Novel Tacrine-Benzofuran Hybrids as Potent Multitarget-Directed Ligands for the Treatment of Alzheimers Disease: Design, Synthesis, Biological Evaluation, and X-ray Crystallography

Article abstract of DOI:10.1021/acs.jmedchem.5b01119

Twenty-six new tacrine-benzofuran hybrids were designed, synthesized, and evaluated in vitro on key molecular targets for Alzheimers disease. Most hybrids exhibited good inhibitory activities on cholinesterases and β-amyloid self-aggregation. Selected compounds displayed significant inhibition of human β-secretase-1 (hBACE-1). Among the 26 hybrids, 2e showed the most interesting profile as a subnanomolar selective inhibitor of human acetylcholinesterase (hAChE) (IC₅₀ = 0.86 nM) and a good inhibitor of both β-amyloid aggregation (hAChE- and self-induced, 61.3% and 58.4%, respectively) and hBACE-1 activity (IC₅₀ = 1.35 μM). Kinetic studies showed that 2e acted as a slow, tight-binding, mixed-type inhibitor, while X-ray crystallographic studies highlighted the ability of 2e to induce large-scale structural changes in the active-site gorge of Torpedo californica AChE (TcAChE), with significant implications for structure-based drug design. In vivo studies confirmed that 2e significantly ameliorates performances of scopolamine-treated ICR mice. Finally, 2e administration did not exhibit significant hepatotoxicity.

Full text of DOI:10.1021/acs.jmedchem.5b01119

Article
pubs.acs.org/jmc
Novel Tacrine−Benzofuran Hybrids as Potent Multitarget-Directed
Ligands for the Treatment of Alzheimer’s Disease: Design, Synthesis,
Biological Evaluation, and X‑ray Crystallography
Xiaoming Zha,*,†,¶ Doriano Lamba,‡,¶ Lili Zhang,†,§ Yinghan Lou,†,∥ Changxu Xu,†,§ Di Kang,†,∥
Li Chen,§ Yungen Xu,∥ Luyong Zhang,† Angela De Simone,⊥ Sarah Samez,‡,# Alessandro Pesaresi,‡
Jure Stojan,∇ Manuela G. Lopez,○ Javier Egea,○ Vincenza Andrisano,⊥ and Manuela Bartolini*,◆
†State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, P. R. China
‡Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Area Science Park - Basovizza, S.S. no. 14 Km 163.5, I-34149 Trieste,
Italy
∥
§Department of Natural Medicinal Chemistry, Department of Medicinal Chemistry, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, P. R. China
⊥Department for Life Quality Studies, University of Bologna, Corso d’Augusto 237, I-47921 Rimini, Italy
#Dipartimento di Scienze Chimiche e Farmaceutiche, Universita
̀
di Trieste, Via L. Giorgieri 1, I-34127 Trieste, Italy
∇Institute of Biochemistry, Medical Faculty, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia
○Instituto Teofi
lo Hernando, Department of Pharmacology, Universidad Autonoma de Madrid, C/Arzobispo Morcillo 4, 28029
Madrid, Spain
́
́
◆Department of Pharmacy and Biotechnology, University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy
S
* Supporting Information
ABSTRACT: Twenty-six new tacrine−benzofuran hybrids were
designed, synthesized, and evaluated in vitro on key molecular targets
for Alzheimer’s disease. Most hybrids exhibited good inhibitory
activities on cholinesterases and β-amyloid self-aggregation. Selected
compounds displayed significant inhibition of human β-secretase-1
(hBACE-1). Among the 26 hybrids, 2e showed the most interesting
profile as a subnanomolar selective inhibitor of human acetylcholines-
terase (hAChE) (IC50 = 0.86 nM) and a good inhibitor of both β-
amyloid aggregation (hAChE- and self-induced, 61.3% and 58.4%,
respectively) and hBACE-1 activity (IC50 = 1.35 μM). Kinetic studies
showed that 2e acted as a slow, tight-binding, mixed-type inhibitor,
while X-ray crystallographic studies highlighted the ability of 2e to
induce large-scale structural changes in the active-site gorge of Torpedo
californica AChE (TcAChE), with significant implications for structure-
based drug design. In vivo studies confirmed that 2e significantly ameliorates performances of scopolamine-treated ICR mice.
Finally, 2e administration did not exhibit significant hepatotoxicity.
stabilize some symptoms of early to-midstage forms of AD for a
limited period of time. Although several research strategies have
been envisaged in recent decades, increasing cholinergic
neurotransmission by inhibiting the enzyme acetylcholinester-
ase (AChE) still represents one of the mainstream treatment
options for AD. Cholinesterase inhibitors (ChEIs) temporally
restore native levels of the neurotransmitter acetylcholine
(ACh), which compensates for the cholinergic impairment
caused by extensive degeneration of the cholinergic neurons
INTRODUCTION
■
Alzheimer’s disease (AD) is an age-related neurodegenerative
disorder with a known multifactorial etiopathology in which
genetic, environmental, and endogenous factors, including
protein misfolding and aggregation, oxidative stress, mitochon-
drial abnormalities, and neuroinflammation, all play a role. The
impact of this pathology in developed countries with increasing
aging population is tremendous.1 According to the Alzheimer’s
Association, AD is the only one of the top 10 causes of death in
the United States without a means to prevent, cure, or slow its
progression. Notwithstanding the clear need for an effective
therapeutic treatment, the drugs currently on the market merely
Received: July 16, 2015
Published: December 3, 2015
© 2015 American Chemical Society
114
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
and the loss of cholinergic transmission in brain areas.
However, the cholinergic deficit is just one of the hallmarks
of the AD pathology. The complex interconnection of the
molecular events underpinning AD is thought to be one of the
reasons that the single-target directed drugs that have reached
clinical trials have failed. This complexity implies that the
success of a therapeutic approach is likely to depend on the
simultaneous modulation of more than one pathological target,
which has led to a new paradigm in drug discovery for AD,
namely the development of multifunctional molecules as
multitarget direct ligands (MTDLs). MTDLs with two or
more complementary biological activities have been shown to
offer important advances toward effective treatment, and several
research groups have contributed to their development.2−5
MTDLs may be generated by combining fragments endowed
with complementary properties, each fragment synergistically
contributing to the overall activity profile of the resulting
molecule.
One hallmark of AD is the presence in the hippocampus of
senile plaques, which are primarily formed by the extracellular
deposition of β-amyloid peptides (Aβ). Although there is still
debate on the interconnection with other AD hallmarks, Aβ
oligomerization and aggregation are thought to play a key role
in AD pathogenesis and progression. Aβ is a 39−42 amino acid
peptide, which is generated from the amyloid precursor protein
(APP) via sequential cleavage by two enzymes, β-secretase
(also called β-site APP cleaving enzyme, BACE-1) and γ-
secretase. Thus, inhibiting Aβ generation by hindering BACE-1
activity and/or inhibiting Aβ oligomerization and aggregate
accumulation have both been envisaged as suitable strategies for
AD treatment.
To achieve a new family of MTDL hybrid molecules with
anticholinesterase activity, the framework of the well-known
cholinesterase inhibitor tacrine (1,2,3,4-tetrahydroacridine-9-
amine) was selected. Until recently, AChE inhibition has been
mainly associated with alleviation of AD symptoms, but AChE
seems to also be implicated in AD pathogenesis by influencing
Aβ deposition, as demonstrated both in vitro6 and in vivo.7
Furthermore, a relationship between AChE inhibitors
(AChEIs) and the APP processing has been suggested, based
on evidence that AChEIs possibly affect the expression and/or
the metabolic processing of the APP and, consequently, may
influence Aβ generation.8,9 Finally, recent studies have shown
that selective AChE/butyrylcholinesterase (BChE) inhibitors
can reduce Aβ-induced inflammatory processes,10 thus opening
a new route for anticholinesterase inhibitors in AD treatment.
On the basis of the recently discovered role played by AChE,
developing MTDLs with AChE inhibitory activity is therefore
still of interest. However, when designing hybrids containing a
1,2,3,4-tetrahydroacridine-9-amine unit, the issue of hepatotox-
icity must be taken into consideration. Although hepatotoxicity
associated with the administration of tacrine has limited its
clinical use,11 recent findings have shown that the safety can be
improved by synthesizing tacrine derivatives and homo- and
heterodimers.12−20 Thus, provided that the lack of significant
hepatotoxicity is proven, in our opinion, the tethahydroacridine
scaffold still represents a powerful starting point to achieve
multipotent derivatives.
AD progression as AChE levels decline and BChE levels rise.21
More importantly, in vivo experiments showed that brain-
targeted BChE inhibitors not only improved the cognitive
performance of aged rats without the classic adverse effects
associated with AChE inhibition but they also lowered Aβ brain
levels in transgenic mice overexpressing human mutant APP22
and ameliorated the Aβ-induced cognitive dysfunction in
mice.23
Thus, considering the widened biological profile toward
amyloid aggregation, the benzofuran nucleus (and the
modification thereof) was selected as the framework to build
new MTDLs. Some benzofuran derivatives have previously
been shown to act as an AChEI24 or as an inhibitor of Aβ fibril
formation in vitro25,26 and to reduce aggregated Aβ in the
brain.27,28 Furthermore, some 2-arylbenzofuran derivatives29
and benzofuran−chalcone hybrids30 were recently characterized
in vitro as MTDL agents. Finally, an aminostyrylbenzofuran
derivative was shown to reduce amyloid oligomers and
ameliorate the cognitive deficit in a transgenic mouse model
for AD.31
On the basis of these promising new developments, herein
we report on the design, synthesis, and biological evaluation of
26 new benzofuran hybrids (1a−3h). The rationale behind
designing MTDLs is basically to achieve a balance among the
different structural requirements for the optimal interaction
with each selected target. Therefore, in an attempt to obtain a
suitable activity profile against all the selected targets, i.e.,
BACE-1 as well as ChEs and Aβ aggregation, the influence of
an amido vs amino linkage at the benzofuran unit was
investigated [series 1 (1a−1l) and 2 (2a−2f), respectively].
The importance of a functional moiety able to hydrogen bond
with the Asp dyad (Asp32/Asp228) of BACE-1 has been
demonstrated.32 According to recent work by Viayna et al.,33
compounds bearing an aminoquinoline system may establish
electrostatic interactions with the catalytic dyad that could
stabilize the inhibitor−enzyme complex. Furthermore, while
the presence of a properly spaced protonable amino group and
aromatic residues should facilitate the interaction with the
AChE’s peripheral anionic site (PAS), the introduction of an
amido group coupled with aromatic planar moieties may
enhance the binding properties toward BACE-1.34 Finally,
thanks to the synthetic accessibility, series 3 (3a−3h), which
includes hybrids bearing two benzofuran nuclei, was inves-
tigated. Indeed, the presence of larger substituent may improve
the inhibitory activity against Aβ aggregation as well as against
BACE-1. In fact, the relatively large β-secretase active site can
accommodate bulky molecules and represents an additional
challenge for designing potent nonpeptidic inhibitors.34
Characterizing the biological activity includes: (i) evaluating
the in vitro inhibitory activity against human cholinesterase
enzymes, (ii) investigating the mechanism of action on AChE
via in-solution kinetic studies, (iii) assessing the binding mode
of the most active derivative by elucidating the crystal structure
of the inhibitor−AChE complex via X-ray diffraction (XRD),
(iv) assessing the in vivo activity of the most promising
derivative in scopolamine-treated ICR mice, and (v) evaluating
the in vitro ability to interfere with Aβ production and
aggregation by studying the effects on BACE-1 activity (via a
FRET-based assay) and both Aβ1−42 self-aggregation and
AChE-induced Aβ1−40 aggregation (via fluorescence assays).
Finally, to address the critical hepatotoxicity issue related to the
presence of the tacrine unit, the hepatotoxicity of the most
Furthermore, a number of new lines of evidence have
suggested that inhibitors of both cholinesterase enzymes may
have beneficial functions in AD. Indeed, in a healthy human
brain, BChE mainly plays a secondary role in the hydrolysis of
ACh, while in an AD brain, its role becomes predominant with
115
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of Compounds 1a−1l, 2a−2f, and 3a−3h
a
Reagents and conditions: (a) N,N′-carbonyl diimidazole, THF, rt, 4 h; (b) K2CO3, KI, CH2Cl2, rt.
a
Scheme 2. Synthesis of Intermediate 4f
a
Reagents and conditions: (a) POCl3, reflux, 3 h; (b) pentanol, NH2(CH2)7NH2, reflux, 18 h.
interesting derivative was evaluated by monitoring the plasma
levels of two serum biomarkers for acute liver injury.
compounds tacrine and bis(7)-tacrine, a tacrine dimer with
multiple neuroprotective activities, including concurrent
inhibition of cholinesterase enzymes, Aβ cascade, and related
toxicity and the reduction of hydrogen peroxide-induced cell
injury.42−46 The IC50 values of all the compounds tested against
hAChE and hBChE are summarized in Table 1.
The inhibitory activities of the 26 hybrids ranged from good
to very potent against both cholinesterases. The IC50 values for
hAChE inhibition ranged from micromolar to subnanomolar,
from 7.49 μM to 0.86 nM. Specifically, hybrids with the general
structure 1 had inhibitory activities from 24.4 nM to 1.685 μM.
The presence of either a cyclopentane (1a) or cycloheptane
(1c) ring in place of a cyclohexane ring (1b) only slightly
affected the anti-hAChE activity of the hybrids bearing a six-
carbon tether chain. However, the concomitant introduction of
a methoxy substituent on the benzofuran moiety increased the
inhibitory potency by an order of magnitude for hybrids with
either a cycloheptane [X = (CH2)3] or a cyclohexane [X =
(CH2)2] ring (1b vs 1e and 1c vs 1f). This effect was less
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of the hybrids 1a−3h is depicted
in Scheme 1. The amines 4a−4e18,35−37 and 4f were treated
with the acids 5a and 5b38,39 and N,N′-carbonyl diimidazole in
dry THF at rt to yield series 1 (1a−1l). Derivatives 4a−4f were
reacted with the bromides 5c and 5d40 using KI and K2CO3 in
dry CH2Cl2 to yield series 2 (2a−2f) and 3 (3a−3h).
The compound 4f was obtained from anthranilic acid and
cycloheptanone (6). The product 11-chloro-7,8,9,10-tetrahy-
dro-6H-cyclohepta[b]quinoline (7) was treated with 1,7-
diaminoheptane. The procedure is reported in Scheme 2.
Biological Evaluation. AChE and BChE Inhibition. The
inhibitory potency of compounds 1a−1l, 2a−2f, and 3a−3h
was assessed by Ellman’s assay on recombinant human AChE
(hAChE) and BChE from human serum (hBChE).41 The IC50
values were compared with those obtained for the reference
116
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
Table 1. Inhibition of hAChE and hBChE, Selectivity Ratio, and Inhibition of Aβ1-42 Self-Aggregation
hAChE IC (nM)
hBChE IC (nM)
selectivity
inhibition of Aβ1−42 self-aggregation (%
c
50a
50 a
b
compd
X
R
n
SEM
SEM
ratio
SEM)
1a
1b
1c
1d
1e
1f
CH2
H
H
H
6
6
6
6
6
6
7
7
7
7
7
7
6
6
6
7
7
7
6
6
6
6
7
7
7
7
545 35
424 21
308 34
1,685 33
24.4 0.4
32.3 0.9
48.6 2.7
76.9 6.3
323 25
993 72
44.2 3.3
15.7 1.4
4.68 0.19
10.8 0.6
80.6 5.4
10.2 1.0
30.0 1.9
30.9 2.3
6.90 0.25
11.6 0.7
26.1 1.0
7.35 0.40
31.9 1.4
2.48 0.24
1.30 0.14
0.49 0.03
2.54 0.25
2.18 0.25
1.23 0.07
8.89 0.53
2.30 0.14
0.48 0.06
2.35 0.15
12.8 1.5
6.86 0.56
17.9 2.3
4.87 0.20
45.8 3.0
5.66 0.15
0.029
0.011
0.035
0.048
0.418
0.929
0.636
0.090
0.036
0.026
0.166
0.077
0.053
0.021
0.032
0.235
2.535
0.481
0.004
0.0003
0.002
0.002
0.058
0.059
0.090
0.025
0.108
6.988
18.7 4.3
13.7 0.2
24.2 0.8
32.6 9.8
40.2 5.6
18.7 5.4
10.2 4.3
26.2 0.5
17.3 1.7
19.6 1.1
42.5 2.5
17.7 2.5
9.3 1.8
(CH2)2
(CH2)3
CH2
OCH3
OCH3
OCH3
H
(CH2)2
(CH2)3
CH2
1g
1h
1i
(CH2)2
(CH2)3
CH2
H
H
1j
OCH3
OCH3
OCH3
H
1k
1l
(CH2)2
(CH2)3
CH2
416
5
2a
2b
2c
2d
2e
2f
47.0 5.5
63.1 5.6
15.3 1.1
10.8 0.8
0.86 0.05
2.56 0.16
2,070 225
7,495 345
260 65
CH2
OCH3
OCH3
H
64.1 6.5
52.0 4.5
76.2 5.1
61.3 4.5
31.9 11.3
62.2 3.2
38.5 1.3
44.6 2.1
25.0 2.6
30.7 2.4
44.5 3.5
39.8 9.1
37.3 5.8
<5
(CH2)2
CH2
(CH2)2
(CH2)3
CH2
H
OCH3
H
3a
3b
3c
3d
3e
3f
CH2
OCH3
OCH3
OCH3
H
(CH2)2
(CH2)3
CH2
1,036 115
222 23
(CH2)2
(CH2)3
(CH2)3
H
117
200
8
8
3g
3h
H
OCH3
199 13
424 21
0.81 0.09
tacrine
bis(7)-tacrine
50.1 2.3
a
b
Recombinant human AChE and BChE from human serum were used. Data are the mean of at least three independent determinations. Selectivity
c
ratio = IC50 hBChE/IC50 hAChE. Inhibition of Aβ1-42 self-aggregation investigated by the thioflavin-T fluorescence assay. Assays were carried out in
the presence of 10 μM inhibitor and 50 μM Aβ1-42
.
pronounced for hybrids bearing a seven-carbon tether chain.
Conversely, for the cyclopentane analogues [X = CH2], the
introduction of the methoxy group decreased the inhibitory
activity (1a vs 1d and 1g vs 1j). The varying results can
possibly be ascribed to the various positions of the benzofuran
unit at the entrance to the AChE gorge depending on the
structure of the quinoline fragment (see the X-ray crystallog-
raphy section for the binding mode).
Concerning the effect of the spacer chain length on the anti-
AChE activity, the optimal tether was a seven-carbon chain for
the benzofuran hybrids bearing a tacrine unit or a 2,3-dihydro-
1H-cyclopenta[b]quinoline unit. Indeed, by lengthening the
tether chain from six to seven carbon units, the inhibitory
activity against hAChE increased from 5.5- to 11-fold. However,
for the cycloheptane analogues, the length of the tether chain
did not strongly affect the anti-AChE activity (1c vs 1i).
Concerning the methoxybenzofuran hybrids, a slightly different
trend was observed. Indeed, lengthening the tether chain to
seven carbon units was beneficial for hybrids with the 2,3-
dihydro-1H-cyclopenta[b]quinoline unit (the potency in-
creased by a factor of 1.7, 1d vs 1j) but was detrimental for
hybrids bearing a tacrine unit or a 7,8,9,10-tetrahydro-6H-
cyclohepta[b]quinoline unit (the potency decreased by a factor
of 1.8 and 12.9, respectively).
nanomolar−subnanomolar range. Indeed, the presence of an
amino group in the spacer chain may have enabled π−cation
interaction with the PAS, greatly enhancing the anti-AChE
activity (e.g., 2e is 89 times more potent than its amido
analogue 1h). The optimal linker for anti-AChE activity was a
seven-carbon chain. The most potent hybrid (2e) showed a
subnanomolar inhibitory potency and was approximately 493
times more potent than tacrine and equipotent to the reference
compound bis(7)-tacrine. 2e was also the only derivative
slightly selective for hAChE (2.5-fold).
Finally, in series 3, the introduction of the second benzofuran
unit in hybrids 3a−3h decreased the anti-AChE activity by 1−2
orders of magnitude (e.g., 2a vs 3a or 2d vs 3e). Because AChE
would poorly accommodate bulky inhibitors, this detrimental
effect was more evident for hAChE than for hBChE. Indeed,
the volumes of the upper portions of the Torpedo californica
AChE (TcAChE) and hAChE catalytic gorges are 812 and 790
Å3, respectively, while that of hBChE is as high as 1133 Å3. The
unfavorable effect of the bulkier fragment was worse for hybrids
with a six-carbon tether than for hybrids with a seven-carbon
chain (3a vs 3e or 3d vs 3h). Lengthening the tether chain by
one carbon unit reduced the unfavorable effect of the second
benzofuran unit by almost 1 order of magnitude. For hybrids
within series 3, the presence or absence of a methoxy residue
on the benzofuran moiety had only a weak influence on the
inhibitory activity (3a vs 3b, 3g vs 3h).
The introduction of an amino group in the spacer chain in
series 2 lead to some of the most potent hAChE inhibitors
synthesized in this work, with IC50 values in the low
117
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
To properly rank the anticholinesterase activity of these new
hybrids in the scenario of tacrine-based MTDLs, a comparison
can be made with tacrine heterodimers published within the last
two years. Compared with the recently published tacrine
hybrids, 2e is 17.9 times more potent than the best coumarin−
tacrine derivative reported by Hamulakova S. et al. (15.4
nM),47 25.6 times more potent than the rhein−tacrine hybrid
developed by Li et al. (22.0 nM, on AChE from electric eel),35
slightly more potent than rhein−huprine derivatives reported
by Viayna et al. (2.39 nM),33 103 times more potent than the
best phenothiazine−tacrine hybrid (89 nM),48 and similar in
potency to a recently published quinone−tacrine hybrid (0.72
nM).17
deserves a final comment. Considering that the roles played by
AChE and BChE in the modulation of the central cholinergic
tone vary with the progression of the disease, where the role of
BChE progressively increases as AChE levels decrease, it is
conceivable that selective AChEIs are more effective in the early
stages of AD, when AChE still plays a major role. Conversely,
nonselective and BChE-selective inhibitors may be more
effective in moderate to severe forms of AD. Thus, the
complex scenario of this pathology, together with an
incomplete understanding of the “nonclassical” roles played
by each of these two enzymes, makes the development of
centrally active cholinesterase inhibitors selective for either
AChE or BChE a worthy avenue to investigate.
Kinetic Study of Human AChE Inhibition. In the last two
decades, the ability of cholinesterase inhibitors to interact with
the PAS of AChE has attracted attention for the development
of a new disease-modifying drug able to interfere with the pro-
aggregating action of AChE on β-amyloid peptides, possibly
exerted through the interaction of Aβ with PAS.52 Thus, the
ability of this series of hybrids to act as dual binding site AChE
inhibitors was investigated.
However, it is worth mentioning that, in an MTDL
framework, a strong AChE inhibitory activity is only part of
the rationale behind the development of such molecules.
Because of its role in the AD brain, the inhibitory potency
against hBChE was also evaluated and reported in Table 1.
Within the brain, BChE is primarily expressed and secreted by
glial cells49 and it is thought to play a compensatory role in
response to the decrease of AChE activity in the brain as AD
progresses.50 Indeed, inhibition of central BChE activity has
also been investigated as a potential therapeutic approach to
ameliorate the cholinergic deficit in moderate forms of AD.51
All benzofuran hybrids proved to be potent and selective
hBChE inhibitors (except for 2e). The IC50 values against
hBChE ranged from 0.478 to 80.6 nM. The most selective
hBChE inhibitors belonged to series 3 (3a−3d, with selectivity
ratios from 2 to 3 orders of magnitude) and were characterized
by a hexamethylene chain. Removing one of the 7-
methoxybenzofuran fragments reduced the selectivity for
hBChE by 67-fold (2b vs 3b). However, the lower selectivity
of 2b, which lacks the second benzofuran unit, is a consequence
of a higher inhibitory potency against hAChE rather than a
higher anti-hBChE activity, which can be ascribed to lower
steric hindrance. Indeed, because the volume of the upper
portion of the hBChE catalytic gorge is approximately 340 Å3
larger than the corresponding portion of the hAChE gorge,
hBChE should better accommodate the two benzofuran
fragments. Consequently, the introduction of the second
methoxybenzofuran unit either did not affect (e.g., 2c vs 3c)
or only slightly decreased (e.g., 2a vs 3a, 2b vs 3b) the
inhibitory potency against hBChE. As opposed to what was
observed for the inhibition of hAChE, one of the most potent
hBChE inhibitor of series 3 (hybrid 3c) bore a hexamethylene
chain.
Among the hybrids belonging to series 3 and 2, higher
hBChE inhibitory activity was associated with the presence of a
7-methoxy substituent on the benzofuran nucleus (3a vs 3b, 3g
vs 3h and 2a vs 2b). For compounds within series 1 that carried
an amido group at the interconnection between the spacer
chain and the benzofuran fragment, this relationship was lost.
The results also confirmed that both downsizing and enlarging
the cyclohexane nucleus of the tacrine unit negatively affected
the inhibitory potency against hBChE.
The highest hBChE inhibition was achieved with compounds
2c and 3c, whose IC50 values fall in the subnanomolar range
(0.49 and 0.48 nM, respectively). Both compounds were
characterized by the presence of a tacrine unit, a hexamethylene
chain, and two 7-methoxy-benzofuran units, which were
therefore key structural features for optimal hBChE inhibition.
The importance of the selectivity for one of the two
cholinesterase enzymes by the tacrine−benzofuran hybrids
In vitro studies on the isolated enzyme were carried out using
recombinant hAChE and 2e. The mechanism of action was
evaluated by building a Lineweaver−Burk double reciprocal
plot at increasing inhibitor and substrate concentrations.
Overlaid reciprocal Lineweaver−Burk plots showed both higher
slopes (decreased Vmax) and intercepts (higher Km) at
increasing inhibitor concentrations (Figure 1), a trend that is
Figure 1. Kinetic study on the mechanism of hAChE inhibition by 2e.
Overlaid Lineweaver−Burk reciprocal plots of the AChE initial
velocity at increasing substrate (acetylthiocholine, ATCh) concen-
trations (ATCh < 0.56 mM) in the absence and in the presence of 2e
(0−0.76 nM) are shown. Lines were derived from a weighted least-
squares analysis of the data points.
generally ascribed to a mixed-type inhibition. The mixed-type
mechanism of action was also confirmed by a Cornish−Bowden
plot (data not shown). The inhibitor dissociation constants Ki
and the dissociation constant for the enzyme−substrate−
inhibitor complex K′i were estimated to be 0.72 and 1.04 nM,
respectively.
X-ray Crystallography of TcAChE−2e Complex. To
gain insights into the molecular determinants responsible for
the high activity of 2e (Figure 2A), the crystal structure of the
inhibitor-bound TcAChE (Figure 2B) was determined by X-ray
crystallography at 2.8 Å resolution (summary of Crystallo-
118
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
Figure 2. (A) Molecular structure of compound 2e. (B) Close-up view of the active site of TcAChE complexed with 2e. The final 2Fo − Fc σA-
weighted electron density map is contoured at 1.0σ. 2e is rendered as a ball and stick model with carbon, oxygen, and nitrogen atoms colored orange,
red, and blue, respectively. Selected key protein residues (Cα atoms and side chains) in the vicinity of 2e are rendered in stick format and labeled
appropriately. Hydrogen bonding interactions have been omitted for clarity. Part B was created using PyMOL (http://www.pymol.org).
Figure 3. Conformational plasticity of the Trp279−Phe290 loop. (A) Conformation of the Trp279−Phe290 loop and orientation of the Phe330 side
chain in the TcAChE apo structure (PDB ID 1EA5). (B) Conformational adaptation of the Trp279−Phe290 loop and orientation of the Phe330 side
chain upon binding of 2e to TcAChE. Selected key protein residues (Cα atoms and side chains) are rendered in stick format and labeled
appropriately. Created using PyMOL (http://www.pymol.org).
graphic Data of the TcAChE−2e complex in Supporting
Information, Table S1).
the aged phosphonylated TcAChE (PDB ID 2DFP)54 as well as
in the TcAChE−tacrine55 (PDB ID 1ACJ), TcAChE−bis(5)-
tacrine56 (PDB ID 2CMF) and TcAChE−bis(7)-tacrine56
(PDB ID 2CKM) complexes. In the TcAChE−2e complex,
the swinging gate residue Phe330 closely matches one of the
two alternative conformations observed in the TcAChE−
NF595 [(N-(1,2,3,4-tetrahydroacridin-9-yl)-8-[(1,2,3,4-tetrahy-
droacridin-9-yl)thio]octan-1-amine)] (PDB ID 2CEK) com-
plex (χ1 = 76.1°, χ2 = −72.6°).57
The endocyclic nitrogen of 2e is hydrogen bonded to the
main-chain carbonyl oxygen (2.76 Å) of the catalytic residue
His440 (Figure 2B). It can be inferred from this distance and
the pKa of tacrine (9.8)58 that the tacrine moiety of 2e is
protonated. The distance between Ser200-Oγ and His440-Nε2
of 3.4 Å indicates that the catalytic triad is disrupted. Indeed,
Ser200-Oγ is shifted toward the oxyanion hole and interacts
with the main chain nitrogen atoms of the Ala201 (2.8 Å),
Gly118 (3.4 Å), and Gly119 (3.7 Å) residues, which in turn do
not significantly shift from their position in the apo structure.53
A protonated N-alkyl tacrine would be expected to have
Crystallographic studies of AChE have been carried out on
Torpedo californica, mouse, or human homologues. Of the 165
AChE structures deposited in the Protein Data Bank, there are
82 Torpedo californica, 70 mouse, and 13 human AChE
structures. A comparative amino acid sequence analysis
indicates an overall 57% identity and 75% similarity between
TcAChE and hAChE. The 44 residues of the catalytic gorge
share an even higher sequence identity and a similarity of 77%
and 91%, respectively, which validates TcAChE as an accurate
alternative to hAChE for the present structural study of drug
binding.
The position and orientation of 2e with respect to the key
residues in the TcAChE active-site gorge confirmed the critical
role of the tacrine fragment, which binds at the anionic site by
stacking against the aromatic rings of Trp84 and Phe330. The
conformation of Phe330 in the TcAChE−2e complex (χ1 =
92.6°, χ2 = −68.6°) was found to be significantly different than
those observed in the apo TcAChE53 (PDB ID 1EA5) and in
119
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
maximal resonance stabilization if the N-alkyl substituent were
roughly coplanar with the ring system. In the TcAChE−2e
complex, an intermediate dihedral angle of 133.7° is observed
for the tacrine fragment, i.e., 56.3° out of coplanarity. This value
is consistent with those observed in the crystal structures of
complexes with TcAChE.33 Interestingly, in the TcAChE−2e
complex, the exocyclic NH2 group of 2e is not involved in
hydrogen bonding, with the otherwise structurally conserved
water molecules belonging to the active site water network.
The alkyldiamine linker [Cquinolinium-NH-(CH2)7-NH-
CH2benzofuran] between the quinolinium and the methylbenzo-
furan substructures adopts a conformation not previously
reported, namely a (−)anticlinal (−111.7°), (+)synclinal
(39.5°), (+)anticlinal (137.1°), (+)anticlinal (140.9°), (+)-
synclinal (80.7°), (+)anticlinal (111.1°), (+)anticlinal (127.9°),
antiperiplanar (155.2°), and synperiplanar (8.1°) conformation.
Strikingly, this allows a close interaction (2.39 Å) between the
Ser200-Oγ and the distal nitrogen (NWA) of the alkyldiamine
linker (Supporting Information, Figure S1).
In the TcAChE−2e complex, the displacement of the main-
chain atoms of Phe331 (0.4 Å) toward the opening of the gorge
is not significant compared with that observed in the structure
of the apo TcAChE53 and the above-mentioned com-
plexes,54−57 except for the TcAChE−bis(5)-tacrine complex56
(1.0 Å). On the other hand, the position/orientation of the
Phe331 phenyl ring is significantly different from those
observed in the above-mentioned structures.53−57 This has
dramatic consequences on the position/orientation and the
overall conformation of the loop encompassing the residues
from Trp279 to Phe290, which bears residues of both the PAS
and the acyl pocket (Supporting Information, Figures S2A and
S2B).
The reorientation of the Phe331 side chain (χ1 = 168.0°, χ2 =
−85.6°) compared with those observed in the apo TcAChE,53
TcAChE−tacrine,55 TcAChE−bis(7)-tacrine56 and, to some
extent, in the TcAChE−NF59557 complex, disrupts its stacking
interaction with Phe288, as previously reported for TcAChE−
bis(5)-tacrine56 and for aged phosphonylated TcAChE.54
The largest structural alterations upon the binding of 2e to
TcAChE are observed in the Trp279−Phe290 loop (Figure 3).
The backbone atoms in this region are displaced as much as
6.0 Å (Cα-Phe288) from their native positions,53 with the side
chains dramatically reoriented, while in the TcAChE−bis(5)-
tacrine56 and in the aged phosphonylated TcAChE54 structures
the observed displacements are as high as 4.8 Å (Cα-Asp285)
and 4.6 Å (Cα-Arg289), respectively (Supporting Information,
Figure S2B).
An alternative conformation of the Trp279−Phe290 loop
with respect to that of the apo structure (Figure 3A) was
already observed in TcAChE complexed with a bifunctional
derivative of the alkaloid galanthamine (PDB ID 1W76).59 The
inherent flexibility of this loop has also been demonstrated in
site-directed mutagenesis studies. Indeed, the mutation of the
TcAChE Leu282 to either Ser or Ala disrupted cross-gorge
interactions, which resulted in a substantial decrease in the
enzyme’s stability and, ultimately, the entire structure.60 This
rearrangement may explain the residual activity of the
fasciculin/TcAChE complex,61 although molecular dynamics
simulations have suggested that the occurrence of transient
motions that would open or close alternative routes for the
substrate access to the enzyme’s active site would also maximize
the inhibition of the enzyme.62,63
In the TcAChE−2e complex, the 2-methylbenzofuran
fragment displaces Phe288 outward from its position in the
apo TcAChE structure (Figure 3).53 This shift largely occludes
the entrance to the active-site gorge and accompanies a
significant change in the main-chain conformation of the whole
loop without disrupting the crystal packing. The 2-methyl-
benzofuran fragment is nicely accommodated by a hydrophobic
pocket formed by Trp233, Phe290, Phe330, Phe331, Val395,
Asn399, and Val400. The orientation of the benzofuran ring is
stabilized by a short nonbonded contact (3.3 Å) between the
heterocyclic oxygen (OAY) and the Nε1 of the indole ring of
Trp233.
Interestingly, in the structure of the TcAChE−bis(5)-
tacrine,56 the rearrangement of the Trp279−Phe290 loop shifts
the Phe331 toward the position occupied by Phe288 in the apo
TcAChE structure,53 whereas in the conjugate of TcAChE with
the nerve agent, diisopropyl phosphofluoridate,54 the isopropyl
moiety of the organophosphoryl group, which is covalently
bound to Ser200, displaces both Phe288 and Phe290 from their
positions in the apo structure.53
The backbone atoms of Trp279 do not significantly differ
from their native positions,53 i.e., they differ by 0.4, 0.6, 0.2, and
0.7 Å in the TcAChE−bis(5)-tacrine,56 the aged phosphony-
lated TcAChE,54 the TcAChE-NF595,57 and TcAChE−2e
structures, respectively. On the other hand, the side chain of
Trp279 is dramatically reoriented in the TcAChE−2e complex
(Figure 3B) with dihedral angles of χ1 = 51.3° and χ2 = −82.0°
compared with those in the apo TcAChE53 (−62.3°, 96.7°),
TcAChE−bis(5)-tacrine56 (−76.3°, 95.2°), the aged phospho-
nylated TcAChE54 (−56.2°, 99.4°), and the TcAChE−NF59557
(−118.2°, −131.9°) structures, respectively.
Kinetic Evaluation of Compound 2e on TcAChE. To
confirm the high inhibitory activity and gain further
information about the mechanism of TcAChE inhibition by
2e, kinetic experiments were performed using a stopped-flow
apparatus. The shape of the progress curves (Supporting
Information, Figure S3) and the concentrations of the tested
compound (1−10 nM) and TcAChE (3.4 nM) used in the
assay are typical for slow, tight-binding inhibition. This type of
inhibition was further confirmed by potentiometric titration of
2e (0.3 nM) with increasing enzyme concentrations (Support-
ing Information, Figure S4). The concave-upward shape of the
residual activity plot obtained after 90 min of preincubation
indicates tight-binding inhibition, with the linear portion of the
plot extrapolating exactly to the added 2e concentration of 0.3
nM.
The atypical shape of the progress curves, with the kink
appearing earlier as the inhibitor concentration increases, was
thoroughly tested by several kinetic models. To simplify the
progress curve analysis, an ATCh concentration of 80 μM was
used, at which the TcAChE obeys the Michaelis−Menten
relationship. Among the mechanisms tested, the best fitting was
obtained with a model in which the inhibitor binds to both free
and acetylated TcAChE, confirming a mixed-type inhibition.
It has also been assumed that the observed slow inhibition is
the effect of the slow binding of the inhibitor 2e to the enzyme
active site (kon competitive 1.45
0.70 × 107 M−1 s−1; koff
competitive 0.0320 0.0002 s−1; kon non-competitive 1.45 × 107
M−1 s−1; koff noncompetitive 0.0310 0.0004 s−1). This is the
result of the low concentrations of the enzyme and inhibitor in
a close stoichiometric ratio. Consequently, an instantaneous
TcAChE inhibition was not detected, and the kinetic analysis
required a nonequilibrium treatment. The Ki values calculated
120
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
for AChE inhibition by 2e (Ki competitive = 2.2 nM; Ki
noncompetitive = 2.1 nM) confirmed a nanomolar inhibitory
potency. Similarly, compounds 1e, 1f, 1h, and 3c were assayed
(results in SI, Table S2).
The reported TcAChE−2e crystal structure reveals the
structural basis for the high anti-AChE activity. Indeed, 2e
forms a favorable π−π interaction with Trp84 via the tacrine
fragment and disrupts the catalytic triad as a result of Ser200-Oγ
hydrogen bonding with the distal nitrogen of the alkyldiamine
linker.
Inhibition of AChE-Induced Aβ1-40 Aggregation. Growing
experimental evidence has shown that PAS binders can inhibit
some noncholinergic functions of AChE, including the AChE-
induced Aβ oligomerization and fibrillization.6,64 Given the
ability of 2e to contact PAS, its ability to inhibit AChE-induced
Aβ1−40 aggregation was investigated by a thioflavin T-based
fluorometric assay6 and compared with that of bis(7)-tacrine.
The results demonstrated that 2e was able to reduce hAChE-
induced amyloid fibrillization by 58.4% at 100 μM, similarly to
bis(7)-tacrine (68.0%) and to a recently developed tacrine-6-
ferulic acid (50.27%).65 In the same experimental conditions,
the drug tacrine was not able to inhibit AChE-induced Aβ1−40
aggregation to a significant extent.
On the basis of the binding mode of 2e to AChE highlighted
by X-ray crystallography, the inhibitory activity against AChE-
induced Aβ1−40 aggregation is likely the result of the dramatic
rearrangement of the Trp279−Phe290 loop encompassing the
overall position and main-chain/side-chain conformations/
orientations that occurs to accommodate the benzofuran
moiety. This has dramatic consequences on the physicochem-
ical properties of the affected molecular surface region so that
the aggregation and deposition of the Aβ peptide is expected to
be hampered.
Inhibition of Aβ Production and Self-Aggregation. The
production and accumulation of oligomeric aggregates of Aβ
are considered to play an important role in the pathogenesis of
AD. Thus, the inhibition of Aβ production and oligomerization
have been investigated as attractive therapeutic strategies to
more efficiently combat AD. Two key targets have been
considered: (i) BACE-1 activity and (ii) Aβ1−42 self-
aggregation.
BACE-1 is involved in the rate-limiting step in the proteolytic
cleavage of APP to generate Aβ, and it is considered a key
target to lower cerebral Aβ levels and limit Aβ-associated
neurotoxicity.66 The long-term administration of a BACE-1
inhibitor to transgenic mice significantly suppressed Aβ levels
and halted the age-related cognitive decline.34 Both preclinical
and early phase clinical studies have validated BACE-1 as a
therapeutic target.67 Because of the high cost of substrates and
recombinant enzymes, the inhibitory activity of a small
selection of the hybrids was investigated in vitro using a
fluorometric assay.68 The selection included the most potent
hybrid against hAChE, namely 2e, its analogue 3f with two
benzofuran moieties to investigate the importance of this
additional fragment on the interaction with the enzyme active
site, and its amido analogue 1k. Finally, because of the
interesting inhibitory activity of 3f against BACE-1, the
analogue 3c was also assayed.
Table 2. Inhibition of hBACE-1 Activity by 1k, 2e, 3c, and
3f
a
compd
hBACE-1 (IC50, μM)
1k
0.43 0.22
1.35 0.13
0.19 0.03
0.62 0.18
2e
3c
3f
b
bis(7)-tacrine
7.50 0.40
a
Recombinant hBACE-1 was used; the results are the mean of two
b
independent experiments SEM. From ref 77.
previously shown to be inactive as a BACE-1 inhibitor.69
Moreover, a mild inhibition of BACE-1 may limit toxic effects
associated with the complete suppression of BACE-1 activity in
vivo.70,71 As a final remark, the performance of these hybrids
parallels the known nonpeptidic BACE-1 inhibitors. Indeed, the
most potent BACE-1 inhibitors, including those that have
entered clinical trials,72 are characterized by one- to two-digit
nanomolar potencies.73 Examples of MTDLs with good BACE-
1 inhibitory activity are the quinone-bearing polyamine
memoquin (IC50 = 0.108 μM),74 a rhein−huprine hybrid
(IC50 = 0.08 μM),33 and a recently published dual BACE-1/
GSK-3β inhibitor (18.03 μM).75
Molecular dynamic simulations of the rhein−huprine
hybrids33 suggest that, like the huprine Y moiety, the tacrine
fragment can be accommodated in the binding site
encompassing the BACE-1 catalytic pocket (sheltered by the
highly flexible β-hairpin loop between Val67 and Glu77
commonly known as the “flap”)32 as a consequence of the
likely electrostatic stabilization between the protonated amino-
quinoline system of the hybrids 1k, 2e, 3c, and 3f and the
catalytic dyad Asp32−Asp228. On the other hand, the
benzofuran moiety (similarly to the hydroxyanthraquinone
moiety) may fill the BACE-1 subsites S5−S7, which exhibit a
remarkable preference for Trp and the hydrophobic residues in
the P5−P7 positions of substrates/inhibitors.76
Indeed, the distance between the centroids of these binding
sites is ca. 12 Å, a value that satisfies the geometrical criteria
required for the tether of hybrids 1k, 2e, 3c, and 3f. The
presence of the amido moiety in 1k may account for the slightly
higher inhibitory activity (compared with 2e), which is likely to
be capable to be engaged in an extra hydrogen bond (with the
carbonyl acting as the acceptor).
The good hBACE-1 inhibitory activity of hybrids 3c and 3f,
which both lack the extra hydrogen bonding capability, can be
ascribed to the presence of an extra benzofuran moiety able to
better fill the active site cleft between the N-terminal and C-
terminal lobes of hBACE-1. The presence in 3c of a more
hydrophobic substituent on the benzofuran moiety (i.e., R =
OMe, instead of H) may explain its slightly higher inhibitory
activity against hBACE-1 than that exerted by 3f.
Aβ1−42 is the predominant form of Aβ in senile plaques, and
its accumulation has been associated with increased neuro-
toxicity.78 Previous studies showed that some benzofuran
derivatives were able to reduce aggregated Aβ in the brain.25
Therefore, all synthesized derivatives were screened to assess
the structural elements responsible for the inhibitory activity.
To properly rank the benzofuran−tacrine hybrids, the
inhibitors were screened at a concentration close to the IC50
value of the reference compound bis(7)-tacrine, which is 8.4
1.4 μM,44 i.e., 10 μM, which corresponds to Aβ/inhibitor = 5/
1.
Remarkably, all the selected hybrids were good BACE-1
inhibitors with submicromolar IC50 values (Table 2). They all
clearly showed a much higher inhibitory potency than the
reference compound bis(7)-tacrine, with 3c being 40 times
more potent than bis(7)-tacrine. Note that the drug tacrine was
121
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
An analysis of the results reported in Table 1 for hybrids 1a−
1l reveals that independently of the length of the spacer chain,
the tacrine−methoxybenzofuran hybrids, i.e., hybrids bearing
both the 7-methoxybenzofuran moiety and the 1,2,3,4-
tetrahydroacridine unit, exert the highest inhibitory activity,
i.e., 40.2% and 42.5% for 1e and 1k, respectively. The presence
of both moieties seems to be a key feature for the optimal
inhibitory activity. Indeed, the removal of the methoxy group,
as in 1b and 1h, led to significantly less potent inhibitors
(inhibition of 13.7% vs 40.2% and 26.2% vs 42.5%, for 1b vs 1e
and 1h vs 1k, respectively). Similarly, 7-methoxybenzofuran
hybrids containing a 2,3-dihydro-1H-cyclopenta[b]quinoline
unit or a 7,8,9,10-tetrahydro-6H-cyclohepta[b]quinoline unit
were significantly weaker than the corresponding hybrids
bearing a tacrine unit (e.g., 1k vs 1j and 1l). In hybrids from
series 2, the presence of an amino group enhanced the
inhibitory potency, which led to hybrids with the highest
inhibitory potency. The most potent hybrids were 2b, 2d, and
2e, which showed inhibition between 61.6% and 71.3%. These
three compounds were all more potent than the reference
compound bis(7)-tacrine, which showed 51% inhibition at the
same concentration. Thus, these compounds can definitively be
considered strong inhibitors against β-amyloid self-aggregation.
Finally, the presence of two 7-methoxybenzofuran units
(series 3) led to new derivatives with generally higher inhibitory
activities compared with hybrids bearing a single 7-methox-
ybenzofuran unit. For derivatives within series 3, the benefit of
the methoxy substituent on the benzofuran unit seemed to be
lost, while the importance of having a 1,2,3,4-tetrahydroacridine
unit remained, even if at a lower extent.
higher cytotoxicity at the highest tested concentration (30 μM).
Representative analogues from series 1 (1h) and 3 (3c) were
also assayed. Derivatives 1k and 3c were significantly more
cytotoxic than 2e (Supporting Information, Figure S5), which
therefore confirmed that 2e was the best candidate for further
investigation.
Behavioral Studies. Cognition-improving potency is of
utmost importance for anti-AD agents. Given the interesting
multipotent activity profile of 2e, its ability to ameliorate
scopolamine-induced cognition impairment in ICR mice was
investigated in a behavioral study using a Morris water maze
test.79 Scopolamine-induced cognition-impaired adult mice
were used as an animal model to measure the cognitive
improvement effects of 2e compared with tacrine (in equimolar
doses). Tacrine and 2e (20 μmol/kg body weight) were orally
administered to the ICR mice 30 min before intraperitoneal
(ip) administration of scopolamine (1 mg/kg) or saline
solution for 10 consecutive days to adapt the apparatus. The
test included 5 days of learning and memory training and a
probe trial on the sixth day. Figure 5 shows the mean values of
Note that the lack of activity in the reference compound
tacrine shows that the tetrahydroacridine fragment is unable to
inhibit Aβ oligomerization by itself.
Figure 5. Effects of oral administration of 2e (20 μmol/kg) and tacrine
(20 μmol/kg) on scopolamine-induced memory impairment in ICR
mice evaluated by the Morris water maze test. Data are presented as
the mean SEM (n = 9−10; *p < 0.05, **p < 0.01 vs sham group; ##p
< 0.01, vs scopolamine group).
Cell Viability Assay on the SH-SY5Y Cell Line. The effects of
compound 2e on cell viability were determined at 3, 10, and 30
μM in parallel with tacrine. The experimental data showed that
exposing SH-SY5Y cells for 48 h to 2e or tacrine at either 3 or
10 μM did not significantly reduce cell viability (Figure 4).
However, at 30 μM, both 2e and tacrine significantly affected
viability (p < 0.01), and the cytotoxic effect was higher for 2e
than tacrine (63.0 20.1% vs 33.8 5.1%). In general, the cell
toxicity profile of 2e roughly matches that of tacrine, with
the escape latencies to the hidden platform at day 1 and day 5
(the mean escape latency values for each training day are
provided as Supporting Information, Table S3 and Figure S6).
Sham-operated mice exhibited a reduction of mean escape
latency from 37.4 to 21.1 s (**p < 0.01) over the course of five
training days. Compared with the sham group, the 2e-treated
group had similar but better performance than the tacrine-
treated group, displaying a reduction of mean escape latency
from 33.8 to 22.1 s (*p < 0.05), whereas the tacrine group
showed a reduction from 39.3 to 27.7 s (*p < 0.05).
Furthermore, in the probe trial on day 6, the administration
of 2e significantly improved the overall target quadrant
preference (36.25%, **p < 0.01) compared with the tacrine-
treated group (32.5%, *p < 0.05) (Figure 6 and Supporting
Information, Table S4). These results indicated that 2e
considerably ameliorated the cognition impairment of the
treated mice and also suggested that 2e may penetrate the
blood−brain barrier and target the central nervous system.
Hepatotoxicity Studies. The possible drug-induced hep-
atotoxicity associated with the presence of the tetrahydroacri-
dine nucleus in 2e was investigated. The serious hepatotoxicity
associated with tacrine intake has been the primary limitation
for its clinical use. Approximately half of patients treated with
tacrine showed liver enzyme abnormalities.11 Compound 2e,
tacrine, and bis(7)-tacrine were assayed at equimolar doses in
Figure 4. Cell viability, measured as the reduction of MTT, in SH-
SY5Y cells exposed for 48 h with increasing concentrations of 2e or
tacrine. Data correspond to the mean
SEM of four different
experiments performed in triplicate. **p < 0.01, in comparison with
the basal condition in the absence of the tested compound.
122
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
interesting MTDL profile: they are able to reduce the
hydrolytic activity of hAChE, potentially prevent amyloid
production through the inhibition of hBACE-1 activity, and
interfere with amyloid aggregation. In particular, the most
interesting derivative within this series, the hybrid 2e, showed
an inhibitory potency against human AChE in the sub-
nanomolar range, a mixed-type mechanism of inhibition, and
the ability to partially inhibit AChE-induced Aβ fibril formation.
Moreover, it displayed a significant inhibition of amyloid self-
aggregation (at [I]/Aβ = 1/5) and a good inhibitory potency
(low micromolar range) against hBACE-1.
The X-ray crystal structure of 2e in complex with TcAChE,
besides highlighting the mode of interaction and providing the
molecular basis for interpreting the biological data, provided
some general considerations for future AChEI design. Indeed,
the conformational plasticity of the functional residues lining
the active-site gorge of AChE has significant implications for
structure-based drug design, as it has been observed in the
TcAChE−2e complex as well as in other complexes.54,56−58
The outcomes of the structural study highlight the limitations
of designing new inhibitors using the native enzyme structure
as a rigid template, and they suggest that small-to-large-scale
conformational heterogeneity should be taken into account. An
additional and important outcome of this structural study is the
possible advantage of choosing an appropriate ensemble of
protein structures in docking-based virtual screening rather
than using a single protein conformation.
Figure 6. Effects of oral administration of 2e and tacrine on spatial bias
(% of total distance swum in the training quadrant during the spatial
probe trial). Data are presented as the mean SEM (n = 9−10; *p <
0.05, **p < 0.01, vs sham group; ##p < 0.01, vs scopolamine group). Tacr
stands for tacrine.
adult mice, and their toxicity profiles were compared.
Heparinized serum was collected 8, 22, and 36 h after the
administration of tacrine, bis(7)-tacrine, and 2e, and the levels
of aspartate aminotranferase (ASAT) and alanine amino-
tranferase (ALT) were evaluated (Table 3). The serum levels
of ASAT and ALT are directly related to the extent of the liver
damage.80 Indeed, clinical evidence has shown that tacrine
therapy is associated with an elevation in serum amino-
transferase activity in almost half of patients in addition to acute
liver injury.11 In agreement with that clinical evidence,
significantly elevated ALT and ASAT activities were detected
in tacrine-treated mice, indicating that liver was affected during
the acute exposure. Specifically, 8 h after tacrine administration,
a statistically significant increase in both ALT (1.6-fold, p ≤
0.001) and ASAT (1.5-fold, p ≤ 0.05) was observed. On the
other hand, when 2e was administered, only a slight increase in
ALT (1.16-fold, not statistically significant) and ASAT (1.22-
fold, not statistically significant) was observed. In general, both
bis(7)-tacrine and 2e showed a significantly lower hepatotox-
icity than tacrine when tested at equimolar doses, and 2e
showed the highest safety among the three tested compounds.
Furthermore, ICR male mice treated with scopolamine (1
mg/kg/day) showed significantly ameliorated memory per-
formance in a Morris water maze test when 2e was
administered.
Finally, results from hepatotoxicity studies demonstrated
evidence that modification of the parent scaffold of tacrine may
lead to safer tacrine derivatives. Levels of serum biomarkers for
acute liver toxicity confirmed that 2e was significantly safer than
tacrine. The advantageous biological properties and safety
profile of hybrid 2e, along with its likely ability to reach the
CNS, make it an interesting new lead compound. Thus, 2e can
be used for further studies to help understand the structural
requirements for an optimal MTDL for AD treatment.
The potential in vivo relevance of the multiple activities
shown in the in vitro assays deserves a final comment. For
example, the concentration required to inhibit amyloid
aggregation is much higher (μM range) than that necessary
to inhibit hAChE (in the nanomolar/subnanomolar range).
Nevertheless, to properly compare activities, data from each in
vitro assay need to first be “normalized” on the basis of the
physiological/pathological levels of the molecular target. In fact,
a high concentration of amyloid (50 μM) is required in the in
vitro assay to aggregate in a suitable time frame, while in vivo
CONCLUSIONS
■
Developing a single molecule able to strongly interact with
structurally distinct targets is not an easy task, even using
computer-aided drug design.81 For the series of hybrids
developed and characterized in the present work, the results
from in vitro and in vivo tests confirmed that they have an
Table 3. ALT and ASAT Activity after the Administration of 2e and Reference Compounds Tacrine (30 mg/kg) and bis(7)-
a
tacrine (30 mg/kg)
ALT (U/L)
22 h
ASAT (U/L)
22 h
8 h
36 h
8 h
36 h
control
tacrine
38.8 10.1
62.7 13.9***
49.9 13.0
45.1 7.9
40.1 9.9
38.1 8.2
55.4 16.3*
44.8 10.5
42.0 5.1
113.3 30.0
170.4 57.6*
142.4 23.5*
138.3 21.6
113.9 31.1
151.6 32.7*
132.7 6.0
110.6 23.1
138.1 38.6
113.9 4.5
114.7 16.9
65.7 20.3**
50.8 9.1*
48.2 18.7
bis(7)-tacrine
2e
134.5 33.8
a
Values are expressed as the mean SD (n = 8−9; t test, compared with a no-treatment control group after the same amounts of time, *p ≤ 0.05,
**p ≤ 0.01, ***p ≤ 0.001).
123
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
N-((1,2,3,4-Tetrahydroacridin-9-ylamino)methyl)benzofuran-2-
Aβ concentration is estimated to be in the nanomolar/
subnanomolar range.82 Therefore, because the hybrids were
active at a concentration five times lower than that of Aβ, it
seems plausible that, provided a suitable pharmacokinetic and
proper BBB crossing, the most potent tacrine−benzofuran
hybrids may exert their inhibitory activity at nanomolar
concentrations.
carboxamide (1b). Compound 1b was prepared according to the
1
general procedure I, yellow oil, yield 78%. H NMR (CDCl3, 300
MHz): δ ppm 7.98−7.93 (m, 2H), 7.64 (d, 1H, J = 7.7 Hz), 7.55−7.50
(m, 1H), 7.46 (br s, 1H), 7.46−7.44 (m, 1H), 7.40−7.24 (m, 3H),
6.86 (s, 1H), 3.52−3.43 (m, 4H), 3.06 (br s, 2H), 2.67 (br s, 2H), 1.88
(br s, 4H), 1.67−1.62 (m, 4H), 1.44−1.43 (m, 4H). 13C NMR
(CDCl3, 75 MHz): δ ppm 167.5, 158.9, 154.5, 151.0, 148.7, 146.6,
128.4, 127.8, 127.5, 126.7, 123.6, 123.5, 122.8, 122.5, 119.7, 115.4,
114.5, 110.1, 49.1, 39.2, 33.4, 31.4, 29.4, 26.5, 26.4, 24.6, 22.8, 22.5. IR
(KBr): ν 3366, 2929, 2856, 1653, 1581, 1563, 1298, 749 cm−1. MS
(m/z): 442.1 ([M + H]+). HRMS (m/z): calcd for C28H32N3O2 [M +
H]+, 442.2489; found, 442.2493.
EXPERIMENTAL SECTION
■
Chemistry. All chemicals (reagent grade) were purchased from
Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Reference
compound bis(7)-tacrine was synthesized following the previously
reported protocol.83 Intermediate compounds 4a−4e18,35−37 and 5a−
5d38−40 were also synthesized following previously reported protocols.
Reactions were followed by thin-layer chromatography (TLC) on
silica gel GF254 plates (Qingdao Haiyang Chemical Plant, Qingdao,
China), and the spots were visualized using a UV lamp (λ = 254 nm).
Chromatographic separations were performed on silica gel columns
(90−150 μm; Qingdao Marine Chemical Inc., Qingdao, Shandong,
China) by flash chromatography. Melting points were measured on an
XT-4 micromelting point instrument (Beijing Tech Instrument Corp.,
Beijing, China) and were uncorrected. IR (KBr-disc) spectra were
recorded by Bruker Tensor 27 spectrometer (Bruker Corp., Billerica,
MA, USA). 1H NMR spectra (300 or 500 MHz) and 13C NMR (75 or
125 MHz) were acquired on a Bruker ACF-300 or ACF-500
spectrometer (Bruker Corp., Billerica, MA, USA) at 25 °C and
referenced to tetramethylsilane (TMS). Chemical shifts were reported
in ppm (δ) using the residue solvent line as the internal standard.
Splitting patterns are designated as: s, singlet; br s, broad singlet; d,
doublet; dd, doublet of doublets; t, triplet; dt, doublet of triplets; q,
quartet; and m, multiplet. The identity of the final compounds was
confirmed by acquiring mass spectra on an Agilent 1100 series LC/
MSD high performance ion trap mass spectrometer (Agilent
Technologies, Santa Clara, CA, USA) and a Mariner ESI-TOF
spectrometer (Applied Biosystems, Foster, CA, USA). Compound
purities were evaluated by reverse-phase chromatography using a
Shimadzu Shim-Pack VP-ODS (150 mm × 4.6 mm i.d., 5 μm particle
size) column (Shimadzu Corp., Kyoto, Japan) and DAD detection.
Satisfactory chromatographic analyses were obtained for all new
compounds, confirming >95% purity (see Supporting Information).
Synthesis of 1a−1l. General Procedure I. A mixture of
benzofuran-2-carboxylic acid (5a) or 7-methoxy-1-benzofuran-2-
carboxylic acid (5b) (2 mmol) and 1,1′-carbonyldiimidazole (2.05
mmol) in 25 mL of anhydrous THF was stirred for 3 h at rt before the
dropwise addition of a 5 mL solution of 4a−4f (2 mmol) in anhydrous
THF. The final mixtures were stirred for 4 h. The reaction mixture was
washed with saturated aqueous solution of sodium chloride (3 × 25
mL) and extracted with CH2Cl2 (3 × 25 mL). The combined organic
layers were dried over anhydrous Na2SO4 and evaporated under
vacuum. Purification of the crude product was achieved by column
chromatography (CH2Cl2:MeOH:triethylamine = 30:1:0.5, v:v:v) to
afford 1a−1l.
N-(6-((7,8,9,10-Tetrahydro-6H-cyclohepta[b]quinolin-11-yl)-
amino)hexyl)benzofuran-2-carboxamide (1c). Compound 1c was
prepared according to the general procedure I, yellow oil, yield 65%.
1H NMR (CDCl3, 500 MHz): δ ppm 7.95 (d, 1H, J = 8.3 Hz), 7.89 (d,
1H, J = 8.3 Hz), 7.66 (d, 1H, J = 7.8 Hz), 7.55 (dt, 1H, J = 6.9, 1.2
Hz), 7.47 (t, 1H, J = 8.3 Hz), 7.45 (s, 1H), 7.42−7.38 (m, 2H), 7.30−
7.26 (m, 1H), 6.68 (br s, 1H), 3.89 (br s, 1H), 3.47 (q, 2H, J = 6.9
Hz), 3.27 (q, 2H, J = 7.2 Hz), 3.16 (t, 2H, J = 5.7 Hz), 2.90 (t, 2H, J =
5.5 Hz), 1.87−1.85 (m, 2H), 1.80−1.78 (m, 2H), 1.74−1.63 (m, 2H),
1.49−1.43 (m, 4H), 1.31−1.22 (m, 4H). 13C NMR (CDCl3, 125
MHz): δ ppm 164.9, 159.1, 154.8, 150.3, 148.9, 146.1, 128.6, 127.8,
126.9, 124.9, 123.8, 122.8, 122.1, 118.9, 118.7, 115.2, 111.8, 110.4,
50.5, 39.6, 39.3, 32.0, 31.4, 29.7, 28.3, 27.8, 26.9, 26.8, 26.8. IR (KBr):
ν 3448, 2925, 2853, 1646, 1589, 1569, 1300, 750 cm−1. MS (m/z):
456.3 ([M + H]+). HRMS (m/z): calcd for C29H34N3O2 [M + H]+,
456.2646; found, 456.2655.
N-(6-((2,3-Dihydro-1H-cyclopenta[b]quinolin-9-yl)amino)hexyl)-
7-methoxybenzofuran-2-carboxamide (1d). Compound 1d was
prepared according to the general procedure I, white solid, yield
68%, mp 120−123 °C. 1H NMR (CDCl3, 300 MHz): δ ppm 7.97 (d,
1H, J = 8.3 Hz), 7.86 (d, 1H, J = 8.3 Hz), 7.56 (t, 1H, J = 7.5 Hz), 7.45
(s, 1H), 7.38 (t, 1H, J = 8.3 Hz), 7.24−7.18 (m, 2H), 6.91 (d, 1H, J =
7.6 Hz), 6.79 (br s, 1H), 5.11 (br s, 1H), 4.01 (s, 3H), 3.62 (q, 2H, J =
7.2 Hz), 3.49 (q, 2H, J = 6.8 Hz), 3.19 (t, 2H, J = 7.2 Hz), 3.09 (t, 2H,
J = 7.7 Hz), 2.13 (t, 2H, J = 7.5 Hz), 1.70−1.65 (m, 4H), 1.50−1.44
(m, 4H). 13C NMR (CDCl3, 75 MHz): δ ppm 167.5, 159.0, 149.2,
147.6, 146.7, 145.6, 144.4, 129.4, 128. 9, 127.9, 124.5, 124.4, 124.2,
120.3, 114.9, 113.8, 110.9, 108.4, 56.1, 45.4, 39.1, 34.5, 31.2, 31.1, 29.7,
26.5, 26.2, 23.3. IR (KBr): ν 3355, 2925, 2853, 1658, 1574, 1560,
1301, 751 cm−1. MS (m/z): 458.2 ([M + H]+). HRMS (m/z): calcd
for C28H32N3O3 [M + H]+, 458.2438; found, 458.2454.
7-Methoxy-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-
benzofuran-2-carboxamide (1e). Compound 1e was prepared
according to the general procedure I, yellow oil, yield 70%. 1H
NMR (CDCl3, 300 MHz): δ ppm 7.96 (dd, 1H, J = 8.5, 0.8 Hz), 7.91
(dd, 1H, J = 8.5, 0.7 Hz), 7.54 (dt, 1H, J = 8.2, 1.3 Hz), 7.45 (s, 1H),
7.34 (dt, 1H, J = 8.3, 1.2 Hz), 7.25−7.16 (m, 2H), 6.88 (dd, 1H, J =
7.4, 1.5 Hz), 6.81 (br s, 1H), 3.98 (br s, 1H), 3.98 (s, 3H), 3.48−3.41
(m, 4H), 3.05 (br s, 2H), 2.70 (br s, 2H), 1.91−1.89 (m, 4H), 1.67−
1.60 (m, 4H), 1.43−1.40 (m, 4H). 13C NMR (CDCl3, 75 MHz): δ
ppm 167.5, 158.9, 158.4, 151.0, 149.2, 145.6, 144.3, 129.5, 128.6,
128.5, 124.5, 123.8, 123.0, 120.3, 116.0, 114.9, 110.9, 108.3, 56.1, 49.5,
39.3, 31.8, 30.1, 29.8, 26.7, 26.6, 24.9, 23.2, 22.9. IR (KBr): ν 3421,
2933, 2857, 1652, 1590, 1491, 1316, 763 cm−1. MS (m/z): 472.3 ([M
+ H]+). HRMS (m/z): calcd for C29H34N3O3 [M + H]+, 472.2595;
found, 472.2617.
N-(6-((2,3-Dihydro-1H-cyclopenta[b]quinolin-9-yl)amino)hexyl)-
benzofuran-2-carboxamide (1a). Compound 1a was prepared
according to the general procedure I, white solid, yield 65%, mp
1
118−120 °C. H NMR (CDCl3, 500 MHz): δ ppm 7.96 (d, 1H, J =
8.3 Hz), 7.81 (d, 1H, J = 8.2 Hz), 7.79 (d, 1H, J = 7.8 Hz), 7.58 (dt,
1H, J = 8.4, 1.2 Hz), 7.50 (dt, 1H, J = 8.4, 0.7 Hz), 7.48 (s, 1H), 7.44
(dt, 1H, J = 7.2, 1.2 Hz), 7.40 (dt, 1H, J = 8.2, 1.2 Hz), 7.32 (dt, 1H, J
= 8.0, 0.9 Hz), 6.69 (s, 1H), 4.85 (s, 1H), 3.64 (q, 2H, J = 6.9 Hz),
3.53 (q, 2H, J = 6.8 Hz), 3.22 (t, 2H, J = 7.1 Hz), 3.10 (t, 2H, J = 7.8
Hz), 2.15 (t, 2H, J = 7.5 Hz), 1.75−1.68 (m, 4H), 1.57−1.46 (m, 4H).
13C NMR (CDCl3, 75 MHz): δ ppm 168.3, 159.1, 154.8, 148.9, 147.9,
146.9, 128.8, 128.7, 127.8, 127.0, 124.2, 123.9, 122.9, 120.0, 119.0,
114.1, 111.8, 110.4, 53.1, 39.2, 34.9, 31.2, 31.0, 29.8, 26.6, 26.4, 23.4.
IR (KBr): ν 3359, 2927, 2854, 1657, 1573, 1546, 1305, 748 cm−1. MS
(m/z): 428.2 ([M + H]+). HRMS (m/z): calcd for C27H30N3O2 [M +
H]+, 428.2333; found, 428.2347.
7-Methoxy-N-(6-((7,8,9,10-tetrahydro-6H-cyclohepta[b]quinolin-
11-yl)amino)hexyl)benzofuran-2-carboxamide (1f). Compound 1f
was prepared according to the general procedure I, yellow solid, yield
1
62%, mp 75−78 °C. H NMR (CDCl3, 300 MHz): δ ppm 7.95 (d,
1H, J = 8.3 Hz), 7.90 (d, 1H, J = 8.3 Hz), 7.56 (t, 1H, J = 8.0 Hz), 7.45
(s, 1H), 7.40 (t, 1H, J = 8.1 Hz), 7.27−7.17 (m, 2H), 6.89 (d, 1H, J =
7.3 Hz), 6.79 (br s, 1H), 3.99 (s, 3H), 3.46 (q, 2H, J = 6.7 Hz), 3.27 (t,
2H, J = 7.1 Hz), 3.19−3.15 (m, 2H); 2.93−2.90 (m, 2H), 1.85−1.63
(m, 10H), 1.44−1.43 (m, 4H). 13C NMR (CDCl3, 125 MHz): δ ppm
165.3, 158.9, 150.0, 149.2, 146.6, 145.6, 144.3, 129.4, 129.0, 128.4,
124.9, 124.5, 124.0, 122.2, 122.0, 114.9, 110.8, 108.3, 56.1, 50.7, 39.9,
124
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
39.3, 32.1, 31.5, 29.7, 28.4, 27.8, 26.9, 26.8, 26.8. IR (KBr): ν 3391,
2926, 2854, 1655, 1590, 1566, 1315, 758 cm−1. MS (m/z): 486 [M +
H]+. HRMS (m/z): calcd for C30H36N3O3 [M + H]+, 486.2751; found,
486.2769.
1.60 (m, 4H), 1.39−1.32 (m, 6H). 13C NMR (CDCl3, 75 MHz): δ
ppm 167.5, 159.0, 149.2, 147.3, 145.6, 144.3, 139.8, 131.4, 129.2,
128.8, 124.9, 124.6, 124.0, 122.2, 119.2, 114.9, 110.8, 108.4, 56.1, 49.0,
39.3, 31.3, 30.1, 29.8, 29.7, 28.9, 26.7, 24.0, 22.1, 21.0. IR (KBr): ν
3423, 2931, 2856, 1650, 1592, 1491, 1315, 761 cm−1. MS (m/z): 486.1
([M + H]+). HRMS (m/z): calcd for C30H36N3O2 [M + H]+,
486.2751; found, 486.2729.
N-(7-((2,3-Dihydro-1H-cyclopenta[b]quinolin-9-yl)amino)heptyl)-
benzofuran-2-carboxamide (1g). Compound 1g was prepared
according to the general procedure I, yellow solid, yield 68%, mp
80−83 °C. 1H NMR (CDCl3, 500 MHz): δ ppm 7.91 (d, 1H, J = 8.4
Hz), 7.74 (d, 1H, J = 8.3 Hz), 7.65 (d, 1H, J = 7.8 Hz), 7.54−7.51 (m,
1H), 7.47−7.45 (m, 1H), 7.45 (s, 1H), 7.39 (dd, 1H, J = 7.3, 1.0 Hz),
7.35−7.32 (m, 1H), 7.29−7.26 (m, 1H), 6.74 (br s, 1H), 4.71 (br s,
1H), 3.57 (q, 2H, J = 6.8 Hz), 3.47 (q, 2H, J = 6.7 Hz), 3.19−3.13 (m,
2 H), 3.05−3.02 (m, 2H), 2.16−2.08 (m, 2H), 1.66−1.62 (m, 4H),
1.47−1.41 (m, 6H). 13C NMR (CDCl3, 125 MHz): δ ppm 168.5,
158.9, 154.6, 148.8, 148.2, 146.4, 128.9, 128.1, 126.7, 123.8, 123.6, 12
2.6, 122.1, 119.7, 118.8, 114.0, 111.6, 110.2, 45.6, 39.2, 34.9, 31.1, 30.8,
29.5, 28.9, 26.7, 26.5, 23.2. IR (KBr): ν 3362, 2927, 2852, 1656, 1574,
1546, 1302, 749 cm−1. MS (m/z): 442.3 [M + H]+. HRMS (m/z):
calcd for C28H32N3O2 [M + H]+, 442.2489; found, 442.2476.
N-(7-((1,2,3,4-Tetrahydroacridin-9-yl)amino)heptyl)benzofuran-
2-carboxamide (1h). Compound 1h was prepared according to the
N-(7-(7,8,9,10-Tetrahydro-6H-cyclohepta[b]quinolin-11-
ylamino)heptyl)-7-methoxybenzofuran-2-carboxamide (1l). Com-
pound 1l was prepared according to the general procedure I, yellow
1
oil, yield 75%. H NMR (CDCl3, 300 MHz): δ ppm 8.48 (d, 1H, J =
8.4 Hz), 8.27 (d, 1H, J = 8.6 Hz), 7.61 (t, 1H, J = 7.5 Hz), 7.45−7.42
(m, 2H), 7.45 (br s, 1H), 7.24−7.16 (m, 2H), 6.94−6.87 (m, 2H),
6.74 (s, 1H), 3.99 (s, 1H), 3.73 (t, 2H, J = 6.8 Hz), 3.46−3.39 (m,
4H), 2.88−2.85 (m, 2H), 1.81−1.74 (m, 6H), 1.70−1.59 (m, 4H),
1.34−1.27 (m, 6H). 13C NMR (CDCl3, 75 MHz): δ ppm 167.8, 158.8,
149.0, 147.5, 145.4, 144.1, 137.6, 129.3, 128.8, 125.9, 124.3, 124.0,
123.3, 121.0, 119.5, 114.6, 110.5, 108.2, 55.9, 48.9, 38.6, 39.1, 33.6,
31.1, 30.9, 29.6, 29.4, 28.6, 27.1, 25.7, 22.4. IR (KBr): ν 3423, 2924,
2850, 1640, 1561, 1540, 1289, 749 cm−1. MS (m/z): 500.3 ([M +
H]+). HRMS (m/z): calcd for C31H38N3O3 [M + H]+, 500.2908;
found, 500.2886.
1
general procedure I, yellow oil, yield 48%. H NMR (CDCl3, 300
MHz): δ ppm 7.97−7.91 (m, 2H), 7.65 (d, 1H, J = 8.0 Hz), 7.56−7.48
(m, 2H), 7.46 (s, 1H), 7.42−7.25 (m, 3H), 6.76 (s, 1H), 3.51−3.43
(m, 4H), 3.06 (br s, 2H), 2.69 (br s, 2H), 1.91−1.89 (m, 4H), 1.66−
1.60 (m, 4H), 1.39 (br s, 6H). 13C NMR (CDCl3, 75 MHz): δ ppm
167.7, 159.1, 154.9, 153.4, 148.7, 146.7, 128.7, 127.8, 127.0, 124.8,
124.6, 123.9, 123.7, 122.9, 118.1, 113.3, 111.8, 110.3, 49.1, 39.5, 33.3,
31.5, 31.2, 29.7, 28.9, 26.8, 24.3, 22.6, 21.8. IR (KBr): ν 3424, 2930,
2856, 1644, 1591.9, 1524, 1299, 751 cm−1. MS (m/z): 456.1 ([M +
H]+). HRMS (m/z): calcd for C29H34N3O2 [M + H]+, 456.2646;
found, 456.2630.
Synthesis of 2a−2f and 3a−3h. General Procedure II. To a
mixture of 4a−4f (1 mmol) and 2-(bromomethyl)benzofuran (5c) or
2-(bromomethyl)-7-methoxybenzofuran (5d) (1 mmol) in 20 mL of
CH2Cl2 was added with K2CO3 (0.8 g) and KI (0.08g), and the
reaction mixture was stirred for 3 days at rt until the completion
monitored by TLC. Then 200 mL of CH2Cl2 were added to the
mixture and then washed with a saturated aqueous solution of sodium
chloride. The organic layer was dried over anhydrous Na2SO4 and
evaporated under vacuum. Purification was achieved by column
chromatography (CH2Cl2:EtOAc:MeOH = 30:1:1, v:v:v) to afford
2a−2f and 3a−3h.
N-(7-(7,8,9,10-Tetrahydro-6H-cyclohepta[b]quinolin-11-
ylamino)heptyl)benzofuran-2-carboxamide (1i). Compound 1i was
prepared according to the general procedure I, pale-brown oil, yield
N1-(Benzofuran-2-ylmethyl)-N6-(2,3-dihydro-1H-cyclopenta[b]-
quinolin-9-yl)hexane-1,6-diamine (2a). Compound 2a was prepared
1
1
68%. H NMR (CDCl3, 500 MHz): δ ppm 8.44 (d, 1H, J = 7.2 Hz),
according to the general procedure II, yellow oil, yield 30%. H NMR
8.12 (d, 1H, J = 8.2 Hz), 7.65 (d, 1H, J = 7.7 Hz), 7.62 (t, 1H, J = 7.7
Hz), 7.49−7.43 (m, 2H), 7.46 (s, 1H), 7.40 (t, 1H, J = 7.5 Hz), 7.31−
7.29 (m, 1H), 6.79 (br s, 1H), 5.30 (s, 1H), 3.63 (br s, 2H), 3.47−3.46
(m, 2H), 3.38 (br s, 2H), 2.88 (br s, 2H), 1.87−1.71 (m, 4H), 1.71−
1.63 (m, 2H), 1.40−1.33 (m, 6H), 1.26−1.20 (m, 4H). 13C NMR
(CDCl3, 125 MHz): δ ppm 164.0, 158.9, 154.7, 151.2, 148.8, 144.5,
129.2, 127.7, 127.2, 126.8, 125.1, 123.7, 122.7, 122.3, 122.0, 121.0,
111.7, 110.3, 50.3, 39.3, 38.3, 31.8, 31.4, 29.7, 29.4, 28.9, 28.0, 27.5,
26.8, 26.0. IR (KBr): ν 3424, 2924, 2851, 1640, 1584, 1298, 748 cm−1.
MS (m/z): 470.3 ([M + H]+). HRMS (m/z): calcd for C29H36N3O2
[M + H]+, 470.2802; found, 470.2798.
N-(7-(2,3-Dihydro-1H-cyclopenta[b]quinolin-9-ylamino)heptyl)-
7-methoxybenzofuran-2-carboxamide (1j). Compound 1j was
prepared according to the general procedure I, yellow solid, yield
72%, mp 127−129 °C. 1H NMR (CDCl3, 500 MHz): δ ppm 7.95 (d,
1H, J = 8.3 Hz), 7.79 (d, 1H, J = 8.4 Hz), 7. 55 (t, 1H, J = 7.4 Hz),
7.45 (s, 1H), 7.35 (t, 1H, J = 7.6 Hz), 7.24−7.19 (m, 2H), 6.90 (d, 1H,
J = 7.6 Hz), 6.75 (br s, 1H), 5.04 (br s, 1H), 4.01 (s, 3H), 3.62 (t, 2H,
J = 7.0 Hz), 3.46 (q, 2H, J = 6.8 Hz), 3.19 (t, 2H, J = 7.2 Hz), 3.09 (t,
2H, J = 7.8 Hz), 2.14 (q, 2H, J = 6.8 Hz), 1.69−1.62 (m, 4H), 1.41−
1.34 (m, 6H). 13C NMR (CDCl3, 125 MHz): δ ppm 167.6, 158.7,
149.0, 147.1, 146.9, 145.4, 144.1, 129.2, 128.6, 128.0, 124.3, 124.1,
120.0, 118.6, 114.7, 113.7, 110.7, 108.1, 55.9, 45.5, 39.2, 34.5, 31.1,
30.9, 29.5, 28.9, 26.7, 26.6, 23.1. IR (KBr): ν 3356, 2925, 2851, 1657,
1573, 1458, 1308, 759 cm−1. MS (m/z): 472.3 ([M + H]+). HRMS
(m/z): calcd for C29H34N3O3 [M + H]+, 472.2595; found, 472.2577.
7-Methoxy-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)-
benzofuran-2-carboxamide (1k). Compound 1k was prepared
according to the general procedure I, yellow oil, yield 67%. 1H
NMR (CDCl3, 300 MHz): δ ppm 7.98 (dd, 1H, J = 8.6, 0.8 Hz), 7.91
(dd, 1H, J = 8.5, 0.7 Hz), 7.55 (dt, 1H, J = 8.2, 1.3 Hz), 7.45 (s, 1H).
7.34 (dt, 1H, J = 8.3, 1.2 Hz), 7.25−7.16 (m, 2H), 6.88 (dd, 1H, J =
7.4, 1.5 Hz), 6.77 (br s, 1H), 4.12 (br s, 1H), 4.01 (s, 3H), 3.54−3.42
(m, 4H), 3.08 (br s, 2H), 2.69 (br s, 2H), 1.93−1.89 (m, 4H), 1.68−
(CDCl3, 300 MHz): δ ppm 8.25 (d, 1H, J = 8.3 Hz), 7.93 (d, 1H, J =
8.0 Hz), 7.55−7.53 (m, 2H), 7.40−7.36 (m, 2H), 7.24−7.17 (m, 2H),
6.79 (s, 1H), 4.10 (s, 2H), 3.55 (t, 2H, J = 7.0 Hz), 3.11 (t, 2H, J = 7.9
Hz), 2.93 (t, 2H, J = 7.0 Hz), 2.81 (t, 2H, J = 6.8 Hz), 2.09−2.02 (m,
2H), 1.67−1.65 (m, 4H), 1.47−1.39 (m, 4H). 13C NMR (CDCl3, 75
MHz): δ ppm 158.7, 157.2, 155.3, 151.3, 147.6, 144.9, 128.7, 128.5,
128.3, 124.9, 123.8, 122.6, 121.9, 120.7, 116.4, 111.2, 105.5, 53.4, 50.6,
47.0, 34.2, 31.4, 30.2, 29.2, 26.9, 26.7, 23.4. IR (KBr): ν 3420, 2930,
2856, 1587, 1490, 1270, 751 cm−1. MS (m/z): 414.1 ([M + H]+).
HRMS (m/z): calcd for C27H32N3O [M + H]+, 414.2540; found,
414.2521.
N1-(2,3-Dihydro-1H-cyclopenta[b]quinolin-9-yl)-N6-((7-methoxy-
benzofuran-2-yl)methyl)hexane-1,6-diamine (2b). Compound 2b
was prepared according to the general procedure II, yellow solid, yield
27%, mp 82−85 °C. 1H NMR (CDCl3, 300 MHz): δ ppm 7.90−7.86
(m, 2H), 7.54 (t, 1H, J = 7.6 Hz), 7.35 (t, 1H, J = 7.4 Hz), 7.13−7.11
(m, 2H), 6.79−6.73 (m, 1H), 6.56 (s, 1H), 3.99 (s, 3H), 3.94 (s, 2H),
3.64 (t, 2H, J = 7.1 Hz), 3.13−3.03 (m, 4H), 2.64 (t, 2H, J = 7.0 Hz),
2.17−2.04 (m, 2H), 1.66−1.64 (m, 2H), 1.55−1.48 (m, 2H), 1.41−
1.39 (m, 4H). 13C NMR (CDCl3, 75 MHz): δ ppm 156.8, 156.6,
152.1, 145.1, 145.0, 144.1, 128.9, 126.7, 123.9, 123.6, 122.7, 119.7,
114.2, 113.2, 111.6, 106.4, 104.2, 55.7, 49.2, 48.7, 45.6, 34.9, 31.1, 30.8,
29.6, 28.9, 26.7, 23.2. IR (KBr): ν 3422, 2927, 2854, 1581, 1488, 1271,
758 cm−1. MS (m/z): 444.1 ([M + H]+). HRMS (m/z): calcd for
C28H34N3O2 [M + H]+, 444.2789; found, 444.2781.
N1-((7-Methoxybenzofuran-2-yl)methyl)-N6-(1,2,3,4-tetrahydroa-
cridin-9-yl)hexane-1,6-diamine (2c). Compound 2c was prepared
1
according to the general procedure II, yellow oil, yield 29%. H NMR
(CDCl3, 500 MHz): δ ppm 8.05 (d, 1H, J = 8.5 Hz), 7.99 (d, 1H, J =
8.3 Hz), 7.58 (t, 1H, J = 7.1 Hz), 7.36 (t, 1H, J = 7.2 Hz), 7.14−7.10
(m, 2H), 6.76 (dd, 1H, J = 6.3, 2.7 Hz), 6.55 (s, 1H), 3.99 (s, 3H),
3.93 (s, 2H), 3.59−3.56 (m, 2H), 3.12 (s, 2H), 2.65−2.62 (m, 4H),
1.90−1.89 (m, 4H), 1.72−1.66 (m, 2H), 1.55−1.49 (m, 2H), 1.41−
1.36 (m, 4H). 13C NMR (CDCl3, 125 MHz): δ ppm 156.8, 156.5,
125
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
152.1, 145.1, 145.0, 144.1, 130.1, 129.4, 126.5, 124.0, 123.3, 123.1,
119.0, 114.4, 113.1, 106.0, 104.1, 55.9, 49.1, 48.7, 46.4, 31.5, 29.8, 29.7,
26.9, 26.7, 24.4, 22.7, 22.1. IR (KBr): ν 3419, 2924, 2852, 1583, 1490,
1261, 749 cm−1. MS (m/z): 458.3 ([M + H]+). HRMS (m/z): calcd
for C29H35N3O2 [M + H]+, 458.2749; found, 458.2762.
4H), 1.48−1.43 (m, 2H). 13C NMR (CDCl3, 125 MHz): δ ppm 156.8,
155.7, 152.1, 145.2, 145.0, 144.2, 130.9, 129.1, 128.8, 126.7, 124.3,
123.3, 120.3, 118.2, 113.1, 106.1, 105.5, 56.0, 53.1, 50.6, 45.4, 34.9,
31.2, 30.6, 29.7, 27.3, 26.8, 26.4, 26.0. IR (KBr): ν 3444, 2927, 2855,
1567, 1493.0, 1270, 758 cm−1. MS (m/z): 604.4 ([M + H]+). HRMS
(m/z): calcd for C38H42N3O4 [M + H]+, 604.3170; found, 604.3146.
N1,N1-Bis(7-methoxybenzofuran-2-yl)methyl)-N6-(1,2,3,4-tetrahy-
droacridin-9-yl)hexane-1,6-diamine (3c). Compound 3c was pre-
N1-(Benzofuran-2-ylmethyl)-N7-(2,3-dihydro-1H-cyclopenta[b]-
quinolin-9-yl)heptane-1,7-diamine (2d). Compound 2d was pre-
pared according to the general procedure II, pale-brown oil, yield 28%.
1H NMR (CDCl3, 300 MHz): δ ppm 7.93 (d, 2H, J = 8.3 Hz), 7.55−
1
pared according to the general procedure II, yellow oil, yield 40%. H
NMR (CDCl3, 300 MHz): δ ppm 7.95 (d, 2H, J = 8.9 Hz), 7.55 (t,
1H, J = 7.2 Hz), 7.32 (t, 1H, J = 7.2 Hz), 7.12−7.03 (m, 4H), 6.78−
6.69 (m, 2H), 6.63 (s, 2H), 3.97 (s, 6H), 3.91 (s, 4H), 3.45 (t, 2H, J =
7.3 Hz), 3.07 (br s, 2H), 2.65−2.57 (m, 4H), 1.90−1.88 (m, 4H),
1.63−1.58 (m, 4H), 1.36−1.35 (m, 4H). 13C NMR (CDCl3, 75 MHz):
δ ppm 156.9, 155.7, 152.1, 151.1, 145.2, 145.2, 144.1, 130.1, 128.6,
128.0, 123.7, 123.3, 123.1, 122.9, 112.8, 112.8, 106.1, 105.5, 55.9, 53.1,
50.6, 49.3, 33.5, 29.7, 27.3, 26.8, 26.7, 24.6, 22.9, 22.6. IR (KBr): ν
3451, 2924, 2853, 1589, 1492, 1262, 750 cm−1. MS (m/z): [M + H]+
618.2. HRMS (m/z): calcd for C39H44N3O4 [M + H]+, 618.3326;
found, 618.3302.
7.49 (m, 2H), 7.43 (d, 1H, J = 7.7 Hz), 7.35 (t, 1H, J = 7.5 Hz), 7.23−
7.16 (m, 2H), 6.56 (s, 1H), 5.80 (br s, 1H), 3.92 (s, 2H), 3.61 (br s,
2H), 3.11 (q, 4H, J = 7.1 Hz), 2.65 (t, 2H, J = 7.0 Hz), 2.14−2.09 (m,
2H), 1.67−1.64 (m, 2H), 1.53−1.51 (m, 2H), 1.37−1.30 (m, 6H). 13C
NMR (CDCl3, 75 MHz): δ ppm 158.7, 157.5, 155.2, 151.2, 147.8,
144.7, 129.2, 128.4, 126.0, 124.4, 123.7, 122.6, 120.6, 118.2, 113.1,
111.0, 103.8, 49.0, 46.6, 45.3, 33.7, 31.2, 31.0, 29.7, 29.6, 29.1, 27.0,
26.5. IR (KBr): ν 3422, 2926, 2853, 1583, 1492, 1268, 754 cm−1. MS
(m/z): 428.7 ([M + H]+). HRMS (m/z): calcd for C28H34N3O [M +
H]+, 428.2696; found, 428.2679.
N1-(Benzofuran-2-ylmethyl)-N7-(1,2,3,4-tetrahydroacridin-9-yl)-
heptane-1,7-diamine (2e). Compound 2e was prepared according to
the general procedure II, yellow oil, yield 30%. 1H NMR (CDCl3, 500
MHz): δ ppm 7.95−7.90 (m, 2H), 7.56−7.50 (m, 2H), 7.44 (d, 1H, J
= 8.0 Hz), 7.35−7.32 (m, 1H), 7.25−7.17 (m, 2H), 6.54 (s, 1H), 3.92
(s, 2H), 3.48 (t, 2H, J = 7.2 Hz), 3.06 (br s, 2H), 2.70 (br s, 2H), 2.64
(t, 2H, J = 7.2 Hz), 1.91−1.90 (m, 4H), 1.67−1.61 (m, 2H), 1.51−
1.49 (m, 2H), 1.32−1.26 (m, 6H). 13C NMR (CDCl3, 75 MHz): δ
ppm 158.6, 157.2, 155.2, 151.0, 147.7, 129.0, 128.7, 128.5, 124.0,
123.8, 123.0, 122.8, 120.9, 120.5, 116.1, 111.3, 103.9, 49.7, 49.4, 47.0,
34.2, 31.9, 30.2, 29.5, 27.4, 27.1, 25.0, 23.3, 23.0. IR (KBr): ν 3418,
2930, 2855, 1581, 1490, 1271, 753 cm−1. MS (m/z): 442.3 ([M +
H]+). HRMS (m/z): calcd for C29H36N3O [M + H]+, 442.2858;
found, 442.2868.
N1,N1-Bis((7-methoxybenzofuran-2-yl)methyl)-N6-(7,8,9,10-tetra-
hydro-6H-cyclohepta[b]quinolin-11-yl)hexane-1,6-diamine (3d).
Compound 3d was prepared according to the general procedure II,
1
yellow oil, yield 57%. H NMR (CDCl3, 300 MHz): δ ppm 7.96 (d,
2H, J = 8.4 Hz), 7.87 (dd, 1H, J = 8.4, 0.8 Hz), 7.59−7.53 (m, 1H),
7.42−7.36 (m, 1H), 7.15−7.08 (m, 4H), 6.78−6.69 (m, 2H), 6.63 (s,
2H), 3.98 (s, 6H), 3.91 (s, 4H), 3.25−3.15 (m, 4H), 2.89 (t, 2H, J =
5.3 Hz), 2.62−2.46 (m, 2H), 1.87−1.79 (m, 4H), 1.72−1.59 (m, 6H),
1.36−1.33 (m, 4H). 13C NMR (CDCl3, 75 MHz): δ ppm 156.9, 155.8,
152.1, 145.4, 145.0, 144.4, 130.3, 129.1, 128.4, 124.9, 124.0, 123.7,
123.4, 122.1, 113.2, 106.2, 105.6, 56.1, 53.4, 50.8, 50.7, 40.1, 32.1, 31.6,
29.8, 28.4, 27.8, 27.5, 27.1, 27.0. IR (KBr): ν 3445, 2926, 2853, 1586,
1462, 1266, 752 cm−1. MS (m/z): 632.3 ([M + H]+). HRMS (m/z):
calcd for C40H44N3O4 [M + H]+, 632.3483; found, 632.3474.
N1-((7-Methoxybenzofuran-2-yl)methyl)-N7-(7,8,9,10-tetrahydro-
6H-cyclohepta[b]quinolin-11-yl)heptane-1,7-diamine (2f). Com-
pound 2f was prepared according to the general procedure II, yellow
N1,N1-Bis(benzofuran-2-ylmethyl)-N7-(2,3-dihydro-1H-
cyclopenta[b]quinolin-9-yl)heptane-1,7-diamine (3e). Compound
3e was prepared according to the general procedure II, yellow oil,
1
oil, yield 43%. H NMR (CDCl3, 500 MHz): δ ppm 8.29 (br s, 1H),
1
yield 61%. H NMR (CDCl3, 300 MHz): δ ppm 8.02 (d, 1H, J = 8.3
7.95 (d, 1H, J = 8.5 Hz), 7.64 (t, 1H, J = 7.4 Hz), 7.46 (t, 1H, J = 7.4
Hz), 7.14−7.12 (m, 2H), 6.77 (dd, 2H, J = 6.2, 2.4 Hz), 6.59 (s, 1H),
4.00 (s, 3H), 3.96 (s, 2H), 3.48 (br s, 2H), 3.32 (br s, 2H), 2.88−2.86
(m, 2H), 2.65 (t, 2H, J = 7.1 Hz), 1.89 (br s, 2H), 1.82−1.78 (m, 2H),
1.72−1.69 (m, 4H), 1.51−1.47 (m, 2H), 1.39−1.26 (m, 10H). 13C
NMR (CDCl3, 125 MHz): δ ppm 156.8, 156.2, 152.6, 145.2, 145.0,
144.2, 130.1, 128.9, 125.4, 125.3, 123.4, 122.6, 122.5, 122.4, 113.2,
106.1, 104.5, 56. 0, 49.9, 48.8, 46.3, 39.2, 31.6, 31.2, 29.7, 29.5, 29.0,
27.6, 27.0, 26.7, 26.3. IR(KBr): ν 3424, 2923, 2852, 1586, 1494, 1265,
752 cm−1. MS (m/z): 486.3 ([M + H]+). HRMS (m/z): calcd for
C31H40N3O2 [M + H]+, 486.3115; found, 486.3099.
Hz), 7.95 (d, 1H, J = 8.5 Hz), 7.57−7.52 (m, 3H), 7.47 (d, 2H, J = 7.7
Hz), 7.37 (t, 1H, J = 7.6 Hz), 7.26−7.17 (m, 4H), 6.62 (s, 2H), 6.32
(s, 1H), 3.88−3.83 (m, 4H), 3.65−3. 59 (m, 2H), 3.14 (q, 4H, J = 7.9
Hz), 2.59 (t, 2H, J = 7.2 Hz), 2.14 (q, 2H, J = 7.8 Hz), 1.67−1.60 (m,
4H), 1.34−1.26 (m, 6H). 13C NMR (CDCl3, 75 MHz): δ ppm 158.9,
157.3, 155.3, 151.2, 147.4, 129.3, 128.3, 126.1, 124.5, 123.8, 123.0,
122.6, 120.6, 118.1, 113.2, 111.2, 105.6, 53.4, 50.6, 45.4, 33.7, 31.2,
31.0, 29.7, 27.2, 27.1, 22.6, 23.0. IR(KBr): ν 3421, 2927, 2853, 1583,
1454, 1271, 752 cm−1. MS (m/z): 558.3 ([M + H]+). HRMS (m/z):
calcd for C37H40N3O2 [M + H]+, 558.3115; found, 558.3114.
N1,N1-Bis(benzofuran-2-ylmethyl)-N7-(1,2,3,4-tetrahydroacridin-
9-yl)heptane-1,7-diamine (3f). Compound 3f was prepared according
N1,N1-Bis(benzofuran-2-ylmethyl)-N6-(2,3-dihydro-1H-
cyclopenta[b]quinolin-9-yl)hexane-1,6-diamine (3a). Compound 3a
was prepared according to the general procedure II, yellow oil, yield
1
to the general procedure II, yellow oil, yield 42%. H NMR (CDCl3,
1
300 MHz): δ ppm 8.33 (d, 1H, J = 8.4 Hz), 8.08 (d, 1H, J = 8.6 Hz),
7.64 (t, 1H, J = 7.5 Hz), 7.53 (d, 2H, J = 7.0 Hz), 7.46 (d, 2H, J = 7.0
Hz), 7.39 (t, 1H, J = 7.8 Hz), 7.26−7.17 (m, 4H), 6.62 (s, 2H), 3.89
(s, 4H), 3.74 (t, 2H, J = 7.1 Hz), 3.22 (s, 2H), 2.63−2.57 (m, 4H),
1.89−1.81 (m, 4H), 1.72−1.69 (m, 4H), 1.34−1.26 (m, 6H). 13C
NMR (CDCl3, 75 MHz): δ ppm 158.5, 157.1, 155.2, 151.2, 147.5,
131.1, 128.3, 124.7, 123.8, 123.7, 123.3, 122.6, 120.7, 117.0, 112.1,
111.2, 105.6, 53.3, 50.6, 48.9, 31.3, 30.1, 29.9, 27.2, 27.1, 26.6, 23.9,
22.2, 21.2. IR (KBr): ν 3421, 2929, 2855, 1583, 1453, 1254, 752 cm−1.
MS (m/z): 572.2 ([M + H]+). HRMS (m/z): calcd for C38H42N3O2
[M + H]+, 572.3272; found, 572.3257.
40%. H NMR (CDCl3, 300 MHz): δ ppm 7.93 (d, 1H, J = 8.3 Hz),
7.71 (d, 1H, J = 8.4 Hz), 7.53 (d, 2H, J = 7.1 Hz), 7.47 (d, 2H, J = 7.8
Hz), 7.41−7.32 (m, 2H), 7.23−7.13 (m, 4H), 6.62 (s, 2H), 4.74 (s,
1H), 3.89 (s, 4H), 3.54 (s, 2H), 3.17 (t, 2H, J = 7.2 Hz), 3.06 (t, 2H, J
= 7.8 Hz), 2.60 (t, 2H, J = 7.1 Hz), 2.19−2.06 (m, 2H), 1.63 (s, 4H),
1.40−1.38 (m, 4H). 13C NMR (CDCl3, 75 MHz): δ ppm 158.7, 157.4,
155.4, 151.5, 147.9, 130.0, 128.9, 128.5, 125.0, 124.0, 122.8, 121.0,
120.8, 119.4, 118.0, 111.3, 105.7, 53.4, 50.8, 45.5, 33.4, 31.4, 31.2, 29.8,
27.4, 27.0, 26.6. IR (KBr): ν 3446, 2927, 2854, 1567, 1453, 1252, 752
cm−1. MS (m/z): 544.1 ([M + H]+). HRMS (m/z): calcd for
C36H38N3O2 [M + H]+, 544.2959; found, 544.2940.
N1-(2,3-Dihydro-1H-cyclopenta[b]quinolin-9-yl)-N6,N6-bis((7-me-
thoxybenzofuran-2-yl)methyl)hexane-1,6-diamine (3b). Compound
3b was prepared according to the general procedure II, yellow oil, yield
N1,N1-Bis(benzofuran-2-ylmethyl)-N7-(7,8,9,10-tetrahydro-6H-
cyclohepta[b]quinolin-11-yl)heptane-1,7-diamine (3g). Compound
3g was prepared according to the general procedure II, pale-brown oil,
yield 47%. 1H NMR (CDCl3, 300 MHz):): δ ppm 8.04 (d, 1H, J = 8.3
Hz), 7.89 (d, 1H, J = 8.4 Hz), 7.61−7.39 (m, 6H), 7.26−7.17 (m, 4H),
6.62 (s, 2H), 3.89 (s, 4H), 3.30 (t, 2H, J = 7.2 Hz), 3.23−3.20 (m,
2H), 2.91−2.88 (m, 2H), 2.59 (t, 2H, J = 7.3 Hz), 1.88−1.72 (m, 6H),
1.61−1.59 (m, 4H), 1.32−1.25 (m, 6H). 13C NMR (CDCl3, 75 MHz):
1
42%. H NMR (CDCl3, 500 MHz): δ ppm 7.96 (d, 1H, J = 8.3 Hz),
7.80 (d, 1H, J = 8.5 Hz), 7.55−7.51 (m, 1H), 7.34 (t, 1H, J = 7.9 Hz),
7.12−7.09 (m, 4H), 6.77−6.73 (m, 2H), 6.63 (s, 2H), 3.98 (s, 6H),
3.90 (s, 4H), 3.57 (t, 2H, J = 7.1 Hz), 3.15−3.08 (m, 4H), 2.61 (t, 2H,
J = 7.2 Hz), 2.14−2.08 (m, 2H), 1.75−1.69 (m, 2H), 1.64−1.58 (m,
126
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
δ ppm 156.9, 156.6, 152.2, 145.3, 145.0, 144.2, 128.3, 125.1, 123.8,
122.6, 122.0, 120.6, 111.2, 106.3, 105.5, 53.4, 50.6, 50.3, 38.8, 31.7,
31.4, 29.7, 29.1, 27.9, 27.5, 27.3, 27.1, 26.9, 26.6, 26.2. IR (KBr): ν
3420, 2925, 2854, 1589, 1271, 751 cm−1. MS (m/z): 586.5 ([M +
H]+). HRMS (m/z): calcd for C39H44N3O2 [M + H]+, 586.3428;
found, 586.3415.
Laboratory Animals.93 The procedures were approved by the Animal
Care and Use Committee of the China Pharmaceutical University. The
mice (ICR male mice, 8−10 weeks old, weight 20−25 g) were orally
administered with tacrine and 2e (20 μmol/kg body weight) 30 min
before ip injection with scopolamine (1 mg/kg) or saline for 10
consecutive days. From the 11th day onward, the Morris water maze
test was carried out 30 min after scopolamine injection.94 Data for the
escape latency, the distances traveled and the number of platform
location crossings were collected by video equipment and processed
by a computer equipped with an analysis-management system
(Viewer2 Tracking Software, Ji Liang Instruments, Shanghai, China).
Behavioral Studies. The experiments were performed with adult
male ICR mice (8−10 weeks old, weight 20−25 g), which were
purchased from the Yangzhou University Medical Center (Yangzhou,
China). Scopolamine hydrobromide was obtained from Aladdin
Reagents (Shanghai, China). Tacrine was synthesized in the lab with
>95% purity as determined by HPLC.
N1,N1-Bis((7-methoxybenzofuran-2-yl)methyl)-N7-(7,8,9,10-tetra-
hydro-6H-cyclohepta[b]quinolin-11-yl)heptane-1,7-diamine (3h).
Compound 3h was prepared according to the general procedure II,
1
pale-brown oil, yield 38%. H NMR (CDCl3, 300 MHz): δ ppm 8.21
(d, 1H, J = 8.1 Hz), 7.91 (d, 1H, J = 8.4 Hz), 7.62 (t, 1H, J = 7.7 Hz),
7.43 (t, 1H, J = 8.0 Hz), 7.12−7.11 (m, 4H), 6.77−6.74 (m, 2H), 6.63
(s, 2H), 3.99 (s, 6H), 3.91 (s, 4H), 3.39 (t, 2H, J = 7.0 Hz), 3.30−3.27
(m, 2H), 2.89−2.86 (m, 2H), 2.60 (t, 2H, J = 7.1 Hz), 1.88−1.81 (m,
4H), 1.71−1.54 (m, 6H), 1.32−1.25 (m, 6H). 13C NMR (CDCl3, 75
MHz): δ ppm 156.8, 156.6, 152.3, 145.1, 144.9, 144.1, 130.2, 129.8,
125.7, 125.3, 123.3, 122.6, 122.4, 122.2, 113.1, 106.2, 105.5, 56.0, 53.3,
50.6, 50.2, 36.5, 31.6, 31.3, 29.7, 29.0, 27.7, 27.3, 26.9, 26.8, 26.4.
IR(KBr): ν 3421, 2923, 2851, 1582, 1493, 1266, 750 cm−1. MS(m/z):
646.5 ([M + H]+). HRMS (m/z): calcd for C41H48N3O4 [M + H]+,
646.3649; found, 646.3616.
Determination of the 2e−TcAChE Crystal Structure. a. Crys-
tallization. TcAChE was isolated, purified, and crystallized as
previously described,84 except for the affinity chromatography ligand,
mono(aminocaproyl)-p-aminophenyltrimethylammonium. Because of
its relatively limited solubility in water, 2e was dissolved in DMSO
(100 mM). The crystals of the complex were obtained by soaking the
native crystals at 4 °C for 12 h in 2 mM 2e, 30% PEG [poly(ethylene
glycol)] 200, 8% DMSO, and 100 mM 2-(N-morpholino)-
ethanesulfonic acid (MES) at pH 6.0.
b. Structure Determination. X-ray diffraction data were collected at
the XRD-1 beamline of the Italian Synchrotron Facility ELETTRA
(Trieste, Italy).85 A PILATUS 2 M detector (Dectris Ltd., Baden,
Switzerland) and focusing optics were employed for the measure-
ments. The crystals were flash-cooled in a nitrogen stream at 100 K
using an Oxford Cryosystems cooling device (Oxford, UK). Data were
processed with MOSFLM version 7.0.786,87 and the CCP4 package
version 6.3.0 software.88 The enzyme−inhibitor structure was
determined by Patterson search methods with the PHASER package
version 2.3.089 using the refined coordinates of the native TcAChE
(PDB ID 1EA5)53 after removal of the water molecules. Crystallo-
graphic refinement of the complex was performed with REFMAC
version 5.7.90 All data within the resolution range were included with
no σ-cutoff. Bulk solvent correction and anisotropic scaling were
applied. The Fourier maps were computed with σ-A weighted (2|Fo| −
|Fc|, Φc) and (|Fo| − |Fc|, Φc) coefficients91 after initial refinement of
the native protein by the rigid body followed by maximum likelihood
positional and individual isotropic temperature factor refinements. A
prominent electron density feature in the catalytic gorge of TcAChE
and along the gorge itself allowed unambiguous fitting of the inhibitor.
The initial Fourier difference map clearly indicated that a change
occurred in the main-chain conformation of the loop encompassing
residues Phe284 to Phe290 relative to its conformation in native
TcAChE. These residues were therefore removed from the model
during the early rounds of refinement and refitted in stages as the
refinement progressed, along with the respective side-chain
orientations of residues within the Trp279 to Phe290 tract.
Carbohydrates (N-acetyl β-D-glucosamine linked at Asn59, Asn416,
and Asn457) were built in by inspecting electron density maps. Peaks
in the difference Fourier maps that were greater than 1.8 rmsd were
automatically added as water molecules to the atomic model and
retained if they met stereochemical requirements, and their B factor
were less than 80 Å2 after refinement. Map inspection and model
correction during refinement were based on the graphics program
COOT version 0.7.92 The crystal parameters, data collection, and
refinement statistics are summarized in Supporting Information, Table
S1. Figures were created using PyMOL version 1.7.2.1 (http://www.
pymol.org).
The mice were divided into four groups, and compounds were given
to each group as follows: (i) vehicle, (ii) scopolamine, (iii) tacrine plus
scopolamine, and (iv) compound 2e plus scopolamine. Mice in groups
(iii) and (iv) were orally administered with tacrine and 2e (20 μmol/
kg body weight), respectively, 30 min before the ip administration of
scopolamine (1 mg/kg) or saline for 10 consecutive days.
Cognitive function was evaluated by the Morris water maze. The
apparatus and test procedure have been previously described.94 The
test included 5 days of learning and memory training and a probe trial
on day 6. Mice were individually trained in a circular pool (110 cm
diameter, 60 cm height) filled to a depth of 40 cm with water at 25 °C.
The maze was located in a lit room with visual cues. An escape
platform (10 cm diameter) was placed in the center of one quadrant of
the pool. The platform position was fixed, and the animal starting
positions, facing the pool wall, were pseudorandomized for each trial.
Each mouse was separately tested on both visible-platform (days 1−2)
and hidden-platform (days 3−5) versions of the water maze. The
hidden-platform version evaluates the spatial learning and was used to
determine the retention of memory to find the platform. During the
hidden-platform training trials, the escape platform was placed 1 cm
below the surface of the water. On each day, the animal was subjected
to three trials with a 20 min interval between trials. Each trial lasted for
90 s, unless the animal reached the platform before that time elapsed.
The time taken for the mouse to reach the platform (a successful
escape) was recorded. If an animal failed to find the platform within 90
s, the test ended and the animal was gently navigated to the platform
by hand. Whether a mouse found or failed to find the platform within
90 s, it was kept on the platform for 30 s. On the last day (day 6), the
platform was removed from its location and the animals were given a
probe trial in which they had 90 s to search for the platform. The time
taken to reach the missing platform and the number of times the
animals crossed the platform location were recorded. All animals
underwent nonspatial pretraining during the first two training days,
which prepared them for the spatial learning test. During the visible
trials of the water maze, mice were trained to find the platform, which
was made visible to them by a small flag (5 cm tall).
Data for the escape latency, the distances traveled, and the number
of platform location crossings were recorded and processed by an
analysis-management system (Viewer2 Tracking Software, Ji Liang
Instruments, China).
Hepatotoxicity Studies. The experiments were carried out on adult
male ICR mice (weighing 18−22 g), which were purchased from the
Yangzhou University Medicine Centre (Yangzhou, China). Tacrine
was dissolved in a sodium carboxymethyl cellulose (CMC-Na)
solution (0.5 g CMC-Na in 100 mL distilled water) and 30 mg/kg
body wt, corresponding to 151.3 μmol/kg body wt., were given
intragastrically (ig). Test compounds were dissolved in CMC-Na
solution, and an equimolar dose corresponding to tacrine was
administered ig. Heparinized serum was obtained 8, 22, and 36 h
after dosing from the retrobulbar plexus to determine aspartate
aminotransferase (ASAT) and alanine aminotransferase (ALT)
activity, two indicators of liver damage, using routine colorimetric
methods.18,95
Animal Studies. All experiments were performed in accordance
with the National Institutes of Health Guide for the Care and Use of
127
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
Statistics. The number of animals investigated per group for the
biochemical investigations comprised n = 8−9. For behavioral studies,
9−10 mice per group were used. Results are expressed as the
Gregory E. Garcia, Research Division, U.S. Army Medical
Research Institute of Chemical Defense (Aberdeen Proving
Ground, MD, USA) for providing us with purified TcAChE
used for the structural study and for a fruitful long-term
collaboration.
arithmetic means
SD or means
SEM. For statistical analyses,
Student’s t test was applied.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01119.
■
ABBREVIATIONS USED
■
S
AD, Alzheimer’s disease; ACh, acetylcholine; ChEs, cholines-
terases; AChE, acetylcholinesterase; BChE, butyrylcholinester-
ase; CAS, catalytic active site; PAS, peripheral anionic site; Aβ,
β-amyloid; BACE-1, beta-secretase 1; ChEIs, cholinesterase
inhibitors; MTDLs, multitarget-directed ligands; LAH, lithium
aluminum hydride; THF, tetrahydrofuran; TLT, transfer
latency time; ASAT, aspartate aminotransferase; ALT, alanine
aminotransferase
Synthetic procedures and chemical characterization of
the intermediates 4f and 7; hAChE and hBChE
inhibition assay; kinetic analysis of hAChE inhibition;
TcAChE inhibition by compound 2e; potentiometric
titration of compound 2e by TcAChE; hAChE-induced
Aβ1−40 aggregation inhibition assay; inhibition of Aβ1−42
self-aggregation. hBACE-1 inhibition assay; cell culture of
the human neuroblastoma cell line SH-SY5Y; cell
viability assay measured as reduction of MTT for
derivatives 1h and 3c, crystallographic data collection
and refinement statistics, kinetic studies for derivatives
1e, 1f, 1h, and 3c on TcAChE; full and detailed results
from animal studies; chromatographic determination of
REFERENCES
■
(1) Murphy, S. L.; Xu, J. Q.; Kochanek, K. D. Deaths: Final Data for
2010; National Center for Health Statistics: Hyattsville, MD, 2013.
(2) Rosini, M. Polypharmacology: the rise of multitarget drugs over
combination therapies. Future Med. Chem. 2014, 6, 485−487.
(3) Leon, R.; Garcia, A. G.; Marco-Contelles, J. Recent advances in
the multitarget-directed ligands approach for the treatment of
Alzheimer’s disease. Med. Res. Rev. 2013, 33, 139−189.
(4) Agis-Torres, A.; Sollhuber, M.; Fernandez, M.; Sanchez-Montero,
J. M. Multi-target-directed ligands and other therapeutic strategies in
the search of a real solution for Alzheimer’s disease. Curr.
Neuropharmacol. 2014, 12, 2−36.
1
compounds purity; H NMR, 13C NMR, and HRMS
spectra for N1-(benzofuran-2-ylmethyl)-N7-(1,2,3,4-tetra-
hydroacridin-9-yl)heptane-1,7-diamine (2e) (PDF)
Molecular formula strings (CSV)
(5) Bolea, I.; Gella, A.; Unzeta, M. Propargylamine-derived
multitarget-directed ligands: fighting Alzheimer’s disease with mono-
amine oxidase inhibitors. J. Neural Transm. 2013, 120, 893−902.
(6) Bartolini, M.; Bertucci, C.; Cavrini, V.; Andrisano, V. Beta-
amyloid aggregation induced by human acetylcholinesterase: inhibition
studies. Biochem. Pharmacol. 2003, 65, 407−416.
Accession Codes
Atomic coordinates and structure factor amplitudes of the
TcAChE−2e complex have been deposited in the Brookhaven
Protein Data Bank under the PDB ID code 4W63.
(7) Chacon, M. A.; Reyes, A. E.; Inestrosa, N. C. Acetylcholinesterase
induces neuronal cell loss, astrocyte hypertrophy and behavioral
deficits in mammalian hippocampus. J. Neurochem. 2003, 87, 195−204.
(8) Nitsch, R. M.; Slack, B. E.; Wurtman, R. J.; Growdon, J. H.
Release of Alzheimer amyloid precursor derivatives stimulated by
activation of muscarinic acetylcholine receptors. Science 1992, 258,
304−307.
AUTHOR INFORMATION
Corresponding Authors
*For X.Z.: phone, +86-25-83271057; fax, +86-25-83271142; e-
mail, xmzha@cpu.edu.cn.
*For M.B.: phone, +39-051-2099729; fax, 39-051-2099734; e-
mail, manuela.bartolini3@unibo.it.
■
(9) Peng, Y.; Jiang, L.; Lee, D. Y.; Schachter, S. C.; Ma, Z.; Lemere,
C. A. Effects of huperzine A on amyloid precursor protein processing
and beta-amyloid generation in human embryonic kidney 293 APP
Swedish mutant cells. J. Neurosci. Res. 2006, 84, 903−911.
(10) Reale, M.; Di Nicola, M.; Velluto, L.; D’Angelo, C.; Costantini,
E.; Lahiri, D. K.; Kamal, M. A.; Yu, Q. S.; Greig, N. H. Selective acetyl-
and butyrylcholinesterase inhibitors reduce amyloid-beta ex vivo
activation of peripheral chemo-cytokines from Alzheimer’s disease
subjects: exploring the cholinergic anti-inflammatory pathway. Curr.
Alzheimer Res. 2014, 11, 608−622.
(11) Watkins, P. B.; Zimmerman, H. J.; Knapp, M. J.; Gracon, S. I.;
Lewis, K. W. Hepatotoxic effects of tacrine administration in patients
with Alzheimer’s disease. JAMA, J. Am. Med. Assoc. 1994, 271, 992−
998.
(12) Tumiatti, V.; Minarini, A.; Bolognesi, M. L.; Milelli, A.; Rosini,
M.; Melchiorre, C. Tacrine derivatives and Alzheimer’s disease. Curr.
Med. Chem. 2010, 17, 1825−1838.
(13) Lupp, A.; Appenroth, D.; Fang, L.; Decker, M.; Lehmann, J.;
Fleck, C. Tacrine-NO donor and tacrine-ferulic acid hybrid molecules
as new anti-Alzheimer agents: hepatotoxicity and influence on the
cytochrome P450 system in comparison to tacrine. Arzneim. Forsch.
2010, 60, 229−237.
(14) Romero, A.; Cacabelos, R.; Oset-Gasque, M. J.; Samadi, A.;
Marco-Contelles, J. Novel tacrine-related drugs as potential candidates
for the treatment of Alzheimer’s disease. Bioorg. Med. Chem. Lett. 2013,
23, 1916−1922.
Author Contributions
¶X.Z. and D.L. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
X.Z. acknowledges the National Natural Science Foundation of
China (nos. 30801428, 81273378), Specialized Research Fund
for the Doctoral Program of Higher Education (no.
200803161011), the Natural Science Foundation of Jiangsu
Province (no. BK2011622), and the Fundamental Research
Funds for the Central Universities (no. ZJPZ2013001). M.B.
and V.A. thank UniRimini S.p.A. and CIRI (POR FESR
project). M.G.L. thanks the Spanish Ministry of Economy and
Competence ref. SAF2013-32222 for the financial support. We
also thank Dr. Andrea Milelli for useful discussion. X.Z. thanks
Prof. Hao Hong (China Pharmaceutical University) and Dr.
Susu Tang (China Pharmaceutical University) for their help in
behavioral study. The authors also thank the staff of the XRD-1
beamline at Elettra-Sincrotrone Trieste S.C.p.A., Trieste, Italy,
for assistance during data collection, Professor Israel Silman,
Weizmann Institute of Science, Rehovot, Israel, for providing us
with the purified TcAChE used for the kinetics study, and Dr.
128
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
(15) de Aquino, R. A. N.; Modolo, L. V.; Alves, R. B.; de Fatima, A.
Design of new drugs for the treatment of Alzheimer’s disease based on
tacrine structure. Curr. Drug Targets 2013, 14, 378−397.
(30) Sashidhara, K. V.; Modukuri, R. K.; Jadiya, P.; Dodda, R. P.;
Kumar, M.; Sridhar, B.; Kumar, V.; Haque, R.; Siddiqi, M. I.; Nazir, A.
Benzofuran-chalcone hybrids as potential multifunctional agents
against Alzheimer’s disease: synthesis and in vivo studies with
transgenic caenorhabditis elegans. ChemMedChem 2014, 9, 2671−
2684.
(31) Lee, S. H.; Kim, Y.; Kim, H. Y.; Kim, Y. H.; Kim, M. S.; Kong, J.
Y.; Lee, M. H.; Kim, D. J.; Ahn, Y. G. Aminostyrylbenzofuran directly
reduces oligomeric amyloid-beta and reverses cognitive deficits in
Alzheimer transgenic mice. PLoS One 2014, 9, e95733.
(32) Yuan, J.; Venkatraman, S.; Zheng, Y.; McKeever, B. M.; Dillard,
L. W.; Singh, S. B. Structure-based design of beta-site APP cleaving
enzyme 1 (BACE1) inhibitors for the treatment of Alzheimer’s disease.
J. Med. Chem. 2013, 56, 4156−4180.
(33) Viayna, E.; Sola, I.; Bartolini, M.; De Simone, A.; Tapia-Rojas,
C.; Serrano, F. G.; Sabate, R.; Juarez-Jimenez, J.; Perez, B.; Luque, F. J.;
Andrisano, V.; Clos, M. V.; Inestrosa, N. C.; Munoz-Torrero, D.
Synthesis and multitarget biological profiling of a novel family of rhein
derivatives as disease-modifying anti-Alzheimer agents. J. Med. Chem.
2014, 57, 2549−2567.
(34) Ghosh, A. K.; Brindisi, M.; Tang, J. Developing beta-secretase
inhibitors for treatment of Alzheimer’s disease. J. Neurochem. 2012,
120 (Suppl s1), 71−83.
(35) Li, S. Y.; Jiang, N.; Xie, S. S.; Wang, K. D.; Wang, X. B.; Kong, L.
Y. Design, synthesis and evaluation of novel tacrine-rhein hybrids as
multifunctional agents for the treatment of Alzheimer’s disease. Org.
Biomol. Chem. 2014, 12, 801−814.
(36) Szymanski, P.; Laznickova, A.; Laznicek, M.; Bajda, M.;
Malawska, B.; Markowicz, M.; Mikiciuk-Olasik, E. 2,3-Dihydro-1H-
cyclopenta[b]quinoline derivatives as acetylcholinesterase inhibitors-
synthesis, radiolabeling and biodistribution. Int. J. Mol. Sci. 2012, 13,
10067−10090.
(37) Ronco, C.; Jean, L.; Outaabout, H.; Renard, P. Y. Palladium-
catalyzed preparation of N-alkylated tacrine and huprine compounds.
Eur. J. Org. Chem. 2011, 2011, 302−310.
(38) Baillie, S. E.; Bluemke, T. D.; Clegg, W.; Kennedy, A. R.; Klett,
J.; Russo, L.; de Tullio, M.; Hevia, E. Potassium-alkyl magnesiates:
synthesis, structures and Mg-H exchange applications of aromatic and
heterocyclic substrates. Chem. Commun. 2014, 50, 12859−12862.
(39) Bondar, G.; Coish, P.; Lowe, D.; O’connor, S. J.; Shelekhin, T.;
Smith, R. Preparation and use of biphenyl amino acid derivatives for
the treatment of obesity. WO 2007016538 A2, 2007.
(40) Yu, W. Y.; Tsoi, Y. T.; Zhou, Z.; Chan, A. S. Palladium-catalyzed
cross coupling reaction of benzyl bromides with diazoesters for
stereoselective synthesis of (E)-alpha,beta-diarylacrylates. Org. Lett.
2009, 11, 469−472.
(41) Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Feather-Stone, R.
M. A new and rapid colorimetric determination of acetylcholinesterase
activity. Biochem. Pharmacol. 1961, 7, 88−95.
(42) Pang, Y. P.; Quiram, P.; Jelacic, T.; Hong, F.; Brimijoin, S.
Highly potent, selective, and low cost bis-tetrahydroaminacrine
inhibitors of acetylcholinesterase. Steps toward novel drugs for treating
Alzheimer’s disease. J. Biol. Chem. 1996, 271, 23646−23649.
(43) Wang, H.; Carlier, P. R.; Ho, W. L.; Wu, D. C.; Lee, N. T.; Li, C.
P.; Pang, Y. P.; Han, Y. F. Effects of bis(7)-tacrine, a novel anti-
Alzheimer’s agent, on rat brain AChE. NeuroReport 1999, 10, 789−
793.
(44) Minarini, A.; Milelli, A.; Tumiatti, V.; Rosini, M.; Simoni, E.;
Bolognesi, M. L.; Andrisano, V.; Bartolini, M.; Motori, E.; Angeloni,
C.; Hrelia, S. Cystamine-tacrine dimer: a new multi-target-directed
ligand as potential therapeutic agent for Alzheimer’s disease treatment.
Neuropharmacology 2012, 62, 997−1003.
(16) Minarini, A.; Milelli, A.; Simoni, E.; Rosini, M.; Bolognesi, M. L.;
Marchetti, C.; Tumiatti, V. Multifunctional tacrine derivatives in
Alzheimer’s disease. Curr. Top. Med. Chem. 2013, 13, 1771−1786.
(17) Nepovimova, E.; Uliassi, E.; Korabecny, J.; Pena-Altamira, L. E.;
Samez, S.; Pesaresi, A.; Garcia, G. E.; Bartolini, M.; Andrisano, V.;
Bergamini, C.; Fato, R.; Lamba, D.; Roberti, M.; Kuca, K.; Monti, B.;
Bolognesi, M. L. Multitarget drug design strategy: quinone-tacrine
hybrids designed to block amyloid-beta aggregation and to exert
anticholinesterase and antioxidant effects. J. Med. Chem. 2014, 57,
8576−8589.
(18) Chen, Y.; Sun, J.; Fang, L.; Liu, M.; Peng, S.; Liao, H.; Lehmann,
J.; Zhang, Y. Tacrine-ferulic acid-nitric oxide (NO) donor trihybrids as
potent, multifunctional acetyl- and butyrylcholinesterase inhibitors. J.
Med. Chem. 2012, 55, 4309−4321.
(19) Benchekroun, M.; Bartolini, M.; Egea, J.; Romero, A.; Soriano,
E.; Pudlo, M.; Luzet, V.; Andrisano, V.; Jimeno, M. L.; Lopez, M. G.;
Wehle, S.; Gharbi, T.; Refouvelet, B.; de Andres, L.; Herrera-
Arozamena, C.; Monti, B.; Bolognesi, M. L.; Rodriguez-Franco, M. I.;
Decker, M.; Marco-Contelles, J.; Ismaili, L. Novel tacrine-grafted Ugi
adducts as multipotent anti-Alzheimer drugs: a synthetic renewal in
tacrine-ferulic acid hybrids. ChemMedChem 2015, 10, 523−539.
(20) Esquivias-Perez, M.; Maalej, E.; Romero, A.; Chabchoub, F.;
Samadi, A.; Marco-Contelles, J.; Oset-Gasque, M. J. Nontoxic and
neuroprotective beta-naphthotacrines for Alzheimer’s disease. Chem.
Res. Toxicol. 2013, 26, 986−992.
(21) Ballard, C. G.; Greig, N. H.; Guillozet-Bongaarts, A. L.; Enz, A.;
Darvesh, S. Cholinesterases: roles in the brain during health and
disease. Curr. Alzheimer Res. 2005, 2, 307−318.
(22) Greig, N. H.; Utsuki, T.; Ingram, D. K.; Wang, Y.; Pepeu, G.;
Scali, C.; Yu, Q. S.; Mamczarz, J.; Holloway, H. W.; Giordano, T.;
Chen, D.; Furukawa, K.; Sambamurti, K.; Brossi, A.; Lahiri, D. K.
Selective butyrylcholinesterase inhibition elevates brain acetylcholine,
augments learning and lowers Alzheimer beta-amyloid peptide in
rodent. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 17213−17218.
(23) Furukawa-Hibi, Y.; Alkam, T.; Nitta, A.; Matsuyama, A.;
Mizoguchi, H.; Suzuki, K.; Moussaoui, S.; Yu, Q. S.; Greig, N. H.;
Nagai, T.; Yamada, K. Butyrylcholinesterase inhibitors ameliorate
cognitive dysfunction induced by amyloid-beta peptide in mice. Behav.
Brain Res. 2011, 225, 222−229.
(24) Baharloo, F.; Moslemin, M. H.; Nadri, H.; Asadipour, A.;
Mahdavi, M.; Emami, S.; Firoozpour, L.; Mohebat, R.; Shafiee, A.;
Foroumadi, A. Benzofuran-derived benzylpyridinium bromides as
potent acetylcholinesterase inhibitors. Eur. J. Med. Chem. 2015, 93,
196−201.
(25) Howlett, D. R.; Perry, A. E.; Godfrey, F.; Swatton, J. E.;
Jennings, K. H.; Spitzfaden, C.; Wadsworth, H.; Wood, S. J.; Markwell,
R. E. Inhibition of fibril formation in beta-amyloid peptide by a novel
series of benzofurans. Biochem. J. 1999, 340, 283−289.
(26) Byun, J. H.; Kim, H.; Kim, Y.; Mook-Jung, I.; Kim, D. J.; Lee, W.
K.; Yoo, K. H. Aminostyrylbenzofuran derivatives as potent inhibitors
for Abeta fibril formation. Bioorg. Med. Chem. Lett. 2008, 18, 5591−
5593.
(27) Ono, M.; Kung, M. P.; Hou, C.; Kung, H. F. Benzofuran
derivatives as Abeta-aggregate-specific imaging agents for Alzheimer’s
disease. Nucl. Med. Biol. 2002, 29, 633−642.
(28) Allsop, D.; Gibson, G.; Martin, I. K.; Moore, S.; Turnbull, S.;
Twyman, L. J. 3-p-Toluoyl-2-[4′-(3-diethylaminopropoxy)-phenyl]-
benzofuran and 2-[4′-(3-diethylaminopropoxy)-phenyl]-benzofuran
do not act as surfactants or micelles when inhibiting the aggregation
of beta-amyloid peptide. Bioorg. Med. Chem. Lett. 2001, 11, 255−257.
(29) Rizzo, S.; Tarozzi, A.; Bartolini, M.; Da Costa, G.; Bisi, A.;
Gobbi, S.; Belluti, F.; Ligresti, A.; Allara, M.; Monti, J. P.; Andrisano,
V.; Di Marzo, V.; Hrelia, P.; Rampa, A. 2-Arylbenzofuran-based
molecules as multipotent Alzheimer’s disease modifying agents. Eur. J.
Med. Chem. 2012, 58, 519−532.
(45) Fu, H.; Li, W.; Lao, Y.; Luo, J.; Lee, N. T.; Kan, K. K.; Tsang, H.
W.; Tsim, K. W.; Pang, Y.; Li, Z.; Chang, D. C.; Li, M.; Han, Y. Bis(7)-
tacrine attenuates beta amyloid-induced neuronal apoptosis by
regulating L-type calcium channels. J. Neurochem. 2006, 98, 1400−
1410.
(46) Xiao, X. Q.; Lee, N. T.; Carlier, P. R.; Pang, Y.; Han, Y. F.
Bis(7)-tacrine, a promising anti-Alzheimer’s agent, reduces hydrogen
129
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
peroxide-induced injury in rat pheochromocytoma cells: comparison
with tacrine. Neurosci. Lett. 2000, 290, 197−200.
an induced-fit mechanism. Proc. Natl. Acad. Sci. U. S. A. 2006, 103,
15451−15456.
(47) Hamulakova, S.; Janovec, L.; Hrabinova, M.; Spilovska, K.;
Korabecny, J.; Kristian, P.; Kuca, K.; Imrich, J. Synthesis and biological
evaluation of novel tacrine derivatives and tacrine-coumarin hybrids as
cholinesterase inhibitors. J. Med. Chem. 2014, 57, 7073−7084.
(48) Hui, A.-L.; Chen, Y.; Zhu, S.-J.; Gan, C.-S.; Pan, J.; Zhou, A.
Design and synthesis of tacrine-phenothiazine hybrids as multitarget
drugs for Alzheimer’s disease. Med. Chem. Res. 2014, 23, 3546−3557.
(49) Mesulam, M.; Guillozet, A.; Shaw, P.; Quinn, B. Widely spread
butyrylcholinesterase can hydrolyze acetylcholine in the normal and
Alzheimer brain. Neurobiol. Dis. 2002, 9, 88−93.
(64) Castro, A.; Martinez, A. Targeting beta-amyloid pathogenesis
through acetylcholinesterase inhibitors. Curr. Pharm. Des. 2006, 12,
4377−4387.
(65) Pi, R.; Mao, X.; Chao, X.; Cheng, Z.; Liu, M.; Duan, X.; Ye, M.;
Chen, X.; Mei, Z.; Liu, P.; Li, W.; Han, Y. Tacrine-6-ferulic acid, a
novel multifunctional dimer, inhibits amyloid-beta-mediated Alz-
heimer’s disease-associated pathogenesis in vitro and in vivo. PLoS
One 2012, 7, e31921.
(66) Yan, R.; Vassar, R. Targeting the beta secretase BACE1 for
Alzheimer’s disease therapy. Lancet Neurol. 2014, 13, 319−329.
(67) Ghosh, A. K.; Tang, J. Prospects of β-secretase inhibitors for the
treatment of Alzheimer’s Disease. ChemMedChem 2015, 10, 1463−
1466.
(68) Hanessian, S.; Yun, H.; Hou, Y.; Yang, G.; Bayrakdarian, M.;
Therrien, E.; Moitessier, N.; Roggo, S.; Veenstra, S.; Tintelnot-
Blomley, M.; Rondeau, J. M.; Ostermeier, C.; Strauss, A.; Ramage, P.;
Paganetti, P.; Neumann, U.; Betschart, C. Structure-based design,
synthesis, and memapsin 2 (BACE) inhibitory activity of carbocyclic
and heterocyclic peptidomimetics. J. Med. Chem. 2005, 48, 5175−
5190.
(69) De Simone, A.; Mancini, F.; Real Fernandez, F.; Rovero, P.;
Bertucci, C.; Andrisano, V. Surface plasmon resonance, fluorescence,
and circular dichroism studies for the characterization of the binding of
BACE-1 inhibitors. Anal. Bioanal. Chem. 2013, 405, 827−835.
(70) Willem, M.; Garratt, A. N.; Novak, B.; Citron, M.; Kaufmann, S.;
Rittger, A.; DeStrooper, B.; Saftig, P.; Birchmeier, C.; Haass, C.
Control of peripheral nerve myelination by the beta-secretase BACE1.
Science 2006, 314, 664−666.
(71) Savonenko, A. V.; Melnikova, T.; Laird, F. M.; Stewart, K. A.;
Price, D. L.; Wong, P. C. Alteration of BACE1-dependent NRG1/
ErbB4 signaling and schizophrenia-like phenotypes in BACE1-null
mice. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 5585−5590.
(72) Vassar, R. BACE1 inhibitor drugs in clinical trials for
Alzheimer’s disease. Alzheimer's Res. Ther. 2014, 6, 89.
(73) Ghosh, A. K.; Osswald, H. L. BACE1 (beta-secretase) inhibitors
for the treatment of Alzheimer’s disease. Chem. Soc. Rev. 2014, 43,
6765−6813.
(74) Cavalli, A.; Bolognesi, M. L.; Capsoni, S.; Andrisano, V.;
Bartolini, M.; Margotti, E.; Cattaneo, A.; Recanatini, M.; Melchiorre,
C. A small molecule targeting the multifactorial nature of Alzheimer’s
disease. Angew. Chem., Int. Ed. 2007, 46, 3689−3692.
(75) Prati, F.; De Simone, A.; Bisignano, P.; Armirotti, A.; Summa,
M.; Pizzirani, D.; Scarpelli, R.; Perez, D. I.; Andrisano, V.; Perez-
Castillo, A.; Monti, B.; Massenzio, F.; Polito, L.; Racchi, M.; Favia, A.
D.; Bottegoni, G.; Martinez, A.; Bolognesi, M. L.; Cavalli, A.
Multitarget drug discovery for Alzheimer’s disease: triazinones as
BACE-1 and GSK-3beta inhibitors. Angew. Chem., Int. Ed. 2015, 54,
1578−1582.
(50) Venneri, A.; McGeown, W. J.; Shanks, M. F. Empirical evidence
of neuroprotection by dual cholinesterase inhibition in Alzheimer’s
disease. NeuroReport 2005, 16, 107−110.
(51) Greig, N. H.; Lahiri, D. K.; Sambamurti, K. Butyrylcholinester-
ase: an important new target in Alzheimer’s disease therapy. Int.
Psychogeriatr. 1999, 14 (Suppl S1), 77−91.
(52) Inestrosa, N. C.; Dinamarca, M. C.; Alvarez, A. Amyloid-
cholinesterase interactions. Implications for Alzheimer’s disease. FEBS
J. 2008, 275, 625−632.
(53) Dvir, H.; Jiang, H. L.; Wong, D. M.; Harel, M.; Chetrit, M.; He,
X. C.; Jin, G. Y.; Yu, G. L.; Tang, X. C.; Silman, I.; Bai, D. L.; Sussman,
J. L. X-ray structures of Torpedo californica acetylcholinesterase
complexed with (+)-huperzine A and (−)-huperzine B: structural
evidence for an active site rearrangement. Biochemistry 2002, 41,
10810−10818.
(54) Millard, C. B.; Kryger, G.; Ordentlich, A.; Greenblatt, H. M.;
Harel, M.; Raves, M. L.; Segall, Y.; Barak, D.; Shafferman, A.; Silman,
I.; Sussman, J. L. Crystal structures of aged phosphonylated
acetylcholinesterase: nerve agent reaction products at the atomic
level. Biochemistry 1999, 38, 7032−7039.
(55) Harel, M.; Schalk, I.; Ehret-Sabatier, L.; Bouet, F.; Goeldner, M.;
Hirth, C.; Axelsen, P. H.; Silman, I.; Sussman, J. L. Quaternary ligand
binding to aromatic residues in the active-site gorge of acetylcholi-
nesterase. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 9031−9035.
(56) Rydberg, E. H.; Brumshtein, B.; Greenblatt, H. M.; Wong, D.
M.; Shaya, D.; Williams, L. D.; Carlier, P. R.; Pang, Y. P.; Silman, I.;
Sussman, J. L. Complexes of alkylene-linked tacrine dimers with
Torpedo californica acetylcholinesterase: Binding of Bis5-tacrine
produces a dramatic rearrangement in the active-site gorge. J. Med.
Chem. 2006, 49, 5491−5500.
(57) Colletier, J. P.; Sanson, B.; Nachon, F.; Gabellieri, E.; Fattorusso,
C.; Campiani, G.; Weik, M. Conformational flexibility in the peripheral
site of Torpedo californica acetylcholinesterase revealed by the complex
structure with a bifunctional inhibitor. J. Am. Chem. Soc. 2006, 128,
4526−4527.
(58) Desai, M. C.; Thadeio, P. F.; Lipinski, C. A.; Liston, D. R.;
Spencer, R. W.; Williams, I. H. Physical parameters for brain uptake:
optimizing log P, log D, and pKa of THA. Bioorg. Med. Chem. Lett.
1991, 1, 411−414.
(59) Greenblatt, H. M.; Guillou, C.; Guenard, D.; Argaman, A.; Botti,
S.; Badet, B.; Thal, C.; Silman, I.; Sussman, J. L. The complex of a
bivalent derivative of galanthamine with torpedo acetylcholinesterase
displays drastic deformation of the active-site gorge: implications for
structure-based drug design. J. Am. Chem. Soc. 2004, 126, 15405−
15411.
(60) Morel, N.; Bon, S.; Greenblatt, H. M.; Van Belle, D.; Wodak, S.
J.; Sussman, J. L.; Massoulie, J.; Silman, I. Effect of mutations within
the peripheral anionic site on the stability of acetylcholinesterase. Mol.
Pharmacol. 1999, 55, 982−992.
(61) Radic, Z.; Taylor, P. Interaction kinetics of reversible inhibitors
and substrates with acetylcholinesterase and its fasciculin 2 complex. J.
Biol. Chem. 2001, 276, 4622−4633.
(76) Turner, R. T., 3rd; Hong, L.; Koelsch, G.; Ghosh, A. K.; Tang, J.
Structural locations and functional roles of new subsites S5, S6, and S7
in memapsin 2 (beta-secretase). Biochemistry 2005, 44, 105−112.
(77) Fu, H.; Li, W.; Luo, J.; Lee, N. T.; Li, M.; Tsim, K. W.; Pang, Y.;
Youdim, M. B.; Han, Y. Promising anti-Alzheimer’s dimer bis(7)-
tacrine reduces beta-amyloid generation by directly inhibiting BACE-1
activity. Biochem. Biophys. Res. Commun. 2008, 366, 631−636.
(78) Butterfield, D. A.; Swomley, A. M.; Sultana, R. Amyloid β-
peptide (1−42)-induced oxidative stress in Alzheimer disease:
importance in disease pathogenesis and progression. Antioxid. Redox
Signaling 2013, 19, 823−835.
(79) Morris, R. Developments of a water-maze procedure for the
studying spatial learning in the rat. J. Neurosci. Methods 1984, 11, 47−
60.
(62) Bui, J. M.; Tai, K.; McCammon, J. A. Acetylcholinesterase:
enhanced fluctuations and alternative routes to the active site in the
complex with fasciculin-2. J. Am. Chem. Soc. 2004, 126, 7198−7205.
(63) Bui, J. M.; McCammon, J. A. Protein complex formation by
acetylcholinesterase and the neurotoxin fasciculin-2 appears to involve
(80) Ozer, J.; Ratner, M.; Shaw, M.; Bailey, W.; Schomaker, S. The
current state of serum biomarkers of hepatotoxicity. Toxicology 2008,
245, 194−205.
(81) Dominguez, J. L.; Fernandez-Nieto, F.; Castro, M.; Catto, M.;
Paleo, M. R.; Porto, S.; Sardina, F. J.; Brea, J.; Carotti, A.; Villaverde,
130
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131

Journal of Medicinal Chemistry
Article
M. C.; Sussman, F. S. Computer aided structure based design of
multitarget leads for Alzheimer’s disease. J. Chem. Inf. Model. 2014, 55,
135−148.
(82) Seubert, P.; Vigo-Pelfrey, C.; Esch, F.; Lee, M.; Dovey, H.;
Davis, D.; Sinha, S.; Schiossmacher, M.; Whaley, J.; Swindlehurst, C.;
et al. Isolation and quantification of soluble Alzheimer’s beta-peptide
from biological fluids. Nature 1992, 359, 325−327.
(83) Hu, M. K.; Wu, L. J.; Hsiao, G.; Yen, M. H. Homodimeric
tacrine congeners as acetylcholinesterase inhibitors. J. Med. Chem.
2002, 45, 2277−2282.
(84) Sussman, J. L.; Harel, M.; Frolow, F.; Varon, L.; Toker, L.;
Futerman, A. H.; Silman, I. Purification and crystallization of a dimeric
form of acetylcholinesterase from Torpedo californica subsequent to
solubilization with phosphatidylinositol-specific phospholipase C. J.
Mol. Biol. 1988, 203, 821−823.
(85) Lausi, A.; Polentarutti, M.; Onesti, S.; Plaisier, J. R.; Busetto, E.;
Bais, G.; Barba, L.; Cassetta, A.; Campi, G.; Lamba, D.; Pifferi, A.;
Mande, S. C.; Sarma, D. D.; Sharma, S. M.; Paolucci, G. Status of the
crystallography beamlines at Elettra. Eur. Phys. J. Plus 2015, 130, 43.
(86) Leslie, A. G. The integration of macromolecular diffraction data.
Acta Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 48−57.
(87) Battye, T. G.; Kontogiannis, L.; Johnson, O.; Powell, H. R.;
Leslie, A. iMOSFLM: a new graphical interface for diffraction-image
processing with MOSFLM. Acta Crystallogr., Sect. D: Biol. Crystallogr.
2011, 67, 271−281.
(88) Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.;
Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie, A. G.;
McCoy, A.; McNicholas, S. J.; Murshudov, G. N.; Pannu, N. S.;
Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin, A.; Wilson, K. S.
Overview of the CCP4 suite and current developments. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 235−242.
(89) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M.
D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. J. Appl.
Crystallogr. 2007, 40, 658−674.
́
(90) Murshudov, G. N.; Skubak, P.; Lebedev, A. A.; Pannu, N. S.;
Steiner, R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A.
REFMAC5 for the refinement of macromolecular crystal structures.
Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 355−367.
(91) Read, R. J. Improved Fourier coefficients for maps using phases
from partial structures with errors. Acta Crystallogr., Sect. A: Found.
Crystallogr. 1986, 42, 140−149.
(92) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and
development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010,
66, 486−501.
(93) National Research Council (US), Committee for the Update of
the Guide for the Care and Use of Laboratory Animals Guide for the
Care and Use of Laboratory Animals, 8th Ed.; The National Academies
Press: Washington, DC, 2011.
(94) Xiang, G. Q.; Tang, S. S.; Jiang, L. Y.; Hong, H.; Li, Q.; Wang,
C.; Wang, X. Y.; Zhang, T. T.; Yin, L. PPARgamma agonist
pioglitazone improves scopolamine-induced memory impairment in
mice. J. Pharm. Pharmacol. 2012, 64, 589−596.
(95) Doumas, B.; Biggs, H. G. A colorimetric method for assaying
serum aspartate aminotransferase activities. Clin. Chim. Acta 1969, 23,
75−82.
131
DOI: 10.1021/acs.jmedchem.5b01119
J. Med. Chem. 2016, 59, 114−131