI-MS 31.22, 27.94, 27.70, 26.97, 26.32; ESI-MS m/z: 548.2 [M − H] ;
−
−
−
m/z: 548.2 [M − H] ; HRMS: calcd for C33
48.2191, found 548.2192.
N-(4-((6-Chloro-2,3-dihydro-1H-cyclopenta[b]quinolin-9-yl)amino)-
H
30
N
3
O
5
[M − H] , HRMS: calcd for C33
H
30
N
3
O
5
[M − H] , 548.2191, found
5
548.2189.
4,5-Dihydroxy-9,10-dioxo-N-(4-((7,8,9,10-tetrahydro-6H-cyclo-
butyl)-4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carbox- hepta[b]quinolin-11-yl)amino)butyl)-9,10-dihydroanthracene-2-
amide (10i). Intermediate 9 was treated with 8h according to carboxamide (10l). Intermediate 9 was treated with 8m accord-
the general procedure to give the desired product 10i as a ing to the general procedure to give the desired product 10l as
yellow solid in 55.0% yield, m.p. 153–155 °C; IR (KBr) ν 3456.3, a red solid in 60.0% yield, m.p. 152–154 °C; IR (KBr) ν 3444.9,
2
1
1
1
1
5
2
7
951.8, 1783.0, 1763.7, 1677.2, 1637.5, 1566.9, 1400.7, 1366.5, 2922.8, 1633.2, 1566.1, 1472.8, 1453.1, 1400.8, 1270.9, 1199.3,
193.6, 1022.3 cm− ; H NMR (500 MHz, CDCl ) δ 8.39 (d, J = 1080.1 cm
1
1
−1
; H NMR (500 MHz, DMSO-d ) δ 8.86 (t, J =
6
1
3
.9 Hz, 1H), 8.16 (dd, J = 7.9, 1.3 Hz, 1H), 7.89 (d, J = 1.8 Hz, 5.6 Hz, 1H), 8.16 (dd, J = 8.4, 1.3 Hz, 1H), 8.10 (d, J = 1.7 Hz,
H), 7.81–7.75 (m, 2H), 7.74–7.70 (m, 1H), 7.41 (dd, J = 8.0, 1H), 7.82 (t, J = 7.9 Hz, 1H), 7.75–7.72 (m, 3H), 7.56–7.53
.3 Hz, 1H), 7.23 (dd, J = 9.1, 2.2 Hz, 1H), 7.09–6.99 (m, 1H), (m, 1H), 7.42–7.39 (m, 2H), 5.42 (t, J = 6.9 Hz, 1H), 3.33–3.24
.05 (s, 1H), 3.61 (q, J = 6.1, 5.6 Hz, 2H), 3.53 (q, J = 6.3 Hz, (m, 4H), 3.10–3.00 (m, 2H), 2.96–2.87 (m, 2H), 1.80 (q, J =
1
3
6
H), 3.13 (t, J = 7.3 Hz, 2H), 2.98 (t, J = 7.8 Hz, 2H), 2.10 (p, J = 6.0 Hz, 2H), 1.71–1.56 (m, 8H). C NMR (125 MHz, DMSO-d )
1
3
.6 Hz, 3H), 1.81–1.70 (m, 4H). C NMR (125 MHz, CDCl3) δ 191.32, 181.06, 164.38, 163.81, 161.41, 161.25, 150.09,
δ 191.47, 180.28, 169.49, 169.36, 164.72, 150.75, 150.40, 145.76, 141.78, 137.40, 133.50, 133.29, 127.98, 127.93, 124.46,
1
1
4
47.04, 140.19, 135.02, 134.65, 134.52, 134.40, 130.77, 129.57, 124.10, 122.76, 122.71, 122.38, 121.75, 119.29, 117.41, 116.05,
27.41, 125.71, 124.88, 122.65, 121.96, 117.37, 114.46, 45.28, 49.44, 36.36, 31.31, 30.35, 27.97, 27.87, 27.05, 26.47, 26.38;
+
0.04, 34.76, 29.85, 27.02, 23.18, 21.18; ESI-MS m/z: 556.2 ESI-MS m/z: 578.3 [M + H] ; HRMS: calcd for C H N O
35
36
3
5
+
+
+
[M + H] ; HRMS: calcd for C31
H
27ClN
3
O
5
[M + H] , 556.1634, [M + H] , 578.2649, found 578.2651.
4,5-Dihydroxy-9,10-dioxo-N-(6-((7,8,9,10-tetrahydro-6H-cyclo-
found 556.1633.
N-(6-((6-Chloro-2,3-dihydro-1H-cyclopenta[b]quinolin-9-yl)amino)- hepta[b]quinolin-11-yl)amino)hexyl)-9,10-dihydroanthracene-2-
hexyl)-4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carbox- carboxamide (10m). Intermediate 9 was treated with 8l accord-
amide (10j). Intermediate 9 was treated with 8j according to ing to the general procedure to give the desired product 10m
the general procedure to give the desired product 10j as a red as a red solid 52.8% yield, m.p. 146–148 °C; IR (KBr) ν 3456.9,
solid in 53.6% yield, m.p. 118–120 °C; IR (KBr) ν 3452.3, 2925.6, 1629.3, 1566.1, 1477.0, 1453.7, 1403.0, 1271.1, 1209.5,
1
1
2
1
5
7
1
6
3
924.1, 1635.7, 1499.6, 1483.5, 1400.7, 1286.8, 1198.3, 1085.0 cm−
;
H NMR (500 MHz, DMSO-d
6
) δ 8.82 (t, J =
080.4 cm−
1
; H NMR (500 MHz, DMSO-d ) δ 8.85 (t, J = 5.6 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 8.09–8.08 (m, 1H),
1
6
.6 Hz, 1H), 8.18 (d, J = 9.0 Hz, 1H), 8.10 (d, J = 1.7 Hz, 1H), 7.82–7.78 (m, 1H), 7.76–7.68 (m, 3H), 7.53 (t, J = 7.5 Hz, 1H),
.82 (t, J = 7.9 Hz, 1H), 7.75–7.72 (m, 2H), 7.60 (d, J = 2.3 Hz, 7.43–7.34 (m, 2H), 5.31 (t, J = 6.6 Hz, 1H), 3.31–3.24 (m, 2H),
H), 7.40 (d, J = 8.4 Hz, 1H), 7.30 (dd, J = 9.0, 2.4 Hz, 1H), 3.22 (q, J = 7.5, 6.8 Hz, 2H), 3.08–2.98 (m, 2H), 2.93–2.85 (m,
.69–6.59 (m, 1H), 3.49 (q, J = 6.8 Hz, 2H), 3.29 (q, J = 6.6 Hz, 2H), 1.83–1.78 (m, 2H), 1.68–1.59 (m, 4H), 1.59–1.56 (m, 2H),
H), 3.14 (t, J = 7.2 Hz, 2H), 2.82 (t, J = 7.8 Hz, 2H), 1.99 (p, 1.56–1.50 (m, 2H), 1.37–1.27 (m, 4H). C NMR (125 MHz,
) δ 191.25, 181.04, 164.49, 163.78, 161.44, 161.33,
1
3
J = 8.8, 8.2 Hz, 2H), 1.63–1.50 (m, 4H), 1.41–1.34 (m, 4H). DMSO-d
6
1
3
C NMR (125 MHz, DMSO-d ) δ 191.43, 181.05, 163.81, 150.07, 145.93, 141.82, 137.35, 133.48, 133.27, 128.14, 127.82,
6
1
1
1
61.35, 161.12, 141.88, 137.46, 134.23, 133.50, 133.31, 132.46, 124.46, 124.02, 122.74, 122.69, 122.42, 121.81, 119.25, 117.37,
26.35, 124.44, 123.88, 123.15, 122.28, 119.35, 117.47, 116.07, 116.03, 49.64, 31.30, 30.37, 28.68, 27.92, 27.06, 26.50, 26.18,
+
12.28, 43.98, 34.05, 30.54, 28.64, 26.10, 25.75, 22.52, 20.72; 26.09; ESI-MS m/z: 584.2 [M
+
H] ; HRMS: calcd for
+
+
ESI-MS m/z: 584.2 [M + H] ; HRMS: calcd for C33
H
31ClN
3
O
5
C
33
H
31ClN
N-(6-((3-Chloro-7,8,9,10-tetrahydro-6H-cyclohepta[b]quinolin-
N-(4-((3-Chloro-7,8,9,10-tetrahydro-6H-cyclohepta[b]quinolin-11-yl)- 11-yl)amino)hexyl)-4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthra-
amino)butyl)-4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene- cene-2-carboxamide (10n). Intermediate 9 was treated with 8n
-carboxamide (10k). Intermediate 9 was treated with 8k according to the general procedure to give the desired product
according to the general procedure to give the desired product 10n as a red solid in 51.5% yield, m.p. 139–141 °C; IR (KBr)
3 5
O [M + H] , 584.1947, found 584.1950.
+
[M + H] , 584.1947, found 584.1945.
2
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ν 3454.9, 2924.5, 1630.5, 1559.1, 1472.9, 1452.6, 1400.8, weighted analysis and Ki was determined as the intercept on
−
1
1
1
268.5, 1200.0, 1111.0 cm
;
H NMR (500 MHz, CDCl3) the negative x-axis. Data analysis was performed with Graph-
δ 8.05–7.98 (m, 1H), 7.91–7.88 (m, 1H), 7.86–7.79 (m, 2H), Pad Prism 4.03 software (GraphPad Software Inc.).
7
6
2
1
.76–7.66 (m, 2H), 7.36–7.29 (m, 2H), 6.45 (s, 1H), 3.49 (q, J =
.7 Hz, 2H), 3.27 (t, J = 7.1 Hz, 2H), 3.17–3.10 (m, 2H),
4
.4. Molecular modeling studies
.91–2.82 (m, 2H), 1.90–1.85 (m, 2H), 1.79–1.75 (m, 2H), Molecular modeling calculations and docking studies were
1
3
.73–1.69 (m, 2H), 1.69–1.64 (m, 4H), 1.50–1.42 (m, 4H).
C
performed using Molecular Operating Environment (MOE)
NMR (125 MHz, CDCl ) δ 192.72, 181.25, 166.54, 165.10, software version 2008.10 (Chemical Computing Group, Mon-
3
1
1
1
2
62.97, 162.88, 149.95, 142.72, 137.75, 135.97, 134.11, 133.57, treal, Canada). The X-ray crystallographic structure of AChE
28.08, 125.58, 125.18, 123.82, 123.53, 122.46, 120.62, 120.49, complexed with bis(7)-tacrine (PDB code 2CKM) was obtained
17.46, 117.18, 115.94, 50.71, 40.37, 32.02, 31.47, 29.84, 29.58, from the Protein Data Bank. All water molecules in PDB files
+
8.31, 27.67, 26.90, 26.84, 26.78; ESI-MS m/z: 612.2 [M + H] ; were removed and hydrogen atoms were subsequently added
+
HRMS: calcd for C35
H
35ClN
3
O
5
[M + H] , 612.2260, found to the protein. The compound 10b was built using the builder
interface of the MOE program and the energy minimized
6
12.2262.
using the MMFF94x force field. Then the 10b was docked into
the active site of the protein by the “Triangle Matcher”
4
.2. In vitro inhibition studies on AChE and BuChE
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from the electric eel), method, which generated poses by aligning the ligand triplet
butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from equine of atoms with the triplet of alpha spheres in cavities of tight
serum), 5,5′-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, atomic packing. The Dock scoring in MOE software was done
DTNB), S-butyrylthiocholine iodide (BTCI), acetylthiocholine using the ASE scoring function and Forcefield was selected as
iodide (ATCI), and tarcine hydrochloride were purchased from the refinement method. The best 10 poses of molecules were
Sigma–Aldrich. The capacity of the test compounds (1 and retained and scored. After docking, the geometry of the result-
1
0a–n) to inhibit AChE and BuChE activities was assessed by ing complex was studied using MOE’s pose viewer utility.
3
8
Ellman’s method. A stock solution of test compounds was
dissolved in a minimum volume of DMSO (1%) and was
4.5. Inhibition of AChE-induced Aβ(1–40) aggregation
diluted using the buffer solution (50 mM Tris-HCl, pH = 8.0, Inhibition of AChE-induced Aβ aggregation was measured
4
1,42
0
1
5
.1 M NaCl, 0.02 M MgCl
2
·6H
2
O). In 96-well plates, 160 μL of using a Thioflavin T (ThT)-binding assay.
Aliquots of 2 μL
−
1
−1
.5 mM DTNB, 50 μL of AChE (0.22 U mL prepared in of Aβ(1–40) (Anaspec Inc.), lyophilized from 2 mg mL HFIP
0 mM Tris-HCl, pH = 8.0, 0.1% w/v bovine serum albumin, (1,1,1,3,3,3-hexafluoro-2-propanol) and dissolved in DMSO,
BSA) or 50 μL of BuChE (0.12 U mL prepared in 50 mM Tris- were incubated for 24 h at room temperature in 0.215 M
HCl, pH = 8.0, 0.1% w/v BSA) were incubated with 10 μL of sodium phosphate buffer (pH 8.0) at a final concentration of
−
1
various concentrations of test compounds (0.001–100 μM) at 230 μM. For co-incubation experiments, aliquots of AChE
7 °C for 6 min followed by the addition of the substrates (16 μL, 0.6 U mL− , final concentration) and AChE in the pres-
1
3
(30 μL) acetylthiocholine iodide (15 mM) or S-butyrylthiocho- ence of the tested compound (2 μL, 100 μM, final concen-
line iodide (15 mM) and the absorbance was measured at tration) were added. Blanks containing Aβ, AChE, Aβ plus the
different time intervals (0, 60, 120, and 180 s) at a wavelength tested compound in 0.215 M sodium phosphate buffer (pH
of 405 nm. The concentration of the compound producing 8.0) were prepared. After incubation, the samples were diluted
5
0% of enzyme activity inhibition (IC ) was calculated by non- to a final volume of 200 μL with 50 mM glycine–NaOH buffer
5
0
linear regression analysis of the response–concentration (log) (pH 8.0) containing thioflavin T (1.5 μM). Then the fluo-
curve, using the Graph-Pad Prism program package (GraphPad rescence intensities were measured on a SpectraMax M5 (Mole-
Software; San Diego, CA). Results are expressed as the mean ± cular Devices, Sunnyvale, CA, USA) multi-mode plate reader
SEM of at least three different experiments performed in with excitation and emission wavelengths at 446 nm and
triplicate.
490 nm, respectively. Each inhibitor was examined in tripli-
cate. The percent inhibition of the AChE-induced aggregation
due to the presence of the inhibitor was calculated by the fol-
4
.3. Kinetic analysis of ChE inhibition
i o i o
To obtain the mechanism of action of 10b, reciprocal plots of lowing formula: 100 − (IF /IF × 100), where IF and IF are the
1
/velocity versus 1/[substrate] were constructed at different con- fluorescence intensities obtained for Aβ plus AChE in the pres-
centrations of the substrate thiocholine iodide (0.05–0.5 mM) ence and in the absence of the inhibitor, respectively, minus
by using Ellman’s method. Three concentrations of 10b were the fluorescent intensities due to the respective blanks.
selected for the studies: 13.6, 27.3 and 54.6 nM for the kinetic
4
.6. Spectrophotometric measurement of the complex with
analysis of AChE inhibition, and 100, 200, 400 nM for the
kinetic analysis of BuChE inhibition, respectively. The plots
2
+
2+
Cu and Fe
were assessed by a weighted least-squares analysis that The study of metal chelation was performed in methanol at
assumed the variance of velocity (v) to be a constant percen- 298 using UV-vis spectrophotometer (SHIMADZU
tage of v for the entire data set. Slopes of these reciprocal plots UV-2450PC) with wavelength ranging from 200 to 500 nm.
were then plotted against the concentration of 10b in a The difference UV-vis spectra due to complex formation were
K
a
4
4
812 | Org. Biomol. Chem., 2014, 12, 801–814
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obtained by numerical subtraction of the spectra of the metal
alone and the compound alone (at the same concentration
used in the mixture) from the spectra of the mixture. A fixed
4 R. T. Bartus, R. L. Dean 3rd, B. Beer and A. S. Lippa,
Science, 1982, 217, 408.
5 D. Pratico, Ann. N. Y. Acad. Sci., 2008, 1147, 70.
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7 M. Villarroya, A. G. Garcia, J. Marco-Contelles and
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A. Samadi, J. Marco-Contelles and M. J. Oset-Gasque,
Chem. Res. Toxicol., 2013, 26, 986.
−
1
amount of 10b (25 μmol L ) was mixed with growing amounts
of copper ions (2–50 μmol L− ) and the difference UV-vis
spectra were tested to investigate the ratio of the ligand/metal
in the complex.
1
2
+
The stoichiometry of the complex 10b–Cu was measured
4
5
by using Job’s method. From separation of hybrid 10b and
CuCl in Tris buffer, both of the same concentration (25 μmol
), 17 solutions were obtained under the condition that the
sum of concentrations of both species was a constant in all
2
−
1
L
9 Y. Chen, J. Sun, L. Fang, M. Liu, S. Peng, H. Liao,
J. Lehmann and Y. Zhang, J. Med. Chem., 2012, 55, 4309.
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2
+
that revealed a stoichiometry of 1 : 1 for complex 10b–Cu .
2003, 22, 1.
1
1
2 A. E. Reyes, M. A. Chacon, M. C. Dinamarca, W. Cerpa,
C. Morgan and N. C. Inestrosa, Am. J. Pathol., 2004, 164,
4
.7. Hepatotoxicity studies
The experiments were carried out on adult male ICR mice
weighing 18–22 g), which were purchased from the Compara-
2
163.
3 A. Castro and A. Martinez, Curr. Pharm. Des., 2006, 12,
377.
(
4
tive Medicine Centre, Yangzhou University. Tacrine hydrochlo-
ride hydrate was dissolved in CMC-Na solution (0.5 g CMC-Na
in 100 mL of distilled water) and 3 mg per 100 g b wt, corres-
ponding to 11.86 μmol per 100 g b wt, were administered id.
Test compounds were dissolved in CMC-Na solution, and an
equimolar dose corresponding to tacrine was administered id.
Heparinized serum was obtained 8, 20, and 30 h after dosing
from the retrobulbar plexus to determine aspartate amino-
transferase (ASAT) and alanine aminotransferase (ALT) activity,
two indicators of a liver damage, using routine methods.
For morphological studies, tacrine and tested compound
treated rats were sacrificed in ether anesthesia 30 h after
dosing, and livers were harvested. Two 3 mm sections of each
liver extending from the hilus to the margin of the left lateral
lobe were immediately placed in 10% buffered formaldehyde,
fixed for two days, and embedded together in one paraffin
block. Subsequently, 5 μm sections were prepared from these
paraffin blocks. Paraffin sections of each block were deparaffi-
nated and stained with hematoxylin and eosin or by means of
the periodic acid–Schiff procedure for glycogen.
1
1
1
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1
7 F. Belluti, M. Bartolini, G. Bottegoni, A. Bisi, A. Cavalli,
V. Andrisano and A. Rampa, Eur. J. Med. Chem., 2011, 46,
1682.
1
1
8 A. I. Bush, J. Alzheimers Dis., 2008, 15, 223.
9 D. G. Smith, R. Cappai and K. J. Brnham, Biochim. Biophys.
Acta, 2007, 1768, 1976.
2
0 X. Huang, R. D. Moir, R. E. Tanzi, A. I. Bush and
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2
1999, 38, 7609.
2
2
2
2 P. B. Watkins, H. J. Zimmerman, M. J. Knapp, S. I. Gracon
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Acknowledgements
1825.
This research work was financially supported by the Innovative 25 M. I. Rodriguez-Franco, M. I. Fernandez-Bachiller, C. Perez,
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